U.S. patent application number 15/946634 was filed with the patent office on 2018-08-09 for seca inhibitors and methods of making and using thereof.
The applicant listed for this patent is Arpana S. Chaudhary, Weixuan Chen, Jianmei Cui, Chaofeng Dai, Krishna Damera, Yinghsin Hsieh, Jinshan Jin, Ying-Ju Ritter, Phang-Cheng Tai, Binghe Wang. Invention is credited to Arpana S. Chaudhary, Weixuan Chen, Jianmei Cui, Chaofeng Dai, Krishna Damera, Yinghsin Hsieh, Jinshan Jin, Ying-Ju Ritter, Phang-Cheng Tai, Binghe Wang.
Application Number | 20180222880 15/946634 |
Document ID | / |
Family ID | 48672815 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180222880 |
Kind Code |
A1 |
Wang; Binghe ; et
al. |
August 9, 2018 |
SecA INHIBITORS AND METHODS OF MAKING AND USING THEREOF
Abstract
Inhibitors of SecA, and methods of making and using thereof, are
described herein. The compounds described herein can be used to
treat or prevent microbial infections, such as bacterial
infections.
Inventors: |
Wang; Binghe; (Marietta,
GA) ; Tai; Phang-Cheng; (Atlanta, GA) ; Jin;
Jinshan; (Atlanta, GA) ; Hsieh; Yinghsin;
(Atlanta, GA) ; Ritter; Ying-Ju; (Marietta,
GA) ; Cui; Jianmei; (Kennesaw, GA) ;
Chaudhary; Arpana S.; (Atlanta, GA) ; Dai;
Chaofeng; (Atlanta, GA) ; Damera; Krishna;
(Smyrna, GA) ; Chen; Weixuan; (Atlanta,
GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wang; Binghe
Tai; Phang-Cheng
Jin; Jinshan
Hsieh; Yinghsin
Ritter; Ying-Ju
Cui; Jianmei
Chaudhary; Arpana S.
Dai; Chaofeng
Damera; Krishna
Chen; Weixuan |
Marietta
Atlanta
Atlanta
Atlanta
Marietta
Kennesaw
Atlanta
Atlanta
Smyrna
Atlanta |
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA |
US
US
US
US
US
US
US
US
US
US |
|
|
Family ID: |
48672815 |
Appl. No.: |
15/946634 |
Filed: |
April 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14406085 |
Dec 5, 2014 |
9957247 |
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PCT/US13/44243 |
Jun 5, 2013 |
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15946634 |
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61656793 |
Jun 7, 2012 |
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61824857 |
May 17, 2013 |
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61826345 |
May 22, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 239/60 20130101;
C07D 239/56 20130101; C07D 413/14 20130101; A61P 31/00 20180101;
G01N 2333/4706 20130101; C07D 493/10 20130101; G01N 33/6872
20130101; C07D 239/22 20130101; A61P 31/04 20180101; C07D 497/04
20130101; C07D 471/10 20130101; A61P 31/10 20180101; C07D 311/58
20130101; C07D 311/86 20130101; C07D 311/82 20130101; C07D 249/12
20130101; C07C 63/331 20130101; C07D 491/20 20130101; C07D 417/14
20130101; A61K 31/496 20130101; C07D 239/20 20130101; C07D 403/14
20130101; C07D 495/04 20130101; Y02A 50/30 20180101; C07D 311/74
20130101; C09B 11/24 20130101; Y02A 50/473 20180101; A61P 31/12
20180101; C07D 239/38 20130101; C07D 401/14 20130101 |
International
Class: |
C07D 311/86 20060101
C07D311/86; C07D 239/20 20060101 C07D239/20; C07D 401/14 20060101
C07D401/14; C07D 311/82 20060101 C07D311/82; C07D 403/14 20060101
C07D403/14; C07D 239/38 20060101 C07D239/38; C07D 417/14 20060101
C07D417/14; C07D 249/12 20060101 C07D249/12; C07D 471/10 20060101
C07D471/10; C07D 491/20 20060101 C07D491/20; C07D 495/04 20060101
C07D495/04; C07D 497/04 20060101 C07D497/04; C07D 493/10 20060101
C07D493/10; C07D 311/74 20060101 C07D311/74; C07D 239/60 20060101
C07D239/60; C07D 239/56 20060101 C07D239/56; C07C 63/331 20060101
C07C063/331; G01N 33/68 20060101 G01N033/68; C09B 11/24 20060101
C09B011/24; C07D 413/14 20060101 C07D413/14; C07D 239/22 20060101
C07D239/22; A61K 31/496 20060101 A61K031/496; C07D 311/58 20060101
C07D311/58 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] This invention was made with government support under
Agreement Nos. CA123329, CA 88343, GM34766, and GM 084933 awarded
by the National Institutes of Health. The government has certain
rights in the invention.
Claims
1. A compound, or a pharmaceutically acceptable salt thereof,
wherein the compound has the following formula: ##STR00085##
wherein X of Formula VI is O, S, SO, SO.sub.2, NR.sub.11, or
CR.sub.12R.sub.13; and R.sub.1-R.sub.13 of Formula VI are
independently absent or selected from hydrogen, ethyl, n-propyl,
isopropyl, n-, sec-, iso-, or t-butyl, pentyl, hexyl, heptyl,
substituted or unsubstituted cyclopentanyl; substituted or
unsubstituted, linear, branched, or hetero alkenyl or alkynyl;
substituted or unsubstituted aryl or heteroaryl; halogen;
substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl,
carboxylic acid, carboxylate, --CONH.sub.2, --CONHR.sub.14,
--CONR.sub.14R.sub.14, --OCONHR.sub.14, --NHCOOR.sub.14,
--OCONR.sub.14R.sub.14, --NR.sub.14COOR.sub.14, NHCONHR.sub.14,
--NR.sub.14CONHR.sub.14, --NHCONR.sub.14R.sub.14,
--NR.sub.14CONR.sub.14R.sub.14, --CH.sub.2OH; --CHR.sub.14OH,
--CR.sub.14R.sub.14OH, --COOR.sub.14), thiol, --NH.sub.2,
--NHR.sub.14, --NR.sub.14R.sub.14, --SR.sub.14, --SOR.sub.14, and
--SOOR.sub.14, wherein R.sub.14 of Formula VI is defined the same
as R.sub.1-R.sub.13 of Formula VI; ##STR00086## wherein X of
Formula VII is O, S, SO, SO.sub.2, NR.sub.9, CR.sub.10R.sub.11; and
R.sub.1-R.sub.11 of Formula VII are independently absent or
selected from hydrogen, substituted or unsubstituted, linear,
branched, hetero, or cyclic alkyl, alkenyl, or alkynyl; substituted
or unsubstituted aryl or heteroaryl; halogen; substituted or
unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic
acid, carboxylate, --CONH.sub.2, CONHR.sub.12,
--CONR.sub.12R.sub.12, --OCONHR.sub.12, --NHCOOR.sub.12,
--OCONR.sub.12R.sub.12, --NR.sub.14COOR.sub.12, NHCONHR.sub.12,
--NR.sub.12CONHR.sub.12, --NHCONR.sub.12R.sub.12,
--NR.sub.14CONR.sub.12R.sub.12, --CH.sub.2OH, --CHR.sub.12OH,
--CR.sub.12R.sub.12OH, --COOR.sub.12, thiol, --NH.sub.2,
--NHR.sub.12, --NR.sub.12R.sub.12, --SR.sub.12, --SOR.sub.12, and
--SOOR.sub.12, wherein R.sub.12 of Formula VII is defined the same
as R.sub.1-R.sub.11 of Formula VII; and wherein the compound of
Formula VII is not Rose Bengal; ##STR00087## wherein A and B of
Formula I are independently S, SO.sub.2, SO, O, NR.sub.6, or
CR.sub.7R.sub.8; W and Z of Formula I are independently N or
CR.sub.9; X and Y of Formula I are independently S, O, or
CR.sub.10R.sub.11; and R.sub.1-R.sub.11 of Formula I are
independently absent or selected from hydrogen, substituted or
unsubstituted, linear, branched, hetero, or cyclic alkyl, alkenyl,
or alkynyl; substituted or unsubstituted aryl or heteroaryl;
halogen, substituted or unsubstituted alkoxy; hydroxy, cyano,
formyl, acyl, carboxylic acid, carboxylate, --CONH.sub.2,
--CONHR.sub.12, --CONR.sub.12R.sub.12, --OCONHR.sub.12,
--NHCOOR.sub.12, --OCONR.sub.12R.sub.12, --NR.sub.12COOR.sub.12,
NHCONHR.sub.12, --NR.sub.12CONHR.sub.12, --NHCONR.sub.12R.sub.12,
--NR.sub.12CONR.sub.12R.sub.12, --CH.sub.2OH, --CHR.sub.12OH,
--CR.sub.12R.sub.12OH, --COOR.sub.12, thiol, --NH.sub.2,
--NHR.sub.12, --NR.sub.12R.sub.12, --SR.sub.12, --SOR.sub.12, and
--SOOR.sub.12, wherein R.sub.12 of Formula I is defined the same as
R.sub.1-R.sub.11 of Formula I; ##STR00088## wherein X of Formula II
is S, SO, SO.sub.2, NHR.sub.4, O, or CR.sub.5R.sub.6; Y of Formula
II is N or CR.sub.7; Z of Formula II is S, O, NR.sub.8, or
CR.sub.9R.sub.10; and R.sub.1-R.sub.10 of Formula II is
independently absent or selected from hydrogen, substituted or
unsubstituted, linear, branched, hetero, or cyclic alkyl, alkenyl,
or alkynyl; substituted or unsubstituted aryl or heteroaryl;
halogen; substituted or unsubstituted alkoxy; hydroxy, cyano,
formyl, acyl, carboxylic acid, carboxylate, --CONH.sub.2,
--CONHR.sub.11, --CONR.sub.11R.sub.11, --OCONHR.sub.11,
--NHCOOR.sub.11, --OCONR.sub.11R.sub.11, --NR.sub.11COOR.sub.11,
NHCONHR.sub.11, --NR.sub.10CONHR.sub.11, --NHCONR.sub.11R.sub.11,
--NR.sub.11CONR.sub.11R.sub.11, --CH.sub.2OH, --CHR.sub.11OH,
--CR.sub.11R.sub.11OH, --COOR.sub.11, thiol, --NH.sub.2,
--NHR.sub.11, --NR.sub.11R.sub.11, --SR.sub.11, --SOR.sub.11, and
--SOOR.sub.11, wherein R.sub.11 of Formula II is defined the same
as R.sub.1-R.sub.10 of Formula II; ##STR00089## wherein X of
Formula IV is O, S, NR.sub.10, or CR.sub.11R.sub.12;
R.sub.1-R.sub.12 of Formula IV are independently absent or selected
from hydrogen, substituted or unsubstituted, linear, branched,
hetero, or cyclic alkyl, alkenyl, or alkynyl; substituted or
unsubstituted aryl or heteroaryl; halogen; substituted or
unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic
acid, carboxylate, --CONH.sub.2, --CONHR.sub.13,
--CONR.sub.13R.sub.13, --OCONHR.sub.13, --NHCOOR.sub.13,
--OCONR.sub.13R.sub.13, --NR.sub.14COOR.sub.13, NHCONHR.sub.13,
--NR.sub.14CONHR.sub.13, --NHCONR.sub.13R.sub.13,
--NR.sub.17CONR.sub.13R.sub.13, --CH.sub.2OH, --CHR.sub.13OH,
--CR.sub.13R.sub.13OH, --COOR.sub.13, thiol, --NH.sub.2,
--NHR.sub.13, --NR.sub.13R.sub.13, --SR.sub.13, --SOR.sub.13, and
--SOOR.sub.13, wherein R.sub.13 of Formula IV is defined the same
as R.sub.1-R.sub.12 of Formula IV; and the dotted lines represent
optional double bonds; ##STR00090## wherein X and Y of Formula V
are independently O, S, NR.sub.13, or CR.sub.14R.sub.15; and
R.sub.1-R.sub.15 of Formula V are independently absent or selected
from hydrogen, substituted or unsubstituted, linear, branched,
hetero, or cyclic alkyl, alkenyl, or alkynyl; substituted or
unsubstituted aryl or heteroaryl; halogen; substituted or
unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic
acid, carboxylate, --CONH.sub.2, --CONHR.sub.16,
--CONR.sub.16R.sub.16, --OCONHR.sub.16, --NHCOOR.sub.16,
--OCONR.sub.16R.sub.16, --NR.sub.16COOR.sub.16, NHCONHR.sub.16,
--NR.sub.16CONHR.sub.16, --NHCONR.sub.16R.sub.16,
--NR.sub.16CONR.sub.16R.sub.16, --CH.sub.2OH, --CHR.sub.16OH,
--CR.sub.16R.sub.16OH, --COOR.sub.16, thiol, --NH.sub.2,
--NHR.sub.16, --NR.sub.16R.sub.16, --SR.sub.16, --SOR.sub.16, and
--SOOR.sub.16, wherein R.sub.16 of Formula V is defined the same as
R.sub.1-R.sub.15 of Formula V; ##STR00091## wherein Z of Formula
VIII is O, S, SO, SO.sub.2, NR.sub.6, or CR.sub.7R.sub.8; X and Y
of Formula VIII are independently N, NR.sub.9, or
CR.sub.10R.sub.11; R.sub.1-R.sub.11 of Formula VIII are
independently absent or selected from hydrogen, substituted or
unsubstituted, linear, branched, hetero, or cyclic alkyl, alkenyl,
or alkynyl; substituted or unsubstituted aryl or heteroaryl;
halogen; substituted or unsubstituted alkoxy; hydroxy, cyano,
formyl, acyl, carboxylic acid, carboxylate, --CONH.sub.2,
--CONHR.sub.12, --CONR.sub.12R.sub.12, --OCONHR.sub.12,
--NHCOOR.sub.12, --OCONR.sub.12R.sub.12, --NR.sub.14COOR.sub.12,
NHCONHR.sub.12, --NR.sub.12CONHR.sub.12, --NHCONR.sub.12R.sub.12,
--NR.sub.14CONR.sub.12R.sub.12, --CH.sub.2OH, --CHR.sub.12OH,
--CR.sub.12R.sub.12OH, --COOR.sub.12, thiol, --NH.sub.2,
--NHR.sub.12, --NR.sub.12R.sub.12, --SR.sub.12, --SOR.sub.12, and
--SOOR.sub.12, wherein R.sub.12 of Formula VIII is defined the same
as R.sub.1-R.sub.11 of Formula VIII; and the dotted lines represent
optional double bonds; or ##STR00092## wherein Z of Formulas IX and
IXa is O, S, SO, SO.sub.2, NR.sub.7, or CR.sub.8R.sub.9; X and Y of
Formulas IX and IXa are independently N, NR.sub.10, or
CR.sub.11R.sub.12; R.sub.1-R.sub.12 of Formulas IX and IXa are
independently absent or selected from hydrogen, substituted or
unsubstituted, linear, branched, hetero, or cyclic alkyl, alkenyl,
or alkynyl; substituted or unsubstituted aryl or heteroaryl;
halogen; substituted or unsubstituted alkoxy; hydroxy, cyano,
formyl, acyl, carboxylic acid, carboxylate, --CONH.sub.2,
--CONHR.sub.13, --CONR.sub.13R.sub.13, --OCONHR.sub.13,
--NHCOOR.sub.13, --OCONR.sub.13R.sub.13, --NR.sub.13COOR.sub.13,
NHCONHR.sub.13, --NR.sub.13CONHR.sub.13, --NHCONR.sub.13R.sub.13,
--NR.sub.13CONR.sub.13R.sub.13, --CH.sub.2OH, --CHR.sub.13OH,
--CR.sub.13R.sub.13OH, --COOR.sub.13, thiol, --NH.sub.2,
--NHR.sub.13, --NR.sub.13R.sub.13, --SR.sub.13, --SOR.sub.13, and
--SOOR.sub.13, wherein R.sub.13 of Formulas IX and IXa is defined
the same as R.sub.1-R.sub.12 of Formulas IX and IXa.
2-11. (canceled)
12. The compound of claim 1, wherein the compound is a compound of
Formula VI, wherein R.sub.1-R.sub.2 are independently absent or
selected from the group consisting of hydrogen; halogen; hydroxyl;
ethyl, n-propyl, isopropyl, n-, sec-, iso-, or t-butyl, pentyl,
hexyl, heptyl; substituted or unsubstituted cyclopentanyl;
substituted or unsubstituted alkoxy; substituted or unsubstituted
alkenyl, cycloalkenyl, heterocycloalkyl, and heteroaryl; wherein
R.sub.1-R.sub.2 are optionally substituted with one or more
substituents independently selected from the group consisting of
hydrogen; halogen; hydroxyl; ethyl, n-propyl, isopropyl, n-, sec-,
iso-, or t-butyl, pentyl, hexyl, or heptyl; substituted or
unsubstituted cyclopentanyl; substituted or unsubstituted alkoxy;
substituted or unsubstituted alkenyl, cycloalkenyl,
heterocycloalkyl, heteroaryl; or R.sub.1-R.sub.2 taken together is
O, S, SO, SO.sub.2, NR.sub.11, or CR.sub.12R.sub.13; wherein
R.sub.3-R.sub.13 are independently absent or selected from the
group consisting of hydrogen; halogen; hydroxyl; substituted or
unsubstituted alkoxy; substituted or unsubstituted alkyl; of
--OR.sup.14; cycloalkyl; cycloalkenyl; primary amine; secondary
amine; tertiary amine; --C(O)R.sup.14, --C(O)OR.sup.14,
--C(O)NR.sup.14R.sup.14, --NR.sup.14R.sup.14,
--NR.sup.14S(O).sub.2R.sup.14, --NR.sup.14C(O)R.sup.14,
--S(O).sub.2R.sup.14, --SR.sup.14, and
--S(O).sub.2NR.sup.14R.sup.14; and R.sub.14 is independently
selected from the group consisting of hydrogen, halogen, cyano,
--OR.sup.14, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl,
heterocycloalkyl, and heteroaryl.
13. The compound of claim 12, wherein X is O and wherein
R.sub.3-R.sub.13 are independently absent or selected from the
group consisting of hydrogen; halogen; hydroxyl; alkoxy;
substituted or unsubstituted alkyl; primary amine, secondary amine,
and tertiary amine.
14. The compound of claim 1, wherein the compound is a compound of
Formula VI, wherein the compound of Formula VI is a compound
selected from the group consisting of: ##STR00093## ##STR00094##
##STR00095## ##STR00096##
15. The compound of claim 1, wherein the compound is a compound of
Formula VII, wherein the compound of Formula VII is a compound
selected from the group consisting of: ##STR00097##
16. (canceled)
17. A pharmaceutical composition comprising one or more compounds
of claim 1 and one or more pharmaceutically acceptable
carriers.
18. A method of treating an infection comprising administering or
the composition of claim 17 in an amount effective to inhibit
SecA.
19. The method according to claim 18, wherein the infection is a
fungal, bacterial, or viral infection.
20. The method according to claim 19, wherein the infection is a
bacterial infection.
21. The method according to claim 18, wherein the composition is
administered by one or more routes selected from the group
consisting of buccal, sublingual, intravenous, subcutaneous,
intradermal, transdermal, intraperitoneal, oral, eye drops,
parenteral and topical administration.
22. (canceled)
23. A method for assessing the inhibitory effect of any one of the
compounds of claim 1 on ATPase membrane channel activities, the
method comprising: injecting a SecA-liposome and various
concentrations of the compound into the membrane of oocytes, and
determining the IC.sub.50 value of the compound.
24. The method of claim 23, wherein the liposome further comprises
a protein selected from the group consisting of SecYEG and
SecYEG/DF.YajC.
25. The compound of claim 1, wherein the compound is a compound of
Formula VI.
26. The compound of claim 1, wherein the compound is a compound of
Formula VII.
27. The compound of claim 1, wherein the compound is a compound of
Formula I.
28. The compound of claim 1, wherein the compound is a compound of
Formula II.
29. The compound of claim 1, wherein the compound is a compound of
Formula IV.
30. The compound of claim 1, wherein the compound is a compound of
Formula V.
31. The compound of claim 1, wherein the compound is a compound of
Formula VIII.
32. The compound of claim 1, wherein the compound is a compound of
Formula IX or IXa.
33. The compound of claim 1, wherein the compound is a compound of
Formula I, wherein the compound of Formula I is a compound selected
from the group consisting of: ##STR00098## ##STR00099##
34. The compound of claim 1, wherein the compound is a compound of
Formula II, wherein the compound of Formula II is a compound
selected from the group consisting of: ##STR00100## ##STR00101##
##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106##
##STR00107##
35. The pharmaceutical composition of claim 17, wherein the
composition comprises one or more compounds of Formula VI and one
of more compounds of Formula I.
36. The pharmaceutical composition of claim 17, wherein the
composition comprises one or more compounds of Formula VII and one
of more compounds of Formula I.
37. The pharmaceutical composition of claim 17, wherein the
composition comprises one or more compounds of Formula VI and one
of more compounds of Formula II.
38. The pharmaceutical composition of claim 17, wherein the
composition comprises one or more compounds of Formula VII and one
of more compounds of Formula II.
39. The pharmaceutical composition of claim 17, further comprising
one or more further compounds, or a pharmaceutically acceptable
salt thereof, wherein the further compounds have, independently,
the following formula: ##STR00108## wherein X and Y of Formula III
are independently N or C; D and G of Formula III are independently
NR.sub.7, CR.sub.8R.sub.9, O, or S; A, B, E, and F of Formula III
are independently N or CR.sub.10; L and M of Formula III are
independently S, SO, SO.sub.2, O, NR.sub.11, or CR.sub.12R.sub.13 J
of Formula III is O, S, SO, SO.sub.2, NR.sub.14, or
CR.sub.15R.sub.16; and R.sub.1-R.sub.16 of Formula III are
independently absent or selected from hydrogen, substituted or
unsubstituted, linear, branched, hetero, or cyclic alkyl, alkenyl,
or alkynyl; halogen; substituted or unsubstituted alkoxy; hydroxy,
cyano, formyl, acyl, carboxylic acid, carboxylate, --CONH.sub.2,
--CONHR.sub.17, --CONR.sub.17R.sub.17, --OCONHR.sub.17,
--NHCOOR.sub.17, --OCONR.sub.17R.sub.17, --NR.sub.14COOR.sub.17,
NHCONHR.sub.17; --NR.sub.14CONHR.sub.17, --NHCONR.sub.17R.sub.17,
--NR.sub.17CONR.sub.17R.sub.17, --CH.sub.2OH, --CHR.sub.17OH,
--CR.sub.17R.sub.17OH, --COOR.sub.17, thiol, --NH.sub.2,
--NHR.sub.17, --NR.sub.17R.sub.17, --SR.sub.17, --SOR.sub.17, and
--SOOR.sub.17, wherein R.sub.17 of Formula III is defined the same
as R.sub.1-R.sub.16 of Formula III; or ##STR00109## wherein Z and W
of Formula X or Xa are O, S, SO, SO.sub.2, NR.sub.5, or
CR.sub.6R.sub.7; X and Y of Formula X or Xa are independently N,
NR.sub.8, or CR.sub.9R.sub.10; Cy of Formula X or Xa is substituted
or unsubstituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl
group; and R.sub.1-R.sub.10 of Formula X or Xa are independently
absent or selected from hydrogen, substituted or unsubstituted,
linear, branched, hetero, or cyclic alkyl, alkenyl, or alkynyl;
substituted or unsubstituted aryl or heteroaryl; halogen;
substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl,
carboxylic acid, carboxylate, --CONH.sub.2, --CONHR.sub.11,
--CONR.sub.11R.sub.11, --OCONHR.sub.11, --NHCOOR.sub.11,
--OCONR.sub.11R.sub.11, --NR.sub.14COOR.sub.11, NHCONHR.sub.11,
--NR.sub.11CONHR.sub.11, --NHCONR.sub.11R.sub.11,
--NR.sub.14CONR.sub.11R.sub.11, --CH.sub.2OH, --CHR.sub.11OH,
--CR.sub.11R.sub.11OH, --COOR.sub.11, thiol, --NH.sub.2,
--NHR.sub.11, --NR.sub.11R.sub.11, --SR.sub.11, --SOR.sub.11, and
--SOOR.sub.11, wherein R.sub.11 of Formula X or Xa is defined the
same as R.sub.1-R.sub.10 of Formula X or Xa.
40. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula I and one of
more compounds of Formula III.
41. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula I and one of
more compounds of Formula X or Xa.
42. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VI and one
of more compounds of Formula III.
43. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VI and one
of more compounds of Formula X or Xa.
44. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VII and one
of more compounds of Formula III.
45. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VII and one
of more compounds of Formula X or Xa.
46. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VI, one or
more compounds of Formula I, and one of more compounds of Formula
III.
47. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VII, one or
more compounds of Formula I, and one of more compounds of Formula
III.
48. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VI, one or
more compounds of Formula I, and one of more compounds of Formula X
or Xa.
49. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VII, one or
more compounds of Formula I, and one of more compounds of Formula X
or Xa.
50. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula II and one
of more compounds of Formula III.
51. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula II and one
of more compounds of Formula X or Xa.
52. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VI, one or
more compounds of Formula II, and one of more compounds of Formula
III.
53. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VII, one or
more compounds of Formula II, and one of more compounds of Formula
III.
54. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VI, one or
more compounds of Formula II, and one of more compounds of Formula
X or Xa.
55. The pharmaceutical composition of claim 39, wherein the
composition comprises one or more compounds of Formula VII, one or
more compounds of Formula II, and one of more compounds of Formula
X or Xa.
Description
FIELD OF THE INVENTION
[0002] This invention is in the field of inhibitors of SecA, and
methods of making and using thereof.
BACKGROUND OF THE INVENTION
[0003] Due to the widespread emergence of drug-resistance, diseases
caused by bacterial pathogens have become a major public health
concern in recent years. There is an urgent need for the
development of new antimicrobials, especially those that have a new
target, in order to overcome drug resistance.
[0004] Bacteria generally develop drug resistance in three ways:
production of metabolizing enzymes for the degradation of the
drugs, modification of their targets to render the drugs
ineffective, and expression of high levels of efflux proteins that
"pump" the drug out of cells resulting in the lowering of drug
concentration inside. Therefore, the most promising approaches to
finding new antimicrobials include (1) searching for new targets,
(2) inhibiting or overcoming efflux, and (3) inhibiting metabolic
enzymes.
[0005] SecA, an indispensable ATPase of the protein translocation
machinery is present in all bacteria. SecA is responsible for the
secretion of many vital proteins, important toxins and other
virulence factors, and is essential for bacterial survival. SecA
has no counterpart in mammalian cells, thus providing an ideal
target for developing antimicrobial agents. SecA functions as a
membrane protein, forming a transmembrane channel and thus provides
the possibility for antimicrobial agents to reach this target
without entering into the cells. In such a case, the drug efflux
pump would have less negative effects on the inhibitor's ability to
exert antimicrobial activity. In addition, because SecA is present
in all bacteria, this is a target for the development of
broad-spectrum antimicrobials.
[0006] Inhibitors of SecA can be potential antimicrobial agents.
However, inhibitor development for SecA had not been an active area
of research until recently, presumably due to the difficulty in
working with this membrane protein and the active translocation
complex. To date, inorganic azide was the only known SecA inhibitor
with an IC.sub.50 at the mM range. However, azide is also an
inhibitor of many other enzymes such as cytochrome c oxidase,
superoxide dismutase, alcohol dehydrogenase, and ceruloplasmin.
Additional SecA inhibitors with potencies in the high .mu.M to low
mM range have been reported.
[0007] There exists a need for new SecA inhibitors which have
activity in the low or high nanomolar to low micromolar range.
