Design, Synthesis, and Evaluation of Thiophene[3,2-d]pyrimidine Derivatives as HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors with Significantly Improved Drug Resistance Profiles

Article abstract of DOI:10.1021/acs.jmedchem.6b00738

We designed and synthesized a series of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a piperidine-substituted thiophene[3,2-d]pyrimidine scaffold, employing a strategy of structure-based molecular hybridization and substituent decorating. Most of the synthesized compounds exhibited broad-spectrum activity with low (single-digit) nanomolar EC₅₀ values toward a panel of wild-type (WT), single-mutant, and double-mutant HIV-1 strains. Compound 27 was the most potent; compared with ETV, its antiviral efficacy was 3-fold greater against WT, 5-7-fold greater against Y181C, Y188L, E138K, and F227L+V106A, and nearly equipotent against L100I and K103N, though somewhat weaker against K103N+Y181C. Importantly, 27 has lower cytotoxicity (CC₅₀ > 227 μM) and a huge selectivity index (SI) value (ratio of CC₅₀/EC₅₀) of >159101. 27 also showed favorable, drug-like pharmacokinetic and safety properties in rats in vivo. Molecular docking studies and the structure-activity relationships provide important clues for further molecular elaboration.

Full text of DOI:10.1021/acs.jmedchem.6b00738

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Article
Design, Synthesis and Evaluation of Thiophene[3,2-d]pyrimidine
Derivatives as HIV-1 Non-nucleoside Reverse Transcriptase
Inhibitors with Significantly Improved Drug Resistance Profiles
Dongwei Kang, Zengjun Fang, Zhenyu Li, Boshi Huang, Heng Zhang, Xueyi Lu, Haoran Xu, Zhongxia
Zhou, Xiao Ding, Dirk Daelemans, Erik De Clercq, Christophe Pannecouque, Peng Zhan, and Xinyong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00738 • Publication Date (Web): 19 Aug 2016
Downloaded from http://pubs.acs.org on August 20, 2016
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Design, Synthesis and Evaluation of Thiophene[3,2ꢀd]pyrimidine
Derivatives as HIVꢀ1 Nonꢀnucleoside Reverse Transcriptase
Inhibitors with Significantly Improved Drug Resistance Profiles
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†
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†
Dongwei Kang, Zengjun Fang, Zhenyu Li, Boshi Huang, Heng Zhang, Xueyi
†
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†
†
§
§
Lu, Haoran Xu, Zhongxia Zhou, Xiao Ding, 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, Bꢀ3000
Leuven, Belgium
|
The Second Hospital of Shandong University, No. 247 Beiyuan Avenue, Jinan 250033,
China.
ABSTRACT:
We designed and synthesized a series of human immunodeficiency virus type 1
(
HIVꢀ1) nonꢀnucleoside reverse transcriptase inhibitors (NNRTIs) with
a
piperidineꢀsubstituted thiophene[3,2ꢀd]pyrimidine scaffold, employing a strategy of
structureꢀbased molecular hybridization and substituent decorating. Most of the
synthesized compounds exhibited broadꢀspectrum activity with low (single digit)
nanomolar EC50 values towards a panel of wildꢀtype (WT), singleꢀmutant and
doubleꢀmutant HIVꢀ1 strains. Compound 27 was the most potent; compared with ETV,
its antiviral efficacy was 3ꢀfold greater against WT, 5ꢀ to 7ꢀfold greater against Y181C,
Y188L, E138K and F227L+V106A, and nearly equipotent against L100I and K103N,
though somewhat weaker against K103N+Y181C. Importantly, 27 has lower
cytotoxicity (CC50 >227 ꢁM) and a huge selectivity index (SI) value (ratio of
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CC50/EC50) of >159,101. 27 also showed favorable, drugꢀlike pharmacokinetic and
safety properties in rats in vivo. Molecular docking studies and the structureꢀactivity
relationships provide important clues for further molecular elaboration.
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INTRODUCTION
Nonꢀnucleoside reverse transcriptase inhibitors (NNRTIs) are a component of
Highly Active Antiretroviral Therapy (HAART) regimens used to treat human
immunodeficiency virus type 1 (HIVꢀ1), the main causative agent of acquired immune
1
ꢀ5
deficiency syndrome (AIDS), due to their potent antiviral activity, high selectivity,
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modest toxicity and favorable pharmacokinetics. NNRTIs distort the enzyme’s active
site and perturb the alignment of the primer terminus by interacting in a
noncompetitive manner with the allosteric site (NNRTIs binding pocket, NNIBP)
about 10 Å distant from the polymerase active site of RT, resulting in inhibition of the
6
reverse transcription step of HIVꢀ1 replication. Nevirapine (1, NVP), delavirdine (2,
DLV) and efavirenz (3, EFV) are firstꢀgeneration NNRTIs for AIDS therapy,
exhibiting potent antiꢀHIVꢀ1 activity, but some toxicities (for example, hepatotoxicity
and severe rash are associated with the use of nevirapine, while efavirenz has central
7
nervous system side effects.). However, there is a low genetic barrier for viral
resistance, and drugꢀresistant mutants rapidly emerge, including K103N, Y181C,
Y188L single mutants and K103N/Y181C double mutant (RES056); among them,
K103N and Y181C are the two most prevalent mutations in vivo, primarily selected
by nevirapine and efavirenz, and this has limited the clinical usefulness of the
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firstꢀgeneration NNRTIs. Secondꢀgeneration NNRTIs include the recently approved
drugs etravirine (4, ETV) and rilpivirine (5, RPV), both of which are the members of
10
the diarylpyrimidine (DAPY) family. Another DAPY derivative, dapivirine (6,
TMC120) has emerged as a microbicide with great potential for the prevention of
transmission of HIVꢀ1 infections, showing favorable pharmacokinetics when
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delivered through gels or vaginal rings. Structureꢀactivity relationship (SAR) studies
led to the identification of three regions (A, B, C) of the DAPY scaffold (Figure 1).
The DAPY inhibitors adopt a conformationally flexible horseshoe shape in the NNIBP,
showing both torsional flexibility (‘‘wiggling’’) and repositioning and reorientation
(
‘‘jiggling’’), which may serve to minimize the loss of binding stabilization caused by
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mutations. Consequently, they exhibit a broad spectrum of activity against wildꢀtype
WT) and clinically relevant HIVꢀ1 mutant strains, with nanomolar EC50 values. The
(
genetic barrier to emergence of drug resistance is higher than with the firstꢀgeneration
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NNRTIs. However, hypersensitivity reactions or other adverse effects have been
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reported with secondꢀgeneration NNRTIs. Also, although drug resistance is delayed,
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it continues to emerge in patients receiving secondꢀgeneration NNRTIs regimens.
Among resistance mutations, K103N and E138K are most frequently selected by
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etravirine and rilpivirine. Therefore, considerable effort has been directed to find
new DAPY analogues that retain activity against NNRTIsꢀresistant mutants, including
K103N, Y181C, Y188L and E138K.
