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Article Synthesis and Biological Evaluation of Disubstituted Pyrimidines as Selective 5-HT2C Agonists

1,2, 3, 4,5 4,5 1,4 Juhyeon Kim † , Yoon Jung Kim †, Ashwini M. Londhe , Ae Nim Pae , Hyunah Choo , Hak Joong Kim 2 and Sun-Joon Min 3,6,*

1 Center for Neuro-Medicine, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea 2 Department of Chemistry, Korea University, Seoul 02841, Korea 3 Department of Applied Chemistry, Hanyang University, Ansan, Gyeonggi-do 15588, Korea 4 Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Korea 5 Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seoul 02792, Korea 6 Department of Chemical & Molecular Engineering, Hanyang University, Ansan, Gyeonggi-do 15588, Korea * Correspondence: [email protected]; Tel.: +82-31-400-5502 These authors contributed equally to this work. †


Received: 1 August 2019; Accepted: 3 September 2019; Published: 5 September 2019


Abstract: Here, we describe the synthesis of disubstituted pyrimidine derivatives and their biological evaluation as selective 5-HT2C agonists. To improve selectivity for 5-HT2C over other subtypes, we synthesized two series of disubstituted pyrimidines with fluorophenylalkoxy groups at either the 5-position or 4-position and varying cyclic amines at the 2-position. The in vitro cell-based assay and binding assay identified compounds 10a and 10f as potent 5-HT2C agonists. Further studies on selectivity to 5-HT subtypes and drug-like properties indicated that 2,4-disubstituted pyrimidine 10a showed a highly agonistic effect on the 5-HT2C receptor, with excellent selectivity, as well as exceptional drug-like properties, including high plasma and microsomal stability, along with low CYP inhibition. Thus, pyrimidine 10a could be considered a viable lead compound as a 5-HT2C selective agonist.

Keywords: disubstituted pyrimidine; 5-HT2C receptor; cell-based assay; binding affinity; selectivity

1. Introduction Serotonin (5-hydroxytryptamine, 5-HT), a monoamine neurotransmitter, plays a critical role in the regulation of various neurological functions, including mood, sleep, cognition, anxiety, sexual behavior, and appetite [1,2]. There are 14 variants of serotonin receptors (5-HT receptors), which mostly belong to G-protein-coupled receptors (GPCRs), except for the 5-HT3 receptor, a ligand-gated ion channel. Activation of the 5-HT receptors induces both inhibitory and excitatory signal transduction by modulating the release of many neurotransmitters, such as glutamate, dopamine, epinephrine, and acetylcholine. Therefore, they have been considered crucial therapeutic targets for a variety of neurological diseases, including depression, psychiatric disorders, sexual dysfunction, obesity, and urinary incontinence [3–5]. The 5-HT2 receptors comprise three subtypes: 5-HT2A, 5-HT2B, and 5-HT2C. Recent studies revealed that activation of 5-HT2C receptors in the central nerve system (CNS) is a potential drug target for effective treatment of schizophrenia, as well as obesity [6–10]. One of the crucial problems faced during the development of 5-HT2C agonists is selectivity over the structurally related 5-HT2A and 5-HT2B receptors. 5-HT2A agonism in humans is implicated in acute adverse effects, such as

Molecules 2019, 24, 3234; doi:10.3390/molecules24183234 www.mdpi.com/journal/molecules Molecules 2019, 24, 3234 2 of 21

Molecules 2019, 24, x FOR PEER REVIEW 2 of 21 hallucination and cardiovascular effects. Stimulation of the 5-HT2B receptor is related to other side hallucination and cardiovascular effects. Stimulation of the 5-HT2B receptor is related to other side effects, including chronic cardiac valvulopathy and pulmonary hypertension. effects, including chronic cardiac valvulopathy and pulmonary hypertension. Potent and selective 5-HT2C agonists have been developed, as shown in Figure1. Lorcaserin Potent and selective 5-HT2C agonists have been developed, as shown in Figure 1. Lorcaserin 1 1 was approved by the FDA (Food and Drug Administration) in 2012 for treating obesity [11], and was approved by the FDA (Food and Drug Administration) in 2012 for treating obesity [11], and vabicaserin 2 was clinically evaluated as a potential antipsychotic [12]. Numerous 5-HT2C agonists that vabicaserin 2 was clinically evaluated as a potential antipsychotic [12]. Numerous 5-HT2C agonists have undergone preclinical or early clinical studies have been reported [13–19]. In addition, several that have undergone preclinical or early clinical studies have been reported [13–19]. In addition, 5-HT2C-selective positron emission tomography (PET) radioligands (5 and 6) have been developed as several 5-HT2C-selective positron emission tomography (PET) radioligands (5 and 6) have been chemical tools for in vivo imaging of the 5-HT2C receptor [20,21]. developed as chemical tools for in vivo imaging of the 5-HT2C receptor [20,21].

Figure 1. The representative 5-HT2C selective agonists and PET radioligands. Figure 1. The representative 5-HT2C selective agonists and PET radioligands.

Despite the discovery of potential 5-HT2C agonists, the selectivity of these compounds over other Despite the discovery of potential 5-HT2C agonists, the selectivity of these compounds over other 5-HT2 subtypes is still moderate; therefore, the search for highly selective 5-HT2C agonists remains 5-HT2 subtypes is still moderate; therefore, the search for highly selective 5-HT2C agonists remains challenging. In this regard, our group has previously reported optically active pyrimidine derivative challenging. In this regard, our group has previously reported optically active pyrimidine derivative 8 as a potent and selective 5-HT2C agonist (Figure2)[ 22]. The structure activity relationship (SAR) 8 as a potent and selective 5-HT2C agonist (Figure 2) [22]. The structure activity relationship (SAR) between related pyrimidine derivatives and 5-HT2 subtypes revealed that a subtle change in the between related pyrimidine derivatives and 5-HT2 subtypes revealed that a subtle change in the fluorophenylalkoxy moiety attached to pyrimidine 8 was essential to control the activation of selectivity fluorophenylalkoxy moiety attached to pyrimidine 8 was essential to control the activation of for 5-HT2C over 5-HT2A and 5-HT2B. Although compound 8 was discovered as a lead compound with selectivity for 5-HT2C over 5-HT2A and 5-HT2B. Although compound 8 was discovered as a lead excellent potency and good selectivity for the 5-HT2 subtypes, further structural modification of these compound with excellent potency and good selectivity for the 5-HT2 subtypes, further structural pyrimidine derivatives is required to improve selectivity over other 5-HT subtypes. Furthermore, modification of these pyrimidine derivatives is required to improve selectivity over other 5-HT selective 5-HT2C agonists could be applied in the development of chemical probes such as PET subtypes. Furthermore, selective 5-HT2C agonists could be applied in the development of chemical radiotracersMolecules 2019 [23, 24–, 26x FOR]. PEER REVIEW 3 of 21 probes such as PET radiotracers [23–26]. We first planned to alter the substitution pattern of pyrimidines for the structural adjustment of compound 8 toward the 5-HT2C binding pocket. Recent studies demonstrated that pyridazine-fused azepine 4 containing a remote phenyl group (Figure 1) showed high potency and selectivity to 5-HT2C over 5-HT2B with a good stability profile [19]. Accordingly, we hypothesized that the linear form of 2,5-disubstituted pyrimidine 9, possessing a flexible fluorophenylalkoxy group at the 5-position, might be suitable for exploring the relationship between structural change and selectivity (Figure 2). Based on the molecular docking study of several 5-HT2C agonists published in literature [27,28], we also assumed that the terminal-free amine of the piperazine moiety could be a key functional group binding to a conserved residue, D134, in 5-HT2C protein. Taken together with the fact that compound 8 derived from (R)-isomer of secondary benzyl alcohol showed better binding affinity and selectivity to 5-HT2C than its diastereomer [22], we designed an alternative series of pyrimidine derivatives 10 possessing a variety of cyclic amines to examine their agonistic effect on 5-HT receptor activation. Herein, we report the synthesis of disubstituted pyrimidine derivatives with different substitution Figure 2. Design of new pyrimidine derivatives as 5-HT2C agonists. patterns and their biologicalFigure 2. evaluation Design of new as pyrimidiselectivene 5-HT derivatives2C receptor as 5-HT agonists.2C agonists.

2. Results and Discussion

2.1. Synthesis of 2,5-Disubstituted Pyrimidine Derivatives Initially, we synthesized 2,5-disubstituted pyrimidines with flexible phenylalkoxy groups at the

5-position, as shown in Scheme 1. Starting from 2,5-dibromopyrimidine 13, three kinds of 2- aminopyrimidines 11a–c were prepared through SNAr type amination. Pyrimidine derivatives 11a, 11b, and 11c were reacted with 3- or 4-fluoro-phenols, benzyl alcohols, and phenethyl alcohols in the presence of copper catalyst and 1,10-phenanthroline ligands 14 or 15 [29–31] to afford 2,5- disubstituted pyrimidines possessing an N-Boc-protecting group at the terminal amine. It is noted that the use of 15 as a ligand in this transformation led to formation of the desired products in higher yields. Finally, the 2-amino-5-alkoxypyrimidines 9a–r were obtained after removal of the Boc group under acidic conditions.

Scheme 1. Synthesis of 2,5-disubstituted pyrimidine derivatives 9a–r. Reagents and conditions: (a) N-

Boc-protected amines, K2CO3, CH3CN, 80 °C, 88–97%; (b) 3- or 4-fluorophenol/benzyl alcohol/phenethyl alcohol, CuI or CuO2, 14 or 15, Cs2CO3, toluene, 110 °C, 42–85%; (c) TFA, CH2Cl2, 0 °C, 52–67%.

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We first planned to alter the substitution pattern of pyrimidines for the structural adjustment of compound 8 toward the 5-HT2C binding pocket. Recent studies demonstrated that pyridazine-fused azepine 4 containing a remote phenyl group (Figure1) showed high potency and selectivity to 5-HT 2C over 5-HT2B with a good stability profile [19]. Accordingly, we hypothesized that the linear form of 2,5-disubstituted pyrimidine 9, possessing a flexible fluorophenylalkoxy group at the 5-position, might be suitable for exploring the relationship between structural change and selectivity (Figure2). Based on the molecular docking study of several 5-HT2C agonists published in literature [27,28], we also assumed that the terminal-free amine of the piperazine moiety could be a key functional group binding to a conserved residue, D134, in 5-HT2C protein. Taken together with the fact that compound 8 derived from (R)-isomer of secondary benzyl alcohol showed better binding affinity and selectivity to 5-HT2C than its diastereomer [22], we designed an alternative series of pyrimidine derivatives 10 possessing a variety of cyclic amines to examine their agonistic effect on 5-HT receptor activation. Herein, we report the synthesis of disubstituted pyrimidine derivatives with different substitution patterns and their biological evaluation as selective 5-HT2C receptor agonists. Figure 2. Design of new pyrimidine derivatives as 5-HT2C agonists. 2. Results and Discussion 2. Results and Discussion 2.1. Synthesis of 2,5-Disubstituted Pyrimidine Derivatives 2.1. Synthesis of 2,5-Disubstituted Pyrimidine Derivatives Initially, we synthesized 2,5-disubstituted pyrimidines with flexible phenylalkoxy groups at the 5-position,Initially, we as synthesized shown in 2,5-disubstituted Scheme1. Starting pyrimidines from 2,5-dibromopyrimidine with flexible phenylalkoxy13, three groups kinds at the of 5-position,2-aminopyrimidines as shown11a–c in Schemewere prepared 1. Starting through from S N2,5-dibromopyrimidineAr type amination. Pyrimidine 13, three derivatives kinds of11a, 2- aminopyrimidines11b, and 11c were reacted11a–c were with prepared 3- or 4-fluoro-phenols, through SNAr benzyl type amination. alcohols, and Pyrimidine phenethyl derivatives alcohols in 11a, the 11b,presence and 11c of copper were reacted catalyst with and 3- 1,10-phenanthroline or 4-fluoro-phenols, ligands benzyl14 alcohols,or 15 [29 –and31] phenethyl to afford 2,5-disubstituted alcohols in the presencepyrimidines of possessingcopper catalyst an N -Boc-protectingand 1,10-phenanthroline group at the ligands terminal 14 amine.or 15 [29–31] It is noted to thatafford the 2,5- use disubstitutedof 15 as a ligand pyrimi indines this transformation possessing an ledN-Boc-protecting to formation ofgroup the desiredat the terminal products amine. in higher It is yields.noted thatFinally, the use the 2-amino-5-alkoxypyrimidinesof 15 as a ligand in this transformation9a–r were led obtained to formation after of removal the desired of the products Boc group in higher under yields.acidic conditions.Finally, the 2-amino-5-alkoxypyrimidines 9a–r were obtained after removal of the Boc group under acidic conditions.

SchemeScheme 1. 1. SynthesisSynthesis of of2,5-disubstituted 2,5-disubstituted pyrimidine pyrimidine derivatives derivatives 9a–r.9a–r Reagents. Reagents and conditions: and conditions: (a) N-

Boc-protected(a) N-Boc-protected amines, amines, K2CO K3, 2COCH3,3CN, CH 3CN,80 °C, 80 ◦C,88–97%; 88–97%; (b) (b)3- 3-or or 4-fluorophenol/benzyl 4-fluorophenol/benzyl alcohol/phenethylalcohol/phenethyl alcohol, alcohol, CuI CuI or or CuO CuO22, ,1414 oror 1515, ,Cs Cs2CO2CO3,3 toluene,, toluene, 110 110 °C,◦C, 42–85%; 42–85%; (c) ( cTFA,) TFA, CH CH2Cl2Cl2, 02 , °C,0 ◦ C,52–67%. 52–67%.

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Molecules 2019, 24, x FOR PEER REVIEW 4 of 21 2.2. Synthesis of 2,4-Disubstituted Pyrimidine Derivatives 2.2. Synthesis of 2,4-Disubstituted Pyrimidine Derivatives The synthesis of a series of 2,4-disubstituted pyrimidines 10, possessing different cyclic amines, is describedThe synthesis in Scheme of2 .a Accordingseries of 2,4-disubstituted to our protocol [pyrimidines22], we efficiently 10, possessing synthesized different optically cyclic active amines, 1-(3- oris described 4-fluorophenyl)ethan-1-ol in Scheme 2. According (R)-16a andto our (R )-protocol16b using [22], an we enzymatic efficiently kinetic synthesized resolution optically method. active As 1-(3- or 4-fluorophenyl)ethan-1-ol (R)-16a and (R)-16b using an enzymatic kinetic resolution method. per the procedure previously reported in literature [32], nucleophilic aromatic substitution (SNAr) of 2,4-dichloropyrimidineAs per the procedure previously with alcohols reported (R)-16a inand literature (R)-16b [32],selectively nucleophilic produced aromatic 4-alkoxypyrimidines substitution (SN17aAr) of 2,4-dichloropyrimidine with alcohols (R)-16a and (R)-16b selectively produced 4- and 17b, which were subjected to an additional SNAr reaction to afford 2-alkoxy-4-aminopyrimidines asalkoxypyrimidines free or N-Boc-protected 17a and amine17b, which forms. were The subjected latter were to furtheran additional treated SN withAr reaction a strong to acid, afford such 2- asalkoxy-4-aminopyrimidines HCl or TFA, to give the desiredas free or disubstituted N-Boc-protected pyrimidines. amine forms. Besides The latter pyrimidine were further analogues treated10, wewith synthesized a strong acid, compounds such as 20aHCland or 20bTFA,possessing to give the a directdesired connection disubstituted between pyrimidines. the fluorophenyl Besides grouppyrimidine and pyrimidineanalogues 10 via, we ether synthesized linkage using compounds a similar 20a experimental and 20b possessing process. a Indirect this case,connection these compoundsbetween the20a fluorophenyl/b might allow group us to and examine pyrimidine the optimal via sizeether of linkage the fluorophenylalkoxy using a similar groupexperimental through aprocess. comparison In this of theircase, inthese vitro compounds activity with 20a/b that ofmight8. allow us to examine the optimal size of the fluorophenylalkoxy group through a comparison of their in vitro activity with that of 8.

