Note: Descriptions are shown in the official language in which they were submitted.
CA 02559998 2007-08-28
-1-
RAPAMYC_IN CONiLIGATIE AND ANTIBOD S
BACKGROUND OF THE INVENTION
This invention relates to derivatives of rapamycin which are useful as
immunogenic molecules for the geaexation of antibodies specific for rapamycin
or ring
opened derivatives thereof, for weasuring levels of rapamycin or derivadves
thercof,
for isolating rapamycin binding proteins; and detecting antibodies specific
for
rapamycin or derivatives thereof.
Rapamycin is a macrocyclic triene antibiotic produced by Streptomvices
hy oscoaicus. which was found to have antifungal activity, particularly
against
C+ndida albicans. both ja vitro and jn viv0 [C. Vezina et al., J. Antibiot.
28, 721
(1975); S.N. Schgal et al., J. Antibiot. 28, 727 (1975); I-L A. Bakes et al.,
J. Andbiot.
31, 539 (1978); U.S. Patent 3,929,992; and U.S. Patent 3,993,749].
Rapamycin alone (U.S. Patent 4,885,171) or in combination with picibanil
(U.S. Patent 4,401,653) has been shown to have antitnmor activity. R. Martel
et al.
[Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed that rapamycin is
effective in
the expcrimcntal allcrgic cncephalomyelitis model, a model for multiple
sclerosis; in the
adjuvant arthritis model, a model for rheumatoid arthritis; and effectively
inhibited the
formation of IgE-like antibodies.
The immunosuppressive effects of rapamycin have been disclosed in FASEB 3,
3411 (1989). Cyclosporin A and FK-506, other maerocyclic molecules, also have
been shown to be effective as immunosuppressive agents, therefore useful in
preventing transplant rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989);
R.
Y. Caine et al., Lancet 1183 (1978); and U.S. Patent 5,100,899].
Rapamycin has also been shown to be useful in preventing or treating systetnic
lupus crythematosus [U.S. Patent 5,078,999], pulmonary inflammation [U.S.
Patent
5,080,899], insulin dependent diabetes mcllitus [Ffth Int. Conf. Inflamm. Res.
Assoc.
121(Abstract), (1990)], adult T-cell leukenua/lymphoma [EP0525960A1],
and smooth muscle cell proliferation and intimal thickening following
vascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 2): 197 (1992)].
CA 02559998 2006-10-05
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Mono- and diacylated derivatives of rapamycin (esterified at the 28 and 43
positions) have been shown to be useful as antifungal agents (U.S. Patent
4,316,885)
and used to make water soluble prodrugs of rapamycin (U.S. Patent 4,650,803).
Recently, the numbering convention for rapamycin has been changed; therefore
according to Chemical Abstracts nonienclature, the esters described above
would be at
'the 31- and 42- positions. U.S. Patent 5,100,883 discloses fluorinated esters
of rapamycin. U.S. Patent 5,118,677 discloses amide esters of rapamycin. U.S.
Patent
5,118,678 discloses carbamates of rapamycin. U.S. Patent 5,130, 307 discloses
aminoesters of rapamycin. U.S. Patent 5,177,203 discloses sulfonates and
sulfamates
of rapamycin. U.S. Patent 5,194,447 discloses sulfonylcarbamates of rapamycin.
PCT Pubtication WO 92/05179 discloses carboxylic acid esters of rapamycin.
Yatscoff has reported that rapamycin levels can be quantitated using HPLC
method with a sensitivity of I ng/ml [Ther. 'Dnig Monitoring 14: 138 (1992)]
This
method is time consuming and each sample must be assayed individually.
Immunoassays have been developed for numerous proteins as well as various
drugs including cyclosporin A [Morris, R.G., Ther. Drug Monitoring 14: 226-
(1992)], and FK506 [Tamura, Transplant Proc. 19: 23 (1987); Cadoff, Transplant
Proc. 22: 50 (1990)]. Numerous types of immunoassays, that have been developed
to
measure proteins or compounds, have been based on competitive inhibition, dual
antibodies, receptor-antibody interactions, antigen capture, dipstick,
antibody or
receptor trapping, or on affinity chromatography. Affinity columns with
rapamycin
have been reported in which a rapamycin analog was covalently attached to a
matrix
[Fretz J. Am. Chem. Soc. 113: 1409 (1991)]. These columns have been used to
isolate rapamycin binding proteins.
DESCRIP'ITON OF THE INVENTION
This invention provides a rapamycin conjugate of formula 1, having the
strucmre
CA 02559998 2006-10-05
-3-
ORl (Carria)x
42
= ~
' OMe
. ,.
O O OR2 (C~~)y I
31
O O Me0'~ O
OMe
z
wherein Rl and R2 are each, independently, hydrogen or -(R3-L-R4)a- ;
L is a linldng group;
R3 is selected from the group consisting of carbonyl, -S(O)- ,-S(O)2 ,-P(O)2-
5 5 -P(O)(CH3)-, -C(S)- , and -CH2C(O)- ;
R4 is a selected from the group consisting of carbonyl, -NH- , -S- ,-CH2- ,
and -O- ;
a=1-5;
x=0-1;
y=0-1;
z is from about 1 to about 120;
and Carrier is immunogenic carrier material, detector canier material, or a
solid matrix,
or a salt thereof with the proviso that Rl and R2 are both not hydrogen; and
further provided that when a is greater than 1, each L group can be the same
or
different; and still further provided that x is 0 if R 1 is hydrogen and y is
0 if R2
is hydrogen, and if x and y are both 1, the Carrier moiety is the same in both
cases.
The linking group, L, is any moiety that contains the group R3 on one end and
R4 on other end, therefore enabling the linking group to be connected to the
42- and/or
31-hydroxyl groups of rapamycin on one end and connected to another linking
group
or the immunogenic carrier material, detector material, or matrix on the other
end.
When a is greater than 1, each L group can be the same or different. In such
cases, the
first L group is designated as Ll, the second L group designated as L2 and so
on. The
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rapamycin conjugates of the present invention may be prepared in such ways as
to
encompass a wide range of linking groups (L) and terminal functional groups
R4. For
example, L may be linear or branched alkylenes comprising from 1 to as many as
15,
more usually 10 or less, and normally less than 6 carbon atoms (i.e.,
methylene,
ethylene, n-propylene, iso-propylene, n-butylene, and so forth). In addition,
such
alkylenes can contain other substituent groups such as cyano, amino (including
substituted amino), acylamino, halogen, thiol, hydroxyl, carbonyl groups,
carboxyl
(including substituted carboxyls such as esters, amides, and substituted
amides). The
linking group L can also contain or consist of substituted or unsubstituted
aryl, arallcyl,
or heteroaryl groups (e.g., phenylene, phenethylene, and so forth).
Additionally, such
linkages can contain one or more heteroatoms selected from nitrogen, sulfur
and
oxygen in the form of ether, ester, amido, amino, thio ether, amidino,
sulfone, or
sulfoxide. Also, such linkages can include unsaturated groupings such as
olefuiic or
acetylenic bonds, disulfide, imino, or oximino groups. Preferably L will be a
chain,
usually aliphatic comprising between 1 and about 20 atoms, more usually
between 1
and 10, excluding hydrogen, of which between 0 and 5 are heteroatoms
preferrably
selected from nitrogen, oxygen, and sulfur. Therefore, the choice of linking
group L is
not critical to the present invention and may be selected by one of ordinary
skill takfng
normal pra;audons to assure that stable compounds are produced.
A preferred embodiment of this invention provides a conjugate of formula U,
having the structure
%
OR1 (Carria)x
42
.=
OMe
.,. ;
0 N O 0 ORZ (C.an ier)y II
31
,.
CA 02559998 2006-10-05
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Rl and R2 are each, independently, hydrogen or -R3-I~R4- ;
L is -A-(CR5R6)b[B-(CR7R8)d]e-
A is -CH2- or -NR9- ;
B is -0- , -NR9- , -S- , -S(O)- , or -S(O)Z- ;
R3 is selected from the group consisting of carbonyl, -S(O)- ,-S(O)2 ,-P(O)2-
,
-P(O)(CH3)-, -C(S)- , and -CH2C(O)- ;
R4 is selected from the group consisting of carbonyl, -NH- ,-S- ,-CH2- , and -
O- ;
R5, R6, R7, and R8 are each, independently, hydrogen, alkyl of 1-6 carbon
atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, halo, hydroxy,
trifluoromethyl, arylalkyl of 7-10 carbon atoms, aminoalkyl of 1-6 carbon
atoms, hydroxyalkyl of 1-4 carbon atoms, alkoxy of 1-6 carbon atoms,
carbalkoxy of 2-7 carbon atoms, cyano, amino, -CO2H, or phenyl which is
optionally mono-, di-, or tri-substituted with a substituent selected 5nm
alkyl of
1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro,
carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or -CO2H;
R9 is hydrogen, alkyl of 1-6 carbon atoms, or arallcyl of 7-10 carbon atoms;
b=0-10;
d=0-10;
e = 0-2;
x=0-1;
y=0-1;
z is from about I to about 120;
and Carrier is immunogenic carrier material, detector carrier material, or a
solid matrix,
or a salt thereof with the proviso that Ri and R2 are both not hydrogen; and
further provided that when b is greater than 1, each of the CR5R6 groups can
be
the same or different, and when d is greater than 1, each of the CR7R8 groups
can be the same or different; and still further provided that x is 0 if R1 is
hydrogen and y is 0 if R2 is hydrogen, and if x and y are both 1, the Carrier
moiety is the same in both cases.