[0008] Therefore, it is an object of the invention to provide SecA
inhibitors which have activity in the low or high nanomolar to low
micromolar range and methods of making and using thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a bar graph showing the inhibition of ATPase in E.
coli NR68 for Rose Bengal and selected Rose Bengal analogs.
[0010] FIG. 2 is a bar graph showing the bactericidal effects of
Rose Bengal and selected Rose Bengal analogs against B. subtilis
168. The compounds were tested at concentrations ranging from 0
.mu.M (labeled `a`); 10 .mu.M (labeled `b`); 20 .mu.M (labeled
`c`); and 30 .mu.M (labeled `d`).
[0011] FIGS. 3A-3D are line graphs showing the inhibition kinetics
of Rose Bengal in SecA translocation ATPase and channel activity.
FIG. 3A shows non-competitive inhibition of EcSecA translocation
ATPase by Rose Bengal. FIGS. 3B-3D shows non-competitive inhibition
of channel activity in the oocytes with EcSecA-liposomes (FIG. 3B),
PaSecA-liposomes (FIG. 3C), and SaSecA1-liposomes (FIG. 3D).
[0012] FIG. 4 shows the structures of selected Rose Bengal
analogs.
[0013] FIG. 5 shows the bactericidal effects of SCA-50 against S.
aureus for 1 hour at 37.degree. C. SCA-50 was tested at
concentrations ranging from 0 .mu.g/ml (labeled `a`); 3 .mu.g/ml
(labeled `b`); 6 .mu.g/ml (labeled `c`); 9 .mu.g/ml (labeled `d`);
and 12 .mu.g/ml (labeled `e`).
[0014] FIG. 6 shows the inhibition of Rose Bengal analogs on the
secretion of S. aureus toxins over time.
[0015] FIG. 7 shows the structure of selected Rose Bengal
analogs.
[0016] FIG. 8-1 through 8-120 is a table showing compounds within
the genus described herein that were synthesized or will be
synthesized. Some of the compounds were evaluated in vitro for
inhibition activity and/or toxicity.
SUMMARY OF THE INVENTION
[0017] Compounds having Formula I-X, and methods of making and
using are described herein.
##STR00001##
wherein [0018] A and B are independently S, SO.sub.2, SO, O,
NR.sub.6, or CR.sub.7R.sub.8; [0019] W and Z are independently N or
CR.sub.9; [0020] X and Y are independently S, O, or
CR.sub.10R.sub.11; and R.sub.1-R.sub.11 are independently absent or
selected from hydrogen, substituted or unsubstituted, linear,
branched, hetero, or cyclic alkyl, alkenyl, or alkynyl; substituted
or unsubstituted aryl or heteroaryl, halogen, substituted or
unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid
(--COOH), carboxylate (--COO.sup.-), primary amide (e.g.,
--CONH.sub.2), secondary amide (e.g., --CONHR.sub.12), tertiary
amide (e.g., --CONR.sub.12R.sub.12), secondary carbamate (e.g.,
--OCONHR.sub.12; --NHCOOR.sub.12), tertiary carbamate (e.g.,
--OCONR.sub.12R.sub.12; --NR.sub.12COOR.sub.12), urea (e.g.,
NHCONHR.sub.12; --NR.sub.12CONHR.sub.12; --NHCONR.sub.12R.sub.12,
--NR.sub.12CONR.sub.12R.sub.12), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.12OH, --CR.sub.12R.sub.12OH), ester (e.g.,
--COOR.sub.12), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.12), tertiary amine (e.g.,
--NR.sub.12R.sub.12), thioether (e.g., --SR.sub.12), sulfinyl group
(e.g., --SOR.sub.12), and sulfonyl group (e.g., --SOOR.sub.12),
wherein R.sub.12 is defined the same as R.sub.1-R.sub.11.
[0021] In some embodiments, A and B are S.
[0022] In some embodiments, A and B are S and W and Z are N.
[0023] In some embodiments, A and B are S, W and Z are N, and X and
Y are NR, wherein R is hydrogen or lower alkyl.
[0024] In some embodiments, A and B are S, W and Z are N, X and Y
are NR, wherein R is hydrogen or lower alkyl, and R.sub.1 and
R.sub.3 are C.ident.N.
[0025] In some embodiments, A and B are S, W and Z are N, X and Y
are NR, wherein R is hydrogen or lower alkyl, R.sub.1 and R.sub.3
are C.ident.N, and R.sub.2 and R.sub.4 are aryl, such as
substituted or unsubstituted phenyl or naphthyl. In some
embodiments, the phenyl ring is substituted with a lower alkyl,
such as methyl, ethyl, n-propyl, or isopropyl, at the ortho, meta,
or para position. In other embodiments, the phenyl ring is
substituted with a lower alkoxy, such as methoxy, at the ortho,
meta, or para position. In still other embodiments, the phenyl ring
is substituted with a halogen, such as chloro, bromo, or iodo at
the ortho, meta, or para position. In still other embodiments, the
phenyl ring is substituted with an aryl group, such as a
substituted or unsubstituted phenyl.
##STR00002##
wherein
[0026] X is S, SO, SO.sub.2, NHR.sub.4, O, or CR.sub.5R.sub.6;
[0027] Y is N or CR.sub.7;
[0028] Z is S, O, NR.sub.8, or CR.sub.9R.sub.10; and
[0029] R.sub.1-R.sub.10 is independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.11), tertiary amide (e.g.,
--CONR.sub.11R.sub.11), secondary carbamate (e.g., --OCONHR.sub.11;
--NHCOOR.sub.11), tertiary carbamate (e.g., --OCONR.sub.11R.sub.11;
--NR.sub.11COOR.sub.11), urea (e.g., NHCONHR.sub.11;
--NR.sub.10CONHR.sub.11; --NHCONR.sub.11R.sub.11,
--NR.sub.11CONR.sub.11R.sub.11), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.11OH, --CR.sub.11R.sub.11OH), ester (e.g.,
--COOR.sub.11), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.11), tertiary amine (e.g.,
--NR.sub.11R.sub.11), thioether (e.g., --SR.sub.11), sulfinyl group
(e.g., --SOR.sub.11), and sulfonyl group (e.g., --SOOR.sub.11),
wherein R.sub.11 is defined the same as R.sub.1-R.sub.10.
[0030] In some embodiments, X is S.
[0031] In some embodiments, X is S and Y is N.
[0032] In some embodiments, X is S, Y is N, and Z is NR, wherein R
is hydrogen or lower alkyl.
[0033] In some embodiments, X is S, Y is N, Z is NR, wherein R is
hydrogen or lower alkyl, and R.sub.3 is substituted or
unsubstituted aryl, such as phenyl. In some embodiments, R.sub.3 is
unsubstituted phenyl. In other embodiments, R.sub.3 is phenyl
substituted with amino or azide at the ortho, meta, or para
position. In still other embodiments, R.sub.3 is phenyl,
substituted at the para position by
##STR00003##
wherein R.sub.12 is as defined above. In some embodiments, R.sub.12
is amino.
[0034] In some embodiments, X is S, Y is N, Z is NR, wherein R is
hydrogen or lower alkyl, and R.sub.3 is substituted or
unsubstituted aryl as described above, and R.sub.2 is substituted
or unsubstituted aryl, such as phenyl or naphthyl. In some
embodiments R.sub.2 is phenyl substituted with lower alkyl, such as
methyl, ethyl, n-propyl, or isopropyl at the ortho, meta, or para
position. In other embodiments, R.sub.2 is phenyl substituted with
a halogen, such as chloro, bromo, or iodo, at the ortho, meta, or
para position. In still other embodiments, the phenyl ring is
substituted with an aryl group, such as a substituted or
unsubstituted phenyl.
[0035] In some embodiments, X is S, Y is N, Z is NR, wherein R is
hydrogen or lower alkyl, and R.sub.3 is substituted or
unsubstituted aryl as described above, R.sub.2 is substituted or
unsubstituted aryl as described above, and R.sub.1 is
C.ident.N.
##STR00004##
wherein
[0036] X and Y are independently N or C;
[0037] D and G are independently NR.sub.7, CR.sub.5R.sub.9, O, or
S;
[0038] A, B, E, and F are independently N or CR.sub.10;
[0039] L and M are independently S, SO, SO.sub.2, O, NR.sub.11, or
CR.sub.12R.sub.13
[0040] J is O, S, SO, SO.sub.2, NR.sub.14, or CR.sub.15R.sub.16;
and
[0041] R.sub.1-R.sub.16 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.17), tertiary amide (e.g.,
--CONR.sub.17R.sub.17), secondary carbamate (e.g., --OCONHR.sub.17;
--NHCOOR.sub.17), tertiary carbamate (e.g., --OCONR.sub.17R.sub.17;
--NR.sub.14COOR.sub.17), urea (e.g., NHCONHR.sub.17;
--NR.sub.14CONHR.sub.17; --NHCONR.sub.17R.sub.17,
--NR.sub.17CONR.sub.17R.sub.17), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.17OH, --CR.sub.17R.sub.17OH), ester (e.g.,
--COOR.sub.17), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.17), tertiary amine (e.g.,
--NR.sub.17R.sub.17), thioether (e.g., --SR.sub.17), sulfinyl group
(e.g., --SOR.sub.17), and sulfonyl group (e.g., --SOOR.sub.17),
wherein R.sub.17 is defined the same as R.sub.1-R.sub.16.
[0042] In some embodiments, J is S.
[0043] In some embodiments, J is S and X and Y are N.
[0044] In some embodiments, J is S, X and Y are N, and L and M are
S.
[0045] In some embodiments, J is S, X and Y are N, L and M are S,
and D and G are NR, where R is hydrogen or lower alkyl.
[0046] In some embodiments, J is S, X and Y are N, L and M are S, D
and G are NR, where R is hydrogen or lower alkyl, and A, B, E, and
F are N.
[0047] In some embodiments, J is S, X and Y are N, L and M are S, D
and G are NR, where R is hydrogen or lower alkyl, A, B, E, and F
are N, and R.sub.1 is lower alkyl, such as methyl.
[0048] In some embodiments, J is S, X and Y are N, L and M are S, D
and G are NR, where R is hydrogen or lower alkyl, A, B, E, and F
are N, and R.sub.1 is lower alkyl, such as methyl.
[0049] In some embodiments, J is S, X and Y are N, L and M are S, D
and G are NR, where R is hydrogen or lower alkyl, A, B, E, and F
are N, R.sub.1 is lower alkyl, such as methyl, and R.sub.5 and
R.sub.6 are substituted or unsubstituted aryl, such as phenyl. In
some embodiments, R.sub.5 and R.sub.6 are phenyl, substituted with
chloro or trifluoromethyl at the two meta positions.
##STR00005##
[0050] wherein
[0051] X is O, S, NR.sub.10, or CR.sub.11R.sub.12;
[0052] R.sub.1-R.sub.12 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.13), tertiary amide (e.g.,
--CONR.sub.13R.sub.13), secondary carbamate (e.g., --OCONHR.sub.13;
--NHCOOR.sub.13), tertiary carbamate (e.g., --OCONR.sub.13R.sub.13;
--NR.sub.14COOR.sub.13), urea (e.g., NHCONHR.sub.13;
--NR.sub.14CONHR.sub.13; --NHCONR.sub.13R.sub.13,
--NR.sub.17CONR.sub.13R.sub.13), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.13OH, --CR.sub.13R.sub.13OH), ester (e.g.,
--COOR.sub.13), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.13), tertiary amine (e.g.,
--NR.sub.13R.sub.13), thioether (e.g., --SR.sub.13), sulfinyl group
(e.g., --SOR.sub.13), and sulfonyl group (e.g., --SOOR.sub.13),
wherein R.sub.13 is defined the same as R.sub.1-R.sub.12.
[0053] The dotted lines represent optional double bonds.
[0054] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond.
[0055] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl, such as
phenyl. In some embodiments, R.sub.9 is phenyl substituted with a
carboxylic acid group at the meta, ortho or para position.
[0056] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, and R.sub.3 is hydroxy.
[0057] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, and R.sub.3 is hydroxy.
[0058] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, R.sub.3 is hydroxy, and R.sub.2 and/or R.sub.4 are
halogen, such as chloro, bromo, or iodo.
[0059] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, R.sub.3 is hydroxy, R.sub.2 and/or R.sub.4 are
halogen, such as chloro, bromo, or iodo, and R.sub.1 is
hydrogen.
[0060] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, R.sub.3 is hydroxy, R.sub.2 and/or R.sub.4 are
halogen, such as chloro, bromo, or iodo, R.sub.1 is hydrogen, and
R.sub.5 is halogen, such as chloro, bromo, or iodo.
##STR00006##
[0061] wherein
[0062] X and Y are independently O, S, NR.sub.13, or
CR.sub.14R.sub.15; and
[0063] R.sub.1-R.sub.15 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.16), tertiary amide (e.g.,
--CONR.sub.16R.sub.16), secondary carbamate (e.g., --OCONHR.sub.16;
--NHCOOR.sub.16), tertiary carbamate (e.g., --OCONR.sub.16R.sub.16;
--NR.sub.16COOR.sub.16), urea (e.g., NHCONHR.sub.16;
--NR.sub.16CONHR.sub.16; --NHCONR.sub.16R.sub.16,
--NR.sub.16CONR.sub.16R.sub.16), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.16OH, --CR.sub.16R.sub.16OH), ester (e.g.,
--COOR.sub.16), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.16), tertiary amine (e.g.,
--NR.sub.16R.sub.16), thioether (e.g., --SR.sub.16), sulfinyl group
(e.g., --SOR.sub.16), and sulfonyl group (e.g., --SOOR.sub.16),
wherein R.sub.16 is defined the same as R.sub.1-R.sub.15. In some
embodiments, X is O.
[0064] In some embodiments, X is O and Y is O.
[0065] In some embodiments, X is O, Y is O, and R.sub.2 and/or
R.sub.4 are halogen, such as chloro, bromo, and/or iodo.
[0066] In some embodiments, X is O, Y is O, R.sub.2 and/or R.sub.4
are halogen, such as chloro, bromo, and/or iodo, and R.sub.3 is
hydroxy.
[0067] In some embodiments, X is O, Y is O, R.sub.2 and/or R.sub.4
are halogen, such as chloro, bromo, and/or iodo, R.sub.3 is
hydroxy, and R.sub.9-R.sub.12 are hydrogen.
##STR00007##
[0068] wherein
[0069] X is O, S, SO, SO.sub.2, NR.sub.11, or CR.sub.12R.sub.13;
and
[0070] R.sub.1-R.sub.13 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.14), tertiary amide (e.g.,
--CONR.sub.14R.sub.14), secondary carbamate (e.g., --OCONHR.sub.14;
--NHCOOR.sub.14), tertiary carbamate (e.g., --OCONR.sub.14R.sub.14;
--NR.sub.14COOR.sub.14), urea (e.g., NHCONHR.sub.14;
--NR.sub.14CONHR.sub.14; --NHCONR.sub.14R.sub.14,
--NR.sub.14CONR.sub.14R.sub.14), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.14OH, --CR.sub.14R.sub.14OH), ester (e.g.,
--COOR.sub.14), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.14), tertiary amine (e.g.,
--NR.sub.14R.sub.14), thioether (e.g., --SR.sub.14), sulfinyl group
(e.g., --SOR.sub.14), and sulfonyl group (e.g., --SOOR.sub.14),
wherein R.sub.14 is defined the same as R.sub.1-R.sub.13.
[0071] In some embodiments, X is O.
[0072] In some embodiments, X is O and R.sub.1 is lower alkyl, such
as methyl, ethyl, n-propyl, or isopropyl.
[0073] In some embodiments, X is O, R.sub.1 is lower alkyl, such as
methyl, ethyl, n-propyl, or isopropyl, and one or more of R.sub.4,
R.sub.6, R.sub.7, and R.sub.11 are halogen, such as chloro, bromo,
iodo, or combinations thereof.
[0074] In some embodiments, X is O, R.sub.1 is lower alkyl, such as
methyl, ethyl, n-propyl, or isopropyl, one or more of R.sub.4,
R.sub.6, R.sub.7, and R.sub.11 are halogen, such as chloro, bromo,
iodo, or combinations thereof, and one or more of R.sub.5 and
R.sub.8 are hydroxy.
[0075] In some embodiments, X is O, R.sub.1 is substituted or
unsubstituted aryl, such as phenyl, R.sub.2, R.sub.5, and R.sub.8
are hydroxy and R.sub.3-R.sub.10 are hydrogen or as defined in the
various embodiments above.
[0076] In some embodiments, R.sub.1 is substituted or unsubstituted
cycloalkyl, such as cyclopentyl or cyclohexyl, R.sub.5 and R.sub.8
are hydroxy or lower alkoxy, such methoxy or ethoxy, and one or
more of R.sub.2-R.sub.4, R.sub.6, R.sub.7, and R.sub.8-R.sub.10 are
hydrogen, halogen (chloro, bromo, iodo), hydroxy, or combinations
thereof.
[0077] In some embodiments, R.sub.1 is substituted or unsubstituted
cycloalkyl, such as cyclopentyl or cyclohexyl or alkyl, such as
methyl, ethyl, n-propyl, isopropyl, butyl (n-, sec-, iso-, t-),
pentyl, hexyl, or heptyl, R.sub.5 and R.sub.8 are hydroxy, lower
alkoxy, such methoxy or ethoxy, or primary, secondary, or tertiary
amino, one or more of R.sub.4, R.sub.6, R.sub.7, and R.sub.9 are
halogen, such as chloro, bromo, iodo, or combinations thereof, and
one or more of R.sub.2, R.sub.3, R.sub.4, R.sub.6, R.sub.7,
R.sub.9, and R.sub.10 are hydrogen. In some embodiments, R.sub.1 is
cyclopentyl substituted with a carboxylic acid group at the 2
position.
[0078] In some embodiments, R.sub.1 and R.sub.2 together are .dbd.O
or .dbd.CR.sub.12R.sub.13, X is O, and R.sub.3-R.sub.10 are defined
in the various embodiments above. In some embodiments, R.sub.1 is a
substituted or unsubstituted cycloalkyl, such as cyclopentyl or
cyclohexyl, and R.sub.2 and the valence on C1 of the cycloalkyl
ring is a double bond, X is O, and R.sub.3-R.sub.10 are as defined
in the various embodiments above.
##STR00008##
[0079] wherein X is O, S, SO, SO.sub.2, NR.sub.9,
CR.sub.10R.sub.11; and R.sub.1-R.sub.11 are independently absent or
selected from hydrogen, substituted or unsubstituted, linear,
branched, hetero, or cyclic alkyl, alkenyl, or alkynyl; substituted
or unsubstituted aryl or heteroaryl; halogen, substituted or
unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid
(--COOH), carboxylate (--COO.sup.-), primary amide (e.g.,
--CONH.sub.2), secondary amide (e.g., --CONHR.sub.12), tertiary
amide (e.g., --CONR.sub.12R.sub.12), secondary carbamate (e.g.,
--OCONHR.sub.12; --NHCOOR.sub.12), tertiary carbamate (e.g.,
--OCONR.sub.12R.sub.12; --NR.sub.14COOR.sub.12), urea (e.g.,
NHCONHR.sub.12; --NR.sub.12CONHR.sub.12; --NHCONR.sub.12R.sub.12,
--NR.sub.14CONR.sub.12R.sub.12), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.12OH, --CR.sub.12R.sub.12OH), ester (e.g.,
--COOR.sub.12), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.12), tertiary amine (e.g.,
--NR.sub.12R.sub.12), thioether (e.g., --SR.sub.12), sulfinyl group
(e.g., --SOR.sub.12), and sulfonyl group (e.g., --SOOR.sub.12),
wherein R.sub.12 is defined the same as R.sub.1-R.sub.11, wherein
the compound of formula VII is not Rose Bengal.
[0080] In some embodiments, X.dbd.O, R.sub.1 is substituted or
unsubstituted cycloalkyl, such as cyclopentyl or cyclohexyl, and
one or more of R.sub.2-R.sub.7 are hydrogen, hydroxy, halogen
(chloro, bromo, iodo), or combinations thereof.
[0081] In some embodiments, R.sub.1 is substituted or unsubstituted
aryl, such as phenyl. In some embodiments, R.sub.1 is
2,3,4,5-tetrachloro-2-benzoic acid.
##STR00009##
wherein Z is O, S, SO, SO.sub.2, NR.sub.6, or CR.sub.7R.sub.8; X
and Y are independently N, NR.sub.9, or CR.sub.10R.sub.11;
[0082] R.sub.1-R.sub.11 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.12), tertiary amide (e.g.,
--CONR.sub.12R.sub.12), secondary carbamate (e.g., --OCONHR.sub.12;
--NHCOOR.sub.12), tertiary carbamate (e.g., --OCONR.sub.12R.sub.12;
--NR.sub.14COOR.sub.12), urea (e.g., NHCONHR.sub.12;
--NR.sub.12CONHR.sub.12; --NHCONR.sub.12R.sub.12,
--NR.sub.14CONR.sub.12R.sub.12), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.12OH, --CR.sub.12R.sub.12OH), ester (e.g.,
--COOR.sub.12), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.12), tertiary amine (e.g.,
--NR.sub.12R.sub.12), thioether (e.g., --SR.sub.12), sulfinyl group
(e.g., --SOR.sub.12), and sulfonyl group (e.g., --SOOR.sub.12),
wherein R.sub.12 is defined the same as R.sub.1-R.sub.11; and the
dotted lines represent optional double bonds.
[0083] In some embodiments, Z is S.
[0084] In some embodiments, Z is S, X is N, and Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl.
[0085] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, and
R.sub.1 is C.ident.N.
[0086] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, and R.sub.2 and R.sub.5 are aryl, such as phenyl.
[0087] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 3 or 4
position and the phenyl ring at the 3 or 4 position is optionally
substituted with OH at any position or --NH--COOalkyl, such as
methyl, ethyl, propyl, butyl (e.g., t-butyl) at any position.
[0088] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 3 or 4
position and the phenyl ring at the 3 or 4 position and R.sub.5 is
phenyl substituted with --COOH or B(OH).sub.2. In other
embodiments, R.sub.5 is pyridinyl.
[0089] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 4 position,
and R.sub.5 is phenyl substituted at the 4 position with
##STR00010##
[0090] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 4 position,
and R.sub.5 is phenyl substituted at the 4 position with
##STR00011##
wherein R.sub.13 is --(CH.sub.2).sub.n--OCOalkyl, where alkyl is a
lower alkyl, --(CH.sub.2).sub.n--COOH, --(CH.sub.2).sub.n--OH,
wherein n is at least 1, such as 1, 2, 3, 4, 5, or 6.
[0091] In still other embodiments, In some embodiments, Z is S, X
is N, Y is NR, where R is hydrogen or lower alkyl, such as methyl,
ethyl, or propyl, R.sub.1 is C.ident.N, R.sub.2 is aryl, such as
phenyl, wherein R.sub.2 is phenyl substituted with a phenyl ring at
the 3 or 4 position, and R.sub.5 is --(CH.sub.2).sub.n--OCOalkyl,
where alkyl is a lower alkyl, --(CH.sub.2).sub.n--COOH,
--(CH.sub.2).sub.n--OH, wherein n is at least 1, such as 1, 2, 3,
4, 5, or 6.
[0092] In still other embodiments, In some embodiments, Z is S, X
is N, Y is NR, where R is hydrogen or lower alkyl, such as methyl,
ethyl, or propyl, R.sub.1 is C.ident.N, R.sub.2 is aryl, such as
phenyl, substituted with trifluoromethyl at the 4 position or
wherein R.sub.2 is phenyl substituted with a phenyl ring at the 3
or 4 position which is optionally substituted with trifluoromethyl,
and R.sub.5 is --(CH.sub.2).sub.n--OCOalkyl, where alkyl is a lower
alkyl, --(CH.sub.2).sub.n--COOH, --(CH.sub.2).sub.n--OH, wherein n
is at least 1, such as 1, 2, 3, 4, 5, or 6.
[0093] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 3 or 4
position and R.sub.5 is phenyl substituted with --COOalkyl, where
alkyl is lower alkyl, at the 4 position.
[0094] In still other embodiments, Z is S, R.sub.3-R.sub.5 are
hydrogen, and the remaining variables are defined as in the
embodiments above.
[0095] In still other embodiments, Z is S, the bond between the
ring and Z is a double bond, the bond between N and the carbon
bound to Z is a single bond, and the remaining variables are
defined as in the embodiments above.
[0096] In still other embodiments, Z is O, and the remaining
variables are defined as in the embodiments above.
##STR00012##
wherein Z is O, S, SO, SO.sub.2, NR.sub.7, or CR.sub.5R.sub.9; X
and Y are independently N, NR.sub.10, or CR.sub.11R.sub.12;
[0097] R.sub.1-R.sub.12 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.13), tertiary amide (e.g.,
--CONR.sub.13R.sub.13), secondary carbamate (e.g., --OCONHR.sub.13;
--NHCOOR.sub.13), tertiary carbamate (e.g., --OCONR.sub.13R.sub.13;
--NR.sub.13COOR.sub.13), urea (e.g., NHCONHR.sub.13;
--NR.sub.13CONHR.sub.13; --NHCONR.sub.13R.sub.13,
--NR.sub.13CONR.sub.13R.sub.13), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.13OH, --CR.sub.13R.sub.13OH), ester (e.g.,
--COOR.sub.13), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.13), tertiary amine (e.g.,
--NR.sub.13R.sub.13), thioether (e.g., --SR.sub.13), sulfinyl group
(e.g., --SOR.sub.13), and sulfonyl group (e.g., --SOOR.sub.13),
wherein R.sub.13 is defined the same as R.sub.1-R.sub.12.
[0098] In some embodiments, Z is O or S, X is N, Y is NH, R.sub.2
is CN or COOalkyl, R.sub.1 is --NH--OH, NH(CH.sub.2).sub.nOH, where
n is 1, 2, 3, 4, 5, or 6, halogen (Cl, Br, or I), alkoxy (e.g.,
methoxy), --NHR, where R is alkyl, or oligo- or polyethylglycol, or
--NH--NH.sub.2, and the remaining variables are defined as in the
embodiments above.
[0099] In still other embodiments, the compound has the formula
##STR00013##
wherein the variable positions are as defined above for Formula
IX.
##STR00014##
[0100] wherein
[0101] Z and W are O, S, SO, SO.sub.2, NR.sub.5, or
CR.sub.6R.sub.7;
[0102] X and Y are independently N, NR.sub.8, or
CR.sub.9R.sub.10;
[0103] Cy is substituted or unsubstituted aryl, heteroaryl,
cycloalkyl, or heterocycloalkyl group; and
[0104] R.sub.1-R.sub.10 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.11), tertiary amide (e.g.,
--CONR.sub.11R.sub.11), secondary carbamate (e.g., --OCONHR.sub.11;
--NHCOOR.sub.11), tertiary carbamate (e.g., --OCONR.sub.11R.sub.11;
--NR.sub.14COOR.sub.11), urea (e.g., NHCONHR.sub.11;
[0105] --NR.sub.11CONHR.sub.11; --NHCONR.sub.11R.sub.11,
--NR.sub.14CONR.sub.11R.sub.11), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.11OH, --CR.sub.11R.sub.11OH), ester (e.g.,
--COOR.sub.11), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.11), tertiary amine (e.g.,
--NR.sub.11R.sub.11), thioether (e.g., --SR.sub.11), sulfinyl group
(e.g., --SOR.sub.11), and sulfonyl group (e.g., --SOOR.sub.11),
wherein R.sub.11 is defined the same as R.sub.1-R.sub.10.
[0106] In some embodiments, Z and W are O or S, X and Y are N, Cy
is a triazole ring, substituted at the two position with a
substituted or unsubstituted aryl, such as phenyl (e.g.,
3,5-dimethylphenyl, 3,5-di(trifluoromethyl)), and R.sub.2 is
halogen.
