U.S. patent application number 09/459840 was filed with the patent office on 2001-08-16 for hiv integrase inhibitors.
Invention is credited to DOMBROWSKI, ANNE W., FELOCK, PETER J., HAZUDA, DARIA JEAN, POLISHOOK, JON DAVID, SINGH, SHEO BUX, ZINK, DEBORAH L..
Application Number | 20010014748 09/459840 |
Document ID | / |
Family ID | 22342452 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014748 |
Kind Code |
A1 |
SINGH, SHEO BUX ; et
al. |
August 16, 2001 |
HIV INTEGRASE INHIBITORS
Abstract
Compounds useful in the inhibition of HIV integrase, the
prevention or treatment of infection by HIV and the treatment of
AIDS, either as compounds, pharmaceutically acceptable salts,
pharmaceutical composition ingredients, whether or not in
combination with other antivirals, immunomodulators, antibiotics or
vaccines are described. Methods of treating AIDS and methods of
preventing or treating infection by HIV are also described.
Further, the culture Fusarium sp., MF6381 (ATCC 74469) is also
disclosed, as well as processes for making compounds of structural
formula I employing the culture Fusarium sp., MF6381 (ATCC
74469).
Inventors: |
SINGH, SHEO BUX; (EDISON,
NJ) ; ZINK, DEBORAH L.; (MANALAPAN, NJ) ;
HAZUDA, DARIA JEAN; (DOYLESTOWN, PA) ; FELOCK, PETER
J.; (DOYLESTOWN, PA) ; POLISHOOK, JON DAVID;
(CRANFORD, NJ) ; DOMBROWSKI, ANNE W.; (EAST
BRUNSWICK, NJ) |
Correspondence
Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
Family ID: |
22342452 |
Appl. No.: |
09/459840 |
Filed: |
December 13, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60112168 |
Dec 14, 1998 |
|
|
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Current U.S.
Class: |
552/544 |
Current CPC
Class: |
C12P 33/20 20130101;
C12P 33/00 20130101 |
Class at
Publication: |
552/544 |
International
Class: |
C07J 009/00 |
Claims
What is claimed:
1. A compound of the formula I 21wherein: "a" is selected from a
single bond or a double bond; R.sup.1 is selected from: (a) --OH,
(b) --OC(O)CH.sub.3, (c)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H, (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (f)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.s- ub.3, (g)
--OC(O)(CH.sub.2).sub.2CONHOH, (h) --OCH.sub.2OCH.sub.3, (i)
--OC(O)C.sub.6H.sub.5, (j)
--OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3, (k) --OSO.sub.2CH.sub.3,
(l) --OC(O)CH.sub.2NH.sub.2, (m) --OC(O)--(CH.sub.2).sub.15--OH,
and (n) H; R.sup.2 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) .dbd.O, (d) --OC(O)(CH.sub.2).sub.2CO.sub.- 2H, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3, (f)
--OC(O)(CH.sub.2).sub.2CONHOH, (g) --OCH.sub.2OCH.sub.3, (h)
--OC(O)C.sub.6H.sub.5, (i) --OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3,
(j) --OSO.sub.2CH.sub.3, (k) --OSO.sub.2OH, and (l)
--OC(O)CH.sub.2NH.sub.2; or R.sup.1 and R.sup.2 are joined to form:
22R.sup.3 is selected from: (a) --H, (b) --OH, and (c)
--OC(O)CH.sub.3; R.sup.4 is selected from: (a) --H, (b) --OH, and
(c) --OC(O)CH.sub.3; R.sup.5 and R.sup.6 are independently selected
from: (a) --H, (b) --OH, and (c) --CH.sub.3, or together form: (c)
.dbd.CH.sub.2, or (d) --CH.sub.2O--; R.sup.7 is selected from: (a)
H, and (b) OH; or a pharmaceutically acceptable salt thereof;
provided that: when R.sup.1 is --H, --OH,
--OC(O)--(CH.sub.2).sub.15--OH, or --OC(O)CH.sub.3, and R.sup.2 is
--H, --OH, --OSO.sub.2OH, --OC(O)CH.sub.3, or
--OC(O)(CH.sub.2).sub.2CO.sub.2H- , and R.sup.3 is --OH, and
R.sup.4 is --OH or --OC(O)CH.sub.3, then R.sup.5 and R.sup.6 do not
together form (a) .dbd.CH.sub.2, or (b) --CH.sub.2O--.
2. The compound according to claim 1, wherein: R.sup.1 is selected
from: (a) --OH, (b) --OC(O)CH.sub.3, (c)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2- CO.sub.2H, (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (f)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.s- ub.3, (g)
--OC(O)(CH.sub.2).sub.2CONHOH, (h) --OCH.sub.2OCH.sub.3, (i)
--OC(O)C.sub.6H.sub.5, (j)
--OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3, (k) --OSO.sub.2CH.sub.3,
(l) --OC(O)CH.sub.2NH.sub.2, (m) --OC(O)--(CH.sub.2).sub.15--OH,
and (n) H; R.sup.2 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) .dbd.O, (d) --OC(O)(CH.sub.2).sub.2CO.sub.- 2H, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3, (f) --OCH.sub.2OCH.sub.3,
(g) --OC(O)C.sub.6H.sub.5, (h)
--OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3, (i) --OSO.sub.2OH, and (j)
--OC(O)CH.sub.2NH.sub.2; R.sup.3 is --OH; R.sup.4 is
--OC(O)CH.sub.3; R.sup.5 and R.sup.6 are independently selected
from: (a) --H, (b) --OH, and (c)--CH.sub.3, or together form: (c)
.dbd.CH.sub.2, or (d) --CH.sub.2O--; R.sup.7 is H; or a
pharmaceutically acceptable salt thereof; provided that: when
R.sup.1 is --H, --OH, --OC(O)--(CH.sub.2).sub.15--OH, or
--OC(O)CH.sub.3, and R.sup.2 is --H, --OH, --OSO.sub.2OH,
--OC(O)CH.sub.3, or --OC(O)(CH.sub.2).sub.2CO.sub.2H, then R.sup.5
and R.sup.6 do not together form (a) .dbd.CH.sub.2, or (b)
--CH.sub.2O--.
3. The compound according to claim 2 wherein: R.sup.1 is selected
from: (a) --OH, (b)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H, (c)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3, (d)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (e)
--OC(O)(CH.sub.2).sub.2CONHOH, (f) --OC(O)CH.sub.2NH.sub.2, and (g)
--OC(O)--(CH.sub.2).sub.15--OH; R.sup.2 is selected from: (a) --OH,
(b) .dbd.O, (c) --OC(O)(CH.sub.2).sub.2CO.sub- .2H, (d)
--OSO.sub.2OH, and (e) --OC(O)CH.sub.2NH.sub.2; R.sup.5 and R.sup.6
are independently selected from: (a) --H, and (b) --OH, or together
form: (c) .dbd.CH.sub.2, or (d) --CH.sub.2O--; or a
pharmaceutically acceptable salt thereof; provided that: when
R.sup.1 is --OH, and R.sup.2 is --OH, --OSO.sub.2OH, or
--OC(O)(CH.sub.2).sub.2CO.su- b.2H, then R.sup.5 and R.sup.6 do not
together form (a) .dbd.CH.sub.2, or (b) --CH.sub.2O--.
4. The compound according to claim 1 selected from: 23
5. The compound according to claim 1 selected from: 24
6. A composition useful for inhibiting HIV integrase, comprising an
effective amount of a compound according to claim 1 and a
pharmaceutically acceptable carrier.
7. The composition of claim 6, useful for treating infection by
HIV, or for treating AIDS or ARC.
8. A composition made by combining the compound of claim 1 and a
pharmaceutically acceptable carrier.
9. A process for making a pharmaceutical composition comprising
combining a compound of claim 1 and a pharmaceutically acceptable
carrier.
10. A method of inhibiting HIV integrase, comprising the
administration to a mammal in need of such treatment a
therapeutically effective amount of a compound of formula I:
25wherein: "a" is selected from a single bond or a double bond;
R.sup.1 is selected from: (a) --OH, (b) --OC(O)CH.sub.3, (c)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H, (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (f)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.s- ub.3, (g)
--OC(O)(CH.sub.2).sub.2CONHOH, (h) --OCH.sub.2OCH.sub.3, (i)
--OC(O)C.sub.6H.sub.5, (j)
--OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3, (k) --OSO.sub.2CH.sub.3,
(l) --OC(O)CH.sub.2NH.sub.2, (m) --OC(O)--(CH.sub.2).sub.15--OH,
and (n) H; R.sup.2 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) .dbd.O, (d) --OC(O)(CH.sub.2).sub.2CO.sub.- 2H, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3, (f)
--OC(O)(CH.sub.2).sub.2CONHOH, (g) --OCH.sub.2OCH.sub.3, (h)
--OC(O)C.sub.6H.sub.5, (i) --OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3,
(j) --OSO.sub.2CH.sub.3, (k) --OSO.sub.2OH, and (l)
--OC(O)CH.sub.2NH.sub.2; or R.sup.1 and R.sup.2 are joined to form:
26R.sup.3 is selected from: (a) --H, (b) --OH, and (c)
--OC(O)CH.sub.3; R.sup.4 is selected from: (a) --H, (b) --OH, and
(c) --OC(O)CH.sub.3; R.sup.5 and R.sup.6 are independently selected
from: (a) --H, (b) --OH, and (c) --CH.sub.3, or together form: (c)
.dbd.CH.sub.2, or (d) --CH.sub.2O--; R.sup.7 is selected from: (a)
H, and (b) OH; or a pharmaceutically acceptable salt thereof.
11. The method of inhibiting HIV integrase according to claim 10,
wherein: R.sup.1 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) --OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H, (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (f)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.s- ub.3, (g)
--OC(O)(CH.sub.2).sub.2CONHOH, (h) --OCH.sub.2OCH.sub.3, (i)
--OC(O)C.sub.6H.sub.5, (j)
--OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3, (k) --OSO.sub.2CH.sub.3,
(l) --OC(O)CH.sub.2NH.sub.2, (m) --OC(O)--(CH.sub.2).sub.15--OH,
and (n) H; R.sup.2 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) .dbd.O, (d) --OC(O)(CH.sub.2).sub.2CO.sub.- 2H, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3, (f) --OCH.sub.2OCH.sub.3,
(g) --OC(O)C.sub.6H.sub.5, (h)
--OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3, (i) --OSO.sub.2OH, and (j)
--OC(O)CH.sub.2NH.sub.2; R.sup.3 is --OH; R.sup.4 is
--OC(O)CH.sub.3; R.sup.5 and R.sup.6 are independently selected
from: (a) --H, (b) --OH, and (c) --CH.sub.3, or together form: (c)
.dbd.CH.sub.2, or (d) --CH.sub.2O--; R.sup.7 is H; or a
pharmaceutically acceptable salt thereof.
12. The method of inhibiting HIV integrase according to claim 10,
wherein the compound of structural formula I is selected from:
27
13. The method of inhibiting HIV integrase according to claim 10,
wherein the compound of structural formula I is selected from:
28
14. A method of treating infection by HIV, or of treating AIDS or
ARC, comprising the administration to a mammal in need of such
treatment a therapeutically effective amount of a compound of
formula I: 29wherein: "a" is selected from a single bond or a
double bond; R.sup.1 is selected from: (a) --OH, (b)
--OC(O)CH.sub.3, (c) --OC(O)CH.sub.2C(CH.sub.3)(OH)CH-
.sub.2CO.sub.2H, (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.- 3, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (f)
--OC(O)(CH.sub.2).sub.2CO.sub- .2CH.sub.3, (g)
--OC(O)(CH.sub.2).sub.2CONHOH, (h) --OCH.sub.2OCH.sub.3, (i)
--OC(O)C.sub.6H.sub.5, (j)
--OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3, (k) --OSO.sub.2CH.sub.3,
(l) --OC(O)CH.sub.2NH.sub.2, (m) --OC(O)--(CH.sub.2).sub.15--OH,
and (n) H; R.sup.2 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) .dbd.O, (d) --OC(O)(CH.sub.2).sub.2CO.sub.- 2H, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3, (f)
--OC(O)(CH.sub.2).sub.2CONHOH, (g) --OCH.sub.2OCH.sub.3, (h)
--OC(O)C.sub.6H.sub.5, (i) --OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3,
(j) --OSO.sub.2CH.sub.3, (k) --OSO.sub.2OH, and (l)
--OC(O)CH.sub.2NH.sub.2; or R .sup.1 and R.sup.2 are joined to
form: 30R.sup.3 is selected from: (a) --H, (b) --OH, and (c)
--OC(O)CH.sub.3; R.sup.4 is selected from: (a) --H, (b) --OH, and
(c) --OC(O)CH.sub.3; R.sup.5 and R.sup.6 are independently selected
from: (a) --H, (b) --OH, and (c) --CH.sub.3, or together form: (c)
.dbd.CH.sub.2, or (d) --CH.sub.2O--; R.sup.7 is selected from: (a)
H, and (b) OH; or a pharmaceutically acceptable salt thereof.
15. The method of treating infection by HIV, or of treating AIDS or
ARC according to claim 14, wherein: R.sup.1 is selected from: (a)
--OH, (b) --OC(O)CH.sub.3, (c)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H, (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (f)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.s- ub.3, (g)
--OC(O)(CH.sub.2).sub.2CONHOH, (h) --OCH.sub.2OCH.sub.3, (i)
--OC(O)C.sub.6H.sub.5, (j)
--OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3, (k) --OSO.sub.2CH.sub.3,
(l) --OC(O)CH.sub.2NH.sub.2, (m) --OC(O)--(CH.sub.2).sub.15--OH,
and (n) H; R.sup.2 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) .dbd.O, (d) --OC(O)(CH.sub.2).sub.2CO.sub.- 2H, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3, (f) --OCH.sub.2OCH.sub.3,
(g) --OC(O)C.sub.6H.sub.5, (h)
--OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3, (i) --OSO.sub.2OH, and (j)
--OC(O)CH.sub.2NH.sub.2; R.sup.3 is --OH; R.sup.4 is
--OC(O)CH.sub.3; R.sup.5 and R.sup.6 are independently selected
from: (a) --H, (b) --OH, and (c) --CH.sub.3, or together form: (c)
.dbd.CH.sub.2, or (d) --CH.sub.2O--; R.sup.7 is H; or a
pharmaceutically acceptable salt thereof.
16. The method of treating infection by HIV, or of treating AIDS or
ARC according to claim 14, wherein the compound of structural
formula I is selected from: 31
17. The method of treating infection by HIV, or of treating AIDS or
ARC according to claim 14,, wherein the compound of structural
formula I is selected from: 32
18. The method according to claim 14 additionally comprising the
administration of a therapeutically effective amount of another
AIDS treatment agent selected from: (a) an AIDS antiviral agent,
(b) an immunomodulator, and (c) an anti-infective agent.
19. The method according to claim 18 wherein the AIDS antiviral
agent is:
N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3-p-
yridylmethyl)-2(S)-N'-(t-butylcarboxamido)-piperazinyl))-pentaneamide;
or a pharmaceutically acceptable salt thereof.
