Novel P2-P4 macrocyclic inhibitors of HCV NS3/4A protease by P3 succinamide fragment depeptidization strategy

Article abstract of DOI:10.1016/j.bmcl.2009.11.005

Hepatitis C represents a serious worldwide health-care problem. Recently, we have disclosed a novel class of P2-P4 macrocyclic inhibitors of NS3/4A protease containing a carbamate functionality as capping group at the P3 N-terminus. Herein we report our work aimed at further depeptidizing the P3 region by replacement of the urethane function with a succinamide motif. This peptidomimetic approach has led to the discovery of novel P2-P4 macrocyclic inhibitors of HCV NS3/4A protease with sub-nanomolar enzyme affinities. In addition to being potent inhibitors of HCV subgenomic replication, optimized analogues within this series have also presented attractive PK properties and showed promising liver levels in rat following oral administration.

Full text of DOI:10.1016/j.bmcl.2009.11.005

Bioorganic & Medicinal Chemistry Letters 20 (2010) 168–174
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel P2–P4 macrocyclic inhibitors of HCV NS3/4A protease by P3
succinamide fragment depeptidization strategy
b
a
c
a
Marco Pompei a, , Maria Emilia Di Francesco , Silvia Pesci , Uwe Koch , Sue Ellen Vignetti ,
*
Maria Veneziano a, Paola Pace a, Vincenzo Summa a
a IRBM, Rome, Italy
b Merck Research Laboratories, Boston, United States
c Lead Discovery Center, Dortmund, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatitis C represents a serious worldwide health-care problem. Recently, we have disclosed a novel
class of P2–P4 macrocyclic inhibitors of NS3/4A protease containing a carbamate functionality as capping
group at the P3 N-terminus. Herein we report our work aimed at further depeptidizing the P3 region by
replacement of the urethane function with a succinamide motif. This peptidomimetic approach has led to
the discovery of novel P2–P4 macrocyclic inhibitors of HCV NS3/4A protease with sub-nanomolar
enzyme affinities. In addition to being potent inhibitors of HCV subgenomic replication, optimized ana-
logues within this series have also presented attractive PK properties and showed promising liver levels
in rat following oral administration.
Received 10 October 2009
Revised 1 November 2009
Accepted 3 November 2009
Available online 10 November 2009
Keywords:
Hepatitis C
HCV
Ó 2009 Elsevier Ltd. All rights reserved.
NS3
Protease inhibitor
Bioisosters
Succinamide depeptidization
MK-7009
Hepatitis C virus (HCV) infects an estimated 300 million people
world-wide. This infection is the major causative agent of end-
stage liver disease and failure.1 Current therapy involves a combi-
and further reduce the peptide character of our proprietary
macrocycles.8
Working in that direction, we report herein the results of our
initial studies aimed at the evaluation of the succinamide fragment
as a novel replacement of the P3 capping group (Fig. 2). Amongst
peptidomimetic approaches, the succinic acid fragment depeptidi-
zation has been extensively reported in various unrelated exam-
ples of protease inhibitors as an effective tool to combine
potency with suitable PK properties.9,10 In the specific case of
NS3/4A inhibitors, these macrocyclic systems interact within the
substrate binding site of the protease through a combination of
hydrogen bonds and side-chain hydrophobic interactions. Both
crystal structures of natural cleavage products bound to the en-
zyme,11 and molecular modeling studies of the P2–P4 macrocyclic
inhibitors docked within the substrate binding site,5a suggest that
the NH and carbonyl groups of the P3 residue are engaged in
hydrogen bonds with the carbonyl and NH groups of the backbone
aminoacid Ala157. In light of these data, we decided to investigate
the tolerability in our class of inhibitors for the replacement of the
P3-carbamate capping group with the succinic acid fragment. De-
spite the elimination of the key P3–NH hydrogen bond to the
Ala157 residue, we were nevertheless intrigued by the possibility
of preserving the overall pattern of interactions of these P3 succi-
namide analogues within the protease. To support our hypotheses,
we specifically modeled carbamate 2 in the context of the full
nation of pegylated interferon agents (pegIFN)-
a with ribavirin, a
broad spectrum nucleoside analogue antiviral.2
Since NS3/4A has been identified to be essential in HCV replica-
tion,3 several research groups have actively worked toward the
identification of compounds targeted to inhibit this chymotryp-
sin-like serine protease. Both noncovalent inhibitors containing a
C-terminal carboxylic acid (e.g., BILN-2061) or an acid bioisoster
(e.g., ITMN-191 and TMC-435) and covalent reversible serine-traps
(e.g., VX-950 and SCH-503034) have reached advanced phase clin-
ical studies and validated HCV NS3/4A as target for anti-hepatitis C
therapeutics.4
Novel P2–P4 macrocyclic inhibitors of HCV protease have been
reported by our organization to be potent inhibitors of this en-
zyme5,6 (Fig. 1), and MK-7009, belonging to the same structural
class, is currently in clinical investigation.7
As part of a broader discovery program, we have investigated a
number of modifications of the capping group at the P3 N-terminus
in order to identify valuable replacements of the urethane function
* Corresponding author. Tel.: +39 328 4537 440.