[0107] In some embodiments, Z and W are O or S, X and Y are N, Cy
is a triazole or oxadiazole ring, substituted at the two position
with a substituted or unsubstituted aryl, such as phenyl (e.g.,
3,5-dimethylphenyl, 3,5-di(trifluoromethyl)), R.sub.2 is halogen,
and R.sub.1 is aryl, such as phenyl.
[0108] In some embodiments, Z and R.sub.1 and/or W are absent and
the remaining variables are as defined above.
[0109] In some embodiments, the compound has the formula below,
wherein the variables are as defined above for Formula X.
##STR00015##
[0110] The compounds can be combined with one or more
pharmaceutically acceptable excipients to prepare pharmaceutical
compositions. The compositions can be administered by any route of
administration, such as enteral, parenteral, topical, or
transmucosal. The compositions may be useful for treating or
preventing infections, such as microbial (bacteria, fungi, etc.)
infections.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0111] "Analog" and "Derivative", are used herein interchangeably,
and refer to a compound that possesses the same core as a parent
compound, but differs from the parent compound in bond order, in
the absence or presence of one or more atoms and/or groups of
atoms, and combinations thereof. The derivative can differ from the
parent compound, for example, in one or more substituents present
on the core, which may include one or more atoms, functional
groups, or substructures. The derivative can also differ from the
parent compound in the bond order between atoms within the core. In
general, a derivative can be imagined to be formed, at least
theoretically, from the parent compound via chemical and/or
physical processes. For example, derivatives of celastrol include
compounds possessing one or more substituents affixed to the
core.
[0112] "Co-administration", as used herein, includes simultaneous
and sequential administration. An appropriate time course for
sequential administration may be chosen by the physician, according
to such factors as the nature of a patient's illness, and the
patient's condition.
[0113] "Pharmaceutically acceptable", as used herein, refers to
those compounds, materials, compositions, and/or dosage forms which
are, within the scope of sound medical judgment, suitable for use
in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other
problems or complications commensurate with a reasonable
benefit/risk ratio.
[0114] "Prodrug", as used herein, refers to a pharmacological
substance (drug) that is administered to a subject in an inactive
(or significantly less active) form. Once administered, the prodrug
is metabolized in the body (in vivo) into a compound having the
desired pharmacological activity.
[0115] "Alkyl", as used herein, refers to the radical of saturated
or unsaturated aliphatic groups, including straight-chain alkyl,
heteroalkyl, alkenyl, or alkynyl groups, branched-chain alkyl,
alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or
cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl,
cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted
alkyl, alkenyl, or alkynyl groups. Unless otherwise indicated, a
straight chain or branched chain alkyl has 30 or fewer carbon atoms
in its backbone (e.g., C.sub.1-C.sub.30 for straight chain,
C.sub.3-C.sub.30 for branched chain), more preferably 20 or fewer
carbon atoms, more preferably 12 or fewer carbon atoms, and most
preferably 8 or fewer carbon atoms. Likewise, preferred cycloalkyls
have from 3-10 carbon atoms in their ring structure, and more
preferably have 5, 6 or 7 carbons in the ring structure. The ranges
provided above are inclusive of all values between the minimum
value and the maximum value.
[0116] The term "alkyl" includes "heteroalkyls", "unsubstituted
alkyls", and "substituted alkyls", the latter of which refers to
alkyl moieties having one or more substituents replacing a hydrogen
on one or more carbons of the hydrocarbon backbone. Such
substituents include, but are not limited to, halogen, hydroxyl,
carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),
thiocarbonyl (such as a thioester, a thioacetate, or a
thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a
phosphinate, amino, amido, amidine, imine, cyano, nitro, azido,
sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido,
sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic
moiety.
[0117] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Preferred alkyl
groups are lower alkyls.
[0118] The alkyl groups may also contain one or more heteroatoms
within the carbon backbone. Preferably the heteroatoms incorporated
into the carbon backbone are oxygen, nitrogen, sulfur, and
combinations thereof. In certain embodiments, the alkyl group
contains between one and four heteroatoms.
[0119] "Alkenyl" and "Alkynyl", as used herein, refer to
unsaturated aliphatic groups containing one or more double or
triple bonds analogous in length (e.g., C.sub.2-C.sub.30) and
possible substitution to the alkyl groups described above.
[0120] "Aryl", as used herein, refers to 5-, 6- and 7-membered
aromatic ring. The ring may be a carbocyclic, heterocyclic, fused
carbocyclic, fused heterocyclic, bicarbocyclic, or biheterocyclic
ring system, optionally substituted by halogens, alkyl-, alkenyl-,
and alkynyl-groups. Broadly defined, "Ar", as used herein, includes
5-, 6- and 7-membered single-ring aromatic groups that may include
from zero to four heteroatoms, for example, benzene, pyrrole,
furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "heteroaryl", "aryl heterocycles", or
"heteroaromatics". The aromatic ring can be substituted at one or
more ring positions with such substituents as described above, for
example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,
alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,
heterocyclyl, aromatic or heteroaromatic moieties, --CF.sub.3,
--CN, or the like. The term "Ar" also includes polycyclic ring
systems having two or more cyclic rings in which two or more
carbons are common to two adjoining rings (the rings are "fused
rings") wherein at least one of the rings is aromatic, e.g., the
other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocycles. Examples of heterocyclic
ring include, but are not limited to, benzimidazolyl, benzofuranyl,
benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl,
carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl,
furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,
indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,
isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl,
isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,
methylenedioxyphenyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,
phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,
4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,
pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,
pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl and xanthenyl.
[0121] "Alkylaryl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or hetero
aromatic group).
[0122] "Heterocycle" or "heterocyclic", as used herein, refers to a
cyclic radical attached via a ring carbon or nitrogen of a
monocyclic or bicyclic ring containing 3-10 ring atoms, and
preferably from 5-6 ring atoms, consisting of carbon and one to
four heteroatoms each selected from the group consisting of
non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H,
O, (C.sub.1-4) alkyl, phenyl or benzyl, and optionally containing
one or more double or triple bonds, and optionally substituted with
one or more substituents. The term "heterocycle" also encompasses
substituted and unsubstituted heteroaryl rings. Examples of
heterocyclic ring include, but are not limited to, benzimidazolyl,
benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,
benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,
benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl,
4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,
decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,
dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl,
imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl,
indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,
isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl,
isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,
methylenedioxyphenyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,
phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,
4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,
pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,
pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl and xanthenyl.
[0123] "Heteroaryl", as used herein, refers to a monocyclic
aromatic ring containing five or six ring atoms consisting of
carbon and 1, 2, 3, or 4 heteroatoms each selected from the group
consisting of non-peroxide oxygen, sulfur, and N(Y) where Y is
absent or is H, O, (C.sub.1-C.sub.5) alkyl, phenyl or benzyl.
Non-limiting examples of heteroaryl groups include furyl,
imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl,
isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl,
(or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl,
isoquinolyl (or its N-oxide), quinolyl (or its N-oxide) and the
like. The term "heteroaryl" can include radicals of an ortho-fused
bicyclic heterocycle of about eight to ten ring atoms derived
therefrom, particularly a benz-derivative or one derived by fusing
a propylene, trimethylene, or tetramethylene diradical thereto.
Examples of heteroaryl can be furyl, imidazolyl, triazolyl,
triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl,
pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide),
thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or
its N-oxide), quinolyl (or its N-oxide), and the like.
[0124] "Halogen", as used herein, refers to fluorine, chlorine,
bromine, or iodine.
[0125] The term "substituted" as used herein, refers to all
permissible substituents of the compounds described herein. In the
broadest sense, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, but are not limited to,
halogens, hydroxyl groups, or any other organic groupings
containing any number of carbon atoms, preferably 1-14 carbon
atoms, and optionally include one or more heteroatoms such as
oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic
structural formats. Representative substituents include alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, phenyl, substituted phenyl, aryl, substituted
aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy,
substituted alkoxy, phenoxy, substituted phenoxy, aroxy,
substituted aroxy, alkylthio, substituted alkylthio, phenylthio,
substituted phenylthio, arylthio, substituted arylthio, cyano,
isocyano, substituted isocyano, carbonyl, substituted carbonyl,
carboxyl, substituted carboxyl, amino, substituted amino, amido,
substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,
phosphoryl, substituted phosphoryl, phosphonyl, substituted
phosphonyl, polyaryl, substituted polyaryl, C.sub.3-C.sub.20
cyclic, substituted C.sub.3-C.sub.20 cyclic, heterocyclic,
substituted heterocyclic, aminoacid, peptide, and polypeptide
groups.
[0126] Heteroatoms such as nitrogen may have hydrogen substituents
and/or any permissible substituents of organic compounds described
herein which satisfy the valences of the heteroatoms. It is
understood that "substitution" or "substituted" includes the
implicit proviso that such substitution is in accordance with
permitted valence of the substituted atom and the substituent, and
that the substitution results in a stable compound, i.e. a compound
that does not spontaneously undergo transformation such as by
rearrangement, cyclization, elimination, etc.
II. Compounds
[0127] Compounds having Formula I-X, and methods of making and
using are described herein.
##STR00016##
wherein
[0128] A and B are independently S, SO.sub.2, SO, O, NR.sub.6, or
CR.sub.7R.sub.8;
[0129] W and Z are independently N or CR.sub.9;
[0130] X and Y are independently S, O, or CR.sub.10R.sub.11;
and
[0131] R.sub.1-R.sub.11 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.12), tertiary amide (e.g.,
--CONR.sub.12R.sub.12), secondary carbamate (e.g., --OCONHR.sub.12;
--NHCOOR.sub.12), tertiary carbamate (e.g., --OCONR.sub.12R.sub.12;
--NR.sub.12COOR.sub.12), urea (e.g., NHCONHR.sub.12;
--NR.sub.12CONHR.sub.12; --NHCONR.sub.12R.sub.12,
--NR.sub.12CONR.sub.12R.sub.12), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.12OH, --CR.sub.12R.sub.12OH), ester (e.g.,
--COOR.sub.12), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.12), tertiary amine (e.g.,
--NR.sub.12R.sub.12), thioether (e.g., --SR.sub.12), sulfinyl group
(e.g., --SOR.sub.12), and sulfonyl group (e.g., --SOOR.sub.12),
wherein R.sub.12 is defined the same as R.sub.1-R.sub.11.
[0132] In some embodiments, A and B are S.
[0133] In some embodiments, A and B are S and W and Z are N.
[0134] In some embodiments, A and B are S, W and Z are N, and X and
Y are NR, wherein R is hydrogen or lower alkyl.
[0135] In some embodiments, A and B are S, W and Z are N, X and Y
are NR, wherein R is hydrogen or lower alkyl, and R.sub.1 and
R.sub.3 are C.ident.N.
[0136] In some embodiments, A and B are S, W and Z are N, X and Y
are NR, wherein R is hydrogen or lower alkyl, R.sub.1 and R.sub.3
are C.ident.N, and R.sub.2 and R.sub.4 are aryl, such as
substituted or unsubstituted phenyl or naphthyl. In some
embodiments, the phenyl ring is substituted with a lower alkyl,
such as methyl, ethyl, n-propyl, or isopropyl, at the ortho, meta,
or para position. In other embodiments, the phenyl ring is
substituted with a lower alkoxy, such as methoxy, at the ortho,
meta, or para position. In still other embodiments, the phenyl ring
is substituted with a halogen, such as chloro, bromo, or iodo at
the ortho, meta, or para position. In still other embodiments, the
phenyl ring is substituted with an aryl group, such as a
substituted or unsubstituted phenyl.
##STR00017##
wherein
[0137] X is S, SO, SO.sub.2, NHR.sub.4, O, or CR.sub.5R.sub.6;
[0138] Y is N or CR.sub.7;
[0139] Z is S, O, NR.sub.8, or CR.sub.9R.sub.10; and
[0140] R.sub.1-R.sub.10 is independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.11), tertiary amide (e.g.,
--CONR.sub.11R.sub.11), secondary carbamate (e.g., --OCONHR.sub.11;
--NHCOOR.sub.11), tertiary carbamate (e.g., --OCONR.sub.11R.sub.11;
--NR.sub.11COOR.sub.11), urea (e.g., NHCONHR.sub.11;
--NR.sub.10CONHR.sub.11; --NHCONR.sub.11R.sub.11,
--NR.sub.11CONR.sub.11R.sub.11), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.11OH, --CR.sub.11R.sub.11OH), ester (e.g.,
--COOR.sub.11), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.11), tertiary amine (e.g.,
--NR.sub.11R.sub.11), thioether (e.g., --SR.sub.11), sulfinyl group
(e.g., --SOR.sub.11), and sulfonyl group (e.g., --SOOR.sub.11),
wherein R.sub.11 is defined the same as R.sub.1-R.sub.10.
[0141] In some embodiments, X is S.
[0142] In some embodiments, X is S and Y is N.
[0143] In some embodiments, X is S, Y is N, and Z is NR, wherein R
is hydrogen or lower alkyl.
[0144] In some embodiments, X is S, Y is N, Z is NR, wherein R is
hydrogen or lower alkyl, and R.sub.3 is substituted or
unsubstituted aryl, such as phenyl. In some embodiments, R.sub.3 is
unsubstituted phenyl. In other embodiments, R.sub.3 is phenyl
substituted with amino or azide at the ortho, meta, or para
position. In still other embodiments, R.sub.3 is phenyl,
substituted at the para position by
##STR00018##
wherein R.sub.12 is as defined above. In some embodiments, R.sub.12
is amino.
[0145] In some embodiments, X is S, Y is N, Z is NR, wherein R is
hydrogen or lower alkyl, and R.sub.3 is substituted or
unsubstituted aryl as described above, and R.sub.2 is substituted
or unsubstituted aryl, such as phenyl or naphthyl. In some
embodiments R.sub.2 is phenyl substituted with lower alkyl, such as
methyl, ethyl, n-propyl, or isopropyl at the ortho, meta, or para
position. In other embodiments, R.sub.2 is phenyl substituted with
a halogen, such as chloro, bromo, or iodo, at the ortho, meta, or
para position. In still other embodiments, the phenyl ring is
substituted with an aryl group, such as a substituted or
unsubstituted phenyl.
[0146] In some embodiments, X is S, Y is N, Z is NR, wherein R is
hydrogen or lower alkyl, and R.sub.3 is substituted or
unsubstituted aryl as described above, R.sub.2 is substituted or
unsubstituted aryl as described above, and R.sub.1 is
C.ident.N.
##STR00019##
wherein
[0147] X and Y are independently N or C;
[0148] D and G are independently NR.sub.7, CR.sub.8R.sub.9, O, or
S;
[0149] A, B, E, and F are independently N or CR.sub.10;
[0150] L and M are independently S, SO, SO.sub.2, O, NR.sub.11, or
CR.sub.12R.sub.13
[0151] J is O, S, SO, SO.sub.2, NR.sub.14, or CR.sub.15R.sub.16;
and
[0152] R.sub.1-R.sub.16 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, or cyclic
alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or
heteroaryl; halogen, substituted or unsubstituted alkoxy; hydroxy,
cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.17), tertiary amide (e.g.,
--CONR.sub.17R.sub.17), secondary carbamate (e.g., --OCONHR.sub.17;
--NHCOOR.sub.17), tertiary carbamate (e.g., --OCONR.sub.17R.sub.17;
--NR.sub.14COOR.sub.17), urea (e.g., NHCONHR.sub.17;
--NR.sub.14CONHR.sub.17; --NHCONR.sub.17R.sub.17,
--NR.sub.17CONR.sub.17R.sub.17), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.17OH, --CR.sub.17R.sub.17OH), ester (e.g.,
--COOR.sub.17), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.17), tertiary amine (e.g.,
--NR.sub.17R.sub.17), thioether (e.g., --SR.sub.17), sulfinyl group
(e.g., --SOR.sub.17), and sulfonyl group (e.g., --SOOR.sub.17),
wherein R.sub.17 is defined the same as R.sub.1-R.sub.16.
[0153] In some embodiments, J is S.
[0154] In some embodiments, J is S and X and Y are N.
[0155] In some embodiments, J is S, X and Y are N, and L and M are
S.
[0156] In some embodiments, J is S, X and Y are N, L and M are S,
and D and G are NR, where R is hydrogen or lower alkyl.
[0157] In some embodiments, J is S, X and Y are N, L and M are S, D
and G are NR, where R is hydrogen or lower alkyl, and A, B, E, and
F are N.
[0158] In some embodiments, J is S, X and Y are N, L and M are S, D
and G are NR, where R is hydrogen or lower alkyl, A, B, E, and F
are N, and R.sub.1 is lower alkyl, such as methyl.
[0159] In some embodiments, J is S, X and Y are N, L and M are S, D
and G are NR, where R is hydrogen or lower alkyl, A, B, E, and F
are N, and R.sub.1 is lower alkyl, such as methyl.
[0160] In some embodiments, J is S, X and Y are N, L and M are S, D
and G are NR, where R is hydrogen or lower alkyl, A, B, E, and F
are N, R.sub.1 is lower alkyl, such as methyl, and R.sub.5 and
R.sub.6 are substituted or unsubstituted aryl, such as phenyl. In
some embodiments, R.sub.5 and R.sub.6 are phenyl, substituted with
chloro or trifluoromethyl at the two meta positions.
##STR00020##
[0161] wherein
[0162] X is O, S, NR.sub.10, or CR.sub.11R.sub.12;
[0163] R.sub.1-R.sub.12 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.13), tertiary amide (e.g.,
--CONR.sub.13R.sub.13), secondary carbamate (e.g., --OCONHR.sub.13;
--NHCOOR.sub.13), tertiary carbamate (e.g., --OCONR.sub.13R.sub.13;
--NR.sub.14COOR.sub.13), urea (e.g., NHCONHR.sub.13;
--NR.sub.14CONHR.sub.13; --NHCONR.sub.13R.sub.13,
--NR.sub.17CONR.sub.13R.sub.13), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.13OH, --CR.sub.13R.sub.13OH), ester (e.g.,
--COOR.sub.13), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.13), tertiary amine (e.g.,
--NR.sub.13R.sub.13), thioether (e.g., --SR.sub.13), sulfinyl group
(e.g., --SOR.sub.13), and sulfonyl group (e.g., --SOOR.sub.13),
wherein R.sub.13 is defined the same as R.sub.1-R.sub.12.
[0164] The dotted lines represent optional double bonds.
[0165] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond.
[0166] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl, such as
phenyl. In some embodiments, R.sub.9 is phenyl substituted with a
carboxylic acid group at the meta, ortho or para position.
[0167] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, and R.sub.3 is hydroxy.
[0168] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, and R.sub.3 is hydroxy.
[0169] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, R.sub.3 is hydroxy, and R.sub.2 and/or R.sub.4 are
halogen, such as chloro, bromo, or iodo.
[0170] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, R.sub.3 is hydroxy, R.sub.2 and/or R.sub.4 are
halogen, such as chloro, bromo, or iodo, and R.sub.1 is
hydrogen.
[0171] In some embodiments, X is O or CR, wherein R is defined as
above for R.sub.1-R.sub.13 and wherein the bond between X and the
carbon containing R.sub.7 and R.sub.8 is a double bond and the bond
between the carbons containing R.sub.5 and R.sub.6 and R.sub.9 is a
double bond, R.sub.9 is substituted or unsubstituted aryl as
described above, R.sub.3 is hydroxy, R.sub.2 and/or R.sub.4 are
halogen, such as chloro, bromo, or iodo, R.sub.1 is hydrogen, and
R.sub.5 is halogen, such as chloro, bromo, or iodo.
##STR00021##
[0172] wherein
[0173] X and Y are independently O, S, NR.sub.13, or
CR.sub.14R.sub.15; and
[0174] R.sub.1-R.sub.15 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.16), tertiary amide (e.g.,
--CONR.sub.16R.sub.16), secondary carbamate (e.g., --OCONHR.sub.16;
--NHCOOR.sub.16), tertiary carbamate (e.g., --OCONR.sub.16R.sub.16;
--NR.sub.16COOR.sub.16), urea (e.g., NHCONHR.sub.16;
--NR.sub.16CONHR.sub.16; --NHCONR.sub.16R.sub.16,
--NR.sub.16CONR.sub.16R.sub.16), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.16OH, --CR.sub.16R.sub.16OH), ester (e.g.,
--COOR.sub.16), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.16), tertiary amine (e.g.,
--NR.sub.16R.sub.16), thioether (e.g., --SR.sub.16), sulfinyl group
(e.g., --SOR.sub.16), and sulfonyl group (e.g., --SOOR.sub.16),
wherein R.sub.16 is defined the same as R.sub.1-R.sub.15. In some
embodiments, X is O.
[0175] In some embodiments, X is O and Y is O.
[0176] In some embodiments, X is O, Y is O, and R.sub.2 and/or
R.sub.4 are halogen, such as chloro, bromo, and/or iodo.
[0177] In some embodiments, X is O, Y is O, R.sub.2 and/or R.sub.4
are halogen, such as chloro, bromo, and/or iodo, and R.sub.3 is
hydroxy.
[0178] In some embodiments, X is O, Y is O, R.sub.2 and/or R.sub.4
are halogen, such as chloro, bromo, and/or iodo, R.sub.3 is
hydroxy, and R.sub.9-R.sub.12 are hydrogen.
##STR00022##
[0179] wherein
[0180] X is O, S, SO, SO.sub.2, NR.sub.11, or CR.sub.12R.sub.13;
and
[0181] R.sub.1-R.sub.13 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.14), tertiary amide (e.g.,
--CONR.sub.14R.sub.14), secondary carbamate (e.g., --OCONHR.sub.14;
--NHCOOR.sub.14), tertiary carbamate (e.g., --OCONR.sub.14R.sub.14;
--NR.sub.14COOR.sub.14), urea (e.g., NHCONHR.sub.14;
--NR.sub.14CONHR.sub.14; --NHCONR.sub.14R.sub.14,
--NR.sub.14CONR.sub.14R.sub.14), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.14OH, --CR.sub.14R.sub.14OH), ester (e.g.,
--COOR.sub.14), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.14), tertiary amine (e.g.,
--NR.sub.14R.sub.14), thioether (e.g., --SR.sub.14), sulfinyl group
(e.g., --SOR.sub.14), and sulfonyl group (e.g., --SOOR.sub.14),
wherein R.sub.14 is defined the same as R.sub.1-R.sub.13.
[0182] In some embodiments, X is O.
[0183] In some embodiments, X is O and R.sub.1 is lower alkyl, such
as methyl, ethyl, n-propyl, or isopropyl.
[0184] In some embodiments, X is O, R.sub.1 is lower alkyl, such as
methyl, ethyl, n-propyl, or isopropyl, and one or more of R.sub.4,
R.sub.6, R.sub.7, and R.sub.11 are halogen, such as chloro, bromo,
iodo, or combinations thereof.
[0185] In some embodiments, X is O, R.sub.1 is lower alkyl, such as
methyl, ethyl, n-propyl, or isopropyl, one or more of R.sub.4,
R.sub.6, R.sub.7, and R.sub.11 are halogen, such as chloro, bromo,
iodo, or combinations thereof, and one or more of R.sub.5 and
R.sub.8 are hydroxy.
[0186] In some embodiments, X is O, R.sub.1 is substituted or
unsubstituted aryl, such as phenyl, R.sub.2, R.sub.5, and R.sub.8
are hydroxy and R.sub.3-R.sub.10 are hydrogen or as defined in the
various embodiments above.
[0187] In some embodiments, R.sub.1 is substituted or unsubstituted
cycloalkyl, such as cyclopentyl or cyclohexyl, R.sub.5 and R.sub.8
are hydroxy or lower alkoxy, such methoxy or ethoxy, and one or
more of R.sub.2-R.sub.4, R.sub.6, R.sub.7, and R.sub.8-R.sub.10 are
hydrogen, halogen (chloro, bromo, iodo), hydroxy, or combinations
thereof.
[0188] In some embodiments, R.sub.1 is substituted or unsubstituted
cycloalkyl, such as cyclopentyl or cyclohexyl or alkyl, such as
methyl, ethyl, n-propyl, isopropyl, butyl (n-, sec-, iso-, t-),
pentyl, hexyl, or heptyl, R.sub.5 and R.sub.8 are hydroxy, lower
alkoxy, such methoxy or ethoxy, or primary, secondary, or tertiary
amino, one or more of R.sub.4, R.sub.6, R.sub.7, and R.sub.9 are
halogen, such as chloro, bromo, iodo, or combinations thereof, and
one or more of R.sub.2, R.sub.3, R.sub.4, R.sub.6, R.sub.7,
R.sub.9, and R.sub.10 are hydrogen. In some embodiments, R.sub.1 is
cyclopentyl substituted with a carboxylic acid group at the 2
position.
[0189] In some embodiments, R.sub.1 and R.sub.2 together are .dbd.O
or .dbd.CR.sub.12R.sub.13, X is O, and R.sub.3-R.sub.10 are defined
in the various embodiments above. In some embodiments, R.sub.1 is a
substituted or unsubstituted cycloalkyl, such as cyclopentyl or
cyclohexyl, and R.sub.2 and the valence on C1 of the cycloalkyl
ring is a double bond, X is O, and R.sub.3-R.sub.10 are as defined
in the various embodiments above.
##STR00023##
[0190] wherein X is O, S, SO, SO.sub.2, NR.sub.9,
CR.sub.10R.sub.11; and
[0191] R.sub.1-R.sub.11 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.12), tertiary amide (e.g.,
--CONR.sub.12R.sub.12), secondary carbamate (e.g., --OCONHR.sub.12;
--NHCOOR.sub.12), tertiary carbamate (e.g., --OCONR.sub.12R.sub.12;
--NR.sub.14COOR.sub.12), urea (e.g., NHCONHR.sub.12;
--NR.sub.12CONHR.sub.12; --NHCONR.sub.12R.sub.12,
--NR.sub.14CONR.sub.12R.sub.12), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.12OH, --CR.sub.12R.sub.12OH), ester (e.g.,
--COOR.sub.12), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.12), tertiary amine (e.g.,
--NR.sub.12R.sub.12), thioether (e.g., --SR.sub.12), sulfinyl group
(e.g., --SOR.sub.12), and sulfonyl group (e.g., --SOOR.sub.12),
wherein R.sub.12 is defined the same as R.sub.1-R.sub.11;
[0192] wherein the compound of formula VII is not Rose Bengal.
[0193] In some embodiments, X.dbd.O, R.sub.1 is substituted or
unsubstituted cycloalkyl, such as cyclopentyl or cyclohexyl, and
one or more of R.sub.2-R.sub.7 are hydrogen, hydroxy, halogen
(chloro, bromo, iodo), or combinations thereof.
[0194] In some embodiments, R.sub.1 is substituted or unsubstituted
aryl, such as phenyl. In some embodiments, R.sub.1 is
2,3,4,5-tetrachloro-2-benzoic acid.
##STR00024##
wherein Z is O, S, SO, SO.sub.2, NR.sub.6, or CR.sub.7R.sub.8; X
and Y are independently N, NR.sub.9, or CR.sub.10R.sub.11;
[0195] R.sub.1-R.sub.11 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.12), tertiary amide (e.g.,
--CONR.sub.12R.sub.12), secondary carbamate (e.g., --OCONHR.sub.12;
--NHCOOR.sub.12), tertiary carbamate (e.g., --OCONR.sub.12R.sub.12;
--NR.sub.14COOR.sub.12), urea (e.g., NHCONHR.sub.12;
--NR.sub.12CONHR.sub.12; --NHCONR.sub.12R.sub.12,
--NR.sub.14CONR.sub.12R.sub.12), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.12OH, --CR.sub.12R.sub.12OH), ester (e.g.,
--COOR.sub.12), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.12), tertiary amine (e.g.,
--NR.sub.12R.sub.12), thioether (e.g., --SR.sub.12), sulfinyl group
(e.g., --SOR.sub.12), and sulfonyl group (e.g., --SOOR.sub.12),
wherein R.sub.12 is defined the same as R.sub.1-R.sub.11; and
[0196] the dotted lines represent optional double bonds.
