Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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RAFAMYCIN ANALOGS AND METHODS FOR MAKING SAME
BACKGROUND OF THE INVENTION
[OH The compound cyclosporine (cyclosporin A) has found wide use since its
introduction in
the fields of organ transplantation and immunomodulation, and has brought
about a significant
increase in the success rate for transplantation procedures. Recently, several
classes of
macrocyclic compounds having potent immunomodulatory activity have been
discovered.
Okuhara et al.. in European Patent Application No. 184. 162, published Jun. I
I. 1986, discloses
a number of macrocyclic compounds isolated from the genus Streptomyces,
including the
immunosuppressant FK-506. a 23-membered macrocyclic lactone. which was
isolated from a
strain of S. tsukuhaensis.
1021 Other related natural products. such as FR-900520 and FR-900523, which
differ from
FK-506 in their alkyl substituent at C-2 I. have been isolated from S.
hygroscopicus
yakushimnaensis. Another analog, FR-900525. produced by S. tsukubaensis,
differs from FK-
506 in the replacement of a pipecolic acid moiety with a proline group.
Unsatisfactory side-
effects associated with cyclosporine and FK-506, such as nephrotoxicity, have
led to a continued
search for immunosuppressant compounds having improved efficacy and safety.
including an
immunosupressive agent which is effective topically. but ineffective
systemically (U.S. Pat.
5A57.I I I ).
103] Rapamycin. as illustrated below, is a macrocyclic triene antibiotic
produced by
Streptomyces hygroscopicus. which was found to have antifungal activity,
particularly against
Candida alhicans. both in vitro and in vivo (US 3.929.992 and US 3.993.749).
1041 Rapamycin alone (US 4.885,171) or in combination with picibanil (US
4,401,653) has
been shown to have antitumor activity. In 1977, rapamycin was also shown to be
effective as an
immunosuppressant in the experimental allergic encephalomyelitis model, a
model for multiple
sclerosis: in the adjuvant arthritis model, a model for rheumatoid arthritis:
and was shown to
effectively inhibit the formation of IgE-like antibodies.
1051 The immunosuppressive effects of rapamycin have also been disclosed in
FASEB in
1989, as has its ability to prolong survival time of organ gratis in
histoincompatible rodents.
These and other biological effects of rapamycin are reviewed in
Trunsplcmiution Reviews.. 1992.
6. 39-87. Mono-ester and di-ester derivatives of rapamycin (esterilication at
positions 31 and 42)
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have been shown to be useful as antifungal agents (US 4.316.885) and as water
soluble prodrugs
of rapamycin (US 4,650,803).
1061 Mono-ester and di-ester derivatives of rapamycin (esterification at
positions 31 and 42)
have been shown to be useful as antifungal agents (US 4,316,885) and as water
soluble prodrugs
of rapamycin (US 4,650.803).
1071 Numerous chemical modifications of rapamycin have been attempted.
These include the
preparation of mono- and di-ester derivatives of rapamycin (WO 92/05179). 27-
oximes of
rapamycin (FPO 467606): 42-oxo analog of rapamycin (US 5.023,262); bicyclic
rapamycins (US
5.120.725): rapamvein dimers (US 5,120.727): silyl ethers of rapamycin (US
5.120.842); and
arvIsullonates and sulfamates (US 5.177.203). Rapamycin was recently
synthesized in its
naturally occurring enantiomeric form (K. C. Nicolaou et al.. J. Am. Chem.
Soc., 1993, 115,
4419-4420:S. L. Schreiber. .1. Am. Chem. Soc.. 1993. 115.7906-7907: S. J.
DanishefskyõI. Am.
Chem. Soc.. 1993, 115. 9345-9346). One recent example of a rapamycin analog is
a tetrazole
containing rapamycin analog (US 6,015,815). The tetrazole heterocyclic ring is
used to replace
the hydroxyl group to effect the analog.
1081 Although some of these modified compounds exhibit immunosuppressive
activity, anti-
restenotic activities in suppressing the migration and growth of vascular
smooth muscles.
especially when used in a stent coating, the need remains for rapamycin
analogs which possess
potentially enhanced efficacy against broad spectrum of cancers such as renal
cell carcinoma,
breast cancers, head and neck cancers, and potentially better lipophilicity.
longer half live in the
blood or in local tissues. or resistance to oxidative forces and better
stability in a formulation.
One way to achieve these goals is through introduction of a triazole moiety to
the side chain of a
rapamycin which may impart a better lipophilicity, better stability, better
bioavailability, better
tissue and cellular uptake. better efficacy compared to the known and existing
modified
rapamycin analogs or derivatives. The efficacy of the modified rapamycin may
also have better
potency against a variety of cancers, and potentially reduced toxicities.
SUMMARY OF THE INVENTION
1091 Accordingly. one object of the present invention is to provide novel
semi-synthetic
rapamycin analogs which possess a desired triazole moiety attached to either
or both to 31C-,
and or 42C-position of a rapamycin molecule.
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1101 In accordance with one aspect. the present invention is directed to
compounds
represented by the structural formula illustrated below.
[II] In accordance with one aspect. the present invention is directed to
compounds
represented by the structural formula illustrated below.
1121 In accordance with yet another aspect. a compound of the present
invention may contain
two such substitutes at both the 42C and 3 IC-positions of a rapamycin.
1131 The triazole moiety of the present invention may be introduced via a
variety of reaction
schemes, the typical ones are illustrated below:
, I 0 I ,
H OHt
0 0 N
o- o
0 OH 0
A'
0 0
- 0
Series A
wherein A' is one of the following structures:
NH
N \tr
N=-=rc' '
A3 A4 A5 A6
011
, N . OH
0
N -N NN OH
N- N A7 N- N OH
A8 ,A9 A 0 OH
- N - .
N
N 0 N -OH
N
N- N N,N0
N
All Al2 A13 A14
Series B:
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=-=
N N¨
NJ N' N N = N N'
IV > õ HN
N b N
(Th ,r4
82 83 B4 135
R N- N N _N CON .N N N N
0 r 0 I., N N co,H N
- = N
H OH.
O ONR6R7Bg 89 1310
0 '0
N N N
N .N. N- N
0 OH N = = CO211 N ' 0 , N , = ,
= SOMH, N = CO/H
= r' ' 0
- - B11 1312 1313 B14
1141
Another object of the present invention is to provide a synthetic processes
for the
preparation of such compounds from starting materials obtained by
fermentation, as well as
chemical intermediates useful in such synthetic processes.
[15] A
further object of the present invention is to provide pharmaceutical
compositions
containing, as an active ingredient. at least one of the above compounds.
1161 Yet
another object of the present invention is to provide a method of treating a
variety of
disease states. including restenosis. post-transplant tissue rejection. immune
and autoimmune
dysfunction. fungal growth. and cancer,
[17] In
addition. the compounds of the present invention may be employed as an oral
tablet.
oral solid or oral liquid, oral immediate or sustained release dosage,
intravenous injection
dosages. parenteral dosages. cream or solutions by formulation with
pharmaceutically acceptable
vehicles.
1181
Also within the scope of this invention includes pharmaceutical compositions
for
immediate release or sustained release of its active ingredient. each
comprising a compound of
this invention and pharmaceutically acceptable excepient.
1191
Still further related to this invention are medical devices, each comprising a
compound of
this invention. [xamples of the medical deices include drug-eluting coronary
or peripheral.
esophageal. urinary. ovar, or neurovascular stem.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Graph for Renal cell carcinoma tumor cell inhibition studies;
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FIG. 2 Graph for Renal cell carcinoma tumor cell inhibition studies;
FIG. 3 Graph for Lung Cancer A549 cell inhibition studies;
FIG. 4 Graph for Lung Cancer A549 cell inhibition studies;
FIG. 5 Graph for Lung Cancer A549 cell inhibition studies;
FIG. 6 Graph for Melanoma SK-MEL-28 cell inhibition studies;
FIG. 7 Graph for Melanoma SK-MEL-28 cell inhibition studies;
FIG. 8 Graph for Melanoma SK-ME1.-28 cell inhibition studies:
FIG. 9 Graph for Epidermal cancer A431 tumor cell model;
FIG. 10 Graph for Epidermal cancer A43I tumor cell model:
FIG. 11 Graph for Epidermal cancer A431 tumor cell model:
FIG. 12 Graph for Glioblastoma U87 MG Tumor model studies;
FIG. 13 Graph for Glioblastoma U87 MG Tumor model studies;
FIG. 14 Graph for Glioblastoma U87 MG Tumor model studies;
FIG. 15 Graph for liuman colorectal tumor HCT 116 model studies;
FIG. 16 Graph for Human colorectal tumor HCT 116 model studies;
FIG. 17 Graph for Human colorectal tumor I ICI- 116 model studies;
FIG. 18 Graph for Breast cancer MDA-MB-231 tumor model:
FIG. 19 Graph for Breast cancer MDA-M13-231 tumor model:
FIG. 20 Graph for Breast cancer MDA-MB-23 I tumor model;
FIG. 21 Graph for Breast cancer mcr-7 tumor model;
FIG. 22 Graph for Breast cancer MCE-7 tumor model;
FIG. 23 Graph for Breast cancer MCF-7 tumor model;
FIG, 24 Graph for Prostate cancer PC-3 tumor studies:
FIG. 25 Graph for Prostate cancer PC-3 tumor studies;
FIG. 26 Graph for Prostate cancer PC-3 tumor studies:
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FIG. 27 Efficacy of rapamycin analog of the present invention in treating HOT
116.
DETAIITD DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definition of Terms
1201 The term "prodrug." as used herein. refers to compounds which are
rapidly transformed
in vivo to the parent compound of the above formula, for example, by
hydrolysis in blood. A
thorough discussion is provided in T. Higuchi and V. Stella, "Pro-drugs as
Novel Delivery
Systems." Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed.,
"Bioreversible
Carriers in Drug Design." American Pharmaceutical Association and Pergamon
Press, 1987.
both of which are hereby incorporated by reference.
1211 The term "pharmaceutically acceptable prodrugs." as used herein,
refers to those
prodrugs of the compounds of the present invention which are. within the scope
of sound
medical judgment. suitable for use in contact with the tissues of humans and
lower mammals
without undue toxicity, irritation, and allergic response. are commensurate
with a reasonable
benefit/risk ratio, and are effective for their intended use, as well as the
zwitterionic forms, where
possible. of the compounds of the present invention. Particularly preferred
pharmaceutically
acceptable prodrugs of the present invention are prodrug esters of the C-3I
hydroxyl group of
compounds of the present invention.
1221 The term "prodrug esters." as used herein, refers to any of several
ester-forming groups
that are hydrolyzed under physiological conditions. Examples of prodrug ester
groups include
acetyl, ethanoyl, pivaloyl. pivaloyloNvmethyl. acetoxymethyl. phthalidyl.
methoxymethyl.
indanyl. and the like, as well as ester groups derived from the coupling of
naturally or
unnaturally-occurring amino acids to the C-31 hydroxyl group of compounds of
the present
invention.
1231 The term -isomer- as used herein, refers to a compound having the
identical chemical
formula but different structural or optical configurations.
1241 The term "epimer" as used herein, refers to a compound having the
identical chemical
formula but a different optical configuration at a particular position. In the
case of a rapamycin, a
42-Epi rapamycin refers to the compound that has the opposite optical rotation
compared to the
rapamycin obtained by a fermentation process.
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1251 The
term "15-isomer- as used herein, refers to the analog of rapamycin that
contains a 7-
member ring at the 15-position as opposed to a regular rapamycin obtained from
a fermentation
process which contains a six-member ring. This kind of conversion is also
called
"tautomerization-. The 15-isomer- as used herein. may also be referred to as a
15 tautomer of a
rapamycin.
Preparation of Compounds
1261 The
compounds and processes of the present invention will be better understood in
connection w.t.1 the 2o. .ow .ng synthetic schemes w hich illustrate the
methods by which the
compounds of the present invention may he prepared.
1271 The compounds of the present invention may' be prepared by a variety
of synthetic routes.
Most of the common conjugation reactions of rapamycin at 42- and/or 31-
hydroxyl positions are
found in the rapamycin patents mentioned above, the contents of which are
incorporated herein
by reference in their entireties.
EXAMPLES
Synthesis of Rapamyein Derivatives. The parent rapamycin structure is shown
below.
