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Synthesis, antiplasmodial activity and in silico molecular docking study of pinocembrin and its analogs

Synthesis, antiplasmodial activity and in silico molecular docking study of pinocembrin and its... Background: Malaria remains the major health problem responsible for many mortality and morbidity in developing countries. Because of the development of resistance by Plasmodium species, searching effective antimalarial agents becomes increasingly important. Pinocembrin is a flavanone previously isolated as the most active antiplasmodial compound from the leaves of Dodonaea angustifolia. For a better understanding of the antiplasmodial activity, the synthesis of pinocembrin and a great number of analogs was undertaken. Methods: Chalcones 5a-r were synthesized via Claisen-Schmidt condensation using 2,4-dibenzyloxy-6-hydroxy- acetophenone and aromatic aldehydes as substrates under basic conditions. Cyclization of compounds 5a-r to the corresponding dibenzylated pinocembrin analogs 6a-r was achieved using NaOAc in EtOH under reflux. Catalytic hydrogenation using 10% Pd/C as catalyst in an H-Cube Pro was used for debenzylation to deliver 7a-l. The structures of the synthesized compounds were characterized using various physical and spectroscopic methods, including mp, UV, IR, NMR, MS and HRMS. The synthesized dibenzylated flavanones 6a-d, 6i and 7a were evaluated for their in vivo antiplasmodial activities against Plasmodium berghei infected mice. Molecular docking simulation and drug likeness properties of compounds 7a-l were assessed using AutoDock Vina and SwissADME, respectively. Results: A series of chalcones 5a-r has been synthesized in yields ranging from 46 to 98%. Treatment of the chal- cones 5a-r with NaOAc refluxing in EtOH afforded the dibenzylated pinocembrin analogs 6a-r with yields up to 54%. Deprotection of the dibenzylated pinocembrin analogs delivered the products 7a-l in yields ranging from 78 to 94%. The dibenzylated analogs of pinocembrin displayed percent inhibition of parastaemia in the range between 17.4 and 87.2% at 30 mg/kg body weight. The parastaemia inhibition of 87.2 and 55.6% was obtained on treatment of the infected mice with pinocembrin (7a) and 4’-chloro-5,7-dibenzylpinocembrin (6e), respectively. The mean survival times of those infected mice treated with these two compounds were beyond 14 days indicating that the samples suppressed P. berghei and reduced the overall pathogenic effect of the parasite. The molecular docking analysis of the chloro derivatives of pinocembrin revealed that compounds 7a-l show docking affinities ranging from – 8.1 to – 8.4 kcal/mol while it was -7.2 kcal/mol for chloroquine. Conclusion: Pinocembrin (7a) and 4’-chloro-5,7-dibenzyloxyflavanone (6e) displayed good antiplasmodial activ- ity. The in silico docking simulation against P. falciparum dihydrofolate reductase-thymidylate synthase revealed that *Correspondence: yalemtshay.mekonnen@aau.edu.et Biology Department, Addis Ababa University, Addis Ababa, Ethiopia Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Melaku et al. BMC Chemistry (2022) 16:36 Page 2 of 30 pinocembrin (7a) and its chloro analogs 7a-l showed better binding affinity compared with chloroquine that was used as a standard drug. This is in agreement with the drug-like properties of compounds 7a-l which fulfill Lipinski’s rule of five with zero violations. Therefore, pinocembrin and its chloro analogs could serve as lead compounds for further antiplasmodial drug development. Keywords: Catalytic hydrogenation, Claisen-Schmidt Condensation, Malaria, Plasmodium berghei, Pinocembrin, Chloropinocembrin Introduction Results and discussion Malaria remains one of the major health problems in Synthesis of Pinocembrin (7a) and its analogs 7b-l developing countries with high recorded rates of mor- The synthesis of various chalcones was performed after tality and morbidity [1]. The most predominant species protection of trihydroxyacetophenone (1) with benzyl responsible for 90% of malarial cases worldwide is Plas- chloride (2) using potassium carbonate as base in DMF modium falciparum [2]. Nearly half of the world’s pop- (Fig. 1) [10]. The product 3 was obtained as a white solid ulation resides in regions where malaria is endemic and in 79% yield. In the next step, the desired chalcones 5a- are thus at risk of infection [3]. The burden is severe r were prepared via Claisen-Schmidt condensation [11, in sub-Saharan Africa which account for 90% of the 12] of 2,4-dibenzyloxy-6-hydroxyacetophenone (3) with deaths where 5% of children die from the disease before various aromatic aldehydes 4a-r with electron deficient reaching 5 years of age [4]. Efforts to reduce the spread or electron donating properties using KOH as base in have been exacerbated by the increasing resistance of EtOH. The chalcones 5a-r was isolated from 46 to 98% the mosquito to insecticides, and of the parasite to the in yields (Fig.  2). The lowest yields were obtained with currently available drugs [4]. This necessitates search 2-bromobenzaldehyde 4f and 3,4,5-trimethoxybenzalde- for alternative drugs at reasonable cost for use against hyde 4 h as substrates. Low yields of chalcones with ben- malaria. zaldehydes containing a methoxy group in the substrate Flavonoids are phenolic compounds possessing enor- are documented in the literature [13]. mous pharmacological activities. They play signifi - Having secured the synthesis of the intended chal- cant roles in promoting health and preventing chronic cones, we focused our attention towards the synthesis of degenerative diseases. Among flavonoids, pinocem - pinocembrin and its analogs. Previous work has shown brin (7a) is a flavanone with wide arrays of biological that the cyclization of chalcones to flavanones can be activities including antimicrobial, antiinflammatory, effected employing various methods using acids, bases antioxidant, anticancer [5] and against ischemic stroke and celite supported potassium fluoride in methanol [14, [6]. Pinocembrin (7a) has also been reported having 15]. In the present work, the transformation of chalcones antifibrotic effects in addition to its ability to decrease to flavanones was attempted with acetic acid, methane proinflammatory cytokines production [7 ] and protec- sulphonic acid and sodium acetate as catalysts. The lat - tive capacity against gastric tissue damage [8]. In our ter reagent was found to deliver the desired products in previous antiplasmodial study, we isolated pinocembrin higher yields than with the two other reagents. Upon (7a) as the most active compound from the leaves of D. refluxing the chalcones 5a-r (1  mmol) with sodium ace - angustifolia [9]. To establish its antiplasmodial activity tate (8  mmol) in ethanol, they were transformed into an large quantities of this compound are required which equilibrium mixture made up of the chalcones 5a-r and however is present only as a minor constituent in the the flavanones 6a-r (Fig.  2). This is evident as approxi - leaves of D. angustifolia. The synthesis of pinocembrin mately 40% of the starting material chalcone was also analogs with improved antimalarial activity are there- recuperated. Therefore, the low yields of the flavanones fore encouraged for further advances. Hence, to further obtained in the present work can therefore be attributed optimize the antiplasmodial activities of pinocembrin to the reversibility of the chalcone cyclization. This is in (7a), the synthesis of this natural product and analogs agreement with the finding reported by Urgaonkar et al., thereof were undertaken. In this paper, we report the (2005) [16]. synthesis and antiplasmodial activities of pinocem- Compounds 6a-e and 6i were subjected to in vivo anti- brin (7a) and its analogs against Plasmodium berghei plasmodial activity with the better result achieved with in infected mice. Furthermore, the in silico molecular compounds 6a and 6e. The activity displayed by the lat - docking analysis against P. falciparum dihydrofolate ter compound might be due to the presence of a chlo- reductase-thymidylate synthase and the drug-like prop- rine atom. The antiplasmodial activity of dibenzylated erties of compounds 7a-l are also presented herein. pinocembrin 6a was compared with pinocembrin (7a). M elaku et al. BMC Chemistry (2022) 16:36 Page 3 of 30 R R H R BnO OH BnO OBn HO OH O R Cl K CO 2 3 4a-r DMF, 70 C, 3h KOH, EtOH OBn O OH O OH O 1 2 3 5a-r F OMe MeO BnO OH OH BnO OH BnO OH BnO OMe OMe OBn O OBn O OBn O OBn O 5a,80% 5b,68% 5c,67% 5d,67% OMe Cl OMe CF BnO OH OH BnO OH BnO BnO OH OMe Br OBn O OBn O OBn O OBn O 5f,57% 5g,71% 5h,46% 5e,72% CN BnO OH OH BnO OH BnO BnO OH Cl Cl Br Cl OBn O OBn O OBn O OBn O 5i,77% 5l,77% 5j, 97% 5k,67% Cl Cl Cl BnO OH BnO OH BnO OH BnO OH Cl Cl Cl Cl OBn O OBn O OBn O OBn O 5m,92% 5n,96% 5o,70% 5p,69% Cl Cl BnO OH BnO OH Cl Cl OBn O OBn O 5q,70% 5r,98% Fig. 1 Synthesis of chalcones 5a-r Melaku et al. BMC Chemistry (2022) 16:36 Page 4 of 30 2 2 R R 1 5 1 5 R R R BnO OH BnO O 4 NaOAc, EtOH 4 R R R reflux,48h R OBn O OBn O 5a-r 6a-r F OMe MeO BnO O BnO O BnO O BnO O OMe OMe OBn O OBn O OBn O OBn O 6a,50% 6b,48% 6d,52% 6c,45% OMe OMe Cl CF BnO O BnO O O O BnO BnO OMe Br OBn O OBn O OBn O OBn O 6e,40% 6h,38% 6f,36% 6g,8% CN BnO O BnO O BnO O BnO O Cl Cl Br Cl OBn O OBn O OBn O OBn O 6j,26% 6i,48% 6k,14% 6l,42% Cl Cl Cl BnO O BnO O BnO O BnO O Cl Cl Cl Cl OBn O OBn O OBn O OBn O 6p,32% 6m,50% 6n,54% 6o,26% Cl Cl BnO BnO O Cl Cl OBn O OBn O 6q,43% 6r,44% Fig. 2 Synthesis of benzylated flavanones 6a-r M elaku et al. BMC Chemistry (2022) 16:36 Page 5 of 30 The result showed a dramatic decrease in activity of 6a Pd/C as catalyst and a flow rate of 1 mL/min at 70 °C and compared with 7a. This is probably accounted to the 1  bar. This method was found highly attractive as it fur - presence of benzyl group. Hence, we found it neces- nished the corresponding debenzylated products in yields sary to undertake the debenzylation of the pinocembrin ranging between 78 and 94% and in short reaction times analogs. To achieve this goal, the debenzylation of the (Fig.  3). The use of the H-Cube Pro has also eliminated dibenzylated flavanones 6a-r (Fig.  3) was achieved by the dangers associated with hydrogenation by generating catalytic hydrogenation using an H-Cube. To find out hydrogen in situ and the handling of pyrophoric catalysts optimal parameters for the deprotection, different sol - by filling them in sealed catalyst cartridges. This is advan - vents including EtOH, CHCl , EtOAc and EtOH:EtOAc tageous over the conventional debenzylation protocol (1:1) were tested using the conversion of 6a to 7a as an which succeeded to give the desired product neither with example. The latter solvent system was found efficient Pd/C (5%, 10% and Pd black) nor Pd(OH) /C (20%) under in furnishing the product in 93% yield. The reaction various conditions (Up to 15  kg hydrogen pressure and temperature of the deprotection was also optimized. It 70 °C in temperature and varieties of solvents) [17]. was found that the debenzylation of both benzyl groups The structures of the compounds synthesized in the occurred at 70  °C while the undesired monobenzylated present work were characterized using various spectro- product was obtained at 60  °C exclusively. Hence, the scopic methods, including UV, IR and NMR as well as debenzylation of compounds 6b-r to pinocembrin ana- by mass spectrometry. logs 7b-r was performed using an H-Cube Pro with 10% 5 1 5 R R HO O BnO O H ,10% Pd/C,EtOAc:EtOH(1:1) 4 2 0.01M, 1mL/min, 70 C, 1bar OH O OBn O 7a-l 6a,d,e,h,j,l,m,n,o,p,q,r OMe MeO Cl OMe HO O HO O HO O HO O OMe OMe OH O OH O OH O OH O 7a, 93% 7b,78% 7c,91.2% 7d,89% Cl HO O HO O HO O HO O Cl Cl Cl Cl Cl OH O OH O OH O OH O 7e,88% 7f,91.5% 7g,78% 7h,87% Cl Cl Cl Cl HO O HO O HO O HO O Cl Cl Cl Cl OH O OH O OH O OH O 7i, 89% 7j, 91% 7l,88% 7k,94% Fig. 3 Synthesis of flavanones 7a-l Melaku et al. BMC Chemistry (2022) 16:36 Page 6 of 30 Antiplasmodial activity of pinocembrin and its dose 20 mg/kg and the results were turned out to be sig- dibenzylated analogs nificant compared with the NC group (P < 0.05). The in vivo antiplasmodial activities of pinocembrin (7a) In some cases, compounds isolated from natural prod- and some of its dibenzylated analogs (6a, 6b, 6c, 6e and ucts may show instability on exposure to high tempera- 6i) were evaluated at different doses using Peter’s four ture, hence synthesizing analogs could help to increase day suppressive assay against P. berghei infected mice the chemical stability and biological activities of the [18]. The ability to reduce parasitaemia density is an indi - compounds. However, the results in the present study cator of the presence of antimalarial activities in a sample showed that the benzylated analogs exhibited less bio- [19]. A drug/sample which suppresses the parasitaemia logical activity compared with pinocembrin (7a). There - beyond 30% is considered as active against the parasites fore, this study is parallel with the work of Werbel and [20]. In view of this, pinocembrin and its analogs showed Degnan (1987) in which the original quinazoline amino antiplasmodial activities against P. berghei infected mice group depicted a better antiplasmodial activity compared as evidenced from the percentage of parasite inhibition with its analogs [21]. In contrast, Silva et al. [22] reported (Table 1). Pinocembrin (7a) had shown significant in vivo that the analogs of 4-nerolidylcatechol showed a better antiplasmodial activities compared with both control parastaemia inhibition effect (72% suppression at dose groups (P < 0.05). It inhibits the parasitaemia by 74.4% 600 mg/kg b. wt) compared with isolated compound (60% and 87.2% at 20  mg/kg and 30  mg/kg, respectively. The suppression at dose 600 mg/kg b. wt) [22]. Furthermore, result obtained herein is superior to the suppression of flavanone7a inhibits the parasites in a dose dependent parasitaemia observed for pinocembrin isolated from manner with the higher dose causing a higher parastae- D. angustifolia which inhibited the parasites by 80.0% mia inhibition (Table  1). This is in close agreement with at 40  mg/kg [9]. A group of mice treated by 6b and 6e the work of Silva et al. [23] whereby some indole alkaloids showed 25.5% and 55.6% suppression of parasitaemia at a displayed better activity as the dose increases [23]. Table 1 In vivo suppressive effect of pinocembrin and its analogs against P. berghei in Swiss albino mice No. Doses (mg/kg) Parasitological effect MST ± SEM Body Weight ± SEM %Parasitaemia ± SEM %Suppression D0 D4 6a NC 59.00 ± 0.78 0.00 7.00 ± 0.32 24.00 ± 0.63 22.92 ± 0.84 b b 15 57.00 ± 1.28 21.69 9.60 ± 0.40 24.38 ± 0.91 23.38 ± 0.93 bc bc 20 40.00 ± 0.79 32.20 10.40 ± 0.87 22.98 ± 0.80 22.38 ± 0.57 c c 25 46.92 ± 1.17 35.92 12.00 ± 0.55 23.90 ± 0.78 23.36 ± 0.54 c c 30 38.08 ± 0.56 38.04 13.60 ± 0.87 23.44 ± 0.63 22.72 ± 0.53 d d 35 34.80 ± 0.86 49.09 11.60 ± 0.40 23.70 ± 0.56 22.87 ± 0.69 6b NC 39.85 ± 0.80 0.00 7.60 ± 0.25 24.38 ± 0.56 23.60 ± 0.58 b b 20 29.62 ± 1.21 25.52 7.60 ± 0.68 23.92 ± 0.74 22.74 ± 0.64 6c NC 77.40 ± 1.60 0.00 6.80 ± 0.66 23.68 ± 0.98 22.26 ± 0.93 b b 20 51.50 ± 1.14 33.46 11.80 ± 0.37 23.44 ± 0.78 22.74 ± 0.87 6d NC 39.85 ± 0.80a 0.00 7.60 ± 0.25 24.38 ± 0.56 23.60 ± 0.58 30 36.23 ± 2.20a 8.90 a 6.60 ± 0.30 24.32 ± 0.45 22.60 ± 0.65 6e NC 59.00 ± 0.78 0.00 7.00 ± 1.28 24.00 ± 0.63 22.92 ± 0.84 b b 20 26.14 ± 0.93 55.57 14.60 ± 0.51 24.52 ± 0.58 24.52 ± 0.56 6i NC 73.22 ± 1.25 0.00 7.80 ± 0.37 23.66 ± 0.57 22.04 ± 0.46 b b 15 60.46 ± 0.78 17.42 8.80 ± 0.49 23.06 ± 0.60 22.06 ± 0.59 c c 25 51.30 ± 0.44 29.05 8.00 ± 0.45 23.12 ± 0.47 22.90 ± 1.02 d d 30 43.58 ± 0.62 40.45 13.6 ± 1.03 23.70 ± 0.68 23.50 ± 0.65 7a NC 33.25 ± 1.16 0.00 7.40 ± 0.51 24.24 ± 0.64 23.50 ± 0.65 b b 20 8.5 0 ± 0.41 74.41 13.60 ± 1.25 23.70 ± 0.47 23.30 ± 0.49 c c 30 4.26 ± 0.13 87.19 14.40 ± 0.87 24.54 ± 0.58 24.06 ± 0.63 CQ 30 0.00 100.00 21 < NC Negative control; Means with different letters are significantly different (P < 0.05); P-value is set for the comparison between treated and NC groups; CQ: Chloroquine; has been used as positive control for the test of all compounds M elaku et al. BMC Chemistry (2022) 16:36 Page 7 of 30 Significant (p < 0.05) parasitaemia suppression was also of parasitaemia [26]. Those mice treated with most of the observed on mice treated with compound 6i with per- samples showed a slight decrease in PCV at D4 (Table 2). cent inhibition of 40.4% at 35  mg/kg with the effect sta - However those mice treated with 6e at dose 30  mg/kg tistically significant compared with NC (P < 0.05). At day and 7a at dose 20  mg/kg and 30  mg/kg showed no sig- four of post infection, compound 6c exerted 33.5% para- nificant change in PCV reduction compared with the sitaemia inhibition. The parasitaemia suppression in all positive control indicating the ability of the samples in treated groups was significant (P < 0.05) compared with reducing the parasites. the NC groups. Body weight loss is another common feature in P. Molecular docking simulation of compounds 7a-i berghi infected mice with the effect reduced by an effec - against Plasmodium falciparum dihydrofolate tive antimalarial agent. A significant body weight reduc - reductase-thymidylate synthase (PfDHFR-TS) (PDB ID 1J3I) tion was not observed in most of the group treated P. falciparum  has an unmatched track record of gain- with the samples (Table  1) compared with NC groups ing resistance to drugs currently existing in the mar- (p < 0.005). Compound 6e, 6a (20, 25 and 30 mg/kg b. wt), ket. Hence, it is necessary to search for compounds that 6i (25 and 35  mg/kg), 6c and 7a were shown to be sig- can halt enzyme that play a key role in the biosynthesis nificant in preventing body weight reduction (p < 0.005) of precursors for the DNA of the parasites. One of the of the mice (Table  1). However, significant body weight enzymes responsible for the production of folates as reduction was observed on mice treated with 6i and 6a well as thymidylate required for DNA synthesis is P. fal- at dose 15  mg/kg indicating that these two compounds ciparum dihydrofolate reductase-thymidylate synthase couldn’t prevent a body weight loss as the dose decreases. [27]. This enzyme is a key target for searching for anti - Furthermore, those treated with compound 6a at dose malarial drugs. Molecular docking is used in drug design 35  mg/kg (highest given dose) also showed significant because of its ability to substantially predict the confor- body weight reduction (p < 0.05). Thus, this compound mation of ligands within the appropriate target binding likely has a property that causes depression and loss of site [28]. Among the synthesized dibenzylated analogs of appetite following the dose increment. pinocembrin in this work, the chloroderivatives exhib- A material which induces longer survival time in P. ited better antiplasmodial activity. Hence in the present berghei infected mice compared with the NC group is investigation, a molecular docking study was carried out considered as a good antimalarial agent [24]. In this study, mice treated with pinocembrin and its benzylated analogs showed prolonged MST compared with the NC Table 2 Eec ff t of pinocembrin and its analogs on PCV of P. groups (P < 0.05) indicating that the samples suppressed berghei infected mice P. berghei and reduced the overall pathogenic effect of No. Dose PCV ± SME % Change P value the parasite. The survival time in compound 7a treated groups were found to be in a dose dependent manner D 0 D 4 with the group treated in a higher dose (30  mg/kg) sur- 6a NC 51.11 ± 0.63 48.47 ± 0.42 − 5.16 0.01 vived longer (14.40 days) compared with the one treated 15 51.25 ± 0.21 50.83 ± 0.52 − 0.82 0.10 at 20 mg/kg which survived for 13.60 days. A better MST 25 51.13 ± 0.15 49.93 ± 0.41 − 2.35 0.06 was observed in a group treated with 6e (14.60) at dose 30 51.02 ± 0.13 50.04 ± 0.31 − 1.92 0.10 20  mg/kg. These two compounds also induced a better 35 50.75 ± 0.24 50.55 ± 0.32 − 0.40 0.30 parasitaemia inhibition compared with the other com- 6b NC 51.38 ± 0.25 48.76 ± 0.40 − 5.10 0.01 pounds tested in this study. This result is in agreement 20 51.20 ± 0.20 50.19 ± 0.27 − 2.00 0.08 with the work of Nardos and Mekonnen (2017) in which 6c NC 51.11 ± 0.63 48.47 ± 0.42 − 5.16 0.01 a group treated by hydro-ethanol crude extract of leaves 20 50.84 ± 0.40 51.00 ± 0.31 0.32 0.31 of Ajuga remota at dose 100 mg/kg showed a higher para- 6d NC 51.38 ± 0.25 48.76 ± 0.31 − 5.10 0.01 sitemia inhibition (77.54%) and with a longer survival 30 51.16 ± 0.35 49.79 ± 0.40 − 2.67 0.04 time [25]. 6i NC 51.38 ± 0.31 47.98 ± 0.56 − 6.62 0.00 P. berghei infected mice suffer from anaemia because 15 51.09 ± 0.19 49.89 ± 0.24 − 2.35 0.06 of RBC destruction, either by parasite multiplication or 25 50.78 ± 0.29 50.00 ± 0.35 − 1.54 0.10 by spleen reticuloendotelial cell action as the presence 35 51.26 ± 0.21 51.09 ± 0.23 − 0.33 0.37 of many abnormal RBC stimulates the spleen to pro- 7a NC 50.98 ± 0.64 49.68 ± 0.20 − 2.55 0.04 duce many phagocytes [19]. An effective antimalarial 20 50.38 ± 0.40 50.66 ± 0.45 0.56 0.21 agent prevents reduction of packed cell volume (PCV) 30 50.76 ± 0.16 51.00 ± 0.12 0.47 0.30 which is caused by hemolysis of RBC following the rise Melaku et al. BMC Chemistry (2022) 16:36 Page 8 of 30 using AutoDock Vina in order to elucidate which of the standard clinical drug indicating that compounds 7a-l chloro derivatives of pinocembrin has the best binding are potential antimalarial agents. The binding affinity, affinity against the P. falciparum dihydrofolate reduc - H-bond and hydrophobic, pi-cation and Van der Waals tase-thymidylate synthase. The synthesized compounds interactions of the synthesized compounds were summa- 7a-l were found to have minimum binding energy var- rized in Table 3. ied from −  8.1 to −  8.4  kcal/mol (Table  3). The results The 2D and 3D binding interactions of compounds 7k, demonstrated that the compounds have a better docking 7l and chloroquine against P. falciparum dihydrofolate affinity within the binding pocket of PfDHFR-TS than the reductase-thymidylate synthase were depicted in Figs.  4, standard drug. The key amino acid residues within the 5, 6. Ribbon model shows the binding pocket structure active sites of PfDHFR-TS are Ala16, Ser-108, Phe-58, of PfDHFR-TS with compounds 7f, 7k and 7l. Hydrogen Asp-54, Ile-14, Met-55, Trp-48, and Thr-185. The com - bonds between compounds and amino acids are shown pounds 7a, 7b, 7c, 7d, 7f, 7g, 7i, 7j and 7l have shown as green dash lines, hydrophobic interactions are shown at least one hydrogen bonding interaction within the as pink lines. The molecular docking analysis of com - active site of PfDHFR-TS with the key amino acid resi- pounds 7a-j are given as supplementary material (Addi- dues. The compounds 7e (Gly-44), 7h (Ser-111, Ser-167), tional file 2). 