[0197] In some embodiments, Z is S.
[0198] In some embodiments, Z is S, X is N, and Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl.
[0199] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, and
R.sub.1 is C.ident.N.
[0200] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, and R.sub.2 and R.sub.5 are aryl, such as phenyl.
[0201] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 3 or 4
position and the phenyl ring at the 3 or 4 position is optionally
substituted with OH at any position or --NH--COOalkyl, such as
methyl, ethyl, propyl, butyl (e.g., t-butyl) at any position.
[0202] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 3 or 4
position and the phenyl ring at the 3 or 4 position and R.sub.5 is
phenyl substituted with --COOH or B(OH).sub.2. In other
embodiments, R.sub.5 is pyridinyl.
[0203] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 4 position,
and R.sub.5 is phenyl substituted at the 4 position with
##STR00025##
[0204] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 4 position,
and R.sub.5 is phenyl substituted at the 4 position with
##STR00026##
wherein R.sub.13 is --(CH.sub.2).sub.n--OCOalkyl, where alkyl is a
lower alkyl, --(CH.sub.2).sub.n--COOH, --(CH.sub.2).sub.n--OH,
wherein n is at least 1, such as 1, 2, 3, 4, 5, or 6.
[0205] In still other embodiments, In some embodiments, Z is S, X
is N, Y is NR, where R is hydrogen or lower alkyl, such as methyl,
ethyl, or propyl, R.sub.1 is C.ident.N, R.sub.2 is aryl, such as
phenyl, wherein R.sub.2 is phenyl substituted with a phenyl ring at
the 3 or 4 position, and R.sub.5 is --(CH.sub.2).sub.n--OCOalkyl,
where alkyl is a lower alkyl, --(CH.sub.2).sub.n--COOH,
--(CH.sub.2).sub.n--OH, wherein n is at least 1, such as 1, 2, 3,
4, 5, or 6.
[0206] In still other embodiments, In some embodiments, Z is S, X
is N, Y is NR, where R is hydrogen or lower alkyl, such as methyl,
ethyl, or propyl, R.sub.1 is C.ident.N, R.sub.2 is aryl, such as
phenyl, substituted with trifluoromethyl at the 4 position or
wherein R.sub.2 is phenyl substituted with a phenyl ring at the 3
or 4 position which is optionally substituted with trifluoromethyl,
and R.sub.5 is --(CH.sub.2).sub.n--OCOalkyl, where alkyl is a lower
alkyl, --(CH.sub.2).sub.n--COOH, --(CH.sub.2).sub.n--OH, wherein n
is at least 1, such as 1, 2, 3, 4, 5, or 6.
[0207] In some embodiments, Z is S, X is N, Y is NR, where R is
hydrogen or lower alkyl, such as methyl, ethyl, or propyl, R.sub.1
is C.ident.N, R.sub.2 and R.sub.5 are aryl, such as phenyl, wherein
R.sub.2 is phenyl substituted with a phenyl ring at the 3 or 4
position and R.sub.5 is phenyl substituted with --COOalkyl, where
alkyl is lower alkyl, at the 4 position.
[0208] In still other embodiments, Z is S, R.sub.3-R.sub.5 are
hydrogen, and the remaining variables are defined as in the
embodiments above.
[0209] In still other embodiments, Z is S, the bond between the
ring and Z is a double bond, the bond between N and the carbon
bound to Z is a single bond, and the remaining variables are
defined as in the embodiments above.
[0210] In still other embodiments, Z is O, and the remaining
variables are defined as in the embodiments above.
##STR00027##
wherein Z is O, S, SO, SO.sub.2, NR.sub.7, or CR.sub.5R.sub.9; X
and Y are independently N, NR.sub.10, or CR.sub.11R.sub.12;
[0211] R.sub.1-R.sub.12 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.13), tertiary amide (e.g.,
--CONR.sub.13R.sub.13), secondary carbamate (e.g., --OCONHR.sub.13;
--NHCOOR.sub.13), tertiary carbamate (e.g., --OCONR.sub.13R.sub.13;
--NR.sub.13COOR.sub.13), urea (e.g., NHCONHR.sub.13;
--NR.sub.13CONHR.sub.13; --NHCONR.sub.13R.sub.13,
--NR.sub.13CONR.sub.13R.sub.13), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.13OH, --CR.sub.13R.sub.13OH), ester (e.g.,
--COOR.sub.13), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.13), tertiary amine (e.g.,
--NR.sub.13R.sub.13), thioether (e.g., --SR.sub.13), sulfinyl group
(e.g., --SOR.sub.13), and sulfonyl group (e.g., --SOOR.sub.13),
wherein R.sub.13 is defined the same as R.sub.1-R.sub.12.
[0212] In some embodiments, Z is O or S, X is N, Y is NH, R.sub.2
is CN or COOalkyl, R.sub.1 is --NH--OH, NH(CH.sub.2).sub.nOH, where
n is 1, 2, 3, 4, 5, or 6, halogen (Cl, Br, or I), alkoxy (e.g.,
methoxy), --NHR, where R is alkyl, or oligo- or polyethylglycol, or
--NH--NH.sub.2, and the remaining variables are defined as in the
embodiments above.
[0213] In still other embodiments, the compound has the formula
##STR00028##
wherein the variable positions are as defined above for Formula
IX.
##STR00029##
[0214] wherein
[0215] Z and W are O, S, SO, SO.sub.2, NR.sub.5, or
CR.sub.6R.sub.7;
[0216] X and Y are independently N, NR.sub.8, or
CR.sub.9R.sub.10;
[0217] Cy is substituted or unsubstituted aryl, heteroaryl,
cycloalkyl, or heterocycloalkyl group; and
[0218] R.sub.1-R.sub.10 are independently absent or selected from
hydrogen, substituted or unsubstituted, linear, branched, hetero,
or cyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted
aryl or heteroaryl; halogen, substituted or unsubstituted alkoxy;
hydroxy, cyano, formyl, acyl, carboxylic acid (--COOH), carboxylate
(--COO.sup.-), primary amide (e.g., --CONH.sub.2), secondary amide
(e.g., --CONHR.sub.11), tertiary amide (e.g.,
--CONR.sub.11R.sub.11), secondary carbamate (e.g., --OCONHR.sub.11;
--NHCOOR.sub.11), tertiary carbamate (e.g., --OCONR.sub.11R.sub.11;
--NR.sub.14COOR.sub.11), urea (e.g., NHCONHR.sub.11;
--NR.sub.11CONHR.sub.11; --NHCONR.sub.11R.sub.11,
--NR.sub.14CONR.sub.11R.sub.11), carbinol (e.g., --CH.sub.2OH;
--CHR.sub.11OH, --CR.sub.11R.sub.11OH), ester (e.g.,
--COOR.sub.11), thiol (--SH), primary amine (--NH.sub.2), secondary
amine (e.g., --NHR.sub.11), tertiary amine (e.g.,
--NR.sub.11R.sub.11), thioether (e.g., --SR.sub.11), sulfinyl group
(e.g., --SOR.sub.11), and sulfonyl group (e.g., --SOOR.sub.11),
wherein R.sub.11 is defined the same as R.sub.1-R.sub.10.
[0219] In some embodiments, Z and W are O or S, X and Y are N, Cy
is a triazole ring, substituted at the two position with a
substituted or unsubstituted aryl, such as phenyl (e.g.,
3,5-dimethylphenyl, 3,5-di(trifluoromethyl)), and R.sub.2 is
halogen.
[0220] In some embodiments, Z and W are O or S, X and Y are N, Cy
is a triazole or oxadiazole ring, substituted at the two position
with a substituted or unsubstituted aryl, such as phenyl (e.g.,
3,5-dimethylphenyl, 3,5-di(trifluoromethyl)), R.sub.2 is halogen,
and R.sub.1 is aryl, such as phenyl.
[0221] In some embodiments, Z and R.sub.1 and/or W are absent and
the remaining variables are as defined above.
[0222] In some embodiments, the compound has the formula below,
wherein the variables are as defined above for Formula X.
##STR00030##
[0223] In another embodiment, the compounds of formula I, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00031## ##STR00032##
[0224] In another embodiment, the compounds of formula II, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037##
##STR00038## ##STR00039## ##STR00040## ##STR00041##
[0225] In another embodiment, the compounds of formula III, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00042##
[0226] In another embodiment, the compounds of formula IV, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00043## ##STR00044##
[0227] In another embodiment, the compounds of formula V, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00045##
[0228] In another embodiment, the compounds of formula VI, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051## ##STR00052##
[0229] In another embodiment, the compounds of formula VII, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00053##
[0230] In another embodiment, the compounds of formula VIII, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##
##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063##
[0231] In another embodiment, the compounds of formula IX, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00064## ##STR00065## ##STR00066##
[0232] In another embodiment, the compounds of formula X, or a
pharmaceutically acceptable salt or a prodrug thereof, is a
compound selected from the group consisting of:
##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071##
##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076##
##STR00077## ##STR00078## ##STR00079## ##STR00080##
##STR00081##
[0233] The compounds described herein may have one or more chiral
centers, and thus exist as one or more stereoisomers. Such
stereoisomers can exist as a single enantiomer, a mixture of
enantiomers, a mixture of diastereomers, or a racemic mixture.
[0234] As used herein, the term "stereoisomers" refers to compounds
made up of the same atoms having the same bond order but having
different three-dimensional arrangements of atoms that are not
interchangeable. The three-dimensional structures are called
configurations. As used herein, the term "enantiomers" refers to
two stereoisomers that are non-superimposable mirror images of one
another. As used herein, the term "optical isomer" is equivalent to
the term "enantiomer". As used herein the term "diastereomer"
refers to two stereoisomers which are not mirror images but also
not superimposable. The terms "racemate", "racemic mixture" or
"racemic modification" refer to a mixture of equal parts of
enantiomers. The term "chiral center" refers to a carbon atom to
which four different groups are attached. Choice of the appropriate
chiral column, eluent, and conditions necessary to effect
separation of the pair of enantiomers is well known to one of
ordinary skill in the art using standard techniques (see e.g.
Jacques, J. et al., "Enantiomers, Racemates, and Resolutions", John
Wiley and Sons, Inc. 1981).
[0235] The compounds can also be a pharmaceutically acceptable salt
of any of the compounds described above. In some cases, it may be
desirable to prepare the salt of a compound described above due to
one or more of the salt's advantageous physical properties, such as
enhanced stability or a desirable solubility or dissolution
profile.
[0236] Generally, pharmaceutically acceptable salts can be prepared
by reaction of the free acid or base forms of a compound described
above with a stoichiometric amount of the appropriate base or acid
in water, in an organic solvent, or in a mixture of the two.
Generally, non-aqueous media including ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
20th ed., Lippincott Williams & Wilkins, Baltimore, Md., 2000,
p. 704; and "Handbook of Pharmaceutical Salts: Properties,
Selection, and Use," P. Heinrich Stahl and Camille G. Wermuth,
Eds., Wiley-VCH, Weinheim, 2002.
[0237] Suitable pharmaceutically acceptable acid addition salts
include those derived from inorganic acids, such as hydrochloric,
hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric,
metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and
organic acids such as acetic, benzenesulfonic, benzoic, citric,
ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic,
lactobionic, maleic, malic, methanesulfonic,
trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and
trifluoroacetic acids.
[0238] Suitable organic acids generally include, for example,
aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,
carboxylic, and sulfonic classes of organic acids. Specific
examples of suitable organic acids include acetate,
trifluoroacetate, formate, propionate, succinate, glycolate,
gluconate, digluconate, lactate, malate, tartaric acid, citrate,
ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate,
glutamate, benzoate, anthranilic acid, mesylate, stearate,
salicylate, p-hydroxybenzoate, phenylacetate, mandelate, embonate
(pamoate), methanesulfonate, ethanesulfonate, benzenesulfonate,
pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate,
sufanilate, cyclohexylaminosulfonate, algenic acid,
.beta.-hydroxybutyric acid, galactarate, galacturonate, adipate,
alginate, butyrate, camphorate, camphorsulfonate,
cyclopentanepropionate, dodecylsulfate, glycoheptanoate,
glycerophosphate, heptanoate, hexanoate, nicotinate,
2-naphthalesulfonate, oxalate, palmoate, pectinate,
3-phenylpropionate, picrate, pivalate, thiocyanate, tosylate, and
undecanoate.
[0239] In some cases, the pharmaceutically acceptable salt may
include alkali metal salts, including sodium or potassium salts;
alkaline earth metal salts, e.g., calcium or magnesium salts; and
salts formed with suitable organic ligands, e.g., quaternary
ammonium salts. Base salts can also be formed from bases which form
non-toxic salts, including aluminum, arginine, benzathine, choline,
diethylamine, diolamine, glycine, lysine, meglumine, olamine,
tromethamine and zinc salts.
[0240] Organic salts may be made from secondary, tertiary or
quaternary amine salts, such as tromethamine, diethylamine,
N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and
procaine. Basic nitrogen-containing groups may also be quaternized
with agents such as lower alkyl (C.sub.1-C.sub.6) halides (e.g.,
methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides),
dialkyl sulfates (e.g., dimethyl, diethyl, dibuytl, and diamyl
sulfates), long chain halides (e.g., decyl, lauryl, myristyl, and
stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g.,
benzyl and phenethyl bromides), and others.
[0241] The compound can also be a pharmaceutically acceptable
prodrug of any of the compounds described above. Prodrugs are
compounds that, when metabolized in vivo, undergo conversion to
compounds having the desired pharmacological activity. Prodrugs can
be prepared by replacing appropriate functionalities present in the
compounds described above with "pro-moieties" as described, for
example, in H. Bundgaar, Design of Prodrugs (1985). Examples of
prodrugs include ester, ether or amide derivatives of the compounds
described above, polyethylene glycol derivatives of the compounds
described above, N-acyl amine derivatives, dihydropyridine pyridine
derivatives, amino-containing derivatives conjugated to
polypeptides, 2-hydroxybenzamide derivatives, carbamate
derivatives, N-oxides derivatives that are biologically reduced to
the active amines, and N-mannich base derivatives. For further
discussion of prodrugs, see, for example, Rautio, J. et al. Nature
Reviews Drug Discovery. 7:255-270 (2008).
III. Pharmaceutical Formulations
[0242] Pharmaceutical formulations are provided containing a
therapeutically effective amount of a compound described herein, or
a pharmaceutically acceptable salt or prodrug thereof, in
combination with one or more pharmaceutically acceptable
excipients. Representative excipients include solvents, diluents,
pH modifying agents, preservatives, antioxidants, suspending
agents, wetting agents, viscosity modifiers, tonicity agents,
stabilizing agents, and combinations thereof. Suitable
pharmaceutically acceptable excipients are preferably selected from
materials that are generally recognized as safe (GRAS), and may be
administered to an individual without causing undesirable
biological side effects or unwanted interactions.
[0243] A. Additional Therapeutics
[0244] The compounds described herein can be formulated with one or
more additional active agents, such as anti-infectious agents,
analgesic, etc.
[0245] Pharmaceutical formulations can also include one or more
vitamins, minerals, dietary supplements, nutraceutical agents, such
as proteins, carbohydrates, amino acids, fatty acids, antioxidants,
and plant or animal extracts, or combinations thereof. Suitable
vitamins, minerals, nutraceutical agents, and dietary supplements
are known in the art, and disclosed, for example, in Roberts et
al., (Nutraceuticals: The Complete Encyclopedia of Supplements,
Herbs, Vitamins, and Healing Foods, American Nutriceutical
Association, 2001). Nutraceutical agents and dietary supplements
are also disclosed in Physicians' Desk Reference for Nutritional
Supplements, 1st Ed. (2001) and The Physicians' Desk Reference for
Herbal Medicines, 1st Ed. (2001).
[0246] B. Enteral Formulations
[0247] Suitable oral dosage forms include tablets, capsules,
solutions, suspensions, syrups, and lozenges. Tablets can be made
using compression or molding techniques well known in the art.
Gelatin or non-gelatin capsules can prepared as hard or soft
capsule shells, which can encapsulate liquid, solid, and semi-solid
fill materials, using techniques well known in the art.
[0248] Formulations may be prepared using one or more
pharmaceutically acceptable excipients, including diluents,
preservatives, binders, lubricants, disintegrators, swelling
agents, fillers, stabilizers, and combinations thereof.
[0249] Excipients, including plasticizers, pigments, colorants,
stabilizing agents, and glidants, may also be used to form coated
compositions for enteral administration. Delayed release dosage
formulations may be prepared as described in standard references
such as "Pharmaceutical dosage form tablets", eds. Liberman et. al.
(New York, Marcel Dekker, Inc., 1989), "Remington--The science and
practice of pharmacy", 20th ed., Lippincott Williams & Wilkins,
Baltimore, Md., 2000, and "Pharmaceutical dosage forms and drug
delivery systems", 6th Edition, Ansel et al., (Media, Pa.: Williams
and Wilkins, 1995). These references provide information on
excipients, materials, equipment and process for preparing tablets
and capsules and delayed release dosage forms of tablets, capsules,
and granules.
[0250] Examples of suitable coating materials include, but are not
limited to, cellulose polymers such as cellulose acetate phthalate,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate and hydroxypropyl
methylcellulose acetate succinate; polyvinyl acetate phthalate,
acrylic acid polymers and copolymers, and methacrylic resins that
are commercially available under the trade name EUDRAGIT.RTM. (Roth
Pharma, Westerstadt, Germany), zein, shellac, and
polysaccharides.
[0251] Diluents, also referred to as "fillers," are typically
necessary to increase the bulk of a solid dosage form so that a
practical size is provided for compression of tablets or formation
of beads and granules. Suitable diluents include, but are not
limited to, dicalcium phosphate dihydrate, calcium sulfate,
lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline
cellulose, kaolin, sodium chloride, dry starch, hydrolyzed
starches, pregelatinized starch, silicone dioxide, titanium oxide,
magnesium aluminum silicate and powdered sugar.
[0252] Binders are used to impart cohesive qualities to a solid
dosage formulation, and thus ensure that a tablet or bead or
granule remains intact after the formation of the dosage forms.
Suitable binder materials include, but are not limited to, starch,
pregelatinized starch, gelatin, sugars (including sucrose, glucose,
dextrose, lactose and sorbitol), polyethylene glycol, waxes,
natural and synthetic gums such as acacia, tragacanth, sodium
alginate, cellulose, including hydroxypropylmethylcellulose,
hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic
polymers such as acrylic acid and methacrylic acid copolymers,
methacrylic acid copolymers, methyl methacrylate copolymers,
aminoalkyl methacrylate copolymers, polyacrylic
acid/polymethacrylic acid and polyvinylpyrrolidone.
[0253] Lubricants are used to facilitate tablet manufacture.
Examples of suitable lubricants include, but are not limited to,
magnesium stearate, calcium stearate, stearic acid, glycerol
behenate, polyethylene glycol, talc, and mineral oil.
[0254] Disintegrants are used to facilitate dosage form
disintegration or "breakup" after administration, and generally
include, but are not limited to, starch, sodium starch glycolate,
sodium carboxymethyl starch, sodium carboxymethylcellulose,
hydroxypropyl cellulose, pregelatinized starch, clays, cellulose,
alginine, gums or cross linked polymers, such as cross-linked PVP
(Polyplasdone.RTM. XL from GAF Chemical Corp).
[0255] Stabilizers are used to inhibit or retard drug decomposition
reactions that include, by way of example, oxidative reactions.
Suitable stabilizers include, but are not limited to, antioxidants,
butylated hydroxytoluene (BHT); ascorbic acid, its salts and
esters; Vitamin E, tocopherol and its salts; sulfites such as
sodium metabisulphite; cysteine and its derivatives; citric acid;
propyl gallate, and butylated hydroxyanisole (BHA).
[0256] 1. Controlled Release Formulations
[0257] Oral dosage forms, such as capsules, tablets, solutions, and
suspensions, can for formulated for controlled release. For
example, the one or more compounds and optional one or more
additional active agents can be formulated into nanoparticles,
microparticles, and combinations thereof, and encapsulated in a
soft or hard gelatin or non-gelatin capsule or dispersed in a
dispersing medium to form an oral suspension or syrup. The
particles can be formed of the drug and a controlled release
polymer or matrix. Alternatively, the drug particles can be coated
with one or more controlled release coatings prior to incorporation
in to the finished dosage form.
[0258] In another embodiment, the one or more compounds and
optional one or more additional active agents are dispersed in a
matrix material, which gels or emulsifies upon contact with an
aqueous medium, such as physiological fluids. In the case of gels,
the matrix swells entrapping the active agents, which are released
slowly over time by diffusion and/or degradation of the matrix
material. Such matrices can be formulated as tablets or as fill
materials for hard and soft capsules.
[0259] In still another embodiment, the one or more compounds, and
optional one or more additional active agents are formulated into a
sold oral dosage form, such as a tablet or capsule, and the solid
dosage form is coated with one or more controlled release coatings,
such as a delayed release coatings or extended release coatings.
The coating or coatings may also contain the compounds and/or
additional active agents.
Extended Release Formulations
[0260] The extended release formulations are generally prepared as
diffusion or osmotic systems, for example, as described in
"Remington--The science and practice of pharmacy" (20th ed.,
Lippincott Williams & Wilkins, Baltimore, Md., 2000). A
diffusion system typically consists of two types of devices, a
reservoir and a matrix, and is well known and described in the
art.
[0261] The matrix devices are generally prepared by compressing the
drug with a slowly dissolving polymer carrier into a tablet form.
The three major types of materials used in the preparation of
matrix devices are insoluble plastics, hydrophilic polymers, and
fatty compounds. Plastic matrices include, but are not limited to,
methyl acrylate-methyl methacrylate, polyvinyl chloride, and
polyethylene. Hydrophilic polymers include, but are not limited to,
cellulosic polymers such as methyl and ethyl cellulose,
hydroxyalkylcelluloses such as hydroxypropyl-cellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and
Carbopol.RTM. 934, polyethylene oxides and mixtures thereof. Fatty
compounds include, but are not limited to, various waxes such as
carnauba wax and glyceryl tristearate and wax-type substances
including hydrogenated castor oil or hydrogenated vegetable oil, or
mixtures thereof.
[0262] In certain preferred embodiments, the plastic material is a
pharmaceutically acceptable acrylic polymer, including but not
limited to, acrylic acid and methacrylic acid copolymers, methyl
methacrylate, methyl methacrylate copolymers, ethoxyethyl
methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate
copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic
acid alkylamine copolymer poly(methyl methacrylate),
poly(methacrylic acid)(anhydride), polymethacrylate,
polyacrylamide, poly(methacrylic acid anhydride), and glycidyl
methacrylate copolymers.
[0263] In certain preferred embodiments, the acrylic polymer is
comprised of one or more ammonio methacrylate copolymers. Ammonio
methacrylate copolymers are well known in the art, and are
described in NF XVII as fully polymerized copolymers of acrylic and
methacrylic acid esters with a low content of quaternary ammonium
groups.
[0264] In one preferred embodiment, the acrylic polymer is an
acrylic resin lacquer such as that which is commercially available
from Rohm Pharma under the tradename Eudragit.RTM. In further
preferred embodiments, the acrylic polymer comprises a mixture of
two acrylic resin lacquers commercially available from Rohm Pharma
under the tradenames Eudragit.RTM. RL30D and Eudragit.RTM. RS30D,
respectively. Eudragit.RTM. RL30D and Eudragit.RTM.. RS30D are
copolymers of acrylic and methacrylic esters with a low content of
quaternary ammonium groups, the molar ratio of ammonium groups to
the remaining neutral (meth)acrylic esters being 1:20 in
Eudragit.RTM. RL30D and 1:40 in Eudragit.RTM. RS30D. The mean
molecular weight is about 150,000. Edragit.RTM. S-100 and
Eudragit.RTM. L-100 are also preferred. The code designations RL
(high permeability) and RS (low permeability) refer to the
permeability properties of these agents. Eudragit.RTM. RL/RS
mixtures are insoluble in water and in digestive fluids. However,
multiparticulate systems formed to include the same are swellable
and permeable in aqueous solutions and digestive fluids.
[0265] The polymers described above such as Eudragit.RTM. RL/RS may
be mixed together in any desired ratio in order to ultimately
obtain a sustained-release formulation having a desirable
dissolution profile. Desirable sustained-release multiparticulate
systems may be obtained, for instance, from 100% Eudragit.RTM. RL,
50% Eudragit.RTM. RL and 50% Eudragit.RTM. RS, and 10%
Eudragit.RTM. RL and 90% Eudragit.RTM. RS. One skilled in the art
will recognize that other acrylic polymers may also be used, such
as, for example, Eudragit.RTM. L.
[0266] Alternatively, extended release formulations can be prepared
using osmotic systems or by applying a semi-permeable coating to
the dosage form. In the latter case, the desired drug release
profile can be achieved by combining low permeable and high
permeable coating materials in suitable proportion.
[0267] The devices with different drug release mechanisms described
above can be combined in a final dosage form comprising single or
multiple units. Examples of multiple units include, but are not
limited to, multilayer tablets andcapsules containing tablets,
beads, or granules. An immediate release portion can be added to
the extended release system by means of either applying an
immediate release layer on top of the extended release core using a
coating or compression process or in a multiple unit system such as
a capsule containing extended and immediate release beads. Extended
release tablets containing hydrophilic polymers are prepared by
techniques commonly known in the art such as direct compression,
wet granulation, or dry granulation. Their formulations usually
incorporate polymers, diluents, binders, and lubricants as well as
the active pharmaceutical ingredient. The usual diluents include
inert powdered substances such as starches, powdered cellulose,
especially crystalline and microcrystalline cellulose, sugars such
as fructose, mannitol and sucrose, grain flours and similar edible
powders. Typical diluents include, for example, various types of
starch, lactose, mannitol, kaolin, calcium phosphate or sulfate,
inorganic salts such as sodium chloride and powdered sugar.
Powdered cellulose derivatives are also useful. Typical tablet
binders include substances such as starch, gelatin and sugars such
as lactose, fructose, and glucose. Natural and synthetic gums,
including acacia, alginates, methylcellulose, and
polyvinylpyrrolidone can also be used. Polyethylene glycol,
hydrophilic polymers, ethylcellulose and waxes can also serve as
binders. A lubricant is necessary in a tablet formulation to
prevent the tablet and punches from sticking in the die. The
lubricant is chosen from such slippery solids as talc, magnesium
and calcium stearate, stearic acid and hydrogenated vegetable
oils.
[0268] Extended release tablets containing wax materials are
generally prepared using methods known in the art such as a direct
blend method, a congealing method, and an aqueous dispersion
method. In the congealing method, the drug is mixed with a wax
material and either spray-congealed or congealed and screened and
processed.
Delayed Release Formulations
[0269] Delayed release formulations can be created by coating a
solid dosage form with a polymer film, which is insoluble in the
acidic environment of the stomach, and soluble in the neutral
environment of the small intestine.
