Synthesis and biological evaluation of [D-lysine]8cyclosporin A analogs as potential anti-HCV agents

Article abstract of DOI:10.1016/j.bmcl.2010.09.036

An efficient synthesis of [d-lysine]⁸cyclosporin A has been developed. Several analogs of [d-lysine]⁸cyclosporin A have been synthesized and show promising anti-HCV activity, particularly compounds 39 and 43, which each exhibit an anti-HCV EC₅₀ <200 nM, and are each ≥50-fold less immunosuppressive than cyclosporin A.

Full text of DOI:10.1016/j.bmcl.2010.09.036

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6542–6546
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of [D-lysine]8cyclosporin A analogs
as potential anti-HCV agents
Andrew Scribner a, , David Houck a, Zhuhui Huang b, Sarah Mosier a, Michael Peel a, Bernard Scorneaux a
⇑
a SCYNEXIS, Inc., PO Box 12878, Research Triangle Park, NC 27709-2878, USA
b Southern Research Institute, 431 Aviation Way, Frederick, MD 21701, USA
a r t i c l e i n f o
a b s t r a c t
An efficient synthesis of [D D
-lysine]8cyclosporin A has been developed. Several analogs of [ -lysine]8cyclo-
Article history:
Received 7 August 2010
Revised 3 September 2010
Accepted 8 September 2010
Available online 21 September 2010
sporin A have been synthesized and show promising anti-HCV activity, particularly compounds 39 and
43, which each exhibit an anti-HCV EC50 <200 nM, and are each ꢀ50-fold less immunosuppressive than
cyclosporin A.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cyclosporin
Cyclophilin
Hepatitis C
Nearly 200 million people worldwide are infected with hepati-
tis C, a disease of the liver caused by the hepatitis C virus (HCV),
which can lead to liver fibrosis, liver cirrhosis, liver cancer, and
ultimately death.1 There is presently no vaccine for HCV, and cur-
rent interferon-based therapeutics are successful in only 40–50% of
patients treated,1 prompting the need for new drugs with alterna-
tive modes of action. While there are several drugs in development
that target key HCV proteins, most notably NS3-4A serine protease
and NS5B RNA-dependent RNA polymerase,1,2 there remains a
need to find additional drug targets to increase options for drug
cocktails that would combat HCV drug resistance.
Cyclosporins are cyclic undecapeptides that exhibit a broad
spectrum of biological activities.3 Cyclosporin A (1, CsA, shown in
Fig. 1), isolated from the fungus Tolypocladium inflatum, is a power-
ful immunosuppressant of T-cells, and is the active ingredient of
SandimmuneÒ and NeoralÒ for preventing organ transplant rejec-
tion, as well as RestatisÒ for treating the autoimmune disease dry
eye.3 CsA exerts its immunosuppressive activity by binding to
two proteins sequentially to form a ternary complex.4 The first of
these is a cyclophilin (Cyp), which is a cis–trans proline isomerase,
the most predominant in humans being cyclophilin A (CypA).5 The
binary CsA–CypA complex is a potent inhibitor of the phosphatase
activity expressed by calcineurin (CN), a calcium-dependent ser-
ine/threonine phosphatase that promotes the synthesis of T-cell
lymphokines such as interleukin-2 (IL-2).6 Thus, CN inhibition ulti-
mately suppresses immune response.6
X-ray crystallography has revealed which residues of CsA bind
to CypA and CN, respectively.4 Figure 1 shows that residues 9,
10, 11, 1, and 2 form the ‘cyclophilin binding domain’ that binds
to CypA, while residues 4–7 comprise the ‘calcineurin binding do-
main’ that binds to CN. Residues 3 and 8 are at the interfaces be-
tween these two binding domains, and can potentially have an
impact on both CypA and CN binding. Figure 2 depicts how CsA
binds to CypA and CN from X-ray crystallographic data, with key
CypA and CN residues that form hydrogen bonds shown.
