U.S. patent application number 15/164183 was filed with the patent office on 2017-04-20 for polycyclic tetracycline compounds.
The applicant listed for this patent is Tetraphase Pharmaceuticals, Inc.. Invention is credited to Roger B. Clark, Trudy Grossman, Minsheng He, Diana Katharine Hunt, Louis Plamondon, Magnus P. Ronn, Joyce A. Sutcliffe, Xiao-Yi Xiao.
Application Number | 20170107179 15/164183 |
Document ID | / |
Family ID | 43929063 |
Filed Date | 2017-04-20 |
United States Patent
Application |
20170107179 |
Kind Code |
A1 |
Xiao; Xiao-Yi ; et
al. |
April 20, 2017 |
Polycyclic Tetracycline Compounds
Abstract
The present invention is directed to a compound represented by
Structural Formula (I): ##STR00001## or a pharmaceutically
acceptable salt thereof. The variables for Structural Formula (I)
are defined herein. Also described is a pharmaceutical composition
comprising the compound of Structural Formula (I) and its
therapeutic use.
Inventors: |
Xiao; Xiao-Yi; (Lexington,
MA) ; Clark; Roger B.; (Lexington, MA) ; Hunt;
Diana Katharine; (Cambridge, MA) ; Ronn; Magnus
P.; (Melrose, MA) ; Plamondon; Louis;
(Belmont, MA) ; He; Minsheng; (Watertown, MA)
; Sutcliffe; Joyce A.; (Newton, MA) ; Grossman;
Trudy; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tetraphase Pharmaceuticals, Inc. |
Watertown |
MA |
US |
|
|
Family ID: |
43929063 |
Appl. No.: |
15/164183 |
Filed: |
May 25, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13075886 |
Mar 30, 2011 |
9371283 |
|
|
15164183 |
|
|
|
|
61319614 |
Mar 31, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 405/06 20130101;
C07D 243/10 20130101; C07D 498/04 20130101; C07D 405/04 20130101;
C07D 471/04 20130101; A61P 31/00 20180101; C07D 403/06 20130101;
A61P 11/00 20180101; C07D 487/04 20130101; A61P 17/00 20180101;
A61P 31/16 20180101; C07D 221/18 20130101; A61P 31/10 20180101;
C07D 209/58 20130101; A61P 31/04 20180101 |
International
Class: |
C07D 209/58 20060101
C07D209/58; C07D 405/04 20060101 C07D405/04; C07D 498/04 20060101
C07D498/04; C07D 487/04 20060101 C07D487/04; C07D 221/18 20060101
C07D221/18; C07D 243/10 20060101 C07D243/10; C07D 471/04 20060101
C07D471/04; C07D 405/06 20060101 C07D405/06; C07D 403/06 20060101
C07D403/06 |
Claims
1. A compound of Structural Formula I: ##STR00348## or a
pharmaceutically acceptable salt thereof, wherein: X is selected
from halo, --R, --OR, --SR, --S(O).sub.mR, --N(R).sub.2,
--N(R)C(O)R, N(R)C(O)OR', and N(R)S(O).sub.mR', wherein: each R is
independently selected from H, (C.sub.1-C.sub.6)alkyl, carbocyclyl,
or heterocyclyl; or two R groups taken together with the atom or
atoms to which they are bound form a 4-7 membered non-aromatic
heterocyclyl; and R' is (C.sub.1-C.sub.6)alkyl, carbocyclyl, or
heterocyclyl; ring A is a 5-7 membered non-aromatic heterocyclic
ring optionally containing 1-2 heteroatoms independently selected
from N, S and O in addition to the indicated nitrogen atom,
wherein: R.sup.1 is selected from hydrogen,
--(C.sub.1-C.sub.8)alkyl, --(C.sub.0-C.sub.6)alkylene-carbocyclyl,
--(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.1-C.sub.6)alkylene-O--(C.sub.1-C.sub.6)alkyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl,
--C(O)--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
--C(O)--(C.sub.1-C.sub.6)alkyl, --C(O)-heterocyclyl,
--C(O)-carbocyclyl,
--S(O).sub.m--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
and --S(O).sub.m--(C.sub.1-C.sub.4)alkylene-carbocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkylene-heterocyclyl, or R.sup.1
taken together with a ring atom adjacent to the nitrogen atom to
which R.sup.1 is bound forms a saturated heterocyclic ring fused to
ring A; each of R.sup.2 and R.sup.3 is independently selected from
hydrogen, (C.sub.1-C.sub.8)alkyl, --(C.sub.0-C.sub.6)
alkylene-carbocyclyl, --(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl, and
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl; or R.sup.2 and
R.sup.3, taken together with the nitrogen atom to which they are
bound form a heterocyclyl, wherein the heterocyclyl optionally
comprises 1 to 4 additional heteroatoms independently selected from
N, S and O; each R.sup.4 is independently selected from hydrogen,
(C.sub.1-C.sub.6)alkyl, carbocyclyl, heterocyclyl or a naturally
occurring amino acid side chain moiety, or two R.sup.4 taken
together with a common carbon atom to which they are bound form a
3-7 membered non-aromatic carbocyclyl or a 4-7 membered
non-aromatic heterocyclyl, wherein the heterocyclyl formed by two
R.sup.4 comprises one to three heteroatoms independently selected
from N, S and O; any substitutable carbon atom on ring A is
optionally: (i) substituted with one to two substituents
independently selected from --(C.sub.1-C.sub.4)alkyl, and
--(C.sub.0-C.sub.4)alkylene-carbocyclyl; or (ii) substituted with
.dbd.O; (iii) taken together with an adjacent ring atom to form a
3-7 membered saturated carbocyclyl or a 4-7 membered saturated
heterocyclyl ring; or (iv) spyrofused to a 3-7 membered saturated
carbocyclyl; any additional N heteroatom on ring A is substituted
with hydrogen, C.sub.1-C.sub.6 alkyl, carbocyclyl, or heterocyclyl;
each alkyl or alkylene in Structural Formula I is optionally and
independently substituted with one or more substituents
independently selected from halo, --OH, .dbd.O,
--O--(C.sub.1-C.sub.4)alkyl,
fluoro-substituted-(C.sub.1-C.sub.4)alkyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl and --N(R.sup.5)(R.sup.5);
each carbocyclyl or heterocyclyl portion of a substituent of ring A
or the saturated heterocyclic ring fused to ring A is optionally
and independently substituted with one or more substituents
independently selected from halo, --(C.sub.1-C.sub.4)alkyl, --OH,
.dbd.O, --O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
halo-substituted-(C.sub.1-C.sub.4)alkyl,
halo-substituted-O--(C.sub.1-C.sub.4)alkyl,
--C(O)--(C.sub.1-C.sub.4)alkyl,
--C(O)-(fluoro-substituted-(C.sub.1-C.sub.4)alkyl),
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl, --N(R.sup.5)(R.sup.5) and CN;
each R.sup.5 is independently selected from hydrogen and
(C.sub.1-C.sub.4)alkyl, wherein each alkyl in the group represented
by R.sup.5 is optionally and independently substituted with one or
more substituents independently selected from
--(C.sub.1-C.sub.4)alkyl, (C.sub.3-C.sub.6)cycloalkyl, halo, --OH,
--O--(C.sub.1-C.sub.4)alkyl, and
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl; and each m
is independently 1 or 2, with the proviso that when X is hydrogen,
ring A is not an unsubstituted bivalent piperidine radical.
2. A compound of Structural Formula I: ##STR00349## or a
pharmaceutically acceptable salt thereof, wherein: X is selected
from halo, --R', --OR, --SR, --S(O).sub.mR, --N(R).sub.2,
--N(R)C(O)R, N(R)C(O)OR', and N(R)S(O).sub.mR', wherein: each R is
independently selected from H, (C.sub.1-C.sub.6)alkyl, carbocyclyl,
or heterocyclyl; or two R groups taken together with the atom or
atoms to which they are bound form a 4-7 membered non-aromatic
heterocyclyl; and R' is (C.sub.1-C.sub.6)alkyl, carbocyclyl, or
heterocyclyl; ring A is a 5-7 membered non-aromatic heterocyclic
ring optionally containing 1-2 heteroatoms independently selected
from N, S and O in addition to the indicated nitrogen atom,
wherein: R.sup.1 is selected from hydrogen,
--(C.sub.1-C.sub.8)alkyl, --(C.sub.0-C.sub.6)alkylene-carbocyclyl,
--(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.1-C.sub.6)alkylene-O--(C.sub.1-C.sub.6)alkyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl,
--C(O)--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
--C(O)--(C.sub.1-C.sub.6)alkyl, --C(O)-heterocyclyl,
--C(O)-carbocyclyl,
--S(O).sub.m--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
and --S(O).sub.m--(C.sub.1-C.sub.4)alkylene-carbocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkylene-heterocyclyl; or R.sup.1
taken together with a ring atom adjacent to the nitrogen atom to
which R.sup.1 is bound forms a saturated heterocyclic ring fused to
ring A; each of R.sup.2 and R.sup.3 is independently selected from
hydrogen, (C.sub.1-C.sub.8)alkyl, --(C.sub.0-C.sub.6)
alkylene-carbocyclyl, --(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl, and
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl; or R.sup.2 and
R.sup.3, taken together with the nitrogen atom to which they are
bound form a heterocyclyl, wherein the heterocyclyl optionally
comprises 1 to 4 additional heteroatoms independently selected from
N, S and O; each R.sup.4 is independently selected from hydrogen,
(C.sub.1-C.sub.6)alkyl, carbocyclyl, heterocyclyl or a naturally
occurring amino acid side chain moiety, or two R.sup.4 taken
together with a common carbon atom to which they are bound form a
3-7 membered non-aromatic carbocyclyl or a 4-7 membered
non-aromatic heterocyclyl, wherein the heterocyclyl formed by two
R.sup.4 comprises one to three heteroatoms independently selected
from N, S and O; any substitutable carbon atom on ring A is
optionally: (i) substituted with one to two substituents
independently selected from --(C.sub.1-C.sub.4)alkyl, and
--(C.sub.0-C.sub.4)alkylene-carbocyclyl; or (ii) substituted with
.dbd.O; (iii) taken together with an adjacent ring atom to form a
3-7 membered saturated carbocyclyl or a 4-7 membered saturated
heterocyclyl ring; or (iv) spyrofused to a 3-7 membered saturated
carbocyclyl; any additional N heteroatom on ring A is substituted
with hydrogen, C.sub.1-C.sub.6 alkyl, carbocyclyl, or heterocyclyl;
each alkyl or alkylene in Structural Formula I is optionally and
independently substituted with one or more substituents
independently selected from halo, --OH, .dbd.O,
--O--(C.sub.1-C.sub.4)alkyl,
fluoro-substituted-(C.sub.1-C.sub.4)alkyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl and --N(R.sup.5)(R.sup.5);
each carbocyclyl or heterocyclyl portion of a substituent of ring A
or the saturated heterocyclic ring fused to ring A is optionally
and independently substituted with one or more substituents
independently selected from halo, --(C.sub.1-C.sub.4)alkyl, --OH,
.dbd.O, --O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
halo-substituted-(C.sub.1-C.sub.4)alkyl,
halo-substituted-O--(C.sub.1-C.sub.4)alkyl,
--C(O)--(C.sub.1-C.sub.4)alkyl,
--C(O)-(fluoro-substituted-(C.sub.1-C.sub.4)alkyl),
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl, --N(R.sup.5)(R.sup.5) and CN;
each R.sup.5 is independently selected from hydrogen and
(C.sub.1-C.sub.4)alkyl, wherein each alkyl in the group represented
by R.sup.5 is optionally and independently substituted with one or
more substituents independently selected from
--(C.sub.1-C.sub.4)alkyl, (C.sub.3-C.sub.6)cycloalkyl, halo, --OH,
--O--(C.sub.1-C.sub.4)alkyl, and
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl; and each m
is independently 1 or 2.
3. The compound of claim 1, wherein X is selected from fluoro,
chloro, hydrogen, methoxy, methyl, trifluoromethyl,
trifluoromethoxy and dimethylamino.
4. The compound of claim 2, wherein X is selected from fluoro,
chloro, methoxy, methyl, trifluoromethyl, trifluoromethoxy and
dimethylamino.
5. The compound of claim 1, wherein R.sup.1 is selected from
hydrogen, --(C.sub.1-C.sub.8)alkyl,
--(C.sub.2-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.0-C.sub.3)alkylene-(saturated heterocycle),
--(C.sub.0-C.sub.3)alkylene-(C.sub.3-C.sub.7)cycloalkyl,
--C(O)--(C.sub.1-C.sub.3)alkylene-N(R.sup.2)(R.sup.3), or R.sup.1
taken together with a ring atom adjacent to the nitrogen atom to
which R.sup.1 is bound forms a saturated heterocyclic ring fused to
ring A; wherein: any alkyl or alkylene portion of R.sup.1 or the
saturated heterocyclic ring fused to ring A is optionally
substituted with fluoro or hydroxy; R.sup.2 is selected from
hydrogen and (C.sub.1-C.sub.3)alkyl; R.sup.3 is selected from
(C.sub.1-C.sub.3)alkyl and (C.sub.3-C.sub.7)cycloalkyl, or R.sup.2
and R.sup.3, taken together with the nitrogen atom to which they
are bound form a 4-7 membered saturated heterocyclyl, wherein the
heterocyclyl is optionally substituted with fluoro.
6. The compound of claim 5, wherein R.sup.1 is selected from
hydrogen; (C.sub.1-C.sub.3)straight alkyl optionally substituted
with one or more of: 1 to 5 methyl groups, a single hydroxy group,
a single methoxy group, 1 to 3 fluoro groups, a single saturated
heterocycle, and a single (C.sub.3-C.sub.7)cycloalkyl group;
(C.sub.3-C.sub.7)cycloalkyl; tetrahydrofuranyl; and
--C(O)--CH.sub.2--N(R.sup.2)(R.sup.3), wherein R.sup.2 and R.sup.3
are simultaneously methyl; R.sup.2 is hydrogen and R.sup.3 is
C.sub.3-C.sub.7 cycloalkyl; or R.sup.2 and R.sup.3, taken together
with the nitrogen atom to which they are bound form a pyrrolidinyl
ring optionally substituted with fluoro, or R.sup.1 taken together
with a ring atom adjacent to the nitrogen atom to which R.sup.1 is
bound forms a pyrrolidinyl or piperidinyl ring fused to ring A,
wherein the pyrrolidinyl or piperidinyl ring fused to ring A is
optionally substituted with hydroxy or fluorine.
7. The compound of claim 1, wherein: ring A is selected from
##STR00350## R.sup.6a is selected from hydrogen and methyl; and
R.sup.6 is selected from hydrogen, (C.sub.1-C.sub.4)alkyl
optionally substituted with hydroxy or phenyl; or R.sup.6 taken
together with R.sup.1 and the nitrogen atom and the carbon atom to
which they are respectively bound form a pyrrolidinyl or
piperidinyl ring fused to ring A, wherein the pyrrolidinyl or
piperidinyl ring is optionally substituted with --OH or --F; or
R.sup.6 and R.sup.6a are taken together with the carbon atom to
which they are both bound to form a cyclopropyl ring; and R.sup.7a
and R.sup.7b are each hydrogen or are taken together to form
.dbd.O.
8. The compound of claim 1, wherein: ring A is ##STR00351## X is
selected from fluoro, chloro, methoxy, trifluoromethyl, and
dimethylamino; and R.sup.1 is selected from ethyl, propyl,
(C.sub.3-C.sub.5)branched alkyl, (C.sub.3-C.sub.5)cycloalkyl,
(C.sub.1-C.sub.3)alkylene-cyclopropyl,
--C(O)CH.sub.2NH-cyclopentyl, and --C(O)CH.sub.2-pyrrolidin-1-yl,
wherein R.sup.1 is optionally substituted with fluoro.
9. The compound of claim 8, wherein: X is selected from fluoro,
chloro, methoxy, trifluoromethyl, and dimethylamino; and R.sup.1 is
selected from 3-fluoroethyl, propyl, isopropyl, sec-butyl,
tert-butyl, (C.sub.3-C.sub.5)cycloalkyl,
--C(CH.sub.3).sub.2-cyclopropyl, --C(O)CH.sub.2NH-cyclopentyl,
--C(O)CH.sub.2-(3-fluoropyrrolidin-1-yl); and when X is methoxy or
dimethylamino, R.sup.1 is further selected from tert-pentyl.
10. The compound of claim 1, wherein: ring A is ##STR00352## X is
fluoro; and R.sup.1 is selected from hydrogen,
(C.sub.1-C.sub.4)alkyl.
11. The compound of claim 10, wherein R.sup.1 is selected from
isopropyl, propyl or ethyl.
12. The compound of claim 1, wherein: ring A is ##STR00353## X is
fluoro; R.sup.1 is selected from hydrogen, (C.sub.1-C.sub.4)alkyl;
R.sup.6 is selected from hydrogen, (R)-(C.sub.1-C.sub.4)alkyl, or
--CH.sub.2-phenyl, or R.sup.1 and R.sup.6 taken together with the
nitrogen atom and the carbon atom to which they are respectively
bound form a pyrrolidinyl ring fused to ring A; R.sup.7a and
R.sup.7b are each hydrogen or are taken together to form .dbd.O;
wherein at least one of R.sup.1, and R.sup.6 is other than
hydrogen.
13. The compound of claim 12, wherein: R.sup.1 is selected from
hydrogen, methyl, isobutyl, and tert-butyl; and R.sup.6 is selected
from hydrogen, (R)-methyl, (R)-isobutyl, (R)-sec-butyl,
(R)-isopropyl, and --CH.sub.2-phenyl, or R.sup.1 and R.sup.6 taken
together with the nitrogen atom and the carbon atom to which they
are respectively bound form a pyrrolidinyl ring fused to ring
A.
14. A compound of claim 1 selected from any one of Compounds 100,
103, 110, 112, 113, 114, 115, 118, 119, 120, 121, 123, 124, 125,
126, 127, 128, 129, 130, 132, 135, 138, 141, 142, 143, 144, 145,
148, and 149 or a pharmaceutically acceptable salt thereof.
15. A compound of claim 1 selected from any one of Compounds 300,
304, and 307 or a pharmaceutically acceptable salt thereof.
16. A compound of claim 1 selected from any one of Compounds 400,
404, 405, 406, 407, 408, 409, 410, 412, 413, 416, 417, 419, 421,
422, 423, 424, 427, 428, and 429 or a pharmaceutically acceptable
salt thereof.
17. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier or diluent and a compound of claim 1.
18. A method for treating or preventing an infection or
colonization in a subject comprising administering to the subject
an effective amount of a compound of claim 1 or a pharmaceutically
acceptable salt thereof.
19. (canceled)
20. The method of claim 18, wherein the infection is caused by a
Gram-positive organism.
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. The method of claim 18, wherein the infection is caused by a
Gram-negative organism.
26-47. (canceled)
48. A method for treating or preventing a respiratory infection in
a subject comprising administering to the subject an effective
amount of a compound of claim 1 or a pharmaceutically acceptable
salt thereof.
49-50. (canceled)
51. A method for treating or preventing a skin infection in a
subject comprising administering to the subject an effective amount
of a compound of claim 1 or a pharmaceutically acceptable salt
thereof.
52-53. (canceled)
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 13/075,886, filed on Mar. 30, 2011, which claims the benefit of
U.S. Provisional Application No. 61/319,614, filed on Mar. 31,
2010. The entire teachings of the above application are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The tetracyclines are broad spectrum anti-microbial agents
that are widely used in human and veterinary medicine. The total
production of tetracyclines by fermentation or semi-synthesis is
measured in the thousands of metric tons per year.
[0003] The widespread use of tetracyclines for therapeutic purposes
has led to the emergence of resistance to these antibiotics, even
among highly susceptible bacterial species. Therefore, there is
need for new tetracycline analogs with improved antibacterial
activities and efficacies against other tetracycline responsive
diseases or disorders.
SUMMARY OF THE INVENTION
[0004] A first embodiment of the present invention is directed to a
compound represented by Structural Formula (I):
##STR00002##
[0005] or a pharmaceutically acceptable salt thereof, wherein:
[0006] X is selected from halo, --R, --OR, --SR, --S(O).sub.mR,
--N(R).sub.2, --N(R)C(O)R, N(R)C(O)OR', and N(R)S(O).sub.mR',
wherein: [0007] each R is independently selected from H,
(C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl, or [0008] two
R groups taken together with the atom or atoms to which they are
bound form a 4-7 membered non-aromatic heterocyclyl; and [0009] R'
is (C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl; [0010]
ring A is a 5-7 membered non-aromatic heterocyclic ring optionally
containing 1-2 heteroatoms independently selected from N, S and O
in addition to the indicated nitrogen atom, wherein: [0011] R.sup.1
is selected from hydrogen, --(C.sub.1-C.sub.8)alkyl,
--(C.sub.0-C.sub.6)alkylene-carbocyclyl,
--(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.1-C.sub.6)alkylene-O--(C.sub.1-C.sub.6)alkyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl,
--C(O)--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
--C(O)--(C.sub.1-C.sub.6)alkyl, --C(O)-heterocyclyl,
--C(O)-carbocyclyl,
--S(O).sub.m--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
and --S(O).sub.m--(C.sub.1-C.sub.4)alkylene-carbocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkylene-heterocyclyl, or [0012]
R.sup.1 taken together with a ring atom adjacent to the nitrogen
atom to which R.sup.1 is bound forms a saturated heterocyclic ring
fused to ring A; [0013] each of R.sup.2 and R.sup.3 is
independently selected from hydrogen, (C.sub.1-C.sub.8)alkyl,
--(C.sub.0-C.sub.6)alkylene-carbocyclyl,
--(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl, and
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl; or [0014]
R.sup.2 and R.sup.3, taken together with the nitrogen atom to which
they are bound form a heterocyclyl, wherein the heterocyclyl
optionally comprises 1 to 4 additional heteroatoms independently
selected from N, S and O; [0015] each R.sup.4 is independently
selected from hydrogen, (C.sub.1-C.sub.6)alkyl, carbocyclyl,
heterocyclyl or a naturally occurring amino acid side chain moiety,
or [0016] two R.sup.4 taken together with a common carbon atom to
which they are bound form a 3-7 membered non-aromatic carbocyclyl
or a 4-7 membered non-aromatic heterocyclyl, wherein the
heterocyclyl formed by two R.sup.4 comprises one to three
heteroatoms independently selected from N, S and O; [0017] any
substitutable carbon atom on ring A is optionally: [0018] (i)
substituted with one to two substituents independently selected
from --(C.sub.1-C.sub.4)alkyl, and
--(C.sub.0-C.sub.4)alkylene-carbocyclyl; or [0019] (ii) substituted
with .dbd.O; [0020] (iii) taken together with an adjacent ring atom
to form a 3-7 membered saturated carbocyclyl or a 4-7 membered
saturated heterocyclyl ring; or [0021] (iv) spyrofused to a 3-7
membered saturated carbocyclyl; [0022] any additional N heteroatom
on ring A is substituted with hydrogen, C.sub.1-C.sub.6 alkyl,
carbocyclyl, or heterocyclyl; [0023] each alkyl or alkylene in
Structural Formula I is optionally and independently substituted
with one or more substituents independently selected from halo,
--OH, .dbd.O, --O--(C.sub.1-C.sub.4)alkyl,
fluoro-substituted-(C.sub.1-C.sub.4)alkyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl and --N(R.sup.5)(R.sup.5);
[0024] each carbocyclyl or heterocyclyl portion of a substituent of
ring A or the saturated heterocyclic ring fused to ring A is
optionally and independently substituted with one or more
substituents independently selected from halo,
--(C.sub.1-C.sub.4)alkyl, --OH, .dbd.O,
--O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
halo-substituted-(C.sub.1-C.sub.4)alkyl,
halo-substituted-O--(C.sub.1-C.sub.4)alkyl,
--C(O)--(C.sub.1-C.sub.4)alkyl,
--C(O)-(fluoro-substituted-(C.sub.1-C.sub.4)alkyl),
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl, --N(R.sup.5)(R.sup.5) and CN;
[0025] each R.sup.5 is independently selected from hydrogen and
(C.sub.1-C.sub.4)alkyl, wherein each alkyl in the group represented
by R.sup.5 is optionally and independently substituted with one or
more substituents independently selected from
--(C.sub.1-C.sub.4)alkyl, (C.sub.3-C.sub.6)cycloalkyl, halo, --OH,
--O--(C.sub.1-C.sub.4)alkyl, and
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl; and [0026]
each m is independently 1 or 2, with the proviso that when X is
hydrogen, ring A is not an unsubstituted bivalent piperidine
radical.
[0027] In one aspect of the first embodiment, [0028] X is selected
from halo, --R', --OR, --SR, --S(O).sub.mR, --N(R).sub.2,
--N(R)C(O)R, N(R)C(O)OR', and N(R)S(O).sub.mR'; and [0029] R' is
(C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl, wherein the
values for the remaining variables are as defined in the first
embodiment.
[0030] Another embodiment of the present invention is directed to a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier or diluent and a compound represented by Structural Formula
(I) or a pharmaceutically acceptable salt thereof. The
pharmaceutical composition is used in therapy, such as treating an
infection (e.g., a bacterial infection) in a subject.
[0031] Another embodiment of the present invention is a method of
treating an infection (e.g., a bacterial infection) in a subject
comprising administering to the subject an effective amount of a
compound represented by Structural Formula (I) or a
pharmaceutically acceptable salt thereof.
[0032] Another embodiment of the present invention is a method of
preventing an infection (e.g., a bacterial infection) in a subject
comprising administering to the subject an effective amount of a
compound represented by Structural Formula (I) or a
pharmaceutically acceptable salt thereof.
[0033] Another embodiment of the present invention is the use of a
compound represented by Structural Formula (I) or a
pharmaceutically acceptable salt thereof for the manufacture of a
medicament for treating an infection (e.g., a bacterial infection)
in a subject.
[0034] Another embodiment of the present invention is the use of a
compound represented by Structural Formula (I) or a
pharmaceutically acceptable salt thereof for the manufacture of a
medicament for preventing an infection (e.g., a bacterial
infection) in a subject.
[0035] Another embodiment of the present invention is the use of a
compound represented by Structural Formula (I) or a
pharmaceutically acceptable salt thereof in therapy, such as
treating or preventing an infection (e.g., a bacterial infection)
in a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The foregoing will be apparent from the following more
particular description of example embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating embodiments of the present invention.
[0037] FIG. 1 is a bar graph that demonstrates the efficacy of
Compounds 102, 143, 130, 126, and 135 at 10 mg/kg IV, BID and 30
mg/kg, BID orally in a S. pneumoniae SP160 lung model. Linezolid at
5 mg/kg IV, BID and 30 mg/kg, BID orally served as a control.
[0038] FIG. 2 is a bar graph demonstrating the Compound 102 in the
immunocompetent mouse lung infection model with S. pneumoniae
SP514, oral dosing.
[0039] FIG. 3 is a bar graph demonstrating efficacy of Compounds
102, 143, and 130 in the MRSA SA191 lung model. Compounds 102, 143,
and 130 and linezolid were evaluated at 10 mg/kg IV, BID. All
compounds were tested at 50 mg/kg, BID orally except linezolid.
Linezolid was evaluated at 30 mg/kg, BID orally.
[0040] FIG. 4 is a bar graph demonstrating the efficacy of Compound
102 in a Rat lung infection model with H. influenzae HI551.
DETAILED DESCRIPTION OF THE INVENTION
Values and Alternative Values for Variables
[0041] The present invention is directed to a compound represented
by Structural Formula (I) or a pharmaceutically acceptable salt
thereof. Values and alternative values for the variables in
Structural Formula I and for each of the embodiments described
herein are provided in the following paragraphs. It is understood
that the invention encompasses all combinations of the substituent
variables (i.e., R.sup.1, R.sup.2, R.sup.3, etc.) defined
herein.
[0042] X is selected from halo, --R, --OR, --SR, --S(O).sub.mR,
--N(R).sub.2, --N(R)C(O)R, --N(R)C(O)OR', and --N(R)S(O).sub.mR',
wherein each R is independently selected from H,
(C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl; or two R
groups taken together with the atom or atoms to which they are
bound form a 4-7 membered non-aromatic heterocyclyl; and R' is
(C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl.
[0043] Alternatively, X is selected from halo, --R', --OR, --SR,
--S(O).sub.mR, --N(R).sub.2, --N(R)C(O)R, --N(R)C(O)OR', and
--N(R)S(O).sub.mR', wherein each R is independently selected from
H, (C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl, or two R
groups taken together with the atom or atoms to which they are
bound form a 4-7 membered non-aromatic heterocyclyl; and R' is
(C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl.
[0044] Further, X is selected from fluoro, chloro, hydrogen,
methoxy, methyl, trifluoromethyl, trifluoromethoxy and
dimethylamino. Alternatively, X is selected from fluoro, chloro,
methoxy, methyl, trifluoromethyl, trifluoromethoxy and
dimethylamino.
[0045] X is selected from fluoro, chloro, methoxy, trifluoromethyl,
and dimethylamino. Alternatively, X is methoxy or dimethylamino.
Specifically, X is fluoro.
[0046] Ring A is a 5-7 membered non-aromatic heterocyclic ring
optionally containing 1-2 heteroatoms independently selected from
N, S and O in addition to the indicated nitrogen atom.
[0047] Ring A is selected from
##STR00003##
Specifically, ring A is
##STR00004##
Alternatively, ring A is
##STR00005##
Alternatively, ring A is
##STR00006##
[0048] R.sup.1 is selected from hydrogen, --(C.sub.1-C.sub.8)alkyl,
--(C.sub.0-C.sub.6) alkylene-carbocyclyl,
--(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.1-C.sub.6)alkylene-O--(C.sub.1-C.sub.6)alkyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl,
--C(O)--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
--C(O)--(C.sub.1-C.sub.6)alkyl, --C(O)-heterocyclyl,
--C(O)-carbocyclyl,
--S(O).sub.m--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
and --S(O).sub.m--(C.sub.1-C.sub.4)alkylene-carbocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkylene-heterocyclyl, or R.sup.1
taken together with a ring atom adjacent to the nitrogen atom to
which R.sup.1 is bound forms a saturated heterocyclic ring fused to
ring A.
[0049] Alternatively, R.sup.1 is selected from hydrogen,
--(C.sub.1-C.sub.8)alkyl,
--(C.sub.2-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.0-C.sub.3)alkylene-(saturated heterocycle),
--(C.sub.0-C.sub.3)alkylene-(C.sub.3-C.sub.7)cycloalkyl,
--C(O)--(C.sub.1-C.sub.3)alkylene-N(R.sup.2)(R.sup.3), or R.sup.1
taken together with a ring atom adjacent to the nitrogen atom to
which R.sup.1 is bound forms a saturated heterocyclic ring fused to
ring A; wherein any alkyl or alkylene portion of R.sup.1 or the
saturated heterocyclic ring fused to ring A is optionally
substituted with fluoro or hydroxy.
[0050] Further, R.sup.1 is selected from hydrogen;
(C.sub.1-C.sub.3)straight alkyl optionally substituted with one or
more of: 1 to 5 methyl groups, a single hydroxy group, a single
methoxy group, 1 to 3 fluoro groups, a single saturated
heterocycle, and a single (C.sub.3-C.sub.7)cycloalkyl group;
(C.sub.3-C.sub.7)cycloalkyl; tetrahydrofuranyl; and
--C(O)--CH.sub.2--N(R.sup.2)(R.sup.3); or R.sup.1 taken together
with a ring atom adjacent to the nitrogen atom to which R.sup.1 is
bound forms a pyrrolidinyl or piperidinyl ring fused to ring A,
wherein the pyrrolidinyl or piperidinyl ring fused to ring A is
optionally substituted with hydroxy or fluorine.
[0051] Alternatively, R.sup.1 is selected from ethyl, propyl,
(C.sub.3-C.sub.5)branched alkyl, (C.sub.3-C.sub.5)cycloalkyl,
(C.sub.1-C.sub.3)alkylene-cyclopropyl,
--C(O)CH.sub.2NH-cyclopentyl, and --C(O)CH.sub.2-pyrrolidin-1-yl,
wherein R.sup.1 is optionally substituted with fluoro.
Alternatively, R.sup.1 is selected from 3-fluoroethyl, propyl,
isopropyl, sec-butyl, tert-butyl, (C.sub.3-C.sub.5)cycloalkyl,
--C(CH.sub.3).sub.2-cyclopropyl, --C(O)CH.sub.2NH-cyclopentyl, and
--C(O)CH.sub.2-(3-fluoropyrrolidin-1-yl). Alternatively, R.sup.1 is
further selected from tert-pentyl. In another alternative, R.sup.1
is selected from hydrogen and (C.sub.1-C.sub.4)alkyl.
Alternatively, R.sup.1 is selected from hydrogen, methyl, isobutyl,
and tert-butyl.
[0052] Each of R.sup.2 and R.sup.3 is independently selected from
hydrogen, (C.sub.1-C.sub.8)alkyl, --(C.sub.0-C.sub.6)
alkylene-carbocyclyl, --(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl, and
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl; or
[0053] R.sup.2 and R.sup.3, taken together with the nitrogen atom
to which they are bound form a heterocyclyl, wherein the
heterocyclyl optionally comprises 1 to 4 additional heteroatoms
independently selected from N, S and O.
[0054] Alternatively, R.sup.2 is selected from hydrogen and
(C.sub.1-C.sub.3)alkyl and R.sup.3 is selected from
(C.sub.1-C.sub.3)alkyl and (C.sub.3-C.sub.7)cycloalkyl, or R.sup.2
and R.sup.3, taken together with the nitrogen atom to which they
are bound form a 4-7 membered saturated heterocyclyl, wherein the
heterocyclyl is optionally substituted with fluoro.
[0055] In another alternative, R.sup.2 and R.sup.3 are
simultaneously methyl; R.sup.2 is hydrogen and R.sup.3 is
C.sub.3-C.sub.7 cycloalkyl; or R.sup.2 and R.sup.3, taken together
with the nitrogen atom to which they are bound form a pyrrolidinyl
ring optionally substituted with fluoro.
[0056] Each R.sup.4 is independently selected from hydrogen,
(C.sub.1-C.sub.6)alkyl, carbocyclyl, heterocyclyl or a naturally
occurring amino acid side chain moiety.
[0057] Alternatively, two R.sup.4 taken together with a common
carbon atom to which they are bound form a 3-7 membered
non-aromatic carbocyclyl or a 4-7 membered non-aromatic
heterocyclyl, wherein the heterocyclyl formed by two R.sup.4
comprises one to three heteroatoms independently selected from N, S
and O.
[0058] Any substitutable carbon atom on ring A is optionally:
[0059] (i) substituted with one to two substituents independently
selected from --(C.sub.1-C.sub.4)alkyl, and
--(C.sub.0-C.sub.4)alkylene-carbocyclyl; or [0060] (ii) substituted
with .dbd.O; [0061] (iii) taken together with an adjacent ring atom
to form a 3-7 membered saturated carbocyclyl or a 4-7 membered
saturated heterocyclyl ring; or [0062] (iv) spyrofused to a 3-7
membered saturated carbocyclyl.
[0063] Any additional N heteroatom on ring A is substituted with
hydrogen, (C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl.
[0064] Each alkyl or alkylene in Structural Formula I is optionally
and independently substituted with one or more substituents
independently selected from halo, --OH, .dbd.O,
--O--(C.sub.1-C.sub.4)alkyl,
fluoro-substituted-(C.sub.1-C.sub.4)alkyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl and --N(R.sup.5)(R.sup.5).
[0065] Each carbocyclyl or heterocyclyl portion of a substituent of
ring A or the saturated heterocyclic ring fused to ring A is
optionally and independently substituted with one or more
substituents independently selected from halo,
--(C.sub.1-C.sub.4)alkyl, --OH, .dbd.O,
--O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
halo-substituted-(C.sub.1-C.sub.4)alkyl,
halo-substituted-O--(C.sub.1-C.sub.4)alkyl,
--C(O)--(C.sub.1-C.sub.4)alkyl,
--C(O)-(fluoro-substituted-(C.sub.1-C.sub.4)alkyl),
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl, --N(R.sup.5)(R.sup.5) and
CN.
[0066] Each R.sup.5 is independently selected from hydrogen and
(C.sub.1-C.sub.4)alkyl, wherein each alkyl in the group represented
by R.sup.5 is optionally and independently substituted with one or
more substituents independently selected from
--(C.sub.1-C.sub.4)alkyl, (C.sub.3-C.sub.6)cycloalkyl, halo, --OH,
--O--(C.sub.1-C.sub.4)alkyl, and
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl.
[0067] In one alternative, when X is hydrogen, ring A is not an
unsubstituted bivalent piperidine radical.
[0068] Each m is independently 1 or 2.
[0069] R.sup.6a is selected from hydrogen and methyl.
[0070] R.sup.6 is selected from hydrogen, (C.sub.1-C.sub.4)alkyl
optionally substituted with hydroxy or phenyl; or R.sup.6 taken
together with R.sup.1 and the nitrogen atom and the carbon atom to
which they are respectively bound form a pyrrolidinyl or
piperidinyl ring fused to ring A, wherein the pyrrolidinyl or
piperidinyl ring is optionally substituted with --OH or --F; or
R.sup.6 and R.sup.6a are taken together with the carbon atom to
which they are both bound to form a cyclopropyl ring.
[0071] Alternatively, R.sup.6 is selected from hydrogen,
(R)-(C.sub.1-C.sub.4)alkyl, or --CH.sub.2-phenyl, or R.sup.1 and
R.sup.6 taken together with the nitrogen atom and the carbon atom
to which they are respectively bound form a pyrrolidinyl ring fused
to ring A. Further, R.sup.6 is selected from hydrogen, (R)-methyl,
(R)-isobutyl, (R)-sec-butyl, (R)-isopropyl, and --CH.sub.2-phenyl.
Further, at least one of R.sup.1 and R.sup.6 is other than
hydrogen.
[0072] R.sup.7a and R.sup.7b are each hydrogen. Alternatively,
R.sup.7a and R.sup.7b are taken together to form .dbd.O.
[0073] A first embodiment of the present invention is directed to a
compound represented by Structural Formula (I):
##STR00007##
or a pharmaceutically acceptable salt thereof, wherein: [0074] X is
selected from halo, --R, --OR, --SR, --S(O).sub.mR, --N(R).sub.2,
--N(R)C(O)R, N(R)C(O)OR', and N(R)S(O).sub.mR', wherein: [0075]
each R is independently selected from H, (C.sub.1-C.sub.6)alkyl,
carbocyclyl, or heterocyclyl, or [0076] two R groups taken together
with the atom or atoms to which they are bound form a 4-7 membered
non-aromatic heterocyclyl; and [0077] R' is (C.sub.1-C.sub.6)alkyl,
carbocyclyl, or heterocyclyl; [0078] ring A is a 5-7 membered
non-aromatic heterocyclic ring optionally containing 1-2
heteroatoms independently selected from N, S and O in addition to
the indicated nitrogen atom, wherein: [0079] R.sup.1 is selected
from hydrogen, --(C.sub.1-C.sub.8)alkyl,
--(C.sub.0-C.sub.6)alkylene-carbocyclyl,
--(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.1-C.sub.6)alkylene-O--(C.sub.1-C.sub.6)alkyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl,
--C(O)--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
--C(O)--(C.sub.1-C.sub.6)alkyl, --C(O)-heterocyclyl,
--C(O)-carbocyclyl,
--S(O).sub.m--[C(R.sup.4)(R.sup.4)].sub.0-4--N(R.sup.2)(R.sup.3),
and --S(O).sub.m--(C.sub.1-C.sub.4)alkylene-carbocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkylene-heterocyclyl, or [0080]
R.sup.1 taken together with a ring atom adjacent to the nitrogen
atom to which R.sup.1 is bound forms a saturated heterocyclic ring
fused to ring A; [0081] each of R.sup.2 and R.sup.3 is
independently selected from hydrogen, (C.sub.1-C.sub.8)alkyl,
--(C.sub.0-C.sub.6) alkylene-carbocyclyl,
--(C.sub.0-C.sub.6)alkylene-heterocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-carbocyclyl,
--(C.sub.2-C.sub.6)alkylene-O-heterocyclyl,
--S(O).sub.m--(C.sub.1-C.sub.6)alkyl, --S(O).sub.m-carbocyclyl,
--S(O).sub.m-heterocyclyl,
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-carbocyclyl, and
--(C.sub.2-C.sub.4)alkylene-S(O).sub.m-heterocyclyl; or [0082]
R.sup.2 and R.sup.3, taken together with the nitrogen atom to which
they are bound form a heterocyclyl, wherein the heterocyclyl
optionally comprises 1 to 4 additional heteroatoms independently
selected from N, S and O; [0083] each R.sup.4 is independently
selected from hydrogen, (C.sub.1-C.sub.6)alkyl, carbocyclyl,
heterocyclyl or a naturally occurring amino acid side chain moiety,
or [0084] two R.sup.4 taken together with a common carbon atom to
which they are bound form a 3-7 membered non-aromatic carbocyclyl
or a 4-7 membered non-aromatic heterocyclyl, wherein the
heterocyclyl formed by two R.sup.4 comprises one to three
heteroatoms independently selected from N, S and O; [0085] any
substitutable carbon atom on ring A is optionally: [0086] (i)
substituted with one to two substituents independently selected
from --(C.sub.1-C.sub.4)alkyl, and
--(C.sub.0-C.sub.4)alkylene-carbocyclyl; or [0087] (ii) substituted
with .dbd.O; [0088] (iii) taken together with an adjacent ring atom
to form a 3-7 membered saturated carbocyclyl or a 4-7 membered
saturated heterocyclyl ring; or [0089] (iv) spyrofused to a 3-7
membered saturated carbocyclyl; [0090] any additional N heteroatom
on ring A is substituted with hydrogen, C.sub.1-C.sub.6 alkyl,
carbocyclyl, or heterocyclyl; [0091] each alkyl or alkylene in
Structural Formula I is optionally and independently substituted
with one or more substituents independently selected from halo,
--OH, .dbd.O, --O--(C.sub.1-C.sub.4)alkyl,
fluoro-substituted-(C.sub.1-C.sub.4)alkyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl and --N(R.sup.5)(R.sup.5);
[0092] each carbocyclyl or heterocyclyl portion of a substituent of
ring A or the saturated heterocyclic ring fused to ring A is
optionally and independently substituted with one or more
substituents independently selected from halo,
--(C.sub.1-C.sub.4)alkyl, --OH, .dbd.O,
--O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
halo-substituted-(C.sub.1-C.sub.4)alkyl,
halo-substituted-O--(C.sub.1-C.sub.4)alkyl,
--C(O)--(C.sub.1-C.sub.4)alkyl,
--C(O)-(fluoro-substituted-(C.sub.1-C.sub.4)alkyl),
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl, --N(R.sup.5)(R.sup.5) and CN;
[0093] each R.sup.5 is independently selected from hydrogen and
(C.sub.1-C.sub.4)alkyl, wherein each alkyl in the group represented
by R.sup.5 is optionally and independently substituted with one or
more substituents independently selected from
--(C.sub.1-C.sub.4)alkyl, (C.sub.3-C.sub.6)cycloalkyl, halo, --OH,
--O--(C.sub.1-C.sub.4)alkyl, and
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl; and [0094]
each m is independently 1 or 2, with the proviso that when X is
hydrogen, ring A is not an unsubstituted bivalent piperidine
radical.
[0095] In one aspect of the first embodiment, [0096] X is selected
from fluoro, chloro, hydrogen, methoxy, methyl, trifluoromethyl,
trifluoromethoxy and dimethylamino, wherein the values for the
remaining variables are as defined in the first embodiment or in
the values or alternative values described above.
[0097] In a second aspect of the first embodiment, [0098] X is
selected from halo, --R', --OR, --SR, --S(O).sub.mR, --N(R).sub.2,
--N(R)C(O)R, N(R)C(O)OR', and N(R)S(O).sub.mR'; and [0099] R' is
(C.sub.1-C.sub.6)alkyl, carbocyclyl, or heterocyclyl, wherein the
values for the remaining variables are as defined in the first
embodiment or in the values or alternative values described
above.
[0100] In a third aspect of the first embodiment: [0101] X is
selected from fluoro, chloro, methoxy, methyl, trifluoromethyl,
trifluoromethoxy and dimethylamino; wherein the values for the
remaining variables are as defined in the second aspect of the
first embodiment or in the values or alternative values described
above.
[0102] In a fourth aspect of the first embodiment,
[0103] R.sup.1 is selected from hydrogen, --(C.sub.1-C.sub.8)alkyl,
--(C.sub.2-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.0-C.sub.3)alkylene-(saturated heterocycle),
--(C.sub.0-C.sub.3)alkylene-(C.sub.3-C.sub.7)cycloalkyl,
--C(O)--(C.sub.1-C.sub.3)alkylene-N(R.sup.2)(R.sup.3), or
[0104] R.sup.1 taken together with a ring atom adjacent to the
nitrogen atom to which R.sup.1 is bound forms a saturated
heterocyclic ring fused to ring A; wherein: [0105] any alkyl or
alkylene portion of R.sup.1 or the saturated heterocyclic ring
fused to ring A is optionally substituted with fluoro or hydroxy;
[0106] R.sup.2 is selected from hydrogen and
(C.sub.1-C.sub.3)alkyl; [0107] R.sup.3 is selected from
(C.sub.1-C.sub.3)alkyl and (C.sub.3-C.sub.7)cycloalkyl, or
[0108] R.sup.2 and R.sup.3, taken together with the nitrogen atom
to which they are bound form a 4-7 membered saturated heterocyclyl,
wherein the heterocyclyl is optionally substituted with fluoro,
wherein the values for the remaining variables are as defined in
the first embodiment or in the values or alternative values
described above.