[0270] The delayed release dosage units can be prepared, for
example, by coating a drug or a drug-containing composition with a
selected coating material. The drug-containing composition may be,
e.g., a tablet for incorporation into a capsule, a tablet for use
as an inner core in a "coated core" dosage form, or a plurality of
drug-containing beads, particles or granules, for incorporation
into either a tablet or capsule. Preferred coating materials
include bioerodible, gradually hydrolyzable, gradually
water-soluble, and/or enzymatically degradable polymers, and may be
conventional "enteric" polymers. Enteric polymers, as will be
appreciated by those skilled in the art, become soluble in the
higher pH environment of the lower gastrointestinal tract or slowly
erode as the dosage form passes through the gastrointestinal tract,
while enzymatically degradable polymers are degraded by bacterial
enzymes present in the lower gastrointestinal tract, particularly
in the colon. Suitable coating materials for effecting delayed
release include, but are not limited to, cellulosic polymers such
as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl
cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl
cellulose acetate succinate, hydroxypropylmethyl cellulose
phthalate, methylcellulose, ethyl cellulose, cellulose acetate,
cellulose acetate phthalate, cellulose acetate trimellitate and
carboxymethylcellulose sodium; acrylic acid polymers and
copolymers, preferably formed from acrylic acid, methacrylic acid,
methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl
methacrylate, and other methacrylic resins that are commercially
available under the tradename Eudragit.RTM. (Rohm Pharma;
Westerstadt, Germany), including Eudragit.RTM. L30D-55 and L100-55
(soluble at pH 5.5 and above), Eudragit.RTM. L-100 (soluble at pH
6.0 and above), Eudragit.RTM. S (soluble at pH 7.0 and above, as a
result of a higher degree of esterification), and Eudragits.RTM.
NE, RL and RS (water-insoluble polymers having different degrees of
permeability and expandability); vinyl polymers and copolymers such
as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate,
vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate
copolymer; enzymatically degradable polymers such as azo polymers,
pectin, chitosan, amylose and guar gum; zein and shellac.
Combinations of different coating materials may also be used.
Multi-layer coatings using different polymers may also be
applied.
[0271] The preferred coating weights for particular coating
materials may be readily determined by those skilled in the art by
evaluating individual release profiles for tablets, beads and
granules prepared with different quantities of various coating
materials. It is the combination of materials, method and form of
application that produce the desired release characteristics, which
one can determine only from the clinical studies.
[0272] The coating composition may include conventional additives,
such as plasticizers, pigments, colorants, stabilizing agents,
glidants, etc. A plasticizer is normally present to reduce the
fragility of the coating, and will generally represent about 10 wt.
% to 50 wt. % relative to the dry weight of the polymer. Examples
of typical plasticizers include polyethylene glycol, propylene
glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl
phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate,
triethyl acetyl citrate, castor oil and acetylated monoglycerides.
A stabilizing agent is preferably used to stabilize particles in
the dispersion. Typical stabilizing agents are nonionic emulsifiers
such as sorbitan esters, polysorbates and polyvinylpyrrolidone.
Glidants are recommended to reduce sticking effects during film
formation and drying, and will generally represent approximately 25
wt. % to 100 wt. % of the polymer weight in the coating solution.
One effective glidant is talc. Other glidants such as magnesium
stearate and glycerol monostearates may also be used. Pigments such
as titanium dioxide may also be used. Small quantities of an
anti-foaming agent, such as a silicone (e.g., simethicone), may
also be added to the coating composition.
Pulsatile Release
[0273] The formulation can provide pulsatile delivery of the one or
more of the compounds disclosed herein. By "pulsatile" is meant
that a plurality of drug doses are released at spaced apart
intervals of time. Generally, upon ingestion of the dosage form,
release of the initial dose is substantially immediate, i.e., the
first drug release "pulse" occurs within about one hour of
ingestion. This initial pulse is followed by a first time interval
(lag time) during which very little or no drug is released from the
dosage form, after which a second dose is then released. Similarly,
a second nearly drug release-free interval between the second and
third drug release pulses may be designed. The duration of the
nearly drug release-free time interval will vary depending upon the
dosage form design e.g., a twice daily dosing profile, a three
times daily dosing profile, etc. For dosage forms providing a twice
daily dosage profile, the nearly drug release-free interval has a
duration of approximately 3 hours to 14 hours between the first and
second dose. For dosage forms providing a three times daily
profile, the nearly drug release-free interval has a duration of
approximately 2 hours to 8 hours between each of the three
doses.
[0274] In one embodiment, the pulsatile release profile is achieved
with dosage forms that are closed and preferably sealed capsules
housing at least two drug-containing "dosage units" wherein each
dosage unit within the capsule provides a different drug release
profile. Control of the delayed release dosage unit(s) is
accomplished by a controlled release polymer coating on the dosage
unit, or by incorporation of the active agent in a controlled
release polymer matrix. Each dosage unit may comprise a compressed
or molded tablet, wherein each tablet within the capsule provides a
different drug release profile. For dosage forms mimicking a twice
a day dosing profile, a first tablet releases drug substantially
immediately following ingestion of the dosage form, while a second
tablet releases drug approximately 3 hours to less than 14 hours
following ingestion of the dosage form. For dosage forms mimicking
a three times daily dosing profile, a first tablet releases drug
substantially immediately following ingestion of the dosage form, a
second tablet releases drug approximately 3 hours to less than 10
hours following ingestion of the dosage form, and the third tablet
releases drug at least 5 hours to approximately 18 hours following
ingestion of the dosage form. It is possible that the dosage form
includes more than three tablets. While the dosage form will not
generally include more than a third tablet, dosage forms housing
more than three tablets can be utilized.
[0275] Alternatively, each dosage unit in the capsule may comprise
a plurality of drug-containing beads, granules or particles. As is
known in the art, drug-containing "beads" refer to beads made with
drug and one or more excipients or polymers. Drug-containing beads
can be produced by applying drug to an inert support, e.g., inert
sugar beads coated with drug or by creating a "core" comprising
both drug and one or more excipients. As is also known,
drug-containing "granules" and "particles" comprise drug particles
that may or may not include one or more additional excipients or
polymers. In contrast to drug-containing beads, granules and
particles do not contain an inert support. Granules generally
comprise drug particles and require further processing. Generally,
particles are smaller than granules, and are not further processed.
Although beads, granules and particles may be formulated to provide
immediate release, beads and granules are generally employed to
provide delayed release.
[0276] C. Parenteral Formulations
[0277] The compounds described herein can be formulated for
parenteral administration. "Parenteral administration", as used
herein, means administration by any method other than through the
digestive tract or non-invasive topical or regional routes. For
example, parenteral administration may include administration to a
patient intravenously, intradermally, intraperitoneally,
intrapleurally, intratracheally, intramuscularly, subcutaneously,
by injection, and by infusion.
[0278] Parenteral formulations can be prepared as aqueous
compositions using techniques is known in the art. Typically, such
compositions can be prepared as injectable formulations, for
example, solutions or suspensions; solid forms suitable for using
to prepare solutions or suspensions upon the addition of a
reconstitution medium prior to injection; emulsions, such as
water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and
microemulsions thereof, liposomes, or emulsomes.
[0279] The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, one or more polyols (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol), oils,
such as vegetable oils (e.g., peanut oil, corn oil, sesame oil,
etc.), and combinations thereof. The proper fluidity can be
maintained, for example, by the use of a coating, such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and/or by the use of surfactants. In many cases, it will
be preferable to include isotonic agents, for example, sugars or
sodium chloride.
[0280] Solutions and dispersions of the active compounds as the
free acid or base or pharmacologically acceptable salts thereof can
be prepared in water or another solvent or dispersing medium
suitably mixed with one or more pharmaceutically acceptable
excipients including, but not limited to, surfactants, dispersants,
emulsifiers, pH modifying agents, and combination thereof.
[0281] Suitable surfactants may be anionic, cationic, amphoteric or
nonionic surface active agents. Suitable anionic surfactants
include, but are not limited to, those containing carboxylate,
sulfonate and sulfate ions. Examples of anionic surfactants include
sodium, potassium, ammonium of long chain alkyl sulfonates and
alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate;
dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene
sulfonate; dialkyl sodium sulfosuccinates, such as sodium
bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as
sodium lauryl sulfate. Cationic surfactants include, but are not
limited to, quaternary ammonium compounds such as benzalkonium
chloride, benzethonium chloride, cetrimonium bromide, stearyl
dimethylbenzyl ammonium chloride, polyoxyethylene and coconut
amine. Examples of nonionic surfactants include ethylene glycol
monostearate, propylene glycol myristate, glyceryl monostearate,
glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose
acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene
monolaurate, polysorbates, polyoxyethylene octylphenylether,
PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene
glycol butyl ether, Poloxamer.RTM. 401, stearoyl
monoisopropanolamide, and polyoxyethylene hydrogenated tallow
amide. Examples of amphoteric surfactants include sodium
N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,
myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
[0282] The formulation can contain a preservative to prevent the
growth of microorganisms. Suitable preservatives include, but are
not limited to, parabens, chlorobutanol, phenol, sorbic acid, and
thimerosal. The formulation may also contain an antioxidant to
prevent degradation of the active agent(s).
[0283] The formulation is typically buffered to a pH of 3-8 for
parenteral administration upon reconstitution. Suitable buffers
include, but are not limited to, phosphate buffers, acetate
buffers, and citrate buffers.
[0284] Water soluble polymers are often used in formulations for
parenteral administration. Suitable water-soluble polymers include,
but are not limited to, polyvinylpyrrolidone, dextran,
carboxymethylcellulose, and polyethylene glycol.
[0285] Sterile injectable solutions can be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent or dispersion medium with one or more of the
excipients listed above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the various sterilized active ingredients into a sterile vehicle
which contains the basic dispersion medium and the required other
ingredients from those listed above. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum-drying and freeze-drying
techniques which yield a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered
solution thereof. The powders can be prepared in such a manner that
the particles are porous in nature, which can increase dissolution
of the particles. Methods for making porous particles are well
known in the art.
[0286] 1. Controlled release formulations
[0287] The parenteral formulations described herein can be
formulated for controlled release including immediate release,
delayed release, extended release, pulsatile release, and
combinations thereof.
[0288] Nano- and Microparticles
[0289] For parenteral administration, the compounds, and optionally
one or more additional active agents, can be incorporated into
microparticles, nanoparticles, or combinations thereof that provide
controlled release. In embodiments wherein the formulations
contains two or more drugs, the drugs can be formulated for the
same type of controlled release (e.g., delayed, extended,
immediate, or pulsatile) or the drugs can be independently
formulated for different types of release (e.g., immediate and
delayed, immediate and extended, delayed and extended, delayed and
pulsatile, etc.).
[0290] For example, the compounds and/or one or more additional
active agents can be incorporated into polymeric microparticles
that provide controlled release of the drug(s). Release of the
drug(s) is controlled by diffusion of the drug(s) out of the
microparticles and/or degradation of the polymeric particles by
hydrolysis and/or enzymatic degradation. Suitable polymers include
ethylcellulose and other natural or synthetic cellulose
derivatives.
[0291] Polymers that are slowly soluble and form a gel in an
aqueous environment, such as hydroxypropyl methylcellulose or
polyethylene oxide may also be suitable as materials for drug
containing microparticles. Other polymers include, but are not
limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy
acids, such as polylactide (PLA), polyglycolide (PGA),
poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and
copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers
thereof, polycaprolactone and copolymers thereof, and combinations
thereof.
[0292] Alternatively, the drug(s) can be incorporated into
microparticles prepared from materials which are insoluble in
aqueous solution or slowly soluble in aqueous solution, but are
capable of degrading within the GI tract by means including
enzymatic degradation, surfactant action of bile acids, and/or
mechanical erosion. As used herein, the term "slowly soluble in
water" refers to materials that are not dissolved in water within a
period of 30 minutes. Preferred examples include fats, fatty
substances, waxes, wax-like substances and mixtures thereof.
Suitable fats and fatty substances include fatty alcohols (such as
lauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty
acids and derivatives, including, but not limited to, fatty acid
esters, fatty acid glycerides (mono-, di- and tri-glycerides), and
hydrogenated fats. Specific examples include, but are not limited
to hydrogenated vegetable oil, hydrogenated cottonseed oil,
hydrogenated castor oil, hydrogenated oils available under the
trade name Sterotex.RTM., stearic acid, cocoa butter, and stearyl
alcohol. Suitable waxes and wax-like materials include natural or
synthetic waxes, hydrocarbons, and normal waxes. Specific examples
of waxes include beeswax, glycowax, castor wax, carnauba wax,
paraffins and candelilla wax. As used herein, a wax-like material
is defined as any material that is normally solid at room
temperature and has a melting point of from about 30 to 300.degree.
C.
[0293] In some cases, it may be desirable to alter the rate of
water penetration into the microparticles. To this end,
rate-controlling (wicking) agents may be formulated along with the
fats or waxes listed above. Examples of rate-controlling materials
include certain starch derivatives (e.g., waxy maltodextrin and
drum dried corn starch), cellulose derivatives (e.g.,
hydroxypropylmethyl-cellulose, hydroxypropylcellulose,
methylcellulose, and carboxymethyl-cellulose), alginic acid,
lactose and talc. Additionally, a pharmaceutically acceptable
surfactant (for example, lecithin) may be added to facilitate the
degradation of such microparticles.
[0294] Proteins that are water insoluble, such as zein, can also be
used as materials for the formation of drug containing
microparticles. Additionally, proteins, polysaccharides and
combinations thereof that are water soluble can be formulated with
drug into microparticles and subsequently cross-linked to form an
insoluble network. For example, cyclodextrins can be complexed with
individual drug molecules and subsequently cross-linked.
[0295] Encapsulation or incorporation of drug into carrier
materials to produce drug containing microparticles can be achieved
through known pharmaceutical formulation techniques. In the case of
formulation in fats, waxes or wax-like materials, the carrier
material is typically heated above its melting temperature and the
drug is added to form a mixture comprising drug particles suspended
in the carrier material, drug dissolved in the carrier material, or
a mixture thereof. Microparticles can be subsequently formulated
through several methods including, but not limited to, the
processes of congealing, extrusion, spray chilling or aqueous
dispersion. In a preferred process, wax is heated above its melting
temperature, drug is added, and the molten wax-drug mixture is
congealed under constant stirring as the mixture cools.
Alternatively, the molten wax-drug mixture can be extruded and
spheronized to form pellets or beads. Detailed descriptions of
these processes can be found in "Remington--The science and
practice of pharmacy", 20th Edition, Jennaro et. al., (Phila,
Lippencott, Williams, and Wilkens, 2000).
[0296] For some carrier materials it may be desirable to use a
solvent evaporation technique to produce drug containing
microparticles. In this case drug and carrier material are
co-dissolved in a mutual solvent and microparticles can
subsequently be produced by several techniques including, but not
limited to, forming an emulsion in water or other appropriate
media, spray drying or by evaporating off the solvent from the bulk
solution and milling the resulting material.
[0297] In some embodiments, drug in a particulate form is
homogeneously dispersed in a water-insoluble or slowly water
soluble material. To minimize the size of the drug particles within
the composition, the drug powder itself may be milled to generate
fine particles prior to formulation. The process of jet milling,
known in the pharmaceutical art, can be used for this purpose. In
some embodiments drug in a particulate form is homogeneously
dispersed in a wax or wax like substance by heating the wax or wax
like substance above its melting point and adding the drug
particles while stirring the mixture. In this case a
pharmaceutically acceptable surfactant may be added to the mixture
to facilitate the dispersion of the drug particles.
[0298] The particles can also be coated with one or more modified
release coatings. Solid esters of fatty acids, which are hydrolyzed
by lipases, can be spray coated onto microparticles or drug
particles. Zein is an example of a naturally water-insoluble
protein. It can be coated onto drug containing microparticles or
drug particles by spray coating or by wet granulation techniques.
In addition to naturally water-insoluble materials, some substrates
of digestive enzymes can be treated with cross-linking procedures,
resulting in the formation of non-soluble networks. Many methods of
cross-linking proteins, initiated by both chemical and physical
means, have been reported. One of the most common methods to obtain
cross-linking is the use of chemical cross-linking agents. Examples
of chemical cross-linking agents include aldehydes (gluteraldehyde
and formaldehyde), epoxy compounds, carbodiimides, and genipin. In
addition to these cross-linking agents, oxidized and native sugars
have been used to cross-link gelatin (Cortesi, R., et al.,
Biomaterials 19 (1998) 1641-1649). Cross-linking can also be
accomplished using enzymatic means; for example, transglutaminase
has been approved as a GRAS substance for cross-linking seafood
products. Finally, cross-linking can be initiated by physical means
such as thermal treatment, UV irradiation and gamma
irradiation.
[0299] To produce a coating layer of cross-linked protein
surrounding drug containing microparticles or drug particles, a
water soluble protein can be spray coated onto the microparticles
and subsequently cross-linked by the one of the methods described
above. Alternatively, drug containing microparticles can be
microencapsulated within protein by coacervation-phase separation
(for example, by the addition of salts) and subsequently
cross-linked. Some suitable proteins for this purpose include
gelatin, albumin, casein, and gluten.
[0300] Polysaccharides can also be cross-linked to form a
water-insoluble network. For many polysaccharides, this can be
accomplished by reaction with calcium salts or multivalent cations
that cross-link the main polymer chains. Pectin, alginate, dextran,
amylose and guar gum are subject to cross-linking in the presence
of multivalent cations. Complexes between oppositely charged
polysaccharides can also be formed; pectin and chitosan, for
example, can be complexed via electrostatic interactions.
[0301] Depot Formulations
[0302] Active agents can be formulated for depot injection. In a
depot injection, the active agent is formulated with one or more
pharmaceutically acceptable carriers that provide for the gradual
release of active agent over a period of hours or days after
injection. The depot formulation can be administered by any
suitable means; however, the depot formulation is typically
administered via subcutaneous or intramuscular injection.
[0303] A variety of carriers may be incorporated into the depot
formulation to provide for the controlled release of the active
agent. In some cases, depot formulations contain one or more
biodegradable polymeric or oligomeric carriers. Suitable polymeric
carriers include, but are not limited to poly(lactic acid) (PLA),
poly(lactic-co-glycolic acid) (PLGA), poly(lactic
acid)-polyethyleneglycol (PLA-PEG) block copolymers,
polyanhydrides, poly(ester anhydrides), polyglycolide (PGA),
poly-3-hydroxybutyrate (PHB) and copolymers thereof,
poly-4-hydroxybutyrate (P4HB), polycaprolactone, cellulose,
hydroxypropyl methylcellulose, ethylcellulose, as well as blends,
derivatives, copolymers, and combinations thereof.
[0304] In depot formulations containing a polymeric or oligomeric
carrier, the carrier and active agent can be formulated as a
solution, an emulsion, or suspension. One or more compounds, and
optionally one or more additional active agents, can also be
incorporated into polymeric or oligomeric microparticles,
nanoparticles, or combinations thereof.
[0305] In some cases, the formulation is fluid and designed to
solidify or gel (i.e., forming a hydrogel or organogel) upon
injection. This can result from a change in solubility of the
composition upon injection, or for example, by injecting a
pre-polymer mixed with an initiator and/or crosslinking agent. The
polymer matrix, polymer solution, or polymeric particles entrap the
active agent at the injection site. As the polymeric carrier is
gradually degraded, the active agent is released, either by
diffusion of the agent out of the matrix and/or dissipation of the
matrix as it is absorbed. The release rate of the active agent from
the injection site can be controlled by varying, for example, the
chemical composition, molecular weight, crosslink density, and/or
concentration of the polymeric carrier. Examples of such systems
include those described in U.S. Pat. Nos. 4,938,763, 5,480,656 and
6,113,943.
[0306] Depot formulations can also be prepared by using other
rate-controlling excipients, including hydrophobic materials,
including acceptable oils (e.g., peanut oil, corn oil, sesame oil,
cottonseed oil, etc.) and phospholipids, ion-exchange resins, and
sparingly soluble carriers.
[0307] The depot formulation can further contain a solvent or
dispersion medium containing, for example, water, ethanol, one or
more polyols (e.g., glycerol, propylene glycol, and liquid
polyethylene glycol), oils, such as vegetable oils (e.g., peanut
oil, corn oil, sesame oil, etc.), and combinations thereof. The
proper fluidity can be maintained, for example, by the use of a
coating, such as lecithin, by the maintenance of the required
particle size in the case of dispersion and/or by the use of
surfactants. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride.
[0308] Solutions and dispersions of the compounds as the free acid
or base or pharmacologically acceptable salts thereof can be
prepared in water or another solvent or dispersing medium suitably
mixed with one or more pharmaceutically acceptable excipients
including, but not limited to, surfactants, dispersants,
emulsifiers, pH modifying agents, and combination thereof.
[0309] Suitable surfactants may be anionic, cationic, amphoteric or
nonionic surface active agents. Suitable anionic surfactants
include, but are not limited to, those containing carboxylate,
sulfonate and sulfate ions. Examples of anionic surfactants include
sodium, potassium, ammonium of long chain alkyl sulfonates and
alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate;
dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene
sulfonate; dialkyl sodium sulfosuccinates, such as sodium
bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as
sodium lauryl sulfate. Cationic surfactants include, but are not
limited to, quaternary ammonium compounds such as benzalkonium
chloride, benzethonium chloride, cetrimonium bromide, stearyl
dimethylbenzyl ammonium chloride, polyoxyethylene and coconut
amine. Examples of nonionic surfactants include ethylene glycol
monostearate, propylene glycol myristate, glyceryl monostearate,
glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose
acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene
monolaurate, polysorbates, polyoxyethylene octylphenylether,
PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene
glycol butyl ether, Poloxamer.RTM. 401, stearoyl
monoisopropanolamide, and polyoxyethylene hydrogenated tallow
amide. Examples of amphoteric surfactants include sodium
N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,
myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
[0310] The formulation can contain a preservative to prevent the
growth of microorganisms. Suitable preservatives include, but are
not limited to, parabens, chlorobutanol, phenol, sorbic acid, and
thimerosal. The formulation may also contain an antioxidant to
prevent degradation of the active agent(s).
[0311] The formulation is typically buffered to a pH of 3-8 for
parenteral administration upon reconstitution. Suitable buffers
include, but are not limited to, phosphate buffers, acetate
buffers, and citrate buffers.
[0312] Water soluble polymers are often used in formulations for
parenteral administration. Suitable water-soluble polymers include,
but are not limited to, polyvinylpyrrolidone, dextran,
carboxymethylcellulose, and polyethylene glycol.
[0313] Sterile injectable solutions can be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent or dispersion medium with one or more of the
excipients listed above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the various sterilized active ingredients into a sterile vehicle
which contains the basic dispersion medium and the required other
ingredients from those listed above. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum-drying and freeze-drying
techniques which yield a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered
solution thereof. The powders can be prepared in such a manner that
the particles are porous in nature, which can increase dissolution
of the particles. Methods for making porous particles are well
known in the art.
[0314] Implants
[0315] Implantation of a slow-release or sustained-release system,
such that a constant level of dosage is maintained is also
contemplated herein. In such cases, the active agent(s) provided
herein can be dispersed in a solid matrix optionally coated with an
outer rate-controlling membrane. The compound diffuses from the
solid matrix (and optionally through the outer membrane) sustained,
rate-controlled release. The solid matrix and membrane may be
formed from any suitable material known in the art including, but
not limited to, polymers, bioerodible polymers, and hydrogels.
[0316] C. Pulmonary Formulations
[0317] The compounds described herein can be formulated for
parenteral administration. Pharmaceutical formulations and methods
for the pulmonary administration are known in the art.
[0318] The respiratory tract is the structure involved in the
exchange of gases between the atmosphere and the blood stream. The
respiratory tract encompasses the upper airways, including the
oropharynx and larynx, followed by the lower airways, which include
the trachea followed by bifurcations into the bronchi and
bronchioli. The upper and lower airways are called the conducting
airways. The terminal bronchioli then divide into respiratory
bronchioli which then lead to the ultimate respiratory zone, the
alveoli, or deep lung, where the exchange of gases occurs.
[0319] The alveolar surface area is the largest in the respiratory
system and is where drug absorption occurs. The alveoli are covered
by a thin epithelium without cilia or a mucus blanket and secrete
surfactant phospholipids. Effective delivery of therapeutic agents
via pulmonary routes requires that the active agent be formulated
so as to reach the alveoli.
[0320] In the case of pulmonary administration, formulations can be
divided into dry powder formulations and liquid formulations. Both
dry powder and liquid formulations can be used to form aerosol
formulations. The term aerosol as used herein refers to any
preparation of a fine mist of particles, which can be in solution
or a suspension, whether or not it is produced using a
propellant.
[0321] Useful formulations, and methods of manufacture, are
described by Caryalho, et al., J Aerosol Med Pulm Drug Deliv. 2011
April; 24(2):61-80. Epub 2011 Mar. 16, for delivery of
chemotherapeutic drugs to the lungs.
[0322] 1. Dry Powder Formulations
[0323] Dry powder formulations are finely divided solid
formulations containing one or more active agents which are
suitable for pulmonary administration. In dry powder formulations,
the one or more active agents can be incorporated in crystalline or
amorphous form.
[0324] Dry powder formulations can be administered via pulmonary
inhalation to a patient without the benefit of any carrier, other
than air or a suitable propellant. Preferably, however, the dry
powder formulations include one or more pharmaceutically acceptable
carriers.
[0325] The pharmaceutical carrier may include a bulking agent, such
as carbohydrates (including monosaccharides, polysaccharides, and
cyclodextrins), polypeptides, amino acids, and combinations
thereof.
[0326] Suitable bulking agents include fructose, galactose,
glucose, lactitol, lactose, maltitol, maltose, mannitol,
melezitose, myoinositol, palatinite, raffinose, stachyose, sucrose,
trehalose, xylitol, hydrates thereof, and combinations thereof.
[0327] The pharmaceutical carrier may include a lipid or
surfactant. Natural surfactants such as
dipalmitoylphosphatidylcholine (DPPC) are the most preferred. This
is commercially available for treatment of respiratory distress
syndrome in premature infants. Synthetic and animal derived
pulmonary surfactants include:
[0328] Synthetic Pulmonary Surfactants
Exosurf--a mixture of DPPC with hexadecanol and tyloxapol added as
spreading agents Pumactant (Artificial Lung Expanding Compound or
ALEC)--a mixture of DPPC and PG KL-4--composed of DPPC,
palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined
with a 21 amino acid synthetic peptide that mimics the structural
characteristics of SP-B. Venticute--DPPC, PG, palmitic acid and
recombinant SP-C
[0329] Animal Derived Surfactants
Alveofact--extracted from cow lung lavage fluid Curosurf--extracted
from material derived from minced pig lung Infasurf--extracted from
calf lung lavage fluid Survanta--extracted from minced cow lung
with additional DPPC, palmitic acid and tripalmitin Exosurf,
Curosurf, Infasurf, and Survanta are the surfactants currently FDA
approved for use in the U.S.
[0330] The pharmaceutical carrier may also include one or more
stabilizing agents or dispersing agents. The pharmaceutical carrier
may also include one or more pH adjusters or buffers. Suitable
buffers include organic salts prepared from organic acids and
bases, such as sodium citrate or sodium ascorbate. The
pharmaceutical carrier may also include one or more salts, such as
sodium chloride or potassium chloride.
[0331] Dry powder formulations are typically prepared by blending
one or more active agents with a pharmaceutical carrier.
Optionally, additional active agents may be incorporated into the
mixture. The mixture is then formed into particles suitable for
pulmonary administration using techniques known in the art, such as
lyophilization, spray drying, agglomeration, spray coating,
extrusion processes, hot melt particle formation, phase separation
particle formation (spontaneous emulsion particle formation,
solvent evaporation particle formation, and solvent removal
particle formation), coacervation, low temperature casting,
grinding, milling (e.g., air-attrition milling (jet milling), ball
milling), high pressure homogenization, and/or supercritical fluid
crystallization.
[0332] An appropriate method of particle formation can be selected
based on the desired particle size, particle size distribution, and
particle morphology. In some cases, the method of particle
formation is selected so as to produce a population of particles
with the desired particle size, particle size distribution for
pulmonary administration. Alternatively, the method of particle
formation can produce a population of particles from which a
population of particles with the desired particle size, particle
size distribution for pulmonary administration is isolated, for
example by sieving.
