U.S. patent application number 12/961367 was filed with the patent office on 2011-03-31 for triptolide derivatives for modulation of apoptosis and immunosuppression.
Invention is credited to Dongcheng Dai, Edwin S. Lennox, John H. Musser.
Application Number | 20110076293 12/961367 |
Document ID | / |
Family ID | 29712040 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110076293 |
Kind Code |
A1 |
Dai; Dongcheng ; et
al. |
March 31, 2011 |
TRIPTOLIDE DERIVATIVES FOR MODULATION OF APOPTOSIS AND
IMMUNOSUPPRESSION
Abstract
Variously substituted carbonate and carbamate derivatives of
triptolide compounds have good aqueous solubility and convert to
biologically active compounds in vivo, at a rate which can be
modulated by varying the substitution on the prodrug. The prodrugs
are useful as immunosuppressive, anti-inflammatory and anticancer
agents.
Inventors: |
Dai; Dongcheng; (Mountain
View, CA) ; Musser; John H.; (San Carlos, CA)
; Lennox; Edwin S.; (Stanford, CA) |
Family ID: |
29712040 |
Appl. No.: |
12/961367 |
Filed: |
December 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10478777 |
Jun 24, 2004 |
7847109 |
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PCT/US03/17177 |
May 29, 2003 |
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12961367 |
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60384480 |
May 31, 2002 |
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Current U.S.
Class: |
424/184.1 ;
514/468 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 35/00 20180101; C07D 493/22 20130101; A61P 9/10 20180101; A61P
25/00 20180101; A61K 31/365 20130101; A61P 3/10 20180101; A61P
37/08 20180101; A61P 7/06 20180101; A61P 37/06 20180101; A61P 17/06
20180101; A61P 11/06 20180101; A61P 35/02 20180101 |
Class at
Publication: |
424/184.1 ;
514/468 |
International
Class: |
A61K 31/365 20060101
A61K031/365; A61P 35/00 20060101 A61P035/00; A61P 35/02 20060101
A61P035/02; A61P 37/06 20060101 A61P037/06; A61P 3/10 20060101
A61P003/10; A61P 7/06 20060101 A61P007/06; A61P 25/00 20060101
A61P025/00; A61P 17/06 20060101 A61P017/06; A61P 19/02 20060101
A61P019/02; A61P 37/08 20060101 A61P037/08; A61P 9/10 20060101
A61P009/10; A61P 11/06 20060101 A61P011/06 |
Claims
1. A method of inducing cell death, comprising administering to a
subject in need of such treatment, in a pharmaceutically acceptable
vehicle, an effective amount of a triptolide prodrug, or a
pharmaceutically acceptable salt thereof, having the structure I:
##STR00023## where X.sup.1 is OR.sup.1, and X.sup.2 and X.sup.3 are
H; and OR.sup.1 is O--(C.dbd.O)--Z, where Z is selected from the
group consisting of: --OR.sup.2, --O--Y--(C.dbd.O)--OR.sup.3,
--O--Y--NR.sup.4R.sup.5, --NR.sup.4R.sup.5,
--NR.sup.3--Y--(C.dbd.O)--OR.sup.3, and
--NR.sup.3--Y--NR.sup.4N.sup.5; wherein Y is a divalent alkyl,
alkenyl or alkynyl group having up to six carbon atoms; R.sup.2 is
selected from alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,
aralkyl, hydroxyalkyl, alkoxyalkyl, aryloxyalkyl, and acyloxyalkyl;
each R.sup.3 is independently selected from hydrogen and R.sup.2;
and R.sup.4 and R.sup.5 are independently selected from hydrogen,
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl,
hydroxyalkyl, alkoxyalkyl, aryloxyalkyl, and acyloxyalkyl, or
R.sup.4 and R.sup.5 taken together form a 5- to 7-member
heterocyclic ring whose ring atoms are selected from the group
consisting of carbon, nitrogen, oxygen and sulfur, wherein said
ring atoms include at most 3 heteroatoms.
2. The method of claim 1, wherein OR.sup.1 is-selected from the
group consisting of: O--(C.dbd.O)--NR.sup.4R.sup.5,
O--(C.dbd.O)--NR.sup.3--Y--(C.dbd.O)--OR.sup.3, and
O--(C.dbd.O)--NR.sup.3--Y--NR.sup.4N.sup.5.
3. The method of claim 1, wherein Z is --NR.sup.4R.sup.5.
4. The method of claim 1, wherein each of the groups defined as
R.sup.2, R.sup.3, R.sup.4, and R.sup.5, when selected from alkyl,
alkenyl, and alkynyl, have up to six carbon atoms, with the proviso
that R.sup.2 is not alkyl; when defined as cycloalkyl, have 3 to 7
carbon atoms; when defined as cycloalkenyl, have 5 to 7 carbon
atoms; and when selected from aralkyl, hydroxyalkyl, alkoxyalkyl,
aryloxyalkyl, and acyloxyalkyl, have alkyl components with up to
six carbon atoms.
5. The method of claim 4, wherein each of R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 is independently selected from the group
consisting of alkyl having up to six carbon atoms, aryl, aralkyl,
and alkoxyalkyl.
6. The method of claim 5, wherein R.sup.2 has up to six carbon
atoms.
7. The method of claim 1, wherein said treatment is treatment of
leukemia.
8. The method of claim 1, wherein said treatment is treatment of a
solid tumor selected from the group consisting of pancreatic cancer
and melanoma.
9. A method of effecting immunosuppression, comprising
administering to a subject in need of such treatment, in a
pharmaceutically acceptable vehicle, an effective amount of a
compound having the structure I as defined in claim 1.
10. The method of claim 9, wherein said immunosuppression comprises
inhibition of transplant rejection.
11. The method of claim 10, wherein said immunosuppression
comprises inhibition of a kidney transplant rejection.
12. The method of claim 10, wherein said immunosuppression
comprises inhibition of a bone marrow transplant rejection.
13. The method of claim 9, wherein said immunosuppression comprises
inhibition of graft-versus-host disease (GVHD).
14. The method of claim 9, wherein said immunosuppression comprises
treatment of an autoimmune disease.
15. The method of claim 14, wherein said autoimmune disease is
selected from the group consisting of Addison's disease, autoimmune
hemolytic anemia, autoimmune thyroiditis, Crohn's disease, diabetes
(Type I), Graves' disease, Guillain-Barre syndrome, systemic lupus
erythematosus (SLE), lupus nephritis, multiple sclerosis,
myasthenia gravis, psoriasis, primary biliary cirrhosis, rheumatoid
arthritis and uveitis, asthma, atherosclerosis, Type I diabetes,
psoriasis, and various allergies.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of currently
pending U.S. application Ser. No. 10/478,777, a 35 U.S.C. .sctn.371
National Stage of International Application No. PCT/US03/17177,
filed 29 May 2003, which claims the benefit under 35 U.S.C.
.sctn.119(e) of Provisional Application No. 60/384,480, filed 31
May 2002, the contents of each of which are expressly incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to prodrugs useful as
immunosuppressive, anti-inflammatory and anticancer agents, and
methods of their use. The compounds have good aqueous solubility
and convert to biologically active compounds in vivo, at a rate
which can be modulated by varying the substitution on the
prodrug.
REFERENCES
[0003] Bagshawe, K. D. Antibody directed enzymes revive anti-cancer
prodrugs concept. Br J Cancer 56:531-532 (1987). [0004] Bagshawe,
K. D. Antibody-directed enzyme prodrug therapy (ADEPT). Adv
Pharmacol. 24:99-121 (1993). [0005] Bagshawe, K. D, Springer, C.
J., Searle, F., Antoniw, P., Sharma, S. K., Melton, R. G., Sherwood
R F. A cytotoxic agent can be generated selectively at cancer
sites. Br J Cancer 58:700-703 (1988). [0006] Bagshawe, K. D.
Towards generating cytotoxic agents at cancer sites. Br J Cancer
60:275-281 (1989). [0007] Boyd, G. V. and Heatherington, K., J.
Chem. Soc. Perkin I 2523-2531 (1973). [0008] Ferrier, R. J., in
CARBOHYDRATE CHEMISTRY, Kennedy, J. F., Ed., Clarendon Press,
Oxford (1990). [0009] Garver, L. C. et al., J. Am. Chem. Soc.
104:867 (1982). [0010] Gleichmann, E. et al., Immunol. Today 5:324
(1984). [0011] Hormi, O. E. O. and Nasman, J. H., Syn. Commun.
16:69 (1986). [0012] Kocienski, P. J., PROTECTING GROUPS, Georg
Thieme Verlag, Stuttgart (1994). [0013] Korngold, R. and Sprent,
J., J. Exp. Med. 148:1687 (1978). [0014] Kupchan, S. M. et al., J.
Am. Chem. Soc. 94:7194 (1972). [0015] Kupchan, S. M. et al., U.S.
Pat. No. 3,005,108 (1977). [0016] Lipsky, P. E. et al., U.S. Pat.
No. 5,294,443 (1994). [0017] Ma, P-C. et al., J. Chin. Pharm. Sci.
1:12 (1992). [0018] Mori, S. et al., Tetrahedron 47(27):5051-5070
(1991). [0019] Morris, R. E., Transplant Proc. 23(6):2722-2724
(1991). [0020] Morris, R. E. et al., Transplant Proc. 23(1):238-240
(1991). [0021] Murase, N. et al., Transplantation 55:701 (1993).
[0022] Ono and Lindsey, J. Thor. Cardiovasc. Surg. 57(2):225-29
(1969). [0023] Pu, L. et al., Zhongguo Yaoli Xuebao 11:76 (1990).
[0024] Wang, J. and Morris, R. E., Transplantation Proc. 23:699
(1991). [0025] Wentworth. P., Datta, A., Blakey, D., Boyle, T.,
Partridge, L. J., Blackburn, G. M. Proc. Natl. Acad. Sci. USA
93:799-803 (1996). [0026] Yu et al., Acta Pharmaceutica Sinica
27(11):830-836 (1992). [0027] Zheng, J. et al., Zhongguo Yixue
Kexueyuan Xuebao 13:391 (1991). [0028] Zheng, J. et al., Zhongguo
Yixue Kexueyuan Xuebao 16:24 (1994).
BACKGROUND OF THE INVENTION
[0029] Immunosuppressive agents are widely used in the treatment of
autoimmune disease and in treating or preventing transplantation
rejection, including the treatment of graft-versus-host disease
(GVHD), a condition in which transplanted marrow cells attack the
recipient's cells. Common immunosuppressive agents include
azathioprine, corticosteroids, cyclophosphamide, methotrexate,
6-mercaptopurine, vincristine, and cyclosporin A. In general, none
of these drugs are completely effective, and most are limited by
severe toxicity. For example, cyclosporin A, a widely used agent,
is significantly toxic to the kidney. In addition, doses needed for
effective treatment may increase the patient's susceptibility to
infection by a variety of opportunistic invaders.
[0030] A number of compounds derived from the Chinese medicinal
plant Tripterygium wilfordii (TW) have been identified as having
immunosuppressive activity, e.g. in the treatment of autoimmune
disease, and in treating or preventing transplantation rejection,
including the treatment of graft-versus-host disease (GVHD), a
condition in which transplanted marrow cells attack the recipient's
cells. See, for example, coowned U.S. Pat. No. 6,150,539
(Triptolide prodrugs having high aqueous solubility), U.S. Pat. No.
5,962,516 (Immunosuppressive compounds and methods), U.S. Pat. No.
5,843,452 (Immunotherapy composition and method), U.S. Pat. No.
5,759,550 (Method for suppressing xenograft rejection), U.S. Pat.
No. 5,663,335 (Immunosuppressive compounds and methods), and U.S.
