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A Concise Synthesis towards Antimalarial Quinazolinedione TCMDC-125133 and Its Anti-Proliferative Activity against MCF-7

A Concise Synthesis towards Antimalarial Quinazolinedione TCMDC-125133 and Its Anti-Proliferative... molbank Communication Communication A Concise Synthesis towards Antimalarial Quinazolinedione A Concise Synthesis towards Antimalarial Quinazolinedione TCMDC-125133 and Its Anti-Proliferative Activity TCMDC-125133 and Its Anti-Proliferative Activity against MCF-7 against MCF-7 1 , 2 , 3 , 1 2 1,2,3, 1 2 Sitthivut Charoensutthivarakul * , Duangporn Lohawittayanan , Phongthon Kanjanasirirat , Sitthivut Charoensutthivarakul *, Duangporn Lohawittayanan , Phongthon Kanjanasirirat , 2 2 2 , 4 5 2 2 2,4 5 Kedchin Jearawuttanakul , Sawinee Seemakhan , Suparerk Borwornpinyo and Matthew Phanchana Kedchin Jearawuttanakul , Sawinee Seemakhan , Suparerk Borwornpinyo and Matthew Phanchana 1 1 Innovative Molecular Discovery Laboratory (iMoD), School of Bioinnovation and Bio-Based Product Innovative Molecular Discovery Laboratory (iMoD), School of Bioinnovation and Bio-Based Product Intelligence, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; duangporn.loha@gmail.com Intelligence, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; duangporn.loha@gmail.com 2 2 Excellent Center for Drug Discovery (ECDD), Faculty of Science, Mahidol University, Excellent Center for Drug Discovery (ECDD), Faculty of Science, Mahidol University, Ba Bangkok ngkok 110400, 0400, TThailand; hailand; pphongku1232@gmail.com hongku1232@gmail.com (P (P .K .K.); .); o o-nion@hotmail.com -nion@hotmail.com ((K.J.); K.J.); sawinee.ecdd@gmail.com (S.S.); bsuparerk@gmail.com (S.B.) sawinee.ecdd@gmail.com (S.S.); bsuparerk@gmail.com (S.B.) 3 3 Center for Neuroscience, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Center for Neuroscience, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 4 4 Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 5 5 Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, M Bangkok ahidol U 10400, niversi Thailand; ty, Bangkmatthew ok 10400.pha@mahidol.edu , Thailand; matthew.pha@mahidol.edu * Correspondence: sitthivut.cha@mahidol.ac.th; Tel.: +66-2201-5899 * Correspondence: sitthivut.cha@mahidol.ac.th; Tel.: +66-2201-5899 Abstract: Quinazolinedione is one of the most notable pharmacophores in drug discovery due to its Abstract: Quinazolinedione is one of the most notable pharmacophores in drug discovery due broad spectrum of biological activities including antimalarial, anticancer, anti-inflammatory, and to its broad spectrum of biological activities including antimalarial, anticancer, anti-inflammatory, others. TCMDC-125133, whose structure features a quinazolinedione core, exhibits promising anti- and others. TCMDC-125133, whose structure features a quinazolinedione core, exhibits promising malarial activity and low toxicity as described in the GlaxoSmithKline (GSK) report. Herein, a con- antimalarial activity and low toxicity as described in the GlaxoSmithKline (GSK) report. Herein, cise four-step synthesis towards quinazolinedione TCMDC-125133 is described using low cost a concise four-step synthesis towards quinazolinedione TCMDC-125133 is described using low cost Citation: Citation: Char Charoensutthivarakul, oensutthivarakul, S.; S.; goods and greener alternatives where possible. All synthesized compounds were characterized us- goods and greener alternatives where possible. All synthesized compounds were characterized Lohawittayanan, Lohawittayanan, D.; D.; Kanjanasirirat, Kanjanasirirat, ing polarimetry, IR, NMR, and mass spectrometry. The in-house synthesized TCMDC-125133 was using polarimetry, IR, NMR, and mass spectrometry. The in-house synthesized TCMDC-125133 was P P. .;; Jearawuttanakul, Jearawuttanakul, K.; K.;Seemakhan, Seemakhan, evaluated for its antimalarial activity against P. falciparum 3D7 and antiproliferative activity against evaluated for its antimalarial activity against P. falciparum 3D7 and antiproliferative activity against S.; S.; Borwornpinyo, Borwornpinyo, S.; S.; Phanchana, Phanchana, M. M. MCF-7 cell line. A Concise Synthesis towards A Concise Synthesis towards MCF-7 cell line. Antimalarial Quinazolinedione Antimalarial Quinazolinedione TCMDC-125133 and Its Anti- Keywords: quinazolinedione derivatives; antimalarial activity; antiproliferative activity TCMDC-125133 and Its Keywords: quinazolinedione derivatives; antimalarial activity; antiproliferative activity Proliferative Activity against MCF-7. Anti-Proliferative Activity against Molbank 2022, 2022, M1358. https:// MCF-7. Molbank 2022, 2022, x. doi.org/10.3390/M1358 https://doi.org/10.3390/xxxxx 1. 1. Introduction Introduction Academic Editor: Stanislav Kafka Academic Editor: Stanislav Kafka Quinazolinedione is a remarkable heterocycle which is widely used as a functional ma- Quinazolinedione is a remarkable heterocycle which is widely used as a functional Received: Received: 25 25 Febr February uary 2022 2022 terial materfor ial f synthetic or synthechemistry tic chemi.str Ityis . It also is apr lso esent presin envarious t in vario active us acti pharmaceutical ve pharmaceuti molecules cal mole- Accepted: Accepted: 18 18 April April 2022 2022 along with its diverse range of biological activities, including antimalarial, anticancer, an- cules along with its diverse range of biological activities, including antimalarial, anti- Published: Published: 21 21 April April 2022 2022 timicrobial, antihypertensive, antiviral, anti-inflammatory, and others [1–5]. The examples cancer, antimicrobial, antihypertensive, antiviral, anti-inflammatory, and others [1–5]. Publisher Publisher’ ’s s Note: Note:MDPI MDPI stays stayneutral s neu- shown in Figure 1 are some representatives of commercial drugs and biologically active The examples shown in Figure 1 are some representatives of commercial drugs and bio- with tral r w egar ith d rto egju arrisdictional d to jurisd claims ictional in molecules with quinazolinedione moiety [6]. logically active molecules with quinazolinedione moiety [6]. published maps and institutional affil- claims in published maps and institu- iations. tional affiliations. Copyright: © 2022 by the authors. Copyright: © 2022 by the authors. Submitted for possible open access Licensee MDPI, Basel, Switzerland. publication under the terms and con- This article is an open access article ditions of the Creative Commons At- distributed under the terms and tribution (CC BY) license (https://cre- conditions of the Creative Commons Figure 1. Examples of quinazolinedione-based drugs and its biological activity. Figure 1. Examples of quinazolinedione-based drugs and its biological activity. ativecommons.org/licenses/by/4.0/). Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ One of the interesting quinazolinedione-containing drugs is ketanserin, as displayed 4.0/). in Figure 2a. This drug is used clinically as an antihypertensive agent selectively targeting Molbank 2022, 2022, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/molbank Molbank 2022, 2022, M1358. https://doi.org/10.3390/M1358 https://www.mdpi.com/journal/molbank Molbank 2022, 2022, x FOR PEER REVIEW 2 of 9 Molbank 2022, 2022, M1358 2 of 9 One of the interesting quinazolinedione-containing drugs is ketanserin, as displayed in Figure 2a. This drug is used clinically as an antihypertensive agent selectively targeting at 5-HT2A receptor, a subtype of the 5-HT2 receptor that belongs to the serotonin receptor at 5-HT receptor, a subtype of the 5-HT receptor that belongs to the serotonin receptor 2A 2 family (5-HT). Several studies have reported that serotonin was involved in the regulation family (5-HT). Several studies have reported that serotonin was involved in the regulation of cell proliferation, survival, and metastasis [7]. Moreover, it is reported that 5-HT plays of cell proliferation, survival, and metastasis [7]. Moreover, it is reported that 5-HT plays a mitogenic role and exerts its growth effect in human breast cancer cell line (MCF-7) [8]. a mitogenic role and exerts its growth effect in human breast cancer cell line (MCF-7) [8]. Recently, Seyed H Hejazi and his group studied the pattern of serotonin receptor gene Recently, Seyed H Hejazi and his group studied the pattern of serotonin receptor gene expression in MCF-7. Their result showed that the ketanserin had suppression effects on expression in MCF-7. Their result showed that the ketanserin had suppression effects MCF-7 cell’s proliferation of 72.36% at the ketanserin concentration of 25 µM [9]. In a pre- on MCF-7 cell’s proliferation of 72.36% at the ketanserin concentration of 25 M [9]. In a lim prieliminary nary drugdr re ug pur repurposing posing studstudy y, ket,aketanserin nserin surp surprisingly risingly poss possesses esses googood d antiantimal malaria alr a ial c- tivity with the IC50 against multidrug-resistant P. falciparum K1 at around 10 µM [10]. activity with the IC against multidrug-resistant P. falciparum K1 at around 10 M [10]. Figure 2. Chemical structures of (a) Ketanserin and (b) TCMDC-125133 with quinazolinedione core Figure 2. Chemical structures of (a) Ketanserin and (b) TCMDC-125133 with quinazolinedione core highlighted highlighted in in blue. blue. In 2010, GlaxoSmithKline (GSK) published the Tres Cantos Antimalarial Set (TCAMS) In 2010, GlaxoSmithKline (GSK) published the Tres Cantos Antimalarial Set containing 13,533 compounds that are the result of screening nearly 2 million compounds (TCAMS) containing 13,533 compounds that are the result of screening nearly 2 million from the GSK corporate collection [11]. With the result openly available in the public compounds from the GSK corporate collection [11]. With the result openly available in the domain, one of the compounds identified from the screen as a singleton is TCMDC-125133 public domain, one of the compounds identified from the screen as a singleton is TCMDC- whose structure featured a quinazolinedione core as displayed in Figure 2b. The preliminary 125133 whose structure featured a quinazolinedione core as displayed in Figure 2b. The in vitro antimalarial activity of this compound against asexual blood stage and gametocytes preliminary in vitro antimalarial activity of this compound against asexual blood stage of P. falciparum 3D7 has shown excellent IC s at 0.27 and 0.54 M, respectively [12]. This, and gametocytes of P. falciparum 3D7 has shown excellent IC50s at 0.27 and 0.54 µM, re- combined with the drug-like property and low toxicity (cytotoxicity HG2; IC > 25 M) of spectively [12]. This, combined with the drug-like property and low toxicity (cytotoxicity this compound, has made it an excellent target for further lead optimization. HG2; IC50 > 25 µM) of this compound, has made it an excellent target for further lead The main structural feature of these compounds is quinazolinedione; however, the optimization. synthesis of quinazolinedione core has been reported to achieve this in multiple steps The main structural feature of these compounds is quinazolinedione; however, the using harsh conditions and tedious workup procedures with low yields [13,14], while synthesis of quinazolinedione core has been reported to achieve this in multiple steps us- there is no previous report on the synthesis towards TCMDC-125133. This present work, ing harsh conditions and tedious workup procedures with low yields [13,14], while there therefore, described a concise synthetic route towards the quinazolinedione TCMDC- is no previous report on the synthesis towards TCMDC-125133. This present work, there- 125133 using commercially available starting materials with a low cost of goods. Several fore, described a concise synthetic route towards the quinazolinedione TCMDC-125133 spectroscopic techniques were employed to confirm the structure of in-house synthesized using commercially available starting materials with a low cost of goods. Several spectro- TCMDC-125133 and the synthetic intermediates. The final compound was assayed for scopic techniques were employed to confirm the structure of in-house synthesized its antimalarial activity against 3D7 and for antiproliferative activity against MCF-7. The TCMDC-125133 and the synthetic intermediates. The final compound was assayed for its resulting synthetic route will be crucial for further structure-activity relationships (SARs) antimalarial activity against 3D7 and for antiproliferative activity against MCF-7. The re- study in lead optimization and development of this class of compounds. sulting synthetic route will be crucial for further structure-activity relationships (SARs) study in lead optimization and development of this class of compounds. 