U.S. patent application number 14/306778 was filed with the patent office on 2014-10-02 for peptidomimetic protease inhibitors.
The applicant listed for this patent is Vertex Pharmaceuticals Incorporated. Invention is credited to Robert Edward Babine, Shu Hui Chen, Ivan Collado-Cano, Luc J. Farmer, Victor Frantz, Maria Cristina Garcia Paredes, John Irvin Glass, Deqi Guo, Ling Jin, Jason Eric Lamar, Raymond Samuel Parker, III, Robert B. Perni, Nancy June Snyder, Xicheng David Sun, Mark Joseph Tebbe, May Q. Wang, Yvonne Yee Mai Yip.
Application Number | 20140294763 14/306778 |
Document ID | / |
Family ID | 26923261 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140294763 |
Kind Code |
A1 |
Babine; Robert Edward ; et
al. |
October 2, 2014 |
PEPTIDOMIMETIC PROTEASE INHIBITORS
Abstract
The present invention relates to peptidomimetic compounds useful
as protease inhibitors, particularly as serine protease inhibitors
and more particularly as hepatitis C NS3 protease inhibitors;
intermediates thereto; their preparation including novel
steroselective processes to intermediates. The invention is also
directed to pharmaceutical compositions and to methods for using
the compounds for inhibiting HCV protease or treating a patient
suffering from an HCV infection or physiological condition related
to the infection. Also provided are pharmaceutical combinations
comprising, in addition to one or more HCV serine protease
inhibitors, one or more interferons exhibiting anti-HCV activity
and/or one or more compounds having anti HCV activity and a
pharmaceutically acceptable carrier, and methods for treating or
preventing a HCV infection in a patient using the compositions. The
present invention is also directed to a kit or pharmaceutical pack
for treating or preventing HCV infection in a patient.
Inventors: |
Babine; Robert Edward;
(Franklin, MA) ; Chen; Shu Hui; (Carmel, IN)
; Collado-Cano; Ivan; (Madrid, ES) ; Garcia
Paredes; Maria Cristina; (Madrid, ES) ; Glass; John
Irvin; (Indianapolis, IN) ; Jin; Ling;
(Carmel, IN) ; Lamar; Jason Eric; (Indianapolis,
IN) ; Parker, III; Raymond Samuel; (Doylestown,
PA) ; Snyder; Nancy June; (Lizton, IN) ; Sun;
Xicheng David; (Superior, CO) ; Guo; Deqi;
(Carmel, IN) ; Yip; Yvonne Yee Mai; (Indianapolis,
IN) ; Wang; May Q.; (Indianapolis, IN) ;
Frantz; Victor; (Indianapolis, IN) ; Tebbe; Mark
Joseph; (Hamburg, DE) ; Perni; Robert B.;
(Marlborough, MA) ; Farmer; Luc J.; (Montreal,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vertex Pharmaceuticals Incorporated |
Boston |
MA |
US |
|
|
Family ID: |
26923261 |
Appl. No.: |
14/306778 |
Filed: |
June 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13955249 |
Jul 31, 2013 |
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14306778 |
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13541436 |
Jul 3, 2012 |
8529882 |
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13955249 |
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12231982 |
Sep 8, 2008 |
8252923 |
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13541436 |
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10344112 |
Dec 17, 2004 |
7820671 |
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PCT/US2001/026008 |
Aug 31, 2001 |
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12231982 |
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60277641 |
Mar 21, 2001 |
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60229398 |
Aug 31, 2000 |
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Current U.S.
Class: |
424/85.2 ;
424/85.4; 424/85.6; 424/85.7; 435/184; 435/375; 514/4.3;
530/323 |
Current CPC
Class: |
A61P 31/18 20180101;
C07K 5/06052 20130101; A61P 43/00 20180101; C12N 2770/24222
20130101; A61K 38/215 20130101; A61P 31/12 20180101; C07K 5/0205
20130101; A61K 38/212 20130101; C07K 5/1016 20130101; A61K 38/095
20190101; C07K 5/101 20130101; C07D 209/02 20130101; A61K 38/21
20130101; A61P 31/14 20180101; A61K 38/08 20130101; A61K 38/07
20130101; A61K 45/06 20130101; C07K 5/0808 20130101; C07D 209/52
20130101; C07K 5/0202 20130101; Y02P 20/55 20151101; C07K 7/02
20130101; C07K 7/06 20130101; C07K 14/005 20130101; A61P 1/16
20180101; A61K 38/21 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.2 ;
530/323; 514/4.3; 424/85.4; 424/85.7; 424/85.6; 435/184;
435/375 |
International
Class: |
C07K 7/02 20060101
C07K007/02; A61K 38/21 20060101 A61K038/21; A61K 45/06 20060101
A61K045/06; C07K 5/103 20060101 C07K005/103; A61K 38/07 20060101
A61K038/07; A61K 38/08 20060101 A61K038/08; C07D 209/52 20060101
C07D209/52 |
Claims
1. A compound of formula 1 ##STR00445## wherein: R.sup.0 is a bond
or difluoromethylene; R.sup.1 is hydrogen, optionally substituted
aliphatic group, optionally substituted cyclic group or optionally
substituted aromatic group; R.sup.2 and R.sup.9 are each
independently optionally substituted aliphatic group, optionally
substituted cyclic group or optionally substituted aromatic group;
R.sup.3, R.sup.5 and R.sup.7 are each independently; optionally
substituted (1,1- or 1,2-)cycloalkylene; or optionally substituted
(1,1- or 1,2-)heterocyclylene; or methylene or ethylene,
substituted with one substituent selected from the group consisting
of an optionally substituted aliphatic group, an optionally
substituted cyclic group or an optionally substituted aromatic
group, and wherein the methylene or ethylene is further optionally
substituted with an aliphatic group substituent; or R.sup.4,
R.sup.6, R.sup.8 and R.sup.10 are each independently is hydrogen or
optionally substituted aliphatic group; ##STR00446## is substituted
monocyclic azaheterocyclyl or optionally substituted multicyclic
azaheterocyclyl, or optionally substituted multicyclic
azaheterocyclenyl wherein the unsaturatation is in the ring distal
to the ring bearing the
R.sup.9-L-(N(R.sup.8)--R.sup.7--C(O)--).sub.nN(R.sup.6)--R.sup.5--C(O)--N
moiety and to which the
--C(O)--N(R.sup.4)--R.sup.3--C(O)C(O)NR.sup.2R.sup.1 moiety is
attached; L is --C(O)--, --OC(O)--, --NR.sup.10C(O)--,
--S(O).sub.2--, or --NR.sup.10S(O).sub.2--; and n is 0 or 1, or a
pharmaceutically acceptable salt or prodrug thereof, or a solvate
of such a compound, its salt or its prodrug, provided when
##STR00447## is substituted ##STR00448## then L is --OC(O)-- and
R.sup.9 is optionally substituted aliphatic; or at least one of
R.sup.3, R.sup.5 and R.sup.7 is ethylene, substituted with one
substituent selected from the group consisting of an optionally
substituted aliphatic group, an optionally substituted cyclic group
or an optionally substituted aromatic group and wherein the
ethylene is further optionally substituted with an aliphatic group
substituent; or R.sup.4 is optionally substituted aliphatic.
2. A compound of claim 1 wherein R.sup.0 is a bond.
3. A compound of claim 1 or 2 wherein: optionally substituted
aliphatic groups are alkyl, alkenyl or alkynyl, optionally
substituted with one or more aliphatic group substituent;
optionally substituted cyclic groups are cycloalkyl, cycloalkenyl,
heterocyclyl or heterocyclenyl groups optionally substituted with
one or more ring group substituents; optionally substituted
aromatic groups are aryl or heteroaryl groups optionally
substituted with one or more ring group substituents; optionally
substituted (1,1- or 1,2) cycloalkylene groups are (1,1- or 1,2)
cycloalkylene groups optionally substituted with one or more ring
group substituents; optionally substituted (1,1- or 1,2)
heterocyclylene groups are (1,1- or 1,2) heterocyclylene groups
optionally substituted with one or more ring group substituents;
##STR00449## as substituted monocyclic azaheterocyclyl is a
monocyclic azaheterocyclyl group substituted directly or through a
linker group by at least one substituent selected from aryl,
heteroaryl, aryloxy, heteroaryloxy, aroyl or its thio analogue,
heteroaryl or its thioxo analogue, aroyloxy, heteroaroyloxy,
aryloxycarbonyl, heteroaryloxycarbonyl, arylsulfonyl,
heteroarylsulfonyl, arylsulfinyl, heteroarylsulfinyl, arylthio,
heteroarylthio, aryldiazo, heteroaryldiazo, Y.sup.1Y.sup.2N--,
Y.sup.1Y.sup.2NC(O)--, Y.sup.1Y.sup.2NC(O)O--,
Y.sup.1Y.sup.2NC(O)NY.sup.3-- or Y.sup.1Y.sup.2NSO.sub.2-- wherein
at least one of Y.sup.1 and Y.sup.2 is aryl or heteroaryl, wherein
said linker group is selected from the group consisting of
--C(O)--, --OC(O)--, lower alkyl, lower alkoxy, lower alkenyl,
--O--, --S--, --C(O)C(O)--, --S(O)--, --S(O).sub.2--,
--NR.sup.80--, where R.sup.80 is hydrogen, alkyl, cycloalkyl, aryl,
aralkyl, heterocyclyl or heteroaryl; optionally substituted
multicyclic azaheterocyclyl is a multicyclic azaheterocyclyl group
optionally substituted by one or more ring group substituents;
optionally substituted multicyclic azaheterocyenyll is a
multicyclic azaheterocyclenyll group optionally substituted by one
or more ring group substituents; wherein: aliphatic group
substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy,
aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl
or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl
or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy,
heteroaroyloxy, halo, nitro, cyano, carboxy (acid), --C(O)--NHOH,
--C(O)--CH.sub.2OH, --C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho,
phosphono, alkylsulphonylcarbamoyl, tetrazolyl,
arylsulphonylcarbamoyl, N-methoxycarbamoyl,
heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,
3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as
3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,
alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl,
alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl,
alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl; ring group substituents means aryl, heteroaryl,
hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its
thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or
its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), acid biostere, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl, or when a ring system is saturated or partially
saturated, the "ring group substituents" further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.); aryl means an
aromatic monocyclic or multicyclic ring system of 6 to 14 carbon
atoms; cycloalkyl means a non-aromatic mono- or multicyclic ring
system of 3 to 10 carbon atoms; cycloalkenyl means a non-aromatic
mono- or multicyclic ring system of 3 to 10 carbon atoms which
contain at least one carbon-carbon double bond; cyclyl means
cycloalkyl, cycloalkenyl, heterocyclyl or heterocyclenyl;
heterocyclyl means a non-aromatic saturated monocyclic or
multicyclic ring system of about 3 to about 10 carbon atoms in
which one or more of the carbon atoms in the ring system is/are
hetero element(s) other than carbon; heterocyclenyl means a
non-aromatic monocyclic or multicyclic hydrocarbon ring system of
about 3 to about 10 carbon atoms in which one or more of the carbon
atoms in the ring system is/are hetero element(s) other than carbon
and which contains at least one carbon-carbon double bond or
carbon-nitrogen double bond; and heteroaryl means an aromatic
monocyclic or multicyclic ring system of about 5 to about 14 carbon
atoms, in which one or more of the carbon atoms in the ring system
is/are hetero element(s) other than carbon.
4. A compound of any one of claims 1 to 3 wherein R.sup.0 is
difluoromethylene.
5. A compound of any one of claims 1 to 4 wherein R.sup.1 is
hydrogen or optionally substituted lower aliphatic group.
6. A compound of any one of claims 1 to 5 wherein R.sup.1 is
hydrogen or lower alkyl.
7. A compound of any one of claims 1 to 6 wherein R.sup.1 is
hydrogen.
8. A compound of any one of claims 1 to 7 wherein R.sup.2 is
optionally substituted lower aliphatic group or optionally
substituted monocyclic group.
9. A compound of any one of claims 1 to 8 wherein R.sup.2 is
optionally substituted lower alkyl, optionally substituted lower
alkenyl, or optionally substituted monocyclic cycloalkyl.
10. A compound of any one of claims 1 to 9 wherein R.sup.2 is
carboxymethyl, 1-carboxy-2-phenylethyl, cyclopropyl, cyclobutyl,
1-cyclohexylethyl, 1-phenylethyl, but-2-yl, 1-pyrid-4-ylethyl,
propen-3-yl or 3-methylbut-2-yl.
11. A compound of any one of claims 1 to 10 wherein R.sup.3 is
optionally substituted lower aliphatic group methylene.
12. A compound of any one of claims 1 to 11 wherein R.sup.3 is
optionally halo substituted lower (alkyl or alkenyl)methylene.
13. A compound as claimed in any one of claims 1 to 12 wherein
R.sup.3 is propylmethylene, 2,2-difluoroethylmethylene,
2,2,2-trifluoromethylene or propen-3-ylmethylene;
14. A compound of claim 13 wherein R.sup.3 is propylmethylene or
2,2-difluoroethylmethylene.
15. A compound of claim 14 wherein R.sup.3 is propylmethylene.
16. A compound of any one of claims 1 to 15 wherein R.sup.4 is
hydrogen or optionally substituted lower aliphatic group.
17. A compound of claim 16 wherein R.sup.4 is hydrogen.
18. A compound of any one of claims 1 to 17 wherein R.sup.5 is
optionally substituted lower aliphatic group methylene.
19. A compound of any one of claims 1 to 18 wherein R.sup.5 is
optionally (phenyl, carboxy, carboxamido or alkoxycarbonyl)
substituted lower (alkyl or alkenyl) methylene.
20. A compound of any one of claims 1 to 19 wherein R.sup.5 is
methylmethylene, isopropylmethylene, t-butylmethylene,
but-2-ylmethylene, butylmethylene, benzylmethylene,
3-methylbutylmethylene, 2-methylpropylmethylene,
carboxymethylmethylene, carboxamidomethylmethylene,
benzyloxycarbonylmethylmethylene, benzyloxycarbonylpropylmethylene
or phenylpropen-3-ylmethylene.
21. A compound of claim 20 wherein R.sup.5 is isopropylmethylene or
t-butylmethylene.
22. A compound of any one of claims 1 to 21 wherein R.sup.6 is
hydrogen or optionally substituted lower aliphatic group.
23. A compound of any one of claims 1 to 22 wherein R.sup.6 is
hydrogen or lower alkyl.
24. A compound of claim 23 wherein R.sup.6 is hydrogen.
25. A compound of any one of claims 1 to 24 wherein R.sup.7 is
optionally substituted lower aliphatic group methylene, optionally
substituted lower cyclic group methylene or optionally substituted
monocyclic (aryl or heteroaryl) methylene.
26. A compound of any one of claims 1 to 25 wherein R.sup.7 is
optionally substituted lower alkylmethylene, optionally substituted
lower cycloalkylmethylene or optional substituted
phenylmethylene.
27. A compound of any one of claims 1 to 26 wherein R.sup.7 is
methylmethylene, isopropylmethylene, n-propylmethylene,
phenylmethylene, cyclohexylmethylene, cyclopentylmethylene,
t-butylmethylene, s-butylmethylene, cyclohexylmethylmethylene, or
phenylmethylmethylene.
28. A compound of claim 27 wherein R.sup.7 is isopropylmethylene,
cyclohexylmethylene, cyclopentylmethylene, t-butylmethylene or
s-butylmethylene.
29. A compound of any one of claims 1 to 28 wherein each of
R.sup.3, R.sup.5, and R.sup.7 is mono substituted methylene.
30. A compound of any one of claims 1 to 29 wherein R.sup.3 is mono
substituted methylene and has an (S) configuration on the carbon
attached to the --C(O)--R.sup.0--C(O)--NR.sup.1R.sup.2 moiety.
31. A compound of any one of claims 1 to 30 wherein R.sup.8 is
hydrogen or optionally substituted lower aliphatic group.
32. A compound of claim 31 wherein R.sup.8 is hydrogen or lower
alkyl.
33. A compound of claim 32 wherein R.sup.8 is hydrogen.
34. A compound of any one of claims 1 to 33 wherein R.sup.9 is
optionally substituted lower aliphatic group or optionally
substituted monocyclic aromatic group.
35. A compound of claim 34 wherein R.sup.9 is optionally
substituted lower alkyl or optionally substituted monocyclic
heteroaryl.
36. A compound of any one of claims 1 to 34 wherein R.sup.9 is
optionally (carboxy, (loweralkyl)SO.sub.2NH--, (lower alkyl)HNCO--,
hydroxy, phenyl, heteroaryl, or (lower alkyl)OC(O)NH--)-substituted
lower alkyl, or optionally substituted monocyclic heteroaryl.
37. A compound of any one of claims 1 to 33 wherein R.sup.9 is
lower alkyl substituted by (mono- or di-)MeOC(O)NH--.
38. A compound of any one of claims 1 to 33 wherein R.sup.9 is
(carboxy, (lower alkyl)HNCO-- or tetrazolyl) substituted lower
alkyl.
39. A compound of any one of claims 1 to 33 wherein R.sup.9 is
3-carboxypropyl, 2-tetrazol-5ylpropyl,
3-(N-methylcarboxamido)propyl or 3-carboxy-2,2-dimethylpropyl.
40. A compound of any one of claims 1 to 33 wherein R.sup.9 is
3-carboxypropyl, 2-tetrazol-5ylpropyl or
3-(N-methylcarboxamido)propyl.
41. A compound of any one of claims 1 to 33 wherein R.sup.9 is
optionally substituted lower alkyl.
42. A compound of any one of claims 1 to 33 wherein R.sup.9 is
1-hydroxy-2-phenylethyl, isopropyl or t-butyl.
43. A compound of any one of claims 1 to 33 wherein R.sup.9 is
isopropyl or t-butyl.
44. A compound of any one of claims 1 to 33 wherein R.sup.9 is
selected from the group consisting of ##STR00450## ##STR00451##
45. A compound of any one of claims 1 to 33 wherein R.sup.9 is
pyrazinyl.
46. A compound of any one of claims 1 to 45 wherein R.sup.10 is
hydrogen or optionally substituted lower aliphatic group.
47. A compound of any one of claims 1 to 46 wherein R.sup.10 is
hydrogen or lower alkyl.
48. A compound of any one of claims 1 to 47 wherein R.sup.10 is
hydrogen.
49. A compound of any one of claims 1 to 48 wherein ##STR00452## as
a substituted monocyclic azaheterocyclyl is substituted
pyrrolidinyl.
50. A compound of any one of claims 1 to 48 wherein ##STR00453## as
a substituted monocyclic azaheterocyclyl is optionally substituted
##STR00454## or optionally substituted ##STR00455## wherein Ar is
R.sup.2 that comprises an aromatic moiety.
51. A compound of any one of claims 1 to 48 wherein ##STR00456## as
a substituted monocyclic azaheterocyclyl is optionally substituted
##STR00457##
52. A compound of any one of claims 1 to 48 wherein
##STR00458##
53. A compound of any one of claims 1 to 48 wherein ##STR00459## as
an optionally substituted multicyclic azaheterocyclyl is optionally
substituted ##STR00460##
54. A compound of any one of claims 1 to 48 wherein ##STR00461## as
an optionally substituted multicyclic azaheterocyclyl is optionally
substituted ##STR00462##
55. A compound of any one of claims 1 to 48 wherein ##STR00463## as
an optionally substituted multicyclic azaheterocyclenyl is
optionally substituted ##STR00464##
56. A compound of any one of claims 1 to 48 wherein ##STR00465## as
an optionally substituted multicyclic azaheterocyclenyl is
optionally substituted ##STR00466##
57. A compound of any one of claims 1 to 48 wherein ##STR00467## as
an optionally substituted multicyclic azaheterocyclenyl is
optionally substituted ##STR00468##
58. A compound of any one of claims 1 to 57 wherein the
--C(O)--N(R.sup.4)--R.sup.3--C(O)R.sup.0C(O)NR.sup.2R.sup.1 moiety
attached to ##STR00469## is attached a carbon .alpha. to the
nitrogen atom.
59. A compound of any one of claims 1 to 58 wherein L is --C(O)--
or --OC(O)--.
60. A compound of any one of claims 1 to 59 wherein n is 0.
61. A compound of any one of claims 1 to 59 wherein n is 1.
62. A compound as claimed in claim 1 selected from the group
consisting of: ##STR00470## ##STR00471## ##STR00472## ##STR00473##
##STR00474## ##STR00475## ##STR00476## ##STR00477## ##STR00478##
##STR00479## ##STR00480## ##STR00481## ##STR00482## ##STR00483##
##STR00484## or a pharmaceutically acceptable salt or prodrug
thereof, or a solvate of such a compound, its salt or its
prodrug.
63. A pharmaceutical compositions comprising a pharmaceutically
acceptable amount of the compound of any one of the preceeding
claims and a pharmaceutically acceptable carrier.
64. A method for inhibiting HCV protease comprising contacting the
protease with a compound of any one of claims 1 to 62.
65. A method for treating a patient suffering from an HCV infection
or physiological conditions related to the infection comprising
administering to the patient a pharmaceutically effective amount of
a compound of any one of claims 1 to 62.
66. A method for treating a patient suffering from an HCV infection
or physiological conditions related to the infection comprising
administering to the patient a pharmaceutically effective amount of
a compound of any one of claims 1 to 62 in combination with a
pharmaceutically effective amount of another anti-HCV
therapeutic.
67. The method of claim 66 wherein the anti-HCV therapeutic is
interferon or derivatized interferon.
68. A pharmaceutical composition, comprising a hepatitis C virus
serine protease inhibitor, an interferon having anti-hepatitis C
virus activity, and a pharmaceutically acceptable carrier.
69. The pharmaceutical composition of claim 68, further comprising
a compound having anti-hepatitis C virus activity, wherein said
compound is other than an interferon.
70. A pharmaceutical composition, comprising a hepatitis C virus
serine protease inhibitor, a compound having anti-hepatitis C virus
activity, and a pharmaceutically acceptable carrier, wherein said
compound is other than an interferon.
71. The pharmaceutical composition of claim 69, wherein said
hepatitis C virus serine protease inhibitor, said interferon, and
said compound having anti-hepatitis C virus activity are each
present in an amount selected from the group consisting of a
pharmaceutically effective amount, a subclinical pharmaceutically
effective amount, and a combination thereof.
72. The pharmaceutical composition of claim 71, wherein said
hepatitis C virus serine protease inhibitor is a compound of any
one of claims 1 to 62; said interferon is selected from the group
consisting of interferon alpha 2B, pegylated interferon alpha,
consensus interferon, interferon alpha 2A, lymphoblastoid
interferon, and interferon tau; and said compound having
anti-hepatitis C virus activity is selected from the group
consisting of interleukin 2, interleukin 6, interleukin 12, a
compound that enhances the development of a type 1 helper T cell
response, double stranded RNA, double stranded RNA complexed with
tobramycin, Imiquimod, ribavirin, an inosine 5'-monophosphate
dehydrogenase inhibitor, amantadine, and rimantadine.
73. A method of treating or preventing a hepatitis C virus
infection in a patient in need thereof, comprising administering to
said patient a pharmaceutically effective amount of a combination
of a hepatitis C virus serine protease inhibitor and an interferon
having anti-hepatitis C virus activity.
74. The method of claim 73, wherein said pharmaceutically effective
amount of said combination further comprises a compound having
anti-hepatitis C virus activity, wherein said compound is other
than an interferon.
75. A method of treating or preventing a hepatitis C virus
infection in a patient in need thereof, comprising administering to
said patient a pharmaceutically effective amount of a combination
of a hepatitis C virus serine protease inhibitor and a compound
having anti-hepatitis C virus activity, wherein said compound is
other than an interferon.
76. The method of any one of claim 74, wherein said hepatitis C
virus serine protease inhibitor, said interferon, and said compound
having anti-hepatitis C virus activity are each present in an
amount selected from the group consisting of a pharmaceutically
effective amount, a subclinical pharmaceutically effective amount,
and a combination thereof.
77. The method of claim 76, wherein said hepatitis C virus serine
protease inhibitor is a compound of any one of claims 1 to 62; said
interferon is selected from the group consisting of interferon
alpha 2B, pegylated interferon alpha, consensus interferon,
interferon alpha 2A, lymphoblastoid interferon, and interferon tau;
and said compound having anti-hepatitis C virus activity is
selected from the group consisting of interleukin 2, interleukin 6,
interleukin 12, a compound that enhances the development of a type
1 helper T cell response, double stranded RNA, double stranded RNA
complexed with tobramycin, Imiquimod, ribavirin, an inosine
5'-monophosphate dehydrogenase inhibitor, amantadine, and
rimantadine.
78. Use of a hepatitis C virus serine protease inhibitor in
combination with an interferon having anti-hepatitis C virus
activity to prepare a medicament for the treatment or prevention of
hepatitis C virus infection in a patient in need thereof.
79. Use of a hepatitis C virus serine protease inhibitor in
combination with a compound having anti-hepatitis C virus activity
to prepare a medicament for the treatment or prevention of
hepatitis C virus infection in a patient in need thereof, wherein
said compound is other than an interferon.
80. Use of a hepatitis C virus serine protease inhibitor in
combination with an interferon having anti-hepatitis C virus
activity and a compound having anti-hepatitis C virus activity to
prepare a medicament for the treatment or prevention of hepatitis C
virus infection in a patient in need thereof, wherein said compound
is other than an interferon.
81. The use according to claim 80, wherein said hepatitis C virus
serine protease inhibitor, said interferon, and said compound
having anti-hepatitis C virus activity are each present in said
medicament in an amount selected from the group consisting of a
pharmaceutically effective amount, a subclinical pharmaceutically
effective amount, and a combination thereof.
82. The use according to claim 81, wherein said hepatitis C virus
serine protease inhibitor is a compound of any one of claims 1 to
62; said interferon is selected from the group consisting of
interferon alpha 2B, pegylated interferon alpha, consensus
interferon, interferon alpha 2A, lymphoblastoid interferon, and
interferon tau; and said compound having anti-hepatitis C virus
activity is selected from the group consisting of interleukin 2,
interleukin 6, interleukin 12, a compound that enhances the
development of a type 1 helper T cell response, double stranded
RNA, double stranded RNA complexed with tobramycin, Imiquimod,
ribavirin, an inosine 5'-monophosphate dehydrogenase inhibitor,
amantadine, and rimantadine.
83. A kit or pharmaceutical pack, comprising a plurality of
separate containers, wherein at least one of said containers
contains a hepatitis C virus serine protease inhibitor and at least
another of said containers contains an interferon having
anti-hepatitis C virus activity.
84. A kit or pharmaceutical pack, comprising a plurality of
separate containers, wherein at least one of said containers
contains a hepatitis C virus serine protease inhibitor and at least
another of said containers contains a compound having
anti-hepatitis C virus activity, wherein said compound is other
than an interferon.
85. A kit or pharmaceutical pack, comprising a plurality of
separate containers, wherein at least one of said containers
contains a hepatitis C virus serine protease inhibitor, at least
another of said containers contains an interferon having
anti-hepatitis C virus activity, and at least another of said
containers contains a compound having anti-hepatitis C virus
activity, wherein said compound is other than an interferon.
86. The kit or pharmaceutical pack of claim 85, wherein said
hepatitis C virus serine protease inhibitor, said interferon, and
said compound having anti-hepatitis C virus activity are each
present in an amount selected from the group consisting of a
pharmaceutically effective amount, a subclinical pharmaceutically
effective amount, and a combination thereof.
87. The kit or pharmaceutical pack of claim 86 wherein said
hepatitis C virus serine protease inhibitor is a compound of any
one of claims 1 to 62; said interferon is selected from the group
consisting of interferon alpha 2B, pegylated interferon alpha,
consensus interferon, interferon alpha 2A, lymphoblastoid
interferon, and interferon tau; and said compound having
anti-hepatitis C virus activity is selected from the group
consisting of interleukin 2, interleukin 6, interleukin 12, a
compound that enhances the development of a type 1 helper T cell
response, double stranded RNA, double stranded RNA complexed with
tobramycin, Imiquimod, ribavirin, an inosine 5'-monophosphate
dehydrogenase inhibitor, amantadine, and rimantadine.
88. A method of inhibiting hepatitis C virus replication in a cell,
comprising contacting said cell, a hepatitis C virus serine
protease inhibitor, and an interferon having anti-hepatitis C virus
activity.
89. The method of claim 88, further comprising contacting said cell
and a compound having anti-hepatitis C virus activity, wherein said
compound is other than an interferon.
90. A method of inhibiting hepatitis C virus replication in a cell,
comprising contacting said cell, a hepatitis C virus serine
protease inhibitor, and a compound having anti-hepatitis C virus
activity, wherein said compound is other than an interferon.
91. The method of claim 88, wherein said hepatitis C virus serine
protease inhibitor, said interferon, and said compound having
anti-hepatitis C virus activity are each present in an amount
selected from the group consisting of a pharmaceutically effective
amount, a subclinical pharmaceutically effective amount, and a
combination thereof.
92. The method of claim 91, wherein said hepatitis C virus serine
protease inhibitor is a compound of any one of claims 1 to 62; said
interferon is selected from the group consisting of interferon
alpha 2B, pegylated interferon alpha, consensus interferon,
interferon alpha 2A, lymphoblastoid interferon, and interferon tau;
and said compound having anti-hepatitis C virus activity is
selected from the group consisting of interleukin 2, interleukin 6,
interleukin 12, a compound that enhances the development of a type
1 helper T cell response, double stranded RNA, double stranded RNA
complexed with tobramycin, Imiquimod, ribavirin, an inosine
5'-monophosphate dehydrogenase inhibitor, amantadine, and
rimantadine.
93. A compound of formula 24 ##STR00485## wherein: ##STR00486## is
optionally substituted cycloalkyl or optionally substituted fused
arylcycloalkyl; R.sup.11 is --CO.sub.2R.sup.13; R.sup.12 is an
iminic glycinimide derivative adduct; and R.sup.13 is acid
protecting group or optionally substituted aliphatic group.
94. A compound of claim 93 wherein: optionally substituted
cycloalkyl means a non-aromatic mono- or multicyclic ring system of
3 to 10 carbon atoms optionally substituted with one or more ring
group substituents; optionally substituted fused arylcycloalkyl
means a fused arylcycloalkyl optionally substituted with one or
more ring group substituents; optionally substituted aliphatic
group are alkyl, alkenyl, or alkynyl optionally substituted with an
aliphatic group substituent; an iminic glycinimide derivative
adduct is a compound selected from the group consisting of
##STR00487## wherein: R.sup.16 is an acid protecting group,
optionally substituted aryl, or optionally substituted aliphatic
group; R.sup.17 is optionally substituted aryl, optionally
substituted aliphatic group, ##STR00488## R.sup.18 is hydrogen,
alkyl, or alkylthio; or optionally substituted aryl; wherein; ring
group substituents mean substituents attached to aromatic or
non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy,
alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo
analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its
thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl or when the ring system is saturated or partially
saturated, the ring group substituents further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.); and aliphatic
group substituents means aryl, heteroaryl, hydroxy, alkoxy,
cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,
cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo
analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl; and aryl means an aromatic monocyclic or
multicyclic ring system of 6 to 14 carbon atoms.
95. A compound according to claim 93 or 94 where R.sup.11 is
--CO.sub.2R.sup.13.
96. A compound according to any one of claim 93 or 95 where
R.sup.13 is an optionally substituted aliphatic group.
97. A compound according to any one of claims 93 to 96 where
R.sup.13 is an alkyl group.
98. A compound according to any one of claims 93 to 97 where
R.sup.13 is lower alkyl.
99. A compound according to any one of claims 93 to 98 where
R.sup.13 is methyl.
100. A compound according to any one of claims 93 to 99 where
R.sup.12 is ##STR00489## wherein: R.sup.14 is
--CONR.sup.15R.sup.15, --CN; ##STR00490## or --CO.sub.2R.sup.16;
R.sup.15 is optionally substituted aliphatic group; R.sup.16 is
acid protecting group, optionally substituted aryl, or optionally
substituted aliphatic group; R.sup.17 is optionally substituted
aryl, optionally substituted aliphatic group, ##STR00491## R.sup.18
is hydrogen, alkyl, or alkylthio; or optionally substituted aryl;
R.sup.17 and R.sup.18 taken together with the carbon to which
R.sup.17 and R.sup.18 are attached ##STR00492## and {circle around
(S)} is a solid phase.
101. A compound according to claim 100 where R.sup.14 is
--CO.sub.2R.sup.16.
102. A compound according to claim 100 or 101 where R.sup.16 is
optionally substituted aliphatic.
103. A compound according to any one of claims 100 to 102 where
R.sup.16 is alkyl.
104. A compound according to any one of claims 100 to 103 where
R.sup.16 is lower alkyl.
105. A compound according to any one of claims 100 to 104 where
R.sup.16 is t-Bu.
106. A compound according to any one of claims 100 to 105 where
R.sup.17 is optionally substituted aryl.
107. A compound according to any one of claims 100 to 106 where
R.sup.17 is phenyl.
108. A compound according to any one of claims 100 to 107 where
R.sup.18 is optionally substituted aryl.
109. A compound according to any one of claims 100 to 108 where
R.sup.18 is phenyl.
110. A compound of formula 25 ##STR00493## wherein: R.sup.14 is
--CONR.sup.15R.sup.15, --CN; ##STR00494## or --CO.sub.2R.sup.16;
R.sup.15 is optionally substituted aliphatic group; and R.sup.16 is
acid protecting group, optionally substituted aryl, or optionally
substituted aliphatic group.
111. A compound according to claim 110 wherein: optionally
substituted aliphatic groups are alkyl, alkenyl, or alkynyl
optionally substituted with one or more aliphatic group
substituents; optionally substituted aryl means an aromatic
monocyclic or multicyclic ring systems of 6 to 14 carbon atoms
optionally substituted with one or more ring group substituents;
wherein; ring group substituents mean substituents attached to
aromatic or non-aromatic ring systems inclusive of aryl,
heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,
acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,
aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,
acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,
cyano, carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl or when the ring system is saturated or partially
saturated, the "ring group substituents" further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.); and aliphatic
group substituents means aryl, heteroaryl, hydroxy, alkoxy,
cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,
cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo
analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl.
112. A compound according to claim 110 or 111 where R.sup.14 is
--CO.sub.2R.sup.16.
113. A compound according to any one of claims 110 to 112 where
R.sup.16 is optionally substituted aliphatic.
114. A compound according to any one of claims 110 to 113 where
R.sup.16 is alkyl.
115. A compound according to any one of claims 110 to 114 where
R.sup.16 is lower alkyl.
116. A compound according to any one of claims 110 to 115 where
R.sup.16 is t-Bu.
117. A compound of formula 26 ##STR00495## wherein: p.sup.O is
amide protecting group; R.sup.14 is --CONR.sup.15R.sup.15, --CN;
##STR00496## or --CO.sub.2R.sup.16; R.sup.15 is optionally
substituted aliphatic group; and R.sup.16 is acid protecting group,
optionally substituted aryl, or optionally substituted aliphatic
group.
118. A compound according to claim 117 wherein: optionally
substituted aliphatic groups are alkyl, alkenyl, or alkynyl
optionally substituted with one or more aliphatic group
substituents; optionally substituted aryl means an aromatic
monocyclic or multicyclic ring systems of 6 to 14 carbon atoms
optionally substituted with one or more ring group substituents;
wherein; ring group substituents mean substituents attached to
aromatic or non-aromatic ring systems inclusive of aryl,
heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,
acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,
aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,
acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,
cyano, carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfonyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl or when the ring system is saturated or partially
saturated, the ring group substituents further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.); and aliphatic
group substituents means aryl, heteroaryl, hydroxy, alkoxy,
cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,
cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo
analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl.
119. A compound according to claim 117 or 118 where R.sup.14 is
--CO.sub.2R.sup.16.
120. A compound according to any one of claims 117 to 119 where
R.sup.16 is optionally substituted aliphatic.
121. A compound according to any one of claims 117 to 120 where
R.sup.16 is alkyl.
122. A compound according to any one of claims 117 to 121 where
R.sup.16 is lower alkyl.
123. A compound according to any one of claims 117 to 122 where
R.sup.16 is t-Bu.
124. A compound according to any one of claims 117 to 123 where
p.sup.0 is selected from the group consisting of BOC, CBz, and
--CO.sub.2alkyl.
125. A compound according to claim 124 where p.sup.0 is BOC.
126. A compound of formula 27 ##STR00497## wherein: p.sup.O is
amide protecting group; R.sup.14 is --CONR.sup.15R.sup.15, --CN;
##STR00498## or --CO.sub.2R.sup.16; R.sup.15 is optionally
substituted aliphatic group; and R.sup.16 is acid protecting group,
optionally substituted aryl, or optionally substituted aliphatic
group.
127. A compound according to claim 126 wherein: optionally
substituted aliphatic groups are alkyl, alkenyl, or alkynyl
optionally substituted with one or more aliphatic group
substituents; optionally substituted aryl means an aromatic
monocyclic or multicyclic ring systems of 6 to 14 carbon atoms
optionally substituted with one or more ring group substituents;
wherein; ring group substituents mean substituents attached to
aromatic or non-aromatic ring systems inclusive of aryl,
heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,
acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,
aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,
acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,
cyano, carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl or when the ring system is saturated or partially
saturated, the ring group substituents further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.); and aliphatic
group substituents means aryl, heteroaryl, hydroxy, alkoxy,
cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,
cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo
analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl.
128. A compound according to any one of claim 126 or 127 where
R.sup.14 is --CO.sub.2R.sup.16.
129. A compound according to any one of claims 126 to 128 where
R.sup.16 is optionally substituted aliphatic.
130. A compound according to any one of claims 126 to 129 where
R.sup.16 is alkyl.
131. A compound according to any one of claims 126 to 130 where
R.sup.16 is lower alkyl.
132. A compound according to any one of claims 126 to 131 where
R.sup.16 is t-Bu.
133. A compound according to any one of claims 126 to 132 where
p.sup.0 is selected from the group consisting of BOC, CBz, and
--CO.sub.2alkyl.
134. A compound according to claim 133 where p.sup.0 is BOC.
135. A process for preparing a chiral bicycloprolinate compound of
formula 28 ##STR00499## comprising the steps of: (a) cleaving and
cyclizing a compound of formula 24 ##STR00500## wherein:
##STR00501## is optionally substituted cycloalkyl or optionally
substituted fused arylcycloalkyl; R.sup.11 is --CO.sub.2R.sup.13;
R.sup.12 is an iminic glycinimide derivative adduct; R.sup.13 is
acid protecting group or optionally substituted aliphatic group;
under cleaving and cyclizing conditions to form a compound of
formula 25 ##STR00502## wherein: R.sup.14 is --CONR.sup.15R.sup.15,
--CN; ##STR00503## or --CO.sub.2R.sup.16; R.sup.15 is optionally
substituted aliphatic group; R.sup.16 is acid protecting group,
optionally substituted aryl, or optionally substituted aliphatic
group; and (b) protecting the nitrogen of the lactam moiety in the
compound of formula 25 with an amide protecting group to form a
compound of formula 26 ##STR00504## wherein: p.sup.O is amide
protecting group; R.sup.14 is as described herein; and (c) reducing
the compound of formula 26 under reducing conditions to form a
compound of formula 27 ##STR00505## wherein: p.sup.O and R.sup.14
are as described herein; and (d) deprotecting the compound of
formula 27 under deprotecting conditions to form a compound of
formula 28 ##STR00506## wherein: R.sup.14 is as described
herein.
136. A compound according to claim 135 wherein: optionally
substituted aliphatic groups are alkyl, alkenyl, or alkynyl
optionally substituted with one or more aliphatic group
substituents; optionally substituted aryl means an aromatic
monocyclic or multicyclic ring systems of 6 to 14 carbon atoms
optionally substituted with one or more ring group substituents;
optionally substituted cycloalkyl means a non-aromatic mono- or
multicyclic ring system of 3 to 10 carbon atoms optionally
substituted with one or more ring group substituents; optionally
substituted fused arylcycloalkyl means a fused arylcycloalkyl
optionally substituted with one or more ring group substituents; an
iminic glycinimide derivative adduct is a compound selected from
the group consisting of ##STR00507## wherein: R.sup.16 is an acid
protecting group, optionally substituted aryl, or optionally
substituted aliphatic group; R.sup.17 is optionally substituted
aryl, optionally substituted aliphatic group, ##STR00508## R.sup.18
is hydrogen, alkyl, or alkylthio; or optionally substituted aryl;
wherein; ring group substituents mean substituents attached to
aromatic or non-aromatic ring systems inclusive of aryl,
heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,
acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,
aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,
acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,
cyano, carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl or when the ring system is saturated or partially
saturated, the ring group substituents further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.); and aliphatic
group substituents means aryl, heteroaryl, hydroxy, alkoxy,
cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,
cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo
analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl.
137. The process of claim 136 further comprising the step wherein
the compound of formula 24 is prepared by effecting a Michael
addition with an iminic glycinimide compound on a compound of
formula 29 ##STR00509## wherein: ##STR00510## is optionally
substituted cycloalkenyl or optionally substituted fused
arylcycloalkenyl; wherein: the compound of formula 29 may be
prepared by esterifying a compound of formula 29a ##STR00511##
wherein: R.sup.11a is --CHO, --COR.sup.15, --C.ident.N, or
--CONR.sup.15R.sup.15.
138. The process of claim 137 wherein the process is carried out at
a temperature between 0.degree. C. and -78.degree. C.
139. The process of claim 138 wherein the process is carried out at
-60.degree..
140. The process of claim 139 wherein the process is catalyzed by a
chiral phase transfer catalyst.
141. The process of claim 139 wherein the process is catalyzed by a
nonchiral phase transfer catalyst.
142. The process of any one of claims 135 to 141 wherein the
protecting group is BOC.
143. The process of claim 142 wherein the iminic glycinimide is
(N-diphenylmethylene)-glycine tert-butyl ester.
143. The process of claim 142 wherein the compound of formula 29 is
1-carboxy-1-cyclopentene methyl ester.
144. A compound of claim 1 having the structural formula:
##STR00512## wherein: R.sup.1 is hydrogen, optionally substituted
aliphatic group, optionally substituted cyclic group or optionally
substituted aromatic group; R.sup.2 and R.sup.9 are each
independently optionally substituted aliphatic group, optionally
substituted cyclic group or optionally substituted aromatic group;
R.sup.3, R.sup.5 and R.sup.7 are each independently methanediyl or
ethanediyl, substituted with one substituent selected from the
group consisting of an optionally substituted aliphatic group, an
optionally substituted cyclic group or an optionally substituted
aromatic group and wherein the methanediyl or ethanediyl is further
optionally substituted with an aliphatic group substituent;
R.sup.4, R.sup.6, R.sup.8 and R.sup.10 are each independently is
hydrogen or optionally substituted aliphatic group; ##STR00513## is
substituted monocyclic azaheterocyclyl or optionally substituted
multicyclic azaheterocyclyl, or optionally substituted multicyclic
azaheterocyclenyl wherein the unsaturatation is in the ring distal
to the ring bearing the
R.sup.9-L-(N(R.sup.8)--R.sup.7--C(O)--).sub.nN(R.sup.6)--R.sup.5--C(O)--N
moiety and to which the
--C(O)--N(R.sup.4)--R.sup.3--C(O)C(O)NR.sup.2R.sup.1 moiety is
attached; L is --C(O)--, --OC(O)--, --NR.sup.10C(O)--,
--S(O).sub.2--, or --NR.sup.10S(O).sub.2--; and n is 0 or 1, or a
pharmaceutically acceptable salt or prodrug thereof, or a solvate
of such a compound, its salt or its prodrug, provided when
##STR00514## is substituted ##STR00515## then L is --OC(O)-- and
R.sup.9 is optionally substituted aliphatic, or at least one of
R.sup.3, R.sup.5 and R.sup.7 is methanediyl or ethanediyl,
substituted with at least one substituent selected from the group
consisting of an optionally substituted aliphatic group, an
optionally substituted cyclic group or an optionally substituted
aromatic group and wherein the methanediyl or ethanediyl is further
optionally substituted with an aliphatic group substituent, or
R.sup.4 is optionally substituted aliphatic.
145. A compound of claim 144 wherein: optionally substituted
aliphatic groups are alkyl, alkenyl or alkynyl, optionally
substituted with one or more aliphatic group substituent;
optionally substituted cyclic groups are cycloalkyl, cycloalkenyl,
heterocyclyl or heterocyclenyl groups optionally substituted with
one or more ring group substituents; optionally substituted
aromatic groups are aryl or heteroaryl groups optionally
substituted with one or more ring group substituents; optionally
substituted (1,1- or 1,2) cycloalkylene groups are (1,1- or 1,2)
cycloalkylene groups optionally substituted with one or more ring
group substituents; optionally substituted (1,1- or 1,2)
heterocyclylene groups are (1,1- or 1,2) heterocyclylene groups
optionally substituted with one or more ring group substituents;
##STR00516## as substituted monocyclic azaheterocyclyl is a
monocyclic azaheterocyclyl group substituted directly or through a
linker group by at least one substituent selected from aryl,
heteroaryl, aryloxy, heteroaryloxy, aroyl or its thio analogue,
heteroaryl or its thioxo analogue, aroyloxy, heteroaroyloxy,
aryloxycarbonyl, heteroaryloxycarbonyl, arylsulfonyl,
heteroarylsulfonyl, arylsulfinyl, heteroarylsulfinyl, arylthio,
heteroarylthio, aryldiazo, heteroaryldiazo, Y.sup.1Y.sup.2N--,
Y.sup.1Y.sup.2NC(O)--, Y.sup.1Y.sup.2NC(O)O--,
Y.sup.1Y.sup.2NC(O)NY.sup.3-- or Y.sup.1Y.sup.2NSO.sub.2-- wherein
at least one of Y.sup.1 and Y.sup.2 is aryl or heteroaryl, wherein
said linker group is selected from the group consisting of
--C(O)--, --OC(O)--, lower alkyl, lower alkoxy, lower alkenyl,
--O--, --S--, --C(O)C(O)--, --S(O)--, --S(O).sub.2--,
--NR.sup.80--, where R.sup.8.degree. is hydrogen, alkyl,
cycloalkyl, aryl, aralkyl, heterocyclyl or heteroaryl; optionally
substituted multicyclic azaheterocyclyl is a multicyclic
azaheterocyclyl group optionally substituted by one or more ring
group substituents; optionally substituted multicyclic
azaheterocyenyl is a multicyclic azaheterocyclenyll group
optionally substituted by one or more ring group substituents;
wherein: aliphatic group substituents means aryl, heteroaryl,
hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its
thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or
its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl; ring group substituents means aryl, heteroaryl,
hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its
thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or
its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), acid biostere, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfonyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl, or when a ring system is saturated or partially
saturated, the "ring group substituents" further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.); aryl means an
aromatic monocyclic or multicyclic ring system of 6 to 14 carbon
atoms; cycloalkyl means a non-aromatic mono- or multicyclic ring
system of 3 to 10 carbon atoms; cycloalkenyl means a non-aromatic
mono- or multicyclic ring system of 3 to 10 carbon atoms which
contain at least one carbon-carbon double bond; cyclyl means
cycloalkyl, cycloalkenyl, heterocyclyl or heterocyclenyl;
heterocyclyl means a non-aromatic saturated monocyclic or
multicyclic ring system of about 3 to about 10 carbon atoms in
which one or more of the carbon atoms in the ring system is/are
hetero element(s) other than carbon; heterocyclenyl means a
non-aromatic monocyclic or multicyclic hydrocarbon ring system of
about 3 to about 10 carbon atoms in which one or more of the carbon
atoms in the ring system is/are hetero element(s) other than carbon
and which contains at least one carbon-carbon double bond or
carbon-nitrogen double bond; and heteroaryl means an aromatic
monocyclic or multicyclic ring system of about 5 to about 14 carbon
atoms, in which one or more of the carbon atoms in the ring system
is/are hetero element(s) other than carbon.
146. The compound of any one of claims 143 to 145 wherein R.sup.2
is 1-carboxy-2-phenylethyl, cyclopropyl, carboxymethyl, cyclobutyl,
1-phenylethyl, but-2-yl, 1-pyrid-4-ylethyl, or prop en-3-yl.
147. The compound of any one of claims 143 to 146 wherein R.sup.3
is optionally substituted lower aliphatic group methanediyl.
148. The compound of any one of claims 143 to 147 wherein R.sup.3
is optionally halo substituted lower (alkyl or alkenyl) group
methanediyl.
149. The compound of any one of claims 143 to 148 wherein R.sup.3
is propylmethanediyl, 2,2-difluoroethylmethanediyl,
2,2,2-trifluoromethanediyl or propen-3-ylmethanediyl; more
preferred R.sup.3 is propylmethanediyl,
2,2-difluoroethylmethanediyl.
150. The compound of any one of claims 143 to 149 wherein R.sup.5
is optionally substituted lower aliphatic group methanediyl.
151. The compound of any one of claims 143 to 150 wherein R.sup.5
is optionally (phenyl, carboxy, carboxamido or alkoxycarbonyl)
substituted lower (alkyl or alkenyl) methanediyl.
152. The compound of any one of claims 143 to 151 wherein R.sup.5
is methylmethanediyl, isopropylmethanediyl, t-butylmethanediyl,
but-2-ylmethanediyl, butylmethanediyl, benzylmethanediyl,
3-methylbutylmethanediyl, 2-methylpropylmethanediyl,
carboxymethylmethanediyl, carboxamidomethylmethanediyl,
benzyloxycarbonylmethyl-methanediyl,
benzyloxycarbonylpropylmethanediyl,
phenylpropen-3-ylmethanediyl.
153. The compound of any one of claims 143 to 152 wherein R.sup.7
is optionally substituted lower aliphatic group methanediyl or
optionally substituted lower cyclic group methanediyl.
154. The compound of any one of claims 143 to 153 wherein R.sup.7
is optionally substituted lower alkylmethanediyl or optionally
substituted lower cycloalkylmethanediyl.
155. The compound of any one of claims 143 to 154 wherein R.sup.7
is isopropylmethanediyl or cyclohexylmethanediyl.
156. The compound of any one of claims 143 to 155 wherein R.sup.9
is optionally (carboxy, (lower alkyl)HNCO--, hydroxy, phenyl or
heteroaryl) substituted lower alkyl or optionally substituted
monocyclic heteroaryl.
157. The compound of any one of claims 143 to 1556 wherein .sup.9
is isopropyl or t-butyl.
158. The compound of any one of claims 143 to 157 wherein R.sup.9
is selected from the group consisting of: ##STR00517##
##STR00518##
159. The compound of any one of claims 143 to 158 wherein
##STR00519## as a substituted monocyclic azaheterocyclyl is
optionally substituted ##STR00520## or optionally subtituted
##STR00521## wherein Ar is R.sup.2 that comprises an aromatic
moiety/substituent.
160. The compound of claim 144 selected from the following
compounds: ##STR00522## ##STR00523## ##STR00524## ##STR00525##
##STR00526## ##STR00527## ##STR00528## ##STR00529## ##STR00530##
##STR00531## ##STR00532## ##STR00533## ##STR00534##
161. The compound of claim 144 selected from the group consisting
of: ##STR00535## ##STR00536## ##STR00537## ##STR00538##
##STR00539## ##STR00540## ##STR00541## ##STR00542## ##STR00543##
##STR00544## ##STR00545## ##STR00546## ##STR00547## ##STR00548##
##STR00549## ##STR00550## ##STR00551## ##STR00552## ##STR00553##
##STR00554## ##STR00555## ##STR00556## ##STR00557##
##STR00558##
161. A compound of the formula ##STR00559## or a pharmaceutically
acceptable salt or prodrug thereof, or a solvate of such a
compound, its salt or its prodrug.
162. A compound of claim 1 or 2 wherein: R.sup.0 is a bond; R.sup.1
is hydrogen; R.sup.2 is lower alkyl optionally substituted with 1
to 3 aliphatic group substituents; or lower cycloalky optionally
substituted with 1 to 3 cyclic group substituents; R.sup.3 and
R.sup.5 are each independently methylene optionally substituted
with 1 to 3 aliphatic group substitutents; R.sup.4, R.sup.6,
R.sup.8 and R.sup.1.degree. are hydrogen; R.sup.7 is methylene
substituted with cycloalkyl, lower alkyl or aryl; or or (1,1- or
1,2-)cycloalkenyl optionally substituted with cycloalkyl, lower
alkyl or aryl; R9 is lower alkyl optionally substituted with 1 to 3
aliphatic group substituents; or heteroaryl optionally substituted
with 1 to 3 cyclic group substituents; or heterocyclic optionally
substituted with 1 to 3 cyclic group substituents; ##STR00560## is
monocyclic azaheterocyclyl, multicyclic azaheterocyclyl, or
multicyclic azaheterocyclenyl optionally substituted with from 1 to
3 cyclic group substituents; and L is --C(O)--, --OC(O)--.
163. A compound of claim 163 wherein: aliphatic group substituents
means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy,
heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its
thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its
thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy,
heteroaroyloxy, halo, nitro, cyano, carboxy (acid), --C(O)--NHOH,
--C(O)--CH.sub.2OH, --C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho,
phosphono, alkylsulphonylcarbamoyl, tetrazolyl,
arylsulphonylcarbamoyl, N-methoxycarbamoyl,
heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,
3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as
3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,
alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl,
alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl,
alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl; ring group substituents means aryl, heteroaryl,
hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its
thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or
its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy,
cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano,
carboxy (acid), acid biostere, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl, or when a ring system is saturated or partially
saturated, the "ring group substituents" further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.); aryl means an
aromatic monocyclic or multicyclic ring system of 6 to 14 carbon
atoms; cycloalkyl means a non-aromatic mono- or multicyclic ring
system of 3 to 10 carbon atoms; cycloalkenyl means a non-aromatic
mono- or multicyclic ring system of 3 to 10 carbon atoms which
contain at least one carbon-carbon double bond; cyclyl means
cycloalkyl, cycloalkenyl, heterocyclyl or heterocyclenyl;
heterocyclyl means a non-aromatic saturated monocyclic or
multicyclic ring system of about 3 to about 10 carbon atoms in
which one or more of the carbon atoms in the ring system is/are
hetero element(s) other than carbon; and heteroaryl means an
aromatic monocyclic or multicyclic ring system of about 5 to about
14 carbon atoms, in which one or more of the carbon atoms in the
ring system is/are hetero element(s) other than carbon.
164. Any compound or synthetic intermediate as substantially
disclosed herein.
165. A compound selected from the group consisting of: ##STR00561##
##STR00562## ##STR00563## ##STR00564## ##STR00565## ##STR00566##
##STR00567## or a pharmaceutically acceptable salt or prodrug
thereof, or a solvate of such a compound, its salt or its
prodrug.
166. A compound of any one of claim 1, 2, 162 or 163 wherein the
optionally substituted aliphatic group, optionally substituted
cyclic group or optionally substituted aromatic group of R.sup.9 is
substituted with at least one heteroaryl substituent.
167. A compound of any one of claim 1, 2, 162 or 163 wherein the
optionally substituted aromatic group of R.sup.9 is optionally
substituted heteroaryl.
168. A compound of claim 166 wherein the optionally substituted
aliphatic group of R.sup.9 is optionally substituted
alkylheteroaryl.
Description
[0001] This invention is directed to peptidomimetic compounds and
intermediates thereto, their preparation including stereoselective
synthetic processes to intermediates, pharmaceutical compositions
containing the peptidomimetic compounds, and the use of the
peptidomimetic compounds or compositions thereof as protease
inhibitors, particularly as serine protease inhibitors, and more
particularly as hepatitis C virus ("HCV") NS3 protease inhibitors.
The peptidomimetic compounds, as HCV NS3 protease inhibitors, are
particularly useful in interfering with the life cycle of the
hepatitis C virus and in treating or preventing an HCV infection or
physiological conditions associated therewith. The present
invention is also directed to methods of combination therapy for
inhibiting HCV replication in cells, or for treating or preventing
an HCV infection in patients using the peptidomimetic compounds or
pharmaceutical compositions, or kits and pharmaceutical packs
therefor. According to the present invention included as
pharmaceutical compositions are those comprising an inhibitor of
HCV serine protease in combination with an interferon having
anti-HCV activity; an inhibitor of HCV serine protease in
combination with a compound, other than an interferon, having
anti-HCV activity; or an inhibitor of HCV serine protease in
combination with both an interferon having anti-HCV activity and a
compound, other than an interferon, having anti-HCV activity.
Further the present invention is directed to stereoselective
methods for preparing chiral bicycloprolinate intermediates useful
in the synthesis of the peptidomimetic compounds.
BACKGROUND OF THE INVENTION
[0002] Infection by the HCV is a compelling human medical problem
and is now recognized as the causative agent for most cases of
non-A, non-B hepatitis.
[0003] The HCV is thought to infect chronically 3% of the world's
population [A. Alberti et al., "Natural History of Hepatitis C," J.
Hepatology, 31, (Suppl. 1), 17-24 (1999)]. In the United States
alone the infection rate is 1.8% or 3.9 million people [M. J.
Alter, "Hepatitis C Virus Infection in the United States," J.
Hepatology, 31, (Suppl. 1), 88-91 (1999)]. Of all patients infected
over 70% develop a chronic infection that is believed to be a major
cause of cirrhosis and hepatocellular carcinoma. [D. Lavanchy,
"Global Surveillance and Control of Hepatitis C," J. Viral
Hepatitis, 6, 35-47 (1999)]
[0004] The replication of the HCV encompasses genomic encoding a
polyprotein of 3010-3033 amino acids [Q.-L. Choo, et al., "Genetic
Organization and Diversity of the Hepatitis C Virus", Proc. Natl.
Acad. Sci. USA, 88, 2451-2455 (1991); N. Kato et al., "Molecular
Cloning of the Human Hepatitis C Virus Genome From Japanese
Patients with Non-A, Non-B Hepatitis", Proc. Natl. Acad. Sci. USA,
87, 9524-9528 (1990); A. Takamizawa et al., "Structure and
Organization of the Hepatitis C Virus Genome Isolated From Human
Carriers", J. Virol., 65, 1105-1113 (1991)]. The HCV nonstructural
(NS) proteins are presumed to provide the essential catalytic
machinery for viral replication. The NS proteins are derived by
proteolytic cleavage of the polyprotein [R. Bartenschlager at al.,
"Nonstructural Protein 3 of the Hepatitis C Virus Encodes a
Serine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5
Junctions", J. Virol., 67, 3835-3844 (1993); A. Grakoui et al.
