Novel quinoline-based P2-P4 macrocyclic derivatives as pan-genotypic HCV NS3/4a protease inhibitors

Article abstract of DOI:10.1021/ml400466p

We have previously reported the discovery of our P2-P4 macrocyclic HCV NS3/4a protease inhibitor MK-5172, which in combination with the NS5a inhibitor MK-8742 recently received a breakthrough therapy designation from the US FDA for treatment of chronic HCV infection. Our goal for the next generation NS3/4a inhibitor was to achieve pan-genotypic activity while retaining the pharmacokinetic profile of MK-5172. One of the areas for follow-up investigation involved replacement of the quinoxaline moiety in MK-5172 with a quinoline and studying the effect of substitution at 4-position of the quinoline. The rationale for this effort was based on molecular modeling, which indicated that such modifications would improve interactions with the S2 subsite, in particular with D79. We wish to report herein the discovery of highly potent inhibitors with pan-genotypic activity and an improved profile over MK-5172, especially against gt-3a and A156 mutants.

Full text of DOI:10.1021/ml400466p

Letter
pubs.acs.org/acsmedchemlett
Novel Quinoline-Based P2−P4 Macrocyclic Derivatives As Pan-
Genotypic HCV NS3/4a Protease Inhibitors
Unmesh Shah,*,† Charles Jayne,† Samuel Chackalamannil,† Francisco Velazquez,† Zhuyan Guo,†
́
Alexei Buevich,† John A. Howe,† Robert Chase,† Aileen Soriano,† Sony Agrawal,† Michael T. Rudd,‡
John A. McCauley,‡ Nigel J. Liverton,‡ Joseph Romano,‡ Kimberly Bush,‡ Paul J. Coleman,‡
Christiane Grise-Bard,§ Marie-Christine Brochu,§ Sylvie Charron,§ Virender Aulakh,§ Benoit Bachand,§
́
Patrick Beaulieu,§ Helmi Zaghdane,§ Sathesh Bhat,§ Yongxin Han,§ Joseph P. Vacca,† Ian W. Davies,†
Ann E. Weber,† and Srikanth Venkatraman†
†Merck Research Laboratories, Kenilworth, New Jersey 07033, United States
‡Merck Research Laboratories, West Point, Pennsylvania 19486, United States
§Merck Research Laboratories, Kirkland, Quebec H9H3L1, Canada
S
* Supporting Information
ABSTRACT: We have previously reported the discovery of
our P2−P4 macrocyclic HCV NS3/4a protease inhibitor MK-
5172, which in combination with the NS5a inhibitor MK-8742
recently received a breakthrough therapy designation from the
US FDA for treatment of chronic HCV infection. Our goal for
the next generation NS3/4a inhibitor was to achieve pan-
genotypic activity while retaining the pharmacokinetic profile
of MK-5172. One of the areas for follow-up investigation
involved replacement of the quinoxaline moiety in MK-5172
with a quinoline and studying the effect of substitution at 4-
position of the quinoline. The rationale for this effort was
based on molecular modeling, which indicated that such modifications would improve interactions with the S2 subsite, in
particular with D79. We wish to report herein the discovery of highly potent inhibitors with pan-genotypic activity and an
improved profile over MK-5172, especially against gt-3a and A156 mutants.