Pat. No. 5,648,376 (Immunosuppressant diterpene compound), all of
which are incorporated herein by reference, and references cited
therein. Such compounds have also been reported to show anticancer
activity. See, for example, Kupchan et al., 1972, 1977, cited
above, as well as co-owned PCT Publication No. WO 02/56835, which
is incorporated herein by reference.
[0031] The administration and therapeutic effectiveness of these
compounds have been limited, however, by their low water
solubility. This problem has been addressed by formulating the
compounds in mixtures of ethanol and polyethoxylated castor oil
(e.g., "CREMOPHOR EL.TM."), allowing subsequent dilution in saline
for intravenous administration. However, such formulations have
suffered from high toxicity, due to the high concentration of
solubilizing agent required to dissolve these compounds. For
example, the ratio of solubilizing agent (ethanol plus "CREMOPHOR
EL.TM.") to triptolide in such formulations is typically on the
order of 1000:1 or greater, due to the poor solubility of
triptolide (Morris, 1991; Morris et al., 1991). Standardization of
dosage amounts is also more problematic with a suspension than with
a solution.
[0032] It is therefore desirable to provide immunosuppressive
compounds having comparatively low toxicity and improved water
solubility. It is also desirable to provide prodrug compounds which
are convertible to an immunosuppressive form in vivo at a rate
which can be controlled by selection of substituents on the
prodrug.
SUMMARY OF THE INVENTION
[0033] In one aspect, the invention provides a method of inducing
cell death, as in treatment of cancer, particularly in treatment of
treatment of colon cancer, breast cancer, lung cancer, or prostate
cancer. In another aspect, the invention provides a method of
effecting immunosuppression, as in inhibition of transplant
rejection, prevention or treatment of graft-versus-host disease, or
treatment of an autoimmune disease. In accordance with the
invention, a subject in need of such treatment is treated with an
effective amount of a triptolide prodrug, or a pharmaceutically
acceptable salt thereof, having the structure I, below, in a
pharmaceutically acceptable vehicle.
##STR00001##
In the structure I, the variables are defined as follows:
[0034] X.sup.1 is OH or OR.sup.1, and X.sup.2 and X.sup.3 are
independently OH, OR.sup.1 or H, with the proviso that at least one
of X.sup.1, X.sup.2 and X.sup.3 is OR.sup.1, and at least one of
X.sup.2 and X.sup.3 is H; and
[0035] OR.sup.1 is O--(C.dbd.O)--Z, where Z is selected from the
group consisting of: --OR.sup.2, --O--Y--(C.dbd.O)--OR.sup.3,
--O--Y--NR.sup.4R.sup.5, --NR.sup.4R.sup.5,
--NR.sup.3--Y--(C.dbd.O)--OR.sup.3, and
--NR.sup.3--Y--NR.sup.4N.sup.5;
[0036] wherein
[0037] Y is a divalent alkyl, alkenyl or alkynyl group having up to
six carbon atoms;
[0038] R.sup.2 is selected from alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, aryl, aralkyl, hydroxyalkyl, alkoxyalkyl,
aryloxyalkyl, and acyloxyalkyl;
[0039] each R.sup.3 is independently selected from hydrogen and
R.sup.2; and
[0040] R.sup.4 and R.sup.5 are independently selected from
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,
aralkyl, hydroxyalkyl, alkoxyalkyl, aryloxyalkyl, and acyloxyalkyl,
or R.sup.4 and R.sup.5 taken together form a 5- to 7-member
heterocyclic ring whose ring atoms are selected from the group
consisting of carbon, nitrogen, oxygen and sulfur, wherein the ring
atoms include at most 3 heteroatoms.
[0041] The groups defined as R.sup.2, R.sup.3, R.sup.4, and
R.sup.5, when selected from alkyl, alkenyl, and alkynyl, preferably
have up to six carbon atoms. When selected from cycloalkyl or
cycloalkenyl, they preferably have 3 to 7, or, for cycloalkenyl, 5
to 7 carbon atoms. When selected from aralkyl, hydroxyalkyl,
alkoxyalkyl, aryloxyalkyl, and acyloxyalkyl, the alkyl components
of these groups preferably have up to six carbon atoms. In one
embodiment, each of these groups is independently selected from
alkyl, aryl, aralkyl, and alkoxyalkyl.
[0042] In selected embodiments, X.sup.2.dbd.X.sup.3.dbd.H, and Y is
--CH.sub.2-- or --CH.sub.2CH.sub.2--. In further embodiments,
OR.sup.1 is selected from the group consisting of
O--(C.dbd.O)--OR.sup.2, O--(C.dbd.O)--O--Y--(C.dbd.O)--OR.sup.3,
and O--(C.dbd.O)--O--Y--NR.sup.4R.sup.5(carbonate derivatives). In
other embodiments, OR.sup.1 is-selected from the group consisting
of O--(C.dbd.O)--NR.sup.4R.sup.5,
O--(C.dbd.O)--NR.sup.3-Y--(C.dbd.O)--OR.sup.3, and
O--(C.dbd.O)--NR.sup.3--Y--NR.sup.4N.sup.5(carbamate
derivatives).
[0043] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 is a graph showing apoptosis induction by invention
compound PG666 (14-ethyl carbamate), in comparison to triptolide
(PG490), its 14-succinyl ester (PG490-88), and its 14-glutamyl
ester (PG661); see also Table 3.
[0045] FIG. 2 is a graph showing apoptosis induction by invention
compounds PG666 (14-ethyl carbamate), PG671 (14-phenyl carbamate)
and PG 672 (N-methylpiperazinecarbonyl) (carbamate), in comparison
to triptolide (PG490), its 14-succinyl ester (PG490-88), and its
14-glutamyl ester (PG661); see also Table 4.
[0046] FIG. 3 is a graph showing apoptosis induction by invention
compounds PG666 (14-ethyl carbamate) and PG688
(14-dimethylaminoethyl carbamate), in comparison to triptolide
(PG490) and its 14-succinyl ester (PG490-88); see also Table 5.
[0047] FIG. 4 is a graph showing IL-2 inhibition by invention
compounds PG666 (14-ethyl carbamate) and PG688
(14-dimethylaminoethyl carbamate), in comparison to triptolide
(PG490) and its 14-succinyl ester (PG490-88); see also Table 7.
[0048] FIG. 5 is a graph showing IL-2 inhibition by invention
compounds PG666 (14-ethyl carbamate), PG671 (14-phenyl carbamate)
and PG672 (14-N-methylpiperazinecarbonyl) (carbamate), in
comparison to triptolide (PG490), its 14-succinyl ester (PG490-88),
and its isoglutamyl ester (PG661); see also Table 8.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0049] The terms below have the following meanings unless indicated
otherwise.
[0050] "Triptolide derivatives" or "triptolide analogs" refers to
derivatives of triptolide, 16-hydroxytriptolide, or tripdiolide
(2-hydroxytriptolide) which are derivatized at one or more hydroxyl
groups.
[0051] "Alkyl" refers to a fully saturated acyclic moiety
consisting of carbon and hydrogen, which may be linear or branched.
Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl,
n-heptyl, and isopropyl. Generally preferred are lower alkyl
groups, having one to six carbon atoms, as exemplified by methyl,
ethyl, n-butyl, i-butyl, t-butyl, isoamyl, n-pentyl, and
isopentyl.
[0052] "Cycloalkyl" refers to a fully saturated cyclic moiety
consisting of carbon and hydrogen, having three to eight carbon
atoms, preferably three to six carbons atoms; e.g. cyclopropyl or
methylcyclopentyl. "Cycloalkenyl" refers to an unsaturated cyclic
moiety consisting of carbon and hydrogen, having five to eight
carbon atoms, preferably five or six carbon atoms.
[0053] "Alkenyl" refers to an unsaturated acyclic moiety consisting
of carbon and hydrogen, which may be linear or branched, having one
or more double bonds. Generally preferred are lower alkenyl groups,
having two to six carbon atoms. "Alkynyl" refers to an unsaturated
acyclic moiety consisting of carbon and hydrogen, which may be
linear or branched, containing one or more triple bonds. Generally
preferred are lower alkynyl groups, having two to six carbon
atoms.
[0054] "Aryl" refers to a substituted or unsubstituted monovalent
aromatic radical, generally having a single ring (e.g., benzene) or
two condensed rings (e.g., naphthyl), where monocyclic aryl groups
are preferred. The term includes heteroaryl groups, which are
aromatic ring groups having one or more nitrogen, oxygen, or sulfur
atoms in the ring, such as furyl, pyrrole, pyridyl, and indole. By
"substituted" is meant that one or more ring hydrogens in the aryl
group, preferably one or two ring hydrogens, is replaced with a
group preferably selected from fluorine, chlorine, bromine, methyl,
ethyl, hydroxy, hydroxymethyl, nitro, amino, methylamino,
dimethylamino, methoxy, halomethoxy, and halomethyl.
[0055] "Acyloxyalkyl" refers to a substituent of the form
--R--O--(C.dbd.O)--R', where R is alkyl, preferably having up to
six carbon atoms, and R' is selected from alkyl, alkenyl, alkynyl,
aryl, and aralkyl, where R' preferably comprises lower alkyl, lower
alkenyl, or lower alkynyl (i.e. C.sub.2-C.sub.6) groups and
monocyclic aryl groups.
[0056] "Aralkyl" refers to an alkyl, preferably lower
(C.sub.1-C.sub.4, more preferably C.sub.1-C.sub.2) alkyl,
substituent which is further substituted with an aryl group,
preferably a monocyclic aryl group; examples are benzyl and
phenethyl. Also included is fluorenylmethyl, a component of the
widely employed Fmoc (fluorenylmethoxycarbonyl) protecting
group.
[0057] The term "pharmaceutically acceptable salt" encompasses
carboxylate salts having organic and inorganic cations, such as
alkali and alkaline earth metal cations (for example, lithium,
sodium, potassium, magnesium, barium and calcium); ammonium; or
organic cations, for example, dibenzylammonium, benzylammonium,
2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium,
phenylethylbenzylammonium, dibenzylethylene diammonium, and the
like. Other cations encompassed by the above term include the
protonated form of procaine, quinine and N-methylglucosamine, and
the protonated forms of basic amino acids such as glycine,
ornithine, histidine, phenylglycine, lysine, and arginine.
[0058] The term also includes salts formed by standard acid-base
reactions with basic groups, such as amino groups, having a
counterion derived from an organic or inorganic acid. Such
counterions include chloride, sulfate, phosphate, acetate,
succinate, citrate, lactate, maleate, fumarate, palmitate, cholate,
glutamate, glutarate, tartrate, stearate, salicylate,
methanesulfonate, benzenesulfonate, sorbate, picrate, benzoate,
cinnamate, and the like.
[0059] For the purposes of the current disclosure, the following
numbering scheme is used for triptolide and triptolide analogs:
##STR00002##
II. Triptolide Analogs
[0060] Compounds as represented by structure I, below, are
derivatives of triptolide having hydrophilic substituents, possess
greater water solubility than the non-derivatized starting
compound, and are effective to hydrolyze and convert in vivo to the
parent compound. The compounds are useful as prodrugs for
immunosuppressive, anti-inflammatory and anticancer
applications.
[0061] A. Structure
[0062] In compounds of formula I:
##STR00003##
X.sup.1 is OH or OR.sup.1, and X.sup.2 and X.sup.3 are
independently OH, OR.sup.1 or hydrogen, with the proviso that at
least one of X.sup.1, X.sup.2 and X.sup.3 is OR.sup.1, and at least
one of X.sup.2 and X.sup.3 is hydrogen.
