Discovery of phenylaminopyridine derivatives as novel HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1021/ml300146q

We identified a novel class of aryl-substituted triazine compounds as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) during a high-throughput screening campaign that evaluated more than 200000 compounds for antihuman immunodeficiency virus (HIV) activity using a cell-based full replication assay. Herein, we disclose the optimization of the antiviral activity in a cell-based assay system leading to the discovery of compound 27, which possessed excellent potency against wild-type HIV-1 (EC₅₀ = 0.2 nM) as well as viruses bearing Y181C and K103N resistance mutations in the reverse transcriptase gene. The X-ray crystal structure of compound 27 complexed with wild-type reverse transcriptase confirmed the mode of action of this novel class of NNRTIs. Introduction of a chloro functional group in the pyrazole moiety dramatically improved hERG and CYP inhibition profiles, yielding highly promising leads for further development.

Full text of DOI:10.1021/ml300146q

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Letter
Discovery of Phenylaminopyridine Derivatives as Novel
HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors
Junwon Kim, Doohyun Lee, Changmin Park, Wonyoung So, Mina Jo, Taedong Ok, Jeongjin Kwon,
Sunju Kong, Suyeon Jo, Youngmi Kim, Jihyun Choi, Hyoung Cheul Kim, Yoonae Ko, Inhee Choi,
Youngsam Park, Jaewan Yoon, Moon Kyeong Ju, Junghwan Kim, Sung-Jun Han, Tae-Hee Kim,
Jonathan Cechetto, Jiyoun Nam, Peter Sommer, Michel Liuzzi, Jinhwa Lee, and Zaesung No
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml300146q • Publication Date (Web): 11 Jul 2012
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Platforms Group
Nam, Jiyoun; Institut Pasteur Korea, DMPK Group
Sommer, Peter; Institut Pasteur Korea, Cell Biology of Retroviruses Group
Liuzzi, Michel; Institut Pasteur Korea, Early Discovery Program
Lee, Jinhwa; Institut Pasteur Korea, Late Discovery Program
No, Zaesung; Institut Pasteur Korea, Late Discovery Program
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Discovery of Phenylaminopyridine Derivatives as Novel HIV-1
Non-Nucleoside Reverse Transcriptase Inhibitors
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Junwon Kim,a Doohyun Lee,a Changmin Park,a Wonyoung So,a Mina Jo,a Taedong Ok,a
Jeongjin Kwon,a Sunju Kong,c Suyeon Jo,a Youngmi Kim,b Jihyun Choi,a Hyoung Cheul Kim,a
Yoonae Ko,b Inhee Choi,b Youngsam Park,d Jaewan Yoon,d Moon Kyeong Ju,d Junghwan Kim,d
Sung-Jun Han,d Tae-Hee Kim,e,j Jonathan Cechetto,e Jiyoun Nam,f Peter Sommer,g Michel
Liuzzi,h Jinhwa Lee,i Zaesung No*,i
a Medicinal Chemistry 2, b Medicinal Chemistry 1, c Medicinal Chemistry 3, d Drug Biology Group, e Screening
Technology Platforms Group, f DMPK Group, g Cell Biology of Retroviruses Group, h Early Discovery Program, i
Late Discovery Program, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si,
Gyeonggi-do 463-400, Republic of Korea
j Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu,
Seoul 120-749, Republic of Korea
KEYWORDS: HIV-1, Reverse transcriptase, Non-nucleoside reverse transcriptase inhibitors, synthesis, X-ray
crystal structure
Supporting Information Placeholder
ABSTRACT: We identified a novel class of aryl substituted triazine compounds as potent non-nucleoside reverse tran-
scriptase inhibitors (NNRTIs) during a high-throughput screening campaign that evaluated more than 200,000 com-
pounds for anti-human immunodeficiency virus (HIV) activity using a cell-based full replication assay. Herein, we dis-
close the optimization of the antiviral activity in a cell-based assay system leading to the discovery of compound 27
which possessed excellent potency against wild-type HIV-1 (EC50 = 0.2 nM) as well as viruses bearing Y181C and K103N
resistance mutations in the reverse transcriptase gene. The X-ray crystal structure of compound 27 complexed with
wild-type reverse transcriptase confirmed the mode of action of this novel class of NNRTIs. Introduction of a chloro
functional group in the pyrazole moiety dramatically improved hERG and CYP inhibition profiles, yielding highly prom-
ising leads for further development.
