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Molecules 2017, 22(9), 1416; doi:10.3390/molecules22091416

Article
Identification of Optically Active Pyrimidine Derivatives as Selective 5-HT2C Modulators
Juhyeon Kim 1,2Orcid, Hanbyeol Jo 3, Hyunseung Lee 3, Hyunah Choo 1,4, Hak Joong Kim 2, Ae Nim Pae 4,5, Yong Seo Cho 1,4,* and Sun-Joon Min 3,*Orcid
1
Center for Neuro-Medicine, Korea Institute of Science and Technology (KIST), 5 Hwarangno 14-gil, Seongbuk-gu, Seoul 02792, Korea
2
Department of Chemistry, Korea University, Seoul 02841, Korea
3
Department of Chemical & Molecular Engineering/Applied Chemistry, Hanyang University, Ansan, Gyeonggi-do 15588, Korea
4
Department of Biological Chemistry, Korea University of Science and Technology (UST), 217 Gajungro, Yuseong-gu, Daejeon 34113, Korea
5
Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, KIST, Seoul 02792, Korea
*
Correspondence: Tel.: +82-2-958-5156 (Y.S.C.); +82-31-400-5502 (S.-J.M.)
Received: 31 July 2017 / Accepted: 23 August 2017 / Published: 26 August 2017

Abstract

:
A series of pyrimidine derivatives 4ai were synthesized and evaluated for their binding affinities towards 5-HT2C receptors. With regard to designed molecules 4ai, the influence of the size of alkyl ether and the absolute configuration of a stereogenic center on the 5-HT2C binding affinity and selectivity was studied. The most promising diasteromeric mixtures 4d and 4e were selected in the initial radioligand binding assay and they were further synthesized as optically active forms starting from optically active alcohols 5d and 5e, prepared by an enzymatic kinetic resolution. Pyrimidine analogue (R,R)-4e displayed an excellent 5-HT2C binding affinity with good selectivity values against a broad range of other 5-HT receptor subtypes.
Keywords:
pyrimidine; optically active; enzymatic kinetic resolution; 5-HT2C receptor; binding affinity; selectivity

1. Introduction

Serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are mainly located in the central nervous system (CNS) and play an important role in mediating both excitatory and inhibitory neurotransmission. There are 14 different 5-HT receptors classified into seven major subfamilies (5-HT1–7). They have been known to regulate various physiological functions such as mood, depressive behavior, appetite, biorhythm, and feeding [1,2]. According to recent studies, the 5-HT2C receptor (5-HT2CR) is expected to be a potential drug target for the diagnosis and treatment of a number of CNS disorders including schizophrenia, depression, substance abuse, and Parkinson diseases, as well as obesity and urinary incontinence [3,4,5,6,7,8]. In particular, 5-HT2C-specific modulators may have few undesired side effects on peripheral tissues because this receptor is exclusively expressed in the CNS [9,10]. However, the 5-HT2C receptor belongs to the 5-HT2 receptor family, together with 5-HT2A and 5-HT2B, which have a high similarity in terms of their amino acid sequences [11]. It has been reported that the activation of 5-HT2A and 5-HT2B is strongly implicated in hallucinations and valvular heart disease [12,13]. Thus, the discovery of 5-HT2C agonists with a high specificity and subtype selectivity for 5-HT2A and 5-HT2B receptors is important for avoiding side effects.
To date, several compounds, including lorcaserin 1 and vabicaserin 2, have been identified as 5-HT2C agonists with significant selectivity against 5-HT2A and 5-HT2B (Figure 1) [14,15,16,17,18]. Lorcaserin (ADP-356) was developed for the treatment of obesity and was approved for clinical use by FDA in 2012 [19]. Vabicaserin is a drug developed for the treatment of acute schizophrenia or appetite suppressants, but has not been shown to be effective in clinical trial studies [20,21]. In addition, other small molecule 5-HT2C agonists have been reported to be in preclinical or early clinical development [15,16,17,18].
During our efforts toward the development of 5-HT receptor modulators, we have initiated a program to discover 5-HT2C agonists as potential therapeutic and diagnostic agents for CNS diseases. Recently, pyrimidine analogue 3 with good pharmacological and pharmacokinetic properties has been reported as a potential 5-HT2C agonist [22]. Our in vitro study of this molecule proved that it has a high potency against the 5-HT2C receptor and relative good selectivity against the 5-HT2A receptor [23]. However, the binding affinity of 3 to the 5-HT2B receptor is still too high, which prompted us to investigate this compound for the development of 5-HT2C selective agonists. Based on the report of the activation selectivity of 5-HT2C over 5-HT2B [11], we postulated that altering the chain length between the phenyl group and pyrimidine may induce a subtle structural change in the molecule, which can make a large difference in its interaction with 5-HT2C and 5-HT2B because the binding site of 5-HT2C is slightly deeper than that of 5-HT2B [11]. Thus, pyrimidine derivatives 4 with hydrocarbon chains shorter or longer than that of the parent molecule 3 were designed to examine the effect of the structural modification of compound 3 on the target selectivity toward 5-HT2C over 5-HT2B (Figure 2). In this paper, we report our progress in the synthesis and biological evaluation of pyrimidine derivatives 4 as selective 5-HT2C agonists.

2. Results and Discussion

2.1. Synthesis of Pyrimidine Derivatives

The synthesis of compounds 4ai is described in Scheme 1. Following the literature procedure [22,24], a series of pyrimidine derivatives 4ai were synthesized starting from either primary or secondary alcohols 5ai. The alcohols 5ae were obtained from commercial sources, whereas the others 5fi were easily prepared via the reduction/oxidation of phenyl propanoic acids 8 and 9. Thus, alcohols 5ai were first reacted with 2,4-dichloro-5-fluoropyrimidine in the presence of NaOtBu to afford 4-alkoxypyrmidines 6ai. A nucleophilic aromatic substitution reaction (SNAr) of 6ai with (R)-(+)-1-Boc-3-methylpiperazine gave 2-amino-4-alkoxypiperazine 7ai. Finally, the BOC group in 7ai was removed in the presence of trifluoroacetic acid or 4 M hydrochloric acid to furnish the corresponding pyrimidine derivatives 4ai.

2.2. Biological Evaluation

The serotonin receptor binding affinity of our synthesized compounds 4ai was examined by a radioligand binding assay in transfected CHO-K1 cell lines using [3H]mesulergine as a radioligand. Practically, a displacement of radioligand with compounds 4ai was first evaluated at a concentration of 10 μM, and then their Ki values were determined on the basis of the dose-response curves. As summarized in Table 1, compounds 4d and 4e showed the highest binding affinities to 5-HT2C and good selectivity values for 5-HT2A. However, the binding affinities of 4d and 4e to 5-HT2B were comparable to the value of 3. At this moment, we assumed that both diastereomeric mixtures 4d and 4e might have a negative influence on the selectivity for 5-HT2B. Thus, we planned to synthesize each diastereomer derived from 4d and 4e as an optically pure form to test its in vitro activity against 5-HT2 receptors.

2.3. Synthesis and In Vitro Evaluation of Optically Active Pyrimidines

In order to synthesize 4d and 4e as optically active diastereomers, optically active secondary alcohols 5d and 5e should be prepared. For this purpose, we initially attempted the separation of diastereomers 10 and 11, which were synthesized by the reaction of 5d and 5e with (R)-(−)-O-acetylmadelic acid using EDCI as a coupling reagent. However, diastereomeric mixtures 10 and 11 were not completely separated by column chromatography on silica gel. Alternatively, the enzymatic kinetic resolution was applied to separate racemic 5d and 5e, as shown in Scheme 2. It has been reported that CAL-B lipase can selectively acetylate the (R)-form of secondary benzyl alcohols using vinyl acetate as an acyl transfer reagent (Scheme 2) [25,26]. According to the literature procedure, the selective acetylation reactions of racemic secondary alcohols 5d and 5e were performed using 0.5 equivalent of vinyl acetate, pyridine, and the CAL-B enzyme in hexane. Enantiomeric pure acetate 12 and 13 were separated and then hydrolyzed to afford the desired (R)-forms of 5d and 5e. It should be noted that we obtained each diasteromeric 5d and 5e with a high optical purity when hexane was used as a solvent, although an ionic liquid such as [bmim][PF6] and [bmim][BF4] was used to enhance the enantiomeric selectivity of lipases in the literature. Additionally, (S)-5d and (S)-5e were successfully obtained by further acetylation of the remaining alcohols 5d and 5e with an excess of vinyl acetate and CAL-B enzyme followed by chromatographic separation.
For determining the optical purity of (R)/(S)-5d and 5e, they were converted to the corresponding mandelic ester (R)/(S)-10 and 11, respectively. A 1H-NMR analysis of diastereomeric protons in (R)/(S)-10 and 11 indicated that the ee’s of (R/(S)-5d and 5e were greater than 93%. We also confirmed that the optical rotation values of our compounds (R/(S)-5d and 5e are almost identical to those of the compounds reported in the literature.
With each enantiomer (R)- or (S)-5d and 5e in hand, the optically active pyrimidine derivatives 4d4e were synthesized following the same reaction sequences (Scheme 3). Finally, compounds 4d and 4e were assessed for their binding affinity to 5-HT2 receptor subtypes by radioligand binding assays. The in vitro assay results are demonstrated in Table 2. In general, (R,R)-4d and 4e prepared from secondary alcohols (R)-5d and (R)-5e showed excellent binding affinities to 5-HT2C, whereas (S,R)-forms of 4d and 4e exhibited low potencies for 5-HT2A and 5-HT2B. A further evaluation of these compounds for other 5-HT receptor subtypes was also performed and is provided in the supplementary data (Table S2). These results combined with the in vitro data in Table 1 suggested that pyrimidine derivatives 4 with a short alkyl chain could maintain their binding affinity to 5-HT2C comparable to that of 3 and the binding affinity for other 5-HT subtypes could be significantly influenced by an absolute configuration of the stereogenic methyl group in 4. Considering the potencies and selectivities of 4, we can conclude that (R,R)-4e would be a viable candidate for a selective 5-HT2C modulator.

3. Materials and Methods

3.1. General Methods

All reactions were conducted under oven-dried glassware under an atmosphere of nitrogen. All commercially available reagents were purchased and used without further purification. Solvents and gases were dried according to standard procedures. Organic solvents were evaporated with reduced pressure using a rotary evaporator. Reactions were followed by analytical thin layer chromatography (TLC) analysis using glass plates precoated with silica gel (0.25 mm). TLC plates were visualized by exposure to UV light (UV), and were then visualized with a KMnO4 or p-anisaldehyde stain followed by brief heating on a hot plate. Flash column chromatography was performed using silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany) with the indicated solvents. 1H-NMR spectra were measured with 400MHz and 13C-NMR spectra were measured with 100MHz using CDCl3 and MeOD. 1H-NMR spectra are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration, and coupling constant (J) in Hertz (Hz). 1H-NMR chemical shifts are reported relative to CDCl3 (7.26 ppm). 13C NMR was recorded relative to the central line of CDCl3 (77.0 ppm). High resolution mass spectra (HR-MS) were obtained using positive electrospray ionization and mass/charge (m/z) ratios that are reported as values in atomic mass units.

