pyrimidine Derivatives as Potent HIV-1 Non-nucleosi - ACS Publications

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Structure-Based Optimization of Thiophene[3,2‑d]pyrimidine Derivatives as Potent HIV‑1 Non-nucleoside Reverse Transcriptase Inhibitors with Improved Potency against Resistance-Associated Variants Dongwei Kang,† Zengjun Fang,†,| Boshi Huang,† Xueyi Lu,† Heng Zhang,† Haoran Xu,† Zhipeng Huo,† Zhongxia Zhou,† Zhao Yu,† Qing Meng,† Gaochan Wu,† Xiao Ding,† Ye Tian,† Dirk Daelemans,§ Erik De Clercq,§ Christophe Pannecouque,*,§ Peng Zhan,*,† and Xinyong Liu*,† †

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong P.R. China § Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium | The Second Hospital of Shandong University, no. 247 Beiyuan Avenue, Jinan 250033, China S Supporting Information *

ABSTRACT: This work follows on from our initial discovery of a series of piperidine-substituted thiophene[3,2-d]pyrimidine HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) (J. Med. Chem. 2016, 59, 7991−8007). In the present study, we designed, synthesized, and biologically tested several series of new derivatives in order to investigate previously unexplored chemical space. Some of the synthesized compounds displayed single-digit nanomolar anti-HIV potencies against wild-type (WT) virus and a panel of NNRTIresistant mutant viruses in MT-4 cells. Compound 25a was exceptionally potent against the whole viral panel, affording 3-4-fold enhancement of in vitro antiviral potency against WT, L100I, K103N, Y181C, Y188L, E138K, and K103N+Y181C and 10-fold enhancement against F227L+V106A relative to the reference drug etravirine (ETV) in the same cellular assay. The structure− activity relationships, pharmacokinetics, acute toxicity, and cardiotoxicity were also examined. Overall, the results indicate that 25a is a promising new drug candidate for treatment of HIV-1 infection.

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INTRODUCTION Non-nucleoside reverse transcriptase inhibitors (NNRTIs) comprise an important class of antiretroviral drugs, and combinations of NNRTIs are widely used in highly active antiretroviral therapy (HAART) regimens to treat HIV infection, owing to their potent antiviral activity, high selectivity, favorable pharmacokinetics, and higher genetic barrier to the development of resistance.1−3 NNRTIs are structurally diverse, but mechanistically all of them interact with the allosteric site (also known as the non-nucleoside inhibitory binding pocket, NNIBP) of HIV-1 RT in a noncompetitive manner, inducing conformational changes of the catalytic domain that lead to inhibition of its DNA polymerase activity.4−6 Nevirapine (NVP), delavirdine (DLV), and efavirenz (EFV) are first-generation NNRTIs for AIDS therapy; they exhibit potent anti-HIV-1 activity, but their toxicities and low genetic barriers for viral resistance, especially single mutants K103N, Y181C, and double mutant K103N+Y181C, limited their clinical application.7,8 To address this major problem, etravirine (1, ETV) and rilpivirine (2, RPV), two representative compounds of the diarylpyrimidine (DAPY) family, were approved as second-generation NNRTIs by the © 2017 American Chemical Society

U.S. Food and Drug Administration (FDA) in 2008 and 2011, respectively.9,10 Both of them display nanomolar EC50 values against wild-type (WT) HIV-1 and a panel of clinically relevant HIV-1 mutant strains.11,12 However, 1 must be given twice daily and is not recommended as part of an initial treatment regimen. On the other hand, the guidelines recommend 2 as an alternative NNRTI for treatment initiation. However, 2 was shown to have suboptimal efficacy in patients with viral loads of 100000 copies/mL or CD4 cell counts below 200 copies/mL at baseline.13,14 In addition, although these compounds exhibited higher genetic barriers to emergence of drug resistance than first-generation NNRTIs, drug resistance still emerges in patients receiving second-generation NNRTIs regimens, and K103N and E138K are the mutant strains most frequently selected by ETV and RPV.6,15−18 Therefore, new NNRTIs that offer a combination of improved potency against these mutants, a distinct resistance profile, dosing convenience, and a favorable safety and tolerability profile would provide physicians with additional choices for effective treatment of HIV-1 patients. Received: March 9, 2017 Published: May 8, 2017 4424

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

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Figure 1. Chemical structures of next-generation NNRTI drugs and thiophene[3,2-d]pyrimidine lead 3 (K-5a2).

strains of HIV-1, being more potent than the approved drug ETV. A pharmacokinetic study of the most potent compound, 25a, in rats showed that it has favorable, drug-like pharmacokinetics. We also carried out a detailed SARs study of derivatives in all six series as well as examining acute toxicity and hERG activity. This work led to the identification of highly potent compounds with significantly improved drug resistance profiles. Among them, 25a proved to be particularly promising.

Previous research efforts in our lab led to the identification of a series of thiophene[3,2-d]pyrimidine derivatives with low nanomolar anti-HIV activity against HIV-1 WT and a panel of NNRTI-resistant single- and double-mutant strains.19 It was noteworthy that the antiviral efficacy of the most potent compound 3 (K-5a2) was 1.7−3-fold greater against WT and L100I (EC50 = 1.43 and 3.38 nM) and 5−7-fold greater against Y181C, Y188L, E138K, and F227L+V106A (EC50 = 3.25, 3.00, 2.91, and 4.25 nM, respectively) compared with ETV (Figure 1). However, 3 did not exhibit improved activity against the most common single mutant, K103N (EC50 = 2.91 nM). Moreover, 3 showed nearly 2-fold decreased potency (EC50 = 30.6 nM) for the most challenging variant strain, K103N +Y181C, which has double mutation in the allosteric pocket of the HIV-1 RT. Therefore, further optimization of 3 was initiated with the aim of obtaining improved potency against NNRTI-resistant viruses, especially K103N and K103N+Y181C. Our strategy was to explore every region of the structure of compound 3 (namely, the left wing, central ring, and the substituted piperidine ring of the right wing) and identify structure−activity relationships (SARs) that could be utilized to improve the potency against both K103N and K103N+Y181C while retaining the good potency against other mutants already manifested by compound 3 (Figure 2). Toward this end, six

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CHEMISTRY The synthetic protocols for the newly designed derivatives are outlined in Schemes 1−4.19 All derivatives were synthesized by well-established methods from commercially available 2,4-dichlorothiophene[3,2-d]pyrimidine (4) or 2,4-dichlorothiophene[2,3-d]pyrimidine (16) as a starting material. Treatment of 4 with 3,5-dimethyl-4-hydroxybenzonitrile and 2-substituted-4-(tert-butoxycarbonyl) aminopiperidine afforded the key intermediates 6a or 6b, which was directly deprotected with trifluoroacetic acid in dichloromethane to afford the corresponding analogues, 7a or 7b, respectively. Then, 7a or 7b was converted into 8a or 8b by nucleophilic substitution with 4-bromomethylbenzenesulfonamide. Subsequently, 8b was hydrolyzed in the presence of LiOH to give the corresponding acid 8c. The target compounds 11a−b and 14a−k were prepared via similar procedures from 7a−b, except that intermediate 5 was treated with N-(tert-butoxycarbonyl)-3aminopiperidine and tert-butyl(4-aminocyclohexyl)carbamate (Scheme 1). The oxidative product 15 was prepared by treating the lead compound 3 with m-CPBA in dichloromethane at ambient temperature (Scheme 2). The synthetic procedures to 20a−f were also similar to those used for 8a−b, starting from 2,4-dichlorothiophene[2,3-d]pyrimidine (16) (Scheme 3). Similarly, treatment of 4 with 3,5-dimethyl-4-hydroxybenzaldehyde gave 21. Subsequent reaction with diethyl cyanomethylphosphonate via Wittig−Horner reaction afforded cyanovinyl compound 22, which was treated with N-(tert-butoxycarbonyl)4-aminopiperidine, trifluoroacetic acid, and substituted benzyl chloride (or bromide) to give the target compounds 25a−w (Scheme 4). All novel derivatives were fully characterized by electrospray ionization mass spectrometry (HRMS), proton nuclear magnetic resonance (1H NMR) spectroscopy, and carbon nuclear magnetic resonance (13C NMR) spectroscopy. The purity of all compounds was >95% as determined by analytical HPLC (Table S1 in Supporting Information).

Figure 2. Strategy for further structure optimization of thiophene[3,2d]pyrimidine 3.

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new series (8, 11, 14, 15, 20, and 25) of thiophene[3,2d]pyrimidine and thiophene[2,3-d]pyrimidine derivatives were synthesized. Anti-HIV activity evaluation demonstrated that series 20 and series 25 showed potent inhibitory activity in the low nanomolar range against both WT and resistant mutant

RESULTS AND DISCUSSION Antiviral potency were evaluated in MT-4 cell cultures infected with WT HIV-1 strain (IIIB) as well as cells infected with a 4425

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

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Scheme 1. Synthesis of 8a−c, 11a−b, and 14a−ka

a Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, rt; (ii) 2-substituted-4-(tert-butoxycarbonyl)aminopiperidine or N-(tert-butoxycarbonyl)-3-aminopiperidine or tert-butyl(4-aminocyclohexyl)carbamate, DMF, K2CO3, reflux; (iii) TFA, DCM, rt; (iv) substituted benzyl bromide, DMF, K2CO3, rt; (v) LiOH, CH3OH, rt.

Scheme 2. Synthesis of 15a

a

(EFV), etravirine (ETV), and azidothymidine (AZT) were selected as control drugs, and the lead compound 3 was selected as the reference drug. The values of EC50 (anti-HIV potency), CC50 (cytotoxicity) as well as SI (selectivity index, CC50/EC50 ratio) of the synthesized compounds are summarized in Tables 1−6. First, we focused our attention on novel piperidinesubstituted thiophene[3,2-d]pyrimidine derivatives (8a−c and 11a−b) in which the 4-aminopiperidine of 3 is replaced by substituted 4-aminopiperidine and 3-aminopiperidine, respectively. Because we considered that a fluorine atom might interact favorably with backbone and side chain amide groups in the enzyme pocket, as previously observed in some other studies,20,21 we introduced a fluorine atom at the C2 position of the aminopiperidine. At the same time, −CO2C2H5 and −COOH were also introduced, anticipating that they might

Reagents and conditions: (i) m-CPBA, DCM, rt.

panel of NNRTI-resistant single- and double-mutant strains, such as L100I, K103N, Y181C, Y188L, E138K, F227L+V106A, and K103N+Y181C (RES056). Nevirapine (NVP), efavirenz Scheme 3. Synthesis of 20a−fa

a

Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, rt; (ii) 4-(tert-butoxycarbonyl)aminopiperidine, DMF, K2CO3, reflux; (iii) TFA, DCM, rt; (iv) substituted benzyl chloride (or bromide), DMF, K2CO3, rt. 4426

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

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Scheme 4. Synthesis of 25a−wa

develop additional interactions with Lys103 as hydrogenbonding donor or receptor groups. Analysis of the SARs demonstrated that the substituent group and the position of the N atom in the aminopiperidine significantly influenced antiHIV activity (Table 1 and Table 2). Among the three derivatives 8a−c, compound 8a (R = F) exhibited the most potent activity against WT HIV-1 (EC50 = 5.69 nM), being as potent as the reference drug ETV (EC50 = 4.07 nM), but its activity was about 3-fold lower than that of 3 (EC50 = 1.43 nM). Substituting the fluorine atom for −CO2C2H5 or −COOH significantly reduced the antiviral activity (8b, EC50 = 134 nM; 8c, EC50 = 723 nM). The order of potency of compounds with various substituents at the R-position in aminopiperidine was: H (3) > F > −CO2C2H5 > −COOH. Thus, a bulky substituent group appeared to have a negative effect on the potency, possibly because it would change the conformation of the piperidine ring and decrease the binding affinity with the NNIBP. Furthermore, 8a was a highly potent inhibitor of mutants Y188L (EC50 = 13.9 nM) and F227L+V106A (EC50 = 8.73 nM), showing greater potency than ETV (EC50 = 20.5 and 29.4 nM, respectively). In the case of the L100I and K103N mutants, 8a also provided single-digit nanomolar activity

a

Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzaldehyde, DMF, K2CO3, rt; (ii) (EtO)2P(O)CH2CN, t-BuOK, THF, 0 °C to rt; (iii) 4-(tert-butoxycarbonyl)aminopiperidine, DMF, K2CO3, reflux; (iv) TFA, DCM, rt; (v) substituted benzyl chloride (or bromide) or 4-picolyl chloride hydrochloride, DMF, K2CO3, rt.

Table 1. Structures and anti-HIV Activity of 8a−c, 11a−b, 14a−k, and 15

EC50 (nM)a compd 8a 8b 8c 11a 11b 14a 14b 14c 14d 14e 14f 14g 14h 14i 14j 14k 15 3d NVP EFV ETV AZT

1

R

4-SO2NH2 4-CONH2 4-SO2NH2 4-CONH2 3-CONH2 4-NO2 4-SO2CH3 H 4-Br 4-F 4-OH 3-CF3 4-CN

SIc b

IIIB (nM)

ROD (μM)

CC50 (μM)

5.69 ± 1.17 134 ± 29.3 723 ± 38.1 >1.17 × 103 3.22 × 103 ± 841 42.2 ± 5.50 121 ± 8.5 135 ± 36 100 ± 110 57.3 ± 14.2 230 ± 15 339 ± 224 553 ± 181 1.31 × 103 ± 224 174 ± 58 3.81 × 103 3.78 ± 1.25 1.43 ± 0.427 250 ± 65.6 5.03 ± 2.12 4.07 ± 0.149 5.81 ± 2.41

>5.15 >4.24 >27.6 >1.17 6.63 ± 0.135 >3.69 >5.92 >9.21 >54.4 >75.2 >2.97 >3.45 >2.76 >20.1 >707 >5.15 >40.4 ≥227

5.15 ± 1.53 4.24 ± 0.318 27.6 ± 2.18 1.17 ± 0.279 19.5 ± 4.04 3.69 ± 1.53 7.72 ± 5.86 9.21 ± 5.27 54.4 ± 46.4 75.2 ± 83.4 2.97 ± 0.478 3.45 ± 2.07 2.76 ± 0.491 20.1 ± 2.81 0.707 ± 0.411 5.15 ± 3.87 40.4 ± 10.7 >227 >0.015 >0.00633 >0.00459 >0.00748

IIIB 906 32 38 159101 >60 >1258 >1128 625

a

EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method. bCC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method. c SI: selectivity index, the ratio of CC50/EC50. dData were reported in ref 19. 4427

