Novel oxidation of Cyclosporin A: Preparation of Cyclosporin methyl vinyl ketone (Cs-MVK)

Article abstract of DOI:10.1055/s-0029-1218011

Cyclosporin A (CsA) was converted into cyclosporin methyl vinyl ketone (Cs-MVK) by either a biocatalytic method utilizing 1-hydroxybenzotriazole- mediated laccase oxidation or by a chemical oxidation using t-butyl hydroperoxide and potassium -periodate as

Full text of DOI:10.1055/s-0029-1218011

LETTER
2935
Novel Oxidation of Cyclosporin A: Preparation of Cyclosporin Methyl Vinyl
Ketone (Cs-MVK)
N
ovel Oxidation
h
of
C
yclosp
i
orin A cai Yang,a Kevin Pattamana,a Bruce F. Molino,*a Simon N. Haydar,a Yeyu Cao,a Frederic Bois,a Jun-Ho Maeng,b
Michael S. Hemenway,b Joseph O. Rich,b Yuri L. Khmelnitsky,b Thomas D. Friedrich,c Denise Peace,c
Peter C. Michelsb
a
Medicinal Chemistry, AMRI, 26 Corporate Circle, P.O. Box 15098, Albany, NY, 12212-5098, USA
Fax +1(518)5122079; E-mail: bruce.molino@amriglobal.com
Discovery Research and Development, AMRI, 26 Corporate Circle, P.O. Box 15098, Albany, NY, 12212-5098, USA
Center for Immunology & Microbial Disease, Albany Medical College, 43 New Scotland Ave., Albany, NY, 12208
b
c
Received 14 July 2009
of O2.8a The use of laccase in synthesis is not well-known.
Abstract: Cyclosporin A (CsA) was converted into cyclosporin
methyl vinyl ketone (Cs-MVK) by either a biocatalytic method uti-
lizing 1-hydroxybenzotriazole-mediated laccase oxidation or by a
chemical oxidation using t-butyl hydroperoxide and potassium
One of the few published reports indicates that a laccase
mediator-assisted oxidation is useful for the transforma-
tion of a benzyl methyl group or an allylic alcohol into the
periodate as co-oxidants. Cs-MVK is a novel, versatile synthetic in- corresponding alcohol and/or aldehyde, respectively.8b
termediate that can be used for the preparation of many novel cy-
closporin analogues possessing therapeutic potential as
immunosuppressive agents.
Key words: cyclosporin A, cyclosporin methyl vinyl ketone, lac-
case oxidation, allylic oxidation, immunosuppression
HO
H
N
N
N
O
N
N
N
O
1
O
O
O
O
N
O
O
Cyclosporin A (1; Figure 1), a naturally occurring cyclic
peptide isolated from the fungus Tolypocladium infla-
tum,1 possesses potent immunosuppressive activity and
continues to be an important first-line agent prescribed for
the prevention of organ transplantation rejection.2,3 In ad-
dition, cyclosporin A and its analogues have demonstrated
potential utility in other therapeutic areas, such as psoria-
sis, asthma and other autoimmune diseases.3 The reactiv-
ity of a variety of biocatalysts on CsA was explored in our
laboratory with the goal of introducing functionality onto
the CsA scaffold that could lead to novel and medicinally
useful CsA analogues.
N
O
H
N
H
N
H
N
O
O
1
cyclosporin A (CsA) is abbreviated as
HO
Cs
It has been well established that the side chain of the first
amino acid of cyclosporin A [N,4-dimethyl-4(R)-[2(E)-
butenyl]-L-threonine (MeBmt, 2); Figure 2] plays an es-
sential role in the immunosuppressive activity of CsA.4
The presence of the trans carbon–carbon double bond and
the hydroxy substituent on the MeBmt side chain makes
this a readily accessible part of the molecule for synthetic
modification. Therefore, many medicinal chemistry ef-
forts have focused on modification of the side chain of this
amino acid.4–7
Figure 1 Cyclosporin A (CsA)
O
HO
HO
Me
HO2C
N
H
Cs
2
3
One of the interesting reactions that was found to take
place during the biocatalyst screening strategy was the 1-
hydroxybenzotriazole (HOBt)-mediated laccase oxida-
tion of CsA 1. Laccase (EC 1.10.3.2) is characterized as a
multi-copper oxidase that can catalyze the oxidation of a
range of reducing substances with concomitant reduction
OH
CHO
HO
HO
Cs
Cs
4
5
SYNLETT 2009, No. 18, pp 2935–2938
x
x
.x
x
.2
0
0
9
Advanced online publication: 02.10.2009
DOI: 10.1055/s-0029-1218011; Art ID: S08009ST
© Georg Thieme Verlag Stuttgart · New York
Figure 2 MeBmt (2), Cs-MVK (3), and other cyclosporin ana-
logues

