Inhibitors of hepatitis C virus NS3/4A: α-Ketoamide based macrocyclic inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.02.079

A novel series of hepatitis C virus (HCV) NS3/4A protease inhibitors bearing a P2-P4 macrocycle and a P1-P1′ α-ketoamide serine trap is reported. The NS3 protease, which is essential for viral replication, is considered one of the most attractive targets

Full text of DOI:10.1016/j.bmcl.2009.02.079

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2295–2298
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of hepatitis C virus NS3/4A:
macrocyclic inhibitors
a-Ketoamide based
a
a
b
a
Salvatore Avolio a, , Keith Robertson , Josè Ignacio Martin Hernando , Jillian DiMuzio , Vincenzo Summa
*
a IRBM—Merck Research Laboratories Rome, Via Pontina km 30,600, Pomezia 00040, Rome, Italy
b Merck Research Laboratories, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of hepatitis C virus (HCV) NS3/4A protease inhibitors bearing a P2-P4 macrocycle and a P1-
P10 a-ketoamide serine trap is reported. The NS3 protease, which is essential for viral replication, is con-
sidered one of the most attractive targets for developing novel anti-HCV therapies. The optimization of
both the macrocycle and the warhead portions led to the discovery of compounds 8b and 8g with a good
activity both in the enzyme as well as in the cell based (replicon) assays with favorable PK profile in a
preclinical species.
Received 22 January 2009
Revised 19 February 2009
Accepted 20 February 2009
Available online 25 February 2009
Keywords:
Hepatitis C
Ó 2009 Elsevier Ltd. All rights reserved.
NS3 serine protease
a-Ketoamide
Macrocycle
PK properties
Hepatitis C virus (HCV) is a major cause of acute hepatitis and
VX-950 (1: telaprevir, Fig. 1)7 and SCH 503034 (2: boceprevir)8
and (ii) the non-covalent reversible macrocyclic inhibitors
exemplified by the prototypical compound, now discontinued,
BILN-2061 (3: ciluprevir)9 and the more recent ITMN-191 (phase
Ib)10 and TMC435350 (phase IIa).11 Telaprevir (1) and boceprevir
(2) are the most advanced inhibitors, and both recently entered
phase III clinical trials.12
chronic liver disease, including cirrhosis and liver cancer. Globally,
an estimated 170 million persons, 3% of the world’s population, are
chronically infected with HCV and 3–4 million people are newly
infected each year.1 A significant fraction of these develop serious
liver disease including cirrhosis and hepatocellular carcinoma.2 No
vaccine is currently available to prevent hepatitis C, while the cur-
rent standard of care—pegylated interferon-
a
in combination with
A new series of structurally distinct NS3/4A inhibitors bearing a
P2-P4 macrocyclization was recently reported by our colleagues13
and, the most advanced compound is MK-7009 (4, Fig. 2) currently
in phase II with POC achieved.14
In this Letter, we report our efforts to evaluate the NS3/4A inhibi-
tion ability of a hybrid series of molecules designed by the
ribavirin—is poorly tolerated and not highly efficacious, particu-
larly in patients infected with HCV genotypes 1 and 4–6.3 Conse-
quently, there is an intense interest in the development of orally
active, small-molecule inhibitors of HCV replication that may offer
enhanced efficacy and less adverse effects.
Following the successful paradigm established for HIV, initial
efforts to develop HCV-directed antiviral agents have focused on
inhibition of essential virally-encoded enzymes.4 Among these,
the serine protease NS3 is one of the validated targets that plays
an essential role in the viral replication cycle.5 The HCV NS3 pro-
tein is noncovalently associated with the viral protein NS4A in a
heterodimeric complex (NS3/4A) responsible for the proteolytic
cleavage of the viral polyprotein into its components and it is,
therefore, essential in the process of HCV replication.6
N
O
H
N
N
O
O
N
N
H
H
N
H
N
S
N
O
O
NH
N
O
MeO
O
O
O
1
(VX-950)
O
N
H
N
H
CO2H
HN
NH2
O
N
O
Currently there are several NS3/4A protease inhibitors in clini-
cal trials, which can be divided into two main classes: (i) the cova-
N
H
N
H
N
O
O
O
O
lent reversible
a-ketoamide serine trap inhibitors, represented by
2
3
(SCH 503034)
(BILN 2061)
* Corresponding author. Tel. +39 06 91093360; fax: +39 06 91093654.