20. A composition comprising a therapeutically effective amount of
a compound of structural formula I: 33wherein: "a" is selected from
a single bond or a double bond; R.sup.1 is selected from: (a) --OH,
(b) --OC(O)CH.sub.3, (c)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H, (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (f)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.s- ub.3, (g)
--OC(O)(CH.sub.2).sub.2CONHOH, (h) --OCH.sub.2OCH.sub.3, (i)
--OC(O)C.sub.6H.sub.5, (i)
--OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3, (k) --OSO.sub.2CH.sub.3,
(l) --OC(O)CH.sub.2NH.sub.2, (m) --OC(O)--(CH.sub.2).sub.15--OH,
and (n) H; R.sup.2 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) .dbd.O, (d) --OC(O)(CH.sub.2).sub.2CO.sub.- 2H, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3, (f)
--OC(O)(CH.sub.2).sub.2CONHOH, (g) --OCH.sub.2OCH.sub.3, (h)
--OC(O)C.sub.6H.sub.5, (i) --OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3,
(j) --OSO.sub.2CH.sub.3, (k) --OSO.sub.2OH, and (l)
--OC(O)CH.sub.2NH.sub.2; or R.sup.1 and R.sup.2 are joined to form:
34R.sup.3 is selected from: (a) --H, (b) --OH, and (c)
--OC(O)CH.sub.3; R.sup.4 is selected from: (a) --H, (b) --OH, and
(c) --OC(O)CH.sub.3; R.sup.5 and R.sup.6 are independently selected
from: (a) --H, (b) --OH, and (c) --CH.sub.3, or together form: (c)
.dbd.CH.sub.2, or (d) --CH.sub.2O--; R.sup.7 is selected from: (a)
H, and (b) OH; or a pharmaceutically acceptable salt thereof; in
combination with a therapeutically effective amount of an AIDS
treatment agent selected from: (a) an AIDS antiviral agent, (b) an
immunomodulator, and (c) an anti-infective agent, and a
pharmaceutically acceptable carrier.
21. The composition according to claim 20 wherein the AIDS
antiviral agent is:
N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4--
(3-pyridylmethyl)-2(S)-N'-(t-butylcarboxamido)-piperazinyl))-pentaneamide,
or a pharmaceutically acceptable salt thereof.
22. A biologically pure culture of MF6381 (ATCC 74469).
23. A culture of MF6381 (ATCC 74469).
24. A biologically pure culture of claim 22, or a mutant thereof
capable of producing a compound of structural formula (I)
35wherein: "a" is selected from a single bond or a double bond;
R.sup.1 is selected from: (a) --OH, (b)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (c)
--OC(O)--(CH.sub.2).sub.15--OH, and (d) H; R.sup.2 is selected
from: (a) --OH, and (b) --OSO.sub.2OH; R.sup.3 is --OH; R.sup.4 is
selected from: (a) --OH, and (b) --OC(O)CH.sub.3; R.sup.5 and
R.sup.6 together form: (a) .dbd.CH.sub.2, or (b) --CH.sub.2O--;
R.sup.7 is selected from: (a) H, and (b) OH; in recoverable
amounts.
25. A culture of claim 23 or a mutant thereof capable of producing
a compound of structural formula (I) 36wherein: "a" is selected
from a single bond or a double bond; R.sup.1 is selected from: (a)
--OH, (b) --OC(O)(CH.sub.2).sub.2CO.sub.2H, (c)
--OC(O)--(CH.sub.2).sub.15--OH, and (d) H; R.sup.2 is selected
from: (a) --OH, and (b) --OSO.sub.2OH; R.sup.3 is --OH; R.sup.4 is
selected from: (a) --OH, and (b) --OC(O)CH.sub.3; R.sup.5 and
R.sup.6 together form: (a) .dbd.CH.sub.2, or (b) --CH.sub.2O--;
R.sup.7 is selected from: (a) H, and (b) OH; in recoverable
amounts.
26. A process of making a compound of structural formula I
37wherein: "a" is selected from a single bond or a double bond;
R.sup.1 is selected from: (a) --OH, (b) --OC(O)CH.sub.3, (c)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH- .sub.2CO.sub.2H, (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.- 3, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2H, (f)
--OC(O)(CH.sub.2).sub.2CO.sub- .2CH.sub.3, (g)
--OC(O)(CH.sub.2).sub.2CONHOH, (h) --OCH.sub.2OCH.sub.3, (i)
--OC(O)C.sub.6H.sub.5, (j)
--OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3, (k) --OSO.sub.2CH.sub.3,
(l) --OC(O)CH.sub.2NH.sub.2, (m) --OC(O)--(CH.sub.2).sub.15--OH,
and (n) H; R.sup.2 is selected from: (a) --OH, (b) --OC(O)CH.sub.3,
(c) .dbd.O, (d) --OC(O)(CH.sub.2).sub.2CO.sub.- 2H, (e)
--OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3, (f)
--OC(O)(CH.sub.2).sub.2CONHOH, (g) --OCH.sub.2OCH.sub.3, (h)
--OC(O)C.sub.6H.sub.5, (i) --OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3,
(j) --OSO.sub.2CH.sub.3, (k) --OSO.sub.2OH, and (l)
--OC(O)CH.sub.2NH.sub.2; or R.sup.1 and R.sup.2 are joined to form:
38R.sup.3 is selected from: (a) --H, (b) --OH, and (c)
--OC(O)CH.sub.3; R.sup.4 is selected from: (a) --H, (b) --OH, and
(c) --OC(O)CH.sub.3; R.sup.5 and R.sup.6 are independently selected
from: (a) --H, (b) --OH, and (c) --CH.sub.3, or together form: (c)
.dbd.CH.sub.2, or (d) --CH.sub.2O--; R.sup.7 is selected from: (a)
H, and (b) OH; comprising cultivating MF6381 (ATCC 74469) or a
mutant thereof under conditions suitable for formation of the
compound and recovering the compound.
27. The process according to claim 26, additionally comprising the
step of performing synthetic organic chemistry on the recovered
compound, and recovering the chemically-modified compound.
28. The process according to claim 26, wherein: "a" is selected
from a single bond or a double bond; R.sup.1 is selected from: (a)
--OH, (b) --OC(O)(CH.sub.2).sub.2CO.sub.2H, (c)
--OC(O)--(CH.sub.2).sub.15--OH, and (d) H; R.sup.2 is selected
from: (a) --OH, and (b) --OSO.sub.2OH; R.sup.3 is --OH; R.sup.4 is
selected from: (a) --OH, and (b) --OC(O)CH.sub.3; R.sup.5 and
R.sup.6 together form: (a) .dbd.CH.sub.2, or (b) --CH.sub.2O--;
R.sup.7 is selected from: (a) H, and (b) OH.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of United States
provisional patent application Ser. No. 60/112,168, filed Dec. 14,
1998, the disclosure of which is hereby incorporated by reference
in its entirety.
BACKGROUND OF THE INVENTION
[0002] A retrovirus designated human immunodeficiency virus (HIV)
is the etiological agent of the complex disease that includes
progressive destruction of the immune system (acquired immune
deficiency syndrome; AIDS) and degeneration of the central and
peripheral nervous system. This virus was previously known as LAV,
HTLV-III, or ARV. A common feature of retrovirus replication is the
insertion by virally-encoded integrase of proviral DNA into the
host cell genome, a required step in HIV replication in human
T-lymphoid and monocytoid cells. Integration is believed to be
mediated by integrase in three steps: assembly of a stable
nucleoprotein complex with viral DNA sequences; cleavage of two
nucleotides from the 3' termini of the linear proviral DNA;
covalent joining of the recessed 3' OH termini of the proviral DNA
at a staggered cut made at the host target site. The fourth step in
the process, repair synthesis of the resultant gap, may be
accomplished by cellular enzymes.
[0003] Nucleotide sequencing of HIV shows the presence of a pol
gene in one open reading frame [Ratner, L. et al., Nature, 313,
277(1985)]. Amino acid sequence homology provides evidence that the
pol sequence encodes reverse transcriptase, integrase and an HIV
protease [Toh, H. et al., EMBO J. 4, 1267 (1985); Power, M. D. et
al., Science, 231, 1567 (1986); Pearl, L. H. et al., Nature, 329,
351 (1987)]. All three enzymes have been shown to be essential for
the replication of HIV.
[0004] It is known that some antiviral compounds which act as
inhibitors of HIV replication are effective agents in the treatment
of AIDS and similar diseases, e.g., azidothymidine or AZT.
Applicants demonstrate that the compounds of this invention are
inhibitors of HIV integrase and inhibitors of HIV replication. The
applicants additionally demonstrate that inhibition of integrase in
vitro and HIV replication in cells is a direct result of inhibiting
the strand transfer reaction catalyzed by the recombinant integrase
in vitro and integrase as a component of the preintegration complex
in HIV infected cells. The particular advantage of the present
invention is highly specific inhibition of HIV integrase and HIV
replication. The compounds of the present invention inhibit
integrases of closely related lentiviruses such as HIV 2 and SIV,
but not integrases from more distantly related retroviruses, for
example RSV. These compounds do not inhibit binding or catalysis of
other nucleic acid binding proteins, including enzymatic reactions
such as those catalyzed by HIV reverse transcriptase, HIV Rnase H,
Influenza transcriptase, Hepatitis C polymerase, Yeast DNA
polymerase, DNase I, Eco RI endonuclease, or mammalian polymerase
II.
[0005] Zhao et al., (J. Med Chem. vol. 40, pp. 937-941 and
1186-1194 (1997)) describe hydrazide and arylamide HIV integrase
inhibitors. Bis-catechols useful for inhibiting HIV integrase are
described in LaFemina et al. (Antimicrobial Agents &
Chemotherapy, vol. 39, no. 2, pp. 320-324, February 1995).
[0006] U.S. Pat. No. 4,871,727 to Burg et al. describes
anti-inflammatory and degenerative compounds isolated from the soil
microorganism ATCC 20858 of structural formula A below: 1
[0007] wherein:
[0008] R is OH or H;
[0009] R .sup.1 and R.sup.2 together form .dbd.CH.sub.2 or
--CH.sub.2O--;
[0010] R.sup.3 is H, OH, HSO.sub.3O,
HOCOCH.sub.2CH.sub.2CO.sub.2;
[0011] R.sup.4 is OH, HOC.sub.15H.sub.30CO.sub.2, AcO or is H
except that when R.sup.4 is H, the double bond in the cyclopentane
ring is absent;
[0012] R.sup.5 is O.dbd., OH, AcO; and
[0013] R.sup.6 is OH or --O--C(O)CH.sub.3.
[0014] Brill et al. (J. Antibiotics, 49(6): 541-546 (1996)),
describe particular triterpene sulfates from Fusarium
compactum.
[0015] PCT publication WO 98/31371 (Application No. PCT/US98/00766)
describe the use of androst-5-ene-3.beta.-ol-7,17 dione and
metabolizable precursors thereof, such as
androst-5-ene-3.beta.-acetoxy-7,17-dione, for the treatment of
HIV-related weight loss, HIV-related cachexia and HIV-related
wasting syndrome.
[0016] Applicants have discovered that certain natural product
compounds derived from Fusarium sp. MF6381 (ATCC 74469) and
derivatives thereof are potent inhibitors of HIV integrase. These
compounds are useful for the treatment of AIDS or HIV
infections.
BRIEF DESCRIPTION OF THE INVENTION
[0017] Compounds of formula I, as herein defined, are disclosed.
These compounds are useful in the inhibition of HIV integrase, the
prevention of infection by HIV, the treatment of infection by HIV
and in the treatment of AIDS and/or ARC, either as compounds,
pharmaceutically acceptable salts or hydrates (when appropriate),
pharmaceutical composition ingredients, whether or not in
combination with other antivirals, anti-infectives,
immunomodulators, antibiotics or vaccines. Methods of treating
AIDS, methods of preventing infection by HIV, and methods of
treating infection by HIV are also disclosed.
[0018] Further, the culture Fusarium sp., MF6381 (ATCC 74469) is
also disclosed, as well as processes for making compounds of
structural formula I employing the culture Fusarium sp., MF6381
(ATCC 74469).
DETAILED DESCRIPTION OF THE INVENTION
[0019] This invention is concerned with compounds of formula I,
combinations thereof, or pharmaceutically acceptable salts thereof,
in the inhibition of HIV integrase, the prevention or treatment of
infection by HIV and in the treatment of the resulting acquired
immune deficiency syndrome (AIDS). Compounds of formula I are
defined as follows: 2
[0020] wherein:
[0021] "a" is selected from a single bond or a double bond;
[0022] R.sup.1 is selected from:
[0023] (a) --OH,
[0024] (b) --OC(O)CH.sub.3,
[0025] (c) --OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H,
[0026] (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3,
[0027] (e) --OC(O)(CH.sub.2).sub.2CO.sub.2H,
[0028] (f) --OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3,
[0029] (g) --OC(O)(CH.sub.2).sub.2CONHOH,
[0030] (h) --OCH.sub.2OCH.sub.3,
[0031] (i) --OC(O)C.sub.6H.sub.5,
[0032] (j) --OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3,
[0033] (k) --OSO.sub.2CH.sub.3,
[0034] (l) --OC(O)CH.sub.2NH.sub.2,
[0035] (m) --OC(O)--(CH.sub.2).sub.15--OH, and
[0036] (n) H;
[0037] R.sup.2 is selected from:
[0038] (a) --OH,
[0039] (b) --OC(O)CH.sub.3,
[0040] (c) .dbd.O,
[0041] (d) --OC(O)(CH.sub.2).sub.2CO.sub.2H,
[0042] (e) --OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3,
[0043] (f) --OC(O)(CH.sub.2).sub.2CONHOH,
[0044] (g) --OCH.sub.2OCH.sub.3,
[0045] (h) --OC(O)C.sub.6H.sub.5,
[0046] (i) --OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3,
[0047] (j) --OSO.sub.2CH.sub.3,
[0048] (k) --OSO.sub.2OH, and
[0049] (l) --OC(O)CH.sub.2NH.sub.2;
[0050] or R.sup.1 and R.sup.2 are joined to form: 3
[0051] R.sup.3 is selected from:
[0052] (a) --H,
[0053] (b) --OH, and
[0054] (c) --OC(O)CH.sub.3;
[0055] R.sup.4 is selected from:
[0056] (a) --H,
[0057] (b) --OH, and
[0058] (c) --OC(O)CH.sub.3;
[0059] R.sup.5 and R.sup.6 are independently selected from:
[0060] (a) --H,
[0061] (b) --OH, and
[0062] (c) --CH.sub.3,
[0063] or together form:
[0064] (c) .dbd.CH.sub.2, or
[0065] (d) --CH.sub.2O--;
[0066] R.sup.7 is selected from:
[0067] (a) H, and
[0068] (b) OH;
[0069] or a pharmaceutically acceptable salt thereof.