Scheme 2. Synthesis of 2,4-disubstituted pyrimidine derivatives 10a–j and 20ab. Reagents and Scheme 2. Synthesis of 2,4-disubstituted pyrimidine derivatives 10a–j and 20ab. Reagents and conditions: (a) (i) Vinyl acetate (0.4 eq), CAL-B, pyridine, hexane, rt, 66–67%; (ii) 1 M NaOH, MeOH, rt, conditions: (a) (i) Vinyl acetate (0.4 eq), CAL-B, pyridine, hexane, rt, 66–67%; (ii) 1 M NaOH, MeOH, 78%; (b) 2,4-dichloro-5-fluoropyrimidine, t-BuONa, toluene, 0 ◦C, 51–93%; (c) amines, Et3N, toluene rt, 78%; (b) 2,4-dichloro-5-fluoropyrimidine, t-BuONa, toluene, 0 °C, 51–93%; (c) amines, Et3N, toluene (0.5 M), 100 ◦C, 12 h, 61–98%; (d) (i) N-Boc-protected amines, Et3N, DMSO (0.5 M), 100 ◦C, 12 h, 44–94%; (0.5 M), 100 °C, 12 h, 61–98%; (d) (i) N-Boc-protected amines, Et3N, DMSO (0.5 M), 100 °C, 12 h, 44– (ii) HCl (4.0 M in dioxane), CH3CN, 0 ◦C or TFA, CH2Cl2, 0 ◦C. 94%; (ii) HCl (4.0 M in dioxane), CH3CN, 0 °C or TFA, CH2Cl2, 0 °C. 2.3. In Vitro Evaluation of Pyrimidine Derivatives 2.3. In Vitro Evaluation of Pyrimidine Derivatives In vitro agonistic activities of pyrimidines against 5-HT2C were evaluated using a fluorescence-basedIn vitro agonistic receptor activities functional of pyrimidines assay, and against the results 5-HT2C are were summarized evaluated in using Table a1 .fluorescence- Among the firstbased series receptor of pyrimidines functional possessingassay, and dithefferent results sizes are of summarized phenylalkoxy in groupsTable 1. at Among the 5-position, the first we series found of thatpyrimidines the cellular possessing agonistic different effect of sizes only of compound phenylalko9bxywas groups higher at thanthe 5-position, 50% at a concentration we found that of the 10 µcellularM. Regarding agonistic the effect effect of of only cyclic compound amines at the9b 2-positionwas higher of than pyrimidine, 50% at onlya concentration compounds of9g–l 10 withμM. methylRegarding piperazine the effect showed of cyclic low amines to moderate at th activities,e 2-position with of uppyrimi to 48%dine, activation. only compounds Based on this9g–l result, with wemethyl concluded piperazine that showed the substitution low to moderate pattern activities, of cyclic amineswith up and to 48% the activation. fluorophenylalkoxy Based on this group result, on we concluded that the substitution pattern of cyclic amines and the fluorophenylalkoxy group on the the pyrimidine ring is important for 5-HT2C agonism. Thus, we decided not to proceed with further pyrimidine ring is important for 5-HT2C agonism. Thus, we decided not to proceed with further biological evaluation of this first series of compounds because an activation value higher than 50% is considered to represent significant agonistic effects of the compounds.

Molecules 2019, 24, 3234 5 of 21 Molecules 2019, 24, x FOR PEER REVIEW 5 of 21 Molecules 2019, 24, x FOR PEER REVIEW 5 of 21 Moleculesbiological 2019Table, 24, xevaluation FOR 1. In PEER vitro REVIEW agonistic of this first activity series of of2,5-disubstituted compounds because pyrimidine an activation9a–r against value 5-HT2C higher. 5 than of 21 50% is Molecules 2019, 24, x FOR PEER REVIEW 5 of 21 consideredTable to1. In represent vitro agonistic significant activity agonistic of 2,5-disubstituted effects of the pyrimidine compounds. 9a–r against 5-HT2C.

Table 1. In vitro agonistic activity of 2,5-disubstituted pyrimidine 9a–r against 5-HT2C. Table 1. In vitro agonistic activity of 2,5-disubstituted pyrimidine 9a–r against 5-HT2C. Table 1. In vitro agonistic activity of 2,5-disubstituted pyrimidine 9a–r against 5-HT2C.

1 2 %activation Entry Compds n R R %activation(10 μM) Entry Compds n R1 R2 1 9a 0 3-F %activation%activation(10 7μ M) Entry EntryCompds Compdsn n R1 R1 R2 %activation 12 Entry 9b9a Compds 01 n 3-F R1 R2 (10(10µM) 617μ M) (10 μM) 231 9b9a9c 120 3-F 61157 1 1 9a 9a 0 0 3-F3-F 7 7 342 9d9b9c 201 3-F4-F 1561 5 2 2 9b 9b 1 1 3-F3-F 61 61 453 3 9d9e9c 9c 012 24-F3-F 3-F 15 1815 5 3 9c 2 3-F 15 564 4 9d9e9f 9d 120 04-F 4-F 5 18 45 4 9d 0 4-F 5 65 5 9e9f 9e 21 14-F 4-F 18 18 4 5 9e 1 4-F 18 76 6 9g9f 9f 02 23-F4-F 4-F 4 35 4 6 9f 2 4-F 4 78 9h9g 01 3-F 35 48 7 9g 0 3-F 35 897 8 9h9g9i 9h 120 13-F 3-F 48 484035 7 9g 0 3-F 35 1098 9 9h9i9j 9i 201 23-F4-F 3-F 40 402648 8 9h 1 3-F 48 10119 10 9k9j9i 9j 012 04-F3-F 4-F 26 264840 9 9i 2 3-F 40 111210 11 9k9l9j 9k 120 14-F 4-F 48 483226 10 9j 0 4-F 26 1211 12 9k9l 9l 21 24-F 4-F 32 3248 11 9k 1 4-F 48 1312 9m9l 9m 02 3-F4-F 16 32 12 13 9l 2 0 3-F4-F 16 32 1314 14 9m9n 9n 01 13-F 3-F 116−1 − 141513 15 9m9n9o 9o 120 23-F 3-F 3 −16 31 13 9m 0 3-F 16 151614 16 9p9n9o 9p 201 03-F4-F 4-F 14 14− 31 14 9n 1 3-F −1 161715 17 9p9q9o 9q 012 14-F3-F 4-F 16 1416 3 15 18 9o 9r 2 2 4-F3-F 1 3 171816 9q9p9r 120 4-F − 16−141 16 19 Lorcaserin9p (1) 0 4-F 94a 14 181917 Lorcaserin9q9r (1) 21 4-F 94− 161a 17 9q 1 4-F 16 a µ a 1918 Lorcaserin9r (1)a %activation2 value at4-F1 M, EC50 = 14 nM. 94−1 18 9r %activation value2 at 1 μM, EC504-F = 14 nM. −1 a 19 Lorcaserin (1)a %activation value at 1 μM, EC50 = 14 nM. 94 a Next, the19 in vitro biologicalLorcaserin activities (1) of 2,4-disubstituted pyrimidines 10a–j and 20a–b against 94 a %activation value at 1 μM, EC50 = 14 nM. a %activation value at 1 μM, EC50 = 14 nM. the 5-HTNext,2C Next, thereceptor in the vitro inwere vitrobiological evaluated,biological activities as activities shown of 2,4-disubstituted ofin 2,4-disubstitutedTable 2. At this pyrimidines point, pyrimidines the ligand10a–j10a–j and bindingand 20a–b20a–b assay, againstagainst as the thewell 5-HT Next,as5-HT the2CNext, 2C thereceptorcell-basedreceptor in the vitro inwere functional were vitrobiological evaluated, evaluated, biological assay, activities as as activities wereshown shown of performed 2,4-disubstituted in inof Table Table2,4-disubstituted 2 2.to. At Atidentify thisthis pyrimidines point,point, more pyrimidines the the potent ligand ligand10a–j and 10a–j bindingand binding selective 20a–b and assay, assay,20a–b against5-HT as asagainst well2C as wellagonists.the 5-HT asthe the 2C cell-basedAmong cell-basedreceptor the functionalwere functionaltested evaluated, compounds, assay, assay, wereas wereshown performed four performed in pyrimidine Table to identify2.to Atidentify derivativesthis more point, more potent the potentshowed ligand and and selective greater binding selective 5-HTthan assay, 5-HT 50%agonists. as2C the 5-HT2C receptor were evaluated, as shown in Table 2. At this point, the ligand binding2C assay, as agonists.activationwell asAmong the Among against cell-based the tested the5-HT functionaltested compounds,2C in thecompounds, assay,cell-based four were pyrimidine fourassay. performed pyrimidine Studies derivatives to onidentify derivativesthe showed binding more greater potentshowed affinity thanand ofgreater 50% selectivethis activationseries than 5-HT to50% 5-2C against well as the cell-based functional assay, were performed to identify more potent and selective 5-HT2C activationHTagonists.2C agonists.5-HTindicated Amongagainst2C in Among thethat 5-HTthe cell-based compounds tested 2Cthe in tested thecompounds, assay. cell-based 10a compounds, Studiesand 10ffourassay. on, with thepyrimidinefour Studies binding1,4-diazepane pyrimidine on a derivativesffithenity bindingderivatives at of the this showed affinity2-position series showedto ofgreater 5-HT thisof greaterpyrimidine, 2Cseries thanindicated tothan50% 5- 50% that HTexhibitedactivation2C compoundsindicated excellentagainst that 10a5-HT K compoundsi andvalues2C in10f the ,of with cell-based 7.910a nM 1,4-diazepane and and 10f assay. 19.0, with nM,Studies at 1,4-diazepane the respecti 2-positionon thevely. binding atAlthough of the pyrimidine, affinity2-position the ofresult exhibited thisof pyrimidine, seriesof the excellent tocell- 5- Ki activation against 5-HT2C in the cell-based assay. Studies on the binding affinity of this series to 5- exhibitedbasedHT2C values indicatedassay excellent is of important7.9 that nM K compoundsi andvalues to 19.0 explore of nM, 7.9 10a the respectively.nM and cellular and 10f 19.0, functionwith Although nM, 1,4-diazepane respecti of the the 5-HT resultvely. 2C atAlthough of receptor, the the 2-position cell-based bindingthe result assayof affinity pyrimidine, of isthe important plays cell- to HT2C indicated that compounds 10a and 10f, with 1,4-diazepane at the 2-position of pyrimidine, basedaexhibited key exhibitedexplorerole assay inexcellent isdetermining the important excellent cellular Ki values to K functionwhetheri explorevalues of 7.9 theof theof nM the compound7.9 cellular and 5-HT nM 19.02Cand functionreceptor, is nM,19.0 suitable respecti nM,of bindingthe forrespecti 5-HTvely. the a2Cdevelopment ffively.Although receptor,nity Although plays bindingthe aof keyresult athe 5-HT role affinity result of2C in -specificthe determining ofplays cell- the cell- achemicalbased key whetherrole assay probe.in isdetermining important the Thus, compound we to whether exploreselected is suitable the the compounds compound cellular for the function development 10ais suitable and of the10f for 5-HT offor the a further 5-HTdevelopment2C receptor, -specificexamination binding of chemicala 5-HT affinityof 2Cthe-specific probe. plays5-HT Thus, based assay is important to explore the cellular function of the 5-HT2C2C receptor, binding affinity plays chemicalsubtypea key werole selectivity selected probe.in determining Thus, compounds and drug-likewe whether selected10a properties. theand compounds compound10f for further 10ais suitable and examination 10f for for the further development of the examination 5-HT of subtype a 5-HT of 2C selectivitythe-specific 5-HT and a key role in determining whether the compound is suitable for the development of a 5-HT2C-specific subtype chemicalchemicaldrug-like selectivity probe. probe. properties. Thus, and Thus, drug-likewe selected we selectedproperties. compounds compounds 10a and 10a 10fand for 10f further for further examination examination of the of 5-HT the 5-HT subtypesubtype selectivity selectivity and drug-like and drug-like properties. properties.

Molecules 2019,, 24,, 3234x FOR PEER REVIEW 66 of of 21 MoleculesMoleculesMolecules 2019 2019 ,2019 ,24 24,, x ,x 24FOR FOR, x FORPEER PEER PEER REVIEW REVIEW REVIEW 66 ofof 62121 of 21 Molecules 2019, 24, x FOR PEER REVIEW 6 of 21 MoleculesMoleculesMolecules 2019 2019 ,2019 ,24 24, ,x ,x 24FOR FOR, x FORPEER PEER PEER REVIEW REVIEW REVIEW 66 ofof 62121 of 21 Molecules 2019, 24, x FOR PEER REVIEW 6 of 21 MoleculesMolecules 2019 2019, 24, ,x 24 FOR, x FOR PEER PEER REVIEW REVIEW 6 of 621 of 21 Table 2. In vitro activity of 2,4-disubsitutued pyrimidine against 5-HT2C.2C. 2C. TableTable 2. In 2. vitro In vitro activity activity of 2,4-disubs of 2,4-disubsitutueditutued pyrimidine pyrimidine against against 5-HT 5-HT2C. TableTableTable 2. 2.In2. InIn vitro vitrovitro activity activityactivity of ofof 2,4-disubsitutued 2,4-disubs2,4-disubsitutueditutued pyrimidine pyrimidinepyrimidine against againstagainst 5-HT 5-HT5-HT2C. Table 2. In vitro activity of 2,4-disubsitutued pyrimidine against 5-HT2C.2C. 2C. TableTableTable 2.2. InIn 2. vitrovitro In vitro activityactivity activity ofof 2,4-disubs2,4-disubs of 2,4-disubsitutueditutueditutued pyrimidinepyrimidine pyrimidine againstagainst against 5-HT5-HT 5-HT2C. Table 2. In vitro activity of 2,4-disubsitutued pyrimidine against 5-HT2C. TableTable 2. In 2. vitro In vitro activity activity of 2,4-disubs of 2,4-disubsitutueditutued pyrimidine pyrimidine against against 5-HT 5-HT2C. 2C.

