Chiral Resolution, Absolute Configuration Assignment, and Genotoxicity Evaluation of Racemic 3,4-Dihydroquinazoline as a Novel Anticancer Agent

Ahn, Junseong; Ko, Dohyeong; Yang, Seyoung; Moon, Kwang H.; Woo, Jiwon; Yoo, Ho; Ahn, Joohoon; Lee, Jeong H.; Chung, Kyung S.; Lee, Kyung-T.; Lee, Jae Y.

doi:https://doi.org/10.1155/2021/6169055

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Abstract Introduction Materials and Methods Results and Discussion Conclusions Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2021 | Article ID 6169055 | https://doi.org/10.1155/2021/6169055

Chiral Resolution, Absolute Configuration Assignment, and Genotoxicity Evaluation of Racemic 3,4-Dihydroquinazoline as a Novel Anticancer Agent

Junseong Ahn,¹Dohyeong Ko,¹Seyoung Yang,¹Kwang H. Moon,¹Jiwon Woo,¹Ho Yoo,²Joohoon Ahn,²Jeong H. Lee,^3,4Kyung S. Chung,³Kyung-T. Lee,^3,4and Jae Y. Lee^1,5

Academic Editor: Joaquin Campos

Received12 Sept 2021

Accepted01 Oct 2021

Published25 Oct 2021

Abstract

If a new drug candidate will be a mixture of enantiomers, both enantiomers should be separately studied for at least latent genotoxicity as early as possible since the thalidomide tragedy. Our group has recently reported that KCP-10043F (OZ-001) as a racemate (±)-3,4-dihydroquinazoline derivative strongly represses the proliferation of human A549 lung cancer cells by caspase-mediated apoptosis via STAT3 inactivation. To investigate the possible teratological effects of the two enantiomers of a racemic KCP-10043F, therefore chiral resolution of (±)-KCP-10043F was performed and subsequently followed by a series of chemical processes to afford the corresponding chiral diastereomers. By using ¹H NMR anisotropy method, the absolute configuration (+)-KCP-10043F and (−)-KCP-10043F could be assigned as S and R configuration, respectively. The bacterial reverse mutation test (Ames test) for racemate (±)-KCP-10043F and its two enantiomers exhibited that all three stereoisomers were found to be nongenotoxic against five bacterial strains with/without metabolic activation. In addition, (R)-(−)-KCP-10043F displayed almost equal anticancer activity to (S)-(+)-KCP-10043F against three cancer cell lines. Based on these overall results, racemate KCP-10043F (OZ-001) could be used for our ongoing preclinical and clinical studies without the expensive asymmetric process and/or chiral separation.

1. Introduction

The significance of chirality in drug design and development cannot be understated [1]. About more than half of the drugs currently in use are chiral compounds and near 90% of the last ones are marketed as racemates (or racemic mixtures) [2]. Although the enantiomers of chiral drugs have the same chemical connectivity of atoms, they would exhibit marked differences in their pharmacology, toxicology, pharmacokinetics, metabolism, etc.: while one enantiomer is beneficial to the body, the other enantiomer can be highly toxic to the body [3]_. A well-known example of enantiomer-related toxicity is the R- and S-enantiomers of thalidomide [4]. The R-enantiomer is an effective sedative, but the S-enantiomer is known to cause teratogenic birth defects. During the late 1950s and early 1960s, thalidomide produced birth defects in children born to women who took the racemic mixture during pregnancy [5]. According to the United States Food and Drug Administration (US FDA) guidelines, it is mandatory to separate chiral drugs before they are marketed [6]. Rigorous justification is needed for the market approval of racemic drugs. FDA has approved the marketing of some racemic drugs such as flecainide, ketorolac, and prilocaine [7]. In this case, both enantiomers have equal therapeutic potency without any toxicity or they are known to racemize in the body.