[0063] OR.sup.1 is a carbamate or carbonate group, which may be
further substituted, e.g. with an ester or amine. In particular,
where OR.sup.1 is represented as O--(C.dbd.O)--Z, Z is selected
from the group consisting of:
[0064] --OR.sup.2,
[0065] --O--Y--(C.dbd.O)--OR.sup.3,
[0066] --O--Y--NR.sup.4R.sup.5,
[0067] --NR.sup.4R.sup.5,
[0068] --NR.sup.3--Y--(C.dbd.O)--OR.sup.3, and
[0069] --NR.sup.3--Y--NR.sup.4N.sup.5,
where Y is a divalent alkyl, alkenyl or alkynyl group having up to
six carbon atoms; R.sup.2 is selected from alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, aryl, aralkyl, hydroxyalkyl, alkoxyalkyl,
aryloxyalkyl, and acyloxyalkyl; and each R.sup.3 is independently
selected from hydrogen and R.sup.2. R.sup.4 and R.sup.5 are
independently selected from hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, aryl, aralkyl, hydroxyalkyl, alkoxyalkyl,
aryloxyalkyl, and acyloxyalkyl. Alternatively, R.sup.4 and R.sup.5
taken together may form a 5- to 7-member heterocyclic ring whose
ring atoms are selected from the group consisting of carbon,
nitrogen, oxygen and sulfur, where the ring atoms include at most 3
heteroatoms. Examples include, but are not limited to, piperidine,
piperazine, pyrrolidine, and morpholine.
[0070] The groups defined as R.sup.2, R.sup.3, R.sup.4, and
R.sup.5, when selected from alkyl, alkenyl, and alkynyl, preferably
have up to six carbon atoms. When selected from cycloalkyl or
cycloalkenyl, they preferably have 3 to 7, or, for cycloalkenyl, 5
to 7 carbon atoms. When selected from aralkyl, hydroxyalkyl,
alkoxyalkyl, aryloxyalkyl, and acyloxyalkyl, the alkyl components
of these groups preferably have up to six carbon atoms. In one
embodiment, each of these groups is independently selected from
alkyl, aryl, aralkyl, and alkoxyalkyl.
[0071] In one embodiment, X.sup.1 is OR.sup.1, and each of X.sup.2
and X.sup.3 is hydrogen. In another embodiment, Y is methylene
(--CH.sub.2--) or ethylene (--CH.sub.2CH.sub.2--).
[0072] B. Preparation
[0073] The compounds of structure I may be prepared from
triptolide, as obtained from the root xylem of the Chinese
medicinal plant Tripterygium wilfordii (TW) or from other known
sources. The TW plant is found in the Fujiang Province and other
southern provinces of China; TW plant material can generally be
obtained in China or through commercial sources in the United
States. Methods for preparing triptolide and some of its
derivatives (e.g. tripdiolide and 16-hydroxytriptolide) are known
in the art and are described, for example, in Kupchan et al. (1972,
1977); Lipsky et al. (1994); Pu et al. (1990); and Ma et al.
(1992).
[0074] The hydroxyl group(s) of triptolide or its derivatives can
be converted to the carbamates of structure I by reaction with an
appropriately substituted isocyanate, as shown in Examples 1
(General Procedure A), 5 and 6, or by reaction with phosgene and an
appropriately substituted amine, as shown in Examples 2 (General
Procedure B) and 7.
[0075] Similarly, the hydroxyl group(s) of triptolide or its
derivatives can be converted to the carbonates of structure I by
reaction with an appropriately substituted chloroformate, as shown
in Examples 3 (General Procedure C), 8 and 9, or by reaction with
phosgene and an appropriately substituted alcohol, as shown in
Examples 4 (General Procedure D), 10-13 and 15. As shown in
Examples 7 and 11-15, further functionality on a carbonate or
carbamate alkyl group can be incorporated. Metal salts and amine
salts are readily prepared by reaction or exchange with an
appropriate counterion (Examples 14, 16, 17).
[0076] In cases where all available hydroxyl groups on the starting
material are to be derivatized, an excess of reagent can be used to
drive the reaction to completion. The compound 16-hydroxytriptolide
contains two free hydroxyl groups, one secondary (at C-14) and one
primary (at C-16). Since the hydroxyl group at the 16-position is
more reactive than the 14-hydroxyl group for steric reasons, mono-
and diester derivatives can be selectively made using appropriate
reaction conditions. Reaction with a stoichiometric amount of a
selected reagent yields primarily the compound monoderivatized at
the 16-position, with the 14-hydroxyl group remaining free.
Monoderivatives substituted at the more hindered (secondary)
hydroxyl group can be prepared by first selectively protecting the
less hindered (primary) hydroxyl group, carrying out the
derivatization at the unprotected position, and then removing the
protecting group. Suitable hydroxyl protecting groups are well
known, and are described, for example, by Kocienski (1994).
[0077] Various compounds of the invention, prepared as described
above and in the Examples, are given in the Table below. All are
substituted at the 14-hydroxyl of triptolide with a carbonate or
carbamate substituent. Also included are reference ester
substituted compounds, PC490-88 and PG661, as well as the parent
compound, designated herein as PG490.
TABLE-US-00001 TABLE 1 Exemplary Carbamate- and
Carbonate-Substituted Triptolide Derivatives Designation Name
(Triptolide derivative) 14-O--(C.dbd.O)X substituent Controls PG490
Triptolide PG490-88 14-succinyl ester CH.sub.2CH.sub.2COOH PG661
14-isoglutamyl ester CH.sub.2CH.sub.2CH(NH.sub.2)COOH Compounds
PG666 14-ethyl carbamate NHCH.sub.2CH.sub.3 PG671 14-phenyl
carbamate NH(C.sub.6H.sub.5) PG672 N-methylpiperazinecarbonyl
(carbamate) ##STR00004## PG674 14-ethyl carbonate OCH.sub.2CH.sub.3
PG676 14-phenyl carbonate O(C.sub.6H.sub.5) PG679 14-ethoxyethyl
carbonate OCH.sub.2CH.sub.2OCH.sub.2CH.sub.3 PG680
14-methoxycarbonylmethyl OCH.sub.2(C.dbd.O)OCH.sub.3 carbonate
PG681 14-(R)-.alpha.-methyl-tert-
OC*H(CH.sub.3)(C.dbd.O)OC(CH.sub.3).sub.3 butoxycarbonylmethyl
carbonate PG682 14-dimethylaminoethyl
OCH.sub.2CH.sub.2N(CH.sub.3).sub.2 carbonate PG682 PTSA
14-dimethylaminoethyl OCH.sub.2CH.sub.2N.sup.+H(CH.sub.3).sub.2
.sup.-OTs carbonate, p-toluenesulfonate salt PG687
14-hydroxycarbonylmethyl OCH.sub.2COOH carbonate PG687 Na
14-hydroxycarbonylmethyl OCH.sub.2COO.sup.-+Na carbonate, sodium
salt PG687 tris 14-hydroxycarbonylmethyl OCH.sub.2COO.sup.-
.sup.+NH.sub.3C(CH.sub.2OH).sub.3 carbonate, tris(hydroxy-
methyl)aminomethane salt PG688 14-dimethylaminoethyl
NHCH.sub.2CH.sub.2N(CH.sub.3).sub.2 carbamate PG695 14-tert-butyl
carbonate OC(CH.sub.3).sub.3
III. Prodrug Conversion and Apoptosis Inducing Activity
[0078] A. Conversion Assays
[0079] The compounds of formula I provide the advantage of
different and sometimes widely varying rates of conversion to
parent compound, as demonstrated below. Accordingly, prodrugs of
formula I can be selected for different desired conversion rates in
human serum/plasma by choosing different structural constituents
linked via a carbonate or carbamate linkage to triptolide.
[0080] Compounds of formula I, as shown in Table 1 above and in the
Examples, were assayed for their capacity to induce apoptosis in
cells from the Jurkat human T lymphocyte cell line, after
incubation with pooled human serum for varying periods of time at
37.degree. C. (see Example 19). An ester prodrug,
triptolide-14-succinate, designated PG490-88, was included for
comparison. The extent of conversion to triptolide after such
incubation was also independently determined by HPLC analysis.
[0081] The results of the apoptosis assay are presented in Table 2.
The ED.sub.50 values (column 3) are calculated directly from the
data in each experiment, and the % conversion values (column 4) are
calculated as percent of the ED.sub.50 value produced by
triptolide, designated PG490, incubated in the same plasma (i.e. in
the same experiment). This procedure gives the most valid direct
comparison of each compound to triptolide under the same
experimental conditions.
[0082] Comparison of the percent conversion at each of the
incubation times shows a broad range of values among the compounds.
The percent conversion varied from 7% (PG681;
14-(R)-.alpha.-methyl-tert-butoxycarbonylmethyl carbonate) to 98%
(PG674; 14-ethyl carbonate) after 1 hour, and from 6% (PG687-tris;
14-hydroxycarbonylmethyl carbonate, tris salt) to 100% (PG674,
PG695; 14-tert-butyl carbonate) or greater (PG682;
14-dimethylaminoethyl carbonate, calculated as >100% compared to
PG490) after 48 hours.
TABLE-US-00002 TABLE 2 ED.sub.50 (nM) Conversion, Incubation in
apoptosis as relative Time in assay after ED.sub.50 compared
t.sub.1/2 in human Serum incubation to triptolide plasma (min)
Cmpd. (hours) with serum (%) by HPLC Control PG490-88 0.5 2584 2
max. 26% PG490-88 1 2268 2 conversion PG490-88 24 328 18 at 48 hr
PG490-88 48 147 43 (Na salt) Compounds PG674 0.5 27 188 12 PG674 1
55 98 PG674 24 60 97 PG674 48 65 100 PG676 48 56 96 15 PG679 0.5 39
128 11 PG680 48 58 85 9 PG681 1 684 7 max. 20% PG681 48 139 40
conversion at 48 hr PG682 1 66 76 n.d. PG682 48 32 146 PG682PTSA 1
57 89 17 PG687tris 48 960 6 max. 10% at 48 hr (Na salt) PG695 48 59
100 n.d.
[0083] Prodrug conversions to triptolide as determined
independently by HPLC are given in column 4. As with the bioassay
data, a comparison of the t.sub.1/2 values for conversion to
triptolide shows a broad range of values among the compounds. The
t.sub.1/2 values range from 9 minutes (PG680;
14-methoxycarbonylmethyl carbonate), 11 minutes (PG679;
14-ethoxyethyl carbonate) and 12 minutes (PG674; 14-ethyl
carbonate) to incomplete conversion (10%) in 48 hours of incubation
(PG687Na; 14-hydroxycarbonylmethyl carbonate, sodium salt). PG681
(14-(R)-.alpha.-methyl-tert-butoxycarbonylmethyl carbonate), which
exhibits the lowest percent conversion in the bioassay (7%),
converts incompletely (20%) in 48 hours as assessed by HPLC. PG687
(14-hydroxycarbonylmethyl carbonate) converts only 6% within 48
hours when evaluated in the apoptosis assay, and only 10% in this
time span when assayed by HPLC. PG674 (14-ethyl carbonate) converts
98% in 1 hour and 100% in 48 hours in the bioassay, and exhibits a
t.sub.1/2 of 12 minutes by HPLC analysis. PG682
(14-dimethylaminoethyl carbonate) displays conversion calculated as
>100% in is apoptosis induction, and a 17 minute t.sub.1/2 by
HPLC evaluation.
[0084] There is a large measure of consistency between the results
of prodrug conversion in human serum to a biologically active,
apoptosis-inducing compound (presumably triptolide) and the
conversion in human plasma and expressed in minutes as the
t.sub.1/2 of prodrug conversion to triptolide assessed by HPLC.