[0333] It is known in the art that particle morphology affects the
depth of penetration of a particle into the lung as well as uptake
of the drug particles. As discussed above, drug particles should
reach the alveoli to maximize therapeutic efficacy. Accordingly,
dry powder formulations is processed into particles having the
appropriate mass median aerodynamic diameter (MMAD), tap density,
and surface roughness to achieve delivery of the one or more active
agents to the deep lung. Preferred particle morphologies for
delivery to the deep lung are known in the art, and are described,
for example, in U.S. Pat. No. 7,052,678 to Vanbever, et al.
[0334] Particles having a mass median aerodynamic diameter (MMAD)
of greater than about 5 microns generally do not reach the lung;
instead, they tend to impact the back of the throat and are
swallowed. Particles having diameters of about 3 to about 5 microns
are small enough to reach the upper-to mid-pulmonary region
(conducting airways), but may be too large to reach the alveoli.
Smaller particles, (i.e., about 0.5 to about 3 microns), are
capable of efficiently reaching the alveolar region. Particles
having diameters smaller than about 0.5 microns can also be
deposited in the alveolar region by sedimentation, although very
small particles may be exhaled.
[0335] The precise particle size range effective to achieve
delivery to the alveolar region will depend on several factors,
including the tap density of particles being delivered. Generally
speaking, as tap density decreases, the MMAD of particles capable
of efficiently reaching the alveolar region of the lungs increases.
Therefore, in cases of particles with low tap densities, particles
having diameters of about 3 to about 5 microns, about 5 to about 7
microns, or about 7 to about 9.5 microns can be efficiently
delivered to the lungs. The preferred aerodyanamic diameter for
maximum deposition within the lungs can be calculated. See, for
example, U.S. Pat. No. 7,052,678 to Vanbever, et al.
[0336] In some embodiments, the dry powder formulation is composed
of a plurality of particles having a median mass aerodynamic
diameter between about 0.5 to about 10 microns, more preferably
between about 0.5 microns to about 7 microns, most preferably
between about 0.5 to about 5 microns. In some embodiments, the dry
powder formulation is composed of a plurality of particles having a
median mass aerodynamic diameter between about 0.5 to about 3
microns. In some embodiments, the dry powder formulation is
composed of a plurality of particles having a median mass
aerodynamic diameter between about 3 to about 5 microns. In some
embodiments, the dry powder formulation is composed of a plurality
of particles having a median mass aerodynamic diameter between
about 5 to about 7 microns. In some embodiments, the dry powder
formulation is composed of a plurality of particles having a median
mass aerodynamic diameter between about 7 to about 9.5 microns.
[0337] In some cases, there may be an advantage to delivering
particles larger than about 3 microns in diameter. Phagocytosis of
particles by alveolar macrophages diminishes precipitously as
particle diameter increases beyond about 3 microns. Kawaguchi, H.,
et al., Biomaterials 7: 61-66 (1986); and Rudt, S. and Muller, R.
H., J. Contr. Rel, 22: 263-272 (1992). By administering particles
with an aerodynamic volume greater than 3 microns, phagocytic
engulfment by alveolar macrophages and clearance from the lungs can
be minimized.
[0338] In some embodiments, at least about 80%, more preferably at
least about 90%, most preferably at least about 95% of the
particles in dry powder formulation have aerodynamic diameter of
less than about 10 microns, more preferably less than about 7
microns, most preferably about 5 microns. In some embodiments, at
least about 80%, more preferably at least about 90%, most
preferably at least about 95%, of the particles in dry powder
formulation have aerodynamic diameter of greater than about 0.5
microns. In some embodiments, at least about 80%, more preferably
at least about 90%, most preferably at least about 95%, of the
particles in dry powder formulation have an aerodynamic diameter of
greater than about 0.1 microns.
[0339] In some embodiments, at least about 80%, more preferably at
least about 90%, most preferably at least about 95%, of the
particles in dry powder formulation have aerodynamic diameter of
greater than about 0.5 microns and less than about 10 microns, more
preferably greater than about 0.5 microns and less than about 7
microns, most preferably greater than about 0.5 microns and less
than about 5 microns. In some embodiments, at least about 80%, more
preferably at least about 90%, most preferably at least about 95%
of the particles in dry powder formulation have aerodynamic
diameter of greater than about 0.5 microns and less than about 3
microns. In some embodiments, at least about 80%, more preferably
at least about 90%, most preferably at least about 95% of the
particles in dry powder formulation have aerodynamic diameter of
greater than about 3 microns and less than about 5 microns. In some
embodiments, at least about 80%, more preferably at least about
90%, most preferably at least about 95% of the particles in dry
powder formulation have aerodynamic diameter of greater than about
5 microns and less than about 7 microns. In some embodiments, at
least about 80%, more preferably at least about 90%, most
preferably at least about 95% of the particles in dry powder
formulation have aerodynamic diameter of greater than about 7
microns and less than about 9.5 microns.
[0340] In some embodiments, the particles have a tap density of
less than about 0.4 g/cm.sup.3, more preferably less than about
0.25 g/cm.sup.3, most preferably less than about 0.1 g/cm.sup.3.
Features which can contribute to low tap density include irregular
surface texture and porous structure.
[0341] In some cases, the particles are spherical or ovoid in
shape. The particles can have a smooth or rough surface texture.
The particles may also be coated with a polymer or other suitable
material to control release of one or more active agents in the
lungs.
[0342] Dry powder formulations can be administered as dry powder
using suitable methods known in the art. Alternatively, the dry
powder formulations can be suspended in the liquid formulation s
described below, and administered to the lung using methods known
in the art for the delivery of liquid formulations.
[0343] 2. Liquid Formulations
[0344] Liquid formulations contain one or more compounds dissolved
or suspended in a liquid pharmaceutical carrier.
[0345] Suitable liquid carriers include, but are not limited to
distilled water, de-ionized water, pure or ultrapure water, saline,
and other physiologically acceptable aqueous solutions containing
salts and/or buffers, such as phosphate buffered saline (PBS),
Ringer's solution, and isotonic sodium chloride, or any other
aqueous solution acceptable for administration to an animal or
human.
[0346] Preferably, liquid formulations are isotonic relative to
physiological fluids and of approximately the same pH, ranging
e.g., from about pH 4.0 to about pH 7.4, more preferably from about
pH 6.0 to pH 7.0. The liquid pharmaceutical carrier can include one
or more physiologically compatible buffers, such as a phosphate
buffers. One skilled in the art can readily determine a suitable
saline content and pH for an aqueous solution for pulmonary
administration.
[0347] Liquid formulations may include one or more suspending
agents, such as cellulose derivatives, sodium alginate,
polyvinylpyrrolidone, gum tragacanth, or lecithin. Liquid
formulations may also include one or more preservatives, such as
ethyl or n-propyl p-hydroxybenzoate.
[0348] In some cases the liquid formulation may contain one or more
solvents that are low toxicity organic (i.e., nonaqueous) class 3
residual solvents, such as ethanol, acetone, ethyl acetate,
tetrahydofuran, ethyl ether, and propanol. These solvents can be
selected based on their ability to readily aerosolize the
formulation. Any such solvent included in the liquid formulation
should not detrimentally react with the one or more active agents
present in the liquid formulation. The solvent should be
sufficiently volatile to enable formation of an aerosol of the
solution or suspension. Additional solvents or aerosolizing agents,
such as a freon, alcohol, glycol, polyglycol, or fatty acid, can
also be included in the liquid formulation as desired to increase
the volatility and/or alter the aerosolizing behavior of the
solution or suspension.
[0349] Liquid formulations may also contain minor amounts of
polymers, surfactants, or other excipients well known to those of
the art. In this context, "minor amounts" means no excipients are
present that might adversely affect uptake of the one or more
active agents in the lungs.
[0350] 3. Aerosol Formulations
[0351] The dry powder and liquid formulations described above can
be used to form aerosol formulations for pulmonary administration.
Aerosols for the delivery of therapeutic agents to the respiratory
tract are known in the art. The term aerosol as used herein refers
to any preparation of a fine mist of solid or liquid particles
suspended in a gas. In some cases, the gas may be a propellant;
however, this is not required. Aerosols may be produced using a
number of standard techniques, including as ultrasonication or high
pressure treatment.
[0352] Preferably, a dry powder or liquid formulation as described
above is formulated into aerosol formulations using one or more
propellants. Suitable propellants include air, hydrocarbons, such
as pentane, isopentane, butane, isobutane, propane and ethane,
carbon dioxide, chlorofluorocarbons, fluorocarbons, and
combinations thereof. Suitable fluorocarbons include 1-6 hydrogen
containing fluorocarbons, such as CHF.sub.2CHF.sub.2,
CF.sub.3CH.sub.2F, CH.sub.2F.sub.2CH.sub.3, and CF.sub.3CHFCF.sub.3
as well as fluorinated ethers such as CF.sub.3--O--CF.sub.3,
CF.sub.2H--O--CHF.sub.2, and
CF.sub.3--CF.sub.2--O--CF.sub.2--CH.sub.3. Suitable fluorocarbons
also include perfluorocarbons, such as 1-4 carbon perfluorocarbons
including CF.sub.3CF.sub.3, CF.sub.3CF.sub.2CF.sub.3, and
CF.sub.3CF.sub.2CF.sub.2CF.sub.3.
[0353] Preferably, the propellants include, but not limited to, one
or more hydrofluoroalkanes (HFA). Suitable HFA propellants, include
but are not limited to, 1,1,1,2,3,3,-heptafluoro-n-propane (HFA
227), 1,1,1,2-tetrafluoroethane (HFA 134) 1,1,1,2, 25
3,3,3-heptafluoropropane (Propellant 227), or any mixture of these
propellants.
[0354] Preferably, the one or more propellants have sufficient
vapor pressure to render them effective as propellants. Preferably,
the one or more propellants are selected so that the density of the
mixture is matched to the density of the particles in the aerosol
formulation in order to minimize settling or creaming of the
particles in the aerosol formulation.
[0355] The propellant is preferably present in an amount sufficient
to propel a plurality of the selected doses of the aerosol
formulation from an aerosol canister.
[0356] 4. Devices for Pulmonary Administration
[0357] In some cases, a device is used to administer the
formulations to the lungs. Suitable devices include, but are not
limited to, dry powder inhalers, pressurized metered dose inhalers,
nebulizers, and electrohydrodynamic aerosol devices.
[0358] Inhalation can occur through the nose and/or the mouth of
the patient. Administration can occur by self-administration of the
formulation while inhaling, or by administration of the formulation
via a respirator to a patient on a respirator.
[0359] Dry Powder Inhalers
[0360] The dry powder formulations described above can be
administered to the lungs of a patient using a dry powder inhaler
(DPI). DPI devices typically use a mechanism such as a burst of gas
to create a cloud of dry powder inside a container, which can then
be inhaled by the patient.
[0361] In a dry powder inhaler, the dose to be administered is
stored in the form of a non-pressurized dry powder and, on
actuation of the inhaler, the particles of the powder are inhaled
by the subject. In some cases, a compressed gas (i.e., propellant)
may be used to dispense the powder, similar to pressurized metered
dose inhalers (pMDIs). In some cases, the DPI may be breath
actuated, meaning that an aerosol is created in precise response to
inspiration. Typically, dry powder inhalers administer a dose of
less than a few tens of milligrams per inhalation to avoid
provocation of cough.
[0362] DPIs function via a variety of mechanical means to
administer formulations to the lungs. In some DPIs, a doctor blade
or shutter slides across the dry powder formulation contained in a
reservoir, culling the formulation into a flowpath whereby the
patient can inhale the powder in a single breath. In other DPIs,
the dry powder formulation is packaged in a preformed dosage form,
such as a blister, tabule, tablet, or gelcap, which is pierced,
crushed, or otherwise unsealed to release the dry powder
formulation into a flowpath for subsequent inhalation. Still others
DPIs release the dry powder formulation into a chamber or capsule
and use mechanical or electrical agitators to keep the dry powder
formulation suspended in the air until the patient inhales.
[0363] Dry powder formulations may be packaged in various forms,
such as a loose powder, cake, or pressed shape for insertion in to
the reservoir of a DPI.
[0364] Examples suitable DPIs for the administration of the
formulations described above include the Turbohaler.RTM. inhaler
(Astrazeneca, Wilmington, Del.), the Clickhaler.RTM. inhaler
(Innovata, Ruddington, Nottingham, UK), the Diskus.RTM. inhaler
(Glaxo, Greenford, Middlesex, UK), the EasyHaler.RTM. (Orion,
Expoo, FI), the Exubera.RTM. inhaler (Pfizer, New York, N.Y.), the
Qdose.RTM. inhaler (Microdose, Monmouth Junction, N.J.), and the
Spiros.RTM. inhaler (Dura, San Diego, Calif.).
Pressurized Metered Dose Inhalers
[0365] The liquid formulations described above can be administered
to the lungs of a patient using a pressurized metered dose inhaler
(pMDI).
[0366] Pressurized Metered Dose Inhalers (pMDIs) generally include
at least two components: a canister in which the liquid formulation
is held under pressure in combination with one or more propellants,
and a receptacle used to hold and actuate the canister. The
canister may contain a single or multiple doses of the formulation.
The canister may include a valve, typically a metering valve, from
which the contents of the canister may be discharged. Aerosolized
drug is dispensed from the pMDI by applying a force on the canister
to push it into the receptacle, thereby opening the valve and
causing the drug particles to be conveyed from the valve through
the receptacle outlet. Upon discharge from the canister, the liquid
formulation is atomized, forming an aerosol.
[0367] pMDIs typically employ one or more propellants to pressurize
the contents of the canister and to propel the liquid formulation
out of the receptacle outlet, forming an aerosol. Any suitable
propellants, including those discussed above, may be utilized. The
propellant may take a variety of forms. For example, the propellant
may be a compressed gas or a liquefied gas. Chlorofluorocarbons
(CFC) were once commonly used as liquid propellants, but have now
been banned. They have been replaced by the now widely accepted
hydrofluororalkane (HFA) propellants.
[0368] pMDIs are available from a number of suppliers, including 3M
Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories,
Glaxo-Wellcome, Schering Plough and Vectura. In some cases, the
patient administers an aerosolized formulation by manually
discharging the aerosolized formulation from the pMDI in
coordination with inspiration. In this way, the aerosolized
formulation is entrained within the inspiratory air flow and
conveyed to the lungs.
[0369] In other cases, a breath-actuated trigger, such as that
included in the Tempo.RTM. inhaler (MAP Pharmaceuticals, Mountain
View, Calif.) may be employed that simultaneously discharges a dose
of the formulation upon sensing inhalation. These devices, which
discharge the aerosol formulation when the user begins to inhale,
are known as breath-actuated pressurized metered dose inhalers
(baMDIs).
Nebulizers
[0370] The liquid formulations described above can also be
administered using a nebulizer. Nebulizers are liquid aerosol
generators that convert the liquid formulation described able,
usually aqueous-based compositions, into mists or clouds of small
droplets, preferably having diameters less than 5 microns mass
median aerodynamic diameter, which can be inhaled into the lower
respiratory tract. This process is called atomization. The droplets
carry the one or more active agents into the nose, upper airways or
deep lungs when the aerosol cloud is inhaled. Any type of nebulizer
may be used to administer the formulation to a patient, including,
but not limited to pneumatic (jet) nebulizers and electromechanical
nebulizers.
[0371] Pneumatic (jet) nebulizers use a pressurized gas supply as a
driving force for atomization of the liquid formulation. Compressed
gas is delivered through a nozzle or jet to create a low pressure
field which entrains a surrounding liquid formulation and shears it
into a thin film or filaments. The film or filaments are unstable
and break up into small droplets that are carried by the compressed
gas flow into the inspiratory breath. Baffles inserted into the
droplet plume screen out the larger droplets and return them to the
bulk liquid reservoir. Examples of pneumatic nebulizers include,
but are not limited to, PARI LC Plus.RTM., PARI LC Sprint.RTM.,
Devilbiss PulmoAide.RTM., and Boehringer Ingelheim
Respima.RTM..
[0372] Electromechanical nebulizers use electrically generated
mechanical force to atomize liquid formulations. The
electromechanical driving force can be applied, for example, by
vibrating the liquid formulation at ultrasonic frequencies, or by
forcing the bulk liquid through small holes in a thin film. The
forces generate thin liquid films or filament streams which break
up into small droplets to form a slow moving aerosol stream which
can be entrained in an inspiratory flow.
[0373] In some cases, the electromechanical nebulizer is an
ultrasonic nebulizer, in which the liquid formulation is coupled to
a vibrator oscillating at frequencies in the ultrasonic range. The
coupling is achieved by placing the liquid in direct contact with
the vibrator such as a plate or ring in a holding cup, or by
placing large droplets on a solid vibrating projector (a horn). The
vibrations generate circular standing films which break up into
droplets at their edges to atomize the liquid formulation. Examples
of ultrasonic nebulizers include DuroMist.RTM., Drive Medical
Beetle Neb.RTM., Octive Tech Densylogic.RTM., and John Bunn
Nano-Sonic.RTM..
[0374] In some cases, the electromechanical nebulizer is a mesh
nebulizer, in which the liquid formulation is driven through a mesh
or membrane with small holes ranging from 2 to 8 microns in
diameter, to generate thin filaments which break up into small
droplets. In certain designs, the liquid formulation is forced
through the mesh by applying pressure with a solenoid piston driver
(for example, the AERx.RTM. nebulizer), or by sandwiching the
liquid between a piezoelectrically vibrated plate and the mesh,
which results in a oscillatory pumping action (for example
EFlow.RTM., AerovectRx.RTM., or TouchSpray.RTM. nebulizer). In
other cases, the mesh vibrates back and forth through a standing
column of the liquid to pump it through the holes. Examples of such
nebilzers include the AeroNeb Go.RTM., AeroNeb Pro.RTM.. PARI
EFlow.RTM., Omron 22UE.RTM.; and Aradigm AERx.RTM..
Electrohydrodynamic Aerosol Devices
[0375] The liquid formulations described above can also be
administered using an electrohydrodynamic (EHD) aerosol device. EHD
aerosol devices use electrical energy to aerosolize liquid drug
solutions or suspensions. Examples of EHD aerosol devices are known
in the art. See, for example, U.S. Pat. No. 4,765,539 to Noakes et
al. and U.S. Pat. No. 4,962,885 to Coffee, R. A.
[0376] The electrochemical properties of the formulation may be
important parameters to optimize when delivering the liquid
formulation to the lung with an EHD aerosol device and such
optimization is routinely performed by one of skill in the art.
V. Methods of Treatment
[0377] Pharmaceutical formulations containing one or more of the
compounds described herein can be administered to treat microbial
infections, such as bacterial infection. Assays have been developed
to assess compounds for their ability to inhibit enzyme activity,
protein transport (using a vesicle or whole cell system), and
bacterial viability.
[0378] SecA exerts its transporter functions while integrated into
membrane in a bound form with the SecYEG complex. However, SecA's
ATPase is functional in solution alone or in a membrane. In
addition, SecA itself has a C-terminal regulatory sequence. Thus
there are several ways to test SecA inhibitory activities. The
ATPase activity can be examined using SecA alone in solution
(intrinsic/regulated ATPase), truncated SecA without the C-terminal
inhibitory sequence in solution (e.g., EcSecAN68, unregulated
ATPase), SecA in membrane (membrane ATPase), and SecA in complex
with SecYEG in membrane (translocation ATPase).
[0379] For functional assays, the in vitro translocation of proOmpA
into E. coli membrane vesicles (protein translocation), can be
used. A sensitive semi-physiological assay for electrophysiological
measurement of protein-channel activity in the oocytes has also
been developed. This assay is valuable, because of the ease of use,
the small amount of materials (nanograms) needed, and the ability
to study individual oocytes. The large size of oocytes can easily
accommodate various manipulations and electrode penetration. The
recording noise is very low from a large number of channels
(calculated to be 200-1,000,000 channels). The activity is strictly
dependent on the injection of exogenous SecA and membrane vesicles.
Liposomes have been developed for measuring SecA activity that
allows for easy demonstration that SecA alone can form a
protein-conducting channel. The liposome system in the oocytes
allows the sensitive detection of channel activities of various
SecA (SecA2 has no channel activity) including S. aureus SecA1
(SaSecA1) and S. pyogenes SecA1 (SpSecA1).
[0380] To evaluate antimicrobial activity, the initial enzyme
screening was done with the truncated form (unregulated ATPase) or
soluble SecA2 because of its ease of use and sensitivity. The
truncated EcSecAN68 SecA ATPase, membrane SecA ATPase, and membrane
transport experiments revealed the intrinsic ability for the
compounds to bind and inhibit the most relevant forms of the
transporter/ATPase.
[0381] In one embodiment, membrane channel activities may be
monitored by introducing a proteo-liposome, such as SecA-liposomes
in oocytes. Preferably, the proteo-liposomes are purified
reconstituted proteo-liposomes. In this embodiment, the expression
of the SecA-liposome is very efficient, reaching up to 80%,
preferably up to 90%, more preferably up to 95% of the expression
rate, within hours of the injected oocytes. The oocytes can be
reconstituted with membrane protein complexes, such as SecYEG and
SecDF YajC, to achieve more specific and efficient ion-channel
activities. This method shortens the channel expression time and
increases the expression rate, and allows for monitoring channel
activities for protein-protein interaction in the oocytes. The
injection of liposomes having encapsulated therein SecA homologs
also allows similar assessments for other bacterial systems, which
otherwise lack the homologs assays due to the strain specificity
for translocation ATPase or protein translocation. The inhibitory
effect of various SecA inhibitors may be evaluated by injecting
liposomes containing either SecA or SecA coupled to SecYEG at
various concentrations of a SecA inhibitor. Example 3 demonstrates
the inhibitory effect.
[0382] Three structural classes of nM inhibitors of SecA have been
developed.
##STR00082##
The inhibitors identified include (1) Rose Bengal (RB) analogs
(Class A), (2) pyrimidine analogs (Class B), and (3) triazole
analogs (Class C). Kinetic studies using selected analogs against
EcSecA clearly suggest competitive inhibition against ATP at low
ATP concentrations indicating the binding pocket being that of ATP.
Such knowledge is critical to the computational work. At high ATP
concentrations, the inhibition is non-competitive, presumably
because of the existence of a secondary low-affinity ATP binding
site.
[0383] A number of SecA inhibitors have shown potent inhibition of
protein translocation at high nM concentrations in an in vitro
(vesicle) model and in vivo oocyte model. For example, RB inhibits
protein translocation at IC.sub.50 of 250 nM. In the oocyte assay,
RB (Class A) showed IC.sub.50 of 400 nM in inhibiting SecA (S.
pyogenes, S. aureus, and E. coli); SCA-8 (Class B) and SCA-107
(Class C) showed IC.sub.50 of 500-900 nM. The inhibitory
sensitivity of these assays parallels that of bacterial growth
inhibition.
[0384] Selected inhibitors have shown potent antimicrobial effects
including against drug-resistant bacteria such as S. aureus Mu50.
In side-by-side comparisons, the inhibition potency for some SecA
inhibitors surpasses that of commonly used antibiotics such as
tetracycline (by more than 200 fold) and vancomycin (by up to
12-fold). For example, against drug resistant S. aureus Mu50 (MRSA
and vancomycin-resistant), the MIC.sub.95 values are 1.7 and 2.4
.mu.M for RB analogs SCA-41 and SCA-50, 4.5 .mu.M for pyrimidine
analog SCA-93, and 1.5, 0.5, and 0.4 .mu.M for triazole analogs
SCA-21, SCA-107, and SCA-112. In contrast, the MIC.sub.95 values
are 5 .mu.M for vancomycin, and over 100 .mu.M for kanamycin,
gentamycin, tetracycline, erythromycin and other antibiotics
tested. For a highly virulent strain of S. pyogenes, MGAS5005, the
situation is similar. The MIC.sub.95 values for RB, SCA-15, SCA-21,
SCA-50, SCA-93, SCA-107, and SCA-112 are 6.25, 3.13, 0.39, 6.25,
0.78 .mu.M and 0.19 .mu.M respectively.
[0385] SecA functions in the membrane as a protein-conducting
channel. It is possible that SecA is accessible from the
extracellular matrix and thus not susceptible to the effect of
efflux, which is a common multidrug resistance (i.e., MDR)
mechanism in general and in S. aureus and S. pyogenes,
specifically. I Interestingly, most SecA1 in S. pyogenes is present
in the membranes as micro-domain `ExPortal`, and it was found that
80-90% of SecA1 are in the membranes of S. pyogenes and S. aureus.
Experimental evidence suggests that expression of various efflux
pumps has no effect on the antimicrobial effects of the SecA
inhibitors that were tested. For example, it was found that the MIC
(bacteriostatic) did not increase and bactericidal (killing) effect
was not attenuated for SCA-41 (Class A), SCA-15 (Class B), and
SCA-21 (Class C) with the over expression of efflux pumps in S.
aureus. Bacterial strains used include wild type (S. aureus Mu50,
8325 or 6538), deletion strains (NorA-, MepA-) and strains (NorA++,
MepA++) with over-expressed efflux pumps. Such results strongly
support the hypothesis that SecA inhibitors can overcome the effect
of efflux and thus may not be subjected to multi-drug resistance
problems.
[0386] It has also been demonstrated that SecA inhibition results
in inhibition of virulence factor secretion. Specifically, SecA
inhibitors such as SCA-15 can inhibit the secretion of hemolysin,
enterotoxin B, and toxic shock syndrome toxin (TSST) by the MRSA
Mu50 strain.
[0387] A summary of the in vitro inhibition effects is shown in
Table 1:
TABLE-US-00001 TABLE 1 Summary of in vitro inhibition effects.
IC.sub.50(.mu.M) Protein RB SCA-41 SCA-50 SCA-8 SCA-15 SCA-21
SCA-107 SCA-112 Intrincsic BsSECA 20 30 33 8 >100 >100
>200 >200 ATPase BsSecA2 15 30 20 7 20 45 65 ND SaSecA2 1 6
ND 3 13 43 50 ND EcSecA 1 8 4 2 8 18 30 20 N68 EcSecA 60 30 60
>100 30 32 28 ND Translocation EcSecA 1 15 60 6 30 20 28 ND
ATPase Protein EcSecA 1 55 38 50 >100 21 25 5 Translocation Ion
Channel EcSecA 0.4 3.4 2.3 1.5 4.2 2.4 1.6 1.3 activity SaSecA1 0.4
3.4 1.1 0.5 2 1.6 0.6 1 BGaSecA1 0.4 3.8 1 0.9 2.8 1.5 0.7 1 PASecA
0.3 3.6 3 1.5 3.2 1.5 1.3 1.1 BsSecA 0.3 3 2.5 1.2 3 2.6 2.1 2.3
MsSecA 0.4 3.5 2.5 1.3 3 2 2.5 2.3 MtbSecA 0.5 3.2 3 1.7 3.1 2 2 2
SpSecA 0.9 3 1.9 1.5 3.5 1 0.7 1.3
[0388] A comparison of the activities of the compounds described
herein with other antibiotics is shown in Table 2:
TABLE-US-00002 TABLE 2 Comparison of the activities of RB analogs
and known antibiotics against SecA inhibition. Strains S. aureus
Mu50 B. anthracis Sterne Bacteriostatic Bacteriostatic MIC.sub.95
MIC.sub.95 Antbiotics (.mu.g/ml) Bactericidal (.mu.g/ml)
Bacteriostatic RB & analogs RB 40.7 + 12.2 ND SCA-41 1.7 ND 1.1
+ SCA-50 2.4 + 1.7 + Pyrimidine SCA-15 10.9 + 2.2 + analogs SCA-93
4.5 ND 1.6 ND Bistriazole SCA-21 1.5 + 3.0 + analogs SCA-112 0.4 ND
0.8 ND Glycopeptides Vancomycin 5 + 2.5 + Penicillins Ampicillin
7.8 + >20 + Aminoclycosides Gentamycin >500 + 5 +
Polypeptides Polymyxin B 15 + 10 + Tetracyclines Tetracycline 200 -
0.1 - Macrolides Erythromycin >500 - 0.3 - Other Chloramphenic
>40 - 10 -
[0389] A. Dosages
[0390] The precise dosage administered to a patient will depend on
many factors, including the physical characteristics of the patient
(e.g., weight), the degree of severity of the disease or disorder
to be treated, and the presence or absence of other complicating
diseases or disorders and can be readily determined by the
prescribing physician.