In addition to the immune diseases discussed above that can
be addressed by CN inhibition, there are a variety of diseases that
are treated by Cyp inhibition alone, most notably the infectious
diseases caused by human immunodeficiency virus 1 (HIV-1)7
and HCV.8 At the moment, there is some debate regarding what
role Cyp plays in HCV inhibition, and in fact which Cyp (CypA or
CypB) is the operative agent, although recently a consensus has
emerged indicating that CypA is the predominant if not exclusive
cyclophilin
binding
domain
HO
H
N
N
N
O
N
H
N
10
O
11
7
1
6
2
5
O
O
O
N
O
3
O
9
N
N
4
O
O
H
N
H
N
H
N
8
O
O
calineurin
binding
domain
Cyclosporin A (CsA)
1
⇑
Corresponding author. Tel.: +1 919 544 8610.
E-mail address: andrew.scribner@scynexis.com (A. Scribner).
Figure 1. The binding domains of CsA.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.036

A. Scribner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6542–6546
6543
Although there is a published total synthesis of [
D
-Lys]8CsA16 in
20 steps in an overall yield of 7.0–11.4%, it does not include the
synthesis of the tripeptide composing residues 9–11, nor the total
synthesis of the (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine
(MeBmt) residue at the 1-position. The former requires six steps
in 68% yield,17 while the highest yield reported to date for the lat-
ter is 22% in 12 steps,18 bringing the overall yield of [ -Lys]8CsA to
D
1.0–1.6% in a prohibitive 38 steps. In the years since these publica-
tions, technology has allowed for the industrial-scale biosynthesis
of CsA, which can now serve as a viable starting material. Our syn-
thesis (Scheme 1) starts from CsA and combines regioselective
ring-opening methodology first reported by Seebach19 and further
exemplified by Eberle,20 Edman degradation and amino acid cou-
pling chemistry to replace D-alanine with N-Boc-D-lysine at the
8-position,20,16 and then ring closure as reported by Rich.16
CsA (1) was first treated with acetic anhydride to protect the
hydroxyl group of MeBmt.21 The resulting acetate (5) was then
treated with 1.0 equiv of Lawesson’s reagent, which regioselec-
tively reacted with the amide carbonyl groups of residues 4 and
7 to give a statistical mixture of 7-thioamide (6), 4-thioamide (7),
4,7-bisthioamide (8), and unreacted 5, all of which were partially
separated by chromatography, and identified by reported proton
NMR spectra.19,20 A mixture of 6 and 8 was then treated with benzyl
bromide and DBU to give the corresponding benzyl thioimidates.17
The 4,7-bisbenzyl thioimidate (10) was considerably less polar than
the desired 7-benzyl thioimidate (9), and the components of
the resulting mixture were more readily separated by chromatogra-
phy than then the corresponding mixture of thioamides 6 and 8.
Hydrolysis of 9 with HCl afforded 7,8-secocyclosporins 11, which
was then subjected to Edman degradation conditions to first
prepare thiourea 12 and then decapeptide 13.20 Installation of
Figure 2. CsA in its active conformation with hydrogen bonds to both CypA and CN.
CsA residues are labeled in white; CypA residues are labeled in green; CN residues
are labeled in red.
Cyp that supports HCV-specific RNA replication and protein
expression in human cells.9 In any event, a drug that inhibited
a host Cyp protein would clearly complement a drug that tar-
geted a viral protein10; however, a CsA-based drug used to treat
such an infectious disease must be devoid of immunosuppressive
activity, as this would antagonize host immune mechanisms for
clearing the virus.
Fmoc-protected
D-Lys and related
D-amino acids afforded
undecapeptides 14–19.16 Treatment with base hydrolyzed the
acetate at [MeBmt]1, the thioester at [Ala]7, and the Fmoc group
of the 8-position to give 7,8-secocyclosporin 20–25.16 Cyclization
with n-propylphosphonic anhydride under dilute conditions gave
cyclic undecapeptides 26–3116; in the case of amides 24 and 25,
cyclization occurred with concomitant amide dehydration to give
nitriles 30 and 31. Subsequent treatment of Boc-protected amino
acids 26–29 with CF3CO2H yielded primary amines 32–35,16
Cyclosporins that have been developed for treating HIV and/or
HCV are shown in Figure 3. Each of these compounds possess a
modification at the 4-position that interferes with CN binding,
but not Cyp binding. NIM-81111 (2) has an N-methylisoleucine at
the 4-position instead of an N-methylleucine; Debio-02512 (3)
has a D-alanine at the 3-position and an N-ethylvaline at the 4-po-
sition; our clinical candidate SCY-63513 (4) has a dimethylamino-
ethyl-thioether at the 3-position and a 40-hydroxymethylleucine
at the 4-position.
including [D
-Lys]8CsA (35), which was prepared in 6.3% overall
yield in 10 steps, and compares favorably to the previously
reported 1.0–1.6% overall yield in 38 steps. We then treated
primary amines 32–35 with formaldehyde or acetaldehyde under
reductive amination conditions to ultimately afford dimethylam-
ines 36–39 and diethylamines 40–43, respectively.