There is a broad range of values for the conversion of prodrugs,
whether the conversion is evaluated in the apoptosis induction
bioassay or by HPLC identification and quantification of
triptolide. This broad range of conversion values in human serum or
plasma indicates that the compounds of formula I do not share a
similar conversion rate under these circumstances. This unexpected
difference in conversion rates from these triptolide prodrugs to
triptolide shows that different and widely varying rates of
conversion can be obtained by making differently substituted
prodrugs as described herein.
[0085] In general, the carbamate derivatives of the invention, as a
class, were found to convert in human serum less readily than the
carbonate derivatives, as a class. As discussed further below,
derivatives which are resistant to hydrolysis by human esterases
and proteases may be useful in antibody directed enzyme prodrug
therapy.
[0086] B. Dose-Response Data
[0087] Dose-response data on apoptosis induction by invention
compound PG666 (14-ethyl carbamate), in comparison to triptolide
(PG490), its 14-succinyl ester (PG490-88), and its 14-glutamyl
ester (PG661), is given in Table 3. The dose-response data is also
represented graphically in FIG. 1.
TABLE-US-00003 TABLE 3 Apoptotic Induction by Triptolide Esters and
Carbamate PG666 in the Presence of Human Serum % apoptotic cells at
given concentration (nM) 1 3 10 30 100 300 1000 3000 10000 Human
serum, 48 hr PG490 (cntrl) 8.5 8.5 8.5 26.2 91.7 93.4 94.6 95.2
95.4 PG490-88 (cntrl) 7.8 8.1 7.8 9.1 51.5 92.1 93.9 94.1 95.1
PG661 (cntrl) 11.3 10.4 10.3 10.5 10.3 10.5 10.2 9.7 9.4 PG666 13.4
14.4 14.0 10.1 11.0 20.6 91.0 92.9 93.5 Human serum, 0 hr PG490
(cntrl) -- -- -- -- -- -- -- -- 94.9 PG490-88 (cntrl) -- -- -- --
-- -- -- -- 89.2 PG661 (cntrl) -- -- -- -- -- -- -- -- 7.9 PG666 --
-- -- -- -- -- -- -- 93.6 Medium only PG490 (cntrl) -- -- -- -- --
-- -- -- 96.5 PG490-88 (cntrl) -- -- -- -- -- -- -- -- 93.8 PG661
(cntrl) -- -- -- -- -- -- -- -- 13.6 PG666 + medium -- -- -- -- --
-- -- -- 94.3 Other controls DMSO + Hu -- -- -- -- -- -- -- -- 7.5
PBS + Hu -- -- -- -- -- -- -- -- 7.5 Medium -- -- -- -- -- -- -- --
7.8
[0088] Dose-response data on apoptosis induction by invention
compounds PG666 (14-ethyl carbamate), PG671 (14-phenyl carbamate)
and PG 672 (N-methylpiperazinecarbonyl) (carbamate), in comparison
to triptolide (PG490), its 14-succinyl ester (PG490-88), and its
14-glutamyl ester (PG661), is given in Table 4. The dose-response
data is also represented graphically in FIG. 2. (Some assays gave a
higher apparent background apoptosis than is usually seen, which is
assumed to be an artifact isolated to this experiment.)
TABLE-US-00004 TABLE 4 Apoptotic Induction by Triptolide Esters and
Carbamates in the Presence of Human Serum % apoptotic cells at
given concentration (nM) 0.03 0.1 0.3 1 3 10 30 100 300 1000 3000
10000 Serum, 48 hr PG666 39.9 40.7 40.8 41.3 39.8 39.8 39.1 40.2
44.5 88.6 96.8 92.3 PG671 42.5 43.3 43.9 44.2 43.2 42.4 44.7 43.5
43.1 43.0 43.3 15.4 PG672 42.7 45.2 45.4 45.9 45.3 46.0 46.8 46.6
42.3 43.7 44.9 63.2 Controls PG490 7.0 6.9 6.8 6.4 7.0 7.1 29.4
90.9 93.0 93.2 94.6 94.2 PG490-88 6.6 6.5 7.0 6.0 6.2 6.2 6.8 30.5
89.6 92.6 92.8 93.9 PG661 38.8 38.2 39.0 39.2 39.1 39.2 40.3 38.6
36.3 28.7 28.2 5.2 Serum, 0 hr PG666 -- -- -- -- -- -- -- -- -- --
-- 87.2 PG671 -- -- -- -- -- -- -- -- -- -- -- 10.2 PG672 -- -- --
-- -- -- -- -- -- -- -- 47.6 Controls PG490 -- -- -- -- -- -- -- --
-- -- -- 92.1 PG490-88 -- -- -- -- -- -- -- -- -- -- -- 82.9 PG661
-- -- -- -- -- -- -- -- -- -- -- 5.2 Medium PG666 -- -- -- -- -- --
-- -- -- -- -- 92.6 PG671 -- -- -- -- -- -- -- -- -- -- -- 13.7
PG672 -- -- -- -- -- -- -- -- -- -- -- 48.6 Controls PG490 -- -- --
-- -- -- -- -- -- -- -- 93.4 PG490-88 -- -- -- -- -- -- -- -- -- --
-- 89.7 PG661 -- -- -- -- -- -- -- -- -- -- -- 7.3 DMSO + Hu -- --
-- -- -- -- -- -- -- -- -- 6.7 PBS + Hu -- -- -- -- -- -- -- -- --
-- -- 6.6
[0089] Dose-response data on apoptosis induction by invention
compounds PG666 (14-ethyl carbamate) and PG688
(14-dimethylaminoethyl carbamate), in comparison to triptolide
(PG490) and its 14-succinyl ester (PG490-88), is given in Table 5.
The dose-response data is also represented graphically in FIG.
3.
TABLE-US-00005 TABLE 5 Apoptotic Induction by Triptolide Esters and
Carbamates in the Presence of Human Serum (48 hrs) % apoptotic
cells at given concentration (nM) 1 3 10 30 100 300 1000 3000 Human
serum PG490 (cntrl) 8.8 8.5 8.6 15.9 86.5 90.7 91.7 93.1 PG490-88
8.6 9.3 8.7 7.8 23.3 88.4 90.5 91.7 (cntrl) PG688 9.2 9.8 9.4 9.5
9.4 8.7 8.6 8.2 PG666 9.8 10.9 10.4 10.2 9.5 14.9 87.7 91.0 Medium
PG490 (cntrl) -- -- -- -- -- -- -- 93.7 PG490-88 -- -- -- -- -- --
-- 64.8 (cntrl) PG688 13.0 14.0 12.1 10.9 11.2 11.3 12.1 10.9 PG666
13.9 13.1 13.4 13.0 13.2 14.2 14.1 92.1 Other controls Medium -- --
-- -- -- -- -- 9.1 DMSO + Hu -- -- -- -- -- -- -- 9.2 PBS + Hu --
-- -- -- -- -- -- 9.1
[0090] Inspection of the dose-response data for these compounds
shows PG666 (14-ethyl carbamate) to be more active than PG688
(4-dimethylaminoethyl carbamate) and PG671 (14-phenyl carbamate)
after 48 hr. incubation in human serum. PG666 showed equal
apoptotic activity to PG490 (triptolide) at roughly a 10-fold
higher concentration. The N-methylpiperazinecarbamate (PG672)
showed activity at high concentrations (FIG. 2), while to the
isoglutamyl ester (PG661) showed essentially no activity (FIGS.
1-2).
III. Anticancer Treatment
[0091] Triptolide prodrugs have shown effectiveness in cancer
treatment in vivo. See, for example, coowned PCT Publication No. WO
02/56835, which is incorporated herein by reference. This document
describes high efficacy of a triptolide prodrug, in comparison to
5-FU and CPT-11, in studies with tumor xenografts of the LIT-29
human colon cancer cell line. The triptolide prodrug (a
14-succinate derivative of triptolide) strongly inhibited tumor
growth, to a significantly greater degree than 5-FU and CPT-11, and
induced tumor regression.
[0092] The invention thus includes the use of a composition as
described herein to treat cancers, including cancers involving
cells derived from reproductive tissue (such as Sertoli cells, germ
cells, developing or more mature spermatogonia, spermatids or
spermatocytes and nurse cells, germ cells and other cells of the
ovary), the lymphoid or immune systems (such as Hodgkin's disease
and non-Hodgkin's lymphomas), the hematopoietic system, and
epithelium (such as skin and gastrointestinal tract), solid organs,
the nervous system, and musculo-skeletal tissue. The triptolide
prodrugs may be used for treatment of various cancer cell types,
including, but not limited to, breast, colon, small cell lung,
large cell lung, prostate, malignant melanoma, liver, kidney,
pancreatic, esophogeal, stomach, ovarian, cervical or lymphoma
tumors. Treatment of breast, colon, lung, and prostate tumors is
particularly contemplated. Treatment of leukemias is also
contemplated. The composition may be administered to a patient
afflicted with cancer and/or leukemia by any conventional route of
administration, as discussed above.
[0093] The method is useful to slow the growth of tumors, prevent
tumor growth, induce partial regression of tumors, and induce
complete regression of tumors, to the point of complete
disappearance. The method is also useful in preventing the
outgrowth of metastases derived from solid tumors.
[0094] The compositions of formula I may be administered as sole
therapy or with other supportive or therapeutic treatments not
designed to have anti-cancer effects in the subject. The method
also includes administering the compounds of formula I in
combination with one or more conventional anti-cancer drugs or
biologic protein agents, where the amount of drug(s) or agent(s)
is, by itself, ineffective to induce the appropriate suppression of
cancer growth, in an amount effective to have the desired
anti-cancer effects in the subject. Such anti-cancer drugs include
actinomycin D, camptothecin, carboplatin, cisplatin,
cyclophosphamide, cytosine arabinoside, daunorubicin, doxorubicin,
etoposide, fludarabine, 5-fluorouracil, hydroxyurea, gemcitabine,
irinotecan, methotrexate, mitomycin C, mitoxantrone, paclitaxel,
taxotere, teniposide, topotecan, vinblastine, vincristine,
vindesine, and vinorelbine. Anti-cancer biologic protein agents
include tumor necrosis factor (TNF), TNF-related apoptosis inducing
ligand (TRAIL), other TNF-related or TRAIL-related ligands and
factors, interferon, interleukin-2, other interleukins, other
cytokines, chemokines, and factors, antibodies to tumor-related
molecules or receptors (such as anti-HER2 antibody), and agents
that react with or bind to these agents (such as members of the TNF
super family of receptors, other receptors, receptor antagonists,
and antibodies with specificity for these agents).
IV. Prodrug Conversion and Cytokine Inhibiting Activity
[0095] A. Conversion Assays
[0096] As discussed above, the compounds of formula I provide the
advantage of different and sometimes widely varying rates of
conversion to parent compound. Accordingly, prodrugs of formula I
can be selected for different conversion rates in human
serum/plasma by choosing different structural constituents linked
via a carbonate or carbamate linkage to triptolide.
[0097] Several compounds of formula I were analyzed for their
capacity to inhibit IL-2 production in Jurkat human T lymphocyte
cells, after incubation with pooled human serum for 48 hours at
37.degree. C. (see Example 20). An ester prodrug,
triptolide-14-succinate, designated PG490-88, was included for
comparison.
[0098] The results of the immunosuppression assay are presented in
Table 6. The IC.sub.50 values (column 1) are calculated directly
from the data in each experiment. The % conversion values (column
2) are calculated as the percent of the IC.sub.50 value produced by
triptolide, designated PG490, incubated in the same plasma (i.e. in
the same experiment).
