Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DEUTERATED CYCLOSPORIN ANALOGS AND THEIR USE AS
IMMUNOMODULATING AGENTS
This application is a divisional application of Canadian Patent Application
2,372,639 issued
November 1, 2005, which in turn is a divisional application of Canadian Patent
Application 2,298,572, filed
October 8, 1998, issued April 9, 2002.
Cyclosporin derivatives of the present invention are disclosed which possess
enhanced efficacy and
reduced toxicity over naturally occurring and other presently known
cyclosporins and cyclosporine
derivatives. The cyclosporin derivatives of the present invention are produced
by chemical and isotopic
substitution of the cyclosporine A (CsA) molecule by:
1. Chemical substitution and optionally deuterium substitution of amino acid
1; and
2. Deuterium substitution at key sites of metabolism of the cyclosporine A
molecule such as amino
acids 1, 4, 9.
The most active derivatives of the invention were those possessing both
chemical and deuterium substitution.
The cyclosporins are a family of, neutral, hydrophobic cyclic undecapeptides,
containing a novel
nine-carbon amino acid (MeBmt) at position 1 of the ring that exhibit potent
immunosuppressive,
antiparasitic, fungicidal, and chronic anti-inflammatory properties. The
naturally occurring members of this
family of structurally related compounds are produced by various fungi
imperfecti. Cyclosporines A and C,
are the major components. Cyclosporine A, which is discussed further below, is
a particularly important
member of the cyclosporin family of compounds. Twenty four minor metabolites,
also oligopeptides, have
been identified: Lawen et al., J. Antibiotics 42, 1283 (1989); Traber et al.,
Helv. Chim. Acta 70, 13 (1987);
Von Wartburg and Traber Prog. Med. Chem., 25, 1 (1988).
Isolation of cyclosporines A and C, as well as the structure of A were
reported by A. Ruegger et al.,
Helv. Chim. Acta 59, 1075 (1976); M. Dreyfuss et al., J. Appl. Microbiol. 3,
125 (1976). Crystal and
molecular structures of the iodo derivative of A have been reported by T.J.
Petcher et al., Helv. Chim. Acta
59, 1480 (1976). The structure of C was reported by R. Traber et al., ibid.
60, 1247 (1977). Production of A
and C has been reported by E. Harri et al., U.S. Pat. No. 4,117,118 (1978 to
Sandoz). Isolation,
characterization and antifungal activity of B, D, E, as well as the structures
of A through D have been
reported by R. Traber et al., Helv. Chim. Acta 60, 1568 (1977). Isolation and
structures of E, F, G, H, I:
eidem, ibid. 65, 1655 (1982). Preparation of [2-Deutero-3-fluoro-D-Ala]8-CsA
is disclosed by Patchett et. al.
in GB 2,206,199A which was published on Dec. 29, 1988.
Cyclosporin was discovered to be immunosuppressive when it was observed to
suppress antibody
production in mice during the screening of fungal extracts. Specifically, its
suppressive effects appear to be
related to the inhibition of T-cell receptor-mediated activation events. It
accomplishes this by interrupting
calcium dependent signal transduction during T-cell activation by inactivating
calmodulin and cyclophilin, a
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peptidly propyl isornerase. It also inhibits lymphokine production by T-helper
cells in vitro and arrests the
development of mature CD8 and CD4 cells in the thymus. Other in vitro
properties include inhibition of IL-2
producing T-lymphocytes and cytotoxic T-lymphocytes, inhibition of IL-2
released by activated T-cells,
inhibition of restin; T-lymphocytes in response to alloantigen and exogenous
lymphokine, inhibition of IL-1
production, and inhibition of tnitogen activation of IL-2 producing T-
lymphocytes. Further evidence indicates
that the above effects involve the T-lymphocytes at the activation and
maturarion stages.
Stimulation of TCR (T cell receptor) by foreign antigen on a major
histocompatibility (MHC)
molecule on the surface of the T cell results in the adtivation of a TCR
signal transmission pathway (exact
method of transmission unknown) through the cytoplasm causing the signal
results in the activation of nuclear
transcription factors, i.e. nuclear factors of activated T-cells (NF-AT) which
regulate transcription of T-cell
activation genes. These genes include that of lymphokine interleukin-2 (IL-2).
Translation of the message is
followed by secretion of IL-2. T-cell activation also involves the appearance
of the lymphokine receptor IL-2R
on the cell srface. After iL-2 binds to IL-2R, a lymphokine receptor (LKR)
signal transmission pathway is
activated. The iminunosuppressive drug, rapamycin, inhibits this pathway.
CsA is a potent inhibitor of TCR-mediated signal transduction pathway. It
inhibits binding of NF-AT
to the IL-2 enhancer, and thus inhibits transcriptional activation. CsA binds
to cyclophilin, which binds to
calcineurin, which is a key enzyme in the T-cell signal transduction cascade.
Cyclophilin is found in prokaryotic and eukarotic organisms and is ubiquitous
and abundant.
Cyclophilin is a single polypeptide chain with 165 amino acid residues. It has
a molecular mass of 17.8 kD. A
roughly spherical molecule with a radius of 17 angstroms, cyclophilin has a
eight-stranded antiparallel beta
barrel. inside the barrel, the tightly packed core contains mostly hydrophobic
side chains. CsA has numerous
hydrophobic side chains which allow it to fit into the cyclophilin beta barreL
Cyclophillin catalyzes the
interconversion of the cis and trans-rotamers of the peGlFdyl-prolyl amide
bond of peptide and protein
substrates. Cyclophilin is identical in structure with peptidyl prolyl cis-
trans isomerase and bears structural
resemblance to the superfamily of proteins that transports ligands such as
retinol-binding protein (RBP). These
proteins cany the ligand in the barrel core. But cyclophilin actually carries
the ligand binding site on the
outside of the barrel. The tetrapeptide ligand binds in a lon; deep groove on
the protein siirface between one
face of the beta barrel and the Thrl 16-G1y130 loop.
Further properties have also been reported in studies of the biological
activity of CsA: J. F. Borel et
al., Agents Actions 6,468 (1976). Pharmacology: Eidem. Immunology 32, 1017
(1977); R. Y. Calne, Clin.
Exp. Immunol. 35, 1(1979). Human studies: R. Y. Calne et al., Lancet 2,
1323(1978); R. L. Powles et al., ibid.
1327; R L. Powles et al., ibid 1, 327 (1980): In vitro activity (porcine T-
cells): D. J. White et al.,
Transplantation 27, 55 (1979). Effects on human lymphoid and myeloid cells: M.
Y. Gordon, J. W. Singer,
Nature 279, 43 3(1979). Clinical study of CsA in graft-versus-host disease: P.
J. Tutschka et al.,'Blood 61,
318(1983).
Mechanism of Cvclosporine A Action
Cyclosporine A-Cyclophilin A complex
CsA, as discussed above, binds to the cyclophilin beta barreL Thirteen CyP A
residues define the
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CsA binding site. These residues are Arg 55, Phe 60, Met 61, Gln 63, Gly 72,
Ala 101, Asn 102, Ala 103, GIn
111, Phe 113, Trp 121, Leu 122, His 126. The largest side-chain movements are
1.3 A for Arg 55 and up to
0.7 A for Phe 60, Gln 63, and Trp 121. There are four direct hydrogen bonds
between the CyP A and CsA.
Residues 4, 5, 6, 7, 8 of CsA protrude out into the solvent and are thought to
be involved in binding the
effector protein, calcineurin (Pflugl, G., Kallen, J., Schitmer, T.,
Jansonius, J.N., Zurini, M.G.M., &
Walkinshaw, M.D. (1993) Nature 361, 91-94.)
Function of CsA-CyP A complex..
The C$A-CyP A complex inhibits the phosphatase activity of the heterodimeric
protein serine/
threonine phosphatase or calcineurin (Liu, J., Farmer, J.D., Lane, W.S.,
Friedman, J., Weissman, I., &
Schreiber, S.L. (1991) Cel166, 807-15.; Swanson, S.K., Born, T., Zydowsky,
C.D., Cho, H., Chang, H.Y., &
Walsh, C.T. (1992) Proc. Nat1. Acad: Sci.USA 89, 3686-90). CyP A binds CsA
with an affinity of ca. 10 nM.
The complex is then presented to calcineurin (Liu, J., Farmer, J.D., Lane,
W.S., Friedman, J., Weissman, I., &
Schreiber, S.L. (1991) Cell 66, 807-15.).
Calcineurin dephosphorylates the transcription factor NFAT found in the
cytoplasm of T-cells.
Dephosphorylation.allows NFAT to translocate to the nucleus, combine with jun/
fos genes and activate the
transcription of the IL-2 gene responsible for cell cycle progression, leading
to immune response. CsA-CyP A
complex inhibits the phospbatase activity of calcineurin and ultimately
immunosuppression (Etzkorn, F. A.,
Chang, Z., Stolz, L.A., &Walsh, C.T. (1994) Biochemistry 33, 2380-2388.).
Neither CsA or CyP A alone are
important immunologgically. Only their complex is important (Liu, J., Farmer,
J.D., Lane, W.S., Friedman, J.,
Weissman, L, & Schreiber, S.L. (1991) Cell 66, 807-15).