7k (Gly-44 Ser-167), and 7l (Ser-167) displayed hydro- gen bond interaction with non-key residual amino acids In silico pharmacokinetics (drug-likeness) properties within the active site of PfDHFR-TS. On the other hand, In order to get a potential drug for drug development, compound 7i had no hydrogen bonding interaction with forecasting of ADME (absorption, distribution, metab- residual amino acids. Overall, the in silico docking analy- olism and excretion) profiles of compounds including sis revealed that the synthesized compounds shown bet- their pharmacokinetic and drug-like properties using ter binding affinity compared with chloroquine used as Swiss ADME is vital. In this investigation, compounds Table 3 Molecular docking simulation of compounds 7a-l against P. falciparum dihydrofolate reductase-thymidylate synthase No. Formula Binding H-bond Residual interactions affinity Hydrophobic/Pi-Cation Van der Waals (Kcal/mol) 7a C H O − 8.1 Ala-16 Leu-46, Leu-40, Phe-58, Met-55, Leu-119, Cys-15, Ser-167, Gly-166, Tyr-170 15 12 4 Ile-112, 7b C H O − 8.1 Asp-54, Ala-16 Leu-40, Leu-46, Phe-58, Met-55 Cys-15, Gly-44, Val-45, Ile-164, Ser-111, Gly- 17 16 6 41, Ser-167, Tyr-170 7c C H ClO − 8.2 Ser-111, Ser-167, Ile-164 Ala-16, Ile-14, Cys-15, Leu-40, Phe-58 Asp-54, Gly-44, Ser-108, Gly-41, Gly-166, 15 11 4 Tyr-170 7d C H O − 8.2 Ala-16, Leu-40, Ser-108, Leu-46, Ile-112, Val-195 Lys-43, Val-45, Gly-41, Val-168, Ser-167, Ile- 18 18 7 Asn-42, Gly-44, Thr-107 164, Tyr-170 7e C H Cl O − 8.2 Gly-44 Phe-58, Ser-167 Ala-16, Asp-54, Ile-164, Ser-108, Thr-107, Val- 15 10 2 4 45, Gly-166, Tyr-170 7f C H ClO − 8.4 Ser-111, Ile-164 Ala-16, Cys-15, Leu-46, Trp-48, Leu-40 Ile-14, Phe-58, Asp-54, Ser-108, Gly-44, Gly- 15 11 4 166, Ser-167, Val-195, Tyr-170 7g C H ClO − 8.3 Ser-108, Ser-111, Ser-167 Ala-16, Leu-40, Phe-58, Met-55 Asp-54, Cys-15, Leu-46, Thr-107, Gly-41, 15 11 4 Gly-166 7h C H Cl O − 8.3 Ser-111, Ser-167 Ala-16, Leu-46, Trp-48, Ile-14, Cys-15, Phe- Gl-41, Gly-166, Ser-108, Ile-164, Thr-185, 15 10 2 4 58, Leu-40 Tyr-170 7i C H Cl O − 8.3 – Ala-16, Phe-58, Ser-167 Asp-54, Leu-46, Cys-15, Ser-108, Ser-111, 15 10 2 4 Gly-44, Thr-107, Val-195, Gly-166, Leu-40, Ile-164,Tyr-170 7j C H O − 8.1 Ser-108, Ile-164 Val-195, Thr-107 Asn-42, Val-45, Gly-41, Ser-111, leu-40, Gly- 15 12 4 165, Gly-166, Ser-167, Val-168 7k C H O − 8.4 Gly-44, Ser-167 Ala-16, Leu-46, Phe-58, Ile-112, Leu-119, Ser-108, Ser-111, Thr-107, Val-45, Val-195, 17 16 6 Ile-164 Ggly-166, Tyr-170 7l C H ClO − 8.4 Ser-167 Ala-16, Leu-46, Phe-58, Ile-165 Cys-15, Asp-54, Ser-108, Ser-111, Gly-166, 15 11 4 Tyr-170 CQ C H ClN − 7.2 Ile-164 Ala-16, Ile-14, Phe-58 Asp-54, Trp-48, Cys-15, Leu-46, Ser-108, Ser- 18 26 3 111, Val-45, Gly-44, Asn-42, Gly-41, Leu-40, Gly-166, Gly-165, Tyr-170 M elaku et al. BMC Chemistry (2022) 16:36 Page 9 of 30 Fig. 4 2D and 3D binding interactions of 7k against Plasmodium falciparum dihydrofolate reductase-thymidylate synthase Fig. 5 2D and 3D binding interactions of 7l against Plasmodium falciparum dihydrofolate reductase-thymidylate synthase 7a-l were assessed for their drug-like properties using parameters showed that all the compounds have high SwissADME. The results indicates that compounds GI absorption, high blood brain barrier (BBB) permea- 7a-l satisfy Lipinski’s rule of five with zero violations tion (except compounds 7b and 7d) and compound (Table  4) [29]. The Kp values of the synthesized com - 7d is a substrate of permeability glycoprotein (P-gp) pounds were within the range of − 5.29 to − 6.35 cm/s (Table 5). Overall, these prediction results indicate that while it was −  4.96  cm/s for chloroquine inferring low the compounds 7e, 7f, 7h, 7i, 7j and 7l can be better skin permeability (Table  5). The predicted logP values active pharmacological agent compared to the other of the compounds synthesized also indicate that all the reported compounds in this study. This is in agreement compounds had optimal lipophilicity (ranging from from the observed good binding affinity of these com - 2.11 to 2.9). This was inferior to chloroquine which pounds with P. falciparum dihydrofolate reductase-thy- had logP value of 3.95. The SwissADME prediction midylate synthase. Melaku et al. BMC Chemistry (2022) 16:36 Page 10 of 30 Fig. 6 2D and 3D binding interactions of CQ against Plasmodium falciparum dihydrofolate reductase-thymidylate synthase Table 4 Drug-likeness predictions of compounds 7a-l computed by SwissADME °2 S. No. Formula Mol.Wt. (g/mol) NHD NHA NRBTPSA (A ) LogP (cLogP) Lipinski’s rule of Five Violation 7a C H O 256.25 2 4 1 66.76 2.11 0 15 12 4 7b C H O 316.31 2 6 3 85.22 2.71 0 17 16 6 7c C H ClO 290.7 2 4 1 66.76 2.37 0 15 11 4 7d C H O 346.33 2 7 4 94.45 2.9 0 18 18 7 7e C H Cl O 325.14 2 4 1 66.76 2.43 0 15 10 2 4 7f C H ClO 290.7 2 4 1 66.76 2.36 0 15 11 4 7g C H ClO 290.7 2 4 1 66.76 2.39 0 15 11 4 7h C H Cl O 325.14 2 4 1 66.76 2.58 0 15 10 2 4 7i C H Cl O 325.14 2 4 1 66.76 1.91 0 15 10 2 4 7j C H O 325.14 2 4 1 66.76 2.53 0 15 12 4 7k C H O 325.14 2 4 1 66.76 2.57 0 17 16 6 7l C H ClO 325.14 2 4 1 66.76 2.56 0 15 11 4 CQ C H ClN 319.87 1 2 8 28.16 3.95 0 18 26 3 NHD Number of Hydrogen donor, NHA Number of Hydrogen acceptor, NRB Number of rotatable bonds, TPSA total polar surface area, CQ chloroquine in capillary tubes with a digital electrothermal melt- Experimental ing point apparatus. IR spectra were measured on a General Bruker Alpha FT-IR spectrometer. UV/Vis spectra All reagents used in the present work were used with- were recorded on a Cary 4E spectrophotometer. H and out further purification. Glassware used was dried for C NMR spectra were recorded at 300 (75) MHz and 24  h at 120  °C in an oven. Solvents used in reactions 500 (125) MHz on Varian Unity Inova spectrometers. were distilled over appropriate drying agents prior to Coupling constants J [Hz] were directly taken from the use while those used for extraction and purification spectra and are not averaged. Splitting patterns are des- were distilled prior to use. Thin-layer chromatography ignated as s (singlet), d (doublet), t (triplet), q (quar- (TLC) was performed on precoated aluminum plates tet), and m (multiplet). Low-resolution electron impact (silica gel Merck 60 F) and visualized under UV light mass spectra [MS (EI)] and exact mass electron impact (254 nm) and/or by dipping in vanillin/H SO followed 2 4 mass spectra [HRMS (EI)] were obtained at 70  eV by heating. Products were purified by column chro - using a double focusing sector field mass spectrometer matography over silica gel (MN 60, 0.04–0.063  mm; Finnigan MAT 95, for the measurement of exact mass Marcherey & Nagel). Melting points were determined M elaku et al. BMC Chemistry (2022) 16:36 Page 11 of 30 Table 5 ADME predictions of compounds 7a-l computed by SwissADME and PreADMET No. Chemical Formula Skin Permeation GI Absorption BBB Inhibitor Interaction (SwissADME/PreADMET) Value (log Kp) cm/s Permeability P-gp substrate CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 inhibitor inhibitor inhibitor inhibitor inhibitor 7a C H O − 5.82 High Yes No Yes Yes No No No 15 12 4 7b C H O − 6.23 High No No Yes No Yes No Yes 17 16 6 7c C H ClO − 5.59 High Yes No Yes Yes Yes Yes Yes 15 11 4 7d C H O − 6.35 High No Yes Yes No Yes No Yes 18 18 7 7e C H Cl O − 5.29 High Yes No Yes Yes Yes Yes Yes 15 10 2 4 7f C H ClO − 5.52 High Yes No Yes Yes Yes Yes Yes 15 11 4 7g C H ClO − 5.52 High Yes No Yes Yes Yes Yes Yes 15 11 4 7h C H Cl O − 5.35 High Yes No Yes Yes Yes Yes Yes 15 10 2 4 7i C H Cl O − 5.29 High Yes No Yes Yes Yes Yes Yes 15 10 2 4 7j C H O − 5.35 High Yes No Yes Yes Yes Yes Yes 15 12 4 7k C H O − 5.29 High Yes No Yes Yes Yes Yes Yes 17 16 6 7l C H ClO − 5.45 High Yes No Yes Yes Yes Yes Yes 15 11 4 CQ C H ClN − 4.96 High Yes No Yes No No Yes Yes 18 26 3 CQ Chloroquine, GI Gastro-Intestinal, BBB Blood Brain Barrier, P-gp P-glycoprotein, CYP Cytochrome-P Melaku et al. BMC Chemistry (2022) 16:36 Page 12 of 30 electrospray ionization mass spectra [ESI (HRMS)] a reaction mixture was extracted with EtOAc (3 × 30  mL). Bruker Daltonik spectrometer micrOTOF-Q was used. The combined organic phases were dried over anhydrous MgSO , filtered and concentrated in vacuo. Column Synthesis and characterization of starting materials chromatography of the crude product on silica gel using and chalcones petroleum ether:EtOAc (4:1) as eluent furnished the cor- Synthesis and analytical data responding chalcones 5a-r as yellow solids. Compounds of 2,4‑dibenzyloxy‑6‑hydroxyacetophenone (3) [10] 5a-r were synthesized according to general procedure I. Synthesis and analytical data O 4 O of 2’,4’‑dibenzyloxy‑6’‑hydroxychalcone (5a) 6 4 OH O BnO 4' OH 7 2 2' OBn O A 250  mL 2-neck round bottom flask was charged with N,N-dimethylformamide (24  mL) under argon, 5a heated to 35  °C, then trihydroxyacetophenone (1) (5  g, 29.7 mmol) was added in one portion, followed by more According to general procedure I, 2,4-dibenzyloxy-6-hy- DMF (16  mL). Potassium carbonate (8.5  g, 55.9  mmol) droxyacetophenone (3) (1  g, 2.8  mmol) was condensed was added and the mixture was heated to 65  °C. Benzyl with benzaldehyde (4a) (0.3  g, 2.8  mmol) in the pres- chloride (2) (7.5 g, 59.4 mmol) was added in one portion ence of aqueous KOH (60%, 1.5 mL) in ethanol (8.5 mL). and the mixture was heated to 70  °C for 3  h, cooled to The product 5a was obtained in 80% yield as a yellow room temperature and filtered. The filter cake was rinsed solid. mp 124–125  °C); UV (MeOH) λ : 278, 345  nm; max with dichloromethane. The combined filtrates were con - H NMR (CDCl , 300  MHz) δ 5.01 (2H, s, OCH ), 5.06 3 2 centrated in vacuo and the residual orange oil was taken (2H, s, OCH ), 6.18 (1H, d, J = 2.3 Hz, H-5’), 6.23 (1H, d, up in dichloromethane (50 mL), stirred for five min, and J = 2.3 Hz, H-3’), 7.06 (2H, d, J = 7.6 Hz, H-2,6), 7.19 (3H, filtered. The filtrate was column filtered over silica gel t, J = 8.1  Hz, H-3,4,5), 7.25–7.52 (10H, m, H-2’’– H-6’’, with dichloromethane as eluent. It was concentrated, H-2’’’– H-6’’’), 7.71 (1H, d, J = 16  Hz, H-8), 7.89 (1H, d, dichloromethane (5  mL) and cyclohexane (8  mL) were J = 16  Hz, H-9), 14.50 (1H, s, OH); C NMR (CDCl , added and the mixture was stirred for 20 min. The result - 75  MHz) δ 70.3 (OCH ), 71.4 (OCH ), 92.5 (C-3’), 95.0 2 2 ant white crystalline solid was collected by suction fil - (C-5’), 106.3 (C-1’), 127.4 (C-8), 127.6 (C-2, C-6), 128.31 tration, washed with cyclohexane and air dried to yield (C-2’’’, C-6’’’), 128.4 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.62 5.7 g (79%) of a white crystalline solid which was identi- (C-4’’), 128.64 (C-4), 128.7 (C-3’’’,C-5’’’), 128.8 (C-3, C-5), fied as 2,4-dibenzyloxy-6-hydroxyacetophenone (3). H 128.9 (C-3’’,C-5’’),129.8 (C-1), 135.2 (C-1’’’), 135.3 (C-1’’), NMR (CDCl , 300  MHz) δ 2.56 (3H, s, H-2’), 5.06 (4H, 3 135.8 (C-7), 161.7 (C-2’), 165.2 (C-6’), 168.8 (C-4’), 192.6 br s, benzylic H), 6.09 (1H, d, J = 2.3 Hz, H-3), 6.16 (1H, (C-9). d, J = 2.3 Hz, H-5), 7.34–7.43 (10H, m, aromatic H), 14.01 (1H, s, OH); C NMR (CDCl , 75  MHz) δ 33.1 (C-2’), Synthesis and analytical data 70.3 (C-1’’’), 71.1 (C-1’’), 92.3 (C-5), 94.6 (C-3), 106.3 of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑4‑fluorochalcone (5b) (C-1), 127.6 (C-2’’’, C-6’’’, C-4’’’,), 127.9 (C-2’’’’, C-4’’’’, C-6’’’’), 128.3 (C-3’’’, C-5’’’), 128.4 (C-3’’’’, C-5’’’’), 135.5 (C-1’’’), 4 F 135.8 (C-1’’’’), 161.9 (C-2), 165.0 (C-6), 167.5 (C-4), 203.2 4' BnO OH (C-2’). 7 2 2' 9 General procedure I for the synthesis of chalcones 5a‑r [30] OBn O 2,4-Dibenzyloxy-6-hydroxyacetophenone (3) (2.8  mmol) was condensed with substituted benzaldehydes 4a- 5b r (2.8  mmol) in the presence of aqueous KOH (60%, 1.5  mL) in ethanol (8.5  mL). The reaction mixture was According to general procedure I, 2,4-dibenzyloxy-6-hy- stirred for 24  h at room temperature and poured into droxyacetophenone (3) (0.4  g, 1.6  mmol) was condensed water (30  mL). After neutralization with 10% HCl, eth- with 4-fluorobenzaldehyde (4b) (0.19  g, 1.6  mmol) in anol was removed using a rotary evaporator and the the presence of aqueous KOH (60%, 1.5  mL) in ethanol M elaku et al. BMC Chemistry (2022) 16:36 Page 13 of 30 Synthesis and analytical data (8.5 mL). The product was obtained in 68% yield. mp 126– −1 of 2’,4’‑dibenyloxy‑6’‑hydroxy‑2,6‑dimethoxychalcone (5d) 127  °C; UV(MeOH) λ : 276, 337  nm; IR ν ̃ [cm ]: 1660 max (conjugated C = O), 1571 (arom. C = C), and 1023 (C-O); H CO 4 H NMR (CDCl , 300 MHz) δ 5.0 (2H, s, O CH ), 5.1 (2H, 3 3 2 s), 5.56 (1H, s, OCH ), 6.18 (1H, d, J = 2.4  Hz, H-5’), 6.2 1 2 4' BnO OH (1H, d, J = 2.4 Hz, H-3’), 6.8 (2H, m, H-3,H-5), 7.01 (2H, m, 2' 9 OCH H-2,H-6), 7.50–7.39 (10H, m, H-2’’– H-6’’, H-2’’’– H-6’’’), 7.64 (1H, d, J = 15  Hz, H-8), 7.78 (1H, d, J = 15  Hz, H-7), OBn O 14.5 (1H, s, 6’-OH); C NMR (CDCl , 75  MHz) δ 70.3 5d (OCH ), 71.4 (OCH ), 92.5 (C-3’), 95.0 (C-5’), 106.2 (C-1’), 2 2 115.5 (C-3,C-5), 115.8 (C-8), 127.2 (C-2’’’, C-6’’’), 127.6 (C-2’’, According to general procedure I, 2,4-dibenzyloxy-6-hy- C-6’’), 128.3 (C-4’’’), 128.7 (C-4’’), 128.9 (C-2, C-6), 129.3 droxyacetophenone (3) (0.4 g, 1.1 mmol) was condensed (C-3’’’, C-5’’’), 130.2 (C-3’’, C-5’’), 131.5 (C-1), 132.7 (C-1’’’), with 3′4’-dimethoxybenzaldehyde (4d) (0.19 g, 1.1 mmol) 135.4 (C-1’’), 135.8 (C-7), 141.3 (d, C-4), 161.7 (C-2’), 165.3 in the presence of aqueous KOH (60%, 1.5  mL) in etha- (C-6’), 168.8 (C-4’), 192.4 (C-9). nol (8.5  mL). The product 5d was obtained in 67%. mp −1 99–100  °C; UV(MeOH) λ : 275, 332  nm; IR ν̃ [cm ]: max Synthesis 1671 (conjugated C = O), 1603 (C = C), 1560 (C = C), of 2’,4’‑dibenyloxy‑6’‑hydroxy‑3,4‑dimethoxychalcone (5c) 1347, 1206, and 1027 (C-O); H NMR (C D COCD , 3 3 300 MHz) δ 3.6 (3H, s, OMe), 3.8 (3H, s, OMe), 5.22 (2H, OCH s, OCH ), 5.29 (2H, s, OCH ), 6.23 (1H, d, J = 2.2  Hz, 2 2 4' BnO OH H-5’), 6.36 (1H, d, J = 2.2  Hz, H-3’), 6.96 (1H, m, H-4), OCH 7 2 6.98 (2H, br d, H-3, H-5), 7.54–7.30 (10H, m, H-2’’’– H-6’’’, 2' H-2’’– H-6’’), 14.1 (1H, s, 6’OH); C NMR (C D COCD , 3 3 OBn O 75  MHz) δ 55.8 (OMe), 56.3 (OMe), 70.8 (OCH ), 71.8 (OCH ), 93.5 (C-3’), 95.8 (C-5’), 107.3 (C-1’), 113.3 (C-3), 5c 113.5 (C-5), 118.0 (C-1), 125.2 (C-8), 128.5 (C-2’’’, C-6’’’), 128.6 (C-2’’, C-6’’), 128.7 (C-4’’’), 128.8 (C-4’’), 128.9 (C-3’’’, According to general procedure I, 2,4-dibenzyloxy-6-hy- C-5’’’), 128.91 (C-3’’, C-5’’), 129.3 (C-4), 129.4 (C-1’’’), droxyacetophenone (3) (0.4  g, 1.1  mmol) was condensed 137.1 (C-1’’), 137.8 (C-7), 153.8 (C-2), 154.5 (C-6), 162.6 with 3′4’-dimethoxybenzaldehyde (4c) (0.19  g, 1.1  mmol) (C-2’), 166.2 (C-6’), 168.7 (C-4’), 193.7 (C-9). in the presence of aqueous KOH (60%, 1.5 mL) in ethanol (8.5 mL). The product 5c was obtained in 67% as a yellow - Synthesis and analytical data ish solid. mp 102–103  °C; UV(MeOH) λ : 276, 331  nm; max −1 of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑4‑chlorochalcone (5e) IR ν ̃ [cm ]: 1677 (conjugated C = O), 1630 (C = C), and 1040 (C-O); H NMR (CDCl , 300  MHz) δ 3.62 (1H, s, 4 Cl OMe), 3.91 (1H, s, OMe), 5.10 (4H, s, OCH ), 6.17 (1H, d, J = 2.4  Hz, H-5’), 6.23 (1H, d, J = 2.4  Hz, H-3’), 6.72 (1H, 1 2 4' BnO OH d, J = 9 Hz, H-5), 6.77 (1H, d, J = 3 Hz, H-2), 6.81 (1H, dd, 2' J = 3  Hz and 9  Hz, H-6), 7.47–7.31 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.70 (1H, d, J = 15.6  Hz, H-8), 7.80 (1H, d, OBn O J = 15.6  Hz, H-7), 14.4 (1H, s, 6’OH); C NMR (CDCl , 5e 75  MHz) δ 55.6 (OMe), 55.9 (OMe), 70.2 (O CH ), 71.1 (OCH ), 92.7 (C-3’), 95.0 (C-5’), 106.6 (C-1’), 110.8 (C-2), According to general procedure I, 2,4-dibenzyloxy-6-hy- 110.9 (C-5), 122.4 (C-6), 125.5 (C-8), 127.6 (C-2’’’, C-6’’’), droxyacetophenone (3) (0.5 g, 1.4 mmol) was condensed 127.8 (C-2’’, C-6’’), 128.2 (C-4’’’), 128.3 (C-4’’), 128.6 (C-1), with 4-chlorobenzaldehyde (4e) (0.21  g, 1.4  mmol) in 128.7 (3’’’, C-5’’’), 128.8 (C-3’’, C-5’’),, 135.7 (C-1’’’), 135.8 the presence of aqueous KOH (60%, 1.5  mL) in etha- (C-1’’), 142.8 (C-7), 148.8 (C-4), 150.8 (C-3), 161.5 (C-2’), nol (8.5  mL). The product 5e was obtained as a white 165.0 (C-6’), 168.3 (C-4’), 192.5 (C-9). Melaku et al. BMC Chemistry (2022) 16:36 Page 14 of 30 Synthesis of 2’,4’‑dibenyloxy‑6’‑hydroxy‑4‑(trifluoromethyl) solid in 72% yield. mp 142–144  °C; R 0.63 (petroleum chalcone (5g) ether:EtOAc 4:1); UV(MeOH) λ : 274, 336  nm; IR ν̃ max −1 [cm ]: 1613 (C = C), 1487, 1162, 1114, and 1023 (C-O); 4 CF H NMR (CDCl , 300  MHz) δ 5.05 (2H, s, OCH ), 5.11 3 3 2 (2H, s, OCH ), 6.18 (1H, d, J = 2.2 Hz, H-5’), 6.23 (1H, d, 1 2 4' BnO OH J = 2.2  Hz, H-3’), 6.91 (2H, d, J = 7.0  Hz, H-3, H-5), 7.13 2' 9 (2H, d, J = 7.0  Hz, H-2, H-6), 7.4 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.62 (1H, d, J = 15.4  Hz, H-8), 7.82 (1H, OBn O d, J = 15.4  Hz, H-7); C-NMR (CDCl , 75  MHz) δ 70.3 5g (OCH ), 71.2 (OCH ), 92.6 (C-3’), 95.2 (C-5’), 106.2 2 2 (C-1’), 127.2 (C-8), 127.3 (C-2’’’, C-6’’’), 127.6 (C-2’’, C-6’’), According to general procedure I, 2,4-dibenzyloxy-6-hy- 128.0 (C-4’’’), 128.3 (C-4’’), 128.7 (C-2, C-6), 128.8 (C-3, droxyacetophenone (3) (0.6 g, 1.7 mmol) was condensed C-5), 128.9 (C-3’’’, C-5’’’), 129.4 (C-3’’, C-5’’), 133.8 (C-1), with 4-(trifluoromethyl)benzaldehyde (4  g) (0.32  g, 135.3 (C-4), 135.5 (C-1’’’), 135.7 (C-1’’), 141.0 (C-7), 161.6 1.7 mmol) in the presence of aqueous KOH (60%, 1.5 mL) (C-2’), 165.4 (C-6’), 168.8 (C-4’), 192.3 (C-9). in ethanol (8.5 mL). The product 5 g was obtained in 71% yield. H NMR (C DCl , 300 MHz) δ 5.05 (2H, s, O CH ), 3 2 Synthesis and analytical data 5.10 (2H, s, OCH ), 6.18 (1H, d, J = 2.2  Hz, H-5’), 6.23 of 2’,4’‑dibenyloxy‑6’‑hydroxy‑2‑bromochalcone (5f ) (1H, d, J = 2.2  Hz, H-3’), 7.09 (2H, d, J = 7.0, H-2, H-6), 7.48 (2H, d, J = 7.0  Hz, H-3, H-5), 7.42 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.63 (1H, d, J = 15.4  Hz, H-8), 7.88 1 2 4' 13 BnO OH (1H, d, J = 15.4  Hz, H-7); C NMR (CDCl , 75  MHz) δ 2' 970.2 (OCH ), 71.3 (OCH ), 92.6 (C-3’), 95.0 (C-5’), 106.2 Br 2 2 (C-1’), 125.4 (C-7), 125.5 (q, C F ), 127.6 (C-3, C-5), 128.2 OBn O (C-2, C-6), 128.5 (C-2’’’, C-6’’’), 128.7 (C-2’’, C-6’’), 128.9 5f (C-4’’’), 129.0 (C-4’’), 130.1 (C-3’’’, C-5’’’), 130.8 (C-3’’, C-5’’), 131.2 (C-4), 135.2 (C-1), 135.7 (C-1’’’), 138.7 (C-1’’), According to general procedure I, 2,4-dibenzyloxy-6-hy- 140.2 (C-7), 161.8 (C-2’), 165.8 (C-6’), 168.8 (C-4’), 192.3 droxyacetophenone (3) (0.6 g, 1.7 mmol) was condensed (C-9). with 2-bromobenzaldehyde (4f) (0.32  g, 1.7  mmol) in the presence of aqueous KOH (60%, 1.5  mL) in ethanol (8.5  mL). The product 5f was obtained in 57% yield. mp Synthesis −1 111–112 °C; UV(MeOH) λ : 276, 338 nm; IR ν̃ [cm ]: of 2’,4’‑dibenyloxy‑6’‑hydroxy‑3,4,5‑trimethoxychalcone (5h) max : 1673 (conjugated C = O), 1618 (C = C), 1552 (C = C), 1453, 1226, 1156, and 1021 (C-O); H NMR (CDCl , OCH 300 MHz) δ 5.04 (2H, s, OCH ), 5.10 (2H, s, OCH ), 6.17 4 OCH 2 2 (1H, d, J = 2.2  Hz, H-5’), 6.22 (1H, d, J = 2.2  Hz, H-3’), 4' BnO OH OCH 6.96 (1H, t, J = 7.5  Hz, H-4), 7.14 (1H, dd, J = 7.2  Hz, 2' 9 1.8  Hz, H-3), 7.41 (12H, m, H-5, H-6, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.77 (1H, d, J = 15.4  Hz, H-8), 8.05 (1H, OBn O d, J = 15.4  Hz, H-7); C NMR (CDCl , 75  MHz) δ 70.2 5h (OCH ), 71.2 (OCH ), 92.2 (C-3’), 95.1 (C-5’), 106.2 2 2 (C-1’), 125.8 (C-2), 127.4 (C-8), 127.6 (C-2’’’, C-6’’’), 128.3 According to general procedure I, 2,4-dibenzyloxy- (C-2’’, C-6’’), 128.5 (C-4’’’), 128.6 (C-4’’), 128.7 (C-5), 128.8 6-hydroxyacetophenone (3) (0.6  g, 1.7  mmol) was (C-6), 128.9 (C-3’’’, C-5’’’), 130.0 (C-3’’, C-5’’), 130.5 (C-4), condensed with 3,4,5-trimethoxybenzaldehyde (4  h) 133.2 (C-3), 135.2 (C-1), 135.5 (C-1’’’), 135.7 (C-1’’), 140.5 (0.345  g, 1.7  mmol) in the presence of aqueous KOH (C-7), 161.6 (C-2’), 164.4 (C-6’), 168.7 (C-4’), 192.2 (C-9). M elaku et al. BMC Chemistry (2022) 16:36 Page 15 of 30 Synthesis of 2’ ,4’‑dibenyloxy‑6’‑hydroxy‑2,3‑dichlorochalcone (60%, 1.5  mL) in ethanol (8.5  mL). The product 5  h (5j) was obtained in 46% yield. mp 94–96  °C; UV(MeOH) −1 λ : 275, 324  nm; IR ν̃ [cm ]: 1611 (C = C ), 1577 max (C = C), 1266, 1168, and 1095 (C-O); H NMR (CDCl , 300  MHz) δ 3.64 (6H, s, OMe), 3.87 (3H, s, OMe), 4' BnO OH 5.10 (2H, s, OCH ), 5.11 (2H, s, OCH ), 6.16 (1H, d, 2 2 Cl J = 2.2  Hz, H-5’), 6.23 (1H, d, J = 2.2  Hz, H-3’), 7.44 2' Cl (12H, m, H-2, H-6, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.68 (1H, d, J = 15.4 Hz, H-8), 7.79 (1H, d, J = 15.4 Hz , H-7); OBn O C NMR (CDCl , 75 MHz) δ 56.0 (OMe), 60.9 (OMe), 5j 70.3 (OCH ), 71.3 (O CH ), 92.9 (C-3’), 95.6 (C-5’), 2 2 105.7 (C-1’), 106.8 (C-2, C-6), 127.1 (C-8), 127.3 (C-2’’’, According to general procedure I, 2,4-dibenzyloxy-6-hy- C-6’’’), 127.6 (C-2’’, C-6’’), 127.9 (C-4’’’), 128.2 (C-4’’), droxyacetophenone (3) (0.5 g, 1.4 mmol) was condensed 128.3 (C-3’’’, C-5’’’), 128.7 (C-3’’, C-5’’), 130.7 (C-1), with 2,3-dichlorobenzaldehyde (4j) (0.25  g, 1.4  mmol) 135.6 (C-1’’’), 135.8 (C-1’’), 140.0 (C-4), 142.5 (C-7), in the presence of aqueous KOH (60%, 1.5  mL) in etha- 153.1 (C-3,C-5), 161.4 (C-2’), 165.2 (C-6’), 168.1 (C-4’), nol (8.5  mL). The product 5j was obtained in 97% yield. 192.5 (C-9). mp 121–123  °C; R 0.60 (petroleum ether:EtOAc 4:1); −1 UV(MeOH); λ : 274, 328  nm; IR ν̃ [cm ]: 1605 max (C = C), 1560 (C = C), 1337, 1204, 1154, and 1044 (C-O); Synthesis and analytical data H NMR (CDCl , 300  MHz) δ 5.01 (2H, s, OCH ), 5.12 of 2’,4’‑dibenyloxy‑6’‑hydroxy‑2‑bromo‑4‑fluorochalcone (5i) 3 2 (2H, s, OCH ), 6.18 (1H, d, J = 2.2 Hz, H-5’), 6.23 (1H, d, 4 F J = 2.2  Hz, H-3’), 6.62 (1H, dd, J = 7.5  Hz and J = 2.4  Hz, H-6), 6.84 (1H, t, J = 8.1  Hz, H-5), 7.40 (11H, m, H-4, BnO 4' OH H-2’’’– H-6’’’, H-2’’– H-6’’), 7.75 (1H, d, J = 15.4  Hz), 8.06 2' 9 Br (1H, d, J = 15.4  Hz); C NMR (CDCl , 75  MHz) δ 70.3 (OCH ), 71.3 (OCH ), 92.5 (C-3’), 95.1 (C-5’), 106.3 2 2 OBn O (C-1’), 125.5 (C-8), 127.1, 127.6 (C-2’’’, C-6’’’), 128.3 (C-2’’, 5i C-6’’), 128.4 (C-4’’’), 128.5 (C-4’’), 128.6 (C-2), 128.7 (C-5), 128.8 (3’’’, C-5’’’), 130.8 (C-3’’, C-5’’), 133.1 (C-4), 133.6 According to general procedure I, 2,4-dibenzyloxy- (C-3), 135.2 (C-1), 135.7 (C-1’’’), 135.8 (C-1’’), 137.6 (C-7), 6-hydroxyacetophenone (3) (0.5  g, 1.4  mmol) was 161.6 (C-2’), 165.5 (C-6’), 168.7 (C-4’), 192.1 (C-9). condensed with 2-bromo-4-chlorobenzaldehyde (4i) (0.32  g, 1.4  mmol) in the presence of aqueous KOH (60%, 1.5 mL) in ethanol (8.5 mL). The product 5i was Synthesis of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑4‑cyanochalcone obtained in 77% yield. mp 132–133  °C; UV(MeOH) (5k) −1 λ : 271, 333  nm; IR ν̃ [cm ]: 1670 (conjugated max C = O), 1603 (C = C), 1154, and 1025 (C-O); H NMR 4 CN (CDCl , 300  MHz) δ 5.01 (2H, s, O CH ), 5.11 (2H, 3 2 1 2 s, OCH ), 6.17 (1H, d, J = 2.2  Hz, H-5’), 6.22 (1H, d, BnO 4' OH J = 2.2 Hz, H-3’), 6.57 (1H, dd, J = 9.3  Hz and J = 3  Hz , 2' H-3), 6.87 (1H, m, H-5), 7.44 (11H, m, H-6, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.71 (1H, d, J = 15.4 Hz, H-8), 7.96 OBn O (1H, d, J = 15.4  Hz , H-7); C NMR (CDCl , 75  MHz) δ 70.3 (OCH ), 71.6 (OCH ), 92.6 (C-3’), 95.0 (C-5’), 2 2 5k 106.3 (C-1’), 113.9 (C-5), 114.2 (C-2), 117.8 (C-3), 119.9 (C-8), 127.6 (C-2’’’, C-6’’’), 128.7 (C-2’’, C-6’’), According to general procedure I, 2,4-dibenzyloxy-6-hy- 128.8 (C-4’’’), 129.0 (C-4’’), 131.0 (C-3’’’, C-5’’’), 134.2 droxyacetophenone (3) (0.5 g, 1.4 mmol) was condensed (C-3’’, C-5’’), 134.3 (C-6), 135.0 (C-1), 135.7 (C-1’’’), 136.7 (C-1’’), 139.3 (C-7), 159.9 (d, C-4), 161.6 (C-2’), 165.5 (C-6’), 168.5 (C-4’), 192.3 (C-9). Melaku et al. BMC Chemistry (2022) 16:36 Page 16 of 30 Synthesis of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑3‑chlorochalcone with 4-cyanobenzaldehyde (4  k) (0.19  g, 1.4  mmol) in (5m) the presence of aqueous KOH (60%, 1.5  mL) in ethanol (8.5 mL). The product 5 k was obtained in 67% yield. mp 148–150  °C; H NMR (C DCl , 300  MHz) δ 5.03 (2H, s, OCH ), 5.11 (2H, s, OCH ), 6.18 (1H, d, J = 2.2 Hz, H-5’), 2 2 2 BnO 4' OH Cl 6.23 (1H, d, J = 2.2 Hz, H-3’), 7.02 (2H, d, J = 7.0 Hz, H-3, 7 2' 9 H-5), 7.43 (12H, m, H-2, H-6, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.58 (1H, d, J = 15.4  Hz), 7.88 (1H, d, J = 15.4  Hz); C OBn O NMR (CDCl , 75  MHz) δ 70.5 (O CH ), 71.4 (OCH ), 3 2 2 5m 92.6 (C-3’), 95.2 (C-5’), 106.3 (C-1’), 112.4 (C-4), 118.6 (CN), 127.6 (C-8), 127.8 (C-2, C-6), 127.9 (C-2’’’, C-6’’’), According to general procedure I, 2,4-dibenzyloxy-6-hy- 128.7 (C-2’’, C-6’’), 128.8 (C-4’’’), 129.0 (C-4’’), 129.1 (C-3’’’, droxyacetophenone (3) (0.7 g, 2.0 mmol) was condensed C-5’’’), 130.9 (C-3’’, C-5’’), 132.3 (C-3, C-5), 135.2 (C-1), with 3-chlorobenzaldehyde (4  m) (0.28  g, 2.0  mmol) in 135.7 (C-1’’’), 139.5 (C-1’’), 139.7 (C-7), 161.6 (C-2’), 165.8 the presence of aqueous KOH (60%, 3  mL) in ethanol (C-6’), 168.8 (C-4’), 192.3 (C-9). (15  mL). The product 5  m was obtained in 92% yield. mp 135–136  °C; R 0.60 (petroleum ether:EtOAc 4:1); Synthesis and analytical data −1 UV(MeOH) λ : 280, 334 nm; IR ν̃ [cm ]: 1607 (C = C), max of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑2‑chlorochalcone (5l) 1573 (C = C), 1269, 1226, 1116, and 1024 (C-O); H NMR (CDCl , 300  MHz) δ 5.0 (2H, s, OCH ), 5.1 (2H, 3 2 s, OCH ), 6.18 (1H, d, J = 2.4  Hz, H-5’), 6.22 (1H, d, 1 2 4' BnO OH J = 2.4 Hz, H-3’), 6.89 (1H, d, J = 7.8 Hz, H-4), 7.10 (1H, t, J = 7.8 Hz, H-5), 7.2 (1H, br s, H-2), 7.25 (1H, dd, J = 7.8, 2' Cl J = 3.6 Hz, H-6), 7.45 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), OBn O 7.62 (1H, d, J = 15.9, H-8), 7.83 (1H, d, J = 15.9, H-7), 14.3 (1H, s, 6’OH); C NMR (C DCl , 75 MHz) δ 70.3 (O CH ), 5l 3 2 71.4 (OCH ), 92.6 (C-3’), 95.0 (C-5’), 106.3 (C-1’), 126.1, 127.6 (C-6), 128.2 (C-2), 128.3 (C-2’’’-C-6’’’), 128.4 (C-2’’- According to general procedure I, 2,4-dibenzyloxy-6-hy- C-6’’), 128.7 (C-4’’’), 128.8 (C-4’’), 128.85 (C-4), 128.9 droxyacetophenone (3) (1  g, 2.8  mmol) was condensed (C-3’’’, C-5’’’), 129.6 (C-3’’, C-5’’), 129.8 (C-5), 134.5 (C-3), with 2-chlorobenzaldehyde (4  l) (0.4  g, 2.8  mmol) in 135.2 (C-1), 135.7 (C-1’’’), 137.1 (C-1’’), 140.7 (C-7), 161.6 the presence of aqueous KOH (60%, 3  mL) in etha- (C-2’), 165.4 (C-6’), 168.6 (C-4’), 192.3 (C-9). nol (15  mL). The product 5  l was obtained in 77% yield. mp 138–139  °C; R 0.59 (petroleum ether:EtOAc −1 4:1); UV(MeOH) λ : 270, 338  nm; IR ν̃ [cm ]: 1608 max (C = C),1578 (C = C), 1554, 1425, 11, 1156, and 1040 Synthesis and analytical data (C-O); H NMR (CDCl , 300 MHz) δ 5.04 (2H, s, O CH ), of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑2,4‑dichlorochalcone (5n) 3 2 5.10 (2H, s, OCH ), 6.17 (1H, d, J = 2.4  Hz, H-5’), 6.23 Cl (1H, d, J = 2.4 Hz, H-3’), 6.75 (1H, dd, J = 7.8 and 2.4 Hz, H-3), 6.92 (1H, d, J = 8.7  Hz, H-5), 7.19 (2H, m, H-4, 4' BnO OH H-6), 7.41 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.81 (1H, 2' 13 9 Cl d, J = 15.6  Hz, H-8), 8.10 (1H, d, J = 15.6  Hz, H-7); C NMR (CDCl , 75  MHz) δ 70.3 (O CH ), 71.4 (OCH ), 3 2 2 OBn O 92.6 (C-3’), 95.0 (C-5’), 106. 3 (C-1’), 126.1 (C-8), 127.6 5n (C-5), 128.2 (C-2’’’, C-6’’’), 128.3 (C-2’’, C-6’’), 128.4 (C-4’’’), 128.7 (C-4’’), 128.8 (C-6), 128.9 (C-3), 129.6 (C-3’’’-C-5’’’), According to general procedure I, 2,4-dibenzyloxy- 129.8 (C-3’’-C-5’’), 128.85 (C-4), 134.5(C-2), 135.2 (C-1), 6-hydroxyacetophenone (3) (0.5  g, 1.4  mmol) was 135.7 (C-1’’’), 137.1 (C-1’’), 140.7 (C-7), 161.6 (C-2’), 165.4 (C-6’), 168.6 (C-4’), 192.3 (C-9). M elaku et al. BMC Chemistry (2022) 16:36 Page 17 of 30 Synthesis and analytical data condensed with 2,4-dichlorobenzaldehyde (4n) (0.25  g, of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑2,6‑dichlorochalcone (5p) 1.4 mmol) in the presence of aqueous KOH (60%, 3 mL) in ethanol (15 mL). The product 5n was obtained in 97% Cl 4 yield. mp 127–129  °C; R 0.65 (petroleum ether:EtOAc −1 4:1); UV(MeOH) λ : 281, 330  nm; IR ν̃ [cm ]: 1623 1 2 max 4' BnO OH (C = C), 1559 (C = C), 1464, 1299, 1101, and 1049 (C-O); 2' 9 Cl H NMR (CDCl , 300  MHz) δ 5.03 (2H, s, OCH ), 5.10 3 2 (2H, s, OCH ), 6.17 (1H, dd, J = 2.1  Hz, H-5’), 6.22 (1H, OBn O dd, J = 2.1  Hz, H-3’), 6.59 (1H, d, J = 8.4  Hz, H-6), 6.85 5p (1H, dd, J = 8.7, J = 2.4  Hz, H-5), 7.32 (1H, d, J = 2.4  Hz, H-3), 7.45 (10H, m, H-2’’’– H-6’’’, H-2’’–H-6’’), 7.75 (1H, According to general procedure I, 2,4-dibenzyloxy-6-hy- d, J = 15.6  Hz, H-8), 7.99 (1H, d, J = 15.6  Hz, H-7), 14.3 droxyacetophenone (3) (1  g, 2.8  mmol) was condensed (1H, s, 6’OH); C NMR (C DCl , 75 MHz) δ 70.4 (O CH ), 3 2 with 2,6-dichlorobenzaldehyde (4p) (0.54  g, 2.8  mmol) 71.5 (OCH ), 92.7 (C-3’), 95.0 (C-5’), 106.4 (C-1’), 126.3 in the presence of aqueous KOH (60%, 3  mL) in etha- (C-8), 127.6 (C-5), 128.0 (C-2’’’,C-6’’’), 128.2 (C-2’’,C-6’’), nol (15  mL). The product 5p was obtained in 69% yield. 128.4 (C-4’’’), 128.6 (C-4’’), 128.7 (C-3’’’,C-5’’’), 128.9 (C-3’’, mp 121–122  °C; R 0.59 (petroleum ether:EtOAc 4:1); C-5’’), 129.0 (C-6), 129.4 (C-3), 130.1 (C-1), 135.0 (C-2), −1 UV(MeOH) λ : 276, 331 nm; IR ν̃ [cm ]: 1618 (C = C), max 135.2 (C-4), 135.7 (C-1’’’), 138.2 (C-1’’), 139.0 (C-7), 161.6 1577(C = C), 1497, 1453, 1284, 1174, and 1026 (C-O); (C-2’), 165.6 (C-6’), 168.5 (C-4’), 192.0 (C-9). H NMR (CDCl , 300  MHz) δ 5.01 (2H, s, OCH ), 5.07 3 2 (2H, s, OCH ), 6.09 (1H, d, J = 2.1 Hz, H-5’), 6.21 (1H, d, Synthesis and analytical data J = 2.1 Hz, H-3’), 7.14 (3H, m, H-3, H-4, H-5), 7.28 (10H, of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑3,5‑dichlorochalcone (5o) m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.71 (1H, d, J = 16.2  Hz, H-8), 7.94 (1H, d, J = 16.2, H-7), 14.0 (1H, s); C NMR Cl (CDCl , 75  MHz) δ 70.3 (OCH ), 71.1 (OCH ), 92.8 3 2 2 (C-3’), 94.9 (C-5’), 106.6 (C-1’), 127.5 (C-8), 127.6 (C-3, 1 2 4' BnO OH C-5), 128.0 (C-2’’’, C-6’’’), 128.3 (C-2’’, C-6’’), 128.4 (C-4’’’), Cl 128.5 (C-4’’), 128.7 (C-3’’’, C-5’’’), 129.2 (C-3’’, C-5’’), 133.0 2' 9 (C-4), 134.8 (C-2, C-6), 135.0 (C-1), 135.3 (C-1’’’), 135.7 OBn O (C-1’’), 136.1 (C-7), 161.6 (C-2’), 165.5 (C-6’), 168.2 (C-4’), 192.5 (C-9). 