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

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.

1. 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 at 5-HT_2A receptor, a subtype of the 5-HT₂ receptor that belongs to the serotonin receptor 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 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.

2. 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 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 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 (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 IC₅₀ (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 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.

3. Materials and Methods

3.1. 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 ¹H/¹³C and 500/125 MHz ¹H/¹³C) 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: ¹H: δ 3.31, ¹³C: δ 49.1; DMSO-d₆: ¹H: δ 2.50, ¹³C: δ 39.5; CDCl₃: ¹H: δ 7.26, ¹³C: δ 77.23). For the ¹H-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, 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).

3.2. Synthesis

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 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 Na₂CO₃ solution for an hour and the mixture was extracted with CH₂Cl₂. The organic phase was collected, dried with anhydrous MgSO₄, 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 (2-aminobenzoyl)-L-valinate 1. (1.39 g, 55% 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 mL/min, t_r = 21.73 min, [α]_D = −53.1 (c = 1.5, DMSO), Rf: 0.46 (30% EtOAc/Hexanes), FTIR (cm⁻¹): 3457.7 (NH-C=O), 3380.8 and 3353.5 (NH₂), 2967.7, 2925.7 and 2873.8 (=C-H aromatic), 1735.4 (O-C=O), and 1638.2 (O=C-NH), ¹H-NMR (400 MHz, CDCl₃): δ = 7.40 (d, J = 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), 4.26-4.13 (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 = 6.9 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃); δ = 172.17, 168.99, 148.66, 132.39, 127.35, 117.18, 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)

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 MgSO₄. 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).

(–)-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 (Chiralpak^® ADH), gradient hexane:i-PrOH 95:5 to 98:2, 1.0 mL/min, t_r = 61.92 min. [α]_D = −156.3 (c = 1.5, DMSO), R_f: 0.26 (30% EtOAc/Hexanes), FTIR (cm⁻¹): 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), ¹H-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), 4.21-4.07 (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); ¹³C-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 calculated 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)

A solution of LiOH (0.3 g, 12.9 mmol, 2.5 eq) in water (6 mL) was added into a solution of compound 2 (1.49 g, 5.1 mmol, 1 eq) in THF (20 mL). The reaction mixture was heated and stirred at 85 °C for 18 h. After that the mixture was allowed to cool down to room temperature and was concentrated under a reduced pressure. The residue was dissolved in 10 mL of H₂O and acidified with 1 M HCl. The white precipitate was filtered off and washed successively with MeOH to afford the desired acid 3 without further purification (1.26 g, 93% yield).

(–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-3-methylbutanoic acid (3): white solid, Enantiomeric excess (65%) was determined by chiral HPLC analysis (Chiralpak^® ADH), hexane:i-PrOH 90:10, 1.0 mL/min, major enantiomer t_r = 20.22 min, minor enantiomer t_r = 31.99 min, [α]_D = −30.9 (c = 1.4, DMSO), Rf: 0.08 (20% MeOH/EtOAc), m.p.: >305.4 °C (decomposed) FTIR (cm⁻¹): 3250.1 (NH-C=O), 3078.9 (OH) 2970.3, 2924.8 and 2850.6 (=C-H aromatic), 1751 (O=C-OH), 1682.4 (O=C-N-R), and 1640.6 (O=C-NH), ¹H-NMR (400 MHz, DMSO-d₆): δ = 12.61 (br(-OH), 1H), 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 = 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); ¹³C-NMR (100 MHz, DMSO); δ = 172.21, 161.95, 150.22, 139.53, 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]⁺ (calcd. for C₁₃H₁₅N₂O₄⁺ 263.1026).

3.2.4. (–)-(S)-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(3-methoxyphenyl)-3-methyl-butanamide (4) [TCMDC-125133]

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 Na₂CO₃ solution and washed with water. The organic layer was collected, dried over MgSO₄, 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_r = 45.21 min, minor enantiomer t_r = 36.36 min, [α]_D = −57.8 (c = 1.1, DMSO), Rf: 0.11 (30% EtOAc/Hexanes), m.p.: 72.8–73.4 °C, FTIR (cm⁻¹): 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), ¹H-NMR (500 MHz, CDCl₃): δ = 10.51 (s, 1H), 8.87 (s, 1H), 8.06 (d, J = 10.0 Hz, 1H), 7.55 (td, 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); ¹³C-NMR (125 MHz, CDCl₃); δ = 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 C₂₀H₂₂N₃O₄⁺ 368.1602).

3.3. Antimalarial Assay against P. falciparum 3D7

Plasmodium falciparum strain 3D7 was cultured in complete medium (RPMI-1640 supplemented with 10% Albumax II) using O Rh+ red blood cell in microaerobic environment (5% CO₂, 5% O₂, 90% N₂). IC50 assay plates were prepared by four-fold serially diluted test compounds in complete medium to a final volume of 50 µL. Artemisinin at 1 µM and complete medium were used as positive and negative controls, respectively. Then, 50 µL 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 µL of lysis buffer supplemented with 1X DNA fluorescent dye (UltraPower, Gellex, Tokyo, Japan) and fluorescent signal was measured at 495/530 nm. The IC₅₀ 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 × 10³ 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% CO₂ 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 (µM) and additional incubation for 72 h at 37 °C, 5% CO₂. 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% CO₂. 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 IC₅₀ value was calculated using GraphPad. Doxorubicin at 10 µM 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 conditions. 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.