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

>3.69 × 10

827 ± 25.0

>1.17 × 103

6.81 × 103 ± 13.7

152 ± 4.24

1.64 × 103 ± 492

>1.17 × 103

5.27 × 103 ± 675

8c

11a

11b

4428

>3.45 × 103 >2.77 × 103 >2.01 × 10

754 ± 59.1

≥766

883 ± 2.83

4.60 × 10 ± 169

>707

>5.15 × 103

5.14 ± 0.803

2.91

983 ± 81.6

≥1.40 × 103

919 ± 70.8

>2.01 × 10

>707

6.42 × 103 ± 347

23.1 ± 4.74

3.38 ± 0.657

14f

14g

14h

14i

14j

14k

15

5.24 ± 0.265

3.75 ± 2.10

AZT

6.68 ± 1.35

15.7 ± 2.12

8.20 ± 1.03 6.34 ± 0.715

20.5 ± 2.92

514 ± 51.5

>1.50 × 104

3.00 ± 0.129

3.25 ± 0.477 >1.50 × 104

24.6 ± 3.65

≥7.85 × 103

>707

>2.01 × 10

7.30 ± 2.06

14.4 ± 2.27

5.19 ± 0.627

271 ± 4.78

2.91

23.1 ± 1.82

>5.15 × 103

>707

4.02 × 10 ± 56.8 3

≥1.31 × 103

4

>2.77 × 103

891 ± 17.4

135 ± 19.2

158 ± 19.6

935 ± 181

946 ± 244

3

857 ± 56.1

175 ± 43.2

5.32 × 103 ± 386

>1.17 × 103

3.34 × 103 ± 214

607 ± 25.0

17.4 ± 7.56

E138k

>3.45 × 103

>2.98 × 103

≥1.96 × 105

1.11 × 10 ± 3.65 × 10 4

4.59 × 103 ± 618

13.6 ± 1.64

5.68 × 103 ± 83.2

>707

4

4

5.91 × 103 ± 429

>3.69 × 10 ≥4.48 × 103

3

≥7.70 × 103

>1.17 × 103

2.97 × 103 ± 417

>4248

13.9 ± 4.16

Y188L

>3.69 × 10

4.79 ± 0.741

29.4 ± 7.79

348 ± 58.2

>1.50 × 104

4.25 ± 1.27

32.4 ± 2.31

>5.15 × 103

>707

>2.01 × 10 4

>2.77 × 103

>3.45 × 103

1.05 × 103 ± 298

1.13 × 105 ± 2.15 × 104

646 ± 108

≥5.26 × 103

≥4.16 × 103

3

5.00 × 103 ± 41.3

>1.17 × 103 >3.69 × 10

11.1 ± 5.16

17.0 ± 1.78

308 ± 164

>1.50 × 104

30.6 ± 12.5

189 ± 59.0

>5.15 × 103

>707

>2.01 × 104

>2.77 × 103

>3.45 × 103

>2.98 × 103

>7.52 × 104

≥5.44 × 104

5.91 × 103 ± 1.81 × 103

3.88 × 103 ± 1.72 × 103

3

>1.95 × 104

>1.17 × 103

>2.76 × 104

≥918

2.53 × 103 ± 274

37.2 ± 8.11

8.73 ± 0.549

RES056

1.27 × 103 ± 308

F227L+V106A

7860

2332 5446 >159101 >60 >1258 >1128 625

a

EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method. bCC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method. c SI: selectivity index, the ratio of CC50/EC50.

antiresistance potency (RF = 1.6 and 2.4) than 14a against E138K. The sulfone derivative (15) of 3 exhibited an EC50 value of 3.78 nM against WT HIV-1, which was superior to those of the reference drugs (NVP, EFV, ETV, and AZT). 15 was also effective against K103N (EC50 = 5.14 nM). Moreover, it turned out to be a potent inhibitor with EC50 values of 13.6−32.5 nM against the other mutant strains, except RES056. However, its activity toward the double mutant strain RES056 (EC50 = 190 nM) was reduced compared to that of ETV (EC50 = 17.0 nM). Considering that switching of the pharmaceutical core pyrimidine in two approved NNRTIs to thiophene[2,3-d]pyrimidine was well tolerated, we envisioned that further modifications on the biheterocyclic ring might lead to the discovery of new chemical entities (NCEs) with anti-HIV activity. Thus, the bioisosterism strategy was employed to design thiophene[2,3d]pyrimidine replacements for the thiophene[3,2-d]pyrimidine central ring, with the aim of improving the antiviral potency against HIV mutant strains, especially K103N+Y181C. Indeed, 20a−b, 20d, and 20f were potent inhibitors of WT HIV-1 with EC50 values from 1.97 to 5.26 nM (Table 3). Among them, 20a (EC50 = 1.97 nM) was more potent than all the control drugs, with comparable activity to 3 (EC50 = 1.43 nM). It also showed low cytotoxicity (CC50 = 24.1 μM). Replacement of the SO2NH2 group at the C4 position in the benzene ring with CONH2 yielded 20c, which showed sharply decreased potency (EC50 = 152 nM). Elaboration with a methyl formate group at the para-position of the phenyl ring yielded 20f with a potency of 5.26 nM, while ethyl formate at this position (20e) reduced the potency to 98.9 nM, suggesting that the antiviral activity is very sensitive to the length of the ester chain. However, 20e exhibited no cytotoxicity at the concentration of 230 μM. The order of potency of compounds with para-substitution at the right phenyl ring was as follows: para-SO2NH2 > paraSO2Me > meta-CONH2 > para-COOMe > para-COOEt > para-CONH2; this is consistent with our previously established SAR.19

(EC50 = 8.94 and 7.89 nM, respectively). However, it was only weakly effective against the K103N+Y181C double mutant (EC50 = 37.2 nM). Regarding fold resistance (RF, ratio of EC50 against mutant strain/EC50 against WT strain), 8a exhibited low RF values (RF = 1.4−6.5) toward these mutant HIV-1 strains, comparable to those of ETV (RF = 0.6−7.2) (see Table S2 in Supporting Information). Notably, replacing 4-aminopiperidines with 3-aminopiperidines resulted in sharply reduced antiviral activity against WT and mutant HIV-1 strains. Compound 11a completely lacked anti-HIV potency even at the CC50 (1.17 μM). On the basis of X-ray cocrystal information, 4-aminopiperidine could interact with Lys103 via a bound water molecule.22 Aiming to provide a novel back-up series, we decided to design and synthesize compounds that might interact directly with Lys103, and we examined how this interaction affects potency. To achieve direct access to Lys103, we further replaced the 4-aminopiperidine of 3 with cyclohexanediamine, obtaining compounds 14a−k. However, only 14a (R = −SO2NH2) and 14e (R = −SO2CH3) exhibited acceptable anti-HIV-1 (WT) activity (EC50 = 42.2 and 57.3 nM, respectively). Modification at position 3 or 4 of the benzene ring (R = −CONH2, NO2, F, Br, H, OH, CN, and CF3) considerably decreased the activity toward WT HIV-1 (EC50 = 100−3817 nM), which was consistent with established structure−activity relationships (SAR). In general, compounds that exhibited potent activity against WT HIV-1 strain also displayed potent activity against these mutant HIV-1 strains. Compared to the lead 3, 14a displayed sharply reduced antiviral activity against WT and mutant HIV-1 strains, concomitantly with greatly increased cytotoxicity (CC50 = 3.69 μM). Also, 14a was inactive toward L100I, Y181C, Y188L, F227L+V106A, and K103N+Y181C, although it exhibited moderate activity (EC50 = 152 and 175 nM) and low RF values (RF = 3.6 and 4.2) toward K103N and E138K, respectively. In particular, 14d and 14e bearing NO2 and −SO2CH3 substituents showed more potent activity (EC50 = 158 and 135 nM) and higher 4429

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry >675

>270

EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method. SI: selectivity index, the ratio of CC50/EC50.

b a

>1563

156 >318 >224 >291

>1429 >1120 >1180 >1026

>1952 >853

>1994 11.1 ± 5.16

17.0 ± 1.78 29.4 ± 7.79

4.79 ± 0.741 7.30 ± 2.06

14.4 ± 2.27 20.5 ± 2.92

6.34 ± 0.715 6.68 ± 1.35 5.24 ± 0.265

15.7 ± 2.12 2.35 ± 0.668 5.38 ± 2.06

3.75 ± 2.10

ETV

AZT

X1

>21 >18

X1 >55

>1220 >12

X1 X1

>772 >79

>3 >7

>55 308 ± 164 348 ± 58.2 5.19 ± 0.627 514 ± 51.5 8.20 ± 1.03 80.1 ± 2.68 115 ± 8.28 EFV

>1.50 × 104 >1.50 × 104 271 ± 4.78 >1.50 × 104 >1.50 × 104 2.10 × 103 ± 196 5.39 × 103 ± 3.73 × 103

2.91 3.38 ± 0.657

NVP

19

>159101 >67385 >78125 >70028 >75758 >78125 >53533 30.6 ± 12.5 4.25 ± 1.27 2.91 3.00 ± 0.129

>6

3

3.25 ± 0.477

109 805 181 316 982 214 1.53 × 104 ± 583 263 ± 88.4 35.6 ± 3.75 158 ± 2.81 29.1 ± 9.91 134 ± 80.9 20f

90.7 ± 21.1

>10 >1934

>63 >463 >124 >207

>5488 >5302 >2867 >5612

>625 >160

>4634 4.69 × 10 ± 1.72 × 10 25.2 ± 27.5 8.69 ± 0.01 17.0 ± 10.9

369 ± 49.6

9.19 ± 0.89 8.88 ± 0.841 10.5 ± 5.91

1.44 × 103 ± 254

20d

20e

438 ± 71.7 529 ± 86.9

3

3

>4.93 × 103 805 ± 24.8 >4935

38.7 ± 7.81

20c

250 ± 31.7

8.68 ± 0.735

8.85 ± 0.412 7.60 ± 0.695

7.26 9.60 ± 1.73

7.63 ± 1.54

7.85 ± 7.90 20b

7.64 ± 0.68 3.28 ± 1.15

1.64 ± 0.070

4.44 ± 2.37 20a

5.96 ± 0.761

RES056 F227L+V106A E138K Y188L Y181C K103N L100I compd

EC50 (nM)a

Table 4. Anti-HIV-1 Activity of Compounds 20a−f against HIV-1 Mutant Strains 4430

1.11 × 103 ± 69.1 1.85 × 103 ± 610 498 ± 248 3.68 × 103 ± 1.73 × 103 4.09 × 104 ± 3.75 × 104

28

3 SO2Me > CN ≈ F > NO2 ≈ CH3 > COOEt > Cl ≈ Br. Change of the substituent position did not greatly influence antiviral activity. None of these compounds showed activity against HIV-2 ROD in MT-4 cells. As for mutant HIV-1 strain L100I, 25a−d, 25r,s, and 25u,v provided single-digit nanomolar activity, and 25a−c (EC50 = 1.34, 2.97, and 2.97 nM, respectively) were more active than ETV (EC50 = 5.38 nM) and AZT (EC50 = 3.75 nM). Furthermore, 25a−b had higher antiresistance potency (RF = 1.1 and 1.3) than ETV (RF = 1.3) and the lead 3 (RF = 2.4). Against K103N, 25a was 2.4-fold more potent than ETV (EC50 = 0.958 vs 2.35 nM, respectively). Replacement of the para-SO2NH2 with para- and meta-CO2NH2 resulted in reduced antiviral activity (25c, EC50 = 2.97 nM; 25d, EC50 = 2.97 nM), but these compounds still displayed comparable activity to ETV. Modification at this position with other substituent groups resulted in reduced activity (EC50 = 4.43− 29.9 nM) toward K103N, but all the derivatives gave low RF values (0.8−2.4) with respect to WT HIV-1. In addition, 25a−c and 25v inhibited Y181C, Y188L, and E138K, with up to 3.7-fold higher potency than ETV; again, 25a was the most effective (3.1, 3.7, and 3.0 times more potent than ETV, respectively).

F227L K103N Y181C Y188L E138k +V106A RES056

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DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

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Table 5. Structures and Anti-HIV Activity of 25a−w

EC50a compd

R

25a 25b 25c 25d 25e 25f 25g 25h 25i 25j 25k 25l 25m 25n 25o 25p 25q 25r 25s 25t 25u 25v 25w 3 NVP EFV ETV AZT

4-SO2NH2 4-SO2CH3 4-CONH2 3-CONH2 4-COOC2H5 4-F 3-F 2-F 4-Br 3-Br 2-Br 4-Cl 3-Cl 2-Cl 4-NO2 2-CH3 4-CH3 4-CN 3-CN 2-CN pyridine-4-yl pyridine-3-yl pyridine-2-yl

SIc

IIIB (nM)

ROD (μM)

CC50 (μM)b

IIIB

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

>2.30 >1.80 ≥1.78 >3.53 >3.61 >2.58 >4.76 >2.48 >6.79 >7.95 >5.61 >3.98 >4.88 >4.93 >3.52 >2.83 >2.91 >2.68 >2.83 >2.28 >4.78 >3.86 >3.85 ≥227

2.30 ± 0.468 1.804 ± 0.461 ≥3.15 3.53 ± 0.223 ≥3.61 2.58 ± 0.223 4.76 ± 2.50 2.48 ± 0.302 6.79 ± 4.02 7.95 ± 3.86 5.61 ± 2.32 3.98 ± 0.999 4.88 ± 1.57 4.93 ± 2.33 3.52 ± 0.418 2.83 ± 0.706 2.91 ± 0.468 2.67 ± 0.310 2.82 ± 0.490 2.28 ± 0.569 4.78 ± 0.971 3.86 ± 0.682 3.85 ± 0.667 >227 >0.0150 >0.00633 >0.00459 >0.00748

1882 775 ≥2504 1084 ≥247 441 489 392 240 308 140 156 188 179 456 180 326 509 639 236 1429 1199 1146 >159101 >60 >1258 >1128 625

1.22 2.32 1.26 3.26 14.6 5.86 9.73 6.34 28.3 25.8 40.2 25.6 25.9 27.4 7.73 15.7 8.91 5.26 4.43 9.66 3.34 3.22 3.36 1.43 250 5.03 4.07 5.81

0.26 0.876 0.118 2.40 9.46 0.716 6.46 0.565 8.76 3.45 8.02 0.935 2.00 3.07 0.890 5.48 3.33 0.544 0.965 4.25 1.05 2.56 1.85 0.427 65.6 2.12 0.149 2.41

a

EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method. bCC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method. c SI: selectivity index, the ratio of CC50/EC50.

