Novel potent macrocyclic inhibitors of the hepatitis C virus NS3 protease: Use of cyclopentane and cyclopentene P2-motifs

Article abstract of DOI:10.1016/j.bmc.2007.07.027

Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-acyl-(4R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of different ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields. It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and 16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV protease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a K_i value of 0.41 nM and an EC₅₀ value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors against the HCV protease.

Full text of DOI:10.1016/j.bmc.2007.07.027

Bioorganic & Medicinal Chemistry 15 (2007) 7184–7202
Novel potent macrocyclic inhibitors of the hepatitis C virus
NS3 protease: Use of cyclopentane and cyclopentene P2-motifs
a
a
a
a
˚
Marcus Ba¨ck, Per-Ola Johansson, Fredrik Wangsell, Fredrik Thorstensson,
Ingemar Kvarnstro¨m,a Susana Ayesa,b Horst Wa¨hling,b Mikael Pelcman,b
Katarina Jansson,b Stefan Lindstro¨m,b Hans Wallberg,b Bjo¨rn Classon,b
b
b
b
c
˚
Christina Rydergard, Lotta Vrang, Elizabeth Hamelink, Anders Hallberg,
a,b,
b,d,
*
˚
*
Asa Rosenquist
and Bertil Samuelsson
aDepartment of Chemistry, Linko¨ping University, S-581 83 Linko¨ping, Sweden
bMedivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden
cDepartment of Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, S-751 23 Uppsala, Sweden
dDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 8 February 2007; revised 2 July 2007; accepted 6 July 2007
Available online 22 August 2007
Abstract—Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either
a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-
acyl-(4R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of dif-
ferent ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target
molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields.
It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and
16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior
potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV pro-
tease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a
Ki value of 0.41 nM and an EC50 value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our
knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon
assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors
against the HCV protease.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
some cases hepatocellular carcinoma.3 HCV infection is
now the leading cause for liver transplantation in Wes-
tern countries,4 and with infected individuals harboring
the virus for a decade or more before symptoms emerge,
the population requiring medical treatment will increase
dramatically over the next 10–20 years.2
Hepatitis C virus (HCV) infection is predominantly a
chronic viral disease afflicting over 170 million individu-
als worldwide,1 representing a human epidemic nearly
five times more prevalent than infections caused by the
human immunodeficiency virus (HIV-1).2
At present there is no vaccine against HCV infection and
the current main therapy, PEGylated interferon-a in com-
bination with ribavirin,5 is associated with side effects and
is effective in less than 50% of patients infected by the pre-
dominant HCV genotype 1.6 Non-responders and relaps-
ers are currently left with few treatment options.
The disease is associated with slowly progressive liver in-
jury with the risk of patients developing cirrhosis and in
Keywords: HCV; NS3 protease; Macrocyclic inhibitors; Cyclopentane
and Cyclopentene derived P2 templates.
The virally encoded NS3/4A serine protease has
emerged as one of the most promising and intensively
studied HCV drug targets. The NS3 protease is respon-
*
Corresponding authors. Tel.: +46
8 54683100; fax: +46 8
54683199; e-mail addresses: asa.rosenquist@medivir.se; bertil.
samuelsson@medivir.se
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.027

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7185
sible for cleavages of the downstream region of the poly-
protein; thereby making it essential for viral replica-
tion.7 The development of drug-like inhibitors has
however proven to be a challenging task. Recently,
previr; Boehringer Ingelheim)9,10,14–17 (Fig. 1), a mac-
rocyclic mimic of the peptide product inhibitor.
BILN 2061 was the first HCV protease inhibitor to enter
clinical trials, and early results were highly promising
showing a rapid and significant decline in plasma
HCV RNA virus load in all treated patients infected
with HCV genotype 1.15 Subsequent clinical trials were
halted apparently due to cardiac toxicity observed at
high doses in rhesus monkeys.18
`
highly promising inhibitors were reported by Llinas-
Brunet and co-workers8–11 exemplified by the tetrapep-
tide inhibitor A11 (Fig. 1), displaying promising low
nanomolar activity against the NS3/4A protease.
This inhibitor incorporates a P2 N-acyl-(4R)-alkoxypro-
line moiety, a motif subsequently adopted in many re-
ported and potent inhibitors.12,13 Further refinement14
of inhibitor A, and analogs thereof, mainly through
optimization of the P2 quinoline substituent, and mac-
rocyclization led to the discovery of BILN 2061 (Cilu-
Currently there are two product-derived linear pepti-
domimetics, VX-95019–22 and SCH 503034,23–25 re-
ported to be in phase II clinical trials. Previous
work26 from our laboratory on linear HCV NS3/4A
HN
N
S
MeO
N
O
MeO
O
O
O
O
H
H
N
N
O
H
N
N
O
N
N
OH
OH
H
O
O
O
N
H
O
O
A
BILN 2061
IC50 = 1 nM
Ki = 0.30 nM (HCV 1a)
Ki = 0.66 nM (HCV 1b)
EC50 = 4 nM (HCV 1a)
EC50 = 3 nM (HCV 1b)
N
N
O
O
O
O
NH
NH
NH
NH
O
O
O
O
N
NH2
HN
NH
NH
O
O
N
O
VX-950
SCH 503034
Ki = 14 nM
EC50 = 200 nM
Ki = 47 nM (HCV 1a)
Ki = 100 nM (HCV 1b)
EC50 = 400 nM
MeO
N
O
O
O
H
H
N
N
H
OH
N
N
O
O
H
O
B
Ki = 22 nM
EC50 > 10000 nM
Figure 1. Two potent product-based HCV NS3 protease inhibitors incorporating a 4-hydroxyproline moiety and a C-terminal carboxylic acid (A and
BILN 2061), and two promising linear peptidomimetics comprising the electrophilic a-ketoamide motif (VX-950 and SCH 503034). B is a previously
synthesized and promising compound in our series of cyclopentane-based inhibitors.

7186
M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
protease inhibitors has highlighted that a trisubstituted
cyclopentane dicarboxylic acid could be a novel P2
mimic of the frequently used N-acyl-(4R)-hydroxypro-
line, exemplified by inhibitor B (Fig. 1). Encouraged
by the observation that the sp2-hybridized proline
nitrogen could be replaced by an sp3-hybridized car-
bon in the cyclopentane proline mimic series, we
now report on the further optimization of this series
with particular emphasis on macrocyclization studies
and P4 truncations, aiming to improve both potency
and cell based activity.
available 5-bromo-1-pentene (1a) and 6-bromo-1-hex-
ene (1b) with tert-butyl carbazate at 100 ꢁC in DMF
afforded the alkylated products 2a and 2b in 72%
and 75% yields, respectively.28 In the second approach
a two-step reductive amination protocol was em-
ployed. Commercial 6-heptenol (3a) and 7-octenol
(3b) were oxidized, using a catalytic amount of tetra-
˚
propylammonium perruthenate (TPAP) and 4 A
molecular sieves in dichloromethane (DCM) with N-
methylmorpholine N-oxide as reoxidizing agent.29
The resulting volatile aldehydes 4a and 4b, obtained
in 74% and 98% yields, respectively, were treated with
In another report27 we have shown that a trisubstituted
cyclopentene dicarboxylic acid, in which the 1-position
of the cyclopentene adopts a planar configuration,
may also be effective as a P2 N-acyl-(4R)-hydroxypro-
line mimic. Consequently we also report on the effect
of macrocyclization and P4 truncation in this series.
tert-butyl carbazate in MeOH containing 3 A molecu-
˚
lar sieves furnishing the hydrazones, which were sub-
sequently reduced to their corresponding hydrazine
cyanoborane adducts using sodium cyanoborohydride
in acetic acid/THF 1:1. Final hydrolysis of the borane
adducts with sodium hydroxide in MeOH provided
hydrazines 5a and 5b in 45% and 38% total yields,
respectively, over the three steps.30
The SAR from HCV NS3 protease inhibitors contain-
ing a P1 carboxylic acid with either a P3 hydrazine- or
P4 NH-Boc-functionalized macrocyclic moiety clearly
indicated a preference for 14-membered rings, exempli-
fied by the P2 cyclopentane inhibitors 14b (Scheme 2
and Table 1) and 16b (Scheme 2 and Table 1), dis-
playing Ki values of 31 nM and 6 nM, respectively,
and the P2 cyclopentene inhibitor 24a (Scheme 3
and Table 3) displaying a Ki value of 15 nM. Inhibi-
tors 14b and 16b were subsequently converted into
the corresponding acyl sulfonamides 17 (Scheme 2
and Table 2) and 18 (Fig. 2, Scheme 2, Table 2),
exhibiting Ki values of 0.07 nM and 0.19 nM, and
EC50 values, replicon assay genotype 1b, of 530 nM
and 33 nM, respectively. After having determined the
14-membered ring size as the optimal we proceeded
to investigate the inhibitory effects of compounds lack-
ing a P4 substituent or just containing small P3 cap-
ping groups. Among these inhibitors 14e (–H) and
14f (–Me) (Table 1) delivered Ki values of 260 nM
and 44 nM, respectively. Converting inhibitors 14e
and 14f into their corresponding P1 acyl sulfonamides
19 (Scheme 2 and Table 2) and 20 (Fig. 2, Scheme 2
and Table 2) furnished Ki values of promising 2.2 and
0.41 nM. More impressive, inhibitor 20 delivered an
EC50 replicon value of 9.1 nM. To the best of our
knowledge this is the first reported example of a
HCV protease inhibitor lacking a P4 substituent,
and still maintaining a high potency in the replicon
assay. Moreover inhibitor 20 exhibits good selectivity
against the human serine proteases cathepsin B, chy-
motrypsin, and elastase.
The direct alkylation protocol using a large excess of
tert-butyl carbazate was found to work surprisingly well
in our hands delivering the monoalkylated hydrazine
building blocks 2a and 2b in good yields.
Treating commercial 5-hexenol (6) with pyridine and
methanesulfonyl chloride in DCM gave mesylate 7
(91%), which was subjected to two different reaction
conditions. Stirring 7 in a solution of aqueous ammonia
and MeOH gave amine 8 in 96% yield, whereas adding
the mesylate to a solution of N-Boc-methylamine (9)
and sodium hydride in DMF gave compound 10 in
69% yield (Scheme 1).
The synthesis of the macrocyclic inhibitors containing
the P2 cyclopentane moiety is depicted in Scheme 2,
where the synthesis of building block 11 has previously
been reported.26,31
Hydrolysis of the tert-butyl ester in 11 using TFA and
triethylsilane in DCM followed by subsequent coupling
to amines 2a, 2b, 5a, 5b, 8 or deprotected 10 (subjected
to HCl in dioxane prior to coupling) employing O-(7-
azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HATU) and N,N-diisopropyl-
ethylamine (DIPEA) in DMF furnished dienes 12a–f
in yields ranging from 64 to 85%. Dienes 12a–f were
then subjected to ring-closing olefin metathesis, using
2nd generation Hoveyda-Grubbs ruthenium catalyst in
refluxing DCM,9 providing compounds 13a–f in 10–
79% yield. The yields of compounds 13a–f depended
on the ring size of the products, with the 13-membered
ring (13a) being the most difficult to form.
2. Results and discussion
2.1. Chemistry
Ethyl esters 13a–f were subsequently hydrolyzed with
lithium hydroxide in refluxing THF/MeOH/H2O 2:1:1
providing target compounds 14a–f (Table 1) in 32–
100% yields. Treating compounds 13b–d with TFA
and triethylsilane in DCM afforded hydrazines 15b–d
in yields ranging from 63 to 74%. Hydrolysis of the ethyl
esters, vide supra, produced inhibitors 16b–d (Table 1) in
46–71% yields (Scheme 2).
Scheme 1 depicts the synthesis of six P3 olefin building
blocks employed in the coupling steps generating 12a–f
(Scheme 2) and 22a–b (Scheme 3).
Two procedures were used in order to obtain the
hydrazine building blocks. Heating commercially

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7187
Table 1. Effect of ring size and P3 capping groups on inhibition of NS3/4A and EC50 in the replicon assay for macrocyclic P2 cyclopentane P1
carboxylic acid inhibitors
MeO
N
R1:
Compound
Structure
R2 P3 capping group
Ring size
Ki (nM) HCV NS3 1a
EC50 (lM) HCV NS3 1b
R1
O
14a
Boc-NH
13
14
15
16
14
15
16
14
130
>10
O
O
O
O
O
O
O
O
H
N
N
N
N
N
N
N
N
N
R2
R2
R2
R2
R2
R2
R2
R2
OH
OH
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
(0)
R1
O
14b
14c
14d
16b
16c
16d
14e
Boc-NH
Boc-NH
Boc-NH
H2N
31
>10
>10
>10
7.6
H
N
O
(1)
R1
O
710
H
N
O
(2)
R1
O
>10,000
H
N
O
(3)
R1
O
6
H
N
O
(1)
R1
O
H2N
120
>10
H
N
O
(2)
R1
O
H2N
>10,000
>10
H
N
O
(3)
R1
O
H
260
>10
H
N
O
(continued on next page)
(1)

7188
M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
Table 1 (continued)
Compound
Structure
R2 P3 capping group
Ring size
Ki (nM) HCV NS3 1a
EC50 (lM) HCV NS3 1b
R1
O
14f
Me
14
44
2.2
O
H
N
N
R2
OH
O
O
(1)
Table 2. Effect of ring size and P3 capping groups on inhibition of NS3/4A and EC50 in the replicon assay for macrocyclic P2 cyclopentane P1
cyclopropyl acyl sulfonamide inhibitors
MeO
N
R1:
Compound
Structure
R2 P3 capping group
Ring size
Ki (nM) HCV NS3 1a
EC50 (lM) HCV NS3 1b
R1
O
17
Boc-NH
14
0.07
0.53
O
O
O
O
H
N
O
S
N
N
N
N
O
O
O
O
R2
R2
R2
R2
N
H
O
O
O
O
O
(1)
R1
O
18
H2N
14
14
14
0.19
0.033
H
N
O
S
N
H
O
(1)
R1
O
19
H
2.2
4.4
H
N
O
S
N
H
O
(1)
R1
O
20a
Me
0.41
0.0091
H
N
O
S
N
H
O
(1)
a Selectivity data: Ki = 2200, >5000, and >5000 nM for the human serine proteases cathepsin B, chymotrypsin, and elastase, respectively.
For the introduction of the cyclopropylsulfonamide car-
boxylic acid moiety, compounds 14b, 14e, and 14f were
preactivated with N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide hydrochloride (EDC) in DCM. Subse-
quent reaction of the preactivated compounds with
cyclopropanesulfonic acid amide and DBU in DCM
gave target compounds 17, 19, and 20 in 23–80% yields.
Boc-removal of 17, vide supra, furnished target com-
pound 18 in 95% yield (Scheme 2 and Table 2).
81% and 79% yields, respectively. Ring-closing metathe-
sis using 2nd generation Hoveyda-Grubbs catalyst pro-
duced macrocycles 23a and 23b in 81% and 53% yields,
respectively. Final hydrolysis of both the tert-butyl ester
and the Boc group in 23a and 23b with TFA and trieth-
ylsilane in DCM generated inhibitors 24a and 24b
(Scheme 3 and Table 3) (diastereomeric mixtures) in
38% and 47% yields, respectively.
2.1.1. Biological data and structure–activity relationships.
All target compounds, summarized in Tables 1–3, were
screened against the HCV NS3 1a protease to determine
Ki values and in the cellular subgenomic genotype 1b rep-
licon assay to determine EC50 values. All the inhibitors
synthesized have Z configured double bonds, as deter-
For the synthesis of the inhibitors incorporating the P2
cyclopentene moiety the diastereomeric building block 21
(Scheme 3), synthesized as previously reported,27,31–33 was
employed. Coupling of carboxylic acid 21 to amines 2b
and 5a, vide supra, furnished dienes 22a and 22b in