According to global estimates of the WHO/UNAIDS in
2009, more than 33 million people were infected with
the human immunodeficiency virus (HIV), the causative
agent of the acquired immunodeficiency syndrome
(AIDS), which currently accounts for the highest number
of deaths by any single infectious agent.1 The global
HIV/AIDS pandemic triggered intensive drug discovery
efforts and the first FDA-approved antiretroviral drug,
Zidovudine (AZT), was available in 1987. Currently, 26
drugs belonging to six different inhibitor classes have
been approved for the treatment of HIV infection: nucle-
oside and nucleotide reverse transcriptase inhibitors
(NRTIs/NtRTIs), non-nucleoside reverse transcriptase
inhibitors (NNRTIs), protease inhibitors (PIs), integrase
inhibitors (INIs), entry (CCR5 coreceptor antagonist)
and fusion inhibitors (FIs).2 Highly active antiretroviral
therapy (HAART)—a regimen combining 3–4 antiretro-
virals from different inhibitor classes—has dramatically
improved the life quality of infected people by delaying
the progression of the disease and reducing disabilities,
transforming HIV/AIDS into a chronic manageable dis-
ease.3,4 Non-nucleoside reverse transcriptase inhibitors
(NNRTIs) that interact non-competitively with an allo-
steric binding pocket in the vicinity of the RT’s polymer-
ase active site are an important component of first-line
regimens.5 Although there are five-FDA approved NNR-
TIs for clinical use (Figure 1), alternatives are still needed
due to the tendency of HIV-1 to rapidly mutate. Pro-
longed HAART treatment leads to the emergence of
drug-resistant viral mutants.6 Also, the undesired side
effects of combination therapy have limited their clinical
effectiveness.7 Therefore, further development of novel
NNRTIs with acceptable toxicity and improved drug-
resistance profiles is undoubtedly required.8
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Scheme 1. Synthesis of compound 27.a
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Figure 1. Structures of the FDA-approved NNRTI drugs
In a high-throughput screening campaign for the dis-
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covery of novel antiretrovirals employing a HIV full rep-
lication assay based on reporter cells harboring an EGFP
expression cassette under the control of the HIV promot-
er, we identified hit compound 6 containing a triazine
scaffold (Figure 2) that exhibited inhibitory activities
against HIV replication at submicromolar concentra-
tions. Compound 6 also showed moderate inhibitory
activity in an in vitro RT polymerase assay, indicating
that HIV RT is the potential target. Here, we report a
detailed structure-activity relationship (SAR) study of
this series of compounds that led to compound 27 which
has excellent antiviral activity against wild-type (WT)
and NNRTI-resistant HIV-1. Also, its x-ray co-crystal
structure in complex with WT RT is included.
a
Reagents and conditions: (a) (i) NaOEt, EtOH, diethyl
oxalate , 25 °C, 20 h; (ii) H2NNH2, H2O/EtOH, reflux, 2 h,
69% over 2 steps; (b) NCS, CCl4, DMF, 55 °C, 15 h, 86%; (c)
DHP, TFA, CH3CN/toluene, 100 °C, 6 h, 42%; (d) LAH, THF,
0 °C, 30 min, 58%; (e) Dess-Martin Periodinane, CH2Cl2, 25
°C, 3 h, 91%; (f) Pd(dppf)Cl2, Na2CO3, DME/H2O, 140 °C, 2
h, 70%; (g) 1s, NaBH(OAc)3, AcOH, 1,2-dichloroethane, 25
°C, 24 h, 50%; (h) HCl, MeOH, 40 °C, 15 h, 82%
The first series of LHS modifications (7−21) were eval-
uated for their inhibitory activity against HIV-1 WT rep-
lication in a cell-based assay and Nevirapine was used as
a positive control. Assay results of compounds (7−21) are
summarized in Table 1.