[0070] In one class of compounds of the present invention, R.sup.1
is selected from:
[0071] (a) --OH,
[0072] (b) --OC(O)CH.sub.3,
[0073] (c) --OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H,
[0074] (d)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3,
[0075] (e) --OC(O)(CH.sub.2).sub.2CO.sub.2H,
[0076] (f) --OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3,
[0077] (g) --OC(O)(CH.sub.2).sub.2CONHOH,
[0078] (h) --OCH.sub.2OCH.sub.3,
[0079] (i) --OC(O)C.sub.6H.sub.5,
[0080] (j) --OC(O)CH.sub.2NH--C(O)OC(CH.sub.3).sub.3,
[0081] (k) --OSO.sub.2CH.sub.3,
[0082] (l) --OC(O)CH.sub.2NH.sub.2,
[0083] (m) --OC(O)--(CH.sub.2).sub.15--OH, and
[0084] (n) H.
[0085] In a subclass of compounds of the present invention, R.sup.1
is selected from:
[0086] (a) --OH,
[0087] (b) --OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2H,
[0088] (c)
--OC(O)CH.sub.2C(CH.sub.3)(OH)CH.sub.2CO.sub.2CH.sub.3,
[0089] (d) --OC(O)(CH.sub.2).sub.2CO.sub.2H,
[0090] (e) --OC(O)(CH.sub.2).sub.2CONHOH,
[0091] (f) --OC(O)CH.sub.2NH.sub.2, and
[0092] (g) --OC(O)--(CH.sub.2).sub.15--OH.
[0093] In one class of compounds of the present invention, R.sup.2
is selected from:
[0094] (a) --OH,
[0095] (b) --OC(O)CH.sub.3,
[0096] (c) .dbd.O,
[0097] (d) --OC(O)(CH.sub.2).sub.2CO.sub.2H,
[0098] (e) --OC(O)(CH.sub.2).sub.2CO.sub.2CH.sub.3,
[0099] (f) --OCH.sub.2OCH.sub.3,
[0100] (g) --OC(O)C.sub.6H.sub.5,
[0101] (h) --OC(O)CH.sub.2NHC(O)OC(CH.sub.3).sub.3,
[0102] (i) --OSO.sub.2OH, and
[0103] (j) --OC(O)CH.sub.2NH.sub.2.
[0104] In a subclass of compounds of the present invention, R.sup.2
is selected from:
[0105] (a) --OH,
[0106] (b) .dbd.O,
[0107] (c) --OC(O)(CH.sub.2).sub.2CO.sub.2H,
[0108] (d) --OSO.sub.2OH, and
[0109] (e) --OC(O)CH.sub.2NH.sub.2.
[0110] In one class of compounds of the present invention, R.sup.4
is --OC(O)CH.sub.3.
[0111] In another class of compounds of the present invention,
R.sup.5 and R.sup.6 independently are selected from:
[0112] (a) --H, and
[0113] (b) --OH,
[0114] or together form:
[0115] (c) .dbd.CH.sub.2, or
[0116] (d) --CH.sub.2O--.
[0117] In still another class of compounds of the present
invention, R.sup.7 is hydrogen.
[0118] Also included within the present invention are
pharmaceutical compositions useful for inhibiting HIV integrase,
comprising an effective amount of a compound of this invention, and
a pharmaceutically acceptable carrier. Pharmaceutical compositions
useful for treating infection by HIV, or for treating AIDS or ARC,
are also encompassed by the present invention, as well as a method
of inhibiting HIV integrase, and a method of treating infection by
HIV, or of treating AIDS or ARC. Additionally, the present
invention is directed to a pharmaceutical composition comprising a
therapeutically effective amount of a compound of the present
invention in combination with a therapeutically effective amount of
an AIDS treatment agent selected from:
[0119] (1) an AIDS antiviral agent,
[0120] (2) an anti-infective agent, and
[0121] (3) an immunomodulator.
[0122] The compounds of the present invention may have asymmetric
centers and may occur, except when specifically noted, as mixtures
of stereoisomers or as individual diastereomers, or enantiomers,
with all isomeric forms being included in the present
invention.
[0123] Some of the compounds of the present invention are made by
chemical modification of Compound A: 4
[0124] U.S. Pat. No. 4,871,727 describes the production of Compound
A by cultivation from a soil microorganism, ATCC 20858. Compound A
is therein described as an elastase inhibitor.
[0125] U.S. Pat. No. 4,871,727 also describes the production of the
following compounds by cultivation from the soil microorganism ATCC
20858, or alternatively by cultivation from the soil microorganism
ATCC 20858 followed by chemical modification: 5
[0126] This invention also discloses the culture MF6381 (ATCC
74469) identified as Fusarium sp.
[0127] In addition, compounds of the present invention, including
Compound A, may be prepared by fermentation of the culture MF6381,
ATCC 74469.
[0128] Compounds of the present invention may be prepared by
chemical modification of Compound A, or Compound B.
[0129] The present invention also relates to the preparation of
compounds of structural formula I comprising:
[0130] (a) fermenting a culture of MF6381 (ATCC 74469), Fusarium
sp. or a mutant thereof to produce a fermentation broth,
[0131] (b) extracting the fermentation broth with an organic
solvent,
[0132] (c) isolating the compounds of structural formula I.
[0133] Alternatively, an additional step may be performed:
[0134] (d) chemically modifying the isolated compound of formula
I.
[0135] The compounds of structural formula I are preferably
isolated by partitioning the fermentation extract between the
organic solvent and water, followed by size exclusion
chromatography and normal or reverse-phase chromatography.
[0136] When any variable (e.g., R.sup.1, R.sup.2, etc.) occurs more
than one time in any constituent or in formula I, its definition on
each occurrence is independent of its definition at every other
occurrence. Also, combinations of substituents and/or variables are
permissible only if such combinations result in stable
compounds.
[0137] The compounds of the present inventions are useful in the
inhibition of HIV integrase, the prevention or treatment of
infection by human immunodeficiency virus (HIV) and the treatment
of consequent pathological conditions such as AIDS. Treating AIDS
or preventing or treating infection by HIV is defined as including,
but not limited to, treating a wide range of states of IV
infection: AIDS, ARC (AIDS related complex), both symptomatic and
asymptomatic, and actual or potential exposure to HIV. For example,
the compounds of this invention are useful in treating infection by
HIV after suspected past exposure to HIV by e.g., blood
transfusion, exchange of body fluids, bites, accidental needle
stick, or exposure to patient blood during surgery.
[0138] The compounds of this invention are useful in the
preparation and execution of screening assays for antiviral
compounds. For example, the compounds of this invention are useful
for isolating enzyme mutants, which are excellent screening tools
for more powerful antiviral compounds. Furthermore, the compounds
of this invention are useful in establishing or determining the
binding site of other antivirals to HIV integrase, e.g., by
competitive inhibition. Thus, the compounds of this invention are
commercial products to be sold for these purposes.
[0139] The present invention also provides for the use of a
compound of structural formula (I) to make a pharmaceutical
composition useful for inhibiting HIV integrase and in the
treatment of AIDS or ARC.
[0140] Applicants have discovered that compounds of structural
formula (I), are useful for inhibiting HIV integrase. The compounds
of formula (I) are prepared by an aerobic fermentation.
[0141] ATCC Deposit of MF6381 (ATCC 74469), Identified as Fusarium,
sp..
[0142] Before the U.S. filing date of the present application, a
sample of MF6381 (ATCC 74469), Fusarium sp., was deposited at the
American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, United States of America under
the terms of the Budapest Treaty. The culture access designation is
ATCC 74469. This deposit will be maintained in the ATCC for at
least 30 years and will be made available to the public upon the
grant of a patent disclosing it. It should be understood that the
availability of a deposit does not constitute a license to practice
the subject invention in derogation of patent rights granted by
government action.
[0143] General Characteristics and Description of MF6381 (ATCC
74469) Fusarium sp.
[0144] MF6381 was isolated from soil collected in Africa.
[0145] In the following description, MF6381 was edge inoculated
with a 5 mm diameter plug on 2,100 mm petri dishes for each the
following growth media. All cultures were incubated for 10 days at
25.degree. C. and 67% relative humidity in 12 hr photoperiod in
fluorescent light unless otherwise indicated. In addition, all
capitalized color names are from Ridgway, Color Standards and
Nomenclature, (Published by author, Washington D.C., 1912) 43p.+53
pl.
[0146] On oatmeal agar (Difco) colony mat attaining a diameter of
55 mm. Culture mat thickly woolly, forming distinct tufts. Area of
the inoculation point yellow (Buff Yellow, Pinard Yellow), at
colony center and margin light red to pink (Venetian Pink, Chatenay
Pink, Alizarine Pink). Margin white, entire. Reverse brown (Apricot
Buff). Exudate and soluble pigment absent.
[0147] On potato-dextrose agar (Difco), colony mat attaining a
diameter of 67 mm. Culture mat cottony to flat, consistent
throughout. Area of the inoculation point yellow (Antimony Yellow,
Ochraceous Buff). Area of colony center pink (Flesh Pink, Chatenay
Pink, Flesh Color). Margin light brown (Salmon-Buff), entire.
Reverse dark brown at inoculation point (Hazel, Cinnamon-Rufous) to
light brown near margin (Salmon-Buff). Exudate and soluble pigment
absent.
[0148] On cornmeal agar (Difco), colony mat attaining a diameter of
70 mm. Colony mat cottony, forming sparse, white tufts of mycelium,
otherwise hyaline. Margin entire, hyaline. Exudate, reverse and
soluble pigment absent.
[0149] On YME agar (malt extract, 10.0 g; yeast extract, 4.0 g;
dextrose, 4.0 g; agar, 20.0 g, distilled water, 1 L) attaining a
diameter of 67 mm. Culture mat cottony, at colony center hyphae
aggregated into tufts. Culture mat at inoculation point light
yellow (Light Buff, Cream Color) and light pink near margin (Flesh
Pink, Chatenay Pink). Reverse, exudate and soluble pigment absent.
At 37.degree. C., in the dark and no humidity control, culture mat
attaining a diameter of 10 mm. Culture mat mostly appressed to
slight cottony, mostly with light brown sections (Pale
Ochraceous-Buff, Warm Buff), sulcate. Reverse, exudate and soluble
pigment absent.
[0150] Microscopic: Hyphae hyaline, usually 2-3 .mu.m wide, up to 4
.mu.m wide. No conidiophores observed. Conidia, hyaline,
elliptical, 5-6 .times.2-3 .mu.m. Large swollen cells, 10 .mu.m
diameter, singly or in clusters.
[0151] Although MF6381 is easily placed into the genus Fusarium
(Ascomycotina, Hypocreales) by typical growth characteristics
observed in plate cultures and certain microscopic features, it is
difficult to speciate this fungus without additional
characteristics to distinguish it from the numerous described
species of Fusarium.
[0152] For a more complete description, another characteristic of
MF6581 included below is the ribosomal DNA sequence of the Internal
Transcribed Spacer (ITS) region using the primers ITS 1 and ITS4.
The primers and the techniques used to recover the sequence are
described in White, et. al., Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics, in PCR Protocols: a
Guide to Methods and Applications 315-322 (Innis, M. A., et al.,
eds., Academic Press 1990).
[0153] By comparing the sequence from MF6381 using a BLAST search
of GenBank, it was determined that MF6381 is closely related (99%
match of 502 base pairs) to another Fusarium sp. (NRRL 25483,
GenBank accession: u61695, NID g3320369), isolated from Pennisetum
typhoideum collected in Namibia. It has been cited in O'Donnell et
al., Molecular systematics and phylogeography of the Gibberella
fujikuroi species complex, Mycologia 90, 465 (1998).
[0154] Sequence from Nuclear ribosomal DNA (rDNA) Internal
Transcribed Spacer (ITS) region using primers ITS1/ITS4 (SEQ. D.
NO.: 1)
[0155] TTTACAACTCCCAAACCCCTGTGAACATACCTATACGTTGCCTCGGC
GGATCAGCCCGCGCCCCGTAAAACGGGACGGCCCGCCGCAGGACCC
ATAAACCCTGAATTTTATTGTAACTT- CTGAGTTTAAAAAACAAATAA
ATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGA
ACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAA
TCATCGAATCTTTGAACGCACATTGC- GCCCGCCAGTATTCTGGCGGG
CATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCCCGGGTTTGGT
GTTGGGGATCGGGCTGCGGTTCTACCGCGTCCCGGCCCCGAAATCT
AGTGGCGGTCTCGCTGCAGCCTCCAT- TGCGTAGTAGCTAACACCTCG
CAACTGGAACGCGGCGCGGCCAAGCCGTTAAACCCCCAACTTCTGA
ATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAA
[0156] In general, MF6381 (ATCC 74469) is strain cultured on a
solid medium, or in an aqueous nutrient medium containing sources
of assimilable carbon and nitrogen. For example, the cultures can
be grown under submerged aerobic conditions (e.g., shaking culture,
submerged culture, etc.) The aqueous medium is preferably
maintained at a pH of about 6-8 at the initiation of the
fermentation process. The desired pH may be maintained by the
choice of nutrient materials which inherently possess buffering
properties, or alternatively by the use of a buffer such as
morpholinoethanesulfonic acid (MES), morpholino-propanesulfonic
acid (MOPS), and the like.
[0157] The sources of carbon in the nutrient medium are
carbohydrates such as glucose, xylose, galactose, glycerin, starch,
sucrose, dextrin, and the like. Other sources which may be included
are maltose, rhamnose, raffinose, arabinose, mannose, sodium
succinate, and the like.
[0158] The sources of nitrogen are yeast extract, meat extract,
peptone, gluten meal, cottonseed meal, soybean meal and other
vegetable meals (partially or totally defatted), casein
hydrolysates, soybean hydrolysates, and yeast hydrolysates, corn
steep liquor, dried yeast, wheat germ, feather meal, peanut powder,
distiller's solubles, etc., as well as inorganic and organic
nitrogen compounds such as ammonium salts (e.g., ammonium nitrate,
ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and
the like.
[0159] The carbon and nitrogen sources, though advantageously
employed in combination, need not be used in their pure form,
because less pure materials which contain traces of growth factors
and considerable quantities of mineral nutrients, are also suitable
for use. When desired, there may be added to the medium mineral
salts such as sodium or calcium carbonate, sodium or potassium
phosphate, sodium or potassium chloride, sodium or potassium
iodide, magnesium salts, copper salts, cobalt salts, and the like.
If necessary, especially when the culture medium foams seriously, a
defoaming agent, such as liquid paraffin, fatty oil, plant oil,
mineral oil or silicone may be added.
[0160] Agitation and aeration of the culture mixture may be
accomplished in a variety of ways. Agitation may be provided by a
propeller or similar mechanical agitation equipment, by revolving
or shaking the fermentor or growth flask, by various pumping
equipment, or by the passage of sterile air through the medium.
Aeration may be effected by passing sterile air through the
fermentation mixture.
[0161] The fermentation is usually conducted at a temperature
between about 20.degree. C. and 30.degree. C., preferably
22-25.degree. C., for a period of about 14-21 days, which may be
varied according to fermentation conditions and scales.