3.2. Synthesis of Pyrimidines 4ai

3.2.1. General Procedure for Preparing Compounds 5f and 5g

To a solution of 3-(3 or 4-fluorophenyl)propanoic acid 8 or 9 (1.78 mmol) in THF (8.90 mL), borane-dimethyl sulfide (3.57 mmol) was added dropwise. The reaction mixture was allowed to stir at room temperature for 1 h. After completion of the reaction (monitored by TLC), the mixture was slowly quenched with MeOH until bubbling ceased, extracted with EtOAc. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:4) to afford propanol 5f or 5g.
3-(3-Fluorophenyl)propan-1-ol (5f): Yield: 84%; 1H-NMR (400 MHz, CDCl3) δ 7.26–7.21 (m, 1H), 6.97 (d, J = 7.6 Hz, 1H), 6.92–6.86 (m, 2H), 3.68 (q, J = 5.8 Hz, 2H), 2.72 (t, J = 7.7 Hz, 2H), 1.92–1.85 (m, 2H). 13C-NMR (100 MHz, CDCl3) δ 162.9 (d, 1J = 243 Hz), 144.4 (d, 3J = 7 Hz), 129.8 (d, 3J = 8 Hz), 124.1 (d, 4J = 3 Hz), 115.2 (d, 2J = 21 Hz), 112.7 (d, 2J = 21 Hz), 62.0, 33.9, 31.7 (d, 4J = 2 Hz).
3-(4-Fluorophenyl)propan-1-ol (5g): Yield: 94%; 1H-NMR (400 MHz, CDCl3) δ 7.17–7.14 (m, 2H), 7.00–6.95 (m, 2H), 3.67 (t, J = 6.4 Hz, 2H), 2.69 (t, J = 7.8 Hz, 2H), 3.67 (t, J = 6.4 Hz, 2H), 1.91–1.84 (m, 2H). 13C-NMR (100 MHz, CDCl3) δ 161.6 (d, 1J = 241.6 Hz), 137.7 (d, 4J = 3.1 Hz), 130.0 (d, 3J = 7.6 Hz), 115.4 (d, 2J = 21 Hz), 62.3, 34.6, 31.5.

3.2.2. General Procedure for Preparing Compounds 5f’ and 5g’

To a solution of propanol 5f or 5g (0.482 mmol) in CH2Cl2 (4.80 mL), PCC (0.964 mmol) was added at 0 °C. The reaction mixture was allowed to stir at room temperature for 1 h. After completion of the reaction (monitored by TLC), the mixture was filtered with silica gel and a celite pad, and extracted with ether. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:4) to afford propanal 5f’ or 5g’.
3-(3-Fluorophenyl)propanal (5f’): Yield: 77%; 1H-NMR (400 MHz, CDCl3) δ 9.82 (t, J = 1.2 Hz, 1H), 7.27–7.22 (m, 1H), 6.97 (d, J = 7.8 Hz, 1H), 6.91–6.88 (m, 2H), 2.95 (t, J = 7.5 Hz, 2H), 2.80–2.77 (m, 2H). 13C-NMR (100 MHz, CDCl3) δ 201.0, 162.9 (d, 1J = 242 Hz), 142.9 (d, 3J = 8 Hz), 130.4 (d, 3J = 8 Hz), 124.0 (d, 4J = 2 Hz), 115.2 (d, 2J = 21 Hz), 113.2 (d, 2J = 21 Hz), 44.9, 27.8 (d, 4J = 2 Hz).
3-(4-Fluorophenyl)propanal (5g’): Yield: 73%; 1H-NMR (400 MHz, CDCl3) δ 9.77 (t, J = 1.2 Hz, 1H), 7.13–7.09 (m, 2H), 6.96–6.91 (m, 2H), 2.91–2.87 (m, 2H), 2.72 (t, J = 7.5 Hz, 2H). 13C-NMR (100 MHz, CDCl3) δ 201.2, 161.5 (d, 1J = 243 Hz), 136.0 (d, 4J = 3 Hz), 129.7 (d, 3J = 8 Hz), 115.3 (d, 2J = 21 Hz), 45.4, 27.3.

3.2.3. General Procedure for Preparing Compounds 5h and 5i

To a solution of propanal 5f’ or 5g’ (0.204 mmol) in THF (2.00 mL), MeMgBr (3.0 M in ether: 0.245 mmol) was added dropwise at 0 °C. The reaction mixture was allowed to stir at room temperature for 1 h. After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NH4Cl, extracted with EtOAc and washed with brine. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:2) to afford butan-2-ol 5h or 5i.
4-(3-Fluorophenyl)butan-2-ol (5h): Yield: 85%; 1H-NMR (400 MHz, CDCl3) δ 7.26–7.20 (m, 1H), 6.97 (d, J = 7.7 Hz, 1H), 6.92–6.85 (m, 2H), 3.82 (d, J = 2.3 Hz, 1H), 2.80–2.63 (m, 2H), 1.79–1.73 (m, 2H), 1.24 (d, J = 6.2 Hz, 3H).
4-(4-Fluorophenyl)butan-2-ol (5i): Yield: 72%; 1H-NMR (400 MHz, CDCl3) δ 7.12 (dd, J = 5.6, 8.2 Hz, 2H), 6.93 (t, J = 8.7 Hz, 2H), 3.82–3.75 (m, 1H), 2.74–2.58 (m, 2H), 1.75–1.68 (m, 2H), 1.20 (d, J = 6.2 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 161.3 (d, 1J = 241 Hz), 137.7 (d, 4J = 3 Hz), 129.7 (d, 3J = 8 Hz), 115.1 (d, 2J = 21 Hz), 67.4, 40.9, 31.3, 23.7.

3.2.4. General Procedure for Preparing Compounds 6ai

A solution of sodium tert-butoxide (2.72 mmol) in toluene (18.2 mL) was treated with primary or secondary alcohol (1.82 mmol) dropwise at 0 °C. After 5 min, 2,4-dichloro-5-fluoropyrimidine (2.18 mmol) was added to the mixture. The reaction mixture was allowed to stir at room temperature for 1 h. After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NH4Cl, extracted with EtOAc, and washed with brine. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford pyrimidine 6.
2-Chloro-5-fluoro-4-((2-fluorobenzyl)oxy)pyrimidine (6a): Yield: 91%; 1H-NMR (400 MHz, CDCl3) δ 8.21 (d, J = 2.2 Hz, 1H), 7.53–7.49 (m, 1H), 7.49–7.34 (m, 1H), 7.20–7.09 (m, 2H), 5.57 (s, 2H). 13C-NMR (100 MHz, CDCl3) δ 161.2 (d, 1J = 247 Hz), 159.0 (d, 2J = 12 Hz), 153.2 (d, 4J = 5 Hz), 145.9 (d, 1J = 263 Hz), 144.3 (d, 2J = 20 Hz), 131.1 (d, 3J = 4 Hz), 131.0 (d, 3J = 8 Hz), 124.3 (d, 4J = 4 Hz), 121.8 (d, 2J = 14 Hz), 115.7 (d, 2J = 20 Hz), 63.9 (d, 3J = 4 Hz).
2-Chloro-5-fluoro-4-((3-fluorobenzyl)oxy)pyrimidine (6b): Yield: 90%; 1H-NMR (400 MHz, CDCl3) δ 8.23 (d, J = 2.2 Hz, 1H), 7.40–7.34 (m, 1H), 7.25 (d, J = 7.26 Hz, 1H), 7.19 (d, J = 9.4 Hz, 1H), 7.09–7.04 (m, 1H), 5.50 (s, 2H). 13C-NMR (100 MHz, CDCl3) δ 162.9 (d, 1J = 245 Hz), 158.9 (d, 2J = 11 Hz), 153.2 (d, 4J = 5 Hz), 145.8 (d, 1J = 245 Hz), 144.4 (d, 2J = 20 Hz), 136.9 (d, 3J = 8 Hz), 130.3 (d, 3J = 8 Hz), 123.9 (d, 4J = 3 Hz), 115.7 (d, 2J = 21 Hz), 115.3 (d, 2J = 22 Hz), 68.9 (d, 4J = 2 Hz).
2-Chloro-5-fluoro-4-((4-fluorobenzyl)oxy)pyrimidine (6c): Yield: 75%; 1H-NMR (400 MHz, CDCl3) δ 8.20 (d, J = 2.2 Hz, 1H), 7.47 (dd, J = 5.4 Hz, 8.7Hz, 2H), 7.08 (t, J = 8.7 Hz, 2H), 5.47 (s, 2H). 13C-NMR (100 MHz, CDCl3) δ 163 (d, 1J = 246 Hz), 159.03 (d, 2J = 12 Hz), 153.2 (d, 4J = 5 Hz), 145 (d, 1J = 263 Hz), 144.3 (d, 2J = 20 Hz), 130.8 (d, 3J = 9 Hz), 130.3 (d, 3J = 4 Hz), 115.2 (d, 2J = 21 Hz), 69.2.
2-Chloro-5-fluoro-4-(1-(3-fluorophenyl)ethoxy)pyrimidine (6d): Yield: 79%; 1H-NMR (400 MHz, CDCl3) δ 8.17 (d, J = 2.2 Hz, 1H), 7.35–7.30 (m, 1H), 7.22 (d, J = 7.7 Hz, 1H), 7.17–7.14 (m, 1H), 7.02–6.97 (m, 1H), 6.31–6.27 (m, 1H), 1.70 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.9 (d, 1J = 245 Hz), 158.6 (d, 2J = 11 Hz), 153.2 (d, 4J = 5 Hz), 146.0 (d, 1J = 263 Hz), 144.4 (d, 2J = 20 Hz), 142.9 (d, 3J = 7 Hz), 130.2 (d, 3J = 8 Hz), 122.0 (d, 4J = 2 Hz), 115.3 (d, 2J = 21 Hz), 113.3 (d, 2J = 22 Hz), 75.7 (d, 4J = 2 Hz), 22.2.
2-Chloro-5-fluoro-4-(1-(4-fluorophenyl)ethoxy)pyrimidine (6e): Yield: 89%; 1H-NMR (400 MHz, CDCl3) δ 8.35 (d, J = 2.2 Hz, 1H), 7.66 (dd, J = 5.3, 8.7 Hz, 2H), 7.29–7.25 (m, 2H), 6.54–6.49 (m, 1H), 1.92 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.6 (d, 1J = 245 Hz), 158.6 (d, 2J = 11 Hz), 153.1 (d, 4J = 5 Hz), 146.0 (d, 1J = 262 Hz), 144.3 (d, 2J = 20 Hz), 136.1 (d, 4J = 3 Hz), 128.4 (d, 3J = 8 Hz), 115.6 (d, 2J = 22 Hz), 75.9, 22.1.
2-Chloro-5-fluoro-4-(3-(3-fluorophenyl)propoxy)pyrimidine (6f): Yield: 86%; 1H-NMR (400 MHz, CDCl3) δ 8.19 (d, J = 2.2 Hz, 1H), 7.28–7.22 (m, 1H), 6.98 (d, J = 7.7 Hz, 1H), 6.92–6.87 (m, 2H), 4.48 (t, J = 6.4 Hz, 2H), 2.80 (t, J = 7.6 Hz, 2H), 2.19–2.12 (m, 2H). 13C-NMR (100 MHz, CDCl3) δ 163.0 (d, 1J = 244 Hz), 159.4 (d, 2J = 11 Hz), 153.3 (d, 4J = 5 Hz), 145.9 (d, 1J = 263 Hz), 144.0 (d, 2J = 20 Hz), 143.3 (d, 3J = 7 Hz), 123.0 (d, 3J = 8 Hz), 124.1 (d, 4J = 3 Hz), 115.3 (d, 2J = 21 Hz), 113.1 (d, 2J = 21 Hz), 67.6, 31.6 (d, 4J = 1 Hz), 29.6.
2-Chloro-5-fluoro-4-(3-(4-fluorophenyl)propoxy)pyrimidine (6g): Yield: 93%; 1H-NMR (400 MHz, CDCl3) δ 8.17 (d, J = 4.2 Hz, 1H), 7.16–7.12 (m, 2H), 6.99–6.93 (m, 2H), 4.45 (t, J = 6.4 Hz, 2H), 2.75 (t, J = 7.6 Hz, 2H), 2.14–2.10 (m, 2H). 13C-NMR (100 MHz, CDCl3) δ 161.4 (d, 1J = 246 Hz), 159.4 (d, 2J = 11 Hz), 153.3 (d, 4J = 5 Hz), 145.9 (d, 1J = 262 Hz), 144.0 (d, 2J = 20 Hz), 136.3 (d, 4J = 3 Hz), 129.8 (d, 3J = 8 Hz), 115.3 (d, 2J = 21 Hz), 67.6, 31.1, 30.0.
2-Chloro-5-fluoro-4-((4-(3-fluorophenyl)butan-2-yl)oxy)pyrimidine (6h): Yield: 70%; 1H-NMR (400 MHz, CDCl3) δ 8.17 (d, J = 2.3 Hz, 1H), 7.26–7.20 (m, 1H), 6.95–6.87 (m, 3H), 5.40–5.35 (m, 1H), 2.79–2.69 (m, 2H), 2.18–2.11 (m, 1H), 2.02–1.96 (m, 1H), 1.43 (d, J = 6.2 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 163.0 (d, 1J = 244 Hz), 159.2 (d, 2J = 11 Hz), 153.3 (d, 4J = 5 Hz), 146.0 (d, 1J = 262 Hz), 144.1 (d, 2J = 20 Hz), 143.6 (d, 3J = 7 Hz), 129.9 (d, 3J = 8 Hz), 124.0 (d, 4J = 3 Hz), 115.2 (d, 2J = 21 Hz), 113.0 (d, 2J = 21 Hz), 75.1, 37.0, 31.4 (d, 4J = 1 Hz), 19.6.
2-Chloro-5-fluoro-4-((4-(4-fluorophenyl)butan-2-yl)oxy)pyrimidine (6i): Yield: 79%; 1H-NMR (400 MHz, CDCl3) δ 8.17 (d, J = 2.3 Hz, 1H), 7.14–7.11 (m, 2H), 6.99–6.94 (m, 2H), 5.40–5.35 (m, 1H), 2.78–2.65 (m, 2H), 2.17–2.09 (m, 1H), 2.00–1.92 (m, 1H), 1.42 (d, J = 6.2 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 161.4 (d, 1J = 242 Hz), 159.2 (d, 2J = 11 Hz), 153.3 (d, 4J = 5 Hz), 146.0 (d, 1J = 262 Hz), 144.0 (d, 2J = 21 Hz), 136.6 (d, 4J = 3 Hz), 129.7 (d, 3J = 8 Hz), 115.2 (d, 2J = 21 Hz), 75.2, 37.4, 30.8, 19.6.