In our drug discovery program focused on expanding SAR
around SCY-635, we were curious if a water-solubilizing amine-
bearing tether at the 8-position would have a similar anti-HCV ef-
fect as it does at the 3-position of SCY-635. Furthermore, we won-
dered if lengthening an amine sidechain at position 8 would
attenuate CN binding without reducing Cyp binding, as is reported
Biological activity of [D
-Lys]8CsA analogs is presented in Table 1.
for 8-position modified cyclosporins,14 particularly [
analogs.14a,b In this study, we report a synthesis of [ -lysine]8cyclo-
sporin A ([
-Lys]8CsA), a known precursor to a reagent used in an
ELISA assay to measure a compound’s binding affinity for Cyp.15
We then present the synthesis of several [
-Lys]8CsA analogs and
D
-Ser]8CsA
Compound binding affinity to CypA and CypB was measured
using an ELISA assay, as described by Quesniaux.15 In most cases,
binding to CypA was within fivefold of that of CsA and SCY-635
themselves, although there were exceptions (26, 32, 33, 34, 37,
40, 41) which showed weaker Cyp binding affinity. For most com-
pounds, CypB binding was within twofold of that of CypA binding;
however, two of the four compounds with a lengthy 4-carbon
D
D
D
evaluate their anti-HCV activity along with their CypA binding,
CypB binding, and immunosuppressive activity.
HO
H
HO
H
HO
H
N
S
N
N
N
O
N
N
N
N
O
N
N
N
N
O
N
N
H
N
N
H
N
O
N
H
N
O
O O
O
O O
O
O O
O
O
N
O
O
N
O
O
N
O
O
O
O
N
N
N
H
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
O
O
O
HO
O
NIM-811 (2)
Debio-025 (3)
SCY-635 (4)
Figure 3. Non-immunosuppressive cyclosporins.

6544
A. Scribner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6542–6546
O
O
O
HO
H
O
H
N
O
H
N
N
N
O
N
N
H
N
O
N
N
N
S
N
H
N
O
N
N
N
O
N
N
H
N
O
a
b
O
O
O
O
N
O
O
O
O
N
O
O
O
O
O
N
O
O
O
O
N
N
N
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
O
O
O
1
6
5
+ 4-thioamide (7)
+ 4,7-bisthioamide (8)
c
O
O
O
O
O
O
H
N
O
H
N
O
H
N
N
N
N
H
N
O
N
N
N
N
N
N
N
N
H
N
O
N
H
N
O
e
d
O
O
O
O
N
O
O
O
O
O
O
N
O
O
O
N
O
O
N
O
O
O
N
SBn
O
O
N
N
H
N
SBn
O
O
SBn
O
O
H
N
O
H
N
H
N
H
N
H
N
O
O
NH
NH2
S
NHPh
12
11
9
+ 4,7-bisthioimidate (10)
f
O
O
O
O
O
H
N
O
H
N
HO
H
N
N
N
H
N
O
N
O
N
N
H
N
O
g
N
N
N
N
N
H
N
O
h
O
O
O
O
N
O
O
O
O
O
N
O
O
O
O
O
O
N
O
O
O
N
N
N
N
SBn
O
O
N
H
SBn
O
O
OH
O
O
H
N
H
N
H
N
H
N
H
N
H
N
O
R
O
R
NH
NH2
fmoc
13
14 R = CH2NHBoc
15 R = CH2CH2NHBoc
16 R = CH2CH2CH2NHBoc
20 R = CH2NHBoc
21 R = CH2CH2NHBoc
22 R = CH2CH2CH2NHBoc
17 R = CH2CH2CH2CH2NHBoc
18 R = CH2C(=O)NH2
23 R = CH2CH2CH2CH2NHBoc
24 R = CH2C(=O)NH2
19 R = CH2CH2C(=O)NH2
25 R = CH2CH2C(=O)NH2
i
HO
H
HO
H
HO
H
N
O
N
O
N
N
H
N
O
N
N
N
O
N
N
H
N
O
N
N
N
O
N
N
H
N
O
k
j
O
O
O
N
O
O
O
O
O
N
O
O
O
O
O
N
O
O
O
O
N
N
N
N
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
O
O
O
R
R
R
36 R = CH2NMe2
37 R = CH2CH2NMe2
38 R = CH2CH2CH2NMe2
39 R = CH2CH2CH2CH2NMe2
40 R = CH2NEt2
32 R = CH2NH2
33 R = CH2CH2NH2
34 R = CH2CH2CH2NH2
35 R = CH2CH2CH2CH2NH2
