The discovery of potent nonstructural protein 5A (NS5A) inhibitors with a unique resistance profile - Part 2

Article abstract of DOI:10.1002/cmdc.201400045

In ongoing studies towards novel hepatitis C virus (HCV) therapeutics, inhibitors of nonstructural protein 5A (NS5A) were evaluated. Specifically, starting from previously reported lead compounds, peripheral substitution patterns of a series of biaryl-linked pyrrolidine NS5A replication complex inhibitors were probed and structure-activity relationships were elucidated. Using molecular modelling and a supercritical fluid chromatographic (SFC) technique, intramolecular H-bonding and peripheral functional group topology were evaluated as key determinants of activity and membrane permeability. The novel compounds exhibited retained potency as compared with the lead compounds, and also showed promising results against a panel of resistance viruses. Together, the results of the study take us a step closer towards understanding the potency of daclatasvir, a clinical candidate upon which the compounds were based, and to designing improved analogues as second-generation antiviral agents targeting NS5A. Refining the resistance: An investigation into the anti-hepatitis C virus (HCV) replicon activity of a series of biaryl-linked prolinamide NS5A inhibitors explored intramolecular H-bonding and peripheral functional group topology as key determinants of activity and membrane permeability. Studies of several peripheral group molecular designs resulted in compounds with improved aqueous solubility and a unique resistance profile.

Full text of DOI:10.1002/cmdc.201400045

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DOI: 10.1002/cmdc.201400045
The Discovery of Potent Nonstructural Protein 5A (NS5A)
Inhibitors with a Unique Resistance Profile—Part 1
Thien Duc Tran,[a] Florian Wakenhut,[a] Chris Pickford,[b] Stephen Shaw,[b] Mike Westby,[b]
Caroline Smith-Burchnell,[b] Lesa Watson,[a] Michael Paradowski,[a] Jared Milbank,[a]
Rebecca A. Brimage,[d] Rebecca Halstead,[d] Rebecca Glen,[d] Craig P. Wilson,[d] Fiona Adam,[a]
Duncan Hay,[a] Jean-Yves Chiva,[a] Carly Nichols,[a] David C. Blakemore,[a] Iain Gardner,[c]
Satish Dayal,[c] Andrew Pike,[c] Rob Webster,[c] and David C. Pryde*[a]
Nonstructural protein 5A (NS5A) represents a novel target for
the treatment of hepatitis C virus (HCV). Daclatasvir, recently
reported by Bristol–Myers–Squibb, is a potent NS5A inhibitor
currently under investigation in phase 3 clinical trials. While
the performance of daclatasvir has been impressive, the emer-
gence of resistance could prove problematic and as such, im-
proved analogues are being sought. By varying the biphenyl-
imidazole unit of daclatasvir, novel inhibitors of HCV NS5A
were identified with an improved resistance profile against
mutant strains of the virus while retaining the picomolar po-
tency of daclatasvir. One compound in particular, methyl ((S)-1-
((S)-2-(4-(4-(6-(2-((S)-1-((methoxycarbonyl)-l-valyl)pyrrolidin-2-
yl)-1H-imidazol-5-yl)quinoxalin-2-yl)phenyl)-1H-imidazol-2-yl)-
pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (17), ex-
hibited very promising activity and showed good absorption
and a long predicted human pharmacokinetic half-life. This
compound represents a promising lead that warrants further
evaluation.
fected with, and other genetic factors.[3] Genotype 1 (gt1), the
most prevalent genotype in Europe and the US, responds only
modestly to interferon therapy with a sustained virologic re-
sponse in approximately half of the treated population, and
the treatment is associated with severe side effects and signifi-
cant discontinuation rates. Recent approvals of drugs that
target alternative antiviral mechanisms include the protease in-
hibitors telaprevir, boceprevir and simeprevir, and the poly-
merase inhibitor sofosbuvir, bringing the prospect of interfer-
on-sparing treatment regimens closer.[4]
To make this a reality, new effective antivirals are required
that will allow a phasing out of interferon-based regimens to
be replaced with better tolerated medicines. HCV is also
a highly mutable virus, and as resistance mutations emerge,
new and effective antiviral mechanisms will be needed.