5o According to general procedure I, 2,4-dibenzyloxy-6-hy- droxyacetophenone (3) (1  g, 2.8  mmol) was condensed Synthesis and analytical data with 3,5-dichlorobenzaldehyde (4o) (0.54  g, 2.8  mmol) of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑2,5‑dichlorochalcone (5q) in the presence of aqueous KOH (60%, 3  mL) in etha- nol (15  mL). The product 5o was obtained in 70% yield. Cl mp: 125–127  °C; R 0.66 (petroleum ether:EtOAc 4:1); −1 UV(MeOH) λ : 278, 329 nm; IR ν̃ [cm ]: 1620 (C = C), 1 2 max 4' BnO OH 1574 (C = C), 1552, 1366, 1201, 1156, and 1045 (C-O); 2' 9 Cl H NMR (CDCl , 300  MHz) δ 5.05 (2H, s, OCH ), 5.10 3 2 (2H, s, OCH ), 6.18 (1H, d, J = 3.3 Hz, H-5’), 6.22 (1H, d, OBn O J = 2.4  Hz, H-3’), 7.05 (2H, br s, H-2, H-6), 7.28 (1H, m, 5q H-4), 7.42 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.50 (1H, d, J = 15.9  Hz, H-8), 7.75 (1H, d, J = 15.6  Hz, H-7); C According to general procedure I, 2,4-dibenzyloxy- NMR (CDCl , 75 MHz) δ 70.4 (OCH ), 71.5 (OCH ), 92.7 3 2 2 6-hydroxyacetophenone (3) (0.7  g, 2.0  mmol) was con- (C-3’), 95.0 (C-5’), 106.4 (C-1’), 126.4 (C-8), 127.6 (C-2, densed with 2,5-dichlorobenzaldehyde (4q) (0.37  g, C-6), 127.6 (C-2’’’, C-6’’’), 128.0 (C-2’’, C-6’’), 128.4 (C-4’’’), 2.0 mmol) in the presence of aqueous KOH (60%, 3 mL) 128.7 (C-4’’), 128.9 (C-3’’’, C-5’’’), 129.0 (C-3’’, C-5’’), 130.1 in ethanol (15 mL). The product 5q was obtained in 96% (C-4), 135.0 (C-3, C-5), 135.2 (C-1), 135.7 (C-1’’’), 138.2 yield. mp 128–129  °C; R 0.63 (petroleum ether:EtOAc (C-1’’), 139.0 (C-7), 161.6 (C-2’), 165.6 (C-6’), 168.5 (C-4’), −1 4:1); UV(MeOH) λ : 275, 338  nm; IR ν̃ [cm ]: 1617 max 192.0 (C-9). Melaku et al. BMC Chemistry (2022) 16:36 Page 18 of 30 Synthesis of the dibenzylated flavanones 6a-r (C = C), 1574 (C = C), 1552, 1423, 1201, 1165, and 1045 General procedure II for synthesis of the dibenzylated (C-O); H NMR (CDCl , 300 MHz) δ 5.06 (2H, s, O CH ), 3 2 flavanones 6a‑r [16, 30] 5.10 (2H, s, OCH ), 6.17 (1H, d, J = 2.4  Hz, H-5’), 6.22 A solution of a chalcone 5 (1  mmol) in ethanol was (1H, d, J = 2.4  Hz, H-3’), 7.08 (1H, d, J = 2.4  Hz, H-6), treated with sodium acetate (8  mmol) and the solution 7.20 (1H, dd, J = 8.1, J = 2.4  Hz, H-4), 7.29 (1H, m, H-3), was refluxed for 48  h, cooled to room temperature and 7.30–7.41 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.73 (1H, then diluted with water (60  mL/mmol). After extraction d, J = 15.9  Hz, H-8), 7.98 (1H, d, J = 15.9  Hz, H-7), 14.1 with CH Cl (3 × 30  mL/mmol), the combined organic (1H, s, 6’OH); C NMR (C DCl , 75 MHz) δ 70.3 (O CH ), 2 2 3 2 extracts were dried over anhydrous MgSO , filtered and 71.4 (OCH ), 92.7 (C-3’), 95.0 (C-5’), 106.4 (C-1’), 127.0 concentrated in vacuo. Purification was achieved by col - (C-8), 127.6 (C-2’’’, C-6’’’), 127.7 (C-2’’, C-6’’), 127.9 (C-4’’’), umn chromatography over silica gel using petroleum 128.3 (C-4’’), 128.7 (C-6), 128.8 (C-3’’’, C-5’’’), 130.3 (C-3’’, ether:EtOAc (4:1) as eluent to furnish the product 6 as a C-5’’), 130.9 (C-2), 131.1 (C-4), 132.7 (C-3), 133.4 (C-5), white solid. 134.9 (C-1), 135.0 (C-1’’’), 135.7 (C-1’’), 136.2 (C-7), 161.6 (C-2’), 165.6 (C-6’), 168.4 (C-4’), 192.1 (C-9). Synthesis and analytical data of 5,7‑dibenzyloxyflavanone (6a) Synthesis and analytical data of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑3,4‑dichlorochalcone (5r) 4' BnO 79 O 2 4 Cl 1' 1 4 BnO 4' OH Cl 5 OBn O 2' 9 6a OBn O According to the general procedure II, 2′4’-dibenzy- 5r loxy-6’-hydroxychalcone (5a) (700  mg, 1.6  mmol) was According to general procedure I, 2,4-dibenzyloxy- dissolved in ethanol (75  mL). Sodium acetate (1.05  g, 6-hydroxyacetophenone (3) (0.7  g, 2.0  mmol) was con- 12.8 mmol) was added and the solution was refluxed for densed with 3,4-dichlorobenzaldehyde (4r) (0.37  g, 48  h. After workup, the product 6a was obtained as a 2.0 mmol) in the presence of aqueous KOH (60%, 3 mL) white solid in 50% yield. mp 119–120  °C; R 0.70 (petro- in ethanol (15 mL). The product 5r was obtained in 98% leum ether:EtOAc 4:1); UV(MeOH) λ : 270, 330  nm; max −1 yield. mp 130–131  °C; R 0.64 (petroleum ether:EtOAc IR ν̃ [cm ]: 1674 (conjugated C = O), 1599 (C = C), −1 1 4:1); UV(MeOH) λ : 276, 329  nm; IR ν̃ [cm ]: 1618 1551(C = C), 1062, and 1028 (C-O); H NMR (CDCl , max 3 (C = C), 1578 (C = C), 1420, 1202, 1160, and 1022(C- 300  MHz) δ 2.82 (1H, dd, J = 2.7, J = 16.5  Hz, H-3), 3.06 O); H NMR (CDCl , 300  MHz) δ 5.05 (2H, s, OCH ), (1H, dd, J = 13.2, J = 16.5 Hz, H-3), 5.0 (2H, s, O CH ), 5.2 3 2 2 5.11 (2H, s, OCH ), 6.18 (1H, d, J = 2.4  Hz, H-5’), 6.22 (2H, s, OCH ), 5.43 (1H, dd, J = 2.7, J = 13.2  Hz, H-2), 2 2 (1H, d, J = 2.4 Hz, H-3’), 6.74 (1H, dd, J = 8.4, J = 1.8  Hz, 6.25 (1H, s, H-8), 6.26 (1H, s, H-6), 7.59–7.30 (15H, m, H-6), 7.20 (1H, d, J = 8.1 Hz, H-5), 7.28 (1H, d, J = 2.4 Hz, H-2’–H-6’, H-2’’–H-6’’, H-2’’’–H-6’’’); C NMR (CDCl , H-2), 7.43 (10H, m, H-2’’’–H-6’’’, H-2’’– H-6’’), 7.54 (1H, 75  MHz) δ 45.7 (C-3), 70.2 (OCH ), 70.3 (OCH ), 2 2 d, J = 15.6  Hz, H-8), 7.79 (1H, d, J = 15.6  Hz, H-7); C 79.3 (C-2), 94.7 (C-6), 95.1 (C-8), 106.4 (C-10), 126.1 NMR (CDCl , 75  MHz) δ 70.2 (O CH ), 70.6 (OCH ), (C-2’,C-6’), 126.5 (C-2’’’, C-6’’’), 127.5 (C-2’’, C-6’’), 127.6 3 2 2 92.6 (C-3’), 95.0 (C-5’), 106.5 (C-1’), 126.7 (C-8), 127.6 (C-4’), 128.3 (C-4’’’), 128.5 (C-4’’), 128.6 (C-3’,C-5’), (C-6), 127.7 (C-2’’’, C-6’’’), 127.9 (C-2’’, C-6’’), 128.4 (C-4’’’), 128.7 (C-3’’’, C-5’’’), 128.8 (C-3’’, C-5’’), 135.7 (C-1’), 136.3 128.5 (C-4’’), 128.8 (C-2), 128.9 (C-3’’’, C-5’’’), 129.2 (C-3’’, (C-1’’’), 138.7 (C-1’’), 161.0 (C-9), 164.8 (C-5), 164.9 (C-7), C-5’’), 130.1 (C-5), 130.5 (C-4), 132.7 (C-3), 135.2 (C-1), 188.8 (C-4); MS (EI, 70  eV) m/z (%) 436 (19) [M] , 346 135.3 (C-1’’’), 135.7 (C-1’’), 139.4 (C-7), 161.9 (C-2’), 165.9 (7), 317 (4), 240 (3), 91 (100). (C-6’), 192.8 (C-9). M elaku et al. BMC Chemistry (2022) 16:36 Page 19 of 30 Synthesis and analytical data J = 16.5 H-3), 3.90 (1H, s, OMe), 3.92 (1H, s, OMe), of 5,7‑dibenzyloxy‑4’‑fluoroflavanone (6b) 5.04 (2H, s, OCH ), 5.10 (2H, s, OCH ), 5.38 (1H, dd, 2 2 2.7, J = 16.5  Hz, H-2), 6.24 (2H, br s, H-8), 6.90 (1H, 4' F br s, H-6), 7.0 (2H, m, H-2’, H-5’), 7.18 (1H, m, H-6’), 7.40–7.58 (10H, m, H-2’’’–H-6’’’, H-2’’–H-6’’); C NMR BnO O 2 1' (CDCl , 75 MHz) δ 45.6 (C-3), 55.8 (OMe), 55.9 (OMe), 70.2 (OCH ), 70.4 (OCH ), 79.3 (C-2), 94.7 (C-6), 95.1 2 2 (C-8), 106.6 (C-10), 109.4 (C-2’), 118.8 (C-5’), 126.5 OBn O (C-6’), 127.2 (C-2’’’, C-6’’’), 127.6 (C-2’’, C-6’’), 128.0 6b (C-4’’’), 128.3 (C-4’’), 128.6 (C-3’’’, C-5’’’), 128.7 (C-3’’, C-5’’), 131.1(C-1’), 135.7 (C-1’’’), 136.3 (C-1’’), 149.2 According to the general procedure II, 2′4’-dibenzy- (C-4’), 149.4 (C-3’), 158.2 (C-9), 161.0 (C-5), 164.9 loxydibenzyloxy-6’-hydroxy-4-fluorochalcone (5b) (C-7), 189.0 (C-4); MS (EI, 70  eV) m/z (%) 496 (16) (250  mg, 0.5  mmol) was dissolved in ethanol (60  mL). [M] , 406 (24), 348 (9), 227 (13), 151 (27), 91 (100); Sodium acetate (0.33  g, 4  mmol) was added and the HRMS (EI, M ) calculated for C H O 496.1886 solution was refluxed for 48  h. After workup, the 31 28 6 found 496.1880. product 6b was obtained in 48% yield. mp 76–78  °C; UV(MeOH) λ : 272, 335  nm; H NMR (CDCl , max 3 300 MHz) δ 2.69 (1H, dd, J = 2.7, J = 16.2 Hz, H-3), 3.00 Synthesis and analytical data of 5,7‑dibenzyloxy‑2′6’‑dimeth (1H, dd, J = 12.6, J = 16.2 Hz, H-3), 5.20 (2H, s, O CH ), 2 oxyflavanone (6d) 5.22 (2H, s, OCH ), 5.56 (1H, dd, J = 2.7, J = 12.6  Hz , H-2), 6.32 (1H, d, J = 2.4  Hz, H-8), 6.42 (1H, d, MeO 4' J = 2.4 Hz, H-6), 7.69–7.18 (14H, m, H-2’,H-6’, H-3’, H-5’, BnO O 2 H-2’’’–H-6’’’, H-2’’–H-6’’); C NMR (CDCl , 75  MHz) δ 1' 2' 46.2 (C-3), 70.7 (OCH ), 70.8 (OCH ), 79.2 (C-2), 95.6 OMe 2 2 (C-6), 95.7 (C-8), 107.1 (C-10), 115.9 (C-3’, C-5’), 116.2 OBn O (C-2’, C-6’), 127.4 (C-2’’’, C-6’’’), 128.1 (C-2’’, C-6’’), 128.5 6d (C-4’’’), 128.9 (C-4’’), 129.0 (C-3’’’, C-5’’’), 129.3 (C-3’’, C-5’’), 129.4 (C-1’), 137.4 (C-1’’’), 138.8 (C-1’’), 161.8 (d, According to the general procedure II, 2’,4’-dibeny- C-4’), 165.0 (C-9), 165.4 (C-5), 165.6 (C-7), 187.8 (C-4). loxy-6’-hydroxy-2,6-dimethoxychalcone (5d) (250  mg, 0.5  mmol) was dissolved in ethanol (75  mL). Sodium acetate (0.32 g, 4 mmol) was added and the solution was Synthesis and analytical data of 5,7‑dibenzyloxy‑3′4’‑dimeth refluxed for 48  h. The product 6d was obtained in 52%. oxyflavanone (6c) −1 mp 87  °C; UV(MeOH) λ : 275, 344  nm; IR ν̃ [cm ]: max 1667 (conjugated C = O), 1607 (C = C), 1572 (C = C), 4' OMe 1051, and 1025 (C-O); H NMR (CDCl , 300  MHz) BnO 79 O 2 δ 2.88–2.80 (2H, m, H-3), 3.7 (3H, s, OMe), 3.8 (3H, s, 1' 3' OMe OMe), 5.0 (2H, s, OCH ), 5.1 (2H, s, OCH ), 5.77 (1H, 2 2 5 dd, J = 4.8, J = 11.1  Hz, H-2), 6.24 (1H, d, J = 2.4  Hz, OBn O H-8), 6.28 (1H, d, J = 2.4  Hz, H-6), 6.83 (1H, br d, H-4), 6c 7. 20–7.29 (10H, m, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.60 (2H, br d, J = 7.5  Hz, H-3’, H-5’); C NMR (CDCl , 75  MHz) According to the general procedure II, 2’,4’-dibeny- δ 44.8 (C-3), 55.8 (OMe), 55.9 (OMe), 70.2 (OCH ), 70.3 loxy-6’-hydroxy-3,4-dimethoxychalcone (5c) (200  mg, (OCH ), 74.3 (C-2), 94.7 (C-6), 95.0 (C-8), 106.5 (C-10), 0.4  mmol) was dissolved in ethanol (70  mL). Sodium 111.5 (C-1’), 112.5 (C-3’), 113.5 (C-5’), 126.5 (C-2’’’, C-6’’’), acetate (0.26  g, 3.2  mmol) was added and the solution 127.5 (C-2’’, C-6’’), 128.3 (C-4’’), 128.5 (C-4’’), 128.6 (C-3’’’, was refluxed for 48  h. The product 6c was obtained C-5’’’), 128.7 (C-3’’, C-5’’), 128.9 (C-4’), 135.8 (C-4’’’), 136.6 in 40% yield. mp 91–92  °C; UV(MeOH) λ : 276, max (C-4’’), 149.9 (C-2’), 153.5 (C-6’), 161.1 (C-5), 164.1 (C-9), −1 345  nm; IR ν̃ [cm ]: 1672 (conjugated C = O), 1595 165.2 (C-7), 189.5 (C-4); MS (EI, 70 eV) m/z (%) 496 (24) (C = C), 1571 (C = C), 1257, 1117, 1075, and 1012 [M] , 465 (14), 405 (29), 348 (9), 91 (100); HRMS (EI, (C-O); H NMR(CDCl , 300  MHz) δ 2.77 (1H, dd, 3 M ) calculated for C H O 496.1886 found 496.1870. 31 28 6 J = 2.7, J = 16.5  Hz, H-3), 3.08 (1H, dd, J = 13.2  Hz , Melaku et al. BMC Chemistry (2022) 16:36 Page 20 of 30 Synthesis and analytical data 7.76  mmol) was added and the solution was refluxed of 5,7‑dibenzyloxy‑4’‑chloroflavanone (6e) for 48  h. The product 6f was obtained in 36% yield. mp −1 89–91  °C; UV(MeOH) λ : 273, 338  nm; IR ν̃ [cm ]: max 4' Cl 1672 (conjugated C = O), 1603 (C = C), 1569 (C = C), 1068, and 1024 (C-O); H NMR (CDCl , 300  MHz) δ BnO O 2 1' 2.82 (1H, dd, J = 13.2  Hz, J = 16.5 Hz, H-3), 2.98 (1H, dd, J = 16.2  Hz, J = 2.7  Hz, H-3), 5.06 (2H, s, OCH ), 5.17 (2H, s, OCH ), 5.8 (1H, dd, J = 10.5  Hz, J = 2.4  Hz, H-2), OBn O 6.27 (2H, br s, H-6, H-8), 7.20–7.70 (14H, m, H-3’–H-6’, 6e H-2’’’–H-6’’’, H-2’’–H-6’’); C NMR (CDCl , 75  MHz) δ 44.6 (C-3), 70.3 (O CH ), 70.4 (OCH ), 78.4 (C-2), 94.7 2 2 According to the general procedure II, 2’,4’-dibenzyloxy- (C-6), 95.3 (C-8), 106.4 (C-10), 121.5 (C-2), 126.5 (C-2’’’, 6’-hydroxy-4-chlorochalcone (5e) (300  mg, 0.6  mmol) C-6’’’), 127.3 (C-2’’, C-6’’), 127.5 (C-4’’’), 127.6 (C-4’’), 127.9 was dissolved in ethanol (60 mL). Sodium acetate (0.39 g, (C-5’), 128.3 (C-3’’’, C-5’’’), 128.5 (C-3’’, C-5’’), 128.7 (C-6’), 4.8  mmol) was added and the solution was refluxed for 129.8 (C-4’), 132.9 (C-3’), 135.7 (C-1’’’), 136.3 (C-1’’), 48 h. The product 6e was obtained in 40% yield. mp 129– 138.3 (C-1’’), 161.3 (C-9), 164.9 (C-5), 165.0 (C-7), 188.4 131 °C; R 0.56 (petroleum ether:EtOAc 4:1); UV(MeOH) −1 (C-4); MS (EI, 70  eV) m/z (%) 516 (16) [M + 2] , 514 λ : 271, 336  nm; IR ν̃ [cm ]:1671 (conjugate C = O), max [M] (18), 435 (12), 425 (14), 423 (12), 256 (4), 91 (100); 1606 (C = C), 1571(C = C), 1064, and 1035 (C-O); H HRMS (EI, M ) calculated for C H O Br 514.0780 29 23 4 NMR (CD COCD , 300 MHz) δ 2.75 (1H, dd, J = 3.0  Hz, 3 3 found 514.0777. J = 16.2  Hz, H-3), 3.00 (1H, dd, J = 12.6  Hz, J = 16.2  Hz, H-3), 5.20 (1H, s, OCH ), 5.21 (1H, s, OCH ), 5.57 (1H, 2 2 Synthesis of 5,7‑dibenyloxy‑4‑(trifluoromethyl)flavanone (6g) dd, J = 3  Hz, J = 12.6  Hz, H-2), 6.23 (1H, d, J = 2.2  Hz, H-8), 6.43 (1H, d, J = 2.2  Hz, H-6), 7.40 (10H, m, H-2’’’– 4' CF H-6’’’, H-2’’–H-6’’), 7.59 (2H, d, J = 7.0, H-3’,H-5’), 7.6 (2H, d, J = 7.0  Hz, H-2’, H-6’); C NMR (CD COCD , 79 BnO O 2 3 3 1' 75  MHz) δ 46.1 (C-3), 70.7 (O CH ), 70.8 (O CH ), 79.1 2 2 (C-2), 95.7 (C-6), 95.8 (C-8), 107.0 (C-10), 127.4 (C-2’’’, OBn O C-6’’’), 128.1 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.9 (C-4’’), 129.0, (C-3’’’, C-5’’’) 129.1 (C-3’’, C-5’’), 129.3 (C-2’, C-6’), 6g 129.4 (C-3’, C-5’), 134.4 (C-4’), 137.4 (C-1’’’), 138.0 (C-1’’), According to the general procedure II, 2’,4’-dibenyloxy- 139.3 (C-1’), 161.9 (C-9), 165.3 (C-5), 165.5 (C-7), 188.8 6’-hydroxy-4-(trifluoromethyl)chalcone (5g) (500  mg, (C-4); MS (EI,70 eV) m/z (%) 470 (11) [M] , 379 (8), 348 0.97  mmol) was dissolved in ethanol (75  mL). Sodium (11), 257 (6), 180 (4), 91 (100); HRMS (EI, M ) calculated acetate (0.64  g, 7.8  mmol) was added and the solution for C H O Cl 470.1285 found 470.1260. 29 23 4 was refluxed for 48  h. The product 6g was obtained in 8% yield. H NMR (CD COCD , 300  MHz) δ 2.78 (1H, 3 3 Synthesis and analytical data dd, J = 12.5 Hz, J = 3.3 Hz, H-3), 3.00 (1H, dd, J = 12.5 Hz, of 5,7‑dibenzyloxy‑2’‑bromoflavanone (6f ) J = 13.3 Hz, H-3), 5.20 (2H, s, OCH ), 5.23 (2H, s, OCH ), 2 2 5.68 (1H, dd, J = 12.5  Hz, J = 2.5  Hz, H-2), 6.36 (1H, d, 4' J = 2.2  Hz, H-8), 6.44 (1H, d, J = 2.2  Hz, H-6), 7.31–7.77 BnO 79 O 2 1' (14H, m, H-2’, H-3’, H-5’, H-6’, H-2’’’–H-6’’’, H-2’’–H-6’’); Br C NMR (CD COCD , 75  MHz) δ 45.7 (C-3), 70.2 3 3 (OCH ), 70.9 (O CH ), 78.7 (C-2), 95.7 (C-6), 95.6, 107.2 2 2 OBn O (C-10), 126.3 (CF ), 127.5 (C-3’, C-5’), 127.7 (C-2’’’, C-6’’’), 6f 128.2 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.9 (C-4’’), 129.0 (C-2’, C-6’), 129.3 (C-3’’’, C-5’’’), 129.4 (C-3’’, C-5’’), 132.0 (C-4’), According to the general procedure II, 2’,4’-dibenyloxy- 137.4 (C-1’’’), 138.0 (C-1’’), 144.8 (C-1’), 161.9 (C-9), 165.2 6’-hydroxy-2-bromochalcone (5f) (500  mg, 0.97  mmol) (C-5), 165.8 (C-7), 187.0 (C-4). was dissolved in ethanol (75 mL). Sodium acetate (0.64 g, M elaku et al. BMC Chemistry (2022) 16:36 Page 21 of 30 1154, and 1025 (C-O); H NMR (CDCl , 300  MHz) δ Synthesis and analytical data of 5,7‑dibenzyloxy‑3′4’5’‑trime 3 2.88 (1H, dd, J = 12.9  Hz, J = 3.3  Hz, H-3), 2.98 (1H, dd, thoxyflavanone (6h) J = 12.9  Hz, J = 16.8  Hz, H-3), 5.05 (2H, s, O CH ), 5.17 (2H, s, OCH ), 5.44 (1H, dd, J = 12.6, J = 3.3  Hz, H-2), OMe 4' OMe 6.21 (2H, br s, H-6 and H-8), 7.28–7.66 (10H, m, H-2’’’– H-6’’’, H-2’’–H-6’’), 7.61 (2H, d, J = 7.5 Hz, H-2’ and H-3’), BnO O 2 1' OMe 7.72 (1H, d, J = 9.3 Hz, H-5’); C NMR (CDCl , 75 MHz) δ 44.5 (C-3), 70.3 (OCH ), 70.4 (OCH ), 76.2 (C-2), 94.7 2 2 (C-6), 95.2 (C-8), 106.4 (C-2’, C-6’), 107.4 (C-10), 126.5 OBn O (C-2’’’, C-6’’’), 127.1 (C-2’’, C-6’’), 127.3 (C-4’’’), 127.5 6h (C-4’’), 128.5 (C-3’’’, C-5’’’), 129.3 (C-3’’, C-5’’), 131.7 (C-1’), 135.7 (C-4’), 136.3 (C-1’’’), 136.7 (C-1’’), 161.6 (C-9), 164.9 According to the general procedure II, 2’,4’-dibenyloxy- (C-5), 164.9 (C-7), 188.5 (C-4); MS (EI, 70  eV) m/z (%) 6’-hydroxy-3,4,5-trimethoxychalcone (5h) (400  mg, + + 534 (10) [M + 2] , 532 (9) [M] , 442 (6), 359 (4), 256 (3), 0.76  mmol) was dissolved in ethanol (70  mL). Sodium 91 (100); HRMS (EI, M ) calculated for C H O BrF 29 22 4 acetate (0.5  g, 6.08  mmol) was added and the solution 532.0685 found 532.0671. was refluxed for 48  h. The product 6h was obtained in 38% yield. mp 84–86  °C; UV(MeOH) λ : 274, 351  nm; max Synthesis and analytical data −1 IR ν̃ [cm ]: 1671 (conjugated C = O), 1607 (C = C), of 5,7‑dibenzyloxy‑2’‑chloroflavanone (6l) 1569, 1116, and 1032 (C-O); H NMR (CD COCD , 3 3 300  MHz) δ 2.68 (1H, dd, J = 16.5  Hz, J = 3  Hz, H-3), 3.06 (1H, dd, J = 12.9  Hz, J = 16.5  Hz, H-3), 3.75 (3H, s, BnO 79 O 2 3' OMe), 3.85 (6H, s, OMe), 5.19 (2H, s, O CH ), 5.22 (2H, 1' s, OCH ), 5.44 (1H, dd, J = 12.9, J = 3 Hz, H-2), 6.32 (1H, 4 Cl d, J = 2.2  Hz, H-8), 6.41 (1H, d, J = 2.2  Hz), 7.28–7.66 OBn O (12H, m, H-2’, H-6’, H-2’’’–H-6’’’, H-2’’–H-6’’); C NMR 6l (CD COCD , 75  MHz) δ 46.3 (C-3), 56.3 (OMe), 60.5 3 3 (OMe), 70.3 (O CH ), 70.8 (O CH ), 80.3 (C-2), 95.7 (C-6), 2 2 According to the general procedure II, 2’,4’-dibenzyloxy- 95.8 (C-8), 104.8 (C-2’, C-6’), 107.4 (C-10), 127.4 (C-2’’’, 6’-hydroxy-2-chlorochalcone (5l) (1.3  g, 2.9  mmol) was C-6’’’), 128.2 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.9 (C-4’’), 129.0 dissolved in ethanol (100  mL). Sodium acetate (1.9  g, (C-3’’’, C-5’’’), 129.3 (C-3’’, C-5’’), 135.7 (C-1’), 137.5 (C-4’), 23  mmol) was added and the solution was refluxed 138.1 (C-1’’’), 139.2 (C-1’’), 154.4 (C-3’, C-5’), 161.6 (C-9), for 48  h. The product 6l was obtained in 42% yield. 165.5 (C-5), 165.6 (C-7), 188.7 (C-4); MS (EI, 70 eV) m/z mp 127–128  °C; R 0.55 (petroleum ether:EtOAc 4:1); (%) 526 (40) [M] , 435 (28), 407(16), 257 (16), 181 (26), −1 UV(MeOH) λ : 272, 337  nm; IR ν̃ [cm ]: 1672 (con- max 91 (100). jugated C = O), 1604 (C = C), 1570 (C = C), 1162, 1068, and 1033 (C-O); H NMR (CDCl , 300  MHz) δ 2.85 Synthesis and analytical data (1H, dd, J = 16.5, J = 3.3  Hz, H-3), 2.94 (1H, dd, J = 15.5, of 5,7‑dibenzyloxy‑2’‑bromo‑4’‑fluoroflavanone (6i) J = 3.3 Hz, H-3), 5.06 (2H, s, O CH ), 5.17 (2H, s, O CH ), 2 2 5.80 (1H, dd, J = 12.9 J = 3.3 Hz, H-2), 6.27 (2H, br s, H-6, 4' H-8), 7.38 (11H, m, H-5’, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.58 BnO O 2 (1H, br s, H-6’), 7.60 (1H, d, J = 9.3 Hz, H-4’), 7.71 (1H, d, 1' J = 9.3 Hz, H-3’); C NMR (C DCl , 75 MHz) δ 44.5 (C-3), Br 3 570.3 (OCH ), 70.4 (OCH ), 76.4 (C-2), 94.7 (C-6), 95.2 2 2 OBn O (C-8), 106.4 (C-10), 126.3 (C-5’), 127.3 (C-2’’’, C-6’’’), 127.5 6i (C-2’’, C-6’’), 127.6 (C-4’’’), 128.3 (C-4’’), 128.5 (C-6’), 128.7 (C-3’’’, C-5’’’), 128.8 (C-3’’, C-5’’), 129.4 (C-4’), 129.6 (C-3’), According to the general procedure II, 2’,4’-dibenyloxy- 131.7 (C-2’), 135.7 (C-1’), 136.3 (C-1’’’), 136.7 (C-1’’), 6’-hydroxy-2-bromo-4-fluorochalcone (5i) (400  mg, 161.1 (C-9), 164.9 (C-5), 165.2 (C-7), 188.5 (C-4); MS 0.75  mmol) was dissolved in ethanol (70  mL). Sodium + (EI, 70 eV) m/z (%) 470 (16) [M] , 435 (4), 379 (11), 255 acetate (0.49 g, 6.0 mmol) was added and the solution was + (4), 165 (3); HRMS (EI, M ) calculated for C H O Cl 29 23 4 refluxed for 48  h. The product 6i was obtained as 48% 470.1285 found 470.1264. −1 yield. IR ν̃ [cm ]: 1668 (conjugated C = O), 1603 (C = C), Melaku et al. BMC Chemistry (2022) 16:36 Page 22 of 30 Synthesis and analytical data (C = C), 1570 (C = C), 1430, 1163, 1116, and 1040 (C-O); of 5,7‑dibenzyloxy‑3’‑chloroflavanone (6m) H NMR (CDCl , 300  MHz) δ 2.79 (1H, dd, J = 12.9  Hz, J = 16.5  Hz, H-3), 2.93 (1H, dd, J = 16.5  Hz, J = 3.6  Hz, H-3), 5.08 (2H, s, OCH ), 5.17 (2H, s, OCH ), 5.77 (1H, 2 2 3' BnO O 2 dd, J = 12.9  Hz, J = 3.0  Hz, H-2), 6.25 (1H, d, J = 2.4  Hz, 1' Cl H-8), 6.27 (1H, d, J = 2.4  Hz, H-6), 7.39 (10H, m, H-2’’’– H-6’’’, H-2’’–H-6’’), 7.59 (2H, d, J = 7.2 Hz, H-3, H-5’), 7.64 OBn O (1H, d, J = 8.4  Hz, H-3’); C-NMR (CDCl , 75  MHz) δ 6m 44.4 (C-3), 70.3 (O CH ), 70.4 (OCH ), 75.7 (C-2), 94.7 2 2 (C-6), 95.3 (C-8), 106.4 (C-10), 126.5 (C-5’), 127.5 (C-2’’’, According to the general procedure II, 2’,4’-dibenzyloxy- C-6’’’), 127.7 (C-3’’, C-6’’), 128.1 (C-4’’’), 128.3 (C-4’’), 128.5 6’-hydroxy-3-chlorochalcone (5m) (0.8 g, 1.8 mmol) was (C-3’’’, C-5’’’), 128.7 (C-3’’, C-5’’), 128.9 (C-3’), 129.4 (C-6’), dissolved in ethanol (100  mL). Sodium acetate (1.2  g, 132.3 (C-2’), 134.7 (C-4’), 135.4 (C-1’), 135.6 (C-1’’’), 136.2 14.4  mmol) was added and the solution was refluxed (C-1’’), 161.1 (C-9), 164.6 (C-5), 164.9 (C-7), 188.0 (C-4); + + for 48 h. The product 6m was obtained in 50% yield. mp MS (EI, 70 eV) m/z (%) 506 (6) [M + 2] , 504 (12) [M] , 129 °C; R 0.56 (petroleum ether:EtOAc 4:1); UV(MeOH) 470 (3), 413 (7), 256 (3), 199 (6), 91 (100); HRMS (EI, M ) −1 λ : 274, 334 nm; IR ν̃ [cm ]: 1673 (conjugated C = O), calculated for C H O Cl 504.0895 found 504.0908. max 29 22 4 2 1604 (C = C), 1571 (C = C), 1428, 1163, 1117, and 1025 (C-O); H NMR (CDCl , 300  MHz) δ 2.78 (1H, dd, Synthesis and analytical data J = 16.5  Hz, J = 3  Hz, H-3), 2.98 (1H, dd, J = 16.5  Hz, of 5,7‑dibenzyloxy‑3’,5’‑dichloroflavanone (6o) J = 13.2 Hz, H-3), 5.06 (2H, s, OCH ), 5.16 (2H, s, OCH ), 2 2 5.40 (1H, dd, J = 3 Hz, J = 12.9 Hz, H-2), 6.25 (2H, s, H-6, Cl H-8), 7.36 (11H, m, H-6’, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.49 (1H, br s, H-2’), 7.59 (2H, d, J = 7.8  Hz, H-4’, H-5’); C BnO O 2 1' 3' NMR (CDCl , 75  MHz) δ 45.6 (C-3), 70.3 (OCH ), 70.3 Cl 3 2 (OCH ), 78.4 (C-2), 94.7 (C-6), 95.2 (C-8), 106.3 (C-10), 124.1 (C-6’), 126.2 (C-2’), 126.4 (C-2’’’, C-6’’’), 127.5 (C-2’’, OBn O C-6’’), 127.6 (4’’’), 128.1 (C-4’’), 128.3 (C-4’), 128.5 (C-3’’’, 6o C-6’’’), 128.7 (C-3’’, C-5’’), 130.0 (C-5’), 134.7 (C-3’), 135.6 (C-1’’’), 136.2 (C-1’’), 140.8 (C-1’), 161.0 (C-9), 164.5 According to the general procedure II, 2’,4’-dibenzyloxy- (C-5), 164.9 (C-7), 188.1 (C-4); MS (EI, 70  eV) m/z (%) 6’-hydroxy-3,5-dichlorochalcone (5o) (0.7  g, 1.4  mmol) 470 (26) [M] , 379 (9), 348 (12), 306 (6), 257 (5), 215 was dissolved in ethanol (100 mL). Sodium acetate (0.9 g, (6), 91 (100); HRMS (EI, M ) calculated for C H O Cl 29 23 4 11.2  mmol) was added and the solution was refluxed for 470.1285 found 470.1270. 48 h. The product 6o was obtained in 36% yield. mp 115– 116  °C; R 0.58 (petroleum ether:EtOAc 4:1); UV(MeOH) −1 λ : 271, 338  nm; IR ν ̃ [cm ]: 1668 (conjugated C = O), max Synthesis and analytical data 1614 (C = C), 1572 (C = C), 1374, 1216, 1068, and 1018 of 5,7‑dibenzyloxy‑2’,4’‑dichloroflavanone (6n) (C-O); H NMR (CDCl , 300  MHz) δ 2.80 (1H, dd, J = 3.3  Hz, J = 16.5  Hz, H-3), 2.95 (1H, dd, J = 16.5  Hz, 4' Cl J = 12.9 Hz), 5.06 (2H, s, OCH ), 5.16 (2H, s, OCH ), 5.36 2 2 BnO O 2 (1H, dd, J = 12.9  Hz, J = 3.3 Hz, H-2), 6.21 (2H, br s, H-6, 1' H-8), 7.33 (11H, m, H-4’, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.57 Cl (2H, d, J = 7.5 Hz, H-2’, H-6’); C NMR (C DCl , 75 MHz) 5 3 OBn O δ 45.5 (C-3), 70.4 (OCH ), 70.4 (OCH ), 77.7 (C-2), 94.7 2 2 6n (C-6), 95.5 (C-8), 106.3 (C-10), 124.5 (C-2’, C-6’), 126.5 (C-2’’’, C-6’’’), 127.6 (C-2’’, C-6’’), 127.7 (C-4’’’), 128.4 (C-4’’), According to the general procedure II, 2’,4’-dibenzyloxy- 128.6 (C-3’’’, C-5’’’), 128.7 (C-3’’, C-5’’), 128.8 (C-4’), 135.4 6’-hydroxy-2,4-dichlorochalcone (5n) (0.6  g, 1.2  mmol) (C-3’, C-5’), 135.6 (C-1’’’), 136.2 (C-1’’), 142.2 (C-1’), 161.1 was dissolved in ethanol (100 mL). Sodium acetate (0.8 g, (C-9), 164.2 (C-5), 165.0 (C-7), 187.6 (C-4); MS (EI, 70 eV) + + 9.6  mmol) and the solution was refluxed for 48  h. The m/z (%) 506 (9) [M + 2] , 504 (16) [M] , 413(7), 384 (4), product 6n was obtained in 54% yield. mp 117–118  °C; 359 (3), 303 (4), 256 (4), 199 (5), 91 (100); HRMS (EI, M ) R 0.57 (petroleum ether:EtOAc 4:1); UV(MeOH) λ : calculated for C H O Cl 504.0895 found 504.0889. f max 29 22 4 2 −1 275, 339 nm; IR ν̃ [cm ]: 1669 (conjugated C = O), 1604 M elaku et al. BMC Chemistry (2022) 16:36 Page 23 of 30 Synthesis and analytical data 11.2 mmol) was added and the solution was refluxed for of 5,7‑dibenzyloxy‑2’,6’‑dichloroflavanone (6p) 48 h. The product 6q was obtained in 43% yield. mp 121– 124 °C; R 0.55 (petroleum ether:EtOAc 4:1); UV(MeOH) −1 Cl λ : 273, 338  nm; IR ν̃ [cm ]:1671 (conjugated C = O), max 1606 (C = C), 1570 (C = C), 1466, 1209, 1108, and 1032 BnO O 2 1' 2' (C-O); H NMR (CDCl , 500  MHz) δ 2.78 (1H, dd, Cl J = 13.5  Hz, J = 17.0  Hz, H-3), 2.96 (1H, dd, J = 16.5  Hz, J = 2.5 Hz, H-3), 5.07 (2H, s, O CH ), 5.17 (2H, s, O CH ), 2 2 OBn O 5.75 (1H, dd, J = 13.5  Hz, J = 3  Hz, H-2), 6.28 (1H, d, 6p J = 2  Hz, H-8), 6.29 (1H, d, J = 2  Hz, H-6), 7.29 (1H, dd, J = 8.5  Hz, J = 2.5  Hz, H-4’), 7.39 (10H, m, H-2’’’–H-6’’’, According to the general procedure II, 2’,4’-diben- H-2’’–H-6’’), 7.59 (1H, d, J = 7.5  Hz, H-3’), 7.72 (1H, d, zyloxy-6’-hydroxy-2,6-dichlorochalcone (5p) (0.7  g, J = 2.5  Hz, H-6’); C NMR (C DCl , 125  MHz) δ 44.4 1.4  mmol) was dissolved in ethanol (100  mL). Sodium (C-3), 70.4 (OCH ), 70.4 (OCH ), 75.9 (C-2), 94.8 (C-6), 2 2 acetate (0.9  g, 11.2  mmol) was added and the solution 95.5 (C-8), 106.4 (C-10), 126.5 (C-2’’’, C-6’’’), 126.5 (C-2’’, was refluxed for 48  h. The product 6p was obtained C-6’’), 127.2 (C-4’’’), 127.5 (C-4’’), 127.7 (C-6’), 128.4 in 32% yield. mp 109–111  °C; R 0.53 (petroleum (C-3’’’, C-5’’’), 128.6 (C-3’’, C-5’’), 129.4 (C-4’), 129.6 (C-3’), ether:EtOAc 4:1); UV(MeOH) λ : 275, 339  nm; IR ν̃ max −1 130.8 (C-2’), 133.4 (C-5’), 135.7 (C-1’), 136.2 (C-1’’’), [cm ]: 1663 (conjugated C = O), 1607 (C = C), 1570 138.5 (C-1’’), 161.2 (C-9), 164.5 (C-5), 165.0 (C-7), 187.9 (C = C), 1241, 1055, and 1029 (C-O); H NMR (C DCl , (C-4); MS (EI, 70 eV) m/z (%) 506 (34) [M + 2] , 504 (49) 500  MHz) δ 2.56 (1H, dd, J = 3.3  Hz , J = 16.5  Hz , H-3), [M] , 469 (16), 413 (31), 385 (9), 359 (12), 305 (15), 256 3.68 (1H, dd, J = 14.4  Hz , J = 16.8  Hz, H-3), 5.0 (2H, (10), 199 (14), 91 (100); HRMS (EI, M ) calculated for s, OCH ), 5.13 (2H, s, OCH ), 6.19 (1H, d, J = 1.8  Hz , 2 2 C H O Cl 504.0895 found 504.0863. 29 22 4 2 H-8), 6.20 (1H, d, J = 1.8  Hz, H-6), 7.20 (1H, m, H-4), 7.35 (10H, m, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.56 (2H, d, J = 7.5  Hz, H-3’, H-5’); C NMR (C DCl , 125  MHz) δ Synthesis and analytical data 40.5 (C-3), 70.3 (OCH ), 70.4 (OCH ), 75.7 (C-2), 94.5 2 2 of 5,7‑dibenzyloxy‑3’,4’‑dichloroflavanone (6r) (C-6), 95.1 (C-8), 106.2 (C-10), 126.5 (C-3’, C-5’), 127.6 (C-2’’’, C-6’’’), 128.3 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.6 4' Cl (C-4’’), 129.6 (C-3’’’, C-5’’’), 130.1 (C-3’’, C-5’’), 132.4 BnO O 2 (C-4’), 132.5 (C-2’, C-6’), 135.1 (C-1’), 135.7 (C-1’’’), 1' Cl 3' 136.3 (C-1’’), 161.2 (C-9), 164.6 (C-5), 164.9 (C-7), 4 188.2 (C-4); MS (EI, 70  eV) m/z (%) 506 (10) [M + 2] , OBn O 504 (17) [M] , 469 (6), 413 (11), 359 (14), 305 (6), 256 6r (4), 199 (16), 91 (100); HRMS (EI, M ) calculated for C H O Cl 504.0895 found 504.0881. 