E-mail address: marco.pompei@gmail.com (M. Pompei).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.005

M. Pompei et al. / Bioorg. Med. Chem. Lett. 20 (2010) 168–174
169
P2
N
Ph
N
P1P1'
O
O
O
O
O
O
O
O
O
S
Linker
S
N
HN
N
HN
N
N
H
H
N
H
N
O
O
H
O
O
O
O
O
O
P3
1
2
Figure 1. NS3/4A protease inhibitors.
These results encouraged the evaluation of a preliminary num-
ber of derivatives. Initially, the exploration was focused on ana-
logues bearing the isoindoline moiety in P2 and introducing a
limited number of structural variations. In particular, the succina-
mide fragments, 2-isopropyl and 2-cyclohexyl, were selected on
the basis of previously developed SAR, which had identified valine
and cyclohexylglycine (c-Hex) residues amongst the preferred
P3.12
N
O
O
O
O
O
S
N
HN
N
O
H
HN
O
Moreover, we intended to investigate if the effect of the substi-
tution at the succinamide nitrogen could be exploited to engage
the enzyme in further hydrophobic interactions with possibly po-
sitive effects on potency and on pharmacokinetic properties of
the corresponding analogues. The substitution on the N-amide
atom was initially evaluated by a direct comparison between the
N-iPr and N–H compounds. The choice of this small aliphatic sub-
stituent derived both from previously developed SAR on acyclic
systems13 as well as from studies on related P2–P4 macrocyclic
inhibitors.14
The synthetic approach toward the proposed targets relied on
ring closing metathesis (RCM) to perform the key macrocyclization
step and is illustrated in Scheme 1, starting with the preparation of
the N-iPr substituted analogues 14a–b and 15a–b.
The commercially available succinic acids 4a and 4b were first
converted into the corresponding acyl chlorides and then coupled
with the amine 5 in presence of triethylamine. Hydrolysis of the
methyl esters and coupling of the corresponding carboxylic acids
with the proline derivative 814 gave intermediates 9a and 9b; pre-
cursors to the key macrocyclization step.
O
3
Figure 2. Example of targeted P3-succinamide analogues: compound 3.
length NS3/4A protease and superimposed it to the succinamide
derivative 3 (Figs. 2 and 3).
Pleasingly, the modeling study suggested that the general con-
formation of the proposed succinamide macrocyclic inhibitor re-
mained mostly unchanged despite the replacement of the P3
aminoacid NH with a methylene group. The introduction of the
succinamide moiety was tolerated and allowing a global interac-
tion within the substrate-binding site of the protease comparable
to that predicted for the inhibitor 2.
The latter were then progressed to 10a and 10b via a microwave
assisted RCM reaction with Zhan-1 catalyst.15 In both cases only
the E-isomers were isolated after chromatography, and the trans
geometry was assessed measuring the vicinal coupling constant
of the two protons on the double bond (15–17 Hz). The synthesis
was then completed by hydrogenation, hydrolysis of the proline
methyl ester and final amide coupling with 1316 in presence of
TBTU to obtain the saturated compounds 14a–b after purification
by reverse-phase high performance liquid chromatography
(RP-HPLC). The corresponding unsaturated analogues 15a–b were
obtained in similar way from the E-macrocycles 12a–b.17
Turning next the attention toward the synthesis of the N-unsub-
stituted succinamide analogues, and according to the previous
synthetic approach, amine 1718 was at first coupled with the succi-
nic acid 4b via the corresponding acyl chloride. Surprisingly, a 3:1
inseparable mixture of the positional isomers 18 and 19 was
obtained in this case (Scheme 2).