[0109] In a fifth aspect of the first embodiment, wherein: R.sup.1
is selected from hydrogen; (C.sub.1-C.sub.3)straight alkyl
optionally substituted with one or more of: 1 to 5 methyl groups, a
single hydroxy group, a single methoxy group, 1 to 3 fluoro groups,
a single saturated heterocycle, and a single
(C.sub.3-C.sub.7)cycloalkyl group; (C.sub.3-C.sub.7)cycloalkyl;
tetrahydrofuranyl; and --C(O)--CH.sub.2--N(R.sup.2)(R.sup.3),
wherein R.sup.2 and R.sup.3 are simultaneously methyl; R.sup.2 is
hydrogen and R.sup.3 is C.sub.3-C.sub.7 cycloalkyl; or R.sup.2 and
R.sup.3, taken together with the nitrogen atom to which they are
bound form a pyrrolidinyl ring optionally substituted with fluoro,
or
[0110] R.sup.1 taken together with a ring atom adjacent to the
nitrogen atom to which R.sup.1 is bound forms a pyrrolidinyl or
piperidinyl ring fused to ring A, wherein the pyrrolidinyl or
piperidinyl ring fused to ring A is optionally substituted with
hydroxy or fluorine wherein the values for the remaining variables
are as defined in the first embodiment or in the values or
alternative values described above.
[0111] In a second embodiment, the compound of the present
invention is represented by Structural Formula (I), or a
pharmaceutically acceptable salt thereof, wherein:
[0112] ring A is selected from
##STR00008##
[0113] R.sup.6a is selected from hydrogen and methyl; and
[0114] R.sup.6 is selected from hydrogen, (C.sub.1-C.sub.4)alkyl
optionally substituted with hydroxy or phenyl; or
[0115] R.sup.6 taken together with R.sup.1 and the nitrogen atom
and the carbon atom to which they are respectively bound form a
pyrrolidinyl or piperidinyl ring fused to ring A, wherein the
pyrrolidinyl or piperidinyl ring is optionally substituted with
--OH or --F; or
[0116] R.sup.6 and R.sup.6a are taken together with the carbon atom
to which they are both bound to form a cyclopropyl ring; and
[0117] R.sup.7a and R.sup.7b are each hydrogen or are taken
together to form .dbd.O; wherein the values for the remaining
variables are as defined in the first embodiment or aspects thereof
or in the values or alternative values described above.
[0118] For example, the compounds of the second embodiment are
represented by Structural Formula (II), (IIIa), (IVa), (Va) or
(VIa):
##STR00009##
or pharmaceutically acceptable salt thereof, wherein the values for
the remaining variables are as defined in the first embodiment or
aspects thereof, the second embodiment, or in the values or
alternative values described above.
[0119] In a third embodiment, the compound of the present invention
is represented by Structural Formula (I), or a pharmaceutically
acceptable salt thereof, wherein:
[0120] ring A is
##STR00010##
[0121] X is selected from fluoro, chloro, methoxy, trifluoromethyl,
and dimethylamino; and
[0122] R.sup.1 is selected from ethyl, propyl,
(C.sub.3-C.sub.5)branched alkyl, (C.sub.3-C.sub.5)cycloalkyl,
(C.sub.1-C.sub.3)alkylene-cyclopropyl,
--C(O)CH.sub.2NH-cyclopentyl, and --C(O)CH.sub.2-pyrrolidin-1-yl,
wherein R.sup.1 is optionally substituted with fluoro, wherein the
values for the remaining variables are as defined in the first or
second embodiments or aspects thereof or in the values or
alternative values described above.
[0123] In a specific aspect of the third embodiment, X is selected
from fluoro, chloro, methoxy, trifluoromethyl, and dimethylamino;
and
[0124] R.sup.1 is selected from 3-fluoroethyl, propyl, isopropyl,
sec-butyl, tert-butyl, (C.sub.3-C.sub.5)cycloalkyl,
--C(CH.sub.3).sub.2-cyclopropyl, --C(O)CH.sub.2NH-cyclopentyl,
--C(O)CH.sub.2-(3-fluoropyrrolidin-1-yl); and when X is methoxy or
dimethylamino, R.sup.1 is further selected from tert-pentyl,
wherein the values for the remaining variables are as defined in
the first or second embodiments or aspects thereof or in the values
or alternative values described above.
[0125] In a fourth embodiment, the compound of the present
invention is represented by Structural Formula (I), or a
pharmaceutically acceptable salt thereof, wherein:
[0126] ring A is
##STR00011##
[0127] X is fluoro; and
[0128] R.sup.1 is selected from hydrogen, (C.sub.1-C.sub.4)alkyl,
wherein the values for the remaining variables are as defined in
the first or second embodiments or aspects thereof or in the values
or alternative values described above.
[0129] In a specific aspect of the fourth embodiment, R.sup.1 is
selected from isopropyl, propyl or ethyl, wherein the values for
the remaining variables are as defined in the first or second
embodiments or aspects thereof or in the values or alternative
values described above.
[0130] In a fifth embodiment, the compound of the present invention
is represented by Structural Formula (I), or a pharmaceutically
acceptable salt thereof, wherein:
[0131] ring A is
##STR00012##
[0132] X is fluoro;
[0133] R.sup.1 is selected from hydrogen,
(C.sub.1-C.sub.4)alkyl;
[0134] R.sup.6 is selected from hydrogen,
(R)-(C.sub.1-C.sub.4)alkyl, or --CH.sub.2-phenyl, or
[0135] R.sup.1 and R.sup.6 taken together with the nitrogen atom
and the carbon atom to which they are respectively bound form a
pyrrolidinyl ring fused to ring A;
[0136] R.sup.7a and R.sup.7b are each hydrogen or are taken
together to form .dbd.O, wherein at least one of R.sup.1, and
R.sup.6 is other than hydrogen, wherein the values for the
remaining variables are as defined in the first or second
embodiments or aspects thereof or in the values or alternative
values described above.
[0137] In a specific aspect of the fifth embodiment, R.sup.1 is
selected from hydrogen, methyl, isobutyl, and tert-butyl; and
[0138] R.sup.6 is selected from hydrogen, (R)-methyl, (R)-isobutyl,
(R)-sec-butyl, (R)-isopropyl, and --CH.sub.2-phenyl. "(R)"
signifies the chirality at the carbon atom to which R.sup.6 is
attached. Specific structures are as follows:
##STR00013##
[0139] Alternatively, R.sup.1 and R.sup.6 taken together with the
nitrogen atom and the carbon atom to which they are respectively
bound form a pyrrolidinyl ring fused to ring A. The values for the
remaining variables are as defined in the first or second
embodiments or aspects thereof or in the values or alternative
values described above.
[0140] Exemplary compounds represented by Structural Formula (II)
are shown in Tables 1-4 below:
TABLE-US-00001 TABLE 1 (II) ##STR00014## Compound X R.sup.1 100 F
##STR00015## 101 F ##STR00016## 102 F ##STR00017## 103 N(CH3).sub.2
##STR00018## 104 F ##STR00019## 105 F ##STR00020## 106 F
##STR00021## 107 F ##STR00022## 108 F ##STR00023## 109 H
##STR00024## 110 Cl ##STR00025## 111 F ##STR00026## 112 CF.sub.3
##STR00027## 113 CF.sub.3 ##STR00028## 114 F ##STR00029## 115
N(CH3).sub.2 ##STR00030## 116 CF.sub.3 ##STR00031## 117 Cl
##STR00032## 118 F ##STR00033## 119 OCH.sub.3 ##STR00034## 120 F
##STR00035## 121 F ##STR00036## 122 F ##STR00037## 123 F
##STR00038## 124 N(CH3).sub.2 ##STR00039## 125 OCH.sub.3
##STR00040## 126 CF.sub.3 ##STR00041## 127 N(CH3).sub.2
##STR00042## 128 CF.sub.3 ##STR00043## 129 F ##STR00044## 130 F
##STR00045## 131 F ##STR00046## 132 F ##STR00047## 133 F
##STR00048## 134 F ##STR00049## 135 N(CH3).sub.2 ##STR00050## 136 F
##STR00051## 137 F ##STR00052## 138 OCH.sub.3 ##STR00053## 139 F
##STR00054## 140 F ##STR00055## 141 CF.sub.3 ##STR00056## 142 F
##STR00057## 143 F ##STR00058## 144 Cl ##STR00059## 145 OCH.sub.3
##STR00060## 146 F ##STR00061## 147 F ##STR00062## 148 OCH.sub.3
##STR00063## 149 Cl ##STR00064## 150 Cl ##STR00065##
TABLE-US-00002 TABLE 2 Exemplary Compounds of Formula III (III)
##STR00066## Compound R.sup.1 200 ##STR00067## 201 ##STR00068## 202
##STR00069##
TABLE-US-00003 TABLE 3 Exemplary Compounds of Formula IV. (IV)
##STR00070## Compound R.sup.1 300 ##STR00071## 301 ##STR00072## 302
##STR00073## 303 ##STR00074## 304 ##STR00075## 305 ##STR00076## 306
##STR00077## 307 ##STR00078## 308 ##STR00079##
TABLE-US-00004 TABLE 4 Exemplary Compounds of Formula V or VI. (V)
##STR00080## (VI) ##STR00081## Compound ring A 400 ##STR00082## 401
##STR00083## 402 ##STR00084## 403 ##STR00085## 404 ##STR00086## 405
##STR00087## 406 ##STR00088## 407 ##STR00089## 408 ##STR00090## 409
##STR00091## 410 ##STR00092## 411 ##STR00093## 412 ##STR00094## 413
##STR00095## 414 ##STR00096## 415 ##STR00097## 416 ##STR00098## 417
##STR00099## 418 ##STR00100## 419 ##STR00101## 420 ##STR00102## 421
##STR00103## 422 ##STR00104## 423 ##STR00105## 424 ##STR00106## 425
##STR00107## 426 ##STR00108## 427 ##STR00109## 428 ##STR00110## 429
##STR00111##
[0141] In a sixth embodiment, the compound of the invention is
represented by any one of the structural formulas described in
Tables 1, 2, 3 or 4, or a pharmaceutically acceptable salt
thereof.
[0142] In a seventh embodiment, the compound of the invention is a
compound selected from any one of Compounds 100, 103, 110, 112,
113, 114, 115, 118, 119, 120, 121, 123, 124, 125, 126, 127, 128,
129, 130, 132, 135, 138, 141, 142, 143, 144, 145, 148, and 149 or a
pharmaceutically acceptable salt thereof.
[0143] In an eighth embodiment, the compound of the invention is a
compound selected from any one of Compounds 300, 304, and 307 or a
pharmaceutically acceptable salt thereof.
[0144] In a ninth embodiment, the compound of the invention is a
compound selected from any one of Compounds 400, 404, 405, 406,
407, 408, 409, 410, 412, 413, 416, 417, 419, 421, 422, 423, 424,
427, 428, and 429 or a pharmaceutically acceptable salt
thereof.
DEFINITIONS
[0145] "Alkyl" means an optionally substituted saturated aliphatic
branched or straight-chain monovalent hydrocarbon radical having
the specified number of carbon atoms. Thus, "(C.sub.1-C.sub.6)
alkyl" means a radical having from 1-6 carbon atoms in a linear or
branched arrangement. "(C.sub.1-C.sub.6)alkyl" includes methyl,
ethyl, propyl, butyl, pentyl and hexyl.
[0146] "Alkylene" means an optionally substituted saturated
aliphatic branched or straight-chain divalent hydrocarbon radical
having the specified number of carbon atoms. Thus,
"(C.sub.1-C.sub.6)alkylene" means a divalent saturated aliphatic
radical having from 1-6 carbon atoms in a linear arrangement, e.g.,
--[(CH.sub.2).sub.n]--, where n is an integer from 1 to 6,
"(C.sub.1-C.sub.6)alkylene" includes methylene, ethylene,
propylene, butylene, pentylene and hexylene. Alternatively,
"(C.sub.1-C.sub.6)alkylene" means a divalent saturated radical
having from 1-6 carbon atoms in a branched arrangement, for
example: --[(CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH(CH.sub.3)]--,
--[(CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(CH.sub.3).sub.2]--,
--[(CH.sub.2C(CH.sub.3).sub.2CH(CH.sub.3))]--, and the like. A
specific branched C.sub.3-alkylene is
##STR00112##
and a specific C.sub.4-alkylene is
##STR00113##
[0147] Each alkyl or alkylene in Structural Formula I is optionally
and independently substituted with one or more substituents
independently selected from halo, --OH, .dbd.O,
--O--(C.sub.1-C.sub.4)alkyl,
fluoro-substituted-(C.sub.1-C.sub.4)alkyl,
--S(O).sub.m--(C.sub.1-C.sub.4)alkyl and --N(R.sup.5)(R.sup.5).
[0148] "Aryl" or "aromatic" means an aromatic monocyclic or
polycyclic (e.g. bicyclic or tricyclic) carbocyclic ring system. In
one embodiment, "aryl" is a 6-12 membered monocylic or bicyclic
system. Aryl systems include, but not limited to, phenyl,
naphthalenyl, fluorenyl, indenyl, azulenyl, and anthracenyl.
[0149] "Carbocyclyl" means a cyclic group with only ring carbon
atoms. "Carbocyclyl" includes 3-12 membered saturated or
unsaturated aliphatic cyclic hydrocarbon rings or 6-12 membered
aryl rings. A carbocyclyl moiety can be monocyclic, fused bicyclic,
bridged bicyclic, spiro bicyclic, or polycyclic.
[0150] Monocyclic carbocyclyls are saturated or unsaturated
aliphatic cyclic hydrocarbon rings or aromatic hydrocarbon rings
having the specified number of carbon atoms. Monocyclic
carbocyclyls include cycloalkyl, cycloalkenyl, cycloalkynyl and
phenyl.
[0151] A fused bicyclic carbocyclyl has two rings which have two
adjacent ring atoms in common. The first ring is a monocyclic
carbocyclyl and the second ring is a monocyclic carbocyclyl or a
monocyclic heterocyclyl.
[0152] A bridged bicyclic carbocyclyl has two rings which have
three or more adjacent ring atoms in common. The first ring is a
monocyclic carbocyclyl and the second ring is a monocyclic
carbocyclyl or a monocyclic heterocyclyl.
[0153] A spiro bicyclic carbocyclyl has two rings which have only
one ring atom in common. The first ring is a monocyclic carbocyclyl
and the second ring is a monocyclic carbocyclyl or a monocyclic
heterocyclyl.
[0154] Polycyclic carbocyclyls have more than two rings (e.g.,
three rings resulting in a tricyclic ring system) and adjacent
rings have at least one ring atom in common. The first ring is a
monocyclic carbocyclyl and the remainder of the ring structures are
monocyclic carbocyclyls or monocyclic heterocyclyls. Polycyclic
ring systems include fused, bridged and spiro ring systems. A fused
polycyclic ring system has at least two rings that have two
adjacent ring atoms in common. A spiro polycyclic ring system has
at least two rings that have only one ring atom in common. A
bridged polycyclic ring system has at least two rings that have
three or more adjacent ring atoms in common.
[0155] "Cycloalkyl" means a saturated aliphatic cyclic hydrocarbon
ring. Thus, "C.sub.3-C.sub.7 cycloalkyl" means a hydrocarbon
radical of a (3-7 membered) saturated aliphatic cyclic hydrocarbon
ring. A C.sub.3-C.sub.7 cycloalkyl includes, but is not limited to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
cycloheptyl.
[0156] "Cycloalkene" means an aliphatic cyclic hydrocarbon ring
having one or more double bonds in the ring.
[0157] "Cycloalkyne" means an aliphatic cyclic hydrocarbon ring
having one or more triple bonds in the ring.
[0158] "Hetero" refers to the replacement of at least one carbon
atom member in a ring system with at least one heteroatom selected
from N, S, and O. "Hetero" also refers to the replacement of at
least one carbon atom member in a acyclic system. A hetero ring
system or a hetero acyclic system may have 1, 2, 3 or 4 carbon atom
members replaced by a heteroatom.
[0159] "Heterocyclyl" means a cyclic 4-12 membered saturated or
unsaturated aliphatic or aromatic ring containing 1, 2, 3, 4 or 5
heteroatoms independently selected from N, O or S. When one
heteroatom is S, it can be optionally mono- or di-oxygenated (i.e.
--S(O)-- or --S(O).sub.2--). The heterocyclyl can be monocyclic,
fused bicyclic, bridged bicyclic, spiro bicyclic or polycyclic.
[0160] "Saturated heterocyclyl" means an aliphatic heterocyclyl
group without any degree of unsaturation (i.e., no double bond or
triple bond). It can be monocyclic, fused bicyclic, bridged
bicyclic, spiro bicyclic or polycyclic.
[0161] Examples of monocyclic saturated heterocyclyls include, but
are not limited to, azetidine, pyrrolidine, piperidine, piperazine,
azepane, hexahydropyrimidine, tetrahydrofuran, tetrahydropyran,
morpholine, thiomorpholine, thiomorpholine 1,1-dioxide,
tetrahydro-2H-1,2-thiazine, tetrahydro-2H-1,2-thiazine 1,1-dioxide,
isothiazolidine, isothiazolidine 1,1-dioxide.
[0162] A fused bicyclic heterocyclyl has two rings which have two
adjacent ring atoms in common. The first ring is a monocyclic
heterocyclyl and the second ring is a monocyclic carbocycle (such
as a cycloalkyl or phenyl) or a monocyclic heterocyclyl. For
example, the second ring is a (C.sub.3-C.sub.6)cycloalkyl, such as
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Alternatively,
the second ring is phenyl. Examples of fused bicyclic heterocyclyls
include, but are not limited to, octahydrocyclopenta[c]pyrrolyl,
indoline, isoindoline, 2,3-dihydro-1H-benzo[d]imidazole,
2,3-dihydrobenzo[d]oxazole, 2,3-dihydrobenzo[d]thiazole,
octahydrobenzo[d]oxazole, octahydro-1H-benzo[d]imidazole,
octahydrobenzo[d]thiazole, octahydrocyclopenta[c]pyrrole,
3-azabicyclo[3.1.0]hexane, and 3-azabicyclo[3.2.0]heptane.
[0163] A spiro bicyclic heterocyclyl has two rings which have only
one ring atom in common. The first ring is a monocyclic
heterocyclyl and the second ring is a monocyclic carbocycle (such
as a cycloalkyl or phenyl) or a monocyclic heterocyclyl. For
example, the second ring is a (C.sub.3-C.sub.6)cycloalkyl.
Alternatively, the second ring is phenyl. Example of spiro bicyclic
heterocyclyl includes, but are not limited to, azaspiro[4.4]nonane,
7-azaspiro[4.4]nonane, azasprio[4.5]decane, 8-azaspiro[4.5]decane,
azaspiro[5.5]undecane, 3-azaspiro[5.5]undecane and
3,9-diazaspiro[5.5]undecane.
[0164] A bridged bicyclic heterocyclyl has two rings which have
three or more adjacent ring atoms in common. The first ring is a
monocyclic heterocyclyl and the other ring is a monocyclic
carbocycle (such as a cycloalkyl or phenyl) or a monocyclic
heterocyclyl. Examples of bridged bicyclic heterocyclyls include,
but are not limited to, azabicyclo[3.3.1]nonane,
3-azabicyclo[3.3.1]nonane, azabicyclo[3.2.1]octane,
3-azabicyclo[3.2.1]octane, 6-azabicyclo[3.2.1]octane and
azabicyclo[2.2.2]octane, 2-azabicyclo[2.2.2]octane.
[0165] Polycyclic heterocyclyls have more than two rings, one of
which is a heterocyclyl (e.g., three rings resulting in a tricyclic
ring system) and adjacent rings having at least one ring atom in
common. Polycyclic ring systems include fused, bridged and spiro
ring systems. A fused polycyclic ring system has at least two rings
that have two adjacent ring atoms in common. A spiro polycyclic
ring system has at least two rings that have only one ring atom in
common. A bridged polycyclic ring system has at least two rings
that have three or more adjacent ring atoms in common.
[0166] "Heteroaryl" or "heteroaromatic ring" means a 5-12 membered
monovalent heteroaromatic monocyclic or bicylic ring radical. A
herteroaryl contains 1, 2, 3 or 4 heteroatoms independently
selected from N, O, and S. Heteroaryls include, but are not limited
to furan, oxazole, thiophene, 1,2,3-triazole, 1,2,4-triazine,
1,2,4-triazole, 1,2,5-thiadiazole 1,1-dioxide, 1,2,5-thiadiazole
1-oxide, 1,2,5-thiadiazole, 1,3,4-oxadiazole, 1,3,4-thiadiazole,
1,3,5-triazine, imidazole, isothiazole, isoxazole, pyrazole,
pyridazine, pyridine, pyridine-N-oxide, pyrazine, pyrimidine,
pyrrole, tetrazole, and thiazole. Bicyclic heteroaryl rings
include, but are not limited to, bicyclo[4.4.0] and bicyclo[4.3.0]
fused ring systems such as indolizine, indole, isoindole, indazole,
benzimidazole, benzthiazole, purine, quinoline, isoquinoline,
cinnoline, phthalazine, quinazoline, quinoxaline,
1,8-naphthyridine, and pteridine.
[0167] In a particular embodiment, each carbocyclyl or heterocyclyl
portion of a substituent of ring A or the saturated heterocyclic
ring fused to ring A is optionally and independently substituted.
Exemplary substituents include halo, --(C.sub.1-C.sub.4)alkyl,
--OH, .dbd.O, --O--(C.sub.1-C.sub.4)alkyl,
--(C.sub.1-C.sub.4)alkylene-O--(C.sub.1-C.sub.4)alkyl,
halo-substituted-(C.sub.1-C.sub.4)alkyl,
halo-substituted-O--(C.sub.1-C.sub.4)alkyl, and
--C(O)--(C.sub.1-C.sub.4)alkyl.
[0168] "Halogen" used herein refers to fluorine, chlorine, bromine,
or iodine.
[0169] "Alkoxy" means an alkyl radical attached through an oxygen
linking atom. "(C.sub.1-C.sub.6)-alkoxy" includes methoxy, ethoxy,
propoxy, butoxy, pentoxy and hexoxy.
[0170] Haloalkyl and halocycloalkyl include mono, poly, and
perhaloalkyl groups where each halogen is independently selected
from fluorine, chlorine, and bromine.
[0171] "Halogen" and "halo" are interchangeably used herein and
each refers to fluorine, chlorine, bromine, or iodine.
[0172] "Fluoro" means --F.
[0173] As used herein, fluoro-substituted-(C.sub.1-C.sub.4)alkyl
means a (C.sub.1-C.sub.4)alkyl substituted with one or more --F
groups. Examples of fluoro-substituted-(C.sub.1-C.sub.4)alkyl
include, but are not limited to, --CF.sub.3, --CH.sub.2CF.sub.3,
--CH.sub.2CF.sub.2H, --CH.sub.2CH.sub.2F and
--CH.sub.2CH.sub.2CF.sub.3.
[0174] "Naturally occurring amino acid side chain moiety" refers to
any amino acid side chain moiety present in a natural amino
acid.
[0175] Another embodiment of the present invention is a
pharmaceutical composition comprising one or more pharmaceutically
acceptable carrier and/or diluent and a compound disclosed herein
or a pharmaceutically acceptable salt thereof.
[0176] "Pharmaceutically acceptable carrier" and "pharmaceutically
acceptable diluent" means non-therapeutic components that are of
sufficient purity and quality for use in the formulation of a
composition of the invention that, when appropriately administered
to an animal or human, typically do not produce an adverse
reaction, and that are used as a vehicle for a drug substance (i.e.
a compound of the present invention).
[0177] Pharmaceutically acceptable salts of the compounds of the
present invention are also included. For example, an acid salt of a
compound of the present invention containing an amine or other
basic group can be obtained by reacting the compound with a
suitable organic or inorganic acid, resulting in pharmaceutically
acceptable anionic salt forms. Examples of anionic salts include
the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate,
bromide, calcium edetate, camsylate, carbonate, chloride, citrate,
dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,
glyceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate,
iodide, isethionate, lactate, lactobionate, malate, maleate,
mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate,
pamoate, pantothenate, phosphate/diphosphate, polygalacturonate,
salicylate, stearate, subacetate, succinate, sulfate, tannate,
tartrate, teoclate, tosylate, and triethiodide salts.
[0178] Salts of the compounds of the present invention containing a
carboxylic acid or other acidic functional group can be prepared by
reacting with a suitable base. Such a pharmaceutically acceptable
salt may be made with a base which affords a pharmaceutically
acceptable cation, which includes alkali metal salts (especially
sodium and potassium), alkaline earth metal salts (especially
calcium and magnesium), aluminum salts and ammonium salts, as well
as salts made from physiologically acceptable organic bases such as
trimethylamine, triethylamine, morpholine, pyridine, piperidine,
picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine,
2-hydroxyethylamine, bis-(2-hydroxyethyl)amine,
tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine,
dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine,
N-methylglucamine, collidine, quinine, quinoline, and basic amino
acids such as lysine and arginine.
[0179] The invention also includes various isomers and mixtures
thereof. Certain of the compounds of the present invention may
exist in various stereoisomeric forms. Stereoisomers are compounds
which differ only in their spatial arrangement. Enantiomers are
pairs of stereoisomers whose mirror images are not superimposable,
most commonly because they contain an asymmetrically substituted
carbon atom that acts as a chiral center. "Enantiomer" means one of
a pair of molecules that are mirror images of each other and are
not superimposable. Diastereomers are stereoisomers that are not
related as mirror images, most commonly because they contain two or
more asymmetrically substituted carbon atoms. "R" and "S" represent
the configuration of substituents around one or more chiral carbon
atoms. When a chiral center is not defined as R or S, either a pure
enantiomer or a mixture of both configurations is present.
[0180] "Racemate" or "racemic mixture" means a compound of
equimolar quantities of two enantiomers, wherein such mixtures
exhibit no optical activity; i.e., they do not rotate the plane of
polarized light.
[0181] The compounds of the invention may be prepared as individual
isomers by either isomer-specific synthesis or resolved from an
isomeric mixture. Conventional resolution techniques include
forming the salt of a free base of each isomer of an isomeric pair
using an optically active acid (followed by fractional
crystallization and regeneration of the free base), forming the
salt of the acid form of each isomer of an isomeric pair using an
optically active amine (followed by fractional crystallization and
regeneration of the free acid), forming an ester or amide of each
of the isomers of an isomeric pair using an optically pure acid,
amine or alcohol (followed by chromatographic separation and
removal of the chiral auxiliary), or resolving an isomeric mixture
of either a starting material or a final product using various well
known chromatographic methods.
[0182] When the stereochemistry of a disclosed compound is named or
depicted by structure, the named or depicted stereoisomer is at
least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to
the other stereoisomers. When a single enantiomer is named or
depicted by structure, the depicted or named enantiomer is at least
60%, 70%, 80%, 90%, 99% or 99.9% by weight optically pure. Percent
optical purity by weight is the ratio of the weight of the
enantiomer that is present divided by the combined weight of the
enantiomer that is present and the weight of its optical
isomer.
[0183] The present invention also provides a method of treating or
preventing a subject with a tetracycline-responsive disease or
disorder comprising administering to the subject an effective
amount of a compound of the present invention or a pharmaceutically
acceptable salt thereof.
[0184] "Tetracycline-responsive disease or disorder" refers to a
disease or disorder that can be treated, prevented, or otherwise
ameliorated by the administration of a tetracycline compound of the
present invention. Tetracycline-responsive disease or disorder
includes infections, cancer, inflammatory disorders, autoimmune
disease, arteriosclerosis, corneal ulceration, emphysema,
arthritis, osteoporosis, osteoarthritis, multiple sclerosis,
osteosarcoma, osteomyelitis, bronchiectasis, chronic pulmonary
obstructive disease, skin and eye diseases, periodontitis,
osteoporosis, rheumatoid arthritis, ulcerative colitis,
prostatitis, tumor growth and invasion, metastasis, diabetes,
diabetic proteinuria, panbronchiolitis, aortic or vascular
aneurysms, skin tissue wounds, dry eye, bone, cartilage
degradation, malaria, senescence, diabetes, vascular stroke,
neurodegenerative disorders, cardiac disease, juvenile diabetes,
acute and chronic bronchitis, sinusitis, and respiratory
infections, including the common cold, Wegener's granulomatosis;
neutrophilic dermatoses and other inflammatory diseases such as
dermatitis herpetiformis, leukocytoclastic vasculitis, bullous
lupus erythematosus, pustular psoriasis, erythema elevatum
diutinum; vitiligo, discoid lupus erythematosus; pyoderma
gangrenosum, pustular psoriasis, blepharitis, or meibomianitis,
Alzheimer's disease, degenerative maculopathy; acute and chronic
gastroenteritis and colitis; acute and chronic cystitis and
urethritis; acute and chronic dermatitis; acute and chronic
conjunctivitis, acute and chronic serositis, uremic pericarditis;
acute and chronic cholecystis, cystic fibrosis, acute and chronic
vaginitis, acute and chronic uveitis, drug reactions, insect bites,
burns and sunburn, bone mass disorder, acute lung injury, chronic
lung disorders, ischemia, stroke or ischemic stroke, skin wound,
aortic or vascular aneurysm, diabetic retinopathy, hemorrhagic
stroke, angiogenesis, and other states for which tetracycline
compounds have been found to be active (see, for example, U.S. Pat.
Nos. 5,789,395; 5,834,450; 6,277,061 and 5,532,227, each of which
is expressly incorporated herein by reference).
[0185] In addition, a method to treat any disease or disease state
that could benefit from modulating the expression and/or function
of nitric oxide, metalloproteases, proinflammatory mediators and
cytokines, reactive oxygen species, components of the immune
response, including chemotaxis, lymphocyte transformation, delayed
hypersensitivity, antibody production, phagocytosis, and oxidative
metabolism of phagocytes. A method to treat any disease or disease
state that could benefit from modulating the expression and/or
function of C-reactive protein, signaling pathways (e.g., FAK
signaling pathway), and/or augment the expression of COX-2 and
PGE.sub.2 production is covered. A method to treat any disease or
disease state that could benefit from inhibition of
neovascularization is covered.
[0186] Compounds of the invention can be used to prevent or treat
important mammalian and veterinary diseases such as diarrhea,
urinary tract infections, infections of skin and skin structure
including wounds, cellulitis, and abscesses, ear, nose and throat
infections, mastitis and the like. In addition, methods for
treating neoplasms using tetracycline compounds of the invention
are also included (van der Bozert et al., Cancer Res., 48:
6686-6690 (1988)).
[0187] Infections that can be treated using compounds of the
invention or a pharmaceutically acceptable salt thereof include,
but are not limited to, skin infections, GI infections, urinary
tract infections, genito-urinary infections, respiratory tract
infections, sinuses infections, middle ear infections, systemic
infections, intra-abdominal infections, pyelonephritis, pneumonia,
bacterial vaginosis, streptococcal sore throat, chronic bacterial
prostatitis, gynecological and pelvic infections, sexually
transmitted bacterial diseases, ocular and otic infections,
cholera, influenza, bronchitis, acne, psoriasis, rosacea, impetigo,
malaria, sexually transmitted disease including syphilis and
gonorrhea, Legionnaires' disease, Lyme disease, Rocky Mountain
spotted fever, Q fever, typhus, bubonic plague, gas gangrene,
hospital acquired infections, leptospirosis, whooping cough,
anthrax and infections caused by the agents responsible for
lymphogranuloma venereum, inclusion conjunctivitis, or psittacosis.
Infections can be bacterial, fungal, parasitic and viral infections
(including those which are resistant to other tetracycline
compounds).
[0188] In one embodiment, the infection is a respiratory infection.
In a particular aspect, the respiratory infection is
Community-Acquired Bacterial Pneumonia (CABP). In a more particular
embodiment, the respiratory infection, for example, CABP is caused
by a bacterium selected from S. aureus, S. pneumoniae, S. pyogenes,
H. influenza, M. catarrhalis and Legionella pneumophila.
[0189] In another embodiment, the infection is a skin infection. In
a particular aspect the skin infection is an acute bacterial skin
and skin structure infection (ABSSSI). In a more particular
embodiment, the skin infection, for example ABSSSI is caused by a
bacterium selected from S. aureus, CoNS, S. pyogenes, S.
agalactiae, E. faecalis and E. faecium.
[0190] In one embodiment, the infection can be caused by a
bacterium (e.g. an anaerobic or aerobic bacterium).
[0191] In another embodiment, the infection is caused by a
Gram-positive bacterium. In a specific aspect of this embodiment,
the infection is caused by a Gram-positive bacterium selected from
class Bacilli, including, but not limited to, Staphylococcus spp.,
Streptococcus spp., Enterococcus spp., Bacillus spp., Listeria
spp.; phylum Actinobacteria, including, but not limited to,
Propionibacterium spp., Corynebacterium spp., Nocardia spp.,
Actinobacteria spp., and class Clostridia, including, but not
limited to, Clostridium spp.
[0192] In another embodiment, the infection is caused by a
Gram-positive bacterium selected from S. aureus, CoNS, S.
pneumoniae, S. pyogenes, S. agalactiae, E. faecalis and E.
faecium.
[0193] In another embodiment, the infection is caused by a
Gram-negative bacterium. In one aspect of this embodiment, the
infection is caused by a phylum Proteobacteria (e.g.,
Betaproteobacteria and Gammaproteobacteria), including Escherichia
coli, Salmonella, Shigella, other Enterobacteriaceae, Pseudomonas,
Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic
acid bacteria, Legionella or alpha-proteobacteria such as
Wolbachia. In another aspect, the infection is caused by a
Gram-negative bacterium selected from cyanobacteria, spirochaetes,
green sulfur or green non-sulfur bacteria. In a specific aspect of
this embodiment, the infection is caused by a Gram-negative
bacteria selected from Enterobactericeae (e.g., E. coli, Klebsiella
pneumoniae including those containing extended-spectrum
.beta.-lactamases and/or carbapenemases), Bacteroidetes (e.g.,
Bacteroides fragilis), Vibrionaceae (Vibrio cholerae),
Pasteurellaceae (e.g., Haemophilus influenzae), Pseudomonadaceae
(e.g., Pseudomonas aeruginosa), Neisseriaceae (e.g. Neisseria
meningitidis), Rickettsiae, Moraxellaceae (e.g., Moraxella
catarrhalis), any species of Proteeae, Acinetobacter spp.,
Helicobacter spp., and Campylobacter spp. In a particular
embodiment, the infection is caused by Gram-negative bacterium
selected from the group consisting of Enterobactericeae (e.g., E.
coli, Klebsiella pneumoniae), Pseudomonas, and Acinetobacter spp.
In another embodiment, the infection is caused by an organism
selected from the group consisting of K. pneumoniae, Salmonella, E.
hirae, A. baumanii, M. catarrhais, H. influenzae, P. aeruginosa, E.
faecium, E. coli, S. aureus, and E. faecalis.
[0194] In another embodiment, the infection is cause by a gram
negative bacterium selected from H. influenza, M. catarrhalis and
Legionella pneumophila.
[0195] In one embodiment, the infection is caused by an organism
that grows intracellularly as part of its infection process.
[0196] In another embodiment, the infection is caused by an
organism selected from the group consisting of order Rickettsiales;
phylum Chlamydiae; order Chlamydiales; Legionella spp.; class
Mollicutes, including, but not limited to, Mycoplasma spp. (e.g.
Mycoplasma pneumoniae); Mycobacterium spp. (e.g. Mycobacterium
tuberculosis); and phylum Spriochaetales (e.g. Borrelia spp. and
Treponema spp.).
[0197] In another embodiment, the infection is caused by a Category
A Biodefense organism as described at
http://www.bt.cdc.gov/agent/agentlist-category.asp, the entire
teachings of which are incorporated herein by reference. Examples
of Category A organisms include, but are not limited to, Bacillus
anthracis (anthrax), Yersinia pestis (plague), Clostridium
botulinum (botulism) or Francisella tularensis (tularemia). In
another embodiment the infection is a Bacillus anthracis infection.
"Bacillus anthracis infection" includes any state, diseases, or
disorders caused or which result from exposure or alleged exposure
to Bacillus anthracis or another member of the Bacillus cereus
group of bacteria.
[0198] Additional infections that can be treated using compounds of
the invention or a pharmaceutically acceptable salt thereof
include, but are not limited to, anthrax, botulism, bubonic plague,
and tularemia.
[0199] In another embodiment, the infection is caused by a Category
B Biodefense organism as described at
http://www.bt.cdc.gov/agent/agentlist-category.asp, the entire
teachings of which are incorporated herein by reference. Examples
of Category B organisms include, but are not limited to, Brucella
spp, Clostridium perfringens, Salmonella spp., Escherichia coli
O157:H7, Shigella spp., Burkholderia mallei, Burkholderia
pseudomallei, Chlamydia psittaci, Coxiella burnetii, Staphylococcal
enterotoxin B, Rickettsia prowazekii, Vibrio cholerae, and
Cryptosporidium parvum.
[0200] Additional infections that can be treated using compounds of
the invention or a pharmaceutically acceptable salt thereof
include, but are not limited to, Brucellosis, Clostridium
perfringens, food-borne illnesses, Glanders, Melioidosis,
Psittacosis, Q fever, and water-borne illnesses.
[0201] In yet another embodiment, the infection can be caused by
one or more than one organism described above. Examples of such
infections include, but are not limited to, intra-abdominal
infections (often a mixture of a gram-negative species like E. coli
and an anaerobe like B. fragilis), diabetic foot (various
combinations of Streptococcus, Serratia, Staphylococcus and
Enterococcus spp., anaerobes (S. E. Dowd, et al., PloS one 2008;
3:e3326, the entire teachings of which are incorporated herein by
reference) and respiratory disease (especially in patients that
have chronic infections like cystic fibrosis--e.g., S. aureus plus
P. aeruginosa or H. influenzae, atypical pathogens), wounds and
abscesses (various gram-negative and gram-positive bacteria,
notably MSSA/MRSA, coagulase-negative staphylococci, enterococci,
Acinetobacter, P. aeruginosa, E. coli, B. fragilis), and
bloodstream infections (13% were polymicrobial (H. Wisplinghoff, et
al., Clin. Infect. Dis. 2004; 39:311-317, the entire teachings of
which are incorporated herein by reference)).
[0202] In one embodiment, the infection is caused by an organism
resistant to one or more antibiotics.
[0203] In another embodiment, the infection is caused by an
organism resistant to tetracycline or any member of first and
second generation of tetracycline antibiotics (e.g., doxycycline or
minocycline).
[0204] In another embodiment, the infection is caused by an
organism resistant to methicillin.
[0205] In another embodiment, the infection is caused by an
organism resistant to vancomycin.
[0206] In another embodiment, the infection is caused by an
organism resistant to a quinolone or fluoroquinolone.
[0207] In another embodiment, the infection is caused by an
organism resistant to tigecycline or any other tetracycline
derivative. In a particular embodiment, the infection is caused by
an organism resistant to tigecycline.
[0208] In another embodiment, the infection is caused by an
organism resistant to a .beta.-lactam or cephalosporin antibiotic
or an organism resistant to penems or carbapenems.
[0209] In another embodiment, the infection is caused by an
organism resistant to an antimicrobial peptide or a biosimilar
therapeutic treatment. Antimicrobial peptides (also called host
defense peptides) are an evolutionarily conserved component of the
innate immune response and are found among all classes of life. In
this case, antimicrobial peptide refers to any naturally occurring
molecule or any semi/synthetic molecule that are analogs of anionic
peptides, linear cationic .alpha.-helical peptides, cationic
peptides enriched for specific amino acids (i.e. rich in proline,
arginine, phenylalanine, glycine, tryptophan), and anionic and
cationic peptides that contain cystein and form disulfide
bonds.
[0210] In another embodiment, the infection is caused by an
organism resistant to macrolides, lincosamides, streptogramin
antibiotics, oxazolidinones, and pleuromutilins.
[0211] In another embodiment, the infection is caused by an
organism resistant to PTK0796 (7-dimethylamino,
9-(2,2-dimethyl-propyl)-aminomethylcycline).
[0212] In another embodiment, the infection is caused by a
multidrug-resistant pathogen (having intermediate or full
resistance to any two or more antibiotics).
[0213] In a further embodiment, the tetracycline responsive disease
or disorder is not a bacterial infection. In another embodiment,
the tetracycline compounds of the invention are essentially
non-antibacterial. For example, non-antibacterial compounds of the
invention may have MIC values greater than about 4 .mu.g/ml (as
measured by assays known in the art and/or the assay given in
Example 151. In another embodiment, the tetracycline compounds of
the invention have both antibacterial and non-antibacterial
effects.
[0214] Tetracycline responsive disease or disorder also includes
diseases or disorders associated with inflammatory process
associated states (IPAS). The term "inflammatory process associated
state" includes states in which inflammation or inflammatory
factors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO),
TNF, interleukins, plasma proteins, cellular defense systems,
cytokines, lipid metabolites, proteases, toxic radicals, adhesion
molecules, etc.) are involved or are present in an area in aberrant
amounts, e.g., in amounts which may be advantageous to alter, e.g.,
to benefit the subject. The inflammatory process is the response of
living tissue to damage. The cause of inflammation may be due to
physical damage, chemical substances, micro-organisms, tissue
necrosis, cancer or other agents. Acute inflammation is
short-lasting, lasting only a few days. If it is longer lasting
however, then it may be referred to as chronic inflammation.
[0215] IPASs include inflammatory disorders. Inflammatory disorders
are generally characterized by heat, redness, swelling, pain and
loss of function. Examples of causes of inflammatory disorders
include, but are not limited to, microbial infections (e.g.,
bacterial and fungal infections), physical agents (e.g., burns,
radiation, and trauma), chemical agents (e.g., toxins and caustic
substances), tissue necrosis and various types of immunologic
reactions.
[0216] Examples of inflammatory disorders can be treated using the
compounds of the invention or a pharmaceutically acceptable salt
thereof include, but are not limited to, osteoarthritis, rheumatoid
arthritis, acute and chronic infections (bacterial and fungal,
including diphtheria and pertussis); acute and chronic bronchitis,
sinusitis, and upper respiratory infections, including the common
cold; acute and chronic gastroenteritis and colitis; inflammatory
bowel disorder; acute and chronic cystitis and urethritis;
vasculitis; sepsis; nephritis; pancreatitis; hepatitis; lupus;
inflammatory skin disorders including, for example, eczema,
dermatitis, psoriasis, pyoderma gangrenosum, acne rosacea, and
acute and chronic dermatitis; acute and chronic conjunctivitis;
acute and chronic serositis (pericarditis, peritonitis, synovitis,
pleuritis and tendinitis); uremic pericarditis; acute and chronic
cholecystis; acute and chronic vaginitis; acute and chronic
uveitis; drug reactions; insect bites; burns (thermal, chemical,
and electrical); and sunburn.
[0217] IPASs also include matrix metalloproteinase associated
states (MMPAS). MMPAS include states characterized by aberrant
amounts of MMPs or MMP activity. Examples of matrix
metalloproteinase associated states ("MMPAS's") can be treated
using compounds of the invention or a pharmaceutically acceptable
salt thereof, include, but are not limited to, arteriosclerosis,
corneal ulceration, emphysema, osteoarthritis, multiple sclerosis
(Liedtke et al., Ann. Neurol. 1998, 44: 35-46; Chandler et al., J.
Neuroimmunol. 1997, 72: 155-71), osteosarcoma, osteomyelitis,
bronchiectasis, chronic pulmonary obstructive disease, skin and eye
diseases, periodontitis, osteoporosis, rheumatoid arthritis,
ulcerative colitis, inflammatory disorders, tumor growth and
invasion (Stetler-Stevenson et al., Annu. Rev. Cell Biol. 1993, 9:
541-73; Tryggvason et al., Biochim. Biophys. Acta 1987, 907:
191-217; Li et al., Mol. Carcillog. 1998, 22: 84-89)), metastasis,
acute lung injury, stroke, ischemia, diabetes, aortic or vascular
aneurysms, skin tissue wounds, dry eye, bone and cartilage
degradation (Greenwald et al., Bone 1998, 22: 33-38; Ryan et al.,
Curr. Op. Rheumatol. 1996, 8: 238-247). Other MMPAS include those
described in U.S. Pat. Nos. 5,459,135; 5,321,017; 5,308,839;
5,258,371; 4,935,412; 4,704,383, 4,666,897, and RE 34,656,
incorporated herein by reference in their entirety.
[0218] In a further embodiment, the IPAS includes disorders
described in U.S. Pat. Nos. 5,929,055; and 5,532,227, incorporated
herein by reference in their entirety.
[0219] Tetracycline responsive disease or disorder also includes
diseases or disorders associated with NO associated states. The
term "NO associated states" includes states which involve or are
associated with nitric oxide (NO) or inducible nitric oxide
synthase (iNOS). NO associated state includes states which are
characterized by aberrant amounts of NO and/or iNOS. Preferably,
the NO associated state can be treated by administering
tetracycline compounds of the invention. The disorders, diseases
and states described in U.S. Pat. Nos. 6,231,894; 6,015,804;
5,919,774; and 5,789,395 are also included as NO associated states.
The entire contents of each of these patents are hereby
incorporated herein by reference.
[0220] Examples of diseases or disorders associated with NO
associated states can be treated using the compounds of the present
invention or a pharmaceutically acceptable salt thereof include,
but are not limited to, malaria, senescence, diabetes, vascular
stroke, neurodegenerative disorders (Alzheimer's disease and
Huntington's disease), cardiac disease (reperfusion-associated
injury following infarction), juvenile diabetes, inflammatory
disorders, osteoarthritis, rheumatoid arthritis, acute, recurrent
and chronic infections (bacterial, viral and fungal); acute and
chronic bronchitis, sinusitis, and respiratory infections,
including the common cold; acute and chronic gastroenteritis and
colitis; acute and chronic cystitis and urethritis; acute and
chronic dermatitis; acute and chronic conjunctivitis; acute and
chronic serositis (pericarditis, peritonitis, synovitis, pleuritis
and tendonitis); uremic pericarditis; acute and chronic
cholecystis; cystic fibrosis, acute and chronic vaginitis; acute
and chronic uveitis; drug reactions; insect bites; burns (thermal,
chemical, and electrical); and sunburn.