E-mail address: salvatore_avolio@merck.com (S. Avolio).
Figure 1. NS3/4A protease reference inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.079

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S. Avolio et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2295–2298
Table 2
P1-P10 optimisation
O
N
O
O
N
N
O
N
O
H
N
O
O
H
N
H
N
O
N
S
H
N
N
H
P'
N
N
H
O
O
O
H
NH
O
O
O
N
O
O
R1
N
O
O
H
O
O
N
O
O
NH
O
4
1
1b Replicon*
(MK-7009)
Compd
R1
P0
1b
O
Ki* (nM)
EC50 (nM)
*
N
H
N
O
O
H
N
8b
8d
8e
8f
57
140
78
380
940
O
N
H
O
O
N
O
n
O
1050
520
n = 2; 8b
Figure 2. Strategy leading to the discovery of 8b.
66
8g
8h
8i
71
550
combination of the P2-P4 macrocycle portion of MK-7009 (4, in blue
in Fig. 2) and the P1-P10 warhead of the VX-950 (1, in red in Fig. 2)
inhibitor.
242
140
1320
>500
An initial effort was aimed at determinining the best macrocy-
cle size and the optimal P3 residue. Driven by our recent results in
the field, attention was focused on 19- and 20-member macrocy-
les.13 As shown in Table 1, the coupling of a P2-P4 19-member
macrocycle moiety with the VX-950 (1) P1-P10 warhead resulted
in compound 8a that showed similar enzymatic potency
(Ki = 83 nM vs Ki = 110 nM for VX-950)15 and improved activity in
the cell based assay (EC50 = 600 nM vs EC50 = 1600 nM for VX-
950).16 An incremental lengthening of the linker afforded a further
enhancement of both the enzymatic and the cell based replicon
activity (8b; Ki = 57 nM; EC50 = 380 nM) compared to VX-950 (1).
Replacement of the t-Leu residue of 8b with one of the best known
P3 residues previously identified, the c-pentyl, gave 8c that
resulted in a reduction of both the enzymatic inhibition and the
8j
176
790
N
8k
8l
ꢀ2400
>12,500
>6250
F
>2900
F
*
See note a, b and c in Table 1.
cell based replicon activity (Table 1). Compound 8b was then
selected for the exploration of the P0 region.
The first moiety studied was the ring-size in the P0 ketoamide
contained in 8b. Towards this end, cyclobutyl and cyclopentyl were
incorporated (as P10) to yield 8d and 8e (Table 2). When compared
to 8b, both compounds showed a decreased activity in the enzy-
matic assay and in the replicon assay (8d: Ki = 140 nM,
EC50 = 940 nM; 8e: Ki = 78 nM, EC50 1050 nM). Moving from cyclic
to acyclic derivatives, the ( )-sec-butyl
a-ketoamide 8f retained
the enzymatic activity (Ki = 66 nM) but exhibited a 1.4-fold
reduced replicon activity when compared to 8b (EC50 = 520 nM
vs 380 nM for 8f and 8b, respectively). A similar shift in replicon
activity was obtained when the cyclopropyl moiety was replaced
with the allyl group yielding 8g. When compared with 8b, the
newly synthesized inhibitor exhibited similar enzyme activity
(Ki = 71 nM vs 57 nM for 8g and 8b, respectively) and a 1.4-fold
shift in the replicon assay (EC50 = 550 nM vs 380 nM for 8g and
8b, respectively). When benzyl 8h, ( )-1-phenylethyl 8i and 2-pyr-
idylmethyl 8j were introduced in place of the cyclopropyl amide
Table 1
Effect of the macrocycle size and P3 substitution on enzymatic and cellular potencies
O
N
H
N
O
O
H
N
O
N
H
O
O
N
O
n
O
R3
Compd
n
R3
1b
1b replicona
Kib (nM)
EC50 (nM)
c
moiety of 8b,
a reduced potency was observed, the three
VX-950 (1)
SCH 503034 (2)
—
—
—
—
110
35
1600d
200e
8a
8b
1
2
2
83
57
95
600
380
670
Table 3
Liver levels and pharmacokinetic profile of 8b and 8da
Compd
Cmax
M)
Plasma AUC
4 h liver
F
(%)
(
l
0–1 (
lM h)
Concnb
(
lM)
8c
1 (VX-950)c
0.63
0.13
0.02
0.16
1.54d
0.21c
0.08
0.81e
34.8
3.5
a
On the basis of the CC50 values determined for all the inhibitors listed in Tables
2 (SCH 503034)c
NAf
1 and 2 (mostly > 50–100 lM), none of these compounds was considered as
8b
8g
0.17
0.48
10
cytotoxic.