"Characterization of the Hepatitis C Virus-Encoded Serine
Proteinase: Determination of Proteinase-Dependent Polyprotein
Cleavage Sites", J. Virol., 67, 2832-2843 (1993); A. Grakoui at
al., Expression and Identification of Hepatitis C Virus Polyprotein
Cleavage Products", J. Virol., 67, 1385-1395 (1993); L. Tomei at
al., "NS3 is a serine protease required for processing of hepatitis
C virus polyprotein", J. Virol., 67, 4017-4026 (1993)]. In fact, it
is the first 181 amino acids of NS3 (residues 1027-1207 of the
viral polyprotein) have been shown to contain the serine protease
domain of NS3 that processes all four downstream sites of the HCV
polyprotein [C. Lin at al., "Hepatitis C Virus NS3 Serine
Proteinase: Trans-Cleavage Requirements and Processing Kinetics",
J. Virol., 68, 8147-8157 (1994)].
[0005] The HCV NS protein 3 (NS3) contains a serine protease
activity that helps in the processing of the majority of the viral
enzymes, and thus is considered essential for viral replication and
infectivity. The essentiality of the NS3 protease was inferred from
the fact that mutations in the yellow fever virus NS3 protease
decreases viral infectivity [T. J. Chambers et al., "Evidence that
the N-terminal Domain of Nonstructural Protein NS3 From Yellow
Fever Virus is a Serine Protease Responsible for Site-Specific
Cleavages in the Viral Polyprotein", Proc. Natl. Acad. Sci. USA,
87, 8898-8902 (1990)]. More recently, it was demonstrated that
mutations at the active site of the HCV NS3 protease could
completely abolish the HCV infection in a chimpanzee model [C. M.
Rice at al. "Hepatitis C virus-encoded enzymatic activities and
conserved RNA elements in the 3'-nontranslated region are essential
for virus replication in vivo." J. Virol., 74(4) 2046-51 (2000)].
The HCV NS3 serine protease is also considered essential for viral
replication as it and its associated cofactor, NS4A, help in the
processing of all of the viral enzymes. This processing appears to
be analogous to that carried out by the human immunodeficiency
virus ("HIV") aspartyl protease. In addition, the demonstrated use
of HIV protease inhibitors as potent antiviral agents in man
demonstrates that interrupting a protease protein processing stage
in the viral life cycle does result in therapeutically active
agents. Consequently, the protease enzyme is an attractive target
for drug discovery.
[0006] Several potential HCV protease inhibitors have been
described. PCT Publications Numbers WO 00/09558, WO 00/09543, WO
99/64442, WO 99/07733, WO 99/07734, WO 99/50230, WO98/46630, WO
98/17679 and WO 97/43310, U.S. Pat. No. 5,990,276, M. Llinas-Brunet
et al., Bioorg. Med. Chem. Lett., 8, 1713-1718 (1998), W. Han at
al., Bioorg. Med. Chem. Lett., 10, 711-713 (2000), R. Dunsdon et
al., Bioorg. Med. Chem. Len., 10, 1571-1579 (2000), M.
Llinas-Brunet et al., Bioorg. Med. Chem. Lett., 10, 2267-2270
(2000), and S. LaPlante at al., Bioorg. Med. Chem. Lett., 10,
2271-2274 (2000) each describe potential HCV NS3 protease
inhibitors. Unfortunately, there are no serine protease inhibitors
available currently as anti-HCV agents.
[0007] In fact, there are no anti-HCV therapies except
interferon-.alpha., interferon-.alpha./ribavirin combination and
more recently pegylated interferon-.alpha.. The sustained response
rates for the interferon-.alpha. therapies and
interferon-.alpha./ribavirin however tend to be low (<50%) and
the side effects exhibited by the therapies tend to be significant
and severe [M. A. Walker, "Hepatitis C Virus: an Overview of
Current Approaches and Progress," DDT, 4, 518-529 (1999); D.
Moradpour at al., "Current and Evolving Therapies for Hepatitis C,"
Eur. J. Gastroenterol. Hepatol., 11, 1199-1202 (1999); H. L. A.
Janssen et al., "Suicide Associated with Alfa-Interferon Therapy
for Chronic Viral Hepatitis," J. Hepatol., 21, 241-243 (1994); and
P. F. Renault et al., "Side effects of alpha interferon", Seminars
in Liver Disease 9, 273-277, (1989)]. Furthermore, the interferon
therapies only induce long term remission in only a fraction
(.about.25%) of cases [O. Weiland, "Interferon Therapy in Chronic
Hepatitis C Virus Infection", FEMS Microbiol. Rev., 14, 279-288
(1994)]. The aforesaid problems with the interferon-.alpha.
therapies has even led to the development and clinical study of
pegylated derivatized interferon-.alpha. compounds as improved
anti-HCV therapeutics.
[0008] In view of the current situation regarding anti-HCV
therapeutics, it is clear that there is a need for more effective
and better tolerated therapies.
[0009] Furthermore, synthesis of complex peptidomimetic compounds
has long been hampered by the nonstereoselective nature of most
synthetic organic processes. It is well known that the therapeutic
activity of enantiomers of peptidomimetic compounds varies widely.
It is therefore of great benefit to provide such stereospecific
synthetic processes.
[0010] Previous attempts to synthesize chirally specific
bicycloprolinate intermediates, useful in the synthesis of the
present therapeutic peptidomimetic protease inhibitors have
suffered from being non enatioselective, or diasteroselective, or
long encompassing synthetic pathways, or being unsuitable for
preparing large quantities of product. Thus, there is also a need
for a means of preparing large quantities of bicycloprolmates in a
diastereoselective manner and enantiomerically enriched form.
SUMMARY OF THE INVENTION
[0011] The present invention relates to a peptidomimetic compound
of formula 1
##STR00001##
wherein: R.sup.0 is a bond or difluoromethylene; R.sup.1 is
hydrogen, optionally substituted aliphatic group, optionally
substituted cyclic group or optionally substituted aromatic group;
R.sup.2 and R.sup.9 are each independently optionally substituted
aliphatic group, optionally substituted cyclic group or optionally
substituted aromatic group; R.sup.3, R.sup.5 and R.sup.7 are each
independently (optionally substituted aliphatic group, optionally
substituted cyclic group or optionally substituted aromatic group)
(optionally substituted methylene or optionally substituted
ethylene), optionally substituted (1,1- or 1,2-)cycloalkylene or
optionally substituted (1,1- or 1,2-)heterocyclylene; R.sup.4,
R.sup.6, R.sup.8 and R.sup.10 are each independently hydrogen or
optionally substituted aliphatic group;
##STR00002##
is substituted monocyclic azaheterocyclyl or optionally substituted
multicyclic azaheterocyclyl, or optionally substituted multicyclic
azaheterocyclenyl wherein the unsaturatation is in the ring distal
to the ring bearing the
R.sup.9-L-(N(R.sup.8)--R.sup.7--C(O)--).sub.nN(R.sup.6)--R.sup.5--C(O)--N
moiety and to which the
--C(O)--N(R.sup.4)--R.sup.3--C(O)C(O)NR.sup.2R.sup.1 moiety is
attached;
L is --C(O)--, --OC(O)--, --NR.sup.10C(O)--, --S(O).sub.2--, or
--NR.sup.10S(O).sub.2--; and
[0012] n is 0 or 1, or a pharmaceutically acceptable salt or
prodrug thereof, or a solvate of such a compound, its salt or its
prodrug, provided when
##STR00003##
is substituted
##STR00004##
then L is --OC(O)-- and R.sup.9 is optionally substituted
aliphatic, or at least one of R.sup.3, R.sup.5 and R.sup.7 is
(optionally substituted aliphatic group, optionally substituted
cyclic group or optionally substituted aromatic group) (optionally
substituted ethanediyl), or R.sup.4 is optionally substituted
aliphatic. This inventions also provides a compound having the
structural formula:
##STR00005##
wherein: R.sup.1 is hydrogen, optionally substituted aliphatic
group, optionally substituted cyclic group or optionally
substituted aromatic group; R.sup.2 and R.sup.9 are each
independently optionally substituted aliphatic group, optionally
substituted cyclic group or optionally substituted aromatic group;
R.sup.3, R.sup.5 and R.sup.7 are each independently (optionally
substituted aliphatic group, optionally substituted cyclic group or
optionally substituted aromatic group) (optionally substituted
methanediyl or optionally substituted ethanediyl); R.sup.4,
R.sup.6, R.sup.8 and R.sup.10 are each independently is hydrogen or
optionally substituted aliphatic group;
##STR00006##
is substituted monocyclic azaheterocyclyl or optionally substituted
multicyclic azaheterocyclyl, or optionally substituted multicyclic
azaheterocyclenyl wherein the unsaturatation is in the ring distal
to the ring bearing the
R.sup.9-L-(N(R.sup.8)--R.sup.7--C(O)--).sub.nN(R.sup.6)--R.sup.5--C(O)--N
moiety and to which the
--C(O)--N(R.sup.4)--R.sup.3--C(O)C(O)NR.sup.2R.sup.1 moiety is
attached;
L is --C(O)--, --OC(O)--, --NR.sup.10C(O)--, --S(O).sub.2--, or
--NR.sup.10S(O).sub.2--; and
[0013] n is 0 or 1, or a pharmaceutically acceptable salt or
prodrug thereof, or a solvate of such a compound, its salt or its
prodrug, provided when
##STR00007##
is substituted
##STR00008##
then L is --OC(O)-- and R.sup.9 is optionally substituted
aliphatic, or at least one of R.sup.3, R.sup.5 and R.sup.7 is
(optionally substituted aliphatic group, optionally substituted
cyclic group or optionally substituted aromatic group) (optionally
substituted ethanediyl), or R.sup.4 is optionally substituted
aliphatic.
[0014] The invention is also directed to a pharmaceutical
composition comprising a compound of formula 1, and method for
using the compound of formula 1 for inhibiting HCV protease, or
treating or preventing an HCV infection in patients or
physiological condition related to the infection.
[0015] The invention is also directed to a stereoselective process
for preparing a chiral bicycloprolinate compound that is an
intermediate useful in preparing a compound of formula 1. The
synthetic process comprises the steps of:
[0016] (a) cleaving and cyclizing a compound of formula 24
##STR00009##
[0017] wherein:
##STR00010##
is optionally substituted cycloalkyl or optionally substituted
fused arylcycloalkyl;
[0018] R.sup.11 is --CO.sub.2R.sup.13;
[0019] R.sup.12 is an iminic glycinimide adduct;
[0020] R.sup.13 is acid protecting group or optionally substituted
aliphatic group;
[0021] under cleaving and cyclizing conditions to form a compound
of formula 25
##STR00011## [0022] wherein: [0023] R.sup.14 is
--CONR.sup.15R.sup.15, --CN;
##STR00012##
[0023] or --CO.sub.2R.sup.16;
[0024] R.sup.15 is optionally substituted aliphatic group;
[0025] R.sup.16 is acid protecting group, optionally substituted
aryl, or optionally substituted aliphatic group; and
[0026] (b) protecting the nitrogen of the lactam moiety in the
compound of formula 25 with an amide protecting group to form a
compound of formula 26
##STR00013##
[0027] wherein:
[0028] p.sup.O is amide protecting group;
[0029] R.sup.14 is as described herein; and
[0030] (c) reducing the compound of formula 26 under reducing
conditions to form a compound of formula 27
##STR00014##
[0031] wherein:
[0032] p.sup.O and R.sup.14 are as described herein; and
[0033] (d) deprotecting the compound of formula 27 under
deprotecting conditions to form a compound of formula 28
##STR00015##
[0034] wherein:
[0035] R.sup.14 is as described herein.
[0036] The invention is also directed to the above synthetic
process further comprising the step wherein the compound of formula
24 is prepared by effecting a Michael addition with an iminic
glycinimide compound on a compound of formula 29
##STR00016##
[0037] wherein:
##STR00017##
is optionally substituted cycloalkenyl or optionally substituted
fused arylcycloalkenyl;
[0038] R.sup.11 is --CO.sub.2R.sup.13;
[0039] wherein:
[0040] the compound of formula 29 may be prepared by esterifying a
compound of formula 29a
##STR00018##
[0041] wherein:
##STR00019##
is optionally substituted cycloalkenyl or optionally substituted
fused arylcycloalkenyl;
[0042] R.sup.11a is --CHO, --COR.sup.15, --C.ident.N, or
--CONR.sup.15R.sup.15; and
[0043] R.sup.15 is as described herein.
[0044] Notably, one skilled in the art would know that conversion
of ketones to esters may be accomplished, for example, by a
Bayer-Villiger reaction. Conversion of nitriles and amides to
esters may be accomplished, for example, by aqueous hydrolysis
followed by further esterification. Conversion of aldehydes to
esters may be accomplished, for example, by oxidation of the
aldehyde followed by esterification.
[0045] Another aspect of the invention is a compound of formula 1
wherein the substituents are selected from a combination of
preferred or particular embodiments as defined herein.
[0046] Another aspect of the invention is a compound of formulae
24-29 wherein the substituents are selected from a combination of
preferred or particular embodiments as defined herein.
[0047] Another aspect of the invention are pharmaceutical
compositions comprising, in addition to one or more HCV serine
protease inhibitors, one or more interferons or compounds that
induce the production of interferons that exhibit anti-HCV activity
and/or one or more compounds having anti HCV activity, including
immunomodulatory compounds such as immunostimulatory cytokines
exhibiting HCV antiviral activity, and a pharmaceutically
acceptable carrier.
[0048] Another aspect of the invention are methods of treating or
preventing a HCV infection in a patient in need thereof, comprising
administering to said patient a pharmaceutically effective amount
of a combination of one or more HCV serine protease inhibitors; one
or more interferons or compounds that induce the production of an
interferon that exhibit anti-HCV activity; and/or one or more
compounds having anti-HCV activity, including immunomodulatory
compounds such as inununostimulatory cytokines exhibiting HCV
antiviral activity.
[0049] The invention is also directed to the use of one or more HCV
serine protease inhibitors in combination with one or more
interferons or compounds that induce the production of an
interferon that exhibit anti-HCV activity and/or one or more
compounds having anti-HCV activity, including immunomodulatory
compounds such as immunostimulatory cytokines exhibiting HCV
antiviral activity, to prepare a medicament for treating or
preventing a HCV infection in a patient in need thereof.
[0050] The present invention is also directed to a kit or
pharmaceutical pack for treating or preventing HCV infection in a
patient, wherein the kit or pharmaceutical pack comprises a
plurality of separate containers, wherein at least one of said
containers contains one or more HCV serine protease inhibitors
(alone or in combination with a pharmaceutically acceptable carrier
or diluent), at least another of said containers contains one or
more interferons or compounds that induce the production of an
interferon that exhibit anti-HCV activity, (alone or in combination
with a pharmaceutically acceptable carrier or diluent) and,
optionally, at least another of said containers contains one or
more compounds having anti-HCV activity (alone or in combination
with a pharmaceutically acceptable carrier or diluent), including
immunomodulatory compounds such as immunostimulatory cytokines
exhibiting HCV antiviral activity.
[0051] The amount of the HCV serine protease inhibitor(s),
interferon(s), or anti-HCV compound(s) in any of the foregoing
applications can be a pharmaceutically effective amount, a
subclinical anti-HCV effective amount, or combinations thereof, so
long as the final combination of HCV serine protease inhibitor(s),
interferon(s), or compounds that induce the production of an
interferon that exhibit anti-HCV activity, and/or anti-HCV
compound(s) comprises a pharmaceutically effective amount of
compounds that is effective in treating or preventing HCV infection
in a patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The above and other aspects, features, and advantages of the
present invention will be better understood from the following
detailed description taken in conjunction with the accompanying
drawings, all of which are given by way of illustration only, and
are not limitative of the present invention, in which:
[0053] FIG. 1 shows the inhibition of HCV replicon RNA accumulation
after 48 hour treatment of replicon-containing cells with Compound
CU and interferon-alpha 2B, individually or in combination.
[0054] FIG. 2 graphically shows the isobol concavity exhibited by
compounds used in combination that are antagonistic, additive, and
synergistic according to the synergy calculation methods of Greco,
Park and Rustom ((1990) Application of a New Approach for the
Quantitation of Drug Synergism to the Combination of
cis-Diamminedichloroplatinum and
1-.beta.-D-Arabinofuranosylcytosine. Cancer Research, 50,
5318-5327).
[0055] FIG. 3 shows the geometric relationship between a and the
amount of curvature in the isobol. A hypothetical isobol at the
E=50% effect level is displayed with a straight line isobol that
would be expected under additivity. M is the point of intersection
of the line y=x and the hypothetical isobol. N is the point of
intersection of the line y=x and the straight line isobol. O is the
origin (0,0). S gives a measure of the amount of curvature in the
isobol, where S=ON/OM. ON is the distance from O to N and OM is the
distance from O to M. The parameter .alpha. is related to S by the
equation .alpha.=4(S.sup.2-S).
[0056] FIG. 4 shows the isobol calculations using the method of
Greco et al., supra, for the combination of compound CU and
interferon alpha-2B (Schering-Plough) using 6 dilutions of each
compound in Experiment 1
[0057] FIG. 5 shows the isobol calculations using the method of
Greco et al., supra, for the combination of compound CU and
interferon alpha-2A using 6 dilutions of each compound in
Experiment 2.
[0058] FIG. 6 shows the isobol calculations using the method of
Greco et al., supra, for the combination of compound CU and
interferon alpha-2B (Schering-Plough) using 8 dilutions of each
compound in Experiment 3.
[0059] FIG. 7 shows the isobol calculations using the method of
Greco et al., supra, for the combination of compound CU and
interferon alpha-2A using 8 dilutions of each compound in
Experiment 4.
[0060] FIG. 8 shows the isobol calculations using the method of
Greco et al., supra, for the combination of compound CU and ovine
interferon tau using 8 dilutions of each compound in Experiment
5.
[0061] FIG. 9 shows the isobol calculations using the method of
Greco at al., supra, for the combination of compound EC and
interferon alpha-2B (Schering-Plough) using 8 dilutions of each
compound in Experiment 6.
[0062] FIG. 10 shows the isobol calculations using the method of
Greco at al., supra, for the combination of compound EC and
interferon alpha-2A using 8 dilutions of each compound in
Experiment 7.
[0063] FIG. 11 shows the isobol calculations using the method of
Greco at al., supra, for the combination of compound CU and
interferon beta using 8 dilutions of each compound in Experiment
8.
[0064] FIG. 12 shows the isobol calculations using the method of
Greco et al., supra, for the combination of compound EP and
interferon alpha-2B (Schering-Plough) using 8 dilutions of each
compound in Experiment 9.
[0065] FIG. 13 shows the isobol calculations using the method of
Greco et al., supra, for the combination of Ribavirin and
interferon alpha-2B (Schering-Plough) using 8 dilutions of each
compound in Experiment 10.
[0066] FIG. 14 shows inhibition of HCV replicon RNA accumulation
caused by treatment of replicon cells with either (A) Ribavirin
alone or (B) interferon alpha-2B alone. In both panels, the
measured inhibition as well as the inhibition corrected for
cytotoxicity of the compounds is shown.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The contents of each of the patent documents and other
references cited herein are herein incorporated by reference in
their entirety.
[0068] As used above, and throughout the description of the
invention, the following abbreviations, unless otherwise indicated,
shall be understood to have the following meanings: --
TABLE-US-00001 Designation Reagent or Fragment ACN acetonitrile
AIBN 2,2'-azobisisobutyronitrile BOC or Boc tert-butyl carbamate
BOP benzotriazol-1-yl-oxytris (dimethylamino)phosphonium
hexafluorophosphate n-Bu.sub.3SnH tri-n-butyltin hydride t-Bu
tert-butyl Cbz benzyl carbamate chiral PTC chiral phase transfer
catalyst ##STR00020## DAST (diethylamino)sulfur trifluoride
(Et.sub.2NSF.sub.3) DCC dicyclocarbodiimide DCM dichloromethane
(CH.sub.3Cl.sub.2) DIBAL-H Diisobutylaluminum hydride DIC
1,3-diisopropylcarbodiimide DIPEA diisopropylethylamine DMAP
4-(N,N-dimethylamino)pyridine DMP reagent Dess-Martin Periodinane
reagent ##STR00021## DMF dimethylformamide DMSO dimethylsulfoxide
EA elemental analysis EDCI 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide HCl eq equivalent(s) Et ethyl Et.sub.2O diethyl ether
EtOH ethanol EtOAc ethyl acetate Et.sub.3Si triethylsilane FMOC
9-fluorenylmethoxycarbonyl H-Chg-OH ##STR00022## HOAt
1-hydroxy-7-azabensotriazole HOBT 1-hydroxybenztriazole HOSu
N-hydroxysuccinamide HPLC high performance liquid chromatography
LAH lithium aluminum anhydride Me methyl MeI methyliodide MeOH
methanol MeOC(O)Cl methyl chloroformate MOMCl methoxymethylchloride
MOM methoxymethyl MS mass spectroscopy NaBH.sub.4 sodium
borohydride Na.sub.2C.sub.4H.sub.4O.sub.6 sodium tartrate NMP
N-methyl pyrrolidinone NMR nuclear magnetic resonance P- Polymer
bond PyBOP benzotriazole-1-yl-oxytris-pyrrolidino-phosphonium
hexafluorophosphate TBD 1,5,7-triazabicyclo[4.4.0]-dec-5-ene
RP-HPLC reverse phase - high pressure liquid chromatography TBSCl
tert-butyldimethylsilyl chloride TCA trichloroacetic acid TFA
trifluoroacetic acid Tf.sub.2O triflate anhydride THF
tetrahydrofuran THP tetrahydropyran TLC thin layer
chromatography
[0069] As used above, and throughout the description of the
invention, the following terms, unless otherwise indicated, shall
be understood to have the following meanings: --
[0070] "Acid bioisostere" means a group which has chemical and
physical similarities producing broadly similar biological
properties to a carboxy group (see Lipinski, Annual Reports in
Medicinal Chemistry, "Bioisosterism In Drug Design" 21, 283 (1986);
Yun, Hwahak Sekye, "Application Of Bioisosterism To New Drug
Design" 33, 576-579, (1993); Zbao, Huaxue Tongbao, "Bioisosteric
Replacement And Development Of Lead Compounds In Drug Design"
34-38, (1995); Graham, Theochem, "Theoretical Studies Applied To
Drug Design: ab initio Electronic Distributions In Bioisosteres"
343, 105-109, (1995)). Exemplary acid bioisosteres include
--C(O)--NHOH, --C(O)--CH.sub.2OH, --C(O)--CH.sub.2SH;
--C(O)--NH--CN, sulpho, phosphono, alkylsulphonylcarbamoyl,
tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl,
heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,
3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as
3-hydroxyisoxazolyl, 3-hydroxy-1-methylpyrazolyl and the like.
[0071] "Acidic functional group" means a moiety bearing an acidic
hydrogen. Exemplary acid functional groups include carboxyl
(--C(O)OH), --C(O)--NHOH, --C(O)--CH.sub.2OH, --C(O)--CH.sub.2SH,
--C(O)--NH--CN, sulpho, phosphono, alkylsulphonylcarbamoyl,
tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl,
heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,
3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as
3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, imidazolyl,
mercapto, and the like, and an appropriate hydroxy such as an
aromatic hydroxy, e.g., hydroxyphenyl.
[0072] "Acid protecting group" means an easily removable group that
is known in the art to protect an acidic hydrogen of a carboxyl
group against undesirable reaction during synthetic procedures,
e.g., to block or protect the acid functionality while the
reactions involving other functional sites of the compound are
carried out, and to be selectively removable. Such acid protecting
groups are well known to those skilled in the art, having been
extensively used in the protection of carboxyl groups, as described
in U.S. Pat. Nos. 3,840,556 and 3,719,667, the disclosures of which
are hereby incorporated herein by reference. For suitable acid
protecting groups, see T. W. Green and P. G. M. Wuts in "Protective
Groups in Organic Chemistry" John Wiley and Sons, 1991. Acid
protecting group also includes hydrogenation labile acid protecting
group as defined herein. Exemplary acid protecting groups include
esters such as substituted and unsubstituted C.sub.1-8 lower alkyl,
e.g., methyl, ethyl, t-butyl, methoxymethyl, methylthiomethyl,
2,2,2-trichloroethyl and the like, tetrahydropyranyl, substituted
and unsubstituted phenylalkyl such as benzyl and substituted
derivatives thereof such as alkoxybenzyl or nitrobenzyl groups and
the like, cinnamyl, dialkylaminoalkyl, e.g., dimethylaminoethyl and
the like, trimethylsilyl, substituted and unsubstituted amides and
hydrazides, e.g., amides and hydrazides of N,N-dimethylamine,
7-nitroindole, hydrazine, N-phenylhydrazine and the like,
acyloxyalkyl groups such as pivaloyloxymethyl or propionyloxymethyl
and the like, aroyloxyalkyl such as benzoyloxyethyl and the like,
alkoxycarbonylalkyl such as methoxycarbonylmethyl,
cyclohexyloxycarbonylmethyl and the like, alkoxycarbonyloxyalkyl
such as 1-butyloxycarbonyloxymethyl and the like,
alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and
the like, alkylaminocarbonylaminoalkyl, such as
methylaminocarbonylaminomethyl and the like, acylaminoalkyl such as
acetylaminomethyl and the like, heterocyclylcarbonyloxyalkyl such
as 4-methylpiperazinyl-carbonyloxymethyl and the like,
dialkylarminocarbonylalkyl such as dimethylaminocarbonyl-methyl and
the like, (5-(lower alkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as
(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like, and
(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as
(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like.
[0073] "Acid labile amine protecting group" means an amine
protecting group as defined herein which is readily removed by
treatment with acid while remaining relatively stable to other
reagents. A preferred acid labile amine protecting group is
BOC.
[0074] "Aliphatic" means alkyl, alkenyl or alkynyl as defined
herein.
[0075] "Aliphatic group substituent(s)" mean substituents attached
to an aliphatic group as defined herein inclusive of aryl,
heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,
acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,
aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,
acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,
cyano, carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfonyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, methylene (H.sub.2C.dbd.), oxo (O.dbd.),
thioxo (S.dbd.), Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3--,
Y.sup.1Y.sup.2NSO.sub.2--, or Y.sup.3SO.sub.2NY.sup.1-- wherein
R.sup.2 is as defined herein, Y.sup.1 and Y.sup.2 are independently
hydrogen, alkyl, aryl or heteroaryl, and Y.sup.3 is alkyl,
cycloalkyl aryl or heteroaryl, or for where the substituent is
Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2 may be acyl,
cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl. Acidic/amide aliphatic group substituents are
carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxylsoxazolyl,
3-hydoxy-1-methylpyrazolyl and Y.sup.1Y.sup.2NCO--. Non-acidic
polar aliphatic group substituents are hydroxy, oxo (O.dbd.),
thioxo (S.dbd.), acyl or its thioxo analogue, cyclylcarbonyl or its
thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its
thioxo analogue, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, acyloxy, cyclylcarbonyloxy,
aroyloxy, heteroaroyloxy, alkylsulfonyl, cyclylsulfonyl,
arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl,
arylsulfinyl, heteroarylsulfinyl, thiol, Y.sup.1Y.sup.2N--,
Y.sup.1Y.sup.2NC(O)--, Y.sup.1Y.sup.2NC(O)O--,
Y.sup.1Y.sup.2NC(O)NY.sup.3-- or Y.sup.1Y.sup.2NSO.sub.2--.
Exemplary aliphatic groups bearing an aliphatic group substituent
include methoxymethoxy, methoxyethoxy, ethoxyethoxy, (methoxy-,
benzyloxy-, phenoxy-, or ethoxy-)carbonyl(methyl or ethyl),
benzyloxycarbonyl, pyridylmethyloxy-carbonylmethyl, methoxyethyl,
ethoxymethyl, n-butoxymethyl, cyclopentylmethyloxyethyl,
phenoxypropyl, phenoxyallyl, trifluoromethyl, cyclopropyl-methyl,
cyclopentylmethyl, carboxymethyl or ethyl), 2-phenethenyl,
benzyloxy, 1- or 2-naphthyl-methoxy, 4-pyridyl-methyloxy,
benzyloxyethyl. 3-benzyloxyallyl, 4-pyridylmethyl-oxyethyl,
4-pyridylmethyl-oxyallyl, benzyl, 2-phenethyl, naphthylmethyl,
styryl, 4-phenyl-1,3-pentadienyl, phenyl-propynyl,
3-phenylbut-2-ynyl, pyrid-3-ylacetylenyl and
quinolin-3-ylacetylenyl, 4-pyridyl-ethynyl, 4-pyridylvinyl,
thienylethenyl, pyridylethenyl, imidazolyl-ethenyl,
pyrazinylethenyl, pyridylpentenyl, pyridyihexenyl and
pyridylheptenyl, thienyl-methyl, pyridylmethyl, imidazolylmethyl,
pyrazinylmethyl, tetrahydropyranylmethyl,
tetrahydropyranyl-methyloxymethyl, and the like.
[0076] "Acyl" means an H--CO-- or (aliphatic or cyclyl)-CO-- group
wherein the aliphatic group is as herein described. Preferred acyls
contain a lower alkyl. Exemplary acyl groups include formyl,
acetyl, propanoyl, 2-methylpropanoyl, butanoyl, palmitoyl,
acryloyl, propynoyl, cyclohexylcarbonyl, and the like.
[0077] "Alkenyl" means an alkenyl-CO-- group wherein alkenyl is as
defined herein.
[0078] "Alkenyl" means an aliphatic hydrocarbon group containing a
carbon-carbon double bond and which may be straight or branched
having about 2 to about 15 carbon atoms in the chain. Preferred
alkenyl groups have 2 to about 12 carbon atoms in the chain; and
more preferably about 2 to about 4 carbon atoms in the chain.
Branched means that one or more lower alkyl groups such as methyl,
ethyl or propyl are attached to a linear alkenyl chain. "Lower
alkenyl" means about 2 to about 4 carbon atoms in the chain that
may be straight or branched. Exemplary alkenyl groups include
ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl,
n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl, decenyl, and the
like. "Substituted alkenyl" means an alkenyl group as defined above
which is substituted with one or more "aliphatic group
substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein. Exemplary alkenyl aliphatic
group substituents include halo or cycloalkyl groups
[0079] "Alkenyloxy" means an alkenyl-O-- group wherein the alkenyl
group is as herein described. Exemplary alkenyloxy groups include
allyloxy, 3-butenyloxy, and the like.
[0080] "Alkoxy" means an alkyl-O-- group wherein the alkyl group is
as herein described. Exemplary alkoxy groups include methoxy,
ethoxy, n-propoxy, i-propoxy, n-butoxy, heptoxy, and the like.
[0081] "Alkoxycarbonyl" means an alkyl-O--CO-- group, wherein the
alkyl group is as herein defined. Exemplary alkoxycarbonyl groups
include methoxycarbonyl, ethoxycarbonyl, s-butyloxycarbonyl, and
the like.
[0082] "Alkyl" means an aliphatic hydrocarbon group which may be
straight or branched having about 1 to about 20 carbon atoms in the
chain. Preferred alkyl groups have 1 to about 12 carbon atoms in
the chain, more preferred is lower alkyl as defined herein.
Branched means that one or more lower alkyl groups such as methyl,
ethyl or propyl are attached to a linear alkyl chain. "Lower alkyl"
means about 1 to about 4 carbon atoms in the chain that may be
straight or branched. "Substituted alkyl" means an alkyl group as
defined above which is substituted with one or more "aliphatic
group substituents" (preferably 1 to 3) which may be the same or
different, and are as defined herein.
[0083] "Alkylsulfinyl" means an alkyl-SO-- group wherein the alkyl
group is as defined above. Preferred groups are those wherein the
alkyl group is lower alkyl.
[0084] "Alkylsulfonyl" means an alkyl-SO.sub.2-group wherein the
alkyl group is as defined above. Preferred groups are those wherein
the alkyl group is lower alkyl.
[0085] "Alkylsulphonylcarbamoyl" means an
alkyl-SO.sub.2--NH--C(.dbd.O)-- group wherein the alkyl group is as
herein described. Preferred alkylsulphonylcarbamoyl groups are
those wherein the alkyl group is lower alkyl.
[0086] "Alkylthio" means an alkyl-S-- group wherein the alkyl group
is as herein described. Exemplary alkylthio groups include
methylthio, ethylthio, i-propylthio and heptylthio.
[0087] "Alkynyl" means an aliphatic hydrocarbon group containing a
carbon-carbon triple bond and which may be straight or branched
having about 2 to about 15 carbon atoms in the chain. Preferred
alkynyl groups have 2 to about 12 carbon atoms in the chain; and
more preferably about 2 to about 4 carbon atoms in the chain.
Branched means that one or more lower alkyl groups such as methyl,
ethyl or propyl are attached to a linear alkynyl chain. "Lower
alkynyl" means about 2 to about 4 carbon atoms in the chain that
may be straight or branched. The alkynyl group may be substituted
by one or more halo. Exemplary alkynyl groups include ethynyl,
propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl,
heptynyl, octynyl, decynyl, and the like. "Substituted alkynyl"
means alkynyl as defined above which is substituted with one or
more "aliphatic group substituents" (preferably 1 to 3) which may
be the same or different, and are as defined herein.
[0088] "Amine protecting group" means an easily removable group
that is known in the art to protect a nitrogen moiety of an amino
or amide group against undesirable reaction during synthetic
procedures and to be selectively removable. The use of amine/amide
protecting groups is well known in the art for protecting groups
against undesirable reactions during a synthetic procedure and many
such protecting groups are known, for example, T. W. Greene and P.
G. M. Wuts, Protective Groups in Organic Synthesis, 2nd edition,
John Wiley & Sons, New York (1991), incorporated herein by
reference. Amine/amide protecting group also includes "acid labile
amine/amide protecting group" and "hydrogenation labile amine/amide
protecting group". Exemplary amine/amide protecting groups are
acyl, including formyl, acetyl, chloroacetyl, trichloroacetyl,
o-nitrophenylacetyl, o-nitrophenoxy-acetyl, trifluoroacetyl,
acetoacetyl, 4-chlorobutyryl, isobutyryl, o-nitrocinnamoyl,
picolinoyl, acylisothiocyanate, aminocaproyl, benzoyl and the like,
and acyloxy including methoxy-carbonyl, 9-fluorenylmethoxycarbonyl,
2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl,
vinyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl (BOC),
1,1-dimethyl-propynyloxycarbonyl, benzyloxycarbonyl (CBZ),
p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl, and the
like.
[0089] "Amide protecting group" means an easily removable group
that is known in the art to protect a nitrogen moiety of an amide
group against undesirable reaction during synthetic procedures and
to be selectively removable after its conversion to the amine. The
use of amide protecting groups is well known in the art for
protecting groups against undesirable reactions during a synthetic
procedure and many such protecting groups are known, for example,
T. W. Greene and P. G. M. Wuts, Protective Groups in Organic
Synthesis, 2nd edition, John Wiley & Sons, New York (1991),
incorporated herein by reference. Amide protecting group also
includes "acid labile amide protecting group" and "hydrogenation
labile amide protecting group". Exemplary amide protecting groups
are o-nitrocinnamoyl, picolinoyl, aminocaproyl, benzoyl and the
like, and acyloxy including methoxy-carbonyl,
9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl,
2-trimethylsilylethoxy-carbonyl, vinyloxycarbonyl,
allyloxycarbonyl, t-butyloxycarbonyl (BOC),
1,1-dimethyl-propynyloxycarbonyl, benzyloxycarbonyl (CBZ),
p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl, and the
like.
[0090] "Amino acid" means an amino acid selected from the group
consisting of natural and unnatural amino acids as defined herein.
Amino acid is also meant to include -amino acids having L or D
stereochemistry at the .alpha.-carbon. Preferred amino acids are
those possessing an .alpha.-amino group. The amino acids may be
neutral, positive or negative depending on the substituents in the
side chain. "Neutral amino acid" means an amino acid containing
uncharged side chain substituents. Exemplary neutral amino acids
include alanine, valine, leucine, isoleucine, proline,
phenylalanine, tryptophan, methionine, glycine, serine, threonine
and cysteine. "Positive amino acid" means an amino acid in which
the side chain substituents are positively charged at physiological
pH. Exemplary positive amino acids include lysine, arginine and
histidine. "Negative amino acid" means an amino acid in which the
side chain substituents bear a net negative charge at physiological
pH. Exemplary negative amino acids include aspartic acid and
glutamic acid. Preferred amino acids are .alpha.-amino acids.
Exemplary natural amino acids are isoleucine, proline,
phenylalanine, tryptophan, methionine, glycine, serine, threonine,
cysteine, tyrosine, asparagine, glutamine, lysine, arginine,
histidine, aspartic acid and glutamic acid. Unnatural amino acid"
means an amino acid for which there is no nucleic acid codon.
Exemplary unnatural amino acids include, for example, the D-isomers
of the natural .alpha.-amino acids as indicated above; Aib
(aminobutyric acid), .beta.Aib (3-amino-isobutyric acid), Nva
(norvaline), .beta.-Ala, Aad (2-aminoadipic acid), .beta.Aad
(3-aminoadipic acid), Abu (2-aminobutyric acid), Gaba
(.gamma.-aminobutyric acid), Acp (6-aminocaproic acid), Dbu
(2,4-diaminobutryic acid), .alpha.-aminopimelic acid, TMSA
(trimethylsilyl-Ala), aIle (allo-isoleucine), Nle (norleucine),
tert-Leu, Cit (citrulline), Orn, Dpm (2,2'-diaminopimelic acid),
Dpr (2,3-diaminopropionic acid), .alpha.- or .beta.-Nal, Cha
(cyclohexyl-Ala), hydroxyproline, Sar (sarcosine), and the like;
cyclic amino acids; N.sup.a-alkylated amino acids such as MeGly
(N.sup.a-methylglycine). EtGly (N.sup.a-ethylglycine) and EtAsn
(N.sup.a-ethylasparagine); and amino acids in which the
.alpha.-carbon bears two side-chain substituents. The names of
natural and unnatural amino acids and residues thereof used herein
follow the naming conventions suggested by the IUPAC Commission on
the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission
on Biochemical Nomenclature as set out in "Nomenclature of a-Amino
Acids (Recommendations, 1974)" Biochemistry, 14(2), (1975). To the
extent that the names and abbreviations of amino acids and residues
thereof employed in this specification and appended claims differ
from those noted, differing names and abbreviations will be made
clear.
[0091] "Amino acid protecting group" mean a group that protects an
acid or amine moiety of the amino acid or other reactive moiety on
the side chain of an amino acid, e.g., hydroxy or thiol. For
examples of "corresponding protected derivatives" of amino acid
side chains, see T. W. Green and P. G. M. Wuts in "Protective
Groups in Organic Chemistry" John Wiley and Sons, 1991. Protecting
groups for an acid group in an amino acid are described herein, for
example in the sections "acidic functional group" and
"hydrogenation labile acid protecting group". Protecting groups for
an amine group in an amino acid are described herein, for example
in the sections "amine protecting group", "acid labile amine
protecting group" and "hydrogenation labile amine protecting
group".
[0092] "Amino acid residue" means the individual amino acid units
incorporated into the compound of the invention.
[0093] "Amino acid side chain" means the substituent found on the
carbon between the amino and carboxy groups in .alpha.-amino acids.
Exemplary .cndot.-amino acid side chains include isopropyl, methyl,
and carboxymethyl for valine, alanine, and aspartic acid,
respectively.
[0094] "Amino acid equivalent" means an amino acid that may be
substituted for another amino acid in the peptides according to the
invention without any appreciable loss of function. In making such
changes, substitutions of like amino acids are made on the basis of
relative similarity of side chain substituents, for example
regarding size, charge, hydrophilicity, hydropathicity and
hydrophobicity as described herein.
[0095] "Aromatic group" means aryl or heteroaryl as defined herein.
Exemplary aromatic groups include phenyl, halo substituted phenyl,
azaheteroaryl, and the like.
[0096] "Aroyl" means an aryl-CO-- group wherein the aryl group is
as herein described. Exemplary aroyl groups include benzoyl, 1- and
2-naphthoyl, and the like.
[0097] "Aryl" means an aromatic monocyclic or multicyclic ring
system of about 6 to about 14 carbon atoms, preferably of about 6
to about 10 carbon atoms. Encompassed by aryl are fused
arylcycloalkenyl, fused arylcycloalkyl, fused arylheterocyclenyl
and fused arylheterocyclyl as defined herein when bonded through
the aryl moiety thereof. The aryl is optionally substituted with
one or mom "ring group substituents" which may be the same or
different, and are as defined herein. Exemplary aryl groups include
phenyl or naphthyl, or phenyl substituted or naphthyl substituted.
"Substituted aryl" means an aryl group as defined above which is
substituted with one or more "ring group substituents" (preferably
1 to 3) which may be the same or different and are as defined
herein.
[0098] "Aryldiazo" means an aryl-diazo-group wherein the aryl and
diazo groups are as defined herein.
[0099] "Arylene" means an optionally substituted 1,2-, 1,3-, 1,4-,
bivalent aryl group, wherein the aryl group is as defined herein.
Exemplary arylene groups include optionally substituted phenylene,
naphthylene and indanylene. A particular arylene is optionally
substituted phenylene. "Substituted arylene" means an arylene group
as defined above which is substituted with one or more "ring group
substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein.
[0100] "Aryloxy" means an aryl-O-- group wherein the aryl group is
as defined herein. Exemplary aryloxy groups include phenoxy and
2-naphthyloxy.
[0101] "Aryloxycarbonyl" means an aryl-O--CO-- group wherein the
aryl group is as defined herein. Exemplary aryloxycarbonyl groups
include phenoxycarbonyl and naphthoxycarbonyl.
[0102] "Arylsulfonyl" means an aryl-SO.sub.2-- group wherein the
aryl group is as defined herein.
[0103] "Arylsulphonylcarbamoyl" means an
aryl-SO.sub.2--NH--C(.dbd.O)-- group wherein the aryl group is as
herein described. An exemplary arylsulphonylcarbamoyl group is
phenylsulphonylcarbamoyl.
[0104] "Arylsulfinyl" means an aryl-SO-- group wherein the aryl
group is as defined herein.
[0105] "Arylthio" means an aryl-S-- group wherein the aryl group is
as herein described. Exemplary arylthio groups include phenylthio
and naphthylthio.
[0106] "Basic nitrogen atom" means a sp.sup.2 or sp.sup.3
hybridized nitrogen atom having a non-bonded pair of electrons
which is capable of being protonated. Exemplary basic nitrogen
atoms include optionally substituted imino, optionally substituted
amino and optionally substituted amidino groups.
[0107] "Carboxy" means an HO(O)C-- (carboxylic acid) group.
[0108] "Coupling agent" means a compound that reacts with the
hydroxyl moiety of a carboxy moiety thereby rendering it
susceptible to nucleophilic attack. Exemplary coupling agents
include DIC, EDCI, DCC, and the like.
[0109] "Cycloalkenyl" means a non-aromatic mono- or multicyclic
ring system of about 3 to about 10 carbon atoms, preferably of
about 5 to about 10 carbon atoms, and which contains at least one
carbon-carbon double bond. Encompassed by cycloalkenyl are fused
arylcycloalkenyl and fused heteroarylcycloalkenyl as defined herein
when bonded through the cycloalkenyl moiety thereof. Preferred ring
sizes of rings of the ring system include about 5 to about 6 ring
atoms; and such preferred ring sizes are also referred to as
"lower". "Substituted cycloalkenyl" means an cycloalkyenyl group as
defined above which is substituted with one or more "ring group
substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein. Exemplary monocyclic
cycloalkenyl include cyclopentenyl, cyclohexenyl, cycloheptenyl,
and the like. An exemplary multicyclic cycloalkenyl is
norbomylenyl.
[0110] "Cycloalkyl" means a non-aromatic mono- or multicyclic ring
system of about 3 to about 10 carbon atoms, preferably of about 5
to about 10 carbon atoms. Preferred ring sizes of rings of the ring
system include about 5 to about 6 ring atoms; and such preferred
ring sizes are also referred to as "lower". Encompassed by
cycloalkyl are fused arylcycloalkyl and fused heteroarylcycloalkyl
as defined herein when bonded through the cycloalkyl moiety
thereof. "Substituted cycloalkyl" means a cycloalkyl group as
defined above which is substituted with one or more "ring group
substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein. Exemplary monocyclic
cycloalkyl include cyclopentyl, cyclohexyl, cycloheptyl, and the
like. Exemplary multicyclic cycloalkyl include 1-decalin,
norbornyl, adamant-(1- or 2-)yl, and the like.
[0111] "Cycloalkylene" means a bivalent cycloalkyl group as defined
herein having about 4 to about 8 carbon atoms. Preferred ring sizes
of the cycloalkylene include about 5 to about 6 ring atoms; and
such preferred ring sizes are also referred to as "lower". The
points of binding on the cycloalkylene group include 1,1-, 1,2-,
1,3-, or 1,4-binding patterns, and where applicable the
stereochemical relationship of the points of binding is either cis
or trans. Exemplary cycloalkylene groups include (1,1-, 1,2- or
1,3-)cyclohexylene and (1,1- or 1,2-)cyclopentylene. "Substituted
cycloalkylene" means an cycloalkylene group as defined above which
is substituted with one or more "ring group substituents"
(preferably 1 to 3) which may be the same or different and are as
defined herein
[0112] "Cyclic" or "Cyclyl" means cycloalkyl, cycloalkenyl,
heterocyclyl or heterocyclenyl as defined herein. The term "lower"
as used in connection with the term cyclic is the same as noted
herein regarding the cycloalkyl, cycloalkenyl, heterocyclyl or
heterocyclenyl.
[0113] "Cyclyloxy" means a cyclyl-O-- group wherein the cyclyl
group is as herein described. Exemplary cycloalkoxy groups include
cyclopentyloxy, cyclohexyloxy, quinuclidyloxy,
pentamethylenesulfideoxy, tetrahydropyranyloxy,
tetrahydrothiophenyloxy, pyrrolidinyloxy, tetrahydrofuranyloxy or
7-oxabicyclo[2.2.1]heptanyloxy, hydroxytetrahydropyranyloxy,
hydroxy-7-oxabicyclo[2.2.1]heptanyloxy, and the like.
[0114] "Cyclylsulfinyl" means a cyclyl-S(O)-- group wherein the
cyclyl group is as herein described.
[0115] "Cyclylsulfonyl" means a cyclyl-S(O).sub.2-- group wherein
the cyclyl group is as herein described.
[0116] "Cyclylthio" means a cyclyl-S-- group wherein the cyclyl
group is as herein described.
[0117] "Diazo" means a bivalent --N.dbd.N-- radical.
[0118] "Displaceable moiety" means a group that where associated
with Las defined herein is subject to being displaced by
nucleophilic attack by a mono- or di-substituted amine moiety with
or without the presence of an agent that facilitates said attack,
e.g., coupling agent. Exemplary displaceable moieties include
hydroxy, aliphatic oxy, halo, N-oxysuccinimide, acyloxy, and the
like.
[0119] "Effective amount" is means an amount of a
compound/composition according to the present invention effective
in producing the desired therapeutic effect.
[0120] "Fused arylcycloalkenyl" means a fused aryl and cycloalkenyl
as defined herein. Preferred fused arylcycloalkenyls are those
wherein the aryl thereof is phenyl and the cycloalkenyl consists of
about 5 to about 6 ring atoms. A fused arylcycloalkenyl as a
variable may be bonded through any atom of the ring system thereof
capable of such. "Substituted fused arylcycloalkenyl" means a fused
arylcycloalkenyl group as defined above which is substituted with
one or more "ring group substituents" (preferably 1 to 3) which may
be the same or different and are as defined herein. Exemplary fused
arylcycloalkenyl include 1,2-dihydronaphthylene, indene, and the
like.
[0121] "Fused arylcycloalkyl" means a fused aryl and cycloalkyl as
defined herein. Preferred fused arylcycloalkyls are those wherein
the aryl thereof is phenyl and the cycloalkyl consists of about 5
to about 6 ring atoms. A fused arylcycloalkyl as a variable may be
bonded through any atom of the ring system thereof capable of such.
"Substituted fused arylcycloalkyl" means a fused arylcycloalkyl
group as defined above which is substituted with one or more "ring
group substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein. Exemplary fused arylcycloalkyl
includes 1,2,3,4-tetrahydro-naphthylene, and the like.
[0122] "Fused arylheterocyclenyl" means a fused aryl and
heterocyclenyl as defined herein. Preferred fused
arylheterocyclenyls are those wherein the aryl thereof is phenyl
and the heterocyclenyl consists of about 5 to about 6 ring atoms. A
fused arylheterocyclenyl as a variable may be bonded through any
atom of the ring system thereof capable of such. The designation of
the aza, oxa or thia as a prefix before heterocyclenyl portion of
the fused arylheterocyclenyl define that at least a nitrogen,
oxygen or sulfur atom is present, respectively, as a ring atom.
"Substituted fused arylheterocyclenyl" means a fused
arylheterocyclenyl group as defined above which is substituted with
one or more "ring group substituents" (preferably 1 to 3) which may
be the same or different and are as defined herein. The nitrogen
atom of a fused arylheterocyclenyl may be a basic nitrogen atom.
The nitrogen or sulfur atom of the heterocyclenyl portion of the
fused arylheterocyclenyl may also be optionally oxidized to the
corresponding N-oxide, S-oxide or S,S-dioxide. Exemplary fused
arylheterocyclenyl include 3H-indolinyl, 1H-2-oxoquinolyl,
2H-1-oxoisoquinolyl, 1,2-di-hydroquinolinyl, 3,4-dihydroquinolinyl,
1,2-dihydroisoquinolinyl, 3,4-dihydroisoquinolinyl, and the
like.
[0123] "Fused arylheterocyclyl" means a fused aryl and heterocyclyl
as defined herein. Preferred fused arylheterocyclyls are those
wherein the aryl thereof is phenyl and the heterocyclyl consists of
about 5 to about 6 ring atoms. A fused arylheterocyclyl as a
variable may be bonded through any atom of the ring system thereof
capable of such. The designation of the aza, oxa or thia as a
prefix before heterocyclyl portion of the fused arylheterocyclyl
define that at least a nitrogen, oxygen or sulfur atom is present,
respectively, as a ring atom. "Substituted fused arylheterocyclyl"
means a fused arylheterocyclyl group as defined above which is
substituted with one or more "ring group substituents" (preferably
1 to 3) which may be the sane or different and are as defined
herein. The nitrogen atom of a fused arylheterocyclyl may be a
basic nitrogen atom. The nitrogen or sulfur atom of the
heterocyclyl portion of the fused arylheterocyclyl may also be
optionally oxidized to the corresponding N-oxide, S-oxide or
S,S-dioxide. Exemplary fused arylheterocyclyl ring systems include
indolinyl, 1,2,3,4-tetrahydroisoquinoline,
1,2,3,4-tetrahydroquinoline, 1H-2,3-dihydroisoindol-2-yl,
2,3-dihydrobenz[f]isoindol-2-yl,
1,2,3,4-tetrahydrobenz[g]-isoquinolin-2-yl, and the like.
[0124] "Fused heteroarylcycloalkenyl" means a fused heteroaryl and
cycloalkenyl as defined herein. Preferred fused
heteroarylcycloalkenyls are those wherein the heteroaryl thereof is
phenyl and the cycloalkenyl consists of about 5 to about 6 ring
atoms. A fused heteroaryl-cycloalkenyl as a variable may be bonded
through any atom of the ring system thereof capable of such. The
designation of the aza, oxa or thia as a prefix before heteroaryl
portion of the fused heteroarylcycloalkenyl define that at least a
nitrogen, oxygen or sulfur atom is present, respectively, as a ring
atom. "Substituted fused heteroarylcycloalkenyl" means a fused
heteroarylcycloalkyenyl group as defined above which is substituted
with one or more "ring group substituents" (preferably 1 to 30
which may be the same or different and are as defined herein. The
nitrogen atom of a fused heteroarylcycloalkenyl may be a basic
nitrogen atom. The nitrogen atom of the heteroaryl portion of the
fused heteroarylcycloalkenyl may also be optionally oxidized to the
corresponding N-oxide. Exemplary fused heteroarylcyclo-alkenyl
include 5,6-dihydroquinolyl, 5,6-dihydroisoquinolyl,
5,6-dihydroquinoxalinyl, 5,6-dihydroquinazolinyl,
4,5-dihydro-1H-benzimidazolyl, 4,5-di-hydrobenzoxazolyl, and the
like.
[0125] "Fused heteroarylcycloalkyl" means a fused heteroaryl and
cycloalkyl as defined herein. Preferred fused heteroarylcycloalkyls
are those wherein the heteroaryl thereof consists of about 5 to
about 6 ring atoms and the cycloalkyl consists of about 5 to about
6 ring atoms. A fused heteroarylcycloalkyl as a variable may be
bonded through any atom of the ring system thereof capable of such.
The designation of the aza, oxa or thia as a prefix before
heteroaryl portion of the fused heteroarylcycloalkyl define that at
least a nitrogen, oxygen or sulfur atom is present, respectively,
as a ring atom. "Substituted fused heteroarylcycloalkyl" means a
fused heteroarylcycloalkyl group as defined above which is
substituted with one or more "ring group substituents" (preferably
1 to 3) which may be the same or different and are as defined
herein. The nitrogen atom of a fused heteroarylcycloalkyl may be a
basic nitrogen atom. The nitrogen atom of the heteroaryl portion of
the fused heteroarylcycloalkyl may also be optionally oxidized to
the corresponding N-oxide. Exemplary fused heteroarylcycloalkyl
include 5,6,7,8-tetrahydroquinolinyl,
5,6,7,8-tetra-hydroisoquinolyl, 5,6,7,8-tetrahydroquinoxalinyl,
5,6,7,8-tetrahydroquinazolyl,
4,5,6,7-tetrahydro-1H-benzimidazolyl,
4,5,6,7-tetrahydrobenzoxazolyl,
1H-4-oxa-1,5-diazanaphthalen-2-onyl,
1,3-dihydroimidizole-[4,5]-pyridin-2-onyl, and the like.
[0126] "Fused heteroarylheterocyclenyl" means a fused heteroaryl
and heterocyclenyl as defined herein. Preferred fused
heteroarylheterocyclenyls are those wherein the heteroaryl thereof
consists of about 5 to about 6 ring atoms and the heterocyclenyl
consists of about 5 to about 6 ring atoms. A fused
heteroarylheterocyclenyl as a variable may be bonded through any
atom of the ring system thereof capable of such. The designation of
the aza, oxa or thia as a prefix before the heteroaryl or
heterocyclenyl portion of the fused heteroarylhetero-cyclenyl
define that at least a nitrogen, oxygen or sulfur atom is present,
respectively, as a ring atom. "Substituted fused
heteroarylbeterocyclenyl" means a fused heteroarylheterocyclenyl
group as defined above which is substituted with one or more "ring
group substituents" (preferably L to 3) which may be the same or
different and are as defined herein. The nitrogen atom of a fused
heteroarylazaheterocyclenyl may be a basic nitrogen atom. The
nitrogen or sulfur atom of the heteroaryl portion of the fused
heteroarylheterocyclyl may also be optionally oxidized to the
corresponding N-oxide. The nitrogen or sulfur atom of the
heteroaryl or heterocyclyl portion of the fused
heteroarylheterocyclyl may also be optionally oxidized to the
corresponding N-oxide, S-oxide or S,S-dioxide. Exemplary fused
heteroarylheterocyclenyl include 7,8-dihydro[1,7]naphthyridinyl,
1,2-dihydro[2,7]-naphthyridinyl,
6,7-dihydro-3H-imidazo[4,5-c]pyridyl,
1,2-dihydro-1,5-naphthyridinyl, 1,2-dihydro-1,6-naphthyridinyl,
1,2-dihydro-1,7-naphthyridinyl, 1,2-dihydro-1,8-naphthyridinyl,
1,2-dihydro-2,6-naphthyridinyl, and the like.
[0127] "Fused heteroarylheterocyclyl" means a fused heteroaryl and
heterocyclyl as defined herein. Preferred fused
heteroarylheterocyclyls are those wherein the heteroaryl thereof
consists of about 5 to about 6 ring atoms and the heterocyclyl
consists of about 5 to about 6 ring atoms. A fused
heteroarylheterocyclyl as a variable may be bonded through any atom
of the ring system thereof capable of such. The designation of the
aza, oxa or thia as a prefix before the heteroaryl or heterocyclyl
portion of the fused heteroarylheterocyclyl define that at least a
nitrogen, oxygen or sulfur atom is present, respectively, as a ring
atom. "Substituted fused heteroarylheterocyclyl" means a fused
heteroarylheterocyclyl group as defined above which is substituted
with one or more "ring group substituents" (preferably 1 to 3)
which may be the same or different and are as defined herein The
nitrogen atom of a fused heteroarylheterocyclyl may be a basic
nitrogen atom. The nitrogen or sulfur atom of the heteroaryl
portion of the fused heteroarylheterocyclyl may also be optionally
oxidized to the corresponding N-oxide. The nitrogen or sulfur atom
of the heteroaryl or heterocyclyl portion of the fused
heteroarylheterocyclyl may also be optionally oxidized to the
corresponding N-oxide, S-oxide or S,S-dioxide. Exemplary fused
heteroarylheterocyclyl include 2,3-dihydro-1H
pyrrol[3,4-b]quinolin-2-yl,
1,2,3,4-tetrahydrobenz[b][1,7]naphthyridin-2-yl,
1,2,3,4-tetrahydrobenz[b][1,6]naphthyridin-2-yl,
1,2,3,4-tetra-hydro-9H-pyrido(3,4-b)indol-2yl,
1,2,3,4-tetrahydro-9H-pyrido[4,3-b]indol-2yl,
2,3-dihydro-1H-pyrrolo[3,4-b]indol-2-yl,
1H-2,3,4,5-tetrahydroazepino[3,4-b]indol-2-yl,
1H-2,3,4,5-tetrahydroazepino[4,3-b]indol-3-yl,
1H-2,3,4,5-tetrahydroazepino[4,5-b]indol-2 yl,
5,6,7,8-tetrahydro[1,7]napthyridyl,
1,2,3,4-tetrhydro[2,7]naphthyridyl,
2,3-dihydro[1,4]dioxino[2,3-b]pyridyl,
2,3-dihydro-[1,4]dioxino[2,3-b]pyridyl,
3,4-dihydro-2H-1-oxa[4,6]diazanaphthalenyl,
4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridyl,
6,7-dihydro[5,8]diazanaphthalenyl,
1,2,3,4-tetrahydro[1,5]-napthyridinyl,
1,2,3,4-tetrahydro[1,6]napthyridinyl,
1,2,3,4-tetrahydro[1,7]napthyridinyl,
1,2,3,4-tetrahydro[1,8]napthyridinyl,
1,2,3,4-tetra-hydro[2,6]napthyridinyl, and the like.
[0128] "Halo" means fluoro, chloro, bromo, or iodo. Preferred are
fluoro, chloro or bromo, and more preferred are fluoro or
chloro.