Metabolism of C}=closparine:
Cyclosporine is metabolized in liver, small intestine and kidney ta morc than
30 metabolites. The
.25, structure of 13 metabolites and 2 phase 11 metabolites have been
identified and at least,23 fiuther metabolites
have been isolated by HPLC arid their structures characterized by mass
spectrometry. The reactions involved in
phase I metabolism of cyclosporine are hydroxylation, demethylation as well as
oxidation and cyclisation at
amino acid 1. Several clinical studies and reports showed an association
between blood concentrations of
cyclosporine metabolites and neuro- or nephrotoxicity. In vitro experiments
indicate that metabolites are
considerably less immunosupiessive and more toxic than CsA.
As exemplified by the ever expanding list of indications for which CsA has
been found useful, the
cyctosporin family of compounds find utility in the prevention of rejection or
organ and bone marrow
transplants;'and in the treatment of psbriasis, and a number of autoimmune
disorders such as type I diabetes
mellitus, multiple sclerosfs, autoitnmune uveitis, andrheumatoid arthritis.
Additional indications are discussed
infra.
As is generally accepted by: those of skill in the art, inhibition of
secretion of interleuicin-2 (IL-2) and
other lymphokines from lymphocytes, is a useful indicator of intrinsic
imtnttnosuppressive activity of a
cyclosporin analoo. For a recent review of cyclosporin uses and mechanisms of
action see Wenger et al
Cyclosporine: Chemistry, Structure-Activity Relationships and Mode of Action,
Progress in Clinical
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Biochemistry and Medicine, vol. 2, 176 (1986).
Cyclosporin A is a cyclic peptide which contains several N-methyl amino acids
and, at position-8,
contains a D-alanine. The structure of Cyclosporin A' is given below:
H3C,, H
i
C
MaL 9 MeLeu 10 MeVal 11 I MeBmt 1
CHN /CF{3 CH3\ /CH3 H/C \IH2 CH CH CH CH3
~ ~ CH3, ~~ H0~ / ~CH3I
cH2 eHZ CH iH iH2
I I
CH--CO -N --CH -CO-N-CH-CO-N-CH-CO-N-CH
( I I I Abu 2
~
CHa- N CH3 CH3 CH3 H CO
c0
~
H3C-7H CH3- N
H-N CH, iH3 iH2MeGly3
D-Ala 8 I
CO--CH-N-CO- i H-N-COiH-N-CO-iH-N-CD
CH3 H CH2 CH H "2
CH H~O CH3 CH
t{,C CH3 H3C \ CH3
Ala 7 MeLeu 6 Val5 MeLeu 4
'Unless otherwise specified, each of the amino acids of the disclosed
cyclosporin is of the L-configuration.
As is the practice in the field, a particular cyclosporin analog may be named
using a shorthand
notation identifying how the analog differs from cyclosporin A. Thus,
cyclosporin C which differs from
cyclosporin A by the threonine at positioin-2 may be identified as ['11u]'-
cyclosporin or [Thr]2-CsA. Similarly,
.25 cyclosporin B is [Ala]'--CsA; cyclosporin D is [Val]2-CsA; cyclosporin E
is [Val]"-CsA; cyclosporin F is [:r
DesoxyMeBmt]'-CsA; cyclosporin G is [NVa]2-CsA; and cyclosporin H is [D-
MeVal]"-CsA.
D-Serine and D-Threonine have been introduced into the 8-position of
cyclosporin A by biosynthesis
resulting in active compounds. See R. Traber et al. J. Antibiotics 42, 591
(1989). D-Chloroalanine has also
been introduced into position-8 of Cyclosporin A by biosynthesis. See A. Lawen
et al J. Antibiotics 52, 1283
(1989).
Indications for Cyclosnorine Therany
Immunoregulatory abnormalities have been shown to exist in a wide variety of
autoimmune and
chronic inflanunatory diseases, including systemic lupus erythematosis,
chronic rheumatoid arthritis, type I
diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis,
multiple sclerosis and other disorders
such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis,
psoriasis, ichthyosis, and Graves
ophthalmopathy. Although the underlying pathogenesis of each of these
conditions may be quite different, they
have in common the appearance. of a variety of autoantibodies and self-
reactive lymphocytes. Such self-
reactivity may be due, in part, to a loss of the horneostatic controls under
which the normal immune system
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operates.
Similarly, following a bone marrow or an organ transplantation, the host
lymphocytes recognize the
foreign tissue antigens and begin to produce antibodies which lead to graft
rejection.
One end result of an autoimmune or a rejection process is tissue destruction
caused by inflanunatory
5 cells and the mediators they release. Aiiti-infIaminatory agents, such as
NSAID's (Non-Steroidal Anti-
inflammatory Drugs), and corticosteroids act principally by blocking the
effect of, or secretion ot these
mediators, but do nothing to modify the imrnunologic basis of the disease. On
the other hand, cytotoxic agents,
such as cyclophosphainide, act in such a nonspecific fashion that both the
normal and autoimmune responses
are shut off. Indeed, patients treated with such nonspecific immunosuppressive
agents are as likely to succumb
to infection as they are to their autoimmune disease.
Generally, a cyclosporin, such as cyclosporine A, is not cytotoxic nor
myelotoxic. It does not inhibit
migration of monocytes nor does it inhibit granulocytes and macrophage action.
Its action is specific and
leaves most established immune responses intact. However, it is nephrotoxic
and is known to cause the
following undes'u-able side effects:
(1) abnormal liver function;
(2) hirsutism;
(3) gum hypertrophy;
(4) tremor;
(5) neurotoxicity;
(6) hyperaesthesia; and
(7) gastrointestinal discomfort.
A number of cyclosporines and analogs have been described in the patent
literature:
U.S: Pat. No. 4,108,985 issued to Ruegger, et al. on Aug. 22, 1978 entitled,
"Dihydrocyclosporin C",
discloses dihydrocyclosporin C, which can be produced by hydrogenation of
cyclospori.n C.
U.S. Pat. No. 4,117,118 issued to Harri, et al. on Sep. 26, 1978 entitled,
"Organic Compounds",
discloses cyclosporins A and B, and the production thereof by fermentation.
U.S. Pat..No. 4,210,581 issued to Ruegger, et al. on Ju1.1, 1980 entitled,
"Organic Compounds",
discloses cyclosporin C and diltydrocyclosporin C which can be produced by
hydrogenation of cyclosporin C.
U.S. Pat. No. 4,220,641, issued to Traber, et aI. on Sep. 2, 1980 entitled,
"Organic Compounds",
discloses cyclosporin D, dihydrocyclosporin D, and isocyclosporin D.
U.S. Pat. No. 4,288,431 issued to Traber, et aL on Sep. 8, 1981 entitled,
"Cyclosporin Derivatives,
Their Production and Pharmaceutical Compositions Containing Them", discloses
cyclosporin G,
dihydrocylospotin G, and isocyciosporin G.
U.S. Pat. No. 4,289,851, issued to Traber, et al. on Sep. 15, 1981 entitled,
"Process for Producing
Cyclosporin Derivatives", discloses cyciosporin D, dihydrocyclosporin D, and
isocyclosporin D, and a process
for producing same.
U.S. Pat. No. 4,384,996, issued to Bollinger, et al. on May 24, 1983 entitled
"Novel Cyclosporins",
discloses cyclosporins having a(i-vinylene-a-amino acid residue at the 2-
position and/or a(3-hydroxy-
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x-amino acid residue at the 8-position. The cyclosporins disclosed included
either MeBtnt or dihydro-MeBmt
at the 1-position.
U.S. Pat. No. 4,396,542, issued to Wenger on Aug. 2, 1983 entitled, "Method
for the Total Synthesis
of Cyclosporins, Novel Cyclosporins and Novel Intermediates and Methods for
their Production", discloses the
synthesis of cyclosporins, wherein the residue at the 1-position is either
MeBmt, dihydro-MeBmt, and
protected intennediates.
U.S. Pat. No. 4,639,434, issued to Wenger, etal on Jan. 27, 1987, entitled
"Novel Cyclosporins",
discloses cyclosporins with substituted residues at positions 1, 2, 5 and 8.
U.S. Pat. No. 4,681,754, issued to Siegel on Jul. 21, 1987 entitled,
"Counteractinb Cyclosporin Organ
] 0 Toxicity", discloses methods of use of cyclosporin coinprisitrg co-
dergocrine.
U.S. Pat. No. 4,703,033. issued to Seebach on Oct. 27, 1987 entitled, "Novel
Cyclbsporins", discloses
cyclosporins with substituted residues at positions 1, 2 and 3. The
substitutions at position-3 include halogen.
H. Kobel and R. Traber, Directed Biosynthesis ojCydosporirrs, European J.
Appin: Microbiol
Biotechnol., 14, ?37B240 (1982), discloses the biosynthesis of cyclosporins A,
B, C, D & G by fermentation.
15. Additional cyclosporin analogs are disclosed in U.S. Pat. No.
4,798,823,.issued to Witzel, entitled,
New Cyclosporin Analogs with Modified "C-9 amino acids", which discloses
cyclosporin analogs with sulfur-
containing amino acids at positio6-1.
