U.S. patent application number 10/705022 was filed with the patent office on 2004-05-20 for triterpenes having antibacterial activity.
This patent application is currently assigned to Regents of the University of Minnesota. Invention is credited to Carlson, Robert M., Karim, Raj, Krasutsky, Pavel A..
Application Number | 20040097436 10/705022 |
Document ID | / |
Family ID | 22889812 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040097436 |
Kind Code |
A1 |
Krasutsky, Pavel A. ; et
al. |
May 20, 2004 |
Triterpenes having antibacterial activity
Abstract
Methods of treating a bacterial infection and of killing or
inhibiting bacteria are disclosed. The methods use derivatives of
triterpenes that are abundant in birch bark and other plants. The
triterpenes include betulin, allobetulin, and lupeol.
Inventors: |
Krasutsky, Pavel A.;
(Duluth, MN) ; Carlson, Robert M.; (Duluth,
MN) ; Karim, Raj; (Duluth, MN) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Regents of the University of
Minnesota
|
Family ID: |
22889812 |
Appl. No.: |
10/705022 |
Filed: |
November 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10705022 |
Nov 11, 2003 |
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09969556 |
Oct 1, 2001 |
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6689767 |
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60236510 |
Sep 29, 2000 |
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Current U.S.
Class: |
514/33 ; 514/106;
514/143; 514/169; 514/451; 514/613; 514/63; 514/640; 514/691 |
Current CPC
Class: |
A61K 31/56 20130101;
A61K 31/704 20130101; A61K 31/695 20130101; Y02A 50/30 20180101;
Y02A 50/473 20180101; C07J 63/008 20130101 |
Class at
Publication: |
514/033 ;
514/106; 514/143; 514/063; 514/451; 514/613; 514/640; 514/691;
514/169 |
International
Class: |
A61K 031/704; A61K
031/695; A61K 031/66; A61K 031/56 |
Claims
What is claimed is:
1. A therapeutic method of treating a mammal afflicted with a
bacterial infection comprising administering to the mammal an
effective anti-bacterial amount of a triterpene of formula (I):
49wherein R.sub.1 is hydrogen or hydroxy; R.sub.2 is a direct bond,
carbonyl, oxy, thio, carbonyl oxy, oxy carbonyl,
(C.sub.6-C.sub.10)aryl, or (C.sub.1-C.sub.6)alkyl; R.sub.3 is
hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl, O.dbd.P(OH).sub.2,
O.dbd.P(OH).sub.2OP(O)(OH)--, (C.sub.1-C.sub.5)alkanoyl,
Si(R).sub.3 wherein each R is H, phenyl or (C.sub.1-C.sub.6)alkyl,
C(O)N(R).sub.2, benzyl, benzoyl, tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, or a glycoside;
R.sub.4 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoy- l, Si(R).sub.3 wherein each R is H,
phenyl or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, a glycoside, or
amino; or R.sub.4 and R.sub.5 together are oxo or (.dbd.NOH); and
R.sub.5 is direct bond, carbonyl, oxy, thio, carbonyl oxy, oxy
carbonyl, (C.sub.6-C.sub.10)aryl, or (C.sub.1-C.sub.6)alkyl; or
R.sub.4 and R.sub.5 together are oxo or (.dbd.NOH); wherein any
alkyl can optionally be substituted with one or more halo, hydroxy,
(C.sub.6-C.sub.10)aryl, nitro, cyano, (C.sub.1-C.sub.6)alkoxy,
trifluoromethyl, polyethyleneimine, poly(ethylene glycol), oxo,
NR.sub.7R.sub.8, wherein R.sub.7 and R.sub.8 are each independently
hydrogen, (C.sub.1-C.sub.6)alkyl or polyethyleneimine;
--OP(.dbd.O)(OH).sub.2; or C(.dbd.O)OR.sub.9, wherein R.sub.9 is
hydrogen, (C.sub.1-C.sub.6)alkyl, or polyethyleneimine; each of the
bonds represented by--is independently absent or is present;
wherein any alkyl is optionally interrupted on carbon with one or
more oxy, thio, sulfinyl, sulfonyl, polyethyleneimine, or
poly(ethylene glycol); wherein any alkyl is optionally partially
unsaturated; wherein any aryl can optionally be substituted with
one or more halo, hydroxy, nitro, cyano, (C.sub.1-C.sub.6)alkoxy,
trifluoromethyl, polyethyleneimine, poly(ethylene glycol), oxo,
NR.sub.7R.sub.8, wherein R.sub.7 and R.sub.8 are each independently
hydrogen, (C.sub.1-C.sub.6)alkyl or polyethyleneimine; or
C(.dbd.O)OR.sub.9, wherein R.sub.9 is hydrogen,
(C.sub.1-C.sub.6)alkyl, or polyethyleneimine; or a pharmaceutically
acceptable salt thereof.
2. The method of claim 1 wherein R.sub.4 is not amino; R.sub.4 and
R.sub.5 are not together (.dbd.NOH); and alkyl is not substituted
with --OP(.dbd.O)(OH).sub.2.
3. The method of claim 1 wherein the bond between carbons 1 and 2
is a single bond.
4. The method of claim 1 wherein the bond between carbons 1 and 2
is a double bond.
5. The method of claim 1 wherein R.sub.1 is hydrogen.
6. The method of claim 1 wherein R.sub.1 is hydroxy.
7. The method of claim 1 wherein R.sub.2 is a direct bond.
8. The method of claim 7 wherein R.sub.3 is (C.sub.1-C.sub.6)alkyl;
wherein any alkyl can optionally be substituted with one or more
oxo, carboxy, amino, (C.sub.6-C.sub.10)aryl, or
--OP(.dbd.O)(OH).sub.2,; any alkyl is optionally interrupted on
carbon with one or more oxy or thio; any alkyl is optionally
partially unsaturated; and any aryl can optionally be substituted
with one or more hydroxy or carboxy.
9. The method of claim 8 wherein R.sub.3 is
3-carboxypropenoyloxymethyl, aminoacetoxymethyl,
(carboxymethoxy)acetoxymethyl, 4-carboxybutanoyloxymethyl,
2-carboxybenzoyloxymethyl, butanoyloxymethyl, or
--CH.sub.2OC(.dbd.O)OP(.dbd.O)(OH).sub.2.
10. The method of claim I wherein R.sub.4 is
(C.sub.1-C.sub.6)alkyl; wherein any alkyl can optionally be
substituted with one or more oxo, carboxy, amino,
(C.sub.6-C.sub.10)aryl, or --OP(.dbd.O)(OH).sub.2,
(C.sub.6-C.sub.10)aryl; any alkyl is optionally interrupted on
carbon with one or more oxy or thio; any alkyl is optionally
partially unsaturated; and any aryl can optionally be substituted
with one or more hydroxy or carboxy.
11. The method of claim 1 wherein R.sub.4 is 2-carboxybenzoyl,
2-amino-3-methylbutanoyl, 3-carboxypropenoyl, aminoacetyl,
4-carboxybutanoyl, (carboxymethoxy)acetyl,
3-(3,4-dihydroxyphenyl)propeno- yl, carboxymethylenethioacetyl,
3-carboxy-3-methylbutanoyl, amino, --P(.dbd.O)(OH).sub.2, oxo, or
(.dbd.NOH).
12. The method of claim 1 wherein R.sub.5 is oxy or a direct
bond.
13. The method of claim 1 wherein R.sub.1 is hydrogen or hydroxy;
R.sub.2 is a direct bond; R.sub.3 is (C.sub.1-C.sub.5)alkoxymethyl
or hydroxymethyl; R.sub.4 is hydrogen, phosphono, sulfo, or
(C.sub.1-C.sub.6)alkyl, and R.sub.5 is oxy; or R.sub.4 is amino and
R.sub.5 is a direct bond; or R.sub.4 and R.sub.5 together are oxo
or (.dbd.NOH); wherein any alkyl, or alkyl segment of an R group,
is optionally interrupted on carbon with one or more oxy, thio, or
imido; wherein any alkyl, or alkyl segment of an R group, can
optionally be substituted with one or more oxo, carboxy, amino,
--OP(.dbd.O)(OH).sub.2, or phenyl; wherein phenyl can optionally be
substituted with one or more hydroxy or carboxy.
14. The method of claim 1 wherein R.sub.1 is hydrogen or hydroxy;
R.sub.2 is a direct bond; R.sub.3 is 3-carboxypropenoyloxymethyl,
aminoacetoxymethyl, (carboxmethox)acetoxymethyl,
4-carboxybutanoyloxymeth- yl, 2-carboxybenzoyloxymethyl,
butanoyloxymethyl, or --CH.sub.2OC(.dbd.O)OP(.dbd.O)(OH).sub.2;
R.sub.4 is 2-carboxybenzoyl, 2-amino-3-methylbutanoyl,
3-carboxypropenoyl, aminoacetyl, 4-carboxybutanoyl,
(carboxymethoxy)acetyl, 3-(3,4-dihydroxyphenyl)propeno- yl,
carboxymethylenethioacetyl, 3-carboxy-3-methylbutanoyl, amino,
--P(.dbd.O)(OH).sub.2, oxo, or (.dbd.NOH); and R.sub.5 is oxy or a
direct bond.
15. The method of claim 1 wherein the triterpene is betulin;
betulin-3,28-diglycine; betulin-28-glycerol oxalate;
betulin-28-glycine; betulin-28-oxalate; betulin arabinose galactan;
betulin-3,28didiglycolate- ; betulin-3,28-diglycine;
betulin-3-maleate; betulin-3,28-di-(L-glutamic acid
.gamma.-benzylester) ester; betulin-3,28-di-L-alanine;
betulin-3,28-di-L-proline; betulin-3,28-dioxalate;
betulin-1-ene-2-ol; betulin-3,28-diphenylalanine;
betulin-3-28-dioxalate-polyethylene amine;
betulin-3,38-diphosphate; betulin-3-caffeate;
betulin-3,28-(3',3'-dimethy- l) glutarate; betulin-28-diglycolate;
betulin-28-glutarate; betulin-28-maleate; betulin-28-phthalate;
betulin-3,28-di(3',3'-dimethyl)- glutarate;
betulin-3,28-didiglycolate; betulin-3,28-di(thiodiglycolate);
betulin-3,28-diglutarate; betulin-3,28-dimaleate;
betulin-3,28-diglycolat- e; betulin-3,28-diphthalate;
betulin-3,28-di-L-phenylalanine; betulin-3,28--di-L-valine;
betulin-28-succinate; betulin-3,28-disuccinate- ;
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=1448);
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=906 crude);
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=906 pure); betulinic
acid; betulon-1-ene-2-ol; betulin-3,28-(dipoly(ethylene glycol)bis
(carboxymethylester); allobetulin-3,28-(dipoly(ethylene glycol)bis
(carboxymethyl allobetulin ester); hederin hydrate; lupeol;
lupeol-3-glutarate; lupeol-3-succinate; lupeol-3-thiodiglycolate;
lupeol-3-phthalate; oleanolic acid; ursolic acid; or uvaol.
16. The method of claim 1 wherein the triterpene is betulin;
betulin-28-glycerol oxalate; betulin-28-oxalate; betulin arabinose
galactan; betulin-3,28-didiglycolate; betulin-3,28-diglycine;
betulin-3,28-di-(L-glutamic acid .gamma.-benzylester) ester;
betulin3,28-di-L-proline ester; betulin-3,28-dioxalate;
betulin-1-ene-2-ol; betulin-3,28-dioxalate-polyethylene amine;
betulin-3,28-diphosphate; betulin-3-caffeate;
betulin-28-diglycolate; betulin-28-glutarate; betulin-28-maleate;
betulin-28-phthalate; betulin-3,28-dithiodiglycolate;
betulin-3,28-diglutarate; betulin-3,28-dimaleate;
betulin-3,28-diglycolate; betulin-3,28-diphthalat- e;
betulin-3,28-di-L-phenylalanine; betulin-di-L-valine;
betulin-28-succinate; betulin-3,28-disuccinate;
betulin-3,28-di-(polyethy- lene glycol)-COOH (Mw=906 pure);
betulinic acid; betulon-1-ene-2-ol; betulin-3,28-(dipoly(ethylene
glycol)bis (carboxymethylester); hederin hydrate;
lupeol-3-glutarate; lupeol-3-succinate; lupeol-3-thiodiglycolate- ;
lupeol-3-phthalate; oleanolic acid; uvaol.
17. The method of claim 1 wherein the triterpene is
betulin-3-caffeate; betulin-28-diglycolate;
betulin-3,28-diglutarate; betulin-3,28-diglycine;
betulin-3,28-didiglycolate; betulin-3,28-dimaleate;
betulin-3,28-diphosphate; betulin-3,28-diphthalate;
betulin-3,28-di-L-valine; lupeol; lupeol-3-amine;
lupeol-3-(3',3'-dimethy- l)succinate; lupeol-3-maleate;
lupeol-3-phosphate; lupeol-3-thiodiglycolat- e; lupenone;
lupenon-1,2-ene-2-ol; lupenon-3-oxime.
18. The method of claim 1 wherein the bacterial infection is caused
by Escherichia coli, Staphylococcus sp., Enterococcus faecalis, or
a combination thereof.
19. The method of claim 18 wherein the bacterial infection is
caused by Staphylococcus aureus.
20. A therapeutic method of treating a mammal afflicted with a
bacterial infection comprising administering to the mammal an
effective anti-bacterial amount of a triterpene of formula (II):
50wherein one of R.sub.1 and R.sub.2 is --O--Y and the other is
hydrogen or (C.sub.1-C.sub.6)alkyl optionally substituted by
hydroxy, (C.sub.1-C.sub.6)alkoxy, halo, halo(C.sub.1-C.sub.6)alkoxy
or NR.sub.jR.sub.k wherein R.sub.j and R.sub.k are independently H,
(C.sub.1-C.sub.6)alkyl or (C.sub.1-C.sub.6)alkonyl; or R.sub.1 and
R.sub.2 together are oxo (.dbd.O); R.sub.3 is hydrogen, halo,
carboxy, mercapto, (C.sub.1-C.sub.6)alkyl,
(C.sub.3-C.sub.8)cycloalkyl, or --O--Y; R.sub.4 and R.sub.5 are
each independently hydrogen, (C.sub.1-C.sub.6)alkyl, or
hydroxy(C.sub.1-C.sub.6)alkyl; R.sub.6 is hydrogen or is absent
when the adjacent--is a bond; R.sub.7 is hydrogen or
(C.sub.1-C.sub.6)alkyl; R.sub.8 is hydrogen,
(C.sub.1-C.sub.6)alkyl, or hydroxy(C.sub.1-C.sub.6)alkyl and
R.sub.11 is hydrogen, (C.sub.1-C.sub.6)alkyl, carboxy, or
hydroxy(C.sub.1-C.sub.6)alkyl; or R.sub.8 and R.sub.11 together are
--O--C(.dbd.X)--; R.sub.9 and R.sub.10, are each independently
hydrogen or (C.sub.1-C.sub.6)alkyl; each of the bonds represented
by--is independently absent or is present; X is two hydrogens, oxo
(.dbd.O) or thioxo (.dbd.S); each Y is independently H, aryl,
P(O)(Cl).sub.2, (C.sub.3-C.sub.8)cycloalkyl, adamantyl,
--SO.sub.2R.sub.a O.dbd.P(R.sub.b).sub.2,
O.dbd.P(R.sub.c).sub.2OP(O)(R.s- ub.d)--, Si(R.sub.e).sub.3,
tetrahydropyran-2-yl, an amino acid, a peptide, a glycoside, or a 1
to 10 membered branched or unbranched carbon chain optionally
comprising 1, 2, or 3 heteroatoms selected from non-peroxide oxy,
thio, and --N(R.sub.f)--; wherein said chain may optionally be
substituted on carbon with 1, 2, 3, or 4 oxo (.dbd.O), hydroxy,
carboxy, halo, mercapto, nitro, --N(R.sub.g)(R.sub.h),
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.3-C.sub.8)cycloalkyloxy, aryl,
aryloxy, adamantyl, adamantyloxy, hydroxyamino,
trifluoroacetylamino, a glycoside, an amino acid, or a peptide; and
wherein said chain may optionally be saturated or unsaturated (e.g.
containing one, two, three or more, double or triple bonds);
R.sub.a is (C.sub.1-C.sub.6)alkyl or aryl; R.sub.b, R.sub.c, and
R.sub.d are each independently hydroxy, (C.sub.1-C.sub.6)alkoxy,
hydroxy(C.sub.2-C.sub.6)alkoxy, adamantyloxy,
adamantyl(C.sub.1-C.sub.6)alkoxy, norbornyloxy,
1,1-di(hydroxymethyl)-2-h- ydroxyethoxy,
carboxy(C.sub.1-C.sub.6)alkoxy, 2,3-epoxypropyloxy, benzyloxy,
(C.sub.3-C.sub.8)cycloalkyloxy, NR.sub.xR.sub.y, or aryloxy;
R.sub.e is H, aryl or (C.sub.1-C.sub.6)alkyl; R.sub.f is hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkanoyl, phenyl or
benzyl; R.sub.g and R.sub.h are each independently selected from
the group consisting of hydrogen, (C.sub.1-C.sub.6)alkyl,
hydroxy(C.sub.1-C.sub.6)a- lkyl, adamantyl,
adamantyl(C.sub.1-C.sub.6)alkyl, amino(C.sub.1-C.sub.6)al- kyl,
aminosulfonyl, (C.sub.1-C.sub.6)alkanoyl, aryl and benzyl; or
R.sub.b and R.sub.c together with the nitrogen to which they are
attached form a pyrrolidino, piperidino, or morpholino radical; and
R.sub.x and R.sub.y are each independently hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkanoyl, aryl or benzyl;
wherein each aryl of Y, R.sub.a--R.sub.d, R.sub.g--R.sub.h,
R.sub.x, and R.sub.y may optionally be substituted by 1, 2, or 3
aminosulfonyl, carboxy, NR.sub.iR.sub.j, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxy, hydroxy, halo, nitro, cyano, mercapto,
carboxy, hydroxy(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkyl,
trifluoromethoxy, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkoxycarbonyl, (C.sub.1-C.sub.6)alkylthio, or
(C.sub.1-C.sub.6)alkanoyloxy; wherein R.sub.i and R.sub.j are each
independently hydrogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl- , phenyl, or benzyl; wherein any alkyl
can optionally be substituted with one or more polyethyleneimine or
poly(ethylene glycol); and wherein any alkyl can optionally be
interrupted with one or more polyethyleneimine or poly(ethylene
glycol); or a pharmaceutically acceptable salt thereof.
21. The method of claim 20 wherein the triterpenoid is
3-.beta.-acetoxy-19.alpha.H-19,28 lactone oleanan; allobetulin;
allobetulin-3-succinate; allobetulin-3-glycine ester; allobetulin
lactone; allobetulin lactone-3-acetate; allobetulin
lactone-3-phosphate; allobetulin-3-L-alanine;
allobetulin-3-L-valine; allobetulin-3-L-proline;
allobetulin-3-succinate; allobetulin-3-diglycolate;
allobetulin-3-glutarate; allobetulin-3-phthalate;
allobetulin-3-methylena- mine; allobetulin-3-ethanolamine;
allobetulin-3-ethanolamine hydrochloride; allobetulin-3-glycolate;
allobetulin-3-glutarate; allobetulin-28-glutarate;
allobetulin-3-methylamine HCl; allobetulin-3-phosphate;
allobetulin-3-(polyethylene glycol)-COOH (Mw=674); allobetulon;
allobetulon lactone-1-ene-2-ol; allobetulon
lactone-1-en-2-succinate; allobetulon-1-ene-2-ol;
allobetulon-1-ene-2-dig- lycolate; 3-allobetulon-1-ene-2-succinate;
or 3-allobetulon-1-ene-2-diglyc- olate.
22. The method of claim 20 wherein the triterpenoid is
3-.beta.-acetoxy-19.alpha.H-19,28 lactone oleanan; allobetulin;
allobetulin-3-glycine ester; allobetulin lactone-3-phosphate;
allobetulin-3-succinate; allobetulin-3-ethanolamine;
allobetulin-3-glutarate; allobetulin-28-glutarate;
allobetulin-3-methylamine HCl; allobetulin-3-phosphate;
allobetulon; allobetulon lactone-1-ene-2-ol;
3-allobetulon-1-ene-2-succinate; or ursolic acid.
23. A method of inhibiting or killing a bacterium, comprising
contacting the bacterium with an effective antibacterial amount of
a triterpene of formula (I): 51wherein R.sub.1 is hydrogen or
hydroxy; R.sub.2 is a direct bond, carbonyl, oxy, thio, carbonyl
oxy, oxy carbonyl, (C.sub.6-C.sub.10)aryl, or
(C.sub.1-C.sub.6)alkyl; R.sub.3 is hydrogen, hydroxy,
(C.sub.1-C.sub.6)alkyl, O.dbd.P(OH).sub.2,
O.dbd.P(OH).sub.2OP(O)(OH)--, (C.sub.1-C.sub.5)alkanoyl,
Si(R).sub.3 wherein each R is H, phenyl or (C.sub.1-C.sub.6)alkyl,
C(O)N(R).sub.2, benzyl, benzoyl, tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.- 1-C.sub.4)alkyl, or a
glycoside; R.sub.4 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoyl, Si(R).sub.3 wherein each R is H, phenyl
or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, a glycoside, or
amino; or R.sub.4 and R.sub.5 together are oxo or (.dbd.NOH); and
R.sub.5 is direct bond, carbonyl, oxy, thio, carbonyl oxy, oxy
carbonyl, (C.sub.6-C.sub.10)aryl, or (C.sub.1-C.sub.6)alkyl; or
R.sub.4 and R.sub.5 together are oxo or (.dbd.NOH); wherein any
alkyl can optionally be substituted with one or more halo, hydroxy,
(C.sub.6-C.sub.10)aryl, nitro, cyano, (C.sub.1-C.sub.6)alkoxy,
trifluoromethyl, polyethyleneimine, poly(ethylene glycol), oxo,
NR.sub.7R.sub.8, wherein R.sub.7 and R.sub.8 are each independently
hydrogen, (C.sub.1-C.sub.6)alkyl or polyethyleneimine;
--OP(.dbd.O)(OH).sub.2; or C(.dbd.O)OR.sub.9, wherein R.sub.9 is
hydrogen, (C.sub.1-C.sub.6)alkyl, or polyethyleneimine; each of the
bonds represented by - - - is independently absent or is present;
wherein any alkyl is optionally interrupted on carbon with one or
more oxy, thio, sulfinyl, sulfonyl, polyethyleneimine, or
poly(ethylene glycol); wherein any alkyl is optionally partially
unsaturated; wherein any aryl can optionally be substituted with
one or more halo, hydroxy, nitro, cyano, (C.sub.1-C.sub.6)alkoxy,
trifluoromethyl, polyethyleneimine, poly(ethylene glycol), oxo,
NR.sub.7R.sub.8, wherein R.sub.7 and R.sub.8 are each independently
hydrogen, (C.sub.1-C.sub.6)alkyl or polyethyleneimine; or
C(.dbd.O)OR.sub.9, wherein R.sub.9 is hydrogen,
(C.sub.1-C.sub.6)alkyl, or polyethyleneimine; or a pharmaceutically
acceptable salt thereof.