1 2 a Entry Compds 1 1 R 1 R2 2 2 %activation a a %bindinga Ki (nM) Entry Compds R 1 R 2 %activation a %binding Ki (nM) EntryEntry CompdsCompds R1 R R2 R %activation %activationa %binding%binding Ki (nM)Ki (nM) EntryEntry CompdsCompds R R1 1 RR2 2 %activation %activationa a %binding%binding KKii (nM)(nM) EntryEntry CompdsCompds R 1 R R 2 R %activation%activationa %binding%binding Ki (nM)Ki (nM) EntryEntry CompdsCompds R R1 RR2 %activation%activationa %binding%binding KKii (nM)(nM) Entry Compds R1 1 R2 2 %activationa a %binding Ki (nM) EntryEntry CompdsCompds R1 R1 R2 R2 %activation%activationa a %binding%binding Ki (nM)Ki (nM) 1 1 1 10a10a 10a10a 81.081.081.0 81.0 81.381.381.3 81.3 7.9 7.97.9 7.9 11 10a10a 81.081.0 81.381.3 7.97.9 11 1 10a10a 10a 81.081.0 81.0 81.381.3 81.3 7.97.9 7.9 1 1 10a 10a 81.081.0 81.381.3 7.9 7.9 1 1 10a 10a 81.081.0 81.381.3 7.9 7.9

2 2 2 10b10b 10b 78.278.2 78.2 47.647.6 47.6 NDND ND 22 2 10b10b 10b 78.278.278.2 47.647.647.6 ND NDND 22 2 10b10b 10b 78.278.2 78.2 47.647.6 47.6 NDND ND 2 2 10b 10b 78.278.2 47.647.6 ND ND 2 2 10b 10b 78.278.2 47.647.6 ND ND

3 33 3 10c10c10c 10c 29.529.529.5 29.5 73.673.673.6 73.6 295.0295.0295.0 295.0 3 3 10c 10c10c 29.529.5 29.5 73.673.6 73.6 295.0 295.0295.0 33 10c10c 29.529.5 73.673.6 295.0295.0 3 3 10c 10c 29.529.5 73.673.6 295.0295.0

4 44 4 10d10d10d 10d 16.416.416.4 16.4 88.688.688.6 88.6 119.0119.0119.0 119.0 4 4 10d 10d10d 16.416.4 16.4 88.688.6 88.6 119.0 119.0119.0 44 10d10d 16.416.4 88.688.6 119.0119.0 4 4 10d 10d 16.416.4 88.688.6 119.0119.0

5 55 5 10e10e10e 10e 7.77.77.7 7.7 61.461.461.4 61.4 1255.01255.01255.01255.0 5 5 10e 10e10e 7.77.7 7.7 61.461.4 61.4 1255.0 1255.01255.0 55 10e10e 7.77.7 61.461.4 1255.01255.0 5 5 10e 10e 7.7 7.7 61.461.4 1255.01255.0 5 5 10e 10e 7.7 7.7 61.461.4 1255.01255.0

6 6 6 10f10f 10f 52.452.4 52.4 93.993.9 93.9 19.019.0 19.0 66 10f10f 52.452.4 93.993.9 19.019.0 66 6 10f10f 10f10f 52.452.452.4 52.4 93.993.993.9 93.9 19.0 19.019.0 19.0 6 6 10f 10f 52.452.4 93.993.9 19.019.0 6 6 10f 10f 52.452.4 93.993.9 19.019.0

7 7 7 10g10g 10g NDND ND 78.178.1 78.1 232.0232.0 232.0 77 10g10g NDND 78.178.1 232.0232.0 77 7 10g10g 10g10g NDNDND ND 78.178.178.1 78.1 232.0 232.0232.0232.0 7 7 10g 10g ND ND 78.178.1 232.0232.0 7 7 10g 10g ND ND 78.178.1 232.0232.0

8 88 8 10h10h10h 10h 10.110.110.1 10.1 50.850.850.8 50.8 466.0466.0466.0 466.0 8 8 10h 10h 10.1 10.1 50.8 50.8 466.0466.0 88 8 10h10h 10h 10.110.110.1 50.850.850.8 466.0 466.0466.0 8 8 10h 10h 10.110.1 50.850.8 466.0466.0

9 99 9 10i10i10i 10i NDNDND ND 85.985.985.9 85.9 120.0120.0120.0 120.0 9 9 10i 10i ND ND 85.9 85.9 120.0120.0 99 9 10i10i 10i NDNDND 85.985.985.9 120.0 120.0120.0 9 9 10i 10i ND ND 85.985.9 120.0120.0

101010 10 10j10j10j 10j 0.10.10.1 0.1 26.226.226.2 26.2 NDNDND ND 10 10 10j 10j 0.1 0.1 26.2 26.2 ND ND 1010 10j10j 0.10.1 26.226.2 NDND 10 1010 10j 10j10j 0.10.1 0.1 26.226.226.2 ND ND ND 10 10 10j 10j 0.1 0.1 26.226.2 ND ND

1111 11 20a20a 20a 56.056.0 56.0 64.864.8 64.8 660.0660.0 660.0 1111 20a20a 56.056.0 64.864.8 660.0660.0 1111 11 20a20a 20a 56.056.0 56.0 64.864.8 64.8 660.0660.0660.0 11 20a 56.0 64.8 660.0 11 1111 20a 20a20a 56.056.056.0 64.864.864.8 660.0 660.0660.0

1212 12 20b20b 20b 34.034.0 34.0 54.854.8 54.8 1107.01107.01107.0 1212 20b20b 34.034.0 54.854.8 1107.01107.0 1212 12 20b20b 20b 34.034.0 34.0 54.854.8 54.8 1107.01107.01107.0 12 20b 34.0 54.8 1107.0 12 1212 20b 20b20b 34.034.0b 34.0b b 54.854.854.8 1107.0 1107.01107.0 13 8 N/A b 89.0 5.9 1313 13 88 8 N/AN/AN/Ab 89.089.0 89.0 5.95.9 5.9 13 8 N/Ab b 89.0 5.9 13 13 a a 8 a 8 N/AN/Abb b b b 89.0 89.0 5.9 5.9 1313 Serotonina88 was used as a reference compound (EC50 50= 2.6N/AN/A50 nM).b Notb available.89.089.0 5.95.9 13 a 8Serotonin Serotonin was wasused used as a asreference a reference compound compound (EC (EC50 =N/A 2.6 = nM).b 2.6 bnM). b Not Notavailable.89.0 available. 5.9 13 13 a 8 SerotoninSerotonin a8 waswas usedused as as aa referencereference compoundcompound (EC(EC50 ==N/A 2.62.6b N/A nM).nM). b NotNotb available.available.89.089.0 5.9 5.9 13 1313 a 8Serotonin 8 Serotonin 8 was wasused used as a referenceas a reference compound compound (ECN50 (EC /=AN/A 2.650 = N/AnM). 2.6 nM). bNot 89.0 Notavailable.89.0 available.89.0 5.9 5.9 5.9 a SerotoninSerotonin waswas usedused asas aa referencereference compoundcompound (EC(EC50 == 2.62.6 nM).nM). b NotNot available.available. a Serotonina was used as a reference compound (EC50 = 2.6 nM). b Notb available. TheThe evaluationThe evaluation evaluationa Serotonina a ofSerotonin of compounds compounds of was compounds was used used as10a 10a a as referenceand 10aanda reference 10f and 10f forcompound 10ffor bindingcompound bindingfor binding (EC affinity affinity (EC50 = affinity2.650 = to nM).2.6 bto the nM).the tob Not5-HT 5-HTthe b Not available. 5-HTreceptor available.receptor receptor subtypes subtypes subtypes is is is TheThe evaluationevaluationSerotonin ofof compoundscompounds was used as 10a a10a reference andand 10f10f compound forfor bindingbinding (EC50 = affinityaffinity2.6 nM). totoNot thethe available. 5-HT5-HT receptorreceptor subtypessubtypes isis TheThe The evaluationevaluation evaluation ofof compoundscompounds of compounds 10a10a 10a andand and 10f10f for10ffor bindingforbinding binding affinityaffinity affinity toto thethe to 5-HTthe5-HT 5-HT receptorreceptor receptor subtypessubtypes subtypes2C isis is illustratedillustratedillustratedThe evaluationin in Table Table in Table 3. 3. Weof We 3.compounds calculated Wecalculated calculated the 10athe selectivity selectivityandthe selectivity 10f for index index binding index (SI) (SI) of affinity(SI)of binding binding of binding to affinities the affinities 5-HT affinities between receptorbetween between 5-HT subtypes5-HT 5-HT2C 2Cand and 2Cis and illustratedillustratedTheThe evaluation inin evaluation TableTable 3.3. of WeWe compounds of calculatedcalculated compounds 10a thethe 10a selectivityandselectivity and 10f 10ffor indexindex forbinding binding (SI)(SI) affinity ofof bindingbindingaffinity to the affinitiestoaffinities the5-HT 5-HT receptor betweenbetween receptor subtypes 5-HT5-HT subtypes2C andand is is illustratedillustratedTheThe evaluation in evaluation Table in Table 3. of We compounds3. of calculatedWe compounds calculated 10athe 10a selectivityandthe andselectivity 10f 10ffor index forbinding index binding (SI) affinityof(SI) binding affinity of binding to the affinitiesto the5-HT affinities 5-HT receptorbetween receptor between subtypes5-HT subtypes 5-HT2C and 2Cis and is otherillustratedotherillustratedother subtypes subtypes subtypes in in Table Tableby by divi divi 3.by 3. dingWe Wedividing calculated calculatedtheding the given giventhe given the subtypethe subtype selectivity selectivity subtype K Ki valuei value Kindex indexi value with with (SI) (SI) thewith ofthe of 5-HTbinding binding 5-HTthe 2C5-HT2C K affinities Kiaffinities value.i2C value. Ki value. The betweenThebetween results The results results5-HT 5-HT indicate indicate2C2C indicateand and illustratedotherillustrated subtypes in Table in by Table divi3. We 3.ding Wecalculated thecalculated given the subtype theselectivity selectivity Ki value index index with (SI) (SI) ofthe binding of 5-HT binding2C2C affinities Ki affinitiesvalue. between The between results 5-HT 5-HTindicate2C and2C and otherillustratedillustrated subtypes in Table in by Table divi 3. We ding3. We calculated the calculated given the subtype theselectivity selectivity Ki value index index with (SI) (SI) theof binding of5-HT binding2C affinitiesKi2C value.affinities betweenThe between results 5-HT indicate5-HT2C and2C and thatotherother compoundother subtypessubtypes subtypes 10a byby displayed divi diviby dividingdingding thethe higher giventhegiven given selectivity subtypesubtype subtype KforKii valuevalue mostKi value with5-HTwith with the thesubtypes 5-HTthe5-HT 5-HT2C2C compared KKii value.value. Ki value. TheTheto compound The resultsresults results indicateindicate 10f indicate, otherthatthatThethat compoundcompound subtypes evaluationcompound by 10a10a diviof 10a displayeddisplayed compoundsding displayed the higherhigher given higher10a selectivitysubtypeselectivityand selectivity10f Kfor i for forvalue binding mostformost withmost 5-HT5-HT a ffi the5-HTnity subtypessubtypes 5-HT subtypes to the2C K comparedcompared 5-HTi value. compared receptor The toto results compoundcompound to subtypes compound indicate 10f10f is , , 10f , thatotherother compound subtypes subtypes by10a diviby displayed dividingding the higher thegiven given selectivitysubtype subtype K ifor value K imost value with 5-HT with the subtypes the5-HT 5-HT2C K compared2Ci value. Ki value. The to The resultscompound results indicate indicate 10f, thatthatthat compoundcompound compound1B 10a10a 10a displayeddisplayed displayed1E higherhigher higher selectivityselectivity selectivity forfor mostformost most 5-HT5-HT 5-HT subtypessubtypes subtypes comparedcompared compared toto compoundcompound to compound 10f10f, , 10f , exceptthatexceptexcept compound for for 5-HT 5-HTfor 5-HT 10a1B 1Band and displayed1B 5-HTand 5-HT 5-HT1E,1E to,higher to 1Ewhich ,which to selectivitywhich the the binding thebinding for binding most affinities affinities 5-HTaffinities of subtypesof both both of bothcompounds compounds compared compounds towere were compound werenegligible. negligible. negligible. 10f , illustratedthatexceptexceptthat compound forforcompound in 5-HT5-HT Table 10a1B3 . and and10a Wedisplayed calculated5-HTdisplayed5-HT1E ,higher, toto higher thewhichwhich selectivity selectivity thethe bindingbinding indexfor formost affinities (SI)affinities most 5-HT of 5-HT binding subtypes ofof subtypesbothboth a ffi compounds compoundscomparednities compared between to werewere compound to 5-HT compound negligible.negligible.2C and 10f ,10f , exceptthatthat compound for compound 5-HT 10a1B and 10a displayed1B 5-HTdisplayed1E ,higher to1E higherwhich selectivity selectivitythe binding for formost affinities most 5-HT 5-HT subtypesof subtypesboth compounds compared compared to were compound to compound negligible. 10f ,10f , exceptexceptexcept forfor 5-HTfor5-HT 5-HT1B andand and 5-HT5-HT 5-HT1E,, toto , which whichto which thethe thebindingbinding binding affinitiesaffinities affinities ofof 2Cboth bothof both compoundscompounds compounds2A werewere were negligible.2Bnegligible. negligible. MostexceptMost Mostimportantly, importantly, for importantly, 5-HT1B compound andcompound compound5-HT 1E10a ,10a to showed which10ashowed showed thegood good binding goodselectivity selectivity selectivity affinities for for 5-HT 5-HT forof both5-HT2C 2Cover over 2Ccompounds 5-HTover 5-HT 5-HT2A 2Aand and 2Awere 5-HT and 5-HT negligible.5-HT2B,2B which, which2B, which otherexceptMostMostexcept subtypes importantly,importantly, for for5-HT by5-HT1B dividing and compoundcompound1B and 5-HT the5-HT1E given,10a 10ato1E , which showedtoshowed subtype which the goodgood theKbindingi value selectivitybindingselectivity withaffinities affinities the forfor 5-HT 5-HT 5-HTof bothof2C2C both overKover compoundsi value. 5-HTcompounds5-HT The2A andand resultswere 5-HT 5-HTwere negligible. indicate2B ,negligible., whichwhich Mostexceptexcept importantly, for for5-HT 5-HT1B andcompound1B and 5-HT 5-HT1E ,10a to1E, whichshowedto which the good thebinding selectivitybinding affinities affinities for 5-HT of bothof2C bothover 2Ccompounds 5-HTcompounds2A and 2Awere 5-HT were negligible.2B ,negligible. which2B MostMostMost importantly,importantly, importantly, compoundcompound compound 10a10a 10a showedshowed showed goodgood good selectivityselectivity selectivity forfor 5-HTfor5-HT2C 5-HT2C overover over 5-HT5-HT 5-HT2A andand and 5-HT5-HT 5-HT2B,, whichwhich, which areMostare associated associatedare importantly, associated with with thewithcompound the primary primarythe primary 10a side side showed effectsside effects effects goodof of non-specific non-specific ofselectivity non-specific 5-HT for5-HT 5-HT 5-HT2C 2Cagonists. agonists.2C2C over agonists. 5-HT 2A and 5-HT2B, which thatMostareare compoundMost associatedassociated importantly, importantly, 10awithwith displayedcompound thethe compound primaryprimary higher 10a sideside 10a showed selectivity effectseffectsshowed good ofof good non-specific fornon-specific selectivity most selectivity 5-HT for 5-HT5-HT subtypes for5-HT2C 5-HT agonists.agonists.2C over2C compared over 5-HT 5-HT2A toand2A compound and 5-HT 5-HT2B, which2B10f, which, areMost Mostassociatedare importantly, associated importantly, with withcompound the compound primarythe primary 10a side 10a showed side effects showed effects good of goodnon-specific ofselectivity non-specific selectivity for5-HT for5-HT 5-HT2C 5-HT agonists.2C2C over agonists.2C over 5-HT 5-HT 2A and2A and 5-HT 5-HT2B, which2B, which are are associated associated withwith the the primaryprimary side side effectseffects ofof non-specificnon-specific 5-HT 5-HT2C2C agonists. agonists. exceptare areassociated for associated 5-HT with1B andwith the 5-HT theprimary primary1E, to side which side effects theeffects of binding non-specific of non-specific affinities 5-HT of5-HT2C both agonists.2C agonists. compounds were negligible. are areassociated associated with with the theprimary primary side side effects effects of non-specific of non-specific 5-HT 5-HT2C agonists.2C agonists. Most importantly, compound 10a showed good selectivity for 5-HT over 5-HT and 5-HT , which 2C 2A 2B are associated with the primary side effects of non-specific 5-HT2C agonists.