The single-stranded RNA genome of HCV encodes for
a single polyprotein of approximately 3000 amino acids that is
processed by the viral HCV protease enzyme into three struc-
tural proteins (HCV core, E1 and E2), six nonstructural proteins
(NS2, NS3, NS4A, NS4B, NS5A, NS5B), and the p7 ion channel.[5]
The nonstructural proteins are a combination of enzymes and
cofactors that are responsible for proper viral replication. Much
of the direct-acting antiviral drug development to date has fo-
cussed on the functionally well-characterised HCV protease
(NS3) and polymerase (NS5B) enzymes, for which crystal struc-
tures are available.[6] The nonstructural protein 5A (NS5A) exists
as basally and hyperphosphorylated forms that participate in
viral replication via a replication complex and indeed has been
shown to interact with a wide variety of cellular proteins. How-
ever, NS5A has no known enzymatic function, confounding ef-
forts to accurately determine its function in viral replication.[7,8]
Several companies have identified inhibitors of NS5A as
promising direct acting antivirals.[9] The most advanced of
these is daclatasvir (1; BMS790052), a highly potent NS5A in-
hibitor that was discovered and developed by Bristol–Myers–
Squibb and is currently in phase 3 clinical trials (Figure 1).[10,11]
The impressive efficacy of daclatasvir was confirmed in a single
ascending dose study with a mean 3.3 log10 drop in viral load
in HCV patients 24 h post administration of a 100 mg dose and
a mean 1.95 log10 drop in viral load just 6 h after dosing. This
was confirmed in a phase 2 trial that confirmed the efficacy of
a once daily regimen of daclatasvir in combination with pegy-
lated interferon and ribavirin.[12] Recently, daclatasvir has been
proposed to block both the synthesis of new viral genomes
It is estimated that some 3% of the world’s population is cur-
rently infected with hepatitis C virus (HCV).[1] Approximately
70% of infected individuals develop a chronic infection, a por-
tion of whom go on to develop chronic liver disease.[2] While
the rate of new infections has declined sharply in the last two
decades, the need for broadly efficacious and well-tolerated
therapies remains high. Standard of care therapy for many
years was a combination of injected pegylated interferon and
ribavirin, which can eradicate the virus with varying degrees of
success depending on the genotype of virus the patient is in-
[a] T. D. Tran, F. Wakenhut, L. Watson, M. Paradowski, Dr. J. Milbank, F. Adam,
D. Hay, J.-Y. Chiva, C. Nichols, Dr. D. C. Blakemore, Dr. D. C. Pryde
Worldwide Medicinal Chemistry, Pfizer Global Research & Development
Ramsgate Road, Sandwich, Kent CT13 9NJ (UK)
E-mail: david.pryde@pfizer.com
[b] Dr. C. Pickford, S. Shaw, Dr. M. Westby, C. Smith-Burchnell
Antivirals Virology, Pfizer Global Research & Development
Ramsgate Road, Sandwich, Kent CT13 9NJ (UK)
[c] I. Gardner, S. Dayal, Dr. A. Pike, R. Webster
Pharmacokinetics, Dynamics & Metabolism
Pfizer Global Research & Development
Ramsgate Road, Sandwich, Kent CT13 9NJ (UK)
[d] Dr. R. A. Brimage, R. Halstead, Dr. R. Glen, Dr. C. P. Wilson
Peakdale Molecular Ltd.
Sheffield Road, Chapel-en-le-Frith, High Peak, SK23 0PG (UK)
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able in a novel linking structure, but that we had not identified
an optimal linker as yet.
With this in mind, we then embarked upon a more focussed
study of spacing groups that separated one of the imidazole
rings from the remainder of the central linker in a number of
different ways, as shown in Table 2. At this stage, we based all
our subsequent designs on the prolyl-valinyl carbamate cap-
ping group, which we found to be uniformly more potent and
in many cases offered improved permeability and oral bioavail-
ability.[15] The 1,5-naphthyl analogue (8), an isomer of the 1,4-
naphthyl system (7), was completely inactive against both gt1a
and gt1b but, most intriguingly, the related 2,6-naphthyl ana-
logue 13 was very potent, particularly against gt1b lending fur-
ther weight to the importance of the molecular cores spatially
presenting the remainder of the molecule correctly for bal-
anced activity. Compound 13 was, however, both lipophilic
and very poorly soluble, and we continued to seek analogues
with better physicochemical properties. The level of potency
of 13 was not reproduced in saturated ring-containing cores
such as 9, 10 and 12 or in alicyclic ether linkers such as 11.
Saturated versions of the 2,6-naphthyl ring system were also
investigated, for example, the tetrahydro-isoquinoline ring
system in compound 14 that retained a reasonably balanced
genotype profile but were significantly less active than the cor-
responding aromatic systems. The fully aromatic 2,6-naphthyl
ring system (13) therefore became the most promising lead
that was now pursued aggressively. Analogues of 13 are
shown in Table 3.
Figure 1. Daclatasvir (1), a clinically precedented HCV NS5A inhibitor.
and also to prevent the release of virus from infected cells, and
it is this dual mode of action that is thought to be responsible
for its high efficacy.[13] Given the exciting efficacy of NS5A in-
hibitors such as daclatasvir, we initiated our own programme
aimed at finding a novel and highly potent agent, starting
from daclatasvir itself. In our typical screening cascade, com-
pound potency was assessed in subgenomic replicon cell lines
within both a gt1b and gt1a background and for cytotoxicity
in the same gt1b cell line.
At first glance, the most interesting aspects of 1 are its very
high molecular size (MWt=739) and dimeric topology. Molecu-
lar properties based on this type of structure would be predict-
ed to be in higher risk chemical space for both absorption and
solubility.[14] For our own programme, we were interested in
evaluating the pharmacokinetic properties of analogues from
this series as we progressed. Firstly, however, we em-
barked on scoping the structure–activity relationships
Table 1. Structure and activities of phenylglycine-pyrrolidine analogues of the biphen-
yl-imidazole unit of 1.[a]
(SARs) of the central biphenyl-imidazole unit of 1,
and in particular, monitored structural changes for
their effects on both gt1b and gt1a potency in the
replicon system.
