U.S. patent application number 10/339043 was filed with the patent office on 2003-11-06 for novel substituted succinic acid metallo-beta-lactamase inhibitors and their use in treating bacterial infections.
Invention is credited to Balkovec, James M., Greenlee, Mark L., Olson, Steven H., Rouen, Gregory P..
Application Number | 20030207859 10/339043 |
Document ID | / |
Family ID | 22585340 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207859 |
Kind Code |
A1 |
Balkovec, James M. ; et
al. |
November 6, 2003 |
Novel substituted succinic acid metallo-beta-lactamase inhibitors
and their use in treating bacterial infections
Abstract
This invention relates to novel substituted succinic acid
metallo-.beta.-lactamase inhibitors which are useful potentiators
of .beta.-lactam antibiotics. Accordingly, the present invention
provides a method of treating bacterial infections in animals or
humans which comprises administering, together with a b-lactam
antibiotic, a therapeutically effective amount of a compound of
formula I: 1 including pharmaceutically acceptable salts, prodrugs,
anhydrides, and solvates thereof.
Inventors: |
Balkovec, James M.;
(Martinsville, NJ) ; Greenlee, Mark L.; (Rahway,
NJ) ; Olson, Steven H.; (Metuchen, NJ) ;
Rouen, Gregory P.; (New Brunswick, NJ) |
Correspondence
Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
Family ID: |
22585340 |
Appl. No.: |
10/339043 |
Filed: |
January 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10339043 |
Jan 9, 2003 |
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09697415 |
Oct 26, 2000 |
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60162370 |
Oct 28, 1999 |
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Current U.S.
Class: |
514/210.09 ;
514/249; 514/400; 514/447; 514/468; 514/534; 544/351; 548/335.5;
549/461; 549/74; 560/169 |
Current CPC
Class: |
A61P 43/00 20180101;
C07C 69/612 20130101; C07D 333/20 20130101; C07C 57/58 20130101;
C07D 307/91 20130101; C07C 69/734 20130101; C07D 409/06 20130101;
C07D 233/56 20130101; C07D 405/06 20130101; C07C 57/38 20130101;
C07D 249/08 20130101; C07C 59/64 20130101; A61P 31/04 20180101;
C07D 231/12 20130101 |
Class at
Publication: |
514/210.09 ;
544/351; 548/335.5; 560/169; 549/461; 514/249; 514/400; 514/534;
514/447; 514/468; 549/74 |
International
Class: |
A61K 031/498; A61K
031/407; A61K 031/4172; A61K 031/381; A61K 031/343; C07D 43/14;
C07D 333/20 |
Claims
What is claimed is:
1. A compound represented by formula I: 230including
pharmaceutically acceptable salts, prodrugs, anhydrides, and
solvates thereof, wherein: M.sup.1 and M.sup.2 are independently
selected from: (a) Hydrogen, (e) Pharmaceutically acceptable
cation, (e) Pharmaceutically acceptable esterifying group; and (g)
A negative charge; R.sup.1 and R.sup.2 are independently selected
from the following: (d) Hydrogen, provided that R.sup.1 and R.sup.2
are not hydrogen at the same time; (e) a C.sub.1 to C.sub.16
straight, branched or unsaturated alkyl group substituted with 0 to
2 R.sup.q groups and substituted with 0 to 3 R.sub.x groups and
optionally interrupted by one of the following O, S, SO.sub.2,
--C(O)--, (f) --C(O)--NR.sup.a--, --CO.sub.2--; (c) a group of the
formula: 231 wherein --A-- represents a single bond, C.sub.1 to
C.sub.8 straight, branched or unsaturated alkyl group optionally
substituted with 1 to 2 R.sub.x groups and optionally interrupted
by one of the following O, S, SO.sub.2, --C(O)--,
--C(O)--NR.sup.a--, --CO.sub.2--; 232represents: (1) a C.sub.6 to
C.sub.14 aryl group; (2) a C.sub.3 to C.sub.10 alicyclic group; (3)
a C.sub.3 to C.sub.14 heteroaryl group, which contains 1 to 3
heteroatoms, 0 to 3 of which heteroatoms are nitrogen and 0 to 1 of
which are oxygen or sulfur; (4) a C.sub.3 to C.sub.10 heterocyclic
group, which contains 1 to 2 heteroatoms, 0 to 1 of which
heteroatoms are nitrogen, and 0 to 2 of which are oxygen or sulfur;
or (d) a group of the formula: 233 wherein: --A-- is as defined
above; A' is a single bond, O, S, or a C.sub.1 to C.sub.6 straight,
branched or unsaturated alkyl group optionally substituted with 1-2
R.sub.x groups and optionally interrupted by one of the following
groups O, S, SO.sub.2, --C(O)--, --C(O)--NR.sup.a--, --CO.sub.2--;
234are independently selected from: (1) a C.sub.6 to C.sub.10 aryl
group; (2) a C.sub.3 to C.sub.8 alicyclic group; (3) a C.sub.2 to
C.sub.9 heteroaryl group, which contains 1 to 3 heteroatoms, 0 to 3
of which heteroatoms are nitrogen and 0 to 1 of which are oxygen or
sulfur; (4) a C.sub.3 to C.sub.8 heterocyclic group, which contains
1 to 2 heteroatoms, 0 to 1 of which heteroatoms are nitrogen, and 0
to 2 of which are oxygen or sulfur; provided that at least one
R.sup.q group is present in R.sup.1 or R.sup.2 and that when more
than one R.sup.q is present the total number of cationic nitrogen
atoms does not exceed 8; the total number of cationic nitrogen
atoms can be charged balanced by M.sup.1 and/or M.sup.2 or by
M.sup.1 and/or M.sup.2 in combination with an appropriate number of
Y.sup.-; wherein: R.sup.q is --E--Q.sup.+Y.sup.-; Y.sup.- is a
pharmaceutically acceptable anionic group; E is
--(CH.sub.2).sub.m--X--(CH.sub.2).sub.n--; m is 0 to 6; n is 0 to 6
(but when E is attached to an aromatic ring n is 1-6); X is a bond,
O, S, SO.sub.2, --C(O)--, --C(O)--N(R.sup.a)--, --C(O)O--,
--CH.dbd.CH-- or --C.ident.C--, provided that when X is O, S,
--C(O)--N(R.sup.a)-- or --C(O)O--, then n is 2 to 6 and Q.sup.+,
attached to the (CH.sub.2).sub.n terminus of E is: (1) a cationic
group selected from the following: 235 wherein: R.sup.u and R.sup.v
are independently hydrogen or C.sub.1-6 alkyl optionally
substituted with 1 to 2 R.sup.y; R.sup.w is hydrogen or C.sub.1-6
alkyl optionally substituted with 1 to 2 R.sub.x; R.sup.u and
R.sup.v when bonded to the same nitrogen atom may together be a
C.sub.3-6 alkyl radical, which when taken together with the
intervening atoms form a ring; Two R.sup.u groups on separate
nitrogen atoms may together comprise a C.sub.2-5 alkyl radical,
which when taken together with the intervening atoms form a ring;
R.sup.u, R.sup.v and R.sup.w when bonded to the same nitrogen atom
may together form a C.sub.6-10 tertiary alkyl radical, which with
N.sup.+ forms a bicyclic ring; (2) A dicationic group: 236 wherein:
E.sup.1 is --(CH.sub.2).sub.p--Z--(CH.sub.2).sub.r--; p and r are
independently 1 to 4; Z is a bond, O, S, SO.sub.2, --C(O)--,
--C(O)O--**, --CH.dbd.CH--, --C.ident.C--, or 237Provided that when
Z is O or S, p is 2 to 4 and r is 2 to 4 and when Z is 238or
--C(O)O--**, r is 2 to 4; wherein ** denotes the atom which is
bonded to the --(CH.sub.2).sub.r-- moiety of E.sup.1 above; Q.sup.1
is selected from the following: 239Q.sup.2 is selected from the
following: 240 R.sup.u, R.sup.v and R.sup.w are independently
selected and defined as above, And in addition, in the case where
two R.sup.u groups on separate nitrogen atoms are joined to form a
ring as defined above, two R.sup.v groups on the same two separate
nitrogen atoms may also comprise a C.sub.1-5 alkyl radical to form
together with the intervening atoms a bicyclic ring; an example of
such is: 241(5) A tricationic group selected from the following:
242 wherein: Each E.sup.1 is as defined above, but selected
independently; Each Q.sup.1 is as defined above, but selected
independently; Each Q.sup.2 is as defined above, but selected
independently; R.sup.u, R.sup.v and R.sup.w are defined as in the
definition of Q.sup.+ item (2) above and selected independently; or
(6) A tetracationic group selected from the following: 243 wherein:
Each E.sup.1 is as defined above, but selected independently; Each
Q.sup.1 is as defined above, but selected independently; Each
Q.sup.2 is as defined above, but selected independently; R.sup.u,
R.sup.v and R.sup.w are defined as in the definition of Q.sup.+
item (2) above and selected independently; where each R.sub.x is
independently selected from the group consisting of: (f) F, Cl, Br,
I, (g) CF.sub.3, (h) OR.sup.b, (i) CN, (j) --C(O)--R.sup.c, (f)
--S(O.sub.2)--R.sup.f, (g) --C(O)--OR.sup.a (h) --O--C(O)--R.sup.c,
(i) --S--R.sup.b, (j) --N(R.sup.a)--C(O)--R.sup.c, 244(q)
--N(R.sup.a)--C(O)--OR.sup.f, (r) --S(O)--R.sup.f, (s)
--N(R.sup.a)--S(O.sub.2)--R.sup.f, (t) NO.sub.2, and (u) C.sub.1 to
C.sub.8 straight, branched or unsaturated alkyl optionally
substituted with one of the substituents (a) through (t) above; (v)
--CH.sub.2-aryl wherein the aryl is optionally substituted with one
of the substituents (a) through (t) above; or two adjacent R.sub.x
groups on an aromatic ring may consist of the following divalent
moiety, --O--CH.sub.2--O--; wherein: R.sup.a is H, C.sub.1 to
C.sub.6 alkyl optionally substituted with R.sup.y; R.sup.b is H,
C.sub.1 to C.sub.6 alkyl optionally substituted with R.sup.y,
CH.sub.2-aryl, or aryl, said aryls optionally substituted with 1-2
R.sup.y groups; R.sup.c is H, C.sub.1 to C.sub.6 alkyl optionally
substituted with R.sup.y, CF.sub.3, or aryl, said aryl optionally
substituted with 1-2 R.sup.y groups; R.sup.d and R.sup.e are
independently hydrogen, C.sub.1 to C.sub.4 alkyl optionally
substituted with R.sup.y, or R.sup.d and R.sup.e taken together may
represent a 3 to 5-membered alkyl radical to form a ring, or
R.sup.d and R.sup.e taken together may represent a 2 to 4-membered
alkyl radical interrupted by O, S, SO or SO.sub.2 to form a ring;
R.sup.f is C.sub.1 to C.sub.6 alkyl optionally substituted with
R.sup.y, or aryl, said aryl optionally substituted with 1-2 R.sup.y
groups; and R.sup.y is --OH, --OCH.sub.3, OCONH.sub.2, OCOCH.sub.3,
CHO, COCH.sub.3, CO.sub.2CH.sub.3, CONH.sub.2, CN, SOCH.sub.3,
SO.sub.2CH.sub.3, SO.sub.2NH.sub.2, F, Cl, Br, I or CF.sub.3.
2. A compound in accordance with claim 1 wherein M.sup.1 and
M.sup.2 are independently hydrogen or a negative charge and all
other variables are as defined above.
3. A compound in accordance with claim 1 wherein R.sup.1 and/or
R.sup.2 represents a C.sub.1 to C.sub.16 straight, branched or
unsaturated alkyl group substituted with 0 to 2 R.sup.q, and
substituted with 0 to 3 R.sub.x groups, provided that at least one
of R.sup.1 or R.sup.2 contains an R.sup.q and all other variables
are defined as above.
4. A compound in accordance to claim 1 where R.sup.1 and/or R.sup.2
represents 245wherein at least one R.sup.q group is present on
R.sup.1 or R.sup.2 and all other variables are defined as
above.
5. A compound in accordance to claim 1 where R.sup.1 and/or R.sup.2
represents (d) 246wherein at least one R.sup.q group is present on
R.sup.1 or R.sup.2 and all other variables are defined as
above.
6. A compound in accordance with claim 1 wherein the relative and
absolute stereochemistry is: 247
7. A compound in accordance with claim 6 wherein R.sup.1 and/or
R.sup.2 represents C.sub.4-12 straight, branched or unsaturated
alkyl group optionally substituted with 1 to 2 R.sub.x and
optionally substituted with 1 to 2 R.sup.q groups provided that at
least one of R.sup.1 or R.sup.2 contains an R.sup.q and all other
variables are as described above.
8. A compound in accordance with claim 6 wherein R.sup.1 and/or
R.sup.2 represents a group of the formula: 248wherein A is
(CH.sub.2).sub.1-5 and 249is phenyl, naphthyl, cyclohexyl or
dibenzofuranyl, provided that at least one of R.sup.1 or R.sup.2
contains an R.sup.q and all other variables are as originally
defined.
9. A compound in accordance with claim 6 wherein where R.sup.1 or
R.sup.2 represents a group of the formula: 250wherein A is
(CH.sub.2).sub.1-3, A' is a single bond, --O-- or
(CH.sub.2).sub.1-2 and 251 independently represent phenyl, thienyl,
pyridyl, furanyl or cyclohexyl.
10. A compound in accordance with claim 6 where one of R.sup.1 or
R.sup.2 is C.sub.4-8 straight, branched or unsaturated alkyl
optionally substituted with 1 to 2 R.sub.x or a group of the
formula: 252where A is (CH.sub.2).sub.1-2 and 253is phenyl,
cyclopentyl or cyclohexyl and the other of R.sup.1 or R.sup.2 is:
i) a C.sub.7-12 alkyl group substituted with R.sup.q, ii) a group
of the formula: 254 where A is (CH.sub.2).sub.1-2, A' is a single
bond, 255is phenyl, thienyl or cyclohexyl and 256is phenyl, thienyl
or pyridyl, or iii) a group of the formula: 257 where A is
(CH.sub.2).sub.1-3, 258is phenyl or thienyl and R.sup.q is
--(CH.sub.2).sub.2-6--Q.sup.+ Y.sup.Q; and all other variables are
as originally defined.
11. A compound according to claim 6 wherein R.sup.1 is C.sub.5-7
alkyl substituted with 0 to 2 R.sub.x goups, 259R.sup.2 is
C.sub.7-10 alkyl substituted with 1 R.sup.q groups and 0 to 2
R.sub.x groups, 260and all other variables are as originally
defined.
12. A compound according to claim 11 wherein R.sup.1 is: 261R.sup.2
is: 262and all other variables are as originally defined.
13. A compound according to claim 1 wherein 1 or 2 R.sup.q groups
are present containing a total number of 2 to 6 cationic nitrogen
atoms.
14. A compound according to claim 1 wherein a single R.sup.q
substituent is present containing a tricationic or tetracationic
Q.sup.+ group.
15. A compound according to claim 14 wherein R.sup.q is
--E--Q.sup.+Y.sup.- wherein E is (CH.sub.2).sub.0-6 or
--C(O)--N(R.sup.a)--(CH.sub.2).sub.2-4-- and Q.sup.+ is a
tricationic or tetracationic group and Y.sup.- and R.sup.a are as
originally defined.
16. A compound according to claim 14 wherein the tricationic
Q.sup.+ group is selected from the group consisting of: 263wherein
E.sup.1 is (CH.sub.2).sub.2-4 or
--(CH.sub.2)--C(O)--N(R.sup.a)--(CH.sub.2).sub.2-4-- - and R.sup.a,
Q.sup.1 and Q.sup.2 are as previously defined.
17. A compound according to claim 16 wherein the tricationic
Q.sup.+ group is selected from: 264wherein R.sup.u, R.sup.v, and
R.sup.w are as defined above.
18. A compound according to claim 14 wherein the tetracationic
Q.sup.+ group is selected from the group consisting of: 265wherein
E.sup.1 is (CH.sub.2).sub.2-4 or
--(CH.sub.2)--C(O)--N(R.sup.a)--(CH.sub.2).sub.2-4-- - and R.sup.a,
Q.sup.1, Q.sup.2, and R.sup.w are as defined above.
19. A compound according to claim 18 wherein the tetracationic
Q.sup.+ group is selected from: 266wherein R.sup.a, R.sup.u,
R.sup.v, and R.sup.w are as described above.
20. A compound of the structural formula: 267
21. A compound represented by Tables 3-7:
19TABLE 3 268 Example No. Q.sup..sym. Y.sup..crclbar. 5 269 3
Cl.sup..crclbar. 6 270 3 Cl.sup..crclbar. 7 271 3 Cl.sup..crclbar.
8 272 4 Cl.sup..crclbar. 9 273 4 Cl.sup..crclbar. 10 274 3
Cl.sup..crclbar. 11 275 3 Cl.sup..crclbar. 12 276 3
Cl.sup..crclbar. 13 277 3 Cl.sup..crclbar. 14 278 3
Cl.sup..crclbar.
20TABLE 4 279 Example No. Q.sup..sym. Y.sup..crclbar. 15 280
CF.sub.3CO.sub.2.sup..crclbar. 16 281
CF.sub.3CO.sub.2.sup..crclbar. 17 282 2 Cl.sup..crclbar. 18 283 2
CF.sub.3CO.sub.2.sup..crclbar. 19 284 3
CF.sub.3CO.sub.2.sup..crclbar. 20 285 3
CF.sub.3CO.sub.2.sup..crclbar.
21TABLE 5 286 Example No. Q.sup..sym. Y.sup..crclbar. 21 287
Cl.sup..crclbar. 22 288 2 Cl.sup..crclbar. 23 289 3
Cl.sup..crclbar. 24 290 3 Cl.sup..crclbar.
22TABLE 6 291 Example No. Q.sup..sym. Y.sup..crclbar. 25 292
Cl.sup..crclbar. 26 293 2 Cl.sup..crclbar. 27 294 3
Cl.sup..crclbar. 28 295 3 Cl.sup..crclbar.
23TABLE 7 296 Example No. Q.sup..sym. Y.sup..crclbar. 29 297
Cl.sup..crclbar.
22. A compound represented by Tables 8-18:
24TABLE 8 298 Example No. Q.sup..sym. Y.sup..crclbar. 30 299 3
Cl.sup..crclbar. 31 300 3 CH.sub.3CO.sub.2 32 301 3
Cl.sup..crclbar. 33 302 3 Cl.sup..crclbar. 34 303 3
Cl.sup..crclbar. 35 304 3 Cl.sup..crclbar. 36 305 4
Cl.sup..crclbar. 37 306 4 CH.sub.3CO.sub.2.sup..crclbar. 38 307 4
Cl.sup..crclbar. 39 308 4 Cl.sup..crclbar. 40 309 4
Cl.sup..crclbar. 41 310 4 Cl.sup..crclbar. 42 311 Cl.sup..crclbar.