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O
NH
O
HN
S
O
CF3
O
NH
N
N
HN
Cl
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N
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H
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1 (Nevirapine)
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(Delavirdine)
3 (Efavirenz)
CN
CN
CN
CN
CN
C
A
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A
C
A
O
N
NH
B
HN
N
NH
HN
N
NH
N
Br
B
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N
NH2
(Etravirine)
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5 (Rilpivirine)
6 (Dapivirine)
Figure 1. Chemical structures of five NNRTIs approved by the US FDA and dapivirine, a
microbicide in the DAPY family.
Our prior studies
CONH2
O O
S
N
CN
C
N
O
N
NH
B
O
NH
N
N
HN
(XC-6b3)
NO2
7
8 (LZ-5a6)
EC50 (IIIB) = 4.61 nM,
SI = 5945;
EC50 (IIIB) = 22 nM,
SI > 10770;
EC50 (K103N/Y181C) = 0.56 uM EC50 (K103N/Y181C) > 234 uM
Substituent Decorating
R'
CN
CN
Ar
R
C
O
C
A
N
Molecular Hybridization
Scaffold Hopping
O
N
NH
B
N
NH
N
Br
B
N
NH2
(Etravirine)
S
4
Piperidine-substituted
thiophene[3,2-d]pyrimidine
Isosteric Replacement
This work
CN
A
CN
(PDB code: 3M8P)
C
O
NH
HN
N
NH
B
N
N
N
N
N
N
9
(YT-5a)
10 (LW-7n)
EC50 (IIIB) = 20 nM,
SI = 8986;
EC50 (IIIB) = 70 nM,
SI = 3999;
EC50 (K103N/Y181C) = 7.6 uM
Our prior studies
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Figure 2. Design strategies used in our previous and present studies to obtain structurally
elaborated DAPYꢀbased NNRTIs utilizing the thiophene[3,2ꢀd]pyrimidine scaffold. The
illustration of the coꢀcrystal structure of etravirine/RT was generated using PyMOL
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(
www.pymol.org).
Detailed analysis of a wide range of crystal structures of HIVꢀ1 RT/NNRTI
complexes, together with data on drug resistance mutations, has identified several
factors important for the design of NNRTIs with broadꢀspectrum activity against
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mutant HIVꢀ1 strains; these factors include extensive mainꢀchain hydrogen bonding
16,17
interactions (double hydrogenꢀbonding chelate with the Lys101/Lys103 backbone)
and addition of a heterocyclic motif to the parent compound to maximize interactions
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with highly conserved residues in the HIVꢀ1 RT binding pocket.
Based on the crystallographic studies, it was concluded that NNIBP contains
solventꢀaccessible regions that provide broad regions that are potentially available to
accommodate novel NNRTIs, such as tolerant region I (the Pro236 hairpin loop) and
tolerant region II (the entrance channel, i.e. the largely open region in front of Lys103,
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Glu138 and Val179).
By the classical definition for drug design, an active molecule should efficiently
enter and maximally occupy the binding pocket, thereby interacting effectively with
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the amino acids around the binding site. The availability of crystallographic
structures of RT/DAPYs, together with the results of medicinal chemistry studies,
have enabled structureꢀbased design of various NNRTI subꢀclasses with promising
activity profiles against mutant strains. In our lab, we have made great efforts to
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utilize these two tolerant regions in the NNBIP in order to develop potent and highly
selective DAPYs derivatives as antiviral agents active against the prevalent mutations
in clinical HIVꢀ1 isolates, as shown in Figure 2.
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Regarding the tolerant region I, we recently reported the addition of a
functionalized side tail to the known DAPY NNRTIs. The resulting
piperidineꢀsubstituted triazine/aniline derivatives (exemplified by 7 (XCꢀ6b3) and 8
(LZꢀ5a6)) showed higher potency against WT, but had only weak (7: EC50 = 0.56 ꢁM)
or no activity (8: EC50 > 234 ꢁM) against the K103N/Y181C double mutant resistant
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strain. The aromatic pꢀcyanoaniline moiety (Aꢀring) in ETV and RPV was replaced
with various substituted benzylꢀlinked piperidinꢀ4ꢀylꢀamino moieties, as we
anticipated several advantages of introducing such structures. Firstly, the N atom of
the piperidine can form an additional Hꢀbond with Lys103 through a water bridge,
16,17
which might contribute to an improved resistance profiles.
Thus, the
piperidineꢀlinked NH might serve as a Lys101/Lys103 double backbone chelate motif,
because all NNRTIs can form direct or waterꢀmediated hydrogen bonds with these
amino acids in the protein backbone. Secondly, the substituted benzyls are directed
toward the proteinꢀsolvent interface (the Pro236 hairpin loop) and are available to
participate in additional hydrogen bonds with key residues of the NNIBP. Thirdly, the
polar substituted benzylꢀlinked piperidinꢀ4ꢀylꢀamino moiety could contribute to
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improved physicochemical and pharmacokinetic properties.
On the other hand, the entrance channel is another relatively unexplored open
region in the NNIBP; this region extends into the solventꢀexposed region, and is
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surrounded by several relatively inflexible residues, Leu100, Lys101, Glu138 and
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Val179. The entrance channel therefore represents another attractive tolerant region
that would be available to accommodate diverse groups, providing additional scope
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for the generation of novel inhibitors.
At the beginning of our endeavor to identify new DAPYs by establishing novel
binding interactions with the solventꢀaccessible entrance channel and to further
expand the chemical scope of the core ring system, we adopted an isosteric
replacement strategy to morph the central sixꢀmembered heterocyclic core to several
fused heterocycles bearing bridgehead nitrogen, such as pyrazolo[1,5ꢀa]pyrimidine
and [1,2,4]triazolo[1,5ꢀa]pyrimidine (exemplified by 9 (YTꢀ5a) and 10 (LWꢀ7n),
respectively). Although these representative compounds displayed potent activity
toward WT HIVꢀ1 at low nanomolar concentrations, with high selectivity (Figure 2),
they exhibited weak potency against the K103N/Y181C double mutant HIVꢀ1 strain
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(
for 10: EC = 7.6 ꢁM).