29 22 4 2 According to the general procedure II, 2’,4’-dibenzyloxy- 6’-hydroxy-2,6-dichlorochalcone (5r) (0.9  g, 1.8  mmol) Synthesis and analytical data was dissolved in ethanol (100 mL). Sodium acetate (1.2 g, of 5,7‑dibenzyloxy‑2’,5’‑dichloroflavanone (6q) 14.4 mmol) was added and the solution was refluxed for 48 h. The product 6r was obtained in 44% yield. mp 123– Cl 125 °C; R 0.54 (petroleum ether:EtOAc 4:1); UV(MeOH) −1 λ : 271, 337 nm; IR ν̃ [cm ]: 1666 (conjugated C = O), max BnO 79 O 2 1606 (C = C), 1431, 1165, 1118, and 1028 (C-O); H NMR 1' 2' (CDCl , 500 MHz) δ 2.81 (1H, dd, J = 16.5 Hz, J = 3.0 Hz, Cl 5 H-3), 2.96 (1H, dd, J = 13  Hz, J = 16.5 Hz, H-3), 5.06 (2H, OBn O s, OCH ), 5.16 (2H, s, OCH ), 5.38 (1H, dd, J = 13  Hz, 2 2 6q J = 3  Hz, H-3), 6.24 (1H, d, J = 1.5  Hz, H-8), 6.26 (1H, d, J = 1.5  Hz, H-6), 7.28 (1H, d, J = 2  Hz, H-2’), 7.29–7.39 According to the general procedure II, 2’,4’-dibenzyloxy- (10H, m, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.50 (1H, d, J = 8.5 Hz, 6’-hydroxy-2,6-dichlorochalcone (5q) (0.7  g, 1.4  mmol) H-5’), 7.58 (1H, dd, J = 7.5  Hz, J = 2  Hz, H-6’); C NMR was dissolved in ethanol (100 mL). Sodium acetate (0.9 g, Melaku et al. BMC Chemistry (2022) 16:36 Page 24 of 30 Synthesis and analytical data of 2’,6’‑dimethoxyflavanone (CDCl , 125  MHz) δ 45.5 (C-3), 70.3 (O CH ), 70.4 3 2 (7b) (OCH ), 77.8 (C-2), 94.7 (C-6), 95.4 (C-8), 106.3 (C-10), 125.2 (C-6’), 126.5 (C-2’’’, C-6’’’), 127.5 (C-2’’, C-6’’), 127.6 MeO (C-4’’’), 127.7 (C-4’’), 128.1 (C-2’), 128.4 (C-3’’’, C-5’’’), 4' 128.5 (C-3’’, C-5’’), 128.6 (C-5’), 128.7 (C-4’), 130.7 (C-3’), HO O 2 1' 135.2, (C-1’), 136.2 (C-1’’’), 139.0 (C-1’’), 161.1 (C-9), 2' OMe 164.3 (C-5), 165.0 (C-7), 187.8 (C-4); MS (EI, 70 eV) m/z + + (%) 506 (1) [M + 2] , 504 (2) [M] , 413 (1), 348 (17), 306 OH O (7), 257 (7), 215 (8), 180 (7), 91 (100); HRMS (EI, M ) cal- 7b culated for C H O Cl 504.0895 found 504.0871. 29 22 4 2 Following general procedure III, 5,7-dibenzyloxy-2’,6’- dimethoxyflavanone (6d) (60  mg, 0.12  mmol) was Synthesis of flavanones 7a-l by deprotection debenzylated and the crude product was purified by of the dibenzylated flavanones 6a-r column chromatography over silica gel with petroleum General Procedure III for the synthesis of 7a‑l ether:EtOAc (7:3) as the eluent to furnish 7b in 78% yield A dibenzylated flavanone 6 was dissolved in sufficient as a white solid. mp 193–194  °C; UV(MeOH) λ : 291, max EtOAc:EtOH (1:1) to produce a 0.01  M solution. The 343  nm; H NMR (DMSO-d , 500  MHz) δ 2.78, (1H, resulting solution was subjected to hydrogenolysis using dd, J = 3  Hz, J = 17  Hz, H-3), 3.05 (1H, dd, J = 17.5  Hz, a H-Cube Pro over 10%Pd/C at a flow rate of 1 mL/min at J = 13  Hz, H-3), 3.78 (3H, s, OMe), 3.83 (3H, s, OMe), 70 °C and 1 bar. The solution was concentrated in vacuo 5.75 (1H, dd, J = 2.5  Hz, J = 12.5  Hz, H-2), 5.98 (1H, d, and purified by column chromatography on silica gel. J = 2  Hz, H-6), 6.02 (1H, d, J = 2.5  Hz, H-8), 6.92 (1H, dd, J = 3  Hz, J = 9  Hz, H-3’), 7.01 (1H, d, J = 9  Hz, H-4’), Synthesis and analytical data of pinocembrin (7a) 7.81 (1H, d, J = 3 Hz, H-5’), 12.16 (1H, s, 5-OH); MS (EI, 70  eV) m/z (%) 316 (56) [M] , 285 (100), 179 (11), 164 4' (24), 151 (28), 121 (25), 77 (21), 73 (24), 60 (33). HO O 2 1' Synthesis and analytical data of 4’‑chloroflavanone (7c) Following general procedure III, 5,7-dibenzyloxy- OH O 4’-chloroflavanone (6c) (180 mg, 0.38 mmol) was deben - 7a zylated and the crude product was purified by column chromatography over silica with petroleum ether:EtOAc Following general procedure III, 5,7-dibenzyloxyfla - (7:3) as the eluent to furnish 7c as a white solid in 91% vanone (6a) (100  mg, 0.23  mmol) was debenzylated yield. MS (EI, 70 eV) m/z (%) 290 (33) [M] , 256 (73), 255 and the crude product was purified by column chro - (100), 179 (73), 152 (51), 124 (27). matography over silica gel with petroleum ether:EtOAc (7:3) as the eluent to furnish 7a as white solid in 93% Synthesis and analytical data of 3’,4’,5’‑trimetoxyflavanone yield. mp 193–194  °C; R 0.68 (petroleum ether:EtOAc −1 (7d) 4:1); UV(MeOH) λ : 290, 335  nm; IR ν̃ [cm ]: 1628 max (C = C), 1581 (C = C), 1453, 1300, 1085, and 1063 (C-O); 1 OMe H NMR (C D OD, 300  MHz) δ 2.66 (1H, dd, J = 3.3  Hz, OMe 4' J = 17.4  Hz, H-3), 2.92 (1H, dd, J = 12.9  Hz, J = 17.1  Hz, H-3), 5.25 (2H, dd, J = 3  Hz, J = 12.9  Hz, H-2), 5.96 (1H, 79 HO O 2 1' OMe d, J = 2.4  Hz, H-6), 6.01 (1H, d, J = 2.1  Hz, H-8), 7.30 (5H, m, H-2’–H-6’); C NMR (CD OD, 75  MHz) δ 42.7 3 5 (C-3), 78.9 (C-2), 95.6 (C-8), 96.2 (C-6), 101.6 (C-10), OH O 125.4 (C-3’, C-5’), 128.5 (C-2’, C-4’, C-6’), 137.3 (C-1’), 7d 162.5 (C-9), 163.9 (C-5), 167.4 (C-7), 195.4 (C-4); MS (EI, 70  eV) m/z (%) 256 (100) [M] , 179 (66), 152 (46), 124 Following general procedure III, 5,7-dibenzyloxy- (16). 3’,4’,5’-trimetoxyflavanone (6h) (60  mg, 0.12  mmol) M elaku et al. BMC Chemistry (2022) 16:36 Page 25 of 30 was debenzylated and the crude product was purified J = 12.3  Hz, H-2), 5.83 (1H, d, J = 3  Hz, H-6), 5.86 (1H, by column chromatography over silica gel with petro- d, J = 3  Hz, H-8), 5.97 (4H, m, H-3’–H-6’); C-NMR leum ether:EtOAc (7:3) as the eluent to furnish 7d as a (CD OD, 75  MHz) δ 44.3 (C-3), 79.7 (C-2), 96.2 (C-6), white solid in 89% yield. mp 178–179  °C; UV(MeOH) 96.3 (C-8), 103.3 (C-10), 125.5 (C-5’), 127.5 (C-6’), 129.5 λ : 293, 347  nm; H NMR (DMSO-d , 500  MHz) δ (C-3’), 131.3 (C-4’), 135.6 (C-2’), 142.7 (C-1’), 164.2 (C-9), max 6 2.78, (1H, dd, J = 3  Hz, J = 17.5  Hz, H-3), 3.21 (1H, dd, 165.5 (C-5), 168.3 (C-7), 196.8 (C-4); MS (EI, 70 eV) m/z J = 13  Hz, J = 14  Hz, H-3), 3.74 (3H, s, OMe), 3.86 (6H, (%) 290 (33) [M] , 256 (73), 255 (100), 179 (73), 152 (51), s, OMe), 5.47 (1H, dd, J = 3 Hz, J = 13 Hz, H-2), 5.96 (1H, 124 (27). d, J = 2 Hz, H-6), 5.99 (1H, d, J = 2 Hz, H-8), 6.90 (2H, s, H-2’, H-6’), 12.16 (1H, s, 5-OH); MS (EI, 70  eV) m/z (%) Synthesis and analytical data of 3’‑chloroflavanone (7g) 346 (39) [M] , 303 (4), 194 (12), 181 (100). Synthesis and analytical data of 2’,3’‑dichloroflavanone (7e) HO O 2 1' 3' Cl HO 79 O 2 OH O 1' 3' Cl 7g Cl OH O Following general procedure III, 5,7-dibenzyloxy- 7e 3’-chloroflavanone (6m) (10  mg, 0.02  mmol) was debenzylated and the crude product was purified by Following general procedure III, 5,7-dibenzyloxy- column chromatography over silica gel with petroleum 2’,3’-dichloroflavanone (6j) (80  mg, 0.16  mmol) was ether:EtOAc (7:3) as the eluent to furnish 7g as a white debenzylated and the crude product was purified by solid in 78% yield. R 0.68 (petroleum ether:EtOAc 4:1); column chromatography over silica gel with petro- IR ʋ 1628 (C = C), 1581 (C = C), 1453, 1300, 1085 cm-1: leum ether:EtOAc (7:3) as the eluent to furnish 7e as a and 1063 (C-O); H NMR (CD OD, 300  MHz) δ 2.84, white solid in 88% yield. H NMR (DMSO-d , 500 MHz) (1H, m, H-3), 3.11 (1H, m, H-3), 5.50 (1H, dd, J = 4.5  Hz, δ 2.91 (1H, dd, J = 3  Hz, J = 17.5  Hz, H-3), 3.11 (1H, J = 9.6 Hz, H-2), 5.97 (1H, d, J = 1.8 Hz, H-6), 6.00 (1H, d, dd, J = 13  Hz, J = 17  Hz, H-3), 5.94 (1H, dd, J = 3  Hz, J = 1.8 Hz, H-8), 7.52 (4H, m, H-2’, H-3’–H-6’); C NMR J = 13  Hz, H-2), 6.00 (1H, d, J = 2  Hz, H-6), 6.03 (1H, d, (CD OD, 75  MHz) δ 42.6 (C-3), 78.3 (C-2), 94.7 (C-6), J = 2  Hz, H-8), 7.53 (1H, t, J = 8  Hz, H-5’), 7.65 (1H, dd, 95.8 (C-8), 102.1 (C-10), 124.1 (C-6’), 126.2 (C-2’), 128.7 J = 1.5 Hz, J = 8 Hz, H-4’), 7.78 (1H, dd, J = 1 Hz, J = 7 Hz, (C-4’), 133.9 (C-5’), 138.9 (C-3’), 141.3 (C-1’), 162.9 (C-9), H-6’), 12.05 (1H, s); MS (EI, 70  eV) m/z (%) 326 (31) 164.4 (C-5), 166.8 (C-7), 195.9 (C-4); MS (EI, 70 eV) m/z + + [M + 2] , 324 (51) [M] , 289 (94), 255 (14), 179 (100), 152 (%) 290 (33) [M] , 256 (63), 179 (100), 152 (62), 124 (37). (66), 124 (46). Synthesis and analytical data of 2’,4’‑dichloroflavanone (7h) Synthesis and analytical data of 2’‑chloroflavanone (7f ) Cl HO 79 O 2 3' HO 79 O 2 3' 1' 1' 4 Cl Cl OH O OH O 7h 7f Following general procedure III, 5,7-dibenzyloxy- Following general procedure III, 5,7-dibenzyloxy- 2’,4’-dichloroflavanone (6n) (160  mg, 0.12  mmol) was 2’-chloroflavanone (6  l) (270  mg, 0.57  mmol) was debenzylated and the crude product was purified by debenzylated and the crude product was purified by column chromatography over silica gel with petro- column chromatography over silica gel with petro- leum ether:EtOAc (7:3) as the eluent to furnish 7h as a leum ether:EtOAc (7:3) as the eluent to furnish 7f as a white solid in 87% yield. H NMR (CD OD, 500  MHz) white solid in 92% yield. H NMR (CD OD, 300  MHz) δ 2.87, (1H, dd, J = 2.5  Hz, J = 14  Hz, H-3), 3.02 (1H, δ 2.84 (1H, dd, J = 3  Hz, J = 17.1  Hz, H-3), 3.08 (1H, dd, dd, J = 14  Hz, J = 16  Hz, H-3), 5.80 (1H, dd, J = 2.5  Hz, J = 12.3  Hz, J = 17.1  Hz, H-3), 5.52 (1H, dd, J = 2.4  Hz, Melaku et al. BMC Chemistry (2022) 16:36 Page 26 of 30 solid in 91% yield. H NMR (CD OD/CDCl , 500  MHz) J = 13 Hz, H-2), 5.96 (1H, br s, H-6), 5.99 (1H, br s, H-8), 3 3 δ 2.59, (1H, dd, J = 3.5  Hz, J = 17.5  Hz, H-3), 3.74 (1H, 7.48 (1H, d, J = 8.5  Hz, H-6’), 7.57 (1H, br s, H-3’), 7.77 dd, J = 14.5  Hz, J = 17.5  Hz, H-3), 5.92 (1H, d, J = 2  Hz, (1H, dd, J = 1.2  Hz, J = 8.5  Hz, H-5’); C NMR (C D OD, H-6), 5.94 (1H, d, J = 2 Hz, H-8), 6.22 (1H, dd, J = 3.5 Hz, 125 MHz) δ 42.6 (C-3), 76.9 (C-2), 95.8 (C-6), 97.5 (C-8), J = 14.5  Hz, H-2), 7.35 (1H, dd, J = 7.5  Hz, J = 7.5  Hz, 129.0 (C-5’), 129.8 (C-6’), 130.8 (C-3’), 133.2 (C-2’), 135.7 H-4’), 7.45 (2H, br d, J = 7.5  Hz, H-3’, H-5’); C NMR (C-4’), 136.7 (C-1’), 164.9 (C-9), 165.8 (C-5), 168.2 (C-7), (CD OD/CDCl , 125 MHz) δ 39.2 (C-3), 76.2 (C-2), 95.8 196.2 (C-4); MS (EI, 70  eV) m/z (%) 326 (24) [M + 2] , 3 3 (C-6), 97.2 (C-8), 102.8 (C-10), 126.3 (C-5’, C-5’), 130.5 324 (32) [M] , 289 (74), 256 (68), 179 (100), 152 (83), 124 (C-4’), 131.8 (C-1’), 136.5 (C-2’, C-6’), 164.1 (C-9), 165.2 (52), 69 (54). (C-5), 168.2 (C-7), 195.9 (C-4); MS (EI, 70  eV) m/z (%) 325 (62) [M] , 323 (75), 289 (96), 256 (31), 179 (100), 151 Synthesis and analytical data of 3’,5’‑dichloroflavanone (7i) (70), 124 (36). Cl Synthesis and analytical data of 2’,5’‑dichloroflavanone (7k) HO O 2 1' 3' Cl Cl 4' OH O HO O 2 1' 7i Cl OH O Following general procedure III, 5,7-dibenzyloxy- 3’,5’-dichloroflavanone (6o) (200  mg, 0.4  mmol) was 7k debenzylated and the crude product was purified by Following general procedure III, 5,7-dibenzyloxy- column chromatography over silica gel with petro- 2’,5’-dichloroflavanone (6q) (115  mg, 0.23  mmol) was leum ether:EtOAc (7:3) as the eluent to furnish 7i as a debenzylated and the crude product was purified by white solid in 89% yield. H NMR (CD OD, 500  MHz) column chromatography over silica gel with petroleum δ 2.87, (1H, dd, J = 3  Hz, J = 17  Hz, H-3), 3.06 (1H, dd, ether:EtOAc (7:3) as the eluent to furnish 7k as a white J = 12.5  Hz, J = 17  Hz, H-3), 5.51 (1H, dd, J = 3  Hz, solid in 94% yield. H NMR (C D OD, 500  MHz) δ 2.90, J = 12.5 Hz, H-2), 5.95 (1H, d, J = 2 Hz, H-6), 6.02 (1H, d, (1H, dd, J = 3 Hz, J = 17 Hz, H-3), 3.01 (1H, dd, J = 13 Hz, J = 2.5 Hz, H-8), 7.49 (1H, t, J = 1.75 Hz, H-4’), 7.53 (2H, J = 17  Hz, H-3), 5.80 (1H, dd, J = 3  Hz, J = 13  Hz, H-2), m, H-2’, H-6’); C NMR (CD OD, 125 MHz) δ 42.2 (C-3), 5.98 (1H, d, J = 2  Hz, H-6), 6.04 (1H, d, J = 2.5  Hz, H-8), 77.2 (C-2), 94.7 (C-6), 96.0 (C-8), 124.5 (C-2’, C-6’), 127.9 7.43(1H, dd, J = 2.5  Hz, J = 8.5  Hz, H-4’), 7.50 (1H, d, (C-4’), 135.0 (C-3’, C-5’), 143.0 (C-1’), 162.6 (C-9), 164.1 J = 9  Hz, H-3’), 7.78 (1H, d, J = 2.5  Hz, H-6’); C NMR (C-5), 167.1 (C-7), 196.8 (C-4); MS (EI, 70  eV) m/z (%) (CD OD, 125  MHz) δ 41.2 (C-3), 75.5 (C-2), 95.1 (C-6), 326 (22) [M + 2] , 324 (36) [M] + , 289 (79), 256 (64), 255 96.1 (C-8), 102.1 (C-10), 127.2 (C-2’), 130.8 (C-4’), 131.1 (68), 179 (100), 152 (86), 124 (48),, 69 (52), 57 (97). (C-3’), 133.5 (C-2’), 133.6 (C-5’), 138.8 (C-1’), 163.0 (C-9), 164.1 (C-5), 167.1 (C-7), 194.3 (C-4); MS (EI,7 0 eV) m/z Synthesis and analytical data of 2’,6’‑dichloroflavanone (7j) + + (%) 326 (24) [M + 2] , 324 (32) [M] , 290 (17), 256 (34), 179 (100), 152 (70), 124 (42). Cl 4' HO O 2 Synthesis and analytical data of 3’,4’‑dichloroflavanone (7l) 1' Cl 4' Cl OH O HO O 2 1' 7j Cl Following general procedure III, 5,7-dibenzyloxy- OH O 2’,6’-dichloroflavanone (6p) (140  mg, 0.28  mmol) was 7l debenzylated and the crude product was purified by column chromatography over silica gel with petroleum Following general procedure III, 5,7-dibenzyloxy- ether:EtOAc (7:3) as the eluent to furnished 7j as a white 3’,4’-dichloroflavanone (6r) (120  mg, 0.24  mmol) was M elaku et al. BMC Chemistry (2022) 16:36 Page 27 of 30 In vivo antiplasmodial experiment debenzylated and the crude product purified by col - Standard four days suppression test was used to evaluate umn chromatography over silica gel with petroleum the antiplasmodial activity of the synthetic compounds ether:EtOAc (7:3) as the eluent to furnish 7  l as a white [18]. The mice in this study were grouped into treatment, solid in 88% yield. H NMR (CD OD, 500  MHz) δ positive and negative control groups. Each group contains 2.85, (1H, dd, J = 3.5  Hz, J = 17  Hz, H-3), 3.09 (1H, dd, five mice. On D0, all the mice were weighted on a sensitive J = 12.5  Hz, J = 17  Hz, H-3), 5.52 (1H, dd, J = 3.5  Hz, balance and the packed cell volume (PCV) were measured. J = 13 Hz, H-2), 5.95 (1H, d, J = 2.5 Hz, H-6), 6.00 (1H, d, Then each mouse in the treatment and control group was J = 2 Hz, H-8), 7.47 (1H, dd, J = 2.5  Hz, J = 8.5  Hz, H-6’), injected with 0.2  ml of blood diluted with physiological 7.61 (1H, d, J = 8.5 Hz, H-5’), 7.75 (1H, d, J = 2  Hz, H-2’); + + saline (intraperitoneally) which contains approximately MS (EI, 70 eV) m/z (%) 326 (24) [M + 2] , 324 (32) [M] , 1 × 10 P. berghei parasitized RBCs. 290 (17), 256 (34), 179 (100), 152 (70), 124 (42). Administration of pinocembrin and its analogs Experimental design for antiplasmodial assay After three hours of injection of P. berghei infected blood, This experiment was done on Swiss albino mice of the synthetic pinocembrin and its analogs were administered age of 6–8  weeks and body weight ranges from 22–28  g each separately. Compound 6a was administered at doses which were reared in the Animal House of College of of 15, 20, 25, 30 and 35  mg/kg b. wt. Compounds 6b, 6e Natural and Computational Science, Addis Ababa Uni- and 6c were tested at a dose of 20 mg/kg b. wt each. Com- versity. The mice were handled according to Guide for pound 6i was given at doses of 15, 25 and 35 mg/kg b. wt. the Care and Use of Laboratory Animals [31].They were Compound 7a was given at doses of 20 and 30  mg/kg b. given a standard food and water (ad libitum) and main- wt. Chloroquine (CQ) 25  mg/kg b.wt and 1  ml/100  g of tained in 12  h dark and 12  h light (artificial light). The 10% DMSO were given as a positive and a negative control experiment was done on male mice. The mice have been respectively. The administration continued for 4 days (D0– acclimatized for a week before the experiment started. D3) based on modified procedure of Peters (1965) [18]. The amount of the compounds administration was based on Preparation of Plasmodium parasite the availability of the synthesized quantity. The Plasmodium berghei parasite was obtained from Ethio- pian Public Health Institute (EPHI) and was passaged on Percent parasitaemia and percent suppression a weekly basis from infected mice to healthy mice. For the On D4, blood was taken gently from the tail of each mouse experiment four donor mice were prepared by infecting P. on a slide to make a blood smear. The smear was fixed with berghei through intraperitoneal injection of the blood of methanol and stained with 10% Giemsa stain for 30  min, infected mice with normal saline. When the parasitaemia then washed with water. Each blood smear was examined was confirmed to be 30–40% by preparing a blood smear, under the compound microscope with oil emulsion at blood was taken from the hearts of the donor mice after magnification power of 100 × (together with ocular lens anesthetized them. The blood was taken from each donor 10,000 ×) and each slide was counted for 5–10 times. The mouse and diluted with physiological saline (0.85%) in 1:4 percent parasitaemia and percent suppression was calcu- ratio. lated by the following formula as given in Peters [18]. number of infected RBC % Parasitaemia = × 100. Number of infected RBC + number of uninfected RBC parasitaemia in negative control − parasitaemia in treatment group % Suppression = ×100. Parasitaemia in negative control Melaku et al. BMC Chemistry (2022) 16:36 Page 28 of 30 Determination of mean survival time was setted using graphical user interface program. The All the mice in the treatment and control groups were grid was set so that it surrounds the region of interest followed daily starting from the day of parasite injection in the macromolecule. The best docked conformation and MST was calculated by using the following formula between ligand and protein was searched using the dock- given below. ing algorithm with AutoDock Vina. During the docking process, a maximum of nine conformers were considered sum of survival day of all mice in a group day for each ligand. The conformations with the most favora - MST = Total number of mice in that group ble (least) free binding energy were selected for analyzing the interactions between the target receptor and ligands by Discovery studio visualizer and PyMOL. The ligands are Body weight and PCV determination represented in different color, H-bonds and the interacting Body weight and PCV were measured on D0 and D4 to residues are represented in stick model representation. evaluate the effect of the compounds on the mice. The PCV was measured by taking blood ¾ of heparinized microhematocrit capillary tube (75 mm) from the tail of In silico drug-likeness predictions the mice. The tube was sealed with sealant and centri - In silico Drug-likeness helps to know whether a particu- fuged on microhematocrit centrifuge (MK IV, England) lar pharmacological agent has properties consistent with by 13,000  rpm for 4  min. Then, the total blood volume being an orally active drug. This prediction is based on an and the volume of erythrocyte were measured by using already established rule called Lipinski rule of five [29]. a ruler. Mean body weight was also calculated using the The structures of compounds synthesized (7a-l) were following formula. changed to their canonical simplified molecular input line entry system (SMILE) then submitted to SwissADME Total weight of mice in a group Meanbody weight = tool to estimate in silico pharmacokinetic parameters and Total number of mice in that group other molecular properties based on the methodology The PCV was calculated using the formula given by reported by Amina et  al. 2016. The data obtained were Gilmour and Sykes (1951). compared with chloroquine (standard drug), and only compounds without violation of any of the screenings Volume of erythrocyte in a given blood were used for the molecular docking analysis. PCV = × 100. Total blood volume Ethical consideration Molecular docking simulation of compounds 7a-l To conduct this study, ethical clearance was obtained against Plasmodium falciparum dihydrofolate from Institutional Ethics Review Board of College of Nat- reductase-thymidylate synthase ural Sciences, Addis Ababa University (IRB/024/2017). AutoDock Vina with standard protocol was used to dock The study is reported in accordance with the Animal the proteins (PfDHFR-TS) (PDB ID 1J3I) and compounds Research Reporting of in  vivo Experiments (ARRIVE) synthesized (7a-l) into the active site of proteins [32, 33]. guidelines (https:// arriv eguid elines. org) and were han- ChemOffice tool (Chem Draw 16.0) was used to draw the dled according to the Guide for the Care and Use of chemical structures of the synthesized compounds (7a- Laboratory Animals (https:// grants. nih. gov/ grants/ olaw/ l) while the proper 2D orientation, and energy of each guide- for- the- care- and- use- of- labor atory- anima ls). molecule was minimized using ChemBio3D. The energy minimized ligand molecules were then used as input for AutoDock Vina, in order to carry out the docking simu- Data analysis lation. The crystal structure of Plasmodium falciparum The data generated in this study were analyzed using IBM dihydrofolate reductase-thymidylate synthase was down- SPSS, version 20 statistical package. The results were loaded from protein data bank. The protein preparation presented as mean ± SEM (standard error of the mean). was by removing the co-crystallized ligand, selected water One-way ANOVA (analysis of variance), paired Student’s molecules and cofactors, the target protein file was pre - t-test and Kaplan–Meier analyses were used for the sta- pared by leaving the associated residue with protein by tistical analysis. A statistically significant difference was using Auto preparation of target protein file AutoDock 4.2 taken at P-value less than 0.05 (P < 0.05). (MGL tools1.5.7). The grid box for docking simulations M elaku et al. BMC Chemistry (2022) 16:36 Page 29 of 30 Author details Supplementary Information Chemistry Department, Adama Science and Technology University, The online version contains supplementary material available at https:// doi. 1888 Adama, Ethiopia. Biology Department, Addis Ababa University, Addis org/ 10. 1186/ s13065- 022- 00831-z. Ababa, Ethiopia. Bioorganische Chemie, Institut Für Chemie, Universität Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany. Depar tment Additional file 1. The NMR spectra and molecular docking simulations of Chemical Engineering, FH Münster-University of Applied Sciences, for the synthetic compounds are included within Additional materials Stegerwaldstrasse 39, 48565 Steinfurt, Germany. Department of Biomateri- (Additional files 1 and 2). als, Saveetha Dental College and Hospitals, Saveetha Institute of Meidcal and Technical Sciences (SIMATS), Saveetha University, Chennai 600 077, India. Additional file 2. The NMR spectra and molecular docking simulations for the synthetic compounds are included within Additional materials Received: 23 February 2022 Accepted: 11 May 2022 (Additional file 1 and 2). Acknowledgements The authors thank the Alexander von Humboldt Foundation for the research References grant. We thank Addis Ababa University, Adama Science and Technology 1. Lubis IN. Contribution of Plasmodium knowlesi to multispecies University and Hohenheim University for technical and financial support. human malaria infections in North Sumatera, Indonesia. J Infect Dis. We thank Dr. J. Conrad and Mr. M. Wolf (Institut für Chemie, Universität 2017;215(7):1148–55. Hohenheim) for recording of NMR spectra and Dr. C. Braunberger Institute für 2. Ayoola GA, Coker H, Adesegun S, Adepoju-Bello A, Obaweya K, Ezennia Chemie, Universität Hohenheim for recording of mass spectra. Prof. Ermias E, Atangbayila T. Phytochemical screening and antioxidant activities of Dagne is acknowledged for being part of the research project (Department of some selected medicinal plants used for malaria therapy in southwestern Chemistry, Addis Ababa University, Ethiopia). nigeria. Trop J Pharm Res. 2008;7:1019–24. 3. Cropper ML, Haile M, Lampietti J, Poulos C, Whittington D. The demand Author contributions for malaria vaccine: evidence from Ethiopia. J Dev Econ. 2009;75:303–18. YM and UB were responsible for planning the synthetic experiments. YM and 4. World Health Organization. World Malaria Report. Geneva: World Health VO were responsible for the synthesis and characterization of compounds. Organization; 2012. MS and Y TM were responsible for the in vivo antiplasmodial activity of the 5. Rasul A, Millimouno FM, Eltay WA, Ali M, Li X. Pinocembrin: A Novel Natu- synthesized compounds. RE conducted the molecular docking simulation of ral Compound with Versatile Pharmacological and Biological Activities. compounds 7a-l. The manuscript was written by YM, UB and Y TM. Corre- Biomed Res Int. 2013. https:// doi. org/ 10. 1155/ 2013/ 379850. spondence to Yalemtsehay Mekonnen. All authors read and approved the final 6. Qingyun Y, Yuanfeng Y, Feng C, Yan Q, Wei L, Song W. Identification and manuscript. synthesis of impurities in pinocembrin-A new drug for the treatment of ischemic stroke. Chin J Chem. 2012;30:1315–9. Funding 7. Said MM, Azab SS, Saeed NM, Demerdash EE. Antifibrotic mechanism of This project work was done by the fund obtained from the Alexander von pinocembrin: impact on oxidative stress inflammation and TGF-β/Smad Humboldt Foundation for the research group linkage grant (2014–2017) with inhibition in rats. Ann Hepatol. 2018;17(2):307–17. grant number 3.4-IP-DEU/1074366. 8. Saad AAE, Tadros MG, Elsherbiny DA, Menze ET. Therapeutic effects of pinocembrin on indomethacin-induced gastric ulcer model in rat. Az J Data availability Pharm Sci. 2017;56:1–9. The datasets supporting the findings of this article are presented in the main 9. Melaku Y, Worku T, Tadesse Y, Mekonnen Y, Schmidt J, Arnold N, Dagne E. manuscript. The MS and IR spectra of the synthesized compounds along with Antiplasmodial Compounds from Leaves of Dodonaea angustifolia. Curr the molecular docking simulations of compound 7a-j can be accessed from Bioact Compd. 2017;13:268–73. the corresponding author on reasonable request. "The X-ray structure of Wild- 10. Zhang M, Jagdmann GE, Zandt MV, Beckett P, Schroeter H. Enantioselec- type Plasmodium falciparum dihydrofolate reductase-thymidylate synthase tive synthesis of orthogonally protected (2R,3R)-(-)-epicatechin deriva- (PfDHFR-TS) complexed with WR99210, NADPH, and dUMP (PDB code 1J3I) tives, key intermediates in the de novo chemical synthesis of (-)-epicat- was obtained from the Protein Data Bank. The target protein was downloaded echin glucuronides and sulfates. Tetrahedron. 2013;24:362–73. from the protein data bank and no homology or structural determination was 11. Hull LA. The dibenzalacetone reaction revisited. J Chem Educ. done. The structures are searchable in the protein data bank repository using 2001;78(2):226. (PDB ID: 1J3I) (https:// www. rcsb. org/ struc ture/ 1J3I). The PDB ID was given in 12. Dhar DN, Lal JB. Chalcones. Condensation of aromatic aldehydes with the method section. The NMR spectra and molecular docking interaction of resacetophenone. J Org Chem. 1958;23(8):1159–61. the compounds synthesized against the protein PfDHFR-TS (https:// www. rcsb. 13. Mardiana L, Ardiansah B, Bakri R, Aziza NP. Utilization of eggshell-derived org/ struc ture/ 1J3I) are provided as Additional files 1 and 2, respectively. material as a solid base catalyst for efficient synthesis of substituted chalcones. J Teknol. 2017;79(5):175–82. Declarations 14. Kulkarni P, Wagh P, Zubaidha P. An improved and eco-friendly method for the synthesis of flavanone by the cyclization of 2’-hydroxy chalcone using Ethics approval and consent to participate methane sulphonic acid as catalyst. Chemistry Journal. 2012;2(3):106–10. To conduct this study, ethical clearance was obtained from Institutional 15. Harwood LM, Loftus GC, Oxford A, Thomson C. An improved procedure Ethics Review Board of College of Natural Sciences, Addis Ababa University for cyclisation of chalcones to flavanones using celite supported potas- (IRB/024/2017). The study is reported in accordance with the Animal Research sium fluoride in methanol: total synthesis of bavachinin. Synth Commun. Reporting of in vivo Experiments (ARRIVE) guidelines (https:// arriv eguid elines. 1990;20(5):649–57. org) and were handled according to the Guide for the Care and Use of Labora- 16. Urgaonkar S, Pierre HS, Meir I, Lund H, Chaudhuri DR, Shaw JT. Synthesis tory Animals (https:// grants. nih. gov/ grants/ olaw/ guide- for- the- care- and- use- of Antimicrobial Natural Products Targeting FtsZ:(±)-Dichamanetin and of- labor atory- anima ls). (±)-2′′′-Hydroxy-5′′-benzylisouvarinol-B. Org Lett. 2005;7(25):5609–12. 17. Ali Y, Manickam G, Ghosha A, Subramanian P. More efficient palladium Consent for publication hydrogenolysis of benzyl groups. Synth Commun. 2006;36:925–8. Not applicable. 18. Peters W. Drug resistance in Plasmodium berghei Vincke and Lips. 1948 I. Chloroquine resistance. Exp Parasitol. 1965;17(1):80–9. Competing interests 19. 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Oliveira SQ, de Almeida MTR, Maraslis F, Silva IT, Sincero TCM, Palermo JA, Cabrera CM, Caro MSB, Simoes CMO, Schenkel EP. Isolation of three new ent-labdanediterpenes from Dodonaea viscosa Jacq. (Sapindaceae): pre- liminary evaluation of antiherpes activity. Phytochem Lett. 2012;5:500–5. 25. Nardos A, Makonnen E. In vivo antiplasmodial activity and toxicological assessment of hydroethanolic crude extract of Ajuga remota. Malar J. 2017;16(1):25. 26. Okokon JE, Effiong I, Ettebong E. In vivo antimalarial activities of ethanolic crude extracts and fractions of leaf and root of Carpolobialutea. Pak J Pharm Sci. 2011;24:57–61. 27. Ivanetich KM, Santi DV. Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa. FASEB J. 1990;4:1591–7. 28. Meng XY, Zhang HX, Mezei M, Cui M. Molecular docking: a powerful approach for structure-based drug discovery. Curr Comput Aided Drug Des. 2011;7:146–57. 29. Lipinski CA. Rule of five in 2015 and beyond: Target and ligand structural limitations, ligand chemistry structure and drug discovery project deci- sions. Adv Drug Deliv Rev. 2016;101:34–41. 30. Zhao L, Jin H, Sun L, Piao H, Quan Z. Synthesis and evaluation of anti- platelet activity of trihydroxychalcone derivatives. Bioorg Med Chem Lett. 2005;15:5027–9. 31. Council NR. Guide for the care and use of laboratory animals. Washing- ton: National Academies Press; 2010. 32. Seeliger D, Groot BL. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des. 2010;24:417–22. 33. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multi- threading. J Comput Chem. 2010;31:455–61. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? 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Synthesis, antiplasmodial activity and in silico molecular docking study of pinocembrin and its analogs