In our previous work, a family of 3,4-dihydroquinazoline derivatives was discovered as a promising anticancer agent [8–10], among which KCP-10043F (OZ-001) represses the proliferation of human A549 lung cancer cells by caspase-mediated apoptosis via STAT3 inactivation [11, 12]. This compound was a racemate (±)-3,4-dihydroquinazoline derivative with a single chiral center (Figure 1). In this work, we separated the racemate (±)-KCP-10043F into two optically pure enantiomers (+)-KCP-10043F and (−)-KCP-10043F, which were assigned for their absolute configuration by the formation of chiral diastereomers and ¹H NMR anisotropy method. The bacterial reverse mutation test (Ames test) for two enantiomers and a racemate of KCP-10043F was performed to investigate their latent genotoxicity as well as their anticancer activities.

2. Materials and Methods

2.1. Chiral Resolution

Two enantiomers of racemate (±)-KCP-10043F (OZ-001) were separated by a Shimadzu LC-20AD (CP-HPLC-08) with CHIRALPAK IA column (2.5 cm I.D. × 25 cm L, 10 μm particle size) using ethanol (100%) as a mobile phase with a flow rate of 25 mL/min and a detection wavelength of 214 nm at a temperature of 38°C. Each separated enantiomer was analyzed by a Shimadzu LC-20AD (CP-HPLC-08) with CHIRALPAK IA-3 (IA30CE-OB011) column (0.46 cm I.D. × 25 cm L.) using ethanol/diethylamine (100/0.1: v/v) as a mobile phase with a flow rate of 0.5 mL/min and a detection wavelength of 214 nm at a temperature of 35°C.

2.2. Chemistry

Analytical thin-layer chromatography (TLC) was performed on silica gel precoated on glass-backed plates (Fluka Kieselgel 60F₂₅₄, Merck). An UV light (λ = 254 nm) was used for the detection. Flash chromatography was performed on silica gel 60 (particle size 230–400 mesh, Merck). Commercially available reactants were supplied by Sigma-Aldrich and used without further purification.

Optical rotations were obtained with an Autopol III Automatic Polarimeter (Rudolph Research Analytical). Experimental electronic circular dichroism (ECD) spectrum was recorded on a Jasco J-715 circular dichroism (CD) spectropolarimeter (Jasco). Melting points were measured on a MEL-TEMP® 3.0 capillary melting point apparatus (Sigma-Aldrich). ¹H NMR spectra on a Bruker Avance DXR 400 (400 MHz) spectrometer (Bruker) and Jeol JNM-ECZR 500 MHz spectrometer were recorded in CDCl₃. Chemical shifts were reported in δ (ppm) units relative to tetramethylsilane (TMS).

2.3. Preparation of Compound (±)-6

To a solution of (±)-5 (1.86 g, 3.57 mmol) in THF/H₂O (1 : 1, v/v, 15 mL) LiOH (0.45 g, 10.71 mmol) was added. The reaction mixture was stirred at room temperature for 4 h and concentrated under reduced pressure. The reaction mixture was dissolved with a mixed solvent of DCM (20 mL) and water (20 mL). The extracted aqueous layer was acidified by 0.5 N HCl (pH = 4) and extracted with DCM (25 mL). The extracted organic layer was dried with MgSO₄ and evaporated under reduced pressure to give the desired product (±)-6 (180 mg, >99%): ¹H NMR (400 MHz, CDCl₃) δ 12.42 (br s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.35–7.31 (m, 1H), 7.18–7.14 (m, 3H), 7.13–7.07 (m, 3H), 6.31 (br s, 1H), 5.18 (dd, J = 10.4 and 4.8 Hz, 1H), 3.59 (br s, 1H), 3.27 (br s, 2H), 2.96 (s, 6H), 2.93–2.89 (m, 3H), 2.68–2.63 (m, 1H), 2.47–2.44 (m, 1H), 2.06 (s, 1H), 1.86–1.81 (m, 5H), 1.76–1.72 (d, J = 13.2 Hz, 2H), 1.38–1.28 (m, 4H), 1.26–1.21 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.8, 171.8, 159.4, 153.4, 147.6, 140.3, 132.4, 129.2, 128.6, 126.1, 125.4, 123.9, 118.8, 61.9, 50.6, 43.8, 40.6, 38.1, 36.5, 34.1, 34.1, 27.3, 26.6, 25.8.

