Exploiting the Tolerant Region i of the Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) Binding Pocket: Discovery of Potent Diarylpyrimidine-Typed HIV-1 NNRTIs against Wild-Type and E138K Mutant Virus with Significantly Improved Water Solubility an

Article abstract of DOI:10.1021/acs.jmedchem.8b01729

Diarylpyrimidine derivatives (DAPYs) exhibit robust anti-HIV-1 potency, although they have been compromised by E138K variant and severe side-effects and been suffering from poor water solubility. In the present work, hydrophilic morpholine or methylsulfon

Full text of DOI:10.1021/acs.jmedchem.8b01729

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Article
Exploiting the Tolerant Region I of Non-nucleoside Reverse Transcriptase
Inhibitor Binding Pocket (NNIBP): Discovery of Potent Diarylpyrimidine
(DAPY)-typed HIV-1 NNRTIs against Wild-Type and E138K Mutant Virus
with Significantly Improved Water Solubility and Favorable Safety Profiles
Boshi Huang, Wenmin Chen, Tong Zhao, Zhenyu Li, Xiangyi Jiang, Tiziana Ginex,
David Vílchez, F. Javier Luque, Dongwei Kang, Ping Gao, Jian Zhang, Ye Tian, Dirk
Daelemans, Erik De Clercq, Christophe Pannecouque, Peng Zhan, and Xinyong Liu
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 05 Feb 2019
Downloaded from http://pubs.acs.org on February 5, 2019
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for Medical Research, Microbiology and Immunology
Zhan, Peng; Shangdong University, Department of Medicinal Chemistry
Liu, Xinyong; School of Pharmaceutical Sciences, Shandong University,
Department of Medicinal Chemistry
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Exploiting the Tolerant Region I of Non-nucleoside Reverse
Transcriptase Inhibitor Binding Pocket (NNIBP): Discovery of
Potent Diarylpyrimidine (DAPY)-typed HIV-1 NNRTIs against
Wild-Type and E138K Mutant Virus with Significantly Improved
Water Solubility and Favorable Safety Profiles
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†
,§
†,&
†
‡
†
Boshi Huang, Wenmin Chen, Tong Zhao, Zhenyu Li, Xiangyi Jiang, Tiziana
&
&
&
†
†
Ginex, David Vílchez, Francisco Javier Luque, Dongwei Kang, Ping Gao, Jian
†
†
#
#
#,*
Zhang, 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.
‡
Department of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong
University, Jinan 250021, Shandong, China.
&Department of Nutrition, Food Science and Gastronomy, Faculty of Pharmacy,
Campus Torribera, Institute of Biomedicine (IBUB) and Institute of Theoretical and
Computational Chemistry (IQTCUB), University of Barcelona, 08921 Santa Coloma
de Gramenet, Spain.
#
Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy,
K.U.Leuven, Herestraat 49 Postbus 1043 (09.A097), B-3000 Leuven, Belgium.
ABSTRACT:
Diarylpyrimidine derivatives (DAPYs) exhibit robust anti-HIV-1 potency, while
they have been compromised by E138K variant and severe side-effects, and been
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suffering from poor water solubility. In the present work, hydrophilic morpholine or
methylsulfonyl and sulfamide substituted piperazine/piperidines were introduced into
the right wing of DAPYs targeting the solvent-exposed tolerant region I. The
anti-HIV-1 activities of 11c (EC50 (WT) = 0.0035 μM, EC50 (E138K) = 0.0075 μM) were
the same as and 2-fold better than that of the lead etravirine against the wild-type and
E138K mutant HIV-1 respectively, with a relative low cytotoxicity (CC ≥ 173 μM).
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Further test showed a significant improvement in the water solubility of 11c. Besides,
1c displayed no significant inhibition on main cytochrome P450 enzymes, and
1
-1
-1
exhibited no acute/subacute toxicities at doses of 2000 mg·kg /50 mg·kg in mice.
Taken together, we consider 11c is a promising lead for further structural
optimization.
INTRODUCTION
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs), which disrupt the
normal functions of RT via binding to NNRTI binding pocket (NNIBP) close to the
1
polymerase active site, have been a vital component of highly active antiretroviral
2
therapy (HAART) for the treatment of HIV-1 infection. Unfortunately, the clinical
application of NNRTIs is seriously compromised by rapid emergence of HIV-1
drug-resistant variants.3
In arduous efforts to develop next-generation NNRTIs with improved anti-drug
resistance profiles, etravirine (ETR, TMC125) and rilpivirine (RPV, TMC278)
4
(
Figure 1) have been approved by U.S. FDA in 2008 and 2011, respectively. Both of
them belong to diarylpyrimidine derivatives (DAPYs) and show robust antiviral
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potency against wild-type (WT) and mutant HIV-1 strains at nanomolar
5
6
concentrations. However, DAPYs have been suffering from poor solubility. ETR is
practically insoluble in water over a wide pH range (<< 1 μg/mL at pH 7.0), leading
to a daily dosage of 400 mg due to extensive formulation work. Besides, the absolute
oral bioavailability of ETR in human is still unknown. RPV is hardly dissolved in
water (20 ng/mL at pH 7.0), which makes it to display an atypical absorption
mechanism involving aggregates. The daily dosage for another NNRTI drug
nevirapine (NVP) is 400 mg as for ETR, whereas its antiviral activity against WT
HIV-1 strain is approximately 100-fold less than that of ETR. Undoubtedly, a crucial
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factor is that the aqueous solubility of NVP is 167 μg/mL. The low solubility of
DAPYs was therefore identified as an intractable issue to address.