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7189
mined by NMR-analysis, and they contain the P2 2-phe-
nyl-7-methoxy-4-quinoline substituent, a motif fre-
quently employed11,14,16,26,27,34 as P2 extension in potent
HCV NS3 protease inhibitors, for example, compounds
A11 and B26 (Fig. 1). Besides being crucial in shielding
the catalytic machinery from exposure to solvent,35,36
the P2 aryl substituent likely interacts favorably with
the helicase domain of the NS3 protease.37–39 The benefi-
cial effects from macrocyclization are likely resulting from
favorable conformational restrictions of these inhibitors
providing a good overall fit into the active site.
MeO
N
O
MeO
N
O
O
O
H
H
O
O
O
O
N
N
N
N
S
H2N
S
N
N
O
O
O
O
H
H
18
Ki = 0.19 nM
EC50 = 33 nM
20
Ki = 0.41 nM
EC50 = 9.1 nM
From examination of the P2 cyclopentane-derived
inhibitors and BILN 2061 it is evident that the former
inhibitors differ from BILN 2061 by, for example, hav-
ing an sp3-hybridized carbon at the 1-position in the
P2 cyclopentane, whereas the corresponding position
in the proline of BILN 2061 is an sp2-hybridized nitro-
gen. Moreover, the cyclopentane-derived inhibitors have
a reversed direction of the amino acid chain extending
from the P2 group. In view of these substantial differ-
ences it was not evident whether potent macrocyclic
inhibitors could be obtained at all from this investiga-
tory series and furthermore which macrocyclic ring size
would be favored. To investigate this we decided to pre-
pare and evaluate each of the 13–16 membered rings.
Figure 2. Two 14-membered ring acyl sulfonamide inhibitors from the
new series of macrocyclic cyclopentane-based inhibitors; P3 hydrazine
compound 18 and P3 N-methyl-capped compound 20 displaying Ki
values of 0.19 nM and 0.41 nM and EC50 replicon 1b values of 33 nM
and 9.1 nM, respectively.
O
H
N
(n)
Br
(n)
a
O
N
H
2a, n = 1 (72%)
2b, n = 2 (75%)
1a, n = 1
1b, n = 2
The 13-membered macrocycle 14a (Table 1) exhibits a
moderate Ki value of 130 nM whereas compound 14b,
containing a 14-membered ring, is over four times more
potent than the corresponding 13-membered ring exhib-
iting a promising Ki value of 31 nM. The 15- and 16-
membered macrocyclic compounds 14c and 14d exhibit
Ki values of 710 nM and >10 lM, respectively, indicat-
ing that the 14-membered rings possess the overall best
fit into the S1–S3 pocket for these P2 cyclopentane-de-
rived inhibitors.
HO
(n)
(n)
b
O
4a, n = 1 (74%)
4b, n = 2 (98%)
3a, n = 1
3b, n = 2
O
c, d, e
H
N
O
N
(n)
H
5a, n = 1 (45%)
5b, n = 2 (38%)
The same trend held true for the P4 truncated series
where the P4 Boc groups of inhibitors 14b–d were re-
placed with the small but polar P3 amino extension in
inhibitors 16b–d. The 14-, 15-, and 16-membered macro-
cyclic inhibitors 16b–d deliver Ki values of 6 nM,
120 nM, and >10 lM, respectively. Furthermore, the
P3 amino 14-membered macrocyclic inhibitor 16b is five
times more potent than the corresponding P4 Boc-ami-
no 14-membered macrocyclic inhibitor 14b, displaying
Ki values of 6 nM and 31 nM, respectively. Whilst nei-
ther of inhibitors 14b and 16b is highly potent it points
to the possibility that potent inhibitors can be delivered
from compounds that do not interact with the S4 sub
pocket of the NS3 protease. Thus to further explore
small P3 substituents in the 14-membered macrocyclic
series, inhibitors 14e, lacking a P4 group, and 14f, hav-
ing a P3 methyl cap, were prepared delivering Ki values
of 260 nM and 44 nM, respectively (Table 1).
g
H2N
8 (96%)
O
S
f
HO
O
O
7 (91%)
6
O
H
O
N
h
O
N
O
10 (69%)
9
In spite of having promising potencies in the enzyme as-
say these P1 carboxylic acid inhibitors give rise to poor
activities in the relevant cell-based HCV replicon assay.
It has previously been reported that extensions of the P1
carboxylic acid, furnishing the corresponding acyl sul-
fonamides, sometimes result in improved activities in
Scheme 1. Reagents and conditions: (a) tert-butyl carbazate, DMF,
100 ꢁC; (b) N-methylmorpholine N-oxide, TPAP, molecular sieves
(4 A), CH2Cl2; (c) tert-butyl carbazate, molecular sieves (3 A), MeOH;
(d) NaBH3CN, AcOH/THF, 1:1; (e) NaOH (2M), MeOH; (f) MsCl,
pyridine, DCM; (g) NH3 (aq), MeOH; (h) NaH, DMF.
˚
˚

7190
M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
MeO
MeO
N
O
N
O
a, b
O
O
H
N
H
N
O
N
R
O
O
O
O
O
O
O
O
(n)
11
12a, n = 0, R = Boc-NH (85%)
12b, n = 1, R = Boc-NH (82%)
12c, n = 2, R = Boc-NH (82%)
12d, n = 3, R = Boc-NH (64%)
12e, n = 1, R = H (70%)
c
12f, n = 1, R = Me (82%)
MeO
MeO
N
O
N
O
MeO
N
O
a
d
O
O
O
H
N
H
N
H
N
N
N
N
R
R
R
OH
O
O
O
O
O
O
O
(n)
(n)
(n)
13a, n = 0, R = Boc-NH (10%)
13b, n = 1, R = Boc-NH (51%)
13c, n = 2, R = Boc-NH (70%)
13d, n = 3, R = Boc-NH (79%)
13e, n = 1, R = H (23%)
15b, n = 1, R = H2N (63%)
15c, n = 2, R = H2N (66%)
15d, n = 3, R = H2N (74%)
14a, n = 0, R = Boc-NH (32%)
14b, n = 1, R = Boc-NH (99%)
14c, n = 2, R = Boc-NH (46%)
14d, n = 3, R = Boc-NH (100%)
14e, n = 1, R = H (68%)
d
e, f
13f, n = 1, R = Me (37%)
14f, n = 1, R = Me (53%)
MeO
N
O
MeO
MeO
N
O
N
O
a
O
H
O
H
O
H
O
O
O
O
N
N
N
N
N
N
S
R
R
S
R
N
OH
N
O
O
O
O
O
H
H
(n)
(n)
(n)
16b, n = 1, R = H2N (52%)
16c, n = 2, R = H2N (46%)
16d, n = 3, R = H2N (71%)
17, n = 1, R = Boc-NH (80%)
19, n = 1, R = H (23%)
20, n = 1, R = Me (29%)
18, n = 1, R = H2N (95%)
Scheme 2. Reagents and conditions: (a) TFA, Et3SiH, CH2Cl2; (b) 2a or 2b or 5a or 5b or 8 or 10 (subjected to HCl in dioxane prior to coupling),
HATU, DIPEA, DMF; (c) Hoveyda-Grubbs Catalyst 2nd Gen. CH2Cl2, reflux; (d) LiOH (1M), THF/MeOH/H2O 2:1:1, reflux; (e) EDC, DCM;
(f) cyclopropanesulfonic acid amide, DBU, DCM.
both enzyme and cell-based assays of HCV NS3
inhibitors.12,34,40,41
tion at the 1-position. Nonetheless, the 14-membered
macrocyclic inhibitor 24a is more potent than the 15-
membered macrocycle 24b following the same trend as
for the P2 cyclopentane inhibitor series. However, inhib-
itor 24a is somewhat less potent than 16b. The inconve-
nient synthesis route for the P2 cyclopentene inhibitors
which furnishes diastereomeric mixtures makes them
less attractive than the corresponding P2 cylopentane
derivatives. Moreover, the potential of irreversible Mi-
chael acceptor properties with increased risk for adverse
events in vivo renders this inhibitor class less attractive
for further development.27
By introducing a cyclopropyl acyl sulfonamide into the
14-membered macrocyclic inhibitors 14b and 16b (Table
2) a substantial improvement in Ki values to 0.07 nM
and 0.19 nM, respectively, was obtained for inhibitors
17 and 18. In addition, moderate to highly potent activ-
ities in the subgenomic replicon assay can be seen, with
EC50 values of 530 nM for 17 and promising 33 nM for
18. For inhibitor 19, lacking a P4 group, and the P3-
methyl-capped inhibitor 20 the corresponding Ki values
were 2.2 nM and 0.41 nM, respectively. In the subge-
nomic cell-based replicon assay an EC50 value of
4.4 lM was obtained for inhibitor 19 and an impressive
EC50 replicon value of 9.1 nM for inhibitor 20 (Table 2).
In contrast, the P2 cyclopentane inhibitor class shows
great promise for further refinements.
Modeling was used to rationalize the SAR observed for
the various P4 substituents and P3 extensions. Confor-
mational analysis indicates that the moderate potency
The diastereomeric P2 cylopentene derivatives 24a and
24b share with the P2 proline the same planar configura-

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7191
MeO
N
O
MeO
N
O
a
O
O
H
H
N
N
HO
N
R
O
O
O
O
O
O
(n)
21 (anti)
22a, n = 1, R = Boc-NH (81%) (anti)
22b, n = 2, R = Boc-NH (79 %) (anti)
b
MeO
N
O
MeO
N
O
c
O
O
O
H
H
N
N
N
N
R
R
OH
O
O
O
O
(n)
(n)
24a, n = 1, R = NH2 (38 %) (anti)
24b, n = 2, R = NH2 (47 %) (anti)
23a, n = 1, R = Boc-NH (81 %) (anti)
23b, n = 2, R = Boc-NH (53 %) (anti)
Scheme 3. Reagents and conditions: (a) 2b or 5a, DIPEA, HATU, DMF; (b) Hoveyda-Grubbs Catalyst 2nd Gen., CH2Cl2, reflux; (c) TFA, Et3SiH,
CH2Cl2.
Table 3. Effect of ring size on inhibition of NS3/4A and EC50 in the replicon assay for macrocyclic P2 cyclopentene P1 carboxylic acid inhibitors
MeO
N
R1:
Compound
Structure
R2 P3 capping group
Ring size
Ki (nM) HCV NS3 1a
EC50 (lM) HCV NS3 1b
R1
O
O
H
N
24a
H2N
14
15
5.4
N
R2
OH
O
O
(1)
(anti)
R1
O
O
H
N
24b
H2N
15
110
4.5
N
R2
OH
O
O
(2)
(anti)
seen with the P3-H in both series (14e and 19) may result
from an internal hydrogen bond, between the P3-H and
the P2 carbonyl, forcing the macrocycle to a conforma-
tional change upon binding to the active site. Conforma-
tional studies were also used to explain how inhibitor
16b having the small P3-NH2 extension is five times
more potent than inhibitor 14b having the larger P4-
NHBoc. Analysis of these inhibitors indicates that the
conformations of the reversed amides may differ. Thus
inhibitor 16b could be docked into the active site in
any of the two rotations around the reversed amide
bond while inhibitor 14b fits only in the higher energy
rotamer state.
Using a previously published crystal structure of the full
length NS339 compound 20 was docked and minimized
in the active site of NS3 (Fig. 3). The model of com-
pound 20 shows interactions with both the protease
and the helicase parts of NS3. The protease interactions
are the most important and the inhibitor forms hydro-
gen bonds to five residues in the active site of the
protease.