[0162] As to the conditions for the production of cells in massive
amounts, submerged aerobic cultural conditions is one method of
culturing the cells. For the production in small amounts, a shaking
or surface culture in a flask or bottle is employed. The use of
fermentors (tanks) is preferred for the generation of large
quantities of materials. Fermentors can be sterilized with the
production medium or can be sterilized empty and the medium sent
through a continuous sterilizer, which is preferred for very large
fermentations (20,000 gallons or larger). Preferably, the pH of the
medium is adjusted to about 6-7, generally using acid or base
additions, preferably made automatically with a pH electrode and a
controller. The parameters for fermenter operation include
agitation, aeration, temperature and pressure. Agitation is
preferably carried out by mixing the medium with a number of
impellers mounted on a rotating agitator shaft located in the midst
of the tank. Aeration may be carried out by a variety of means,
preferably by bubbling sterile air into the medium, preferably at
0.25 v.v.m. to 1.0 v.v.m. (e.g., airflow=7 liters/minute at a
medium volume of 14 liters equals 0.5 v.v.m.) The pressure in the
tank would be maintained between 3 psig to 15 psig. Temperature is
preferably maintained at between about 20.degree. C. and 30.degree.
C., preferably 22-25.degree. C.
[0163] When the growth is carried out in large tanks, vegetative
forms of the organism for inoculation in the production tanks may
be employed in order to avoid growth lag in the process of
production. This requires production of a vegetative inoculum of
the organism by inoculating a relatively small quantity of culture
medium with spores or mycelia of the organism produced in a "slant"
and culturing said inoculated medium, also called the "seed
medium", and then transferring the cultured vegetative inoculum
aseptically to large tanks. The fermentation medium, in which the
inoculum is produced, is generally sterilized prior to inoculation.
The pH of the medium is generally adjusted to about 6-7 prior to
the autoclaving step, generally using acid or base additions,
preferably made automatically with a pH electrode and a
controller.
[0164] Preferred culturing/production media for carrying out the
fermentation are those set forth in the Examples.
[0165] After growth is completed, the cells are harvested by adding
the appropriate solvent, e.g. methylethylketone, to the entire
culture medium and cells. If the culture is grown in a liquid
fermentation, the growth could be harvested by other conventional
methods, e.g., centrifugation and filtration, and then extracted
with the appropriate solvent, e.g., methylethylketone, ethyl
acetate, methylene chloride and the like.
[0166] Preferably, the broth filtrate is diluted with a suitable
solvent such as methanol or acetone and the product is recovered on
resins such as SP207, HP20, amberchrome and the like.
[0167] The product of the present invention can be recovered from
the culture medium by conventional means which are commonly used
for the recovery of other known substances. The substances produced
may be found in either or both the cultured mycelium and broth
filtrate, and accordingly can be isolated and purified from the
mycelium and the filtrate, which are obtained by filtering or
centrifuging the cultured broth, by a conventional method such as
concentration under reduced pressure, lyophilization, extraction
with a conventional solvent, such as methylene chloride or methanol
and the like, pH adjustment, treatment with a conventional resin
(e.g., anion or cation exchange resin, non-ionic adsorption resin,
etc.), treatment with a conventional adsorbent (e.g., activated
charcoal, silicic acid, silica gel, cellulose, alumina, etc.),
crystallization, recrystallization, and the like. A preferred
method is extraction of cultured whole broth with
methylethylketone, followed by filtration of the extract through
filtering aid such as diatomaceous earth. The methylethylketone
layer of the filtrate is separated and concentrated to dryness
initially by evaporating under reduced pressure followed by
lyophilization. The compounds are finally isolated either by
solvent partitioning and crystallization or by size exclusion,
normal, and or reversed-phase HPLC.
[0168] Compounds of formula (I) may be isolated from the aerobic
fermentation of a culture of MF6381 (ATCC 74469). A culture of
MF6381 (ATCC 74469) is defined as substantially free of its natural
soil contaminants and capable of forming compounds of structural
formula (I) in recoverable amounts. The culture employed in the
present invention should be free from viable contaminating
microorganisms deleterious to the production of the compound of
structural formula (I). A biologically pure culture of MF6381 (ATCC
74469) may also be employed.
[0169] The compounds of the present invention may be administered
in the form of pharmaceutically acceptable salts. The term
"pharmaceutically acceptable salt" is intended to include all
acceptable salts such as acetate, lactobionate, benzenesulfonate,
laurate, benzoate, malate, bicarbonate, maleate, bisulfate,
mandelate, bitartrate, mesylate, borate, methylbromide, bromide,
methylnitrate, calcium edetate, methylsulfate, camsylate, mucate,
carbonate, napsylate, chloride, nitrate, clavulanate,
N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate,
edetate, oxalate, edisylate, pamoate (embonate), estolate,
palmitate, esylate, pantothenate, fumarate, phosphate/diphosphate,
gluceptate, polygalacturonate, gluconate, salicylate, glutamate,
stearate, glycollylarsanilate, sulfate, hexylresorcinate,
subacetate, hydrabamine, succinate, hydrobromide, tannate,
hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide,
tosylate, isothionate, triethiodide, lactate, panoate, valerate,
and the like which can be used as a dosage form for modifying the
solubility or hydrolysis characteristics or can be used in
sustained release or pro-drug formulations. Depending on the
particular functionality of the compound of the present invention,
pharmaceutically acceptable salts of the compounds of this
invention include those formed from cations such as sodium,
potassium, aluminum, calcium, lithium, magnesium, zinc, and from
bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,
arginine, ornithine, choline, N,N'-dibenzylethylenediamine,
chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,
diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and
tetramethylammonium hydroxide. These salts may be prepared by
standard procedures, e.g. by reacting a free acid with a suitable
organic or inorganic base. Where a basic group is present, such as
amino, an acidic salt, i.e. hydrochloride, hydrobromide, acetate,
pamoate, and the like, can be used as the dosage form.
[0170] Also, in the case of an acid (--COOH) or alcohol group being
present, pharmaceutically acceptable esters can be employed, e.g.
acetate, maleate, pivaloyloxymethyl, and the like, and those esters
known in the art for modifying solubility or hydrolysis
characteristics for use as sustained release or prodrug
formulations.
[0171] For these purposes, the compounds of the present invention
may be administered orally, parenterally (including subcutaneous
injections, intravenous, intramuscular, intrasternal injection or
infusion techniques), by inhalation spray, or rectally, in dosage
unit formulations containing conventional non-toxic
pharmaceutically-acceptabl- e carriers, adjuvants and vehicles.
[0172] The terms "administration of" and or "administering a"
compound should be understood to mean providing a compound of the
invention or a prodrug of a compound of the invention to the
individual in need of treatment.
[0173] Thus, in accordance with the present invention there is
further provided a method of treating and a pharmaceutical
composition for treating HIV infection and AIDS. The treatment
involves administering to a patient in need of such treatment a
pharmaceutical composition comprising a pharmaceutical carrier and
a therapeutically-effective amount of a compound of the present
invention.
[0174] As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results directly,
or indirectly, from combination of the specified ingredients in the
specified amounts.
[0175] By "pharmaceutically acceptable" it is meant the carrier,
diluent or excipient must be compatible with the other ingredients
of the formulation and not deleterious to the recipient
thereof.
[0176] These pharmaceutical compositions may be in the form of
orally-administrable suspensions or tablets, nasal sprays, sterile
injectible preparations, for example, as sterile injectible aqueous
or oleagenous suspensions or suppositories.
[0177] When administered orally as a suspension, these compositions
are prepared according to techniques well-known in the art of
pharmaceutical formulation and may contain microcrystalline
cellulose for imparting bulk, alginic acid or sodium alginate as a
suspending agent, methylcellulose as a viscosity enhancer, and
sweeteners/flavoring agents known in the art. As immediate release
tablets, these compositions may contain microcrystalline cellulose,
dicalcium phosphate, starch, magnesium stearate and lactose and/or
other excipients, binders, extenders, disintegrants, diluents and
lubricants known in the art.
[0178] When administered by nasal aerosol or inhalation, these
compositions are prepared according to techniques well-known in the
art of pharmaceutical formulation and may be prepared as solutions
in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, and/or other solubilizing or dispersing agents known
in the art.
[0179] The injectible solutions or suspensions may be formulated
according to known art, using suitable non-toxic,
parenterally-acceptable diluents or solvents, such as mannitol,
1,3-butanediol, water, Ringer's solution or isotonic sodium
chloride solution, or suitable dispersing or wetting and suspending
agents, such as sterile, bland, fixed oils, including synthetic
mono- or diglycerides, and fatty acids, including oleic acid.
[0180] When rectally administered in the form of suppositories,
these compositions may be prepared by mixing the drug with a
suitable non-initiating excipient, such as cocoa butter, synthetic
glyceride esters of polyethylene glycols, which are solid at
ordinary temperatures, but liquefy and/or dissolve in the rectal
cavity to release the drug.
[0181] The compounds of this invention can be administered orally
to humans in a dosage range of 1 to 1000 mg/kg body weight in
divided doses. One preferred dosage range is 0.1 to 200 mg/kg body
weight orally in divided doses. Another preferred dosage range is
0.5 to 100 mg/kg body weight orally in divided doses. For oral
administration, the compositions are preferably provided in the
form of tablets containing 1.0 to 1000 milligrams of the active
ingredient, particularly 1.0, 5.0, 10.0, 15.0. 20.0, 25.0, 50.0,
75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0,
750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient
for the symptomatic adjustment of the dosage to the patient to be
treated. It will be understood, however, that the specific dose
level and frequency of dosage for any particular patient may be
varied and will depend upon a variety of factors including the
activity of the specific compound employed, the metabolic stability
and length of action of that compound, the age, body weight,
general health, sex, diet, mode and time of administration, rate of
excretion, drug combination, the severity of the particular
condition, and the host undergoing therapy.
[0182] The present invention is also directed to combinations of
the HIV integrase inhibitor compounds with one or more agents
useful in the treatment of AIDS. For example, the compounds of this
invention may be effectively administered, whether at periods of
pre-exposure and/or post-exposure, in combination with effective
amounts of the AIDS antivirals, imunomodulators, antiinfectives, or
vaccines, such as those in the following table.
1 ANTIVIRALS Drug Name Manufacturer Indication 097 Hoechst/Bayer
HIV infection, AIDS, ARC (non-nucleoside reverse transcriptase (RT)
inhibitor) 141 W94 Glaxo Wellcome HIV infection, AIDS, ARC
(protease inhibitor) 1592U89 Glaxo Wellcome HIV infection, AIDS,
ARC (protease inhibitor) Abacavir Glaxo Wellcome HIV infection,
AIDS, (1592U89) ARC (RT inhibitor) Acemannan Carrington Labs ARC
(Irving, TX) Acyclovir Burroughs HIV infection, AIDS, Wellcome ARC,
in combination with AZT AD-439 Tanox Biosystems HIV infection,
AIDS, ARC AD-519 Tanox Biosystems HIV infection, AIDS, ARC Adefovir
Gilead Sciences HIV infection dipivoxil AL-721 Ethigen ARC, PGL
(Los Angeles, CA) HIV positive, AIDS Alpha Glaxo Wellcome Kaposi's
sarcoma, HIV Interferon in combination w/ Retrovir Ansamycin Adria
Laboratories ARC LM 427 (Dublin, OH) Erbamont (Stamford, CT)
Antibody which Advanced AIDS, ARC neutralizes pH Biotherapy labile
alpha Concepts aberrant (Rockville, MD) Interferon AR177 Aronex
Pharm HIV infection, AIDS, ARC beta-fluoro-ddA Nat'l Cancer
Institute AIDS-associated diseases BMS-232623 Bristol-Myers Squibb/
HIV infection, AIDS, (CGP-73547) Novartis ARC (protease inhibitor)
BMS-234475 Bristol-Myers Squibb/ HIV infection, AIDS, (CGP-61755)
Novartis ARC (protease inhibitor) CI-1012 Warner-Lambert HIV-1
infection Cidofovir Gilead Science CMV retinitis, herpes,
papillomavirus Curdlan sulfate AJI Pharma USA HIV infection
Cytomegalovirus MedImmune CMV retinitis immune globin Cytovene
Syntex sight threatening CMV Ganciclovir peripheral CMV retinitis
Delaviridine Pharmacia-Upjohn HIV infection, AIDS, ARC (RT
inhibitor) Dextran Sulfate Ueno Fine Chem. AIDS, ARC, HIV Ind. Ltd.
positive asymptomatic (Osaka, Japan) ddC Hoffman-La Roche HIV
infection, AIDS, Dideoxycytidine ARC ddI Bristol-Myers Squibb HIV
infection, AIDS, Dideoxyinosine ARC; combination with AZT/d4T
DMP-450 AVID HIV infection, AIDS, (Camden, NJ) ARC (protease
inhibitor) Efavirenz DuPont Merck HIV infection, AIDS, (DMP 266)
ARC (-) 6-Chloro-4(S)- (non-nucleoside RT cyclopropylethynyl-
inhibitor) 4(S)-trifluoro-methyl- 1,4-dihydro-2H-3,1-
benzoxazin-2-one, STOCRINE EL10 Elan Corp, PLC HIV infection
(Gainesville, GA) Famciclovir Smith Kline herpes zoster, herpes
simplex FTC Emory University HIV infection, AIDS, ARC (reverse
transcriptase inhibitor) GS 840 Gilead HIV infection, AIDS, ARC
(reverse transcriptase inhibitor) GW 141 Glaxo Welcome HIV
infection, AIDS, ARC (protease inhibitor) GW 1592 Glaxo Welcome HIV
infection, AIDS, ARC (reverse transcriptase inhibitor) HBY097
Hoechst Marion HIV infection, AIDS, Roussel ARC (non-nucleoside
reverse transcriptase inhibitor) Hypericin VIMRx Pharm. HIV
infection, AIDS, ARC Recombinant Human Triton Biosciences AIDS,
Kaposi's Interferon Beta (Almeda, CA) sarcoma, ARC Interferon
alfa-n3 Interferon Sciences ARC, AIDS Indinavir Merck HIV
infection, AIDS, ARC, asymptomatic HIV positive, also in
combination with AZT/ddI/ddC ISIS 2922 ISIS Pharmaceuticals CMV
retinitis KNI-272 Nat'l Cancer Institute HIV-assoc. diseases
Lamivudine, 3TC Glaxo Wellcome HIV infection, AIDS, ARC (reverse
transcriptase inhibitor); also with AZT Lobucavir Bristol-Myers
Squibb CMV infection Nelfinavir Agouron HIV infection, AIDS,
Pharmaceuticals ARC (protease inhibitor) Nevirapine Boeheringer
Ingleheim HIV infection, AIDS, ARC (RT inhibitor) Novapren
Novaferon Labs, Inc. HIV inhibitor (Akron, OH) Peptide T Peninsula
Labs AIDS Octapeptide (Belmont, CA) Sequence
[0183] It will be understood that the scope of combinations of the
compounds of this invention with AIDS antivirals, immunomodulators,
anti-infectives or vaccines is not limited to the list in the above
Table, but includes in principle any combination with any
pharmaceutical composition useful for the treatment of AIDS.