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Specific nonpolar interactionsꢀbased scaffold decoration has recently
experienced a renaissance, gaining increased recognition as a useful tool in NNRTIs
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discovery. Continuing our research project, we therefore turned our attention to the
introduction of small hydrophobic groups at the 5,6ꢀpositions in the central
pyrimidine ring of DAPY NNRTIs, aiming to establish nonpolar interactions with
hydrophobic side chains of residues in the entrance channel cleft. In the present work,
we further explored privileged and synthetically accessible piperidineꢀsubstituted
thiophene[3,2ꢀd]pyrimidine derivatives, in which the thiophene[3,2ꢀd]pyrimidine
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structure replaces the pyrimidine (Bꢀring) of the lead compound, ETV. In principle,
the sulfur atom of thiophene[3,2ꢀd]pyrimidine occupies the same position as the
bromo atom of ETV. The loneꢀpair electrons and atomic radius (r = 104 pm) of the
sulfur atom are similar to those of the bromo substituent of ETV (r = 114 pm), and
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thiophene[3,2ꢀd]pyrimidine scaffold and Val179. Next, the pꢀcyanoaniline moiety
Aꢀring) was replaced with different substituted benzylꢀlinked piperidinꢀ4ꢀylꢀamino
moieties. In the resulting compounds, the piperidinꢀ4ꢀylꢀamino and the N atom at the
ꢀposition of the thiophene[3,2ꢀd]pyrimidine can form multiple hydrogen bonds with
could
be
a
strong
electrostatic
interaction
between
the
(
1
the backbones of Lys101 and Lys103. The trisubstituted phenoxy ring (Cꢀring), which
plays a crucial role in arylꢀaryl interactions with Tyr181, Tyr188, Phe227 and Trp229,
was retained. We expected that all these factors would allow the novel
thiophene[3,2ꢀd]pyrimidine derivatives to occupy the NNIBP more effectively,
strengthening the binding interactions with residues of the NNIBP.
Thus, we report here the design and synthesis of a series of novel
thiophene[3,2ꢀd]pyrimidine derivatives with different aryl substituents varying in
size, polar group substitution and electronic nature on the piperidinꢀ4ꢀylꢀamino moiety.
AntiꢀHIV activity evaluation showed that these compounds have potent,
broadꢀspectrum profiles against WT and resistant mutant strains of HIVꢀ1, being
superior in these respects to the approved drug ETV. A pharmacokinetics study of the
most potent compound, 27, in rats showed that it has favorable, drugꢀlike
pharmacokinetics. Also, a detailed consideration of the SARs of this series of
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derivatives suggested opportunities for further elaboration of these compounds.
CHEMISTRY
The newly designed thiophene[3,2ꢀd]pyrimidine derivatives were synthesized
according to the an efficient and reliable method, previously reported for the DAPY
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family,
as outlined in Scheme 1. The synthetic work was started from
commercially available 2,4ꢀdichlorothiophene[3,2ꢀd]pyrimidine (11); successive
nucleophilic substitution reactions with substituted phenols (12a and 12b) and
4
ꢀ(tertꢀbutoxycarbonyl)ꢀaminopiperidine (14) in the presence of potassium carbonate
and N,Nꢀdimethylformamide afforded the key intermediates 15a and 15b.
Deprotection with trifluoroacetic acid at room temperature in dichloromethane
afforded the corresponding analogues 16a and 16b, which were led to the target
compounds 17ꢀ37 and 38ꢀ48 by nucleophilic substitution with substituted benzyl
chloride (or bromine) or 4ꢀpicolyl chloride hydrochloride in the presence of potassium
carbonate. All new thiophene[3,2ꢀd]pyrimidines were fully characterized by proton
1
13
nuclear magnetic resonance ( H NMR), carbon nuclear magnetic resonance ( C
NMR), and electrospray ionisation mass spectrometry (ESIꢀMS). Purity of all
compounds was determined to be >95% by analytical HPLC (Table S1 in Supporting
Information).
2 3
Scheme 1. Reagents and conditions: (i) substituted phenols (12a or 12b), DMF, K CO , r.t.; (ii)
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ꢀ(tertꢀbutoxycarbonyl)aminopiperidine (14), DMF, K CO , reflux, 12h; (iii) TFA, DCM, r.t.; (iv)
2
3
substituted benzyl chloride (or bromine) or 4ꢀpicolyl chloride hydrochloride, DMF, K
2
CO
3
, r.t.
RESULTS AND DISCUSSION
Table 1. AntiꢀHIV activity and cytotoxicity of target compounds 17ꢀ25
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
EC50 (nM)
SI
compd
17
R
CC50 (nM)
III
B
RES056
ROD
III
B
RES056
4ꢀCN
2ꢀCN
4ꢀBr
5.86±3.49
5.71±2.89
26.56±3.94
9.40±2.06
4.14±0.21
3.25±2.18
4.40±2.73
8.23±0.36
7.57±0.23
312±56
440±260
810±140
2030±920
780±90
>5370
>5780
>24320
>10360
>4530
>41390
>76740
>189400
>4660
ꢀ
5360±760
914
1014
916
12
7
1
8
5790±2410
24320±2520
10360±8270
4520±310
41390±20000
76740±22030
189400±39480
4660±200
>15020
1
2
9
0
12
13
13
59
267
8
2ꢀCH
3
1101
1091
21
3ꢀCN
360±130
700±140
290±60
2
2
2
2
3
4
4ꢀCOOCH
3
12740
17451
23011
615
3ꢀF
2ꢀF
24200±18490
520±150
≥7590
25
4ꢀNO
2
9
NVP
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
48.08
>39
>orX2ꢀ
ꢀ
3
TC
2240±820
7.1±2.9
ꢀ
8790±2910
6.6±1.4
ꢀ
>87240
AZT
EFV
DLV
ETV
10±9.2
>93550
>13144
>1014
>67
>9149
>41
6.02±2.0
653±623
4.1±0.2
156.1±14.9
>43810
>6340
ꢀ
>43810
X1
25.0±3.0
ꢀ
>4590
>1127
>184
a
EC50: concentration of compound required to achieve 50% protection of MTꢀ4 cell cultures against
HIVꢀ1ꢀinduced cytotoxicity, presented as the mean ± standard deviation (SD) and determined by the
MTT method.
b
CC50: concentration required to reduce the viability of mockꢀinfected cell cultures by 50%, as
determined by the MTT method.
c
SI: selectivity index, the ratio of CC50/EC50
.
The first phase of our new explorations involved determining the suitability of
the thiophene[3,2ꢀd]pyrimidine core. For this purpose, we kept the
2
,6ꢀdimethylꢀ4ꢀcyanophenyl structure constant in the left ring and used a small set of
substituted benzylꢀlinked piperidinꢀ4ꢀylꢀamino moieties as the right ring. It can be
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observed that the nine newly synthesized derivatives 17ꢀ25 were evaluated for
antiꢀHIV activity and cytotoxicity in MTꢀ4 cell cultures infected with WT HIVꢀ1
strain (IIIB), doubleꢀmutant strain RES056 (K103N+Y181C) and a HIVꢀ2 strain
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
ROD). Nevirapine (NVP), lamivudine (3TC), delavidine (DLV), efavirenz (EFV),
etravirine (ETV) and azidothymidine (AZT) were selected as reference drugs. The
values of EC50, CC50 and SI (selectivity index, i.e., CC50/EC50 ratio) are summarized
in Table 1. All nine compounds exhibited high potency against the WT HIVꢀ1 strain
with low nanomolar EC50 values ranging from 3.2 to 26 nM and SI values between
6
15 and 23,011, which are superior to those of the reference drugs (3TC, NVP, EFV,
DLV, AZT) and comparable to those of the secondꢀgeneration NNRTI drug ETV. As
expected, the compounds did not inhibit the HIVꢀ2 strain. 22 turned out to be the most
potent HIVꢀ1 inhibitor with an EC50 value of 3.2 nM and a high SI of 12,740 in this
initial assay, being slightly more potent than ETV.