EC50 = 5.50 nM) and 10-fold (F227L+V106A, EC50 = 2.70 nM) more potent than the reference drug ETV in the same cellular assay. More importantly, 25a potently inhibited the most common NNRTI-resistant mutants K103N and K103N+Y181C, being 2.6- and 3.1-fold more potent than ETV. Compared to the lead 3 (RF = 2.0−21), replacement of the cyano group with cyano vinyl seems to have notably improved the RF values (RF = 0.8−4.5), especially for the double-mutant K103N+Y181C (3, RF = 21; 25a, RF = 4.5). Some representative compounds were performed an enzymatic assay against WT HIV-1 RT enzymes to validate the binding target of these newly designed derivatives, and the results are shown in Table 7. The result indicated that compound 14a showed inferior affinity with an IC50 of 4.02 μM compared to ETV (IC50 = 1.00 μM), which was consistent with its less potent antiviral activities. Compounds 20a, 25a, and 25b showed excellent affinity to HIV-1 RT (IC50 = 0.42, 0.16, and 0.89 μM, respectively), being superior to that of ETV. The reasonable results demonstrated that the newly synthesized representative derivatives showed high affinity to WT HIV-1

In the case of the double-mutant strain F227L+V106A, 25a turned out to be a 2.70 nM inhibitor, being 159- and 10.8-fold more potent than EFV (EC50 = 348 nM) and ETV (EC50 = 29.4 nM) and 1.5-fold more potent than AZT (EC50 = 4.79 nM). Moreover, 25b−d, 25r,s, and 25u−w also inhibited double-mutant strain F227L+V106A (EC50 = 4.67−26.7 nM) more potently than did ETV. The antiviral activities of the novel compounds in this study against double-mutant strain K103N+Y181C are noteworthy. 25a−c and 25v showed significantly improved antiviral activity (EC50 = 5.50, 7.93, 7.62, and 12.6 nM, respectively) and similar fold resistance values (RF = 3.4−6.0) compared to ETV (EC50 = 17.0 nM, RF = 4.2). Thus, we had successfully overcome the weak activity of the lead compound 3 toward double-mutant strain K103N+Y181C. Among all the novel synthesized compounds, 25a showed exceptional antiviral activity against the whole viral panel, being about 3−4-fold (WT, EC50 = 1.22 nM; L100I, EC50 = 1.34 nM; K103N, EC50 = 0.958 nM; Y181C, EC50 = 5.00 nM; Y188L, EC50 = 5.45 nM; E138K, EC50 = 4.74 nM; K103N+Y181C, 4431

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

4432

2.91

5.39 × 103 ± 3.73 × 103

80.1 ± 2.68

2.35 ± 0.668

5.24 ± 0.265

3.38 ± 0.657

2.10 × 103 ± 196

115 ± 8.28

5.38 ± 2.06

3.75 ± 2.10

3

NVP

EFV

ETV 6.68 ± 1.35

15.7 ± 2.12

8.20 ± 1.03

>1.50 × 104

3.25 ± 0.477

9.23 ± 2.28

6.06 ± 0.798

5.79 ± 0.157

34.6 ± 1.08

26.9

85.7 ± 24.8

30.7 ± 2.09

133 ± 3.07

71.1 ± 7.08

77.7 ± 60.8

123 ± 2.34

128 ± 8.39

127 ± 6.41

29.8 ± 3.85

34.5 ± 4.82

27.2 ± 7.16

32.9 ± 1.24

6.34 ± 0.715

20.5 ± 2.92

514 ± 51.5

>1.50 × 104

3.00 ± 0.129

41.7 ± 8.55

10.7 ± 2.12

32.2 ± 8.55

124 ± 71.7

51.0 ± 25.1

33.2 ± 5.70

138 ± 21.5

174 ± 7.22

117 ± 9.81

207 ± 64.6

170 ± 24.4

177 ± 3.47

150 ± 7.03

165 ± 10.6

287 ± 96.2

139 ± 11.5

159 ± 3.85

155 ± 48.0

163 ± 17.3

27.4 ± 2.10

8.14 ± 0.565

7.20 ± 1.69 6.28 ± 0.03

7.93 ± 1.06

4.68 ± 0.111

5.45 ± 0.197

Y188L

7.30 ± 2.06

14.4 ± 2.27

5.19 ± 0.627

271 ± 4.78

2.91

7.46 ± 0.584

6.59 ± 0.06

8.53 ± 0.869

43.2 ± 17.9

27.9 ± 0.408

29.6 ± 1.63

32.3 ± 1.52

128 ± 4.44

40.8 ± 5.62

158 ± 9.48

131 ± 15.6

152 ± 9.88

145 ± 9.00

129 ± 1.23

154 ± 5.92

33.5 ± 2.75

31.6 ± 0.413

34.9 ± 3.16

37.4 ± 1.12

6.41 ± 0.07

8.48 ± 1.09

5.62 ± 0.308

4.74 ± 0.160

E138k

4.79 ± 0.741

29.4 ± 7.79

348 ± 58.2

>1.50 × 104

4.25 ± 1.27

26.7 ± 3.56

5.82 ± 0.2

14.8 ± 1.99

37.8 ± 2.71

26.3 ± 9.51

22.7 ± 6.76

129 ± 1.94

61.4 ± 43.8

106 ± 39.2

72.3 ± 5.61

68.3 ± 20.7

108 ± 18.7

89.5 ± 4.81

93.6 ± 9.99

161 ± 33.5

40.6 ± 7.30

56.9 ± 16.6

103 ± 73.4

85.2 ± 48.9

7.89 ± 1.18

10.8 ± 1.68

4.67 ± 0.395

2.70 ± 1.74

F227L +V106A

11.1 ± 5.16

17.0 ± 1.78

308 ± 164

>1.50 × 104

30.6 ± 12.5

122 ± 20.6

12.5 ± 5.28

29.4 ± 3.03

319 ± 241

115 ± 12.4

59.7 ± 25.7

146 ± 16.1

829 ± 73.1

111 ± 16.4

165 ± 24.1

148 ± 7.47

195 ± 50.4

351 ± 65.5

216 ± 42.0

154 ± 27.2

177 ± 29.9

126 ± 11.2

125 ± 16.0

165 ± 17.7

43.8 ± 11.0

7.62 ± 3.18

7.93 ± 3.05

5.50 ± 0.811

RES056

>1994

>853

>55

>7

>67385

210

866

752

151

352

364

91

60

134

162

163

93

117

279

117

32

216

181

≥134

689

≥1063

607

1723

L100I

>1429

>1952

>79

>3

>78125

724

872

859

130

470

494

258

114

365

169

164

159

124

269

227

361

226

405

≥273

1189

≥1063

400

2411

K103N

>1120

>291

>772

X1

>70028

418

637

825

66

105

86

98

33

115

37

69

51

45

62

53

83

138

95

≥110

563

≥439

385

461

Y181C

b

>1180

>224

>12

X1

>75758

92

358

148

18

55

81

21

16

30

24

29

22

37

48

24

18

30

17

≥22

129

≥388

227

424

Y188L

SIb

>1026

>318

>1220

>55

>78125

517

587

560

53

101

91

90

22

86

31

37

26

39

61

44

74

150

74

>1563

156

>18

X1

>53533

144

664

322

60

107

118

22

46

33

68

72

37

63

85

42

61

84

25

448 ≥42

551

≥291

386

854

F227L +V106A

≥96

≥372

321

487

E138k

>675

>270

>21

X1

>7348

32

308

162

7

24

45

20

3

32

30

33

20

16

37

44

14

38

20

≥22

81

≥414

228

419

RES056

EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method. SI: selectivity index, the ratio of CC50/EC50.

a

AZT

5.33 ± 0.926

18.4 ± 10.7

25w

31.9 ± 4.02

25q

5.56 ± 0.03

11.2 ± 8.10

47.2 ± 1.80

25p

4.43 ± 0.741

24.8 ± 0.694

26.3 ± 9.03

25o

4.46 ± 2.03

9.66 ± 5.78

30.4 ± 1.06

25n

6.35 ± 1.78

29.1 ± 0.802

30.0 ± 0.53

25m

25v

29.8 ± 2.13

42.8 ± 11.6

25l

25u

25.0 ± 0.134

47.8 ± 0.86

25k

17.5 ± 9.46

45.1 ± 12.3

28.5 ± 1.85

25j

15.1 ± 5.57

29.5 ± 0.617

58.2 ± 15.2

25i

25t

31.0 ± 1.22

29.9 ± 0.900

30.4 ± 5.64

25h

5.42 ± 0.03

6.88 ± 0.427

22.0 ± 11.8

25g

6.02 ± 0.313

21.1 ± 7.34

14.3 ± 9.52

25f

7.36 ± 1.41

6.38 ± 0.565

26.8 ± 12.8

25e

8.04 ± 0.612

2.97

13.2 ± 4.46

5.12 ± 1.31

25d

25r

2.97

2.97

25c

25s

29.7 ± 2.36

4.51 ± 0.04

2.97 ± 2.65

25b

5.00 ± 0.111

0.958 ± 0.07

1.34 ± 0.496

25a

Y181C

K103N

L100I

compd

EC50 (nM)a

Table 6. Anti-HIV-1 Activity of Compounds 25a−w against HIV-1 Mutant Strains

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

conducted with SurflexeDock SYBYL-X 2.0 software. PyMOL was used to visualize the results. The docking protocol is described in the computational section. In our previous study, we reported the binding mode of 3 with HIV-1 WT RT. As shown in Figure 3 and Figure 4, all the selected compounds adopt a horseshoe-like conformation, as is typically found for most NNRTIs. The binding modes were similar to that of 3. Especially, the most potent compound 25a exhibited a series of well-known interactions: (i) the position of the 4-cyanovinyl-2,6-dimethylphenyl moiety is stabilized by π-stacking interactions with aromatic side chains of Tyr181, Tyr188, Phe227, and Trp229, (ii) the thiophene ring of 25a effectively occupies the NNIBP entrance channel, and the sulfur atom develops an additional electrostatic interaction with Val179, (iii) the N atom of the thiophene[3,2-d]pyrimidine and NH linker connecting the central pyridine ring and the right ring forms double hydrogen bonding with the main-chain backbone of Lys101, which is important for maintaining effective antiviral activity, (iv) the secondary nitrogen atom of piperidine develops water-mediated hydrogen bonds with

Table 7. Inhibitory Activity of Compounds 14a, 20a, 25a, and 25b against WT HIV-1 RT compd IC50 (μM)

a

14a

20a

25a

25b

ETVb

4.02

0.42

0.16

0.89

1.00

a

IC50: inhibitory concentration of test compound required to inhibit biotin deoxyuridine triphosphate (biotin-dUTP) incorporation into WT HIV-1 RT by 50%. bThe data were obtained from the same laboratory with the same method.23

RT and prove the viewpoint that they are acted as classical NNRTIs.

■

MOLECULAR MODELING STUDIES In an attempt to rationalize the SAR results of the newly synthesized thienopyrimidine derivatives and to understand their key interactions with the NNIBP of HIV-1 RT, molecular docking studies of representative compounds 8a, 8b, 11a, 14a, 25a, 25e, and 25t to the crystal structure of HIV-1 RT extracted from the complex with DAPY (PDB 3M8Q22) were

Figure 3. Predicted binding modes of 8a, 8b, 11a, and 14a with the HIV-1 WT RT crystal structure (PDB 3M8Q). Hydrogen bonds between inhibitors and amino acid residues are indicated with dashed lines (red). Ligand carbon atoms are shown in cyan, and protein carbon atoms in green, yellow, and magenta. Nonpolar hydrogen atoms are not shown for clarity.

Figure 4. Predicted binding modes of 25a, 25e, and 25r with the HIV-1 WT RT crystal structure (PDB 3M8Q). 4433

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

Lys103, and (v) the piperidine-linked benzenesulfonamide structure is directed to the protein−solvent interface, and the sulfonyl group and amino group of the sulfonamide can develop double hydrogen-bonding interactions with hydrogen of Val106 and carbonyl of Lys104 as hydrogen bond receptor and donor, respectively. Interestingly, the peripheral sulfonamide group is ubiquitous among structurally diverse NNRTIs, including diarylpyrimidine,24,25 benzophenone,26 indolylarylsulfones,27 azolylthioacetanilide,28−30 etc. Thus, it appears that the sulfonamide can be used as a basic fragment for the generation of libraries of “privileged scaffolds” that would be capable of providing high-quality NNRTI hits. Of particular note is that the cyanovinyl group of 25a is positioned in a cylindrical tunnel formed by Phe227 and Trp22931 and develops additional interactions with the hydrophobic tunnel. Because Phe227 and Trp229 are highly conserved, these interactions are expected to be retained despite mutations in the binding pocket, and this may be the reason why 25a retains high potency against a variety of mutant strains compared to 3. Compared with 25a, 25r forms only one hydrogen-bonding interaction with Val106 while 25e lacks this hydrogen-bonding ability (Figure 4), providing a rational explanation of the reduced activity of 25e and 25r and suggesting an important role of hydrogen-bonding interactions at the protein−solvent interface. Furthermore, the results of docking of 8a, 8b, 11a, and 14a with the WT RT allosteric pocket indicate that the conformation of the piperidine significantly influences the watermediated hydrogen bonds with Lys103, which are believed to

be important contributors to the binding affinity of RT inhibitors with the enzyme. As shown in Figure 3, introducing a substituent group into the piperidine (8a and 8b) can alter the conformation, causing a shift of the secondary nitrogen atom of piperidine and loss of the water-mediated hydrogen bonds with Lys103. Similarly, replacement of piperidylamine with cyclohexanediamine (14a) also resulted in loss of the watermediated hydrogen bonds. Moreover, 8b and 11a lack critical hydrogen-bonding interactions with Lys101, which may account for their sharply reduced potency toward WT and mutant strains.

■

IN VIVO PHARMACOKINETICS STUDY AND SAFETY ASSESSMENT The in vivo pharmacokinetic profile of compound 25a was examined in Sprague−Dawley rats (Table 8 and Figure 5). When administered at 24 mg/kg orally, compound 25a was rapidly absorbed with a Tmax of 1.7 h, a favorable half-life of 3.93 h, and a mean residence time (MRT) of 5.54 h. The Cmax of 25a was 0.552 μg/mL (552 nM), which is more than 100 times the EC50 value of anti HIV-1 activity in vitro (Table 7). It exhibited a moderate oral bioavailability of 16.2% in this experiment. A single-dose toxicity test of compound 25a was carried out in Kunming mice. After intragastric administration of 25a at a dose of 2000 mg/kg, no death occurred and there was no abnormity of body weight increase over the subsequent 2 weeks. All these results support the potential of 25a as a new, highly potent, orally bioavailable NNRTI drug candidate.

■

ASSESSMENT OF HERG ACTIVITY In vitro manual patch-clamp electrophysiology was used to determine the activity against the hERG potassium channel in order to estimate the potential risk for cardiotoxicity. Terfenadine was used as a reference drug. Compound 25a showed an IC50 of 0.186 μM against the potassium channel; thus, its inhibitory activity is lower than that of terfenadine (IC50 = 0.022 μM), suggesting that cardiotoxicity is not likely to be a major risk for 25a (Figure 6).