[0184] Preferred combinations are simultaneous or alternating
treatments of with a compound of the present invention and an
inhibitor of HIV protease and/or a non-nucleoside inhibitor of HIV
reverse transcriptase. An optional fourth component in the
combination is a nucleoside inhibitor of HIV reverse transcriptase,
such as AZT, 3TC, ddC or ddI. A preferred inhibitor of HIV protease
is indinavir, which is N-(2(R)-hydroxy-1(S)-ind-
anyl)-2(R)-phenylmethyl-4-(S)-hydroxy-5-(1-(4-(3-pyridyl-methyl)-2(S)-N'-(-
t-butylcarboxamido)-piperazinyl))-pentaneamide, and is synthesized
according to U.S. Pat. No. 5,413,999. Indinavir is generally
administered as a sulfate ethanolate salt at a dosage of 800 mg
three times a day. Other preferred protease inhibitors are
nelfinavir and ritonavir. Another preferred inhibitor of HIV
protease is saquinavir which is administered in a dosage of 600 or
1200 mg tid. Preferred non-nucleoside inhibitors of HIV reverse
transcriptase include efavirenz. The preparation of ddC, ddI and
AZT are also described in EPO 0,484,071. These combinations may
have unexpected effects on limiting the spread and degree of
infection of HIV. Preferred combinations include those with the
following (1) indinavir with efavirenz, and, optionally, AZT and/or
3TC and/or ddI and/or ddC; (2) indinavir, and any of AZT and/or ddI
and/or ddC and/or 3TC, in particular, indinavir and AZT and 3TC;
(3) stavudine and 3TC and/or zidovudine; (4) zidovudine and
lamivudine and 141W94 and 1592U89; (5) zidovudine and
lamivudine.
[0185] In such combinations the compound of the present invention
and other active agents may be administered separately or in
conjunction. In addition, the administration of one element may be
prior to, concurrent to, or subsequent to the administration of
other agent(s).
[0186] It will be understood that the scope of combinations of the
compounds of this invention with AIDS antivirals, immunomodulators,
anti-infectives or vaccines is not limited to the list in the above
Table, but includes in principle any combination with any
pharmaceutical composition useful for the treatment of AIDS.
[0187] Indinavir is an inhibitor of HIV protease and is
N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4-(S)-hydroxy-5-(1-(4-(3--
pyridyl-methyl)-2(S)-N'-(t-butylcarboxamido)-piperazinyl))-pentaneamide,
and is synthesized according to U.S. Pat. No. 5,413,999. Indinavir
is generally administered as the sulfate ethanolate salt at a
dosage of 800 mg three times a day.
[0188] The following examples are provided to further illustrate
details for the preparation and use of the compounds of the present
invention. The examples are not intended to be limitations on the
scope of the instant invention in any way, and they should not be
so construed. Furthermore, the compounds described in the following
examples are not to be construed as forming the only genus that is
considered as the invention, and any combination of the compounds
or their moieties may itself form a genus. Those skilled in the art
will readily understand that known variations of the conditions and
processes of the following preparative procedures can be used to
prepare these compounds. All temperatures are in degrees Celsius
unless noted otherwise.
[0189] Abbreviations: Ac represents acetyl; ACN is acetonitrile;
BOC and t-BOC are t-butoxycarbonyl; Bn represents benzyl; Bz
represents benzoyl; DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene; DIEA
is diisopropylethylamine; DMAP is 4-dimethylaminopyridine; DMF is
dimethyl formamide; Fmoc is N-(9-fluorenylmethoxycarbonyl); Et
represents ethyl; HPLC is high pressure liquid chromatography; IPA
is isopropyl alcohol; MEK is methyl ethyl ketone; Me represent
methyl; MOM is methoxymethyl; Ms represents methane sulfonyl; PDA
is photodiode array; TFA is trifluoroacetic acid; THF is
tetrahydrofuran; TLC is thin layer (SiO.sub.2) chromatography.
EXAMPLE 1
[0190] Fermentation of MF6381 (ATCC 74469) Using Solid Medium
[0191] A. Media
2 SEED MEDIUM Component g/L Yeast extract 4.0 Malt extract 8.0
Glucose 4.0 Junlon 1.5
[0192] The medium was prepared with distilled water, the pH
adjusted to 7.0 prior to sterilization, and was dispensed at 50
mL/250 mL unbaffled Erlenmeyer flask. Cotton closures were used.
Sterilization was at 121.degree. C. for 20 minutes.
[0193] PRODUCTION MEDIUM:
[0194] 1. Solid portion:
[0195] 675 cc vermiculite was added to a 2 liter roller bottle
which was plugged with latex closure and autoclaved for 60 minutes,
plus 30 minutes on the dry cycle.
3 2. Liquid portion Component g/L Glycerol 75.0 Glucose 10.0
Ardamine pH 5.0 (NH.sub.4).sub.2SO.sub.4 2.0 Soybean meal 5.0
Tomato paste 5.0 Sodium citrate 2.0 pH to 7.0
[0196] The medium was prepared with distilled water, dispensed at
220 mL in 500 mL bottles and sterilized at 121.degree. C. for 20
minutes.
[0197] B. Inoculum Preparation
[0198] Growth from an agar slant was used to prepare FVMs (frozen
vegetative mycelia). A portion of the agar slant was transferred
aseptically to seed medium. (The composition of the seed medium is
detailed above). The flasks were incubated on a 2-inch throw
gyratory shaker, 220 rpm for 2 days at 25.degree. C., 85% relative
humidity (rh), to obtain biomass. Portions of the biomass were
transferred into sterile vials containing glycerol and frozen (as
FVM). These were maintained in a final concentration of 10-15%
glycerol at -75.degree. C.
[0199] C. Seed Culture
[0200] Frozen vials (FVM) were thawed to room temperature and used
to inoculate seed cultures, at 1.0 mL per 50 mL seed medium. The
cultures were grown on a gyratory shaker (220 rpm) for 2 days at
25.degree. C., 85% rh, until a sufficient amount of biomass was
obtained.
[0201] D. Production
[0202] The composition of the solid substrate fermentation medium
is shown above. An aliquot (12 mL) of each grown seed was placed
into 220 mL of the liquid portion of the production. This was
swirled vigorously to disperse the biomass. The contents were
dispensed by pouring into a 2-liter roller culture vessel which
contained 675 cubic centimeters of steam-sterilized large-particle
vermiculite. The contents of the roller bottle were shaken/mixed to
insure homogeneous inoculation and coverage. The roller bottles
were incubated horizontally, revolving at approximately 4 rpm on a
Wheaton roller apparatus at 22.degree. C., 75% rh for 18 days, to
obtain secondary metabolite production in the fermentation
medium.
EXAMPLE 2
[0203] Fermentation of MF6381 (ATCC 74469) Using Liquid Medium
[0204] A. MEDIA
4 1. KF Seed Medium Component (g/L) Corn steep powder 2.5 Tomato
paste 40.0 Oat flour 10.0 Glucose 10.0 Trace elements solution 10.0
mL/L
[0205] pH to 6.8 NaOH
5 TRACE ELEMENTS SOLUTION Component (g/L) FeSO.sub.4.7H.sub.2O 1.0
MnSO.sub.4.H.sub.2O 1.0 CuCl.sub.2.2H.sub.2O 0.025
CaCl.sub.2.H.sub.2O 0.1 H.sub.3BO.sub.3 0.056
(NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O 0.019
ZnSO.sub.4.7H.sub.2O 0.2 Trace elements prepared in 0.6 N HCl
[0206] The medium was prepared with distilled water, the pH
adjusted to 6.8 prior to sterilization, and was dispensed at 50
mL/250 mL unbaffled Erlenmeyer flask. Cotton closures were used.
Sterilization was at 121.degree. C. for 20 minutes.
6 2. Liquid Production Medium Component g/L Glucose 150.0 Glycerol
20.0 Yeast extract 4.0 NaNO.sub.3 1.0 Monosodium glutamate 3.0
Na.sub.2HPO.sub.4 0.5 MgSO.sub.47H.sub.2O 1.0 CaCO.sub.3 8.0
K-elements 1.0 ml/L
[0207] pH to 7.0. with NaOH. Autoclave 15 min
7 K-elements Component g/L FeCl.sub.3.6H.sub.2O 5.8
MnSO.sub.4.H.sub.2O 0.1 CoCl.sub.2.6H.sub.2O 0.02
CuSO.sub.4.5H.sub.2O 0.015 Na.sub.2MoO.sub.4.2H.sub.2O 0.012
ZnCl.sub.2 0.02 SnCl.sub.2.2H.sub.2O 0.005 H.sub.3BO.sub.3 0.01 KCl
0.02 HCl (concentrated) 2.0 ml/L
[0208] The medium was prepared with distilled water, dispensed at
50 mL per 250 mL flask, and sterilized at 121.degree. C. for 15
minutes.
[0209] B. SEED:
[0210] Frozen vials (FVM) were thawed to room temperature and used
to inoculate KF medium seed cultures, at 1.0 mL per 50 mL seed
medium. The cultures were grown on a gyratory shaker (220 rpm) for
2 days at 25.degree. C., 85% rh, until a sufficient amount of
biomass was obtained.
[0211] C. PRODUCTION:
[0212] The composition of the solid substrate fermentation medium
is shown in the table above. An aliquot (1-2 mL) of each grown seed
was placed into 50 mL of the liquid production medium in 250 mL
flasks. The flasks were incubated at 22.degree. C. for 7-21
days.
EXAMPLE 3
[0213] Isolation of A 6
[0214] The Fusarium culture grown on vermiculite medium was
extracted with 1.2 volume methyl ethyl ketone (MEK) by shaking at a
shaker for 30-60 min. Sixty mL of the MEK extract was concentrated
to dryness and the residual water was removed by lyophilization to
give 180 mg of pale residue. This material was dissolved in 5 mL
methanol-methylene chloride (1:1) and was charged on to a 1 L
SEPHADEX LH-20 column packed in methanol. Twenty mL each fractions
were collected at a flow rate of .about.20 mL/min. The compound
eluted from 400 mL to 900 mL of the elution volume of methanol. The
combined fractions were concentrated to give the title compound as
a colorless powder. .sup.1H NMR (acetone-d.sub.6+10% CD.sub.3OD)
.delta.: 5.56 (1H, brs, H-15), 5.04 (1H, brd, J=1.2 HZ, H-12),4.70,
4.66 (1H each, brs, H-28), 4.24 (1H, brs, H-11), 3.91 (1H, brdt,
J=10, 4 Hz, H-2), 3.82 (1H, d, J=10 Hz, H-3), 2.44 (1H, m, H-16),
2.38 (1H, dd, 12,4 Hz, H-1.beta.), 2.37 (1H, m, H-7.alpha.), 2.30
(1H, m, H-7.beta.), 2.23 (1H, hept, J=6.8 Hz, H-25), 2.14 (1H, m,
H-23), 2.05 (1H, m, H-16), 2.00 (1H, m, H-17), 1.95 (3H, s,
H.sub.3-32), 1.91 (1H, m, H-23), 1.77 (1H, m, H-6), 1.69 (1H, m,
H-6), 1.68 (1H, m, H-20), 1.58 (1H, m, H-22), 1.31 (3H, s,
H.sub.3-19), 1.27 (1H, m, H-1.alpha.), 1.23 (1H, m, H-5), 1.14 (1H,
m, H-22), 1.09 (3H, s, H.sub.3-18), 1.08 (3H, s, H.sub.3-30), 1.01,
1.00 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27), 0.91 (3H, d, J=6.4
Hz, H.sub.3-21), 0.87 (3H, s, H.sub.3-29); .sup.13C NMR
(acetone-d.sub.6+10% CD.sub.3OD) .delta.: 170.70 (C-31), 157.12
(C-24), 148.37 (C-14), 140.00 (C-9), 125.47 (C-8), 120.98 (C-15),
106.71 (C-28), 90.11 (C-3), 78.99 (C-12), 69.14 (C-11), 68.20
(C-2), 51.39 (C-5), 49.80 (C-17), 47.68 (C-13), 44.15 (C-1), 40.49
(C-4), 38.56 (C-10), 35.95 (C-16), 35.30 (C-22), 34.35 (C-25),
34.14 (C-20), 31.62 (C-23), 29.22 (C-30), 27.39 (C-7), 23.37
(C-19), 22.30, 22.15 (C-26, C-27), 21.24 (C-32), 18.92 (C-6), 18.50
(C-21), 17.96 (C-29), 17.10 (C-18); ESIMS: m/z 595 [M+H].sup.30 ,
593 [M-H].sup.-; EIMS: m/z 436 [M--H.sub.2SO.sub.4--
AcOH].sup.+.
EXAMPLE 4
[0215] Preparation of Compound B from A 7
[0216] A solution of Compound A L-155,911 (160 mg) in dioxane (16
mL) was heated at 66.degree. C. for 5 min. After addition of sodium
bicarbonate (300 mg) the reaction mixture was filtered through a
bed of sodium sulfate and washed with ethyl acetate (150 mL). The
combined filtrate was washed once each with 50 mL of 10% aqueous
sodium bicarbonate and 50 mL water, dried over sodium sulfate,
concentrated under reduced pressure and chromatographed over a
silica gel column. Elution with 50% ethyl acetate in hexane gave 13
mg of fraction A, 27 mg of fraction B and 72 mg (33%) of Compound B
L-155,944 as a gum. Lyophilization of Compound B from
acetonitrile-water gave colorless powder. .sup.1H NMR (CDCl.sub.3)
.delta.: 5.61 (1H, t, J=2.5 Hz, H-15), 4.97 (1H, d, J=2.0 Hz,
H-12), 4.73 (1H, brs, H-28), 4.66 (1H, d, J=1.0 Hz, H-28), 4.24
(1H, brs, H-11), 3.81 (1H, ddd, J=11.5, 10, 4 Hz, H-2), 3.20 (1H,
brs, OH), 3.05 (1H, d, J=9.5 Hz, H-3), 2.45 (2H, m, H-16,
H-7.beta.), 2.37 (1H, dd, J=12, 5 Hz, H-1.beta.), 2.32 (1H, dd,
J=17.5, 7 Hz, H-7.alpha.), 2.23 (1H, heptet, J=7 Hz, H-25), 2.10
(1H, m, H-23), 2.06 (1H, m, H-16), 2.05 (3H, s, H.sub.3-32), 1.97
(H, dt, J=10.5, 7.5 Hz, H-17), 1.89 (1H, m, H-23), 1.77 (1H, brdd,
J=13.5, 7.5 Hz, H-6.beta.), 1.69 (1H, m, H-6.alpha.), 1.65 (1H, m,
H-20), 1.57 (1H, m, H-22), 1.31 (3H, s, H.sub.3-19), 1.27 (1H, t,
J=12 Hz, H-1.alpha.), 1.27 (1H, dd, J=12.5, 3.0 Hz, H-5), 1.15 (1H,
m, H-22), 1.08 (3H, s, H.sub.3-18), 1.06 (3H, s, H.sub.3-30), 1.03,
1.01 (6H, d, J=7 Hz, H.sub.3-26, H.sub.3-27), 0.89 (3H, s,
H.sub.3-29), 0.88 (3H, d, J=7 Hz, H.sub.3-21); .sup.13C NMR
(CDCl.sub.3) .delta.: 171.19 (C-31), 156.58 (C-24), 146.73 (C-14),
138.03 (C-9), 125.77 (C-8), 121.45 (C-15), 106.09 (C-28), 83.40
(C-3), 79.23 (C-12), 69.13 (C-2), 68.88 (C-11), 50.11 (C-5), 49.10
(C-17), 46.58 (C-13), 42.76 (C-1), 39.31 (C-4), 38.37 (C-10), 35.30
(C-16), 34.45 (C-22), 33.79 (C-25), 33.25 (C-20), 30.90 (C-23),
28.66 (C-30), 26.77 (C-7), 23.24 (C-19), 21.98, 21.85 (C-26, C-27),
21.28 (C-32), 18.19 (C-21), 18.00 (C-6), 16.74 (C-18), 16.69
(C-29); ESIMS (m/z): 1046 [2M+NH.sub.4].sup.+, 532
[M+NH.sub.4].sup.+, 497 [M+H].sup.+, 437
[M+H--H.sub.2O--AcOH].sup.+, 1141[2M+CF.sub.3CO.sub.2].sup.-, 627
[M+CF.sub.3CO.sub.2].sup.-, HREIMS: m/z 454.3448 ([M --AcOH].sup.+,
calcd for C.sub.30H.sub.46O.sub.3: 454.3447), 439.3219
([M--ACOH--CH.sub.3].sup.+, calcd for
C.sub.29H.sub.43O.sub.3:439.3212), 311.1990
([M--AcOH--H.sub.2O--C-- 17 side chain].sup.+, calcd for
C.sub.21H.sub.27O.sub.2: 311.2010).