Molecules 2019, 24, 3234 7 of 21

Table 3. Evaluation of binding affinity of 10a and 10f to 5-HT receptor subtypes a.

5-HT Subtypes (%Binding at 10 µM/Ki (nM)) 1A 1B 1D 1E 2A 2B 2C 3 5A 6 7 74.1 56.1 71.4 43.1 75.5 98.0 81.3 89.0 22.5 50.7 61.3 10a /578.0 /3237.0 /679.0 /NDb /1284.0 /83.0 /7.9 /323.0 /ND /348.0 /1016.0 SI 73.2 409.7 85.9 - 162.5 10.5 1.0 40.9 23.4 44.1 128.6 67.6 19.2 81.3 31.3 52.9 95.0 98.9 88.0 15.2 56.5 55.4 10f /1066.0 /NDb /937.0 /NDb /2863.0 /118.0 /19.0 /415.0 /ND /467.0 /616.0 SI 56.1 - 49.3 - 150.7 6.2 1.0 21.8 10.3 24.6 32.4 89.1 4.9 83.4 48.0 85.3 98.1 89.0 83.4 18.5 56.7 63.4 8c /271.0 /NDb /1052.0 /NDb /511.0 /38.0 /5.9 /543.0 /NDb /30.0 /528.0 SI 45.9 - 178.3 - 86.6 6.4 1.0 92.0 - 5.1 89.5 a 5-HT receptor binding was determined by competitive binding assay using radioligands and reference compounds in Table S1. b Not determined due to low %binding. c Compound 8 was used as a reference compound. SI, selectivity index.

Considering the 5-HT2C cellular agonistic effect, binding affinity, and subtype selectivity of 10a, we further investigated its drug-like properties, such as plasma stability, microsomal stability, and CYP inhibition, as shown in Table4. For plasma stability, 10 µM of compound 10a was incubated in human plasma; the remaining amount of 10a was measured after incubation for 30 min and 2 h. For microsomal stability, human liver microsomes were treated with 10a at a concentration of 1 µM; the % remaining amount of 10a was analyzed at 30 min after treatment. CYP inhibition values against five human CYP450 isozymes were recorded as % remaining activity values of the given isozymes. We found that compound 10a was stable in both the plasma and liver, indicating that compound 10a would be less affected by hepatic metabolism or plasma decomposition throughout systemic circulation. Furthermore, the inhibitory activity of 10a against all the tested CYP isozymes was significantly low (%remaining activity of isozymes >60%), suggesting that compound 10a might not exert toxic effects due to drug–drug interaction (DDI). Overall, compound 10a exhibited considerably excellent plasma and microsomal stability with low CYP inhibitory activity.

Table 4. The results of plasma stability, microsomal stability, and CYP inhibition.

Plasma Stability HLM CYP Isozymes (%Remaining @ 10 µM) Compd %Remaining @ %Remaining @ 1A2 2D6 2C9 3A4 2C19 10 µM after 0.5 and 2 h 1 µM after 0.5 h 10a 99.9/98.0 96.6 86.5 63.6 >100 86.2 86.3

2.4. Molecular Docking Study

In order to investigate the good selectivity of 10a to 5-HT2C over 5-HT2B, the molecular docking study was performed using Discovery Studio software. Thus, the interaction of 10a and 8 with those receptors were evaluated using the crystal structures of 5-HT2C (PDB ID = 6BQH) and 5-HT2B (PDB ID = 4IB4), which were recently reported in the literatures [28,33]. In the case of 5-HT2C receptor docking study, the binding orientation of both ligands 10a (-CDOCKER score 36.31) and 8 (-CDOCKER score 36.96) are similar but not precisely overlapping over each other (Figure3A). Both of these ligands have shown hydrogen bonds with crucial aspartic acids Asp134 (Figure3B,C). NH groups of piperazine ( 8) and 1,4-diazepane ring (10a) are involved in hydrogen bonds and π-cationic interactions with Trp130 and Asp134. Additionally, 8 has shown two more hydrogen bonds with Tyr358 and Leu209. The hydrogen of the NH group of the piperazine ring has shown a hydrogen bond with Tyr358 and nitrogen of the pyrimidine ring has shown one hydrogen bond with Leu209. In total, we recognized four hydrogen bond interactions between 5-HT2C and 8. Fluorophenyl rings of 8 were involved in π–π stacking interactions with Phe328 residue. In the case of 10a, in addition to hydrogen bonds, strong π–π stacking contacts were observed with Phe327, phe328, Molecules 2019, 24, 3234 8 of 21 and Trp324. Several hydrophobic contacts in both ligands were identified, mediated by Val135 and Leu209Molecules in 201910a, 24and, x FOR Val135, PEER Val208,REVIEW and Leu209 in 8. 9 of 21

Figure 3. Interactions of 10a and 8 inside the 5-HT2C and 5-HT2B receptors. Ligands 10a and 8 are Figure 3. Interactions of 10a and 8 inside the 5-HT2C and 5-HT2B receptors. Ligands 10a and 8 are shown in green and magenta color stick format. 5-HT2C (wheat color) and 5-HT2B (green color) protein shown in green and magenta color stick format. 5-HT2C (wheat color) and 5-HT2B (green color) protein structures are shown in cartoon format. Hydrogen bond, π-cationic, π–π stacking, and hydrophobic structures are shown in cartoon format. Hydrogen bond, π-cationic, π–π stacking, and hydrophobic interactions are shown in green, orange, dark pink, and light pink dash line format. (A) Overlap interactions are shown in green, orange, dark pink, and light pink dash line format. (A) Overlap of of 10a and 8 inside 5-HT2C protein. (B) Interactions between ligand 10a and the 5-HT2C receptor. 10a and 8 inside 5-HT2C protein. (B) Interactions between ligand 10a and the 5-HT2C receptor. (C) (C) Interactions between ligand 8 and the 5-HT receptor. (D) Overlap of 10a and 8 inside 5-HT Interactions between ligand 8 and the 5-HT2C receptor.2C (D) Overlap of 10a and 8 inside 5-HT2B protein. 2B protein. (E) Interactions between ligand 10a and the 5-HT receptor. (F) Interactions between ligand 8 (E) Interactions between ligand 10a and the 5-HT2B receptor.2B (F) Interactions between ligand 8 and the

and5-HT the2B 5-HT receptor.2B receptor.

3. MaterialsIn the case and of 5-HT2BMethods receptor docking study, overlap of ligand 10a (-CDOCKER score 37.40) and 8 (-CDOCKER score 39.53) inside 5-HT2B binding pocket (Figure3D) shows that ligand bind at the same3.1. placeGeneral but Methods differ in pyrimidine, fluorophenyl ring, and diazepane ring/piperidine orientation. Compound 8 has shown three hydrogen bonds with binding site residues. The hydrogen (NH group) All reactions were conducted under oven-dried glassware, under an atmosphere of nitrogen. All of the piperazine ring showed two hydrogen bonds with Asp135 (Figure3F). The fluorine group on commercially available reagents were purchased and used without further purification. Solvents and thegases phenyl were ring dried in according8 has shown to standard an additional procedures. hydrogen Organic bond solvents with Thr114.were evaporated Beside thesewith reduced hydrogen bondingpressure interactions, using a rotary several evaporator. hydrophobic Reactions residues, were followed Val136, byVal208, analytical Leu209, thin layer and chromatography Leu362, hold the molecule(TLC) analysis by several using hydrophobic glass plates precoated interactions. with Compoundsilica gel (0.2510a mmhas). TLC only plates one hydrogenwere visualized bond by with Asp135exposure (Figure to UV3E). light Compared (UV), and to then8, were10a has visualized fewer hydrophobicwith a KMnO4 contacts or p-anisaldehyde with the 5-HTstain 2Bfollowedreceptor bindingby brief pocket heating (Val2018, on a hot Leu209,plate. Flash and column Leu362). chromatography was performed using silica gel 60 (230– 400Although mesh, Merck) both 10awithand the8 indicatedbind in a similarsolvents. way 1H-NMR inside thespectra 5-HT were2B binding measured pocket, with a minor400 MHz diff erenceand in13 aromaticC-NMR ringspectra orientation were measured affects the with hydrophobic 100 MHz using interactions CDCl3 ofand10a CD. The3OD. di ff1Herence NMR in spectra fluorophenyl are ringrepresented orientation as isfollows: responsible Chemical for theshift, additional multiplicity hydrogen (s = singlet, bond d = between doublet, Thr114t = triplet, and q =8 .quartet, These extram 1 hydrogen= multiplet), bonds integration, and hydrophobic and coupling contacts constant inside (J) the in Hertz 5-HT2B (Hz).receptors H NMR make chemical the potency shifts are of reported compound 13 8 superiorrelative to to CDCl that of3 (7.26 compound ppm). 10aC NMRin the was 5-HT recorded2B binding relative assay. to the Compared central line to the of 5-HTCDCl2B3 (77.0receptor, ppm).10a hasHigh stronger resolution interactions mass spectra (hydrogen (LR-MS) bond were and obtain hydrophobic)ed using withpositive the 5-HTelectrospray2C receptor. ionization Overall, and the mass/charge (m/z) ratios are reported as values in atomic mass units. decrease in hydrophobic contacts inside the 5-HT2B receptor makes our molecule 10a more selective towards the 5-HT2C receptor.

Molecules 2019, 24, 3234 9 of 21

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 then were 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) with the indicated solvents. 1H-NMR spectra were measured with 400 MHz 13 1 and C-NMR spectra were measured with 100 MHz using CDCl3 and CD3OD. H 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 13 reported relative to CDCl3 (7.26 ppm). C NMR was recorded relative to the central line of CDCl3 (77.0 ppm). High resolution mass spectra (LR-MS) were obtained using positive electrospray ionization and mass/charge (m/z) ratios are reported as values in atomic mass units.

3.2. Synthesis of 2,5-Disubstituted Pyrimidines

3.2.1. General Procedure for Preparing Compounds 11a–c In a dry sealed tube under argon were placed, to a solution of 1-Boc-piperazine (5.04 mmol), potassium carbonate (13.1 mmol) in acetonitrile (12.6 mL) was added 2,5-dibromo pyrimidine 13 (5.04 mmol). The mixture was allowed to stir at 80 ◦C for 12 h. After completion of the reaction (monitored by TLC), the mixture was then cooled at room temperature, then it was quenched with saturated aqueous NH4Cl (10 mL) 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 piperazine 11a. tert-Butyl 4-(5-bromopyrimidin-2-yl)piperazine-1-carboxylate (11a): Yield: 88%; 1H-NMR (400 MHz, CDCl3) δ 8.30 (s, J = 2.0 Hz, 2H), 3.78—3.75 (t, J = 5.2 Hz, 4H), 3.49–3.47 (t, J = 5.2 Hz, 4H), 1.48 (s, 9H). 13 C-NMR (100 MHz, CD3OD) δ 161.2, 159.2, 156.46, 107.15, 81.5, 44.8, 28.6. tert-Butyl (R)-4-(5-bromopyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (11b): Yield: 92%; 1H-NMR (400 MHz, CDCl3) δ 8.28 (s, J = 2.0 Hz, 1H), 4.81–4.70 (m, 1H), 4.39–4.36 (m, 1H), 4.15–3.90 (m, 2H), 13 3.19–3.08 (m, 1H), 3.08–2.90 (m, 2H), 1.47 (s, 9H), 1.16 (d, J = 6.7 Hz, 3H). C-NMR (100 MHz, CD3OD) δ 160.9, 159.2, 156.9, 107.0, 81.4, 45.1, 44.0, 39.4, 14.5. tert-Butyl 4-(5-bromopyrimidin-2-yl)-1,4-diazepane-1-carboxylate (11c): Yield: 97%; 1H-NMR (400 MHz, CDCl3) δ 8.28 (s, 2H), 3.86–3.80 (m, 2H), 3.73–3.70 (t, J = 6.1 Hz, 2H), 3.55–3.52 (t, J = 5.4 Hz, 2H), 3.38–3.35 (t, J = 5.9 Hz, 1H), 3.28–3.25 (t, J = 6.1 Hz, 1H), 1.96–1.90 (m, 2H), 1.43 (d, J = 8.4 Hz, 9H). 13 C-NMR (100 MHz, CDCl3) δ 159.5, 158.1, 155.3, 105.8, 79.6, 49.0, 48.5, 47.4, 46.9, 46.3, 46.1, 28.5, 25.4.