The position of the substituents in the right phenyl ring has some influence on
the antiꢀHIV activity. For instance, compounds 17ꢀ25 are basically equipotent on WT
HIV with the exception of compound 19. Specifically, addition of a cyano group at
the C
2
or C
4
position in the phenyl ring (18 and 17) resulted in similar EC50 values of
ꢀcyano analogue 21 had an EC50 value
5
.7 nM and 5.8 nM respectively, whereas the C
3
of 4.1 nM. The activity was also influenced by the electronic nature or steric demand
of substituents in the right phenyl ring. For example, introduction of a nitro group at
4
the C position yielded a 7.5 nM inhibitor (25), while bromine at this position (19)
2
reduced the potency to 26.5 nM. Placement of a methyl group at the C ꢀposition
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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
yielded 20, which was found to have similar potency to the 2ꢀfluoro derivative 24.
As regards activity towards the doubleꢀmutant strain RES056, 23 was the most
potent compound with an EC50 value of 290 nM, which is comparable to that of EFV
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
(EC50 = 156.1 nM). It showed a moderate SI value (267) towards WT and RES056
mutant HIVꢀ1 strains. These encouraging data provided some support for our
hypothesis that introduction of thiophene[3,2ꢀd]pyrimidine to replace the central
Bꢀring of DAPYs would be feasible and effective. Interestingly, the 2ꢀfluoro
counterpart of 23, i.e., 24, showed a marked decrease of potency against RES056
mutant strain. Thus, the antiꢀresistance potency of the compounds is also dependent
on the position of the substituents in the right phenyl ring. In addition to influencing
the antiviral activity, the shapes and volumes of substituents on the right phenyl ring
also had a major influence on the cytotoxicity (CC50), so we continued our exploration
of this fused heterocyclic motif.
Based on the preliminary SARs of these novel thienopyrimidine analogues and
our previous investigations in other DAPY series, we decided to introduce some
hydrophilic groups on the aryl moiety directed toward the proteinꢀsolvent interface,
and we synthesized 12 new compounds (26ꢀ37) in the 2,6ꢀdimethylꢀ4ꢀcyanophenyl
series. In addition, based on the structural characteristics of dapivirine in conjunction
24
with previous studies, we replaced the cyano group of the left ring with a methyl
group to obtain a new 2,4,6ꢀtrimethylꢀphenyl series (11 compounds, 38ꢀ48). The
antiꢀHIV activity of these compounds was evaluated in the MTꢀ4 cell line against WT
HIVꢀ1 (IIIB), HIVꢀ2 strain (ROD), and a panel of NNRTIsꢀresistant singleꢀ and
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doubleꢀmutant strains (L100I, K103N, E138K, Y181C, Y188L, K103N+Y181C
(RES056) and F227L+V106A), which are common clinical mutations in HIVꢀinfected
patients. Nevirapine (NVP), efavirenz (EFV), etravirine (ETV) and azidothymidine
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
AZT) were selected as reference drugs. The EC , CC and SI values obtained are
50 50
summarized in Tables 2, 3 and 4.
Table 2. AntiꢀHIV activity and cytotoxicity of 26ꢀ37 and 38ꢀ48
b
c
EC50(nM)
SI
b
compd
26
R
Ar
CC (nM)
50
IIIB
22.1±11.0
1.4±0.4
ROD
>22312
≥227890
≥140940
>20621
>4082
IIIB
1005
>159101
18708
1999
185
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
4ꢀSO
4ꢀSO
4ꢀFꢀPh
2
MeꢀPh
22312±3453
>227890
2
7
2
NH ꢀPh
2
28
7.5±1.3
140940±19443
20621±4744
4081.8±701.2
>227800
2
3
3
3
9
0
1
2
pyridineꢀ4ꢀyl
2,4ꢀdiFꢀPh
10.3±1.2
22.0±0.9
2.4±1.1
3ꢀSO
4ꢀNHSO
4ꢀSO NHMeꢀPh
2
NH
2
ꢀPh
>227800
>68266
>4422
>96006
44221
2789
66867
>19216
≥1436
576
2
MeꢀPh
1.5±0.3
68266±71241
4422±1546
95352±27689
>48760
33
2
1.5±0.4
3
3
3
3
4
5
6
7
4ꢀCONH
2
2
ꢀPh
ꢀPh
1.4±0.07
2.5±0.3
>95350
>48760
>230700
>3027
3ꢀCONH
4ꢀCOOEtꢀPh
4ꢀSO NHCOMeꢀPh
4ꢀSO MeꢀPh
4ꢀCO MeꢀPh
4ꢀFꢀPh
160.7±3.7
5.2±0.9
≥230700
2
>3027
38
CH
CH
CH
CH
CH
CH
3
2
5.7±1.4
>3416
3417±1043
24873±2177
11948±7041
24591±5262
17238±6753
5653±2002
595
3
4
4
4
9
0
1
2
3
3
3
3
3
2
15.4±11.8
25.7±12.7
39.1±6.8
21.1±1.0
7.6±1.3
>24873
>11948
>24591
>17238
>5653
1612
465
3ꢀFꢀPh
3ꢀCNꢀPh
628
819
43
pyridineꢀ4ꢀyl
747
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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
4
4
4
4
5
6
CH
CH
CH
CH
CH
ꢀ
3
3
3
3
3
2,4ꢀdiFꢀPh
36.8±8.8
6.5±2.6
>13958
13958±5207
5814±2294
7638±3891
16460±8026
6394±3429
>15.0207
>6.3355
380
896
4ꢀCONH
2
2
ꢀPh
ꢀPh
>5814
3ꢀCONH
14.4±8.6
3552±848
31.8±5.2
250±65.6
5.0±02.1
4.1±0.1
>7638
531
47
4ꢀSO
2
NH
2
ꢀPh
>16460
2
4
8
4ꢀNO
2
ꢀPh
>6394
201
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
NVP
EFV
ETV
AZT
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
>60
ꢀ
>1258
>1128
>1228
ꢀ
>4.5946
ꢀ
5.8±4.0
>7.4836
a
EC50: concentration of compound required to achieve 50% protection of MTꢀ4 cell cultures against
HIVꢀ1ꢀinduced cytotoxicity, as determined by the MTT method.
b
CC50: concentration required to reduce the viability of mockꢀinfected cell cultures by 50%, as
determined by the MTT method.
c
SI: selectivity index, the ratio of CC50/EC50
.