[0129] "Heteroaroyl" means an heteroaryl-CO-- group wherein the
heteroaryl group is as herein described. Exemplary heteroaroyl
groups include thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, 1- and
2-naphthoyl, pyridinoyl, and the like."
[0130] "Heteroaryl" means an aromatic monocyclic or multicyclic
ring system of about 5 to about 14 carbon atoms, preferably about 5
to about 10 carbon atoms, in which one or more of the carbon atoms
in the ring system is/are hetero element(s) other than carbon, for
example nitrogen, oxygen or sulfur. Preferably the ring system
includes 1 to 3 heteroatoms. Preferred ring sizes of rings of the
ring system include about 5 to about 6 ring atoms. Encompassed by
heteroaryl are fused heteroarylcycloalkenyl, fused
heteroarylcycloalkyl, fused heteroarylheterocyclenyl and fused
heteroarylheterocyclyl as defined herein when bonded through the
heteroaryl moiety thereof. "Substituted heteroaryl" means a
heteroaryl group as defined above which is substituted with one or
more "ring group substituents" (preferably 1 to 3) which may be the
same or different and are as defined herein. The designation of the
aza, oxa or thia as a prefix before heteroaryl define that at least
a nitrogen, oxygen or sulfur atom is present, respectively, as a
ring atom. A nitrogen atom of an heteroaryl may be a basic nitrogen
atom and may also be optionally oxidized to the corresponding
N-oxide. Exemplary heteroaryl and substituted heteroaryl groups
include pyrazinyl, thienyl, isothiazolyl, oxazolyl, pyrazolyl,
furazanyl, pyrrolyl, 1,2,4-thiadiazolyl, pyridazinyl, quinoxalinyl,
phthalazinyl, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl,
benzofurazanyl, azaindolyl, benzimidazolyl, benzothienyl,
thienopyridyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl,
benzoazaindolyl, 1,2,4-triazinyl, benzthiazolyl, furanyl,
imidazolyl, indolyl, indolizinyl, isoxazolyl, isoquinolinyl,
isothiazolyl, oxadiazolyl, pyrazinyl, pyridazinyl, pyrazolyl,
pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl,
1,3,4-thiadiazolyl, thiazolyl, thienyl, triazolyl, and the like. A
preferred heteroaryl group is pyrazinyl.
[0131] "Heteroaryldiazo" means an heteroaryl-azo-group wherein the
heteroaryl and azo groups are as defined herein.
[0132] "Heteroarylidyl" means a bivalent radical derived from a
heteroaryl, wherein the heteroaryl is as described herein. An
exemplary heteroaryldiyl radical is optionally substituted
pyridinediyl.
[0133] "Heteroarylsulphonylcarbamoyl" means a
heteroaryl-SO.sub.2--NH--C(.dbd.O)-- group wherein the heteroaryl
group is as herein described.
[0134] "Heterocyclenyl" means a non-aromatic monocyclic or
multicyclic hydrocarbon ring system of about 3 to about 10 carbon
atoms, preferably about 5 to about 10 carbon atoms, in which one or
more of the carbon atoms in the ring system is/are hetero
element(s) other than carbon, for example nitrogen, oxygen or
sulfur atoms, and which contains at least one carbon-carbon double
bond or carbon-nitrogen double bond. Preferably, the ring includes
1 to 3 heteroatoms. Preferred ring sizes of rings of the ring
system include about 5 to about 6 ring atoms; and such preferred
ring sizes are also referred to as "lower". Encompassed by
heterocyclenyl are fused arylheterocyclenyl and fused
heteroarylheterocyclenyl as defined herein when bonded through the
heterocyclenyl moiety thereof. The designation of the aza, oxa or
thia as a prefix before heterocyclenyl define that at least a
nitrogen, oxygen or sulfur atom is present, respectively, as a ring
atom. "Substituted heterocyclenyl" means a heterocyclenyl group as
defined above which is substituted with one or more "ring group
substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein. The nitrogen atom of an
heterocyclenyl may be a basic nitrogen atom. The nitrogen or sulfur
atom of the heterocyclenyl may also be optionally oxidized to the
corresponding N-oxide, S-oxide or S,S-dioxide. Exemplary monocyclic
azaheterocyclenyl groups include 1,2,3,4-tetrahydrohydropyridine,
1,2-dihydropyridyl, 1,4-dihydropyridyl,
1,2,3,6-tetra-hydropyridine, 1,4,5,6-tetrahydro-pyrimidine,
2-pyrrolinyl, 3-pyrrolinyl, 2-imidazolinyl, 2-pyrazolinyl, and the
like. Exemplary oxaheterocyclenyl groups include
3,4-dihydro-2H-pyran, dihydrofuranyl, and fluorodihydro-furanyl. An
exemplary multicyclic oxaheterocyclenyl group is
7-oxabicyclo[2.2.1]heptenyl. Exemplary monocyclic
thiaheterocyclenyl rings include dihydrothiophenyl and
dihydrothiopyranyl.
[0135] "Heterocyclyl" means a non-aromatic saturated monocyclic or
multicyclic ring system of about 3 to about 10 carbon atoms,
preferably about 5 to about 10 carbon atoms, in which one or more
of the carbon atoms in the ring system is/are hetero element(s)
other than carbon, for example nitrogen, oxygen or sulfur.
Preferably, the ring system contains from 1 to 3 heteroatoms.
Preferred ring sizes of rings of the ring system include about 5 to
about 6 ring atoms; and such preferred ring sizes are also referred
to as "lower". Encompassed by heterocyclyl are fused
arylheterocyclyl and fused heteroarylheterocyclyl as defined herein
when bonded through the heterocyclyl moiety thereof. The
designation of the aza, oxa or this as a prefix before heterocyclyl
define that at least a nitrogen, oxygen or sulfur atom is present
respectively as a ring atom. "Substituted heterocyclyl" means a
heterocyclyl group as defined above which is substituted with one
or more "ring group substituents" (preferably 1 to 3) which may be
the same or different and are as defined herein. The nitrogen atom
of an heterocyclyl may be a basic nitrogen atom. The nitrogen or
sulfur atom of the heterocyclyl may also be optionally oxidized to
the corresponding N-oxide, S-oxide or S,S-dioxide. Exemplary
monocyclic heterocyclyl rings include piperidyl, pyrrolidinyl,
piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl,
1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
##STR00023##
as substituted monocyclic azaheterocyclyl is substituted directly
or through a linker by at least one substituent that is, or
encompasses, or is substituted by an aromatic group as defined
herein; for example aryl, heteroaryl, aryloxy, heteroaryloxy, aroyl
or its thioxo analogue, heteroaroyl or its thioxo analogue,
aroyloxy, heteroaroyloxy, aryloxycarbonyl, heteroaryloxycarbonyl,
arylsulfonyl, heteroarylsulfonyl, arylsulfinyl, heteroarylsulfinyl,
arylthio, heteroarylthio, aryldiazo, heteroaryldiazo,
Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--, Y.sup.1Y.sup.2NC(O)O--,
Y.sup.1Y.sup.2NC(O)NY.sup.3-- or Y.sup.1Y.sup.2NSO.sub.2-- wherein
at least one of Y.sup.1 and Y.sup.2 is, encompasses or is
substituted by an aryl or heteroaryl moiety. Preferred linkers
include --C(O)--, --OC(O)--, lower alkyl, lower alkoxy, lower
alkenyl, --O--, --S--, --C(O)C(O)--, --S(O)--, --S(O).sub.2,
--NR.sup.80--, where R.sup.80 is hydrogen, alkyl, cycloalkyl, aryl,
aralkyl, heterocyclyl or heteroaryl. Particularly preferred linkers
are --C(O)-- and --OC(O)--. "Substituted multicyclic
azaheterocyclyl" means a multicyclic azaheterocyclyl group as
defined above which is substituted with one or more "ring group
substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein. "Substituted multicyclic
azaheterocyclenyl" means a multicyclic azaheterocyclenyl group as
defined above which is substituted with one or more "ring group
substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein.
[0136] "Heterocyclylene" means a bivalent heterocyclyl group as
defined herein having about 4 to about 8 carbon atoms. Preferred
ring sizes of the heterocyclylene include about 5 to about 6 ring
atoms; and such preferred ring sizes are also referred to as
"lower". The points of binding on the cycloalkylene group include
1,1-, 1,2-, 1,3-, or 1,4-binding patterns, and where applicable the
stereochemical relationship of the points of binding is either cis
or trans. Exemplary heterocyclylene groups include (1,1-, 1,2- or
1,3-)piperidinylene and (1,1- or 1,2-)tetrahydrofuranylene.
"Substituted heterocyclylene" means a heterocyclylene group as
defined above which is substituted with one or more "ring group
substituents" (preferably 1 to 3) which may be the same or
different and are as defined herein.
[0137] "Hydrate" means a solvate wherein the solvent molecule(s)
is/are H.sub.2O.
[0138] "Hydrogenation labile amine protecting group" means an amine
protecting group as defined herein which is readily removed by
hydrogenation while remaining relatively stable to other reagents.
A preferred hydrogenation labile amine protecting group is Cbz.
[0139] "Hydrogenation labile acid protecting group" means an acid
protecting group as defined herein which is readily removed by
hydrogenation while remaining relatively stable to other reagents.
A preferred hydrogenation labile acid protecting group is
benzyl.
[0140] "Hygroscopicity" means sorption, implying an acquired amount
or state of water sufficient to affect the physical or chemical
properties of the substance (Eds. J. Swarbrick and J. C. Boylan,
Encyclopedia of Pharmaceutical Technology, 10, 33).
[0141] "Iminic glycinimide derivative" means an iminic Schiff base
of a glycine that is useful in the synthesis of .alpha.-amino
acids, both natural and unnatural. The iminic ester functionality
may contain one or more assymetric centers that may aid in
stereoinduction during the bond formatting process. In addition,
these iminic glycinimide derivatives may be incorporated onto
polymeric supports to facilitate combinatorial synthesis. Iminic
glycinimide derivatives may be prepared by condensing a glycine
ester with the appropropriate ketone in the presence of an acid
catalyst. The reaction is facilitated by the removal of water.
Iminic glycinimide derivatives are well known in the art for use in
Michael Addition synthetic procedures, for example as disclosed by
Guillena, G., et al., J. Org. Chem. 2000, 65, 7310-7322, herein
incorporated by reference. Particular examples of iminic
glycinimide derivatives according to the invention include one
selected from the group of formulae
##STR00024##
wherein:
[0142] M* is a transition metal, preferably CU, more preferably
CU.sup..quadrature..
[0143] R.sup.14 is --CO.sub.2R.sup.16, --CN
##STR00025##
or --CONR.sup.15R.sup.15;
[0144] R.sup.15 is optionally substituted aliphatic group;
[0145] R.sup.16 is acid protecting group, optionally substituted
aryl, or optionally substituted aliphatic group;
[0146] R.sup.17 is optionally substituted aryl, optionally
substituted aliphatic group,
##STR00026##
[0147] R.sup.18 is hydrogen, alkyl, or alkylthio; or optionally
substituted aryl;
[0148] R.sup.17 and R.sup.18 taken together with the carbon to
which R.sup.17 and R.sup.18 are attached
##STR00027##
and
[0149] {circle around (S)} is a solid phase.
[0150] "Iminic glycinimide derivative adduct" means the resulting
compound where an .alpha.-hydrogen to the nitrogen and carbonyl
moiety of the Schiff base portion is removed and used to form an
attachment for the bond formation thereto. Particular examples of
imbue glycinimide derivative adducts according to the invention
include one selected from the group of formulae
##STR00028##
wherein:
[0151] R.sup.14, R.sup.17, and R.sup.18 are defined as described in
the definition of iminic glycinimide derivative herein.
[0152] "N-oxysuccinimide" means a moiety of the following
structure
##STR00029##
[0153] "N-oxide" means a moiety of the following structure
##STR00030##
[0154] "Patient" includes both human and other mammals.
[0155] "Peptidomimetic" mean a polymer encompassing amino acid
residues joined together through amide bonds.
[0156] "Pharmaceutically acceptable ester" refers to esters that
hydrolyze in vivo and include those that break down readily in the
human body to leave the parent compound or a salt thereof. Suitable
ester groups include, for example, those derived from
pharmaceutically acceptable aliphatic carboxylic acids,
particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic
acids, in which each alkyl or alkenyl moiety advantageously has not
more than 6 carbon atoms. Exemplary esters include formates,
acetates, propionates, butyrates, acrylates, ethylsuccinates, and
the like.
[0157] "Pharmaceutically acceptable prodrugs" as used herein refers
to those prodrugs of the compounds of the present invention which
are, within the scope of sound medical judgement, suitable for use
in contact with the tissues of humans and lower animals with undue
toxicity, irritation, allergic response, and the like, commensurate
with a reasonable benefit/risk ratio, and effective for their
intended use, as well as the zwitterionic forms, where possible, of
the compounds of the invention. The term "prodrug" refers to
compounds that are rapidly transformed in vivo to yield the parent
compound of the above formula, for example by hydrolysis in blood.
Functional groups that may be rapidly transformed, by metabolic
cleavage, in vivo form a class of groups reactive with the carboxyl
group of the compounds of this invention. They include, but are not
limited to such groups as alkanoyl (such as acetyl, propanoyl,
butanoyl, and the like), unsubstituted and substituted aroyl (such
as benzoyl and substituted benzoyl), alkoxycarbonyl (such as
ethoxycarbonyl), trialkylsilyl (such as trimethyl- and
triethysilyl), monoesters formed with dicarboxylic acids (such as
succinyl), and the like. Because of the ease with which the
metabolically cleavable groups of the compounds of this invention
are cleaved in vivo, the compounds bearing such groups act as
pro-drugs. The compounds bearing the metabolically cleavable groups
have the advantage that they may exhibit improved bioavailability
as a result of enhanced solubility and/or rate of absorption
conferred upon the parent compound by virtue of the presence of the
metabolically cleavable group. A thorough discussion is provided in
Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985); Methods in
Enzymology; K. Widder et al, Ed., Academic Press, 42, 309-396
(1985); A Textbook of Drug Design and Development,
Krogsgaard-Larsen and H. Bandaged, ed., Chapter 5; "Design and
Applications of Prodrugs" 113-191 (1991); Advanced Drug Delivery
Reviews, H. Bundgard, 1-38, (1992); J. Pharm. Sci., 77, 285 (1988);
Chem. Pharm. Bull., N. Nakeya et al, 22, 692 (1984); Pro-drugs as
Novel Delivery Systems. T. Higuchi and V. Stella, 14 A.C.S.
Symposium Series, and Bioreversible Carriers in Drug Design, E. B.
Roche, ed., American Pharmaceutical Association and Pergamon Press,
1987, which are incorporated herein by reference.
[0158] "Pharmaceutically acceptable salts" refers to the relatively
non-toxic, inorganic and organic acid addition salts, and base
addition salts, of compounds of the present invention. These salts
can be prepared in situ during the final isolation and purification
of the compounds. In particular, acid addition salts can be
prepared by separately reacting the purified compound in its free
base form with a suitable organic or inorganic acid and isolating
the salt thus formed. Exemplary acid addition salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,
nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate,
laurate, borate, benzoate, lactate, phosphate, tosylate, citrate,
maleate, fumarate, succinate, tartrate, naphthylate, mesylate,
glucoheptonate, lactiobionate, sulphamates, malonates, salicylates,
propionates, methylene-bis-.beta.-hydroxynaphthoates, gentisates,
isethionates, di-p-toluoyltartrates, methanesulphonates,
ethanesulphonates, benzenesulphonates, p-toluenesulphonates,
cyclohexylsulphamates and quinateslaurylsulphonate salts, and the
like. See, for example S. M. Berge, et al., "Pharmaceutical Salts,"
J. Pharm. Sci., 66, 1-19 (1977) which is incorporated herein by
reference. Base addition salts can also be prepared by separately
reacting the purified compound in its acid form with a suitable
organic or inorganic base and isolating the salt thus formed. Base
addition salts include pharmaceutically acceptable metal and amine
salts. Suitable metal salts include the sodium, potassium, calcium,
barium, zinc, magnesium, and aluminum salts. The sodium and
potassium salts are preferred. Suitable inorganic base addition
salts are prepared from metal bases which include sodium hydride,
sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum
hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide
and the like. Suitable amine base addition salts are prepared from
amines which have sufficient basicity to form a stable salt, and
preferably include those amines which are frequently used in
medicinal chemistry because of their low toxicity and acceptability
for medical use, ammonia, ethylenediamine, N-methyl-glucamine,
lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine,
chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,
diethylamine, piperazine, tris(hydroxymethyl)-aminomethane,
tetramethylammonium hydroxide, triethylamine, dibenzylamine,
ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine,
tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g.,
lysine and arginine, and dicyclohexylamine, and the like.
[0159] "Ring group substituents" mean substituents attached to
aromatic or non-aromatic ring systems inclusive of aryl,
heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,
acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,
aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,
acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,
cyano, carboxy (acid), --C(O)--NHOH, --C(O)--CH.sub.2OH,
--C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho, phosphono,
alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,
N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,
3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl
or hydroxyheteroaryl such as 3-hydroxyisoxazolyl,
3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl,
cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,
cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio,
cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,
heteroaryldiazo, thiol, Y.sup.1Y.sup.2N--, Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, wherein Y.sup.1, Y.sup.2 and Y.sup.3 are
independently hydrogen, alkyl, aryl or heteroaryl, or for where the
substituent is Y.sup.1Y.sup.2N--, then one of Y.sup.1 and Y.sup.2
may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl,
cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as
defined herein and the other of Y.sup.1 and Y.sup.2 is as defined
previously, or for where the substituent is Y.sup.1Y.sup.2NC(O)--,
Y.sup.1Y.sup.2NC(O)O--, Y.sup.1Y.sup.2NC(O)NY.sup.3-- or
Y.sup.1Y.sup.2NSO.sub.2--, Y.sup.1 and Y.sup.2 may also be taken
together with the N atom through which Y.sup.1 and Y.sup.2 are
linked to form a 4 to 7 membered azaheterocyclyl or
azaheterocyclenyl. When a ring system is saturated or partially
saturated, the "ring group substituents" further include, methylene
(H.sub.2C.dbd.), oxo (O.dbd.) and thioxo (S.dbd.). Acidic/amide
ring group substituents are carboxy (acid), --C(O)--NHOH,
--C(O)--CH.sub.2OH, --C(O)--CH.sub.2SH, --C(O)--NH--CN, sulpho,
phosphono, alkylsulphonylcarbamoyl, tetrazolyl,
arylsulphonylcarbamoyl, N-methoxycarbamoyl,
heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,
3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as
3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazoly and
Y.sup.1Y.sup.2NCO--. Non-acidic polar ring group substituents are
hydroxy, oxo (O.dbd.), thioxo (S.dbd.), acyl or its thioxo
analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its
thioxo analogue, heteroaroyl or its thioxo analogue,
alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl,
heteroaryloxycarbonyl, acyloxy, cyclylcarbonyloxy, aroyloxy,
heteroaroyloxy, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl,
heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl,
heteroarylsulfonyl, thiol, Y.sup.1Y.sup.2N--,
Y.sup.1Y.sup.2NC(O)--, Y.sup.1Y.sup.2NC(O)O--,
Y.sup.1Y.sup.2NC(O)NY.sup.3-- or Y.sup.1Y.sup.2NSO.sub.2--.
[0160] "Solvate" means a physical association of a compound of this
invention with one or more solvent molecules. This physical
association includes hydrogen bonding. In certain instances the
solvate will be capable of isolation, for example when one or more
solvent molecules are incorporated in the crystal lattice of the
crystalline solid. "Solvate" encompasses both solution-phase and
isolable solvates. Exemplary solvates include hydrates,
ethanolates, methanolates, and the like.
EMBODIMENTS
[0161] With reference to inventions described herein, below are
particular and preferred embodiments are related thereto.
[0162] A particular embodiment according to the invention is where
R.sup.0 is a bond.
[0163] Another particular embodiment according to the invention is
where R.sup.0 is difluoromethylene.
[0164] A particular embodiment according to the invention is where
R.sup.1 is hydrogen or optionally substituted lower aliphatic
group.
[0165] Another particular embodiment according to the invention is
where R.sup.1 is hydrogen or lower alkyl.
[0166] A preferred embodiment according to the invention is where
R.sup.1 is hydrogen.
[0167] A particular embodiment according to the invention is where
R.sup.2 is optionally substituted lower aliphatic group or
optionally substituted monocyclic group.
[0168] Another particular embodiment according to the invention is
where R.sup.2 is optionally substituted lower alkyl, optionally
substituted lower alkenyl, or optionally substituted monocyclic
cycloalkyl.
[0169] Further particular embodiment according to the invention is
where R.sup.2 is carboxymethyl, 1-carboxy-2-phenylethyl,
cyclopropyl, cyclobutyl, 1-cyclohexylethyl, 1-phenylethyl,
but-2-yl, 1-pyrid-4-ylethyl, propen-3-yl or 3-methylbut-2-yl; more
preferred cyclopropyl.
[0170] A particular embodiment according to the invention is where
R.sup.3 is optionally substituted lower aliphatic group
methylene.
[0171] Another particular embodiment according to the invention is
where R.sup.3 is optionally halo substituted lower (alkyl or
alkenyl)methylene.
[0172] A preferred embodiment according to the invention is where
R.sup.3 is propylmethylene, 2,2-difluoroethylmethylene,
2,2,2-trifluoromethylene or propen-3-ylmethylene; more preferred
R.sup.3 is propylmethylene or 2,2-difluoroethylmethylene; further
preferred R.sup.3 is propylmethylene.
[0173] A particular embodiment according to the invention is where
R.sup.4 is hydrogen or optionally substituted lower aliphatic
group.
[0174] Another particular embodiment according to the invention is
where R.sup.4 is hydrogen or lower alkyl.
[0175] A preferred embodiment according to the invention is where
R.sup.5 is hydrogen.
[0176] A particular embodiment according to the invention is where
R.sup.5 is optionally substituted lower aliphatic group
methylene.
[0177] Another particular embodiment according to the invention is
where R.sup.5 is optionally (phenyl, carboxy, carboxamido or
alkoxycarbonyl) substituted lower (alkyl or alkenyl) methylene.
[0178] A preferred embodiment according to the invention is where
R.sup.5 is methylmethylene, isopropylmethylene, t-butylmethylene,
but-2-ylmethylene, butylmethylene, benzylmethylene,
3-methylbutylmethylene, 2-methylpropyl-methylene,
carboxymethylmethylene, carboxamidomethylmethylene,
benzyloxycarbonylmethylmethylene, benzyloxycarbonylpropylmethylene,
or phenylpropen-3-ylmethylene; more preferred R.sup.5 is
isopropylmethylene or t-butyl-methylene.
[0179] A particular embodiment according to the invention is where
R.sup.6 is hydrogen or optionally substituted lower aliphatic
group.
[0180] Another particular embodiment according to the invention is
where R.sup.6 is hydrogen or lower alkyl.
[0181] A preferred embodiment according to the invention is where
R.sup.6 is hydrogen.
[0182] A particular embodiment according to the invention is where
R.sup.7 is optionally substituted lower aliphatic group methylene,
optionally substituted lower cyclic group methylene or optionally
substituted monocyclic (aryl or heteroaryl) methylene.
[0183] Another particular embodiment according to the invention is
where R.sup.7 is optionally substituted lower alkylmethylene,
optionally substituted lower cycloalkylmethylene or optional
substituted phenylmethylene.
[0184] A preferred embodiment according to the invention is where
R.sup.7 is methylmethylene, isopropylmethylene, n-propylmethylene,
phenylmethylene, cyclohexylmethylene, cyclopentylmethylene,
t-butylmethylene, s-butylmethylene, cyclohexylmethylmethylene, or
phenylmethylmethylene; more preferred is isopropylmethylene,
cyclohexylmethylene, cyclopentylmethylene, t-butylmethylene or
s-butylmethylene.
[0185] A preferred embodiment according to the invention is also
wherein each of R.sup.3, R.sup.5, and R.sup.7 is mono substituted
methylene.
[0186] A preferred embodiment according to the invention is also
wherein R.sup.3 is mono substituted methylene and has an (S)
configuration on the carbon attached to the
--C(O)--R.sup.0--C(O)--NR.sup.1R.sup.2 moiety.
[0187] A particular embodiment according to the invention is where
R.sup.8 is hydrogen or optionally substituted lower aliphatic
group.
[0188] Another particular embodiment according to the invention is
where R.sup.8 is hydrogen or lower alkyl.
[0189] A preferred embodiment according to the invention is where
R.sup.8 is hydrogen.
[0190] A particular embodiment according to the invention is where
R.sup.9 is optionally substituted lower aliphatic group or
optionally substituted monocyclic aromatic group.
[0191] Another particular embodiment according to the invention is
where R.sup.9 is optionally substituted lower alkyl or optionally
substituted monocyclic heteroaryl.
[0192] Another particular embodiment according to the invention is
where R.sup.9 is optionally (carboxy, (loweralkyl)SO.sub.2NH--,
(lower alkyl)HNCO--, hydroxy, phenyl, heteroaryl, or (lower
alkyl)OC(O)NH--)-substituted lower alkyl, or optionally substituted
monocyclic heteroaryl.
[0193] A further preferred embodiment according to the invention is
where R.sup.9 is lower alkyl substituted by (mono- or
di-)MeOC(O)NH--; more preferred is 1,2-di(MeOC(O)NH)ethyl or
1-(MeOC(O)NH)ethyl.
[0194] A preferred embodiment according to the invention is where
R.sup.9 is (carboxy, (lower alkyl)HNCO-- or tetrazolyl) substituted
lower alkyl; more preferred 3-carboxypropyl, 2-tetrazol-5ylpropyl,
3-(N-methylcarboxamido)propyl or 3-carboxy-2,2-dimethylpropyl;
further preferred is 3-carboxypropyl, 2-tetrazol-5ylpropyl or
3-(N-methylcarboxamido)propyl.
[0195] Another preferred embodiment according to the invention is
where R.sup.9 is optionally substituted lower alkyl; more preferred
is 1-hydroxy-2-phenylethyl, methyl, isopropyl or t-butyl; further
preferred is methyl, isopropyl or t-butyl.
[0196] Another preferred embodiment according to the invention is
where R.sup.9 is selected from the group consisting of
##STR00031## ##STR00032##
[0197] Yet another preferred embodiment according to the invention
is where R.sup.9 is pyrazinyl.
[0198] A particular embodiment according to the invention is where
R.sup.10 is hydrogen or optionally substituted lower aliphatic
group.
[0199] Another particular embodiment according to the invention is
where R.sup.10 is hydrogen or lower alkyl.
[0200] A preferred embodiment according to the invention is where
R.sup.10 is hydrogen.
[0201] A preferred embodiment according to the invention is
where
##STR00033##
as a substituted monocyclic azaheterocyclyl is substituted
pyrrolidinyl.
[0202] A preferred embodiment according to the invention is
where
##STR00034##
as a substituted monocyclic azaheterocyclyl is optionally
substituted
##STR00035##
or optionally substituted
##STR00036##
wherein Ar is R.sup.2 that comprises an aromatic moiety; more
preferred is optionally substituted
##STR00037##
further preferred is optionally substituted
##STR00038##
[0203] Further preferred optionally substituted
##STR00039##
yet further preferred
##STR00040##
[0204] Another preferred embodiment according to the invention is
where
##STR00041##
as an optionally substituted multicyclic azaheterocyclyl is
optionally substituted
##STR00042##
more preferred is optionally substituted
##STR00043##
Particular substituents for
##STR00044##
are hydroxy, fluoro or oxo.
[0205] Another preferred embodiment according to the invention is
where
##STR00045##
as an optionally substituted multicyclic azaheterocyclenyl is
optionally substituted
##STR00046##
more preferred is
##STR00047##
further preferred is
##STR00048##
[0206] Another preferred embodiment according to the invention is
where
##STR00049##
as an optionally substituted multicyclic azaheterocyclenyl is
optionally substituted
##STR00050##
[0207] A preferred embodiment according to the invention is where
the --C(O)--N(R.sup.4)--R.sup.3--C(O)R.sup.0C(O)NR.sup.2R.sup.1
moiety attached to
##STR00051##
is attached a carbon .alpha. to the nitrogen atom.
[0208] A preferred embodiment according to the invention is where L
is --C(O)-- or --OC(O)--.
[0209] A preferred embodiment according to the invention is where n
is 0.
[0210] Another preferred embodiment according to the invention is
where n is 1.
[0211] A preferred embodiment according to the invention is where
R.sup.11 is --CO.sub.2R.sup.13.
[0212] A preferred embodiment according to the invention is where
R.sup.12 is
##STR00052##
[0213] A particular embodiment according to the invention is where
R.sup.13 is an optionally substituted aliphatic group.
[0214] Another particular embodiment according to the invention is
where R.sup.13 is an alkyl group.
[0215] A preferred embodiment according to the invention is where
R.sup.13 is lower alkyl.
[0216] Another preferred embodiment according to the invention is
where R.sup.13 is methyl.
[0217] A preferred embodiment according to the invention is where
R.sup.14 is --CO.sub.2R.sup.16.
[0218] A particular embodiment according to the invention is where
R.sup.15 is alkyl.
[0219] A preferred embodiment according to the invention is where
R.sup.15 is lower alkyl.
[0220] A preferred embodiment according to the invention is where
R.sup.15 is methyl.
[0221] A particular embodiment according to the invention is where
R.sup.16 is optionally substituted aliphatic.
[0222] Another particular embodiment according to the invention is
where R.sup.16 is alkyl.
[0223] A preferred embodiment according to the invention is where
R.sup.16 is lower alkyl.
[0224] A preferred embodiment according to the invention is where
R.sup.16 is t-Bu.
[0225] A particular embodiment according to the invention is where
R.sup.17 is optionally substituted aryl.
[0226] A preferred embodiment according to the invention is where
R.sup.17 is phenyl.
[0227] A particular embodiment according to the invention is where
R.sup.18 is optionally substituted aryl.
[0228] A preferred embodiment according to the invention is where
R.sup.18 is phenyl.
[0229] A particular embodiment according to the invention is where
p.sup.0 is selected from the group consisting of BOC, CBz, and
--CO.sub.2alkyl.
[0230] A preferred embodiment according to the invention is where
p.sup.0 is BOC.
[0231] It is to be understood that this invention covers all
appropriate combinations of the particular and preferred groupings
referred to herein.
[0232] Particular compounds according to the invention are selected
from the group of compounds A-FH consecutively consisting of
##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057##
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##
##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078## ##STR00079## ##STR00080## ##STR00081##
or a pharmaceutically acceptable salt or prodrug thereof, or a
solvate of such a compound, its salt or its prodrug.
[0233] A preferred compound is one selected from the group
consisting of S, U, BW, BX, BY, BZ, CE, CU, CW, CY, DZ, EA, EC, EJ,
FH, EW, EO, EZ, FG and EN a pharmaceutically acceptable salt or
prodrug thereof, or solvate of such compound, its salt or its
prodrug.
[0234] Another preferred embodiment of the invention are selected
from the following group of compounds:
##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086##
##STR00087##
or a pharmaceutically acceptable salt or prodrug thereof, or a
solvate of such a compound, its salt or its prodrug.
[0235] Another preferred embodiment of the invention is a compound
of the formula
##STR00088##
or a pharmaceutically acceptable salt or prodrug thereof, or a
solvate of such a compound, its salt or its prodrug.
[0236] Another preferred embodiment of the invention is compound of
formula 1 wherein:
R.sup.0 is a bond; R.sup.1 is hydrogen; R.sup.2 is lower alkyl
optionally substituted with 1 to 3 aliphatic group substituents; or
lower cycloalky optionally substituted with 1 to 3 cyclic group
substituents; R.sup.3 and R.sup.5 are each independently methylene
optionally substituted with 1 to 3 aliphatic group substitutents;
R.sup.4, R.sup.6, R.sup.8 and R.sup.10 are hydrogen; R.sup.7 is
methylene substituted with cycloalkyl, lower alkyl or aryl; or or
(1,1- or 1,2-)cycloalkenyl optionally substituted with cycloalkyl,
lower alkyl or aryl;
R9 is
[0237] lower alkyl optionally substituted with 1 to 3 aliphatic
group substituents; or
[0238] heteroaryl optionally substituted with 1 to 3 cyclic group
substituents;
[0239] or heterocyclic optionally substituted with 1 to 3 cyclic
group substituents;
##STR00089##
is monocyclic azaheterocyclyl, multicyclic azaheterocyclyl, or
multicyclic azaheterocyclenyl optionally substituted with from 1 to
3 cyclic group substituents; and L is --C(O)--, --OC(O)--.
[0240] Another preferred embodiment of the invention is a compound
selected from the group consisting of:
##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099##
##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104##
##STR00105## ##STR00106## ##STR00107## ##STR00108##
or a pharmaceutically acceptable salt or prodrug thereof, or a
solvate of such a compound, its salt or its prodrug.
[0241] Another preferred embodiment of the invention is a compound
of formula 1 wherein the optionally substituted aliphatic group,
optionally substituted cyclic group or optionally substituted
aromatic group of R.sup.9 is substituted with at least one
heteroaryl substituent.
[0242] Another preferred embodiment of the invention is a compound
of formula 1 wherein the optionally substituted aromatic group of
R.sup.9 is optionally substituted heteroaryl.
[0243] Another preferred embodiment of the invention is a compound
of formula 1 wherein the optionally substituted aliphatic group of
R.sup.9 is optionally substituted alkylheteroaryl.
[0244] Another preferred embodiment of the invention is a compound
wherein the optionally substituted heteroary group of R.sup.9 is
pyrazinyl, tetrazolyl, quinolinyl, imidazolyl, isoxazolyl and
pyradonyl, optionally substituted with a ring group
substituent.
[0245] The compounds of the invention optionally are supplied as
salts. Those salts that are pharmaceutically acceptable are of
particular interest since they are useful in administering the
foregoing compounds for medical purposes. Salts that are not
pharmaceutically acceptable are useful in manufacturing processes,
for isolation and purification purposes, and in some instances, for
use in separating stereoisomeric forms of the compounds of this
invention. The latter is particularly true of amine salts prepared
from optically active amines.
[0246] Where the compound of the invention contains a carboxy
group, or a sufficiently acidic bioisostere, base addition salts
may be formed and are simply a more convenient form for use; and in
practice, use of the salt form inherently amounts to use of the
free acid form.
[0247] Also, where the compound of the invention contains a basic
group, or a sufficiently basic bioisostere, acid addition salts may
be formed and are simply a more convenient form for use; and in
practice, use of the salt form inherently amounts to use of the
free base form.
[0248] A preferred embodiment of a method according to the present
invention is for treating a patient suffering from an HCV infection
or physiological conditions related to the infection comprising
administering to the patient a pharmaceutically effective amount of
a compound of formula 1.
[0249] Another preferred embodiment of a therapeutic method
according to the present invention is for treating a patient
suffering from an HCV infection or physiological conditions related
to the infection comprising administering a pharmaceutically
effective amount of a compound of formula 1 in combination with a
pharmaceutically effective amount of another anti-HCV therapeutic
to the patient.
[0250] Another object of the present invention is to provide
pharmaceutical compositions comprising, in addition to one or more
HCV serine protease inhibitors, one or more interferons exhibiting
anti-HCV activity and/or one or more compounds having anti HCV
activity, including immunomodulatory compounds such as
immunostimulatory cytokines exhibiting HCV antiviral activity, and
a pharmaceutically acceptable carrier or diluent.
[0251] It is another object of the invention to provide a
pharmaceutical composition which is effective, in and of itself,
for utilization in a beneficial combination therapy because it
includes a plurality of active ingredients which may be utilized in
accordance with the invention.
[0252] The invention also provides kits or single packages
combining two or more active ingredients useful in treating or
preventing an HCV infection in a patient. A kit may provide (alone
or in combination with a pharmaceutically acceptable diluent or
carrier), the compound of formula 1 and the additional active
ingredient (alone or in combination with diluent or carrier) other
anti-HCV therapeutic.
[0253] Compounds of formula 1 may be prepared by the application or
adaptation of known methods as used heretofore or described in the
literature, or by methods according to this invention herein.
[0254] Another object of the present invention is to provide
methods of treating or preventing a HCV infection in a patient in
need thereof, comprising administering to said patient a
pharmaceutically effective amount of a combination of one or more
HCV serine protease inhibitors; one or more interferons exhibiting
anti-HCV activity; and/or one or more compounds having anti-HCV
activity, including immunomodulatory compounds such as
immunostimulatory cytokines exhibiting HCV antiviral activity.
[0255] Another object of the present invention is the use of one or
more HCV serine protease inhibitors in combination with one or more
interferons exhibiting anti-HCV activity and/or one or more
compounds having anti-HCV activity, including immunomodulatory
compounds such as immunostimulatory cytokines exhibiting HCV
antiviral activity, to prepare a medicament for treating or
preventing a HCV infection in a patient in need thereof.
[0256] A further object of the present invention is a kit or
pharmaceutical pack for treating or preventing HCV infection in a
patient, wherein the kit or pharmaceutical pack comprises a
plurality of separate containers, wherein at least one of said
containers contains one or more HCV serine protease inhibitors, at
least another of said containers contains one or more interferons
or compounds that induce the production of an interferon that
exhibit anti-HCV activity (alone or in combination with a
pharmaceutically acceptable carrier or diluent), and, optionally,
at least another of said containers contains one or more compounds
having anti-HCV activity (alone or in combination with a
pharmaceutically acceptable carrier or diluent), including
immunomodulatory compounds such as immunostimulatory cytokines
exhibiting HCV antiviral activity.
[0257] Yet another object of the present invention is to provide a
method of inhibiting hepatitis C virus replication in a cell,
comprising contacting said cell, a hepatitis C virus serine
protease inhibitor, and optionally an interferon or compounds that
induce the production of an interferon that have anti-hepatitis C
virus activity.
[0258] The amount of the HCV serine protease inhibitor(s),
interferon(s), or anti-HCV compound(s) in any of the foregoing
applications can be a pharmaceutically effective amount, a
suboptimal anti-HCV effective amount, or combinations thereof, so
long as the final combination of HCV protease inhibitor(s),
interferon(s), and/or anti-HCV compound(s) comprises a
pharmaceutically effective amount of compounds that is effective in
treating or preventing HCV infection in a patient.
[0259] It is a further object of the invention to provide a method
for preparing a chiral bicycloprolinate compound that is useful in
preparing the compound of formula 1.
Preparation of Compounds of the Invention
[0260] The starting materials and intermediates of compounds of the
invention may be prepared by the application or adaptation of known
methods, for example methods as described in the Reference Examples
or their obvious chemical equivalents.
[0261] Compounds of the invention may be prepared by the
application or adaptation of known methods, by which is meant
methods used heretofore or described in the literature, for example
those described by R. C. Larock in Comprehensive Organic
Transformations, VCH publishers (1989).
[0262] A compound of formula 1, wherein the variables and
##STR00109##
moiety thereof are as described herein, may be prepared by treating
a compound of formula 2, wherein the variables
##STR00110##
and
##STR00111##
moiety thereof are as described herein, with an appropriate
oxidizing agent and under appropriate conditions. A particular
oxidizing agent is DMP reagent. Particular conditions include
carrying out the oxidation in an appropriate organic solvent such
as dichloromethane at about room temperature.
[0263] A compound of formula 2, wherein the variables and
##STR00112##
moiety thereof are as described herein, may be prepared by coupling
a compound of formula 3, wherein the variables and
##STR00113##
moiety thereof are as described herein, and a compound of formula
4, wherein the
##STR00114##
variables thereof are as described herein, with an appropriate
coupling agent and under appropriate conditions. Particular
coupling agent and conditions include using DIC and HOAt in an
appropriate organic solvent such as DMF at about 0.degree. C. or
using PyBop and DIPEA in an appropriate organic solvent such as
dichloromethane at about room temperature.
[0264] A compound of formula 3, wherein the variables and
##STR00115##
moiety thereof are as described herein, may be prepared by coupling
a compound of formula 5, wherein the variables thereof are as
described herein, and a compound of formula 6, wherein P.sup.2 is
an acid
##STR00116##
protecting group and the
##STR00117##
moiety thereof is as described herein, with an appropriate coupling
agent and under appropriate coupling conditions, followed by an
appropriate deprotecting agent and under appropriate deprotecting
conditions. Particular coupling agent and conditions include using
DIC or DCC and HOAt in an appropriate organic solvent such as DMF
or dichloromethane at about 0.degree. C. to about room temperature.
The deprotecting is carried out using an appropriate deprotecting
agent that depends on the nature of the protecting agent, i.e.,
whether it is removable (labile) under acid, base, or hydrogenation
conditions, and other reactive moieties in the compound undergoing
deprotection, i.e., a deprotecting agent is chosen to carry out the
deprotection without effecting the other reactive moieties unless a
concomitant reaction is desired. A particular acid protecting agent
is C.sub.1 to C.sub.8 lower alkyl; more particular methyl. A
particular deprotecting agent is an inorganic base such as an
alkali hydroxide; more particular NaOH. Particular deprotection
conditions encompass carrying out the deprotection in an alcoholic
solvent such as methanol or ethanol at about room temperature.
[0265] A compound of formula 5, wherein n is 0 and the other
variables are as described herein, i.e., compound 5a, may be
prepared by deprotecting a compound of formula 7,
##STR00118##
wherein P.sup.2 is an acid protecting group and the other variables
thereof are as described herein, with an appropriate deprotecting
agent and under appropriate conditions. The deprotecting is carried
out using an appropriate deprotecting agent that depends on the
nature of the protecting agent, i.e., whether it is removeable
(labile) under acid, base, or hydrogenation conditions, and other
reactive moieties in the compound undergoing deprotection, i.e., a
deprotecting agent is chosen to carry out the deprotection without
effecting the other reactive moieties unless a concomitant reaction
is desired. A particular acid protecting agent is C.sub.1 to
C.sub.8 lower alkyl; more particular methyl. A particular
deprotecting agent is an inorganic base such as an alkali
hydroxide; more particular NaOH. Particular deprotection conditions
encompass carrying out the deprotection in an alcoholic solvent
such as methanol or ethanol at about room temperature.
[0266] A compound of formula 7, wherein the variables thereof are
as described herein, may be prepared by acylating a compound of
formula 8, wherein the variables thereof are as described herein,
with a compound of formula 9, wherein M is a displaceable moiety
and the other variables
##STR00119##
thereof are as described herein, under appropriate conditions.
Particular coupling conditions use DIC or DCC and HOAt in an
appropriate organic solvent such as DMF or dichloromethane at about
0.degree. C. to about room temperature, or PyBop and DIPEA in an
appropriate organic solvent such as DMF or dichloromethane at about
room temperature; and preferably the latter conditions. A
particular L is carbonyl. A particular M is hydroxy or
N-oxysuccinimide.
[0267] A compound of formula 5, wherein n is 1 and the other
variables are as described herein, i.e., compound 5b, may be
prepared by deprotecting a compound of formula 10, wherein P.sup.2
is an
##STR00120##
acid protecting group and the other variables thereof are as
described herein, with an appropriate deprotecting agent and under
appropriate conditions. The deprotecting is carried out using an
appropriate deprotecting agent that depends on the nature of the
acid protecting agent. i.e., whether it is removeable (labile)
under acid, base, or hydrogenation conditions, and other reactive
moieties in the compound undergoing deprotection, i.e., a
deprotecting agent is chosen to carry out the deprotection without
effecting the other reactive moieties unless a concomitant reaction
is desired. A particular acid protecting agent is C.sub.1 to
C.sub.8 lower alkyl; more particular methyl. A particular
deprotecting agent is an inorganic base such as an alkali
hydroxide; more particular NaOH. Particular deprotection conditions
encompass carrying out the deprotection in an alcoholic solvent
such as methanol or ethanol at about room temperature.
[0268] A compound of formula 10, wherein the variables thereof are
as described herein, may be prepared by acylating a compound of
formula 11, wherein the variables thereof are as described herein,
with a compound of formula 9, wherein the variables thereof are as
described herein,
##STR00121##
under appropriate conditions. Particular coupling conditions use
DIC or DCC and HOAt in an appropriate organic solvent such as DMF
or dichloromethane at about 0.degree. C. to about room temperature,
or PyBop and DIPEA in an appropriate organic solvent such as DMF or
dichloromethane at about room temperature; and preferably the
latter conditions. A particular L is carbonyl. A particular M is
hydroxy or N-oxysuccinimide.
[0269] A compound of formula 11, wherein the variables are as
described herein, may be prepared by deprotecting a compound of
formula 12, wherein P.sup.1 is an amine protecting group
##STR00122##
and the other variables thereof are as described herein, with an
appropriate deprotecting agent and under appropriate conditions.
The deprotecting is carried out using an appropriate deprotecting
agent that depends on the nature of the amine protecting agent,
i.e., whether it is removeable (labile) under acid, base, or
hydrogenation conditions, and other reactive moieties in the
compound undergoing deprotection, i.e., a deprotecting agent is
chosen to carry out the deprotection without effecting the other
reactive moieties unless a concomitant reaction is desired. A
particular amine protecting agent is Cbz or BOC; more particular
Cbz. A particular deprotecting agent is acid such as HCl or
H.sub.2/Pd(OH).sub.2; more particular H.sub.2/Pd(OH).sub.2.
Particular deprotection conditions encompass carrying out the
deprotection in an alcoholic solvent such as methanol or ethanol or
an alkyl alkanoate solvent such as ethyl acetate at about room
temperature.
[0270] A compound of formula 12, wherein the variables thereof are
as described herein, may be prepared by coupling a compound of
formula 13, wherein the variables thereof are as described herein,
with a compound of formula 14, wherein the variables thereof are as
described herein,
##STR00123##
under appropriate conditions. Particular coupling conditions use
HOAt/DIC and DIPEA in an appropriate organic solvent such as THF at
about room temperature.
[0271] A compound of formula 4, wherein the variables are as
described herein, may be prepared by deprotecting a compound of
formula 15, wherein the variables thereof are
##STR00124##
as described herein, with an appropriate deprotecting agent and
under appropriate conditions. The deprotecting is carried out using
an appropriate deprotecting agent that depends on the nature of the
amine protecting agent, i.e., whether it is removeable (labile)
under acid, base, or hydrogenation conditions, and other reactive
moieties in the compound undergoing deprotection, i.e., a
deprotecting agent is chosen to carry out the deprotection without
effecting the other reactive moieties unless a concomitant reaction
is desired. A particular amine protecting agent is Cbz or BOC; more
particular Cbz. A particular deprotecting agent is an acid such as
HCl or H.sub.2/Pd(OH).sub.2; more particular H.sub.2/Pd(OH).sub.2.
Particular deprotection conditions encompass carrying out the
deprotection in an alcoholic solvent such as methanol or ethanol or
an alkyl alkanoate solvent such as ethyl acetate at about room
temperature.
[0272] A compound of formula 15, wherein the variables thereof are
as described herein, may be prepared by coupling a compound of
formula 16, wherein the variables thereof are as described herein,
with a compound of formula 17, wherein the variables thereof are as
described herein.
##STR00125##
under appropriate conditions. A particular amine protecting agent
is Cbz or BOC; more particular Cbz. Particular coupling conditions
use HOBT, PyBop and DIPEA in an appropriate organic solvent such as
dichloromethane at about 0.degree. C. to about room
temperature.
[0273] A compound of formula 16, wherein the variables are as
described herein may be prepared by deprotecting a compound of
formula 18, wherein the other variables thereof are
##STR00126##
as described herein, with an appropriate deprotecting agent and
under appropriate conditions. The deprotecting is carried out using
an appropriate deprotecting agent that depends on the nature of the
acid protecting agent, i.e., whether it is removeable (labile)
under acid, base, or hydrogenation conditions, and other reactive
moieties in the compound undergoing deprotection, i.e., a
deprotecting agent is chosen to carry out the deprotection without
effecting the other reactive moieties unless a concomitant reaction
is desired. A particular amine protecting agent is Cbz. A
particular acid protecting agent is C.sub.1 to C.sub.8 lower alkyl;
more particular methyl. A particular deprotecting agent is an
inorganic base such as an alkali hydroxide; more particular NaOH.
Particular deprotection conditions encompass carrying out the
deprotection in an alcoholic solvent such as methanol or ethanol at
about room temperature.
[0274] A compound of formula 18, wherein R.sup.0 is a bond and the
other variables thereof are as described herein, may be prepared by
protecting a compound of formula 20, wherein the variables thereof
are as described herein, with a compound of formula 19, wherein
the
##STR00127##
variables thereof are as described herein, under appropriate
conditions. A particular amine protecting agent is Cbz or BOC.
Particular coupling conditions use an appropriate organic solvent
such as dichloromethane at about 0.degree. C. to about room
temperature.
[0275] A compound of formula 20, wherein R.sup.4 is hydrogen and
the other variables are as described herein, may be prepared by
hydrogenating a compound of formula 21, wherein the
##STR00128##
variables thereof are as described herein, with an appropriate
hydrogenating agent and under appropriate conditions. A particular
hydrogenating agent is H.sub.2/Pd(OH).sub.2. Particular
hydrogenating conditions encompass carrying out the hydrogenation
in an alcoholic solvent such as methanol or ethanol or an alkyl
alkanoate solvent such as ethyl acetate at about room
temperature.
[0276] A compound of formula 20 wherein R.sup.4 is optionally
substituted aliphatic and the other variables are as described
herein may be prepared by alkylating compound 20' wherein the
variables are as described herein with compound 22 (alkylating
agent) wherein R.sup.4 is optionally substituted aliphatic and Q is
a displaceable group such as a halides, tosylates or sulfonates,
under appropriate conditions.
##STR00129##
Appropriate alkylating agents include aliphatic (halides, tosylates
or sulfonates). Appropriate alkylating conditions encompass
carrying out to alkylation in an appropriate organic solvent such
as an alcoholic solvent, e.g., methanol or ethanol, or etheric
solvent, e.g., ether or tetrahydrofuran at about room temperature
to about reflux.
[0277] A compound of formula 21, wherein the variables are as
described herein, may be prepared by alkylating a compound of
formula 22, wherein the variable thereof is as described herein,
with a compound of formula 23, wherein the R.sup.3's independently
are optionally
##STR00130##
substituted aliphatic group, optionally substituted cyclic group or
optionally substituted aromatic group as described herein, under
appropriate conditions. Particular alkylating conditions encompass
carrying out the alkylation using a strong base such as potassium
t-butoxide in an alcoholic solvent such as methanol or ethanol at
about room temperature.
[0278] A compound of formula 24 wherein the variables thereof are
as described herein, may be prepared by effecting a Michael
addition on a Michael acceptor of formula 29, wherein the variable
thereof is as described herein, with an iminic glycinimide
derivative.
##STR00131##
Michael additions comprise appropriate aprotic polar solvents,
alkali methyl hydroxide bases, and appropriate temperatures. For
Michael additions, see Corey, E. J.; Noe, M. C.; Xu, F. Tetrahedron
Letter 1998, 39, 5347. For the synthesis of chiral phase transfer
catalysts, see Corey, E. J.; Noe, M. C.; Xu, F. J. Am. Chem. Soc.
1997, 119, 12414. Appropriate solvents include DCM, ACN, or THF
depending on the reaction conditions. Appropriate bases include
CsOH, NaOH, KOH, and LiOH. Appropriate temperatures range from
about -78.degree. C. to about 0.degree. C., more particularly at
about -60.degree. C. Iminic glycinimides useful in the invention
are described herein. A preferred iminic glycinimide is
N-(diphenylmethylene)glycine tert-butyl ester. In addition, Michael
addition conditions may be affected with or without a phase
transfer catalyst (PTC) (chiral and nonchiral). A preferred PTC is
O-[9]allyl-N-9-anthracenylmethylcinchonidium bromide.
[0279] A compound of formula 25, wherein the variables thereof are
as described herein, may be prepared by imine cleavage and
cyclizing of the compound of formula 24.
##STR00132##
For cleavage and cyclization procedures, see Javidan, A.; Schfer,
K.; Pyne, S. Synlett 1996, 100; Tatsukawa, A.; Dan, M.; Ohbatake,
M.; Kawatake, K.; Fukata, T.; Wada, E.; Kanemase, S.; Kakei, S. J.
Org. Chem. 1993, 58, 4221. Cleavage and cyclizing conditions
include the use of polar solvents, add reagents, and temperatures
of about room temperature to about 150.degree. C. Preferred
conditions include the use of EtOH, AcONa and NH.sub.2OH.HCl, and a
temperature of about boiling point for the solvent used.
[0280] A compound of formula 26, wherein the variables thereof are
as described herein, may be prepared by protecting the amide of the
compound of formula 25, wherein the variables thereof are as
described herein, with a suitable amide protecting group such as
BOC. Other suitable protecting groups include CBz, --CO.sub.2alkyl.
Also see, Greene, T. W.; P. G. M. in Protective Groups in Organic
Synthesis, Wiley, New York, 1991 for other amine protecting groups.
Protecting conditions include the use of aprotic polar solvents,
organic bases as catalysts, and temperatures of about 0.degree.
C.-100.degree. C. Preferred conditions include the use of ACN,
dimethyl amino pyridine, and a temperature of about room
temperature.
##STR00133##
[0281] A compound of formula 27, wherein the variables thereof are
as described herein, may be prepared by reducing the protected
compound of formula 26, wherein the variables thereof are as
described herein.
##STR00134##
Two reductions are, in fact, done. The first reduction is of the
amide to a hemiaminal using DIBALH or superhydride [LiBEt.sub.3H].
The second reduction is of the hemiaminal to the amine using
Et.sub.3SiH and BF.sub.3.OEt.sub.2. See Collado, J.; Ezquerra, J.;
Mateo, A. I.; Rubio, A., J. Org. Chem. 1998, 63 1995-2001 and
Ezqueera, J.; Pedregal, C.; Yruretagoyena, B.; Rubio, A.; Carreno,
M. C.; Escribano, A.; Garcia Ruano, J. L. J. Org. Chem. 1995,
60,2925 for reducing conditions. Other usual conditions for
converting pyroglutamates into pyrrolidines is the use of
BH.sub.3.SMe.sub.2.
[0282] A compound of formula 28, wherein the variables thereof are
as described herein, may be prepared by deprotecting the compound
of formula 27, wherein the variables thereof are as described
herein.
##STR00135##
See Gibson, F. G.; Bermeier, S. C.; Rapoport, H., J. Org Chem.
1994, 59, 3216-3218 for the conditions for selective removal of
N--BOC protecting group in the presence of tert-butyl ester. One
skilled in the art would know that the deprotecting conditions will
be dependent upon the choice of the protecting group. For example,
if CBz is used, hydrogenation or basic conditions may be used.
Preferably, if BOC is used, 1 N HCl in ethyl acetate may be used.
See, Greene, T. W.; P. G. M. in Protective Groups in Organic
Synthesis, Wiley, New York. 1991.
[0283] The person of ordinary skill in the art will appreciate that
a compound of formula 3 may be prepared by coupling a compound of
formula 5 with a compound of formula 28 under conditions described
above herein.
[0284] Methods for preparing R.sup.3, R.sup.5 or R.sup.7 as
optionally substituted ethanediyl moieties include those known to
those skilled in the art, e.g., those methods described in "The
organic Chemistry of .beta.-Lactams" edited by G. Georg, VCH
Publishers, Inc. (1993), e.g., pages 240-241 and 303-305.
[0285] Schemes 1-11 that follow exemplify assorted methods for
preparing an optionally substituted multicyclic azaheterocyclyl.
The methods in the schemes below are also applicable to other
optionally substituted multicyclic azaheterocyclyls comprising
compatible like substituents.
##STR00136##
##STR00137##
##STR00138##
##STR00139##
##STR00140##
##STR00141##
##STR00142##
##STR00143## ##STR00144##
[0286] A compound of formula 1 including a group containing one or
more nitrogen ring atoms, preferably imine (.dbd.N--), may be
converted to the corresponding compound wherein one or more
nitrogen ring atoms of the group are oxidized to an N-oxide,
preferably by reacting with a peracid, for example peracetic acid
in acetic acid or m-chloroperoxybenzoic acid in an inert solvent
such as dichloromethane, at a temperature from about room
temperature to reflux, preferably at elevated temperature.
[0287] In the reactions described hereinafter it may be necessary
to protect reactive functional groups, for example hydroxy, amino,
imino, thio or carboxy groups, where these are desired in the final
product, to avoid their unwanted participation in the reactions.
Conventional protecting groups may be used in accordance with
standard practice, for examples see T. W. Green and P. G. M. Wuts
in "Protective Groups in Organic Chemistry" John Wiley and Sons
(1991); J. F. W. McOmie in "Protective Groups in Organic Chemistry"
Plenum Press, 1973.
[0288] A compound that is prepared as described herein may be
recovered from the reaction mixture by conventional means. For
example, the compounds may be recovered by distilling off the
solvent from the reaction mixture or, if necessary after distilling
off the solvent from the reaction mixture, pouring the residue into
water followed by extraction with a water-immiscible organic
solvent and distilling off the solvent from the extract.
Additionally, the product can, if desired, be further purified by
various well techniques, such as recrystallization, reprecipitation
or the various chromatography techniques, notably column
chromatography or preparative thin layer chromatography.
[0289] According to a further feature of the present invention,
compounds of the invention may be prepared by interconversion of
other compounds of the invention.
[0290] As an example of the interconversion process, compounds of
formula 1 containing sulphoxide linkages may be prepared by the
oxidation of corresponding compounds containing --S-- linkages. For
example, the oxidation may conveniently be carried out by means of
reaction with a peroxyacid, e.g., 3-chloroperbenzoic acid,
preferably in an inert solvent, e.g., dichloromethane, preferably
at or near room temperature, or alternatively by means of potassium
hydrogen peroxomonosulphate in a medium such as aqueous methanol,
buffered to about pH 5, at temperatures between about 0.degree. C.
and room temperature. This latter method is preferred for compounds
containing an acid-labile group.
[0291] As another example of the interconversion process, compounds
of formula 1 containing sulphone linkages may be prepared by the
oxidation of corresponding compounds containing --S-- or sulphoxide
linkages. For example, the oxidation may conveniently be carried
out by means of reaction with a peroxyacid, e.g.,
3-chloroperbenzoic acid, preferably in an inert solvent, e.g.,
dichloromethane, preferably at or near room temperature.
[0292] It will be understood that designation of aromaticity with
respect to aryls and heteroaryls herein includes any highly
resonant unsaturated ring structure. Alternatively, placement of
double bonds, where indicated, represents one potential structure
for the depicted compound but will be understood to include other
resonant states of the compound as well as protonated and charged
species, only one of which may be shown.