TABLE-US-00006 TABLE 6 Compound IC.sub.50 (nM) Conversion (%)
PG490-88 (cntrl) 9 51 PG682PTSA 2 97 PG680 3 44 PG681 3 55 PG676 6
84 PG679 12 11 PG682 23 78 PG687tris 29 6 PG687 61 2 PG687Na 92 1
PG695 100 2
[0099] Again, a broad range of values is shown for the conversion
of the prodrugs as evaluated in the IL-2 inhibition assay. This
broad range of conversion values in human serum or plasma indicates
that the compounds of formula I do not share a similar conversion
rate under these circumstances. This unexpected difference in
conversion rates from these triptolide prodrugs to triptolide shows
that different and widely varying rates of conversion can be
obtained by making differently substituted prodrugs as described
herein.
[0100] B. Dose-Response Data
[0101] Dose-response data on IL-2 inhibition by invention compounds
PG666 (14-ethyl carbamate) and PG688 (14-dimethylaminoethyl
carbamate), in comparison to triptolide (PG490) and its 14-succinyl
ester (PG490-88), is given in Table 7. The dose-response data is
also represented graphically in FIG. 4.
TABLE-US-00007 TABLE 7 Inhibition of IL-2 production in Jurkat
cells (48 hrs) IL-2 pg/mL at given concentration (nM) 0 0.001 0.01
0.1 1 10 100 1000 10000 Controls PG490 932.4 929.7 908.6 937.8
835.2 556.1 120.7 62.9 59.2 PG490- 838.0 776.4 771.0 809.5 732.4
605.1 317.5 65.9 58.2 88 Com- pounds PG688, 848.5 883.9 754.1 810.4
900.4 796.3 873.3 759.9 459.8 serum PG666, 846.0 844.6 799.8 860.6
773.0 819.1 528.0 180.1 63.5 serum
[0102] Dose-response data on IL-2 inhibition by invention compounds
PG666 (14-ethyl carbamate), PG671 (14-phenyl carbamate) and PG672
(14-N-methylpiperazinecarbonyl) (carbamate), in comparison to
triptolide (PG490), its 14-succinyl ester (PG490-88), and its
isoglutamyl ester (PG661), is given in Table 8. The dose-response
data is also represented graphically in FIG. 5.
TABLE-US-00008 TABLE 8 Inhibition of IL-2 production in Jurkat
cells IL-2 pg/mL at given concentration (nM) 0 0.0001 0.001 0.01
0.1 1 10 100 1000 10000 Compounds PG666 104.5 94.3 105.1 97.0 92.8
80.0 89.8 33.3 10.0 8.0 PG671 117.7 96.4 102.7 99.7 114.7 106.1
90.7 77.9 48.7 8.8 PG672 92.0 103.3 90.8 99.1 117.1 91.4 99.1 64.6
26.8 8.7 Controls PG490 77.4 80.0 97.2 83.2 87.1 75.7 25.5 17.8
12.9 22.3 PG490-88 79.0 96.1 83.0 87.5 86.3 88.2 42.7 14.0 8.4 23.8
PG661 94.9 82.0 102.1 123.6 120.7 98.2 110.3 103.6 74.4 68.7
[0103] Several of the invention compounds (PG666, 14-ethyl
carbamate; PG688, 4-dimethylaminoethyl carbamate; PG671, 14-phenyl
carbamate; and PG672, N-methylpiperazinecarbamate) showed some
level of bioactivity in these assays (FIGS. 4-5). Again, the
isoglutamyl ester (PG661) showed little or no activity (FIG. 5).
PG666 (14-ethyl carbamate) showed equal IL-2 inhibitory activity to
PG490 (triptolide) at about 10-30 times the active concentration of
PG490.
V. Immunomodulating and Antiinflammatory Treatment
[0104] Pharmaceutical compositions comprising compounds of formula
I, which are prodrugs of triptolide, are useful in other
applications for which triptolide has proven effective, e.g. in
immunosuppression therapy, as in treating an autoimmune disease,
preventing transplantation rejection, or treating or preventing
graft-versus-host disease (GVHD).
[0105] The method is useful for inhibiting rejection of a solid
organ transplant, tissue graft, or cellular transplant from an
incompatible human donor, thus prolonging survival and function of
the transplant, and survival of the recipient. This use would
include, but not be limited to, solid organ transplants (such as
heart, kidney and liver), tissue grafts (such as skin, intestine,
pancreas, gonad, bone, and cartilage), and cellular transplants
(such as cells from pancreas, brain and nervous tissue, muscle,
skin, bone, cartilage and liver).
[0106] The method is also useful for inhibiting xenograft
(interspecies) rejection; i.e. in preventing the rejection of a
solid organ transplant, tissue graft, or cellular transplant from a
non-human animal, whether natural in constitution or bioengineered
(genetically manipulated) to express human genes, RNA, proteins,
peptides or other non-native, xenogeneic molecules, or
bioengineered to lack expression of the animal's natural genes,
RNA, proteins, peptides or other normally expressed molecules. The
invention also includes the use of a composition as described above
to prolong the survival of such a solid organ transplant, tissue
graft, or cellular transplant from a non-human animal.
[0107] In another aspect, the invention includes a method of
treatment or prevention of graft-versus-host disease, resulting
from transplantation into a recipient of matched or mismatched bone
marrow, spleen cells, fetal tissue, cord blood, or mobilized or
otherwise harvested stem cells. The dose is preferably in the range
0.25-2 mg/kg body weight/day, preferably 0.5-1 mg/kg/day, given
orally or parenterally.
[0108] Also included are methods of treatment of autoimmune
diseases or diseases having autoimmune manifestations, such as
Addison's disease, autoimmune hemolytic anemia, autoimmune
thyroiditis, Crohn's disease, diabetes (Type I), Graves' disease,
Guillain-Barre syndrome, systemic lupus erythematosus (SLE), lupus
nephritis, multiple sclerosis, myasthenia gravis, psoriasis,
primary biliary cirrhosis, rheumatoid arthritis and uveitis,
asthma, atherosclerosis, Type I diabetes, psoriasis, and various
allergies. In treating an autoimmune condition, the patient is
given the composition on a periodic basis, e.g., 1-2 times per
week, at a dosage level sufficient to reduce symptoms and improve
patient comfort. For treating rheumatoid arthritis, in particular,
the composition may be administered by intravenous injection or by
direct injection into the affected joint. The patient may be
treated at repeated intervals of at least 24 hours, over a several
week period following the onset of symptoms of the disease in the
patient.
[0109] Immunosuppressive activity of compounds in vivo can be
evaluated by the use of established animal models known in the art.
Such assays may be used to evaluate the relative effectiveness of
immunosuppressive compounds and to estimate appropriate dosages for
immunosuppressive treatment. These assays include, for example, a
well-characterized rat model system for allografts, described by
Ono and Lindsey (1969), in which a transplanted heart is attached
to the abdominal great vessels of an allogeneic recipient animal,
and the viability of the transplanted heart is gauged by the
heart's ability to beat in the recipient animal. A xenograft model,
in which the recipient animals are of a different species, is
described by Wang (1991) and Murase (1993). A model for evaluating
effectiveness against GVHD involves injection of normal F.sub.1
mice with parental spleen cells; the mice develop a GVHD syndrome
characterized by splenomegaly and immunosuppression (Korngold,
1978; Gleichmann, 1984). Single cell suspensions are prepared from
individual spleens, and microwell cultures are established in the
presence and absence of concanavalin A to assess the extent of
mitogenic responsiveness.
[0110] For therapy in transplantation rejection, the method is
intended particularly for the treatment of rejection of heart,
kidney, liver, cellular, and bone marrow transplants, and may also
be used in the treatment of GVHD. The treatment is typically
initiated perioperatively, either soon before or soon after the
surgical transplantation procedure, and is continued on a daily
dosing regimen, for a period of at least several weeks, for
treatment of acute transplantation rejection. During the treatment
period, the patient may be tested periodically for
immunosuppression level, e.g., by a mixed lymphocyte reaction
involving allogenic lymphocytes, or by taking a biopsy of the
transplanted tissue.
[0111] In addition, the composition may be administered chronically
to prevent graft rejection, or in treating acute episodes of late
graft rejection. As above, the dose administered is preferably 1-25
mg/kg patient body weight per day, with lower amounts being
preferred for parenteral administration, and higher amounts for
oral administration. The dose may be increased or decreased
appropriately, depending on the response of the patient, and over
the period of treatment, the ability of the patient to resist
infection.
[0112] The compounds are also useful as potentiators when
administered concurrently with another immunosuppressive drug for
immunosuppressive treatments as discussed above. A conventional
immunosuppressant drug, such as cyclosporin A, FK506, azathioprine,
rapamycin, mycophenolic acid, or a glucocorticoid, may thus be
administered in an amount substantially less (e.g. 20% to 50% of
the standard dose) than when the compound is administered alone.
Alternatively, the triptolide analog and immunosuppressive drug are
administered in amounts such that the resultant immunosuppression
is greater than what would be expected or obtained from the sum of
the effects obtained with the drug and triptolide analog used
alone. Typically, the immunosuppressive drug and potentiator are
administered at regular intervals over a time period of at least 2
weeks.
[0113] The compositions of formula I are also useful for the
treatment of inflammatory conditions such as asthma, both intrinsic
and extrinsic manifestations. For treatment of asthma, the
composition is preferably administered via inhalation, but any
conventional route of administration may be useful. The composition
and method may also be used for treatment of other inflammatory
conditions, including traumatic inflammation, inflammation in Lyme
disease, psoriasis, chronic bronchitis (chronic infective lung
disease), chronic sinusitis, sepsis associated acute respiratory
distress syndrome, Behcet's disease, pulmonary sarcoidosis,
pemphigus, pemphigoid inflammatory bowel disease, and ulcerative
colitis. Triptolide and the present analogs are also useful in
reducing male fertility.
[0114] The compositions of formula I may also be administered in
combination with a conventional anti-inflammatory drug (or drugs),
where the drug or amount of drug administered is, by itself,
ineffective to induce the appropriate suppression or inhibition of
inflammation.
[0115] The dose that is administered is preferably in the range of
1-25 mg/kg patient body weight per day, with lower amounts being
preferred for parenteral administration, and higher amounts being
preferred for oral administration. Optimum dosages can be
determined by routine experimentation according to methods known in
the art.
VI. Prodrugs of Triptolide as Substrates for Antibody-Conjugated
Enzymes
[0116] Triptolide derivatives which are resistant to hydrolysis by
human esterases and proteases may be advantageously employed in
antibody directed enzyme prodrug therapy. In this methodology, an
anti-tumor antibody is conjugated to an appropriate enzyme (e.g.,
carboxypeptidase G2) and allowed to localize to a tumor, while
clearing from normal tissues. A non-toxic prodrug is then
delivered, and is activated to a toxic drug specifically by enzyme
at the tumor site. (See e.g. Bagshawe 1987, 1989, 1993; Bagshawe et
al. 1988).
[0117] An enzyme that hydrolyzes an oxygen-carbonyl-nitrogen moiety
may be used to convert the less readily converted carbamates of the
invention (e.g. PG671, PG672, PG688). Antibody-conjugated enzymes
capable of hydrolyzing a carbamate ester bond are known; see e.g.
Wentworth et al. 1996. Accordingly, the above carbamates of
triptolide may be useful as prodrugs that are significantly less
toxic and would be liberated at a tumor site in to the presence of
antibody-conjugated enzymes.
VII. Therapeutic Compositions
[0118] Formulations containing the triptolide analogs of formula I
may take the form of solid, semi-solid, lyophilized powder, or
liquid dosage forms, such as tablets, capsules, powders,
sustained-release formulations, solutions, suspensions, emulsions,
ointments, lotions, or aerosols, preferably in unit dosage forms
suitable for simple administration of precise dosages. The
compositions typically include a conventional pharmaceutical
carrier or excipient and may additionally include other medicinal
agents, carriers, or adjuvants. Preferably, the composition will be
about 0.5% to 75% by weight of a compound or compounds of formula
I, with the remainder consisting of suitable pharmaceutical
excipients. For oral administration, such excipients include
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, gelatin,
sucrose, magnesium carbonate, and the like. If desired, the
composition may also contain minor amounts of non-toxic auxiliary
substances such as wetting agents, emulsifying agents, or
buffers.