Figure 3. Superposition of compound 2 (green) with the succinamide analogue 3
(cyan) into the substrate binding site of the genotype 1b NS3/4A crystal structure.
Both models were obtained via conformational analysis with Batchmin V9.4 using
the OPLS2005 force field. Inhibitors were minimized and docked into the active site
of the genotype 1b NS3/4A crystal structure. Potential hydrogen bonds are shown
by broken lines.
Reasoning the high reactivity of the intermediate acyl chloride,
with the plausible involvement of anhydride 16, could account for

170
M. Pompei et al. / Bioorg. Med. Chem. Lett. 20 (2010) 168–174
O
i, ii
CO2Me
HO
N
+
NH
OR
R2
O
R2
O
6a R2 = i-Pr, R=OMe Y=44%
4a R2 = i-Pr
5
6b R2 = c-Hex, R=OMe Y=41%
4b R2 = c-Hex
7a R2 = i-Pr, R=OH
7b R2 = c-Hex, R=OH
Y=91%
Y=80%
iii
N
8
N
O
iv
O
7a
7b
+
O
O
O
O
O
O
9a
9b
N
H
N
N
O
R2
O
N
N
O
O
O
O
O
O
vi
v
N
N
O
OH
N
N
O
O
R2
R2
O
O
10a R2 = i-Pr, Y=31%
11a R2 = i-Pr
Y=83%
10b R2 = c-Hex Y=44%
Saturated
E-olefin
11b R2 = c-Hex
Y=86%
(yields over 2 steps)
12a R2 = i-Pr
Y=91%
Y=48%
12b R2 = c-Hex
O
Cl
O
O
S
vii
11a-b
12a-b
NH
+
H3N
N
O
O
O
O
O
S
N
HN
13
N
O
H
N
O
R2
O
14a R2 = i-Pr
Y=49%
Saturated
E-olefin
14b R2 = c-Hex
Y=20%
15a R2 = i-Pr
Y=41%
Y=25%
15b R2 = c-Hex
Scheme 1. Synthesis of analogues 14a–b and 15a–b. Reagents and conditions: (i) oxalyl chloride, DMF (cat.), DCM, rt; (ii) TEA, DCM, rt; (iii) LiOH, dioxane/water 1:1, 50 °C;
(iv) BOP, N-methyl morpholine, DCM, rt; (v) Zhan-1 catalyst, DCM, W irradiation, 100 °C, 10 min; (vi) unsaturated analogs: LiOH, THF/dioxane 1:1, rt; saturated analogs:
Pd(C) 10%, H2, MeOH, then LiOH THF/dioxane 1:1, rt; (vii) TBTU, DIPEA, DCM, RP-HPLC purification.
l
the observed outcome, the initial exploration of alternative and
milder coupling conditions was set up with the aim of improving
the selectivity in favor of the required regioisomer 18. Unfortu-
nately, in all the cases evaluated (e.g., 2-bromo-1-ethylpyridinium
tetrafluoroborate, HOBT in presence of diethylamine19 or EDC,
HOBT and triethylamine20) the coupling reactions gave inseparable
mixtures of 18 and 19, with no practical improvements in the iso-
meric ratio. In order to circumvent these problems, a different dis-
connection approach was also screened and based on the amide
coupling step between 8 and (2S)-4-tert-butoxy-2-cyclohexyl-4-
oxobutanoic acid. Unfortunately, complex mixtures of positional
isomers were observed in this case too, independently of the cou-
pling reagent system employed (data not reported).
In contrast with these results, the secondary amine 5, as previ-
ously shown, discriminating between the two carbonyl groups of
anhydride 16, most probably on steric grounds, gave a straightfor-
ward and completely regioselective synthesis of the required suc-
cinamides 6a–b. In light of these observations, the solution to the

M. Pompei et al. / Bioorg. Med. Chem. Lett. 20 (2010) 168–174
171
O
O
O
HO
CO2Me
Cl
CO2Me
i
NH2
+
O
O
17
4b
16
O
H
O
H
N
ii
O
N
+
O
O
O
18
19
Scheme 2. Preparation intermediates 18 and 19. Reagents and conditions: (i) oxalyl chloride, DMF (cat.), DCM, rt; (ii) TEA, DCM, rt.