26 R = CH2NHBoc
27 R = CH2CH2NHBoc
28 R = CH2CH2CH2NHBoc
29 R = CH2CH2CH2CH2NHBoc
30 R = CH2CN
41 R = CH2CH2NEt2
31 R = CH2CH2CN
42 R = CH2CH2CH2NEt2
43 R = CH2CH2CH2CH2NEt2
Scheme 1. Reagents and conditions: (a) Ac2O, DMAP, rt, 12 h, 95%; (b) Lawesson’s reagent, o-xylene, 130 °C, 30 min, 25% (15% as a mixture with 4,7-bisthioamide; 10% as a
mixture with 4-thioamide and unreacted 5); (c) BnBr, DBU, CH2Cl2, rt, 1 h, 74%; (d) 6 N HCl, CH3CN, rt, 1 h; (e) PhNCS, CH3CN, rt, 2.5 h, 86% (two steps); (f) 6 N HCl, CH3CN, rt,
4 h, 87%; (g) Fmoc-amino acid, EDC–HCl, CH2Cl2, rt, 18 h, 89–99%; (h) 2 N NaOH, CH3OH, rt, 2.5 h; (i) (n-PrO2)3, DMAP, 0.2 mM in CH2Cl2, rt, 60 h, 37–54% (two steps);
(j) CF3CO2H, CH2Cl2, 0 °C to rt, 45 min, 73–88%; (k) NaB(OAc)3H, H2C@O in CH3OH or CH3C(@O)H in CH2Cl2, rt, 24 h, 80–90%.
chain at the 8-position (34 and 39) exhibited distinctly greater
binding to CypA than CypB, while the other two compounds of this
series (29 and 43) showed comparable affinity to both CypA and
CypB. A possible explanation for this difference in CypA and CypB
binding is that a key Cyp loop containing an Arg (Arg148 in CypA;
Arg158 in CypB) proximal to the CsA 8-residue is positioned
differently in CypA than it is in CypB.22 X-ray crystal structures
of CypA–CsA and CypB–CsA complexes suggest that the CsA 8-po-
sition a-carbon is closer to this particular Arg guanidine moiety in
CypA (ꢁ6.5 Å) than it is in CypB (ꢁ15.0 Å). This may in turn intro-
duce a distinction between CypA and CypB binding in those CsA
analogs with lengthier 8-position substituents. Beyond this, there

A. Scribner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6542–6546
6545
Table 1
Biological activity of [D-Lys]8CsA analogs
HO
H
N
N
N
O
N
N
H
N
O
O O
O
O
N
O
O
N
O
O
H
N
H
N
H
N
O
R
Compound
R
ELISA CypA IC50 (nM)
ELISA CypB IC50 (nM)
IL-2 EC50 (nM)
HCV EC50/EC90 (nM)
Huh-7 cytotoxicity IC50/IC90 (nM)
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
CH2NHBoc
83.5
31.2
26.7
22.8
9.12
23.0
103
66.8
26.1
30.5
20.6
9.45
24.2
79.7
151
1583
12.7
25.2
59.5
28.9
97.1
158
50
22
21
121
5.1
12
13
90
111
94
20
24
39
332
55
114
134
2168
200/480
140/360
120/310
160/300
40/140
1719/>10,000
2015/>10,000
2069/>10,000
2565/>10,000
2818/>10,000
2371/>10,000
4612/>10,000
5303/>10,000
>10,000/>10,000
>10,000/>10,000
2054/>10,000
3275/>10,000
2154/8733
CH2CH2NHBoc
CH2CH2CH2NHBoc
CH2CH2CH2CH2NHBoc
CH2C„N
CH2CH2C„N
CH2NH2
100/270
300/820
660/2350
1890/6740
1000/2850
190/670
170/300
140/380
140/300
380/950
170/350
150/370
180/530
CH2CH2NH2
218
662
CH2CH2CH2NH2
CH2CH2CH2CH2NH2
CH2NMe2
CH2CH2NMe2
CH2CH2CH2NMe2
CH2CH2CH2CH2NMe2
CH2NEt2
CH2CH2NEt2
CH2CH2CH2NEt2
CH2CH2CH2CH2NEt2
11.1
22.9
46.0
35.2
14.0
140
65.2
21.4
26.6
2807/>10,000
2683/>10,000
1496/8620
1817/8913
2239/7897
55.9
17.2
19.8
1 (CsA)
4 (SCY-635)
8.33
9.48
8.78
10.3
6.7
4209
370/910
50a/240a
5810/18,750
>10,000/>10,000
a
These values were obtained when tested alongside compounds 26–43.