43 312 Cl.sup..crclbar. 44 313 3 Cl.sup..crclbar. 45 314 3
Cl.sup..crclbar. 46 315 4 Cl.sup..crclbar. 47 316 3
Cl.sup..crclbar.
25TABLE 9 317 Example No. Q.sup..sym. Y.sup..crclbar. 48 318
Cl.sup..crclbar. 49 319 2 Cl.sup..crclbar. 50 320 3
Cl.sup..crclbar. 51 321 3 Cl.sup..crclbar. 52 322 3
Cl.sup..crclbar. 53 323 3 Cl.sup..crclbar.
26TABLE 10 324 Example No. Q.sup..sym. Y.sup..crclbar. 54 325
Cl.sup..crclbar. 55 326 2 Cl.sup..crclbar. 56 327 3
Cl.sup..crclbar. 57 328 3 Cl.sup..crclbar. 58 329 3
Cl.sup..crclbar. 59 330 4 Cl.sup..crclbar.
27 331 Example No. Q.sup..sym. Y.sup..crclbar. 60 332
Cl.sup..crclbar. 61 333 2 Cl.sup..crclbar. 62 334 3
Cl.sup..crclbar. 63 335 3 Cl.sup..crclbar. 64 336 3
Cl.sup..crclbar. 65 337 4 Cl.sup..crclbar.
28TABLE 2 338 Example No. Q.sup..sym. Y.sup..crclbar. 66 339
Cl.sup..crclbar. 67 340 2 Cl.sup..crclbar. 68 341 3
Cl.sup..crclbar. 69 342 3 Cl.sup..crclbar. 70 343 3
Cl.sup..crclbar. 71 344 4 Cl.sup..crclbar.
29TABLE 13 345 Example No. Q.sup..sym. Y.sup..crclbar. 72 346
Cl.sup..crclbar. 73 347 2 Cl.sup..crclbar. 74 348 3
Cl.sup..crclbar. 75 349 3 Cl.sup..crclbar. 76 350 3
Cl.sup..crclbar. 77 351 4 Cl.sup..crclbar.
30TABLE 14 352 Example No. Q.sup..sym. Y.sup..crclbar. 78 353
Cl.sup..crclbar. 79 354 2 Cl.sup..crclbar. 80 355 3
Cl.sup..crclbar. 81 356 3 Cl.sup..crclbar. 82 357 3
Cl.sup..crclbar. 83 358 4 Cl.sup..crclbar.
31TABLE 15 359 Example No. Q.sup..sym. Y.sup..crclbar. 84 360
Cl.sup..crclbar. 85 361 2 Cl.sup..crclbar. 86 362 3
Cl.sup..crclbar. 87 363 3 Cl.sup..crclbar. 88 364 3
Cl.sup..crclbar. 89 365 4 Cl.sup..crclbar.
32TABLE 16 366 Example No. Q.sup..sym. Y.sup..crclbar. 90 367
Cl.sup..crclbar. 91 368 2 Cl.sup..crclbar. 92 369 3
Cl.sup..crclbar. 93 370 3 Cl.sup..crclbar. 94 371 3
Cl.sup..crclbar. 95 372 4 Cl.sup..crclbar.
33TABLE 17 373 Example No. Q.sup..sym. Y.sup..crclbar. 96 374
Cl.sup..crclbar. 97 375 2 Cl.sup..crclbar. 98 376 3
Cl.sup..crclbar. 99 377 3 Cl.sup..crclbar. 100 378 3
Cl.sup..crclbar. 101 379 4 Cl.sup..crclbar.
34TABLE 18 Example No. 102 380 103 381 104 382
23. A pharmaceutical composition comprised of a compound in
accordance with claim 1 in combination with a pharmaceutically
acceptable carrier.
24. A pharmaceutical composition in accordance with claim 23 used
in the manufacture of a medicament for the treatment of bacterial
infections.
25. A pharmaceutical composition in accordance with claim 23
further comprising a .beta.-lactam antibiotic.
26. A pharmaceutical composition in accordance with claim 25
wherein the .beta.-lactam is a carbapenem antibiotic.
27. A composition according to claim 25 which further contains a
serine .beta.-lactamase inhibitor.
28. A composition according to claim 26 which further contains a
DHP inhibitor.
29. A method of treating a bacterial infection comprising
administering to a mammalian patient in need of such treatment a
metallo-.beta.-lactamase inhibitor compound as defined in claim 1
in combination with a pharmaceutically acceptable .beta.-lactam
antibiotic in an amount which is effective for treating a bacterial
infection.
30. A method according to claim 29 wherein the .beta.-lactam is a
carbapenem antibiotic.
31. A method according to claim 30, which further contains a DHP
inhibitor.
32. A method according to claim 31 wherein the DHP inhibitor is
cilastatin.
33. A method according to claim 29 which further contains a serine
.beta.-lactamase inhibitor.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to compounds which have
metallo-.beta.-lactamase inhibitory characteristics. The invention
also relates to methods of preparing, pharmaceutical compositions
and uses of the compounds.
[0002] Metallo-.beta.-lactamases are bacterial enzymes which confer
resistance to virtually all clinically relevant .beta.-lactam
antibiotics, including carbapenems and jeopardize the future use of
all such agents. The increased treatment of infections with
carbapenems and other .beta.-lactam antibiotics may lead to the
proliferation of clinical bacterial strains which are able to
produce metallo-.beta.-lactamases and thus resist the effects of
.beta.-lactam antibiotics. In fact, metallo-.beta.-lactamases have
now been identified in a number of pathogenic bacterial species
including Bacillus cereus, Bacteroides fragilis, Aeromonas
hydrophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia
marcescens, Stenotrophomonas maltophilia, Shigella flexneri,
Legionella gormanii, Chryseobacterium meningosepticum,
Chryseobacterium indologenes, Acinetobacter baumannii, Citrobacter
freundii, and Aeromonas veronii.
[0003] Accordingly, there is an increasing need for agents which
when combined with a .beta.-lactam antibiotic, e.g. imipenem, will
restore the effectiveness of the .beta.-lactam antibiotics and
which are at the same time relatively free from undesirable side
effects.
[0004] WO 98/17639, 97/30027, 98/40056,98/39311 and 97/10225 teach
certain beta-thiopropionyl-amino acid derivatives and their use as
inhibitory agents against metallo-.beta.-lactamases. Goto et. al.,
Biol. Pharm. Bull. 20, 1136 (1997), Payne et. al., FEMS
Microbiology Letters 157, 171 (1997), Payne et al., Antimicrob.
Agents Chemother. 41, 135 (1997), Page et. al., Chem. Commun. 1609
(1998) and Page et al., Biochem. J. 331, 703 (1998) also disclose
certain thiols and thioesters as metallo-.beta.-lactamase
inhibitors. Additionally, Toney et al., Chemistry and Biology 5,
185 (1998), Fastrez et al., Tetrahedron Lett. 36, 9313 (1995),
Schofield et al., Tetrahedron 53, 7275 (1997), Schofield et. al.,
Bioorg. & Med. Chem. Lett. 6, 2455 (1996) and WO 97/19681
disclose other metallo-.beta.-lactamase inhibitors. However, the
above noted references do not teach the compounds of the instant
invention.
[0005] Other references which disclosed the general state of the
art are Bush et al., Antimicrob. Agents Chemother. 41, 223 (1997);
Livermore, D. M. J. Antimicrob. Chemother. 1998, 41 (Suppl. D), 25;
Bush, K. Clin. Infect. Dis. 1998, 27 (Suppl 1), S48; Livermore, D.
M. J. Antimicrob. Chemother. 1997, 39, 673 and Payne, D. J. J. Med.
Microbiol. 1993, 39, 93.
SUMMARY OF THE INVENTION
[0006] This invention relates to novel substituted succinic acid
metallo-.beta.-lactamase inhibitors, which are useful potentiators
of .beta.-lactam antibiotics. Accordingly, the present invention
provides a method of treating bacterial infections in animals or
humans which comprises administering, together with a .beta.-lactam
antibiotic, a therapeutically effective amount of a compound of
formula I: 2
[0007] including pharmaceutically acceptable salts, prodrugs,
anhydrides, and solvates thereof, wherein:
[0008] M.sup.1 and M.sup.2 are independently selected from:
[0009] (a) Hydrogen,
[0010] (b) Pharmaceutically acceptable cation,
[0011] (c) Pharmaceutically acceptable esterifying group; and
[0012] (d) A negative charge;
[0013] R.sup.1 and R.sup.2 are independently selected from the
following:
[0014] (a) Hydrogen, provided that R.sup.1 and R.sup.2 are not
hydrogen at the same time;
[0015] (b) a C.sub.1 to C.sub.16 straight, branched or unsaturated
alkyl group substituted with 0 to 2 Rq groups and substituted with
0 to 3 R.sub.x groups and optionally interrupted by one of the
following O, S, SO.sub.2, --C(O)--,
[0016] (c) --C(O)--NR.sup.a--, --CO.sub.2--;
[0017] (c) a group of the formula: 3
[0018] wherein
[0019] --A-- represents a single bond, C.sub.1 to C.sub.8 straight,
branched or unsaturated alkyl group optionally substituted with 1
to 2 R.sub.x groups and optionally interrupted by one of the
following O, S, SO.sub.2, --C(O)--, --C(O)--NR.sup.a--,
--CO.sub.2--; 4
[0020] represents:
[0021] (1) a C.sub.6 to C.sub.14 aryl group;
[0022] (2) a C.sub.3 to C.sub.10 alicyclic group;
[0023] (3) a C.sub.3 to C.sub.14 heteroaryl group, which contains 1
to 3 heteroatoms, 0 to 3 of which heteroatoms are nitrogen and 0 to
1 of which are oxygen or sulfur;
[0024] (4) a C.sub.3 to C.sub.10 heterocyclic group, which contains
1 to 2 heteroatoms, 0 to 1 of which heteroatoms are nitrogen, and 0
to 2 of which are oxygen or sulfur; or
[0025] (d) a group of the formula: 5
[0026] wherein:
[0027] --A-- is as defined above;
[0028] A' is a single bond, O, S, or a C.sub.1 to C.sub.6 straight,
branched or unsaturated alkyl group optionally substituted with 1-2
R.sub.x groups and optionally interrupted by one of the following
groups O, S, SO.sub.2, --C(O)--, --C(O)--NR.sup.a--, --CO.sub.2--;
6
[0029] are independently selected from:
[0030] (1) a C.sub.6 to C.sub.10 aryl group;
[0031] (2) a C.sub.3 to C.sub.8 alicyclic group;
[0032] (3) a C.sub.2 to C.sub.9 heteroaryl group, which contains 1
to 3 heteroatoms, 0 to 3 of which heteroatoms are nitrogen and 0 to
1 of which are oxygen or sulfur,
[0033] (4) a C.sub.3 to C.sub.8 heterocyclic group, which contains
1 to 2 heteroatoms, 0 to 1 of which heteroatoms are nitrogen, and 0
to 2 of which are oxygen or sulfur;
[0034] provided that at least one Rq group is present in R.sup.1 or
R.sup.2 and that when more than one Rq is present the total number
of cationic nitrogen atoms does not exceed 8; the total number of
cationic nitrogen atoms can be charged balanced by M.sup.1 and/or
M.sup.2 or by M.sup.1 and/or M.sup.2 in combination with an
appropriate number of Y.sup.-;
[0035] wherein:
[0036] Rq is --E--Q.sup.+Y.sup.-;
[0037] Y.sup.- is a pharmaceutically acceptable anionic group;
[0038] E is --(CH.sub.2).sub.m--X--(CH.sub.2).sub.n--;
[0039] m is 0 to 6;
[0040] n is 0 to 6 (but when E is attached to an aromatic ring n is
1-6);
[0041] X is a bond, O, S, SO.sub.2, --C(O)--, --C(O)--N(R.sup.a)--,
--C(O)O--, --CH=CH-- or--C--C--, provided that when X is O, S,
--C(O)--N(R.sup.a)-- or --C(O)O--, then n is 2 to 6
[0042] and Q.sup.+, attached to the (CH.sub.2)n terminus of E
is:
[0043] (1) a cationic group selected from the following: 7
[0044] wherein:
[0045] R.sup.u and R.sup.v are independently hydrogen or C.sub.1-6
alkyl optionally substituted with 1 to 2 R.sup.y;
[0046] R.sup.w is hydrogen or C.sub.1-6 alkyl optionally
substituted with 1 to 2 R.sub.x;
[0047] R.sup.u and R.sup.v when bonded to the same nitrogen atom
may together be a C.sub.3-6 alkyl radical, which when taken
together with the intervening atoms form a ring;
[0048] Two R.sup.u groups on separate nitrogen atoms may together
comprise a C.sub.2-5 alkyl radical, which when taken together with
the intervening atoms form a ring;
[0049] R.sup.u, R.sup.v and R.sup.w when bonded to the same
nitrogen atom may together form a C.sub.6-10 tertiary alkyl
radical, which with N.sup.+ forms a bicyclic ring;
[0050] (2) A dicationic group: 8
[0051] wherein:
[0052] E.sup.1 is --(CH.sub.2).sub.p--Z--(CH.sub.2).sub.r--;
[0053] p and r are independently 1 to 4;
[0054] Z is a bond, O, S, SO.sub.2, --C(O)--, --C(O)O--**,
--CH.dbd.CH--, --C.dbd.C--, or 9
[0055] Provided that when Z is O or S, p is 2 to 4 and r is 2 to 4
and when Z is 10
[0056] or --C(O)O--**, r is 2 to 4;
[0057] wherein ** denotes the atom which is bonded to the
--(CH.sub.2).sub.r-- moiety of E.sup.1 above;
[0058] Q.sup.1 is selected from the following: 11
[0059] Q.sup.2 is selected from the following: 12
[0060] R.sup.u, R.sup.v and R.sup.w are independently selected and
defined as above,
[0061] And in addition, in the case where two R.sup.u groups on
separate nitrogen atoms are joined to form a ring as defined above,
two R.sup.v groups on the same two separate nitrogen atoms may also
comprise a C.sub.1-5 alkyl radical to form together with the
intervening atoms a bicyclic ring; an example of such is: 13
[0062] (3) A tricationic group selected from the following: 14
[0063] wherein:
[0064] Each E.sup.1 is as defined above, but selected
independently;
[0065] Each Q.sup.1 is as defined above, but selected
independently;
[0066] Each Q.sup.2 is as defined above, but selected
independently;
[0067] R.sup.u, R.sup.v and R.sup.w are defined as in the
definition of Q.sup.+ item (2) above and selected independently;
or
[0068] (4) A tetracationic group selected from the following:
15
[0069] wherein:
[0070] Each E.sup.1 is as defined above, but selected
independently;
[0071] Each Q.sup.1 is as defined above, but selected
independently;
[0072] Each Q.sup.2 is as defined above, but selected
independently;
[0073] R.sup.u, R.sup.v and R.sup.w are defined as in the
definition of Q.sup.+ item (2) above and selected
independently;
[0074] where each R.sub.x is independently selected from the group
consisting of:
[0075] (a) F, Cl, Br, I,
[0076] (b) CF.sub.3,
[0077] (c) OR.sup.b,
[0078] (d) CN,
[0079] (e) --C(O)--R.sup.c,
[0080] (f) --S(O.sub.2)--R.sup.f,
[0081] (g) --C(O)--OR.sup.a
[0082] (h) --O--C(O)--R.sup.c,
[0083] (i) --S--R.sup.b,
[0084] (j) --N(R.sup.a)--C(O)--R.sup.c, 16
[0085] (q) --N(R.sup.a)--C(O)--OR.sup.f,
[0086] (r) --S(O)--R.sup.f,
[0087] (s) --N(R.sup.a)--S(O.sub.2)--R.sup.f,
[0088] (t) NO.sub.2, and
[0089] (u) C.sub.1 to C.sub.8 straight, branched or unsaturated
alkyl optionally substituted with one of the substituents (a)
through (t) above;
[0090] (v) --CH.sub.2-aryl wherein the aryl is optionally
substituted with one of the substituents (a) through (t) above;
[0091] or two adjacent R.sub.x groups on an aromatic ring may
consist of the following divalent moiety, --O--CH.sub.2--O--;
[0092] wherein:
[0093] R.sup.a is H, C.sub.1 to C.sub.6 alkyl optionally
substituted with R.sup.y;
[0094] R.sup.b is H, C.sub.1 to C.sub.6 alkyl optionally
substituted with R.sup.y, CH.sub.2-aryl, or aryl, said aryls
optionally substituted with 1-2 R.sup.y groups;
[0095] R.sup.c is H, C.sub.1 to C.sub.6 alkyl optionally
substituted with R.sup.y, CF.sub.3, or aryl, said aryl optionally
substituted with 1-2 R.sup.y groups;
[0096] R.sup.d and R.sup.e are independently hydrogen, C.sub.1 to
C.sub.4 alkyl optionally substituted with R.sup.y, or R.sup.d and
R.sup.e taken together may represent a 3 to 5-membered alkyl
radical to form a ring, or R.sup.d and R.sup.e taken together may
represent a 2 to 4-membered alkyl radical interrupted by O, S, SO
or SO.sub.2 to form a ring;
[0097] R.sup.f is C.sub.1 to C.sub.6 alkyl optionally substituted
with R.sup.y, or aryl, said aryl optionally substituted with 1-2
R.sup.y groups; and
[0098] R.sup.y is --OH, --OCH.sub.3, OCONH.sub.2, OCOCH.sub.3, CHO,
COCH.sub.3, CO.sub.2CH.sub.3, CONH.sub.2, CN, SOCH.sub.3,
SO.sub.2CH.sub.3, SO.sub.2NH.sub.2, F, Cl, Br, I or CF.sub.3.
[0099] The invention is intended to include all of the isomeric
forms of the compounds of formula I, including racemic,
enantiomeric and diastereomeric forms.
DETAILED DESCRIPTION OF THE INVENTION
[0100] The invention is described herein in detail using the terms
defined below unless otherwise specified.
[0101] The term "alkyl" refers to a monovalent alkane (hydrocarbon)
derived radical containing from 1 to 16 carbon atoms unless
otherwise defined. It may be straight or branched. Preferred alkyl
groups include methyl, ethyl, propyl, isopropyl, butyl and hexyl.
