Discovery of potent HIV-1 non-nucleoside reverse transcriptase inhibitors by exploring the structure–activity relationship of solvent-exposed regions I

Article abstract of DOI:10.1111/cbdd.13429

Two novel series of human immunodeficiency virus-1 (HIV-1) non-nucleoside reverse transcriptase inhibitors (NNRTIs) bearing a thiophene[3,2-d]pyrimidine scaffold and sulfonamide linker in the right wing have been identified, which demonstrated activity against the wild-type (WT) HIV-1 strain in MT-4 cells with inhibitory concentrations ranging from micromolar to submicromolar. Especially, against the mutant strains K103N and E138K, most compounds exhibited more potent activity than against WT HIV-1. Compound 7 (EC₅₀?=?0.014, 0.031?μM) achieved the most potent activity against the two mutants, being more effective than that of nevirapine (NVP, EC₅₀?=?7.572, 0.190?μM) and comparable to that of etravirine (ETV, EC₅₀?=?0.004, 0.014?μM). Molecular docking experiments on the novel analogs have also suggested that the extensive network of main chain hydrogen bonds are important in the binding mode, which may provide valuable insights for further optimization.

Full text of DOI:10.1111/cbdd.13429

DR ZHAO WANG (Orcid ID : 0000-0001-5431-1785)
DR PENG ZHAN (Orcid ID : 0000-0002-9675-6026)
PROFESSOR XINYONG LIU (Orcid ID : 0000-0002-5833-3807)
Article type
: Research Article
Correponding author mail id: xinyongl@sdu.edu.cn
Discovery of potent HIV-1 non-nucleoside reverse transcriptase inhibitors
by exploring the structure-activity relationship of solvent-exposed regions I
Dongwei Kang,† Zhao Wang,† Meng Chen, | Da Feng,† Gaochan Wu,† Zhongxia Zhou,†
Lanlan Jing,† Xiaofang Zuo, † Xiangyi Jiang, † 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
|Shandong Center for Disease Control and Prevention, 250014 Jinan, Shandong, PR China
§Rega Institute for Medical Research, K.U.Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
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doi: 10.1111/cbdd.13429
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ABSTRACT:
Two novel series of human immunodeficiency virus-1 (HIV-1) non-nucleoside reverse
transcriptase inhibitors (NNRTIs) bearing a thiophene[3,2-d]pyrimidine scaffold and
sulfonamide linker in the right wing have been identified, which demonstrated with active
potency against wild-type (WT) HIV-1 strain in MT-4 cells with inhibitory concentrations
ranging from micromolar to submicromolar. Especially, against the mutant strains K103N
and E138K, most compounds exhibited more potent activity than against WT HIV-1.
Compound 7 (EC50 = 0.014, 0.031 µM) achieved the most potent activity against the two
mutations, being more effective than that of nevirapine (NVP, EC50 = 7.572, 0.190 µM) and
comparable to that of etravirine (ETV, EC50 = 0.004, 0.014 µM). Molecular docking
experiments on the novel analogues have also suggested that the extensive network of main
chain hydrogen bonds are important in the binding mode, which may provide valuable
insights for further optimization.
Keywords: HIV-1, NNRTIs, DAPY, thiophene[3,2-d]pyrimidine, solvent-exposed region I
1. Introduction
HIV-1 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 in view of their potent
antiviral activity, high selectivity, and favorable pharmacokinetics.[1, 2] Currently, there are
five FDA-approved NNRTIs administered as a combination therapy in HAART, including
the first generation NNRTIs nevirapine (NVP), efavirenz (EFV), delavirdine (DLV) and the
second generation NNRTIs etravirine (ETV, 1) and rilpivirine (PRV, 2)[3, 4] (Figure 1).
Although HAART has greatly reduced the morbidity and mortality of AIDS, the increasing
incidence of drug resistant continues being a major cause of treatment failure[5]. Among all
the mutant strains, K103N and Y181C are the two most prevalent mutations selected by NVP
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and EFV[6], E138K are the mutant strains most frequently selected by RPV[7]. In addition,
hypersensitivity reactions and some other drug toxicity have been reported with second
generation NNRTIs[8]. Therefore, searching for novel chemical skeletons with improved drug
resistance profiles is actively pursed.
