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

Article abstract of DOI:10.1021/acs.jmedchem.7b00332

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 NNRTI-resistant 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.

Full text of DOI:10.1021/acs.jmedchem.7b00332

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Article
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
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 May 2017
Downloaded from http://pubs.acs.org on May 9, 2017
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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
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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, PR China
§ Rega Institute for Medical Research, K.U.Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
|The Second Hospital of Shandong University, No. 247 Beiyuan Avenue, Jinan 250033, China.
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 NNRTI-resistant 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,
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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 non-competitive
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
U.S. Food and Drug Administration (FDA) in 2008 and 2011, respectively.9,10 Both of
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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 100,000
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.
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Figure 1. Chemical structures of next-generation NNRTI drugs and thiophene[3,2-d]pyrimidine
lead 3 (K-5a2).
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
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HIV-1 WT and a panel of NNRTI-resistant single- and double-mutant strains19. 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. 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.
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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. Towards this end, six new
series (8, 11, 14, 15, 20 and 25) of thiophene[3,2-d]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 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
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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.
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Figure 2. Strategy for further structure optimization of thiophene[3,2-d]pyrimidine 3.
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 intermediate 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
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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)-3-aminopiperidine 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).
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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).
Scheme 1. Synthesis of 8a-c, 11a-b and 14a-ka
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a
Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, r.t.; (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, r.t.; (iv) substituted benzyl bromide, DMF, K2CO3, r.t.; (v) LiOH,
CH3OH, r.t.
Scheme 2. Synthesis of 15a
a Reagents and conditions: (i) m-CPBA, DCM, r.t.
Scheme 3. Synthesis of 20a-f a
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Ar
CN
O
CN
O
Boc
N
CN
O
CN
O
H
N
N
Cl
N
Cl
i
ii
iii
iv
N
N
Cl
N
NH
N
NH
N
NH
91%
67%
40-66%
S
N
N
N
N
8
9
16
S
S
S
S
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19
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a
Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, r.t.; (ii)
4-(tert-butoxycarbonyl)aminopiperidine, DMF, K2CO3, reflux; (iii) TFA, DCM, r.t.; (iv)
substituted benzyl chloride (or bromide), DMF, K2CO3, r.t.
Scheme 4. Synthesis of 25a-wa
a
Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzaldehyde, DMF, K2CO3, r.t.; (ii)
(EtO)2P(O)CH2CN, t-BuOK, THF, 0°C to r.t.; (iii) 4-(tert-butoxycarbonyl)aminopiperidine, DMF,
K2CO3, reflux; (iv) TFA, DCM, r.t.; (v) substituted benzyl chloride (or bromide) or 4-picolyl
chloride hydrochloride, DMF, K2CO3, r.t.
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 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 (EFV),
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etravirine (ETV), 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
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synthesized compounds are summarized in Tables 1−6.
Table 1. Structures and anti-HIV activity of 8a-c, 11a-b, 14a-k and 15.
EC50(nM)a
SIc
R1
compd
CC50(µM)b
IIIB(nM)
5.69 1.17
134 29.3
723 38.1
>1.17e3
ROD(µM)
IIIB
906
32
-
-
>5.15
>4.24
>27.6
>1.17
6.63 .135
>3.69
>5.92
>9.21
>54.4
>75.2
>2.97
>3.45
>2.76
>20.1
>707
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
8a
8b
-
38
8c
4-SO2NH2
4-CONH2
4-SO2NH2
4-CONH2
3-CONH2
4-NO2
4-SO2CH3
H
<1
11a
11b
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
15
3.22e3 841
42.2 5.50
121 8.5
6
87
63
135 36
68
100 110
57.3 14.2
230 15
544
1315
13
4-Br
339 224
553 181
1.31e3 224
174 58
10
4-F
5
4-OH
3-CF3
4-CN
-
15
4
3.81e3
>5.15
>40.4
≥227
1.3
10706
>159101
>60
3.78 1.25
1.43 0.427
250 65.6
3d
-
-
>0.015
NVP
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-
-
-
5.03 2.12
4.07 0.149
5.81 2.41
-
-
-
>0.00633
>0.00459
>0.00748
>1258
>1128
625
EFV
ETV
AZT
7
8
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against
9
HIV-1-induced cytotoxicity, as determined by the MTT method.
b
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as
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determined by the MTT method.
c SI: selectivity index, the ratio of CC50/EC50.
d Data were reported in ref 19.
Firstly, we focused our attention on novel piperidine-substituted
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. Since 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 develop additional interactions with
Lys103 as hydrogen-bonding 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 anti-HIV 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
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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 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 (EC50 = 8.94 and 7.89 nM, respectively).
However, it was only weakly effective against the K103N+Y181C double mutant
(EC50 = 37.2 nM). As regards 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).
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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).
Based on X-ray co-crystal 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. In order 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
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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
towards 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 towards L100I, Y181C, Y188L,
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F227L+V106A and K103N+Y181C, though it exhibited moderate activity (EC50
=
152 and 175 nM) and low RF values (RF = 3.6 and 4.2) towards 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 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 towards the double mutant strain RES056 (EC50
= 190 nM) was reduced compared to that of ETV (EC50 = 17.0 nM).