Firstly, we will discuss the activity of the 2,6ꢀdimethylꢀ4ꢀcyanophenyl series
towards WT HIVꢀ1. These twelve newly synthesized compounds clearly showed more
potent inhibition than the initial nine compounds in this series. In particular, 27 and
31ꢀ35 displayed prominent inhibitory activity with EC50 values of 1.4 nM to 2.5 nM,
being more potent than the reference drugs AZT (EC50 = 5.8 nM), EFV (EC50 = 5.0
nM) and ETV (EC50 = 4.1 nM). On the other hand, 26, 28, 29, 30 and 31 (EC50 = 5.2
nM) were equipotent with ETV, or less potent. It was concluded that the activity is
very sensitive to substitution at the paraꢀposition. The order of potency of the
paraꢀsubstitution at the right phenyl ring was as follows: ꢀSO
nM) = ꢀCONH (34, EC50 = 1.4 nM) ≈ ꢀNHSO Me (32, EC50 = 1.5 nM) ≈
SO NHMe (33, EC = 1.5 nM) ≈ ꢀSO NHCOMe (37, EC = 5.2 nM) > ꢀF (28,
2 2
NH (27, EC50 = 1.4
2
2
ꢀ
2
50
2
50
EC50 = 7.5 nM) > ꢀCOOEt (36, EC50 = 160.7 nM), indicating that polar groups, such
as unsubstituted sulfamide and amide, are preferred over groups with lower polarity,
such as substituted sulfamide, fluorine or ester.
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It was particularly noteworthy that when the ꢀCO
2
Me group of the 22 was
replaced with ꢀCO Et (36), the increased length of the carbochain reduced the activity
2
about 50ꢀfold (EC50 = 160.7 nM), but 36 exhibited no cytotoxicity at the
concentration of 230 µM. Detailed comparison of the activities of 26 with 27 and 34
with 36 suggested that a hydrogenꢀbond donor group at the 4ꢀposition in the phenyl
ring strengthens the interaction with the NNRTI binding site. Subsequent Nꢀacylation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2 2
of SO NH of 27 gave the 5.2 nM inhibitor 37, which is as potent as the reference
drug ETV (EC50 = 4.1 nM); however, 37 shows reduced activity and increased
cytotoxicity compared with 27, suggesting that increased hydrophobicity and steric
bulk at the paraꢀposition impact negatively on the activity and cytotoxicity.
In addition, it is noteworthy that fluorine substitution at the C
.5 nM compound 28, which is equipotent with the C ꢀfluorine compound (24, EC50
8.2 nM), whereas the simultaneous presence of fluorines at C and C in the phenyl
4
ꢀposition gave the
7
2
=
2
4
ring was significantly detrimental: the potency was reduced to 22 nM and the
cytotoxicity was increased 4.08 ꢁM. On the whole, para substitution of the phenyl
moiety on the right ring, relative to the metaꢀposition, seems favorable for antiviral
potency, as suggested by comparison of 27/31 and 34/35.
The cytotoxicity is also strongly dependent on the nature of the substituents at
the paraꢀposition of the phenyl ring. For instance, 27 and 31 with ꢀSO NH
2
2
4 3
substitution at C and C in the phenyl ring showed no cytotoxicity (CC50) up to the
maximum tested concentration of 227 µM, while addition of a methyl group to
ꢀSO
2
NH
2
(33) sharply increased the cytotoxicity to 4.4 µM (>51.7ꢀfold increase).
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5
6
7
8
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
Compared to 27 and 31, 34 and 35 bearing a ꢀCONH
cytotoxicity. Replacement of the SO NH group at C in the phenyl ring with SO
yielded 26, which showed reduced cytotoxicity and decreased potency.
2
group showed moderate
2
2
4
2
Me
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
Furthermore, the pyridine derivative 29 showed an acceptable antiꢀHIVꢀ1 profile,
with an EC50 of 10.3 nM against WT HIVꢀ1 (Table 2). These results support the idea
that introduction of structurally diverse heterocycles in this region could be a valid
strategy to discover novel molecules with appreciable antiviral potency.
Next, we turned our attention to the SAR of the R substituent at the
paraꢀposition of the left ring. The activities of the 2,4,6ꢀtrimethylphenyl series (38ꢀ48)
were much lower than those of the 2,6ꢀdimethylꢀ4ꢀcyanophenyl series, except for 38
and 43, which showed an improvement of EC50 to 5.7 nM and 7.6 nM compared with
2
6 and 29, respectively. Moreover, nearly all of the 2,4,6ꢀtrimethylphenyl series
compounds exhibited higher cytotoxicity than the 2,6ꢀdimethylꢀ4ꢀcyanophenyl series.
All these results indicate that the cyano group plays a key role in enhancing the
antiviral potency and reducing cytotoxicity. Preliminary SAR of the paraꢀsubstituent
group in the phenyl ring indicated the following order of activity towards WT HIVꢀ1:
ꢀSO Me (38: EC = 5.7 nM) ≈ ꢀCO NH (45: EC = 6.5 nM) > ꢀCO Me (39: EC
2
50
2
2
50
2
50
=
2
15.4 nM) > F (40: EC50 = 25.7 nM) > NO (48: EC50 = 31.8 nM). 47 bearing the
SO NH substituent showed sharply weaker activity than the corresponding derivative
2
2
2
7. This seems to be a good example of an activity cliff (in particular, a 3D activity
cliff), where pairs of compounds with similar structure exhibit a large difference in
29,30
activity profile.
High sensitivity of the activity profile to molecular configuration
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is related to the flexibility of the binding pocket and the inducedꢀfit mechanism of the
NNRTIs (vide supra).
Similarly, variation of the position of the substituent in the right ring (from the
paraꢀposition to the metaꢀposition) resulted in slightly reduced potency (40/41, 45/46),
which is consistent with the SAR conclusions in the 2,6ꢀdimethylꢀ4ꢀcyanophenyl
series.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The activity of the synthesized derivatives was further evaluated against a panel
of clinical relevant HIVꢀ1 mutant strains, including L100I, K103N, Y181C, Y188L,
E138K, F227L+V106A and RES056, in MTꢀ4 cells. The results are summarized in
Table 3. Generally speaking, compounds that displayed potent activity against WT
HIVꢀ1 virus also showed potent activity against mutant HIVꢀ1 strains. Nearly all of
the compounds in the 2,6ꢀdimethylꢀ4ꢀcyanophenyl series exhibited high potency
against mutant HIVꢀ1 replication, with nanomolar EC50 values and much higher SI
values than EFV, comparable to those of ETV and AZT, with the exception of 36.
Regarding the fold resistance (RF, ratio of EC50 against mutant strain/EC50 against
WT strain), most compounds exhibited low RF values towards L100I, K103N, Y181C,
Y188L, E138K and F227L+V106A, as shown in Table S2 (see Supporting
Information). Indeed, the most potent compound 27 showed nearly the lowest RF
value (< 3) towards these mutants with the exception of RES056, and was comparable
to ETV in this regard.