According to a wide range of co-crystal structures of HIV-1 reverse transcriptase
(RT)/NNRTIs complexes[9, 10], the diarylpyrimidines (DAPY) derivatives featured a similar
pharmacodynamics interactions: 1) the left aryl wing develops hydrophobic interaction with
Tyr181, Tyr188, Phe227 and Trp229; 2) the NH linker between the central pyrimidine and
the right wing and the N atom of the pyrimidine participates in the conserved hydrogen bonds
with the backbone of Lys101; 3) the right wing forms extensive interactions with the
surrounding lipophilic sub-pocket defined by Val106, Pro236, and Tyr318, which was named
the “solvent-exposed region I”. In the structure-based lead optimization phase of drug
development, solvent-exposed protein regions are known as prospective sites for
modification for the reason that they have larger spaces to accommodate diverse structures
with different structures and electrical properties, which contribute to achieve additional and
specific protein-ligand interactions with the target protein. Thus, exploiting solvent-exposed
regions is becoming more conventional strategy for enhancing binding affinity, improving
potency, and overcoming drug resistance profiles.
Previous research efforts in our lab for exploiting the solvent-exposed regions of HIV-1
RT achieved the discovery of piperidine-substituted thiophene[3,2-d]pyrimidine K-5a2,
which exhibited broad-spectrum activity with single-digit nanomolar EC50 values against a
panel of wild-type (WT) and mutant HIV-1 strains[11-13]. Furthermore, subsequent structural
exploration was focus on the solvent-exposed region I of the NNRTIs binding pocket
(NNIBP) and the 4-aminopiperidine moiety of the K-5a2 was replaced with
cyclohexanediamine, resulting in a conclusion that the linker between the
cyclohexanediamine and aryl group exhibited significant influence to anti-HIV-1 activity
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(Figure 2). The sulfamide linker was turned to be the most efficient privilege structure [14, 15]
Compound ACS-9d proved to be the most effective inhibitor and exhibited
.
single-figure-nanomolar activity (EC50 = 7.1 nM) against WT HIV-1 strain. However,
ACS-9d showed decreased activities against other single and double mutant strains compared
to ETV. With the aim to further explore the structure-activity relationship (SAR) of the right
wing and generate more effective inhibitors with improved potency against
resistance-associated variants, we designed subseries-5 with the strategy of molecular
hybridization by introducing the sulfonamide linker to the aminopiperidine of the K-5a2. The
subseries-8 was designed based on the strategy of scaffold hopping, by replacing the
cyclohexane ring with benzene ring. Substituents with structural diversity were introduced to
the sulfamide linker and point to the solvent-exposed region I. We hope the newly introduce
sulfamide linker and substituents could develop additional interactions with the amino acid in
solvent-exposed region I and improve activities against mutant HIV-1 strains.
In this study, we report the synthesis, antiviral activity and molecular simulation of the
newly designed compounds. Preliminary SARs are also discussed in detail to gain further
insights into this series of analogues.
2. Results and discussion
2.1 Chemistry
The synthetic protocols for the novel designed derivatives are outlined in Schemes 1−2.
As depicted in Scheme 1, the synthetic sequence started from
2,4-dichlorothiophene[3,2-d]pyrimidine with 3,5-dimethyl-4-hydroxybenzonitrile in dimethyl
formamide (DMF) to give 2. Subsequently, treatment of 2 with
4-(tert-butoxycarbonyl)aminopiperidine brought 3 via nucleophilic substitution. Remove the
t-butyloxycarboryl (Boc) protective group of 3 with trifluoroacetic acid (TFA) in
dichloromethane (DCM) gave 4, followed by acylation reaction in the presence of substituted
sulfonyl chloride to achieve the target compounds 5a-k. The synthetic routes for target
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compounds 8a-i was similar to that of 5a-k, with the difference that the intermediate 6 was
obtained by Buchwald-Hartwig cross coupling reaction of 2 with tert-butyl
(4-aminophenyl)carbamate in the presence of
(±)-2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (BINAP) and PdCl2(PPh3)2 (Scheme 2).