Table 8. Pharmacokinetic Profile of 25aa parameter

unit

pob

ivc

AUC(0−t) AUC(0−∞) AUMC(0−t) AUMC(0−∞) MRT(0−t) MRT(0−∞) VRT(0−t) VRT(0−∞) t1/2z Tmax CLz/F Vz/F Cmax F

μg/L·h μg/L·h

2316.8 ± 520.2 2692.2 ± 872.1 8755.2 ± 2972.5 15978.8 ± 11014.0 3.71 ± 0.53 5.54 ± 1.87 8.11 ± 1.44 35.67 ± 27.1 3.93 ± 1.54 1.7 ± 0.67 0.008 ± 0.002 0.044 ± 0.019 552.9 ± 88.9 16.19

1367.3 ± 97.9 1386.0 ± 100.6 2153.9 ± 209.2 2340.2 ± 236.0 1.57 ± 0.04 1.69 ± 0.05 2.78 ± 0.07 3.72 ± 0.14 1.34 ± 0.03

h h h2 h2 h h L/h/kg L/kg μg/L %

■

CONCLUSION We previously reported 3 as a potent inhibitor of various mutant HIV-1 viruses, but it showed weaker potency than ETV toward the most common single-mutant K103N and doublemutant K103N+Y181C. The aim of this work was to overcome this weakness through structural modification studies, focusing on detailed optimization of the cyan group on the left wing, the central thiophene[3,2-d]pyrimidine ring, and the piperidylamine moiety of the right wing. All the changes in the right wing, including introduction of substituents at 4-aminopiperidine

0.0145 ± 0.002 0.0275 ± 0.002 2217.1 ± 148.6

PK parameters (mean ± SD, n = 5). bDosed orally at 24 mg/kg. Dosed intravenously at 2 mg/kg.

a c

Figure 5. Plasma 25a concentration−time profiles in rats following oral administration (24 mg·kg−1) and intravenous administration (2 mg·kg−1). 4434

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

chromatography was performed on columns packed with silica gel (200−300 mesh), purchased from Qingdao Haiyang Chemical Company. Solvents were purified and dried by means of standard methods when necessary. Organic solutions were dried over anhydrous sodium sulfate and concentrated with a rotary evaporator under reduced pressure. Other reagents were obtained commercially and were used without further purification. Sample purity was analyzed on a Shimadzu SPD-20A/20AV HPLC system with a Inertsil ODS-SP, 5 μm C18 column (150 mm × 4.6 mm). HPLC conditions: methanol/ water with 0.1% formic acid 80:20; flow rate, 1.0 mL/min; UV detection, from 210 to 400 nm; temperature, ambient; injection volume, 20 μL. Purity of all tested compounds was >95%. 4-((4-((4-(4-Cyano2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)amino)piperidin-1yl)methyl)benzenesulfonamide (3) and 4-((2-chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (5) were prepared as previously reported.19 4-((2-((3-Fluoropiperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4yl)oxy)-3,5-dimethylbenzonitrile (7a). A solution of 5 (1.0 g, 3.17 mmol), tert-butyl 4-amino-3-fluoropiperidine-1-carboxylate (0.83 g, 3.80 mmol), and anhydrous K2CO3 (0.87 g, 6.33 mmol) in 15 mL of DMF was heated at 120 °C under magnetic stirring for 12 h. Then the solution was cooled to ambient temperature and 50 mL of ice−water was added to it. Stirring was continued for another 15 min, and the resulting precipitate was collected by filtration and dried to give crude 6a, which was used directly in the next step without further purification. To a solution of 6a (1.26 g, 2.53 mmol) in dichloromethane (DCM) (5 mL) was added trifluoroacetic acid (TFA) (2.22 mL, 30 mmol) at ambient temperature, and the solution was stirred for 3−5 h (monitored by TLC). Then the reaction solution was alkalized to pH 9 with saturated sodium bicarbonate solution and washed with saturated salt water (20 mL). The aqueous phase was extracted with DCM (3 × 5 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give 7a as a white solid. Yield: 73%, mp 123−126 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.49 (d, 1H, J = 7.3 Hz, C6-thienopyrimidineH), 8.21 (d, 1H, J = 5.4 Hz, C7-thienopyrimidine-H), 7.73 (s, 2H, C3,C5-Ph-H), 4.79 (d, 1H, J = 6.5 Hz, NH), 3.73−3.76 (m, 1H), 2.75− 2.78 (m, 2H), 2.09 (s, 6H), 1.93−1.99 (m, 2H), 1.34−1.67 (m, 4H). HRMS m/z C20H20FN5OS: calcd 397.1373, found 398.1454 [M + H]+. HPLC purity: 98.52%. Ethyl 4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)amino) piperidine-3-carboxylate (7b). The synthetic method was similar to that described for 7a, except that the starting material 5 (1.0 g, 3.17 mmol) was reacted with 1-(tert-butyl) 3-ethyl 4-aminopiperidine-1,3-dicarboxylate (1.03 g, 3.80 mmol). White solid, 68% yield, mp 154−156 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.47 (d, J = 7.1 Hz, 1H, C6-thienopyrimidine-H), 8.19 (d, 1H, J = 5.7 Hz, C7-thienopyrimidine-H), 7.72 (d, J = 8.1 Hz, 2H, C3,C5-Ph-H), 4.67 (s, 1H, NH), 4.26 (q, J = 6.7 Hz, 2H, CO2CH2), 3.71 (s, 1H), 2.71− 2.73 (m, 2H), 2.09 (s, 6H), 2.01−2.04 (m, 2H), 1.51−1.65 (m, 3H), 1.30 (t, J = 7.1 Hz, 3H, CH2CH3). HRMS m/z C23H25N5O3S: calcd 451.1678, found 452.1923 [M + H]+. HPLC purity: 98.71%. General Procedure for the Preparation of Final Compounds 8a,b, 11a,b, 14a−k, 20a−f, and 25a−w. Compound 7a (or 10, 13, 19, or 24) was dissolved in anhydrous DMF (10 mL) in the presence of anhydrous K2CO3 (1.2 equal), followed by addition of the appropriate substituted benzyl chloride (bromide) (1.1 equiv). The reaction mixture was stirred at ambient temperature for 3−8 h (monitored by TLC), and then the solvent was removed under reduced pressure. The residue was taken up in water (20 mL) and extracted with ethyl acetate (3 × 10 mL). The organic phase was washed with saturated sodium chloride (2 × 10 mL), dried over anhydrous Na2SO4, filtered, and purified by flash column chromatography. The product was recrystallized from ethyl acetate/petroleum ether to afford the target compounds 8a,b, 11a,b, 14a−k, 20a−f, and 25a−w. 4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin2-yl)amino)-3-fluoropiperidin-1-yl)methyl)benzenesulfonamide (8a). Recrystallized from EA/PE as a white solid, 69% yield, mp 236− 238 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.21 (d, J = 5.3 Hz, 1H,

Figure 6. Activity of 25a and terfenadine against hERG potassium channel in HEK293 cells.

(8a−c) and replacement of 4-aminopiperidine with 3-aminopiperidine (11a,b) or cyclohexanediamine (14a−k), had negative effects on the antiviral activity. Although the oxidation product (15) of the lead 3 and the thiophene[2,3-d]pyrimidine derivatives (20a−f) exhibited high potency against WT and mutant HIV-1 strains, they showed decreased activity compared to 3. On the other hand, 25a−w exhibited significant inhibitory activity toward WT HIV-1 in MT-4 cells (EC50 < 50 nM), and 25a and 25c proved to be single-figure nanomolar inhibitors. In addition, 25a showed picomolar-level inhibition of K103N. Notably, 25a was superior to all the reference drugs against double-mutant strains F227L+V106A and K103N+Y181C. Molecular docking experiments indicated that double hydrogen bonding with the main-chain backbone of Lys101, watermediated hydrogen bonds with Lys103. and double hydrogenbonding interactions with Val106 and Lys104 play roles in the binding of these compounds with RT. These findings are in accordance with the in vitro experimental data. Overall, 25a proved to be exceptionally potent, with EC50 values of 1.22 nM (WT), 1.34 nM (L100I), 0.958 nM (K103N), 5.00 nM (Y181C), 5.45 nM (Y188L), 4.74 nM (E138K), 2.70 nM (F227L+V106A), and 5.50 nM (RES056) in MT-4 cells; indeed, it was more potent than ETV against all of these NNRTI-resistant strains. Compound 25a also displayed favorable in vivo pharmacokinetic properties and acceptable oral bioavailability in rats. Furthermore, it exhibited weaker inhibitory activity than terfenadine against the hERG channel. On the basis of these excellent in vitro and in vivo results, we consider that 25a is a promising new drug candidate for the treatment of HIV-1 and further profiling is in progress.

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EXPERIMENTAL SECTION

Chemistry. All melting points were determined on a micromelting point apparatus (RY-1G, Tianjin TianGuang Optical Instruments) and are uncorrected. Proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) spectra were recorded in DMSO-d6 or CDCl3 on a Bruker AV-400 spectrometer with tetramethylsilane (TMS) as the internal standard. Coupling constants are given in hertz, and chemical shifts are reported in δ values (ppm) from TMS; signals are abbreviated as s (singlet), d (doublet), t (triplet), q (quarter), and m (multiplet). A G1313A Standard LC autosampler (Agilent) was used to collect samples for measurement of mass spectra. The temperature of the reaction mixture was monitored with a mercury thermometer. All reactions were routinely monitored by thin layer chromatography (TLC) on silica gel GF254 for TLC (Merck), and spots were visualized with iodine vapor or by irradiation with UV light (λ = 254 nm). After completion of each reaction, the mixture was brought to ambient temperature via air-jet cooling. Flash column 4435

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

109.3, 62.4, 52.3, 31.4, 16.2. HRMS m/z C28H28N6O2S: calcd 512.1994, found 513.2139 [M + H]+. HPLC purity: 98.32%. 4-((2-((4-Aminocyclohexyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (13). The synthetic method was similar to that described for 7a except that the starting material 5 (1.0 g, 3.17 mmol) was reacted with tert-butyl (4-aminocyclohexyl)carbamate (0.82 g, 3.80 mmol). White solid, 56% yield, mp 185−190 °C. 1 H NMR (400 MHz, CDCl3): δ 7.72 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.36 (s, 2H, C3,C5-Ph-H), 7.13 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 4.78 (s, 1H, NH), 3.06 (q, J = 7.3 Hz, 1H), 2.80−2.96 (m, 3H), 2.09 (s, 6H), 1.95−1.97 (m, 4H), 1.07−1.35 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 164.2, 161.7, 158.9, 152.4, 132.0, 131.3, 122.2, 117.6, 116.8, 113.9, 108.2, 59.3, 48.9, 44.7, 35.5, 29.4, 28.1, 15.3. HRMS m/z C21H23N5OS: calcd 393.1623, found 394.1740 [M + H]+. 4-(((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin2-yl)amino)cyclohexyl)amino)methyl)benzenesulfonamide (14a). Recrystallized from EA/PE as a white solid, 78% yield, mp 131− 133 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.19 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.76 (d, J = 8.0 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.52 (d, J = 8.1 Hz, 2H, C2,C6-Ph′-H), 7.27−7.29 (m, 3H), 6.75 (s, 1H, pyrimidine-NH), 4.36 (s, 1H, cyclohexane-NH), 3.79 (s, 2H, N-CH2), 3.24−3.39 (m, 2H), 2.11 (s, 6H), 1.74−1.91 (m, 4H), 1.12−1.20 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 170.8, 160.6, 142.7, 133.2, 132.9, 128.6, 125.9, 119.0, 109.0, 60.2, 56.5, 55.5, 49.9, 32.0, 31.0, 21.2, 19.0, 16.2, 14.5. HRMS m/z C28H30N6O3S2: calcd 562.1681, found 563.1935 [M + H]+. HPLC purity: 99.25%. 4-(((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin2-yl)amino)cyclohexyl)amino)methyl)benzamide (14b). Recrystallized from EA/PE as a white solid, 75% yield, mp 115−118 °C. 1 H NMR (400 MHz, CDCl3): δ 8.17 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.76 (d, J = 8.3 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.29−7.35 (m, 4H), 7.27 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.77 (s, 1H, pyrimidine-NH), 4.36 (s, 1H, cyclohexane-NH), 3.79 (s, 2H, N-CH2), 3.27−3.35 (m, 2H), 2.09 (s, 6H), 1.96−2.08 (m, 4H), 1.19−1.35 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 165.4, 160.1, 141.9, 133.2, 132.4, 127.7, 125.9, 118.6, 109.0, 60.1, 56.7, 55.5, 48.6, 32.2, 31.0, 21.2, 19.0, 16.2, 14.5. HRMS m/z C29H30N6O2S: calcd 526.2151, found 527.2281 [M + H]+. HPLC purity: 97.67%. 3-(((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin2-yl)amino)cyclohexyl)amino)methyl)benzamide (14c). Recrystallized from EA/PE as a white solid, 51% yield, mp 193−196 °C. 1 H NMR (400 MHz, CDCl3): δ 8.17 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.75−7.78 (m, 2H), 7.72 (s, 2H, C3,C5-Ph″H), 7.51−7.53 (m, 2H), 7.27 (s, 2H), 7.27 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.77 (s, 1H, pyrimidine-NH), 4.39 (s, 1H, cyclohexane-NH), 3.79 (s, 2H, N-CH2), 3.21−3.31 (m, 2H), 2.11 (s, 6H), 1.85−2.01 (m, 4H), 1.21−1.35 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 165.1, 160.6, 142.3, 133.7, 131.5, 128.1, 124.6, 119.2, 109.7, 60.0, 57.3, 55.5, 49.0, 32.7, 31.0, 21.6, 18.3, 16.7, 14.5. HRMS m/z C29H30N6O2S: calcd 526.2151, found 527.2299 [M + H]+. HPLC purity: 96.39%. 3,5-Dimethyl-4-((2-((4-((4-nitrobenzyl)amino)cyclohexyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (14d). Recrystallized from EA/PE as a white solid, 48% yield, mp 214−217 °C. 1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 9.2 Hz, 2H, C3,C5-Ph′-H), 7.79 (d, J = 5.4, 1H, C6-thienopyrimidine-H), 7.53 (d, J = 8.6 Hz, 1H, C2,C6-Ph′-H), 7.43 (s, 1H, C3,C5-Ph″-H), 7.21 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.87 (s, 1H, pyrimidine-NH), 4.73 (s, 1H, cyclohexane-NH), 3.92 (s, 2H, N-CH2), 2.54−2.66 (m, 2H), 2.17 (s, 6H), 1.94−2.05 (m, 4H), 1.12−1.42 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 165.5, 160.3, 153.3, 147.0, 134.9, 133.1, 132.2, 128.6, 123.6, 123.3, 118.7, 109.5, 60.4, 55.8, 50.3, 31.8, 31.4, 28.4, 27.9, 16.4, 14.2. HRMS m/z C28H28N6O3S: calcd 528.1944, found 529.2076 [M + H]+. HPLC purity: 97.25%. 3,5-Dimethyl-4-((2-((4-((4-(methylsulfonyl)benzyl)amino)cyclohexyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (14e). Recrystallized from EA/PE as a white solid, 61% yield, mp 131− 134 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.4 Hz, 1H,

