Finger-loop inhibitors of the HCV NS5b polymerase. Part 1: Discovery and optimization of novel 1,6- and 2,6-macrocyclic indole series

Article abstract of DOI:10.1016/j.bmcl.2012.03.097

Novel conformationaly constrained 1,6- and 2,6-macrocyclic HCV NS5b polymerase inhibitors, in which either the nitrogen or the phenyl ring in the C2 position of the central indole core is tethered to an acylsulfamide acid bioisostere, have been designed and tested for their anti-HCV potency. This transformational route toward non-zwitterionic finger loop-directed inhibitors led to the discovery of derivatives with improved cell potency and pharmacokinetic profile.

Full text of DOI:10.1016/j.bmcl.2012.03.097

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4431–4436
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Finger-loop inhibitors of the HCV NS5b polymerase. Part 1: Discovery
and optimization of novel 1,6- and 2,6-macrocyclic indole series
⇑
David McGowan , Sandrine Vendeville, Tse-I Lin, Abdellah Tahri, Lili Hu, Maxwell D. Cummings,
Katie Amssoms, Jan Martin Berke, Maxime Canard, Erna Cleiren, Pascale Dehertogh, Stefaan Last,
Els Fransen, Elisabeth Van Der Helm, Iris Van den Steen, Leen Vijgen, Marie-Claude Rouan,
Gregory Fanning, Origène Nyanguile, Kristof Van Emelen, Kenneth Simmen, Pierre Raboisson
Janssen Infectious Diseases BVBA, 30 Turnhoutseweg, B-2340 Beerse, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel conformationaly constrained 1,6- and 2,6-macrocyclic HCV NS5b polymerase inhibitors, in which
either the nitrogen or the phenyl ring in the C2 position of the central indole core is tethered to an acy-
lsulfamide acid bioisostere, have been designed and tested for their anti-HCV potency. This transforma-
tional route toward non-zwitterionic finger loop-directed inhibitors led to the discovery of derivatives
with improved cell potency and pharmacokinetic profile.
Received 24 February 2012
Revised 27 March 2012
Accepted 28 March 2012
Available online 3 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C virus
NS5b polymerase
Allosteric inhibitor
Macrocycle
One hundred and seventy million people worldwide are cur-
rently infected with hepatitis C virus (HCV).1 Chronic infection is
the leading cause of liver disease, the largest indication for trans-
plantation in Europe and the United States, eventually leading to
liver cirrhosis.2 Hepatocellular carcinoma (HCC) related to HCV
infection has become the fastest growing cause of cancer-related
death in the US, and the incidence of HCC has tripled over the past
20 years.3 Increased survival rates and improved clinical outcome
site or an allosteric site, respectively.12 Allosteric finger loop inhib-
itors based on the 3-cyclohexyl indole structure, or analogs thereof,
that target the Thumb Pocket 1 site of the HCV NS5b polymerase
have been described (Fig. 1).13 The first indole inhibitors suffered
from poor aqueous solubility (e.g., amide analogs of compound
1)14 which hampered their development as drug candidates.
To address this drawback, scientists developed zwitterionic
derivatives exemplified by 2,15 exhibiting improved water solubil-
ity and drug-like properties. However, this series of compounds
has been reported to form glucoronide conjugates on the carbox-
ylic acid, which might eventually be responsible for toxicity linked
to their acylating potential.15 Moreover, zwitterionic drugs are of-
ten absorbed in the GI tract at specific locations (pH driven), which
may lead to higher patient variability. In this context, we envi-
sioned a different strategy dealing with the introduction of an un-
charged polar solubilizing group in a macrocycle, which might
eventually counterbalance the very lipophilic nature of the
3-cyclohexyl-2-phenylindole moiety.