SUMMARY OF THE INVENTION
20 The present invention concerns chemically substituted and deuterated
analogs of cyclosporine A and
related cyclosporines.
An object of the present invention is to provide new cyclosporine analogs
which have enhanced
efficacy and altered phannacokinetic and pharmacodynarnic parameters. Another
object of the present
invention is to provide a cyclosporine analog for the care of
imtitunoregulatory disorders and diseases,
including the prevention, control and treatment thereof. An additional oliject
of the present invention is to
provide pharmaceutical compositions for administering to a patient in, need of
the treatment one or more of the
active immunosuppressive agents of the present invention. Still a further
object of this invention is to provide
a method of controlling graft rejection, autoinunune and chronic inflammatory
diseases by administ.ering a
sufficient amount of one or more of the novel immunosuppressive agents in a
mammaliau species in need of
30 such treatment. Finally, it is the object of this invention to provide
processes for the preparation of the active
compounds of the present invention.
Substitution and deuteration of the cyclosporme molecule results in altered
physicocheniical and
phartnacokinetic properties which enhance its usefulness in the treatment of
transplantation rejection, host vs.
graft disease, graft vs. host disease, aplastic anemia, focal and segmental
glomerulosclerosis, myasthenia
35 gravis, psoriatic arthrids, relapsing polychondritis and ulcerative
colitis.
Embodiments of the invention include CsA derivatives wherein one or more
hydrogen atoms in the 1,
3 and 9 amino acid positions are substituted with a deuterium atom and wherein
the cyclosporine A derivatives
are optionally chemically substituted at the amino acid 9 position. A further
specific embodiment of the
invention is the CsA derivative represented by formula l:
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~
CHR
n
CH
H3C~( H3 H3C CH3 CHZ
CH H3C CH3 R. CH-CH3 CH3
CH2 CH2 CH CH CHZ
i H-CO-N-CH-CO-N-CH-CO-N--CH-CO-N- i H
H3C-N CH3 CH3 CH3 H CO (n
CO l
, H3C-NY
H3C-CH C, ~
H-N CH3 Z
CO-CH-N-CO-CH-N-CO-CH-N-CO-CH-N -CO
CH3 H CH CH H CHZ CH3
2 H3C.1 CH3 -CH
H3CIC CH3 H3C CH3
where R is (i) a deuterium or (ii) a saturated or unsaturated straight or
branched aliphatic chain of
from I to 16 carbon atoms and containing one or more deuterium atoms or an
ester, ketone or
alcohol of the carbon chain and optionally containing one or more substituents
selected from
halogen, nitro, amino, amindo, aromatic, and heterocyclic, or (iii) R is an
aromatic or
heterocyclic group optionally containing a deuterium atom or (iv) R is a
methyl group; and X, Y,
and Z are hydrogen or deuterium provided that at least one of X, Y, or Z is
deuterium; and R' is
an OH or an ester or is an 0 and together with a carbon adjacent to a double
bond on amino acid
1 form a heterocyclic ring such as 5-membered rings where the heteroatom is
oxygen.
Preferably, R is an unsaturated straight or branched aliphatic carbon chain of
from 2 to 3
carbons, X, Y and Z are hydrogen or deuterium, and R' is an -OH or acetoxy.
Other specific
embodiments of the present invention include the CsA derivative of formula I
where R is a
saturated or unsaturated carbon chain of from 2 to 3 carbons containing one or
more deuterium
atoms.
CA 02511888 1998-10-08
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The invention also specifically contemplates the compounds, and mixtures
thereof,
represented by the structures A and B:
R
HO,~j
MeLeu-MeVal- i Abu-Sar
O
MeLeu -D-AIa -Ala -MeLeu -VaI-MeLeu
(A)
R
HO,
MeLeu-MeVal- i Abu-Sar
0
MeLeu -D-AIa-AIa-MeLeu -Val-MeLeu
(B)
wherein R is selected from the group consisting of :(i)-CH=CH-CH3, (ii)-
CH=CHz, and
(iii)-CD=CD2.
Further specific embodiments include those of formulas 5g and 5e below:
CD2
~(~~,
(5g) (5e)
HO/ HO/
MeLeu-MeVal- i Abu=Sar MeLeu-MeVal-N Abu-Sar
I
O 1 p
MeLeu -D-A1a -Ala -MeLeu -Val -MeLeu Meleu-D-Ata-Ala-Meleu - Val-MeLeu
CA 02511888 1998-10-08
9
DESCRIPTION OF THE FIGURES
Figure 1 is the structure of cyclosporine A showing a site of deuteration at
the amino acid 3 position.
Figure 2 is the structure of cyclosporine A showing a site of deuteration at
the amino acid 9 position.
Figtire 3 is scheme I of the synthesis of the cyclosporine derivatives.
Figure 4 is scheme II of the synthesis of the cyclosporine derivatives.
Figure 5 is a graph of the results of the calcineurin assay of Example 9.
Figure 6 is a graph of the results of a mixed lymphocyte reaction assay of
Example 10.
DETAILED DESCRIPTION OF THE INVENTION
Substitution of deuterium for ordinary hydrogen and deuterated substrates for
protio metabolites can
produce profound changes in biosystems. Isotopically altered dru&s have shown
widely divcrgent
pharmacological effects.. Pettersen et al., found increased anti-cancer effect
with deuterated 5,6-benzylidene-dl-
L-ascorbic acid (tilascorb) [Anticancer Res. 12, 33 (1992)].
Substitution of deuterium in methyl groups of cyclosporine will result in a
slower rate-of oxidation of
the C-D bond relative to the rate of oxidation of a non-deuterium substituted
C-H bond. The isotopic effect
acts to reduce formation of demethylated metabolites and thereby alters the
pharmacokinetic parameters of the
drug. Lower rates of oxidation, metabolism and clearance result in greater and
more sustained biological
activity. Deuteration is targeted at various sites of the cyclosporin molecule
to increase the potency of drug,
reduce toxicity of the drug, reduce the clearance of the pharmacologically
active moiety and improve the
stability of the molecule.
Isotopic Substitution:
Stable isotopes (e.g., deuterium, 13C, 13N,18 0) are nonradioacdve isotopes
which contain one
additional neutron than the normally abundant isotope of the respective atom.
Deuterated compounds have
been used in pharmaceutical research to investigate the in vfvo metabolic fate
of the compounds by evaluation
of the mechanistn of action and metabolic pathway of the non deuterated parent
compound. (Blake et al. J.
Phami. Sci. 64, 3, 367-391,1975). Such metabolic studies ire important in the
design of safe, effective
therapeutic drugs, either because the in vivo active compound administered to
the patient or because the
-30 metabolites produced from the parent compound prove to be toxic or
carcinogenic (Foster et al., Advances in
Drug Research Vol. 14, pp. 2-36, Academic press, London, 1985).
Incorporation of a heavy atom particularly substitution of deuterium for
hydrogen, can give iise to an
isotope effect that could alter the pharmacolcinetics of the drug. This effect
is usually insignificant if the label is
placed at a metabolically inert posidon of the molecule.
Stable isotope labeling of a drt:g can alter its physico-chemical properties
such as pKa and lipid
solubility. These changes may influence the fate of the drug at different
steps along its passage through the
body. Absorption, distribution, metabolism or excretion can be changed.
Absorption and distn'bution are
processes that depend primarily on the molecular size and the lipophilicity of
the substanc~. These effects and
alterations can affect the pharmacodynamic response of the drug molecule if
the isotopic substitution affects a
CA 02511888 1998-10-08
region involved in a ligand-receptor interaction.
Drug rrietabolism can give rise to large isotopic effect if the breaking of a
chemical bond to a
deuterium atom is the rate lintiting step in the process. While some of the
physical properties of a stable
isotope-labeled molecule are different from those of the unlabeled one, the
chemical and biological properties
5 are the same, with one important exception: because of the increased mass of
the heavy isotope, any bond
involving the heavy isotope and another atom will be stronger than the same
bond between the light isotope
and that atom. In any reaction in which the breaking of this bond is the rate
limiting step, the reaction will
proceed slower for the molecule with the heavy isotope due to "kinetic isotope
effect" A reaction involving
breaking a C-D bond can be up to 700 per cent slower than a similar reaction
involving breaking a C-H bond.
10 If the C-D bond isnot involved in any of the steps leading to the
metabolite, there may not be any effect to
alter the behavior of the drug. If a deuterium is placed at a site involved in
the metabolism of a drug, an
isotope effect will be observed only if breaking of the C-D bond is the rate
limiting step There is evidence to
suggest that whenever cleavage of an aliphatic C-H bond occurs, usually by
oxidation catalyzed by a mixed-
function oxidase, replacement of the hydrogen by deuterium will lead to
observable isotope effect. lt is also
important to understand that the incorporation of deuterium at the site of
metabolism slows its rate to the point
where another metabolite produced by attack at a carbon atom not substituted
by deuterium becomes the major
pathway a process called "metabolic switching". It is also observed that one
of the most important metabolic
pathways of compounds containing aromatic systems is hydroxylation leading to
a phenolic group in the 3 or 4
position to carbon substituents. Although this pathway involves cleavage of
the C-H bond, it is often not
accompanied by an isotope effect, because the cleavage of this bond mostly not
involved in the rate limiting
step. The substitution of hydrogen by deuterium at the stereo center will
induce a greater effect on the activity
of the drug.