2936
Z. Yang et al.
LETTER
Application of the laccase mediator-assisted oxidation This reaction was run several times on a 5 g scale under
conditions to CsA 1 (Scheme 1) afforded the unexpected the conditions described (Table 1, entry 5) to provide the
cyclosporin methyl vinyl ketone 3 (Cs-MVK) as the major desired product in yields consistently in the 50–70%
product, instead of the expected products from the allylic range.9
oxidation of the terminal methyl group to a Cs-alcohol 4
The precise mechanism of the oxidation of cyclosporin A
by the HOBt-mediated laccase oxidation or under the
or aldehyde 59 shown in Figure 2, which were only ob-
served in trace amounts. Cs-MVK 3 is a novel CsA deriv-
KIO4/t-BuOOH/18-crown-6 system remains unclear;
ative that has neither been reported previously in the
however, the synthetic utility of the novel Cs-MVK 3 was
readily demonstrated (Scheme 2). The novel Cs-aldehyde
6 was prepared in excellent yield by ozonolysis of Cs-
MVK 3 followed by reductive workup. Acetylation of the
literature as an oxidative metabolite from human liver me-
tabolism nor as a reaction product from biocatalytic or
chemical reaction.
secondary alcohol of Cs-MVK 3 followed by catalytic hy-
drogenation of the double bond afforded the saturated Cs-
O
ketone 7. Direct hydrogenation of compound 3 resulted in
the formation of the stable Cs-cyclic hemi-ketal 9.
a
HO
HO
or b
O
Cs
Cs
a
c
1
3
HO
3
Scheme 1 Reagents and conditions: (a) laccase, HOBt, methyl
t-butyl ether, buffer pH 5.6 (biocatalytic method); (b) 70% t-butyl hy-
droperoxide, KIO4, 18-crown-6, acetone, benzene, H2O, r.t., 3 d (che-
mical method).
Cs
6
OH
The novel structure and potential synthetic utility of Cs-
MVK 3 prompted us to look for a more convenient chem-
ical method for the preparation of this compound. A sur-
vey of the chemistry literature indicated that co-oxidants
have been used successfully to effect oxidation of allylic
methyl groups. 1-Hydroxyphthalimide/benzoyl peroxide
(PhCOO)2,10 sodium periodate (NaIO4)/tert-butyl hydro-
peroxide (t-BuOOH),11 and chromium trioxide (CrO3)/t-
BuOOH12 have been successfully applied in the oxidation
of the allylic carbon atoms on steroids. However, when
CsA 1 was treated under these reaction conditions, only a
trace (<5% yield) of the desired Cs-MVK 3 was observed
along with unreacted CsA.
O
b
Cs
9
O
O
c
AcO
AcO
Cs
Cs
8
7
Scheme 2 Reagents and conditions: (a) O3, CH2Cl2, –78 °C, 20
min, then Me2S, –78 °C to r.t., 90%; (b) Ac2O, pyridine, DMAP,
CH2Cl2, r.t., 95%; (c) H2 (30 psi), 10% Pd/C, MeOH, r.t., 85%.
Attempts to drive the reaction to completion at higher
reaction temperatures led to decomposition of the desired
product that formed (Table 1, entries 1 and 3). Treatment
of CsA with NaIO4/t-BuOOH at room temperature in the
presence of a phase-transfer catalyst (BnNEt3Cl, Table 1,
entry 4), however, provided a modest improvement (10%
yield). Variation of the co-oxidizing agents led to the find-
ing that potassium periodate (KIO4)/t-BuOOH with 18-
crown-6 as the phase-transfer catalyst in a solvent system
of benzene–acetone–water (1:1:1) for three days at room
temperature was optimal (Scheme 1, Table 1, entry 5).