CN
CN
CN
CN
NH
O
N
NH
HN
N
N
Br
N
NH2
ETR (TMC125, 1)
RPV (TMC278, 2)
Figure 1. Chemical structures of DAPY-typed NNRTI drugs.
Moreover, Steegen et. al. found that only about one-third of patients could retain
full susceptibility to the two DAPY drugs (for ETR, 36.5%; for RPV, 27.3%).8
Among the NNRTI resistance-associated mutations (RAMs), E138K substitution in
RT is the first of these ETR selected mutations in B and non-B HIV-1 subtypes
(non-B subtype infections represent > 90% of the global HIV problem), and is also
the most prevalent single mutation found in patients who failed RPV-containing
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therapy.9,10 E138K mutation confers phenotypic resistance to the first-generation
NNRTIs NVP and efavirenz (EFV) as well.11 The broad cross-resistance profiles
against almost all of the NNRTI drugs indicate the development of E138K must be
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restrained whenever possible. In addition, ETR shows severe side-effects, such as
peripheral neuropathy, skin rash and hepatotoxicity, which was ever listed as
“dangerous drugs” by US FDA in 2008. Thus, it is still urgently needed to identify
novel DAPY-typed HIV-1 NNRTIs endowed with high potency against WT and
resistance-associated variants especially E138K, as well as with improved water
solubility and favorable safety profiles.
The crystallographic studies of DAPYs showed that the right aminobenzonitrile
motif pointed to a rather plastic “groove” of NNIBP, termed as tolerant region I. This
open tolerant region, composed of V106, F227, Y318 and P236 residues of the
binding pocket, is extended to the RT/solvent interface and could well accommodate
1
3
diverse substituents (Figure 2). Introduction of polar or hydrophilic substituents,
like morpholine, piperazine and piperidine, into the right wing of DAPYs around the
tolerant region I may contribute to improvement of solubility or pharmacokinetic
properties. Based on this analysis, we previously synthesized a series of morpholine
substituted pyridine-type DAPY derivatives. However, the most active inhibitor
WC-II-4b exhibited decreased activity against WT HIV-1 strain compared to that of
ETR and was totally inactive against the most frequently encountered K103N/Y181C
double mutant HIV-1 strain, though it demonstrated enhanced solubility.13 We
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speculated that lack of polar interactions between the right wing of the inhibitor and
the surrounding residues of tolerant region I lead to unsatisfactory anti-HIV activity.
Additionally, we found that methylsulfonyl and sulfamide functional groups were
adaptive substituents around the tolerant region I according to previously extensive
studies.14,15 They are highly polar and prone to accepting or donating a proton (for
sulfamide), resulting in readily forming potential hydrogen bonding interactions with
RT.
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In the present study, hydrophilic morpholine or methylsulfonyl and sulfamide
substituted piperazine/piperidines were introduced into the right wing of DAPYs
targeting the tolerant region I of NNIBP for the first time. The privileged pyrimidine
or triazine was employed as the core ring in these new derivatives, which was
expected to anchor function groups and act as a hydrogen bond acceptor to form
crucial hydrogen bonding interaction with K101. Meanwhile, the preferred
2
,4,6-trisubstitued moiety of DAPYs was installed at the left-wing portion. We
expected the newly introduced hydrophilic motif could form additional interactions
most possibly hydrogen bonding interactions) with the surrounding residues of the
(
tolerant region I, aiming at the identification of compounds with maintained or even
higher potency against WT and drug-resistant HIV-1 strains as well as improved
water solubility (Figure 2). Herein, we report the multidimensional optimization,
preliminary structure-activity relationships (SARs), modeling simulation study,
solubility measurement, inhibitory effects on CYP enzymes, and acute/subacute
toxicity assessment results of the newly designed DAPY-typed derivatives.
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Tolerant region I
CN
O
CN
NH
O
N
O
X
N
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R1
O
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Br
NH2
O
N
NH
ETR
N
NH
NH
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N
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N
N
NH2
CN
NH
1
1
0a-f R1 = CN
1a-d R1 = CV
17
Cl
WC-II-4b
O2
S
H
N
S
X= eg.
N
HN
N
O2
N
N
N
H
N
NH2
RPV
O
S
O2
N
N
Figure 2. The illustration of the tolerant region I in NNIBP (co-crystal structure of ETR/RT, PDB
code: 3MEC) and flowchart summarizing the discovery and optimization strategy for novel
DAPY-typed HIV-1 NNRTIs targeting the tolerant region I of NNIBP. The figure of the
co-crystal structure of ETR/RT was generated using PyMOL (www.pymol.org).
CHEMISTRY
The synthetic protocols for the newly designed derivatives are depicted in Scheme
1
4
, Scheme 2, Scheme 3 and Scheme 4. First, intermediates 5a-d were prepared from
-nitrobenzyl bromide (3) via a nucleophilic substitution with morpholine or
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substituted piperazine/piperidines and followed by reduction of the nitro group to the
amino group in the presence of stannous chloride.13 Intermediate 7 (tert-butyl
(chlorosulfonyl)carbamate) was obtained through treating chlorosulfonyl isocyanate
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with t-BuOH. Next, the starting material 2,4-dichloropyrimidine (8) was undergone
a nucleophilic substitution reaction with 4-hydroxy-3,5-dimethylbenzonitrile or
(E)-3-(4-hydroxy-3,5-dimethylphenyl)acrylonitrile to give the key intermediates 9a-b,
followed by a Buchwald-Hartwig reaction with prepared 5a-d, providing part of the
target compounds 10a-c and 11a-b.17 Subsequent deprotection of the Boc group
afforded intermediates 12a-d,18 which were finally treated with methanesulfonyl
1
9
chloride to offer the target compounds 10d-e and 11c-d. Additionally, treatment of
12b with intermediate 7 in the presence of triethylamine yielded the Boc-protected
intermediate 13, which was also deprotected to supply the final product 10f. Similarly,
the starting material 2,4,6-trichloro-1,3,5-triazine (14) was undergone successive
2
0
nucleophilic substitution reactions and one-step aminating reaction to provide the
target compound 17. All the novel DAPY-typed derivatives were fully characterized
by means of high resolution mass spectrometry (HRMS), proton nuclear magnetic
1
13
resonance ( H NMR) and carbon nuclear magnetic resonance ( C NMR).