For synthetic expedience, we initially based this
study on the phenylglycine-pyrrolidine series shown
in Table 1 in which simple modifications to the core
structure resulted in sharp drops in cell-based poten-
cy, but also illustrated that these drops were not con-
sistent for the two gt1 subtypes tested. For example,
biphenyl parent structure 2 showed a balanced gt1
profile, albeit significantly weaker than 1, while oxa-
zole analogue 3 resulted in a significantly greater loss
of activity against gt1a, but showed improved poten-
cy against gt1b. A number of linker groups were
then inserted between the two phenyl rings, and
again, resulted in a general loss of activity against
the subtypes through incorporating a hydrocarbon
(4), ether (6) or a contracted, fused naphthyl system
(7). Interestingly, rigid unsaturated linking groups
such as the alkynyl group in 5 did show a reduction
in potency against both gt1 subtypes but did still
retain a balanced activity profile. Our conclusion from
this exercise was that the correct spatial arrangement
of analogues of the biphenyl-imidazole system was
key to achieving balanced gt1 activity, and that the
initial data generated suggested this could be achiev-
Compd
Structure
IC50 [pm][a]
Replicon 1b Replicon 1a
Cytotoxicity
[nm][b]
2
3
4
5
6
490
6
1190
117000
5700
>500
6020
387
93
>500
>500
>500
304
706
158000
7
5020
500000
>500
Data were determined in [a] a replicon cell-based assay or [b] a WST-1 cell prolifera-
tion assay and are the mean of at least two replicates.
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arating the rings of a 6,5-bicycle as in phenyl oxadia-
zole 21 was not tolerated and was significantly
weaker at both genotypes. Returning to very potent
quinoxaline 17, the rings of the 6,6-bicyclic system
were separated and the phenyl ring fused to the ad-
jacent imidazole. Similar strategies have recently
been reported by others.[9a,15,17] Resulting pyrazine-
benzimidazole 22 was of similar potency, albeit
slightly weaker against genotype 1a, while the direct
pyrimidine analogue 23 was slightly weaker again
against genotype 1a. Pyridine analogues 24 and 25
were some fivefold less potent against genotype 1a
than the phenyl equivalent. Moving the extra nitro-
gen atom onto the benzimidazole ring resulted in
compounds that were less potent at both subtypes,
with the 7-aza analogue (27) more balanced than the
4-aza analogue (26).
Table 2. Structure and activities of analogues of the biphenyl-imidazole unit of 1.[a]
Compd
Structure
IC50 [pm][a]
Replicon 1b Replicon 1a
Cytotoxicity
[nm][b]
1
10
500000
1200
47
500000
257
>500
>500
8
9
>40000
From the analogues prepared and profiled, a selec-
tion of the more potent and balanced agents were
investigated alongside 1 for potency against several
of the most common reported NS5A replicon-derived
mutants[18] to emerge as shown in Table 4. In our sys-
tems, compound 1 displayed significant drops in ac-
tivity, particularly within a gt1a background, with the
M28T mutation showing a 350-fold drop in potency.
Interestingly, the core-modified compounds de-
scribed herein were all uniformly less sensitive to the
gt1a mutations tested than 1 but against a gt1b
background showed mixed results. While compound
17 showed a similar gt1b sensitivity as compound 1,
all other compounds tested were more sensitive to
the L31V and the Y93H mutations. Whilst we have
not generated any de novo resistance virus with any
of the compounds described herein, our data are en-
tirely consistent with the proposed mode of interac-
tion of similar chemotypes across an NS5A dimer in-
terface with the core structure proximal to the L31
amino acid location and the cap region to the Y93
amino acid location.[19]
10
11
211
558
8840
>10
44200
>24800
12[c]
13
113
2
4470
22
>10
>40000
14
39
136
23200
[a] Data were determined in [a] a replicon cell-based assay or [b] a WST-1 cell prolifera-
tion assay and are the mean of at least two replicates. [c] Compound 12 was prepared
and evaluated as a mixture of cis and trans diastereoisomers at the cyclobutyl ring.
We were mindful of the high lipophilicity of compound 13
These data were intriguing in that the peripheral substitu-
(cLogP=5.9) and sought changes that lowered overall lipophi-
licity whilst retaining activity. Inserting nitrogen atoms into the
naphthyl ring of 13 to decrease LogP gave compounds such
as quinoline 16 (cLogP=4.8) and quinazoline 15 (cLogP=3.9)
that both retained the excellent potency and subtype balance
of 13. Quinoxaline 17 (cLogP=4.1) was particularly potent,
with a gt1b IC50 value of 8 pm balanced with a similar gt1a IC50
value of 13 pm. Through adding the N atoms to this structure,
the overall cLogP value of 17 was almost two units lower com-
pared with starting naphthyl 13 and had an enhanced aque-
ous solubility (thermodynamic solubility of approximately
6 mgmLꢀ1, similar to that of 1). Fused 6,5-bicyclic heterocycles
displayed very subtle SAR as has been observed by other-
s.[9a,15,16] While benzoxazole 18 and indazole 20 showed similar
levels of potency and genotype balance to that of the starting
naphthyl compound 13, closely related benzothiazole 19 was
at least two orders of magnitude less potent against gt1a. Sep-
tion pattern of the analogues prepared in our study was iden-
tical to that of 1, but significant differences in mutant sensitivi-
ty were seen.