[0221] In another embodiment, the tetracycline responsive disease
or disorder is cancer. Examples of cancers that can be treated
using the compounds of the invention or a pharmaceutically
acceptable salt thereof include all solid tumors, i.e., carcinomas
e.g., adenocarcinomas, and sarcomas. Adenocarcinomas are carcinomas
derived from glandular tissue or in which the tumor cells form
recognizable glandular structures. Sarcomas broadly include tumors
whose cells are embedded in a fibrillar or homogeneous substance
like embryonic connective tissue. Examples of carcinomas which may
be treated using the methods of the invention include, but are not
limited to, carcinomas of the prostate, breast, ovary, testis,
lung, colon, and breast. The methods of the invention are not
limited to the treatment of these tumor types, but extend to any
solid tumor derived from any organ system. Examples of treatable
cancers include, but are not limited to, colon cancer, bladder
cancer, breast cancer, melanoma, ovarian carcinoma, prostate
carcinoma, lung cancer, and a variety of other cancers as well. The
methods of the invention also cause the inhibition of cancer growth
in adenocarcinomas, such as, for example, those of the prostate,
breast, kidney, ovary, testes, and colon. In one embodiment, the
cancers treated by methods of the invention include those described
in U.S. Pat. Nos. 6,100,248; 5,843,925; 5,837,696; or 5,668,122,
incorporated herein by reference in their entirety.
[0222] Alternatively, the tetracycline compounds may be useful for
preventing or reducing the likelihood of cancer recurrence, for
example, to treat residual cancer following surgical resection or
radiation therapy. The tetracycline compounds useful according to
the invention are especially advantageous as they are substantially
non-toxic compared to other cancer treatments.
[0223] In a further embodiment, the compounds of the invention are
administered in combination with standard cancer therapy, such as,
but not limited to, chemotherapy.
[0224] Examples of tetracycline responsive states can be treated
using the compounds of the invention or a pharmaceutically
acceptable salt thereof also include neurological disorders which
include both neuropsychiatric and neurodegenerative disorders, but
are not limited to, such as Alzheimer's disease, dementias related
to Alzheimer's disease (such as Pick's disease), Parkinson's and
other Lewy diffuse body diseases, senile dementia, Huntington's
disease, Gilles de la Tourette's syndrome, multiple sclerosis,
amyotrophic lateral sclerosis (ALS), progressive supranuclear
palsy, epilepsy, and Creutzfeldt-Jakob disease; autonomic function
disorders such as hypertension and sleep disorders, and
neuropsychiatric disorders, such as depression, schizophrenia,
schizoaffective disorder, Korsakoff's psychosis, mania, anxiety
disorders, or phobic disorders; learning or memory disorders, e.g.,
amnesia or age-related memory loss, attention deficit disorder,
dysthymic disorder, major depressive disorder, mania,
obsessive-compulsive disorder, psychoactive substance use
disorders, anxiety, phobias, panic disorder, as well as bipolar
affective disorder, e.g., severe bipolar affective (mood) disorder
(BP-1), bipolar affective neurological disorders, e.g., migraine
and obesity.
[0225] Further neurological disorders include, for example, those
listed in the American Psychiatric Association's Diagnostic and
Statistical manual of Mental Disorders (DSM), the most current
version of which is incorporated herein by reference in its
entirety.
[0226] In another embodiment, the tetracycline responsive disease
or disorder is diabetes. Diabetes that can be treated using the
compounds of the invention or a pharmaceutically acceptable salt
thereof include, but are not limited to, juvenile diabetes,
diabetes mellitus, diabetes type I, or diabetes type II. In a
further embodiment, protein glycosylation is not affected by the
administration of the tetracycline compounds of the invention. In
another embodiment, the tetracycline compound of the invention is
administered in combination with standard diabetic therapies, such
as, but not limited to insulin therapy.
[0227] In another embodiment, the tetracycline responsive disease
or disorder is a bone mass disorder. Bone mass disorders that can
be treated using the compounds of the invention or a
pharmaceutically acceptable salt thereof include disorders where a
subjects bones are disorders and states where the formation, repair
or remodeling of bone is advantageous. For examples bone mass
disorders include osteoporosis (e.g., a decrease in bone strength
and density), bone fractures, bone formation associated with
surgical procedures (e.g., facial reconstruction), osteogenesis
imperfecta (brittle bone disease), hypophosphatasia, Paget's
disease, fibrous dysplasia, osteopetrosis, myeloma bone disease,
and the depletion of calcium in bone, such as that which is related
to primary hyperparathyroidism. Bone mass disorders include all
states in which the formation, repair or remodeling of bone is
advantageous to the subject as well as all other disorders
associated with the bones or skeletal system of a subject which can
be treated with the tetracycline compounds of the invention. In a
further embodiment, the bone mass disorders include those described
in U.S. Pat. Nos. 5,459,135; 5,231,017; 5,998,390; 5,770,588; RE
34,656; 5,308,839; 4,925,833; 3,304,227; and 4,666,897, each of
which is hereby incorporated herein by reference in its
entirety.
[0228] In another embodiment, the tetracycline responsive disease
or disorder is acute lung injury. Acute lung injuries that can be
treated using the compounds of the invention or a pharmaceutically
acceptable salt thereof include adult respiratory distress syndrome
(ARDS), post-pump syndrome (PPS), and trauma. Trauma includes any
injury to living tissue caused by an extrinsic agent or event.
Examples of trauma include, but are not limited to, crush injuries,
contact with a hard surface, or cutting or other damage to the
lungs.
[0229] The tetracycline responsive disease or disorders of the
invention also include chronic lung disorders. Examples of chronic
lung disorders that can be treated using the compounds of the
invention or a pharmaceutically acceptable salt thereof include,
but are not limited, to asthma, cystic fibrosis, chronic
obstructive pulmonary disease (COPD), and emphysema. In a further
embodiment, the acute and/or chronic lung disorders that can be
treated using the compounds of the invention or a pharmaceutically
acceptable salt thereof include those described in U.S. Pat. Nos.
5,977,091; 6,043,231; 5,523,297; and 5,773,430, each of which is
hereby incorporated herein by reference in its entirety.
[0230] In yet another embodiment, the tetracycline responsive
disease or disorder is ischemia, stroke, or ischemic stroke.
[0231] In a further embodiment, the tetracycline compounds of the
invention or a pharmaceutically acceptable salt thereof can be used
to treat such disorders as described above and in U.S. Pat. Nos.
6,231,894; 5,773,430; 5,919,775 and 5,789,395, incorporated herein
by reference.
[0232] In still a further embodiment, the tetracycline compounds of
the invention or a pharmaceutically acceptable salt thereof can be
used to treat pain, for example, inflammatory, nociceptive or
neuropathic pain. The pain can be either acute or chronic.
[0233] In another embodiment, the tetracycline responsive disease
or disorder is a skin wound. The invention also provides a method
for improving the healing response of the epithelialized tissue
(e.g., skin, mucosae) to acute traumatic injury (e.g., cut, burn,
scrape, etc.). The method includes using a tetracycline compound of
the invention or a pharmaceutically acceptable salt thereof to
improve the capacity of the epithelialized tissue to heal acute
wounds. The method may increase the rate of collagen accumulation
of the healing tissue. The method may also decrease the proteolytic
activity in the epithelialized tissue by decreasing the
collagenolytic and/or gellatinolytic activity of MMPs. In a further
embodiment, the tetracycline compound of the invention or a
pharmaceutically acceptable salt thereof is administered to the
surface of the skin (e.g., topically). In a further embodiment, the
tetracycline compound of the invention or a pharmaceutically
acceptable salt thereof is used to treat a skin wound, and other
such disorders as described in, for example, U.S. Pat. Nos.
5,827,840; 4,704,383; 4,935,412; 5,258,371; 5,308,839, 5,459,135;
5,532,227; and 6,015,804; each of which is incorporated herein by
reference in its entirety.
[0234] In yet another embodiment, the tetracycline responsive
disease or disorder is an aortic or vascular aneurysm in vascular
tissue of a subject (e.g., a subject having or at risk of having an
aortic or vascular aneurysm, etc.). The tetracycline compound or a
pharmaceutically acceptable salt thereof may be effective to reduce
the size of the vascular aneurysm or it may be administered to the
subject prior to the onset of the vascular aneurysm such that the
aneurysm is prevented. In one embodiment, the vascular tissue is an
artery, e.g., the aorta, e.g., the abdominal aorta. In a further
embodiment, the tetracycline compounds of the invention are used to
treat disorders described in U.S. Pat. Nos. 6,043,225 and
5,834,449, incorporated herein by reference in their entirety.
[0235] The compounds of the invention or a pharmaceutically
acceptable salt thereof can be used alone or in combination with
one or more therapeutic agent in the methods of the invention
disclosed herein.
[0236] The language "in combination with" another therapeutic agent
or treatment includes co-administration of the tetracycline
compound and with the other therapeutic agent or treatment as
either a single combination dosage form or as multiple, separate
dosage forms, administration of the tetracycline compound first,
followed by the other therapeutic agent or treatment and
administration of the other therapeutic agent or treatment first,
followed by the tetracycline compound.
[0237] The other therapeutic agent may be any agent that is known
in the art to treat, prevent, or reduce the symptoms of a
tetracycline-responsive disease or disorder. The choice of
additional therapeutic agent(s) is based upon the particular
tetracycline-responsive disease or disorder being treated. Such
choice is within the knowledge of a treating physician.
Furthermore, the other therapeutic agent may be any agent of
benefit to the patient when administered in combination with the
administration of a tetracycline compound.
[0238] The compounds of the invention or a pharmaceutically
acceptable salt thereof can be used alone or in combination with
one or more antibiotics and/or immunomodulators (e.g. Deoxycholic
acid, Macrokine, Abatacept, Belatacept, Infliximab, Adalimumab,
Certolizumab pegol, Afelimomab, Golimumab, and
FKBP/Cyclophilin/Calcineurin: Tacrolimus, Ciclosporin,
Pimecrolimus).
[0239] As used herein, the term "subject" means a mammal in need of
treatment or prevention, e.g., companion animals (e.g., dogs, cats,
and the like), farm animals (e.g., cows, pigs, horses, sheep, goats
and the like) and laboratory animals (e.g., rats, mice, guinea pigs
and the like). Typically, the subject is a human in need of the
specified treatment.
[0240] As used herein, the term "treating" or "treatment" refers to
obtaining desired pharmacological and/or physiological effect. The
effect can include achieving, partially or substantially, one or
more of the following results: partially or totally reducing the
extent of the disease, disorder or syndrome; ameliorating or
improving a clinical symptom or indicator associated with the
disorder; delaying, inhibiting or decreasing the likelihood of the
progression of the disease, disorder or syndrome.
[0241] As used herein, "preventing" or "prevention" refers to
reducing the likelihood of the onset or development of disease,
disorder or syndrome.
[0242] "Effective amount" means that amount of active compound
agent that elicits the desired biological response in a subject. In
one embodiment, the effective amount of a compound of the invention
is from about 0.01 mg/kg/day to about 1000 mg/kg/day, from about
0.1 mg/kg/day to about 100 mg/kg/day, or from about 0.5 mg/kg/day
to about 50 mg/kg/day.
[0243] The invention further includes the process for making the
composition comprising mixing one or more of the present compounds
and an optional pharmaceutically acceptable carrier; and includes
those compositions resulting from such a process, which process
includes conventional pharmaceutical techniques.
[0244] The compositions of the invention include ocular, oral,
nasal, transdermal, topical with or without occlusion, intravenous
(both bolus and infusion), inhalable, and injection
(intraperitoneally, subcutaneously, intramuscularly,
intratumorally, or parenterally) formulations. The composition may
be in a dosage unit such as a tablet, pill, capsule, powder,
granule, liposome, ion exchange resin, sterile ocular solution, or
ocular delivery device (such as a contact lens and the like
facilitating immediate release, timed release, or sustained
release), parenteral solution or suspension, metered aerosol or
liquid spray, drop, ampoule, auto-injector device, or suppository;
for administration ocularly, orally, intranasally, sublingually,
parenterally, or rectally, or by inhalation or insufflation.
[0245] Compositions of the invention suitable for oral
administration include solid forms such as pills, tablets, caplets,
capsules (each including immediate release, timed release, and
sustained release formulations), granules and powders; and, liquid
forms such as solutions, syrups, elixirs, emulsions, and
suspensions. Forms useful for ocular administration include sterile
solutions or ocular delivery devices. Forms useful for parenteral
administration include sterile solutions, emulsions, and
suspensions.
[0246] The compositions of the invention may be administered in a
form suitable for once-weekly or once-monthly administration. For
example, an insoluble salt of the active compound may be adapted to
provide a depot preparation for intramuscular injection (e.g., a
decanoate salt) or to provide a solution for ophthalmic
administration.
[0247] The dosage form containing the composition of the invention
contains an effective amount of the active ingredient necessary to
provide a therapeutic effect. The composition may contain from
about 5,000 mg to about 0.5 mg (preferably, from about 1,000 mg to
about 0.5 mg) of a compound of the invention or salt form thereof
and may be constituted into any form suitable for the selected mode
of administration. The composition may be administered about 1 to
about 5 times per day. Daily administration or post-periodic dosing
may be employed.
[0248] For oral administration, the composition is preferably in
the form of a tablet or capsule containing, e.g., 500 to 0.5
milligrams of the active compound. Dosages will vary depending on
factors associated with the particular patient being treated (e.g.,
age, weight, diet, and time of administration), the severity of the
condition being treated, the compound being employed, the mode of
administration, and the strength of the preparation.
[0249] The oral composition is preferably formulated as a
homogeneous composition, wherein the active ingredient is dispersed
evenly throughout the mixture, which may be readily subdivided into
dosage units containing equal amounts of a compound of the
invention. Preferably, the compositions are prepared by mixing a
compound of the invention (or pharmaceutically acceptable salt
thereof) with one or more optionally present pharmaceutical
carriers (such as a starch, sugar, diluent, granulating agent,
lubricant, glidant, binding agent, and disintegrating agent), one
or more optionally present inert pharmaceutical excipients (such as
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents, and syrup), one or more optionally present
conventional tableting ingredients (such as corn starch, lactose,
sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate, and any of a variety of gums), and an optional
diluent (such as water).
[0250] Binder agents include starch, gelatin, natural sugars (e.g.,
glucose and beta-lactose), corn sweeteners and natural and
synthetic gums (e.g., acacia and tragacanth). Disintegrating agents
include starch, methyl cellulose, agar, and bentonite.
[0251] Tablets and capsules represent an advantageous oral dosage
unit form. Tablets may be sugarcoated or filmcoated using standard
techniques. Tablets may also be coated or otherwise compounded to
provide a prolonged, control-release therapeutic effect. The dosage
form may comprise an inner dosage and an outer dosage component,
wherein the outer component is in the form of an envelope over the
inner component. The two components may further be separated by a
layer which resists disintegration in the stomach (such as an
enteric layer) and permits the inner component to pass intact into
the duodenum or a layer which delays or sustains release. A variety
of enteric and non-enteric layer or coating materials (such as
polymeric acids, shellacs, acetyl alcohol, and cellulose acetate or
combinations thereof) may be used.
[0252] Compounds of the invention may also be administered via a
slow release composition; wherein the composition includes a
compound of the invention and a biodegradable slow release carrier
(e.g., a polymeric carrier) or a pharmaceutically acceptable
non-biodegradable slow release carrier (e.g., an ion exchange
carrier).
[0253] Biodegradable and non-biodegradable slow release carriers
are well known in the art. Biodegradable carriers are used to form
particles or matrices which retain an active agent(s) and which
slowly degrade/dissolve in a suitable environment (e.g., aqueous,
acidic, basic and the like) to release the agent. Such particles
degrade/dissolve in body fluids to release the active compound(s)
therein. The particles are preferably nanoparticles or
nanoemulsions (e.g., in the range of about 1 to 500 nm in diameter,
preferably about 50-200 nm in diameter, and most preferably about
100 nm in diameter). In a process for preparing a slow release
composition, a slow release carrier and a compound of the invention
are first dissolved or dispersed in an organic solvent. The
resulting mixture is added into an aqueous solution containing an
optional surface-active agent(s) to produce an emulsion. The
organic solvent is then evaporated from the emulsion to provide a
colloidal suspension of particles containing the slow release
carrier and the compound of the invention.
[0254] The compound disclosed herein may be incorporated for
administration orally or by injection in a liquid form such as
aqueous solutions, suitably flavored syrups, aqueous or oil
suspensions, flavored emulsions with edible oils such as cottonseed
oil, sesame oil, coconut oil or peanut oil and the like, or in
elixirs or similar pharmaceutical vehicles. Suitable dispersing or
suspending agents for aqueous suspensions, include synthetic and
natural gums such as tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone, and
gelatin. The liquid forms in suitably flavored suspending or
dispersing agents may also include synthetic and natural gums. For
parenteral administration, sterile suspensions and solutions are
desired. Isotonic preparations, which generally contain suitable
preservatives, are employed when intravenous administration is
desired.
[0255] The compounds may be administered parenterally via
injection. A parenteral formulation may consist of the active
ingredient dissolved in or mixed with an appropriate inert liquid
carrier. Acceptable liquid carriers usually comprise aqueous
solvents and other optional ingredients for aiding solubility or
preservation. Such aqueous solvents include sterile water, Ringer's
solution, or an isotonic aqueous saline solution. Other optional
ingredients include vegetable oils (such as peanut oil, cottonseed
oil, and sesame oil), and organic solvents (such as solketal,
glycerol, and formyl). A sterile, non-volatile oil may be employed
as a solvent or suspending agent. The parenteral formulation is
prepared by dissolving or suspending the active ingredient in the
liquid carrier whereby the final dosage unit contains from 0.005 to
10% by weight of the active ingredient. Other additives include
preservatives, isotonizers, solubilizers, stabilizers, and
pain-soothing agents. Injectable suspensions may also be prepared,
in which case appropriate liquid carriers, suspending agents and
the like may be employed.
[0256] Compounds of the invention may be administered intranasally
using a suitable intranasal vehicle.
[0257] In another embodiment, the compounds of this invention may
be administered directly to the lungs by inhalation.
[0258] Compounds of the invention may also be administered
topically or enhanced by using a suitable topical transdermal
vehicle or a transdermal patch.
[0259] For ocular administration, the composition is preferably in
the form of an ophthalmic composition. The ophthalmic compositions
are preferably formulated as eye-drop formulations and filled in
appropriate containers to facilitate administration to the eye, for
example a dropper fitted with a suitable pipette. Preferably, the
compositions are sterile and aqueous based, using purified water.
In addition to the compound of the invention, an ophthalmic
composition may contain one or more of: a) a surfactant such as a
polyoxyethylene fatty acid ester; b) a thickening agents such as
cellulose, cellulose derivatives, carboxyvinyl polymers, polyvinyl
polymers, and polyvinylpyrrolidones, typically at a concentration
in the range of about 0.05 to about 5.0% (wt/vol); c) (as an
alternative to or in addition to storing the composition in a
container containing nitrogen and optionally including a free
oxygen absorber such as Fe), an anti-oxidant such as butylated
hydroxyanisol, ascorbic acid, sodium thiosulfate, or butylated
hydroxytoluene at a concentration of about 0.00005 to about 0.1%
(wt/vol); d) ethanol at a concentration of about 0.01 to 0.5%
(wt/vol); and e) other excipients such as an isotonic agent,
buffer, preservative, and/or pH-controlling agent. The pH of the
ophthalmic composition is desirably within the range of 4 to 8.
[0260] In certain embodiments, the composition of this invention
includes one or more additional agents. The other therapeutic agent
may be ay agent that is capable of treating, preventing or reducing
the symptoms of a tetracycline-responsive disease or disorder.
Alternatively, the other therapeutic agent may be any agent of
benefit to a patient when administered in combination with the
tetracycline compound in this invention.
[0261] While this invention has been particularly shown and
described with references to example embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
EXEMPLIFICATION
[0262] The following abbreviations are used in throughout the
application. [0263] Ac acetyl [0264] AIBN
2,2'-azobis(2-methylpropionitrile) [0265] aq aqueous [0266] Bn
benzyl [0267] Boc tert-butoxycarbonyl [0268] Bu butyl [0269] Cbz
benzyloxycarbonyl [0270] Cy tricyclohexylphosphine [0271] dba
dibenzylideneacetone [0272] DIBAL-H diisobutylaluminum hydride
[0273] DIEA N,N-diisopropylethylamine [0274] DMAP
4-(dimethylamino)pyridine [0275] DME 1,2-dimethoxyethane [0276] DMF
N,N-dimethylformamide [0277] DMPU
1,3-dimethyl-3,4-5,6-tetrahydro-2(1H)-pyrimidone [0278] DMSO
dimethyl sulfoxide [0279] EDC
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide [0280] ESI
electrospray ionization [0281] Et ethyl [0282] EtOAc ethyl acetate
[0283] HPLC high performance liquid chromatography [0284] HOBt
1-hydroxybenzotriazole [0285] i iso [0286] IBX 2-iodoxybenzoic acid
[0287] LDA lithium diisopropylamide [0288] LHMDS lithium
bis(trimethylsilyl)amide [0289] LTMP lithium
2,2,6,6-tetramethylpiperidide [0290] MeOH methanol [0291] Ms
methanesulfonyl [0292] MS mass spectrometry [0293] MTBE methyl
tert-butyl ether [0294] MW molecular weight [0295] NBS
N-bromosuccinimide [0296] NCS N-chlorosuccinimide [0297] NMR
nuclear magnetic resonance spectrometry [0298] Ph phenyl [0299] Pr
propyl [0300] s secondary [0301] t tertiary [0302] TMEDA
N,N,N'N'-tetramethylethylenediamine [0303] TBS
tert-butyldimethylsilyl [0304] TEA triethylamine [0305] Tf
trifluoromethanesulfonyl [0306] TFA trifluoroacetic acid [0307]
TFAA trifluoroacetic anhydride [0308] THF tetrahydrofuran [0309]
TLC thin layer chromatography [0310] Ts para-toluenesulfonyl [0311]
TsOH para-toluenesulfonic acid [0312] Xantphos
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
[0313] Detailed procedures for each of the steps depicted in the
following Schemes 1-13 are set forth in the Examples section.
[0314] Compounds of Formula II were prepared according to one of
Schemes 7-9, depending upon the actual structure. Intermediates
used in Scheme 7-9 were prepared by one of Schemes 1-6, as was
appropriate for the final structure of the compound.
[0315] Compounds of Formula II, wherein X is fluoro were
synthesized using a common N-substituted phenyl
4-(benzyloxy)-7-fluoro-6-methylisoindoline-5-carboxylate
intermediate, which is prepared according to Scheme 1.
##STR00114##
[0316] An alternate route to certain N-substituted phenyl
4-(benzyloxy)-7-fluoro-6-methylisoindoline-5-carboxylate
intermediates is shown in Scheme 2
##STR00115##
[0317] Compounds of Formula II, wherein X is chloro were
synthesized using a common N-substituted phenyl
4-(benzyloxy)-7-chloro-6-methylisoindoline-5-carboxylate
intermediate, which is prepared according to Scheme 3.
##STR00116##
[0318] Compounds of Formula II, wherein X is CF.sub.3 were
synthesized using a common N-substituted phenyl
4-(benzyloxy)-7-trifluoromethyl-6-methylisoindoline-5-carboxylate
intermediate, which is prepared according to Scheme 4.
##STR00117##
[0319] Compounds of Formula II, wherein X is OCH.sub.3 were
synthesized using a common N-substituted phenyl
4-(benzyloxy)-7-methoxy-6-methylisoindoline-5-carboxylate
intermediate, which is prepared according to Scheme 5.
##STR00118##
[0320] Compounds of Formula II, wherein X is N(CH.sub.3).sub.2 were
synthesized using a common N-substituted phenyl
4-(benzyloxy)-7-dimethylamino-6-methylisoindoline-5-carboxylate
intermediate, which is prepared according to Scheme 6.
##STR00119##
[0321] Compounds of Formula II were synthesized by combining any of
the intermediates S1-11, S2-1, S3-13, S4-10, S5-9, or S6-2
described above in Schemes 1-6 with an enone S7-1, followed by
deprotection and reduction according to Scheme 7.
##STR00120##
[0322] Compounds of Formula II wherein X is fluoro and R.sup.1 is
--C(O)CH.sub.2N(R.sup.2)(R.sup.3) or hydrogen were prepared
according to Scheme 8.
##STR00121##
[0323] Compound of Formula II wherein X is hydrogen are prepared by
reduction of the corresponding compounds wherein X is chloro
according to Scheme 9
##STR00122##
[0324] Compounds of Formula III are synthesized through a common
N-substituted phenyl
8-(benzyloxy)-5-fluoro-6-methyl-1,2,3,4-tetrahydroisoquinoline-7-carboxyl-
ate intermediate (S10-3) according to Scheme 10, below
##STR00123##
[0325] Compounds of Formula IV were prepared using a common
N-substituted phenyl
5-(benzyloxy)-8-fluoro-7-methyl-1,2,3,4-tetrahydroisoquinoline-6-c-
arboxylate intermediate according to Scheme 11
##STR00124##
[0326] Compounds of Formula V, wherein R.sup.7a and R.sup.7b are
taken together to form .dbd.O are synthesized according to Scheme
12.
##STR00125##
[0327] Compounds of Formula V, wherein R.sup.7a and R.sup.7b are
hydrogen are prepared according to Scheme 13
##STR00126## ##STR00127##
Example 1. Preparation of phenyl
4-(benzyloxy)-2-tert-butyl-7-fluoro-6-methylisoindoline-5-carboxylate
(S1-11-1)
Synthesis of S1-2
##STR00128##
[0329] To a THF solution of 5-fluoro-2-methoxybenzoic acid (S1-1,
500 mg, 2.94 mmol, Aldrich 523097) cooled at -78.degree. C. was
added a THF solution of s-BuLi (4.60 mL, 1.40 M, 6.44 mmol, 2.2 eq)
and TMEDA (0.97 mL, 6.47 mmol, 2.2 eq). The reaction was stirred at
-78.degree. C. for 2 h. Methyl iodide (1.10 mL, 17.64 mmol, 6 eq)
was added to the reaction mixture dropwise. The reaction was
allowed to warm to 25.degree. C. over 1 h and stirred at 25.degree.
C. for 1 h. NaOH (6 N, 20 mL) was added. The resulting mixture was
extracted with t-butylmethyl ether (20 mL.times.2). The aqueous
layer was acidified with HCl (6 N) to pH 1 and extracted with EtOAc
(20 mL.times.4). The combined EtOAc extracts were dried
(Na.sub.2SO.sub.4) and concentrated to give 510 mg of crude product
S1-2: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.06 (dd, J=9.8,
8.5 Hz, 1H), 6.75 (dd, J=9.8, 3.7 Hz, 1H), 3.86 (s, 3H), 2.34 (d,
J=2.4 Hz, 3H); MS (ESI) m/z 185.12 (M+H).
Synthesis of S1-3
##STR00129##
[0331] Oxalyl chloride (0.95 mL, 11.10 mmol, 5.5 eq) was added to
CH.sub.2Cl.sub.2 solution (15 mL, anhydrous) of S1-2 (510 mg, 2.00
mmol). DMF (0.1 mL) was added to the resulting mixture. The
reaction was stirred at 25.degree. C. for 1 h and concentrated. The
resulting solid was re-dissolved in 15 mL of anhydrous
CH.sub.2Cl.sub.2. Phenol (520 mg, 5.50 mmol, 2.8 eq), DMAP (670 mg,
5.6 mmol, 2.8 eq), and triethylamine (1.90 mL, 13.90 mmol, 7.0 eq)
were added to the reaction mixture. The reaction was stirred at
25.degree. C. for 12 h and concentrated. EtOAc and H.sub.2O were
added to the residue. The organic layer was washed with NaOH (1 N),
H.sub.2O, and brine, dried (Na.sub.2SO.sub.4), and concentrated.
Flash chromatography on silica gel (40:1 hexanes/EtOAc) yielded 400
mg of compound S1-3 (52% for 2 steps): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.47-7.41 (m, 2H), 7.31-7.24 (m, 3H), 7.08 (dd,
J=9.2, 9.2 Hz, 1H), 6.77 (dd, J=9.2, 3.7 Hz, 1H), 3.88 (s, 3H),
2.36 (d, J=2.3 Hz, 3H); MS (ESI) m/z 261.12 (M+H).
Synthesis of S1-4
##STR00130##
[0333] BBr.sub.3 (1.85 mL, 1 M, 1.85 mmol, 1.2 eq) was added to a
CH.sub.2Cl.sub.2 solution (8 mL) of S1-3 (400 mg, 1.54 mmol) at
-78.degree. C. The reaction was stirred from -78.degree. C. to
25.degree. C. for 1.5 h, quenched with saturated NaHCO.sub.3 and
concentrated. EtOAc and H.sub.2O were added to the reaction
mixture. The aqueous layer was extracted with EtOAc. The combined
EtOAc extracts were dried (Na.sub.2SO.sub.4) and concentrated to
yield 360 mg of crude S1-4: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 10.66 (s, 1H), 7.50-7.44 (m, 2H), 7.36-7.31 (m, 1H),
7.26-7.18 (m, 3H), 6.86 (dd, J=9.3, 4.9 Hz, 1H), 2.60 (d, J=2.4 Hz,
3H); MS (ESI) m/z 245.11 (M-H).
Synthesis of S1-5
##STR00131##
[0335] Compound S1-4 (4.92 g, 95% purity, 20 mmol) was dissolved in
acetic acid (50 mL) and bromine (1.54 mL, 30 mmol) was added via
syringe at room temp. After stirred at room temp for 2 hour, LC/MS
indicated that the starting material was consumed. This reaction
mixture was dilute with ethyl acetate, wash with water (3.times.100
mL) and brine. The organics were dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. This gave 7.06 g
of compound S1-5 as light yellow solid: .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 11.14 (s, 1H), 7.52 (d, J=9.2 Hz, 1H),
7.49-7.43 (m, 2H), 7.36-7.30 (m, 1H), 7.21-7.16 (m, 2H), 2.55 (d,
J=2.3 Hz, 3H).
Synthesis of S1-6
##STR00132##
[0337] Compound S1-5 (crude, 1.06 g, 2.97 mmol) was dissolve in
acetone (20 mL) with potassium carbonate (821 mg, 5.94 mmol, 2.0
eq) and cooled to 0.degree. C. in an ice-bath. Benzyl bromide (540
.mu.L, 4.45 mmol, 1.5 eq) was added dropwise. After 2 hrs, LC/MS
indicated that the starting material was consumed 40%. The reaction
mixture was heated to 50.degree. C. for another hour and the
starting material was all consumed. The reaction mixture was
diluted with ethyl acetate (100 mL) and washed with water and
brine. The organics were dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure. This gave 2.2 g of the crude
S1-6, which was purified by column chromatography (Biotage 10 g
column, 2 to 5% ethyl acetate in hexane gradient), yielding 1.03 g
(84% for two steps) of the pure compound S1-6 as an colorless oil:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.50-7.47 (m, 2H),
7.40-7.33 (m, 6H), 7.25 (t, J=7.3 Hz, 1H), 7.04 (d, J=8.6 Hz, 2H),
5.09 (s, 2H), 2.32 (d, J=1.8 Hz, 3H).
Synthesis of S1-7
##STR00133##
[0339] LDA solution was prepared by adding n-BuLi (1.6 M, 5.1 mL,
8.16 mmol, 1.5 eq) to diisopropylamine (1.15 mL, 8.16 mmol) in THF
(15 mL) at -78.degree. C. The reaction mixture was warmed up to
-20.degree. C. and stirred for 15 min. After LDA solution was
cooled to -78.degree. C., compound S1-6 (2.26 g, 5.44 mmol) in THF
(5 mL) was added dropwise, forming an orange-red solution. After 10
min, DMF (1.26 mL, 16.3 mmol, 3 eq) was added dropwise. The
reaction solution was allowed to warm up to -20.degree. C. in 1
hour and was quenched with NH.sub.4Cl (aq. Solution). LC/MS
indicated that the starting material was all consumed. The reaction
mixture was diluted with ethyl acetate (100 mL) and washed with
water and brine. The organics were dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. This gave 2.42 g
of the crude S1-7, which was purified by column chromatography
(Biotage 24 g column, 5 to 10% ethyl acetate in hexane gradient),
yielding 2.23 g (92%) of the pure compound S1-7 as light yellow
solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.37 (s, 1H),
7.51-7.47 (m, 2H), 7.40-7.33 (m, 5H), 7.27 (t, J=7.3 Hz, 1H),
7.06-7.02 (m, 2H), 5.12 (s, 2H), 2.37 (d, J=2.3 Hz, 3H).
Synthesis of S1-8
##STR00134##
[0341] Compound S1-7 (416 mg, 0.94 mmol) was dissolved in methanol
(5 mL) and sodium borohydride (75.6 mg, 2 mmol) was added in
several portions. During the addition, gas evolution was observed.
After stirring at rt for 30 min, LC/MS indicated that the starting
material was consumed. This reaction mixture was diluted with ethyl
acetate and washed with water (2.times.20 mL) and brine. The
organics were dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure. The crude material was
purified by column chromatography (Biotage 10 g column, 5 to 20%
ethyl acetate in hexane gradient), yielding 367 mg (87.7%) of the
pure compound S1-8 as a colorless oil. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.37 (s, 1H), 7.49 (dd, 1=7.8, 2.3 Hz, 2H),
7.40-7.33 (m, 5H), 7.25 (t, J=7.8 Hz, 1H), 7.07-7.02 (m, 2H), 5.10
(s, 2H), 4.91 (dd, J=6.9, 2.3 Hz, 2H), 2.35 (d, J=2.3 Hz, 3H); MS
(ESI) m/z 467.10, 469.08 (M+Na).
Synthesis of S1-9
##STR00135##
[0343] i-Propyl magnesium chloride/lithium chloride solution
(Chemetall Foote Corporation, 1.2 M solution in THF, 4.4 mL, 5.3
mmol) was added to a -78.degree. C. solution of compound S1-8 (472
mg, 1.06 mmol) in THF (10 mL). The reaction mixture was allowed to
warm to 0.degree. C. over 1 hour. Paraformaldehyde (318 mg, 10.6
mmol) was added, and the reaction was allowed to warm to rt. After
1 hour, the reaction mixture was heated to 40.degree. C. After 1
hour, the reaction mixture was quenched with ammonium chloride
(saturated, aqueous solution) and was extracted with EtOAc
(2.times.). The combined extracts were dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. The crude
material was purified by column chromatography (Biotage 10 g
column, 10 to 35% EtOAc in hexane gradient), yielding 337 mg (80%)
of S1-9 as a thick oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.45-7.34 (m, 7H), 7.30-7.23 (m, 1H), 7.10 (d, J=7.8 Hz, 2H), 5.08
(s, 2H), 4.85 (s, 2H), 4.76 (s, 2H), 2.39 (d, J=2.3 Hz, 3H); MS
(ESI) m/z 419.19 (M+Na).
Synthesis of S1-10
##STR00136##
[0345] To a solution of compound S1-9 (2.98 g, 7.52 mmol, 1 eq) in
1,2-dichloroethane (20 mL) was added thionyl chloride (2.18 mL,
30.1 mmol, 4 eq) and tetrabutylammonium chloride (174 mg, 0.76
mmol, 0.1 eq). The reaction vessel was sealed and the mixture
heated to 80.degree. C. for 2 h, then concentrated under reduced
pressure. Purification of the resulting crude oil via flash column
chromatography on silica gel (Redisep, 80 g, 4 to 6% EtOAc in
hexane gradient) provided 2.66 g of S1-10 (81%) as a waxy white
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.42 (m, 2H),
7.41-7.34 (m, 4H), 7.29-7.24 (m, 1H), 7.10-7.05 (m, 2H), 5.13 (s,
2H), 4.81 (s, 4H), 2.44-2.39 (m, 3H); MS (ESI) m/z 431.14, 433.16
(M+H).
Synthesis of S1-11-1
##STR00137##
[0347] Compound S1-10 (120 mg, 0.277 mmol), t-butylamine (0.032 mL,
0.305 mmol) and diisopropylethylamine (0.096 mL, 0.554 mmol) were
heated to 110.degree. C. in 1,2-dimethoxyethane (1 mL). After 2
hours, additional t-butylamine (0.100 mL, 0.95 mmol) was added.
After 2 more hours, additional t-butylamine (0.500 mL, 4.75 mmol)
was added, and the reaction mixture was heated overnight. The
reaction mixture was concentrated under reduced pressure and was
purified by column chromatography (Biotage 10 g column, 5 to 20%
EtOAc in hexane gradient), yielding 64.1 mg (53%) of the product.
R.sub.f=0.25 in 20% EtOAc in hexane; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.42-7.30 (m, 7H), 7.27-7.20 (m, 1H), 7.04 (d,
J=7.8 Hz, 2H), 5.02 (s, 2H), 4.08 (s, 2H), 4.04 (s, 2H), 2.33 (d,
J=1.8 Hz, 3H), 1.15 (s, 9H); MS (ESI) m/z 434.29 (M+H).
[0348] The following compounds were prepared by methods similar to
those described for S1-11-1.
Example 2. S1-11-2
##STR00138##
[0350] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41-7.30 (m, 7H),
7.25-7.20 (m, 1H), 7.05-7.00 (m, 2H), 5.01 (s, 2H), 4.67 (t, J=4.9
Hz, 1H), 4.55 (t, J=4.9 Hz, 1H), 4.08 (s, 4H), 3.08 (t, J=4.9 Hz,
1H), 3.01 (t, J=4.9 Hz, 1H), 2.34-2.32 (m, 3H); MS (ESI) m/z 424.63
(M+H).
Example 3. S1-11-3
##STR00139##
[0352] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.43-7.31 (m, 7H),
7.25-7.20 (m, 1H), 7.07-7.01 (m, 2H), 5.03 (s, 2H), 4.07 (s, 4H),
3.57 (t, J=5.5 Hz, 2H), 3.41 (s, 3H), 2.95 (t, J=5.5 Hz, 2H),
2.36-2.34 (m, 3H); MS (ESI) m/z 436.38 (M+H).
Example 4. S1-11-4
##STR00140##
[0354] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.37-7.31 (m, 7H),
7.29-7.23 (m, 1H), 7.05-6.99 (m, 2H), 5.01 (s, 2H), 3.95 (s, 3H),
2.47 (d, J=6.1 Hz, 2H), 2.33 (s, 3H), 1.83-1.72 (m, 1H), 0.95 (d,
J=5.5 Hz, 6 Hz); MS (ESI) m/z 434.27 (M+H).
Example 5. S1-11-5
##STR00141##
[0356] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41-7.32 (m, 7H),
7.25-7.20 (m, 1H), 7.07-7.02 (m, 2H), 5.03 (s, 2H), 4.16-4.01 (m,
5H), 3.96-3.87 (m, 1H), 3.84-3.76 (m, 1H), 3.37-3.27 (m, 1H),
2.89-2.77 (m, 2H), 2.35 (s, 3H), 1.98-1.83 (m, 2H), 1.66-1.54 (m,
1H); MS (ESI) m/z 462.82 (M+H).
Example 6. S1-11-6
##STR00142##
[0358] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41-7.32 (m, 7H),
7.25-7.20 (m, 1H), 7.07-7.02 (m, 2H), 5.03 (s, 2H), 4.16-4.01 (m,
5H), 3.96-3.87 (m, 1H), 3.84-3.76 (m, 1H), 3.37-3.27 (m, 1H),
2.89-2.77 (m, 2H), 2.35 (s, 3H), 1.98-1.83 (m, 2H), 1.66-1.54 (m,
1H); MS (ESI) m/z 462.80 (M+H).
Example 7. S1-11-7
##STR00143##
[0360] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.44-7.30 (m, 7H),
7.25-7.20 (m, 1H), 7.08-7.00 (m, 2H), 5.04 (s, 2H), 4.06-3.95 (m,
4H), 2.82-2.71 (m, 1H), 2.35 (s, 3H), 1.18 (d, J=6.1 Hz, 6H); MS
(ESI) m/z 420.62 (M+H).
Example 8. S1-11-8
##STR00144##
[0362] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.43-7.30 (m, 7H),
7.25-7.20 (m, 1H), 7.08-7.01 (m, 2H), 5.04 (s, 2H), 4.06-3.95 (m,
4H), 2.67-2.56 (m, 1H), 2.35 (s, 3H), 1.72-1.57 (m, 1H), 1.51-1.37
(m, 1H), 1.13 (d, J=6.1 Hz, 3H), 0.94 (t, J=7.0 Hz, 3H); MS (ESI)
m/z 434.00 (M+H).
Example 9. S1-11-9
##STR00145##
[0364] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.43-7.29 (m, 7H),
7.25-7.20 (m, 1H), 7.08-7.00 (m, 2H), 5.04 (s, 2H), 4.05-3.96 (m,
4H), 2.66-2.55 (m, 1H) 2.34 (s, 3H), 1.72-1.57 (m, 1H), 1.51-1.37
(m, 1H), 1.13 (d, J=6.1 Hz, 3H), 0.95 (t, J=7.3 Hz, 3H); MS (ESI)
m/z 434.64 (M+H).
Example 10. S1-11-10
##STR00146##
[0366] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.43-7.29 (m, 7H),
7.25-7.20 (m, 1H), 7.08-7.00 (m, 2H), 5.04 (s, 2H), 4.05-3.96 (m,
4H), 2.66-2.55 (m, 1H) 2.34 (s, 3H), 1.72-1.57 (m, 1H), 1.51-1.37
(m, 1H), 1.13 (d, J=6.1 Hz, 3H), 0.95 (t, J=7.3 Hz, 3H); MS (ESI)
m/z 434.60 (M+H).
Example 11. S1-11-11
##STR00147##
[0368] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.34 (m, 7H),
7.29-7.22 (m, 1H), 7.06-6.99 (m, 2H), 5.04 (s, 2H), 4.02-3.95 (m,
4H), 2.51-2.42 (m, 1H), 2.34 (s, 3H), 1.98-1.87 (m, 1H), 1.01 (d,
J=6.1 Hz, 3H), 0.95 (d, J=6.7 Hz, 3H), 0.89 (d, J=6.7 Hz, 3H): MS
(ESI) m/z 448.85 (M+H).
Example 12. S1-11-12
##STR00148##
[0370] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.34 (m, 7H),
7.29-7.22 (m, 1H), 7.06-6.99 (m, 2H), 5.04 (s, 2H), 4.02-3.95 (m,
4H), 2.51-2.42 (m, 1H), 2.34 (s, 3H), 1.98-1.87 (m, 1H), 1.01 (d,
J=6.1 Hz, 3H), 0.95 (d, J=6.7 Hz, 3H), 0.89 (d, J=6.7 Hz, 3H): MS
(ESI) m/z 446.48 (M-H).
Example 13. S1-11-13
##STR00149##
[0372] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41-7.3 (m, 7H),
7.28-7.19 (m, 1H), 7.05-7.00 (m, 2H), 5.01 (s, 2H), 3.99-3.94 (m,
4H), 2.93-2.91 (m, 1H), 2.33 (s, 3H), 1.93-1.80 (m, 2H), 1.80-1.67
(m, 2H), 1.66-1.45 (m, 4H); MS (ESI) m/z 446.61 (M+H).
Example 14. S1-11-14
##STR00150##
[0374] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41-7.32 (m, 7H),
7.25-7.20 (m, 1H), 7.07-7.02 (m, 2H), 5.03 (s, 2H), 4.04-3.94 (m,
5H), 3.93-3.81 (m, 2H), 3.77-3.70 (m, 1H), 3.37-3.27 (m, 1H),
2.37-2.31 (m, 3H), 2.10-2.05 (m, 1H), 2.02-2.10 (m, 1H); MS (ESI)
m/z 448.80 (M+H).
Example 15. S1-11-15
##STR00151##
[0376] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.40-7.20 (m, 7H),
7.28-7.25 (m, 1H), 7.16-7.02 (m, 2H), 5.02 (s, 2H), 4.05 (s, 2H),
4.00 (s, 2H), 2.33-2.32 (m, 3H), 1.52 (s, 3H), 1.49 (q, J=7.3 Hz,
2H), 1.05 (s, 6H), 0.90 (t, J=7.3 Hz, 3H); MS (ESI) m/z 448.25
(M+H).
Example 16. S1-11-16
##STR00152##
[0378] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.40-7.23 (m, 7H),
7.28-7.25 (m, 1H), 7.16-7.02 (m, 2H), 5.03 (s, 2H), 4.17 (s, 2H),
4.12 (s, 2H), 2.34-2.32 (m, 3H), 1.03-0.98 (m, 7H), 0.47-0.40 (m,
2H), 0.31-0.26 (m, 2H); MS (ESI) m/z 460.28 (M+H).
Example 17. S1-11-17
##STR00153##
[0380] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.28 (m, 7H),
7.25-7.20 (m, 1H), 7.08-7.00 (m, 2H), 5.03 (s, 2H), 4.09 (s, 2H),
4.03 (s, 2H), 2.35 (s, 3H), 1.46 (s, 2H), 1.19 (s, 6H), 1.02 (s,
9H); MS (ESI) m/z 490.34 (M+H).
Example 18. S1-11-18
##STR00154##
[0382] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.28 (m, 7H),
7.25-7.20 (m, 1H), 7.08-7.00 (m, 2H), 5.04 (s, 2H), 4.15 (s, 2H),
4.13 (s, 2H), 2.35 (s, 3H), 2.10-2.02 (m, 1H), 0.60-0.48 (m, 4H);
MS (ESI) m/z 416.41 (M-H).
Example 19. S1-11-19
##STR00155##
[0384] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.28 (m, 7H),
7.25-7.20 (m, 1H), 7.08-7.00 (m, 2H), 5.03 (s, 2H), 3.96 (s, 2H),
3.94 (s, 2H), 3.35-3.22 (m, 1H), 2.35 (s, 3H), 2.10-1.98 (m, 4H),
1.80-1.70 (m, 2H); MS (ESI) m/z 430.46 (M-H).
Example 20. S1-11-20
##STR00156##
[0386] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41-7.31 (m, 7H),
7.27-7.21 (m, 1H), 7.08-7.03 (m, 2H), 5.03 (s, 2H), 4.05 (s, 2H),
3.94 (s, 2H), 3.40 (m, 2H), 2.35 (s, 3H), 1.11 (s, 6H); MS (ESI)
m/z 448.35 (M-H).