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Abstract

Background: Malaria remains the major health problem responsible for many mortality and morbidity in developing countries. Because of the development of resistance by Plasmodium species, searching effective antimalarial agents becomes increasingly important. Pinocembrin is a flavanone previously isolated as the most active antiplasmodial compound from the leaves of Dodonaea angustifolia. For a better understanding of the antiplasmodial activity, the synthesis of pinocembrin and a great number of analogs was undertaken. Methods: Chalcones 5a-r were synthesized via Claisen-Schmidt condensation using 2,4-dibenzyloxy-6-hydroxy- acetophenone and aromatic aldehydes as substrates under basic conditions. Cyclization of compounds 5a-r to the corresponding dibenzylated pinocembrin analogs 6a-r was achieved using NaOAc in EtOH under reflux. Catalytic hydrogenation using 10% Pd/C as catalyst in an H-Cube Pro was used for debenzylation to deliver 7a-l. The structures of the synthesized compounds were characterized using various physical and spectroscopic methods, including mp, UV, IR, NMR, MS and HRMS. The synthesized dibenzylated flavanones 6a-d, 6i and 7a were evaluated for their in vivo antiplasmodial activities against Plasmodium berghei infected mice. Molecular docking simulation and drug likeness properties of compounds 7a-l were assessed using AutoDock Vina and SwissADME, respectively. Results: A series of chalcones 5a-r has been synthesized in yields ranging from 46 to 98%. Treatment of the chal- cones 5a-r with NaOAc refluxing in EtOH afforded the dibenzylated pinocembrin analogs 6a-r with yields up to 54%. Deprotection of the dibenzylated pinocembrin analogs delivered the products 7a-l in yields ranging from 78 to 94%. The dibenzylated analogs of pinocembrin displayed percent inhibition of parastaemia in the range between 17.4 and 87.2% at 30 mg/kg body weight. The parastaemia inhibition of 87.2 and 55.6% was obtained on treatment of the infected mice with pinocembrin (7a) and 4’-chloro-5,7-dibenzylpinocembrin (6e), respectively. The mean survival times of those infected mice treated with these two compounds were beyond 14 days indicating that the samples suppressed P. berghei and reduced the overall pathogenic effect of the parasite. The molecular docking analysis of the chloro derivatives of pinocembrin revealed that compounds 7a-l show docking affinities ranging from – 8.1 to – 8.4 kcal/mol while it was -7.2 kcal/mol for chloroquine. Conclusion: Pinocembrin (7a) and 4’-chloro-5,7-dibenzyloxyflavanone (6e) displayed good antiplasmodial activ- ity. The in silico docking simulation against P. falciparum dihydrofolate reductase-thymidylate synthase revealed that *Correspondence: yalemtshay.mekonnen@aau.edu.et Biology Department, Addis Ababa University, Addis Ababa, Ethiopia Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Melaku et al. BMC Chemistry (2022) 16:36 Page 2 of 30 pinocembrin (7a) and its chloro analogs 7a-l showed better binding affinity compared with chloroquine that was used as a standard drug. This is in agreement with the drug-like properties of compounds 7a-l which fulfill Lipinski’s rule of five with zero violations. Therefore, pinocembrin and its chloro analogs could serve as lead compounds for further antiplasmodial drug development. Keywords: Catalytic hydrogenation, Claisen-Schmidt Condensation, Malaria, Plasmodium berghei, Pinocembrin, Chloropinocembrin Introduction Results and discussion Malaria remains one of the major health problems in Synthesis of Pinocembrin (7a) and its analogs 7b-l developing countries with high recorded rates of mor- The synthesis of various chalcones was performed after tality and morbidity [1]. The most predominant species protection of trihydroxyacetophenone (1) with benzyl responsible for 90% of malarial cases worldwide is Plas- chloride (2) using potassium carbonate as base in DMF modium falciparum [2]. Nearly half of the world’s pop- (Fig. 1) [10]. The product 3 was obtained as a white solid ulation resides in regions where malaria is endemic and in 79% yield. In the next step, the desired chalcones 5a- are thus at risk of infection [3]. The burden is severe r were prepared via Claisen-Schmidt condensation [11, in sub-Saharan Africa which account for 90% of the 12] of 2,4-dibenzyloxy-6-hydroxyacetophenone (3) with deaths where 5% of children die from the disease before various aromatic aldehydes 4a-r with electron deficient reaching 5 years of age [4]. Efforts to reduce the spread or electron donating properties using KOH as base in have been exacerbated by the increasing resistance of EtOH. The chalcones 5a-r was isolated from 46 to 98% the mosquito to insecticides, and of the parasite to the in yields (Fig.  2). The lowest yields were obtained with currently available drugs [4]. This necessitates search 2-bromobenzaldehyde 4f and 3,4,5-trimethoxybenzalde- for alternative drugs at reasonable cost for use against hyde 4 h as substrates. Low yields of chalcones with ben- malaria. zaldehydes containing a methoxy group in the substrate Flavonoids are phenolic compounds possessing enor- are documented in the literature [13]. mous pharmacological activities. They play signifi - Having secured the synthesis of the intended chal- cant roles in promoting health and preventing chronic cones, we focused our attention towards the synthesis of degenerative diseases. Among flavonoids, pinocem - pinocembrin and its analogs. Previous work has shown brin (7a) is a flavanone with wide arrays of biological that the cyclization of chalcones to flavanones can be activities including antimicrobial, antiinflammatory, effected employing various methods using acids, bases antioxidant, anticancer [5] and against ischemic stroke and celite supported potassium fluoride in methanol [14, [6]. Pinocembrin (7a) has also been reported having 15]. In the present work, the transformation of chalcones antifibrotic effects in addition to its ability to decrease to flavanones was attempted with acetic acid, methane proinflammatory cytokines production [7 ] and protec- sulphonic acid and sodium acetate as catalysts. The lat - tive capacity against gastric tissue damage [8]. In our ter reagent was found to deliver the desired products in previous antiplasmodial study, we isolated pinocembrin higher yields than with the two other reagents. Upon (7a) as the most active compound from the leaves of D. refluxing the chalcones 5a-r (1  mmol) with sodium ace - angustifolia [9]. To establish its antiplasmodial activity tate (8  mmol) in ethanol, they were transformed into an large quantities of this compound are required which equilibrium mixture made up of the chalcones 5a-r and however is present only as a minor constituent in the the flavanones 6a-r (Fig.  2). This is evident as approxi - leaves of D. angustifolia. The synthesis of pinocembrin mately 40% of the starting material chalcone was also analogs with improved antimalarial activity are there- recuperated. Therefore, the low yields of the flavanones fore encouraged for further advances. Hence, to further obtained in the present work can therefore be attributed optimize the antiplasmodial activities of pinocembrin to the reversibility of the chalcone cyclization. This is in (7a), the synthesis of this natural product and analogs agreement with the finding reported by Urgaonkar et al., thereof were undertaken. In this paper, we report the (2005) [16]. synthesis and antiplasmodial activities of pinocem- Compounds 6a-e and 6i were subjected to in vivo anti- brin (7a) and its analogs against Plasmodium berghei plasmodial activity with the better result achieved with in infected mice. Furthermore, the in silico molecular compounds 6a and 6e. The activity displayed by the lat - docking analysis against P. falciparum dihydrofolate ter compound might be due to the presence of a chlo- reductase-thymidylate synthase and the drug-like prop- rine atom. The antiplasmodial activity of dibenzylated erties of compounds 7a-l are also presented herein. pinocembrin 6a was compared with pinocembrin (7a). M elaku et al. BMC Chemistry (2022) 16:36 Page 3 of 30 R R H R BnO OH BnO OBn HO OH O R Cl K CO 2 3 4a-r DMF, 70 C, 3h KOH, EtOH OBn O OH O OH O 1 2 3 5a-r F OMe MeO BnO OH OH BnO OH BnO OH BnO OMe OMe OBn O OBn O OBn O OBn O 5a,80% 5b,68% 5c,67% 5d,67% OMe Cl OMe CF BnO OH OH BnO OH BnO BnO OH OMe Br OBn O OBn O OBn O OBn O 5f,57% 5g,71% 5h,46% 5e,72% CN BnO OH OH BnO OH BnO BnO OH Cl Cl Br Cl OBn O OBn O OBn O OBn O 5i,77% 5l,77% 5j, 97% 5k,67% Cl Cl Cl BnO OH BnO OH BnO OH BnO OH Cl Cl Cl Cl OBn O OBn O OBn O OBn O 5m,92% 5n,96% 5o,70% 5p,69% Cl Cl BnO OH BnO OH Cl Cl OBn O OBn O 5q,70% 5r,98% Fig. 1 Synthesis of chalcones 5a-r Melaku et al. BMC Chemistry (2022) 16:36 Page 4 of 30 2 2 R R 1 5 1 5 R R R BnO OH BnO O 4 NaOAc, EtOH 4 R R R reflux,48h R OBn O OBn O 5a-r 6a-r F OMe MeO BnO O BnO O BnO O BnO O OMe OMe OBn O OBn O OBn O OBn O 6a,50% 6b,48% 6d,52% 6c,45% OMe OMe Cl CF BnO O BnO O O O BnO BnO OMe Br OBn O OBn O OBn O OBn O 6e,40% 6h,38% 6f,36% 6g,8% CN BnO O BnO O BnO O BnO O Cl Cl Br Cl OBn O OBn O OBn O OBn O 6j,26% 6i,48% 6k,14% 6l,42% Cl Cl Cl BnO O BnO O BnO O BnO O Cl Cl Cl Cl OBn O OBn O OBn O OBn O 6p,32% 6m,50% 6n,54% 6o,26% Cl Cl BnO BnO O Cl Cl OBn O OBn O 6q,43% 6r,44% Fig. 2 Synthesis of benzylated flavanones 6a-r M elaku et al. BMC Chemistry (2022) 16:36 Page 5 of 30 The result showed a dramatic decrease in activity of 6a Pd/C as catalyst and a flow rate of 1 mL/min at 70 °C and compared with 7a. This is probably accounted to the 1  bar. This method was found highly attractive as it fur - presence of benzyl group. Hence, we found it neces- nished the corresponding debenzylated products in yields sary to undertake the debenzylation of the pinocembrin ranging between 78 and 94% and in short reaction times analogs. To achieve this goal, the debenzylation of the (Fig.  3). The use of the H-Cube Pro has also eliminated dibenzylated flavanones 6a-r (Fig.  3) was achieved by the dangers associated with hydrogenation by generating catalytic hydrogenation using an H-Cube. To find out hydrogen in situ and the handling of pyrophoric catalysts optimal parameters for the deprotection, different sol - by filling them in sealed catalyst cartridges. This is advan - vents including EtOH, CHCl , EtOAc and EtOH:EtOAc tageous over the conventional debenzylation protocol (1:1) were tested using the conversion of 6a to 7a as an which succeeded to give the desired product neither with example. The latter solvent system was found efficient Pd/C (5%, 10% and Pd black) nor Pd(OH) /C (20%) under in furnishing the product in 93% yield. The reaction various conditions (Up to 15  kg hydrogen pressure and temperature of the deprotection was also optimized. It 70 °C in temperature and varieties of solvents) [17]. was found that the debenzylation of both benzyl groups The structures of the compounds synthesized in the occurred at 70  °C while the undesired monobenzylated present work were characterized using various spectro- product was obtained at 60  °C exclusively. Hence, the scopic methods, including UV, IR and NMR as well as debenzylation of compounds 6b-r to pinocembrin ana- by mass spectrometry. logs 7b-r was performed using an H-Cube Pro with 10% 5 1 5 R R HO O BnO O H ,10% Pd/C,EtOAc:EtOH(1:1) 4 2 0.01M, 1mL/min, 70 C, 1bar OH O OBn O 7a-l 6a,d,e,h,j,l,m,n,o,p,q,r OMe MeO Cl OMe HO O HO O HO O HO O OMe OMe OH O OH O OH O OH O 7a, 93% 7b,78% 7c,91.2% 7d,89% Cl HO O HO O HO O HO O Cl Cl Cl Cl Cl OH O OH O OH O OH O 7e,88% 7f,91.5% 7g,78% 7h,87% Cl Cl Cl Cl HO O HO O HO O HO O Cl Cl Cl Cl OH O OH O OH O OH O 7i, 89% 7j, 91% 7l,88% 7k,94% Fig. 3 Synthesis of flavanones 7a-l Melaku et al. BMC Chemistry (2022) 16:36 Page 6 of 30 Antiplasmodial activity of pinocembrin and its dose 20 mg/kg and the results were turned out to be sig- dibenzylated analogs nificant compared with the NC group (P < 0.05). The in vivo antiplasmodial activities of pinocembrin (7a) In some cases, compounds isolated from natural prod- and some of its dibenzylated analogs (6a, 6b, 6c, 6e and ucts may show instability on exposure to high tempera- 6i) were evaluated at different doses using Peter’s four ture, hence synthesizing analogs could help to increase day suppressive assay against P. berghei infected mice the chemical stability and biological activities of the [18]. The ability to reduce parasitaemia density is an indi - compounds. However, the results in the present study cator of the presence of antimalarial activities in a sample showed that the benzylated analogs exhibited less bio- [19]. A drug/sample which suppresses the parasitaemia logical activity compared with pinocembrin (7a). There - beyond 30% is considered as active against the parasites fore, this study is parallel with the work of Werbel and [20]. In view of this, pinocembrin and its analogs showed Degnan (1987) in which the original quinazoline amino antiplasmodial activities against P. berghei infected mice group depicted a better antiplasmodial activity compared as evidenced from the percentage of parasite inhibition with its analogs [21]. In contrast, Silva et al. [22] reported (Table 1). Pinocembrin (7a) had shown significant in vivo that the analogs of 4-nerolidylcatechol showed a better antiplasmodial activities compared with both control parastaemia inhibition effect (72% suppression at dose groups (P < 0.05). It inhibits the parasitaemia by 74.4% 600 mg/kg b. wt) compared with isolated compound (60% and 87.2% at 20  mg/kg and 30  mg/kg, respectively. The suppression at dose 600 mg/kg b. wt) [22]. Furthermore, result obtained herein is superior to the suppression of flavanone7a inhibits the parasites in a dose dependent parasitaemia observed for pinocembrin isolated from manner with the higher dose causing a higher parastae- D. angustifolia which inhibited the parasites by 80.0% mia inhibition (Table  1). This is in close agreement with at 40  mg/kg [9]. A group of mice treated by 6b and 6e the work of Silva et al. [23] whereby some indole alkaloids showed 25.5% and 55.6% suppression of parasitaemia at a displayed better activity as the dose increases [23]. Table 1 In vivo suppressive effect of pinocembrin and its analogs against P. berghei in Swiss albino mice No. Doses (mg/kg) Parasitological effect MST ± SEM Body Weight ± SEM %Parasitaemia ± SEM %Suppression D0 D4 6a NC 59.00 ± 0.78 0.00 7.00 ± 0.32 24.00 ± 0.63 22.92 ± 0.84 b b 15 57.00 ± 1.28 21.69 9.60 ± 0.40 24.38 ± 0.91 23.38 ± 0.93 bc bc 20 40.00 ± 0.79 32.20 10.40 ± 0.87 22.98 ± 0.80 22.38 ± 0.57 c c 25 46.92 ± 1.17 35.92 12.00 ± 0.55 23.90 ± 0.78 23.36 ± 0.54 c c 30 38.08 ± 0.56 38.04 13.60 ± 0.87 23.44 ± 0.63 22.72 ± 0.53 d d 35 34.80 ± 0.86 49.09 11.60 ± 0.40 23.70 ± 0.56 22.87 ± 0.69 6b NC 39.85 ± 0.80 0.00 7.60 ± 0.25 24.38 ± 0.56 23.60 ± 0.58 b b 20 29.62 ± 1.21 25.52 7.60 ± 0.68 23.92 ± 0.74 22.74 ± 0.64 6c NC 77.40 ± 1.60 0.00 6.80 ± 0.66 23.68 ± 0.98 22.26 ± 0.93 b b 20 51.50 ± 1.14 33.46 11.80 ± 0.37 23.44 ± 0.78 22.74 ± 0.87 6d NC 39.85 ± 0.80a 0.00 7.60 ± 0.25 24.38 ± 0.56 23.60 ± 0.58 30 36.23 ± 2.20a 8.90 a 6.60 ± 0.30 24.32 ± 0.45 22.60 ± 0.65 6e NC 59.00 ± 0.78 0.00 7.00 ± 1.28 24.00 ± 0.63 22.92 ± 0.84 b b 20 26.14 ± 0.93 55.57 14.60 ± 0.51 24.52 ± 0.58 24.52 ± 0.56 6i NC 73.22 ± 1.25 0.00 7.80 ± 0.37 23.66 ± 0.57 22.04 ± 0.46 b b 15 60.46 ± 0.78 17.42 8.80 ± 0.49 23.06 ± 0.60 22.06 ± 0.59 c c 25 51.30 ± 0.44 29.05 8.00 ± 0.45 23.12 ± 0.47 22.90 ± 1.02 d d 30 43.58 ± 0.62 40.45 13.6 ± 1.03 23.70 ± 0.68 23.50 ± 0.65 7a NC 33.25 ± 1.16 0.00 7.40 ± 0.51 24.24 ± 0.64 23.50 ± 0.65 b b 20 8.5 0 ± 0.41 74.41 13.60 ± 1.25 23.70 ± 0.47 23.30 ± 0.49 c c 30 4.26 ± 0.13 87.19 14.40 ± 0.87 24.54 ± 0.58 24.06 ± 0.63 CQ 30 0.00 100.00 21 < NC Negative control; Means with different letters are significantly different (P < 0.05); P-value is set for the comparison between treated and NC groups; CQ: Chloroquine; has been used as positive control for the test of all compounds M elaku et al. BMC Chemistry (2022) 16:36 Page 7 of 30 Significant (p < 0.05) parasitaemia suppression was also of parasitaemia [26]. Those mice treated with most of the observed on mice treated with compound 6i with per- samples showed a slight decrease in PCV at D4 (Table 2). cent inhibition of 40.4% at 35  mg/kg with the effect sta - However those mice treated with 6e at dose 30  mg/kg tistically significant compared with NC (P < 0.05). At day and 7a at dose 20  mg/kg and 30  mg/kg showed no sig- four of post infection, compound 6c exerted 33.5% para- nificant change in PCV reduction compared with the sitaemia inhibition. The parasitaemia suppression in all positive control indicating the ability of the samples in treated groups was significant (P < 0.05) compared with reducing the parasites. the NC groups. Body weight loss is another common feature in P. Molecular docking simulation of compounds 7a-i berghi infected mice with the effect reduced by an effec - against Plasmodium falciparum dihydrofolate tive antimalarial agent. A significant body weight reduc - reductase-thymidylate synthase (PfDHFR-TS) (PDB ID 1J3I) tion was not observed in most of the group treated P. falciparum  has an unmatched track record of gain- with the samples (Table  1) compared with NC groups ing resistance to drugs currently existing in the mar- (p < 0.005). Compound 6e, 6a (20, 25 and 30 mg/kg b. wt), ket. Hence, it is necessary to search for compounds that 6i (25 and 35  mg/kg), 6c and 7a were shown to be sig- can halt enzyme that play a key role in the biosynthesis nificant in preventing body weight reduction (p < 0.005) of precursors for the DNA of the parasites. One of the of the mice (Table  1). However, significant body weight enzymes responsible for the production of folates as reduction was observed on mice treated with 6i and 6a well as thymidylate required for DNA synthesis is P. fal- at dose 15  mg/kg indicating that these two compounds ciparum dihydrofolate reductase-thymidylate synthase couldn’t prevent a body weight loss as the dose decreases. [27]. This enzyme is a key target for searching for anti - Furthermore, those treated with compound 6a at dose malarial drugs. Molecular docking is used in drug design 35  mg/kg (highest given dose) also showed significant because of its ability to substantially predict the confor- body weight reduction (p < 0.05). Thus, this compound mation of ligands within the appropriate target binding likely has a property that causes depression and loss of site [28]. Among the synthesized dibenzylated analogs of appetite following the dose increment. pinocembrin in this work, the chloroderivatives exhib- A material which induces longer survival time in P. ited better antiplasmodial activity. Hence in the present berghei infected mice compared with the NC group is investigation, a molecular docking study was carried out considered as a good antimalarial agent [24]. In this study, mice treated with pinocembrin and its benzylated analogs showed prolonged MST compared with the NC Table 2 Eec ff t of pinocembrin and its analogs on PCV of P. groups (P < 0.05) indicating that the samples suppressed berghei infected mice P. berghei and reduced the overall pathogenic effect of No. Dose PCV ± SME % Change P value the parasite. The survival time in compound 7a treated groups were found to be in a dose dependent manner D 0 D 4 with the group treated in a higher dose (30  mg/kg) sur- 6a NC 51.11 ± 0.63 48.47 ± 0.42 − 5.16 0.01 vived longer (14.40 days) compared with the one treated 15 51.25 ± 0.21 50.83 ± 0.52 − 0.82 0.10 at 20 mg/kg which survived for 13.60 days. A better MST 25 51.13 ± 0.15 49.93 ± 0.41 − 2.35 0.06 was observed in a group treated with 6e (14.60) at dose 30 51.02 ± 0.13 50.04 ± 0.31 − 1.92 0.10 20  mg/kg. These two compounds also induced a better 35 50.75 ± 0.24 50.55 ± 0.32 − 0.40 0.30 parasitaemia inhibition compared with the other com- 6b NC 51.38 ± 0.25 48.76 ± 0.40 − 5.10 0.01 pounds tested in this study. This result is in agreement 20 51.20 ± 0.20 50.19 ± 0.27 − 2.00 0.08 with the work of Nardos and Mekonnen (2017) in which 6c NC 51.11 ± 0.63 48.47 ± 0.42 − 5.16 0.01 a group treated by hydro-ethanol crude extract of leaves 20 50.84 ± 0.40 51.00 ± 0.31 0.32 0.31 of Ajuga remota at dose 100 mg/kg showed a higher para- 6d NC 51.38 ± 0.25 48.76 ± 0.31 − 5.10 0.01 sitemia inhibition (77.54%) and with a longer survival 30 51.16 ± 0.35 49.79 ± 0.40 − 2.67 0.04 time [25]. 6i NC 51.38 ± 0.31 47.98 ± 0.56 − 6.62 0.00 P. berghei infected mice suffer from anaemia because 15 51.09 ± 0.19 49.89 ± 0.24 − 2.35 0.06 of RBC destruction, either by parasite multiplication or 25 50.78 ± 0.29 50.00 ± 0.35 − 1.54 0.10 by spleen reticuloendotelial cell action as the presence 35 51.26 ± 0.21 51.09 ± 0.23 − 0.33 0.37 of many abnormal RBC stimulates the spleen to pro- 7a NC 50.98 ± 0.64 49.68 ± 0.20 − 2.55 0.04 duce many phagocytes [19]. An effective antimalarial 20 50.38 ± 0.40 50.66 ± 0.45 0.56 0.21 agent prevents reduction of packed cell volume (PCV) 30 50.76 ± 0.16 51.00 ± 0.12 0.47 0.30 which is caused by hemolysis of RBC following the rise Melaku et al. BMC Chemistry (2022) 16:36 Page 8 of 30 using AutoDock Vina in order to elucidate which of the standard clinical drug indicating that compounds 7a-l chloro derivatives of pinocembrin has the best binding are potential antimalarial agents. The binding affinity, affinity against the P. falciparum dihydrofolate reduc - H-bond and hydrophobic, pi-cation and Van der Waals tase-thymidylate synthase. The synthesized compounds interactions of the synthesized compounds were summa- 7a-l were found to have minimum binding energy var- rized in Table 3. ied from −  8.1 to −  8.4  kcal/mol (Table  3). The results The 2D and 3D binding interactions of compounds 7k, demonstrated that the compounds have a better docking 7l and chloroquine against P. falciparum dihydrofolate affinity within the binding pocket of PfDHFR-TS than the reductase-thymidylate synthase were depicted in Figs.  4, standard drug. The key amino acid residues within the 5, 6. Ribbon model shows the binding pocket structure active sites of PfDHFR-TS are Ala16, Ser-108, Phe-58, of PfDHFR-TS with compounds 7f, 7k and 7l. Hydrogen Asp-54, Ile-14, Met-55, Trp-48, and Thr-185. The com - bonds between compounds and amino acids are shown pounds 7a, 7b, 7c, 7d, 7f, 7g, 7i, 7j and 7l have shown as green dash lines, hydrophobic interactions are shown at least one hydrogen bonding interaction within the as pink lines. The molecular docking analysis of com - active site of PfDHFR-TS with the key amino acid resi- pounds 7a-j are given as supplementary material (Addi- dues. The compounds 7e (Gly-44), 7h (Ser-111, Ser-167), tional file 2). 