Discovery of Novel Diarylpyrimidine Derivatives as Potent HIV-1 NNRTIs Targeting the "nNRTI Adjacent" Binding Site

Article abstract of DOI:10.1021/acsmedchemlett.7b00524

A novel series of diarylpyrimidine derivatives, which could simultaneously occupy the classical NNRTIs binding pocket (NNIBP) and the newly reported "NNRTI Adjacent" binding site, were designed, synthesized, and evaluated for their antiviral activities in

Full text of DOI:10.1021/acsmedchemlett.7b00524

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Letter
Discovery of Novel Diarylpyrimidine Derivatives as Potent
HIV-1 NNRTIs Targeting the “NNRTI Adjacent” Binding Site
Zhipeng Huo, Heng Zhang, Dongwei Kang, Zhongxia Zhou, Gaochan Wu, Samuel
Desta, Xiaofang Zuo, Zhao Wang, Lanlan Jing, Xiao Ding, Dirk Daelemans,
Erik De Clercq, Christophe Pannecouque, Peng Zhan, and Xinyong Liu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00524 • Publication Date (Web): 27 Feb 2018
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Discovery of Novel Diarylpyrimidine Derivatives as Potent HIV-1
NNRTIs Targeting the “NNRTI Adjacent” Binding Site
Zhipeng Huo,† Heng Zhang,† Dongwei Kang,† Zhongxia Zhou,† Gaochan Wu,† Samuel Desta,† Xiaofang
Zuo,† Zhao Wang,† Lanlan Jing,† Xiao Ding,† Dirk Daelemans,§ Erik De Clercq,§ Christophe
Pannecouque,§ Peng Zhan,†,* and Xinyong Liu†,*
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† Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceu-
tical Sciences, Shandong University, 44 West Culture Road, 250012 Ji’nan, Shandong, PR China
§ Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, K.U. Leuven, Herestraat 49 Postbus
1043 (09.A097), B-3000 Leuven, Belgium.
KEYWORDS: HIV-1, AIDS, NNRTIs, drug design, “NNRTI Adjacent” binding site.
ABSTRACT: A novel series of diarylpyrimidine derivatives, which could simultaneously occupy the classical NNRTIs binding
pocket (NNIBP) and the newly reported “NNRTI Adjacent” binding site, were designed, synthesized and evaluated for their antivi-
ral activities in MT-4 cell cultures. The results demonstrated that six compounds (20, 27 and 31-34) showed excellent activities
against wild-type (WT) HIV-1 strain (EC50 = 2.4-3.8 nM), which were more potent than that of ETV (EC50 = 4.0 nM). Furthermore,
20, 27, 33 and 34 showed more potent or equipotent activity against single mutant HIV-1 strains compared to that of ETV. Espe-
cially, 20 showed marked antiviral activity, which was 1.5-fold greater against WT and 1.5−3-fold greater against L100I, K103N,
Y181C, Y188L, and E138K when compared with ETV. In addition, all compounds showed lower toxicity (CC50 = 5.1-149.2 μM)
than that of ETV (CC50 = 2.2 μM). The HIV-1 RT inhibitory assay was further conducted to confirm their binding target. Prelimi-
nary structure-activity relationships (SARs), molecular modeling as well as calculated physicochemical properties of selected com-
pounds were also discussed comprehensively.
in recent years (Figure 2).7-10 Notably, series of triazine deriva-
Attributing to their potent activity, modest toxicity, high
tives and piperidine-substituted thiophene[3,2-d]pyrimidine deriv-
atives were the most remarkable, with a prominent antiviral ac-
tivity against the clinical prevalent mutations. The most potent
compounds DCS-a4 (6) and K-5a2 (7) exhibited EC50 values of
7.8 nM and 2 nM against WT HIV-1, respectively.8,11 Encourag-
ingly, 7 showed improved potency against resistance-associated
variants.