[0293] It will be appreciated that compounds of the present
invention may contain asymmetric centers. These asymmetric centers
may independently be in either the R or S configuration. It will be
apparent to those skilled in the art that certain compounds of the
invention may also exhibit geometrical isomerism. It is to be
understood that the present invention includes individual
geometrical isomers and stereoisomers and mixtures thereof,
including racemic mixtures, of compounds according to the
invention. Such isomers can be separated from their mixtures, by
the application or adaptation of known methods, for example
chromatographic techniques and recrystallization techniques, or
they are separately prepared from the appropriate isomers of their
intermediates.
[0294] For the purpose herein it is understood that tautermeric
forms are included in the recitation of a given group, e.g.,
thioxo/mercapto or oxo/hydroxyl.
[0295] Acid additional salts are formed with the compounds of the
invention in which a basic function such as an amino, alkylamino,
or dialkylamino group is present. The pharmaceutically acceptable,
i.e., nontoxic, acid addition salts are preferred. The salts chosen
are chosen optimally to be compatible with the customary
pharmaceutical vehicles and adapted for oral or parenteral
administration. Acid addition salts of the compounds of this
invention may be prepared by reaction of the free base with the
appropriate acid, by the application or adaptation of known
methods. For example, the acid addition salts of the compounds of
this invention may be prepared either by dissolving the free base
in water or aqueous alcohol solution or other suitable solvents
containing the appropriate acid and isolating the salt by
evaporating the solution, or by reacting the free base and acid in
an organic solvent, in which case the salt separates directly or
can be obtained by concentration of the solution. Some suitable
acids for use in the preparation of such salts are hydrochloric
acid, hydrobromic acid, phosphoric acid, sulfuric acid, various
organic carboxylic and sulfonic acids, such as acetic acid, citric
acid, propionic acid, succinic acid, benzoic acid, tartaric acid,
fumaric acid, mandelic acid, ascorbic acid, malic acid,
methanesulfonic acid, toluenesulfonic acid, fatty acids, adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
cyclopentanepropionate, digluconate, dodecylsulfate, bisulfate,
butyrate, lactate, laurate, lauryl sulfate, malate, hydroiodide,
2-hydroxy-ethanesulfonate, glycerophosphate, picrate, pivalate,
pamoate, pectinate, persutfate, 3-phenylpropionate, thiocyanate,
2-naphthalenesulfonate, undecanoate, nicotinate, hemisulfate,
heptonate, hexanoate, camphorate, camphersulfonate, and others.
[0296] The acid addition salts of the compounds of this invention
can be regenerated from the salts by the application or adaptation
of known methods. For example, parent compounds of the invention
can be regenerated from their acid addition salts by treatment with
an alkali, e.g., aqueous sodium bicarbonate solution or aqueous
ammonia solution.
[0297] Compounds of this invention can be regenerated from their
base addition salts by the application or adaptation of known
methods. For example, parent compounds of the invention can be
regenerated from their base addition salts by treatment with an
acid, e.g., hydrochloric acid.
[0298] Base addition salts may be formed where the compound of the
invention contains a carboxy group, or a sufficiently acidic
bioisostere. The bases which can be used to prepare the base
addition salts include preferably those which produce, when
combined with the free acid, pharmaceutically acceptable salts,
that is, salts whose cations are non-toxic to the patient in
pharmaceutical doses of the salts, so that the beneficial
inhibitory effects inherent in the free base are not vitiated by
side effects ascribable to the cations. Pharmaceutically acceptable
salts, including those derived from alkali and alkaline earth metal
salts, within the scope of the invention include those derived from
the following bases: sodium hydride, sodium hydroxide, potassium
hydroxide, calcium hydroxide, aluminum hydroxide, lithium
hydroxide, magnesium hydroxide, zinc hydroxide, ammonia,
ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine,
choline, N,N'-dibenzylethylenediamine, chloroprocaine,
diethanolamine, procaine, N-benzylphenethylamine, diethylamine,
piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium
hydroxide, and the like.
[0299] Compounds of the present invention may be conveniently
prepared, or formed during the process of the invention, as
solvates (e.g., hydrates). Hydrates of compounds of the present
invention may be conveniently prepared by recrystallization from an
aqueous/organic solvent mixture, using organic solvents such as
dioxan, tetrahydrofuran or methanol.
[0300] The starting materials and intermediates may be prepared by
the application or adaptation of known methods, for example methods
as described in the Reference Examples or their obvious chemical
equivalents.
[0301] The compounds of the invention, their methods or preparation
and their biological activity will appear more clearly from the
examination of the following examples which are presented as an
illustration only and are not to be considered as limiting the
invention in its scope.
[0302] Samples were analyzed by TLC, NMR, RP-HPLC or EA.
The compounds of the invention, their methods or preparation and
their biological activity will appear more clearly from the
examination of the following examples which are presented as an
illustration only and are not to be considered as limiting the
invention in its scope.
[0303] Samples were analyzed by TLC, NMR, RP-HPLC or EA.
Example 1
Compounds A-E
[0304] Toa DCM solution (10 mL) of compound xi (310 mg, 0.39 mmol)
is added TFA (4 mL). The reaction is stirred at about room
temperature for 5 hours. At this point, the solvent is removed in
vacuo. The resulting residue is purified by reverse phase HPLC to
give 195 mg (68%) of compound A,
##STR00145##
[0305] Following the above method and using the appropriate
starting materials, the following consecutive compounds B-E are
prepared:
##STR00146##
Example 2
Compounds F-M
[0306] To a DCM solution (10 mL) of compound xii (350 mg, 0.56
mmol) is added DMP reagent (307 mg, 0.73 mmol). The reaction is
stirred at about room temperature for 2 hours and then quenched
with 10% Na.sub.2SO.sub.3 for 30 minutes. The reaction mixture is
then extracted with EtOAc (75 mL) and washed with brine. The
organic layer is dried and concentrated in vacuo. The resulting
residue is purified with silica gel chromatography (80-90%
EtOAc/Hexanes) to give 248 mg (71%) of compound F
##STR00147##
[0307] Following the above method and using the appropriate
starting materials, the following consecutive compounds G-M are
prepared:
##STR00148## ##STR00149##
Example 3
Compounds N-R
[0308] To a DCM solution (4 mL) of compound xix (.about.0.22 mmol)
is added DMP reagent (146 mg, 0.34 mmol). After stirring at about
room temperature for 2 hours, the reaction is quenched with 10%
Na.sub.3SO.sub.3. The reaction mixture is then diluted with DCM.
The organic layer is separated and washed with 10% Na.sub.2SO.sub.3
twice and brine. The resulting organic layer is dried and
concentrated in vacuo to give a residue, which is purified by
silica gel chromatography (5% EtOH/EtOAc) to provide 78 mg (56%) of
the desired compound N
##STR00150##
[0309] Following the above method and using the appropriate
starting materials, the following consecutive compounds O-R are
prepared:
##STR00151##
Example 4
Compounds S-W
[0310] To a DCM solution (10 mL) of compound xxv (320 mg, 0.5 mmol)
is added DMP reagent reagent (272 mg, 0.65 mmol). The reaction is
stirred at about room temperature for 2 hours and quenched with 10%
Na.sub.2SO.sub.3 for 20 minutes. The resulting mixture is then
extracted with EtOAc. The organic layer is washed with brine, dried
and concentrated in vacuo. The resulting residue is purified by
silica gel chromatography (80% EtOAc/Hexanes) to give 170 mg (53%)
of compound S,
##STR00152##
[0311] Following the above method and using the appropriate
starting materials, the following consecutive compounds T-W are
prepared:
##STR00153##
Example 5
Compounds X-AD
[0312] To a DCM solution (20 mL) of compound xxvi (400 mg. 0.6
mmol) is added DMP reagent (329 mg, 0.78 mmol). The reaction is
stirred at about room temperature for 1.5 hours and quenched with
10% Na.sub.2SO.sub.3 for 20 minutes. The resulting mixture is then
extracted with EtOAc. The organic layer is washed with brine, dried
and concentrated in vacuo. The resulting residue is purified by
silica gel chromatography (70-100% EtOAc/Hexanes) to give 210 mg
(53%) of compound X,
##STR00154##
[0313] Following the above method and using the appropriate
starting materials, the following consecutive compounds Y-AD are
prepared:
##STR00155##
Example 6
Compounds AE-AI
[0314] Compound xxxiii (150 mg; 0.076 mmol) is dissolved in 5 mL
TFA and stirred for two days. The product is purified by RP-HPLC to
yield 40 mg (33% yield) of compound AE,
##STR00156##
[0315] Following the above method and using the appropriate
starting materials, the following consecutive compounds AF-AI are
prepared:
##STR00157##
Example 7
Compound AJ
[0316] Compound xxxviii (180 mg, 0.21 mmol) is dissolved in neat
TFA (5 mL) and left for 3 day at about room temperature. At this
point, the reaction mixture is concentrated in vacuo to give a
residue, which is purified by reverse phase HPLC to give 50 mg
(32%) of the compound AJ.
##STR00158##
Example 8
Compounds AK-AM
[0317] Compound xxxxiii (150 mg; 0.16 mmol) is dissolved in 4.5 mL
TFA and stirred for three days. The product is purified by RP-HPLC
to yield 70 mg (54% yield) compound AK.
##STR00159##
[0318] Following the above method and using the appropriate
starting materials, the following consecutive compounds AL-AM are
prepared:
##STR00160##
Example 9
Compounds AN
[0319] Compound lii (80 mg) is dissolved in 3 mL TFA and 3 mL DCM.
The mixture is stirred at about room temperature for 5 hours. The
solvent is removed by evaporation. The resulting residue is
purified by HPLC to yield 62 mg (83%) of compound AN,
##STR00161##
Example 10
Compounds AO
[0320] Compound liii (160 mg; 0.2 mmol) is dissolved in 5 mL DCM
and DMP reagent (170 mg; 0.4 mmol) is added. The mixture is stirred
at about room temperature for three hours. The solvent is removed
by evaporation and the residue is dissolved in 50%
acetonitrile/water and purified by RP-HPLC to yield 51 mg (32%) of
compound AO,
##STR00162##
Example 11
Compounds AP
[0321] Compound lix (162 mg; 0.22 mmol) is dissolved in 8 mL of DCM
and DMP reagent reagent (189 mg; 0.44 mmol) is added. The mixture
is stirred at about room temperature for 3 hours. The solvent is
removed by evaporation and product is purified by RP-HPLC to yield
41 mg (25%) of compound AP,
##STR00163##
Example 12
Compounds AQ
[0322] Compound lx (70 mg; 0.09 mmol) is dissolved in 5 mL TFA and
5 mL DCM. The mixture is stirred at about room temperature for 3
hours. The solvent is removed by vacuum and the residue is
dissolved in 50% acetonitrile/water and lyophilized to yield
compound AQ as a powder,
##STR00164##
Example 13
Compounds AR-BG
[0323] Compound lxvi (223 mg, 0.326 mmol) is stirred in a solution
of TFA (5 mL) and DCM (5 mL) for 4 hours. TLC (silica gel: 2%
MeOH/EtOAc) showed complete conversion to the slower product. The
solvent is removed under reduced pressure and the product
lyophilized to give 198 mg (97%) compound AR,
##STR00165##
[0324] Following the above method and using the appropriate
starting materials, the following consecutive compounds AS-BG are
prepared:
##STR00166## ##STR00167## ##STR00168##
Example 14
Compounds BH-BS
[0325] Compound lxxiii (150 mg, 0.15 mmol) is taken up in DCM (3
mL). To this solution is added TFA (1.5 mL). The resulting solution
is stirred overnight. At this point, the reaction is concentrated
in vacuo to give a residue. The residue is purified by reverse
phase HPLC and lyophilized to give 60 mg (50%) of compound BH,
##STR00169##
[0326] Following the above method and using the appropriate
starting materials, the following consecutive compounds BI-BS are
prepared:
##STR00170## ##STR00171##
Example 15
Compounds BT-BU
[0327] Following the method of Example 12 and using the appropriate
starting materials, the following consecutive compounds BT-BU are
prepared:
##STR00172##
Example 16
Compound BV
[0328] To a dichloromethane solution (4.2 mL) of compound lxxvii
(143 mg, 0.21 mmol) is added DMP reagent (165 mg, 0.39 mmol). The
reaction is stirred at about room temperature for 2 hours and
quenched with 10% Na.sub.2SO.sub.3 (aq.) for 20 minutes. The
resulting mixture is extracted with EtOAc. The organic layer is
washed with brine, dried over MgSO.sub.4 and concentrated to a
yellow oil. Purification by silica get chromatography (5%
EtOH/EtOAc) yielded 124 mg (79%) of compound BV,
##STR00173##
Example 17
Compounds BW-CA
[0329] To a dichloromethane solution (20 mL) of compound lxxxix
(420 mg, 0.62 mmol) is added DMP reagent (342 mg, 0.81 mmol). The
reaction is stirred at about room temperature for 1 hour and
quenched with 10% Na.sub.2SO.sub.3 for 20 minutes. The resulting
mixture is then extracted with EtOAc. The organic layer is washed
with brine, dried and concentrated in vacuo. The resulting residue
is purified by silica gel chromatography (80% EtOAc/Hexanes) to
give 208 mg (50%) of compound BW,
##STR00174##
[0330] Following the above method but using the appropriate
starting materials, the following consecutive compounds BX-CA are
prepared:
##STR00175##
Example 18
Compounds CB-CC
[0331] To a dichloromethane solution (6.5 mL) of compound lxxxvii
(200 mg, 0.3 mmol) is added DMP reagent (227 mg, 0.54 mmol). The
reaction is stirred at about room temperature for 2 hours and
quenched with 10% Na.sub.2SO.sub.3 (aq.) for 20 minutes. The
resulting mixture is extracted with EtOAc. The organic layer is
washed with brine, dried over MgSO.sub.4 and concentrated to a
yellow oil. Purification by silica gel chromatography (5%
EtOH/EtOAc) yields 138 mg (70%) of compound CB,
##STR00176##
[0332] Following the above method but using the appropriate
starting materials, the following compound CC is prepared:
##STR00177##
Example 19
Compound CD
[0333] Compound lxxxxviii (40 mg, 0.05 mmol) is taken up in TFA (3
mL). The solution stirred over two nights and is concentrated. The
residue is purified on reverse phase HPLC to give 25 mg (74%) of
compound CD,
##STR00178##
Example 20
Compound CE
[0334] Following the method of Example 17 and using the appropriate
starting materials, the following compound CE is prepared:
##STR00179##
Example 21
Compounds CF-CG
[0335] Following the method of Example 14 and using the appropriate
starting materials, the following consecutive compounds CF-CG are
prepared:
##STR00180##
Example 22
Compound CH
[0336] Following the method of Example 16 and using the appropriate
starting materials, the following compound CH is prepared:
##STR00181##
Example 23
Compounds CI-CM
[0337] Compound cxi (490 mg. 0.75 mmol) is dissolved in DCM (6 mL).
DMP reagent (380 mg, 0.9 mmol) is added to this solution and
stirred 1 hour. The reaction mixture is quenched with a 10%
Na.sub.2SO.sub.3 solution, and then the organic phase is washed
with saturated NaHCO.sub.3 and brine. Following the concentration
of the organic phase, the resultant residue is chromatographically
purified by 70% EtOAc/hexanes to yield compound CI (325 mg,
66.4%).
##STR00182##
[0338] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following consecutive
compounds CJ-CM are prepared:
##STR00183##
Example 24
Compounds CN
[0339] To a DCM/THF solution (3 mL/3 mL) of compound cxviii (335
mg, 0.46 mmol) is added DMP reagent (300 mg, 0.69 mmol). The
reaction mixture is stirred at room temperature for 2 hours and
quenched with 10% Na.sub.2SO.sub.3(aq.) for 20 minutes. The
resulting mixture is extracted with EtOAc. The organic phase is
washed with brine, dried over MgSO.sub.4 and concentrated to yield
a yellow oil. Purification by silica gel chromatography (80%
EtOAc/hexanes) yields compound CN (220 mg, 67%).
##STR00184##
Example 25
Compounds CO-CR
[0340] To a DCM/THF solution (1.5 mL/1.5 mL) of compound cxix (164
mg, 0.25 mmol) is added DMP reagent (159 rag, 0.38 mmol). The
reaction mixture is stirred at room temperature for 2 hours and
quenched with 10% Na.sub.2SO.sub.3(aq.) for 20 minutes. The
resulting mixture is extracted with EtOAc. The organic phase is
washed with brine, dried over MgSO.sub.4 and concentrated to yellow
oil. Purification by silica gel chromatography (70% EtOAc/hexanes)
yields compound CO (100 mg, 61%).
##STR00185##
[0341] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following consecutive
compounds CP-CR are prepared:
##STR00186##
Example 26
Compounds CS-CT
[0342] Compound cxx is dissolved in DCM (3 mL). DMP reagent (180
mg. 0.41 mmol) is added to the solution, and then stirred for 1
hour. The reaction mixture is quenched with 10% Na.sub.2SO.sub.3,
and then the organic phase is washed with saturated NaHCO.sub.3 and
brine. Following the concentration of the organic phase, the
residue is chromatographically purified by 100% EtOAc to yield
compound CS (95 mg, 43.7%).
##STR00187##
[0343] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following compound CT
is prepared:
##STR00188##
Example 27
Compounds CU, EL, EK-EM, EO-EZ, and FA-FG
[0344] Compound cxxviii (356 mg, 0.52 mmol) is dissolved in DCM (5
mL). DMP reagent reagent (270 mg, 0.63 mmol) is added to this
solution and stirred 1 hour. The reaction mixture is quenched with
10% Na.sub.2SO.sub.3, and then the organic phase is separated and
washed with saturated NaHCO.sub.3 and brine. Following the
concentration of the organic solvent, the residue is
chromatographically purified by 100% EtOAc to yield compound CU
(200 mg, 56.3%). mp 225-235.degree. C.
##STR00189##
[0345] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following consecutive
compounds EI, EK-EM, EO-EZ and FA-FH are prepared:
##STR00190## ##STR00191## ##STR00192## ##STR00193##
Example 28
Compounds CV-DC
[0346] Compound cxxx (330 mg, 0.46 mmol) is dissolved in DCM (5
mL). DMP reagent reagent (240 mg, 0.56 mmol) is added to this
solution and stirred 1 hour. The reaction mixture is quenched with
a 10% Na.sub.2SO.sub.3, and the organic phase washed with saturated
NaHCO.sub.3 and brine.
[0347] Following the concentration of the organic phase, the
resulting residue is chromatographically purified by 100% EtOAc to
yield compound cxxx (280 mg, 85.9%).
##STR00194##
[0348] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following consecutive
compounds CW-DC are prepared:
##STR00195## ##STR00196##
Example 29
Compounds DD-DE
[0349] To a DCM solution (6 mL) of compound cxxxviii (400 mg, 0.57
mmol) is added DMP reagent (362 mg, 0.85 mmol). The reaction
mixture is stirred at room temperature for 2 hours and quenched
with 10% Na.sub.2SO.sub.3 (aq.) for 20 minutes. The resulting
mixture is extracted with EtOAc. The extracted organic phase is
washed with brine, dried over MgSO.sub.4 and concentrated to yield
yellow oil. Purification by silica gel (70% EtOAc/hexanes) yields
compound DD (201 mg, 51%).
##STR00197##
[0350] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following compound DE
is prepared:
##STR00198##
Example 30
Compound DF
[0351] Compound cxxxxiii (165 mg, 0.24 mmol) is dissolved in DCM (5
DMP reagent reagent (125 mg, 0.29 mmol) is added to the solution
and stirred 1 hour. The reaction mixture is quenched with a 10%
Na.sub.2SO.sub.3, and the organic phase washed with saturated
NaHCO.sub.3 and brine. Following the concentration of the organic
phase, the resultant residue is purified chromatographically by 70%
EtOAc/hexanes to yield compound DF (108 mg, 65.6%).
##STR00199##
Example 31
Compounds DG-DJ
[0352] To a solution of compound cil (0.350 g, 0.516 mmol) in DCM
(15 mL) cooled by an ice bath is added DMP reagent (0.281 g, 0.671
mmol). The mixture is stirred at room temperature for 2 hours, then
quenched with 10% Na.sub.2SO.sub.3 solution and stirred for 20
minutes. The resulting mixture is extracted with DCM (3.times.20
mL) and the organic extract is dried (MgSO.sub.4). After filtration
to remove MgSO.sub.4, the filtrate is concentrated and purified by
column chromatography (70% Ethyl acetate/Hexanes) to yield the
final compound DG (0.265 g, 76%) as white solid.
##STR00200##
[0353] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following consecutive
compounds DH-DJ are prepared:
##STR00201##
Example 32
Compounds DK-DN
[0354] A DCM solution of compound clx (108 mg, 0.123 mmol) is
treated with DMP reagent (78 mg, 0.185 mmol). After stirring at
room temperature for 1 hour, the reaction mixture is diluted with
EtOAc (50 mL), and then quenched with 10% Na.sub.2SO.sub.3. After
stirring for 30 minutes, the organic phase is separated and washed
with NaHCO.sub.3 and brine. The organic phase is dried and
concentrated in vacuo to give a residue that is purified by silica
gel chromatography (80% EtOAc/hexanes) to yield compound DK (84 mg,
78%).
##STR00202##
[0355] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following compounds
DL-DN are prepared.
##STR00203##
Example 33
Compounds DO-DS
[0356] An EtOH solution (10 mL) of compound clxii (174 mg, 0.189
mmol) is hydrogenated using Pd/C (30% eq., 60 mg, 10% palladium
content) for 2.5 hours. The catalyst is then filtered off. The
resulting filtrate is concentrated in vacuo to yield a residue that
is purified by semi-preparative reverse phase chromatography and
lyophilized to afford compound DO in 70% yield.
##STR00204##
[0357] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following consecutive
compounds DP-DS are prepared.
##STR00205##
Example 34
Compound CW
[0358] Compound clxiii (175 mg, 0.24 mmol) is taken up in DCM (3
mL). DMP reagent (120 mg, 0.28 mmol) is added to this solution and
stirred 1 hour. The reaction is quenched with a 10%
Na.sub.2SO.sub.3 and washed with saturated NaHCO.sub.3 and brine.
Purification by 70% EtOAc yields compound CW (134 mg, 75%).
Example 35
Compounds CY and DT-DX
[0359] To a DCM solution (15 mL) of clxxii (290 mg. 0.43 mmol) is
added DMP reagent (239 mg, 0.56 mmol). The reaction is stirred at
room temperature for 1 hour and quenched with 10% Na.sub.2SO.sub.3
for 20 minutes. The resulting mixture is then extracted with EtOAc.
The organic layer is washed with brine, dried and concentrated in
vacuo. The resulting residue is purified by silica gel
chromatography (8-100% EtOAc/Hexanes) to give compound CY (151 mg,
52%).
[0360] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following consecutive
compounds DT-DX are prepared.
##STR00206##
Example 36
Compounds DY
[0361] Compound lxxxv (1.17 mmol) is taken up in DCM (5 mL). DMP
reagent (545 mg. 1.3 mmol) is added to this solution and stirred 1
hour. The reaction is quenched with a P--Na.sub.2SO.sub.3 (1.5
mmol/g resin) and stirred one hour. P-TBD scavenger resin* (2.5
mmol/g resin) is added and stirred 45 minutes. The resulting
mixture is filtered and purified by 50% EtOAc to give compound DY
(440 mg, 50.2% over two steps). *Reference for P-TBD scavenger
resin: J. Parlow et al. Tetrahedron. 55, 6785-6796 (1999).
##STR00207##
Example 37
Compound DZ
[0362] The starting material compound clxxxxi (94 mg, 0.14 mmole)
is dissolved in a mixture of THF (10 mL) and DCM (20 mL). The DMP
reagent (118 mg, 0.28 mmol) is then added. After stirring at room
temperature for 2 hours. The reaction is dumped in a separatory
funnel containing Dri Solv THF (120 mL). The reaction is washed
with 10% Na.sub.2SO.sub.3 (50 mL), and then brine (75 mL). The
organic layer is then separated, dried over MgSO.sub.4 and the
solvent removed under reduced pressure. After chromatography
(silica gel: elution with 50% Dri Solv THF/EtOAc, and then 4%
MeOH/THF). Fractions are checked by MS. Appropriate fractions are
lyopholized to yield compound DZ (38.8 mg, 41%).
##STR00208##
Example 38
Compounds EA-EB
[0363] The starting compound clxxxxv (185 mg, 0.26 mmol) is
dissolved in THF (20 mL). The DMP reagent (219 mg, 0.52 mmol) is
then added. After stirring at room temperature for 1 hour. TLC
shows complete conversion to ketone (5% MeOH/THF). The reaction is
dumped in a separatory funnel containing Dri Solv THF (120 mL). The
reaction is washed with 10% Na.sub.2SO.sub.3 (50 mL), and then
brine (75 mL). The organic layer is then separated, dried over
MgSO.sub.4 and solvent removed by reduced pressure to yield a
residue that is purified by chromatography (silica gel: elution
with 50% Dri Solv THF/EtOAc, and then 4% MeOH/THF) and fractions
are checked by UV and MS. The appropriate fractions are lyopholized
to yield compound EA (159 mg, 88%).
##STR00209##
[0364] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following compound EB
is prepared:
##STR00210##
Example 39
Compounds EC-ED
[0365] To a solution of compound clxxxxviii (0.341 g, 0.503 mmol)
in DCM (15 mL) cooled in an ice bath is added DMP reagent (0.277 g,
0.654 mmol). The mixture is stirred at room temperature for 2
hours, then quenched with 10% Na.sub.2SO.sub.3 solution and stirred
for 20 minutes. The resulting mixture is extracted with DCM
(3.times.20 mL) and the organic extract is dried (MgSO.sub.4).
After filtration to remove MgSO.sub.4, the filtrate is concentrated
and purified by column chromatography (70% EtOAc/Hexane) to give
compound EC (0.183 g, 54%) as white solid.
##STR00211##
[0366] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following compound ED
is prepared:
##STR00212##
Example 40
Compounds EE-EG
[0367] Compound ccii (290 mg, 0.37 mmol) is taken up in DCM (5 mL).
DMP reagent (175 mg, 0.41 mmol) is added to this solution and
stirred 1 hour. The reaction is quenched with P--Na.sub.2SO.sub.3
(1.5 mmol/g resin) and stirred 1 hour. Quenched DMP reagent is
scavenged with P-TBD (2.5 mmol/g resin) and stirred 1 hour. The
resulting mixture is filtered, rinsed with DCM, before being
concentrated to a residue. The resulting residue is purified by 50%
EtOAc/Hex to yield compound EE (440 mg, 28%).
##STR00213##
[0368] Following the above method for preparing the above compound
and methods related to preparing the intermediate thereto, but
using the appropriate starting materials the following compounds
EF-EG are prepared:
##STR00214##
Example 41
Compound EH
[0369] To a DCM solution (3 mL) of compound cciii (140 mg 0.2 mmol)
is added DMP reagent (133 mg, 0.3 mmol). The reaction is stirred at
room temperature for 2 hours and quenched with 10% Na.sub.2SO.sub.3
(aq.) for 20 minutes. The resulting mixture is extracted with
EtOAc. The organic layer is washed with brine, dried over
MgSO.sub.4, concentrated to a yellow oil that is purified by silica
gel (70% EtOAc/hexane), and after lyophilized to yield compound EH
(50 mg, 38%).
##STR00215##
Example 42
Compound EJ
[0370] Compound ixxxiii (520 mg, 1 mmol) is taken up in DCM (5 mL).
PyBOP (624 mg 1.2 mmol) is added to the above solution and stirred
for 5 minutes. Compound cdviii (300 mg 1.2 mmol) in THF (5 mL) is
added drop-wise to this solution, followed by DIPEA (0.22 ml, 1.2
mmol). The reaction is stirred at room temperature overnight under
nitrogen. At this point, the reaction is diluted with EtOAc, washed
with saturated NaHCO.sub.3, and brine. The organic phase is dried
with MgSO.sub.4, filtered, and concentrated to give the crude
coupled intermediate cdix.
##STR00216##
This intermediate cdix (.about.1 mmol) is taken up in DCM (10 mL).
Dess-Martin Periodinane (466 mg, 1.1 mmol) is added to this
solution. After stirring for 1 hour at room temperature, the
reaction is quenched with a polymer bound Na.sub.2SO.sub.3 (740 mg,
1.5 mmol DMP/g resin) and stirred 45 minutes. Then, the reaction
mixture is scavenged with polymer bound TBD resin (440 mg, 2.5 mmol
DMP/g resin). The resulting mixture is stirred for 45 minutes and
then filtered. Purification is achieved in 5% EtOH/EtOAc to yield
compound EJ (245 mg, 32% over 2 steps). Literature reference for
the work-up procedure can be found in Tetrahedron 55 (1999)
6785-6796.
##STR00217##
Example 43
Compound EN
[0371] Intermediate compound cdvii (415 mg, 0.59 mmol) is taken up
in DCM (10 mL) and THF (10 mL), t-BuOH (300 uL) is added followed
by Dess-Martin Periodinane (750 mg, 1.77 mmol). The reaction is
stirred 50 minutes and then quenched with P--Na.sub.2SO.sub.3 (1.5
mmol DMP/g resin). After stirring for 20 minutes at room
temperature, the reaction mixture is scavenged with P-TBD (2.5 mmol
DMP/g resin). After stirring for 1 hour, the resulting mixture was
filtered and concentrated. Product was purified by silica gel
chromatography (50% to 70% EtOAc/Hexanes) to yield compound EN (220
ing, 53%).
##STR00218##
[0372] Mass Spectra [M] were obtained for the following compounds
as shown in Table 1 below.
TABLE-US-00002 TABLE 1 LY# Example Mass Found A 733.3 B 747.2 C
657.2 D 769.4 E 733.4 F 625.4 G 639.3 H 661.4 I 643.4 J 707.3 K
641.3 L 689.3 M 639.3 N 639.4 O 731.4 P 687.4 Q 653.4 R 701.4 S
639.3 T 747.1 U 655.4 V 653.4 W 703.4 X 661.3 Y 647.3 Z 663.3 AA
667.4 AB 711.4 AC 725.4 AD 647.3 AE 779.4 AF 689.3 AG 671.4 AK
806.4 AH 687.5 AI 735.4 AJ 736.5 AM 870.4 AN 813.3 AP 724.4 AQ
653.4 AR 628.2 AW 642.2 AX 614.2 AY 628.3 BD 570.3 BE 520.2 BF
534.3 BG 584.3 BU 890.3 BV 685.4 BW 679.3 BX 695.3 BY 697.3 BZ
787.4 CA 701.3 CB 669.4 CC 733.5 CD 643.3 CE 653.5 CH 749.4 CI
653.3 CJ 717.5 CK 683.4 CL 669.3 CM 675.2 CN 717.2 CO 653.3 CP
683.3 CQ 669.3 CR 675.2 CT 661.8 CS 639.3 CU 679.2 CV 709.3 CW
743.3 CX 695.3 CY 665.2 CZ 681.3 DA 695.3 DB 701.2 DC 673.3 DD
693.3 DE 757.4 DF 682.3 DG 676.3 DH 676.2 DI 692.5 DJ 605.2 DK
874.4 DL 924.5 DM 924.2 DN 952.7 DO 830 DP 842.5 DT 667.4 DU 639.2
DV 740.3 DW 684.2 DX 678.5 DY 749.3 DZ 685.3 EA 649.3 EB 700.3 EC
702.3 ED 730.3 EE 775.3 EF 749.3 EG 722.3 EH 665.2 EI 796.4 EJ
744.3 EK 730.5 EL 730.5 EM 757.3 EN 703.5 EO 715.5 EP 679.2 EQ
651.3 ER 715.3 ES 668.5 ET 732.5 EU 743.3 EV 683.3 EW 750.4 EX
786.4 EY 744.5 EZ 780.4 FB 693.4 FC 655.3 FD 655.3 FE 774.4 FF
681.5 FG 667.5
High Resolution Mass Spectra (HRMS) of the following compounds were
obtained as shown in Table 2.
TABLE-US-00003 TABLE 2 Molecular Formula Calculated MS Mass Found
Example (M + 1) (M + 1) (M + 1) L C37H52N7O6 690.3979 690.3986 M
C33H50N7O6 640.3822 640.3822 Z C32H48F2N7O6 664.3634 664.3627 AB
C36H48F2N7O6 712.3634 712.3649 CE C34H52N7O6 654.3979 654.3967 EN
C35H52N7O6F2 704.3947 704.3945 EK C37H63N6O8S 751.4428 750.4350 (M)
EC C36H59N6O8 703.4395 703.4382 CA C35H50N7O6F2 702.3790 702.3801
EZ C40H55N8O6F2 781.4213 781.4196 EU C36H52N7O6F2 716.3947 716.3929
CY C35H52N7O6 666.3979 666.3966 BX C37H58N7O6 696.4448 696.4432 S
C33H50N7O6 640.3823 640.3831 BW C36H54N7O6 680.4136 680.4126 CU
C36H54N7O6 680.4136 680.4128 EJ C40H57N8O6 745.4401 745.4417 EM
C35H54N7O6 668.4136 668.4139 None C41H58N7O6 744.4448 744.4691
Intermediate Example 1
Compound ii
[0373] To an ethanol solution (40 mL) of compound i (8.1 g, 24.4
mmol) is added NaBH.sub.4
##STR00219##
(924 mg, 24.4 mmol) at -10.degree. C. The reaction is stirred at
that temperature for 30 minutes, and then quenched with AcOH (3
mL). The reaction mixture is diluted with EtOAc (250 mL), and
washed with NaHCO.sub.3 and brine. The organic layer is dried and
concentrated in vacuo to yield a residue that is purified by silica
gel chromatography (50% EtOAc/Hexanes) to provide 7.85 g (97%) of
compound ii,
##STR00220##
Intermediate Example 2
Compound iii
[0374] To a THF solution (70 mL) of compound ii (4.48 g, 13.4 mmol)
is added at 0.degree. C. of NaH (699 mg, 60%, 17.42 zonal). After
stirring at that temperature for 40 minutes, neat MeI (1.25 mL,
20.1 mmol) is added. The reaction is stirred at about room
temperature overnight. At this point, the reaction is quenched
carefully with saturated solution of NH.sub.4Cl at 0.degree. C. The
reaction mixture is extracted with Et.sub.2O and EtOAc. The organic
layer is washed with water, brine and dried with Na.sub.2SO.sub.4.
The organic layer thus obtained is concentrated in vacuo to provide
the xanthate compound iii,
##STR00221##
Intermediate Example 3
Compound iv
[0375] The xanthate compound iii (.about.13.4 mmol) is dissolved in
toluene (100 mL). To this solution is added AIBN (216 mg, 1.34
mmol). The resulting solution is degassed with dry nitrogen and
then treated with n-Bu.sub.3SnH (5.4 mL, 20.1 mmol). The reaction
mixture is heated at 90.degree. C. for 3 hours. At this point, the
reaction is cooled to room temperature and concentrated in vacuo.
The resulting residue is purified with silica gel chromatography
(15-20% EtOAc/Hexanes) to provide 2.8 g (66% overall from compound
ii) of compound iv,
##STR00222##
Intermediate Example 4
Compound v
[0376] To an ethanol solution (21 mL) of compound iv (1 g, 3.15
mmol) is added Pd(OH).sub.2/C (655 mg, 20%, 0.95 mmol) under a
stream of nitrogen. The resulting reaction mixture is subjected to
standard hydrogenation (1.5 atm). After 5 hours, the hydrogen
source is removed and the reaction is filtered. The filtrates are
concentrated in vacuo to provide the free amine compound v,
##STR00223##
Intermediate Example 5
Compound vi
[0377] To a DCM solution (10 mL) of compound vii (629 mg, 1.95
mmol) is added at about
##STR00224##
room temperature HOAt (265 mg, 1.95 mmol) and followed by 1 M DCC
solution in DCM (1.95 mL, 1.95 mmol). After stirring for 30
minutes, a DCM solution (3 mL) of compound v (1.5 mmol) is added to
the above HOAt-activated acid. The reaction is stirred at about
room temperature overnight. At this point, the reaction is filtered
through Celite. The filtrates are diluted with EtOAc (75 mL) and
washed with water and brine. The organic layer is dried and
concentrated in vacuo. The resulting residue is purified by silica
gel chromatography (70-80% EtOAc/Hexanes) to afford 620 mg (85%) of
compound vi,
##STR00225##
Intermediate Example 6
Compound viii
[0378] To an ethanol solution (10 mL) of compound vi (615 mg, 1.26
mmol) is added 2 N NaOH aqueous solution (1.26 mL, 2.52 mmol). The
reaction is stirred overnight at about room temperature and then
acidified to pH 3 using Dowex acidic resins. The solids are
filtered off and the filtrates are concentrated in vacuo to give a
residue that is redissolved in 1:1 CH.sub.3CN/H.sub.2O. This
solution is subjected to lyophilization to provide 495 mg (85%) of
compound viii,
##STR00226##
Intermediate Example 7
Compound ix
[0379] To a DCM solution (10 mL) of compound viii (230 mg, 0.5
mmol) is added PyBop (417 mg, 0.8 mmol). The reaction is stirred at
about room temperature for 30 minutes. To this solution is then
added a THP solution (5.25 mL) of compound x (263 mg, 0.75 mmol)
and followed by
##STR00227##
DIPEA (0.174 mL, 1 mmol). The reaction is stirred at about room
temperature overnight and then quenched with water (30 mL) for 30
minutes. The reaction mixture is extracted with EtOAc (100 mL). The
organic layer is washed with brine and dried and concentrated in
vacuo to afford a residue that is purified via silica gel
chromatography (5% EtOH/EtOAc) to give .about.400 mg (100%) of
compound ix,
##STR00228##
Intermediate Example 8
Compound xi
[0380] To a DCM solution (10 mL) of compound ix (396 mg, 0.5 mmol)
is added DMP reagent reagent (278 mg, 0.65 mmol). The reaction is
stirred at about room temperature for 1 hour and then quenched with
10% Na.sub.2SO.sub.3 for 30 minutes. The reaction mixture is then
extracted with EtOAc (75 mL) and washed with brine. The organic
layer is dried and concentrated in vacuo. The resulting residue is
purified with silica gel chromatography (70% EtOAc/Hexanes) to give
320 mg (81%) of compound xi,
##STR00229##
Intermediate Example 9
Compound xii
[0381] Toa DCM solution (10 mL) of compound viii (230 mg, 0.5 mmol)
is added PyBop (417 mg, 0.8 mmol). The reaction is stirred at about
room temperature for 30 minutes. To this solution is then added a
THF solution (3.5 mL) of compound xiii (140 mg, 0.75 mmol) and
##STR00230##
followed by DIPEA (0.174 mL, 1 mmol). The reaction is stirred at
about room temperature overnight and then quenched with water (30
mL) for 30 minutes. The reaction mixture is extracted with EtOAc
(75 mL). The organic layer is washed with brine and dried and
concentrated in vacuo to afford a residue that is purified via
silica gel chromatography (5% EtOH/EtOAc) to give in quantitative
yield compound xii,
##STR00231##
Intermediate Example 10
Compound i'
[0382] To a methanol solution (30 mL) of compound i (5 g, 15.1
mmol) is added (BOC).sub.2O (3.3 g, 15.1 mmol) and
H.sub.2/Pd(OH).sub.2/C (1.6 g, 10% Pd content). The reaction is
stirred at about room temperature for 2 hours and then filtered
through Celite twice. The Celite bed is rinsed with DCM. The
combined filtrates are concentrated in vacuo to yield an oily
residue that is purified by silica gel chromatography (40%
EtOAc/Hexanes) to give 3.8 g (85%) of compound i',
##STR00232##
Intermediate Example 11
Compound ii'
[0383] To a methanol solution (111 mL) of compound i' (3.7 g, 12.5
mmol) is added at 0.degree. C. NaBH.sub.4 (0.805 g. 21 mmol). After
stirring at 0.degree. C. for 2.5 hours, the reaction solvent is
evaporated slowly in vacuo to yield a residue that is diluted with
EtOAc. This solution is then washed with water twice. The aqueous
layer is extracted with EtOAc. The combined organic layers are
dried with MgSO.sub.4 and filtered and concentrated in vacuo to
yield a residue that is purified with chromatography to provide
3.76 g (99%) of compound ii',
##STR00233##
Intermediate Example 12
Compound xiv
[0384] To a DCM solution (180 mL) of compound ii' (3.76 g, 12.3
mmol) is added at 0.degree. C. DMAP (5 g, 40.1 mmol) and then
followed by Tf.sub.2O (4 mL, 23.7 mmol). The reaction is stirred at
0.degree. C. for 1 hour and at about room temperature for
additional 1.5 hours. The reaction mixture is then washed twice
with 5% NaNCO.sub.3 and dried with MgSO.sub.4. The organic layer
thus obtained is concentrated in vacuo to provide the crude
triflate. The resulting triflate (2.7 g, 6 mmol) is dissolved in
DCM (120 mL). To this solution is added DMAP (2.5 g, 2.0.5 mmol).
The resulting reaction mixture is heated to reflux overnight. At
this point, the reaction is cooled to room temperature and washed
with 5% NaHCO.sub.3 twice. The reaction mixture is dried with
MgSO.sub.4 filtered and concentrated in vacuo to yield a brownish
oily residue that is purified (1% MeOH/DCM) to give 500 mg (30%) of
compound xiv,
##STR00234##
Intermediate Example 13
Compound xv
[0385] Compound xiv (500 mg, 1.8 mmol) is dissolved in 4 N HCl in
dioxane (6.75 mL). The reaction is stirred at about room
temperature for .about.4 hours. At this point, the solvent is
removed in vacuo. The resulting residue is titrated with
diethylether twice to give in almost quantitative yield the HCl
salt of compound xv,
##STR00235##
Intermediate Example 14
Compound xvi
[0386] To a THF solution (7 mL) of compound vii (579 mg, 1.8 mmol)
is added HOAt (245 mg, 1.8 mmol) and DCC (1.8 mL, 1 M in DCM). A
suspension is resulted. After stirring at about room temperature
for 15 minutes, a THF solution (6 mL) of compound xv (1.8 mmol) and
DIPEA (0.63 mL, 3.6 mmol) is added to the above suspension.
Additional DIPEA (0.8 mL) is added later. The reaction mixture is
stirred overnight at about room temperature. At that point, the
white solids so formed are filtered off. The white solids are
rinsed with THF. The combined filtrates and washings are
concentrated in vacuo to give the crude product that is purified by
silica gel chromatography (100% EtOAc) to provide 665 mg (76%) of
compound xvi,
##STR00236##
Intermediate Example 15
Compound xvii
[0387] To an ethanol solution (8 mL) of 7 (665 mg, 1.37 mmol) is
added 1 N aqueous NaOH (2.4 mmol) at 0.degree. C. The reaction is
stirred overnight at about room temperature, and then acidified to
pH 3 using Dowex acidic resins. The solids are filtered. The
resulting filtrates are concentrated in vacuo to give a pale yellow
residue that is redissolved in 1:1 CH.sub.3CN/H.sub.2O and
lyophilized to give 467 mg (74%) of compound xvii.
##STR00237##
Intermediate Example 16
Compound xix
[0388] A DCM solution (4 mL) of compound xvii (100 mg, 0.22 mmol)
is treated with PyBop (207 mg, 0.4 mmol) at about room temperature
for 20 minutes. At this point, the above solution is treated with a
THF solution (2.6 mL) of compound xviii (65 mg, 0.32 mmol),
followed by DIPEA
##STR00238##
(0.076 mL). After stirring at about room temperature for 7 hours,
the reaction is quenched with water. The reaction mixture is
diluted with DCM (60 mL). The organic layer is separated and washed
twice with brine and dried with MgSO.sub.4. Upon filtration,
concentrated and silica gel chromatography (5% EtOH/EtOAc), 148 mg
(.about.100%) of compound xix is obtained.
##STR00239##
Intermediate Example 17
Compound xx
[0389] To a THF solution (100 mL) of N-Cbz-L-valine (14.4 g, 57.2
mmol) is added HOBT (7.72 g, 57.2 mmol) and EDCI (10.98 g, 57.2
mmol). After stirring at about room temperature for 20 minutes, a
THF solution (50 mL) containing tert-L-Leucine methyl
ester-hydrochloride (10.4 g. 57.2 mmol) and DIPEA (11.9 mL, 68.7
mmol) is added to the above solution. The reaction is stirring at
about room temperature overnight Upon standard aqueous work-up and
silica gel chromatography (30% EtOAc/Hexanes) 14 g (64%) of
compound xx is afforded.
##STR00240##
Intermediate Example 18
Compound xxi
[0390] To a methanol solution (80 mL) of xx (6.71 g, 17.7 mmol) is
added (under a stream of N.sub.2) Pd/C (1.88 g, 10% Pd content).
The reaction vessel is subjected to hydrogenation (1 atm H.sub.2)
overnight at about room temperature. At this point, the reaction
mixture is filtered through a pad of Celite and concentrated in
vacuo to provide the corresponding crude free amine for next step.
A THF solution of this amine (.about.17.7 mmol) is added to a THF
(46 mL) and DMF (5 mL) solution containing 2-pyrazinecarboxylic
acid (2.85 g, 23 mmol), Hobbit (3.12 g. 23 mmol) and EDCI (4.41 g,
23 mmol). To the resulting mixture is then added DIPEA (3.08 g,
17.7 mmol). The reaction is stirred overnight at about room
temperature and then quenched with water. The reaction mixture is
extracted with EtOAc. The organic layer is washed with brine and
concentrated in vacuo to provide a residue that is purified by
silica gel chromatography (40-50% EtOAc/Hexanes) to provide 3.9 g
(63%) of compound xxi,
##STR00241##
Intermediate Example 19
Compound xxii
[0391] To a methanol solution (40 mL) of compound xxi (4.67 g.
13.34 mmol) is added 2 N NaOH (10 mL, 20 mmol). The reaction is
stirred at about room temperature for 2 hours. At this time, an
additional amount of 2 N NaOH (3.3 mL, 6.67 mmol) is added to the
reaction mixture. After stirring at about room temperature
overnight, the reaction is acidified to pH 3 using acidic resin.
The reaction is then filtered and the filtrates are concentrated in
vacuo to yield a residue that is dissolved in 1:1
CH.sub.3CN/H.sub.2O for lyophilization. 4.15 g (93%) of compound
xxii is obtained.
##STR00242##
Intermediate Example 20
Compound xxiii
[0392] A DCM solution (10 mL) of compound xxii (917 mg, 2.73 mmol)
is treated with HOAt (371 mg, 2.73 mmol) and DCC (2.73 mL, 1 M,
2.73 mmol). After stirring for 30 minutes, the reaction mixture is
treated with a THF solution (10 mL) of compound v (500 mg, 2.73
mmol). After stirring at about room temperature overnight, the
white solids (urea) are filtered. The filtrates are concentrated in
vacuo to give a residue that is purified by silica gel
chromatography (60-70% EtOAc/Hexanes) to provide 1.06 g (77%) of
compound xxiii,
##STR00243##
Intermediate Example 21
Compound xxiv
[0393] An ethanol solution (20 mL) of compound xxiii (1.06 g, 2.11
mmol) is treated with 2 N NaOH (2.11 mL, 4.23 mmol). After stirring
at about room temperature overnight, the reaction mixture is
acidified to pH 3 with acidic resin. The solids are filtered off.
The resulting filtrates are concentrated in vacuo to give a residue
that is lyophilized to give .about.1 g (100%) of compound xxiv,
##STR00244##
Intermediate Example 22
Compound xxv
[0394] A DCM solution (10 mL) of compound xxiv (236.7 mg, 0.5 mmol)
is treated with PyBop (417 mg, 0.8 mmol). After stirring at about
room temperature for 20 minutes, the reaction mixture is treated
with a DMF solution (5.6 mL) of compound xiii (139.5 mg, 0.75
mmol), followed by DIPEA (0.174 mL, 1 mmol). After stirring at
about room temperature for 8 hours, the reaction is quenched with
water and extracted with EtOAc. The resulting organic layer is
washed with brine and dried and concentrated in vacuo to give a
residue that is purified by silica gel chromatography (5%
EtOH/EtOAc) to afford .about.320 mg (100%) of compound xxv,
##STR00245##
Intermediate Example 23
Compound xxvi
[0395] A DCM solution (15 mL) of compound xxiv (355 mg, 0.75 mmol)
is treated with PyBop (622 mg, 1.2 mmol). After stirring at about
room temperature for 20 minutes, the reaction mixture is treated
with a THF solution (10 mL) of compound xxvii' (156 mg, 0.75
##STR00246##
mmol), followed by DIPEA (0.26 mL, 1.5 mmol). After stirring at
about room temperature overnight, the reaction is quenched with
water and extracted with EtOAc. The resulting organic layer is
washed with brine and dried and concentrated in vacuo to give a
residue that is purified by silica gel chromatography (2%
EtOH/EtOAc) to afford .about.400 mg (80%) of compound xxvi,
##STR00247##
Intermediate Example 24
Methyl 5-cyanopentanoate
[0396] Potassium Cyanide (4 g, 61.44 mmol) is dissolved in 70 mL
water and 200 mL methanol. To the solution 10 g (51.2 mmol) of
methyl 5-bromopentanoate is added and the mixture is refluxed
overnight. The reaction mixture is concentrated to dryness. To the
residue, 100 mL of EtOAc is added to extract the product. The
organic is washed with water three times, dried and concentrated to
yield 5.37 g (74%) of methyl 5-cyanopentanoate as an oil.
Intermediate Example 25
Methyl 5-tetrazol-5-ylpentanoate
[0397] Methyl 5-cyanopentanoate (4.8 g, 34 mmol) is dissolved in
toluene, triethylammonium chloride (14 g, 102 mmol) and sodium
azide (6.63, 102 mmol) is added. The mixture is heated to reflux
for overnight. The reaction mixture is cooled to room temperature,
water is added to extract (3.times.100 mL) methyl
5-tetrazol-5-ylpentanoate from the organic. To the aqueous phase,
concentrate HCl is added to adjust pH to 2. The product is
extracted from the aqueous solution with EtOAc (3.times.50 mL). The
organic is combined, dried and concentrated to yield 4.25 g (68%)
of methyl 5-tetrazol-5-ylpentanoate.
Intermediate Example 26
Methyl 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate
[0398] Methyl 5-tetrazol-5-ylpentanoate (4.23 g, 23 mmol) and
trichloroacetic acid (8.69 g, 53 mmol) are dissolved in 50 mL of
CHCl.sub.3. .alpha.-Methylstyrene (2.72, 23 mmol) is added to the
solution dropwise, and the reaction mixture is allowed to stirred
at about room temperature for overnight. The reaction mixture is
diluted with EtOAc to 200 mL, and organic layer is washed with 10%
aqueous KOH and brine. The organic layer is dried, concentrated.
The product is purified by flash column chromatography to yield 6.6
g (95%) methyl
5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate.
Intermediate Example 27
5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoic acid
[0399] Methyl 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate
(6.6 g, 21.8 mmol) is dissolved in methanol (100 mL) and 23 mL of 1
N aqueous NaOH is added. The mixture is stirred overnight and is
concentrated to remove methanol. The residue is dissolved in water
(100 mL) and the solution is neutralized by adding the same
equivalent of 1 N aqueous HCl. The product is extracted with EtOAc
(3.times.50 mL). The organic is dried and concentrated to yield
4.75 g (75%) 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoic
acid.
Intermediate Example 28
Compound xxviii
[0400] 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoic acid (4.75
g, 16.5 mmol) is dissolved in DCM (100 mL), 4.8 g (24.8 mmol) of
EDCI and 6 mL of DIPEA are added. To the mixture,
N-hydroxylsuccinimide (3.8 g, 33 mmol) is added. The reaction
mixture is stirred for three hours at about room temperature. The
mixture is diluted with DCM to 200 mL and the solution is washed
with water three times. The organic is dried and concentrated to
yield 4.79 g (75%) of compound xxviii,
##STR00248##
Intermediate Example 29
Compound xxix
[0401] The dipeptide H-Val-Val-OH (3.22 g, 14.9 mmol) is suspended
in 50 mL, of NV-dimethylformamide (DMF) and 4.75 g (12.42 mmol) of
compound xxviii is added followed the addition of 3.4 mL (18.63
mmol) of diisopropylethylamine (DIPEA). The mixture is warmed up to
40.degree. C. and stirred overnight. The solvent is evaporated
under high vacuum. The residue is dissolved in EtOAc and washed
with 1 N HCl and brine to yield 5.52 g (91%) of compound xxix,
##STR00249##
Intermediate Example 30
Compound xxx
[0402] 1.6 g (3.29 mmol) of compound xxix is dissolved in 20 mL of
DCM, 3.3 mL of 1 M solution of DCC in THF is added. To the mixture,
500 mg (2.73 mmol) of compound v is added. The mixture is stirred
at about room temperature overnight. The mixture is diluted with
EtOAc to 100 mL and washed with 1 N HCl, NaHCO.sub.3 and brine.
Purified by column chromatography (50% EtOAc/hexane) to yield 1.02
g (58%) compound xxx,
##STR00250##
Intermediate Example 31
Compound xxxi
[0403] Compound xxx (1.02 g, 1.57 mmol) is dissolved in 10 mL MeOH
and 2 mL of 1 N aqueous NaOH is added. The mixture is stirred
overnight. The methanol is removed by evaporation and the residue
is dissolved in water and neutralized with 2 mL HCl. Following
extraction with EtOAc, 1.00 g (.about.100%) of compound xxxi is
afforded.
##STR00251##
Intermediate Example 32
Compound xxxii
[0404] Compound xxxi (300 mg, 0.48 mmol) and PyBop (300 mg, 0.58
mmol) are dissolved in 10 mL DCM. To the solution, compound x (201
mg, 0.58 mmol) is added and then DIPEA (104 .mu.l) is added. The
mixture is stirred at about room temperature overnight. The
reaction mixture is then diluted with EtOAc to 100 mL and washed
twice with 1 N HCl, twice with NaHCO.sub.3 and thrice with brine.
The organic is dried and concentrated. The residue is purified by
column chromatography (100% EtOAc) to yield 450 mg (98%) of
compound xxxii,
##STR00252##
Intermediate Example 33
Compound xxxiii
[0405] Compound xxxii 360 mg (0.38 mmol) is dissolved in 8 mL DCM
and 240 mg (0.57 mmol) of DMP reagent is added. The mixture is
stirred at about room temperature for three hours. The mixture is
diluted with EtOAc to 50 ml, and washed with brine three times. The
product is purified by column chromatography (25% ethanol/EtOAc) to
yield 300 mg (83%) of compound xxxiii,
##STR00253##
Intermediate Example 34
Compound xxxiv
[0406] To a DCM solution (10 mL) of xxxv (790 mg, 2.80 mmol) is
added PyBop (1.7 g,
##STR00254##
3.36 mmol) and Hobbit (450 mg, 3.36 mmol). The resulting solution
is cooled to 0.degree. C. and treated with a DCM solution (3 mL) of
(s)-.alpha.-(4-pyridyl)ethylamine (410 mg, 3.36 mmol). This is
followed by the addition of DIPEA (0.5 mL, 3.36 mmol). The reaction
is stirred overnight at about room temperature. At this point, the
reaction mixture is diluted with EtOAc. The whole is washed with
saturated NaHCO.sub.3 and brine. The organic layer thus obtained is
dried and concentrated in vacuo. The resulting residue is purified
by silica gel chromatography (5% EtOH/EtOAc) to provide 630 mg
(58%) of compound xxxiv,
##STR00255##
Note: (s)-.alpha.-(4-pyridyl)ethylamine is obtained from its
D-tartrate salt by base wash (1 N NaOH) and subsequent EtOAc
extraction. The recovery rate is 89%.
Intermediate Example 35
Compound xxxvi
[0407] To a methanol solution (15 mL) of compound xxxiv (630 mg,
1.64 mmol) is added under N.sub.2Pd/C (150 mg, 10% palladium
content). The reaction is stirred under H.sub.2 overnight. The
reaction mixture is filtered through a pad of Celite.RTM. 521. The
filtrates are concentrated in vacuo to provide 420 mg (.about.100%)
of compound xxxvi,
##STR00256##
Intermediate Example 36
Compound xxxvii
[0408] To a DCM solution (3 mL) of compound xxxi (270 mg, 0.43
mmol) is added PyBop (270 mg, 0.52 mmol). This is followed by
addition of compound xxxvi (160 mg, 0.64 mmol) and DIPEA (0.09 mL,
0.52 mmol). The reaction is stirred at about room temperature
overnight. At this point, the reaction is diluted with EtOAc and
washed with 0.1N HCl, followed by saturated NaHCO.sub.3 and brine.
The resulting organic layer is dried and concentrated to give
compound xxxvii (430 mg total mass) for next step
##STR00257##
Intermediate Example 37
Compound xxxviii
[0409] To a DCM solution (3 mL) of compound xxxvii (370 mg, 0.43
mmol) is added DMP reagent (280 mg, 0.65 mmol). The reaction is
stirred at about room temperature for 2 hours and then quenched
with 10% Na.sub.2SO.sub.3. After stirring for 30 minutes, the
reaction is extracted with EtOAc. The organic layer is washed with
saturated NaHCO.sub.3 and brine. The resulting organic layer is
dried and concentrated in vacuo to give a residue that is purified
by silica gel chromatography (5% EtOH/EtOAc) to provide 180 mg (49%
for 2-steps) of compound xxxviii,
##STR00258##
Intermediate Example 38
Compound xxxx
[0410] Compound xxix (2.5 g, 5 mmol) is dissolved in 40 mL of DCM,
5.1 mL of 1 M solution of DCC in THF is added to the solution. To
the mixture, 1.08 g (3.53 mmol) of compound xxxix is added. The
mixture is stirred at about room temperature overnight.