7k (Gly-44 Ser-167), and 7l (Ser-167) displayed hydro- gen bond interaction with non-key residual amino acids In silico pharmacokinetics (drug-likeness) properties within the active site of PfDHFR-TS. On the other hand, In order to get a potential drug for drug development, compound 7i had no hydrogen bonding interaction with forecasting of ADME (absorption, distribution, metab- residual amino acids. Overall, the in silico docking analy- olism and excretion) profiles of compounds including sis revealed that the synthesized compounds shown bet- their pharmacokinetic and drug-like properties using ter binding affinity compared with chloroquine used as Swiss ADME is vital. In this investigation, compounds Table 3 Molecular docking simulation of compounds 7a-l against P. falciparum dihydrofolate reductase-thymidylate synthase No. Formula Binding H-bond Residual interactions affinity Hydrophobic/Pi-Cation Van der Waals (Kcal/mol) 7a C H O − 8.1 Ala-16 Leu-46, Leu-40, Phe-58, Met-55, Leu-119, Cys-15, Ser-167, Gly-166, Tyr-170 15 12 4 Ile-112, 7b C H O − 8.1 Asp-54, Ala-16 Leu-40, Leu-46, Phe-58, Met-55 Cys-15, Gly-44, Val-45, Ile-164, Ser-111, Gly- 17 16 6 41, Ser-167, Tyr-170 7c C H ClO − 8.2 Ser-111, Ser-167, Ile-164 Ala-16, Ile-14, Cys-15, Leu-40, Phe-58 Asp-54, Gly-44, Ser-108, Gly-41, Gly-166, 15 11 4 Tyr-170 7d C H O − 8.2 Ala-16, Leu-40, Ser-108, Leu-46, Ile-112, Val-195 Lys-43, Val-45, Gly-41, Val-168, Ser-167, Ile- 18 18 7 Asn-42, Gly-44, Thr-107 164, Tyr-170 7e C H Cl O − 8.2 Gly-44 Phe-58, Ser-167 Ala-16, Asp-54, Ile-164, Ser-108, Thr-107, Val- 15 10 2 4 45, Gly-166, Tyr-170 7f C H ClO − 8.4 Ser-111, Ile-164 Ala-16, Cys-15, Leu-46, Trp-48, Leu-40 Ile-14, Phe-58, Asp-54, Ser-108, Gly-44, Gly- 15 11 4 166, Ser-167, Val-195, Tyr-170 7g C H ClO − 8.3 Ser-108, Ser-111, Ser-167 Ala-16, Leu-40, Phe-58, Met-55 Asp-54, Cys-15, Leu-46, Thr-107, Gly-41, 15 11 4 Gly-166 7h C H Cl O − 8.3 Ser-111, Ser-167 Ala-16, Leu-46, Trp-48, Ile-14, Cys-15, Phe- Gl-41, Gly-166, Ser-108, Ile-164, Thr-185, 15 10 2 4 58, Leu-40 Tyr-170 7i C H Cl O − 8.3 – Ala-16, Phe-58, Ser-167 Asp-54, Leu-46, Cys-15, Ser-108, Ser-111, 15 10 2 4 Gly-44, Thr-107, Val-195, Gly-166, Leu-40, Ile-164,Tyr-170 7j C H O − 8.1 Ser-108, Ile-164 Val-195, Thr-107 Asn-42, Val-45, Gly-41, Ser-111, leu-40, Gly- 15 12 4 165, Gly-166, Ser-167, Val-168 7k C H O − 8.4 Gly-44, Ser-167 Ala-16, Leu-46, Phe-58, Ile-112, Leu-119, Ser-108, Ser-111, Thr-107, Val-45, Val-195, 17 16 6 Ile-164 Ggly-166, Tyr-170 7l C H ClO − 8.4 Ser-167 Ala-16, Leu-46, Phe-58, Ile-165 Cys-15, Asp-54, Ser-108, Ser-111, Gly-166, 15 11 4 Tyr-170 CQ C H ClN − 7.2 Ile-164 Ala-16, Ile-14, Phe-58 Asp-54, Trp-48, Cys-15, Leu-46, Ser-108, Ser- 18 26 3 111, Val-45, Gly-44, Asn-42, Gly-41, Leu-40, Gly-166, Gly-165, Tyr-170 M elaku et al. BMC Chemistry (2022) 16:36 Page 9 of 30 Fig. 4 2D and 3D binding interactions of 7k against Plasmodium falciparum dihydrofolate reductase-thymidylate synthase Fig. 5 2D and 3D binding interactions of 7l against Plasmodium falciparum dihydrofolate reductase-thymidylate synthase 7a-l were assessed for their drug-like properties using parameters showed that all the compounds have high SwissADME. The results indicates that compounds GI absorption, high blood brain barrier (BBB) permea- 7a-l satisfy Lipinski’s rule of five with zero violations tion (except compounds 7b and 7d) and compound (Table  4) [29]. The Kp values of the synthesized com - 7d is a substrate of permeability glycoprotein (P-gp) pounds were within the range of − 5.29 to − 6.35 cm/s (Table 5). Overall, these prediction results indicate that while it was −  4.96  cm/s for chloroquine inferring low the compounds 7e, 7f, 7h, 7i, 7j and 7l can be better skin permeability (Table  5). The predicted logP values active pharmacological agent compared to the other of the compounds synthesized also indicate that all the reported compounds in this study. This is in agreement compounds had optimal lipophilicity (ranging from from the observed good binding affinity of these com - 2.11 to 2.9). This was inferior to chloroquine which pounds with P. falciparum dihydrofolate reductase-thy- had logP value of 3.95. The SwissADME prediction midylate synthase. Melaku et al. BMC Chemistry (2022) 16:36 Page 10 of 30 Fig. 6 2D and 3D binding interactions of CQ against Plasmodium falciparum dihydrofolate reductase-thymidylate synthase Table 4 Drug-likeness predictions of compounds 7a-l computed by SwissADME °2 S. No. Formula Mol.Wt. (g/mol) NHD NHA NRBTPSA (A ) LogP (cLogP) Lipinski’s rule of Five Violation 7a C H O 256.25 2 4 1 66.76 2.11 0 15 12 4 7b C H O 316.31 2 6 3 85.22 2.71 0 17 16 6 7c C H ClO 290.7 2 4 1 66.76 2.37 0 15 11 4 7d C H O 346.33 2 7 4 94.45 2.9 0 18 18 7 7e C H Cl O 325.14 2 4 1 66.76 2.43 0 15 10 2 4 7f C H ClO 290.7 2 4 1 66.76 2.36 0 15 11 4 7g C H ClO 290.7 2 4 1 66.76 2.39 0 15 11 4 7h C H Cl O 325.14 2 4 1 66.76 2.58 0 15 10 2 4 7i C H Cl O 325.14 2 4 1 66.76 1.91 0 15 10 2 4 7j C H O 325.14 2 4 1 66.76 2.53 0 15 12 4 7k C H O 325.14 2 4 1 66.76 2.57 0 17 16 6 7l C H ClO 325.14 2 4 1 66.76 2.56 0 15 11 4 CQ C H ClN 319.87 1 2 8 28.16 3.95 0 18 26 3 NHD Number of Hydrogen donor, NHA Number of Hydrogen acceptor, NRB Number of rotatable bonds, TPSA total polar surface area, CQ chloroquine in capillary tubes with a digital electrothermal melt- Experimental ing point apparatus. IR spectra were measured on a General Bruker Alpha FT-IR spectrometer. UV/Vis spectra All reagents used in the present work were used with- were recorded on a Cary 4E spectrophotometer. H and out further purification. Glassware used was dried for C NMR spectra were recorded at 300 (75) MHz and 24  h at 120  °C in an oven. Solvents used in reactions 500 (125) MHz on Varian Unity Inova spectrometers. were distilled over appropriate drying agents prior to Coupling constants J [Hz] were directly taken from the use while those used for extraction and purification spectra and are not averaged. Splitting patterns are des- were distilled prior to use. Thin-layer chromatography ignated as s (singlet), d (doublet), t (triplet), q (quar- (TLC) was performed on precoated aluminum plates tet), and m (multiplet). Low-resolution electron impact (silica gel Merck 60 F) and visualized under UV light mass spectra [MS (EI)] and exact mass electron impact (254 nm) and/or by dipping in vanillin/H SO followed 2 4 mass spectra [HRMS (EI)] were obtained at 70  eV by heating. Products were purified by column chro - using a double focusing sector field mass spectrometer matography over silica gel (MN 60, 0.04–0.063  mm; Finnigan MAT 95, for the measurement of exact mass Marcherey & Nagel). Melting points were determined M elaku et al. BMC Chemistry (2022) 16:36 Page 11 of 30 Table 5 ADME predictions of compounds 7a-l computed by SwissADME and PreADMET No. Chemical Formula Skin Permeation GI Absorption BBB Inhibitor Interaction (SwissADME/PreADMET) Value (log Kp) cm/s Permeability P-gp substrate CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 inhibitor inhibitor inhibitor inhibitor inhibitor 7a C H O − 5.82 High Yes No Yes Yes No No No 15 12 4 7b C H O − 6.23 High No No Yes No Yes No Yes 17 16 6 7c C H ClO − 5.59 High Yes No Yes Yes Yes Yes Yes 15 11 4 7d C H O − 6.35 High No Yes Yes No Yes No Yes 18 18 7 7e C H Cl O − 5.29 High Yes No Yes Yes Yes Yes Yes 15 10 2 4 7f C H ClO − 5.52 High Yes No Yes Yes Yes Yes Yes 15 11 4 7g C H ClO − 5.52 High Yes No Yes Yes Yes Yes Yes 15 11 4 7h C H Cl O − 5.35 High Yes No Yes Yes Yes Yes Yes 15 10 2 4 7i C H Cl O − 5.29 High Yes No Yes Yes Yes Yes Yes 15 10 2 4 7j C H O − 5.35 High Yes No Yes Yes Yes Yes Yes 15 12 4 7k C H O − 5.29 High Yes No Yes Yes Yes Yes Yes 17 16 6 7l C H ClO − 5.45 High Yes No Yes Yes Yes Yes Yes 15 11 4 CQ C H ClN − 4.96 High Yes No Yes No No Yes Yes 18 26 3 CQ Chloroquine, GI Gastro-Intestinal, BBB Blood Brain Barrier, P-gp P-glycoprotein, CYP Cytochrome-P Melaku et al. BMC Chemistry (2022) 16:36 Page 12 of 30 electrospray ionization mass spectra [ESI (HRMS)] a reaction mixture was extracted with EtOAc (3 × 30  mL). Bruker Daltonik spectrometer micrOTOF-Q was used. The combined organic phases were dried over anhydrous MgSO , filtered and concentrated in vacuo. Column Synthesis and characterization of starting materials chromatography of the crude product on silica gel using and chalcones petroleum ether:EtOAc (4:1) as eluent furnished the cor- Synthesis and analytical data responding chalcones 5a-r as yellow solids. Compounds of 2,4‑dibenzyloxy‑6‑hydroxyacetophenone (3) [10] 5a-r were synthesized according to general procedure I. Synthesis and analytical data O 4 O of 2’,4’‑dibenzyloxy‑6’‑hydroxychalcone (5a) 6 4 OH O BnO 4' OH 7 2 2' OBn O A 250  mL 2-neck round bottom flask was charged with N,N-dimethylformamide (24  mL) under argon, 5a heated to 35  °C, then trihydroxyacetophenone (1) (5  g, 29.7 mmol) was added in one portion, followed by more According to general procedure I, 2,4-dibenzyloxy-6-hy- DMF (16  mL). Potassium carbonate (8.5  g, 55.9  mmol) droxyacetophenone (3) (1  g, 2.8  mmol) was condensed was added and the mixture was heated to 65  °C. Benzyl with benzaldehyde (4a) (0.3  g, 2.8  mmol) in the pres- chloride (2) (7.5 g, 59.4 mmol) was added in one portion ence of aqueous KOH (60%, 1.5 mL) in ethanol (8.5 mL). and the mixture was heated to 70  °C for 3  h, cooled to The product 5a was obtained in 80% yield as a yellow room temperature and filtered. The filter cake was rinsed solid. mp 124–125  °C); UV (MeOH) λ : 278, 345  nm; max with dichloromethane. The combined filtrates were con - H NMR (CDCl , 300  MHz) δ 5.01 (2H, s, OCH ), 5.06 3 2 centrated in vacuo and the residual orange oil was taken (2H, s, OCH ), 6.18 (1H, d, J = 2.3 Hz, H-5’), 6.23 (1H, d, up in dichloromethane (50 mL), stirred for five min, and J = 2.3 Hz, H-3’), 7.06 (2H, d, J = 7.6 Hz, H-2,6), 7.19 (3H, filtered. The filtrate was column filtered over silica gel t, J = 8.1  Hz, H-3,4,5), 7.25–7.52 (10H, m, H-2’’– H-6’’, with dichloromethane as eluent. It was concentrated, H-2’’’– H-6’’’), 7.71 (1H, d, J = 16  Hz, H-8), 7.89 (1H, d, dichloromethane (5  mL) and cyclohexane (8  mL) were J = 16  Hz, H-9), 14.50 (1H, s, OH); C NMR (CDCl , added and the mixture was stirred for 20 min. The result - 75  MHz) δ 70.3 (OCH ), 71.4 (OCH ), 92.5 (C-3’), 95.0 2 2 ant white crystalline solid was collected by suction fil - (C-5’), 106.3 (C-1’), 127.4 (C-8), 127.6 (C-2, C-6), 128.31 tration, washed with cyclohexane and air dried to yield (C-2’’’, C-6’’’), 128.4 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.62 5.7 g (79%) of a white crystalline solid which was identi- (C-4’’), 128.64 (C-4), 128.7 (C-3’’’,C-5’’’), 128.8 (C-3, C-5), fied as 2,4-dibenzyloxy-6-hydroxyacetophenone (3). H 128.9 (C-3’’,C-5’’),129.8 (C-1), 135.2 (C-1’’’), 135.3 (C-1’’), NMR (CDCl , 300  MHz) δ 2.56 (3H, s, H-2’), 5.06 (4H, 3 135.8 (C-7), 161.7 (C-2’), 165.2 (C-6’), 168.8 (C-4’), 192.6 br s, benzylic H), 6.09 (1H, d, J = 2.3 Hz, H-3), 6.16 (1H, (C-9). d, J = 2.3 Hz, H-5), 7.34–7.43 (10H, m, aromatic H), 14.01 (1H, s, OH); C NMR (CDCl , 75  MHz) δ 33.1 (C-2’), Synthesis and analytical data 70.3 (C-1’’’), 71.1 (C-1’’), 92.3 (C-5), 94.6 (C-3), 106.3 of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑4‑fluorochalcone (5b) (C-1), 127.6 (C-2’’’, C-6’’’, C-4’’’,), 127.9 (C-2’’’’, C-4’’’’, C-6’’’’), 128.3 (C-3’’’, C-5’’’), 128.4 (C-3’’’’, C-5’’’’), 135.5 (C-1’’’), 4 F 135.8 (C-1’’’’), 161.9 (C-2), 165.0 (C-6), 167.5 (C-4), 203.2 4' BnO OH (C-2’). 7 2 2' 9 General procedure I for the synthesis of chalcones 5a‑r [30] OBn O 2,4-Dibenzyloxy-6-hydroxyacetophenone (3) (2.8  mmol) was condensed with substituted benzaldehydes 4a- 5b r (2.8  mmol) in the presence of aqueous KOH (60%, 1.5  mL) in ethanol (8.5  mL). The reaction mixture was According to general procedure I, 2,4-dibenzyloxy-6-hy- stirred for 24  h at room temperature and poured into droxyacetophenone (3) (0.4  g, 1.6  mmol) was condensed water (30  mL). After neutralization with 10% HCl, eth- with 4-fluorobenzaldehyde (4b) (0.19  g, 1.6  mmol) in anol was removed using a rotary evaporator and the the presence of aqueous KOH (60%, 1.5  mL) in ethanol M elaku et al. BMC Chemistry (2022) 16:36 Page 13 of 30 Synthesis and analytical data (8.5 mL). The product was obtained in 68% yield. mp 126– −1 of 2’,4’‑dibenyloxy‑6’‑hydroxy‑2,6‑dimethoxychalcone (5d) 127  °C; UV(MeOH) λ : 276, 337  nm; IR ν ̃ [cm ]: 1660 max (conjugated C = O), 1571 (arom. C = C), and 1023 (C-O); H CO 4 H NMR (CDCl , 300 MHz) δ 5.0 (2H, s, O CH ), 5.1 (2H, 3 3 2 s), 5.56 (1H, s, OCH ), 6.18 (1H, d, J = 2.4  Hz, H-5’), 6.2 1 2 4' BnO OH (1H, d, J = 2.4 Hz, H-3’), 6.8 (2H, m, H-3,H-5), 7.01 (2H, m, 2' 9 OCH H-2,H-6), 7.50–7.39 (10H, m, H-2’’– H-6’’, H-2’’’– H-6’’’), 7.64 (1H, d, J = 15  Hz, H-8), 7.78 (1H, d, J = 15  Hz, H-7), OBn O 14.5 (1H, s, 6’-OH); C NMR (CDCl , 75  MHz) δ 70.3 5d (OCH ), 71.4 (OCH ), 92.5 (C-3’), 95.0 (C-5’), 106.2 (C-1’), 2 2 115.5 (C-3,C-5), 115.8 (C-8), 127.2 (C-2’’’, C-6’’’), 127.6 (C-2’’, According to general procedure I, 2,4-dibenzyloxy-6-hy- C-6’’), 128.3 (C-4’’’), 128.7 (C-4’’), 128.9 (C-2, C-6), 129.3 droxyacetophenone (3) (0.4 g, 1.1 mmol) was condensed (C-3’’’, C-5’’’), 130.2 (C-3’’, C-5’’), 131.5 (C-1), 132.7 (C-1’’’), with 3′4’-dimethoxybenzaldehyde (4d) (0.19 g, 1.1 mmol) 135.4 (C-1’’), 135.8 (C-7), 141.3 (d, C-4), 161.7 (C-2’), 165.3 in the presence of aqueous KOH (60%, 1.5  mL) in etha- (C-6’), 168.8 (C-4’), 192.4 (C-9). nol (8.5  mL). The product 5d was obtained in 67%. mp −1 99–100  °C; UV(MeOH) λ : 275, 332  nm; IR ν̃ [cm ]: max Synthesis 1671 (conjugated C = O), 1603 (C = C), 1560 (C = C), of 2’,4’‑dibenyloxy‑6’‑hydroxy‑3,4‑dimethoxychalcone (5c) 1347, 1206, and 1027 (C-O); H NMR (C D COCD , 3 3 300 MHz) δ 3.6 (3H, s, OMe), 3.8 (3H, s, OMe), 5.22 (2H, OCH s, OCH ), 5.29 (2H, s, OCH ), 6.23 (1H, d, J = 2.2  Hz, 2 2 4' BnO OH H-5’), 6.36 (1H, d, J = 2.2  Hz, H-3’), 6.96 (1H, m, H-4), OCH 7 2 6.98 (2H, br d, H-3, H-5), 7.54–7.30 (10H, m, H-2’’’– H-6’’’, 2' H-2’’– H-6’’), 14.1 (1H, s, 6’OH); C NMR (C D COCD , 3 3 OBn O 75  MHz) δ 55.8 (OMe), 56.3 (OMe), 70.8 (OCH ), 71.8 (OCH ), 93.5 (C-3’), 95.8 (C-5’), 107.3 (C-1’), 113.3 (C-3), 5c 113.5 (C-5), 118.0 (C-1), 125.2 (C-8), 128.5 (C-2’’’, C-6’’’), 128.6 (C-2’’, C-6’’), 128.7 (C-4’’’), 128.8 (C-4’’), 128.9 (C-3’’’, According to general procedure I, 2,4-dibenzyloxy-6-hy- C-5’’’), 128.91 (C-3’’, C-5’’), 129.3 (C-4), 129.4 (C-1’’’), droxyacetophenone (3) (0.4  g, 1.1  mmol) was condensed 137.1 (C-1’’), 137.8 (C-7), 153.8 (C-2), 154.5 (C-6), 162.6 with 3′4’-dimethoxybenzaldehyde (4c) (0.19  g, 1.1  mmol) (C-2’), 166.2 (C-6’), 168.7 (C-4’), 193.7 (C-9). in the presence of aqueous KOH (60%, 1.5 mL) in ethanol (8.5 mL). The product 5c was obtained in 67% as a yellow - Synthesis and analytical data ish solid. mp 102–103  °C; UV(MeOH) λ : 276, 331  nm; max −1 of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑4‑chlorochalcone (5e) IR ν ̃ [cm ]: 1677 (conjugated C = O), 1630 (C = C), and 1040 (C-O); H NMR (CDCl , 300  MHz) δ 3.62 (1H, s, 4 Cl OMe), 3.91 (1H, s, OMe), 5.10 (4H, s, OCH ), 6.17 (1H, d, J = 2.4  Hz, H-5’), 6.23 (1H, d, J = 2.4  Hz, H-3’), 6.72 (1H, 1 2 4' BnO OH d, J = 9 Hz, H-5), 6.77 (1H, d, J = 3 Hz, H-2), 6.81 (1H, dd, 2' J = 3  Hz and 9  Hz, H-6), 7.47–7.31 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.70 (1H, d, J = 15.6  Hz, H-8), 7.80 (1H, d, OBn O J = 15.6  Hz, H-7), 14.4 (1H, s, 6’OH); C NMR (CDCl , 5e 75  MHz) δ 55.6 (OMe), 55.9 (OMe), 70.2 (O CH ), 71.1 (OCH ), 92.7 (C-3’), 95.0 (C-5’), 106.6 (C-1’), 110.8 (C-2), According to general procedure I, 2,4-dibenzyloxy-6-hy- 110.9 (C-5), 122.4 (C-6), 125.5 (C-8), 127.6 (C-2’’’, C-6’’’), droxyacetophenone (3) (0.5 g, 1.4 mmol) was condensed 127.8 (C-2’’, C-6’’), 128.2 (C-4’’’), 128.3 (C-4’’), 128.6 (C-1), with 4-chlorobenzaldehyde (4e) (0.21  g, 1.4  mmol) in 128.7 (3’’’, C-5’’’), 128.8 (C-3’’, C-5’’),, 135.7 (C-1’’’), 135.8 the presence of aqueous KOH (60%, 1.5  mL) in etha- (C-1’’), 142.8 (C-7), 148.8 (C-4), 150.8 (C-3), 161.5 (C-2’), nol (8.5  mL). The product 5e was obtained as a white 165.0 (C-6’), 168.3 (C-4’), 192.5 (C-9). Melaku et al. BMC Chemistry (2022) 16:36 Page 14 of 30 Synthesis of 2’,4’‑dibenyloxy‑6’‑hydroxy‑4‑(trifluoromethyl) solid in 72% yield. mp 142–144  °C; R 0.63 (petroleum chalcone (5g) ether:EtOAc 4:1); UV(MeOH) λ : 274, 336  nm; IR ν̃ max −1 [cm ]: 1613 (C = C), 1487, 1162, 1114, and 1023 (C-O); 4 CF H NMR (CDCl , 300  MHz) δ 5.05 (2H, s, OCH ), 5.11 3 3 2 (2H, s, OCH ), 6.18 (1H, d, J = 2.2 Hz, H-5’), 6.23 (1H, d, 1 2 4' BnO OH J = 2.2  Hz, H-3’), 6.91 (2H, d, J = 7.0  Hz, H-3, H-5), 7.13 2' 9 (2H, d, J = 7.0  Hz, H-2, H-6), 7.4 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.62 (1H, d, J = 15.4  Hz, H-8), 7.82 (1H, OBn O d, J = 15.4  Hz, H-7); C-NMR (CDCl , 75  MHz) δ 70.3 5g (OCH ), 71.2 (OCH ), 92.6 (C-3’), 95.2 (C-5’), 106.2 2 2 (C-1’), 127.2 (C-8), 127.3 (C-2’’’, C-6’’’), 127.6 (C-2’’, C-6’’), According to general procedure I, 2,4-dibenzyloxy-6-hy- 128.0 (C-4’’’), 128.3 (C-4’’), 128.7 (C-2, C-6), 128.8 (C-3, droxyacetophenone (3) (0.6 g, 1.7 mmol) was condensed C-5), 128.9 (C-3’’’, C-5’’’), 129.4 (C-3’’, C-5’’), 133.8 (C-1), with 4-(trifluoromethyl)benzaldehyde (4  g) (0.32  g, 135.3 (C-4), 135.5 (C-1’’’), 135.7 (C-1’’), 141.0 (C-7), 161.6 1.7 mmol) in the presence of aqueous KOH (60%, 1.5 mL) (C-2’), 165.4 (C-6’), 168.8 (C-4’), 192.3 (C-9). in ethanol (8.5 mL). The product 5 g was obtained in 71% yield. H NMR (C DCl , 300 MHz) δ 5.05 (2H, s, O CH ), 3 2 Synthesis and analytical data 5.10 (2H, s, OCH ), 6.18 (1H, d, J = 2.2  Hz, H-5’), 6.23 of 2’,4’‑dibenyloxy‑6’‑hydroxy‑2‑bromochalcone (5f ) (1H, d, J = 2.2  Hz, H-3’), 7.09 (2H, d, J = 7.0, H-2, H-6), 7.48 (2H, d, J = 7.0  Hz, H-3, H-5), 7.42 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.63 (1H, d, J = 15.4  Hz, H-8), 7.88 1 2 4' 13 BnO OH (1H, d, J = 15.4  Hz, H-7); C NMR (CDCl , 75  MHz) δ 2' 970.2 (OCH ), 71.3 (OCH ), 92.6 (C-3’), 95.0 (C-5’), 106.2 Br 2 2 (C-1’), 125.4 (C-7), 125.5 (q, C F ), 127.6 (C-3, C-5), 128.2 OBn O (C-2, C-6), 128.5 (C-2’’’, C-6’’’), 128.7 (C-2’’, C-6’’), 128.9 5f (C-4’’’), 129.0 (C-4’’), 130.1 (C-3’’’, C-5’’’), 130.8 (C-3’’, C-5’’), 131.2 (C-4), 135.2 (C-1), 135.7 (C-1’’’), 138.7 (C-1’’), According to general procedure I, 2,4-dibenzyloxy-6-hy- 140.2 (C-7), 161.8 (C-2’), 165.8 (C-6’), 168.8 (C-4’), 192.3 droxyacetophenone (3) (0.6 g, 1.7 mmol) was condensed (C-9). with 2-bromobenzaldehyde (4f) (0.32  g, 1.7  mmol) in the presence of aqueous KOH (60%, 1.5  mL) in ethanol (8.5  mL). The product 5f was obtained in 57% yield. mp Synthesis −1 111–112 °C; UV(MeOH) λ : 276, 338 nm; IR ν̃ [cm ]: of 2’,4’‑dibenyloxy‑6’‑hydroxy‑3,4,5‑trimethoxychalcone (5h) max : 1673 (conjugated C = O), 1618 (C = C), 1552 (C = C), 1453, 1226, 1156, and 1021 (C-O); H NMR (CDCl , OCH 300 MHz) δ 5.04 (2H, s, OCH ), 5.10 (2H, s, OCH ), 6.17 4 OCH 2 2 (1H, d, J = 2.2  Hz, H-5’), 6.22 (1H, d, J = 2.2  Hz, H-3’), 4' BnO OH OCH 6.96 (1H, t, J = 7.5  Hz, H-4), 7.14 (1H, dd, J = 7.2  Hz, 2' 9 1.8  Hz, H-3), 7.41 (12H, m, H-5, H-6, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.77 (1H, d, J = 15.4  Hz, H-8), 8.05 (1H, OBn O d, J = 15.4  Hz, H-7); C NMR (CDCl , 75  MHz) δ 70.2 5h (OCH ), 71.2 (OCH ), 92.2 (C-3’), 95.1 (C-5’), 106.2 2 2 (C-1’), 125.8 (C-2), 127.4 (C-8), 127.6 (C-2’’’, C-6’’’), 128.3 According to general procedure I, 2,4-dibenzyloxy- (C-2’’, C-6’’), 128.5 (C-4’’’), 128.6 (C-4’’), 128.7 (C-5), 128.8 6-hydroxyacetophenone (3) (0.6  g, 1.7  mmol) was (C-6), 128.9 (C-3’’’, C-5’’’), 130.0 (C-3’’, C-5’’), 130.5 (C-4), condensed with 3,4,5-trimethoxybenzaldehyde (4  h) 133.2 (C-3), 135.2 (C-1), 135.5 (C-1’’’), 135.7 (C-1’’), 140.5 (0.345  g, 1.7  mmol) in the presence of aqueous KOH (C-7), 161.6 (C-2’), 164.4 (C-6’), 168.7 (C-4’), 192.2 (C-9). M elaku et al. BMC Chemistry (2022) 16:36 Page 15 of 30 Synthesis of 2’ ,4’‑dibenyloxy‑6’‑hydroxy‑2,3‑dichlorochalcone (60%, 1.5  mL) in ethanol (8.5  mL). The product 5  h (5j) was obtained in 46% yield. mp 94–96  °C; UV(MeOH) −1 λ : 275, 324  nm; IR ν̃ [cm ]: 1611 (C = C ), 1577 max (C = C), 1266, 1168, and 1095 (C-O); H NMR (CDCl , 300  MHz) δ 3.64 (6H, s, OMe), 3.87 (3H, s, OMe), 4' BnO OH 5.10 (2H, s, OCH ), 5.11 (2H, s, OCH ), 6.16 (1H, d, 2 2 Cl J = 2.2  Hz, H-5’), 6.23 (1H, d, J = 2.2  Hz, H-3’), 7.44 2' Cl (12H, m, H-2, H-6, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.68 (1H, d, J = 15.4 Hz, H-8), 7.79 (1H, d, J = 15.4 Hz , H-7); OBn O C NMR (CDCl , 75 MHz) δ 56.0 (OMe), 60.9 (OMe), 5j 70.3 (OCH ), 71.3 (O CH ), 92.9 (C-3’), 95.6 (C-5’), 2 2 105.7 (C-1’), 106.8 (C-2, C-6), 127.1 (C-8), 127.3 (C-2’’’, According to general procedure I, 2,4-dibenzyloxy-6-hy- C-6’’’), 127.6 (C-2’’, C-6’’), 127.9 (C-4’’’), 128.2 (C-4’’), droxyacetophenone (3) (0.5 g, 1.4 mmol) was condensed 128.3 (C-3’’’, C-5’’’), 128.7 (C-3’’, C-5’’), 130.7 (C-1), with 2,3-dichlorobenzaldehyde (4j) (0.25  g, 1.4  mmol) 135.6 (C-1’’’), 135.8 (C-1’’), 140.0 (C-4), 142.5 (C-7), in the presence of aqueous KOH (60%, 1.5  mL) in etha- 153.1 (C-3,C-5), 161.4 (C-2’), 165.2 (C-6’), 168.1 (C-4’), nol (8.5  mL). The product 5j was obtained in 97% yield. 192.5 (C-9). mp 121–123  °C; R 0.60 (petroleum ether:EtOAc 4:1); −1 UV(MeOH); λ : 274, 328  nm; IR ν̃ [cm ]: 1605 max (C = C), 1560 (C = C), 1337, 1204, 1154, and 1044 (C-O); Synthesis and analytical data H NMR (CDCl , 300  MHz) δ 5.01 (2H, s, OCH ), 5.12 of 2’,4’‑dibenyloxy‑6’‑hydroxy‑2‑bromo‑4‑fluorochalcone (5i) 3 2 (2H, s, OCH ), 6.18 (1H, d, J = 2.2 Hz, H-5’), 6.23 (1H, d, 4 F J = 2.2  Hz, H-3’), 6.62 (1H, dd, J = 7.5  Hz and J = 2.4  Hz, H-6), 6.84 (1H, t, J = 8.1  Hz, H-5), 7.40 (11H, m, H-4, BnO 4' OH H-2’’’– H-6’’’, H-2’’– H-6’’), 7.75 (1H, d, J = 15.4  Hz), 8.06 2' 9 Br (1H, d, J = 15.4  Hz); C NMR (CDCl , 75  MHz) δ 70.3 (OCH ), 71.3 (OCH ), 92.5 (C-3’), 95.1 (C-5’), 106.3 2 2 OBn O (C-1’), 125.5 (C-8), 127.1, 127.6 (C-2’’’, C-6’’’), 128.3 (C-2’’, 5i C-6’’), 128.4 (C-4’’’), 128.5 (C-4’’), 128.6 (C-2), 128.7 (C-5), 128.8 (3’’’, C-5’’’), 130.8 (C-3’’, C-5’’), 133.1 (C-4), 133.6 According to general procedure I, 2,4-dibenzyloxy- (C-3), 135.2 (C-1), 135.7 (C-1’’’), 135.8 (C-1’’), 137.6 (C-7), 6-hydroxyacetophenone (3) (0.5  g, 1.4  mmol) was 161.6 (C-2’), 165.5 (C-6’), 168.7 (C-4’), 192.1 (C-9). condensed with 2-bromo-4-chlorobenzaldehyde (4i) (0.32  g, 1.4  mmol) in the presence of aqueous KOH (60%, 1.5 mL) in ethanol (8.5 mL). The product 5i was Synthesis of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑4‑cyanochalcone obtained in 77% yield. mp 132–133  °C; UV(MeOH) (5k) −1 λ : 271, 333  nm; IR ν̃ [cm ]: 1670 (conjugated max C = O), 1603 (C = C), 1154, and 1025 (C-O); H NMR 4 CN (CDCl , 300  MHz) δ 5.01 (2H, s, O CH ), 5.11 (2H, 3 2 1 2 s, OCH ), 6.17 (1H, d, J = 2.2  Hz, H-5’), 6.22 (1H, d, BnO 4' OH J = 2.2 Hz, H-3’), 6.57 (1H, dd, J = 9.3  Hz and J = 3  Hz , 2' H-3), 6.87 (1H, m, H-5), 7.44 (11H, m, H-6, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.71 (1H, d, J = 15.4 Hz, H-8), 7.96 OBn O (1H, d, J = 15.4  Hz , H-7); C NMR (CDCl , 75  MHz) δ 70.3 (OCH ), 71.6 (OCH ), 92.6 (C-3’), 95.0 (C-5’), 2 2 5k 106.3 (C-1’), 113.9 (C-5), 114.2 (C-2), 117.8 (C-3), 119.9 (C-8), 127.6 (C-2’’’, C-6’’’), 128.7 (C-2’’, C-6’’), According to general procedure I, 2,4-dibenzyloxy-6-hy- 128.8 (C-4’’’), 129.0 (C-4’’), 131.0 (C-3’’’, C-5’’’), 134.2 droxyacetophenone (3) (0.5 g, 1.4 mmol) was condensed (C-3’’, C-5’’), 134.3 (C-6), 135.0 (C-1), 135.7 (C-1’’’), 136.7 (C-1’’), 139.3 (C-7), 159.9 (d, C-4), 161.6 (C-2’), 165.5 (C-6’), 168.5 (C-4’), 192.3 (C-9). Melaku et al. BMC Chemistry (2022) 16:36 Page 16 of 30 Synthesis of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑3‑chlorochalcone with 4-cyanobenzaldehyde (4  k) (0.19  g, 1.4  mmol) in (5m) the presence of aqueous KOH (60%, 1.5  mL) in ethanol (8.5 mL). The product 5 k was obtained in 67% yield. mp 148–150  °C; H NMR (C DCl , 300  MHz) δ 5.03 (2H, s, OCH ), 5.11 (2H, s, OCH ), 6.18 (1H, d, J = 2.2 Hz, H-5’), 2 2 2 BnO 4' OH Cl 6.23 (1H, d, J = 2.2 Hz, H-3’), 7.02 (2H, d, J = 7.0 Hz, H-3, 7 2' 9 H-5), 7.43 (12H, m, H-2, H-6, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.58 (1H, d, J = 15.4  Hz), 7.88 (1H, d, J = 15.4  Hz); C OBn O NMR (CDCl , 75  MHz) δ 70.5 (O CH ), 71.4 (OCH ), 3 2 2 5m 92.6 (C-3’), 95.2 (C-5’), 106.3 (C-1’), 112.4 (C-4), 118.6 (CN), 127.6 (C-8), 127.8 (C-2, C-6), 127.9 (C-2’’’, C-6’’’), According to general procedure I, 2,4-dibenzyloxy-6-hy- 128.7 (C-2’’, C-6’’), 128.8 (C-4’’’), 129.0 (C-4’’), 129.1 (C-3’’’, droxyacetophenone (3) (0.7 g, 2.0 mmol) was condensed C-5’’’), 130.9 (C-3’’, C-5’’), 132.3 (C-3, C-5), 135.2 (C-1), with 3-chlorobenzaldehyde (4  m) (0.28  g, 2.0  mmol) in 135.7 (C-1’’’), 139.5 (C-1’’), 139.7 (C-7), 161.6 (C-2’), 165.8 the presence of aqueous KOH (60%, 3  mL) in ethanol (C-6’), 168.8 (C-4’), 192.3 (C-9). (15  mL). The product 5  m was obtained in 92% yield. mp 135–136  °C; R 0.60 (petroleum ether:EtOAc 4:1); Synthesis and analytical data −1 UV(MeOH) λ : 280, 334 nm; IR ν̃ [cm ]: 1607 (C = C), max of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑2‑chlorochalcone (5l) 1573 (C = C), 1269, 1226, 1116, and 1024 (C-O); H NMR (CDCl , 300  MHz) δ 5.0 (2H, s, OCH ), 5.1 (2H, 3 2 s, OCH ), 6.18 (1H, d, J = 2.4  Hz, H-5’), 6.22 (1H, d, 1 2 4' BnO OH J = 2.4 Hz, H-3’), 6.89 (1H, d, J = 7.8 Hz, H-4), 7.10 (1H, t, J = 7.8 Hz, H-5), 7.2 (1H, br s, H-2), 7.25 (1H, dd, J = 7.8, 2' Cl J = 3.6 Hz, H-6), 7.45 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), OBn O 7.62 (1H, d, J = 15.9, H-8), 7.83 (1H, d, J = 15.9, H-7), 14.3 (1H, s, 6’OH); C NMR (C DCl , 75 MHz) δ 70.3 (O CH ), 5l 3 2 71.4 (OCH ), 92.6 (C-3’), 95.0 (C-5’), 106.3 (C-1’), 126.1, 127.6 (C-6), 128.2 (C-2), 128.3 (C-2’’’-C-6’’’), 128.4 (C-2’’- According to general procedure I, 2,4-dibenzyloxy-6-hy- C-6’’), 128.7 (C-4’’’), 128.8 (C-4’’), 128.85 (C-4), 128.9 droxyacetophenone (3) (1  g, 2.8  mmol) was condensed (C-3’’’, C-5’’’), 129.6 (C-3’’, C-5’’), 129.8 (C-5), 134.5 (C-3), with 2-chlorobenzaldehyde (4  l) (0.4  g, 2.8  mmol) in 135.2 (C-1), 135.7 (C-1’’’), 137.1 (C-1’’), 140.7 (C-7), 161.6 the presence of aqueous KOH (60%, 3  mL) in etha- (C-2’), 165.4 (C-6’), 168.6 (C-4’), 192.3 (C-9). nol (15  mL). The product 5  l was obtained in 77% yield. mp 138–139  °C; R 0.59 (petroleum ether:EtOAc −1 4:1); UV(MeOH) λ : 270, 338  nm; IR ν̃ [cm ]: 1608 max (C = C),1578 (C = C), 1554, 1425, 11, 1156, and 1040 Synthesis and analytical data (C-O); H NMR (CDCl , 300 MHz) δ 5.04 (2H, s, O CH ), of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑2,4‑dichlorochalcone (5n) 3 2 5.10 (2H, s, OCH ), 6.17 (1H, d, J = 2.4  Hz, H-5’), 6.23 Cl (1H, d, J = 2.4 Hz, H-3’), 6.75 (1H, dd, J = 7.8 and 2.4 Hz, H-3), 6.92 (1H, d, J = 8.7  Hz, H-5), 7.19 (2H, m, H-4, 4' BnO OH H-6), 7.41 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.81 (1H, 2' 13 9 Cl d, J = 15.6  Hz, H-8), 8.10 (1H, d, J = 15.6  Hz, H-7); C NMR (CDCl , 75  MHz) δ 70.3 (O CH ), 71.4 (OCH ), 3 2 2 OBn O 92.6 (C-3’), 95.0 (C-5’), 106. 