0.58
NDg
b
Inhibition of the full-length HCV NS3/4A protease measured by the inhibiton
constants (Ki values);15 Ki and EC50 values are the average of at least three inde-
pendent determinations, standard deviation 10%.
a
Compounds dosed at 5 mg/kg P.O. in PEG 400; data are geometric means from 3
animals, standard deviation 10%.
c
b
Inhibition of HCV replication in Huh-7 cell expressing a stably-replicating
It is assumed that density of liver tissue is 1 g/mL.
Data adapted from Ref. 19.
0–8 h.
Measured at 8 h; data adapted from Ref. 7.
NA = not available.
ND = not done.
c
subgenomic HCV RNA; compounds were incubated at 37 °C for 96 h in the presence
of 10% FCS.16,17
d
d
e
In house data; the literature data (from Ref. 7) is: EC50 = 354 nM 35 nM
f
obtained after 48 h incubation in the presence of 2% FBS.
e
g
Data from Ref. 8b, obtained after 72 h incubation in the presence of 5% FBS.

S. Avolio et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2295–2298
2297
OH
.HCl
H2N
OH
OH
O
OH
H
N
H
N
H
N
H
N
a or b
H
N
c
H
N
H
N
H
N
a or b or c
R2
Boc
R2
Boc
COOH
Macrocycle
Macrocycle
R1
O
R1
O
R1
O
O
5a-i
cpd# R1
R2
Yield (%)
Scheme 3. Alternative oxidations. Reagents and conditions: (a) TEMPO, BAIB, DCM,
rt to 40 °C;23 (b) MnO2, acetone, rt to reflux;24 (c) PS-2-iodylbenzamide, DCM, rt to
40 °C, 72 h.25
5a
5b
5c
5d
5e
5f
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
c
c
c
-Pr
72
70
72
63
80
60
-Bu
-Pent
( )-sec-Bu
allyl
HATU- or EDC-mediated amide coupling followed by the HCl-med-
iated removal of the Boc protection (Scheme 1).
CH2Ph
5g
5h
5i
( )-CH(CH3)Bn 75
CH2Pyr
59
77
The key macrocycles 6a–c (Scheme 2) were obtained in turn
starting from acyclic precursors using an high yielding ring closing
metathesis (RCM) performed according to the published patent lit-
erature.21 Subsequently, Pd on charcoal catalyzed hydrogenation of
the styryl olefin followed by the hydrolysis of the proline ester,
using standard basic conditions (LiOH/THF), provided the acids
(R/S)-diFAbu ( )-sec-Bu
Scheme 1. Synthesis of the key
a-hydroxyamides 5a–i. Reagents and conditions:
(a) HOBt, EDC, DIPEA, DMF, R–NH2; (b) HATU, DIPEA, DMF, R–NH2; (c) 4 M HCl,
dioxane.
compounds exhibited
enzymatic (2.5–4-fold) and cell based activity (up to fourfold)
when compared with the best compound of the series.