EXAMPLE 5
[0217] Isolation of Compounds A and B
[0218] A 9 L fermentation broth (pH=7.0) grown for 19 days on
liquid production media was filtered through CELITE.TM.
diatomaceous earth. The filtrate contained small amounts of
Compounds A and B and was discarded. The mycelia was extracted
twice each with 4 L methanol followed by 8 L of acetone. The
combined acetone extract was concentrated almost to dryness and
then combined with the methanol extract. The combined extracts were
diluted with 8 L of water and charged over a 2 L SP207 column at a
flow rate of 100 mL/min. The column was thoroughly washed with 50%
aqueous methanol until the eluent became almost colorless. Elution
with 70% aqueous methanol (16 L) gave fraction A which contained
almost exclusively Compound A. Subsequent elution with 100%
methanol (6 L) and acetone (4 L) gave fraction B that possessed a
mixture of Compounds B and A. The fraction B also contained minor
amounts of related congeners. An aliquot (2.8 g) of fraction B was
dissolved in 8 mL methanol and 1 mL each was chromatographed, in
eight equal runs, on a reverse phase HPLC (ZORBAX RX C-8,
22.times.250 mm, a 40 min gradient of 30 to 70% aqueous CH.sub.3CN
at a flow rate of 8 mL/min). Lyophilization of fractions between
24-28 min gave Compound A and lyopholization of fractions between
65-77 gave Compound B (0.26 g) both as colorless powders. Numerous
fractions eluted between 4-24 min, 28-65 min and after 77 min
contain a number of related minor compounds. The structure and
biological activity of these compounds is under active
investigation.
EXAMPLE 6
[0219] Preparation of methoxymethyl ethers C, D, and E 8
[0220] To a cold (0.degree. C.) solution of Compound B (10 mg,
0.019 mmol) in 1 mL CH.sub.2Cl.sub.2 was added
diisopropylethylamine (DIEA, 15.5 .mu.L, 0.114 mmol) and
methoxymethyl chloride (MOMCl, 7.4 .mu.L, 0.095 mmol). The solution
was stirred at 0.degree. C. for 2 h followed by stirring at room
temperature overnight. The reaction mixture was quenched with ice
and 50 mL EtOAc was added. The organic layer was washed
sequentially with 50 mL each of water, 10% aqueous citric acid,
water, 10% aqueous NaHCO.sub.3 followed by water and dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and products were purified by preparative TLC (SiO.sub.2) using
hexane-EtOAc (1:1). The three bands were eluted with EtOAc to give
bis-MOMether Compound C, 2-MOMether Compound D and 3-MOMether
Compound E as amorphous powder. Compound C: .sup.1H NMR
(CDCl.sub.3) (only distinct signals are presented) .delta.: 5.65
(1H, brs, H-15), 5.01 (1H, d, J=2.0 Hz, H-12), 4.95 (1H, d, J=6.4
Hz, OCH.sub.2O), 4.77 (2H, brs, OCH.sub.2O), 4.76 (1H, d, J=6.4 Hz,
OCH.sub.2O), 4.75 (1H, brs, H-28), 4.69 (1H, d, J=1.0 Hz, H-28),
4.26 (1H, bd, J.sub.H,OH=5.6 Hz, H-11), 3.86 (1H, ddd, J=12, 10, 4
Hz, H-2), 3.46 (3H, s, OCH.sub.3), 3.43 (3H, s, OCH.sub.3), 3.06
(1H, d, J=10 Hz, H-3), 2.07 (3H, s, H.sub.3-32), 1.77 (1H, d,
J.sub.H,OH=5.6 Hz, 11--OH), 1.32 (3H, s, H.sub.3-19), 1.10 (3H, s,
H.sub.3-18), 1.08 (3H, s, H.sub.3-30), 1.05, 1.04 (6H, d, J= 6.8
Hz, H.sub.3-26, H.sub.3-27), 0.94 (3H, s, H.sub.3-29), 0.92 (3H, d,
J=6.4 Hz, H.sub.3-21); ESIMS (m/z): 620 (100%, M+NH.sub.4).sup.+,
603 (5%, M+H).sup.+, 585 (30%, M-H.sub.2O+H).sup.+. Compound D:
.sup.1H NMR (CDCl.sub.3) (only distinct signals are presented)
.delta.: 5.65 (1H, t, J=2.0 Hz, H-15),5.01 (1H, d, J=1.2 Hz,
H-12),4.83 (1H, d, J=6.8 Hz, OCH.sub.2O),4.76 (1H, d, J=6.8 Hz,
OCH.sub.2O), 4.75 (1H, brs, H-28), 4.69 (1H, d, J=1.2 Hz, H-28),
4.27 (1H, bd, J.sub.H,OH=5.2 Hz, H-11), 3.68 (1H, ddd, J=13.6, 9.6,
4 Hz, H-2), 3.47 (3H, s, OCH.sub.3), 3.31 (1H, d, J.sub.H,OH=2 Hz,
3--OH), 3.12 (1H, dd, J.sub.2,3=10 Hz, J.sub.H,OH=2 Hz, H-3), 2.07
(3H, s, H.sub.3-32), 1.80 (1H, d, J.sub.H,OH=6.0 Hz, 11--OH), 1.31
(3H, s, H.sub.3-19), 1.12 (3H, s, H.sub.3-18), 1.10 (3H, s,
H.sub.3-30), 1.05, 1.04 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27),
0.93 (3H, s, H.sub.3-29), 0.91 (3H, d, J=6.4 Hz, H.sub.3-21); ESIMS
(m/z): 576 (100%, M+NH.sub.4).sup.+, 541 (40%, M-H.sub.2O+H).sup.+.
Compound E: .sup.1H NMR (CDCl) (only distinct signals are
presented) .delta.: 5.64 (1H, t, J=2.0 Hz, H-15), 5.02 (1H, d,
J=1.2 Hz, H-12), 4.87 (1H, d, J=6.4 Hz, OCH.sub.2O), 4.75 (1H, brs,
H-28), 4.69 (1H,brs, H-28), 4.66 (1H, d, J=6.4 Hz, OCH.sub.2O),
4.31 (1H, bd, J.sub.H,OH=4.4 Hz, H-11), 3.84 (1H, ddd, J=12.8, 9.6,
4 Hz, H-2),3.49 (3H, s, OCH.sub.3),2.85 (1H, dd, J.sub.2,3=9.6 Hz,
J.sub.H,OH=2 Hz, H-3), 2.05 (3H, s, H.sub.3-32), 1.95 (1H, d,
J.sub.H,OH=5.2 Hz, 11--OH), 1.33 (3H, s, H.sub.3-19), 1.10 (3H, s,
H.sub.3-18), 1.05, 1.04 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27),
1.02 (3H, s, H.sub.3-30), 0.93 (3H, s, H.sub.3-29), 0.92 (3H, d,
J=6.4 Hz, H.sub.3-21); ESIMS (m/z): 576 (60%, M+NH.sub.4).sup.+,
541 (90%, M-H.sub.2O+H).sup.+, 481 (100%, M+H-H.sub.2O-
AcOH).sup.+.
EXAMPLE 7
[0221] Preparation of methoxymethyl ethers F, G, and H 9
[0222] Triethylamine (20 .mu.L), DMAP (5 mg) and benzoic anhydride
(17.2 mg, 0.076 mmol) was added to a stirred solution of Compound B
(10 mg, 0.019 mmol) in anhydrous THF (1 mL). The reaction mixture
was stirred overnight under nitrogen. Ice (5 g) was added to quench
the reaction and the mixture was diluted with EtOAc (50 mL). The
organic layer was separated and sequentially washed with 20 mL each
of water, 10% aqueous citric acid, water, 10% aqueous NaHCO.sub.3
followed by water, dried (Na.sub.2SO.sub.4), and EtOAc was
evaporated under reduced pressure. The mixture was chromatographed
by preparative TLC (SiO.sub.2, hexane-EtOAc, 7:3) to give Compounds
F, G, and H all as colorless amorphous powders. Compound F: .sup.1H
NMR (CDCl.sub.3) (only distinct signals are presented) .delta.:
7.99 (2H, dd, J=7.6, 1.6 Hz, ArH), 7.92 (2H, dd, J=8.4, 1.2 Hz,
ArH), 7.47 (2H, m, ArH), 7.37 (2H, t, J=8 Hz, ArH), 7.35 (2H, t,
J=7.6 Hz, ArH), 5.68 (1H, brs, H-15), 5.62 (1H, dt, J=11.6, 4.4 Hz,
H-2), 5.26 (1H, d, J=10.4 Hz, H-3), 4.99 (1H, d, J=2.0 Hz, H-12),
4.7 5 (1H, brs, H-28), 4.69 (1H, d, J=1.0 Hz, H-28), 4.23 (1H, bd,
J.sub.H,OH=5.0 Hz, H-11), 2.10 (3H, s, H.sub.3-32), 1.83 (1H, d,
J.sub.H,OH=5.6 Hz, 11--OH), 1.52 (3H, s, CH.sub.3), 1.20 (3H, s,
CH.sub.3), 1.11 (3H, s, CH.sub.3), 1.06 (3H, s, CH.sub.3), 1.06,
1.05 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27), 0.92 (3H, d, J=6.4
Hz, H.sub.3-21); ESIMS (m/z): 1462 (85%, 2M+NH.sub.4).sup.+, 740
(100%, M+NH.sub.4).sup.+, 705 (35%, M-H.sub.2O+H).sup.+. Compound
G: .sup.1H NMR (CDCl.sub.3) (only distinct signals are presented)
.delta.: 8.10 (2H, dd, J=7.6, 1.6 Hz, ArH), 7.60 (1H, t, J= 7.6 Hz,
ArH), 7.48 (2H, t, J=8 Hz, ArH), 7.35 (2H, t, J=7.6 Hz, ArH), 5.67
(1H, brs, H-15),5.36 (1H, dt, J=11.2, 4.4 Hz, H-2),4.99 (1H, brs,
H-12),4.75 (1H, brs, H-28),4.69 (1H, d, J=1.0 Hz, H-28),4.23 (1H,
brs, H-11), 3.41 (1H, d, J=10 Hz, H-3), 2.09 (3H, s, H.sub.3-32),
1.43 (3H, s, CH.sub.3), 1.14 (3H, s, CH.sub.3), 1.10 (3H, s,
CH.sub.3), 1.05, 1.04 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27),
1.02 (3H, s, CH.sub.3), 0.91 (3H, d, J=6.4 Hz, H.sub.3-21); ESIMS
(m/z): 1254 (10%, 2M+NH.sub.4).sup.+, 636 (100%, M+NH.sub.4).sup.+,
601 (30%, M-H.sub.2O+H).sup.+. Compound H: .sup.1H NMR (CDCl.sub.3)
(only distinct signals are presented) .delta.: 8.12 (2H, d, J=7.2,
ArH), 7.61 (1H, t, J=7.6 Hz, ArH), 7.49 (2H, t, J= 8 Hz, ArH), 7.35
(2H, t, J=7.6 Hz, ArH), 5.67 (1H, brs, H-15), 5.02 (1H, brs, H-12),
4.84 (1H, d, J=10 Hz, H-3),4.76 (1H, brs, H-28),4.70 (1H, d, J=1.0
Hz, H-28), 4.32 (1H, brs, H-11), 4.11 (1H, dt, J=10, 3.6 Hz, H-2),
2.10 (3H, s, H.sub.3-32), 1.39 (3H, s, CH.sub.3), 1.12 (3H, s,
CH.sub.3), 1.11 (3H, s, CH.sub.3), 1.06, 1.05 (6H, d, J=6.8 Hz,
H.sub.3-26, H.sub.3-27), 1.01 (3H, s, CH.sub.3), 0.92 (3H, d, J=6.4
Hz, H.sub.3-21); ESIMS (m/z): 1254 (10%, 2M+NH.sub.4).sup.+, 636
(100%, M+NH.sub.4).sup.+, 601 (30%, M-H.sub.2O+H).sup.+.
EXAMPLE 8
[0223] Preparation of hemisuccinates J and K 10
[0224] To a THF (2 mL) solution of Compound B (10 mg, 0.019 mmol)
was added triethylamine (60 .mu.L), DMAP (5 mg) and succinic
anhydride (22 mg). The mixture was stirred at room temperature
overnight and heated at 50 .degree. C. for 2 h. The reaction
mixture was allowed to cool down and then ice followed by addition
of EtOAc (50 mL). The layers were separated and the organic layer
was sequentially washed with 2.times.20 mL each of water, 10%
aqueous citric acid, water, and dried (Na.sub.2SO.sub.4). EtOAc was
removed under reduced pressure. Chromatography of the mixture on
reverse phase HPLC (ZORBAX RX C-8, 22.times.250 mm, gradient of 60%
to 75% CH.sub.3CN in H.sub.2O, both containing 0.05% TFA, flow rate
8 mL/min) followed by lyophilization gave Compound J and Compound K
as colorless amorphous powders. Compound J: .sup.1H NMR
(CDCl.sub.3) (only distinct signals are presented) .delta.: 5.64
(1H, brs, H-15),5.04 (1H, brs, H-12),4.75 (1H, brs, H-28),4.69 (1H,
d, J=1.0 Hz, H-28), 4.65 (1H, d, J=9.6 Hz, H-3), 4.23 (1H, brs,
H-11), 4.00 (1H, m, H-2), 2.73 (4H, m, 2.times.CH.sub.2CO), 2.07
(3H, s, H.sub.3-32), 1.34 (3H, s, CH.sub.3), 1.11 (3H, s,
CH.sub.3), 1.05, 1.04 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27),
0.97 (3H, s, CH.sub.3), 0.93 (3H, s, CH.sub.3), 0.91 (3H, d, J=6.4
Hz, H.sub.3-21); ESIMS (m/z): 1246 (10%, 2M+NH.sub.4).sup.+, 632
(100%, M+NH.sub.4).sup.+, 597 (35%, M-H.sub.2O+H).sup.+. Compound
K: .sup.1H NMR (CDCl.sub.3) (only distinct signals are presented)
.delta.: 5.65 (1H, brs, H-15), 5.15 (1H, dt, J=11.6, 4.4 Hz, H-2),
4.98 (1H, brs, H-12), 4.76 (1H, brs, H-28), 4.69 (1H, d, J=1.0 Hz,
H-28), 4.22 (1H, brs, H-11), 3.29 (1H, d, J=10 Hz, H-3), 2.76 (2H,
m, 2.times.CH.sub.2CO), 2.68 (2H, m, 2.times.CH.sub.2CO), 2.10 (3H,
s, H.sub.3-32), 1.36 (3H, s, CH.sub.3), 1.10 (3H, s, CH.sub.3),
1.09 (3H, s, CH.sub.3), 1.05, 1.04 (6H, d, J=6.8 Hz, H.sub.3-26,
H.sub.3-27), 0.95 (3H, s, CH.sub.3), 0.91 (3H, d, J=6.4 Hz,
H.sub.3-21); ESIMS (m/z): 1246 (10%, 2M+NH.sub.4).sup.+, 632 (100%,
M+NH.sub.4).sup.+, 597 (35%, M-H.sub.2O+H).sup.+.