4(4C)
, \
'0 N
0 0
0 OH ,OH
0 Rapa
Li- 0
Rap,'
The synthetic scheme of series A of rapamycin analogs of the present invention
is shown below:
-
I
Y>,
N A
Cin A I Series A
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1281 Shown below are additional rapamvcin analogs of this invention that
were synthesized
similarly:
wherein A. CO,H
NH
- N N N N N N N
A6
.A5
.k3 OH
0N OH
Q. '0 csi N=
"` NN N-N H
WN A8 OH
0 0H N N A7 A9 A10 "
- ,"
AN
-`y=OH N -
NN
Series A NN NN
NN
k I I A 12 Al3 \14
Example 1: Synthesis of Compound Al
1291 To a stirred solution of Rapamycin (3 g. 3.2 mmol) and Cs2CO3 (3.2 g.
9.6 mmol) in
dried WI' (90 ml.) \vas added Nat (1.5 g. 9.6 mmol) and 3-bromoprop-1-yne (1.2
g. 9.6 mmol).
The reaction mixture was stirred at rt for 30 hours. Upon the completion of
reaction. 300 mt.
water was added in and extracted with ethyl acetate (200 m1_ x 3). The
combined organic layer
was washed by brine (300 ml.) and dried over anhydrous Na2SO4. After
concentration, the
residue was purified with silica gel chromatography (50% to 100% of ethyl
acetate in petroleum
ether as eluent) to give the compound Al (2.1 g. 68%) as a light green oil.
LCMS (m/z) ES- 950 (M-I )-.
Example 2: Synthesis of Compound A3
1301 "To a solution of 40-0-(prop-2-ynylox rapamycin A I (200 mi.:. 0.2
mmol) and 1-azido-
Admantane (100 mg. 0.6 mmol) in anhydrous THE (9 ml.) was added DIPEA (100
af,. 0.6
mmol) and Cul (20mg. 0.1 mmol) under N,. The solution was stirred at rt
overnight. Then, 20
water was added and extracted with ethyl acetate (20 ml. x 3). The combined
organic layer
\\ as washed by brine and dried over anh.\ drous Na2501. After concentration,
the residue \\ as
purified with silica gel chromatography (25% to 50% of ethyl acetate in
petroleum ether as eluet)
to give white solid which was further purified by prep-HPLC to give Compound
A3 (26 mg.
10%) as a white solid. 'H NMR (300 MHz. CDC13) 6 7.71 (s, 1H), 6.74 (m, H),
6,39-6.02 (m.
5H), 5.62-5.36 (m. 510: LCMS (m/z)F.S- I I 28(M-1 ).
Example 3: Synthesis of Compound A4
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1311 To a solution of 40-0-(prop-2-ynyloxy) rapamycin Al (200 mg,. 0.2
mmol) and 4-
azidobenzoic acid (100 mg. 0.6 mmol) in anhydrous THE (9 mL) was added D1PEA
(100 L.
0.6 mmol) and Cut (20 mg. 0.1 mmol) under N,. The solution was stirred at rt
for 3 hours. Then.
20 ml. ater was added and extracted with ethyl acetate (20 nil. x 3). The
combined organic
layer was washed by brine and dried over anhydrous Na2804. After
concentration. the residue
was purified with silica gel chromatography (5% to 10% of methanol in
dichloromethane as
eluent) to give µYliite solid which was further purified by PREP-I IPI,C to
give Compound A4 (29
mg. 12%) as a white solid. II NNIR (300 Ml ii. CDC13) 68.27 (m. III). 7.90 (m.
11-1). 7.73 (m.
I H). 7.56 (m. I H). 6.74 (m. 11-1). 6.55-6.00 (m, 51-1), 5.60-5.36 Om 5H):
LCMS (m/z) ES-
11 14(M-1)1.
Example 4: Synthesis of Compound AS
Preparation of Intermediate 2
1
(110 NaN3, Tf2O, acetonitrile , rt, 2h N
a
H2N 2 CuSO4, H20. TEA, rt, 6h N3
1 2
132] To a stirred suspension of NaN:, (2.0 g. 30.8 mmol) in acetonitrile
(20 ml.) was added
1120 (7.3 g. 25.8 mmol) via syringe slowly at 0 C. The mixture was stirred
for another 2 h at
this temperature. The insoluble solids were removed through filtration. At 0
C. the filtrate was
added dropwise into the mixture of Compound I (2.0 g, 13 mmol). CuSO4 (160 mg.
1 mmol).
H,0 (6 ml..) and Ft3N (3.6 mL. 25.8 mmol). The reaction mixture was stirred
for 6 h at room
temperature. The mixture was diluted with Et0Ac and washed with brine. The
organic layer was
dried over Na2SO4 and evaporated to yield brown solid which was purified with
silica 2e1
chromatography (30% to 50% of 110Ac in petroleum ether as eluent) to give
Intermediate 2 (1.1
g. 48%) as a w lute solid. 11-1 NMR (300 Mlii,. CDC1,1) 6 9.93 (s. III). 7.63
(m. 21I). 7.03 (m. 211).
2.02 (s. 311): I.CMS (m/z) F.S4- 177 (NI+ 1
1.331 To a solution of 40-0-(prop-2-ynyloxy) rapamycin A I (200 mg. 0.2
mmol) and N-(4-
azido- phenyl) acetainicle. Intermediate 2 ( I 00 nm. 0.6 mmol) in anhydrous
THE (9 ml.,) was
added Dl PEA (100 1.., 0.6 mmol) and Cul (20 tug. 0.1 mmol) under N,. The
solution was stirred
at rt for 4 hours. Then. 20 mL. water was added and the mixture was extracted
with Et0Ac (20
ml, x 3). The combined organic layer was washed by brine and dried over
anhydrous Na2SO4.
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Alter concentration. the residue was purified with silica gel chromatography
(30% to 100% of
Et0Ac in petroleum as eluent) to give white solid which was further purified
by prep-HPLC to
give Compound AS (56 mg, 25%) as a white solid. 1H NMR (300 MHz, CDCI3) 6 8.13
(m, 1H),
7.73 (m. 41-1), 6.74 (m,
6.49-6.00 (m. 511), 5.65-5.37 (m, 5H); LCMS (m/z) ES- 1127 (M-1)-.
Example 5: Synthesis of Compound A6
R
TMSN3. Na2CO3. Fri 0 H
OuS0,. sodium ascorbate R - TF3AF THE- it, 7h ^
t-BuOH. H20 rt. 3h "o-
N TMS
'
Al A2NNA6
1341 To
a solution of 40-0-(prop-2-ynyloxy) rapamycin A I (200 mu. 0.2 mmol) and TMS-
N1
(IOU mg. 0.9 mmol) in t-13u011 (6 mi.) and 1-120 (6 ml.) was added Na2CO3 (100
mg, 1 mmol).
CuSO4 (20 mg, 0.13 mmol) and sodium ascorbate (40 mg, 0.2 mmol) under N,. The
solution
was stirred at rt for 3 hours. Then. 20 mL water was added and extracted with
Et0Ac (20 mL x
3). The combined organic layer was washed by brine and dried over anhydrous
Na2SO4. After
concentration, the residue was purified with silica t.,,e1 chromatography (25%
of Et0Ac in
petroleum ether as eluent) to give compound A2 (189 mg, 82%) as a white solid
which was
dissolved in IBM' in THF (10 ml.) at 0 C and stirred at rt for 7 hours. Then
the reaction
mixture was partitioned between Ft0Ac and water. The aqueous phase was
extracted with
Lt0Ac (25 ml. x 3). The combined organic laver was \\ ashed with brine and
dried over
anhydrous Na2S0.4. After concentration. the residue was purl tied with silica
gel chromatography
(5% to 10% of methanol in dichloromethane as eluent) to give white solid which
was further
purified by prep-FIPLC to give compound A6 (38 mg, 24%) as a white solid. 1H
NMR (300 MHz.
CDCI3) 6 7.75-7.55 (m. 114 6.76 (m, 1 I-1). 6.49-6.08 (m. 511). 5.53-5.35 (m,
3H); LCMS (m/z)
ES- 1012 (M-1-f-18)'.
Example 6: Synthesis of Compound A7
Preparation of ntermed kites 5 and 6:
Br N HO-
NaN3, H20 TsCI, TEA Cs2003, morpholine
3
1,-
refluxing, DCM. it, 4h DMF, it, overnight
3 4 5
overnight 6
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135i To
a solution of 3 ( 5 u. 41 mmol) in 100 ml.. of water was added NaN1 (5g. 83
mmol)
and was refluxed overnight. Then 100 ml. [)CM was added in after reaction
mixture was cooled
to rt. The organic phase separated was dried over Na2S0.1. filtered. To the
solution was added
Et3N (5.05 u. 50 mmol) and IsCI (9.55 g. 50 mmol) at 0 deg. The reaction
mixture was stirred at
rt for 4 hours. 100 ml.water \\ as added. The organic phase was separated and
dried over Na2S0.2.
Filtration and concentration in vacuo gave the crude product. Purification by
column
chromatography (10% of Et0Ac in petroleum ether as eluent) gave intermediate 5
(5.7 g, 58%)
as a colorless oil. IFl NMR (300 MHz, CDC13) 6 7.81 (d. 2H), 7.39 (d, 211).
4.14 (m,2H), 3.48 (m.
2I-1).2.43(s. 3H): I.CMS (m/z) FS+ 242(M+1)-'.
1361 To
a solution of intermediate 5 (I g. 4,1 mmol) and Cs2CO3 (2.8 g. 8.2 mmol) in
30 ml.
of anhydrous DM': was added morpholine (0.71 g. 8,2 mmol) at 0 C. Then it was
stirred at rt
overnight. The reaction mixture \\ as partitioned between 50 ml of Ft0Ae and
60 mL of water.
The organic phase was dried over Na?S0.4. Illtration and concentration in
VaCLIO gave the crude
product. Purification by column chromatography (50% of It0Ac in petroleum
ether as eluent)
gave intermediate 6 (0.4 u. 72%) as a colorless oil. [.CMS (m/i.) FS 4 157 (M4-
1)'.
1371 To
a solution of 40-0-(prop-2-ynylox) rapamycin AI (200 mg. 0.2 mmol) and 4-(2-
azido- ethyl)morpholine. Intermediate 6 (100 mg, 0.6 mmol) in anhydrous THE (9
mL) was
added DIPEA (100 L. 0.6 mmol) and Cu! (20 mg, 0.1 mmol) under N-). The
solution was stirred
at rt for 3 hours. Then. 20 ml. water was added and extracted with Et0Ac (20
mL x 3). The
combined organic laver was washed by brine and dried over anhydrous Na2SO4.
After
concentration, the residue \\ as purified with silica gel chromatography (30%
of Nt0Ac in
petroleum ether as eluent) to give white solid which was further purified by
prep-HPLC to give
compound A7 (45 mg. 20%) as a white solid. IF! NMR (300 Ml ii. CDCL,) 6 7.89
(m. 1H). 6.72
(m. 1 II). 6.44-6,05 (m. 511). 5.60-5.37 ( m. 511): [('MS (m/i) [5- 1107 (NI-
1 f.
Example 7: Synthesis of Compound A9:
1381 To
a solution of 40-0-(prop-2-ynyloxy) rapamycin A 1 (200 mu. 0.2 mmol) and 2-
azidoethanol (100 mg. 1.2 mmol) in anhydrous TI IF (9 mL) was added DIPEA (100
L. 0.6
mmol) and Cul (20 mg. 0.1 mmol) under The
solution was stirred at rt overnight. Then, 20
ml.. water was added and extracted with Et0Ac (20 ml, x 3). The combined
organic layer was
washed by brine and dried over anhydrous Na-,.S0.1. After concentration. the
residue was purified
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with silica gel chromatography (5% to 10% of methanol in dichloromethane as
eluent) to give
white solid which was further purified by prep-HPLC to give compound A9 (26
mg, 11%) as a
white solid. IH NMR (300 MI lz. CDCI3) 6 8.03-7.78 (m. 1H), 6.70 (m, I H),
6.46-6.00 (m. 5H).
5.61-5.39 (m, 511): I.CMS (m/i.) FS- 1038 (M-1)-.