may be associated with sustained virologic response (SVR).4
A
higher overall SVR could be achieved when direct anti-virals, re-
cently approved by the FDA,5,6 compliment the precedent standard
of care treatment, that is, using ribavirin and pegylated interferon.7
Novel medicaments like these are especially important for the dif-
ficult to treat population including those with genotype 1 and pa-
tients with liver cirrhosis.5 HCV can develop resistance to anti-viral
monotherapy within a matter of days. Future therapies will evalu-
ate the combination of newly targeted agents, that could be used to
enhance the anti-viral activity and potentially translate into higher
cure rates in shorter time.8 One of these potential targets is the
RNA-dependant RNA-polymerase (NS5b), essential for viral repli-
cation.9 HCV NS5b inhibitors can be divided into two classes;
nucleoside10 and non-nucleoside inhibitors,11 targeting the active
Previously reported X-ray structures with indoles bound to the
HCV polymerase suggest that a solvent exposed tether from the C6
carbonyl to the indole nitrogen or to the C2 aromatic ring would
not hinder the binding affinity of these macrocycles. Indeed, when
the carboxylic acid in C6 is replaced by an acyl sulfamide bioisoste-
re,16 (e.g., 3, Fig. 1) the key interaction with Arg-50317 is main-
tained, resulting in NS5b inhibition. Furthermore, a tether to the
⇑
Corresponding author. Tel.: +32 14 64 10 19; fax: +32 14 60 61 21.
E-mail address: dmcgowan@its.jnj.com (D. McGowan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.097

4432
D. McGowan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4431–4436
N
N
N
O
O
S
O
OH
OH
NH
O
N
N
N
O
O
O
O
O
Cl
1
2
3
µ
M
µ
M
µ
EC50 = 4.2
EC50 = 0.18
EC50 = 1.5 M
Figure. 1. Indole based finger loop inhibitors of NS5b polymerase.14–16
C2 aromatic ring would increase the affinity by holding the 46o
dihedral angle between the phenyl and indole moiety as previously
reported.13c
compound that caused a 50% reduction in signal as compared to
the control. Enzymatic activity (IC50) was measured against puri-
fied HCV NS5b
10a and 10b (Table 1) displayed, relatively low activity in the
HCV replicon (EC50 = 14 M and 10 M, respectively). Subsequent
ring-closed products 11a and 11b, displayed activities that in-
creased by fourfold (EC50 = 3.4 and 2.6 M, respectively)
D
21C isolate.20 The open amide intermediates
The synthesis of the macrocyclic indole derivatives 11a–d, via
ring closing metathesis (RCM), started with the 2-bromoindole
derivative 414,18 following the five step procedure outlined in
Scheme 1. Aryl bromide 4 reacted with 3-furanboronic acid under
standard Suzuki–Miyaura conditions in ethanol/toluene to give
intermediate 5 in 90% yield. Subsequent alkylation of 514 with bro-
momethylacetate with NaH in DMF led to acetate 6 in 90% yield.
Regioselective ester cleavage of intermediate 6 at 0 °C in THF/
methanol and aqueous LiOH, was followed by standard aminoacid
coupling with the alkenylamines 7a,b (Table 1, in blue) using HATU
in DMF afforded amides 8a,b. Basic hydrolysis of the second ester
group and subsequent coupling of the alkenes 9a–c (Table 1, in
red) using standard aminoacid coupling conditions in DMF pro-
vided dialkenes 10a–d in good yields (>80%). Final ring closing
metathesis using Hoveyda–Grubbs 1st generation catalyst
(5 mol %) in dichloroethane at 80 °C for 15 h, afforded macrocyclic
products 11a–d in 20–30% yield. The saturated macrocycle targets
l
l
l
M
l
potentially as a result of the entropic gain in macrocyclization. Acyl
sulfonamide 11c exhibits decreased cell potency for a comparable
ring size, attributed to its poor permeability, measured in CACO-2
cells (Papp <1 Â 10À6 cm/s), and a high efflux ratio >25.21 In con-
trast, a marked increase in cell potency was observed with the acy-
lsulfamide derivative 11d, which was found to be 10 times more
potent than 11c (EC50 = 0.72 lM, and 7.5 lM, respectively). 11c,d
and 12b displayed moderate metabolism in liver microsomes
(e.g., 12b: 63% rat, 67% human),22 in contrast to the rapid metabo-
lism seen for the macrocyclic diamides 11a–b, and 12a (>95%
metabolized in both rat and human liver microsomes).22
With these encouraging results in hand, we embarked on the
synthesis of additional macrocyclic indole derivatives incorporat-
ing the acyl sulfamide via the five step protocol outlined in
Scheme 2. The indole nitrogen of 514 and 1323 were then alkylated
with
12a (IC50 = 9.5
tion of 11a (IC50 = 2.2
l
M), and 12b were reached by catalytic hydrogena-
M) and 11d, respectively (Table 1).