PMB
N
O
i
6a
ii
17
NH
+
O
6b
R2
Y=47%
O
21a
R
2 = i-Pr
20
21b R2 = c-Hex Y=72%
O
N
O
PMB
N
O
iii
iv, v
O
O
O
8
+
OH
R2
O
N
22a
R
2 = i-Pr
Y=90%
22b R2 = c-Hex Y=92%
O
N
PMB
R2
2 = i-Pr
23b R2 = c-Hex Y=65%
O
23a
R
Y=55%
N
N
O
O
vii
vi
O
O
O
O
O
13
+
N
N
OH
HN
HN
O
O
R2
R2
O
O
2 = i-Pr
Y=62%
24a
R
Y=71%
Y=80%
24b R2 = c-Hex Y=86%
25a
25b
R
R
2 = i-Pr
2
= c-Hex
N
O
O
O
viii
O
O
N
HN
S
N
H
O
HN
O
R2
O
R2 = i-Pr
Y=39%
Y=35%
3
26b R2 = c-Hex
Scheme 3. Synthesis analogues 3 and 26b. Reagents and conditions: (i) p-anisaldehyde, NaBH4, DCM, rt; (ii) HOBT, EDC, TEA, DCM, rt; (iii) LiOH, dioxane/water 1:1, 50 °C; (iv)
BOP, N-methyl morpholine, DCM, rt; (v) Zhan-1 catalyst, DCM, lW irradiation, 100 °C, 10 min; (vi) TFA 0.1 N, rt; (vii) LiOH, dioxane/water 1:1, rt, then Pd(C) 10%, H2, MeOH,
rt; (viii) TBTU, DIPEA, DCM, RP-HPLC purification.

172
M. Pompei et al. / Bioorg. Med. Chem. Lett. 20 (2010) 168–174
regioselectivity issue was finally identified in the installation of a
transient N-protecting group on the amine fragment, exploiting
the potential steric bias observed. The choice fell on the N-4-
methoxybenzyl group (PMB) in view of its facile cleavage in mild
acidic conditions.21 Indeed, by modifying the synthetic sequence
as depicted in Scheme 3, the required N-substituted succinamide
building blocks 22a–b were generated from amine 2022 in high
yields and with complete regioselectivity, as confirmed by NMR
structural elucidation.23
Amides 22a–b were then progressed to the macrocyclic succi-
namide derivatives 23a–b according to the previously described
synthetic route. Deprotection of the N-PMB group to give interme-
diates 24a–b was accomplished with TFA at room temperature. Fi-
nally, in a similar fashion to the procedure already described in
Scheme 1, the targeted compounds 3 and 26b were obtained in
overall acceptable yields after RP-HPLC purification.
distinct P2-heterocyclic cores such as the 7-methoxy-2-phenylquin-
oline, also previously employed by optimization studies within re-
lated series of macrocyclic inhibitors.5 The synthesis of analogues
32a and 32b are described in Scheme 4.
All the succinamide analogues prepared were tested against
full-length genotype 1b NS3/4A24 and in subgenomic replication
assay against genotype 1b in Huh-7 cells with a luciferase reporter
read-out.25 The corresponding data are showed in Table 1.