24. The method of claim 23 wherein R.sub.4 is not amino; R.sub.4
and R.sub.5 are not together (.dbd.NOH); and alkyl is not
substituted with --OP(.dbd.O)(OH).sub.2.
25. The method of claim 23 wherein the bond between carbons 1 and 2
is a single bond.
26. The method of claim 23 wherein the bond between carbons 1 and 2
is a double bond.
27. The method of claim 23 wherein R.sub.1 is hydrogen.
28. The method of claim 23 wherein R.sub.1 is hydroxy.
29. The method of claim 23 wherein R.sub.2 is a direct bond.
30. The method of claim 29 wherein R.sub.3 is
(C.sub.1-C.sub.6)alkyl, wherein any alkyl can optionally be
substituted with one or more oxo, carboxy, amino,
(C.sub.6-C.sub.10)aryl, or --OP(.dbd.O)(OH).sub.2,; wherein any
alkyl is optionally interrupted on carbon with one or more oxy or
thio; wherein any alkyl is optionally partially unsaturated;
wherein any aryl can optionally be substituted with one or more
hydroxy or carboxy.
31. The method of claim 30 wherein R.sub.3 is aminoacetoxymethyl,
(carboxymethoxy)acetoxymethyl, 4-carboxybutanoyloxymethyl,
2-carboxybenzoyloxymethyl, 3-carboxypropanoyloxymethyl,
butanoyloxymethyl, or --CH.sub.2OC(.dbd.O)OP(.dbd.O)(OH).sub.2.
32. The method of claim 23 wherein R.sub.4 is
(C.sub.1-C.sub.6)alkyl, wherein any alkyl can optionally be
substituted with one or more oxo, carboxy, amino,
(C.sub.6-C.sub.10)aryl, or --OP(.dbd.O)(OH).sub.2,; wherein any
alkyl is optionally interrupted on carbon with one or more oxy or
thio; wherein any alkyl is optionally partially unsaturated;
wherein any aryl can optionally be substituted with one or more
hydroxy or carboxy.
33. The method of claim 23 wherein R.sub.4 is 2-carboxybenzoyl,
2-amino-3-methylbutanoyl, 3-carboxypropenoyl, aminoacetyl,
4-carboxybutanoyl, (carboxymethoxy)acetyl,
3-(3,4-dihydroxyphenyl)propeno- yl, carboxymethylenethioacetyl,
3-carboxy-3-methylbutanoyl, amino, --P(.dbd.O)(OH).sub.2, oxo, or
(.dbd.NOH).
34. The method of claim 23 wherein R.sub.5 is oxy or a direct
bond.
35. The method of claim 23 wherein R.sub.1 is hydrogen or hydroxy;
R.sub.2 is a direct bond; R.sub.3 is (C.sub.1-C.sub.5)alkoxymethyl
or hydroxymethyl; R.sub.4 is hydrogen, phosphono, sulfo, or
(C.sub.1-C.sub.6)alkyl, and R.sub.5 is oxy; or R.sub.4 is amino and
R.sub.5 is a direct bond; or R.sub.4 and R.sub.5 together are oxo
or (.dbd.NOH); wherein any alkyl, or alkyl segment of an R group,
is optionally interrupted on carbon with one or more oxy, thio, or
imido; wherein any alkyl, or alkyl segment of an R group, can
optionally be substituted with one or more oxo, carboxy, amino,
--OP(.dbd.O)(OH).sub.2, or phenyl; wherein phenyl can optionally be
substituted with one or more hydroxy or carboxy.
36. The method of claim 23 wherein R.sub.1 is hydrogen or hydroxy;
R.sub.2 is a direct bond; R.sub.3 is aminoacetoxymethyl,
(carboxmethox)acetoxymet- hyl, 4-carboxybutanoyloxymethyl,
2-carboxybenzoyloxymethyl, 3-carboxypropanoyloxymethyl,
butanoyloxymethyl, or --CH.sub.2OC(.dbd.O)OP(.dbd.O)(OH).sub.2;
R.sub.4 is 2-carboxybenzoyl, 2-amino-3-methylbutanoyl,
3-carboxypropenoyl, aminoacetyl, 4-carboxybutanoyl,
(carboxymethoxy)acetyl, 3-(3,4-dihydroxyphenyl)propeno- yl,
carboxymethylenethioacetyl, 3-carboxy-3-methylbutanoyl, amino,
--P(.dbd.O)(OH).sub.2, oxo, or (.dbd.NOH); and R.sub.5 is oxy or a
direct bond.
37. The method of claim 23 wherein the triterpene is betulin;
betulin-3,28-diglycine; betulin-28-glycerol oxalate;
betulin-28-glycine; betulin-28-oxalate; betulin arabinose galactan;
betulin-3,28didiglycolate- ; betulin-3,28-diglycine;
betulin-3-maleate; betulin-3,28-di-(L-glutamic acid
.gamma.-benzylester) ester; betulin-3,28-di-L-alanine;
betulin-3,28-di-L-proline; betulin-3,28-dioxalate;
betulin-1-ene-2-ol; betulin-3,28-diphenylalanine;
betulin-3-28-dioxalate-polyethylene amine;
betulin-3,38-diphosphate; betulin-3-caffeate;
betulin-3,28-(3',3'-dimethy- l) glutarate; betulin-28-diglycolate;
betulin-28-glutarate; betulin-28-maleate; betulin-28-phthalate;
betulin-3,28-di(3',3'-dimethyl)- glutarate;
betulin-3,28-didiglycolate; betulin-3,28-di(thiodiglycolate);
betulin-3,28-diglutarate; betulin-3,28-dimaleate;
betulin-3,28-diglycolat- e; betulin-3,28-diphthalate;
betulin-3,28-di-L-phenylalanine; betulin-3,28-di-L-valine;
betulin-28-succinate; betulin-3,28-disuccinate;
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=1448);
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=906 crude);
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=906 pure); betulinic
acid; betulon-1-ene-2-ol; 3,28 (dipoly(ethylene glycol)bis
(carboxymethylester); 3,28 (dipoly(ethylene glycol)bis
(carboxymethyl allobetulin ester); hederin hydrate; lupeol;
lupeol-3-glutarate; lupeol-3-succinate; lupeol-3-thiodiglycolate;
lupeol-3-phthalate; lupeol-3-succinate; oleanolic acid; ursolic
acid; or uvaol.
38. The method of claim 23 wherein the triterpene is betulin;
betulin-28-glycerol oxalate; betulin-28-oxalate; betulin arabinose
galactan; betulin-3,28-didiglycolate; betulin-3,28-diglycine;
betulin-3,28-di-(L-Glutamic acid r-benzylester) ester;
betulin3,28-di-L-proline ester; betulin-3,28-dioxalate;
betulin-1-ene-2-ol; betulin-3,28-dioxalate-polyethylene amine;
betulin-3,28-diphosphate; betulin-3-caffeate;
betulin-28-diglycolate; betulin-28-glutarate; betulin-28-maleate;
betulin-28-phthalate; betulin-3,28-dithiodiglycolate;
betulin-3,28-diglutarate; betulin-3,28-dimaleate;
betulin-3,28-diglycolate; betulin-3,28-diphthalat- e;
betulin-3,28-di-L-phenylalanine; betulin-di-L-valine;
betulin-28-succinate; betulin-3,28-disuccinate;
betulin-3,28-di-(polyethy- lene glycol)-COOH (Mw=906 pure);
betulinic acid; betulon-1-ene-2-ol; betulin-3,28-(dipoly(ethylene
glycol)bis (carboxymethylester); hederin hydrate;
lupeol-3-glutarate; lupeol-3-succinate; lupeol-3-thiodiglycolate- ;
lupeool-3-phthalate; oleanolic acid; uvaol.
39. The method of claim 23 wherein the triterpene is
betulin-3-caffeate; betulin-28-diglycolate;
betulin-3,28-diglutarate; betulin-3,28-diglycine;
betulin-3,28-didiglycolate; betulin-3,28-dimaleate;
betulin-3,28-diphosphate; betulin-3,28-diphthalate;
betulin-3,28-di-L-valine; lupeol; lupeol-3-amine;
lupeol-3-(3',3'-dimethy- l)succinate; lupeol-3-maleate;
lupeol-3-phosphate; lupeol-3-thiodiglycolat- e; lupenone;
lupenon-1,2-ene-2-ol; lupenon-3-oxime.
40. The method of claim 23 wherein the bacterium is Escherichia
coli, Staphylococcus sp., Enterococcus faecalis, or a combination
thereof.
41. The method of claim 40 wherein the bacterium is Staphyloccoccus
aureus.
42. The method of claim 23 wherein the contacting is in vitro.
43. The method of claim 23 wherein the contacting is in vivo.
44. A method of inhibiting or killing a bacterium, comprising
contacting the bacterium with an effective antibacterial amount of
a triterpene of formula (II): 52wherein one of R.sub.1 and R.sub.2
is --O--Y and the other is hydrogen or (C.sub.1-C.sub.6)alkyl
optionally substituted by hydroxy, (C.sub.1-C.sub.6)alkoxy, halo,
halo(C.sub.1-C.sub.6)alkoxy or NR.sub.jR.sub.k wherein R.sub.j and
R.sub.k are independently H, (C.sub.1-C.sub.6)alkyl or
(C.sub.1-C.sub.6)alkonyl; or R.sub.1 and R.sub.2 together are oxo
(.dbd.O); R.sub.3 is hydrogen, halo, carboxy, mercapto,
(C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl, or --O--Y;
R.sub.4 and R.sub.5 are each independently hydrogen,
(C.sub.1-C.sub.6)alkyl, or hydroxy(C.sub.1-C.sub.6)alkyl; R.sub.6
is hydrogen or is absent when the adjacent--is a bond; R.sub.7 is
hydrogen or (C.sub.1-C.sub.6)alkyl; R.sub.8 is hydrogen,
(C.sub.1-C.sub.6)alkyl, or hydroxy(C.sub.1-C.sub.6)alkyl and
R.sub.11 is hydrogen, (C.sub.1-C.sub.6)alkyl, carboxy, or
hydroxy(C.sub.1-C.sub.6)alkyl; or R.sub.8 and R.sub.11 together are
--O--C(.dbd.X)--; R.sub.9 and R.sub.10, are each independently
hydrogen or (C.sub.1-C.sub.6)alkyl; each of the bonds represented
by--is independently absent or is present; X is two hydrogens, oxo
(.dbd.O) or thioxo (.dbd.S); each Y is independently H, aryl,
P(O)(Cl).sub.2, (C.sub.3-C.sub.8)cycloalkyl, adamantyl,
--SO.sub.2R.sub.a O.dbd.P(R.sub.b).sub.2,
O.dbd.P(R.sub.c).sub.2OP(O)(R.s- ub.d)--, Si(R.sub.e).sub.3,
tetrahydropyran-2-yl, an amino acid, a peptide, a glycoside, or a 1
to 10 membered branched or unbranched carbon chain optionally
comprising 1, 2, or 3 heteroatoms selected from non-peroxide oxy,
thio, and --N(R.sub.f)--; wherein said chain may optionally be
substituted on carbon with 1, 2, 3, or 4 oxo (.dbd.O), hydroxy,
carboxy, halo, mercapto, nitro, --N(R.sub.g)(R.sub.h),
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.3-C.sub.8)cycloalkyloxy, aryl,
aryloxy, adamantyl, adamantyloxy, hydroxyamino,
trifluoroacetylamino, a glycoside, an amino acid, or a peptide; and
wherein said chain may optionally be saturated or unsaturated (e.g.
containing one, two, three or more, double or triple bonds);
R.sub.a is (C.sub.1-C.sub.6)alkyl or aryl; R.sub.b, R.sub.c, and
R.sub.d are each independently hydroxy, (C.sub.1-C.sub.6)alkoxy,
hydroxy(C.sub.2-C.sub.6)alkoxy, adamantyloxy,
adamantyl(C.sub.1-C.sub.6)alkoxy, norbornyloxy,
1,1-di(hydroxymethyl)-2-h- ydroxyethoxy,
carboxy(C.sub.1-C.sub.6)alkoxy, 2,3-epoxypropyloxy, benzyloxy,
(C.sub.3-C.sub.8)cycloalkyloxy, NR.sub.xR.sub.y, or aryloxy;
R.sub.e is H, aryl or (C.sub.1-C.sub.6)alkyl; R.sub.f is hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkanoyl, phenyl or
benzyl; R.sub.g and R.sub.h are each independently selected from
the group consisting of hydrogen, (C.sub.1-C.sub.6)alkyl,
hydroxy(C.sub.1-C.sub.6)a- lkyl, adamantyl,
adamantyl(C.sub.1-C.sub.6)alkyl, amino(C.sub.1-C.sub.6)al- kyl,
aminosulfonyl, (C.sub.1-C.sub.6)alkanoyl, aryl and benzyl; or
R.sub.b and R.sub.c together with the nitrogen to which they are
attached form a pyrrolidino, piperidino, or morpholino radical; and
R.sub.x and R.sub.y are each independently hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkanoyl, aryl or benzyl;
wherein each aryl of Y, R.sub.a--R.sub.d, R.sub.g--R.sub.h,
R.sub.x, and R.sub.y may optionally be substituted by 1, 2, or 3
aminosulfonyl, carboxy, NR.sub.iR.sub.j, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxy, hydroxy, halo, nitro, cyano, mercapto,
carboxy, hydroxy(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkyl,
trifluoromethoxy, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkoxycarbonyl, (C.sub.1-C.sub.6)alkylthio, or
(C.sub.1-C.sub.6)alkanoyloxy; wherein R.sub.i and R.sub.j are each
independently hydrogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl- , phenyl, or benzyl; wherein any alkyl
can optionally be substituted with one or more polyethyleneimine or
poly(ethylene glycol); and wherein any alkyl can optionally be
interrupted with one or more polyethyleneimine or poly(ethylene
glycol); or a pharmaceutically acceptable salt thereof.
45. The method of claim 44 wherein the triterpenoid is
3-.beta.-acetoxy-19.alpha.H-19,28 lactone oleanan; allobetulin;
allobetulin-3-succinate; allobetulin-3-glycine ester; allobetulin
lactone; allobetulin lactone-3-acetate; allobetulin
lactone-3-phosphate; allobetulin-3-L-alanine;
allobetulin-3-L-valine; allobetulin-3-L-proline;
allobetulin-3-succinate; allobetulin-3-diglycolate;
allobetulin-3-glutarate; allobetulin-3-phthalate;
allobetulin-3-methylena- mine; allobetulin-3-ethanolamine;
allobetulin-3-ethanolamine hydrochloride; allobetulin-3-glycolate;
allobetulin-3-glutarate; allobetulin-28-glutarate;
allobetulin-3-methylamine HCl; allobetulin-3-phosphate;
allobetulin-3-(polyethylene glycol)-COOH (Mw=674); allobetulon;
allobetulon lactone-1-ene-2-ol; allobetulon
lactone-1-en-2-succinate; allobetulon-1-ene-2-ol;
allobetulon-1-ene-2-dig- lycolate; 3-allobetulon-1-ene-2-succinate;
or 3-allobetulon-1-ene-2-diglyc- olate.
46. The method of claim 44 wherein the triterpenoid is
3-.beta.-acetoxy-19.alpha.H-19,28 lactone oleanan; allobetulin;
allobetulin-3-glycine ester; allobetulin lactone-3-phosphate;
allobetulin-3-succinate; allobetulin-3-ethanolamine;
allobetulin-3-glutarate; allobetulin-28-glutarate;
allobetulin-3-methylamine HCl; allobetulin-3-phosphate;
allobetulon; allobetulon lactone-1-ene-2-ol;
3-allobetulon-1-ene-2-succinate; or ursolic acid.
47. The method of claim 44 wherein the contacting is in vitro.
48. The method of claim 44 wherein the contacting is in vivo.
Description
RELATED REFERENCES
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/969,556, filed Oct. 1, 2001, which claims priority from
U.S. Provisional Application No. 60/236510, filed on Sep. 29, 2000,
which applications are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Betulin is a pentacyclic triterpenoid derived from the outer
bark of paper birch trees (Betula paperifera). It can be present at
concentrations of up to about 24% of the bark of white birch. Merck
Index, twelfth edition, page 1236, 1996. Lupeol is a related
compound also found in birch bark and in other plant sources.
Lupeol is present at concentrations of about 1.5-3% of birch bark
and at up to about 8.2% in Canavalia ensiformis, a plant widespread
in the humid tropics of Asia, India, and Africa. Allobetulin is
another triterpenoid found in birch bark. A typical pulp mill that
processes birch produces enough bark waste to allow for the
inexpensive isolation of significant quantities of these
triterpenoids.
[0003] Several triterpenoids have been found to have utility. For
example, betulin and related compounds have been shown to have
anti-viral activity against herpes simplex virus. Carlson et al.,
U.S. Pat. No. 5,750,578.
[0004] Bacteria are very common pathogens of humans. Among the
bacterial species that cause serious disease are the gram negative
bacterium Escherichia coli and gram positive bacteria of the genus
Staphylococcus. Staphylococcus aureus is the most serious pathogen
of the Staphylococcus bacteria. It is estimated to causes 13% of
the 2 million hospital infections each year, and result in 80,000
deaths in the United States. Staphylococcal infections occur most
commonly in persons weakened by poor health or
immunodeficiency.
[0005] Antibiotic resistance of bacteria is a growing problem. New
agents active against bacteria are needed. A need particularly
exists for agents that will act against a range of species,
including gram-negative and gram-positive species. Ideally, new
agents would also be inexpensive to manufacture. New anti-bacterial
agents would be less expensive to manufacture if they were abundant
natural products or were easily synthesized from abundant natural
products.
SUMMARY OF THE INVENTION
[0006] The present invention provides a therapeutic method of
treating a mammal afflicted with a bacterial infection, the method
comprising administering to the mammal an effective antibacterial
amount of a triterpene of formula (I): 1
[0007] wherein
[0008] R.sub.1 is hydrogen or hydroxy;
[0009] R.sub.2 is a direct bond, carbonyl, oxy, thio, carbonyl oxy,
oxy carbonyl, (C.sub.6-C.sub.10)aryl, or (C.sub.1-C.sub.6)alkyl; or
(C.sub.1-C.sub.6)alkyl;
[0010] R.sub.3 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoy- l, Si(R).sub.3 wherein each R is H,
phenyl or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, or a
glycoside;
[0011] R.sub.4 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoyl, Si(R).sub.3 wherein each R is H, phenyl
or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, a glycoside, or
amino; or R.sub.4 and R.sub.5 together are oxo or (.dbd.NOH);
and
[0012] R.sub.5 is direct bond, carbonyl, oxy, thio, carbonyl oxy,
oxy carbonyl, (C.sub.6-C.sub.10)aryl, or (C.sub.1-C.sub.6)alkyl; or
R.sub.4 and R.sub.5 together are oxo or (.dbd.NOH);
[0013] wherein any alkyl can optionally be substituted with one or
more halo, hydroxy, (C.sub.6-C.sub.10)aryl, nitro, cyano,
(C.sub.1-C.sub.6)alkoxy, trifluoromethyl, polyethyleneimine,
poly(ethylene glycol), oxo, NR.sub.7R.sub.8, wherein R.sub.7 and
R.sub.8 are each independently hydrogen, (C.sub.1-C.sub.6)alkyl or
polyethyleneimine; --OP(.dbd.O)(OH).sub.2; or C(.dbd.O)OR.sub.9,
wherein R.sub.9 is hydrogen, (C.sub.1-C.sub.6)alkyl, or
polyethyleneimine;
[0014] each of the bonds represented by--is independently absent or
is present;
[0015] wherein any alkyl is optionally interrupted on carbon with
one or more oxy, thio, sulfinyl, sulfonyl, polyethyleneimine, or
poly(ethylene glycol);
[0016] wherein any alkyl is optionally partially unsaturated;
[0017] wherein any aryl can optionally be substituted with one or
more halo, hydroxy, nitro, cyano, (C.sub.1-C.sub.6)alkoxy,
trifluoromethyl, polyethyleneimine, poly(ethylene glycol), oxo,
NR.sub.7R.sub.8, wherein R.sub.7 and R.sub.8 are each independently
hydrogen, (C.sub.1-C.sub.6)alkyl or polyethyleneimine; or
C(.dbd.O)OR.sub.9, wherein R.sub.9 is hydrogen,
(C.sub.1-C.sub.6)alkyl, or polyethyleneimine;
[0018] or a pharmaceutically acceptable salt thereof.