specificity and favorable pharmacokinetic properties, HIV-1
non-nucleoside reverse transcriptase inhibitors (NNRTIs)
function as an essential component of highly active antiretro-
viral therapy (HAART).1,2 Nevirapine (1, NVP), delavirdine (2,
DLV) and efavirenz (3, EFV) are first generation NNRTIs
approved by U.S. FDA (Figure 1), but the rapid emergence of
drug-resistant compromised their clinical application, among
which K103N and Y181C are the two most prevalent NNRTI
resistance-associated mutations selected by NVP and EFV.3
Although second generation NNRTIs, etravirine (4, ETV) and
rilpivirine (5, RPV), which belongs to the diarylpyrimidine
(DAPY) family, showed better anti-resistance profiles than the
first generation NNRTIs, they still suffer from bad pharmaco-
kinetics and cross-drug resistance with long-term clinical ther-
apy.3 Among them, E138K was the most commonly observed
resistance mutation in the treatment-emergent of second gen-
eration NNRTIs. Therefore, there is an urgent need to exploit
novel NNRTI drugs with improved potency against resistance-
associated variants.4
Recently, an underexploited sites named as the “NNRTI Adja-
cent” binding site (PDB code: 4KFB), which formed by Thr139
(p51), Pro140 (p51), Thr165, Leu168, Lys172, and Ile180, was
found as an additional opening tunnel-like pocket. It is located at
the p66/p51 interface in the palm sub-domain adjacent to Glu138
of p51, separated from the NNIBP by β9 strand. Gratifyingly, the
highly conserve residues Pro140, Ile180, and Gln182 were related
in pivotal interactions, indicating that it is a promising site for the
design of novel DAPY derivatives.12 In this paper, we firstly at-
tempt to design novel diarylpyrimidine derivatives target both
NNIBP and the “NNRTI Adjacent” binding site simultaneously.
The novel target compounds incorporated the privilege skeleton
of ETV and the structure characteristics of K-5a2 (Figure 2). The
linkers with different length and substituents varying in size and
electronic feature were primarily designed based on the chemistry
space around the original fragment at NNRTI adjacent site. We
also proposed that the privileged aromatic/heterocyclic substitu-
ents would improve physicochemical properties.13 Herein, we
report the synthesis, biological evaluation and RT inhibition assay
of novel diarylpyrimidine derivatives. Furthermore, preliminary
SARs, molecular modeling and physicochemical properties study
are also discussed.
Based on the recent X-ray crystallographic studies, solvent-
accessible regions such as tolerant region I (the Pro236 hairpin
loop) and tolerant region II (the entrance channel, namely, the
largely open region in front of Leu100, Lys101, Glu138,
andVal179) in the NNIBP are revealed as the two broad regions
which could accommodate the newly designed NNRTIs.5,6 Modi-
fications of the DAPY scaffold such as fused heterocycles and/or
introducing other groups like piperidine-substituted groups target-
ing these two tolerant regions have made breakthrough in our lab
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The synthetic protocol for 5-substituted diarylpyrimidine deriv-
inhibit E138K mutant HIV-1 strain in lower or equal
concentration than ETV (EC50 = 16.9 nM).
atives is depicted in Scheme 1.14-17 Firstly, treatment of commer-
cially available 2,4-dichloro pyrimidine (8) with 4-hydroxy-3,5-
dimethylbenzonitrile (9) afforded intermediate 10. Then, 12 was
obtained by Buchwald-Hartwig reaction of 10 and 4-
aminobenzonitrile (11). With the presence of N-iodosuccinimide
(NIS) and CF3COOH, key intermediate 13 was obtained through
electrophilic reaction. Subsequently, 13 reacted with methyl thio-
glycolate or methyl mercaptopropionate to provide important
intermediate 14 or 15 via cross-coupling reaction. The oxidative
product 16 and 17 was prepared by treating 14 and 15 with m-
CPBA. Finally, 14 was hydrolyzed with lithium hydroxide to
afford 18 and then yield the target compounds 19-34 by amide
condensation reaction. Both analytical and spectral data of the
newly synthesized compounds were found in full agreement with
the proposed structures.
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3) As for single-mutant strains L100I, Y181C, and Y188L,
it was noted that compounds 20 and 33 had single-digit
nanomolar activity (EC50 = 6.5 and 7.7 nM, respective-
ly) against L100I being slightly more potent compared
to AZT (EC50 = 8.3 nM), and compounds 20, 27, and 33
exhibited an EC50 values ranging from 11.1 to 13.8 nM
against Y181C, which were somewhat greater than
AZT (EC50 = 14.1 nM).
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4) Against the double-mutant strains F227L/V106A and
Y181C/K103N, the most potent compounds 20, 27, 33,
and 34 exhibited a moderate activity (EC50 > 100 nM ).
It was concluded that the inflexible conformation of
compounds caused by bulky groups may be account for
their moderate activity.
Antiviral activity were evaluated in MT-4 cell cultures infected
with WT HIV-1 strain (IIIB), single-mutant strains L100I, K103N,
Y181C, Y188L, E138K, and double-mutant strains
F227L+V106A and K103N+Y181C (RES056). NVP, EFV, ETV,
and azidothymidine (AZT) were selected as reference drugs. The
values of EC50 (anti-HIV potency), CC50 (cytotoxicity) and SI
(selectivity index, CC50/EC50 ratio) are summarized in Table 1
and Table 2.