2. Results and Discussion The synthesis towards the quinazolidinone TCMDC-125133 begins with the reaction 2. Results and Discussion between commercially available isatoic anhydride and corresponding L-valine ethyl ester The synthesis towards the quinazolidinone TCMDC-125133 begins with the reaction hydrochloride in the presence of K CO in CH CN to afford compound 1 (55%) [15]. This 2 3 3 between commercially available isatoic anhydride and corresponding L-valine ethyl ester step involves the use of low-cost isatoic anhydride and a mild reaction condition with hydrochloride in the presence of K2CO3 in CH3CN to afford compound 1 (55%) [15]. This a capability to be performed at a multi-gram scale. Although the racemization of the step involves the use of low-cost isatoic anhydride and a mild reaction condition with a -carbon of the valine moiety could be of concern in this step, the working reagent and capability to be performed at a multi-gram scale. Although the racemization of the α-car- condition did not affect the stereochemistry of this optically active molecule as shown by bon of the valine moiety could be of concern in this step, the working reagent and condi- its distinctive levorotatory property and a chiral HPLC trace (>99% ee). For the synthesis of compound tion did no2 t ,athe ffeccyclocarbonylation t the stereochemistrr y eaction of this of op compound tically activ 1ecould molecbe ule performed as shown using by its various distincticarbonylating ve levorotatory reagents property such andas a phosgene chiral HPL or C its traequivalent ce (>99% eederivatives ). For the sy(i.e., ntheethyl sis of chloroformate, diphosgene or triphosgene), however, those reagents are toxic and difficult compound 2, the cyclocarbonylation reaction of compound 1 could be performed using to handle, and some require harsh conditions [6]. 1,1-carbonyldiimidazole (CDI) was chosen as a greener alternative for this reaction owing to its safety and ease of handling. Molbank 2022, 2022, x FOR PEER REVIEW 3 of 9 various carbonylating reagents such as phosgene or its equivalent derivatives (i.e., ethyl chloroformate, diphosgene or triphosgene), however, those reagents are toxic and difficult Molbank 2022, 2022, M1358 3 of 9 to handle, and some require harsh conditions [6]. 1,1-carbonyldiimidazole (CDI) was cho- sen as a greener alternative for this reaction owing to its safety and ease of handling. The resulting intermediate 1 was reacted with CDI in THF to yield the quinazolinedione 2 in The resulting intermediate 1 was reacted with CDI in THF to yield the quinazolinedione 2 97% yield (>99% ee) [15]. in 97% yield (>99% ee) [15]. As shown in Scheme 1 (Route A), it is proposed that the final product 4 (TCMDC- As shown in Scheme 1 (Route A), it is proposed that the final product 4 (TCMDC- 125133) could be made using a tert-butoxide-assisted amidation reaction [16], however, 125133) could be made using a tert-butoxide-assisted amidation reaction [16], however, the reaction was not successful. An alternative synthesis was employed instead starting the reaction was not successful. An alternative synthesis was employed instead starting with hydrolysis of the ethyl ester followed by an amide formation as described in Scheme with hydrolysis of the ethyl ester followed by an amide formation as described in Scheme 1 1 (Route B). The ethyl ester 2 was subsequently hydrolyzed using LiOH in THF/H2O mix- (Route B). The ethyl ester 2 was subsequently hydrolyzed using LiOH in THF/H O mix- ture to give the carboxylic acid 3 without any purification (93%) [15]. It is worth noting ture to give the carboxylic acid 3 without any purification (93%) [15]. It is worth noting that the levorotatory effect can still be observed in compound 3 suggesting the majority that the levorotatory effect can still be observed in compound 3 suggesting the majority of product still retains its configuration (65% ee determined by chiral HPLC) (see Supple- of product still retains its configuration (65% ee determined by chiral HPLC) (see Sup- mentary Materials). To generate the amide 4, it is important to maintain that any harsh plementary Materials). To generate the amide 4, it is important to maintain that any condition could affect the stereochemistry of the α-carbon of the valine moiety, hence, 1- harsh condition could affect the stereochemistry of the -carbon of the valine moiety, [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro- hence, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hex- phosphate (HATU) is employed as it is one of the commonly used reagents in amide cou- afluorophosphate (HATU) is employed as it is one of the commonly used reagents in pling chemistry. Although it is not ‘low-cost’ as originally proposed, HATU was chosen amide coupling chemistry. Although it is not ‘low-cost’ as originally proposed, HATU was because of its mild condition, high coupling efficiencies, fast reaction rates, and its by- chosen because of its mild condition, high coupling efficiencies, fast reaction rates, and product can be easily removed [17]. In the final step, the corresponding acid 3 was then its by-product can be easily removed [17]. In the final step, the corresponding acid 3 was coupled to the m-anisidine side chain using HATU with the presence of triethylamine then coupled to the m-anisidine side chain using HATU with the presence of triethylamine (Et (Et3N) N) in in DMF DMF[ 15 [15 ]]to toaf a for ffo d rd the thquinazolidinone e quinazolidinon 4e(TCMDC-125133) 4 (TCMDC-12513 in 3)77% in 7yield 7% yiwith eld w 76% ith ee 76determined % ee determby inechiral d by cHPLC hiral HP (see LC Supplementary (see SupplemeMaterials). ntary Mater All ials synthesized ). All synthe compounds sized com- described here were fully characterized using polarimetry, IR, NMR, and mass spectrometry. pounds described here were fully characterized using polarimetry, IR, NMR, and mass This newly discovered synthetic route will allow further structural modification around spectrometry. This newly discovered synthetic route will allow further structural modifi- this pharmacophore core. cation around this pharmacophore core. Scheme Scheme 1 1.. R Reagents eagents aand nd C Conditions: onditions: (i( ) iK ) K 2CO CO 3, C ,H CH 3CN CN, , 60 60 °C, 1 C, 8 h 18 ; (i h; i) ( C ii DI ) CDI, , THF THF , 85 ,°C 85 , 18 C, h18 ; (iih; i) 2 3 3 m-anisidine, t-BuOK, THF, room temperature, overnight; (iv) LiOH, THF/H2O, 85 °C, 18 h; (v) m- (iii) m-anisidine, t-BuOK, THF, room temperature, overnight; (iv) LiOH, THF/H O, 85 C, 18 h; anisidine, HATU, Et3N, DMF, room temperature, 18 h. (v) m-anisidine, HATU, Et N, DMF, room temperature, 18 h. Compound Compound 4 4 d derived erived f fr ro om m th this is s synthesis ynthesis w was as a assayed ssayed ffor or iin n v vitr itro o a antimalarial ntimalarial a activ- ctiv- ity ity against against the the blood blood stage stage P P.. falciparum falciparum 3D7 3D7 strain. strain. The The r results esults show show that that the the in-house in-house synthesised compound 4 possesses a promising IC50 (3D7) of 219 nM (Figure 3) which is synthesised compound 4 possesses a promising IC (3D7) of 219 nM (Figure 3) which is com comparable parable to to thethe priprimary mary scre scr eneening ing resul result t by T by CA TCAMS. MS. FurFurther ther leadlead optioptimisation misation is un is - underway derway to e to nh enhance ance thethe anti antimalarial malarial acti activity vity of of thithis s claclass ss of of com compound. pound. As previously mentioned, quinazolinedione containing molecules could potentially be repurposed as an anti-breast cancer agent. In this regard, compound 4 was assessed for in vitro antiproliferative activity against MCF-7 and additionally HCT-116 (human colorectal cancer) (Figure 4). These data show that compound 4 possesses a moderate anti- MCF-7 activity with an IC of 17.5 M, but it also demonstrates a mild antiproliferative activity against HCT-116 (IC = 58.0 M). The result shown here may suggest some selectivity within this class of compound over a specific type of cancer. Further research is ongoing to study the SARs around this pharmacophore. Molbank 2022, 2022, x FOR PEER REVIEW 4 of 9 Molbank 2022, 2022, x FOR PEER REVIEW 4 of 9 Molbank 2022, 2022, M1358 4 of 9 Figure 3. Dose-dependent antimalarial (3D7) activity of compound 4 (TCMDC-125133). The graph shows the reduction in parasitemia of blood stage 3D7 P. falciparum laboratory strain in human red blood cell at serial concentrations of compound 4. As previously mentioned, quinazolinedione containing molecules could potentially be repurposed as an anti-breast cancer agent. In this regard, compound 4 was assessed for in vitro antiproliferative activity against MCF-7 and additionally HCT-116 (human colo- rectal cancer) (Figure 4). These data show that compound 4 possesses a moderate anti- MCF-7 activity with an IC50 of 17.5 µM, but it also demonstrates a mild antiproliferative activity against HCT-116 (IC50 = 58.0 µM). The result shown here may suggest some selec- Figure 3. Dose-dependent antimalarial (3D7) activity of compound 4 (TCMDC-125133). The graph Fti ig v u ir ty e 3w . Do ithsie n- d th ep is en cd la en ss t a o nft ic m oa m lap rio aun l (3d D7 o) v ae cr t iv ai ts yp o ef cc if o im c p ty op ue n d o 4 f ( cTC anMDC cer. F -1 ur 25 th 13 e3 r). r Th ese e a g rrcah p h is shows the reduction in parasitemia of blood stage 3D7 P. falciparum laboratory strain in human red sh oo nw go s itn hg e r ted o s u tud ctioy n th in e p S ar A as R it sem ario aun of d b lth ooid s s p ta hg ae rm 3D7 ac o Pp . h fao lcr ip ea . rum laboratory strain in human red blood cell at serial concentrations of compound 4. blood cell at serial concentrations of compound 4. As previously mentioned, quinazolinedione containing molecules could potentially be repurposed as an anti-breast cancer agent. In this regard, compound 4 was assessed for in vitro antiproliferative activity against MCF-7 and additionally HCT-116 (human colo- rectal cancer) (Figure 4). These data show that compound 4 possesses a moderate anti- MCF-7 activity with an IC50 of 17.5 µM, but it also demonstrates a mild antiproliferative activity against HCT-116 (IC50 = 58.0 µM). The result shown here may suggest some selec- tivity within this class of compound over a specific type of cancer. Further research is ongoing to study the SARs around this pharmacophore. Figure 4. Dose-dependent anti-MCF-7 (human breast cancer; in red) and anti-HCT-116 (human Figure 4. Dose-dependent anti-MCF-7 (human breast cancer; in red) and anti-HCT-116 (human col- colorectal cancer; in blue) activities of compound 4 (TCMDC-125133). The curves show the cell orectal cancer; in blue) activities of compound 4 (TCMDC-125133). The curves show the cell viability p viability ercentagper e ocentage f each caof nceach er celcancer l line acell t serline ial cat onserial centraconcentrations tions of compoof uncompound d 4. 4. 3. Materials and Methods 3. Materials and Methods 3.1. General 3.1. General All reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich, All reagents and solvents were purchased from commercial suppliers (Sigma-Al- Merck, or Tokyo Chemical Industry (TCI)) and were used without further purification. drich, Merck, or Tokyo Chemical Industry (TCI)) and were used without further purifica- 1 13 NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz H/ C tion. NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz 1 13 and 500/125 MHz H/ C) spectrometer (Bruker, Billerica, MA, USA) and chemical shifts 1 13 1 13 H/ C and 500/125 MHz H/ C) spectrometer (Bruker, Billerica, MA, USA) and chemical (, ppm) were downfield from TMS. The chemical shifts are reported relative to residual the Figure 4. Dose-dependent anti-MCF-7 (human breast cancer; in red) and anti-HCT-116 (human col- shifts (δ, ppm) were downfield from TMS. Th 1e chemica13 l shifts are reported re 1lative to solvent signal in part per million () (CD OD: H:  3.31, C:  49.1; DMSO-d : H:  2.50, orectal cancer; in blue) activities of compound 4 3 (TCMDC-125133). The curves show the c 6ell viability 1 13 r13 esidual the solvent 1signal in p 13 art per million (δ) (C 1D3OD: H: δ 3.31, C: δ 49.1; DMSO- C:  39.5; CDCl : H:  7.26, C:  77.23). For the H-NMR spectrum, data are assumed percentage of each ca 3ncer cell line at serial concentrations of compound 4. 