Notably, derivatives with hydrophilic substituents in the phenyl ring, such as
4
2 2 2 2 2 2 2 2
ꢀSO NH , 3ꢀSO NH , 4ꢀNHSO Me, 4ꢀSO NHMe, 4ꢀSO NHCOMe and 4ꢀCONH ,
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1
1
1
1
1
1
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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
were as potent as ETV or more potent towards nearly all the mutants, with the notable
2
exception of the 3ꢀCONH substituent (35, 36), which resulted in substantially
reduced antiviral activity. Replacement of hydrophilic groups in the phenyl ring with
hydrophobic groups (such as 27 vs. 28, 36; 45 vs. 40, 41 and 42) markedly reduced
the activity towards the mutants, as well as towards WT HIVꢀ1 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
In the case of the L100I mutant HIVꢀ1 strain, 27 and 33 exhibited potency of 3.4
nM and 4.5 nM, respectively, being far more potent than EFV, showing the same
potency as ETV, and comparable to AZT. As for K103N, which is the most common
mutation emerging in HIV patients treated with EFV, eleven compounds provided
singleꢀdigitꢀnanomolar activity. In particular, 33 (EC50 = 1.5 nM) was notably more
active than ETV and AZT. Further, compounds 27 and 32ꢀ34 showed
singleꢀdigitꢀnanomolar inhibitory activity towards the single mutants Y181C, Y188L
and E138K, with EC50 values ranging from 2.9 nM to 8.2 nM (at least 2ꢀ7 fold more
potent than ETV). 31 and 37 were also highly potent inhibitors toward Y188L and
E138K, with greater potency than ETV; however, both derivatives exhibited reduced
inhibition of the Y181C strain. Intriguingly, seven compounds (27, 31ꢀ35 and 37) in
the 2,6ꢀdimethylꢀ4ꢀcyanophenyl series displayed greater potency than ETV against
E138K, a major mutation conferring resistance to the newꢀgeneration drug rilpivirine
(5, RPV). In particular, 27, 31 and 34 had high antiꢀresistance potency, with RF =
23
2
.0ꢀ3.1 for E138K.
Against the doubleꢀmutant strain F227L+V106A, derivatives with hydrophilic
substituents in the phenyl ring were all superior to ETV, except for 32, indicating that
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ꢀNHSO
2
Me is unfavorable for activity towards this mutant strain. Among all the
derivatives, 27 and 33 showed the greatest potency against the double mutation
RES056 (EC50 = 30.6 and 32.4 nM), being slightly inferior to ETV (EC50 = 17 nM),
but they were less potent towards K103N and Y181C.
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However, the 2,4,6ꢀtrimethylphenyl series showed weaker activity than the
2
2 3
,6ꢀdimethylꢀ4ꢀcyanophenyl series, with the exception of 38 bearing a SO CH
substituent. 38 exhibited remarkable potency against several key mutant strains.
Indeed, it turned out to be a 23.9 nM inhibitor of F227L+V106A, being more potent
than ETV. 45 was also effective against E138K. In contrast, 47 with the SO NH
2
2
substituent showed greatly reduced activity compared to 27, which may represent
another example of the activity cliff concept. 44 with fluorine substitution at both C
and C of the phenyl ring also showed reduced potency compared with
monoꢀfluorineꢀsubstituted 40 and 41.
2
4
Among all the compounds examined, 27 showed outstanding antiretroviral
potency against most of the viral panel, being about 3ꢀfold (WT, EC50 = 0.14 nM),
1
.6ꢀfold (L100I, EC50 =3.4 nM), 5ꢀfold (Y181C, EC50 = 3.2 nM; E138K, EC50 = 2.9
nM) and 7ꢀfold (Y188L, EC50 = 3.0 nM; F227L+V106A, EC50 = 4.2 nM) more potent
than the reference drug ETV in the same cellular assay, and remarkably superior to
that of the reference drugs NVP and EFV. Importantly, 27 showed no cytotoxicity at
concentrations up to 227ꢁM, resulting in a huge SI value (CC50/EC50) of >159,101
compared with that of ETV (CC50 > 4.59 ꢁM; SI > 1128). Since this novel chemotype
has activity against a broad range of NNRTIsꢀresistant mutant viruses, we believe that
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compounds of this class have enormous potential as antiꢀHIV drug candidates.
To validate the binding target of these novel thienopyrimidine derivatives, some
representative compounds were also tested for ability to inhibit recombinant WT and
K103N/Y181C doubleꢀmutant HIVꢀ1 RT enzymes; the results are shown in Table 4.
In this assay, all the tested compounds exhibited potent inhibitory activities toward
WT with IC50 values ranging from 0.21 to 1.68 ꢁM, being superior to the reference
drug NVP. These compounds were also comparable in potency to EFV against
doubleꢀmutant RT, with the IC50 values ranging from 0.36 to 46.37 ꢁM. Generally, the
SAR for inhibitory potency appeared to be consistent between HIVꢀ1 replication and
RT activity. Hydrophilic substituents on the phenyl ring were beneficial for inhibitory
activities against both WT and double mutant RT (27, 29, 38 and 43).
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Compound 43 with a pyridineꢀ4ꢀyl substituent in the phenyl ring displayed the
most potent activity towards WT RT with an IC50 value of 0.21 ꢁM, while the
inhibitory activity against doubleꢀmutant RT was reduced to 1.26 ꢁM (FR = 6.0).
4 2 2
Another potent inhibitor of WT RT (IC50 = 0.30 ꢁM), the C ꢀSO NH derivative 27,
also showed more potent inhibitory activity against doubleꢀmutant RT than the
reference drug EFV, and it also showed a low FR value (1.20) with respect to WT RT.
It seems that the binding mode of 27 was not affected by the K103N/Y181C mutation
and was consistent with the interactions reported for the WT strain. These results
demonstrated that representative compounds displayed high affinity for WT and
doubleꢀmutant HIVꢀ1 RT, and support the view that the target of the newly
synthesized compounds is HIVꢀ1 RT.
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It is noteworthy that the antiviral activities of the best compounds in this study
(
the pyridineꢀ4ꢀyl 43 and the sulfonamide 27) were inconsistent with their
enzymeꢀinhibitory potencies to some extent. These differences are considered to be
due to templateꢀspecific variation in the relationship between HIVꢀRTꢀRNA binding
affinity and polymerase processivity, and have been observed in most NNRTI
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26
27
28
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32
33
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38
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43
44
45
46
47
48
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31
series. Besides, since these molecules have multiple hydrogen bond donor and
acceptor sites, there is a high probability of formation of cocrystals or salts. Therefore,
the differences between the two assay systems can be attributed at least in part to
differences in the physiochemical properties (especially the intrinsic solubility).