Synthesis of Cyclosnorine Derivatives:
The staring material for the preparation of the compounds of this invention is
cyclosporine A. The
process for preparing the compounds of the present invention are illustrated
as shown in scheme I in figure 3.
It will be readily apparent to one of ordinary skill in the art reviewing the
synthetic route depicted below that
other compounds with formula I can be synthesized by substitution of
appropriate reactants and agents in the
synthesis shown below.
The first step in the process for making deuterated cyclosporin analogs is the
preparation of the key
intemnediate 3 and 6. This can be achieved by the oxidation of the double bond
in the amino acid 1.
Treatment of cyclospornt with acetic anhydride and excess of
dimethylaminopyridine provided the hydroxyl
protected acetyl cyclosporin. 2.Although cleavage of the double bond could
then be accomplished by
treatment of 2 with ozone, or KIvInO4l NaIO4,, it was found out that
OsO4/NdIO4 was the reagent of choice
for the transforination to the aldehyde product 3. The reaction was generally
found to be cleaner, producing
the required material and to proceed in higher yield. The drawback to this
reaction is that Os04, is expensive
and highly toxic, so that its use is limited . But the results can be
accomplished more economically by the use
of H20, with Os04 present in catalytic amounts. t-butyl hydroxide in alkaline
solution and N-
methylmorpholine-N-oxide can be substituted for H202 in this process. The
aldehyde compound 3 was further
CA 02511888 1998-10-08
11
treated with various deuterated.alkyl or aryl triphenyl phosphoniurn
derivatives(wittig reagents) and hydrolysis
by alkaline solution provided the final derivatives ( S a-h). We also
developed a general procedure to obtain
various compounds as shown in Scheme 11 in figure 4.
In this approach, the aldehyde derivative 3 was treated with the Wittig
reagent prepared by using
standard procedure. The resultant product on mild acid hydrolysis provided the
key intermediate aldehyde
product 6 This was futther treated with second deuterated alkyl or aryl
triphenylphosphonium halide reagents
and on mild acid hydrolysis yielded the. required products. This method
provides control over the extension of
the diene system. By using this approch, olefinic double bonds can be
introduced step by step.
A third approach to prepare the deuterated compounds 5a-h- is by heating non
deuterated cyclosporin
analogs descnbed earlier, in a deuerated solvent such as deuterated water,
deuterated acetic acid in the
presence of acid or base catalyst.
Preferred cyclosporins of the present invention are those which both contain a
deuterium and a chemical
substitution on aritino acid I sueh as those of formula II: .
X-Abu-Sar-MeLeu-Vat -MeLeu-Ala-(D)Ata-MeLeu-MeLeu -MeVal
GI) 1 2 3 4 S 6 7 a 9 10 11
Where X is
R\
CH2
CH
HO'~'C/
H ~CH3
-N-CH-CO-
And R= --CHO, -CDO, -CH=CD-CD,,-CD=CD-CD; ,-CH=CH-CH=CD-CDz ; CD=CH-CD=CD.CD;
,
-CH=CH-CH=CD; CD=CH-CD=CDt; CH=CD,,-CH=CHzand-CD=CDZ.
EXAMPLES:
Ezample 1.
To a stirred solution of cyclosporine 1(1.01 g, 0.84rntnol) in acetic
anhydride (20mL) at room temperature was
added DMAP (150mg, 123mmol, 1.5eq). After stirring overnight, the reaction
mixture was partitioned
between EtOAc (50ml) and water (25m1). The separated EtOAc layer was-then
washed with water (50mL) and
brine (50mL), dried (MgSO4) and the solvent removed in vacuo to give the crude
product as a glassy solid.
Purification by flash chromatography through a short column of silica (2%
MeOH/DCM) and lyophilisation
from benzene yielded 2(1.044g, 0.84mmol, quant.) as a fluffy, eolourless
solid; [Cx]o -305.7 (c. 0.3, CHC13);
CA 02511888 1998-10-08
12
v,n. (CHCl3 cast)/cni l 3328m, 2963m, 1746m, 1627s, 1528m; 1472m, 1233m; SH
(600MHz, C6D6) 8.73 (1H,
d, J= 9.5Hz NH), 8.30 (1 H, d, J= 7.OHz, NM,?.92 (1 H, d, J= 7SHz; NH
), 7.49 (1 H, d, J= 7.5Hz, NH~,
6.05 (1 H, d, J= 11.5Hz), 5.88 (1 H, dd,J = 3.5, 11 SHz), 5.82 (1 H, d, J=
11.SHz), 5:65 (1 H; dd, J 4.0,
12.0Hz), 5.60 (1H, dd, J= 3.5, 12.5Hz), 5.63-5.57 (1H, m), 5.51-5.45 (1H, m),
537 (lii, dd, J= 5.5, 8.5Hz),
5.05-5.01 (2H, complex), 4.99 (1H, d, J=11.0Hz), 4.76 (1H, p,J= 7.0Hz), 4.58
(IH, p, J= 7.0Hz), 4.02 (IH,
_d, J=13.5Hz), 3.47 (3H, s), 3.30 (3H, s), 337 (3H, s), 3.11 (3H, s), 2.98
(3H, s), 2.68-2.62 (IH, m), 2.63 (31-1,
s), 2.51-2.39 (2H, complex), 2.34-2.25 (8H, complex), 2.03 (3H, s), 1.97-1.85
(2H, complex), 1.83 (3H, dd, J
= 1.0, 6.SHz), 1.82-1.77 (211, complex), 1.68-1.61 (3H, complex), 1.55 (3H,,
d, J= 7.0Hz), 1.55-1.51 (1H, m),
1.441.38 (1H, m), 1.32-1.20 (5H, complex), 129 (3H, d, J= 7.0Hz), 1.21 (3H, d,
J= 6.5Hz), 1.17 (3H, d, J=
6.5Hz), 1.14 (3H, d, J= 6.5Hz), 1.08 (3H, d,.J= 6.5Hz), 1.04 (3H, d, J=
6.0Hz), 1.03 (31L d, J=:7.0Hz),
1.00 (3H, d, J= 7.0Hz), 0.93 (3H, d,J= 6.0Hi), 0.92 (3H, d, J= 6SHz), 0.88-
0.84 (9H, complex), 0.76 (3H,
d, J= 6.5Hz),-0.57 (3H, d, J= 6.5Hz); Sc (75MHz, C6D6) 173.6, 173.2, 172.8,
172.6, 171.3, 171.1, 170.71,
170.67, 170.4, 1702, 169.8, 167.9 (C=O), 129.0, 126:2 (C=C), 73.1 (COAc),
58.1, 57.1, 56.0, 55.0, 54.6,
542, 50.3, 49.9, 48.6, 48.1, 47.8, 44.5, 40.8, 39.1, 35.7, 33.6, 32.9, 32.1,
31.5, 312, 30.0, 29.7, 29.5, 29.3,
24.9,24.6, 24.4, 24.0, 23.6, 23.4, 233, 21.7, 21.1, 21.0, 20.6, 20.3, 19.5,
18.5, 18.0, 17.7, 17.5, 17.4, 14.9,
9.7; m/. (Electrospray)
Example 2
To a solution of compound 2(289mg, 023mmol) in a 1:1 mixture of dioxane and
water (5mL) was added
firstly sodium metaperiodate (100mg, 0.47mmol, 2eq) and secondly a solution of
osmium tetraoxide (5mL;
0.5g OsO4 in 250mL of solvent). Two-phase work-up, purif cation by flash
colunui chromatography (40%
acetone in petroleum ether) and lyophilisation from benzene gave compound 3.
(226mg, 0.18mmol, 80%) as a
fluffy, colourless solid; [[zf -260.0 (c. 0.1, CHCI3); võa (CHCh cast)/cni l
3325m, 2962m, 1748w, 1724w,
1677m, 1626s, 1228m, 755m; SH (300MHz, C6D6) 8.63 (IH, d,J=9.5Hz, NLD, 8.16
(IH, d, J 7.0Hz, NH,,
7.95(1H,d,J=7.5Hz,NH,,7.48'(1H,d,J=9.0Hz,NH~,5.93(1H,d,J7.SHz),5.84(1H,dd,J=4.0
,
11.5Hz), 5.70 (1H, d, J= 11.5Hz), 5.56-5.54 (1H, m), 5.32 (lH, dd, J= 5.5,
8.0Hz), 5.07-4.88 (3H, complex),
4.72 (IH, p, J= 7.0Hz), 4.49 (IH, p, J= 7.0Hz), 3.98 (IH, d,J= 14.0Hz), 3.42
(3H,:s, CH3N), 3.27 (3H, s,
CH N), 3.12 (3H, s, CH N), 3.07 (3H, s, CH N), 2.91 (3H, s, CH N), 2.79 (3H,
s, CH N), 2.59 (3H, s, CH N)22.42 2.08 (]OH, complex), 1.94 (3H, s, CH COZ),
1.47 (3H, d, J= 7.0Hz), 124 (3H, 7.0Hz), 1.I4-1.09 (9H,
complex), 1.04 (3H, d, J= 6.5Hz), 1.01 (3H, d, J= 7.01Iz), 0.96 (3H, d, J=
6.5Hz), 0.92 (3H, d, J= 6.5Hz),
0.91 (3H, d, J= 6.5Hz), 0.89 (3H, d, J= 6.0Hz), 0.83 (6H, d, J= 6.5Hz), 0.74
(3H, d,J= 6.5Hz), 0.59 (3H, d,
J= 6.5Hz); Sc (75M1-Iz, C6D6) 202.5 (CHO), 174.4, 174.0, 173.7,172.8, 171.6,
171.5, 171.2, 171.1, 170.6,
170.2, 170.2, 168.1, 73.0, 58.7, 57.6, 56.7, 55.5, 55.0, 54.5, 49.4, 48.9,
48.5, 48.1, 45.0, 44.6, 41,3, 39.8, 38.8,
37.7, 36.2, 32.5, 32.0, 31.6, 30.9, 30.3, 30.0, 29.8, 29.6,25.6, 25.3, 25.0,
24.8, 24.5, 24.0, 23.8, 23.4, 22.0,
35- 21.7, 21.2, 20.5, 20.0, 19.8, 18.8, 185, L8:1, 17.4, 152, 10.0; m/z
(Electrospray) 1232.8 (MH`, 100%).