The SAR then moved on the critical moiety represented by the
P1 fragment. In particular the Norvaline (Nva) was replaced by
the 2-amino-4,4-difluorobutyric acid (difluoroAbu), an amino acid
that was designed and successfully implemented in our laboratory
as a unique replacement of the natural cysteine residue (as P1) in
a
substantial reduction in both the
7a–c that were coupled with the a-hydroxy-b-aminoamide P1-
P10 residues (5a–i) using a TBTU-mediated peptide coupling to lead
to diastereomeric mixtures of the corresponding macrocyclic
hydroxyamides. In a final step, oxidation of the latter with the
Dess–Martin periodinane22 afforded the desired
a-ketoamides
macrocycles 8a–l.
The moderate yields obtained in the oxidation step led us to con-
sider alternatives to the Dess–Martin protocol. A summary of differ-
ent approaches tried is reported in the Scheme 3. None of them
resulted in a clear improvement of the synthetic yield and all were
discarded in favour of the already in place Dess–Martin procedure.
The structures and purity of the final products 8a–l, obtained
after HPLC purification as powders via lyophilization, were con-
firmed on the basis of their proton NMR, HPLC–MS traces and
HRMS.26
In conclusion, we have prepared a new series of HCV NS3/4A
inhibitors designed as hybrids of the slowly reversible serine trap
P1-P10 a-ketoamide warhead contained in the VX-950 (1) with the
P2-P4 portion of our recently disclosed reversible macrocyclic inhib-
itor MK-7009 (4). Two compounds 8b, 8g showed improved in vitro
activity when compared to the clinical candidate VX-950 (1). In par-
ticular, compound 8b showed a >4-fold increase of the inhibition in
the cell based replicon assay. Compound 8b showed a comparable
level of inhibition also with the other clinical candidate SCH
503034 (2). Unfortunately 8b showed an inferior PK profile in rat
with respect to VX-950 (1) although it was comparable with SCH
503034 (2) and 8g showed similar exposure and liver level with
respect to both VX-950 and SCH 503034. Encouraged by the preli-
minary results a further evaluation of the series is ongoing.
the substrate-, product-like and
This replacement was applied to the
a
-ketoacid inhibitors of NS3/4A.18
-ketoamide series, and
a
resulted in compounds 8k and 8l (obtained as a mixture of diaste-
reoisomers) that showed a significant loss of both the enzymatic
activity (8k: Ki ꢀ 2400 nM; 8l: Ki > 2900 nM) and the cell based
activity (EC50 in the lM range).
Two different compounds (8b and 8g) with a good in vitro
potency profile, were selected for further evaluate of their proper-
ties in vivo. To this end, compounds 8b and 8g were dosed orally to
male Wistar rats at 5 mg/kg and results compared with reported
data for compounds 1 and 2 (Table 3).19 After oral administration,
both compounds 8b and 8g showed low plasma exposure (barely
detectable for 8b). The oral bioavailability F (%) of 8b was slightly
higher than the reported value for SCH 503034 (2) (F% = 10 vs 3.5
for 8b and 2, respectively) but significantly lower than the reported
value for VX-950 (1) (F% = 34.8). The plasma exposure and Cmax of
8g were favorable with respect to 2, and poorer in comparison to 1.
It is interesting to note that the liver level of 8g is not significantly
less than VX-950 (1) in spite of the fact that the AUC is three fold
lower than 1.