3.2.2. General Procedure for Preparing Compounds 9a–r In a dry sealed tube under argon were placed 11b (0.56 mmol), 3-fluorobenzyl alcohol (1.68 mmol), cesium carbonate (0.84 mmol), copper iodide (0.056 mmol) (or copper(I) oxide), 1,10-phenanthroline (0.11 mmol) (or 3,4,7,8-tetramethyl-1,10-phenanthroline) in toluene (1.1 mL) and the mixture was heated at 130 ◦C for 20 h. After completion of the reaction (monitored by TLC), the mixture was then cooled at room temperature and it was quenched with saturated aqueous NH Cl (3 50 mL), extracted 4 × 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: Acetone: Hexane = 1:1:15) to afford intermediate pre-9h. Molecules 2019, 24, 3234 10 of 21

To a solution of methyl piperazine carboxylate pre-9h (0.136 mmol) in dichloromethane (1.3 mL) was added trifluoroacetic acid (0.21 mL). The mixture was allowed to stir at 0 ◦C for 3 h. After completion of the reaction (monitored by TLC), then neutralized by sodium hydrogen carbonate until the mixture to be pH 8 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 (MeOH:DCM = 1:15) to afford pyrimidine 9h, which was isolated as HCl salt form after treating with 4 M HCl in diethyl ether. tert-Butyl 4-(5-((3-fluorophenoxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9a): Yield: 21%; 1 H-NMR (400 MHz, CDCl3) δ 8.17 (s, 2H), 7.27–7.21 (m, 1H), 6.77–6.73 (m, 1H), 6.71–6.62 (m, 2H), 3.80 (t, J = 5.1 Hz, 4H), 3.52 (t, J = 5.0 Hz, 4H), 1.49 (s, 9H). 4-(5-((3-fluorophenoxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9a): Yield: 21%; 1H-NMR · (400MHz, CDCl3) δ 8.17 (s, 2H), 7.24 (dd, J = 15.4, 7.5 Hz, 1H), 6.75 (m, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 10.2 Hz, 1H), 3.89 (t, J = 4.5 Hz, 4H), 3.07 (t, J = 4.7 Hz, 4H). 13C-NMR (100 MHz, 1 3 4 CD3OD) δ 162.4 (d, J = 259 Hz), 159.7, 152.3, 152.0, 144.5, 132.2 (d, J = 9 Hz), 113.4 (d, J = 3 Hz), 110.9 2 2 + (d, J = 21 Hz), 105.4 (d, J = 25 Hz), 44.4, 42.5; LRMS-EI (m/z): [M–Cl] calcd for C14H15FN4O: 274.30, found: 274.30 tert-Butyl 4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9b): Yield: 79%; 1H-NMR (400 MHz, CDCl3) δ 8.11 (s, 2H), 7.37–7.32 (m, 1H), 7.17–7.11 (m, 2H), 7.05–6.99 (m, 1H), 5.01 (s, 2H), 3.71 (t, J = 5.2 Hz, 4H), 3.49 (t, J = 5. 2 Hz, 4H), 1.48 (s, 9H). 4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9b): Yield: 58%; 1H-NMR · (400 MHz, CD3OD) δ 8.23 (s, 1H), 7.42–7.37 (m, 1H), 7.25–7.17 (m, 2H), 7.06 (td, J = 8.5, 2.4 Hz, 1H), 7.07–7.02 (m, 1H), 5.12 (s, 2H), 3.95 (t, J = 5.3 Hz, 4H), 3.23 (t, J = 5.3 Hz, 4H). 13C-NMR (100 MHz, 1 3 3 4 CD3OD) δ 164.3 (d, J = 243 Hz), 158.2, 147.7, 147.4, 140.8 (d, J = 7 Hz), 131.5 (d, J = 8 Hz), 124.4 (d, J = 3 Hz), 115.8 (d, 2J = 21 Hz), 115.3 (d, 2J = 22 Hz), 71.9, 44.3, 42.6; LRMS-EI (m/z): [M–Cl]+ calcd for C15H17FN4O: 288.33, found: 288.33 tert-Butyl 4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9c): Yield: 46%; 1 H-NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.24–7.20 (m, 2H), 7.02–6.98 (m, 2H), 4.12 (t, J = 6.8 Hz, 2H), 3.69 (t, J = 5.2 Hz, 2H), 3.48 (t, J = 5.2 Hz, 2H), 3.03 (t, J = 6.8 Hz, 2H), 1.48 (s, 9H). 4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9c): Two-step yield: 46%; 1 · H-NMR (400 MHz, CD3OD) δ 8.17 (s, 2H), 7.34–7.29 (m, 1H), 7.13 (d, J = 8 Hz, 1H), 7.07 (d, J = 10.1 Hz, 1H), 6.96 (td, J = 8.7, 2.3 Hz, 1H), 4.25 (t, J = 6.5 Hz, 2H), 3.97 (t, J = 5.2 Hz, 4H), 3.27 (t, J = 5.2 Hz, 13 1 4H), 3.09 (t, J = 6.5 Hz, 2H). C-NMR (100 MHz, CD3OD) δ 164.3 (d, J = 242 Hz), 158.1, 148.2, 146.8, 142.5 (d, 3J = 7 Hz), 131.1 (d, 3J = 8 Hz), 125.9 (d, 4J = 3 Hz), 116.7 (d, 2J = 21 Hz), 114.3 (d, 2J = 21 Hz), + 71.28, 44.3, 42.7, 36.4, 36.4; LRMS-EI (m/z): [M–Cl] calcd for C16H19FN4O: 302.35, found: 302.35 tert-Butyl 4-(5-((4-fluorophenoxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9d): Yield: 21%; 1 H-NMR (400 MHz, CDCl3) δ 8.17 (s, 2H), 7.03–6.99 (m, 2H), 6.92–6.89 (m, 2H), 3.80 (t, J = 5.2 Hz, 4H), 3.52 (t, J = 5.2 Hz, 4H), 1.49 (s, 9H). 4-(5-((4-fluorophenoxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9d): Yield: 60%; 1H-NMR (400 · MHz, CDCl3) δ 8.15 (s, 2H), 7.02-6.98 (m, 2H), 6.91–6.88 (m, 2H), 3.77 (t, J = 5.2 Hz, 4H), 3.51 (t, J = 5.1 Hz, 13 1 3 4H). C-NMR (100 MHz, CD3OD) δ 158.9 (d, J = 241 Hz), 157.9, 153.8, 150.1, 144.4, 118.6 (d, J = 8 Hz), 2 + 116.6 (d, J = 23 Hz), 43.4, 41.5; LRMS-EI (m/z): [M–Cl] calcd for C14H15FN4O: 274.30, found: 274.30. tert-Butyl 4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9e): Yield: 76%; 1 H-NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.37 (dd, J = 8.7, 5.3 Hz, 2H), 7.09–7.05 (m, 2H), 4.98 (s, 2H), 3.71 (t, J = 5.2 Hz, 4H), 3.49 (t, J = 5.2 Hz, 4H), 1.48 (s, 9H). 4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9e): Two-step yield: 67%; 1 · H-NMR (400 MHz, CD3OD) δ 8.22 (s, 2H), 7.47–7.43 (m, 2H), 7.13–7.08 (m, 2H), 5.08 (s, J = 2.5 Hz, 13 1 2H), 3.95 (t, J = 5.3 Hz, 4H), 3.24 (t, J = 5.2 Hz, 4H). C-NMR (100 MHz, CD3OD) δ 164.0 (d, J = 243 Hz), 158.4, 147.4, 134.0 (d, 4J = 3 Hz), 132.3, 131.0 (d, 3J = 8 Hz), 116.3 (d, 2J = 22 Hz), 72.1, 44.7, 43.3; + LRMS-EI (m/z): [M–Cl] calcd for C15H17FN4O: 288.33, found: 288.33. Molecules 2019, 24, 3234 11 of 21

tert-Butyl 4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9f): Yield: 54%; 1 H-NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.24–7.20 (m, 2H), 7.02–6.98 (m, 2H), 4.12 (t, J = 6.8 Hz, 2H), 3.70 (t, J = 5.2 Hz, 2H), 3.48 (t, J = 5.2 Hz, 2H), 3.03 (t, J = 6.8 Hz, 2H), 1.48 (s, 9H). 4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9f): Two-step yield: 53%; 1 · H-NMR (400 MHz, CD3OD) δ 8.16 (s, 2H), 7.32–7.28 (m, 2H), 7.04–6.99 (m, 2H), 4.21 (t, J = 6.6 Hz, 2H), 13 3.95 (t, J = 5.1 Hz, 4H), 3.27 (t, J = 5.1 Hz, 4H), 3.05 (t, J = 6.6 Hz, 2H). C-NMR (100 MHz, CD3OD) δ 163.1 (d, 1J = 241 Hz), 158.6, 147.7, 146.8, 135.51, 131.7 (d, 3J = 8 Hz), 116.0 (d, 2J = 21 Hz), 71.7, 45.3, + 44.5, 35.9; LRMS-EI (m/z): [M–Cl] calcd for C16H19FN4O: 302.35, found: 302.35. tert-Butyl (R)-4-(5-((3-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9g): Yield: 1 17%; H-NMR (400 MHz, CDCl3) δ 8.18 (s, 2H), 7.27–7.22 (m, 1H), 6.78–6.70 (m, 2H), 6.65–6.63 (m, 1H), 4.90–4.84 (m, 1H), 4.43–4.40 (m, 1H), 4.18–3.93 (m, 2H), 3.24–3.18 (m, 1H), 3.13–2.94 (m, 2H), 1.49 (s, 9H), 1.20 (d, J = 6.8 Hz, 3H). (R)-4-(5-((3-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-ium hydrochloride (9g): Yield: 21%; 1 · H-NMR (400 MHz, CDCl3) δ 8.18 (s, 2H), 7.27–7.21 (m, 1H), 6.77–6.65 (m, 2H), 6.63–6.62 (m, 1H), 4.78–4.75 (m, 1H), 4.41–4.38 (m, 1H), 3.16–3.08 (m, 2H), 3.04–3.00 (m, 1H), 2.92–2.89 (m, 1H), 2.83–2.76 13 (m, 1H), 1.28 (d, J = 6.8 Hz, 3H). C-NMR (100 MHz, CD3OD) δ 159.3, 152.1, 146.9, 144.3, 132.2 (d, 3J = 9 Hz), 131.8, 113.4, 110.8 (d, 2J = 21 Hz), 105.4 (d, 2J = 25 Hz), 45.8, 44.8, 36.9, 35.9, 13.7; LRMS-EI + (m/z): [M–Cl] calcd for C15H17FN4O: 288.33, found: 288.33. tert-Butyl (R)-4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9h): 1 Yield: 43%; H-NMR (400 MHz, CDCl3) δ 8.11 (s, 2H), 7.37–7.32 (m, 1H), 7.15–7.11 (m, 2H), 7.04–6.99 (m, 1H), 5.00 (s, 2H), 4.75 (s, 1H), 4.31 (d, J = 12.5 Hz, 1H), 4.10 (d, J = 12.5 Hz, 1H, broad), 3.17–3.10 (m, 1H), 3.10–2.91 (brs, 1H), 1.48 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H). (R)-4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-ium hydrochloride (9h): Yield: 70%; 1 · H-NMR (400 MHz, CDCl3) δ 8.12 (s, 2H), 7.37–7.33 (m, 1H), 7.17–7.11 (m, 2H), 7.05–7.00 (td, J = 8.4, 2.3 Hz, 1H), 5.01 (s, 1H), 4.85–4.82 (m, 1H), 4.45–4.40 (m, 1H), 3.23–3.17 (m, 2H), 3.10–3.01 (m, 2H), 13 1 2.89–2.82(m, 1H), 1.30 (d, J = 6.9 Hz, 3H). C-NMR (100 MHz, CD3OD) δ 165.6 (d, J = 244 Hz), 157.8, 147.4, 140.8, 131.4 (d, 3J = 9 Hz), 124.3, 115.8 (d, 2J = 20 Hz), 115.3 (d, 2J = 22 Hz), 71.9, 45.8, 44.8, 37.2, + 13.4; LRMS-EI (m/z): [M–Cl] calcd for C16H19FN4O: 302.35, found: 302.35 tert-Butyl (R)-4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9i): 1 Yield: 24%; H-NMR (400 MHz, CDCl3) δ 8.06 (s, 2H), 7.30–7.26 (m, 1H), 7.04–7.02 (m, 1H), 6.98–6.91 (m, 2H), 4.98 (s, 2H), 4.80–4.70 (m, 1H), 4.31–4.28 (m, 1H), 4.14 (t, J = 6.7 Hz, 2H), 3.97–3.90 (m, 2H), 3.17–3.10 (m, 2H), 3.06 (t, J = 6.7 Hz, 2H), 3.00–2.80 (m. 2H), 1.48 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H). (R)-4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium hydrochloride (9i): Two-step 1 · yield: 37%; H-NMR (400 MHz, CDCl3) δ 8.06 (s, 2H), 7.28 (dd, J = 13.9, 7.9 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 6.98–6.91 (m, 2H), 4.86 (s, 2H), 4.45 (d, J = 12.4 Hz, 1H), 4.15 (t, J = 6.7 Hz, 2H), 3.28–3.14 (m, 2H), 3.11–3.08 (m, 1H), 3.06 (t, J = 6.7 Hz, 2H), 2.87 (td, J = 12.6, 4.1 Hz, 2H), 1.26 (d, J = 6.9 Hz, 2H). 13 1 3 C-NMR (100 MHz, CD3OD) δ 163.10 (d, J = 245 Hz), 157.6, 147.9, 146.1, 142.5 (d, J = 7 Hz), 131.1 (d, 3J = 8 Hz), 125.9 (d, 4J = 3 Hz), 116.7 (d, 2J = 21 Hz), 114.2 (d, 2J = 21 Hz), 71.3, 45.6, 44.6, 36.7, 36.4, 30.9, + 13.3; LRMS-EI (m/z): [M–Cl] calcd for C17H21FN4O: 316.38, found: 316.38. tert-Butyl (R)-4-(5-((4-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9j): Yield: 1 27%; H-NMR (400 MHz, CDCl3) δ 8.17 (s, 2H), 7.02–6.98 (m, 2H), 6.91–6.89 (m, 2H), 4.90–4.83 (m, 1H), 4.43–4.39 (m, 1H), 4.18–3.92 (m, 3H), 3.25–3.18 (m, 1H), 3.15–2.93 (m, 2H), 1.49 (s, 9H), 1.20 (d, J = 6.7 Hz, 3H). (R)-4-(5-((4-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-ium hydrochloride (9j): Yield: 39%; 1 · H-NMR (400 MHz, CDCl3) δ 8.15 (s, 2H), 7.01–6.96 (m, 2H), 6.91–6.87 (m, 2H), 4.79–4.76 (m, 1H), 4.42–4.38 (m, 1H), 3.16–3.09 (m, 2H), 3.05–3.01 (m, 1H), 2.94–2.91 (m, 1H), 2.84–2.77 (m, 1H), 2.77–2.70 (m, 1H), 1.28 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz, CD3OD) δ 160.1 (d, 1J = 239 Hz), 159.0, 155.5, 155.5 (d, 4J = 2 Hz), 145.5, 119.8 (d, 3J = 8 Hz), 117.4 (d, 2J = 24 Hz), 45.9, 44.9, 37.2, 30.9, 30.7, 13.5; + LRMS-EI (m/z): [M–Cl] calcd for C15H17FN4O: 288.33, found: 288.33. Molecules 2019, 24, 3234 12 of 21