CA 02511888 1998-10-08
13
Example 3
Method A: To a solution of compound 3( 315mg, 0.26mmol) in THF (5mL) at 0 C
was added a solution of the
deutero-phosphorus ylid (2.67mmol, -10eq), prepared from ds-
ethyltriphenylphosphonium iodide. After work-
up, purification by flash column chromatography (30% to 60% acetone in PE) and
HPLC (60% to 65% MeCN
in water), then lyophilisation from benzene yielded compound 4(153mg,
0.12tnmol, 47%) as a flnffy,
colourless sol.id
Method B: To a stirred solution of compound 3 (287mg, 0.23tnmol) in THF (5mL)
under Ar at -78 C was
carefully added a solution of phosphorus ylid (formed by the addition of
sodium hexamethyldisilylamide
(1.OM; 2.25m1w 2.25mmoL, -l0eq) to a suspension of ds-
ethyltriphenylphosphonium iodide (480mg,
1.13mmol; --5eq) in THF (IUmL) under Ar at room temperature). After stirring
for 2hr with gradual warming
to room temperatui=e; the reaction mixture was cooled to 0 C and was quenched
by the addition of 10%
AcOH/CHF (l OmL). The reaction mixture was concentrated in vacuo and
partitioned between water (20mL)
and EtOAc (20mL). The aqueous layer was fitrther extracted with EtOAc (20tnL)
and the combined organic
extracts were then washed with IN HCI (20mL) and water (20mL), dried (MgSO4)
and the solvent removed in
vacuo to give the crude product. Purification by flash column chromatography
(40% acetone in petroleum
ether) and lyophilisation from benzene yielded compound 4d (84mg, 671imo1,
29%) as a fluffy, colourless
solid; ~a]D -283.0 (c. 0.1, CHCI3); vmõ (CHCl3 cast)!cm' 3320m, 3010m, 2959s,
2924s, 2871rn, 2853m,
1743m, 1626s, 756s; SH (600MHz, C6D6) 8.78 (IH, d, J= 9.5Hz), 8.33 (1H, d, J =
7.011z), 7.99 (1H, d, J
7.51-z), 7.59 (1H, d, J= 9.0Hz), 6.09 (IH, d, J= 11.5Hz), 5.92 (1H, dd, J=4.0,
11.014z), 5.86 (IH, d, J=
11.5Hz), 5.72-5.64 (2H, complex), 5.62 (11-I, dd, J= 3.5, 12.5Hz), 5.40 (1 H,
dd, J = 5.5, 8.5Hz), 5.10-5.02
(3H, complex), 4.80 (1H, q, J= 7.0Hz), 4.60 (IH, q, J= 7.0Hz), 4.05 (IH, d,
J=14.0Hz), 3.51 (3H, s), 331
(3H, s), 3.20 (3H, s), 3.13 (3H, s), 3.01 (3H, s), 2.87 (31I, s), 2.64 (3H,
s), 2.45 (1H, dt, J= 4.0, 12.5Hz), 236-
2.20 (IOH, complex), 2.06 (3H, s), 1.93-1.79 (3H, complex); Sp (84MHz, C6H6)
Sc (125MHz,"C6D6) 174.5, 173.7, 173.6,.173.1, 171.7, 171.4, 170.9; 170.7,
170.6, 170.3, 170.0, 168.4, 130.2
(C=C), 123.8 (C=C), 73.8 (MeBmt C-3), 58.7, 58.1, 57.6, 57.1, 55.5, 55.0,
54.5, 49.4, 49.0, 48.6, 48.2, 45.0,
41.4, 39.9, 39.0, 37.8, 34.2, 33.9, 32.6, 32.3, 32.0, 31.4, 30.9, 30.8, 30.2,
30.1, 30.0, 29.9, 29.8, 29.6, 28.5,
25.6, 25.3, 25.0, 24.9, 24.8, 24.1, 23.9, 23.8, 23.6, 23.1, 22.1, 21.7, 21.4,
20.7, 20.0, 19.9, 19.9, 18.9, 18.7,
18.6, 18.3, 17.4, 15.3, 14.3, 10.2; nriz (Electrospray) 1270 ([M+Na]+, 100%),
1286 QM+K]', 20).
4
Example
To a stirred solution of 4d (84mg, 67 mol) in MeOH (5mL) and water (2.5mL) at
room temperature was
added potassium carbonate (99mg; 0.72mmol, -l0eq). After stirring overnight,
the MeOH was removed in
vacuo and the aqueous residue was partitioned between EtOAc (IOmL) and 5%
citric acid solution (10mL).
The EtOAc layer was then washed with w.ater (IOmL) and brine (]OmL), dried
(MgSO4) and the solvent
removed in vacuo to give the cttide product. HPLC purification (60% to 65%
MeCN in water) and
lyophilisation from benzene yielded compound Sd (59mg, 49 mol, 70%) as a
fluffy, colourless solid; [a]D
262.0 (c. 0.05, CHCh); v,,,,., (CHClt cast)/cm' 3318m, 3008m, 2960s, 2872in,
1627s, 1519m, 1470m, 1411 m,
CA 02511888 1998-10-08
14
1295m, 1095m, 754m; SH (600MHz, C6H6) 8.27 (1H, d, J= 9.5Hz), 7.96 (1H, d, J=
7.5Hz), 7.63 (1H, d, J=
8.0Hz), 7.45 (1H, d, J= 9.OHz),.5.87 (1H, dd, J= 3.5, 11.0Hz), 5.74 (iH, d, J=
7.5Hz); 5.73-5.69 (1H, m),
5.66-5.64 (1H, br.d, J= I 1,0Hz), 5.79 (1H, dd, J= 4:0, 11.511z), 3.39 (1H,
dd, J= 5.5, 10.5Hz), 5.33 (1H, dd,
J= 5.5, 8.5Hz), 5.24.(1H, d, J= 11.0Hz), 5.12 (1H, dt, J= 7.5, 10.0Hz), 4.88-
4.79 (3H, complex), 4.22 (I H,
dd, J= 5.5, 7.5Hz), 4.00 (1H, d, 13.5Hz), 3.72 (3H, s), 322 (3H, s); 3.06 (3H,
s), 2.97 (3H, s), 2.92 (3H, s),
2.85 (3H; s), 2.67-2.60 (1H, m), 2.58 (3H, s), 2.56-2.50 (IH, br m), 2.33-223
(4H, complex), 2.20-2.07 (4H,
complex), 1.80-1.74 (3H, complex), 1.67 (3H, d, J= 7.0Hz), 1.56-1.50 (2H,
complex), 1.46-1.23 (9H,
cornpiex), 1.17-1.13 (16H, complex), 1.06 (3H, d, J= 6.5Hz), 1.02 (31d, d, J=
7.0Hz), 0.98 (3H, d, J=
6.5Hz), 0.96 (3H, d, J= 7.0Hz), 0.92-0.89 (9H complex), 0.86 (3H, t, J=
7.5Hz), 0.83 (311, d, J = 6.0Hz),
0.64 (3H, d, J= 6.5Hz); So (84MHz, C6H6) 1.64 (CD ); Sc (75MHz, C6H6) 1742,
174.1, 174.0, 173.7, 171.8,
171.4, 171.2, 170:5, 170.4, 170.3, 169.8,130.2,124.1. (99.2,) 74.3, (67.1,)
66.3, 66.1, 61.0, 59.5, 58.3, 57.8,
55.7, 55.5, 55,4, 49.4, 49.0, 48.4, 453, 41.4, 39.6, 39.0, 37.8, 36.5, 36.1,
35.8, 33.7, 31.6, 30.8, 30.4, 30.1,
29.9, 29.5, 29.4, 25.5, 25.2, 25.0, 24.9,.24.5, 24.2, 23.8, 23.7, 23.6,22.0,
21.4, 20.0,18..8, 18.5, 17.8, 16.0,
10.1; m/z (Electrospray) 1206 ([M+H]', 30%),.1228 ([M+Na]', 100), 1244
([M+K]', 25).