Replacement of the P3-carbamate with the succinamide back-
bone produced analogues with intrinsic potency in the low-nano-
molar range. In particular, this insertion proved partially
detrimental, as apparent from the direct comparison amongst 226
(Ki = 0.06 nM) and 3 (Ki = 1.4 nM). In the unsubstituted series the
introduction of the cyclohexyl-succinamide fragment as in 26b re-
sulted in a marginal improvement of the observed Ki value
(Ki = 0.76 nM) but with a rather significant boost in the replicon
potency (EC50 = 12 nM). The presence of the iPr group on the succi-
namide nitrogen, as in analogues 14a and 14b, although not asso-
ciated with a further boost in enzyme affinity (Ki = 0.72 nM and
Having identified an efficient synthetic route enabling access to
both the N-substituted and the N-unsubstituted succinamide
analogues, the investigation was extended to evaluate structurally
MeO
N
O
MeO
N
O
ii
i
7b
+
22b
CO2Me
CO2Me
N
R1
N
N
H
O
27
O
1
28a
28b
R =PMB Y=46%
1
R =i-Pr
Y=47%
MeO
N
O
MeO
N
O
iv
iii
CO2Me
CO2Me
N
N
R1
N
R1
N
O
O
O
O
1
1
29a
29b
30a
R =PMB Y=82%
R =H
Y=45%
1
R =i-Pr
Y=44%
MeO
N
MeO
N
O
v
13
+
O
O
CO2H
O
S
O
N
R1
N
N
O
HN
R1
N
N
O
H
O
O
O
32a R1=H
Y=17%
Y=20%
1
31a
31b
R =H
Y=62%
1
1
32b
R =-iPr
R =i-Pr Y=86%
Scheme 4. Synthesis of analogues 32a–b. Reagents and conditions: (i) BOP, N-methyl morpholine, DCM, rt; (ii) Zhan-1 catalyst, DCM,
lW irradiation, 100 °C 10 min; (iii) TFA
0.1 N, rt; (iv) Pd(C) 10%, H2, MeOH, then LiOH THF/dioxane 1:1, rt; (v) TBTU, DIPEA, DCM, RP-HPLC purification.

M. Pompei et al. / Bioorg. Med. Chem. Lett. 20 (2010) 168–174
173
Table 1
Table 2
HCV NS3 protease inhibition constants and EC50 values
Rat PK data (P.O., 4mpk) for selected compounds
P2
Compda
Cmax
(l
M)
Plasma AUC0–1
(
l
M h)
4 h liver concn (lM)
O
14a
14b
26b
32a
32b
0.08 0.01
0.09 0.02
0.05 0.02
BLQ
0.12 0.01
0.22 0.05b
0.09 0.05
—
2.2 0.1
P2
A
B
3
1
N
N
0.38 0.08
0.10 0.04
9.3 0.1
O
O
O
O
S
0.38 0.01
1.1 0.1
N
HN
a
N
H
Compounds dosed at 4 mg/kg P.O. in PEG 400; data are means of three animals.
0–4 h.
O
b
N
O
R1
O
R2
O
favorable profile. In particular, the plasma exposure was remark-
ably improved compared to all the other analogues in the series
Compd
P2
Linkera
R1
R2
Ki (nM)b 1b
EC50 (nM)c 10% FCS
26
2d,
A
A
A
A
A
A
A
B
B
db
—
H
H
iPr
0.06
1.4
0.76
0.72
0.61
16
14
0.56
0.60
12
100
12
35
11
284
276
7
(Cmax = 0.38
lM, AUC = 1.1
lM h), with a significant increase also
3
Satd
Satd
Satd
Satd
db
db
Satd
Satd
iPr
in liver levels (9.3 l
M). Despite the variability of the in vivo data
26b
14a
14b
15a
15b-
32a
32b
c-Hex
iPr
within this series, compound 32b showed a very promising profile
and the need for further investigation of closely related analogues.
In summary, this study has explored the possibility of replacing
the P3 carbamate with a succinamide motif in the context of P2–P4
macrocyclic inhibitors of HCV protease. The initial evaluation of
this depeptidization strategy, generated analogues such as 14b
and 26b with sub-nanomolar enzyme potencies and good cell
based activities (Ki = 0.61 nM and Ki = 0.76 nM, EC50 = 11 nM and
EC50 = 12 nM 10% FCS, respectively), suggesting that the succina-
mide motif might be considered a suitable peptidomimetic ap-
proach also in the case of P2–P4 inhibitors. These data confirmed
that the introduction of substitution on the succinamide nitrogen
did not prevent the interaction of this class of inhibitors within
the substrate binding site of the enzyme. Moreover, this substitu-
tion showed favorable effects on plasma exposure and liver levels
of the corresponding derivatives as consequence of a possible mod-
ulation of their physicochemical properties. Extension of the initial
investigation to other P2 heterocyclic moieties resulted in the
identification of the 7-methoxy-2-phenylquinoline analogue 32b,
a sub-nanomolar enzyme inhibitor characterized by encouraging
levels of replicon potency and remarkable liver levels when dosed
orally in rat, a key feature for any potential treatment for HCV
(Ki = 0.60 nM, EC50 = 37 nM; liver concentration at 4 h from oral
iPr
iPr
iPr
iPr
H
c-Hex
iPr
c-Hex
c-Hex
c-Hex
iPr
37
a
Satd = saturated analogs, db = E double bond.