Table 1. Cell-based antiviral activity of PAP de-
rivatives 7−21 with LHS modifications
Figure 2. Hit compound from the cell-based HIV-1 replica-
tion assay
The target phenylaminopyridine (PAP) compounds
(7−27 in Tables 1 and 2) were synthesized according to
the general routes.9 The lead compound 27 was prepared
efficiently in a convergent manner by coupling two subu-
nits as depicted in Scheme 1. The formylated pyrazole 1s
required for the reductive amination was prepared from
1-(pyridin-2-yl)ethanone. Addition of enolate to diethyl
oxalate, followed by the cyclization with hydrazine for
the pyrazole ring formation under refluxing condition
gave the desired ester 1o in 69% yield over 2 steps.10 Hal-
ogenation on the pyrazole with NCS produced chlorinat-
ed pyrazole 1p in good yield and the resulting pyrazole
was protected with THF group to give intermediate 1q.
The ester moiety was then converted into the aldehyde 1s
in a typical two-step reduction with LAH and oxidation
with Dess-Martin reagent sequence. After the Suzuki
coupling reaction to produce compound 1t, the resulting
LHS subunit was then subjected to the final coupling step
with compound 1s involving reductive amination, fol-
lowed by deprotection of the THF group under acidic
conditions to give the desired lead compound 27 in good
yield.
a
EC50 is the concentration of compound that inhibits
HIV-1 replication by 50%. For compounds 7−21, the values
are the geometric mean of two determinations; all individual
values are within 25% of the mean. CC50 is the cytotoxic
concentration of compound that reduces viability of unin-
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c
fected cells by 50%. Nevirapine (NVP) was used as a posi-
tive control.
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As seen in Table 1, it was clear that the anti-HIV activi-
ties of PAP compounds are sensitive to structural pertur-
bations. Compounds 7−10 with common triazine core B
ring exhibited considerable variation in the inhibitory
activity (EC50
= 0.124 ꢀM to > 10 ꢀM). Ortho-
substitution on the A ring markedly improved their po-
tency, indicating that increasing the angle between the
phenyl A ring and the triazine B ring is favorable. The
effect of ortho-substitution is further exemplified by
compound 11, which showed over 400-fold improved
antiviral activity compared to compound 7. We next in-
vestigated the effect of nitrogen atoms in the B ring in
PAP derivatives 12−17 and the results showed a broad
range of EC50 values. Compound 15 with a pyridine B
ring is the most active among these analogues. However,
the complete removal of nitrogen atoms from the B ring
induced a significant reduction in inhibitory activity in
compound 12. Pyrimidine 16 and pyrazine 17 derivatives
displayed 2- or 6-fold reduced potencies compared to
compound 15. Further optimization on the A ring was
based on a computational approach by overlaying with
known NNRTIs (Etravirine and MK-4965). Gratifyingly,
the introduction of a cyano group in the para-position on
the A ring significantly enhanced HIV-1 inhibitory activi-
ty more than 75-fold in derivatives 18−21 compared with
derivatives lacking the cyano group (8, 13, 15, and 16).11
This dramatic effect was not seen in ortho- and meta-
positions (data not shown). These results indicate that
the binding mode of PAP derivatives might be similar to
that of diarylpyrimidine (DAPY) compounds (e.g., et-
ravirine 4, rilpivirine 5).12,13
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a
EC50 is the concentration of compound that inhibits
HIV-1 replication by 50%. For compounds 22−27, the val-
ues are the geometric mean of two determinations; all indi-
vidual values are within 25% of the mean. b CC50 is the cyto-
toxic concentration of compound that reduces viability of
uninfected cells by 50%. c Nevirapine (NVP) was used as a
positive control. d Solubility of HCl salt in distilled water.