tert-Butyl (R)-4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9k): 1 Yield: 41%; H-NMR (400 MHz, CDCl3): δ 8.10 (s, 2H), 7.37 (dd, J = 8.5, 5.4 Hz, 2H), 7.09–7.05 (m, 2H), 4.97 (s, 2H), 4.80–4.74 (m, 1H), 4.32–4.28 (m, 1H), 4.15–3.89 (m, 3H), 3.17–3.10 (m, 1H), 3.10–2.90 (m, 2H), 1.48 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H). (R)-4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium hydrochloride (9k): Yield: 73%; 1 · H-NMR (400 MHz, CDCl3) δ 8.11 (s, 2H), 7.39–7.35 (m, 2H), 7.09–7.05 (m, 2H), 4.98 (s, 2H), 4.94–4.89 (m, 1H), 4.64–4.61 (m, 1H), 3.48–3.39 (m, 2H), 3.28–3.25 (m, 1H), 3.20–3.12 (m, 1H), 3.01–2.94 (m, 1H), 13 1 1.42 (d, J = 7.0 Hz, 3H). C-NMR (100 MHz, CD3OD) δ 162.8 (d, J = 245 Hz), 156.4, 146.4, 146.1, 131.9, 129.7 (d, 3J = 8 Hz), 115.8 (d, 2J = 21 Hz), 71.5, 47.2, 44.3, 43.3, 35.7, 14.0; LRMS-EI (m/z): [M–Cl]+ calcd for C16H19FN4O: 302.35, found: 302.35. tert-Butyl (R)-4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9l): 1 Yield: 39%; H-NMR (400MHz, CDCl3) δ 8.05 (s, 2H), 7.23–7.20 (m, 2H), 7.02–6.98 (m, 2H), 4.80–4.70 (m, 1H), 4.30–4.27 (m, 1H), 4.12 (t, J = 6.9 Hz, 2H), 3.98–3.87 (m, 2H), 3.17–3.09 (m, 1H), 3.03 (t, J = 6.8 Hz, 2H), 3.01–2.89 (m, 2H), 1.48 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H). (R)-4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium hydrochloride (9l): Yield: 85%; 1 · H-NMR (400 MHz, CDCl3): δ 8.06 (s, 2H), 7.22 (dd, J = 8.4, 5.5 Hz, 2H), 7.00 (t, J = 8.7 Hz, 2H), 4.87–4.85 (m, 1H), 4.46–4.42 (m, 1H), 4.12 (t, J = 6.8 Hz, 2H), 3.27–3.16 (m, 2H), 3.09–3.05 (m, 2H), 3.03 (t, J = 6.7 Hz, 2H), 2.90–2.84 (m, 1H), 1.37 (d, J = 7.0 Hz, 3H). 13C-NMR (100 MHz, CD3OD): δ 163.08 (d, 1J = 241 Hz), 157.7, 147.8, 146.9, 135.5 (d, 4J = 3 Hz), 131.7 (d, 3J = 8 Hz), 116.0 (d, 2J = 21 Hz), 71.7, 45.8, + 44.8, 37.2, 35.9, 30.9, 30.7, 13.3; LRMS-EI (m/z): [M–Cl] calcd for C17H21FN4: 316.38, found: 316.38. tert-Butyl 4-(5-((3-fluorophenoxy)pyrimidin-2-yl)-1,4-diazepane-1-carboxylate (pre-9m): Yield: 19%; 1 H-NMR (400 MHz, CDCl3) δ 8.17 (s, 2H), 7.28–7.22 (m, 1H), 6.78–6.70 (m, 2H), 6.71 (m, 1H), 6.65–6.60 (m, 1H), 3.90–3.88 (m, 2H), 3.80–3.78 (m, 2H), 3.59–3.58 (m, 2H), 3.40–3.39 (m, 1H), 3.33–3.30 (m, 1H), 1.99–1.96 (m, 2H), 1.44 (s, 9H). 4-(5-((3-fluorophenoxy)pyrimidin-2-yl)-1,4-diazepan-1-ium hydrochloride (9m): Yield: 40%; 1H-NMR · (400 MHz, CDCl3) δ 8.17 (s, 2H), 7.26–7.22 (m, 1H), 6.77–6.73 (m, 1H), 6.70–6.67 (m, 1H), 6.65–6.62 (m, 1H), 4.08–4.01 (s, 2H), 3.90 (t, J = 6.3 Hz, 2H), 3.36–3.27 (m, 2H), 3.16–3.15 (m, 2H), 2.16–2.14 (m, 2H). 13 1 3 C-NMR (100 MHz, CD3OD) δ 165.0 (d, J = 244 Hz), 159.6, 152.3, 143.9, 132.1 (d, J = 10 Hz), 131.6 (d, 3J = 8 Hz), 113.3, 110.7 (d, 2J = 21 Hz), 105.2 (d, 2J = 25 Hz), 46.8, 46.5, 44.7, 26.8; LRMS-EI (m/z): + [M–Cl] calcd for C15H17FN4O: 288.33, found: 288.33. tert-Butyl 4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-1,4-diazepane-1-carboxylate (pre-9n): Yield: 79%; 1 H-NMR (400 MHz, CDCl3): δ 8.09 (s, 2H), 7.37–7.32 (m, 1H), 7.17–7.11 (m, 2H), 7.04–6.99 (m, 1H), 4.99 (s, 2H), 3.84–3.78 (m, 2H), 3.71–3.68 (m, 2H), 3.53 (s, 2H), 3.35 (t, J = 5.8 Hz, 1H), 3.25 (t, J = 5.9 Hz, 1H), 1.96–1.90 (m, 2H), 1.42 (s, 9H, a mixture of rotamers). 4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-1,4-diazepan-1-ium hydrochloride (9n): Two-step yield: 53%; 1 · H-NMR (400 MHz, CDCl3) δ 8.10 (s, 2H), 7.38–7.32 (m, 1H), 7.17–7.11 (m, 2H), 7.05–7.01 (m, 1H), 5.01 (s, 2H), 3.92–3.90 (m, 2H), 3.81–3.77 (m, 2H), 3.76–3.71 (m, 2H), 3.58 (t, J = 6.0 Hz, 1H), 3.52 (t, J = 6.0 13 1 Hz, 1H), 2.07–1.97 (m, 2H). C-NMR (100 MHz, CD3OD) δ 164.4 (d, J = 243 Hz), 157.9, 147.8, 147.1, 140.9 (d, 3J = 7 Hz), 131.4 (d, 3J = 8 Hz), 124.3 (d, 4J = 3 Hz), 115.8 (d, 2J = 21 Hz), 115.3 (d, 2J = 22 Hz), + 72.1, 72.1, 47.0, 46.6, 44.6, 26.9; LRMS-EI (m/z): [M–Cl] calcd for C16H19FN4O: 302.35, found: 302.35. tert-Butyl 4-(5-((4-fluorophenoxy)pyrimidin-2-yl)-1,4-diazepane-1-carboxylate (pre-9p): Yield: 31%; 1 H-NMR (400 MHz, CDCl3) δ 8.14 (s, 2H), 7.01–6.96 (m, 2H), 6.87–6.75 (m, 2H), 3.89–3.84 (m, 2H), 3.76–3.73 (m, 2H), 3.58–5.55 (m, 2H), 3.39 (t, J = 6.0 Hz, 1H), 3.31 (d, J = 6.0 Hz, 1H), 3.31 (t, J = 6.0 Hz, 1H), 1.98–1.92 (m, 2H), 1.44 (s, 9H). 4-(5-((4-fluorophenoxy)pyrimidin-2-yl)-1,4-diazepan-1-ium hydrochloride (9p): Yield: 34%; 1H-NMR · (400 MHz, CDCl3) δ 8.14 (s, 2H), 7.00–6.96 (m, 2H), 6.90–6.87 (m, 2H), 4.10–4.01 (m, 2H), 3.98–3.88 (m, 1H), 3.38–3.30 (m, 2H), 3.27–3.20 (m, 2H), 2.20–2.15 (m, 2H). 13C-NMR (100 MHz, CD3OD) δ 163.9 (d, 1J = 244 Hz), 157.9, 147.9, 147.1, 134.1, 131.0 (d, 3J = 8 Hz), 116.3 (d, 2J = 21 Hz), 72.4, 47.1, 46.6, 46.5, + 44.5, 26.8; LRMS-EI (m/z): [M–Cl] calcd for C15H17FN4O: 288.33, found: 288.33. Molecules 2019, 24, 3234 13 of 21

4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)-1,4-diazepan-1-ium hydrochloride (9o): Yield: 6.5% (2 steps); 1 · H-NMR (400 MHz, CDCl3) δ 8.04 (s, 2H), 7.30–7.28 (m, 2H), 7.04–7.03 (m, 1H), 6.99–6.94 (m, 2H), 4.15 (t, J = 6.6 Hz, 2H), 3.91–3.89 (m, 2H), 3.79–3.77 (m, 2H), 3.75–3.72 (m, 2H), 3.57–3.55(m, 1H), 3,50–3.49 13 (m, 1H), 3.06 (t, J = 6.5 Hz, 2H), 2.05–1.98 (m, 2H). C-NMR (100 MHz, CD3OD) δ 147.4, 147.3, 147.2, 131.1, 131.0, 125.9, 125.9, 116.8, 116.6, 114.2, 114.0, 71.59, 47.7, 46.66, 46.5, 36.5, 25.9; LRMS-EI (m/z): + [M–Cl] calcd for C17H21FN4O: 316.38, found: 316.38. 4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)-1,4-diazepan-1-ium hydrochloride (9q): Two-step yield: 7%; 1 · H-NMR (400 MHz, CDCl3) δ 8.11 (s, 2H), 7.37 (dd, J = 8.4, 5.4 Hz, 2H), 7.08 (t, J = 8.6 Hz, 2H), 4.97 (s, 2H), 4.07–4.03 (s, 2H), 3.89–3.86 (m, 2H), 3.35–3.29 (m, 2H), 3.20–3.17 (m, 2H), 2.23–2.22 (m, 2H). 13 1 3 C-NMR (100 MHz, CD3OD) δ 165.4 (d, J = 243 Hz), 155.0, 147.0, 146.8, 133.5, 131.2 (d, J = 8 Hz), 2 + 116.4 (d, J = 21 Hz), 72.5, 47.2, 46.9, 46.6, 27.0; LRMS-EI (m/z): [M–Cl] calcd for C16H19FN4O: 302.35, found: 302.35. 4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)-1,4-diazepan-1-ium hydrochloride (9r): Two-step yield: 3.5%; 1 · H-NMR (400 MHz, CDCl3) δ 8.04 (s, 2H), 7.22 (dd, J = 8.4, 5.5 Hz, 2H), 7.01 (t, J = 8.6 Hz, 2H), 4.12 (t, J = 6.8 Hz, 2H), 3.92–3.89 (m, 2H), 3.81–3.77 (m, 2H), 3.75–3.71 (m, 2H), 3.57 (t, J = 6.0 Hz, 1H), 3.51 (t, J = 6.0 Hz, 1H), 3.03 (t, J = 6.7 Hz, 2H), 2.07–1.96 (m, 2H). 13C-NMR (100 MHz, CD3OD) δ 156.4, 146.2, 145.8, 134.2, 130.3 (d, 3J = 8 Hz), 114.6 (d, 2J = 21 Hz), 70.5, 60.1, 45.6, 45.2, 43.2, 34.6, 29.5, 25.5; + LRMS-EI (m/z): [M–Cl] calcd for C17H21FN4O: 316.38, found: 316.38.

3.3. Synthesis of 2,4-Disubstituted Pyrimidines 10a–j

3.3.1. General Procedure for Preparing Compound (R)-(+)-16 To a solution of 1-(3 or 4-fluorophenyl)ethanol (6.67 mmol) in n-hexane (22.2 mL) was added CAL-B (147 mg), vinyl acetate (3.34 mmol), and triethylamine (0.667 mmol). The reaction mixture was allowed to stir at the 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 acetate (1.73 mmol) in MeOH (3.45 mL) was added 1M NaOH (2.59 mmol). The reaction mixture was allowed to stir at the room temperature for 1 h. After completion of the reaction (monitored by TLC), it was quenched with distilled 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)-(+)-16. 1 (R)-1-(3-Fluorophenyl)ethan-1-ol ((R)-(+)-16a): Yield: 17%; H-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, 13 1 3 3 3H). C-NMR (100 MHz, CDCl3) δ 163.0 (d, J = 244 Hz), 148.5 (d, J = 6 Hz), 130.0 (d, J = 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)-(+)-16a: 26 [α]D + 43.7 ◦ (c 0.7, CHCl3). 1 (R)-1-(4-Fluorophenyl)ethan-1-ol ((R)-(+)-16b): Yield: 32%; H-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 1 4 3 2 MHz, CDCl3) δ 162.1 (d, J = 244 Hz), 141.6 (d, J = 3 Hz), 127.1 (d, J = 8 Hz), 115.2 (d, J = 21 Hz), 27 69.7, 25.3; optical rotation for (R)-(+)-16b:[α]D + 51.9 ◦ (c 0.5, CHCl3).

3.3.2. General Procedure for Preparing Compound 17 A solution of sodium tert-butoxide (1.43 mmol) in toluene (7.10 mL) was treated with primary or secondary alcohol 16 (0.713 mmol) dropwise at 0 ◦C. After 5 min, 2,4-dichloro-5-fluoropyrimidine (0.713 mmol) was added to the mixture. The reaction mixture was allowed to stir at the 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 Molecules 2019, 24, 3234 14 of 21

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 17. (R)-2-Chloro-5-fluoro-4-(1-(3-fluorophenyl)ethoxy)pyrimidine ((R)-17a): 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), 13 7.03–6.99 (m, 1H), 6.30 (q, J = 6.5 Hz, 1H), 1.71 (d, J = 6.6 Hz, 3H). C-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), 4 27 75.6 (d, J = 2 Hz), 22.2; optical rotation for (R)-6d:[α]D +178.3 ◦ (c 0.7, CHCl3). (R)-2-Chloro-5-fluoro-4-(1-(4-fluorophenyl)ethoxy)pyrimidine ((R)-17b): 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), 13 1 2 1.71 (d, J = 6.6 Hz, 3H). C-NMR (100 MHz, CDCl3) δ 162.6 (d, J = 245 Hz), 158.7 (d, J = 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 2 27 (d, J = 22 Hz), 75.9, 22.2; optical rotation for (R)-6e:[α]D +197.3 ◦ (c 0.8, CHCl3). 1 2-Chloro-5-fluoro-4-(3-fluorophenoxy)pyrimidine (19a): Yield: 51%; H-NMR (400MHz, CDCl3) δ 8.38 (d, J = 2.0 Hz, 1H), 7.45–7.39 (m, 1H), 7.06–7.01 (m, 2H), 6.99–6.96 (m, 1H). 13C-NMR (100 MHz, 1 2 4 3 CD3OD) δ 164.5 (d, J = 245 Hz), 159.8 (d, J = 11 Hz), 154.2 (d, J = 4 Hz), 153.7 (d, J = 11 Hz), 147.4 (d, 1J = 262 Hz), 147.4 (d, 2J = 20 Hz), 132.0 (d, 3J = 9 Hz), 118.5 (d, 4J = 3 Hz), 114.4 (d, 2J = 21 Hz), 110.5 (d, 2J = 25 Hz). 1 2-Chloro-5-fluoro-4-(4-fluorophenoxy)pyrimidine (19b): Yield: 52%; H-NMR (400MHz, CDCl3) 13 1 δ 8.36 (d, J = 2.0 Hz, 1H), 7.19–7.11 (m, 4H). C-NMR (100 MHz, CD3OD) δ 162.0 (d, J = 242 Hz), 160.2 (d, 2J = 11 Hz), 154.2 (d, 4J = 5 Hz), 148.7 (d, 4J = 2 Hz), 147.2 (d, 2J = 20 Hz), 146.10, 124.3 (d, 3J = 9 Hz), 117.4 (d, 2J = 24 Hz).