When substituted, alkyl groups may be substituted with up to 3
substituent groups selected from R.sub.x, as defined, and up to 2
substituent groups selected from Rq, as defined, at any available
point of attachment. When the alkyl group is said to be substituted
with an alkyl group, this is used interchangeably with "branched
alkyl group". When the alkyl chain is interrupted by a group, eg.
O, this may occur between any two saturated carbons of the alkyl
chain.
[0102] The term unsaturated alkyl refers to "alkenyl" or "atkynyl".
The term "alkenyl" refers to an unsaturated alkyl such as a
hydrocarbon radical, straight or branched containing from 2 to 16
carbon atoms and at least one carbon to carbon double bond.
Preferred alkenyl groups include propenyl, hexenyl and butenyl. The
term "alkynyl" refers to an unsaturated alkyl such as a hydrocarbon
radical straight or branched, containing from 2 to 16 carbon atoms
and at least one carbon to carbon triple bond. Preferred alkynyl
groups include propynyl, hexynyl and butynyl.
[0103] The term "alicyclic" refers to non-aromatic monocyclic or
bicyclic C.sub.3-C.sub.10 hydrocarbons, including unsaturated,
which can be substituted with 0-3 groups of R.sub.x. Examples of
said groups include cycloalkyls such as cyclohexyl, cyclopentyl,
bicyclo[2.2.1]heptyl, bicyclo[2.2.1]hepta-2,5-dienyl,
bicyclo[2.2.2]octyl, bicyclo[2.2.2]octa-2,5-dienyl.
[0104] The term "alkylidene" refers to an alkyl group which is
attached through two bonds on the same carbon atom of the alkyl
group to a single attachment atom Examples of said groups include
methylene, ethylidene, isopropylidene and the like.
[0105] Examples of when R.sup.d and R.sup.e are taken together
along with the adjacent nitrogen atom to represent a 3 to 5
membered alkyl radical forming a ring or a 2 to 4 membered alkyl
radical interrupted by O, S, SO, SO.sub.2, to form a ring are
pyrrolidinyl, piperidinyl, morpholinyl and the like.
[0106] The term "heterocyclic" refers to a monocyclic non-aromatic
moiety containing 3-8 ring atoms or a bicyclic non-aromatic moiety
containing 6-10 ring atoms, at least one of which ring atoms is a
heteroatom selected from nitrogen, oxygen and sulfur and where one
additional ring atom may be oxygen or sulfur. Examples of
heterocyclic groups are furanyl, pyranyl, morpholinyl, dioxanyl and
quinuclidinyl: 17
[0107] Aryl refers to aromatic rings e.g., phenyl, substituted
phenyl and the like, as well as rings which are fused, e.g.,
naphthyl, phenanthrenyl fluorenonyl and the like. An aryl group
thus contains at least one ring having at least 6 atoms, with up to
three such rings being present, containing up to 14 atoms therein,
with alternating (resonating) double bonds between adjacent carbon
atoms. The preferred aryl groups are phenyl, naphthyl, and
fluorenone. Aryl groups may likewise be substituted as defined.
Preferred substituted aryls include phenyl, fluorenonyl and
naphthyl.
[0108] The term "heteroaryl" (Het) refers to a monocyclic aromatic
group having 5 or 6 ring atoms, a bicyclic aromatic group having 8
to 10 atoms, or tricyclic having 12-14 ring atoms, containing at
least one heteroatom, O, S or N, in which a carbon or nitrogen atom
is the point of attachment, and in which one or two additional
carbon atoms is optionally replaced by a heteroatom selected from O
or S, and in which from 1 to 3 additional carbon atoms are
optionally replaced by nitrogen heteroatoms, said heteroaryl group
being optionally substituted as described herein. Examples of this
type are pyrrole, pyridine, oxazole, thiazole, dibenzofuran,
dibenzothiophene, carbazole, phenanthrene, anthracene,
dibenzothiophene sulfone, fluorenone, quinoline and oxazine.
Additional nitrogen atoms may be present together with the first
nitrogen and oxygen or sulfur, giving, e.g., thiadiazole. Examples
include the following: 1819
[0109] Heteroarylium refers to heteroaryl groups bearing a
quaternary nitrogen atom and thus a positive charge. Non-limiting
examples include the following: 20
[0110] When a charge is shown on a particular nitrogen atom in a
ring which contains one or more additional nitrogen atoms, it is
understood that the charge may reside on a different nitrogen atom
in the ring by virtue of charge resonance that occurs. 21
[0111] Similar charge resonance may occur in amidinium and
guanidimum groups: 22
[0112] The term "heteroatom" means O, S or N, selected on an
independent basis.
[0113] Halogen and "halo" refer to bromine, chlorine, fluorine and
iodine.
[0114] The term "pro-drug" refers to compounds with a removable
group attached to one or both of the carboxyl groups of compounds
of formula I (e.g. biolabile esters). Groups which are useful in
forming pro-drugs should be apparent to the medicinal chemist from
the teachings herein. Examples include pivaloyloxymethyl,
acetoxymethyl, phthalidyl, indanyl and methoxymethyl, and others
described in detail in U.S. Pat. No. 4,479,947. These are also
referred to as "biolabile esters".
[0115] The term "hydrate" is used in the conventional sense to
include the compounds of formula I in physical association with
water.
[0116] When a group is termed "substituted", unless otherwise
indicated, this means that the group contains from 1 to 3
substituents thereon.
[0117] A bond terminated by a wavy line is used herein to signify
the point of attachment of a substituent group. This usage is
illustrated by the following example: 23
[0118] The terms "quaternary nitrogen" and "cationic nitrogen"
refer to tetravalent, positively charged nitrogen atoms including,
e.g., the positively charged nitrogen in a tetraalkylammonium group
(e. g. --N.sup.+R.sup.uR.sup.vR.sup.w), heteroarylium, (e.g.,
N-methyl-imidazolium), amidinium, guanidinium, basic nitrogens
which are protonated at physiological pH, and the like. A "cationic
group" is a moiety which contains at least one such quaternary
nitrogen atom. Cationic groups thus encompass positively charged
nitrogen-containing groups, as well as basic nitrogens which are
protonated at physiologic pH. The terms dicationic, tricationic and
tetracationic refer to groups which contain 2, 3 or 4 positively
charged nitrogen atoms, respectively.
[0119] When a functional group is termed "protected", this means
that the group is in modified form to preclude undesired side
reactions at the protected site. Suitable protecting groups for the
compounds of the present invention will be recognized from the
present application taking into account the level of skill in the
art, and with reference to standard textbooks, such as Greene, T.
W. et al. Protective Groups in Organic Synthesis Wiley, New York
(1991). Examples of suitable protecting groups are contained
throughout the specification.
[0120] In some of the compounds of the present invention suitable
protecting groups represents hydroxyl-protecting, amine-protecting
or carboxyl-protecting groups. Such conventional protecting groups
consist of groups, which are used to protectively block the
hydroxyl, amine or carboxyl group during the synthesis procedures
described herein. These conventional blocking groups are readily
removable, i.e., they can be removed, if desired, by procedures
which will not cause cleavage or other disruption of the remaining
portions of the molecule. Such procedures include chemical and
enzymatic hydrolysis, treatment with chemical reducing or oxidizing
agents under mild conditions, treatment with a transition metal
catalyst and a nucleophile and catalytic hydrogenation.
[0121] Examples of carboxyl protecting groups include allyl,
benzhydryl, 2-naphthylmethyl, benzyl, silyl such as
t-butyldimethylsilyl (TBDMS), phenacyl, p-methoxybenzyl,
o-nitrobenzyl, p-methoxyphenyl, p-nitrobenzyl, 4-pyridylmethyl and
t-butyl.
[0122] Examples of suitable amine protecting groups include
9-fluorenylmethoxycarbonyl, p-nitrobenzyloxycarbonyl,
benzyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl,
2,2,2-trichloroethyloxycarbonyl and the like.
[0123] Examples of suitable hydroxyl protecting groups include
triethylsilyl, t-butyldimethylsilyl, o-nitrobenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl,
t-butyloxycarbonyl, 2,2,2-trichloroethyloxycarbonyl and the
like.
[0124] With respect to M.sup.1 and/or M.sup.2, this represents a
carboxylic hydrogen, a carboxylate anion (M represents a negative
charge), a pharmaceutically acceptable ester (M represents an ester
forming group) or a pharmaceutically acceptable cation. When
M.sup.1 and/or M.sup.2 is a negative charge it can be used to
provide the necessary charge balance in a compound with one or more
positive charges. Likewise, when M.sup.1 and/or M.sup.2 is a
negative charge it can be balanced by the appropriate number of
counterions, e.g., an alkali metal cation such as sodium or
potassium. Other pharmaceutically acceptable counterions may be
calcium, magnesium, zinc, ammonium, or alkylammonium cations such
as tetramethylammonium, tetrabutylammonium, choline,
triethylhydroammonium, meglumine, triethanolhydroammonium, etc.
[0125] For the purposes of this invention, all compounds have at
least one Rq substituent containing at least one cationic nitrogen.
Preferably 2 to 8 cationic nitrogens, more preferably 2 to 4
cationic nitrogens and most preferably 3 to 4 cationic nitrogens
are present. The compounds are balanced with one or more, as
necessary, of a charge balancing group Y.sup.-. Alternatively, the
compounds can be balanced using M.sup.1 and/or M.sup.2 as the
charge balancing group with or without the use of Y.sup.-. Examples
of cases where a charge balancing group is required are quaternized
substituents such as heteroarylium, --N.sup.+R.sup.uR.sup.v-E-
.sup.1-Q.sup.1, --N.sup.+R.sup.uR.sup.vR.sup.w, and the like.
[0126] Additionally, all compounds having one or more anions are
counter balanced with one or more, as necessary, charge balancing
cations.
[0127] The compounds of the present invention are useful per se and
in their pharmaceutically acceptable salt and ester forms are
potentiators for the treatment of bacterial infections in animal
and human subjects. The term "pharmaceutically acceptable ester,
salt or hydrate", refers to those salts, esters and hydrated forms
of the compounds of the present invention which would be apparent
to the pharmaceutical chemist. For example, those which are
substantially non-toxic and which may favorably affect the
pharmacokinetic properties of said compounds, such as palatability,
absorption, distribution, metabolism and excretion. Other factors,
more practical in nature, which are also important in the
selection, are cost of the raw materials, ease of crystallization,
yield, stability, solubility, hygroscopicity and flowability of the
resulting bulk drug. Conveniently, pharmaceutical compositions may
be prepared from the active ingredients in combination with
pharmaceutically acceptable carriers. Thus, the present invention
is also concerned with pharmaceutical compositions and methods of
treating bacterial infections utilizing as an active ingredient the
novel carbapenemase compounds.
[0128] The pharmaceutically acceptable salts referred to above also
include acid addition salts. Thus, the Formula I compounds can be
used in the form of salts derived from inorganic or organic acids.
Included among such salts are the following:
[0129] acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,
glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, methanesulfonate, 2-naphthalenesulfonate,
nicotinate, oxalate, pamoate, pectinate, persulfate,
3-phenylpropionate, picrate, pivalate, propionate, succinate,
tartrate, thiocyanate, tosylate and undecanoate.
[0130] Acid addition salts of the compounds of formula I include
compounds that contain a protonated, basic moiety in R.sup.q.
Compounds containing a basic moiety in R.sup.q are capable of
protonation in aqueous media near pH 7, so that the basic moiety
can exist as an equilibrium mixture of its neutral form and acid
addition (protonated) form. The more basic the group, the greater
the degree of protonation near pH 7. All such compounds are
included in the present invention.
[0131] The pharmaceutically acceptable cations which can form a
salt with one or both of the carboxyls (CO.sub.2M.sup.1 and
CO.sub.2M.sup.2) of the compounds of formula I are known to those
skilled in the art. Examples include those where M.sup.1 and
M.sup.2 independently can be alkali metals such as sodium,
potassium and the like, ammonium and the like.
[0132] The pharmaceutically acceptable esterifying groups are such
as would be readily apparent to a medicinal chemist, and include,
for example, those described in detail in U.S. Pat. No. 4,309,438.
Included within such pharmaceutically acceptable esters are those
which are hydrolyzed under physiological conditions, such as
pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and
methoxymethyl, and others described in detail in U.S. Pat. No.
4,479,947. These are also referred to as "biolabile esters".
[0133] Biolabile esters are biologically hydrolizable, and may be
suitable for oral administration, due to good absorption through
the stomach or intestinal mucosa, resistance to gastric acid
degradation and other factors. Examples of biolabile esters include
compounds in which M.sup.1 and/or M.sup.2 represents an
alkoxyalkyl, alkylcarbonyloxyalkyl, alkoxycarbonyloxyalkyl,
cycloalkoxylalkyl, alkenyloxyalkyl, aryloxyalkyl, alkoxyaryl,
alkylthioalkyl, cycloalkylthioalkyl, alkenylthioalkyl,
arylthioalkyl or alkylthioaryl group. The following M.sup.1 and/or
M.sup.2 species are examples of biolabile ester forming moieties:
acetoxymethyl, 1-acetoxyethyl, 1-acetoxypropyl, pivaloyloxymethyl,
1-isopropyloxycarbonyloxyethyl, 1-cyclohexyloxycarbonyloxyethyl,
phthalidyl and (2-oxo-5-methyl-1,3-dioxolen-4-yl)methyl.
[0134] Y.sup.- can be present or absent as necessary to maintain
the appropriate charge balance. When present, these represent
pharmaceutically acceptable counterions. Most anions derived from
inorganic or organic acids are suitable. Representative examples of
such counterions are the following: acetate, adipate,
aminosalicylate, anhydromethylenecitrate, ascorbate, aspartate,
benzoate, benzenesulfonate, bromide, citrate, camphorate,
camphorsulfonate, chloride, estolate, ethanesulfonate, fumarate,
glucoheptanoate, gluconate, glutamate, lactobionate, malate,
maleate, mandelate, methanesulfonate, pantothenate, pectinate,
phosphate/diphosphate, polygalacturonate, propionate, salicylate,
stearate, succinate, sulfate, tartrate and tosylate. Other suitable
anionic species will be apparent to the ordinarily skilled
chemist.
[0135] Likewise, when more than one negative charge is necessary to
maintain charge neutrality, the counterion indicator may represent
a specie with more than one negative charge, such as malonate,
tartrate or ethylenediaminetetraacetate (EDTA), or two or more
monovalent anions, such as chloride, etc. When a multivalent
negatively charged counterion is present with a compound of formula
I which bears a net single positive charge, an appropriate number
of molar equivalents of the anionic species can be found in
association therewith to maintain the overall charge balance and
neutrality.
[0136] Some of the compounds of formula I may be crystallized or
recrystallized from solvents such as organic solvents. In such
cases solvates may be formed. This invention includes within its
cope stoichiometric solvates including hydrates as well as
compounds containing variable amounts of solvents such as water
that may be produced by processes such as lyophilization. The
compounds of formula I may be prepared in crystalline form by for
example dissolution of the compound in water, preferably in the
minimum quantity thereof, followed by admixing of this aqueous
solution with a water miscible organic solvent such as a lower
aliphatic ketone such as a di-(C.sub.1-6) alkyl ketone, or a
(C.sub.1-6) alcohol, such as acetone or ethanol.
[0137] A subset of compounds of formula I which is of interest
relates to those compounds where M.sup.1 and M.sup.2 are
independently hydrogen or negative charge, said negative charge(s)
balanced by the appropriate number of counter balancing ions, and
all other variables are as described above.
[0138] Another subset of compounds of formula I which is of
interest relates to those compounds where R.sup.1 and/or R.sup.2
represents a C.sub.1 to C.sub.16 straight, branched or unsaturated
alkyl group substituted with 0 to 2 R.sup.q, and substituted with 0
to 3 R.sub.x groups and optionally interrupted by one of the
following O, S, SO.sub.2, --C(O)--, --C(O)--NR.sup.a-- and
--CO.sub.2-, provided that at least one of R.sup.1 or R.sup.2
contains an R.sup.q and all other variables are described as
above.
[0139] Another subset of compounds of formula I which is of
interest relates to those compounds where R.sup.1 and/or R.sup.2
represents C.sub.4-12 straight, branched or unsaturated alkyl group
optionally substituted with 1-2 R.sub.x and optionally substituted
with 1-2 R.sup.q groups, provided that at least one of R.sup.1 or
R.sup.2 contains an R.sup.q, wherein all other variables are as
described above.
[0140] Another subset of compounds of formula I which is of
interest relates to those compounds where R.sup.1 and/or R.sup.2
represents a group of the formula: 24
[0141] wherein at least one R.sup.q is present on R.sup.1 or
R.sup.2 and all other variables are defined as above.
[0142] Another subset of compounds of formula I which is of
interest relates to those compounds where R.sup.1 and/or R.sup.2
represents a group of the formula: 25
[0143] wherein at least one R.sup.q group is present on R.sup.1 or
R.sup.2 and all other variables are defined as above.
[0144] Another subset of compounds of formula I which is of
interest relates to those compounds where the relative and absolute
stereochemistry is: 26
[0145] Still another subset of compounds of formula I which is of
interest relates to those compounds where R.sup.1 and/or R.sup.2
represents a group of the formula: 27
[0146] wherein A is (CH.sub.2).sub.1-5 and 28
[0147] is phenyl, naphthyl, cyclohexyl or dibenzofuranyl, provided
that at least one of R.sup.1 or R.sup.2 contains an R.sup.q and all
other variables are as originally defined.
[0148] Still another subset of compounds of formula I that is of
interest relates to those compounds where R.sup.1 or R.sup.1
represents a group of the formula: 29
[0149] wherein
[0150] A is (CH.sub.2).sub.1-3, A' is a single bond, --O-- or
(CH.sub.2).sub.1-2 30
[0151] independently represent phenyl, thienyl, pyridyl, furanyl or
cyclohexyl
[0152] Yet another subset of compounds of formula I, that is of
interest relates to those compounds where one of R.sup.1 or R.sup.2
is C.sub.4-8 straight, branched or unsaturated alkyl optionally
substituted with 1 to 2 R.sub.x or a group of the formula: 31
[0153] where A is (CH.sub.2).sub.1-2 and 32
[0154] is phenyl, cyclopentyl or cyclohexyl and the other of
R.sup.1 or R.sup.2 is
[0155] i) a C.sub.7-12 alkyl group substituted with R.sup.q,
[0156] ii) a group of the formula: 33
[0157] where A is (CH.sub.2).sub.1-2, A' is a single bond, 34
[0158] is phenyl, thienyl or cyclohexyl and 35
[0159] is phenyl, thienyl or pyridyl, or
[0160] iii) a group of the formula: 36
[0161] where A is (CH.sub.2.sub.1-3, 37
[0162] is phenyl or thienyl and R.sup.q is
--(CH.sub.2).sub.2-6-Q.sup.+Y.s- up.- and all other variables are
as originally defined.