Both Cs-aldehyde 6 and Cs-ketone 7 were further trans-
formed into novel cyclosporin analogues as outlined in
Scheme 3. Treatment of Cs-ketone 7 with ten equivalents
of Wittig reagent prepared from methyltriphenylphospho-
nium bromide and sodium bis(trimethylsilyl)amide, pro-
vided Cs-alkene 10, which was deacylated under mild
basic conditions (potassium carbonate/methanol) to give
the novel analogue 11.
Table 1 Oxidation of Cyclosporin A 1 to Cs-MVK 3
Entry
Co-oxidants
Conditions
Yield of 3 (%)
1
2
3
4
5
1-hydroxyphthalimide/(PhCO2)2
NaIO4/t-BuOOH
NaIO4/t-BuOOH
NaIO4/t-BuOOH
KIO4/t-BuOOH
acetone, reflux, 24 h
<5
acetone, r.t., 3 d
<5
acetone, 50 °C, 12 h
decomposed
10
BnNEt3Cl, benzene, acetone, H2O, r.t., 3 d
18-crown-6, benzene, acetone, H2O, r.t., 3 d
50–70
Synlett 2009, No. 18, 2935–2938 © Thieme Stuttgart · New York

LETTER
Novel Oxidation of Cyclosporin A
2937
O
shown to be a synthetically useful intermediate that was
transformed into several new cyclosporin analogues via
standard methodology. The discovery of this chemistry
provides a facile approach to a variety of novel cyclospor-
in derivatives from commercially available CsA that were
not readily accessible prior to the existence of this meth-
odology.
a
b
AcO
AcO
HO
Cs
Cs
Cs
7
10
11
CN
O
References and Notes:
d
c
HO
HO
HO
(1) Dreyfuss, M. H.; Hofmann, H.; Kobel, H.; Pache, W.;
Tscherter, H. Eur. J. Appl. Microbiol. 1976, 3, 125.
(2) Borel, J. F.; Feurer, C.; Gubler, H. V.; Stahelin, H. Agents
Actions 1976, 6, 468.
Cs
Cs
13
Cs
12
6
e
(3) Faulds, D.; Goa, K. L.; Benfiled, P. Drugs 1993, 45, 953.
(4) (a) Rich, D. H.; Sun, C. Q.; Guillaume, D.; Dunlap, B.;
Evans, D. A.; Weber, A. E. J. Med. Chem. 1989, 32, 1982.
(b) Aebi, J. D.; Deyo, D. T.; Sun, C. Q.; Guillaume, D.;
Dunlap, B.; Rich, D. H. J. Med. Chem. 1990, 33, 999.
(c) Huai, Q.; Kim, H.-Y.; Liu, Y.; Zhao, Y.; Mondragon, A.;
Liu, J. O.; Ke, H. Proc. Natl. Acad. Sci. USA 2002, 12037.
(5) (a) Lazarova, T.; Chen, J. S.; Hamann, B.; Kang, J. M.;
Homuth-Trombino, D.; Han, F.; Hoffmann, E.; McClure,
J. E.; Or, Y. S. J. Med. Chem. 2003, 46, 674. (b) Lazarrova,
T.; Weng, Z. Expert Opin. Ther. Pat. 2003, 13, 1327.
(6) (a) Dumont, F. J. Curr. Opin. Invest. Drugs 2004, 5, 542.
(b) Birsan, T.; Dambrin, C.; Freitag, D. G.; Yatscoff, R. W.;
Morris, R. E. Transplant International 2005, 17, 767.
(7) Papp, K.; Bissonnette, R.; Rosoph, L.; Wasel, N.; Lynde,
C. W.; Searles, G.; Shear, N. H.; Huizinga, R. B.;
f
O
Me
O
NH2
NH
N
g
HO
HO
HO
Cs
15
Cs
16
Cs
14
Scheme 3 Reagents and conditions: (a) CH3PPh3Br, NaHMDS,
THF, 0 °C, 30 min, 12%; (b) K2CO3, MeOH, r.t., 12 h, 35%; (c)
MeCH2PPh3Br, NaHMDS, THF, 0 °C, 30 min, 30%; (d)
(EtO)2P(O)CH2CN, NaHMDS, THF, 0 °C, 30 min, 49%; (e)
MeONH2·HCl, pyridine, MeOH, r.t., 20%; (f) NH4OAc, NaBH3CN,
HOAc, MeOH, r.t., 20%; (g) benzoyl chloride, pyridine, CH2Cl2, r.t.,
26%.