Example 21. S1-11-21
##STR00157##
[0388] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.43-7.30 (m, 7H),
7.25-7.20 (m, 1H), 7.07-7.01 (m, 2H), 6.00-5.87 (m, 1H), 5.33-5.24
(m, 1H), 5.19 (d, J=10.4, 1 H), 5.02 (s, 2H), 4.00 (s, 4H), 3.36
(d, J=6.1, 3 H), 2.35 (s, 3H); MS (ESI) m/z 418.26 (M+H).
Example 22. Synthesis of S1-11-22
##STR00158##
[0390] A solution of alcohol S1-11-20 (92.1 mg, 0.205 mmol, 1 eq)
in CH.sub.2Cl.sub.2 (1 mL) was added dropwise to a solution of
pyridine (33.2 .mu.L, 0.410 mmol, 2 eq) and diethylaminosulfur
trifluoride (30.1 .mu.L, 0.246 mmol, 1.2 eq) in CH.sub.2Cl.sub.2 (2
mL) at 0.degree. C. The resulting solution was allowed to warm to
ambient temperature and stirred for 2 h. The reaction was diluted
with saturated aqueous NH.sub.4Cl solution (2 mL), and extracted
with EtOAc (2.times.30 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4), filtered, and concentrated under reduced
pressure. Purification of the resulting oil via flash column
chromatography on silica gel (Biotage, 25 g, 5 to 30% EtOAc in
hexanes gradient) provided 40.0 mg of S1-11-22 (43%) as a clear
oil: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41-7.32 (m, 7H),
7.25-7.20 (m, 1H), 7.07-7.02 (m, 2H), 5.03 (s, 2H), 4.12 (s, 4H),
2.89 (s, 1H), 2.82 (s, 1H), 2.34 (s, 3H), 1.44 (s, 3H), 1.39 (s,
3H); MS (ESI) m/z 450.45 (M-H).
Example 23. Synthesis of S1-11-23
##STR00159##
[0392] To a solution of DMSO (23.9 .mu.L, 0.337 mmol, 2 eq) in
CH.sub.2Cl.sub.2 (1 mL) at -70.degree. C. was added oxalyl chloride
(17.3 .mu.L, 0.201 mmol, 1.2 eq). After 15 minutes, alcohol
S1-11-20 (75.8 mg, 0.168 mmol, 1 eq) in CH.sub.2Cl.sub.2 (500
.mu.L) was added dropwise. After an additional 20 minutes at
-70.degree. C., DIEA (147 .mu.L, 0.84 mmol, 5 eq) was added and the
solution removed from the cold bath. After 5 minutes, saturated
aqueous NH.sub.4Cl solution (800 .mu.L) was added and the mixture
was allowed to warm. The solution was further diluted with
saturated aqueous NH.sub.4Cl solution (4 mL) and extracted with
CH.sub.2Cl.sub.2 (2.times.7 mL). The combined organic layers were
washed with brine (2 mL), dried (Na.sub.2SO.sub.4), filtered, and
concentrated under reduced pressure. The resulting crude oil was
dissolved in CH.sub.2Cl.sub.2 (1 mL) and pyrrolidine (69.7 .mu.L,
0.84 mmol, 5 eq) and acetic acid (48 .mu.L, 0.84 mmol, 5 eq) were
added. After 40 minutes, sodium triacetoxyborohydride (178.4 mg,
0.84 mmol, 5 eq) was added. After 50 minutes, the reaction was
poured into saturated aqueous NaHCO.sub.3 solution (8 mL) and
extracted with EtOAc (2.times.30 mL). The combined organic layers
were dried (Na.sub.2SO.sub.4), filtered, and concentrated under
reduced pressure. Purification of the resulting oil via flash
column chromatography on silica gel (Biotage, 10 g, 1 to 12%
methanol in CH.sub.2Cl.sub.2 gradient) provided 30.3 mg of S1-11-23
(36%) as a white solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.44-7.31 (m, 7H), 7.26-7.21 (m, 1H), 7.09-7.02 (m, 2H), 5.04 (s,
2H), 4.16 (s, 2H), 4.12 (s, 2H), 2.77-2.52 (m, 4H), 2.35 (s, 3H),
1.75 (s, 4H), 1.15 (s, 6H); MS (ESI) m/z 503.38 (M+H).
Example 24. Synthesis of S2-1-1
##STR00160##
[0394] Methanesulfonyl chloride (0.0446 mL, 0.575 mmol) was added
dropwise to a solution of compound S1-9 (76.0 mg, 0.192 mmol) and
triethylamine (0.107 mL, 0.768 mmol) in dichloromethane (2 mL).
After 1 hour, the reaction mixture was diluted with EtOAc and was
washed with water (2.times.) and brine (1.times.). The organics
were dried over Na.sub.2SO.sub.4, filtered, and were concentrated
under reduced pressure. The material was dissolved in DMF (2 mL),
diisopropylethylamine (0.100 mL, 0.575 mmol) and neopentylamine
(16.7 mg, 0.192 mmol) were added, and the reaction mixture was
heated to 60.degree. C. After heating overnight, the reaction
mixture was purified by column chromatography (Biotage 5 g column,
0 to 8% EtOAc in hexane gradient), yielding 26.5 mg (31%) of the
product S2-1-1 as a white solid. R.sub.f=0.42 in 10% EtOAc in
hexane; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.44-7.30 (m,
7H), 7.28-7.21 (m, 1H), 7.05 (d, J=7.8 Hz, 2H), 5.02 (s, 2H), 4.12
(br s, 4H), 2.53 (s, 2H), 2.34 (d, J=1.8 Hz, 3H), 0.96 (s, 9H); MS
(ESI) m/z 448.32 (M+H).
Example 25. Synthesis of phenyl
4-(benzyloxy)-7-chloro-6-methyl-2-tert-pentylisoindoline-5-carboxylate
(S3-13-1)
##STR00161##
[0395] Synthesis of S3-2
##STR00162##
[0397] To an ice-cooled solution of 2-methoxy-6-methylaniline
(S3-1, 25.12 g, 183.1 mmol) in methanol (79 mL) and acetic acid (25
mL) was added a solution of bromine (9.41 mL, 183.1 mmol) in of
Acetic acid (79 mL) dropwise via addition funnel. The reaction
mixture was allowed to stand for 2 h after complete addition. EtOAc
(150 mL) was added, and the solid was collected by filtration and
washed with EtOAc, yielding 37.2 g of the HBr salt of compound S3-2
as an off-white solid.
Synthesis of S3-3
##STR00163##
[0399] 4-Bromo-2-methoxy-6-methylaniline (S3-2, 20 g, 92.7 mmol)
was suspended in concentrated HCl (22 mL) and crushed ice (76 g)
and cooled in an ice-bath. A solution of NaNO.sub.2 (6.52 g, 94.6
mmol) in H.sub.2O (22 mL) was added dropwise. The resulting mixture
was stirred at 0.degree. C. for 30 min and then neutralized with
Na.sub.2CO.sub.3. A suspension of CuCN (10.4 g 115.9 mmol) in
H.sub.2O (44 mL) was mixed with a solution of NaCN (14.4 g, 294.8
mmol) in H.sub.2O (22 mL) and cooled in an ice-bath. The initial
diazonium salt mixture was added to the CuCN and NaCN solution
along with toluene (180 mL) with vigorous stirring. The reaction
mixture was stirred at 0.degree. C. for 1 h, rt for 2 h, and
50.degree. C. for 1 h. After cooling to rt, the layers were
separated. The aqueous layer was further extracted with toluene.
The combined organic layers were washed with brine, dried over
MgSO.sub.4, and concentrated. The residue was passed through a
silica gel plug, washed with toluene, and concentrated to give 14.5
g of compound S3-3 as a light yellow solid.
Synthesis of S3-4
##STR00164##
[0401] To a solution of S3-3 (11.34 g, 50.2 mmol) in THF (100 mL)
was added DIBAL-H (1.5 M solution in toluene, 40.1 mL, 60.2 mmol)
slowly at -78.degree. C. The reaction mixture was allowed to warm
to rt gradually and was stirred overnight. After cooling to
0.degree. C., the reaction was carefully quenched with 1N HCl, and
the resulting mixture was stirred at rt for 1 h. The mixture was
extracted three times with EtOAc. The combined EtOAc layers were
washed with H.sub.2O, saturated, aqueous NaHCO.sub.3, and brine,
dried over MgSO.sub.4 and concentrated to provide compound S3-4 as
a yellow solid, which was used directly for the next step.
Synthesis of S3-5
##STR00165##
[0403] To a suspension of S3-4 (assumed 50.2 mmol) in t-BuOH (200
mL) was added a solution of NaClO.sub.2 (11.34 g, 100.3 mmol) and
NaH.sub.2PO.sub.4 (34.6 g, 250.8 mmol) in H.sub.2O (100 mL) via
addition funnel. After complete addition, 2-methyl-2-butene was
added. The resulting homogenous solution was stirred at rt for 30
min, and then the volatiles were removed. The residue was suspended
in 150 mL of H.sub.2O. The solution was acidified to pH.about.1
with 1N HCl, and was extracted three times with tert-butyl methyl
ether. The combined organic solution was extracted three times with
1N NaOH. The combined aqueous solution was acidified with 6N HCl
and was extracted three times with EtOAc. The combined EtOAc
extracts were washed with brine, dried over MgSO.sub.4, and
concentrated to provide 6.84 g benzoic acid (S3-4-a) as an
off-white solid. This was pure enough to use directly for the next
step.
[0404] To a solution of the above benzoic acid (8.64 g, 35.2 mmol)
in dichloromethane (70 mL) was added oxalyl chloride (3.76 mL, 42.3
mmol, 1.2 eq), followed by a couple of drops of DMF (caution, gas
evolution). The mixture was stirred at rt for 30 min and the
reaction mixture was concentrated under reduced pressure. The
residue was further dried under high vacuum. The crude benzoyl
chloride was re-dissolved in dichloromethane (70 mL). Triethylamine
(12.3 mL, 88.1 mmol, 2.5 eq), phenol (3.98 g, 42.3 mmol, 1.2 eq)
and DMAP (0.43 g, 3.52 mmol, 0.1 eq) were added. The mixture was
stirred at rt for 1 h at which point LC-MS showed all SM was
consumed. The solvent was evaporated. The residue was suspended in
EtOAc, and the precipitate was filtered off. The organic solution
was then washed with 1 N HCl (three times), H.sub.2O, sat. aq.
NaHCO.sub.3, and brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated. Purification of the residue by Biotage flash
chromatography gave compound S3-5 (10.05 g) as an off-white solid:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.42 (s, 3H), 3.87 (s,
3H), 6.97 (d, J=0.9 Hz, 1H), 7.04 (d, J=0.9 Hz, 1H), 7.22-7.27 (m,
3H), 7.41-7.45 (m, 2H); MS (electrospray) m/z 319.0 (M-H), calcd
for C.sub.15H.sub.12BrO.sub.3 319.0.
Synthesis of S3-6
##STR00166##
[0406] To a solution of compound S3-5 (2.52 g, 7.87 mmol) in
CH.sub.3CN (16 mL) was added NCS (1.104 g, 8.27 mmol, 1.05 eq) in
one portion. The resulting mixture was heated to 60.degree. C. for
45 h. The solvent was evaporated. The residue was suspended in
Et.sub.2O (400 mL) and was washed with 1 N NaOH, H.sub.2O, and
brine, dried over Na.sub.2SO.sub.4, and concentrated to provide
2.76 g of compound S3-6 as a white solid. This material was used
directly for the next step without further purification: .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 2.51 (s, 3H), 3.87 (s, 3H), 7.13
(s, 1H), 7.22-7.28 (m, 3H), 7.44 (dd, J=7.8, 7.8 Hz, 2H); MS
(electrospray) m/z 353.0 (M-H), calcd for
C.sub.15H.sub.11BrClO.sub.3 352.97.
Synthesis of S3-7
##STR00167##
[0408] Compound S3-6 (2.76 g, 7.76 mmol) was dissolved in anhydrous
dichloromethane (78 mL) and a solution of boron tribromide (1.0 M
in dichloromethane, 7.76 mL, 7.76 mmol, 1.0 eq) was added at
-78.degree. C. The resulting yellow solution was stirred at
-78.degree. C. for 15 min and then at 0.degree. C. for 30 min
whereupon sat. aq. NaHCO.sub.3 was added. The mixture was stirred
at rt for 10 min. and was extracted with EtOAc three times. The
combined organic layers were washed with brine, dried over
Na.sub.2SO.sub.4, and concentrated to provide 2.69 g of the phenol
intermediate as an off-white solid. This material was used directly
for the next step without further purification: .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 2.83 (s, 3H), 7.19 (d, J=7.8 Hz, 2H), 7.27
(s, 1H), 7.32 (dd, J=7.8, 7.8 Hz, 1H), 7.46 (dd, J=7.8, 7.8 Hz,
2H); MS (electrospray) m/z 339.0 (M-H), calcd for
C.sub.14H.sub.9BrClO.sub.3 338.95.
[0409] The above phenol (2.65 g, 7.76 mmol) was dissolved in
acetone (40 mL), and K.sub.2CO.sub.3 (2.14 g, 15.5 mmol, 2 eq) was
added followed by benzylbromide (0.97 mL, 8.15 mmol, 1.05 eq).
After stirring overnight at rt, the solution was filtered through a
bed of Celite. The solid cake was further washed with three
portions of EtOAc. The combined organic solution was concentrated.
The residue was purified by Biotage flash chromatography to yield
2.97 g of compound S3-7 as a white solid: .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 2.51 (s, 3H), 5.11 (s, 2H), 7.05 (d, J=7.8 Hz,
2H), 7.19-7.26 (m, 2H), 7.33-7.43 (m, 7H); MS (electrospray) m/z
429.0 (M-H), calcd for C.sub.21H.sub.15BrClO.sub.3 429.00.
Synthesis of S3-8
##STR00168##
[0411] To a solution of compound $3-7 (1.98 g, 4.59 mmol) in
anhydrous THF (23 mL) was added i-PrMgCl.LiCl (1.2 M in THF, 7.65
mL, 9.18 mmol, 2 eq) dropwise at -78.degree. C. under N.sub.2
atmosphere. After 10 min, the temperature was raised to 0.degree.
C. After stirring for another 1 h at 0.degree. C., DMF (1.80 mL,
22.9 mmol, 5 eq) was added. Stirring was maintained for 30 min at
rt. The reaction was quenched by the addition of saturated, aqueous
NH.sub.4Cl. The layers were separated, and the aqueous layer was
further extracted twice with EtOAc. The combined organic layers
were washed with brine, dried over Na.sub.2SO.sub.4, filtered, and
concentrated. Purification of the residue by Biotage flash
chromatography gave compound S3-8 (1.45 g) as a white solid:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.51 (s, 3H), 5.19 (s,
2H), 7.05 (d, J=7.8 Hz, 2H), 7.25-7.27 (m, 1H), 7.33-7.44 (m, 8H)
10.51 (s, 1H); MS (electrospray) m/z 379.1 (M-H), calcd for
C.sub.22H.sub.16ClO.sub.4 379.08.
Synthesis of S3-9
##STR00169##
[0413] Compound S3-8 (2.51 g, 6.59 mmol) was suspended in methanol
(25 mL) and sodium borohydride (373 mg, 9.88 mmol) was added in
several portions. After gas evolution ceased and complete solution
was achieved, the reaction mixture was quenched with NaHCO.sub.3
(saturated, aqueous solution) and was extracted with EtOAc
(3.times.). The organics were dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. This gave 2.49 g
(99%) of S3-9 as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.46-7.32 (m, 7H), 7.27-7.21 (m, 1H), 7.13 (s, 1H), 7.07
(d, J=8.7 Hz, 2H), 5.16 (s, 2H), 4.77 (d, J=6.4 Hz, 2H), 2.46 (s,
3H), 2.06 (t, J=6.4 Hz, 1H), MS (ESI) m/z 405.15 (M+H).
Synthesis of S3-10
##STR00170##
[0415] 10% Palladium on carbon (Degussa, 50 mg) was added to a
solution of compound S3-9 (1.85 g, 4.84 mmol) in EtOAc (10 mL),
Methanol (10 mL), and chlorobenzene (1.5 mL) and an atmosphere of
hydrogen was introduced. After 5 hours, the reaction mixture was
purged with nitrogen and was filtered through Celite. The filtrate
was concentrated under reduced pressure, yielding the phenol
intermediate as a white solid. The intermediate was dissolved in
Acetic acid (15 mL) and sodium acetate (0.595 g, 7.26 mmol) was
added. Bromine (0.372 mL, 7.26 mmol) was added dropwise over
.about.3 min. After 10 min, the reaction mixture was quenched with
Na.sub.2S.sub.2O.sub.3 (5% aqueous solution) and was diluted with
EtOAc. The layers were separated, and the EtOAc layer was washed
with water (3.times.) and brine (1.times.). The organics were dried
over Na.sub.2SO.sub.4, filtered, and concentrated under reduced
pressure. The material was dissolved in acetone (30 mL), and
K.sub.2CO.sub.3 (1.34 g, 9.68 mmol) and benzyl bromide (0.633 mL,
5.32 mmol) were added. The reaction mixture was heated to
50.degree. C. overnight. Upon cooling to rt, the reaction mixture
was diluted with EtOAc and was washed with water (3.times.) and
brine (1.times.). The organics were dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. The material was
purified by column chromatography (Biotage 50 g column, 7 to 60%
EtOAc in hexane gradient), yielding 2.03 g (91%) of S3-10. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.51-7.47 (m, 2H), 7.41-7.31 (m,
5H), 7.30-7.23 (m, 1H), 7.03 (d, J=8.2 Hz, 2H), 5.12-5.05 (m, 4H),
2.48 (s, 3H), 2.18 (t, J=7.1 Hz, 1H); MS (ESI) m/z 482.99, 484.99,
486.99 (M+Na).
Synthesis of S3-11
##STR00171##
[0417] i-Propyl magnesium chloride/lithium chloride solution
(Chemetall Foote Corporation, 1.2 M solution in THF, 4.4 mL, 5.3
mmol) was added to a -78.degree. C. solution of compound S3-10 (490
mg, 1.06 mmol) in THF (10 mL). The reaction mixture was allowed to
warm to 0.degree. C. over 1 hour. Paraformaldehyde (318 mg, 10.6
mmol) was added, and the reaction was heated to 40.degree. C. After
1 hour, the reaction mixture was quenched with ammonium chloride
(saturated, aqueous solution) and was extracted with EtOAc
(3.times.). The combined extracts were washed with water (3.times.)
and brine (1.times.), and were dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. The material was
purified by column chromatography (Biotage 25 g column, 7 to 80%
EtOAc in hexane gradient), yielding 238 mg (54%) of S3-11 as a
thick oil. R=0.22 in 30% EtOAc in hexane; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.45-7.30 (m, 7H), 7.28-7.22 (m, 1H), 7.09 (d,
J=8.3 Hz, 2H), 5.09 (s, 2H), 5.00 (d, J=6.4 Hz, 2H), 4.80 (d, J=6.0
Hz, 2H), 2.73 (t, J=6.4 Hz, 1H), 2.52 (s, 3H), 2.48 (t, J=6.0 Hz,
1H); MS (ESI) m/z 435.12 (M+Na).
Synthesis of S3-12
##STR00172##
[0419] To a solution of S3-11 (2.76 g, 6.67 mmol, 1 eq) in
1,2-dichloroethane (25 mL) was added thionyl chloride (1.93 mL,
26.6 mmol, 4 eq) and tetrabutylammonium chloride (154.3 mg, 0.67
mmol, 0.1 eq). The reaction vessel was sealed and the mixture
heated to 80.degree. C. for 2 h, then concentrated under reduced
pressure. Purification of the resulting crude oil via flash column
chromatography on silica gel (Biotage, 100 g, 2 to 18% EtOAc in
hexane gradient) provided 2.47 g of S3-12 (82%) as a waxy white
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.37 (m, 7H),
7.35-7.324 (m, 1H), 7.10-7.06 (m, 2H), 5.15 (s, 2H), 4.96 (s, 2H),
4.83 (s, 2H), 2.53 (s, 3H); MS (ESI) m/z 447.28, 449.30 (M+H).
Synthesis of S3-13-1
##STR00173##
[0421] Compound S3-12 (150 mg, 0.334 mmol), t-amylamine (0.041 mL,
0.35 mmol) and diisopropylethylamine (0.233 mL, 1.34 mmol) were
heated to 60.degree. C. in 1,2-dimethoxyethane (0.8 mL). After 1
hour, the reaction mixture was heated to 80.degree. C. overnight.
Upon cooling to rt, the reaction mixture was diluted with EtOAc (20
mL) and was washed with NaHCO.sub.3 (saturated, aqueous solution,
2.times.) and brine (1.times.). The organics were dried over
Na.sub.2SO.sub.4, filtered, and concentrated under reduced
pressure. The material was purified by column chromatography
(Biotage 25 g column, 2 to 20% EtOAc in hexane gradient), yielding
62.8 mg (40%) of the product. R.sub.f=0.42 in 15% EtOAc in hexane;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.30 (m, 7H),
7.28-7.20 (m, 1H), 7.01 (d, J=7.8 Hz, 2H), 5.05 (s, 2H), 4.15-4.04
(m, 4H), 2.43 (s, 3H), 1.49 (q, J=7.8 Hz, 2H), 1.07 (s, 6H), 0.91
(t, 7.8 Hz, 3H); MS (ESI) m/z 464.24, 466.24 (M+H).
[0422] The following compounds were prepared by methods similar to
those described for S3-13-1.
Example 26. Synthesis of S3-13-2
##STR00174##
[0424] R.sub.f=0.19 in 15% EtOAc in hexane; MS (ESI) m/z 450.21,
452.20 (M+H).
Example 27. Synthesis of S3-13-3
##STR00175##
[0426] R.sub.f=0.18 in 15% EtOAc in hexane; MS (ESI) m/z 436.21,
438.19 (M+H).
Example 28. Synthesis of S3-13-4
##STR00176##
[0428] R.sub.f=0.22 in 15% EtOAc in hexane; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.42-7.28 (m, 7H), 7.26-7.18 (m, 1H), 7.01 (d,
J=7.3 Hz, 2H), 5.05 (s, 2H), 4.15-4.00 (m, 4H), 2.43 (s, 3H),
1.74-1.62 (m, 1H), 1.50-1.36 (m, 2H), 1.12 (d, J=6.4 Hz, 3H), 0.94
(t, 7.6 Hz, 3H); MS (ESI) m/z 450.26, 452.26 (M+H).
Example 29. Synthesis of S3-13-5
##STR00177##
[0430] R.sub.f=0.22 in 15% EtOAc in hexane; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.44-7.30 (m, 7H), 7.28-7.20 (m, 1H), 7.03 (d,
J=7.3 Hz, 2H), 5.07 (s, 2H), 4.10 (s, 2H), 4.04 (s, 2H), 2.45 (s,
3H), 1.74-1.62 (m, 1H), 1.50-1.38 (m, 2H), 1.14 (d, J=6.4 Hz, 3H),
0.96 (t, 7.6 Hz, 3H); MS (ESI) m/z 450.21, 452.21 (M+H).
Example 30. Synthesis of phenyl
4-(benzyloxy)-2-isopropyl-6-methyl-7-(trifluoromethyl)isoindoline-5-carbo-
xylate (S4-10-1)
Synthesis of S4-1
##STR00178##
[0432] Compound S3-5 (20 g, 62.5 mmol, 1.0 eq), 2, 4,
6-trivinyl-cyclotriboroxane-pyridine complex (7.8 g, 31.25 mmol,
0.50 eq), Pd(PPh.sub.3).sub.4 (2.2 g, 1.88 mmol, 0.030 eq) and
K.sub.2CO.sub.3 (17.25 g, 125 mmol, 2.0 eq) was added to vessel in
1,4-dioxane:H.sub.2O (3:1, V:V). The mixture was bubbled with
N.sub.2 to remove O.sub.2 for 6 times. The mixture was heated to
reflux for 19 h. The mixture was concentrated. The residue
partitioned between EtOAc and water. The organic layer was dried
over Na.sub.2SO.sub.4 and evaporated to dryness. The crude compound
was purified by column chromatography on silica gel eluting with
(petroleum ether:EtOAc=200:1.fwdarw.100:1.fwdarw.50:1) to yield
14.8 g of compound S4-1 (88%) as a light yellow solid.
Synthesis of S4-2
##STR00179##
[0434] An ozone-enriched stream of oxygen was bubbled through a
cold (-78.degree. C.) solution of compound S4-1 (21 g, 78.3 mmol,
1.0 eq) in anhydrous CH.sub.2Cl.sub.2, and the reaction was
monitored by TLC until the starting material was consumed. The
solution was purged with argon at -78 C for 10 min to remove the
excess O.sub.3. CH.sub.3SCH.sub.3 (50 mL) was added into the
reaction mixture and stirred for 1 hour from -78.degree. C. to
25.degree. C. The reaction mixture was concentrated. The crude
compound was purified by column chromatography on silica gel elute
with (petroleum ether:EtOAc=100:1.fwdarw.50:.fwdarw.30:1) to yield
13 g of compound 4-2 (62%) as a light yellow solid.
Synthesis of S4-3
##STR00180##
[0436] Compound S4-2 (1.8 g, 6.62 mmol, 1 eq) was dissolved in
HOAc. Bromine (1.6 mL, 26.5 mmol, 4 eq) was added dropwise into the
solution. The reaction mixture was stirred for 1 hour at rt. The
mixture was concentrated. The residue was extracted with EtOAc and
a saturated NaHCO.sub.3. The organic layer was washed with brine
and water in return, dried over Na.sub.2SO.sub.4 and concentrated
to dryness. To afford 1.9 g compound S4-3 as a light yellow
solid.
Synthesis of S4-4
##STR00181##
[0438] BBr.sub.3 (4.9 g, 1.9 mL, 19.5 mmol, 1.5 eq) was added to a
CH.sub.2Cl.sub.2 solution (30 mL) of S4-3 (3.5 g, 13.0 mmol, 1.0
eq) at -78.degree. C. The reaction was stirred from -78.degree. C.
to 25.degree. C. for 1.5 h, quenched with saturated NaHCO.sub.3 and
the reaction mixture was extracted with EtOAc. The combined EtOAc
extracts were dried (Na.sub.2SO.sub.4) and concentrated to yield
3.3 g of the crude phenol intermediate.
[0439] K.sub.2CO.sub.3 (3.6 g, 26.0 mmol, 2.0 eq) and BnBr (4.2 g,
26.0 mmol, 2.0 eq) were added to a solution of the above crude
phenol (3.3 g, 13.0 mmol, 1.0 eq) in DMF (15 mL). The reaction
mixture was stirred at rt for 2 h. The reaction mixture was
filtered and washed with EtOAc. Water (150 mL) was added into it
and extracted with EtOAc. The organic layer was dried over
Na.sub.2SO.sub.4 and concentrated. The crude compound was purified
by column chromatography on silica gel elute with (petroleum
ether:EtOAc=100:1.fwdarw.50:1) to yield 3.5 g of compound S4-4 (62%
for 3 steps) as a light yellow solid.
Synthesis of S4-5
##STR00182##
[0441] A DMF (50 mL) solution of compound S4-4 (5 g, 11.8 mmol, 1.0
eq), MeO.sub.2CCF.sub.2SO.sub.2F (11.3 g, 59 mmol, 5.0 eq) and CuI
(4.5 g, 23.6 mmol, 2.0 eq) in a sealed tube was heated to
100.degree. C. for 20 h. The mixture was filtered and the solid was
washed with EtOAc. The solution was concentrated and partitioned
with EtOAc and water. The organic layer was separated and dried
over Na.sub.2SO.sub.4, concentrated to give 7 g of the crude
compound S4-5 as brown oil.
Synthesis of S4-6
##STR00183##
[0443] To a stirred suspension of S4-5 (3.24 g, 7.81 mmol, 1 eq) in
methanol (40 mL) was added sodium borohydride (389 mg, 10.2 mmol,
1.3 eq). Gas evolution was evident; the solution was homogeneous
after 5 min. After 2 h the reaction mixture was poured into a
saturated aqueous NH.sub.4Cl solution (95 mL), water (5 mL), and
extracted with EtOAc (2.times.80 mL). The combined organic layers
were dried (Na.sub.2SO.sub.4), filtered, and concentrated under
reduced pressure. MS (ESI) m/z 415.39 (M-H).
Synthesis of S4-7
##STR00184##
[0445] Compound S4-6 (crude, 7.81 mmol) was dissolved in
methanol:dioxane (40 mL, 15:1). Palladium on carbon (10%, 160 mg)
was added, and the vessel was fitted with a septum and evacuated
and back-filled with hydrogen gas three times, and then stirred at
ambient temperature under a hydrogen balloon. After 2 h, another
100 mg of palladium catalyst was added and the evacuation and
back-fill procedure repeated. After 16 h, another 500 mg of
palladium catalyst was added, and the reaction vessel, the
evacuation and back-fill procedure repeated, and the solution
degassed with bubbling hydrogen for 5 min. After an additional 3 h,
the suspension was filtered through Celite to remove the palladium
catalyst and concentrated under reduced pressure. The resulting oil
was suspended in acetic acid (30 mL). Following addition of sodium
acetate (958 mg, 11.7 mmol, 1.5 eq) the solution became homogenous.
Bromine (602 .mu.L, 11.7 mmol, 1.5 eq) was added dropwise over six
minutes. After 1 h, a solution of sodium thiosulfate (5% aqueous,
40 mL) was added and the solution stirred vigorously for 15
minutes. The reaction solution was extracted with EtOAc (2.times.45
mL) and the combined organic layers washed with water (2.times.20
mL), brine (20 mL), dried (Na.sub.2SO.sub.4), filtered, and
concentrated under reduced pressure. To this crude intermediate in
acetone (35 mL), were added benzyl bromide (1.02 mL, 8.59 mmol, 1.1
eq) and potassium carbonate (2.16 g, 15.6 mmol, 2 eq). The flask
was fitted with a reflux condenser and heated to 50.degree. C. for
6 h. The reaction solution was diluted with water (30 mL) and
extracted with EtOAc (2.times.100 mL). The combined organic layers
were dried (Na.sub.2SO.sub.4), filtered, and concentrated under
reduced pressure. Purification of the resulting crude oil via flash
column chromatography on silica gel (Biotage, 100 g, 7 to 55% EtOAc
in hexane gradient) provided 2.13 g of intermediate
8-benzylalcohol-9-bromo compound S4-7 (55%, 4 steps) as a waxy
yellow solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.53-7.48
(m, 2H), 7.42-7.32 (m, 5H), 7.29-7.24 (m, 1H), 7.10-6.95 (m, 2H),
5.14 (s, 2H), 5.05-4.95 (m, 4H), 2.58-2.53 (m, 3H), 2.20-2.13 (m,
1H); MS (ESI) m/z 493.39, 495.27 (M-H).
Synthesis of S4-8
##STR00185##
[0447] Compound S4-7 (2.13 g, 4.30 mmol, 1 eq) was azeotropically
dried from toluene three times and dried under vacuum for 18 h. To
a solution of this bromide in THF (35 mL) under N.sub.2 at
-50.degree. C. was added isopropyl magnesium chloride-lithium
chloride complex (1.2 M solution in THF, 17.9 mL, 21.5 mmol, 5 eq)
dropwise over 10 minutes. The resulting dark yellow solution was
allowed to warm to 0.degree. C. over 1 h. Paraformaldehyde (1.27 g,
43.1 mmol, 10 eq) was added as a solid at 0.degree. C., the
reaction flask was fitted with a reflux condenser, and the vessel
was heated to 40.degree. C. in an oil bath for 2 h. After cooling,
the resulting slurry was poured into saturated aqueous NH.sub.4Cl
solution (40 mL) and water (15 mL), and extracted with EtOAc
(2.times.90 mL). The combined organic layers were washed with brine
(30 mL), dried (Na.sub.2SO.sub.4), filtered, and concentrated under
reduced pressure. Purification of the resulting crude oil via flash
column chromatography on silica gel (Biotage, 100 g, 6 to 55% EtOAc
in hexane gradient) provided 1.47 g of S4-8 (76%) as a white solid:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.35 (m, 7H),
7.29-7.23 (m, 1H), 7.10-7.03 (m, 2H), 5.14 (s, 2H), 4.92-4.83 (m,
4H), 2.96 (t, J=6.7 Hz, 1H), 2.78 (t, J=6.7 Hz, 1H), 2.62-2.55 (m,
3H): MS (ESI) m/z 445.38 (M-H).
Synthesis of S4-9
##STR00186##
[0449] To a solution of S4-8 (1.47 g, 3.29 mmol, 1 eq) in
1,2-dichloroethane (13 mL) was added thionyl chloride (956 .mu.L,
13.2 mmol, 4 eq) and tetrabutylammonium chloride (75 mg, 0.33 mmol,
0.1 eq). The reaction vessel was sealed and the mixture heated to
80.degree. C. for 3 h, then concentrated under reduced pressure.
Purification of the resulting crude oil via flash column
chromatography on silica gel ((Biotage, 50 g, 2 to 20% EtOAc in
hexane gradient) provided 1.41 g of S4-9 (89%) as a waxy white
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.35 (m, 7H),
7.29-7.23 (m, 1H), 7.10-7.03 (m, 2H), 5.20 (s, 2H), 4.94-4.86 (m,
4H), 2.64-2.58 (m, 3H); MS (ESI) m/z 481.31, 483.30 (M+H).
Synthesis of S4-10-1
##STR00187##
[0451] To a solution of S4-9 (862 mg, 1.78 mmol, 1 eq) in
1,2-dimethoxyethane (10 mL) was added DIEA (930 .mu.L, 5.34 mmol, 3
eq) and isopropylamine (152 .mu.L, 1.78 mmol, 1 eq). The reaction
was sealed and heated to 110.degree. C. for 2.5 h. The solution was
cooled and another 85 .mu.L isopropylamine (0.99 mmol, 0.55 eq) was
added and the reaction replaced in the heating bath. After an
additional 15 h, the solution was concentrated under reduced
pressure. Purification of the resulting oil via flash column
chromatography on silica gel (Biotage 100 g, 5 to 40% EtOAc in
hexanes gradient) provided 696 mg of S4-10-1 (83%) as a white
solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.29 (m, 7H),
7.23-7.19 (m, 1H), 7.00-6.96 (m, 2H), 5.10 (s, 2H), 4.13 (s, 2H),
4.02 (s, 2H), 2.81-2.72 (m, 1H), 2.53-2.48 (m, 3H), 1.17 (d, J=6.1
Hz, 6H): MS (ESI) m/z 468.39 (M-H).
[0452] The following compounds were prepared from S4-9 and the
corresponding amines by methods similar to those described for
S4-10-1.
Example 31. S4-10-2
##STR00188##
[0454] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.32 (m, 7H),
7.28-7.21 (m, 1H), 5.13 (s, 2H), 4.16 (m, 2H), 4.05 (s, 2H),
2.65-2.60 (s, 1H), 2.53 (s, 3H), 1.75-1.62 (m, 1H), 1.51-1.40 (m,
1H), 1.14 (d, J=6.7 Hz, 3H), 0.96 (t, J=7.3 Hz, 3H): MS (ESI) m/z
482.47 (M-H).
Example 32. S4-10-3
##STR00189##
[0456] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.31 (m, 7H),
7.29-7.21 (m, 1H), 7.03-6.98 (m, 2H), 5.13 (s, 2H), 4.15 (s, 2H),
4.05 (s, 2H), 2.66-2.59 (m, 1H), 2.53 (s, 3H), 1.75-1.62 (m, 1H),
1.51-1.40 (m, 1H), 1.14 (d, J=6.7 Hz, 3H), 0.96 (t, J=7.3 Hz, 3H);
MS (ESI) m/z 482.48 (M-H).
Example 33. S4-10-4
##STR00190##
[0458] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.31 (m, 7H),
7.29-7.19 (m, 1H), 7.02-6.96 (m, 2H), 5.10 (s, 2H), 4.20 (s, 2H),
4.07 (s, 2H), 2.51 (s, 3H), 1.17 (s, 9H); MS (ESI) m/z 482.48
(M-H).
Example 34. S4-10-5
##STR00191##
[0460] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.31 (m, 7H),
7.28-7.19 (m, 1H), 7.02-6.96 (m, 2H), 5.13 (s, 2H), 4.25 (s, 2H),
4.19 (s, 2H), 2.53 (s, 3H), 2.07-1.98 (m, 1H), 0.60-0.50 (m, 4H);
MS (ESI) m/z 466.43 (M-H).
Example 35. S4-10-6
##STR00192##
[0462] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.31 (m, 7H),
7.28-7.21 (m, 1H), 7.02-6.97 (m, 2H), 5.12 (s, 2H), 4.11 (s, 2H),
4.03 (s, 2H), 2.68 (t, J=8.6 Hz, 2H), 2.53 (s, 3H), 1.65-1.55 (m,
2H), 0.99 (t, J=7.3 Hz, 3H); MS (ESI) m/z 481.28 (M-H).
Example 36. Preparation of phenyl
4-(benzyloxy)-7-methoxy-6-methyl-2-tert-pentylisoindoline-5-carboxylate
(S5-9-1)
Synthesis of S5-1
##STR00193##
[0464] BBr.sub.3 (1.0 M solution in CH.sub.2Cl.sub.2, 28.0 mL, 28.0
mmol) was added to a solution of compound S3-5 (8.98 g, 28.0 mmol)
in CH.sub.2Cl.sub.2 (100 mL) at -78.degree. C. The resulting
reaction mixture was stirred at -78.degree. C. for 20 min and at
0.degree. C. for 15 min. NaHCO.sub.3 (saturated, aqueous solution,
120 mL) was added slowly. The resulting mixture was stirred at rt
for 20 min, and the CH.sub.2Cl.sub.2 was evaporated. The residue
was extracted with ethyl acetate (250 mL), and the combined
extracts were dried over MgSO.sub.4, filtered, and concentrated
under reduced pressure. The material was purified by
recrystallization from EtOAc/Hexanes to give 6.76 g of the desired
product S5-1 as a white solid. The mother liquor was concentrated
and purified by column chromatography (2-10% ethyl acetate in
hexanes gradient) to afford an additional 973 mg of product (90%
combined yield). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 11.13
(s, 1H), 7.47-7.43 (m, 2H), 7.33-7.29 (m, 1H), 7.19-7.16 (m, 2H),
7.08 (d, J=1.8 Hz, 1H), 6.96 (d, J=1.8 Hz, 1H), 2.66 (s, 3H); MS
(ESI) m/z 305.05, 307.05 (M-H).
Synthesis ofS5-2
##STR00194##
[0466] A solution of PhI(OAc).sub.2 (3.77 g, 11.72 mmol) in
Methanol (20 mL) was added slowly to a solution of S5-1 (1.71 g,
5.58 mmol) in a mixture of Methanol (30 mL) and 1,4-dioxane (10 mL)
at 0.degree. C. The reaction mixture was stirred at rt for 17 h.
Acetic acid (6 mL) was added to the reaction mixture. Zinc dust
(1.09 g, 16.74 mmol) was added (exothermic), and the reaction
mixture was stirred at rt for 20 min. The reaction mixture was
filtered through a pad of Celite, and the Celite was washed
thoroughly with EtOAc (100 mL). The filtrate was concentrated under
reduced pressure. The residue was partitioned between EtOAc (120
mL) and sat. NaHCO.sub.3/brine solution. The organic layer was
separated and dried (MgSO.sub.4). The dried solution was filtered,
and the filtrate was concentrated. The residue was purified by
flash-column chromatography (0-4% ethyl acetate-hexanes gradient)
to afford 763 mg (41%) of the desired product S5-2. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 10.70 (s, 1H), 7.47-7.43 (m, 2H),
7.33-7.30 (m, 1H), 7.20-7.17 (m, 2H), 7.16 (s, 1H), 3.75 (s, 3H),
2.67 (s, 3H); MS (ESI) m/z 335.11, 337.14 (M-H).
Synthesis of S5-3
##STR00195##
[0468] Di-tert-butyl dicarbonate (543 mg, 2.49 mmol) and
4-N,N-dimethylaminopyridine (28 mg, 0.226 mmol) were added to a
solution of S5-2 (763 mg, 2.26 mmol) in CH.sub.2Cl.sub.2 (20 mL).
The resulting mixture was stirred for 20 min at rt and was
concentrated under reduced pressure. The residue was purified by
flash-column chromatography (0-5% ethyl acetate-hexanes gradient)
to afford 783 mg (79%) of compound S5-3 as a white solid. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.41 (m, 2H), 7.38 (s, 1H),
7.30-7.26 (m, 1H), 7.24-7.22 (m, 2H), 3.81 (s, 3H), 2.47 (s, 3H),
1.43 (s, 9H); MS (ESI) m/z 435.14, 437.15 (M-H).
Synthesis of S5-4
##STR00196##
[0470] Isopropylmagnesium chloride/lithium chloride (Chemetall
Foote Corporation, 1.2 M solution in THF, 0.547 mL, 0.657 mmol) was
added dropwise to a solution of compound S5-3 (143.6 mg, 0.328
mmol) in THF (3.3 mL) at 0.degree. C. The resulting yellow reaction
mixture was then stirred at 0.degree. C. for 1 h. DMF (0.127 mL,
1.64 mmol) was added, and the resulting mixture was stirred at
0.degree. C. for 10 min and then at rt for 20 min. Saturated,
aqueous NH.sub.4Cl and brine were added. The resulting mixture was
extracted with EtOAc (50 mL), and the organics were dried
(MgSO.sub.4), filtered, and concentrated under reduced pressure.
The crude product S5-4 was used directly in the next step. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 10.38 (s, 1H), 7.61 (s, 1H),
7.46-7.42 (m, 2H), 7.32-7.28 (m, 1H), 7.26-7.24 (m, 2H), 3.91 (s,
3H), 2.46 (s, 3H), 1.45 (s, 9H); MS (ESI) m/z 385.24 (M-H).
Synthesis of S5-5
##STR00197##
[0472] Compound S5-4 (3.09 g, 8 mmol) was dissolved in dry
dichloromethane (20 mL). TFA (10 mL) was slowly added at 0.degree.
C. The solution was stirred at 10.degree. C. for 1 h. LC-MS
analysis showed the complete consumption of starting material. The
reaction mixture was concentrated under reduced pressure. The
material was dissolved in acetic acid (30 mL) and sodium acetate
(1.31 g, 16.0 mmol) was added. Bromine (0.49 mL, 9.6 mmol) was
added via syringe at 10.degree. C. After stirring at rt for 10 min,
LC/MS indicated that the starting material was consumed. Most of
the acetic acid was removed under reduced pressure. The material
was diluted with EtOAc, was washed with water (3.times.50 mL) and
brine, was dried over sodium sulfate, filtered, and concentrated
under reduced pressure. This gave 3.23 g (110% crude yield) of
compound S5-5 as an orange oil. MS (ESI) m/z 363.19, 365.21
(M-H).
Synthesis of S5-6
##STR00198##
[0474] Potassium carbonate (2.21 g, 16.0 mmol) was added to a
solution of compound S5-5 (3.23 g, 8.0 mmol) in DMF (20 mL), and
the reaction mixture was cooled to 0.degree. C. in an ice-bath.
Benzyl bromide (1.14 mL, 9.6 mmol) was added dropwise. After 1
hour, LC/MS indicated that the starting material was completely
consumed. The reaction mixture was diluted with EtOAc (100 mL), was
washed with water and brine, and was dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The material was
dissolved in Methanol (50 mL) and was cooled to 0.degree. C. for
the addition of NaBH.sub.4 (0.355 g, 9.6 mmol). The reaction was
stirred at 0.degree. C. for 30 min at which point LC/MS indicated
that the starting material was completely consumed. The reaction
was quenched with water, and the resulting mixture was extracted
with EtOAc. The combined extracts were dried (sodium sulfate) and
concentrated under reduced pressure. Flash chromatography on silica
gel (10:1 to 4:1 hexanes/EtOAc) yielded 3.52 g (96%, 4 steps) of
S5-6. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.52-7.48 (m, 2H),
7.40-7.32 (m, 5H), 7.27-7.22 (m, 1H), 7.07-7.03 (m, 2H), 5.10 (s,
2H), 4.90 (s, 2H), 3.85 (s, 3H), 2.37 (s, 3H); MS (ESI) m/z 479.26,
481.25 (M+Na).
Synthesis of S5-7
##STR00199##
[0476] Isopropylmagnesium chloride/lithium chloride (Chemetall
Foote Corporation, 1.2 M solution in THF, 31.6 mL, 37.9 mmol) was
added to a solution of compound S5-6 (3.47 g, 7.58 mmol) in THF
(100 mL) under nitrogen atmosphere at 0.degree. C. The resulting
solution was warmed to rt and was stirred for 30 min. After the
solution was cooled to 0.degree. C., DMF (5.84 mL, 75.8 mmol) was
added slowly via syringe. The reaction was warmed to rt over 1
hour. The reaction mixture was diluted with ethyl acetate (200 mL),
was washed with water and brine, and was dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The material was
dissolved in Methanol (50 mL) and was cooled to 0.degree. C.
NaBH.sub.4 (0.42 g, 11.4 mmol) was added, and the reaction mixture
was stirred at 0.degree. C. for 30 min. The reaction was quenched
with water and was extracted with EtOAc. The combined EtOAc
extracts were dried (sodium sulfate) and concentrated under reduced
pressure to give 3.02 g of crude S5-7. The material was used
without further purification. MS (ESI) m/z 407.46 (M-H).
Synthesis of S5-8
##STR00200##
[0478] Compound S5-7 (961 mg, 2.35 mmol) was partially dissolved in
1,2-dichloroethane (10 mL) and tetrabutylammonium chloride (64.0
mg, 0.23 mmol) was added. Thionyl chloride (0.683 mL, 9.41 mmol)
was added slowly, forming a clear solution. The reaction mixture
was heated to 80.degree. C. in a sealed tube and was stirred for 1
hour 30 min. The reaction mixture was concentrated under reduced
pressure and was purified by flash chromatography on silica gel
(50:1 to 20:1 hexanes/EtOAc). This gave 1.40 g (80%, 3 steps) of
compound S5-8. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.50-7.43
(m, 2H), 7.43-7.32 (m, 5H), 7.29-7.22 (m, 1H), 7.11-7.06 (m, 2H),
5.15 (s, 2H), 4.89 (s, 2H), 4.86 (s, 2H), 3.89 (d, J=0.72 Hz, 3H),
2.43 (d, J=0.92 Hz, 3H); MS (ESI) m/z 467.35 (M+Na).