[0163] Still another subset of compounds of formula I that is of
interest relates to those compounds where:
[0164] R.sup.1 is C.sub.5-7 alkyl substituted with 0 to 2 R.sub.x
goups, 38
[0165] R.sup.2 is C.sub.7-10 alkyl substituted with 1R.sup.q group
and 0 to 2 R.sub.x groups, 39
[0166] and all other variables are as described above.
[0167] Still another subset of compounds of formula I that is of
interest relates to those compounds where:
[0168] R.sup.1 is: 40
[0169] R.sup.2 is: 41
[0170] and all other variables are as described above.
[0171] A preferred subset of R.sub.x is R.sup.y.
[0172] It is preferred that a total of one or two R.sup.q groups
are present in R.sup.1 and R.sup.2 containing a total of 2 to 6
cationic nitrogen atoms. It is more preferred that a single R.sup.q
substituent is present containing a tricationic or tetracationic
Q.sup.+ group. A more preferred R.sup.q is --E-Q.sup.+Y.sup.-
wherein E is (CH.sub.2).sub.0-6 or
--C(O)--N(R.sup.a)--(CH.sub.2).sub.2-4, and Q.sup.+ is a
tricationic or tetracationic group.
[0173] Preferred tricationic Q.sup.+ groups are: 42
[0174] wherein E.sup.1 is (CH.sub.2).sub.2-4 or
--(CH.sub.2)--C(O)--N(R.su- p.a)--(CH.sub.2).sub.2-4-- and R.sup.a,
Q.sup.1 and Q.sup.2 are as previously defined.
[0175] More preferred tricationic Q.sup.+ groups are: 43
[0176] wherein R.sup.a, R.sup.u, R.sup.v, and R.sup.w are
independently selected as defined above.
[0177] Preferred tetracationic Q.sup.+ groups are: 44
[0178] wherein E.sup.1 is (CH.sub.2).sub.2-4 or
--(CH.sub.2)--C(O)--N(R.su- p.a)--(CH.sub.2).sub.2-4-- and R.sup.a,
Q.sup.1, Q.sup.2, and R.sup.w are as defined above.
[0179] More preferred tetracationic Q.sup.+ groups are: 45
[0180] wherein R.sup.a, R.sup.u, R.sup.v, and R.sup.w are as
defined above.
[0181] Preferred. Y.sup.- groups are chloride, bromide, acetate,
citrate, succinate, phosphate, maleate, tartrate and sulfate.
[0182] The compounds of the invention, which are succinic acids or
derivatives thereof, can be formulated in pharmaceutical
compositions by combining the compound with a pharmaceutically
acceptable carrier. Examples of such carriers are set forth below.
The compounds of formula I have metallo-.beta.-lactamase inhibitory
properties, and are useful when combined with a .beta.-lactam
antibiotic for the treatment of infections in animals, especially
mammals, including humans. The compounds may be used, for example,
in the treatment of infections of, amongst others, the respiratory
tract, urinary tract and soft tissues and blood.
[0183] The compounds may be employed in powder or crystalline form,
in liquid solution, or in suspension. They may be administered by a
variety of means; those of principal interest include: topically,
orally and parenterally by injection (intravenously or
intramuscularly).
[0184] Compositions for injection, a preferred route of delivery,
may be prepared in unit dosage form in ampules, or in multidose
containers. The injectable compositions may take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and may contain various formulating agents. Alternatively, the
active ingredient may be in powder (lyophillized or
non-lyophillized) form for reconstitution at the time of delivery
with a suitable vehicle, such as sterile water. In injectable
compositions, the carrier is typically comprised of sterile water,
saline or another injectable liquid, e.g., peanut oil for
intramuscular injections. Also, various buffering agents,
preservatives and the like can be included.
[0185] Topical applications may be formulated in carriers such as
hydrophobic or hydrophilic bases to form ointments, creams,
lotions, in aqueous, oleaginous or alcoholic liquids to form paints
or in dry diluents to form powders.
[0186] Oral compositions may take such forms as tablets, capsules,
oral suspensions and oral solutions. The oral compositions may
utilize carriers such as conventional formulating agents, and may
include sustained release properties as well as rapid delivery
forms.
[0187] The compounds of the instant invention are
metallo-.beta.-lactamase inhibitors, which are intended for use in
pharmaceutical compositions. Accordingly, it is preferable that the
metallo-.beta.-lactamase inhibitors are provided in substantially
pure form, for example at least about 60% to about 75% pure,
preferably about 85% to about 95% pure and most preferably about
98% or more pure (% are on a weight for weight basis). Impure
preparations of the compounds may be used for preparing the more
pure forms used in pharmaceutical compositions.
[0188] The dosage to be administered depends to a large extent upon
the condition and size of the subject being treated, the route and
frequency of administration, the sensitivity of the pathogen to the
particular compound selected, the virulence of the infection and
other factors. Such matters, however, are left to the routine
discretion of the physician according to principles of treatment
well known in the antibacterial arts. Another factor influencing
the precise dosage regimen, apart from the nature of the infection
and peculiar identity of the individual being treated, is the
molecular weight of the compound.
[0189] The compositions for human delivery per unit dosage, whether
liquid or solid, may contain from about 0.01% to as high as about
99% of active material, the preferred range being from about
10-60%. The composition will generally contain from about 15 mg to
about 2.5 g of the active ingredient; however, in general, it is
preferable to employ dosage amounts in the range of from about 250
mg to 1000 mg. In parenteral administration, the unit dosage will
typically include the pure compound in sterile water solution or in
the form of a soluble powder intended for solution, which can be
adjusted to neutral pH and isotonic.
[0190] The invention described herein also includes a method of
treating a bacterial infection in a mammal in need of such
treatment comprising administering to said mammal a compound of
formula I in conjunction with a .beta.-lactam antibiotic such as a
carbapenem, penicillin or cephalosporin in an effective
combination.
[0191] The preferred methods of administration of the Formula I
compounds include oral and parenteral, e.g., i.v., infusion, i.v.
bolus and i.m. injection.
[0192] The compounds of formula I may suitably be administered to
the patient at a daily dosage of from 0.7 to 50 mg/kg of body
weight. For an adult human (of approximately 70 kg body weight),
from 50 to 3000 mg, preferably from 100 to 1000 mg, of a compound
according to the invention may be administered daily, suitably in
from 1 to 6, preferably from 2 to 4, separate doses. Higher or
lower dosages may, however, be used in accordance with clinical
practice.
[0193] The compounds may be used in combination with antibiotic
agents for the treatment of infections caused by
metallo-.beta.-lactamase producing strains, in addition to those
infections which are subsumed within the antibacterial spectrum of
the antibiotic agent. Metallo-.beta.-lactamase producing strains
include: Bacillus cereus, Bacteroides fragilis, Aeromonas
hydrophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia
marcescens, Stenotrophomonas maltophilia, Shigella flexneri,
Legionella gormanii, Chryseobacterium meningosepticum,
Chryseobacterium indologenes, Acinetobacter baumannii, Citrobacter
freundii, and Aeromonas veronii.
[0194] In accordance with the instant invention, it is generally
advantageous to use a compound of formula I in admixture or
conjuction with a carbapenem, penicillin, cephalosporin or other
.beta.-lactam antibiotic or prodrug. It also advantageous to use a
compound of formula I in combination with one or more .beta.-lactam
antibiotics, because of the metallo-.beta.-lactamase inhibitory
properties of the compounds. In this case, the compound of formula
I and the .beta.-lactam antibiotic can be administered separately
or in the form of a single composition containing both active
ingredients.
[0195] Carbapenems, penicillins, cephalosporins and other
.beta.-lactam antibiotics suitable for co-administration with the
compounds of Formula I, whether by separate administration or by
inclusion in the compositions according to the invention, include
both those known to show instability to or to be otherwise
susceptible to metallo-.beta.-lactamases and also known to have a
degree of resistance to metallo-.beta.-lactamase.
[0196] When the compounds of Formula I are combined with
antibiotics such as carbapenems dehydropeptidase (DHP) inhibitors
may also be combined. Many carbapenems are susceptible to attack by
a renal enzyme known as DHP. This attack or degradation may reduce
the efficacy of the carbapenem antibacterial agent. Inhibitors of
DHP and their use with carbapenems are disclosed in, e.g.,
(European Patent 0007614, filed Jul. 24, 1979 and application
number 82107174.3, filed Aug. 9, 1982. A preferred DHP inhibitor is
7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropane-
carboxamide)-2-heptenoic acid or a useful salt thereof. Thus,
compounds of the present invention in combination with a carbapenem
such as imipenem and a DHP inhibitor such as, cilastatin is
contemplated within the scope of this invention.
[0197] A serine .beta.-lactamase inhibitor such as clavulanic acid,
sulbactam or tazobactam may also be co-administered with the
compound of the invention and .beta.-lactam antibiotics, either by
separate administration, or co-formulation with one, other or both
of the compounds of the invention and the .beta.-lactam
antibiotic.
[0198] Examples of carbapenems that may be co-administered with the
compounds of formula I include imipenem, meropenem, biapenem, (4R,
5S, 6S)-3-[3S,
5S)-5-(3-carboxyphenyl-carbamoyl)pyrrolidin-3-ylthio]-6-(1R)-1-
-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic
acid, (1S, 5R,
6S)-2-(4-(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]-
oct-1-yl)-ethyl(1,8-naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylca-
rbapen-2-em-3-carboxylate chloride, BMS181139
([4R-[4alpha,5beta,6beta(R*)-
]]-4-[2-[(aminoiminomethyl)amino]ethyl]-3-[(2-cyanoethyl)thio]-6-(1-hydrox-
yethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid),
BO2727 ([4R-3[3S*,5S*(R*)],
4alpha,5beta,6beta(R*)]]-6-(1-hydroxyethyl)-3-[[5-[1-
-hydroxy-3-(methylamino)propyl]-3-pyrrolidinyl]thio]-4-methyl-7-oxo-1-azab-
icyclo[3.2.0]hept-2-ene-2-carboxylic acid monohydrochloride), E1010
((1R, 5S,
6S)-6-[1(R)-hydroxymethyl]-2-[2(S)-[1(R)-hydroxy-1-[pyrrolidin-3(R)-y-
l]methyl]pyrrolidin-4(S)-ylsulfanyl]-1-methyl-1-carba-2-penem-3-carboxylic
acid hydrochloride), S4661
((1R,5S,6S)-2-[(3S,5S)-5-(sulfamoylaminomethyl- )
pyrrolidin-3-yl]thio-6-[(1R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-car-
boxylic acid) and
(1S,5R,6S)-1-methyl-2-{7-[4-(aminocarbonylmethyl)-1,4-di-
azoniabicyclo(2.2.2)octan-1yl]-methyl-fluoren-9-on-3-yl}-6-(1R-hydroxyethy-
l)-carbapen-2-em-3-carboxylate chloride.
[0199] Examples of penicillins suitable for co-administration with
the compounds according to the invention include benzylpenicillin,
phenoxymethylpenicillin, carbenicillin, azidocillin, propicillin,
ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin,
pirbenicillin, azloccillin, mezlocillin, sulbenicillin,
piperacillin, and other known penicillins. The penicillins may be
used in the form of pro-drugs thereof; for example as in vivo
hydrolysable esters, for example the acetoxymethyl,
pivaloyloxymethyl, .alpha.-ethoxycarbonyloxy-e- thyl and phthalidyl
esters of ampicillin, benzylpenicillin and amoxycillin; as aldehyde
or ketone adducts of penicillins containing a
6-.alpha.-aminoacetamido side chain (for example hetacillin,
metampicillin and analogous derivatives of amoxycillin); and as
a-esters of carbenicillin and ticarcillin, for example the phenyl
and indanyl .alpha.-esters.
[0200] Examples of cephalosporins that may be co-administered with
the compounds according to the invention include, cefatrizine,
cephaloridine, cephalothin, cefazolin, cephalexin, cephacetrile,
cephapirin, cephamandole nafate, cephradine, 4-hydroxycephalexin,
cephaloglycin, cefoperazone, cefsulodin, ceftazidime, cefuroxime,
cefmetazole, cefotaxime, ceftriaxone, and other known
cephalosporins, all of which may be used in the form of pro-drugs
thereof.
[0201] Examples of .beta.-lactam antibiotics other than penicillins
and cephalosporins that may be co-administered with the compounds
according to the invention include aztreonam, latamoxef
(Moxalactam-trade mark), and other known .beta.-lactam antibiotics
such as carbapenems like imipenem, meropenem or (4R, 5S,
6S)-3-[(3S,5S)-5-(3-carboxyphenylcarbamoy-
l)pyrrolidin-3-ylthio]-6-(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[-
3.2.0]hept-2-ene-2-carboxylic acid, all of which may be used in the
form of pro-drugs thereof
[0202] Preferred carbapenems are imipenem, meropenem and (4R, 5S,
6S)-3-[(3S,5S)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-(1R)-1--
hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic
acid.
[0203] Particularly suitable penicillins for co-administration with
the compounds according to the invention include ampicillin,
amoxycillin, carbenicillin, piperacillin, azlocillin, mezlocillin,
and ticarcillin. Such penicillins may be used in the form of their
pharmaceutically acceptable salts, for example their sodium salts.
Alternatively, ampicillin or amoxycillin may be used in the form of
fine particles of the zwitterionic form (generally as ampicillin
trihydrate or amoxycillin trihydrate) for use in an injectable or
infusable suspension, for example, in the manner described herein
in relation to the compounds of formula I. Amoxycillin, for example
in the form of its sodium salt or the trihydrate, is particularly
preferred for use in compositions according to the invention.
[0204] Particularly suitable cephalosporins for co-administration
with the compounds according to the invention include cefotaxime,
ceftriaxone and ceftazidime, which may be used in the form of their
pharmaceutically acceptable salts, for example their sodium
salts.
[0205] When the compositions according to this invention are
presented in unit dosage form, each unit dose may suitably comprise
from about 25 to about 1000 mg, preferably about from 50 to about
500 mg, of a compound according to the invention. Each unit dose
may, for example, be 62.5, 100, 125, 150, 200 or 250 mg of a
compound according to the invention.
[0206] When the compounds of formula I are co-administered with a
penicillin, cephalosporin, carbapenem or other .beta.-lactam
antibiotic, the ratio of the amount of the compounds of formula I
to the amount of the other .beta.-lactam antibiotic may vary within
a wide range. The said ratio may, for example, be from 100:1 to
1:100; more particularly, it may for example, be from 2:1 to 1:30.
The amount of carbapenem, penicillin, cephalosporin or other
.beta.-lactam antibiotic according to the invention will normally
be approximately similar to the amount in which it is
conventionally used.
[0207] The claimed invention also includes the use of a compound of
formula I, a pharmaceutically acceptable salt, ester, prodrug,
anhydride or solvate thereof, in the manufacture of a medicament
for the treatment of bacterial infections.
[0208] The claimed invention also includes the use of a compound of
formula I as a metallo-.beta.-lactamase inhibitor.
[0209] The claimed invention further includes a method of treating
bacterial infections in humans or animals which comprises
administering, in combination with a .beta.-lactam antibiotic, a
therapeutically effective amount of a metallo-.beta.-lactamase
inhibitor of formula I.
[0210] The claimed invention further includes a method of treating
bacterial infections in humans or animals which comprises
administering, in combination with a carbapenem antibiotic, a
therapeutically effective amount of a metallo-.beta.-lactamase
inhibitor of formula I.
[0211] The claimed invention also includes a composition comprising
a metallo-.beta.-lactamase inhibitor of formula I together with a
.beta.-lactam antibiotic and a pharmaceutically acceptable
carrier.
[0212] The claimed invention also includes a composition comprising
a metallo-.beta.-lactamase inhibitor of formula I together with a
carbapenem antibiotic and a pharmaceutically acceptable
carrier.
[0213] The compositions discussed above may optionally include a
serine .beta.-lactamase inhibitor as described above as well as a
DHP inhibitor.
[0214] Using standard susceptibility tests the compounds of the
instant invention were found to be active against
metallo-.beta.-lactamase enzymes produced by a range of
organisms.
[0215] The compounds of the present invention are synthesized using
the general conditions shown in the accompanying flow charts (A
through F). 46 47 48 49 50 51
[0216] The 2,3-disubstituted succinic acid compounds of the present
invention can be prepared as described in Flow Sheets A-F. The
cationic R.sup.q substituents of the compounds of the present
invention are generally carried through the syntheses in protected
or precursory form and are then deprotected or elaborated near or
at the end of the synthesis. Introduction of the R.sup.q
substituent from a precursor group is described in detail in Flow
Sheet F.
[0217] The synthesis of Flow Sheet A is based on a known literature
procedure (M. J. Crimmin et. al., SynLett 1993, 137). Referring to
Flow Sheet A, the R.sup.1-substituted acetic acid starting
materials A1 are readily available from commercial sources or are
readily prepared by a variety of methods known in the art. Briefly,
the starting material A1 is alkylated with an ester derivative of
bromoacetic acid, employing a chiral auxiliary group to achieve
stereoselectivity in the reaction. After removal of the chiral
auxiliary to give A4, the R.sup.2* group is introduced
stereoselectively by an alkylation reaction to give A5. The
R.sup.2* group may be R.sup.2 as defined above, or may contain
R.sup.x and R.sup.q substituents in precursory or protected form
which require elaboration. Such elaboration may be carried-out at
this point. Removal of the carboxyl protecting group of A5 then
provides the final compound A6.
[0218] The first step of Flow Sheet A is introduction of the chiral
auxiliary. A suggested method is as follows. A mixed anhydride is
formed between the starting carboxylic acid A1 and pivalic acid by
treating A1 with a tertiary amine base such as triethylamine and
pivaloyl chloride in a suitable ethereal solvent such as
tetrahydrofuran at reduced temperature such as between -78.degree.
C. and 0.degree. C. After a suitable reaction time, such as from 30
min to 3 hours, the resulting activated intermediate is then
reacted with a freshly prepared solution of
lithio-(4R)-benzyl-2-oxazolidinone in tetrahydrofuran at reduced
temperature such as between -78.degree. C. and 0.degree. C. After
conventional isolation and purification, intermediate A2 is
obtained. Intermediate A2 is deprotonated with a strong base such
as sodium hexamethyldisilazide in a solvent such as tetrahydrofuran
at reduced temperature such as between -78.degree. C. and
-70.degree. C. The resulting enolate is alkylated by addition of
BrCH.sub.2CO.sub.2P.sup.1, where P.sup.1 is a removable carboxyl
protecting group. After an appropriate reaction period, such as
from 1 to 3 hours, compound A3 is obtained by conventional
isolation and purification techniques. Suitable removable ester
derivatives of bromoacetic acid for this alkylation reaction are
t-butyl bromoacetate, allyl bromoacetate or benzyl
bromoacetate.