A second preferred embodiment of this invention provides a conjugate of
formula III, having the structure
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OR1 (Carrier)x
. . . '
OMe
,=- .
,.
N O 0 OR2 (~~)y m
31
HO O O Me0'. 0
O OMe
.
L Z
Rl and R2 are each, independently, hydrogen or -(R3-Ll-R4)f-(R10-L2-R1 i)g-
Carrier,
L1 is -(CH2)b-CHR12-(CH2)j- ;
L2 is -(CH2)k-D-(CH2)m-E- ;
O
D is -CH2-, -S-S-, or N
S----
O
E is -CH2- or - C-
NH2 Cl
R3 and RlO are each, independently, selected from the group consisting of
carbonyl,
-S(O)-, -S(0)2 , -P(O)2- , -P(O)(CH3)-, -C(S)-, and -CH2C(O)-;
R4 and Ril are each, independently, selected from the group consisting of
carbonyl,
-NH- , -S- , -CH2-, and -O- ;
R12 is hydrogen, alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms,
alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -(CH2)n C O 2R 13 ,
-(CH2)pNR14RI5, carbamylalkyl of 2-3 carbon atoms, aminoalkyl of 1-4
carbon atoms, hydroxyalkyl of 1-4 carbon atoms, guanylalkyl of 2-4 carbon
atoms, mercaptoalkyl of 1-4 carbon atoms, alkylthioalkyl of 2-6 carbon atoms,
indolylmethyl, hydroxyphenylmethyl, imidazoylmethyl, halo, trifluoromethyl,
or phenyl which is optionally mono-, di-, or tri-substituted with a
substituent
selected from allcyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy,
CA 02559998 2006-10-05
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cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or
-CO2K.
R14 , and R15 are each, independently, hydrogen, alkyl of 1-6 carbon atonts,
or
arylalkyl of 7-10 carbon atoms;
R13 is hydrogen, alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms,
alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or phenyl which is optionally
mono-, di-, or tri-substituted with a substituent selected from alkyl of 1-6
carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro,
carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or -C02H;
f = 0-3;
g = 0-1;
h=0-10;
j=0-10;
k = 0-10;
m=0-10;
n = 0-6;
p = 0-6;
x=0-1;
y=0-1;
z is flom about 1 to about 120;
and Carrier is immunogenic carrier material, detector canier material, or a
solid matrix,
or a salt thereof with the proviso that RI and R2 are both not hydrogen; and
further provided that f and g are both not 0 and when f is greater than 1,
each of
the -(R3-Ll-R4)- moieties can be the same or different; and still further
provided
that x is 0 if Rl is hydrogen and y is 0 if R2 is hydrogen, and if x and y are
both
1, the Carrier moiety is the same in both cases.
This invention also provides a conjugate of formula IV, having the smcture
CA 02559998 2006-10-05
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OH
42
OMe
y OH IV
0 R t 31 Carrier
0 Me0''
O
H
O OMe
=. /
z
wherein RI is -OCH2(CH2)qR4-
R4 is selected from the group consisting of carbonyl, -NH-,-S- -S-, -C, and -0-
;
q=0-6;
z is from about 1 to about 120;
and Carrier is immunogenic carrier material, detector carrier material, or a
solid matrix,
or a salt thereof.
The immunogenic carrier material can be selected from any of those
conventionally known. In most cases, the carrier will be a protein or
polypeptide,
although other materials such as carbohydrates, polysaccharides,
lipopolysaccharides,
nucleic acids and the like of sufficient size and immunogenicity can likewise
be used.
For the most part, immunogenic proteins and polypeptides will have molecular
weights
between 5,000 and 10,000,000, preferably greater than 15,000 and more usually
greater than 40,000. Generally, proteins taken from one animal species will be
immunogenic when introduced into the blood stream of another species.
Particularly
useful proteins are those such as albumins, globulins, enzymes, hemocyanins,
glutelins
or proteins having significant non-proteinaceous constituents, e.g.,
glycoproteins, and
the like. Further reference for the state-of-the-art concerning conventional
immunogenic carrier materials and techniques for coupling haptens thereto may
be had
to the following: Parker, Radioimmunoassay of Biologically Active Compounds,
Prendce-HaII (Englewood Cliffs, NJ., USA, 1976), Butler, J. Immunol. Meth. 7:1-
24 (1975) and Phamiacol. Rev. 29(2):103-163 (1978); Weinryb and Shroff, Drug
Metab. Rev. 10:P271-283 (1975); Broughton and Strong, Clin. Chem. 22:726-732
(1976); and Playfair et al., Br. Med. Bull. 30:24-31 (1974). Preferred
immunogenic
CA 02559998 2006-10-05
-9-
carrier materials for use in the present invention are ovalbumin and keyhole
limpet
hemocyanin. Particularly prefemd for use in the present invention is
ovalbumin. The
detector carrier material can be a rapamycin-linking moiety conjugated to an
enzyme
such as horsemdish peroxidase, alkaline phosphatase, luciferase, a fluorescent
moiety
such as fluorescein or fluorescein derivatives, Texas Red, or rhodamine, a
chemiluminescent moiety, and the like. The solid matrix carrier material can
be resin
beads, an ELISA plate, glass beads as conunonly used in a radioimmunoassay,
plastic
beads, solid matrix material typicaIly used in a dipstick-type assay. When
rapamycin is
conjugated to a solid matrix, the resulting conjugate can be used in a
dipstick assay, as
described in this disclosure, for the affinity purification of antibodies, or
for isolating
rapamycin binding proteins.
It should be noted that as used in the formulae above describing the specific
rapamycin conjugates, i represents the number of rapamycin conjugated to the
carrier
material. The value z is sometimes refeaed to as the epitopic density of the
immunogen, detector, or solid matrix and in the usual situation wilt be on the
average
from about I to about 120 and more typically from 1 to 50. The densities,
however,
may vary gneatly depending on the particular canrier material used.
When any of the compounds of this invention contain an aryl or arylalkyl
moiety, it is prefenred that the aryl portion is a phenyl, naphthyl, pyridyl,
quinolyl,
isoquinolyl, quinoxalyl, thienyl, thionaphthyl, furyl, benzofuryl,
benzodioxyl,
benzoxazolyl, benzoisoxazolyl, or benzodioxolyl group that may be optionally
mono-,
di-, or tri- substituted with a group selected from alkyl of 1-6 carbon atoms,
arylalkyl
of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, nitro,
carbalkoxy of 2-
7 carbon atoms, trifluoromethyl, amino, dialkylamino of 1-6 carbon atoms per
alkyl
group, alkylthio of 1-6 carbon atoms, -SO3H and -C02H. It is more preferred
that the
aryl moiety is a phenyl group that is optionally mono-, di-, or tri-
substituted with a
group selected from alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms,
alkoxy
of 1-6 carbon atoms, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms,
trifluoromethyl, amino, diallcylamino of 1-6 carbon atoms per alkyl group,
alkylthio of
1-6 carbon atoms, -SO3H and -C02H.
The salts are those derived from such inorganic cations such as sodium,
potassium, and the like; organic bases such as: mono-, di-, and trialkyl
amines of 1-6
carbon atoms, per alkyl group and mono-, di-, and trihydroxyalkyl amines of 1-
6
carbon atoms per alkyl group, and the like; and organic and inorganic acids
as: acetic,
CA 02559998 2007-08-28
-10-
lactic, citric, tartaric, succinic, maleic, malonic, gluconic, hydrochloric,
hydrobromic,
phosphoric, nitric, sulfuric, methanesulfonic, and similarly lcnown acceptable
acids.
The compounds of this invention can be prepared by reacting the 42- and/or 31-
hydroxyl groups of rapamycin with a suitable electrophilic reagent that will
serve as the
linker moiety. The following patents exemplify the preparation of the 42-
and/or 31-
derivatives of rapamycin that can be used as linking groups for the
preparation of the
compounds of this invention. The preparation of fluorinated esters of
rapamycin is
described in U.S. Patent 5,100,883. The preparation of amide esters is
disclosed in
5,118,677. The pneparation of carbamates of rapamycin is disclosed in U.S.
Patent
5,118,678. The preparation of aminoesters of rapamycin is described in U.S.
Patent
5,130,307. The preparation of sulfonates and sulfamates of rnpamycin are
described in
U.S. Patent 5,177,203. The preparation of sulfonylearbamates of rapamycin are
described in U.S. Patent 5,194,447.