##STR00259##
The mixture is diluted with EtOAc to 100 mL, washed sequentially
with 1 N HCl, NaHCO.sub.3 and brine, and then purified by column
chromatography (80% EtOAc/hexane) to yield 2.59 g (95%) of compound
xxxx,
##STR00260##
Intermediate Example 39
Compound xxxxi
[0411] Compound xxxx (2.59 g, 3.35 mmol) is dissolved in 20 mL MeOH
and 4 mL of 1 N aqueous NaOH is added. The mixture is stirred
overnight and then rotary evaporated to leave a residue. The
residue is dissolved in water and neutralized with 2 mL HCl. The
neutralized solution is then extracted with EtOAc to yield 2.49 g
(.about.100%) of compound xxxxi,
##STR00261##
Intermediate Example 40
Compound xxxxii
[0412] Compound xxxxi (847 mg, 1.16 mmol) and 724 mg (1.39 mmol) of
PyBop are dissolved in 10 mL DCM. To the solution, compound xiii
(260 mg, 1.39 mmol) is added and then followed by the addition of
DIPEA (209 id). The mixture is stirred at about room temperature
overnight. The reaction mixture is then diluted with EtOAc to 100
mL, and washed twice with 1 N HCl, twice with NaHCO.sub.3 and
thrice with brine. The organic is dried and concentrated. The
residue is purified by column chromatography (5% ethanol/EtOAc) to
yield 930 mg (86%) of compound xxxxii,
##STR00262##
Intermediate Example 41
Compound xxxxiii
[0413] Compound xxxxii (350 mg, 0.38 mmol) is dissolved in 10 mL
DCM and 242 mg (0.57 mmol) of DMP reagent is added. The mixture is
stirred at about room temperature for three hours. The mixture is
diluted with EtOAc to 50 mL and washed thrice with brine. The
product is purified by column chromatography (100% EtOAc) to yield
180 mg (51%) of compound xxxxiii,
##STR00263##
Intermediate Example 42
Compound xxxxv
[0414] H-Val-Val-OH (5 g, 23 mmol) is suspended in 100 mL DMF,
compound xxxxiv
##STR00264##
(8.3 g, 27.6 mmol) is added, and then 6.2 mL (35.5 mmol) of DIPEA
is added. The mixture is stirred at 40.degree. C. for two days. The
solvent is removed under high vacuum and the residue is dissolved
in 100 mL EtOAc and washed thrice with 1 N HO and twice with brine.
9.14 g (99%) of compound xxxxv is afforded.
##STR00265##
Intermediate Example 43
Compound xxxxvi
[0415] Compound xxxxv (2.8 g, 7 mmol) and 954 mg (7 mmol) of HOAt
is dissolved in 100 mL DCM. 7 mL of 1 M DCC/DCM is added. To the
reaction mixture, compound xxxix (2.15 g) is added and the reaction
mixture is stirred at about room temperature for overnight. The
mixture is concentrated to dryness and the residue is dissolved in
EtOAc and purified by column chromatography (100% EtOAc) to yield
4.57 g (95%) of compound xxxxvi,
##STR00266##
Intermediate Example 44
Compound xxxxvii
[0416] Compound xxxxvi (4.57 g, 6.65 mmol) is dissolved in 10 mL
TFA and 10 mL DCM. The mixture is stirred at about room temperature
for 4 hours. The solvent is removed by vacuum and the residue is
dissolved in 50:50 acetonitrile/water and lyophilized to yield as a
powder compound xxxxvii,
##STR00267##
Intermediate Example 45
Compound xxxxviii
[0417] Compound xxxxvii (1 g, 1.59 mmol) and 990 mg (2.28 mmol) of
PyBop is dissolved in 20 mL DCM and 1.6 mL of 1 M methylamine in
THF is added. The mixture is stirred at about room temperature for
4 hours. The reaction mixture is diluted to 100 mL with EtOAc and
washed with 1 N HCl, NaHCO.sub.3 and brine. The residue is purified
by flash column chromatography (10% EtOH/EtOAc) to yield 1 g (98%)
of compound xxxxviii,
##STR00268##
Intermediate Example 46
Compound xxxxix
[0418] Compound xxxxviii (1 g, 1.55 mmol) is dissolved in 10 mL of
MeOH and 2 mL 1 N NaOH is added. The mixture is stirred at about
room temperature for overnight. The solvent is removed by
evaporation. The residue is dissolved in water, neutralized and
extracted with EtOAc to yield 960 mg (98%) of compound xxxxix,
##STR00269##
Intermediate Example 47
Compound li
[0419] Compound xxxxix (315 mg, 0.5 mmol) and 312 mg (0.6 mmol) of
PyBop are dissolved in 10 mL DCM. Compound l (56 mg, 0.6 mmol) and
108 .mu.l of DIPEA is added. The mixture
##STR00270##
is stirred at about room temperature overnight, and is diluted to
100 mL with EtOAc and washed with 1 N HCl, NaHCO.sub.3 and brine.
Purified by column chromatography (15% EtOH/EtOAc) to yield 400 mg
(92%) of compound li,
##STR00271##
Intermediate Example 48
Compound lii
[0420] Compound li (400 mg, 0.46 mmol) is dissolved in 10 mL of DCM
and 292 mg (0.69 mmol) DMP reagent is added. The mixture is stirred
at about room temperature for 3 hours. The solvent is removed by
evaporation and product is purified by RP-HPLC to yield 130 mg
(32%) of compound lii,
##STR00272##
Intermediate Example 49
Compound liii
[0421] Compound xxxxix (210 mg, 0.33 mmol) and 208 mg (0.4 mmol) of
PyBop are dissolved in 10 mL DCM. Compound xiii (154 mg, 0.83 mmol)
is added to the solution followed by the addition of DIPEA (72
.mu.l, 0.4 mmol). The mixture is stirred at about room temperature
overnight. The reaction mixture is diluted to 100 mL with EtOAc,
washed with 1 N HCl, NaHCO.sub.3 and brine, and then purified by
flash column chromatography (10% EtOH/EtOAc) to yield 250 mg (95%)
of compound liii,
##STR00273##
Intermediate Example 50
Compound liv
[0422] Compound xxxxv (755 mg, 1.88 mmol) and 255 mg (1.88 mmol) of
HOAt are dissolved in 20 ml DCM. 1.88 mL of 1 M DCC/DCM is added.
To the reaction mixture, compound v (288 mg) is added and the
reaction mixture is stirred at about room temperature for 2 hours.
The mixture is concentrated to dryness and the residue is dissolved
in EtOAc and purified by column chromatography (80% EtOAc/Hexanes)
to yield 800 mg (90%) of compound liv,
##STR00274##
Intermediate Example 51
Compound lv
[0423] Compound liv (800 mg, 1.41 mmol) is dissolved in 10 mL MeOH
and 2 mL NaOH is added. The mixture is stirred at about room
temperature overnight. The solvent is removed by vacuum and the
residue is dissolved in water and neutralized with 2 mL 1 N HCl.
The product is extracted with EtOAc. Evaporation of the extraction
solvent afforded 760 mg (.about.100%) lv,
##STR00275##
Intermediate Example 52
Compound lvii
[0424] Compound lv (760 mg, 1.41 mmol) and 880 mg (1.69 mmol) of
PyBop are dissolved in 5 mL DCM. Compound lvi (530 mg, 2.12 mmol)
is added to the solution and then 0.31
##STR00276##
of DIPEA is added. The mixture is stirred at about room temperature
overnight. The reaction mixture is diluted to 100 mL with EtOAc,
washed with 1 N HCl, NaHCO.sub.3 and brine, and then purified by
flash column chromatography (100% EtOAc) to yield 870 mg (80%) of
compound lvii,
##STR00277##
Intermediate Example 53
Compound lviii
[0425] Compound lvii (350 mg, 0.45 mmol) is dissolved in 5 mL TFA
and 5 mL DCM and the mixture is stirred at about room temperature
for 3 hours. The solvent is removed by evaporation and the product
is purified by RP-HPLC to yield 220 mg (69%) of compound lviii,
##STR00278##
Intermediate Example 54
Compound lix
[0426] Compound lviii (200 mg, 0.28 mmol) and 218 mg (0.42 mmol) of
PyBop are dissolved in 5 mL DCM. Methylamine (0.28 mL of 2 M in
THF) is added. The mixture is stirred at about room temperature
overnight. The mixture is diluted to 100 mL with EtOAc, washed with
1 N HCl, NaHCO.sub.3 and brine, and then purified by column
chromatography (15% EtOH/EtOAc) to yield 168 mg (79%) of lix,
##STR00279##
Intermediate Example 55
Compound lx
[0427] Compound lviii (200 mg, 0.26 mmol) is dissolved in 4 mL of
DCM and 165 mg (0.39 mmol) of DMP reagent is added. The mixture is
stirred at about room temperature for 3 hours. The solvent is
removed by evaporation. The residue is dissolved in 50%
acetonitrile/water, and filtered purified by RP-HPLC to yield 140
mg (70%) of compound lx,
##STR00280##
Intermediate Example 56
Compound ii
[0428] A DCM (30 mL) and EtOH (30 mL) solution of compound i (4 g,
12.1 mmol), under N.sub.2, is cooled down to -10.degree. C.
NaBH.sub.4 (458 mg, 12.1 mmol) is added and the solution is stirred
at -10.degree. C. for 50 minutes. TLC (50% EtOAc/Hexane) showed
total conversion to a slower running spot. The reaction is
carefully quenched with ice and then with a cold saturated solution
of NH.sub.4Cl (10 mL). The mixture is dumped in DCM (300 mL). The
organic layer is washed once with saturated solution of NH.sub.4Cl
(60 mL) and twice with brine (60 mL). The organic layer is then
separated, dried over MgSO.sub.4 and concentrated in vacuo, to
yield 3.5 g of compound ii (87%)
Intermediate Example 57
Compound lxi
[0429] In a 250 mL round bottom flask equipped with a H.sub.2
balloon, an ethanolic solution (50 mL) of compound ii (3.5 g. 10.5
mmol) is subjected to standard hydrogenation conditions [20%
Pd(OH).sub.2/C (1.47 g, 2.1 mmol)] for 5 hours at about room
temperature. The catalyst is filtered off through Celite and washed
with DCM. The solvent is then removed under reduced pressure to
yield 2 g (96%) of compound lxi,
##STR00281##
Intermediate Example 58
Compound lxii
[0430] Under inert atmosphere, a solution of compound lxi (200 mg,
1 mmol), compound lxiii,
##STR00282##
(233 mg, 1.1 mmol), HOAt (1-hydroxy-7-azabenzotriazole) (156 mg,
1.15 mmol) in anhydrous DMF (6 mL) is stirred for 20 minutes. The
temperature is then taken down to 0.degree. C., followed by the
addition of DIC (0.18 mL, 1.15 mmol). The reaction is stirred
overnight at about room temperature. The solution is diluted with
EtOAc and then washed twice with 1 N HCl, twice with saturated
aqueous NaHCO.sub.3, and brine. The organic layer is separated
dried over MgSO.sub.4 and the solvent removed under reduced
pressure. The residue is cleaned by chromatography (silica gel: 70%
EtOAc/DCM) to give in 45% yield compound lxii.
##STR00283##
Intermediate Example 59
Compound lxiv
[0431] A solution of compound lxii (777 mg, 2 mmol) in dioxane (6
mL) and 0.5 M NaOH (6 mL) is stirred for 5 hours at about room
temperature. Examination by TLC (100% EtOAc) shows complete
conversion to a spot at the origin. The reaction is cooled down
with an ice bath followed by the addition of 1 N HCl (4 mL). Solid
NaCl is then added and the whole mixture is extracted twice with
EtOAc (2.times.150 mL). The organic extracts are then combined,
dried over MgSO.sub.4 and the solvent removed under reduced
pressure to give compound lxiv in 92% yield.
##STR00284##
Intermediate Example 60
Compound lxv
[0432] Under an inert atmosphere, a solution of compound x (203 mg,
0.58 mmol), compound lxiv (276 mg, 0.775 mmol), HOAt
(1-hydroxy-7-azabenzotriazole) (126 mg, 0.93 mmol) in anhydrous DMF
(6 mL) is stirred for 20 minutes. The temperature is then taken
down to 0.degree. C., followed by the addition of DIC (0.14 mL,
0.93 mmol). The reaction is stirred overnight at about room
temperature. The solution is diluted with EtOAc and then washed
twice with 1 N HCl, twice with saturated aqueous NaHCO.sub.3, and
brine. The organic layer is separated dried over MgSO.sub.4 and the
solvent removed under reduced pressure. The residue is purified by
chromatography (silica gel: 50% EtOAc/DCM to 80:19:1
EtOAC/DCM/MeOH) to give compound lxv in 62% yield.
##STR00285##
Intermediate Example 61
Compound lxvi
[0433] Under an inert atmosphere, to a solution of compound lxv
(287 mg, 0.42 mmol) in anhydrous DCM (15 mL) is added the DMP
reagent (605 mg, 1.43 mmol) The reaction is stirred for 2 hours at
about room temperature. (Note. --The doubling of the amount of the
DMP reagent and the reaction time is to assure that both alcohol
groups are completely oxidized to the corresponding keto groups).
Examination by TLC (silica gel: 2% MeOH/EtOAc) shows complete
conversion to the faster product. The reaction is diluted with DCM
(150 mL) and then washed twice with a 10% aqueous sodium sulfite
solution (2.times.50 mL), twice with saturated aqueous NaHCO.sub.3,
and with brine. The organic layer is separated dried over
MgSO.sub.4 and the solvent removed under reduced pressure. The
residue is purified by chromatography (silica gel: 50% EtOAC/DCM to
80:19:1 EtOAC/DCM/MeOH) to give in 77% yield compound lxvi.
##STR00286##
Intermediate Example 62
Compound lxvii
[0434] To a DCM solution (60 ml) of L-3 phenyl lactic acid (2 g, 12
mmol) is added PyBOP (7.5 g, 14.4 mmol). To this solution is added
a DCM solution (20 mL) containing L-valine methyl ester HCl (2.4 g,
14.4 mmol) and DIPEA (2.6 mL, 14.4 mmol). The resulting reaction
mixture is stirred overnight at about room temperature. At this
point, the reaction is diluted with EtOAc (30 mL), washed with
NaHCO.sub.3 (30 mL) and brine (15 mL). The organic layer is dried
over Na.sub.2SO.sub.4, filtered and concentrated. Purification is
achieved in 50% EtOAc/Hex on silica gel to give 2.97 g (89%) of
compound lxvii,
##STR00287##
Intermediate Example 63
Compound lxviii
[0435] Compound lxvii (2.97 g, 10.6 mmol) is taken up in DCM (50
mL) and cooled with an ice bath. TBSCl (2.1 g, 13.8 mmol) is added
to this solution followed by imidazole (0.94 g, 13.8 mmol). The
resulting solution is stirred overnight. The reaction is then
diluted with EtOAc (50 mL), washed with NaHCO.sub.3 and brine. The
organic layer is dried over Na.sub.2SO.sub.4, filtered and
concentrated. Purification is achieved in 20% EtOAc/Hexane on
silica gel to give 3.79 g (90%) of compound lxviii,
##STR00288##
Intermediate Example 64
Compound lxix
[0436] To a methanol (50 ml) solution of compound lxviii (3.78 g,
9.6 mmol) is added 1 N aqueous NaOH (14.4 mL, 14.4 mmol). The
resulting solution is stirred overnight. The solvent is partially
removed in vacuo. The pH of the reaction mixture is then lowered to
3 using 1 N HCl aqueous solution. The solution is diluted with
EtOAc and brine. The desired product is extracted with EtOAc
(3.times.50 ml). The organic layers are combined, dried over
Na.sub.2SO.sub.4, filtered and concentrated to give 3.5 g (96%) of
compound lxix,
##STR00289##
Intermediate Example 65
Compound lxx
[0437] To a DCM (15 mL) solution containing compound lxix (1.1 g,
2.9 mmol) is added HOAt (0.44 g, 3.2 mmol) followed by a 1 M
solution of DCC (3.2 mL, 3.2 mmol) in DCM. After stirring at about
room temperature for 20 minutes, a DCM (15 mL) solution of compound
xxxix (970 mg. 3.2 mmol) is added. This reaction is stirred
overnight under N.sub.2. The reaction is then diluted with EtOAc
(30 mL), filtered through a pad of silica gel, washed with 0.1 N
HCl, NaHCO.sub.3, and brine. The organic layer is dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Purification
is achieved in 50% EtOAc/Hex on silica gel to give 1.5 g (77%) of
compound lxx,
##STR00290##
Intermediate Example 66
Compound lxxi
[0438] To a methanol solution (30 mL) of compound xx (1.5 g, 2.4
mmol) is added 1 N aqueous NaOH (3.6 mL, 3.6 mmol). The resulting
solution stirred overnight. At this point, the solvent is partially
removed, and the pH of the reaction mixture is adjusted to 3 using
1 N aqueous HCl. The reaction is then diluted with EtOAc (50 mL)
and brine (20 mL). The aqueous layer is extracted with EtOAc
(3.times.50 mL). The organic layers are combined, dried over
Na.sub.2SO.sub.4, filtered and concentrated to provide 1.3 g (92%)
of compound lxxi,
##STR00291##
Intermediate Example 67
Compound lxxii
[0439] To a solution of DCM (2 mL) containing compound lxxi (180
mg, 0.28 mmol) is added PyBOP (175 mg, 0.34 mmol) and DIPEA (0.06
mL, 0.34 mmol), followed by a DCM solution (3 mL) of compound x
(150 mg, 0.41 mmol). The resulting solution is stirred overnight
under N2. The reaction is then diluted with EtOAc (30 mL), washed
with NaHCO.sub.3 and brine. The organic layer is dried over
Na.sub.2SO.sub.4, filtered and concentrated. Purification is
achieved in 100% EtOAc on silica gel to give 270 mg (98%) of
compound lxxii,
##STR00292##
Intermediate Example 68
Compound lxxiii
[0440] To a DCM (3 mL) solution of compound lxxii (270 mg, 0.27
mmol) is added DMP reagent (140 mg, 0.33 mmol). After stirring at
about room temperature for 1.5 hours, the reaction is quenched with
10% Na.sub.2SO.sub.3 (10 mL). The reaction is diluted with EtOAc
(30 mL) and stirred for 10 minutes. The organic layer is washed
with NaHCO.sub.3 and brine. The organic layer is dried over
Na.sub.2SO.sub.4, filtered and concentrated. Purification is
achieved in 60% EtOAc/Hex, to give 150 mg (56%) of compound
lxxiii,
##STR00293##
Intermediate Example 69
Compound lxi
[0441] To an ethanol solution (50 mL) of compound ii (3.5 g, 10.5
mmol) is added under a stream of nitrogen Pd(OH).sub.2/C (1.47 g,
20% Pd content, 2.1 mmol). The reaction is subjected to
hydrogenation under 1 atm pressure. Upon completion, the catalysts
are filtered through a pad of Celite and washed with
dichloromethane. The filtrates are concentrated in vacuo to give 2
g (96%) of compound lxi.
Intermediate Example 70
Compound lxxiv
[0442] To a DMF solution (60 mL) of compound vii (9.1 g, 28.2 mmol)
is added HOAt (4 g, 29.4 mmol) and 1,3-disiopropylcarbodiimide (3.7
g, 29.4 mmol). After stirring at about room temperature for 30
minutes, a DMF solution (10 mL) of compound lxi (5.1 g, 25.6 mmol)
is added to the above solution. The reaction is stirred at about
room temperature overnight. At this point, the white solids are
filtered off. The filtrates are concentrated in vacuo to give a
residue that is purified by silica gel chromatography to give 9.5 g
(67%) of compound lxxiv,
##STR00294##
Intermediate Example 71
Compound lxxv
[0443] To a solution of compound lxxiv (1.5 g, 3 mmol) in anhydrous
THF (25 mL) is added EtiPr.sub.2N (0.78 mL, 4.5 mmol) at about room
temperature. The mixture is cooled to 0.degree. C. and MOMCl (1.5
mL, 19.7 mmol) is added in a dropwise fashion. The reaction is
allowed to warm to room temperature and stirred overnight. The
solution is then diluted with ether and washed with water (3
times). The aqueous layers are extracted further by ether and all
the organic layers are dried over MgSO.sub.4 before being
concentrated to afford a yellow oil. The desired isomer of compound
lxxv is isolated by silica gel chromatography (EtOAc/Hexanes
##STR00295##
5/2) in 40% yield with clear separation of diastereomers.
Intermediate Example 72
Compound lxxvi
[0444] To a solution of compound lxxv (502 mg, 0.9 mmol) in EtOH (5
mL) is added 2 N aqueous NaOH (0.9 mL, 1.8 mmol) dropwise at
0.degree. C. The reaction is allowed to warm to room temperature
and stirred overnight. Upon completion of the saponification, the
solution is acidified to pH 3 with Dowex 50W8X-200 acidic resin.
The solids are filtered off and the resulting filtrate is
concentrated in vacuo to give a oily residue that is lyophilized to
give 370 mg (80%) compound lxxvi,
##STR00296##
Intermediate Example 73
Compound lxxvii
[0445] A dichloromethane solution (4 mL) of compound lxxvi (110 mg,
0.21 mmol) is treated with PyBOP (200 mg, 0.38 mmol). After
stirring at about room temperature for 30 minutes, the reaction
mixture is charged with a THF solution (3.2 mL) of compound xiii
(60 mg, 0.32 mmol), followed by EtiPr.sub.2N. After stirring
overnight at about room temperature, the reaction is quenched with
water and extracted with EtOAc. The resulting organic layer is
washed with brine and dried over MgSO.sub.4, before being
concentrated to a yellow oil. Purification by silica gel
chromatography (5% EtOH/EtOAc) yields 143 mg (100%) of compound
lxxvii,
##STR00297##
Intermediate Example 74
Compound lxxviii
[0446] To a THF solution (50 mL) of H-Chg-OH 2 (5 g, 19.4 mmol) is
added HOBt (2.63 g, 19.4 mmol) and EDCI (3.72 g, 19.4 mmol). After
stirring at about room temperature for 20 minutes, a THF (19 mL)
and DMF (10 mL) solution containing tert-L-Leucine methyl
ester-hydrochloride (19.4 mmol) and DIPEA (6.75 mL, 38.8 mmol) is
added to the above solution. The reaction is stirred at about room
temperature overnight. Standard aqueous work-up and silica gel
chromatography (15-20% EtOAc/Hexanes) affords 2.27 g (30%) of
compound lxxviii,
##STR00298##
Intermediate Example 75
Compound lxxix
[0447] To a THF solution (12 mL) of compound lxxviii (2.27 g, 5.91
mmol) is added 4 N HO solution in dioxane (7.38 mL, 29.5 mmol). The
reaction is stirred at about room temperature overnight. At this
point, the solvent is removed under reduced pressure to yield the
compound lxxix that is used directly for next reaction.
##STR00299##
Intermediate Example 76
Compound lxxx
[0448] To a THF solution of compound lxxix (5.9 mmol) is added to a
THF (20 mL) solution containing 2-pyrazinecarboxylic acid (878 mg,
7.08 mmol), HOBt (957 mg, 7.08 mmol) and EDCI (1.36 g, 7.08 mmol).
To the resulting mixture is then added DIPEA (2.05 mL, 11.8 mmol).
The reaction is stirred overnight at about room temperature and
then quenched with water. The reaction mixture is extracted with
EtOAc. The organic layer is washed with brine and concentrated in
vacuo to provide a residue that is purified by silica gel
chromatography (40-50% EtOAc/Hexanes) to provide 1 g (36%) of
compound lxxx,
##STR00300##
Intermediate Example 77
Compound lxxxi
[0449] To a methanol solution (20 mL) of compound lxxx (1 g, 2.56
mmol) is added 2 N NaOH 3.2 mL, 6.4 mmol). The reaction is stirred
at about room temperature overnight. At this point, the reaction is
acidified to pH 3 using 5 N HCl. The reaction is diluted with EtOAc
(75 mL), and washed with water and brine. The organic layer thus
obtained is dried and concentrated in vacuo to give a residue that
is dissolved in 1:1 CH.sub.3CN/H.sub.2O for lyophilization. A total
of .about.1 g (100%) of compound lxxxi is obtained.
##STR00301##
Intermediate Example 78
Compound lxxxii
[0450] A dichloromethane solution (10 mL) of compound lxxxi (2.56
mmol) is treated with HOAt (348 mg, 2.56 mmol) and DCC (2.56 mL,
1M. 2.56 mmol). After stirring for 30 minutes, the reaction mixture
is treated with a THF solution (5 mL) of compound v (2.56 mmol).
After stirring at about room temperature overnight, the white
solids (urea) are removed by filtration. The filtrates are
concentrated in vacuo to give a residue that is purified by silica
gel chromatography to provide 1.4 g (100%) of the compound
lxxxii,
##STR00302##
Intermediate Example 79
Compound lxxxiii
[0451] An ethanol solution (15 mL) of compound lxxxii (1.4 g, 2.58
mmol) is treated with 2 N NaOH (2.58 mL, 5.17 mmol). After stirring
at about room temperature overnight, the reaction mixture is
acidified to pH 3 with acidic resin. The solids are filtered off.
The resulting filtrates are concentrated in vacuo to give a residue
that is lyophilized to give 1.32 g (.about.100%) of compound
lxxxiii,
##STR00303##
Intermediate Example 80
Compound lxxxiv
[0452] A dichloromethane solution (15 mL) of compound lxxxiii (360
mg, 0.7 mmol) is treated with PyBOP (582 mg, 1.12 mmol). After
stirring at about room temperature for 20 minutes, the reaction
mixture is treated with a THF solution (10 mL) of compound xiii
(195.6 mg, 1.05 mmol), followed by DIPEA (0.25 mL, 1.40 mmol).
After stirring overnight at about room temperature, the reaction is
quenched with water and extracted with EtOAc. The resulting organic
layer is washed with brine and dried and concentrated in vacuo to
give a residue that is purified by silica gel chromatography (3%
EtOH/EtOAc) to afford 420 mg (88%) of compound lxxxiv,
##STR00304##
Intermediate Example 81
Compound ii'''
[0453] A mixture of anhydrous dichloromethane and ether (20 mL, 20
mL) is cooled to -78.degree. C. under N.sub.2 (g). To the solution
is added TiCl.sub.4 (1 M in dichloromethane, 10 mL, 10 mmol) and
then MeLi (1.4 M in ether, 7.1 mL, 10 mmol) is added subsequently
with stirring for another 30 minutes at -78.degree. C. A solution
of compound i (2 g, 6 mmol) in 10 mL dichloromethane is added to
the mixture dropwise at the same temperature over 15 minutes. The
solution is slowly warmed up to -40.degree. C. for 10 minutes and
then stirred at 0.degree. C. for 2 hours. The reaction is quenched
by pouring the mixture into a water/ether mixture (1:1) and then
the layers are allowed to separate. The aqueous layer is further
extracted by ether twice. All organic layers is washed by water,
brine and dried over MgSO.sub.4 before being concentrated to a
yellow oil. The desired compound ii''' is isolated by silica gel
chromatography (EtOAc/Hexanes 2/1) in 83%
##STR00305##
Intermediate Example 82
Compound lxi'
[0454] To the compound ii''' (1.7 g, 5 mmol) is added to 10 wt % Pd
on C (0.53 g, 0.5 mmol), followed by addition of MeOH (17 mL).
Hydrogen gas is flushed through the reaction mixture and hydrogen
gas is maintained for reaction at 1 atm overnight. The reaction
mixture is then filtered and concentrated to afford 929 mg (87%) of
compound lxi' as a colorless oil.
##STR00306##
Intermediate Example 83
Compound lxxxv
[0455] To a THF solution (16 mL) of compound xxii (1 g, 3 mmol) is
added at about room temperature HOAt (0.41 g, 3 mmol) and followed
by 1 M DCC solution of dichloromethane (3 mL, 3 mmol). After
stirring for 30 minutes at about room temperature, a
dichloromethane solution (6 mL) of compound lxi' is added to the
above HOAt-activated acid. The reaction is stirred at about room
temperature overnight. At this point, the reaction Is filtered
through Celite. The filtrate is diluted with EtOAc (120 mL) and
washed with water and then brine. The organic layer is dried and
concentrated to an yellow oil which is purified by silica gel
chromatography (100% EtOAc) to yield 1 g (65%) of compound
lxxxv,
##STR00307##
Intermediate Example 84
Compound lxxxvi
[0456] To an ethanol solution (8 mL) of compound lxxxv (920 mg. 1.7
mmol) is added 2 N NaOH aqueous solution (1.7 mL, 3.4 mmol). The
reaction is stirred overnight at about room temperature and then
acidified to pH 3 by Dowes acidic resin. The solids are filtered
off and the filtrate is concentrated to give a colorless oil, which
is redissolved in 1:1 CH.sub.3CN/H.sub.2O and lyophilized to
provide 800 mg (93%) of compound lxxxvi. HPLC shows a single
product peak.
##STR00308##
Intermediate Example 85
Compound lxxxvii
[0457] To a dichloromethane solution (4 mL) of compound lxxxvi (150
mg. 0.3 mmol) is added by PyBOP (250 mg, 0.47 mmol). The solution
is stirred at about room temperature for 30 minutes. To this
solution is then added a THF (4.5 mL) solution of compound xiii (84
mg, 0.45 mmol) followed by EtiPr.sub.2N (0.1 mL, 0.6 mmol). The
reaction is stirred at about room temperature overnight and then
quenched with water (25 mL) for 30 minutes. The mixture is then
extracted with EtOAc. The resulting organic layer is washed with
brine and dried over MgSO.sub.4, before being concentrated to a
yellow oil. Purification by silica gel chromatography (5%
EtOH/EtOAc) yields 200 mg (100%) of compound lxxxvii,
##STR00309##
Intermediate Example 86
Compound lxxxix
[0458] Compound lxxxviii, N-Cbz-L-Valine, (2.5 g, 9.9 mmol) is
taken up in THF (30 mL).
##STR00310##
EDCI (2.29 g, 11.9 mmol) and HOBT (1.62 g. 11.9 mmol) are added and
the mixture stirred five minutes. L-tert-Leucine methyl ester
hydrochloride (2.17 g, 11.9 mmol) is added in THF (23.9 mL)
followed by DIPEA (2.1 mL). The reaction mixture is stirred
overnight under nitrogen. The reaction mixture is diluted with
ethyl acetate, washed with 1 N HCl, saturated sodium bicarbonate,
and brine. The organic phase is dried over sodium sulfate, filtered
and concentrated. The concentrate residue is purified in 25% ethyl
acetate/hexane to afford 1.1 g (29%) of compound lxxxix,
##STR00311##
Intermediate Example 87
Compound lxxxx
[0459] Compound lxxxix is hydrolyzed under standard conditions
using methyl alcohol (0.3 M) and 1 N NaOH (1.5 eq) to afford 1.03 g
(95%) of compound lxxxx,
##STR00312##
Intermediate Example 88
Compound lxxxxi
[0460] Compound lxxxx (385 mg, 1.06 mmol) is taken up in
dichloromethane (3 mL). DCC (1.4 mmol) is added followed by HOAt
(190 mg, 1.4 mmol). Compound v (260 mg, 1.4 mmol) is then added in
dichloromethane (3 mL). The resulting mixture is stirred overnight
under nitrogen. The reaction is diluted with ethyl acetate,
filtered through silica gel, and concentrated. The residue is
purified in 50% ethyl acetate/hexane to afford 440 mg (80%) of
compound lxxxxi,
##STR00313##
Intermediate Example 89
Compound lxxxxii
[0461] Compound lxxxxi is hydrolyzed under standard conditions
using ethyl alcohol (0.3 M) and 1 N NaOH (1.5 eq) to afford 390 mg
of compound lxxxxii,
##STR00314##
Intermediate Example 90
Compound lxxxxiii
[0462] Compound lxxxxii (350 mg, 0.7 mmol) is taken up in
dichloromethane (3 mL). PyBOP (480 mg, 0.91 mmol) is added followed
by compound xiii (170 mg, 0.91 mmol). DIPEA (0.16 mL, 0.91 mmol) is
added and reaction mixture stirred overnight. The reaction mixture
is concentrated and purified in 100% ethyl acetate to afford 420 mg
(90%) of compound lxxxxiii,
##STR00315##
Intermediate Example 91
Compound lxxxxiv
[0463] Compound lxxxxiii is hydrogenated using 10% Pd/C (1% mol) in
methyl alcohol under hydrogen to afford 335 mg (100%) of compound
lxxxxiv,
##STR00316##
Intermediate Example 92
Compound lxxxxv
[0464] Ethyl 1H-tetrazole-5-acetate (5 g, 32 mmol) is taken up in
chloroform (80 mL). Trichloroacetic acid (12.03 g, 73.65 trump is
added followed by alpha methyl styrene (3.78 g, 32 mmol). The
reaction mixture is stirred overnight. The next day, the solution
is diluted with ethyl acetate, washed with 10% KOH and brine. The
organic phase is dried over magnesium sulfate, filtered and
concentrated to afford 8 g (96%) of the corresponding N-protected
ethyl tetrazole-5-acetate. This material is subjected to standard
hydrolysis conditions using ethyl alcohol (0.3 M) and 1 N NaOH (3
eq) to afford 7 g (99%) of compound lxxxxv,
##STR00317##
Intermediate Example 93
Compound lxxxxvi
[0465] Compound lxxxxv (3.62 g, 14.7 mmol) is taken up in
dichloromethane (50 mL). EDCI (4.32 g, 22.1 mmol) and DIPEA (5.1
mL, 29.4 mmol) are added and stirred for five minutes. N-hydroxy
succinimide (3.38 g, 29.4 mmol) is added and stirred three hours.
The reaction is diluted with dichloromethane and washed with water
three times. The organic phase is dried over sodium sulfate,
filtered and concentrated to afford 3.66 g (73%) of compound
lxxxxvi,
##STR00318##
Intermediate Example 94
Compound lxxxxvii
[0466] Compound lxxxxiv (335 mg, 0.62 mmol) and compound lxxxxvi
(343 mg, 1 mmol) are taken up in dichloromethane (6 mL). DIPEA
(0.17 mL, 1 mmol) is added and reaction mixture stirred overnight.
The reaction is diluted with ethyl acetate, washed with saturated
sodium bicarbonate, brine and concentrated. The residue is purified
in 5% ethyl alcohol/ethyl acetate to give 80 mg (16%) of compound
lxxxxvii,
##STR00319##
Intermediate Example 95
Compound lxxxxviii
[0467] Compound lxxxxvii (80 mg, 0.11 mmol) is taken up in
dichloromethane (3 mL). DMP reagent (55 mg, 0.13 mmol) is added and
stirred for one hour. The reaction mixture is diluted with ethyl
acetate and quenched with 10% solution of sodium sulfite. The
organic phase is washed with saturated sodium bicarbonate and
brine. The organic phase is concentrated and the resulting residue
is purified in 100% ethyl acetate to afford 40 mg (48%) of compound
lxxxxviii,
##STR00320##
Intermediate Example 96
Compound xxxix
[0468] Compound ic, N-Cbz-4-Hydroxy Pro methyl ester, (2.1 g, 7.9
mmol is prepared in
##STR00321##
quantitative yield from compound c, N-Cbz-4-hydroxy Pro), is
dissolved in DCM (25
##STR00322##
mL). CDI (1.54 g, 9.5 mmol) and DIPEA (1.7 mL, 9.5 mmol) are added
to the solution and stirred for 10 minutes.
1,2,3,4-Tetrahydroisoquinoline (TIQ) (1.2 mL, 9.5 mmol) is added
drop-wise to the reaction mixture and stirred five hours. The
organic phase is washed with water, 1 N HCl, and brine. Following
the concentration of the organic phase, the resultant residue is
chromatographically purified by 40% EtOAc/Hexanes to yield compound
ci, N-Cbz-4-TIQcarbonyloxy-Pro methyl ester, (2.5 g, 75%).
##STR00323##
[0469] Compound ci (2.5 g. 5.9 mmol) is dissolved in MeOH (75 mL).
The solution is flushed with N.sub.2 and Pd/C (10%, 300 mg) is
added. The reaction mixture is flushed with H.sub.2 and stirred
overnight. The reaction mixture is filtered through Celite and
concentrated to yield compound compound xxxix.
4-(TIQ-carbonyloxy)-Pro, methyl ester, (1.49 g, 83%).
Intermediate Example 97
Compound vii
[0470] Compound cii, N-pyrazin-2-ylcarbonyl-Val-Val methyl ester.
(10.9 g. 32.4 mmol) is
##STR00324##
dissolved in THF (80 mL), and then aqueous NaOH (48.6 mL, 48.6
mmol) is added. The resulting mixture is stirred 48 hours, and then
additional NaOH (16.3 mL, 16.3 mmol) is added and mixture is heated
to 40.degree. C. for three hours. The pH of the reaction mixture is
then lowered to 3, and the aqueous phase extracted with EtOAc and
then concentrated to yield crude compound vii,
N-pyrazin-2-ylcarbonyl-Val-Val acid (10.6 g, 100%).
Intermediate Example 98
Compound ciii
[0471] Compound cii (4.1 g, 12.7 mmol) is dissolved in DCM (20 mL).
HOAt (1.73 g. 12.7 mmol) and DCC (12.7 mmol) are added to this
solution, and the solution stirred for one hour. Compound xxxix
(3.22 g, 10.6 mmol) is added to reaction mixture in DCM (10 mL).
The resulting mixture is stirred overnight under N.sub.2. The
reaction mixture is filtered through silica gel and concentrated.
The resulting residue is purified by silica gel chromatography (50%
to 80% EtOAc/Hexanes gradient) to yield compound ciii,
[0472] N-pyrazin-2-ylcarbonyl-Val-Val-4-(TIQcarbonyloxy)-Pro methyl
ester, (5.27 g, 81.7%).
##STR00325##
Intermediate Example 99
Compound civ
[0473] Compound ciii (650 mg, 1.29 mmol) is dissolved in THF (5
mL). Aqueous NaOH (1.42 mL, 1.42 mmol) is added to the solution and
then stirred overnight. The pH of the solution is lowered to 3, and
the organic phase is isolated and concentrated to yield a residue.
The residue is purified using reverse phase HPLC in
acetonitrile/water to yield compound civ,
N-pyrazin-2-ylcarbonyl-Val-Val-4-(TIQcarbonyloxy)-Pro acid, (600
mg, 95%).
##STR00326##
Intermediate Example 100
Compound cv
[0474] N-Boc-L-tert-Leucine (2.3 g, 10 mmol) and L-ten-Leucine
methyl ester hydrochloride (2 g, 11 mmol) are combined in DMF (30
mL). HOAt (1.6 g, 11.5 mmol) is then added to the solution. The
resulting mixture is stirred for 20 minutes under N2 and then
lowered to 0.degree. C. whereupon DIC (1.8 mL, 11.5 mmol) and
2,4,6-collidine (1.45 mL, 11 mmol) are added. The resulting
solution is stirred overnight with warming to room temperature. The
reaction mixture is diluted with EtOAc, and the organic phase
washed with 1 N HCl, saturated NaHCO.sub.3 and brine. Following the
concentration of the organic phase, the resultant residue is
chromatographically purified by 20%-30% EtOAc/hexanes gradient to
yield compound cv (3.3 g, 92%).
##STR00327##
Intermediate Example 101
Compound cvi
[0475] Compound cv (3.3 g, 9.2 mmol) is hydrolyzed using dioxane
(40 mL) and 0.5 N NaOH (37 mL, 18.4 mmol) to yield compound cvi
(2.9 g, 92%).
##STR00328##
Intermediate Example 102
Compound cvii
[0476] Compound cvi (2 g, 5.8 mmol) and compound v (1 g, 5.5 mmol)
are dissolved in DMF (20 mL). HOAt (832 mg, 6.6 mmol) and DIC (1.1
mL, 6.6 mmol) are then added to the solution. The resulting
solution is stirred overnight under N.sub.2. The reaction mixture
is diluted with EtOAc, and the organic phase washed with 1 N HCl,
saturated NaHCO.sub.3 and brine. Following the concentration of the
organic phase, the resultant residue is chromatographically
purified by 20%-30% EtOAc/hexanes gradient to yield compound cvii
(2.4 g, 81%).
##STR00329##
Intermediate Example 103
Compound cviii
[0477] Compound cvii (2.4 g, 4.72 mmol) is dissolved in DCM (10
mL). TFA (10 mL) is added to the solution. The resulting solution
is stirred for 4 hours. The reaction mixture is concentrated,
dissolved in EtOAc, and then the organic phase is washed with 1 N
NaOH and brine. The organic phase is concentrated to yield compound
cviii (1.084 g, 56.1%).
##STR00330##
Intermediate Example 104
Compound cix
[0478] 2-Pyrazinecarboxylic acid (181 mg, 1.46 mmol) and compound
cviii (541 mg, 1.325 mmol) are dissolved in DMF (15 mL). HOAt (207
mg, 1.52 mmol) and DIC (0.24 mL, 1.52 mmol) are added to the
solution. The resulting solution is stirred overnight under
N.sub.2. The reaction mixture is diluted with EtOAc, and the
organic phase washed with 1 N HCl, saturated NaHCO.sub.3 and brine.
Following the concentration of the organic phase, the resultant
residue is chromatographically purified by 20%-30%-35%
EtOAc/hexanes gradient to yield compound cix (430 mg, 63%).
##STR00331##
Intermediate Example 105
Compound cx
[0479] Compound cix is hydrolyzed using EtOH (7 mL) and 1 N NaOH
(4.7 mL, 4.7 mmol) to yield compound cx (700 mg, 91.6%).
##STR00332##
Intermediate Example 106
Compound cxi
[0480] Compound cx (690 mg, 1.42 mmol) is dissolved in DCM (9 mL).
PyBOP (890 mg, 1.7 mmol) is then added to the solution, followed by
the addition of Compound xiii' (320
##STR00333##
mg, 1.7 mmol). To the resulting mixture is added DIPEA (0.3 mL, 1.7
mmol). The reaction mixture is stirred overnight under N.sub.2. The
reaction mixture is then diluted with EtOAc, washed with saturated
NaHCO.sub.3, and brine. Following the concentration of the organic
phase, the resultant residue is chromatographically purified by
100% EtOAc to yield compound cxi (490 mg, 52.7%).
##STR00334##
Intermediate Example 107
Compound cxiv
[0481] Compound cxii (1.2 g, 3.06 mmol) is dissolved in MeOH (12
mL). After thoroughly
##STR00335##
flushing with N.sub.2, 10 wt % Pd(OH).sub.2 on carbon (0.6 g) is
added and the mixture is hydrogenated for overnight, whereupon a
complete reaction mixture is shown by TLC (30% EtOAc/hexanes). The
solution is isolated from solid material by filtration and
concentrated to the corresponding deprotected compound cxiiias a
colorless oil (100%)
##STR00336##
that is used in the next step without further purification.
[0482] 2-Pyrazinecarboxylic acid (400 mg, 3.2 mmol, 1.1 eq) is
dissolved in DCM/THF (4 mL/4 mL), and then HOAt (440 mg, 3.2 mmol)
and DCC (343 mL, 1 M in DCM) are added. After stirring at room
temperature for 20 minutes, the compound cxiii (0.96 g, 3.2 mmol)
obtained previously is dissolved in DCM (6.4 mL) and added to the
activated mixture. After stirring overnight at room temperature,
the reaction mixture is filtered through Celite and compound cxiv
is purified by column chromatography (30% EtOAc/hexanes)
##STR00337##
to yield a white solid (0.8 g, 80%).
Intermediate Example 108
Compound cxv
[0483] Compound cxiv (0.8 g, 2.2 mmol) is dissolved in MeOH (10
mL), and then 2 N NaOH (aq) (3.3 mL, 6.6 mmol) is added. The
solution is stirred at room temperature overnight, whereupon the
completion of the reaction mixture is indicated by TLC (50%
EtOAc/hexanes). Acidification to pH 3 by 5 N HCl and diluted with
EtOAc is followed by extraction of the organic phase. The extracted
organic phase is washed with brine and dried over MgSO.sub.4 to
yield compound cxv (0.74, 95%) upon concentration.
##STR00338##
Intermediate Example 109
Compound cxvi
[0484] To a DCM solution (6 mL) of compound cxv (0.74 g. 2.1 mmol)
at room temperature is added HOAt (290 mg, 2.1 mmol), followed by
the addition of 1 M DCC solution in DCM (2.2 mL, 2.2 mmol). After
stirring for 30 minutes at room temperature, a THF solution (10.5
mL, 0.2 M) of compound v (2.1 mmol) is added to the above
HOAt-activated acid. The reaction mixture is stirred at room
temperature overnight. At this point, the reaction mixture is
filtered through celite. The filtrate is diluted with EtOAc (120
mL) and washed with water and brine. The organic phase is dried and
concentrated to a yellow oil that is purified by silica gel
chromatography (50% EtOAc/hexanes) to yield compound cxvi (0.714 g,
66%).
##STR00339##
Intermediate Example 110
Compound cxvii
[0485] To an EtOH solution of compound cxvi (0.7 g, 1.4 mmol) is
added 2 N NaOH aqueous solution (2 mL, 4 mmol). The reaction
mixture is stirred overnight at room temperature, then acidified to
pH 3 by 5 N HCl and diluted with EtOAc is followed by extraction of
the organic phase. The extracted organic phase is washed with brine
and dried over MgSO.sub.4 to yield compound cxvii (95%) upon
concentration.
##STR00340##
Intermediate Example 111
Compound cxviii
[0486] To a DCM/THF solution (10 mL/2 mL) of compound cvii (300 mg,
0.6 mmol) is added PyBOP (416 mg, 0.8 mmol). The solution is
stirred at room temperature for 30 minutes. To this solution is
then added compound xxxvi' (200 mg, 0.8 mmol), followed by
DIPEA
##STR00341##
(0.22 mL, 1.2 mmol). The reaction mixture is stirred at room
temperature overnight and then quenched with water (25 mL) for 30
minutes. The mixture is then extracted with EtOAc. The resulting
organic phase is washed with brine and dried over MgSO.sub.4,
before being concentrated to yield a yellow oil. Purification by
silica gel chromatography (3-5% EtOH/EtOAc) yields compound cxviii
(335 mg, 76%).
##STR00342##
Intermediate Example 112
Compound cxix
[0487] To a DCM solution (10 mL) of compound cxvii (340 mg, 0.6
mmol) is added PyBOP (470 mg, 0.9 mmol). The solution is stirred at
room temperature for 30 minutes. To this solution is then added
compound xiii' (170 mg. 0.9 mmol), followed by DIPEA (0.24 mL, 1.2
mmol). The reaction mixture is stirred at room temperature
overnight and then quenched with water (25 mL) for 30 minutes. The
mixture is then extracted with EtOAc. The resulting organic phase
is washed with brine and dried over MgSO.sub.4, before being
concentrated to yellow oil. Purification by silica gel
chromatography (3-5% EtOH/EtOAc) yields compound cxix (164 mg,
36%).
##STR00343##
Intermediate Example 113
Compound xx
[0488] N-Cbz-L-Valine (6.28 g, 25 mmol) is dissolved in DCM (30
mL). HOBT (3.38 g, 25 mmol) and DCC (2.5 mL, 1 M solution) are
added to this solution and stirred five minutes. L-tert-Leucine
methyl ester hydrochloride (25 mL, 1 M solution) is added to this
mixture and stirred overnight under N.sub.2. The reaction mixture
is diluted with EtOAc, washed with 1 N HO, saturated NaHCO.sub.3,
and brine. The organic phase is dried over Na.sub.2SO.sub.4,
filtered and concentrated. The residue is chromatographically
purified by 20%-30% EtOAc/hexanes to yield compound xx (2.96 g,
31%).
Intermediate Example 114
Compound xxi
[0489] Compound xx (2.95 g, 7.8 mmol) is hydrogenated using 10%
Pd/C (800 mg) in MeOH (40 mL) under H.sub.2 to yield the below
corresponding free amine (1.9 g, 100%).
##STR00344##
2-Pyrazine-carboxylic acid (970 mg, 7.8 mmol) is dissolved in DCM
(20 mL). PyBOP (4.06 g, 7.8 mmol) is added to this solution. The
free amine (1.9 g. 7.8 mmol) in DCM (15 mL) is added to the
solution, and then DIPEA (1.36 mL. 7.8 mmol) is added. The
resulting mixture is stirred overnight under N.sub.2. The reaction
mixture is diluted with EtOAc, and the organic phase is washed with
saturated NaHCO.sub.3 and brine. Following the concentration of the
organic phase, the residue is chromatographically purified by
30%-40% EtOAc/Hexanes to yield compound xxi (2.07 g, 75.8%).
##STR00345##
Intermediate Example 115
Compound xxii
[0490] Compound xxi is hydrolyzed using MeOH (20 mL) and 1 N NaOH
(3 eq) to yield compound xxii (1.82 g, 93.9%).
##STR00346##
Intermediate Example 116
Compound xxiii
[0491] Compound xxii (895 mg, 2.66 mmol) is dissolved in DCM (10
mL). DCC (3.2 mmol) is added to the solution, and then HOAt (435
mg, 3.2 mmol) is added. Compound v (3.2 mmol) in THF (16 mL) is
then added. The resulting mixture is stirred overnight under
N.sub.2. The reaction mixture is diluted with EtOAc, filtered
through silica gel, and concentrated. The resulting residue is
chromatographically purified by 50% EtOAc/hexanes to yield compound
xxiii (730 mg, 54.8%).
Intermediate Example 117
Compound xxiv
[0492] Compound xxiii is hydrolyzed using EtOH (5 mL) and 1 N NaOH
(1.5 eq) to yield compound xxiv (690 mg, 100%).
Intermediate Example 118
Compound cxx
[0493] Compound xxiv (245 mg, 0.52 mmol) is dissolved in DCM (3
mL). PyBOP (330 mg, 0.62 mmol) is added to the solution, and then
compound xiii' (120 mg, 0.62 mmol) is added. To the resulting
mixture is added DIPEA (0.11 mL, 0.62 mmol). The reaction mixture
is stirred overnight under N.sub.2. The reaction mixture is diluted
with EtOAc, and the organic phase washed with saturated NaHCO.sub.3
and brine. Following the concentration of the organic phase, the
residue is chromatographically purified by 5% EtOH/EtOAc to yield
compound cxx (220 mg, 60%).
##STR00347##
Intermediate Example 119
Compound xiii'
[0494] Boc-NVA-OH (24.96 g. 114.9 mmol) is dissolved in THF (200
mL). CDI (22.35, 137.8
##STR00348##
mmol) is added portion-wise to the solution, and the solution is
stirred for 30 minutes. N,O-Dimethylhydroxylamine hydrochloride
(12.33 g, 126.4 mmol) is dissolved in DMF (50 mL) and then DIPEA
(22 mL, 126.4 mmol) is added to the solution. The DMF solution is
allowed to stir at room temperature for 20 minutes and then added
to THF solution. The resulting mixture is stirred over a weekend
under N.sub.2. The reaction mixture is concentrated in vacuo to 100
mL total volume. This organic phase is washed with 1 N HCl,
saturated NaHCO.sub.3 and brine. The organic phase is concentrated
to yield crude compound cxxi (25.3 g).
##STR00349##
[0495] LAH (107.3 mmol) is added to a dry 1-L round bottom flask
under N.sub.2 in a 1 M Et.sub.2O solution. This solution is lowered
to 0.degree. C., and then compound cxxi (97.5 mmol) is added
drop-wise in Et.sub.2O (100 mL). Upon completion of the addition,
the resulting mixture is stirred for 30 minutes. The reaction
mixture is quenched at 0.degree. C. by slowly adding EtOAc (50 mL),
followed by slowly adding a 5% KHSO.sub.4 (50 mL) solution. This
mixture is stirred for 30 minutes. The organic phase is washed with
1 N HCl, saturated NaHCO.sub.3, and brine. The organic phase is
concentrated to yield crude compound cxxii (22.28 g).
##STR00350##
[0496] Compound cxxii is dissolved in MeOH (100 mL).
Na.sub.2S.sub.2O.sub.4 (16.82 g, 96.6 mmol) is dissolved in water
(100 mL and then added to solution of compound cxxii at 0.degree.
C. This mixture is stored in the refrigerator (5.degree. C.)
overnight. KCN (7.53 g, 115.9 mmol) in water (100 mL) is added to
reaction mixture and stirred for 1.5 hours at room temperature. The
compound is extracted with EtOAc (3.times.100 mL). The organic
phase is washed with brine (3.times.50 mL), dried over MgSO.sub.4,
filtered and concentrated to yield crude compound cxxiii (15.86
g).
##STR00351##
[0497] Compound cxxiii (15.86 g) is dissolved in dioxane (100 mL).
Concentrated HCl (37%, 100 mL) is added to this solution followed
by anisole (10 mL) and reflux is established (110.degree. C.). The
reaction stirred for 1.5 hours. When the reaction mixture is cooled
to room temperature, the solvent is removed in vacuo to yield a dry
paste. The residue is dried overnight under high vacuum to yield
crude compound cxxiv.
##STR00352##
[0498] Compound cxxiv (69.6 mmol) is dissolved in DMF (60 mL) and
THF (60 mL). N-(Benzyl-oxycarbonyloxy)succinimide (17.33 g, 69.6
mmol) is added to the mixture, followed by the addition of DIPEA
(12.1 mL, 69.6 mmol). The reaction mixture is stirred overnight
under N2. The mixture is concentrated to a reduced volume (50 mL)
and diluted with EtOAc. The organic phase is washed with 0.1 N HCl
(2.times.100 mL) and brine to yield compound cxxv (17.5 g. 54.2%
over five steps).
##STR00353##
[0499] Compound cxxv (5.66 g, 20.14 mmol) is dissolved in DCM (60
mL). PyBOP (12.57 g, 24.2 mmol) and HOBT (3.27 g, 24.2 mmol) are
added to this solution and stirred five minutes. The resulting
mixture is lowered to 0.degree. C., and then cyclopropylamine (1.67
mL, 24.2 mmol) and DIPEA (4.2 mL, 24.2 mmol) are added. The
reaction mixture is stirred overnight warming to room temperature.
The reaction mixture is washed with 0.1 N HCl, saturated
NaHCO.sub.3, and brine. The organic phase is then concentrated and
chromatographically purified using 70% EtOAc/Hexanes to yield
compound cxxvi (3.18 g, 49.3%).
##STR00354##
[0500] Compound cxxvi (3.18 g, 9.94 mmol) is hydrogenated using 10%
Pd/C (600 mg) in MeOH (70 mL). The reaction mixture is stirred
overnight under H.sub.2, filtered through celite and concentrated
to yield crude compound xiii' (2.1 g, 100%).
##STR00355##
Intermediate Example 120
Compound cxxvii
[0501] N-Cbz-L-Cyclohexylglycine (3 g, 10.3 mmol) is dissolved in
DCM (36 mL). HOAt (1.5 g, 11.28 mmol) and DCC (11.28 mL, 11.28
mmol) are added to this solution and stirred five minutes.
L-tert-Leucine methyl ester hydrochloride (103 mL, 1 M solution,
10.3 mmol) is added to this mixture and stirred overnight under
N.sub.2. The reaction mixture is filtered through celite, rinsed
with EtOAc and concentrated to a residue that is purified
chromatographically using 20%-30% EtOAc/hexanes to yield compound
cxxvii (2.2 g, 52%).
##STR00356##
Intermediate Example 121
Compound lxxix'
[0502] Compound cxxvii (2.2 g, 5.2 mmol) is hydrogenated using 20%
Pd(OH).sub.2/C (1 g) in MeOH (15 mL) under H.sub.2 to yield
compound lxxix' (1.4 g, 98%).
##STR00357##
Intermediate Example 122
Compound lxxx
[0503] 2-Pyrazinecarboxylic acid (360 mg, 2.9 mmol) is dissolved in
DCM (10 mL). PyBOP (1.81 g, 3.5 mmol) is added to the solution.
Compound lxxix' (825 mg, 2.9 mmol) in THF (10 mL) is then added to
the solution, followed by the addition of DIPEA (0.5 mL, 2.9 mmol).
The resulting mixture is stirred overnight under N.sub.2. The
reaction mixture is diluted with EtOAc, and the organic phase
washed with saturated NaHCO.sub.3 and brine. The residue resulting
from the concentration of the organic phase is chromatographically
purified by 30% EtOAc/Hexanes to yield Compound lxxx (780 mg,
69%).
Intermediate Example 123
Compound lxxxi
[0504] Compound lxxx is hydrolyzed using MeOH (10 mL) and 1 N NaOH
(3 eq) to yield compound lxxxi (615 mg, 81.8%).
Intermediate Example 124
Compound lxxxii
[0505] Compound lxxxi (610 mg, 1.6 mmol) is dissolved in DCM (10
mL). DCC (1.94 mL, 1.94 mmol) is then added to the solution,
followed by the addition of HOAt (270 mg, 1.94 mmol). Compound v
(1.94 mmol) in THF (19.4 mL) is then added to the solution. The
resulting mixture is stirred over two nights under N.sub.a. The
reaction mixture is diluted with EtOAc, filtered through silica
gel, and concentrated. The resulting residue is purified
chromatographically by 40% EtOAc/hexanes to yield compound lxxxii
(450 mg. 83.4%).
Intermediate Example 125
Compound lxxxiii
[0506] Compound lxxxi is hydrolyzed using EtOH (10 mL) and 1 N NaOH
(3 eq) to yield compound lxxxiii (650 mg, 99%).
Intermediate Example 126
Compound cxxviii
[0507] Compound lxxxiii (400 mg, 0.78 mmol) is dissolved in DCM (5
mL). PyBOP (610 mg, 1.2 mmol) is added to the solution, followed by
Compound xiii' (230 mg, 1.2 mmol). To the resulting mixture is
added DIPEA (0.2 mL, 1.2 mmol). The reaction mixture is stirred
overnight under N.sub.2. The reaction mixture is diluted with
EtOAc, and the organic phase washed with saturated NaHCO.sub.3 and
brine. Following the concentration of the organic phase, the
residue is chromatographically purified by 100% EtOAc to 5%
EtOH/EtOAc gradient to yield compound cxxviii (365 mg, 68.7%).
##STR00358##
Intermediate Example 127
Compound cxxx
[0508] Compound lxxxiii (365 mg, 0.7 mmol) is dissolved in DCM (5
mL). PyBOP (440 mg, 0.84 mmol) is added to the solution, followed
by the addition of compound cxxix (0.84
##STR00359##
mmol) in THF (8.4 mL). To the resulting mixture is added DIPEA (0.1
mL, 0.84 mmol). The reaction mixture is stirred overnight under
N.sub.2. The reaction mixture is diluted with EtOAc, and the
organic phase is washed with saturated NaHCO.sub.3 and brine.
Following the concentration of the organic phase, the resulting
residue is chromatographically purified by 100% EtOAc to yield
compound cxxx (350 mg, 70%).
##STR00360##
Intermediate Example 128
Compound cxxxi
[0509] Compound cxxv (2.54 g, 9.05 mmol) is dissolved in DCM (30
PyBOP (5.65 g, 10.9 mmol) and HOBT (1.47 g, 10.9 mmol) are added to
the solution and stirred five minutes. The resulting mixture is
lowered to 0.degree. C., whereupon (S)-(+)-3-Methyl-2-butylamine
(1.27 mL, 10.9 mmol) and DIPEA (1.9 mL, 10.9 mmol) are added. The
reaction mixture is stirred overnight with warming to room
temperature. The organic phase is washed with 0.1 N HCl, saturated
NaHCO.sub.3 and brine. Following the concentration of the organic
phase, the resultant residue is chromatographically purified by 30%
EtOAc/hexanes to yield compound cxxxi (1.44 g, 453%).