[0219] The oxazolidinone chiral auxiliary group of A3 is removed by
a hydrolysis reaction. Aqueous lithium hydroxide and aqueous
hydrogen peroxide are employed for this reaction along with an
organic co-solvent such as tetrahydrofuran. The reaction is
carried-out at a temperature of from 0.degree. C. to 30.degree. C.
for a reaction time of from 30 min to 4 hours. After acidification,
conventional isolation and purification provides intermediate
A4.
[0220] An alternative method of removing the chiral auxilliary
consists of reacting A3 with lithium benzyloxide (LiOCH.sub.2Ph)
followed by cleavage of the resulting benzyl ester to give A4. The
reaction of A3 with lithium benzyloxide is carried-out in
tetrahydrofuran as solvent at a temperature of from -78.degree. C.
to 30.degree. C. for a reaction time of from 30 min to 4 hours.
Cleavage of the resulting benzyl ester is accomplished in
conventional fashion, eg by hydrogenolysis employing a suitable
catalyst such as palladium on carbon in an appropriate solvent such
as ethanol at 1-2 atmospheres pressure of hydrogen. After
conventional isolation and purification, compound A4 is
obtained.
[0221] Alkylation of A4 to give A5 is accomplished by deprotonating
A4 with >2 equivalents of a strong hindered base to give a
dianion which is then reacted with an alkylating agent R.sup.2*L to
give A5, where R.sup.2* is as defined above and L is a displaceable
leaving group such as iodide, bromide or trifluoromethanesulfonate.
The reaction proceeds with high stereoselectivity to give
predominately the stereoisomer shown in Flow Sheet A. The
deprotonation reaction is carried-out in a suitable solvent such as
tetrahydrofuran at a temperature of from -78.degree. C. to
-70.degree. C. for a reaction time of from 30 min to 3 hours.
Preferred bases for this reaction are lithium
bis(trimethylsilyl)amide and lithium diisopropylamide. After
addition of the alkylating agent, the reaction is allowed to
proceed at a temperature of from -78.degree. C. to 25.degree. C.
for a reaction time of from 1 to 12 hours. Progress of the reaction
can be monitored by conventional analytical methods, eg HPLC and
TLC. Preferred alkylating agents for this reaction are alkyl
iodides and alkyl bromides. Other suitable alkylating agents are
well known in the art and include alkyl trifluoromethanesulfonates,
alkyl methanesulfonates and alkyl tosylates. After conventional
isolation and purification, intermediate A5 is obtained. The minor
stereoisomer produced in this reaction can often be separated from
A5 at this stage by conventional chromatographic techniques.
However, it is often preferable to carry-out this separation at the
stage of A6, after removal of the carboxyl protecting group as
described below.
[0222] Deprotection of any R.sup.x or R.sup.q groups which are
present in protected form may be accomplished at this point. For
example, if compound A5 contains a protected hydroxyl or amino
group, said protecting group may conveniently be removed at the
stage of A5. Alternatively, depending on the nature of the
protecting group it may be removed concurrent with or subsequent to
the removal of the. carboxyl protecting group as described
immediately below. Introduction of the cationic R.sup.q group may
also be accomplished at this point from a precursor substituent.
This procedure is described in detail in Flow Sheet F further
below.
[0223] Removal of the carboxyl protecting group of A5 by standard
methods gives the final compound A6. When P.sup.1 is t-butyl, this
is accomplished by treating A5 with a strong acid such as
trifluoroacetic acid in a suitable solvent such as dichloromethane.
The reaction is carried-out at a temperature of from 0.degree. C.
to 30.degree. C. for a reaction time of from 1 to 8 hours. The
final compound A6 is then isolated by conventional techniques.
Other methods of removing tert-butyl ester groups are known in the
art and may also be employed (see e.g. Greene, T. W., et al.
Protective Groups in Organic Synthesis, John Wiley & Sons.
Inc., 1991).
[0224] It will be apparent to one skilled in the art that employing
a chiral auxiliary of the opposite absolute configuration [eg.
lithio-(4S)-benzyl-2-oxazolidinone] in the first step of Flow Sheet
A will make possible the synthesis of compound A3 with the
alternative stereochemistry at the newly created stereocenter. This
will in turn make possible the synthesis of the final compounds A6
of Flow Sheet A, with the opposite absolute configuration. Other
chiral auxiliary groups are also known in the art and may also be
employed.
[0225] Flow Sheet B illustrates a variation of Flow Sheet A which
is preferred in certain cases, for example when Ar.sup.1 is a
heteroaryl group such as pyridyl. In this synthesis the second
substituent on the succinic acid is introduced by an aldol reaction
instead of an alkylation reaction. The synthesis begins with
compound A4, which is prepared as described in Flow Sheet A.
Compound A4 is deprotonated with >2 equivalents of a strong
hindered base to give a dianion which is then reacted with an
aldehyde Ar.sup.1CHO to give B1, where Ar.sup.1 is an optionally
substituted aryl or heteroaryl group, terms which are defined
above. The deprotonation reaction is carried-out in a suitable
solvent such as tetrahydrofuran at a temperature of from
-78.degree. C. to -70.degree. C. for a reaction time of from 30 min
to 3 hours. Preferred bases for this reaction are lithium
bis(trimethylsilyl)amide and lithium diisopropylamide. After
addition of the aldehyde, the reaction is allowed to proceed at a
temperature of from -78.degree. C. to 25.degree. C. for a reaction
time of from 1 to 12 hours. After conventional isolation and
purification, intermediate B1 is obtained.
[0226] Compound B1 is next cyclized to the lactone B2. Suitable
conditions for this cyclization reaction would be exposure of B1 to
acetic anhydride and triethylamine in an inert solvent such as
dichloromethane. Reductive opening of lactone B2, such as by
hydrogenolysis over palladium on carbon in a-suitable solvent such
as methanol, provides compound B3. Deprotection of any R.sup.x or
R.sup.q groups which are present in protected form may be
accomplished at this point. In addition, introduction of a cationic
R.sup.q group from a precursor substituent may be carried-out at,
the stage of B3. This procedure is described in detail in Flow
Sheet F further below. Removal of the carboxyl protecting group of
B3 by conventional methods then gives the final compound B4.
[0227] Flow Sheet C illustrates an extension of the synthesis of
Flow Sheet A which makes possible the introduction of a variety of
preferred biaryl-type R.sup.2 substituents. Briefly, starting with
compound A4 from Flow Sheet A, alkylation with
K--Ar.sup.2--(CH.sub.2).sub.n--L by the method described in Flow
Sheet A gives intermediate C1; where L is a displaceable leaving
group such as iodide, bromide or trifluoromethanesulfonate, n is
1,2,3 or 4, Ar2 is an optionally substituted aryl or heteroaryl
group as defined above, and K is iodide, bromide, chloride or a
protected hydroxyl group which can be converted to a
trifluoromethanesulfonate group by known methods. Protection of the
free carboxyl group of C1 with a removable protecting group p.sup.2
gives C2. When K is a protected hydroxyl group it is deprotected
and converted to a trifluoromethanesulfonate group at this point. A
palladium catalyzed organometallic cross-coupling reaction between
C2 and an organometallic reagent R.sup.3-Met gives compound C3;
where Met is a boronic acid or trialkyltin moiety and R.sup.3 is an
optionally substituted alkenyl, alkynyl, aryl or heteroaryl group
as defined above. Deprotection or elaboration of any R.sup.x or
R.sup.q groups which are present in protected or precursory form is
accomplished at this point. Removal of the two carboxyl protecting
groups of C3 then provides the final compound C4.
[0228] The p.sup.2 carboxyl protecting group is introduced in
conventional fashion. A preferred p.sup.2 group is p-methoxybenzyl
which can be introduced employing p-methoxybenzyl alcohol, a
carbodiimide reagent such as 1,3-diisopropylcarbodiimide and
N,N-dimethylaminopyridine catalyst in a suitable inert solvent such
as dichloromethane. Other suitable ester protecting groups known in
the art could also be employed (see e.g. Greene, T. W., et al.
Protective Groups in Organic Synthesis, John Wiley & Sons.
Inc., 1991).
[0229] The palladium catalyzed cross-coupling reaction between C2
and R.sup.3-Met is carried-out by procedures known in the
scientific and patent literature. When Met is a boronic acid moiety
[--B(OH).sub.2] the reaction is commonly known as a Suzuki reaction
(see Suzuki, Chem. Rev. 1995, 95, 2457). Compound C2 is combined
with the boronic acid R.sup.3--B(OH).sub.2 in a coupling solvent
such as 1,2-dimethoxyethane, N,N-dimethylformamide or toluene,
optionally with water as a co-solvent, with a base such as sodium
carbonate and a palladium catalyst such as
tetrakis(triphenylphosphine)-palladium(0). The reaction is
carried-out at a temperature of from 20.degree. C. to 125.degree.
C. for a reaction time of from 1 to 48 hours. The coupled product
C3 is then isolated by conventional techniques. When Met is a
trialkyltin moiety, the reaction is commonly known as a Stille
reaction and the cross-coupling is carried-out by procedures well
known in the literature (T. N. Mitchell, Synthesis 1992, 803).
[0230] Deprotection of any R.sup.x or R.sup.q groups which are
present in protected form may be accomplished at this point. In
addition, introduction of a cationic R.sup.q group from a precursor
substituent may be carried-out at the stage of C3. This procedure
is described in detail in Flow Sheet F further below.
[0231] Removal of the carboxyl protecting groups of C3 by standard
methods provides the final compound C4. It is often convenient for
the protecting groups p.sup.1 and p.sup.2 to be selected such that
they can both be removed under the same reaction conditions. For
example, when p.sup.1 is tert-butyl and p.sup.2 is p-methoxybenzyl,
both esters of C3 can be removed in a single step by treating C3
with a strong acid such as trifluoroacetic acid in a suitable
solvent such as dichloromethane. It is sometimes advantageous to
include a trapping agent such as triethylsilane or anisole in the
reaction mixture. The reaction is carried-out at a temperature of
from 0.degree. C. to 30.degree. C. for a reaction time of from 1 to
8 hours. The final compound C4 is then isolated by conventional
techniques. Other methods of removing tert-butyl and
p-methoxybenzyl ester groups are known in the art and may also be
employed (see e.g. Greene, T. W., et al. Protective Groups in
Organic Synthesis, John Wiley & Sons. Inc., 1991). Flow Sheet D
illustrates an alternative synthesis of compounds of the present
invention. The R.sup.1-substituted acetic acid starting materials
D1 (M=H) and the esterified derivatives thereof (M=esterifying
group) are readily available from commercial sources or are readily
prepared by a variety of methods known in the art. The synthesis of
Flow Sheet D is based on known literature procedures (see for
example J. L. Belletire and D. F. Fry, J. Org. Chem. 1987, 52,
2549). Briefly, starting material D1 is deprotonated with a strong
base and the resulting dianion (M=H) or anion (M=esterifying group)
is oxidatively coupled with a suitable oxidizing reagent. In the
case of M=H, acidic work-up and conventional isolation and
purification gives the final compound D2. In the case of
M=esterifying group, an additional deprotection step is also
needed. A preferred strong base for the deprotonation reaction is
lithium diisopropylamide. Suitable oxidizing agents for the
synthesis of Flow Sheet D include iodine, copper(II) salts such as
CuBr2, and titanium tetrachloride.
[0232] In the synthesis of Flow Sheet D, protection or elaboration
of any R.sup.x or R.sup.q groups which are present in protected or
precursory form is best accomplished where M=esterifying group,
prior to removal of said estenrfying group. In addition,
introduction of a cationic R.sup.q group from a precursor
substituent may be carried-out as described in detail in Flow Sheet
F further below.
[0233] Since the synthesis of Flow Sheet D is based on a
dimerization-type reaction, it is best suited for the synthesis of
symmetrically 2,3-disubstituted succinic acids (R.sup.1=R.sup.2).
For this reason, it is generally less preferred than the syntheses
of Flow Sheets A, B and C. The synthesis of Flow Sheet D also
generally produces a racemic mixture of stereoisomers., However, it
is possible to employ a chiral auxiliary in the synthesis of Flow
Sheet D in order to achieve high stereoselectivity and optical
purity (see for example N. Kise et. al. J. Org. Chem. 1995, 60,
1100). Such use of a chiral auxiliary is illustrated in Flow Sheet
E.
[0234] Flow Sheet F illustrates a suggested method for the
introduction of the cationic substituents of the compounds of the
present invention from a precursor substituent. The starting
material F1 of Flow Sheet F is substituted with a precursor
substituent which can be elaborated into the desired cationic
substitutent, R.sup.q. A preferred precursor substituent is a
protected hydroxymethyl group, P.sup.3OCH.sub.2--, where p.sup.3 is
a removable hydroxyl protecting group. In Flow Sheet F, R.sup.4 is
defined such that the moiety [--R.sup.4--CH.sub.2--Q.sup.+Y.sup.-]
represents an R.sup.2 group as defined above. Examples of
representative R.sup.4 groups are shown in Table 1.
[0235] The starting material F1 of Flow Sheet F is synthesized by
one the methods described in Flow Sheets A, B, C, D, and E. When F1
is synthesized according to Flow Sheet A, it is derived from
intermediate A5, through protection of the free carboxyl group with
an appropriate carboxyl protecting group p.sup.2. In this case, the
precursor substituent is present in the R.sup.1 or R.sup.2*
substituent of A5. When F1 is synthesized according to Flow Sheet
B, it is derived from intermediate B3, through protection of the
free carboxyl group with an appropriate carboxyl protecting group
p.sup.2. In this case, the precursor substituent is present in the
R.sup.1 or Ar.sup.1 substituent of B3. When F1 is synthesized
according to Flow Sheet C, it is derived from or corresponds to,
intermediate C3. In this case, the precursor substituent is present
in the R.sup.1, Ar.sup.2 or R.sup.3 substituent of C3. Starting
material F1 may also be prepared by appropriate modification of the
syntheses of Flow Sheets D and E as would be apparent to those
skilled in the art.
[0236] Referring to Flow Sheet F, the first step is removal of the
hydroxyl protecting group p.sup.3. This is accomplished by
conventional methods. Hydroxyl protecting group p.sup.3 is
generally selected such that it may be selectively removed in the
presence of the carboxyl protecting groups P.sup.1 and p.sup.2 A
preferred p.sup.3 is t-butyldimethylsilyl. Removal of the preferred
t-butyldimethylsilyl p.sup.3 is accomplished by treating F1 with
tetra-n-butylammonium fluoride and acetic acid in tetrahydrofuran
as solvent. Other hydroxyl protecting groups are well known in the
art and may also be employed (see e.g. Greene, T. W., et al.
Protective Groups in Organic Synthesis, John Wiley & Sons.
Inc., 1991).
[0237] Introduction of the cationic substituent is accomplished by
an activation-dispacement process. Briefly, the hydroxyl group of
F2 is converted into a suitable leaving group, G, which is
thereafter displaced with a nucleophilic nitrogen. compound Q*, to
yield F4. With certain Q* groups, additional steps may also be
needed such as removal of amino protecting groups or conversion of
an amine precursor such azide into an amino group. The protecting
groups are removed from F4 in conventional fashion and then in an
optional step a pharmaceutically acceptable counterion Y.sup.- may
be introduced to provide compound F5.
[0238] The following are examples of suitable leaving groups G:
alkyl and substituted alkylsulfonates, aryl and substituted
arylsulfonates and halides. The common sulfonate leaving groups
are: methanesulfonyloxy, trifluoromethanesulfonyloxy,
fluorosulfonyloxy, p-toluenesulfonyloxy, and
2,4,6-triisopropylbenzenesulfonyloxy. The preferred halogen leaving
groups are bromide and iodide.
[0239] Compound Q* represents a precursor group to the cationic
group Q.sup.+ as defined above. As such, it may require further
modification after its reaction with F3. The nucleophilic nitrogen
moiety of Q* is generally the nitrogen of a primary, secondary or
tertiary amino group or a ring nitrogen of a heteroaryl group such
as a 1-substituted-imidazole. In addition to its nucleophilic
nitrogen atom, Q* may include 1,2 or 3 of the following moieties:
positively charged nitrogen atoms, protected amino groups, amine
precursor groups such as azido. Suitable protecting groups for
amino groups present in Q* would be t-butyloxycarbonyl-,
allyloxycarbonyl- and p-nitrobenzyloxycarbonyl-. The Q* groups may
be prepared by standard methods known in the scientific and patent
literature. Suitable Q* groups are listed in Table 2.
[0240] Referring to Flow Sheet F, the hydroxyl group of F2 may be
converted into a suitable alkyl- or arylsulfonate leaving group by
treating with an appropriate agent such as an alkyl- or
arylsulfonyl chloride or an alkyl- or arylsulfonic anhydride in the
presence of a hindered organic base such as triethylamine or
2,6-lutidine. A suitable solvent such as dichloromethane is
employed and the reaction is carried out at reduced temperature,
such as from about -70.degree. C. to 0.degree. C.
[0241] The preferred halogen leaving groups may be introduced by
displacing an alkyl- or arylsulfonate leaving group with an
appropriate metal halide. Thus, compound F3, where G is an alkyl-
or arylsulfonate group, is reacted with a suitable metal halide
such as sodium iodide or potassium bromide in a suitable solvent
such as acetone, acetonitrile, tetrahydrofuran,
1-methyl-2-pyrrolidinone and the like, at from about 0.degree. C.
to 50.degree. C. Alternatively, the hydroxyl group of F2 may be
directly converted into an iodide group by reaction with an
appropriate reagent, eg. by treatment of F2 with methyl
triphenoxyphosphonium iodide in a suitable solvent, such as
N,N-dimethylformamide, at reduced or ambient temperatures.
Introduction of the cationic substituent is accomplished by
reacting F3 with a nucleophilic nitrogen compound Q* in a suitable
solvent, such as acetonitrile, tetrahydrofuran,
1-methyl-2-pyrrolidinone and the like, at about 0.degree. C. to
50.degree. C. to provide F4. When the leaving group, G, is iodide
or bromide, this displacement reaction may also be facilitated by
the addition of silver trifluoromethanesulfonate to the reaction
mixture.