[0391] In certain embodiments, the compound(s) is administered at a
dosage equivalent to an oral dosage of between about 0.005 mg and
about 500 mg per kg of body weight per day, more preferably between
about 0.05 mg and about 100 mg per kg of body weight per day, most
preferably between about 0.1 mg and about 10 mg per kg of body
weight per day.
[0392] B. Therapeutic Administration
[0393] Pharmaceutical formulations may be administered, for
example, in a single dosage, as a continuous dosage, one or more
times daily, or less frequently, such as once a week. The
pharmaceutical formulations can be administered once a day or more
than once a day, such as twice a day, three times a day, four times
a day or more. In certain embodiments, the formulations are
administered orally, once daily or less.
[0394] The pharmaceutical formulations are administered in an
effective amount and for an effective period of time to elicit the
desired therapeutic benefit. In certain embodiments, the
pharmaceutical formulation is administered for a period of at least
one week, two weeks, three weeks, four weeks, one month, two
months, three months, four months, five months, six months, seven
months, eight months, nine months, ten months, eleven months, one
year, or longer.
[0395] The pharmaceutical formulations may also be administered
prophylactically, e.g., to patients or subjects who are at risk for
infection.
[0396] The exact amount of the formulations required will vary from
subject to subject, depending on the species, age, sex, weight and
general condition of the subject, extent of the disease in the
subject, route of administration, whether other drugs are included
in the regimen, and the like. Thus, it is not possible to specify
an exact dosages for every formulation. However, an appropriate
dosage can be determined by one of ordinary skill in the art using
only routine experimentation. For example, effective dosages and
schedules for administering the compositions may be determined
empirically, and making such determinations is within the skill in
the art.
[0397] Dosage can vary, and can be administered in one or more dose
administrations daily, for one or several days. Guidance can be
found in the literature for appropriate dosages for given classes
of pharmaceutical products.
[0398] 1. Co-Administration with Active Agents
[0399] In other embodiments, the compounds disclosed herein can be
co-administered with one or more additional therapeutic,
prophylactic, or diagnostic agents. Co-administration, as used
herein, includes administration within the same dosage form or
within different dosage forms. For those embodiments where the
compounds described herein and the one or more additional
therapeutic, prophylactic, or diagnostic agents are administered in
different dosage forms, the dosage forms can be administered
simultaneously (e.g., at the same time or essentially at the same
time) or sequentially. "Essentially at the same time" as used
herein generally means within ten minutes, preferably within five
minutes, more preferably within two minutes, most preferably within
in one minute. Dosage forms administered sequentially can be
administered within several hours of each other, e.g., with ten
hours, nine hours, eight hours, seven hours, six hours, five hours,
four hours, three hours, two hours, one hour, 30 minutes, 20
minutes, or 15 minutes.
EXAMPLES
Example 1. Model SecA Inhibitors
[0400] General
[0401] Strains and plasmids used in this study were: E. coli K-12
strain MC4100, NR698 (MC4100 imp4213), a leaky mutant with
increased outer membrane permeability supplied by Thomas J. Slhavy
(Princeton University, USA); BA13 (MC4100 secA13(am) supF(ts)),
pT7-SecA and pT7div supplied by D. B. Oliver; pIMBB28 obtained from
Prof. Anastasios Economou (University of Athens, Greece);
F1F0-proton ATPase-enriched membrane of E. coli strain KY7485
supplied by Prof. William S. Brusilow (Wayne State University,
USA); B. subtilis strain 168 (lab stock). Luria-Bertani (LB) liquid
and solid (1.5% agar) media with glucose (0.2%) were used for
bacterial growth.
[0402] Fluorescein analogues were purchased from Sigma-Aldrich (St.
Louis, Mo., USA) and were dissolved in H.sub.2O (for Rose Bengal,
erythrosin B, and fluorescein) or DMSO (for diiodofluorescein,
eosin Y, and dinitrofluorescein).
[0403] Bacteriostatic and Bactericidal Effects
[0404] Plate assay: A 0.5 mL culture of bacterial cells
(exponential phase, OD600=0.5) was mixed with LB (4 mL)
supplemented with glucose (0.2%) and soft agar (0.75%) and then
poured into petri dishes. After the soft agar solidified, test
compound (1 mL) was spotted on the surface of the culture.
Bacteriostatic effects were judged by the appearance of a clear
zone of growth inhibition after overnight incubation at 37.degree.
C.
[0405] Liquid culture assay: Bacterial cells of exponential phase
(OD600=0.5-0.8) were diluted to an OD600 value of 0.05 with LB
supplemented with glucose (0.2%). The diluted culture (90 mL) was
incubated with inhibitor or H.sub.2O as control (10 mL) at
37.degree. C. with shaking (1000 rpm, Eppendorf Thermomixer R,
Eppendorf, Germany). After 14 h of incubation, the OD600 value was
determined. The inhibition of cell growth (or bacteriostatic
effects) was evaluated using the relative decrease in the OD600
value.
[0406] Bactericidal effect assay: The inhibitor or H.sub.2O as
control (40 .mu.L) was added to bacteria cultures (360 .mu.L,
exponential phase, OD600=0.5). After 1 h of incubation at
37.degree. C., cultures were spread on LB agar plates after serial
dilution, and the colony forming units (CFU) of surviving cells
were counted after overnight incubation at 37.degree. C.
[0407] Protein Preparation
[0408] The N-terminal catalytic domain of SecA from E. coli (EcN68)
was overexpressed from pIMBB28. EcN68 was used for the early and
initial screening because it has higher intrinsic activity and is
more sensitive to inhibitors. The full-length SecA from E. coli
(EcSecA) and B. subtilis (BsSecA) were overexpressed from pT7-SecA
and pT7div, respectively. SecA proteins were purified as previously
described. F1F0-proton ATPase-enriched membrane of E. coli strain
KY7485 was prepared as described in the literature. F1F0-proton
ATPase was partially purified by sucrose-gradient fractionation and
then reconstituted into liposomes by dialysis. Non-radiolabeled and
[.sup.35S]-labeled proOmpA were purified as previously described.
SecA-depleted BA13 membrane vesicles were prepared as described in
the literature, [32] and washed with 6M urea to reduce endogenous
ATPase activity.
[0409] In Vitro ATPase Activity Assay
[0410] ATPase activity assays were performed as described
previously with minor modifications. For the intrinsic ATPase
assay, the reaction mixture (50 .mu.L) contained EcN68 (1.8 .mu.g),
EcSecA (1.5 .mu.g), or BsSecA (1.5 .mu.g), ovalbumin (20 .mu.g),
ATP (1.2 mM), Tris-HCl (50 mM, pH 7.6), KCl (20 mM), NH.sub.4Cl (20
mM), Mg(OAc).sub.2 (2 mM), and DTT (1 mM). For the membrane ATPase
assay, the reaction mixture (50 .mu.L) was supplemented with
urea-washed E. coli BA13 membrane (3 rig). The reaction mixture for
the translocation ATPase assay also contained proOmpA (1 .mu.g) in
addition to the BA13 membrane. For the proton ATPase activity,
reconstituted liposomes containing partially purified
F.sub.1F.sub.0-proton ATPase were assayed using the same conditions
as in the intrinsic ATPase assay. All reactions were carried out at
40.degree. C. for an appropriate time in the linear ranges of the
activity assay that was determined by the release of inorganic
phosphate detected by the photometric method, with absorption
measured at 660 nm (SmartSpec Plus, Bio-Rad Laboratories, Inc.,
Hercules, Calif., USA). The inhibitory effects are given as the
percentage (%) of remaining ATPase activity relative to the
controls in the absence of test compounds. All assays were
performed at least in triplicate, and the results are expressed as
the mean.+-.standard error of the mean (SEM).
[0411] In Vitro Protein Translocation Assay
[0412] The assay was performed as previously described using
[.sup.35S]-labeled proOmpA as a marker.[34] The protease-resistant
translocated proteins were analyzed by SDS-PAGE, autoradiographed,
and quantified by a densitometer (GS-800 Calibrated Densitometer,
Bio-Rad, Hercules, Calif., USA).
[0413] Molecular Simulation of Docking Complexes
[0414] The structures of DI, EB, RB and CJ-21058 were docked into
the ATP site of EcSecA using DOCK 6 to generate their predicted
binding pose. Residues within a radius of 6 angstroms around the
center of ATP were defined as the active site to construct a grid.
The active site included residues Gly 80, Met81, Arg82, His 83,
Phe84, Gln 87, Arg103, Thr 104, Gly 105, Glu 106, Gly 107, Lys 108,
Thr 109, Leu110, Arg138, Asp209, Glu 210, Arg 509, and Gln 578. The
subsequent computational work was conducted as described
previously. Briefly, the docked complexes were solvated by using
the TIP3P water model, and then subjected to 500 steps of molecular
mechanics minimization and molecular dynamics simulations at 300 K
for 1.5 ns using the SANDER module in the AMBER 8 program.
[0415] Results
[0416] A series of fluorescein analogues were screened against
EcSecA using the intrinsic ATPase of the truncated N-terminal
catalytic domain EcN68 (unregulated ATPase). Those fluorescein
analogues with significant IC50 values are shown in Table 3.
TABLE-US-00003 TABLE 3 Screen of fluorescein analogs using EcN68
SecA ATPase Compound IC.sub.50 [.mu.m] Rose bengal (RB) 0.5
Erythrosin B (EB) 2 Dildofluorescein (DI) 30 Eosin Y (EY) 25
Dinitrofluorescein (DN) 50 Sodium azide >10 [a] Fluorescein
analogues were applied to the intrinsic ATPase assay of EcN68 as
described in the Experimental Section.
[0417] Among the screened compounds, RB and EB were the most
effective with IC50 values of 0.5 .mu.M and 2 .mu.M, respectively.
Since RB and EB are known to inhibit a number of ATPases from
animal tissues, we tested whether these compounds inhibit other E.
coli ATPases, such as the F.sub.1F.sub.0-proton ATPase. The
IC.sub.50 values of RB and EB against F.sub.1F.sub.0-proton ATPase
are approximately 10 .mu.M and 30 .mu.M, respectively. The data
indicate that RB and EB could be general ATPase inhibitors.
However, they are more effective on the catalytic SecA ATPase. It
has been previously reported that some ATPases from animal tissues
can be inhibited by RB and EB through photo-oxidation and
subsequent reactions.
[0418] In order to fully understand the ability of these
fluorescein analogues to inhibit the biological relevant SecA
ATPase, the effect of these compounds on all three forms of the
SecA ATPase was investigated. The inhibitory effects on the
full-length SecA alone (regulated intrinsic ATPase) were evaluated.
As expected, the IC.sub.50 values (.about.20-30 .mu.M) for RB and
EB are higher than those measured against the unregulated ATPase
(truncated SecA, EcN68). The inhibitory effects of RB and EB on the
membrane and translocation ATPase activities of EcSecA was also
investigated. It is interesting to note that both RB and EB show
the following trends in terms of their affinity for the different
forms of SecA ATPase: unregulated ATPase (EcN68), translocation
ATPase, membrane ATPase and intrinsic ATPase. RB showed IC.sub.50
values of 0.5, 0.9 and 5 .mu.M for unregulated, translocation and
membrane ATPase activities respectively. In the presence of the
C-terminal domain (i.e., the native regulated form of SecA ATPase),
the IC50 value is higher (25 .mu.M). EB shows a similar trend in
inhibiting the different forms of SecA ATPase, that is, higher
potency against unregulated ATPase (truncated SecA), translocation
and membrane ATPase than the regulated intrinsic ATPase
(full-length SecA) activities. However, the potency of EB is lower
than that of RB with IC.sub.50 values of approximately 10-20 .mu.M.
The significant differences in sensitivities of the three ATPase
forms of EcSecA also indicate that conformational changes of SecA
induced by the interaction with membranes and precursors can
influence the accessibility of the enzyme to inhibitors. In
addition, the inhibition profile of RB and EB onSecA from
Gram-positive B. subtilis (BsSecA), which has a high homology (51%
identity) to EcSecA and much higher intrinsic ATPase activity, was
also determined. As expected, both RB and EB show inhibitory
effects on the intrinsic ATPase activity of BsSecA, with RB as the
more potent inhibitor.
[0419] The inhibition of ATPase activity is only relevant if it
also results in the inhibition of protein translocation. Therefore,
the effects of RB and EB on the SecA-dependent protein
translocation in vitro were investigated. It was found that the in
vitro translocation of precursor proOmpA into membrane vesicles is
severely inhibited by RB and EB. Interestingly, the SecA-dependent
protein translocation is about three- to four-times more sensitive
to RB and EB than the translocation ATPase activity. Consistent
with the result against translocation ATPase activity, RB shows a
stronger inhibitory effect on protein translocation (IC50=0.25
.mu.M) than EB (IC50=4 .mu.M). Sodium azide is a well-known SecA
ATPase inhibitor; however, the intrinsic ATPase of SecA is not
inhibited by sodium azide at concentrations as high as 10 mM.
According to a previous report, the inhibitory effects of sodium
azide against the translocation ATPase activity of SecA
(IC.sub.50=5 mM) and the in vitro protein translocation
(IC.sub.50=0.6 mM) are moderate. On the other hand, RB inhibits
both the translocation ATPase activity and in vitro protein
translocation very efficiently, with IC.sub.50 values of 0.9 .mu.M
and 0.25 .mu.M, respectively, which are approximately several
thousand-times more effective than sodium azide.
[0420] The SecA-dependent protein translocation is essential for
maintaining the normal physiology of bacteria. The above-mentioned
fluorescein analogues inhibit bacterial growth in plate assays. E.
coli .mu.MC4100 (wild-type), a Gram-negative bacteria, is very
resistant to the fluorescein analogues, while its permeable leaky
mutant NR698 shows high sensitivity. Such results suggest that the
outermembrane barrier could be the reason for the observed
difference in activity. Among the tested fluorescein analogues,
diiodofluorescein (DI), eosin Y (EY), and dinitrofluorescein (DN)
show a MIC values in the millimolar range, while RB and EB exhibit
stronger inhibitions with MIC values in the micromolar range. RB
also completely inhibits the growth of E. coli NR698 in liquid
culture at low concentrations (50 mm, data not shown). RB
demonstrates the same level of bacteriostatic activity with or
without 0.2% glucose supplemented to the media, suggesting that
F1F0-proton ATPase is not the primary target of the inhibition. The
observed inhibition effect against bacterial growth validates the
idea that SecA inhibitors can be used as antimicrobial agents. The
inhibitory potency of RB is in the single-digit micromolar range,
which is similar to the IC.sub.50 values obtained using truncated
SecA and SecA in the presence of membrane and precursor proteins.
In the case of EB, the MIC value is much higher than the IC.sub.50
values obtained in the ATPase inhibition assays. As seen with the
results obtained using the wild-type strain of E. coli, minimal
inhibition is observed. However, when the leaky mutant NR698 was
used, the inhibitory potency increased substantially.
[0421] It is interesting to note that sodium azide has been
reported to inhibit the translocation ATPase activity of SecA and
the transport of a Gram-negative bacteria, is very resistant to the
fluorescein analogues, while its permeable leaky mutant NR698 shows
high sensitivity. Such results suggest that the outermembrane
barrier could be the reason for the observed difference in
activity. Among the tested fluorescein analogues, diiodofluorescein
(DI), eosin Y (EY), and dinitrofluorescein (DN) show a MIC values
in the millimolar range, while RB and EB exhibit stronger
inhibitions with MIC values in the micromolar range. RB also
completely inhibits the growth of E. coli NR698 in liquid culture
at low concentrations (50 .mu.M). RB demonstrates the same level of
bacteriostatic activity with or without 0.2% glucose supplemented
to the media, suggesting that F.sub.1F.sub.0-proton ATPase is not
the primary target of the inhibition.
[0422] The observed inhibition effect against bacterial growth
validates the idea that SecA inhibitors can be used as
antimicrobial agents. The inhibitory potency of RB is in the
single-digit micromolar range, which is similar to the IC.sub.50
values obtained using truncated SecA and SecA in the presence of
membrane and precursor proteins. In the case of EB, the MIC value
is much higher than the IC.sub.50 values obtained in the ATPase
inhibition assays. Many reasons could contribute to such results. A
key consideration is permeability. As seen with the results
obtained using the wild-type strain of E. coli, minimal inhibition
is observed. However, when the leaky mutant NR698 was used, the
inhibitory potency increased substantially.
[0423] It is interesting to note that sodium azide has been
reported to inhibit the translocation ATPase activity of SecA and
the transport of precursor proteins across the inner membrane
vesicles in vitro. SecA mutants that lack the stimulated
translocation ATPase activity show defects of preprotein
translocation in vitro. The in vitro translocation of precursor
protein proOmpA into membrane vesicles is also inhibited by RB and
EB. The in vitro translocation is even more sensitive to RB and EB
than the translocation ATPase of EcSecA. Similar differences are
also reported for sodium azide, but the in vitro protein
translocation and the cell growth show similar sensitivities. In
the case of RB and EB, in vivo growth is significantly less
sensitive than in vitro protein translocation. This again could be
due to the different membrane permeability of inhibitors. While
sodium azide is a small inorganic molecule, RB and EB are much
larger organic molecules that presumably exhibit lower permeability
through bacterial membranes.
[0424] Since the permeability is important for the antibacterial
effect of RB and EB, Gram-positive bacteria B. subtilis without the
barrier of the outer-membrane were also examined. B. subtilis shows
high sensitivities toward fluorescein analogues similar to the
leaky E. coli mutant NR698. Indeed, RB and EB are very effective
against Gram-positive bacteria where permeability is not a major
problem.
[0425] In addition to the bacteriostatic studies, bactericidal
effects were also investigated. After a one-hour treatment of
exponential-phase cells, the colony-forming units (CFU) were
determined after overnight incubation. RB showed strong
bactericidal effects in a concentration dependent manner. With 100
.mu.M of RB, cell survival decreased about 10 log units in leaky
mutant E. coli NR698 and 8 log units in B. subtilis. The cell
density did not decrease in the presence of 100 .mu.M RB up to
incubation times of 90 min, indicating that the bactericidal
effects of RB on both bacteria were not caused by cell lysis. It
has been reported that RB can inhibit the growth and kill
Staphylococcus aureus in dark with unknown mechanisms, while some
halogenated fluoresceins work as the photosensitizer in
antimicrobial actions to kill various other bacteria, mainly
through photo-oxidation. As discussed earlier, under the
experimental condition in this study, photo-oxidation was not
likely the primary mechanism of the bacteriostatic and bactericidal
effects. Taken together, the results suggest that SecA could be the
target of fluorescein analogues, and the inhibition of ATPase and
SecA-dependent protein translocation might contribute to the
antibacterial effects.
[0426] Because of the literature reports of other fluorescein
analogues binding to enzymes containing nucleotide binding sites,
in silico modeling was performed. Results from kinetic experiments
suggest that RB and EB are competitive inhibitors against ATP at
low ATP concentrations. Such results indicate that these compounds
bind to the high-affinity ATP binding site. Thus, the structures of
RB, EB, and DI were docked into the high-affinity ATP binding site.
RB and EB show very similar predicted binding profiles, while DI
shows a different conformation because of the lack of the diiodo
moiety. For comparison, the binding mode of translocation
activities of SecA, and bacterial growth might lead to alternative
antimicrobial strategies. The fluorescein analogues used in this
study are hydroxyxanthenes. Xanthene derivatives are well known and
have been used as food additives for some time. Although some
xanthene dyes have safety concerns, ten of those dyes could be
approved by the US Food and Drug Administration (FDA) for food,
drug, or cosmetic use RB is reportedly in phase II clinical trials
for the treatment of metastatic melanoma. EB is at present the only
xanthene derivative with FDA-approval for use in food. These
fluorescein analogues have several advantages as SecA inhibitors:
the convenience of commercial availability, high solubility in
water, known chemical structure for further modification, and
relatively low or no toxicity for food and drug use.
Example 2: Rose Bengal Analogs as SecA Inhibitors
General
Bacterial Strain and Growth Conditions
[0427] An outer membrane leaky mutant strain, E. coli NR698 (Ruiz
et al., Cell, 2005, 121:307-317; provided by Thomas J Silhavy of
Princeton University) and B. subtilis 168 (lab stock) were grown in
Luria-Bertani (LB) medium at 37.degree. C.
[0428] Protein Preparation
[0429] EcSecAN68, a truncated mutant of EcSecA containing the
N-terminal catalytic domain, EcSecA, and BsSecA were used to study
the in vitro inhibition effect of RB analogs. These proteins were
purified as previously described (Chen et al., J. Biol. Chem. 1996,
271:29698-29706; Chen et al., J. Bacteriol. 1998, 180:527-537).
[0430] In Vitro ATPase Activity Assay
[0431] The malachite green colorimetric assay was used to determine
the inhibition effect of RB analogs against the ATPase activity of
SecA proteins. In this assay, ATPase assays were carried out at
different concentrations of the inhibitor, and IC.sub.50 was
defined as the concentration of the compound, which could inhibit
50% ATPase activity of the enzyme. Because RB analogs were
dissolved in 100% DMSO, there was 5% DMSO in the final assay.
[0432] Bacteriostatic Effect
[0433] Bacteriostatic effects were tested by a liquid microdilution
method according to the guidelines of the Clinical and Laboratory
Standards Institute (Performance standards for antimicrobial
susceptibility testing. M100-S21; 21st informational supplement.
Clinical and Laboratory Standards Institute, Wayne, Pa. 2011). This
assay was performed in a 96-well microtiter tray under normal room
light condition. All bacteria were grown in LB broth, and when the
OD.sub.600 reach 0.5, the culture was diluted to
OD.sub.600.apprxeq.0.05. 97.5 .mu.l diluted culture and 2.5 .mu.l
of compound were added to each well. Cells were incubated at
37.degree. C. with shaking (250 rpm) for 24 hr. MIC is the lowest
concentration of inhibitors at which cells were not able to
grow.
[0434] Bactericidal Effect
[0435] B. subtilis 168 was grown in LB broth. When OD.sub.600
reached 0.5, 97.5 .mu.l culture and 2.5 .mu.l compound were added
into a 1.5-mL Eppendorf tube. After incubation at 37.degree. C.
with shaking (1000 rpm) for 1 hr, cultures were serially diluted
with LB and spread on LB plate. Bactericidal effect was determined
by counting the number of reduced viable colonies. This assay was
performed under normal room light condition.
[0436] SecA-Lipsomes Ion-Channel Activity Assays in the Oocytes
[0437] The liposomes were prepared as described previously (Hsieh
et al., J. Biol. Chem. 2011, 286, 44702-44709; Lin et al., J.
Membr. Biol. 2006, 214, 103-113; Lin et al., J. Membr. Biol. 2012,
245, 747-757). E. coli total lipids (Avanti) were dried,
re-suspended in 150 mM KCl, and sonicated in an ice water bath
until the solution became clear (usually for 3-5 mins). Samples of
the liposomes were stored at -80.degree. C. and thawed only once
before use. Oocytes were obtained from live frog Xenopus laevis
(Xenopus Express, Inc) and injected with sample mixtures as
described. 50 nl of the sample mixtures were injected into dark
pole site of oocytes using Nanoject II injector (Drummond
Scientific Co., Broomall, Pa.). The ion current was recorded three
hours after injection. The amount for each component is 120 ng
liposomes, 120 ng SecA, 14 ng proOmpA, 2 mM ATP, and 1 mM
Mg.sup.2+. The effective concentration of each component in the
oocytes was based on the average volume of oocytes of 500 nl.
[0438] Synthesis of Rose Bengal Analogs
3-Bromo-1-(2-hydroxyphenyl) propan-1-one (3)
[0439] To a mixture of resorcinol 1 (10 g, 91 mmol) and
3-chloropropionic acid 2 (10 g, 92 mmol) was added trifluoromethane
sulfonic acid (29.6 mL) in one portion. After stirring at
80.degree. C. for 30 min, the reaction mixture was cooled to room
temperature and poured into 40 mL dichloromethane (DCM) and 40 mL
water. The organic layer was separated and the aqueous layer was
extracted with DCM twice. The combined organic layers was washed
with water and brine, dried over Na2SO4, then filtered, and
evaporated under reduced pressure. The crude product 3 (11.4 g) was
used directly for the next step.
7-Hydroxychroman-4-one (4)
[0440] To a solution of 2 N NaOH 400 mL was added crude product 3
(11.4 g) at 0-5.degree. C. in one portion. The solution was warmed
up to room temperature over 2 hr, then acidified with 6N
H.sub.2SO.sub.4 to pH.about.4, and finally extracted with ethyl
acetate. The combined organic layers was washed with water and
brine, dried over Na.sub.2SO.sub.4, filtered, and evaporated under
reduced pressure to give the crude product 4, which was dried under
vacuum overnight and used directly for the next step.
7-Methoxychroman-4-one (5)
[0441] To a solution of 4 in 200 mL acetone was added
K.sub.2CO.sub.3 (10 g, 72.5 mmol) and excess amount of iodomethane
(5 mL, 80.1 mmol). Then the reaction mixture was heated at reflux
for 3 hr. The solid was filtered off and solvent was evaporated
under reduced pressure. The resulting residue was purified by
silica gel column chromatography (hexane: ethyl acetate 5:1) to
give 5 (8.5 g, 53% for 3 steps). 1H-NMR (CDCl3): .delta. 7.86-7.83
(d, J=8.8 Hz, 1H), 6.60-6.58 (d, J=8.8 Hz, 1H), 6.42 (s, 1H),
4.54-4.52 (t, J=5.2 Hz, 1H), 3.85 (s, 3H), 2.78-2.75 (t, J=4.8 Hz,
1H); ESIMS: 179.1 [M+H].sup.+.
7-Methoxy-3'-H-spiro[chroman-4,1'-isobenzofuran]-3'-imine (6)
[0442] To a solution of 2-bromobenzonitrile (250 mg, 1.37 mmol) in
5 mL THF was added 2.5 M n-BuLi (0.55 mL, 1.37 mmol) at -78.degree.
C., The reaction mixture was kept stirring under this condition for
40 min. Then 5 (150 mg, 0.91 mmol) in 4 mL THF was added slowly and
the reaction mixture was stirred for another 30 min at the same
temperature, before the reaction temperature was warmed up to room
temperature over a period of 1 hr. The reaction was stopped with
the addition of saturated NH.sub.4Cl and the mixture extracted with
DCM. The DCM solution was washed with water and brine, and dried
over Na.sub.2SO.sub.4. The solid was filtered off and the filtrate
was evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (hexane: ethyl acetate 5:1) to
give 6 (165 mg, 64%). .sup.1H-NMR (CDCl.sub.3): .delta. 7.95-7.94
(d, J=6.8 Hz, 1H), 7.59-7.52 (m, 2H), 7.19-7.17 (d, J=6.8 Hz 1H),
6.52-6.46 (m, 2H), 6.40-6.37 (dd, J=2.8, 8.8 Hz, 1H), 4.49-4.47
(dd, J=2.4, 7.2 Hz, 2H), 3.78 (s, 3H), 2.56-2.50 (m, 1H), 2.19-2.15
(d, J=14.8 Hz, 1H); .sup.13C-NMR (CDCl.sub.3): .delta. 166.3,
161.4, 156.4, 149.6, 132.6, 130.0, 129.6, 129.2, 123.8, 122.0,
113.7, 108.4, 101.3, 84.0, 63.1, 55.2, 36.0; ESI-MS: 282.1
[M+H].sup.+.