Br
X
X
i
ii
O N
2
O N
2
H N
2
3
4a-d
5a-d
H
N
N
N
4a, 5a: X=
4b, 5b: X=
4d,5d: X=
Boc
N
N
Boc
H
N
4c, 5c: X=
O
N
Boc
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Scheme 1. Reagents and conditions: (i) TEA, DCM, room temperature; (ii) SnCl ·2H O, EtOH,
2
2
N , r.t.
2
O
1
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O2
S
i
O=C=N-SO Cl
Cl
N
H
O
2
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7
Scheme 2. Reagents and conditions: (i) t-BuOH, DCM, 0°C.
Y
Z
X
N
N
R1
O
R1
O
R1
O
R1
O
Cl
N
Cl
i
ii
iii
iv
N
Cl
N
NH
N
NH
N
NH
5a-d
N
N
N
N
N
10d, R = CN Z= NSO CH
1 2 3
1
0a-c, R = CN
12a, R1= CN Y=NH
8
9a, R1 = CN
b, R = CV
1
11a-b, R = CV
12b, R = CN Y=CHNH
10e, R1= CN Z= CHNHSO2CH3
11c, R1= CV Z= NSO2CH3
9
1
1
2
1
12c, R = CV Y=NH
1
1
2d, R = CV Y=CHNH
11d, R1= CV Z= CHNHSO2CH3
1
2
7
v
H
W
N
NH
S
O
N
2
CN
O
N
NH
N
13, W=Boc
iii
10f, W=H
Scheme 3. Reagents and conditions: (i) 4-hydroxy-3,5-dimethylbenzonitrile or
E)-3-(4-hydroxy-3,5-dimethylphenyl)acrylonitrile, K CO , DMF, 50°C; (ii) 5a-d, Pd(OAc) ,
(
2
3
2
XantPhos, Cs CO , N atmosphere, 1,4-dioxane, 90°C; (iii) TFA, DCM, r.t.; (iv) ClSO CH , TEA,
2
2
3
3
2
DCM, r.t. (v) 7, TEA, DCM, r.t.
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6
7
8
9
0
N
N
N
N
N
N
Cl
Cl
15
Cl
NH2
14
16
17
Scheme 4. Reagents and conditions: (i) 2,4,6-trimethylphenol, K CO , DMF, 50°C; (ii) 5c,
2
3
NaHCO , THF/acetone/water, 30°C; (iii) NH H O, 1,4-dioxane, sealed tube, 100°C.
3
3·
2
RESULTS AND DISCUSSION
Antiviral and HIV-1 RT Inhibitory Activities and SAR Analysis.
Table 1. Antiviral Activity Against HIV-1 III and RES056 Strains and Cytotoxicity of the
B
Target Compounds in MT-4 Cells.
O
N
X
R1
O
N
NH
O
N
NH
N
N
N
NH2
10a-f
11a-d
1
7
EC50 (μM)a
SI c
CC50 (μM)b
Compds
R
1
X
III
B
RES056
III
B
RES056
Boc
NH
1
0a
CN
CN
CN
CN
CN
0.20 ± 0.019
0.19 ± 0.011
> 4. 34
4. 34 ± 1.09
21
19
<1
N
H
1
0b
N
> 3.51
> 23.0
> 3.43
> 18.1
3.51 ± 1.47
23.0 ± 7.30
3.43 ± 2.03
18.1 ± 6.98
<1
<1
<1
<1
N
Boc
10c
0.011 ± 0.0015
0.0072 ± 0.0029
0.055 ± 0.014
2042
475
331
O
N
O2
S
1
0d
N
N
H
10e
N
S
O2
N
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H
N
1
0f
CN
CVd
CV
0.092 ± 0.045
0.015 ± 0.0088
0.026 ± 0.017
> 3.72
> 3.04
> 3.55
3.72 ± 1.37
3.04 ± 1.05
3.55 ± 1.15
40
<1
<1
<1
NH2
S
O2
N
1
1a
Boc
209
138
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1b
Boc
NH
N
O2
S
11c
CV
CV
0.0035 ± 0.00075
0.0069 ± 0.0015
0.79 ± 0.026
> 6.99
≥ 173
≥ 48,774
≥ 219
N
N
H
1
1d
6.99 ± 5.93
1019
<1
N
S
O2
N
1
7
0.12 ± 0.034
0.93 ± 0.6
> 12.89
> 6.9
≥ 12.89
7.0 ± 2.9
> 87.2
≥ 105
7
<orX1e
<1
WC-II-4bf
3TC
6.41 ± 0.37
NDg
> 14
ND
AZT
0.011 ± 0.0016
0.24 ± 0.024
0.015 ± 0.0044
> 15.0
> 7.48
>704
> 62
>515
X1
NVP
> 15.0
EFV
0.0013 ± 0.00032
0.0035 ± 0.00018
0.0013 ± 0.0004
0.24 ± 0.032
0.050 ± 0.000
_
> 6.34
> 4969
> 1320
3490
> 26
> 92
_
ETR
> 4.59
RPVh
4.38 ± 1.42
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
CV: cyanovinyl
.
d
e
f
X1: ≥1 or <1
Used for comparison. The data was obtained from the same laboratory with the same method (Prof.