Synthesis of S5-9-1
##STR00201##
[0480] Diisopropylethylamine (2.39 mL, 13.73 mmol) and t-amylamine
(0.294 mL, 2.52 mmol) were added to a solution of compound $5-8
(1.02 g, 2.29 mmol) in 1,2-dimethoxyethane (15 mL). The reaction
mixture was heated to 110.degree. C. overnight in a sealed tube.
The reaction mixture was concentrated under reduced pressure and
was purified by flash chromatography on silica gel (20:1 to 1:1
hexanes/EtOAc), yielding 623 mg (59%) of compound S5-9-1. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.42-7.38 (m, 2H), 7.37-7.30 (m,
5H), 7.23-7.19 (m, 1H), 7.06-7.02 (m, 2H), 5.02 (s, 2H), 4.10 (s,
2H), 4.03 (s, 2H), 3.76 (s, 3H), 2.34 (s, 3H), 1.86 (q, J=7.3 Hz,
2H), 1.08 (s, 6H), 0.91 (t, J=7.3 Hz, 3H); MS (ESI) m/z 460.45
(M+H).
[0481] The following compounds were prepared from S5-8 and the
corresponding amines by methods similar to those described for
S5-9-1.
Example 37. S5-9-2
##STR00202##
[0483] R.sub.f=0.20 in 33% EtOAc in Hexane; MS (ESI) m/z 432.48
(M+H).
Example 38. S5-9-3
##STR00203##
[0485] MS (ESI) m/z 446.45 (M+H).
Example 39. S5-9-4
##STR00204##
[0487] MS (ESI) m/z 446.48 (M+H).
Example 40. S5-9-5
##STR00205##
[0489] R.sub.f=0.25 in 33% EtOAc in Hexane; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.42-7.38 (m, 2H), 7.37-7.28 (m, 5H), 7.23-7.19
(m, 1H), 7.06-7.01 (m, 2H), 5.02 (s, 2H), 4.10 (s, 2H), 4.04 (s,
2H), 3.75 (s, 3H), 2.34 (s, 3H), 1.16 (s, 9H); MS (ESI) m/z 446.48
(M+H).
Example 41. S5-9-6
##STR00206##
[0491] MS (ESI) m/z 432.48 (M+H).
Example 42. S5-9-7
##STR00207##
[0493] R.sub.f=0.31 in 33% EtOAc in Hexane; MS (ESI) m/z 472.51
(M+H).
Example 43. S6-1-1
##STR00208##
[0495] To a solution of S3-13-2 (221 mg, 0.491 mmol, 1 eq) in
dioxane:methanol:0.5 N HCl in methanol (1:1:1, 4 mL) was added
palladium on carbon (10%, 146 mg). The vessel was evacuated and
back-filled with hydrogen gas three times, then degassed with
bubbling hydrogen for 4 min, and stirred at ambient temperature
under a hydrogen balloon. After 16.5 h, another 80 mg palladium
catalyst was added, and the evacuation and degassing procedure
repeated. After an additional 4 h, the reaction suspension was
filtered through Celite to remove the palladium catalyst and
concentrated under reduced pressure. Purification of the resulting
crude oil via flash column chromatography on silica gel (Silicycle,
25 g, 1 to 8% methanol in dichloromethane gradient) provided 112.6
mg of compound S6-1-1 (70%) as a waxy white solid: .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 11.42-11.10 (brs, 1H), 7.37 (t, J=8.3 Hz,
2H), 7.28-7.20 (m, 1H), 7.11 (d, J=7.4 Hz, 2H), 6.66 (s, 1H),
4.43-4.32 (m, 4H), 2.61 (s, 3H), 1.35 (s, 9H); MS (ESI) m/z 326.94
(M+H).
Example 44. S6-2-1
##STR00209##
[0497] To a solution of S6-1-1 (113 mg, 0.346 mmol, 1 eq) in
trifluoroacetic acid (4 mL) at 0.degree. C. was added potassium
nitrate (67.4 mg, 0.667 mmol, 1.92 eq). The mixture was allowed to
warm to ambient temperature at which point the solution turned
orange. After 30 min, the solvent was removed under reduced
pressure. To a solution of this crude oil in methanol:THF (1:1, 2.5
mL) was added formaldehyde (37% aq, 64 .mu.L, 0.87 mmol, 2.5 eq)
and palladium on carbon (10%, 101 mg). The reaction vessel was
evacuated and back-filled with hydrogen gas three times, and the
solution stirred at ambient temperature under a hydrogen balloon.
After 18 h, the reaction mixture was filtered through Celite and
concentrated under reduced pressure. This crude oil was dissolved
in dimethylformamide (2 mL), and diisopropylethylamine (241 .mu.L,
1.38 mmol, 4 eq), di-tert-butylcarbonate (226 mg, 1.04 mmol, 3 eq)
and a catalytic amount of dimethylaminopyridine were added. The
reaction mixture was placed under nitrogen and stirred at ambient
temperature. After 2 h, the reaction solution was diluted with
saturated aqueous sodium bicarbonate (10 mL) and water (30 mL) and
extracted with EtOAc (2.times.30 mL). The combined organic extracts
were washed with brine, dried (Na.sub.2SO.sub.4), filtered, and
concentrated under reduced pressure. Purification of the resulting
crude oil via flash column chromatography on silica gel (Silicycle,
12 g, 5 to 30% EtOAc in hexane gradient) provided 72 mg of S6-2-1
(44%) as a white solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.45-7.38 (m, 2H), 7.29-7.20 (m, 3H), 4.15 (s, 2H), 3.93 (s, 3H),
2.73 (s, 6H), 2.40 (s, 3H), 1.42 (s, 9H), 1.19 (s, 9H); MS (ESI)
m/z 467.47 (M-H).
[0498] The following compounds were prepared by methods similar to
those described for S6-2-1.
Example 45. S6-2-2
##STR00210##
[0500] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.35 (m, 2H),
7.28-7.20 (m, 3H), 4.08 (s, 2H), 3.86 (s, 2H), 2.88-2.80 (7H), 2.40
(s, 3H), 1.41 (s, 9H), 1.19 (d, J=4.9 Hz, 6H); MS (ESI) m/z 455.01
(M+H).
Example 46. S6-2-3
##STR00211##
[0502] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.38 (m, 2H),
7.29-7.20 (m, 3H), 4.09 (s, 2H), 3.87 (s, 2H), 2.73 (s, 6H),
2.64-2.54 (m, 1H), 2.40 (s, 3H), 1.78-1.60 (m, 2H), 1.42 (s, 9H),
1.14 (d, J=8.0 Hz, 3H), 0.94 (t, J=7.6 Hz, 3H); MS (ESI) m/z 467.51
(M-H).
Example 47. S6-2-4
##STR00212##
[0504] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.38 (m, 2H),
7.29-7.20 (m, 3H), 4.09 (s, 2H), 3.86 (s, 2H), 2.73 (s, 6H),
2.64-2.54 (m, 1H), 2.39 (s, 3H), 1.78-1.60 (m, 2H), 1.42 (s, 9H),
1.14 (d, J=8.0 Hz, 3H), 0.94 (t, J=7.6 Hz, 3H); MS (ESI) m/z 467.55
(M-H).
Example 48. S6-2-5
##STR00213##
[0506] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.49-7.35 (m, 2H),
7.29-7.20 (m, 3H), 4.13 (s, 2H), 3.91 (s, 2H), 2.73 (s, 6H), 2.40
(s, 3H), 1.59-1.48 (m, 2H), 1.42 (s, 9H), 1.09 (s, 6H), 0.92 (t,
J=7.3 Hz, 3H); MS (ESI) m/z 481.48 (M-H).
Example 49. Compound 102
Synthesis of S7-2-1
##STR00214##
[0508] Lithium diisopropylamide was prepared at -40.degree. C. from
n-butyllithium (2.5 M solution in hexane, 0.118 mL, 0.294 mmol) and
diisopropylamine (0.0416 mL, 0.294 mmol) in THF (5 mL). The
reaction mixture was cooled to -78.degree. C. and TMEDA (0.114 mL,
0.762 mmol) was added followed by the dropwise addition of a
solution of compound S1-11-1 (66.5 mg, 0.153 mmol) in THF (2 mL).
This resulted in an orange-red colored solution. After 5 min, a
solution of enone S7-1 (61.3 mg, 0.127 mmol) in THF (1 mL) was
added. After complete addition, the reaction mixture was allowed to
warm to -20.degree. C. over 1 h. The reaction was quenched by the
addition of ammonium chloride (saturated, aqueous solution) and was
extracted with EtOAc (2.times.). The combined extracts were dried
over Na.sub.2SO.sub.4, filtered, and concentrated under reduced
pressure. The material was purified on a Waters Autopurification
system equipped with a Sunfire Prep C18 OBD column [5 .mu.m,
19.times.50 mm; flow rate, 20 mL/min; Solvent A: H.sub.2O with 0.1%
HCO.sub.2H; Solvent B: CH.sub.3CN with 0.1% HCO.sub.2H; gradient:
20.fwdarw.100% B; mass-directed fraction collection], yielding 17.2
mg (17%) of the desired product S7-2-1 as a yellow solid. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 16.0 (s, 1H), 7.52-7.44 (m, 2H),
7.42-7.26 (m, 8H), 5.35 (s, 2H), 4.92 (s, 2H), 4.32-4.20 (m, 2H),
4.06-3.90 (m, 3H), 3.21 (dd, J=15.6, 4.6 Hz, 1H), 3.03-2.91 (m,
1H), 2.58-2.36 (m, 9H), 2.13 (d, J=14.6 Hz, 1H), 1.18 (s, 9H), 0.82
(s, 9H), 0.27 (s, 3H), 0.12 (s, 3H); MS (ESI) m/z 822.51 (M+H).
Synthesis of Compound 102
##STR00215##
[0510] Aqueous HF (0.4 mL, 48%) was added to a solution of S7-2-1
(17.2 mg, 0.0209 mmol) in 1,4-dioxane (0.8 mL) in a plastic vial.
After 4 h, the reaction mixture was poured into a solution of
K.sub.2HPO.sub.4 (4.8 g) in water (15 mL). The mixture was
extracted with EtOAc (3.times.). The combined EtOAc extracts were
dried over Na.sub.2SO.sub.4, filtered and concentrated under
reduced pressure. The material was dissolved in Methanol (1 mL),
1,4-dioxane (1 mL) and 0.5 M HCl in Methanol (0.5 mL), and
palladium on carbon (Degussa, 10 wt %, .about.5 mg) was added. An
atmosphere of hydrogen was introduced, and the reaction mixture was
stirred for 2 h. The reaction mixture was filtered through Celite,
and the filtrate was concentrated under reduced pressure. The
material was purified on a Waters Autopurification system equipped
with a Phenomenex Polymerx 10.mu. RP 100A column [10 .mu.m,
30.times.21.20 mm; flow rate, 20 mL/min; Solvent A: 0.05N HCl in
water; Solvent B: CH.sub.3CN; gradient: 0.fwdarw.70% B;
mass-directed fraction collection]. Fractions with the desired MW
were collected and freeze-dried to yield 8.7 mg (69%, 2 steps) of
the desired product Compound 102 as a yellow solid. .sup.1H NMR
(400 MHz, CD.sub.3OD with 1 drop DCl) .delta. 4.85 (q, J=15.1 Hz,
2H), 4.73 (s, 2H), 4.16 (s, 1H), 3.22-2.95 (m, 9H), 2.36-2.24 (m,
2H), 1.72-1.56 (m, 1H), 1.53 (s, 9H); MS (ESI) m/z 530.35
(M+H).
[0511] The following compounds were prepared by methods similar to
that for Compound 102, substituting the appropriate isoindoline
S1-11, S2-1, S3-13, 54-10, S5-9, or S6-2 for S1-11-1.
Example 50. Compound 101
##STR00216##
[0513] Prepared from S2-1-1, yellow solid: .sup.1H NMR (400 MHz,
CD.sub.3OD with 1 drop DCl) .delta. 5.17 (d, J=14.7 Hz, 1H), 5.08
(d, J=14.2 Hz, 1H), 4.81 (d, J=14.7 Hz, 1H), 4.67 (d, J=14.2 Hz,
1H), 4.15 (s, 1H), 3.52 (s, 2H), 3.34-2.95 (m, 9H), 2.38-2.22 (m,
2H), 1.61 (q, J=12.5 Hz, 1H), 1.19 (s, 9H); MS (ESI) m/z 544.35
(M+H).
Example 51. Compound 150
##STR00217##
[0515] Prepared from S3-13-1, yellow solid: .sup.1H NMR (400 MHz,
CD.sub.3OD with 1 drop DCl) .delta. 4.94-4.67 (m, 4H), 4.18 (s,
1H), 3.18-2.95 (m, 9H), 2.40-2.26 (m, 2H), 1.91 (q, J=7.3 Hz, 2H),
1.63 (q, J=12.4 Hz, 1H), 1.48 (s, 6H), 1.08 (t, J=7.3 Hz, 3H); MS
(ESI) m/z 560.26, 562.27 (M+H).
Example 52. Compound 144
##STR00218##
[0517] Prepared from S3-13-2, yellow solid: 1H NMR (400 MHz,
CD.sub.3OD with 1 drop DCl) .delta. 4.90-4.73 (m, 4H), 4.16 (s,
1H), 3.17-2.95 (m, 9H), 2.41-2.24 (m, 2H), 1.68-1.56 (m, 1H), 1.53
(s, 9H); MS (ESI) m/z 546.20, 548.29 (M+H).
Example 53. Compound 149
##STR00219##
[0519] Prepared from S3-13-3, yellow solid: .sup.1H NMR (400 MHz,
CD.sub.3OD with 1 drop DCl) .delta. 5.05-4.95 (m, 2H), 4.71 (d,
J=15.1 Hz, 1H), 4.62 (d, J=14.2 Hz, 1H), 4.16 (s, 1H), 3.50-3.42
(m, 2H), 3.17-2.94 (m, 9H), 2.42-2.24 (m, 2H), 1.94-1.82 (m, 2H),
1.63 (q, J=12.8 Hz, 1H), 1.07 (t, J=7.3 Hz, 3H); MS (ESI) m/z
532.23, 534.20 (M+H).
Example 54. Compound 110
##STR00220##
[0521] Prepared from S3-13-4, yellow solid: .sup.1H NMR (400 MHz,
CD.sub.3OD with 1 drop DCl) .delta. 4.98-4.86 (m, 2H), 4.78 (d,
J=16.0 Hz, 1H), 4.70 (d, J=14.2 Hz, 1H), 4.15 (s, 1H), 3.70-3.57
(m, 1H), 3.17-2.92 (m, 9H), 2.43-2.24 (m, 2H), 2.08-1.96 (m, 1H),
1.79-1.56 (m, 2H), 1.50-1.42 (m, 3H), 1.08 (t, J=7.3 Hz, 3H); MS
(ESI) m/z 546.21, 548.23 (M+H).
Example 55. Compound 117
##STR00221##
[0523] Prepared from S3-13-5, yellow solid: .sup.1H NMR (400 MHz,
CD.sub.3OD with 1 drop DCl) .delta. 4.98-4.88 (m, 2H), 4.84-4.64
(m, 2H), 4.15 (s, 1H), 3.70-3.57 (m, 1H), 3.15-2.94 (m, 9H),
2.43-2.24 (m, 2H), 2.09-1.96 (m, 1H), 1.77-1.55 (m, 2H), 1.45 (d,
J=6.4 Hz, 3H), 1.07 (t, J=7.3 Hz, 3H); MS (ESI) m/z 546.48, 548.48
(M+H).
Example 56. Compound 119
##STR00222##
[0525] Prepared from S5-9-1, yellow solid: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 4.87 (s, 2H), 4.71 (s, 2H), 4.08 (s, 1H), 3.76
(d, J=4.1 Hz, 3H), 3.27-3.19 (m, 1H), 3.03 (s, 3H), 2.95 (s, 3H),
3.06-2.92 (m, 2H), 2.37-2.18 (m, 2H), 1.88 (q, J=7.3 Hz, 2H),
1.70-1.58 (m, 1H), 1.47 (s, 6H), 1.08 (t, J=7.3 Hz, 3H); MS (ESI)
m/z 556.53 (M+H).
Example 57. Compound 138
##STR00223##
[0527] Prepared from S5-9-2: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.87 (s, 2H), 4.69 (s, 2H), 4.09 (s, 1H), 3.76 (d, J=3.2
Hz, 3H), 3.27-3.19 (m, 1H), 3.04 (s, 3H), 2.96 (s, 3H), 3.10-2.91
(m, 4H), 2.36-2.18 (m, 2H), 2.09-1.97 (m, 1H), 1.77-1.57 (m, 2H),
1.08 (t, J=7.3 Hz, 3H); MS (ESI) m/z 528.51 (M+H).
Example 58. Compound 145
##STR00224##
[0529] Prepared from S5-9-3: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.00-4.76 (m, 2H), 4.59 (d, J=14.2 Hz, 1H), 4.12 (d, J=3.3
Hz, 1H), 3.76 (d, J=6.0 Hz, 1H), 3.66-3.55 (m, 1H), 3.28-3.20 (m,
1H), 3.10-2.91 (m, 9H), 2.35-2.19 (m, 2H), 2.09-1.97 (m, 1H),
1.77-1.57 (m, 2H), 1.46 (d, J=6.4 Hz, 3H), 1.08 (t, J=7.1 Hz, 3H);
MS (ESI) m/z 542.54 (M+H).
Example 59. Compound 148
##STR00225##
[0531] Prepared from S5-9-4: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.00-4.76 (m, 2H), 4.58 (d, J=14.2 Hz, 1H), 4.10 (s, 1H),
3.75 (d, J=6.0 Hz, 1H), 3.64-3.55 (m, 1H), 3.27-3.19 (m, 1H),
3.09-2.90 (m, 9H), 2.35-2.19 (m, 2H), 2.09-1.95 (m, 1H), 1.77-1.57
(m, 2H), 1.45 (dd, J=6.4, 3.7 Hz, 3H), 1.07 (t, J=7.2 Hz, 3H); MS
(ESI) m/z 542.52 (M+H).
Example 60. Compound 125
##STR00226##
[0533] Prepared from S5-9-5: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.87 (s, 2H), 4.70 (s, 2H), 4.09 (s, 1H), 3.76 (d, J=3.2
Hz, 3H), 3.27-3.19 (m, 1H), 3.04 (s, 3H), 2.96 (s, 3H), 3.10-2.91
(m, 2H), 2.36-2.18 (m, 2H), 1.70-1.58 (m, 1H), 1.53 (s, 9H); MS
(ESI) m/z 542.56 (M+H).
Example 61. Compound 107
##STR00227##
[0535] Prepared from S1-11-2: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.99-4.94 (m, 1H), 4.88-4.82 (m, 1H), 4.10 (s, 1H),
3.97-3.92 (m, 1H), 3.90-3.85 (m, 1H), 3.25-3.16 (m, 1H), 3.15-2.92
(m, 11H), 2.41-2.28 (m, 1H), 2.28-2.17 (m, 1H), 1.72-1.59 (m, 1H);
MS (ESI) m/z 520.24 (M+H).
Example 62. Compound 134
##STR00228##
[0537] Prepared from S1-11-3: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.07-4.92 (m, 1H), 4.80-4.55 (m, 1H), 4.10 (s, 1H),
3.85-3.75 (m, 2H), 3.75-3.65 (m, 2H), 3.46 (s, 3H), 3.23-3.14 (m,
1H), 3.13-2.92 (m, 9H), 2.39-2.19 (m, 2H), 1.70-1.56 (m, 1H); MS
(ESI) m/z 532.24 (M+H).
Example 63. Compound 121
##STR00229##
[0539] Prepared from S1-11-4: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.78-4.68 (m, 1H), 4.63-4.51 (m, 1H), 4.08 (s, 1H),
3.38-3.34 (m, 2H), 3.23-3.14 (m, 1H), 3.14-2.89 (m, 10H), 2.41-2.28
(m, 1H), 2.25-2.13 (m, 2H), 1.72-1.58 (m, 1H), 1.11 (d, J=6.7 Hz,
6H); MS (ESI) m/z 530.19 (M+H).
Example 64. Compound 104
##STR00230##
[0541] Prepared from S1-11-5: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.08-4.70 (m, 3H), 4.69-4.58 (m, 1H), 4.37-4.27 (m, 1H),
4.09 (s, 1H), 4.01-3.92 (m, 1H), 3.91-3.82 (m, 1H), 3.67-3.57 (m,
1H), 3.53-3.43 (m, 1H), 3.23-3.14 (m, 1H), 3.14-2.92 (m, 8H),
2.40-2.27 (m, 1H), 2.27-2.13 (m, 2H), 2.05-1.92 (m, 2H), 1.72-1.57
(m, 2H); MS (ESI) m/z 558.26 (M+H).
Example 65. Compound 108
##STR00231##
[0543] Prepared from S1-11-6: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.07-4.70 (m, 3H), 4.69-4.58 (m, 1H), 4.37-4.27 (m, 1H),
4.09 (s, 1H), 4.01-3.92 (m, 1H), 3.91-3.82 (m, 1H), 3.67-3.57 (m,
1H), 3.53-3.43 (m, 1H), 3.23-3.14 (m, 1H), 3.14-2.92 (m, 8H),
2.40-2.27 (m, 1H), 2.27-2.13 (m, 2H), 2.05-1.92 (m, 2H), 1.72-1.57
(m, 2H); MS (ESI) m/z 558.21 (M+H).
Example 66. Compound 143
##STR00232##
[0545] Prepared from S1-11-7: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.05-4.81 (m, 2H), 4.80-4.70 (m, 1H), 4.68-4.55 (m, 1H),
4.08 (s, 1H), 3.85-3.72 (m, 1H), 3.24-3.13 (m, 1H), 3.13-2.90 (m,
8H), 2.40-2.26 (m, 1H), 2.25-2.16 (m, 1H), 1.71-1.56 (m, 1H), 1.47
(d, J=6.7 Hz, 6H); MS (ESI) m/z 516.32 (M+H).
Example 67. Compound 120
##STR00233##
[0547] Prepared from S1-11-8: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.10-4.74 (m, 3H), 4.70-4.58 (m, 1H), 4.09 (s, 1H),
3.69-3.54 (m, 1H), 3.24-2.88 (m, 9H), 2.40-2.28 (m, 1H), 2.28-2.19
(m, 1H), 2.07-1.94 (m, 1H), 1.77-1.57 (m, 2H), 1.45 (d, J=6.1 Hz,
3H), 1.08 (t, J=7.9 Hz, 3H); MS (ESI) m/z 530.27 (M+H).
Example 68. Compound 130
##STR00234##
[0549] Prepared from S1-11-9: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.03-4.74 (m, 3H), 4.68-4.58 (m, 1H), 4.10 (s, 1H),
3.67-3.55 (m, 1H), 3.23-2.90 (m, 9H), 2.37-2.18 (m, 2H), 2.07-1.94
(m, 1H), 1.76-1.56 (m, 2H), 1.44 (d, J=6.1 Hz, 3H), 1.07 (t, J=7.3
Hz, 3H); MS (ESI) m/z 530.26 (M+H).
Example 69. Compound 123
##STR00235##
[0551] Prepared from S1-11-10: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.05-4.73 (m, 3H), 4.68-4.58 (m, 1H), 4.09 (s, 1H),
3.66-3.54 (m, 1H), 3.23-2.91 (m, 9H), 2.38-2.28 (m, 1H), 2.28-2.19
(m, 1H), 2.07-1.94 (m, 1H), 1.75-1.57 (m, 2H), 1.44 (d, J=6.1 Hz,
3H), 1.07 (t, J=7.3 Hz, 3H); MS (ESI) m/z 530.26 (M+H).
Example 70. Compound 137
##STR00236##
[0553] Prepared from S1-11-11: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.08-4.73 (m, 3H), 4.72-4.52 (m, 1H), 4.09 (s, 1H),
3.67-3.55 (m, 1H), 3.23-2.90 (m, 9H), 2.44-2.27 (m, 2H), 2.27-2.18
(m, 1H), 1.70-1.57 (m, 1H), 1.37 (d, J=6.7 Hz, 3H), 1.09 (d, J=6.7
Hz, 3H), 1.07-1.01 (m, 3H); MS (ESI) m/z 544.32 (M+H).
Example 71. Compound 106
##STR00237##
[0555] Prepared from S1-11-12: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.10-4.73 (m, 3H), 4.72-4.58 (m, 1H), 4.09 (s, 1H),
3.66-3.56 (m, 1H), 3.24-2.87 (m, 9H), 2.45-2.29 (m, 2H), 2.27-2.19
(m, 1H), 1.71-1.58 (m, 1H), 1.38 (d, J=6.7 Hz, 3H), 1.10 (d, J=7.3
Hz, 3H), 1.05 (d. J=6.7 Hz, 3H); MS (ESI) m/z 544.31 (M+H).
Example 72. Compound 100
##STR00238##
[0557] Prepared from S1-11-13: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.10-4.91 (m, 2H), 4.78-4.69 (m, 1H), 4.65-4.53 (m, 1H),
4.10 (s, 1H), 4.03-3.90 (m, 1H), 3.24-2.90 (m, 9H), 2.39-2.18 (m,
4H), 1.98-1.70 (m, 6H), 1.70-1.56 (m, 1H); MS (ESI) m/z 542.27
(M+H).
Example 73. Compound 140
##STR00239##
[0559] Prepared from S1-11-14: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.15-5.43 (broad, 4H), 4.41-4.33 (m, 1H), 4.27-4.19 (m,
1H), 4.17-4.10 (m, 1H), 4.08 (s, 1H), 3.90-3.83 (m, 1H), 3.80-3.71
(m, 1H), 3.23-3.14 (m, 1H), 3.13-2.91 (m, 8H), 2.57-2.44 (m, 1H),
2.40-2.17 (m, 3H), 1.71-1.57 (m, 1H); MS (ESI) m/z 544.21
(M+H).
Example 74. Compound 129
##STR00240##
[0561] Prepared from S1-11-15: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.96-4.63 (m, 4H), 4.10 (s, 1H), 3.28-2.85 (m, 9H),
2.41-2.16 (m, 2H), 1.92-1.82 (m, 2H), 1.70-1.57 (m, 1H), 1.46 (s,
6H), 1.12-1.02 (m, 3H); MS (ESI) m/z 569.26 (M+H).
Example 75. Compound 118
##STR00241##
[0563] Prepared from S1-11-16: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.02-4.74 (m, 4H), 4.09 (s, 1H), 3.23-2.91 2.39-2.27 (m,
1H), 2.27-2.18 (m, 1H), 1.71-1.57 (m, 1H), 1.37 (s, 6H), 1.34-1.25
(m, 1H), 0.78-0.68 (m, 2H), 0.68-0.61 (m, 2H); MS (ESI) m/z, 556.36
(M+H).
Example 76. Compound 133
##STR00242##
[0565] Prepared from S1-11-17: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.99-4.79 (m, 2H), 4.79-4.69 (m, 2H), 4.10 (s, 1H),
3.24-2.92 (m, 9H), 2.39-2.27 (m, 1H), 2.27-2.19 (m, 1H), 1.86 (s,
2H), 1.70-1.56 (m, 7H), 1.13 (s, 9H); MS (ESI) m/z 586.38
(M+H).
Example 77. Compound 114
##STR00243##
[0567] Prepared from S1-11-18: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.09-4.80 (m, 4H), 4.10 (s, 1H), 3.28-2.94 (m, 10H),
2.40-2.29 (m, 1H), 2.28-2.21 (m, 1H), 1.72-1.59 (m, 1H), 1.20-1.28
(m, 2H), 1.18-1.03 (m, 2H); MS (ESI) m/z 514.47 (M+H).
Example 78. Compound 132
##STR00244##
[0569] Prepared from S1-11-19: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.04-4.84 (m, 2H), 4.64-4.56 (m, 1H), 4.53-4.42 (m, 1H),
4.18-4.04 (m, 2H), 3.22-3.15 (m, 1H), 3.14-2.95 (m, 8H), 2.50-2.29
(m, 5H), 2.28-2.20 (m, 1H), 2.05-1.85 (m, 2H), 1.71-1.58 (m, 1H);
MS (ESI) m/z 528.49 (M+H).
Example 79. Compound 136
##STR00245##
[0571] Prepared from S1-11-20: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.97-4.81 (m, 1H), 4.80-4.65 (m, 3H), 4.09 (s, 1H), 3.69
(s, 2H), 3.23-2.91 (m, 9H), 2.39-2.27 (m, 1H), 2.27-2.19 (m, 1H),
1.70-1.57 (m, 1H), 1.44 (s, 6H); MS (ESI) m/z 546.33 (M+H).
Example 80. Compound 142
##STR00246##
[0573] Prepared from S1-11-21: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.08-4.81 (m, 2H), 4.75-4.47 (m, 2H), 4.08 (s, 1H),
3.50-3.37 (m, 2H), 3.21-2.84 (m, 9H), 2.40-2.27 (m, 1H), 2.26-2.17
(m, 1H), 1.92-1.76 (m, 2H), 1.71-1.57 (m, 1H), 1.07 (t. J=7.3 Hz,
3H); MS (ESI) m/z 516.24 (M+H).
Example 81. Compound 122
##STR00247##
[0575] Prepared from S1-11-22: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.96-4.82 (m, 4H), 4.10 (s, 1H), 3.89 (m, 1H), 3.83 (m,
1H), 3.23-3.15 (m, 1H), 3.14-2.91 (m, 8H), 2.40-2.29 (m, 1H),
2.28-2.20 (m, 1H), 1.72-1.54 (m, 7H); MS (ESI) m/z 548.53
(M+H).
Example 82. Compound 146
##STR00248##
[0577] Prepared from S1-11-23: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.92-4.78 (m, 2H), 4.78-4.66 (m, 2H), 4.09 (s, 1H),
3.98-3.85 (m, 2H), 3.85-3.78 (m, 2H), 3.22-3.12 (m, 1H), 3.14-2.90
(m, 8H), 2.40-2.27 (m, 1H), 2.27-2.01 (m, 7H), 1.74-1.56 (m, 7H);
MS (ESI) m/z 599.29 (M+H).
Example 83. Compound 126
##STR00249##
[0579] Prepared from S4-10-1: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.13-4.96 (m, 1H), 4.64-4.51 (m, 1H), 4.11 (s, 1H),
3.86-3.74 (m, 1H), 3.24-2.89 (m, 11H), 2.66-2.52 (m, 1H), 2.27-2.18
(m, 1H), 1.69-1.59 (m, 1H), 1.47 (s, 6H); MS (ESI) m/z 566.26
(M+H).
Example 84. Compound 113
##STR00250##
[0581] Prepared from S4-10-2: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.08-4.93 (m, 1H), 4.80-4.60 (m, 1H), 4.12 (s, 1H),
3.67-3.55 (m, 1H), 3.27-3.17 (m, 1H), 3.16-2.85 (m, 10H), 2.65-2.52
(m, 1H), 2.28-2.19 (m, 1H), 2.08-1.95 (m, 1H), 1.77-1.58 (m, 2H),
1.45 (d, J=6.7 Hz, 3H), 1.07 (t, J=7.6 Hz, 3H), MS (ESI) m/z 580.26
(M+H).
Example 85. Compound 128
##STR00251##
[0583] Prepared from S4-10-3: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.08-4.91 (m, 1H), 4.70-4.51 (m, 1H), 4.13 (s, 1H),
3.66-3.56 (m, 1H), 3.26-3.17 (m, 1H), 3.16-2.86 (m, 10H), 2.66-2.53
(m, 1H), 2.28-2.19 (m, 1H), 2.09-1.94 (m, 1H), 1.77-1.57 (m, 2H),
1.45 (d, J=6.1 Hz, 3H), 1.07 (t, J=7.3 Hz, 3H); MS (ESI) m/z 580.26
(M+H).
Example 86. Compound 112
##STR00252##
[0585] Prepared from S4-10-4: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.98-4.86 (m, 1H), 4.78-4.66 (m, 1H), 4.12 (s, 1H),
3.25-2.89 (m, 12H), 2.68-2.52 (m, 1H), 2.27-2.18 (m, 1H), 1.72-1.59
(m, 1H), 1.53 (s, 9H); MS (ESI) m/z 580.26 (M+H).
Example 87. Compound 116
##STR00253##
[0587] Prepared from S4-10-5: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.17-5.01 (m, 2H), 4.12 (s, 1H), 3.27-3.19 (2H), 3.16-2.84
(m, 10H), 2.66-2.54 (m, 1H), 2.27-2.19 (m, 1H), 1.72-1.59 (m, 1H),
1.20-1.13 (m, 2H), 1.09-1.02 (m, 2H); MS (ESI) m/z 564.17
(M+H).
Example 88. Compound 141
##STR00254##
[0589] Prepared from S4-10-6: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.20-5.07 (m, 1H), 4.58-4.47 (m, 1H), 4.13 (s, 1H),
3.51-3.38 (m, 2H), 3.28-3.17 (m, 1H), 3.16-2.90 (m, 10H), 2.67-2.51
(m, 1H), 2.28-2.19 (m, 1H), 1.94-1.80 (m, 2H), 1.72-1.59 (m, 1H),
1.08 (t, J=7.4 Hz, 3H); MS (ESI) m/z 566.26 (M+H).
Example 89. Compound 115
##STR00255##
[0591] Prepared from S6-2-1: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.16-4.96 (m, 2H), 4.78-4.62 (m, 2H), 4.16 (s, 1H),
3.28-2.92 (m, 15H), 2.61-2.40 (m, 1H), 2.36-2.27 (m, 1H), 1.75-1.53
(m, 10H); MS (ESI) m/z 555.27 (M+H).
Example 90. Compound 135
##STR00256##
[0593] Prepared from S6-2-2: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.19-5.03 (m, 1H), 4.60-4.46 (m, 1H), 4.13 (s, 1H),
3.88-3.75 (m, 1H), 3.13-2.82 (m, 17H), 2.48-2.21 (m, 2H), 1.73-1.59
(m, 1H), 1.57-1.44 (m, 6H); MS (ESI) m/z 541.24 (M+H).
Example 91. Compound 124
##STR00257##
[0595] Prepared from S6-2-3: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.10-4.96 (m, 1H), 4.58-4.46 (m, 1H), 4.10 (s, 1H),
3.68-3.55 (m, 1H), 3.10-2.68 (m, 18H), 2.40-2.18 (m, 1H), 2.11-1.98
(m, 1H), 1.78-1.57 (m, 2H), 1.46 (d, J=6.1 Hz, 3H), 1.09 (t, J=6.7
Hz, 3H); MS (ESI) m/z 555.33 (M+H).
Example 92. Compound 127
##STR00258##
[0597] Prepared from S6-2-4: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.14-4.96 (m, 1H), 4.58-4.44 (m, 1H), 4.16 (s, 1H),
3.66-3.54 (m, 1H), 3.10-2.69 (m, 18H), 2.38-2.19 (m, 1H), 2.14-1.99
(m, 1H), 1.76-1.57 (m, 1H), 1.53-1.40 (m, 3H), 1.08 (t, J=7.3 Hz,
3H); MS (ESI) m/z 555.39 (M+H).
Example 93. Compound 103
##STR00259##
[0599] Prepared from S6-2-5: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.12-4.98 (m, 2H), 4.71 (s, 2H), 4.16 (s, 1H), 3.25-2.91
(m, 15H), 2.61-2.38 (m, 1H), 2.35-2.25 (m, 1H), 1.99-1.89 (m, 2H),
1.73-1.60 (m, 1H), 1.52 (s, 6H), 1.10 (t, J=7.3 Hz, 3H); MS (ESI)
m/z 569.26 (M+H).
Example 94. Compound 105
Synthesis of S8-1
##STR00260##
[0601] To a solution of lithium diisopropylamide (1.8 M in hexanes,
446 .mu.L, 0.804 mmol, 2.2 eq) and TMEDA (328 .mu.L, 2.19 mmol, 6
eq) in THF (8 mL) at -78.degree. C. was added a solution of
compound S1-11-21 (168 mg, 0.402 mmol, 1.1 eq) in THF (1 mL) by
dropwise addition. This resulted in a dark red colored solution.
After 30 min, a solution of enone S7-1 (175 mg, 0.362 mmol, 1 eq)
in THF (1.2 mL) was added. After complete addition, the reaction
mixture was allowed to warm to -15.degree. C. over 1 h. The
reaction was quenched by the addition of ammonium chloride
(saturated, aqueous solution, 15 mL) and was extracted with EtOAc
(2.times.30 mL). The combined organic extracts were dried over
Na.sub.2SO.sub.4, filtered, and concentrated under reduced
pressure. Purification of the resulting oil via flash column
chromatography on silica gel (Silicycle, 25 g, 10 to 25% EtOAc in
hexanes gradient) provided 208 mg of S8-1 (71%) as a white solid:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 16.05 (s, 1H), 7.53-7.43
(m, 2H), 7.42-7.28 (m, 8H), 5.95-5.79 (m, 1H), 5.35 (s, 2H),
5.27-5.12 (m, 2H), 4.90 (q, J=10.4 Hz, 2H), 4.01-3.74 (m, 4H), 3.29
(d, J=6.1 Hz, 1H), 3.25-3.18 (m, 1H), 3.03-2.92 (m, 1H), 2.58-2.34
(m, 9H), 2.13 (d, J=14.7 Hz, 1H), 0.82 (s, 9H), 0.27 (s, 3H), 0.12
(s, 3H); MS (ESI) m/z 806.38 (M+H).
Synthesis of S8-2
##STR00261##
[0603] A flame-dried vial was charged with N,N-dimethylbarbituric
acid (103 mg, 0.66 mmol, 2.6 eq) and
tetrakis(triphenylphosphine)palladium(0) (20.1 mg, 0.017 mmol, 0.07
eq). The vial was evacuated and back-filled with nitrogen three
times. A solution of S8-1 (205 mg, 0.254 mmol, 1 eq) in
dichloromethane (degassed, 4 mL) under nitrogen was transferred via
syringe to the prepared vial. The resulting heterogeneous solution
was placed in a 35.degree. C. heating block. After 1 h, the
reaction mixture was concentrated under reduced pressure.
Purification of the resulting oil via flash column chromatography
on silica gel (Silicycle, 12 g, 20 to 60% EtOAc in hexanes
gradient) provided 176 mg of S8-2 (90%) as an orange solid: .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 7.52-7.45 (m, 2H), 7.41-7.28 (m,
8H), 5.36 (s, 2H), 4.91 (s, 2H), 4.34-4.20 (m, 2H), 4.19-3.99 (m,
2H), 3.96 (d, J=10.4 Hz, 1H), 3.36-3.27 (m, 1H), 3.23 (dd, J=4.9,
15.2 Hz, 1H), 3.04-2.93 (m, 1H), 2.59-2.36 (m, 9H), 2.14 (d, J=14.7
Hz, 1H), 0.82 (s, 9H), 0.27 (s, 3H), 0.13 (s, 3H); MS (ESI) m/z
766.33 (M+H).
Synthesis of Compound 105
##STR00262##
[0605] To a solution of S8-2 (9.6 mg, 0.012 mmol, 1 eq) in
1,4-dioxane (1 mL) was added an aqueous solution of HF (50%, 150
.mu.L). After two hours, the reaction mixture was poured into an
aqueous K.sub.2HPO.sub.4 solution (2.4 g in 25 mL) and extracted
with EtOAc (2.times.30 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4), filtered, and concentrated under reduced
pressure. Palladium on carbon (10%, 8 mg) was added to a solution
of this crude oil in dioxane:MeOH:0.5 N HCl in Methanol (5:4:1, 1
mL). The flask was fitted with a septum and evacuated and
back-filled three times with hydrogen gas, and then the solution
was degassed with bubbling hydrogen for 3 minutes. The reaction was
stirred under an atmosphere (balloon) of hydrogen gas for 2 h. The
reaction mixture was filtered through Celite to remove the
palladium catalyst and concentrated under reduced pressure.
Preparative reverse phase HPLC of the resulting oil was performed
on a Waters Autopurification system using a Polymerx 10.mu.
RP-.gamma. 100 R column [30.times.21.20 mm, 10 micron, solvent A:
0.05 N HCl in water, solvent B: Methanol; injection volume: 1.5 mL
(0.05 N HCl in water); gradient: 20.fwdarw.80% B over 20 min;
mass-directed fraction collection]. Fractions with the desired MW,
eluting at 6.75-7.5 min, were collected and freeze-dried to provide
2.0 mg of the desired compound Compound 105 (33%): .sup.1H NMR (400
MHz, CD.sub.3OD) 4.74 (s, 2H), 4.64 (s, 2H), 4.09 (s, 1H),
3.25-3.14 (m, 1H), 3.14-2.88 (m, 8H), 2.40-2.28 (m, 1H), 2.27-2.18
(m, 1H), 1.71-1.59 (m, 1H); MS (ESI) m/z 474.13 (M+H).
Example 95. Compound 111
Synthesis of S8-4-1
##STR00263##
[0607] To a solution of S8-2 (30.3 mg, 0.040 mmol, 1 eq) in THF (1
mL) was added bromoacetylbromide (3.6 .mu.L, 0.041 mmol, 1.05 eq).
After 5 min, 0.75 .mu.L bromoacetylbromide (0.008 mmol, 0.2 eq) was
added, followed by cyclopentylamine (19.5 .mu.L, 0.197 mmol, 5 eq).
After 1 h, the reaction was complete, and the mixture was
concentrated under reduced pressure to produce crude S8-4-1, which
was used without further purification
Synthesis of Compound 111
##STR00264##
[0609] To a solution of this crude oil in 1,4-dioxane (1.8 mL) was
added an aqueous solution of HF (50%, 250 .mu.L). After 1.5 h, the
reaction mixture was poured into an aqueous K.sub.2HPO.sub.4
solution (3.6 g in 30 mL) and extracted with EtOAc (2.times.30 mL).
The combined organic layers were dried (Na.sub.2SO.sub.4),
filtered, and concentrated under reduced pressure. Palladium on
carbon (10%, 15.1 mg) was added to a solution of this crude oil in
dioxane:MeOH (1:1, 1 mL). The flask was fitted with a septum and
evacuated and back-filled three times with hydrogen gas. The
reaction was stirred under an atmosphere (balloon) of hydrogen gas
for 3 h. The reaction mixture was filtered through Celite to remove
the palladium catalyst and concentrated under reduced pressure.
Preparative reverse phase HPLC of the resulting oil was performed
on a Waters Autopurification system using a Polymerx 10.mu.
RP-.gamma. 100 R column [30.times.21.20 mm, 10 micron, solvent A:
0.05 N HCl in water, solvent B: CH.sub.3CN; injection volume: 2.4
mL (0.05 N HCl in water); gradient: 20.fwdarw.80% B over 20 min;
mass-directed fraction collection]. Fractions with the desired MW,
eluting at 11.0-12.5 min, were collected and freeze-dried to
provide 2.4 mg of the desired compound Compound 111 (9%): .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 5.04-4.75 (m, 4H), 4.17-4.06 (m,
3H), 3.68-3.56 (m, 1H), 3.24-290 (m, 9H), 2.38-2.26 (m, 1H),
2.26-2.04 (m, 3H), 1.91-1.57 (m, 7H); MS (ESI) m/z 599.28
(M+H).
Example 96. Compound 131
##STR00265##
[0611] To a solution of S8-2 (20.1 mg, 0.026 mmol, 1 eq) in THF (1
mL) was added dimethylaminoacetyl chloride hydrochloride (85%, 7.4
mg, 0.039 mmol, 1.5 eq). After 2.5 h, the reaction mixture was
diluted with sodium bicarbonate solution (saturated, aqueous, 3 mL)
and extracted with EtOAc (2.times.7 mL). The combined organic
layers were washed with brine (2 mL), dried (Na.sub.2SO.sub.4),
filtered, and concentrated under reduced pressure to produce S8-4-2
(not shown). To a solution of this crude oil in 1,4-dioxane (1.5
mL) was added an aqueous solution of HF (50%, 300 .mu.L). After 1.5
h, the reaction mixture was poured into an aqueous K.sub.2HPO.sub.4
solution (3.6 g in 30 mL) and extracted with EtOAc (2.times.25 mL).
The combined organic layers were dried (Na.sub.2SO.sub.4),
filtered, and concentrated under reduced pressure. Palladium on
carbon (10%, 12 mg) was added to a solution of this crude oil in
dioxane:MeOH (1:1, 1 mL). The flask was fitted with a septum and
evacuated and back-filled three times with hydrogen gas. The
reaction mixture was stirred under an atmosphere (balloon) of
hydrogen gas for 2.5 h, then was filtered through Celite to remove
the palladium catalyst and concentrated under reduced pressure.
Preparative reverse phase HPLC of the resulting oil was performed
on a Waters Autopurification system using a Polymerx 10.mu.
RP-.gamma. 100 R column [30.times.21.20 mm, 10 micron, solvent A:
0.05 N HCl in water, solvent B: Methanol; injection volume: 2.0 mL
(20% Methanol in 0.05 N HCl in water); gradient: 20.fwdarw.80% B
over 20 min; mass-directed fraction collection]. Fractions with the
desired MW, eluting at 8.0-10.2 min, were collected and
freeze-dried to provide 7.0 mg of the desired compound Compound 131
(42%): .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.99-4.73 (m, 4H),
4.37-4.27 (m, 2H), 4.09 (s, 1H), 3.22-2.91 (m, 15H), 2.37-2.16 (m,
2H), 1.71-1.56 (m, 1H); MS (ESI) m/z 559.19 (M+H).
Example 97. Compound 139
##STR00266##
[0613] To a solution of S8-2 (21.0 mg, 0.027 mmol, 1 eq) in THF (1
mL) was added pyrrolidineacetylchloride hydrochloride (8.4 mg,
0.045 mmol, 1.7 eq). After 1 h, the reaction mixture was diluted
with sodium bicarbonate solution (saturated, aqueous, 3.5 mL) and
extracted with EtOAc (2.times.7 mL). The combined organic layers
were washed with brine (2 mL), dried (Na.sub.2SO.sub.4), filtered,
and concentrated under reduced pressure to produce S8-4-3 (not
shown). To a solution of this crude oil in 1,4-dioxane (1.7 mL) was
added an aqueous solution of HF (50%, 300 .mu.L). After 1.5 h, the
reaction mixture was poured into an aqueous K.sub.2HPO.sub.4
solution (3.6 g in 30 mL) and extracted with EtOAc (2.times.25 mL).