Example 5
To a vigorously stirred mixture of compound 3(49mg; 39.81imol) and deuterated
d3-
allyltriphenylphosphonium bromide (311mg, 812pmol, -20eq) in benzene (3mL) at
room temperature was
added IN NaOH (3mL). Stirring was continued at room temperature for 5days,
afier which time the 2 layers
were separated, the benzene layer was washed with water (5mL), dried (Mg-SOa)
and the solvent removed in
vacuo to give the crude product. Purification by HPLC (20% to 60% MeCN in
water) and lyophilisation from
benzene yielded compound 4g (23mg, 18.3ptnol, 47%) as a fluffy, colourless
solid; [af -264.2 (c. 024,
CHC13); v,,,,x (CHC13 cast)lcm'1 3322m, 2959m, 1744m, 1626s, 1231m, 754m; Sn
(300MHz, C6D6) complex
due to 1:1 ratio of geometrical isomers 8.73 (d, J= 9.5Hz, NH), 8.72 (d, J=
9.5Hz, NH), 8.29 (d, J= 6.5Hz,
NH_), 8.26 (d, J= 6.5Hz, Nll, 7.92 (d, J= 7.5Hz, NH), 7.86 (d, J = 7.5Hz, NH);
7.53 (d, J = 9.0Hz, NI-_)1, 7.49
(d, J= 9.0Hz, NH), 7.10-6.70 (complex), 6.33 (br t, J= 11.0Hz), 6.18 (d, J=
10.5Hz), 6.12 (d, J= 10.5Hz),
6.05 (d, J= 11.0Hz), 6.03 (d, J= 11.01-Iz), 5.90-5.53 (complex), 537 (dd, J
6.0, 8.0Hz), 5.20 (d, J=
12.0Hz), 5.14 (d, J= 12.0Hz), 5.07-4.97 (complex), 4.80-4.70 (complex), 4S7
(p, J= 7.0Hz), 4.02 (d, J
14.0Hz), 4.01 (d, J= 14.0Hz), 3.47 (s), 3.46 (s), 328 (s), 3.26 (s), 3.16 (s),
3.15 (s), 3.09 (s), 2.97 (s), 2.96 (s),
2.84 (s), 2.62 (s), 2.48-2.23 (complex),2.05 (s), 2.03 (s), 1.95-1.59
(complex),:1.54 (d, J= 7.0Hz), 1.53-0.80
(complex), 0.77 (d, J= 6.5Hz), 0.58 (d, J= 6.5Hz), 0.57 (d, J= 6.5Hz); Sc
(75MHz, C6D6) 174.5, 174.0,
173.9, 173.6, 173.5, 173.1, 171.7, 171.6, 171.4, 170.9, 170.8, 170.6, 170.6,
170.3,169.8, 169.7, 168.4, 137.9,
133.9, 133.5, 132.8, 132.3, 131.6, 130.1, 116.9, 115.0, 73.6, 58.6, 57.6,
57.0, 56.8, 55.7, 55.5, 55.0, 54.9,
54.7, 54.5, 49.4, 48.9, 48.5, 48.2, 48.1, 44.9, 41.5, 39.9, 39.0, 38.9, 37.8,
37.6, 36.6, 36.3, 34.1, 33.7, 32.7,
32.1, 32.0, 31.5, 30.9, 30.7, 30.0, 29.8, 29.6, 25.6, 25.5, 253, 25.2, 25.0,
24.9, 24.1, 23.9, 23.7, 23.6, 22.1,
21.7, 21.6, 21.4, 21.3, 20.7, 20.0, 19.9, 18.9, 18.6, 13.3, 17.6, 15.3, 10.2;
m/a (Electrospray) 1258.8 (MH',
100%).
CA 02511888 1998-10-08
Example 6
To a vigorously stirred mixture of compound 3 (56mg, 45.5 mol) and deuterated
d4-
crotyltriphenylphosphonium bromide (360mg, 907 mol, --20eq) in benzene (3mL)
at room temperature was
added 1N NaOH (3mL). Stirring was cotitinued at room temperature for Sdays,
after which time the 2 layers
5 were separated, the benzene layer was washed with water (5mL), dried (MgSO4)
and the solvent removed in
vacuo to give the crude product. Purification by HPLC (20% to 60% MeCN in
water) and lyophilisation from
benzene yielded compound 4e (23mg, 18.l mol, 40%) as a fluffy, colourless
solid; ~cz]o -236.0 (c. 0.25
,
CHCI;); v,. (CHCh cast)/cm" 3324m, 2959m, 2871m, 1745w, 1626s, 1231m; SN
(300MHz, C6D6) complex
due to presence of 4 isomers 8.76 (d, J= 6.0Hz), 8.73 (d, J 6.0Hz), 8.29 (d,
J= 7.0Hz), 7.93 (d, J= 7.5Hz),
10 7.88 (d, J= 7.5Hz), 7.53 (d, J = 9.5Hz), 7.62-7.31 (1 H, complex), 7.16-
6.88 (2H, complex), 6.59-6.39
(complex), 6.28 (t, J= I 1.011z), 6.15 (d, J=10.5Hz), 6.09 (d, J= 10;5Hz),
6,05 (d, J= 11.51-1z), 6.03 (d, J
11.5Hz), 5.90-5.82 (complex), 5.68-5.35 (complex), 5.08-4.97 (complex), 4.81-
4.72 (complex), 4.63-4.53.
(complex), 4.03 (d, J= 14.0Hz), 3.47 (s), 3.46 (s), 3.28 (s), 3.26 (s), 3.17
(s), 3.15 (s), 3.09 (s), 2.98 (s), 2.97
(s), 2.83 (s), 2.63 (s), 2.62 (s), 2.71-2.56 (complex), 2.47-2.23 (complex),
2.05 (s), 2.04 (s), 2.03 (s),'2.02 (s),
15 1.98-0.82 (complex), 0.77 (d, J= 6.5Hz), 0.58 (d, J= 6.5Hz), 0.58 (d, J=
6.5Hz); m/a (Electrospray) 1273.8
(MH+, 100%).
Example 7
To a stirred solution of compound 4g (20mg, 15.9 mo1) in methanol (5mL) and
water (1mL) at room
temperature was added potassium carbonate (30mg, 217 mol). After stirring
overnight, the reaction mixture
was partitioned between EtOAc (10mL)=and 5% aqueous citric acid (10mL). The
aqueous layer was ftuther
extracted with EtOAc (5mL), the combined organic layers were then washed with
5% citric acid (I OmL) and
brine (l OmL), dried (MgSO.4) and the solvent iemoved in vacuo to give the
crude product. Purification by
HPLC (65% MeCN) and lyophilisation from benzene yielded compound 5g (10mg,
8.21imol, 52%) as a fluffy,
colourless solid; [a]o -285.2 (c. 0.29, CHCI3); vm,x (CHCI; cast)/cm '.3500-
3200br, 3319m, 2958m,.2927m,
1626s, 1520m, 1468m, 754m; SH (300MHz, C6D6) complex due to the presence of 2
isomers 825 (d, J=
10.0Hz, NI-~I, 8.13 (d, J= 10.0Hz, Nl-i~, 793 (d, J= 7.0Hz, NH), 7.84 (d, J=
7.0Hz, NI-~1 , 7.67 (d, J= 8.0Hz,
NH), 7.61 (d, J= 8.0Hz, NID, 7.55 (d, J= 8.5Hz, Nl-1, 7.54 (d, J= 8.5Hz, NID,
6.84 (t,.J = 10.5Hz), 6.79 (t,
J= 10.5Hz), 6.58 (t, J= 10.5Hz), 6.52 (t, J=10SHz), 6.30-6.14 (complex), 5.88-
5.78 (complex), 5.75-5.66
(complex), 5.44-4.74 (complex), 422-4.15 (complex), 3.95. (d, J= 14.0Hz), 3.93
(d, J= 14.0Hz), 3.72 (s),
3.68 (s), 3.19 (s), 3.17 (s), 3.05 '(s), 3.03 (s), 2.94 (s), 2.93 (s), 2.89
(s), 2.86 (s), 2.82 (s), 2.81 (s), 2.72-2.53
(complex), 2.55 (s), 2.54 (s), 2.49-2.36 (complex), 2.32-2.03 (complex), 1.81-
0.81 (complex), 0.65 (d, J=
6.5Hz)), nm/z (Electrospray) 1216.8 (MH', 100%), 607.9 ([M+2H]2+, 1_._
CA 02511888 1998-10-08
16
Example 8
To a stirred solution of compound 4e (18mg, 142pmo1) in methanol (5mL) and
water (lmL) at room
temperature was added potassium carbonate (35mg, 254pmo1). After stirring
overnight, the reaction mixture
was partitioned between EtOAc (10mL) and 5% aqueous citric acid (10mL). The
aqueous layer was fimher
extracted with EtOAc (5mL), the combined organic layers were then washed with
5% citric acid (IOnil.) and
brine ( l OmL), dried (MgSO4) and the solvent removed in vacuo to give the
crude product. Purification by
HPLC (65% MeCN) and lyophilisation from benzene yielded compound 5e (10mg, 8.I
pmol, 57%) as a fluffy,
colourless sotid; [a]D -2855(c. 0.11, CHCII); 6H (300MHz, C6)6) complex due to
presence of 4 isomers.
