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 Activity against MCF-7

: Quinazolinedione is one of the most notable pharmacophores in drug discovery due to its broad spectrum of biological activities including antimalarial, anticancer, anti-inﬂammatory, and others. TCMDC-125133, whose structure features a quinazolinedione core, exhibits promising antimalarial activity and low toxicity as described in the GlaxoSmithKline (GSK) report. Herein, a concise four-step synthesis towards quinazolinedione TCMDC-125133 is described using low cost goods and greener alternatives where possible. All synthesized compounds were characterized using polarimetry, IR, NMR, and mass spectrometry. The in-house synthesized TCMDC-125133 was evaluated for its antimalarial activity against P. falciparum 3D7 and antiproliferative activity against MCF-7 cell line. Abstract: Quinazolinedione is one of the most notable pharmacophores in drug discovery due to its broad spectrum of biological activities including antimalarial, anticancer, anti-inflammatory, and others. TCMDC-125133, whose structure features a quinazolinedione core, exhibits promising antimalarial activity and low toxicity as described in the GlaxoSmithKline (GSK) report. Herein, a concise four-step synthesis towards quinazolinedione TCMDC-125133 is described using low cost goods and greener alternatives where possible. All synthesized compounds were characterized using polarimetry, IR, NMR, and mass spectrometry. The in-house synthesized TCMDC-125133 was evaluated for its antimalarial activity against P. falciparum 3D7 and antiproliferative activity against MCF-7 cell line.


Introduction
Quinazolinedione is a remarkable heterocycle which is widely used as a functional material for synthetic chemistry. It is also present in various active pharmaceutical molecules along with its diverse range of biological activities, including antimalarial, anticancer, antimicrobial, antihypertensive, antiviral, anti-inflammatory, and others [1][2][3][4][5]. The examples shown in Figure 1 are some representatives of commercial drugs and biologically active molecules with quinazolinedione moiety [6].

Introduction
Quinazolinedione is a remarkable heterocycle which is widely used as a functional material for synthetic chemistry. It is also present in various active pharmaceutical molecules along with its diverse range of biological activities, including antimalarial, anticancer, antimicrobial, antihypertensive, antiviral, anti-inflammatory, and others [1][2][3][4][5]. The examples shown in Figure 1 are some representatives of commercial drugs and biologically active molecules with quinazolinedione moiety [6].   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 2 of 9 at 5-HT 2A receptor, a subtype of the 5-HT 2 receptor that belongs to the serotonin receptor family . 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 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 expression in MCF-7. Their result showed that the ketanserin had suppression effects on MCF-7 cell's proliferation of 72.36% at the ketanserin concentration of 25 µM [9]. In a preliminary drug repurposing study, ketanserin surprisingly possesses good antimalarial activity with the IC 50 against multidrug-resistant P. falciparum K1 at around 10 µM [10].
Molbank 2022, 2022, x FOR PEER REVIEW 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 family . 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 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 expression in MCF-7. Their result showed that the ketanserin had suppression effects on MCF-7 cell's proliferation of 72.36% at the ketanserin concentration of 25 µM [9]. In a preliminary drug repurposing study, ketanserin surprisingly possesses good antimalarial activity with the IC50 against multidrug-resistant P. falciparum K1 at around 10 µM [10]. In 2010, GlaxoSmithKline (GSK) published the Tres Cantos Antimalarial Set (TCAMS) containing 13,533 compounds that are the result of screening nearly 2 million compounds from the GSK corporate collection [11]. With the result openly available in the public domain, one of the compounds identified from the screen as a singleton is TCMDC-125133 whose structure featured a quinazolinedione core as displayed in Figure 2b. The preliminary in vitro antimalarial activity of this compound against asexual blood stage and gametocytes of P. falciparum 3D7 has shown excellent IC50s at 0.27 and 0.54 µM, respectively [12]. This, combined with the drug-like property and low toxicity (cytotoxicity HG2; IC50 > 25 µM) of this compound, has made it an excellent target for further lead optimization.
The main structural feature of these compounds is quinazolinedione; however, the synthesis of quinazolinedione core has been reported to achieve this in multiple steps using harsh conditions and tedious workup procedures with low yields [13,14], while there is no previous report on the synthesis towards TCMDC-125133. This present work, therefore, described a concise synthetic route towards the quinazolinedione TCMDC-125133 using commercially available starting materials with a low cost of goods. Several spectroscopic techniques were employed to confirm the structure of in-house synthesized TCMDC-125133 and the synthetic intermediates. The final compound was assayed for its antimalarial activity against 3D7 and for antiproliferative activity against MCF-7. The resulting synthetic route will be crucial for further structure-activity relationships (SARs) study in lead optimization and development of this class of compounds.