2.4. Preparation of Compound 8a-8b

To a solution of (±)-6 (1.05 g, 2.08 mmol), 7 (0.75 mL, 6.23 mmol), and DMAP (0.13 g, 1.04 mmol) in dry dichloromethane (20 mL) a solution of EDC·HCl (0.48 g, 2.49 mmol) in dry dichloromethane (5 mL) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 3 h in argon atmosphere and concentrated under reduced pressure. The reaction mixture was dissolved with a mixed solvent of EtOAc (30 mL) and water (3 × 10 mL). The extracted combined organic layer was dried with MgSO₄ and evaporated under reduced pressure. The crude product was then purified by flash column chromatography (EA:DCM:hexane:EtOH = 2:2:12:1) to afford (S, 4R)-8a and (S, 4S)-8b (1:1 ratio, 899 mg, 71%), for (S, 4R)-8a. ¹H NMR (500 MHz, CDCl₃) δ 7.36–7.35 (m, 1H), 7.35–7.33 (m, 2H), 7.32–7.31 (m, 1H), 7.29–7.27 (m, 1H), 7.15 (td, J = 7.9 and 1.3 Hz, 1H), 7.02–6.89 (m, 3H), 6.94–6.93 (m, 1H), 6.88–6.86 (m, 3H), 5.99 (q, J = 6.6 Hz, 1H), 5.04 (dd, J = 11.2 and 4.4 Hz, 1H), 4.28 (br s, 1H), 3.66 (br s, 1H), 2.97 (s, 6H), 2.81 (d, J = 13.2 Hz, 1H), 2.72–2.67 (m, 2H), 2.54–2.50 (m, 1H), 2.41–2.36 (m, 1H), 2.07 (s, 3H), 1.81–1.78 (m, 4H), 1.72–1.70 (m, 1H), 1.57 (d, J = 6.6 Hz, 4H), 1.35–1.28 (m, 4H), 1.25–1.23 (m, 2H), 1.21–1.15 (m, 1H).

2.5. Preparation of Compound (R)-6

To a solution of (S, 4R)-8a (0.17 g, 0.28 mmol) in THF/H₂O (1 : 1, v/v, 8 mL) LiOH (70 mg, 1.67 mmol) was added. The reaction mixture was stirred at room temperature for 72 h and concentrated under reduced pressure. The reaction mixture was dissolved with a mixed solvent of DCM (10 mL) and water (10 mL). The extracted aqueous layer was acidified with 0.5 N HCl until the pH of the aqueous layer was 2, extracted with DCM (15 mL). The extracted organic layer was dried with MgSO₄ and evaporated under reduced pressure to give desired product (R)-6 (140 mg, >99%): ¹H NMR (500 MHz, CDCl₃) δ 12.86 (br s, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.35–7.28 (m, 1H), 7.18–7.12 (m, 3H), 7.11–7.08 (m, 3H), 6.27 (br s, 1H), 5.18 (dd, J = 10.8 and 5.0 Hz, 1H), 3.52 (br s, 1H), 3.28 (br s, 2H), 2.96 (s, 6H), 2.92–2.87 (m, 3H), 2.64–2.60 (m, 1H), 2.48–2.42 (m, 1H), 1.96 (br s, 1H), 1.86–1.78 (m, 5H), 1.73 (d, J = 12.7 Hz, 2H), 1.38–1.28 (m, 4H), 1.25–1.19 (m, 2H).