3.3.3. General Procedure for Preparing Compounds pre-10b–e and pre-10g–j To a solution of pyrimidine 17 (0.154 mmol) in toluene (0.300 mL) was added cyclic amine (0.231 mmol) and triethylamine (0.231 mmol). The reaction mixture was allowed to stir at 90 ◦C for 12 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 = 1:3) to afford carboxylate pre-10b–e and pre-10g–j. tert-Butyl 7-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane-2- 1 carboxylate (pre-10b): Yield: 92%; H-NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.94 (s, 1H), 7.31–7.26 (m, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.11 (d, J = 9.6 Hz, 1H) 6.95 (t, J = 8.3 Hz, 1H), 6.15–6.10 (m, 1H), 3.59–3.23 (m, 8H), 1.94–1.83 (m, 4H), 1.66 (d, J = 6.6 Hz, 3H), 1.45 (s, 9H). 13C-NMR (100 MHz, 1 2 3 CDCl3, a mixture of rotamers) δ 162.9 (d, J = 244 Hz), 157.1 (d, J = 11 Hz), 156.0, 154.6, 145.0 (d, J = 7 Hz), 143.4 (d, 2J = 20 Hz), 140.0 (d, 1J = 245 Hz), 130.0 (d, 3J = 8 Hz), 121.6, 114.6 (d, 2J = 21 Hz), 113.1 (d, 2J = 22 Hz), 79.4, 73.7, 55.9, 55.3, 54.7, 54.6, 48.6, 47.8, 46.0, 45.2, 35.3, 34.9, 34.5, 28.5, 22.7. tert-Butyl (3aR,6aS)-5-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl) hexahydropyrrolo 1 [3,4-c]pyrrole-2(1H)-carboxylate (pre-10c): Yield: 82%; H-NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.94 (d, J = 3.1 Hz, 1H), 7.32–7.28 (m, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.11 (dd, J = 2.0, 9.7 Hz, 1H), 6.95 (td, J = 2.0, 8.4 Hz, 1H), 6.14 (q, J = 6.6 Hz, 1H), 3.69–3.59 (m, 4H), 3.40–3.21 (m, 4H), 2.94 (bs, 2H), 1.66 13 1 (d, J = 6.6 Hz, 3H), 1.47 (s, 9H). C-NMR (100 MHz, CDCl3, a mixture of rotamers) δ 162.9 (d, J = 244 Hz), 157.1 (d, 2J = 11 Hz), 156.0 (d, 4J = 3 Hz), 154.5, 145.0 (d, 3J = 10 Hz), 143.4 (d, 2J = 20 Hz), 140.1 (d, 1J = 246 Hz), 130.0 (d, 3J = 8 Hz), 121.6, 114.6 (d, 2J = 21 Hz), 113.0 (d, 2J = 21 Hz), 79.5, 73.6, 50.8, 50.1, 49.8, 42.2, 41.2, 28.5, 22.7. tert-Butyl (R)-3-((5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)aminopyrrolidine-1- 1 carboxylate (pre-10d): Yield: 68%; H-NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.89 (s, 1H), 7.31–7.28 (m, 1H), 7.14 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 9.6 Hz, 1H) 6.94 (t, J = 7.9 Hz, 1H), 6.08 (d, J = 4.4 Hz, 1H), 5.12 (bs, 1H), 4.24–4.16 (m, 1H), 3.71–3.65 (m, 1H), 3.43–3.37 (m, 2H), 3.28–3.12 (m, 1H), 13 2.01–1.98 (m, 1 H), 1.65 (d, J = 6.6 Hz, 3H), 1.45 (s, 9H). C-NMR (100 MHz, CDCl3, a mixture of Molecules 2019, 24, 3234 15 of 21 rotamers) δ 162.9 (d, 1J = 245 Hz), 157.6 (d, 2J = 11 Hz), 157.3, 154.6, 144.7 (d, 3J = 6 Hz), 143.7 (d, 2J = 20 Hz), 140.6 (d, 1J = 248 Hz), 130.1 (d, 3J = 8 Hz), 121.3, 114.6 (d, 2J = 22 Hz), 112.8 (d, 2J = 22 Hz), 79.5, 73.9, 51.8, 51.7, 51.4, 51.0, 44.1, 43.7, 31.8, 31.1, 28.5, 22.8. tert-Butyl (S)-3-((5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)aminopyrrolidine-1- 1 carboxylate (pre-10e): Yield: 52%; H-NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.90 (d, J = 2.9 Hz, 1H), 7.33–7.26 (m, 1H), 7.15 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 9.6 Hz, 1H) 6.98–6.94 (m, 1H), 6.11 (q, J = 6.5 Hz, 1H), 5.12 (bs, 1H), 4.28 (bs, 1H), 3.47–3.42 (m, 3H), 3.21–3.04 (m, 1H), 2.20–2.12 (m, 1H), 1.86 13 (bs, 1H), 1.65 (d, J = 6.6 Hz, 3H), 1.45 (s, 9H). C-NMR (100 MHz, CDCl3, a mixture of rotamers) δ 162.9 (d, 1J = 245 Hz), 157.6 (d, 2J = 11 Hz), 157.2, 154.6, 143.6 (d, 2J = 20 Hz), 143.3 (d, 1J = 271 Hz), 139.4, 130.1 (d, 3J = 8 Hz), 121.4, 114.7 (d, 2J = 21 Hz), 112.8 (d, 2J = 22 Hz), 79.5, 73.8, 51.9, 51.6, 51.4, 51.0, 44.1, 43.8, 31.9, 31.1, 28.5, 22.8. tert-Butyl 7-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane-2- 1 carboxylate (pre-10g): Yield: 98%; H-NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.93 (s, 1H), 7.40–7.37 (m, 2H), 7.03–6.99 (m, 2H), 6.18–6.13 (m, 1H), 3.61–3.22 (m, 8H), 1.95–1.82 (m, 4H), 1.66 (d, J = 13 1 6.6 Hz, 3H), 1.45 (s, 9H). C-NMR (100 MHz, CDCl3, a mixture of rotamers) δ 162.2 (d, J = 244 Hz), 157.2 (d, 2J = 11 Hz), 156.0, 154.6, 143.3 (d, 2J = 20 Hz), 140.1 (d, 1J = 246 Hz), 138.0, 127.9 (d, 3J = 8 Hz), 115.3 (d, 2J = 21 Hz), 79.4, 73.6, 55.9, 55.4, 54.8, 54.6, 48.6, 47.8, 46.0, 45.2, 44.9, 35.3, 34.9, 34.5, 28.5, 22.8. tert-Butyl (3aR,6aS)-5-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)hexahydropyrrolo 1 [3,4-c]pyrrole-2(1H)-carboxylate (pre-10h): Yield: 93%; H-NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.92 (d, J = 3.1 Hz, 1H), 7.40–7.36 (m, 2H), 7.00 (t, J = 8.6 Hz, 2H), 6.15 (d, J = 5.7 Hz, 1H), 3.73–3.60 (m, 4H), 3.41–3.21 (m, 4H), 2.91 (bs, 2H), 1.64 (d, J = 6.6 Hz, 3H), 1.44 (s, 9H). 13C-NMR (100 1 2 4 MHz, CDCl3, a mixture of rotamers) δ 162.2 (d, J = 245 Hz), 157.2 (d, J = 11 Hz), 156.0 (d, J = 3 Hz), 154.5, 143.3 (d, 2J = 20 Hz), 140.1 (d, 1J = 245 Hz), 138.0, 127.8 (d, 3J = 8 Hz), 115.3 (d, 2J = 21 Hz), 79.5, 73.6, 50.8, 50.1, 49.8, 42.2, 41.2, 28.5, 22.8. tert-Butyl (R)-3-((5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)amino)pyrrolidine-1- 1 carboxylate (pre-10i): Yield: 52%; H-NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.88 (d, J = 2.0 Hz, 1H), 7.38–7.34 (m, 2H), 7.01 (t, J = 8.5 Hz, 2H), 6.14–6.09 (m, 1H), 5.13 (bs, 1H), 4.26–4.19 (m, 1H), 3.69–3.66 (m, 1H), 3.40 (bs, 2H), 3.29–3.16 (m, 1H), 2.09–2.01 (m, 1H), 1.74–1.73 (m, 1H), 1.64 (d, J = 6.6 13 1 Hz, 3H), 1.46 (s, 9H). C-NMR (100 MHz, CDCl3, a mixture of rotamers) δ 162.2 (d, J = 245 Hz), 157.7 (d, 2J = 11 Hz), 157.3, 154.6, 143.5 (d, 2J = 21 Hz), 140.6 (d, 1J = 246 Hz), 137.8 (d, 2J = 20 Hz), 127.6 (d, 3J = 12 Hz), 115.4 (d, 2J = 21 Hz), 79.5, 73.9, 51.8, 51.7, 51.5, 51.0, 44.1, 43.8, 31.8, 31.1, 28.5, 22.8. tert-Butyl (S)-3-((5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)aminopyrrolidine-1- 1 carboxylate (pre-10j): Yield: 44%; H-NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.89 (d, J = 3.0 Hz, 1H), 7.39–7.36 (m, 2H), 7.03 (t, J = 8.6 Hz, 2H), 6.14 (q, J = 6.5 Hz, 1H), 4.98 (bs, 1H), 4.31 (m, 1H), 3.62–3.44 (m, 3H), 3.24–3.06 (m, 1H), 2.28-2.13 (m, 1H), 1.86 (bs, 1H), 1.64 (d, J = 6.6 Hz, 3H), 1.45 (s, 13 1 2 9H). C-NMR (100 MHz, CDCl3, a mixture of rotamers) δ 162.3 (d, J = 244 Hz), 157.7 (d, J = 11 Hz), 157.2, 154.6, 143.5 (d, 2J = 19 Hz), 140.8 (d, 1J = 247 Hz), 137.7, 127.7, 130.1, 115.4 (d, 2J = 21 Hz), 79.5, 73.8, 52.0, 51.7, 51.5, 51.0, 44.1, 43.8, 31.9, 31.1, 29.8, 28.5, 22.8. tert-Butyl (R)-4-(5-fluoro-4-(3-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate 1 (pre-20a): Yield: 74%; H-NMR (400MHz, CDCl3) δ 8.13 (d, J = 2.7 Hz, 1H), 7.39–7.33 (m, 1H), 6.99–6.94 (m, 3H), 4.44 (m, 1H), 4.05–3.80 (m, 3H), 3.06–3.2.99 (m, 2H), 2.99–2.82 (m, 1H), 1.45 (s, 9H), 1.04 (d, J = 6.6 Hz, 3H). tert-Butyl (R)-4-(5-fluoro-4-(4-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate 1 (pre-20b): Yield: 61%; H-NMR (400MHz, CDCl3) δ 8.11 (d, J = 2.7 Hz, 1H), 7.15–7.12 (m, 2H), 7.11–7.06 (m, 2H), 4.50–4.30 (m, 1H), 4.03–3.80 (m, 3H), 3.04–2.97 (m, 2H), 2.97–2.81 (m, 1H), 1.45 (s, 9H), 1.03 (d, J = 6.6 Hz, 3H).

3.3.4. General Procedure for Preparing Compounds 10a and 10f To a solution of pyrimidine 17 (0.115 mmol) in toluene (0.250 mL) was added 1,4-diazepane (0.230 mmol) and triethylamine (0.230 mmol). The reaction mixture was allowed to stir at 90 ◦C for 12 h. Molecules 2019, 24, 3234 16 of 21

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 (DCM/MeOH = 10:1) to afford 2,4-disubstituted pyrimidine 10a and 10f. (R)-1-(5-fluoro-4-(1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-1,4-diazepane (10a): Yield: 68%; 1H-NMR (400 MHz, CD3OD) δ 8.06 (d, J = 3.2 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.15 (d, J = 9.8 Hz, 1H), 7.00 (td, J = 2.1, 8.5 Hz, 1H), 6.11 (q, J = 6.5 Hz, 1H), 3.90–3.77 (m, 4H), 3.26–3.03 (m, 4H), 2.07–1.94 (m, 2H), 1.66 13 1 2 (d, J = 6.6 Hz, 3H). C-NMR (100 MHz, CD3OD) δ 163.0 (d, J = 244 Hz), 157.7 (d, J = 11 Hz), 155.9, 145.5 (d, 3J = 7 Hz), 142.5 (d, 2J = 21 Hz), 140.3 (d, 1J = 246 Hz), 130.3 (d, 3J = 8 Hz), 121.1 (d, 4J = 3 Hz), 114.1 (d, 2J = 21 Hz), 112.0 (d, 2J = 22 Hz), 74.7, 45.5, 45.3, 45.0, 43.3, 25.3, 22.0. LRMS-EI (m/z): [M+H]+ calcd for C17H21F2N4O: 335.17, found: 335.10. (R)-1-(5-fluoro-4-(1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-1,4-diazepane (10f): Yield: 79%; 1H-NMR (400 MHz, CD3OD) δ 8.05 (d, J = 3.2 Hz, 1H), 7.45 (dd, J = 5.4, 8.4 Hz, 1H), 7.11–7.07 (m, 2H), 6.16 (q, J = 6.5 Hz, 1H), 4.10–3.72 (m, 4H), 3.42–3.15 (m, 4H), 2.21–1.98 (m, 2H), 1.66 (d, J = 6.5 Hz, 3H). 13C-NMR 1 2 2 (100 MHz, CD3OD) δ 162.3 (d, J = 243 Hz), 158.0 (d, J = 12 Hz), 155.60, 141.7 (d, J = 20 Hz), 140.3 (d, 1J = 246 Hz), 138.3 (d, 4J = 3 Hz), 127.4 (d, 3J = 8 Hz), 115.0 (d, 2J = 22 Hz), 75.0, 45.6, 45.4, 45.0, 43.4, + 25.3, 22.1. LRMS-EI (m/z): [M+H] calcd for C17H21F2N4O: 335.17, found: 335.10.

3.3.5. General Procedure for Preparing Compounds 10b–e and 10g–j

To a solution of carboxylate 17 (0.144 mmol) in CH3CN (1.45 mL) was added 4M HCl in dioxane (1.44 mmol) 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 compound 10. 2-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane (10b): Yield: 66%; 1 H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 8.00 (d, J = 3.4 Hz, 1H), 7.35–7.30 (m, 1H), 7.21 (d, J = 7.7 Hz, 1H), 7.13 (d, J = 9.8 Hz, 1H), 6.97 (td, J = 1.7, 8.4 Hz, 1 H), 6.23–6.16 (m, 1H), 3.59–3.37 13 (m, 6H), 3.30–3.23 (m, 2H), 2.11–1.98 (m, 4H), 1.64 (d, J = 6.6 Hz, 3H). C-NMR (100 MHz, CD3OD, a mixture of rotamers) δ 162.9 (d, 1J = 243 Hz), 158.3 (d, 2J = 12 Hz), 154.39, 144.7 (d, 3J = 9 Hz), 140.0 (d, 2J = 21 Hz), 139.8 (d, 1J = 246 Hz), 130.1 (d, 3J = 8 Hz), 121.5 (d, 4J = 3 Hz), 114.3 (d, 2J = 21 Hz), 112.5 (d, 2J = 22 Hz), 75.0, 74.9, 55.3, 55.5, 46.0, 44.8, 44.7, 34.3, 34.2, 33.8, 33.7, 21.5, 21.5. LRMS-EI (m/z): + [M+H] calcd for C19H23F2N4O: 361.18, found: 361.10. (3aR,6aS)-2-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)octahydropyrrolo[3,4-c]pyrrole 1 (10c): Yield: 92%; H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 8.26 (d, J = 4.4 Hz, 1H), 7.44–7.38 (m, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.24 (dd, J = 1.9, 9.7 Hz, 1H), 7.06 (td, J = 2.3, 8.4 Hz, 1H), 3.91 (bs, 1H), 3.80 (bs, 2H), 3.68–3.58 (m, 3H), 3.37–3.28 (m, 4H), 1.75 (d, J = 6.5 Hz, 3H). 13C-NMR (100 1 2 MHz, CD3OD, a mixture of rotamers) δ 162.9 (d, J = 244 Hz), 157.8 (d, J = 12 Hz), 152.12, 149.9, 143.9 (d, 3J = 7 Hz), 139.6 (d, 1J = 248 Hz), 135.6 (d, 2J = 26 Hz), 130.3 (d, 3J = 8 Hz), 121.7 (d, 4J = 3 Hz), 114.6 (d, 2J = 21 Hz), 112.6 (d, 2J = 22 Hz), 79.6, 50.9, 50.8, 50.7, 49.6, 49.4, 41.6, 41.5, 21.3. LRMS-EI (m/z): + [M+H] calcd for C18H21F2N4O: 347.17, found: 347.05. 5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)-N-((R)-pyrrolidin-3-yl)pyrimidin-2-amine (10d): Yield: 71%; 1 H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 7.97 (d, J = 3.1 Hz, 1H), 7.36–7.29 (m, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 9.8 Hz, 1H), 6.96 (td, J = 2.3, 8.5 Hz, 1H), 6.17 (q, J = 6.5 Hz, 1H), 4.38–4.33 (m, 1H), 3.49–3.30 (m, 1H), 3.26–3.25 (m, 3H), 2.30–2.14 (m, 1H), 2.09–1.93 (m, 1 H), 1.61 (d, J = 6.6 Hz, 13 1 2 3H). C-NMR (100 MHz, CD3OD, a mixture of rotamers) δ 163.0 (d, J = 243 Hz), 158.0 (d, J = 12 Hz), 156.9, 144.9 (d, 3J = 7 Hz), 142.2 (d, 2J = 17 Hz), 140.6 (d, 1J = 247 Hz), 130.2 (d, 3J = 8 Hz), 121.2 (d, 4J = 3 Hz), 114.1 (d, 2J = 21 Hz), 112.1 (d, 2J = 22 Hz), 74.1, 51.0, 50.3, 50.1, 44.1, 29.7, 29.5, 21.74, 21.66. + LRMS-EI (m/z): [M+H] calcd for C16H19F2N4O: 321.15, found: 321.05. Molecules 2019, 24, 3234 17 of 21