KEYWORDS: Antiviral, HCV, NS3/4a, macrocycle, pan-genotypic, genotype 3a
additional classes of HCV direct acting antivirals offer
promising all-oral interferon sparing treatment regimens.7
We have previously reported the discovery of our P2−P4
macrocyclic HCV NS3/4a protease inhibitor MK-5172 (Figure
1), which is currently undergoing clinical trials.8,9 Preclinically,
MK-5172 demonstrated broad genotype and mutant enzyme
potency and cellular activity. One of the areas for follow-up
investigation involved replacement of the quinoxaline moiety in
MK-5172 with a quinoline and studying the effect of
substitution at 4-position of the quinoline. The rationale for
this effort was based on molecular modeling, which indicated
that such modifications would improve interactions with the S2
subsite, in particular with D79 (Figure 2). Although in the NS3
protease structure of the catalytic domain D79 points toward
the solvent, in the full-length enzyme (pdb 1cu1) it is at the
n estimated 180 million people worldwide are chronically
Ainfected with the hepatitis C virus (HCV) and about 20%
of this population is at a risk of developing liver cirrhosis, which
can lead to end stage liver disease and hepatocellular
carcinoma.1−3 The majority of infections in the developed
world are caused by HCV genotypes 1, 2, and 3. While standard
of care treatment with pegylated IFN-α (P) and ribavirin (R)
results in a cure rate of ∼70−90% in patients infected with
genotypes 2 and 3, the cure rate is only ∼45% for genotype 1
infected patients.4 The addition of direct acting HCV serine
protease NS3/4a inhibitors such as boceprevir or telaprevir to
PR treatment regimens has significantly improved the sustained
̈
virological response (SVR) rate to up to 75% for naıve HCV
genotype 1 patients (representing more than 70% of all cases of
chronic HCV infection in the United States).5,6 Furthermore,
side effects associated with PR and first generation NS3/4a
inhibitors, the rapid emergence of drug resistance, and
suboptimal SVR have led to the development of more potent
NS3/4a protease inhibitors with a higher barrier to resistance.
These drug candidates in combination with compounds from
Special Issue: HCV Therapies
Received: November 15, 2013
Accepted: January 9, 2014
Published: January 9, 2014
© 2014 American Chemical Society
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Scheme 1. Synthesis of an Advanced P2−P4 Macrocyclic
a
Intermediate
Figure 1. Structures of MK-5172 and quinoline-based P2−P4
macrocyclic core.
Figure 2. Model of quinoline-based P2−P4 macrocycle bound to the
HCV NS3 gt-1b protease active site. The protein is shown as the
surface (white carbon) and the inhibitor as a stick (cyan carbon).
interface of NS3 protease and helicase domains.10,11 We
hypothesized that having a basic group in this region would
offer charge complementarity to D79 and improve inhibitor
binding energetics. In addition, from sequence alignment D79
is conserved across most of the known genotypes, and
therefore, we rationalized that targeting this interaction would
be beneficial to maintain/improve the overall gt profile. On the
basis of some of our previous SAR we also knew that
replacement of the t-butyl group in the P3 region of MK-5172
was tolerated, and the cyclopropyl group in the sulfonamide
region could be replaced with a methylcyclopropyl group
without losing activity; these modifications had resulted in
better rodent pharmacokinetics. This letter will describe our
SAR findings in the series represented by macrocycle I (Figure
1), where the R group contains a basic side-chain capable of
interacting with D79 (Figure 2).
On the basis of our modeling rationale, we explored a series
of amine analogues linked to the quinoline via different spacers.
These analogues were prepared from a common 4-hydrox-
yquinoline-based macrocyclic core, which was synthesized as
shown in Scheme 1. Treatment of (R),(R)-alkynol 1 with N,N′-
discuccinimidyl carbonate (DSC) and coupling the resulting
DSC-adduct with P3 cyclohexyl-glycine 2 provided the alkyne-
carboxylic acid 3.12 Mitsunobu reaction between the
commercially available hydroxyproline derivative 4 and
bromoquinolinone 512 furnished the proline-containing bro-
moquinoline fragment 6, which underwent Sonagashira
coupling with alkyne 3 to provide intermediate 7. Hydrolysis
of the NBoc group followed by an intramolecular amide
a
Reagents and conditions: (a) DSC, pyridine, DMAP, MeCN, 40 °C;
(b) 2, Et3N, MeCN; (c) DIAD, Ph3P, THF; (d) 3, PdCl2(MeCN)2
tBu3P·BF4, K2CO3, Bn2NH, MeCN, 75 °C; (e) TFA, CH2Cl2; (f)
HATU, DIPEA, DMF; (g) 10% Pd−C, H2, MeOH, THF, 1 atm.
coupling resulted in macrocycle 8, and subsequent hydro-
genation provided the macrocyclic intermediate 9, which was
utilized for further derivatization as shown in Scheme 2.