Based on the above results, the preliminary SARs could be
concluded in terms of the length of the chain and different termi-
nal substitution group. For the former, detailed comparison of 14
(EC50 = 65.9 nM) with 15 (EC50 = 60.2 nM), 16 (EC50 = 68.8 nM)
with 17 (EC50 = 1563.9 nM), 19 (EC50 = 6.2 nM) with 20 (EC50
=
2.6 nM) and 27 (EC50 = 2.4 nM) with 31 (EC50 = 3.8 nM) for their
activity against WT HIV-1 strain suggested that the length of
carbon chain couldn’t govern the antiviral activity neither propor-
tionally nor inversely. It was anticipated that compounds bearing
different length of carbon chain would present distinct confor-
mations and so that had distinguished antiviral potency.
All the synthesized compounds were found to be active against
WT HIV-1 with an EC50 values of 2.4 nM-216.5 nM and SI val-
ues between 152-15413, with an exception of 17 (EC50 = 1563.9
nM, SI = 9). Notably, 11 compounds (19, 20, 25-27, 29-34) dis-
played superior activity (EC50 < 10 nM), among which six com-
pounds (20, 27, 31-34) exhibited more potent or equipotent antivi-
ral activity (EC50 = 2.4−3.8 nM) compared to ETV (EC50 = 4.0
nM). The most potent compound 27 (EC50 = 2.4 nM) and 33
(EC50 = 2.4 nM) exhibited a 1.6-fold greater potency than ETV.
Meanwhile, all the compounds showed much lower cytotoxicity
(CC50 = 5.1-149.2 μM) than that of ETV (CC50 = 2.2 μM).
For the latter, when we focused on 14 (EC50 = 65.9 nM) and 15
(EC50 = 60.2 nM) comparing with their sulfur oxidation products
16 (EC50 = 68.8 nM) and 17 (EC50 = 1563.9 nM), the result sug-
gested that sulfone group have a negative impact on the antiviral
activity. Next, comparison of activity of 34 (methoxy group, 3.8
nM), 19 (morpholinyl, 2.6 nM), and 21 (Boc-protected pyrazinyl,
32.6 nM) with that of 22 (Boc-protected pyrazinyl, 88.5 nM), 23
(Boc-protected 3-aminopyrrolyl, 33.9 nM) and 24 (thiomor-
pholinyl, 38.3 nM) indicated that polar groups are much preferred
than groups with lower polarity distinctly. By comparing the ac-
tivity between 31-33 and 29-30, it was suggested that compounds
with groups in similar size with short linker had similar activity,
and small group with short linker (31-33) displayed better activity
than those of bulky groups, such as phenol (29) and tetrahydrofu-
ran (30). It was also found that the terminal t-butyloxycarbonyl
group was an inferior group that impeded antiviral potency when
compared 21, 22 and 23 to 28.
Table 2 showed that compounds with potent activity against
WT HIV-1 strain also exhibited potent activity against mutant
HIV-1 strains. Among all the target compounds, 20, 27, 33, and
34 were demonstrated with excellent activity against HIV-1 single
mutations L100I, K103N, Y181C, Y188L and E138K, being more
potent or equipotent compared to ETV. Particularly, 20 was the
most potent inhibitor and its antiviral efficacy was 1.5−3-fold
greater against these single mutations than ETV. But for the dou-
ble-mutant strains F227L+V106A and K103N+Y181C, 20
showed less potent activity. It is noteworthy that our newly de-
signed compounds showed an overall and excellent activity for a
panel of single mutants and the results are summarized as follows:
In general, these data indicated that modifications at 5-position
of pyrimidine ring contribute to improve antiviral activity against
WT and a panel of single mutant strains, especially K103N and
E138K. The preliminary SARs will support information for the
future structure modification. In addition, the compounds did not
inhibit HIV-2.
1) In case of the K103N mutant strain, all the thirteen test-
ed compounds showed lower EC50 values (1.4−44.9 nM)
and FR values (0.4-1.9), being more potent than EFV
(EC50 = 81.0 nM, FR = 16.3). Nine compounds (19, 20,
25, 27, 29, 30 and 32-34) provided a single-digit nano-
molar activity (EC50 = 1.4-8.3 nM). In particular, 19, 20,
27 and 32-34 displayed prominent inhibitory activity,
being 1.3−2.3-fold superior to ETV (EC50 = 3.3 nM).