2.2 Biological evaluations
The antiviral potency of the newly synthesized derivatives were evaluated in MT-4 cell
cultures infected with WT HIV-1 strain (IIIB), NNRTI-resistant single-mutant strains L100I,
K103N, Y181C, Y188L, E138K and double-mutant strains F227L+V106A and
K103N+Y181C (RES056). Nevirapine (NVP) and Etravirine (ETV) were selected as control
drugs[12]. The results were expressed as EC50 (anti-HIV potency), CC50 (cytotoxicity) and SI
(selectivity index, CC50/EC50 ratio) (Tables 1 and 2).
As depicted in Table 1, all the compounds exhibited effective potency against WT
HIV-1 with EC50 value from micromolar to submicromolar (EC50 = 0.058-3.60 µM), with an
exception of 8f (EC50 = 12.55 µM). Among them, 7 (EC50 = 0.058 µM) and 8b (EC50 = 0.066
µM) were proved to be the most potent inhibitors, being about 5-fold more potent than NVP
(EC50 = 0.31 µM), but their activities less potent than that of ETV (EC50 = 0.004 µM). All
compounds were inactive to HIV-2.
Preliminary SAR analysis demonstrated that incorporation of sulfuryl group to the right
wing (aniline and piperidine) could result in a decreased activity of the compounds and the
bulky substituent group appeared to have a more negative effect on the potency, such as
compounds 8f (EC50 = 12.55 μM) and 8h (EC50 = 3.60 μM).
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Next, the anti-mutant HIV-1 activities were investigated. As depicted in Table 2, all the
target compounds exhibited moderate potency against single mutant HIV-1 strains (L100I,
K103N, Y181C, Y188L, and E138K) and double mutant HIV-1 strain F227L+V106A, but all
of them lost their activity to double mutant strain RES056. A brief investigation of SARs
revealed that the activity of the compounds against mutant HIV-1 strains was consistent with
that against WT HIV-1 virus. Compounds 7 (EC50 = 0.014-0.77 µM) and 8b (EC50 =
0.044-2.77 µM) inhibited most common single mutations and double mutations
F227L+V106A in the micromolar concentration range. More encouragingly, the results
demonstrated that their activity to mutant strains K103N and E138K, was superior to the
activity against WT HIV-1 strain. Especially, the activity of 7 (EC50 = 0.014, 0.031 µM) was
more effective than that of NVP (EC50 = 7.57, 0.19 µM) and comparable to that of ETV
(EC50 = 0.004, 0.014 µM) toward the two mutations, which could be regarded as promising
lead compound for drug discovery overcoming the drug resistance profiles.
2.3 Molecular modeling analysis
To shed light on the binding modes of these novel thiophene[3,2-d]pyrimidine
derivatives, the representative compound 8b was docked into the WT HIV-1 NNRTIs
binding pocket (PDB code: 3MEC[10]) and K103N mutant NNRTIs binding pocket (PDB
code: 3MED[10]) using the software SurflexeDock SYBYL-X 2.0. PyMOL was used to
visualize the results. The docking protocol was same with our established method
previously[12].
As shown in Figure 3A, 8b adopted a typically horseshoe-like conformation in the
NNIBP, the binding mode was similar to that of the lead compound, featuring mainly
well-known pharmacophoric interactions: (i) The left wing structure
(4-cyano-2,6-dimethylphenoxy) of 8b occupied the hydrophobic subpocket formed by
aromatic amino acid residues Tyr181, Tyr188, Phe227 and Trp229, developing hydrophobic
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interactions with these amino acid residues; (ii) The right piperidine ring of 8b formed close
van der Waals interactions with the lipophilic side chains of Lys103, Val106, Phe227, and
Pro236; (iii) NH linker connecting the central pyridine ring and the piperidine ring formed a
conserved hydrogen bond with the backbone of Lys101.