[0019] The present invention also provides a therapeutic method of
treating a mammal afflicted with a bacterial infection, the method
comprising administering to the mammal an effective anti-bacterial
amount of a triterpene of formula (II): 2
[0020] wherein
[0021] one of R.sub.1 and R.sub.2 is --O--Y and the other is
hydrogen or (C.sub.1-C.sub.6)alkyl optionally substituted by
hydroxy, (C.sub.1-C.sub.6)alkoxy, halo, halo(C.sub.1-C.sub.6)alkoxy
or NR.sub.jR.sub.k wherein R.sub.j and R.sub.k are independently H,
(C.sub.1-C.sub.6)alkyl or (C.sub.1-C.sub.6)alkanoyl; or R.sub.1 and
R.sub.2 together are oxo (.dbd.O);
[0022] R.sub.3 is hydrogen, halo, carboxy, mercapto,
(C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl, or --O--Y;
[0023] R.sub.4 and R.sub.5 are each independently hydrogen,
(C.sub.1-C.sub.6)alkyl, or hydroxy(C.sub.1-C.sub.6)alkyl;
[0024] R.sub.6 is hydrogen or is absent when the adjacent--is a
bond;
[0025] R.sub.7 is hydrogen or (C.sub.1-C.sub.6)alkyl;
[0026] R.sub.8 is hydrogen, (C.sub.1-C.sub.6)alkyl, or
hydroxy(C.sub.1-C.sub.6)alkyl and R.sub.11 is hydrogen,
(C.sub.1-C.sub.6)alkyl, carboxy, or hydroxy(C.sub.1-C.sub.6)alkyl;
or R.sub.8 and R.sub.11 together are --O--C(.dbd.X)--;
[0027] R.sub.9 and R.sub.10, are each independently hydrogen or
(C.sub.1-C.sub.6)alkyl;
[0028] each of the bonds represented by--is independently absent or
is present;
[0029] X is two hydrogens, oxo (.dbd.O) or thioxo (.dbd.S);
[0030] each Y is independently H, aryl, P(O)(Cl).sub.2,
(C.sub.3-C.sub.8)cycloalkyl, adamantyl, --SO.sub.2R.sub.a
O.dbd.P(R.sub.b).sub.2, O.dbd.P(R.sub.c).sub.2OP(O)(R.sub.d)--,
Si(R.sub.e).sub.3, tetrahydropyran-2-yl, an amino acid, a peptide,
a glycoside, or a 1 to 10 membered branched or unbranched carbon
chain optionally comprising 1, 2, or 3 heteroatoms selected from
non-peroxide oxy, thio, and --N(R.sub.f)--; wherein said chain may
optionally be substituted on carbon with 1, 2, 3, or 4 oxo
(.dbd.O), hydroxy, carboxy, halo, mercapto, nitro,
--N(R.sub.g)(R.sub.h), (C.sub.3-C.sub.8)cycloalkyl- ,
(C.sub.3-C.sub.8)cycloalkyloxy, aryl, aryloxy, adamantyl,
adamantyloxy, hydroxyamino, trifluoroacetylamino, a glycoside, an
amino acid, or a peptide; and wherein said chain may optionally be
saturated or unsaturated (e.g. containing one, two, three or more,
double or triple bonds);
[0031] R.sub.a is (C.sub.1-C.sub.6)alkyl or aryl;
[0032] R.sub.b, R.sub.c, and R.sub.d are each independently
hydroxy, (C.sub.1-C.sub.6)alkoxy, hydroxy(C.sub.2-C.sub.6)alkoxy,
adamantyloxy, adamantyl(C.sub.1-C.sub.6)alkoxy, norbornyloxy,
1,1-di(hydroxymethyl)-2-h- ydroxyethoxy,
carboxy(C.sub.1-C.sub.6)alkoxy, 2,3-epoxypropyloxy, benzyloxy,
(C.sub.3-C.sub.8)cycloalkyloxy, NR.sub.xR.sub.y, or aryloxy;
[0033] R.sub.e is H, aryl or (C.sub.1-C.sub.6)alkyl;
[0034] R.sub.f is hydrogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl, phenyl or benzyl;
[0035] R.sub.g and R.sub.h are each independently selected from the
group consisting of hydrogen, (C.sub.1-C.sub.6)alkyl,
hydroxy(C.sub.1-C.sub.6)a- lkyl, adamantyl,
adamantyl(C.sub.1-C.sub.6)alkyl, amino(C.sub.1-C.sub.6)al- kyl,
aminosulfonyl, (C.sub.1-C.sub.6)alkanoyl, aryl and benzyl; or
R.sub.b and R.sub.c together with the nitrogen to which they are
attached form a pyrrolidino, piperidino, or morpholino radical;
and
[0036] R.sub.x and R.sub.y are each independently hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkanoyl, aryl or
benzyl;
[0037] wherein each aryl of Y, R.sub.a--R.sub.d, R.sub.g--R.sub.h,
R.sub.x, and R.sub.y may optionally be substituted by 1, 2, or 3
aminosulfonyl, carboxy, NR.sub.iR.sub.j, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxy, hydroxy, halo, nitro, cyano, mercapto,
carboxy, hydroxy(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkyl,
trifluoromethoxy, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkoxycarbo- nyl, (C.sub.1-C.sub.6)alkylthio, or
(C.sub.1-C.sub.6)alkanoyloxy; wherein R.sub.i and R.sub.j are each
independently hydrogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl, phenyl, or benzyl;
[0038] wherein any alkyl can optionally be substituted with one or
more polyethyleneimine or poly(ethylene glycol); and wherein any
alkyl can optionally be interrupted with one or more
polyethyleneimine or poly(ethylene glycol);
[0039] or a pharmaceutically acceptable salt thereof.
[0040] The present invention also provides a method of inhibiting
or killing a bacterium, the method comprising contacting the
bacterium with an effective antibacterial amount of a triterpene of
formula (I): 3
[0041] wherein
[0042] R.sub.1 is hydrogen or hydroxy;
[0043] R.sub.2 is a direct bond, carbonyl, oxy, thio, carbonyl oxy,
oxy carbonyl, (C.sub.6-C.sub.10)aryl, or
(C.sub.1-C.sub.6)alkyl;
[0044] R.sub.3 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoy- l, Si(R).sub.3 wherein each R is H,
phenyl or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, or a
glycoside;
[0045] R.sub.4 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoy- l, Si(R).sub.3 wherein each R is H,
phenyl or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, a glycoside, or
amino; or R.sub.4 and R.sub.5 together are oxo or (.dbd.NOH);
and
[0046] R.sub.5 is direct bond, carbonyl, oxy, thio, carbonyl oxy,
oxy carbonyl, (C.sub.6-C.sub.10)aryl, or (C.sub.1-C.sub.6)alkyl; or
R.sub.4 and R.sub.5 together are oxo or (.dbd.NOH);
[0047] wherein any alkyl can optionally be substituted with one or
more halo, hydroxy, (C.sub.6-C.sub.10)aryl, nitro, cyano,
(C.sub.1-C.sub.6)alkoxy, trifluoromethyl, polyethyleneimine,
poly(ethylene glycol), oxo, NR.sub.7R.sub.8, wherein R.sub.7 and
R.sub.8 are each independently hydrogen, (C.sub.1-C.sub.6)alkyl or
polyethyleneimine; --OP(.dbd.O)(OH).sub.2; or C(.dbd.O)OR.sub.9,
wherein R.sub.9 is hydrogen, (C.sub.1-C.sub.6)alkyl, or
polyethyleneimine;
[0048] each of the bonds represented by--is independently absent or
is present;
[0049] wherein any alkyl is optionally interrupted on carbon with
one or more oxy, thio, sulfinyl, sulfonyl, polyethyleneimine, or
poly(ethylene glycol);
[0050] wherein any alkyl is optionally partially unsaturated;
[0051] wherein any aryl can optionally be substituted with one or
more halo, hydroxy, nitro, cyano, (C.sub.1-C.sub.6)alkoxy,
trifluoromethyl, polyethyleneimine, poly(ethylene glycol), oxo,
NR.sub.7R.sub.8, wherein R.sub.7 and R.sub.8 are each independently
hydrogen, (C.sub.1-C.sub.6)alkyl or polyethyleneimine; or
C(.dbd.O)OR.sub.9, wherein R.sub.9 is hydrogen,
(C.sub.1-C.sub.6)alkyl, or polyethyleneimine;
[0052] or a pharmaceutically acceptable salt thereof.
[0053] The present invention also provides a method of inhibiting
or killing a bacterium, the method comprising contacting the
bacterium with an effective antibacterial amount of a triterpene of
formula (II): 4
[0054] wherein
[0055] one of R.sub.1 and R.sub.2 is --O--Y and the other is
hydrogen or (C.sub.1-C.sub.6)alkyl optionally substituted by
hydroxy, (C.sub.1-C.sub.6)alkoxy, halo, halo(C.sub.1-C.sub.6)alkoxy
or NR.sub.jR.sub.k wherein R.sub.j and R.sub.k are independently H,
(C.sub.1-C.sub.6)alkyl or (C.sub.1-C.sub.6)alkanoyl; or R.sub.1 and
R.sub.2 together are oxo (.dbd.O);
[0056] R.sub.3 is hydrogen, halo, carboxy, mercapto,
(C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.8)cycloalkyl, or --O--Y;
[0057] R.sub.4 and R.sub.5 are each independently hydrogen,
(C.sub.1-C.sub.6)alkyl, or hydroxy(C.sub.1-C.sub.6)alkyl;
[0058] R.sub.6 is hydrogen or is absent when the adjacent--is a
bond;
[0059] R.sub.7 is hydrogen or (C.sub.1-C.sub.6)alkyl;
[0060] R.sub.8 is hydrogen, (C.sub.1-C.sub.6)alkyl, or
hydroxy(C.sub.1-C.sub.6)alkyl and R.sub.11 is hydrogen,
(C.sub.1-C.sub.6)alkyl, carboxy, or hydroxy(C.sub.1-C.sub.6)alkyl;
or R.sub.8 and R.sub.11 together are --O--C(.dbd.X)--;
[0061] R.sub.9 and R.sub.10, are each independently hydrogen or
(C.sub.1-C.sub.6)alkyl;
[0062] each of the bonds represented by--is independently absent or
is present;
[0063] X is two hydrogens, oxo (.dbd.O) or thioxo (.dbd.S);
[0064] each Y is independently H, aryl, P(O)(Cl).sub.2,
(C.sub.3-C.sub.8)cycloalkyl, adamantyl, --SO.sub.2R.sub.a
O.dbd.P(R.sub.b).sub.2, O.dbd.P(R.sub.c).sub.2OP(O)(R.sub.d)--,
Si(R.sub.e).sub.3, tetrahydropyran-2-yl, an amino acid, a peptide,
a glycoside, or a 1 to 10 membered branched or unbranched carbon
chain optionally comprising 1, 2, or 3 heteroatoms selected from
non-peroxide oxy, thio, and --N(R.sub.f)--; wherein said chain may
optionally be substituted on carbon with 1, 2, 3, or 4 oxo
(.dbd.O), hydroxy, carboxy, halo, mercapto, nitro,
--N(R.sub.g)(R.sub.h), (C.sub.3-C.sub.8)cycloalkyl- ,
(C.sub.3-C.sub.8)cycloalkyloxy, aryl, aryloxy, adamantyl,
adamantyloxy, hydroxyamino, trifluoroacetylamino, a glycoside, an
amino acid, or a peptide; and wherein said chain may optionally be
saturated or unsaturated (e.g. containing one, two, three or more,
double or triple bonds);
[0065] R.sub.a is (C.sub.1-C.sub.6)alkyl or aryl;
[0066] R.sub.b, R.sub.c, and R.sub.d are each independently
hydroxy, (C.sub.1-C.sub.6)alkoxy, hydroxy(C.sub.2-C.sub.6)alkoxy,
adamantyloxy, adamantyl(C.sub.1-C.sub.6)alkoxy, norbornyloxy,
1,1-di(hydroxymethyl)-2-h- ydroxyethoxy,
carboxy(C.sub.1-C.sub.6)alkoxy, 2,3-epoxypropyloxy, benzyloxy,
(C.sub.3-C.sub.8)cycloalkyloxy, NR.sub.xR.sub.y, or aryloxy;
[0067] R.sub.e is H, aryl or (C.sub.1-C.sub.6)alkyl;
[0068] R.sub.f is hydrogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl, phenyl or benzyl;
[0069] R.sub.g and R.sub.h are each independently selected from the
group consisting of hydrogen, (C.sub.1-C.sub.6)alkyl,
hydroxy(C.sub.1-C.sub.6)a- lkyl, adamantyl,
adamantyl(C.sub.1-C.sub.6)alkyl, amino(C.sub.1-C.sub.6)al- kyl,
aminosulfonyl, (C.sub.1-C.sub.6)alkanoyl, aryl and benzyl; or
R.sub.b and R.sub.c together with the nitrogen to which they are
attached form a pyrrolidino, piperidino, or morpholino radical;
and
[0070] R.sub.x and R.sub.y are each independently hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkanoyl, aryl or
benzyl;
[0071] wherein each aryl of Y, R.sub.a--R.sub.d, R.sub.g--R.sub.h,
R.sub.x, and R.sub.y may optionally be substituted by 1, 2, or 3
aminosulfonyl, carboxy, NR.sub.iN.sub.j, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxy, hydroxy, halo, nitro, cyano, mercapto,
carboxy, hydroxy(C.sub.1-C.sub.6)alkyl, halo(C.sub.1-C.sub.6)alkyl,
trifluoromethoxy, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkoxycarbo- nyl, (C.sub.1-C.sub.6)alkylthio, or
(C.sub.1-C.sub.6)alkanoyloxy; wherein R.sub.i and R.sub.j are each
independently hydrogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkanoyl, phenyl, or benzyl;
[0072] wherein any alkyl can optionally be substituted with one or
more polyethyleneimine or poly(ethylene glycol); and wherein any
alkyl can optionally be interrupted with one or more
polyethyleneimine or poly(ethylene glycol);
[0073] or a pharmaceutically acceptable salt thereof.
[0074] The present invention provides a therapeutic method of
treating a mammal afflicted with a bacterial infection, the method
comprising administering to the mammal an effective anti-bacterial
amount of a triterpene of formula (I): 5
[0075] wherein
[0076] R.sub.1 is hydrogen or hydroxy;
[0077] R.sub.2 is a direct bond, carbonyl, oxy, thio, carbonyl oxy,
oxy carbonyl, (C.sub.6-C.sub.10)aryl, or
(C.sub.1-C.sub.6)alkyl;
[0078] R.sub.3 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoy- l, Si(R).sub.3 wherein each R is H,
phenyl or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, or a
glycoside;
[0079] R.sub.4 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoy- l, Si(R).sub.3 wherein each R is H,
phenyl or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, or a glycoside;
or R.sub.4 and R.sub.5 together oxo; and
[0080] R.sub.5 is direct bond, carbonyl, oxy, thio, carbonyl oxy,
oxy carbonyl, (C.sub.6-C.sub.10)aryl, or (C.sub.1-C.sub.6)alkyl; or
R.sub.4 and R.sub.5 together are oxo;
[0081] wherein any alkyl can optionally be substituted with one or
more halo, hydroxy, (C.sub.6-C.sub.10)aryl, nitro, cyano,
(C.sub.1-C.sub.6)alkoxy, trifluoromethyl, polyethyleneimine,
poly(ethylene glycol), oxo, NR.sub.7R.sub.8, wherein R.sub.7 and
R.sub.8 are each independently hydrogen, (C.sub.1-C.sub.6)alkyl or
polyethyleneimine; --OP(.dbd.O)(OH).sub.2; or C(.dbd.O)OR.sub.9,
wherein R.sub.9 is hydrogen, (C.sub.1-C.sub.6)alkyl, or
polyethyleneimine;
[0082] each of the bonds represented by--is independently absent or
is present;
[0083] wherein any alkyl is optionally interrupted on carbon with
one or more oxy, thio, sulfinyl, sulfonyl, polyethyleneimine, or
poly(ethylene glycol);
[0084] wherein any alkyl is optionally partially unsaturated;
[0085] wherein any aryl can optionally be substituted with one or
more halo, hydroxy, nitro, cyano, (C.sub.1-C.sub.6)alkoxy,
trifluoromethyl, polyethyleneimine, poly(ethylene glycol), oxo,
NR.sub.7R.sub.8, wherein R.sub.7 and R.sub.8 are each independently
hydrogen, (C.sub.1-C.sub.6)alkyl or polyethyleneimine; or
C(.dbd.O)OR.sub.9, wherein R.sub.9 is hydrogen,
(C.sub.1-C.sub.6)alkyl, or polyethyleneimine;
[0086] or a pharmaceutically acceptable salt thereof.
[0087] The present invention also provides a method of inhibiting
or killing a bacterium, the method comprising contacting the
bacterium with an effective antibacterial amount of a triterpene of
formula (I): 6
[0088] wherein
[0089] R.sub.1 is hydrogen or hydroxy;
[0090] R.sub.2 is a direct bond, carbonyl, oxy, thio, carbonyl oxy,
oxy carbonyl, (C.sub.6-C.sub.10)aryl, or
(C.sub.1-C.sub.6)alkyl;
[0091] R.sub.3 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoy- l, Si(R).sub.3 wherein each R is H,
phenyl or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, or a
glycoside;
[0092] R.sub.4 is hydrogen, hydroxy, (C.sub.1-C.sub.6)alkyl,
O.dbd.P(OH).sub.2, O.dbd.P(OH).sub.2OP(O)(OH)--,
(C.sub.1-C.sub.5)alkanoy- l, Si(R).sub.3 wherein each R is H,
phenyl or (C.sub.1-C.sub.6)alkyl, C(O)N(R).sub.2, benzyl, benzoyl,
tetrahydropyran-2-yl,
1-[(C.sub.1-C.sub.4)alkoxy](C.sub.1-C.sub.4)alkyl, or a glycoside;
or R.sub.4 and R.sub.5 together are oxo; and
[0093] R.sub.5 is direct bond, carbonyl, oxy, thio, carbonyl oxy,
oxy carbonyl, (C.sub.6-C.sub.10)aryl, or (C.sub.1-C.sub.6)alkyl; or
R.sub.4 and R.sub.5 together are oxo;
[0094] wherein any alkyl can optionally be substituted with one or
more halo, hydroxy, (C.sub.6-C.sub.10)aryl, nitro, cyano,
(C.sub.1-C.sub.6)alkoxy, trifluoromethyl, polyethyleneimine,
poly(ethylene glycol), oxo, NR.sub.7R.sub.8, wherein R.sub.7 and
R.sub.8 are each independently hydrogen, (C.sub.1-C.sub.6)alkyl or
polyethyleneimine; --OP(.dbd.O)(OH).sub.2; or C(.dbd.O)OR.sub.9,
wherein R.sub.9 is hydrogen, (C.sub.1-C.sub.6)alkyl, or
polyethyleneimine;
[0095] each of the bonds represented by--is independently absent or
is present;
[0096] wherein any alkyl is optionally interrupted on carbon with
one or more oxy, thio, sulfinyl, sulfonyl, polyethyleneimine, or
poly(ethylene glycol);
[0097] wherein any alkyl is optionally partially unsaturated;
[0098] wherein any aryl can optionally be substituted with one or
more halo, hydroxy, nitro, cyano, (C.sub.1-C.sub.6)alkoxy,
trifluoromethyl, polyethyleneimine, poly(ethylene glycol), oxo,
NR.sub.7R.sub.8, wherein R.sub.7 and R.sub.8 are each independently
hydrogen, (C.sub.1-C.sub.6)alkyl or polyethyleneimine; or
C(.dbd.O)OR.sub.9, wherein R.sub.9 is hydrogen,
(C.sub.1-C.sub.6)alkyl, or polyethyleneimine;
[0099] or a pharmaceutically acceptable salt thereof.
[0100] The invention provides novel compounds of formula (I) and
formula (II), intermediates for the synthesis of compounds of
formula (I) and formula (II), as well as methods of preparing
compounds of formula (I) and (II). The invention also provides
compounds of formula (I) and (II) that are useful as intermediates
for the synthesis of other useful compounds. The invention provides
for the use of compounds of formula (I) and formula (II) for the
manufacture of medicaments useful for the treatment of bacterial
infections in a mammal, such as a human.
DETAILED DESCRIPTION
[0101] The following definitions are used, unless otherwise
described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy,
alkenyl, etc. denote both straight and branched groups; but
reference to an individual radical such as "propyl" embraces only
the straight chain radical, a branched chain isomer such as
"isopropyl" being specifically referred to. Aryl denotes a phenyl
radical or an ortho-fused bicyclic carbocyclic radical having about
nine to ten ring atoms in which at least one ring is aromatic.
[0102] It will be appreciated by those skilled in the art that
compounds useful in the invention having a chiral center may exist
in and be isolated in optically active and racemic forms. Some
compounds may exhibit polymorphism. It is to be understood that the
present invention encompasses any racemic, optically-active,
polymorphic, or stereoisomeric form, or mixtures thereof, of a
compound useful in the invention, which possess the useful
properties described herein, it being well known in the art how to
prepare optically active forms (for example, by resolution of the
racemic form by recrystallization techniques, by synthesis from
optically-active starting materials, by chiral synthesis, or by
chromatographic separation using a chiral stationary phase) and how
to determine antibacterial activity using the standard tests
described herein, or using other similar tests which are well known
in the art.