2.6. Preparation of Compound 1 [(R)-(-)-KCP-10043F (OZ-001)]

To a solution of (R)-6 (0.26 g, 0.50 mmol) and TEA (0.09 mL, 0.64 mmol) in DMF (20 mL) pivaloyl chloride (0.073 mL, 0.60 mmol) was added dropwise. The reaction mixture was stirred for 3 h at room temperature. The reaction mixture was treated with 4-fluorobenzylamine (0.053 mL, 0.46 mmol) and TEA (0.07 mL, 0.50 mmol), further stirred for 2 h at room temperature. The reaction mixture was dissolved with a mixed solvent of DCM (20 mL) and water (20 mL). The extracted combined organic layer was dried with MgSO₄ and evaporated under reduced pressure. The crude product was then purified by flash column chromatography (EA:DCM:hexane:EtOH = 2:2:12:1) to provide the final product 1 [(R)-(-)-KCP-10043F (OZ-001)] as a white solid (245 mg, 80%): mp 107–116°C;¹H NMR (400 MHz, CDCl₃) δ 7.29–7.26 (br m, 2H), 7.16–7.14 (m, 2H), 7.07–7.03 (m, 3H), 6.98–6.93 (m, 5H), 6.90–6.88 (m, 1H), 5.21 (dd, J = 10.4 and 4.8 Hz, 1H), 4.45–4.37 (m, 2H), 4.16 (br, 1H), 3.41 (br s, 1H), 3.02–2.88 (m, 2H), 2.85 (s, 6H), 2.58 (dd, J = 14.4 and 10.4 Hz, 1H), 2.43–2.38 (m, 5H), 1.83–1.81 (m, 4H), 1.75–1.73 (m, 3H), 1.40–1.32 (m, 4H), 1.28–1.21 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 170.0, 158.9, 154.5, 144.0, 143.7, 143.4, 134.7, 129.9, 129.8, 127.9, 127.6, 126.6, 125.1, 121.9, 121.8, 115.2, 115.0, 61.2, 43.8, 42.8, 41.4, 39.6, 37.2, 36.3, 34.5, 34.4, 27.1, 26.9, 26.1; low MS (ES): m/z 613.4 [M+H]+; HRMS (FAB): m/z calcd for C₃₆H₄₆N₆O₂F [M+H]⁺ 613.3661, found 613.3660; of −261° (c 0.003, CHCl₃).

2.7. Time-Dependent Density Functional Theory (TDDFT) Calculation

The simulated ECD spectrum of (R)-(−)-KCP-10043F was calculated with TDDFT approach at B3LYP/TZVP level based on the structure optimized at the same level in Gaussian09 program package [13].

2.8. Ames Test

The Ames test for mutagenicity evaluation was performed by using Ames MPF™ Penta 1 Mutagenicity Assay Kit (Xenometrix; B10-513-S1-P) [14, 15]. Each compound was studied at concentrations of 20∼5,000 μg/mL in the presence or absence of metabolic activation (S9 fraction).

2.9. MTT Assay (Anticancer Activity)

Human lung adenocarcinoma A549 cells, human colorectal adenocarcinoma HT-29 cells, and human pancreatic cancer PANC-1 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin sulfate (100 μg/mL). Cells were cultured at 37°C in an atmosphere of 5% CO₂.

The cancer cells (5 × 10⁴/mL) were seeded in each well containing 100 μL of RPMI medium supplemented with 10% FBS in a 96-well plate. Various concentrations of each compound were added and incubated for 24 h. MTT (5 mg/mL stock solution) was added and the plates were incubated for an additional 4 h. The medium was discarded and formazan blue, which was formed in the cells, was dissolved with 100 μL DMSO. The optical density was measured at 540 nm by an automatic microplate reader (Molecular Devices Corp).