The combined organic layers were dried (Na.sub.2SO.sub.4),
filtered, and concentrated under reduced pressure. Palladium on
carbon (10%, 15 mg) was added to a solution of this crude oil in
dioxane:MeOH (5:4, 0.90 mL). The flask was fitted with a septum and
evacuated and back-filled three times with hydrogen gas. The
reaction mixture was stirred under an atmosphere (balloon) of
hydrogen gas for 2.5 h, then was filtered through Celite to remove
the palladium catalyst and concentrated under reduced pressure.
Preparative reverse phase HPLC of the resulting oil was performed
on a Waters Autopurification system using a Polymerx 10.mu.
RP-.gamma. 100 R column [30.times.21.20 mm, 10 micron, solvent A:
0.05 N HCl in water, solvent B: Methanol; injection volume: 2.0 mL
(20% Methanol in 0.05 N HCl in water); gradient: 20.fwdarw.80% B
over 20 min; mass-directed fraction collection]. Fractions with the
desired MW, eluting at 9.4-11.1 min, were collected and
freeze-dried to provide 3.5 mg of the desired compound Compound 139
(19%): .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 5.00-4.74 (m, 4H),
4.43-4.35 (m, 2H), 4.09 (s, 1H), 3.84-3.73 (m, 2H), 3.27-2.90 (m,
11H), 2.37-2.00 (m, 6H), 1.70-1.56 (m, 1H); MS (ESI) m/z 585.28
(M+H).
Example 98. Compound 147
##STR00267##
[0615] To a solution of S8-2 (33.0 mg, 0.043 mmol, 1 eq) in THF (1
mL) was added bromoacetylbromide (4.1 .mu.L, 0.047 mmol, 1.1 eq).
After 40 min, (S)-(+)-3-fluoropyrrolidine hydrochloride salt (15.6
mg, 0.124 mmol, 3 eq) was added, followed by triethylamine (18
.mu.L, 0.126 mmol, 3 eq). After an additional 19 h, additional
pyrrolidine salt (32 mg, 0.254 mmol, 6 eq) and triethylamine (54
.mu.L, 0.387 mmol, 9 eq) were added. After 20 h, the mixture was
diluted with brine (8 mL), water (1.5 mL), and extracted with EtOAc
(2.times.30 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4), filtered, and concentrated under reduced
pressure to produce S8-4-4 (not shown). To a solution of this crude
oil in 1,4-dioxane (1 mL) was added an aqueous solution of HF (50%,
250 .mu.L). After 1.5 h, the reaction mixture was poured into an
aqueous K.sub.2HPO.sub.4 solution (3 g in 30 mL) and extracted with
EtOAc (2.times.30 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4), filtered, and concentrated under reduced
pressure. Palladium on carbon (10%, 16.5 mg) was added to a
solution of this crude oil in dioxane:MeOH (1:1, 1 mL). The flask
was fitted with a septum and evacuated and back-filled three times
with hydrogen gas. The reaction was stirred under an atmosphere
(balloon) of hydrogen gas for 2 h. The reaction mixture was
filtered through Celite to remove the palladium catalyst and
concentrated under reduced pressure. Preparative reverse phase HPLC
of the resulting oil was performed on a Waters Autopurification
system using a Polymerx 10.mu. RP-.gamma. 100 R column
[30.times.21.20 mm, 10 micron, solvent A: 0.05 N HCl in water,
solvent B: CH.sub.3CN; injection volume: 2.4 mL (0.05 N HCl in
water); gradient: 10-60% B over 15 min; mass-directed fraction
collection]. Fractions with the desired MW, eluting at 6.3-7.3 min,
were collected and freeze-dried to provide 7.8 mg of the desired
compound Compound 147 (27%): .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 5.61-5.34 (m, 1H), 5.02-4.77 (m, 4H), 4.58-4.38 (m, 2H),
4.18-3.90 (m, 3H), 3.74-3.38 (m, 2H), 3.24-2.89 (m, 9H), 2.59-2.28
(m, 4H), 2.27-2.18 (m, 1H), 1.71-1.58 (m, 1H); MS (ESI) m/z 603.35
(M+H).
Example 99. Compound 109
##STR00268##
[0617] Compound 150 (7.9 mg, 0.013 mmol) was dissolved in Methanol
(1 mL) and 1,4-dioxane (1 mL) and 0.5 M HCl in Methanol (0.2 mL),
and palladium on carbon (Degussa, 10 wt %, .about.2 mg) was added.
An atmosphere of hydrogen was introduced, and the reaction mixture
was stirred overnight. The reaction mixture was filtered through
Celite, and the filtrate was concentrated under reduced pressure.
The material was dissolved in Methanol (1 mL) and palladium on
carbon (Degussa, 10 wt %, .about.20 mg) was added. An atmosphere of
hydrogen was introduced, and the reaction mixture was stirred
overnight. The reaction mixture was filtered through Celite, and
the filtrate was concentrated under reduced pressure. The material
was purified on a Waters Autopurification system equipped with a
Phenomenex Polymerx 10.mu. RP 100A column [10 .mu.m, 30.times.21.20
mm; flow rate, 20 mL/min; Solvent A: 0.05N HCl in water; Solvent B:
Methanol; gradient: 20.fwdarw.100% B; mass-directed fraction
collection]. Fractions with the desired MW were collected and
freeze-dried to yield 1.2 mg (16%, 2 steps) of the desired product
Compound 109 as a yellow solid. .sup.1H NMR (400 MHz, CD.sub.3OD
with 1 drop DCl) .delta. 6.84 (s, 1H), 4.85-4.65 (m, 4H), 4.13 (s,
1H), 3.15-2.88 (m, 9H), 2.61-2.50 (m, 1H), 2.28-2.20 (m, 1H),
1.92-1.82 (m, 2H), 1.65-1.50 (m, 1H), 1.44 (s, 6H), 1.06 (t, J=7.3
Hz, 3H); MS (ESI) m/z 526.30 (M+H).
Example 100. Compound 201
Synthesis of S10-1
##STR00269##
[0619] (Methoxymethyl)triphenylphosphonium chloride (1.55 g, 4.51
mmol) was added to a suspension of potassium t-butoxide (0.506 g,
4.51 mmol) in THF (15 mL), giving an immediate red colored
solution. After 15 min, a solution of compound S1-7 (1.00 g, 2.26
mmol) in THF (5 mL) was added. After 2 h, the reaction mixture was
quenched with water and was extracted with EtOAc (2.times.). The
combined extracts were dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure. The material was purified by
column chromatography (Biotage 20 g column, 0 to 6% EtOAc in hexane
gradient), yielding 986 mg (93%) of the compound S10-1 as a mixture
of two isomers. MS (ESI) m/z 493.04, 495.04 (M+Na).
Synthesis of S10-2
##STR00270##
[0621] i-Propyl magnesium chloride/lithium chloride solution
(Chemetall Foote Corporation, 1.2 M solution in THF, 8.5 mL, 10.2
mmol) was added to a -50.degree. C. solution of compound S10-1 (956
mg, 2.03 mmol) in THF (20 mL). The reaction mixture was allowed to
warm to 0.degree. C. over 1 h. N,N-Dimethylformamide (1.25 mL, 16.2
mmol) was added, and the reaction was allowed to warm to rt. After
1 hour, the reaction mixture was quenched with ammonium chloride
(saturated, aqueous solution) and was extracted with EtOAc
(2.times.). The combined extracts were dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. The material was
purified by column chromatography (Biotage 25 g column, 5 to 40%
EtOAc in hexane gradient), yielding 205 mg (24%) of compound S10-2.
R.sub.f=0.23 in 20% EtOAc in hexane; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.3 (s, 1H), 7.45-7.30 (m, 7H), 7.28-7.24 (m,
1H), 7.10-7.02 (m, 3H), 6.67 (d, J=12.8 Hz, 1H), 5.09 (s, 2H), 3.77
(s, 3H), 2.43 (d, J=4.6 Hz, 3H); MS (ESI) m/z 443.18 (M+Na).
Synthesis of S10-3-1
##STR00271##
[0623] Neopentylamine (0.077 mL, 0.66 mmol) was added to a solution
of compound S10-2 (55.5 mg, 0.132 mmol) in CH.sub.2Cl.sub.2 (5 mL)
and Acetic acid (0.038 mL, 0.66 mmol). After 5 min, sodium
triacetoxyborohydride (83.9 mg, 0.396 mmol) was added. After 1
hour, the reaction mixture was diluted with EtOAc and was washed
with NaHCO.sub.3 (saturated, aqueous solution, 2.times.). The
organics were dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure, yielding 53.3 mg (88% crude)
of compound S10-3-1. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.46-7.30 (m, 7H), 7.26-7.20 (m, 1H), 7.10-7.04 (m, 2H), 4.96 (s,
2H), 3.72 (s, 2H), 2.86-2.75 (m, 4H), 2.35 (d, J=1.8 Hz, 3H), 2.23
(s, 2H), 0.89 (s, 9H); MS (ESI) m/z 462.28 (M+H).
Synthesis of S10-4-1
##STR00272##
[0625] Lithium diisopropylamide was prepared at -40.degree. C. from
n-butyllithium (2.5 M solution in hexane, 0.045 mL, 0.11 mmol) and
diisopropylamine (0.016 mL, 0.11 mmol) in THF (2 mL). The reaction
mixture was cooled to -78.degree. C. and TMEDA (0.040 mL, 0.27
mmol) was added followed by the dropwise addition of a solution of
compound S10-3-1 (24.9 mg, 0.0539 mmol) in THF (1 mL). No color
change was observed, so additional lithium diisopropylamide (2.0M
solution in THF, 0.060 mL, 0.12 mmol) was added until a deep red
colored solution persisted. After 15 min, a solution of enone S7-1
(21.7 mg, 0.045 mmol) in THF (0.5 mL) was added. After complete
addition, the reaction mixture was allowed to warm to -20.degree.
C. over 1 h. The reaction was quenched by the addition of ammonium
chloride (saturated, aqueous solution) and was extracted with EtOAc
(2.times.). The combined extracts were dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. The material was
purified on a Waters Autopurification system equipped with a
Sunfire Prep C18 OBD column [5 .mu.m, 19.times.50 mm; flow rate, 20
mL/min; Solvent A: H.sub.2O with 0.1% HCO.sub.2H; Solvent B:
CH.sub.3CN with 0.1% HCO.sub.2H; gradient: 50.fwdarw.100% B;
mass-directed fraction collection], yielding 18.9 mg (49%) of the
desired product S10-4-1 as a yellow solid. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 16.0 (s, 1H), 7.52-7.44 (m, 2H), 7.40-7.28 (m,
8H), 5.36 (s, 2H), 4.94 (d, J=11.0 Hz, 1H), 4.78 (d, J=10.4 Hz,
1H), 4.10-3.89 (m, 3H), 3.29-3.15 (m, 2H), 3.06-2.96 (m, 2H),
2.65-2.40 (m, 11H), 2.15 (d, J=14.6 Hz, 1H), 0.98 (s, 9H), 0.82 (s,
9H), 0.27 (s, 3H), 0.12 (s, 3H); MS (ESI) m/z 850.39 (M+H).
Synthesis of Compound 201
##STR00273##
[0627] Aqueous HF (0.4 mL, 48%) was added to a solution of S10-4-1
(18.9 mg, 0.022 mmol) in 1,4-dioxane (1 mL) in a plastic vial.
After stirring overnight, the reaction mixture was poured into a
solution of K.sub.2HPO.sub.4 (4.8 g) in water (15 mL). The mixture
was extracted with EtOAc (3.times.). The combined EtOAc extracts
were dried over Na.sub.2SO.sub.4, filtered and concentrated under
reduced pressure. The material was dissolved in Methanol (2 mL),
1,4-dioxane (2 mL) and 0.5M HCl in Methanol (0.5 mL), and palladium
on carbon (Degussa, 10 wt %, .about.5 mg) was added. An atmosphere
of hydrogen was introduced, and the reaction mixture was stirred
for 2 h. The reaction mixture was filtered through Celite, and the
filtrate was concentrated under reduced pressure. The material was
purified on a Waters Autopurification system equipped with a
Phenomenex Polymerx 10.mu. RP 100A column [10 .mu.m, 30.times.21.20
mm; flow rate, 20 mL/min; Solvent A: 0.05N HCl in water; Solvent B:
CH.sub.3CN; gradient: 0.fwdarw.70% B; mass-directed fraction
collection]. Fractions with the desired MW were collected and
freeze-dried to yield 7.8 mg (57%, 2 steps) of the desired product
Compound 201 as a yellow solid. .sup.1H NMR (400 MHz, CD.sub.3OD
with 1 drop DCl) .delta. 4.60 (t, J=14.4 Hz, 1H), 4.32 (dd, J=16.0,
7.8 Hz, 1H), 4.15 (s, 1H), 3.88-3.79 (m, 1H), 3.62-3.50 (m, 1H),
3.36-3.16 (m, 5H), 3.15-2.96 (m, 8H), 2.35-2.24 (m, 2H), 1.61 (q,
J=12.7 Hz, 1H), 1.20 (s, 9H); MS (ESI) m/z 558.26 (M+H).
[0628] The following compounds were prepared by methods similar to
that for Compound 201, substituting the appropriate
tetrahydroisoquinoline for S10-3-1. The appropriate
tetrahydroisoquinolines were prepared by methods similar to that
for S10-3-1, substituting the appropriate amine for
neopentylamine.
Example 101. Compound 200
##STR00274##
[0630] Yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD with 1 drop
DCl) .delta. 4.53 (t, J=15.8 Hz, 1H), 4.24 (dd, J=16.0, 3.7 Hz,
1H), 4.14 (s, 1H), 4.04-3.96 (m, 1H), 3.34-3.14 (m, 4H), 3.14-2.90
(m, 8H), 2.34-2.23 (m, 2H), 1.69-1.52 (m, 10H); MS (ESI) m/z 544.27
(M+H).
[0631] Prepared from S10-3-2,
##STR00275##
[0632] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.30 (m, 7H),
7.29-7.22 (m, 1H), 7.12-7.08 (m, 2H), 4.96 (s, 2H), 3.70 (s, 2H),
2.86-2.80 (m, 2H), 2.78-2.72 (m, 2H), 2.33 (s, 3H), 1.11 (s, 9H);
MS (ESI) m/z 448.31 (M+H).
Example 102. Compound 202
##STR00276##
[0634] Yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD with 1 drop
DCl) .delta. 4.59 (t, J=15.3 Hz, 1H), 4.22 (dd, J=16.3, 5.7 Hz,
1H), 4.14 (s, 1H), 3.94-3.86 (m, 1H), 3.86-3.75 (m, 1H), 3.44-3.34
(m, 1H), 3.33-2.96 (m, 11H), 2.35-2.22 (m, 4H), 2.00-1.84 (m, 4H),
1.80-1.70 (m, 2H), 1.68-1.55 (m, 1H); MS (ESI) m/z 556.26
(M+H).
[0635] Prepared from S10-3-3,
##STR00277##
[0636] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.30 (m, 7H),
7.29-7.22 (m, 1H), 7.12-7.08 (m, 2H), 4.96 (s, 2H), 3.66 (s, 2H),
2.90-2.83 (m, 2H), 2.78-2.72 (m, 2H), 2.71-2.62 (m, 1H), 2.34 (d,
J=1.4 Hz, 3H), 1.96-1.86 (m, 2H), 1.76-1.64 (m, 2H), 1.63-1.42 (m,
4H); MS (ESI) m/z 460.54 (M+H).
Example 103. Preparation of phenyl
5-(benzyloxy)-8-fluoro-7-methyl-2-propyl-1,2,3,4-tetrahydroisoquinoline-6-
-carboxylate (S11-4-1)
Synthesis of S11-1
##STR00278##
[0638] To a stirred suspension of compound S1-7 (3.99 g, 8.99 mmol,
1 eq) in methanol (50 mL) was added sodium borohydride (420 mg,
11.1 mmol, 1.3 eq). Gas evolution was evident; the solution was
homogeneous after 5 min. After 40 min the reaction was complete.
The mixture was poured into a saturated aqueous NH.sub.4Cl solution
(40 mL), water (10 mL), and extracted with EtOAc (3.times.75 mL).
The combined organic layers were dried (Na.sub.2SO.sub.4),
filtered, and concentrated under reduced pressure. The crude
material (2.13 g, 4.30 mmol, 1 eq) was azeotropically dried from
toluene three times and dried under vacuum for 2 h. To a solution
of this bromide in THF (90 mL) under N.sub.2 at -50.degree. C. was
added isopropyl magnesium chloride-lithium chloride complex (1.2 M
solution in THF, 37.4 mL, 44.9 mmol, 5 eq) dropwise over 10
minutes. The resulting dark yellow solution was allowed to warm to
0.degree. C. over 1 h. Dimethylformamide (5.57 mL, 71.9 mmol, 8 eq)
was added dropwise, and the solution was heated to 40.degree. C.
for 1.5 h. The reaction solution was poured into a saturated
aqueous NH.sub.4Cl solution (45 mL), water (20 mL), and extracted
with EtOAc (2.times.100 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4), filtered, and concentrated under reduced
pressure. MS (ESI) m/z 393.32 (M-H).
Synthesis of S11-2
##STR00279##
[0640] A flame-dried flask was cooled under nitrogen and charged
with potassium tert-butoxide (1.78 g, 15.8 mmol, 2 eq), evacuated
and back-filled with N.sub.2, charged with THF (80 mL), and cooled
to 0.degree. C. To this solution was added
(methoxymethyl)triphenylphosphonium chloride (5.43 g, 15.8 mmol, 2
eq). The resulting red solution was allowed to warm to room
temperature for 30 min, and a solution of S11-1 (3.11 g, 7.88 mmol,
1 eq) in THF (15 mL) was added slowly. After 1.5 h, the reaction
was diluted with water (45 mL) and extracted with EtOAc (2.times.75
mL). The combined organic layers were washed with brine, dried
(Na.sub.2SO.sub.4), filtered, and concentrated under reduced
pressure. Purification of the resulting crude oil via flash column
chromatography on silica gel (Redisep, 220 g, 5 to 40% EtOAc in
hexane gradient) provided 1.57 g and 949 mg of the E and Z isomers
of S11-2, respectively (75% total, 1.65:1 E:Z) as yellow oils:
.sup.1H NMR (E-isomer, 400 MHz, CDCl.sub.3) .delta. 7.45-7.30 (m,
7H), 7.28-7.20 (m, 1H), 7.14-7.03 (m, 3H), 5.88 (d, J=13.4 Hz, 1H),
5.05 (s, 2H), 4.76 (s, 2H), 3.63 (s, 3H), 2.35 (s, 3H); MS (ESI)
m/z 421.37 (E-isomer, M-H); .sup.1H NMR (Z-isomer, 400 MHz,
CDCl.sub.3) .delta. 7.42-7.29 (m, 7H), 7.04 (d, J=7.3 Hz, 2H), 6.31
(d, J=7.3 Hz, 1H), 5.48 (d, J=7.3 Hz, 1H), 4.97 (s, 2H), 4.65 (s,
2H), 3.70 (s, 3H), 2.36 (s, 3H); MS (ESI) m/z 421.34 (Z-isomer,
M-H).
Synthesis of S11-3
##STR00280##
[0642] To a solution of S11-2 (196 mg, 0.464 mmol, 1 eq) in
dichloromethane (4.6 mL) was added Dess-Martin periodinane (239 mg,
0.563 mmol, 1.2 eq). After 1 h, the solution was diluted with
saturated aqueous sodium bicarbonate (25 mL) and extracted with
EtOAc (2.times.30 mL). The combined organic layers were washed with
saturated aqueous sodium bicarbonate (10 mL), brine (20 mL), dried
(Na.sub.2SO.sub.4), filtered, and concentrated under reduced
pressure. The material was used immediately in the next reaction
without further purification or characterization.
Synthesis of S11-4-1
##STR00281##
[0644] To the crude compound S11-3 (0.116 mmol) in dichloromethane
(1.5 mL) was added acetic acid (33 .mu.L, 0.58 mmol, 5 eq) and
propylamine (48 .mu.L, 0.58 mmol, 5 eq) were added. After 50 min,
the solution was deep red in color. After 2 h, sodium
triacetoxyborohydride (123 mg, 0.58 mmol, 5 eq) was added to the
reaction mixture. The solution color faded to yellow. After an
additional 17.5 h, the reaction mixture was diluted with saturated
aqueous sodium bicarbonate (4 mL) and extracted with EtOAc
(2.times.8 mL). The combined organic layers were washed with brine
(3 mL), dried (Na.sub.2SO.sub.4), filtered, and concentrated under
reduced pressure. Purification of the resulting crude oil via flash
column chromatography on silica gel (Biotage, 10 g, 2 to 20% EtOAc
in hexane gradient) provided 29 mg of S11-4-1 (57%) as a clear oil:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.47-7.40 (m, 2H),
7.40-7.30 (m, 5H), 7.27-7.21 (m, 1H), 7.07 (d, J=7.3 Hz, 2H), 4.97
(s, 2H), 3.66 (s, 2H), 2.99-2.89 (m, 2H), 2.76-2.63 (m, 2H),
2.58-2.48 (m, 5H), 2.38 (s, 3H) 1.72-1.58 (m, 2H), 0.97 (d, J=7.3
Hz, 3H); MS (ESI) m/z 432.40 (M-H).
[0645] The following intermediates were prepared according to the
methods used to synthesize S11-4-1.
Example 104. S11-4-2
##STR00282##
[0647] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.30 (m, 7H),
7.28-7.21 (m, 1H), 7.06 (d, J=7.3 Hz, 2H), 4.97 (s, 2H), 3.66 (s,
2H), 2.98-292 (m, 2H), 2.73-2.60 (m, 4H), 2.35 (s, 3H) 1.21 (t,
J=7.3 Hz, 3H); MS (ESI) m/z 418.41 (M-H).
Example 105. S11-4-3
##STR00283##
[0649] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.47-7.40 (m, 2H),
7.40-7.30 (m, 5H), 7.27-7.21 (m, 1H), 7.07 (d, J=7.3 Hz, 2H), 4.98
(s, 2H), 3.61 (s, 2H), 2.96-2.85 (m, 2H), 2.70-2.60 (m, 2H),
2.38-2.25 (m, 5H), 1.91-1.85 (m, 1H), 0.95 (d, J=6.1 Hz, 6H); MS
(ESI) m/z 446.40 (M-H).
Example 106. S11-4-4
##STR00284##
[0651] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45-7.40 (m, 2H),
7.40-7.30 (m, 5H), 7.28-7.22 (m, 1H), 7.07 (d, J=7.3 Hz, 2H), 5.00
(s, 2H), 3.73 (s, 2H), 2.92-2.85 (m, 2H), 2.79-2.70 (m, 2H), 2.34
(s, 3H), 2.28 (s, 2H), 0.92 (s, 9H); MS (ESI) m/z 460.41 (M-H).
Example 107. S11-4-5
##STR00285##
[0653] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.29 (m, 7H),
7.28-7.20 (m, 1H), 7.06 (d, J=8.6 Hz, 2H), 4.97 (s, 2H), 3.76 (s,
2H), 3.04-2.87 (m, 3H), 2.80-2.69 (m, 2H), 2.35 (s, 3H), 1.16 (d,
J=6.7 Hz, 6H); MS (ESI) m/z 432.39 (M-H).
Example 108. S11-4-6
##STR00286##
[0655] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.40 (m, 2H),
7.40-7.29 (m, 5H), 7.27-7.22 (m, 1H), 7.07 (d, J=7.3 Hz, 2H), 4.97
(s, 2H), 3.85-3.67 (m, 2H), 3.00-2.85 (m, 2H), 2.81-2.65 (m, 3H),
2.34 (s, 3H), 1.75-1.60 (m, 1H), 1.49-1.36 (m, 1H), 1.09 (d, J=6.7
Hz, 3H), 0.95 (d, J=7.3 Hz, 3H); MS (ESI) m/z 446.43 (M-H).
Example 109. S11-4-7
##STR00287##
[0657] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.40 (m, 2H),
7.40-7.29 (m, 5H), 7.27-7.22 (m, 1H), 7.07 (d, J=7.3 Hz, 2H), 4.97
(s, 2H), 3.85-3.67 (m, 2H), 3.00-2.85 (m, 2H), 2.81-2.65 (m, 3H),
2.34 (s, 3H), 1.75-1.60 (m, 1H), 1.49-1.36 (m, 1H), 1.09 (d, J=6.7
Hz, 3H), 0.95 (d, J=7.3 Hz, 3H); MS (ESI) m/z 446.46 (M-H).
Example 110. S11-4-8
##STR00288##
[0659] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.48-7.40 (m, 2H),
7.40-7.29 (m, 5H), 7.27-7.22 (m, 1H), 7.07 (d, J=7.3 Hz, 2H), 4.97
(s, 2H), 3.85-3.67 (m, 2H), 3.00-2.85 (m, 2H), 2.81-2.65 (m, 3H),
2.34 (s, 3H), 1.75-1.60 (m, 1H), 1.49-1.36 (m, 1H), 1.09 (d, J=6.7
Hz, 3H), 0.95 (d, J=7.3 Hz, 3H); MS (ESI) m/z 446.46 (M-H).
Example 111. S11-4-9
##STR00289##
[0661] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.47-7.40 (m, 2H),
7.40-7.30 (m, 5H), 7.28-7.22 (m, 1H), 7.07 (d, J=7.3 Hz, 2H), 4.97
(s, 2H), 3.00-2.92 (m, 2H), 2.81-2.70 (m, 2H), 2.34 (s, 3H), 1.20
(s, 9H); MS (ESI) m/z 446.47 (M-H).
Example 112. Compound 304
Synthesis of S11-5-1
##STR00290##
[0663] To a solution of lithium diisopropylamide (1.8 M in hexanes,
73 .mu.L, 0.132 mmol, 2.4 eq) and TMEDA (41 .mu.L, 0.275 mmol, 6
eq) in THF (2 mL) at -78.degree. C. was added a solution of
compound S11-4-1 (29 mg, 0.065 mmol, 1.1 eq) in THF (400 .mu.L) by
dropwise addition. This resulted in a dark red colored solution.
After 10 min, a solution of enone S7-1 (27 mg, 0.055 mmol, 1 eq) in
THF (400 .mu.L) was added. After complete addition, the reaction
mixture was allowed to warm to -20.degree. C. over 1 h. The
reaction was quenched by the addition of ammonium chloride
(saturated, aqueous solution, 800 .mu.L) and was extracted with
EtOAc (2.times.30 mL). The combined organic extracts were dried
over Na.sub.2SO.sub.4, filtered, and concentrated under reduced
pressure. Purification of the resulting oil via flash column
chromatography on silica gel (Biotage, 10 g, 5 to 40% EtOAc in
hexanes gradient) provided 25 mg of S11-5-1 (55%): .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.51-7.46 (m, 2H), 7.46-7.41 (m, 2H),
7.40-7.29 (m, 6H), 5.35 (s, 2H), 4.90-4.75 (m, 2H), 3.96 (d, J=11.0
Hz, 1H), 3.80-3.42 (m, 2H), 3.26-3.16 (m, 1H), 3.02-2.64 (m, 3H),
2.62-2.40 (m, 10H), 2.14 (d, J=14.0 Hz, 1H), 0.97-0.92 (3H),
0.89-0.77 (m, 10H), 0.27 (s, 3H), 0.12 (s, 3H); MS (ESI) m/z 820.71
(M-H).
Synthesis of Compound 304
##STR00291##
[0665] To a solution of S11-5-1 (25 mg, 0.030 mmol, 1 eq) in
1,4-dioxane (1 mL) was added an aqueous solution of HF (50%, 300
.mu.L). After 15.5 h, the reaction mixture was poured into an
aqueous K.sub.2HPO.sub.4 solution (3.6 g in 30 mL) and extracted
with EtOAc (2.times.30 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4), filtered, and concentrated under reduced
pressure. Palladium on carbon (10%, 16 mg) was added to a solution
of this crude oil in dioxane:MeOH (1:1, 1 mL). The flask was fitted
with a septum and evacuated and back-filled three times with
hydrogen gas. The reaction was stirred under an atmosphere
(balloon) of hydrogen gas for 1 h. The reaction mixture was
filtered through Celite to remove the palladium catalyst and
concentrated under reduced pressure. Preparative reverse phase HPLC
of the resulting oil was performed on a Waters Autopurification
system using a Polymerx 10.mu. RP-.gamma. 100 R column
[30.times.21.20 mm, 10 micron, solvent A: 0.05 N HCl in water,
solvent B: Methanol; injection volume: 1.5 mL (0.05 N HCl in
water); gradient: 30.fwdarw.70% B over 15 min; mass-directed
fraction collection]. Fractions with the desired MW, eluting at
6.0-8.3 min, were collected and freeze-dried to provide 8.4 mg of
the desired compound Compound 304 (45%): .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 4.73-4.62 (m, 1H), 4.41-4.27 (m, 1H), 4.10 (s,
1H), 3.93-3.81 (m, 1H), 3.43-3.24 (m, 1H), 3.24-2.88 (m, 13H),
2.36-2.18 (m, 2H), 1.97-1.83 (m, 2H), 1.70-1.54 (m, 1H), 1.07 (t,
J=7.3 Hz, 3H); MS (ESI) m/z 530.34 (M-H).
[0666] The following compounds of Formula IV were prepared
according to the methods of Compound 304, using the appropriate
N-substituted phenyl
5-(benzyloxy)-8-fluoro-7-methyl-1,2,3,4-tetrahydroisoquinoline-6-carboxyl-
ate intermediate in place of S11-4-1
Example 113. Compound 307
##STR00292##
[0668] Prepared from S11-4-2: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.74-4.62 (m, 1H), 4.37-4.26 (m, 1H), 4.09 (s, 1H),
3.92-3.83 (m, 1H), 3.49-3.34 (m, 4H), 3.23-2.92 (m, 10H), 2.38-2.27
(m, 1H), 2.26-2.18 (m, 1H), 1.72-1.58 (m, 1H), 1.48 (t, J=7.3 Hz,
3H); MS (ESI) m/z 516.31 (M-H).
Example 114. Compound 306
##STR00293##
[0670] Prepared from S11-4-3: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.72-4.61 (m, 1H), 4.40-4.29 (m, 1H), 4.08 (s, 1H),
3.93-3.83 (m, 1H), 3.42-3.30 (m, 1H), 3.24-2.92 (m, 13H), 2.37-2.26
(m, 3H), 1.70-1.58 (m, 1H), 1.10 (t, J=6.7 Hz, 6H); MS (ESI) m/z
544.36 (M-H).
Example 115. Compound 306
##STR00294##
[0672] Prepared from S11-4-4: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.71-4.61 (m, 1H), 4.51-4.40 (m, 1H), 4.09 (s, 1H),
3.91-3.82 (m, 1H), 3.59-3.49 (m, 1H), 3.27-2.92 (m, 12H), 2.38-2.17
(m, 2H), 1.71-1.59 (m, 1H), 1.19 (s, 9H); MS (ESI) m/z 558.35
(M-H).
Example 116. Compound 300
##STR00295##
[0674] Prepared from S11-4-5: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.57-4.39 (m, 2H), 4.09 (s, 1H), 3.88-3.75 (m, 2H),
3.39-3.26 (m, 1H), 3.24-2.92 (m, 11H), 2.37-2.18 (m, 2H), 1.70-1.58
(m, 1H), 1.48 (d, 5.9 Hz, 6H); MS (ESI) m/z 530.32 (M-H).
Example 117. Compound 301
##STR00296##
[0676] Prepared from S11-4-6: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.51-4.41 (m, 2H), 4.09 (s, 1H), 3.84-3.74 (m, 1H),
3.61-3.49 (m, 1H), 3.43-3.39 (m, 1H), 3.24-2.89 (m, 11H), 2.36-2.17
(m, 2H), 2.06-1.92 (m, 1H), 1.83-1.57 (m, 2H), 1.48-1.41 (m, 3H),
1.09 (t, J=7.3 Hz, 3H); MS (ESI) m/z 544.36 (M-H).
Example 118. Compound 305
##STR00297##
[0678] Prepared from S11-4-7: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.56-4.41 (m, 2H), 4.08 (s, 1H), 3.84-3.74 (m, 1H),
3.61-3.50 (m, 1H), 3.43-3.39 (m, 1H), 3.24-2.89 (m, 11H), 2.36-2.17
(m, 2H), 2.04-1.90 (m, 1H), 1.81-1.57 (m, 2H), 1.48-1.40 (m, 3H),
1.09 (t, J=7.3 Hz, 3H); MS (ESI) m/z 544.36 (M-H).
Example 119. Compound 302
##STR00298##
[0680] Prepared from S11-4-8: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.56-4.41 (m, 2H), 4.08 (s, 1H), 3.84-3.74 (m, 1H),
3.61-3.52 (m, 1H), 3.43-3.39 (m, 1H), 3.24-2.92 (m, 11H), 2.36-2.17
(m, 2H), 2.04-1.91 (m, 1H), 1.81-1.54 (m, 2H), 1.48-1.40 (m, 3H),
1.10 (t, J=7.3 Hz, 3H), MS (ESI) m/z 544.43 (M-H).
Example 120. Compound 308
##STR00299##
[0682] Prepared from S11-4-9: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 4.61-4.37 (m, 2H), 4.07-3.99 (m, 2H), 3.27-2.91 (m, 12H),
2.37-2.18 (m, 2H), 1.72-1.49 (m, 10H); MS (ESI) m/z 544.3
(M-H).
Example 121. Compound 400
Synthesis of S12-1
##STR00300##
[0684] To a solution of compound S1-7 (10 g, 22.60 mmol, 1.0 equiv)
in MeOH was added trimethylorthoformate (4.8 g, 45.20 mmol, 2.0
equiv) and TsOH.H.sub.2O (0.13 g, 0.68 mmol, 0.03 equiv) at rt. The
reaction mixture was heated to reflux overnight and concentrated
under reduced pressure. The residue was diluted with H.sub.2O and
extracted with EtOAc. The organic layer was dried over sodium
sulfate and evaporated to dryness. The crude product was purified
by column chromatography on silica gel (petroleum ether:EtOAc from
100:1 to 30:1) to afford compound S12-1 as a light yellow solid (10
g, 91%): .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41-7.45 (m,
2H), 7.25-7.35 (m, 5H), 7.16-7.21 (m, 1H), 6.98 (d, J=8.0 Hz, 2H),
5.71 (s, 1H), 5.04 (s, 2H), 3.46 (s, 6H), 2.29 (d, J=2.4 Hz,
3H).
Synthesis of S12-2
##STR00301##
[0686] To bromide S12-1 (500 mg, 1.02 mmol, 1 eq) in anhydrous
1,4-dioxanl (5 mL) was added benzylamine (0.165 mL, 1.50 mmol, 1.5
eq), cesium carbonate (0.585 g, 1.80 mmol, 1.8 eq), XantPhos (70
mg, 0.12 mmol, 0.12 eq), and Pd.sub.2(dba).sub.3 (20 mg, 0.02 mmol,
0.02 eq). The mixture was sealed, degassed by bubbling dry nitrogen
through for 5 min with gentle stirring, and heated at 160.degree.
C. in a Biotage microwave reactor for 25 min, and cooled to room
temperature. LC/MS analysis indicated complete consumption of the
starting material and the appearance of the desired secondary amine
S12-2 as the major product.
[0687] A total of 2.45 g of bromide S12-1 was processed in 500 mg
batches per the above procedure. The reaction mixtures were
combined, diluted with saturated aqueous sodium bicarbonate (100
mL), and extracted with EtOAc (200 mL.times.1, 50 mL.times.2). The
EtOAc extracts were combined, dried over sodium sulfate, and
concentrated under reduced pressure. Flash column chromatography on
silica gel using 0% to 10% EtOAc/hexane yielded the desired product
S12-2 as an orange oil (1.68 g, 65%): R.sub.f 0.70 (20%
EtOAc/hexane); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.20-7.45
(m, 13H), 7.05 (d. J=8.6 Hz, 2H), 5.55 (s, 1H), 5.24 (br t, J=6.1
Hz, 1H), 5.14 (s, 2H), 4.43 (d, J=6.1 Hz, 2H), 3.37 (s, 6H), 2.26
(s, 3H); MS (ESI) m/z 516.3 (M+H), calcd for
C.sub.31H.sub.31FNO.sub.5 516.2.
Synthesis of S12-3
##STR00302##
[0689] To secondary amine S12-1 (1.47 g, 2.85 mmol, 1 eq) in
anhydrous DMF (6 mL) was added NaH (250 mg, 60% in mineral oil,
6.30 mmol, 2.2 eq). The yellow suspension was stirred at rt for 30
min. NaI (43 mg, 0.28 mmol, 0.1 eq) and benzyl bromide (0.82 mL,
6.90 mmol, 2.4 eq) were added. The reaction (deep-orange) was
stirred at rt for 24 h, diluted with EtOAc (100 mL), washed with
saturated aqueous sodium bicarbonate (100 mL.times.2) and brine (50
mL.times.1), dried over sodium sulfate, and concentrated in under
reduced pressure. Flash column chromatography on silica gel using
0% to 10% EtOAc/hexane yielded the desired tertiary amine S12-3 as
a pale oil (1.16 g, 67%): R.sub.f 0.33 (10% EtOAc/hexane); .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.20-7.40 (m, 18H), 6.99 (d,
J=8.0 Hz, 2H), 5.72 (s, 1H), 4.68 (s, 2H), 4.20-4.40 (br m, 4H),
3.32 (s, 6H), 2.34 (s, 3H), MS (ESI) m/z 606.3 (M+H), calcd for
C.sub.38H.sub.37FNO.sub.5 606.3. The compound was contaminated with
the corresponding benzyl ester (instead of phenyl ester), which was
not removed prior to the next step.
Synthesis of S12-4
##STR00303##
[0691] The diisopropylamine (0.30 mL, 2.12 mmol, 1.1 eq) in
anhydrous THF (10 mL) at -78.degree. C. was added n-BuLi (1.33 mL,
1.6 M/hexane, 2.12 mmol, 1.1 eq) dropwise. The pale solution was
stirred at 0.degree. C. for 30 min and cooled to -78.degree. C.
TMEDA (0.35 mL, 2.33 mmol, 1.2 eq) was added, followed by the
addition of compound S12-3 (1.16 g, 1.92 mmol, 1 eq, in 30 mL THF)
dropwise over a period of 5 min. The deep-red solution was stirred
at -78.degree. C. for 30 min. LHMDS (2.12 mL, 1.0 M/THF, 1.1 eq)
was added, followed by the addition of enone S7-1 (0.96 g, 1.92
mmol, in 10 mL THF) dropwise over a period of 2 min. The resulting
yellow solution was slowly warm up to 0.degree. C. over a period of
3 h, diluted with EtOAc (200 mL) and saturated aqueous sodium
bicarbonate (100 mL). The EtOAc layer was collected. The aqueous
layer was extracted with more EtOAc (50 mL.times.2). The combined
EtOAc solution was dried over sodium sulfate and concentrated in
under reduced pressure. Flash column chromatography on silica gel
using 0% to 15% EtOAc/hexane yielded the desired product as a
yellow solid (0.77 g, 40): R.sub.f 0.50 (20/o EtOAc/hexane);
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 15.82 (s, 1 H), 7.00-7.50
(m, 20H), 5.79 (s, 1H), 5.38 (s, 2H), 5.04 (d, J=10.4 Hz, 1H), 4.50
(d, J=10.4 Hz, 1H), 4.00-4.40 (m, 4H), 3.95 (d, J=10.4 Hz, 1H),
3.35 (s, 3H), 3.20-3.30 (m, 3H), 3.13 (s, 3H), 2.95-3.05 (m, 1H),
2.55-2.65 (m, 1H), 2.50 (s, 6H), 2.15-2.20 (m, 1H), 0.85 (s, 9H),
0.30 (s, 3H), 0.14 (s, 3H); MS (ESI) m/z 994.5 (M+H), calcd for
C.sub.58H.sub.65FN.sub.3O.sub.9Si 994.6.
[0692] 0.52 g of compound S12-3 was also recovered.
Synthesis of S12-5
##STR00304##
[0694] To compound S12-4 (0.77 g, 0.78 mmol, 1 eq) in THF (10 mL)
was added 3 N HCl/water (2 mL, final [HCl]=0.5 M). The deep yellow
solution was stirred at rt for 2 h, diluted with EtOAc (100 mL),
washed with saturated aqueous sodium bicarbonate (100 mL.times.2)
and brine (50 mL.times.1), dried over sodium sulfate, and
concentrated in under reduced pressure to yield the crude product
as a deep-orange solid (0.72 g, 97%): MS (ESI) m/z 948.4 (M+H),
calcd for C.sub.56H.sub.59FN.sub.3O.sub.8Si 948.4.
Synthesis of S2-7-1
##STR00305##
[0696] To aldehyde S12-5 (95 mg, 0.10 mmol, 1 eq) in
1,2-dichloroethane (2 mL) was added glycine benzyl ester (50 mg,
TsOH salt, 0.15 mmol, 1.5 eq), triethylamine (0.022 mL, 0.16 mmol,
1.6 eq), HOAc (0.024 mL, 0.42 mmol, 4 eq), and Na(OAc).sub.3BH (32
mg, 0.15 mmol, 1.5 eq). The deep-red solution became yellow and was
stirred at rt for 1 h. Isobutyraldehyde (0.032 mL, 0.35 mmol, 3.5
eq) and Na(OAc).sub.3BH (82 mg, 0.40 mmol, 4 eq) were added. The
reaction was stirred at rt for 1 h, diluted with EtOAc (20 mL),
washed with saturated aqueous sodium bicarbonate (10 mL.times.1)
and brine (10 mL.times.1), dried over sodium sulfate, and
concentrated in under reduced pressure to yield the crude product
(S12-7-1) as a yellow residue: MS (ESI) m/z 1153.5 (M+H), calcd for
C.sub.69H.sub.78FN.sub.4O.sub.9Si 1153.6.
Synthesis of S12-8-1
##STR00306##
[0698] Crude compound S12-7-1 was dissolved in THF (1.5 mL) and
added with 50% HF/water (0.5 mL). The yellow solution was stirred
at rt for 2 h and added into K.sub.2HPO.sub.4/water (5 g in 20 mL
water) with stirring. The mixture was extracted with EtOAc (20
mL.times.3). The EtOAc extracts were combined, dried over sodium
sulfate, and concentrated in under reduced pressure to yield the
crude product as a yellow residue: MS (ESI) m/z 1039.5 (M+H), calcd
for C.sub.63H.sub.63FN.sub.4O.sub.9 1038.5.
[0699] The above crude product (0.10 mmol, 1 eq) was dissolved in
methanol (3 mL) and 1,4-dioxane (1 mL). 10% Pd--C (21 mg, 0.01
mmol, 0.1 eq) and 0.5 N HCl/methanol (1 mL) were added. The mixture
was purged with hydrogen and stirred under 1 atm hydrogen at rt for
1 h. The catalyst was filtered off with a small Celite pad and
washed with methanol (2 mL.times.3). The yellow methanol solution
was concentrated in under reduced pressure to afford the crude
product, which was purified with HPLC to yield the desired product
S12-8-1) as a yellow solid (26 mg, HCl salt, 37% overall): .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 4.52 (s, 2H), 4.08 (s, 1H), 4.02
(s, 2H), 2.90-3.50 (m, 8H), 2.10-2.30 (m, 3H), 1.55-1.70 (m, 1H),
1.00 (d, J=6.1 Hz, 6H): MS (ESI) m/z 591.4 (M+H), calcd for
C.sub.28H.sub.36FN.sub.4O.sub.9 591.3.
Synthesis of Compound 400
##STR00307##
[0701] The above amino acid S12-8-1 (20 mg, HCl salt, 0.029 mmol, 1
eq) was dissolved in anhydrous DMF (5 mL). DIEA (0.0067 mL, 0.039
mmol, 1.3 eq) and DCC (12 mg, 0.058 mmol, 2 eq) were added. The
reaction was stirred at rt for 24 h. 0.5 N HCl/methanol (0.5 mL)
was added. The reaction mixture was added dropwise into ether (500
mL) with vigorous stirring. The yellow precipitates were collected
onto a small Celite pad, washed with more ether (10 mL.times.3),
and eluted with methanol (10 mL.times.3). The yellow methanol
solution was concentrated in under reduced pressure to afford the
crude product, which was purified by HPLC to yield the desired
product Compound 400 as an orange solid (8 mg, 43%): .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 4.52 (s, 2H), 4.10 (s, 1H), 3.86 (br
s, 2H), 2.90-3.50 (m, 8H), 2.37 (t, J=14.6 Hz, 1H), 2.15-2.30 (m,
2H), 1.60-1.70 (m, 1H), 1.08 (d, J=6.7 Hz, 6H); MS (ESI) m/z 573.5
(M+H), calcd for C.sub.28H.sub.34FN.sub.4O.sub.8 573.2.
[0702] The following compounds were prepared similarly to Compound
400 using the appropriate intermediate S12-6 or S12-7.
Example 122. Compound 426
##STR00308##
[0704] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.43 (s, 2H), 4.10
(s, 1H), 3.80 (s, 2H), 2.90-3.40 (m, 9H), 2.31-2.41 (m, 1H),
2.22-2.30 (m, 1H), 1.60-1.72 (m, 1H); MS (ESI) m/z 517.4 (M+H),
calcd for C.sub.24H.sub.26FN.sub.4O.sub.8 517.2.
Example 123. Compound 416
##STR00309##
[0706] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.53 (br s, 2H),
4.17 (s, 1H), 3.87 (br, s, 2H), 2.90-3.30 (m, 12H), 2.32-2.42 (m,
1H), 2.23-2.30 (m, 1H), 1.60-1.72 (m, 1H); MS (ESI) m/z 531.3
(M+H), calcd for C.sub.25H.sub.28FN.sub.4O.sub.8 531.2.