We next turned our attention to evaluating the physico-
chemistry and pharmacokinetics of the series. When various
properties of the compounds investigated were plotted in
Figure 2, it appeared that microsomal metabolic stability
across the LogD range was uniformly good in general. Howev-
er, it also appeared that targeting a clogP value of around 4 or
less would be beneficial for both permeability and for aqueous
solubility. This focussed our attention on some of the less lipo-
philic analogues we had made, and we looked more closely at
quinoxaline 17.
Encouraged by the mutant profile and in vitro data of 17,
further in vitro and in vivo pharmacokinetic evaluation was un-
dertaken as shown in Table 5. Compound 17 showed good
passive permeability and low turnover in both human micro-
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Table 3. Structure and activities of aromatic analogues of the 2,6-naphthyl lead (13).[a]
Compd
Structure
IC50 [pm][a]
Replicon 1b Replicon 1a
Cytotoxicity
[nm][b]
Compd
Structure
IC50 [pm][a]
Replicon 1b Replicon 1a
Cytotoxicity
[nm][b]
13
2
22
>40000
21
582
10000
>10
15
16
17
6
9
8
36
75
13
>24300
>40000
16000
22
23
24
7
8
32
70
9800
>13300
>1000
10
163
18
19
20
10
45
16
132
10000
52
>12700
>10
25
26
27
15
27
22
176
146
54
>40000
12000
18900
20200
[a] Data were determined in [a] a replicon cell-based assay or [b] a WST-1 cell proliferation assay and are the mean of at least two replicates.
indicated that any metabolite formation was over-
whelmingly driven by CYP3A4 oxidative metabolism.
In the rat, the compound showed low total clearance,
was well absorbed, and had a short half-life.
Table 4. Activity of selected compounds against mutant replicon cell lines, expressed
as fold shift relative to wild type activity.
Compd
IC50 [pm]
Shift (gt1b background) Shift (gt1a background)
Replicon 1b Replicon 1a
L31V
Y93H
M28T
Q30H
The human pharmacokinetics of 17 were predicted
to show low clearance (CL<0.3 mLminꢀ1 kgꢀ1) and
a long terminal half-life in excess of 24 h. These phar-
macokinetic parameters were then used to translate
into dose predictions based on a gt1a antiviral IC90
value for 17 of approximately 20 pm. It was predicted
that a 10 mg dose would be sufficient to provide
a free exposure at 24 h post-dosing of approximately
200 pm, some tenfold the antiviral IC90, and 17 would
therefore be suitable for once daily dosing. Com-
pound 17 was evaluated in a rat exploratory toxicolo-
gy study over 7 days at doses up to 500 mgkgꢀ1 with
1
10
8
16
7
47
13
52
32
70
163
176
146
54
175
101
671
1160
3050
393
1270
182
48.8
120
350
24.1
38.8
33.1
9.95
138
197
98.1
115
314
88.8
263
66.6
62
94.5
64.5
51.3
30.6
17
20
22
23
24
25
26
27
2180
1410
1110
1330
3180
318
8
10
15
27
22
814
448
[a] Data were determined in a replicon cell based assay and are the mean of at least
two replicates. Assay details are described in the Experimental Section.
somes and human hepatocytes. It was highly protein bound in
all species and demonstrated significant efflux in a P-glycopro-
tein (Pgp)-overexpressing MDR-1 cell line. Metabolite studies
no adverse findings reported or histopathology abnormalities
observed.
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Table 5. Selected in vitro parameters and in vivo rat pharmacokinetic pa-
rameters for compound 17.
Parameter [unit]
Value
In vitro parameters[a]
MW
791
clogP (logD)
4.1 (1.7)
<2
16/13
<8
<8
0.3
hHeps [mLminꢀ1 millionꢀ1
]
RRCK[20] permeability AB/BA [10ꢀ6 cmsꢀ1
]
HLM[21] [mLminꢀ1 mgꢀ1
]
RLM [mLminꢀ1 mgꢀ1
Human fu [%]
]
MDR-1[22] (AB/BA)
3.5/41
Rat pharmacokinetic parameters[b]
CL [mLminꢀ1 kgꢀ1
CLu [mLminꢀ1 kgꢀ1
dss [Lkgꢀ1
]
14
3889
2
]
V
]
t1/2 [h]
F [%]
2
31
[a] All metabolism assays were conducted as described in Reference [21].
Permeability assays were conducted as described in References [20] and
[22], as indicated. [b] Dose: 1 mgkgꢀ1 (i.v.); 2 mgkgꢀ1 (p.o.); cremaphor
vehicle.
5. For compound 17, the molecule was synthesised from three key
fragments as shown in Scheme 1.