1 13 1 13 1 d6: H: δ 2.50, C: δ 39.5; CDCl3: H: δ 7.26, C: δ 77.23). For the H-NMR spectrum, data to be first order with apparent singlet, doublet, triplet, quartets and multiplet reported as are assumed to be first order with apparent singlet, doublet, triplet, quartets and multiplet 3.s, Ma d,tt, erq, ialand s and m, Me respectively thods . Doublet of doublet was reported as dd, triplet of doublet reported as s, d, t, q, and m, respectively. Doublet of doublet was reported as dd, triplet was reported as td, and the resonance that appears broad was designated as br. High 3.1. General of doublet was reported as td, and the resonance that appears broad was designated as resolution mass spectral measurements were performed on a Thermo Scientific Orbitrap All reagents and solvents were purchased from commercial suppliers (Sigma-Al- br. High resolution mass spectral measurements were performed on a Thermo Scientific Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). TLC drich, Merck, or Tokyo Chemical Industry (TCI)) and were used without further purifica- Orbitrap Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, was performed on TLC aluminium sheet coated with silica gel 60 F (Merck, Darmstadt, tion. NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz USA). TLC was performed on TLC aluminium sheet coated with silica gel 60 F254 (Merck, Germany). To visualize spots on the TLC sheet, UV lamps were used. Melting points were 1 13 1 13 H/ C and 500/125 MHz H/ C) spectrometer (Bruker, Billerica, MA, USA) and chemical measured on a Buchi melting point M-565 (Büchi, Flawil, Switzerland). Fourier Transform shifts (δ, ppm) were downfield from TMS. The chemical shifts are reported relative to Infrared (FTIR) spectroscopy was performed on a Bruker ALPHA FTIR model (Bruker, 1 13 residual the solvent signal in part per million (δ) (CD3OD: H: δ 3.31, C: δ 49.1; DMSO- Billerica, MA, USA). Optical rotation was recorded on a Biobase Bk-P2s Digital Automatic 1 13 1 13 1 d6: H: δ 2.50, C: δ 39.5; CDCl3: H: δ 7.26, C: δ 77.23). For the H-NM ® R spectrum, data Polarimeter (Jinan, China). The purification was performed on a Biotage Selekt Automated are assumed to be first order with apparent singlet, doublet, triplet, quartets and multiplet flash column chromatography (Biotage, Uppsala, Sweden) where indicated. Chiral HPLC reported as s, d, t, q, and m, respectively. Doublet of doublet was reported as dd, triplet analysis was run on a Waters Alliance e2695 HPLC system with a Waters 2489 UV/Vis of doublet was reported as td, and the resonance that ap ®pears broad was designated as Detector (Waters, Milford, MA, USA) using a Chiralpak ADH column (dimension (Ø) of br. High resolution mass spectral measurements were performed on a Thermo Scientific 4.6 mm  250 mm) (Daicel, Osaka, Japan). Orbitrap Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). TLC was performed on TLC aluminium sheet coated with silica gel 60 F254 (Merck, Molbank 2022, 2022, x FOR PEER REVIEW 5 of 9 Molbank 2022, 2022, x FOR PEER REVIEW 5 of 9 Darmstadt, Germany). To visualize spots on the TLC sheet, UV lamps were used. Melting points were measured on a Buchi melting point M-565 (Büchi, Flawil, Switzerland). Fou- Darmstadt, Germany). To visualize spots on the TLC sheet, UV lamps were used. Melting rier Transform Infrared (FTIR) spectroscopy was performed on a Bruker ALPHA FTIR points were measured on a Buchi melting point M-565 (Büchi, Flawil, Switzerland). Fou- model (Bruker, USA). Optical rotation was recorded on a Biobase Bk-P2s Digital Auto- rier Transform Infrared (FTIR) spectroscopy was performed on a Bruker ALPHA ® FTIR matic Polarimeter (Shandong, China). The purification was performed on a Biotage Sel- model (Bruker, USA). Optical rotation was recorded on a Biobase Bk-P2s Digital Auto- ekt Automated flash column chromatography (Biotage, Uppsala, Sweden) where indi- matic Polarimeter (Shandong, China). The purification was performed on a Biotage Sel- cated. Chiral HPLC analysis was run on a Waters Alliance e2695 HPLC system with a ekt Automated flash column chromatography (Biotage, Uppsala, Sweden) where indi- Waters 2489 UV/Vis Detector (Waters, USA) using a Chiralpak ADH column (dimension cated. Chiral HPLC analysis was run on a Waters Alliance e2695 HPLC system with a (Ø) of 4.6 mm × 250 mm) (Daicel, Japan). Waters 2489 UV/Vis Detector (Waters, USA) using a Chiralpak ADH column (dimension Molbank 2022, 2022, M1358 5 of 9 (Ø) of 4.6 mm × 250 mm) (Daicel, Japan). 3.2. Synthesis 3.2.1. (–)-Ethyl (2-aminobenzoyl)-L-valinate (1) 3.2. Synthesis 3.2. Synthesis 3.2.1. (–)-Ethyl (2-aminobenzoyl)-L-valinate (1) 3.2.1. (–)-Ethyl (2-aminobenzoyl)-L-valinate (1) To a solution of acetonitrile (75 mL) in a round bottom flask, isatoic anhydride (1.6 g, 10 mmol, 1 eq), L-valine ethyl ester hydrochloride (1.8 g, 10 mmol, 1 eq), and potassium To a solution of acetonitrile (75 mL) in a round bottom flask, isatoic anhydride (1.6 g, carbonate (3.4 g, 25 mmol, 2.5 eq) were added. The reaction was allowed stirred and To a solution of acetonitrile (75 mL) in a round bottom flask, isatoic anhydride (1.6 g, 10 mmol, 1 eq), L-valine ethyl ester hydrochloride (1.8 g, 10 mmol, 1 eq), and potassium heated to 60 °C for 18 h. After that the mixture was allowed to cool to room temperature 10 mmol, 1 eq), L-valine ethyl ester hydrochloride (1.8 g, 10 mmol, 1 eq), and potassium carbonate (3.4 g, 25 mmol, 2.5 eq) were added. The reaction was allowed stirred and carbonate (3.4 g, 25 mmol, 2.5 eq) wer an ed added. evapoThe rated reaction to remwas ove th allowed e solvestirr nt. T ed he and resul heated ting residue was then stirred in a 0.4 M heated to 60 °C for 18 h. After that the mixture was allowed to cool to room temperature Na2CO3 solution for an hour and the mixture was extracted with CH2Cl2. The organic to 60 C for 18 h. After that the mixture was allowed to cool to room temperature and and evaporated to remove the solvent. The resulting residue was then stirred in a 0.4 M evaporated to remove the solvent. The pha resulting se was co residue llectedwas , drie then d wistirr th aed nhy in da ro0.4 us M Mg Na SO4 CO , and evaporated to dryness by a rotary 2 3 Na2CO3 solution for an hour and the mixture was extracted with CH2Cl2. The organic solution for an hour and the mixture evwas aporextracted ator. Puriwith ficatio CH n wCl as .pThe erfor or mganic ed usph ing ase colwas umn chromatography (CC) over silica 2 2 phase was collected, dried with anhydrous MgSO4, and evaporated to dryness by a rotary collected, dried with anhydrous MgSO , and evaporated to dryness by a rotary evaporator. gel (30–50% EtOAc/Hexanes) to yield ethyl (2-aminobenzoyl)-L-valinate 1. (1.39 g, 55% evaporator. Purification was performed using column chromatography (CC) over silica Purification was performed using column chromatography (CC) over silica gel (30–50% yield). gel (30–50% EtOAc/Hexanes) to yield ethyl (2-aminobenzoyl)-L-valinate 1. (1.39 g, 55% EtOAc/Hexanes) to yield ethyl (2-aminobenzoyl)-L-valinate (–)-Ethyl (2-aminoben 1.zo (1.39 yl)-Lg, -v55% alinayield). te (1): yellow oil, Enantiomeric excess (>99%) was yield). ® (–)-Ethyl (2-aminobenzoyl)-L-valinate (1): yellow oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chiralpak ADH), hexane:i-PrOH 90:10, 1.0 (–)-Ethyl (2-aminobenzoyl)-L-valinate (1): yellow oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chiralpak ADH), hexane:i-PrOH 90:10, 1.0 mL/min, mL/min, tr = 21.73 min, [α]D = −53.1 (c = 1.5, DMSO), Rf: 0.46 (30% EtOAc/Hexanes), FTIR 1 ® −1 t = 21.73 min, [ ] = 53.1 (c = 1.5,dDMSO), etermine Rf: d b 0.46 y c(30% hiral EtOAc/Hexanes), HPLC analysis ( FTIR Chira (cm lpak ): ADH), hexane:i-PrOH 90:10, 1.0 r D (cm ): 3457.7 (NH-C=O), 3380.8 and 3353.5 (NH2), 2967.7, 2925.7 and 2873.8 (=C-H aro- 3457.7 (NH-C=O), 3380.8 and 3353.5 mL(NH /min,), tr 2967.7, = 21.73 2925.7 min, [α and ]D = 2873.8 −53.1 (c (=C-H = 1.5, D arM omatic), SO), Rf: 0.46 (30% EtOAc/Hexanes), FTIR matic), 2 1735.4 (O-C=O), and 1638.2 (O=C-NH), H-NMR (400 MHz, CDCl3): δ = 7.40 (d, J = −1 1735.4 (O-C=O), and 1638.2 (O=C-NH), H-NMR (400 MHz, CDCl ):  = 7.40 (d, J = 7.8 Hz, (cm ): 3457.7 (NH-C=O), 3380.8 and 3353.5 (NH2), 2967.7, 2925.7 and 2873.8 (=C-H aro- 7.8 Hz, 1H), 7.17 (td, J = 5.4, 7.7 Hz, 1H), 6.65–6.61 (m, 2H), 4.57 (dd, J = 5.0, 8.5, 1H), 4.26- 1H), 7.17 (td, J = 5.4, 7.7 Hz, 1H), 6.65–6.61 matic), 1 (m, 7352H), .4 (O4.57 -C=O (dd, ), anJd = 1 5.0, 6388.5, .2 (O 1H), =C-NH) 4.26-4.13 , H-NM (m,R (400 MHz, CDCl3): δ = 7.40 (d, J = 4.13 (m, 2H), 2.27-2.19 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 2H), 2.27-2.19 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.9 Hz, 7.8 Hz, 1H), 7 13.17 (td, J = 5.4, 7.7 Hz, 1H), 6.65–6.61 (m, 2H), 4.57 (dd, J = 5.0, 8.5, 1H), 4.26- 6.9 Hz, 3H); C-NMR (100 MHz, CDCl3); δ = 172.17, 168.99, 148.66, 132.39, 127.35, 117.18, 3H); C-NMR (100 MHz, CDCl );  = 172.17, 168.99, 148.66, 132.39, 127.35, 117.18, 116.51, + 4.13 (m, 2H), 2.27-2.19 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 116.51, 115.62, 61.24, 57.01, 31.40, 18.91, 17.88, 14.13. ESI-HRMS (m/z): 265.1541 [M + H] 115.62, 61.24, 57.01, 31.40, 18.91, 17.88, 14.13. ESI-HRMS ( +m/z): 265.1541 [M + H] (calcd. 6.9 Hz, 3H); C-NMR (100 MHz, CDCl3); δ = 172.17, 168.99, 148.66, 132.39, 127.35, 117.18, (calcd. for C14H21N2O3 265.1547). for C H N O 265.1547). 14 21 2 3 116.51, 115.62, 61.24, 57.01, 31.40, 18.91, 17.88, 14.13. ESI-HRMS (m/z): 265.1541 [M + H] (calcd. for C14H21N2O3 265.1547). 3.2.2. (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2) 3.2.2. (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2) 3.2.2. (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2) To a solution of compound 1 (1.39 g, 5.3 mmol, 1 eq) in THF (40 mL), CDI (1.71 g, 10.5 To a solution of compound 1 (1.39 g, 5.3 mmol, 1 eq) in THF (40 mL), CDI (1.71 g, mmol, 2 eq) was added. The reaction was stirred for 18 h at 85 °C. When completed, the 10.5 mmol, 2 eq) was added. The reaction was stirred for 18 h at 85 C. When completed, the To a solution of compound 1 (1.39 g, 5.3 mmol, 1 eq) in THF (40 mL), CDI (1.71 g, 10.5 reaction was concentrated by a rotary evaporator. The resulting residue was then dis- reaction was concentrated by a rotary evaporator. The resulting residue was then dissolved mmol, 2 eq) was added. The reaction was stirred for 18 h at 85 °C. When completed, the solved in EtOAc, washed with water, and dried over MgSO4. The organic portion was in EtOAc, washed with water, and dried over MgSO . The organic portion was filtered and reaction was concentrated by a rotary evaporator. The resulting residue was then dis- filtered and concentrated to give a crude product. Purification was performed using CC concentrated to give a crude product. Purification was performed using CC over silica gel solved in EtOAc, washed with water, and dried over MgSO4. The organic portion was over silica gel (30–50% EtOAc/Hexanes) to obtain the desired cyclized product 2 (1.49 g, (30–50% EtOAc/Hexanes) to obtain the desired cyclized product 2 (1.49 g, 97% yield). filtered and concentrated to give a crude product. Purification was performed using CC 97% yield). (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2): yel- over silica gel (30–50% EtOAc/Hexanes) to obtain the desired cyclized product 2 (1.49 g, low oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chiralpak 97% yield). ADH), gradient hexane:i-PrOH 95:5 to 98:2, 1.0 mL/min, t = 61.92 min. [ ] = 156.3 (c = 1.5, DMSO), R : 0.26 (30% EtOAc/Hexanes), FTIR (cm ): 3253.5 (NH-C=O), 2965.5, 2928.6 and 2873.6 (=C-H aromatic), 1746.6 (O-C=O), 1716.9 (O=C-N-R), and 1656.4 (O=C-NH), H-NMR (400 MHz, MeOD-d ):  = 8.02 (dd, J = 1.2, 8.0 Hz, 1H), 7.67 (td, J = 1.4, 7.8 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 5.12 (d, J = 9.3 Hz, 1H), 4.21-4.07 (m, 2H), 2.77–2.68 (m, 1H), 1.67 (d, J = 6.5 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H), 0.76 (d, J = 6.9 Hz, 3H); C-NMR (100 MHz, MeOD-d );  = 171.22, 164.09, 152.11, 140.84, 136.79, 129.16, 124.43, 116.33, 114.95, 62.25, 60.12, 28.69, 22.51, 19.26, 14.45. ESI-HRMS: m/z calculated for C H N O ([M + H] ) 291.1339 found 291.1335. 15 18 2 4 Molbank 2022, 2022, x FOR PEER REVIEW 6 of 9 Molbank 2022, 2022, x FOR PEER REVIEW 6 of 9 (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2): (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2): yellow oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chi- yellow oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chi- ralpak AD ®H), gradient hexane:i-PrOH 95:5 to 98:2, 1.0 mL/min, tr = 61.92 min. [α]D = ralpak ADH), gradient hexane:i-PrOH 95:5 to 98:2, 1.0 mL/min, tr = 61.92 min. [α]D = −1 −156.3 (c = 1.5, DMSO), Rf: 0.26 (30% EtOAc/Hexanes), FTIR (cm ): 3−1 253.5 (NH-C=O), −156.3 (c = 1.5, DMSO), Rf: 0.26 (30% EtOAc/Hexanes), FTIR (cm ): 3253.5 (NH-C=O), 2965.5, 2928.6 and 2873.6 (=C-H aromatic), 1746.6 (O-C=O), 1716.9 (O=C-N-R), and 1656.4 2965.5, 2928.6 and 2873.6 (=C-H aromatic), 1746.6 (O-C=O), 1716.9 (O=C-N-R), and 1656.4 (O=C-NH), H-NMR (400 MHz, MeOD-d4): δ = 8.02 (dd, J = 1.2, 8.0 Hz, 1H), 7.67 (td, J = (O=C-NH), H-NMR (400 MHz, MeOD-d4): δ = 8.02 (dd, J = 1.2, 8.0 Hz, 1H), 7.67 (td, J = 1.4, 7.8 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 5.12 (d, J = 9.3 Hz, 1H), 4.21- 1.4, 7.8 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 5.12 (d, J = 9.3 Hz, 1H), 4.21- 4.07 (m, 2H), 2.77–2.68 (m, 1H), 1.67 (d, J = 6.5 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H), 0.76 (d, J = 4.07 (m, 2H), 2.77–2.68 (m, 1H), 1.67 (d, J = 6.5 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H), 0.76 (d, J = 6.9 Hz, 3H); C-NM 13 R (100 MHz, MeOD-d4); δ = 171.22, 164.09, 152.11, 140.84, 136.79, 6.9 Hz, 3H); C-NMR (100 MHz, MeOD-d4); δ = 171.22, 164.09, 152.11, 140.84, 136.79, 129.16, 124.43, 116.33, 114.95, 62.25, 60.12, 28.69, 22.51, 19.26, 14.45. ESI-HRMS: m/z calcu- 129.16, 124.43, 116.33, 114.95, 62.25, 60.12, 28.69, 22.51, 19.26, 14.45. ESI-HRMS: m/z calcu- Molbank 2022, 2022, M1358 6 of 9 lated for C15H18N2O4 ([M + H] ) 291.1339 found 291.1335. lated for C15H18N2O4 ([M + H] ) 291.1339 found 291.1335. 3.2.3. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3) 3.2.3. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3) 3.2.3. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3) A solution of LiOH (0.3 g, 12.9 mmol, 2.5 eq) in water (6 mL) was added into a solu- A solution of LiOH (0.3 g, 12.9 mmol, 2.5 eq) in water (6 mL) was added into a solu- A solution of LiOH (0.3 g, 12.9 mmol, 2.5 eq) in water (6 mL) was added into a solution tion of compound 2 (1.49 g, 5.1 mmol, 1 eq) in THF (20 mL). The reaction mixture was tion of compound 2 (1.49 g, 5.1 mmol, 1 eq) in THF (20 mL). The reaction mixture was of compound 2 (1.49 g, 5.1 mmol, 1 eq) in THF (20 mL). The reaction mixture was heated heated and stirred at 85 °C for 18 h. After that the mixture was allowed to cool down to heated and stirred at 85 °C for 18 h. After that the mixture was allowed to cool down to and stirred at 85 C for 18 h. After that the mixture was allowed to cool down to room room temperature and was concentrated under a reduced pressure. The residue was dis- room temperature and was concentrated under a reduced pressure. The residue was dis- temperature and was concentrated under a reduced pressure. The residue was dissolved solved in 10 mL of H2O and acidified with 1 M HCl. The white precipitate was filtered off solved in 10 mL of H2O and acidified with 1 M HCl. The white precipitate was filtered off in 10 mL of H O and acidified with 1 M HCl. The white precipitate was filtered off and and washed successively with MeOH to afford the desired acid 3 without further purifi- and washed successively with MeOH to afford the desired acid 3 without further purifi- washed successively with MeOH to afford the desired acid 3 without further purification cation (1.26 g, 93% yield). cation (1.26 g, 93% yield). (1.26 g, 93% yield). (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3): (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3): (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3): white white solid, Enantiomeric excess (65%) was determined® by chiral HPLC analysis (Chi- white solid, Enantiomeric excess (65%) was determined by chiral HPLC analysis (Chi- solid, Enantiomeric excess (65%) was determined by chiral HPLC analysis (Chiralpak ralpak AD ®H), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer tr = 20.22 min, minor ralpak ADH), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer tr = 20.22 min, minor ADH), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer t = 20.22 min, minor enan- enantiomer tr = 31.99 min, [α]D = −30.9 (c = 1.4 , DMSO), Rf: 0.08 (20% MeOH/EtOAc), m.p.: enantiomer tr = 31.99 min, [α]D = −30.9 (c = 1.4 , DMSO), Rf: 0.08 (20% MeOH/EtOAc), m.p.: tiomer t = 31.99 min, [ ] = 30.9 (c = 1.4, DMSO), Rf: 0.08 (20% MeOH/EtOAc), m.p.: r D −1 >305.4 °C (decomposed) FTIR (cm ): 3−1 250.1 (NH-C=O), 3078.9 (OH) 2970.3, 2924.8 and >305.4 °C (decomposed) FTIR (cm ): 3250.1 (NH-C=O), 3078.9 (OH) 2970.3, 2924.8 and >305.4 C (decomposed) FTIR (cm ): 3250.1 (NH-C=O), 3078.9 (OH) 2970.3, 2924.8 and 1 1 2850.6 (=C-H aromatic), 1751 (O=C-OH), 1682.4 (O=C-N-R), and 1640.6 (O=C-NH), H- 2850.6 (=C-H aromatic), 1751 (O=C-OH), 1682.4 (O=C-N-R), and 1640.6 (O=C-NH), H- 2850.6 (=C-H aromatic), 1751 (O=C-OH), 1682.4 (O=C-N-R), and 1640.6 (O=C-NH), H- NMR (400 MHz, DMSO-d6): δ = 12.61 (br(-OH), 1H), 11.61 (s(-NH), 1H), 7.95 (d, J = 7.7 Hz, NMR (400 MHz, DMSO-d ):  = 12.61 NM (br(-OH), R (400 M 1H), Hz, 11.61 DMSO (s(-NH), -d6): δ =1H), 12.61 7.95 (br( (- d, OH) J =, 7.7 1H) Hz , 11 , .61 (s(-NH), 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.70 (td, J = 1.1, 7.7 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 4.96 (d, J 1H), 7.70 (td, J = 1.1, 7.7 Hz, 1H), 7.25 1H),(d, 7.70J = (td 7.6 , J = Hz, 1.11H), , 7.7 Hz 7.22 , 1(d, H), J7= .25 8.0 (d,Hz, J = 7 1H), .6 Hz 4.96 , 1H), 7.22 (d, J = 8.0 Hz, 1H), 4.96 (d, J = 9.3 Hz, 1H), 2.66–2.57 (m, 1H) 1.17 (d, J = 6.4 Hz, 3H), 0.68 (d, J = 6.9 Hz, 3H); C-NM 13 R (d, J = 9.3 Hz, 1H), 2.66–2.57 (m, 1H) = 9.1.17 3 Hz(d, , 1H) J ,= 26.4 .66–Hz, 2.57 3H), (m, 10.68 H) 1.(d, 17 (Jd= , J 6.9 = 6.Hz, 4 Hz3H); , 3H), 0.68 (d, J = 6.9 Hz, 3H); C-NMR (100 MHz, DMSO); δ = 172.21, 161.95, 150.22, 139.53, 135.19, 127.59, 122.64, 115,19, 113.48, C-NMR (100 MHz, DMSO);  = (172.21, 100 MHz 161.95, , DMS150.22, O); δ = 1 139.53, 72.21, 1135.19, 61.95, 15 127.59, 0.22, 13 122.64, 9.53, 135.19, 127.59, 122.64, 115,19, 113.48, + + 59.22, 26.93, 22.75, 19.28. ESI-HRMS (m/z): 263.1025 [M + H] (ca+ lcd. for C13H15N2O4 + 115,19, 113.48, 59.22, 26.93, 22.75, 19.28. ESI-HRMS (m/z): 263.1025 [M + H] (calcd. for 59.22, 26.93, 22.75, 19.28. ESI-HRMS (m/z): 263.1025 [M + H] (calcd. for C13H15N2O4 263.1026). C H N O 263.1026). 263.1026). 13 15 2 4 3.2.4. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3- 3.2.4. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3-me- 3.2.4. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3-me- methyl-butanamide (4) [TCMDC-125133] thyl-butanamide (4) [TCMDC-125133] thyl-butanamide (4) [TCMDC-125133] To a solution of acid 3 (0.26 g, 1.0 mmol, 1 eq) in DMF (4 mL), triethylamine (TEA) To a solution of acid 3 (0.26 g, 1.0 mmol, 1 eq) in DMF (4 mL), triethylamine (TEA) To a solution of acid 3 (0.26 g, 1.0 mmol, 1 eq) in DMF (4 mL), triethylamine (TEA) (0.14 mL, 1 mmol, 1 eq) and HATU (0.38 g, 1 mmol, 1 eq) were added. The mixture was (0.14 mL, 1 mmol, 1 eq) and HATU (0.38 g, 1 mmol, 1 eq) were added. The mixture was (0.14 mL, 1 mmol, 1 eq) and HATU (0.38 g, 1 mmol, 1 eq) were added. The mixture was left stirring for 1 h at room temperature, after which m-anisidine (0.17 mL, 1.5 mmol, 1.5 left stirring for 1 h at room temperature, after which m-anisidine (0.17 mL, 1.5 mmol, 1.5 left stirring for 1 h at room temperature, after which m-anisidine (0.17 mL, 1.5 mmol, eq) was added and the reaction was left stirring at room temperature for 18 h. After the eq) was added and the reaction was left stirring at room temperature for 18 h. After the 1.5 eq) was added and the reaction was left stirring at room temperature for 18 h. After the reaction was completed, the solvent was removed under a reduced pressure. The residue reaction was completed, the solvent was removed under a reduced pressure. The residue reaction was completed, the solvent was removed under a reduced pressure. The residue was dissolved in EtOAc, and the solution was extracted with 0.4 M Na CO solution and 2 3 washed with water. The organic layer was collected, dried over MgSO , and evaporated under a reduced pressure. Purification was performed using automated flash column chromatography (Biotage , gradient system of 10–50% EtOAc/Hexanes) to afford the desired quinazolinedione product 4 (TCMDC-125133) (0.29 g, 77% yield). (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3-methylbuta- namide (4) [TCMDC-125133]: light brown solid, Enantiomeric excess (76%) was determined by chiral HPLC analysis (Chiralpak ADH), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer t = 45.21 min, minor enantiomer t = 36.36 min, [ ] = 57.8 (c = 1.1, DMSO), r r Rf: 0.11 (30% EtOAc/Hexanes), m.p.: 72.8–73.4 C, FTIR (cm ): 3206.5 and 3142.1 (NH- C=O), 2959.7, 2928.0 and 2872.3 (=C-H aromatic), 1715.7 and 1648.9 (O=C-N-H), H-NMR (500 MHz, CDCl ):  = 10.51 (s, 1H), 8.87 (s, 1H), 8.06 (d, J = 10.0 Hz, 1H), 7.55 (td, 3 Molbank 2022, 2022, M1358 7 of 9 J = 1.8, 9.5 Hz, 1H), 7.32 (s, 1H), 7.19 (td, J = 1.1, 9.6 Hz, 1H), 7.13 (t, J = 10.0 Hz, 2H), 6.98 (d, J = 10.0 Hz, 1H), 6.59 (dd, J = 2.34, 10.3 Hz, 1H), 5.30 (d, J = 13.4 Hz, 1H), 3.73 (s, 3H), 3.14-3.05 (m, 1H), 1.22 (d, J = 8.2 Hz, 3H), 0.85 (d, J = 8.4 Hz, 3H); C-NMR (125 MHz, CDCl );  = 167.36, 163.56, 160.07, 152.11, 139.08, 138.57, 135.76, 129.56, 123.75, 115.42, 114.08, 112.22, 110.26, 105.69, 64.37, 55.28, 26.80, 21.00, 19.10. ESI-HRMS (m/z): 368.1602 + + [M + H] (calcd. for C H N O 368.1602). 20 22 3 4 3.3. Antimalarial Assay against P. falciparum 3D7 Plasmodium falciparum strain 3D7 was cultured in complete medium (RPMI-1640 sup- plemented with 10% Albumax II) using O Rh+ red blood cell in microaerobic environment (5% CO , 5% O , 90% N ). IC50 assay plates were prepared by four-fold serially diluted 2 2 2 test compounds in complete medium to a final volume of 50 L. Artemisinin at 1 M and complete medium were used as positive and negative controls, respectively. Then, 50 L of parasite inoculum of 2% parasitemia of ring stage and 1% hematocrit was added to each well and incubated for 48 h in a microaerobic environment. The assay was terminated by freezing at 20 C before growth measurement. Parasite growth was measured adding 100 L of lysis buffer supplemented with 1X DNA fluorescent dye (UltraPower, Gellex, Tokyo, Japan) and fluorescent signal was measured at 495/530 nm. The IC value was calculated by GraphPad Prism 9.0 software (La Jolla, CA, USA) using the dose response (four parameter) function. 3.4. Antiproliferative Assay against MCF-7 and HCT-116 Human breast cancer cells (MCF-7) or human colorectal cancer cells (HCT-116 cells) purchased from ATCC were seeded at 2  10 cells/well on a 96-well plate and were cultured by DMEM (Dulbecco’s Modified Eagle Medium) high glucose supplemented with 10% FBS and 1% penicillin/streptomycin. The culture was incubated at 37 C, 5% CO for 24 h. After the incubation period, TCMDC-125133 was added into the cell plate as a dose- response manner at: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10 (M) and additional incubation for 72 h at 37 C, 5% CO . After the 72-h incubation, the cultured medium containing compound was removed and the serum-free media containing MTT was added into the same well with additional incubation for 3 h at 37 C, 5% CO . After 3 h incubation, the serum-free media containing MTT was removed and DMSO was added into the same well and the resulting-coloured solution was measured for its absorbance at 570 nm using a Multi-Mode Microplate Reader (ENVISION) (PerkinElmer, Waltham, MA, USA). The IC value was calculated using GraphPad. Doxorubicin at 10 M was used as a positive control in both assays. 4. Conclusions In conclusion, TCMDC-125133 can be prepared by employing the concise four-step synthesis reported here with good overall yields, low cost of goods, and mild reaction con- ditions. The in-house synthetic TCMDC-125133 was assayed against the P. falciparum 3D7 strain and its highly potent antimalarial activity corresponded to that of those previously published. The anti-proliferative activity of TCMDC-125133 was also performed and it exhibited moderate activity against the MCF-7 cell line. The result of this work confirmed the integrity of the TCMDC-125133 that was synthesized in-house. The presented synthesis would contribute to a future lead optimization campaign for this class of compounds as either antimalarial or antiproliferative agents. 1 13 Supplementary Materials: The following data are available online: H-NMR, C-NMR, IR spectra, mass spectra, and chiral HPLC traces of compound 1–4. Author Contributions: S.C. initiated the overall concept, designed the synthetic route, and secured funding. D.L., S.C. performed the chemical synthesis. D.L. carried out compound characterization. M.P. carried out antimalarial assay and its interpretation. P.K., K.J., S.S., S.B. carried out anti- Molbank 2022, 2022, M1358 8 of 9 proliferative assay and its interpretation. S.C., D.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: S.C. was funded by the New Researcher Grant (A13/2563), Mahidol University. This research was partially supported by the Faculty of Science, Mahidol University, the Ramathibodi Foundation, and the Thailand Center of Excellence for Life Sciences (TCELS). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article and supporting supplementary material. Acknowledgments: The authors wish to thank Suttiporn Pikulthong, Samreang Bunteang and the Department of Chemistry, Faculty of Science, Mahidol University for access to the NMR, FTIR and melting point instruments, Tawatchai Thongkongkaew (Chulabhorn Graduate Institute) for access to the high-resolution mass spectrometry facility and optical activity measurement, and Torsak Luanphaisarnnont, the Central Instrument Facility and Napason Chabang for their help in chiral HPLC analysis. This research project is supported by the Faculty of Science and Mahidol University. Conflicts of Interest: The authors declare no conflict of interest. References 1. Birhan, Y.S.; Bekhit, A.A.; Hymete, A. In Vivo antimalarial evaluation of some 2,3-disubstituted-4(3H)-quinazolinone derivatives. BMC Res. Notes 2015, 8, 589. [CrossRef] [PubMed] 2. Gouhar, R.S.; Kamel, M.M. Synthesis and Reactions of Some New Quinazoline Derivatives forIn VitroEvaluation as Anticancer and Antimicrobial Agents. J. Heterocycl. Chem. 2018, 55, 2082–2089. [CrossRef] 3. Tsuchihashi, H.; Nagatomo, T. Alpha-blocking potencies of antihypertensive agents (prazosin, terazosin, bunazosin, SGB-1534 and ketanserin) having with quinazoline or quinazolinedione as assessed by radioligand binding assay methods in rat brain. J. Pharmacobiodyn. 1989, 12, 170–174. [CrossRef] [PubMed] 4. Matharu, D.S.; Flaherty, D.P.; Simpson, D.S.; Schroeder, C.E.; Chung, D.; Yan, D.; Noah, J.W.; Jonsson, C.B.; White, E.L.; Aubé, J.; et al. Optimization of Potent and Selective Quinazolinediones: Inhibitors of Respiratory Syncytial Virus That Block RNA-Dependent RNA-Polymerase Complex Activity. J. Med. Chem. 2014, 57, 10314–10328. [CrossRef] [PubMed] 5. Baraka, M.M. Synthesis of novel 2,4 (1H, 3H)-quinazolinedione derivatives with analgesic and anti-inflammatory activities. Boll. Chim. Farm. 2001, 140, 90–96. [PubMed] 6. Zhang, X.; Ding, Q.; Wang, J.; Yang, J.; Fan, X.; Zhang, G. Pd(ii)-Catalyzed [4 + 1 + 1] cycloaddition of simple o-aminobenzoic acids, CO and amines: Direct and versatile synthesis of diverse N-substituted quinazoline-2,4(1H,3H)-diones. Green Chem. 2021, 23, 526–535. [CrossRef] 7. Banasr, M.; Hery, M.; Printemps, R.; Daszuta, A. Serotonin-induced increases in adult cell proliferation and neurogene- sis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone. Neuropsychopharmacology 2004, 29, 450–460. [CrossRef] [PubMed] 8. Sonier, B.; Arseneault, M.; Lavigne, C.; Ouellette, R.J.; Vaillancourt, C. The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin. Biochem. Biophys. Res. Commun. 2006, 343, 1053–1059. [CrossRef] [PubMed] 9. Hejazi, S.H.; Ahangari, G.; Deezagi, A. Alternative Viewpoint Against Breast Cancer Based on Selective Serotonin Receptors 5HTR3A and 5HTR2A Antagonists that can Mediate Apoptosis in MCF-7 Cell Line. Curr. Drug Discov. Technol. 2015, 12, 240–249. [CrossRef] [PubMed] 10. Matthews, H. Accelerating Antimalarial Drug Discovery through Repositioning; University of Salford: Salford, UK, 2015. 