Fioin these patents, it can be seen that reactive
clectrophiles such as isocyanates, used in the preparation of carbamates, or
sulfonyl
chlorides, used in the preparation of sulfonates, can be tcacted with the
hydroxyl
groups of rapamycin without the need for an activating agent. For the
esterification of
the rapamycin hydroxyl groups with a carboxylic acid, activation is usually
required
thrnugh the use of a coupling reagent such as DCC, or a water soluble analog
thereof,
such as dimethylaminopropyl)-3-ethyl carbodiimide (DAEC). Representative
examples
of the preparation of rapamycin-linking group moieties are provided as
examples
below. The preparation of ether derivatives of rapamycin can be accomplished
using
the methodology disclosed in Example 18.
For the compounds of this invention in which the linker group is attached to
the
42- or the 31,42-hydroxyls, the clectrophile (or activated electtophile) is
reacted with
rapamycin to typically provide a mixture of the 42- and 31,42-derivatized
raparnycin
that can be separatcd by chromatography. For the compounds of this invention
in
which the linker group is attached to the 31-hydroxyl of rapamycin, the 42-
hydroxyl
group must be protected with a suitable protecting group, such as with a tert-
butyldimethyl silyl group. Tbe 31-hydroxyl can then be reacted with a suitable
clectrnphile to provlde the derivatized rapamycin, followed by deprotection of
the 42-
hydroxyl group. The preparation of 42-0-silyl ethers of rapamycin and
subsequent
deprotection is described in U.S. Patent 5,120,842.
Preparation of compounds containing different linkers at the 31- and 42-
positions can be accomplished by fsrst preparing the 42-derivatized compound
and then
using a diffcrent linker to derivatize the 31-position. The preparation of the
27-oxime
CA 02559998 2006-10-05
-11-
linldng groups can be accomplished using the methodology disclosed in U.S.
Patent
5,023,264, which is hereby incorporated by reference; and as described in
Example 21.
The linker group attached to rapamycin can be coupled to a second linker group
using standard methodology described in the peptide literature; typically by
activating
the electrophilic moiety, with DCC type coupling reagent, or with N-
hydroxysuccinimide, or as an activated ester or anhydride. The activated
electrophilic
end of one linldng moiety can then be reacted with the nucleophilic end of the
other
linker moiety.
The coupling of the rapamycin linking group moiety to the immunogenic carrier
can be accomplished under standard literature conditions. In general, for
reaction with
a nucleophilic gi+oup on the immunogenic carrier material, an electrophilic
moiety, such
as a carboxylic acid, on the linking group is activated with a suitable
activating agent
such as N-hydroxysuccinimide, and then reacted with the nucleophilic moiety on
the
immunogenic carrier material. Examples 2 and 3 specifically exemplify this
technique.
Similar methodology is employed for the coupling of a nucleophilic moiety on
the
linking group to an electrophilic moiety on the immunogenic carrier material.
In such
cases, the electrophilic moiety on the immunogenic carrier material is
activated as
described above, and then reacted with the nucleophilic end of the linking
group.
The reagents used to prepare the compounds of the invention are commercially
available or can be prepared by methods that are disclosed in the literanu+e.
This invention also covers analogous conjugates of other rapamycins such as,
but not limited to, 29-demethoxyrapamycin, [U.S. Patent 4,375,464, 32-
demethoxyrapamycin under C.A. nomenclature]; rapamycin derivatives in which
the
double bonds in the 1-, 3-, and/or 5-positions have been reduced [U.S. Patent
5,023,262]; 42-oxorapamycin [U.S. Patent 5,023,262]; 27-oximes of rapamycin
[U.S. Patent 5,023,264]; 27-hydrazones of rapamycin [U.S. Patent 5,120,726];
29-desmethyirapamycin [U.S. Patent 5,093,339, 32-desmethylrapamycin under C.A.
nomenclature]; 7,29-bisdesmethyirapamycin [U.S. Patent 5,093,338, 7,32-
dcsmethylrapamycin under C.A. nomenclature]; and 15-hydroxy- and 15,27-
bishydroxy- rapamycin [U.S. Patent 5,102,876]. The disclosures in the above
cited
U.S. Patents are hereby incorporated by reference. Also covered are conjugates
of the
rapamycin 1,3-Diels Alder adduct with diethyl azidodicarboxylate and rapamycin
1,3-Diels Alder adduct with phenyltriazoline dione. The preparation of these
compounds is described in Examples 14 and 15.
CA 02559998 2006-10-05
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The compounds of this invention are rapamycin immunogen, detector, and
matrix bound conjugates that are useful for the generation and detection of
antibodies
specific for rapamycin and derivatives thereof, for measuring levels of
rapamycin or a
derivative thereof in biological or laboratory fluids, and for isolating
rapamycin binding
proteins. Rapamycin derivatives as defined here are compounds containing a
rapamycin nucleus, a metabolite of rapamycin, or a ring opened rapamycin (such
as
secorapamycin, described in U. S. Patent 5,252,579, which is hereby
incorporated by
refe,rence), in which one or more of the hydroxyl groups has been esterified
into a
carboxylic ester, a carbamate, a sulfonate ester, an amide, or the like, or
one or more of
the ketones has been reduced to a hydroxyl group, or one or more of the double
bonds
has been reduced, or one ketones has been converted to an oxime or a
hydrazone.
Other rapamycin derivatives for which the compounds of this invention can be
used for
measuring levels of or generating antibodies to will be apparent to one
skilled in the art
based on this disclosure.
Antibodies specific for rapamycin or a derivative thereof using the rapamycin
immunogen conjugates of this invention may be generated by standard techniques
that
are known in the art. Typically, a host animal is inoculated at one or more
sites with
the immunogen conjugate, either alone or in combination with an adjuvant. The
typical
host mammals include, but are not limited to, mice, goats, rabbits, guinea
pigs, sheep,
or horses. Subsequent injections can be made until a sufficient titer of
antibodies are
produced. The antibodies generated from the rapamycin immunogen conjugates of
this invention can be used in numerous immunoassays, for determining rapamycin
levels, in ELISAs, radioimmunoassays, in chemiluminesence immunoassays, and in
fluorescent immunoassays. Although many variations of the immunoassay can be
used
(antigen capture, antibody capture, competitive inhibition, or two antibody
immunoassay), a basic competitive inhibition immunoassay can be performed as
follows: Antibody specific for the ligand is usually bound to a matrix. A
solution is
applied to decrease nonspecific binding of the ligand to the matrix. After
rinsing the
excess away, the antibody coupled matrix may be treated in some cases so it
can be
stored. In a competitive inhibition assay, the ligand standard curve is made
and added
with the rapamycin detector conjugate to compete for binding to the rapamycin-
specific
antibody. If necessary, the excess is removed. The detector molecule is
detected by
the standard methods used by one skilled in the art. Different formats can be
used,
which include but are not limited to, dipstick assays, FPIA, ENIIT, ELISA,
VISTA,
RIA, and MEIA. Detector conjugates of the present invention can be prepared to
use in
the above assays. For example, the detector conjugates can be Carrier material
with
labeled fluorescent, chemiluminescent, or enrymatic moieties.
CA 02559998 2007-08-28
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This invention also provides for the use of the rapamycin immunogen
conjugates or antibodies specific for rapamycin or a derivative thereof in a
test !dt that
can be commercially marketed. The test kit may be used for measuring levels of
--rapamycin--ia-biological-or-laboratory fluids.---Test k-it--components may
include
antibodies to rapamycin or a derivative thereof, antisera, or rapamycin
carrier
conjugates. The conjugates or antibodies may be bound to a solid matrix, and
rapamycin derivadves or antibodies may be radiolabeled if the assay so
requires.
Standard concentrations of rapamycin can be included so that a standard
concxntration
curve can be generated. Suitable containers, microdter plates, solid supports,
test
tubes, trays, can also be included in any such ldt. Many variations of
reagents can be
included in the kit depending on the type of assay used.
The following is illustrative of the use of a rapamycin immunogen conjugate of
this
invention .to generate andbodies specific for rapamycin or a derlvative
thereof and detect
them using an ELISA format immunoassay. Five mice were immunized with 50 g
rapamycin 31,42-diester with glutaric acid conjugate with keyhole limpet
hemocyanin
in Complete Freund's Adjuvant) intrasplenically and after about one month were
boosted with 50 g of rapamycin 31,42-diester with glutaric acid conjugate
with
keyhole limpet hemocyanin in incomplete Freund's Adjuvant) into the footpads.
Microtiter plates (ImmunolonTM I) were coated overnight with 100 l of goat
anti-mouse
antibody (10 g/ml in 10 mM potassium phosphate buffer, pH 7.2) at 4' C. The
plates
were flicked and blocked with 100 l of 196 bovine sera albumin in phosphate
buffered
saline overnight at 4' C. After flicking and washing the plates thrice with 10
mM
phosphate buffer, pH 7.05, 30 mM NaCI, 0.02% ' TritonTM X-100, and 0.004%
thimerosal wash buffer, 100 l of each mouse sera diluted with phosphate
buffer
solution was added to a well and incubated at ioom temperature for overnight.
After
flicking and washing the plates thrice with wash buffer, rapamycin 31,42-
diester with
glutaric acid conjugate with horseradish peroxidase (compound of Example 10
(100 l,
0.5 ng/ml) was added and incubated for 1 hour at room temperature in the dark.
