Synthesis and neurotrophic activity of nonimmunosuppressant cyclosporin A derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.06.028

In order to exploit cyclophilin as a potential target for neurological drug design, we demonstrate in this presentation that several nonimmunosuppressant analogues of cyclosporin A, modified at the various positions in the 'effector' domain, are equipotent nerve growth agents compared to cyclosporin A. Our results suggest that neurotrophic activity of cyclosporin A and its derivatives resides in the binding domain, and binding to cyclophilin and/or inhibiting rotamase activity may be a necessity for neurotrophic effects of cyclophilin ligands.

Full text of DOI:10.1016/j.bmcl.2004.06.028

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4549–4551
Synthesis and neurotrophic activity of nonimmunosuppressant
cyclosporin A derivatives
Ling Wei, Joseph P. Steiner, Gregory S. Hamilton and Yong-Qian Wu*
Department of Research, Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MD 21224, USA
Received 12 June 2003; accepted 8 June 2004
Available online 2 July 2004
Abstract—In order to exploit cyclophilin as a potential target for neurological drug design, we demonstrate in this presentation that
several nonimmunosuppressant analogues of cyclosporin A, modified at the various positions in the ‘effector’ domain, are equi-
potent nerve growth agents compared to cyclosporin A. Our results suggest that neurotrophic activity of cyclosporin A and its
derivatives resides in the binding domain, and binding to cyclophilin and/or inhibiting rotamase activity may be a necessity for
neurotrophic effects of cyclophilin ligands.
Ó 2004 Elsevier Ltd. All rights reserved.
The term immunophilin refers to a number of proteins
that serve as receptors for the principal immunosup-
pressant drugs, cyclosporin A (CsA), FK506 and rapa-
side effect. Therefore, in order to exploit cyclophilin as
potential target for neurological drug design, the critical
question whether the neurotrophic effect of cyclophilin
ligands is separable from the immunosuppressive activ-
ity needs to be answered. We have shown previously
that a nonimmunosuppressant analogue of cyclosporin
A (6-MeVal-CsA) promotes neurite outgrowth in neu-
1;2
mycin.
cyclophilins (CyP), and FK506 binding proteins
FKBP). Cyclosporin A binds to cyclophilin while
Known classes of immunophilins are
(
FK506 and rapamycin bind to FKBP. Immunophilins
are also known to possess peptidyl-prolyl isomerase
6
ronal cultures with potency resembling CsA. In this
(
the interconversion of the cis and trans rotamers of
amide bonds adjacent to proline residues in peptidic
3
substrates. Immunophilins were originally discovered
PPIase) or rotamase enzyme activity, which catalyzes
report, we wish to extend and further validate the pre-
vious findings with several additional nonimmunosup-
pressant cyclosporin A analogues, which are modified at
various positions and with various amino acid residues
(Fig. 1).
and characterized in immune tissue. It was shown that
the inhibition of cyclophilin rotamase activity by
cyclosporin A, in and of itself, is not sufficient for
immunosuppressant activity. Instead immunosuppres-
sion appears to stem from the formation of CsA-CyP
complexes that inhibit the intrinsic target protein––
calcineurin, a calcium/calmodulin dependent protein
Cyclosporin A is a cyclic undecapeptide, and it pos-
sesses two distinct binding domains: a cyclophilin-
binding domain, which expends about five amino acids
4
phosphatase.
3
H C
HO
O
CH
N
3
O
CH
3
Recent findings that cyclophilin A is present at high
levels in the CNS and that cyclosporin A possesses
neurotrophic effects suggest a potential therapeutic
utility for cyclophilin ligands in treating neurological
disorders. However, at levels where cyclosporin A
exhibits neurotrophic activity, the known immunosup-
pressant activity of cyclosporin A becomes an unwanted
N
N
O
N
H
N
CH3 11
2
5
1
10
3
4
9
N
O
O
CH
3
5
CH
3
O
H
3
C
N
H
N
O
H
O
O
6
7
8
N
N
N
H
CH
3
O
CH
3
O
CH
3
*
0
Corresponding author. Fax: +1-410-631-6823; e-mail: wuy@
guildfordpharm.com
Figure 1. Structure of cyclosporin A.
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.028