[0103] Specific and preferred values listed below for radicals,
substituents, and ranges, are for illustration only; they do not
exclude other defined values or other values within defined ranges
for the radicals and substituents
[0104] Specifically, (C.sub.1-C.sub.6)alkyl can be methyl, ethyl,
propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl,
or hexyl; partially unsaturated (C.sub.2-C.sub.6)alkyl or
(C.sub.2-C.sub.6)alkenyl can be vinyl, 1-propenyl, 2-propenyl,
1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl,
3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,
or 5-hexenyl; (C.sub.1-C.sub.5)alkanoyl can be carbonyl, acetyl,
propanoyl, butanoyl, isopropanoyl, or pentenoyl;
(C.sub.1-C.sub.6)alkoxy can be methoxy, ethoxy, propoxy,
isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 2-pentoxy,
3-pentoxy, or hexyloxy; halo(C.sub.1-C.sub.6)alkoxy can be
trifluoromethyloxy, 2-chloroethyloxy, 3,3-dichloropropyloxy, or
4,4,4-trifluorobutyloxy; (C.sub.3-C.sub.8)cycloalkyl can be
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or
cyclooctyl; (C.sub.3-C.sub.8)cycloalkyloxy can be cyclopropyloxy,
cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, or
cyclooctyloxy; hydroxy(C.sub.1-C.sub.6)alkoxy can be
hydroxymethoxy, 1-hydroxyethoxy, 2-hydroxyethoxy, 1-hydroxypropoxy,
2-hydroxypropoxy, 3-hydroxypropoxy, 1-hydroxybutoxy,
4-hydroxybutoxy, 1-hydroxypentoxy, 5-hydroxypentoxy,
1-hydroxyhexoxy, or 6-hydroxyhexoxy; amino(C.sub.1-C.sub.6)alkyl
can be aminomethyl, 1-aminoethyl, 2-aminoethyl, 1-aminopropyl,
2-aminopropyl, 3-aminopropyl, 1-aminobutyl, 2-aminobutyl,
3-aminobutyl, 4-aminobutyl, 1-aminopentyl, 2-aminopentyl,
3-aminopentyl, 5-aminopentyl, 1-aminohexyl, 2-aminohexyl,
3-aminohexyl, or 6-aminohexyl; (C.sub.1-C.sub.6)alkoxycarbonyl can
be methoxycarbonyl, ethoxycarbonyl, propyloxycarbonyl,
isopropyloxycarbonyl, 2-methylpropyloxycarbonyl, butyloxycarbonyl,
pentyloxycarbonyl, or hexyloxycarbonyl;
(C.sub.1-C.sub.6)alkanoyloxy can be carbonyloxy, acetyloxy,
propanoyloxy, butanoyloxy, 2-methylpropanoyloxy,
2-methylbutanoyloxy, 3-methylbutanoyloxy, pentanoyloxy, or
hexanoyloxy.
[0105] "3-carboxypropenoyloxymethyl" refers to the group
--CH.sub.2OC(.dbd.O)CH.dbd.CHCOOH;
[0106] "aminoacetoxymethyl" refers to the group
--CH.sub.2OC(.dbd.O)CH.sub- .2NH.sub.2;
[0107] "(carboxymethoxy)acetoxymethyl" refers to the group
--CH.sub.2OC(.dbd.O)CH.sub.2OCH.sub.2COOH;
[0108] "4-carboxybutanoyloxymethyl" refers to the group
--CH.sub.2OC(.dbd.O)CH.sub.2CH.sub.2CH.sub.2COOH;
[0109] "2-carboxybenzoyloxymethyl" refers to the group 7
[0110] "butanoyloxymethyl" refers to the group
--CH.sub.2OC(.dbd.O)CH.sub.- 2CH.sub.2CH.sub.3;
[0111] "2-carboxybenzoyl" refers to the group 8
[0112] "2-amino-3-methylbutanoyl" refers to the group
--C(.dbd.O)CH.sub.2(NH.sub.2)CH.sub.2(CH.sub.3).sub.2;
[0113] "3-carboxypropenoyl" refers to the group
--C(.dbd.O)CH.dbd.CHCOOH;
[0114] "aminoacetyl" refers to the group
--C(.dbd.O)CH.sub.2NH.sub.2;
[0115] "4-carboxybutanoyl" refers to the group
--C(.dbd.O)CH.sub.2CH.sub.2- CH.sub.3COOH,
[0116] "(carboxymethoxy)acetyl" refers to the group
--C(.dbd.O)CH.sub.2OCH.sub.2COOH,
[0117] "3-(3,4-dihydroxyphenyl)propenoyl" refers to the group 9
[0118] "carboxymethylenethioacetyl" refers to the group
--C(.dbd.O)CH.sub.2SCH.sub.2COOH;
[0119] "3-carboxy-3-methylbutanoyl" refers to the group
--C(.dbd.O)CH.sub.2C(COOH)(CH.sub.3).sub.2;
[0120] "oxime" refers to the group (.dbd.NOH) that is substituted
directly on a carbon atom, thereby providing the group
C.dbd.N--OH.
[0121] The term "amino acid," comprises the residues of the natural
amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl,
Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in
D or L form, as well as unnatural amino acids (e.g. phosphoserine,
phosphothreonine, phosphotyrosine, hydroxyproline,
gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic
acid, statine, 1,2,3,4,-tetrahydroisoquinoli- ne-3-carboxylic acid,
penicillamine, ornithine, citruline, .alpha.-methyl-alanine,
para-benzoylphenylalanine, phenylglycine, propargylglycine,
sarcosine, and tert-butylglycine). The term also comprises natural
and unnatural amino acids bearing a conventional amino protecting
group (e.g. acetyl or benzyloxycarbonyl), as well as natural and
unnatural amino acids protected at the carboxy terminus (e.g. as a
(C.sub.1-C.sub.6)alkyl, phenyl or benzyl ester or amide; or as an
.alpha.-methylbenzyl amide). Other suitable amino and carboxy
protecting groups are known to those skilled in the art (See for
example, T. W. Greene, Protecting Groups In Organic Synthesis;
Wiley: New York, Third Edition, 1999, and references cited
therein). An amino acid can be linked to the remainder of a
compound of formula I or II through the carboxy terminus, the amino
terminus, or through any other convenient point of attachment, such
as, for example, through the sulfur of cysteine.
[0122] The term "peptide" describes a sequence of 2 to 25 amino
acids (e.g. as defined herein) or peptidyl residues. The sequence
may be linear or cyclic. For example, a cyclic peptide can be
prepared or may result from the formation of disulfide bridges
between two cysteine residues in a sequence. A peptide can be
linked to the remainder of a compound of formula I or II through
the carboxy terminus, the amino terminus, or through any other
convenient point of attachment, such as, for example, through the
sulfur of a cysteine. Preferably a peptide comprises 3 to 25, or 5
to 21 amino acids. Peptide derivatives can be prepared as disclosed
in U.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620.
[0123] Glycosides are formed by reacting mono-, di- and
polysaccharides with 1-2 hydroxyl groups of the compound of formula
(I) or formula (II), including glucose, glucuronic acid, mannose,
galactose, sorbase, ribose, maltose, sucrose, modified cellulosics,
dextrans, modified starches and the like. These derivatives can
advantageously exhibit improved water solubility over betulin
itself. See, Remington's Pharmaceutical Sciences, A. R. Gennaro,
ed., Mack Pub. Co. (18th ed., 1990) at pages 384-386. Glycoside
derivatives can be prepared as described in PCT Applications WO
96/34005 and 97/03995.
[0124] "Polyethyleneimine" refers to the group
(--NHCH.sub.2CH.sub.2--).su-
b.x[--N(CH.sub.2CH.sub.2NH.sub.2)CH.sub.2CH.sub.2--].sub.y.
Polyethyleneimine can be attached to a compound of formula I or II
through either of the nitrogen atoms marked with hash marks.
"Poly(ethylene glycol)" refers to the compound
H(OCH.sub.2CH.sub.2).sub.n- OH. It can be attached to a compound of
formula I or II through the terminal hydroxyl group.
[0125] The term "partially unsaturated" refers to a linear or
branched hydrocarbon having one or more carbon-carbon double
bonds.
[0126] The term "phosphono" refers to O.dbd.P(OH).sub.2--.
[0127] The term "direct bond" refers to a group being absent.
[0128] Combinations of substituents and/or variables are
permissible only if such combinations result in stable compounds.
By "stable compound" is meant herein a compound that is
sufficiently robust to survive isolation to a useful degree of
purity from a reaction mixture, and formulation into an efficacious
antibacterial agent.
[0129] The term "bacterium" or "bacteria" refers to any prokaryotic
organism.
[0130] The structure and carbon numbering of three exemplary
compounds of the present invention are shown below. 10
[0131] Specific values for compounds of formula (I) are as follows:
A specific value for the bond between carbons 1 and 2 is a single
bond.
[0132] Another specific value for the bond between carbons 1 and 2
is a double bond.
[0133] A specific value for R.sub.1 is hydrogen.
[0134] Another specific value for R.sub.1 is hydroxy.
[0135] A specific value for R.sub.2 is a direct bond.
[0136] A specific value for R.sub.3 is(C.sub.1-C.sub.6)alkyl;
wherein any alkyl can optionally be substituted with one or more
oxo, carboxy, amino, (C.sub.6-C.sub.10)aryl, or
--OP(.dbd.O)(OH).sub.2; any alkyl is optionally interrupted on
carbon with one or more oxy or thio; any alkyl is optionally
partially unsaturated; and any aryl can optionally be substituted
with one or more hydroxy or carboxy.
[0137] Another specific value for R.sub.3 is
3-carboxypropenoyloxymethyl, aminoacetoxymethyl,
(carboxymethoxy)acetoxymethyl, 4-carboxybutanoyloxymethyl,
2-carboxybenzoyloxymethyl, butanoyloxymethyl, or
--CH.sub.2OC(.dbd.O)OP(.dbd.O)(OH).sub.2.
[0138] A specific value for R.sub.4 is (C.sub.1-C.sub.6)alkyl;
wherein any alkyl can optionally be substituted with one or more
oxo, carboxy, amino, (C.sub.6-C.sub.10)aryl, or
--OP(.dbd.O)(OH).sub.2; any alkyl is optionally interrupted on
carbon with one or more oxy or thio; any alkyl is optionally
partially unsaturated; and any aryl can optionally be substituted
with one or more hydroxy or carboxy.
[0139] Another specific value for R.sub.4is 2-carboxybenzoyl,
2-amino-3-methylbutanoyl, 3-carboxypropenoyl, aminoacetyl,
4-carboxybutanoyl, (carboxymethoxy)acetyl,
3-(3,4-dihydroxyphenyl)propeno- yl, carboxymethylenethioacetyl,
3-carboxy-3-methylbutanoyl, amino, --P(.dbd.O)(OH).sub.2, oxo, or
(.dbd.NOH).
[0140] A specific value for R.sub.5 is oxy or a direct bond.
[0141] A specific group of compounds are compounds of formula (I)
wherein R.sub.1 is hydrogen or hydroxy; R.sub.2 is a direct bond;
R.sub.3 is (C.sub.1-C.sub.5)alkoxymethyl or hydroxymethyl; R.sub.4
is hydrogen, phosphono, sulfo, or (C.sub.1-C.sub.6)alkyl, and
R.sub.5 is oxy; or R.sub.4 is amino and R.sub.5 is a direct bond;
or R.sub.4and R.sub.5 together are oxo or (.dbd.NOH); wherein any
alkyl, or alkyl segment of an R group, is optionally interrupted on
carbon with one or more oxy, thio, or imido; wherein any alkyl, or
alkyl segment of an R group, can optionally be substituted with one
or more oxo, carboxy, amino, --OP(.dbd.O)(OH).sub.2, or phenyl;
wherein phenyl can optionally be substituted with one or more
hydroxy or carboxy.
[0142] Another specific group of compounds are compounds of formula
(I) wherein R.sub.1 is hydrogen or hydroxy; R.sub.2 is a direct
bond; R.sub.3 is 3-carboxypropenoyloxymethyl, aminoacetoxymethyl,
(carboxmethoxy)acetoxymethyl, 4-carboxybutanoyloxymethyl,
2-carboxybenzoyloxymethyl, butanoyloxymethyl, or
--CH.sub.2OC(.dbd.O)OP(.- dbd.O)(OH).sub.2; R.sub.4 is
2-carboxybenzoyl, 2-amino-3-methylbutanoyl, 3-carboxypropenoyl,
aminoacetyl, 4-carboxybutanoyl, (carboxymethoxy)acetyl,
3-(3,4-dihydroxyphenyl)propenoyl, carboxymethylenethioacetyl,
3-carboxy-3-methylbutanoyl, amino, --P(.dbd.O)(OH).sub.2, oxo, or
(.dbd.NOH); and R.sub.5 is oxy or a direct bond.
[0143] Another specific group of compounds of formula (I) is
betulin; betulin-3,28-diglycine; betulin-28-glycerol oxalate;
betulin-28-glycine; betulin-28-oxalate; betulin arabinose galactan;
betulin-3,28didiglycolate- ; betulin-3,28-diglycine;
betulin-3-maleate; betulin-3,28-di-(L-Glutamic acid r-benzylester)
ester; betulin-3,28-di-L-alanine; betulin-3,28-di-L-proline;
betulin-3,28-dioxalate; betulin-1-ene-2-ol;
betulin-3,28-diphenylalanine; betulin-3-28-dioxalate-polyethylene
amine; betulin-3,38-diphosphate; betulin-3-caffeate;
betulin-3,28-(3',3'-dimethy- l) glutarate; betulin-28-diglycolate;
betulin-28-glutarate; betulin-28-maleate; betulin-28-phthalate;
betulin-3,28-di(3',3'-dimethyl)- glutarate;
betulin-3,28-didiglycolate; betulin-3,28-di(thiodiglycolate);
betulin-3,28-diglutarate; betulin-3,28-dimaleate;
betulin-3,28-diglycolat- e; betulin-3,28-diphthalate;
betulin-3,28-di-L-phenylalanine; betulin-3,28--di-L-valine;
betulin-28-succinate; betulin-3,28-disuccinate- ;
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=1448);
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=906 crude);
betulin-3,28-di-(polyethylene glycol)-COOH (Mw=906 pure); betulinic
acid; betulon-1-ene-2-ol; betulin-3,28-(dipoly(ethylene glycol)bis
(carboxymethylester); allobetulin-3,28-(dipoly(ethylene glycol)bis
(carboxymethyl ester); hederin hydrate; lupeol; lupeol-3-glutarate;
lupeol-3-succinate; lupeol-3-thiodiglycolate; lupeol-3-phthalate;
lupeol-3-succinate; oleanolic acid; ursolic acid; or uvaol.
[0144] Another specific group of compounds of formula (I) is
betulin; betulin-28-glycerol oxalate; betulin-28-oxalate; betulin
arabinose galactan; betulin-3,28-didiglycolate;
betulin-3,28-diglycine; betulin-3,28-di-(L-glutamic acid
.gamma.-benzylester) ester; betulin3,28-di-L-proline ester;
betulin-3,28-dioxalate; betulin-1-ene-2-ol;
betulin-3,28-dioxalate-polyethylene amine;
betulin-3,28-diphosphate; betulin-3-caffeate;
betulin-28-diglycolate; betulin-28-glutarate; betulin-28-maleate;
betulin-28-phthalate; betulin-3,28-dithiodiglycolate;
betulin-3,28-diglutarate; betulin-3,28-dimaleate;
betulin-3,28-diglycolate; betulin-3,28-diphthalat- e;
betulin-3,28-di-L-phenylalanine; betulin-di-L-valine;
betulin-28-succinate; betulin-3,28-disuccinate;
betulin-3,28-di-(polyethy- lene glycol)-COOH (Mw=906 pure);
betulinic acid; betulon-1-ene-2-ol; betulin-3,28-(dipoly(ethylene
glycol)bis (carboxymethylester); hederin hydrate;
lupeol-3-glutarate; lupeol-3-succinate; lupeol-3-thiodiglycolate- ;
lupeol-3-phthalate; oleanolic acid; or uvaol.
[0145] Another specific group of compounds of formula (I) is
betulin-3-caffeate; betulin-28-diglycolate;
betulin-3,28-diglutarate; betulin-3,28-diglycine;
betulin-3,28-didiglycolate; betulin-3,28-dimaleate;
betulin-3,28-diphosphate; betulin-3,28-diphthalat- e;
betulin-3,28-di-L-valine; lupeol; lupeol-3-amine;
lupeol-3-(3',3'-dimethyl)succinate; lupeol-3-maleate;
lupeol-3-phosphate; lupeol-3-thiodiglycolate; lupenone;
lupenon-1,2-ene-2-ol; or lupenon-3-oxime.
[0146] A specific group of compounds of formula (II) is
3-.beta.-acetoxy-19.alpha.H-19,28 lactone oleanan; allobetulin;
allobetulin-3-succinate; allobetulin-3-glycine ester; allobetulin
lactone; allobetulin lactone-3-acetate; allobetulin
lactone-3-phosphate; allobetulin-3-L-alanine;
allobetulin-3-L-valine; allobetulin-3-L-proline;
allobetulin-3-succinate; allobetulin-3-diglycolate;
allobetulin-3-glutarate; allobetulin-3-phthalate;
allobetulin-3-methylena- mine; allobetulin-3-ethanolamine;
allobetulin-3-ethanolamine hydrochloride; allobetulin-3-glycolate;
allobetulin-3-glutarate; allobetulin-28-glutarate;
allobetulin-3-methylamine HCl; allobetulin-3-phosphate;
allobetulin-3-(polyethylene glycol)-COOH (Mw=674); allobetulon;
allobetulon lactone-1-ene-2ol; allobetulon
lactone-1-en-2-succinate; allobetulon-1-ene-2-ol;
allobetulon-1-ene-2-dig- lycolate; 3-allobetulon-1-ene-2-succinate;
or 3-allobetulon-1-ene-2-diglyc- olate.
[0147] Another specific group of compounds of formula (II) are
3-.beta.-acetoxy-19.alpha.H-19,28 lactone oleanan; allobetulin;
allobetulin-3-glycine ester; allobetulin lactone-3-phosphate;
allobetulin-3-succinate; allobetulin-3-ethanolamine;
allobetulin-3-glutarate; allobetulin-28-glutarate;
allobetulin-3-methylamine HCl; allobetulin-3-phosphate;
allobetulon; allobetulon lactone-l-ene-2-ol;
3-allobetulon-1-ene-2-succinate; or ursolic acid.
[0148] A specific method of the invention is the method of treating
a mammal afflicted with a bacterial infection comprising
administering to the mammal an effective anti-bacterial amount of a
compound of formula (I) or formula (II), wherein the bacterial
infection is caused by Escherichia coli, Staphylococcus sp.,
Enterococcus faecalis, or a combination thereof.
[0149] Another specific method of the invention is the method of
treating a mammal afflicted with a bacterial infection comprising
administering to the mammal an effective anti-bacterial amount of a
compound of formula (I) or formula (II), wherein the bacterial
infection is caused by Staphylococcus aureus.
[0150] Another specific method of the invention is the method of
inhibiting or killing a bacterium or bacteria comprising contacting
the bacterium with an effective antibacterial amount of a
triterpene of formula (I) or formula (II), wherein the bacterium is
Escherichia coli, Staphylococcus sp., Enterococcus faecalis, or a
combination thereof.
[0151] Another specific method of the invention is the method of
inhibiting or killing a bacterium or bacteria comprising contacting
the bacterium with an effective antibacterial amount of a
triterpene of formula (I) or formula (II) wherein the bacterium is
Staphylococcus aureus.
[0152] Another specific method of the invention is the method of
inhibiting or killing a bacterium or bacteria comprising contacting
the bacterium with an effective antibacterial amount of a
triterpene of formula (I) wherein the contacting is in vivo.
[0153] Another specific method of the invention is the method of
inhibiting or killing a bacterium or bacteria comprising contacting
the bacterium with an effective antibacterial amount of a
triterpene of formula (I) wherein the contacting is in vitro.
[0154] Another specific method of the invention is the method of
inhibiting or killing a bacterium or bacteria comprising contacting
the bacterium with an effective antibacterial amount of a
triterpene of formula (II) wherein the contacting is in vivo.
[0155] Another specific method of the invention is the method of
inhibiting or killing a bacterium or bacteria comprising contacting
the bacterium with an effective antibacterial amount of a
triterpene of formula (II) wherein the contacting is in vitro.
[0156] Specific triterpenes of formula (I) having antibacterial
activity are shown below in Table 1 below.
1TABLE 1 Specific compounds of formula (I) having anti-bacterial
activity. Name R.sub.1 R.sub.2 R.sub.3 R.sub.5 R.sub.4 Active
against Betulin-3- caffeate H -- 11 --O-- 12 E. coli Betulin-28- H
-- --CH.sub.2OC(.dbd.O)CH.sub.2OCH.sub.2COOH --O-- H E. coli
diglycolate Betulin-3,28- H -- --CH.sub.2OC(.dbd.O)CH.sub.2CH.sub.-
2CH.sub.2COOH --O-- --C(.dbd.O)CH.sub.2CH.sub.2CH.sub.2COOH E. coli
diglutarate Betulin-3,28- H -- --CH.sub.2OC(.dbd.O)CH.sub.2NH.sub.-
2 --O-- --C(.dbd.O)CH.sub.2NH.sub.2 E. coli diglycine Betulin-3,28-
H -- --CH.sub.2OC(.dbd.O)CH.sub.2OCH.sub.2COOH --O--
--C(.dbd.O)CH.sub.2OCH.sub.2COOH E. coli didiglycolate
Betulin-3,28- H -- --CH.sub.2OC(.dbd.O)CH.dbd.CHCOOH --O--
--C(.dbd.O)CH.dbd.CHCOOH S. aureus, dimaleate S. aureus methicillin
resistant Betulin-3,28- H -- --CH.sub.2O--P(.dbd.O)(OH).sub.2 --O--
--P(.dbd.O)(OH).sub.2 E. coli diphosphate Betulin-3,28- diphthalate
H -- 13 --O-- 14 S. aureus Betulin-3,28-di- H --
--CH.sub.2OC(.dbd.O)-- --O-- --C(.dbd.O)-- S. aureus L-valine
CH.sub.2(NH.sub.2)CH(CH.sub.3).- sub.2
CH.sub.2(NH.sub.2)CH(CH.sub.3).sub.2 Lupeol H -- --CH.sub.3 --O-- H
S. aureus, S. epidermidis Enterococcus faecalis Lupeol-3-amine H --
--CH.sub.3 -- H S. aureus, E. faecalis Lupeol-3-(3',3', H --
--CH.sub.3 --O-- --CH.sub.2OC(.dbd.O)CH.sub.2C(CH.sub.3).sub.2COOH
S. aureus dimethyl) succinate Lupeol-3- H -- --CH.sub.3 --O--
--P(.dbd.O)(OH).sub.2 S. aureus, phosphate E. faecalis Lupeol-3- H
-- --CH.sub.3 --O-- --C(.dbd.O)CH.sub.2SCH.sub.2COOH S. aureus,
thiodiglycolate S. epidermidis, E. faecalis Lupeol-3- H --
--CH.sub.3 --O-- --C(.dbd.O)CH.dbd.CHCOOH S. aureus, maleate S.
epidermidis, E. faecalis Lupenone H -- --CH.sub.3 (.dbd.O) S.
aureus, E. faecalis Lupenon-3- H -- --CH.sub.3 -- (.dbd.NOH) S.
aureus, oxime E. faecalis Lupenon-1,2- H -- --CH.sub.3 -- (.dbd.O)
S. aureus, ene-2-ol S. epidermidis, E. faecalis In addition,
lupenon-1,2-ene-2-ol has a double bond between carbons 1 and 2. The
other compounds in Table 1 have a single bond at that position.