3.2.5. General Procedure for Preparing Compounds 7ai

To a solution of pyrimidine 6 (0.569 mmol) in CH3CN (2.90 mL), (R)-(+)-1-Boc-3-methylpiperazine (1.14 mmol) and N,N-diisopropylethylamine (1.14 mmol) were added. The reaction mixture was allowed to stir at 110 °C for 14 h. After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NH4Cl, extracted with EtOAc, and washed with brine. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc/CHCl3/n-hexane = 1:4:8) to afford methyl piperazine carboxylate 7.
(R)-tert-Butyl-4-(5-fluoro-4-((2-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7a): Yield: 14%; 1H-NMR (400 MHz, CDCl3) δ 7.98 (d, J = 2.8 Hz, 1H), 7.49–7.45 (m, 1H), 7.35–7.29 (m, 1H), 7.17–7.13 (m, 1H), 7.11–7.06 (m, 1H), 5.53–5.44 (m, 2H), 4.73 (bs, 1H), 4.32 (d, J =13.3, 1H), 4.14–3.90 (m, 2H), 3.17–2.89 (m, 3H), 1.49 (s, 9H), 1.12 (d, J = 6.7 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 160.7 (d, 1J = 246 Hz), 157.6 (d, 2J = 11 Hz), 156.8 (d, 4J = 2 Hz), 155.2, 143.5 (d, 2J = 19 Hz), 139.9 (d, 1J = 247 Hz), 130.08 (d, 3J = 4 Hz), 130.05 (d, 3J = 9 Hz), 124.2 (d, 3J = 3 Hz), 123.4 (d, 2J = 14 Hz), 115.4 (d, 2J = 21 Hz), 79.8, 67.6 (d, 3J = 5 Hz), 48.5, 47.2, 44.0, 42.9, 38.7, 28.4, 14.1.
(R)-tert-Butyl-4-(5-fluoro-4-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7b): Yield: 27%; 1H-NMR (400 MHz, CDCl3) δ 7.99 (d, J = 2.8 Hz, 1H), 7.36–7.30 (m, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.15–7.09 (m, 1H), 7.03–6.98 (m, 1H), 5.43–5.36 (m, 2H), 4.69 (bs, 1H), 4.29 (d, J = 13.0 Hz, 1H), 4.14–3.84 (m, 2H), 3.16–2.87 (m, 3H), 1.48 (s, 9H), 1.11 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.9 (d, 1J = 245 Hz), 157.5 (d, 2J = 10 Hz), 156.8 (d, 4J = 2 Hz), 155.2, 143.6 (d, 2J = 19 Hz), 139.9 (d, 1J = 240 Hz), 138.7 (d, 3J = 7 Hz), 130.1 (d, 3J = 8 Hz), 123.2 (d, 4J = 3 Hz), 115.1 (d, 2J = 21 Hz), 114.6 (d, 2J = 22 Hz), 79.9, 67.1 (d, 4J = 1 Hz), 48.5, 47.2, 44.0, 42.9, 38.8, 28.4, 14.1.
(R)-tert-Butyl-4-(5-fluoro-4-((4-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7c): Yield: 21%; 1H-NMR (400 MHz, CDCl3) δ 7.98 (d, J = 2.8 Hz, 1H), 7.43–7.39 (m, 2H), 7.07–7.03 (m, 2H), 5.40–5.34 (m, 2H), 4.72 (bs, 1H), 4.30 (d, J = 12.8 Hz, 1H), 4.15–3.78 (m, 2H), 3.17–2.87 (m, 3H), 1.48 (s, 9H), 1.13 (d, J = 6.7 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.7 (d, 1J = 245 Hz), 157.7 (d, 2J = 11 Hz), 156.8 (d, 4J = 2 Hz), 155.2, 143.5 (d, 2J = 20 Hz), 139.9 (d, 1J = 247 Hz), 131.9 (d, 4J = 3 Hz), 129.9 (d, 3J = 8 Hz), 115.5 (d, 2J = 21 Hz), 79.9, 67.2, 48.5, 47.2, 44.0, 42.9, 38.8, 28.4, 14.1.
(R)-tert-Butyl-4-(5-fluoro-4-(1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7d): Yield: 44%; 1H-NMR (400 MHz, CDCl3, Diastereomeric mixture) δ 7.94–7.93 (m, 1H, 1H′), 7.32–7.25 (m, 1H, 1H′), 7.16–7.06 (m, 2H, 2H′), 6.97–6.90 (m, 1H, 1H′), 6.09–6.00 (m, 1H, 1H′), 4.57 (bs, 1H, 1H′), 4.22–3.85 (m, 3H, 3H′), 3.08–2.74 (m, 3H, 3H′), 1.65 (d, J = 6.6 Hz, 3H, 3H′), 1.46 (d, J = 2.4 Hz, 9H, 9H′), 1.13–0.90 (m, 3H, 3H′). 13C-NMR (100 MHz, CDCl3, Diastereomeric mixture) δ 162.9 (d, 1J = 244 Hz), 157.14 (d, 3J = 11 Hz), 157.11 (d, 3J = 11 Hz), 156.7 (d, 4J = 3 Hz), 155.20, 155.18, 145.0 (d, 2J = 23 Hz), 144.91 (d, 2J = 23 Hz), 143.47 (d, 2J = 20 Hz), 143.44 (d, 2J = 20 Hz), 140.0 (d, 1J = 245 Hz), 130.11 (d, 3J = 8 Hz), 130.07 (d, 3J = 9 Hz), 121.42 (d, 4J = 3 Hz), 121.17 (d, 4J = 3 Hz), 114.6 (d, 2J = 21 Hz), 114.5 (d, 2J = 21 Hz), 112.8 (d, 2J = 22 Hz), 112.6 (d, 2J = 22 Hz), 79.8, 79.8, 73.92, 73.90, 73.80, 73.79, 48.4, 47.1, 44.0, 42.8, 38.7, 28.4, 23.0, 22.8, 13.9.
(R)-tert-Butyl-4-(5-fluoro-4-(1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7e): Yield: 40%; 1H-NMR (400 MHz, CDCl3, Diastereomeric mixture) δ 7.95–7.94 (m, 1H, 1H′), 7.94–7.33 (m, 2H, 2H′), 7.06–6.98 (m, 2H, 2H′), 6.12–6.05 (m, 1H, 1H′), 4.60 (bs, 1H, 1H′), 4.25–3.88 (m, 3H, 3H′), 3.10–2.87 (m, 3H, 3H′), 1.66 (d, J = 6.6 Hz, 3H, 3H′), 1.48 (d, J = 2.0 Hz, 9H, 9H′), 1.15–0.96 (m, 3H, 3H′). 13C-NMR (100 MHz, CDCl3, Diastereomeric mixture) δ 162.3 (d, 1J = 245 Hz), 162.2 (d, 1J = 245 Hz), 157.3 (d, 2J = 11 Hz), 157.2 (d, 2J = 11 Hz), 156.7, 155.2, 143.4 (d, 2J = 20 Hz), 143.3 (d, 2J = 19 Hz), 140.0 (d, 1J = 247 Hz), 138.1 (d, 4J = 3 Hz), 137.9 (d, 4J = 3 Hz), 127.7 (d, 3J = 8 Hz), 127.4 (d, 3J = 8 Hz), 115.5 (d, 2J = 22 Hz), 115.4 (d, 2J = 21 Hz), 79.9, 73.9, 73.8, 48.5, 47.2, 43.8, 42.9, 38.71, 38.68, 28.4, 23.0, 22.8, 14.0.
(R)-tert-Butyl-4-(5-fluoro-4-(3-(3-fluorophenyl)propoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7f): Yield: 53%; 1H-NMR (400 MHz, CDCl3) δ 7.94 (d, J = 3.0 Hz, 1H), 7.26–7.20 (m, 1H), 6.96 (d, J = 7.7 Hz, 1H), 6.90–6.85 (m, 2H), 4.65 (bs, 1H), 4.35 (t, J = 6.5 Hz, 2H), 4.25–4.21 (m, 1H), 4.18–3.88 (m, 2H), 3.13–2.87 (m, 3H), 2.77 (t, J = 7.6 Hz, 2H), 2.13–2.06 (m, 2H), 1.47 (s, 9H), 1.12 (d, J = 6.7 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 163.6 (d, 1J = 244 Hz), 158.7 (d, 2J = 11 Hz), 157.5 (d, 4J = 2 Hz), 155.9, 144.4 (d, 3J = 7 Hz), 143.8 (d, 2J = 20 Hz), 140.6 (d, 1J = 247 Hz), 130.5 (d, 3J = 9 Hz), 124.8 (d, 4J = 3 Hz), 115.9 (d, 2J = 21 Hz), 113.6 (d, 2J = 21 Hz), 80.4, 66.0, 49.1, 47.8, 44.6, 43.5, 39.4, 32.5, 30.6, 29.1, 14.6.
(R)-tert-Butyl-4-(5-fluoro-4-(3-(4-fluorophenyl)propoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7g): Yield: 45%; 1H-NMR (400 MHz, CDCl3) δ 7.96 (d, J = 2.9 Hz, 1H), 7.16–7.13 (m, 2H), 6.99–6.94 (m, 2H), 4.66 (bs, 1H), 4.39–4.33 (m, 2H), 4.25–3.89 (m, 3H) 3.20–3.06 (m, 2H), 3.02–2.98 (m, 1H), 2.88–2.73 (m, 2H), 2.12–2.05 (m, 2H), 1.48 (s, 9H), 1.12 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 161.4 (d, 1J = 242 Hz), 158.1 (d, 2J = 11 Hz), 156.9 (d, 4J = 3 Hz), 155.2, 143.1 (d, 2J = 20 Hz), 140.0 (d, 1J = 247 Hz), 136.8 (d, 4J = 3 Hz), 129.8 (d, 3J = 8 Hz), 115.2 (d, 2J = 21 Hz), 79.8, 65.4, 48.5, 47.1, 44.0, 42.9, 38.7, 31.3, 30.4, 28.4, 14.0.
(R)-tert-Butyl-4-(5-fluoro-4-((4-(3-fluorophenyl)butan-2-yl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7h): Yield: 45%; 1H-NMR (400 MHz, CDCl3, Diastereomeric mixture) δ 7.96 (d, J = 2.9 Hz, 1H, 1H′), 7.24–7.18 (m, 1H, 1H′), 6.94–6.84 (m, 3H, 3H′), 5.27–5.19 (m, 1H, 1H′), 4.64–4.61 (m, 1H, 1H′), 4.22–3.88 (m, 3H, 3H′), 3.13–3.05 (m, 2H, 2H′), 2.88–2.67 (m, 3H, 3H′), 2.17–2.08 (m, 1H, 1H′), 1.96–1.87 (m, 1H, 1H′), 1.48 (d, J = 0.7 Hz, 9H, 9H′), 1.38 (d, J = 6.2 Hz, 3H, 3H′), 1.13–1.10 (m, 3H, 3H′). 13C-NMR (100 MHz, CDCl3, Diastereomeric mixture) δ 162.9 (d, 1J = 244 Hz), 157.8 (d, 2J = 11 Hz), 156.9 (d, 4J = 3 Hz), 155.2, 144.06 (d, 3J = 7 Hz), 144.04 (d, 3J = 3 Hz), 143.2 (d, 2J = 19 Hz), 140.1 (d, 1J = 246 Hz), 129.8 (d, 3J = 7 Hz), 124.1 (d, 4J = 3 Hz), 124.1 (d, 4J = 4 Hz), 115.3 (d, 2J = 20 Hz), 115.2 (d, 2J = 21 Hz), 113.8 (d, 2J = 22 Hz), 79.8, 72.1, 72.2, 48.4, 47.2, 44.0, 42.9, 38.7, 37.3, 31.5, 28.4, 19.8, 19.8, 14.0.
(R)-tert-Butyl-4-(5-fluoro-4-((4-(4-fluorophenyl)butan-2-yl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (7i): Yield: 40%; 1H-NMR (400 MHz, CDCl3, Diastereomeric mixture) δ 7.96–7.95 (m, 1H, 1H′), 7.12–7.07 (m, 2H, 2H′), 6.96–6.91 (m, 2H, 2H′), 5.26–5.18 (m, 1H, 1H′), 4.64 (bs, 1H, 1H′), 4.22–3.89 (m, 3H, 3H′), 3.14–3.05 (m, 2H, 2H′), 2.89–2.62 (m, 3H, 3H′), 2.13–2.05 (m, 1H, 1H′) 1.94–1.85 (m, 1H, 1H′), 1.48 (d, J = 0.6 Hz, 9H, 9H′), 1.38 (d, J = 6.2 Hz, 3H, 3H′), 1.13–1.11 (m, 3H, 3H′). 13C-NMR (100 MHz, CDCl3, Diastereomeric mixture) δ 161.3 (d, 1J = 242 Hz), 157.9 (d, 2J = 10 Hz), 156.9, 155.2, 143.1 (d, 2J = 20 Hz), 140.2 (d, 1J = 246 Hz), 137.07 (d, 4J = 3 Hz), 137.06 (d, 4J = 2 Hz), 129.76 (d, 3J = 7 Hz), 129.72 (d, 3J = 7 Hz), 115.14 (d, 2J = 21 Hz), 115.12 (d, 2J = 21 Hz), 79.8, 72.20, 72.17, 48.5, 47.2, 44.0, 42.9, 38.7, 37.7, 30.99, 30.96, 28.4, 19.9, 19.8, 13.9.