4550
L. Wei et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4549–4551
fragments A were constructed by coupling MeBmt res-
MeBmt-Abu-Sar-Y-Val-MeLeu-Ala-Bzl
Boc-X-MeLeu-MeLeu-MeVal
1
2
3
4
5
6
7
+
8
9
10
11
idue as an acetonide derivative to hexapeptide precur-
sors, followed by acid deprotection. Fragments A, with
various amino acids at position 4, correspond to the
residues 1–7 of CsA. Fragments B, which were also
synthesized stepwise, correspond to the residues 8–11 of
A
B
2 2
BOP/NMM/CH Cl
Boc-X- MeLeu-MeLeu-MeVal-MeBmt-Abu-Sar-Y-Val-MeLeu-Ala-Bzl
CsA. Position 8 contains either
D-Ala or dehydroAla
a. NaOH/EtOH/H
b. TFA/CH Cl
2
O
residue. The free amines of fragments A were then
coupled to Boc-protected tetrapeptide fragments B,
leading to desired linear undecapeptides. Double de-
protection of both C-terminals and N-terminals of un-
decapeptides, followed by cyclization with BOP in
dilution condition (<0.25 mM concentration) gave four
CsA analogues with yields between 40% and 50%. Sep-
arately, dihydroCsA was obtained from CsA by 10% Pd/
C catalyzed hydrogenation of double bond at position 1.
Synthesized analogues were purified by HPLC, and
2
2
X- MeLeu-MeLeu-MeVal-MeBmt-Abu-Sar-Y-Val-MeLeu-Ala
2 2
BOP/NMM/CH Cl
3
4
days/RT
0-50% yield
MeLeu-MeLeu-MeVal-MeBmt-Abu-Sar-Y-Val-MeLeu
4
X
Ala
8
1
characterized by H NMR and mass spectroscopy, and
most of the analogues (Table 1) were reported
10a;b;c
and known in the literature.
When X = D-Ala, Y = MeVal, MehomoAla or Me(α-methyl)Thr
When X = dehydroAla, Y = MeLeu
Scheme 1. Total synthesis of cyclosporin A analogues.
The synthesized cyclosporin A analogues were evaluated
for cyclophilin A rotamase inhibition by a method
previously described. This is a chymotrypsin-coupled
assay using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide
11
(
MeLeu9, MeLeu10, MeVal1, MeBmt1 and Abu2), and
an ‘effector domain’, which interacts with calcineurin
after the formation of the CsA-CyP complex (Sar3,
as a substrate. The measured inhibition constants k are
i
MeLeu4, Val5, MeLeu6, Ala7).
D
-Ala8 residue of CsA
the mean of at least three independent estimations with
variation generally less than 20% (Table 1). Modifica-
tion at position 4 resulted in several analogues, which
are inhibitors for rotamase as potent as cyclosporin A,
essentially independent of different amino acid substi-
tutions. However, analogue modified at position 8,
which is closer to the binding domain, has threefold less
potency than that of CsA against rotamase. Not sur-
prisingly, the saturated dihydroCsA, modified at bind-
ing domain, is fivefold less potent than CsA. On the
other hand, the analogues modified at position 4 lose
immunosuppressive activity (>10 lM) in a standard T-
contacts with neither cyclophilin nor calcineurin di-
rectly, suggested by recently solved X-ray crystal struc-
7
ture of CyP-CsA-CN complex. It appears that there is
an open, presumably water filled cavity around residue 8
in the ternary complex. It has been known that modifi-
cation in the ‘effector domain’ would interrupt activity
towards calcineurin, resulting in nonimmunosuppressive
compounds. As shown in Scheme 1, we synthesized
several cyclosporin A analogues modified at both 4 and
8 positions employing a solution-phase fragment cou-
pling strategy, which was originally developed by
Wenger in the first synthesis of CsA. Heptapeptide
8;9
12
cell proliferation inhibition assay, shown in Table 1.
Table 1. Biological activity of cyclosporin A analogues
Compounds
Modified residue
––
k
i
(nM)/CyP
ED50 (nM) T-cell prolif.
50
Neutrite outgrowth
+++
10a
CsA
20
HO
O
N
10a
DihydroCsA
100
130
+
10b
O
N
[
DehydroAla]8-CsA
75
10
>10,000
>10,000
+
H
10c
[
MeVal]4-CsA
O
N
+++
1
0c
O
N
[
[
MeAbu]4-CsA
24
18
>10,000
>10,000
+++
+++
OH
Me(a-methyl)Thr]4-CsA
O
N