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M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
Figure 3. Model of compound 20 in the active site of the NS3 protease. The NS3 crystal structure 1cu1 from PDB was used for modeling and Arg 155
was rotated to accommodate the P2 quinoline substituent.
The cyclopropyl acyl sulfonamide has been shown to add
potency to the inhibitors and this can be explained from
the interactions of this group to the NS3 protease. In this
model the sulfone oxygens of the acyl sulfonamide bind to
the oxyanion hole, that is, the NH of both Gly 137 and Ser
139. The flexible side chain of Lys 136 has possibilities to
interact both with one of the sulfone oxygens and with the
amide carbonyl of the acyl sulfonamide. The cyclopropyl
group has close contact interactions with Gln 41 and the
catalytic His 57 in the S10 sub pocket.
Utilizing ring-closing metathesis, inhibitors of different
ring sizes were synthesized and evaluated. The ring size
with the best fit, 14-membered macrocycles, delivered
inhibitors with sub nanomolar activity, for example,
inhibitors 17, 18, and 20, with Ki values of 0.07, 0.19,
and 0.41 nM, respectively and with EC50 values of
530, 33, and 9.1 nM, respectively, in the subgenomic
replicon assay.
The excellent potencies found, together with the obser-
vation that this class of inhibitors can display potent
activities in the replicon assay in spite of P4 truncation,
make further refinements of the compounds presented
herein highly warranted.
The amides flanking the P2 cyclopentyl have main chain
hydrogen bonds to Arg 155 and Ala 157. The aliphatic
P1–P3 linker of the macrocycle has close contact interac-
tions with the side chain of Val 132.
With the introduction of an extending P2 substituent
there seems to be an inhibitor induced conformational
change of the Arg 155 and Asp 168 side chains leading
to the formation of a salt bridge.16 This conformational
change makes it possible for the methoxy quinoline to
make a favorable interaction with the guanidine of
Arg 155. The quinoline is also shielding the catalytic
interaction between His 57 and Asp 81, while the
extending phenyl seems to add interactions and stabilize
the position of the P2 substituent by aromatic stacking
with Tyr 56. The P2 substituent also has interactions
with some residues from the NS3 helicase, mainly the
side chains of Pro 482, Met 485, Val 524, and Gln 526.
4. Experimental
4.1. HCV NS3 protease enzyme assay42
The enzyme-based inhibition assay with HCV protease
was performed using recombinant full length NS3 en-
zyme (Poliakov et al.)43 and NS4A (KKGSVVIV-
GRIVLSGK, Gunnar Lindeberg, Department of
Medicinal Chemistry, Uppsala University, Sweden) in
final concentrations of 3.5 nM and 14 lM, respectively.
The test compounds were dissolved and diluted in
DMSO and were added to the assay buffer containing
50 mM Hepes, pH 7.5, 40% glycerol, 0.1% CHAPS,
and 10 mM DTT. The maximum final DMSO concen-
tration in the assay was 1%. After a pre-incubation for
30 min at room temperature, the enzyme reaction was
started by adding the FRET substrate Ac-Asp-Glu-As-
p(EDANS)-Glu-Glu-Abu-w-[COO)Ala-Ser-Lys(DAB-
CYL)-NH2 (RET S1, AnaSpec, San Jose, CA. USA) to
a final concentration of 2 lM. The enzyme activity was
continously measured over time (20 min) in a fluores-
cence reader (Fluorocan Ascent, ThermoLab systems,
3. Conclusion
Trisubstituted cyclopentane- and cyclopentene dicar-
boxylic acid moieties have successfully been used to
replace the commonly employed N-acyl-(4R)-hydroxy-
proline, and have been incorporated in the P2 position
of macrocyclic functionalized HCV NS3 inhibitors.

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7193
Stockholm, Sweden) with 355 nm as excitation and
500 nm as emission wavelengths, respectively.
performed on a preparative C-18 column. Gradient
HPLC-MS was performed on a Gilson system (Column:
Phenomenex C-18 250 · 15 mm and Phenomenex C-18
150 · 4.6 mm for preparative and analytical runs,
respectively; Pump: Gilson gradient pump 322; UV/
vis-detector: Gilson 155; MS detector: Thermo Finnigan
Surveyor MSQ; Gilson Fraction Collector FC204) using
methanol with 0.1% formic acid and deionized water
with 0.1% formic acid as mobile phase. MALDI-TOF-
spectra were recorded on a Voyager-DE STR Biospect-
rometry Workstation using a-cyano-4-hydroxycinnamic
acid as a matrix and reference.
IC50 values were calculated by non-linear fitting into the
equation (1 ꢀ vi/vo) = (I)/((I) + IC50) and the Ki value
was calculated from the IC50 value using the equation
Ki = IC50/(1 + S/Km) assuming a competitive enzyme
inhibition.
4.2. HCV replicon assay
The Huh-7 cell line containing HCV subgenomic geno-
type 1b replicon with firefly luciferase (Lohmann et al.
1999) was cultured in DMEM (Dulbecco’s minimal
essential medium, Life Technologies) supplemented with
10% fetal calf serum (Life Technologies), 100 U/ml pen-
icillin, 100 lg/ml streptomycin, and 0.5 mg/ml G418
(Geneticin, Life Technologies). Replicon cells were tryp-
sinized and seeded into 96-well microplates (1500 cells/
well). After 24 h, test compounds, fivefold serially di-
luted in cell culture medium, were added to the subcon-
fluent cells. The compound stock solutions were made in
DMSO and the final concentration of DMSO in the as-
say never exceeded 1%.
4.5. LC-MS purity measurements
4.5.1. Chromatography system A. Column: Phenomenex
C18 150 · 4.6 mm; Pump: Gilson gradient pump 322;
UV/VIS-detector: Gilson 155; MS detector: Thermo
Finnigan Surveyor MSQ; Software: Gilson UniPoint
4.0 and Xcalibur 1.3. Gradient: methanol 40–100% over
10 min at 1 mL/min followed by 100% for 5 min at
1 mL/min. To all solvents formic acid (0.1% v/v) was
added. Peaks were detected at 254 nm.
4.5.2. Chromatography system B. As system A except:
Gradient: acetonitrile 0–100% over 10 min at 1 mL/
min followed by 100% for 5 min at 1 mL/min.
The cells were incubated with compounds for 48 h and
were then lysed and the luciferase activity in each well
was determined using Luciferase Assay HTS Kit (Bio
Thema) and a Berthold luminometer.
4.5.3. Chromatography system C. As system A except:
Gradient: acetonitrile 5–99% over 3 min followed by
100% for 3 min. Ammonium acetate buffer was used in-
stead of formic acid.
The EC50 value represents the concentration of the com-
pound that decreases the luciferase signal with 50%
compared to the untreated control.
4.5.4. Chromatography system D. As system A except:
Gradient: acetonitrile 30–80% over 3 min followed by
100% for 3 min. Ammonium acetate buffer was used in-
stead of formic acid.
4.3. Modeling
All modeling experiments were performed using SYBYL
7.3 (Tripos Inc. 1699 South Hanley Road, St. Louis,
Missouri, 63144, USA). Inhibitor 20 was modeled in
the full length NS3 crystal structure 1cu139 from PDB,
where Arg 155 had been rotated to mimic the inhibitor
induced conformational change taking place upon
inhibitor binding.16
4.6. General synthetic procedures
4.6.1. General procedure A: tert-butyl ester deprotection
and amide bond formation. Scaffold molecule 11 (135 mg,
0.225 mmol) and triethylsilane (71 lL, 0.45 mmol) were
dissolved in DCM (2 mL) and trifluoroacetic acid (TFA)
(2 mL) was added. The mixture was stirred for 2 h and
thereafter concentrated and co-concentrated with tolu-
ene. The residue was dissolved in DMF (3 mL) and
the amine (60 mg, 0.26 mmol) and DIPEA (118 lL,
0.677 mmol) were added. The temperature was lowered
to 0 ꢁC and the coupling reagent O-(7-azabenzotriazol-
1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophos-
phate (HATU) (94 mg, 0.25 mmol) was added. The
solution was allowed to stir for 30 min at 0 ꢁC and then
for ꢁ16 h (overnight) at room temperature. The solvent
was removed by heating the reaction flask in a water
bath under diminished pressure. The residue was there-
after dissolved in ethyl acetate and the organic phase
was washed with brine, dried, filtered, and concentrated.
The crude product was purified by isocratic HPLC.
4.4. General methods
NMR-spectra were recorded on a Varian 300 MHz
instrument using CDCl3, CD3OD or (CD3)2SO as sol-
vents. TMS was used as reference. In recorded NMR-
spectra with diasteromeric mixtures the presumed dias-
teromeric peaks were put into brackets with the general
formula [n.nn and n.nn, (x, yH)] for 1H NMR and [nn.n
and nn.n] for 13C NMR, respectively. Optical rotations
were measured using a Perkin-Elmer 141 polarimeter.
TLC was carried out on Merck precoated 60F254 plates
using UV-light and charring with ethanol/sulfuric acid/
p-anisaldehyde/acetic acid 90:3:2:1, and a solution of
0.5% ninhydrin in ethanol for visualization. Flash col-
umn chromatography was performed using silica gel
60 (0.040–0.063 mm, Merck). Organic phases were dried
over anhydrous magnesium sulfate. Concentrations
were performed under diminished pressure (1–2 kPa)
at a bath temperature of 40 ꢁC. Isocratic HPLC was
4.6.2. General procedure B: olefin ring-closing metathesis
(RCM). A solution of the diene (158 mg, 0.209 mmol) in
dry DCM (25 mL) was bubbled with argon for 5 min.

7194
M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
To the stirred solution under argon atmosphere was
then added a solution of 2nd Generation Hoveyda-
Grubbs Catalyst (14 mg, 0.022 mmol) in dry DCM
(5 mL). The mixture was stirred at reflux under argon
atmosphere for ꢁ16 h and concentrated. The crude
product was purified by isocratic HPLC.
oborane adduct was hydrolyzed by treatment with 2 M
NaOH (1.9 mL) in MeOH (3 mL). The mixture was stir-
red for 2 h and the MeOH was evaporated. H2O (5 mL)
and DCM (5 mL) were added and the organic phase was
separated. The water phase was extracted three addi-
tional times with DCM. The combined organic phases
were dried, filtered, concentrated, and the crude residue
was purified by flash column chromatography.
4.6.3. General procedure C: deprotection of Boc group.
The Boc protected hydrazine (33 mg, 0.046 mmol) and
triethylsilane (15 lL, 0.094 mmol) were dissolved in
DCM (2 mL) after which was added trifluoroacetic acid
(TFA) (2 mL). The mixture was stirred for 3 h and was
subsequently concentrated and co-concentrated with tol-
uene. The crude product was purified by isocratic HPLC.
4.6.8. General procedure G2: synthesis of olefin hydra-
zine. The bromo olefin (200 lL, 1.604 mmol) and tert-
butyl carbazate (1.50 g, 11.4 mmol) were stirred at
100 ꢁC in DMF for 5 h. The solvent was removed by
heating the reaction flask in a water bath under dimin-
ished pressure. The residue was thereafter dissolved in
ethyl acetate and the organic phase was washed with
brine, dried, filtered, concentrated and the crude residue
was purified by flash column chromatography.
4.6.4. General procedure D: ester hydrolysis. To a solu-
tion of the ethyl ester (27 mg, 0.037 mmol) in THF/
MeOH/H2O 2:1:1 (5 mL) was added 1 M LiOH (296
lL, 0.296 mmol). The solution was stirred at reflux for
ꢁ5 h. After acidification to approximately pH 3–4 with
1 M HCl and concentration the residue was purified by
isocratic HPLC.
4.7. Synthetic experimentals
4.7.1. N0-Pent-4-enyl-hydrazinecarboxylic acid tert-butyl
ester (2a). Hydrazine 2a was synthesized according to
General procedure G2 using 5-bromo-1-pentene (1a).
Purification by flash column chromatography (toluene/
ethyl acetate 6:1 + 1% triethylamine) gave the product
4.6.5. General procedure E: sulfonamide coupling. To a
solution of the acid (36 mg, 0.053 mmol) in dry DCM
(1.5 mL) was added EDC (21 mg, 0.11 mmol). The mix-
ture was stirred at room temperature overnight and di-
luted with DCM, washed with water, dried, filtered
and concentrated. The residual was dissolved in dry
DCM (1.5 mL) and cyclopropanesulfonic acid amide
(14 mg, 0.12 mmol) and DBU (20 lL, 0.13 mmol) were
added. The mixture was stirred at room temperature
overnight and diluted with DCM, washed with 10%
aqueous citric acid and brine, dried, filtered, and con-
centrated. The crude product was purified by isocratic
HPLC.
1
(72%) as a colorless oil. H NMR (300 MHz, CDCl3)
d 1.46 (s, 9H), 1.49–1.62 (m, 2H), 2.05–2.16 (m, 2H),
2.80–2.89 (m, 2H), 3.93 (bs, 1H), 4.91–5.08 (m, 2H),
5.72–5.88 (m, 1H), 6.81 (bs, 1H); 13C NMR
(75.5 MHz, CDCl3) d 27.1, 28.3, 31.2, 51.4, 80.2,
114.8, 138.3, 156.9.
4.7.2. N0-Hex-5-enyl-hydrazinecarboxylic acid tert-butyl
ester (2b). Hydrazine 2b was synthesized according to
General procedure G2 using 6-bromo-1-hexene (1b).
Purification by flash column chromatography (toluene/
ethyl acetate 6:1 + 1% triethylamine) gave the product
4.6.6. General procedure F: oxidation of olefin alcohol. To
a solution of the olefin alcohol (1.00 mL, 7.44 mmol)
and N-methylmorpholine N-oxide (1.31 g, 11.2 mmol)
in DCM (17 mL) were added ground molecular sieves
1
(75%) as a colorless oil. H NMR (300 MHz, CDCl3)
d 1.36–1.56 (m, 4H), 1.46 (s, overlapped, 9H), 2.01–
2.12 (m, 2H), 2.79–2.88 (m, 2H), 3.91 (bs, 1H), 4.90–
5.06 (m, 2H), 5.71–5.88 (m, 1H), 6.62 (bs, 1H); 13C
NMR (75.5 MHz, CDCl3) d 26.2, 27.1, 28.2, 33.4,
51.7, 80.1, 114.4, 138.4, 156.7.
˚
(3.5 g, 4 A). The mixture was stirred for 10 min at room
temperature under nitrogen atmosphere before tetrapro-
pylammonium
perruthenate
(TPAP)
(131 mg,
0.370 mmol) was added. After stirring for an additional
2.5 h the solution was filtered through Celite. The sol-
vent was carefully evaporated and the remaining liquid
was purified by flash column chromatography.
4.7.3. Hept-6-enal (4a). Alcohol 3a was oxidized accord-
ing to General procedure F. Flash column chromatogra-
phy (CH2Cl2) gave the crude and volatile aldehyde 4a
1
(74%) as a colorless oil. H NMR (300 MHz, CDCl3)
4.6.7. General procedure G1: synthesis of olefin hydra-
zine. To a mixture of the volatile aldehyde (68 mg,
0.61 mmol) and tert-butyl carbazate (81 mg, 0.61 mmol)
in MeOH (5 mL) was added ground molecular sieves
(115 mg, 3 A). The mixture was stirred for 3 h, filtered
through Celite and concentrated.
d 1.35–1.50 (m, 2H), 1.59–1.72 (m, 2H), 2.01–2.14 (m,
2H), 2.39–2.49 (m, 2H), 4.91–5.08 (m, 2H), 5.71–5.88
(m, 1H), 9.76 (t, J = 1.8 Hz, 1H); 13C NMR
(75.5 MHz, CDCl3) d 21.5, 28.3, 33.4, 43.7, 114.8,
138.2, 202.5.
˚
4.7.4. Oct-7-enal (4b). Alcohol 3b was oxidized accord-
ing to General procedure F. Flash column chromatogra-
phy (CHCl3) gave the volatile aldehyde 4b (98%) as a
The residue was dissolved in dry THF (3 mL) and
AcOH (3 mL). NaBH3CN (95 mg, 1.5 mmol) was added
and the solution was stirred overnight. The reaction
mixture was diluted with saturated aqueous NaHCO3
(6 mL) and ethyl acetate (6 mL). The organic phase
was washed with brine and saturated aqueous NaHCO3,
dried over MgSO4, filtered, and concentrated. The cyan-
1
colorless oil. H NMR (300 MHz, CDCl3) d 1.26–1.48
(m, 4H), 1.56–1.71 (m, 2H), 1.97–2.11 (m, 2H), 2.36–
2.47 (m, 2H), 4.87–5.05 (m, 2H), 5.70–5.89 (m, 1H),
9.77 (t, J = 1.9 Hz, 1H); 13C NMR (75.5 MHz, CDCl3)
d 21.9, 28.6, 33.5, 43.9, 114.5, 138.7, 202.8.