[0119] The composition may be administered to a subject orally,
transdermally or parenterally, e.g., by intravenous, subcutaneous,
intraperitoneal, or intramuscular injection. For use in oral liquid
preparation, the composition may be prepared as a solution,
suspension, emulsion, or syrup, being supplied either in liquid
form or a dried form suitable for hydration in water or normal
saline. For parenteral administration, an injectable composition
for parenteral administration will typically contain the triptolide
analog in a suitable intravenous solution, such as sterile
physiological salt solution.
[0120] Liquid compositions can be prepared by dissolving or
dispersing the triptolide analog (about 0.5% to about 20%) and
optional pharmaceutical adjuvants in a carrier, such as, for
example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to
form a solution or suspension. The high water solubility of the
compounds of formula I make them particularly advantageous for
administering in aqueous solution, e.g. by intraperitoneal
injection. Although aqueous solutions are preferred, compositions
in accordance with formula I may also be formulated as a suspension
in a lipid (e.g., a triglyceride, a phospholipid, or a
polyethoxylated castor oil such as "CREMOPHOR EL.TM."), in a
liposomal suspension, or in an aqueous emulsion.
[0121] The compound may also be administered by inhalation, in the
form of aerosol particles, either solid or liquid, preferably of
respirable size. Such particles are sufficiently small to pass
through the mouth and larynx upon inhalation and into the bronchi
and alveoli of the lungs. In general, particles ranging from about
1 to 10 microns in size, and preferably less than about 5 microns
in size, are respirable. Liquid compositions for inhalation
comprise the active agent dispersed in an aqueous carrier, such as
sterile pyrogen free saline solution or sterile pyrogen free water.
If desired, the composition may be mixed with a propellant to
assist in spraying the composition and forming an aerosol.
[0122] Methods for preparing such dosage forms are known or will be
apparent to those skilled in the art; for example, see Remington's
Pharmaceutical Sciences (19th Ed., Williams & Wilkins, 1995).
The composition to be administered will contain a quantity of the
selected compound in a pharmaceutically effective amount for
effecting immunosuppression in a subject.
EXAMPLES
[0123] The following examples illustrate but are not intended in
any way to limit the invention.
Example 1
Preparation of a Triptolide Carbamate by Reaction with an
Isocyanate (General Procedure A)
##STR00005##
[0125] A mixture of triptolide, 1 (0.20 mmol, 1.0 eq) and an
isocyanate (3.0 mmol, 15.0 eq) in N,N-dimethylformamide (DMF, 7.0
ml) is sealed and heated in 54.degree. C. oil bath with stirring.
The reaction is monitored with TLC. After the starting material is
completely consumed, the reaction mixture is concentrated under
vacuum, and the crude product is purified with preparative TLC.
Example 2
Preparation of a Triptolide Carbamate by Reaction with Phosgene and
an Amine (General Procedure B)
##STR00006##
[0127] To a solution of triptolide, 1 (0.325 mmol, 1.0 eq) and
4-dimethylaminopyridine (DMAP, 0.0377 mmol, 0.12 eq) in 1,4-dioxane
(15 ml) is added with stirring pyridine (1.0 ml) and phosgene
(.about.20% in toluene, 1.19 ml, 2.25 mmol, 6.92 eq) at room
temperature under nitrogen. After 1 hour of stirring at room
temperature, the reaction mixture is concentrated under vacuum. To
the residue is added dichloromethane (DCM, 15.0 ml) and then the
amine (R.sup.6R.sup.7NH, 1.0 ml). After 10 minutes of stirring at
room temperature, the reaction mixture is concentrated under
vacuum, and the crude product is purified with preparative TLC.
Example 3
Preparation of a Triptolide Carbonate by Reaction with a
Chloroformate (General Procedure C)
##STR00007##
[0129] To a solution of triptolide, 1 (0.33 mmol, 1.0 eq) and
4-dimethylaminopyridine (DMAP, 3.92 mmol, 11.9 eq) in
dichloromethane (DCM, 15 ml) is added with stirring a chloroformate
(2.15 mmol, 6.5 eq) at room temperature under nitrogen. After 24
hours of stirring at room temperature, the reaction mixture is
concentrated under vacuum, and the crude product is purified with
preparative TLC.
Example 4
Preparation of a Triptolide Carbonate by Reaction with Phosgene and
an Alcohol (General Procedure D)
##STR00008##
[0131] To a solution of triptolide, 1 (0.30 mmol, 1.0 eq) and
4-dimethylaminopyridine (DMAP, 3.60 mmol, 12.0 eq) in 1,4-dioxane
(15 ml) is added with stirring phosgene (-20% in toluene, 0.79 ml,
1.50 mmol, 5.0 eq) at room temperature under nitrogen. After 1 hour
of stirring at room temperature, the reaction mixture is
concentrated under vacuum. To the residue is added dichloromethane
(DCM, 15 ml) and then the alcohol (R.sup.2OH, 1.0 ml). After
stirring at room temperature overnight, the reaction mixture is
concentrated under vacuum, and the crude product is purified with
preparative TLC.
Example 5
Synthesis of Triptolide 14-Ethyl Carbamate (PG666)
##STR00009##
[0133] Using General Procedure A, the product was obtained in 98.5%
yield from ethyl isocyanate and triptolide. Analytical TLC Rf=0.44
(ethyl acetate/hexanes/methanol 1:1:0.1). IR (KBr): 3369.6, 2975.6,
2937.6, 2878.0, 1753.0, 1719.0, 1686.1, 1676.5, 1524.0, 1517.7,
1509.0, 1458.7, 1448.8, 1245.8, 1142.5, 1076.3, 1030.8, 988.1,
944.4, 866.9, 722.6, 560.5 cm.sup.-1. H.sup.1 NMR (300 MHz,
CDCl.sub.3): .delta.=4.94 (1H, s, 14-CH), 4.68 (2H, s,
19-CH.sub.2), 3.83 (1H, d, 11-CH), 3.51 (1H, d, 12-CH), 3.48 (1H,
d, 7-CH), 3.26 {2H, m, 22-CH.sub.2 (--NCH.sub.2CH.sub.3)}, 2.70
(1H, m, 5-CH), 2.32 (1H, m, 2-CHb), 2.13 (2H, m, 6-CHb and 2-CHa),
1.93 (2H, m, 15-CH and 6-CHa), 1.57 (1H, dd, 1-CHb), 1.22 (1H, m,
1-CHa), 1.16 {3H, t, 23-CH.sub.3 (--NCH.sub.2CH.sub.3)}, 1.07 (3H,
s, 20-CH.sub.3), 0.99 (3H, d, 17-CH.sub.3), 0.86 (3H, d,
16-CH.sub.3) ppm. HRMS (FAB) m/z calcd for
C.sub.23H.sub.30NO.sub.7.sup.+ (MH.sup.+) 432.2022. found
432.2016.
Example 6
Synthesis of Triptolide 14-Phenyl Carbamate (PG671)
##STR00010##
[0135] Using General Procedure A, the product was obtained in 87.0%
yield from phenyl isocyanate and triptolide. Analytical TLC Rf=0.51
(ethyl acetate/hexanes/methanol 1:1:0.1). IR (KBr): 3315.3, 2970.4,
2939.5, 2878.3, 1751.9, 1676.6, 1600.8, 1543.0, 1534.7, 1443.6,
1314.6, 1215.3, 1063.3, 1028.5, 761.0, 693.1 cm.sup.-1. H.sup.1 NMR
(300 MHz, CDCl.sub.3): .delta.=7.43 (2H, d, Ar--H), 7.30 (2H, dd,
Ar--H), 7.07 (1H, t, Ar--H), 5.04 (1H, s, 14-CH), 4.82 (2H, s,
19-CH.sub.2), 3.88 (1H, d, 11-CH), 3.57 (1H, d, 12-CH), 3.52 (1H,
d, 7-CH), 2.67 (1H, m, 5-CH), 2.33 (1H, d, 2-CHb), 2.17 (2H, m,
6-CHb and 2-CHa), 1.96 (2H, m, 15-CH and 6-CHa), 1.58 (1H, dd,
1-CHb), 1.25 (1H, m, 1-CHa), 1.08 (3H, s, 20-CH.sub.3), 1.02 (3H,
d, 17-CH.sub.3), 0.88 (3H, d, 16-CH.sub.3) ppm.
Example 7
Synthesis of Triptolide 14-Dimethylaminoethyl Carbamate (PG688)
##STR00011##
[0137] Using General Procedure B, the product was obtained in 79.9%
yield from triptolide, phosgene and N,N-dimethylethylenediamine.
Analytical TLC Rf=0.45 (ethyl
acetate/hexanes/methanol/triethylamine 1.2:0.8:0.2:0.1). IR (KBr):
3380.1, 2969.6, 2827.3, 2780.3, 1753.4, 1720.3, 1675.8, 1560.7,
1542.0, 1523.8, 1459.4, 1448.6, 1388.3, 1348.3, 1254.3, 1132.9,
1069.9, 1023.7, 888.2, 773.7, 561.0, 522.3 cm.sup.-1. H.sup.1 NMR
(300 MHz, CDCl.sub.3): .delta.=5.57 (1H, t, CONH--), 4.94 (1H, s,
14-CH), 4.68 (2H, s, 19-CH.sub.2), 3.83 (1H, d, 11-CH), 3.52 (1H,
d, 12-CH), 3.48 (1H, d, 7-CH), 3.31 {2H, m,
22-CH.sub.2(CONHCH.sub.2CH.sub.2)}, 2.69 (1H, m, 5-CH), 2.48 {2H,
dd, 23-CH.sub.2 (CONHCH.sub.2CH.sub.2--)}, 2.34 (1H, m, 2-CHb),
2.27 {6H, s, --N(CH.sub.3).sub.2}, 2.23-2.13 (2H, m, 2-CHa and
6-CHb), 2.03-1.84 (2H, m, 15-CH and 6-CHa), 1.58 (1H, dd, 1-CHb),
1.21 (1H, m, 1-CHa), 1.07 (3H, s, 20-CH.sub.3), 0.99 (3H, d,
17-CH.sub.3), 0.85 (3H, d, 16-CH.sub.3) ppm.
Example 8
Synthesis of Triptolide 14-Ethyl Carbonate (PG674)
##STR00012##
[0139] Using General Procedure C, the product was obtained in 89.6%
yield from ethyl chloroformate and triptolide. Analytical TLC
Rf=0.58 (ethyl acetate/hexanes/methanol 1:1:0.1). IR (KBr): 2972.3,
2938.3, 2879.4, 1474.3, 1677.0, 1448.0, 1372.0, 1253.5, 1170.1,
1092.8, 1068.5, 1004.4, 962.3, 912.3, 864.6, 786.0, 560.1
cm.sup.-1. H.sup.1 NMR (300 MHz, CDCl.sub.3): .delta.=4.83 (1H, s,
14-CH), 4.68 (2H, q, 19-CH.sub.2), 4.25 {2H, qd, 22-CH.sub.2
(--OCH.sub.2CH.sub.3)}, 3.82 (1H, d, 11-CH), 3.55 (1H, dd, 12-CH),
3.49 (1H, d, 7-CH), 2.70 (1H, m, 5-CH), 2.32 (1H, m, 2-CHb), 2.19
(2H, m, 6-CHb and 2-CHa), 1.96 (2H, m, 15-CH and 6-CHa), 1.61 (1H,
m, 1-CHb), 1.37 {3H, t, 23-CH.sub.3 (--OCH.sub.2CH.sub.3)}, 1.21
(1H, m, 1-CHa), 1.07 (3H, s, 20-CH.sub.3), 0.99 (3H, d,
17-CH.sub.3), 0.86 (3H, d, 16-CH.sub.3) ppm.