[0242] When the hydroxyl group of F2 is located at a benzylic
position, and the reactive trifluoromethanesulfonate group is
employed as the leaving group G in F3, the activation and
displacement steps must be carried-out in situ, since in this case
F3 cannot be isolated by conventional techniques due to its
instability. Thus, treatment of F2 with a slight excess of
trifluoromethanesulfonic anhydride in the presence of a hindered,
non-nucleophilic base such as 2,6-lutidine, 2,4,6-collidine, or
2,6-di-tert-butyl-4-methyl-pyridine in a suitable solvent, such as
dichloromethane or acetonitrile, at from about -78.degree. C. to
-20.degree. C. provides for the generation of the
trifluoromethanesulfonate activating group. Introduction of the
cationic group is then accomplished by reacting the above
trifluoromethanesulfonat- e intermediate in situ with Q* at reduced
temperature. It is also possible in certain instances to use the
nucleophilic nitrogen compound Q* as the base for the formation of
the trifluoromethanesulfonate activating group. In this case,
treatment of F2 with trifluoromethanesulfonic anhydride in the
presence of at least two equivalents of Q* at reduced temperature
such as from -78.degree. C. to 0.degree. C. provides intermediate
F4. Examples of Q* which are suitable for use in this manner are
1-methylimidazole and 1,4-diazabicyclo(2.2.2)octane.
[0243] Removal of the carboxyl protecting groups of F4 by standard
methods provides the final compound F5. If Q* includes one or more
protected amino groups, these are removed either before, after or
simultaneous with the carboxyl protecting groups depending on the
exact nature of the protecting groups. If Q* includes one or more
amine precursor groups, these may be converted to the desired amine
or amines either before or after removal of the carboxyl protecting
groups depending on the nature of the protecting groups. In the
case of an azido amine precursor group, this may be accomplished by
hydrogenation over a suitable catalyst such as rhodium on carbon.
After the protecting groups are removed from F4, and the cationic
group Q.sup.+ has been fully elaborated, the final compound F5 is
isolated by conventional techniques. As an optional final step, a
pharmaceutically acceptable counterion Y.sup.-, which may differ
from G.sup.-, may be introduced by standard techniques, e.g. by
employing an anion exchange resin. Suitable negatively charged
counterions are listed above under the description of
pharmaceutically acceptable salts.
[0244] Compound F5 is electronically balanced. If more than one
positive charge is present in the cationic Q.sup.+ group of F5, it
is understood that an appropriate amount of negative counterion is
Y.sup.- present to result in overall electronic balance in the
final compound F5. Likewise, it is understood that when the
counterion Y.sup.- is an anionic species possessing more than one
negative charge, then an appropriate amount of Y.sup.- is present
to result in overall electronic balance in the final compound of
Formula I. For example, when Y.sup.- is a dianionic species, then
one-half of a molar equivalent of Y.sup.- is present relative to
the succinate moiety.
[0245] Representative examples of R.sup.4 and Q* are found below in
Tables 1 and 2 respectively:
1TABLE 1 Representative P.sup.3OCH.sub.2--R.sup.4 Groups 52 53 54
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
[0246]
2TABLE 2 Representative Q* Groups 76 77 78 79 80 81 82 83 84 85 86
87 88 89 90 91 92
[0247] The invention is further described in connection with the
following non-limiting examples.
PREPARATION 1
[0248] 93
[0249] A solution of (4R)-benzyl-2-oxazolidinone (2.44 g, 13.77
mmol) in 100 mL of THF was cooled to -70.degree. C. and metalated
by the dropwise addition of a 2.5M solution of n-butyllithium in
hexanes (5.52 mL, 13.77 mmol). After 20 min, neat hydrocinnamoyl
chloride (2.05 ml, 13.79 mmol) was added. After 15 min, the
reaction mixture was warmed by placing in an ice bath and kept at
0.degree. C. C for 1 hr. The reaction was hydrolyzed by the
addition of sat. aqueous NH.sub.4Cl and most of the THF was removed
by rotary evaporation. The residue was partitioned between ethyl
acetate and sat. aqueous NH.sub.4Cl and the organic phase was
washed with sat. aqueous NaHCO.sub.3, water and brine. The organic
layer was dried over Na.sub.2SO.sub.4 and evaporated in vacuo to
give a solid. Flash chromatography through 500 g of silica gel
(50:40:10 hexane/CH.sub.2Cl.sub.2/EtOAc) yielded 3.89 g of the
title compound as a white solid.
[0250] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 2.79 (dd, J=13.5,
9.4 Hz, 1H), 3.02-3.13 (m, 2H), 3.24-3.41 (m, 3H), 4.16-4.21 (m,
2H), 4.65-4.74 (m, 1H), 7.16-7.40 (m, 10H). MS (CI): m/z=385.2
(MH.sup.+).
PREPARATION 2
[0251] 94
[0252] A stirred solution of compound 1 (3.283 g, 10.612 mmol) in
35 mL of THF was cooled to -78.degree. C. and a 1.0M solution of
NaN(TMS).sub.2 in THF (11.67 mL, 11.67 mmol) was added dropwise
during 15 min. After 30 min, a solution of t-butyl bromoacetate
(2.04 mL, 13.82 mmol) in 2 mL of THF was added dropwise during 5
min. The solution was stirred at -78.degree. C. for 1 h and then
the ice bath was removed and stirring was continued for 1 h. The
reaction was hydrolyzed by the addition of sat. aqueous NH4Cl and
most of the THF was removed by rotary evaporation. The residue was
partitioned between ethyl acetate and sat. aqueous NH4Cl and the
organic phase was washed with water and brine. The organic layer
was dried over Na2SO.sub.4 and evaporated in vacuo to give a solid.
Flash chromatography through 410 g of silica gel (35:60:5
hexane/CH.sub.2Cl.sub.2/EtOAc) yielded 2.86 g of the title compound
as a white foam.
[0253] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 1.43 (s, 9H), 2.41
(dd, J=17.0, 4.1 Hz, 1H), 2.64-2.80 (m, 2H), 2.88 (dd, J=17.0, 11.0
Hz, 1H), 3.04 (dd, J=13.0, 6.3 Hz, 1H), 3.34 (dd, J=13.5, 3.2 Hz,
1H), 3.95 (t, J=8.4 Hz, 1H), 4.08-4.12 (m, 1H), 4.5-4.6 (m, 2H),
7.21-7.40 (m, 10H). MS (ESI): m/z=441.3 (M+NH.sub.4.sup.+).
PREPARATION 3
[0254] 95
[0255] A stirred solution of compound 2 (2.860 g, 6.753 mmol) in 70
mL of 4:1 THF/H.sub.2O was cooled to 0.degree. C. and 30% aq.
hydrogen peroxide (2.8 mL, 27.01 mmol) was added dropwise during 5
min. After 5 min, a 1.0M solution of LiOH.H.sub.2O in H.sub.2O
(13.51 ml, 13.51 mmol) was added dropwise during 10 min. The
reaction was kept at 0.degree. C. for 1.75 hr. and then a 1.5M
solution of Na.sub.2SO.sub.3 in H.sub.2O (18.0 ml, 27.01 mmol) was
added. The ice bath was removed and the reaction mixture was
allowed to warm towards room temperature over 30 min. A solution of
1.0N NaHCO.sub.3 in H.sub.2O was added until the reaction mixture
had a pH=9 by pH paper (.about.5 ml). Most of the THF was removed
by rotary evaporation and the residue was partitioned between
CH.sub.2Cl.sub.2 and H.sub.2O. The aqueous layer was washed
3.times.CH.sub.2Cl.sub.2 and then acidified with 2N HCl until pH=3
by pH paper. The aqueous layer was extracted
4.times.CH.sub.2Cl.sub.2 and the combined organic extracts were
dried over Na2SO4 and evaporated in vacuo to give an oil. Flash
chromatography through 100 g of silica gel (94:6
CH.sub.2Cl.sub.2/MeOH+0.- 5% HOAc) yielded 1.74 g of the title
compound as a white solid.
[0256] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 1.45 (s, 9H), 2.38
(dd, J=16.6, 4.4 Hz, 1H), 2.58 (dd, J=16.6, 8.5 Hz, 1H), 2.80 (dd,
J=15.3, 10.3 Hz, 1H), 3.10-3.20 (m, 2H), 7.20-7.40 (m, 5H), 11.99
(bs, 1H). MS (ESI): m/z=430.2 (M+NH.sub.4.sup.+).
PREPARATION 4
[0257] 96
[0258] To a stirred solution of 3-(4-biphenyl)-propionic acid
(1.805 g, 7.977 mmol) in 40 mL of THF was added Et.sub.3N (1.28 mL,
9.17 mmol) and the solution was cooled to -70.degree. C. Neat
pivaloyl chloride (1.0 ml, 8.1 mmol) was added and a thick white
suspension resulted. After 15 min, the reaction mixture was warmed
by placing in an ice bath and kept at 0.degree. C. for 40 min. The
mixture was then re-cooled to -70.degree. C. In a separate flask, a
solution of (4R)-benzyl-2-oxazolidinone (1.44 g, 8.13 mmol) in 35
mL of THF was cooled to -70.degree. C. and metalated by the
dropwise addition of a 2.5M solution of n-butyllithium in hexanes
(3.25 mL, 8.13 mmol). The resulting anion solution was added to the
re-cooled suspension via a cannula, rinsing with an additional 3 mL
of THF. After 15 min, the reaction mixture was warmed by placing in
an ice bath and kept at 0.degree. C. for 30 min. The reaction was
hydrolyzed by the addition of sat. aqueous NH.sub.4Cl and most of
the THF was removed by rotary evaporation. The residue was
partitioned between ethyl acetate and sat. aqueous NH.sub.4Cl and
the organic phase was washed with sat. aqueous NaHCO.sub.3, water
and brine. The organic layer was dried over Na.sub.2SO.sub.4 and
evaporated in vacuo to give a solid. Flash chromatography through
240 g of silica gel (CH.sub.2Cl.sub.2) yielded 2.45 g of the title
compound as a white solid.
[0259] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 2.79 (dd, J=13.3,
9.4 Hz, 1H), 3.05-3.15 (m, 2H), 3.25-3.41 (m, 3H), 4.15-4.25 (m,
2H), 4.65-4.75 (m, 1H), 7.15-7.65 (m, 14H). MS (EI): m/z=385.2
(M.sup.+).
PREPARATION 5
[0260] 97
[0261] A stirred solution of compound 4 (1.000 g, 2.594 mmol) in 40
mL of THF was cooled to -78.degree. C. and a 1.0M solution of
NaN(TMS).sub.2 in THF (2.85 mL, 2.85 mmol) was added dropwise
during 5 min. After 30 min, a solution of t-butyl bromoacetate
(0.500 mL, 3.37 mmol) in 4 mL of THF was added dropwise during 5
min. The solution was stirred at -78.degree. C. for 1 h and then
the reaction was hydrolyzed by the addition of sat. aqueous
NH.sub.4Cl. The reaction mixture was partitioned between ethyl
acetate and sat. aqueous NH.sub.4Cl and the organic phase was
washed with water and brine. The organic layer was dried over
Na.sub.2SO.sub.4 and evaporated in vacuo to give a solid. Flash
chromatography through 150 g of silica gel (65:30:5
hexane/CH.sub.2Cl.sub.2/EtOAc) yielded 1.12 g of the title compound
as a white solid.
[0262] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 1.43 (s, 9H), 2.45
(dd, J=16.9, 4.1 Hz, 1H), 2.65-2.80 (m, 2H), 2.89 (dd, J=16.9, 10.8
Hz, 1H), 3.07 (dd, J=13.0, 6.1 Hz, 1H), 3.34 (dd, J =13.5, 3.0 Hz,
1H), 3.94 (t, J=8.4 Hz, 1H), 4.08-4.11 (m, 1H), 4.5-4.6 (m, 2H),
7.25-7.60 (m, 14H). MS (ESI): m/z=517.5 (M+NH.sub.4.sup.+).
PREPARATION 6
[0263] 98
[0264] A stirred solution of compound 5 (0.907 g, 1.815 mmol) in 10
mL of THF was cooled to -70.degree. C. and a freshly prepared 0.27
M solution of LiOBn in THF (10 mL, 2.7 mmol) was added dropwise
during 10 min. The reaction was allowed to warm gradually to
-10.degree. C. during 2 h and was then placed in an ice bath and
kept at 0.degree. C. for 50 min. The reaction mixture was
partitioned between ethyl acetate and sat. aqueous NH.sub.4Cl and
the organic phase was washed with water and brine. The organic
layer was dried over Na.sub.2SO.sub.4 and evaporated in vacuo to
give an oil. Flash chromatography through 125 g of silica gel
(75:20:5 hexane/CH.sub.2Cl.sub.2/EtOAc) yielded 0.696 g of the
title compound as a white solid.
[0265] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 1.42 (s, 9H), 2.43
(dd, J=16.6, 5.1 Hz, 1H), 2.67 (dd, J=16.6, 9.1 Hz, 1H), 2.85 (dd,
J=13.6, 7.9 Hz, 1H), 3.07 (dd, J=13.5, 6.9 Hz, 1H), 3.15-3.25 (m,
1H), 5.09 (d, J=12.4 Hz, 1H), 5.15 (d, J=12.4 Hz, 1H), 7.20-7.65(m,
14H). MS (EI): m/z=430.2 (M.sup.+).
PREPARATION 7
[0266] 99
[0267] A solution of compound 6 (0.696 g, 1.617 mmol) in 10 mL of
EtOH and 5 mL of THF was hydrogenated at atmospheric pressure at
room temperature over 70 mg of 10% Pd/C. After 20 h, the mixture
was filtered and evaporated to give a solid. Flash chromatography
through 50 g of silica gel (5:2:2:1
hexane/CH.sub.2Cl.sub.2/EtOAc/MeOH+0.05% HOAc) yielded 0.540 g of
the title compound as a white solid.
[0268] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 1.45 (s, 9H), 2.43
(dd, J=16.6, 5.1 Hz, 1H), 2.62 (dd, J=16.8, 8.6 Hz, 1H), 2.84 (dd,
J=15.5, 10.4 Hz, 1H), 3.1-3.2 (m, 2H), 7.25-7.60(m, 9H). MS (EI):
m/z=340.2 (M.sup.+).
PREPARATION 8
[0269] 100
[0270] STEP A:
[0271] A stirred solution of compound 3 from Preparation 3 (1.011
g, 3.825 mmol) in 15.5 mL of THF was cooled to -70.degree. C. and a
1.0M solution of LiN(TMS).sub.2 in hexane (8.42 mL, 8.42 mmol) was
added dropwise. After 1 h, a freshly prepared 1.14M solution of
p-iodobenzyl iodide in THF (6.0 mL, 6.84 mmol) was added dropwise.
The solution was stirred at -70.degree. C. for 30 min and was then
allowed to warm gradually to 10.degree. C. during 90 min. The
reaction was hydrolyzed by the addition of sat. aqueous NH.sub.4Cl
and most of the THF was removed by rotary evaporation. The residue
was partitioned between ethyl acetate and sat. aqueous NH.sub.4Cl
and the organic phase was washed with water and brine. The organic
layer was dried over Na.sub.2SO.sub.4 and evaporated in vacuo to
give a solid. Flash chromatography through 450 g of silica gel
(98:2 CH.sub.2Cl.sub.2/MeOH+0.1% HOAc) yielded 1.78 g of compound 8
as a .about.6:1 mixture of (S,S:R,S) diastereomers (major isomer
depicted).
[0272] .sup.1H-NMR (500 Mz, CDCl.sub.3): 1.32 (s, 9H), 2.74-3.05
(m, 6H), 6.89 (d, J=8.2 Hz, 2H), 7.12-7.28 (m, 5H), 7.56 (d,
J=8.4Hz, 2H). MS (EI): m/z=480.4 (M.sup.+).
[0273] STEP B:
[0274] To a solution of compound 8 (182.0 mg, 0.3789 mmol) in 0.6
mL of CH.sub.2Cl.sub.2 was added neat trifluoroacetic acid (0.2
mL). The solution was stirred at room temperature for 4 h, and was
then evaporated in vacuo to give an oil. Separation by reverse
phase medium pressure chromatography on RP-18 (40:60 MeCN/0.1%
aqueous TFA) gave after lyophilization 103.7 mg of the title
compound as a white solid.
[0275] .sup.1H-NMR (500 Mz, CD.sub.3OD): 2.84-2.91 (m, 2H),
2.96-3.06 (m, 4H), 6.89 (d, J=8.2 Hz, 2H), 7.11-7.25 (m, 5H), 7.55
(d, J=8.2 Hz, 2H). MS (EI): m/z=424.2 (M.sup.+).
PREPARATION 9
[0276] 101
[0277] STEP A:
[0278] A stirred solution of compound 7 (26.2 mg, 0.0770 mmol) in
0.7 mL of THF was cooled to -70.degree. C. and a freshly prepared
1.0M solution of LiN(i-Pr).sub.2 in THF (0.17 mL, 0.17 mmol) was
added dropwise. After 1 h, neat benzyl bromide (0.015 mL, 0.12
mmol) was added dropwise. The solution was stirred at -70.degree.
C. for 20 min and was then allowed to warm gradually to 10.degree.
C. during 90 min. The reaction was hydrolyzed by the addition of
sat. aqueous NH.sub.4Cl. The reaction mixture was partitioned
between ethyl acetate and sat. aqueous NH.sub.4Cl and the organic
phase was washed with water and brine. The organic layer was dried
over Na.sub.2SO.sub.4 and evaporated in vacuo to give an oil.
Purification by preparative layer chromatography on silica gel
(93:7 CH.sub.2Cl.sub.2/MeOH+0.1% HOAc) yielded 28 mg of compound 10
as an .about.8:1 mixture of (S,S:R,S) diastereomers (major isomer
depicted).
[0279] .sup.1H-NMR (500 Mz, CDCl.sub.3): 1.33 (s, 9H, isomer B,
minor), 1.37 (s, 9H, isomer A, major), 2.85-3.20 (m, 6H, isomers A
& B), 7.10-7.65 (m, 14H, isomers A & B). MS (EI): m/z=430.3
(M.sup.+).
[0280] STEP B:
[0281] To a solution of compound 10 from Step A (10.3 mg, 0.0239
mmol) in 0.3 mL of CH.sub.2Cl.sub.2 was added neat trifluoroacetic
acid (0.1 mL). The solution was stirred at room temperature for 4
h, and was then evaporated in vacuo to give an oil. Separation by
reverse phase medium pressure liquid chromatography on RP-18 (45:55
MeCN/0.1% aqueous TFA) gave after lyophilization 5.0 mg of compound
11 and 0.7 mg of compound 12 as white solids.
[0282] Compound 11:
[0283] .sup.1H-NMR (500 Mz, CD.sub.3OD): 2.90-2.97 (m, 2H), 3.0-3.1
(m, 4H), 7.14 (d, J=7.1 Hz, 3H), 7.15-7.25 (m, 5H), 7.41 (t, J=7.7
Hz, 2H), 7.52 (d, J=8.0 Hz, 2H), 7.58 (d, J=7.8Hz, 2H). MS (EI):
m/z=374.2 (M.sup.+).