Intrinsic enzymatic inhibitory activity against WT and
key mutant RTs (K103N, Y181C) and also hERG inhibi-
tion profiles of PAP derivatives (22−27) are summarized
in Table 3. In the enzymatic assay, the majority of the
tested compounds except compound 26 showed single
digit nanomolar IC50 values against WT RT and also dis-
played significant inhibitory activity against the K103N
and Y181C mutated RTs, with only minimal changes in
IC50 values with respect to WT RT. Results from hERG
inhibition assays revealed the intriguing characteristics
of PAP derivatives. Although rather moderate inhibition
values were observed in azaindazoles 22−24, the halo-
genated pyrazole derivatives (25, 27) exhibited accepta-
ble safety margins, which could be beneficial for long
term treatment.14
After the evaluation of LHS subunits in the PAP scaf-
fold, we investigated the modification of the RHS subu-
nits by synthesizing derivatives 22−27 and the results
are presented in Table 2. Structural modification on the
RHS subunits was directed toward the improvement of
physicochemical properties, especially solubility of this
series by the introduction of fused heteroaryls or substi-
tuted pyrazoles with a polar moiety. Azaindazole 22−24
and pyrazole 25−27 derivatives maintained excellent
HIV-1 inhibitory activities along with good solubility pro-
files. As shown in derivatives 24 and 27, the formation of
HCl salt could be utilized to further increase absorption
for pharmacokinetic studies.
Table 3. HIV-1 RT Inhibitory Activity and hERG
Inhibition of PAP derivatives 22−27
Table 2. Cell-based antiviral activity of PAP de-
rivatives 22−27 with RHS modifications
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tions with Y181. The nitrogen linker made a hydrogen
a Compounds were evaluated in a standard SPA assay. In-
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bond (H-bond) interaction with the main chain carbonyl
oxygen of K101. One additional H-bond was found be-
tween the same atom of K101 and one of the nitrogens in
the chloropyrazole C ring as the RHS subunit was posi-
tioned in the flexible loop region. The other nitrogen in
the pyrazole formed an H-bond with the main chain ni-
trogen of K103. The chloropyrazole C ring also makes
van der Waals interactions with Y318, and the chloride of
this ring is pointing in the same direction as the hydroxyl
group of Y318. The pyridine D ring is surrounded by
V106, P225 and P236 (Figure 3).
hibition of RNA-dependent DNA polymerase activity using
the WT, K103N and Y181C polymerases. For compounds
22−27, the values are the geometric mean of two determi-
nations; all individual values are within 25% of the mean.
Assay protocols are detailed in Supporting Information.
Fold resistance is defined by IC50 (mutant) /IC50 (WT). hERG
inhibition was measured in CHO cells stably expressing the
recombinant human hERG channel subunit (IKr) using an
automated patch clamp platform. (QPatch-Sophion Biosci-
ences). d MK-4965 was used as a positive control.15
b
c
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The pharmacokinetic parameters of key compounds
(24, 25, and 27) were measured in rats and are tabulated
in Table 4. All tested compounds showed bioavailability
above 70% and moderate clearance rates and half-lives.
Notably, compound 27 exhibited an improved AUC value
compared to other analogues, which suggested that once
daily dosing in humans might be achievable after further
optimization. Also, the in vitro drug interaction profile
showed that compound 27 was not inhibitory against a
panel of five isoforms of the cytochrome P450 (CYP) en-
zymes and did not induce CYP 3A4 at concentrations up
to 10 ꢀ
M. 16,17
Table 4. Pharmacokinetic Parameters for 24, 25,
and 27 in Rat
Figure 3. Co-crystal structure of HIV-1 wild-type RT with
compound 27. Three H-bonds (red dotted line) were formed
between residues K101 and K103 (atom colored blue stick)
and compound 27 (atom colored yellow stick).