831(d, J= 9.5Hz), V8 (d, J= 9SHz), 8.16 (d, J= 9.SHz), 8.14 (d, J= 9.5Hz),
7.96 (d, J= 7.5Hz), 7.95 (d,
J = 7.5Hz), 7.86 (d, J= 7.5Hz), 7.85 (d, J= 7SHz), 7.63 (d, J= 7SHz), 7.59 (d,
J= 7.5Hz), 7.50-7.44
(complex), 6.60-6.49 (complex), 6.32-6.11(complex), 5.88-5.83 (complex), 5.76-
5.71 (complex), 5.64-5.22
(complex), 5.17-5.08 (complex), 4.91-4.77 (complex), 426-4.18 (complex), 3.99
(d, J= 14.0Hz), 3.97 (d, J=
14.0Hz), 3.74 (s), 3.73 (s), 3.71 (s), 3.69 (s), 3.22 (s), 3.21 (s), 320 (s),
3.19 (s), 3.07 (s), 3.06 (s), 3.05 (s),
2.97 (s), 2.96 (s), 2.95 (s), 2.92 (s), 2.91 (s); 2.89 (s), 2.84 (s), 2.83
(s), 2.69-2.07 (complex), 2.58 (s), 2.57 (s),
1.84-0.81 (complex), 0.64 (d, J= 6.5Hz); m/_ (Electrospray) 1269.8 ([M+K]`,
5%), 1253.8 ([M+Na]t, 30),
1231.8 (MH')
Example 9
The immunosupressive activity was tested for deuterated cyclosporin analogs as
descn'bed below.
Compoimd 5e and compound 5g are more potent than the parent cyclosporin.
Calcineurin activity was
assayed using a modification of the method previously described by Fruman et
al. (Proc Natl Acad Sci
USA, 89,3686-3690, 1992). Whole blood lysates were evaluated for their ability
to dephosphorylate a
32P-labelled 19 amino acid peptide substrate in the presence of okadaic acid,
a phosphatase type I and 2
inhibitor. Background phosphatase 2C activity (CsA and okadaic acid resistant
activity) was determined
and subtracted from each sample, with the assay performed in the presence and
absence of excess added
CsA. The remaining phosphatase activity was taken as calcineurin activity. The
results of the calcineurin
assay are presented in figure 5. The results are expressed as means the
standard error of the mean. The
results are plotted as CsA derivative concentration in ug/L versus percentage
of calcineurin inhibitation.
The structrues of the compounds assayed include:
H
O
MeLeu-MeVa! i Abu--Sar
I O !
MeLeu---O-AIa-AIa--MeLeu~Val---MeLeu (Compound 2)
CA 02511888 1998-10-08
17
H2OH
H4ti
MeLeu--MeVai i Abu-+Sar
I I
MeLeu-D-Ala-Ala--Mel..eu-Val--MeLeu (Componnd 3)
Ffb
ACL3~~
MeLeu-MeVal ~ Abu-Sar
.1 I
MeLeu--D-AIa-AIa-MeLeu-Val-MeLeu (Compound 6)
Example 10
A mixed lymphocyte reaction (MLR) assay was performed with cyclosporine and
compounds 5e and
5g. The results are presented in figure 6 and are plotted as the means of four
experiments showing
concenvation of cyclosporine or derivative versus percent inhibition.
The MLR assay is useful for identifying CsA derivatives with biological
('mimunosuppressive) activity
and to quantify this activity relative to the immunosuppressive activity of
the parent CsA molecule.
Art example of a lymphocyte proliferation assay procedure useful for this
purpose is as follows:
TM
1. Collect blood from two individuals (20m1s each) and isolate lymphocytes
using 1 icoll-Paque (Pharmacia
Biotech).
2. - Count lymphocytes at 1:10 dilution in 2 % acetic acid (v/v):
3. Prepara 10rnls of cadt lymphocyte populations (A + B) at Ix106 cells/ml in
DMEM 120 % FCS (v/v).
4. Set up a 96 weII sterile tissue culture plate, flat bottom (Sarstedt, cat #
83.1835). To each wetl add:
5. AGquot 100 1 per well lymphocyte population A
6. Aliquot I 00}cl per well lymphocyte population B
7. Aliquot 20pI per weII of drug (CSA and CSA derivatives) at 0, 25, 5, 10,
25, 50 and lOOpg/L in triplicate in
DMEM with no supplements.
8. To measure the effect of drug on proliferation,,incubate the plate for 5
days at 37 C in S% CO2 atmosphere.
9. On day 6, prepare 3.?mIs of 1:50 dilution ofMethyl-sH-Thymid'me'(Amersham
Life Science, cat # TRK 120)
in DMEM with no supplements. Add 301e1 per well and incubate for 18 hours at
37 C in 5% CGZ
atmosphere.
10: On day 7 cells are.harvested onto glass microfiber filters GF/A (Whatinan,
cat # 1820024) using a CeII-
TM
1-Iaivestor (Skatron, cat # 11019). Wash cells 3x with 1-0-mI sterile
distilIed water.
Note: All procxdures are done using sterile teclmiques in a bioIogical flow
hood.
TM
1 I. Place fitters in Scinrilaiion vials and add 1.5mis of SciniSafe Plus 50 %
scinti7ation fluid (Fisher, c.at # SX-25-
CA 02511888 1998-10-08
18
5).
12, Measure the amount of radioactivity incorporated in the lymphocytes using
a beta counter (Micromedic
System Inc., TAURUS Automatic Liquid Scintilation Counter) for 1.0 minute.
13. Calculate averages and standard deviations for each drug and express
results as:
% Inhibitian =[1- Ave CPM of test dru~ ] x 100
Ave CPM of zero drug
% Proliferation =100 - % Inhtbition
The MLR assay can be utilized to select antibodies of the invention which bind
biologically active
CSA metabolites and the parent CSA molecule. Antibodies could also be selected
for reactivity to biologically
inactive metabolites.
From the results of the calcineurin assay and the mixed lymphocyte reaction
assay, it was found that
cyclosporines that have been chemically substituted and deuterated at the
amino acid I position can possess
significant immunosupressant activity. In the case of the derivatives 5e and
5g, immunasupressant activity that
is significantly greater than CsA was obtained.
Example 11
Other cyclosporine derivatives of th'e invention which have been prepared
include the following:
STRUCTURE
H
O
MeLeu-MeVal i Abt.l--Sar
I 0 1
MeLeu-D-Ala-Ala-MeLeu-Val--MeLeu
AcO-q
MeLeu-MeVal - i Abu-Sar
I 0 1
MeLeu-D-Ala-AI a--(U1eLeu-Val-MeLeu
CA 02511888 1998-10-08
19
HO
Aca,/
. MeLeu--MeVaI i Abu-Sar
~ O I
MeLeu--0-AIa-AIa-MeLeu-Val-MeLeu
O
4~~
MeLeu=MeVaI i Abu-Sar
I O I
MeLeu-D-Ala-Ala-MeLeu-Val-MeLeu
HZOH
H4il
MeLeu-MeVal i Abu-Sar
I O l
MeLeu-D AIa--.41a-MeLeu-Val--MeLeu
AcOl,
MeLeu-MeVal i Abu-Sar
( O 1
Me1Leu-D-Ala-Ata-MeLeu-Vat-MeLeu
CA 02511888 1998-10-08
(H)D CD3
Ac4//
MeLeu-MeVai N Abu-Sar
+ I
I
MeLeu--D'-A1a-Ala--Meleu-Val-MeLeu
(H) D3
Ha
MeLeu-MeVal - i Abu-Sar
I 0 1
MeLeu-D-Ala Ala-MeLeu-VaF-MeLeu
D-~, ~-D
Aca,/
MeLeu-=-MeVat - i Abu-Sac
I 0 1
M eLeu-D-AIa--Aia-M eLeu-Val-Me Leu
/ CQ3
AcOj/
MeLeu-MeVa! -- i Abu-Sar
~ Q 1
MeLeu-QAIa -Ala-Mel.eu-Vai--MeLeu
CA 02511888 1998-10-08
21
D2
H4,j
MeLeu-MeVal N Abu-Sar
1 1 O
1
MeLeu-D=Ala-Ala-MeLeu=Val-MeLeu
~CD3
HQi~
MeLeu--MeVaI Abu-Sar
MeLeu-D-Ala-Ala-MeLeu--Val-MeLeu
NOtBu
{
Ac4l/
MeLeu-MeVal--- i Abu-Sar
I 0 1
MeLeu-D-Ala-Ala-MeLeu-Vaf-MeLeu
CA 02511888 1998-10-08
22
N NH2
o
Ac0
MeLeu-MeVa! i Abu-Sar
I O 1
MeLeu--D-AIa-AIa--MeLeu-Val--MeLeu
NOMe
Ac4,f/
MeLeu--MeVal i Abu-Sar
I 0 I
MeLeu-D Ala-Aia-MeLeu-Val-MeLeu
ACO//
MeLeu-=MeVal i Abu--Sar
I Q 1
MeLeu-D-Ala AIa-MeLeu-Val-MeLeu
\
AcO
MeLeu-MeVal Abu-Sar
( O `
MeLeu--D-AIa--Aia-MeLeu-Vai--MeLe u
CA 02511888 1998-10-08
23
HN
O
HO/I
MeLeu-MeVa! Abu-Sar
1 0 `
MeLeu-D-Ala-Ala-MeLeu-Val-MeLeu
HN-I
O
HaI,
MeLeu-MeVai i Abu-Sar
I 0 I
Me Leu-D-Ala-Aia-MeLeu--Val-MeLeu
HN^
O
H4,l
MeLeu-MeVai L) Abu-Sar
f f
MeLeu-(]-Ala-Ala-MeLeu-Val-MeLeu
IN N~
O
HOi/
MeLeu-MeVal N Abu-Sar
I 1 0 1
MeLeu-D-Ala-Aia-MeLeu-Vai-MeLeu
CA 02511888 1998-10-08
24
4'i,
MeLeu-MeVal Abu-Sar
0 MeLeu-D-AIa-Ala-MeLeu--Val-MeLeu
Drvl! Composition Formulation and Elicitation of Immunosunression
Determination of the physicochemical, pharmacodynamic, toxicological and
phatmacokinetic
properties of the cyclosporine derivatives disclosed can be made using
standard chemical and biological assays
and through the use of mathematical modeling techniques which are known in the
chemical and
pharmacological/toxicological arts. The therapeutic utility and dosing regimen
can be extrapolated from the
results of such techniques and through the use of appropriate pharmacokinetic
and/or pharmacodynamic
models.