[0157] Processes for preparing compounds of formula (I) and formula
(II) are provided as further embodiments of the invention and are
illustrated by the following procedures in which the meanings of
the generic radicals are as given above unless otherwise qualified.
Specifically, the compounds of formula (I) or formula (II) can be
prepared from convenient starting materials, employing procedures
(e.g., reagents and reaction conditions) known to those of skill in
the art. For example, suitable reagents and reaction conditions are
disclosed, e.g., in Advanced Organic Chemistry, Part B: Reactions
and Synthesis, Second Edition, Carey and Sundberg (1983); Advanced
Organic Chemistry, Reactions, Mechanisms, and Structure, Second
Edition, March (1977); Greene, T. W.; Wutz, P. G. M. Protecting
Groups In Organic Synthesis, Second Edition, 1991, New York, John
Wiley & sons, Inc.; and Comprehensive Organic Transformations,
Second Edition, Larock (1999). Additionally, specific exemplary
procedures are shown in the examples herein below.
[0158] In cases where compounds are sufficiently basic or acidic to
form stable nontoxic acid or base salts, administration of the
compounds as salts may be appropriate. Examples of pharmaceutically
acceptable salts are organic acid addition salts formed with acids
which form a physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorbate, .alpha.-ketoglutarate, and
.alpha.-glycerophosphate. Suitable inorganic salts may also be
formed, including hydrochloride, sulfate, nitrate, bicarbonate, and
carbonate salts.
[0159] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made.
[0160] The compounds of formula I or II can be formulated as
pharmaceutical compositions and administered to a mammalian host,
such as a human patient in a variety of forms adapted to the chosen
route of administration, i.e., orally or parenterally, by
intravenous, intramuscular, topical or subcutaneous routes.
[0161] Thus, the present compounds may be systemically
administered, e.g., orally, in combination with a pharmaceutically
acceptable vehicle such as an inert diluent or an assimilable
edible carrier. They may be enclosed in hard or soft shell gelatin
capsules, may be compressed into tablets, or may be incorporated
directly with the food of the patient's diet. For oral therapeutic
administration, the active compound may be combined with one or
more excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. Such compositions and preparations should contain at
least 0.1% of active compound. The percentage of the compositions
and preparations may, of course, be varied and may conveniently be
between about 2 to about 60% of the weight of a given unit dosage
form. The amount of active compound in such therapeutically useful
compositions is such that an effective dosage level will be
obtained.
[0162] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound may be incorporated into sustained-release
preparations and devices.
[0163] The active compound may also be administered intravenously
or intraperitoneally by infusion or injection. Solutions of the
active compound or its salts can be prepared in water, optionally
mixed with a nontoxic surfactant. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, triacetin, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0164] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form should be
sterile, fluid and stable under the conditions of manufacture and
storage. The liquid carrier or vehicle can be a solvent or liquid
dispersion medium comprising, for example, water, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the formation of liposomes, by the
maintenance of the required particle size in the case of
dispersions or by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, buffers or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the use in
the compositions of agents delaying absorption, for example,
aluminum monostearate and gelatin.
[0165] Sterile injectable solutions are prepared by incorporating
the active compound in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0166] For topical administration, the present compounds may be
applied in pure form, i.e., when they are liquids. However, it will
generally be desirable to administer them to the skin as
compositions or formulations, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0167] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the present compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[0168] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0169] Examples of useful dermatological compositions which can be
used to deliver the compounds of formula I or II to the skin are
known to the art; for example, see Jacquet et al. (U.S. Pat. No.
4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S.
Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
[0170] Useful dosages of the compounds of formula I or II can be
determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949.
[0171] Generally, the concentration of the compound(s) of formula I
or II in a liquid composition, such as a lotion, will be from about
0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration
in a semi-solid or solid composition such as a gel or a powder will
be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
[0172] The amount of the compound, or an active salt or derivative
thereof, required for use in treatment will vary not only with the
particular salt selected but also with the route of administration,
the nature of the condition being treated and the age and condition
of the patient and will be ultimately at the discretion of the
attendant physician or clinician.
[0173] In general, however, a suitable dose will be in the range of
from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75
mg/kg of body weight per day, such as 3 to about 50 mg per kilogram
body weight of the recipient per day, preferably in the range of 6
to 90 mg/kg/day, most preferably in the range of 15 to 60
mg/kg/day.
[0174] The compound is conveniently administered in unit dosage
form; for example, containing 5 to 1000 mg, conveniently 10 to 750
mg, most conveniently, 50 to 500 mg of active ingredient per unit
dosage form.
[0175] Ideally, the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.5 to about 75 .mu.M, preferably, about 1 to 50 .mu.M, most
preferably, about 2 to about 30 .mu.M. This may be achieved, for
example, by the intravenous injection of a 0.05 to 5% solution of
the active ingredient, optionally in saline, or orally administered
as a bolus containing about 1-100 mg of the active ingredient.
Desirable blood levels may be maintained by continuous infusion to
provide about 0.01-5.0 mg/kg/hr or by intermittent infusions
containing about 0.4-15 mg/kg of the active ingredient(s).
[0176] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[0177] The ability of a compound of the invention to act as an
antibacterial agent may be determined using pharmacological models
which are well known to the art, including the tests described in
the Examples below.
[0178] The compounds of the invention may be also be useful as
pharmacological tools for the further investigation of the
mechanism of their antibacterial action.
[0179] The compounds of the invention can also be administered in
combination with other therapeutic agents that are effective to
treat bacterial infections, or to inhibit or kill a bacteria.
[0180] The system used to name the compounds of the invention will
be clear to one of skill in the art based on the following
examples. Names generally consist of the base structure, e.g.,
betulin, allobetulin, or lupeol, followed by a substituent. For
example, betulin-28-succinate, with the structure shown in Example
1, consists of a succinic acid molecule esterified to the hydroxyl
at carbon 28 of betulin. If no number is given for the substituent,
the substitent is attached to the hydroxyl at carbon 3 on the base
structure.
[0181] Betulin-3-glycerol oxalate is a compound of formula (I),
wherein R.sub.4 and R.sub.5 together are hydroxyl, R.sub.2 and
R.sub.3 together are
--OC(.dbd.O)C(.dbd.O)OCH.sub.2CH(OH)CH.sub.2OH, and R.sub.1 is
hydrogen. Betulin-1-ene-2-ol is a compound of formula (I), wherein
the bond between carbons 1 and 2 is a double bond, R.sub.1 is
hydroxyl, R.sub.2 and R.sub.3 together are hydroxymethyl, and
R.sub.4 and R.sub.5 together are oxo. Uvaol is a compound of
formula (II), wherein R.sub.10 is methyl, R.sub.9 is hydrogen,
R.sub.8 is methyl, R.sub.7 is hydrogen, R.sub.11 is hydroxymethyl,
R.sub.6 is absent and the bond between carbons 12 and 13 is double,
R.sub.3 is hydrogen, R.sub.4 and R.sub.5 are methyl, R.sub.2 is
hydrogen, and R.sub.1 is hydroxy. Oleanolic acid has the same
structure as uvaol, except it has a carboxy at R.sub.11 instead of
hydroxymethyl. The structure of hederin hydrate is disclosed at
page 871 of the Aldrich Chemical Co. 2000-2001 catalog. The
structure of other named compounds can be found in standard sources
such as the Merck Index. "Betulin arabinose galactan" refers to
betulin in a solution of arabino-galactan.
[0182] Unless otherwise stated, amino acid substituents are
attached to the compounds of the invention through their carboxyl
groups via ester linkages. Thus, betulin-3,28-diglycine is the same
compound as betulin-3,28-diglycine ester.
[0183] The invention will now be illustrated by the following
non-limiting Examples.
EXAMPLES
Example 1
Betulin-28-succinate
[0184] 15
[0185] Betulin-28-succinate
[0186] m=0.200 g
[0187] C.sub.34H.sub.54O.sub.5
[0188] Exact Mass: 542.40
[0189] Mol. Wt.: 542.79
[0190] C, 75.23; H, 10.03; O, 14.74
[0191] Place Betulin 1.00 g (1 equivalent) along with Succinic
anhydride 0.249 g (1.1 equivalent) and imidazole 0.462 g (3
equivalent) in a 25 ml flask. Add 20 ml dried dichloromethane, stir
and reflux for 24 hours. After the reaction completes, add 10 ml 3%
HCl, shake gently. The pH should be 2. Separate the organic part.
Use dichloromethane (3.times.5 ml) to extract the water layer.
Combine the organic part and use 3% HCI (2.times.10 ml) to wash it.
Use Na.sub.2SO.sub.4 (anhy.) to dry the organic part. Evaporate the
solvent, get white powder 1.10 g. Use small amount of acetone to
tritrate the white product. After drying, get 0.90 g white granular
solid with yield 73.2%. M.P.: 234.1-235.5.degree. C.; 1R (KBr):
3355.76, 2953.19, 1734.29, 1708.63, 1264.63, 1174.11 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3): .delta.4.69 (S, 1H), 4.59 (S, 1H), 4.32
(D, J=11.1 Hz, 1H), 3.91 (D, J=11.1 Hz), 3.22 (M, 1H), 2.68 (M,
4H), 2.44 (M, 1H), 1.68 (S, 3H), 0.76, 0.82, 0.97, 1.02 (All S,
4.times.3H), 0.71-2.1 (complex, 28H); .sup.13C NMR (CDCl.sub.3):
172.43, 167.96, 145.61, 105.38, 74.56, 58.71, 50.80, 45.88, 44.32,
43.21, 41.94, 38.21, 36.38, 34.36, 34.21, 33.12, 32.66, 30.01,
29.68, 25.23, 25.08, 24.57, 24.37, 23.50, 22.87, 22.54, 20.71,
16.30, 14.67, 13.79, 11.62, 11.54, 10.89, 10.30;
Example 2
Betulin-3,28-disuccinate
[0192] 16
[0193] Betulin-3,28-disuccinate
[0194] m=0.200 g
[0195] C.sub.38H.sub.58O.sub.8
[0196] Exact Mass: 642.41
[0197] Mol Wt.: 642.86
[0198] C, 71.00; H, 9.09; O, 19.91
[0199] Place 0.5 g Betulin along with 0.34 g succinic anhydride and
0.46 g imidazole in a 25 ml flask. Add 15 ml CH.sub.2Cl.sub.2
(dried) and reflux for 12 hours. Add 10 ml 3% HCl, separate the
organic part, use CH.sub.2Cl.sub.2 (3.times.5 ml) to wash, and
combine the organic parts. Use 3% HCl (2.times.10 ml) wash the
organic part, then use Na.sub.2SO.sub.4 to dry it. Evaporating the
solvent gives 0.73 g yellow powder. Useing CHCl.sub.3-hexane to
crystalize gives 0.65 g yellow powder. Or stiring the solid with 3%
HCl in the warm condition for 12 hours, followed by filtration and
drying, gives 0.60 g powder with yield 82.5%. M.P. (decomp.)
116.1-117.8.degree. C.; IR(KBr): 2954.83, 1726.44, 1169.46
cm.sup.-1; .sup.1H NMR (CDCl.sub.3): 4.69 (S, 1H), 4.59 (S, 1H),
4.51 (M, 1H), 4.31 (D, J=11.4 Hz, 1H), 3.88 (D, J=10.8 Hz, 1H),
2.67 (M, 8H), 2.44 (M, 1H), 1.68 (S, 3H), 1.3, 0.98, 0.85, 0.86,
0.79 (all S, 5.times.3H), 1.06-2.1 (complex, 24H); .sup.13C NMR
(CDCl.sub.3): 173.73, 173.65, 167.98, 167.37, 145.63, 105.44,
77.09, 58.72, 50.91, 45.77, 44.30, 43.25, 41.94, 38.22, 36.40,
33.88, 33.36, 33.10, 32.56, 29.97, 29.61, 25.18, 24.87, 24.61,
23.41, 22.54, 20.67, 19.13, 16.34, 14.65, 13.69, 12.04, 11.68,
11.55, 1031;
Example 3
Betulin-28-phthalate
[0200] 17
[0201] Betulin-28-phthalate
[0202] C.sub.38H.sub.54O.sub.5
[0203] Exact Mass: 590.40
[0204] Mol. Wt.: 590.83
[0205] C, 77.25; H, 9.21; O, 13.54
[0206] Place Betulin 1 g (1 equivalent) and imidazole 0.31 g (4
equivalents) with phthalic anhydride 0.35 g (1.05 equivalents) in a
15 mL flask. Add 5 mL of 1-methyl-2-pyrrolidinone and stir at room
temperature for 48 hours. Pour the mixture into the water with
strong stirring and adjust pH around 3. Stir for 2-3 hours. All the
chunks should become small particles. After filtration, use water
to wash three times, and then dry it in the oven. Get 1.25 g white
solid. Use ethyl acetate: Hexane (1:4) to elute the product from
the silica gel column and get 0.69 g white prism solid. Yield is
51.5%. M.P.: 205.2-206.9.degree. C. JR(cm.sup.-1): 3500.0, 2957.0,
2876.4, 1719.7, 1458.2, 1386.5, 1289.8, 1136.8, 1072.4; .sup.1H NMR
(CDCl.sub.3, ppm): 7.93 (D, 1H, J=6.9 Hz), 7.73 (D, 1H, J=6.6 Hz),
7.59 (M, 2H), 4.71 (S, 1H), 4.60 (S, 1H), 4.53 (D, 1H, J=8.4 Hz),
4.14 (D, 1H, J=10.8 Hz), 3.22 (M, 1H), 2.51 (M, 1H), 1.69 (S, 3H),
1.05, 0.97, 0.95, 0.82, 0.76 (all S, 5.times.3H), 2.2-0.6 (Complex,
26H); .sup.13C NMR (CDCl.sub.3, ppm): 171.85, 169.03, 150.46,
133.90, 132.46, 131.13, 130.35, 129.19, 110.25, 79.43, 66.23,
64.90, 55.60, 50.69, 49.26, 48.08, 46.83, 43.07, 41.12, 39.18,
39.04, 38.01, 37.48, 34.92, 34.41, 30.11, 29.92, 28.34, 27.69,
27.37, 25.54, 21.14, 19.53, 18.58, 16.45, 16.38, 15.75, 15.61,
15.14.
Example 4
Lupeol-3-phthalate
[0207] 18
[0208] Lupeol-3-phthalate
[0209] C.sub.38H.sub.54O.sub.4
[0210] Exact Mass: 574.40
[0211] Mol. Wt.: 574.83
[0212] C, 79.40; H, 9.47; 0, 11.13
[0213] Place Lupeol 0.100 g and imidazole 0.96 g with 0.069 g
phthalic anhydride in a 25 mL flask, add dried dichloromethane 10
mL and reflux for 24 hours. Then use 3% HCl (3.times.5 mL) to wash
the organic part, which is followed by drying with, sodium sulfate
(anhy.). After evaporate the solvent, receive white powder, which
is followed by stirring with 3% HCl for 12 hours. Then filter and
dry the white solid in the oven. This results in 0.128 g white
product with 94.8% yield. M.P.: 160.2-162.1.degree. C. .sup.1H NMR
(CDCl.sub.3, ppm): 7.96 (D, 1H, J-6.9 Hz), 7.79 (D, 1H, J=6.0 Hz),
7.63 (M, 2H), 4.79 (M, 1H), 4.74 (S, 1H), 4.63 (S, 1H), 2.44 (M,
1H), 1.74 (S, 3H), 1.46, 1.08 1.00, 0.93, 0.91, 0.84 (S,
6.times.3H), 2.1-0.7 (Complex, 25H). .sup.13C NMR (CDCl.sub.3,
ppm): 172.05, 168.19, 151.29, 133.74, 132.25, 131.23, 130.79,
130.35, 129.37, 109.73, 83.59, 55.91, 50.73, 48.67, 48.37, 43.37,
43.21, 41.24, 40.37, 38.81, 38.43, 37.48, 35.94, 34.59, 30.21,
28.40, 27.81, 25.47, 23.51, 21.35, 19.66, 18.56, 18.37, 16.99,
16.51, 16.35, 14.91.
Example 5
Lupeol-3-succinate
[0214] 19
[0215] Lupeol-3-succinate
[0216] C.sub.34H.sub.54O.sub.4
[0217] Exact Mass: 526.40
[0218] Mol. Wt.: 526.79
[0219] C, 77.52; H, 10.33; O, 12.15
[0220] Place Lupeol 100 mg (1 equivalence) and succinic anhydride
0.070 g (3 equivalence) with imidazole 0.016 g (1 equivalence) in a
25 mL flask. Add dried dichloromethane 10 mL, then reflux for 48
hours. After the reaction is done, add sodium bicarbonate saturated
water solution 10 mL, separate the organic part, and extract the
water phase with dichloromethane (3.times.5 mL). Then use 3% HCl
(3.times.10 mL) to wash the organic part, which is followed by
drying with sodium sulfate (anhy.). Evaporating the solvent gives a
white powder, which is stirred with 3% HCl 15 mL overnight, which
is followed by filtration and drying in the oven. 0.12 g white
powder is obtained with 97.6% yield. M.P.: 224.7-226.3.degree. C.
.sup.1H NMR; 4.69 (S, 1H), 4.57 (S, 1H) 4.501 (M, 1H), 2.66 (M,
4H), 2.39 (M, 1H), 1.68 (S, 3H), 1.36, 1.03, 0.94, 0.85, 0.83, 0.79
(S, 6.times.3H), 1.8-0.7 (Complex, 25H), .sup.13C NMR (CDCl.sub.3,
ppm): 174.13, 168.54, 147.54, 105.94, 78.14, 51.94, 46.87, 44, 83,
44358, 39.56, 39.39, 37.40, 36.57, 34.91, 34.59, 34.41, 33.62,
32.13, 30.75, 26.39, 26.00, 25.72, 24.47, 23.99, 21.64, 20.21,
17.51, 15.86, 14.75, 14.58, 13.09, 12.74, 12.54, 11.09.
Example 6
3-Allobetulon-1-en-2-succinate
[0221] 20
[0222] 3-Allobetulon-1-en-2-succinate
[0223] C.sub.34H.sub.50O.sub.6
[0224] Exact Mass: 554.36
[0225] Mol. Wt.: 554.76
[0226] C, 73.61; H, 9.08; O, 17.30
[0227] Place 0.5 g 3-Allobetulon-1-en-2-ol (1 equivalent) and 0.33
g succinic anhydride (3 equivalents) with 0.13 g
4-(dimethylamino)-pyridine (1 equivalent) in a 25 mL flask. Add 10
mL acetonitrile and reflux for 48 hours, which is followed by
adding 15 mL chloroform. Use 10 mL 3% HCl to wash the organic part
three times, which is followed by drying with sodium sulfate
(anhy.). Evaporating the solvent gives 0.55 g crude product. Use
silica gel column to separate the crude product with solvent
hexane: diethyl ether (3:1), which results in 0.303 g white
amorphous solid with yield 49.7%. M.P.: 178.1-180.4.degree. C. IR
(cm.sup.-1); 2944.6, 2866.3, 1764.8, 1695.4, 1139.5; .sup.1H NMR
(CDCl.sub.3, ppm): 6.83 (S, 1H) 3.80 (D, 1H, J=7.8 Hz), 3.59 (S
1H), 3.49 (D, 1H, J=7.8 Hz), 2.82 (M 4H), 1.19, 1.17, 1.13, 1.05,
0.91, 0.82 (all S, 6.times.3H), 1.8-0.8 (Complex, 23H); .sup.13C
NMR (CDCl.sub.3, ppm): 198.10, 176.47, 170.86, 145.43, 143.16,
88.23, 71.52, 53.44, 46.99, 45.82, 45.40, 41.82, 41.84, 41.37,
40.07, 36.98, 36.58, 34.57, 33.54, 33.00, 29.10, 28.89, 28.79,
28.21, 26.66, 26.51, 24.89, 21.67, 20.52, 19.26, 16.49, 13.74.
Example 7
Allobetulin-3-diglycolate
[0228] 21
[0229] Allobetulin-3-diglycolate
[0230] m=0.300 g
[0231] C.sub.34H.sub.54O.sub.6
[0232] Exact Mass: 558.39
[0233] Mol. Wt.: 558.79
[0234] C, 73.08; H, 9.74; O, 17.18
[0235] In 25-mL flask, stir diglycolic anhydride 0.39 g and 0.5 g
allobetulin in 15 mL CHCl.sub.3. Then reflux for 24 hours. Add 10
mL saturated NaHCO.sub.3, shake gently. Then separate the organic
part, use the CHCl.sub.3 (2.times.5 ml) to wash, and combine the
organic parts. Use 3% HCl (10 ml) and water (2.times.10 ml) to wash
it. Then use Na.sub.2SO.sub.4 (anhy.) to dry the organic part.