L. Wei et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4549–4551
4551
derivatives resides in the binding domain, and binding to
cyclophilin and/or inhibiting rotamase activity may be a
necessity for neurotrophic effects of cyclophilin ligands.
At this point, the molecular mechanism of such effects
remains elusive, although several possibilities are worth
mentioning. One possibility is that interaction of the
compounds with cyclophilin or a cyclophilin-like protein
results in formation of an active complex, leading to a
gain of function for the cyclophilin. Another possibility
is that other cyclophilins (e.g., cyclophilin D in mito-
chondrial), present in lower concentrations in nerve cells,
15
mediate the actions of these compounds. Despite many
unanswered questions, cyclophilin may well, in our
opinion, serve as an attractive target for small-molecule
intervention against neuro-degenerative disorders such
as Alzheimer’s disease and Parkinson’s disease.
Figure 2. Dose–response curve of cyclosporin A in promoting neurite
outgrowth.
References and notes
Although there are published examples of other
amino acid substituents at position 8 that retain
immunosuppressive activity, dehydroAla8 CsA ana-
logue failed to inhibit T-cell proliferation up to 10 lM,
D-
1
. Handschumacher, R. E.; Harding, M. W.; Rice, J.;
Drugge, R. J.; Speicher, D. W. Science 1984, 226, 544.
13
2. Siekierka, J. J.; Hung, S. H.; Poe, M.; Lin, C. S.; Sigal,
N. H. Nature 1989, 341, 755.
3. Fisher, G.; Wittmann-Liebold, B.; Lang, K.; Kiefhaber,
T.; Schmid, F. X. Nature 1989, 340, 351.
suggesting maintaining a
D
-configuration at this posi-
tion may be important for the inhibition of calcineurin
activity.
4
. Liu, J. Cell 1991, 66, 807.
5
. Dawson, T. M.; Steiner, J. P.; Dawson, V. L.; Dinerman,
J. L.; Uhl, G. R.; Snyder, S. H. Neuroscience 1994, 62, 569.
. Steiner, J. P.; Connolly, M. A.; Valentine, H. L.; Ham-
ilton, G. S.; Dawson, T. M.; Hester, L.; Snyder, S. H.
Nature Med. 1997, 3, 421.
To demonstrate their potential neurotrophic effects,
cyclosporin A and its analogues were evaluated in a
neurite outgrowth assay using chick dorsal root ganglia
6
7
6;14
(
DRG).
Neurite outgrowth was quantified by the
. (a) Huai, Q.; Kim, H. Y.; Liu, Y.; Zhao, Y.; Monderagon,
A.; Liu, J. O.; Ke, H. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 12037; (b) Jin, L.; Harrison, S. C. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 13522.
photomicrographs of explants. It was observed that the
maximal increase in the number of processes, their
length and branching is quite similar at maximally
effective concentrations of cyclosporin A and NGF
8
9
. Wenger, R. M. Helv. Chim. Acta 1984, 67, 502.
. Lee, J. P.; Dunlap, B.; Rich, D. H. Int. J. Pept. Protein
Res. 1990, 35, 481.
(
100 ng/mL). Shown in Figure 2, the ED50 of cyclosporin
A, the dose at which 50% of the maximal response was
elicited, was obtained from dose–response curves, and
calculated to be 5 nM. The potencies of the other ana-
logues at concentration of 100 nM (Table 1) are given
the relative number of ‘+’ marks denoting, when com-
pared to cyclosporin A.
1
0. (a) von Wartburg, A.; Traber, R. Prog. Allergy 1986, 38,
8; (b) Patchett, A. A.; Taub, D.; Goegelman, R. T.
J. Antibiot. 1992, 45, 94; (c) Papageorgiou, C.; Borer, X.;
French, R. R. Bioorg. Med. Chem. Lett. 1994, 4, 267.
1. Kofron, J. L.; Kuzmic, P.; Kishore, V.; Colon-Bonilla, E.;
Rich, D. H. Biochemistry 1991, 30, 6127.
2. Janossy, G.; Greaves, M. F. Clin. Exp. Immunol. 1971, 9,
483–498.
3. (a) Nelson, P. A.; Akselband, Y.; Kawamura, A.; Su, M.;
Tung, R. D.; Rich, D. H.; Kishor, V.; Rosborough, S. L.;
DeCenzo, M. T.; Livingston, D. J. J. Immunol. 1993, 150,
2
1
1
1
The data demonstrate that cyclosporin A and its deriv-
atives, which bind to cyclophilin and inhibit rotamase
activity, whether immunosuppressive or nonimmuno-
suppressive, are capable of promoting neurite outgrowth
in cultured neurons, and are capable of achieving maxi-
mal effects comparable to nerve growth factor itself. In
addition, the neurite outgrowth effect of these derivatives
is apparently insensitive towards amino acid residue
alterations in the ‘effector domain’. The combined data
imply that neurotrophic activity of cyclosporin A and its
2
139; (b) Baumann, G.; Andersen, E.; Quesniaux, V.;
Eberle, M. K. Transplant. Proc. 1992, 24, 43.
4. Tonge, D. A.; Golding, J. P.; Gordon-Weeks, P. R.
Neuroscience 1996, 73, 541.
5. Waldmeier, P. C.; Zimmermann, K.; Qian, T.; Tintelnot-
Blomley, M.; Lemasters, J. J. Curr. Med. Chem. 2003, 10,
1485.
1
1