After
flicking and washing the plates thrice with wash buffer, tetramethyl benzidine
(TMB)
substrate with H202 was added and the plates were incubated covered for 30
min. at
room temperature in the dark. The optical density was read on a
spectrophotometer at
450 nm. As shown in Table I, five of the five mice had antibodies reactive for
rapamycin 31,42-diester with glutaric acid conjugate with horseradish
peroxidase
(compound of Example 10).
CA 02559998 2007-08-28
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TABLE I
MOUSE# DII,UTION8 O.D.
----..----.6902 1r300 0.199
6903 1/100 0.231
6904 1/500 0.412
6905 1/100 1 0.121
6906 1/300 0.321
background - 0:076
a Dilution of mouse sera in PBS
The results in Table 1 show that mouse 6904 produced the most antibodies to
the
compound of Example 10. Hybridomas were generated using standard methodology.
Following a splenectomy of a mouse immunized and boosted 3 times with the
compound of Example 4, spleen cells were fused to SP20 cells to produce
hybridomas.
The hybridomas were evaluated for the production of antibodies specific for
rapamycin
or a derivative thercof using an ELISA assay as briefly described below.
Microtiter plates (ImmunolonTM 1) were coated overnight with 100 l of goat
anti-
mouse antibody (10 ghnl in lOmM potassium phosphate buffer, pH 7.2) at 4' C.
The
plates were flicked and blocked with 100 gl of 196 = bovine sera albuniin in
phosphate
buffered saline (PBS) overnight at 4' C. After flicking and washing the plates
thrice
with 0.2x PBS containing 0.02% TritonTM X-1 00 and 0.004% thimerosal,100 l of
each
hybridoma supernatant was added to a well and incubated at room temperature
for
overnight. After flicking and washing the plates thrice with 0.2x PBS
containing
0.02%TritonTMX-100and 0.004% thimerosal, the compound of Example 22 (100 l,
0.17 M) was added and incubated for 1 hour at 4' C. After flicking and
washing the
plates thrice with 0.2x PBS containing 0.02% TritonTM X-100 and 0.004%
thimerosal,
strepavidin or avidin conjugated to horseradish peroxidase (100 1, 0.2 g/ml)
was
added and incubated at room temperature for I hour in the dark. After flicldng
and
washing the plates thrice with 0.2x PBS containing 0.02% TritonTM X-100 and
0.004%
thimerosal, TMB substrate and H202 was added and the plates were incubated
covered
for 30 min. at room temperature in the dark. The optical density was read on a
spectrophotometer at 450 nm. An optical density reading of 0.25 - 3 indicates
specific
antibody binding. The results in Table 2 show that the hybridoma from well
P4G1 is
CA 02559998 2006-10-05
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positive for binding to the compound of Example 22, and is therefore specific
for
rapamycin or a derivative thereof.
TABLE 2
Screening for Monoclonal Antibodies Specific for
Rapamycin or a Derivative Thereof
WELL OPTICAL DENSITY
P3H4 0.120
P3H5 0.105
P4Gl 1.940
The hybridoma cell line in P4G1 was cloned by limiting dilution and is
designated as hybridoma cell line, RAP-42-OVAF2#lhc-. In a Fluorescent
Polarization
Immunoassay (FPIA), rapamycin 42-ester with succinic acid conjugate with 5-
glycinylfluoresceinamine (Example 23a) was used as a tracer at a concentration
of
lOnM and showed a polarization of 77mP in 100mM sodium phosphate pH 7.5. After
addition of an excess of FKBP12, the polarization measured 195mP whereas the
addition of excess of RAP-42-OVAF2#1MoAb yielded 84mP. The ring opened non-
enzymatically transformed product of the above tracer (secorapamycin 42-ester
with
succinic acid conjugate with 5-glyeinylfluoresceinamine; Example 24) was
isolated on
TLC plate (50 chloroform : 4 methanol : 0.5 acetic acid; migrated slowest of
three
components).
The rapamycin- and secorapamycin-fluorescein derivatives of Examples 23 and
24 were characterized by demonstrating their binding to FKBP12 and RAP-42-
OVAF2#1 MoAb. On addition of O.Olug FKBP12 to 1.OOmL lOnM rapamycin-
fluorescein the fluorescence polarization increased from 57mP to 148mP, while
the
fluorescence polarization of the secorapamycin-fluoreseein derivative
increased from
49mP only to 58mP, indicating much weaker binding of the seco derivative. On
addition of 5ug/mL RAP-42-OVAF2#1 MoAb to the rapamycin derivative the
fluorescence polarizadon was unchanged at 58mP, while the secorapamycin
derivative
fluorescence polarization increased from 44mP to 136mP. This shows that RAP-42-
OVAF2#1 MoAb binds specifically to the secorapamycin-fluorescein derivative,
but not
to its rapamycin-fluorescein precursor.
CA 02559998 2006-10-05
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Specificity of the two systems was also demonstrated in an assay format. To
500uL i0ug/mL RAP-42-OVAF2#1 was added rapamycin or secorapamycin to give
final concentrations ranging from 0 to 100nM. Addition of 500uL 20nM
secorapamycin-fluorescein resulted in a fluorescence polarization of 124mP in
the
absence of analyte, I20mP in the presence of lOOnM rapamycin, and 74mP in the
presence of lOOnM secorapamycin. Secorapamycin analyte thus inhibits binding
of the
secorapamycin fluorescein derivative to RAP-42-OVAF2#1, while rapamycin has no
effect. In the converse experiment, to 500uL 0.02ug/mL FKBP12 was added
rapamycin or secoiapamycin to give final concentrations ranging from 0 to
lOQnM.
Addition of 500uL 2OnM rapamycin-fluorescein resulted in a fluorescence
polarization
of 128mP in the absence of analyte, 6lmP in the presence of lOOnM rapamycin,
and
122mP in the presence of lOOnM secorapamycin. Here rapamycin inhibited binding
while secoraparnycin has no effect.
A second antibody designated 34294163MoAb, that is specific for rapamycin
was prepared as follows. A female, 6-8 week old, RBF/DnJ mouse (Jackson
Laboratories, Bar Harbor, Maine) was immunized with rapamycin conjugated at
the 42
position to bovine serum albumin with a hemisuccinate linker (designated as
RAPA-42-
HS-BSA) which was emulsified in Freund's Adjuvant (Difco, Detroit, Michigan).
The
primary immunization was administered with Freund's Complete Adjuvant and
subsequent boosts with Freund's Incomplete Adjuvant. The animal boosting
interval
for this long term immunized animal was at weeks 1, 3, 9, and 19, with the
respective
dosage level at 50, 25, 25, and 100 g per animal at two subcutaneous
locations each
time. The animal was allowed a 14 week rest period before a 5 g prefusion
boost was
administered to the spleen 3 days prior to fusion.
On the day of the fusion, the mouse was euthanized by a quick cervical
dislocation and the spleen was removed. The splenocytes are washed one time in
Iscove's Modified Dulbecco's Medium (IIVIDM) (GIBCO, Grand Island, New York)
and centrifuged 1000 RPM for 10 minutes. The pelleted splenocytes are combined
with SP2J0 myeloma cells (Dr. Milstein, Cambridge, United Kingdom) at a 1:1
ratio,
washed in IlvIDM, and centrifuged. The supernatant was removed and 1 ml of 50%
polyethylene glycol (PEG) (American Type Culture Collection, Rockville,
Maryland)
was added to the pellet for 1 minute as the pellet was gently being dispersed
by tapping
and swirling. Thirty mLs of IIvIDM was added to the mixture and centrifuged as
previously described. Supernate was decanted, the pellet was resuspended in
IMDM
with hypoxanthine, aminopterin, thymidine (HAT) (GIBCO) and 10% Fetal Bovine
Serum (FBS) (Hyclone Laboratories, Logan, Utah). To enhance fusion frequency,
CA 02559998 2007-08-28
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0.5% Salmonella tvnhimurium mitogen v/v (STM; RIBI Immunochem Research, Inc.,
Hamilton, Montana) and 1 R'o v/v ORIGEN (Igen, Rockville, Maryland) were added
to
the fusion cell suspension plated, into 96-well tissue culture plates.
The primary scrcening of the fusion occured on day 10 confluent cultures. An
ELA was used to detect anti-rapamycin rcactivity in the supernate samples.
Microtiter
wells were coated with 100 l of a 2 g/m1 solution of 42-HS-BSA in phosphate
buffered saline (PBS) and incubated at room temperatm for 2 hours. The plates
were
blocked for 1 hour with 200 l per well of 3% bovine serum albumin (BSA) in
PBS.
After washing the plates 3 times with distilled water, 100 l of culture
supernate was
added per well and incubated 30 minutes. The plates were washed 3 times and
100 l
per well of goat anti-mouse lgG+M-HRPO conjugate (Kirkegaard Penry
Laboratories,
Gaithcrsburg, Maryland) diluted in the block solution was added to the plate
for a 30
n-jinute incubation period The plate was washed a final time and the color
development
utilizes O-phenylenediamine:2HC3 (OPD) (Abbott Laboratories, Abbott Park,
Illinois).