Example 8: Synthesis of Compound A10:
Preparation of Intermediate 8
F-0 HBr, rt, 1 h NaN3, DMSO,
Br 80deg, 2 days N3
HO
OH OH
7 8 9
1391 A
solution of compound 7 (1.3 g. 12.7 mmol) in 40% HBr (10 mL) was stirred at rt
for I
hour. Then. 20 mL water was added and extracted with Ft0Ac (20 mL x 3). The
combined
organic laver was washed by brine, dried over anhydrous Na2SO4. After
concentration, the
residue was purified with silica gel chromatography (50% of Et0Ac in petroleum
ether as eluent)
to give intermediate 8 (0.7 g. 31%) as a white solid.
I.CMS (m/z) LS+ 183 (M
Preparation of Intermediate 9
1401 A
solution of' intermediate 8 (0.7 g. 3.9 mmol) and NEIN.; (1.13 g. 15 mmol) in
DMS0 (16
ml..) was stirred at 80 deg for 2 days. Then. 20 mt. water was added and
extracted with Et0Ac
(20 nil. x 3). The combined organic layer was washed by brine, dried over
anhydrous Na250.1.
After concentration, the residue \vas purified with silica gel chromatography
(50% to 100% of
Ft0Ac in petroleum ether as eluent) to give intermediate 9 (0.25 g. 45%) as a
white solid.
LCMS (m/z) ES+ 146 (M-1.1)'.
Preparation of Compound A 10
1411 to
a solution of' 40-O-( prop-2-ynyloxy) rapamycin A I (200 mg. 0.2 mmol) and 2-
(azidomethyl) 2-methylpropane-1.3-diol Intermediate 9 (100 mu. 0.7 mmol) in t-
Bu011 (6 m1.)
and H20 (6 ml.) was added Na2CO3 (100 mg. 1 mmol), CuSO4 (20 mg, 0.13 mmol)
and sodium
ascorhate (40 mu. 0.2 mmol) under The
solution was stirred at rt for 6 hours. Then. 20 nil.
water was added and extracted \\ ith Et0Ac (20 ml. x 3). 'Hie combined organic
layer was
washed by brine and dried over anhydrous Na250.1. After concentration, the
residue was purified
with silica gel chromatography (5% to 10% of methanol in dichloromethane as
eluent) to give
SUBSTITUTE SHEET (RULE 26)
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13
while solid w hich was further purified 11\ prep-IIPI.0 to give compound A10
(15 mg. 7%) as a
white solid. I H NMR (300 Nil It. cDci,) 6 7.76 (m. III). 6.69 (m. 111), 6.55-
6.00 (m, 511). 5.63-
5.33 (m. 511). LCMS (m/z) ES- 1096 (M-1)-.
Example 9: Synthesis of Compound Al2
Preparation of Intermediate II
Li0H, THE, MeON
0 ' 0
H20, rt, 3h
N3H
N3
1
1
1421 To a mixture of compound 10 (I 2. 7.8 mmol) in of Me01-1/TIIF (10 mL/I
0 iii L) was
added a solution oftAA 1 (0.9 LI. 39 mmol) in 10 ml of w.ater. The resulting
solution is stirred at
room temperature for 3 hours. I he mixture was acidified bv 2N 11(1 to PH -4.
and extracted with
Et0Ac (25 nil. ,,2). The combine organic laver was concentrated under vacuum
to give
intermediate 11 (0.7 g, 910/0) as a colorless oil. 11-1 NMR (300 MHz, CDC13) 6
2.34 (s,2H):
LCMS (m/z) ES+ 102 (M+1)'.
Preparation of Compound Al2
1431 To a solution of 40-0-(prop-2-ynyloxy) rapamycin A 1 (200 mu. 0.2
mmol) and 2-
azidoacetic acid Intermediate 11 ( IOU mg. I mmol) in t-1.3u0H (6 ml,) and 110
(6 ml,) was
added Na:CO3 (100 mg. 1 mmol). CuSO4 (20 mg. 0.13 mmol) and sodium ascorbate
(40 mg, 0.2
mmol) under N. The solution was stirred at rt for 2 hours. 'Ben. 20 ml water
was added and
extracted \\ id) FIOAc (20 3). 'Hie combined organic layer was washed by
brine and dried
over anhydrous Na:SO4. After concentration. the residue was purified with
silica gel
chromatography (5% to 200/0 of methanol in dichloromethane as eluent) to give
white solid
which was further purified by prep-HP1.0 to give compound Al2 (15 mg. 7%) as a
white solid.
H NMR (300 MHz. CDCI3) 6 7.89 (m. I H). 6.72 (m, 1H). 6.49-6.08 (m. 5H), 5.60-
5.35 (m, 511):
LCMS (m/z) ES- 1052 (M-1).
Example 10: Synthesis of Compound A13
Preparation of intermediate 13
SUBSTITUTE SHEET (RULE 26)
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14
\N_ 1 NaN3, Tf20, acetonitrile , it, 2h
H2N
N3 -N N-
2 CuSO4, H20, TEA, it, 6h
12
13
1441 to
a stirred suspension of NaN,, (2.0 g. 30.8 mmol) in acetonitrile (20 mL) was
added
ILO (7.3 g. 25.8 mmol) s\
rinee SIO\\ 1\ at 0 deg.. The mixture was stirred for another 2 h at
this temperature. The insoluble solids wel'e. removed through filtration. At 0
deg. the filtrate was
added dropwise into the mixture of compound 12 (2.0 g, I() mmol), CuSO4 (160
mg, 1 mmol).
11,0 (6 nil.) and Ft3N (3.6 ml.. 25.8 mmol). The reaction mixture was stirred
for 6 11 at room
temperature. The mixture was diluted with Et0Ac and washed with brine. The
organic layer was
dried over Na2SO4 and evaporated to yield brown solid which was purified with
silica gel
chromatography (30% to 50% of Ft0Ac in petroleum ether as eluent) to give
brown solid which
was further purified by prep-I IPEC to give intermediate 13 (0.4 LI. 17%) as a
brown solid. 111
NMR (300 MI It. CDCL) 6 6.94 (m, 411). 3.19 (m. 41I). 2.60 (m. 411). 2.36 (s.
ECMS
(m/z)ES4- 218 (M1 I )'.
Preparation of Compound Al3
1451 In a solution or 40-0-(prop-2-ynylox
rapamycin Al (200 mg,. 0.2 rnmol) and 1-(4-
azido- pheny1)-4-methylpiperaiine Intermediate 13 (100 mg. 0.5 mmol) in t-Bu01-
1 (6 inE) and
1120 (6 111E) was added Na2CO3 (100 mg. 1 mmol), CuSO4 (20 mg, 0.13 mmol) and
sodium
ascorbate (40 mg. 0.2 mmol) under N,. The solution was stirred at rt for 3
hours. Then, 20 niL
water was added and extracted with Ft0Ac (20 ml. x 3). The combined organic
layer was
washed by brine and dried over anhydrous Na2SO4. Alter concentration. the
residue was purified
with silica gel chromatography (5% to 20% of methanol in diehloromethane as
eluent) to give
white solid which was further purified by prep-111TC to give compound Al3 (33
mg. 14%) as a
white solid. '11 NMR (300 MI It. CDCI;) 6 8.11 (m. 111). 7.71 (m. 211). 7.06
(m. 214 6.70 (m.
111). 6.45-6.00 (m. 511). 5.66-5.36 (m. 511): I.CMS (m/i.) ES- 1168 (M-1)-.
Example 11: Synthesis of Compound Al4
1461 To
a solution of 40-0-(prop-2-ynyloxy) rapamvcin AI (200 Inv,. 0.2 mmol) and 1-
(azido-
methyl)-4-fluorobenzene (100 mg. 0.6 mmol) in anhydrous TI IF (9 mL) was added
D1PEA (100
E. 0.6 mmol) and Cut (20 mg. 0.1 mmol) under N,. The solution was stirred at
rt for 3 hours.
SUBSTITUTE SHEET (RULE 26)
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Then. 20 ml, water \\. as added and extracted with Pt0Ae (20 ml. x 3). The
combined organic
layer as washed by brine and dried over anhydrous Na2SO4. Alter concentration,
the residue
was purified with silica gel chromatography (30% to 100% of Et0Ac in petroleum
ether as
eluent) to give white solid which was further purified by prep-11PLC to give
compound A 14 (32
mg. 14%) as a white solid. '11 NMR (300 MHz. CDCI3) 6 7.58 (m. 1 LI). 7.26 (m.
211). 7.06 (m.
211), 6.75 (m. 1 II). 6.50-6.00 (m. 511). 5.60-5.36 (m. 511): I.CMS(m/z)FS--
1102(M-1).
Example 12: Synthesis of Compound A15
TBDPSCI, TEA, DMAP DIEA. 1120, DCM
TBDPS
DCM, rt, overnight rt, overnight
14 15 16
RO H
16, DIEA, toluene, R HF/pyridine. THF C)
\>",
'OH 80 deg, 2h
rt, 4h
17 A15
Preparation of Intermediate 15
1471 to a solution of compound 14 g.
-18 mmol) and kt (5 g. 50 mmol) in DCM (100 m1.)
\vas added DMA P (0.6 g. 5 mmol) and drop w ised TI3DPS-C1 (4.4 g. 16 mmol) at
0 deg and
stirred at rt overnight. Then. I 00 1111, water was added and extracted \vith
DCM (80 m L x 3). The
combined organic laver \\ as washed by brine and dried over anhydrous Na,SO4.
After
concentration, the residue \\ as purified \\ ith silica gel chromatography
(30% of Et0Ac in
petroleum ether as eluent) to give intermediate 15 (1.7 g. 12%) as a colorless
oil. 111 NMR (300
MI lz. CDCI3) 6 7,63 (lit -1r1). 7.36 (m. 6H), 3.74 (m.21-1), 3.66 (m, 211),
1.04 (s.91-1); LCMS (m/z)
ES+ 301 (M+1)'.
Preparation of Intermediate 16
1481 To a solution of intermediate 15 (1.7 LI. 5.7 mmol) and Dl PEA (1.5 g.
11.4 mmol) in
DCM (40 ml.) was added 1 10 (1.7 g. 6 mmol) at 0 deg and stirred at rt
overnight. Then. 50 m1.
water was added and extracted \\ ith DCM (40 ml. x 3). The combined organic
layer was washed
by brine and dried over anhydrous Na:SOA. After concentration, the residue was
purified with
silica gel chromatograph ( of Ft0.Ac in petroleum ether as cluent) to give
intermediate 16
SUBSTITUTE SHEET (RULE 26)
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16
(1.5 g. 63%) as a colorless oil. 11 NMR (300 MHz. CDCF) 6 7.63 (m. 411). 7.36
(m. 611). 4.58
(m,211). 3.95 (m. 211). 1.04 (s.91I): 1,CMS (m,/z) ES-I- 433 (M+1)..
Preparation of Intermediate 17
1491 .lo a solution of Rapamycin (400 mg. 0.43 mmol) and DIPFA (278 mg.
2.15 mmol) in
toluene (30 ml was added intermediate 16 (0.93 g. 2.15 mmol) at rt and stirred
at 80 deg for 2
hours. Then. 50 ml, water was added and extracted with 1/t0Ac (30 m1_, x 3).
The combined
organic layer was washed by 0.5 N 1-ICI, saturated NaHCO3 and brine, dried
over anhydrous
Na2SO4. After concentration, the residue was purified with silica gel
chromatography (25% to
40% of Ft0Ac in petroleum ether as cluent) to give intermediate 17 (280 mg.
53%) give as a
\\Ilite solid. 1.CMS (iniz) Es- I 94 (M-11.
Preparation of Compound Al5
1501 To a solution of intermediate 17 (280 mg. 0.23 mmol) in THE (10 ml_.)
was added 2 ml.
HE in pyridine at 0 dog and stirred at rt for 3 hours. Then. 20 ml water was
added and extracted
with It0Ac (20 nil. x 3). Ihe combined organic la\ or was washed b) 0.5N 11C1.
saturated
NalICOt and brine, dried over anhydrous NaSO4. Alter concentration, the
residue was purified
with silica gel chromatography (30% to 100 % of Ft0Ac in petroleum ether as
eluent) to give
white solid which was further purified by prep-I !PLC to give compound Al5 (34
mg. 15%) as a
white solid. 111 NMR (300 MI Ii. CDCF) 6 6.40-6.00 (m. 511). 5.53-5.25 (m.
4H), 4.83 (s. 11-1).
4,13 (m. I.CMS (m/z) FS- 957 (M-1)-.