3.2.6. General Procedure for Preparing Compounds 4ai

Methods A: To a solution of methyl piperazine carboxylate 7 (0.161 mmol) in CH2Cl2 (1.60 mL), trifluoro acetic acid (4.01 mmol) was added at 0 °C. The reaction mixture was allowed to stir at the same temperature for 1 h. After completion of the reaction (monitored by TLC), the mixture was diluted with saturated aqueous NaHCO3, and extracted with EtOAc. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (DCM/MeOH = 10:1) to afford methyl piperazine 4.
Methods B: To a solution of methyl piperazine carboxylate 7 (0.144 mmol) in dioxane (1.45 mL), 1 M HCl in ether (1.44 mmol) was added at 0 °C. The reaction mixture was allowed to stir at the same temperature for 2.5 h. After completion of the reaction (monitored by TLC), the mixture was diluted with saturated aqueous NaHCO3, and extracted with EtOAc. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (DCM/MeOH = 10:1) to afford methyl piperazine 4.
(R)-5-Fluoro-4-((2-fluorobenzyl)oxy)-2-(2-methylpiperazin-1-yl)pyrimidine (4a): Methods A: Yield: 52%; 1H-NMR (400 MHz, MeOD) δ 8.16 (d, J = 3.1 Hz, 1H), 7.67–7.63 (m, 1H), 7.56–7.50 (m, 1H), 7.37–7.27 (m, 2H), 5.71–5.61 (m, 2H), 4.86–4.83 (m, 1H), 4.49–4.45 (m, 1H), 3.24–3.17 (m, 2H), 3.09–3.01 (m, 2H), 2.87–2.80 (m, 1H), 1.35 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz, MeOD) δ 160.9 (d, 1J = 245 Hz), 157.6 (d, 2J = 11 Hz), 156.9 (d, 4J = 2 Hz), 143.0 (d, 2J = 20 Hz), 139.6 (d, 1J = 246 Hz), 130.1 (d, 4J = 3 Hz), 130.0 (d, 3J = 8 Hz), 124.1 (d, 4J = 3 Hz), 123.4 (d, 2J = 14 Hz), 115.0 (d, 2J = 21 Hz), 61.5 (d, 3J = 5 Hz), 49.1, 46.3, 44.9, 38.8, 12.4; HRMS-ESI (m/z): [M + H]+ calcd. for C16H19F2N4O: 321.1521 ; found: 321.1525; HPLC purity, 6.7 min, 98.8%.
(R)-5-Fluoro-4-((3-fluorobenzyl)oxy)-2-(2-methylpiperazin-1-yl)pyrimidine (4b): Methods A: Yield: 72%; 1H-NMR (400 MHz, MeOD) δ 8.18 (d, J = 2.9 Hz, 1H), 7.67–7.54 (m, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 9.6 Hz, 1H), 7.31–7.21 (m, 1H), 5.66–5.58 (m, 2H), 4.83 (bs, 1H), 4.47 (d, J =12.6 Hz, 1H), 3.25–3.18 (m, 2H), 3.10–3.02 (m, 2H), 2.88–2.81 (m, 1H), 1.35 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz, MeOD) δ 162.9 (d, 1J = 243 Hz), 157.6 (d, 2J = 11 Hz), 156.9 (d, 4J = 2 Hz), 143.1 (d, 2J = 20 Hz), 139.6 (d, 1J = 245 Hz), 139.3 (d, 3J = 8 Hz), 130.0 (d, 3J = 8 Hz), 123.0 (d, 4J = 3 Hz), 114.4 (d, 2J = 21 Hz), 114.0 (d, 2J = 22 Hz), 66.9 (d, 4J = 1 Hz), 49.2, 46.4, 44.8, 38.8, 12.4; HRMS-ESI (m/z): [M + H]+ calcd. for C16H19F2N4O: 321.1521; found: 321.1523; HPLC purity, 6.9 min, 97.1%.
(R)-5-Fluoro-4-((4-fluorobenzyl)oxy)-2-(2-methylpiperazin-1-yl)pyrimidine (4c): Methods A: Yield: 57%; 1H-NMR (400 MHz, MeOD) δ 8.19 (d, J = 3.1 Hz, 1H), 7.68–7.65 (m, 2H), 7.30 (t, J = 8.8 Hz, 2H), 5.65–5.57 (m, 2H), 4.89–4.86 (m, 1H), 4.53–4.48 (m, 1H), 3.28–3.21 (m, 2H), 3.14–3.06 (m, 2H), 2.92–2.84 (m, 1H), 1.38 (d, J = 6.9 Hz, 3H). 13C-NMR (100 MHz, MeOD) δ 162.6 (d, 1J = 243 Hz), 157.7 (d, 2J = 11 Hz), 156.9 (d, 4J = 2 Hz), 143.0 (d, 2J = 20 Hz), 139.7 (d, 1J = 246 Hz), 132.5 (d, 4J = 4 Hz), 129.8 (d, 3J = 9 Hz), 114.9 (d, 2J = 22 Hz), 67.0, 49.1, 46.3, 44.8, 38.7, 12.4; HRMS-ESI (m/z): [M + H]+ calcd. for C16H19F2N4O: 321.1521; found: 321.1524; HPLC purity, 7.4 min, 97.4%.
5-Fluoro-4-(1-(3-fluorophenyl)ethoxy)-2-((R)-2-methylpiperazin-1-yl)pyrimidine (4d): Methods A: Yield: 71%; 1H-NMR (400 MHz, MeOD, Diastereomeric mixture) δ 8.13 (s, 1H, 1H′), 7.52–7.49 (m, 1H, 1H′), 7.39–7.36 (m, 1H, 1H′), 7.32–7.29 (m, 1H, 1H′), 7.16–7.14 (m, 2H), 6.31–6.22 (m, 1H, 1H′), 4.71 (bs, 1H, 1H′), 4.43–4.33 (m, 1H, 1H′), 3.18–2.99 (m, 4H, 4H′), 2.86–2.72 (m, 1H, 1H′), 1.83–1.81 (m, 3H, 3H′), 1.39–1.12 (m, 3H, 3H′). 13C-NMR (100 MHz, MeOD, Diastereomeric mixture) δ 162.93 (d, 1J = 245 Hz), 162.91 (d, 1J = 245 Hz), 157.13 (d, 2J = 11 Hz), 157.10 (d, 2J = 11 Hz), 156.8, 145.6 (d, 3J = 7 Hz), 145.3 (d, 3J = 7 Hz), 143.1 (d, 2J = 20 Hz), 143.0 (d, 2J = 19 Hz), 139.7 (d, 1J = 246 Hz), 130.1 (d, 3J = 8 Hz), 130.0 (d, 3J = 8 Hz), 121.14 (d, 2J = 27 Hz), 121.11 (d, 2J = 27 Hz), 114.0 (d, 2J = 22 Hz), 113.9 (d, 2J = 21 Hz), 74.05 (d, 4J = 2 Hz), 73.97 (d, 4J = 1 Hz), 49.1, 49.0, 46.3, 44.8, 44.7, 38.72, 38.68, 22.0, 21.8, 12.5, 12.4; HRMS-ESI (m/z): [M + H]+ calcd. for C17H21F2N4O: 335.1678 ; found: 335.1680; HPLC purity, 7.5 min, 98.1%.