l
The cell-based activity was measured as the inhibition of HCV
RNA replication in Huh-7 cells, based on a bicistronic expression
construct.19 Inhibition was calculated as the concentration of
t-butyl bromoacetate using sodium hydride in DMF at room tem-
perature. Regioselective unmasking of the t-butylesters 14a,b via
O
O
O
O
H
N
O
H
N
O
O
i
N
O
ii
O
O
Br
6
5
4
n
n
HN
O
NH
n
n
O
N
O
n
X
HN
O
O
HN
X
iii
iv
9a-c
v
N
N
N
O
O
O
O
O
H
7a,b
11a-f
12a,b
8a,b
10a-d
H2, Pd/C
Scheme 1. Synthesis of macrocyclic indoles. Reagents and conditions: (i) 3-furanylboronic acid, Pd(PPh3)4, Na2CO3, LiCl, ethanol/toluene, 80 °C, 3.5 h; (ii) NaH, methyl
bromoacetate, DMF, 0 °C; (iii) (a) LiOH, water/THF/methanol, 0 °C, 15 h, (b) 7a,b, HATU, DMF, rt, 6 h; (iv) (a) NaOH, water/THF/methanol, rt, 24 h, (b) for 9a,b: HATU, DIPEA,
DMF; for 9c: EDC, DMAP, DMF, rt, 24 h; (v) Hoveyda–Grubbs 1st generation catalyst (5 mol %), DCE, 80 °C, 15 h. Subsequent hydrogenation of the double bond: 10% Pd/C,
methanol, 1 atm H2, rt, 6 h.

D. McGowan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4431–4436
4433
M)
Table 1
1,6-macrocylic indoles via ring closing metathesis
Alkenylamine
Alkene
Open analog
RCM product
Macrocycle structure
REP Huh-7 EC50 (l
10a
11a (CH@CH)
12a (CH2–CH2)
3.4
4.3
NH2
NH2
O
HN
O
N
N
H
O
NH2
NH2
O
HN
O
N
N
O
10b
11b
2.6
H
O
S
O
NH2
O
NH2
HN
O
O
O
S
N
N
N
O
O
O
10c
10d
11c
7.5
H
NH2
O
S
N
O
NH2
11d (CH@CH)
12b (CH2–CH2)
0.72
0.79
N
H
N
O
O
S
N
H
O
O
O
O
O
O
H
N
N
i
ii
O
4
R1
R1
5: R1 = 3-furan
14a: R1 = 3-furan
13: R1 = 4-CH3OPh
14b: R1 = 4-CH3OPh
O
H2N
O
Linker
O
Linker
O
Boc
S
Linker
O
O
S
O
O
O
HN
N
N
R1
O
iv
v
iii
R1
N
O
O
R1
15a-d
18a-e
(Table 2)
17a-e
16a-e
Boc-Linker
H
H
N
N
N
NH2
NH2
N
N
Boc
N
Boc
Boc
NH2
N
Boc
15a
15b
15c
15d
Scheme 2. Synthesis of 1,6-macrocyclic indoles. Reagents and conditions: (i) 1 M aq Na2CO3, R1-B(OH)2, LiCl, EtOH/toluene, Pd(PPh3)4, 80 °C, 12 h; (ii) NaH, t-butyl
bromoacetate, DMF, rt; (iii) (1) TFA, DCM, rt, (2) HATU, 15a–d, DCM, DIPEA, rt; (iv) (1) TFA,DCM, 15 h, rt, (2) Sulfamide, dioxane 100 °C, 40 min; (v) (1) aq NaOH, methanol,
THF, (2) CDI, CH3CN, rt, then DBU, CH3CN, rt.