11. Calderon, F.; Barros, D.; Bueno, J.M.; Coteron, J.M.; Fernandez, E.; Gamo, F.J.; Lavandera, J.L.; Leon, M.L.; Macdonald, S.J.; Mallo, A.; et al. An Invitation to Open Innovation in Malaria Drug Discovery: 47 Quality Starting Points from the TCAMS. ACS Med. Chem. Lett. 2011, 2, 741–746. [CrossRef] [PubMed] 12. Almela, M.J.; Lozano, S.; Lelievre, J.; Colmenarejo, G.; Coteron, J.M.; Rodrigues, J.; Gonzalez, C.; Herreros, E. A New Set of Chemical Starting Points with Plasmodium falciparum Transmission-Blocking Potential for Antimalarial Drug Discovery. PLoS ONE 2015, 10, e0135139. [CrossRef] [PubMed] 13. Goto, S.; Tsuboi, H.; Kanoda, M.; Mukai, K.; Kagara, K. The Process Development of a Novel Aldose Reductase Inhibitor, FK366. Part 1. Improvement of Discovery Process and New Syntheses of 1-Substituted Quinazolinediones. Org. Process Res. Dev. 2003, 7, 700–706. [CrossRef] 14. Ismail, M.A.H.; Barker, S.; Abou El Ella, D.A.; Abouzid, K.A.M.; Toubar, R.A.; Todd, M.H. Design and Synthesis of New Tetrazolyl- and Carboxy-biphenylylmethyl-quinazolin-4-one Derivatives as Angiotensin II AT1 Receptor Antagonists. J. Med. Chem. 2006, 49, 1526–1535. [CrossRef] [PubMed] Molbank 2022, 2022, M1358 9 of 9 15. Nencini, A.; Pratelli, C.; Quinn, J.M.; Salerno, M.; Tunici, P.; De Robertis, A.; Valensin, S.; Mennillo, F.; Rossi, M.; Bakker, A.; et al. Structure-activity relationship and properties optimization of a series of quinazoline-2,4-diones as inhibitors of the canonical Wnt pathway. Eur. J. Med. Chem. 2015, 95, 526–545. [CrossRef] [PubMed] 16. Yoon, Y.-J.; Park, J.; Kim, B.; Lee, H.-G.; Kang, S.-B.; Sung, G.; Kim, J.-J.; Lee, S.-G. tert-Butoxide-Assisted Amidation of Esters under Green Conditions. Synthesis 2011, 44, 42–50. [CrossRef] 17. Carpino, L.A.; Imazumi, H.; Foxman, B.M.; Vela, M.J.; Henklein, P.; El-Faham, A.; Klose, J.; Bienert, M. Comparison of the Effects of 5- and 6-HOAt on Model Peptide Coupling Reactions Relative to the Cases for the 4- and 7-Isomers. Org. Lett. 2000, 2, 2253–2256. [CrossRef] [PubMed] http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molbank Multidisciplinary Digital Publishing Institute

A Concise Synthesis towards Antimalarial Quinazolinedione TCMDC-125133 and Its Anti-Proliferative Activity against MCF-7

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molbank Communication Communication A Concise Synthesis towards Antimalarial Quinazolinedione A Concise Synthesis towards Antimalarial Quinazolinedione TCMDC-125133 and Its Anti-Proliferative Activity TCMDC-125133 and Its Anti-Proliferative Activity against MCF-7 against MCF-7 1 , 2 , 3 , 1 2 1,2,3, 1 2 Sitthivut Charoensutthivarakul * , Duangporn Lohawittayanan , Phongthon Kanjanasirirat , Sitthivut Charoensutthivarakul *, Duangporn Lohawittayanan , Phongthon Kanjanasirirat , 2 2 2 , 4 5 2 2 2,4 5 Kedchin Jearawuttanakul , Sawinee Seemakhan , Suparerk Borwornpinyo and Matthew Phanchana Kedchin Jearawuttanakul , Sawinee Seemakhan , Suparerk Borwornpinyo and Matthew Phanchana 1 1 Innovative Molecular Discovery Laboratory (iMoD), School of Bioinnovation and Bio-Based Product Innovative Molecular Discovery Laboratory (iMoD), School of Bioinnovation and Bio-Based Product Intelligence, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; duangporn.loha@gmail.com Intelligence, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; duangporn.loha@gmail.com 2 2 Excellent Center for Drug Discovery (ECDD), Faculty of Science, Mahidol University, Excellent Center for Drug Discovery (ECDD), Faculty of Science, Mahidol University, Ba Bangkok ngkok 110400, 0400, TThailand; hailand; pphongku1232@gmail.com hongku1232@gmail.com (P (P .K .K.); .); o o-nion@hotmail.com -nion@hotmail.com ((K.J.); K.J.); sawinee.ecdd@gmail.com (S.S.); bsuparerk@gmail.com (S.B.) sawinee.ecdd@gmail.com (S.S.); bsuparerk@gmail.com (S.B.) 3 3 Center for Neuroscience, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Center for Neuroscience, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 4 4 Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 5 5 Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, M Bangkok ahidol U 10400, niversi Thailand; ty, Bangkmatthew ok 10400.pha@mahidol.edu , Thailand; matthew.pha@mahidol.edu * Correspondence: sitthivut.cha@mahidol.ac.th; Tel.: +66-2201-5899 * Correspondence: sitthivut.cha@mahidol.ac.th; Tel.: +66-2201-5899 Abstract: Quinazolinedione is one of the most notable pharmacophores in drug discovery due to its Abstract: Quinazolinedione is one of the most notable pharmacophores in drug discovery due broad spectrum of biological activities including antimalarial, anticancer, anti-inflammatory, and to its broad spectrum of biological activities including antimalarial, anticancer, anti-inflammatory, others. TCMDC-125133, whose structure features a quinazolinedione core, exhibits promising anti- and others. TCMDC-125133, whose structure features a quinazolinedione core, exhibits promising malarial activity and low toxicity as described in the GlaxoSmithKline (GSK) report. Herein, a con- antimalarial activity and low toxicity as described in the GlaxoSmithKline (GSK) report. Herein, cise four-step synthesis towards quinazolinedione TCMDC-125133 is described using low cost a concise four-step synthesis towards quinazolinedione TCMDC-125133 is described using low cost Citation: Citation: Char Charoensutthivarakul, oensutthivarakul, S.; S.; goods and greener alternatives where possible. All synthesized compounds were characterized us- goods and greener alternatives where possible. All synthesized compounds were characterized Lohawittayanan, Lohawittayanan, D.; D.; Kanjanasirirat, Kanjanasirirat, ing polarimetry, IR, NMR, and mass spectrometry. The in-house synthesized TCMDC-125133 was using polarimetry, IR, NMR, and mass spectrometry. The in-house synthesized TCMDC-125133 was P P. .;; Jearawuttanakul, Jearawuttanakul, K.; K.;Seemakhan, Seemakhan, evaluated for its antimalarial activity against P. falciparum 3D7 and antiproliferative activity against evaluated for its antimalarial activity against P. falciparum 3D7 and antiproliferative activity against S.; S.; Borwornpinyo, Borwornpinyo, S.; S.; Phanchana, Phanchana, M. M. MCF-7 cell line. A Concise Synthesis towards A Concise Synthesis towards MCF-7 cell line. Antimalarial Quinazolinedione Antimalarial Quinazolinedione TCMDC-125133 and Its Anti- Keywords: quinazolinedione derivatives; antimalarial activity; antiproliferative activity TCMDC-125133 and Its Keywords: quinazolinedione derivatives; antimalarial activity; antiproliferative activity Proliferative Activity against MCF-7. Anti-Proliferative Activity against Molbank 2022, 2022, M1358. https:// MCF-7. Molbank 2022, 2022, x. doi.org/10.3390/M1358 https://doi.org/10.3390/xxxxx 1. 1. Introduction Introduction Academic Editor: Stanislav Kafka Academic Editor: Stanislav Kafka Quinazolinedione is a remarkable heterocycle which is widely used as a functional ma- Quinazolinedione is a remarkable heterocycle which is widely used as a functional Received: Received: 25 25 Febr February uary 2022 2022 terial materfor ial f synthetic or synthechemistry tic chemi.str Ityis . It also is apr lso esent presin envarious t in vario active us acti pharmaceutical ve pharmaceuti molecules cal mole- Accepted: Accepted: 18 18 April April 2022 2022 along with its diverse range of biological activities, including antimalarial, anticancer, an- cules along with its diverse range of biological activities, including antimalarial, anti- Published: Published: 21 21 April April 2022 2022 timicrobial, antihypertensive, antiviral, anti-inflammatory, and others [1–5]. The examples cancer, antimicrobial, antihypertensive, antiviral, anti-inflammatory, and others [1–5]. Publisher Publisher’ ’s s Note: Note:MDPI MDPI stays stayneutral s neu- shown in Figure 1 are some representatives of commercial drugs and biologically active The examples shown in Figure 1 are some representatives of commercial drugs and bio- with tral r w egar ith d rto egju arrisdictional d to jurisd claims ictional in molecules with quinazolinedione moiety [6]. logically active molecules with quinazolinedione moiety [6]. published maps and institutional affil- claims in published maps and institu- iations. tional affiliations. Copyright: © 2022 by the authors. Copyright: © 2022 by the authors. Submitted for possible open access Licensee MDPI, Basel, Switzerland. publication under the terms and con- This article is an open access article ditions of the Creative Commons At- distributed under the terms and tribution (CC BY) license (https://cre- conditions of the Creative Commons Figure 1. Examples of quinazolinedione-based drugs and its biological activity. Figure 1. Examples of quinazolinedione-based drugs and its biological activity. ativecommons.org/licenses/by/4.0/). Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ One of the interesting quinazolinedione-containing drugs is ketanserin, as displayed 4.0/). in Figure 2a. This drug is used clinically as an antihypertensive agent selectively targeting Molbank 2022, 2022, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/molbank Molbank 2022, 2022, M1358. https://doi.org/10.3390/M1358 https://www.mdpi.com/journal/molbank Molbank 2022, 2022, x FOR PEER REVIEW 2 of 9 Molbank 2022, 2022, M1358 2 of 9 One of the interesting quinazolinedione-containing drugs is ketanserin, as displayed in Figure 2a. This drug is used clinically as an antihypertensive agent selectively targeting at 5-HT2A receptor, a subtype of the 5-HT2 receptor that belongs to the serotonin receptor at 5-HT receptor, a subtype of the 5-HT receptor that belongs to the serotonin receptor 2A 2 family (5-HT). Several studies have reported that serotonin was involved in the regulation family (5-HT). Several studies have reported that serotonin was involved in the regulation of cell proliferation, survival, and metastasis [7]. Moreover, it is reported that 5-HT plays of cell proliferation, survival, and metastasis [7]. Moreover, it is reported that 5-HT plays a mitogenic role and exerts its growth effect in human breast cancer cell line (MCF-7) [8]. a mitogenic role and exerts its growth effect in human breast cancer cell line (MCF-7) [8]. Recently, Seyed H Hejazi and his group studied the pattern of serotonin receptor gene Recently, Seyed H Hejazi and his group studied the pattern of serotonin receptor gene expression in MCF-7. Their result showed that the ketanserin had suppression effects on expression in MCF-7. Their result showed that the ketanserin had suppression effects MCF-7 cell’s proliferation of 72.36% at the ketanserin concentration of 25 µM [9]. In a pre- on MCF-7 cell’s proliferation of 72.36% at the ketanserin concentration of 25 M [9]. In a lim prieliminary nary drugdr re ug pur repurposing posing studstudy y, ket,aketanserin nserin surp surprisingly risingly poss possesses esses googood d antiantimal malaria alr a ial c- tivity with the IC50 against multidrug-resistant P. falciparum K1 at around 10 µM [10]. activity with the IC against multidrug-resistant P. falciparum K1 at around 10 M [10]. Figure 2. Chemical structures of (a) Ketanserin and (b) TCMDC-125133 with quinazolinedione core Figure 2. Chemical structures of (a) Ketanserin and (b) TCMDC-125133 with quinazolinedione core highlighted highlighted in in blue. blue. In 2010, GlaxoSmithKline (GSK) published the Tres Cantos Antimalarial Set (TCAMS) In 2010, GlaxoSmithKline (GSK) published the Tres Cantos Antimalarial Set containing 13,533 compounds that are the result of screening nearly 2 million compounds (TCAMS) containing 13,533 compounds that are the result of screening nearly 2 million from the GSK corporate collection [11]. With the result openly available in the public compounds from the GSK corporate collection [11]. With the result openly available in the domain, one of the compounds identified from the screen as a singleton is TCMDC-125133 public domain, one of the compounds identified from the screen as a singleton is TCMDC- whose structure featured a quinazolinedione core as displayed in Figure 2b. The preliminary 125133 whose structure featured a quinazolinedione core as displayed in Figure 2b. The in vitro antimalarial activity of this compound against asexual blood stage and gametocytes preliminary in vitro antimalarial activity of this compound against asexual blood stage of P. falciparum 3D7 has shown excellent IC s at 0.27 and 0.54 M, respectively [12]. This, and gametocytes of P. falciparum 3D7 has shown excellent IC50s at 0.27 and 0.54 µM, re- combined with the drug-like property and low toxicity (cytotoxicity HG2; IC > 25 M) of spectively [12]. This, combined with the drug-like property and low toxicity (cytotoxicity this compound, has made it an excellent target for further lead optimization. HG2; IC50 > 25 µM) of this compound, has made it an excellent target for further lead The main structural feature of these compounds is quinazolinedione; however, the optimization. synthesis of quinazolinedione core has been reported to achieve this in multiple steps The main structural feature of these compounds is quinazolinedione; however, the using harsh conditions and tedious workup procedures with low yields [13,14], while synthesis of quinazolinedione core has been reported to achieve this in multiple steps us- there is no previous report on the synthesis towards TCMDC-125133. This present work, ing harsh conditions and tedious workup procedures with low yields [13,14], while there therefore, described a concise synthetic route towards the quinazolinedione TCMDC- is no previous report on the synthesis towards TCMDC-125133. This present work, there- 125133 using commercially available starting materials with a low cost of goods. Several fore, described a concise synthetic route towards the quinazolinedione TCMDC-125133 spectroscopic techniques were employed to confirm the structure of in-house synthesized using commercially available starting materials with a low cost of goods. Several spectro- TCMDC-125133 and the synthetic intermediates. The final compound was assayed for scopic techniques were employed to confirm the structure of in-house synthesized its antimalarial activity against 3D7 and for antiproliferative activity against MCF-7. The TCMDC-125133 and the synthetic intermediates. The final compound was assayed for its resulting synthetic route will be crucial for further structure-activity relationships (SARs) antimalarial activity against 3D7 and for antiproliferative activity against MCF-7. The re- study in lead optimization and development of this class of compounds. sulting synthetic route will be crucial for further structure-activity relationships (SARs) study in lead optimization and development of this class of compounds. 