The syntheses of the three fragments are shown in Scheme 2. Bro-
moimidazole 28 was synthesised starting from Boc-l-proline 31.
The proline was converted to aldehyde 33 via reduction to the al-
cohol 32 followed by TEMPO/bleach oxidation. The aldehyde was
sensitive to epimerisation but this sequence proved most effective
for maintaining chiral integrity. The aldehyde was converted to imi-
dazole 34 using ammonia and glyoxal, and the imidazole NH was
protected with a [2-(trimethylsilyl)ethoxy]methyl (SEM) group to
give imidazole 35. Bromination occurred selectively at the 5-posi-
tion of the imidazole, with the presence of the electron-withdraw-
ing SEM group preventing over bromination to give 28. Imidazole
boronate 29 was synthesised starting from Boc-l-proline 31 and
bromoketone 36. The resulting ketoester (37) was rearranged to
imidazole 38 using ammonium acetate in xylene; this was convert-
ed to boronate ester 29 under palladium-mediated conditions with
bis(pinacolato)diboron. Finally, quinoxaline fragment 30 was syn-
thesised starting from quinoxalinone 39. Selective bromination at
the 6-position of the quinoxaline could be accomplished under
silver-mediated conditions to give 40; this was then treated with
POCl3 giving the desired quinoxaline fragment (30).
Figure 2. Physicochemical data correlate for compounds described in this
paper, where data were measured: a) human liver microsomal (HLM) stability
(mLminꢀ1 mgꢀ1) versus measured logD in the top panel; b) aqueous solubili-
ty (mgmLꢀ1) plotted against calculated logP in the middle panel; c) passive
permeability measured in a canine kidney cell line (10ꢀ6 cmsꢀ1) plotted
against calculated logP. Solubility was measured from a DMSO stock solu-
tion using a kinetic miniaturized shake flask method.
In conclusion, through this study, we have identified novel
core structures with a distinct resistance profile. Through
a careful examination of in vitro physicochemical parameters,
we have identified a compound that showed good absorption
and a long predicted human pharmacokinetic half-life. Further
investigations with compound 17 will be reported in due
course.
To put the three key fragments together, the approach shown in
Scheme 3 was followed. Quinoxaline 30 was Suzuki coupled with
imidazole boronate 29 to give bromoquinoxaline 41. Key to the
success of our strategy was the selective coupling at the 2-chloro
position rather than the 6-bromo position, and we found that Pd-
(dppf)Cl2 at room temperature effected this transformation with
excellent regioselectivity. Subsequent boronate ester formation
was followed by a second Suzuki coupling with bromoimidazole
28 to give 43. To access compound 17, all that remained was to
deprotect the SEM and Boc groups and carry out a double amide
bond formation using 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide (EDCI), hydroxybenzotriazole (HOBt) and protected valine 45.
This sequence gave 17 without loss of chiral integrity.[23]
Experimental Section
Discussion of synthesis
For compound 22, a similar strategy was utilised with the molecule
All compounds were synthesised in a similar manner. Representa-
being built from three key fragments (Scheme 4). Imidazoleboro-
tive syntheses of compounds 17 and 22 are shown in Schemes 1–
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Scheme 1. Synthetic strategy towards compound 17.
Scheme 2. Synthesis of key fragments for making 17. Reagents and conditions: a) BH3·THF, THF, 08C!RT, 100%; b) TEMPO, NaOCl, NaBr, NaHCO3, CH2Cl2/H2O,
08C, 86%; c) NH3(aq), MeOH, RT, 65%; d) NaH, N-methylpyrrolidine, 08C then SEMCl, 80%; e) NBS, CH2Cl2, RT, 99%; f) DIPEA, CH2Cl2, RT, 100%; g) NH4OAc,
xylene, 1508C, 79%; h) Pd(dppf)Cl2·CH2Cl2, KOAc, dioxane, 1008C, 73%; i) Br2, Ag2SO4, H2SO4, CHCl3, 90%; j) POCl3, DMF, 90%.
nate 29 was synthesised as for the previous compound. Benzimid-
azole boronate 46 was synthesised from Boc-l-proline 31 as
shown in Scheme 5. Amide coupling to generate 48 was followed
by ring closure under acidic conditions to give bromobenzimida-
zole 49. This was converted to boronate ester 46 under palladium-
mediated conditions with bis(pinacolato)diboron.
Screening methods
Determination of HCV 1a replicon inhibitory activity: Genotype 1a
(H77, licensed from Apath LLC) HCV replicon cells were resuspend-
ed to a concentration of 1.4ꢁ105 cellsmLꢀ1 by addition of pre-
warmed assay medium (DMEM + 10% FCS). A 45 mL aliquot of this
suspension was added to each well of a 384-well assay plate (Lumi-
trac, Greiner) already containing 0.5 mL of test compound. All
plates were covered with gas-permeable seals and incubated at
378C, 5% CO2 for 48 h. After 48 h, the assay plate was removed
from the incubator and left to cool to RT for 15–30 min. Medium
was removed from the wells, and 5 mL lysis buffer (Renilla Lucifer-
ase Assay Kit, Promega) was added to each well. The plate was in-
cubated at RT on a rocker for 15 min, then 15 mL Assay Substrate
was added to each well. Luminescence was read immediately
using an EnVision plate reader.