TFA in dichloromethane, and subsequent reaction with mono-boc
protected diamines (15a–d) under standard aminoacid coupling
conditions afforded 16a–e. Acid mediated deprotection of the
boc-protected linkers in TFA/DCM, formation of the free base, then
subsequent heating with excess sulfamide in dioxane at 100 °C af-
fords 17a–e in yields above 50%. Methyl ester hydrolysis proceeded

4434
D. McGowan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4431–4436
Table 2
in good yield (>90%) to afford the corresponding acid
HCV inhibition of the 1,6-macrocyclic indole series
intermediates, which were then treated with carbonyl diimidazole
(CDI) to form an acyl imidazole intermediate purified by flash chro-
matography. Following isolation, the compound was reconstituted
in acetonitrile, then DBU was added to effect macrocyclization at
room temperature giving rise to products 18a–e (Table 2). Com-
pounds 18f–h (Table 2) have been synthesized from 2-(4-chloro-
phenyl)-3-cyclopentyl-6-carboxylic acid methyl ester (18f and
18h) following a similar pathway as reported for the synthesis of
12a,b (Scheme 1). The dehalogenated 18g was obtained as a side
product of 18f during the final hydrogenation over 10% Pd/C in
methanol at room temperature.
Compd R1
Linker
IC50
(l
M) REP Huh-7 EC50 (lM)
18a
18b
3-Furan
4-
0.11
0.18
1.58
1.76
N
N
H
CH3OPh
H
N
H
N
4-
CH3OPh
18c
0.44
3.47
N
4-
CH3OPh
N
18d
18e
NA
3.5
N
N
N
4-
CH3OPh
4-ClPh
H
N
0.19
0.35
The 16-membered ring compound 18e displayed more favor-
able replicon inhibition over the 15-membered ring analog 18b.
Introduction of a basic nitrogen in the linker (18c, 18d) decreased
potency by 10-fold versus the carbon chain linker of the same
18f
0.19
NA
0.59
0.43
18g
Ph
N
18h
4-ClPh
0.44
0.53
N
length (18e) (EC50 = 3.47 lM, 3.5 lM, 0.35 lM, respectively).
Moreover, the potency is maintained when the phenyl ring is
substituted with a chlorine (18f, 18h) or unsubstituted (18g).
NA = not available.
R
O
R1
N
R1
N
O
O
H
HO
N
i
ii
O
O
O
R3
Br
Br
R2
21a-f
1. KOH, THF
2. DMF-DBA
4: R = CH3
20a: R1 = CH3
20b: R1 = iPr
19: R = -butyl
t
20c: R1 = cyclopentyl
O
H2N
O
Linker
O
O
O
Linker
Linker
S
R
S
O
O
HN
iv
v
iii
R1
O
N
O
O
R1
O
R1
N
O
O
22a-e
N
O
O
R3
R3
R3
R2
R2
R2
24a-k
25a-k
23a-k
(Table 3)
R-Linker (R = H or Ns = 4-nitrobenzenesulfonyl)
H
N
H
N
H
N
HN
O
N
H
NH2
22b
22a
22c
N
Ns
N
N
NH2
Ns
NH
22e
22d
Scheme 3. Synthesis of 2,6-macrocyclic indoles. Reagents and conditions: (i) iodoalkane (I-R1), NaH, THF, rt; (ii) 2-hydroxyphenyl-(R2,R3)-boronic acid, Pd(PPh3)4, K2CO3,
DME/water, 100 °C, 12 h; (iii) (1) methyl bromoacetate, K2CO3, CH3CN, rt, (2) LiOH(aq), THF/methanol, (3) amine, HATU, DIPEA, THF. For 22d–e; Ns deprotection via Cs2CO3,
thiophenol, DMF, rt; (iv) sulfamide, dioxane 100 °C, 45 min.; (v) (1) TFA, DCM, rt, (2) CDI, CH3CN then DBU.
Table 3
HCV inhibition and metabolic stability of compounds in the 2,6-macrocyclic indole series
Compd
R1, R2, R3
Linker
IC50
(
lM)
REP Huh-7 EC50
7.56
lM
RLM22
—
HLM22
—
N
25a
CH3, H, H
1.9
N
H
25b
25c
CH3, H, H
CH3, H, OCH3
0.097
0.054
0.31
0.17
60
64
92
96
N
N
N
25d
25e
CH3, H, H
CH3, F, H
0.033
0.088
0.18
0.68
44
56
71
59
H
N
25f
CH3, H, F
0.110
0.46
58
81
25g
25h
25i
25j
CH3, H, OCH3
CH3, H, H
iPr, H, H
0.056
0.11
1.66
1.49
0.15
0.19
11.9
15.7
52
50
35
40
69
65
64
74
N
N
N
O
N
Cyclopentyl, H, H
25k
CH3, H, H
0.14
0.24
35
56
N

D. McGowan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4431–4436
4435
Table 4
Pharmacokinetic profile of compounds 12b, 25b and 25h in rata
Acknowledgments
c
d
Compd
CLb (L/h/kg)
Vdss (L/kg)
T1/2 (h)
Fe (%)
We thank the discovery biology, analytical chemistry and
ADME/PK teams for their technical assistance.