Christophe Pannecouque, Rega Institute for Medical Research, KU Leuven, Belgium).13
g
ND: not determined.
h
Used for comparison. The data was obtained from the same laboratory with the same method (Prof.
Christophe Pannecouque, Rega Institute for Medical Research, KU Leuven, Belgium).21
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Antiviral Activity against Several HIV-1 Mutant Strains in MT-4 Cells.
EC50 (μM)a
Compds
E138K
L100I
K103N
Y181C
Y188L
F227L+V106A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0c
0.14 ± 0.037
0.089 ± 0.0023
0.38 ± 0.029
0.41 ± 0.030
0.11 ± 0.073
0.70 ± 0.14
0.068 ± 0.016
0.23 ± 0.20
0.91 ± 0.069
0.47 ± 0.088
2.60 ± 0.37
4.93 ± 0.0099
> 5.22
1.80 ± 0.19
1.03 ± 0.13
3.23 ± 1.40
≥ 6.82
1
0d
1
0e
0.46 ± 0.017
≥ 7.80
1
0f
0.45 ± 0.070
2.15 ± 1.76
0.50 ± 0.071
6.54 ± 0.76
> 6.97
1
1a
0.13 ± 0.015
0.20 ± 0.053
0.28 ± 0.13
0.020 ± 0.0095
0.068 ± 0.0050
0.0036 ± 0.00014
0.025 ± 0.0046
0.011 ± 0.0019
10.9 ± 1.53
0.42 ± 0.0069
0.57 ± 0.044
0.025 ± 0.0031
0.33 ± 0.045
0.0056 ± 0.0016
> 15.0
> 4.03
1.30 ± 0.10
≥ 1.14
11b
11c
0.15 ± 0.032
> 4.27
0.0075 ± 0.0016
0.036 ± 0.0040
0.014 ± 0.0071
0.16 ± 0.087
0.010 ± 0.0041
0.13 ± 0.0090
0.0036 ± 0.00026
1.64 ± 0.47
1.62 ± 0.24
8.35 ± 0.52
0.0051 ± 0.0013
> 15.0
0.11 ± 0.025
0.47 ± 0.011
0.0062 ± 0.0013
> 15.0
1
1d
AZT
NVP
EFV
ETR
0.0060 ± 0.00045
0.015 ± 0.0049
0.052 ± 0.000
0.0074 ± 0.00065
0.089 ± 0.019
0.0039 ± 0.00032
0.0060 ± 0.0018
0.021 ± 0.0047
0.24 ± 0.046
0.027 ± 0.0054
0.27 ± 0.027
0.026 ± 0.0026
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.
All synthesized compounds were evaluated for their anti-HIV activity and
cytotoxicity using the MTT method in MT-4 cells infected with WT HIV-1 strain
(
III ) and K103N/Y181C double mutant HIV-1 strain (RES056), as well as HIV-2
B
strain (ROD).22 Selected compounds were further biologically evaluated for their
inhibitory activity against E138K and a panel of other drug-resistant single- and
double-mutant strains, including L100I, K103N, Y181C, Y188L and F227L+V106A.
Five approved drugs, lamividine (3TC), zidovudine (AZT), NVP, EFV and ETR,
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were used as controls. The antiviral evaluation results, interpreted as EC (anti-HIV
5
0
potency), CC50 (cytotoxicity) and SI (selectivity index, CC /EC50 ratio), are
50
summarized in Table 1 and Table 2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All compounds exhibited moderate to excellent potency against WT HIV-1 strain
with EC50 values ranging from 0.20 to 0.0035 μM, which were superior to the
reference drugs 3TC (EC = 6.41 μM) and NVP (EC = 0.24 μM) , as well as the
5
0
50
previously reported compound WC-II-4b (EC = 0.93 μM). Among them, 11c turned
5
0
out to be the most active inhibitor with an EC value of 0.0035 μM, which was much
5
0
more lower than those of 3TC and NVP, 3-fold lower than that of AZT (EC = 0.011
5
0
μM) and equivalent to that of ETR. As expected, none of the target compounds
exhibited inhibitory activity against the HIV-2 strain at subtoxic concentrations (data
was not shown in this article).
Moreover, compound 11c showed activity against the K103N/Y181C double mutant
HIV-1 strain (RES056) at a submicromolar concentration (EC = 0.79 μM), which
5
0
was more active than the reference drug NVP (EC > 15.0 μM) and the precursor
5
0
compound WC-II-4b (EC > 6.9 μM), though still less potent than AZT, EFV and
5
0
ETR.
Table 2 showed that these selected compounds with potent activity against WT
HIV-1 strain also demonstrated moderate to prominent activity against the mutant
HIV-1 strains. Among them, 11c demonstrated most excellent activity against HIV-1
single mutations E138K and K103N with EC values as 0.0075 μM and 0.0036 μM,
50
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
respectively. The antiviral potency against these HIV-1 variants are summarized as
follows:
1
) In case of the E138K mutant strain which is the key drug-resistant HIV-1 strain
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to DAPYs, three compounds 10d, 11c and 11d exhibited potent activity at two- or
single- digit nanomolar concentrations, and the others showed inhibitory activity at
submicromolar concentrations. To our delight, 11c was 2 times more potent than ETR
(EC = 0.015 μM).