Results and Discussion
The synthesis towards the quinazolidinone TCMDC-125133 begins with the reaction between commercially available isatoic anhydride and corresponding L-valine ethyl ester hydrochloride in the presence of K2CO3 in CH3CN to afford compound 1 (55%) [15]. This step involves the use of low-cost isatoic anhydride and a mild reaction condition with a capability to be performed at a multi-gram scale. Although the racemization of the α-carbon of the valine moiety could be of concern in this step, the working reagent and condition did not affect the stereochemistry of this optically active molecule as shown by its distinctive levorotatory property and a chiral HPLC trace (>99% ee). For the synthesis of compound 2, the cyclocarbonylation reaction of compound 1 could be performed using In 2010, GlaxoSmithKline (GSK) published the Tres Cantos Antimalarial Set (TCAMS) containing 13,533 compounds that are the result of screening nearly 2 million compounds from the GSK corporate collection [11]. With the result openly available in the public domain, one of the compounds identified from the screen as a singleton is TCMDC-125133 whose structure featured a quinazolinedione core as displayed in Figure 2b. The preliminary in vitro antimalarial activity of this compound against asexual blood stage and gametocytes of P. falciparum 3D7 has shown excellent IC 50 s at 0.27 and 0.54 µM, respectively [12]. This, combined with the drug-like property and low toxicity (cytotoxicity HG2; IC 50 > 25 µM) of this compound, has made it an excellent target for further lead optimization.
The main structural feature of these compounds is quinazolinedione; however, the synthesis of quinazolinedione core has been reported to achieve this in multiple steps using harsh conditions and tedious workup procedures with low yields [13,14], while there is no previous report on the synthesis towards TCMDC-125133. This present work, therefore, described a concise synthetic route towards the quinazolinedione TCMDC-125133 using commercially available starting materials with a low cost of goods. Several spectroscopic techniques were employed to confirm the structure of in-house synthesized TCMDC-125133 and the synthetic intermediates. The final compound was assayed for its antimalarial activity against 3D7 and for antiproliferative activity against MCF-7. The resulting synthetic route will be crucial for further structure-activity relationships (SARs) study in lead optimization and development of this class of compounds.

Results and Discussion
The synthesis towards the quinazolidinone TCMDC-125133 begins with the reaction between commercially available isatoic anhydride and corresponding L-valine ethyl ester hydrochloride in the presence of K 2 CO 3 in CH 3 CN to afford compound 1 (55%) [15]. This step involves the use of low-cost isatoic anhydride and a mild reaction condition with a capability to be performed at a multi-gram scale. Although the racemization of the α-carbon of the valine moiety could be of concern in this step, the working reagent and condition did not affect the stereochemistry of this optically active molecule as shown by its distinctive levorotatory property and a chiral HPLC trace (>99% ee). For the synthesis of compound 2, the cyclocarbonylation reaction of compound 1 could be performed using various carbonylating reagents such as phosgene or its equivalent derivatives (i.e., ethyl chloroformate, diphosgene or triphosgene), however, those reagents are toxic and difficult 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.
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, 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 1 (Route B). The ethyl ester 2 was subsequently hydrolyzed using LiOH in THF/H 2 O mixture 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 of product still retains its configuration (65% ee determined by chiral HPLC) (see Supplementary Materials). To generate the amide 4, it is important to maintain that any harsh condition could affect the stereochemistry of the α-carbon of the valine moiety, hence, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) is employed as it is one of the commonly used reagents in amide coupling chemistry. Although it is not 'low-cost' as originally proposed, HATU was chosen because of its mild condition, high coupling efficiencies, fast reaction rates, and its by-product can be easily removed [17]. In the final step, the corresponding acid 3 was then coupled to the m-anisidine side chain using HATU with the presence of triethylamine (Et 3 N) in DMF [15] to afford the quinazolidinone 4 (TCMDC-125133) in 77% yield with 76% ee determined by chiral HPLC (see Supplementary Materials). All synthesized compounds described here were fully characterized using polarimetry, IR, NMR, and mass spectrometry. This newly discovered synthetic route will allow further structural modification around this pharmacophore core. various carbonylating reagents such as phosgene or its equivalent derivatives (i.e., ethyl chloroformate, diphosgene or triphosgene), however, those reagents are toxic and difficult 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. The resulting intermediate 1 was reacted with CDI in THF to yield the quinazolinedione 2 in 97% yield (>99% ee) [15]. 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, 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 1 (Route B). The ethyl ester 2 was subsequently hydrolyzed using LiOH in THF/H2O mixture 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 of product still retains its configuration (65% ee determined by chiral HPLC) (see Supplementary Materials). To generate the amide 4, it is important to maintain that any harsh condition could affect the stereochemistry of the α-carbon of the valine moiety, hence, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) is employed as it is one of the commonly used reagents in amide coupling chemistry. Although it is not 'low-cost' as originally proposed, HATU was chosen because of its mild condition, high coupling efficiencies, fast reaction rates, and its byproduct can be easily removed [17]. In the final step, the corresponding acid 3 was then coupled to the m-anisidine side chain using HATU with the presence of triethylamine (Et3N) in DMF [15] to afford the quinazolidinone 4 (TCMDC-125133) in 77% yield with 76% ee determined by chiral HPLC (see Supplementary Materials). All synthesized compounds described here were fully characterized using polarimetry, IR, NMR, and mass spectrometry. This newly discovered synthetic route will allow further structural modification around this pharmacophore core. Compound 4 derived from this synthesis was assayed for in vitro antimalarial activity against the blood stage P. falciparum 3D7 strain. The results show that the in-house synthesised compound 4 possesses a promising IC 50 (3D7) of 219 nM ( Figure 3) which is comparable to the primary screening result by TCAMS. Further lead optimisation is underway to enhance the antimalarial activity of this class of compound.
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 50 of 17.5 µM, but it also demonstrates a mild antiproliferative activity against HCT-116 (IC 50 = 58.0 µM). 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 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 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 selectivity within this class of compound over a specific type of cancer. Further research is ongoing to study the SARs around this pharmacophore.