2. Results and Discussion The synthesis towards the quinazolidinone TCMDC-125133 begins with the reaction 2. Results and Discussion between commercially available isatoic anhydride and corresponding L-valine ethyl ester The synthesis towards the quinazolidinone TCMDC-125133 begins with the reaction hydrochloride in the presence of K CO in CH CN to afford compound 1 (55%) [15]. This 2 3 3 between commercially available isatoic anhydride and corresponding L-valine ethyl ester step involves the use of low-cost isatoic anhydride and a mild reaction condition with hydrochloride in the presence of K2CO3 in CH3CN to afford compound 1 (55%) [15]. This a capability to be performed at a multi-gram scale. Although the racemization of the step involves the use of low-cost isatoic anhydride and a mild reaction condition with a -carbon of the valine moiety could be of concern in this step, the working reagent and capability to be performed at a multi-gram scale. Although the racemization of the α-car- condition did not affect the stereochemistry of this optically active molecule as shown by bon of the valine moiety could be of concern in this step, the working reagent and condi- its distinctive levorotatory property and a chiral HPLC trace (>99% ee). For the synthesis of compound tion did no2 t ,athe ffeccyclocarbonylation t the stereochemistrr y eaction of this of op compound tically activ 1ecould molecbe ule performed as shown using by its various distincticarbonylating ve levorotatory reagents property such andas a phosgene chiral HPL or C its traequivalent ce (>99% eederivatives ). For the sy(i.e., ntheethyl sis of chloroformate, diphosgene or triphosgene), however, those reagents are toxic and difficult compound 2, the cyclocarbonylation reaction of compound 1 could be performed using to handle, and some require harsh conditions [6]. 1,1-carbonyldiimidazole (CDI) was chosen as a greener alternative for this reaction owing to its safety and ease of handling. Molbank 2022, 2022, x FOR PEER REVIEW 3 of 9 various carbonylating reagents such as phosgene or its equivalent derivatives (i.e., ethyl chloroformate, diphosgene or triphosgene), however, those reagents are toxic and difficult Molbank 2022, 2022, M1358 3 of 9 to handle, and some require harsh conditions [6]. 1,1-carbonyldiimidazole (CDI) was cho- sen as a greener alternative for this reaction owing to its safety and ease of handling. The resulting intermediate 1 was reacted with CDI in THF to yield the quinazolinedione 2 in The resulting intermediate 1 was reacted with CDI in THF to yield the quinazolinedione 2 97% yield (>99% ee) [15]. in 97% yield (>99% ee) [15]. As shown in Scheme 1 (Route A), it is proposed that the final product 4 (TCMDC- As shown in Scheme 1 (Route A), it is proposed that the final product 4 (TCMDC- 125133) could be made using a tert-butoxide-assisted amidation reaction [16], however, 125133) could be made using a tert-butoxide-assisted amidation reaction [16], however, the reaction was not successful. An alternative synthesis was employed instead starting the reaction was not successful. An alternative synthesis was employed instead starting with hydrolysis of the ethyl ester followed by an amide formation as described in Scheme with hydrolysis of the ethyl ester followed by an amide formation as described in Scheme 1 1 (Route B). The ethyl ester 2 was subsequently hydrolyzed using LiOH in THF/H2O mix- (Route B). The ethyl ester 2 was subsequently hydrolyzed using LiOH in THF/H O mix- ture to give the carboxylic acid 3 without any purification (93%) [15]. It is worth noting ture to give the carboxylic acid 3 without any purification (93%) [15]. It is worth noting that the levorotatory effect can still be observed in compound 3 suggesting the majority that the levorotatory effect can still be observed in compound 3 suggesting the majority of product still retains its configuration (65% ee determined by chiral HPLC) (see Supple- of product still retains its configuration (65% ee determined by chiral HPLC) (see Sup- mentary Materials). To generate the amide 4, it is important to maintain that any harsh plementary Materials). To generate the amide 4, it is important to maintain that any condition could affect the stereochemistry of the α-carbon of the valine moiety, hence, 1- harsh condition could affect the stereochemistry of the -carbon of the valine moiety, [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro- hence, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hex- phosphate (HATU) is employed as it is one of the commonly used reagents in amide cou- afluorophosphate (HATU) is employed as it is one of the commonly used reagents in pling chemistry. Although it is not ‘low-cost’ as originally proposed, HATU was chosen amide coupling chemistry. Although it is not ‘low-cost’ as originally proposed, HATU was because of its mild condition, high coupling efficiencies, fast reaction rates, and its by- chosen because of its mild condition, high coupling efficiencies, fast reaction rates, and product can be easily removed [17]. In the final step, the corresponding acid 3 was then its by-product can be easily removed [17]. In the final step, the corresponding acid 3 was coupled to the m-anisidine side chain using HATU with the presence of triethylamine then coupled to the m-anisidine side chain using HATU with the presence of triethylamine (Et (Et3N) N) in in DMF DMF[ 15 [15 ]]to toaf a for ffo d rd the thquinazolidinone e quinazolidinon 4e(TCMDC-125133) 4 (TCMDC-12513 in 3)77% in 7yield 7% yiwith eld w 76% ith ee 76determined % ee determby inechiral d by cHPLC hiral HP (see LC Supplementary (see SupplemeMaterials). ntary Mater All ials synthesized ). All synthe compounds sized com- described here were fully characterized using polarimetry, IR, NMR, and mass spectrometry. pounds described here were fully characterized using polarimetry, IR, NMR, and mass This newly discovered synthetic route will allow further structural modification around spectrometry. This newly discovered synthetic route will allow further structural modifi- this pharmacophore core. cation around this pharmacophore core. Scheme Scheme 1 1.. R Reagents eagents aand nd C Conditions: onditions: (i( ) iK ) K 2CO CO 3, C ,H CH 3CN CN, , 60 60 °C, 1 C, 8 h 18 ; (i h; i) ( C ii DI ) CDI, , THF THF , 85 ,°C 85 , 18 C, h18 ; (iih; i) 2 3 3 m-anisidine, t-BuOK, THF, room temperature, overnight; (iv) LiOH, THF/H2O, 85 °C, 18 h; (v) m- (iii) m-anisidine, t-BuOK, THF, room temperature, overnight; (iv) LiOH, THF/H O, 85 C, 18 h; anisidine, HATU, Et3N, DMF, room temperature, 18 h. (v) m-anisidine, HATU, Et N, DMF, room temperature, 18 h. Compound Compound 4 4 d derived erived f fr ro om m th this is s synthesis ynthesis w was as a assayed ssayed ffor or iin n v vitr itro o a antimalarial ntimalarial a activ- ctiv- ity ity against against the the blood blood stage stage P P.. falciparum falciparum 3D7 3D7 strain. strain. The The r results esults show show that that the the in-house in-house synthesised compound 4 possesses a promising IC50 (3D7) of 219 nM (Figure 3) which is synthesised compound 4 possesses a promising IC (3D7) of 219 nM (Figure 3) which is com comparable parable to to thethe priprimary mary scre scr eneening ing resul result t by T by CA TCAMS. MS. FurFurther ther leadlead optioptimisation misation is un is - underway derway to e to nh enhance ance thethe anti antimalarial malarial acti activity vity of of thithis s claclass ss of of com compound. pound. As previously mentioned, quinazolinedione containing molecules could potentially be repurposed as an anti-breast cancer agent. In this regard, compound 4 was assessed for in vitro antiproliferative activity against MCF-7 and additionally HCT-116 (human colorectal cancer) (Figure 4). These data show that compound 4 possesses a moderate anti- MCF-7 activity with an IC of 17.5 M, but it also demonstrates a mild antiproliferative activity against HCT-116 (IC = 58.0 M). The result shown here may suggest some selectivity within this class of compound over a specific type of cancer. Further research is ongoing to study the SARs around this pharmacophore. Molbank 2022, 2022, x FOR PEER REVIEW 4 of 9 Molbank 2022, 2022, x FOR PEER REVIEW 4 of 9 Molbank 2022, 2022, M1358 4 of 9 Figure 3. Dose-dependent antimalarial (3D7) activity of compound 4 (TCMDC-125133). The graph shows the reduction in parasitemia of blood stage 3D7 P. falciparum laboratory strain in human red blood cell at serial concentrations of compound 4. As previously mentioned, quinazolinedione containing molecules could potentially be repurposed as an anti-breast cancer agent. In this regard, compound 4 was assessed for in vitro antiproliferative activity against MCF-7 and additionally HCT-116 (human colo- rectal cancer) (Figure 4). These data show that compound 4 possesses a moderate anti- MCF-7 activity with an IC50 of 17.5 µM, but it also demonstrates a mild antiproliferative activity against HCT-116 (IC50 = 58.0 µM). The result shown here may suggest some selec- Figure 3. Dose-dependent antimalarial (3D7) activity of compound 4 (TCMDC-125133). The graph Fti ig v u ir ty e 3w . Do ithsie n- d th ep is en cd la en ss t a o nft ic m oa m lap rio aun l (3d D7 o) v ae cr t iv ai ts yp o ef cc if o im c p ty op ue n d o 4 f ( cTC anMDC cer. F -1 ur 25 th 13 e3 r). r Th ese e a g rrcah p h is shows the reduction in parasitemia of blood stage 3D7 P. falciparum laboratory strain in human red sh oo nw go s itn hg e r ted o s u tud ctioy n th in e p S ar A as R it sem ario aun of d b lth ooid s s p ta hg ae rm 3D7 ac o Pp . h fao lcr ip ea . rum laboratory strain in human red blood cell at serial concentrations of compound 4. blood cell at serial concentrations of compound 4. As previously mentioned, quinazolinedione containing molecules could potentially be repurposed as an anti-breast cancer agent. In this regard, compound 4 was assessed for in vitro antiproliferative activity against MCF-7 and additionally HCT-116 (human colo- rectal cancer) (Figure 4). These data show that compound 4 possesses a moderate anti- MCF-7 activity with an IC50 of 17.5 µM, but it also demonstrates a mild antiproliferative activity against HCT-116 (IC50 = 58.0 µM). The result shown here may suggest some selec- tivity within this class of compound over a specific type of cancer. Further research is ongoing to study the SARs around this pharmacophore. Figure 4. Dose-dependent anti-MCF-7 (human breast cancer; in red) and anti-HCT-116 (human Figure 4. Dose-dependent anti-MCF-7 (human breast cancer; in red) and anti-HCT-116 (human col- colorectal cancer; in blue) activities of compound 4 (TCMDC-125133). The curves show the cell orectal cancer; in blue) activities of compound 4 (TCMDC-125133). The curves show the cell viability p viability ercentagper e ocentage f each caof nceach er celcancer l line acell t serline ial cat onserial centraconcentrations tions of compoof uncompound d 4. 4. 3. Materials and Methods 3. Materials and Methods 3.1. General 3.1. General All reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich, All reagents and solvents were purchased from commercial suppliers (Sigma-Al- Merck, or Tokyo Chemical Industry (TCI)) and were used without further purification. drich, Merck, or Tokyo Chemical Industry (TCI)) and were used without further purifica- 1 13 NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz H/ C tion. NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz 1 13 and 500/125 MHz H/ C) spectrometer (Bruker, Billerica, MA, USA) and chemical shifts 1 13 1 13 H/ C and 500/125 MHz H/ C) spectrometer (Bruker, Billerica, MA, USA) and chemical (, ppm) were downfield from TMS. The chemical shifts are reported relative to residual the Figure 4. Dose-dependent anti-MCF-7 (human breast cancer; in red) and anti-HCT-116 (human col- shifts (δ, ppm) were downfield from TMS. Th 1e chemica13 l shifts are reported re 1lative to solvent signal in part per million () (CD OD: H:  3.31, C:  49.1; DMSO-d : H:  2.50, orectal cancer; in blue) activities of compound 4 3 (TCMDC-125133). The curves show the c 6ell viability 1 13 r13 esidual the solvent 1signal in p 13 art per million (δ) (C 1D3OD: H: δ 3.31, C: δ 49.1; DMSO- C:  39.5; CDCl : H:  7.26, C:  77.23). For the H-NMR spectrum, data are assumed percentage of each ca 3ncer cell line at serial concentrations of compound 4. 