The relative intensity of optical density readings idcntified hybrid 34-294 at
least 5
times that of the negative control, normal mouse serum (NMS) (Organon Teknika-
Cappel, Malvern, Pennsylvania) and the hybrid was selected as a candidate for
cloning
and further evaluation.
Hybrid #34-294 was cloned by limiting dilutions from 1-100 to 1-100,000.
The cloning media used was IIvIDM with 10% v/v FBS and 196 v/v HT Supplement
(GIBCO). A 200 l cell suspension was added to each 96 well in the tissue
culture
Plate-
Clone #34-294-163 (designated as 34-294-163hc) was selected for further
evaluation based on additional EIA screening of the clone supernate of
confluent
cultutcs. The EIA scrcening protocol used was described previously.
The Isotype of the monoclonal antibody secreted from the cell Iines identified
as
34-294-163 (designated as 34-294163MoAb) was detcnnined on a Mouse monoclonal
antibody isotyping kit, RPN 29, (Amersham Life Science, Arlington Heights,
11).
The assay was performed according to the vendor recommendations and the
results
indicate an isotype of IgG 1, kappa light chain.
The compounds of Examples 12 and 13 can be used in an assay for the detection
of polyclonal antibodies and monoclonal antibodies specific for rapamycin or a
derivative thereof as described below.
Microtiter plates (ImmunolonTM 1) were coated overnight with 100 l of goat
anti-
mouse antibody (10 g/ml in 10mM potassium phosphate buffer, pH 7.2) at 4' C.
The
plates were flicked and blocked with 100 l of 1% bovine sera albumin in
phosphate
CA 02559998 2007-08-28
18
buffes-ed saline overnight at 4' C. After flicking and washing the plates
thrice with
wash buffer,100 l of rabbit sera diluted 1:5 in phosphate buffered saline was
added to
a well and incubated at room temperatune for ovemight After flicldrig and
washing tlne
_ plates .thrice with wash buffer, rapamycin 42-ester with 3-j3-(4-imin4D_
butylthio)succinimidyI)phenacylglyciiie conjugate with horseradish peroxidatse
(compound of Example 12) (100 1, 0.5 ng4ml) or rapamycin 42 esta with (N-C3_
carboxyphenyl)-3-thiosuecinimidyl)glycine conjugate with horseradish
peroxidaLw
(compound of Example 13) (100 l, 0.5 ng/ml) was added and incubated for 1
hour at
room temperature in the dark. After flicking and washing the plates thricx
with wash
buffer, TMB substrate with H202 was added and the plates wera incubated
covered for
30 min. at room temperature in the dark. The optical density was read ori a
spec,-trophotometer at 450 nm. The results are shown in Table M.
TABLE 3
Comparison of Anti-rapamycin Antibody Levels in Rabbits
Immunized with the Compound of Example 3 vs. Naive
Rabbits Using a Capture ELISA Assay
Prebleed AA450 (3rd Bleed-Prcbleed)
Rabbit No. Example 10 Example 10 Example 12 Example 13
81 0.119 0.713 0.217 0.114
89 0.136 0.037 0.026 0.020
The data in Table 3 show that the compounds of Examples 12 and 13 can be
used to detect antibodies specific for rapamycin or a derivative thereof in a
mammal, as
seen in rabbit number 81.
Thc following is an example of the measurement of rapamycin concentrations
using a competitive inhibition assay for rapamycin with an ELISA format using
an
antibody specific for rapamycin. Microtiter plates (ImmunolonTM I) were coated
overnight with 100 1 of goat anti-mouse antibody (10 g/ml in 10 mM potassium
phosphate buffer, pH 7.2) at 4- C. The plates were flicked and blocked with
100 W of
1 % bovine sera albumin in phosphate buffered saline overnight at 4' C. After
flicking
and washing the plates thrice with wash buffer, the rapamycin spccific
antibody
describcd above (100 1 of 1 g/ml) was added to each well and incubated at
room
temperature for 1-4 hour. After flicking and washing the plates thrice with
wash
buffer, rapamycin 31,42-bis(hemiglutaratc) conjugate with horseradish
peroxidase (200
CA 02559998 2007-08-28
-19
l, 0.5 ng/ml) was added and incubated for 1 hour at room temperature in the
dark.
After flicking and washing the plates thrice with wash buffer, TMB substrate
was
added and the plates were incubated covered for 5 min at room tcmperature in
the dark.
The optical density was - read on a spectrophotometer at 450 nm. - Results of
the
competition between rapamycin and rapamycin 31,42-diester with glutaric acid
conjugate with horseradish peroxidase binding to mouse sera are shown in Table
4.
From these results, a standard curve can be constructed and the concentration
of
rapamycin in a sample can be deterrained.
TABLE 4
Free OPTICAL DENSITY x1000 % Inhibition
BAPAMYSIN
-L . AYf
10 M 158 158 158 74.1
5 182 194 188 69.2
0.5 304 322 313 48.6
0.05 494 501 498 18.4
0.005 528 546 537 11.9
0.0005 601 611 606 0.6
0 583 636 610 --
The compound of Example 11 (rapamycin 42-ester with N-[9H-fluoren-9-
ylmethoxy)carbonyl]glycine) can be deprotccted by the procedure used in
Example 12
(to give rapamycin 42-ester with glycine) and conjugated to a solid matrix. It
can bind
antibodies specific for rapamycin or a derivative thereof as used in some
dipstick
immunoassay methods or to isolate rapamycin binding proteins. The following
example illustrates that 803 resonance units (RU) of the compound of Example
11 can
be immobilized on a solid matrix using the BIAcoreTM standard. protocol based
on EDC
and NHS used in a BIAcoreTM. This matrix bound 1401 RU units of rapamycin
specific
antibody. The kinetics of association and dissociation were determined for
each
concentration of antibody tested (0.625, 1.25, 2.5, 5.0, 10.0 ug/ml). These
data show
that the compound of Example 11, even when bound to a matrix was accessible to
binding by a ring opened rapamycin-specific antibody and the interaction could
be
characterized. Siniilar procedures can be used to bind a rapamycin-binding
protein to
deprotected rapamycin 42-ester with N-19H-fluoren-9-ylmethoxy)carbonyl]glycine
.
CA 02559998 2006-10-05
-20-
conjugated matrix. This matrix can also be used for the isolation of novel
binding
proteins, as practiccd by one skilled in the art. Deprutected rapamycin 42-
ester with N-
[9H-fluoren-9-ylmethoxy)carbonyl]glycine can be used to isolate binding
proteins of
rapamycin-FKBP complex by one of the following methods. In one approach,
tissue
or cell lysates containing the appropriate protease inhibitors are incubated
with FKBP
which has been incubated with a deprotected-rapamycin 42-ester with N-[9H-
fluonrn-
9-ylmethoxy)carbonyl]glycine conjugated matrix for a sufficient time to allow
binding.
Various buffers are used to rinse the proteins which are nonspecifically
bound.
Proteins are released by the addition of additional buffers which disrupt the
bond
between the rapamycin nucleus-FKBP and the binding proteins.
The hybridoma cell line, RAP-42-OVAF2#1hc, was deposited under the terms
of the Budapest Treaty at the American Type Culture Collection (ATCC) of 12301
Parklawn Drive, Rockville, Maryland, 20852, USA, on March 10, 1994, and was
granted accession number HB 11568.
The hybridoma cell line 34-294-163hc has also been deposited under the terms
of the Budapest Treaty at the ATCC on Apri16, 1994, and was granted accession
number HB 11606.
The following examples represent the preparation of representative compounds
of this invention.
Example 1
Rana ycin 42-ester with succinic acid
1.1 g(11mmo1) of succinic anhydride and 400 mg of dimethylaminopyridine
(DMAP) were added to a stin-ing solution of 5g (5.5mmo1) of rapamycin and 880
l
of pyridine in 15 mi of inethylene chloride. The reaction mixture was stirred
for 2 days
at room temperature, diluted with methylene chloride and washed with three 50
nil
portions of 1N HCI. The organic layer was then dried over Na2SO4 and
concentrated
in vacuo affording crude product. Pure material was obtained by reverse phase
HPLC
with 55% acetonitrilelwater as eluant affording lg (18%) of the title
compound.
Spectral data follows: 1H NMR (CDC13, 300 MHz) 4.650 (m,1H, H2COC=O), 4.168
(d, 1H, H2COH), 2.795 (s, 4H, OC-OCH2CH2C=O).
CA 02559998 2006-10-05
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Example 2
Ragamvcin 42-estg,r with (N-hvd, roxysuccinimide(hemisuccinate))
21 mg (0.098 mmol) of DCC and 12 mg (0.098 mmol) of N-
hydn,xysuccinimide were added to a stirring solution of 100 mg of rapamycin 42-
ester
with succinic acid in 3 ml ethyl acetate. The reaction mixture was stirred
overnight at
room temperature, filtered, and concentrated in vacuo affording crude product.