Example 124. Compound 403
##STR00310##
[0708] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.65 (d, J=14.4
Hz, 1H), 4.05-4.15 (m, 2H), 3.80 (dd, J=4.3, 9.8 Hz, 1H), 2.90-3.30
(m, 9H), 2.32-2.42 (m, 1H), 2.23-2.30 (m, 1H), 2.10-2.20 (m, 1H),
1.60-1.73 (m, 2H), 1.38-1.45 (m, 1H), 0.92 (d, J=6.7 Hz, 3H), 0.87
(d, J=6.7 Hz, 3H); MS (ESI) m/z 573.4 (M+H), calcd for
C.sub.28H.sub.34FN.sub.4O.sub.8 573.2.
Example 125. Compound 411
##STR00311##
[0710] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.22 (br s, 1H),
4.11 (s, 1H), 3.96 (br s, 1H), 2.95-3.45 (m, 12H), 2.35-2.45 (m,
1H), 2.20-2.30 (m, 2H), 1.61-1.72 (m, 1H), 1.52-1.60 (m, 1H),
1.42-1.50 (m, 1H), 0.93 (d, J=6.7 Hz, 3H), 0.85 (d, J=6.7 Hz, 3H);
MS (ESI) m/z 587.5 (M+H), calcd for C.sub.29H.sub.36FN.sub.4O.sub.8
587.2.
Example 126. Compound 419
##STR00312##
[0712] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.66 (d, J=14.0
Hz, 1H), 4.11 (s, 1H), 4.09 (d, J=14.0 Hz, 1H), 3.78 (dd, J=4.3,
9.2 Hz, 1H), 2.85-3.30 (m, 9H), 2.30-2.42 (m, 1H), 2.21-2.30 (m,
1H), 2.10-2.20 (m, 1H), 1.58-1.70 (m, 2H), 1.37-1.46 (m, 1H), 0.91
(d, J=6.7 Hz, 3H), 0.85 (d, J=6.7 Hz, 3H); MS (ESI) m/z 573.3
(M+H), calcd for C.sub.28H.sub.34FN.sub.4O.sub.8 573.2.
Example 127. Compound 428
##STR00313##
[0714] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.20 (br s, 1H),
4.11 (s, 1H), 3.85 (br s, 1H), 2.95-3.30 (m, 12H), 2.35-2.45 (m,
1H), 2.20-2.30 (m, 2H), 1.61-1.72 (m, 1H), 1.52-1.60 (m, 1H),
1.43-1.51 (m, 1H), 0.93 (d, J=6.7 Hz, 3H), 0.85 (d, J=6.7 Hz, 3H);
MS (ESI) m/z 587.3 (M+H), calcd for C.sub.29H.sub.36FN.sub.4O.sub.8
587.2.
Example 128. Compound 410
##STR00314##
[0716] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.58 (d, J=13.6
Hz, 1H), 4.40 (d. J=14.4 Hz, 1H), 4.12 (s, 1H), 3.81 (d, J=9.2 Hz,
1H), 4.41 (d, J=9.2 Hz, 1H), 3.17-2.99 (m, 10H), 2.43-2.35 (m, 1H),
2.29-2.26 (m, 1H), 2.05-1.89 (m, 6H), 1.69-1.65 (m, 1H); MS (ESI)
m/z 571.1 (M+H), calcd for C.sub.28H.sub.32FN.sub.4O.sub.8
571.2.
Example 129. Compound 418
##STR00315##
[0718] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.55 (d, J=14.4
Hz, 1H), 4.41 (d, J=14.4 Hz, 1H), 4.14 (s, 1H), 3.83 (d, J=10.4 Hz,
1H), 4.41 (d, J=10.4 Hz, 1H), 3.13-2.98 (m, 10H), 2.43-2.36 (m,
1H), 2.29-2.26 (m, 1H), 1.99-1.90 (m, 6H), 1.72-1.61 (m, 1H); MS
(ESI) m/z 571.1 (M+H), calcd for C.sub.28H.sub.32FN.sub.4O.sub.8
571.2.
Example 130. Compound 401
##STR00316##
[0720] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.13 (s, 1H), 3.86
(d, J=8.4 Hz, 1H), 3.22-2.99 (m, 13H), 2.41-2.15 (m, 3H), 1.68-1.62
(m, 1H), 1.06 (d, J=6.4 Hz, 3H), 0.99 (d, J=4.4 Hz, 3H); MS (ESI)
m/z 573.0 (M+H), calcd for C.sub.28H.sub.34FN.sub.4O.sub.8
573.2.
Example 131. Compound 402
##STR00317##
[0722] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.58 (s, 2H), 4.12
(s, 1H), 3.21-2.86 (m, 13H), 2.42-2.34 (m, 1H), 2.27-2.18 (m, 1H),
1.74-1.62 (m, 1H), 1.30 (s, 6H); MS (ESI) m/z 559.1 (M+H), calcd
for C.sub.27H.sub.32FN.sub.4O.sub.8 559.2.
Example 132. Compound 422
##STR00318##
[0724] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.64-4.63 (m, 2H),
4.12 (s, 1H), 3.21-2.98 (m, 12H), 2.40-2.33 (m, 1H), 2.28-2.25 (m,
1H), 1.71-1.62 (m, 1H), 1.32-1.29 (m, 4H); MS (ESI) m/z 557.0
(M+H), calcd for C.sub.27H.sub.30FN.sub.4O.sub.8 557.2.
Example 133. Compound 425
##STR00319##
[0726] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.57 (s, 2H), 4.11
(s, 1H), 3.06-2.98 (m, 12H), 2.43-2.25 (m, 3H), 1.84-1.55 (m, 6H),
1.32-1.29 (m, 2H); MS (ESI) m/z 585.1 (M+H), calcd for
C.sub.29H.sub.34FN.sub.4O.sub.8 585.2.
Example 134. Compound 407
##STR00320##
[0728] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.12 (s, 1H), 3.83
(d, J=8.4 Hz, 1H), 3.35-2.84 (m, 14H), 2.40-2.33 (m, 3H), 1.71-1.61
(m, 1H), 1.07-1.06 (d, J=6.4 Hz, 3H), 0.99 (d, J=6.4 Hz, 3H); MS
(ESI) m/z 573.0 (M+H), calcd for C.sub.28H.sub.34FN.sub.4O.sub.8
573.2.
Example 135. Compound 413
##STR00321##
[0730] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.11 (s, 1H), 3.85 (d,
J=10.0 Hz, 1H), 3.24-2.91 (m, 14H), 2.40-2.16 (m, 3H), 1.70-1.56
(m, 2H), 1.07-1.06 (m, 1H), 0.98-0.83 (m, 6H); MS (ESI) m/z 587.1
(M+H), calcd for C.sub.29H.sub.36FN.sub.4O.sub.8 581.2.
Example 136. Compound 424
##STR00322##
[0732] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.26-7.25 (m, 5H),
4.23-4.14 (m, 2H), 4.09 (s, 1H), 3.53 (t, J=10.8 Hz, 1H), 3.14-2.97
(m, 14H), 2.39-2.23 (m, 2H), 1.67-1.60 (m, 1H); MS (ESI) m/z 621.0
(M+H), calcd for C.sub.32H.sub.34FN.sub.4O.sub.8 621.2.
Example 137. Compound 421
##STR00323##
[0734] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.45 (s, 2H), 4.02
(s, 1H), 3.89 (s, 2H), 3.04-2.87 (m, 9H), 2.60-2.52 (m, 1H),
2.31-2.14 (m, 2H), 1.49 (s, 9H); MS (ESI) m/z 573.2 (M+H), calcd
for C.sub.28H.sub.34FN.sub.4O.sub.8 573.2.
Example 138. Compound 415
##STR00324##
[0736] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.11 (s, 1H),
3.36-3.25 (m, 5H), 3.05-2.97 (m, 9H), 2.48-2.36 (m, 1H), 2.27-2.24
(m, 1H), 1.74-1.62 (m, 1H), 1.48 (d, J=6.0 Hz, 3H); MS (ESI) m/n
545.0 (M+H), calcd for C.sub.26H.sub.30FN.sub.4O.sub.8 545.2.
Example 139. Compound 406
##STR00325##
[0738] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.12 (s, 1H),
3.25-2.86 (m, 14H), 2.43-2.25 (m, 2H), 1.71-1.61 (m, 1H), 1.49 (d,
J=6.0 Hz, 3H); MS (ESI) m/z 545.0 (M+H), calcd for
C.sub.26H.sub.30FN.sub.4O.sub.8 545.2.
Example 140. Compound 423
##STR00326##
[0740] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.11 (s, 3H), 3.90
(d, J=7.6 Hz, 1H), 3.25-2.97 (m, 14H), 2.41-2.25 (m, 2H), 1.71-1.61
(m, 1H); MS (ESI) m/z 561.4 (M+H), calcd for
C.sub.26H.sub.30FN.sub.4O.sub.9 561.2.
Example 141. Compound 420
##STR00327##
[0742] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.09 (s, 1H), 3.85
(d, J=9.6 Hz, 1H), 3.19-2.95 (m, 12H), 2.39-2.32 (m, 2H), 2.24-2.19
(m, 1H), 1.69-1.52 (m, 4H), 1.51-1.28 (m, 1H), 1.16-1.14 (m, 2H),
0.97-0.95 (m, 6H); MS (ESI) m/z 587.3 (M+H), calcd for
C.sub.29H.sub.36FN.sub.4O.sub.8 587.2.
Example 142. Compound 409
##STR00328##
[0744] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.26-7.25 (m, 5H),
4.17-4.11 (m, 3H), 3.53 (t, J=10.8 Hz, 1H), 3.15-2.97 (m, 14H),
2.38-2.24 (m, 2H), 1.66-1.63 (m, 1H); MS (ESI) m/z 621.0 (M+H),
calcd for C.sub.32H.sub.34FN.sub.4O.sub.8 621.2.
Example 143. Compound 405
##STR00329##
[0746] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.51 (d, J=12.8
Hz, 1H), 4.20 (d, J=12.8 Hz, 1H), 4.11 (s, 1H), 3.84 (t, J=11.2 Hz,
1H), 3.21-2.81 (m, 11H), 2.37-2.33 (m, 4H), 2.06-2.04 (m, 2H),
1.71-1.64 (m, 1H); MS (ESI) m/z 557.3 (M+H), calcd for
C.sub.27H.sub.30FN.sub.4O.sub.8 557.2.
Example 144. Compound 412
##STR00330##
[0748] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.48-4.46 (m, 1H),
4.18 (d, J=13.6 Hz, 1H), 4.12 (s, 1H), 3.86-3.83 (m, 1H), 3.35-3.29
(m, 2H), 3.24-2.97 (m, 9H), 2.81-2.77 (m, 2H), 2.38-2.24 (m, 3H),
2.12-2.01 (m, 2H), 1.66 (m, 1H); MS (ESI) m/z 557.0 (M+H), calcd
for C.sub.27H.sub.30FN.sub.4O.sub.8 557.2.
Example 145. Compound 404
##STR00331##
[0750] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 5.52, 5.40 (m, 1H
Total), 4.63 (d, J=14.0 Hz, 1H), 4.52 (d, J=14.0 Hz, 1H), 4.10 (s,
1H), 4.06-3.97 (m, 1H), 3.86-3.81 (m, 1H), 3.04-2.96 (m, 10H),
2.60-2.48 (m, 1H), 2.49-2.26 (m, 3H), 1.69-1.59 (m, 1H); MS (ESI)
m/z 575.1 (M+H), calcd for C.sub.27H.sub.29F.sub.2N.sub.4O.sub.8
575.2.
Example 146. Compound 414
##STR00332##
[0752] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.72-4.62 (m, 2H),
4.28-4.17 (m, 1H), 4.12 (s, 1H), 3.75-3.67 (m, 1H), 3.49-3.40 (m,
1H), 3.28-2.94 (m, 10H), 2.42-2.33 (m, 1H), 2.31-2.22 (m, 1H),
2.09-1.99 (m, 1H), 1.71-1.60 (m, 1H), 1.39-1.34 (m, 1H); MS (ESI)
m/z 573.1 (M+H), calcd for C.sub.27H.sub.30F.sub.2N.sub.4O.sub.9
573.2.
Example 147. Compound 417
##STR00333##
[0754] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.72-4.62 (m, 2H),
4.21 (d, J=13.2 Hz, 1H), 4.13 (s, 1H), 3.72 (d, J=13.2 Hz, 1H),
3.49-3.40 (m, 1H), 3.27-2.94 (m, 10H), 2.40-2.22 (m, 2H), 2.10-1.99
(m, 2H), 1.71-1.60 (m, 1H); MS (ESI) m/z 573.0 (M+H), calcd for
C.sub.27H.sub.30F.sub.2N.sub.4O.sub.9 573.2.
Example 148. Compound 427
Synthesis of S13-1
##STR00334##
[0756] To a 250 mL round bottom flask was added compound S1-4
(14.47 g, 56.30 mmol, 1.0 equiv, crude), tetrabutylammonium bromide
(0.90 g, 2.80 mmol, 0.05 equiv), 1,2-dichloroethane (60 mL), and
water (60 mL). The clear bi-layer was cooled in a 20.degree. C.
water bath. Nitric acid (7.2 mL, 70 wt %, 112.60 mmol, 2.0 equiv)
was added. After the addition, the reaction temperature slowly rose
to 26.degree. C. The reaction was stirred at room temperature
overnight (19 hrs). TLC (heptane/EtOAc=9.5/0.5) showed the reaction
was complete. The organic layer was separated, washed with water
(60 mL.times.2) and brine, and dried over anhydrous sodium sulfate.
The solvent was removed to give compound S13-1 as a brown oil,
which solidified on standing (17.71 g, quantitative). The crude
product was used directly for the next step.
Synthesis of S13-2
##STR00335##
[0758] To a 250 mL round bottom flask was added compound S13-1
(17.7 g, 56.30 mmol 1.0 equiv), acetone (177 mL), anhydrous
potassium carbonate (15.6 g, 113.00 mmol, 2.0 equiv), and potassium
iodide (0.47 g, 2.80 mmol, 0.05 equiv). To the stirred suspension
at room temperature was added benzyl bromide (7.03 mL, 59.10 mmol,
1.05 equiv). The suspension was then heated to 56.degree. C. for 4
hrs. TLC (heptane/EtOAc=9/1) showed the reaction was complete. The
solid was removed by filtration and washed with acetone (30 mL).
The filtrated was concentrated to give a paste. The paste was
partitioned between methyl t-butyl ether (MTBE, 120 mL) and water
(80 mL). The organic layer was washed with water (80 mL) and brine,
dried over anhydrous sodium sulfate, and concentrated to give
compound S13-2 as a brown oil (21.09 g, 98%). The crude product was
used directly for the next step.
Synthesis of S13-3
##STR00336##
[0760] To a 1 L round bottom flask was added compound S13-2 (21.08
g, 55.40 mmol, 1.0 equiv) and THF (230 mL). The solution was cooled
in a cold water bath to 10.degree. C. To another 500 mL round
bottom flask containing water (230 mL), sodium hydrosulfite
(Na.sub.2S.sub.2O.sub.4, 56.7 g, 276.80 mmol, 5.0 equiv) was added
slowly with stirring. The aqueous solution of sodium hydrosulfite
was added to the THF solution of compound S13-2. The temperature
quickly rose from 10.degree. C. to 20.4.degree. C. after the
addition. The yellow suspension was stirred while the cold water
bath slowly warmed up to room temperature overnight to give an
orange cloudy solution. The reaction temperature during this period
was between 15.degree. C. to 19.degree. C. TLC (heptane/EtOAc=9/1)
showed the reaction was complete. The orange cloudy solution was
diluted with EtOAc (460 mL). The organic layer was washed with
water (150 mL.times.2) and brine, dried over anhydrous sodium
sulfate, and concentrated under reduced pressure to give the crude
product as a brown oil. The crude product was purified by flash
silica gel column eluted with heptane/EtOAc 9/1 to yield the
desired product 513-3 (15.83 g, 80%, 3 steps).
Synthesis of S13-4
##STR00337##
[0762] To compound S13-3 5.50 g, 16.65 mmol, 1 eq in DMF (30 mL)
was added Boc.sub.2O (8.54 g, 39.13 mmol, 2.5 eq), DIEA (8.18 mL,
46.96 mmol, 3 eq), and DMAP (102 mg, 0.84 mmol, 0.05 eq). The
reaction solution was stirred at rt for overnight, diluted with
ethyl acetate (300 mL), washed with water (500 mL), saturated
aqueous sodium bicarbonate (100 mL) and brine (100 mL), dried over
sodium sulfate, and concentrated under reduced pressure. Flash
column chromatography on silica gel (0%.fwdarw.5% ethyl
acetate/hexanes) yielded the desired product S13-4 as a white solid
(6.12 g, 71%): R.sub.f 0.80 (20% ethyl acetate/hexanes); MS
(electrospray) m/z 574.3 (M+Na), calcd for
C.sub.31H.sub.34FNNaO.sub.7 574.2.
Synthesis of S13-5
##STR00338##
[0764] To diisopropylamine (1.70 mL, 12.00 mmol, 1.2 eq) in THF (10
mL) at -78.degree. C. was added nBuLi (4.80 mL, 2.5 M/hexane, 12.00
mmol, 1.2 eq) dropwise. The reaction was stirred at 0.degree. C.
for 10 min and re-cooled to -78.degree. C. Compound S13-4 (5.52 g,
10.00 mmol, 1 eq) in THF (10 mL) was added dropwise over a period
of 5 min. The resulting deep orange solution was stirred at
-78.degree. C. for 30 min. Anhydrous DMF (0.98 mL, 12.50 mmol, 1.25
eq) was added dropwise. The resulting light yellow solution was
stirred at -78.degree. C. for 30 min. Acetic acid (0.90 mL) was
added at -78.degree. C. The reaction was warmed to rt, diluted with
saturated aqueous sodium bicarbonate (100 mL), and extracted with
ethyl acetate (50 mL.times.3). The organic extracts were combined,
dried over sodium sulfate, and concentrated under reduced pressure.
Flash column chromatography with ethyl acetate/hexanes
(0%.fwdarw.10%) yielded the desired product S13-5 as an orange foam
(2.04 g, 43%): R.sub.f 0.45 (20% ethyl acetate/hexane); MS
(electrospray) m/z 534.3 (M+CH.sub.3OH+Na), calcd for
C.sub.28H.sub.30FNNaO.sub.7 534.2.
Synthesis of S13-6-1
##STR00339##
[0766] To compound S13-5 (1.00 g, 2.08 mmol, 1 eq) in
1,2-dichloroethane (10 mL) was added (R)-(-)-leucinol (0.27 g, 2.30
mmol, 1.1 eq), acetic acid (0.30 mL, 5.24 mmol, 2.5 eq), and sodium
triacetoxyborohydride (0.66 g, 3.11 mmol, 1.5 eq). The reaction
mixture was stirred at rt for 1 h, diluted with ethyl acetate (50
mL), washed with saturated aqueous sodium bicarbonate (50 mL) and
brine (50 mL), dried over sodium sulfate, and concentrated under
reduced pressure to give the crude product as a yellow solid
(quantitative): R.sub.f 0.55 (ethyl acetate); MS (electrospray) m/z
581.1 (M+H), calcd for C.sub.33H.sub.42FN.sub.2O.sub.6 581.3.
Synthesis of S13-7-1
##STR00340##
[0768] To compound S13-6-1 (0.52 g, 0.90 mmol) in acetonitrile (20
mL) was added sodium bicarbonate (0.16 g, 1.95 mmol, 2.2 eq), allyl
bromide (0.15 mL, 1.80 mmol, 2.0 eq), and tetrabutylammonium iodide
(33 mg, 0.09 mmol, 0.1 eq). The reaction mixture was heated at
70.degree. C. for 24 h, cooled to rt, diluted with water (100 mL),
and extracted with ethyl acetate (100 mL.times.1, 50 mL.times.2).
The ethyl acetate extracts were combined, dried over sodium
sulfate, and concentrated under reduced pressure. Flash column
chromatography on silica gel (0%.fwdarw.60% ethyl acetate/hexanes)
yielded the desired product S13-7-1 as a white solid (0.37 g, 66%):
R.sub.f 0.60 (30% ethyl acetate/hexane); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.25-7.35 (m, 8H), 7.06 (d, J=8.6 Hz, 2H),
5.70-5.81 (m, 1H), 5.18 (d, J=17.1 Hz, 1H), 5.10 (d, J=10.4 Hz,
1H), 5.00 (d, J=10.4 Hz, 1H), 4.85 (d, J=10.4 Hz, 1H), 3.45-3.80
(m, 4H), 3.10-3.28 (m, 1H), 2.99 (dd, J=8.0, 14.0 Hz, 1H),
2.80-2.90 (m, 1H), 2.33 (d, J=2.4 Hz, 3H), 1.43 (s, 9H), 1.35-1.60
(m, 2H), 1.05-1.15 (m, 1H), 0.90 (d, J=6.7 Hz, 3H), 0.87 (d, J=6.7
Hz, 3H); MS (electrospray) m/z 621.5 (M+H), calcd for
C.sub.36H.sub.46FN.sub.2O.sub.6 621.3.
Synthesis of S13-8-1
##STR00341##
[0770] To compound S13-7-1 (0.35 g, 0.56 mmol, 1 eq) in methylene
chloride (10 mL) was added triethylamine (0.16 mL, 1.15 mmol, 2
eq), DMAP (14 mg, 0.11 mmol, 0.2 eq), and methanesulfonyl chloride
(65 .mu.L, 0.84 mmol, 1.5 eq). The reaction solution was stirred at
rt for 1 h, diluted with ethyl acetate (100 mL), washed with
saturated aqueous sodium bicarbonate (50 mL.times.2) and brine (50
mL), dried over sodium sulfate, and concentrated under reduced
pressure. Flash column chromatography on silica gel (0%.fwdarw.0.60
(20% ethyl acetate/hexane); .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.97, 7.83 (br s, 1H, combined), 7.20-7.50 (m, 8H), 7.05
(d, J=7.3 Hz, 2H), 5.90-6.08 (m, 1H), 5.19-5.20 (m, 2H), 4.92-5.03
(m, 2H), 3.94-4.02, 3.45-3.75, 3.15-3.30, 3.00-3.10, 2.55-2.80 (m,
7H combined), 2.33 (d, J=1.8 Hz, 3H), 1.30-1.90 (m, 3H), 1.46 (s,
9H), 0.80-0.92 (m, 6H); MS (electrospray) m/z 639.2 (M+H), calcd
for C.sub.36H.sub.45ClFN.sub.2O.sub.5 639.3.
Synthesis of S13-9-1
##STR00342##
[0772] To compound S13-8-1 (0.22 g, 0.34 mmol, 1 eq) in anhydrous
DMF (15 mL) was added tetrabutylammonium iodide (25 mg, 0.068 mmol,
0.2 eq) and sodium hydride (27 mg, 60% in mineral oil, 0.68 mmol, 2
eq). The reaction mixture was stirred at rt for 5 h, diluted with
ethyl acetate (200 mL), washed with saturated aqueous sodium
bicarbonate (200 mL), water (200 mL) and brine (100 mL), dried over
sodium sulfate, and concentrated under reduced pressure. Flash
column chromatography on silica gel (0%.fwdarw.8% ethyl
acetate/hexanes) yielded the desired product S13-9-1 as a colorless
oil (85 mg, 42%): R.sub.f 0.75 (15% ethyl acetate/hexane); .sup.1H
NMR (400 MHz, CDCl.sub.3) mixture of tautomers, complex; MS
(electrospray) m/z 603.5 (M+H), calcd for
C.sub.36H.sub.44FN.sub.2O.sub.5 603.3.
Synthesis of S13-10-1
##STR00343##
[0774] To diisopropylamine (44 .mu.L, 0.31 mmol, 2.2 eq) in
anhydrous THF (1 mL) at -78.degree. C. was added nBuLi (0.20 mL,
1.6 M/hexanes, 0.32 mmol, 2.2 eq) dropwise. The reaction solution
was stirred at 0.degree. C. for 10 min and re-cooled to -78.degree.
C. TMEDA (53 .mu.L, 0.35 mmol, 2.5 eq) was added, followed by
dropwise addition of compound S13-9-1 (85 mg, 0.14 mmol, 1 eq) in
anhydrous THF (2 mL) over a period of 3 min. The resulting deep red
solution was stirred at -78.degree. C. for 30 min. Enone S7-1 (68
mg, 0.14 mmol) in anhydrous THF (2 mL) was added dropwise. The
resulting light brown solution was gradually warmed up with
stirring from -78.degree. C. to -20.degree. C. over a period of 1
h. Acetic acid (0.1 mL) was added. The reaction mixture was diluted
with ethyl acetate, washed with saturated aqueous sodium
bicarbonate (50 mL) and brine (50 mL), dried over sodium sulfate,
and concentrated under reduced pressure. Flash column
chromatography on silica gel (0%.fwdarw.20% ethyl acetate/hexanes)
yielded the desired product S13-10-1 as a yellow oil (103 mg, 74%):
R.sub.f 0.20 (10% ethyl acetate/hexane); .sup.1H NMR (400 MHz,
CDCl.sub.3) mixture of tautomers, complex; MS (electrospray) m/z
991.8 (M+H), calcd for C.sub.56H.sub.72FN.sub.4O.sub.9Si 991.5.
Synthesis of Compound 427
##STR00344##
[0776] To compound S13-10-1 (21 mg, 0.021 mmol) in THF (1 mL) was
added 48% aqueous HF (1 mL). After stirring at rt for overnight,
the yellow reaction solution was slowly added to 25% aqueous
K.sub.2HPO.sub.4 (40 mL) with rapid stirring. The mixture was
extracted with ethyl acetate (20 mL.times.3). The organic extracts
were combined, dried over sodium sulfate, and concentrated under
reduced pressure to give the crude product as a yellow residue: MS
(electrospray) m/z 777.6 (M+H), calcd for
C.sub.56H.sub.72FN.sub.4O.sub.9Si 777.4.
[0777] To the above intermediate in methanol (3 mL) and 1,4-dioxane
(1 mL) was added 0.5 M HCl/methanol (1 mL) and 10% Pd--C (9 mg,
0.004 mmol, 0.2 eq). The mixture was purged with hydrogen and
stirred under 1 atm hydrogen atmosphere at rt for 2 h. The catalyst
was filtered off with a small Celite pad and washed with methanol
(1 mL.times.3). The filtrate was concentrated under reduced
pressure. Preparative HPLC purification yielded the desired product
Compound 427 as a bright yellow solid (4.1 mg, 33% overall):
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.83 (s, 1H), 4.66 (s,
1H), 4.08 (s, 1H), 2.80-3.70 (m, 15H), 2.05-2.30 (m, 1H), 1.70-1.90
(m, 3H), 1.45-1.75 (m, 1H), 0.97-1.10 (m, 9H); MS (electrospray)
m/z 601.5 (M+H), calcd for C.sub.31H.sub.42FN.sub.4O.sub.7
601.3.
Example 149. Compound 408
Synthesis of S13-12-2-1
##STR00345##
[0779] To compound S13-10-1 (80 mg, 0.081 mmol, 1 eq) in methylene
chloride (2 mL) was added N,N-dimethylbarbituric acid (31 mg, 0.25
mmol, 3 eq) and Pd(PPh.sub.3).sub.4 (4.7 mg, 0.004 mmol, 0.05 eq).
The reaction mixture was degassed by bubbling nitrogen through for
2 min and heated at 40.degree. C. with stirring for 24 h. Stirring
was continued at rt for another 24 h. Saturated aqueous sodium
bicarbonate (10 mL) was added. The mixture was extracted with ethyl
acetate (10 mL.times.3). The organic extracts were combined, dried
over sodium sulfate, and concentrated under reduced pressure to
yield the crude product as a yellow solid: MS (electrospray) m/z
951.8 (M+H), calcd for C.sub.53H.sub.68FN.sub.4O.sub.9Si 951.5.
Synthesis of Compound 408
##STR00346##
[0781] Prepared from compound S13-12-1 (0.027 mmol) using similar
procedures for Compound 427 (orange solid, 4.6 mg, 30% overall):
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.56 (d, J=15.9 Hz, 1H),
4.36 (d, J=15.9 Hz, 1H), 4.08 (s, 1H), 3.75 (dd, J=3.6, 15.3 Hz,
1H), 3.60-3.68 (m, 1H), 2.85-3.15 (m, 11H), 2.15-2.25 (m, 1H),
1.50-1.85 (m, 4H), 1.03 (d, J=6.7 Hz, 3H), 1.00 (d, J=6.7 Hz, 3H);
MS (electrospray) m/z 559.5 (M+H), calcd for
C.sub.28H.sub.36FN.sub.4O.sub.7 559.3.
Example 150. Compound 429
##STR00347##
[0783] To compound S13-12-1 (0.054 mmol) in 1,2-dichloroethane (5
mL) was added acetic acid (10 .mu.L, 0.17 mmol, 3 eq), formaldehyde
(8 .mu.L, 36.5% aqueous solution, 0.11 mmol, 2 eq), and sodium
triacetoxyborohydride (27 mg, 0.13 mmol, 2.5 eq). The reaction
mixture was stirred at rt for 4 h. Additional formaldehyde (8
.mu.L, 36.5% aqueous solution, 0.11 mmol, 2 eq) and sodium
triacetoxyborohydride (10 mg, 0.048 mmol, 0.9 eq) were added. The
reaction mixture was stirred at rt for another 20 min. Saturated
aqueous sodium bicarbonate (20 mL) was added. The mixture was
extracted with ethyl acetate (20 mL.times.3). The organic extracts
were combined, dried over sodium sulfate, and concentrated under
reduced pressure to yield the crude product as a yellow solid: MS
(electrospray) m/z 965.4 (M+H), calcd for
C.sub.54H.sub.70FN.sub.4O.sub.9Si 965.5.
[0784] The above intermediate was then deprotected using similar
procedures for Compound 427 to give the desired product Compound
429 as an orange solid (5.6 mg, 15% overall): .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 4.55-5.00 (m, 2H), 4.09 (s, 1H), 3.45-3.85 (m,
4H), 2.85-3.20 (m, 12H), 2.05-2.30 (m, 2H), 1.50-1.85 (m, 3H),
1.00-1.10 (m, 6H); MS (electrospray) m/z 573.5 (M+H), calcd for
C.sub.29H.sub.38FN.sub.4O.sub.7 573.3.
Example 151. Antibacterial Activity
[0785] The antibacterial activities for the compounds of the
invention were studied according to the following protocols.
Minimum Inhibitory Concentration (MIC) Assay
[0786] MICs were determined according to the Clinical and
Laboratory Standards Institute (CLSI) guidances (e.g., CLSI.
Performance standards for antimicrobial susceptibility testing;
nineteenth information supplement. CLSI document M100-S19, CLSI,
940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898, USA,
2009). Briefly, frozen bacterial strains were thawed and
subcultured onto Mueller Hinton Broth (MHB) or other appropriate
media (Streptococcus requires blood and Haemophilus requires hemin
and NAD). Following incubation overnight, the strains were
subcultured onto Mueller Hinton Agar and again incubated overnight.
Colonies were observed for appropriate colony morphology and lack
of contamination. Isolated colonies were selected to prepare a
starting inoculum equivalent to a 0.5 McFarland standard. The
starting inoculum was diluted 1:125 (this is the working inoculum)
using MHB for further use. Test compounds were prepared by dilution
in sterile water to a final concentration of 5.128 mg/mL.
Antibiotics (stored frozen, thawed and used within 3 hours of
thawing) and compounds were further diluted to the desired working
concentrations.
[0787] The assays were run as follows. Fifty .mu.L of MHB was added
to wells 2-12 of a 96-well plate. One hundred .mu.L of
appropriately diluted antibiotics was added to well 1. Fifty .mu.L
of antibiotics was removed from well 1 and added to well 2 and the
contents of well 2 mixed by pipetting up and down five times. Fifty
.mu.L of the mixture in well 2 was removed and added to well 3 and
mixed as above. Serial dilutions were continued in the same manner
through well 12. Fifty .mu.L was removed from well 12 so that all
contained 50 .mu.L. Fifty .mu.L of the working inoculum was then
added to all test wells. A growth control well was prepared by
adding 50 .mu.L of working inoculum and 50 .mu.L of MHB to an empty
well. The plates were then incubated at 37.degree. C. overnight,
removed from the incubator and each well was read on a plate
reading mirror. The lowest concentration (MIC) of test compound
that inhibited the growth of the bacteria was recorded.
Example
TABLE-US-00005 [0788] 1 2 3 4 5 6 7 8 9 10 11 12 [Abt] 32 16 8 4 2
1 0.5 0.25 0.125 0.06 0.03 0.015 Growth - - - - - + + + + + + +
[abt] = antibiotic concentration in the well in .mu.g/ml Growth =
bacterial growth (cloudiness) Interpretation: MIC = 2 .mu.g/mL
Protocol for Determining Inoculum Concentration (Viable Count)
[0789] Fifty 50 .mu.l of the inoculum was pipetted into well 1.
Ninety .mu.l of sterile 0.9% NaCl was pipetted into wells 2-6 of a
96-well microtiter plate. Ten .mu.L from was removed from well 1
and added it to well 2 followed by mixing. Ten .mu.L was removed
from well two and mixed with the contents of well 3 and so on
creating serial dilutions through well 6. Ten .mu.L was removed
from each well and spotted onto an appropriate agar plate. The
plate was placed into an incubator overnight. The colonies in spots
that contain distinct colonies were counted. Viable count was
calculated by multiplying the number of colonies by the dilution
factor.
TABLE-US-00006 Spot from Well 1 2 3 4 5 6 Dilution 10.sup.2
10.sup.3 10.sup.4 10.sup.5 10.sup.6 10.sup.7 Factor
Bacterial Strains
[0790] The following bacterial strains, listed below, were examined
in minimum inhibitory concentration (MIC) assays.
TABLE-US-00007 STRAIN ORGANISM DESIGNATION KEY PROPERTIES
Staphylococcus aureus SA100 ATCC 13709, MSSA, Smith strain
Staphylococcus aureus SA101 ATCC 29213, CLSI quality control
strain, MSSA Staphylococcus aureus SA191 HA-MRSA,
tetracycline-resistant, lung infection model isolate Staphylococcus
aureus SA161 HA-MRSA, tetracycline-resistant, tet(M) Staphylococcus
aureus SA158 Tetracycline-resistant tet(K) aaaureusaureus
Staphylococcus epidermidis SE164 ATCC 12228, CLSI quality control
strain, tetracycline-resistant Enterococcus faecalis EF103 ATCC
29212, tet-I/R, control strain Enterococcus faecalis EF159
Tetracycline-resistant, tet(M) Streptococcus pneumoniae SP106 ATCC
49619, CLSI quality control strain Streptococcus pneumoniae SP160
Tetracycline-resistant, tet(M) Streptococcus pyogenes SP312 2009
clinical isolate, tet(M) Streptococcus pyogenes SP193 S. pyogenes
for efficacy models; tetS; sensitive to sulfonamides Haemophilus
influenzae HI262 Tetracycline-resistant, ampicillin- resistant
Moraxella catarrhalis MC205 ATCC 8176, CLSI quality control strain
Escherichia coli EC107 ATCC 25922, CLSI quality control strain
Escherichia coli EC155 Tetracycline-resistant, tet(A) Enterobacter
cloacae EC108 ATCC 13047, wt Klebsiella pneumoniae KP109 ATCC
13883, wt Klebsiella pneumoniae KP153 Tetracycline-resistant,
tet(A), MDR, ESBL.sup.+ Klebsiella pneumoniae KP457 2009
ESBL.sup.+, CTX-M, OXA Proteus mirabilis PM112 ATCC 35659
Pseudomonas aeruginosa PA111 ATCC 27853, wt, control strain
Pseudomonas aeruginosa PA169 Wt, parent of PA170-173 Pseudomonas
aeruginosa PA173 PA170 .DELTA.mexX; MexXY-(missing a functional
efflux pump) Pseudomonas aeruginosa PA555 ATCC BAA-47, wild type
strain PAO1 Pseudomonas aeruginosa PA556 Multiple-Mex efflux pump
knockout strain Acinetobacter baumannii AB110 ATCC 19606, wt
Acinetobacter baumannii AB250 Cystic fibrosis isolate, MDR
Stenotrophomonas maltophilia SM256 Cystic fibrosis isolate, MDR
Burkholderia cenocepacia BC240 Cystic fibrosis isolate, MDR *MDR,
multidrug-resistant; MRSA, methicillin-resistant S. aureus; MSSA,
methicillin-sensitive S. aureus; HA-MRSA, hospital-associated MRSA;
tet(K), major gram-positive tetracycline efflux mechanism; tet(M),
major gram-positive tetracycline ribosome-protection mechanism;
ESBL.sup.+, extended spectrum .beta.-lactamase
Results
[0791] Values of minimum inhibition concentration (MIC) for the
compounds of the invention are provided in Tables 5-7.
TABLE-US-00008 TABLE 5 MIC Values for Compounds of the Invention
Compared to Sancycline, Minocycline and Tigecycline. A = lower than
or equal to lowest MIC among three control compounds; B = greater
than lowest MIC among three control compounds, but lower than
highest MIC among three control compounds; C = greater than MIC of
all three control compounds. Cmpd SA101 SA100 SA161 SA158 EF103
EF159 SP106 SP160 No. 29213 13709 MRSAtetM tetK 29212 tetM 49619
tetM 100 B B B B B B B B 101 B B B B B B C B 102 C B B B B B B B
104 C C B B B B B B 105 B B C B B B B B 106 B B B B B B B B 107 B B
B B B B B B 108 C C B B B B B B 109 C C B B B B B B 110 B B B B B B
B B 111 C B B B B B B B 117 C C B B B B B B 118 B B B B B B B B 120
B B B B B B B B 121 B B B B B B B B 123 B B B B B B B B 129 B B B B
B B B B 130 B B B B B B B B 131 B B B B B B B B 133 C B B B B B C B
134 B B B B B B B B 136 C B B B B B B B 137 B B B B B B B B 139 B B
B B B B B B 140 B B B B B B B B 142 B B B B B B B B 143 B B B B B B
B B 144 B B B B B B B B 146 C C B B B B B B 147 B B B B B B B B 149
B B B B B B B B 150 B B B B B B B B 200 C C B NT B B B B 201 B B B
B B B C B 202 C B B B B B B B Cmpd EC107 EC155 AB110 PA111 EC108
KP109 KP153 No. 25922 tetA 19608 27853 13047 13883 tetA 100 B B A B
B B B 101 B B C B C B B 102 B B B B B B B 104 B B C B B B B 105 B C
C B B B B 106 B B B B B B B 107 B B B B B B B 108 B B C B C B B 109
B B C B B B B 110 B B A B B B B 111 B B C B B B B 117 B B B B B B B
118 B B A B B B B 120 B B A B B B B 121 B B A B B B B 123 B B A B B
B B 129 B B A B B B B 130 B B A B B B B 131 B B C B B B B 133 B C C
B C B C 134 B B B B B B B 136 B C C B B B B 137 B B B B B B B 139 B
B C B B B B 140 B C C B C B C 142 B B A B B B B 143 B B A B B B B
144 B B A B B B B 146 B B C B B B B 147 B B C B B B B 149 B B A B B
B B 150 B B C B B B B 200 B B C B B B B 201 B B C B B B B 202 B B C
B C B B
TABLE-US-00009 TABLE 6 MIC Values for Compounds of the Invention
Compared to Sancycline, Minocycline and Tigecycline. A = lower than
or equal to lowest MIC among three control compounds; B = greater
than lowest MIC among three control compounds, but lower than
highest MIC among three control compounds; C = greater than MIC of
all three control compounds. Cmpd SA101 SA100 SA161 SA158 SE164
EF159 SP106 SP160 SP193 HI262 No. 29213 13703 tetM tetK 12228 tetM
49619 tetM 8668 33923 112 C B B B A B A B B C 113 C B B B B B A B B
B 114 B B B B B B A B B C 115 B B B B A B A B B B 116 C B B B B B B
B C C 119 B B B B B B A B B C 122 C B B B B B B B C C 125 B B B B A
B A B B A 126 B B B B B B A B B A 128 B B B B B B A B A B 132 B B B
B A B A B A B 138 B B B B B B A B A A 141 C A B B B B A B A A 145 B
B B B A B A B A B 148 B B B B A B A B A B 300 C B B B B B NT B C C
301 C B B B B B B B C C 302 C C C B B B C B C C 303 C B B B B B NT
B C NT 304 C B B B B B B B B C 305 C C B B B B B B C C 306 C B B B
B B C B C C 307 C B B B B B B B B C 308 C B B B B B B B B C 400 B A
B B NT A B B NT NT 403 C B B B NT B B B NT NT 405 B A B B B B A B B
A 406 B A B A B B A B B A 407 C NT B B B B A B NT C 408 B B B B A B
B B B C 409 C NT B B B B A B NT C 411 B B B B NT B B B NT NT 412 A
A B A A B A A A A 413 C NT B B B B A B NT C 415 B B C B NT C B B NT
NT 416 A A B B NT B B B NT NT 419 A A B B NT B B A NT NT 420 C B B
B NT B B B NT NT 421 B A B A A B A A A B 423 C C B B B B B B C A
424 C B B B B B A B B C 426 B B B B NT B B B NT NT 427 B B B B B B
B B B C 428 A A A A NT B B A NT NT 429 B B B B B B B B B C Cmpd
MC205 EC107 EC155 KP153 PM112 No. 8176 25922 tetA tetA 35659 PA169
PA173 AB250 SM256 BC240 112 C B B B B C B A C C 113 C B B B C C B A
C B 114 B B B B B C B A C C 115 C B B B C C B B C C 116 C C B C C C
B B C C 119 C B B B B C B A C C 122 C C C C C C B C C C 125 B B B B
B C NT A B B 126 B B B B B B B A B B 128 C B B B B C B A C B 132 B
B B B B B B A C B 138 B B B B B C B A C B 141 B B B B B C B A C B
145 B B B B B C B A C B 148 C B B B B C B A C B 300 C B B B B B B A
C C 301 B B B B C B B B C C 302 C C B B C B B C C C 303 B C B B C B
B C C C 304 C B B B B B B A C C 305 C B B B C B B C C C 306 B B B B
C B B C C C 307 C B B B B B B A C C 308 C B B B C B B C C C 400 NT
B B B NT NT NT NT NT NT 403 NT B B B NT NT NT NT NT NT 405 B B C C
B C B B C C 406 B B C C B A B C C C 407 C C C C C NT NT C C C 408 C
B B B B B B A C B 409 C C C C C NT NT C C C 411 NT B B C NT NT NT
NT NT NT 412 A B C C B C B A C B 413 C C C C C NT NT C C C 415 NT B
C C NT NT NT NT NT NT 416 NT B C C NT NT NT NT NT NT 419 NT B B B
NT NT NT NT NT NT 420 NT C C C NT NT NT NT NT NT 421 B B B B C B B
A C C 423 C C C C C C B C C C 424 C C C C C C B C C C 426 NT B C C
NT NT NT NT NT NT 427 C B B B B C B A C C 428 NT B B C NT NT NT NT
NT NT 429 B B B B C C B A C B
TABLE-US-00010 TABLE 7 MIC Values for Compounds of the Invention
Compared to Sancycline, Minocycline and Tigecycline. A = lower than
or equal to lowest MIC among three control compounds; B = greater
than lowest MIC among three control compounds, but lower than
highest MIC among three control compounds; C = greater than MIC of
all three control compounds. Cmpd SA101 SA161 SA158 SE164 EF159
SP106 SP160 SP312 HI262 MC205 No. 29213 SA191 tetM tetK 12228 tetM
49619 tetM tetM 33929 8176 103 C B B B A B A B B C C 124 C B B B A
B A B B B C 127 C B B B A B A B B B C 135 B B B B A B A B B A B 401
C B B B B B B B B C C 402 C B C B B B B B B C C 404 C B C B B B A B
B A C 410 B A B A A A A B B A B 414 C C C B C C C C C C C 417 C C C
B B B C B B A C 418 C C C B B C B B B C C 422 B B B A A B A B B B B
425 C B B B B B B B B C C Cmpd EC107 EC155 KP153 PM112 No. 25922
tetA tetA KP457 35659 PA555 PA556 AB250 SM256 BC240 103 B B B NT C
C C B C C 124 B B B NT C C C A C C 127 B B B NT C C C A C C 135 B B
B NT B C B A C C 401 C C C C C C C C C C 402 C B B C C B C C C C
404 B C C C B C C C C C 410 B B B C C C B C C C 414 C C C C C C C C
C C 417 C C C C C C C C C C 418 C C C C C C C C C C 422 B C C C C C
C B C C 425 C C C C C C C C C C
Example 152. In Vivo Models
A. Mouse Systemic Infection Protocol
[0792] Compounds were screened for antibacterial activity in vivo
in a mouse systemic infection (septicemia) model. In the model,
CD-1 female mice (18-22 grams) were injected IP with a S. aureus
Smith inoculum that results in 0%, survival within 24 to 48 hours.
The bacterial dose required to achieve this effect was previously
been established through virulence studies. At one hour post
infection, mice received either 3 mg/ml IV or 30 mg/ml PO.
Typically, six mice were treated per dose group. Animal survival
was assessed and recorded for 48 hours. Percent survival at 48
hours was recorded for each compound in Table 8.
TABLE-US-00011 TABLE 8 Percent survival at 48 hours for tested
compounds. Cmpd No. IV (3 mg/kg) PO (30 mg/kg) 102 100% 83% 143 83%
100% 130 33% 83% 123 33% 67% 132 50% 50% 106 17% 20% 137 33% 33%
131 100% 17% 147 83% 0% 118 17% 50% 129 50% 0% 150 0 0 144 50% 0%
110 33% 50% 149 17% 0% 125 100% 33% 119 83% 75% 112 100% 20% 126
83% 100% 128 17% 0% 115 100% 100% 103 83% 100% 135 100% 100% 304 0%
17% 410 100% 50% 419 100% 40% 416 100% 20% 400 100% 0% 428 50% 0%
412 100% 40% 406 100% 40% 408 100% 0%
B. Neutropenic Respiratory Infection Models for S. pneumoniae
[0793] Compounds were tested in a neutropenic BALB/c murine model
of lung infection challenged with tetracycline-resistant tet(M) S.
pneumoniae strain SP160. Mice were made neutropenic by
pre-treatment with cyclophosphamide and infected with SP160 via
intranasal administration. Mice were dosed orally with 30 mg/kg
compound or IV with 10 mg/kg compound at 2 and 12 hours
post-infection. At 24 hours following initiation of treatment, mice
were euthanized and bacterial reduction in the lung was quantified
by plating lung homogenates. Data was recorded as log.sub.10
reduction in lung colony forming units versus an untreated control
group. The results of the testing are shown in FIG. 1.