1 13 1 13 1 d6: H: δ 2.50, C: δ 39.5; CDCl3: H: δ 7.26, C: δ 77.23). For the H-NMR spectrum, data to be first order with apparent singlet, doublet, triplet, quartets and multiplet reported as are assumed to be first order with apparent singlet, doublet, triplet, quartets and multiplet 3.s, Ma d,tt, erq, ialand s and m, Me respectively thods . Doublet of doublet was reported as dd, triplet of doublet reported as s, d, t, q, and m, respectively. Doublet of doublet was reported as dd, triplet was reported as td, and the resonance that appears broad was designated as br. High 3.1. General of doublet was reported as td, and the resonance that appears broad was designated as resolution mass spectral measurements were performed on a Thermo Scientific Orbitrap All reagents and solvents were purchased from commercial suppliers (Sigma-Al- br. High resolution mass spectral measurements were performed on a Thermo Scientific Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). TLC drich, Merck, or Tokyo Chemical Industry (TCI)) and were used without further purifica- Orbitrap Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, was performed on TLC aluminium sheet coated with silica gel 60 F (Merck, Darmstadt, tion. NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz USA). TLC was performed on TLC aluminium sheet coated with silica gel 60 F254 (Merck, Germany). To visualize spots on the TLC sheet, UV lamps were used. Melting points were 1 13 1 13 H/ C and 500/125 MHz H/ C) spectrometer (Bruker, Billerica, MA, USA) and chemical measured on a Buchi melting point M-565 (Büchi, Flawil, Switzerland). Fourier Transform shifts (δ, ppm) were downfield from TMS. The chemical shifts are reported relative to Infrared (FTIR) spectroscopy was performed on a Bruker ALPHA FTIR model (Bruker, 1 13 residual the solvent signal in part per million (δ) (CD3OD: H: δ 3.31, C: δ 49.1; DMSO- Billerica, MA, USA). Optical rotation was recorded on a Biobase Bk-P2s Digital Automatic 1 13 1 13 1 d6: H: δ 2.50, C: δ 39.5; CDCl3: H: δ 7.26, C: δ 77.23). For the H-NM ® R spectrum, data Polarimeter (Jinan, China). The purification was performed on a Biotage Selekt Automated are assumed to be first order with apparent singlet, doublet, triplet, quartets and multiplet flash column chromatography (Biotage, Uppsala, Sweden) where indicated. Chiral HPLC reported as s, d, t, q, and m, respectively. Doublet of doublet was reported as dd, triplet analysis was run on a Waters Alliance e2695 HPLC system with a Waters 2489 UV/Vis of doublet was reported as td, and the resonance that ap ®pears broad was designated as Detector (Waters, Milford, MA, USA) using a Chiralpak ADH column (dimension (Ø) of br. High resolution mass spectral measurements were performed on a Thermo Scientific 4.6 mm  250 mm) (Daicel, Osaka, Japan). Orbitrap Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). TLC was performed on TLC aluminium sheet coated with silica gel 60 F254 (Merck, Molbank 2022, 2022, x FOR PEER REVIEW 5 of 9 Molbank 2022, 2022, x FOR PEER REVIEW 5 of 9 Darmstadt, Germany). To visualize spots on the TLC sheet, UV lamps were used. Melting points were measured on a Buchi melting point M-565 (Büchi, Flawil, Switzerland). Fou- Darmstadt, Germany). To visualize spots on the TLC sheet, UV lamps were used. Melting rier Transform Infrared (FTIR) spectroscopy was performed on a Bruker ALPHA FTIR points were measured on a Buchi melting point M-565 (Büchi, Flawil, Switzerland). Fou- model (Bruker, USA). Optical rotation was recorded on a Biobase Bk-P2s Digital Auto- rier Transform Infrared (FTIR) spectroscopy was performed on a Bruker ALPHA ® FTIR matic Polarimeter (Shandong, China). The purification was performed on a Biotage Sel- model (Bruker, USA). Optical rotation was recorded on a Biobase Bk-P2s Digital Auto- ekt Automated flash column chromatography (Biotage, Uppsala, Sweden) where indi- matic Polarimeter (Shandong, China). The purification was performed on a Biotage Sel- cated. Chiral HPLC analysis was run on a Waters Alliance e2695 HPLC system with a ekt Automated flash column chromatography (Biotage, Uppsala, Sweden) where indi- Waters 2489 UV/Vis Detector (Waters, USA) using a Chiralpak ADH column (dimension cated. Chiral HPLC analysis was run on a Waters Alliance e2695 HPLC system with a (Ø) of 4.6 mm × 250 mm) (Daicel, Japan). Waters 2489 UV/Vis Detector (Waters, USA) using a Chiralpak ADH column (dimension Molbank 2022, 2022, M1358 5 of 9 (Ø) of 4.6 mm × 250 mm) (Daicel, Japan). 3.2. Synthesis 3.2.1. (–)-Ethyl (2-aminobenzoyl)-L-valinate (1) 3.2. Synthesis 3.2. Synthesis 3.2.1. (–)-Ethyl (2-aminobenzoyl)-L-valinate (1) 3.2.1. (–)-Ethyl (2-aminobenzoyl)-L-valinate (1) To a solution of acetonitrile (75 mL) in a round bottom flask, isatoic anhydride (1.6 g, 10 mmol, 1 eq), L-valine ethyl ester hydrochloride (1.8 g, 10 mmol, 1 eq), and potassium To a solution of acetonitrile (75 mL) in a round bottom flask, isatoic anhydride (1.6 g, carbonate (3.4 g, 25 mmol, 2.5 eq) were added. The reaction was allowed stirred and To a solution of acetonitrile (75 mL) in a round bottom flask, isatoic anhydride (1.6 g, 10 mmol, 1 eq), L-valine ethyl ester hydrochloride (1.8 g, 10 mmol, 1 eq), and potassium heated to 60 °C for 18 h. After that the mixture was allowed to cool to room temperature 10 mmol, 1 eq), L-valine ethyl ester hydrochloride (1.8 g, 10 mmol, 1 eq), and potassium carbonate (3.4 g, 25 mmol, 2.5 eq) were added. The reaction was allowed stirred and carbonate (3.4 g, 25 mmol, 2.5 eq) wer an ed added. evapoThe rated reaction to remwas ove th allowed e solvestirr nt. T ed he and resul heated ting residue was then stirred in a 0.4 M heated to 60 °C for 18 h. After that the mixture was allowed to cool to room temperature Na2CO3 solution for an hour and the mixture was extracted with CH2Cl2. The organic to 60 C for 18 h. After that the mixture was allowed to cool to room temperature and and evaporated to remove the solvent. The resulting residue was then stirred in a 0.4 M evaporated to remove the solvent. The pha resulting se was co residue llectedwas , drie then d wistirr th aed nhy in da ro0.4 us M Mg Na SO4 CO , and evaporated to dryness by a rotary 2 3 Na2CO3 solution for an hour and the mixture was extracted with CH2Cl2. The organic solution for an hour and the mixture evwas aporextracted ator. Puriwith ficatio CH n wCl as .pThe erfor or mganic ed usph ing ase colwas umn chromatography (CC) over silica 2 2 phase was collected, dried with anhydrous MgSO4, and evaporated to dryness by a rotary collected, dried with anhydrous MgSO , and evaporated to dryness by a rotary evaporator. gel (30–50% EtOAc/Hexanes) to yield ethyl (2-aminobenzoyl)-L-valinate 1. (1.39 g, 55% evaporator. Purification was performed using column chromatography (CC) over silica Purification was performed using column chromatography (CC) over silica gel (30–50% yield). gel (30–50% EtOAc/Hexanes) to yield ethyl (2-aminobenzoyl)-L-valinate 1. (1.39 g, 55% EtOAc/Hexanes) to yield ethyl (2-aminobenzoyl)-L-valinate (–)-Ethyl (2-aminoben 1.zo (1.39 yl)-Lg, -v55% alinayield). te (1): yellow oil, Enantiomeric excess (>99%) was yield). ® (–)-Ethyl (2-aminobenzoyl)-L-valinate (1): yellow oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chiralpak ADH), hexane:i-PrOH 90:10, 1.0 (–)-Ethyl (2-aminobenzoyl)-L-valinate (1): yellow oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chiralpak ADH), hexane:i-PrOH 90:10, 1.0 mL/min, mL/min, tr = 21.73 min, [α]D = −53.1 (c = 1.5, DMSO), Rf: 0.46 (30% EtOAc/Hexanes), FTIR 1 ® −1 t = 21.73 min, [ ] = 53.1 (c = 1.5,dDMSO), etermine Rf: d b 0.46 y c(30% hiral EtOAc/Hexanes), HPLC analysis ( FTIR Chira (cm lpak ): ADH), hexane:i-PrOH 90:10, 1.0 r D (cm ): 3457.7 (NH-C=O), 3380.8 and 3353.5 (NH2), 2967.7, 2925.7 and 2873.8 (=C-H aro- 3457.7 (NH-C=O), 3380.8 and 3353.5 mL(NH /min,), tr 2967.7, = 21.73 2925.7 min, [α and ]D = 2873.8 −53.1 (c (=C-H = 1.5, D arM omatic), SO), Rf: 0.46 (30% EtOAc/Hexanes), FTIR matic), 2 1735.4 (O-C=O), and 1638.2 (O=C-NH), H-NMR (400 MHz, CDCl3): δ = 7.40 (d, J = −1 1735.4 (O-C=O), and 1638.2 (O=C-NH), H-NMR (400 MHz, CDCl ):  = 7.40 (d, J = 7.8 Hz, (cm ): 3457.7 (NH-C=O), 3380.8 and 3353.5 (NH2), 2967.7, 2925.7 and 2873.8 (=C-H aro- 7.8 Hz, 1H), 7.17 (td, J = 5.4, 7.7 Hz, 1H), 6.65–6.61 (m, 2H), 4.57 (dd, J = 5.0, 8.5, 1H), 4.26- 1H), 7.17 (td, J = 5.4, 7.7 Hz, 1H), 6.65–6.61 matic), 1 (m, 7352H), .4 (O4.57 -C=O (dd, ), anJd = 1 5.0, 6388.5, .2 (O 1H), =C-NH) 4.26-4.13 , H-NM (m,R (400 MHz, CDCl3): δ = 7.40 (d, J = 4.13 (m, 2H), 2.27-2.19 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 2H), 2.27-2.19 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.9 Hz, 7.8 Hz, 1H), 7 13.17 (td, J = 5.4, 7.7 Hz, 1H), 6.65–6.61 (m, 2H), 4.57 (dd, J = 5.0, 8.5, 1H), 4.26- 6.9 Hz, 3H); C-NMR (100 MHz, CDCl3); δ = 172.17, 168.99, 148.66, 132.39, 127.35, 117.18, 3H); C-NMR (100 MHz, CDCl );  = 172.17, 168.99, 148.66, 132.39, 127.35, 117.18, 116.51, + 4.13 (m, 2H), 2.27-2.19 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 116.51, 115.62, 61.24, 57.01, 31.40, 18.91, 17.88, 14.13. ESI-HRMS (m/z): 265.1541 [M + H] 115.62, 61.24, 57.01, 31.40, 18.91, 17.88, 14.13. ESI-HRMS ( +m/z): 265.1541 [M + H] (calcd. 6.9 Hz, 3H); C-NMR (100 MHz, CDCl3); δ = 172.17, 168.99, 148.66, 132.39, 127.35, 117.18, (calcd. for C14H21N2O3 265.1547). for C H N O 265.1547). 14 21 2 3 116.51, 115.62, 61.24, 57.01, 31.40, 18.91, 17.88, 14.13. ESI-HRMS (m/z): 265.1541 [M + H] (calcd. for C14H21N2O3 265.1547). 3.2.2. (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2) 3.2.2. (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2) 3.2.2. (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2) To a solution of compound 1 (1.39 g, 5.3 mmol, 1 eq) in THF (40 mL), CDI (1.71 g, 10.5 To a solution of compound 1 (1.39 g, 5.3 mmol, 1 eq) in THF (40 mL), CDI (1.71 g, mmol, 2 eq) was added. The reaction was stirred for 18 h at 85 °C. When completed, the 10.5 mmol, 2 eq) was added. The reaction was stirred for 18 h at 85 C. When completed, the To a solution of compound 1 (1.39 g, 5.3 mmol, 1 eq) in THF (40 mL), CDI (1.71 g, 10.5 reaction was concentrated by a rotary evaporator. The resulting residue was then dis- reaction was concentrated by a rotary evaporator. The resulting residue was then dissolved mmol, 2 eq) was added. The reaction was stirred for 18 h at 85 °C. When completed, the solved in EtOAc, washed with water, and dried over MgSO4. The organic portion was in EtOAc, washed with water, and dried over MgSO . The organic portion was filtered and reaction was concentrated by a rotary evaporator. The resulting residue was then dis- filtered and concentrated to give a crude product. Purification was performed using CC concentrated to give a crude product. Purification was performed using CC over silica gel solved in EtOAc, washed with water, and dried over MgSO4. The organic portion was over silica gel (30–50% EtOAc/Hexanes) to obtain the desired cyclized product 2 (1.49 g, (30–50% EtOAc/Hexanes) to obtain the desired cyclized product 2 (1.49 g, 97% yield). filtered and concentrated to give a crude product. Purification was performed using CC 97% yield). (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2): yel- over silica gel (30–50% EtOAc/Hexanes) to obtain the desired cyclized product 2 (1.49 g, low oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chiralpak 97% yield). ADH), gradient hexane:i-PrOH 95:5 to 98:2, 1.0 mL/min, t = 61.92 min. [ ] = 156.3 (c = 1.5, DMSO), R : 0.26 (30% EtOAc/Hexanes), FTIR (cm ): 3253.5 (NH-C=O), 2965.5, 2928.6 and 2873.6 (=C-H aromatic), 1746.6 (O-C=O), 1716.9 (O=C-N-R), and 1656.4 (O=C-NH), H-NMR (400 MHz, MeOD-d ):  = 8.02 (dd, J = 1.2, 8.0 Hz, 1H), 7.67 (td, J = 1.4, 7.8 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 5.12 (d, J = 9.3 Hz, 1H), 4.21-4.07 (m, 2H), 2.77–2.68 (m, 1H), 1.67 (d, J = 6.5 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H), 0.76 (d, J = 6.9 Hz, 3H); C-NMR (100 MHz, MeOD-d );  = 171.22, 164.09, 152.11, 140.84, 136.79, 129.16, 124.43, 116.33, 114.95, 62.25, 60.12, 28.69, 22.51, 19.26, 14.45. ESI-HRMS: m/z calculated for C H N O ([M + H] ) 291.1339 found 291.1335. 15 18 2 4 Molbank 2022, 2022, x FOR PEER REVIEW 6 of 9 Molbank 2022, 2022, x FOR PEER REVIEW 6 of 9 (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2): (–)-Ethyl (S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoate (2): yellow oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chi- yellow oil, Enantiomeric excess (>99%) was determined by chiral HPLC analysis (Chi- ralpak AD ®H), gradient hexane:i-PrOH 95:5 to 98:2, 1.0 mL/min, tr = 61.92 min. [α]D = ralpak ADH), gradient hexane:i-PrOH 95:5 to 98:2, 1.0 mL/min, tr = 61.92 min. [α]D = −1 −156.3 (c = 1.5, DMSO), Rf: 0.26 (30% EtOAc/Hexanes), FTIR (cm ): 3−1 253.5 (NH-C=O), −156.3 (c = 1.5, DMSO), Rf: 0.26 (30% EtOAc/Hexanes), FTIR (cm ): 3253.5 (NH-C=O), 2965.5, 2928.6 and 2873.6 (=C-H aromatic), 1746.6 (O-C=O), 1716.9 (O=C-N-R), and 1656.4 2965.5, 2928.6 and 2873.6 (=C-H aromatic), 1746.6 (O-C=O), 1716.9 (O=C-N-R), and 1656.4 (O=C-NH), H-NMR (400 MHz, MeOD-d4): δ = 8.02 (dd, J = 1.2, 8.0 Hz, 1H), 7.67 (td, J = (O=C-NH), H-NMR (400 MHz, MeOD-d4): δ = 8.02 (dd, J = 1.2, 8.0 Hz, 1H), 7.67 (td, J = 1.4, 7.8 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 5.12 (d, J = 9.3 Hz, 1H), 4.21- 1.4, 7.8 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 5.12 (d, J = 9.3 Hz, 1H), 4.21- 4.07 (m, 2H), 2.77–2.68 (m, 1H), 1.67 (d, J = 6.5 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H), 0.76 (d, J = 4.07 (m, 2H), 2.77–2.68 (m, 1H), 1.67 (d, J = 6.5 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H), 0.76 (d, J = 6.9 Hz, 3H); C-NM 13 R (100 MHz, MeOD-d4); δ = 171.22, 164.09, 152.11, 140.84, 136.79, 6.9 Hz, 3H); C-NMR (100 MHz, MeOD-d4); δ = 171.22, 164.09, 152.11, 140.84, 136.79, 129.16, 124.43, 116.33, 114.95, 62.25, 60.12, 28.69, 22.51, 19.26, 14.45. ESI-HRMS: m/z calcu- 129.16, 124.43, 116.33, 114.95, 62.25, 60.12, 28.69, 22.51, 19.26, 14.45. ESI-HRMS: m/z calcu- Molbank 2022, 2022, M1358 6 of 9 lated for C15H18N2O4 ([M + H] ) 291.1339 found 291.1335. lated for C15H18N2O4 ([M + H] ) 291.1339 found 291.1335. 