EXAMPLE 9
[0225] Preparation of hemisuccinate methyl esters L and M 11
[0226] A mixture of 16 mg of hemisuccinates (Compounds J and K),
described in the Example 7, was dissolved in 0.1 mL
CH.sub.2Cl.sub.2 and cooled to 0.degree. C. An ethereal solution of
freshly prepared diazomethane was added and the solution was kept
at 0.degree. C. overnight. Volatile material was evaporated under a
stream of nitrogen and the methyl esters were purified by
preparative TLC (SiO.sub.2, hexane-EtOAc, 3:1). Elution of the
bands with EtOAc gave mono methyl esters Compounds M and L as
amorphous powders. Compound M: .sup.1H NMR (CDCl.sub.3) (only
distinct signals are presented) .delta.: 5.63 (1H, t, J=2.8 Hz,
H-15), 5.15 (1H, ddd, 11.6, 10, 4.4 Hz, H-2), 4.97 (1H, d, J=1.6
Hz, H-12), 4.73 (1H, brs, H-28), 4.67 (1H, d, J=1.0 Hz, H-28), 4.18
(1H, brs, H-11), 3.70 (3H, s, OCH.sub.3), 3.23 (1H, d, 10 Hz, H-3),
2.68 (4H, m, 2.times.CH.sub.2CO), 2.07 (3H, s, H.sub.3-32), 1.34
(3H, s, CH.sub.3), 1.09 (3H, s, CH.sub.3), 1.07 (3H, s, CH.sub.3),
1.03, 1.02 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27), 0.94 (3H, s,
CH.sub.3), 0.89 (3H, d, J= 6.4 Hz, H.sub.3-21); ESIMS (m/z): 629
(100%, M+H).sup.+, HREIMS (m/z): 628.3978 (calcd for
C.sub.37H.sub.56O.sub.8: 428.3975). Compound L: .sup.1H NMR
(CDCl.sub.3) (only distinct signals are presented) .delta.:
5.63(1H, t, J=2.8 Hz, H-15), 4.98 (1H, d, J=1.6 Hz, H-12), 4.73
(1H, brs, H-28), 4.67 (1H, d, J=1.6 Hz, H-28), 4.62 (1H, d, J=9.6
Hz, H-3), 4.27 (1H, brs, H-11), 3.95 (1H, ddd, J=11.6, 10, 4.4 Hz,
H-2), 3.70 (3H, s, OCH.sub.3), 2.68 (4H, m, 2.times.CH.sub.2CO),
2.05 (3H, s, H.sub.3-32), 1.32 (3H, s, CH.sub.3), 1.08 (3H, s,
CH.sub.3), 1.03, 1.02 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27),
0.94 (3H, s, CH.sub.3), 0.92 (3H, s, CH.sub.3), 0.89 (3H, d, J=6.4
Hz, H.sub.3-21); ESIMS (m/z): 629 (50%, M+H).sup.+, HREIMS (m/z):
628.4009 (calcd for C.sub.37H.sub.56O.sub.8: 428.3975).
EXAMPLE 10
[0227] Preparation of succinic acid hydroxymate N 12
[0228] To a cooled (-40 .degree. C.) solution of Compound K (10 mg,
0.016 mmol, Example 7) in THF (0.5 mL) was added N-methylmorpholine
(15 .mu.L) followed by allyl chloroformate (10 .mu.L). The reaction
mixture was allowed to warm to room temperature. After stirring for
30 min under nitrogen it was re-cooled at -23 .degree. C. and an
aqueous solution of hydroxylamine was added via a syringe. The
mixture was stirred at 0.degree. C. for 30 min and then quenched
with ice and diluted with EtOAc (50 mL). The ethyl acetate layer
was sequentially washed with 20 mL each of 10% aqueous citric acid,
water, 10% aqueous NaHCO.sub.3 and water. EtOAc extract was dried
(Na.sub.2SO.sub.4), concentrated under reduced pressure and
chomatographed by reverse phase HPLC (ZORBAX RX C-8, 22.times.250
mm, 20 to 80% CH.sub.3CN in H.sub.2O (+0.1%TFA) gradient in 40 min,
at 8 mL/min). The fractions containing the product were lyophilized
to give hydroxymate Compound N (4 mg) as an amorphous powder.
.sup.1H NMR (CDCl.sub.3) .delta.: (only distinct signals are
listed, spectrum was very broad) 5.64 (1H, brs, H-15), 5.14 (1H, m,
H-2), 4.98 (1H, brs, H-12), 4.75 (1H, brs, H-28), 4.69 (1H, brs,
H-28), 4.19 (1H,brs, H-11), 3.30 (4H, m, 2.times.CH.sub.2),2.75
(1H, H-3), 2.23 (1H, heptet, J=7.2 Hz, H-25), 2.09 (3H, s,
COCH.sub.3), 1.35 (3H, s, CH.sub.3), 1.09 (6H, s,
2.times.CH.sub.3), 1.05, 1.04 (6H, d, J=6.8 Hz, H.sub.3-26,
H.sub.3-27), 0.94 (3H, s, CH.sub.3), 0.90 (3H, d, J=6 Hz,
H.sub.3-21), ESIMS m/z: 630 (M+H).sup.+.
EXAMPLE 11
[0229] Preparation of Compound O 13
[0230] To a solution of Compound B (10 mg) in dioxane-water (2:1,
1.5 mL) was added LiOH (13.4 mg) and the yellowish solution was
stirred at room temperature overnight. EtOAc (50 mL) was added and
the solution was washed with 2.times. 20 mL of water. The EtOAc
layer was dried (Na.sub.2SO.sub.4), evaporated under reduced
pressure and chromatographed on preparative TLC (SiO.sub.2,
hexane-EtOAc, 3:7). Elution of the major band gave Compound O as
colorless amorphous powder. .sup.1H NMR (CDCl.sub.3+CD.sub.3OD,
10:1) .delta.: 5.59 (1H, brs, H-15), 4.66 (1H, brs, H-28), 4.60
(1H, brs, H-28), 4.23 (1H, brs, H-11), 3.69 (1H, dt, J=11.2, 4 Hz,
H-2), 3.69 (1H, d, J= 1.6 Hz, H-12), 2.90 (1H, d, J=9.2 Hz, H-3),
2.36 (2H, m, H-16, H-7.beta.), 2.27 (1H, dd, J=11, 6 Hz,
H-1.beta.), 2.20 (1H, m, H-7.alpha.), 2.20 (1H, m, H-17), 2.19 (1H,
m, H-25), 2.07 (1H, m, H-23), 1.97 (1H, m, H-16), 1.86 (1H, m,
H-23), 1.69 (1H, m, H-6.beta.), 1.61 (1H, m, H-6.alpha.), 1.61 (1H,
m, H-20), 1.51 (1H, m, H-22), 1.27 (1H, t, J=12 Hz, H-1.alpha.),
1.23 (3H, s, H.sub.3-19), 1.20 (2H, m, H-5, H-22), 0.97 (3H, s,
H.sub.3-18), 0.97 (3H, d, J=6.4 Hz, H.sub.3-21), 0.96 (3H, s,
H.sub.3-30), 0.96, 0.95 (6H, d, J=5.6 Hz, H.sub.3-26, H.sub.3-27),
0.81 (3H, s, H.sub.3-29), HREIMS (m/z): 472.3543 (M.sup.+, calcd
for C.sub.30H.sub.48O.sub.4: 472.3552).
EXAMPLE 12
[0231] Preparation of triacetate Compound P 14
[0232] To a solution of Compound B (10 mg) in pyridine (0.5 mL) was
added acetic anhydride (0.3 mL) and the solution was stirred at
room temperature overnight under an inert atmosphere followed by
heating at 50.degree. C. for 3 h. Methanol was added to consume
excess acetic anhydride. The solvent and volatile material was
removed under a stream of N.sub.2. The product was purified by
preparative TLC (SiO.sub.2, hexane-EtOAc, 7:3). The band was eluted
with EtOAc. Evaporation of EtOAc under reduced pressure afforded
the triacetate Compound P as a colorless foam. .sup.1H NMR
(CDCl.sub.3) .delta.: 5.73 (1H, brs, H-15), 5.33 (1H, brs, H-11),
5.18 (1H, brdt, J=11.6, 4.4 Hz, H-2), 5.13 (1H, d, J=2.0 Hz, H-12),
4.77 1H, d, J=10 Hz, H-3), 4.74 (1H, brs, H-28), 4.68 (1H, brs,
H-28), 2.50 (1H, m, H-16), 2.48 (1H, m, H-7.beta.), 2.40 (1H, brdd,
J=18.4, 6.8 Hz, H-7.alpha.), 2.24 (1H, doublet of heptet, J=6.4,
0.8 Hz, H-25), 2.13 (2H, m, H-23, H-16), 2.10 (3H, s, COCH.sub.3),
2.09 (3H, s, COCH.sub.3), 2.08 (3H, s, COCH.sub.3), 2.03 (3H, s,
COCH.sub.3), 1.95 (1H, dd, J=12, 4 Hz, H-1), 1.86 (1H, m, H-23),
1.85 (1H, m, H-23), 1.82 (1H, m, H-5), 1.81 (1H, m, H-6), 1.70 (1H,
m, H-6), 1.61 (1H, m, H-20), 1.56 (1H, m, H-22), 1.41 (1H, t, J=12
Hz, H-1.alpha.), 1.38 (1H, m, H-5), 1.27 (3H, s, H.sub.3-19), 1.16
(2H, m, H-22), 1.05 (3H, s, CH.sub.3), 1.04, 1.03 (6H, d, J=6.8 Hz,
H.sub.3-26, H.sub.3-27), 0.99 (3H, s, CH.sub.3), 0.95 (3H, s,
CH.sub.3), 0.89 (3H, d, J=6.4 Hz, H.sub.3-21), ESIMS (m/z): 658
(M+NH.sub.4).sup.+, HREIMS (m/z): 598.3837 (M-COCH.sub.2, calcd for
C.sub.36H.sub.54O.sub.7: 598.3869).
EXAMPLE 13
[0233] Preparation of 2,3-acetonide Compound Q 15
[0234] To a solution of Compound B (10 mg) in CH.sub.2Cl.sub.2 (1
mL) was added 2,2-dimethoxy propane (0.1 mL) and pyridinium
p-toluenesulfonic acid (5 mg) and the solution was stirred at room
temperature for 30 min. Water (20 mL) and EtOAc (50 mL) was added
and the layers were separated. The organic layer was sequentially
washed with 20 mL each of 10% aqueous citric acid, water, 10%
aqueous NaHCO.sub.3 and water. EtOAc extract was dried
(Na.sub.2SO.sub.4), concentrated under reduced pressure and
chomatographed over a preparative TLC (SiO.sub.2, hexane-EtOAc,
7:3). Elution of the band with EtOAc and evaporation of the solvent
gave acetonide Compound Q as an amorphous powder. .sup.1H NMR
(CDCl.sub.3) .delta.: 5.65 (1H, brs, H-15), 5.01 (1H, d, J=2.0 Hz,
H-12), 4.75 (1H, brs, H-28), 4.69 (1H, d, J=1.6 Hz, H-28),4.28 (1H,
brd, J.sub.H,OH=4.8 HZ, H-11), 3.85 (1H, ddd, J=12.8, 9.6, 3.6 Hz,
H-2), 3.11 (1H, d, J=9.6 Hz, H-3), 2.54 (1H, dd, J=10.8, 3.2 Hz,
H-1), 2.49 (1H, m, H-7), 2.47 (1H, m, H-16), 2.37 (1H, brdd, J=18,
7.2 Hz, H-7.alpha.), 2.25 (1H, heptet, J=6.8 Hz, H-25), 2.09 (1H,
m, H-16), 2.08 (3H, s, COCH.sub.3), 2.06 (1H, m, H-23), 1.95 (1H,
m, H-17), 1.92 (1H,m, H-23), 1.91 (1H, d, J=5.2 Hz, OH), 1.83 (1H,
m, H-6), 1.71 (1H, m, H-6), 1.65 (1H, m, H-20), 1.57 (1H, m, H-22),
1.47, 1.45 (3H each, s, 2.times.CH.sub.3), 1.45 (1H, t, J=13.2 Hz,
H-1.alpha.), 1.36 (3H, s, CH.sub.3), 1.29 (1H, dd, J=12.8, 2.4 Hz,
H-5), 1.20 (1H, m, H-22), 1.10 (6H, s, 2.times.CH.sub.3), 1.05,
1.04 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27), 0.98 (3H, s,
CH.sub.3), 0.91 (3H, d, J=6.4 Hz, H.sub.3-21), HREIMS (m/z):
554.3947 (M.sup.+, calcd for C.sub.35H.sub.54O.sub.5:
554.3971).
EXAMPLE 14
[0235] 16
[0236] To a cooled (-40 .degree. C.) solution of Compound B (42 mg,
0.08 mmol) in CH.sub.2Cl.sub.2 (1 mL) was added
diisopropylethylamine (33 .mu.L), dimethylaminopyridine (5 mg) and
methane sulfonyl chloride (14 .mu.L). The reaction mixture was
stirred for 20 min and was allowed to warm to room temperature and
quenched by addition of ice. EtOAc (50 mL) was added and the layers
were separated. The organic layer was sequentially washed with 20
mL each of water, 10% aqueous citric acid, water, 10% aqueous
NaHCO.sub.3 and finally with water, dried (Na.sub.2SO.sub.4),
evaporated under reduced pressure to give clean 2-mesolate Compound
R as a foam. .sup.1H NMR (CDCl.sub.3) .delta.: only distinct
signals are listed. 5.64 (1H, brs, H-15), 5.00 (1H, d, J=0.8 Hz,
H-12), 4.81 (1H, ddd, J=11.6, 9.6, 4.0 Hz, H-2), 4.75 (1H, brs,
H-28), 4.68 (1H, d, J=0.2 Hz, H-28), 4.19 (1H, brs, H-11), 3.27
(1H, d, J=9.6 Hz, H-3), 3.16 (3H, brs, SOCH.sub.3), 2.11 (3H, s,
COCH.sub.3), 1.30 (3H, s, CH.sub.3), 1.11 (3H, s, CH.sub.3), 1.09
(3H, s, CH.sub.3), 1.05, 1.04 (6H, d, J=6.8 Hz, H.sub.3-26,
H.sub.3-27), 0.96 (3H, s, CH.sub.3),0.90 (3H, d, J=6.4 Hz,
H.sub.3-21), ESIMS (m/z): 610 (M+NH.sub.4).sup.+. The mesolate
Compound R (12 mg) in 1 mL of toluene and 50 .mu.L of DBU was
heated at 50.degree. C. for 30 min. Ice followed by EtOAc (50 mL)
was added to the reaction after it was cooled to room temperature.