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7195
4.7.5. N0-Hept-6-enyl-hydrazinecarboxylic acid tert-butyl
ester (5a). Hydrazine 5a was synthesized according to
General procedure G1 using aldehyde 4a. Purification
by flash column chromatography (toluene/ethyl acetate
9:1 + 1% triethylamine) gave the product (45%) as a col-
2H), 2.82 (s, 3H), 3.19 (t, J = 7.2 Hz, 2H), 4.91–5.03
(m, 2H), 5.71–5.85 (m, 1H).
4.7.10. (1R,2R,4R)-2-((1R,2S)-1-Ethoxycarbonyl-2-vinyl-
cyclopropylcarbamoyl)-4-(7-methoxy-2-phenyl-quinolin-
4-yloxy)-cyclopentanecarboxylic acid tert-butyl ester
1
orless oil. H NMR (300 MHz, CDCl3) d 1.25–1.51 (m,
6H), 1.43 (s, overlapped, 9H), 1.96–2.07 (m, 2H), 2.75–
2.85 (m, 2H), 3.90 (bs, 1H), 4.86–5.04 (m, 2H), 5.69–
5.85 (m, 1H), 6.31 (bs, 1H); 13C NMR (75.5 MHz,
CDCl3) d 26.5, 27.6, 28.3, 28.7, 33.6, 51.9, 80.2, 114.3,
138.8, 156.7.
(11). Compound 11 was synthesized according to Refs.
22
D
26 and 31. NMR in accordance with Ref. 26. ½aꢂ ꢀ 22
(c 1.0, CHCl3); HPLC-MS: (M+H)+ calcd: 601.3, found:
601.4.
4.7.11. (1R,2S)-1-{[(1R,2R,4R)-2-(N0-tert-Butoxycarbonyl-
N-pent-4-enyl-hydrazinocarbonyl)-4-(7-methoxy-2-phenyl-
quinolin-4-yloxy)-cyclopentanecarbonyl]-amino}-2-vinyl-
cyclopropanecarboxylic acid ethyl ester (12a). Scaffold
molecule 11 was coupled to hydrazine 2a according to
General procedure A. Purification by HPLC (MeOH/
H2O 85:15 + 0.2% triethylamine) gave tripeptide diene
12a (85%) as a colorless solid. 1H NMR (300 MHz,
CDCl3) d 1.23 (t, J = 7.1 Hz, 3H), 1.30–1.52 (m, 1H),
1.46 (s, overlapped, 9H), 1.57–1.73 (m, 2H), 1.82–1.92
(m, 2H), 1.96–2.15 (m, 4H), 2.17–2.36 (m, 2H), 2.40–
2.56 (m, 1H), 2.57–2.74 (m, 1H), 3.30–3.54 (m, 2H),
3.68–3.86 (b, 1H), 3.92 (s, 3H), 4.15 (q, J = 7.1 Hz, 2H),
4.92–5.05 (m, 2H), 5.06–5.19 (m, 2H), 5.20–5.32 (m,
1H), 5.62–5.84 (m, 2H), 6.58 (bs, 1H), 6.94 (s, 1H),
7.06–7.15 (m, 1H), 7.36–7.55 (m, 4H), 7.99–8.14 (m,
3H); 13C NMR (75.5 MHz, CDCl3) d 14.2, 23.0, 25.7,
28.1, 30.7, 32.8, 35.2, 37.3, 40.3, 44.5, 47.0, 47.8, 55.4,
61.5, 77.9, 81.7, 98.2, 107.3, 115.0, 115.2, 118.0, 123.0,
127.4, 128.6, 129.0, 133.1, 137.5, 140.3, 151.1, 154.2,
159.0, 160.6, 161.1, 170.5, 174.0, 176.2. MALDI-TOF:
(M+H)+ calcd: 727.4, found: 727.5.
4.7.6. N0-Oct-7-enyl-hydrazinecarboxylic acid tert-butyl
ester (5b). Hydrazine 5b was synthesized according to
General procedure G1 using aldehyde 4b. Purification
by flash column chromatography (toluene/ethyl acetate
9:1 + 1% triethylamine) gave the product (38%) as a col-
1
orless oil. H NMR (300 MHz, CDCl3) d 1.24–1.57 (m,
8H), 1.46 (s, overlapped, 9H), 1.98–2.10 (m, 2H), 2.78–
2.88 (m, 2H), 3.91 (bs, 1H), 4.88–5.06 (m, 2H), 5.71–
5.89 (m, 1H), 6.49 (bs, 1H); 13C NMR (75.5 MHz,
CDCl3) d 26.8, 27.7, 28.3, 28.7, 28.9, 33.6, 51.9, 80.1,
114.1, 138.9, 156.7.
4.7.7. Methanesulfonic acid hex-5-enyl ester (7). To a
stirred solution of 5-hexenol (6) (500 lL, 4.24 mmol) in
dry pyridine (1.25 mL, 15.5 mmol) and dry CH2Cl2
(25 mL) at 0 ꢁC was added methanesulfonyl chloride.
After stirring for 24 h at room temperature the mixture
was concentrated and co-concentrated with toluene.
Flash column chromatography (toluene/ethyl acetate
9:1) gave 7 (91%) as a colorless oil. 1H NMR
(300 MHz, CDCl3) d 1.46–1.58 (m, 2H), 1.71–1.83 (m,
2H), 2.05–2.17 (m, 2H), 3.00 (s, 3H), 4.23 (t,
J = 6.5 Hz, 2H), 4.95–5.08 (m, 2H), 5.71–5.86 (m, 1H);
13C NMR (75.5 MHz, CDCl3) d 24.7, 28.5, 33.0, 37.4,
69.9, 115.2, 137.9.
4.7.12. (1R,2S)-1-{[(1R,2R,4R)-2-(N0-tert-Butoxycarbonyl-
N-hex-5-enyl-hydrazinocarbonyl)-4-(7-methoxy-2-phenyl-
quinolin-4-yloxy)-cyclopentanecarbonyl]-amino}-2-vinyl-
cyclopropanecarboxylic acid ethyl ester (12b). Scaffold
molecule 11 was coupled to hydrazine 2b according to
General procedure A. Purification by HPLC (MeOH/
H2O 85:15 + 0.2% triethylamine) gave tripeptide diene
12b (82%) as a colorless solid. 1H NMR (300 MHz,
CDCl3) d 1.23 (t, J = 7.1 Hz, 3H), 1.32–1.50 (m, 3H),
1.46 (s, overlapped, 9H), 1.51–1.64 (m, 2H), 1.83–1.92
(m, 2H), 1.99–2.14 (m, 4H), 2.18–2.36 (m, 2H), 2.43–
2.58 (m, 1H), 2.59–2.73 (m, 1H), 3.35–3.52 (m, 2H),
3.70–3.86 (b, 1H), 3.93 (s, 3H), 4.15 (q, J = 7.1 Hz,
2H), 4.89–5.03 (m, 2H), 5.08–5.20 (m, 2H), 5.21–5.32
(m, 1H), 5.64–5.83 (m, 2H), 6.51 (bs, 1H), 6.95 (s,
1H), 7.09 (dd, J = 2.5, 9.1 Hz, 1H), 7.37–7.52 (m, 4H),
8.00–8.10 (m, 3H); 13C NMR (75.5 MHz, CDCl3) d
14.1, 23.0, 25.9, 25.9, 28.1, 32.5, 32.9, 33.2, 35.2, 37.3,
40.3, 44.5, 47.1, 48.0, 55.3, 61.4, 77.9, 81.7, 98.2, 107.2,
114.7, 115.2, 117.9, 123.0, 127.4, 128.6, 129.0, 133.2,
138.2, 140.2, 151.1, 154.2, 159.0, 160.6, 161.1, 170.5,
174.0, 176.2. MALDI-TOF: (M+H)+ calcd: 741.4,
found: 741.4.
4.7.8. Hex-5-enylamine (8). A solution of 7 (250 mg,
1.40 mmol), ꢁ28% NH3 aqueous solution (10 mL), and
MeOH (10 mL) was stirred for 48 h. Thereafter H2O
(10 mL) was added and the solution was extracted with
CH2Cl2 (3 · 15 mL). The combined organic extracts were
dried, filtered, and carefully concentrated. Purification by
flash column chromatography (ethyl acetate/methanol
4:1) gave the volatile amine 8 (134 mg, 96%) as a yellowish
1
oil. H NMR (300 MHz, CDCl3) d 1.29 (bs, 2H), 1.35–
1.54 (m, 4H), 2.02–2.12 (m, 2H), 2.66–2.73 (m, 2H),
4.91–5.06 (m, 2H), 5.74–5.89 (m, 1H); 13C NMR
(75.5 MHz, CDCl3) d 26.2, 33.3, 33.6, 42.1, 114.5, 138.8.
4.7.9. Hex-5-enyl-methyl-carbamic acid tert-butyl ester
(10). A cooled (0 ꢁC) solution of N-boc-methylamine (9)
(7.55 g, 57.6 mmol) and sodium hydride (60.5 mmol) in
DMF (80 mL) was stirred for 1 h. To the cooled solu-
tion was slowly added compound
7
(11.28 g,
63.30 mmol) and the mixture was allowed to stir over-
night. EtOAc was added and the organic phase was
washed with citric acid and brine, dried, filtered, and
concentrated. Purification by flash column chromatog-
raphy (toluene/ethyl acetate 9:1) gave 10 (69%) as a col-
4.7.13.
(1R,2S)-1-{[(1R,2R,4R)-2-(N0-tert-Butoxycar-
bonyl-N-hept-6-enyl-hydrazinocarbonyl)-4-(7-methoxy-2-
phenyl-quinolin-4-yloxy)-cyclopentanecarbonyl]-amino}-
2-vinyl-cyclopropanecarboxylic acid ethyl ester (12c).
Scaffold molecule 11 was coupled to hydrazine 5a
1
orless oil H NMR (300 MHz, CDCl3) d 1.32–1.40 (m,
2H), 1.44 (s, 9H), 1.46–1.54 (m, 2H), 2.02–2.10 (m,