5-Fluoro-4-(1-(4-fluorophenyl)ethoxy)-2-((R)-2-methylpiperazin-1-yl)pyrimidine (4e): Methods B: Yield: 53%; 1H-NMR (400 MHz, MeOD, Diastereomeric mixture) δ 8.16–8.14 (m, 1H, 1H′), 7.64–7.59 (m, 2H, 2H′), 7.28–7.23 (m, 2H, 2H′), 6.33–6.28 (m, 1H, 1H′), 4.78–4.76 (m, 1H, 1H′), 4.45–4.36 (m, 1H, 1H′), 3.21–3.10 (m, 2H, 2H′), 3.08–2.99 (m, 2H, 2H′), 2.89–2.75 (m, 1H, 1H′), 1.84–1.82 (m, 3H, 3H′), 1.41–1.17 (m, 3H, 3H′). 13C-NMR (100 MHz, MeOD, Diastereomeric mixture) δ 162.3 (d, 1J = 243 Hz), 162.2 (d, 1J = 243 Hz), 157.27 (d, 2J = 11 Hz), 157.25 (d, 2J = 11 Hz), 156.8, 142.93 (d, 2J = 20 Hz), 142.87 (d, 2J = 20 Hz), 139.81 (d, 1J = 243 Hz), 139.78 (d, 1J = 244 Hz), 138.4 (d, 4J = 3 Hz), 127.5 (d, 3J = 8 Hz), 127.3 (d, 3J = 8 Hz), 114.9 (d, 2J = 22 Hz), 114.8 (d, 2J = 22 Hz), 74.1, 74.0, 48.98, 48.94, 46.2, 44.71, 44.68, 38.6, 38.5, 22.0, 21.8, 12.4; HRMS-ESI (m/z): [M + H]+ calcd. for C17H21F2N4O: 335.1678 ; found: 335.1680; HPLC purity, 7.4 min, 98.3%.
(R)-5-Fluoro-4-(3-(3-fluorophenyl)propoxy)-2-(2-methylpiperazin-1-yl)pyrimidine (4f): Methods A: Yield: 62%; 1H-NMR (400 MHz, CDCl3) δ 7.99 (d, J = 3.0 Hz, 1H), 7.30–7.24 (m, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.95–6.89 (m, 2H), 4.67–4.61 (m, 1H), 4.42–4.36 (m, 2H), 4.29–4.25 (m, 1H), 3.09–2.98 (m, 3H), 2.90 (d, J = 12.2 Hz, 1H), 2.83–2.77 (m, 3H), 2.18–2.11 (m, 2H), 1.24 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz, MeOD) δ 163.0 (d, 1J = 243 Hz), 158.3 (d, 2J = 11 Hz), 156.2, 144.1 (d, 3J = 8 Hz), 142.9 (d, 2J = 21 Hz), 140.3 (d, 1J = 247 Hz), 129.7 (d, 3J = 8 Hz), 124.1 (d, 3J = 4 Hz), 114.8 (d, 2J =21 Hz), 112.3 (d, 2J = 21 Hz), 65.5, 44.6, 43.3, 35.8, 31.3, 29.7, 12.3; HRMS-ESI (m/z): [M + H]+ calcd. for C18H23F2N4O: 349.1834; found: 347.1837; HPLC purity, 5.1 min, 98.7%.
(R)-5-Fluoro-4-(3-(4-fluorophenyl)propoxy)-2-(2-methylpiperazin-1-yl)pyrimidine (4g): Methods A: Yield: 63%; 1H-NMR (400 MHz, CDCl3) δ 7.93 (d, J = 3.0 Hz, 1H), 7.14–7.11 (m, 2H), 6.96–6.92 (m, 2H), 4.58 (t, J = 5.0 Hz, 1H), 4.35–4.31 (m, 2H), 4.23–4.19 (m, 1H), 3.03–2.92 (m, 3H), 2.84 (d, J = 12.3 Hz, 1H), 2.75–2.71 (m, 3H), 2.10–2.03 (m, 2H), 1.18 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz, MeOD) δ 161.4 (d, 1J = 241 Hz), 158.4 (d, 2J = 11 Hz), 156.0, 142.8 (d, 2J = 20 Hz), 140.5 (d, 1J = 247 Hz), 137.1, 129.8 (d, 3J = 8 Hz), 114.6 (d, 2J = 21 Hz), 65.7, 46.9, 44.3, 43.1, 35.3, 30.7, 30.1, 12.4; HRMS-ESI (m/z): [M + H]+ calcd. for C18H23F2N4O: 349.1834; found: 347.1836; HPLC purity, 5.2 min, 95.5%.
5-Fluoro-4-((4-(3-fluorophenyl)butan-2-yl)oxy)-2-((R)-2-methylpiperazin-1-l)pyrimidine (4h): Methods A: Yield: 68%; 1H-NMR (400 MHz, MeOD, Diastereomeric mixture) δ 8.16–8.15 (m, 1H, 1H′), 7.46–7.40 (m, 1H, 1H′), 7.16–7.15 (m, 1H, 1H′), 7.09–7.05 (m, 2H, 2H′), 5.48–5.38 (m, 1H, 1H′), 4.80–4.72 (m, 1H, 1H′), 4.42–4.37 (m, 1H, 1H′), 3.24–3.15 (m, 2H, 2H′), 3.12–3.03 (m, 1H, 1H′), 2.99–2.83 (m, 3H, 3H′), 2.31–2.24 (m, 1H, 1H′), 2.18–2.09 (m, 1H, 1H′), 1.57–1.56 (m, 3H, 3H′), 1.39–1.35 (m, 3H, 3H′). 13C-NMR (100 MHz, MeOD, Diastereomeric mixture) δ 162.9 (d, 1J = 242 Hz), 157.86 (d, 2J = 11 Hz), 157.84 (d, 2J = 11 Hz), 156.9, 144.3 (d, 3J = 7 Hz), 142.7 (d, 2J = 20 Hz), 139.8 (d, 1J = 245 Hz), 129.68 (d, 3J = 9 Hz), 129.66 (d, 3J = 9 Hz), 124.02 (d, 4J = 3 Hz), 123.98 (d, 4J = 3 Hz), 114.8 (d, 2J = 21 Hz), 114.7 (d, 2J = 21 Hz), 112.3 (d, 2J = 22 Hz), 112.2 (d, 2J = 22 Hz), 72.01, 71.92, 49.12, 49.10, 46.24, 46.22, 44.82, 44.80, 38.70, 38.68, 36.9, 31.0, 18.66, 18.64, 12.4, 12.3; HRMS-ESI (m/z): [M + H]+ calcd. for C19H25F2N4O: 363.1991; found: 363.1992; HPLC purity, 8.0 min, 98.4%.
5-Fluoro-4-((4-(4-fluorophenyl)butan-2-yl)oxy)-2-((R)-2-methylpiperazin-1-yl)pyrimidine (4i): Methods A: Yield: 53%; 1H-NMR (400 MHz, MeOD, Diastereomeric mixture) δ 8.12 (d, J = 2.4 Hz, 1H, 1H′), 7.32–7.29 (m, 2H, 2H′), 7.14–7.08 (m, 2H, 2H′), 5.43–5.34 (m, 1H, 1H′), 4.74–4.67 (m, 1H, 1H′), 4.35–4.31 (m, 1H, 1H′), 3.18–3.11 (m, 2H, 2H′), 3.08–2.99 (m, 2H, 2H′), 2.93–2.79 (m, 1H, H′), 2.28–2.19 (m, 1H, 1H′) 2.13–2.03 (m, 1H, 1H′), 1.54–1.51 (m, 3H, 3H′), 1.35–1.31 (m, 3H, 3H′). 13C-NMR (100 MHz, MeOD, Diastereomeric mixture) δ 161.3 (d, 1J = 241 Hz), 157.87 (d, 2J = 11 Hz), 157.85 (d, 2J = 11 Hz), 156.9, 142.7 (d, 2J = 20 Hz), 139.9 (d, 1J = 246 Hz), 137.3 (d, 4J = 4 Hz), 129.72 (d, 3J = 7 Hz), 129.67 (d, 3J = 7 Hz), 114.57 (d, 2J = 21 Hz), 114.55 (d, 2J = 21 Hz), 72.00, 71.95, 49.13, 49.09, 46.24, 46.22, 44.83, 44.79, 38.71, 38.68, 37.3, 30.5, 30.4, 18.66, 18.62, 12.4, 12.3; HRMS-ESI (m/z): [M + H]+ calcd. for C19H25F2N4O: 363.1991; found: 363.1993; HPLC purity, 8.2 min, 96.9%.