Table 3. Structures and anti-HIV activity of 20a-f.
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a
EC50
SIc
compd
R
CC50(µM)b
IIIB(nM)
1.97 0.886
3.56 2.29
152 33.4
ROD(µM)
IIIB
12262
335
20a
20b
20c
4-SO2NH2
4-SO2CH3
4-CONH2
3-CONH2
4-COOC2H5
4-COOCH3
>24.1
>1.19
>4.93
>48.7
>230
>28.6
≥227
-
24.1 26.0
1.19 0.290
4.93 0.372
>48.7
32
20d
20e
5.17 1.50
98.9 34.9
5.26 1.50
1.43 0.427
250 65.6
>9416
>2332
5446
>230
20f
28.6 7.70
>227
3
>159101
>60
NVP
EFV
ETV
AZT
-
-
-
-
>0.0150
>0.00633
>0.00459
>0.00748
5.03 2.12
4.07 0.149
5.81 2.41
-
>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.
b
CC50: 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.
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 bi-heterocyclic ring might lead to the discovery of
new chemical entities (NCEs) with anti-HIV activity. Thus, the bioisosterism strategy
was employed to design thiophene[2,3-d]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.
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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 > para-SO2Me > meta-CONH2 >
para-COOMe > para-COOEt > para-CONH2; this is consistent with our previously
established SAR.19
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Next, the anti-mutant HIV-1 activities of 20a-f were investigated (Table 4). In
general, SAR of the compounds against mutant HIV-1 strains was consistent with that
against WT HIV-1 virus. Compounds 20a-b turned out to be single-figure-nanomolar
inhibitors of L100I, K103N, Y181C, Y188L, E138K, and F227L+V106A mutant
strains, with low RF values. More importantly, 20b inhibited the K103N mutant strain
with EC50 = 1.64 nM, being 3370-, 50- and 1.5-fold more potent than NVP, EFV and
ETV, respectively, and 1.8-fold more potent than 3. However, 20a and 20b exhibited
weak efficacy (EC50 = 38.7 and 42.3 nM, respectively) towards double-mutant strain
K103N+Y181C, compared with ETV (EC50 = 17.0 nM) and were disappointing in this
respect.
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Table 5. Structures and anti-HIV activity of 25a-w.
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60
a
EC50
SIc
compd
R
CC50(µM)b
IIIB(nM)
1.22 0.26
2.32 0.876
1.26 0.118
3.26 2.40
14.6 9.46
5.86 0.716
9.73 6.46
6.34 0.565
28.3 8.76
25.8 3.45
40.2 8.02
25.6 0.935
25.9 2.00
27.4 3.07
7.73 0.890
15.7 5.48
8.91 3.33
5.26 0.544
4.43 0.965
9.66 4.25
3.34 1.05
3.22 2.56
3.36 1.85
1.43 0.427
250 65.6
ROD(µM)
>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
-
IIIB
1882
775
25a
25b
25c
25d
25e
25f
4-SO2NH2
4-SO2CH3
4-CONH2
3-CONH2
4-COOC2H5
4-F
2.30 0.468
1.804 0.461
≥3.15
≥2504
1084
≥247
441
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
25g
25h
25i
3-F
489
2-F
392
4-Br
240
25j
3-Br
308
25k
25l
2-Br
140
4-Cl
156
25m
25n
25o
25p
25q
25r
25s
25t
3-Cl
188
2-Cl
179
4-NO2
456
2-CH3
180
4-CH3
326
4-CN
509
3-CN
639
2-CN
236
25u
25v
25w
3
pyridine-4-yl
pyridine-3-yl
pyridine-2-yl
1429
1199
1146
>159101
>60
NVP
EFV
ETV
-
-
-
>0.0150
5.03 2.12
4.07 0.149
-
>0.00633
>0.00459
>1258
>1128
-
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AZT
-
5.81 2.41
-
>0.00748
625
4
5
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.
b
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as
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determined by the MTT method.
c SI: selectivity index, the ratio of CC50/EC50.
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Next, we set out to improve the activity by replacing the cyano group in the right
wing with cyano vinyl, affording derivatives 25a-25w, based on the widely employed
strategy of targeting highly conserved residues (see the following section). The
performance of these derivatives toward WT and mutant strains of HIV-1 is
summarized in Tables 5 and 6. Generally speaking, this chemical modification
resulted in a considerable increase of antiviral activity. All the compounds displayed
potent activity against WT HIV-1 with EC50 values ranging from 1.22 to 40.2 nM,
being superior to the reference drug NVP. Compounds 25a-d and 25u-w with
hydrophilic polar groups showed more potent activity than EFV and ETV, and 25a
and 25c proved to be the most potent, with EC50 values of 1.22 and 1.26 nM,
respectively.
Further variations in antiviral activity were observed when the para-SO2NH2 and
para-CO2NH2 substituents in 25a and 25c were replaced with other substituents.