3. Results and Discussion

3.1. Chiral Resolution

Firstly, we separated racemate (±)-KCP-10043F with the aid of the chiral technique of supercritical fluid chromatography (SFC) to get optically pure enantiomers (+)-KCP-10043F and (−)-KCP-10043F with >99% enantiomeric excess (ee) and purity >99% (Figure 2) [16, 17]. The specific optical rotations of (+)-KCP-10043F (Figure 2(b)) and (−)-KCP-1-170043F (Figure 2(c)) were determined to be +273° (c 0.01, CHCl₃) and −281° (c 0.01, CHCl₃), respectively.

(a)

(b)

(c)

3.2. Absolute Configuration Assignment

Our method for absolute configuration assignment of each enantiomer was based on the direct covalent diastereomer formation between a racemic compound and a chiral selector [18]. For this matter, racemic ester (±)-5 was routinely prepared according to our previously reported method (Scheme 1) [11]. Treatment of (±)-5 with LiOH in THF-H₂O (1 : 1) solvent at room temperature produced racemic acid (±)-6 in quantitative yield. Subsequent esterification of (±)-6 with chiral (S)-(−)-1-phenylethanol 7 (purity ≥99.0%) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 4-dimethylaminopyridine (DMAP) coupling condition yielded a mixture of diastereomeric esters 8a and 8b with a ratio of 1 : 1 (¹H NMR) in 71% yield (Supplemental data: Figure S1) [19]. The mixture was easily separated by column chromatography on normal silica gel (EtOAc : CH₂Cl₂:hexane:EtOH = 2 : 2:12 : 1) to provide the first eluted ester 8a (Supplemental data: Figure S2) and the second one 8b (Supplemental data: Figure S3) as shown in Scheme 1. To prepare the enantiopure amide 1 (KCP-10043F : OZ-001), the first eluted ester 8a was treated with LiOH in THF-H₂O (1 : 1) to afford chirally pure acid 5 in quantitative yield. This acid 5 was treated with pivaloyl chloride and triethylamine (TEA) in DMF to provide a mixed carboxylic acid anhydride in situ [20], which was directly coupled with 4-fluorobenzylamine 9 in the presence of TEA to afford the enantiopure amide 1, (−)-KCP-10043F (OZ-001) in 80% yield: of -261^o (c 0.003, CHCl₃) and 95.57 ± 0.02% ee based on the same chiral HPLC analysis (Supplemental data: Figure S4). The (+)-KCP-10043F (OZ-001) could be also prepared in 78% yield starting from the second eluted ester 8b by using the same procedure.

To assign the absolute configuration at C-4 position of the quinazoline scaffold, we decided to investigate the ¹H NMR data of the separated esters 8a and 8b, respectively. Before the analysis of the chemical shift data of ¹H NMR data, molecular mechanics (MM2) calculation and 3D-conformation analysis showed the considerable energy difference (ΔE = 1.02 kcal/mol) between (S, 4R)-8a and (S, 4S)-8b as shown in Figure 3. N-methyl group inside the white-colored dotted circle in (S, 4R)-8a was completely located above the diamagnetic (shielding) zone of benzene ring (4 Å: yellow-colored dotted circle) as shown in Figure 3(a), while the corresponding methyl group of (S, 4S)-8b was located within the paramagnetic (deshielding) zone (Figure 3(b)). According to the calculated anisotropic effect zone of benzene ring (Supplemental data: Figure S5) [21], the protons of the methyl groups of (S, 4R)-8a were anticipated to be largely upfield-shifted by the diamagnetic anisotropy effect of the phenyl moiety compared to those of (S, 4S)-8b. In experimental ¹H NMR data, the chemical shifts of N-methyl groups in (S, 4R)-8a and (S, 4S)-8b exhibited 2.07 and 2.57 ppm, respectively (Figures 4(b) and 4(c)). Based on the consistency between the theoretical data and experimental data, the chiral center of (−)-KCP-10043F can be designated R configuration and thus (+)-KCP-10043F can be readily assigned as S configuration. Additionally, the ECD spectrum of (−)-KCP-10043F was further measured to afford a positive band at 268 nm and a negative band at 303 nm, which was consistent with Gaussian TDDFT-calculated R configuration of KCP-10043F as shown in Figure 5 [22].