Table 4. Inhibitory activity of compounds 26ꢀ29, 38ꢀ44 against HIVꢀ1 RT (WT and
K103N/Y181C double mutant).
a
b
IC50(ꢁM) (fold resistance)
Compd
Ar
WT
K103N/Y181C double
mutant
1
.51±0.09 (1)
2.83±0.39 (1.87)
2
2
2
6
7
8
4ꢀSO
4ꢀSO
2
MeꢀPh
0.30±0.06(1)
.81±0.06 (1)
0.36±0.06 (1.20)
2.57±0.16 (3.17)
3.74±0.06 (2.87)
NH ꢀPh
2
2
0
4ꢀFꢀPh
pyridineꢀ4ꢀyl
4ꢀSO MeꢀPh
4ꢀCO MeꢀPh
4ꢀFꢀPh
1.30±0.22 (1)
0.65±0.08 (1)
2
3
9
8
2
2.50±0.40 (3.84)
12.3±0.21 (9.92)
1.24±0.15 (1)
1.23±0.07 (1)
0.79±0.02 (1)
1.06±0.01 (1)
0.21±0.01 (1)
39
2
6.57±1.80 (5.34)
40.4±25.3 (51.2)
5.57±0.67 (5.25)
1.26±0.27 (6.0)
46.37± 5.18(30.0)
ꢀ
4
0
41
3ꢀFꢀPh
3ꢀCNꢀPh
4
2
43
pyridineꢀ4ꢀyl
2
,4ꢀdiFꢀPh
ꢀ
1.6±0.21 (1)
2.32 (1)
4
4
NVP
EFV
ETV
ꢀ
ꢀ
0.03 (1)
1.42 (47.3)
0.09
c
0.011±0.000
a
IC50: inhibitory concentration of test compound required to inhibit biotin deoxyuridine
triphosphate (biotinꢀdUTP) incorporation into HIVꢀ1 (WT, K103N/Y181C double mutant) RT by
5
b
0%.
fold resistance (FR): IC50(K103N/Y181C mutant RT)/IC50(WT RT) ratio.
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c
The data were obtained from the same laboratory (Prof. Erik De Clercq, Rega Institute for
25e
Medical Research, K.U. Leuven, Belgium).
MOLECULAR MODELING STUDIES
To obtain further insight into the allosteric binding of the thienopyrimidine
derivatives to the NNIBP of RT and to gain a deeper understanding of key structural
aspects of their interaction, we performed molecular docking studies for
representative compounds 27, 32, 33, 34 and 43 by using the software SurflexeDock
SYBYLꢀX 2.0. Docking results were visualized with PyMOL. The starting point to
establish the most suitable settings for docking experiments was the crystallographic
structure of the coꢀcrystal of WT RT with an analogue (PDB ID: 3M8Q). The docking
protocol is described in the computational section.
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8
9
0
(
a)
Figure 3. Predicted binding modes of 27 with the HIVꢀ1 WT RT crystal structure (PDB: 3M8Q).
a) Overview; (b) View looking inward along the enzymeꢀsolvent interface (tolerant region I, the
(b)
(
Pro236 hairpin loop), showing the solventꢀexposed lower surface of the benzene sulfonamide
group in 27. The hydrogen bonds between the inhibitors and amino acid residues are indicated
with dashed lines (yellow). Ligand carbon atoms are shown in cyan, and protein carbon atoms in
green. Hydrogen atoms are not shown for clarity.
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(
a)
(b)
(
c)
(d)
Figure 4. Predicted binding modes of 32 (a), 33 (b), 34 (c) and 43 (d) with the HIVꢀ1 WT RT
crystal structure (PDB: 3M8Q). The hydrogen bonds between the inhibitors and amino acid
residues are indicated with dashed lines (yellow). Ligand carbon atoms are shown in cyan, and
protein carbon atoms in green. Hydrogen atoms are not shown for clarity.
The docking simulations of these molecules with HIVꢀ1 WT RT indicated that
the binding mode resembles those of known and approved NNRTI drugs (Figure 3
and 4). In particular, the SYBYLꢀX 2.0ꢀpredicated binding mode of these compounds
resembles that of known and approved NNRTI drugs of the DAPY family. Detailed
computational analysis of the pharmacophoric interactions revealed the following
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features. Firstly, the left 4ꢀcyanoꢀ2,6ꢀdimethylphenyl or 2,4,6ꢀtrimethylphenyl group
of these compounds occupies the subꢀpocket formed by hydrophobic aromatic amino
acid residues Tyr181, Tyr188, Phe227 and Trp229, exhibiting πꢀπ interaction with
these residues. Secondly, the thiophene[3,2ꢀd]pyrimidine heterocycle effectively
occupies the NNIBP entrance channel, and the sulphur atom of the thiophene
heterocycle has an electrostatic interaction with Val179. Thirdly, the NH linker
connecting the central pyridine ring and the right ring forms the “signature” hydrogen
bond with the mainꢀchain backbone of Lys101, as seen with many NNRTIs. Fourthly,
the piperidineꢀlinked aryl structure is directed to the proteinꢀsolvent interface, and can
develop extensive interactions with surrounding lipophilic amino acids, thereby
improving the stability of the RTꢀinhibitor complex. Notably, the hydrophilic sulfonyl
group of 27 or methanesulfonamide of 32 (Figure 4a) provides two hydrogenꢀbond
receptors, thus enabling double hydrogenꢀbonding (chelate type) interactions with the
main chain of Lys104 and Val106, which would increase the stability of the
RT/NNRTI complex; In addition, the NH of the Nꢀmethylbenzenesulfonamide group
in 33 (Figure 4b) participates in one hydrogen bond with the backbone C=O group of
Lys104. Compared with 32, the disappeared hydrogen bond between 33 and Val106
may explain the very distinct inhibition profile against F227L+V106A mutant strain
(EC50 = 4.1 nM), which is much better than that of 32 (EC50 = 32.2 nM), probably
because the binding mode of 33 was slightly altered by the V106A mutation. In
contrast, the pyridine ring in 34 does not establish any hydrogen bond interactions
with residues in the enzymeꢀsolvent interface, in accordance with the eclipsed
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antiꢀresistance profiles (Table 4). More importantly, only for 27, additional
hydrogenꢀbond(s) were formed with a bridging water molecule. This provides a
rational explanation of the improved inhibitory potency of 27 towards WT and
drugꢀresistant HIVꢀ1 mutant strains, as compared with ETV (Figure 2) and the other
four counterparts (Figure 4aꢀd). The combination of the above four features is unique
to 27, and this may be the reason why 27 possessed the most potent activity against
WT and mutant HIVꢀ1 strains.
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45
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In summary, the docking simulations indicated that 27 appears to have achieved
the binding mode and the key pharmacophoric interactions that we had targeted in our
original design. Further optimization of backꢀup derivatives is in progress, guided by
these docking simulation results.
IN VIVO PHARMACOKINETICS STUDY AND SAFETY ASSESSMENT
To examine the drug potential of 27, we evaluated the singleꢀdose oral
ꢀ1
pharmacokinetics of 27 in rats after at two dosage levels (8 and 16 mg.kg ). As
shown in Table 5 and Figure 5, the pharmacokinetics of 27 is characterized by a large
volume of distribution (Vz/F of 38.2 and 50.1 L/kg), fast Tmax (1 h, 0.8 h), moderate
plasma clearance (CLz/F of 10.3 and 9.3 L/h/kg), favorable halfꢀlife (t1/2 of 2.51 h and
3
.60 h) and mean residence time (MRT) of 7.7 h and 9.29 h at these two dose levels.