3.3. Synthesis of Optically Active Pyrimidines 4d and 4e

3.3.1. General Procedure for Preparing Compound (R)-(+)-5d and (R)-(+)-5e

To a solution of 1-(3 or 4-fluorophenyl)ethanol (6.67 mmol) in n-hexane (22.2 mL), CAL-B (147 mg), vinyl acetate (3.34 mmol), and triethylamine (0.667 mmol) were added. The reaction mixture was allowed to stir at room temperature for 1 h. After completion of the reaction (monitored by TLC), the mixture was filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford acetate intermediate (315 mg) as a colorless oil. To a solution of acetate (1.73 mmol) in MeOH (3.45 mL), 1 M NaOH (2.59 mmol) was added. The reaction mixture was allowed to stir at room temperature for 1 h. After completion of the reaction (monitored by TLC), it was quenched with ditilled water and extracted with EtOAc. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford alcohol (R)-(+)-5d and (R)-(+)-5e.
(R)-1-(3-Fluorophenyl)ethan-1-ol ((R)-(+)-5d): Yield: 17%; 1H-NMR (400 MHz, CDCl3) δ 7.32–7.26 (m, 1H), 7.12–7.07 (m, 2H), 6.97–6.92 (m, 1H), 4.87 (q, J = 6.4 Hz, 1H), 2.18 (s, 1H), 1.47 (d, J = 6.4 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 163.0 (d, 1J = 244 Hz), 148.5 (d, 3J = 6 Hz), 130.0 (d, 3J = 8 Hz), 121.0 (d, 4J = 3 Hz), 114.2 (d, 2J = 21 Hz), 112.3 (d, 2J = 21 Hz), 69.8, 25.2; Optical rotation for (R)-(+)-5d: [ α ]   D 26 +43.7° (c 0.7, CHCl3).
(R)-1-(4-Fluorophenyl)ethan-1-ol ((R)-(+)-5e): Yield: 32%; 1H-NMR (400 MHz, CDCl3) δ 7.32–7.26 (m, 2H), 7.03–6.97 (m, 2H), 4.84 (q, J = 6.1 Hz, 1H), 2.34 (s, 1H), 1.44 (d, J = 6.4 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.1 (d, 1J = 244 Hz), 141.6 (d, 4J = 3 Hz), 127.1 (d, 3J = 8 Hz), 115.2 (d, 2J = 21 Hz), 69.7, 25.3; Optical rotation for (R)-(+)-5e: [ α ] D 27 +51.9° (c 0.5, CHCl3).

3.3.2. General Procedure for Preparing Compound (S)-(−)-5d and (S)-(−)-5e

To a solution of 1-(3 or 4-fluorophenyl)ethanol (6.67 mmol) in n-hexane (22.2 mL), CAL-B (147 mg), vinyl acetate (13.3 mmol), and triethylamine (0.667 mmol) were added. The reaction mixture was allowed to stir at room temperature for 12 h. After completion of the reaction (monitored by TLC), the mixture was filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford alcohol (S)-(−)-5d and (S)-(−)-5e.
(S)-1-(3-Fluorophenyl)ethan-1-ol ((S)-(−)-5d): Yield: 44%; 1H-NMR (400 MHz, CDCl3) δ 7.31–7.26 (m, 1H), 7.11–7.06 (m, 2H), 6.96–6.91 (m, 1H), 4.85 (td, J = 5.5, 7.5 Hz, 1H), 2.31 (s, 1H), 1.46 (d, J = 6.5 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 163.0 (d, 1J = 245 Hz), 148.6 (d, 3J = 6 Hz), 130.0 (d, 3J = 8 Hz), 121.0 (d, 4J = 3 Hz), 114.2 (d, 2J = 21 Hz), 112.3 (d, 2J = 22 Hz), 69.8 (d, 4J = 2 Hz), 25.2; Optical rotation for (S)-(−)-5d: [ α ]   D 27 −46.9° (c 0.4, CHCl3).
(S)-1-(4-Fluorophenyl)ethan-1-ol ((S)-(−)-5e): Yield: 46%; 1H-NMR (400 MHz, CDCl3) δ 7.33–7.30 (m, 2H), 7.04–6.98 (m, 2H), 4.85 (q, J = 6.4 Hz, 1H), 2.16 (s, 1H), 1.45 (d, J = 6.4 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.1 (d, 1J = 243 Hz), 141.5 (d, 4J = 3 Hz), 127.1 (d, 3J = 8 Hz), 115.2 (d, 2J = 21 Hz), 69.8, 25.3; Optical rotation for (S)-(−)-5e: [ α ]   D 27 –49.7° (c 0.6, CHCl3).

3.3.3. General Procedure for Preparing Compounds (R)/(S)-10 and 11

To a solution of (R) or (S)-secondary alcohol (0.0749 mmol) in CH2Cl2 (0.400 mL), (R)-2-acetoxy-2-phenylacetic acid (0.112 mmol), EDCI (0.112 mmol), and DMAP (0.112 mmol) were added. The reaction mixture was allowed to stir at room temperature for 12 h. After completion of the reaction (monitored by TLC), the mixture was filtered, extracted with CH2Cl2, and washed with brine. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford mandelate (R)/(S)-10 and 11.
(R)-1-(3-Fluorophenyl)ethyl (R)-2-acetoxy-2-phenylacetate ((R)-10): Yield: 86%; 1H-NMR (400 MHz, CDCl3) δ 7.41–7.34 (m, 3H), 7.18–7.12 (m, 1H), 6.91–6.86 (m, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.67 (dd, J = 1.9, 9.8 Hz, 1H), 5.97 (s, 1H), 5.85 (q, J = 6.6 Hz, 1H), 2.19 (s, 3H), 1.52 (d, J = 6.6 Hz, 3H).
(S)-1-(3-Fluorophenyl)ethyl (R)-2-acetoxy-2-phenylacetate ((S)-10): Yield: 84%; 1H-NMR (400 MHz, CDCl3) δ 7.50–7.47 (m, 2H), 7.44–7.40 (m, 3H), 7.33–7.26 (m, 1H), 7.09 (d, J = 7.7 Hz, 1H ), 7.04–6.96 (m, 2H), 5.96 (s, 1H), 5.88 (q, J = 6.6 Hz, 1H), 2.19 (s, 3H), 1.41 (d, J = 6.6 Hz, 3H).
(R)-1-(4-Fluorophenyl)ethyl (R)-2-acetoxy-2-phenylacetate ((R)-11): Yield: 68%; 1H-NMR (400 MHz, CDCl3) δ 7.39–7.31 (m, 5H), 6.99–6.95 (m, 2H), 6.89–6.83 (m, 2H), 5.94 (s, 1H), 5.84 (q, J = 6.6 Hz, 1H), 2.17 (s, 3H), 1.52 (d, J = 6.6 Hz, 3H).
(S)-1-(4-Fluorophenyl)ethyl (R)-2-acetoxy-2-phenylacetate ((S)-11): Yield: 84%; 1H-NMR (400 MHz, CDCl3) δ 7.48–7.45 (m, 2H), 7.41–7.37 (m, 3H), 7.31–7.25 (m, 2H), 7.05–7.00 (m, 2H), 5.93 (s, 1H), 5.87 (q, J = 6.6 Hz, 1H), 2.18 (s, 3H), 1.40 (d, J = 6.6 Hz, 3H).