Synthesis or series 13 of rapamvcin derivatives oldie present invention
1511 13 series of rapam. cin derivati\ es \\ ere prepared according to the
rollo\\ ing reaction
scheme:
R 0 Tr-i0NaN R - 0
0 1
- =
'OH DCM
'OTf NN.I3 'Click Reaction'
Rapamycin Compound I3 Compound III
Series [3
1521 In the formula of the schemes shown above. R and 13' have the
following structures in
some examples of the 13 series of rapamycin derivatives.:
SUBSTITUTE SHEET (RULE 26)
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17
HQ
, f -OH
N N. N - =
N N,
NN N¨
R 13' = N 'OH N- 4
N N õ:" HN
-
kO (!),,,, - --N
132 B3 134 135
..,
-,1
0 I
H '
N-- N N NN N ---N --N
9 - 0 NA N N'N, -
0O211
0 oh , / N
N N CO2E1 N
I
136 137 138 139 1310
N- N -NJ NN N N-
N " '
CO2H N- .
_ SO2N112 N -_ ---, - SO2Nfi2 N ,--- CO7H
N 0 , : ,
1311 1312 1313 B14
Example 13: Synthesis of Compound 131
R0T0 R NaN3
..Ø0 R 0
f2 -,
______________________ .. _______________________ t
'OH DCM "OTf 4...CICN:
compound B compound B1
1531 lo
a solution or !warm chi (5 ti,. 5.5 mmol) and 2.6-di-tert-buty1-4-meth
lpyridine (3.4 g.
3.4 mmol) in dried DCM (150 ml.). trilluoromethanesullonic anhydride (1.55 g.
5.5 mmol) was
added at 0 oC. Alter the mixture was stirred for 2 h at room temperature. NaN3
(3.6 g, 55 mmol)
was added and [WS() (60 ml ) was added at -10 oC. the mixture \\ as stirred
Col- at 40 oC lor 5 h.
The mixture \\ as quenched 1-).\ addition of water. extracted \\ ith DCM (200
ml. x 2). and the
combined extracts were washed \\ ith water. dried over Na2S01. and evaporated
to dryness in
vacuo. The crude product was purified by column chromatography (25% of Et0Ac
in petroleum
ether as eluent) to give compound B (1.5 g. 300/) as a white solid. LCMS (m/z)
ES- 937 (M-HY.
Example 14: Synthesis of Compound B2
0¨
compound B1
,0.---7-'0H - - R /----c_. /-------(OH
CuSO4 =-\
/ N-N
Na2CO3 'N-
SM1 Me0H/H20 Compound B2
1541 to
a solution of compound li 1 (ISO nut. 0.16 mmol) and SMI (27 mg. 0.48 mmol) in
Me011/11,0 (4 ml 12 ml.) was added vitamine C sodium salt (63 mg. 0.32 mmol).
followed \\ ith
the addition of. CuSO4 (51 mg. 0.32 mnfol) and Na:,.001 (51 mt.t. 0.48 mmol).
Aker stirred
overniat, the mixture \\ as littered. the filtrate \\ as concentrated and
purified by column
SUBSTITUTE SHEET (RULE 26)
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18
chromatography (0 to 2% of methanol in dichloromethane as cluent) to give
Compound 132 (33.2
mg. 21%) as yellow solid. II
N (300 MIlt. CD(13) 6 7.83 (m. 111). 6.37-6.01 (m. 41-1). 5.40-
5.31 (m. 411): ELMS (W') ES- 093 (M-I
Example 15: Synthesis of Compound 133
compound B1
Na2CO3 R"-<
CuSO4
SM1
vitamine C sodium salts compound B3
1551 To
a solution of compound 131 (150 rrq..!õ 0.16 mmol) and SM1 (40 mg. 0.48 mmol)
in
Me011/1+0 (4 ml /2 ml \\ as added vitaminc C sodium salt (63 11144. 0.32 mmol)
followed \\ ith
the addition of CuS0.1 (51 mg. 0.32 mmol) and Na2C0t (51 mg. 0.48 mmol). After
stirred
overnight. the mixture \\ as filtered. the filtrate was concentrated and was
purified by column
chromatography (0 to 2% of methanol in dichloromethane as cluent) to give
Compound 133 (20.7
mg. 13%) as \\ hitc solid. 'II NM R (300 M117. CDCE) 6 8.55 (s. 11). 7.80 (m.
111). 6.39-6.02 (m.
411). 5.46-4.83 (m. 411): ELMS (m/z) ES- 1020
Example 16: Synthesis of Compound 114
o- OH
DCMcompound B1
E3r H2N
0"C 1h
Step 1 Step 2
SM1 1 2 compound 84
1561 To
a solution of I (2 g. 32.8 mmol) in DCM (100 ml.) was added SM1 (2 g. 16.4
mmol)
at 0 oC dropw ise over I hour. rhe mixture \Vas concentrated and the residue
was purified by
column chromatograph\ (0 to 200 of methanol in dichloromethane as eluent to
giVe intermediate
2 (0.0 (2. 54%) as yellow oil. ELMS (m/z) [54- 100 NH
1571 To
a solution of compound 131 (150 mg. 0.16 mmol) and intermediate 2 (48 mg,.
0.48
mmol) in Me011/1120 (4 ml ./2 ml,) was added vitamine C sodium salt (63 mg.
0.32 mmol)
followed with the addition of CuSO4 (51 mg. 0.32 mmol) and Na2CO3 (51 mg, 0.48
mmol).
After stirred overnight, the mixture was filtered, the filtrate \\ as
concentrated and purified by
column chromatOgraphy (0 to 2% of methanol in diehloromethane as eluent) to
give Compound
134 (17.1 mg. I 1"/0) as white solid. 11 1 NMR (300 lV111/. ('DCE) 6 8.40 (s.
111). 6.39-6.02 (m. 411).
5.37-4.94 (m. 411): ELMS (m/i) ES- 1036 (M-11)-.
SUBSTITUTE SHEET (RULE 26)
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19
Example 17: Synthesis of Compound 135
N OH compound 61 0--
. H K2CO3 1- 1,
õOHNOH
1111L0F1 Step 2 10--\
Step 1
SM1 2 compound B5
1581 To a solution of I (2 u. 19.0 0111101) in TI IF (40 ml .1 was added
Kr,CO3 (5.2 g, 38 mmol)
and SM I (2.2 g. 19 mmol ) at 0 C. After stirred or 4 h at r.t. the mixture
was filtered, the filtrate
was concentrated and purified by column chromatography (0 to 3% of methanol in
dichloromethane as eluent) to give intermediate 2 (1.2 g, 44%) as yellow oil.
11-1 NMR (300 Mfiz.
CDC1.1) 63.65 (m. 411). 3.49 (m. 21-1), 2.75 (m. 41-1), 2.22 (m.
1591 'lo a solution of compound B 1 (150 01g. 0.16 mmol) and intermediate 2
(69 mg, 0.48
mmol) in Me01-1/1120 (4 ml .12 ml.) was added vitamine C sodium salt (63 mg,
0.32 mmol)
followed with the addition of CuSai (51 mu. 0.32 mmol) and Na2CO3 (51 mg, 0.48
mmol).
After stirred overnight. the mixture was filtered, the filtrate was
concentrated and purified hy
column chromatography 10 to 2% of methanol in dichloromethane as eluent) to
give Compound
135 (26.7 mg. 11%) as white solid. 11 1 NMR (300 MI lz. C'DCI3) 6 7.92 (s,
111). 6.39-5.99 (m.
411). 5.45-4.84 (m. 411): I,Cl\/1S (m/z) 1080 (M-I If .
Example 18: Synthesis of Compound B6
cs2co, compound B1
4
THF Step 2 RNNO
Step 1
SM1 1 2 compound 66
1601 To a solution of 1(0.5 g. 5.8 mmol) in TI IF (40 ml.) was added Ki,CO3
(1.6g. 11.6 mmol)
and SM I (0.69 i, 5.8 mmol) at 0 oC. Alter stirred for 4 h at It. the mixture
was filtered, the
filtrate \\ as concentrated and purified by column chromatography (3% of
methanol in
dichloromcthane as eluent) to give 2 (0.3 u. 42%) as yellow oil. [CMS (m/z)
[S+ 125 (M+1-I)
1611 lo a solution of compound 111 (ISO mg. 0.16 11111101) and 2 (60 mu.
0.48 mmol) iii
Me01 I/1120 (4 101/2 ml,) \\ as added vitamine C sodill111 SaIl (63 fig. 0.32
mmol) followed \\ ith
the addition of CuS01 (51 mg. 0.32 mmol) and Na:,CO: (51 mg. 0,48 mmol). After
stirred
oyerniuht, the mixture \\ as filtered. the filtrate \vas concentrated and
purified by column
chromatography (0 to 2% of methanol in dichloromethane as eluent) to give
Compound 136 (25.8
SUBSTITUTE SHEET (RULE 26)
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WO 2014/082286 PCT/CN2012/085631
mg. 15%) as white solid. if1 NMl (300 MI lz. CDCI3) 6 7.81 (s. III), 6.40-6.02
(m, 41-1). 5.45-
4.81 (m. 411): 1.CMS (mi.) [5- 1062 (M-11)-.
Example 19: Synthesis of Compound 137
compound B1 0¨
Cs2CO3
THF Step 2 N N
Stop 1
SM1 1 2 compound B7
1621 To a solution of 1 (0.5 u. 5 mmol) in T111: (40 ml.) was added K.2CO3
(1.4 g. 10 mmol)
and SM I (0.6 g. 5 mmol) at 0 C. After stirred for 4 h at r.t. the mixture was
filtered, the filtrate
\vas concentrated and purified by column chromatography (3% of methanol in
dichloromethane
as eluent) to give intermediate 2 (0.5 g. 72%) as \ am\ oil. I.(MS (m/z) LS+
139 (M4-11)'.
1631 to a solution of compound 131 (150 mu. 0.16 mmol) and 2 (60 mu. 0.48
mmol) in
Me011/1120 (4 m112 ml,) was added vitamine C sodium salt (63 mu. 0.32 mmol)
followed with
the addition of CuS0.4 (51 mu. 0.32 mmol) and Na2CO3 (51 111g, 0.48 mmol).
After stirred
overniuht, the mixture was filtered. the filtrate was concentrated and
purified by column
chromatography (2% of methanol in dichloromethane as eluent) to give Compound
137 (12 mg.
7%) as white solid. II NMR (300 MHz. CDC[) 6 8.39 (s. 11-1). 7.76 (s. 111).
6.37-6.01 (m. 411).
5,41-4.78 (m. 411): 1.CMS (et) FS- 1075 (M-I-1)..
Example 20: Synthesis of Compouml 139
compound B1 0- - \i
N
N CuSO4
Na2CO3 N
Me0H11-120
SM1 compound B9
1641 To a solution of compound B I (150 mg. 0.16 mmol) and SM1 (50 mg, 0.48
mmol) in
Me011/1120 (4 m1/2 ml.) was added vitamine C sodium salt (63 mu. 0.32 mmol)
followed with
the addition of CuSO., (51 mu. 0..32 mmol) and Na:CO; (51 mifõ 0.48 mmol).
After stirred
overniat, the mixture was filtered, the filtrate was concentrated and purified
by column
chromatography (2% of methanol in dichloromethane as eluent) to give Compound
B9 (37.1 mu.
_
SUBSTITUTE SHEET (RULE 26)
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WO 2014/082286 PCT/CN2012/085631
21
22%) as white solid. III NMR (300 MI lz. CDCI3)69.37-7.89 (m. 511). 6.39-6.01
Om 411). 5.42-
4,99 (m. 411): "CMS (m/i.) ES- 1040 (M-1-1)-.
Example 21: Synthesis of Compound 1111
B Si
TBAF
Pd(Ph3)2C12, Cul. Et3N THE
0 80 degrees. o/n Step 2
0
2
SM1 Step 1
0
LOH compound B1 0 OH
-
._/
Me0H OH Step 3
0 >¨N
µt\IN
3
compound B11
1651 To a solution of SM1 (2.1 e. 10 mmol) ill Dioxane (20 ml.) was added
ethynyltrimethylsilune (2 g. 20 mmol). Cul (191 mg. 1 mmol) and I'd(PPIO:C12
(730 me. 1
mmol) under N. then 1..t,N ( 10 g. 100 mmol) was added dropw Ise. Aker stirred
at 100 oC
overnieht, the mixture was quenched by water. extracted with Nt0Ac (50 mi. x
2). The combine
organic layer was dried over anhydrous Na:SO4. concentrated to give crude
intermediate 1.