7196
M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
according to General procedure A. Purification by
HPLC (MeOH/H2O 90:10 + 0.2% triethylamine) gave
tripeptide diene 12c (82%) as a colorless solid. 1H
NMR (300 MHz, CDCl3, 40 ꢁC) d 1.22 (t, J = 7.1 Hz,
3H), 1.28–1.42 (m, 6H), 1.46 (s, 9H), 1.52–1.62 (m,
2H), 1.82–1.91 (m, 1H), 1.96–2.16 (m, 3H), 2.18–2.34
(m, 2H), 2.42–2.56 (m, 1H), 2.58–2.72 (m, 1H), 3.42
(m, 3H), 3.66–3.84 (m, 1H), 3.92 (s, 3H), 4.15 (q,
J = 7.1 Hz, 2H), 4.88–5.02 (m, 2H), 5.07–5.18 (m, 2H),
5.20–5.32 (m, 1H), 5.63–5.84 (m, 2H), 6.62 (bs, 1H),
6.94 (s, 1H), 7.09 (dd, J = 2.6, 9.2 Hz, 1H), 7.36–7.51
(m, 4H), 7.99–8.10 (m, 3H); 13C NMR (75.5 MHz,
CDCl3) d 14.3, 23.0, 26.4, 26.6, 28.3, 28.6, 33.2, 33.5,
35.6, 37.6, 40.6, 44.7, 47.1, 48.6, 55.5, 61.5, 81.9, 98.4,
107.9, 114.5, 115.6, 118.1, 123.2, 127.6, 128.3, 128.7,
129.1, 133.5, 138.7, 140.7, 151.5, 154.5, 159.2, 160.9,
161.5, 170.5, 174.2, 176.3. MALDI-TOF: (M+H)+
calcd: 755.4, found: 755.6.
4.7.16. (1R,2S)-1-{[(1R,2R,4R)-2-(Hex-5-enyl-methyl-car-
bamoyl)-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclop-
entanecarbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
ethyl ester (12f). Scaffold molecule 11 was coupled to
amine 10 (subjected to HCl in dioxane prior to coupling)
according to General procedure A. Purification by
HPLC (MeOH/H2O 80:20 + 0.2% triethylamine) gave
diene 12f (82%) as a colorless solid. 1H NMR
(300 MHz, CDCl3) d 1.17 (t, J = 7.1 Hz, 3H), 1.26–
1.46 (m, 4H), 1.47–1.67 (m, 2H), 1.78–1.88 (m, 1H),
1.95–2.17 (m, 3H), 2.31–2.35 (m, 1H), 2.43–2.59 (m,
1H), 2.71–2.91 (m, 1H), 2.98 (s, 3H), 3.17–3.49 (m,
3H), 3.54–3.73 (m, 1H), 3.92 (s, 3H), 4.01–4.18 (m,
2H), 4.85–5.12 (m, 3H), 5.20–5.36 (m, 2H), 5.63–5.83
(m, 2H), 6.92–7.02 (m, 1H), 7.04 (s, 1H), 7.13 (d,
J = 9.1 Hz, 1H), 7.23-7.43 (m, 3H), 7.50 (s, 1H), 7.92
(d, J = 8.0 Hz, 2H), 8.00–8.08 (m, 1H); 13C NMR
(75.5 MHz, CDCl3) d 14.3, 23.2, 26.0, 26.5, 28.1, 33.4,
35.4, 37.2, 40.2, 45.6, 45.8, 48.3, 49.9, 55.9, 61.2, 79.2,
99.1, 104.5, 115.1, 115.2, 117.8, 119.4, 123.7, 128.0,
128.9, 130.3, 133.7, 138.1, 138.4, 147.8, 157.8, 162.7,
162.9, 170.0, 173.1, 174.2. HPLC-MS: (M+H)+ calcd:
640.3, found: 640.3.
4.7.14. (1R,2S)-1-{[(1R,2R,4R)-2-(N0-tert-Butoxycarbonyl-
N-oct-7-enyl-hydrazinocarbonyl)-4-(7-methoxy-2-phenyl-
quinolin-4-yloxy)-cyclopentanecarbonyl]-amino}-2-vinyl-
cyclopropanecarboxylic acid ethyl ester (12d). Scaffold
molecule 11 was coupled to hydrazine 5b according to
General procedure A. Purification by HPLC (MeOH/
H2O 90:10 + 0.2% triethylamine) gave tripeptide diene
12d (64%) as a colorless solid. 1H NMR (300 MHz,
CDCl3) d 1.14–1.55 (m, 13H), 1.43 (s, overlapped,
9H), 1.80–1.89 (m, 1H), 1.90–2.02 (m, 3H), 2.04–2.16
(m, 1H), 2.18–2.33 (m, 3H), 2.38–2.52 (m, 1H), 2.53–
2.69 (m, 1H), 3.25–3.54 (m, 2H), 3.88 (s, 3H), 4.11 (q,
J = 7.1 Hz, 2H), 4.84–4.98 (m, 2H), 5.03–5.16 (m, 2H),
5.18–5.29 (m, 1H), 5.58–5.81 (m, 2H), 6.82–6.96 (m,
2H), 7.04 (dd, J = 2.6, 9.2 Hz, 1H), 7.34–7.48 (m, 4H),
7.94–8.06 (m, 3H); 13C NMR (75.5 MHz, CDCl3) d
14.1, 22.9, 26.5, 28.0, 28.5, 28.6, 32.5, 33.5, 35.3, 37.2,
40.2, 44.3, 45.7, 47.0, 48.3, 55.3, 61.3, 77.9, 98.2, 107.3,
114.1, 115.2, 117.8, 123.0, 127.4, 128.5, 128.8, 129.0,
133.2, 138.7, 140.2, 151.1, 154.2, 159.0, 160.6, 161.1,
170.4, 174.0, 176.1. HPLC-MS: (M+H)+ calcd: 769.4,
found: 769.5.
4.7.17. (Z)-(1R,4R,6S,14R,16R)-12-tert-Butoxycarbon-
ylamino-16-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,13-
dioxo-3,12-diaza-tricyclo[12.3.0.04,6]heptadec-7-ene-4-
carboxylic acid ethyl ester (13a). Diene 12a was cyclized
according to General procedure B. Purification by
HPLC (MeOH/H2O 90:10 + 0.2% triethylamine) pro-
vided macrocyclic compound 13a (10%) as a colorless
solid. 1H NMR (300 MHz, CDCl3) d 1.14–1.30 (m,
4H), 1.32–1.58 (m, 2H), 1.51 (s, overlapped, 9H), 1.64–
1.80 (m, 2H), 1.83–2.12 (m, 2H), 2.13–2.28 (m, 1H),
2.28–2.41 (m, 1H), 2.66–2.80 (m, 2H), 3.00–3.17 (m,
1H), 3.40–3.64 (m, 1H), 3.65–3.80 (m, 1H), 3.96 (s,
3H), 4.04–4.28 (m, 2H), 4.34–4.49 (m, 1H), 5.10–5.24
(m, 1H), 5.20–5.36 (m, 1H), 5.53–5.70 (m, 1H), 6.27–
6.39 (m, 1H), 6.90–7.02 (m, 1H), 7.08–7.18 (m, 1H),
7.36–7.58 (m, 4H), 7.82–7.92 (m, 1H), 7.93–8.15 (m,
3H). MALDI-TOF: (M+H)+ calcd: 699.3, found: 699.3.
4.7.15. (1R,2S)-1-{[(1R,2R,4S)-2-Hex-5-enylcarbamoyl-
4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-cyclopentane-
4.7.18. (Z)-(1R,4R,6S,15R,17R)-13-tert-Butoxycarbon-
ylamino-17-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,14-
dioxo-3,13-diaza-tricyclo[13.3.0.04,6]octadec-7-ene-4-car-
boxylic acid ethyl ester (13b). Diene 12b was cyclized
according to General procedure B. Purification by
HPLC (MeOH/H2O 90:10 + 0.2% triethylamine) pro-
vided macrocyclic compound 13b (51%) as a colorless
carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic
acid
ethyl ester (12e). Scaffold molecule 11 was coupled to
amine 8 according to General procedure A. Purification
by HPLC (MeOH/H2O 80:20 + 0.2% triethylamine)
gave diene 12e (70%) as a colorless solid. 1H NMR
(300 MHz, CDCl3) d 1.21 (t, J = 7.1 Hz, 3H), 1.29–
1.50 (m, 6H), 1.83–1.92 (m, 1H), 1.93–2.06 (m, 2H),
2.09–2.20 (m, 1H), 2.34–2.47 (m, 1H), 2.48–2.62 (m,
1H), 2.92–3.07 (m, 1H), 3.08–3.19 (m, 1H), 3.20–3.33
(m, 2H), 3.89 (s, 3H), 4.06–4.20 (m, 2H), 4.89–5.03 (m,
2H), 5.07–5.19 (m, 2H), 5.23–5.33 (m, 1H), 5.65–5.82
(m, 2H), 6.28 (b, 1H), 6.93 (s, 1H), 7.02–7.12 (m, 1H),
7.37 (b, 1H), 7.40–7.54 (m, 4H), 7.94–8.06 (m, 3H);
13C NMR (75.5 MHz, CDCl3) d 14.8, 23.6, 26.7, 29.4,
33.8, 36.4, 36.7, 40.3, 40.9, 47.9, 48.1, 56.0, 61.9, 78.7,
98.8, 108.0, 115.4, 115.8, 118.6, 118.6, 126.6, 128.1,
129.3, 129.8, 134.1, 138.8, 140.9, 151.8, 159.7, 161.2,
161.8, 170.8, 173.7, 175.2. HPLC-MS: (M+H)+ calcd:
626.3, found: 626.2.
solid. 1H NMR (300 MHz, CDCl3)
d 1.23 (t,
J = 7.1 Hz, 3H), 1.30–1.56 (m, 3H), 1.49 (s, overlapped,
9H), 1.60–1.80 (m, 2H), 1.81–1.96 (m, 1H), 1.96–2.08
(m, 2H), 2.10–2.35 (m, 3H), 2.53–2.77 (m, 2H), 2.96–
3.14 (m, 1H), 3.50–3.68 (m, 2H), 3.95 (s, 3H), 4.07–
4.24 (m, 2H), 4.40–4.56 (m, 1H), 5.08–5.20 (m, 1H),
5.20–5.29 (m, 1H), 5.68–5.80 (m, 1H), 6.06–6.24 (bs,
1H), 6.95 (s, 1H), 7.11 (d, J = 9.1 Hz, 1H), 7.40–7.54
(m, 4H), 7.95 (bs, 1H), 8.03 (d, J = 6.6 Hz, 2H), 8.08
(d, J = 9.1 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d
14.3, 22.6, 25.6, 26.1, 26.3, 27.9, 28.2, 32.8, 37.4, 42.0,
45.3, 47.3, 47.5, 55.5, 61.5, 77.8, 81.8, 98.4, 107.4,
115.3, 118.1, 123.2, 125.7, 127.6, 128.7, 129.1, 135.6,
140.5, 151.5, 154.1, 159.2, 160.9, 161.3, 170.4, 173.5,

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7197
177.8. MALDI-TOF: (M+H)+ calcd: 713.4, found:
713.4.
1H), 7.46–7.58 (m, 3H), 8.05 (d, J = 9.1 Hz, 1H), 8.19–
8.29 (m, 2H); 13C NMR (75.5 MHz, (CD3)2SO) d 14.1,
22.1, 25.7, 26.5, 27.8, 28.3, 34.9, 35.8, 37.7, 47.3, 47.9,
55.4, 60.3, 78.2, 98.1, 107.3, 114.8, 117.6, 123.0, 126.7,
127.2, 128.5, 129.4, 132.5, 139.1, 150.4, 157.7, 160.4,
160.8, 169.8, 172.0, 172.9. MALDI-TOF: (M+H)+
calcd: 598.3, found: 598.5.
4.7.19. (Z)-(1R,4R,6S,16R,18R)-14-tert-Butoxycarbon-
ylamino-18-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,15-
dioxo-3,14-diaza-tricyclo[14.3.0.04,6]nonadec-7-ene-4-
carboxylic acid ethyl ester (13c). Diene 12c was cyclized
according to General procedure B. Purification by
HPLC (MeOH/H2O 90:10 + 0.2% triethylamine) pro-
vided macrocyclic compound 13c (70%) as a colorless
4.7.22. (Z)-(1R,4R,6S,15R,17R)-17-(7-Methoxy-2-phe-
nyl-quinolin-4-yloxy)-13-methyl-2,14-dioxo-3,13-diaza-
tricyclo[13.3.0.04,6]octadec-7-ene-4-carboxylic acid ethyl
ester (13f). Diene 12f was cyclized according to General
procedure B. Purification by HPLC (MeOH/H2O
85:15 + 0.2% triethylamine) provided macrocyclic com-
pound 13f (37%) as a green-white solid. 1H NMR
(300 MHz, CDCl3) d 1.19 (t, J = 7.1 Hz, 3H), 1.23–
1.33 (m, 4H), 1.34–1.41 (m, 2H), 1.54–1.69 (m, 1H),
1.70–1.89 (m, 1H), 1.91–2.03 (m, 1H), 2.04–2.16 (m,
1H), 2.18–2.30 (m, 1H), 2.31–2.50 (m, 1H), 2.58–2.76
(m, 2H), 2.80–2.96 (m, 1H), 3.08 (s, 3H), 3.31–3.49 (m,
1H), 3.75–3.89 (m, 1H), 3.99 (s, 3H), 4.02–4.19 (m,
1H), 4.58–4.73 (m, 1H), 5.15–5.25 (m, 1H), 5.27–5.41
(m, 1H), 5.58–5.71 (m, 1H), 6.98 (s, 1H), 7.09 (s, 1H),
7.21 (d, J = 6.1 Hz, 1H), 7.33–7.54 (m, 3H), 7.89 (b,
1H), 7.96–8.11 (m, overlapped, 1H), 8.07 (d, overlapped,
J = 9.3 Hz, 2H); 13C NMR (75.5 MHz, CDCl3) d 14.6,
21.2, 22.5, 24.8, 25.1, 26.5, 28.5, 29.8, 32.4, 34.1, 36.6,
42.1, 45.2, 45.9, 47.2, 56.2, 61.1, 79.2, 99.3, 104.2,
115.0, 119.9, 123.7, 125.2, 128.4, 129.2, 129.5, 130.9,
135.7, 157.7, 163.2, 169.4, 173.7, 174.6. HPLC-MS:
(M+H)+ calcd: 612.3, found: 612.3.
1
solid. H NMR (300 MHz, CD3OD) d 1.03–1.22 (m,
1H), 1.28 (t, J = 7.1 Hz, 3H), 1.32–1.44 (m, 4H), 1.49
(s, 9H), 1.55–1.73 (m, 2H), 1.81–1.91 (m, 1H), 2.04–
2.28 (m, 3H), 2.30–2.52 (m, 3H), 2.53–2.70 (m, 1H),
2.86–3.00 (m, 1H), 3.34–3.44 (m, 1H), 3.46–3.62 (m,
1H), 3.95 (s, 3H), 4.19 (q, J = 7.1 Hz, 2H), 4.32–4.48
(m, 1H), 5.20–5.33 (m, 1H), 5.34 (bs, 1H), 5.58–5.70
(m, 1H), 7.10 (s, 1H), 7.14 (dd, J = 2.5, 9.1 Hz, 1H),
7.39 (d, J = 2.5 Hz, 1H), 7.45–7.55 (m, 3H), 8.00 (d,
J = 8.0 Hz, 2H), 8.17 (d, J = 9.3 Hz, 1H); 13C NMR
(75.5 MHz, CD3OD) d 14.6, 23.4, 27.5, 27.7, 28.0,
28.5, 30.7, 36.1, 38.1, 42.5, 45.6, 56.0, 62.7, 79.9, 82.8,
100.2, 107.4, 116.6, 119.1, 124.5, 126.5, 128.9, 129.8,
130.5, 135.8, 141.5, 152.2, 156.4, 161.3, 162.5, 163.1,
171.9, 175.8, 179.0. MALDI-TOF: (M+H)+ calcd:
727.4, found: 727.5.
4.7.20. (Z)-(1R,4R,6S,17R,19R)-15-tert-Butoxycarbon-
ylamino-19-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,16-
dioxo-3,15-diaza-tricyclo[15.3.0.04,6]icos-7-ene-4-carbox-
ylic acid ethyl ester (13d). Diene 12d was cyclized accord-
ing to General procedure B. Purification by HPLC
(MeOH/H2O 90:10 + 0.2% triethylamine) provided
macrocyclic compound 13d (79%) as a colorless solid.
1H NMR (300 MHz, CDCl3) d 1.06–1.30 (m, 6H),
1.31–1.68 (m, 6H), 1.48 (s, overlapped, 9H), 1.89–2.06
(m, 2H), 2.09–2.38 (m, 4H), 2.54–2.69 (m, 2H), 3.00–
3.14 (m, 1H), 3.40–3.55 (m, 2H), 3.93 (s, 3H), 4.04–
4.24 (m, 2H), 4.28–4.44 (m, 1H), 5.07–5.25 (m, 2H),
5.37–5.50 (m, 1H), 6.39 (b, 1H), 6.94 (s, 1H), 7.09 (dd,
J = 2.6, 9.1 Hz, 1H), 7.38–7.51 (m, 4H), 7.94–8.06 (m,
4H); 13C NMR (75.5 MHz, CDCl3) d 14.3, 23.8, 25.9,
26.7, 26.9, 27.6, 28.1, 28.6, 34.0, 37.7, 41.0, 44.9, 46.8,
47.3, 55.4, 61.4, 77.9, 81.6, 98.3, 107.4, 115.3, 118.0,
123.1, 125.5, 127.5, 128.6, 129.0, 135.3, 140.5, 151.3,
153.8, 159.2, 160.7, 161.2, 170.9, 173.8, 177.0. HPLC-
MS: (M+H)+ calcd: 741.4, found: 741.5.
4.7.23. (Z)-(1R,4R,6S,14R,16R)-12-tert-Butoxycarbon-
ylamino-16-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,13-
dioxo-3,12-diaza-tricyclo[12.3.0. 04, 6]heptadec-7-ene-4-
carboxylic acid (14a). Compound 13a was hydrolyzed
according to General procedure D. Purification by
HPLC (MeOH/H2O 70:30 + 0.2% trifluoroacetic acid)
22
provided 14a (32%) as a colorless solid. ½aꢂ þ 15:0 (c
D
0.1, MeOH); 1H NMR (300 MHz, CD3OD) d 1.18–
1.38 (m, 2H), 1.49 (s, 9H), 1.66–1.86 (m, 1H), 1.91–
2.01 (m, 1H), 2.04–2.22 (m, 3H), 2.24–2.38 (m, 2H),
2.48–2.65 (m, 1H), 2.66–2.81 (m, 1H), 2.90–3.02 (m,
1H), 3.46–3.58 (m, 1H), 3.59–3.70 (m, 1H), 4.07 (s,
3H), 4.28–4.43 (m, 1H), 5.34–5.41 (m, 1H), 5.60–5.72
(m, 2H), 7.43–7.58 (m, 3H), 7.68–7.82 (m, 3H), 7.98–
8.09 (m, 2H), 8.42 (d, J = 9.1 Hz, 1H); HRMS calcd
(M+H)+: 671.3081; found 671.3079. LC-MS Purity Sys-
tem A: tR = 7.78 min, 97%; System B: tR = 7.85 min,
99%.
4.7.21. (Z)-(1R,4R,6S,15R,17S)-17-(7-Methoxy-2-phenyl-
quinolin-4-yloxy)-2,14-dioxo-3,13-diaza-tricyclo[13.3.0. 04,6]-
octadec-7-ene-4-carboxylic acid ethyl ester (13e). Diene
12e was cyclized according to General procedure B.
Purification by HPLC (MeOH/H2O 85:15 + 0.2% tri-
ethylamine) provided macrocyclic compound 13e
(23%) as a green-white solid. 1H NMR (300 MHz,
(CD3)2SO) d 1.13 (t, J = 7.1 Hz, 3H), 1.18–1.30 (m,
1H), 1.36–1.46 (m, 3H), 1.47–1.60 (m, 2H), 1.68–1.86
(m, 1H), 2.00–2.10 (m, 2H), 2.11–2.28 (m, 4H), 2.52–
2.64 (m, 1H), 2.66–2.79 (m, 1H), 2.81–2.95 (m, 1H),
2.95–3.10 (m, 1H), 3.44–3.58 (m, 1H), 3.92 (s, 3H),
4.03 (q, J = 7.1 Hz, 2H), 5.34–5.45 (m, 2H), 5.62–5.75
(m, 1H), 7.18 (dd, J = 2.2, 9.1 Hz, 1H), 7.33 (s, 1H),
7.35–7.45 (m, 1H), 7.38 (d, J = 2.5 Hz, overlapped,
4.7.24. (Z)-(1R,4R,6S,15R,17R)-13-tert-Butoxycarbon-
ylamino-17-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,14-
dioxo-3,13-diaza-tricyclo[13.3.0.04,6]octadec-7-ene-4-car-
boxylic acid (14b). Compound 13b was hydrolyzed
according to General procedure D. Purification by
HPLC (MeOH/H2O 70:30 + 0.2% trifluoroacetic acid)
22
provided 14b (99%) as a colorless solid. ½aꢂ þ 15:3 (c
D
0.2, MeOH); 1H NMR (300 MHz, CD3OD) d 1.22–
1.42 (m, 4H), 1.47 (s, 9H), 1.61–1.75 (m, 2H), 1.75–2.02
(m, 2H), 2.08–2.20 (m, 1H), 2.20–2.29 (m, 1H), 2.30–
2.54 (m, 2H), 2.62–2.78 (m, 1H), 2.80–2.93 (m, 1H),
3.37–3.51 (m, 1H), 3.52–3.70 (m, 1H), 4.06 (s, 3H),