5
0
2
) For K103N that is the most prevalent single mutant strain, 10c and 11a-d
displayed low EC values ranging from 0.068 μM to 0.0036 μM, being more potent
5
0
than EFV (EC = 0.089 μM). Again, 11c possessed slightly improved activity than
5
0
that of ETR (EC = 0.0039 μM).
5
0
3
) As for the other two single-mutant strains L100I and Y181C, it was noted that
only compound 11c exhibited EC values at nanomolar levels (EC = 0.010 and
5
0
50
0
.025 μM, respectively), which were both mildly higher than those of ETR (EC =
50
0
.0074 and 0.021 μM, respectively). 11c was much more active against L100I variant
than EFV (EC = 0.052 μM) while less potent against Y181C mutant strain than EFV
50
(
EC = 0.0060 μM).
50
4
) All of the selected compounds showed moderate or lost activity against Y188L
and F227L+V106A mutant strains.
Based on the biological evaluation results (Table 1), preliminary SARs were
investigated:
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The contribution of R substituents of these new DAPY-typed derivatives to the
1
anti-HIV activities was firstly assessed. Through pairwise comparison on potency,
namely, 10a (EC = 0.20 μM) and 11b (EC = 0.026 μM), 10d (EC = 0.0072 μM)
5
0
50
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and 11c (EC = 0.0035 μM), and 10e (EC = 0.055 μM) and 11d (EC = 0.0069
5
0
50
50
μM), it could be concluded that the cyanovinyl group was more favorable for activity
than the cyano group. Next, we focused our attention on the fragments introduced into
the tolerant region I. It was obvious that this region was indeed highly tolerated by
various substituents. And also, the size of the chemical groups connected to the
terminal of piperazine/piperidine was a major determinant for the anti-HIV-1 activity,
which was illustrated in Table 1. Smaller and hydrophilic groups, like methylsulfonyl
and sulfamide, were favorable to the activity against WT HIV-1 strain (10e, 10f
versus 10a, 11c versus 11a, 11d versus 11b). In addition, compound 17 exhibited
anti-HIV-1 (III ) potency at a submicromolar concentration, though inferior to most
B
compounds in 10a-f subseries, indicating the triazine ring was an alternative core for
DAPY-typed derivatives.
Moreover, it is noteworthy that the morpholine-bearing compound 10c had a CC50
value of 23.0 μM, demonstrating a relative lower cytotoxicity. Most importantly,
compound 11c was found to possess the lowest cytotoxicity (CC ≥ 173 μM), which
5
0
2
1
was much lower than that of RPV (CC = 4.38 μM), and thus had a high selectivity
5
0
index (SI ≥ 48,774). As it has been extensively reported that the cyanovinyl
containing HIV-1 NNRTIs including RPV possess high cytotoxicity in MT-4 cells,
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound 11c featuring a cyanovinyl function group was truly an interesting
molecule.
In general, these biological results demonstrated that introducing hydrophilic and
polar functional groups, including morpholine or methylsulfonyl and sulfamide
substituted piperazine/piperidines, into the right wing of DAPYs to target the tolerant
region I was acceptable and may improve antiviral potency, especially to the E138K
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and K103N mutant HIV-1 strains.
Table 3. Inhibitory Activity of the New Compounds Against HIV-1 RT (WT)
Compds
EC50 (μM)a
IC50 (μM)b
pEC50 c
pIC50 d
10a
10b
0.20 ± 0.019
0.19 ± 0.011
0.413 ± 0.012
0.277 ± 0.001
0.042 ± 0.002
0.049 ± 0.000
0.079 ± 0.011
0.152 ± 0.011
0.297 ± 0.041
0.499 ± 0.025
0.067 ± 0.030
0.076 ± 0.023
0.442 ± 0.138
0.595 ± 0.146
0.007 ± 0.003
0.022 ± 0.004
0.011 ± 0.000f
6.70
6.72
7.96
8.14
7.26
7.04
7.82
7.59
8.46
8.16
6.92
6.62
8.89
8.89
8.46
6.38
6.56
7.38
7.31
7.10
6.82
6.53
6.30
7.17
7.12
6.35
6.23
8.15
7.66
7.96
1
0c
0.011 ± 0.0015
0.0072 ± 0.0029
0.055 ± 0.014
0.092 ± 0.045
0.015 ± 0.0088
0.026 ± 0.017
0.0035 ± 0.00075
0.0069 ± 0.0015
0.12 ± 0.034
1
0d
1
0e
1
0f
1
1a
11b
11c
1
1d
1
7
NVP
EFV
RPV
ETR
0.24 ± 0.024
0.0013 ± 0.00032
0.0013 ± 0.0004e
0.0035 ± 0.00018
a
B
EC50: The anti-HIV-1 (III ) activity in MT-4 cells, as indicated in the Table 1.
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b
IC50: The RT inhibition assay is performed with the EnzCheck Reverse Transcriptase Assay kit
(
Molecular Probes, Invitrogen).
c
pEC50: -logEC50
.
d
pIC50: -logIC50
.
e
Used for comparison. The data was obtained from the same laboratory with the same method (Prof.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Christophe Pannecouque, Rega Institute for Medical Research, KU Leuven, Belgium).21
f
Used for comparison. The data was obtained from the same laboratory with the same method (Prof.
Christophe Pannecouque, Rega Institute for Medical Research, KU Leuven, Belgium).13
In order to confirm the mode of action of these newly synthesized DAPY-typed
derivatives, we further tested all the compounds for their ability to inhibit
recombinant WT HIV-1 RT, with NVP, EFV and RPV as reference drugs (Table 3).