1661 The crude intermediate I was dissolved treated with 'IBM' in THE (20
mL.. 20 mmol) at
r.t. for 2 hours, then quenched by water and extracted with Et0Ac (50 ml. x
2). The combine
organic layer was dried over anhydrOLIS Na:Sat. concentrated to give crude
intermediate 2 which
was purified bv column chromatography to eive 2 (0.9 g. 58%) as yellow solid.
III NMR (300
MHz. DM50-d6) 7.96 (d. 211). 7.62 (d. 211). 4.48 (S. 114 3.87 (s. 311).
1671 To a solution of 2 (0.5 g. 3.13 mmol) in Me011 (10 ml.) was added
1.i01-1 (0.312g. 12.52
mmol) in water (10 ml.). l'lie mixture was stirred for 2 h. then quenched by
[ICI solution (2N).
extracted with Et0Ac (30 ml.*3). The combined organic was dried over anhydrous
Na:S0.1.
concentrated to give the desired intermediate 3 (0,35 g, 77%) as Yellow solid.
1681 To a solution of compound 111 (130 me. 0.16 mmol) and intermediate 3
(70 me. 0.48
mmol) in Me011/11:0 (6 m1.13 mt.) was added vitamine C sodium salt (63 mg.
0.32 mmol)
followed with the addition of CuSai (51 mg. 0.32 mmol) and Na:CO3 (51 me, 0.48
mmol).
After stirred overnight, the mixture was adjusted to pll about 3-4. filtered.
the filtrate was
SUBSTITUTE SHEET (RULE 26)
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WO 2014/082286 PCT/CN2012/085631
22
concentrated and purified b> column chromatography (1.5% of methanol in
dichloromethane as
eluent) to give Compound RI 1 ( 15.5 mg. 9%) as white solid. 11-1 NMR (300
MHz. CDCF)68.19
Om 3H). 7.98 (d. 21-I). 6.77-6.1 1 (m. 411). 5.49-4.51 (m. 411): 1.CMS (m/z)
FS- 108; (M-1-1)-.
Example 22: Synthesis of Compound 1112
0
HO -
Br 0
T 7
Naai Di\AF
SO,Na CH2Cl2 SO,CI
Step 1
SM1 1 Step 2 2
R
0 compound B1
11-3\2\
Step3
2NH2 Step4
O--(-SO2NH2
3
compound B11
1691 fo a solution of SM I 2.
23 mmol) in propan-2-ol (20 ml.) was added NaOH (2.8 g. 69
mmol) in water and 3-bromoprop-Hne (2.4 g. 20 mmol). Alter stirred for 411 at
70 C. the
mixture \\ as concentrated. filtered. the filter cake was washed by water.
dried to give
intermediate I (2 g. 37%) as xellow solid. 1.CMS (m/z) ES 234 (M+Na)'.
1701 bo a solution of intermediate 1 (I g. 4.3 mmol) in Mil (8
\\35 added ()vit.\ I
dichloride (1.1 g. 8.6 mmol) in DCM (4 )
dropw ise at 0 C. :1 fter stirred oµernight at Ft. the
mixture \\ as quenched by \\ ater. extracted \\ ith DCM (30 ml. x 3). The
combined organic was
dried over anhydrous Na2SO4. concentrated. purified by column chromatography
(0 to 10% of
Ft0Ac in petroleum ether as cluent) to give the desired intermediate 2 (0.35
g. 77%) as yellow
solid.
Intermediate 2 (0.35 g. 1.5 mmol) was added into ammonia water (5 ml.), the
mixture was stirred
for 1 h at r.t.. quenched by addition of water. extracted with Ft0Ac (20 ml. x
2). The combined
organic \\ aS dried over an 11\ drous Na2S0.1. concentrated. pained by column
chromatography (0
to 50% of Ft0Ac in petroleum ether as eluent) to gi \ e intermediate 3 (0.2 g.
20% for two steps)
as yellow solid. II NMR (300 MI It. DM SO-d6) 6 7.77 (d. 211). 7.24 (s. 211).
7.14 (d. 211). 4.90
(d. 211). 3.63 (m. II I).
1711
'1(1 a solution of compound RI ( 150 mg. 0.16 mmol) and intermediate 3 (101
mg. 0.48
mmol) in Vleoll/IFO (6 iii 3 ml.) as
added µitamine C sodium salt (63 rrhl. 0.32 mmol)
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followed with the addition of CuS0.1 (51 mu,. 0.32 mmol) and Na2C01 (51 mu.
0.48 mmol).
After stirred overnight. the mixture \\ as filtered. and the filtrate was
concentrated and purified by
column chromatouraphy (0 to 2.5% of methanol in dichloromethane as eluent) to
give
Compound B12 (13.4 mg. 7.3%) as white solid. '11 NMR (300 MI lz. CDCI3)6 7.88
(m, 311). 7.12
(m. 211). 6.39-6.01 (m. 4H). 5.42-4.63 (m. 414): ',CMS (m/z) ES- 1148 (M-H)-.
Example 23: Synthesis of Compound 1113
Br.
Si TI3AF
Pd(Ph3)202, Cul, Et3N
TN=
80 degrees. o/n `SO2NH2 Step 2
SM1 Step 1 1
TI
.S02N Step 3
1-1-2
N
N
2
compound B13
1721 "ro a solution of SM I (2.3 g. 10 mmol) in dioxane (20 mL) was added
ethynyltrimethylsilane (2 g. 20 mmol). Cu! (191 mg, I mmol) and Pd(PPh3)2C12
(730 mg. 1
mmol) under N. then Et31\1 (10 u. 100 mmol) was added drop\vis. Alter stirred
overnight at I 00
C. the mixture \\ as quenched H addition of water. the mixture \\ as extracted
with 11t0Ac (50
ml. x 2). The combine organic layer was dried over anhydrous Na2SO4.
concentrated to give
crude intermediate I.
[731 The crude intermediate I\\ as treated w ith 'IBA I in T111. (20 nil..
20 mmol) and stirred
2h at rt.. then quenched H. water and extracted w ith 11t0Ac (50 ml. x 2). The
combine
organic la.\ er \\ as dried Over anhydrous Na2S01. concentrated to give crude
intermediate 2.
which was purified by column chromatography to give pure intermediate 2 (1.5
g. 84%) as
,vellow solid. 1H NMR (300 MIlz. DMSO-d6) Ci 7.82 (d. 21.1). 7.68 (d. 2H).
7.46 (s. 2E1). 4.45 (s.
111).
1741 To a solution of compound 131 (150 mu. 0.16 mmol) and intermediate 2
(87 mg. 0.48
mmol) in Me011/11,0 (6 ml /3 ml.) \vas added vitamin C sodium salt (63 mu.
0.32 mmol)
followed w ith the addition of CuSO4 (51 mg. 0;12 mmol). Na2CO3 (51 mu. 0.48
mmol). After
stirred at r.t. overnight. the mi \t are \\ as Filtered. 'Hie Filtrate was
concentrated and purified H
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column chromatography (0 to 1.5% of methanol in (Iichloromethane as eluent) to
give
Compound B13 (48.7 mg. 27%) as white solid. II I NMR (300 MHz. (DCI3) 6 8.21
(m. II). 7.93
(m, 41I). 6.37-6.00 (m. 411). 5.44-5.30 (m. 511). 1.[MS (m/7.) NS- I 118 (M-I
Example 24: Synthesis of Compound 1314
/OH
0--
OH
NaOH
0 compound B1
R
0 H20St 2 -N
Et0H ep HOOC
SM1 Step 1
1 B14
1751 To
a solution of SM1 (2.3 g. 20 mmol) in Nt01-1 (20 nil.) was added Na01-1 (1.6
g. 40
mmol) in water. then 3-bromoprop- I -yne (2.4 g. 20 mmol) was added at 0 C.
After stirred for
4h at r.t, the mixture \vas quenched by 11C1 solution (2N) and p1! was
adjusted to 3-4. extracted
\vith l'AOAc (50 ml *5)= The combined organic was dried over anhydrous Na2SO4.
concentrated.
purified H column chromatography (0-3% of methanol in dichloromethane as
eluent) to give
intermediate I (1.2 g. 40%) as colorless cr\ stal. !.CMS (m/i.) NS- 154 (NI -
11)'.
1761 ft
a solution of compound 131 (150 mg, 0.16 mmol) and intermediate I (73 mg. 0.48
mm(1l) in Me01-1/110 (4 iii! .2 ml,) was added vitamine C sodium salt (63 mg.
0.32 111111(11)
followed \\ ith the addition of CuS0.1 (5 I mg. 0.32 mmol) and Na-,.001 (51
mg, 0.48 mmol). After
stirred overnight, the mixture \vas filtered. the fl I trate was concentrated
and purified by column
chromatography (2% of methanol in dichloromethane as eluent) to give Compound
B14 (12.6
mg. 7.2%) as white solid. 'ii NMR (300 MHz. CDCI3) 6 8.17 (m. 11-1). 6.39-5.99
Om 41-1). 5.56-
4.87 (m. 411): [[MS (m//) 1/5- 1090 (M-11) .
Nnivmatic activities of rapaniNcin analogs of the present invention
1771
The m'IµOR is a serine/threonine protein kinase that has been shown to
regulate multiple
cellular responses including cell growth. proliferation. motility. survival
and protein synthesis.
mTOR kinase activity is regulated by several upstream signaling pathways and
its dysregulation
has been implicated in several Corms of cancer. Now we use a l'erbillin
labeled anti-
phosphor lated 4N-13P I anti both to detect phosphorylation of the GI /P-
labeled substrate H
mTOR. This TR-FR[]' based assay can be used to screen inhibitors of mIOR in
vitro.
1781 Materials: Assay buffer components: I M IIFPES p117.5. GII3CO.
Cat#15630 I NI
MgCI:. Sigma. Cat) i MI028: 0.5M [DTA. G1BCO. CoOt 15575: DTT. Sigma Catg
43819:
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EGTA. Sigma CaO( [3889: Triton X100. Sigma. Cattl 18787: BSA CALBIOCHFNI Catii
126575.
1791 Enzyme. substrate and detection reagents: mTOR: Invitrogen. Cat4
PV4753: GI2P-4L-
BP I : Invitrogen. CatII PV4759: FKBP 12: SinoBiological, Catit 10268-H08E:
ATP: Sigma Cat#
A26209: 'lb-anti-1)4E-101: Invitrogen. Cat4 PV4755: TR-FRET Dilution butler
Invitrogen. Cat#
PV3574.
1801 Plate: Compounds preparation plate: 384-well. Corning cat4 3657: Assay
plate: Hack low
volume 384\\ ell microtiter plate (Greiner Cat4 784076).
1811 Procedure: Compounds dosage gradient solution preparation:
Compounds were 3-fold serial diluted in 100% DM50 in a microtiter plate
(Corning 3674) at 11
different concentrations in the range of I ()OLIN] to I .7nM (100 01. 33 0M. I
1pM. 3.7pM.
1.20M. 411 nM. 137 nM. 46 nM. 15 nM. 5 nM. 1.7 nM). Then the diluted compounds
in 100%
DMSO was 10-fold diluted \\ ith ddH20. so the compounds were in 10% DMSO.
1821 A typical assay protocol of measuring the mTOR inhibitory ability of
the rapamycin
derivatives of the invention is as follows:
Assay protocol:
0.5 pl diluted compounds in 10% DMSO was pipetted into a black low volume
384well
microtiter plate (Greiner 13io-One. Frickenhausen. Germany, ca0,' 784076):
:Vora solution of ml OR in aqueous assa \ buffer 150 mM I IEPESIXa011 pll 7.5.
5 mM
MgC12. 1 .0 infV1 clithiothreitol. 1 mM EGTA. 0.0% \/v) Triton-X100 (Sigma).
0.01 % (w/y1
bovine serum albumine ( NSA )1 (mTOR. 0.3125 nu/p > final cone. in the 5 pI
assay volume is
0.125 ng:p I) were added to the assay plate and the compound-enn me mixture
was incubated for
15 min at 22'(' to allow pre-binding of the test compounds to the enzyme
before the start of the
kinase reaction:
1831 The kinase reaction was started by the addition of 2.5 pl of a
solution of ATP (ATP. 200
pM >
final conc. in the 5 01 assa.\ volume is 10 pM) and substrate (0.8 pM ¨> final
conc. in the
5 01 assa \ volume is 0.4 0M) in assay bulThr and the resulting. mixture \\ as
incubated for 18 min
at 22'12.