12b
25b
25h
7.4
7.7
6.9
2.4
2.6
7.0
0.4
0.3
1
40
9
14
Supplementary data
a
Administration: iv 2 mg/kg in PEG400/saline (70:30); po 10 mg/kg PEG400/2%
Vitamin E TPGS.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
03.097. These data include MOL files and InChiKeys of the most
important compounds described in this article.
b
Clearance from plasma.
Volume of distribution.
Half-life.
c
d
e
Oral bioavailability.
References and notes
Attention then turned to installing the linker between the C2
aromatic, holding the aryl group in its preferred conformation,
and the acid bioisostere, described in Scheme 3. Basic hydrolysis
of methyl ester 4, followed by reaction with dimethylformamide
di-t-butylacetal (DMF-DBA) afforded the indole t-butylester 19.
Alkylation of 19 over sodium hydride in the presence of alkyl
iodide in DMF gave rise to 20a–c (R1 = methyl, isopropyl,
cyclopentyl in yields of 90%, 60%, and 20%, respectively). Typical
Suzuki–Miyaura conditions were used in the coupling of
2-bromoindoles 20a–c with boronic acids to afford intermediates
21a–f. The phenol oxygen was alkylated with methyl bromoace-
tate in DMF over potassium carbonate. Subsequent regioselective
deprotection of the intermediate methyl esters was carried out in
basic media, followed by standard aminoacid coupling conditions
in DMF, employing linkers 22a–e, (the nitrogen of the newly
formed bond is shown in bold, Scheme 3, Table 3), allowed the for-
mation of the corresponding amide intermediates 23a–k in 70–
90% yield. Formation of the sulfamides 24a–k, and subsequent ring
closure was effected as in Scheme 2 to give the 2,6-indole macro-
cycles 25a–k (3).
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Stammers, T.; Thauvette, L.; Warrington, J.; Kukolj, G. Bioorg. Med. Chem. Lett.
2006, 16, 4987.
15. Giuliano, C.; Fiore, F.; Di Marco, A.; Velazquez, J. P.; Bishop, A.; Bonelli, F.;
Gonzalez-Paz, O.; Marcucci, I.; Harper, S.; Narjes, F.; Pacini, B.; Monteagudo, E.;
Migliaccio, G.; Rowley, M.; Laufer, R. Xenobiotica 2005, 10/11, 1035.
16. Stansfield, I.; Pompei, M.; Conte, I.; Ercolani, C.; Migliaccio, G.; Jairaj, M.;
Giuliano, C.; Rowley, M.; Narjes, F. Bioorg. Med. Chem. Lett. 2007, 17, 5143.
17. Cummings, M.D.; Lin, T.-I.; Hu, L.; Tahri, A.; McGowan, D.; Amssoms, K.; Last,
S.; Devogelaere, B.; Rouan, M.-C.; Vijgen, L.; Berke, J.M.; Dehertogh, P.; Fransen,
E.; Cleiren, E.; van der Helm, L.; Fanning, G.; Van Emelen, K.; Nyanguile, O.;
Simmen, K.; Raboisson, P.; Vendeville, S. Angew. Chem. Int. Ed. 2012, 51, 1.
Alkylation of the indole nitrogen proved detrimental to activity
(e.g., 25i, EC50 = 11.9 lM, IC50 = 1.66 lM), the augmented alkyl size
potentially displaces the linker to clash with the protein, or alter-
natively, alters the dihedral angle of the 2-aryl group. Contrary to
the 1,6-macrocyclic indoles, this series tolerated a tether contain-
ing a basic amine (25k, EC50 = 0.24 lM, IC50 = 0.14 lM).