To complete the synthesis, all that was needed was the selective
coupling of boronates 29 and 46 with the pyrazine (Scheme 6).
Benzimidazole boronate 46 was coupled with 2-bromo-5-iodopyra-
zine 47 under palladium-mediated conditions to give 50 in good
yield; use of the 2-bromo-5-iodopyrazine proved optimal for mini-
mising double addition in this reaction. Bromopyrazine 50 was
then Suzuki coupled with boronate 29 and, following deprotection
and double amide coupling, the desired product 22 was ob-
tained.[24] Again, no loss of chiral integrity was seen in this se-
quence.
Determination of HCV 1b replicon inhibitory activity: Genotype 1b
(con1, licensed from Reblikon GmbH) HCV replicon cells were re-
suspended to a concentration of 1.4ꢁ105 cellsmLꢀ1 by addition of
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Scheme 3. End-game for the synthesis of 17. Reagents and conditions: a) Pd(dppf)Cl2, Na2CO3, DME, RT, 76%; b) Pd(dppf)Cl2, KOAc, 1,4-dioxane, reflux, 71%;
c) Pd(dppf)Cl2, Na2CO3, DME, reflux, 60%; d) 4n HCl, dioxane, EtOH, 758C, 92%; e) EDCI, HOBt, DIPEA, CH3CN, RT, 71%.
Scheme 4. Synthetic strategy towards 22.
plates were covered with gas-permeable seals and incubated at
378C, 5% CO2 for 48 h. After 48 h, the plate was removed from the
incubator and left to cool to RT for 15–30 min. An equal volume of
reconstituted Lyophilised Britelite Plus Substrate (PerkinElmer) to
medium was added to each well. Luminescence was read immedi-
ately on an EnVision (PerkinElmer) plate reader.
Determination of inhibitor induced cytotoxicity in HCV replicon cell
lines: Genotype 1b (con1) HCV replicon cells were resuspended to
a concentration of 1.4ꢁ105 cellsmLꢀ1 by addition of pre-warmed
medium (DMEM +10% FCS). A 45 mL aliquot of this suspension
was added to each well of a 384-well assay plate (Greiner) already
containing 0.5 mL of test compound. All plates were covered with
gas-permeable seals and incubated at 378C, 5% CO2 for 48 h. After
48 h, 5 mL of WST-1 cell proliferation reagent (Roche) was added to
each well, and the plate returned to the incubator for 1 h. After
this incubation period, absorbance was read at 450 nm on an EnVi-
sion (PerkinElmer) plate reader.
Scheme 5. Synthesis of benzimidazole boronate 46. Reagents and conditions:
a) 2-Amino-4-bromo-aniline, HBTU, DIPEA, CH2Cl2, RT, 100%; b) AcOH, 708C,
75%; c) Pd(dppf)Cl2, KOAc, dioxane, reflux, 79%.
Determination of resistant HCV replicon inhibitory activity: Resistance
mutations were introduced to genotype 1b (con1, licenced from
Apath LLC) and 1a (H77, Apath LLC) replicons using the Quik-
pre-warmed medium (DMEM + 10% FCS). A 45 mL aliquot of this
suspension was added to each well of a 384-well assay plate (Lumi-
trac, Greiner) already containing 0.5 mL of test compound. All
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Scheme 6. End-game for the synthesis of 22. Reagents and conditions: a) Pd(dppf)Cl2, Na2CO3, toluene/EtOH/H2O, 608C, 72%; b) Pd2(dba)3, PCy3, K3PO4, diox-
ane, 1008C, 55%; c) HCl, EtOH, 908C, 87%; d) HOBt, EDCI, CH3CN, DIPEA, 08C!RT, 42%.
[4] T. J. Liang, M. G. Ghany, N. Engl. J. Med. 2013, 368, 1907–1917.
[5] T. P. Holler, T. Parkinson, D. C. Pryde, Expert Opin. Drug Discovery 2009, 4,
293–314.
[6] J. J. Kiser, C. Flexner, Annu. Rev. Pharmacol. Toxicol. 2013, 53, 427–449.
[7] O. Belda, P. Targett-Adams, Virus Res. 2012, 170, 1–14.
[8] M. Belema, O. D. Lopez, J. A. Bender, J. L. Romine, D. R. St. Laurent, D. R.
Langley, J. A. Lemm, D. R. O’Boyle, J-H. Sun, C. Wang, R. A. Fridell, N. A.
Meanwell, J. Med. Chem, 2014, 57, 1643–1672.
[9] For recent disclosures of NS5A inhibitors, see a) C. A. Coburn, P. T.
Meinke, W. Chang, C. M. Fandozzi, D. J. Graham, B. Hu, Q. Huang, S.
Kargman, J. Kozlowski, R. Liu, J. A. McCauley, A. A. Nomeir, R. M. Soll,
J. P. Vacca, D. Wang, H. Wu, B. Zhong, D. B. Olsen, S. W. Ludmerer, Chem-
MedChem 2013, 8, 1930–1940; b) M. Belema, V. N. Nguyen, D. R. St.