Inhibition of the full-length HCV NS3/4A protease measured by the inhibition
constants (Ki values).
b
c
Inhibition of HCV replication in Huh-7-cells measured by 50% effective con-
centration (EC50) in the presence of 10% Fetal Calf Serum (FCS) or 50% Normal
Human Serum (NHS). Results are the mean of at least three independent experi-
ments. SD was 10% of the value.
Compound 2 is the carbamate-based inhibitor used as comparator within the
succinamide series.
d
0.61 nM, respectively), maintained the good levels of replicon po-
tency observed in 10% FCS (EC50 = 35 nM and 11 nM, respectively).
The features observed for these analogues seemed to be quite
severely affected by the introduction of an E-double bond within
the linker chain, as shown by derivatives 15a–b. These compounds
lost up to ca. 10-fold in enzyme potency and showed unfavorable
replicon profile in comparison with the saturated analogues
14a–b and 3–26b. These findings were rather unexpected, espe-
cially given that in related macrocyclic series the presence of an
E-double bond within the linker was generally well tolerated.5
The introduction of the 7-methoxy-2-phenylquinoline as P2
residue resulted in compounds 32a–b with enzyme affinity in
the sub-nanomolar range and equipotent to 14b and 26b
(Ki = 0.56 nM and Ki = 0.60 nM). In terms of replicon potency, the
methoxy-phenyl-quinoline core in P2 also proved to be well toler-
ated as shown by analogue 32a with comparable level of activity to
the reference compound 2 in the replicon assay (EC50 = 7 nM). In
view of these results, compounds 32b, 14a, 14b, 26b and 32a, were
selected to be further profiled in rat, with the initial aim to evalu-
ate plasma and liver levels following oral administration. The com-
pounds were dosed orally in Wistar rats at 4 mg/kg and the data
are reported in Table 2. Analogues 14a and 14b showed compara-
dosing at 4 mg/kg = 9.3 lM).
In view of these encouraging initial data, further preclinical
exploration is underway within this series and aimed to expand
the combinations of P2 heterocyclic cores with substituents on the
succinamide nitrogen optimized at the S4 pocket of the protease.
Acknowledgments
We thank Sergio Altamura, Jillian DiMuzio, Monica Bisbocci,
Ottavia Cecchetti and Nadia Gennari for biological testing. Edith
S. Monteagudo, Fabio Bonelli, Fabrizio Fiore and Anna Alfieri for
in vivo studies.
References and notes
ble plasma exposures (AUC = 0.12
respectively) in combination with encouraging levels of liver con-
centration at 4 h, in both cases in the micromolar range (2.2
and 3 M, respectively). However, the favorable profile observed
for these first two analogues was not paralleled by the N-unsubsti-
tuted compound 26b.
lM h and AUC = 0.22 lM h,
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lM
l
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2559.
nificant drop in liver levels (AUC = 0.09
0.38 M).
A similar trend was also observed for the quinoline derivatives
32a and 32b. While 32a, the N-unsubstituted analogue, had unde-
tectable plasma levels and minimal liver concentration, 32b, the
corresponding N-iPr substituted compound, showed a much more
lM h and [liver] =
l

174
M. Pompei et al. / Bioorg. Med. Chem. Lett. 20 (2010) 168–174
Representative analytical data for compound 15a: 1H NMR (600 MHz, DMSO-d6,
T = 300 K) 10.67 (s, 1H), 9.26 (s, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.26 (t,
5. (a) Liverton, N. J.; Holloway, K.; McCauley, J.; Butcher, J. 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.; 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.