Evaporating the solvent yields 0.57 g of white granular solid with
yield 90.2%. M.P.: 285.2 (decompose). IR (KBr): 2964.07, 1753.33,
1223.67, 1110.16 cm.sup.-1; .sup.1H NMR (CDCl.sub.3); .delta. 4.64
(DD, 1H), 4.32 (S, 4H), 3.66 (D, 1H, J=9 Hz), 3.54 (S, 1H), 3.46
(D, 1H, J=9 Hz), 0.97, 0.926, 0.891, 0.866, 0.852, 0.828, 0.796
(all S, 7.times.3H), 1.1-1.9 (complex CH--, CH.sub.2, 24H);
.sup.13C NMR (CDCl.sub.3): .delta. 171.28, 88.342, 83.431, 71.597,
69.498, 55.872, 51.339, 47.164, 41.837, 41.094, 40.985, 38.886,
38.289, 37.509, 37.079, 36.627, 34.478, 34.157, 33.057, 29.166,
28.408, 26.775, 26.601, 24.917, 24.072, 21.391, 18.476, 16.916,
16.064, 13.87.
Example 8
Allobetulin-3-glutarate
[0236] 22
[0237] Allobetulin-3-glutarate
[0238] C.sub.35H.sub.56O.sub.5
[0239] Exact Mass: 556.41
[0240] Mol. Wt.: 556.82
[0241] C, 75.50; H, 10.14; O, 14.37
[0242] Place 1 g Allobetulin (1 equivalent) and 0.52 g glutaric
anhydride (2 equivalents) with imidazole 0.92 g (6 equivalence) in
a 15 mL flask. Add 4.5 mL 1-methyl-2-pyrrolidinone and stir for 48
hours at 70.degree. C. Pour the reaction mixture into 150 mL water.
Adjust the pH to around 2. Stir for 3-4 hours and all the chunks
should be broken into small particles. After filtration, dry the
crude product in the oven. Crystalizing the crude product with
chloroform and hexane yields 1.11 g of white amorphous product with
yield 88.1%. M.P.: 283.2-284.9.degree. C. IR (cm.sup.-1): 2948.9,
1724.7, 1458.9, 1281.8, 1217.4; .sup.1H NMR (CDCl.sub.3, ppm): 4.50
(M, 1H), 3.80 (D, 1H, J=8.1 Hz), 3.55 (S, 1H), 3.46 (D, 1H, J=7.8
Hz), 2.46 (M, 4H), 1.99 (M, 2H), 0.98, 0.93, 0.92, 0.87, 0.84, 0.80
(all S, 6.times.3H), 1.8-0.8 (Complex, 28H); .sup.13C NMR
(CDCl.sub.3, ppm): 177.86, 173.08, 88.34, 81.44, 71.60, 55.92,
51.35, 47.18, 41.84, 41.09, 40.99, 38.93, 38.22, 37.53, 37.09,
36.63, 34.49, 34.20, 34.06, 33.31, 33.06, 29.17, 28.36, 26.79,
26.62, 24.92, 24.10, 21.38, 20.43, 18.51, 16.95, 16.07, 13.87.
Example 9
Allobetulin-3-phthalate
[0243] 23
[0244] Allobetulin-3-phthalate
[0245] m=0.300 g
[0246] C.sub.38H.sub.54O.sub.5
[0247] Exact Mass: 590.40
[0248] Mol. Wt.: 590.83
[0249] C, 77.25; H, 9.21; O, 13.54
[0250] In 25-ml flask, stir phthalic anhydride 0.20 g and imidazole
0.38 g in 10 ml CH.sub.2Cl.sub.2, add the 0.5 g Allobetulin into
the flask, and then reflux for 6 hours. Add 10 ml saturated sodium
bicarbonate water solution into the flask, dissolve the solid,
separate the organic part, use the CH.sub.2Cl.sub.2 (3.times.5 ml)
wash and combine the organic parts. Use 3% HCl (3.times.10 ml) wash
again. Use Na.sub.2SO.sub.4 (anhy.) to dry the organic part.
Evaporate the solvent, get white solid 0.60 g with yield 89.6%.
M.P.: 252.3-253.9.degree. C.; IR (KBr): 2948.90, 2868.36, 1724.74,
1660.31, 1458.97, 1289.84, 1136.82, 1072.39, 975.75 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3): .delta. 7.91 (D, J=6.6 Hz, 1H), 7.73 (D,
J=6.9 Hz, 1H), 7.58 (M, 2H), 4.78 (M, 1H), 3.80 (D, 1H, J=7.8 Hz),
3.62 (S, 1H), 3.48 (D, J=7.8 Hz, 1H), 2.0-0.8 (complex, 45H);
.sup.13C NMR (CDCl.sub.3): 166.47, 163.55, 129.22, 127.32, 126.21,
125.98, 125.44, 124.28, 83.47, 78.42, 66.75, 51.23, 46.53, 42.31,
37.02, 36.25, 36.16, 34.15, 33.60, 32.72, 32.24, 31.77, 29.66,
29.37, 28.24, 24.31, 23.57, 21.96, 21.77, 20.08, 18.62, 16.60,
13.67, 12.19, 12.12, 11.26, 9.06.
Example 10
Allobetulin-3-succinate
[0251] 24
[0252] Allobetulin-3-succinate
[0253] m=0.300 g
[0254] C.sub.34H.sub.54O.sub.5
[0255] Exact Mass: 542.40
[0256] Mol. Wt.: 542.79
[0257] C, 75.23; H, 10.03; O, 14.74
[0258] In 25-ml flask, stir succinic anhydride 0.23 g and imidazole
0.46 g in 15 ml CH.sub.2CH.sub.2, add 0.5 g allobetulin into the
flask, and then reflux for 24 hours. Add 10 ml saturated sodium
bicarbonate to dissolve the solid, then separate the organic part,
use CH.sub.2Cl.sub.2 (2.times.5 ml) to wash and combine the organic
parts. Use 3% HCl (2.times.10 ml) to wash the organic part. Use
Sodium sulfate (anhy.) to dry the organic part. Evaporating the
solvent results in a white granular solid. Stir the crude product
in 3% HCl for 12 hours, after filtration, which gives 0.48 g of a
white solid, with yield 78.7%. M.P.: (decomp.) 258.1-259.5.degree.
C.; IR (KBr): 2940.85, 2868.36, 1732.79, 1450.91, 1386.49, 1225.41,
1169.04 cm.sup.-1; .sup.1H NMR (CDCl.sub.3): .delta. 4.52 (M, 1H),
3.78 (D, J=7.5 Hz, 1H), 3.55 (S, 1H), 3.45 (D, J=7.5 Hz, 1H), 2.65
(M, 4H), 0.76, 0.78, 0.84, 0.86, 0.90, 0.92, 1.0 (all S,
7.times.3H), 1.1-1.9 (complex, 24H); .sup.13C NMR (CDCl.sub.3):
172.78, 167.44, 83.50, 77.00, 66.73, 51.10, 46.50, 42.31, 36.99,
36.14, 34.08, 33.38, 32.67, 32.23, 31.77, 29.62, 29.35, 28.21,
24.93, 24.56, 24.32, 23.41, 21.94, 21.76, 20.08, 19.14, 16.54,
13.65, 12.07, 11.22, 9.04.
Example 11
Betulin-3,28-didiglycolate
[0259] 25
[0260] Betulin-3,28-didiglycolate
[0261] m=0.200 g
[0262] C.sub.38H.sub.58O.sub.10
[0263] Exact Mass: 674.40
[0264] Mol. Wt.: 674.86
[0265] C, 67.63; H, 8.66; O, 23.71
[0266] In 15-ml flask, stir diglycolic anhydride 0.78 g and
imidazole 0.92 g in 4.5ml 1-methyl-2-pyrrolidinone at 70.degree. C.
After they dissolve add 1 g Betulin. Stir for 24 hours. Pour
mixture slowly into 180 ml water, adjust the pH to 2, stir the
water solution until all the precipitate forms small granules.
After the filtration, use 1% HCl, water to wash the product. Drying
gives 1.45 g granular product (little brown color) with yield
94.8%. M.P. (decomp.) 137.8-139.2.degree. C.; IR (KBr): 2961.07,
1747.02, 1220.45, 1144.87 cm.sup.-1, .sup.1H NMR (CDCl.sub.3):
.delta. 4.71 (S, 1H), 4.61 (complex, 2H), 4.2-4.45 (complex, 9H),
3.96 (D, J-11.4 Hz), 2.45 (M, 1H), 1.70 (S, 3H), 0.83, 0.85, 0.97,
1.04, (S, 4.times.3H), 1.05-2.10 (complex, 28H); .sup.13C NMR
(CDCl.sub.3): 168.46, 168.29, 166.50, 166.17, 145.34, 105.65,
78.48, 64.66, 64.39, 64.28, 59.60, 50.83, 45.75, 44.28, 43.20,
41.94, 38.23, 36.40, 33.83, 33.43, 33.14, 32.56, 29.99, 29.58,
25.16, 23.57, 22.52, 20.61, 19.23, 16.31, 14.62, 13.66, 12.02,
11.68, 11.54, 10.27.
Example 12
Betulin-28-diglycolate
[0267] 26
[0268] Betulin-28-diglycolate
[0269] C.sub.37H.sub.54O.sub.6
[0270] Exact Mass: 558.39
[0271] Mol. Wt.: 558.79
[0272] C, 73.08; H, 9.74; O, 17.18
[0273] Place Betulin 0.5 g (1 equivalent) and diglycolic anhydride
0.14 g (1.02 equivalents) with imidazole 0.31 g (4 equivalents) in
a 15 mL flask. Add 4 mL 1-methyl-2-pyrrolidinone and stir 48 hours
at room temperature. Pour the mixture into 150 mL water, which is
followed by adjusting pH to around 2. Stir for 2-3 hours. All the
chunks should be broken to small particles. After filtration, dry
the crude product in the oven, which is followed by passing through
a silica gel column with hexane: diethyl ether (3:1). This yielded
0.43 g white prizm solid with yield 68.3%. M.P.:
219.2-220.2.degree. C. IR (cm.sup.-1): 3454.5, 2941.1, 1759.5,
1729.44, 1216.3, 1136.8, .sup.1H NMR (CDCl.sub.3, ppm): 4.74 (S,
1H), 4.65 (S, 1H), 4.48 (D, 1H, J=11.1 Hz), 4.33 (S, 4 h), 4.05 (D,
1H, J=11.1 Hz), 3.27 (M, 1H), 2.49 (M, 1H), 1.73 (S, 3H), 1.08,
1.02, 1.01, 0.87, 0.81 (all S, 5.times.3H), 2.2-0.6 (Complex, 25H);
.sup.13C NMR (CDCl.sub.3, ppm): 172.16, 171.55, 150.19, 110.46,
79.44, 69.60, 69.32, 64.58, 55.64, 50.69, 49.15, 48.02, 46.79,
43.06, 41.23, 39.21, 39.05, 38.01, 37.50, 34.83, 34.53, 30.01,
29.82, 28.34, 27.68, 27.36, 25.52, 21.11, 19.49, 18.64, 16.47,
16.39, 15.74, 15.14.
Example 13
Betulin-3,28-diglutarate
[0274] 27
[0275] Betulin-3,28-diglutarate
[0276] m=0.300 g
[0277] C.sub.40H.sub.62O.sub.8
[0278] Exact Mass: 670.44
[0279] Mol. Wt.: 670.92
[0280] C, 71.61; H, 9.31; O, 19.08
[0281] In 15-ml flask, stir glutaric anhydride 1.29 g and imidazole
1.54 g in 4.5 ml 1-methyl-2-pyrrolidinone at 70.degree. C. After
they dissolve add 1 g betulin. Stir for 48 hours. Pour mixture
slowly into 180 ml water, adjust the pH to 2, and stir the water
solution until all the precipitate forms small granules. After the
filtration, use 1% HCl in water to wash the product. Drying results
in 1.22 g gray solid powder with yield 80.3%. M.P. (decomp.):
104.5-106.2.degree. C.; IR (KBr): 2956.95, 2876.42, 1732.79,
1458.97, 1386.49, 1201.25, 991.85 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3): .delta. 4.69 (S, 1H), 4.49 (S,1H), 4.50 (M, 1H), 4.29
(D, 1H, J=10.5 Hz), 3.85 (D, 1H, J=11.1 Hz), 2.42 (M, 9H), 1.98 (M,
5H),1.68 (S, 3H), 0.75-1.9 (complex, 39H); .sup.13C NMR
(CDCl.sub.3): 178.947, 173.693, 173.059, 150.463, 110.297, 81.478,
63.217, 55.712, 50.618, 49.132, 48.082, 46.741, 43.054, 41.247,
38.711, 38.194, 37.924, 37.414, 34.937, 34.449, 34.026, 33.669,
33.370, 30.120, 29.916, 28.371, 27.388, 25.493, 24.087, 21.157,
20.385, 20.254, 19.481, 18.519, 16.938, 16.516, 16.392, 15.109.
Example 14
Betulin-28-glutarate
[0282] 28
[0283] Betulin-28-glutarate
[0284] C.sub.35H.sub.56O.sub.5
[0285] Exact Mass: 556.41
[0286] Mol Wt.: 556.82
[0287] C, 75.50; H, 10.14; O, 14.37
[0288] Place 1 g of Betulin (1 equivalent) and 0.271 g glutaric
anhydride (1.05 equivalents) with 0.615 g imidazole (4 equivalents)
in a 25 mL flask, add 4 mL 1-methyl-2-pyrrolidinone and stir for 48
hours at room temperature. Pour the mixture in 150 mL water, while
stirring. Then adjust pH to around 3. Break the big chunks to small
particles, which is followed by filtration and drying in the oven.
The crude products are passed through the silica gel column with
diethyl ether: hexane (1:3). This results in 0.765 g white prism
solid with a yield of 60.7%. M.P.: 204.3-206.1.degree. C. IR
(cm.sup.-1): 3438.7, 2962.4, 2870.5, 1741.7, 1717.1, 1463.0,
1395.2; .sup.1H NMR (CDCl.sub.3, ppm): 4.73 (S, 1H), 4.64 (S, 1H),
4.35 (D, 1H, J=11.1 Hz), 3.93 (D, 1H, J=11.1 Hz), 3.25 (M, 1H),
2.50 (M, 5H), 1.73 (S, 3H), 1.08, 1.02, 0.87, 0.81 (all S,
4.times.3H), 2.2-0.8 (Complex, 30H); .sup.13C NMR (CDCl.sub.3,
ppm): 177.72, 173.67, 150.49, 110.25, 79.39, 63.202, 55.65, 50.72,
49.15, 48.06, 46.75, 43.06, 41.23, 39.22, 39.05, 37.94, 37.51,
34.94, 33.68, 33.16, 30.14, 29.94, 28.35, 27.73, 27.40, 25.55,
21.14, 20.27, 19.51, 18.65, 16.47, 16.40, 15.74, 15.14.
Example 15
Betulin-3,28-dimaleate
[0289] 29
[0290] Betulin-3,28-dimaleate
[0291] m=0.300 g
[0292] C.sub.38H.sub.54O.sub.8
[0293] Exact Mass: 638.38
[0294] Mol. Wt.: 638.83
[0295] C, 71.44; H, 8.52; O, 20.04
[0296] In 50-ml flask, stir maleic anhydride 11.09 g and Betulin 5
g in 20 ml 1-methyl-2-pyrrolidinone at 70.degree. C. for 48 hours.
Pour the mixture slowly into 800 ml water, adjust the pH to 3, and
stir the water solution until all the precipitation forms small
granules. After the filtration, use 1% HCl, in water to wash the
product. Drying gives 6.50 g gray solid granules with yield 90.1%.
M.P.: 181.4-182.9.degree. C.; IR (KBr): 2952.27, 1738.94, 1700.43,
1635.34, 1239.03, 994, 826 cm.sup.-1; .sup.1H NMR (CDCl.sub.3):
6.55-6.40 (M, 4H), 4.76-4.67 (complex, 3H), 4.56 (D, J=11.1 Hz,
1H), 4.09 (D, J=10.5 Hz, 1H), 2.45 (M, 1H), 1.70 (S, 3H), 0.78,
0.81, 0.90, 1.02, 1.10 (all S, 5.times.3H), 1.12-2.1 (complex,
24H); .sup.13C NMR (CDCl.sub.3): 163.80, 163.41, 159.92, 159.76,
145.10, 132.64, 132.20, 125.13, 124.76, 105.80, 80.56, 61.43,
50.83, 45.73, 44.30, 43.19, 41.94, 38.26, 36.40, 34.38, 33.80,
33.45, 33.23, 32.57, 29.90, 29.55, 25.04, 24.92, 23.50, 22.48,
20.59, 18.89, 16.30, 14.63, 13.63, 11.97, 11.68, 11.55, 10.90,
10.30.
Example 16
Betulin-28-maleate
[0297] 30
[0298] Betulin-28-maleate
[0299] m=0.300 g
[0300] C.sub.34H.sub.52O.sub.5
[0301] Exact Mass: 540.38
[0302] Mol. Wt.: 540.77
[0303] C, 75.51; H, 9.69; O, 14.79
[0304] In 500-ml flask, stir maleic anhydride 3.33 g and 10 g
Betulin in 200 ml CHCl.sub.3. Reflux for 40 hours. Add 50 mL 3%
HCl, separate the organic part, use CHCl.sub.3 (3.times.20 mL) to
wash the aqueous phase, and combine the organic parts. Use 3% HCl
(2.times.50 mL) to wash the organic phase, which is followed by
using Na.sub.2SO.sub.4 (anhydrous) to dry organic part. After
evaporating the solvent, use THF-hexane to crystalize the crude
product. This gives 9.2 g white product with yield 75.2%, M.P.:
242.5-243.6.degree. C.; IR (KBr): 3416.01, 2948.90, 2868.36,
1716.69, 1652.26, 1265.68, 1233.47 cm.sup.-1; .sup.1H
NMR(CDCl.sub.3): .delta. 6.5 (Q, 2H), 4.78 (S, 1H), 4.68 (S, 1H),
4.56 (D J=11.1 Hz, 1H), 4.12 (D, J=11.1 Hz, 1H), 3.26 (M, 1H), 2.50
(M, 1H), 1.76 (S, 3H), 0.84, 0.90, 1.05, 1.06, 1.10 (S,
5.times.3H), 2.1-0.8 (complex, 25H); .sup.13C NMR (CDCl.sub.3):
163.901, 159.57, 145.15, 132.58, 124.62, 105.75, 74.52, 61.44,
50.81, 45.85, 44.35, 43.18, 41.96, 38.25, 36.40, 34.39, 34.23,
33.27, 32.67, 29.91, 29.69, 25.09, 24.94, 23.51, 22.88, 22.50,
20.68, 16.27, 14.65, 13.80, 11.64, 11.56, 10.90, 10.33.
Example 17
Betulin-3,28-diphthalate
[0305] 31
[0306] Betulin-3,28-diphthalate
[0307] m=0.300 g
[0308] C.sub.46H.sub.58O.sub.8
[0309] Exact Mass: 738.41
[0310] Mol. Wt.: 738.95
[0311] C, 74.77; H, 7.91; O, 17.32
[0312] In 50-ml flask, stir phthalic anhydride 8.37 g and imidazole
7.69 g in 20 ml 1-methyl-1-pyrrolidinone at 70.degree. C. After
they dissolve, add 5 g betulin. Stir for 48 hours. Pour mixture
slowly into 800 ml water, adjust the pH to 2, and stir the water
solution until all the precipitate forms small granules. After the
filtration, use 1% HCl in water to wash the product. Drying gives
7.59 g granules (light yellow color) with yield 90.8%. M.P.
(decomp.) 166.8-168.6.degree. C.; IR (KBr): 2956.95, 2876.42,
1716.69, 1394.54, 1281.79, 1128.77, 1088.50, 991.85, 742.19
cm.sup.-1; .sup.1H NMR (CDCl.sub.3): .delta. 7.87 (M 2H), 7.77 (M,
2H), 7.58 (M, 4H), 4.76-4.53 (complex, 4H), 4.08 (D, J=10.2 Hz,
1H), 2.50 (M, 1H), 1.68 (S, 3H), 0.82, 0.84, 0.90, 1.02, 1.08 (all
S, 5.times.3H), 1.1-2.2 (complex, 24H); .sup.13C NMR (CDCl.sub.3):
168.57, 168.33, 163.68, 162.99, 145.58, 128.79, 128.23, 127.37,
126.56, 126.39, 126.32, 126.19, 125.22, 125.08, 124.69, 124.50,
105.47, 78.49, 60.12, 51.04, 45.80, 44.46, 43.36, 42.09, 39.22,
38.27, 36.40, 33.99, 33.56, 33.24, 32.62, 29.96, 29.53, 25.12,
23.55, 22.58, 20.68, 18.72, 16.42, 14.62, 13.66, 12.20, 11.72,
11.56, 10.48.
Example 18
Allobetulin,
oleanan-3.beta.-ol-28 19-.beta.-ether
[0313] 32
[0314] In 100-ml flask stir 2 g of Betulin in 50 ml of
CH.sub.2Cl.sub.2 at 0.degree. C. Add 5 ml of 99% CF.sub.3COOH and
stir for 30 minutes. Pour reaction mixture in 100 ml of cracked ice
and separate the organic part. Extract with CH.sub.2Cl.sub.2
(3.times.10 ml) and wash combined organic extracts with conc.
NaHCO.sub.3 (2.times.20 ml) and water (2.times.20 ml), and dry the
extract over Na.sub.2SO.sub.4 (anh.). Evaporation of solvent gives
1.98 g of Allobetulin, which was recrystallized from
hexane-dichloromethane to yield white needles mp. 268-269.degree.
C. [lit. 265-268], IR (KBr) 3448.5, 2941.5, 2866.6, 1780.7, 1456.6,
1384.4, 1168.9, 1035.0, cm.sup.-1; .sup.1H NMR (CDCl.sub.3) d 3.75
(D, J=10.3 Hz, 1H, 28-H), 3.51 (S, 1-H, 19-H), 3.41 (D, J=10.3 Hz,
1H, 28-H), 3.18 (DD, 1H, 3-H), 0.74, 0.76, 0.81, 0.88, 0.89, 0.94,
0.94 (all S, 7.times.3H, 27-, 23-, 24-, 25-, 26-, 29-, 30-Me),
1.01-1.74 (complex CH--, CH.sub.2, 25 H,); .sup.13C NMR
(CDCl.sub.3) d 88.41, 79.38, 71.49, 55.77, 51.35, 47.07, 41.78,
40.99, 40.88, 40.87, 39.16, 37.52, 36.95, 36.51, 34.40, 34.18,
32.96, 29.08, 28.27, 27.57, 26.72, 26.72, 26.50, 24.82, 21.26,
18.54, 16.79, 15.96, 15.72, 13.82; MS (EI) 442, 424, 411, 371, 355,
303, 273, 257, 245, 231, 220, 207, 203, 189, 177, 162, 149,135,
121,107.