3.3.4. General Procedure for Preparing Compounds (R,R)- or (S,R)-4d, and 4e

The title compounds (R,R) or (S,R)-4d and 4e were prepared from (R)-(+)-5d/5e and (S)-(+)-5d/5e following the same procedures described for the synthesis of 4d and 4e.
(R)-2-Chloro-5-fluoro-4-(1-(3-fluorophenyl)ethoxy)pyrimidine ((R)-6d): Yield: 72%; 1H-NMR (400 MHz, CDCl3) δ 8.19 (d, J = 2.2 Hz, 1H), 7.37–7.31 (m, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.18–7.15 (m, 1H), 7.03–6.99 (m, 1H), 6.30 (q, J = 6.5 Hz, 1H), 1.71 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.9 (d, 1J = 245 Hz), 158.6 (d, 2J = 11 Hz), 153.2 (d, 4J = 5 Hz), 146.0 (d, 1J = 263 Hz), 144.4 (d, 2J = 20 Hz), 142.9 (d, 3J = 7 Hz), 130.3 (d, 3J = 8 Hz), 122.0 (d, 4J = 3 Hz), 115.3 (d, 2J = 21 Hz), 113.3 (d, 2J = 22 Hz), 75.6 (d, 4J = 2 Hz), 22.2; Optical rotation for (R)-6d: [ α ]   D 27 +178.3° (c 0.7, CHCl3).
(S)-2-Chloro-5-fluoro-4-(1-(3-fluorophenyl)ethoxy)pyrimidine ((S)-6d): Yield: 55%; 1H-NMR (400 MHz, CDCl3) δ 8.18 (d, J = 2.2 Hz, 1H), 7.36–7.31 (m, 1H), 7.22 (d, J = 7.7 Hz, 1H), 7.18–7.14 (m, 1H), 7.03–6.98 (m, 1H), 6.30 (q, J = 6.5 Hz, 1H), 1.71 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.9 (d, 1J = 245 Hz), 158.6 (d, 2J = 11 Hz), 153.2 (d, 4J = 4 Hz), 146.0 (d, 1J = 262 Hz), 144.4 (d, 2J = 20 Hz), 142.9 (d, 3J = 7 Hz), 130.3 (d, 3J = 8 Hz), 122.0 (d, 4J = 3 Hz), 115.3 (d, 2J = 21 Hz), 113.3 (d, 2J = 22 Hz), 75.7 (d, 4J = 1 Hz), 22.2; Optical rotation for (S)-6d: [ α ]   D 28 −182.7° (c 0.7, CHCl3).
(R)-2-Chloro-5-fluoro-4-(1-(4-fluorophenyl)ethoxy)pyrimidine ((R)-6e): Yield: 68%; 1H-NMR (400 MHz, CDCl3) δ 8.16 (d, J = 2.2 Hz, 1H), 7.47–7.42 (m, 2H), 7.08–7.02 (m, 2H), 6.30 (q, J = 6.6 Hz, 1H), 1.71 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.6 (d, 1J = 245 Hz), 158.7 (d, 2J = 11 Hz), 153.1 (d, 4J = 4 Hz), 146.0 (d, 1J = 263 Hz), 144.3 (d, 2J = 20 Hz), 136.1 (d, 4J = 4 Hz), 128.4 (d, 3J = 9 Hz), 115.6 (d, 2J = 22 Hz), 75.9, 22.2; Optical rotation for (R)-6e: [ α ]   D 27 +197.3° (c 0.8, CHCl3).
(S)-2-Chloro-5-fluoro-4-(1-(4-fluorophenyl)ethoxy)pyrimidine ((S)-6e): Yield: 65%; 1H-NMR (400 MHz, CDCl3) δ 8.16 (d, J = 2.2 Hz, 1H), 7.47–7.42 (m, 2H), 7.08–7.02 (m, 2H), 6.30 (q, J = 6.6 Hz, 1H), 1.71 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.6 (d, 1J = 245 Hz), 158.7 (d, 2J = 11 Hz), 153.1 (d, 4J = 4 Hz), 146.0 (d, 1J = 263 Hz), 144.3 (d, 2J = 20 Hz), 136.1 (d, 4J = 3 Hz), 128.4 (d, 3J = 8 Hz), 115.7 (d, 2J = 21 Hz), 75.9, 22.2; Optical rotation for (S)-6e: [ α ]   D 27 −204.7° (c 0.8, CHCl3).
tert-Butyl-(R)-4-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate ((R,R)-7d): Yield: 50%; 1H-NMR (400 MHz, CDCl3) δ 7.94 (d, J = 2.8 Hz, 1H), 7.31–7.26 (m, 1H), 7.14 (d, J = 7.7 Hz, 1H), 7.07 (d, J = 9.6 Hz, 1H) 6.96–6.91 (m, 1H), 6.03 (q, J = 6.6 Hz, 1H), 4.58 (bs, 1H), 4.18–3.86 (m, 3H), 3.08–3.01 (m, 2H), 2.84–2.74 (m, 1H), 1.66 (d, J = 6.6 Hz, 3H), 1.47 (s, 9H), 0.91 (d, J = 6.5 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.9 (d, 1J = 245 Hz), 157.1 (d, 2J = 11 Hz), 156.7 (d, 4J = 2 Hz), 155.2, 145.1 (d, 3J = 7 Hz), 143.4 (d, 2J = 20 Hz), 140.0 (d, 1J = 246 Hz), 130.1 (d, 3J = 8 Hz), 121.2 (d, 4J = 3 Hz), 114.5 (d, 2J = 21 Hz), 112.7 (d, 2J = 22 Hz), 79.8, 73.9, 48.4, 47.1, 43.9, 42.8, 38.7, 28.4, 23.0, 13.9; Optical rotation for (R,R)-7d: [ α ]   D 27 +105.0° (c 0.4, CHCl3).
tert-Butyl-(R)-4-(5-fluoro-4-((S)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate ((S,R)-7d): Yield: 57%; 1H-NMR (400 MHz, CDCl3) δ 7.95 (d, J = 2.9 Hz, 1H), 7.32–7.26 (m, 1H), 7.16 (d, J = 7.7 Hz, 1H), 7.11–7.09 (m, 1H) 6.97–6.93 (m, 1H), 6.06 (q, J = 6.6 Hz, 1H), 4.55 (bs, 1H), 4.22–3.84 (m, 3H), 3.07–2.87 (m, 3H), 1.66 (d, J = 6.6 Hz, 3H), 1.47 (s, 9H), 1.13 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 163.0 (d, 1J = 245 Hz), 157.1 (d, 2J = 11 Hz), 156.7 (d, 4J = 2 Hz), 155.2, 144.9 (d, 3J = 7 Hz), 143.5 (d, 2J = 19 Hz), 140.0 (d, 1J = 247 Hz), 130.0 (d, 3J = 8 Hz), 121.4 (d, 4J = 3 Hz), 114.6 (d, 2J = 21 Hz), 112.8 (d, 2J = 22 Hz), 79.8, 73.8, 48.4, 47.1, 43.9, 42.9, 38.7, 28.4, 22.8, 14.0; Optical rotation for (S,R)-7d: [ α ]   D 27 –215.3° (c 0.7, CHCl3).
tert-Butyl-(R)-4-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate ((R,R)-7e): Yield: 44%; 1H-NMR (400 MHz, CDCl3) δ 7.93 (d, J = 2.8 Hz, 1H), 7.39–7.33 (m, 2H), 7.04–6.98 (m, 2H), 6.07 (d, J = 6.6 Hz, 1H), 4.60 (bs, 1H), 4.23–3.86 (m, 3H), 3.09–2.77 (m, 3H), 1.65 (d, J = 6.6 Hz, 3H) 1.46 (s, 9H), 0.95 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.2 (d, 1J = 245 Hz), 157.2 (d, 2J = 11 Hz), 156.7 (d, 4J = 2 Hz), 155.2, 143.3 (d, 2J = 19 Hz), 140.0 (d, 1J = 247 Hz), 138.1 (d, 4J = 3 Hz), 127.4 (d, 3J = 8 Hz), 115.4 (d, 2J = 21 Hz), 79.8, 73.9, 73.9, 48.4, 47.1, 43.7, 42.8, 38.7, 28.4, 23.0, 14.0; Optical rotation for (R,R)-7e: [ α ] D 28 +77.9° (c 0.4, CHCl3).
tert-Butyl-(R)-4-(5-fluoro-4-((S)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate ((S,R)-7e): Yield: 52%; 1H-NMR (400 MHz, CDCl3) δ 7.93 (d, J = 2.8 Hz, 1H), 7.39–7.32 (m, 2H), 7.04–6.95 (m, 2H), 6.09 (d, J = 6.5 Hz, 1H), 4.59 (bs, 1H), 4.24–3.85 (m, 3H), 3.09–2.88 (m, 3H), 1.65 (d, J = 6.6 Hz, 3H) 1.47 (s, 9H), 1.13 (d, J = 6.4 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.3 (d, 1J = 245 Hz), 157.2 (d, 2J = 11 Hz), 156.7 (d, 4J = 3 Hz), 155.2, 143.4 (d, 2J = 19 Hz), 140.0 (d, 1J = 247 Hz), 137.9 (d, 4J = 3 Hz), 127.7 (d, 3J = 8 Hz), 115.4 (d, 2J = 22 Hz), 79.9, 73.8, 48.4, 47.2, 43.7, 42.9, 38.7, 28.4, 22.8, 14.0; Optical rotation for (S,R)-7e: [ α ]   D 28 −212.4° (c 0.5, CHCl3).
5-Fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)-2-((R)-2-methylpiperazin-1-yl)pyrimidine ((R,R)-4d): Methods A: Yield: 67%; 1H-NMR (400 MHz, CDCl3) δ 7.99 (d, J = 2.4 Hz, 1H), 7.32–7.26 (m, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 9.5 Hz, 1H), 6.97–6.93 (m, 1H), 5.99 (q, J = 6.5 Hz, 1H), 4.88 (bs, 1H), 4.47 (d, J = 13.7 Hz, 1H), 3.41–3.14 (m, 4H), 2.84 (bs, 1H), 1.68 (d, J = 6.6 Hz, 3H), 1.12 (d, J = 6.9 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 163.0 (d, 1J = 245 Hz), 157.6 (d, 2J = 11 Hz), 145.2 (d, 4J = 2 Hz), 144.8 (d, 3J = 7 Hz), 143.1 (d, 2J = 20 Hz), 140.6 (d, 1J = 246 Hz), 130.3 (d, 3J = 8 Hz), 121.0 (d, 4J = 3 Hz), 114.7 (d, 2J = 21 Hz), 112.5 (d, 2J = 23 Hz), 74.7, 47.2, 44.4, 43.1, 35.5, 23.0, 13.2; HRMS-ESI (m/z): [M + H]+ calcd for C17H21F2N4O: 335.1678; found: 335.1681; HPLC purity, 9.4 min, 98.4%; Optical rotation for (R,R)-4d: [ α ] D 26 +118.2° (c 0.3, MeOH).
5-Fluoro-4-((S)-1-(3-fluorophenyl)ethoxy)-2-((R)-2-methylpiperazin-1-yl)pyrimidine ((S,R)-4d): Methods A: Yield: 71%; 1H-NMR (400 MHz, CDCl3) δ 7.99 (d, J = 2.4 Hz, 1H), 7.34–7.26 (m, 1H), 7.15 (d, J = 7.7 Hz, 1H), 7.09 (d, J = 9.6 Hz, 1H), 7.00–6.95 (m, 1H), 6.03 (q, J = 6.5 Hz, 1H), 4.88–4.85 (m, 1H), 4.53 (d, J = 13.6 Hz, 1H), 3.43 (d, J = 12.1 Hz, 1H), 3.34–2.97 (m, 4H), 1.68 (d, J = 6.6 Hz, 3H), 1.36 (d, J = 7.1 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 163.0 (d, 1J = 245 Hz), 157.7 (d, 2J = 11 Hz), 155.5 (d, 4J = 3 Hz), 144.5 (d, 3J = 7 Hz), 143.1 (d, 2J = 21 Hz), 140.5 (d, 1J = 249 Hz), 130.2 (d, 3J = 8 Hz), 121.2 (d, 4J = 3 Hz), 114.8 (d, 2J = 21 Hz), 112.7 (d, 2J = 22 Hz), 74.6, 47.0, 44.4, 43.2, 35.7, 22.3, 13.5; HRMS-ESI (m/z): [M + H]+ calcd for C17H21F2N4O: 335.1678; found: 335.1682; HPLC purity, 7.3 min, 97.4%; Optical rotation for (S,R)-4d: [ α ] D 27 −228.6° (c 0.4, MeOH).
5-Fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)-2-((R)-2-methylpiperazin-1-yl)pyrimidine ((R,R)-4e): Methods B: Yield: 82%; 1H-NMR (400 MHz, CDCl3) δ 7.94 (d, J = 2.8 Hz, 1H), 7.40–7.34 (m, 2H), 7.04–6.99 (m, 2H), 6.08 (q, J = 6.6 Hz, 1H), 4.60–4.55 (m, 1H), 4.18 (d, J = 3.5 Hz, 1H), 3.06–2.86 (m, 4H), 2.72–2.65 (m, 1H), 2.54 (bs, 1H), 1.65 (d, J = 6.6 Hz, 1H), 1.04 (d, J = 6.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ 162.2 (d, 1J = 244 Hz), 157.2 (d, 2J = 11 Hz), 156.9 (d, 4J = 2 Hz), 143.3 (d, 2J = 19 Hz), 139.9 (d, 1J = 246 Hz), 138.2 (d, 4J = 3 Hz), 127.5 (d, 3J = 8 Hz), 115.4 (d, 2J = 21 Hz), 73.8, 50.3, 16.6, 45.8, 39.5, 23.0, 13.6; HRMS-ESI (m/z): [M + H]+ calcd for C17H21F2N4O: 335.1678; found: 335.1680; HPLC purity, 7.5 min, 95.7%; Optical rotation for (R,R)-4e: [ α ] D 28 +132.0° (c 0.3, MeOH).
5-Fluoro-4-((S)-1-(4-fluorophenyl)ethoxy)-2-((R)-2-methylpiperazin-1-yl)pyrimidine ((S,R)-4e): Methods B: Yield: 81%; 1H-NMR (400 MHz, CDCl3) δ 7.95 (d, J = 2.9 Hz, 1H), 7.40–7.37 (m, 2H), 7.05–7.00 (m, 2H), 6.11 (q, J = 6.6 Hz, 1H), 4.61–4.57 (m, 1H), 4.27–4.23 (m, 1H), 3.09–2.86 (m, 4H), 2.78–2.71 (m, 1H), 2.59 (bs, 1H), 1.66 (d, J = 6.6 Hz, 1H), 1.23 (d, J = 6.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ 162.3 (d, 1J = 244 Hz), 157.2 (d, 2J = 11 Hz), 156.9 (d, 4J = 2 Hz), 143.4 (d, 2J = 20 Hz), 140.0 (d, 1J = 246 Hz), 137.9 (d, 4J = 3 Hz), 127.7 (d, 3J = 8 Hz), 115.4 (d, 2J = 21 Hz), 73.7, 50.2, 46.5, 45.8, 39.5, 22.7, 13.6; HRMS-ESI (m/z): [M + H]+ calcd for C17H21F2N4O: 335.1678; found: 335.1678; HPLC purity, 7.4 min, 97.7%; Optical rotation for (S,R)-4e: [ α ] D 28 –226.5° (c 0.5, MeOH).