Example 9
Synthesis of Triptolide 14-Phenyl Carbonate (PG676)
##STR00013##
[0141] Using General Procedure C, the product was obtained in 78.8%
yield from phenyl chloroformate and triptolide. Analytical TLC
Rf=0.53 (ethyl acetate/hexanes/methanol 1:1:0.1). IR (KBr): 2969.7,
2937.6, 1752.1, 1676.5, 1442.6, 1265.6, 1210.6, 1021.5, 961.8,
910.7, 774.3, 560.6 cm.sup.-1. H.sup.1 NMR (300 MHz, CDCl.sub.3):
.delta.=7.42-7.20 (5H, m, Ar--H), 4.83 (1H, s, 14-CH), 4.68 (2H, q,
19-CH.sub.2), 3.83 (1H, d, 11-CH), 3.55 (1H, dd, 12-CH), 3.49 (1H,
d, 7-CH), 2.68 (1H, m, 5-CH), 2.32 (1H, m, 2-CHb), 2.19 (1H, m,
6-CHb and 2-CHa), 1.96 (2H, m, 15-CH and 6-CHa), 1.49 (1H, m,
1-CHb), 1.24 (1H, m, 1-CHa), 1.07 (3H, s, 20-CH.sub.3), 0.99 (3H,
d, 17-CH.sub.3), 0.86 (3H, d, 16-CH.sub.3) ppm.
Example 10
Synthesis of Triptolide 14-Ethoxyethyl Carbonate (PG679)
##STR00014##
[0143] Using General Procedure D, the product was obtained in 90.2%
yield from triptolide, phosgene and ethoxyethanol. Analytical TLC
Rf=0.49 (ethyl acetate/hexanes/methanol 1:1:0.1). IR (KBr): 2974.0,
2935.3, 2876.5, 1750.8, 1676.4, 1458.6, 1448.6, 1388.7, 1375.8,
1122.3, 1023.0, 962.3, 910.8, 866.1, 784.1, 751.6, 559.7 cm.sup.-1.
H.sup.1 NMR (300 MHz, CDCl.sub.3): .delta.=4.83 (1H, s, 14-CH),
4.80 (2H, q, 19-CH.sub.2), 4.40 {1H, m, 22-CHb
(OCOOCHaHbCH.sub.2)}, 4.27 {1H, m, 22-CHa (OCOOCHaHbCH.sub.2--)},
3.82 (1H, d, 11-CH), 3.68 {2H, m, 23-CH.sub.2
(OCOOCH.sub.2CH.sub.2--)}, 3.54 {3H, m, 12-CH and 24-CH.sub.2
(--OCH.sub.2CH.sub.3)}, 3.48 (1H, d, 7-CH), 2.68 (1H, m, 5-CH),
2.31 (1H, m, 2-CHb), 2.18 (2H, m, 2-CHa and 6-CHb), 1.96 (2H, m,
15-CH and 6-CHa), 1.58 (1H, dd, 1-CHb), 1.21 {4H, 1-CHa and
25-CH.sub.3 (OCH.sub.2CH.sub.3)}, 1.07 (3H, s, 20-CH.sub.3), 0.99
(3H, d, 17-CH.sub.3), 0.85 (3H, d, 16-CH.sub.3) ppm.
Example 11
Synthesis of Triptolide
14-(R)-.alpha.-Methyl-tert-butoxycarbonylmethyl Carbonate
(PG681)
##STR00015##
[0145] Using General Procedure D, the product was obtained in 76.2%
yield from triptolide, phosgene and tert-butyl (R)-(+)-lactate.
Analytical TLC Rf=0.62 (ethyl acetate/hexanes/methanol 1:1:0.1). IR
(KBr): 2979.5, 2938.3, 2880.6, 1754.6, 1676.9, 1474.0, 1458.1,
1370.1, 1351.6, 1318.0, 1264.2, 1165.7, 1136.2, 1116.3, 1074.7,
1031.2, 962.5, 912.6, 866.8, 843.6, 786.2, 560.6 cm.sup.-1. H.sup.1
NMR (300 MHz, CDCl.sub.3): .delta.=4.85 {1H, q, 22-CH
[OCOOCH(CH.sub.3)CO]}, 4.83 (1H, s, 14-CH), 4.68 (2H, q,
19-CH.sub.2), 3.83 (1H, d, 11-CH), 3.56 (1H, dd, 12-CH), 3.48 (1H,
d, 7-CH), 2.65 (1H, m, 5-CH), 2.31 (1H, m, 2-CHb), 2.23-2.04 (3H,
m, 6-CHb, 2-CHa and 15-CH), 1.93 (1H, dd, 6-CHa), 1.59 (1H, dd,
1-CHb), 1.52 {3H, d, 28-CH.sub.3 [OCOOCH(CH.sub.3)CO]}, 1.45 {9H,
s, OC(CH.sub.3).sub.3}, 1.19 (1H, m, 1-CHa), 1.08 (3H, s,
20-CH.sub.3), 1.01 (3H, d, 17-CH.sub.3), 0.87 (3H, d, 16-CH.sub.3)
ppm.
Example 12
Synthesis of Triptolide 14-Methoxycarbonylmethyl Carbonate
(PG680)
##STR00016##
[0147] Using General Procedure D, the product was obtained in 82.4%
yield from triptolide, phosgene and methyl glycolate. Analytical
TLC Rf=0.45 (ethyl acetate:hexanes:methanol 1:1:0.1). IR (KBr):
2967.9, 2882.3, 1751.9, 1676.6, 1439.5, 1383.0, 1283.9, 1245.5,
1213.3, 1022.0, 1005.0, 962.1, 910.7, 783.1, 560.6, 547.9, 530.6,
521.5, 478.7 cm.sup.-1. H.sup.1 NMR (300 MHz, CDCl.sub.3):
.delta.=4.84 (1H, s, 14-CH), 4.80 {1H, d, 22-CHb (OCOOCHaHbCO)},
4.68 (2H, q, 19-CH.sub.2), 4.57 {1H, d, 22-CHa (OCOOCHaHbCO)}, 3.83
(1H, d, 11-CH), 3.79 {3H, s, 24-CH.sub.3 (--OCH.sub.3)}, 3.56 (1H,
dd, 12-CH), 3.49 (1H, d, 7-CH), 2.70 (1H, m, 5-CH), 2.32 (1H, m,
2-CHb), 2.23-2.14 (2H, m, 2-CHa and 6-CHb), 2.07-1.89 (2H, m, 15-CH
and 6-CHa), 1.59 (1H, m, 1-CHb), 1.23 (1H, m, 1-CHa), 1.08 (3H, s,
20-CH.sub.3), 1.02 (3H, d, 17-CH.sub.3), 0.89 (3H, d, 16-CH.sub.3)
ppm.
Example 13
Synthesis of Triptolide 14-Dimethylaminoethyl Carbonate (PG682)
##STR00017##
[0149] Using General Procedure D, the product was obtained in 71.2%
yield from triptolide, phosgene and dimethylaminoethanol.
Analytical TLC Rf=0.24 (ethyl
acetate:hexanes:methanol:triethylamine 1:1:0.1:0.02). IR (KBr):
2969.4, 2824.8, 2772.3, 1751.1, 1676.2, 1671.3, 1655.0, 1473.9,
1466.0, 1375.1, 1254.9, 1020.4, 992.6, 962.7, 910.6, 778.9, 557.4,
517.1, 472.4, 440.7 cm.sup.-1. H.sup.1 NMR (300 MHz, CDCl.sub.3):
.delta.=4.83 (1H, s, 14-CH), 4.68 (2H, s, 19-CH.sub.2), 4.34 {2H,
m, 22-CH.sub.2 (OCOOCH.sub.2CH.sub.2N)}, 3.82 (1H, d, 11-CH), 3.55
(1H, d, 12-CH), 3.49 (1H, d, 7-CH), 2.75-2.62 {3H, m, 23-CH.sub.2
(OCOOCH.sub.2CH.sub.2N) and 5-CH}, 2.37 (6H, s,
--N(CH.sub.3).sub.2}, 2.31 (1H, m, 2-CHb), 2.23-2.15 (2H, m, 2-CHa
and 6-CHb), 2.04-1.89 (2H, m, 15-CH and 6-CHa), 1.58 (1H, dd,
1-CHb), 1.21 (1H, m, 1-CHa), 1.06 (3H, s, 20-CH.sub.3), 0.99 (3H,
d, 17-CH.sub.3), 0.85 (3H, d, 16-CH.sub.3) ppm. HRMS (FAB) m/z
calcd for C.sub.25H.sub.34NO.sub.8.sup.+ (MH.sup.+) 476.2284. found
476.2289.
Example 14
Synthesis of p-Toluenesulfonate Salt of Triptolide
14-Dimethylaminoethyl Carbonate (PG682 PTSA)
##STR00018##
[0151] With stirring, to a solution of p-toluenesulfonic acid (19.0
mg, 0.10 mmol) in H.sub.2O (8.0 ml) was slowly added triptolide
14-dimethylaminoethyl carbonate (PG682) (47.6 mg, 0.10 mmol). After
the addition, the solution was stirred for another 30 minutes and
then lyophilized to yield 60.5 mg (93.4%) of white powder. IR
(KBr): 3445.0, 3035.8, 2972.1, 2730.1, 1750.9, 1676.1, 1671.9,
1664.8, 1655.4, 1638.1, 1459.3, 1256.2, 1169.3, 1121.7, 1033.3,
1010.3, 961.0, 910.7, 817.9, 683.0, 570.4, 479.0 cm.sup.-1. H.sup.1
NMR (300 MHz, DMSO-d.sub.6): .delta.=7.47 (2H, d, Ar--H), 7.10 (2H,
d, Ar--H), 4.82 (2H, q, 19-CH.sub.2), 4.80 (1H, s, 14-CH), 4.46
{2H, m, 22-CH.sub.2 (OCOOCH.sub.2CH.sub.2N)}, 3.97 (1H, d, 11-CH),
3.73 (1H, d, 12-CH), 3.67 (1H, d, 7-CH), 3.44 {2H, m, 23-CH.sub.2
(OCOOCH.sub.2CH.sub.2N)}, 2.83 (3H, s, Ar--CH.sub.3), 2.63 (1H, m,
5-CH), 2.28 {6H, s, --N(CH.sub.3).sub.2}, 2.22 (1H, m, 6-CHb), 2.15
(1H, m, 2-CHb), 2.09 (1H, m, 2-CHa), 1.98 (1H, m, 1-CHb), 1.90 (1H,
m, 15-CH), 1.81 (1H, dd, 6-CHa), 1.30 (1H, m, 1-CHa), 0.91 (3H, s,
20-CH.sub.3), 0.90 (3H, d, 17-CH.sub.3), 0.78 (3H, d, 16-CH.sub.3)
ppm.
Example 15
Synthesis of Triptolide 14-Hydroxycarbonylmethyl Carbonate
(PG687)
##STR00019##
[0153] Using General Procedure D, the product was obtained in 47.8%
yield from triptolide, phosgene and glycolic acid. Analytical TLC
Rf=0.32 (ethyl acetate:hexanes:methanol:acetic acid 1:1:0.1:0.1).