C6-thienopyrimidine-H), 7.80 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.73 (s, 2H, C3,C5-Ph″-H), 7.45 (d, J = 8.0 Hz, 2H, C2,C6-Ph′-H), 7.31 (s, 2H, SO2NH2), 7.26 (s, 1H, C7-thienopyrimidine-H), 6.98 (s, 1H, NH), 3.68 (s, 1H), 3.56 (s, 2H, N-CH2), 2.72−2.76 (m, 2H), 2.11 (s, 6H), 1.97−2.06 (m, 2H), 1.59−1.73 (m, 3H). 13C NMR (100 MHz, DMSO-d6): δ 175.0, 169.6, 162.4, 160.6, 153.3, 143.2, 142.8, 138.4, 135.8, 133.2, 133.0, 129.4, 126.2, 123.8, 119.1, 109.5, 61.1, 60.5, 49.8, 30.9, 16.7, 14.2. HRMS m/z C27H27FN6O3S2: calcd 566.1570, found 567.1371 [M + H]+. HPLC purity: 97.36%. Ethyl4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)amino)-1-(4-sulfamoylbenzyl)piperidine-3-carboxylate (8b). Recrystallized from EA/PE as a white solid, 58% yield, mp 212−215 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.20 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.80 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.47 (d, J = 8.0 Hz, 2H, C2,C6-Ph′-H), 7.32 (s, 2H, SO2NH2), 7.27 (s, 1H, C7-thienopyrimidine-H), 6.99 (s, 1H, NH), 4.28 (q, J = 6.9 Hz, 2H, CO2CH2), 3.73 (s, 1H), 3.48 (s, 2H, N-CH2), 2.72 (s, 2H), 2.11 (s, 6H), 1.95−2.04 (m, 2H), 1.54−1.77 (m, 3H), 1.32 (t, J = 7.1 Hz, 3H, CH2CH3). 13C NMR (100 MHz, DMSO-d6): δ 172.5, 165.4, 162.4, 160.6, 153.3, 143.2, 142.8, 138.4, 133.1, 133.0, 129.5, 129.4, 126.1, 123.7, 119.0, 109.1, 61.3, 60.1, 49.9, 30.9, 16.1, 14.2. HRMS m/z C30H32N6O5S2: calcd 620.1876, found 621.2023 [M + H]+. HPLC purity: 97.32%. 4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2yl)amino)-1-(4-sulfamoylbenzyl)piperidine-3-carboxylic Acid (8c). Recrystallized from EA as a white solid, 79% yield, mp 198−200 °C. 1 H NMR (400 MHz, DMSO-d6): δ 8.19 (d, J = 4.9 Hz, 1H, C6thienopyrimidine-H), 7.78 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.47 (d, J = 8.1 Hz, 2H, C2,C6-Ph′-H), 7.28 (s, 2H, SO2NH2), 7.24 (s, 1H, C7-thienopyrimidine-H), 6.97 (s, 1H, NH), 3.72 (s, 1H), 3.48 (s, 2H, N-CH2), 2.72−2.74 (m, 2H), 2.11 (s, 6H), 1.97−2.05 (m, 2H), 1.51−1.69 (m, 3H). 13C NMR (100 MHz, DMSO-d6): δ 175.5, 161.0, 153.6, 143.4, 143.0, 135.2, 133.2, 132.8, 129.6, 126.0, 122.2, 119.0, 109.0, 62.1, 56.5, 52.6, 25.7, 16.2. HRMS m/z C28H28N6O5S2: calcd 592.1563, found 593.1646 [M + H]+. HPLC purity: 96.36%. 3,5-Dimethyl-4-((2-(piperidin-3-ylamino)thieno[3,2-d]pyrimidin4-yl)oxy)benzonitrile (10). The synthetic method was similar to that described for 7a except that the starting material 5 (1.0 g, 3.17 mmol) was reacted with tert-butyl 3-aminopiperidine-1-carboxylate (0.76 g, 3.80 mmol). White solid, 72% yield, mp 123−125 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.47 (d, J = 7.1 Hz, 1H, C6-thienopyrimidine-H), 8.15 (d, 1H, J = 5.9 Hz, C7-thienopyrimidine-H), 7.72 (d, J = 8.1 Hz, 2H, C3,C5-Ph-H), 4.96 (s, 1H, NH), 3.71 (s, 1H), 2.71−2.73 (m, 2H), 2.09 (s, 6H), 1.95−2.03 (m, 2H), 1.38−1.62 (m, 4H). HRMS m/z C20H21N5OS: calcd 379.14673, found 380.1587 [M + H]+. HPLC purity: 99.12%. 4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (11a). Recrystallized from EA/PE as a white solid, 73% yield, mp 214−216 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.75 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.43 (d, J = 8.1 Hz, 2H, C2,C6-Ph′-H), 7.27 (s, 2H, SO2NH2), 7.23 (s, 1H, C7-thienopyrimidine-H), 6.86 (s, 1H, NH), 3.7−3.73 (m, 1H), 3.48 (s, 2H, N-CH2), 2.71−2.72 (m, 1H), 2.10 (s, 6H), 1.92−2.01 (m, 3H), 1.76−1.78 (s, 2H), 1.41−1.43 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 165.7, 161.9, 160.7, 153.1, 143.2, 143.7, 136.6, 133.1, 132.7, 129.3, 125.7, 123.2, 123.4, 118.5, 108.7, 61.4, 52.2, 31.1, 16.8. HRMS m/z C27H28N6O3S2: calcd 548.1664, found 549.2087 [M + H]+. HPLC purity: 99.35%. 4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin2-yl)amino)piperidin-1-yl)methyl)benzamide (11b). Recrystallized from EA/PE as a white solid, 65% yield, mp 206−209 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.21 (d, J = 5.2 Hz, 1H, C6-thienopyrimidineH), 7.90 (d, J = 8.0 Hz, 2H, C3,C5-Ph′-H), 7.73 (s, 2H, C3,C5-Ph″-H), 7.42 (d, J = 8.1 Hz, 2H, C2,C6-Ph′-H), 7.32 (s, 2H, CONH2), 7.28 (s, 1H, C7-thienopyrimidine-H), 6.91 (s, 1H, NH), 3.72 (s, 1H), 3.46 (s, 2H, N-CH2), 2.71−2.73 (s, 1H), 2.10 (s, 6H), 1.71−1.93 (m, 3H), 1.42−1.63 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 168.2, 162.1, 160.4, 150.9, 139.7, 136.6, 134.2, 133.1, 132.3, 128.7, 126.8, 119.8, 4436

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

C6-thienopyrimidine-H), 7.74 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.52 (d, J = 8.1 Hz, 2H, C2,C6-Ph′-H), 7.27 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.71 (s, 1H, pyrimidineNH), 4.35 (s, 1H, cyclohexane-NH), 3.74 (s, 2H, N-CH2), 3.21−3.27 (m, 2H), 3.06 (s, 3H, SO2CH3), 2.11 (s, 6H), 1.73−1.98 (m, 4H), 1.17−1.26 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 165.2, 162.6, 159.9, 147.7, 139.1, 133.1, 132.2, 128.2, 127.4, 127.2, 118.7, 63.8, 44.5, 36.6, 31.4, 29.6, 28.8, 19.1, 16.4. HRMS m/z C29H31N5O3S2: calcd 561.1868, found 562.2016 [M + H]+. HPLC purity: 99.02%. 4-((2-((4-(Benzylamino)cyclohexyl)amino)thieno[3,2-d]pyrimidin4-yl)oxy)-3,5-dimethylbenzonitrile (14f). Recrystallized from EA/PE as a white solid, 65% yield, mp 153−156 °C. 1H NMR (400 MHz, CDCl3): δ 7.79 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.49− 7.53 (m, 2H), 7.45 (s, 2H, C3,C5-Ph″-H), 7.32−7.42 (m, 3H), 7.23 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.73 (s, 1H, pyrimidine-NH), 4.37 (s, 1H, cyclohexane-NH), 3.74 (s, 2H, N-CH2), 3.23−3.25 (m, 2H), 2.12 (s, 6H), 1.77−1.92 (m, 5H), 1.25−1.27 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 165.4, 162.6, 160.0, 153.3, 134.9, 133.1, 132.4, 130.0, 129.2, 129.1, 123.4, 118.7, 109.5, 60.4, 55.5, 48.7, 30.7, 27.9, 16.4, 14.2. HRMS m/z C28H29N5OS: calcd 483.2093, found 484.2135 [M + H]+. HPLC purity: 97.39%. 4-((2-((4-((4-Bromobenzyl)amino)cyclohexyl)amino)thieno[3,2d]pyrimidin-4-yl) oxy)-3,5-dimethylbenzonitrile (14g). Recrystallized from EA/PE as a white solid, 62% yield, mp 193−197 °C. 1H NMR (400 MHz, CDCl3): δ 7.79 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.44−7.46 (m, 2H), 7.43 (s, 2H, C3,C5-Ph″-H), 7.26 (d, J = 7.0 Hz, 2H, C2,C6-Ph′-H), 7.21 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.75 (s, 1H, pyrimidine-NH), 4.37 (s, 1H, cyclohexane-NH), 3.79 (s, 2H, N-CH2), 3.21−3.25 (m, 2H), 2.17 (s, 6H), 1.95−2.05 (m, 4H), 1.21− 1.42 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 165.4, 162.7, 160.2, 153.3, 134.9, 133.1, 132.2, 131.6, 130.3, 123.4, 118.7, 109.5, 106.3, 55.5, 49.9, 31.3, 31.1, 27.8, 16.4. HRMS m/z C28H28BrN5OS: calcd 561.1198, found 564.1245 [M + 3]+. HPLC purity: 98.61%. 4-((2-((4-((4-Fluorobenzyl)amino)cyclohexyl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (14h). Recrystallized from EA/PE as a white solid, 59% yield, mp 177−180 °C. 1H NMR (400 MHz, CDCl3): δ 7.81 (d, J = 5.4 Hz, 1H, C6-thienopyrimidineH), 7.48−7.50 (m, 2H), 7.47 (s, 2H, C3,C5-Ph″-H), 7.26−7.28 (m, 2H), 7.23 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.77 (s, 1H, pyrimidine-NH), 4.35 (s, 1H, cyclohexane-NH), 3.75 (s, 2H, N-CH2), 3.23−3.26 (m, 2H), 2.17 (s, 6H), 1.93−2.07 (m, 5H), 1.25−1.38 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 165.5, 161.9, 160.7, 154.1, 134.0, 133.7, 132.1, 131.3, 130.3 (d, J = 8.3 Hz), 123.8, 118.4, 115.2 (d, J = 20.7 Hz), 109.9, 106.3, 55.8, 49.1, 31.3, 30.8, 27.8, 16.2. HRMS m/z C28H28FN5OS: calcd 501.1999, found 502.2145 [M + H]+. HPLC purity: 97.36%. 4-((2-((4-((4-Hydroxybenzyl)amino)cyclohexyl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (14i). Recrystallized from EA/PE as a white solid, 53% yield, mp 192−195 °C. 1H NMR (400 MHz, CDCl3): δ 9.08 (s, 2H), 7.81 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.43 (s, 2H, C3,C5-Ph″-H), 7.31−7.35 (m, 2H), 7.23 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.94−6.97 (m, 2H), 6.73 (s, 1H, pyrimidine-NH), 4.34 (s, 1H, cyclohexane-NH), 3.77 (s, 2H, N-CH2), 3.21−3.25 (m, 2H), 2.17 (s, 6H), 1.91−2.02 (m, 3H), 1.27−1.45 (m, 5H). 13C NMR (100 MHz, CDCl3): δ 165.6, 161.3, 160.7, 154.1, 134.0, 133.8, 132.9, 132.3, 131.3, 130.3, 123.8, 117.8, 109.2, 107.0, 55.8, 49.4, 31.7, 30.8, 27.3, 16.1. HRMS m/z C28H29N5O2S: calcd 499.2042, found 500.2213 [M + H]+. HPLC purity: 97.19%. 3,5-Dimethyl-4-((2-((4-((4-(trifluoromethyl)benzyl)amino)cyclohexyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (14j). Recrystallized from EA/PE as a white solid, 58% yield, mp 166− 173 °C. 1H NMR (400 MHz, CDCl3): δ 7.79 (d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.51−7.61 (m, 4H), 7.45−7.46 (m, 1H), 7.43 (s, 2H, C3,C5-Ph″-H), 7.21 (d, J = 5.3 Hz, 1H, C7-thienopyrimidine-H), 6.74 (s, 1H, pyrimidine-NH), 4.73 (s, 1H, cyclohexane-NH), 3.88 (s, 2H, N-CH2), 3.21−3.25 (m, 2H), 2.18 (s, 6H), 1.98−2.09 (m, 4H), 1.15−1.43 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 165.4, 162.7, 160.3, 153.3, 141.0, 134.9, 133.1, 132.2, 131.5, 130.8, 130.5, 128.9, 124.8, 123.9, 123.4, 118.7, 109.5, 106.3, 58.4, 55.8, 50.5, 31.6, 27.9,

16.4. HRMS m/z C29H28F3N5OS: calcd 551.1967, found 552.2084 [M + H]+. HPLC purity: 98.73%. 4-((2-((4-((4-Cyanobenzyl)amino)cyclohexyl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (14k). Recrystallized from EA/PE as a white solid, 55% yield, mp 213-216 °C. 1H NMR (400 MHz, CDCl3): δ 8.15 (d, J = 5.4 Hz, 1H, C6-thienopyrimidineH), 7.76 (d, J = 8.2 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.53 (d, J = 8.0 Hz, 2H, C2,C6-Ph′-H), 7.26 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.68 (s, 1H, pyrimidine-NH), 4.32 (s, 1H, cyclohexane-NH), 3.71 (s, 2H, N-CH2), 3.23−3.26 (m, 2H), 2.11 (s, 6H), 1.87−2.06 (m, 3H), 1.19−1.31 (m, 5H). 13C NMR (100 MHz, CDCl3): δ 165.1, 162.0, 159.5, 146.3, 138.5, 133.7, 131.8, 128.2, 127.9, 127.1, 117.4, 63.0, 45.2, 36.6, 31.1, 29.3, 27.2, 19.8, 16.2. HRMS m/z C29H28N6OS: calcd 508.2045, found 509.2123 [M + H]+. HPLC purity: 97.79%. 4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,5-dioxidothieno[3,2d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (15). A mixture of 3 (0.3 g, 0.55 mmol) and m-CPBA (0.12 g, 0.66 mmol) in dichloromethane (10 mL) was stirred at ambient temperature for 5 h. Then sodium sulfite solution was added and the mixture was extracted with dichloromethane (3 × 5 mL). The organic solution was dried over anhydrous Na2SO4 and filtered. The filtrate was purified by flash column chromatography, and the residue was recrystallized from ethyl acetate to afford 15. White solid, 68% yield, mp 251−253 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.74 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.48 (d, J = 8.2 Hz, 2H, C2,C6-Ph′-H), 7.21 (s, 2H, SO2NH2), 7.23 (s, 1H, C7-thienopyrimidine-H), 6.87 (s, 1H, NH), 6.57 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 3.73 (s, 1H), 3.48 (s, 2H, N-CH2), 2.71− 2.75 (m, 2H), 2.09 (s, 6H), 1.79−1.95 (m, 4H), 1.41−1.44 (m, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 165.9, 162.4, 160.8, 153.4, 146.7, 143.6, 143.1, 136.9, 133.4, 129.1, 123.8, 123.4, 121.6, 119.1, 109.0, 61.7, 52.8, 31.3, 16.1. HRMS m/z C27H28N6O5S2: calcd 580.1563, found 597.1669 [M + NH3]+. HPLC purity: 96.32%. 4-((2-Chlorothieno[2,3-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (17). A mixture of 4-hydroxy-3,5-dimethylbenzonitrile (0.86 g, 5.85 mmol) and potassium carbonate (1.35 g, 9.75 mmol) in 15 mL of DMF was stirred at 25 °C for 15 min, and then 2,4-dichlorothiopheno[2,3-d]pyrimidine (16, 1.0 g, 4.88 mmol) was added to it. The mixture was stirred for another 2 h (monitored by TLC), then poured into ice−water (30 mL) and left to stand for 1 h. The precipitated white solid was collected by filtration, washed with cold water, and recrystallized from DMF−H2O to provide 17 as a white solid in 91% yield, mp 269−271 °C. HRMS m/z C15H10ClN3OS: calcd 315.0233, found 316.0347 [M + H]+. 3,5-Dimethyl-4-((2-(piperidin-4-ylamino)thieno[2,3-d]pyrimidin4-yl)oxy)benzonitrile (19). The synthetic method was similar to that described for 7a except that the starting material 17 (1.0 g, 3.17 mmol) was reacted with tert-butyl 4-aminopiperidine-1-carboxylate (0.76 g, 3.80 mmol). White solid, 67% yield, mp 131−134 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.35 (d, J = 5.5 Hz, 1H, C7-thienopyrimidine-H), 7.31 (s, 2H, C3,C5-Ph-H), 7.27 (d, J = 5.9 Hz, 1H, C6-thienopyrimidine-H), 3.77 (s, 1H, NH), 3.72−3.74 (m, 1H), 2.69−2.71 (m, 2H), 2.10 (s, 6H), 1.70−1.86 (m, 2H), 1.18−1.48(m, 4H). HRMS m/z C20H21N5OS: calcd 379.1467, found 380.1586 [M + H]+. 4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[2,3-d]pyrimidin2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (20a). Recrystallized from EA/PE as a white solid, 54% yield, mp 241− 243 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.78 (d, J = 8.0 Hz, 2H, C3,C5-Ph′-H), 7.71 (s, 2H, C3,C5-Ph″-H), 7.46 (d, J = 8.0 Hz, 2H, C2,C6-Ph′-H), 7.35 (d, J = 5.5 Hz, 1H, C7-thienopyrimidine-H), 7.31 (s, 2H, SO2NH2), 7.27 (d, J = 5.9 Hz, 1H, C6-thienopyrimidine-H), 7.08 (s, 1H, NH), 3.72 (s, 1H), 3.49 (s, 2H, N-CH2), 2.69−2.72 (m, 2H), 2.10 (s, 6H), 1.70−1.86 (m, 2H), 1.18−1.48 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.2, 159.2, 143.3, 143.1, 133.1, 132.7, 129.4, 128.3, 126.9, 126.0, 119.2, 119.0, 108.8, 61.9, 60.2, 52.6, 31.6, 21.2, 16.3, 14.5. HRMS m/z C27H28N6O3S2: calcd 548.1664, found 549.1693 [M + H]+. HPLC purity: 99.25%. 3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)amino)thieno[2,3-d]pyrimidin-4-yl)oxy)benzonitrile (20b). Recrystallized from EA/PE as a white solid, 63% yield, mp 232−234 °C. 4437