7198
M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
4.35–4.48 (m, 1H), 5.34–5.45 (m, 1H), 5.60–5.70 (m, 1H),
5.71–5.83 (m, 1H), 7.41–7.51 (m, 2H), 7.53 (s, 1H), 7.66–
7.81 (m, 3H), 7.92–8.07 (m, 2H), 8.42 (d, J = 9.3 Hz, 1H);
13C NMR (75.5 MHz, CD3OD) d 23.4, 26.6, 27.6, 28.5,
28.7, 28.8, 35.8, 37.0, 42.7, 46.7, 56.9, 82.7, 83.4, 100.5,
102.3, 116.1, 121.7, 126.4, 127.3, 129.8, 130.8, 133.3,
133.8, 134.9, 143.5, 156.7, 158.0, 166.5, 168.3, 173.3,
175.9, 178.2. HRMS calcd (M+H)+: 684.3237; found
684.3251. LC-MS Purity System A: tR = 8.27 min, 97%;
System B: tR = 7.99 min, 98%.
70:30 + 0.2% trifluoroacetic acid) provided 14e (68%)
22
D
as a colorless solid. ½aꢂ þ 25:3 (c 0.2, MeOH); 1H
NMR (300 MHz, CD3OD) d 1.21–1.41 (m, 1H),
1.42–1.54 (m, 2H), 1.55–1.71 (m, 2H), 1.75–1.85 (m,
1H), 1.86–2.01 (m, 1H), 2.12–2.24 (m, 1H), 2.25–
2.35 (m, 1H), 2.37–2.56 (m, 3H), 2.60–2.74 (m, 1H),
2.80–2.95 (m, 1H), 3.01–3.15 (m, 1H), 3.16–3.29 (m,
1H), 3.62–3.78 (m, 1H), 4.07 (s, 3H), 5.41–5.53 (m,
1H), 5.60–5.70 (m, 1H), 5.76–5.89 (m, 1H), 7.46 (d,
J = 9.3 Hz, 1H), 7.50–7.58 (m, 2H), 7.66–7.82 (m,
3H), 8.00–8.10 (m, 2H), 8.44 (d, J = 9.3 Hz, 1H); 13C
NMR (75.5 MHz, CD3OD) d 23.5, 27.7, 28.2, 29.0,
29.3, 36.1, 36.8, 39.7, 42.6, 56.9, 83.8, 100.4, 102.3,
116.2, 121.7, 126.6, 127.8, 129.8, 130.8, 133.4, 133.8,
134.9, 143.5, 158.1, 166.6, 168.6, 173.5, 175.1, 175.7.
HRMS calcd (M+H)+: 570.2604; found 570.2603.
LC-MS Purity System A: tR = 4.76 min, 98%; System
B: tR = 7.08 min, 98%.
4.7.25. (Z)-(1R,4R,6S,16R,18R)-14-tert-Butoxycarbon-
ylamino-18-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,15-
dioxo-3,14-diaza-tricyclo[14.3.0.04,6]nonadec-7-ene-4-car-
boxylic acid (14c). Compound 13c was hydrolyzed
according to General procedure D. Purification by
HPLC (MeOH/H2O 80:20 and MeOH/H2O 90:10) pro-
22
vided 14c (46%) as a colorless solid. ½aꢂ þ 7:9 (c 0.2,
D
MeOH); 1H NMR (300 MHz, CD3OD) d 1.06–1.24
(m, 1H), 1.26–1.42 (m, 3H), 1.48 (s, 9H), 1.52–1.73 (m,
3H), 1.80–1.90 (m, 1H), 2.02–2.15 (m, 1H), 2.15–2.40
(m, 4H), 2.43–2.54 (m, 1H), 2.54–2.68 (m, 1H), 2.88–
3.00 (m, 1H), 3.35–3.48 (m, 1H), 3.49–3.66 (m, 1H),
3.96 (s, 3H), 4.32–4.48 (m, 1H), 5.25–5.42 (m, 2H),
5.56–5.68 (m, 1H), 7.14 (s, 1H), 7.17 (dd, J = 2.5,
9.1 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.46–7.58 (m,
3H), 8.00 (d, J = 8.0 Hz, 2H), 8.19 (d, J = 9.1 Hz, 1H);
13C NMR (75.5 MHz, CD3OD) d 23.6, 26.8, 27.8,
28.3, 28.5, 30.5, 35.8, 38.1, 43.0, 45.5, 56.0, 80.2, 82.7,
100.4, 106.9, 116.6, 119.2, 124.7, 127.4, 129.0, 129.8,
130.7, 134.8, 140.9, 151.6, 156.5, 161.1, 163.0, 163.4,
173.8, 175.7, 179.3. HRMS calcd (M+H)+: 699.3394;
found 699.3409. LC-MS Purity System A:
tR = 8.50 min, 100%; System B: tR = 8.23 min, 100%.
4.7.28. (Z)-(1R,4R,6S,15R,17R)-17-(7-Methoxy-2-phe-
nyl-quinolin-4-yloxy)-13-methyl-2,14-dioxo-3,13-diaza-
tricyclo[13.3.0.04,6]octadec-7-ene-4-carboxylic acid (14f).
Compound 13f was hydrolyzed according to General
procedure D. Purification by HPLC (MeOH/H2O
70:30 + 0.2% trifluoroacetic acid) provided 14f (53%)
22
D
as a colorless solid. ½aꢂ þ 47:1 (c 0.2, MeOH); 1H
NMR (300 MHz, CD3OD) d 1.25–1.37 (m, 2H), 1.45
(dd, J = 5.1, 9.8 Hz, 1H), 1.55–1.64 (m, 1H), 1.66–1.72
(m, 1H), 1.73–1.83 (m, 2H), 1.84–1.94 (m, 1H), 2.10
(app. q, J = 8.9 Hz, 1H), 2.24–2.31 (m, 1H), 2.32–2.44
(m, 2H), 2.64–2.75 (m, 1H), 2.84–2.98 (m, 1H), 3.08 (s,
3H), 3.37–3.45 (m, 1H), 3.49–3.60 (m, 1H), 3.94 (s,
3H), 4.40–4.49 (m, 1H), 5.29–5.40 (m, 2H), 5.60–5.70
(m, 1H), 7.13 (s, 1H), 7.15 (d, J = 9.4 Hz, 1H), 7.28 (s,
1H), 7.49–7.58 (m, 3H), 8.00 (d, J = 7.8 Hz, 2H), 8.10
(d, J = 9.0 Hz, 1H); 13C NMR (75.5 MHz, CD3OD) d
21.9, 24.6, 26.2, 27.6, 27.9, 33.7, 34.0, 35.4, 45.4, 46.4,
54.8, 78.6, 99.1, 104.9, 115.0, 118.1, 123.3, 125.7,
127.7, 128.6, 129.7, 133.2, 138.6, 149.2, 159.3, 162.0,
162.2, 174.4, 175.4. HRMS calcd (M+H)+: 584.2761;
found 584.2763. LC-MS Purity System C: tR = 2.74 min,
97%; System D: tR = 2.37 min, 95%.
4.7.26. (Z)-(1R,4R,6S,17R,19R)-15-tert-Butoxycarbon-
ylamino-19-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,16-
dioxo-3,15-diaza-tricyclo[15.3.0.04,6]icos-7-ene-4-carbox-
ylic acid (14d). Compound 13d was hydrolyzed accord-
ing to General procedure D. Purification by HPLC
(MeOH/H2O 70:30 + 0.2% trifluoroacetic acid) pro-
22
vided 14d (100%) as a colorless solid. ½aꢂ þ 25:2 (c
D
0.3, MeOH); 1H NMR (300 MHz, CD3OD) d 1.04–
1.51 (m, 6H), 1.46 (s, overlapped, 9H), 1.55–1.69 (m,
2H), 1.70–1.79 (m, 2H), 2.10–2.34 (m, 4H), 2.35–2.53
(m, 2H), 2.63–2.80 (m, 1H), 2.86–2.98 (m, 1H), 3.38–
3.54 (m, 1H), 3.55–3.71 (m, 1H), 4.06 (s, 3H), 4.19–
4.38 (m, 1H), 5.26–5.36 (m, 1H), 5.52–5.70 (m, 2H),
7.40–7.50 (m, 2H), 7.54 (s, 1H), 7.66–7.80 (m, 3H),
8.02 (d, J = 6.6 Hz, 2H), 8.41 (d, J = 9.3 Hz, 1H); 13C
NMR (75.5 MHz, CD3OD) d 24.2, 27.3, 27.5, 28.1,
28.5, 29.0, 30.6, 37.0, 41.6, 45.4, 47.6, 56.9, 82.7, 83.6,
100.5, 102.3, 116.2, 121.7, 126.4, 127.0, 129.8, 130.8,
133.5, 133.8, 135.6, 143.6, 156.6, 158.0, 166.6, 168.3,
169.7, 173.7, 175.5, 178.2. HRMS calcd (M+H)+:
713.3550; found 713.3541. LC-MS Purity System A:
tR = 8.43 min, 100%; System B: tR = 7.68 min, 100%.
4.7.29. (Z)-(1R,4R,6S,15R,17R)-13-Amino-17-(7-meth-
oxy-2-phenyl-quinolin-4-yloxy)-2,14-dioxo-3,13-diaza-tri-
cyclo[13.3.0.04,6]octadec-7-ene-4-carboxylic acid ethyl
ester (15b). The Boc group of 13b was removed accord-
ing to General procedure C. Purification by HPLC
(MeOH/H2O 90:10 + 0.2% triethylamine) gave 15b
1
(63%) as a colorless solid. H NMR (300 MHz, CDCl3)
d 1.08–1.25 (m, 2H), 1.21 (t, J = 7.1 Hz, overlapped,
3H), 1.28–1.45 (m, 2H), 1.55–1.70 (m, 1H), 1.71–1.85
(m, 1H), 1.86–2.02 (m, 3H), 2.03–2.12 (m, 1H), 2.20–
2.32 (m, 1H), 2.33–2.45 (m, 1H), 2.62–2.72 (m, 1H),
2.73–2.85 (m, 1H), 2.85–2.97 (m, 1H), 3.60–3.73 (m,
1H), 3.85 (bs, 2H), 3.96 (s, 3H), 4.02–4.16 (m, 2H),
4.67–4.82 (m, 1H), 5.12–5.25 (m, 2H), 5.58–5.70 (m,
1H), 6.95–7.01 (m, 2H), 7.11 (d, J = 9.2 Hz, 1H), 7.41–
7.60 (m, 4H), 8.02–8.10 (m, 3H); 13C NMR
(75.5 MHz, CDCl3) d 14.6, 22.5, 25.0, 25.1, 25.4, 28.2,
32.7, 37.3, 42.2, 45.7, 46.7, 47.3, 55.8, 61.2, 78.1, 98.8,
107.2, 115.5, 118.6, 123.4, 125.6, 127.9, 128.4, 129.0,
129.6, 135.6, 140.0, 150.9, 159.1, 161.5, 161.8, 169.6,
4.7.27. (Z)-(1R,4R,6S,15R,17S)-17-(7-Methoxy-2-phe-
nyl-quinolin-4-yloxy)-2,14-dioxo-3,13-diaza-tricyclo-
[13.3.0.04,6]octadec-7-ene-4-carboxylic acid (14e).
Compound 13e was hydrolyzed according to General
procedure D. Purification by HPLC (MeOH/H2O