(a)

(b)

3.3. Genotoxicity Evaluation

The Ames test was performed for mutagenicity evaluation of racemate (±)-KCP-10043F and its two enantiomers by using Ames MPF™ Penta 1 Mutagenicity Assay Kit (Xenometrix; B10-513-S1-P) [14, 15]. The pre-incubation method, system metabolic activation (rat liver S9 fraction), seven positive controls (mutagens; refer to Figures S6–S10 of supplemental data), negative control (DMSO), and five bacterial strains {Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA[pKM101]} were employed for the Ames test, in compliance with OECD Guideline 471 [23].

Each compound was studied at concentrations of 20∼5,000 μg/mL in the presence or absence of metabolic activation (S9 fraction). As shown in Table 1, a revertant analysis showed no significant differences between the treatment doses (20∼5,000 μg/mL) and the negative control (DMSO) regardless of +S9 and −S9, when compared to the positive control and included all strains of S. typhimurium and E. coli EP2 (Supplemental data: Figures S6–S10). All of these Ames test results showed that none of all stereoisomers of KCP10043F were genotoxic regarding frameshift mutations (TA98 and TA1537) and base pair substitutions {TA100, TA1535 and E. coli WP2 uvrA[pKM101]} regardless of the treatment dose [24].

3.4. Anticancer Evaluation

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method was used to evaluate the cytotoxic effects of (R)-(−)-KCP-10043F and (S)-(+)-KCP-10043F on three human cancer cell lines: lung cancer (A549), colon cancer (HT-29) and pancreatic cancer (PANC-1) [25]. As shown in Table 2, both enantiomers displayed almost equal cytotoxicity toward three cancer cell lines with micromolar IC₅₀ values of PANC-1 (10–12 μM) > A549 (9 μM) > HT-29 (5 μM) together with the reported data (IC₅₀ = 7 μM) of racemate (±)-KCP-10043F against A549 [11]. Therefore, it could be concluded that the anticancer activity of racemate (±)-KCP-10043F was equally derived from both enantiomers.

4. Conclusions

(R)-(−)-KCP10043F and (S)-(+)-KCP10043F were successfully separated from racemate (±)-KCP-10043F (OZ-001) by the chiral technique of supercritical fluid chromatography and assigned to their absolute configuration by the preparation of chiral diastereomers and ¹H NMR anisotropy method as well as the experimental ECD. The bacterial reverse mutation test (Ames test) for racemate KCP-10043F and its two enantiomers was performed to investigate their latent genotoxicities. All stereoisomers were fortunately found to be non-genotoxic against five bacterial strains with/without metabolic activation. In addition, (R)-(−)-KCP-10043F displayed almost equal cytotoxic activity to (S)-(+)-KCP-10043F against three cancer cell lines. Based on these overall results, racemate (±)-KCP-10043F (OZ-001) could be used for our ongoing preclinical and clinical studies without the expensive asymmetric process and/or chiral separation.

Data Availability

The supplementary materials (NMR spectrum, HPLC data, and Ames test data) used to support the findings of this study are included within the supplemental file.

Conflicts of Interest

The authors declare that there is no conflicts of interest regarding the publication of this paper.

Authors’ Contributions

JunseongAhn, Dohyeong Ko. The contribution of the authors is equal.

Acknowledgments

This work was supported by ONCOZEN Co., Ltd., and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1F1A1059332, No. 2020R1A5A2019413 and No. 2017R1A5A2014768).

Supplementary Materials

The supplementary materials (NMR spectrum, HPLC data, and Ames test data) used to support the findings of this study are included within the supplemental file. (Supplementary Materials)

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Copyright © 2021 Junseong Ahn et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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