IR (KBr): 3416.0, 2975.4, 1752.6, 1701.4, 1685.8, 1638.3, 1559.5,
1415.9, 1257.7, 1021.3, 810.3, 643.0, 528.0 cm.sup.-1. H.sup.1 NMR
(300 MHz, MeOH-d.sub.4): .delta.=4.85 (1H, s, 14-H), 4.82 {2H, q,
22-CH.sub.2 (OCOOCH.sub.2CO)}, 4.46 (2H, q, 19-CH.sub.2), 3.95 (1H,
d, 11-CH), 3.65 (1H, d, 12-CH), 3.50 (1H, d, 7-CH), 2.78 (1H, m,
5-CH), 2.34-2.20 (2H, m, 6-CHb and 2-CHb), 2.08 (1H, m, 15-CH),
1.99-1.62 (2H, m, 2-CHa and 6-CHa), 1.50 (1H, dd, 1-CHb), 1.34 (1H,
td, 1-CHa), 1.04 (3H, s, 20-CH.sub.3), 0.98 (3H, d, 17-CH.sub.3),
0.85 (3H, d, 16-CH.sub.3) ppm. HRMS (FAB) m/z calcd for
C.sub.23H.sub.26NaO.sub.10.sup.+ (MNa.sup.+) 485.1424. found
485.1434.
Example 16
Synthesis of Sodium Salt of Triptolide 14-Hydroxycarbonylmethyl
Carbonate (PG687 Na)
##STR00020##
[0155] To a solution of NaHCO.sub.3 (5.18 mg, 0.0616 mmol) in
H.sub.2O (3.9 ml) was slowly added triptolide
14-hydroxycarbonylmethyl carbonate (PG687) (28.5 mg, 0.0616 mmol)
with stirring. After the addition, the solution was stirred for
another 30 minutes and then lyophilized to yield 29.7 mg (99.3%) of
white powder. IR (KBr): 2961.4, 2877.2, 1638.1, 1560.8, 1551.2,
1412.0, 1261.8, 1021.0, 803.4, 641.0, 530.6, 523.5 cm.sup.-1.
H.sup.1 NMR (300 MHz, DMSO-d.sub.6): .delta.=4.82 {2H, q,
22-CH.sub.2 (OCOOCH.sub.2CO)}, 4.70 (1H, s, 14-CH), 4.16 (1H, d,
19-CHb), 3.98 (1H, d, 19-CHa), 3.94 (1H, d, 11-CH), 3.69 (1H, d,
12-CH), 3.57 (1H, d, 7-CH), 2.58 (1H, m, 5-CH), 2.22 (1H, m,
6-CHb), 2.11 (1H, m, 2-CHb), 1.97 (2H, m, 2-CHa and 15-CH), 1.81
(1H, m, 6-CHa), 1.49 (1H, m, 1-CHb), 1.30 (1H, m, 1-CHa), 0.92 (3H,
s, 20-CH.sub.3), 0.90 (3H, d, 17-CH.sub.3), 0.75 (3H, d,
16-CH.sub.3) ppm.
Example 17
Synthesis of Tris(hydroxymethyl)aminomethane Salt of Triptolide
14-Hydroxycarbonyl-methyl Carbonate (PG687 tris)
##STR00021##
[0157] To a suspension of triptolide 14-hydroxycarbonylmethyl
carbonate (PG687) (8.3 mg, 0.018 mmol) was added a solution of
tris(hydroxymethyl)aminomethane (2.17 mg, 0.018 mmol) in H.sub.2O
(0.75 ml) with stirring. After the addition, the solution was
stirred for another 30 minutes and then lyophilized. The powder was
dissolved in H.sub.2O (2.0 ml) and filtered through a pad of cotton
to remove the fine particles. The filtrate was then lyophilized to
yield 10.1 mg (96.5%) of white powder. IR (KBr): 3364.5 (br),
2975.8, 1750.1, 1581.1, 1413.7, 1349.8, 1255.7, 1019.0, 968.5,
911.1, 648.9, 619.6, 561.2, 497.8 cm.sup.-1. H.sup.1 NMR (300 MHz,
DMSO-ds): .delta.=4.82 {2H, q, 22-CH.sub.2 (OCOOCH.sub.2CO)}, 4.70
(1H, s, 14-CH), 4.20 (1H, d, 19-CHb), 4.02 (1H, d, 19-CHa), 3.94
(1H, d, 11-CH), 3.69 (1H, d, 12-CH), 3.56 (1H, d, 7-CH), 3.20 (6H,
s, (HOCH.sub.2).sub.3CNH.sub.2}, 2.61 (1H, m, 5-CH), 2.22 (1H, m,
6-CHb), 2.11 (1H, m, 2-CHb), 1.96 (2H, m, 2-CHa and 15-CH), 1.81
(1H, m, 6-CHa), 1.30 (2H, m, 1-CH.sub.2), 0.91 (3H, s,
20-CH.sub.3), 0.90 (3H, d, 17-CH.sub.3), 0.76 (3H, d, 16-CH.sub.3)
ppm.
Example 18
Synthesis of Triptolide 14-tert-Butyl Carbonate (PG695)
##STR00022##
[0159] To a solution of triptolide (108.1 mg, 0.30 mmol, 1.0 eq)
and 4-DMAP (367.0 mg, 3.0 mmol, 10.0 eq) in dichloromethane (15 ml)
was added with stirring di-tert-butyl dicarbonate (393.0 mg 1.80
mmol, 6.0 eq) at room temperature under nitrogen. After 48 hours of
stirring at room temperature, methyl alcohol (1.0 ml) was added.
The reaction mixture was concentrated under vacuum and the crude
product was purified with preparative TLC (ethyl
acetate/hexanes/methanol 1:1:0.1) to give 131.3 mg (95.1%) of the
desired product. Analytical TLC Rf=0.66 (ethyl
acetate/hexanes/methanol 1:1:0.1). IR (KBr): 2976.7, 2938.5,
1738.2, 1676.7, 1444.6, 1394.6, 1370.5, 1335.2, 1278.4, 1254.5,
1160.1, 1118.2, 1091.8, 1020.2, 991.6, 962.9, 912.0, 854.4, 786.4,
751.5, 607.2, 558.2, 529.3, 478.2 cm.sup.-1. H.sup.1 NMR (300 MHz,
CDCl.sub.3): .delta.=4.80 (1H, s, 14-CH), 4.68 (2H, q,
19-CH.sub.2), 3.81 (1H, d, 11-CH), 3.53 (1H, d, 12-CH), 3.46 (1H,
d, 7-CH), 2.69 (1H, m, 5-CH), 2.35 (1H, m, 2-CHb), 2.18 (2H, m,
6-CHb and 2-CHa), 1.96 (2H, m, 15-CH and 6-CHa), 1.61 (1H, m,
1-CHb), 1.51 {9H, s, --OC(CH.sub.3).sub.3}, 1.24 (1H, m, 1-CHa),
1.08 (3H, s, 20-CH.sub.3), 0.99 (3H, d, 17-CH.sub.3), 0.86 (3H, d,
16-CH.sub.3) ppm.
Example 19
Apoptosis Assays
[0160] A. Incubation of compounds with human serum. Pooled human
serum was stored in aliquots at -80.degree. C. Test compounds were
added at 20 mM to thawed human serum in 1.5 ml microfuge tubes and
incubated at 37.degree. C. in a water bath for varying periods of
time. The test samples were placed on ice until dilution for the
bioassay. Controls consisted of the compounds incubated in complete
tissue culture medium (RPMI 1640 medium plus 5% heat-inactivated
fetal calf serum, 1% HEPES, 1% pen/strep, 1% glutamine) rather than
human serum.
[0161] B. Apoptosis assay of compounds incubated with human serum.
Test samples were diluted to 1 mM in complete tissue culture
medium. Aliquots were placed in microculture plates and serial
dilutions were prepared so that the final concentration would
encompass the range of 1 to 10,000 nM with half-log increments.
Cells from an exponentially expanding culture of the Jurkat human T
lymphocyte cell line (#TIB-152 obtained from American Type Culture
Collection, Manassas, Va.) were harvested, washed once by
centrifugation and dilution in complete tissue culture medium, and
diluted to a concentration of 1.times.10.sup.6 cells/ml. A volume
of 100 .mu.l of Jurkat cells (1.times.10.sup.5 cells) was added to
wells containing 100 .mu.l of the diluted compounds, and the plates
were incubated at 37.degree. C. in a 5% CO.sub.2 incubator. After
24 hours, the plates were centrifuged to pellet the cells, and the
cells were washed twice with 2% heat-inactivated fetal calf serum
in PBS. To each well, 500 ul of binding buffer was added according
to the Annexin V assay procedure (BioVision, Inc., Mountain View,
Calif.). Next, 5 .mu.l of the fluorescein isothiocyanate (FITC)
conjugate of Annexin V (BioVision, Inc.) was added to each well,
followed by 5 minutes of incubation in the dark. In some assays,
propidium iodide (BioVision, Inc.) was added at this stage to check
for necrotic cells. The contents of the wells were individually
transferred into test tubes, and apoptosis was analyzed using a
FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose,
Calif.). Cells positive for Annexin V binding were considered to be
apoptotic, and the data were calculated as percent apoptotic
cells.
[0162] C. Comparison of bioactivities after incubation of compounds
in human serum. The data were plotted as the concentration of
compound incubated in serum versus percent apoptotic cells. The
concentration of compound inducing 50% apoptosis (ED.sub.50) was
calculated from these dose response curves. The percent conversion
of the test compounds to bioactive compounds (assumed to be
triptolide) was calculated in reference to the result with
triptolide incubated in parallel in human plasma in the same
experiment, as the percent of the ED.sub.50 of the compound
compared to that for triptolide, which was taken as 100%. This
percentage conversion was used to compare the bioactivity of
various compounds after incubation in human serum.
Example 20
Immunosuppression Assays
[0163] A. IL-2 production assay for activity of compounds incubated
with human serum. Test samples were diluted to 1 mM in complete
tissue culture medium. Aliquots were placed in microculture plates
that had been coated with anti-CD3 antibody (used to stimulate the
production of IL-2 by Jurkat cells) and serial dilutions were
prepared so that the final concentration would encompass the range
of 0.001 to 10,000 nM in log increments. Cells from an
exponentially expanding culture of the Jurkat human T lymphocyte
cell line (#TIB-152 obtained from American Type Culture Collection,
Manassas, Va.) were harvested, washed once by centrifugation and
dilution in complete tissue culture medium, and diluted to a
concentration of 2.times.10.sup.6 cells/ml. A volume of 50 .mu.l of
Jurkat cells (1.times.10.sup.5 cells) was added to wells containing
100 .mu.l of the diluted compounds, 50 .mu.l of PMA (10 ng/ml) was
added to each well, and the plates were incubated at 37.degree. C.
in a 5% CO.sub.2 incubator. After 24 hours, the plates were
centrifuged to pellet the cells, 150 .mu.l of supernatant was
removed from each well, and the samples were stored at -20.degree.
C. The stored supernatants were analyzed for human IL-2
concentration using the Luminex 100 (Luminex Corporation, Austin,
Tex.), Luminex microspheres coupled with anti-IL-2 capture
antibody, and fluorochrome-coupled anti-IL-2 detection antibody.
The data were expressed as ng/ml of IL-2.
[0164] B. Comparison of bioactivities after incubation of compounds
in human serum. The data were plotted as the concentration of
compound incubated in serum versus IL-2 concentration. The
concentration of compound inducing a 50% decrease in the IL-2
concentration (IC.sub.50) was calculated from these dose response
curves. The percent conversion of the test compounds to bioactive
compounds (assumed to be triptolide) was calculated in reference to
the result with triptolide incubated in parallel in human plasma in
the same experiment, as the percent of the IC.sub.50 of the
compound compared to that for triptolide, which was taken as 100%.
This percentage conversion was used to compare the bioactivity of
various compounds after incubation in human serum.
* * * * *