In addition, the newly introduced thiophene ring project into the entrance tunnel
surrounded by Val179 and Glu138, and the sulphur atom establish p-π interactions with them;
the benzene-linked sulfuric diamide group develops hydrogen-bonding interactions with the
backbone of His235 as hydrogen bond donor, providing a rational explanation of the
relatively improved activity of 8b compared to other compounds. However, compared with
ETV, 8b lost the crucial hydrogen-bonding interaction between the N atom of the
thiophene[3,2-d]pyrimidine and the backbone of Lys101, which account for its decreased
potency.
When the Lys103 mutates into Asn103 (K103N) (Figure 3B), the binding mode of 8b
with K103N RT was similar to that with WT RT, only with the difference that sulfuric
diamide group develops double hydrogen-bonding interactions with the backbone of His235
and Tyr318, which probably plays an essential role in improving its activity against the
K103N mutant than WT HIV-1.
2.4 In silico prediction of physicochemical properties
Furthermore, the potent compounds 7 and 8b were predicted their druglikeness features
using the free on-line software molinspiration (http://www.molinspiration.com/)[13]. As
depicted in Table 3, the molecular weight (MW), hydrogen bond acceptors (nON), hydrogen
bond donors (nOHNH), rotatable bonds (nrotb), and Logp of the compounds 7 and 8b abide
by the Lipinski's rule of five, with an exception of the Log P of the 7 over the limit value
(Log P = 5.07). In addition, Lipophilic parameter ligand efficiency (LE) and Ligand
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efficiency dependent lipophilic (LELP) were also calculated and conformed well to the
rule[16]. Therefore, it is supposed that 7 and 8b have the desired physicochemical properties
for further lead optimization.
3. Conclusion
We have reported herein efforts to exploit the chemical space of solvent-exposed regions
I of the NNIBP toward generate more effective inhibitors with improved drug resistance
profiles. Based on the molecular hybridization and scaffold hopping strategy, a series of
novel DAPY derivatives was designed. Most of them showed moderate to excellent potency
against WT HIV-1 strain, compounds 7 and 8b proved to be the most potent inhibitors with
EC50 value of 0.058 and 0.066µM, respectively. Notably, compound 7 was demonstrated with
more active potency against the single mutant HIV-1 strains K103N and E138K (EC50 =
0.014 and 0.031 µM, respectively), being comparable to that of ETV (EC50 = 0.004, 0.014
µM). Furthermore, molecular simulation analysis suggested that the double
hydrogen-bonding interactions between the sulfuric diamide group and the backbone of
His235 and Tyr318 contribute to the improved drug resistance profiles. Further optimization
of the scaffold is currently underway and will be reported in due course.
Supporting information
Supportinng Information including the synthetic produce and spectral data of the target
compounds.
Notes
The authors declare no competing financial interest.
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Acknowledgements
We gratefully acknowledge financial support from the National Natural Science
Foundation of China (NSFC Nos. 81420108027, 81573347), Young Scholars Program of
Shandong University (YSPSDU No. 2016WLJH32), the Fundamental Research Funds of
Shandong University (No. 2017JC006), and Key research and development project of
Shandong Province (No. 2017CXGC1401) and KU Leuven (GOA 10/014).
List of the figure legends
Figure 1: Chemical structures of next-generation NNRTI drugs.
Figure 2. Rational design of target compounds by employing molecular hybridization and
scaffold hopping strategy.
Figure 3. (A) Predicted binding mode of 8b with the HIV-1 WT RT (PDB: 3MEC); (B) Predicted
binding mode of 8b with the HIV-1 K103N RT (PDB: 3MED); Hydrogen bonds are indicated with
dashed lines (yellow). Nonpolar-hydrogen atoms are not shown for clarity.
Scheme 1. Synthesis of target compounds 5a-k. Reagents and conditions: (i)
3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, r.t.; (ii) 4-(tert-butoxycarbonyl)aminopiperidine,
DMF, K2CO3, 120°C; (iii) TFA, DCM, r.t.; (iv) substituted sulfonyl chloride, DCM, Et3N, 0°C to r.t.