Pure
material was obtained by reverse phase HPLC with 80% acetonitrilelwater as
eluant
affording 75 mg (69%) of the title compound. Spectral data follows: 1H NMR
(CDC13,
300 MHz) 4.650 (m, 1H, H2COC=O), 4.168 (d, 1H, H2COH), 2.951 (m, 2H,
OC=OCH2), 2.795 (m, 411, OC=OCH2CH2C=O), 2.705 (m, 2H, OC=OCH2); MS
(neg.ion FAB) 1110 (M-), 1056, 1012, 913, 148 (100).
Example 3
Raaamvcin 42-ester with succinic acid coniuPate with kevhoie limpet
hemocyanin
197 mg of keyhole limpet hemocyanin in 6 ml of 0.05 M phosphate buffer was
added to a stirring solution of 37 mg of rapamycin 42-ester with (N-
hydroxysuccinimide(hemisuccinate)) in 3 ml of 1,4 dioxane and the reaction was
left
stirring for 3 days at 4'C. The reaction mixture was then dialyzed for 24 hr
at 4'C in
1500 ml of 0.05 M phosphate buffer to give the title compound which could be
used
without further purification. The number of rapamycin 42-ester with succinic
acid
moieties per keyhole limpet hemocyanin was approximately 42:1.
Example 4
$agamygin 42-ester with succinic acid conjugate with ovalbumin
197 mg of ovalbumin in 6 ml of 0.05 M phosphate buffer was added to a
stirring solution of 37 mg of rapamycin 42-ester with (N-hydroxysuccinimide-
(hemisuccinate)) in 3 ml of 1,4 dioxane and the reaction was left stirring for
3 days at
4'C. The reaction mixture was then dialyzed for 24 hr at 4'C in 1500 ml of
0.05 M
phosphate buffer to give the title compound which could be used without
further
purification.
Example 5
Ranamvcin 42-ester with succinic acid conjugate with horseradish
peroxidase
16 mg of horseradish peroxidase in a solution of 0.4 ml of 1,4 dioxane and 0.4
ml of 0.5% sodium bicarbonate was added to I mg of rapamycin 42-ester with (N-
CA 02559998 2007-08-28
-2Z--
NMR (CDC13) S 6.36 (q, 2H); 5.24 (s, IH), 3.39 (s,3H), 3.32 (s,3H), 3.12
(s,3H),
0.65 (q,IH)
MS (-FAB) 1085 (M-)
Example 19
$gU21Uvcin 42-(4-nitronhenvl)carbonate and Ranamvcin 31.42-bis(4-
nitronhenv, llcarbonate
Rapamycin (0.450 g, 0.49 mmol) was dissolved in dry dichloromethane
.(10 ml) and cooled to 0'C. To this solution was added pyridine (0.4 ml, 5.7
mmol)
and a crystal of 4-dimethyl aminopyridine. A soludon of 4-nitrophenyl
chloroformate
(0.3 g 1.49 mmol) in dichloromethane (3 n-9) was added. The solution was
allowed to
warm to room temperature ovenzight and was stinrod at room temperature for 24
hours.
The reaction was quenched into 0.1N HCJ (5 ml) and the aqueous layer was
washed
with dichloromethane. The organic layer was dried over MgSO4; filtered, and
evaporated in vacuo to afford a yellow solid. Chromatography over silica gel
with 75%
Ethyl acetate in hexane afforded 180 mg of the 42-monocarbonate and 47 mg of
the.
31,42-dicarbonate as yellow solids.
Example 20
42-0-(Phenog,vthiocarbon 1~)-rapamvcin
Rapamycin (1.030 g, 1.12 mmol) was dissolved in dry dichlotomethane (100
ml) and was cooled to 0'C. To this solution was added pyridine (0.27 ml, 3.33
mmol)
and a crystal of 4-dimethyl aminopyridine. A solution of thiophenyl
chloroformate
(0.47 ml 1.49 mmol) in dichloromethane (5 ml) was added to the reaction
mixture. The
solution was allowed to warm to room temperature overnight and was stirred at
room
temperature for 24 hours. The reaction was quenched into 0.IN HCI (5 ml) and
the
aqueous layer was washed with dichloromethane. The organic layer was dried
over
MgSO4, filtered and evaporated jp yacuo to afford a yellow solid.
Chromatography on
a 4 mm silica gel ChromatotronTM plate with a gradient of 40% to 70% ethyl
acetate in
hexane afforded 520 mg of the title compound as a yellow foam.
Analysis Calc for CS8H83NOS 14: C, 66.32; H, 7.97; N, 1.33. Found: C, 66.48;
H,
8.05; N, 1.12
IR (KBr, cm' 1) 3420, 1715
NMR (CDp3) S 7.41 (t, 1H), 7.25 (t, 2H), 7.12 (d, 1H), 3.45 (s,3H), 3.33
(s,3H),
3.13 (s,311)
MS (-FAB) 1049 (M')
CA 02559998 2006-10-05
-23-
separated from the title compound on a G-25 column with phosphate buffer
solution.
The conjugate was mixed with glycerol at 50% and stored at -70'C.
Example 10
Ranamvcin 31.42-diester with g]utaric acid conjyeate with borseradish
neroxidase
To 10 mg of horseradish peroxidase in 1 mL of 0.1 M NaHCO3 was added
105 L of rapamycin 31,42-diester with (N-hydroxysuccinimide(hemiglutarate))
in 10
L incrcments over a 30 min period. The solution was gently shaken until
complete,
centrifuged at 6000 rpm for 20 min, and eluted from a G-25 column with
phosphate
buffer solution. The conjugate was mixed with glycerol at 5017o and stored at -
20'C.
Example 11
Raeamvcin 42-ester with N-f9H.flLQren-9-yjmgjhoxv)carbonvllg tnp
To a chilled (0'C) solution of rapamycin (0.73 g, 0.08 mmol) in methylene
chloride (5 mL) was added 0.6 g (1.19 mmol) of N-[(9H-fluoren-9-
ylmethoxy)carbonyl]glycine pentafluorophenyl ester, followed by pyridine (0.85
mL,
10.5 mmol) and dimethylaminopyridine (18 mg, 0.14 mmol) to form a
heterogeneous
solution, which became homogeneous upon warming to room temperature. The
reaction mixture was stirred at room temperature oveirrnight. A large excess
of EtOAc
was added. The organic layer was washed with 0.5 N HCI (2x) and brine, dried
(MgSO4), and concentrated to yield an off-white foam. Flash chromatography (30-
509'v hexane/EtOAc) yielded the title compound in 71% yield (0.679 g, 0.57
mmol).
Mass spec (negative ion FAB) M- at m/z 1192.
Example 12
Ranam_vcin 42-ester with 3.13.(4-iminob utylthio)succinimidvllnhenacvl-
glvcine conjugate with horseradish neroxidase
To a solution of rapamycin 42-ester with N-[9H-fluoren-9-ylmethoxy)-
carbonyl]glycine (10 mg, 8.4 mol) in acetonitrile (84 L) was added 10 L (in
acetonitrile at 0.84 M) of diethylamine. The reaction mixture was stinred at
room
temperature for 60 minutes and the solvent was removed with a sttcam of
nitrogen.
The residue was dissolved in acetonitrile (100 pL) and washed with hexane (5
times,
200 L), followed by concentration of the solvent with a nitrogen stream. The
resulting rapamycin 42-ester with glycine was taken up in a solution of
m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) (2 mg) in DMF (200 pL) and
allowed to incubate for two hours at 4'C, followed by the addition of 50 nM.
CA 02559998 2007-08-28
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ethanolamine (20 L) in 50 mM Tris HCI, pH 8Ø Horseradish peroxidase (5 mg)
and
Rabbit IgG (10 mg) were treated with 2-iminothiolane and purified with
SephadexTM G-
25, followed by the addition of the MBS-rapamycin glycine ester adduct. The
niixture
was incubated overnight at 4'C and purified .by gel filtration on SephadexTM G-
25 to
provide the title compound.
Example 13
Rana~Ycin 42 ester with (N-0-carboxvnhenvl)-3-thiosuccinimidJL
Qlvci~, 'n conjugate with horseradish neroxida_e
To a solution of rapamycin 42-ester with N-[9H-fluoren-9-ylmethoxy)-
carbonyl]glycine (10 mg, 8.4 mol) in acetonitrile (84 L) was added 10 L (in
acctonitrile at 0.84 M) of diethylamine. The reaction mixture was stirred at
room
temperature for 60 minutes and the solvent was removed with a stream of
nitrogen.
Thc residue was dissolved in acetonitrile (100 L) and washed with hexane (5
times,
200 L), followed by concentration of the solvent with a nitrogen stream. The
izsulting rapamycin 42-ester with glycinc was taken up in a solution of N-
succinimidyl
S-acctylthioacxtate (2 mg) in DMF (200 L). The reaction mixturz was stined at
room
tcmpcraturc for 15 minutes and then at 4'C ovemight. A soludon of
hydroxylamine
HA (7 mg in 50 L DMF) was added to the solution of rapamycin rcaction
mixture,
incubated for one hour, followed by the addition of MBS-horseradish peroxidase
adduct and MBS-Rabbit IgG to give the title compound which was purified by
Sephade)TM G-25 gel filtration.