1841 The reaction \\ as stopped by the addition of Sul of 30m NI [DTA
(FDTA. 30 mM
final cone. in the 10 01 assa volume is 15mM) and 4nM Th-chelate labeled anti-
4F-BP I pT461
phosphospecific ant ibod
ln\ itrogen Cat!: PV47551 (lb-labeled antibock . 4 nM linal conc.
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in the 10 t.t1 assay volume is 2 nM) in TR-FRn- dilution huller, the resulting
mixture was
incubated 1 hour at 22 C to allow the formation of complex of the
phosphorylated substrate and
the [b-chelate labeled antibody.
1851 l
he amount of phosphorylated substrate was evaluated by measurement of the
resonance
energ\ transfer from the Th-chelate to the ()11). Therefore. the fluorescence
emission at 495 nm
and 520 nm after excitation at 340 nm was measured on envision 2104 multi
label reader (Perkin-
Idmer). The ratio of the emission at 520 nm and at 495 nm was taken as the
measure for the
amount of phosphorylated substrate. The data were normalised (enzyme reaction
without
inhibitor - 0 % inhibition, all other assay components but no enzyme = 100 %
inhibition) and
IC50 values were calculated by a 4 parameter fit (equation (1)) using ID13S
Milt software (II)
I3usiness Solutions lad.. )
Y-13ottom 1- (TOP-Bottom )/( 1 10A(( 1 .ogIC50-X )*h i 1 Islope)) equation
(1)
1861 In
this equation. Y was the normalized %inhibition value. X was the log value of
the test
compound concentration. 1050 was the concentration of compound w here half of
maximal
inhibition as achio.ed.
1871 The testing results of in l(
inhibitory effects of the rapamycin analogs/derivatives of
the invention are shown below:
Compound ID 1C50(nM) Compound ID IC50(A1)
1-5.09
Rapam! cin
Compound A 1 5 6.-13 32 ! 18.43
Compound 14 ' 6604.427021 ! 133 ; 15.40
Compound A7 >10000 ' 131 28.52
Compound A 10 = >10000 135 ! 19.38
Compound 15 ! >10000 136 l2.96
Compound A6 ! 91-13.20 137 23.77
Compound A3 r:>10000 39 27.16
Compound A 13 ! >10000 ! 311 ! 446.00
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Compound A 12 1 >10000 B12 35.19
Compound A 14 >10000 1313 29.03
Compound A9 - t >10000 B14 6.80
Tumor cell inhibition studies:
Cell proliferation assay
1881 The effect of different compounds on the cellular activities was
quantitated through
determining the number of living cells in a culture by a homogeneous detection
method For
quantitative determination of cell viability by the CellTiter-Glo
chemiluminescence detection
kit for ATP. ATP is an indicator of the metabolism of living cells.
Homogeneous detection step
is added directly to the single reagent (Cell-liter-GI 'R Reagent) in serum-
containing cultured
cells. without washing. the cells or removing the medilffil. After adding
reagent and mixing in a
96-well or 384-well plates. the number of cells that can be quantified H the
system within 10
minutes. is as low as IS cells in each well.
Preparation of the reagents
1891 Different cell types \\ ere cultured using a medium. containing. 10%
1:13S plus I%
penicillin streptomycin double antibiotics, and the follow ing appropriate
additives: DMIAI
medium (Cibco. Item No. I 19)5073) for culturing colorectal cancer cells I ICT
I 16. breast cancer
cells MCF-7 and MDA-MB-231 melanoma cells SK-MEL-28. A549 and epidermal
squamous
cell carcinoma cell A431: RPM 1-1640 medium (containing 2 niM L-glutamine. 1.5
g / 1. sodium
bicarbonate. 4.5 u / I glucose. 10 RIM 11EITS. 1.0 mM sodium pyruvate ( Item
#1 72400-120
from Giber)) fOr culturing r87/VIG and kidney caneer786-0: HI-2K mixed medium
(Item
#21127 From Gibe()) For culturing prostate cancer cell line PC-3,
Instrumentation
Multi-label Micro-plate Reader rnvison 21-1 From Perkin rimer
Cell Culture conditions
1901 Al! 9 cell lines were cultured in the wells in the plates at a cell
density of 3000 cells/well
alter 9 passages.
Preparation or culture media and cell culture conditions:
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1911
Prepare the compounds and condition the cells the next day: each chemical
compound to
be aSSaNed was diluted to 10 niM stock solution with 100% DM SO. followed by
additional
dilution with 100% DiX150 diluted to 2 mN1. followed b\ serial 5X dilution
using serum-free cell
culture medium to a final 10 di fferent diluted concentrations points (2000.
400. 80. 16, 3.2. 0.64.
0.128, 0.0256, 0.00512. 0.00102 [AM). plus 0.5% {WS (no compound) as a
maximum control
and 10WV1 Rapamycin as a minimum control. A solution of 0.5til of each diluted
compound is
added to the 100W 0i-cell culture plate. the final compound concentration of
10 points (10. 1 0.4.
0.08. 0,016. 0.0032. 0.00064. 0.000128. 0.0000256. .00000512 [AM). '11he cells
were then
cultured in 37 'V incubator for 72 hours. In order to ensure die reliability
of the experiments of
determining. the inhibition of each compound. a duplicate was used for each
compound
concentration gradient w ill do two repeated (lable 1). and the determination
of each compound
was repeated tw ice.
Plate reading
1921
After 72-hr of cell culture. 50 ul of CellTiter Glo was added to each well on
the plate. and
shaken for 5 min on a shaker followed by 10 min at room temperature. The cell
number was
analyzed by the Micro-Plate reader.
Data analysis:
1931
Cell viability was obtained through the reading by the multi-label micro-plate
reader. The
effect of dilution value on the % as calculated using the follow ing
fomula:
% cell inhibition- 100-100, I Signal-low control )/( High control-low control.
in which signal low
control. and high control are the test compound. minimal value. and maximal
value respectively.
[941
The 1050 value of each test compound in inhibiting the cells is obtained by
formula 2
(below ):
Bottom OP-
liottom rt 1 0 lC50/X hillslopco. in w hich X and v are known values. IC50.
1 lillslope. Top and I3ottom 4 parameters generated by the analysis software.
V as the %
inhibition. X as the test compound concentration, and IC50 as the
concentration of the compound
needed to inhbit 50% of the cells. 1 lillslope is the slope of curve fitting.
usually around I.
1951 All experimental data w Cie analyzed by IDI3S X1.111.5 (II) 13LISinCSS
Solutions ).
Experimental results and conclusion
1961
All the potency of each test compound is shown in one of the Hlow kW, graphs
for each
of the cancer cell models tested. The lower the ellrve in the graph. the more
potent each
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compound is. From the data shown in each
the graphs. it is clear that all the series 13
compounds showed varying levels of high potency against the cancer cell
tested. Some of the 13
series compounds were extremely potent. reaching a potency level of nM
concentration range.
Renal cell carcinoma tumor cell inhibition studies:
Renal cell carcinoma tumor cell in studies: Figl and Fig2.
Lung Cancer A549 cell inhibition studies: Fig3, Eig4 and Fig5.
Melanoma SK-MEL-28 cell in studies: Eig6, Fig7 and Fig8.
Epidermal cancer A431 tumor cell model: Fig9, Eig10 and Fig11.
Glioblastoma 1187 MG Tumor model studies: Fig12, Fig13 and Fig14.
Human colorectal tumor HCT 116 model studies: Fig15, Fig16 and Fig17.
Breast cancer MDA-MB-231 tumor model: Fig18, Fig19 and Fig20.
Breast cancer MC7F-7 tumor model: Fig21, Fii,422 and Fig23.
Prostate cancer PC-3 tumor studies: Fig24, Fig25 and Fig26.
Efficacy studies of raparilvein derivatives in Human Colon Tumor (11CT116)
model
1971
Purpose: The objecti\ e or this stud\ is to evaluate preclinicallv the in vivo
therapeutic
ellicac\ 011,:\ IS (positive control) and a lead compound from the 13 series
administrated as per os
(p.o.) in the slowing or eliminating tumor development in subcutaneous I IC F-
116 human colon
cancer model.
1981
Animals: 13a1b/c nude mice. female. 6-8 weeks. \vcighinu approximately 18-20g.
A total
or 70 will be needed For the study. which µvill be purchased From Vital River
Laboratory Animal
Technology Co. ltd.
1991
Tumor Inoculation: 1:.ach mouse will be inoculated subcutaneously at the right
[lank with
IR-1-116 tumor cells 0\10() in O. I ml or P1115 For tumor development. The
treatments w ill be
started when the tumor size reaches approximately -150 mm3. The test article
administration
and the animal numbers in each group are shown in the Follow im2 experiment
design table,
Groups and Treatments
1 Dose Dosing )osi 7
Group 11 I1.Catillt2nt Schedule
(mg/kg) Rout volume
Vehicle p.o. 10 pl/v, QD x21
10 A 5 9 p.o. I 0 W/g QD x 21
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j 3 10 B p.o. I 0 1.11/g QD x 21
4 10 B 9 p.o. 10 tAl/g QD x 21
5 10 18 p.o. 10 1.1.1/g QD x 21
Note: n: animal numher: Dosing volume: adjust dosing volume based on body
weight 10 pi/g),
Treatment schedule may be adjusted i hocky \\ eight loss > 15%.
11001 Assignment to Groups: Before commencement of treatment. all animals will
be weighed
and the tumor volumes will he measured. Since the tumor vollillle can affect
the effectiveness of
any given treatment. mice \\ ill he assigned into groups using randomized
block design based
upon their tumor kolumes, This ensures that all the groups are comparable at
the baseline,
11011 1:.ndpoints: The major endpoint is to see if the tumor growth can he
delayed or mice can
be cured. I umor sites will he measured twice weekly in two dimensions using a
caliper. and the
volume will he expressed in mm3 using the formula: V --- 0.5 a x b2 where a
and h are the long
and short diameters or the tumor. respectively. The tumor sizes are then used
for the calculations
or both T-C and T/C values. T-C is calculated with T as the median time (in
days) required for
the treatment group tumors to reach a predetermined site (e.g.. 500 mm3). and
C is the median
time (in days) for the control group tumors to reach the same site. 'file T/C
value (in percent) is
an indication of antitumor effectiveness. T and C are the mean volume of the
treated and control
groups. respectivek. on a gi\ en (LIV. lumor tiStilleti will he collected 16r
the tumor weight and
photo at the end of the stud\ .
11021 Termination:Th I is stuck. \\ ill he terminated when the mean tumor size
of the control
group reach the volume of 600- 1000 mm3. Animals that are observed to he in a
continuing
deteriorating condition \\ ill be euthanized prior to death, or before
reaching a comatose state.
Animals showing obvious signs of severe distress and/or pain should he
humanely sacrificed. In
case of following situations. the animals will be euthanized:
11031 Animals have lost significant body mass (emaciated). Obvious body weight
loss > 2094):
11041 Animals cannot get to adequate I6od or water.
11051 the study \\ ill he terminated \\ ith all animals in all groups being
sacrificed \\ hen the
mean tumor burden in the \ chicle treated control group reaches a value of
2000 mm3.
11061 Statistical Analysis: I=or comparison between two groups. an independent
sample t-test
\\ill he used. For comparison among three or more groups. a one-\\ a.) ANOVA
will he
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performed. II a signilicant I -statistics (a ratio of treatment variance to
the error variance) is
obtained, multiple comparison procedures will be applied after ANOVA. The
potential
synergistic effect between treatments will he anakted by LSD or Dunnett's 13.
All data will he
anal \ zed using, I 7.() solk\ are. p 0.0 is considered to he
statisticall significant.
11071 Summar\.: As shown in the follo\\ ng figure. after 22 days of treatment.
the 9 mg/kg/day
of A I 5 positive control compound (A In tor from Novartis) significantly
inhibited tumor growth
by 63% in Sub() LICT I 16 resistant colon cancer xenograft model(* P<0.05),
which is consistent
with the reports from literature. After 22 days treatment. 11 compound
suppressed the tumor
growth by 42%. 57% and 64% (**. P<0.01). at 3. 9. and 18 mg/kg/day,
respectively. compared
to the vehicle control. No obvious toxicity was observed, These data indicate
that B compound is
very potent in vivo and ma\ O\ ercome the colon cancer resistance.