3.2.3. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3) 3.2.3. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3) 3.2.3. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3) A solution of LiOH (0.3 g, 12.9 mmol, 2.5 eq) in water (6 mL) was added into a solu- A solution of LiOH (0.3 g, 12.9 mmol, 2.5 eq) in water (6 mL) was added into a solu- A solution of LiOH (0.3 g, 12.9 mmol, 2.5 eq) in water (6 mL) was added into a solution tion of compound 2 (1.49 g, 5.1 mmol, 1 eq) in THF (20 mL). The reaction mixture was tion of compound 2 (1.49 g, 5.1 mmol, 1 eq) in THF (20 mL). The reaction mixture was of compound 2 (1.49 g, 5.1 mmol, 1 eq) in THF (20 mL). The reaction mixture was heated heated and stirred at 85 °C for 18 h. After that the mixture was allowed to cool down to heated and stirred at 85 °C for 18 h. After that the mixture was allowed to cool down to and stirred at 85 C for 18 h. After that the mixture was allowed to cool down to room room temperature and was concentrated under a reduced pressure. The residue was dis- room temperature and was concentrated under a reduced pressure. The residue was dis- temperature and was concentrated under a reduced pressure. The residue was dissolved solved in 10 mL of H2O and acidified with 1 M HCl. The white precipitate was filtered off solved in 10 mL of H2O and acidified with 1 M HCl. The white precipitate was filtered off in 10 mL of H O and acidified with 1 M HCl. The white precipitate was filtered off and and washed successively with MeOH to afford the desired acid 3 without further purifi- and washed successively with MeOH to afford the desired acid 3 without further purifi- washed successively with MeOH to afford the desired acid 3 without further purification cation (1.26 g, 93% yield). cation (1.26 g, 93% yield). (1.26 g, 93% yield). (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3): (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3): (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3): white white solid, Enantiomeric excess (65%) was determined® by chiral HPLC analysis (Chi- white solid, Enantiomeric excess (65%) was determined by chiral HPLC analysis (Chi- solid, Enantiomeric excess (65%) was determined by chiral HPLC analysis (Chiralpak ralpak AD ®H), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer tr = 20.22 min, minor ralpak ADH), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer tr = 20.22 min, minor ADH), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer t = 20.22 min, minor enan- enantiomer tr = 31.99 min, [α]D = −30.9 (c = 1.4 , DMSO), Rf: 0.08 (20% MeOH/EtOAc), m.p.: enantiomer tr = 31.99 min, [α]D = −30.9 (c = 1.4 , DMSO), Rf: 0.08 (20% MeOH/EtOAc), m.p.: tiomer t = 31.99 min, [ ] = 30.9 (c = 1.4, DMSO), Rf: 0.08 (20% MeOH/EtOAc), m.p.: r D −1 >305.4 °C (decomposed) FTIR (cm ): 3−1 250.1 (NH-C=O), 3078.9 (OH) 2970.3, 2924.8 and >305.4 °C (decomposed) FTIR (cm ): 3250.1 (NH-C=O), 3078.9 (OH) 2970.3, 2924.8 and >305.4 C (decomposed) FTIR (cm ): 3250.1 (NH-C=O), 3078.9 (OH) 2970.3, 2924.8 and 1 1 2850.6 (=C-H aromatic), 1751 (O=C-OH), 1682.4 (O=C-N-R), and 1640.6 (O=C-NH), H- 2850.6 (=C-H aromatic), 1751 (O=C-OH), 1682.4 (O=C-N-R), and 1640.6 (O=C-NH), H- 2850.6 (=C-H aromatic), 1751 (O=C-OH), 1682.4 (O=C-N-R), and 1640.6 (O=C-NH), H- NMR (400 MHz, DMSO-d6): δ = 12.61 (br(-OH), 1H), 11.61 (s(-NH), 1H), 7.95 (d, J = 7.7 Hz, NMR (400 MHz, DMSO-d ):  = 12.61 NM (br(-OH), R (400 M 1H), Hz, 11.61 DMSO (s(-NH), -d6): δ =1H), 12.61 7.95 (br( (- d, OH) J =, 7.7 1H) Hz , 11 , .61 (s(-NH), 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.70 (td, J = 1.1, 7.7 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 4.96 (d, J 1H), 7.70 (td, J = 1.1, 7.7 Hz, 1H), 7.25 1H),(d, 7.70J = (td 7.6 , J = Hz, 1.11H), , 7.7 Hz 7.22 , 1(d, H), J7= .25 8.0 (d,Hz, J = 7 1H), .6 Hz 4.96 , 1H), 7.22 (d, J = 8.0 Hz, 1H), 4.96 (d, J = 9.3 Hz, 1H), 2.66–2.57 (m, 1H) 1.17 (d, J = 6.4 Hz, 3H), 0.68 (d, J = 6.9 Hz, 3H); C-NM 13 R (d, J = 9.3 Hz, 1H), 2.66–2.57 (m, 1H) = 9.1.17 3 Hz(d, , 1H) J ,= 26.4 .66–Hz, 2.57 3H), (m, 10.68 H) 1.(d, 17 (Jd= , J 6.9 = 6.Hz, 4 Hz3H); , 3H), 0.68 (d, J = 6.9 Hz, 3H); C-NMR (100 MHz, DMSO); δ = 172.21, 161.95, 150.22, 139.53, 135.19, 127.59, 122.64, 115,19, 113.48, C-NMR (100 MHz, DMSO);  = (172.21, 100 MHz 161.95, , DMS150.22, O); δ = 1 139.53, 72.21, 1135.19, 61.95, 15 127.59, 0.22, 13 122.64, 9.53, 135.19, 127.59, 122.64, 115,19, 113.48, + + 59.22, 26.93, 22.75, 19.28. ESI-HRMS (m/z): 263.1025 [M + H] (ca+ lcd. for C13H15N2O4 + 115,19, 113.48, 59.22, 26.93, 22.75, 19.28. ESI-HRMS (m/z): 263.1025 [M + H] (calcd. for 59.22, 26.93, 22.75, 19.28. ESI-HRMS (m/z): 263.1025 [M + H] (calcd. for C13H15N2O4 263.1026). C H N O 263.1026). 263.1026). 13 15 2 4 3.2.4. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3- 3.2.4. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3-me- 3.2.4. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3-me- methyl-butanamide (4) [TCMDC-125133] thyl-butanamide (4) [TCMDC-125133] thyl-butanamide (4) [TCMDC-125133] To a solution of acid 3 (0.26 g, 1.0 mmol, 1 eq) in DMF (4 mL), triethylamine (TEA) To a solution of acid 3 (0.26 g, 1.0 mmol, 1 eq) in DMF (4 mL), triethylamine (TEA) To a solution of acid 3 (0.26 g, 1.0 mmol, 1 eq) in DMF (4 mL), triethylamine (TEA) (0.14 mL, 1 mmol, 1 eq) and HATU (0.38 g, 1 mmol, 1 eq) were added. The mixture was (0.14 mL, 1 mmol, 1 eq) and HATU (0.38 g, 1 mmol, 1 eq) were added. The mixture was (0.14 mL, 1 mmol, 1 eq) and HATU (0.38 g, 1 mmol, 1 eq) were added. The mixture was left stirring for 1 h at room temperature, after which m-anisidine (0.17 mL, 1.5 mmol, 1.5 left stirring for 1 h at room temperature, after which m-anisidine (0.17 mL, 1.5 mmol, 1.5 left stirring for 1 h at room temperature, after which m-anisidine (0.17 mL, 1.5 mmol, eq) was added and the reaction was left stirring at room temperature for 18 h. After the eq) was added and the reaction was left stirring at room temperature for 18 h. After the 1.5 eq) was added and the reaction was left stirring at room temperature for 18 h. After the reaction was completed, the solvent was removed under a reduced pressure. The residue reaction was completed, the solvent was removed under a reduced pressure. The residue reaction was completed, the solvent was removed under a reduced pressure. The residue was dissolved in EtOAc, and the solution was extracted with 0.4 M Na CO solution and 2 3 washed with water. The organic layer was collected, dried over MgSO , and evaporated under a reduced pressure. Purification was performed using automated flash column chromatography (Biotage , gradient system of 10–50% EtOAc/Hexanes) to afford the desired quinazolinedione product 4 (TCMDC-125133) (0.29 g, 77% yield). (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3-methylbuta- namide (4) [TCMDC-125133]: light brown solid, Enantiomeric excess (76%) was determined by chiral HPLC analysis (Chiralpak ADH), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer t = 45.21 min, minor enantiomer t = 36.36 min, [ ] = 57.8 (c = 1.1, DMSO), r r Rf: 0.11 (30% EtOAc/Hexanes), m.p.: 72.8–73.4 C, FTIR (cm ): 3206.5 and 3142.1 (NH- C=O), 2959.7, 2928.0 and 2872.3 (=C-H aromatic), 1715.7 and 1648.9 (O=C-N-H), H-NMR (500 MHz, CDCl ):  = 10.51 (s, 1H), 8.87 (s, 1H), 8.06 (d, J = 10.0 Hz, 1H), 7.55 (td, 3 Molbank 2022, 2022, M1358 7 of 9 J = 1.8, 9.5 Hz, 1H), 7.32 (s, 1H), 7.19 (td, J = 1.1, 9.6 Hz, 1H), 7.13 (t, J = 10.0 Hz, 2H), 6.98 (d, J = 10.0 Hz, 1H), 6.59 (dd, J = 2.34, 10.3 Hz, 1H), 5.30 (d, J = 13.4 Hz, 1H), 3.73 (s, 3H), 3.14-3.05 (m, 1H), 1.22 (d, J = 8.2 Hz, 3H), 0.85 (d, J = 8.4 Hz, 3H); C-NMR (125 MHz, CDCl );  = 167.36, 163.56, 160.07, 152.11, 139.08, 138.57, 135.76, 129.56, 123.75, 115.42, 114.08, 112.22, 110.26, 105.69, 64.37, 55.28, 26.80, 21.00, 19.10. ESI-HRMS (m/z): 368.1602 + + [M + H] (calcd. for C H N O 368.1602). 20 22 3 4 3.3. Antimalarial Assay against P. falciparum 3D7 Plasmodium falciparum strain 3D7 was cultured in complete medium (RPMI-1640 sup- plemented with 10% Albumax II) using O Rh+ red blood cell in microaerobic environment (5% CO , 5% O , 90% N ). IC50 assay plates were prepared by four-fold serially diluted 2 2 2 test compounds in complete medium to a final volume of 50 L. Artemisinin at 1 M and complete medium were used as positive and negative controls, respectively. Then, 50 L of parasite inoculum of 2% parasitemia of ring stage and 1% hematocrit was added to each well and incubated for 48 h in a microaerobic environment. The assay was terminated by freezing at 20 C before growth measurement. Parasite growth was measured adding 100 L of lysis buffer supplemented with 1X DNA fluorescent dye (UltraPower, Gellex, Tokyo, Japan) and fluorescent signal was measured at 495/530 nm. The IC value was calculated by GraphPad Prism 9.0 software (La Jolla, CA, USA) using the dose response (four parameter) function. 3.4. Antiproliferative Assay against MCF-7 and HCT-116 Human breast cancer cells (MCF-7) or human colorectal cancer cells (HCT-116 cells) purchased from ATCC were seeded at 2  10 cells/well on a 96-well plate and were cultured by DMEM (Dulbecco’s Modified Eagle Medium) high glucose supplemented with 10% FBS and 1% penicillin/streptomycin. The culture was incubated at 37 C, 5% CO for 24 h. After the incubation period, TCMDC-125133 was added into the cell plate as a dose- response manner at: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10 (M) and additional incubation for 72 h at 37 C, 5% CO . After the 72-h incubation, the cultured medium containing compound was removed and the serum-free media containing MTT was added into the same well with additional incubation for 3 h at 37 C, 5% CO . After 3 h incubation, the serum-free media containing MTT was removed and DMSO was added into the same well and the resulting-coloured solution was measured for its absorbance at 570 nm using a Multi-Mode Microplate Reader (ENVISION) (PerkinElmer, Waltham, MA, USA). The IC value was calculated using GraphPad. Doxorubicin at 10 M was used as a positive control in both assays. 4. Conclusions In conclusion, TCMDC-125133 can be prepared by employing the concise four-step synthesis reported here with good overall yields, low cost of goods, and mild reaction con- ditions. The in-house synthetic TCMDC-125133 was assayed against the P. falciparum 3D7 strain and its highly potent antimalarial activity corresponded to that of those previously published. The anti-proliferative activity of TCMDC-125133 was also performed and it exhibited moderate activity against the MCF-7 cell line. The result of this work confirmed the integrity of the TCMDC-125133 that was synthesized in-house. The presented synthesis would contribute to a future lead optimization campaign for this class of compounds as either antimalarial or antiproliferative agents. 1 13 Supplementary Materials: The following data are available online: H-NMR, C-NMR, IR spectra, mass spectra, and chiral HPLC traces of compound 1–4. Author Contributions: S.C. initiated the overall concept, designed the synthetic route, and secured funding. D.L., S.C. performed the chemical synthesis. D.L. carried out compound characterization. M.P. carried out antimalarial assay and its interpretation. P.K., K.J., S.S., S.B. carried out anti- Molbank 2022, 2022, M1358 8 of 9 proliferative assay and its interpretation. S.C., D.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: S.C. was funded by the New Researcher Grant (A13/2563), Mahidol University. This research was partially supported by the Faculty of Science, Mahidol University, the Ramathibodi Foundation, and the Thailand Center of Excellence for Life Sciences (TCELS). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article and supporting supplementary material. Acknowledgments: The authors wish to thank Suttiporn Pikulthong, Samreang Bunteang and the Department of Chemistry, Faculty of Science, Mahidol University for access to the NMR, FTIR and melting point instruments, Tawatchai Thongkongkaew (Chulabhorn Graduate Institute) for access to the high-resolution mass spectrometry facility and optical activity measurement, and Torsak Luanphaisarnnont, the Central Instrument Facility and Napason Chabang for their help in chiral HPLC analysis. This research project is supported by the Faculty of Science and Mahidol University. 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Journal

MolbankMultidisciplinary Digital Publishing Institute

Published: Apr 21, 2022

Keywords: quinazolinedione derivatives; antimalarial activity; antiproliferative activity

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