All these results indicate that 27 as an orally bioavailable candidate for clinical
treatment of human HIVꢀ1 infection.
Single dose toxicity test of compound 27 was carried out in mice. After
intragastric administration of 27 with a dose of 2000 mg/kg, no death of the mice was
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observed and there was no abnormity of the body weight increase for the animals in
one weeks.
a
Table 5. Main pharmacokinetic parameters of 27
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Parameter
AUC(0ꢀt)
Unit
8 mg/kg
1675.8±517.0
1720.9±513.9
5690.8±1589.3
6474.1±1574.9
3.44±0.46
16 mg/kg
1668.8±348.1
1756.7±325.1
5458.8±1341.0
7215.795±1278.4
3.25±0.19
ꢁg/L*h
ꢁg/L*h
AUC(0ꢀ∞)
AUMC(0ꢀt)
AUMC(0ꢀ∞)
MRT(0ꢀt)
h
h
MRT(0ꢀ∞)
VRT(0ꢀt)
3.84±0.51
4.16±0.79
h^2
h^2
h
7.78±1.43
9.29±1.32
VRT(0ꢀ∞)
13.31±2.96
2.51±0.34
25.29±18.57
3.60±1.12
t
1/2
z
T
max
h
1±0.61
0.8±0.27
CLz/F
Vz/F
L/h/kg
L/kg
ꢁg/L
10.3±4.3
9.3±1.8
38.2±19.2
50.1±24.7
C
max
415.1±160.6
471.9±114.8
a
PK parameters (Mean ± SD, n =5).
Figure 5. The plasma concentration–time profiles of 27 in rats following single oral
ꢀ1
ꢀ1
administration at doses of 8 mg.kg (blue) and 16 mg.kg (red).
CONCLUSION
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By exploiting the tolerated regions of the NNIBP, and employing a strategy of
structureꢀbased scaffold hopping and molecular hybridization, we identified a series
of new piperidineꢀlinked thiophene[3,2ꢀd]pyrimidine derivatives having various
substituents at the secondary amine of the piperidine as highly potent HIVꢀ1 inhibitors.
Most of the synthesized compounds exhibited significant inhibitory activity towards
WT HIVꢀ1 strain in MTꢀ4 cells (EC50 < 10 nM). Among them, 17, 20ꢀ23 and 27
exhibited high potency towards WT, with EC50 values of less than 5 nM and were as
potent as, or more potent than, the new NNRTI drug ETV. As for RT HIVꢀ1 mutant
strains, 27ꢀ30, 38 and 43 showed comparable inhibitory activities to those of EFV
against L100I, K103N, Y188L and F227L+V106A. Notably, seven compounds (27,
31ꢀ35 and 37) in the 2,6ꢀdimethylꢀ4ꢀcyanophenyl series displayed greater potency
than ETV against E138K, a major viral mutation conferring resistance to the
newꢀgeneration drug rilpivirine. 38 and 43 exhibited similar inhibitory activities to
ETV against E138K. 27 proved to be exceptionally potent, with EC50 values of 1.4
nM (WT), 3.4 nM (L100I), 2.9 nM (K103N), 3.2 nM (Y181C), 3.0 nM (Y188L), 2.9
nM (E138K), 4.2 nM (F227L+V106A), and 30.6 nM (RES056) in MTꢀ4 cells; it was
more potent than ETV against all these strains except RES056. 27 also has much
lower cytotoxicity (CC50 > 227 ꢁM) and a dramatically higher SI value of >159,101.
In addition, 27 displayed greater inhibitory activities than NVP and EFV against
recombinant WT and K103N/Y181C doubleꢀmutant RT enzymes in RT inhibition
assay. 27 was confirmed to show favorable, drugꢀlike pharmacokinetic properties in
singleꢀdose administration studies in rats. Overall, our results indicate that 27 holds
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great promise as a potential nextꢀgeneration antiꢀHIV drug candidate with
significantly improved activity, high selectivity, an outstanding drug resistance
profiles and favorable pharmacokinetic and safety profile.
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Molecular docking experiments suggested that the strong activity profile of 27
against WT and mutant HIVꢀ1 strains can be attributed at least in part to Hꢀbond
interactions between the hydrophilic sulfonyl group of 27 and the main chain of
Lys104 and Val106, together with the nonpolar interaction between the sulphur atom
of the thiophene heterocycle and Val179. These results are in accordance with
established SAR data and the experimental findings with the panel of WT and mutant
HIVꢀ1 strains. Generally, it seems likely that the extensive network of mainꢀchain
hydrogen bonds is unlikely to be disrupted by side chain mutations and contributes
substantially to the free energy of binding for the inhibitor, and lastly, significantly
improves the resistance profiles of these inhibitors. Therefore, this important design
concept targeting the main chain of a protein and forming additional key interactions
indeed offers a reliable strategy for combating drug resistance. We consider that these
thiophene[3,2ꢀd]pyrimidine compounds are exciting outcomes of the simultaneous
exploitation of tolerated regions I and II of NNIBP. Our findings also provide useful
clues to specific interactions that could be utilized in the design of future HIVꢀ1
NNRTIs. We believe that the thiophene[3,2ꢀd]pyrimidine scaffold can be further
optimized by adjusting the substitution at thiophene, and we are currently focusing
our further medicinal chemistry efforts on this area.
EXPERIMENTAL SECTION
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Page 29 of 65
Journal of Medicinal Chemistry
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Chemistry
All melting points were determined on a micro melting point apparatus (RYꢀ1G,
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Tianjin TianGuang Optical Instruments) and are uncorrected. HꢀNMR and CꢀNMR
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spectra were recorded in CDCl or DMSOꢀd6 on a Bruker AVꢀ400 spectrometer with
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tetramethylsilane (TMS) as the internal standard. Coupling constants are given in
hertz, and chemical shifts are reported in δ values (ppm) from TMS; signals are
abbreviated as s (singlet), d (doublet), t (triplet), q (quarter), and m (multiplet). A
G1313A Standard LC Autosampler (Agilent) was used to collect samples for
measurement of mass spectra. The temperature of the reaction mixture was monitored
with a mercury thermometer. All reactions were routinely monitored by thin layer
chromatography (TLC) on Silica Gel GF254 for TLC (Merck), and spots were
visualized with iodine vapor or by irradiation with UV light (λ = 254 nm). After
completion of each reaction, the mixture was brought to room temperature by means
of airꢀjet cooling. Flash column chromatography was performed on columns packed
with Silica Gel (200ꢀ300 mesh), purchased from Qingdao Haiyang Chemical
Company. Solvents were of reagent grade and 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. Analysis of sample purity was performed on a Shimadzu SPDꢀ20A/20AV
HPLC system with a Inertsil ODSꢀSP, 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
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