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7199
174.2, 177.5. MALDI-TOF: (M+H)+ calcd: 613.3,
found: 613.6.
100.5, 102.3, 116.1, 121.7, 126.4, 127.1, 129.8, 130.8,
133.4, 133.8, 135.0, 143.5, 158.1, 166.6, 168.6, 172.8,
176.5, 177.7. HRMS calcd (M+H)+: 585.2713; found
585.2703. LC-MS Purity System A: tR = 5.14 min,
100%; System B: tR = 6.62 min, 100%.
4.7.30. (Z)-(1R,4R,6S,16R,18R)-14-Amino-18-(7-meth-
oxy-2-phenyl-quinolin-4-yloxy)-2,15-dioxo-3,14-diaza-
tricyclo[14.3.0.04,6]nonadec-7-ene-4-carboxylic acid ethyl
ester (15c). The Boc group of 13c was removed accord-
ing to General procedure C. Purification by HPLC
(MeOH/H2O 90:10 + 0.2% triethylamine) gave 15c
4.7.33. (Z)-(1R,4R,6S,16R,18R)-14-Amino-18-(7-meth-
oxy-2-phenyl-quinolin-4-yloxy)-2,15-dioxo-3,14-diaza-
tricyclo[14.3.0.04,6]nonadec-7-ene-4-carboxylic acid (16c).
Compound 15c was hydrolyzed according to General
procedure D. Purification by HPLC (MeOH/H2O
1
(66%) as a colorless solid. H NMR (300 MHz, CDCl3)
d 1.01–1.20 (m, 3H), 1.24 (t, J = 7.1 Hz, 3H), 1.28–1.49
(m, 3H), 1.52–1.67 (m, 1H), 1.68–1.90 (m, 1H), 1.93–
2.04 (m, 1H), 2.05–2.19 (m, 4H), 2.20–2.31 (m, 1H),
2.62–2.85 (m, 3H), 3.56–3.75 (m, 1H), 3.95 (s, 3H),
4.01 (bs, 2H), 4.09–4.22 (m, 2H), 4.58–4.72 (m, 1H),
5.09–5.22 (m, 2H), 5.50–5.65 (m, 1H), 6.55 (bs, 1H),
6.99 (s, 1H), 7.10 (dd, J = 2.5, 9.1 Hz, 1H), 7.40–7.55
(m, 4H), 8.00–8.11 (m, 3H); 13C NMR (75.5 MHz,
CDCl3) d 14.4, 23.0, 25.6, 26.2, 27.5, 28.8, 33.0, 37.4,
41.7, 45.3, 45.6, 50.4, 55.1, 61.1, 78.0, 98.5, 107.3,
115.4, 118.1, 123.3, 125.8, 127.6, 128.7, 129.2, 134.0,
140.3, 151.1, 159.2, 161.0, 161.3, 170.1, 174.4, 176.9.
MALDI-TOF: (M+H)+ calcd: 627.3, found: 627.6.
70:30 + 0.2% trifluoroacetic acid) provided 16c (46%)
22
D
as a colorless solid. ½aꢂ ꢀ 2:4 (c 0.2, MeOH); 1H
NMR (300 MHz, CD3OD) d 1.05–1.28 (m, 1H), 1.30–
1.48 (m, 2H), 1.44–1.56 (m, 2H), 1.57–1.72 (m, 1H),
1.76–1.91 (m, 2H), 2.05–2.26 (m, 2H), 2.27–2.43 (m,
2H), 2.52–2.66 (m, 1H), 2.81–2.95 (m, 2H), 3.20–3.38
(m, overlapped, 1H), 3.44–3.60 (m, 1H), 4.01 (s, 3H),
4.21–4.35 (m, 1H), 4.45–4.60 (m, 1H), 5.20–5.31 (m,
1H), 5.43–5.75 (m, 2H), 7.46 (d, J = 9.2 Hz, 1H), 7.50
(s, 1H), 7.53 (s, 1H), 7.68–7.82 (m, 3H), 8.00–8.09 (m,
2H), 8.42 (d, J = 9.2 Hz, 1H); 13C NMR (75.5 MHz,
CD3OD) d 23.5, 26.8, 27.3, 27.7, 28.5, 30.3, 35.3, 37.8,
42.6, 45.5, 48.0, 50.5, 56.9, 83.5, 100.4, 102.3, 116.2,
121.7, 126.6, 127.0, 129.8, 130.8, 133.4, 133.8, 135.2,
143.5, 158.1, 160.9, 161.4, 166.6, 168.6, 172.7, 176.0,
178.1. HRMS calcd (M+H)+: 599.2869; found
599.2889. LC-MS Purity System A: tR = 6.09 min,
100%; System B: tR = 7.61 min, 100%.
4.7.31. (Z)-(1R,4R,6S,17R,19R)-15-Amino-19-(7-meth-
oxy-2-phenyl-quinolin-4-yloxy)-2,16-dioxo-3,15-diaza-tri-
cyclo[15.3.0.04,6]icos-7-ene-4-carboxylic acid ethyl ester
(15d). The Boc group of 13d was removed according to
General procedure C. Purification by HPLC (MeOH/
H2O 90:10 + 0.2% triethylamine) gave 15d (74%) as a
colorless solid. 1H NMR (300 MHz, CDCl3) d 1.05–
1.29 (m, 3H), 1.20 (t, J = 7.1 Hz, 3H), 1.30–1.42 (m,
3H), 1.43–1.56 (m, 2H), 1.54–1.80 (m, 1H), 1.85–1.98
(m, 2H), 1.99–2.18 (m, 3H), 2.19–2.34 (m, 2H), 2.53–
2.68 (m, 1H), 2.70–2.83 (m, 1H), 2.85–3.00 (m, 1H),
3.57–3.71 (m, 1H), 3.79 (bs, 2H), 3.94 (s, 3H), 4.00–
4.19 (m, 2H), 4.52–4.66 (m, 1H), 5.10–5.26 (m, 2H),
5.50–5.62 (m, 1H), 6.93–7.01 (m, 2H), 7.09 (dd,
J = 2.5, 9.7 Hz, 1H), 7.38–7.54 (m, 4H), 7.97–8.11 (m,
3H); 13C NMR (75.5 MHz, CDCl3) d 14.3, 23.5, 26.0,
26.1, 26.2, 27.0, 27.5, 29.5, 33.7, 37.8, 41.0, 44.7, 45.9,
49.4, 55.5, 60.9, 77.6, 98.4, 107.4, 115.4, 118.0, 123.2,
125.6, 127.5, 128.7, 129.1, 135.4, 140.5, 151.2, 159.1,
160.8, 161.3, 170.1, 174.2, 176.7. HPLC-MS: (M+H)+
calcd: 641.3, found: 641.4.
4.7.34. (Z)-(1R,4R,6S,17R,19R)-15-Amino-19-(7-meth-
oxy-2-phenyl-quinolin-4-yloxy)-2,16-dioxo-3,15-diaza-tri-
cyclo[15.3.0.04,6]icos-7-ene-4-carboxylic
Compound 15d was hydrolyzed according to General
acid
(16d).
procedure D. Purification by HPLC (MeOH/H2O
70:30 + 0.2% trifluoroacetic acid) provided 16d (71%)
22
D
as a colorless solid. ½aꢂ þ 31:9 (c 0.4, MeOH); 1H
NMR (300 MHz, CD3OD) d 1.08–1.57 (m, 9H), 1.67–
1.75 (m, 1H), 2.02–2.18 (m, 2H), 2.19–2.33 (m, 2H),
2.34–2.50 (m, 2H), 2.81–2.92 (m, 1H), 2.94–3.08 (m,
1H), 3.35–3.52 (m, 1H), 4.06 (s, 3H), 4.11–4.24 (m,
1H), 4.38–4.50 (m, 1H), 5.22–5.32 (m, 1H), 5.53–5.70
(m, 2H), 7.42–7.56 (m, 3H), 7.67–7.80 (m, 3H), 8.00–
8.07 (m, 2H), 8.39 (d, J = 9.3 Hz, 1H); 13C NMR
(75.5 MHz, CD3OD) d 24.1, 27.2, 27.5, 27.7, 28.8,
29.1, 30.7, 36.6, 37.8, 41.2, 46.0, 47.5, 49.9, 56.9, 83.5,
100.5, 102.3, 116.2, 121.7, 126.5, 127.1, 129.8, 130.8,
133.5, 133.8, 135.6, 143.6, 158.1, 166.6, 168.5, 173.5,
176.0, 177.6. HRMS calcd (M+H)+: 613.3026; found
613.3038. LC-MS Purity System A: tR = 6.87 min,
100%; System B: tR = 6.98 min, 100%.
4.7.32. (Z)-(1R,4R,6S,15R,17R)-13-Amino-17-(7-meth-
oxy-2-phenyl-quinolin-4-yloxy)-2,14-dioxo-3,13-diaza-tri-
cyclo[13.3.0.04,6]octadec-7-ene-4-carboxylic acid (16b).
Compound 15b was hydrolyzed according to General
procedure D. Purification by HPLC (MeOH/H2O
70:30 + 0.2% trifluoroacetic acid) provided 16b (52%)
22
D
as a colorless solid. ½aꢂ þ 11:3 (c 0.1, MeOH); 1H
4.7.35. [(Z)-(1R,4R,6S,15R,17R)-4-Cyclopropanesulfonyla-
minocarbonyl-17-(7-methoxy-2-phenyl-quinolin-4-yloxy)-
2,14-dioxo-3,13-diaza-tricyclo[13.3.0.04,6]octadec-7-en-13-
yl]-carbamic acid tert-butyl ester (17). Compound 14b
was coupled to cyclopropanesulfonic acid amide accord-
ing to General procedure E. Purification by HPLC
NMR (300 MHz, CD3OD) d 1.24–1.39 (m, 2H), 1.40–
1.50 (m, 2H), 1.52–1.72 (m, 1H), 1.73–1.84 (m, 1H),
1.86–1.98 (m, 1H), 1.99–2.11 (m, 1H), 2.12–2.24 (m,
1H), 2.30–2.44 (m, 2H), 2.44–2.57 (m, 1H), 2.81–2.92
(m, 1H), 2.95–3.10 (m, 1H), 3.40–3.53 (m, 1H), 4.06 (s,
3H), 4.10–4.25 (m, 1H), 4.48–4.61 (m, 1H), 5.28–5.40
(m, 1H), 5.62–5.84 (m, 2H), 7.41–7.56 (m, 3H), 7.67–
7.82 (m, 3H), 8.00–8.09 (m, 2H), 8.40 (d, J = 9.3 Hz,
1H); 13C NMR (75.5 MHz, CD3OD) d 23.2, 26.4,
26.7, 28.6, 28.9, 35.2, 37.2, 42.8, 47.0, 48.0, 56.9, 83.2,
(MeOH/H2O 70:30 + 0.2% trifluoroacetic acid) pro-
22
vided 17 (80%) as a colorless solid. ½aꢂ þ 35:0 (c 0.1,
D
MeOH); 1H NMR (300 MHz, CD3OD) d 0.84–0.99
(m, 1H), 1.00–1.20 (m, 3H), 1.22–1.40 (m, 4H), 1.46 (s,
9H), 1.54–1.64 (m, 1H), 1.64–1.77 (m, 2H), 1.78–2.00