[0284] Compound 12:
[0285] .sup.1H-NMR (500 Mz, CD.sub.3OD): 2.85-3.00 (m, 6H), 7.19
(d, J=6.8 Hz, 3H), 7.24-7.32 (m, 5H), 7.30 (t, J=7.4 Hz, 1H), 7.41
(dd, J=7.5, 8.0 Hz, 2H), 7.49 (d, J=8.0 Hz, 2H), 7.58 (d, J=7.6 Hz,
2H). MS (EI): m/z=374.2 (M.sup.+).
PREPARATION 10
[0286] 102
[0287] STEP A:
[0288] A stirred solution of compound 8 (830.1 mg, 1.728 mmol) and
p-methoxybenzyl alcohol (0.54 ml, 4.33 mmol) in 14 mL of
CH.sub.2Cl.sub.2 was cooled to 0.degree. C., and a 1.0M solution of
N,N-dimethylaminopyridine in CH.sub.2Cl.sub.2 (0.259 ml, 0.259
mmol) was added, followed by neat 1,3-diisopropylcarbodiimide
(0.541 ml, 3.46 mmol). After 1 hr, the cooling bath was removed.
The reaction mixture was stirred an additional 180 min, and was
then hydrolyzed by the addition of sat. aqueous NH.sub.4Cl. The
reaction mixture was partitioned between ethyl acetate and sat.
aqueoues NH.sub.4Cl and the organic phase was washed with water and
brine. The organic layer was dried over Na.sub.2SO.sub.4 and
evaporated in vacuo to give a semi-solid. This crude material was
triturated with 10 ml CH.sub.2Cl.sub.2 and filtered through a
sintered-glass funnel. Evaporation of the filtrate in vacuo gave an
oil. Flash chromatography through 160 g of silica gel (73:20:7
hexane/CH.sub.2Cl.sub.2/EtOAc) yielded 921.6 mg of compound 13 as a
white solid.
[0289] .sup.1H-NMR (500 Mz, CDCl.sub.3): 1.37 (s, 9H), 2.8-3.1 (m,
6H), 3.83 (s, 3H), 4.98 (dd, J =43.5, 11.9 Hz, 2H), 6.76 (d, J=8.0
Hz, 2H), 6.86 (d, J=8.4 Hz, 2H), 7.07 (d, J=6.6 Hz, 2H), 7.16-7.27
(m, 5H), 7.53 (d, J=8.1 Hz, 2H). MS (ESI): m/z=623.2
(M+Na.sup.+).
[0290] STEP B:
[0291] To a stirred solution of compound 13 (76.6 mg, 0.1276 mmol)
and tetrakis(triphenylphosphine)palladium(0) (7.4 mg, 0.0064 mmol)
in 1.1 ml DME was added a solution of 4-methoxybenzeneboronic acid
(29.1 mg, 0.192 mmol) in 0.2 ml DME. After 10 min, a 2.0M solution
of Na.sub.2CO.sub.3 in H.sub.2O (0.130 ml, 0.260 mmol) was added
and the reaction mixture was heated to 100.degree. C. for 3.5 hr.
The reaction mixture was allowed to cool to RT and then hydrolyzed
by the addition of sat. aqueous NH.sub.4Cl. The reaction mixture
was partitioned between ethyl acetate and sat. aqueous NH.sub.4Cl
and the organic phase was washed with sat. aqueous
NaS.sub.2O.sub.3, water, and brine. The organic layer was dried
over Na.sub.2SO.sub.4 and evaporated in vacuo to give an oil. Flash
chromatography through 18 g of silica gel (73:20:7
hexane/CH.sub.2Cl.sub.2/EtOAc) yielded 37.1 mg of compound 14 as a
white solid.
[0292] .sup.1H-NMR (500 Mz, CDCl.sub.3): 1.36 (s, 9H), 2.90-3.07
(m, 6H), 3.82 (s, 3H), 3.87 (s, 3H), 4.98 (dd, J=34.8, 11.9 Hz,
2H), 6.86 (d, J=8.7 Hz, 2H), 6.99 (d, J=8.7 Hz, 2H), 7.09 (d, J=7.8
Hz, 4H), 7.16-7.26 (m, 5H), 7.42 (d, J=8.0 Hz, 2H), 7.52 (d, J=8.5
Hz, 2H). MS (ESI): m/z=603.3 (M+Na.sup.+).
[0293] STEP C:
[0294] To a solution of compound 14 (37.1 mg, 0.064 mmol) in 0.6 mL
of CH.sub.2Cl.sub.2 was added neat trifluoroacetic acid (0.2 mL).
The solution was stirred at room temperature for 4 h, and was then
evaporated in vacuo to give an oil. Separation by reverse phase
medium pressure chromatography on RP-18 (45:55 MeCN/0.1% aqueous
TFA) gave after lyophilization 9.3 mg of the title compound as a
white solid.
[0295] .sup.1H-NMR (500 Mz, CD.sub.3OD): 2.89-2.96 (m, 2H),
3.01-3.07 (m, 4H), 3.81 (s, 3H), 6.97 (d, J=8.9Hz, 2H), 7.11-7.25
(m, 7H), 7.43 (d, J=8.2Hz, 2H), 7.51 (d, J=8.7 Hz, 2H). MS (ESI):
m/z=427.1 (M+Na.sup.+).
EXAMPLE 1
[0296] Compound 20
[0297] STEP A: 103
[0298] A stirred solution of compound 3 (0.253 g, 0.957 mmol) in 7
mL of THF was cooled to -78.degree. C. and a 1.0M solution of
LiN(TMS).sub.2 in hexanes (2.2 mL, 2.2 mmol) was added dropwise
during 5 min. After 65 min, a solution of
4-(iodomethyl)-4'-(t-butyldimethylsilyloxymethyl)biphenyl (0.630 g,
1.44 mmol) in 1 mL of THF was added dropwise during 5 min. The
solution was stirred at -78.degree. C. for 35 min and was then
allowed to warm gradually to -20.degree. C. during 2 h, at which
point the reaction was judged to be complete by HPLC analysis. The
reaction was hydrolyzed by the addition of sat. aqueous NH.sub.4Cl.
The reaction mixture was then partitioned between ethyl acetate and
sat. aqueous NH.sub.4Cl and the organic phase was washed with water
and brine. The organic layer was dried over Na.sub.2SO.sub.4 and
evaporated in vacuo to give a solid. Flash chromatography on silica
gel (95:3:2 CH.sub.2Cl.sub.2/EtOAc/MeOH) yielded 0.479 g of
compound 16 as an .about.19:1 mixture of (S,S:R,S) diastereomers
(major isomer depicted).
[0299] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 0.15 (s, 6H), 0.99
(s, 9H), 1.39 (s, 9H), 2.85-2.95 (m, 1H), 2.95-3.20 (m, 5H),4.81
(s, 2H), 7.13 (d, J=6.9 Hz, 2H), 7.17 (d, J=8.1 Hz, 2H), 7.20-7.35
(m, 3H), 7.42 (d, J=8.0 Hz, 2H), 7.49 (d, J=8.0 Hz, 2H), 7.57 (d, J
=8.0 Hz, 2H). MS (EI): m/z=574.3 (M.sup.+).
[0300] STEP B: 104
[0301] A stirred solution of compound 16 (0.479 g, 0.835 mmol) and
p-methoxybenzyl alcohol (0.260 mL, 2.08 mmol) in 7.5 mL of
CH.sub.2Cl.sub.2 was cooled to 0.degree. C. and a 1.0 M solution of
N,N-dimethylaminopyridine in CH.sub.2Cl.sub.2 (0.125 mL, 0.125
mmol) was added followed by neat 1,3-diisopropylcarbodiimide (0.260
mL, 1.66 mmol). After 40 min, the cooling bath was removed. The
reaction mixture was stirred for an additional 140 min, and was
then hydrolyzed by the addition of sat. aqueous NH.sub.4Cl. The
reaction mixture was partitioned between ethyl acetate and sat.
aqueous NH.sub.4Cl and the organic phase was washed with water and
brine. The organic layer was dried over Na.sub.2SO.sub.4 and
evaporated in vacuo to give a semi-solid. Flash chromatography on
silica gel (75:21.5:3.5 to 75:20:5 hexane/CH.sub.2Cl.sub.2/EtOAc)
yielded 0.486 g of compound 17 as an oil. The diastereomeric ratio
was>100:1 (S,S): (R,S).
[0302] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 0.16 (s, 6H), 0.99
(s, 9H), 1.37 (s, 9H), 2.90-3.10 (m, 6H), 3.83 (s, 3H), 4.81 (s,
2H), 4.96 (d, J=11.9 Hz, 1H), 5.04 (d, J=11.9 Hz, 1H), 6.87 (d,
J=8.2 Hz, 2H), 7.07-7.15 (m, 4H), 7.18-7.28 (m, 5H), 7.41 (d, J=7.8
Hz, 2H), 7.47 (d, J=7.7 Hz, 2H), 7.56 (d, J=8.0 Hz, 2H). MS (ESI):
m/z=712.6 (M+NH.sub.4.sup.+).
[0303] STEP C: 105
[0304] A stirred solution of compound 17 (0.482 g, 0.694 mmol) in
4.5 mL of THF was cooled to 0.degree. C. and neat acetic acid
(0.120 mL, 2.1 mmol) was added followed by a 1.0 M solution of
tetrabutylammonium fluoride in THF (2.1 mL, 2.1 mmol). After 50
min, the cooling bath was removed. After stirring at room
temperature for 22 h, the reaction was judged to be complete by TLC
on silica gel. The reaction mixture was partitioned between ethyl
acetate and sat. aqueous NH.sub.4Cl and the organic phase was
washed with water and brine. The organic layer was dried over
Na.sub.2SO.sub.4 and evaporated in vacuo to give an oil. Flash
chromatography on silica gel (4:3:3 hexane/CH.sub.2Cl.sub.2/EtOAc)
yielded 0.389 g of compound 18 as an oil.
[0305] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 1.37 (s, 9H),
2.90-3.10 (m, 6H), 3.82 (s, 3H), 4.77 (s, 2H), 4.96 (d, J=11.9 Hz,
1H), 5.03 (d, J=11.9 Hz, 1H), 6.87 (d, J=8.5 Hz, 2H), 7.05-7.30 (m,
9H), 7.4-7.5 (m, 4H), 7.59 (d, J=8.0 Hz, 2H). MS (EST): m/z=598.5
(M+NH.sub.4.sup.+).
[0306] STEP D: 106
[0307] A stirred solution of compound 18 (20.0 mg, 0.0344 mmol) in
0.35 mL of CH.sub.2Cl.sub.2 was cooled to -70.degree. C. and neat
1-methylimidazole (0.0090 mL, 0.11 mmol) was added followed by
trifluoromethanesulfonic anhydride (0.0090 mL, 0.053 mmol). The
temperature was allowed to gradually rise and after 45 min was
-15.degree. C. TLC on silica gel showed some remaining starting
material, so additional 1-methylimidazole (0.0050 mL, 0.063 mmol)
was added. After 25 min more, the temperature was 5.degree. C. and
TLC showed no starting material. The reaction mixture was
partitioned between ethyl acetate and water and the organic phase
was washed once with water. The organic layer was dried over
Na.sub.2SO.sub.4 and evaporated in vacuo to give an oil.
Preparative layer chromatography on silica gel (1:1
MeCN/CH.sub.2Cl.sub.2) yielded 19.0 mg of compound 19 as an
oil.
[0308] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 1.36 (s, 9H),
2.88-3.08 (m, 6H), 3.81 (s, 3H), 3.94 (s, 3H), 4.95 (d, J=12 Hz,
1H), 5.02 (d, J=12 Hz, 1H), 5.39 (s, 2H), 6.86 (d, J=8.6 Hz, 2H),
7.05-7.35 (m, 11H), 7.43 (d, J=8.3 Hz, 2H), 7.48 (d, J=8.0 Hz, 2H),
7.60 (d, J =8.3 Hz, 2H), 9.23 (s, 1H). MS (ESI): m/z=645.5
(M.sup.+).
[0309] STEP E: 107
[0310] To a solution of compound 19 (18.5 mg, 0.0233 mmol) in 0.3
mL of CH.sub.2Cl.sub.2 was added neat trifluoroacetic acid (0.1
mL). The solution was stirred at room temperature for 110 min, and
was then evaporated in vacuo to give a solid. Purification by
reverse phase medium pressure liquid chromatography on RP-18 (40:60
MeCN/0.1% aqueous TFA) gave after lyophilization 10.4 mg of
compound 20 as a white solid.
[0311] .sup.1H-NMR (500 Mz, CD.sub.3OD): .delta. 2.88-2.98 (m, 2H),
3.0-3.1 (m, 4H), 3.93 (s, 3H), 5.44 (s, 2H), 7.10-7.25 (m, 7H),
7.45-7.55 (m, 4H), 7.59 (s, 1H), 7.65 (s, 1H), 7.68 (d, J=8.2 Hz,
2H), 8.99 (s, 1H). MS (ESI): m/z=469.4 (M.sup.+).
EXAMPLE 2
[0312] Compound 23
[0313] STEP A: 108
[0314] A stirred solution of compound 18 (0.262 g, 0.451 mmol) in
4.5 mL of CH.sub.2Cl.sub.2 was cooled to -60.degree. C. and
triethylamine (0.107 mL, 0.768 mmol) was added followed by neat
methanesulfonyl chloride (0.0490 mL, 0.633 mmol). The temperature
was allowed to gradually rise and after 45 min was -25.degree. C.
The reaction was hydrolyzed by the addition of sat. aqueous
NH.sub.4Cl. The reaction mixture was partitioned between ethyl
acetate and sat. aqueous NH.sub.4Cl and the organic phase was
washed with water and brine. The organic layer was dried over
Na.sub.2SO.sub.4 and evaporated in vacuo to give a solid.
[0315] The crude mesylate was dissolved in acetone and cooled to
0.degree. C. Solid sodium iodide was added (0.135 g, 0.901 mmol)
and the mixture was stirred in the dark. After 30 min, the cooling
bath was removed. After stirring for 2 h more, the reaction mixture
was partitioned between ethyl acetate and water. The organic phase
was washed with 51% aqueous Na.sub.2S.sub.2O.sub.3, water and
brine. The organic lawyer was dried over Na.sub.2SO.sub.4 and
evaporated in vacuo to give 313 mg of compound 21 as a solid.
[0316] .sup.1H-NMR (500 Mz, CDCl.sub.3): .delta. 1.36 (s, 9H),
2.9-3.1 (m, 6H), 3.82 (s, 3H), 4.54 (s, 2H), 4.96 (d, J=11.9 Hz,
1H), 5.03 (d, J=11.9 Hz, 1H), 6.87 (d, J=8.5 Hz, 2H), 7.06-7.14 (m,
4H), 7.17-7.28 (m, 5H), 7.42-7.50 (m, 4H), 7.51 (d, J=8.2Hz, 2H).
MS (ESI): m/z=708.1 (M+NH.sub.4.sup.+).
[0317] STEP B: 109
[0318] To a stirred solution of compound 21 (0.0212 g, 0.0307 mmol)
and 1-(aminocarbonylmethyl-4-aza-1-azoniabicyclo(2.2.2)octane
trifluoromethanesulfonate (0.011 g, 0.034 mmol) in 0.35 mL of
acetonitrile and 0.1 mL of THF was added a solution of silver
trifluoromethanesulfonate in acetonitrile (0.845 M, 0.036 mL, 0.030
mmol). A precipitate formed immediately. The mixture was stirred in
the dark for 55 min and was then filtered and evaporated in vacuo
to give 37 mg of compound 22 as a solid.
[0319] .sup.1H-NMR (500 Mz, d.sub.6-acetone): .delta. 1.32 (s, 9H),
2.9-3.1 (m, 6H), 3.80 (s, 3H), 4.38-4.48 (m, 6H), 4.50-4.60 (m,
6H), 4.65 (s, 2H), 4.93 (d, J=12 Hz, 1H), 5.02 (d, J=12 Hz, 1H),
5.14 (s, 2H), 6.90 (d, J=8.7 Hz, 2H), 7.16 (d, J=6.9 Hz, 2H),
7.2-7.3 (m, 8H), 7.63 (d, J=8.0 Hz, 2H), 7.69 (bs, 1H), 7.80 (d,
J=8.2 Hz, 2H), 7.86 (d, J=8.2 Hz, 2H). MS (ESI): m/z=732.5
(M.sup.+2-H.sup.+).
[0320] STEP C: 110
[0321] A solution of compound 22 (37 mg) in 0.3 mL of
CH.sub.2Cl.sub.2 and 0.1 mL of trifluoroacetic acid was stirred at
room temperature for 150 min, and was then evaporated in vacuo to
give a film. Purification by reverse phase medium pressure liquid
chromatography on RP-18 (25:75 MeCN/0.1% aqueous TFA) gave after
lyophilization 21.8 mg of compound 23 as a white solid.
[0322] .sup.1H-NMR (500 Mz, CD.sub.3OD): .delta. 2.88-2.98 (m, 2H),
3.00-3.15 (m, 4H), 3.18-4.08 (m, 6H), 4.2-4.3 (m, 6H), 4.36 (s,
2H), 4.82 (s, 2H), 7.14 (d, J=7.1 Hz, 2H), 7.15-7.30 (m, 5H), 7.56
(d, J=8.0 Hz, 2H), 7.63 (d, J=8.2 Hz, 2H), 7.82 (d, J=8.3 Hz, 2H).
MS (ESI): m/z=556.4 (M.sup.+2-H.sup.+).
EXAMPLE 3
[0323] Compound 25
[0324] STEP A: 111
[0325] To a stirred solution of compound 21 (0.194 g, 0.281 mmol)
in 0.75 mL of THF was added a solution of
1-(1-azidoprop-3-yl)-4-aza-1-azoniabicy- clo(2.2.2)octane
trifluoromethanesulfonate (0.107 g, 0.310 mmol) in 2.25 mL of
acetonitrile. To the resulting solution was added a solution of
silver trifluoromethanesulfonate in acetonitrile (0.845 M, 0.332
mL, 0.281 mmol). A precipitate formed immediately. The mixture was
stirred in the dark for 45 min and was then filtered and evaporated
in vacuo to give 312 mg of compound 24 as a solid. .sup.1H-NMR (500
Mz, d.sub.6-acetone): .delta. 1.32 (s, 9H), 2.25-2.35 (m, 2H),
2.90-3.15 (m, 6H), 3.55-3.65 (m, 2H), 3.80 (s, 3H), 3.93-4.00 (m,
2H), 4.37 (s, 12H), 4.93 (d, J=12 Hz, 1H), 5.02 (d, J=12 Hz, 1H),
5.14 (s, 2H), 6.90 (d, J=8.4 Hz, 2H), 7.16 (d J=6.6 Hz, 2H),
7.2-7.3 (m, 7H), 7.63 (d, J=8.2 Hz, 2H), 7.80 (d, J 8.5 Hz, 2H),
7.85 (d, J=8.2 Hz, 2H). MS (ESI): m/z=872.1
(M.sup.+2+CF.sub.3CO.sub.2--).