Representative synthetic routes employed for the preparation of
the key
protease inhibitors 8a–l are outlined in Schemes 1 and 2. The P1-
P10
-hydroxy-b-aminoamide residues 5a–i (Scheme 1) were
a-hydroxy-b-aminoamide intermediates 5a–i and the HCV
Acknowledgements
a
prepared starting from the
L
-norvaline or ( )-4,4-difluoro-2-ami-
The authors tkank Fabrizio Fiore and Edith S. Monteagudo for
the in vivo studies; Silvia Pesci for structure confirmation by
nobutyric acid (difluoroAbu)-based hydroxyl acids20 through an
cpd#
n
R3
R1
P'
Yield (%)
8a
8b
8c
8d
8e
8f
1
2
2
2
2
2
2
2
2
2
2
2
t
-Bu
(S)-Nva
(S)-Nva
c
c
c
c
c
-Pr
14
20
34
21
36
23
12
20
t
-Bu
-Pr
O
c
-Pent (S)-Nva
-Pr
O
O
O
O
N
O
N
O
N
O
t
t
t
t
t
t
t
t
t
-Bu
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
(S)-Nva
-Bu
-Pent
O
-Bu
-Bu
-Bu
-Bu
-Bu
-Bu
-Bu
-Bu
O
H
N
O R1
f, g
H
N
d, e
( )-sec-Bu
allyl
OH
O
P'
N
8g
8h
8i
N
H
N
H
H
O
O
O
N
O
n
CH2Ph
N
O
n
N
O
n
O
O
O
R3
( )-CH(CH3)Bn 18
R3
7a-c
R3
6a-c
8j
CH2Pyr
20
15
15
8a-l
8k
8l
(R/S)-diFAbu ( )-sec-Bu
(R/S)-diFAbu ( )-sec-Bu
Scheme 2. Synthesis of HCV protease inhibitors 8a–l. Reagents and conditions: (d) H2, 10% Pd/C, MeOH; 83% (7a), 88% (7b), 57% (7c); (e) LiOH, THF, EtOH, H2O; (f) P1
hydroxy-b-aminoamides 5a–i, TBTU, DIPEA, DMF or HATU, DIPEA, DMF; (g) Dess–Martin periodinane, Py, DCM.
a -

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S. Avolio et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2295–2298
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Tomassini, J. E.; Carroll, S. S.; Tomei, L.; Altamura, S.; Bhat, B.; Bartholomew, L.;
Bosserman, M. R.; Ceccacci, A.; Colwell, L. F.; Cortese, R.; De Francesco, R.;
Eldrup, A. B.; Getty, K. L.; Hou, X. S.; LaFemina, R. L.; Ludmerer, S. W.; MacCoss,
M.; McMasters, D. R.; Stahlhut, M. W.; Olsen, D. B.; Hazuda, D. J.; Flores, O. A. J.
Biol. Chem. 2003, 278, 49164.
18. (a) Narjes, F.; Koehler, K. F.; Koch, U.; Gerlach, B.; Colarusso, S.; Steinkühler, C.;
Brunetti, M.; Altamura, S.; De Francesco, R.; Matassa, V. G. Bioorg. Med. Chem.
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Y.; Chovan, L. E.; Guan, Z.; Hernandez, L.; Turner, T. M.; Dandliker, P. J.; Marsh,
K. C. Antiviral Chem. Chemother. 2007, 18, 163.
20. Chen, A.-H.; Lamar, J.; Yip, Y.; Victor, F.; Johnson, R. B.; Wang, Q. M.; Glass, J. I.;
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in combination with [bis(acetoxy)iodo]benzene (BAIB) as stoichiometric
oxidants, as reported in: De Mico, A.; Margarita, R.; Parlanti, L.; Vescovi, A.;
Piancatelli, G. J. Org. Chem. 1997, 62, 6974. afforded the oxidation of only one of
the two hydroxyamide diastereoisomers with moderate conversion.
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assays. We also thank Michael Rowley and Nigel J. Liverton for
helpful discussions.
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Samuelsson, B.; Simmen, K. Bioorg. Med. Chem. Lett. 2008, 18, 4853; (b) Manns,
M. P.; Reesink, H. W.; Moreno, C.; Berg, T.; Benhamou, Y.; Horsmans, Y. J.;
Dusheiko, G. M.; Flisiak, R.; Meyvisch, P.; Lenz, O.; Simmen, K.; Verloes, R.