The compounds of this invention may be administered neat or with a
pharmaceutical carrier to a warm
blooded animal in need thereof. The pharmaceutical camer may be solid or
liquid.
This invention also relates to a method of treatment for patients suffering
from immunoregulatory
abnormalities involving the administration of the disclosed cyclosporines as
the active constituent.
For the treatment of these conditions and diseases caused by
immunoirregularity, a deuterated
cyclosporin may be administered orally, topically, parenterally, by inhalation
spray or reMatly in dosage unit
formulations containing conventional non-toxic pharmaceutically acceptable
carriers, adjuvants and vehicles.
The term parenteral, as used herein, includes subcutaneous injections,
intravenous, intramuscular, intrasternal
injection or infusioq techniques.
The pharmaceutical compositions containing the active ingredient may be in a
form suitable for oral
.use, for example, as tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or 'ganules,
emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended
for oral use may be prepar,ed
according to any method known to the art for the manufacture of pharmaceutical
compositions and such
compositions may contain one or more aftents selected from the group
consisting of sweetening agents,
flavoring agents, coloring a;ents and preserving agents in order to provide
pharmaceutically elegant and
palatable preparation. Tablets containing the active ingredient in admixture
with non-toxic pharmaceutically
acceptable excipients may also be manufactured by known methods. The
ezcipients used may be for example,
(1) inert diluents such as calcium carbonate, lactose, calcium phosphate or
sodium phosphate; (2) granulating
and disintegrating agents such as com starch, or alginic acid; (3) binding
agents such as starch, gelatin or
- acacia, and (4) lubricating agents such as magnesium stearate, stearic acid
or talc. The tablets may be uncoated
CA 02511888 1998-10-08
or they may be coated by known techniques to delay disintegration and
absorption in the gastrointestinal tract
and thereby provide a sustained action over a longer period. For example, a
time delay material such as
glyceryl monostearate or glyceryl distearate may be employed. They may also be
coated by the techniq=
described in the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form
osmotic therapeutic tablets for
5 controlled release.
In some cases, forrnulations for oral use may be in the form of hard gelatin
capsules wherein the
active ingredient is'mixed with an inert solid diluent, for example, calcium
carbonate, calcium phosphate or
kaolin. They may also be in the form of soft gelatin capsules wherein the
active ingredient is mixed with water
or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
10 Aqueous suspensions normally contain the active materials in admixture with
excipients suitable for
the manufacture of aqueous suspensions. Such excipients may be
(1) suspending agents such as sodium carboxymethylcellulose, rnethylcellulose,
hydroxypropylmethy[cellulose, sodium alginate, polyvinylpytrolidone, gum
tragacanth and gum acacia;
(2) dispersing br wetting agents which may be
15 (a) a naturally-occurring phosphatide such as lecithin,
(b) a condensation product of an alkylene oxide with a fatty acid, for
example,
polyoxyethylene stearate,
(c) a condensation product of ethylene oxide with a long chain aliphatic
alcohol, for
example, heptadecaethyleneoxycetanol,
20 (d) a condensation product of ethylene oxide with a partial ester derived
from a fatty acid
and a hexitol such as polyoxyethylene sorbitol monooleate, or
(e) a condensation product of ethylene oxide with a partial ester derived from
a fatty acid and
a hexitol anhydride, for example polyoxyethylene sorbitan monooleate.
The aqueous suspensions may also contain one or more preservatives, for
example, ethyl or n-propyl
25 p-hydroxybenzoate; one or more coloring agents; one or more flavoring
agents; and one or more sweetening
agents such as sucrose, aspartame or saccharin.
Oily suspension may be formulated by suspending the active ingredient in a
vegetable oil, for example
arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as
liquid paraffin. The oily suspensions
may contain a thickening agent, for example beeswax, hard paraffin or cetyl
alcohoI..Sweetening agents and
30' flavoring agents may be added to provide a palatable oral preparation.
These compositions may be preserved
by the addition o[ an antioxidant such as ascorbic acid.
Dispersible powders and granules are suitable for the preparation of an
aqueous suspension. They
provide the active ingredient in admixture with a dispersing or wetting agent,
a suspending agent and one or
more preservatives. Suitable dispersing or wetting agents and suspending
agents are exemplified by those
already mentioned above. Additional excipients, for example, those sweetening,
flavoring and coloring agents
descnbed above may also be present.
The pharmaceutical composidons of the invention may also be in the form of oil-
in-water emulsions.
The oily phase may be a vegetable oil such as olive oil or arachis oils, or a
mineral oil such as liquid paraffin
or a mixture thereof. Suitable emulsifying agents may be (1) naturally-
occurring gums such as gum acacia and
CA 02511888 1998-10-08
26
gum tragacanth, (2) naturally-occurring phosphatides such as soy bean and
lecithin, (3)
esters or partial esters derived from fatty acids and hexitol anhydrides, for
example,
sorbitan monooleate, (4) condensation products of said partial esters with
ethylene oxide,
for example, polyoxyethylene sorbitan monooleate. The emulsions may also
contain
sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example,
glycerol, propylene glycol, sorbitol, aspartame or sucrose. Such formulations
may also
contain a demulcent, a preservative and flavoring and coloring agents.
The pharmaceutical compositions may be in the form of a sterile injectable
aqueous
,
or oleagenous suspension. This suspension may be formulated according to known
methods using those suitable dispersing or wetting agents and suspending
agents which
have been mentioned above. The sterile injectable preparation may also be a
sterile
injectable solution or suspension in a non-toxic parenterally-acceptable
diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents
that may be employed are water, Ringer's solution and isotonic sodium chloride
solution.
In addition, sterile, fixed oils are conventionally employed as a solvent or
suspending
medium. For this purpose any bland fixed oil may be employed including
synthetic mono-
or diglycerides. In addition, fatty acids such as oleic acid find use in the
preparation of
injectables.
The disclosed cyclosporins may also be administered in the form of
suppositories
for rectal administration of the drug. These compositions can be prepared by
mixing the
drug with a suitable non-irritating excipient which is solid at ordinary
temperatures but
liquid at the rectal temperature and will therefore melt in the rectum to
release the drug.
Such materials are cocoa butter and polyethylene glycols.
For topical use, creams, ointments, jellies, solutions or suspensions, etc.,
containing
the disclosed cyclosporins are employed.
Dosage levels of the order from about 0.05 mg to about 50 mg per kilogram of
body
weight per day are useful in the treatment of the above-indicated conditions
(from about 2.5
mg to about 2.5 gms. per patient per day).
CA 02511888 1998-10-08
27
The amount of active ingredient that may be combined with the carrier
materials to
produce a single dosage form will vary depending upon the host treated and the
particular
mode of administration. For example, a formulation intended for the oral
administration of
humans may contain from 2.5 mg to 2.5 gm of active agent compounded with an
appropriate and convenient amount of carrier material which may vary from
about 5 to
about 95 percent of the total composition. Dosage unit forms will generally
contain
between from about 5 mg to about 500 mg of active ingredient.
It will be understood, however, that the specific.dose level for any
particular patient
will depend upon a variety of factors including the activity of the specific
compound
employed, the age, body weight, general health, sex, diet, time of
administration, route of
administration, rate of excretion, drug combination and the severity of the
particular disease
undergoing therapy.
In the case of conflict between the references listed herein and this
application, the
text of the application is controlling. Modifications and changes of the
disclosed
compounds and methods will be apparent to one skilled in the art. Such
modifications and
changes are intended to be encompassed by this disclosure and the claims
appended hereto.