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

solid in 90% yield, mp 285−287 °C. HRMS m/z C15H11ClN2O2S: calcd 318.0230, found 319.0339 [M + H]+. (E)-3-(4-((2-Chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5dimethylphenyl)acrylonitrile (22). A mixture of (EtO)2P(O)CH2CN (0.67 g, 3.76 mmol) and t-BuOK (0.71 g, 6.27 mmol) in THF (10 mL) was stirred for 1 h at 0 °C, and then a solution of 21 (1.0 g, 3.14 mmol) in THF (10 mL) was slowly added to it over 1 h (monitored by TLC). The mixture was poured into ice−water (20 mL), and the precipitate was collected, washed with water, and dried to afford 22 as a white solid in 86% yield. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, 1H, J = 5.4 Hz, C6-thienopyrimidine-H), 7.60 (d, 1H, J = 16.7 Hz, ArCH), 7.49 (s, 2H, C3,C5-Ph-H), 7.45 (d, 2H, J = 8.1 Hz, C2,C6-Ph′-H), 7.32 (s, 2H, SO2NH2), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.45 (d, 1H, J = 16.7 Hz, CHCN), 2.09 (s, 6H). HRMS m/z C17H12ClN3OS: calcd 341.0390, found 342.0498 [M + H]+. (E)-3-(3,5-Dimethyl-4-((2-(piperidin-4-ylamino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (24). The synthetic method was similar to that described for 7a except that the starting material 22 (1.0 g, 2.93 mmol) was reacted with tert-butyl 4-aminopiperidine-1carboxylate (0.71 g, 3.51 mmol). White solid, 63% yield, mp 154− 156 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.16 (d, 1H, J = 5.7 Hz, C6-thienopyrimidine-H), 7.60 (d, 1H, J = 16.7 Hz, ArCH), 7.49 (s, 2H, C3,C5-Ph-H), 7.26 (s, 1H, C7-thienopyrimidine-H), 6.89 (s, 1H, NH), 6.47 (d, 1H, J = 16.7 Hz, CHCN), 3.69 (s, 1H), 2.68− 2.74 (m, 2H), 2.09 (s, 6H), 1.89−2.03 (m, 2H), 1.22−1.40 (m, 4H). HRMS m/z C22H23N5OS: calcd 405.1623, found 406.1716 [M + H]+ (E)-4-((4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)thieno[3,2d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (25a). Recrystallized from EA/PE as a white solid, 53% yield, mp 211−213 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.16 (d, 1H, J = 5.4 Hz, C6-thienopyrimidine-H), 7.77 (d, 2H, J = 8.2 Hz, C3,C5-Ph′-H), 7.60 (d, 1H, J = 16.6 Hz, ArCH), 7.49 (s, 2H, C3,C5-Ph″-H), 7.45 (d, 2H, J = 8.1 Hz, C2,C6-Ph′-H), 7.32 (s, 2H, SO2NH2), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.89 (s, 1H, NH), 6.42 (d, 1H, J = 16.7 Hz, CHCN), 3.67−3.79 (m, 1H), 3.48 (s, 2H, N-CH2), 2.68−2.74 (m, 2H), 2.09 (s, 6H), 1.76−1.83 (m, 2H), 1.16−1.45 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.9, 160.6, 151.8, 150.4, 143.4, 143.1, 136.4, 131.8, 129.4, 128.6, 126.0, 123.6, 119.3, 96.8, 62.0, 60.2, 52.7, 31.7, 21.2, 16.5, 14.5. HRMS m/z C29H30N6O3S2: calcd 574.1821, found 575.1977 [M + H]+. HPLC purity: 99.25%. (E)-3-(3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)piperidin4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25b). Recrystallized from EA/PE as a white solid, 42% yield, mp 113−115 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.16 (d, 1H, J = 5.3 Hz, C6-thienopyrimidine-H), 7.87 (d, 2H, J = 8.2 Hz, C3,C5-Ph′-H), 7.60 (d, 1H, J = 16.7 Hz, ArCH), 7.54 (d, 2H, J = 8.1 Hz, C2,C6-Ph′H), 7.49 (s, 2H, C3,C5-Ph″-H), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.89 (s, 1H, NH), 6.41 (d, 1H, J = 16.7 Hz, CHCN), 3.67−3.78 (m, 1H), 3.53 (s, 2H, N-CH2), 3.34 (s, 3H, SO2CH3), 2.68−2.73 (m, 2H), 2.08 (s, 6H), 1.76−1.83 (m, 2H), 1.26−1.48 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 153.3, 150.4, 145.5, 139.8, 136.4, 131.8, 129.8, 127.2, 123.7, 123.6, 119.3, 96.8, 61.9, 56.5, 52.8, 44.0, 31.7, 16.5. HRMS m/z C30H31N5O3S2: calcd 573.1868, found 574.1973 [M + H]+. HPLC purity: 99.10%. (E)-4-((4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)thieno[3,2d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (25c). Recrystallized from EA/PE as a white solid, 45% yield, mp 221−224 °C. 1 H NMR (400 MHz, DMSO-d6): δ 8.16 (d, 1H, J = 5.3 Hz, C6-thienopyrimidine-H), 7.93 (s, 1H), 7.82 (d, 2H, J = 8.0 Hz, C3,C5Ph′-H), 7.60 (d, 1H, J = 16.7 Hz, ArCH), 7.49 (s, 2H, C3,C5-Ph″H), 7.31−7.35 (m, 3H), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.88 (s, 1H, NH), 6.42 (d, 1H, J = 16.7 Hz, CHCN), 3.67−3.78 (m, 1H)), 3.45 (s, 2H, N-CH2), 2.68−2.73 (m, 2H), 2.08 (s, 6H), 1.88−1.99 (m, 2H), 1.69−1.77 (m, 2H), 1.34−1.53 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 168.2, 162.8, 160.6, 151.8, 150.4, 142.5, 136.5, 136.4, 133.4, 131.8, 128.8, 128.5, 127.8, 123.5, 119.3, 96.8, 62.2, 52.7, 31.7, 16.5. HRMS m/z C30H30N6O2S: calcd 538.2151, found 539.2267 [M + H]+. HPLC purity: 98.69%. (E)-3-((4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)thieno[3,2d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (25d). Recrystallized from EA/PE as a white solid, 35% yield, mp 215−217 °C.

H NMR (400 MHz, DMSO-d6): δ 7.77 (d, J = 8.3 Hz, 2H, C3,C5-Ph′H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.46 (d, J = 8.1 Hz, 2H, C2,C6-Ph′-H), 7.34 (d, J = 5.5 Hz, 1H, C7-thienopyrimidine-H), 7.23 (d, J = 5.9 Hz, 1H, C6-thienopyrimidine-H), 7.01 (s, 1H, NH), 3.71−3.74 (m, 1H), 3.49 (s, 2H, N-CH2), 3.08 (s, 3H, SO2CH3), 2.71−2.73 (m, 2H), 2.10 (s, 6H), 1.87−2.03 (m, 4H), 1.21−1.38 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.7, 160.5, 143.7, 142.9, 133.7, 132.1, 129.4, 128.4, 126.9, 125.3, 119.2, 118.9, 108.8, 61.9, 60.7, 52.6, 31.2, 21.7, 16.3, 14.2. HRMS m/z C28H29N5O3S2: calcd 547.1712, found 548.1809 [M + H]+. HPLC purity: 98.14%. 4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[2,3-d]pyrimidin2-yl)amino) piperidin-1-yl)methyl)benzamide (20c). Recrystallized from EA/PE as a white solid, 40% yield, mp 251−253 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.83 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.56 (d, J = 8.2 Hz, 2H, C2,C6-Ph′-H), 7.34 (d, J = 5.6 Hz, 1H, C7-thienopyrimidine-H), 7.23 (d, J = 5.6 Hz, 1H, C6-thienopyrimidine-H), 7.01 (s, 1H, NH), 3.73 (s, 1H), 3.49 (s, 2H, N-CH2), 2.74−2.76 (m, 2H), 2.09 (s, 6H), 1.81−1.89 (m, 2H), 1.23− 1.34 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.7, 160.1, 143.5, 142.9, 133.2, 131.8, 129.2, 127.5, 126.9, 125.3, 120.4, 118.7, 109.4, 61.9, 60.0, 52.6, 31.7, 21.3, 16.8, 14.2. HRMS m/z C28H28N6O2S: calcd 512.1994, found 513.2218 [M + H]+. HPLC purity: 97.56%. 3-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[2,3-d]pyrimidin2-yl)amino)piperidin-1-yl)methyl)benzamide (20d). Recrystallized from EA/PE as a white solid, 45% yield, mp 216−218 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.89−7.93 (m, 3H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.54−7.55 (m, 1H), 7.34 (d, J = 5.5 Hz, 1H, C7-thienopyrimidine-H), 7.23 (d, J = 5.7 Hz, 1H, C6-thienopyrimidine-H), 7.01 (s, 1H, NH), 3.73 (s, 1H), 3.49 (s, 2H, N-CH2), 2.74−2.76 (m, 2H), 2.09 (s, 6H), 1.81−1.89 (m, 2H), 1.23−1.34 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.7, 160.3, 144.8, 142.1, 133.5, 131.6, 128.7, 127.4, 126.5, 125.1, 121.7, 118.6, 109.5, 62.4, 60.7, 52.6, 31.9, 21.5, 16.8, 14.4. HRMS m/z C28H28N6O2S: calcd 512.1994, found 513.2128 [M + H]+. HPLC purity: 98.32%. Ethyl 4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzoate (20e). Recrystallized from EA/PE as a white solid, 58% yield, mp 224−226 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.87 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.34 (d, J = 5.6 Hz, 1H, C7-thienopyrimidine-H), 7.23 (d, J = 5.6 Hz, 1H, C6-thienopyrimidine-H), 7.13 (d, J = 8.2 Hz, 2H, C2,C6-Ph′-H), 7.03 (s, 1H, NH), 4.32 (q, J = 7.6 Hz, 2H, CO2CH2), 3.73 (s, 1H), 3.49 (s, 2H, N-CH2), 2.74 (s, 2H), 2.09 (s, 6H), 1.96−2.06 (m, 3H), 1.37−1.46 (m, 3H), 1.32 (t, J = 7.2 Hz, 3H, CH2CH3). 13C NMR (100 MHz, DMSO-d6): δ 162.7, 160.1, 143.5, 142.9, 133.2, 131.8, 129.2, 127.5, 126.9, 125.3, 120.4, 118.7, 109.4, 61.9, 60.8, 60.0, 52.6, 31.7, 21.3, 16.8, 14.6, 14.2. HRMS m/z C30H31N5O3S: calcd 541.2148, found 542.2338 [M + H]+. HPLC purity: 99.62%. Methyl 4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[2,3-d]pyrimidin-2-yl) amino)piperidin-1-yl)methyl)benzoate (20f). Recrystallized from EA/PE as a white solid, 66% yield, mp 211−213 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.87 (d, J = 8.2 Hz, 2H, C3,C5-Ph′-H), 7.72 (s, 2H, C3,C5-Ph″-H), 7.36 (d, J = 5.6 Hz, 1H, C7-thienopyrimidine-H), 7.21 (d, J = 5.6 Hz, 1H, C6-thienopyrimidine-H), 7.17 (d, J = 8.1 Hz, 2H, C2,C6-Ph′-H), 7.01 (s, 1H, NH), 3.72−2.75 (m, 1H), 3.49 (s, 2H, N-CH2), 2.74−2.76 (m, 2H), 2.35 (s, 3H, CO2CH3), 2.09 (s, 6H), 1.91−2.00 (m, 3H), 1.22−1.34 (m, 3H). 13 C NMR (100 MHz, DMSO-d6): δ 162.7, 160.0, 143.3, 141.7, 133.7, 131.8, 128.6, 127.5, 126.5, 125.3, 121.2, 118.7, 110.6, 61.9, 60.7, 52.6, 51.1, 31.7, 21.9, 16.8, 14.2. HRMS m/z C29H29N5O3S: calcd 527.1991, found 528.1537 [M + H]+. HPLC purity: 98.33%. 4-((2-Chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzaldehyde (21). A mixture of 4-hydroxy-3,5-dimethylbenzaldehyde (0.88 g, 5.85 mmol) and potassium carbonate (1.35 g, 9.75 mmol) in 15 mL of DMF was stirred at 25 °C for 15 min, and then 2,4-dichlorothiopheno[3,2-d]pyrimidine (4, 1.0 g, 4.88 mmol) was added to it. The mixture was stirred for another 1.5 h (monitored by TLC), then poured into ice−water (30 mL) and left to stand for 1 h. The precipitated white solid was collected by filtration, washed with cold water, and recrystallized from DMF−H2O to provide 21 as a white 1