Hepatology 2008, 48, 1023A.
24. The use of manganese dioxide as an heterogeneous oxidant resulted in no
reaction: Fatiadi, A. J. Synthesis 1976, 65.
25. The polymer-supported 2-iodylbenzamide (IBX) resulted in a sluggish reaction
with only moderate conversion: (a) Chunk, W.-J.; Kim, D.-K.; Lee, Y.-S. Tetrahedron
Lett. 2003, 44, 9251; (b) Chen, J. J.; Aduda, V. Synth. Commun. 2007, 37, 3493.
26. General procedure for the preparation of 8b. Step 1: a solution of 6b (2.44 g,
4.63 mmol; prepared as described in Ref. 21) in MeOH (100 mL) was
hydrogenated at 1 atm over 10% Pd/C (1.20 g) for 4 h at rt. The reaction
mixture was filtered, the filtrate concentrated and crude product purified on
silica gel column chromatography eluting with 10–90% ethyl acetate in
petroleum ether to afford the product as a solid (2.16 g, 88%). 1H NMR (CDCl3,
400 MHz) d 7.21 (t, J = 7.6 Hz, 1 H), 7.10 (d, J = 7.6 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1 H),
5.40 (s, 1H), 4.68–4.81 (m, 2H), 4.51–4.67 (m, 3H), 4.38 (d, J = 9.5 Hz, 1H), 4.20 (d,
J = 11.7 Hz, 1H), 3.85 (dd, J = 3.5, 11.7 Hz, 1H), 3.77 (s, 3H), 3.67–3.74 (m, 1H),
3.15 (s, 1H), 2.98 (s, 1H), 2.69 (dd, J = 7.6, 14.0 Hz, 1H), 2.50–2.61 (m, 1H), 2.39–
2.49 (m, 1H), 2.12–2.22 (m, 1H), 1.22–1.74 (m, 8H), 1.06 (s, 9H). LRMS (ESI) m/z
calcd for C28H39N3O7: 530.3 [M+H]+ found 530.1. Step 2: a solution of product
from the previous step (2.16 g, 4.07 mmol) in THF (46 mL), EtOH (23 mL) and
water (23 mL) was treated with LiOH (0.51 g, 12.22 mmol) for 30 min at 40 °C.
1 M HCl (5 mL) was added, the volatiles evaporated and the residue partitioned
between water and EtOAc (2 ꢁ 100 mL). The combined organic phases were
washed with brine, dried over anhydrous Na2SO4 and solvent evaporated in
vacuo to yield 2.08 g of a solid as crude 7b product which was used with no
further purification. LRMS (ESI) m/z calcd for C27H37N3O7: 516.3 [M+H]+ found
516.1. Step 3: the solid residue from the previous step (0.100 g, 0.194 mmol) was
taken up in DMF (1.0 mL) and treated with (3S)-1-(cyclopropylamino)-2-
hydroxy-1-oxohexan-3-aminium chloride (5a, 0.065 g, 0.291 mmol; prepared
as described in Ref. 20), DIPEA (0.16 mL, 9.116 mmol) and TBTU (0.093 g,
0.291 mmol). After 2 h stirring at rt, the reaction mixture was partitioned
between 1 N HCl and EtOAc (2 ꢁ 25 mL). The combined organic phases were
washed with 1 N HCl (2 ꢁ 10 mL), 1 M NaOH (1 ꢁ 10 mL) and brine, dried over
anhydrous Na2SO4 and solvent removed in vacuo to yield the product (0.125 g as
a mixture of diastereoisomers) as crude solid which was used with no further
purification. Step 4: a solution of the hydroxyamides from the previous step
(0.125 g, 0.183 mmol) in DCM (3.5 mL) was treated with pyridine (0.125 mL,
1.55 mmol) and 1,1,1-Triacetoxy-1,1- dihydro-1,2-benziodoxol-3(1H)-one
(DessꢂMartin periodinane, 0.155 g, 0.366 mmol) at rt. The reaction mixture
was stirred at room temperature for 30 min, treated with aq ss. Na2CO3 and
extracted with DCM (2 ꢁ 15 mL). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4 and solvent removed in vacuo to yield a
crude product that was purified by reverse phase HPLC (Waters XterraÒ PrepMS
12. (a) New Clinical Data Support Broad Profile for Telaprevir in Patients
with Genotype
2008, November 1st.; (b) Boceprevir Phase II Study Showed High Rate of
Sustained Response With 28- and 48- Week Regimens in Genotype
1 Hepatitis C Virus (HCV) Infection, Vertex Press release
1
Treatment-Na Hepatitis
November 1st.