In summary, our search for novel anti-HIV compounds
led to the discovery of a highly potent NNRTI with a PAP
scaffold. The lead compound 27 possessed excellent an-
tiviral activity against wild-type (WT) and key RT mu-
tants. The binding mode of compound 27 was unambig-
uously confirmed by the x-ray co-crystal structure with
WT RT. Introduction of a chloro functional group in the
pyrazole moiety markedly improved hERG and CYP in-
hibition profiles. Altogether, the results presented here
suggested that further development of this series has the
potential to generate a valuable drug candidate for the
treatment of HIV-1 infected patients.
a
IV dosing at 1.10 mg/kg, Oral dosing at 10.7 mg/kg. IV
and PO formulation: 1.0 mg/mL in 50% PEG; b IV dosing at
1.19 mg/kg, Oral dosing at 10.9 mg/kg. IV and PO formula-
tion: 1.0 mg/mL in 40% PEG; IV dosing at 0.94 mg/kg,
Oral dosing at 9.44 mg/kg. IV and PO formulation: 1.0
mg/mL in 20% HP-β-CD
c
To understand the mode of action of PAP compounds,
the lead compound 27 was subjected to co-crystallization
with WT RT.18 Indeed, compound 27 was bound to the
non-nucleoside reverse transcriptase inhibitor binding
pocket (NNIBP) as shown in Figure 3. The x-ray crystal
structure revealed that the RT-bound conformation of
compound 27 resembled a U-shape which is similar to
the binding modes of Etravirine as well as MK-4965.
This U-shape orientation ideally adapts a combination of
torsional flexibility (“wiggling”) and rotational and trans-
lational shifts (“jiggling”) of the inhibitor within the
binding pocket in order to have potency against WT and
a wide range of drug-resistant HIV-1 RTs.19
ASSOCIATED CONTENT
Supporting Information.
Experimental procedures for the synthesis and characteriza-
tion of (27, 7−27) and details for biological methods. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Compound 27 interacted favorably with NNIBP resi-
dues. The LHS subunit of compound 27 is positioned in
the hydrophobic pocket surrounded by aromatic amino
acids such as Y181, Y188, F227, and W229. The 4-cyano-
2-methyl-phenyl A ring makes a π−π interaction with
Y188 and an edge−π interaction with W229. The 2-
methyl on the A ring orients towards the side chain of
L100 contributing to additional hydrophobic interac-
tions. The pyridine B ring makes van der Waals interac-
AUTHOR INFORMATION
Corresponding Author
*E-mail: noxide@ip-korea.org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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S.; Peckham, J.; Watson, C.; Wheelan, P.; Spaltenstein, A.; Wire,
This work was supported by the National Research founda-
tion of Korea (NRF) grant funded by the Korea government
(MEST) (No.2012-00011), Gyeonggi-do and KISTI.
M. B.; Svolto, A.; Youngman, M. Novel 4,4-disubstituted piper-
idine-based C-C chemokine receptor-5 inhibitors with high
potency against human immunodeficiency virus-1 and an im-
proved human ether-a-go-go related gene (hERG) profile. J.
Med. Chem. 2011, 54, 3756-3767.
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ABBREVIATIONS
AIDS, acquired immunodeficiency syndrome; HAART, high-
ly active antiretroviral therapies; NNRTI, non-nucleoside
reverse transcriptase inhibitor; RT, reverse transcriptase;
SAR, structure-activity relationship; WT, wild-type; NNIBP,
non-nucleoside reverse transcriptase inhibitor binding
pocket; PK, pharmacokinetic.
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6
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8
(15) Tucker, T. J.; Sisko, J. T.; Tynebor, R. M.; Williams, T.
M.; Felock, P. J.; Flynn, J. A.; Lai, M.-T.; Liang, Y.; McGaughey,
G.; Liu, M.; Miller, M.; Moyer, G.; Munshi, V.; Perlow-Poehnelt,
R.; Prasad, S.; Reid, J. C.; Sanchez, R.; Torrent, M.; Vacca, J. P.;
Wan, B.-L.; Yan, Y. Discovery of 3-{5-[(6-Amino-1H-
pyrazolo[3,4-b]pyridine-3-yl)methoxy]-2-chlorophenoxy}-5-
chlorobenzonitrile (MK-4965): A Potent, Orally Bioavailable
HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitor with
Improved Potency against Key Mutant Viruses. J. Med. Chem.
2008, 51, 6503-6511.
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