5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)-N-((S)-pyrrolidin-3-yl)pyrimidin-2-amine (10e): Yield: 42%; 1 H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 7.93–7.92 (m, 1H), 7.34–7.28 (m, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.09 (d, J = 9.9 Hz, 1H), 6.97–6.92 (m, 1H), 6.15 (q, J = 6.5 Hz, 1H), 4.34–4.29 (m, 1H), 3.49–3.38 (m, 2H), 3.35–3.25 (m, 2H), 2.98–2.11 (m, 1H), 2.10–1.89 (m, 1 H), 1.59 (d, J = 6.6 Hz, 3H). 13 1 2 C-NMR (100 MHz, CD3OD, a mixture of rotamers) δ 162.9 (d, J = 243 Hz), 157.7 (d, J = 11 Hz), 157.3 (d, 4J = 3 Hz), 145.0 (d, 3J = 7 Hz), 143.0 (d, 2J = 20 Hz), 140.7 (d, 1J = 246 Hz), 130.1 (d, 3J = 8 Hz), 121.3 (d, 4J = 3 Hz), 114.1 (d, 2J = 21 Hz), 112.1 (d, 2J = 22 Hz), 73.8, 51.0, 50.2, 44.1, 29.7, 29.6, 21.8, 21.7. + LRMS-EI (m/z): [M+H] calcd for C16H19F2N4O: 321.15, found: 321.05. 2-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane (10g): Yield: 58%; 1 H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 7.90 (d, J = 3.3 Hz, 1H), 7.44–7.40 (m, 2H), 7.03 (t, J = 8.8 Hz, 2H), 6.22–6.15 (m, 1H), 3.59–3.35 (m, 6H), 3.30–3.19 (m, 2H), 2.09–1.96 (m, 4H), 1.61 (d, J = 13 1 2 6.6 Hz, 3H). C-NMR (100 MHz, CD3OD, a mixture of rotamers) δ 162.3 (d, J = 243 Hz), 157.5 (d, J = 11 Hz), 155.9, 142.7 (d, 2J = 20 Hz), 140.0 (d, 1J = 245 Hz), 138.3 (d, 4J = 7 Hz), 127.8 (d, 3J = 8 Hz), 114.8 (d, 2J = 21 Hz), 74.03, 73.97, 55.2, 52.7, 45.7, 44.8, 34.4, 34.3, 33.9, 33.8, 21.7, 21.6. LRMS-EI (m/z): [M+H]+ calcd for C19H23F2N4O: 361.18, found: 361.10. (3aR,6aS)-2-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)octahydropyrrolo[3,4-c]pyrrole 1 (10h): Yield: 53%; H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 7.96 (d, J = 2.8 Hz, 1H), 7.27 (bs, 2H), 6.86 (t, J = 8.0 Hz, 1H), 6.11 (bs, 1H), 3.66–3.54 (m, 3H), 3.43–3.36 (m, 3H), 3.07–3.03 (m, 4H), 13 1 1.48 (d, J = 3.0 Hz, 3H). C-NMR (100 MHz, CD3OD, a mixture of rotamers) δ 162.8 (d, J = 244 Hz), 161.1 (d, 2J = 12 Hz), 150.0, 139.3 (d, 1J = 250 Hz), 136.4 (d, 4J = 3 Hz), 131.6 (d, 2J = 20 Hz), 128.4 (d, 3J = 8 Hz), 115.2 (d, 2J = 21 Hz), 78.2, 51.4, 51.2, 49.5, 41.7, 41.6, 21.2. LRMS-EI (m/z): [M+H]+ calcd for C18H21F2N4O: 347.17, found: 347.05. 5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)-N-((R)-pyrrolidin-3-yl)pyrimidin-2-amine (10i): Yield: 51%; 1H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 7.93 (d, J = 3.3 Hz, 1H), 7.49–7.45 (m, 2H), 7.10 (t, J = 8.7 Hz, 2H), 6.25–6.22 (m, 1H), 4.31–4.30 (m, 1H), 3.56–3.40 (m, 3H), 3.15–3.06 (m, 1H), 2.23–2.16 + (m, 1H), 1.94–1.90 (m, 1 H), 1.66 (d, J = 6.6 Hz, 3H). LRMS-EI (m/z): [M+H] calcd for C16H19F2N4O: 321.15, found: 321.00. 5-Fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)-N-((S)-pyrrolidin-3-yl)pyrimidin-2-amine (10j): Yield: 27%; 1 H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 7.92–7.91 (m, 1H), 7.42–7.39 (m, 2H), 7.06–7.01 (m, 2H), 6.18 (td, J = 4.8, 6.5 Hz, 1H), 4.39–4.30 (m, 1H), 3.50–3.39 (m, 2H), 3.36–3.28 (m, 2H), 2.39–2.14 13 (m, 1H), 2.10–1.90 (m, 1 H), 1.59 (d, J = 6.6 Hz, 3H). C-NMR (100 MHz, CD3OD, a mixture of rotamers) δ 162.3 (d, 1J = 243 Hz), 157.8 (d, 2J = 11 Hz), 157.3 (d, 4J = 3 Hz), 143.0 (d, 2J = 20 Hz), 140.8 (d, 1J = 233 Hz), 138.1, 127.7 (d, 3J = 8 Hz), 114.9 (d, 2J = 22 Hz), 73.9, 51.0, 50.4, 50.3, 44.2, 29.7, 29.6, 21.8, 21.7. + LRMS-EI (m/z): [M+H] calcd for C16H19F2N4O: 321.15, found: 321.05. (R)-4-(5-Fluoro-4-(3-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium hydrochloride (20a): Yield: 1 · 58%; H-NMR (400MHz, CDCl3) δ 8.13 (s, 1H), 7.37–7.31 (m, 1H), 7.00–6.93 (m, 3H), 4.39–4.38 (m, 1H), 4.07–4.03 (m, 1H), 2.98–2.86 (m, 3H), 2.80–2.77 (m, 1H), 2.70–2.62 (m, 1H), 1.12 (d, J = 6.8 Hz, 13 1 2 3 3H). C-NMR (100 MHz, CD3OD) δ 164.4 (d, J = 245 Hz), 158.7 (d, J = 11 Hz), 157.2 (d, J = 8 Hz), 154.4 (d, 3J = 11 Hz), 146.7 (d, 3J = 20 Hz), 141.4 (d, 1J = 248 Hz), 131.7 (d, 3J = 9 Hz), 118.8 (d, 4J = 3 Hz), 113.7 (d, 2J = 21 Hz), 110.6 (d, 2J = 24 Hz), 45.7, 44.3, 36.6, 13.5. LRMS-EI (m/z): [M-Cl]+ calcd for C15H17F2N4O: 306.32, found: 306.32. (R)-4-(5-Fluoro-4-(4-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium hydrochloride (20b): Yield: 1 · 65%; H-NMR (400MHz, CDCl3): δ 8.08 (d, J = 2.6 Hz, 1H), 7.13 (dd, J = 9.0, 4.5 Hz, 2H), 7.09–7.03 (m, 2H), 4.50–4.30 (s, 1H), 4.03–4.00 (m, 1H), 2.96–2.83 (m, 3H), 2.77–2.74 (m, 1H), 2.68–2.60 (m, 1H), 1.09 (d, 13 1 2 4 J = 6.6 Hz, 3H). C-NMR (100 MHz, CD3OD): δ 161.6 (d, J = 241 Hz), 159.2 (d, J = 11 Hz), 149.3 (d, J = 22 Hz), 146.4 (d, 2J = 20 Hz), 141.5 (d, 1J = 248 Hz), 124.5 (d, 3J = 8 Hz), 117.1 (d, 2J = 23 Hz), 48.0, + 45.6, 44.2, 36.5, 13.5. LRMS-EI (m/z): [M-Cl] calcd for C15H17F2N4O: 306.32, found: 306.32 Molecules 2019, 24, 3234 18 of 21

3.4. Molecular Docking Study

Discovery studio 2019 software were used for modeling study [34]. Crystal structures of 5-HT2B (PDB ID = 4IB4) and 5-HT2C (PDB ID= 6BQH) proteins were downloaded from protein data bank. Ligand 10a and 8 were prepared at 7.4 pH. Proteins and ligands were prepared and minimized using CHARm forcefield. Binding sites were created over co-crystal ligands binding position. Ligands were docked using CDOCKER scoring function. Twenty binding poses were generated for each ligands. Obtained docked poses were analyzed based on docking scores and binding site interactions. Images were rendered using PyMOL software (www.pymol.org).

3.5. In Vitro Assay

3.5.1. Serotonin Receptor Cell-Based Functional Assays

The synthesized compounds were tested in agonist mode with the 5-HT2C receptor assay using recombinant human HEK-293 cells serviced by either Eurofins Cerep or DGMIF (Daegu-Gyeongbuk Medical Innovation Foundation). All the experiments were duplicated. The detailed assay protocols refer to the literature procedure [35].

3.5.2. Serotonin Receptor Binding Affinity Assays Eleven dilutions (5 x 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 S1) was diluted to five times the assay concentration in standard binding buffer. Aliquots (50 µL) of the radioligand were dispensed into the wells of a 96-well plate containing 100 µL of standard binding buffer. Triplicate aliquots (50 µL) of the test and reference compound dilutions were then added. Finally, crude membrane fractions (50 µL) of cells (HEK293 or CHO) expressing human recombinant receptors were dispensed into each well. A total 250 µL of the reaction mixtures was incubated at room temperature and shielded from light for 1.5 h, then harvested by rapid filtration onto Whatman GF/B glass fiber filters presoaked with 0.3% polyethyleneimine, by using a 96-well Brandel harvester. Four rapid washes were performed with chilled standard binding buffer (500 µL) 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, San Diego, CA) and converted into Ki values. Each compound was tested in triplicate at least.

3.6. Drug-Like Properties

3.6.1. Plasma Stability

Human plasma in each culture tube, treated with test compound (10 µM), was incubated 37 ◦C for 0, 30, and 120 min, respectively. In a determined period of time, an internal standard solution of chlorpropamide in acetonitrile was added into each culture tube, which was shaken with a vortex mixer for 5 min and centrifuged for additional 5 min (14,000 rpm, 4 ◦C). The supernatant was then injected into thr LC-MS/MS system to analyze the plasma stability of compound 10a.

3.6.2. Microsomal Stability To human liver microsomes (0.5 mg/mL) were added 0.1 M phosphate buffer (pH 7.4) and tested compounds (1 µM). After incubation at 37 ◦C for 5 min, NADPH generation system solution was also Molecules 2019, 24, 3234 19 of 21

added and incubated at 37 ◦C for 30 min again. To terminate reaction, acetonitrile including internal standard (chlorpropamide) was added, and the solution was centrifuged for 5 min (14,000 rpm, 4 ◦C). The supernatant was then injected into the LC-MS/MS system to analyze the microsomal stability of compound 10a.

3.6.3. CYP Inhibition To human liver microsomes (0.25 mg/mL), 0.1 M phosphate buffer (pH 7.4), a cocktail of five probe substrates (Phenacetin 50 µM, Diclofenac 10 µM, S-mephenytoin 100 µM, Dextromethorphan 5 µM, and Midazolam 2.5 µM), and tested compounds were added at concentrations of 0 µM (as a control) and 10 µM. After incubation at 37 ◦C for 5 min, NADPH generation system solution was also added and incubated at 37 ◦C for 15 min again. To terminate the reaction, acetonitrile including internal standard (Terfenadine) was added, and the solution was centrifuged for 5 min (14,000 rpm, 4 ◦C). The supernatant was then injected into the LC-MS/MS system to simultaneously analyze the metabolites of the probe substrates and evaluate the % CYP inhibition of the tested compound 10a.

4. Conclusions

Thus, we synthesized two series of disubstituted pyrimidine derivatives as potential 5-HT2C selective agonists. Initially, a cell-based assay of 2,5-disubstituted pyrimidines revealed that most compounds did not have good agonistic effects towards the 5-HT2C receptor, although 2-piperazinyl-5-(3-fluorobenzyloxy)pyrimidine 9b showed effective biological activity, with 61% activation. In vitro evaluation of the second series of pyrimidines, possessing different cyclic amines, demonstrated that four compounds showed greater than 50% activation against 5-HT2C in the primary cell-based assay. In the secondary binding affinity assay, compounds 10a and 10f, with 1,4-diazepane at the 2-position of pyrimidine, were identified as the most potent 5-HT2C ligands, with excellent Ki values of 7.9 nM and 19.0 nM, respectively. Compound 10a showed high selectivity profiles for other 5-HT receptor subtypes, with a greater than 10-fold difference in potency. Additional in vitro stability experiments indicated that compound 10a has excellent plasma and microsomal stability, along with low CYP inhibition. Based on these results, 2,4-disubstituted pyrimidine 10a could be considered a potential lead compound as a 5-HT2C selective agonist for further application in chemical probes such as a 5-HT2C PET radiotracer.

Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/18/3234/s1, (NMR spectral data of compounds 9a–9r, 10a–10j, and 20a/b; Table S1: A list of 5-HT receptor radioligands and reference compounds for binding assay). Author Contributions: S.-J.M. initiated the project and designed the experiments; J.K., and Y.J.K. performed the experiments; S.-J.M. and J.K. analyzed the data; H.C. and H.J.K. contributed to analyze drug-like properties; A.M.L. and A.N.P. performed molecular docking studies; S.-J.M., J.K., and Y.J.K. wrote the paper. Acknowledgments: This work was financially supported by the Korea Health Industry Development Institute (KHIDI, HI16C1677, HI17C1037), the National Research Foundation of Korea (NRF-2019R1A2C1008186) and the research fund of Hanyang University (HY-2015-N). Binding affinity data were generously provided by the US National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (HHSN-271-2008-00025-C). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 9a–9r, 10a–10j, and 20a/b are available from the authors.

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