General
All reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich, Merck, or Tokyo Chemical Industry (TCI)) and were used without further purification. NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz 1 H/ 13 C and 500/125 MHz 1 H/ 13 C) spectrometer (Bruker, Billerica, MA, USA) and chemical shifts (δ, ppm) were downfield from TMS. The chemical shifts are reported relative to residual the solvent signal in part per million (δ) (CD3OD: 1 H: δ 3.31, 13 C: δ 49.1; DMSO-d6: 1 H: δ 2.50, 13 C: δ 39.5; CDCl3: 1 H: δ 7.26, 13 C: δ 77.23). For the 1 H-NMR spectrum, data are assumed to be first order with apparent singlet, doublet, triplet, quartets and multiplet reported as s, d, t, q, and m, respectively. Doublet of doublet was reported as dd, triplet of doublet was reported as td, and the resonance that appears broad was designated as br. High resolution mass spectral measurements were performed on a Thermo Scientific 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,  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 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 selectivity within this class of compound over a specific type of cancer. Further research is ongoing to study the SARs around this pharmacophore.

General
All reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich, Merck, or Tokyo Chemical Industry (TCI)) and were used without further purification. NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz 1 H/ 13 C and 500/125 MHz 1 H/ 13 C) spectrometer (Bruker, Billerica, MA, USA) and chemical shifts (δ, ppm) were downfield from TMS. The chemical shifts are reported relative to residual the solvent signal in part per million (δ) (CD3OD: 1 H: δ 3.31, 13 C: δ 49.1; DMSO-d6: 1 H: δ 2.50, 13 C: δ 39.5; CDCl3: 1 H: δ 7.26, 13 C: δ 77.23). For the 1 H-NMR spectrum, data are assumed to be first order with apparent singlet, doublet, triplet, quartets and multiplet reported as s, d, t, q, and m, respectively. Doublet of doublet was reported as dd, triplet of doublet was reported as td, and the resonance that appears broad was designated as br. High resolution mass spectral measurements were performed on a Thermo Scientific 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,