The EtOAc layer was sequentially washed with 20 mL each of water,
10% aqueous citric acid, water, 10% aqueous NaHCO.sub.3 and finally
with water, dried (Na.sub.2SO.sub.4), evaporated under reduced
pressure and chromatographed over a preparative TLC (SiO.sub.2,
hexane-EtOAc, 7:3). The band was eluted with EtOAc to give
2,3-epoxide Compound S as an amorphous powder. .sup.1H NMR
(CDCl.sub.3) .delta.: 5.62 (1H, t, J=2 Hz, H-15),5.01 (1H, d, J=2.4
Hz, H-12),4.73 (1H, brs, H-28),4.67 (1H, d, J=2 Hz, H-28), 4.31
(1H, brd, J.sub.H,OH=6.4 HZ, H-11), 3.34(1H, dt, J=4, 2 Hz, H-2),
2.86(1H, d, J=4.4 Hz, H-3), 2.51(1H, dd, J=14.4, 2 Hz, H-1), 2.45
(1H, ddd, J=16, 6.8, 3.2 Hz, H-16), 2.32 (2H, m, H-7), 2.23 (1H,
heptet, J=7.2 Hz, H-25), 2.10 (1H, m, H-16), 2.07 (1H, m, H-23),
2.06 (3H, s, COCH.sub.3), 1.99 (1H, m, H-17), 1.89 (1H, m, H-23),
1.79 (1H, d, J=5.6 Hz, OH), 1.65 (1H, m, H-20), 1.61 (2H, m, H-6),
1.60, 1.54 (2H, m, H-22), 1.48 (1H, d, J=14 Hz, H-1.alpha.), 1.39
(3H, s, CH.sub.3), 1.16 (1H, m, H-22), 1.084 (6H, s,
2.times.CH.sub.3), 1.078 (3H, s, CH.sub.3), 1.03, 1.02 (6H, d,
J=6.8 Hz, H.sub.3-26, H.sub.3-27), 0.89 (3H, d, J=6.4 Hz,
H.sub.3-21), ESIMS m/z: 514 (M+NH.sub.4).sup.+, HREIMS (m/z):
436.3344 ([M--AcOH].sup.+, calcd for C.sub.30H.sub.44O.sub.2:
436.3341). 17
EXAMPLE 15
[0237] Preparation of t-Boc-glycine esters Compounds T and U
[0238] To an anhydrous solution of Compound B (40 mg, 0.078 mmol)
in a 2:1 mixture of CH.sub.2Cl.sub.2-THF (1.5 mL) was added
N-t-Boc-glycine-succinimide ester (103 mg, 0.39 mmol) followed by
diisopropylethylamine (64 .mu.L) and dimethylaminopyridine (5 mg).
The homogeneous mixture was stirred for overnight under nitrogen
followed by heating at 50.degree. C. for 2 h. The reaction mixture
was quenched by addition of ice and was diluted with EtOAc (50 mL).
The organic layer was sequentially washed with 20 mL each of water,
10% aqueous citric acid, water, 10% aqueous NaHCO.sub.3 and finally
with water, dried (Na.sub.2SO.sub.4), evaporated under reduced
pressure and chromatographed over a preparative TLC (SiO.sub.2,
hexane-EtOAc, 7:3). The two bands were eluted with EtOAc to give
diester Compound T and monoester Compound U; both as amorphous
powders. Compound T: .sup.1H NMR (CDCl.sub.3) .delta.: (only
distinct signals are listed) 5.66 (1H, brs, H-15), 5.48 (1H, brt,
J=6 Hz, NH), 5.37 (1H, dt, J=11.6, 3.6 Hz, H-2), 5.30 (1H, t,
J=6Hz, NH), 4.99 (1H, d, J=0.8 Hz, H-12), 4.81 (1H, d, J=10 Hz,
H-3), 4.75 (1H, brs, H-28),4.69 (1H, d, J= 1.2 Hz, H-28), 4.20 (1H,
brd, J.sub.H,OH=4.8 Hz, H-11), 3.93 (2H, m, gly--CH.sub.2), 3.83
(2H, m, gly--CH.sub.2), 2.26 (1H, heptet, J=7.2 Hz, H-25), 2.10
(3H, s, COCH.sub.3), 1.94 (1H, d, J=5.2 Hz, OH), 1.48, 1.46 (9H
each, s, C(CH.sub.3).sub.3), 1.38 (3H, s, CH.sub.3), 1.09 (3H, s,
CH.sub.3), 1.05, 1.04 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27),
1.00 (3H, s, CH.sub.3), 0.95 (3H, s, CH.sub.3), 0.92 (3H, d, J=6.8
Hz, H.sub.3-21), ESIMS m/z: 846 (M+NH.sub.4).sup.+. Compound U:
.sup.1H NMR (CDCl.sub.3) .delta.: (only distinct signals are
listed) 5.66 (1H, brs, H-15), 5.18 (1H, dt, J=11.6, 4Hz, H-2), 5.10
(1H, t, J=6 Hz, NH), 4.99 (1H, d, J= 0.8 Hz, H-12),4.76 (1H, brs,
H-28),4.69 (1H, d, J=1.6 Hz, H-28),4.19 (1H, brd, J.sub.H,OH=5.2
Hz, H-11), 3.97 (1H, dd, J=17.6, 5.0 Hz, gly--CH), 3.92 (1H, dd, J=
17.6, 5.6 Hz, gly--CH), 3.22 (1H, d, J=7.6 Hz, H-3), 2.26 (1H,
heptet, J=7.2 Hz, H-25), 2.10 (3H, s, COCH.sub.3), 1.93 (1H, d,
J=5.2 Hz, OH), 1.48 (9H, s, C(CH.sub.3).sub.3), 1.37 (3H, s,
CH.sub.3), 1.10 (3H, s, CH.sub.3), 1.09 (3H, s, CH.sub.3), 1.06,
1.05 (6H, d, J=6.8 Hz, H.sub.3-26, H.sub.3-27), 0.96 (3H, s,
CH.sub.3), 0.91 (3H, d, J=6.8 Hz, H.sub.3-21), ESIMS m/z: 689
(M+NH.sub.4).sup.+.
EXAMPLE 16
[0239] 18
[0240] Diisopropylethyl amine (128 .mu.L, 0.7 mmol) and
dimethylaminopyridine (10 mg) followed by
Fmoc-glycine-pentafluorophenyl ester were added to a solution of
Compound B (80 mg, 0.14 mmol) in a 2:3 mixture of
CH.sub.2Cl.sub.2-THF (5 mL). The solution was stirred overnight
under nitrogen. Water followed by EtOAc (50 mL) was added after
completion of the reaction and the layers were separated. The
organic layer was sequentially washed with 2.times.20 mL each of
water, 10% aqueous citric acid, water, 10% aqueous NaHCO.sub.3 and
finally with water, dried (Na.sub.2SO.sub.4), evaporated under
reduced pressure and chromatographed over preparative TLC
(SiO.sub.2, hexane-EtOAc, 7:3). The two bands were eluted with
EtOAc to give diester Compound V and monoester Compound W both as
amorphous pale powders. Compound V: .sup.1H NMR (CDCl.sub.3)
.delta.: (only distinct signals are listed) 7.76 (4H, d, J=7.5 Hz,
ArH), 7.60 (4H, t, J=8 Hz, ArH), 7.38 (4H, t, J=7.5 Hz, ArH), 7.26
(4H, m, ArH), 5.85 (1H, t, J=6 Hz, NH), 5.72 (1H, t, J=6.5 Hz, NH),
5.67 (1H, t, J=2.5 Hz, H-15), 5.37 (1H, ddd, J=12, 10.5, 4.5 Hz,
H-2), 4.99 (1H, d, J=1.5 Hz, H-12), 4.82 (1H, d, J=10.5 Hz, H-3),
4.74 (1H, brs, H-28), 4.69 (1H, d, J=1.5 Hz, H-28), 4.40-4.30
(4H,m, 2.times.CH.sub.2O--), 4.24-4.16 (3H, m, Fmoc--CH, H-11),
3.90 (4H, m, 2.times.gly--CH.sub.2), 2.25 (1H, doublet of heptet,
J=7, 1 Hz, H-25), 2.04 (3H, s, COCH.sub.3), 1.37 (3H, s, CH.sub.3),
1.08 (3H, s, CH.sub.3), 1.04, 1.03 (6H, d, J=6.8 Hz, H.sub.3-26,
H.sub.3-27), 0.98 (3H, s, CH.sub.3), 0.94 (3H, s, CH.sub.3), 0.91
(3H, d, J=6.8 Hz, H.sub.3-21), HRFABMS m/z: 1095.5364 (calcd for
C.sub.66H.sub.76N.sub.2O.sub.11Na: 1095.5347). Compound W: .sup.1H
NMR (CDCl.sub.3) .delta.: (only distinct signals are listed) 7.81
(2H, d, J=7.5 Hz, ArH), 7.65 (2H, m, ArH), 7.43 (2H, brt, J=8 Hz,
ArH), 7.36 (2H, m, ArH), 5.67 (1H, t, J=2.5 Hz, H-15),5.51(1H, t,
J=6.0 Hz, NH),5.14(1H, ddd, J=14, 11.5, 4.0 Hz, H-2), 5.00 (1H, d,
J=1.0 Hz, H-12), 4.76 (1H, d, J=1.5 Hz, H-28), 4.70 (1H, q, J=1.5
Hz, H-28), 4.45 (2H, d, J=7.5 Hz, Fmoc--CH.sub.2), 4.28 (1H, t,
J=7.5 Hz, Fmoc--CH), 4.00 (2H, m, gly--CH.sub.2), 3.19 (1H, d, J=10
Hz, H-3), 2.27 (1H, doublet of heptet, J=7, 1 Hz, H-25), 2.05 (3H,
s, COCH.sub.3), 1.37 (3H, s, CH.sub.3), 1.10 (3H, s, CH.sub.3),
1.06 (3H, s, CH.sub.3), 1.06, 1.05 (6H, d, J =6.8 Hz, H.sub.3-26,
H.sub.3-27), 0.94 (3H, s, CH.sub.3), 0.92 (3H, d, J=6.5 Hz,
H.sub.3-21), HRFABMS m/z: 816.4488 (calcd for
C.sub.49H.sub.63NO.sub.8Na: 816.4452).
EXAMPLE 17
[0241] Preparation of glycine ester Compound X 19
[0242] Piperidine (20 .mu.L) was added to a solution of Compound V
(12 mg) in dimethylformamide (200 .mu.L) and the solution was
stirred at room temperature for 5 min. Volatile material was
removed under a stream of nitrogen and chromatographed over a
reverse phase HPLC (ZORBAX RX C-8, 22.times.250 mm, gradient of 20
to 70% aqueous CH.sub.3CN containing 0.1% TFA, flow rate 8 mL/min).
The product eluted from 50 to 58 min. The combined fractions were
directly lyophilized to yield colorless powder of trifluoroacetate
salt of Compound X. .sup.1H NMR (CD.sub.3CN--CDCl.sub.3, 1:1)
.delta.: (only distinct signals are listed) 5.37 (1H, t, J=2 Hz,
H-15), 5.05 (1H, dt, J=12, 4.5 Hz, H-2), 4.77 (1H, d, J=0.8 Hz,
H-12),4.63 (1H, d, J=10 Hz, H-3),4.48 (1H, brs, H-28), 4.43(1H, d,
J=1.2 Hz, H-28), 3.86 (1H, brs, H-11), 3.65 (4H, m,
2.times.gly--CH.sub.2), 2.01 (1H, heptet, J=7.2 Hz, H-25), 1.81
(3H, s, COCH.sub.3), 1.10 (3H, s, CH.sub.3), 0.81 (3H, s,
CH.sub.3), 0.79, 0.78 (6H, d, J=7 Hz, H.sub.3-26, H.sub.3-27), 0.75
(3H, s, CH.sub.3), 0.71 (3H, s, CH.sub.3), 0.65 (3H, d, J=6.5 Hz,
H.sub.3-21), ESIMS m/z: 629 (M+H).sup.+, HREIMS (m/z): 628.4012
(calcd for C.sub.36H.sub.56N.sub.2O.sub.7: 628.4087).
EXAMPLE 18
[0243] Preparation of glycine esters Compound Y and Z 20
[0244] Compound W (8 mg) is reacted in DMF (100 .mu.L) with
piperidine (20 .mu.L). The products are chromatographed and
lyophilized in a manner similar to the procedure described above.
Compound Y: .sup.1H NMR (CD.sub.3CN--CDCl.sub.3, 1:1) .delta.:
(only distinct signals are listed) 5.35 (1H,brs, H-15), 4.76 (1H,
brs, H-12), 4.48 (1H, brs, H-28), 4.43 (1H, brs, H-28), 4.40 (1H,
d, J=9.5 Hz, H-3), 3.97 (1H,brs, H-11), 3.66 (3H, m, H-2,
gly--CH.sub.2), 2.01 (1H, heptet, J=7.2 Hz, H-25), 1.76 (3H, s,
COCH.sub.3), 1.06 (3H, s, CH.sub.3), 1.03 (3H, s, CH.sub.3), 0.82
(3H, s, CH.sub.3), 0.79, 0.78 (6H, d, J=7 Hz, H.sub.3-26,
H.sub.3-27), 0.70 (3H, s, CH.sub.3), 0.66 (3H, d, J=6.5 Hz,
H.sub.3-21), ESIMS m/z: 572 (M+H).sup.+.
EXAMPLE 19
[0245] HIV Integrase Assay: Strand Transfer Catalyzed by
Recombinant Integrase
[0246] Assays for the strand transfer activity of integrase were
conducted according to Wolfe, A. L. et al., J. Virol. 70, 1424
(1996, hereby incorporated by reference for these purposes. Data
for representative compounds of the present invention follow:
8 Compound IC.sub.50 (.mu.M) A1 14 A3 15 J 16 K 68 X 5
EXAMPLE 20
[0247] Oral Composition
[0248] As a specific embodiment of an oral composition of a
compound of this invention, 50 mg of a compound of the present
invention is formatted with sufficient finely divided lactose to
provide a total amount of 580 to 590 mg to fill a size 0 hard
gelatin capsule.
[0249] While the foregoing specification teaches the principles of
the present invention, with examples provided for the purpose of
illustration, it will be understood that the practice of the
invention encompasses all of the usual variations, adoptions, or
modifications, as come within the scope of the following claims and
their equivalents.
* * * * *