Example 14
Ranamvcin 13. Diels Alder adduct with diethyl azidodicarboxvlate
Rapamycin (lg, 1.093 mmol) and diethyl azodicarboxylate (0.381 g, 2.187
mmol) were dissolved in dichloromethane (10 ml) and heated at 65'C overnight,
TLC
showed that the reaction was complete. The mixture was purified on a silica
gel
column using ethyl acetate as eluant to provide a white solid (0.750 g) which
was
trnwatcd with hexane and air dried to give the title compound (0.666 g) as a
powder.
Anal Calc for C57H89N3017: C, 62.91; H, 8.24; N. 3.86. Found: C, 62.81; H,
8.12; N, 3.91
IR (KBr, cm' 1) 3450, 1720
NMR (CDC13) S 6.15 (m, 1 H), 5.20 (d, 1 H), 3.40 (s, 3H), 3.30 (s, 3H), 3.15
(s,
3H), 0.9 (t, 3H), 0.72 (q, IH)
MS (-FAB) 1087 (M')
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Example 15
Ranamvcin 1.3. Diels Alder adduct with Ph nvltriAznlin dinnn
Rapamycin (0.66g, 721 mmol) was dissolved in dichloroniethane (10 nil) and
cooled to O'C To this was added, dropwise, a solution of phenyltriazolinedione
(0.133
g, 758 mmol) in dichloromethane (10 ml). The solution was stirred overnight,
TLC
showed the reaction was not complete. Additional phenyltriazenedione (0.025g,
27
mmol) was added. The reaction was purified using HPLC (4.1x31cm, Si02) with
ethyl acetate as eluant to provide the title compound as a solid. The solid
was triturated
with 30 mi of hexane and I ml of ethyl acetate filtered and air dried to give
the title
compound as a powder (0.383 g).
Anal Calc for C59H84N4015: C, 65.05; H, 7.77; N, 5.14. Found: C, 65.39; H,
7.98;
N, 4.92
IR (KBr, cm-1) 3450, 1715
NMR (DMSO) S 7.50 (m, 3H), 7.40 (m, 2H), 3.11 (s, 3H), 3.00 (s, 3H) 2.95 (s,
3H), 0.8 (q, 1H)
MS (-FAB) 1088 (M')
The following are repncsentative examples of fluorescent rapamycin derivatives
that can be conjugated via a linker at the 31-position of rapamycin.
Example 16
42-DansYirallamvcin
Rapamycin (200 mg, 0.22 mmol) in dry pyridine (2 ml) was cooled to 0'C and
was treated with dansyl chloride (840 mg, 3.1 mmol). The reaction was warmed
to
room temperature and stitrcd for 24 hours. The rcaction mixture was poured
into cold
2N HCl (30 ml) and was extracted with ethyl acetate (4x25 ml). The ethyl
acetate was
pooled and washed with brine, dried over MgSO4, filtered and concentrated in
vacuo.
The residue was chromatographed on silica with 25% ethyl acetate in benzene.
This
affot+ded 150 mg of the title compound as a yellow powder, mp 101-104'C.
Example 17
Raoamvcin 42-ester with oyre~hutvri acod
Rapamyyein (459 mg, 0.5 mmol) and pyrenebutyric acid (216 mg, 0.75 mmol)
were dissolved in THF/CH2C12 (10 ml, 1:1). 1-(3-Dimethylaminopropyl)-3-ethyl
carbodiimide hydrochloride (146 mg, 0.67 mmol) and 4-dimethylaminopyridine (15
mg) were added to the solution. The reaction was allowed to warm to room
temperature over 15 hours. The reaction was diluted with CH2C12 and washed
with
CA 02559998 2007-08-28
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5% Ha, then brine. The soluaon was dried over MgSO4, filtered and evaporated
to a
solid. The solid was applied to a 3 mm silica gel ChromatronTM plate which was
eluted
with 50% ethyl acetate in hexane to provide 180 mg of the title compound as a
foam.
Tbe rzaction also afforded 100 mg of 31,42-diesterified rapamyein.
IR (KBr, cm71) 3420, 1740
NMR (CDC]3) d 8.3 (d, IH), 8.14 (dd, 2H), 8.10 (d, 2H), 7.85 (d, 1H), 3.34
(s,3H), 3.30 (s, 3H). 3.11 (s, 3H)
MS(FAB)1183(M-)
The following are nepresentative examples of rapamycin derivatives that can be
conjugated to immunogenic carriers by the procedures described above or can be
connected to another linker and then conjugated.
Example 18
$ana ycin 42-carbomethox et 1 ether and Ranamycin 42-
bis(carbomethoxvmet yl ether)
Rapamycin (2.0 g, 2.187 mmol) and rhodium (lI) acetate (0.37 g, 0.08 mmol)
wcre heated to rcflux in benzene and treated with a solution ethyl
diazoacetate (500 ml)
in benzene (10 ml) over 10 minutes. The solution was cooled to room
temperature and
was stirred overnight. TLC showed that the reaction was incomplete. Two
additional
portions of ethyldiazoacetate (3 ml) were added at 24 hour intervals. The
mixture was
concentrated and purified by flash chromatography over silica using ethyl
acetate. This
provided the 42-monoethcr (1 g) and the 31,42 diether (0.850 g) as oils. The
42-
monoether was triturated in a mixturie of hexane, ethyl acetate and
dichloromethane over
the weekend to give the product as a powder. The diether was purified on HP'LC
on a
silica gel column with ethyl acetate as eluant. This provided the product as a
solid.
Analytical data for the monocthei:
Analysis Calc for C55H85NO1S: C, 66.04; H, 8.57; N, 1.40. Found: C, 65.29; H,
8.64; N, 1.60
IR (KBr, cm-1) 3420,1715
NMR (CDC13) d 4.82 (s, IH), 3.41 (s, 3H), 3.33 (s, 3H), 3.13 (s, 3H), 1.28 (t,
3H),
0.70 (q, IH)
MS (-FAB) 999 (M-)
Analytical data for the diether:
Analysis Calc for C59H91 N017: C, 65.23; H. 8.44; N, 1.29. Found: C, 63.29; H,
8.40; N, 1.44
IR (KBr, cm-1) 1740
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NMR (CDC13) 8 6.36 (q, 2H); 5.24 (s, 1H), 3.39 (s,3H), 3.32 (s,3H), 3.12
(s,3H),
0.65 (q,IH)
MS (-FAB)1085 (M-)
Example 19
Ranamycin 42-(4-nitronhenvl)carbonate and Ranamvcin 3],42-bis(4-
nitropjenyl )carbonate
Rapamycin (0.450 g, 0.49 mmol) was dissolved in dry dichloromethane
(10 ml) and cooled to 0'C. To this solution was added pyridine (0.4 ml, 5.7
mmol)
and a crystal of 4-dimethyl aminopyridine. A solution of 4-nitrophenyl
chloroformate
(0.3 g 1.49 mmol) in dichloromethane (3 ml) was added. The solution was
allowed to
warm to room temperature ovennight and was stimed at room temperature for 24
hours.
The reaction was quenched into 0.1N HCI (5 ml) and the aqueous layer was
washed
with dichloromethane. The organic layer was dried over MgSO4q; filtered, and
evaporated in vacuo to afford a yellow solid. Chromatography over silica gel
with 75%
Ethyl acetate in hexane afforded 180 mg of the 42-monocarbonate and 47 mg of
the
31,42-dicarbonate as yellow solids.
Example 20
42-0.(Phenoxythiocarbonyl)-raoa,mvcin
Rapamycin (1.030 g, 1.12 mmol) was dissolved in dry dichloromethane (100
ml) and was cooled to 0'C. To this solution was added pyridine (0.27 ml, 3.33
mmol)
and a crystal of 4-dimethyl aminopyridine. A solution of thiophenyl
chloroformate
(0.47 m11.49 mmol) in dichloromethane (5 ml) was added to the reaction
mixture. The
solution was allowed to warm to room temperature overnight and was stirred at
room
temperature for 24 hours. The reaction was quenched into 0.1N HCl (5 ml) and
the
aqueous layer was washed with dichloromethane. The organic layer was dried
over
MgSO4, filtered and evaporated ja vacuo to afford a yellow solid.
Chromatography on
a 4 mm silica gel Chromatotron plate with a gradient of 40% to 70% ethyl
acetate in
hexane afforded 520 mg of the title compound as a yellow foam.