These compounds displayed HIV-1 RT inhibitory activity in the range of 0.499 μM
- 0.042 μM. Compound 10c exhibited the most active RT inhibitory activity with an
IC value (0.042 μM) in the same magnitude with the reference drug RPV (0.022
5
0
μM). Overall, there was a close correlation between the anti-HIV-1 (III ) activity
B
(
EC ) and the RT inhibitory activity (IC ) of these compounds. This was shown in a
50 50
linear regression curve with both biological results for all new compounds in Table 1
and Tableꢀ3, including the reference drugs NVP and EFV (Figure 3). The results
demonstrated that these inhibitors exhibited antiviral activity by inhibiting the RT
enzyme, and could act as typical HIV-1 NNRTIs.
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1
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1
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1
2
2
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2
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3
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3
3
3
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5
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Regression analysis of pIC and pEC values for the newly synthesized
5
0
50
DAPY-typed derivatives.
Docking and molecular dynamics simulation studies
To better elucidate the anti-HIV potency of these new DAPY derivatives at a
molecular level and to further understand the structural basis of their binding modes
in the NNIBP, a molecular modeling study of a subset of representative compounds
(
11c, 10c and 10a) together with the lead compound ETR was performed using
docking and molecular dynamics (MD) calculations.
2
3
The crystallographic structure (PDB code: 3M8Q) of HIV-1 RT in complex with
the
NNRTI
3,5-dimethyl-4-{[2-({1-[4-(methylsulfonyl)benzyl]piperidin-4-yl}amino)pyrimidin-4-
yl]oxy}benzonitrile (DJZ; Supporting Information Table S1 and Figure S1) was used
to perform a “core-constrained” flexible docking of ligands in the WT enzyme.
Additional docking calculations were performed for the E138K and K103N variants
of the HIV-1 RT, which were built up by replacing E138 and K103 in 3M8Q by Lys
and Asn, respectively, taking into account the proper arrangement of the side chains
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as found upon inspection of the X-ray structures 2HNY and 3MED, which correspond
to the complexes of NVP and ETR with the E138K and K103N mutated HIV-1 RT,
respectively. Finally, the potential influence of the structural water molecule (567),
which mediates the interaction of the protonated piperidine nitrogen of DJZ with
residues K103 and P236 in 3M8Q, was also examined.
1
1
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Docking calculations performed for DJZ and ETR led to poses that reproduced the
crystallographic binding mode (RMSD of the ligand < 0.1 Å; Supporting Information
Figures S1 and S2), irrespective of the introduction of the mutations at positions 138
and 103 as well as of the presence of the structural water 567 (Supporting Information
Table S2), as noted in similar scores obtained for each ligand in the 6 protein models
(3M8Q, 3M8QE138K, and 3M8QK103N, with and without water 567), generally being
comprised between -10.0 and -12.5 kcal/mol. This finding can be attributed to the
large structural resemblance found in the NNRTI binding site for structures 3M8Q,
3
MED and 2HNY (Supporting Information Figure S1). The binding mode predicted
for ligands 11c, 10c and 10a were also consistent with the arrangement of DJZ and
ETR, although the most notable trait was the identification of two orientations of the
piperazine (11c), morpholine (10c), and piperidine (10a) ring in the Tolerant Region I
of the NNRTI binding site (Supporting Information Table S3 and Figure S3). In one
of these arrangements the protonated nitrogen is pointing toward the backbone
carbonyl groups of His235 and Pro236, and in the other the terminal ring is rotated by
almost 180 degrees (denoted reversed hereafter).
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The analysis of the MD simulations revealed that the ligand-protein complexes were
stable in all cases, as noted in the comparison of the time evolution of the root-mean
square deviation (RMSD) profiles determined for the residues that shape the NNRTI
binding site (taken as the set of residues located at 5 Å from the ligand in the
energy-minimized complex) and the ligand, the RMSD being, on average, generally
close to 2.7 Å (Figure 4 for complexes with 11c; see also Supporting Information
Figure S4 for complexes with 10c and ETR).
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Figure 4. Time (ns) evolution of the RMSD (Å) profile determined for the complexes between
1
1c with (A) WT 3M8Q, (B) 3M8QE138K and (C) 3M8QK103N. The RMSD profile determined for
the whole protein backbone is shown in black, and for the heavy atoms of the binding site and
ligand in red.
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Figure 5. (A) Binding mode of 11c in NNIBP of WT HIV-1 RT (PDB code: 3M8Q). (B)
Superposition of the binding mode of 11c in NNIBP of WT 3M8Q, 3M8QE138K and 3M8QK103N
.
(C) Superposition of the binding mode of 11c and 10c in NNIBP of WT 3M8Q. (D) Superposition
of the binding mode of 11c and ETR in NNIBP of WT 3M8Q. The protein backbone is shown as
grey carton, and selected residues are highlighted as sticks (mutations of Glu138 and Lys103 to
Lys and Asn, respectively, are shown as sticks with green-colored carbon atoms; hydrogen atoms
are omitted for the sake of clarity). Ligands are shown as sticks with carbon atoms colored in
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yellow (11c), orange (10c) and magenta (ETR). Hydrogen bonds are indicated as dashed black
lines (red).