Fig 27. In this figure. the top line (diamond) is lot- Vehicle, second line
(triangle) is For 137 at 3
mg /Kg. dose: third line (purple cross) represents 137 at 9gm/Kg dose: rourth
line from the top
(pink square() represent llini(or at 9 mg/Kg dose). the bottom line (blue
cross) represents 137 a
I 8 mg: Kg dose.
Methods offreatment
11081 The compounds of the present invention. including but not limited to
those specified in
the examples, possess immunomodulatory and anti-tumor activity in mammals
(especially
humans). As immunosuppressants. the compounds of the present invention are
useful for the
treatment and prevention of immune-mediated diseases such as the resistance by
transplantation
or organs or tissue such as heart. kidney. liY=er. medulla ossill111. skin.
cornea. lung. pancreas.
intestinum ten ue. limb. muscle, nerves. duodenum, small-bowel. pancreatic-
islet-cell, and the
like: \ ersus-host diseases brought about b\ medulla ossium
transplantation: autoimmune
diseases such as rheumatoid arthritis. s\ stemic lupus er\ theinatosus.
Hashimoto's th
multiple sclerosis. myasthenia gravis. type I diabetes. uveitis. allergic
encephalomyelitis.
glomerulonephritis. and the like. Further uses include the treatment and
prophylaxis ols
intlammator and hyperprolilerative skin diseases and cutaneous manifestations
of
immunologically-mediated illnesses. such as psoriasis. atopic dermatitis,
contact dermatitis and
further eczematous dermatitises. is seborrhoeis dermatitis. lichen plaints.
pemph4n.ts, bulious
pemphigold. epidermic)] \ sis buliosa. urticaria. angioedemas. vasculitides.
ervthemas. cutaneous
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cosinophijias. lupus erythematosus, acne and alopecia areata: various eye
diseases (autoimmune
and otherwise) such as keratoconjunctivitis, vernal conjunctivitis. aveitis
associated with
Behcers disease. keratitis. herpetic keratitis. conical cornea. dystrophia
epithelialis corneae.
corneal leukoma. and Dellini pcmphigus. In addition. reversiHe obstructive
airway disease.
which includes conditions such as asthma (for example. bronchial asthma.
allergic asthma.
intrinsic asthma, extrinsic asthma and dust asthma). particularly chronic or
inveterate asthma (for
example. late asthma and alma hyper-responsk cuss). bronchitis, allergic
rhinitis. and the like
are lar,(4eteci b\ compounds of the present invention. In of
mucosa and blood vessels
such as !.astric ulcers. vascular damage caused lw isehemic diseases and
thrombosis. Moreover.
hyperproliferative vascular diseases such as intimal smooth muscle cell
hyperplasia. restenosis
and vascular occlusion. particularly following biologically- or mechanically-
mediated vascular
injury. may be treated or prevented by the compounds of the present invention.
Other treatable
conditions include ischemic bowel diseases. inflammatory bowel diseases.
necroti/ing
enterocolitis. intestinal in
such as Coeliac diseases. proctitis, eosinophilic
gastroenteritis. mastoevtosis. Crohn's disease and ulcerative colitis: nervous
diseases such as
multiple m ositis. Guillain-Barre syndrome. Meniere's disease. polyneuritis.
multiple neuritis.
mononeuritis and radiculopath\ : endocrine diseases such as 11\ perthyroidism
and Rasedow's
disease: hematie diseases such as pure red cell aplasia. aplastic anemia.
hypoplastic anemia.
idiopathic thrombocytopenie parpura. autoimmune hemolytic anemia.
agranuloeytosis.
pernicious anemia. megaloblastic anemia and anerythroplosia: bone diseases
such as
osteoporosis: respiratory diseases such as sarcoidosis. fibroid lung and
idiopathic interstitial
pneumonia: skin disease such as dermatomyositis. leukoderma vulgaris.
ichthyosis vulgaris.
photoallergie sensitivity and cutaneous T cell lymphoma: circulatory diseases
such as
arteriosclerosis, atherosclerosis. aortitis syndrome. polyarteritis nodosa and
myoeardosis:
collagen diseases such as seleroderma. Wegener's ._?,ranulonta and Siouren's
syndrome: adiposis:
eosinophilic fasciitis: periodontal disease such as lesions of gingiva.
perioclontium. alveolar bone
and substantia ossea dent is: nephrotic syndrome such as glomerulonephritis:
male pattern
alCONCIL1 or alopecia senilis b\ preventinu epilation or pros di in hair
germination and'or
promoting hair generation and hair growth: muscular d stroph\ : l'voderma and
Setar\
s\ ndmme: Addison's disease: aeti\ e oxygen-mediated diseases. as for example
organ injUry sLich
as ischemiLi-reperlusion injur\ 01' organs (such as heart. liver. kidney and
digestive tract) which
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occurs upon preservation. transplantation or ischeme disease (for example.
thrombosis and
cardiac infarction): intestinal diseases such as endotoxin-shock.
pseudomembranous colitis and
colitis caused by drug or radiation: renal diseases such as ischemic acute
renal insufficiency and
chronic renal insufficiency: pulmonary diseases such as toxinosis caused by
lung-oxygen or drug
(for example. paracort and bleonkeins). lung cancer and pulmonary emphysema:
ocular diseases
such as cataracta. siderosis. retinitis, pigmentosa. senile macular
degeneration. vitreal scarring
and conical alkali burn: dermatitis such as erythema multiforme. linear lgA
ballous dermatitis
and cement dermatitis: and others such as gingivitis. periodontitis. sepsis.
pancreatitis. diseases
caused b\. environmental pollution (for example, air pollution). aging.
carcinogenesis. metastasis
of carcinoma and hypobaropathy: diseases caused by histamine or leukotriene-
C.subA release:
fichcers disease such as intestinal-. yasculo- or neuro-Behcers disease. and
also Behcet's which
affects the oral cavik. skin. tne. vulva, articulation. epidick mis. lung.
kidney and so on.
urthermore. the compounds of the present invention ma\ be USCILI I or the
treatment and
prevention of hepatic disease such as immunogenic diseases (for example.
chronic autoimmune
liver diseases such as autoimninune hepatitis. primary biliarv cirrhosis and
sclerosing
cholangitis). partial liver resection, acute liver necrosis (e.g. necrosis
caused by toxin, viral
hepatitis. shock or anoxia). B-virus hepatitis. non-A/non-B hepatitis.
cirrhosis (such as alcoholic
cirrhosis) and hepatic failure such as fulminant hepatic failure. late-onset
hepatic failure and
"acute-on-chronic- liver failure (acute liyer failure on chronic liver
diseases). and moreover are
useful for various diseases because of their usefal act k it
such as augniention of
chemotherapeutic effect. evtomegalovirus infection. particularl I
ICVIV infection. anti-
inflammatory activik, sclerosing and fibrotic diseases such as nephrosis.
scleroderma.
pulmonary fibrosis. arteriosclerosis. congestke heart Failure. ventricular h\
pertrophy. post-
surgical adhesions and scarring. stroke. myocardial infarction and illjUry
associated with
ischemia and reperfusion. and the like.
11091 Additionally. compounds of the present invention possess rk-506
antagonistic properties.
The compounds of the present invention may thus be used in the treatment of
immunodepression
or a disorder involving immunodepression. rxamples of disorders involving
immunodepression
include
cancer. fungal infections. senile dementia. trauma (including wound healing.
surgery and shock) chronic bacterial infection, and certain central nervous
system disorders. The
immunodepression to be treated may be caused by an overdose of an
immunosuppresske
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macrocyclic corn pound.
for example derivatives of 1 2-(2-cyclohexv1- 1 -methylvi ny1)-
1 3.1 9.2 1.27-tetramethyl- 1 1 .28-dioxa4-azatricyclo122.3. 1
Øsup.4.91octacos- 1 8-cue such as 1:1;,-
506 or rapamycin. The overdosing of such medicants by patients is quite common
upon their
realizing that they have forgotten to take their medication at the prescribed
time and may lead to
serious side effects.
11101 The compounds of the present invention, including but not limited to
those specified in
the examples. possess anti-tumor activity in mammals (especially humans). As
an anti-cancer
drug, the compounds of the in\ cut ion can be used to treat brain and
neurovascular tumors, head
and neck cancers, breast cancer. lung cancer. mesothelioma. lymphoid cancer,
stomach cancer.
kidney cancer. renal carcinoma, liver cancer and liver cirrhosis, ovarian
cancer, ovary
endometriosis. testicular cancer, skin cancer. melanoma, neuro and all
endocrine cancers, spleen
cancers. pancreatic cancers. blood proliferative disorders such as Hodgkin's
cancer. lymphoma.
leukemia, and all \ cancer disorders that result from uncontrolled cellular
proliferations.
11 111
Hie COM pounds of the present invention. alaV be mixed \\ itil COM1110111 V
known
pharmaceutical excipients such as rfudragit. sodium carboxymethylcellulose (Na
CM). sodium
carboxyprop\ !cellulose. an\ other naturally derived or s\ IlthetiC C el
pients to effect an
efficacious pharmaceutical hormulation. The formulation comprising the
compounds of the
invention Illa \ be made as a immediate release formulation. or a sustained
release formulation. or
site injection depot formulation. depending on the medical needs. The compound
of the present
invention may also be combined with a medical device, such as a stent. a
balloon, a balloon
catheter. an orthopedic device, to further enhance the efficacy of the medical
device. The
compound of the present invention may he the main 1Mnction component of a
medical treatment
regime. such as a local injection formulation. or an ancillary function. such
as a coating on a
medical de\ ice. or in combination with a low-molecular weight or polymer
excipient. and used
as a coating or filler of' a medical device.
11121 When used to treat restenosis following a balloon angioplasty or stent
placement. the
compounds of the present in
and the native rapamycin. are thought to exhibit their
therapeutic functions through the inhibition of the mammalian target of
rapamycin or mTOR.
he ma \ also bind to 1:1:131' receptors.
11131 When used in the abo\ c or other treatments. a therapeutically effective
amount of one of
the compounds of' the present invention may be employed in pure form or. where
such forms
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exist. in pharmaceutically acceptable salt. ester or prod rug
form..Alternatelv. the compound rria
be administered as a pharmaceutical composition containing the cornpound of
interest in
combination with one or more pharmaceutically acceptable excipients. The
phrase
"therapeutically effective amount- of the compound of the present invention
means a sufficient
amount of the compound to treat disorders. at a reasonable benefit/risk ratio
applicable to any
medical treatment. It will be understood. however. that the total daily isae
of the compounds
and compositions of the present invention will be decided by the attending
physician within the
scope of sound medical judgment. the specific therapeutically effective dose
level for any
particular patient \\ ill depend upon a varlet\ of ractors includ* the
disorder being treated and
the
se\ erit\ of the disorder: activit\ or the specilic compound emplo.\,ed: the
specific
composition employed: the age. body \\ eight. general health. sex and diet of
the patient: the time
of administration. route of administration. and rate or excretion of the
specific compound
employed: the duration of the treatment: drugs used in combination or
coincidental 1\ ith the
specific compound employed: and like factors well known in the medical arts.
For example. it is
well within the skill of the art to start doses of the compound at levels
lower than required to
achieve the desired therapeutic effect and to gradually increase the dosage
until the desired effect
is achieved.
11141
Ihe total dank dose of the compounds of the present in\ ention administered to
a human
or lower mammal may rant:e from about 0.01 to about 20 mg/kg/day. I or
purposes of oral
administration, more prererable doses may be in the range of from about 0.001
to about 3
mg/4.)./da\ . lfdesircd. the effective daily dose ma\ be divided into multiple
doses for purposes of
administration: consequentk. single dose compositions ma\ contain such amounts
or
submultiples thereof to make up the (Jail\ dose. 1 opical administration may
involve doses
ranging from 0.001 to 10 percent mg/kg/clay, depending on the site of
application. When
administered locally to treat restenosis and vulnerable plaque. the dose may
range from about I
microgram:mm stent length to about 100 microgram/mm stem length.
Pharmaceutical Compositions
11151 The pharmaceutical compositions of the present invention comprise a
compound and a
pharmaceuticalk acceptable carrier or e\cipient. which mav be administered
orally. rectalk.
parentera II\ i ntrac isterna ntrayagi na I\
intraperitonealr\ . topical I\ (as b\ pow ders.
ointments, drops or transdermal patch). bucalk. or as an oral or nasal spray.