Example 19
Allobetulinlactone
oleanan-3.beta.-ol-28,19-.beta.-lactone
[0315] 33
[0316] In 100-ml flask boil 2 g of Allobetulin-3-trifluoroacetyl
lactone in 50 ml of CH.sub.3OH in presence of 0.723 g KOH for 4
hours. Evaporate methanol and dilute with 100 mL of cold water.
Filter the precipitate and wash with water (3.times.50 mL). Dry
crystals in oven at 110.degree. C. and recrystallize from
hexane-dichloromethane to yield white needles. mp.
316.3-317.6.degree. C., IR (KBr) 3495, 2940, 2866, 1759, 1447,
1388, 1153, 1118, 967, 923 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) d
3.97 (S, 1H, 19H), 3.22 (DD, 1H, 3H), 1.057, 1.000, 0.987, 0.942,
0.903, 0.87, 0.791 (all S, 7.times.3H, 23-, 24-, 25-, 26-, 27-,
29-, 30-Me), 1.1-1.9 (complex CH--, CH.sub.2--, 24H); .sup.13C NMR
(CDCl.sub.3) d 180.207, 86.316, 79.174, 55.791, 51.528, 47.01,
46.413, 40.853, 40.212, 39.242, 39.177, 37.552, 36.313, 34.018,
33.843, 32.619, 32.232, 29.055, 28.254, 28.188, 27.642, 26.826,
25.842, 24.261, 21.178, 18.46, 16.85, 15.822, 15.669, 13.964; MS
(EI) 456, 438, 423, 395, 356, 329, 261, 234, 206, 189, 175, 161,
147, 135, 121, 107, 95, 81, 69, 55, 43.
Example 20
Allobetulinlactone-3-acetate
oleanan-28,19-.beta.-lactone-3-acetate
[0317] 34
[0318] In 100-ml flask stir 2 g of 3-O-acetyl-betulin in 50 ml of
CH.sub.2Cl.sub.2 at 0.degree. C. Add 10 ml of 99% proof
CF.sub.3COOH stir for 10 minutes and after that add 2.2 g of
powdered NaBrO.sub.3. Stir the mixture for 6 hours and then pour in
100 ml of cracked ice and separate organic part. Extract with
CH.sub.2Cl.sub.2 (3.times.10 ml) and wash combined organic extracts
with 10% aqueous NaHSO.sub.3 (2.times.30 ml), 5% aqueous
NaHCO.sub.3 (2.times.30 ml) and water (2.times.20 ml), and dry the
extract over Na.sub.2SO.sub.4 (anh.). Evaporation of solvent gives
2.08 g of 3-O-acetyl allobetulin-lactone, which was recrystallized
from hexane-dichloromethane to yield white needles. mp.
312.5-315.4.degree. C. (dec.), IR (KBr) 2943, 2878, 1761, 1729,
1502, 1486, 1446, 1374, 1252 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) d
4.50 (DD, 1H, 3H), 3.94 (S, 1H, 19H), 2.03 (S, 3H, Ac-Me), 1.04,
0.97, 0.95, 0.8, 0.8, 0.79, 0.78 (all S, 7.times.3H, 23-, 24-, 24-,
25-, 26-, 29-, 30-Me), 1.02-1.79 (complex CH--, CH.sub.2--, 23H);
.sup.13C NMR (CDCl.sub.3) d 13.899, 15.779, 16.741, 16.879, 18.307,
21.164, 21.601, 23.896, 24.210, 26.745, 25.784, 28.158, 28.159,
29.004, 32.181, 32.568, 33.792, 33.916, 36.284, 37.428, 38.055,
38.878, 40.175, 46.420, 40.831, 46.959, 51.419, 55.835, 180.352,
81.244, 86.381, 171.579; MS (EI) 482, 438, 424, 395, 356, 327, 281,
253, 207, 189, 174, 162, 147, 135, 121,43.
Example 21
Allobetulinlactone-3-phosphate
oleanan-28,19-.beta.-lactone-3-phosphate
[0319] 35
[0320] In 100 mL round bottom flask boil a solution of
allobetulin-3-phosphodichloride in 50 mL of dioxane and 1 mL of
water for 18 hours. Dilute with cold water (50 mL) and filter white
precipitate. Wash on filter with water (3.times.30 mL). Dry in oven
(temperature not higher than 110.degree. C.) to give 3.12 g of
white crystalline compound mp. 226.7-230.1.degree. C. (dec)[lit.
***], IR (KBr) 3414, 2945, 2868, 1760, 167, 1449, 1384, 1524, 1213,
1068, 1025, 967, 495 cm.sup.-1; .sup.1H NMR (CDCl.sub.3/DMSOd6=1:1)
d 5.64 (S, 2H, (OH)2) 3.94 (S, 1H, 19H), 3.81 (M, 1H, 3-H), 1.001,
0.98, 0.98, 0.89, 0.89, 0.87, 0.78 (all S, 7.times.3H, 23-, 24-,
25-, 26-, 27-, 29-, 30-Me), 1.05-1.95 (complex CH--, CH.sub.2--,
23H); .sup.13C NMR (CDCl.sub.3/DMSOd6) d 177.852, 84,195, 82.504,
53.984, 49.510, 44.897, 44.431, 38.995, 38.405, 38.405, 37.275,
37.181, 35.556, 34.608, 32.065, 30.928, 30.243, 27.38, 26.637,
26.345, 24.851, 24.057, 23.758, 22.381, 19.408, 16.712, 15.174,
14.817, 14.059, 12.296; 31P NMR (D.sub.3PO.sub.4 85% in D.sub.2O)
d-0.719.
Example 22
Allobetulin-3-hydroxy-3-aminomethyl
3-aminomethyl-3-hydroxy-28,19-.beta.-epoxy-oleanan
[0321] 36
[0322] In 25 mL round bottom flask boil the mixture of allobetulon
(0.86 g, 1.955 mmol), ZnI.sub.2 (20 mg, 0.063 mmol) and
tret-butyldimethylsilyl- cyanide (0.420 g, 3.78 mmol) in 15 mL of
Toluene for 24 hours. Add the above mentioned mixture to a
suspension of LiAlH.sub.4 (0.37 g, 10 mmol) in 30 mL of THF drop
wise and boil for 2 hours. Next, add 0.5 mL of concentrated KOH,
dilute with 30 mL of THF and filter with diatomaceous earth. Dry
over sodium sulfate and bubble HCl gas through the THF solution and
filter the white precipitate (0.98 g). Dissolve the crystals in 50
mL of chloroform and wash with 1% NaHCO.sub.3 until neutral
reaction of universal paper indicator. Separate organic part and
dry over sodium sulfate. Evaporation of solvent gives 0.89 g (96%
yield) of white crystalline compound mp. 222.0-224.3.degree. C., IR
(KBr) 3414, 2939, 2868, 1617, 1461, 1384, 1036 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3) d 3.67 (D, 1H, 28H, J=7.5 Hz), 3.521 (S, 1H, 19H),
3.437 (D, 1H, 28H, J=7.5 Hz), 2.95 (D, 1H, 31H, J=13.2 Hz), 2.757
(D, 1H, 31H, J=13.2 Hz), 2.523 (S, 3H, OH+NH.sub.2), 0.972, 0.926,
0.911, 0.904, 0.894, 0.824, 0.798 (all S, 7.times.3H, 23-, 24-,
25-, 26-, 27-, 29-, 30-Me), 1.01-1.79 (complex CH--, CH.sub.2--,
24H); .sup.13C NMR (CDCl.sub.3) d 88.13, 75.174, 71.428, 62.517,
53.554, 51.703, 47.018, 43.265, 41.669, 40.911, 40.882, 40.882,
37.683, 37.596, 36.933, 36.459, 34.309, 32.903, 30.265, 29.048,
27.416, 26.643, 26.454, 24.771, 24.166, 21.171, 19.947, 18.905,
17.076, 15.961, 13.811.
Example 22
Allobetulin-3-phosphate
28,19-.beta.-epoxy-oleanan-3-phosphate
[0323] 37
[0324] In 100 mL round bottom flask boil a solution of
Allobetulin-3-phosphodichloride in 50 mL of dioxane and 1 mL of
water for 18 hours. Dilute with cold water (50 mL) and filter white
precipitate. Wash on filter with water (3.times.30 mL). Dry in oven
(temperature not higher than 110.degree. C.) to give 3.12 g of
white crystalline compound mp. 167.0-168.1.degree. C. (dec), IR
(KBr) 3469, 2947, 2868, 1775, 1467, 1388, 1221, 1169, 1022, 884,
585, 505, 481 cm.sup.-1; .sup.1H NMR; .sup.31P NMR (D.sub.3PO.sub.4
85% in D.sub.2O) d-0.684.
Example 23
Allobetulon
oleanan-3-one-28,19-.beta.-ether
[0325] 38
[0326] In 100-mL round bottom two neck flask place 11 mmol (1.397
g) (COCl).sub.2 in 25 ml of dry CH.sub.2Cl.sub.2 at -50-60.degree.
C. (i-Pr alcohol--dry ice bath) and with efficient stirring add 22
mmol (1.76 g) of dry DMSO in 25 ml of dry CH.sub.2Cl.sub.2 in drop
wise in 3-5 minutes. Stir the mixture for additional 5 minutes and
then add crystals of allobetulin (10 mmol, 4.43 g). Stand solution
for 30-45 minutes and after adding with 25 mmol (2.53 g) of
triethylamine, remove cold bath and let temperature to increase up
to 10.degree. C. Pour the mixture in 100 ml of cracked ice, extract
with CH.sub.2Cl.sub.2 (3.times.20 ml) and wash combined organic
extracts with water (5.times.10 ml), 5% HCl (2.times.10 ml), and
H.sub.2O (2.times.10 ml). After drying over sodium sulfate solvent
evaporation gives 4.4 g of crude compound, which after column
chromatography (hexane:ether=80:20) gives 4.31 g of white crystals
mp. 228.8-233.1.degree. C. [lit. 230-235.degree. C.], IR (KBr)
2949, 2859, 1774, 1702, 1457, 1382, 1167, 1034 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3) d 3.74 (D, J=10.3 Hz, 1H, 28-H), 3.48 (S, 1-H,
19H), 3.39 (D, J=10.3 Hz, 1H, 28-H), 2.37 (M, 2H, 2-H,H), 1.85 (M,
1H, 19-H), 0.72, 0.81, 0.815, 0.91, 0.92, 0.99 (all S, 7.times.3H,
27-, 23-, 24-, 25-, 26-, 29-, 30-Me), 1.01-1.54 (complex CH--,
CH.sub.2--, 25H); .sup.13C NMR (CDCl.sub.3) d 218.08, 88.01, 71.39,
55.08, 50.55, 47.43, 46.92, 41.60, 40.91, 40.68, 39.97, 37.11,
36.88, 36.43, 34.41, 34.23, 33.33, 32.86, 29.00, 26.92, 26.60,
26.60, 26.40, 24.73, 21.68, 21.16, 19.79, 16.52, 15.68, 13.63; MS
(EI) 440, 422, 411, 369, 355, 281, 220, 207, 205, 191, 177, 163,
149, 135, 121.
Example 24
Allobetulonlactone-1-ene-2-ol
2-hydroxy-olean-1,2-ene-3-one-28,19-.beta.-lactone
[0327] 39
[0328] To a solution of Allobetulonlactone (1.0 g) in dry
benzene-tert-butyl alcohol (1:1, 40 ml) was added a solution of
potassium tert-butoxide (0.56 g) in tert-butyl (20 ml) and oxygen
was bubbled into the stirred mixture for 3 hours. The mixture was
acidified with 2.0 ml of glacial acetic acid and extracted with
CH.sub.2Cl.sub.2. After washing with water (2.times.15 ml), 5%
aqueous NaHCO.sub.3 (2.times.30 ml) and water (30 ml), the extract
was dried over Na.sub.2SO.sub.4 and evaporated to give crystals
(m=0.983 g, 98%), which after chromatography on silica gel (hexane:
ether=40:60) yields a white crystalline compound mp.
238.8-243.6.degree. C., IR (KBr) 3451, 2944, 2864, 1764, 1663,
1642, 1450, 1405, 1384, 1234, 1055, 967 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) d 6.47 (S, 1H, 2-H), 6.07-5.85 (1H, OH), 3.96 (S, 1H,
19H), 1.207, 1.153, 1.109, 1.037, 0.98, 0.974, 0.877 (all S,
7.times.3H, 23-, 24-, 25-, 26-, 27-, 20-, 30-Me), 1.05-1.91
(complex CH--, CH.sub.2, 22H); .sup.13C NMR (CDCl.sub.3) d 201.36,
180.025 144.217, 128.966, 86.192, 54.501, 46.879, 46.631, 46.376,
44.292, 41.654, 40.525, 38.936, 36.393, 33.836, 33.632, 32.568,
32.174, 29.034, 28.006, 27.365, 26.571, 25.784, 24.232, 21.841,
21.397, 20.923, 18.868, 16.282, 13.768, MS (EI) 468, 454, 441, 425,
407, 369, 340, 313, 303, 269, 259, 234, 215, 207, 189, 176, 165,
153, 151, 135, 128, 124, 108, 95, 78, 69, 55, 43.
Example 25
Allobetulon-1-ene-2-ol
2-hydroxy-28,19-.beta.-epoxy-olean-1(2)-ene-3-one
[0329] 40
[0330] To a solution of allobetulon (1.8 g) in dry
benzene-tert-butyl alcohol (1:1, 40 ml) was added a solution of
potassium tert-butoxide (1.2 g) in tert-butyl (20 ml) and oxygen
was bubbled into the stirred mixture for 1.5 hours. The mixture was
acidified with 2.5 ml of glacial acetic acid and extracted with
CH.sub.2Cl.sub.2. After washing with water (2.times.15 ml), 5%
aqueous NaHCO.sub.3 (2.times.30 ml) and water (30 ml), the extract
was dried over Na.sub.2SO.sub.4 and evaporated to give crystals,
which after chromatography on silica gel (hexane: ether=85:15)
1402, 1234, 1058, 1035 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) d 6.46
(S, 1H, 1-H), 5.9 (S, 1H, 2-OH), 3.75 (D, 1H, 28H), 3.52 (S, 1H,
19-H), 3.52 (S, 1H, 28H), 0.78, 0.91, 0.99, 1.01, 1.09, 1.13, 1.19
(all S, 7.times.3, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me), 1.05-1.78
(complex CH--, CH.sub.2, 25H); .sup.13C NMR (CDCl.sub.3) d 201.43,
144.202, 129.29, 88.16, 71.523, 54.49, 46.99, 44.3, 41.75, 41.312,
38.97, 36.98, 36.56, 34.53, 33.83, 32.97, 29.1, 27.41, 26.6, 26.52,
24.86, 21.89, 21.51, 20.87, 18.96, 16.5, 13.64; MS (EI) 454 383,
327, 281, 245, 215, 207, 191, 177, 151, 137, 136, 123, 109, 95, 81,
69, 55.
Example 26
Betulin
lup-20(29)-ene-3,28-diol
[0331] 41
Isolation of Betulin
[0332] Betulin was isolated from paper birch (B. papyrifera) bark.
Shredded, dry bark (500 g) has been extracted with chloroform on a
Soxhlet apparatus for 10 hours. The extract was evaporated and then
was left overnight at 5-7.degree. C. Crystals were filtered and
washed with hexane and then dried in oven to give 94.5 g of crude
Betulin. Double crystallization from chloroform and then mixture of
chloroform-isopropyl alcohol(4:1) gives 64-68 g of pure Betulin mp.
258-259.degree. C. [lit. mp 256-261.degree. C.]. IR (KBr) 3378,
2942, 2868, 1645, 1453, 1374, 1106, 1031, 880 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3) d 4.68 (S, 1H, 29-H), 4.58 (S, 1H, 29-H), 3.8 (D,
J=10.3 Hz, 1H, 28-H, 3.34 (D, J=10.3 Hz, 1H, 28-H), 3.18 (DD, 1H,
3-H), 2.38 (M, 1H, 19-H), 1.68 (S, 3H, 30-Me), 0.76, 0.82, 0.97,
0.98, 1.02 (all S, 5.times.3H, 27-, 23-, 24-, 25-, 26-Me), 1.01-2.4
(complex CH--, CH.sub.2, 25 H,); .sup.13C NMR (CDCl.sub.3) d
151.249, 110.464, 79.736, 61.278, 56.017, 51.12, 49.48, 48.533,
48.534, 43.454, 41.647, 39.614, 39.432, 38.033, 37.894, 34.958,
34.725, 30.469, 29.901, 28.742, 28.123, 27.773, 25.929, 21.572,
19.845, 19.051, 16.879, 16.726, 16.136, 15.516; MS (EI) 442, 424,
411, 398, 393, 381, 288, 234, 207, 203, 189, 175, 161, 147, 135,
121, 107.
Example 27
Betulon-1-ene-2-ol
lup-1(2),20(29)-diene-2,28-diol-3-one
[0333] 42
[0334] To a solution of betulin-28-acetate (1.0 g) in dry
benzene-tert-butyl alcohol (1:1, 40 ml) was added a solution of
potassium tert-butoxide (1.05 g) in tert-butanol (20 ml). Oxygen
was bubbled into the stirred mixture for 1.5 hours. The mixture was
acidified with 2.2 ml of glacial acetic acid and extracted with
CH.sub.2Cl.sub.2. After washing with water (2.times.15 ml), 5%
aqueous NaHCO.sub.3 (2.times.30 ml) and water (30 ml), the extract
was dried over Na.sub.2SO.sub.4 and evaporated to give crystals,
which after chromatography on silica gel (hexane:ether=80:20)
yielded a white crystalline compound mp. 167-170 (dec) .degree. C.,
IR (KBr) 3446, 2944, 2870, 1717, 1669, 1647, 1457, 1406, 1237,
1032, 882 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) d 6.43(S, 1H, 2-H),
6.12-5.81 (OH), 4.69 (S, 1H, 29H), 4.60 (S, 1H, 29H), 3.79 (DD, 1H,
28-H, J=10.7 Hz), 3.35 (DD, 1H, 28-H, J=10.7 Hz), 2.4 (M, 1H,
19-H), 1.69 (S, 3H, 30-Me), 1.203, 1.124, 1.105, 1.09, 0.976 (all
S, 5.times.3H, 23-, 24-, 25-, 26-, 27-Me), 1.05-2.01 (complex CH--,
CH.sub.2 22H); .sup.13C NMR (CDCl.sub.3) d 201.513, 150.491,
144.188, 129.228, 110.209, 60.768, 54.217, 48.883, 48.045, 48.045,
45.844, 44.278, 43.309, 42.019, 38.893, 37.654, 34 244, 34.149,
29.974, 29.398, 27.409, 27.226, 25.332, 21.907, 21.353, 20.457,
19.364, 19.021, 16.755 114.941; MS (EI) 454, 438, 424, 381, 325,
302, 271, 229, 215, 189, 177, 161, 135,121,95, 81, 55.
Example 28
Betulin-3,28-diphosphaate
Lup-20(29)-ene-3,28-diphosphate
[0335] 43
[0336] In 100 mL round bottom flask boil a solution of
betulin-3,28-diphosphodichloride in 50 mL of dioxane and 1 mL of
water for 18 hours. Dilute with cold water (50 mL) and filtrate
white precipitate. Wash on filter with water (3.times.30 mL). Dry
in oven (temperature not higher than 110.degree. C.).
Example 29
Betulin-3,28-diphosphate Sodium Salt
Lup-20(29)-ene-3,28-sodiumdiphosphate
[0337] 44
[0338] In 100 mL round bottom flask to a suspension of 1 g (1.66
mmol) of betulin-3,28-diphosphate in 40 mL of water a solution of
0.6 g of sodium bicarbonate in 40 ml of water was added dropwise to
maintain pH<=7. Water was evaporated under reduced pressure and
white precipitate was dried in vacuum.
Example 30
Betulinic Acid
3-hydroxy-lup-20(29)-ene-28-oic Acid
[0339] 45
[0340] Betulinic aldehyde (1.5 g) was dissolved in 45 ml of ethyl
acetate and then was placed in a 100 ml heatable column. 0.6 ml of
distilled water and 23 mg of ABIN was added to the solution. Oxygen
has been bubbled through the mixture at 50-60.degree. C. for 6
hours with periodic addition of ABIN (5 mg per hour). Evaporation
of solvent and following crystallization from MeOH gives 1.42 g of
white crystals mp. 288-291.degree. C. [lit. 291-292], IR (KBr)
3449, 2941, 2869, 1686, 1639, 1451, 1376, 1235, 1186, 1043, 886
cm.sup.-1; .sup.1H NMR (CDCl.sub.3), d 4.79 (S, 1H, 29 H), 4.65 (S,
1 H, 29H), 3.22 (DD, 1H. 3-H), 3.02 (T, 1 H, 19H), 1.66 (S, 3 H,
30-Me), 0.79, 0.83, 0.88, 1.0, 1.0 (all S, 5.times.3H, 23-, 24-,
25-, 26-, 27-Me), 1.05-2.24 (complex CH--, CH.sub.2, 25H); .sup.13C
NMR (CDCl.sub.3) d 180.403, 150.542, 109.86, 79.146, 56.433,
55.471, 50.64, 49.401, 47.025, 42.573, 40.824, 39.01, 38.842,
38.529, 37.348, 37.174, 34.456, 32.291, 30.688, 29.85, 28.138,
27.54, 25.631, 20.982, 19.518, 18.417, 16.282, 16.173, 15.495.