General
All reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich, Merck, or Tokyo Chemical Industry (TCI)) and were used without further purification. NMR spectra were recorded on either a Bruker Avance AV400 or 500 (400/100 MHz 1 H/ 13 C and 500/125 MHz 1 H/ 13 C) spectrometer (Bruker, Billerica, MA, USA) and chemical shifts (δ, ppm) were downfield from TMS. The chemical shifts are reported relative to residual the solvent signal in part per million (δ) (CD 3 OD: 1 H: δ 3.31, 13 C: δ 49.1; DMSO-d 6 : 1 H: δ 2.50, 13 C: δ 39.5; CDCl 3 : 1 H: δ 7.26, 13 C: δ 77.23). For the 1 H-NMR spectrum, data are assumed to be first order with apparent singlet, doublet, triplet, quartets and multiplet reported as s, d, t, q, and m, respectively. Doublet of doublet was reported as dd, triplet of doublet was reported as td, and the resonance that appears broad was designated as br. High resolution mass spectral measurements were performed on a Thermo Scientific Orbitrap Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). TLC was performed on TLC aluminium sheet coated with silica gel 60 F 254 (Merck, 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). Fourier Transform Infrared (FTIR) spectroscopy was performed on a Bruker ALPHA FTIR model (Bruker, Billerica, MA, USA). Optical rotation was recorded on a Biobase Bk-P2s Digital Automatic Polarimeter (Jinan, China). The purification was performed on a Biotage ® Selekt Automated flash column chromatography (Biotage, Uppsala, Sweden) where indicated. Chiral HPLC analysis was run on a Waters Alliance e2695 HPLC system with a Waters 2489 UV/Vis Detector (Waters, Milford, MA, USA) using a Chiralpak ® ADH column (dimension (Ø) of 4.6 mm × 250 mm) (Daicel, Osaka, Japan).
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 carbonate (3.4 g, 25 mmol, 2.5 eq) were added. The reaction was allowed stirred and heated to 60 • C for 18 h. After that the mixture was allowed to cool to room temperature and evaporated to remove the solvent. The resulting residue was then stirred in a 0.4 M Na 2 CO 3 solution for an hour and the mixture was extracted with CH 2 Cl 2 . The organic phase was collected, dried with anhydrous MgSO 4 , and evaporated to dryness by a rotary evaporator. Purification was performed using column chromatography (CC) over silica gel (30-50% EtOAc/Hexanes) to yield ethyl ( ekt Automated flash column chromatography (Biotage, Upp cated. Chiral HPLC analysis was run on a Waters Alliance e Waters 2489 UV/Vis Detector (Waters, USA) using a Chiralpak (Ø) of 4.6 mm × 250 mm) (Daicel, Japan).
( To a solution of compound 1 (1.39 g, 5.3 mmol, 1 eq) in TH mmol, 2 eq) was added. The reaction was stirred for 18 h at 8 reaction was concentrated by a rotary evaporator. The resul solved in EtOAc, washed with water, and dried over MgSO4 filtered and concentrated to give a crude product. Purification over silica gel (30-50% EtOAc/Hexanes) to obtain the desired 97% yield).
To a solution of compound 1 (1.39 g, 5.3 mmol, 1 eq) in THF (40 mL), CDI (1.71 g, 10.5 mmol, 2 eq) was added. The reaction was stirred for 18 h at 85 • C. When completed, the reaction was concentrated by a rotary evaporator. The resulting residue was then dissolved in EtOAc, washed with water, and dried over MgSO 4 . The organic portion was filtered and concentrated to give a crude product. Purification was performed using CC over silica gel (30-50% EtOAc/Hexanes) to obtain the desired cyclized product 2 (1.49 g, 97% yield). (

(-)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methy
A solution of LiOH (0.3 g, 12.9 mmol, 2.5 eq) in water (6 mL) w tion of compound 2 (1.49 g, 5.1 mmol, 1 eq) in THF (20 mL). The heated and stirred at 85 °C for 18 h. After that the mixture was all room temperature and was concentrated under a reduced pressure solved in 10 mL of H2O and acidified with 1 M HCl. The white prec and washed successively with MeOH to afford the desired acid 3 w cation (1.26 g, 93% yield). To a solution of acid 3 (0.26 g, 1.0 mmol, 1 eq) in DMF (4 mL (0.14 mL, 1 mmol, 1 eq) and HATU (0.38 g, 1 mmol, 1 eq) were ad left stirring for 1 h at room temperature, after which m-anisidine ( eq) was added and the reaction was left stirring at room temperat reaction was completed, the solvent was removed under a reduced 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 left stirring for 1 h at room temperature, after which m-anisidine (0.17 mL, 1.5 mmol, 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 was dissolved in EtOAc, and the solution was extracted with 0.4 M Na 2 CO 3 solution and washed with water. The organic layer was collected, dried over MgSO 4 , 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).