The potent macrocyclic indoles 12b, 25b and 25h were studied
for their pharmacokinetic properties in Sprague–Dawley rats, and
the data is summarized in Table 4. Plasma kinetics were deter-
mined after a single iv administration of 2 mg/kg compound in
PEG400/saline (70:30) as a vehicle. These data were compared
to the oral dose at 10 mg/kg in PEG400 containing 2% Vitamin E
TPGS. Systemic exposure was attainable, albeit with low to mod-
erate bioavailability ranging from 9% to 40%. High clearance from
plasma and half-lives of 1 h or less were observed. HCV replica-
tion is known to occur in the liver,24 thus the high drug concen-
trations observed in the target organ 7 h post dosing (12b, 25b,
25h = 3743, 1524, 1717 ng/g, respectively), corresponding to
favorable liver to plasma ratios (150, 99, and 64 for 12b, 25b
and 25h, respectively), were encouraging results. No formation
of glutathione conjugates was observed after incubation of 25h
with human liver microsomes, fortified with glutathione
(GSH),25 bolstering the development potential of these macrocy-
clic compounds.
In summary, we have described two series of macrocyclic in-
doles where a tether connects a carboxylic acid bioisostere in the
6-position to either the indole nitrogen or 2-aryl position. Optimi-
zation afforded potent allosteric inhibitors of the HCV NS5b
enzyme, reduction of subgenomic HCV RNA replication in Huh-7
cells, and bioavailability in rats. These findings contribute to
further modifications that will be described in Part 2.

4436
D. McGowan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4431–4436
http://dx.doi.org/10.1002/anie.2012.00.010. Previously reported crystal
2008, 52, 4420. IC50 values reported are quoted as the arithmetic mean for up
to 10 independent determinations.
21. Transepithelial permeability measured through Caco-2 cell monolayers. Value
shown is apical to basal apparent permeability.
structure that aided in the rational design of the compounds described
herein (PDB code 2DXS, Ref. 13c).; See also: Harper, S.; Avolio, S.; Pacini, B.; Di
Filippo, M.; Altamura, S.; Tomei, L.; Paonessa, G.; Di Marco, S.; Carfi, A.;
Giuliano, C.; Padron, J.; Bonelli, F.; Migliaccio, G.; De Francesco, R.; Laufer, R.;
Rowley, M.; Narjes, F. J. Med. Chem. 2005, 48, 4547.
22. Liver microsome stability in rat (RLM) and human (HLM) are expressed as the %
of compound metabolized after 15 min at a concentration of 5 lM.
18. Gharagozloo, P.; Miyauchi, M.; Birdsall, N. J. M. Tetrahedron 1996, 52, 10185.
19. Lohmann, V.; Körner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.; Bartenschlager,
R. Science 1999, 285, 110; with modifications described by Krieger, N.;
Lohmann, V.; Bartenschlager, R. J. Virol. 2001, 75, 4614. EC50 values reported
are quoted as the arithmetic mean for up to 10 independent determinations.
23. Harper, S.; Pacini, B.; Avolio, S.; Di Filippo, M.; Migliaccio, G.; Laufer, R.; De
Francesco, R.; Rowley, M.; Narjes, F. J. Med. Chem. 2005, 48, 1314.
24. Bartenschlager, R.; Lohmann, V. J. Gen. Virol. 2000, 81, 1631.
25. Incubation was performed in 100 mM KPi buffer (pH 7.4) with 5 mM MgCl2
and 1 mM EDTA. Test mixtures contained 1 mg/mL protein from HLM, 2 mM
NADPH and 2 mM GSH. Moreover, compounds were incubated with a mix of
20. (a) Activity (IC50) against the purified NS5bD21C enzyme as described in: WO
2009/080836, WO 2010/018233.; (b) Nyanguile, O.; Pauwels, F.; Van den
Broeck, W.; Boutton, C. W.; Quirynen, L.; Ivens, T.; van der Helm, L.;
Vandercruyssen, G.; Mostmans, W.; Delouvroy, F.; Dehertogh, P.; Cummings,
M. D.; Bonfanti, J.-F.; Simmen, K. A.; Raboisson, P. Antimicrob. Agents Chemother.
GSH/GSH stable-label (1 mM/1 mM) and NADPH. 500 nL from
solution of 25h was added to 250 L of incubation mixture to obtain a final
concentration of 40 M.
a 20 mM
l
l