Analysis Calc for C58H83NOS14: C, 66.32; H, 7.97; N, 1.33. Found: C, 66.48; H,
8.05; N, 1.12
IR (KBr, cm-1) 3420, 1715
NMR (CDC13) 8 7.41 (t, 1H), 7.25 (t, 2H), 7.12 (d, 1H), 3.45 (s,3H), 3.33
(s,3H),
3.13 (s,3H)
MS (-FAB) 1049 (M-)
CA 02559998 2006-10-05
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Example 21
Ragkamycin-O-carboxvmethyl-27-oxime
To a solution of 600 mg (650 M) of rapamycin in 6 mL of methanol was
added at room temperature, 100 mg (1.2 mmol) of anhydrous sodium acetate and
140
mg (660 M) of carboxymethoxylamine hemihydrochloride. After stirring ovemight
at
room temperature, the reaction was complete. The reaction mixture was
concentrated in
vacuo and the residue was triturated with water. The solids were filtered and
washed
thoroughly with water. The product was dried under high vacuum to give 575 mg
(89.7%) of a white solid.13C and 1H NMR indicated a mixture of E and Z isomers
for
the oxime derivative at position 27.
1H NMR (CDC13, 400 MHz): 3.43 and 3.41 (2s, 3H, CH3O), 3.30 (s, 3H, CH3O),
3.18 and 3.12 (2s, 3H, CH3O), 1.82 (s, 3H, CH3C=C), 1.695 and 1.633 (2s, 3H,
CH3C=Q; 13C NMR (CDC13, MHz): 215.8 (C=O), 211.5 (C=O), 194.5 (C=O),
191.0 (C=O). 172.5 (C=O), 169.0 (C=O), 168.5 (C=O), 167.0 (C=O), 161.5
(C=NOC), 160.0 (C=NOC), 140.0; MS (neg. ion FAB: 985 (M-H)-, 590, 167, 128,
97, 75 (100%)
Analysis Calcd for C53H82N2015 - 0.15 HZO : C 63.90; H 8.40; N 2.81
Found C63.81;H8.41;N2.85
The following compound was used in the generadon of antibodies specific for
rapamycin or a derivative thereof.
Example 22
Rangmycin 42-ester with giycvlbiotin
To a solution of biotin (0.83 g, 3.4 mmol) in 60 mL of DMF was added glycine
t-butyl ester hydrochloride (0.57 g, 3.4 mmol), N-methylmorpholine (0.92 mL,
8.36
mmol),1-hydroxybenzotriazole (0.61 g, 3.99 mmol) and 1-(3-Dimethylaminopropyl)-
3-ethylcarbo-diimide hydrochloride (0.65 g, 3.4 mmol). The reaction mixture
was
stirred at room temperature for 7 days. The DMF was concentrated, ethyl
acetate was
added, and the organic layer was washed with water, 0.5 N HCI, saturated
sodium
bicarbonate, and brine. The ethyl acetate layer was dried (MgSO4) and
concentrated to
yield tert-butylgtycylbiotin as a white solid which was primarily one spot on
TLC
(0.611 g, 1.71 mmol, 50%). Mass spec [M+H]+ at m/z 358.
To a solution of tert-butylglycylbiotin (0.271 g, 0.758 mmol) in CH202
(0.5 mL) was added 0.5 mL trifluoroacetic acid. The reaction mixture was
stirred at
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rooin temperature for 2h, concentrated, and triturated with anhydrous diethyl
ether.
The off-white precipitate was coIlected to yield 0.209 g (0.694 nimol, 92%) of
glycylbiotin. Mass spec [M+HJ+ at m/z 302. ,-
To a solution of glycylbiotin (0.65 g, 2.16 mmol) in 1-methylpyrrolidinone
(5 snI-) was added 6 mL of CH2C12, causing a precipitate to fornn which
persisted
even after the addition of 0.33 mL (2.36 mmol) of triethylamine. To this
hetenogenous
solution was added 2 g (2.19 mmol) of rapamycin, 0.43 g (2.24 mmol) of 1-(3-
dinnethylaminoprnpyl)-3-ethylcarbodumide hydrochloride, and 30 nig (2.46 mmol)
of
DIvIAP. After several hours, the reaction mixture became homogenous, and was
stirred
an additional four days. A large excess of ethyl acetate was added and the
organic layer
was washed with water, 0.5 N HCI, saturated sodium bicarbonate, and brine. The
organic layer was dried (MgSO4) and concentrated. The light yellow foam was
triturated with hot anhydrous diethyl ether to yield 1.2 g of impure title
compound as a
light yellow solid. A portion (0.5 g) of this material was flash
chronnatographed in 5%
MeOH/CHC13, and triturated again in hot ether to yield 87 mg of the title
compound
contaminated with a small amount of rapamycin. This material was
rechromatographed
(gradient 0-5% MeOH/CHC13), and triturated a final time with ether to yield 34
mg
(0.028 mmol) of pure title compound as a white solid. Mass spec, negative FAB
M- at
m/z 1196.
Example 23
$1 R._,.sn8mvcin-42-ester with succinic acid conjuQatgwithS
glvcinvlfluoresceinamine
To 4.2mg 5-glycinylfluoresceinamine dissolved in 200uL methanol containing
lOul- triethylamine was added 4mg of the compound of Example 2. After 2 hours
a
portion of the mixture was applied to a silica thin layer chromatography plate
and
developed with 100 parts chloroform, 10 parts methanol, 1 part acetic acid.
After
drying, the band containing product was scraped from the plate, and the
product eluted
from the silica with methanol. The product was characterized by the change in
fluorescence polarization of aqueous solution on addition of FKBP12.
h.) R anamvcin 42-ester with succinic acid conjugate with S.
ami nomethvHluorescein
= By following the method of Example 23a, the title compound was prepared by
substituting 2.5 mg 5-aminomethylfluorescein for 5-glycinylfluorescein.
CA 02559998 2006-10-05
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s) R9namvcin 42-ester with succinic acid coniLg;te with 4'-
aminomethvlfluorescein
By following the nxthod of Example 23a, the title compound was prepared by
substituting 1.1 mg 4'-aminomethylfluorescein for 5-glycinylfluorescein. The
thin
layer chromatography solvent was 100 parts chloroform, 8 parts methanol, and 1
part
scetic acid.
Example 24
Secoranamvcin 42-ester with succinic acid conjuQate with 5-
ElYSi_nviftuoresceinamine
A portion of the product of Example 23 was applied to a silica thin layer
chromatography plate, and eluted with a mixture of 50 parts chloroform, 4
parts
methanol and 0.5 part acetic acid. Three mobile fluorescent bands were
observed, with
unchanged starting material having the highest rf value and the title compound
having
the lowest rf value. The silica containing the desired product was scraped
from the
plate and the dtle compound was eluted with methanol. The product was
charactcrized
by the change in fluorescence polarization of an aqueous solution on addition
of RAP-
42-OVAF2#1 MoAb.
Example 25
a) Ra;amycin 42-ester with adinic acid.
Rapamycin 42-ester with adipic acid was prepared by a variation of the method
of Example 2. Adipic anhydride was prepared by combining 146 mg adipic acid in
1.0
mL dimethylfarmamide with 200 L dicyclohexylcarbodiimide and incubating at
room
temperature 2h. 300 L of the supematant of this reaction was added to 90 mg
rapamycin and 45 mg dimethylaminopyridine dissolved in 200 IlL methylene
chloride.
After 5 min the mixture was centrifuged to sediment precipitated material. The
supernatant was partitioned between 3 mL water with 100 pL acetic acid and 2 X
1 mL
methylene chloride. The organic l.ayers were combined, dried with anhydrous
sodium
sulfate and the solvent evaporated. The residue was dissolved in 1.0 mL
methanol and
0.5 mL ethanol, cooled in ice, and precipitated by adding 5 niL water. This
was
centrifuged, the supernatant discarded and the residue dried in a vacuum
desiccator,
yielding 78 mg white powder.
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b) R;namycin 42-ester with adioic acid, ester with
n-hvdroxvs~ccinimide.
38 mg of the compound of Examle 25a, 38 mg of N-hydroxysuccinimide, and
40 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide were mixed with 0.4 mL
dimethylformamide. After 60 min an additional 40 mg of the carbodiimide was
added.
After an additional 60 min the mixture was cooled in ice, and 4 mL water added
while
vortexing. The precipitate was collected by centrifugation, washed by
resuspending
and centrifuging twice with 5 mL water, and dried in a vacuum desiccator,
yielding 38
mg white powder.
c) Ranamvcin 42-ester with adipic acid coniugate with
5-,gj cinvl uorescein mine.
2.3 mg 5-glycinylfluaresceinamine dissolved in 200 pL methanoI by addition of
6 L triethylamine. 60 pL of a solution of 10.6 mg of the compound of Example
25b
in 200 L methanol was added. After 25 min the mixture was applied to a silica
thin
layer chmmatography plate and developed with a mixture of 100 parts
chloroform,12
parts methanol and 1 part acetic acid. The silica containing the desired
product was
scraped from the plate and the tide compound eluted with methanol.
d) Ranamvcin 42-ester with adiuic acid eoniuQate with
S-ami on methyjAuorescein.
The title compound was prepared as in Example 25c, but using 2.6 mg 5-
aminomethylfluorescein.
e)=amycin 42-ester wit adioic acid coniugate with
4'-aminomethvltluorescejn.
The title compound was prepared as in Example 25c, but using 2.5 mg 4'-
aminomethylfluorescein. The thin layer chromatography solvent was 100 parts
chloroform, 4 parts methanol and 2 parts acetic acid.