C Patients, Schering-Plough Press release 2008,
13. (a) Liverton, N. J.; Holloway, M. K.; McCauley, J. A.; Rudd, M. T.; Butcher, J. W.;
Carroll, S. S.; DiMuzio, J.; Fandozzi, C.; Gilbert, K. F.; Mao, S. –S.; McIntyre, C. J.;
Nguyen, K. T.; Romano, J. J.; Stahlhut, M.; Wan, B. –L.; Olsen, D. B.; Vacca, J. P. J.
Am. Chem. Soc. 2008, 130, 4607; (b) McCauley, J. A.; Rudd, M. T.; Nguyen, K. T.;
McIntyre, C. J.; Romano, J. J.; Bush, K. J.; Varga, S. L.; Ross, C. W., III; Carroll, S. S.;
DiMuzio, J.; Stahlhut, M. W.; Olsen, D. B.; Lyle, T. A.; Vacca, J. P.; Liverton, N. J.
Angew. Chem., Int. Ed. 2008, 47, 9104.
14. McCauley, J. A.; Rudd, M. T.; McIntyre, C. J.; Nguyen, K. T.; Romano, J. J.;
Butcher, J. W.; Holloway, M. K.; Wan, B.-L.; Carroll, S. S.; DiMuzio, J. M.;
Graham, D. J.; Ludmerer, S. W.; Mao, S.-S.; Stahlhut, M.; Fandozzi, C.; Trainor,
N.; Olsen, D. B.; Vacca, J. P.; Liverton, N. J. Abstracts of Papers, 235th ACS
National Meeting, New Orleans, LA, United States, April 6–10, 2008; American
Chemical Society: Washington, DC, 2008; MEDI-018.
19x150 mm, 5 lm column; MeCN/ H2O, 40–90% gradient in 14 min with 0.1%
TFA) to give 8b (0.025 g, 20% yield over three steps) as a white solid, after freeze-
drying. 1H NMR (DMSO, 400 MHz) d 8.72 (d, J = 4.3 Hz, 1H), 8.28 (d, J = 6.7 Hz,
1H), 7.15–7.30 (m, 2H), 7.12 (d, J = 6.8 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 5.23 (s, 1H),
5.02 (br s, 1H), 4.45–4.75 (m, 5H), 4.30–4.42(m, 1H), 4.18 (d, J = 8.6 Hz, 1H), 4.02–
4.11 (m, 1H), 3.69–3.78 (m, 1H), 3.58–3.68 (m, 1H), 2.62–2.81 (m, 1H), 2.30–2.60
(m, 3H, below DMSO), 1.94–2.06 (m, 1H), 1.66–1.77 (m, 1H), 1.21–1.65 (m, 11H),
0.97 (s, 9H), 0.88 (t, J = 6.7 Hz, 3H), 0.64–0.71 (m, 2H), 0.56–0.63 (m, 2H). HRMS
(ESI) m/z calcd for C36H51N5O8: 682.3820 [M+H]+, found: 682.3810.
15. Mao, S. –S.; DiMuzio, J.; McHale, C.; Burlein, C.; Olsen, D.; Carroll, S. S. Anal.
Biochem. 2008, 373, 1.
16. Lohmann, V.; Körner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.; Bartenschlager,
R. Science 1999, 285, 110.