Hydrolysis of the methyl ester in 9 followed by an amide
coupling with the acylsulfonamide-containing amine 1012 gave
11, which upon alkylation with 1,3-dibromopropane resulted in
bromide 12. Displacement of the bromide in 12 with amines of
varying basicity gave the first set of analogues (e.g., 13−18) in
which the basic group was separated from the quinoline moiety
by a propyloxy linker. Enzyme inhibition data against genotypes
1b, 3a, and relevant gt-1b mutants is summarized in Table 1.13
Data summarized in Table 1 shows that replacing the
quinoxaline group in MK-5172 with a quinoline and
incorporating alkoxy-linked amines at 4-position are tolerated.
Similar to MK-5172, most compounds displayed excellent
activity against gt-1b and mutants R155K and D168Y. An
important differentiation point, however, was the improvement
in activity against the key A156 mutants (A156T and A156V)
for several compounds in this series; activity against gt-3a was
variable, although not an improvement over MK-5172. The
more promising compounds, however, were associated with
poor rat pharmacokinetic properties (e.g., 18, AUC = 0.1 μM·h,
5 mpk po).14
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Scheme 2. Synthesis of Amine-Based P2−P4 Macrocyclic
Table 1. SAR of Extended Amine Analogues
a
NS3/4a Protease Inhibitors
a
Reagents and conditions: (a) LiOH·H2O, THF-MeOH; (b) 10,
HATU, DIPEA, DMF; (c) 1,3-dibromopropane, Cs2CO3, DMF, 50
°C; (d) DIPEA/Et3N, KI, HNR1R2; (e) DIAD, Ph3P, 4-hydroxy-1-
methylpiperidine or N-Boc-4-hydroxypiperidine, THF, 40 °C (f) TFA,
CH2Cl2; (g) RX, DIPEA/Et3N, KI, MeCN or DMF, 100 °C or
(methylsulfonyl)ethene, DIPEA, CH2Cl2 for synthesis of 28.
On the basis of the promising activity against A156T and
A156V mutants observed with the 3 carbon-linked amine
analogues, we decided to extend this SAR by cyclizing the
linker and exploring some piperidine-based derivatives. These
targets were synthesized as shown in Scheme 2. Mitsunobu
reaction between quinol 9 and N-substituted 4-hydroxypiper-
idine gave the corresponding ether derivatives (19 and 20).
The acyl sulfonamide motif was then introduced as described
earlier for the synthesis of 11. Hydrolysis of the piperidine
NBoc group followed by N-derivatization gave the desired
targets (Table 2). In the piperidine-ether series (Table 2), the
nonbasic NBoc analogue 22 resulted in a significant drop in
activity; a similar trend was observed with the corresponding
sulfonamide and amide derivatives (data not shown). The free
piperidine and some of the N-alkylated analogues, however,
exhibited excellent overall activity. Unlike the earlier acyclic-
spacer series, the piperidine analogues 21 and 23−25 displayed
subnanomolar IC50 activities not only against the A156 mutants
but also against the critical genotype 3a. Increasing the size of
the alkyl group had a detrimental effect on activity across all
genotypes and mutants (e.g., 26, 27, and 28), and rendering the
piperidine nitrogen less basic (e.g., 29) resulted in a significant
loss of activity. We were able to regain the potency by fine-
tuning the piperidine basicity through incorporation of fewer
fluorine atoms; however, these compounds (e.g., 30) did not
offer additional advantage over other leads. Finally, the
reduction in activity for the pyran analogue 31 confirmed the
importance of a basic nitrogen to make a favorable interaction
with D79 residue in this region.