14.847; MS (EI) (after sililation) 518, 510, 487, 483, 471, 456,
428, 413, 393, 377, 353, 320, 306, 292, 257, 203, 189, 175, 148,
135, 129, 73.
Example 31
Lupeol
monoglynol B, .beta.-viscol, Fagarasterol
lup-20(29)-ene-3.beta.-ol
[0341] 46
[0342] Combined parts after Betulin crystallization and solvent
evaporation were separated on silica gel (eluent hex: ether=10:1).
After 150 ml solvent delay 20 fractions were collected. Fractions
1-7 contain mixture of lower terpenes, fractions 8-13 contained
Lupeol mp 182.7-187.3.degree. C., IR (KBr) 3380, 2920, 1450,1405,
1025, 940 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) d 4.69 (S, 1H, 29-H),
4.55 (S, 1H, 29-H), 3.18 (DD, 1H, 3-H), 2.35 (M, 1H, 19-H), 1.67
(S, 3H, 30-Me), 0.74, 0.76, 0.80, 0.92, 0.94, 1.01 (all S,
6.times.3H, 27-, 23-, 24-, 25-, 26-, 28-Me), 1.01-2.4 (complex
CH--, CH.sub.2, 25 H,); .sup.13C NMR (CDCl.sub.3) d 151.32, 109.67,
79.32, 55.63, 50.77, 48.63, 48.33, 43.34, 43.17, 41.16, 40.34,
39.20, 39.04, 38.38, 37.50, 35.92, 34.61, 30.18, 28.33, 27.77,
26.09, 25.47, 21.26, 19.65, 18.65, 18.35, 16.46, 16.31, 15.72,
14.89; MS (EI) 426, 411, 393, 381, 369, 315, 281, 257, 218, 207,
189, 175, 161, 147, 135, 121, 107.
Example 32
Betulin-28-caffeate
Lup-20(29)-ene-28-ol-3-caffeate
[0343] 47
[0344] In a one liter round bottom one neck flask equipped with
condenser a crude extract of outer birch bark (100 g) was dissolved
in 500 ml of tetrahydrofuran. 10 g of aluminum triisopropoxide was
added. The mixture was boiled for 1 hour, was allowed to cool to
45.degree. C. This formed a precipitate, which was filtered and
washed with tetrahydrofuran (5.times.40 ml) and dried on filter.
Residual powder (34.2 g) was washed with 10% AcOH, dried on filter
and extracted with 1% AcOH in isopropyl alcohol (5.times.50 ml).
Combined extracts were concentrated in vacuum to 50 ml volume and
diluted with water (200 ml) and filtered, and dried in vacuum at
40.degree. C. The resulting 22.7 g of material was treated with a
solution of diazomethane in diethyl ether and solvent was
evaporated after no more nitrogen evolved. The remaining material
was subjected for chromatography on silica gel (hexanes:ether=4:1)
and 30 fractions were collected after 100 ml solvent delay and
analyzed by TLC. Fractions 7, 8, 9 were combined and solvent was
evaporated to give 4.78 g of lite-yellow crystals m.p. 191.1,
198.3.degree. C., IR (KBr) 3426, 2945, 2871, 1708, 1678, 1630,
1604, 1514, 1447, 1376, 1273, 1181, 1109, 1012, 977 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3), 7.602 (1H, DJ=15.9, C3'H from caffeate);
7.1 (1H, D, J=7.8, C2'-H from caffeate); 7.06 (1H, D, J=2); 6.86
(1H, DD, J=7.8, C5'H); 6.314 (1H, D, J=15.9); 4.68 (1H, S, 29-H);
4.62 (1H, M, C3H); 4.59 (1H, S, 29H); 3.82 (1H, D, J=11.5, 28H);
3.35 (1H, D, J=11.5, 28H), 1.69 (3H, S, 30-Me), 1.036, 0.991,
0.927, 0.899, 0.882 (5.times.3H, S, 23-, 24-, 25-, 26-, 27-Me);
1.05-2.24 (complex CH--, CH.sub.2); .sup.13C NMR (CDCl.sub.3)
167.44, 151.29, 150.8, 149.47, 144.54, 127.82, 122.92, 116.83,
111.27, 110.06, 109.78, 81.141, 60.86, 56.28, 55.73, 50.62, 49.06,
48.11, 43.04, 41.264, 38.739, 38.39, 37.61, 37.42, 34.87, 34.30,
30.03, 29.47, 28.33, 27.35, 25.48, 24.165, 21.18, 19.39, 18.53,
17.03, 16.53, 16.31, 15.06.
Example 33
General Procedure for Preparation of
Betulin-3,28-dioxalate-polyethylenimi- ne Amids (Samples 49-51,
100-106):
[0345] In 500 ml round bottom flask to a solution of
polyethylenimine (MW.sub.av 600) (a mmol) in 100 ml of
dichloromethane add a solution of Betulin-3,28-dioxalylchloride (b
mmol) in 300 ml of dichloromethane drop wise while stirring at
21-23.degree. C. The reaction mixture was then stirred for 15
minutes and dichloromethane was evaporated under reduced pressure
at 40.degree. C. Residue (oily amorphous material) was dried in
vacuum.
[0346] 49. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=1:1;
[0347] 50. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=1:3;
[0348] 51. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=5:1;
[0349] 100. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=1:5;
[0350] 101. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=3:1;
[0351] 102. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=1:10;
[0352] 103. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=1:1;
[0353] 104. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=1:2;
[0354] 105. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=2:1;
[0355] 106. Betulin-3,28-dioxalylchloride:polyethylenimine
(MW.sub.av 600) ratio a:b=1:4. 48
[0356] The compounds disclosed herein were tested for growth
inhibition against four strains of bacteria: Escherichia coli,
Staphylococcus aureus, methicillin-resistant Staphylococcus aureus,
and Bacillus subtilis. These three species differ significantly in
size, morphology, environment, and are evolutionarily distant. They
represent three distinct genera. E. coli is a gram-negative,
non-spore forming, rod-shaped, opportunistic pathogen that resides
in the human digestive tract. S. aureus is a gram positive coccus,
is usually found morphologically as a packet, and is an
opportunistic human pathogen, often residing on or in the skin. B.
subtilis is a gram positive spore-forming rod. Its native
environment is the soil. The chemicals were tested for
growth-inhibition of the bacteria by the Kirby-Bauer method. See,
e.g., Microbiology, third edition; Lansing M. Prescott, John P.
Harley, and Donald A. Klein; W m. C. Brown Publishers.
Materials and Methods
[0357] Preparation of media. Mueller-Hinton agar (Difco Lab,
Michigan) was prepared according to the manufacturer's directions,
and 20 ml was dispensed in each sterile petri dish. Sterile
nutrient broth (Difco Lab, Michigan) was also prepared according to
the manufacturer's directions.
[0358] Growth of organism. Bacteria were grown in liquid nutrient
broth for 18 hours at 37.degree. C. until they were turbid.
[0359] Preparation of plates. An aliquot of 0.5 ml of cultured
bacteria was aseptically transferred to the surface of a
Mueller-Hinton agar plat, and spread using a cotton-tipped swab.
Plates were then allowed to dry for 5 minutes. Then a 10 mm
diameter filter paper disk impregnated with the test chemical was
placed with sterile forceps on the plate. The disks were
impregnated with 100 .mu.l of a solution of the test compound in
DMSO or in water, at the indicated concentrations in Table 1.
[0360] Determination of susceptibility. The zones of inhibition
were measured after 18 hours growth at 37.degree. C. Tests were
done in triplicate.
[0361] Determination of Minimum Inhibitory Concentration. Sterile
tryptic soy broth was prepared according to the manufacturer's
instructions (Difco Lab, Michigan). To each tube containing 7 ml of
broth, test compounds dissolved in DMSO were added at different
concentrations in triplicate. The tubes were incubated for 24 hours
at 37.degree. C. and then turbidity was measured using a
colorimeter. The colorimeter was calibrated with sterile broth. The
MIC was the lowest concentration that could prevent an increase in
turbidity.
Results
[0362] Control disks impregnated with DMSO or 0.85% saline produced
no zone of inhibition. Against E. coli, betulin-3,28-diphosphate,
betulin-3,28-diglutarate, and betulin-3,28-didiglycolate were
effective (Tables 1a and 1b). Each had a a minimum inhibitory
concentration of 0.282 .mu.M (data not shown).
[0363] The compounds effective against S. aureus were
betulin-3,28-dimaleate and betulin-3,28-diphthalate (Tables 1a and
1b). Each had a minimum inhibitory concentration of 2.3 .mu.M.
[0364] Against methicillin resistant S. aureus,
betulin-3,28-dimaleate was effective, with an MIC of 0.282
.mu.M.
[0365] No compounds tested were effective against Bacillus
subtilis.
[0366] Betulin-3,28-dimaleate was more effective against the
laboratory strain (not methicillin resistant) S. aureus than
streptomycin. In the disk diffusion assay, S. aureus was inhibited
by 56 ng of betulin-3,28-dimaleate applied to the disk, while
inhibition by streptomycin required 10 .mu.g.
2TABLE 1a Effect of Betulin, Allobetulin, and Derivatives in DMSO
Solution Concentra- Zone of inhibition tion mg/ml Escherichia
Staphylococcus Bacillus Test Compound in DMSO coli aureus subtilis
Betulin 1 mg/ml 0 mm 0 mm 0 mm Allobetulin 1 mg/ml 0 mm 0 mm 0 mm
Betulin 28- 1 mg/ml 0 mm 0 mm 0 mm succinate Betulin 3,28- 1 mg/ml
0 mm 0 mm 0 mm disuccinate Betulin 3,28- 10 mg/ml 0 mm 10.0 mm 0 mm
diphthalate Betulin 3,28- 10 mg/ml 0 mm 0 mm 0 mm diglutarate
Betulin 3,28- 10 mg/ml 0 mm 11.0 mm 0 mm dimaleate Betulin 3,28- 10
mg/ml 0 mm 0 mm 0 mm didiglycolate Allobetulin 3- 5 mg/ml 0 mm 0 mm
0 mm succinate Allobetulin 3- 1 mg/ml 0 mm 0 mm 0 mm glutarate
Betulin 28- 10 mg/ml 0 mm 0 mm 0 mm maleate Betulin 3,28- 20 mg/ml
0 mm 0 mm 0 mm maleate Betulin 28- 1 mg/ml 0 mm 0 mm 0 mm phthalate
DMSO control -- 0 mm 0 mm 0 mm
[0367]
3TABLE 1b Effect of Betulin, Allobetulin, and Derivatives in Water
Solution Con- centration Zone of inhibition mg/ml Escherichia
Staphylococcus Bacillus Test Compound in DMSO coli aureus subtilis
Betulin 28- 125 0 mm 0 mm 0 mm phthalate Betulin 3,28- 125 0 mm 0
mm 0 mm diglycine Betulin ester 125 0 mm Betulin Di-(L- 125 0 mm 0
mm 0 mm glutamic acid .gamma.- benzyl ester) ester Betulin 125 0 mm
0 mm 0 mm dianiline ester Betulin Di-(L- 125 0 mm 0 mm 0 mm
proline) ester Allobetulin 125 0 mm 0 mm 0 mm ethanol amine
Allobetulin 2- 125 0 mm 0 mm 0 mm alanine ester Allobetulin 125 0
mm 0 mm 0 mm lactone Lupeol 125 0 mm 0 mm 0 mm Lupeol-3- 125 0 mm 0
mm 0 mm phthalate Allobetulin 125 0 mm 0 mm 0 mm glycine
Allobetulin 3- 125 0 mm 0 mm 0 mm glutarate Allobetulin ester 125 0
mm 0 mm 0 mm Allobetulin 125 0 mm 0 mm 0 mm ethanolamine
carbohydrate Hederine 0.5 0 mm 0 mm 0 mm hydrate 3-Allobetulon-1-
125 0 mm 0 mm 0 mm ene-2-succinate Betulin 3.3 0 mm 0 mm 0 mm
arabinose galactan Allobetulon- 125 0 mm 0 mm 0 mm lactone-1-ene-2-
ol Betulin-3,28- 125 0 mm 0 mm 0 mm diphenylalanine ester Lupeol-3-
125 0 mm 0 mm 0 mm succinate Allobetulin 2- 125 0 mm 0 mm 0 mm
valine-ester Allobetulin 3- 125 0 mm 0 mm 0 mm phosphate
Allobetulin 125 0 mm 0 mm 0 mm lactone phosphate Allobetulin 3- 125
0 mm 0 mm 0 mm phthalate Betulon-1,2-ene- 125 0 mm 0 mm 0 mm ol
Betulin 28- 125 0 mm 0 mm 0 mm glutarate Poly (ethylene 125 0 mm 0
mm 0 mm glycol) bis (carboxymethyl) ester Allobetulin-3- 125 0 mm 0
mm 0 mm poly (ethylene glycol) bis (carboxymethyl) ether ester
Ursolic Acid 125 0 mm 0 mm 0 mm Betulin 3,28- 125 11.0 mm 0 mm 0 mm
diphosphate Betulin 3,28- 125 12.0 mm 0 mm 0 mm diglutarate Betulin
125 10.0 mm 0 mm 0 mm diglycolate Allobetulon 125 0 mm 0 mm 0 mm
3.beta.-Acetoxy- 125 0 mm 0 mm 0 mm 19.alpha.H-19,28 lactone
oleanan Pencillin G 10 units 0 mm 40.0 mm 25.0 mm control
Streptomycin 10 mcg 20.0 mm 15.0 mm 24.0 mm control Tetracycline 30
mcg 19.0 mm 26.0 mm 31.0 mm control
Example 34
[0368] Additional compounds were tested for activity against E.
coli, laboratory strain S. aureus (not methicillin resistant), and
B. subtilis. The compounds were tested by the disk diffusion
method, as in Example 1, with application of a 10 mg/ml solution of
the test compound in DMSO. The results are shown in Table 2.
[0369] E. coli was sensitive to betulin-3,28-diglycine,
betulin-3-caffeate, and betulin-28-diglycolate. S. aureus was
sensitive to betulin-3,28-di-L-valine, as well as to
betulin-3,28-diphthalate. No compounds were found that inhibited B.
subtilis.
4TABLE 2 List of Compounds Tested Against Bacteria. Escherichia
Stapylococcus Bacillus S. No. Compound coli aureus subtilis
Allobetulin-3-succinate x x x Allobetulin lactone x x x Allobetulin
lactone-3-acetate x x x Allobetulin-3-L-alanine ester x x x
Allobetulin-3-L-valine ester x x x Allobetulin-3-L-proline ester x
x x Allobetulin-3-diglycolate x x x Allobetulin-3-glutarate x x x
Allobetulin-3-phthalate x x x Allobetulin-3-methylenamine x x x
Allobetulin ethanolamine x x x hydrochloride
Allobetulin-3-glycolate x x x Allobetulin-PEG-COOH x x x Mw = 674
Allobetulon lactone-1-en-2- x x x succinate Allobetulon-1-ene-2-ol
x x x Allobetulon-1-ene-2-diglycolate x x x Betulin-3,28-diglycine
ester x x x Betulin-28-glycine ester x x x Betulin arabinose
galactan x x x Betulin-3,28-diglycine ester o x x Betulin-3-maleate
x x x Betulin-3,28-Di-L-alanine ester x x x
Betulin-3-28-diphenylalanine x x x ester Betulin-3,28-di-L-proline
ester) x x x Betulin-3-caffeate o x x Betulin-3,28-di
(3',3'-dimethyl) x x x glutarate Betulin-3,28-didiglycolate x x x
Betulin-28-diglycolate o x x Betulin-3,28-diphthalate x o x
Betulin-3,28-Di-L-phenylalanine x x x ester
Betulin-3,28-Di-L-valine ester x o x Betulin-3,28-di-PEG-COOH x x x
Mw = 1448 Betulin-3,28-di-PEG-COOH x x x Mw = 906 crude
Allobetulin-3,28-(dipoly x x x (ethylene glycol)bis (carboxy-
methyl) Lupeol x x x Lupeol succinate x x x Ursolic acid x x x x =
compound tested o = compound showed anti-microbial activity
Example 35
[0370] Derivatives of lupeol were tested for inhibition of
bacterial growth using the Kirby-Bauer disk diffusion method and by
growth in liquid culture. For the disk diffusion method, compounds
were dissolved in DMSO and 100 .mu.l was added to the 10 mm filter
paper disk. The amount of compound applied to the disk varied. The
amount applied for each compound was as follows: lupeol (50 .mu.g),
lupeol-3-maleate (15 .mu.g), lupeol-3-thiodiglycolate (15 .mu.g),
lupeol-3-dimethyl succinate (60 .mu.g), lupeol-3-phosphate (30
.mu.g), lupenone (50 .mu.g), lupenon-3-oxime (50 .mu.g),
lupeol-3-amine (50 .mu.g), lupenon-1,2-ene-2-ol (50 .mu.g). In
liquid culture, the compounds were dissolved in DMSO at 1 mg/ml and
then 100 .mu.l was added to 7 ml of sterile broth, along with a
1/200 dilution of freshly grown bacterial liquid culture. The
strains tested were Staphylococcus aureus, Staphylococcus
epidemidis, and Enterococcus faecalis.
[0371] The results are shown in Tables 3 and 4.
5TABLE 3 Antibacterial activities against Staphylococcus aureus,
Staphylococcus epidemidis, and Enterococcus faecalis using the
Kirby-Bauer disk diffusion method. Compounds in descending order of
activity Zone of inhibition Compounds in Zone of inhibition
Compounds in Zone of inhibition (amount applied (mm) against S.
descending order (mm) against S. descending order (mm) against E.
to the disk) epiderm. of activity aureus of activity faecalis
Lupenon-1,2-ene-2- 2.65 Ampicillin 6.1 Ampicillin 5.75 ol
Ampicillin 2 Chloramphenicol 2.6 Lupeol 1.4 Lupenone 1 Lupenone
1.25 Chloraphenicol 1.25 Lupeol-3-maleate 0.5 Streptomycin 1.25
Lupeol-3-maleate 1.15 Lupeol-3- 0.5 Lupeol 1 Lupeol-3- 0.75
thiodiglycolate thiodiglycolate Chloramphenicol 0.5
Lupenon-1,2-ene-2- 0.85 Lupenon-1,2-ene-2- 0.6 ol ol Lupeol 0
Lupeol-3-(3',3'- 0.5 Lupenon oxime 0.6 dimethyl)succinate
Lupeol-3-(3',3'- 0 Lupeol-3- 0.5 Lupenone 0.5 dimethyl)succinate
phosphate Lupeol-3- 0 Lupeol-3-maleate 0.4 Lupeol-3- 0.5 phosphate
phosphate Lupenon oxime 0 Lupenon oxime 0.4 Lupeol-3-amine 0.5
Lupeol-3-amine 0 Lupeol-3- 0.3 Lupeol-3-(3',3'- 0 thiodiglycolate
dimethyl)succinate Gentamicin 0 Lupeol-3-amine 0.3 Gentamicin 0
Streptomycin 0 Gentamicin 0.1 Streptomycin 0
[0372]
6TABLE 4 Antibacterial activity against Staphylococcus aureus,
Staphylococcus epidermis, and Enterococcus faecilis using optical
density measurements. Optical density at 600 nm of bacteria in
liquid culture was measured after growth for 24 hours in the
presence of the test compound at 14 .mu.g per ml. Compounds in
Compounds in Compounds in descending order of Optical Density of
descending order of Optical Density of descending order of Optical
Density of activity S. epiderm. activity S. aureus activity E.
faecalis Lupenon-1,2-ene-2- 0.006 Lupenon-1,2-ene-2- 0.04
Lupenon-1,2-ene-2- 0.01 ol ol ol Lupenone 0.03 Lupenone 0.04
Lupeol-3-maleate 0.03 Lupeol-3- 0.035 Lupeol 0.06 Lupeol 0.03
thiodiglycolate Lupeol 0.04 Lupeol-3-maleate 0.06 Lupeol-3- 0.04
phosphate Lupeol-3-maleate 0.04 Lupeol-3- 0.06 Lupenone 0.04
thiodiglycolate Lupeol-3-(3'3'- 0.06 Lupeol-3-(3',3'- 0.06
Lupeol-3-amine 0.04 dimethyl)succinate dimethyl)succinate Lupeol-3-
0.06 Lupeol-3- 0.06 Lupeol-3- 0.04 phosphate phosphate
thiodiglycolate Lupenon oxime 0.06 Lupenon oxime 0.06 Lupenon oxime
0.45 Lupeol-3-amine 0.06 Lupeol-3-amine 0.06 Lupeol-(3',3'- 0.07
dimethyl)succinate None 0.07 None 0.08 None 0.85
[0373] The results with the disk diffusion method and by liquid
growth were consistent with each other. Lupenon-1,2-ene-2-ol was
the most active compound against almost all the bacteria,
especially as measured in liquid growth. Lupenone also had
excellent activity. Against S. epidermidis,
lepeol-3-thiodiglycolate lupeol-3-maleate, and lupeol were active.
Against S. aureus, all the tested compounds showed some activity.
Against E. faecalis, all the compounds except
lupeol-3-(3',3'-dimethyl)su- ccinate showed activity.
[0374] The compounds shown to be active against at least one strain
of bacteria in the assays above are the following:
betulin-3-caffeate; betulin-3,28-diglutarate;
betulin-28-diglycolate; betulin-3,28-diglycine;
betulin-3,28-didiglycolate; betulin-3,28-diphthalate;
betulin-3,28-diphosphate; betulin-3,28-disuccinate;
betulin-3,28-di-L-valine; lupeol; lupeol-3-amine;
lupeol-3-(3',3'-dimethy- l)succinate; lupeol-3-maleate;
lupeol-3-phosphate; lupeol-3-thiodiglycolat- e; lupenone;
lupenon-1,2-ene-2-ol; lupenon-3-oxime.
[0375] All publications, patents, and patent documents are
incorporated by reference herein, as though individually
incorporated by reference. The invention has been described with
reference to various specific and preferred embodiments and
techniques. However, it should be understood that many variations
and modifications may be made while remaining within the spirit and
scope of the invention.
* * * * *