The phrase
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-pharmaceutically acceptable carrier- means a non-toxic solid. semi-solid or
liquid filler. diluent.
encapsulating material or Formulation auxiliar\ of any type. The term -
parcnteral,- as used
herein. refers to modes of administration which include intravenous.
intramuscular.
intraperitoneal. intrasternal. subcutaneous and intraarticular injection and
infusion.
11161 Pharmaceutical compositions of the present invention for parenteral
injection comprise
pharmaceutical l> acceptable sterile aqueous or nonaqueous solutions.
dispersions. suspensions or
emulsions as well as sterile powders iqr reconstitution into sterile
injectable solutions or
dispersions just prior to use. kxamples of suitable aqueous and nonaqueous
carriers, diluents,
solvents or vehicles include water, ethanol, polyols (such as glycerol.
propylene glycol.
polyethylene glycol. and the like), carboxymethylcellulose and Suitable
mixtures thereof*.
vegetable oils (such as olive oil), and injectable organic esters such as
ethyl oleate. Proper
fluidit\ may be maintained, lqr example. bv the use of coating materials such
as lecithin, by the
maintenance of the required particle sin in the case of dispersions. and by
the use of surfactants.
11171 I hese compositions ma\ also contain adju \ ants such as preser\
atives. wetting agents.
emulsifying agents. and dispersing agents. Prevention of the action of
microorganisms ma\ be
ensured H the inclusion of various antibacterial and antilnngal agents. for
example, paraben.
chlorobutanol. phenol sorbic acid. and the like. It may also be desirable to
include isotonic agents
such as stwars, sodium chloride, and the like. Prolonged absorption of the
injectable
pharmaceutical form may be brought about by the inclusion of agents which
delay absorption
such as aluminum monostearate and gelatin.
11181 In some cases. in order to prolong the effect of the drug. it is
desirable to slow the
absorption of the drug from subcutaneous or intramuscular injection. This inay
be accomplished
by the use of a liquid suspension of crystalline or amorphous material \\ ith
poor water solubility.
The rate of absorption or the drug then depends upon its rate of dissolution
which. in turn. inay
depend upon cr\ stal siie and cr\ stall inc form. Alternatek.. delayed
absorption of a parenterall.\
administered drug lqrm is accomplished H dissolving or suspending the drug in
an oil vehicle.
11191 Injectable depot lqrms arc made H. Forming microencapsule matrices of
the drug in
biodegradable polymers such as polylactide-polyglycolide. Depending upon the
ratio of drug to
polymer and the nature of the particular polymer employed, the rate of drug
release may he
controlled. kxamplcs of other biodegradable polymers include poly(orthoesters)
and
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poly(anhydrides). Depot injectable formulations are also prepared by
entrapping the drug in
liposomes or microemulsions which are compatible with body tissues.
11201 The injectable Ibrmulations may be sterilized, for example. by
filtration through a
bacterial-retaining, illter, or bv incorporating sterilizing agents in the
form of sterile solid
compositions which mav be dissolved or dispersed in sterile water or other
sterile injectable
medium just prior to use.
11211 Solid dosage forms or oral administration include capsules. tablets,
pills. powders. and
L2,l'atillles. In such solid dosage forms. the acti \ e compound is mixed with
at least one inert.
pharmaceuticall \ acceptable excipient or carrier such as soditim citrate or
dicalcium phosphate
and/or a) fillers or extenders such as starches. lactose. sucrose. glucose.
mannitol. and silieic acid.
h) binders such as carboxymethvIcellulose. alginates, gelatin.
polyvinylpyrrolidone. sucrose, and
acacia. c) humectants such as glycerol. d) disintegrating agents such as agar-
agar, calcium
carbonate. potato or tapioca starch, alginic acid, certain silicates, and
sodium carbonate, e)
solution retarding agents such as paraffin. r) absorption accelerators such as
quaternary
ammonium colnpounds.
welli119, agents such as cetvl alcohol and glycerol monostearate. h)
absorbents such as kaolin and bentonite clay. and i) lubricants such as talc.
calcium stearate.
magnesium stearate. solid polyethylene glycols. sodillin laUrVI sulfate. and
mixtures thereof. In
the case of capsules. tablets and pills, the dosage form ma\ also comprise
buffering agents.
11221
oIid compositions or a similar type ma \ also be employed as fillers in soft.
semi-solid
and hard-tilled gelatin capsules or liquid-filled capsules using such
excipients as lactose or milk
sugar as well as high molecular wcii2ht pol \ ethylene i.11\ cols and the
like.
11231 The solid dosage forms of tablets. dragees. capsules. pills. and
granules may he prepared
with coatings and shells such as enteric coatings and other coatings well
known in the
pharmaceutical formulating art. They may optionally contain ()pacifying agents
and may also he
or a composition that they release the active ingredient(s) only. or
preferentially. in a certain part
of the intestinal tract. optionall\ . in a dela\ ed manner. Lxamples of
embedding compositions
\vhich lila\ he used include pol\ meric substances and waxes.
11241
lite active compounds ma\ also be in a micro-encapsulated form. i I
'appropriate. with one
or more of the above-mentioned cxcipients.
11251 Liquid dosage forms for oral administration include pharmaceutietill
acceptable
emulsions. solutions, suspensions. syrups and elixirs. In addition to the
active compounds. the
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(lU rd dosa!:,,c forms may contain inert chluents commonly used in the art,
for example. \\ ater Or
other solvents. solubili/ing agents and emulsifiers such as eth \ I alcohol.
isopropyl alcohol. ethyl
carbonate. eth I acetate, ben/y1 alcohol. benn bentoate. propylene glycol. 1.3-
butvlene
dirneth
Ibrmamide, oils (in particular. cottonseed. groundnut. corn, germ. olive.
castor, and
sesame oils). glycerol. tetrahydrofurfuryl alcohol. polyethylene glycols and
fattv acid esters of
sorb tan, and mixtUreti there01.
11261 Besides inert diluents. the oral compositions may also include adjuvants
such as wetting
agents. emulsifying and suspending agents. s\\ eetening. flavoring, and
perfuming agents.
11271 Suspensions. in addition to the active compounds. may contain suspending
agents. for
example. ethox\ kited isostear\ I alcohols, polvox \ ethylene sorbitol and
sorbitan esters.
microcrystalline cellulose. at
metahydroxide. bentonite. agar¨agar. and tragacanth. and
mixtures thereof.
11281 Topical administration includes administration to the skin or mucosa.
including surfaces
of the lung and e\ e. Compositions for topical administration, including those
for inhalation. may
be prepared as a dry powder which may he pressurized or non-pressuri/cd. In
non-pressurized
powder compositions. the active ingredient in finely divided form may he used
in admixture with
a larger-sized pharmaceutically acceptable inert carrier comprising particles
having a size, for
example. of up to 100 micrometers in diameter. Suitable inert carriers include
sugars such as
lactose. Desirabk. at least 95 percent by weight of the particles of the
active ingredient have an
effective particle site in the range of 0.01 to 10 micrometers. Compositions
for topical use on the
skin also include ointments. creams. lotions. and gels.
11291 Alternatek. the composition ma\ be pressuri/ed and contain a compressed
gas. such as
nitrogen or a I killerlCd gas propellant, The liquelied propellant medium and
indeed the total
composition is preferably such that the active ingredient does not dissolve
therein to any
substantial extent. The pressuriyed composition Ma\ also contain a surface
active agent. The
surface active agent ma\ be a liquid or solid non-ionic surface active agent
or may be a solid
anionic surface active agent. It is preferred to use the solid anionic surface
active agent in the
form of a sodium salt.
11301 A further form of topical administration is to the eye, as for the
treatment of immune-
mediated conditions of the c\e such as autoimmunc diseases. allergic or
inflammatory conditions,
and corneal transplants. The compound of the present in is
deli ered in a
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pharmaceutically acceptable ophthalmic vehicle. such that the compound is
maintained in
contact \\ ith the ocular surface for a sufficient time period to allow the
compound to penetrate
the corneal and internal regions of the e\ e. as for example the anterior
chamber. posterior
chamber. \ itreous bock. aqueous humor. \iitreous humor, cornea. iris/eilar.
lens. choroidiretina
and sclera. The pharmaceuticall \ acceptable ophthalmic vehicle may. for
example. be an
ointment. \ egetablc oil or an encapsulatinu material.
[1311 Compositions for rectal or vaginal administration are preferobl \
suppositories or retention
enemas which may be prepared by mixing the compounds of the present invention
with suitable
non-irritating excipients or carriers such as cocoa butter. pol, ethylene
glycol or a suppository
wax which are solid at room temperature but liquid at bed.) temperature and
therefbre melt in the
rectum or \ aginal cavity and release the active compound.
11321 Compounds of the present invention may also be administered in the form
of liposomes.
As is known in the art. liposomes are general IN derived from phospholipids or
other lipid
substances. I iposomes are formed b\ mono- or multi-lamellar Indrated I iquid
crstals that are
dispersed in an aqueous medium. Any non-toxic. physiological l\ acceptable and
mctabolitable
lipid capable of 1brming liposomes can be used. The present compositions in
liposome form ma.
contain. in addition to a compound of the present in\ cntion. stabiliiers.
preservatives. excipients.
and the like. The preferred lipids ore the phospholipids and the phosphatid
cholines (lecithins).
both natural and ti\ ntlICliC. Methods to form liposomes are known in the art.
See. for example.
Prescott. rid.. Methods in Cell Biology. Volume XIV. Academic Press. New York.
N.Y. (1976).
p. 33 et seq.
11331 Compounds of the present invention may also be coadministered with one
or more
immunosuppressant agents. I he immunosuppressant agents w ithin the scope of
the present
invention include IM1
azathioprine sodium. brequinar sodium. SPANIDIN.RTNI.
gusperimus trihvdrochloride (also known as deoxyspergualin). mizoribine (also
known as
brechnin). CH 1 ChiPT.RIAl. mvcophenolate moletil.
Cvlosporin A (also
marketed as di flerent formulation of L\ eloTorin \
under the trademark
PROGIZALIZ VI. tocrolimus (also known as K-506). sirolimus and
RAPAMLN
leflunomide (also known as Il\\ A-186). glucocorticoids. such as
prednisolone and its clerk-A \ es. antibody therapies such as orthoelone (OK
13) and
lenopax.R I Ni.. and antith\ m oc te globulins, such as thYmoglobulins.
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11341 'Ihe local deliver of drug/drug combinations from a stem or other
implantable device
has the follow ino advantages: namek. the pre \ ention of vessel recoil and
remodeling through the
scaffolding action of the stem and the prevention of multiple components of
neointimal
hyperplasia or restenosis as well as a reduction in inflammation and
thrombosis. This local
administration of drugs, agents or compounds to stented coronary arteries may
also have
additional therapeutic benefit. For example. higher tissue concentrations of
the drugs. agents or
compounds ma\ be achic\ ed utilizing local deli\ en. rather than s\ stemic
administration. In
addition, reduced systemic toxicit\ ma\ be achieved utilizing local deliver\
rather than s.stemic
administration while maintaining higher tissue concentrations. Also in
utilizing local delivery
from a stem rather than systemic administration. a sin,,de procedure ma\
suffice with better
patient compliance. An additional benefit of combination drug. agent. and/or
compound theram
ma\ be to reduce the dose of each of the therapeutic drugs. agents or
compounds. thereb
limiting their toxicit\ . while still achic\ Ma a reduction in restenosis.
inflammation and
thrombosis. Local stent-based therapy is therefore a means of improving the
therapeutic ratio
tellicac.0oxicity) of anti-restenosis. anti-illflammator.). antithrombotic
drugs. agents or
compounds.
11351 It is understood that the foregoing detailed description and
accompanying examples are
merel illustrati \ e and are not to be taken as limitations upon the scope of
the invention. which is
defined solek b\ the appended claims and their cqui\ alents. Various changes
and modifications
to the disclosed embodiments will be apparent to those skilled in the art.
Such changes and
modifications. including \\ ithout limitation those relating to the chemical
structures. substituents.
deri\,ati\.es. intermediates. s\ nthescs. formulations andlor methods of use
of the invention. may
he made \\ ithout departing from the spirit and scope thereof.
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