Scheme 2. Synthesis of target compounds 8a-i. Reagents and conditions: (i)
3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, r.t.; (ii) tert-butyl (4-aminophenyl)carbamate,
BINAP, PdCl2(PPh3)2, Cs2CO3, 1,4-dioxane, 120°C; (iii) TFA, DCM, r.t.; (iv) substituted sulfonyl
chloride, DCM, Et3N, 0°C to r.t.
Table 1. Anti-HIV activity against HIV-1 (IIIB) and RES056 strains and cytotoxicity of
5a-k, 7 and 8a-i
Table 2. Anti-HIV-1 activity against mutant strains and cytotoxicity
Table 3. Physicochemical properties and LE, LELP of 7 and 8b
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Graphical Table of Contents
Figure 1: Chemical structures of next-generation NNRTI drugs
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Figure 2. Rational design of target compounds by employing molecular hybridization and
scaffold hopping strategy.
Figure 3. (A) Predicted binding mode of 8b with the HIV-1 WT RT (PDB: 3MEC); (B) Predicted
binding mode of 8b with the HIV-1 K103N RT (PDB: 3MED); Hydrogen bonds are indicated with
dashed lines (yellow). Nonpolar-hydrogen atoms are not shown for clarity.
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Scheme 1. Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, r.t.; (ii)
4-(tert-butoxycarbonyl)aminopiperidine, DMF, K2CO3, 120°C; (iii) TFA, DCM, r.t.; (iv) substituted
sulfonyl chloride, DCM, Et3N, 0°C to r.t.
Scheme 2. Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, r.t.; (ii)
tert-butyl (4-aminophenyl)carbamate, BINAP, PdCl2(PPh3)2, Cs2CO3, 1,4-dioxane, 120°C; (iii) TFA,
DCM, r.t.; (iv) substituted sulfonyl chloride, DCM, Et3N, 0°C to r.t.
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Table 1. Anti-HIV activity against HIV-1 (IIIB) and RES056 strains and cytotoxicity of
5a-k, 7 and 8a-i
EC50 (μM)a
Compds
R
CC50 (μM)b
SI (IIIB)c
IIIB
ROD
CH3
0.58±0.065
>22.21
22.21±7.48
6.19±2.37
39
18
84
67
5a
5b
5c
5d
0.35±0.143
0.24±0.089
0.33±0.109
>6.185
>20.35
>21.98
NH2
20.35±7.119
21.98±13.99
0.74±0.65
0.29±0.11
>219.4
>56.92
>219.4
>295
193
5e
5f
56.92±51.34
0.85±0.45
0.44±0.28
1.10±0.54
0.22±0.12
>81.73
>20.98
>209.8
>236.4
81.73±66.46
20.98±13.94
>209.8
96
48
5g
5h
5i
>191
>1059
>236.4
5j
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1.35±0.59
>233.79
>233.7
>173
5k
-
0.058±0.029
1.77±0.61
>19.39
>268.4
>25.06
19.39±12.60
>268.4
335
>151
378
7
CH3
NH2
8a
8b
0.066±0.019
25.06±5.24
0.58±0.54
>19.65
19.65±11.73
12.66±6.60
34
45
8c
0.28±0.071
>12.664
8d
1.17±0.36
>216.3
>216.3
>185
8e
12.55±2.04
0.32±0.085
3.60±1.11
1.35±0.32
>209.8
>46.73
>207.0
>91.40
>209.8
46.73±24.64
>207.0
>17
148
>57
68
8f
8g
8h
8i
91.40±61.02
-
-
0.31±0.056
>7.590
>4.585
>15.02
>4.585
>48.08
>1818
NVP
ETV
0.004±0.001
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. A smaller number means that the compound has a higher activity.
b CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT
method. A larger number means that the compound has a lower toxicity.
c SI: selectivity index, the ratio of CC50/EC50.