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Compound 11c adopted a binding orientation characterized by the classical “U”
shape conformation in the NNIBP similar to that of ETR (Figure 5A). Distinct traits
characterize the binding mode of 11c in the NNIBP binding site. The
2,6-dimethyl-4-cyanovinylphenoxy moiety filled a hydrophobic sub-pocket composed
by the aromatic side chains of Tyr181, Tyr188 and Trp229, resulting in potential van
der Waals interactions with the aromatic side chains of these residues. Furthermore,
the 1’-N of the pyrimidine ring and the NH linker of the compound formed dual
hydrogen bonds with the backbone carbonyl oxygen and amide NH groups of Lys101,
which appear to be crucial for the binding of the ligand. Thus, the interaction
…
NH O
was very stable along the trajectory with WT 3M8Q, as noted in an
K101
average distance of 2.93 ± 0.04 Å along the last 100 ns of the MD simulation,
…
whereas the interaction 1’-N HNK101 resulted to be a weaker hydrogen bond (average
distance of 3.62 ± 0.30 Å). Finally, the methylsulfonyl piperazine motif located in the
tolerant region I was exposed at the protein/solvent interface, with the piperazine ring
being stacked against Pro236 (in the reversed arrangement), and the methylsulfonyl
group being located close to Pro225 and Phe227, or adopting an alternative
arrangement that enables a transient hydrogen-bond interaction with the backbone of
Val106.
The binding mode of 11c to the WT 3M8Q and to the 3M8QE138K and 3M8QK103N
variants was highly similar, as noted in the superposition of the final snapshots
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collected at the end of the MD simulations for these systems (Figure 5B). The results
…
confirm the relevant contribution of the NH O
interaction (average distance of
K101
…
2
.94 ± 0.04 Å), which is supplemented by the weaker 1’-N HN
hydrogen bond
K101
1
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(average distance of 3.50 ± 0.19 Å, and 3.83 ± 0.31 Å), the occupancy of the
subpocket formed by Tyr181, Tyr188 and Trp229, and the flexibility of the
methylsulfonyl piperazine motif in the tolerant region I, including the conformational
change of the protonated piperazine ring to the reversed arrangement along the
trajectory run for the 11c-3M8QE138K complex.
To further check the preservation of the features that mediate the binding mode of
1
1c, two MD simulations were also run for compound 10c bound to WT 3M8Q,
differing in the orientation of the morpholine ring. The two structures converged to a
similar binding mode (Supporting Information Figure S5), which also matched the
arrangement of 11c (Figure 5C), where the morpholine ring adopted the reversed
arrangement, being stacked against Pro236. On the other hand, the binding of the
central pyrimidine ring was assisted by the two hydrogen bonds mentioned above
…
(
average distances of 2.93 ± 0.05 Å and 3.57 ± 0.26 Å for the NH O
and
K101
…
1’-N HN
interactions, respectively).
K101
As a final remark, let us note that ETR also adopted a stable binding mode in the
WT 3M8Q and the two mutated variants (Supporting Information Figure S6), which
…
…
matches well the binding of 11c (Figure 5D). The NH O
and 1’-N HN
K101
K101
interactions were maintained along the trajectory in all cases, as noted in average
distances of 2.89-2.94 Å and 3.40-3.76 Å, respectively, in the three MD simulations.
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However, it is worth noting that the binding is further assisted by an additional
hydrogen bond formed between ETR and the carboxylate group of Glu138. Overall,
the binding conformation of 11c revealed a remarkable superposition to that of ETR,
giving rise to its potent anti-HIV and RT inhibition activities.
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Taken together, these findings provide a basis to justify that 11c possessed good
activity against the key drug resistant variant E138K to the DAPY-typed HIV-1
NNRTIs. Furthermore, keeping in mind the flexibility of the Tolerant Region I, it can
be speculated that the detrimental effect due to the introduction of the Boc group on
the inhibitory activity (compare 11a and 11b with regard to 11c, and 10a and 10b
relative to 10c) may result from the steric hindrance that impedes an appropriate
"closure" of the loops that shape this region. In turn, this would prevent a proper
packing of the ligand with the surrounding residues, while it would facilitate the
access of water molecules that may promote release of the ligand from the binding
pocket. Overall, present results confirmed our rational drug design as an effective
strategy to overcome the drug-resistance issues.
Water Solubility and Other Drug-like Properties
To validate our hypothesis, we performed water solubility measurement of the
representative compounds 10c and 11c. As revealed in Table 5, both compounds
possessed significantly improved water solubility than that of ETR. Besides, it should
be noted that the morpholine-bearing compound 10c had the best water solubility of
3
3.4 μg/mL. Coincidentally, in lead optimization programs in medicinal chemistry,
the morpholine was commonly introduced into solvent exposed areas as a privileged
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solubilizing group to improve solubility or pharmacokinetic properties of a
prospective drug scaffold, exemplified by the approved drugs copanlisib and gefitinib,
24
as well as many bioactive compounds with greatly improved solubility (including
1
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our previously reported compound WC-II-4b). Additionally, polar surface area (PSA,
a very useful prediction parameter of oral bioavailability) of four representative
compounds 10c, 10d, 11c and 11d as well as of ETR was calculated using free online
software (http://www.molinspiration.com/), and the results were also shown in Table
2
5
. It is suggested that drugs with a PSA < 60 Å are almost completely (more than
2
90%) absorbed, whereas drugs with a PSA > 140 Å are absorbed to less than
1
0%.25,26 Compared with that of ETR, the PSA values of these new derivatives
indicated that our modifications at the right portion of ETR could possibly increase
the oral bioavailability.
In recent years, ligand efficiency (LE, -ΔG/N(non-hydrogen atom)) and ligand-lipophilicity
efficiency (LLE, pEC - cLogP) which combine both “potency and lipophilicity”
5
0
have emerged as important metrics in many drug discovery programs, usefully
directing hit and lead optimization. Desirable leads commonly exhibit LE > 0.3, LLE
> 5 or greater and cLogP < 5. We therefore calculated the metrics of these four
compounds together with ETR (Table 4). Fortunately, all of the selected compounds
met acceptable levels for LE and cLogP. The LLE indices of three compounds 10c,
1
0d and 11c were better than that of ETR. Certainly, they still needed to be improved
to satisfy the standard level.