Laurent, O. D. Lopez, Y, Qiu, A. C. Good, P. T. Nower, L. Valera, D. R.
O’Boyle, J.-H. Sun, M. Liu, R. A. Fridell, J. A. Lemm, M. Gao, J. O. Knipe,
N. A. Meanwell, L. B. Snyder, Bioorg. Med. Chem. Lett. 2013, 23, 4428–
4435.
[10] J. A. Lemm, J. E. Leet, D. R. O’Boyle, J. L. Romine, X. S. Huang, D. R.
Schroeder, J. Alberts, J. L. Cantone, J.-H. Sun, P. T. Nower, S. W. Martin,
M. H. Serrano-Wu, N. A. Meanwell, L. B. Snyder, M. Gao, Antimicrob.
Agents Chemother. 2011, 55, 3795–3802.
change XL (Stratagene). NS5A L31V and Y93H were introduced to
genotype 1b, and the M28T and Q30H mutations were introduced
to genotype 1a replicons. Wild-type or mutant replicons were tran-
siently transfected into Huh-7.5 cells (Licensed from Apath LLC)
using an Amaxa Nucleofector (Lonza group Ltd). Following trans-
fection, the cells were resuspended to a concentration of 5.3ꢁ
104 cellsmLꢀ1 per in assay medium (DMEM + 10% FCS). A 180 mL
aliquot of cell suspension was added to each well of a 96-well
white assay plate (Costar, Corning Inc.), and plates were incubated
overnight at 378C, 5% CO2. Following this incubation, 20 mL of
compound diluted in assay medium were added. All plates were
covered with gas-permeable seals and incubated at 378C, 5% CO2
for 72 h. After 72 h, the assay plate was removed from the incuba-
tor and left to cool to RT for 15–30 min. Medium was removed
from the wells, and 5 mL lysis buffer (Renilla Luciferase Assay Kit,
Promega) was added to each well. The plate was incubated at RT
on a rocker for 15 min, then 15 mL Assay Substrate was added to
each well. Luminescence was read immediately using an EnVision
plate reader.
[11] M. Gao, R. E. Nettles, M. Belema, L. B. Snyder, V. N. Nguyen, R. A. Fridell,
M. H. Serrano-Wu, D. R. Langley, J.-H. Sun, D. R. O’Boyle, J. A. Lemm, C.
Wang, J. O. Knipe, C. Chien, R. J. Colonno, D. M. Grasela, N. A. Meanwell,
L. G. Hamann, Nature 2010, 465, 96–100.
Acknowledgements
[12] S. Pol, R. H. Ghalib, V. K. Rustgi, C. Martorell, G. T. Everson, H. A. Tatum,
C. Hezode, J. K. Lim, J.-P. Bronowicki, G. A. Abrams, N. Brau, D. W. Morris,
P. J. Thuluvath, R. W. Reindollar, P. D. Yin, U. Diva, R. Hindes, F. McPhee,
D. Hernandez, M. Wind-Rotolo, E. A. Hughes, S. Schnittman, Lancet
Infect. Dis. 2012, 12, 671–677.
The authors thank Dr. Gavin Whitlock for helpful discussions
throughout the course of this work, and Ms. Jenny Middleton
and Mr. Malcolm Macartney for help with virology assessments.
[13] J. Guedj, H. Dahari, L. Rong, N. D. Sansone, R. E. Nettles, S. J. Cotler, T. J.
Layden, S. L. Uprichard, A. S. Perelson, Proc. Natl. Acad. Sci. USA 2013,
110, 3991–3996.
Keywords: antiviral agents · drug resistance · HCV · hepatitis C
virus · NS5A · viral proteases
[14] P. D. Leeson, B. Springthorpe, Nat. Rev. Drug Discovery 2007, 6, 881–
890.
[1] D. Lavanchy, Liver Int. 2009, 29, 74–81.
[2] R. S. Brown, Nature 2005, 436, 973–978.
[3] J. H. Hoofnagle, L. B. Seeff, N. Engl. J. Med. 2006, 355, 2444–2451.
[15] M. Belema, V. N. Nguyen, C. Bachand, D. H. Deon, J. T. Goodrich, C. A.
James, R. Lavoie, O. D. Lopez, A. Martel, J. L. Romine, E. H. Ruediger,
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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L. B. Snyder, D. R. St. Laurent, F. Yang, J. Zhu, H. S. Wong, D. R. Langley,
S. P. Adams, G. H. Cantor, A. Chimalakonda, A. Fura, B. M. Johnson, J. O.
Knipe, D. D. Parker, K. S. Santone, R. A. Fridell, J. A. Lemm, D. R. O’Boyle,
R. Colonno, M. Gao, N. A. Meanwell, L. G. Hamann, J. Med. Chem, 2014,
57, 2013–2032.
Feng, C. Rotter, M. V. Varma, S. Kempshall, K. Fenner, A. F. El-Kattan, T. E.