is no obvious trend suggesting that either chain length or nitrogen
basicity has a clear influence on Cyp binding, although primary
amines (32–35) in three of four cases showed distinctly weaker
Cyp binding.
EC50 <100 nM, and a total of 13 out of 18 with an EC50 6 200 nM.
The only sub-series showing weak activity were the primary amines
(32–35), the latter two of which have an EC50 >1000 nM, possibly
due to poor cell permeability. While many of these compounds
exhibited anti-HCV activity comparable to that of SCY-635, they
were also distinctly more cytotoxic in the Huh-7 assay than SCY-
Immunosuppressive activity was determined by an interleukin-
2 (IL-2) inhibition assay, as IL-2 is a key product of T-cell stimula-
tion that is mediated by calcineurin activation. The IL-2 assay mea-
sures inhibition of IL-2 production in Jurkat cells stimulated with
phytohemaglutinin-M and phorbol myristate acetate.23 As a refer-
ence, when SCY-635 (4, IL-2 EC50 = 4209 nM) was administered to
human clinical trial patients at a dose of 3 ꢂ 300 mg per day for
15 days, a subset of four patients analyzed showed no significant
(i.e., <10%) reduction in plasma IL-2 concentrations.24 Of the set
shown in Table 1, three compounds with relatively small substitu-
ents at position 8 (30–32) showed immunosuppressive activity
rivaling that of CsA; several others were within 10-fold of that of
CsA. It is noted that within a given series of compounds with iden-
tical N-substituents, immunosuppression tended to attenuate with
increasing chain size at position 8. The two least immunosuppres-
sive compounds, 39 and 43, include a tertiary amine nitrogen five
atoms away from the ring followed by one or two carbons, suggest-
ing that a basic chain reaching this size threshold may have a
repulsive interaction with CN. Analogs that also possess a nitrogen
five atoms away from the ring but are either less basic (29) or less
bulky (35) were more immunosuppressive. While none of these
compounds were found to be as non-immunosuppressive as SCY-
635 itself, compounds 29, 34, 39, 41, 42, and in particular 43 dem-
onstrate that adding length to the 8-position substituent can re-
duce immunosuppression to within twofold of that of SCY-635.
Anti-HCV activity was assessed in the con 1b derived bi-cistronic
replicon containing a stable luciferase reporter at 72 h.25 Cytotoxic-
ity in Huh-7 cells was determined by measuring the release of lac-
tate dehydrogenase. HCV data reveals one compound (30) with an
635, which exhibited no cytotoxicity up to 10
In summary, we have synthesized [
-Lys]8CsA in 6.3% yield in
10 steps from CsA. We then synthesized [
-Lys]8CsA analogs that
lM.
D
D
show anti-HCV activity greater than or comparable to that of
CsA, several of which are significantly less immunosuppressive
than CsA. In particular, compounds 39 and 43 each exhibit an
anti-HCV EC50 <200 nM, and are each P50-fold less immunosup-
pressive than CsA. While none of these compounds combine the
anti-HCV potency, lack of immunosuppression, and safety profile
exhibited by SCY-635, these results suggest the 8-position of CsA
holds promise as a synthetically feasible site for modification to-
wards potential anti-HCV therapeutics.
Acknowledgments
We thank Sam Hopkins and Paul Kowalczyk for their valuable
comments in preparing this manuscript.
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