3.4. Serotonin Receptor Binding Affinity Assays

Eleven dilutions (5 × assay concentration) of the test and reference compounds (Table S1) were prepared in standard binding buffer (50 mM tris(hydroxymethyl)-aminomethane-HCl (Tris-HCl), 10 mM MgCl2, 1 mM ethylenediaminetetraacetate (EDTA), pH 7.4) by serial dilution: 0.05 nM, 0.5 nM, 1.5 nM, 5 nM, 15 nM, 50 nM, 150 nM, 500 nM, 1.5 μM, 5 μM, and 50 μM. The radioligand (Table S3) was diluted to five times the assay concentration in standard binding buffer. Aliquots (50 mL) of the radioligand were dispensed into the wells of a 96-well plate containing 100 mL of standard binding buffer. Triplicate aliquots (50 mL) of the test and reference compound dilutions were then added. Finally, crude membrane fractions (50 mL) of cells (HEK293 or CHO) expressing human recombinant receptors were dispensed into each well. A total of 250 mL of the reaction mixtures was incubated at room temperature and shielded from light for 1.5 h, and was then harvested by rapid filtration onto Whatman GF/B glass fiber filters presoaked with 0.3% polyethyleneimine, by using a 96-well Brandel harvester (Gaithersburg, MD, USA).
Four rapid washes were performed with chilled standard binding buffer (500 mL) to decrease nonspecific binding. Filters were placed in 6 mL scintillation tubes and allowed to dry overnight. The next day, 4-mL of EcoScint scintillation cocktail (National Diagnostics) was added to each tube. The tubes were capped, labeled, and counted by liquid scintillation counting. The filter mats were dried, and the scintillant was melted onto the filters, then the radioactivity retained on the filters was counted in a Microbeta scintillation counter. The IC50 values were obtained by using the Prism 4.0 program (GraphPad Software, La Jolla, CA, USA) and were converted into Ki values. Each compound was tested at least in triplicate.

4. Conclusions

In summary, we have synthesized a series of pyrimidine derivatives 4ai and evaluated their binding affinities towards 5-HT2C receptors. Our initial biological study indicated that 2-amino-4-alkoxypyrimidines 4b, 4d, and 4e, possessing a short carbon chain between the phenyl group and pyrimidine, have excellent 5-HT2C binding affinities, which are comparable to that of the reported pyrimidine analogue 3. In order to improve the selectivity for other 5-HT2 receptor subtypes, the most potent compounds 4d and 4e were selected and their diastereomeric isomers were synthesized as optically pure forms. For this purpose, optically active secondary alcohols 5d and 5e were also prepared by an enzymatic kinetic resolution. (R,R)-4d and 4e displayed excellent 5-HT2C binding affinities with less selectivity towards 5-HT2A and 5-HT2B, whereas (S,R)-4d and 4e exhibited low potencies for 5-HT2A and 5-HT2B with a slight loss of the 5-HT2C binding affinity. These results suggest that the pyrimidine analogue (R,R)-4e is a potential lead compound for identifying a 5-HT2C selective modulator.

Supplementary Materials

The supplementary materials are available online.

Acknowledgments

This work was financially supported by the Korea Health Industry Development Institute (KHIDI, HI16C1677, HI17C1037) and the National Research Foundation (NRF-2016R1A2B1012277). Binding affinity data were generously provided by the US National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (HHSN-271-2008-00025-C).

Author Contributions

S.-J.M. and Y.S.C. conceived and designed the experiments; J.K., H.J., and H.L. performed the experiments; S.-J.M. and J.K. analyzed the data; H.C. contributed to the evaluation of bioactivity; A.N.P., H.J.K., and Y.S.C. contributed reagents/materials/analysis tools; S.-J.M. and J.K. wrote the paper.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compounds 4a4i are available from the authors.
Figure 1. The chemical structures of representative 5-HT2C receptor ligands and their binding affinities for 5-HT2 receptor subtypes [19,21].
Figure 1. The chemical structures of representative 5-HT2C receptor ligands and their binding affinities for 5-HT2 receptor subtypes [19,21].
Molecules 22 01416 g001
Figure 2. The structures of reported 5-HT2C agonist 3 and designed compounds 4.
Figure 2. The structures of reported 5-HT2C agonist 3 and designed compounds 4.
Molecules 22 01416 g002
Scheme 1. Synthesis of the pyrimidine derivatives 4ai. Reagents and conditions: (a) 2,4-dichloro-5-fluoropyrimidine, NaOtBu, toluene, 0 °C, 70–91%; (b) (R)-(+)-1-Boc-3-methylpiperazine, DIPEA, CH3CN, 110 °C, 14–53%; (c) TFA, CH2Cl2, r.t., 52–72%; (d) 4 M HCl, dioxane, 0 °C, 53%; (e) BH3-Me2S, THF, r.t., 84–94%; (f) PCC, CH2Cl2, r.t., 73–77%; (g) MeMgBr, THF, 0 °C, 72–85%.
Scheme 1. Synthesis of the pyrimidine derivatives 4ai. Reagents and conditions: (a) 2,4-dichloro-5-fluoropyrimidine, NaOtBu, toluene, 0 °C, 70–91%; (b) (R)-(+)-1-Boc-3-methylpiperazine, DIPEA, CH3CN, 110 °C, 14–53%; (c) TFA, CH2Cl2, r.t., 52–72%; (d) 4 M HCl, dioxane, 0 °C, 53%; (e) BH3-Me2S, THF, r.t., 84–94%; (f) PCC, CH2Cl2, r.t., 73–77%; (g) MeMgBr, THF, 0 °C, 72–85%.
Molecules 22 01416 sch001
Scheme 2. The synthesis of (R)/(S)-5d and 5e using enzymatic kinetic resolution. Reagents and conditions: (a) vinyl acetate (0.5 eq), CAL-B, pyridine, hexane, r.t.; (b) 1 M NaOH, MeOH, r.t., 17–32% (two steps); (c) vinyl acetate (1 eq), CAL-B, pyridine, hexane, r.t., then separation, 44–46%; (d) (R)-(−)-O-acetylmadelic acid, DCC, DMAP, CH2Cl2, r.t., 68–86%.
Scheme 2. The synthesis of (R)/(S)-5d and 5e using enzymatic kinetic resolution. Reagents and conditions: (a) vinyl acetate (0.5 eq), CAL-B, pyridine, hexane, r.t.; (b) 1 M NaOH, MeOH, r.t., 17–32% (two steps); (c) vinyl acetate (1 eq), CAL-B, pyridine, hexane, r.t., then separation, 44–46%; (d) (R)-(−)-O-acetylmadelic acid, DCC, DMAP, CH2Cl2, r.t., 68–86%.
Molecules 22 01416 sch002
Scheme 3. Synthesis of the optically active pyrimidine derivatives 4d4e. Reagents and conditions: (a) 2,4-dichloro-5-fluoropyrimidine, NaOtBu, toluene, 0 °C, 55–72%; (b) (R)-(+)-1-Boc-3-methylpiperazine, toluene, 150 °C, 44–57%; (c) TFA, CH2Cl2, r.t., 67–71%; (d) HCl, dioxane, 0 °C, 81–82%.
Scheme 3. Synthesis of the optically active pyrimidine derivatives 4d4e. Reagents and conditions: (a) 2,4-dichloro-5-fluoropyrimidine, NaOtBu, toluene, 0 °C, 55–72%; (b) (R)-(+)-1-Boc-3-methylpiperazine, toluene, 150 °C, 44–57%; (c) TFA, CH2Cl2, r.t., 67–71%; (d) HCl, dioxane, 0 °C, 81–82%.
Molecules 22 01416 sch003
Table 1. Binding affinities of pyrimidine analogues 4ai against 5-HT2 receptor subtypes.
Table 1. Binding affinities of pyrimidine analogues 4ai against 5-HT2 receptor subtypes.
EntryComp.nFR5-HT2A5-HT2B5-HT2C
%BindingKi (nM)%BindingKi (nM)%BindingKi (nM)
14a02–FH93.180.096.956.097.831.0
24b03–FH97.443.098.514.099.05.1
34c04–FH94.651.097.614.098.822.0
44d03–FMe91.9213.098.94.899.32.7
54e04–FMe87.6426.098.410.099.14.5
64f23–FH96.3138.095.624.097.820.0
74g24–FH96.590.097.722.097.922.0
84h23–FMe94.3170.098.716.098.614.0
94i24–FMe96.9173.097.622.095.012.0
10313–FMe95.6128.097.47.998.20.7
Table 2. Binding affinities of optically active pyrimidines 4d and 4e against 5-HT2 receptor subtypes
Table 2. Binding affinities of optically active pyrimidines 4d and 4e against 5-HT2 receptor subtypes
EntryComp.nFR5-HT2A5-HT2B5-HT2C
%BindingKi (nM)%BindingKi (nM)%BindingKi (nM)
1(R,R)-4d03–FMe (R)93.3222.0100.22.698.51.2
2(S,R)-4d03–FMe (S)82.4475.095.267.097.714.0
3(R,R)-4e04–FMe (R)0.9-99.619.094.24.0
4(S,R)-4e04–FMe (S)64.81024.095.3128.097.823.0
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