##STR00361##
Intermediate Example 129
Compound cxxix
[0510] Compound cxxxi (1.3 g. 3.7 mmol) is hydrogenated using 10%
Pd/C (500 mg) in MeOH (40 mL). The reaction mixture is stirred
overnight under H.sub.2. The reaction mixture is filtered through
celite and the organic phase concentrated to yield crude compound
cxxix (800 mg, 100%).
##STR00362##
Intermediate Example 130
Compound cxxxiv
[0511] Compound cxxxii (1.6 g, 3.7 mmol) is dissolved in MeOH (12
mL). After thoroughly
##STR00363##
flushing with N.sub.2, 10 wt % Pd(OH).sub.2 on carbon (0.74 g) is
added and the mixture is hydrogenated overnight, whereupon a
complete reaction mixture is shown by TLC (30% EtOAc/hexanes). The
solution is isolated from solid material by filtration and
concentrated to yield compound cxxxiii as colorless oil (100%) that
is used in the next
##STR00364##
step without further purification. 2-Pyrazinecarboxylic acid (400
mg, 3.2 mmol, 1.1 eq) is dissolved in DCM/THF (4 mL/4 mL), and then
HOAt (440 mg, 3.2 mmol) and DCC (3.3 mL, 1 M in DCM) is added.
After stirring at room temperature for 20 minutes, compound cxxxiii
(0.96 g, 3.2 mmol) obtained previously is dissolved in DCM (6.4 mL)
and added to the activated mixture. After stirring over 2 days at
room temperature, the reaction mixture is filtered through Celite,
and concentrated to a residue that is purified by column
chromatography (50% EtOAc/hexanes) to yield compound cxxxiv as a
white solid (1.06 g, 83%).
##STR00365##
Intermediate Example 131
Compound cxxxv
[0512] Compound cxxxiv (1.06 g, 2.6 mmol) is dissolved in MeOH (10
mL), and then 2 N NaOH (aq) (4 mL, 8 mmol) is added. The solution
is stirred at room temperature overnight, whereupon the completion
of the hydrolysis is indicated by TLC (50% EtOAc/hexanes). The
solution is acidified to pH 3 by 5 N HCl, diluted with EtOAc and
then the organic phase is extracted. The extracted organic phase is
washed with brine and dried over MgSO.sub.4 to yield compound cxxxv
(100%) upon concentration.
##STR00366##
Intermediate Example 132
Compound cxxxvi
[0513] To a DCM solution (8 mL) of compound cxxxv (1.44 g, 3.7
mmol) at room temperature is added HOAt (500 mg, 3.7 mmol), and
then 1 M DCC solution in DCM (3.7 mL, 3.7 mmol) is added. After
stirring for 30 minutes at room temperature, a THF solution (18.5
mL, 0.2 M) of compound v (3.7 mmol) is added to the above
HOAt-activated acid. The reaction mixture is stirred at room
temperature overnight. The reaction mixture is filtered through
Celite. The filtrates are diluted with EtOAc (120 mL) and washed
with water and brine. The organic phase is dried and concentrated
to yield yellow oil that is purified by silica gel chromatography
(70% EtOAc/hexanes) to yield compound cxxxvi (1 g, 71%).
##STR00367##
Intermediate Example 133
Compound cxxxvii
[0514] To an EtOH solution (8 mL) of compound cxxxvi (1 g, 1.8
mmol) is added 2 N NaOH aqueous solution (2.7 mL, 5.4 mmol). The
reaction mixture is stirred overnight at room temperature, then
acidified to pH 3 by 5 N HCl, diluted with EtOAc, and then the
organic phase is extracted. The extracted organic phase is washed
with brine and dried over MgSO.sub.4 to yield compound cxxxvii
(88%) upon concentration.
##STR00368##
Intermediate Example 133
Compound cxxxviii
[0515] To a DCM solution (10 mL) of compound cxxxvii (350 mg, 0.6
mmol) is added PyBOP (450 mg, 0.86 mmol). The solution is stirred
at room temperature for 30 minutes. To this solution is then added
compound xiii' (160 mg, 0.86 mmol) followed by DIPEA (0.23 mL, 1.3
mmol). The reaction mixture is stirred at room temperature
overnight and then quenched with water (25 mL) for 30 minutes. The
mixture is then extracted with EtOAc. The extracted organic phase
is washed with brine and dried over MgSO.sub.4, before being
concentrated to yield yellow oil. Purification by silica gel
chromatography (5% EtOH/EtOAc) yields compound cxxxviii (407 mg,
88%).
##STR00369##
Intermediate Example 134
Compound cxxxix
[0516] 5-Methylisoxazole-3-carboxylic acid (200 mg, 2.05 mmol) is
dissolved in DCM (5 mL). PyBOP (1.07 g, 2.05 mmol) is added to the
solution. Compound lxxix' (582 mg, 2.05 mmol) in DCM (5 mL) is
added to the solution, followed by the addition of DIPEA (0.36 mL,
2.05 mmol). The resulting mixture is stirred overnight under
N.sub.2. The reaction mixture is diluted with EtOAc, and the
organic phase washed with saturated NaHCO.sub.3 and brine. The
organic phase is concentrated, and the resultant residue is
purified chromato-graphically by 30% EtOAc/hexanes to yield
Compound cxxxix (495 mg, 61.4%).
##STR00370##
Intermediate Example 135
Compound cxxxx
[0517] Compound cxxxix is hydrolyzed using MeOH (10 mL) and 1 N
NaOH (3 eq) to yield compound cxxxx (430 mg, 90%).
##STR00371##
Intermediate Example 136
Compound cxxxxi
[0518] Compound cxxxx (380 mg, 1 mmol) is dissolved in DCM (5 mL).
DCC (1.2 mmol) is then added to the solution, followed by the
addition of HOAt (165 mg, 1.2 mmol). Compound v (1.2 mmol) is then
added in THF (12 mL). The resulting mixture is stirred overnight
under N.sub.2. The reaction mixture is diluted with EtOAc, filtered
through silica gel, and concentrated. The resultant residue is
chromatographically purified by 35% EtOAc/hexanes to yield compound
cxxxxi (320 mg. 58%).
##STR00372##
Intermediate Example 137
Compound cxxxxii
[0519] Compound cxxxxi is hydrolyzed using EtOH (10 mL) and 1 N
NaOH (3 eq) to yield compound cxxxxii (730 mg, 94.3%).
##STR00373##
Intermediate Example 138
Compound cxxxxiii
[0520] Compound cxxxxii (240 mg, 0.46 mmol) is dissolved in DCM (5
mL). PyBOP (295 mg 0.56 mmol) is then added to the solution,
followed by the addition of Compound xiii' (110 mg. 0.56 mmol). To
the resulting mixture is added DIPEA (0.1 mL, 0.56 mmol). The
reaction mixture is stirred over two nights under N.sub.2. The
reaction mixture is diluted with EtOAc, and the organic phase
washed with saturated NaHCO.sub.3 and brine. Following the
concentration of the organic phase, the resultant residue is
chromatographically purified by 90% EtOAc/hexanes to yield Compound
cxxxxiii (168 mg, 53%).
##STR00374##
Intermediate Example 139
Compound cxxxxiv
[0521] To a solution of NaOH (2N, 42.1 mL, 84.2 mmol) at 5.degree.
C. is added L-alanine (5.00 g 56.1 mmol). After stirring for 10
minutes, methyl chloroformate (6.5 mL, 84.2 mmol) and NaOH (2 N.
42.1 mL, 84.2 mmol) are added dropwise simultaneously. The solution
is stirred in ice bath for 2 hours, then at room temperature for 1
hour. The mixture is washed with Et.sub.2O (2.times.50 mL), the
aqueous layer is neutralized to pH .about.2 with 5 N HCl, and
extracted with EtOAc (3.times.50 mL). The extracted organic phase
is washed with brine, dried by MgSO.sub.4 and concentrated to yield
compound cxxxxiv,
##STR00375##
N-carbomethoxy-L-alanine. (4.54 g, 54%) as colorless oil.
Intermediate Example 140
Compound cxxxxvi
[0522] A solution of compound cxxxxv (3.57 g, 9.44 mmol) in THF at
5.degree. C. is treated with HOAt
##STR00376##
(1.28 g, 9.44 mmol), and then DCC (9.50 mL, 9.50 mmol) is added.
After stirring in ice bath for 45 minutes, a solution of compound v
(104 mL, 10.4 mmol) in THF is added. The mixture is stirred at room
temperature overnight. The mixture is cooled to 5.degree. C. and
quenched with saturated NaHCO.sub.3. After filtration to remove the
precipitated DCU, the mixture is dissolved in EtOAc (100 mL),
washed with saturated NaHCO.sub.3, brine, and then dried by
MgSO.sub.4 and concentrated to a residue purified by silica column
chromatography (25% EtOAc/Hexanes) to yield compound cxxxxvi (2.91
g, 57%) as gummy foam.
##STR00377##
Intermediate Example 141
Compound cviii
[0523] To a solution of compound cxxxxvi in MeOH (25 mL) cooled by
an ice bath under a stream of N.sub.2 is added slowly Pd/C. The
mixture is hydrogenated at 1 atm overnight. The catalyst is removed
by filtration, the filtrate is combined with 5 mL DMF and dried
under vacuum to yield compound cviii.
Intermediate Example 142
Compound cxxxxvii
[0524] To a solution of compound cxxxxiv (0.298 g, 2.03 mmol) and
HOAt (0.276 g, 2.03 mmol) in THF cooled in ice bath is treated with
DCC (2.05 mL, 2.05 mmol). After stirring in an ice bath for 0.5
hour, a solution of compound cviii in THF is added, and then DIPEA
(0.39 mL, 2.2 mmol) is added. The mixture is stirred at room
temperature overnight, then cooled in an ice bath, and quenched
with saturated NaHCO.sub.3. The precipitated DCU is filtered and
the filtrate is dissolved in EtOAc (100 mL). The organic phase is
washed with saturated NaHCO.sub.3, brine, and then dried by
MgSO.sub.4. After removal of the organic solvent, the residue is
purified by silica column chromatography (60% EtOAc/Hexanes) to
yield compound cxxxxvii (0.47 g, 48%) as gummy foam.
##STR00378##
Intermediate Example 143
Compound cxxxxviii
[0525] To a solution of compound cxxxxvii (0.47 g, 0.847 mmol) in
EtOH (5 mL) at 5.degree. C. is added NaOH (2 N, 1.31 mL, 2.62
mmol). The mixture is stirred at room temperature for 4 hours. The
solution is acidified to pH .about.2 with HCl (1N) and the EtOH is
removed by rotary evaporation. The mixture is extracted with EtOAc
(3.times.30 mL) and the combined extract is washed with brine, and
then dried by MgSO.sub.4. The solvent is removed and the residue is
dried under vacuum to yield compound cxxxxviii (0.366 g, 82%) as
gummy foam.
##STR00379##
Intermediate Example 144
Compound cil
[0526] A solution of compound cxxxxviii (0.366 g, 0.718 mmol) in
DCM is cooled in an ice bath and treated with PyBop (0.599 g, 1.15
mmol). After stirring at room temperature for 0.5 hour, the mixture
is cooled by an ice bath and treated with a solution of compound
xiii' (0.200 g, 1.08 mmol) in THF and DIPEA (0.250 mL, 1.44 mmol).
The mixture is stirred at room temperature overnight and then
quenched with NH.sub.4Cl solution. The solvent is concentrated and
the mixture is dissolved in EtOAc (100 mL). The organic phase is
washed with saturated NaHCO.sub.3, brine, and then dried by
MgSO.sub.4. After removal of the organic solvent, the residue is
purified by column chromatography (5% EtOH/EtOAc) to yield compound
cil (0.35 g. 72%)
##STR00380##
Intermediate Example 145
Compound cxxi
[0527] To a THF solution (85 mL) of N-Boc-Nva-OH (compound 1) (8.68
g, 40 mmol) is added CDI (7.79 g, 48 mmol). After stirring at room
temperature for 30 minutes, the above solution is treated with a
DMF solution (25 mL) containing N,O-dimethyl-hydroxylamine
hydrochloride (4.25 g, 44 mmol) and DIPEA (7.66 mL. 44 mmol). The
reaction mixture is stirred overnight at room temperature. The
reaction mixture is then concentrated in vacuo. The resulting
residue is diluted with EtOAc (300 mL). This solution is washed
sequentially with 0.1 N HCl (50 mL), saturated NaHCO.sub.3
(3.times.50 and brine. The organic phase is concentrated in vacuo
to yield a residue that is purified with silica gel chromatography
(40% EtOAc/Hexanes) to compound cxxi (9.38 g. 94%).
Intermediate Example 146
Compound cxxii
[0528] To a diethyl Et.sub.2O solution (50 mL) of compound cxxi
(9.38 g, 31.9 mmol) cooled to 0.degree. C. is added (slowly) LAH
(34.7 mL, 1 M, 34.7 mmol). The temperature of the reaction flask is
maintained below 5.degree. C. during LAH addition. Upon completion
of the addition, EtOAc (20 mL) is added to the reaction to quench
the excess LAH. Aqueous KHSO.sub.4 (5%, 20 mL) is then added in a
dropwise fashion in order to keep the temperature below 5.degree.
C. The organic phase is separated and then washed sequentially with
1 N HCl (3.times.30 mL), saturated NaHCO.sub.3 (3.times.30 mL) and
brine. The organic phase is concentrated and dried in vacuo to
yield crude compound cxxii (5.18 g, 69%).
Intermediate Example 147
Compound cl
[0529] To a THF (25 mL) suspension of Zn (2.75 g, 42 mmol) is added
at reflux 0.2 mL of EtOC(O)CF.sub.2Br. This is followed by slowly
adding a THF solution (25 mL) of compound cxxii (3.05 g, 15.0 mmol)
and EtOC(O)CF.sub.2Br (4.84 mL, 37.5 mmol). Upon completion of the
addition of both reagents, the reaction mixture is further refluxed
for 30 minutes. The reaction mixture is cooled to room temperature
and diluted with DCM (200 mL). The organic phase is washed with 1 N
KHSO.sub.4. The organic phase is concentrated and dried in vacuo to
yield a residue that is purified by silica gel chromatography (20%
EtOAc/Hexane) to yield compound cl (2.78 g, 57%).
##STR00381##
This preparation is essentially the same as that disclosed by
Thaisrivongs et al., J. Med. Chem., 29, 2080-2087 (1986).
Intermediate Example 148
Compound cli
[0530] A THF solution (40 mL) of compound cl (2.78 g, 8.53 mmol) is
treated with 1 N NaOH (12.8 mL, 12.8 mmol). After stirring at room
temperature overnight, the solvent is partially removed in vacuo.
The remaining reaction mixture is diluted with water (50 mL) and
lyophilized to yield crude compound cli (2.82 g, >100%) as its
sodium salt.
##STR00382##
This preparation is essentially the same as that disclosed by
Thaisrivongs et al., J. Med. Chem., 29, 2080-2087 (1986).
Intermediate Example 149
Compound clii
[0531] A DCM solution (10 mL) of the crude compound cli (516 mg,
1.61 mmol) is treated with HOBT (436 mg, 3.23 mmol) and DIC (0.328
mL, 2.09 mmol). After stirring at room temperature for 30 minutes,
the reaction mixture is treated with a DCM solution (5 mL)
containing glycine benzylester-TsOH salt (815 mg, 2.42 mmol) and
DIPEA (0.422 mL, 2.42 mmol). After stirring at room temperature for
12 hours, the reaction mixture is quenched with water and extracted
with EtOAc. The organic phase is dried and concentrated in vacuo
and purified by silica gel chromatography (40% EtOAc/hexanes) to
yield compound clii (495 mg, 69%).
[0532] .sup.1H NMR of compound clii (400 MHz, CDCl.sub.3): .delta.
7.29-7.21 (m, 5H), 5.16 (bs, 2H), 4.89 (bs, 1H), 4.20-3.90 (m, 4H),
3.80 (bs, 1H), 1.75-1.42 (m, 4H), 1.38 (s, 9H), 0.87 (m, 3H).
##STR00383##
[0533] Starting from crude compound cli, compounds cliii (83%) and
cliv (50%) are prepared in an identical method to that described
for compound clii.
[0534] .sup.1H NMR of compound cliii (400 MHz, CDCl.sub.3): .delta.
7.49 (bs, 1H), 7.34-7.24 (m, 5H), 5.13 (AB q, J=12.2 Hz, J'=23.9
Hz, 2H), 4.88 (bd, J=8.8 Hz, 1H), 4.53 (m, 114), 3.98-3.91 (m, 2H),
3.82 (m, 1H), 1.65-1.20 [m, 16H, including singlet at 1.37 (9H)],
0.86 (t, J=7.3 Hz, 3H).
##STR00384##
[0535] .sup.1H NMR of compound cliv (400 MHz, CDCl.sub.3): .delta.
7.60-7.0 (m, 10H), 5.30-5.00 (m, 2H), 5.00-4.75 (m, 2H), 4.15-3.70
(m, 3H), 3.30-3.00 (m, 2H), 1.75-1.20 [m, 13H, including singlet at
1.36 (9H)], 0.86 (bs, 3H).
##STR00385##
Intermediate Example 150
Compound clv
[0536] To a DCM (10 mL) and THF (5 mL) solution of the crude
compound cli (1 g, 3.13 mmol) is added HOBT (634 mg, 4.69 mmol) and
EDCI (781 mg, 4.07 mmol), and then (s)-.alpha.-methylbenzylamine
(0.604 mL, 4.69 mmol). The reaction mixture is stirred overnight at
room temperature and then quenched with water. The reaction mixture
is extracted with EtOAc. The organic phase is washed with brine and
dried by Na.sub.2SO.sub.4. The organic phase is concentrated in
vacuo to yield a residue that is purified by silica gel
chromatography (20% EtOAc/hexanes) to yield compound clv (459 mg,
37%). .sup.1H NMR of compound clv (400 MHz, CDCl.sub.3): .delta.
7.32-7.21 (m, 6H), 5.00 (m, 1H), 4.75 (m, 1H), 3.94 (m, 2H), 3.70
(m, 1H), 1.65-1.15 [m, 16H, including doublet at 1.51 (J=6.8 Hz,
3H), singlet at 1.39 (9H)], 0.82 (m, 3H).
##STR00386##
Intermediate Example 151
Compound clvi
[0537] Compound clv (220 mg, 0.55 mmol) is dissolved in 4 N HCl in
dioxane (10 mL). The reaction mixture is stirred at room
temperature for 2 hours and then concentrated in vacuo to give the
crude compound clvi (.about.100%) as its HCl salt.
##STR00387##
[0538] Following the procedure described for preparing compound
clvi, compounds clvii, clviii, and clix are prepared in almost
quantitative yield from the crude compound cli.
##STR00388##
Intermediate Example 152
Compound clx
[0539] A DCM solution (4 mL) of the HCl salt of compound vii (96
mg, 0.144 mmol) is treated with PyBOP (120 mg. 0.23 mmol) and DIPEA
(0.1 mL, 0.576 mmol). After stirring at room temperature for 30
minutes, the solution is treated with a THF solution (4 mL)
containing compound clv (0.288 mmol) and DIPEA (0.2 mL, 1.152
mmol). The reaction mixture is stirred at room temperature
overnight. The reaction mixture is then diluted with EtOAc (50 mL),
and the organic phase then washed with NaHCO.sub.3 and brine. The
organic phase is concentrated in vacuo and the residue purified by
silica gel chromatography (80% EtOAc/hexanes) to yield compound clx
(113 mg. 89%).
##STR00389##
Intermediate Example 153
Compound clxi
[0540] A DCM solution (6 mL) of compound vii (140 mg, 0.235 mmol)
is treated with PyBOP (196 mg, 0.376 mmol) for 30 minutes. A THF
solution (6 mL) of compound clvii (.about.0.47 mmol) and DIPEA
(0.327 mL, 1.88 mmol) is then added to the above solution. The
reaction mixture is stirred at room temperature overnight and
quenched with water (30 minutes). The reaction mixture is extracted
with EtOAc (50 mL). The organic phase is washed with NaHCO.sub.3
and brine. The combined aqueous layers are back extracted with
EtOAc (50 mL). The combined organic phases are dried and
concentrated in vacuo. The resultant residue is purified by silica
gel chromatography (80-100 EtOAc/hexanes) to yield compound clxi
(104 mg, 48%).
##STR00390##
Intermediate Example 154
Compound clxii
[0541] To a DCM solution (10 mL) of compound clxi (280 mg, 0.304
mmol) is added DMP reagent (193 mg, 0.456 mmol). The reaction
mixture is stirred at room temperature for 3 hours and quenched
with 10 Na.sub.2SO.sub.3. The organic phase is washed with
NaHCO.sub.3 and brine. The resulting organic phase is dried and
concentrated in vacuo to yield a residue that is purified with
silica gel chromatography (80-100% EtOAc/hexanes) to yield compound
clxii (271 mg, 97%).
##STR00391##
Intermediate Example 155
Compound clxiii
[0542] Compound lxxxiii (220 mg, 0.43 mmol) is taken up in DCM (5
mL). PyBOP (270 mg, 0.51 mmol) is added to the DCM solution and
stirred 5 minutes. Compound xxxvi' (0.51 mmol) in THP (5.1 mL) is
added drop-wise to this solution. DIPEA (0.09 mL, 0.51 mmol) is
added to reaction mixture and stirred overnight under N.sub.2. The
next day, the reaction mixture is diluted with EtOAc, washed with
saturated NaHCO.sub.3, washed with brine. Purification by 70% to
90% EtOAc/Hexane gradient yields compound clxiii (180 mg, 56%).
##STR00392##
Intermediate Example 156
Compound clxiv
[0543] Compound cxxv (2.09 g, 7.4 mmol) is taken up in DCM (20 mL).
PyBOP (4.64 g. 8.9 mmol) and HOBt (1.2 g, 8.9 mmol) are added to
this solution and stirred five minutes. The resulting mixture is
lowered to 0.degree. C. where S(-)-.alpha.-Methylbenzylamine (1.15
mL, 8.9 mmol) and DIPEA (1.55 mL, 8.9 mmol) are added. The reaction
is stirred overnight with warming to room temperature. The reaction
mixture is washed with 0.1 N HCl, sat NaHCO.sub.3, and brine.
Purification by 30% EtOAc/Hexanes yields compound clxiv (1.6 g,
56.3%).
##STR00393##
Intermediate Example 157
Compound xxxvi'
[0544] Compound clxiv (1.48 g, 3.8 mmol) is hydrogenated using 10%
Pd/C (300 mg) in MeOH (50 mL). The reaction mixture stirred
overnight under H.sub.1. The reaction mixture is filtered through
celite and concentrated to give compound xxxvi' (895 mg,
94.2%).
Intermediate Example 158
Compound clxvi
[0545] To a DCM solution (15 mL) of compound clxv (2 g, 8.2 mmol)
is added HOAt (1.34 g,
##STR00394##
9.84 mmol) and DCC (9.84 mL, 1 M, 9.84 mmol). After stirring at
room temperature for 20 minutes, a THF solution (9.84 mL)
containing tert-L-Leucine methyl ester-hydrochloride (9.84 mmol)
and DIPEA (1.72 mL, 9.84 mmol) is added to the above solution. Then
DMAP (1 g, 8.2 mmol) is added at room temperature. The reaction is
stirred at room temperature overnight. Following standard aqueous
work-up and silica gel chromatography (20% EtOAc/Hexanes), compound
clxvi (1.75 g, 58%) is obtained.
##STR00395##
Intermediate Example 159
Compound clxvii
[0546] To a THF solution (35 mL) of compound clxvi (1.75 g, 4.73
mmol) is added 4 N HCl solution in dioxane (11.8 mL, 47.3 mmol).
The reaction is stirred at room temperature overnight. At this
point, the solvent is removed under reduced pressure to yield crude
clxvii (.about.100%), which is redissolved in DMF and used directly
in the next reaction.
##STR00396##
Intermediate Example 160
Compound clxviii
[0547] To a DCM solution (15 mL) containing 2-pyrazinecarboxylic
acid (447 mg, 3.6 mmol), PyBOP (1.87 g, 3.6 mmol) is added a DMF
solution (15 mL) of compound clxvii (811 mg, 3 mmol). To the
resulting mixture is then added DIPEA (0.63 mL, 3.6 mmol). The
reaction is stirred overnight at room temperature and then quenched
with water. The reaction mixture is extracted with EtOAc. The
organic layer is washed with brine and concentrated in vacuo to
provide a residue that is purified by silica gel chromatography
(40% EtOAc/Hexanes) to yield compound clxviii (0.93 g, 82%).
##STR00397##
Intermediate Example 161
Compound clxix
[0548] To a MeOH solution (10 mL) of compound clxviii (0.93 g, 2.47
mmol) is added 2 N NaOH (3.71 mL, 7.41 mmol). The reaction is
stirred at room temperature overnight. Then the reaction is
acidified to pH 3 using 1 N HCl. The reaction is diluted with EtOAc
(75 mL), and washed with water and brine. The organic layer thus
obtained is dried and concentrated in vacuo to give compound clxix
(.about.100%).
##STR00398##
Intermediate Example 162
Compound clxx
[0549] A DCM solution (10 mL) of compound clxix (2.47 mmol) is
treated with HOAt (436 mg. 3.21 mmol) and DCC (3.2 mL, 1 M, 32
mmol). After stirring for 30 minutes, the reaction mixture is
treated with a THF solution (13.6 mL) of compound v (499 mg, 2.72
mmol). After stirring at room temperature overnight, white solids
(urea) are filtered. The filtrates are concentrated in vacuo to
give a residue that is purified by silica gel chromatography to
yield compound clxx (0.99 g, 76%).
##STR00399##
Intermediate Example 163
Compound clxxi
[0550] An EtOH solution (20 mL) of compound clxx (0.99 g, 1.88
mmol) is treated with 2 N NaOH (2.81 mL, 5.63 mmol). After stirring
at room temperature overnight, the reaction mixture is acidified to
pH 3 with 1 N HCl. The reaction mixture is extracted with EtOAc (75
mL). The organic layer is dried and concentrated in vacuo to give
compound clxxi (772 mg, 82%).
##STR00400##
Intermediate Example 164
Compound clxxi
[0551] A DCM solution (10 mL) of compound clxxi (290 mg, 0.58 mmol)
is treated with PyBOP (484 mg, 0.93 mmol). After stirring at room
temperature for 20 minutes, the reaction mixture is treated with a
THF solution (7.5 mL) of compound xiii' (140 mg, 0.75 mmol),
followed by DIPEA (0.13 mL, 0.75 mmol). After stirring overnight at
room temperature, the reaction is quenched with water and extracted
with EtOAc. The resulting organic layer is washed with brine and
dried and concentrated in vacuo. The resulting residue is purified
by silica gel chromatography (5% EtOH/EtOAc) to yield compound
clxxii 290 mg (75%).
##STR00401##
Intermediate Example 165
Compound clxxiv
[0552] Compound lxxxiii (600 mg, 1.17 mmol) is taken up in DCM (4
mL). PyBOP (670 mg, 1.3 mmol) is added, stirred five minutes, and
cooled to 0.degree. C. Compound clxxiii (333 mg, 1.3
##STR00402##
mmol) in THE (13 mL) is added drop-wise to this solution. DIPEA
(0.23 mL, 1.3 mmol) is added to reaction mixture and allowed to
warm to ambient temperature with stirring for two nights. The next
day, the reaction is concentrated and purified by 2% EtOH/EtOAc to
give crude compound clxxiv (900 mg, excess of 100%).
##STR00403##
Intermediate Example 166
Compound clxxxv
[0553] Compound cxxv (3.01 g, 10.7 mmol) is taken up in DCM (30 mL)
and the temperature lowered to -78.degree. C. PyBOP (6.1 g, 11.7
mmol) and HOBT (1.58 g, 11.7 mmol) are added to this solution
followed by (S)-(+)-1-cyclohexylethylamine, compound clxxv, (1.74
mL, 11.7 mmol) and DIPEA (2.1 mL, 11.7 mmol). The resulting mixture
stirred overnight at room temperature. The next day, the reaction
mixture is diluted with EtOAc, washed with 0.1 N HCl, saturated
NaHCO.sub.3, and brine. The product is purified in 40% EtOAc/Hex to
give 2 g (47.8%) of compound clxxvi.
##STR00404##
Intermediate Example 167
Compound clxxiii
[0554] Compound clxxvi (2 g, 5.13 mmol) is hydrogenated using 10%
Pd/C (500 mg) in MeOH (40 mL). The reaction mixture stirred
overnight under H.sub.2. The reaction mixture is filtered through
celite and concentrated to give compound clxxiii (1.31 g,
99.8%).
Intermediate Example 168
Compound clxxix
[0555] In a round bottom flask under inert atmosphere, compound
clxxvii [(S)-(-)-2-oxo 1,5
##STR00405##
imidazoline dicarboxylic acid 1-benzyl ester] (290 mg, 1.1 mmol) is
dissolved in anhydrous DMF (6 mL). HOAt (151 mg, 1.2 mmol) is added
and the reaction is stirred at room temperature for 25 minutes. The
reaction is then cooled down in an ice bath. DIC (0.2 mL, 0.16 g.
1.2 mmol) is then added followed by the addition of compound
clxxviii (1 mmol, 435 mg.) in anhydrous DMF (4 mL). The reaction is
allowed to rise slowly to room temperature and stirred for 2 days.
The reaction is then dumped in a separatory funnel containing 120
mL of EtOAc and washed 2.times. with 1 N HCl (50 mL) and 1.times.
brine. The organic layer is separated, dried over MgSO.sub.4. The
solvent evaporated under reduced pressure and the residue purified
by chromatography on silica gel (load in DCM and elute with 30%
than 50% EtOAc/DCM then 2% MeOH/EtOAc) to yield product clxxix (434
mg, 64%).
##STR00406##
Intermediate Example 169
Compound clxxx
[0556] The starting material clxxix (434 mg, 0.64 mmol) is
dissolved in Dioxane (6 mL) and 0.5 M aqueous NaOH solution (4 mL,
3 eq.). The reaction is run overnight. TLC in 100% EtOAc (using PMA
stain) shows in addition to the expected acid product at the
origin, a faster running product. The reaction mixture is acidified
to pH 2 with 1 N HCl, and then extracted 2.times. with EtOAc. Solid
NaCl is added to the aqueous solution to facilitate the extraction.
The organic extracts are then combined, dried over MgSO.sub.4 and
evaporated under reduced pressure. MS indicates that the CBZ group
is removed by the hydrolysis. The resulting compound clxxx
(quantitative yield) is used as is in the next step.
##STR00407##
Intermediate Example 170
Compound clxxxi
[0557] In a round bottom flask under inert atmosphere, compound
clxxx (279 mg, 0.54 mmol) is dissolved in anhydrous DMF (6 HOAt (82
mg, 0.65 mmol) is added and the reaction is stirred at room
temperature for 25 minutes. The reaction is then cooled down in an
ice bath. DIC (0.11 mL, 0.65 mmol) is then added, followed by the
addition of compound xiii' (0.7 mmol) in anhydrous DMF (4 mL). The
reaction is allowed to rise slowly to room temperature and stirred
for 21 hours. The reaction is then dumped in a separatory funnel
containing 120 mL of EtOAc and washed 2.times. with 1 N Ha (50 mL)
and 1.times. brine. The organic layer is separated, dried over
MgSO.sub.4. The solvent evaporated by reduced pressure and the
product cleaned by chromatography on silica gel (load in DCM and
elute with 50% EtOAc/Hexane, then 3% MeOH/EtOAc, then 20%
EtOH/EtOAc). After removal of solvent, the residue is redissolved
in Dri Solv THF and filtered to remove any silica gel. Removal of
the solvent then yields compound clxxxi (434 mg, 64% yield).
##STR00408##
Intermediate Example 171
Compound clxxxiii
[0558] In a round bottom flask under inert atmosphere, 6-hydroxy
picolinic
##STR00409##
(153 mg, 1.1 mmol) is dissolved in anhydrous DMF (6 mL). HOAt (151
mg, 1.2 mmol) is added and then the reaction is stirred at room
temperature for 25 minutes. The reaction is then cooled down in an
ice bath. DIC (0.2 mL, 0.16 g, 1.2 mmol) is then added followed by
the addition of the compound clxxxii (1.0 mmol, 435 mg.) in
anhydrous DMF (4 mL). The
##STR00410##
reaction is allowed to rise slowly to room temperature and stirred
for 2 days. The reaction is then dumped in a separatory funnel
containing 120 mL of EtOAc and washed 2.times. with 1 N. HCl (50
mL) and 1.times. with brine. The organic layer is separated, dried
over MgSO.sub.4. The solvent is evaporated by reduced pressure and
the product purified by chromatography on silica gel (load in DCM,
elute with 30%, then 50% EtOAc/DCM, and then 2% MeOH/EtOAc) to
yield compound clxxxiii collected (314 mg, 56%).
##STR00411##
Intermediate Example 172
Compound clxxxiv
[0559] The starting material clxxxiii (314 mg, 0.56 mmol) is
dissolved in dioxane (5 mL) and 0.5 M NaOH (3.4 mL, 3 eq). The
reaction is run overnight. TLC in 100% EtOAc (using UV) shows
complete conversion to the slow running acid product at the origin.
The reaction is acidified to pH 2 with 1 N HCl, and then extracted
2.times. with EtOAc. Solid NaCl is added to the aqueous to
facilitate the extraction. The organic extracts are then combined,
dried over MgSO.sub.4, and then evaporated under reduced pressure
to yield compound clxxxiv (0.5 mmol, 89%) that is used as is in the
next step.
##STR00412##
Intermediate Example 173
Compound clxxxv
[0560] In a round bottom flask under inert atmosphere, acid
compound clxxxiv (265 mg, 0.5 mmol) is dissolved in anhydrous DMF
(6 mL). HOAT (75.6 mg, 0.6 mmol) is added and the reaction is
stirred at room temperature for 25 minutes. The reaction is then
cooled down in an ice bath. DIC (0.1 mL, 0.6 mmol) is then added
followed by the addition of the compound xiii' (0.65 mmol) in
anhydrous DMF (4 mL). The reaction is allowed to rise slowly to
room temperature and stirred for 21 hours. The reaction is then
dumped in a separatory funnel containing EtOAc (120 mL) and washed
2.times. with 1 N Ha (50 mL) and 1.times. with brine. The organic
layer is separated, dried over MgSO.sub.4. The solvent is
evaporated by reduced pressure and the product purified by
chromatography on silica gel (load in DCM, elute with 50%
EtOAc/Hexane, then pure EtOAc, and then 4% MeOH/EtOAc) to yield
product compound clxxxv (185 mg. 52%).
##STR00413##
Intermediate Example 174
Compound cxxxxiv'
[0561] To a solution of D-alanine (5 g, 56.1 mmol) in 1 N NaOH (152
mL, 152 mmol) at 0.degree. C. is added a solution of MeOC(O)Cl (6.5
mL, 84.2 mmol) in diethyl ether (30 mL). The mixture is stirred in
ice bath for 3 hours and then adjusted to pH 9 with 1 N NaOH. After
stirring at room temperature for 1 hour, the mixture is washed with
ether (3.times.50 mL), acidified to pH .about.2 with 5 N HCl,
extracted with EtOAc (5.times.50 mL). The organic extract is washed
with water, brine, and then dried (MgSO.sub.4). The solvent is
removed to yield compound cxxxiv, N-methoxycarbonyl-D-alanine, as
colorless oil (6.48 g, 79%).
##STR00414##
Intermediate Example 175
Compound clxxxvi
[0562] To a solution of N-methoxycarbonyl-D-alanine (0.193 g, 1.31
mmol) and HOAt (0.177 g, 1.31 mmol) in DCM (10 mL) cooled in ice
bath is treated with DCC (1.31 mL, 1.31 mmol). After stirring in an
ice bath for 0.5 hour, a solution of prepared compound clxxxii
(0.88 mmol) in THF (8.8 mL) is added. The mixture is warmed up to
room temperature and stirred overnight, then cooled in ice bath,
and quenched with saturated NaHCO.sub.3 solution. The precipitates
are filtered and the filtrate is taken up in EtOAc (100 mL). The
organic layer is washed with saturated NaHCO.sub.3 solution, brine,
and then dried (MgSO.sub.4). After removal of the solvent, the
residue is purified by silica column chromatography (60%
EtOAc/Hexane) to yield compound clxxxvi as gummy foam (0.321 g,
68%).
##STR00415##
Intermediate Example 176
Compound clxxxvii
[0563] To a solution of compound clxxxvi (0.321 g, 0.597 mmol) in
EtOH (5 mL) at 5.degree. C. is added 2 N NaOH (1.05 mL, 2.1 mmol).
The mixture is stirred at room temperature for 4 hours. The
solution is acidified to pH .about.2 with 1 N HCl acid EtOH is
removed by rotary evaporation. The mixture is extracted with EtOAc
(3.times.30 mL) and the combined extract is washed with brine, and
then dried (MgSO.sub.4). Solvent is removed and the residue is
dried under vacuum to give compound clxxxvii as gummy foam (0.235
g. 77%).
##STR00416##
Intermediate Example 177
Compound clxxxviii
[0564] A solution of compound clxxxvii (0.363 g, 0.712 mmol) in DCM
(10 mL) is cooled in an ice bath and treated with PyBOP (0.594 g,
1.14 mmol). After stirring at room temperature for 0.5 hour, the
mixture is cooled in ice bath and treated with a solution of
compound xiii' (1.1 mmol) in THF (11 mL) and DIPEA (0.249 mL, 1.42
mmol). The mixture is stirred at room temperature overnight and
quenched with NH.sub.4Cl solution. The solvent is concentrated and
the mixture is taken up in EtOAc (100 mL). The organic layer is
washed with saturated NaHCO.sub.3 solution, brine, and then dried
(MgSO.sub.4). After removal of the solvent, the residue is purified
by column chromatography (5% EtOH/EtOAc) to give clxxxviii (0.341
g, 71%).
Intermediate Example 178
Compound clxxxix
[0565] Diaminopropionic acid (3 g. 28.7 mmol) is taken up in 1 M
NaOH (86.2 mL, 86.2 mmol) and cooled to 0.degree. C., and then
MeOC(O)Cl (5.54 mL, 71.75 mmol) is added in Et.sub.2O (25 mL). The
resulting mixture stirred overnight warming to room temperature.
The reaction mixture pH is lowered to 2 and aqueous layer is
extracted 3.times. with EtOAc. Extracts are combined and dried over
Na.sub.2SO.sub.4, filtered and concentrated to yield compound
clxxxix (3.09 g, 48.9%).
##STR00417##
Intermediate Example 179
Compound cc
[0566] Compound clxxxix (340 mg, 1.55 mmol) is taken up in DCM (4
mL). DCC (1.7 mmol) and HOAt (235 mg, 1.7 mmol) are added followed
by compound clxxxii (1.7 mmol) in DCM (3.4 mL). The reaction
mixture stirred overnight. The next day, the reaction mixture is
filtered through a pad of silica and concentrated. Purification is
achieved in 75% EtOAc/Hex to give compound clxxxx (715 mg,
72.4%).
##STR00418##
Intermediate Example 180
Compound clxxxxi
[0567] Compound clxxxx (715 mg, 1.12 mmol) is hydrolyzed under
standard conditions using EtOH (4 mL) and 1 N NaOH (3 eq) to yield
compound clxxxxi (600 mg. 88.0%).
##STR00419##
Intermediate Example 181
Compound clxxxxii
[0568] Compound clxxxxi (550 mg, 0.9 mmol) is taken up in DCM (8
mL). PyBOP (675 mg, 1.3 mmol) is added followed by compound xiii'
(1.3 mmol) in THF (1.3 mL). DIPEA (0.23 mL, 1.3 mmol) is added and
the resulting solution stirred overnight. The next day, the
reaction is diluted with EtOAc, washed with saturated NaHCO.sub.3,
and then brine, before being concentrated to yield a residue. The
resulting residue is purified by 5% EtOH/EtOAc to yield compound
clxxxxii (290 mg, 41.5%).
##STR00420##
Intermediate Example 182
Compound clxxxxiii
[0569] Cbz-cyclohexyglycine-ten-leucine methyl ester (7.36 g, 17.6
mmol) is hydrolyzed under standard conditions using MeOH (60 mL)
and 1 N NaOH (52.8 mL, 3 eq) to yield intermediate clxxxxiii
(92%).
##STR00421##
Intermediate Example 183
Compound clxxxxiv
[0570] Compound clxxxxiii (3.82 g, 9.46 mmol) is taken up in DCM
(30 mL). DCC (11.35 mmol) in DCM (11.35 mL) is added, followed by
the addition of HOAt (1.54 g, 11.35 mmol). The resulting mixture
stirred five minutes and compound v (9.46 mmol) in THF (40 mL) is
added. The resulting mixture is stirred overnight. The next day,
the reaction mixture is diluted with EtOAc, washed with 1 N HCl,
saturated NaHCO.sub.3, and then brine, before being concentrated to
yield a residue. The resulting reside is purified by 20% to 30%
gradient on silica gel to give compound clxxxxiv (3.03 g.
56.3%).
##STR00422##
Intermediate Example 183
Compound clxxxii
[0571] Compound clxxxxiv (3.03 g, 5.33 mmol) is hydrogenated using
10% Pd/C (500 mg) in MeOH (30 mL) under H.sub.2 for 4 hours to
yield compound clxxxii (2.3 g, 99%).
Intermediate Example 184
Compound clxxxxv
[0572] To a solution of 1-amino-1-cyclohexanecarboxylic acid (2.86
g, 20 mmol) in MeOH (40 mL) is added dropwise SOCl.sub.2 (3 mL) at
0.degree. C. The mixture is slowly warmed up to room temperature
and then refluxed for 5 hours. Et2O is then added to the clear
solution and the precipitate is isolated. The solid is further
dried over vacuum to yield compound clxxxxv (95%) as white
powder.
##STR00423##
Intermediate Example 185
Compound clxxxxvi
[0573] 2-Pyrazinecarboxylic acid (1 g, 8 mmol, 1 eq) is dissolved
in DCM (15 mL) with addition of HOAt (1.1 g, 8 mmol) and DCC (8 mL,
1 M) in DCM. After stirring at room temperature for 20 minutes,
compound clxxxxv (1.3 g, 8 mmol) is added to the activated mixture.
DIPEA (2 mL, 12 mmol) is added subsequently, followed by DMAP (1.5
g, 12 mmol). After stirring over 3 days at room temperature, the
reaction mixture is filtered through celite, concentrated and the
desired product clxxxxvi is purified by column chromatography (50%
EtOAc/hexane) as yellow oil (2.1 g, 100%).
##STR00424##
Intermediate Example 186
Compound clxxxxvii
[0574] Compound clxxxxvi (1.06 g, 2.6 mmol) is dissolved in MeOH
(30 mL) with addition of 2 N NaOH (aq) (12 mL, 24 mmol). The
solution is stirred at room temperature overnight before TLC (50%
EtOAc/hexane) indicates complete hydrolysis. The solution is then
acidified to pH 3 by 5 N HCl and diluted with EtOAc and followed by
extraction of the organic layer. The organic layer is subsequebtly
washed with brine and dryed over MgSO.sub.4 to yield compound
clxxxxvii (84%) upon concentration.
##STR00425##
Intermediate Example 187
Compound clxxxxviii
[0575] Compound clxxxvii (1.6 g, 6.4 mmol) is dissolved in DCM (18
mL) and then HOAt (0.96 g, 7 mmol) and DCC (7 mL, 1 M in DCM) are
subsequently at room temperature. After stirring at room
temperature for 20 minutes, L-tert-leucine methyl ester
hydrochloride (7 mL, 1 M in THF) is added to the activated mixture.
DIPEA (1.2 mL, 7 mmol) is added subsequently, followed by DMAP (1.2
g, 9.8 mmol). After stirring over 3 days at room temperature, the
reaction mixture is filtered through celite, purified by column
chromatography and concentrated to yield compound clxxxxviii (60%
EtOAc/hexane) as white solid (1.74 g, 72%).
##STR00426##
Intermediate Example 188
Compound cic
[0576] Compound clxxxxviii (1.74 g, 4.6 mmol) is dissolved in MeOH
(22 mL) with addition of 2 N NaOH (aq) (7 mL, 14 mmol). The
solution is stirred at room temperature overnight before TLC (50%
EtOAc/hexane) indicated complete hydrolysis. The solution is
acidified to pH 3 by 5 N HCl and diluted with EtOAc and then the
organic layer is extracted. The organic layer is washed with brine
and dried over MgSO.sub.4 and then concentrated to yield compound
cic (100%).
##STR00427##
Intermediate Example 189
Compound cc
[0577] To a DCM solution (15 mL) of compound cic (1.5 g, 4.1 mmol)
at room temperature is added HOAt (610 mg, 4.5 mmol), followed by 1
M DCC solution in DCM (4.5 mL, 4.5 mmol). After stirring for 30
minutes at room temperature, then a THF solution (20 mL, 0.2 M) of
compound v (4 mmol) is added. The reaction is stirred at room
temperature overnight. Then, the reaction is filtered through
celite. The filtrate is concentrated to a yellow oil which is
purified by silica gel chromatography (50% EtOAc/hexane) to yield
compound cci (660 mg. 32%).
##STR00428##
Intermediate Example 190
Compound cci
[0578] To an EtOH solution (6 mL) of compound cc (600 mg, 1.13
mmol) is added 2 N NaOH (1.7 mL, 3.4 mmol). The reaction is stirred
for 2 hours at room temperature, then acidified to pH 3 by 5 N HCl.
The mixture is then diluted with EtOAc, followed by extraction of
the organic layer. Subsequently, the organic layer is washed with
brine and then dried over MgSO.sub.4 to yield compound cci (92%)
upon concentration.
##STR00429##
Intermediate Example 191
Compound ccii
[0579] To a DCM solution (8 mL) of ccii (310 mg, 0.62 mmol) is
added PyBOP (420 mg, 0.8 mmol). The solution is stirred at room
temperature for 30 minutes. To this solution is then added compound
xiii' (8 mL, 0.1 M) in THF, followed by the addition of DIPEA (0.23
mL, 1.3 mmol). The reaction is stirred at room temperature
overnight and then quenched with water (25 mL) for 30 minutes. The
mixture is then extracted with EtOAc. The resulting organic layer
is washed with brine and then dried over MgSO.sub.4, before being
concentrated to yield a yellow oil. Purification by silica gel
chromatography (3% EtOH/EtOAc) yields compound ccii (140 mg.
33%).
##STR00430##
Intermediate Example 192
Compound ccxiv
[0580] To a solution of compound cciii, tert-butyl
(N-diphenylmethylene)-glycine ester, (6 g, 0.0206 mmol) and chiral
PTC (1.08 g, 0.00206 mmol) in dry DCM (48 mL), under N2 atmosphere,
at -60.degree. C., is added CsOH.H.sub.2O (6.9 g. 0.0412 mmol). To
the reaction mixture is added dropwise 1-carboxy-1-cyclopentene
methyl ester (5.2 mL, 0.0412 mmol) in 10 mL of DCM. The mixture is
stirred for 4 days at -60.degree. C., then diluted with 200 mL of
Et.sub.2O and 15 mL of saturated NH.sub.4Cl aqueous solution is
added. Phases are separated and the organic phase is washed with 15
mL water and 15 mL brine. The aqueous phases are extracted with 100
mL of Et.sub.2O. The organics phases are joined and dried over
Na.sub.2SO.sub.4. Crude product is obtained by removal of the
solvent dissolved in 100 mL of EtOH and then NH.sub.2OH.HCl (1.43
g, 0.0206 mmol) and NaOAc (1.68 g. 0.0206 mmol) are added. The
mixture is refluxed for 48 hours. Then the solvent is removed and
the crude residue obtained is directly purified by flash
chromatography eluting with 30%-50% EtOAc/hexane to yield compound
cciv (65%) as a white solid. C.sub.12H.sub.19NO.sub.3 (MW=225.29);
MS: m/z (M.sup.++1)=226.5. Enantiomeric excess: 18% ee, determined
by Chiral HPLC.
##STR00431##
Intermediate Example 193
Compound ccv
[0581] To a solution of compound cciv (2 g, 0.0088 mmol) in 60 mL
of ACN is added a catalytic amount of DMAP (0.216 g, 0.0017 mmol)
and a solution of di-tert-butyl-di-carbonate (2.49 g, 0.011 mmol)
in 30 mL of ACN. The mixture is stirred for 14 hours at room
temperature, then diluted with 100 mL of DCM, and washed with
saturated NaHCO.sub.3 (10 mL) and with brine (10 mL). The organic
phase is dried over Na.sub.2SO.sub.4. Evaporation of the solvent
yields a crude product that is purified on a silica gel column
eluting with 15% EtOAc/hexane to give compound ccv (86%) as white
solid. C.sub.17H.sub.27NO.sub.3 MW=325.40 MS: ink
(M.sup.++1)=326.2
##STR00432##
Intermediate Example 194
Compound ccvi
[0582] To a solution of compound ccv (1.7 g, 0.0052 mmol) in 50 mL
of THF (0.14 M) at -78.degree. C., is added DIBAL-H (7.8 mL, 0.0078
mmol). The mixture is stirred for 1 hour, then 10 mL of MeOH are
added. The mixture is diluted with 25 mL of EtOAc and 25 mL of
saturated aqueous solution of sodium tartrate, and then stirred at
room temperature for an hour. The phases are separated and the
aqueous phase is extracted once with 50 mL of EtOAc. The organic
phases are combined and dried over Na.sub.2SO.sub.4. Evaporation of
solvent gave a crude residue that is used without any purification.
The crude is dissolved in 25 mL of DCM, Et.sub.3Si (0.84 mL, 0.0052
mmol) is added, and then the mixture is cooled to -78.degree. C.
before the dropwise addition of BF.sub.3OEt.sub.2 (0.71 mL, 0.0061
mmol). After 30 minutes Et.sub.3Si (0.84 mL) and BF.sub.3OEt.sub.2
(0.71 mL) are added and the mixture stirred for 2 hours to
-78.degree. C. The reaction is then quenched with saturated aqueous
NaHCO.sub.3 (10 mL) and extracted with DCM (2.times.20 mL). The
organic phases are combined and dried over Na.sub.2SO.sub.4.
Evaporation of solvent gives a crude residue that is purified by
flash chromatography eluting with 13% EtOAc/hexane to yield
compound ccvi (87%). C.sub.17H.sub.29NO.sub.4 MW=311.42 MS: m/z
(M.sup.++1)=312.6
##STR00433##
Intermediate Example 195
Compound ccvii
[0583] Compound ccvi (0.5 g, 0.0016 mmol) is dissolved in 8 mL of 1
N HCl in EtOAc (prepared by bubbling dry HCl into dry EtOAc then
diluting to 1 N with additional EtOAc). The mixture is stirred for
6 hours at room temperature. Solvent is removed in vacuo and the
resulting precipitate is dissolved in Et.sub.2O. After stirring the
mixture for 15 minutes, the solvent is removed under reduced
pressure. The resulting white solid is washed with Et.sub.2O and
the compound ccvii (0.27 g, 80% yield) is isolated by filtration.
C.sub.12H.sub.21NO.sub.2 MW 211.15 MS: m/z (M.sup.++1)=212.6
##STR00434##
Intermediate Example 196
Compound v
[0584] To a solution of compound ccxvi (0.230 g. 0.74 mmol) in DCM
(3.7 mL) is added TFA (2.85 mL). The mixture is stirred overnight,
then solvent is removed in vacuo to dryness and the residue is
dissolved in EtOH (7.5 mL). The mixture is cooled at 0.degree. C.
and SOCl.sub.2 (0.22 mL, 2.96 mmol) is added dropwise and then
refluxed for 2 hours. EtOH is removed at reduced pressure and the
residue dissolved in DCM (10 mL). The resulting solution is washed
twice with a saturated aqueous solution of NaHCO.sub.3 (5 mL).
Phases are separated and the organic phase is dried over
Na.sub.2SO.sub.4 and solvent removed in vacuo to yield compound v
(80%) as oil. C.sub.10H.sub.17NO.sub.2 M.W.: 183.25 MS: m/z
(M.sup.++1)=184.2
Intermediate Example 197
Compound cd
[0585] 1-Benzylimidazole (6 g, 37.9 mmol) is taken up in Et.sub.2O
(180 mL). The resulting solution is lowered to -60.degree. C. and
treated with n-BuLi (1.6 M, 24 mL). The reaction is stirred for 30
minutes and then CO.sub.2 is bubbled through mixture for 15
minutes. The precipitate is filtered, rinsed with Et.sub.2O and
then taken up in H.sub.2O. This aqueous solution is acidified to pH
3 with 5 N HCl. The desired product, cd, is isolated after
lyophilization as a white solid.
##STR00435##
Intermediate Example 198
Compound cdi
[0586] A DCM solution (100 mL) of compound i (9.25 g, 27.9 mmol) is
treated at 0.degree. C. with DAST (9.2 mL, 69.8 mmol). After
stirring at room temperature overnight, the reaction is quenched
with ice and extracted with DCM (200 mL). The organic layer is
washed with brine and concentrated, in vacuo. The residue is
purified with silica gel chromatography (30% EtOAc/hexanes) to
yield 83 g (86%) of the desired fluorinated intermediate. A portion
of this intermediate (4.5 g, 14.2 mmol) is dissolved in EtOH (75
mL). This solution is subjected to standard hydrogenation
conditions using Pd(OH).sub.3/C (2.98 g, 20% Pd content, 4.26
mmol). After stirring overnight at room temperature, the reaction
mixture is filtered through Celite. The filtrates are concentrated
in vacuo to yield compound cdi (2.5 g, 96%).
##STR00436##
Intermediate Example 199
Compound cdii
[0587] To a solution of compound cd (890 mg, 4.4 mmol) taken up in
DCM (15 mL). HOBT (595 mg, 4.4 mmol) and DCC (4.4 mmol, 1 M in DCM)
are added and stirred for 20 minutes. A DCM solution (15 mL) of
lxxix' (990 mg, 3.5 mmol) is added to this mixture. The resulting
mixture is stirred overnight under nitrogen. The reaction mixture
is diluted with EtOAc, washed with saturated NaHCO.sub.3 and brine.
The organic layer is concentrated in vacuo to give a residue, which
is purified in 30% EtOAc/Hexanes to yield compound cdii (666 mg,
41%).
##STR00437##
Intermediate Example 200
Compound cdiii
[0588] Compound cdiii is prepared from compound cdii under standard
hydrolysis conditions using methyl alcohol (10 mL) and 1 N NaOH (3
eq). 565 mg of compound cdiii are recovered (88%).
##STR00438##
Intermediate Example 201
Compound cdiv
[0589] Compound cdiii (1.24 mmol) is taken up in DCM (5 mL). DCC
(1.6 mmol, 1 M DCM) is added followed by HOAT (1.6 mmol). The
resulting mixture is stirred 20 minutes and
##STR00439##
compound cdi (1.6 mmol) is added dropwise in THF (8 mL). The
reaction is stirred overnight. The reaction is filtered and rinsed
with EtOAc. The combined organic layer is washed with saturated
NaHCO.sub.3, brine, dried over MgSO.sub.4, and concentrated.
Purification is achieved in 30% EtOAc/Hexanes to yield compound
cdiv (565 mg, 70%).
Intermediate Example 202
cdv
[0590] Compound cdv (565 mg, 0.86 mmol) is prepared from compound
cdiv under standard hydrolysis conditions using ethyl alcohol (10
mL) and 1 N NaOH (3 eq). 490 mg (91%) of compound cdv is
recovered.
##STR00440##
Intermediate Example 203
cdvi
[0591] Compound cdv (490 mg, 0.78 mmol) is taken up in DCM (10 mL).
PyBOP (520 mg, 1 mmol) is added to DCM solution followed by a THF
solution (10 mL) of xiii (186 mg. 1 mmol). DIEA (0.18 mL, 1 mmol)
is added to the reaction mixture and stirred overnight under
nitrogen. The next day, the reaction is diluted with EtOAc, washed
with saturated NaHCO.sub.3 and brine. Purification is achieved in
100% EtOAc to yield compound cdvi (478 mg, 77%).
##STR00441##
Intermediate Example 204
cdvii
[0592] Compound cdvi (478 mg, 0.6 mmol) is hydrogenated using
Pd(OH).sub.2/C (20% dry basis, 100 mg) in MeOH (40 mL). The
reaction mixture is stirred overnight under hydrogen. At this
point, the reaction mixture is filtered through Celite and
concentrated to yield compound cdvii (417 mg, 98%).
##STR00442##
Intermediate Example 205
cdx
[0593] Compound cxxv (Purchased from Albany Molecular Research
Inc., 1.5 g, 5.2 mmol) is taken up in DCM (15 mL). PyBOP (2.7 g,
5.2 mmol) and HOBT (700 mg, 5.2 mmol) are added to this solution. A
THF solution (15 mL) of (-)-alpha-(4-pyridyl)ethyl amine (640 mg,
5.2 mmol) is added to above solution, followed by DIEA (0.93 ml,
5.2 mmol). (The (-)-alpha-(4-pyridyl)ethyl amine is obtained from
the tartrate salt of (-)-alpha-(4-pyridyl)ethyl amine (Aldrich) by
stirring with 1 N NaOH (2 eq) for 1 hour followed by extraction
with EtOAc (3.times.) 70% recovery]. The reaction is stirred
overnight at room temperature. The reaction mixture is washed with
saturated NaHCO.sub.3, and brine. The product is purified in 5%
EtOH/EtOAc to yield 2 g (99%) of intermediate compound cdx.
##STR00443##
Intermediate Example 206
cdviii
[0594] Compound cdx (2 g, 5.2 mmol) is hydrogenated using 10% Pd/C
(500 mg) in MEOH (50 mL). The reaction mixture is stirred overnight
under hydrogen. The product is filtered through celite and
concentrated to give compound cdviii (1.3, g 98%).
##STR00444##
Pharmacology
[0595] Compounds according to the invention as described herein as
being useful for being able to inhibit HCV protease, and thus, are
also useful for inhibiting HCV replication.
[0596] Accordingly, an invention herein is directed to a method of
inhibiting HCV protease comprising contacting an anti-HCV protease
inhibitory amount of a compound of formula 1 with a composition
comprising HCV protease.
[0597] Yet another invention herein is directed to a method of
inhibiting replication of HCV comprising contacting HCV with an
effective amount of a compound of formula 1. Furthermore, another
invention herein is directed to a method of treating a patient
suffering from or subject to an HCV infection comprising
administering to the patient a pharmaceutically effective amount of
compound of formula 1. References herein to treating an HCV
infection should be understood to include prophylactic therapy to
prevent or inhibit the infection as well as the treatment of an
established acute or chronic HCV infection or physiological
conditions associated with HCV infection to essentially cure the
patient of the infection, inhibit the degree (amount) of infection
or ameliorate the physiological conditions associated therewith.