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Table 2. Anti-HIV-1 activity against mutant strains and cytotoxicity
EC50 (μM)
Compds
L100I
K103N
Y181C
Y188L
E138K
F227L+V106A
RES056
5a
5b
5c
4.26±0.45
≥8.83
0.31±0.073
0.48±0.077
0.30±0.047
0.48±0.27
0.37±0.01
0.15±0.021
0.54±0.18
0.57±0.13
3.21±0.005
0.57±0.075
1.98±0.541
0.014±0.001
2.11±0.61
0.053±0.007
0.33±0.062
0.14±0.071
0.98±0.089
7.49±1.06
0.20±0.035
2.75±0.30
0.86±0.38
7.53±0.22
0.004±0.001
6.30±0.366
>6.19
6.60±0.97
>6.19
0.31±0.089
0.26±0.056
0.21±0.033
0.40±0.075
0.72±0.039
0.23±0.040
0.77±0.056
0.66±0.095
0.59±0.037
0.056±0.017
3.12±0.024
0.031±0.004
1.18±0.34
2.88±1.13
>6.185
>22.21
>6.185
>20.35
>21.98
>219.4
>56.92
>81.73
>20.98
>209.8
>236.4
>233.79
>19.39
>268.4
>25.06
>19.65
>12.66
>216.3
>209.8
>46.73
>207.0
>91.40
>7.59
4.16±0.99
10.29±9.928
4.09±1.59
1.87±0.005
9.23±1.97
4.32±0.25
191.7±21.50
41.31±25.63
14.18±1.863
0.28±0.061
17.91±0.32
0.71±0.083
2.03±0.48
2.89±2.15
5.53±0.90
108.7±10.70
1.63±0.35
52.79±4.17
3.45±0.11
0.74±0.17
0.010±0.003
4.72±0.82
≥16.73
5.96±1.61
≥15.18
3.67±0.12
≥15.18
5d
5e
4.78±0.40
0.76±0.013
6.17±0.75
3.73±0.045
146.5±44.75
64.55±15.76
17.33±0.005
0.78±0.009
33.79±2.612
1.69±0.27
4.82±0.165
5.86±0.23
110.1±19.63
>209.8
3.42±0.64
1.04±0.107
10.73±3.87
5.78±1.849
171.1±47.31
126.2±9.123
≥35.51
29.43±13.47
≥30.08
5f
5g
5h
5i
7.93±0.51
5.73±1.56
177.7±15.25
59.42±14.48
>233.7
5j
5k
7
0.79±0.043
23.81±1.74
2.77±0.38
7.74±0.98
≥7.15
0.77±0.11
4.19±1.95
0.24±0.092
6.04±0.45
4.60±1.49
57.82±8.26
>209.8
8a
8b
8c
0.044±0.005
0.44±0.20
8d
8e
0.33±0.025
1.58±0.25
127.8±26.30
>209.8
8f
31.15±2.812
0.38±0.12
8g
8h
8i
5.43±0.63
>207.0
6.69±0.54
>207.0
4.14±0.31
≥163.8
4.93±1.74
36.81±5.91
-
41.04±1.16
-
1.93±0.048
0.19±0.087
0.014±0.002
7.86±0.789
-
NVP
ETV
0.019±0.007
0.020±0.003
0.023±0.011
0.25±0.001
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Table 3. Physicochemical properties and LE, LELP of 7 and 8b
Compd
MW
nONa
nOHNHa
nrotba
LogPa
LEb
LELPc
Accepted range
< 500
335.91
466.55
< 10
< 5
3
≤ 10
< 5
5.07
4.61
> 0.3
0.34
0.31
< 16.5
14.53
15.21
6
9
4
6
7
4
8b
a Using free on-line software (http://www.molinspiration.com/);
bLE = calculated by the formula −ΔG/HA (non‑ hydrogen atom), ΔG = RT ln Kd, pre-suming Kd ≈
EC50 (IIIB); R= 1.987 × 10 –3 kcal/K/mol, T = 298 K.
c LELP = Log P/LE
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