Constrained bicyclic piperidine analogues were explored next
as an extension to the above findings. Compounds shown in
Table 3 were prepared following the general synthetic route
shown in Schemes 1 and 2; the corresponding bicyclic alcohols
were either purchased through commercial sources or
synthesized according to known literature procedures.15 The
bicyclic piperidine SAR (Table 3) was conducted first by
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Table 2. SAR of Piperidine-Ether Analogues
a
Compound 31 was synthesized in a similar manner as compound 21; 4-hydroxy-1-methylpiperidine was replaced by tetrahydro-2H-pyran-4-ol.
preparing nortropane analogues in the P3 t-Bu series, and it was
quickly realized that the exo isomer was better tolerated than
the endo isomer (e.g., 33 vs 32, and 35 vs 34); 33 and 35
demonstrated excellent gt-3a and A156 mutant profiles. The
same trend was observed in the P3 cyclohexyl series where
several pan-genotypic compounds were identified (e.g., 36−
38).16
Table 4 shows gt-1b and gt-3a replicon potency and rat
AUCs for selected compounds in the piperidine-ether series.
There was a general improvement in gt-1b replicon activity for
these analogues compared to MK-5172. With regards to gt-3a
activity, compound 37 was highly potent and demonstrated a
∼14-fold improvement in activity over MK-5172. This
compound also exhibited an oral rat pharmacokinetic profile
similar to MK5172. Other compounds in the P3 cyclohexyl
series, with an exception of the free amine 23, also exhibited
good oral AUC in rats (e.g., 21, 24, and 25); compound 35 in
the P3 t-Bu series, however, was associated with inferior rat PK.
In summary, we have discovered a new series of HCV NS3/
4a protease inhibitors based on the P2−P4 macrocyclic scaffold
of our clinical compound MK-5172. Replacing the quinoxaline
moiety with quinoline and incorporating moderately basic
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Table 3. SAR of Bicyclic Piperidine Analogues
a
b
Table 4. Cellular Potency and Rat Pharmacokinetic Profile for Selected Piperidine-Ether Compounds
compd
MK-5172
21
23
24
25
35
37
1b EC50, nM
3a EC50, nM
rat AUC, μM·h
1.5
0.25
3.1
0.21
7.8
0.53
5.2
0.73
13.5
2.8
0.48
1.06
0.01
0.62
0.26
1.2
13.0
0.7
2.7
0.01
2.3
a
Cell-based replicon assays conducted in presence of 10% fetal-bovine serum (FBS); EC50 determination was conducted using a TaqMan-based
b
assay. 5 mg/kg, po, methyl cellulose (PEG400 for MK5172).
groups to provide an interaction with D79 within the S2
subsite, as determined through molecular modeling, provided
highly potent inhibitors. The bicyclic piperidine analogue 37
was identified as the lead compound, which displayed pan-
genotypic activity and an improved profile over MK-5172,
especially against gt-3a and A156 mutants. This compound also
displayed acceptable pharmacokinetic properties in rats upon
oral dosing. Further SAR development and advancement of
lead compounds will be disclosed in the near future.
of primary biological assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(U.S.) Phone: 908-740-7158. Fax: 908-740-2301. E-mail:
unmesh.shah@merck.com.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
■
ACKNOWLEDGMENTS
S
* Supporting Information
■
Synthetic experimental details including H NMR, 13C NMR,
The authors would like to thank Dr. Rebecca Ruck for
providing advanced intermediates for SAR exploration. We also
1
and mass spectral data for selected compounds, and description
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ACS Medicinal Chemistry Letters
Letter
(13) The HCV NS3 protease inhibitory activity was measured using
the protease time-resolved fluorescence (TRF) assay described in WO
Pat. Appl. 2006102087. Also see Supporting Information section for
detailed description of the assay.
(14) Additional SAR work around the spacer length demonstrated
that the corresponding 2C-linked analogues were somewhat inferior to
their 3C-linked counterparts.
(15) Shah, U. G.; Boyle, C. D.; Chackalamannil, S.; Neustadt, B. R.;
Harris, J. M.; Stamford, A.; Greenlee, W. J.; Neelamkavil, S. F.
Preparation of Pyrimidinylazabicyclooctane Derivatives and Analogs
for Use as GPCR Modulators. WO Pat. Appl. 2010075273, 2010.
(16) Changing the bridge to an oxaza bicyclic system or moving the
bridge away from the nitrogen resulted in compounds with inferior
activity, especially against gt-3a.
thank Dr. Mike Rowley for providing valuable input during the
preparation of this manuscript.
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