d
J = 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 6.59 (d, J = 16.4 Hz, 1H), 6.25 (dt,
J1 = 16.4 Hz, J2 = J3 = 5.4 Hz, 1H), 5.64 (ddd, J = 17.2, 10.3, 8.8 Hz, 1H), 5.24–
5.19 (m, 2H), 5.07 (dd, J = 10.3, 1.8 Hz, 1H), 4.87 (d, J = 15.6 Hz, 1H), 4.71 (d,
J = 15.6 Hz, 1H), 4.65–4.60 (m, 2H), 4.20 (d, J = 11.9 Hz, 1H), 4.18 (dd, J = 11.2,
4.9 Hz, 1H), 4.11 (sept, J = 6.7 Hz, 1H), 3.76 (dd, J = 11.9, 3.4 Hz, 1H), 3.40 (td,
J1 = J2 = 12.6 Hz, J3 = 4.4 Hz, 1H), 2.91–2.80 (m, 3H), 2.77–2.73 (m, 1H), 2.46
(dd, J = 16.2, 2.3 Hz, 1H), 2.33–2.27 (m, 2H), 2.20–2.14 (m, 1H), 2.09–2.02 (m,
2H), 1.88–1.76 (m, 2H), 1.70 (dd, J = 8.2, 5.0 Hz, 1H), 1.55–1.48 (m, 1H), 1.32
(dd, J = 9.2, 5.0 Hz, 1H), 1.17 (d, J = 6.7 Hz, 3H), 1.11 (d, J = 6.7 Hz, 3H), 1.08–
0.99 (m, 4H), 0.97 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H). LRMS (ESI) m/z calcd
for C38H51N5O8S: 738.3 [M+1]+ found 738.2.
6. (a) Pompei, M.; Di Francesco, M. E.; Koch, U.; Liverton, N. J.; Summa, V. Bioorg.
Med. Chem. Lett. 2009, 19, 2574; (b) Avolio, S.; Robertson, K.; Hernando, J. M. I.;
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Dessole, G.; Nizi, E.; Pace, P.; Koch, U.; Fiore, F.; Pesci, S.; DiMuzio, J. M.;
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jm9005246.
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ed.; John Wiley & Sons, 2007.
22. Prepared with minor changes and according to the general procedure
described in: Dieltiens, N.; Stevens, C. V.; Masschelein, K.; Hennebel, G.; Van
der Jeught, S. Tetrahedron 2008, 64, 4295. 1H NMR (300 MHz, CDCl3, T = 300 K)
d 7.22 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 5.87–5.73 (m, 1H), 5.03 (br s,
1H), 4.97–4.92 (m, 1H), 3.78 (s, 3H), 3.59 (s, 2H), 2.62 (t, J = 6.9 Hz, 2H), 2.08 (q,
J = 7.2 Hz, 2H), 1.65–1.55 (m, 2H).
23. The structure of 22b (E:Z-isomers at the amide bond, relative ratio = 1:1.3) was
assessed by a 1H–13C HMBC experiment. The two carbonyl carbons of the
amide and the acid moieties (detected at 171.1 ppm and 175.3 ppm,
respectively) were unambiguously distinguished by the pattern of proton–
carbon connectivities observed for the C11–C9 fragment (H8 correlate to both
C9 and C11, while H14 and H20 only correlate to C9); the analysis of the
proton–carbon connectivities from the c-Hex moiety showed the key
correlation H1–C11 (175.3 ppm). The structure of 22a was assigned in
analogous manner.
10. Chatterjee, A. K.; Liu, H.; Tully, D. C.; Guo, J.; Epple, R.; Russo, R.; Williams, J.
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O
14. Holloway, M. K.; Liverton, N. J.; Ludmerer, S. W.; McCauley, J. A.; Olsen, D. B.;
Rudd, M. T.; Vacca, J. P.; McIntyre, C. J. U.S. Patent 027071, 2007.
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Patent 051477, 2008.
20
O
9
8
11
OH
N
16. Wang, X. A.; Sun, L.-Q.; Sit, S. Y.; Sin, Y.; Scola, P. M.; Hewawasam, P.; Good, A.
C.; Chen, Y.; Campbell, J. A. U.S. Patent 6995174, 2006.
14
H1
O
17. The structure of final compounds was assessed by 1H–1H ROESY experiment
(key ROE contacts: H31/H7,H70 and H6/H3).
24. Poliakov, A.; Hubatsch, I.; Shuman, C.; Stenberg, G. Protein Expr. Purif. 2002, 25,
363.
25. Lohmann, V.; Korner, F.; Herian, U. Science 1999, 285, 110.
26. Compound 2 has a Ki = 0.06 nM and EC50 (10% FCS) = 12 nM. The complete
characterization of 2 will be the subject of a separate publication by our
organization.
N
O
3
O
7
O
O
O
S
31
N
HN
N
H
O
N
O
6
R2
O