4438

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.97 (s, 1H), 7.80−7.74 (m, 2H), 7.62 (d, J = 16.6 Hz, 1H, ArCH), 7.49 (s, 2H, C3,C5-Ph″-H), 7.40 (d, J = 6.9 Hz, 2H), 7.35 (s, 1H), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.88 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.73−3.76 (m, 1H), 3.46 (s, 2H, N-CH2), 2.74 (s, 2H), 2.09 (s, 6H), 1.93−2.00 (m, 2H), 1.69−1.77 (m, 2H), 1.35−1.45 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 168.4, 162.8, 160.6, 151.8, 150.4, 139.1, 136.4, 131.8, 128.4, 126.4, 123.7, 119.3, 116.1, 96.8, 62.4, 60.2, 52.7, 31.7, 21.2, 16.5, 14.5. HRMS m/z C30H30N6O2S: calcd 538.2151, found 539.2308 [M + H]+. HPLC purity: 99.36%. Ethyl(E)-4-((4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzoate (25e). Recrystallized from EA/PE as a white solid, 44% yield, mp 194− 199 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.2 Hz, 1H, C6-thienopyrimidine-H), 7.92 (d, J = 8.1 Hz, 2H, C3,C5-Ph′-H), 7.62 (d, J = 16.6 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5-Ph″-H), 7.42 (d, J = 7.9 Hz, 2H, C2,C6-Ph′-H), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.89 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 4.31 (q, J = 7.1 Hz, 2H, CO2CH2), 3.75 (s, 1H), 3.48 (s, 2H, N-CH2), 2.72 (s, 2H), 2.08 (s, 6H), 1.92−2.00 (m, 2H), 1.72−1.76 (m, 2H), 1.42−1.47 (m, 2H), 1.32 (t, J = 7.1 Hz, 3H, CH2CH3). 13C NMR (100 MHz, DMSO-d6): δ 166.1, 162.8, 160.6, 150.4, 144.9, 136.5, 131.9, 129.5, 129.3, 128.9, 128.5, 123.8, 119.3, 96.8, 62.2, 61.0, 56.5, 52.7, 31.7, 19.0, 16.5, 14.6. HRMS m/z C32H33N5O3S: calcd 567.2304, found 568.2497 [M + H]+. HPLC purity: 98.44%. (E)-3-(4-((2-((1-(4-Fluorobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (25f). Recrystallized from EA/PE as a white solid, 58% yield, mp 260−262 °C. 1 H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.62 (d, J = 16.7 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5-Ph″-H), 7.32−7.23 (m, 3H), 7.13 (d, J = 8.8 Hz, 2H, C3,C5-Ph′-H), 6.88 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.71−3.75 (m, 1H), 3.38 (s, 2H, N-CH2), 2.71 (s, 2H), 2.08 (s, 6H), 1.92−1.99(m, 2H), 1.67−1.76(m, 2H), 1.32−1.42 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 165.8, 162.8, 160.4, 151.8, 150.4, 136.6, 135.2, 131.8, 131.7, 130.9 (d, J = 7.9 Hz), 128.5, 123.7, 119.3, 115.1 (d, J = 21.1 Hz), 96.8, 61.7, 52.6, 31.7, 16.5. HRMS m/z C29H28FN5OS: calcd 513.1999, found 514.2151 [M + H]+. HPLC purity: 98.79%. (E)-3-(4-((2-((1-(3-Fluorobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-,5-dimethylphenyl)acrylonitrile (25g). Recrystallized from EA/PE as a white solid, 58% yield, mp 156−158 °C. 1 H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.61 (d, J = 16.6 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5-Ph″-H), 7.36−7.38 (m, 1H), 7.25 (s, 1H, C7-thienopyrimidine-H), 7.13−7.05 (m, 3H), 6.89 (s, 1H, NH), 6.43 (d, J = 16.7 Hz, 1H, CHCN), 3.71−3.75 (m, 1H), 3.43 (s, 2H, N-CH2), 2.72 (s, 2H), 2.09 (s, 6H), 1.77−1.92(m, 4H), 1.34−1.45 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 163.9, 161.4, 160.6, 151.8, 150.4, 142.2, 136.5, 131.8, 131.7, 130.3 (d, J = 8.4 Hz), 128.5, 125.0, 123.7, 119.3, 115.4 (d, J = 21.1 Hz), 113.9 (d, J = 20.9 Hz), 96.8, 61.9, 52.6, 31.7, 16.5. HRMS m/z C29H28FN5OS: calcd 513.1999, found 514.2172 [M + H]+. HPLC purity: 96.96%. (E)-3-(4-((2-((1-(2-Fluorobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (25h). Recrystallized from EA/PE as a white solid, 54% yield, mp 175− 177 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.61 (d, J = 16.7 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5-Ph″-H), 7.41−7.22 (m, 3H), 7.13−7.19 (m, 3H), 6.89 (s, 1H, NH), 6.43 (d, J = 16.7 Hz, 1H, CHCN), 3.72 (s, 1H), 3.47 (s, 2H, N-CH2), 2.74 (s, 2H), 2.09 (s, 6H), 1.63−1.77 (m, 3H), 1.32− 1.44 (m, 3H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 162.4, 160.6, 160.0, 151.8, 150.4, 136.5, 132.0, 131.8 (d, J = 5.0 Hz), 131.7, 129.3 (d, J = 8.0 Hz), 124.5, 124.4, 119.3, 115.4 (d, J = 22.0 Hz), 96.8, 55.0, 52.4, 31.7, 16.5. HRMS m/z C29H28FN5OS: calcd 513.1999, found 514.2119 [M + H]+. HPLC purity: 98.75%. (E)-3-(4-((2-((1-(4-Bromobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (25i). Recrystallized from EA/PE as a white solid, 54% yield, mp 192−194 °C. 1 H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, 1

C6-thienopyrimidine-H), 7.62 (d, J = 16.6 Hz, 1H, ArCH), 7.48− 7.51(m, 4H), 7.22−7.24 (m, 3H), 6.89 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.71−3.75 (m, 1H), 3.37 (s, 2H, N-CH2), 2.71 (s, 2H), 2.08 (s, 6H), 1.92−2.00 (m, 2H), 1.75−1.77 (m, 2H), 1.34−1.42 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 155.7, 151.7, 150.4, 148.5, 138.5, 136.4, 131.8, 131.7, 131.4, 131.3, 128.5, 123.7, 123.6, 120.2, 119.3, 115.6, 96.8, 61.7, 52.6, 31.7, 16.5. HRMS m/z C29H28BrN5OS: calcd 573.1198, found 576.1322 [M + 3]+. HPLC purity: 99.02%. (E)-3-(4-((2-((1-(3-Bromobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (25j). Recrystallized from EA/PE as a white solid, 48% yield, mp 180−182 °C. 1 H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.1 Hz, 1H, C6-thienopyrimidine-H), 7.62 (d, J = 16.5 Hz, 1H, ArCH), 7.49 (s, 2H, C3,C5-Ph″-H), 7.47−7.42 (m, 2H), 7.27−7.29 (m, 3H), 6.90 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.71−3.75 (m, 1H), 3.42 (s, 2H, N-CH2), 2.73 (s, 2H), 2.09 (s, 6H), 1.93−1.99 (m, 2H), 1.75−1.77 (m, 2H), 1.34−1.45 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 150.4, 145.9, 142.1, 136.4, 131.8, 131.7, 131.6, 130.7, 130.6, 130.1, 129.8, 129.4, 128.5, 128.1, 125.7, 122.0, 119.3, 96.8, 62.5, 61.7, 52.6, 50.6, 31.7, 29.0, 16.5. HRMS m/z C29H28BrN5OS: calcd 573.1198, found 576.1309 [M + 3]+. HPLC purity: 98.72%. (E)-3-(4-((2-((1-(2-Bromobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (25k). Recrystallized from EA/PE as a white solid, 56% yield, mp 165− 167 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J = 5.2 Hz, 1H, C6-thienopyrimidine-H), 7.56−7.65 (m, 2H), 7.49 (s, 2H, C3,C5-Ph″H), 7.43 (d, J = 6.8 Hz, 1H), 7.32−7.35 (m, 1H), 7.27 (s, 1H, C7-thienopyrimidine-H), 7.17−7.19 (m, 1H), 6.92 (s, 1H, NH), 6.45 (d, J = 16.7 Hz, 1H, CHCN), 3.76 (s, 1H), 3.49 (s, 2H, N-CH2), 2.78 (s, 2H), 2.09 (s, 6H), 1.93−1.99 (m, 2H), 1.34−1.67 (m, 4H). 13 C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 151.7, 150.4, 148.5, 138.0, 136.3, 132.9, 131.8, 131.2, 129.2, 128.5, 127.9, 124.3, 119.3, 96.8, 61.7, 52.8, 31.7, 29.0, 16.6. HRMS m/z C29H28BrN5OS: calcd 573.1198, found 576.1297 [M + 3]+. HPLC purity: 97.32%. (E)-3-(4-((2-((1-(4-Chlorobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (25l). Recrystallized from EA/PE as a white solid, 56% yield, mp 180−184 °C. 1 H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.62 (d, J = 16.5 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5-Ph″-H), 7.36 (d, J = 8.3 Hz, 2H, C3,C5-Ph′-H), 7.28 (d, J = 8.2 Hz, 2H, C2,C6-Ph′-H), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.88 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.72 (s, 1H), 3.39 (s, 2H, N-CH2), 2.70 (s, 2H), 2.08 (s, 6H), 1.92−2.00 (m, 2H), 1.68−1.76 (m, 2H), 1.37−1.44 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 151.7, 150.4, 148.5, 138.1, 136.5, 131.9, 131.8, 131.7, 130.9, 128.5, 123.7, 119.3, 110.1, 96.8, 61.7, 52.6, 48.4, 31.7, 16.5. HRMS m/z C29H28ClN5OS: calcd 529.1703, found 530.1901 [M + H]+. HPLC purity: 98.12%. (E)-3-(4-((2-((1-(3-Chlorobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (25m). Recrystallized from EA/PE as a white solid, 57% yield, mp 161− 164 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.1 Hz, 1H, C6-thienopyrimidine-H), 7.62 (d, J = 16.5 Hz, 1H, ArCH), 7.49 (s, 2H, C3,C5-Ph″-H), 7.42−7.45 (m, 2H), 7.25−7.27 (m, 3H), 6.90 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.72−3.75 (m, 1H), 3.42 (s, 2H, N-CH2), 2.73 (s, 2H), 2.09 (s, 6H), 1.95−1.99 (m, 2H), 1.74−1.77 (m, 2H), 1.32−1.45 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.9, 160.6, 150.4, 145.7, 142.0, 136.4, 131.8, 131.7, 131.6, 130.7, 130.5, 130.1, 129.8, 129.4, 128.5, 128.1, 125.6, 122.0, 119.3, 96.8, 62.5, 61.6, 52.6, 50.6, 31.4, 29.0, 16.5. HRMS m/z C29H28ClN5OS: calcd 529.1703, found 530.1812 [M + H]+. HPLC purity: 99.24%. (E)-3-(4-((2-((1-(2-Chlorobenzyl)piperidin-4-yl)amino)thieno[3,2d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (25n). Recrystallized from EA/PE as a white solid, 57% yield, mp 165− 168 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J = 5.2 Hz, 1H, C6-thienopyrimidine-H), 7.62 (d, J = 16.6 Hz, 1H, ArCH), 7.49 (s, 2H, C3,C5-Ph″-H), 7.42 (d, J = 8.7 Hz, 2H), 7.34−7.27 (m, 3H), 4439

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

6.91 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.74 (s, 1H), 3.51 (s, 2H, N-CH2), 2.68−2.77 (m, 2H), 2.09 (s, 6H), 1.72−1.78 (m, 2H), 1.33−1.47 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.9, 160.6, 150.4, 136.4, 133.7, 131.8, 131.2, 129.6, 128.9, 128.5, 127.4, 119.3, 96.8, 59.1, 52.8, 31.7, 29.0, 16.5. HRMS m/z C29H28ClN5OS: calcd 529.1703, found 530.1874 [M + H]+. HPLC purity: 98.83%. (E)-3-(3,5-Dimethyl-4-((2-((1-(4-nitrobenzyl)piperidin-4-yl)amino)thieno[3,2-d] pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25o). Recrystallized from EA/PE as a white solid, 47% yield, mp 181− 183 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17−8.20 (m, 3H), 7.62 (d, J = 16.1 Hz, 1H, ArCH), 7.56 (d, J = 8.3 Hz, 2H, C2,C6-Ph′-H), 7.49 (s, 2H, C3,C5-Ph″-H), 7.25 (s, 1H), 6.91 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.74 (s, 1H), 3.56 (s, 2H, N-CH2), 2.74 (s, 2H), 2.09 (s, 6H), 1.71−1.77 (m, 2H), 1.23−1.45 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 150.4, 147.5, 146.9, 136.4, 133.0, 131.9, 130.0, 128.5, 127.4, 123.8, 119.3, 96.8, 61.6, 52.8, 31.7, 16.5. HRMS m/z C29H28N6O3S: calcd 540.1944, found 541.2046 [M + H]+. HPLC purity: 96.36%. (E)-3-(3,5-Dimethyl-4-((2-((1-(2-methylbenzyl)piperidin-4-yl)amino)thieno[3,2-d] pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25p). Recrystallized from EA/PE as a white solid, 57% yield, mp 176− 179 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.62 (d, J = 16.1 Hz, 1H, ArCH), 7.49 (s, 2H, C3,C5-Ph″-H), 7.26 (s, 1H, C7-thienopyrimidine-H), 7.14−7.18 (m, 4H), 6.88 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.75 (s, 1H), 3.44 (s, 2H, N-CH2), 2.63−2.74 (m, 2H), 2.30 (s, 3H, CH3), 2.08 (s, 6H), 1.72−1.77 (m, 2H), 1.22−1.39 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.9, 160.6, 151.8, 150.4, 137.4, 137.1, 131.8, 130.4, 129.9, 128.5, 127.2, 125.8, 123.6, 119.3, 96.8, 60.6, 56.5, 52.9, 31.8, 19.2, 16.5. HRMS m/z C30H31N5OS: calcd 509.2249, found 510.2154 [M + H]+. HPLC purity: 97.89%. (E)-3-(3,5-Dimethyl-4-((2-((1-(4-methylbenzyl)piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25q). Recrystallized from EA/PE as a white solid, 55% yield, mp 185− 191 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.16 (d, J = 5.2 Hz, 1H, C6-thienopyrimidine-H), 7.61 (d, J = 16.4 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5-Ph″-H), 7.24 (s, 1H, C7-thienopyrimidine-H), 7.08−7.14 (m, 4H), 6.85 (s, 1H, NH), 6.43 (d, J = 16.7 Hz, 1H, CHCN), 3.71 (s, 1H), 3.46 (s, 2H, N-CH2), 2.71 (s, 2H), 2.27 (s, 3H, CH3), 2.07 (s, 6H), 1.91−1.99 (m, 2H), 1.71−1.74 (m, 2H), 1.34−1.38 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 151.7, 150.4, 136.4, 136.2, 135.8, 131.8, 129.1, 128.4, 123.6, 119.3, 96.8, 62.4, 56.5, 52.6, 31.7, 21.1, 16.5. HRMS m/z C30H31N5OS: calcd 509.2249, found 510.2388 [M + H]+. HPLC purity: 97.31%. (E)-3-(3,5-Dimethyl-4-((2-((1-(4-cyanobenzyl)piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25r). Recrystallized from EA/PE as a white solid, 48% yield, mp 176− 180 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.78 (d, J = 8.2 Hz, 2H, C3,C5-Ph′-H), 7.62 (d, J = 16.6 Hz, 1H, ArCH), 7.45−7.48 (d, 4H), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.90 (s, 1H, NH), 6.43 (d, J = 16.7 Hz, 1H, CHCN), 3.74 (s, 1H), 3.50 (s, 2H, N-CH2), 2.71 (s, 2H), 2.09 (s, 6H), 1.72−1.76 (m, 2H), 1.33−1.44 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 151.7, 150.4, 149.2, 148.5, 145.3, 136.5, 136.3, 132.5, 131.8, 129.9, 128.5, 123.6, 119.3, 110.0, 96.8, 61.9, 52.7, 48.3, 31.7, 16.5. HRMS m/z C30H28N6OS: calcd 520.2045, found 521.2223 [M + H]+. HPLC purity: 96.85%. (E)-3-(3,5-Dimethyl-4-((2-((1-(3-cyanobenzyl)piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25s). Recrystallized from EA/PE as a white solid, 41% yield, mp 180− 185 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.71 (d, J = 5.9 Hz, 2H), 7.54−7.65 (m, 3H), 7.49 (s, 2H, C3,C5-Ph″-H), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.90 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.75 (s, 1H), 3.48 (s, 2H, N-CH2), 2.71−2.77 (m, 2H), 2.09 (s, 6H), 1.72−1.77 (m, 2H), 1.33−1.44 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 151.8, 150.4, 140.9, 136.5, 134.0, 132.4, 131.8, 131.2, 130.9, 129.8, 128.5, 123.5, 119.3, 111.6, 96.8, 65.4, 61.4, 56.5, 52.6, 31.7, 16.5. HRMS m/z C30H28N6OS: calcd 520.2045, found 521.2232 [M + H]+. HPLC purity: 99.11%.