3 (C-1’), 126.1 (C-8), 127.6 5n (C-5), 128.2 (C-2’’’, C-6’’’), 128.3 (C-2’’, C-6’’), 128.4 (C-4’’’), 128.7 (C-4’’), 128.8 (C-6), 128.9 (C-3), 129.6 (C-3’’’-C-5’’’), According to general procedure I, 2,4-dibenzyloxy- 129.8 (C-3’’-C-5’’), 128.85 (C-4), 134.5(C-2), 135.2 (C-1), 6-hydroxyacetophenone (3) (0.5  g, 1.4  mmol) was 135.7 (C-1’’’), 137.1 (C-1’’), 140.7 (C-7), 161.6 (C-2’), 165.4 (C-6’), 168.6 (C-4’), 192.3 (C-9). M elaku et al. BMC Chemistry (2022) 16:36 Page 17 of 30 Synthesis and analytical data condensed with 2,4-dichlorobenzaldehyde (4n) (0.25  g, of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑2,6‑dichlorochalcone (5p) 1.4 mmol) in the presence of aqueous KOH (60%, 3 mL) in ethanol (15 mL). The product 5n was obtained in 97% Cl 4 yield. mp 127–129  °C; R 0.65 (petroleum ether:EtOAc −1 4:1); UV(MeOH) λ : 281, 330  nm; IR ν̃ [cm ]: 1623 1 2 max 4' BnO OH (C = C), 1559 (C = C), 1464, 1299, 1101, and 1049 (C-O); 2' 9 Cl H NMR (CDCl , 300  MHz) δ 5.03 (2H, s, OCH ), 5.10 3 2 (2H, s, OCH ), 6.17 (1H, dd, J = 2.1  Hz, H-5’), 6.22 (1H, OBn O dd, J = 2.1  Hz, H-3’), 6.59 (1H, d, J = 8.4  Hz, H-6), 6.85 5p (1H, dd, J = 8.7, J = 2.4  Hz, H-5), 7.32 (1H, d, J = 2.4  Hz, H-3), 7.45 (10H, m, H-2’’’– H-6’’’, H-2’’–H-6’’), 7.75 (1H, According to general procedure I, 2,4-dibenzyloxy-6-hy- d, J = 15.6  Hz, H-8), 7.99 (1H, d, J = 15.6  Hz, H-7), 14.3 droxyacetophenone (3) (1  g, 2.8  mmol) was condensed (1H, s, 6’OH); C NMR (C DCl , 75 MHz) δ 70.4 (O CH ), 3 2 with 2,6-dichlorobenzaldehyde (4p) (0.54  g, 2.8  mmol) 71.5 (OCH ), 92.7 (C-3’), 95.0 (C-5’), 106.4 (C-1’), 126.3 in the presence of aqueous KOH (60%, 3  mL) in etha- (C-8), 127.6 (C-5), 128.0 (C-2’’’,C-6’’’), 128.2 (C-2’’,C-6’’), nol (15  mL). The product 5p was obtained in 69% yield. 128.4 (C-4’’’), 128.6 (C-4’’), 128.7 (C-3’’’,C-5’’’), 128.9 (C-3’’, mp 121–122  °C; R 0.59 (petroleum ether:EtOAc 4:1); C-5’’), 129.0 (C-6), 129.4 (C-3), 130.1 (C-1), 135.0 (C-2), −1 UV(MeOH) λ : 276, 331 nm; IR ν̃ [cm ]: 1618 (C = C), max 135.2 (C-4), 135.7 (C-1’’’), 138.2 (C-1’’), 139.0 (C-7), 161.6 1577(C = C), 1497, 1453, 1284, 1174, and 1026 (C-O); (C-2’), 165.6 (C-6’), 168.5 (C-4’), 192.0 (C-9). H NMR (CDCl , 300  MHz) δ 5.01 (2H, s, OCH ), 5.07 3 2 (2H, s, OCH ), 6.09 (1H, d, J = 2.1 Hz, H-5’), 6.21 (1H, d, Synthesis and analytical data J = 2.1 Hz, H-3’), 7.14 (3H, m, H-3, H-4, H-5), 7.28 (10H, of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑3,5‑dichlorochalcone (5o) m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.71 (1H, d, J = 16.2  Hz, H-8), 7.94 (1H, d, J = 16.2, H-7), 14.0 (1H, s); C NMR Cl (CDCl , 75  MHz) δ 70.3 (OCH ), 71.1 (OCH ), 92.8 3 2 2 (C-3’), 94.9 (C-5’), 106.6 (C-1’), 127.5 (C-8), 127.6 (C-3, 1 2 4' BnO OH C-5), 128.0 (C-2’’’, C-6’’’), 128.3 (C-2’’, C-6’’), 128.4 (C-4’’’), Cl 128.5 (C-4’’), 128.7 (C-3’’’, C-5’’’), 129.2 (C-3’’, C-5’’), 133.0 2' 9 (C-4), 134.8 (C-2, C-6), 135.0 (C-1), 135.3 (C-1’’’), 135.7 OBn O (C-1’’), 136.1 (C-7), 161.6 (C-2’), 165.5 (C-6’), 168.2 (C-4’), 192.5 (C-9). 5o According to general procedure I, 2,4-dibenzyloxy-6-hy- droxyacetophenone (3) (1  g, 2.8  mmol) was condensed Synthesis and analytical data with 3,5-dichlorobenzaldehyde (4o) (0.54  g, 2.8  mmol) of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑2,5‑dichlorochalcone (5q) in the presence of aqueous KOH (60%, 3  mL) in etha- nol (15  mL). The product 5o was obtained in 70% yield. Cl mp: 125–127  °C; R 0.66 (petroleum ether:EtOAc 4:1); −1 UV(MeOH) λ : 278, 329 nm; IR ν̃ [cm ]: 1620 (C = C), 1 2 max 4' BnO OH 1574 (C = C), 1552, 1366, 1201, 1156, and 1045 (C-O); 2' 9 Cl H NMR (CDCl , 300  MHz) δ 5.05 (2H, s, OCH ), 5.10 3 2 (2H, s, OCH ), 6.18 (1H, d, J = 3.3 Hz, H-5’), 6.22 (1H, d, OBn O J = 2.4  Hz, H-3’), 7.05 (2H, br s, H-2, H-6), 7.28 (1H, m, 5q H-4), 7.42 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.50 (1H, d, J = 15.9  Hz, H-8), 7.75 (1H, d, J = 15.6  Hz, H-7); C According to general procedure I, 2,4-dibenzyloxy- NMR (CDCl , 75 MHz) δ 70.4 (OCH ), 71.5 (OCH ), 92.7 3 2 2 6-hydroxyacetophenone (3) (0.7  g, 2.0  mmol) was con- (C-3’), 95.0 (C-5’), 106.4 (C-1’), 126.4 (C-8), 127.6 (C-2, densed with 2,5-dichlorobenzaldehyde (4q) (0.37  g, C-6), 127.6 (C-2’’’, C-6’’’), 128.0 (C-2’’, C-6’’), 128.4 (C-4’’’), 2.0 mmol) in the presence of aqueous KOH (60%, 3 mL) 128.7 (C-4’’), 128.9 (C-3’’’, C-5’’’), 129.0 (C-3’’, C-5’’), 130.1 in ethanol (15 mL). The product 5q was obtained in 96% (C-4), 135.0 (C-3, C-5), 135.2 (C-1), 135.7 (C-1’’’), 138.2 yield. mp 128–129  °C; R 0.63 (petroleum ether:EtOAc (C-1’’), 139.0 (C-7), 161.6 (C-2’), 165.6 (C-6’), 168.5 (C-4’), −1 4:1); UV(MeOH) λ : 275, 338  nm; IR ν̃ [cm ]: 1617 max 192.0 (C-9). Melaku et al. BMC Chemistry (2022) 16:36 Page 18 of 30 Synthesis of the dibenzylated flavanones 6a-r (C = C), 1574 (C = C), 1552, 1423, 1201, 1165, and 1045 General procedure II for synthesis of the dibenzylated (C-O); H NMR (CDCl , 300 MHz) δ 5.06 (2H, s, O CH ), 3 2 flavanones 6a‑r [16, 30] 5.10 (2H, s, OCH ), 6.17 (1H, d, J = 2.4  Hz, H-5’), 6.22 A solution of a chalcone 5 (1  mmol) in ethanol was (1H, d, J = 2.4  Hz, H-3’), 7.08 (1H, d, J = 2.4  Hz, H-6), treated with sodium acetate (8  mmol) and the solution 7.20 (1H, dd, J = 8.1, J = 2.4  Hz, H-4), 7.29 (1H, m, H-3), was refluxed for 48  h, cooled to room temperature and 7.30–7.41 (10H, m, H-2’’’– H-6’’’, H-2’’– H-6’’), 7.73 (1H, then diluted with water (60  mL/mmol). After extraction d, J = 15.9  Hz, H-8), 7.98 (1H, d, J = 15.9  Hz, H-7), 14.1 with CH Cl (3 × 30  mL/mmol), the combined organic (1H, s, 6’OH); C NMR (C DCl , 75 MHz) δ 70.3 (O CH ), 2 2 3 2 extracts were dried over anhydrous MgSO , filtered and 71.4 (OCH ), 92.7 (C-3’), 95.0 (C-5’), 106.4 (C-1’), 127.0 concentrated in vacuo. Purification was achieved by col - (C-8), 127.6 (C-2’’’, C-6’’’), 127.7 (C-2’’, C-6’’), 127.9 (C-4’’’), umn chromatography over silica gel using petroleum 128.3 (C-4’’), 128.7 (C-6), 128.8 (C-3’’’, C-5’’’), 130.3 (C-3’’, ether:EtOAc (4:1) as eluent to furnish the product 6 as a C-5’’), 130.9 (C-2), 131.1 (C-4), 132.7 (C-3), 133.4 (C-5), white solid. 134.9 (C-1), 135.0 (C-1’’’), 135.7 (C-1’’), 136.2 (C-7), 161.6 (C-2’), 165.6 (C-6’), 168.4 (C-4’), 192.1 (C-9). Synthesis and analytical data of 5,7‑dibenzyloxyflavanone (6a) Synthesis and analytical data of 2’,4’‑dibenzyloxy‑6’‑hydroxy‑3,4‑dichlorochalcone (5r) 4' BnO 79 O 2 4 Cl 1' 1 4 BnO 4' OH Cl 5 OBn O 2' 9 6a OBn O According to the general procedure II, 2′4’-dibenzy- 5r loxy-6’-hydroxychalcone (5a) (700  mg, 1.6  mmol) was According to general procedure I, 2,4-dibenzyloxy- dissolved in ethanol (75  mL). Sodium acetate (1.05  g, 6-hydroxyacetophenone (3) (0.7  g, 2.0  mmol) was con- 12.8 mmol) was added and the solution was refluxed for densed with 3,4-dichlorobenzaldehyde (4r) (0.37  g, 48  h. After workup, the product 6a was obtained as a 2.0 mmol) in the presence of aqueous KOH (60%, 3 mL) white solid in 50% yield. mp 119–120  °C; R 0.70 (petro- in ethanol (15 mL). The product 5r was obtained in 98% leum ether:EtOAc 4:1); UV(MeOH) λ : 270, 330  nm; max −1 yield. mp 130–131  °C; R 0.64 (petroleum ether:EtOAc IR ν̃ [cm ]: 1674 (conjugated C = O), 1599 (C = C), −1 1 4:1); UV(MeOH) λ : 276, 329  nm; IR ν̃ [cm ]: 1618 1551(C = C), 1062, and 1028 (C-O); H NMR (CDCl , max 3 (C = C), 1578 (C = C), 1420, 1202, 1160, and 1022(C- 300  MHz) δ 2.82 (1H, dd, J = 2.7, J = 16.5  Hz, H-3), 3.06 O); H NMR (CDCl , 300  MHz) δ 5.05 (2H, s, OCH ), (1H, dd, J = 13.2, J = 16.5 Hz, H-3), 5.0 (2H, s, O CH ), 5.2 3 2 2 5.11 (2H, s, OCH ), 6.18 (1H, d, J = 2.4  Hz, H-5’), 6.22 (2H, s, OCH ), 5.43 (1H, dd, J = 2.7, J = 13.2  Hz, H-2), 2 2 (1H, d, J = 2.4 Hz, H-3’), 6.74 (1H, dd, J = 8.4, J = 1.8  Hz, 6.25 (1H, s, H-8), 6.26 (1H, s, H-6), 7.59–7.30 (15H, m, H-6), 7.20 (1H, d, J = 8.1 Hz, H-5), 7.28 (1H, d, J = 2.4 Hz, H-2’–H-6’, H-2’’–H-6’’, H-2’’’–H-6’’’); C NMR (CDCl , H-2), 7.43 (10H, m, H-2’’’–H-6’’’, H-2’’– H-6’’), 7.54 (1H, 75  MHz) δ 45.7 (C-3), 70.2 (OCH ), 70.3 (OCH ), 2 2 d, J = 15.6  Hz, H-8), 7.79 (1H, d, J = 15.6  Hz, H-7); C 79.3 (C-2), 94.7 (C-6), 95.1 (C-8), 106.4 (C-10), 126.1 NMR (CDCl , 75  MHz) δ 70.2 (O CH ), 70.6 (OCH ), (C-2’,C-6’), 126.5 (C-2’’’, C-6’’’), 127.5 (C-2’’, C-6’’), 127.6 3 2 2 92.6 (C-3’), 95.0 (C-5’), 106.5 (C-1’), 126.7 (C-8), 127.6 (C-4’), 128.3 (C-4’’’), 128.5 (C-4’’), 128.6 (C-3’,C-5’), (C-6), 127.7 (C-2’’’, C-6’’’), 127.9 (C-2’’, C-6’’), 128.4 (C-4’’’), 128.7 (C-3’’’, C-5’’’), 128.8 (C-3’’, C-5’’), 135.7 (C-1’), 136.3 128.5 (C-4’’), 128.8 (C-2), 128.9 (C-3’’’, C-5’’’), 129.2 (C-3’’, (C-1’’’), 138.7 (C-1’’), 161.0 (C-9), 164.8 (C-5), 164.9 (C-7), C-5’’), 130.1 (C-5), 130.5 (C-4), 132.7 (C-3), 135.2 (C-1), 188.8 (C-4); MS (EI, 70  eV) m/z (%) 436 (19) [M] , 346 135.3 (C-1’’’), 135.7 (C-1’’), 139.4 (C-7), 161.9 (C-2’), 165.9 (7), 317 (4), 240 (3), 91 (100). (C-6’), 192.8 (C-9). M elaku et al. BMC Chemistry (2022) 16:36 Page 19 of 30 Synthesis and analytical data J = 16.5 H-3), 3.90 (1H, s, OMe), 3.92 (1H, s, OMe), of 5,7‑dibenzyloxy‑4’‑fluoroflavanone (6b) 5.04 (2H, s, OCH ), 5.10 (2H, s, OCH ), 5.38 (1H, dd, 2 2 2.7, J = 16.5  Hz, H-2), 6.24 (2H, br s, H-8), 6.90 (1H, 4' F br s, H-6), 7.0 (2H, m, H-2’, H-5’), 7.18 (1H, m, H-6’), 7.40–7.58 (10H, m, H-2’’’–H-6’’’, H-2’’–H-6’’); C NMR BnO O 2 1' (CDCl , 75 MHz) δ 45.6 (C-3), 55.8 (OMe), 55.9 (OMe), 70.2 (OCH ), 70.4 (OCH ), 79.3 (C-2), 94.7 (C-6), 95.1 2 2 (C-8), 106.6 (C-10), 109.4 (C-2’), 118.8 (C-5’), 126.5 OBn O (C-6’), 127.2 (C-2’’’, C-6’’’), 127.6 (C-2’’, C-6’’), 128.0 6b (C-4’’’), 128.3 (C-4’’), 128.6 (C-3’’’, C-5’’’), 128.7 (C-3’’, C-5’’), 131.1(C-1’), 135.7 (C-1’’’), 136.3 (C-1’’), 149.2 According to the general procedure II, 2′4’-dibenzy- (C-4’), 149.4 (C-3’), 158.2 (C-9), 161.0 (C-5), 164.9 loxydibenzyloxy-6’-hydroxy-4-fluorochalcone (5b) (C-7), 189.0 (C-4); MS (EI, 70  eV) m/z (%) 496 (16) (250  mg, 0.5  mmol) was dissolved in ethanol (60  mL). [M] , 406 (24), 348 (9), 227 (13), 151 (27), 91 (100); Sodium acetate (0.33  g, 4  mmol) was added and the HRMS (EI, M ) calculated for C H O 496.1886 solution was refluxed for 48  h. After workup, the 31 28 6 found 496.1880. product 6b was obtained in 48% yield. mp 76–78  °C; UV(MeOH) λ : 272, 335  nm; H NMR (CDCl , max 3 300 MHz) δ 2.69 (1H, dd, J = 2.7, J = 16.2 Hz, H-3), 3.00 Synthesis and analytical data of 5,7‑dibenzyloxy‑2′6’‑dimeth (1H, dd, J = 12.6, J = 16.2 Hz, H-3), 5.20 (2H, s, O CH ), 2 oxyflavanone (6d) 5.22 (2H, s, OCH ), 5.56 (1H, dd, J = 2.7, J = 12.6  Hz , H-2), 6.32 (1H, d, J = 2.4  Hz, H-8), 6.42 (1H, d, MeO 4' J = 2.4 Hz, H-6), 7.69–7.18 (14H, m, H-2’,H-6’, H-3’, H-5’, BnO O 2 H-2’’’–H-6’’’, H-2’’–H-6’’); C NMR (CDCl , 75  MHz) δ 1' 2' 46.2 (C-3), 70.7 (OCH ), 70.8 (OCH ), 79.2 (C-2), 95.6 OMe 2 2 (C-6), 95.7 (C-8), 107.1 (C-10), 115.9 (C-3’, C-5’), 116.2 OBn O (C-2’, C-6’), 127.4 (C-2’’’, C-6’’’), 128.1 (C-2’’, C-6’’), 128.5 6d (C-4’’’), 128.9 (C-4’’), 129.0 (C-3’’’, C-5’’’), 129.3 (C-3’’, C-5’’), 129.4 (C-1’), 137.4 (C-1’’’), 138.8 (C-1’’), 161.8 (d, According to the general procedure II, 2’,4’-dibeny- C-4’), 165.0 (C-9), 165.4 (C-5), 165.6 (C-7), 187.8 (C-4). loxy-6’-hydroxy-2,6-dimethoxychalcone (5d) (250  mg, 0.5  mmol) was dissolved in ethanol (75  mL). Sodium acetate (0.32 g, 4 mmol) was added and the solution was Synthesis and analytical data of 5,7‑dibenzyloxy‑3′4’‑dimeth refluxed for 48  h. The product 6d was obtained in 52%. oxyflavanone (6c) −1 mp 87  °C; UV(MeOH) λ : 275, 344  nm; IR ν̃ [cm ]: max 1667 (conjugated C = O), 1607 (C = C), 1572 (C = C), 4' OMe 1051, and 1025 (C-O); H NMR (CDCl , 300  MHz) BnO 79 O 2 δ 2.88–2.80 (2H, m, H-3), 3.7 (3H, s, OMe), 3.8 (3H, s, 1' 3' OMe OMe), 5.0 (2H, s, OCH ), 5.1 (2H, s, OCH ), 5.77 (1H, 2 2 5 dd, J = 4.8, J = 11.1  Hz, H-2), 6.24 (1H, d, J = 2.4  Hz, OBn O H-8), 6.28 (1H, d, J = 2.4  Hz, H-6), 6.83 (1H, br d, H-4), 6c 7. 20–7.29 (10H, m, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.60 (2H, br d, J = 7.5  Hz, H-3’, H-5’); C NMR (CDCl , 75  MHz) According to the general procedure II, 2’,4’-dibeny- δ 44.8 (C-3), 55.8 (OMe), 55.9 (OMe), 70.2 (OCH ), 70.3 loxy-6’-hydroxy-3,4-dimethoxychalcone (5c) (200  mg, (OCH ), 74.3 (C-2), 94.7 (C-6), 95.0 (C-8), 106.5 (C-10), 0.4  mmol) was dissolved in ethanol (70  mL). Sodium 111.5 (C-1’), 112.5 (C-3’), 113.5 (C-5’), 126.5 (C-2’’’, C-6’’’), acetate (0.26  g, 3.2  mmol) was added and the solution 127.5 (C-2’’, C-6’’), 128.3 (C-4’’), 128.5 (C-4’’), 128.6 (C-3’’’, was refluxed for 48  h. The product 6c was obtained C-5’’’), 128.7 (C-3’’, C-5’’), 128.9 (C-4’), 135.8 (C-4’’’), 136.6 in 40% yield. mp 91–92  °C; UV(MeOH) λ : 276, max (C-4’’), 149.9 (C-2’), 153.5 (C-6’), 161.1 (C-5), 164.1 (C-9), −1 345  nm; IR ν̃ [cm ]: 1672 (conjugated C = O), 1595 165.2 (C-7), 189.5 (C-4); MS (EI, 70 eV) m/z (%) 496 (24) (C = C), 1571 (C = C), 1257, 1117, 1075, and 1012 [M] , 465 (14), 405 (29), 348 (9), 91 (100); HRMS (EI, (C-O); H NMR(CDCl , 300  MHz) δ 2.77 (1H, dd, 3 M ) calculated for C H O 496.1886 found 496.1870. 31 28 6 J = 2.7, J = 16.5  Hz, H-3), 3.08 (1H, dd, J = 13.2  Hz , Melaku et al. BMC Chemistry (2022) 16:36 Page 20 of 30 Synthesis and analytical data 7.76  mmol) was added and the solution was refluxed of 5,7‑dibenzyloxy‑4’‑chloroflavanone (6e) for 48  h. The product 6f was obtained in 36% yield. mp −1 89–91  °C; UV(MeOH) λ : 273, 338  nm; IR ν̃ [cm ]: max 4' Cl 1672 (conjugated C = O), 1603 (C = C), 1569 (C = C), 1068, and 1024 (C-O); H NMR (CDCl , 300  MHz) δ BnO O 2 1' 2.82 (1H, dd, J = 13.2  Hz, J = 16.5 Hz, H-3), 2.98 (1H, dd, J = 16.2  Hz, J = 2.7  Hz, H-3), 5.06 (2H, s, OCH ), 5.17 (2H, s, OCH ), 5.8 (1H, dd, J = 10.5  Hz, J = 2.4  Hz, H-2), OBn O 6.27 (2H, br s, H-6, H-8), 7.20–7.70 (14H, m, H-3’–H-6’, 6e H-2’’’–H-6’’’, H-2’’–H-6’’); C NMR (CDCl , 75  MHz) δ 44.6 (C-3), 70.3 (O CH ), 70.4 (OCH ), 78.4 (C-2), 94.7 2 2 According to the general procedure II, 2’,4’-dibenzyloxy- (C-6), 95.3 (C-8), 106.4 (C-10), 121.5 (C-2), 126.5 (C-2’’’, 6’-hydroxy-4-chlorochalcone (5e) (300  mg, 0.6  mmol) C-6’’’), 127.3 (C-2’’, C-6’’), 127.5 (C-4’’’), 127.6 (C-4’’), 127.9 was dissolved in ethanol (60 mL). Sodium acetate (0.39 g, (C-5’), 128.3 (C-3’’’, C-5’’’), 128.5 (C-3’’, C-5’’), 128.7 (C-6’), 4.8  mmol) was added and the solution was refluxed for 129.8 (C-4’), 132.9 (C-3’), 135.7 (C-1’’’), 136.3 (C-1’’), 48 h. The product 6e was obtained in 40% yield. mp 129– 138.3 (C-1’’), 161.3 (C-9), 164.9 (C-5), 165.0 (C-7), 188.4 131 °C; R 0.56 (petroleum ether:EtOAc 4:1); UV(MeOH) −1 (C-4); MS (EI, 70  eV) m/z (%) 516 (16) [M + 2] , 514 λ : 271, 336  nm; IR ν̃ [cm ]:1671 (conjugate C = O), max [M] (18), 435 (12), 425 (14), 423 (12), 256 (4), 91 (100); 1606 (C = C), 1571(C = C), 1064, and 1035 (C-O); H HRMS (EI, M ) calculated for C H O Br 514.0780 29 23 4 NMR (CD COCD , 300 MHz) δ 2.75 (1H, dd, J = 3.0  Hz, 3 3 found 514.0777. J = 16.2  Hz, H-3), 3.00 (1H, dd, J = 12.6  Hz, J = 16.2  Hz, H-3), 5.20 (1H, s, OCH ), 5.21 (1H, s, OCH ), 5.57 (1H, 2 2 Synthesis of 5,7‑dibenyloxy‑4‑(trifluoromethyl)flavanone (6g) dd, J = 3  Hz, J = 12.6  Hz, H-2), 6.23 (1H, d, J = 2.2  Hz, H-8), 6.43 (1H, d, J = 2.2  Hz, H-6), 7.40 (10H, m, H-2’’’– 4' CF H-6’’’, H-2’’–H-6’’), 7.59 (2H, d, J = 7.0, H-3’,H-5’), 7.6 (2H, d, J = 7.0  Hz, H-2’, H-6’); C NMR (CD COCD , 79 BnO O 2 3 3 1' 75  MHz) δ 46.1 (C-3), 70.7 (O CH ), 70.8 (O CH ), 79.1 2 2 (C-2), 95.7 (C-6), 95.8 (C-8), 107.0 (C-10), 127.4 (C-2’’’, OBn O C-6’’’), 128.1 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.9 (C-4’’), 129.0, (C-3’’’, C-5’’’) 129.1 (C-3’’, C-5’’), 129.3 (C-2’, C-6’), 6g 129.4 (C-3’, C-5’), 134.4 (C-4’), 137.4 (C-1’’’), 138.0 (C-1’’), According to the general procedure II, 2’,4’-dibenyloxy- 139.3 (C-1’), 161.9 (C-9), 165.3 (C-5), 165.5 (C-7), 188.8 6’-hydroxy-4-(trifluoromethyl)chalcone (5g) (500  mg, (C-4); MS (EI,70 eV) m/z (%) 470 (11) [M] , 379 (8), 348 0.97  mmol) was dissolved in ethanol (75  mL). Sodium (11), 257 (6), 180 (4), 91 (100); HRMS (EI, M ) calculated acetate (0.64  g, 7.8  mmol) was added and the solution for C H O Cl 470.1285 found 470.1260. 29 23 4 was refluxed for 48  h. The product 6g was obtained in 8% yield. H NMR (CD COCD , 300  MHz) δ 2.78 (1H, 3 3 Synthesis and analytical data dd, J = 12.5 Hz, J = 3.3 Hz, H-3), 3.00 (1H, dd, J = 12.5 Hz, of 5,7‑dibenzyloxy‑2’‑bromoflavanone (6f ) J = 13.3 Hz, H-3), 5.20 (2H, s, OCH ), 5.23 (2H, s, OCH ), 2 2 5.68 (1H, dd, J = 12.5  Hz, J = 2.5  Hz, H-2), 6.36 (1H, d, 4' J = 2.2  Hz, H-8), 6.44 (1H, d, J = 2.2  Hz, H-6), 7.31–7.77 BnO 79 O 2 1' (14H, m, H-2’, H-3’, H-5’, H-6’, H-2’’’–H-6’’’, H-2’’–H-6’’); Br C NMR (CD COCD , 75  MHz) δ 45.7 (C-3), 70.2 3 3 (OCH ), 70.9 (O CH ), 78.7 (C-2), 95.7 (C-6), 95.6, 107.2 2 2 OBn O (C-10), 126.3 (CF ), 127.5 (C-3’, C-5’), 127.7 (C-2’’’, C-6’’’), 6f 128.2 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.9 (C-4’’), 129.0 (C-2’, C-6’), 129.3 (C-3’’’, C-5’’’), 129.4 (C-3’’, C-5’’), 132.0 (C-4’), According to the general procedure II, 2’,4’-dibenyloxy- 137.4 (C-1’’’), 138.0 (C-1’’), 144.8 (C-1’), 161.9 (C-9), 165.2 6’-hydroxy-2-bromochalcone (5f) (500  mg, 0.97  mmol) (C-5), 165.8 (C-7), 187.0 (C-4). was dissolved in ethanol (75 mL). Sodium acetate (0.64 g, M elaku et al. BMC Chemistry (2022) 16:36 Page 21 of 30 1154, and 1025 (C-O); H NMR (CDCl , 300  MHz) δ Synthesis and analytical data of 5,7‑dibenzyloxy‑3′4’5’‑trime 3 2.88 (1H, dd, J = 12.9  Hz, J = 3.3  Hz, H-3), 2.98 (1H, dd, thoxyflavanone (6h) J = 12.9  Hz, J = 16.8  Hz, H-3), 5.05 (2H, s, O CH ), 5.17 (2H, s, OCH ), 5.44 (1H, dd, J = 12.6, J = 3.3  Hz, H-2), OMe 4' OMe 6.21 (2H, br s, H-6 and H-8), 7.28–7.66 (10H, m, H-2’’’– H-6’’’, H-2’’–H-6’’), 7.61 (2H, d, J = 7.5 Hz, H-2’ and H-3’), BnO O 2 1' OMe 7.72 (1H, d, J = 9.3 Hz, H-5’); C NMR (CDCl , 75 MHz) δ 44.5 (C-3), 70.3 (OCH ), 70.4 (OCH ), 76.2 (C-2), 94.7 2 2 (C-6), 95.2 (C-8), 106.4 (C-2’, C-6’), 107.4 (C-10), 126.5 OBn O (C-2’’’, C-6’’’), 127.1 (C-2’’, C-6’’), 127.3 (C-4’’’), 127.5 6h (C-4’’), 128.5 (C-3’’’, C-5’’’), 129.3 (C-3’’, C-5’’), 131.7 (C-1’), 135.7 (C-4’), 136.3 (C-1’’’), 136.7 (C-1’’), 161.6 (C-9), 164.9 According to the general procedure II, 2’,4’-dibenyloxy- (C-5), 164.9 (C-7), 188.5 (C-4); MS (EI, 70  eV) m/z (%) 6’-hydroxy-3,4,5-trimethoxychalcone (5h) (400  mg, + + 534 (10) [M + 2] , 532 (9) [M] , 442 (6), 359 (4), 256 (3), 0.76  mmol) was dissolved in ethanol (70  mL). Sodium 91 (100); HRMS (EI, M ) calculated for C H O BrF 29 22 4 acetate (0.5  g, 6.08  mmol) was added and the solution 532.0685 found 532.0671. was refluxed for 48  h. The product 6h was obtained in 38% yield. mp 84–86  °C; UV(MeOH) λ : 274, 351  nm; max Synthesis and analytical data −1 IR ν̃ [cm ]: 1671 (conjugated C = O), 1607 (C = C), of 5,7‑dibenzyloxy‑2’‑chloroflavanone (6l) 1569, 1116, and 1032 (C-O); H NMR (CD COCD , 3 3 300  MHz) δ 2.68 (1H, dd, J = 16.5  Hz, J = 3  Hz, H-3), 3.06 (1H, dd, J = 12.9  Hz, J = 16.5  Hz, H-3), 3.75 (3H, s, BnO 79 O 2 3' OMe), 3.85 (6H, s, OMe), 5.19 (2H, s, O CH ), 5.22 (2H, 1' s, OCH ), 5.44 (1H, dd, J = 12.9, J = 3 Hz, H-2), 6.32 (1H, 4 Cl d, J = 2.2  Hz, H-8), 6.41 (1H, d, J = 2.2  Hz), 7.28–7.66 OBn O (12H, m, H-2’, H-6’, H-2’’’–H-6’’’, H-2’’–H-6’’); C NMR 6l (CD COCD , 75  MHz) δ 46.3 (C-3), 56.3 (OMe), 60.5 3 3 (OMe), 70.3 (O CH ), 70.8 (O CH ), 80.3 (C-2), 95.7 (C-6), 2 2 According to the general procedure II, 2’,4’-dibenzyloxy- 95.8 (C-8), 104.8 (C-2’, C-6’), 107.4 (C-10), 127.4 (C-2’’’, 6’-hydroxy-2-chlorochalcone (5l) (1.3  g, 2.9  mmol) was C-6’’’), 128.2 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.9 (C-4’’), 129.0 dissolved in ethanol (100  mL). Sodium acetate (1.9  g, (C-3’’’, C-5’’’), 129.3 (C-3’’, C-5’’), 135.7 (C-1’), 137.5 (C-4’), 23  mmol) was added and the solution was refluxed 138.1 (C-1’’’), 139.2 (C-1’’), 154.4 (C-3’, C-5’), 161.6 (C-9), for 48  h. The product 6l was obtained in 42% yield. 165.5 (C-5), 165.6 (C-7), 188.7 (C-4); MS (EI, 70 eV) m/z mp 127–128  °C; R 0.55 (petroleum ether:EtOAc 4:1); (%) 526 (40) [M] , 435 (28), 407(16), 257 (16), 181 (26), −1 UV(MeOH) λ : 272, 337  nm; IR ν̃ [cm ]: 1672 (con- max 91 (100). jugated C = O), 1604 (C = C), 1570 (C = C), 1162, 1068, and 1033 (C-O); H NMR (CDCl , 300  MHz) δ 2.85 Synthesis and analytical data (1H, dd, J = 16.5, J = 3.3  Hz, H-3), 2.94 (1H, dd, J = 15.5, of 5,7‑dibenzyloxy‑2’‑bromo‑4’‑fluoroflavanone (6i) J = 3.3 Hz, H-3), 5.06 (2H, s, O CH ), 5.17 (2H, s, O CH ), 2 2 5.80 (1H, dd, J = 12.9 J = 3.3 Hz, H-2), 6.27 (2H, br s, H-6, 4' H-8), 7.38 (11H, m, H-5’, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.58 BnO O 2 (1H, br s, H-6’), 7.60 (1H, d, J = 9.3 Hz, H-4’), 7.71 (1H, d, 1' J = 9.3 Hz, H-3’); C NMR (C DCl , 75 MHz) δ 44.5 (C-3), Br 3 570.3 (OCH ), 70.4 (OCH ), 76.4 (C-2), 94.7 (C-6), 95.2 2 2 OBn O (C-8), 106.4 (C-10), 126.3 (C-5’), 127.3 (C-2’’’, C-6’’’), 127.5 6i (C-2’’, C-6’’), 127.6 (C-4’’’), 128.3 (C-4’’), 128.5 (C-6’), 128.7 (C-3’’’, C-5’’’), 128.8 (C-3’’, C-5’’), 129.4 (C-4’), 129.6 (C-3’), According to the general procedure II, 2’,4’-dibenyloxy- 131.7 (C-2’), 135.7 (C-1’), 136.3 (C-1’’’), 136.7 (C-1’’), 6’-hydroxy-2-bromo-4-fluorochalcone (5i) (400  mg, 161.1 (C-9), 164.9 (C-5), 165.2 (C-7), 188.5 (C-4); MS 0.75  mmol) was dissolved in ethanol (70  mL). Sodium + (EI, 70 eV) m/z (%) 470 (16) [M] , 435 (4), 379 (11), 255 acetate (0.49 g, 6.0 mmol) was added and the solution was + (4), 165 (3); HRMS (EI, M ) calculated for C H O Cl 29 23 4 refluxed for 48  h. The product 6i was obtained as 48% 470.1285 found 470.1264. −1 yield. IR ν̃ [cm ]: 1668 (conjugated C = O), 1603 (C = C), Melaku et al. BMC Chemistry (2022) 16:36 Page 22 of 30 Synthesis and analytical data (C = C), 1570 (C = C), 1430, 1163, 1116, and 1040 (C-O); of 5,7‑dibenzyloxy‑3’‑chloroflavanone (6m) H NMR (CDCl , 300  MHz) δ 2.79 (1H, dd, J = 12.9  Hz, J = 16.5  Hz, H-3), 2.93 (1H, dd, J = 16.5  Hz, J = 3.6  Hz, H-3), 5.08 (2H, s, OCH ), 5.17 (2H, s, OCH ), 5.77 (1H, 2 2 3' BnO O 2 dd, J = 12.9  Hz, J = 3.0  Hz, H-2), 6.25 (1H, d, J = 2.4  Hz, 1' Cl H-8), 6.27 (1H, d, J = 2.4  Hz, H-6), 7.39 (10H, m, H-2’’’– H-6’’’, H-2’’–H-6’’), 7.59 (2H, d, J = 7.2 Hz, H-3, H-5’), 7.64 OBn O (1H, d, J = 8.4  Hz, H-3’); C-NMR (CDCl , 75  MHz) δ 6m 44.4 (C-3), 70.3 (O CH ), 70.4 (OCH ), 75.7 (C-2), 94.7 2 2 (C-6), 95.3 (C-8), 106.4 (C-10), 126.5 (C-5’), 127.5 (C-2’’’, According to the general procedure II, 2’,4’-dibenzyloxy- C-6’’’), 127.7 (C-3’’, C-6’’), 128.1 (C-4’’’), 128.3 (C-4’’), 128.5 6’-hydroxy-3-chlorochalcone (5m) (0.8 g, 1.8 mmol) was (C-3’’’, C-5’’’), 128.7 (C-3’’, C-5’’), 128.9 (C-3’), 129.4 (C-6’), dissolved in ethanol (100  mL). Sodium acetate (1.2  g, 132.3 (C-2’), 134.7 (C-4’), 135.4 (C-1’), 135.6 (C-1’’’), 136.2 14.4  mmol) was added and the solution was refluxed (C-1’’), 161.1 (C-9), 164.6 (C-5), 164.9 (C-7), 188.0 (C-4); + + for 48 h. The product 6m was obtained in 50% yield. mp MS (EI, 70 eV) m/z (%) 506 (6) [M + 2] , 504 (12) [M] , 129 °C; R 0.56 (petroleum ether:EtOAc 4:1); UV(MeOH) 470 (3), 413 (7), 256 (3), 199 (6), 91 (100); HRMS (EI, M ) −1 λ : 274, 334 nm; IR ν̃ [cm ]: 1673 (conjugated C = O), calculated for C H O Cl 504.0895 found 504.0908. max 29 22 4 2 1604 (C = C), 1571 (C = C), 1428, 1163, 1117, and 1025 (C-O); H NMR (CDCl , 300  MHz) δ 2.78 (1H, dd, Synthesis and analytical data J = 16.5  Hz, J = 3  Hz, H-3), 2.98 (1H, dd, J = 16.5  Hz, of 5,7‑dibenzyloxy‑3’,5’‑dichloroflavanone (6o) J = 13.2 Hz, H-3), 5.06 (2H, s, OCH ), 5.16 (2H, s, OCH ), 2 2 5.40 (1H, dd, J = 3 Hz, J = 12.9 Hz, H-2), 6.25 (2H, s, H-6, Cl H-8), 7.36 (11H, m, H-6’, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.49 (1H, br s, H-2’), 7.59 (2H, d, J = 7.8  Hz, H-4’, H-5’); C BnO O 2 1' 3' NMR (CDCl , 75  MHz) δ 45.6 (C-3), 70.3 (OCH ), 70.3 Cl 3 2 (OCH ), 78.4 (C-2), 94.7 (C-6), 95.2 (C-8), 106.3 (C-10), 124.1 (C-6’), 126.2 (C-2’), 126.4 (C-2’’’, C-6’’’), 127.5 (C-2’’, OBn O C-6’’), 127.6 (4’’’), 128.1 (C-4’’), 128.3 (C-4’), 128.5 (C-3’’’, 6o C-6’’’), 128.7 (C-3’’, C-5’’), 130.0 (C-5’), 134.7 (C-3’), 135.6 (C-1’’’), 136.2 (C-1’’), 140.8 (C-1’), 161.0 (C-9), 164.5 According to the general procedure II, 2’,4’-dibenzyloxy- (C-5), 164.9 (C-7), 188.1 (C-4); MS (EI, 70  eV) m/z (%) 6’-hydroxy-3,5-dichlorochalcone (5o) (0.7  g, 1.4  mmol) 470 (26) [M] , 379 (9), 348 (12), 306 (6), 257 (5), 215 was dissolved in ethanol (100 mL). Sodium acetate (0.9 g, (6), 91 (100); HRMS (EI, M ) calculated for C H O Cl 29 23 4 11.2  mmol) was added and the solution was refluxed for 470.1285 found 470.1270. 48 h. The product 6o was obtained in 36% yield. mp 115– 116  °C; R 0.58 (petroleum ether:EtOAc 4:1); UV(MeOH) −1 λ : 271, 338  nm; IR ν ̃ [cm ]: 1668 (conjugated C = O), max Synthesis and analytical data 1614 (C = C), 1572 (C = C), 1374, 1216, 1068, and 1018 of 5,7‑dibenzyloxy‑2’,4’‑dichloroflavanone (6n) (C-O); H NMR (CDCl , 300  MHz) δ 2.80 (1H, dd, J = 3.3  Hz, J = 16.5  Hz, H-3), 2.95 (1H, dd, J = 16.5  Hz, 4' Cl J = 12.9 Hz), 5.06 (2H, s, OCH ), 5.16 (2H, s, OCH ), 5.36 2 2 BnO O 2 (1H, dd, J = 12.9  Hz, J = 3.3 Hz, H-2), 6.21 (2H, br s, H-6, 1' H-8), 7.33 (11H, m, H-4’, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.57 Cl (2H, d, J = 7.5 Hz, H-2’, H-6’); C NMR (C DCl , 75 MHz) 5 3 OBn O δ 45.5 (C-3), 70.4 (OCH ), 70.4 (OCH ), 77.7 (C-2), 94.7 2 2 6n (C-6), 95.5 (C-8), 106.3 (C-10), 124.5 (C-2’, C-6’), 126.5 (C-2’’’, C-6’’’), 127.6 (C-2’’, C-6’’), 127.7 (C-4’’’), 128.4 (C-4’’), According to the general procedure II, 2’,4’-dibenzyloxy- 128.6 (C-3’’’, C-5’’’), 128.7 (C-3’’, C-5’’), 128.8 (C-4’), 135.4 6’-hydroxy-2,4-dichlorochalcone (5n) (0.6  g, 1.2  mmol) (C-3’, C-5’), 135.6 (C-1’’’), 136.2 (C-1’’), 142.2 (C-1’), 161.1 was dissolved in ethanol (100 mL). Sodium acetate (0.8 g, (C-9), 164.2 (C-5), 165.0 (C-7), 187.6 (C-4); MS (EI, 70 eV) + + 9.6  mmol) and the solution was refluxed for 48  h. The m/z (%) 506 (9) [M + 2] , 504 (16) [M] , 413(7), 384 (4), product 6n was obtained in 54% yield. mp 117–118  °C; 359 (3), 303 (4), 256 (4), 199 (5), 91 (100); HRMS (EI, M ) R 0.57 (petroleum ether:EtOAc 4:1); UV(MeOH) λ : calculated for C H O Cl 504.0895 found 504.0889. f max 29 22 4 2 −1 275, 339 nm; IR ν̃ [cm ]: 1669 (conjugated C = O), 1604 M elaku et al. BMC Chemistry (2022) 16:36 Page 23 of 30 Synthesis and analytical data 11.2 mmol) was added and the solution was refluxed for of 5,7‑dibenzyloxy‑2’,6’‑dichloroflavanone (6p) 48 h. The product 6q was obtained in 43% yield. mp 121– 124 °C; R 0.55 (petroleum ether:EtOAc 4:1); UV(MeOH) −1 Cl λ : 273, 338  nm; IR ν̃ [cm ]:1671 (conjugated C = O), max 1606 (C = C), 1570 (C = C), 1466, 1209, 1108, and 1032 BnO O 2 1' 2' (C-O); H NMR (CDCl , 500  MHz) δ 2.78 (1H, dd, Cl J = 13.5  Hz, J = 17.0  Hz, H-3), 2.96 (1H, dd, J = 16.5  Hz, J = 2.5 Hz, H-3), 5.07 (2H, s, O CH ), 5.17 (2H, s, O CH ), 2 2 OBn O 5.75 (1H, dd, J = 13.5  Hz, J = 3  Hz, H-2), 6.28 (1H, d, 6p J = 2  Hz, H-8), 6.29 (1H, d, J = 2  Hz, H-6), 7.29 (1H, dd, J = 8.5  Hz, J = 2.5  Hz, H-4’), 7.39 (10H, m, H-2’’’–H-6’’’, According to the general procedure II, 2’,4’-diben- H-2’’–H-6’’), 7.59 (1H, d, J = 7.5  Hz, H-3’), 7.72 (1H, d, zyloxy-6’-hydroxy-2,6-dichlorochalcone (5p) (0.7  g, J = 2.5  Hz, H-6’); C NMR (C DCl , 125  MHz) δ 44.4 1.4  mmol) was dissolved in ethanol (100  mL). Sodium (C-3), 70.4 (OCH ), 70.4 (OCH ), 75.9 (C-2), 94.8 (C-6), 2 2 acetate (0.9  g, 11.2  mmol) was added and the solution 95.5 (C-8), 106.4 (C-10), 126.5 (C-2’’’, C-6’’’), 126.5 (C-2’’, was refluxed for 48  h. The product 6p was obtained C-6’’), 127.2 (C-4’’’), 127.5 (C-4’’), 127.7 (C-6’), 128.4 in 32% yield. mp 109–111  °C; R 0.53 (petroleum (C-3’’’, C-5’’’), 128.6 (C-3’’, C-5’’), 129.4 (C-4’), 129.6 (C-3’), ether:EtOAc 4:1); UV(MeOH) λ : 275, 339  nm; IR ν̃ max −1 130.8 (C-2’), 133.4 (C-5’), 135.7 (C-1’), 136.2 (C-1’’’), [cm ]: 1663 (conjugated C = O), 1607 (C = C), 1570 138.5 (C-1’’), 161.2 (C-9), 164.5 (C-5), 165.0 (C-7), 187.9 (C = C), 1241, 1055, and 1029 (C-O); H NMR (C DCl , (C-4); MS (EI, 70 eV) m/z (%) 506 (34) [M + 2] , 504 (49) 500  MHz) δ 2.56 (1H, dd, J = 3.3  Hz , J = 16.5  Hz , H-3), [M] , 469 (16), 413 (31), 385 (9), 359 (12), 305 (15), 256 3.68 (1H, dd, J = 14.4  Hz , J = 16.8  Hz, H-3), 5.0 (2H, (10), 199 (14), 91 (100); HRMS (EI, M ) calculated for s, OCH ), 5.13 (2H, s, OCH ), 6.19 (1H, d, J = 1.8  Hz , 2 2 C H O Cl 504.0895 found 504.0863. 