[0794] FIG. 1 shows that Compounds 102 and 135 were as orally
efficacious (reduced the bacterial burden in the lung) as linezolid
in the S. pneumoniae SP160 lung model; and Compounds 143, 130, and
126 did not significantly reduce the lung bacterial burden when
orally administered. Compound 102 was also efficacious when
administered intravenously (IV); linezolid did not substantially
reduce the lung bacterial burden when administered as a control at
5 mg/kg IV. Doxycycline was ineffective, as S. pneumoniae SP160 is
tetracycline-resistant, carrying a tet(M) ribosomal protection
mechanism.
C. Non-Neutropenic Respiratory Infection Model for S.
pneumoniae
[0795] Compound 102 was tested in an immunocompetent CD-1 murine
model of lung infection challenged with S. pneumoniae strain SP514.
Mice were infected with SP514 via intranasal administration and
dosed orally with 30 mg/kg compound at 5, 24 and 36 hours
post-infection. At 48 hours following initiation of treatment, mice
were euthanized and bacterial reduction in the lung was quantified
by plating lung homogenates. Data was recorded as log.sub.10
reduction in lung colony forming units versus an untreated control
group.
[0796] In this model, orally dosed Compound 102 produced a
6.14+/-0.66 log.sub.10 reduction in CFU versus the 48 hour
untreated control (FIG. 2). Linezolid as a comparator produced a
3.56.+-.0.63 log.sub.10 reduction (FIG. 2).
D. Neutropenic Respiratory Infection Model for MRSA
[0797] Compounds were tested in a neutropenic BALB/c murine model
of lung infection challenged with a tetracycline-resistant tet(M)
MRSA strain SA191 infected via intranasal administration. At 2 and
12 hours mice were either dosed orally with 50 mg/kg compound or
via IV administration, at 10 mg/kg. At 24 hours following
initiation of treatment, mice were euthanized and bacterial
reduction in the lung was quantified by plating lung homogenates.
Data was recorded as log.sub.10 reduction in lung colony forming
units versus an untreated control group. The results of the testing
are shown in FIG. 3.
[0798] FIG. 3 shows that Compounds 102, 143 and 130 were as orally
efficacious (reduced the bacterial burden in the lung) as linezolid
in the MRSA SA191 lung model. Compound 102 was more efficacious
when administered intravenously (IV) than linezolid was.
Tetracycline was ineffective as the MRSA strain SA191 is
tetracycline-resistant, carrying a tet(M) ribosomal protection
mechanism.
E. Respiratory Infection Model for H. influenzae
[0799] Compound 102 was tested in a rat lung infection challenged
with H. influenzae via intratracheal administration. At 5, 24, and
48 hours rats were dosed orally with 100 mg/kg compound and
azithromycin was dosed at 50 mg/kg. For IV administration, Compound
102 was dosed at 25 mg/kg at 5, 24 and 48 hours. At 72 hours
following initiation of treatment, rats were euthanized and
bacterial reduction in the lung was quantified by plating lung
homogenates. Data was recorded as log.sub.10 reduction in lung
colony forming units versus an untreated control group. In this
model, orally administered Compound 102 produced a 2.93.+-.0.27
log.sub.10 reduction in CFU versus the 72 hour untreated control
(FIG. 4). Azithromycin, dosed orally, produced 6.24.+-.0.03
reduction. Dosed via the IV route, Compound 102 produced a
3.40.+-.0.31 log.sub.10 reduction in CFU versus the 72 hour
untreated control.
F. In Vitro Activity of Compound 102 for Selected Gram-Negative and
Gram-Positive Pathogens
[0800] The in vitro activity (by broth microdilution MIC) of
Compound 102 against clinically important species of Gram-positive
and Gram-negative pathogens was studied. As part of this study, the
minimum bactericidal concentration (MBC) was also determined
against a subset of the evaluated isolates to determine mode of
action.
Methods
[0801] All isolates were non-duplicate, non-consecutive, clinically
significant isolates and were tested by broth microdilution in
accordance with CLSI M7-A8 (See Clinical and Laboratory Standards
Institute. Methods for dilution antimicrobial susceptibility tests
for bacteria that grow aerobically; approved standard--8.sup.th ed.
CLSI document M7-A8. CLSI, Wayne, Pa. January 2009, the entire
teachings of which are incorporated herein by reference).
[0802] Quality control and interpretations of results were
performed according with CLIS M100-S20, where available (See
Clinical and Laboratory Standards Institute. Performance standards
for antimicrobial susceptibility testing; twentieth informational
supplement. CLSI document M100-S20. CLSI, Wayne, Pa. January 2010,
the entire teachings of which are incorporated herein by
reference).
[0803] A subset of isolates were concurrently tested for MBC in
accordance with CLSI M26-A (See Clinical and Laboratory Standards
Institute. Methods for Determining Bactericidal Activity of
Antimicrobial Agents, Approved Guideline. NCCLS document M26-A
[ISBN 1-56238-384-1]. NCCLS, 940 West Valley Road, Suite 1400,
Wayne, Pa. 19087 USA, 1999.) MBCs were evaluated based on
quantitation of the growth in wells beyond the MIC to determine the
well where a 3-log reduction in CFU relative to the initial
inoculum was observed.
[0804] Results for all MIC testing were within the acceptable
standards based on the CLSI recommended QC ranges for each
comparator agent evaluated and the appropriate ATCC control strains
with the exception of colistin which tested one dilution lower than
the provisional QC breakpoints established by CLSI for E. coli ATCC
25922 and P. aeruginosa ATCC 27853.
Summary of Results
[0805] The data is presented in Tables 9-11. Table 9 is the
antimicrobial susceptibility of all agents tested against all
Gram-negative and Gram-positive organisms. Table 10 is the activity
profile of Compound 102, tigecycline, and tetracycline by
tetracycline resistance phenotype. Table 11 is a summary of MIC and
MBC results for Compound 102 against selected strains.
TABLE-US-00012 TABLE 9 Antimicrobial susceptibility of all agents
tested against all Gram-negative and Gram-positive organisms MIC
(.mu.g/ml) Total Organism Agent n MIC.sub.50 MIC.sub.90 Escherichia
coli.sup.a Compound 102 40 2 4 Tigecycline.sup.b 0.5 2 Tetracycline
>8 >8 Ceftazidime 64 >64 Ceftazidime/clavulanate 4 32
Colistin 0.25 0.5 Ertapenem .ltoreq.1 .ltoreq.1 Gentamicin 2 >8
Levofloxacin .ltoreq.0.25 >4 Piperacillin/Tazobactam 8 >64
Klebsiella pneumonia.sup.a Compound 102 27 4 16 Tigecycline.sup.b 2
4 Tetracycline 8 >8 Ceftazidime >64 >64
Ceftazidime/clavulanate 16 >32 Colistin 0.25 0.5 Ertapenem
.ltoreq.1 8 Gentamicin >8 >8 Levofloxacin 1 >4
Piperacillin/Tazobactam >64 >64 Kiebsielia oxytoca Compound
102 30 1 4 Tigecycline.sup.b 0.5 2 Tetracycline 0.5 4 Ceftazidime
.ltoreq.0.5 .ltoreq.0.5 Ceftazidime/clavulanate .ltoreq.0.25 0.5
Colistin .ltoreq.0.12 0.25 Ertapenem .ltoreq.1 .ltoreq.1 Gentamicin
0.5 2 Levofloxacin .ltoreq.0.25 4 Piperacillin/Tazobactam 2 8
Proteus vulgaris Compound 102 29 8 >16 Tigecycline.sup.b 2 4
Tetracycline 8 >8 Ceftazidime .ltoreq.0.5 >64
Ceftazidime/clavulanate .ltoreq.0.25 .ltoreq.0.25 Colistin >2
>2 Ertapenem .ltoreq.1 .ltoreq.1 Gentamicin 1 >8 Levofloxacin
.ltoreq.0.25 1 Piperacillin/Tazobactam .ltoreq.0.5 2 Enterobacter
Compound 102 30 2 2 aerogenes Tigecycline.sup.b 0.5 0.5
Tetracycline 1 2 Ceftazidime .ltoreq.0.5 16 Ceftazidime/clavulanate
.ltoreq.0.25 16 Colistin .ltoreq.0.12 .ltoreq.0.12 Ertapenem
.ltoreq.1 .ltoreq.1 Gentamicin .ltoreq.0.25 0.5 Levofloxacin
.ltoreq.0.25 .ltoreq.0.25 Piperacillin/Tazobactam 2 16 Enterobacter
cloacae Compound 102 29 4 8 Tigecycline.sup.b 1 4 Tetracycline 4
>8 Ceftazidime >64 >64 Ceftazidime/clavulanate >32
>32 Colistin .ltoreq.0.12 >2 Ertapenem .ltoreq.1 >8
Gentamicin 0.5 >8 Levofloxacin 1 >4 Piperacillin/Tazobactam
>64 >64 Serratia marcescens Compound 102 30 4 8
Tigecycline.sup.b 1 2 Tetracycline >8 >8 Ceftazidime
.ltoreq.0.5 .ltoreq.0.5 Ceftazidime/clavulanate .ltoreq.0.25 0.5
Colistin >2 >2 Ertapenem .ltoreq.1 .ltoreq.1 Gentamicin 0.5 1
Levofloxacin .ltoreq.0.25 2 Piperacillin/Tazobactam 2 8 Morganella
morganii Compound 102 30 8 16 Tigecycline.sup.b 2 4 Tetracycline 2
>8 Ceftazidime .ltoreq.0.5 4 Ceftazidime/clavulanate 4 16
Colistin >2 >2 Ertapenem .ltoreq.1 .ltoreq.1 Gentamicin 1
>8 Levofloxacin .ltoreq.0.25 4 Piperacillin/Tazobactam
.ltoreq.0.5 1 Salmonella species Compound 102 30 2 2
Tigecycline.sup.b 0.25 0.5 Tetracycline 1 >8 Ceftazidime
.ltoreq.0.5 .ltoreq.0.5 Ceftazidime/clavulanate .ltoreq.0.25
.ltoreq.0.25 Colistin .ltoreq.0.12 0.5 Ertapenem .ltoreq.1
.ltoreq.1 Gentamicin 0.5 1 Levofloxacin .ltoreq.0.25 .ltoreq.0.25
Piperacillin/Tazobactam 2 4 Shigella species Compound 102 30 0.5 2
Tigecycline.sup.b 0.25 0.5 Tetracycline >8 >8 Ceftazidime
.ltoreq.0.5 .ltoreq.0.5 Ceftazidime/clavulanate .ltoreq.0.25
.ltoreq.0.25 Colistin .ltoreq.0.12 .ltoreq.0.12 Ertapenem .ltoreq.1
.ltoreq.1 Gentamicin 1 1 Levofloxacin .ltoreq.0.25 0.5
Piperacillin/Tazobactam 2 2 Acinetobacter lwoffii Compound 102 29
0.12 0.5 Tigecycline 0.12 0.5 Tetracycline 0.5 4 Ceftazidime 1 16
Ceftazidime/clavulanate .ltoreq.0.25 4 Colistin .ltoreq.0.12 >2
Ertapenem .ltoreq.1 4 Gentamicin .ltoreq.0.25 1 Levofloxacin
.ltoreq.0.25 .ltoreq.0.25 Piperacillin/Tazobactam .ltoreq.0.5 8
Stenotrophomonas Compound 102 29 0.5 2 maltophilia Tigecycline 0.5
2 Tetracycline 8 >8 Ceftazidime 8 >64 Ceftazidime/clavulanate
32 >32 Colistin 0.25 >2 Ertapenem >8 >8 Gentamicin
>8 >8 Levofloxacin 0.5 >4 Piperacillin/Tazobactam 32
>64 Staphylococcus aureus Compound 102 30 0.25 0.25 (MRSA PVL+)
Tigecycline.sup.b 0.12 0.12 Tetracycline 0.25 0.25 Clindamycin 0.06
0.12 Daptomycin 0.5 1 Ertapenem 4 8 Erythromycin >4 >4
Gentamicin 0.25 0.5 Levofloxacin 0.25 >2 Linezolid 1 2
Vancomycin 1 1 Staphylococcus aureus Compound 102 105 0.5 2
MRSA.sup.c Tigecycline 0.13 0.25 Tetracycline 0.25 >32
Levofloxacin >2 >2 Linezolid 2 4 Vancomycin 1 1 Streptococcus
Compound 102 20 0.06 0.25 anginosus Tigecycline.sup.b 0.03 0.06
Tetracycline 0.12 >4 Clindamycin .ltoreq.0.015 0.03 Daptomycin
0.25 0.25 Ertapenem 0.12 0.25 Erythromycin 0.03 >0.5
Levofloxacin 0.5 0.5 Linezolid 0.5 1 Penicillin .ltoreq.0.12
.ltoreq.0.12 Vancomycin 0.5 0.5 Streptococcus Compound 102 30 0.12
0.12 intermedius Tigecycline.sup.b 0.03 0.12 Tetracycline 0.25
>4 Clindamycin .ltoreq.0.015 0.06 Daptomycin 0.5 1 Ertapenem
0.06 0.5 Erythromycin 0.06 >0.5 Levofloxacin 1 2 Linezolid 1 1
Penicillin .ltoreq.0.12 0.25 Vancomycin 0.5 0.5 Streptococcus mitis
Compound 102 29 0.12 0.25 Tigecycline.sup.b 0.03 0.12 Tetracycline
0.5 >4 Clindamycin 0.03 0.06 Daptomycin 0.5 1 Ertapenem 0.25
>1 Erythromycin >0.5 >0.5 Levofloxacin 1 2 Linezolid 1 1
Penicillin 0.25 2 Vancomycin 0.5 0.5 Streptococcus sanguis Compound
102 18 0.06 0.12 Tigecycline.sup.b 0.03 0.06 Tetracycline 0.25
>4 Clindamycin 0.03 0.06 Daptomycin 0.5 1 Ertapenem 0.12 0.5
Erythromycin 0.03 >0.5 Levofloxacin 0.5 2 Linezolid 0.5 1
Penicillin .ltoreq.0.12 .ltoreq.0.12 Vancomycin 0.5 1 .sup.a37 E.
coli and 24 K. pneumoniae genetically characterized for
beta-lactamase production were tested in a separate laboratory on
the same study panels .sup.bFDA breakpoints for Enterobacteriaceae
were applied: .ltoreq.2 .mu.g/ml (S), 4 .mu.g/ml (I), .gtoreq.8
.mu.g/ml (R); for S. aureus: .ltoreq.0.5 .mu.g/ml (S); for
Streptococcus spp. (other than S. pneumoniae: .ltoreq.0.25 .mu.g/ml
(S) .sup.cStaphylococcus aureus MRSA includes the data from the
Staphylococcus aureus (MRSA PVL+) group.
TABLE-US-00013 TABLE 10 Activity profile of Compound 102,
tigecycline, and tetracycline by tetracycline resistance phenotype
Organism Agent Phenotype Total_n MIC.sub.50 MIC.sub.90
Enterobacteriaceae Compound TET S 168 2 8 102 Compound TET NS 137 4
16 102 Tigecycline TET S 168 1 2 Tigecycline TET NS 137 1 4
Tetracycline TET S 168 1 4 Tetracycline TET NS 137 >8 >8 S.
maltophila Compound TET S 10 0.5 0.5 102 Compound TET NS 19 1 4 102
Tigecycline TET S 10 0.25 0.5 Tigecycline TET NS 19 0.5 2
Tetracycline TET S 10 4 4 Tetracycline TET NS 19 >8 >8
Viridans group Compound TET S 65 0.06 0.12 streptococci 102
Compound TET NS 32 0.12 0.25 102 Tigecycline TET S 65 0.03 0.06
Tigecycline TET NS 32 0.03 0.12 Tetracycline TET S 65 0.25 0.5
Tetracycline TET NS 32 >4 >4 TET S = Tetracycline
susceptible; TET NS = Tetracycline non-susceptible
TABLE-US-00014 TABLE 11 Summary of MIC and MBC results Study
Compound 102 Organism Isolate ID MIC MBC MBC:MIC Acinetobacter
lwoffii 2919857 0.12 0.5 4 Acinetobacter lwoffii 2919860 0.12 16
128 Acinetobacter lwoffii 2919873 0.25 0.25 1 Acinetobacter lwoffii
2919875 0.06 0.25 4 Enterobacter aerogenes 2919897 2 4 2
Enterobacter aerogenes 2919900 1 8 8 Enterobacter aerogenes 2919909
2 8 4 Enterobacter aerogenes 2919913 1 16 16 Enterobacter cloacae
2920072 8 16 2 Enierobacter cloacae 2920082 2 8 4 Enterobacter
cloacae 2920119 2 2 1 Klebsiella oxytoca 2919956 1 8 8 Klebsiella
oxytoca 2919964 1 4 4 Klebsiella oxytoca 2919972 2 4 2 Klebsiella
oxytoca 2919983 1 8 8 Morganella morganii 2919931 4 >16 >4
Morganella morganii 2919935 4 >16 >4 Morganella morganii
2919945 8 >16 >2 Proteus vulgaris 2919822 16 >16 >1
Proteus vulgaris 2919827 8 >16 >2 Proteus vulgaris 2919835 4
>16 >4 Proteus vulgaris 2919836 2 8 4 Salmonella species
2919986 2 8 4 Salmonella species 2919990 2 8 4 Salmonella species
2920006 2 8 4 Salmonella species 2920008 2 8 4 Serratia marcescens
2920092 4 16 4 Serratia marcescens 2920094 4 >16 >4 Serratia
marcescens 2920100 8 16 2 Serratia marcescens 2920109 4 16 4
Shigella species 2919892 1 4 4 Shigella species 2919894 0.25 4 16
Shigella species 2920018 0.25 0.5 2 Shigella species 2920026 0.5 16
32 Shigella species 2920028 0.5 4 8 Staphylococcus aureus 2919648
0.25 >4 >16 Staphylococcus aureus 2919649 0.25 >4 >16
Staphylococcus aureus 2919650 0.25 >4 >16 Stenotrophomonas
maltophilia 2920035 2 >16 >8 Stenotrophomonas maltophilia
2920051 2 16 8 Streptococcus anginosus 2919722 0.25 2 8
Streptococcus anginosus 2919742 0.12 2 16 Streptococcus anginosus
2919797 0.25 2 8 Streptococcus intermedius 2919756 0.06 1 16
Streptococcus intermedius 2919759 0.03 2 64 Streptococcus
intermedius 2919784 0.12 2 16 Streptococcus intermedius 2919819
0.25 1 4 Streptococcus mitis 2919763 0.12 0.5 4 Streptococcus mitis
2919781 0.12 0.12 1 Streptococcus mitis 2919798 0.06 0.06 1
Streptococcus mitis 2919803 0.12 1 8 Streptococcus sanguis 2919749
0.06 0.25 4 Streptococcus sanguis 2919752 0.25 0.5 2 Streptococcus
sanguis 2919758 0.12 2 16 Escherichia coli 2921525 1 >16 >16
Escherichia coli 2921576 2 16 8 Klebsiella pneumonia 2921528 2
>16 >8 Klebsiella pneumoniae 2921529 2 >16 >8
[0806] Against the evaluated Gram-negative and Gram-positive
pathogens, Compound 102 MICs were generally 2-4 fold higher than
those of tigecycline.
[0807] Compound 102 had comparable MICs relative to tetracycline
against the evaluated S. aureus and Enterobacteriaceae excluding
Shigella spp. where Compound 102 was more potent. Compound 102 also
had 2-4 fold lower MICs than tetracycline against Acinetobacter
lwoffli, S. maltophila, and streptococci.
[0808] Compound 102 was more potent by MIC.sub.50/MIC.sub.90
against Gram-positive pathogens relative to Gram-negative
pathogens.
[0809] Compound 102 and tigecycline MICs were not notably altered
against evaluated tetracycline resistant isolates relative to
tetracycline susceptible isolates, and Compound 102 maintained
potency against tetracycline resistant Shigella spp., S.
maltophila, and streptococci.
[0810] MBC:MIC ratios for Compound 102 indicated bacteriostatic
mode of action (ratio >2 for 89.3% of evaluated isolates).
G. In Vitro Activity of Compound 102 for Selected Respiratory
Pathogens
[0811] The in vitro activity (by broth microdilution MIC) of
Compound 102 against clinically important Gram-positive and
Gram-negative species that cause respiratory tract or acute
bacterial skin and skin structure infections was studied.
Methods
[0812] All isolates were non-duplicate, non-consecutive, clinically
significant isolates and were tested by broth microdilution in
accordance with CLSI M7-A8 (See Clinical and Laboratory Standards
Institute. Methods for dilution antimicrobial susceptibility tests
for bacteria that grow aerobically; approved standard--8.sup.th ed.
CLSI document M7-A8. CLSI, Wayne, Pa. January 2009, the entire
teachings of which are incorporated herein by reference); CLSI
M45-A (See Clinical and Laboratory Standards Institute. Methods for
antimicrobial dilution and disk susceptibility testing of
infrequently isolated or fastidious bacteria; approved guideline.
CLSI document M45-A. CLSI, Wayne, Pa. May 2006, the entire
teachings of which are incorporated herein by reference.
[0813] Quality control and interpretations of results were
performed according with CLSI M100-S20, where available. (See
Clinical and Laboratory Standards Institute. Performance standards
for antimicrobial susceptibility testing; twentieth informational
supplement. CLSI document M100-S20. CLSI, Wayne, Pa. January 2010,
the entire teachings of which are incorporated herein by
reference).
[0814] Results for all MIC testing were within the acceptable
standards based on the CLSI recommended QC ranges for each
comparator agent evaluated and the appropriate ATCC control strains
on each day of testing.
Summary of Results
[0815] The activity profiles are presented in Tables 12-14. Table
12 is the activity profile of Compound 102 and other comparator
agents against evaluated Gram-positive pathogens. Table 13 is the
activity profile of Compound 102 and other comparator agents
against evaluated Gram-negative pathogens. Table 14 is the activity
profile of Compound 102 and other comparator agents against
evaluated pathogen by tetracycline phenotype.
TABLE-US-00015 TABLE 12 Activity profile of Compound 102 and other
comparator agents against evaluated Gram-positive pathogens MIC
(mg/mL) Organism Phenotype Drug MIC.sub.50 MIC.sub.90 S. aureus
MSSA (n = 50).sup.1 Compound 102 0.25 0.5 (n = 50)
Tigecycline.sup.3 0.12 0.25 Tetracycline 0.25 0.5 Azithromycin 2
>4 Ceftriaxone 4 4 Clindamycin 0.12 0.12 Gentamicin 0.25 0.5
Imipenem .ltoreq.0.25 .ltoreq.0.25 Levofloxacin 0.25 1 Linezolid 2
4 Vancomycin 1 1 CoNS (n = 52) MSCoNS (n = 26).sup.1 Compound 102
0.25 1 Tigecycline 0.06 0.25 Tetracycline 0.5 2 Azithromycin 0.25
>4 Ceftriaxone 1 2 Clindamycin 0.06 0.06 Gentamicin 0.12 0.25
Imipenem .ltoreq.0.25 .ltoreq.0.25 Levofloxacin 0.25 >4
Linezolid 1 1 Vancomycin 2 2 MRCoNS (n = 26).sup.1 Compound 102
0.25 1 Tigecycline 0.06 0.12 Tetracycline 0.25 2 Azithromycin >4
>4 Ceftriaxone 16 >64 Clindamycin 0.12 >2 Gentamicin 0.25
>8 Imipenem 1 >8 Levofloxacin >4 >4 Linezolid
.ltoreq.0.5 1 Vancomycin 1 2 S. saprophyticus Compound 102 0.25 0.5
(n = 36) Tigecycline 0.12 0.25 Tetracycline 0.5 0.5 Azithromycin 1
>4 Ceftriaxone 8 16 Clindamycin 0.06 0.12 Gentamicin
.ltoreq.0.06 .ltoreq.0.06 Imipenem .ltoreq.0.25 .ltoreq.0.25
Levofloxacin 0.5 0.5 Linezolid 2 4 Vancomycin 1 1 S. pneumoniae PEN
S (n = 39).sup.2 Compound 102 0.06 0.12 (n = 100) Tigecycline 0.06
0.06 Tetracycline 0.12 0.5 Azithromycin 0.12 >4 Ceftriaxone
.ltoreq.0.03 0.06 Clindamycin 0.06 0.06 Imipenem .ltoreq.0.015
.ltoreq.0.015 Levofloxacin 0.5 1 Linezolid 1 1 Penicillin (oral)
.ltoreq.0.12 .ltoreq.0.12 Vancomycin 0.5 0.5 PEN I (n = 11).sup.2
Compound 102 0.12 0.25 Tigecycline 0.06 0.06 Tetracycline 8 32
Azithromycin >4 >4 Ceftriaxone 0.12 0.5 Clindamycin 0.06
>0.5 Imipenem 0.03 0.25 Levofloxacin 0.5 1 Linezolid 1 1
Penicillin (oral) 0.25 1 Vancomycin 0.5 0.5 PEN R (n = 50).sup.2
Compound 102 0.06 0.12 Tigecycline 0.06 0.06 Tetracycline 16 16
Azithromycin >4 >4 Ceftriaxone 1 2 Clindamycin >0.5
>0.5 Imipenem 0.5 0.5 Levofloxacin 0.5 1 Linezolid 1 1
Penicillin (oral) 2 >2 Vancomycin 0.25 0.5 S. pyogenes Compound
102 0.12 0.25 (n = 50) Tigecycline 0.06 0.06 Tetracycline 0.25 32
Azithromycin 0.12 >4 Ceftriaxone .ltoreq.0.03 .ltoreq.0.03
Clindamycin 0.06 0.06 Imipenem .ltoreq.0.015 .ltoreq.0.015
Levofloxacin 0.5 0.5 Linezolid 1 1 Penicillin .ltoreq.0.12
.ltoreq.0.12 Vancomycin 0.5 0.5 S. agalactiae Compound 102 0.5 0.5
(n = 50) Tigecycline 0.12 0.12 Tetracycline 32 >32 Azithromycin
0.06 >4 Ceftriaxone 0.06 0.06 Clindamycin 0.06 >0.5 Imipenem
.ltoreq.0.015 0.03 Levofloxacin 0.5 1 Linezolid 1 1 Penicillin
.ltoreq.0.12 .ltoreq.0.12 Vancomycin 0.5 0.5 E. faecalis VAN S (n =
53) Compound 102 0.5 0.5 (n = 101) Tigecycline 0.12 0.12
Tetracycline >32 >32 Azithromycin >4 >4 Ceftriaxone
>64 >64 Clindarnycin >2 >2 Gentamicin >8 >8
Imipenern 1 1 Levofloxacin 1 >4 Linezolid 2 2 Vancomycin 1 2 VAN
NS (n = 48) Compound 102 0.5 1 Tigecycline 0.06 0.12 Tetracycline
>32 >32 Azithromycin >4 >4 Ceftriaxone >64 >64
Clindamycin >2 >2 Gentamicin >8 >8 Imipenem 1 2
Levofloxacin >4 >4 Linezolid 2 2 Vancomycin >16 >16 E.
faecium VAN S (n = 49) Compound 102 0.12 0.5 (n = 100) Tigecycline
0.06 0.06 Tetracycline 0.25 >32 Azithromycin >4 >4
Ceftriaxone >64 >64 Clindamycin >2 >2 Gentamicin 8
>8 Imipenem >8 >8 Levofloxacin >4 >4 Linezolid 2 2
Vancomycin 0.5 1 VAN NS (n = 51) Compound 102 0.12 0.5 Tigecycline
0.06 0.12 Tetracycline 0.25 >32 Azithromycin >4 >4
Ceftriaxone >64 >64 Clindamycin >2 >2 Gentamicin 8
>8 Imipenem >8 >8 Levofloxacin >4 >4 Linezolid 2 2
Vancomycin >16 >16 .sup.1As oxacillin was not tested as part
of the current study, methicillin phenotype was based off of prior
oxacillin testing performed on these isolates .sup.2Penicillin MICs
from prior testing were utilized to determine penicillin phenotype,
as penicillin was only tested as low as 0.12 mg/mL, and isolates
with MICs of .ltoreq.0.12 mg/mL can not be interpreted as either
susceptible or intermediate .sup.3No CLSI/FDA criteria available
for interpretation of MIC MSSA: methicillin-susceptible S. aureus;
MSCoNS: methicillin-susceptible coagulase-negative staphylococci;
MRCoNS: methicillin-resistant coagulase-negative staphylococci PEN:
penicillin; VAN: vancomycin; S: susceptible; I: intermediate; R:
resistant; NS: non-susceptible; NA: not applicable
TABLE-US-00016 TABLE 13 Activity profile of Compound 102 and other
comparator agents against evaluated Gram-negative pathogens MIC
(mg/mL) Organism Drug MIC.sub.50 MIC.sub.90 H. influenzae (n = 50)
Compound 102 0.5 1 Tigecycline 0.12 0.25 Tetracycline 0.5 0.5
Ampicillin .ltoreq.0.5 8 Azithromycin 1 2 Ceftriaxone .ltoreq.0.03
.ltoreq.0.03 Imipenem 1 2 Levofloxacin 0.03 0.03 M. catarrhalis (n
= 50) Compound 102 0.12 0.12 Tigecycline 0.06 0.12 Tetracycline
0.12 0.25 Azithromycin .ltoreq.0.12 .ltoreq.0.12 Ceftriaxone
.ltoreq.0.5 .ltoreq.0.5 Clindamycin 1 2 Gentamicin 0.12 0.12
Imipenem .ltoreq.0.25 .ltoreq.0.25 Levofloxacin 0.06 0.06
TABLE-US-00017 TABLE 14 Activity profile of Compound 102 and other
comparator agents against evaluated pathogen by tetracycline
phenotype MIC (mg/mL) Organism Drug Phenotype N MIC.sub.50
MIC.sub.90 S. pneumoniae Compound 102 TET S 54 0.06 0.12 TET NS 46
0.12 0.12 Tigecycline TET S 54 0.06 0.06 TET NS 46 0.06 0.06
Tetracycline TET S 54 0.12 0.25 TET NS 46 16 32 S. pyogenes
Compound 102 TET S 44 0.12 0.12 TET NS 6 0.25 NA Tigecycline TET S
44 0.06 0.06 TET NS 6 0.06 NA Tetracycline TET S 44 0.12 0.25 TET
NS 6 32 NA S. agalactiae Compound 102 TET S 11 0.12 0.12 TET NS 39
0.5 0.5 Tigecycline TET S 11 0.12 0.12 TET NS 39 0.12 0.12
Tetracycline TET S 11 0.25 0.25 TET NS 39 32 >32 F. faecalis
Compound 102 TET S 30 0.12 0.25 TET NS 71 0.5 1 Tigecycline TET S
30 0.06 0.12 TET NS 71 0.12 0.12 Tetracycline TET S 30 0.25 0.5 TET
NS 71 >32 >32 E. faecium Compound 102 TET S 60 0.12 0.12 TET
NS 40 0.25 0.5 Tigecycline TET S 60 0.06 0.06 TET NS 40 0.06 0.12
Tetracycline TET S 60 0.25 0.25 TET NS 40 >32 >32 NA: not
applicable; TET: tetracycline; S: susceptible; NS:
non-susceptible
[0816] Against the evaluated Gram-positive aerobic pathogens,
Compound 102 MICs were comparable to those of tetracycline against
staphylococci and were several-fold lower than those of
tetracycline against pneumococci and beta-hemolytic streptococci;
Compound 102 MICs were generally 2-4 fold higher than those of
tigecycline.
[0817] Against the evaluated Gram-negative respiratory pathogens,
Compound 102 had similar MICs to those of tetracycline; Compound
102 MICs were generally 2-4-fold higher than those of
tigecycline.
[0818] There was minimal impact of tetracycline resistance on the
overall activity profile of Compound 102, as Compound 102 MICs were
at most 2-4-fold higher against tetracycline resistant isolates
relative to tetracycline susceptible isolates.
H. Antibacterial Activity Against E. coli DH10B Recombinantly
Expressing Tetracycline-Resistance Genes
[0819] Genes encoding tet(A), tet(B), tet(K), tet(M), tet(X), and
E. coli .beta.-galactosidase (lacZ) as a control were amplified by
PCR from clinical isolates confirmed by gene sequencing to have
these tetracycline-resistance determinants and cloned into an
L-arabinose inducible expression system without any affinity tags
(pBAD-Myc-His, Invitrogen, Carlsbad, Calif.). Plasmids were
transformed and expressed in E. coli DH10B cells (Invitrogen,
Carlsbad, Calif.). Cloned inserts were sequenced to verify the
tetracycline resistance gene sequence and compared to reported
sequences in GenBank (accession numbers; tet(A), AJ419171; tet(B),
AP0961; tet(K), AJ888003; tet(M), X90939.1, tet(X), M37699). Cells
were grown in Mueller Hinton Broth containing ampicillin, 50 mg/ml,
pre-induced for 30 minutes with 1% arabinose (tet(A), tet(B),
tet(M), tet(X)) or 0.1% arabinose (tet(K)) at 30.degree. C. prior
to use as inocula in MIC assays containing ampicillin, 50 mg/ml.
MIC assays were incubated at 35.degree. C. and otherwise followed
Clinical Laboratory Standards Institute guidelines, and the
resultant data is shown in Table 15.
TABLE-US-00018 TABLE 15 MIC values for E. coli DH10B recombinantly
expressing tetracycline-resistance genes. Com- EC971 EC1153 EC969
EC970 EC1082 EC1083 pound LacZ Tet(X) TetM TetK TetA TetB Mino- 0.5
4 64 1 8 16 cycline Tetra- 2 >32 64 64 >128 >128 cycline
Tige- 0.0625 2 0.125 0.0625 1 0.0625 cycline Com- 2 4 1 0.5 2 1
pound 102 Ceftri- 0.125 0.125 0.5 0.0625 0.0625 0.0625 axone tet(X)
encodes an inactivating enzyme for many tetracyclines called a
flavin-dependent monooxygenase. tet(A) and tet(B) encode
tetracycline-specific efflux pumps usually found in gram-negative
bacteria. tet(K) encodes a tetracycline-specific efflux pump found
predominantly in gram-positive bacteria. tet(M) encodes a
tetracycline-specific ribosomal protection mechanism that is
wide-spread in both gram-negatives and gram-positives.
I. Determination of Resistance Development In Vitro
[0820] To estimate resistance development in vivo. Compound 102 was
analyzed for the propensity to select for resistance in vitro. The
spontaneous resistance frequency was determined by plating dense
suspensions of S. aureus SA101 and S. pneumoniae SP106
(.about.10.sup.10 colony forming units (CFU) per plating) in
replicates on Mueller Hinton agar plates containing compound at
5.times. the MIC. Plates were supplemented with 5% defibrinated
sheep blood for SP106 testing. Resistance frequencies were
calculated by dividing the number of colonies that grew at a given
drug concentration divided by the total number of plated CFU. For
SA101 and SP106, the spontaneous resistance frequencies for
Compound 102 were <2.2.times.10.sup.-10 and 1.times.10.sup.-8,
respectively. For SA101 and SP106, the spontaneous resistance
frequencies for the levofloxacin (negative) control were
<2.2.times.10.sup.-10 and <3.13.times.10.sup.-9,
respectively. For SA101 and SP106, the spontaneous resistance
frequencies for the rifampin (positive) control were
2.0.times.10.sup.-8 and 2.88.times.10.sup.-7, respectively. Thus,
neither S. aureus nor S. pneumoniae appear to have large
pre-existing populations that are nonsusceptible to Compound
102.
J. Non-GLP Monkey Pharmacokinetics
[0821] As a result of promising pharmacokinetic data in Sprague
Dawley rats,.sup.a Compound 102 was evaluated in 3 non-na ve
cynomolgus monkeys. Each animal received a single IV dose of 1
mg/kg and after a 7-day washout, and received a single PO dose of
10 mg/kg. Nine to ten plasma samples were drawn for each dosing
route up to 24 hours into heparin-coated vacutainer tubes. Dose
formulations were verified with a 5-point calibration curve. The
plasma concentration of the compound was quantified by LC/MS/MS
using an internal standard. Quality control (QC) samples (low,
medium, high; minimum of 6 standards with LLOQ<3 ng/mL) and
standard curves (in duplicate) were included in the bioanalytical
run. WinNonLin was used to determine individual and mean PK
parameters.+-.standard deviation (F, Cmax, Tmax, T1/2, CL, Vss,
AUC(0-t), AUC(0-.infin.), and MRT). The results are presented in
Table 16.
TABLE-US-00019 TABLE 16 Pharmacokinetic parameters for Compound 102
in non-naive cynomologus monkeys A. IV dosing B. PO dosing
Parameter Average SD Parameter Average SD Dose (mg/kg) 1 Dose
(mg/kg) 10 Co (ng/ml) 3170 1126 Cmax (ng/ml) 1260 497 T1/2 (h)
23.33 3.85 Tmax (h) 4 0 Vdss (L/kg) 3.40 0.38 T1/2 (h) 24.84 8.26
Cl (ml/hr/kg) 111.6 26.46 AUC last 16333 4937 AUC last 4853 551 (ng
h/ml) (ng h/ml) AUC inf 35433 19111 AUC inf 9310 2201 (ng h/ml) (ng
h/ml) % Oral 33.7 9.1 bioavailability .sup.a Initial preliminary
testing in Sprague Dawley rats (n = 3) resulted an oral
bioavailability (% F) of 48.3 .+-. 31.2.
K. Evaluation of Mammalian Phototoxicity
[0822] To estimate its potential to produce phototoxicity in vivo,
Compound 102 was tested in validated in vivo and in vitro models of
acute phototoxic activity at Charles River Laboratories (See
Spielmann, H., et al., The second ECVAM workshop on phototoxicity
testing. The report and recommendations of ECVAM workshop 42.
Altern Lab Anim, 2000. 28(6): p. 777-814; and Peters, B. and H. G.
Holzhutter, In vitro phototoxicity testing: development and
validation of a new concentration response analysis software and
biostatistical analyses related to the use of various prediction
models. Altern Lab Anim, 2002. 30(4): p. 415-32, the entire
teachings of both are incorporated herein by reference). Results
showed that, unlike doxycycline, Compound 102 findings in vitro in
the neutral red uptake 3T3 assay did not translate to a phototoxic
effect in the in vivo model, which is considered to be a better
mimic of clinically-relevant UVA exposure of high-level intradermal
accumulation of compound.
[0823] For in vivo evaluation in the Crl:SKH1-hr hairless mouse
model of phototoxicity, mice (n=3 per group) were injected
intracutaneously along the back (two dorsal injection sites per
mouse) with Compound 102 and control compounds (doxycycline,
minocycline, levofloxacin) at either 0.0375 mg/mouse or 0.375
mg/mouse. A vehicle control group was injected with normal saline.
The pH of compound formulations was adjusted to 6.5.+-.0.5 prior to
injection. Immediately after administration, mice were lightly
anesthetized via intraperitoneal injection of chloral hydrate in
deionized water and then positioned on plastic tubing with
laboratory tape. An aluminum foil mask with a single hole with a
diameter of 1.3 cm (1.3 cm.sup.2) was placed over the mid-dorsum
injection site before UVA exposure. The distal administration site
was shielded from UVA exposure. A UVA dose of no less than 20.0 and
no more than 20.1 J/cm.sup.2 at an intensity of 5.+-.1 mW/cm.sup.2
at the level of the mice was delivered during the exposure period.
Mice were observed before formulation administration, after
completion of administration, 60.+-.10 minutes and 4 hours.+-.30
minutes after the completion of UVR exposure and 1, 2 and 3 days
after UVR exposure for general appearance, clinical observations
and signs of skin responses at the site of UVR exposure and the
non-UVA-exposed site. Results showed that administration of the
positive control, doxycycline, resulted in dosage-dependent
phototoxicity (erythema, edema) at the site of UVA exposure,
validating the assay. Minocycline, administered as a negative
control, produced no skin reaction at either dose. Administration
of levofloxacin resulted in dosage-dependent phototoxicity
(erythema, edema, flaking) in the site of UVA exposure. Compound
102 at either 0.0375 or 0.375 mg/mouse resulted in no skin
reactions indicative of phototoxicity on the day of UVA exposure or
the following three days of observation.
L. In Vitro Susceptibility Study of Compound 102 in Legionella
pneumophila
[0824] Legionella organisms are often associated with respiratory
infections, and Legionella pneumophila results in significant
mortality unless it is promptly and effectively treated. In a
recent FDA workshop on Clinical Trial Design for Community-Acquired
Bacterial Pneumonia (Dec. 9, 2009), the panel voted to include
patients with documented L. pneumophila in non-inferiority
community-acquired bacterial pneumonia (CABP) trials. Because L.
pneumophila can result in an overall case mortality of 15%, it was
important to determine its susceptibility to the compounds of the
invention, such as Compound 102.
Methods
[0825] The in vitro activity of Compound 102 was compared to
tetracycline and erythromycin against a total of 70 L. pneumophila
isolates (serogroup 1 (n=20), 2 (n=10), 3 (n=10), 4 (n=10), 5
(n=10) and 6 (n=10)) by standard agar dilution using buffered yeast
extract agar containing BCYE growth supplement (BYE).
[0826] The Legionella pneumophila strains were isolated from the
respiratory tract from 1992 to 2010 and identified by standard
methods described by Murray et al., Manual of Clinical
Microbiology, 9rd ed., 2007, A.S.M., the entire teachings of which
are incorporated herein by reference. Isolates from six serogroups
were tested for a total number of 70 L. pneumophila. Buffered Yeast
extract (BYE) (with original Legionella BCYE Growth supplement) was
used as the medium to test Legionella strains.
[0827] A pilot test to determine if Compound 102 and tetracycline
activity were impacted artificially by BCYE supplement or iron was
done by testing of Staphylococcus aureus ATCC29213 on BYE (Original
BYE), BYE without ferric pyrophosphate (modified BYE) and
cation-adjusted Mueller-Hinton agar (MH).
Determination of Minimal Inhibitory Concentrations (MICs)
[0828] MICs were determined using the CLSI agar dilution method
((See Performance standards for antimicrobial susceptibility
testing; Seventeenth Informational Supplement; CLSI, M100-S17 VOL
27 number 1, Clinical and Laboratory Standards Institute, Wayne,
Pa., January 2007, the teachings of which are incorporated herein
by reference; and Method for dilution antimicrobial susceptibility
tests for bacteria that grow aerobically; approved standard 17th
edition, M7-A7, Clinical and Laboratory Standards Institute (CLSI),
Wayne, Pa., 2006), the entire teachings of which are incorporated
herein by reference)), with replicate plating of the organisms onto
a series of agar plates of increasing concentrations of compound
from 0.004 mg/L to 64 .mu.g/mL. Erythromycin and tetracycline were
obtained from Sigma Chemicals, Mississauga, Ont.
Results
[0829] Only original BYE supported L. pneumophila growth. The pilot
tests indicated that BYE resulted in a 16- to 64-fold increase in
MICs relative to MH for S. aureus ATCC29213 (Tables 17 and 18).
These results suggest that the MIC values of Compound 102 obtained
in original BYE for L. pneumophila were artificially elevated due
to media effects.
TABLE-US-00020 TABLE 17 Pilot Study with original BYE, Modified BYE
and Cation-adjusted Mueller Hinton media for L. pneumophila
ATCC33152. Compound Incubation Assay Compound Media used Time No.
102 Tetracycline Erythromycin Original 24 hours 1 NG NG NG BYE 2 NG
NG NG 48 hours 1 16 8 0.5 2 16 8 0.25 Modified 24 hours 1 NG NG NG
BYE 2 NG NG NG 48 hours 1 NG NG NG 2 NG NG NG Cation 24 hours 1 NG
NG NG adjusted 2 NG NG NG Mueller- Hinton Expected MIC Range
Unknown Unknown 0.25-0.5*** NG = no growth
TABLE-US-00021 TABLE 18 Pilot Study with original BYE, Modified BYE
and Cation-adjusted Mueller Hinton media for Staphylococcus aureus
ATCC29213. Compound Media Incubation Assay Compound used Time No.
102 Tetracycline Erythromycin Original 24 hours 1 4 2 0.5 BYE 2 4 2
0.5 48 hours 1 >64 32 0.5 2 >64 32 0.5 Modified 24 hours 1 2
0.25 0.5 BYE 2 2 0.25 0.5 48 hours 1 8 0.5 0.5 2 8 0.5 0.5 Cation
24 hours 1 0.25 0.5 1 adjusted 48 hours 1 0.25 0.5 1 Mueller-
Hinton Expected MIC Range 0.25-1* 0.12-1** 0.25-1** NG = No Growth
*Expected MIC Range with Cation adjusted Mueller-Hinton **Expected
MIC Range with Cation Mueller-Hinton. ***Expected MIC Range with
original BYE, data obtained from previous studies.
[0830] The activity of Compound 102, tetracycline and erythromycin
against all Legionella pneumophila serogroups is shown in Table 19.
The MIC.sub.90 values for Compound 102, tetracycline, and
erythromycin against strains from all serogroups of L. pneumophila
were 8, 8, and 0.5 mg/L, respectively, using the original BYE
media.
TABLE-US-00022 TABLE 19 Susceptibility of Legionella pneumophila
all serogroups in original BYE Media MIC (mg/L) Serogroup (no.
tested) Antibiotic MIC.sub.50 MIC.sub.90 All Serogroups (70)
Compound 102 2 8 Tetracycline 4 8 Erythromycin 0.25 0.5
[0831] The teachings of all patents, published applications and
references cited herein are incorporated by reference in their
entirety.
[0832] While this invention has been particularly shown and
described with references to example embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *
References