(E)-3-(3,5-Dimethyl-4-((2-((1-(2-cyanobenzyl)piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25t). Recrystallized from EA/PE as a white solid, 53% yield, mp 122− 125 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.80 (d, J = 7.7 Hz, 1H, C3-Ph′-H), 7.61− 7.68 (m, 2H), 7.54 (d, J = 6.9 Hz, 1H), 7.49 (s, 2H, C3,C5-Ph″-H), 7.46 (d, J = 7.6 Hz, 1H), 7.26 (s, 1H, C7-thienopyrimidine-H), 6.91 (s, 1H, NH), 6.43 (d, J = 16.7 Hz, 1H, CHCN), 3.74 (s, 1H), 3.59 (s, 2H, N-CH2), 2.74 (s, 2H), 2.09 (s, 6H), 1.72−1.76 (m, 2H), 1.33− 1.44 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 151.8, 150.4, 142.7, 136.5, 133.4, 131.8, 130.5, 129.1, 128.3, 123.6, 119.3, 118.1, 112.5, 96.8, 60.3, 52.6, 48.3, 31.6, 16.5. HRMS m/z C30H28N6OS: calcd 520.2045, found 521.2232 [M + H]+. HPLC purity: 98.84%. (E)-3-(3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25u). Recrystallized from EA/PE as a white solid, 45% yield, mp 147− 150 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.46 (d, J = 4.7 Hz, 2H, C3,C5-Py-H), 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.67− 7.69 (m, 1H), 7.62 (d, J = 16.6 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5Ph″-H), 7.35−7.36 (m, 1H), 7.25 (s, 1H, C7-thienopyrimidine-H), 6.89 (s, 1H, NH), 6.44 (d, J = 16.7 Hz, 1H, CHCN), 3.73 (s, 1H), 3.45 (s, 2H, N-CH2), 2.72 (s, 2H), 2.08 (s, 6H), 1.92−2.00 (m, 2H), 1.67−1.76 (m, 2H), 1.36−1.43 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 151.7, 150.4, 148.6, 148.5, 141.8, 136.9, 136.4, 134.3, 131.8, 128.5, 123.8, 119.3, 96.8, 59.7, 52.5, 48.6, 31.6, 16.5. HRMS m/z C28H28N6OS: calcd 496.2045, found 497.2144 [M + H]+. HPLC purity: 97.26%. (E)-3-(3,5-Dimethyl-4-((2-((1-(pyridin-3-ylmethyl)piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25v). Recrystallized from EA/PE as a white solid, 47% yield, mp 156− 159 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.50 (d, J = 4.7 Hz, 2H), 8.17 (d, J = 5.2 Hz, 1H, C6-thienopyrimidine-H), 7.62 (d, J = 16.6 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5-Ph″-H), 7.28 (d, J = 4.1 Hz, 2H), 7.24 (s, 1H, C7-thienopyrimidine-H), 6.90 (s, 1H, NH), 6.43 (d, J = 16.7 Hz, 1H, CHCN), 3.75 (s, 1H), 3.45 (s, 2H, N-CH2), 2.71 (s, 2H), 2.08 (s, 6H), 1.92−1.99 (m,2H), 1.72−1.78 (m, 2H), 1.39−1.47 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.6, 157.1, 150.4, 149.9, 148.9, 148.1, 136.5, 131.9, 128.6, 124.1, 123.6, 119.3, 96.8, 61.2, 52.7, 48.4, 31.6, 16.5. HRMS m/z C28H28N6OS: calcd 496.2045, found 497.2206 [M + H]+. HPLC purity: 97.85%. (E)-3-(3,5-Dimethyl-4-((2-((1-(pyridin-2-ylmethyl)piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (25w). Recrystallized from EA/PE as a white solid, 39% yield, mp 172− 175 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.48 (d, J = 4.6 Hz, 1H), 8.17 (d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 7.75 (t, J = 8.4 Hz, 1H), 7.61 (d, J = 16.6 Hz, 1H, ArCH), 7.48 (s, 2H, C3,C5-Ph″-H), 7.39 (d, J = 7.6 Hz, 1H), 7.21−7.24 (m, 2H), 6.90 (s, 1H, NH), 6.43 (d, J = 16.7 Hz, 1H, CHCN), 3.74 (s, 1H), 3.54 (s, 2H, N-CH2), 2.76 (s, 2H), 2.09 (s, 6H), 1.92−1.97 (m, 2H), 1.68−1.77 (m, 2H), 1.33−1.47 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 162.8, 160.7, 159.1, 151.7, 150.4, 149.1, 136.8, 136.4, 131.9, 131.8, 128.5, 123.0, 122.4, 119.3, 96.8, 64.3, 52.9, 48.3, 31.7, 16.5. HRMS m/z C28H28N6OS: calcd 496.2045, found 497.2092 [M + H]+. HPLC purity: 98.12%. In Vitro Assay of Anti-HIV Activities. The anti-HIV activity and cytotoxicity of the newly synthesized derivatives were evaluated with WT HIV-1 (HIV-IIIB strain), single RT mutant strains (L100I, K103N, Y181C, Y188L, and E138K), double RT mutant strains (F227L+V106A and K103N+Y181C), and HIV-2 (strain ROD) in MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method as described previously.32,33 At the beginning of each experiment, stock solutions (10× final concentration) of test compounds were added in 25 μL volumes to two series of triplicate wells in order to allow simultaneous evaluation of the effects on mock- and HIV-infected cells. Using a Biomek 3000 robot (Beckman Instruments, Fullerton, CA), serial 5-fold dilutions of the test compounds (final 200 μL volume per well) were made directly in flat-bottomed 96-well microtiter trays, including untreated control HIV-1 and mock-infected cell samples for each sample. HIV-1 (IIIB) 4440

DOI: 10.1021/acs.jmedchem.7b00332 J. Med. Chem. 2017, 60, 4424−4443

Journal of Medicinal Chemistry

Article

and mutant HIV-1 strains (K103N+Y181C, F227L+V106A, L100I, K103N, Y181C, Y188L, and E138K) or HIV-2 (ROD) stock (50 μL at 100−300 CCID50) (50% cell culture infectious dose) or culture medium was added to either the infected or mock-infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effect of test compounds on uninfected cells in order to assess cytotoxicity. Exponentially growing MT-4 cells were centrifuged for 5 min at 1000 rpm, and the supernatant was discarded. The MT-4 cells were resuspended at 6 × 105 cells/mL, and 50 μL aliquots were transferred to the microtiter tray wells. At 5 days after infection, the viability of mock- and HIV-infected cells was determined spectrophotometrically by means of MTT assay. The MTT assay is based on the reduction of yellow-colored MTT (Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase of metabolically active cells to form a blue−purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Multiscan Ascent Reader, Labsystems, Helsinki, Finland) at the wavelengths of 540 and 690 nm. All data were calculated using the median optical density (OD) value of three wells. The 50% effective antiviral concentration (EC50) was defined as the concentration of the test compound, affording 50% protection from viral cytopathogenicity. The 50% cytotoxic concentration (CC50) was defined as the compound concentration that reduced the absorbance (OD540) of mock-infected cells by 50%. Recombinant HIV-1 Reverse Transcriptase (RT) Inhibitory Assays. The HIV-1 RT inhibition assay was performed by using an RT assay kit produced by Roche. The procedure for assaying HIV-1 RT inhibition was conducted as described in the kit protocol.34 The reaction mixture containing HIV-1 RT enzyme, reconstituted template, and viral nucleotides [digoxigenin (DIG)-dUTP, biotindUTP, and dTTP] in the incubation buffer with or without inhibitors was incubated for 1 h at 37 °C first. Then the mixture was transferred to a streptavidin-coated microtiter plate (MTP) and incubated for another 1 h at 37 °C. The biotin-labeled dNTPs that were incorporated into the cDNA chain in the presence of RT were bound to streptavidin. The unbound dNTPs were washed with washing buffer, and anti-DIG-POD was added to the MTPs. After incubation for 1 h at 37 °C, the DIG-labeled dNTPs incorporated in cDNA were bound to the anti-DIG-POD antibody. The unbound anti-DIG-PODs were washed out, and the peroxide substrate (ABTS) solution was added to the MTPs. The absorbance of the sample was determined at OD405 nm using a microtiter plate ELISA reader. The IC50 values correspond to the concentrations of the inhibitors required to inhibit biotin-dUTP incorporation by 50%. Molecular Simulations. Molecular simulations was performed with the Tripos molecular modeling package Sybyl-X 2.0. All the molecules for docking were built using standard bond lengths and angles from Sybyl-X 2.0/Base Builder and were optimized using the Tripos force field for 1000 generations two times or more, until the minimized conformers of the ligand were the same. The flexible docking method (Surflex-Dock) docks the ligand automatically into the ligand-binding site of the receptor by using a protocol-based approach and an empirically derived scoring function. The protocol is a computational representation of a putative ligand that binds to the intended binding site and is a unique and essential element of the docking algorithm. The scoring function in Surflex-Dock, containing hydrophobic, polar, repulsive, entropic, and solvation terms, was trained to estimate the dissociation constant (Kd) expressed as −log(Kd)2. The protein was prepared by removing the ligand, water molecules, and other unnecessary small molecules from the crystal structure of the ligand HIV-1 RT complex (PDB 3M8Q) before docking; polar hydrogen atoms and charges were added to the protein. Surflex-Dock default settings were used for other parameters, such as the maximum number of rotatable bonds per molecule (set to 100) and the maximum number of poses per ligand (set to 20). During the docking procedure, all of the single bonds in residue side chains inside the defined RT binding pocket were regarded as rotatable or flexible, and the ligand was allowed to rotate at all single bonds and to move flexibly within the tentative binding pocket. The atomic charges were recalculated using the Kollman all-atom approach for the protein and the Gasteiger−Hückel approach for the ligand. The binding

interaction energy was calculated, including van der Waals, electrostatic, and torsional energy terms defined in the Tripos force field. The structure optimization was performed for 10000 generations using a genetic algorithm, and the 20-best-scoring ligand−protein complexes were kept for further analysis. The −log(Kd)2 values of the 20-bestscoring complexes, representing the binding affinities of ligand with RT, encompassed a wide range of functional classes (10−2−10−9). Therefore, only the highest-scoring 3D structural model of ligandbound RT was chosen to define the binding interaction. Pharmacokinetics Assays. Ten male Sprague−Dawley rats were randomly divided into two groups to receive intravenous (2 mg·kg−1) or oral administration (24 mg·kg−1). A solution of 25a was prepared by dissolving 25a in a mixture of polyethylene glycol (PEG) 400/ normal saline (70/30, V/V) before the experiment. Blood samples of the intravenous group were collected from the sinus jugularis into heparinized centrifugation tubes at 2 min, 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, and 8 h after dosing, and blood samples of the oral administration group were collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h , 14 h, and 24 h after dosing (200 μL of blood each times). All the samples were then centrifuged at 2200g for 10 min to separate plasma, which was stored at −20 °C until analysis. LC-MS analysis was used to determine the concentration of 25a in plasma. Briefly, 50 μL of plasma was added to 50 μL of internal standard and 300 μL of methanol in a 5 mL centrifugation tube, which was centrifuged at 3000g for 10 min. The supernatant layer was collected, and a 20 μL aliquot was injected for LC-MS analysis. Standard curves for 25a in blood were generated by the addition of various concentrations of 25a together with internal standard to blank plasma. All samples were quantified with an Agilent 1200 LC/MSD (Agilent, USA). The mobile phase was methanol/1.5% glacial acetic acid (50:50, V/V) at a flow rate of 1.0 mL/min, and the test wavelength was 225 nm. All blood samples were centrifuged in an Eppendorf 5415D centrifuge and quantified by Agilent 1200 LC/MSD (Agilent, USA). Acute Toxicity Experiment. Kunming mice (18−22 g) were purchased from the animal experimental center of Shandong University. Compound 25a was suspended in PEG-400 at concentrations of 100 and 200 mg·mL−1, respectively, and administered intragastrically by gavage after the mice had been fasted for 12 h. Dosages of 1000 and 2000 mg·kg−1 were administered to 10 mice per group (5 males, 5 females). The death and body weight were monitored in life observations. Assay Procedures for hERG Activity.35 Inhibition of the hERG potassium channel was examined in HEK293 cells (purchased from ATCC) stably transfected with hERG cDNA. HEK239 cells expressing hERG were plated in 35 mm dishes at least 24 h prior to the experiment and maintained at 37 °C under 5% CO2. A micropipette was drawn out from borosilicate glass to give a tip resistance between 3 and 5 MΩ. For each experiment, a single dish of cells was removed from the incubator, washed twice with ECS at room temperature, and then placed on the microscope stage. A commercial patch clamp amplifier was used for the whole-cell recordings. Tail currents were evoked at room temperature once every 30 s by a 3 s, −50 mV repolarizing pulse following a 2 s, +50 mV depolarizing pulse with a hold voltage of −80 mV. A 50 ms depolarizing pulse to −50 mV at the beginning of the voltage protocol served as a baseline for calculating the amplitude of the peak tail current. Only stable cells with recording parameters above threshold were used for experiments. The hERG currents were allowed to stabilize over a 3 min period in the presence of vehicle alone prior to test compound application. The cells were kept in the test solution until the peak tail current was stable (