29 22 4 2 H-8), 6.20 (1H, d, J = 1.8  Hz, H-6), 7.20 (1H, m, H-4), 7.35 (10H, m, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.56 (2H, d, J = 7.5  Hz, H-3’, H-5’); C NMR (C DCl , 125  MHz) δ Synthesis and analytical data 40.5 (C-3), 70.3 (OCH ), 70.4 (OCH ), 75.7 (C-2), 94.5 2 2 of 5,7‑dibenzyloxy‑3’,4’‑dichloroflavanone (6r) (C-6), 95.1 (C-8), 106.2 (C-10), 126.5 (C-3’, C-5’), 127.6 (C-2’’’, C-6’’’), 128.3 (C-2’’, C-6’’), 128.5 (C-4’’’), 128.6 4' Cl (C-4’’), 129.6 (C-3’’’, C-5’’’), 130.1 (C-3’’, C-5’’), 132.4 BnO O 2 (C-4’), 132.5 (C-2’, C-6’), 135.1 (C-1’), 135.7 (C-1’’’), 1' Cl 3' 136.3 (C-1’’), 161.2 (C-9), 164.6 (C-5), 164.9 (C-7), 4 188.2 (C-4); MS (EI, 70  eV) m/z (%) 506 (10) [M + 2] , OBn O 504 (17) [M] , 469 (6), 413 (11), 359 (14), 305 (6), 256 6r (4), 199 (16), 91 (100); HRMS (EI, M ) calculated for C H O Cl 504.0895 found 504.0881. 29 22 4 2 According to the general procedure II, 2’,4’-dibenzyloxy- 6’-hydroxy-2,6-dichlorochalcone (5r) (0.9  g, 1.8  mmol) Synthesis and analytical data was dissolved in ethanol (100 mL). Sodium acetate (1.2 g, of 5,7‑dibenzyloxy‑2’,5’‑dichloroflavanone (6q) 14.4 mmol) was added and the solution was refluxed for 48 h. The product 6r was obtained in 44% yield. mp 123– Cl 125 °C; R 0.54 (petroleum ether:EtOAc 4:1); UV(MeOH) −1 λ : 271, 337 nm; IR ν̃ [cm ]: 1666 (conjugated C = O), max BnO 79 O 2 1606 (C = C), 1431, 1165, 1118, and 1028 (C-O); H NMR 1' 2' (CDCl , 500 MHz) δ 2.81 (1H, dd, J = 16.5 Hz, J = 3.0 Hz, Cl 5 H-3), 2.96 (1H, dd, J = 13  Hz, J = 16.5 Hz, H-3), 5.06 (2H, OBn O s, OCH ), 5.16 (2H, s, OCH ), 5.38 (1H, dd, J = 13  Hz, 2 2 6q J = 3  Hz, H-3), 6.24 (1H, d, J = 1.5  Hz, H-8), 6.26 (1H, d, J = 1.5  Hz, H-6), 7.28 (1H, d, J = 2  Hz, H-2’), 7.29–7.39 According to the general procedure II, 2’,4’-dibenzyloxy- (10H, m, H-2’’’–H-6’’’, H-2’’–H-6’’), 7.50 (1H, d, J = 8.5 Hz, 6’-hydroxy-2,6-dichlorochalcone (5q) (0.7  g, 1.4  mmol) H-5’), 7.58 (1H, dd, J = 7.5  Hz, J = 2  Hz, H-6’); C NMR was dissolved in ethanol (100 mL). Sodium acetate (0.9 g, Melaku et al. BMC Chemistry (2022) 16:36 Page 24 of 30 Synthesis and analytical data of 2’,6’‑dimethoxyflavanone (CDCl , 125  MHz) δ 45.5 (C-3), 70.3 (O CH ), 70.4 3 2 (7b) (OCH ), 77.8 (C-2), 94.7 (C-6), 95.4 (C-8), 106.3 (C-10), 125.2 (C-6’), 126.5 (C-2’’’, C-6’’’), 127.5 (C-2’’, C-6’’), 127.6 MeO (C-4’’’), 127.7 (C-4’’), 128.1 (C-2’), 128.4 (C-3’’’, C-5’’’), 4' 128.5 (C-3’’, C-5’’), 128.6 (C-5’), 128.7 (C-4’), 130.7 (C-3’), HO O 2 1' 135.2, (C-1’), 136.2 (C-1’’’), 139.0 (C-1’’), 161.1 (C-9), 2' OMe 164.3 (C-5), 165.0 (C-7), 187.8 (C-4); MS (EI, 70 eV) m/z + + (%) 506 (1) [M + 2] , 504 (2) [M] , 413 (1), 348 (17), 306 OH O (7), 257 (7), 215 (8), 180 (7), 91 (100); HRMS (EI, M ) cal- 7b culated for C H O Cl 504.0895 found 504.0871. 29 22 4 2 Following general procedure III, 5,7-dibenzyloxy-2’,6’- dimethoxyflavanone (6d) (60  mg, 0.12  mmol) was Synthesis of flavanones 7a-l by deprotection debenzylated and the crude product was purified by of the dibenzylated flavanones 6a-r column chromatography over silica gel with petroleum General Procedure III for the synthesis of 7a‑l ether:EtOAc (7:3) as the eluent to furnish 7b in 78% yield A dibenzylated flavanone 6 was dissolved in sufficient as a white solid. mp 193–194  °C; UV(MeOH) λ : 291, max EtOAc:EtOH (1:1) to produce a 0.01  M solution. The 343  nm; H NMR (DMSO-d , 500  MHz) δ 2.78, (1H, resulting solution was subjected to hydrogenolysis using dd, J = 3  Hz, J = 17  Hz, H-3), 3.05 (1H, dd, J = 17.5  Hz, a H-Cube Pro over 10%Pd/C at a flow rate of 1 mL/min at J = 13  Hz, H-3), 3.78 (3H, s, OMe), 3.83 (3H, s, OMe), 70 °C and 1 bar. The solution was concentrated in vacuo 5.75 (1H, dd, J = 2.5  Hz, J = 12.5  Hz, H-2), 5.98 (1H, d, and purified by column chromatography on silica gel. J = 2  Hz, H-6), 6.02 (1H, d, J = 2.5  Hz, H-8), 6.92 (1H, dd, J = 3  Hz, J = 9  Hz, H-3’), 7.01 (1H, d, J = 9  Hz, H-4’), Synthesis and analytical data of pinocembrin (7a) 7.81 (1H, d, J = 3 Hz, H-5’), 12.16 (1H, s, 5-OH); MS (EI, 70  eV) m/z (%) 316 (56) [M] , 285 (100), 179 (11), 164 4' (24), 151 (28), 121 (25), 77 (21), 73 (24), 60 (33). HO O 2 1' Synthesis and analytical data of 4’‑chloroflavanone (7c) Following general procedure III, 5,7-dibenzyloxy- OH O 4’-chloroflavanone (6c) (180 mg, 0.38 mmol) was deben - 7a zylated and the crude product was purified by column chromatography over silica with petroleum ether:EtOAc Following general procedure III, 5,7-dibenzyloxyfla - (7:3) as the eluent to furnish 7c as a white solid in 91% vanone (6a) (100  mg, 0.23  mmol) was debenzylated yield. MS (EI, 70 eV) m/z (%) 290 (33) [M] , 256 (73), 255 and the crude product was purified by column chro - (100), 179 (73), 152 (51), 124 (27). matography over silica gel with petroleum ether:EtOAc (7:3) as the eluent to furnish 7a as white solid in 93% Synthesis and analytical data of 3’,4’,5’‑trimetoxyflavanone yield. mp 193–194  °C; R 0.68 (petroleum ether:EtOAc −1 (7d) 4:1); UV(MeOH) λ : 290, 335  nm; IR ν̃ [cm ]: 1628 max (C = C), 1581 (C = C), 1453, 1300, 1085, and 1063 (C-O); 1 OMe H NMR (C D OD, 300  MHz) δ 2.66 (1H, dd, J = 3.3  Hz, OMe 4' J = 17.4  Hz, H-3), 2.92 (1H, dd, J = 12.9  Hz, J = 17.1  Hz, H-3), 5.25 (2H, dd, J = 3  Hz, J = 12.9  Hz, H-2), 5.96 (1H, 79 HO O 2 1' OMe d, J = 2.4  Hz, H-6), 6.01 (1H, d, J = 2.1  Hz, H-8), 7.30 (5H, m, H-2’–H-6’); C NMR (CD OD, 75  MHz) δ 42.7 3 5 (C-3), 78.9 (C-2), 95.6 (C-8), 96.2 (C-6), 101.6 (C-10), OH O 125.4 (C-3’, C-5’), 128.5 (C-2’, C-4’, C-6’), 137.3 (C-1’), 7d 162.5 (C-9), 163.9 (C-5), 167.4 (C-7), 195.4 (C-4); MS (EI, 70  eV) m/z (%) 256 (100) [M] , 179 (66), 152 (46), 124 Following general procedure III, 5,7-dibenzyloxy- (16). 3’,4’,5’-trimetoxyflavanone (6h) (60  mg, 0.12  mmol) M elaku et al. BMC Chemistry (2022) 16:36 Page 25 of 30 was debenzylated and the crude product was purified J = 12.3  Hz, H-2), 5.83 (1H, d, J = 3  Hz, H-6), 5.86 (1H, by column chromatography over silica gel with petro- d, J = 3  Hz, H-8), 5.97 (4H, m, H-3’–H-6’); C-NMR leum ether:EtOAc (7:3) as the eluent to furnish 7d as a (CD OD, 75  MHz) δ 44.3 (C-3), 79.7 (C-2), 96.2 (C-6), white solid in 89% yield. mp 178–179  °C; UV(MeOH) 96.3 (C-8), 103.3 (C-10), 125.5 (C-5’), 127.5 (C-6’), 129.5 λ : 293, 347  nm; H NMR (DMSO-d , 500  MHz) δ (C-3’), 131.3 (C-4’), 135.6 (C-2’), 142.7 (C-1’), 164.2 (C-9), max 6 2.78, (1H, dd, J = 3  Hz, J = 17.5  Hz, H-3), 3.21 (1H, dd, 165.5 (C-5), 168.3 (C-7), 196.8 (C-4); MS (EI, 70 eV) m/z J = 13  Hz, J = 14  Hz, H-3), 3.74 (3H, s, OMe), 3.86 (6H, (%) 290 (33) [M] , 256 (73), 255 (100), 179 (73), 152 (51), s, OMe), 5.47 (1H, dd, J = 3 Hz, J = 13 Hz, H-2), 5.96 (1H, 124 (27). d, J = 2 Hz, H-6), 5.99 (1H, d, J = 2 Hz, H-8), 6.90 (2H, s, H-2’, H-6’), 12.16 (1H, s, 5-OH); MS (EI, 70  eV) m/z (%) Synthesis and analytical data of 3’‑chloroflavanone (7g) 346 (39) [M] , 303 (4), 194 (12), 181 (100). Synthesis and analytical data of 2’,3’‑dichloroflavanone (7e) HO O 2 1' 3' Cl HO 79 O 2 OH O 1' 3' Cl 7g Cl OH O Following general procedure III, 5,7-dibenzyloxy- 7e 3’-chloroflavanone (6m) (10  mg, 0.02  mmol) was debenzylated and the crude product was purified by Following general procedure III, 5,7-dibenzyloxy- column chromatography over silica gel with petroleum 2’,3’-dichloroflavanone (6j) (80  mg, 0.16  mmol) was ether:EtOAc (7:3) as the eluent to furnish 7g as a white debenzylated and the crude product was purified by solid in 78% yield. R 0.68 (petroleum ether:EtOAc 4:1); column chromatography over silica gel with petro- IR ʋ 1628 (C = C), 1581 (C = C), 1453, 1300, 1085 cm-1: leum ether:EtOAc (7:3) as the eluent to furnish 7e as a and 1063 (C-O); H NMR (CD OD, 300  MHz) δ 2.84, white solid in 88% yield. H NMR (DMSO-d , 500 MHz) (1H, m, H-3), 3.11 (1H, m, H-3), 5.50 (1H, dd, J = 4.5  Hz, δ 2.91 (1H, dd, J = 3  Hz, J = 17.5  Hz, H-3), 3.11 (1H, J = 9.6 Hz, H-2), 5.97 (1H, d, J = 1.8 Hz, H-6), 6.00 (1H, d, dd, J = 13  Hz, J = 17  Hz, H-3), 5.94 (1H, dd, J = 3  Hz, J = 1.8 Hz, H-8), 7.52 (4H, m, H-2’, H-3’–H-6’); C NMR J = 13  Hz, H-2), 6.00 (1H, d, J = 2  Hz, H-6), 6.03 (1H, d, (CD OD, 75  MHz) δ 42.6 (C-3), 78.3 (C-2), 94.7 (C-6), J = 2  Hz, H-8), 7.53 (1H, t, J = 8  Hz, H-5’), 7.65 (1H, dd, 95.8 (C-8), 102.1 (C-10), 124.1 (C-6’), 126.2 (C-2’), 128.7 J = 1.5 Hz, J = 8 Hz, H-4’), 7.78 (1H, dd, J = 1 Hz, J = 7 Hz, (C-4’), 133.9 (C-5’), 138.9 (C-3’), 141.3 (C-1’), 162.9 (C-9), H-6’), 12.05 (1H, s); MS (EI, 70  eV) m/z (%) 326 (31) 164.4 (C-5), 166.8 (C-7), 195.9 (C-4); MS (EI, 70 eV) m/z + + [M + 2] , 324 (51) [M] , 289 (94), 255 (14), 179 (100), 152 (%) 290 (33) [M] , 256 (63), 179 (100), 152 (62), 124 (37). (66), 124 (46). Synthesis and analytical data of 2’,4’‑dichloroflavanone (7h) Synthesis and analytical data of 2’‑chloroflavanone (7f ) Cl HO 79 O 2 3' HO 79 O 2 3' 1' 1' 4 Cl Cl OH O OH O 7h 7f Following general procedure III, 5,7-dibenzyloxy- Following general procedure III, 5,7-dibenzyloxy- 2’,4’-dichloroflavanone (6n) (160  mg, 0.12  mmol) was 2’-chloroflavanone (6  l) (270  mg, 0.57  mmol) was debenzylated and the crude product was purified by debenzylated and the crude product was purified by column chromatography over silica gel with petro- column chromatography over silica gel with petro- leum ether:EtOAc (7:3) as the eluent to furnish 7h as a leum ether:EtOAc (7:3) as the eluent to furnish 7f as a white solid in 87% yield. H NMR (CD OD, 500  MHz) white solid in 92% yield. H NMR (CD OD, 300  MHz) δ 2.87, (1H, dd, J = 2.5  Hz, J = 14  Hz, H-3), 3.02 (1H, δ 2.84 (1H, dd, J = 3  Hz, J = 17.1  Hz, H-3), 3.08 (1H, dd, dd, J = 14  Hz, J = 16  Hz, H-3), 5.80 (1H, dd, J = 2.5  Hz, J = 12.3  Hz, J = 17.1  Hz, H-3), 5.52 (1H, dd, J = 2.4  Hz, Melaku et al. BMC Chemistry (2022) 16:36 Page 26 of 30 solid in 91% yield. H NMR (CD OD/CDCl , 500  MHz) J = 13 Hz, H-2), 5.96 (1H, br s, H-6), 5.99 (1H, br s, H-8), 3 3 δ 2.59, (1H, dd, J = 3.5  Hz, J = 17.5  Hz, H-3), 3.74 (1H, 7.48 (1H, d, J = 8.5  Hz, H-6’), 7.57 (1H, br s, H-3’), 7.77 dd, J = 14.5  Hz, J = 17.5  Hz, H-3), 5.92 (1H, d, J = 2  Hz, (1H, dd, J = 1.2  Hz, J = 8.5  Hz, H-5’); C NMR (C D OD, H-6), 5.94 (1H, d, J = 2 Hz, H-8), 6.22 (1H, dd, J = 3.5 Hz, 125 MHz) δ 42.6 (C-3), 76.9 (C-2), 95.8 (C-6), 97.5 (C-8), J = 14.5  Hz, H-2), 7.35 (1H, dd, J = 7.5  Hz, J = 7.5  Hz, 129.0 (C-5’), 129.8 (C-6’), 130.8 (C-3’), 133.2 (C-2’), 135.7 H-4’), 7.45 (2H, br d, J = 7.5  Hz, H-3’, H-5’); C NMR (C-4’), 136.7 (C-1’), 164.9 (C-9), 165.8 (C-5), 168.2 (C-7), (CD OD/CDCl , 125 MHz) δ 39.2 (C-3), 76.2 (C-2), 95.8 196.2 (C-4); MS (EI, 70  eV) m/z (%) 326 (24) [M + 2] , 3 3 (C-6), 97.2 (C-8), 102.8 (C-10), 126.3 (C-5’, C-5’), 130.5 324 (32) [M] , 289 (74), 256 (68), 179 (100), 152 (83), 124 (C-4’), 131.8 (C-1’), 136.5 (C-2’, C-6’), 164.1 (C-9), 165.2 (52), 69 (54). (C-5), 168.2 (C-7), 195.9 (C-4); MS (EI, 70  eV) m/z (%) 325 (62) [M] , 323 (75), 289 (96), 256 (31), 179 (100), 151 Synthesis and analytical data of 3’,5’‑dichloroflavanone (7i) (70), 124 (36). Cl Synthesis and analytical data of 2’,5’‑dichloroflavanone (7k) HO O 2 1' 3' Cl Cl 4' OH O HO O 2 1' 7i Cl OH O Following general procedure III, 5,7-dibenzyloxy- 3’,5’-dichloroflavanone (6o) (200  mg, 0.4  mmol) was 7k debenzylated and the crude product was purified by Following general procedure III, 5,7-dibenzyloxy- column chromatography over silica gel with petro- 2’,5’-dichloroflavanone (6q) (115  mg, 0.23  mmol) was leum ether:EtOAc (7:3) as the eluent to furnish 7i as a debenzylated and the crude product was purified by white solid in 89% yield. H NMR (CD OD, 500  MHz) column chromatography over silica gel with petroleum δ 2.87, (1H, dd, J = 3  Hz, J = 17  Hz, H-3), 3.06 (1H, dd, ether:EtOAc (7:3) as the eluent to furnish 7k as a white J = 12.5  Hz, J = 17  Hz, H-3), 5.51 (1H, dd, J = 3  Hz, solid in 94% yield. H NMR (C D OD, 500  MHz) δ 2.90, J = 12.5 Hz, H-2), 5.95 (1H, d, J = 2 Hz, H-6), 6.02 (1H, d, (1H, dd, J = 3 Hz, J = 17 Hz, H-3), 3.01 (1H, dd, J = 13 Hz, J = 2.5 Hz, H-8), 7.49 (1H, t, J = 1.75 Hz, H-4’), 7.53 (2H, J = 17  Hz, H-3), 5.80 (1H, dd, J = 3  Hz, J = 13  Hz, H-2), m, H-2’, H-6’); C NMR (CD OD, 125 MHz) δ 42.2 (C-3), 5.98 (1H, d, J = 2  Hz, H-6), 6.04 (1H, d, J = 2.5  Hz, H-8), 77.2 (C-2), 94.7 (C-6), 96.0 (C-8), 124.5 (C-2’, C-6’), 127.9 7.43(1H, dd, J = 2.5  Hz, J = 8.5  Hz, H-4’), 7.50 (1H, d, (C-4’), 135.0 (C-3’, C-5’), 143.0 (C-1’), 162.6 (C-9), 164.1 J = 9  Hz, H-3’), 7.78 (1H, d, J = 2.5  Hz, H-6’); C NMR (C-5), 167.1 (C-7), 196.8 (C-4); MS (EI, 70  eV) m/z (%) (CD OD, 125  MHz) δ 41.2 (C-3), 75.5 (C-2), 95.1 (C-6), 326 (22) [M + 2] , 324 (36) [M] + , 289 (79), 256 (64), 255 96.1 (C-8), 102.1 (C-10), 127.2 (C-2’), 130.8 (C-4’), 131.1 (68), 179 (100), 152 (86), 124 (48),, 69 (52), 57 (97). (C-3’), 133.5 (C-2’), 133.6 (C-5’), 138.8 (C-1’), 163.0 (C-9), 164.1 (C-5), 167.1 (C-7), 194.3 (C-4); MS (EI,7 0 eV) m/z Synthesis and analytical data of 2’,6’‑dichloroflavanone (7j) + + (%) 326 (24) [M + 2] , 324 (32) [M] , 290 (17), 256 (34), 179 (100), 152 (70), 124 (42). Cl 4' HO O 2 Synthesis and analytical data of 3’,4’‑dichloroflavanone (7l) 1' Cl 4' Cl OH O HO O 2 1' 7j Cl Following general procedure III, 5,7-dibenzyloxy- OH O 2’,6’-dichloroflavanone (6p) (140  mg, 0.28  mmol) was 7l debenzylated and the crude product was purified by column chromatography over silica gel with petroleum Following general procedure III, 5,7-dibenzyloxy- ether:EtOAc (7:3) as the eluent to furnished 7j as a white 3’,4’-dichloroflavanone (6r) (120  mg, 0.24  mmol) was M elaku et al. BMC Chemistry (2022) 16:36 Page 27 of 30 In vivo antiplasmodial experiment debenzylated and the crude product purified by col - Standard four days suppression test was used to evaluate umn chromatography over silica gel with petroleum the antiplasmodial activity of the synthetic compounds ether:EtOAc (7:3) as the eluent to furnish 7  l as a white [18]. The mice in this study were grouped into treatment, solid in 88% yield. H NMR (CD OD, 500  MHz) δ positive and negative control groups. Each group contains 2.85, (1H, dd, J = 3.5  Hz, J = 17  Hz, H-3), 3.09 (1H, dd, five mice. On D0, all the mice were weighted on a sensitive J = 12.5  Hz, J = 17  Hz, H-3), 5.52 (1H, dd, J = 3.5  Hz, balance and the packed cell volume (PCV) were measured. J = 13 Hz, H-2), 5.95 (1H, d, J = 2.5 Hz, H-6), 6.00 (1H, d, Then each mouse in the treatment and control group was J = 2 Hz, H-8), 7.47 (1H, dd, J = 2.5  Hz, J = 8.5  Hz, H-6’), injected with 0.2  ml of blood diluted with physiological 7.61 (1H, d, J = 8.5 Hz, H-5’), 7.75 (1H, d, J = 2  Hz, H-2’); + + saline (intraperitoneally) which contains approximately MS (EI, 70 eV) m/z (%) 326 (24) [M + 2] , 324 (32) [M] , 1 × 10 P. berghei parasitized RBCs. 290 (17), 256 (34), 179 (100), 152 (70), 124 (42). Administration of pinocembrin and its analogs Experimental design for antiplasmodial assay After three hours of injection of P. berghei infected blood, This experiment was done on Swiss albino mice of the synthetic pinocembrin and its analogs were administered age of 6–8  weeks and body weight ranges from 22–28  g each separately. Compound 6a was administered at doses which were reared in the Animal House of College of of 15, 20, 25, 30 and 35  mg/kg b. wt. Compounds 6b, 6e Natural and Computational Science, Addis Ababa Uni- and 6c were tested at a dose of 20 mg/kg b. wt each. Com- versity. The mice were handled according to Guide for pound 6i was given at doses of 15, 25 and 35 mg/kg b. wt. the Care and Use of Laboratory Animals [31].They were Compound 7a was given at doses of 20 and 30  mg/kg b. given a standard food and water (ad libitum) and main- wt. Chloroquine (CQ) 25  mg/kg b.wt and 1  ml/100  g of tained in 12  h dark and 12  h light (artificial light). The 10% DMSO were given as a positive and a negative control experiment was done on male mice. The mice have been respectively. The administration continued for 4 days (D0– acclimatized for a week before the experiment started. D3) based on modified procedure of Peters (1965) [18]. The amount of the compounds administration was based on Preparation of Plasmodium parasite the availability of the synthesized quantity. The Plasmodium berghei parasite was obtained from Ethio- pian Public Health Institute (EPHI) and was passaged on Percent parasitaemia and percent suppression a weekly basis from infected mice to healthy mice. For the On D4, blood was taken gently from the tail of each mouse experiment four donor mice were prepared by infecting P. on a slide to make a blood smear. The smear was fixed with berghei through intraperitoneal injection of the blood of methanol and stained with 10% Giemsa stain for 30  min, infected mice with normal saline. When the parasitaemia then washed with water. Each blood smear was examined was confirmed to be 30–40% by preparing a blood smear, under the compound microscope with oil emulsion at blood was taken from the hearts of the donor mice after magnification power of 100 × (together with ocular lens anesthetized them. The blood was taken from each donor 10,000 ×) and each slide was counted for 5–10 times. The mouse and diluted with physiological saline (0.85%) in 1:4 percent parasitaemia and percent suppression was calcu- ratio. lated by the following formula as given in Peters [18]. number of infected RBC % Parasitaemia = × 100. Number of infected RBC + number of uninfected RBC parasitaemia in negative control − parasitaemia in treatment group % Suppression = ×100. Parasitaemia in negative control Melaku et al. BMC Chemistry (2022) 16:36 Page 28 of 30 Determination of mean survival time was setted using graphical user interface program. The All the mice in the treatment and control groups were grid was set so that it surrounds the region of interest followed daily starting from the day of parasite injection in the macromolecule. The best docked conformation and MST was calculated by using the following formula between ligand and protein was searched using the dock- given below. ing algorithm with AutoDock Vina. During the docking process, a maximum of nine conformers were considered sum of survival day of all mice in a group day for each ligand. The conformations with the most favora - MST = Total number of mice in that group ble (least) free binding energy were selected for analyzing the interactions between the target receptor and ligands by Discovery studio visualizer and PyMOL. The ligands are Body weight and PCV determination represented in different color, H-bonds and the interacting Body weight and PCV were measured on D0 and D4 to residues are represented in stick model representation. evaluate the effect of the compounds on the mice. The PCV was measured by taking blood ¾ of heparinized microhematocrit capillary tube (75 mm) from the tail of In silico drug-likeness predictions the mice. The tube was sealed with sealant and centri - In silico Drug-likeness helps to know whether a particu- fuged on microhematocrit centrifuge (MK IV, England) lar pharmacological agent has properties consistent with by 13,000  rpm for 4  min. Then, the total blood volume being an orally active drug. This prediction is based on an and the volume of erythrocyte were measured by using already established rule called Lipinski rule of five [29]. a ruler. Mean body weight was also calculated using the The structures of compounds synthesized (7a-l) were following formula. changed to their canonical simplified molecular input line entry system (SMILE) then submitted to SwissADME Total weight of mice in a group Meanbody weight = tool to estimate in silico pharmacokinetic parameters and Total number of mice in that group other molecular properties based on the methodology The PCV was calculated using the formula given by reported by Amina et  al. 2016. The data obtained were Gilmour and Sykes (1951). compared with chloroquine (standard drug), and only compounds without violation of any of the screenings Volume of erythrocyte in a given blood were used for the molecular docking analysis. PCV = × 100. Total blood volume Ethical consideration Molecular docking simulation of compounds 7a-l To conduct this study, ethical clearance was obtained against Plasmodium falciparum dihydrofolate from Institutional Ethics Review Board of College of Nat- reductase-thymidylate synthase ural Sciences, Addis Ababa University (IRB/024/2017). AutoDock Vina with standard protocol was used to dock The study is reported in accordance with the Animal the proteins (PfDHFR-TS) (PDB ID 1J3I) and compounds Research Reporting of in  vivo Experiments (ARRIVE) synthesized (7a-l) into the active site of proteins [32, 33]. guidelines (https:// arriv eguid elines. org) and were han- ChemOffice tool (Chem Draw 16.0) was used to draw the dled according to the Guide for the Care and Use of chemical structures of the synthesized compounds (7a- Laboratory Animals (https:// grants. nih. gov/ grants/ olaw/ l) while the proper 2D orientation, and energy of each guide- for- the- care- and- use- of- labor atory- anima ls). molecule was minimized using ChemBio3D. The energy minimized ligand molecules were then used as input for AutoDock Vina, in order to carry out the docking simu- Data analysis lation. The crystal structure of Plasmodium falciparum The data generated in this study were analyzed using IBM dihydrofolate reductase-thymidylate synthase was down- SPSS, version 20 statistical package. The results were loaded from protein data bank. The protein preparation presented as mean ± SEM (standard error of the mean). was by removing the co-crystallized ligand, selected water One-way ANOVA (analysis of variance), paired Student’s molecules and cofactors, the target protein file was pre - t-test and Kaplan–Meier analyses were used for the sta- pared by leaving the associated residue with protein by tistical analysis. A statistically significant difference was using Auto preparation of target protein file AutoDock 4.2 taken at P-value less than 0.05 (P < 0.05). (MGL tools1.5.7). The grid box for docking simulations M elaku et al. BMC Chemistry (2022) 16:36 Page 29 of 30 Author details Supplementary Information Chemistry Department, Adama Science and Technology University, The online version contains supplementary material available at https:// doi. 1888 Adama, Ethiopia. Biology Department, Addis Ababa University, Addis org/ 10. 1186/ s13065- 022- 00831-z. Ababa, Ethiopia. Bioorganische Chemie, Institut Für Chemie, Universität Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany. Depar tment Additional file 1. The NMR spectra and molecular docking simulations of Chemical Engineering, FH Münster-University of Applied Sciences, for the synthetic compounds are included within Additional materials Stegerwaldstrasse 39, 48565 Steinfurt, Germany. Department of Biomateri- (Additional files 1 and 2). als, Saveetha Dental College and Hospitals, Saveetha Institute of Meidcal and Technical Sciences (SIMATS), Saveetha University, Chennai 600 077, India. Additional file 2. The NMR spectra and molecular docking simulations for the synthetic compounds are included within Additional materials Received: 23 February 2022 Accepted: 11 May 2022 (Additional file 1 and 2). Acknowledgements The authors thank the Alexander von Humboldt Foundation for the research References grant. We thank Addis Ababa University, Adama Science and Technology 1. Lubis IN. Contribution of Plasmodium knowlesi to multispecies University and Hohenheim University for technical and financial support. human malaria infections in North Sumatera, Indonesia. J Infect Dis. We thank Dr. J. Conrad and Mr. M. Wolf (Institut für Chemie, Universität 2017;215(7):1148–55. Hohenheim) for recording of NMR spectra and Dr. C. Braunberger Institute für 2. Ayoola GA, Coker H, Adesegun S, Adepoju-Bello A, Obaweya K, Ezennia Chemie, Universität Hohenheim for recording of mass spectra. Prof. Ermias E, Atangbayila T. Phytochemical screening and antioxidant activities of Dagne is acknowledged for being part of the research project (Department of some selected medicinal plants used for malaria therapy in southwestern Chemistry, Addis Ababa University, Ethiopia). nigeria. Trop J Pharm Res. 2008;7:1019–24. 3. Cropper ML, Haile M, Lampietti J, Poulos C, Whittington D. The demand Author contributions for malaria vaccine: evidence from Ethiopia. J Dev Econ. 2009;75:303–18. YM and UB were responsible for planning the synthetic experiments. YM and 4. World Health Organization. World Malaria Report. Geneva: World Health VO were responsible for the synthesis and characterization of compounds. Organization; 2012. MS and Y TM were responsible for the in vivo antiplasmodial activity of the 5. Rasul A, Millimouno FM, Eltay WA, Ali M, Li X. Pinocembrin: A Novel Natu- synthesized compounds. RE conducted the molecular docking simulation of ral Compound with Versatile Pharmacological and Biological Activities. compounds 7a-l. The manuscript was written by YM, UB and Y TM. Corre- Biomed Res Int. 2013. https:// doi. org/ 10. 1155/ 2013/ 379850. spondence to Yalemtsehay Mekonnen. All authors read and approved the final 6. Qingyun Y, Yuanfeng Y, Feng C, Yan Q, Wei L, Song W. Identification and manuscript. synthesis of impurities in pinocembrin-A new drug for the treatment of ischemic stroke. Chin J Chem. 2012;30:1315–9. Funding 7. Said MM, Azab SS, Saeed NM, Demerdash EE. Antifibrotic mechanism of This project work was done by the fund obtained from the Alexander von pinocembrin: impact on oxidative stress inflammation and TGF-β/Smad Humboldt Foundation for the research group linkage grant (2014–2017) with inhibition in rats. Ann Hepatol. 2018;17(2):307–17. grant number 3.4-IP-DEU/1074366. 8. Saad AAE, Tadros MG, Elsherbiny DA, Menze ET. 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Journal

BMC ChemistrySpringer Journals

Published: May 24, 2022

Keywords: Catalytic hydrogenation; Claisen-Schmidt Condensation; Malaria; Plasmodium berghei; Pinocembrin; Chloropinocembrin

References