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Table 4. Water Solubility, tPSA, cLogP, LE and LLE Values of Compounds 10c, 10d, 11c,
1
1d and ETR
Compds
10c
10d
11c
11d
ETR
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<<127
Water Solubility (pH=7) (μg/mL)
33.40 ± 0.04
83.31
-
30.92 ± 0.02
111.45
4.54
-
a
2
tPSA (< 140 Å )
111.45
3.78
0.32
4.36
120.24
4.82
0.29
3.34
120.65
5.03
a
cLogP (< 5)
4.36
b
LE (> 0.3)
0.35
0.31
0.41
c
LEE (> 5)
3.60
3.92
3.43
a
tPSA and cLogP: Calculated by free online software (http://www.molinspiration.com/). tPSA,
topological polar surface area.
b
LE: Calculated by the formula -ΔG/N(non-hydrogen atom), where standard free energy change ΔG =
-RT ln Kd, presuming Kd ≈ EC ; negative logarithm values of potency (pEC ) converted from
5
0
50
experimental data against WT HIV-1 III . R is the Gas constant, and T is 300 K. N
B
(non-hydrogen atom)
is the number of non-hydrogen atoms.
c
LEE: Calculated by the formula pEC - cLogP.
5
0
In Vitro Effects of 11c on CYP Enzymatic Inhibitory Activity
As is well known, metabolizing enzymes in the liver (e.g. cytochrome P450, CYP)
are responsible for the majority of drug metabolism in vivo. Among them, CYP3A,
CYP2D6 and CYP2C subtypes are involved in metabolism of about 90% of the
2
8
marketed drugs. Drugs can also interact with the CYP enzymes, by inhibiting their
enzymatic activity (CYP inhibition) and/or through activating nuclear receptors which
leads to induction of their gene expression (CYP induction). Therefore,
metabolism-mediated drug-drug interactions (DDI) may occur when
co-administrating different drugs, leading to decreased therapeutic effect or adverse
reaction profile. According to disclosed information, etravirine is an inducer of
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CYP3A4 and inhibitor of CYP2C9 and CYP2C19, and it can result in many
drug-drug interactions when co-administrated with other anti-HIV-1 drugs like
integrase strand inhibitors, protease inhibitors, and CCR5 antagonists etc, which
limits the selection of concomitant antiretrovirals. We performed the study of
inhibitory activity of compound 11c on main CYP drug metabolizing enzymes, with
selective inhibitors of these enzymes as positive controls. The results in Table 5
indicated that 11c displayed no significant CYP enzymatic inhibition of CYP2C9,
CYP2C19, CYP2D6, CYP3A4M and CYP3A4T (if one compound inhibits CYP
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
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4
4
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0
1
2
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0
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9
0
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enzymes with IC < 3 μM, it is regarded as a potent CYP enzyme inhibitor ), which
5
0
preliminarily proved that our compound 11c had less potential to induce side effects
in the liver and drug-drug interactions.
Table 5. Inhibitory activity on CYP2C9, CYP2C19, CYP2D6, CYP3A4M and CYP3A4T of
Compound 11c and Positive Controls
CYP subtypes
Selective inhibitors
Inhibitory activity
Compd
Inhibitory activity
(IC50, μM)
(IC50, μM)
CYP2C9
CYP2C19
CYP2D6
Sulfaphenazole
(+)-N-3-benzylnirvanol
Quinidine
0.847
0.124
11c
11c
11c
11c
11c
22.3
27.0
> 50
20.5
7.82
0.263
CYP3A4M
CYP3A4T
Ketoconazole
0.0183
0.0563
Ketoconazole
Acute Toxicity Assessment of 11c in Healthy Mice
Single-dose acute toxicity of 11c in healthy mice was studied regarding to its
relatively low cytotoxicity. After intragastric administration of 11c at a dose of 2000
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1
mg·kg , no mice died, and there was no abnormality of their behaviors (lethargy,
clonic convulsion, anorexia, and ruffled fur). All mice in drug-administration groups
grew normally the same as the mice in control groups. On the whole, the body
weights of both male and female mice increased gradually over the 1-week period
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(
Figure 6), and there was no obvious difference in the weight gain. Therefore, 11c
-1
was well-tolerated at a dose of 2000 mg·kg with no acute toxicity.
(A)
(B)
Figure 6. (A) Body weight of male mice (g)-time (day); (B) body weight of female mice (g)-time
(day).
Subacute Toxicity Assessment of 11c in Healthy Mice
For assessing the in vivo safety of 11c, we further investigated its subacute toxicity
in healthy mice. The mice in test groups were po administered with a dose of 50
mg·kg-1 of 11c every second day for 14 days. Again, no mice died, and no
abnormality of their behaviors (lethargy, clonic convulsion, anorexia, and ruffled fur)
and body weight increase were observed during the treatment period. The body
weight of both males and females in test groups increased gradually and they grew
normally the same as the control groups (Figure 7). Additionally, the five most
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important organs (heart, liver, spleen, lungs, and kidneys) were examined by
hematoxylin-eosin (HE) staining after dissecting all of the mice, and it was observed
that no marked pathological damage occurred after treatment with 11c (Figure 8).
The subacute toxicity evaluation results indicated that 11c possessed favorable in vivo
safety profiles.
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Figure 7. Body weight of all mice in four groups (g)-time (day)
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