Maksymowych, W. P. Lancet 2008, 371, 1337.
Similarly, a mixture of cis/trans-alkenes 12 resulted from
a Wittig reaction between the Cs-aldehyde 6 and an ex-
cess of ethylidene triphenylphosphorane generated in situ.
Horner–Emmons reaction of Cs-aldehyde 6 and diethyl
cyanomethylphosphonate using sodium bis(trimethyl-
silyl)amide as base afforded Cs-a,b-unsaturated nitrile 13
exclusively as the trans-alkene.
(8) (a) Xu, F.; Kulys, J. J.; Duke, K.; Li, K.; Krikstopaitis, K.;
Deussen, H.-J. W.; Abbate, E.; Galinyte, V.; Schneider, P.
Appl. Environ. Microbiol. 2000, 66, 2052. (b) Fritz-
Langhals, E.; Kunath, B. Tetrahedron Lett. 1998, 39, 5955.
(9) Biocatalytic Method: Cyclosporin A (1.0 g) and 1-
hydroxybenzotriazole (500 mg) were dissolved in tert-
butanol (70 mL) in a 500 mL reaction vessel equipped with
a stir bar. Sodium citrate/sodium phosphate buffer (80 mM,
250 mL, pH 5.6) was added while stirring, resulting in a
thick white suspension. Laccase C (1.8 g, ASA
In addition, treatment of Cs-aldehyde 6 with O-methylhy-
droxylamine gave the oxime 14, and reductive amination
of the same aldehyde led to the preparation of a novel Cs-
amine derivative 15, which was benzoylated to give the
Cs-amide 16.
Spezialenzyme) was added as a solution in 35.5 mL of the
same buffer, turning the reaction mixture slightly yellow in
appearance. The reaction was mechanically stirred enough
to create a vortex, open to ambient atmosphere at room
temperature for a period of 20 h, after which time the
reaction mixture became orange in appearance. After
removing a portion of the tert-butanol via rotavapor, the
orange reaction mixture was loaded onto a pre-conditioned
VARIAN Bond-Elut® C8 solid-phase extraction cartridge
(60 cc, 10 g of sorbent). After a wash with water, the
cyclosporin-related products were eluted using acetonitrile.
The acetonitrile eluate was concentrated in vacuo, and the
residue was transferred to a tared scintillation vial and dried
in vacuo inside a Savant dryer to provide 913 mg of crude
product as tan solids. The solids were re-dissolved in a
minimal volume of acetonitrile and purified by reversed-
phase semi-prep chromatography to provide 551 mg of CsA-
MVK.
Novel cyclosporin analogs were tested for activity in a
one-way murine mixed lymphocyte reaction (MLR) as-
say. The MLR assay is designed to measure 3H-thymidine
uptake by murine splenocytes that are undergoing cell
proliferation in an immune response to allogeneic stimu-
lation.13 The Cs-MVK 3 and the Cs-cyclic hemi-ketal 9
retained significant IC50 values (310 and 160 ng/mL, re-
spectively) but attenuated inhibition of 3H-thymidine up-
take relative to CsA 1 (IC50 values of 14 and 16 ng/mL,
respectively), which is run as a positive control in the as-
say.
In conclusion, an HOBt-mediated laccase oxidation of
CsA 1 produced the novel derivative Cs-MVK 3 in good
yield. Efforts undertaken to identify a chemical oxidation
method with which to synthesize Cs-MVK 3 led to iden-
tification of a co-oxidizing system, (KIO4)/t-BuOOH with
18-crown-6 in benzene–acetone–water (1:1:1) that afford-
ed the desired product in good yield. Cs-MVK 3 has been
Chemical Method: Cyclosporin A (5 g, 4.2 mmol) was
dissolved in acetone (25 mL), benzene (25 mL) and H2O (25
mL). tert-Butyl hydroperoxide (31.25 mL of 70% aqueous
solution, 258 mmol), potassium periodate (6.5 g, 28.3
mmol), and 18-crown-6 (4.38 g, 16.5 mmol) were added to
the reaction mixture at room temperature. The resulting
mixture was stirred vigorously at room temperature under N2
Synlett 2009, No. 18, 2935–2938 © Thieme Stuttgart · New York