7200
M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
(m, 2H), 2.21–2.37 (m, 2H), 2.43–2.60 (m, 2H), 2.67–
2.84 (m, 1H), 2.86–3.00 (m, 1H), 3.38–3.53 (m, 1H),
3.53–3.71 (m, 1H), 4.06 (s, 3H), 4.40–4.55 (m, 1H),
5.12–5.25 (m, 1H), 5.58–5.78 (m, 2H), 7.38 (s, 1H),
7.47 (dd, J = 2.5, 9.3 Hz, 1H), 7.55 (s, 1H), 7.65–7.81
(m, 3H), 8.02 (d, J = 6.6 Hz, 2H), 8.37 (d, J = 9.3 Hz,
1H); 13C NMR (75.5 MHz, CD3OD) d 6.5, 6.7, 22.0,
25.7, 26.5, 28.1, 28.5, 31.9, 33.0, 36.0, 36.6, 44.8, 45.1,
56.9, 82.7, 83.3, 100.7, 102.3, 116.0, 121.8, 125.9,
126.2, 129.8, 130.8, 133.5, 133.8, 143.7, 156.9, 158.1,
166.5, 168.1, 171.0, 177.3, 180.4. HRMS calcd
(M+H)+: 788.3329; found 788.3296. LC-MS Purity Sys-
tem A: tR = 6.59 min, 98%; System B: tR = 6.56 min,
97%.
4.7.38. Cyclopropanesulfonic acid [(Z)-(1R,4R,6S,15R,
17R)-17-(7-methoxy-2-phenyl-quinolin-4-yloxy)-13-methyl-
2,14-dioxo-3,13-diaza-tricyclo[13.3.0.04,6]octadec-7-ene-
4-carbonyl]-amide (20). Compound 14f was coupled to
cyclopropanesulfonic acid amide according to General
procedure E. Purification by HPLC (MeOH/H2O
70:30 + 0.2% trifluoroacetic acid) provided 20
22
(29%) as a colorless solid. ½aꢂ þ 59:3 (c 0.1, MeOH);
D
1H NMR (300 MHz, CDCl3) d 0.90–1.01 (m, 1H),
1.01–1.12 (m, 1H), 1.14–1.22 (m, 1H), 1.24–1.35 (m,
2H), 1.36–1.52 (m, 2H), 1.53–1.61 (m, 1H), 1.62–
1.73 (m, 1H), 1.76–1.86 (m, 1H), 1.87–1.97 (m,
2H), 2.03–2.14 (m, 2H), 2.32–2.46 (m, 1H), 2.51–
2.63 (m, 2H), 2.64–2.75 (m, 1H), 2.85–2.95 (m,
1H), 2.98 (s, 3H), 3.27–3.48 (m, 2H), 4.00 (s, 3H),
4.53–4.64 (m, 1H), 4.99–5.09 (m, 2H), 5.58–5.68 (m,
1H), 6.76 (s, 1H), 7.06 (dd, J = 2.5, 9.2 Hz, 1H),
7.41 (b, 1H), 7.43–7.55 (m, 3H), 7.71 (s, 1H), 7.82
(d, J = 9.4 Hz, 1H), 7.92 (d, J = 7.0 Hz, 2H); 13C
NMR (75.5 MHz, CDCl3) d 6.0, 6.6, 21.0, 24.0,
26.0, 27.6, 31.0, 33.8, 34.9, 44.0, 44.9, 46.8, 47.2,
48.1, 55.6, 77.8, 98.4, 106.8, 114.9, 118.3, 123.2,
124.3, 127.5, 128.8, 128.9, 129.6, 133.1, 140.0, 150.9,
159.5, 160.6, 161.5, 172.4, 172.9. HRMS calcd
(M+H)+: 687.2853; found 687.2848. LC-MS Purity
System C: tR = 2.81 min, 100%; System D:
tR = 2.36 min, 99%.
4.7.36. Cyclopropanesulfonic acid [(Z)-(1R,4R,6S,15R,
17R)-13-amino-17-(7-methoxy-2-phenyl-quinolin-4-yloxy)-
2,14-dioxo-3,13-diaza-tricyclo[13.3.0.04,6]octadec-7-ene-
4-carbonyl]-amide (18). The Boc group of 17 was removed
according to General procedure C. Purification by gradi-
ent HPLC-MS gave 18 (95%) as a colorless solid.
22
D
½aꢂ þ 20:0 (c 0.1, MeOH); 1H NMR (300 MHz, CD3OD)
d 0.83–0.98 (m, 1H), 1.00–1.17 (m, 3H), 1.22–1.48 (m,
4H), 1.52–1.61 (m, 1H), 1.64–1.78 (m, 2H), 1.84–1.98
(m, 1H), 1.99–2.14 (m, 1H), 2.23–2.39 (m, 2H), 2.41–
2.59 (m, 2H), 2.76–2.85 (m, 1H), 2.87–2.99 (m, 1H),
3.06–3.19 (m, 1H), 3.53–3.69 (m, 1H), 4.05 (s, 3H),
4.51–4.64 (m, 1H), 5.10–5.22 (m, 1H), 5.58–5.74 (m,
2H), 7.41–7.50 (m, 2H), 7.54 (s, 1H), 7.65–7.80 (m, 3H),
8.03 (d, J = 6.0 Hz, 2H), 8.37 (d, J = 9.6 Hz, 1H); 13C
NMR (75.5 MHz, CD3OD) d 6.5, 6.8, 21.9, 25.6, 25.8,
28.5, 31.8, 33.2, 36.4, 36.7, 45.2, 47.0, 56.9, 83.3, 100.6,
102.3, 116.0, 121.7, 125.9, 126.2, 129.8, 130.8, 133.5,
137.8, 143.7, 158.2, 166.5, 168.3, 170.9, 176.6, 181.1.
HRMS calcd (M+H)+: 688.2805; found 688.2809. LC-
MS Purity System A: tR = 4.59 min, 98%; System B:
tR = 5.85 min, 98%.
4.7.39. ((3R,5R) and (3S,5S))-5-((1R,2S)-1-tert-Butoxy-
carbonyl-2-vinyl-cyclopropylcarbamoyl)-3-(7-methoxy-
2-phenyl-quinolin-4-yloxy)-cyclopent-1-enecarboxylic acid
(21). Compound 21 was synthesized according to
Refs. 27 and 31–33.
4.7.40. (1R,2S)-1-{[((1R,4R) and (1S,4S))-2-(N0-tert-
Butoxycarbonyl-N-hex-5-enyl-hydrazinocarbonyl)-4-(7-
methoxy-2-phenyl-quinolin-4-yloxy)-cyclopent-2-enecar-
bonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid tert-
butyl ester (22a). To a cooled solution (0 ꢁC) of a dia-
stereomeric mixture of compound 21 (28 mg,
0.049 mmol), hydrazine 2b (16 mg, 0.074 mmol),
and DIPEA (13 lL, 0.074 mmol) in DMF (1 mL)
was added HATU (28 mg, 0.074 mmol). The mixture
was stirred for 3 h and concentrated. The residue was
dissolved in DCM and the organic phase was washed
with water, dried with MgSO4, filtered, and concen-
trated. Purification by gradient HPLC-MS gave a
diasteromeric mixture of 22a (31 mg, 81%) as a pale
brown syrup. 1H NMR (300 MHz, CDCl3) d 1.17–
1.61 (m, 24H), 1.68–1.89 (m, 2H), 1.98–2.16 (m,
2H), 2.24–2.41 (m, 1H), 2.99–3.16 (m, 1H), 3.35–
3.52 (m, 1H), 3.65–3.83 (m, 1H), 3.96 (s, 3H),
4.07–4.16 (m, 1H), 4.88–5.04 (m, 2H), 5.11–5.19 (m,
1H), 5.24–5.38 (m, 1H), 5.66–5.85 (m, 2H), 5.93–
6.03 (m, 1H), 6.34–6.46 (m, 1H), 6.71–6.86 (m, 1H),
7.03 (s, 1H), 7.09–7.19 (m, 1H), 7.45–7.64 (m, 4H),
7.93–8.07 (m, 3H); 13C NMR (75.5 MHz, CDCl3) d
22.3, 26.0, 26.5, 27.0, 28.2, 32.6, 33.3, 34.2, 38.8,
39.1, 40.6, 43.9, 45.5, 45.9, 48.5, 51.6, 52.3, 55.8,
83.7, 98.9, 105.1, 114.9, 119.0, 123.4, 128.0, 128.9,
130.1, 133.3, 133.7, 138.3, 149.3, 154.5, 158.9, 162.3,
164.5, 169.5, 173.8. HPLC-MS: (M+H)+ calcd:
767.4, found: 767.4.
4.7.37. Cyclopropanesulfonic acid [(Z)-(1R,4R,6S,15R,
17S)-17-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,14-dioxo-
3,13-diaza-tricyclo[13.3.0. 04,6]octadec-7-ene-4-carbonyl]-
amide (19). Compound 14e was coupled to cyclopro-
panesulfonic acid amide according to General procedure
E. Purification by HPLC (MeOH/H2O 70:30 + 0.2% tri-
fluoroacetic acid) provided 19 (23%) as a colorless solid.
22
D
½aꢂ þ 37:3 (c 0.1, MeOH); 1H NMR (300 MHz,
(CD3)2SO) d 0.95–1.15 (m, 3H), 1.23–1.35 (m, 1H),
1.40–1.60 (m, 4H), 1.67–1.81 (m, 1H), 1.86–1.97 (m,
1H), 1.99–2.10 (m, 1H), 2.11–2.25 (m, 1H), 2.33–2.43
(m, 1H), 2.60–2.69 (m, 2H), 2.70–2.80 (m, 2H), 2.88–
2.95 (m, 1H), 2.96–3.12 (m, 2H), 3.38–3.46 (m, over-
lapped, 1H), 3.47–3.59 (m, 1H), 3.94 (s, 3H), 5.16–5.24
(m, 1H), 5.47–5.56 (m, 1H), 5.61–5.71 (m, 1H), 7.30
(b, 1H), 7.43 (s, 1H), 7.51–7.65 (m, 4H), 7.70 (b, 1H),
8.11 (d, J = 9.4 Hz, 1H), 8.18–8.25 (m, 2H), 8.87 (s,
1H), 11.47 (b, 1H); 13C NMR (75.5 MHz, (CD3)2SO)
d 6.1, 6.3, 21.4, 25.6, 26.7, 28.0, 31.1, 32.4, 35.5, 37.3,
43.1, 43.3, 48.2, 48.5, 56.2, 72.9, 79.6, 98.7, 111.1,
115.0, 119.1, 124.0, 126.0, 128.5, 129.3, 132.3, 141.1,
152.5, 157.6, 169.7, 171.7, 172.0, 175.0, 177.9. HRMS
calcd (M+H)+: 673.2696; found 673.2690. LC-MS Pur-
ity System C: tR = 2.55 min, 99%; System D:
tR = 2.09 min, 99%.

M. Ba¨ck et al. / Bioorg. Med. Chem. 15 (2007) 7184–7202
7201
4.7.41. (1R,2S)-1-{[((1R,4R) and (1S,4S))-2-(N0-tert-
Butoxycarbonyl-N-hept-6-enyl-hydrazinocarbonyl)-4-(7-
methoxy-2-phenyl-quinolin-4-yloxy)-cyclopent-2-enecar-
bonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid tert-
butyl ester (22b). Compound 22b (diastereomeric mix-
ture) (79%) was prepared from 21 according to the
method of the preparation of 22a using hydrazine 5a in-
(11.4 lL, 0.072 mmol) and TFA (1 mL). The mixture
was stirred for 4 h and concentrated. Purification by gra-
dient HPLC-MS gave compound 24a (5.4 mg, 38%)
(diastereomeric mixture) as a white powder after lyoph-
1
ilization. H NMR (300 MHz, CDCl3/MeOD (1:1, v/v))
d 1.24-1.86 (m, 6H), 1.99–2.17 (m, 2H), 2.49–2.68 (m,
2H), 2.84–3.01 (m, 2H), 4.05 (s, 3H), 4.18–4.32 (m,
1H), 5.45–5.73 (m, 2H), 6.15–6.27 (m, 1H), 6.37–6.48
(m, 1H), 6.91–6.99 (m, 1H), 7.07–7.13 (m, 1H), 7.38–
7.84 (m, 5H), 7.98–8.12 (m, 3H). HRMS calcd
(M+H)+: 583.2567; found 583.2557. LC-MS Purity Sys-
tem A: tR = 7.41 min, 100%; System B: tR = 6.58 min,
99%.
1
stead of hydrazine 2b. H NMR (300 MHz, CDCl3) d
1.16–1.63 (m, 24H), 1.69–1.92 (m, 2H), 1.96–2.16 (m,
4H), 2.23–2.38 (m, 1H), 2.99–3.15 (m, 1H), 3.34–3.51
(m, 1H), 3.62–3.83 (m, 1H), 3.95 (s, 3H), 4.06–4.16 (m,
1H), 4.88–5.03 (m, 2H), 5.10–5.18 (m, 1H), 5.25–5.37
(m, 1H), 5.66–5.85 (m, 2H), 5.90–6.00 (m, 1H), 6.37–
6.45 (m, 1H), 6.62–6.73 (m, 1H), 7.02–7.14 (m, 2H),
7.38–7.54 (m, 4H), 7.95–8.08 (m, 3H); 13C NMR
(75.5 MHz, CDCl3) d 22.9, 23.0, 26.3, 26.5, 27.7, 28.2,
28.3, 28.4, 28.6, 32.7, 33.5, 33.6, 34.0, 34.2, 40.7, 41.1,
48.7, 52.2, [55.5 and 55.6], 83.0, 98.2, [107.5 and
107.6], [114.3 and 114.4], [118.1 and 118.2], 123.0,
[127.6 and 127.7], 128.7, 129.2, [133.4 and 133.6],
[138.7 and 138.9], 140.4, 151.3, 154.7, [159.3 and
159.4], 161.0, 161.3, 169.5, 172.5. HPLC-MS: (M+H)+
calcd: 781.4, found: 781.5.
4.7.45. (Z)-(1R,4R,6S,18R) and (1S,4R,6S,18S)-14-Ami-
no-18-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,15-dioxo-
3,14-diaza-tricyclo[14.3.0.04,6]nonadeca-7,16-diene-4-car-
boxylic acid (24b). Compound 24b (47%) was prepared
from 23b according to the method of the preparation
1
of 24a. H NMR (300 MHz, CDCl3/CD3OD (1:1 v/v))
d 1.08–1.71 (m, 8H), 1.96–2.26 (m, 4H), 2.41–2.52 (m,
2H), 2.80–2.95 (m, 2H), 3.94 (s, 3H), 4.18–4.34 (m,
1H), 5.34–5.62 (m, 2H), 5.96–6.05 (m, 1H), 6.51–6.60
(m, 1H), 7.03–7.20 (m, 2H), 7.35–7.56 (m, 3H), 7.71–
7.78 (m, 1H), 7.92–8.07 (m, 2H). HRMS calcd
(M+H)+: 597.2712; found 597.2713. LC-MS Purity Sys-
tem A: tR = 7.22 min, 100%; System B: tR = 6.43 min,
100%.
4.7.42. (Z)-((1R,4R,6S,17R) and (1S,4R,6S,17S))-13-
tert-Butoxycarbonylamino-17-(7-methoxy-2-phenyl-quin-
olin-4-yloxy)-2,14-dioxo-3,13-diaza-tricyclo[13.3.0.04,6
]
octa-deca-7,15-diene-4-carboxylic acid tert-butyl ester
(23a). To solution of compound 22a (23 mg,
a
0.030 mmol) (diastereomeric mixture) in DCM (4 mL)
was added 2nd Generation Hoveyda-Grubbs Catalyst
(1.1 mg, 0.018 mmol) and the reaction mixture was re-
fluxed for 24 h with two extra additions of the catalyst.
The mixture was concentrated and purified by gradient
HPLC-MS to give a diastereomeric mixture of 23a
Acknowledgements
We gratefully thank Medivir AB, the Swedish Founda-
tion for Strategic Research (SSF), and Knut and Alice
Wallenberg’s Foundation for financial support. We
would also like to thank Jussi Kangasmetsa¨, Medivir
UK, and Kurt Benkestock, Medivir SE, for help regard-
ing the HRMS analyses.
1
(18 mg, 81%) as a syrup. H NMR (300 MHz, CD3OD)
d 0.73–0.97 (m, 5H), 1.15–1.74 (m, 20H), 1.95–2.59 (m,
4H), 2.91–3.04 (m, 1H), 3.84–4.05 (m, 4H), 4.18–4.38
(m, 1H), 5.21–5.47 (m, 1H), 5.61–5.79 (m, 1H), 6.01–
6.11 (m, 1H), 6.58–6.70 (m, 1H), 7.10–7.28 (m, 2H),
7.42–7.66 (m, 4H), 7.94–8.11 (m, 3H). HPLC-MS:
(M+H)+ calcd: 739.4, found: 739.4.
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