[0326] STEP B: 112
[0327] A solution of compound 24 (312 mg) in 2.1 mL of
CH.sub.2Cl.sub.2 and 0.7 mL of trifluoroacetic acid was stirred at
room temperature for 125 min, and was then evaporated in vacuo. The
resulting dry film was dissolved in 3 mL of THF, 1 mL of MeOH and 1
mL of water and hydrogenated at atmospheric pressure over 55 mg of
5% rhodium on carbon. After 3 h, the mixture was filtered through a
pad of Celite.RTM.. The tan filtrate was concentrated by rotary
evaporation until it became hazy, and was then frozen and
lyophilized to give a brown solid. Purification by reverse phase
medium pressure liquid chromatography on RP-18 (20:80 MeCN/0.1%
aqueous TFA) gave after lyophilization 164 mg of a white solid. A
portion of this solid (151.4 mg) was dissolved in 2 mL of methanol
and eluted with methanol through a 12 g column of Bio-Rad.RTM.
AG-2-X8 chloride form resin (.about.3 meq/g). Evaporation of the
collected fractions gave a colorless oil which was lyophilized from
water/MeCN to give 109 mg of compound 25 as a white solid.
[0328] .sup.1H-NMR (500 Mz, CD.sub.3OD): .delta. 2.18-2.30 (m, 2H),
2.9-3.0 (m, 2H), 3.00-3.15 (m, 6H), 3.7-3.8 (m, 2H), 4.08 (s, 12H),
4.89 (s, 2H), 7.14 (d, J=7.0 Hz, 2H), 7.15-7.30 (m, 5H), 7.56 (d,
J=8.0 Hz, 2H), 7.67 (d, J=8.0 Hz, 2H), 7.82 (d, J=8.0Hz, 2H). MS
(ESI): m/z=556.4 (M.sup.+3-2H.sup.+).
EXAMPLE 4
[0329] Compound 27
[0330] STEP A: 113
[0331] To a stirred solution of compound 21 (41.2 mg, 0.0597 mmol)
and [3-(dimethylamino)propyl] [3-[(1,1-dimethylethoxy)carbonyl
amino]propyl]carbamic acid, 1,1-dimethylethyl ester (24.7 mg,
0.0687 mmol) in 1.2 mL of acetonitrile was added a solution of
silver trifluoromethanesulfonate in acetonitrile (0.845 M, 0.066
mL, 0.0564 mmol). A precipitate formed immediately. The mixture was
stirred in the dark for 85 min and was then filtered and evaporated
in vacuo to give 66.7 mg of compound 26 as a solid.
[0332] 1H-NMR (500 Mz, d.sub.6-acetone): .delta. 1.33 (s, 9H), 1.40
(s, 9H), 1.45 (s, 9H), 1.74 (bs, 2H), 2.37 (bs, 2H), 2.9-3.2 (m,
8H), 3.26-3.33 (m, 2H), 3.35 (s, 6H), 3.42 (bs, 2H), 3.61 (bs, 2H),
3.80 (s, 3H), 4.85 (s, 2H), 4.97 (dd, J=43.5, 11.9 Hz, 2H), 5.99
(bs, 1H), 6.91 (d, J=8.0 Hz, 2H), 7.16 (d, J=7.3 Hz, 2H), 7.19-7.30
(m, 7H), 7.63 (d, J=7.5 Hz, 2H), 7.8 (dd, J=37.1, 7.9 Hz, 4H). MS
(ESI): m/z=922.5 (M.sup.+).
[0333] STEP B: 114
[0334] To a solution of compound 26 (66.7 mg) in 0.6 mL of
CH.sub.2Cl.sub.2 was added neat trifluoroacetic acid (0.2 mL). The
solution was stirred at room temperature for 5.5 h, and was then
evaporated in vacuo to give an oil. Purification by reverse phase
medium pressure chromatography on RP-18 (30:70 MeCN/0.1% aqueous
TFA) gave after lyophilization 36 mg of a white solid. A portion of
this solid (31.5 mg) was dissolved in 2 mL of methanol and eluted
with methanol through a 3 g column of Bio-Rad.RTM. AG-2-X8 chloride
form resin (.about.3 meq/g). Evaporation of the collected fractions
gave a colorless oil which was lyophilized from water/MeCN to give
22.2 mg of compound 27 as a white solid.
[0335] 1H-NMR (500 Mz, CD.sub.3OD): .delta. 2.07-2.16 (m, 2H),
2.30-2.40 (m, 2H), 2.90-2.97 (m, 2H), 3.03-3.22 (m, 16H), 3.43-3.50
(m, 2H), 4.60 (s, 2H), 7.12-7.28 (m, 7H), 7.56 (d, J=8.2 Hz, 2H),
7.63 (d, J=8.2 Hz, 2H), 7.78 (d, J=8.2 Hz, 2H). MS (ESI): m/z=546.4
(M.sup.+3-2H.sup.+).
EXAMPLES 5-29
[0336] Employing the procedures described herein, additional
compounds of the present invention were prepared. These are
described in Tables 3-7, which additionally include characterizing
data.
3TABLE 3 115 Example No. Q.sup..sym. Y.sup..crclbar. m/z 5 116 3
Cl.sup..crclbar. 542.5(M.sup.+3 - 2H.sup.+); ESI 6 117 3
Cl.sup..crclbar. 660.4(M.sup.+3 - H.sup.+ +
CF.sub.3CO.sub.2.sup.-); ESI 7 118 3 Cl.sup..crclbar.
532.3(M.sup.+3 - 2H.sup.+); ESI 8 119 4 Cl.sup..crclbar.
613.4(M.sup.+4 - 3H.sup.+); ESI 9 120 4 Cl.sup..crclbar. 10 121 3
Cl.sup..crclbar. 649.5(M.sup.+2 - H.sup.+); ESI 11 122 3
Cl.sup..crclbar. 712.3(M.sup.+3 - H.sup.+ + CF.sub.3CO.sub.2--);
ESI 12 123 3 Cl.sup..crclbar. 765.5(M.sup.+3 - H.sup.+ +
CF.sub.3CO.sub.2.sup.-); ESI 13 124 3 Cl.sup..crclbar.
599.3(M.sup.+3 - 2H.sup.+); ESI 14 125 3 Cl.sup..crclbar.
793.5(M.sup.+3 - H.sup.+ + CF.sub.3CO.sub.2.sup.-); ESI
[0337]
4TABLE 4 126 Example No. Q.sup..sym. Y.sup..crclbar. m/z 15 127
CF.sub.3CO.sub.2.sup..crclbar. 393.3(M.sup.+); EI 16 128
CF.sub.3CO.sub.2.sup..crclbar. 328.1(M.sup.+); EI 17 129 2
Cl.sup..crclbar. 437.8(M.sup.+); ESI 18 130 2
CF.sub.3CO.sub.2.sup..crclbar. 480.6(M.sup.+2 - H.sup.+); ESI 19
131 3 CF.sub.3CO.sub.2.sup..crclbar. 480.5(M.sup.+3 - 2H.sup.+);
ESI 20 132 3 CF.sub.3CO.sub.2.sup..crclbar. 523.4(M.sup.+3 -
2H.sup.+); ESI
[0338]
5TABLE 5 133 Example No. Q.sup..sym. Y.sup..crclbar. m/z 21 134
Cl.sup..crclbar. 421.5(M.sup.+); ESI 22 135 2 Cl.sup..crclbar.
508.6(M.sup.+2 - H.sup.+); ESI 23 136 3 Cl.sup..crclbar.
508.3(M.sup.+3 - 2H.sup.+); ESI 24 137 3 Cl.sup..crclbar.
551.3(M.sup.+3 - 2H.sup.+); ESI
[0339]
6TABLE 6 138 Example No. Q.sup..sym. Y.sup..crclbar. m/z 25 139
Cl.sup..crclbar. 387.5(M.sup.+); ESI 26 140 2 Cl.sup..crclbar.
474.7(M.sup.+2 - H.sup.+); ESI 27 141 3 Cl.sup..crclbar.
474.4(M.sup.+3 - 2H.sup.+); ESI 28 142 3 Cl.sup..crclbar.
517.4(M.sup.+3 - 2H.sup.+); ESI
[0340]
7TABLE 7 143 Example No. Q.sup..sym. Y.sup..crclbar. m/z 29 144
Cl.sup..crclbar. 469.3(M.sup.+); ESI
EXAMPLES 30-104
[0341] Additional compounds of the present invention can be
prepared employing the procedures described herein. These are
described in Tables 8-18.
8TABLE 8 145 Example No. Q.sup..sym. Y.sup..crclbar. 30 146 3
Cl.sup..crclbar. 31 147 3 CH.sub.3CO.sub.2.sup..crclbar. 32 148 3
Cl.sup..crclbar. 33 149 3 Cl.sup..crclbar. 34 150 3
Cl.sup..crclbar. 35 151 3 Cl.sup..crclbar. 36 152 4
Cl.sup..crclbar. 37 153 4 CH.sub.3CO.sub.2.sup..crclbar. 38 154 4
Cl.sup..crclbar. 39 155 4 Cl.sup..crclbar. 40 156 4
Cl.sup..crclbar. 41 157 4 Cl.sup..crclbar. 42 158 Cl.sup..crclbar.
43 159 Cl.sup..crclbar. 44 160 3 Cl.sup..crclbar. 45 161 3
Cl.sup..crclbar. 46 162 4 Cl.sup..crclbar. 47 163 3
Cl.sup..crclbar.
[0342]
9TABLE 9 164 Example No. Q.sup..sym. Y.sup..crclbar. 48 165
Cl.sup..crclbar. 49 166 2 Cl.sup..crclbar. 50 167 3
Cl.sup..crclbar. 51 168 3 Cl.sup..crclbar. 52 169 3
Cl.sup..crclbar. 53 170 3 Cl.sup..crclbar.
[0343]
10TABLE 10 171 Example No. Q.sup..sym. Y.sup..crclbar. 54 172
Cl.sup..crclbar. 55 173 2 Cl.sup..crclbar. 56 174 3
Cl.sup..crclbar. 57 175 3 Cl.sup..crclbar. 58 176 3
Cl.sup..crclbar. 59 177 4 Cl.sup..crclbar.
[0344]
11TABLE 11 178 Example No. Q.sup..sym. Y.sup..crclbar. 60 179
Cl.sup..crclbar. 61 180 2 Cl.sup..crclbar. 62 181 3
Cl.sup..crclbar. 63 182 3 Cl.sup..crclbar. 64 183 3
Cl.sup..crclbar. 65 184 4 Cl.sup..crclbar.
[0345]
12TABLE 12 185 Example No. Q.sup..sym. Y.sup..crclbar. 66 186
Cl.sup..crclbar. 67 187 2 Cl.sup..crclbar. 68 188 3
Cl.sup..crclbar. 69 189 3 Cl.sup..crclbar. 70 190 3
Cl.sup..crclbar. 71 191 4 Cl.sup..crclbar.
[0346]
13TABLE 13 192 Example No. Q.sup..sym. Y.sup..crclbar. 72 193
Cl.sup..crclbar. 73 194 2 Cl.sup..crclbar. 74 195 3
Cl.sup..crclbar. 75 196 3 Cl.sup..crclbar. 76 197 3
Cl.sup..crclbar. 77 198 4 Cl.sup..crclbar.
[0347]
14TABLE 14 199 Example No. Q.sup..sym. Y.sup..crclbar. 78 200
Cl.sup..crclbar. 79 201 2 Cl.sup..crclbar. 80 202 3
Cl.sup..crclbar. 81 203 3 Cl.sup..crclbar. 82 204 3
Cl.sup..crclbar. 83 205 4 Cl.sup..crclbar.
[0348]
15TABLE 15 206 Example No. Q.sup..sym. Y.sup..crclbar. 84 207
Cl.sup..crclbar. 85 208 2 Cl.sup..crclbar. 86 209 3
Cl.sup..crclbar. 87 210 3 Cl.sup..crclbar. 88 211 3
Cl.sup..crclbar. 89 212 4 Cl.sup..crclbar.
[0349]
16TABLE 16 213 Example No. Q.sup..sym. Y.sup..crclbar. 90 214
Cl.sup..crclbar. 91 215 2 Cl.sup..crclbar. 92 216 3
Cl.sup..crclbar. 93 217 3 Cl.sup..crclbar. 94 218 3
Cl.sup..crclbar. 95 219 4 Cl.sup..crclbar.
[0350]
17TABLE 17 220 Example No. Q.sup..sym. Y.sup..crclbar. 96 221
Cl.sup..crclbar. 97 222 2 Cl.sup..crclbar. 98 223 3
Cl.sup..crclbar. 99 224 3 Cl.sup..crclbar. 100 225 3
Cl.sup..crclbar. 101 226 4 Cl.sup..crclbar.
[0351]
18TABLE 18 Example No. 102 227 103 228 104 229
BIOLOGICAL ACTIVITY
[0352] IMP-1 metallo-.beta.-lactamase lacking the N-terminal 18
hydrophobic amino acids which encode the putative periplasmic
signal sequence (EMBL access code PACATAAC6) was PCR amplified from
plasmid DNA prepared from a carbapenem-resistant strain of
Pseudomonas aeruginosa (CL5673). The PCR product was cloned into
pET30a+ (Novegen) and expressed in E. coli BL21(DE3) after
induction with 0.5 mM IPTG for 20 hours at room temperature in
minimal media supplemented with casamino acids and 348 .mu.M
ZnSO.sub.4. Soluble IMP-1 was purified from cell extracts by
SP-Sepharose (Pharmacia) ion exchange and Superdex 75 (Pharmacia)
size-exclusion chromatography. Soluble CcrA
metallo-.beta.-lactamase was cloned from an imipenem resistant
clinical isolate of Bacteroides fragilis and was expressed and
purified as described by Toney et al. [Protein Expr. Purif. 9 355
(1997)].
[0353] The IC.sub.50 of succinate derivatives was determined
following a 15 minute incubation at 37.degree. C. with IMP-1 (0.75
nM in 50 mM MOPS, pH 7) or CcrA (4 nM in 50 mM Mops pH 7). Using
initial velocity as a measure of activity, inhibition was monitored
spectrophotometrically at 490 nm in a Molecular Devices
SPECTRAmax.TM. 250 96-well plate reader employing nitrocefin as the
reporter substrate at approximately K.sub.m concentration (60
.mu.M).
[0354] A laboratory strain of E. coli engineered to express IMP-1
was used to evaluate the ability of succinate derivatives to
reverse metallo-.beta.-lactamase-mediated carbapenem resistance in
bacteria. Native IMP-1, which included the N-terminal periplasmic
signal sequence, was PCR amplified from CNA isolated from a
carbapenem resistant P. aeruginosa clinical isolate, CL56673, and
cloned into the pET30a vector. The basal (uninduced) level of IMP-1
expressed when pET30a-IMP-1 was introduced into E. coli BL21(DE3)
resulted in 4-, 64- or 500-fold reduced sensitivity to impenem,
meropenem or (1S,5R,6S)-1-methyl-2-{7[-4-(aminoca-
rbonylmethyl)-1,4-diazoniabicyclo(2.2.2)octan-1-yl]methyl-fluoren-9-on-3-y-
l}-6-(1R-hydroxyethyl)-carbapen-2-em-3-carboxylate chloride (a
carbapenem synthesized at Merck Research Laboratories)
respectively. For example, the minimum inhibitory concentration
(MIC) of (1S,5R,6S)-1-methyl-2-{7-[4-
-(aminocarbonylmethyl)-1,4-diazoniabicyclo(2.2.2)octan-1-yl]methyl-fluoren-
-9-on-3-yl}-6-(1R-hydroxyethyl)-carbapen-2-em-3-carboxylate
chloride, was typically increased from 0.06-0.12 .mu.g/ml to 16-32
.mu.g/ml by the expression of IMP-1. To evaluate IMP-1 inhibitors,
an overnight culture of E. coli BL2(DE3)/pET30a-IMP-1, grown
35.degree. C. in LB broth (Difco) or Mueller Hinton broth (BBL)
supplemented with kanamycin (50 .mu.M/ml), was diluted to a final
concentration of .about.10.sup.5 cells/ml in Mueller Hinton broth
(BBL) containing a subinhibitory concentration (0.25.times.MIC) of
the carbapenem, (1S,5R,6S)-1-methyl-2-{7-[4-(aminocar-
bonylmethyl)-1,4-diazoniabicyclo(2.2.2)octan-1-yl]methyl-fluoren-9-on-3-yl-
}-6-(1R-hydroxyethyl)-carbapen-2-em-3-carboxylate chloride. Various
concentrations of IMP-1 inhibitor were added to the bacterial
growth medium and their capacity to effect a four-fold or greater
increase in sensitivity to the carbapenem was monitored. The
readout for antibacterial activity showed no visible growth after
20 hours incubation at 35.degree. C.
[0355] Representative compounds of Formula I were tested as
inhibitors against purified IMP-1 metallo-.beta.-lactamase and
found to be active in an IC.sub.50 range of from about 0.1 nM to
about 1000 nM. The ability of representative compounds of Formula I
to potentiate the activity of the carbapenem antibiotic
(1S,5R,6S)-1-methyl-2-{7-[4-(aminocarbonylmethyl)-1-
,4-diazoniabicyclo(2.2.2)octan-1yl]-methyl-fluoren-9-on-3-yl}-6-(1R-hydrox-
yethyl)-carbapen-2-em-3-carboxylate chloride against an IMP-1
producing laboratory strain E. coli BL21(DE3)/pET30a-IMP-1 was
tested. Compounds of Formula I in the concentration range of from
about 0.003 .mu.M to about 12.5 .mu.M. were found to produce 4-fold
increase in sensitivity to the carbapenem antibiotic
(1S,5R,6S)-1-methyl-2-{7-[4-(aminocarbonylmethyl)-1-
,4-diazoniabicyclo(2.2.2)octan-1-yl]-methyl-fluoren-9-on-3-yl}-6-(1R-hydro-
xyethyl)-carbapen-2-em-3-carboxylate chloride in an IMP-1 producing
laboratory strain E. Coli. BL21(DE3)/pET30a-IMP-1.
* * * * *