To further validate the binding target, all the newly designed
compounds were tested for their ability to inhibit recombinant WT
HIV-1 RT enzyme. As shown in Table 3, all the tested com-
pounds showed excellent inhibitory activities toward RT with IC50
values ranging from 0.013 to 0.863 μM, being superior to that of
NVP (IC50 = 2.32 μM). Meanwhile, eight compounds (14, 27-29,
and 31-34) had a better inhibitory activity than EFV (IC50 = 0.03
μM). The IC50 values of the most potent compounds 20, 27, 33,
and 34 (IC50 = 0.084, 0.021, 0.023, and 0.026 μM, respectively)
were comparable to that of ETV (IC50 = 0.011 μM). It was identi-
fied that the results of preliminary SARs for IC50 were consistent
2) For E138K, all the tested compounds exhibited more
potent activity (EC50 = 6.0-157 nM) than NVP. The
EC50 values of ten compounds 19, 20, 25, 27-30 and
32-34 (EC50 = 6.0-34.7 nM) were less than 35 nM,
showing more potency than AZT (EC50 = 38.6 nM).
Besides, six compounds (20, 27, 30, 32-34) proved to
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with SARs for EC50 that discussed above. Furthermore, the line
four compounds had a value ranging from 123.73-136.30 Å2 con-
firming their advantageous for intestinal absorption (< 140 Å2)
and inability to penetrate the blood-brain barrier averting the cen-
tral nervous system toxicity (> 60 Å2).
chart was used to observe the consistency between the value of
pEC50 (blue line) and pIC50 (red line) of target compounds in
which the compound is sorted according to their pEC50 value in a
decreasing order as shown in Figure 3. The value of pIC50 showed
a declining trend (purple line) compared with their pEC50 and this
indicated the relative coherence between inhibitory against WT
HIV-1 strain and HIV-1 RT enzyme. However, the EC50 value of
some derivatives such as 14, 15, and 18 were 1.5−5-fold lower
than their values of IC50 presenting an abnormal deviation, poten-
tially owing to the cellular retention for poor membrane permea-
bility or much higher affinity for the cellular full length enzyme
than that of recombinant RT enzyme or metabolism of molecules.
Overall these results illustrated that these new compounds dis-
played high affinity for RT and could be specifically target HIV-1
RT and regarded as typical HIV-1 NNRTIs.
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To the best of our knowledge, we firstly discovered a new se-
ries of dual binding site NNRTIs targeting the NNIBP and the
“NNRTI Adjacent” binding site simultaneously. Encouragingly,
most of the novel derivatives exhibited significant inhibitory ac-
tivity toward WT (EC50 < 10 nM) and a panel of single mutant
HIV-1 strains in MT-4 cells. Compound 20 proved to be the most
potent inhibitor with EC50 values of 2.6 nM (WT), 6.5 nM
(L100I), 1.4 nM (K103N), 11.6 nM (Y181C), 16.2 nM (Y188L),
and 6.0 nM (E138K); it was more potent than ETV against all
single mutant strains, though somewhat weaker against double
mutant strains F227L+V106A and RES056. In addition, 20 has
much lower cytotoxicity (CC50 = 27.2 μM) and higher SI value of
10045. In RT inhibition assay, 20 displayed an IC50 value of 0.086
μM, which was in same magnitude with ETV. The current results
hold great promise for the identification of potential new drug
candidates with high potency and desirable drug-like properties.
Further optimizations focusing to improve the HIV-1 mutant
strains inhibitory activity, especially for double-mutant strains, are
currently in progress and will be reported in due course.
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To obtain further insight into the dual-site binding of the newly
synthesized compounds to the NNIBP and “NNRTI Adjacent”
binding site and rationalize the results of SAR studies, docking
experiments were carried out using representative compounds 19,
20 and 27 into WT HIV-1 RT with bound fragment at NNRTI
adjacent site (PDB code: 4KFB) by using the software Surflexe-
Dock SYBYL-X 2.0. Docking results were visualized with
PyMOL (Figure 4).