"Effective amount" is meant to describe an amount of the compound
of the present invention effective within the scope of reasonable
biological judgement, suitable for use in contact with the cells of
humans and other mammals without undue toxicity, irritation,
allergic response and the like, and are commensurate with a
reasonable benefit/risk ratio in treating an HCV infection and thus
producing the desired therapeutic effect.
[0598] Physiological conditions discussed herein include some, but
not all, of the possible clinical situations where an anti-HCV
treatment is warranted. Those experienced in this field are well
aware of the circumstances requiring either an anti-HCV
treatment.
[0599] A particular aspect of the invention provides for a compound
according to the invention to be administered in the form of a
pharmaceutical composition, though the compound may be administered
alone. "Pharmaceutical composition" means a composition comprising
a compound of formula 1 and at least one component selected from
the group comprising pharmaceutically acceptable carriers,
diluents, coatings, adjuvants, excipients, or vehicles, such as
preserving agents, fillers, disintegrating agents, wetting agents,
emulsifying agents, emulsion stabilizing agents, suspending agents,
isotonic agents, sweetening agents, flavoring agents, perfuming
agents, coloring agents, antibacterial agents, antifungal agents,
other therapeutic agents, lubricating agents, adsorption delaying
or promoting agents, and dispensing agents, depending on the nature
of the mode of administration and dosage forms. The compositions
may be presented in the form of tablets, pills, granules, powders,
aqueous solutions or suspensions, injectable solutions, elixirs or
syrups. Exemplary suspending agents include ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, or mixtures of these substances.
Exemplary antibacterial and antifungal agents for the prevention of
the action of microorganisms include parabens, chlorobutanol,
phenol, sorbic acid, and the like. Exemplary isotonic agents
include sugars, sodium chloride and the like. Exemplary adsorption
delaying agents to prolong absorption include aluminum monosterate
and gelatin. Exemplary adsorption promoting agents to enhance
absorption include dimethyl sulphoxide and related analogs.
Exemplary carriers, diluents, solvents, vehicles, solubilizing
agents, emulsifiers and emulsion stabilizers, include water,
chloroform, sucrose, ethanol, isopropyl alcohol, ethyl carbonate,
ethyl acetate, benzyl alcohol, tetrahydrofurfuryl alcohol, benzyl
benzoate, polyols, propylene glycol, 1,3-butylene glycol, glycerol,
polyethylene glycols, dimethylformamide, Tween.RTM. 60, Span.RTM.
80, cetostearyl alcohol, myristyl alcohol, glyceryl mono-stearate
and sodium lauryl sulfate, fatty acid esters of sorbitan, vegetable
oils (such as cottonseed oil, groundnut oil, corn germ oil, olive
oil, castor oil and sesame oil) and injectable organic esters such
as ethyl oleate, and the like, or suitable mixtures of these
substances. Exemplary excipients include lactose, milk sugar,
sodium citrate, calcium carbonate, dicalcium phosphate. Exemplary
disintegrating agents include starch, alginic acids and certain
complex silicates. Exemplary lubricants include magnesium stearate,
sodium lauryl sulphate, talc, as well as high molecular weight
polyethylene glycols.
[0600] Other therapeutic agents may be used in combination with a
compound of the present invention, including other anti-HCV agents.
Some Exemplary known anti-HCV agents include immunomodulatory
agents, such as .alpha.-, .beta.- or .gamma.-interferons; pegylated
derivatized interferon-.alpha. compounds, other antiviral agents
such as ribavirin and amantadine; other inhibitors of hepatitis C
protease; inhibitors of other targets in the HCV life cycle
including the helicase, polymerase, metalloprotease, internal
ribosome entry, or broad-spectrum antiviral compounds such as
VX-497, an inhibitor of cellular inosine monophosphate
dehydrogenase, IMPDH, covered by U.S. Pat. No. 5,807,876; or
combinations thereof. Therapeutic agents used in combination with a
compound of the present invention may be administered separately,
simultaneously or sequentially.
[0601] The choice of material in the pharmaceutical composition
other than the compound of formula 1 is generally determined in
accordance with the chemical properties of the active compound such
as solubility, the particular mode of administration and the
provisions to be observed in pharmaceutical practice. For example,
excipients such as lactose, sodium citrate, calcium carbonate,
dicalcium phosphate and disintegrating agents such as starch,
alginic acids and certain complex silicates combined with
lubricants such as magnesium stearate, sodium lauryl sulphate and
talc may be used for preparing tablets.
[0602] The pharmaceutical compositions may be presented in assorted
forms such as tablets, pills, granules, powders, aqueous solutions
or suspensions, injectable solutions, elixirs or syrups.
[0603] "Liquid dosage form" means the dose of the active compound
to be administered to the patient is in liquid form, for example,
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups and elixirs. In addition to the active compounds, the liquid
dosage forms may contain inert diluents commonly used in the art,
such solvents, solubilizing agents and emulsifiers.
[0604] Solid compositions may also be employed as fillers in soft
and hard-filled gelatin capsules using such excipients as lactose
or milk sugar as well as high molecular weight polyethylene
glycols, and the like.
[0605] When aqueous suspensions are used they can contain
emulsifying agents or agents which facilitate suspension.
[0606] The oily phase of the emulsion pharmaceutical composition
may be constituted from known ingredients in a known manner. While
the phase may comprise merely an emulsifier (otherwise known as an
emulgent), it desirably comprises a mixture of at least one
emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a
lipophilic emulsifier that acts as a stabilizer. It is also
preferred to include both an oil and a fat. Together, the
emulsifier(s) with or without stabilizer(s) make up the emulsifying
wax, and the way together with the oil and fat make up the
emulsifying ointment base which forms the oily dispersed phase of
the cream formulations.
[0607] If desired, the aqueous phase of the cream base may include,
for example, a least 30% w/w of a polyhydric alcohol, i.e. an
alcohol having two or more hydroxyl groups such as propylene
glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and
polyethylene glycol (including PEG 400) and mixtures thereof. The
topical formulations may desirably include a compound that enhances
absorption or penetration of the active ingredient through the skin
or other affected areas.
[0608] The choice of suitable oils or fats for a formulation is
based on achieving the desired cosmetic properties. Thus the cream
should preferably be a non-greasy, non-staining and washable
product with suitable consistency to avoid leakage from tubes or
other containers. Straight or branched chain, mono- or dibasic
alkyl esters such as di-isopropyl myristate, decyl oleate,
isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a
blend of branched chain esters known as Crodamol CAP may be used.
These may be used alone or in combination depending on the
properties required. Alternatively, high melting point lipids such
as white soft paraffin and/or liquid paraffin or other mineral oils
can be used.
[0609] In practice, a compound/pharmaceutical compositions of the
present invention may be administered in a suitable formulation to
humans and animals by topical or systemic administration, including
oral, inhalational, rectal, nasal, buccal, sublingual, vaginal,
colonic, parenteral (including subcutaneous, intramuscular,
intravenous, intradermal, intrathecal and epidural), intracisternal
and intraperitoneal. It will be appreciated that the preferred
route may vary with for example the condition of the recipient.
[0610] "Pharmaceutically acceptable dosage forms" refers to dosage
forms of the compound of the invention, and includes, for example,
tablets, dragees, powders, elixirs, syrups, liquid preparations,
including suspensions, sprays, inhalants tablets, lozenges,
emulsions, solutions, granules, capsules and suppositories, as well
as liquid preparations for injections, including liposome
preparations. Techniques and formulations generally may be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., latest edition.
[0611] "Formulations suitable for oral administration" may be
presented as discrete units such as capsules, cachets or tablets
each containing a predetermined amount of the active ingredient; as
a powder or granules; as solution or a suspension in an aqueous
liquid or a non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water-in-oil liquid emulsion. The active ingredient
may also be presented as a bolus, electuary or paste.
[0612] A tablet may be made by compression or moulding, optionally
with one or more accessory ingredients. Compressed tables may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binder, lubricant, inert diluent, preservative,
surface active or dispersing agent. Moulded tablets may be made by
moulding in a suitable machine a mixture of the powdered compounds
moistened with an inert liquid diluent. The tablets may optionally
be coated or scored and may be formulated so as to provide slow or
controlled release of the active ingredient therein.
[0613] Solid compositions for rectal administration include
suppositories formulated in accordance with known methods and
containing at least one compound of the invention.
[0614] If desired, and for more effective distribution, the
compounds can be microencapsulated in, or attached to, a slow
release or targeted delivery systems such as a biocompatible,
biodegradable polymer matrices (e.g., poly(d,l-lactide
co-glycolide)), liposomes, and microspheres and subcutaneously or
intramuscularly injected by a technique called subcutaneous or
intramuscular depot to provide continuous slow release of the
compound(s) for a period of 2 weeks or longer. The compounds may be
sterilized, for example, by filtration through a bacteria retaining
filter, or by incorporating sterilizing agents in the form of
sterile solid compositions which can be dissolved in sterile water,
or some other sterile injectable medium immediately before use.
[0615] "Formulations suitable for nasal or inhalational
administration" means formulations which are in a form suitable to
be administered nasally or by inhalation to a patient. The
formulation may contain a carrier, in a powder form, having a
particle size for example in the range 1 to 500 microns (including
particle sizes in a range between 20 and 500 microns in increments
of 5 microns such as 30 microns, 35 microns, etc.) Suitable
formulations wherein the carrier is a liquid, for administration as
for example a nasal spray or as nasal drops, include aqueous or
oily solutions of the active ingredient. Formulations suitable for
aerosol administration may be prepared according to conventional
methods and may be delivered with other therapeutic agents.
Inhalational therapy is readily administered by metered dose
inhalers.
[0616] "Formulations suitable for oral administration" means
formulations which are in a form suitable to be administered orally
to a patient. The formulations may be presented as discrete units
such as capsules, cachets or tablets each containing a
predetermined amount of the active ingredient; as a powder or
granules; as solution or a suspension in an aqueous liquid or a
non-aqueous liquid; or as an oil-in-water liquid emulsion or a
water-in-oil liquid emulsion. The active ingredient may also be
presented as a bolus, electuary or paste.
[0617] "Formulations suitable for parenteral administration" means
formulations that are in a form suitable to be administered
parenterally to a patient. The formulations are sterile and include
emulsions, suspensions, aqueous and non-aqueous injection
solutions, which may contain suspending agents and thickening
agents and anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic, and have a suitably adjusted pH,
with the blood of the intended recipient.
[0618] "Formulations suitable for rectal or vaginal
administrations" means formulations that are in a form suitable to
be administered rectally or vaginally to a patient. The formulation
is preferably in the form of suppositories that can be prepared by
mixing the compounds of this invention with suitable non-irritating
excipients or carriers such as cocoa butter, polyethylene glycol or
a suppository wax, which are solid at ordinary temperatures but
liquid at body temperature and therefore, melt in the rectum or
vaginal cavity and release the active component.
[0619] "Formulations suitable for systemic administration" means
formulations that are in a form suitable to be administered
systemically to a patient. The formulation is preferably
administered by injection, including transmuscular, intravenous,
intraperitoneal, and subcutaneous. For injection, the compounds of
the invention are formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. In addition, the compounds may be formulated in
solid form and redissolved or suspended immediately prior to use.
Lyophilized forms are also included. Systematic administration also
can be by transmucosal or transdermal means, or the compounds can
be administered orally. For transmucosal or transdermal
administration, penetrants appropriate to the bather to be
permeated are used in the formulation. Such penetrants are
generally known in the art, and include, for example, bile salts
and fusidic acid derivatives for transmucosal administration. In
addition, detergents may be used to facilitate permeation.
Transmucosal administration may be through use of nasal sprays, for
example, or suppositories. For oral administration, the compounds
are formulated into conventional oral administration forms such as
capsules, tablets, and tonics.
[0620] "Formulations suitable for topical administration" means
formulations that are in a form suitable to be administered
topically to a patient. The formulation may be presented as a
topical ointment, salves, powders, sprays and inhalants, gels
(water or alcohol based), creams, as is generally known in the art,
or incorporated into a matrix base for application in a patch,
which would allow a controlled release of compound through the
transdermal barrier. When formulated in an ointment, the active
ingredients may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredients
may be formulated in a cream with an oil-in-water cream base.
Formulations suitable for topical administration in the eye include
eye drops wherein the active ingredient is dissolved or suspended
in a suitable carrier, especially an aqueous solvent for the active
ingredient. Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0621] "Solid dosage form" means the dosage form of the compound of
the invention is solid form, for example capsules, tablets, pills,
powders, dragees or granules. In such solid dosage forms, the
compound of the invention is admixed with at least one inert
customary excipient (or carrier) such as sodium citrate or
dicalcium phosphate or (a) fillers or extenders, as for example,
starches, lactose, sucrose, glucose, mannitol and silicic acid, (b)
binders, as for example, carboxymethylcellulose, alignates,
gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants,
as for example, glycerol, (d) disintegrating agents, as for
example, agar-agar, calcium carbonate, potato or tapioca starch,
alginic acid, certain complex silicates and sodium carbonate, (e)
solution retarders, as for example paraffin, (f) absorption
accelerators, as for example, quaternary ammonium compounds, (g)
wetting agents, as for example, cetyl alcohol and glycerol
monostearate, (h) adsorbents, as for example, kaolin and bentonite,
(i) lubricants, as for example, talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate, (j)
opacifying agents, (k) buffering agents, and agents which release
the compound(s) of the 2.5 invention in a certain part of the
intestinal tract in a delayed manner.
[0622] Actual dosage levels of active ingredient(s) in the
compositions of the invention may be varied so as to obtain an
amount of active ingredient(s) that is (are) effective to obtain a
desired therapeutic response for a particular composition and
method of administration for a patient. A selected dosage level for
any particular patient therefore depends upon a variety of factors
including the desired therapeutic effect, on the route of
administration, on the desired duration of treatment, the etiology
and severity of the disease, the patient's condition, weight, sex,
diet and age, the type and potency of each active ingredient, rates
of absorption, metabolism and/or excretion and other factors.
[0623] Total daily dose of the compounds of this invention
administered to a patient in single or divided doses may be in
amounts, for example, of from about 0.001 to about 100 mg/kg body
weight daily and preferably 0.01 to 10 mg/kg/day. For example, in
an adult, the doses are generally from about 0.01 to about 100,
preferably about 0.01 to about 10, mg/kg body weight per day by
inhalation, from about 0.01 to about 100, preferably 0.1 to 70,
more especially 0.5 to 10, mg/kg body weight per day by oral
administration, and from about 0.01 to about 50, preferably 0.01 to
10, mg/kg body weight per day by intravenous administration. The
percentage of active ingredient in a composition may be varied,
though it should constitute a proportion such that a suitable
dosage shall be obtained. Dosage unit compositions may contain such
amounts of such submultiples thereof as may be used to make up the
daily dose. Obviously, several unit dosage forms may be
administered at about the same time. A dosage may be administered
as frequently as necessary in order to obtain the desired
therapeutic effect: Some patients may respond rapidly to a higher
or lower dose and may find much weaker maintenance doses adequate.
For other patients, it may be necessary to have long-term
treatments at the rate of 1 to 4 doses per day, in accordance with
the physiological requirements of each particular patient. It goes
without saying that, for other patients, it will be necessary to
prescribe not more than one or two doses per day.
[0624] The formulations can be prepared in unit dosage form by any
of the methods well known in the art of pharmacy. Such methods
include the step of bringing into association the active ingredient
with the carrier that constitutes one or more accessory
ingredients. In general the formulations are prepared by uniformly
and intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers or both, and then,
if necessary, shaping the product.
[0625] The formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials with elastomeric
stoppers, and may be stored in a freeze-dried (lyophilized)
condition requiring only the addition of the sterile liquid
carrier, for example water for injections, immediately prior to
use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules and tablets of the kind
previously described.
[0626] Compounds within the scope of the present invention exhibit
marked pharmacological activities according to tests described in
the literature and below, which tests results are believed to
correlate to pharmacological activity in humans and other
mammals.
in Vitro Enzyme Assay Procedure
Inhibition of HCV NS3 Serine Protease
[0627] HCV NS3 protease domain was expressed and purified as
described previously (Vertex, PCT publication WO98/17679; which is
incorporated herein by reference). The chromogenic peptide
substrate, EDVVAbuC-p-nitroanilide, and the NS4A cofactor fragment
(-KKGSVVIVGRIVLSGK-) for NS3 protease was custom synthesized by
American Peptide Corn (Ca). The compounds of this invention were
tested for their ability to inhibit HCV NS3 protease activity using
a spectrophotometric assay with EDVVAbuC-p-nitroanilide as
substrate. The assay was run in a 96-well microtiter plate using a
SpectraMax 250 reader (Molecular Devices, Sunnyvale, Calif.) with
kinetic capability. Cleavage of EDVVAbuC-p-nitroanilide (500 .mu.M)
substrate by purified HCV NS3 protease (0.5 .mu.M) was performed at
30.degree. C. in the buffer containing 30 .mu.M NS4A fragment, 46
mM Hepes, pH 8.0, 92 mM NaCl, 18% glycerol, 5 mM DTT, and 7.5% DMSO
in the absence or presence of the testing compound. The reaction
was monitored for pNA (p-nitroaniline) release at 405 nm. The
determination of the kinetic parameters including Vmax, K.sub.m and
V.sub.max/K.sub.m is performed under the conditions as described
above. Ki values are calculated from rate vs. [inhibitor] plots, at
fixed concentrations of enzyme and substrate, by a nonlinear least
squares fit of the data to the equation of Morrison for tight
binding competitive inhibition [J. F. Morrison, Biochim. Biophys.
Acta., 185, 269-286 (1969)]. The Prism program (GraphPad Software,
Inc.) is used for this procedure.
[0628] The HCV serine protease inhibitors disclosed herein can be
used in combination with other molecules that directly exhibit or
indirectly elicit anti-HCV activity either prophylactically in
patients at risk for contracting HCV infection, or to treat
patients that are already infected. The term "anti-HCV activity"
refers to the capacity of a molecule, when present, to completely
inhibit or reduce accumulation of HCV virions compared to HCV
virion accumulation in the absence of such molecule, and/or the
capacity of a molecule to reduce or ameliorate conditions or
symptoms associated with HCV infection or pathogenesis in patients.
Molecules having anti-HCV activity include those that disrupt one
or more steps in HCV infection or replication, as well as those
that evoke immunomodulating and antiproliferative actions in host
cells. Molecules having anti-HCV activity can inhibit HCV-specific
replicative events such as, but not limited to, HCV-directed
nucleic acid or protein synthesis. Stages of HCV replication at
which molecules having anti-HCV activity can act include cell entry
(e.g., attachment; penetration); uncoating and release of the HCV
genome; replication of the HCV genome (e.g., replication of either
strand of the viral RNA genome; transcription of viral messenger
RNA); translation of HCV proteins; posttranslational modification
of HCV proteins (e.g., proteolytic cleavage; glycosylation);
intracellular transport of viral proteins; assembly of virion
components; and release of viral particles (e.g., budding). Classes
of molecules having anti-viral activity include, but are not
limited to, soluble receptor decoys and antireceptor antibodies;
ion channel blockers, capsid stabilizers, and fusion protein
inhibitors; inhibitors of viral polymerases, reverse transcriptase,
helicase, primase, or integrase; antisense oligonucleotides and
ribozymes; immunomodulating and immunostimulating agents, including
cytokines such as interferons, as well as peptide agonists,
steroids, and classic drugs such as levamisole; inhibitors of
regulatory proteins; protease inhibitors; assembly protein
inhibitors; and antiviral antibodies and cytotoxic lymphocytes. The
term "anti-HCV effective amount" or "pharmaceutically effective
amount" refers to an amount of a compound, or combination of
compounds as disclosed herein, effective in reducing or
ameliorating conditions or symptoms associated with HCV infection
or associated pathogenesis in patients, or in reducing viral levels
in vitro or in vivo. In vitro applications include the Replicon
Assay system, described below, where such amounts are effective in
reducing HCV replicon RNA accumulation and/or the accumulation of
proteins encoded by genes contained therein.
[0629] Compounds having anti-HCV activity contemplated for use in
the compositions and methods of combination therapy disclosed
herein include, but are not limited to, immunomodulatory molecules,
including immunostimulatory cytokines, and other compounds known to
have HCV antiviral activity, such as various antiviral nucleosides
and nucleotides. Immunomodulatory molecules contemplated for use in
combination with the HCV serine protease inhibitors disclosed
herein include, but are not limited to, interferon-alpha 2B (Intron
A. Schering Plough); Rebatron (Schering Plough, Interferon-alpha
2B+Ribavirin); pegylated interferon alpha (Reddy, K. R. et al.
Efficacy and safety of pegylated (40-kd) interferon alpha-2a
compared with interferon alpha-2a in noncirrhotic patients with
chronic hepatitis C. Hepatology 33, 433-438 (2001)); consensus
interferon (Kao, J. H., Chen, P. J., Lai, M. Y. & Chen, D. S.
Efficacy of consensus interferon in the treatment of chronic
hepatitis C. J. Gastroenterol. Hepatol. 15, 1418-1423 (2000));
interferon-alpha 2A (Roferon A; Roche); lymphoblastoid or "natural"
interferon; interferon tau (Clayette, P. et al. IFN-tau, a new
interferon type I with antiretroviral activity. Pathol. Biol.
(Paris) 47, 553-559 (1999)); interleukin 2 (Davis, G. L., Nelson,
D. R. & Reyes, G. R. Future options for the management of
hepatitis C. Seminars in Liver Disease 19, 103-112 (1999));
Interleukin 6 (Davis, G. L, Nelson, D. R. & Reyes, G. R. Future
options for the management of hepatitis C. Seminars in Liver
Disease 19, 103-112 (1999)); Interleukin 12 (Davis, G. L., Nelson,
D. R. & Reyes, G. R. Future options for the management of
hepatitis C. Seminars in Liver Disease 19, 103-112 (1999));
Ribavirin; and compounds that enhance the development of a type 1
helper T cell response (Davis, G. L., Nelson, D. R. & Reyes, G.
R. Future options for the management of hepatitis C. Seminars in
Liver Disease 19, 103-112 (1999)). Interferons may ameliorate viral
infections by exerting direct antiviral effects and/or by modifying
the immune response to infection. The antiviral effects of
interferons are often mediated through inhibition of viral
penetration or uncoating, synthesis of viral RNA, translation of
viral proteins, and/or viral assembly and release.
[0630] Compounds that stimulate the synthesis of interferon in
cells (Tazulakhova, E. B., Parshina, O. V., Gusev, T. S. &
Ershov, F. L Russian Experience in Screening, Analysis, and
Clinical Application of Novel Interferon Inducers. J. Interferon
Cytokine Res. 21, 65-73)) include, but are not limited to, double
stranded RNA, alone or in combination with tobramycin, and
Imiquimod (3M Pharmaceuticals) (Sauder, D. N. Immunomodulatory and
pharmacologic properties of imiquimod. J. Am. Acad. Dermatol. 43,
S6-11 (2000)).
[0631] Other compounds known to have, or that may have, HCV
antiviral activity by virtue of non-immunomodulatory mechanisms
include, but are not limited to, Ribavirin (ICN Pharmaceuticals);
inosine 5'-monophosphate dehydrogenase inhibitors (VX-497, being
developed by Vertex Pharmaceuticals); amantadine and rimantadine
(Younossi, A. M. and Perillo, R. P. The roles of amantadine,
rimantadine, ursodeoxycholic acid, NSAIDs, alone or in combination
with alpha interferons, in the treatment of chronic hepatitis C.
Seminars in Liver Disease 19.95-102 (1999)); LY217896 (U.S. Pat.
No. 4,835,168) (Colacino, J. M. et al. Evaluation of the
anti-influenza virus activities of 1,3,4-thiadiazol-2-ylcyanamide
(LY217896) and its sodium salt. Antimicrobial Agents &
Chemotherapy 34, 2156-2163 (1990)); and
9-Hydroxyimino-6-methoxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydro-phen-
anthrene-1-carboxylic acid methyl ester;
6-Methoxy-1,4a-dimethyl-9-(4-methyl-piperazin-1-ylimino)-1,2,3,4,4a,9,10,-
10a-octahydro-phenanthrene-1-carboxylic acid methyl
ester-hydrochloride;
1-(2-Chloro-phenyl)-3-(2,2-diphenyl-ethyl)-urea (U.S. Pat. No.
6,127,422).
[0632] Formulations, doses, and routes of administration for the
foregoing molecules are either taught in the references cited
below, or are well-known in the art as disclosed, for example, in
F. G. Hayden, in Goodman & Gilman's The Pharmacological Basis
of Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill,
New York (1996), Chapter 50, pp. 1191-1223, and the references
cited therein. Alternatively, once a compound that exhibits HCV
antiviral activity has been identified, a pharmaceutically
effective amount of that compound can be determined using
techniques that are well-known to the skilled artisan. Note, for
example, Benet et al., in Goodman & Gilman's The
Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et
al., Eds., McGraw-Hill, New York (1996), Chapter 1, pp. 3-27, and
the references cited therein. Thus, the appropriate formulations,
dose(s) range, and dosing regimens, of such a compound can be
easily determined by routine methods.
[0633] The drug combinations of the present invention can be
provided to a cell or cells, or to a human patient, either in
separate pharmaceutically acceptable formulations administered
simultaneously or sequentially, formulations containing more than
one therapeutic agent, or by an assortment of single agent and
multiple agent formulations. However administered, these drug
combinations form an anti-HCV effective amount of components.
[0634] A large number of other immunomodulators and
immunostimulants that can be used in the methods of the present
invention are currently available and include: AA-2G;
adamantylamide dipeptide; adenosine deaminase, Enzon; adjuvant,
Alliance; adjuvants, Ribi; adjuvants, Vaxcel; Adjuvax;
agelasphin-11; AIDS therapy, Chiron; algal glucan, SRI; algammulin,
Anutech; Anginlyc; anticellular factors, Yeda; Anticort;
antigastrin-17 immunogen, Ap; antigen delivery system, Vac; antigen
formulation, IDBC; antiGnRH immunogen, Aphton; Antiherpin; Arbidol;
azarole; Bay-q-8939; Bay-r-1005; BCH-1393; Betafectin; Biostim;
BL-001; BL-009; Broncostat; Cantastim; CDRI-84-246; cefodizime;
chemokine inhibitors, ICOS; CMV peptides, City of Hope; CN-5888;
cytokine-releasing agent, St; DHEAS, Paradigm; DISC TA-HSV; J07B;
I01A; I01Z; ditiocarb sodium; ECA-10-142; ELS-1; endotoxin,
Novartis; FCE-20696; FCE-24089; FCE-24578; FLT-3 ligand, Immunex;
FR-900483; FR-900494; FR-901235; FTS-Zn; G-proteins, Cadus;
gludapcin; glutaurine; glycophosphopeptical; GM-2; GM-53; GMDP;
growth factor vaccine, EntreM; H-BIG, NABI; H-CIG, NABI; HAB-439;
Helicobacter pylori vaccine; herpes-specific immune factor, HIV
therapy, United Biomed; HyperGAM+CF; ImmuMax; Immun BCG; immune
therapy, Connective; immunomodulator, Evans; Immunomodulators,
Novacell; imreg-1; imreg-2; Indomune; inosine pranobex; interferon,
Dong-A (alpha2); interferon, Genentech (gamma); interferon,
Novartis (alpha); interleukin-12, Genetics Ins; interleukin-15,
Immunex; interleukin-16, Research Cor; ISCAR-1; J005X; L-644257;
licomarasminic acid; LipoTher, LK-409; LK-410; LP-2307; LT (R1926);
LW-50020; MAF, Shionogi; MDP derivatives, Merck; met-enkephalin,
TNI; methylfurylbutyrolactones; MMP; mirimostim; mixed bacterial
vaccine, Tem; MM-1; moniliastat; MPLA, Ribi; MS-705; murabutide;
murabutide, Vacsyn; muramyl dipeptide derivative; muramyl peptide
derivatives myelopid; -563; NACOS-6; NH-765; NISV, Proteus;
NPT-16416; NT-002; PA-485; PEFA-814; peptides, Scios;
peptidoglycan, Pliva; Perthon, Advanced Plant; PGM derivative,
Pliva; Pharmaprojects No. 1099; No. 1426; No. 1549; No. 1585; No.
1607; No. 1710; No. 1779; No. 2002; No. 2060; No. 2795; No. 3088;
No. 3111; No. 3345; No. 3467; No. 3668; No. 3998; No. 3999; No.
4089; No. 4188; No. 4451; No. 4500; No. 4689; No. 4833; No. 494;
No. 5217; No. 530; pidotimod; pimelautide; pinafide; PMD-589;
podophyllotoxin, Conpharm; POL-509; poly-ICLC; poly-ICLC, Yamasa
Shoyu; PolyA-PolyU; Polysaccharide A; protein A, Berlox Bioscience;
PS34WO; Pseudomonas MAbs, Teijin; Psomaglobin; PTL-78419; Pyrexol;
pyriferone; Retrogen; Retropep; RG-003; Rhinostat; rifamaxil;
RM-06; Rollin; romurtide; RU-40555; RU-41821; Rubella antibodies,
ResCo; S-27609; SB-73; SDZ-280-636; SDZ-MRL-953; SK&F-107647;
SL04; SL05; SM-4333; Solutein; SRI-62-834; SRL-172; ST-570; ST-789;
staphage lysate; Stimulon; suppressin; T-150R1; T-LCEF;
tabilautide; temurtide; Theradigm-HBV; Theradigm-HPV;
Theradigm-HSV; THF, Pharm & Upjohn; THF, Yeda; thymalfasin;
thymic hormone fractions; thymocartin; thymolymphotropin;
thymopentin; thymopentin analogues; thymopentin, Peptech; thymosin
fraction 5, Alpha; thymostimulin; thymotrinan; TMD-232; TO-115;
transfer factor, Viragen; tuftsin, Selavo; ubenimex; Ulsastat;
ANGG-; CD-4+; Collag+; COLSF+; COM+; DA-A+; GAST-; GF-TH+; GP-120-;
IF+; IF-A+; IF-A-2+; IF-B+; IF-G+; IF-G-1B+; IL-2+; IL-12+; IL-15+;
IM+; LHRH-; LIPCOR+L LYM-B+; LYM-NK+; LYM-T+; OPI+; PEP+; PHG-MA+;
RNA-SYN-; SY-CW-; TH-A-1+; TH-5+; TNF+; UN.
[0635] Representative nucleoside and nucleotide compounds useful in
the present invention include, but are not limited to:
(+)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosine;
(-)-2'-deoxy-3'-thiocytidine-5'-triphosphate (3TC);
(-)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosine
(FTC); (-) 2',3', dideoxy-3'-thiacytidine[(-)-SddC];
1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine
(FIAC);
1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine
triphosphate (FIACTP);
1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil
(FMAU); 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide;
2',3'-dideoxy-3'-fluoro-5-methyl-dexocytidine (FddMeCyt);
2',3'-dideoxy-3'-chloro-5-methyl-dexocytidine (ClddMeCyt);
2',3'-dideoxy-3'-amino-5-methyl-dexocytidine (AddMeCyt);
2',3'-dideoxy-3'-fluoro-5-methyl-cytidine (FddMeCyt);
2',3'-dideoxy-3'-chloro-5-methyl-cytidine (ClddMeCyt);
2',3'-dideoxy-3'-amino-5-methyl-cytidine (AddMeCyt);
2',3'-dideoxy-3'-fluorothymidine (FddThd);
2',3'-dideoxy-beta-L-5-fluorocytidine (beta-L-FddC)
2',3'-dideoxy-beta-L-5-thiacytidine;
2',3'-dideoxy-beta-L-S-cytidine (beta-L-ddC);
9-(1,3-dihydroxy-2-propoxymethyl) guanine;
2'-deoxy-3'-thia-5-fluorocytosine; 3'-amino-5-methyl-dexocytidine
(AddMeCyt); 2-amino-1,9-[(2-hydroxymethyl-1-(hydroxymethyl)
ethoxy]methyl]-6H-purin-6-one (gancyclovir);
2-[2-(2-amino-9H-purin-9y)ethyl]-1,3-propandil diacetate
(famciclovir);
2-amino-1,9-dihydro-9-[(2-hydroxy-ethoxy)methyl]6H-purin-6-one
(acyclovir); 9-(4-hydroxy-3-hydroxymethyl-but-1-yl) guanine
(penciclovir);
9-(4-hydroxy-3-hydroxymethyl-but-1-yl)-6-deoxy-guanine diacetate
(famciclovir); 3'-azido-3'-deoxythymidine (AZT);
3'-chloro-5-methyl-dexocytidine (ClddMeCyt);
9-(2-phosphoryl-methoxyethyl)-2',6'-diaminopurine-2',3'-dideoxyriboside;
9-(2-phosphonylmethoxyethyl) adenine (PMEA); acyclovir triphosphate
(ACVTP); D-carbocyclic-2'-deoxyguanosine (CdG); dideoxy-cytidine;
dideoxy-cytosine (ddC); dideoxy-guanine (ddG); dideoxy-inosine
(ddI); E-5-(2-bromovinyl)-2'-deoxyuridine triphosphate;
fluoro-arabinofuranosyl-iodouracil;
1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl)-5-iodo-uracil
(FIAU); stavudine; 9-beta-D-arabinofuranosyl-9H-purine-6-amine
monohydrate (Ara-A);
9-beta-D-arabinofuranosyl-9H-purine-6-amine-5'-monophosphate
monohydrate (Ara-AMP); 2-deoxy-3'-thia-5-fluorocytidine;
2',3'-dideoxy-guanine; and 2',3'-dideoxy-guanosine.
[0636] Synthetic methods for the preparation of nucleosides and
nucleotides useful in the present invention are well known in the
art as disclosed in Acta Biochim. Pol., 43, 25-36 (1996); Swed.
Nucleosides Nucleotides 15, 361-378 (1996); Synthesis 12, 1465-1479
(1995); Carbohyd. Chem. 27, 242-276 (1995); Chem. Nucleosides
Nucleotides 3, 421-535 (1994); Ann. Reports in Med. Chem., Academic
Press; and Exp. Opin. Invest. Drugs 4, 95-115 (1995).
[0637] The chemical reactions described in the references cited
above are generally disclosed in terms of their broadest
application to the preparation of the compounds of this invention.
Occasionally, the reactions may not be applicable as described to
each compound included within the scope of compounds disclosed
herein. The compounds for which this occurs will be readily
recognized by those skilled in the art. In all such cases, either
the reactions can be successfully performed by conventional
modifications known to those skilled in the art, e.g., by
appropriate protection of interfering groups, by changing to
alternative conventional reagents, by routine modification of
reaction conditions, and the like, or other reactions disclosed
herein or otherwise conventional will be applicable to the
preparation of the corresponding compounds of this invention. In
all preparative methods, all starting materials are known or
readily preparable from known starting materials.
[0638] While nucleoside analogs are generally employed as antiviral
agents as is, nucleotides (nucleoside phosphates) must sometimes
have to be converted to nucleosides in order to facilitate their
transport across cell membranes. An example of a chemically
modified nucleotide capable of entering cells is
S-1-3-hydroxy-2-phosphonylmethoxypropyl cytosine (HPMPC, Gilead
Sciences). Nucleoside and nucleotide compounds of this invention
that are acids can form salts. Examples include salts with alkali
metals or alkaline earth metals, such as sodium, potassium,
calcium, or magnesium, or with organic bases or basic quaternary
ammonium salts.
[0639] Immunomodulators and immunostimulants useful in the
combination therapy methods of the present invention can be
administered in amounts lower than those conventional in the art.
For example, interferon alpha is typically administered to humans
for the treatment of HCV infections in an amount of from about
1.times.10.sup.6 units/person three times per week to about
10.times.10.sup.6 units/person three times per week (Simon et al.,
Hepatology 25: 445-448 (1997)). In the methods and compositions of
the present invention, this dose can be in the range of from about
0.1.times.10.sup.6 units/person three times per week to about
7.5.times.10.sup.6 units/person three times per week; more
preferably from about 0.5.times.10.sup.6 units/person three times
per week to about 5.times.10.sup.6 units/person three times per
week; most preferably from about 1.times.10.sup.6 units/person
three times per week to about 3.times.10.sup.6 units/person three
times per week. Due to the enhanced hepatitis C virus antiviral
effectiveness of immunomodulators and immunostimulants in the
presence of the HCV serine protease inhibitors of the present
invention, reduced amounts of these
immunomodulators/immunostimulants can be employed in the methods
and compositions disclosed herein. Similarly, due to the enhanced
hepatitis C virus antiviral effectiveness of the present HCV serine
protease inhibitors in the presence of immunomodulators and
immunostimulants, reduced amounts of these HCV serine protease
inhibitors can be employed in the methods and compositions
disclosed herein. Such reduced amounts can be determined by routine
monitoring of hepatitis C virus titers in infected patients
undergoing therapy. This can be carried out by, for example,
monitoring HCV RNA in patients' serum by slot-blot, dot-blot, or
RT-PCR techniques, or by measurement of HCV surface or other
antigens. Patients can be similarly monitored during combination
therapy employing the HCV serine protease inhibitors disclosed
herein and other compounds having anti-HCV activity, for example
nucleoside and/or nucleotide antiviral agents, to determine the
lowest effective doses of each when used in combination.
[0640] In the methods of combination therapy disclosed herein,
nucleoside or nucleotide antiviral compounds, or mixtures thereof,
can be administered to humans in an amount in the range of from
about 0.1 mg/person/day to about 500 mg/person/day; preferably from
about 10 mg/person/day to about 300 mg/person/day; more preferably
from about 25 mg/person/day to about 200 mg/person/day; even more
preferably from about 50 mg/person/day to about 150 mg/person/day;
and most preferably in the range of from about 1 mg/person/day to
about 50 mg/person/day.
[0641] Doses of compounds can be administered to a patient in a
single dose or in proportionate multiple subdoses. In the latter
case, dosage unit compositions can contain such amounts of
submultiples thereof to make up the daily dose. Multiple doses per
day can also increase the total daily dose should this be desired
by the person prescribing the drug.
[0642] The regimen for treating a patient suffering from a HCV
infection with the compounds and/or compositions of the present
invention is selected in accordance with a variety of factors,
including the age, weight, sex, diet, and medical condition of the
patient, the severity of the infection, the route of
administration, pharmacological considerations such as the
activity, efficacy, pharmacokinetic, and toxicology profiles of the
particular compounds employed, and whether a drug delivery system
is utilized. Administration of the drug combinations disclosed
herein should generally be continued over a period of several weeks
to several months or years until virus titers reach acceptable
levels, indicating that infection has been controlled or
eradicated. Patients undergoing treatment with the drug
combinations disclosed herein can be routinely monitored by
measuring hepatitis viral RNA in patients' serum by slot-blot,
dot-blot, or RT-PCR techniques, or by measurement of hepatitis C
viral antigens, such as surface antigens, in serum to determine the
effectiveness of therapy. Continuous analysis of the data obtained
by these methods permits modification of the treatment regimen
during therapy so that optimal amounts of each component in the
combination are administered, and so that the duration of treatment
can be determined as well. Thus, the treatment regimen/dosing
schedule can be rationally modified over the course of therapy so
that the lowest amounts of each of the antiviral compounds used in
combination which together exhibit satisfactory anti-hepatitis C
virus effectiveness are administered, and so that administration of
such antiviral compounds in combination is continued only so long
as is necessary to successfully treat the infection.
[0643] The present invention encompasses the use of the HCV serine
protease inhibitors disclosed herein in various combinations with
the foregoing and similar types of compounds having anti-HCV
activity to treat or prevent HCV infections in patients. For
example, one or more HCV serine protease inhibitors can be used in
combination with: one or more interferons or interferon derivatives
having anti-HCV activity; one or more non-interferon compounds
having anti-HCV activity; or one or more interferons or interferon
derivatives having anti-HCV activity and one or more non-interferon
compounds having anti-HCV activity. When used in combination to
treat or prevent HCV infection in a human patient, any of the
presently disclosed HCV serine protease inhibitors and foregoing
compounds having anti-HCV activity can be present in a
pharmaceutically or anti-HCV effective amount. By virtue of their
additive or synergistic effects, when used in the combinations
described above, each can also be present in a subclinical
pharmaceutically effective or anti-HCV effective amount. i.e., an
amount that, if used alone, provides reduced pharmaceutical
effectiveness in completely inhibiting or reducing the accumulation
of HCV virions and/or reducing or ameliorating conditions or
symptoms associated with HCV infection or pathogenesis in patients
compared to such HCV serine protease inhibitors and compounds
having anti-HCV activity when used in pharmaceutically effective
amounts. In addition, the present invention encompasses the use of
combinations of HCV mine protease inhibitors and compounds having
anti-HCV activity as described above to treat or prevent HCV
infections, where one or more of these inhibitors or compounds is
present in a pharmaceutically effective amount, and the other(s)
is(are) present in a subclinical pharmaceutically effective or
anti-HCV effective amount(s) owing to their additive or synergistic
effects. As used herein, the term "additive effect" describes the
combined effect of two (or more) pharmaceutically active agents
that is equal to the sum of the effect of each agent given alone. A
synergistic effect is one in which the combined effect of two (or
more) pharmaceutically active agents is greater than the sum of the
effect of each agent given alone.
Example 42
[0644] Current standard therapy for hepatitis C virus (HCV)
infection is treatment with the immunomodulator alpha-interferon
(Chronic Hepatitis C: Current Disease Management, U.S. Department
of Health and Human Services, National Institutes of Health, 1999).
This therapy is ineffective in most HCV patients, who show either
no response or a relapse even after prolonged interferon therapy.
Additionally, there are severe side effects associated with
interferon therapy.
[0645] In view of the pressing need for new, more effective
antiviral drugs to treat HCV infected patients, the present
inventors have developed a series of compounds that inhibit the
serine protease of HCV (a complex of HCV viral proteins NS3 and
NS4A). These compounds can be used alone, together with one
another, and in combination with other classes of compounds to
treat or prevent HCV infection. This example describes the testing
of three representative HCV serine protease inhibitors, i.e.,
Compound CU, Compound EP, and Compound EC, alone and in combination
with individual members of a set of interferons (interferon
alpha-2B (Schering-Plough), interferon alpha-2A (PBL Biomedical
Laboratories, New Brunswick, N.J.), interferon beta (Research
Diagnostics Inc, Flanders, N.J.), and ovine-interferon tau
(Research Diagnostics Inc, Flanders, N.J.)) in an HCV subgenomic
RNA replicon assay (Replicon Assay) to determine if the two
compounds act in concert to diminish HCV RNA accumulation. The
Replicon Assay measures the amount of HCV subgenomic RNA (replicon
RNA) remaining in replicon cells (Lohman et al. Science 285:110-113
(1999)) after two days of drug treatment relative to the amount of
replicon RNA in untreated cells. In this assay, the potency of
compounds as HCV antiviral drugs is directly proportional to the
level of inhibition of replicon RNA accumulation.
[0646] The two drugs are tested in combinations in the in vitro
Replicon Assay system to determine whether, when used together,
they exhibit additive or synergistic anti-HCV activity. The
Replicon Assay is employed as a surrogate model for in vitro HCV
infection to evaluate the combined effects of the immunomodulator,
for example interferon-alpha 2B ((Intron A); Schering Plough), and
the HCV serine protease inhibitor, for example Compound CU. As
shown below, the results demonstrate that there is a clear
synergistic anti-HCV effect of these two types of drugs as measured
using formal mathematical determinations of synergy to analyze
their capacity to reduce HCV RNA levels in the Replicon Assay.
The Replicon Assay
[0647] The Replicon Assay employing a cell line containing the
self-replicating HCV subgenomic RNA (replicon) is described in
Lohmann et al. Science 285:110-113 (1999). The Genbank accession
number for the sequence of the replicon used in the experiments
described herein is listed in this reference as AJ242654. This
paper discloses methods for in vitro transcription of RNA from the
replicon cDNA, transfection of the replicon RNA into Huh7 cells by
electroporation, and selection of cells containing the replicon RNA
using the antibiotic G418. Huh7 cells are a hepatoma cell line
obtained from Dr. William Mason at Fox Chase Cancer Research Center
(Philadelphia). These cells are publicly available from Fox Chase,
and have been extensively described in the scientific literature
(Nakabayashi at al. Cancer Res. 42:3858-3863 (1982)). In the
experiments described herein, all of the template DNA is removed
from the in vitro transcribed replicon RNA preparation prior to
electroporation of this RNA into Huh7 cells by multiple treatment
with DNase (three sequential treatments).
[0648] The Replicon Assay is performed as described in detail
below. Briefly, Replicon cells are placed in 96 well trays at a
density of 10,000 cells per well and incubated 37.degree. C. The
cells are incubated in DMEM (Dulbecco's Minimal Essential Media)
supplemented with 10% fetal bovine serum, glutamine, nonessential
amino acids, and the antibiotic G418 (0.25 mg/ml). After overnight
incubation, the medium is replaced with DMEM containing 2% fetal
bovine serum and various concentrations of the serine protease
inhibitor, such as Compound CU, and/or an interferon such as
interferon-alpha 2B (Intron A. Schering Plough). Each compound is
tested at six to eight different concentrations. For one extreme of
the range of concentrations, high concentrations of the compounds
that will result in almost complete inhibition of replicon RNA
accumulation after two days of treatment are selected. From these
starting concentrations, serial dilutions are made so that the
concentration ranges tested in the Replicon Assay include
concentrations at which the compounds are highly effective, as well
as concentrations at which there is no significant effect. Each HCV
serine protease inhibitor concentration is tested without any added
interferon, as well as with the addition of six to eight different
interferon doses. Similarly, interferon is tested without any added
HCV serine protease inhibitor. After a 48-hour incubation with the
compounds, the medium is removed from the plates, and total
cellular RNA is extracted from the cells using the RNeasy-96 kit
manufactured by Qiagen Inc. (Valencia, Calif.). This RNA is then
analyzed by quantitative RT-PCR, or TaqMan.RTM. (Applied
Biosystems, Foster City Calif.). The TaqMan.RTM. RT-PCR target is
the neomycin resistance gene in the replicon RNA. The plates are
configured so that there are 5 replicates of each drug treatment
sample, and 16 replicates of the untreated samples. This permits
greater statistical confidence in the quantitative RT-PCR data.
[0649] Analysis of the Replicon Assay data yields two values that
are useful in assessing the potency of potential HCV antiviral
agents. At each compound concentration tested, the level of
inhibition in replicon RNA accumulation caused by the compound
during two days of treatment relative to the amount of replicon RNA
in untreated cells is determined. This is reported as percent
inhibition. When a series of data points generated by treatments of
cells at a range of concentrations has been obtained, IC.sub.50
values, i.e., the compound concentration at which HCV replicon RNA
accumulation is diminished 50% by the compound, are generated.
Through repeated testing of the HCV serine protease inhibitors in
the Replicon Assay, it is determined that the IC.sub.50 has a
percent coefficient of variation (% CV or 100%.times.standard
deviation in the IC.sub.50/mean IC.sub.50) of about 20%. The
IC.sub.50 is the value used to rank individual compounds tested in
this assay based on their potency as HCV antiviral agents. Simple
IC.sub.50 determinations are inadequate to assess the utility of
compounds used in combination. The most effective analysis of the
array of data generated using all the combinations of different
interferons and serine protease inhibitors requires evaluation of
the percent inhibitions as shown in Table 7 using mathematical
methods described below that are designed to determine if
combination treatments are agonistic, additive, or synergistic.
[0650] Details of the Replicon Assay are as follows:
Procedure for Quantitative Analysis of HCV Replicon RNA in the HCV
Replicon Assay Using TaqMan.RTM..RTM. RT-PCR
[0651] The Replicon Assay is used to measure the capacity of
potential HCV antiviral compounds to inhibit the accumulation of a
HCV subgenomic RNA replicon molecule in a Huh7 cell line (Lohmann
et al. Replication of Subgenomic Hepatitis C Virus RNAs in a
Hepatoma Cell Line. Science 285, 110-113 (1999)). This assay
comprises three operational components: (1) Replicon cell
maintenance, assay plate set up, and compound application: (2)
Extraction of total cellular RNA from replicon cells; and (3) Real
time RT-PCR (TaqMan.RTM.) to measure the mount of replicon RNA in
each sample. The Replicon Assay requires at least 4 days to
perform, however, the process can be interrupted and samples frozen
between steps. Each assay component is described below.
1. Replicon Cell Maintenance, Assay Plate Setup, and Compound
Application
1.1 Replicon Cell Line Maintenance
[0652] The cell line used in the Replicon Assay is produced as
described in Lohmann et al. (Replication of Subgenomic Hepatitis C
Virus RNAs in a Hepatoma Cell Line. Science 285, 110-113 (1999)).
After 150 cm.sup.2 cell culture flasks (Costar) containing Replicon
cells are incubated at 37.degree. C. and 5% CO.sub.2 and become
confluent, the cells are diluted 1:10, v/v, into fresh 150 cm.sup.2
cell culture flasks. The medium is DMEM containing 10% fetal bovine
serum (FBS), 1.times. nonessential amino acids (NEAA), 1.times.
Glutamine (Glu), and 0.25 mg/ml G418. Three serial passages are
performed, each time allowing the cells to become confluent,
followed by dilution of the cells into fresh 150 cm.sup.2 cell
culture flasks. These cells, referred to as "original cells," are
then aliquoted and stored for future use in the Replicon Assay.
TaqMan.RTM.-based analysis is performed to determine the number of
HCV replicon genomes per cell, which reveals the presence of -150
copies of the replicon per cell. This is based on the ratio of
copies of replicon RNA to two times the copies of the human apoB
gene (number of haploid genomes). [0653] 1.1.1 Original cells are
stored in liquid N.sub.2 For cells used in the Replicon Assay,
after 20 serial passages, cells are abandoned, and a fresh lot is
revived from liquid N.sub.2 storage.
1.2 Plating of Cells in 96-Well Trays for the Replicon Assay
[0653] [0654] 1.2.1. For preparation of 96-well plates, a 75%
confluent 75 cm.sup.2 flask of replicon-containing cells are
trypsinized, and resuspended in 10 ml Medium A. Trypsinization is
performed by removing the medium, adding 1 ml of trypsin-EDTA
0.25%, w/v, and then removing the trypsin-EDTA. After 5-10 minutes
the cells release from the flask and are resuspended in medium.
[0655] 1.2.2. Cells are counted using a hemacytometer, and the cell
concentration is adjusted to 10.sup.3 cells/ml. [0656] 1.2.3. Each
well is seeded with a 100 .mu.l cell suspension using an Impact2
multi-channel pipette (Matrix), never plating more than four
96-well plates from a single cell suspension. [0657] 1.2.4. 96-well
plates are incubated at 37.degree. C. overnight.
1.3. Compound Dilution and Application to Replicon Cell Trays
[0657] [0658] 1.3.1. HCV serine protease inhibitor compounds are
dissolved in dimethylsulfoxide (DMSO) to a final concentration of
20 mM. Interferons are suspended in phosphate buffered saline
solution containing 0.1% w/v bovine serum albumin. [0659] 1.3.2.
The 20 mM compound solution is diluted to 1 mM with DMSO. [0660]
1.3.3. 50 .mu.l of compound dissolved in DMSO are added to 10 ml
Medium B (the compound concentration is 5 mM, and the DMSO
concentration is now 0.5%), or 20 .mu.l of 1 mM compound and 30
.mu.l DMSO are added to 10 ml Medium B (compound concentration is 2
.mu.M.) [0661] 1.3.4. Compound dilution to final concentration is
completed by mixing compound/Medium B solution with Medium C
(contains 0.5% DMSO). Serial one to five dilutions of the compound
are made with Medium C in a 2 ml polypropylene 96-well block to
obtain the desired final concentrations of compound. [0662] 1.3.5.
The cell plate is removed from the 37.degree. C. incubator and
labeled on the top right corner of the lid and the right side of
the base. The medium is poured off of the 96-well plates. [0663]
1.3.6. 100 .mu.l compound/medium solutions from each well of the
96-well dilution block are added to the 96-well cell plate using an
Impact2 Pipette. [0664] 1.3.7. 100 .mu.l medium C are added to all
the untreated wells according to Table 3 for testing compounds at
either 1, 3, or 6 different concentrations. "Untx" refers to
mock-treated cells (DMSO added at the same concentration as in
treated cells); "Con." refers to compound concentration.
TABLE-US-00004 [0664] TABLE 3 2 compounds, 6 concentrations, 5
replicates Compound 1 Compound 2 1 2 3 4 5 6 7 8 9 10 11 12 A untx
untx untx untx untx untx untx untx untx untx B con.1 con.1 con.1
con.1 con.1 con.1 con.1 con.1 con.1 con.1 C con.2 con.2 con.2 con.2
con.2 con.2 con.2 con.2 con.2 con.2 D con.3 con.3 con.3 con.3 con.3
con.3 con.3 con.3 con.3 con.3 E con.4 con.4 con.4 con.4 con.4 con.4
con.4 con.4 con.4 con.4 F con.5 con.5 con.5 con.5 con.5 con.5 con.5
con.5 con.5 con.5 G con.6 con.6 con.6 con.6 con.6 con.6 con.6 con.6
con.6 con.6 H untx untx untx untx untx untx untx untx untx untx 4
compounds, 3 concentration, 5 replicates Compound 1 Compound 2 1 2
3 4 5 6 7 8 9 10 11 12 A untx untx untx untx untx untx untx untx
untx untx B con.1 con.1 con.1 con.1 con.1 con.1 con.1 con.1 con.1
con.1 C con.2 con.2 con.2 con.2 con.2 con.2 con.2 con.2 con.2 con.2
D con.3 con.3 con.3 con.3 con.3 con.3 con.3 con.3 con.3 con.3 E
con.1 con.1 con.1 con.1 con.1 con.1 con.1 con.1 con.1 con.1 F con.2
con.2 con.2 con.2 con.2 con.2 con.2 con.2 con.2 con.2 G con.3 con.3
con.3 con.3 con.3 con.3 con.3 con.3 con.3 con.3 H untx untx untx
untx untx untx untx untx untx untx 16 compounds, 1 concentration, 4
replicates. Compound 3 Compound 4 1 2 3 4 5 6 7 8 9 10 11 12 A untx
cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx B untx cpd 1
cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx C untx cpd 1 cpd 2
cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx D untx cpd 1 cpd 2 cpd 3
cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx E untx cpd 9 cpd 10 cpd 11 cpd
12 cpd 13 cpd 14 cpd 15 cpd 16 untx F untx cpd 9 cpd 10 cpd 11 cpd
12 cpd 13 cpd 14 cpd 15 cpd 16 untx G untx cpd 9 cpd 10 cpd 11 cpd
12 cpd 13 cpd 14 cpd 15 cpd 16 untx H untx cpd 9 cpd 10 cpd 11 cpd
12 cpd 13 cpd 14 cpd 15 cpd 16 untx 12 compounds, 1 concentration,
5 replicates. 1 2 3 4 5 6 7 8 9 10 11 12 A untx untx untx untx untx
untx untx untx untx untx B cpd 1 cpd 1 cpd 1 cpd 1 cpd 1 cpd 7 cpd
7 cpd 7 cpd 7 cpd 7 C cpd 2 cpd 2 cpd 2 cpd 2 cpd 2 cpd 8 cpd 8 cpd
8 cpd 8 cpd 8 D cpd 3 cpd 3 cpd 3 cpd 3 cpd 3 cpd 9 cpd 9 cpd 9 cpd
9 cpd 9 E cpd 4 cpd 4 cpd 4 cpd 4 cpd 4 cpd 10 cpd 10 cpd 10 cpd 10
cpd 10 F cpd 5 cpd 5 cpd 5 cpd 5 cpd 5 cpd 11 cpd 11 cpd 11 cpd 11
cpd 11 G cpd 6 cpd 6 cpd 6 cpd 6 cpd 6 cpd 12 cpd 12 cpd 12 cpd 12
cpd 12 H untx untx untx untx untx untx untx untx untx untx
1.4. The Plates are Incubated for 48 Hours at 37.degree. C., and
then Subjected to RNA Extraction.
TABLE-US-00005 TABLE 4 Summary of equipment and supplies used for
cell culture and compound set up 8 channel Impact2 Pipette, 1250
.mu.l cat no 2004 Matrix 2 ml polypropylene deep-well block, cat no
4222 Matrix 96-well, sterile 25 ml Reagent Reservoirs, Sterile cat
no. 8096 Matrix 1250 .mu.l X-tra long pipet tips cat no. 8255
Matrix 96-well plate cat no. 3595 Costar Hemacytometer Bright line
improved Reichert Neubauer 0.1 mm deep DMEM cat no. 51444-79P JRH
L-glutamine (Glu) cat no. 12403-010 GIBCO-BRL Non-essential amino
acids (NEAA) cat no. 11140-050 GIBCO-BRL Fetal Bovine Serum (FBS)
cat no. 16250-078 GIBCO-BRL G418 cat no. 55-0273 Invitrogen DMSO
cat no. D-2650 Sigma Medium A DMEM, 10% FBS, 1X NEAA, 1X Glu, 0.25
mg/ml G418 Medium B DMEM, 2% FBS, 1X NEAA, 1X Glu Medium C DMEM, 2%
FBS, 1X NEAA, 1X Glu, 0.5% DMSO Trypsin-EDTA 0.25% GIBCO-BRL
2. Extraction of Total Cellular RNA from Replicon Cells
2.1 Introduction
[0665] The goal of the procedure is to extract RNA from in vitro
tissue culture samples so that the viral or cellular RNA is
quantitatively recovered and pure enough to be analyzed by
quantitative HCV RT-PCR assay.
[0666] To permit detection of variations in the efficiency of the
RNA extraction, standard amounts of bovine viral diarrhea virus
(BVDV), an RNA virus with some similarity to HCV, are added to each
cell sample before RNA extraction. Thus, the level of BVDV RNA
detected in the final multiplex RT-PCR reaction should be
consistent among all wells within the variability limits associated
with the Replicon Assay. This RNA extraction efficiency internal
control is discussed further in the TaqMan.RTM. section, below.
[0667] The RNA extraction approach used is the RNeasy-96 method
manufactured by Qiagen Inc. (Valencia, Calif.). This method employs
96 silica-based mini-columns that are positioned in an array
compatible with 96-well tissue culture operations. The RNA
extraction technology is a modification of the Boom method, in
which all cellular proteins and nucleic acid, including nucleases,
are first denatured with a strong chaotropic salt (guanidinium
thiocyanate). In this environment, nucleic acids have a strong
affinity for silica, the material in the mini-column discs;
however, proteins and other contaminants do not bind to silica, and
pass through the columns. After washing the columns with chaotropic
ethanol solutions, the samples are partially dried, and the nucleic
acid is then released from the column in a small volume of
water.