Replacement with para-SO2CH3 (25b), meta-CO2NH2 (25d), pyridine (25u-w), CN
(25r-t), F (25f-h), meta-NO2 (25o) and meta-CH3 (25q) slightly decreased the activity
(EC50 < 10 nM). Meta-COOC2H5 (25e), Br (25f-h), Cl (25f-h) and ortho-CH3 (25p)
substituted derivatives showed even weaker activity (EC50 = 17.6－40.2 nM). These
results confirmed that a hydrophilic substituent in this region was essential for high
antiviral potency, and the order of potency of the substitution at the right phenyl ring
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was as follows: SO2NH2 > CONH2 > 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).
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Against K103N, 25a was 2.4-fold more potent than ETV (EC50 = 0.958 nM 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) 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).
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
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F227L+V106A (EC50 = 4.67－26.7 nM) more potently than did ETV.
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9
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
towards double-mutant strain K103N+Y181C.
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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, 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 ꢀM compared to ETV (IC50 = 1.00 ꢀM), which
was consistent with its less potent antiviral activities. Compounds 20a, 25a, and 25b
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showed excellent affinity to HIV-1 RT (IC50 = 0.42, 0.16, and 0.89 ꢀM, respectively)，
being superior to that of ETV. The reasonable results demonstrated that the newly
synthesized representative derivatives showed high affinity to WT HIV-1 RT, and
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prove the viewpoint that they are acted as classical NNRTIs.
Table 7. Inhibitory activity of compounds 14a, 20a, 25a, and 25b against WT HIV-1 RT
Compd.
14a
20a
25a
25b
ETVb
IC50 (µM)a
4.02
0.42
0.16
0.89
1.00
aIC50: 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
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 conducted with SurflexeDock SYBYL-X 2.0 software.
PyMOL was used to visualize the results. The docking protocol is described in the
computational section.
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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 (yellow). 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).
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
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well-known interactions: (i) the position of the 4-cyanovinyl-2,6-dimethylphenyl
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moiety is stabilized by π-stacking interactions with aromatic side chains of Tyr181,
8
9
Tyr188, Phe227 and Trp229; (ii) the thiophene ring of 25a effectively occupies the
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NNIBP entrance channel, and the sulphur 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 Lys103; (v) the piperidine-linked benzene
sulfonamide 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 Trp229,31 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
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variety of mutant strains, compared to 3.
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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.
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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 water-mediated 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
water-mediated hydrogen bonds. Moreover, 8b and 11a lack critical
hydrogen-bonding interactions with Lys101, which may account for their sharply
reduced potency towards 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 ꢀg/mL (552 nM),
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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.
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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 two weeks.
All these results support the potential of 25a as a new, highly potent, orally
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bioavailable NNRTI drug candidate.
Table 8. Pharmacokinetic profile of 25aa
parameter
AUC(0-t)
AUC(0-∞)
AUMC(0-t)
AUMC(0-∞)
MRT(0-t)
MRT(0-∞)
VRT(0-t)
VRT(0-∞)
t1/2z
unit
po b
2316.8 520.2
2692.2 872.1
8755.2 2972.5
15978.8 11014.0
3.71 0.53
iv c
ug/L*h
ug/L*h
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
5.54 1.87
h^2
h^2
h
8.11 1.44
35.67 27.1
3.93 1.54
Tmax
h
1.7 0.67
CLz/F
L/h/kg
L/kg
ug/L
0.008 0.002
0.044 0.019
552.9 88.9
16.19
0.0145 0.002
0.0275 0.002
2217.1 148.6
Vz/F
Cmax
F (%)
a
PK parameters (mean SD, n = 5); b Dosed orally at 24 mg/kg; c Dosed intravenously at 2
mg/kg.
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Figure 5: The plasma 25a concentration–time profiles in rats following oral administration (24
mg.kg-1) and intravenous administration (2 mg.kg-1).
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 ꢀM against the potassium channel; thus, its inhibitory activity is lower
than that of terfenadine (IC50 = 0.022 ꢀM), suggesting that cardiotoxicity is not likely
to be a major risk for 25a.
Figure 6. Activity of 25a and terfenadine against hERG potassium channel in HEK293 cells.
CONCLUSION
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We previously reported 3 as a potent inhibitor of various mutant HIV-1 viruses,
but it showed weaker potency than ETV towards the most common single mutant
K103N and double mutant 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 (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, water-mediated hydrogen bonds
with Lys103 and double hydrogen-bonding 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.
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Overall, 25a proved to be exceptionally potent, with EC50 values of 1.22 nM
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(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. Based on 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 micro melting 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
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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 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 ꢀm 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 ꢀL. Purity of all tested compounds was > 95%.
4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)amino)piperidi
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n-1-yl)methyl)benzenesulfonamide
(3)
and
4-((2-chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile
prepared as previously reported.19
(5)
were
4-((2-((3-fluoropiperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimeth
ylbenzonitrile (7a). solution of (1.0 g, 3.17 mmol), tert-butyl
A
5
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 minutes and the resulting
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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):
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δ
8.49 (d, 1H, J = 7.3 Hz, C6-thienopyrimidine-H), 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 for 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
1
solid, 68% yield, mp: 154-156°C. H 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
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