ASSOCIATED CONTENT
Supporting Information
The binding mode of compound 20 resembled RPV as horse-
shoe conformation in parent scaffold and the morpholine ring
stretched into the NNRTI adjacent site as the fragment located
(Figure 4a). Compound 20 maintained the typical double hydro-
gen bonds with the backbone of Lys101 as described for many
other NNRTIs. Besides, the amide group of the linker formed
additional double hydrogen bonds with Ile180 and Glu138. Com-
pared with 20, the disappearance of hydrogen bond between 19
and Glu138 may account for its reduced activity compared to 20
(Figure 4b). A close-up view inward the “NNRTI Adjacent” bind-
ing site described the morpholine ring stretched into the pocket
and occupied the adjacent site through the linker (Figure 4c). As
shown in Figure 4d, the detailed docking feature of 27 showed
additional hydrogen bonds formed by the NH and C=O of amide
with the backbone C=O of Glu138 and NH of Ile180, respectively.
Noteworthy, although 20 and 27 had different linker, they both
form hydrogen bond with Ile180 and Glu138. It was predicted that
different linkers and various terminal groups determine the con-
formation and then influence the activity. It was also guessed that
various conformations as well as additional binding force played
an important role in their excellent activity against HIV-1 mutant
strains. To sum up, the molecular modeling analysis explained the
theoretical binding mode and potent activity of the designed com-
pounds partially, which was consistent with our original design
intention and would assist further structural optimization.
The figures, tables and scheme except figure 2 are concluded in
supporting information. Experimental protocols for synthesis and
characterization of compounds, in vitro anti-HIV assay and mod-
eling study. The Supporting Information is available free of
charge on the ACS Publications website.
Supporting Information (file type, PDF)
AUTHOR INFORMATION
Corresponding Author
*P.Z.: e-mail, zhanpeng1982@sdu.edu.cn; phone, 086-531-
88382005;
*X.L.: e-mail, xinyongl@sdu.edu.cn; phone, 086-531-88380270.
Funding Sources
Financial support from the National Natural Science Foundation
of China (NSFC Nos. 81273354, 81573347), Key Project of
NSFC for International Cooperation (No. 81420108027), Young
Scholars Program of Shandong University (YSPSDU No.
2016WLJH32), the Fundamental Research Funds of Shandong
University (No. 2017JC006), Key research and development pro-
ject of Shandong Province (No. 2017CXGC1401) and KU Leuven
(GOA 10/014) is gratefully acknowledged.
Furthermore, the preliminary physicochemical properties of
representative compounds 20, 27, 33, and 34 were examined to
evaluate their drug-likeness features by utilizing free on-line
molinspiration software (http://www.molinspiration.com/). Lipo-
philic parameter-ligand efficiency (LE) was also calculated.18,19
The results (Table 4) suggested that parameters like molecular
weight (MW), hydrogen bond acceptors (nON), hydrogen bond
donors (nOHNH), rotatable bonds (nrotb), miLogp, and ligand
efficiency (LE) for all the tested compounds were in coincide with
the Lipinski’s “rule of five” and in an acceptable level except for
slightly deviation for molecular weight and ligand efficiency of
compound 20. Therefore, it is supposed that these four com-
pounds have the desired physicochemical properties. Besides, the
topological polar surface area (tPSA) which is characterized the
absorption and membrane permeability of molecules, showed the
Notes
The authors declare no conflict of interest.
ACKNOWLEDGMENT
We thank K. Erven, K. Uyttersprot and C. Heens for technical
assistance with the HIV assays.
ABBREVIATIONS
AIDS, acquired immunodeficiency syndrome; CC50, 50% cyto-
toxicity concentration; DAPY, diarylpyrimidine; NNRTIs, non-
nucleoside reverse transcriptase inhibitors; NNIBP, NNRTIs bind-
ing pocket; HAART, highly active antiretroviral therapy; EC50,
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the concentration causing 50% inhibition of antiviral activity;
as HIV-1 Non-nucleoside reverse transcriptase inhibitors
with significantly improved drug resistance profiles. J. Med.
Chem. 2016, 59, 7991-8007.
HIV, human immunodeficiency virus; RT, reverse transcriptase;
SAR, structure-activity relationship; SI, selection index; FR, fold
resistance; WT, wild-type; NVP, nevirapine; DLV, delavirdine;
EFV, efavirenz; ETV, etravirine; RPV, rilpivirine; AZT, azido-
thymidine; LE, ligand efficiency.
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Ahmed, S.; Frenkel, Y.; Vijayan, R. S.; Eck, J. T.; Ho, W.
C.; Das, K.; Shatkin, A. J.; Arnold, E. Detecting allosteric
sites of HIV-1 reverse transcriptase by X-ray crystallo-
graphic fragment screening. J. Med. Chem. 2013, 56, 2738-
2746.
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Figure 2. Our prior work and the newly designed compounds targeting the NNIBP and the “NNRTI Adjacent” binding site.
Table of Contents Graphic
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