[0668] To reduce variability in recovering HCV RNA, care is taken
with the column washing and partial drying conditions. The presence
of a small amount of ethanol on a column will contaminate the final
RNA and interfere with the RT-PCR detection system. Caution is
required in all phases of this procedure because the starting
samples may be biohazardous, the chaotropic salt is highly caustic,
and as a thiocyanate, it can generate poisonous cyanide gas if
allowed to come in contact with acidic environments.
TABLE-US-00006 TABLE 5 Summary of Equipment and Supplies Needed for
HCV RNA Extraction Procedures RNeasy 96 Kit (24) cat no. 74183
Qiagen QIAvac 96 manifold cat no. 19504 Qiagen Centrifuge 4-15C,
for 2x96 plates, cat no. 81010 Qiagen 6000 x g plate rotor for 2x96
plates cat no. 81031 Qiagen 200 Proof Ethyl Alcohol 8 channel
Impact2 Pipette, 250 .mu.l cat no 2002 Matrix 8 channel Impact2
Pipette, 1250 .mu.l cat no 2004 Matrix 2 ml polypropylene deep-well
block, cat no 4222 Matrix 96-well, sterile 25 ml Reagent
Reservoirs, Sterile cat no. 8096 Matrix 1250 .mu.l X-tra long pipet
tips cat no. 8255 Matrix 200 .mu.l pipet tips cat no. 7275 Matrix
serum free MEM medium cat no. 11095-80 GIBCOBRL
2.2 Procedure:
[0669] 2.2.1 Cell Lysis [0670] 2.2.1.1. Prepare lysis buffer. For
one 96-well plate, add 150 .mu.l .beta.-mercaptoethanol (.beta.-ME)
and 1 .mu.l BVDV stock (vortex stock before adding) to 15 ml RLT
buffer (a component of the RNeasy kit, Qiagen). This stock is
prepared by infecting MDBK cells (bovine kidney cells, #CCL-22,
available from the American Type Culture Collection, Manassas Va.)
with BVDV and harvesting the culture at peak cytopathic effect
(CPE). This stock has an infectious titer of approximately
1.times.10.sup.7 pfu/ml. This gives BVDV a threshold cycle
(C.sub.t) of about 22 in the TaqMan.RTM. assay. The BVDV stock is
stored in a -80.degree. C. freezer. [0671] 2.2.1.2. Cells are
washed with 150 .mu.l serum-free MEM medium (program 4 on 8 channel
electronic pipette P1250: Fill 1250, Disp 150.times.8). 150 .mu.l
lysis buffer are added to each well (same program). [0672] 2.2.1.3.
RNA is extracted immediately, or cells are frozen at -80.degree. C.
[0673] 2.2.2. Preparation of reagents and materials for RNA
extraction. [0674] 2.2.2.1. Note the lot# of the RPE and RNeasy 96
Kit. [0675] 2.2.2.2. RPE: 720 ml of 100% ethanol are added to one
bottle of RPE (Qiagen), and mixed well; RPE bottles are always
shaken well before use. [0676] 2.2.2.3. 70% Ethanol: 150 ml
diethylpyrocarbonate (DEPC) water are added to 350 ml 100% ethanol
and mixed well. [0677] 2.2.3. Preparation of RNA with RNeasy 96 kit
[0678] 2.2.3.1. Frozen samples are thawed at room temperature for
40 min. At the same time, one column of Extraction Controls is
thawed for each plate (Extraction Controls: The RNeasy Extraction
Controls are a set of 8 tubes all connected together. Inside of
each tube is 170 .mu.l of cell lysate with a certain ratio of HCV
positive and negative cells. From the top to the bottom are two
each of a low, medium, high, and zero number controls,
respectively. (See section 2.3 of the protocol below.) [0679]
2.2.3.2. The samples are mixed by pipetting 100 .mu.l up and down
five times. The entire sample is transferred into columns 1-10 of
the 2 ml Matrix square-well block (program 1 on P250: Mix
100.times.5, Fill 170, Purge). [0680] 2.2.3.3. 150 .mu.l of the
replicon standard is transferred into column 11 (no samples in
column 12). [0681] 2.2.3.4. 150 .mu.l of 70% ethanol (EtOH) are
added to each sample (program 4 on P1250: Fill 1250, Disp 150).
[0682] 2.2.3.5. An RNeasy 96 plate labelled with the appropriate
plate number is placed in the vacuum manifold. Mix and transfer the
lysate/EtOH to the RNeasy 96 plate (program 1 on P1250: Mix 200,
Times 5, Fill 330, and Purge). Any unused wells are sealed with
transparent tape (supplied by Qiagen), usually column 12. [0683]
2.2.3.6. Vacuum (approximately 800 mbar) is applied to load the
sample onto the mini-columns. [0684] 2.2.3.7. The RNeasy-96 plate
is washed with 1000 .mu.l of RW1 buffer (Qiagen)/well (program 2 on
P1250: Fill 1000, Disp 1000). [0685] 2.2.3.8. Vacuum is applied to
the filter through the RW1 buffer, and the flow-through is emptied.
[0686] 2.2.3.9. The RNeasy-96 plate is washed with 1000 .mu.l of
RPE buffer/well (program 2 on P1250). [0687] 2.2.3.10. Vacuum is
applied to filter through the RPE buffer. [0688] 2.2.3.11. Repeat
Step 2.2.3.9 [0689] 2.2.3.12. Vacuum is applied to the filter
through the RPE buffer, keeping the vacuum applied for 3 min.
[0690] 2.2.3.13. Dry the RNeasy 96 plate: The RNeasy-96 plate is
placed in a collection microtube rack (supplied by Qiagen), covered
with the supplied AirPore tape, and the unit is centrifuged for 10
min at 6000.times.g (Qiagen Sigma centrifuge; 4-15.degree. C.).
[0691] 2.2.3.15. Elute the RNA from the RNeasy 96-well plate: The
RNeasy-96 plate is transferred onto the top of a new collection
microtube rack. 70 .mu.l of RNase-free water are added to the
middle of each well (program 3 on P1250: Fill 850, Disp 70). [0692]
2.2.3.16. Incubate 1 min at room temperature, and then place a
fresh AirPore tape over the plate. [0693] 2.2.3.17. The unit is
then centrifuged for 4 min at 6000.times.g in a Sigma 4-15C
centrifuge. The eluted volume measures between 28 .mu.l and 50
.mu.l. [0694] 2.2.3.18. The RNeasy-96 plate is discarded, and the
collection tube rack is sealed with the Qiagen-provided caps (8 per
strip). [0695] 2.2.3.19. The eluted RNA is stored at -80.degree. C.
or immediately analyzed in the TaqMan.RTM. assay.
2.3 Extraction Controls Preparation
Day 1
[0695] [0696] 2.3.1.1. Plate out 2.5.times.10.sup.7
replicon-producing cells in a 150 cm.sup.2 tissue culture flask
(T-150). [0697] 2.3.1.2 Plate out 2.0.times.10.sup.6 Huh7 cells in
a 75 cm.sup.2 tissue culture flask (T-75). [0698] 2.3.1.3 Incubate
overnight at 37.degree. C.
Day 2
[0698] [0699] 2.3.1.4. Lyse the cells with lysis buffer. [0700]
2.3.1.5. Remove the supernatant from the Huh7 and
replicon-producing cells, and wash the monolayer with 10 ml
serum-free medium (MEM). [0701] 2.3.1.6. Add 30 ml of lysis buffer
(with 1 .mu.l of BVDV stock/15 ml of lysis buffer) to the Huh7
cells, mix by repeated pipetting, and place the cell lysate in a 50
ml conical-bottomed tissue culture centrifuge tube. [0702] 2.3.1.7.
Add 10.5 ml of lysis buffer to the replicon-producing cells, mix by
repeated pipetting, and place the cell lysate in a 15 ml
conical-bottomed tissue culture centrifuge tube. [0703] 2.3.2. For
the HIGH Extraction Standard: Aliquot 170 .mu.l of the
replicon-producing cells cell lysate into rows 5 and 6 of two
Matrix 0.75 ml tube racks. [0704] 2.3.3. For the MEDIUM Extraction
Standard: Add 1.0 ml of the replicon-producing cells cell lysate to
9 ml of the Huh7 lysate, and mix well. Aliquot 170 .mu.l of this
mixture to rows 3 and 4 of two Matrix 0.75 ml tube racks. [0705]
2.3.4. For the LOW Extraction Standard: Add 50 .mu.L of the
replicon-producing cells cell lysate to 10 ml of the Huh7 lysate,
and mix well. Aliquot 170 .mu.l of this mixture to rows 1 and 2 of
two Matrix 0.75 ml tube racks. [0706] 2.3.5. ZERO Extraction
Control: Aliquot 170 .mu.L of the Huh7 cell lysate to rows 7 and 8
of two Matrix 0.75 ml tube racks. [0707] 2.3.6. Store controls at
-80.degree. C.
3. TaqMan.RTM. RT-PCR and Data Analysis
3.1 Introduction:
[0708] Real-time quantitative RT-PCR is used to measure the amount
of HCV replicon RNA in each sample. This technology is also
referred to as the PCR-based 5' nuclease assay, and TaqMan.RTM..
The analytic instrument is the Applied Biosystems 7700 Prism
Sequence Detection System (Applied Biosystems, Foster City,
Calif.). This instrument is essentially a time-multiplexed
laser-induced fluorescence spectrograph coupled with a thermal
cycler. It monitors the accumulation of PCR amplicon in each well
of a 96-well sample tray throughout the course of the PCR
process.
3.2. Use of BVDV Internal Control:
[0709] As mentioned in the previous section, an internal positive
control is incorporated into every sample. This serves as a measure
of RNA extraction efficiency, and shows if the sample contains
contaminants that inhibit TaqMan.RTM. PCR. BVDV is mixed with the
chaotropic cell lysis buffer prior to applying the lysis buffer to
the cells. Although the positive control is in every sample, the
BVDV internal positive control assay is only performed when the HCV
replicon RNA assay data fall outside of expected limits, suggesting
that there could be a problem with the samples. The 7700 is capable
of simultaneously monitoring the accumulation of two different PCR
amplicons in the same tube by using detection probes labeled with
two different fluorescent reporter dyes ("multiplexing"). Specific
criteria that elicit a TaqMan.RTM. analysis for the BVDV internal
positive control of a sample plate are described in the section on
data analysis (3.6).
3.3 HCV Replicon RNA TaqMan.RTM. Probe and Primers.
[0710] Because of the expected genetic stability and general lack
of RNA secondary structure in the neomycin resistance gene (neo)
encoded in the replicon, primers and a probe that bind in that
region are employed. This segment of the replicon RNA extends from
bases 342-1193 of the 8001 base pair replicon (SEQ ID NO:1):
TABLE-US-00007 301 gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac
catgagcacg aatcctaaac 361 ctcaaagaaa aaccaaacgt aacaccaacg
ggcgcgccat gattgaacaa gatggattgc 421 acgcaggttc tccggccgct
tgggtggaga ggctattcgg ctatgactgg gcacaacaga 481 caatcggctg
ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt 541
ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
601 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
actgaagcgg 661 gaagggactg gctgctattg ggcgaagtgc cggggcagga
tctcctgtca tctcaccttg 721 ctcctgccga gaaagtatcc atcatggctg
atgcaatgcg gcggctgcat acgcttgatc 781 cggctacctg cccattcgac
caccaagcga aacatcgcat cgagcgagca cgtactcgga 841 tggaagccgg
tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag 901
ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
961 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg CCGCTTTTCT
GGATTCATCG 1021 aCTGTGGCCG GCTGGGTGTG Gcggaccgct atcaggacat forward
primer agcgttggct acccgtgata TaqMan .RTM. probe 1081 ttgctgaaga
gcTTGGCGGC GAATGGGctg accgcttcct cgtgctttac ggtatcgccg reverse
primer 1141 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
tgagtttaaa
3.4. Procedures
3.4.1. Method for Preparing 1.times. Master Mixtures for NEO and
BVDV RT-PCR
TABLE-US-00008 [0711] TABLE 6 Summary of equipment and supplies for
preparing RT-PCR 10-plate Master Mix Equipment and supplies Order
No. Supplier 0.5-10 .mu.l pipette 22 47 005-1 2000 Series Eppendorf
2-20 .mu.l pipette 22 47 015-9 2000 Series Eppendorf 10-100 .mu.l
pipette 22 47 020-5 2000 Series Eppendorf 50-200 .mu.l pipette 22
47 025-6 2000 Series Eppendorf 100-1000 .mu.l pipette 22 47 030-2
2000 Series Eppendorf 1250 .mu.l Matrix tips cat no. 8255 Matrix
200 .mu.l Matrix tips cat no. 7275 Matrix 10 .mu.l ART tips cat no.
2140 Molecular Bioproducts 20 .mu.l ART tips cat no. 2149P
Molecular Bioproducts 100 .mu.l ART tips cat no. 2065E Molecular
Bioproducts 200 .mu.l ART tips cat no. 2069 Molecular Bioproducts
1000 .mu.l ART tips cat no. 2079E Molecular Bioproducts Electronic
pipette, Impact2 cat no. 2001 Matrix 1.5 ml RNase-free microfuge
tubes cat no. 12450 Ambion 14 ml Polypropylene tubes cat no. 352059
Falcon 25 ml reagent reservoir cat no. 8096 Matrix 96-well reaction
plate cat no. N801-0560 Applied Biosystems optical cap strips cat
no. N801-0935 Applied Biosystems Disposable Sterile Gowns cat no.
9515-E Baxter Reagents Order No. Supplier Acid 0.1N HCl Fisher
RNAseZap cat no. 9780 Ambion RNAse away cat no. 7005 Molecular
Bioproducts 10-pak, EZ RT-PCR core reagents cat no. 403028 Applied
Biosystems kit, 5.times. reaction buffer, 25 mM Manganese Acetate,
deoxy NTPs VIC NEO probe, 2 .mu.M (=10.times.), cat no. 450003,
custom, Applied Biosystems 550 .mu.l per aliquot 5'-VIC-CTG TGG CCG
GCT GGG TGT GG-TAMRA-3' (SEQ ID NO: 2) VIC BVDV probe, 2 .mu.M
(=10.times.), cat no. 450003, custom, Applied Biosystems 550 .mu.l
per aliquot 5'-VIC-CCC TCG TCC ACG (Vertex) TGG CAT CTC GA-TAMRA-3'
(SEQ ID NO: 3) NEO forward primer, 3 .mu.M (=10.times.) cat no.
4304972, custom, Applied Biosystems forward/reverse primer mix, 550
.mu.l 5'-CCG CTT TTC TGG ATT per aliquot CAT CG-3' (SEQ ID NO: 4)
NEO reverse primer, 3 .mu.M (=10.times.) cat no. 4304972, custom,
Applied Biosystems forward/reverse primer mix, 550 .mu.l 5'-CCC ATT
CGC CGC CAA-3' per aliquot (SEQ ID NO: 5) BVDV forward primer, 3
.mu.M custom, 5'-CAG GGT AGT Oligos etc (=10.times.)
forward/reverse primer mix, CGT CAG TGG TTC G-3', 550 .mu.l per
aliquot (SEQ ID NO: 6) 1.0 .mu.M scale w/gel purification BVDV
reverse primer, 3 .mu.M custom, 5'-GGC CTC TGC Oligos etc
(=10.times.) forward/reverse primer mix, AGC ACC CTA TC-3', 550
.mu.l per aliquot (SEQ ID NO: 7) 1.0 .mu.M scale w/gel purification
NEO RNA standards In vitro transcribed RNA from a plasmid
containing the neo gene portion of the HCV replicon RNA using T7
RNA polymerase. The in vitro transcribed RNA is quantitated based
on known molecular weight of the transcripts and the UV-absorbance
of the purified transcript solution. This RNA is diluted,
aliquoted, and stored at -80.degree. C. Individual aliquots are
thawed for each TaqMan .RTM. assay. RNA samples to be tested
isolated from HCV replicon cells (section 2 of this Protocol), 10
.mu.l/96-well plate Nuclease-Free Water cat no. 9930 Ambion (Not
DEPC Treated)
3.4.2. Preparation of Reagents for Master Mix
[0712] 3.4.2.1. Clean the bench according to the two steps below,
and wipe the pipettes with RNAse away. [0713] RNAse Zap (Ambion,
Austin, Tex.) [0714] RNAse Away (Molecular Bioproducts, San Diego,
Calif.) [0715] 3.4.2.2. Open core EZ RT-PCR reagents (Applied
Biosystems) and put the 5.times. buffer on ice, thaw frozen
reagents at room temperature for about 15 minutes, and then put
them on ice. One EZ RT-PCR reagents kit can be used to analyze two
96-well RNA extractions. [0716] 3.4.2.3. Take one tube of 2 .mu.M
VIC probe (NEO or BVDV, 550 .mu.l per tube) from -20.degree. C. and
put on ice. [0717] 3.4.2.4. Take one tube 3 AM forward/reverse
primer mix (NEO or BVDV, 550 .mu.l per tube) from -20.degree. C.
and put on ice. [0718] 3.4.2.5. Take one tube (30 .mu.l) of
standards RNA transcript (10.sup.8 copies/10 .mu.l) from
-80.degree. C. and place on ice. [0719] 3.4.2.6. Take one tube of
room temperature Ambion water.
3.43. Assembly of Master Mixture for One 96-Well Plate
Reaction.
[0719] [0720] 3.4.3.1. Use a 1 ml pipette to transfer 5.times.
buffer (Applied Biosystems) to a 14 ml tube; total volume added is
1100 .mu.l. [0721] 3.4.3.2. Use a 1 ml pipette to add 25 mM Mn
(OAc).sub.2 (Applied Biosystems) to a 14 ml tube; total volume
added is 660 .mu.l. [0722] 3.4.3.3. Use a 200 .mu.l pipette to add
165 .mu.l of 10 mM dATP to the 14 ml tube. Do the same for 10 mM
dCTP, 20 mM dUTP, and 10 mM dGTP. [0723] 3.4.3.4. Use a 1 ml
pipette to add 550 .mu.l 10.times.3 .mu.M forward/reverse primer
mix. [0724] 3.4.3.5. Use a 1 ml pipette to add 550 .mu.l 10.times.2
.mu.M probe. [0725] 3.4.3.6. Use a 1 ml pipette to add 220 .mu.l
rTth DNA polymerase (Applied Biosystems). 3.4.3.7. Use a 100 .mu.l
pipette to add 55 .mu.l AmpErase UNG (Applied Biosystems). [0726]
3.4.3.8. Use a 1 ml pipette to add 605 .mu.l Ambion H.sub.2O to the
14 ml tube; the final volume is 4400 .mu.l total. [0727] 3.4.3.9.
Transfer the 4400 .mu.l master mix to a 25 ml reagent reservoir.
[0728] 3.4.3.10. Dispense 40 .mu.l per well for all 96 wells using
an 8-channel pipette. [0729] 3.4.3.11. Transfer 10 .mu.l of
extracted unknown samples to wells of the reaction plate using an
8-channel pipette, column by column, column 1 through column 11.
Cap each column after transfer. [0730] 3.4.3.12. Add 270 .mu.l
Ambion H.sub.2O to the 30 .mu.l 10.sup.8 copies/10 .mu.l RNA
transcript for use in the standard curve and mix. There are now
10.sup.7 copies of the HCV replicon quantitation standard RNA/10
.mu.l.
3.4.4. Setting Up the ABI 7700 for Each Run
[0730] [0731] 3.4.4.1 Before each run, reboot the computer for the
ABI 7700 and rebuild the desktop. [0732] 3.4.4.2 Close and remove
any redundant programs from the hard drive; overuns data to trash.
[0733] 3.4.4.3 Open Sequence Detector v1.7 program (SDS software).
[0734] 3.4.4.5 Open the "Replicon Assay Runs" folder. [0735]
3.4.4.6 Open the "Replicon Assay" template plate. The thermal
cycler conditions programmed into the template are as follows:
[0736] Stage 1: 50.degree. C. for 2 min. [0737] Stage 2: 60.degree.
C. for 30 min. [0738] Stage 3: 95.degree. C. for 5 min. [0739]
Stage 4: 95.degree. C. for 15 sec. [0740] Stage 5: 60.degree. C.
for 60 sec. [0741] Cycle repeat number of stages 4-5: 40. [0742]
Template instrument: diagnosis: advanced options: [0743] Select
views: display mse. [0744] Select views: display best fit. [0745]
Select miscellaneous: reference dye ROX. [0746] 3.4.4.7 "Save" (not
"save as") the file in the "Replicon Assay Runs" folder. [0747]
3.4.4.8 Show setup: hit RUN 3.5 Preparing the ABI7700 Data after a
Run Using SDS Software. [0748] 3.5.1. The assay plates are removed
from the ABI7700 and discarded without ever being opened. This
greatly reduces laboratory problems with PCR cross contamination.
[0749] 3.5.2. The data are analyzed using the Sequence Detector
System software V 1.7. [0750] 3.5.3. The threshold levels are
initially set using default settings. [0751] 3.5.4. Data rejection
criteria: Data points or series of whole plates can be rejected. If
there has been a significant deviation from protocol, reagent
failure or mishap, or ABI 7700 run failure, data can be discarded.
For rejection of any data points from an apparently normal run, one
or more of these criteria must be met. [0752] 3.5.4.1. Threshold
cycle calculations. Normally use the default values for the SDS
software. If the Ct of the most concentrated sample is less than
15, then change the threshold value stop limit as needed to a lower
value so that the Ct of the highest concentration sample is greater
than the stop value. Update calculations after making this change.
[0753] 3.5.3.2. Consider rejecting an entire abnormal TaqMan.RTM.
run as indicated by a deviation from the mean values for the slope
and y-axis intercept of the line generated by analysis of the neo
RNA standards. The acceptable ranges for those values are: [0754]
Slope values should be between 3.0 and 3.6 [0755] y-intercept
cycles should be between 36 and 41 cycles. [0756] 3.5.3.3. Aberrant
individual TaqMan.RTM. wells as indicated by extreme Rn/.DELTA.Rn
can be deleted prior to data analysis so chat they do not affect
the SDS software calculations. [0757] 3.5.3.4. Examine and record
the no-template control Ct values and confirm that they are >7.0
Ct (>100.times.) higher than the Ct for any compound treated
sample. [0758] 3.5.5. The HCV RNA standards Ct values are compared
with previous results. [0759] 3.5.6. The HCV RNA standard curve is
compared with previous results. [0760] 3.5.7. If aberrant
amplification is evident in individual wells, those wells are
identified and noted. [0761] 3.5.8. The "results" file is exported
and transferred from the 7700 computer to another computer for
analysis using Microsoft Excel. [0762] 3.5.9. Any of the following
changes in reagent preparations or dilution used is reported.
[0763] New probe or primer synthesis from vendor. [0764] New probe
or primer dilution and aliquots. [0765] New standards RNA
transcript preparation. [0766] New standards RNA transcript
dilution and aliquots. [0767] New BVDV viral preparation. [0768]
New column 11 standards preparation.
3.6 TaqMan.RTM. Data Analysis.
[0768] [0769] 3.6.1. Copy and paste TaqMan.RTM. HCV Ct number and
copy number from the TaqMan.RTM. results file into the appropriate
cells of the Replicon Assay data analysis Microsoft Excel macro,
and run the macro. [0770] 3.6.2. Copy the TaqMan.RTM. results table
from the macro sheet onto another sheet, input compound serial
number and lot number. [0771] 3.6.3 From this excel sheet, the
mean, standard deviation, and percentage CV of Compound inhibition
activity, as well as HCV copy number, HCV Ct number, and BVDV Ct
number (if available), of all dilution points in 5 replicates and
no-compound control, will be calculated. [0772] 3.6.4. Criteria for
data rejection and implementation of BVDV Control TaqMan.RTM..
Check all the calculations. Data points or series of whole plates
can be rejected. If there is a significant deviation from protocol,
reagent failure or mishap, or ABI 7700 run failure, data can be
discarded. For rejection of any data points from an apparently
normal run, then one or more of these criteria must be met. The
standard deviation of percentage inhibition should be less than 30%
in active compounds. The % CV of HCV copy number should be less
than 30%. The standard deviation of HCV Ct of all samples should be
less than 0.5: this is usually about 0.1 to 0.3 in most samples. If
the HCV Ct standard deviation is more than 0.5, then go back to the
raw data table, and check the Ct numbers of 5 replicates. If the Ct
number of any one well is 2 Ct different from the average Ct number
of 5 replicates, then this well should omitted from the analysis.
If more than 3 wells (not on same column) have unusual Ct numbers,
then the BVDV TaqMan.RTM. internal control assay should be carried
out. If the BVDV data show irregularity, then the compound should
be tested again. [0773] 3.6.5. IC.sub.50 calculation: Copy and
paste the data of average inhibition and standard deviation into a
sigmoid dose response with a variable slope calculator that uses
non-linear regression methods. Using this tool, calculate the
IC.sub.50 by using both of two methods: fixing the top at 100%
inhibition only, or fixing the top at 100% inhibition and the
bottom at 0% inhibition. The method that gives the clearest fit is
then reported for each compound. The most reliable IC.sub.50 comes
from the calculation having the lowest standard error. If
IC.sub.50s calculated from these two curve fit options show more
than one fold difference, or if the IC.sub.50 SD is greater than
the IC.sub.50, the compound should be tested again at adjusted
concentrations. Calculation of the Effect of ACV Serine Protease
Inhibitors in Combination with Interferons
[0774] The effect of a HCV serine protease inhibitor (HSPI) and an
interferon in combination can be assessed in the Replicon Assay by
generating a dose response curve for the HSPI in the presence of
various levels of interferon, or by determining a dose response
curve for an interferon in the presence of various levels of HSPI.
The goal is to assess whether there is more or less inhibition of
viral RNA accumulation than would be expected if the two drugs
produce additive effects on the RNA. More specifically, the
additivity definition of Lowe ((1928) Die Quantitation Probleme der
Pharmakologic, Ergebn. Physiol., 27, 47-187) is used. This is
defined as follows. Let D.sub.E,INF be the concentration of
interferon that results in effect E, and let D.sub.E,HSPI be the
concentration of protease inhibitor that results in effect E.
1 = D 1 D E , INF + D 2 D E , HSPI ( 1 ) ##EQU00001##
[0775] Then no interaction or Lowe additivity is defined by the
following relationship, where the combination of concentration
D.sub.1 of INF and D.sub.2 of HSPI produces the effect E.
[0776] The degree of synergy or antagonism is expressed in terms of
iso-effect curves or Isobols. The combination (D1,D2) is a point on
a graph where the axes are the concentrations of interferon and
HSPI (FIG. 2). All such combinations that produce an effect level E
form the E effect Isobol. It is necessarily the case that
(D.sub.E,INF,0) and (0, D.sub.E,HSPI) are points on the Isobol. The
Isobols are straight lines connecting points (D.sub.E,INF,0) and
(0, D.sub.E,HSPI) when the additivity relationship (1) is
satisfied.
[0777] Concave-up Isobols indicate synergy, and concave-down
Isobols indicate antagonism. Following the guidelines of Berenbaum,
M. C. ((1985) The expected effect of a combination of agents: the
general solution. J. Theor. Biol., 114, 413-431), and Greco, Park
and Rustom ((1990) Application of a New Approach for the
Quantitation of Drug Synergism to the Combination of
cis-Diamminedichloroplatinum and
1-.beta.-D-Arabinofuranosylcytosine, Cancer Research, 50,
5318-5327), add a term to (1) to account for synergy or antagonism.
The equation defines a response surface that can be fit to the
percent control values at all treatment combinations. Contour plots
from this fitted response surface are the Isobols.
[0778] The response surface model assumes a sigmoidal dose response
for each compound defined by (2).
E = E max 1 + ( [ Drug ] IC 50 ) m + B ( 2 ) ##EQU00002##
[0779] The concentrations that give a specified level of activity E
alone are given by (3)
D E , INF = IC 50 INF ( E - B E max - E + B ) 1 / m INF D E , HSPI
= IC 50 HSPI ( E - B E max - E + B ) 1 / m 310 ( 3 )
##EQU00003##
[0780] To satisfy the model of Greco et al. (1990), the combined
action of the drugs must then satisfy equation (4) for every
combination of drugs that produces response level E.
I = [ INF ] IC 50 INF ( E - B E max - E + B ) 1 / m INF + [ HSPI ]
IC 50 HSPI ( E - B E max - E + B ) 1 / m 310 + .alpha. [ INF ] [
HSPI ] IC 50 INF IC 50 HSPI ( E - B E max - E + B ) 1 / 2 m INF ( E
- B E max - E + B ) 1 / 2 m 310 ( 4 ) ##EQU00004##
[0781] The parameter .alpha. measures the amount of interaction. A
zero value of alpha means no interaction or additivity since the
equation reduces to (1) when .alpha.=0. Given IC.sub.50s, Hill
slopes (m), maximum value (Emax), and minimum value (B), this
equation can be solved to give the effect that results from any
treatment combination [INF] and [HSPI]. Therefore, this equation
defines a response surface. Given an experiment where [INF] and
[HSPI] are varied, the parameters can be chosen using nonlinear
weighted least squares regression. The parameter .alpha. can be
related to a synergy measure S (Hewlett, P. S. (1969) Measurement
of potencies of drug mixtures. Biometrics, 25, 477-487), which is
taken directly from the Isobols at a 50% effect. S is the ratio, of
the distance from the origin to the Isobol defining additivity, to
the distance from the origin to Isobol of the fitted data, along
the line at 45 degrees from the axes. (S=ON/OM see FIG. 3). The
relationship is .alpha.=4(S.sup.2-S).
[0782] The method discussed in Greco a al. (1990), above, for
fitting the response surface and determining synergy parameter
.alpha. with its significance level is followed in assessing the
degree of synergy in a series of experiments testing HSPIs in
combination with several different interferons.
[0783] However, there is a need to weight observations with lower
counts more than those with higher counts. The counts relate
directly to the percent control, which is the effect E. Using
methods described in Carroll, R. J. and Rupert, D. ((1988)
(Transformation and Weighting In Regression, Chapman and Hall, New
York), the well to well variability can be seen to increase with
the square of the mean percent control value. Therefore, the
observations are weighted by one over the fitted percent control
value (E) squared. The variance and weighting used to analyze these
experiments is consistent with variability relationships observed
by researchers investigating methods for analyzing Radioligand
assays (Finney, D. J., (1976), Radioligand Assay, Biometrics, 32,
721-740, and Dudley, R. A. Edwards, P., Ekins, R. P., McKinzie, L
G. M., Raab, G. M., Rodbard, D. and Rodgers, R. P. C. (1985),
Guidelines for Immunoassay Data Processing, Clinical Chemistry,
31/8, 1264-1271).
[0784] Results
[0785] In an initial experiment, HCV serine protease inhibitor
Compound CU is tested over a concentration range from 3 .mu.M to
0.0123 .mu.M, i.e., a 244-fold range. The interferon-alpha 2B
concentrations vary from 30 units per sample to 0.0096 units per
sample, i.e., a 3125-fold range. As shown in Table 7, when used as
a single drug treatment, Compound CU exhibits an IC.sub.50 of 0.48
and the interferon IC.sub.50 is 2.19 U. Within the precision of the
Replicon Assay, which is approximately 20%, addition of
interferon-alpha 2B results in an increase in the inhibition of
replicon RNA accumulation in a dose-dependent manner. For example,
treatment of cells with 0.333 .mu.M Compound CU results in 28%
inhibition of replicon RNA accumulation. Treatment of cells with a
combination of 0.333 .mu.M Compound CU, which is 71% of the
IC.sub.50 dose (0.469 .mu.M) and 0.24 U of interferon-alpha 2B,
which is 11% of the interferon-alpha 2B IC.sub.30 (2.05 .mu.M)
results in a 49% inhibition of replicon RNA accumulation. Thus, 71%
of one IC.sub.50 dose in combination with 11% of the other results
in 49% inhibition of replicon RNA accumulation. Using an intuitive
approach to determining if a combination treatment is synergistic
or additive or antagonistic, one could predict that if effect of
combination treatment were only additive, one would expect the
combined fractions of the two IC.sub.50 doses needed to obtain a
49% inhibition of replicon RNA accumulation to be 98%. Our
experimental results demonstrate that the level of inhibition of
replicon RNA accumulation is achieved using 71% plus 11%, i.e. 82%
of the IC.sub.50 dose rather than 98%, as predicted for additive
effects of combination treatment. Thus at these concentrations of
compounds, the effect appears to be synergistic because smaller
fractional doses of the IC.sub.30 dose of each compound are used to
obtain 49% inhibition of HCV replicon RNA than would be required of
either compound alone, where 98% of the IC.sub.50 doses would be
needed. The results of this combination treatment are shown in
Table 8 and graphically in FIG. 1.
TABLE-US-00009 TABLE 7 Inhibition of replicon RNA accumulation
after 48 hour treatment with Compound CU and interferon-alpha 2B,
individually or in combination Compound Interferon-alpha 2B (units)
CU (conc.) 30 U 6 U 1.2 U 0.24 U 0.048 U 0.0096 U 0 U 3 .mu.M 99%
99% 99% 99% 98% 98% 98% 1 .mu.M 99% 98% 96% 95% 92% 93% 88% 0.333
.mu.M 94% 87% 66% 49% 33% 27% 28% 0.1111 .mu.M 93% 79% 46% 29% 12%
15% 11% 0.0370 .mu.M 92% 78% 44% 21% 2% 7% 8% 0.0123 .mu.M 92% 78%
44% 20% 19% 19% 5% 0 .mu.M 89% 73% 38% 16% 8% 12% 0%
[0786] These initial results, derived as stated earlier via use of
the in vitro Replicon Assay and a simple additivity analysis of the
data generated by that assay, demonstrate that combination
treatment of replicon cells with an HCV serine protease inhibitor
and an interferon yields at least an additive antiviral effect, and
likely a synergistic antiviral effect.
[0787] The foregoing data have been reanalyzed using the formal
mathematical tools described above to determine if the relationship
between HCV serine protease inhibitor CU and interferon alpha-2B is
synergistic, additive, or antagonistic. The reanalyzed data are
shown numerically in Table 8, and graphically in FIG. 4.
[0788] Table 8 summarizes further results obtained in the Replicon
Assay after treatment of replicon-containing cells for 48 hours
with various HCV serine protease inhibitors of the present
invention and several different interferons, individually or in
combination. We point out that the standard deviation of values
measured for inhibition of HCV replicon RNA in the Replicon Assay
is -20%. Compounds are tested over a broad concentration range and
at lower compound concentrations that cause no significant
inhibition of HCV replicon RNA concentration. Because of the
.about.20% standard deviation of the assay, some data points will
generate negative numbers. Negative inhibition numbers indicate in
a particular experiment there is on average more HCV replicon RNA
molecules in the compound treated samples than in the mock treated
samples.
TABLE-US-00010 TABLE 8 INHIBITION OF REPLICON RNA ACCUMULATION
AFTER 48 HOUR TREATMENT WITH HCV SERINE PROTEASE INHIBITORS AND
DIFFERENT INTERFERONS, INDIVIDUALLY OR IN COMBINATION EXPERIMENT 1
IFN alpha-2B (units) 30.00 6.00 1.20 0.24 0.048 0.0096 0.000
Compound 0.000 89% 73% 38% 16% 8% 12% 0% CU 0.012 92% 78% 44% 20%
19% 19% 5% (.mu.M) 0.037 92% 78% 44% 21% 2% 7% 8% 0.111 93% 79% 46%
29% 12% 15% 11% 0.333 94% 87% 66% 49% 33% 27% 28% 1.000 99% 98% 96%
95% 92% 93% 88% 3.000 99% 99% 99% 99% 98% 98% 98% EXPERIMENT 2 IFN
alpha-2A (units) 30 6 1.2 0.24 0.048 0.0096 0 Compound 0 86% 61%
27% 4% -7% 5% 0% CU 0.0123 87% 66% 17% -23% 8% 8% 10% (.mu.M) 0.37
85% 62% 13% -2% 0% -1% 1% 0.1111 87% 68% 37% 20% -6% 12% 10% 0.333
92% 77% 58% 41% 26% 25% 44% 1 98% 96% 90% 86% 84% 83% 85% 3 99% 99%
98% 98% 98% 98% 98% EXPERIMENT 3 Compound CU (.mu.M) 3 1.5 0.75
0.375 0.1875 0.0938 0.0469 0.0234 0 Interferon 0 98% 93% 62% 23%
12% -2% -4% -2% 0 alpha-2B 0.049 98% 95% 70% 39% 12% 2% 6% 9% 3%
(units) 0.123 98% 95% 70% 43% 15% 7% 2% 5% 2% 0.307 98% 95% 73% 46%
16% 14% 7% 19% -3% 0.768 98% 95% 82% 56% 43% 34% 28% 32% 28% 1.920
98% 98% 87% 71% 51% 54% 49% 52% 45% 4.8 99% 98% 92% 82% 74% 71% 69%
71% 59% 12.0 99% 98% 96% 89% 87% 85% 85% 85% 80% 30.0 99% 99% 98%
95% 93% 92% 92% 93% 89% EXPERIMENT 4 Compound CU (.mu.M) 3 1.5 0.75
0.375 0.1875 0.0938 0.0469 0.0234 0 Interferon 0 98% 94% 74% 38%
17% 3% -1% 6% 0% alpha-2A 0.049 98% 93% 60% 22% 29% 21% -9% -6% 6%
(units) 0.123 98% 93% 67% 29% 21% 12% 3% 2% -8% 0.307 98% 93% 66%
29% 22% 4% -3% -4% 10% 0.768 98% 95% 67% 46% 24% 21% 20% 9% 15%
1.920 98% 96% 73% 48% 43% 44% 27% 33% 29% 4.8 98% 97% 82% 61% 61%
59% 52% 55% 43% 12.0 99% 98% 91% 75% 76% 72% 71% 74% 73% 30.0 99%
98% 96% 89% 86% 85% 84% 84% 83% EXPERIMENT 5 Compound CU (.mu.M) 3
1.5 0.75 0.375 0.1875 0.0938 0.0469 0.0234 0 Ovine 0.0 98% 95% 65%
24% -1% -14% -14% -12% 0 Interferon 0.9375 97% 95% 72% 41% 17% 11%
12% 6% 17% tau (units) 1.875 97% 95% 71% 40% 31% 18% 18% 11% 4%
3.75 98% 96% 75% 44% 38% 25% 34% 18% 17% 7.5 98% 96% 82% 61% 42%
37% 25% 26% 36% 15 98% 97% 84% 64% 59% 61% 56% 51% 53% 30 98% 98%
90% 79% 72% 68% 65% 68% 68% 60 98% 98% 93% 87% 80% 80% 74% 77% 82%
120 98% 98% 95% 92% 86% 87% 86% 86% 87% EXPERIMENT 6 Compound EC
(.mu.M) 3 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.0234 0 Interferon-
0 96% 93% 81% 56% 29% 23% 19% 1% 0 alpha 0.0492 96% 92% 80% 60% 31%
15% 19% 29% 6% 2B(units) 0.1229 96% 94% 78% 58% 32% 13% 20% 20% 4%
0.3072 97% 95% 82% 60% 38% 32% 34% 42% 23% 0.768 97% 95% 87% 66%
43% 41% 46% 43% 25% 1.92 98% 97% 90% 73% 62% 51% 54% 58% 47% 4.8
98% 97% 94% 87% 76% 73% 78% 76% 69% 12.0 98% 98% 96% 92% 86% 86%
86% 85% 84% 30.0 98% 98% 96% 96% 93% 92% 92% 95% 91% EXPERIMENT 7
Compound EC (.mu.M) 3.0 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.02344
0 Interferon- 0 96% 92% 81% 47% 28% 17% -1% -8% 0 alpha-2A 0.0492
96% 93% 78% 58% 21% 8% -12% 10% -17% (units) 0.1229 95% 93% 79% 64%
14% 5% 14% 7% -22% 0.3072 95% 91% 80% 64% 22% 15% 5% 2% -5% 0.768
96% 95% 81% 64% 34% 21% 19% 20% 4% 1.92 96% 95% 88% 78% 44% 41% 19%
33% 21% 4.8 97% 95% 91% 85% 60% 58% 60% 53% 49% 12.0 97% 97% 95%
91% 77% 72% 76% 70% 71% 30.0 98% 98% 97% 94% 91% 86% 85% 85% 84%
EXPERIMENT 8 Compound CU (.mu.M) 3.0 1.5 0.75 0.375 0.1875 0.0938
0.0469 0.02344 0 Interferon- 0 97% 95% 77% 34% 16% 6% -7% 0% 0 beta
(units) 0.2344 98% 97% 83% 49% 31% 19% -21% -7% 1% 0.4688 98% 96%
84% 56% 39% 27% 10% -3% 21% 0.9375 98% 97% 91% 73% 54% 42% 31% 15%
30% 1.875 98% 98% 95% 80% 65% 58% 65% 60% 60% 3.75 98% 98% 97% 92%
86% 81% 77% 73% 79% 7.5 99% 98% 98% 96% 93% 93% 93% 90% 92% 15.0
99% 99% 99% 97% 97% 96% 97% 95% 96% 30.0 99% 99% 99% 99% 98% 99%
98% 98% 97% EXPERIMENT 9 Compound EP (.mu.M) 8 4 2 1 0.5 0.25 0.125
0.0625 0 Interferon- 0 94% 96% 96% 92% 64% 36% 23% 8% 0 alpha-2B
0.0492 95% 96% 96% 91% 67% 25% 28% 8% 3% (units) 0.1229 95% 97% 96%
91% 65% 44% 4% 11% 4% 0.3072 95% 97% 96% 91% 71% 46% 20% 8% 20%
0.7680 96% 97% 97% 93% 75% 49% 36% 24% 24% 1.92 96% 97% 97% 94% 82%
67% 49% 52% 54% 4.8 96% 98% 97% 96% 90% 79% 75% 75% 70% 12 97% 98%
98% 97% 94% 89% 89% 87% 83% 30 97% 98% 98% 98% 96% 94% 94% 95% 92%
EXPERIMENT 10 Ribavirin (.mu.M) 200 80 32 12.8 5.12 2.048 0.8192
0.3277 0 Interferon- 0 85% 62% 43% 3% -8% -17% -22% -6% 0 alpha-2B
0.0492 87% 66% 48% 44% 11% -4% -10% 11% -7% (units) 0.1229 84% 64%
53% 40% 26% -12% -5% 11% -9% 0.3072 86% 70% 62% 44% 28% 1% 6% 14%
7% 0.7680 90% 80% 72% 65% 38% 30% 28% 44% 29% 1.92 93% 85% 77% 76%
61% 57% 58% 50% 46% 4.8 96% 92% 87% 83% 82% 74% 71% 77% 72% 12 97%
95% 93% 91% 90% 89% 90% 89% 85% 30 98% 97% 96% 95% 94% 94% 93% 95%
94%
[0789] As shown in FIGS. 4-13, which graphically depict the data in
Table 8 plotted using the above-described mathematical method for
measuring synergy, the Isobol curves for all combinations of HCV
serine protease inhibitors and interferons tested are concave-up,
indicating that the antiviral effect of the treatments in the
Replicon Assay is synergistic. These results are tabulated in Table
9, which shows relative levels of synergy for combination treatment
and IC.sub.50 values for antiviral compounds used individually. The
key elements in Table 9 are the .alpha. values, and the p-values
for the determinations. The a term is a measure of the maximum
inflection of the Isobols for each combination treatment. An a
value of zero indicates additivity, a negative value indicates
antagonism, and as is the case in the combination treatments with
the HCV serine protease inhibitors and interferons shown above, a
value greater than one indicates synergy. The larger the .alpha.
parameter, the greater the synergy. As shown in Table 9 for the
combinations of HCV serine protease inhibitors and interferons,
even ignoring significance levels in each experiment, a t test
based on the 9 experiments for the average alpha value being 0 (no
interaction) has a p-value of 0.00014, indicating that the results
are highly significant.
[0790] The calculation of synergy based on the method of Greco
Rustom ((1990) Application of a New Approach for the Quantitation
of Drug Synergism to the Combination of
cis-Diamminedichloroplatinum and
1-.beta.-D-Arabinofuranosylcytosine, Cancer Research, 50,
5318-5327) used in this analysis is an ideal tool for evaluation of
the kind of experimental data that can be generated using the HCV
Replicon Assay. There are other methods that are applied to studies
of antiviral compounds such as Pritchard and Shipman (Prichard, M.
N., and Shipman, C. Jr., (1990) "A three-dimensional model to
analyze drug-drug interactions (review)," Antiviral Res. 14:
181-206). Application of their synergy calculation method to the
data shown in Table 8 also indicates combination treatment of the
replicon cells with an HCV serine protease inhibitor and interferon
will result in a synergistic inhibition of HCV replicon RNA
accumulation (data not shown).
TABLE-US-00011 TABLE 9 RELATIVE LEVELS OF SYNERGY FOR COMBINATION
TREATMENT AND IC.sub.50 VALUES FOR ANTIVIRAL COMPOUNDS USED
INDIVIDUALLY HCV Serine Protease Inhibitor IC.sub.50 INF IC.sub.50
HSPI .alpha. P-value (HSPI) Interferon (units) (.mu.M) (SE).sup.1
.alpha. > 0 Experiment 1 Compound CU IFN alpha-2B 2.05 0.469
0.477 (0.09) <0.0001 Experiment 2 Compound CU IFN alpha-2A 3.72
0.446 0.770 (0.12) <0.0001 Experiment 3 Compound CU IFN alpha-2B
2.36 0.587 0.730 (0.08) <0.0001 Experiment 4 Compound CU IFN
alpha-2A 5.67 0.633 0.438 (0.08) <0.0001 Experiment 5 Compound
CU IFN tau 13.22 0.605 0.328 (0.07) <0.0001 Experiment 6
Compound EC IFN alpha-2B 2.53 0.384 0.516 (0.10) <0.0001
Experiment 7 Compound EC IFN alpha-2A 5.50 0.312 1.24 (0.20)
<0.0001 Experiment 8 Compound CU IFN beta 1.82 0.466 0.551
(0.09) <0.0001 Experiment 9 Compound EP IFN alpha-2B 3.06 0.426
0.490 (0.12) <0.0001 Experiment 10 Ribavirin IFN alpha-2B 1.22
145 -0.24 (0.067) 0.0004 .sup.1(SE) Standard Error
[0791] Another measure for evaluating the synergistic nature of
anti-HCV drug treatment using the present HCV serine protease
inhibitors and interferons is to use the same methods described
above to evaluate the current standard combination therapy for HCV,
i.e., interferon alpha-2B in combination with Ribavirin in the
Replicon Assay. The last line of Table 9 shows that the .alpha.
parameter for a mixture of interferon alpha-2B and Ribavirin is a
negative number, indicating that there is a small amount of
antagonism between these two drugs. This further emphasizes the
significance of the combination treatments disclosed herein
employing the present HCV serine protease inhibitors in combination
with interferons in that these treatments clearly produce synergy,
while the standard combination therapy in use for HCV (interferon
alpha-28 in combination with Ribavirin) is not synergistic in the
Replicon Assay.
[0792] The foregoing comparison of combination treatments employing
the present HCV serine protease inhibitors plus interferons versus
Ribavirin plus interferon in the Replicon Assay clearly indicates
that the former are synergistic, while the latter is not. The
experimental results obtained using the Replicon Assay indicate
that a much lower dose of interferon would be efficacious if the
interferon is used in combination with a HCV serine protease
inhibitor than is needed when interferon alpha-2B is used in
combination with Ribavirin. The Replicon Assay is a useful model
system in which to test potential anti-HCV compounds, and is
currently widely relied upon as an effective predictor of compound
anti-HCV activity. Note, for example, Blight et al. (2000)
Efficient Initiation of HCV RNA Replication in Cell Culture.
Science 8; 290:1972-1974, and Chung et al. (2001) Hepatitis C virus
replication is directly inhibited by IFN-.alpha. in a full-length
binary expression system. Proc. Nat. Acad. Sci. U.S.A.
98(17):9847-52. Ribavirin alone is marginally effective in reducing
the accumulation of HCV replicon RNA in the Replicon Assay (Table
8, Experiment 10 and last line of Table 9). This result is in
apparent conflict with in vivo studies where, when used by itself,
Ribavirin has no significant therapeutic value for the treatment of
HCV. In contrast, in the Replicon Assay, correcting for
cytotoxicity as discussed below, Ribavirin has an IC.sub.50 of 145
.mu.M. This result can be explained by recognizing that the
Replicon Assay permits evaluation of high Ribavirin concentrations
that would not be possible in human therapy due to in vivo
cytotoxicity (Chutaputti A. (2000) Adverse effects and other safety
aspects of the hepatitis C antivirals. Journal of Gastroenterology
and Hepatology. 15 Suppl:E156-63).
[0793] This evaluation necessarily requires assessment of the
cytotoxicity of Ribavirin. Such toxicity occurs in patients and in
cellular assays (Shiffman M. L., Verbeke S. B., Kimball P. M.
(2000) Alpha interferon combined with ribavirin potentiates
proliferative suppression but not cytokine production in
mitogenically stimulated human lymphocytes. Antiviral Research.
48(2):91-9). In the experiments disclosed herein, Ribavirin
cytotoxicity in the Replicon Assay is observed and measured in two
ways. In both the XTT metabolic assay to determine replicon cell
viability (Roehm N. W., Rodgers G. H., Hatfield S. M., Glasebrook
A. L. (1991) An improved colorimetric assay for cell proliferation
and viability utilizing the tetrazolium salt XTT. Journal of
Immunol. Methods. 142(2):257-65) and in the TaqMan.RTM.
quantitative RT-PCR assay that measures glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA levels in treated versus untreated cells
in the Replicon Assay (Brink N., Szamel M., Young A. R., Wittern K.
P., Bergemann J. (2000) Comparative quantification of IL-1beta,
IL-10, IL-10r, TNFalpha and IL-7 mRNA levels in UV-irradiated human
skin in vivo. Inflamation Research. 49(6):290-6), significant
Ribavirin-induced cytotoxicity is observed, but is corrected for as
follows. It is assumed that the level of GAPDH mRNA, which is a
constitutively expressed housekeeping gene, is the same in all
viable cells. It is known from measurements of GAPDH mRNA levels in
cells treated with the transcription inhibitor actinomycin D that
the half life of GAPDH mRNA is only a few hours (data not shown).
Thus, it is postulated, as done by others using TaqMan.RTM.
technology to determine the levels of particular mRNAs in human
cells, that GAPDH mRNA levels are proportional to viable cell
numbers (VCN) in any given sample, with the relationship of
VCN=2.sup. (40-Ct.sub.GAPDH mRNA). The VCN is computed for each of
the Replicon Assay sample wells, and then we divide the HCV
Replicon RNA copy number for a specific well by the VCN for that
well. Once computed, this ratio is used instead of the HCV copy
number to compute inhibition ("Avg. Inh using ratio"; FIG. 14A).
Without correcting the Replicon Assay data for this cytotoxicity,
such cytotoxicity is read as false positive inhibition of HCV RNA
replicon accumulation. In the Replicon Assay, it is assumed that
the measured inhibition of HCV RNA replicon accumulation is the sum
of the actual inhibition of HCV RNA replicon accumulation and the
apparent inhibition of HCV RNA replicon accumulation due to
cytoxicity. It is furthermore assumed that, based on the close
correlation of the XTT and TaqMan.RTM. GAPDH mRNA measures of
cytotoxicity, the inhibition of accumulation of GAPDH mRNA caused
by compounds tested in the Replicon Assay is a reliable measure of
apparent inhibition of HCV RNA replicon accumulation due to
cytoxicity. Thus, the true anti-HCV activity of a compound in the
Replicon Assay corrected for general cytotoxicity can be estimated
by dividing the number of HCV replicon RNA molecules measured in
each sample by the VCN, thus normalizing to the number of viable
cells in each sample. Using this method, FIG. 14A shows an estimate
of true Ribavirin anti-HCV activity in the Replicon Assay ("Avg.
Inh using ratio"). The estimate of the IC.sub.50 for Ribavirin is
best calculated using this method. In FIG. 14A, "Avg. Inh original"
shows the uncorrected IC.sub.50 for Ribavirin, which is
approximately 80 .mu.M, whereas the corrected IC.sub.50 value
calculated from the "Avg. Inh using ratio" curve is approximately
145 .mu.M. Note that the difference between corrected and measured
inhibition of HCV RNA replicon accumulation as a result of
interferon alpha-2B treatment (FIG. 14B) is insignificant in view
of the .about.20% % CV of the Replicon Assay. Like interferon
alpha-2B, the HCV serine protease inhibitors tested in the present
example exhibit no significant cytotoxicity at the concentrations
employed. This is determined using XTT assays, in which the
TC.sub.50 values for the various compounds are: CU=64.7 .mu.M,
EP>10 .mu.M, and EC>50 .mu.M. These TC.sub.50 values are
20-140 fold greater than the IC.sub.50 values shown in Table 9.
Thus, cytotoxicity of these compounds has no significant effect on
HCV RNA accumulation in the Replicon Assay within the precision of
the assay because such cytotoxicity occurs only at HCV serine
protease inhibitor concentrations significantly greater than those
tested in the Replicon Assay.
[0794] Conclusions Regarding the Efficacy of HCV Serine Protease
Inhibitors and Interferons, Individually and in Combination
[0795] The anti-HCV activities of the present HCV serine protease
inhibitors and various interferons when used alone in the HCV
Replicon Assay are shown in the columns and lines of the individual
experiments making up Table 8 that employ only one antiviral agent.
Table 9 lists the IC.sub.50 values measured for each antiviral
compound when tested alone. The foregoing results, derived via use
of the in vitro Replicon Assay, also demonstrate that combination
treatment of replicon cells with HCV serine protease inhibitors of
the present invention and various interferons yields synergistic
antiviral effects. It is fully expected that these effects will
translate into in vivo effectiveness.
[0796] Combination therapy employing HCV serine protease inhibitors
of the present invention possesses several major advantages over
single drug therapy. First, by making treatment possible with lower
doses of the individual drugs than would be possible if used alone,
one would expect a reduction in toxicity and side effects
associated with treatment. This is especially important in the case
of interferon therapy, where the side effects are severe, and have
been shown to be proportional to the dose administered to patients.
The foregoing data indicate that a dose of HCV serine protease
inhibitor such as CU at the IC.sub.95 level could be combined with
a dose of interferon alpha, for example at the IC.sub.50 level, and
the result would be much more effective therapy than could be
achieved with the HCV serine protease inhibitor alone without the
adverse side effects caused by high doses of interferon alpha. A
second major benefit of combination therapy is that because the two
drugs act independently, there is less chance of development of
mutant HCV strains that resist treatment. Development of resistance
is a major concern with RNA viruses like HCV. Because of their high
rate of mutation, such viruses can rapidly adapt to environmental
stresses. A third benefit of combination therapy may be reduced
cost, due to the need for lower amounts of therapeutic agents
required for effective treatment.
[0797] Additional immunomodulators that can be employed in the
methods disclosed herein include, for example, alpha interferon 2A,
consensus interferon, tau interferon, interferon+Ribavirin
(Rebatron), pegylated interferon, and promoters of interferon gene
expression. It is fully anticipated that the anti-HCV activity of
these compounds will be improved when used in combination with HCV
serine protease inhibitors such as those disclosed herein. As
interferons are known to be active in vivo and in the Replicon
Assay, it is expected that the present HCV serine protease
inhibitors will also be active in vivo, and more importantly, be
capable of eliciting synergistic activity when used in combination
with interferons, immune system stimulators thereof, or other
compounds having HCV antiviral activity that act by a mechanism
other than inhibition of the HCV serine protease.
[0798] The best current therapy for HCV employs interferon alpha
and the nucleoside analog Ribavirin. This treatment is only
marginally effective, and results in significant side effects that
diminish patient compliance (Chronic Hepatitis C: Current Disease
Management, U.S. Department of Health and Human Services, National
Institutes of Health, 1999). Additionally, in transplant patients,
it is not clear the Ribavirin-interferon combination works, and may
in fact be worse than interferon alone (Chronic Hepatitis C:
Current Disease Management, U.S. Department of Health and Human
Services, National Institutes of Health, 1999).
[0799] The results presented above demonstrate a synergistic
combination effect when interferons are used with a new class of
HCV antivirals, the serine protease inhibitors of the present
invention. We fully expect that the in vitro results disclosed
herein will lead to more effective treatment of HCV patients than
is currently possible using interferon alone. Sub-therapeutic doses
of interferon could mobilize the patient's immune system to better
fight the virus, and the serine protease inhibitor could attack the
virus directly, dealing the virus a two-pronged attack via
different mechanisms of action. Treatment of HCV infection could
thus be achieved at reduced cost to the patient in terms of both
diminished side effects and lower payments for necessary
pharmaceutical agents as less of both drugs would be needed for
effective HCV antiviral therapy.
[0800] The present invention may be embodied in other specific
forms without departing from the spirit or essential attributes
thereof.
Sequence CWU 1
1
71900DNAUnknownHCV Replicon RNA TaqMan probe and primers
1gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg aatcctaaac
60ctcaaagaaa aaccaaacgt aacaccaacg ggcgcgccat gattgaacaa gatggattgc
120acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
gcacaacaga 180caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
gcaggggcgc ccggttcttt 240ttgtcaagac cgacctgtcc ggtgccctga
atgaactgca ggacgaggca gcgcggctat 300cgtggctggc cacgacgggc
gttccttgcg cagctgtgct cgacgttgtc actgaagcgg 360gaagggactg
gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
420ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
acgcttgatc 480cggctacctg cccattcgac caccaagcga aacatcgcat
cgagcgagca cgtactcgga 540tggaagccgg tcttgtcgat caggatgatc
tggacgaaga gcatcagggg ctcgcgccag 600ccgaactgtt cgccaggctc
aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc 660atggcgatgc
ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
720actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
acccgtgata 780ttgctgaaga gcttggcggc gaatgggctg accgcttcct
cgtgctttac ggtatcgccg 840ctcccgattc gcagcgcatc gccttctatc
gccttcttga cgagttcttc tgagtttaaa 900220DNAUnknownHCV Replicon RNA
TaqMan probe and primers 2ctgtggccgg ctgggtgtgg 20323DNAUnknownHCV
Replicon RNA TaqMan probe and primers 3ccctcgtcca cgtggcatct cga
23420DNAUnknownHCV Replicon RNA TaqMan probe and primers
4ccgcttttct ggattcatcg 20515DNAUnknownHCV Replicon RNA TaqMan probe
and primers 5cccattcgcc gccaa 15622DNAUnknownHCV Replicon RNA
TaqMan probe and primers 6cagggtagtc gtcagtggtt cg
22720DNAUnknownHCV Replicon RNA TaqMan probe and primers
7ggcctctgca gcaccctatc 20
* * * * *