2938
Z. Yang et al.
LETTER
atmosphere for 3 d. Organic solvents were removed from the
reaction mixture in vacuo. The remaining mixture was
poured into ice-water (1 L) and extracted twice with a
mixture of EtOAc–hexanes (200 mL, 1:1). The combined
extracts were stirred in a 10% sodium sulfite solution for 2
h. The organic layer was separated, dried over Na2SO4 and
concentrated. The crude product was purified by either
preparative or semi-preparative HPLC, using acetonitrile
(containing 0.05% TFA)/water (containing 0.05% TFA)
solvent system, to provide CsA-MVK (2.5–3.5 g, 50–70%)
as light-yellow solid.
(s, 3 H), 2.20–1.76 (m, 11 H), 1.75–1.35 (m, 6 H), 1.32 (d,
J = 7.3 Hz, 3 H), 1.26 (d, J = 7.3 Hz, 3 H), 1.10–0.81 (m, 39
H); 13C NMR (90 MHz, CDCl3): d = 198.6, 174.7, 174.1 (2
C), 173.8, 171.7, 171.6, 171.3, 170.6, 170.5, 170.3, 170.2,
148.9, 131.8, 74.9, 59.6, 58.2, 57.8, 55.7 (2 C), 55.2, 50.6,
48.9, 48.6, 48.3, 45.3, 40.7, 39.7, 39.5, 39.2, 38.0, 36.2, 35.0,
31.7, 31.6, 31.3, 30.1 (2 C), 29.8, 29.6, 27.5, 25.3, 25.1, 24.9
(2 C), 24.6, 24.0 (4 C), 23.8, 23.6, 22.0 (2 C), 21.3, 20.8,
20.0, 18.8 (2 C), 18.6, 18.4, 16.1; MS (ESI): m/z = 1216.
(10) Foricher, J.; Fürbringer, C.; Pfoertner, K. US Patent,
US5,030,739, 1991.
Analytical Data of 3: 1H NMR (300 MHz, CDCl3): d = 8.03
(d, J = 9.9 Hz, 1 H), 7.79 (d, J = 7.8 Hz, 1 H), 7.44 (d, J = 8.0
Hz, 1 H), 7.13 (d, J = 8.0 Hz, 1 H), 6.89 (dd, J = 16.1, 7.6
Hz, 1 H), 6.06 (d, J = 16.1 Hz, 1 H), 5.71 (dd, J = 11.0, 3.8
Hz, 1 H), 5.65 (br s, 1 H), 5.22 (dd, J = 11.5, 3.8 Hz, 1 H),
5.10 (d, J = 11.0 Hz, 2 H), 5.05 (dd, J = 15.7, 9.1 Hz, 1 H),
4.96 (dd, J = 10.1, 5.7 Hz, 1 H), 4.85 (q, J = 7.2 Hz, 1 H),
4.73 (d, J = 14.1 Hz, 1 H), 4.65 (q, J = 8.7 Hz, 1 H), 4.55 (q,
J = 7.4 Hz, 1 H), 4.04 (br s, 2 H), 3.52 (s, 3 H), 3.39 (s, 3 H),
3.31 (s, 3 H), 3.20 (d, J = 13.9 Hz, 1 H), 3.12 (s, 3 H), 3.11
(s, 3 H), 2.72 (s, 3 H), 2.68 (s, 3 H), 2.54–2.34 (m, 3 H), 2.26
(11) Marwah, P.; Lardy, H. A. US Patent, US5,869,709, 1999.
(12) Muzart, J. Tetrahedron Lett. 1987, 28, 4665.
(13) The murine system uses the H2 disparate inbred mouse
strains: Balb/c (H2d) and C57B1/6 (H2b). Splenocytes of the
C57B1/6 mice are g-irradiated so as to act as stimulators of
an immune response from the splenocytes from the Balb/c
mice. IC50 values are the concentration of test compound that
inhibit 3H-thymidine uptake by 50% relative to control cells
and are determined from 7 point concentration-response
curves using GraphPad software.
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