Liston, M. D. Troutman, J. Pharm. Sci. 2011, 100, 4974–4985.
[21] G. Allan, J. Davis, M. Dickins, I. Gardner, T. Jenkins, H. Jones, R. Webster,
H. Westgate, Xenobiotica 2008, 38, 620–640.
[22] B. Feng, J. B. Mills, R. E. Davidson, R. J. Mireles, J. S. Janiszewski, M. D.
Troutman, S. M. de Morais, Drug Metab. Dispos. 2008, 36, 268–275.
[16] D. A. DeGoey, J. T. Randolph, D. Liu, J. Pratt, C. Hutchins, P. Donner, A. C.
Krueger, M. Matulenko, S. Patel, C. E. Motter, L. Nelson, R. Keddy, M.
Tufano, D. D. Caspi, P. Krishnan, N. Mistry, G. Koev, T. J. Reisch, R.
Mondal, T. Pilot-Matias, Y. Gao, D. W. A. Beno, C. J. Maring, A. Molla, E.
Dumas, A. Campbell, L. Williams, C. Collins, R. Wagner, W. M. Kati, J.
Med. Chem, 2014, 57, 2047–2057.
[17] J. O. Link, J. G. Taylor, L. Xu, M. Mitchell, H. Guo, H. Liu, D. Kato, T. Kirsch-
berg, J. Sun, N. Squires, J. Parrish, T. Keller, Z-Y. Yang, C. Yang, M. Matles,
Y. Wang, K. Wang, G. Cheng, Y. Tian, E. Mogolian, E. Mondou, M. Corn-
propst, J. Perry, M. C. Desai, J. Med. Chem. 2014, 57, 2033–2046.
[18] R. A. Fridell, D. Qiu, C. Wang, L. Valera, M. Gao, Antimicrob. Agents Che-
mother. 2010, 54, 3641–3650.
[23] Methyl
((S)-1-((S)-2-(4-(4-(6-(2-((S)-1-((methoxycarbonyl)-l-valyl)pyrroli-
din-2-yl)-1H-imidazol-5-yl)quinoxalin-2-yl)phenyl)-1H-imidazol-2-yl)pyrro-
lidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (17): 1H NMR (400 MHz,
CD3OD): d=9.35–9.20 (m, 1H), 8.40–7.60 (m, 7H), 7.55–7.30 (m, 2H),
5.40–5.15 (m, 2H), 4.30–4.20 (m, 2H), 4.15–3.85 (m, 4H), 3.68–3.62 (m,
6H), 2.50–1.90 (m, 10H), 1.00–0.80 ppm (m, 12H); MS (ESI): m/z=791
[M+H]+.
[24] Methyl
((S)-1-((S)-2-(5-(5-(4-(2-((S)-1-((methoxycarbonyl)-l-valyl)pyrroli-
din-2-yl)-1H-imidazol-4-yl)phenyl)pyrazin-2-yl)-1H-benzo[d]imidazol-2-yl)-
pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate
(22):
1H NMR
(400 MHz, CD3OD): d=8.30–7.25 (m, 9H), 7.10–7.00 (m, 1H), 5.35–5.10
(m, 2H), 4.35–4.20 (m, 2H), 4.15–3.85 (m, 4H), 3.68–3.62 (m, 6H), 2.50–
1.90 (m, 10H), 1.00–0.80 ppm (m, 12H); MS (ESI): m/z=791 [M+H]+.
[19] D. R. O’Boyle II, J.-H. Sun, P. T. Nower, J. A. Lemm, R. A. Fridell, C. Wang,
J. L. Romine, M. Belema, V. N. Nguyen, D. R. St. Laurent, M. Serrano-Wu,
L. B. Snyder, N. A. Meanwell, D. R. Langley, M. Gao, Virology 2013, 444,
343–354.
Received: January 20, 2014
[20] L. Di, C. Whitney-Pickett, J. P. Umland, H. Zhang, X, Zhang, D. E. Geb-
hard, Y. Lai, J. J. Federico, R. E. Davidson, R. Smith, E. L. Reyner, C. Lee, B.
Published online on && &&, 0000
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T. D. Tran, F. Wakenhut, C. Pickford,
S. Shaw, M. Westby, C. Smith-Burchnell,
L. Watson, M. Paradowski, J. Milbank,
R. A. Brimage, R. Halstead, R. Glen,
C. P. Wilson, F. Adam, D. Hay, J.-Y. Chiva,
C. Nichols, D. C. Blakemore, I. Gardner,
S. Dayal, A. Pike, R. Webster, D. C. Pryde*
Resisting resistance: An investigation
into the anti-hepatitis C virus (HCV) re-
plicon activity of a series of biaryl-linked
pyrrolidine NS5A inhibitors explored
a diverse range of core structure modifi-
cations as key determinants of antiviral
activity and susceptibility to common
resistance mutations. Further evaluation
of several core structure designs identi-
fied a compound with excellent phar-
macokinetics, suitable for once daily
dosing.
&& – &&
The Discovery of Potent Nonstructural
Protein 5A (NS5A) Inhibitors with
a Unique Resistance Profile—Part 1
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