7-Methoxy-3'H-spiro[chroman-4,1'-isobenzofuran]-3'-one (7)
[0443] To a solution of 6 (205 mg, 0.73 mmol) in 10 mL ethanol and
10 mL water was added NaOH (0.5 g, 12.5 mmol). The reaction mixture
was heated at reflux for 3.5 hr before cooling down to room
temperature and acidification with 4 N HCl to pH.about.5. Then the
reaction mixture was extracted with ethyl acetate, washed with
water and brine, dried over Na2SO.sub.4, and filtered. Solvent
evaporation under reduced pressure gave a residue, which was
purified by silica gel column chromatography (hexane: ethyl acetate
10:1) to yield 7 (110 mg, 54%). .sup.1H-NMR (CDCl.sub.3): .delta.
7.98-7.96 (d, J=7.6 Hz, 1H), 7.71-7.68 (t, J=6.8 Hz 1H), 7.62-7.58
(t, J=7.2 Hz, 1H), 7.29-7.27 (d, J=7.2 Hz, 1H), 6.46-6.35 (m, 3H),
4.52-4.49 (d, J=11.2 Hz, 2H), 3.77 (s, 3H), 2.65-2.57 (m, 1H),
2.18-2.14 (d, J=14.4 Hz, 1H); .sup.13C-NMR (CDCl.sub.3): .delta.
169.3, 161.7, 156.8, 152.4, 134.5, 129.6, 129.5, 126.9, 125.6,
122.3, 112.4, 108.6, 101.4, 82.6, 63.2, 55.3, 35.9; GC-MS: 282
[M].
7-(Methoxychroman-4-yl) benzoic acid (8)
[0444] Compound 8 was synthesized following the same procedure for
the preparation of 5a in 92% yield. .sup.1H-NMR (CDCl.sub.3):
.delta. 8.10-8.08 (dd, J=0.8, 7.6 Hz, 1H), 7.48-7.44 (dt, J=1.2,
7.2 Hz, 1H), 7.35-7.31 (dt, J=1.2, 7.6 Hz, 1H), 7.12-7.09 (t, J=6.0
Hz, 1H), 6.72-6.70 (d, J=8.4 Hz, 1H), 6.47-6.42 (m, 2H), 5.23-5.19
(t, J=6.0 Hz, 1H), 4.22-4.17 (m, 2H), 3.84 (s, 3H), 2.48-2.44 (m,
1H), 2.11-2.04 (m, 1H); .sup.13C-NMR (CDCl.sub.3): .delta. 172.8,
159.3, 156.3, 148.4, 132.7, 131.4, 131.2, 130.8, 128.4, 126.3,
117.1, 107.7, 101.3, 63.9, 55.2, 36.4, 31.4; ESI-MS: 307.2
[M+Na].sup.+; GC-MS: 284 [M].
2-(7-Hydroxychroman-4-yl) benzoic acid (9)
[0445] To a solution of 8 (20 mg, 0.07 mmol) in DCM (2 mL) was
slowly added 1M BBr.sub.3 (0.21 mL, 0.21 mmol) in DCM at
0-5.degree. C. under N.sub.2 atmosphere. After stirring at the same
temperature for 2 hr, the reaction was stopped with the addition of
ice water before extraction with DCM. The combined organic layers
was washed with water and brine, dried over Na2SO.sub.4, and
filtered before solvent evaporation under reduced pressure. The
crude product was purified by silica gel column chromatography
(hexane: acetate 10:1) to afford 9 (12 mg, 64%). .sup.1H-NMR
(CDCl.sub.3): .delta. 8.09-8.07 (d, d, J=1.6, 8.0 Hz, 1H),
7.48-7.44 (m, 1H), 7.35-7.29 (m, 1H), 7.11-7.09 (d, J=7.6 Hz, 1H),
6.67-6.65 (d, J=8.4 Hz, 1H), 6.41-6.41 (d, J=2.4 Hz, 1H), 6.36-6.39
(d, d, J=2.8, 8.4 Hz, 1H), 5.20-5.17 (t, J=9.2 Hz, 1H), 4.19-4.15
(m, 2H), 2.48-2.42 (m, 1H), 2.10-2.05 (m, 1H); .sup.13C-NMR
(CDCl.sub.3): .delta. 156.3, 155.20, 148.3, 132.7, 131.5, 131.3,
130.7, 128.4, 126.3, 117.3, 108.4, 103.2, 63.9, 36.4, 31.4, 30.9;
ESI-MS: 293.4 [M+Na].sup.+.
2-(6,8-dibromo-7-hydroxychroman-4-yl) benzoic acid (10a) and
2-(7-hydroxy-6,8-diiodochroman-4-yl) benzoic acid (10b)
[0446] For 10a: the same procedure for the preparation of 23a was
followed with a 62% yield. .sup.1H-NMR (CDCl.sub.3): .delta.
8.21-8.29 (dd, J=1.2, 7.6 Hz, 1H), 7.77-7.73 (dt, J=1.6, 7.6 Hz,
1H), 7.61-7.56 (dt, J=1.2, 7.6 Hz, 1H), 7.40-7.39 (dd, J=0.8, 7.2
Hz, 1H), 7.00 (s, 1H), 4.71-4.68 (m, 1H), 4.32-4.27 (m, 1H),
3.40-3.36 (t, J=8.0 Hz, 1H), 2.48-2.24 (m, 1H), 2.23-2.17 (m, 1H);
.sup.13C-NMR (CDCl.sub.3): .delta. 172.6, 162.7, 161.9, 139.7,
138.6, 135.8, 131.0, 129.4, 127.9, 126.6, 122.5, 106.8, 68.3, 38.3,
30.9, 30.3; ESI-MS: 429.2, 427.4, 426.0 [M+H].sup.+.
[0447] 10b: the same procedure for the preparation of 23b was used
in 65% yield. .sup.1H-NMR (CDCl.sub.3): .delta. 8.24-8.22 (d, J=8.0
Hz, 1H), 7.77-7.73 (dt, J=1.2, 7.6 Hz, 1H), 7.62-7.58 (dt, J=1.2,
8.0 Hz, 1H), 7.38-7.36 (d, J=7.6 Hz, 1H), 7.30 (s, 1H), 4.74-4.68
(m, 1H), 4.30-4.24 (m, 1H), 3.32-3.28 (t, J=7.6 Hz, 1H), 2.37-2.19
(m, 2H); ESI-MS: 543.0 [M+Na].sup.+.
1-Bromo-6-methoxynaphthalene (12)
[0448] To a suspension of anhydrous CuBr.sub.2 (77 mg, 0.35 mmol)
in anhydrous MeCN was added tert-butyl nitrite in one portion. The
reaction mixture was stirred for 30 min at room temperature under
N.sub.2 atmosphere. A solution of 11 (50 mg, 0.29 mmol) in 2 mL
MeCN was added to the suspension slowly and the resulting mixture
was stirred for 1 hr at room temperature, and then poured into 2 mL
1N HCl. The organic phase was separated and the aqueous layer was
extracted with ethyl acetate. The combined organic layers was
washed with saturated NaHCO.sub.3 and brine, and dried over
Na2SO.sub.4. The solid was filtered off and the solvent was
evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (hexane: acetate 20:1) to give 12
(10 mg, 14%). .sup.1H-NMR (CDCl.sub.3): .delta. 8.22-8.20 (d, J=9.2
Hz, 1H), 7.68-7.66 (d, J=8.0 Hz, 1H), 7.47-7.44 (dd, J=1.2, 7.6 Hz,
1H), 7.38-7.34 (t, J=8.0 Hz, 1H), 7.30-7.27 (dd, J=2.4, 9.2 Hz,
1H), 7.16-7.15 (d, J=2.8 Hz, 1H), 3.95 (s, 3H); .sup.13C-NMR
(CDCl.sub.3): .delta. 158.2, 135.9, 131.9, 126.4, 126.2, 126.0,
126.0, 123.9, 119.7, 106.1, 55.3.
Methyl 2-(6-methoxynaphthalen-1-yl) benzoate (13)
[0449] A solution of 12 (50 mg, 0.2 mmol), (2-(methoxycarbonyl)
phenyl) boronic acid (80 mg, 0.44 mmol), Pd(PPh.sub.3).sub.4 (30
mg, 0.026 mmol), and K.sub.2CO.sub.3 (65 mg, 0.47 mmol) in 3 mL DMF
was heated at 90-100.degree. C. under N.sub.2 atmosphere overnight.
The reaction mixture was cooled to room temperature before water
was added. The reaction mixture was extracted with ethyl acetate.
The combined organic layers were washed with water and brine, and
dried over Na.sub.2SO.sub.4. The solid was filtered off and the
solvent was evaporated under reduced pressure. The crude product
was purified by silica gel column chromatography (hexane: acetate
25:1) to afford 13 (34 mg, 56%). .sup.1H-NMR (CDCl.sub.3): .delta.
8.04-8.02 (dd, J=1.2, 8.0 Hz, 1H), 7.78-7.76 (d, J=8.0 Hz, 1H),
7.64-7.60 (dt, J=1.2, 7.6 Hz 1H), 7.54-7.40 (m, 4H), 7.22-7.19 (m,
2H), 7.07-7.04 (dd, J=2.8, 9.2 Hz, 1H), 3.98 (s, 3H), 3.42 (s, 3H),
2.19-2.15; ESI-MS: 293.2 [M+H].sup.+.
2-(6-Methoxynaphthalen-1-yl) benzoic acid (14)
[0450] To a solution of 13 (130 mg, 0.4 mmol) in 2.5 mL ethanol was
added 1N NaOH (2.2 mL, 2.2 mmol). The reaction mixture was heated
at reflux for 4 hr. The reaction mixture was cooled to room
temperature and acidified with 2N HCl to pH.about.5. The mixture
was extracted with ethyl acetate. The combined organic layers were
washed with water and brine, and dried over Na.sub.2SO.sub.4. The
solid was filtered off and the solvent was evaporated under reduced
pressure to afford 14 (123 mg, 100%). .sup.1H-NMR (CDCl.sub.3):
.delta. 8.10-8.08 (dd, J=1.2, 7.6 Hz, 1H), 7.76-7.74 (d, J=8.0 Hz,
1H), 7.65-7.61 (dt, J=1.2, 7.6 Hz 1H), 7.54-7.36 (m, 4H), 7.20-7.16
(m, 2H), 7.05-7.02 (dd, J=2.8, 9.2 Hz, 1H), 3.98 (s, 3H); ESI-MS:
279.4 [M+H].sup.+, 301.2 [M+Na].sup.+.
2-(6-Hydroxynaphthalen-1-yl) benzoic acid (15)
[0451] To a solution of 14 (24 mg, 0.086 mmol) in DCM (2 mL) was
added 1M BBr.sub.3 (0.26 mL, 0.26 mmol) in DCM slowly at
0-5.degree. C. under N.sub.2 atmosphere. After stirring at the same
temperature for 2 hr, the reaction was stopped with the addition of
ice water. The reaction mixture was extracted with DCM. The
combined organic layers were washed with water and brine, and dried
over Na.sub.2SO.sub.4. The solid was filtered off and the solvent
was evaporated under reduced pressure. The crude product was
purified by silica gel column chromatography (hexane: acetate 10:1)
to afford 15 (15 mg, 67%). .sup.1H-NMR (CD.sub.3OD): .delta.
7.99-7.97 (dd, J=1.2, 7.6 Hz, 1H), 7.63-7.57 (m, 2H), 7.52-7.48
(dt, J=1.2, 7.6 Hz 1H), 7.39-7.31 (m, 3H), 7.15-7.15 (d, J=2.4 Hz,
1H), 7.08-7.06 (dd, J=2.4, 6.8 Hz, 1H), 6.96-6.93 (dd, J=2.8, 9.2
Hz, 1H), 4.93 (s, br, 1H); .sup.13C-NMR (CD.sub.3OD): .delta.
169.8, 154.7, 141.4, 139.6, 135.1, 132.3, 131.5, 131.0, 129.4,
127.1, 127.0, 126.9, 125.5, 125.2, 123.0, 117.7, 108.8; ESI-MS:
263.2 [M-H].sup.-.
2-(6-Hydroxy-5-iodonaphthalen-1-yl) benzoic acid (16)
[0452] Compound 16 was synthesized following the same procedure as
that of 24b in 35% yield. .sup.1H-NMR (CD.sub.3OD): .delta.
8.09-8.06 (d, J=8.8 Hz, 1H), 8.01-7.99 (dd, J=1.2, 7.6 Hz, 1H),
7.63-7.594 (dt, J=1.2, 7.2 Hz, 1H), 7.55-7.47 (m, 2H), 7.33-7.13
(t, J=9.2 Hz, 2H), 7.15-7.13 (dd, J=0.8, 6.8 Hz 1H), 7.00-6.98 (d,
J=9.2 Hz 1H); .sup.13C-NMR (CD.sub.3OD): .delta. 169.4, 155.0,
141.1, 140.2, 135.6, 132.1, 131.5, 131.1, 129.8, 129.6, 127.6,
127.5, 127.3, 126.7, 123.7, 116.0, 83.5; ESI-MS: 389.2
[M-H].sup.-.
3,6-Dihydroxy-9H-xanthen-9-one (18a)
[0453] 2,2',4,4'-Tetrahydroxybenzophenone (5 g, 20.3 mmol) was
heated at 210-220.degree. C. (sand bath) in a 75 mL round-bottom
pressure flask for 4 hr. The yellow powder in the reaction mixture
changed to brown solid. The crude product was used for the next
step without purification. .sup.1H-NMR (DMSO-D.sub.6): .delta.
10.81 (s, 2H), 7.99-7.97 (d, J=8.8 Hz, 2H), 6.87-6.81 (m, 4H);
.sup.13C-NMR (DMSO-D.sub.6): .delta. 174.3, 163.8, 157.9, 128.2,
114.4, 114.1, 102.5; ESI-MS: 229.2 [M+H].sup.+.
2,4,5,7-Tetrabromo-3,6-dihydroxy-9H-xanthen-9-one (18b)
[0454] To a solution of 18 (500 mg, 2.2 mmol) and 49% HBr (1.8 mL,
10.96 mmol) in methanol (11 mL) and water (11 mL) was added 30%
H.sub.2O.sub.2 (1.18 mL, 9.9 mmol) slowly at 0-5.degree. C. The
reaction mixture was allowed to warm to room temperature and
stirred for an additional 4 hr. The solvent was evaporated under
reduced pressure at room temperature, and the crude residue with
brown color was purified by silica gel column chromatography
(hexane: acetate 10:1) to afford 18b (715 mg, 60%). .sup.1H-NMR
(DMSO-D.sub.6): .delta. 8.19-8.19 (d, J=0.8 Hz, 2H); .sup.13C-NMR
(CDCl.sub.3): .delta. 172.3, 157.4, 153.2, 128.9, 115.7, 109.1;
ESI-MS: 540.9, 542.8, 546.9 [M-H].sup.-.
3,6-Dihydroxy-4,5-diiodo-9H-xanthen-9-one (18c)
[0455] To a solution of 18 (500 mg, 2.2 mmol), KI (96 mg, 5.79
mmol) and KIO.sub.3 (619 mg, 2.89 mmol) in methanol (4 mL) and
water (16 mL) was added 1M HCl (8.93 mL, 8.93 mmol) slowly at room
temperature and the reaction mixture was stirred overnight. The
reaction was stopped with the addition of ice water and extracted
with ethyl acetate. The combined ethyl acetate was washed with
water and brine and dried over Na.sub.2SO.sub.4, and filtered.
Solvent evaporation under reduced pressure followed by silica gel
column chromatography (hexane: acetate 20:1) afforded 18c (598 mg,
57%). .sup.1H-NMR (DMSO-D.sub.6): .delta. 11.70 (s, 2H), 8.02-7.97
(dd, J=0.8, 8.4 Hz, 2H), 7.03-7.01 (dd, J=0.8, 8.4 Hz, 2H); ESI-MS:
480.8 [M+H].sup.+.
3,6-Dimethoxy-9H-xanthen-9-one (19)
[0456] In a 100 mL round-bottom flask, 18 (1 g, 4.4 mmol),
K.sub.2CO.sub.3 (0.9 g, 6.6 mmol), MeI (1.1 mL, 17.5 mmol), and 50
mL acetone were added and the reaction mixture was heated at reflux
for 3 hr. The reaction mixture was filtered and washed with ethyl
acetate twice. The combine organic layers were evaporated and
purified by silica gel column chromatography (hexane: acetate 5:1)
to give compound 19 (2.3 g, 45% from 17). .sup.1H-NMR (CDCl.sub.3):
.delta. 8.23-8.20 (dd, J=1.2, 8.8 Hz, 2H), 6.92-6.89 (dt, J=2.0,
8.8 Hz, 1H), 6.83 (s, 6H); .sup.13C-NMR (CDCl.sub.3): .delta.
176.1, 164.7, 158.0, 128.2, 115.7, 112.9, 100.2, 55.8; ESI-MS:
295.2 [M+K].sup.+.
9-Cyclopentylidene-3,6-dimethoxy-9H-xanthene (20a)
[0457] To a suspension of magnesium (307 mg, 12.8 mmol) in 100 mL
anhydrous THF was added cyclopropyl bromide (1.4 mL, 12.5 mmol).
The mixture was maintained at reflux temperature for 3 hr. At that
point, the magnesium was almost completely disappeared. The
reaction was cooled down to room temperature. A solution of 19 (1
g, 4.1 mmol) in 20 mL anhydrous THF was added slowly to the
reaction mixture. The resulting mixture was stirred at room
temperature overnight. Saturated NH.sub.4Cl was added before
extraction with ethyl acetate. The combined organic layers were
washed with water and brine, dried over Na.sub.2SO.sub.4, and
filtered. Solvent evaporation under reduced pressure yields a brown
residue. After purification with silica gel column chromatography
(hexane: acetate 20:1), 20a (780 mg, 65%) was obtained as a light
yellow solid. .sup.1H-NMR (CDCl.sub.3): .delta. 7.41-7.99 (t,
J=4.8, 4.4 Hz, 2H), 6.72-6.70 (m, 4H), 3.86 (s, 6H), 2.69-2.65 (t,
J=6.8 Hz, 4H), 1.72-1.68 (m, 4H); .sup.13C-NMR (CDCl.sub.3):
.delta. 158.9, 154.0, 138.8, 128.6, 119.5, 118.9, 108.8, 101.0,
55.4, 33.5, 25.9; ESI-MS: 309.5 [M+H].sup.+.
3,6-Dimethoxy-9-(propan-2-ylidene)-9H-xanthene (20b)
[0458] Compound 20b was synthesized following the same procedure as
that of 20a in 55% yield. .sup.1H-NMR (CDCl.sub.3): .delta.
7.34-7.32 (d, J=8.4 Hz, 2H), 6.76-6.70 (m, 4H), 3.84 (s, 6H), 2.11
(s, 6H); .sup.13C-NMR (CDCl.sub.3): .delta. 158.8, 155.0, 128.9,
127.6, 121.7, 119.7, 108.8, 101.4, 55.4, 23.3; ESI-MS: 283.5
[M+H].sup.+.
9-Cyclopentyl-3,6-dimethoxy-9H-xanthene (21a)
[0459] To compound 20a (100 mg, 0.32 mmol) in 20 mL methanol was
added a catalytic amount of 10% Pd--C. The reaction was degassed
under vacuum and flushed with hydrogen 3 times. The reaction
mixture was hydrogenated with an H.sub.2 balloon for 2 hr. Then the
reaction mixture was passed through silica gel in a small funnel
and flushed with 2 mL of methanol. After solvent evaporation, the
crude product was purified by silica gel column chromatograph
(hexane: acetate 15:1) to afford 21a (98 mg, 97%). .sup.1H-NMR
(CDCl.sub.3): .delta. 7.11-7.09 (d, J=8.4 Hz, 2H), 6.68-6.65 (m,
4H), 3.83 (s, 3H), 3.76-3.74 (d, J=8.4 Hz, 1H), 1.96-1.94 (d, J=6.0
Hz, 1H), 1.61-1.21 (m, 8H); .sup.13C-NMR (CDCl.sub.3): .delta.
159.0, 153.4, 129.5, 118.1, 109.3, 101.3, 55.4, 50.4, 42.0, 29.4,
24.2; ESI-MS: 311.3 [M+H].sup.+.
9-Isopropyl-3,6-dimethoxy-9H-xanthene (21b)
[0460] Compound 21b was synthesized following the same procedure as
that of 21a in 87% yield. .sup.1H-NMR (CDCl.sub.3): .delta.
7.11-7.08 (dd, J=3.2, 6.8 Hz, 2H), 6.70-6.68 (m, 4H), 3.85 (s, 3H),
3.74 (m, 1H), 1.92 (m, 1H), 0.82-0.80 (dd, J=2.0, 6.8 Hz, 6H);
.sup.13C-NMR (CDCl.sub.3): .delta. 159.1, 153.6, 129.8, 116.6,
109.4, 101.1, 55.3, 44.4, 38.0, 18.8; ESI-MS: 285.2
[M+H].sup.+.
9-Hexyl-3,6-dimethoxy-9H-xanthene (21c)
[0461] Compound 21c was synthesized following the same procedure as
that of 21a in 78% yield. .sup.1H-NMR (CDCl.sub.3): .delta.
7.12-7.12 (d, J=7.6 Hz, 2H), 6.70-6.67 (m, 4H), 3.93 (m, 1H), 3.84
(m, 6H), 1.72-1.70 (m, 2H), 1.25-1.20 (m, J=2.4 Hz, 8H), 0.88-0.85
(t, J=6.4 Hz, 3H); .sup.13C-NMR (CDCl.sub.3): .delta. 159.0, 152.8,
129.1, 117.8, 109.7, 101.2, 55.3, 41.0, 37.5, 31.8, 29.4, 25.2,
22.6, 14.1; ESI-MS: 325.1 [M+H].sup.+.
9-Cyclohexyl-3,6-dimethoxy-9H-xanthene (21d)
[0462] Compound 21d was synthesized following the same procedure as
that of 21a in 79% yield. .sup.1H-NMR (CDCl.sub.3): .delta.
7.09-7.06 (t, J=4.4 Hz, 2H), 6.99-6.66 (m, 4H), 3.8 (s, 6H),
3.70-3.69 (d, J=4.0 Hz, 1H), 1.69-1.58 (m, 6H), 1.14-0.88 (m, 5H);
.sup.13C-NMR (CDCl.sub.3): .delta. 159.0, 153.7, 129.8, 117.0,
109.4, 101.1, 55.3, 48.0, 44.3, 29.3, 26.5, 26.2; ESI-MS: 325.1
[M+H].sup.+.
9-Cyclopentyl-9H-xanthene-3,6-diol (22a)
[0463] To a solution of 20a (440 mg, 1.4 mmol) in DCM (35 mL) was
slowly added 1M BBr.sub.3 (7 mL, 7 mmol) in DCM at 0-5.degree. C.
under N.sub.2 atmosphere. After stirring at the same temperature
for 2 hr, the reaction was stopped with the addition of ice water
and then extracted with DCM. The combined DCM layers was washed
with water and brine, dried over Na2SO.sub.4, and filtered. Solvent
evaporation under reduced pressure followed by purification by
silica gel column chromatography (hexane: acetate 10:1) afforded
22a (254 mg, 64%). .sup.1H-NMR (CD.sub.3OD): .delta. 6.94-6.92 (t,
J=4.4 Hz, 2H), 6.53-6.51 (m, 4H), 3.54-3.52 (d, J=6.4 Hz 1H),
1.81-1.78 (m, 1H), 1.39-1.29 (m, 6H), 1.15-1.10 (m, 2H);
.sup.13C-NMR (CD.sub.3OD): .delta. 156.2, 153.4, 129.5, 117.2,
109.9, 102.4, 50.4, 41.8, 29.0, 23.9; HRMS-ESI Calcd for
C.sub.18H.sub.18O.sub.3: 282.3337. Found: 281.1173 [M-H].sup.-;
ESI-MS: 281.3 [M-H].sup.-.
9-Isopropyl-9H-xanthene-3,6-diol (22b)
[0464] Compound 22b was synthesized following the same procedure as
that of 22a in 63% yield. .sup.1H-NMR (CD.sub.3OD): .delta.
6.98-6.96 (d, J=8.4 Hz, 2H), 6.56-6.49 (m, 4H), 3.61-3.60 (d, J=4.0
Hz 1H), 1.81-1.78 (m, 1H), 0.72-0.70 (d, J=6.4 Hz, 6H);
.sup.13C-NMR (CD.sub.3OD): .delta. 156.4, 153.5, 129.6, 115.5,
109.9, 102.1, 44.1, 37.8, 17.8; ESI-MS: 255.1 [M-H].sup.-.
9-Hexyl-9H-xanthene-3,6-diol (22c)
[0465] Compound 22c was synthesized following the same procedure as
that of 22a in 59.5% yield. .sup.1H-NMR (DMSO): .delta. 9.46 (s,
2H), 7.91-7.89 (d, J=9.6 Hz, 2H), 6.52-6.50 (d, J=8.0 Hz, 2H), 6.43
(s, 2H), 3.81 (m, 1H), 1.98-0.74 (m, 13H); .sup.13C-NMR
(CDCl.sub.3): .delta. 157.1, 152.5, 129.6, 116.1, 111.2, 102.7,
41.0, 36.7, 31.6, 29.1, 24.9, 22.4, 14.3; ESI-MS: 297.3
[M-H].sup.-.
9-Cyclohexyl-9H-xanthene-3,6-diol (22d)
[0466] Compound 22d was synthesized following the same procedure as
that of 22a in 67% yield. .sup.1H-NMR (CD.sub.3OD): .delta.
6.95-6.93 (d, J=8.0 Hz, 2H), 6.54-6.49 (m, 4H), 3.56-3.55 (d, J=4.0
Hz, 1H), 1.62-1.36 (m, 6H), 1.08-0.77 (m, 5H); .sup.13C-NMR
(CD.sub.3OD): .delta. 156.3, 153.6, 129.6, 115.9, 109.9, 102.1,
47.7, 44.0, 29.0, 26.2, 26.1; HRMS-ESI: Calcd for
C.sub.19H.sub.10O.sub.3: 296.36. Found: 295.1346 [M-H].sup.-;
ESI-MS: 295.0 [M-H].sup.-.
2,4,5,7-Tetrabromo-9-cyclopentyl-9H-xanthene-3,6-diol (23a)
[0467] To a solution of 22a (82 mg, 0.29 mmol) and 49% HBr (0.24
mL, 1.45 mmol) in methanol (1 mL) was slowly added 30%
H.sub.2O.sub.2 (0.15 mL, 1.31 mmol) at 0-5.degree. C. Then the
reaction was warmed to room temperature and stirred for an
additional 2 hr. The solvent was evaporated under reduced pressure
at room temperature and the crude orange product was purified by
silica gel column chromatography (hexane: acetate 10:1) afford 23a
(103 mg, 60%). .sup.1H-NMR (CDCl.sub.3): .delta. 7.41 (S, 2H),
3.75-3.73 (d, J=6.8 Hz, 1H), 2.03-0.89 (m, 9H); .sup.13C-NMR
(CDCl.sub.3): .delta. 150.5, 149.3, 130.5, 119.5, 104.5, 100.0,
49.7, 42.4, 29.0, 23.8; ESI-MS: 596.8, 598.7 [M+H].sup.+.
9-Cyclopentyl-2,4,5,7-tetraiodo-9H-xanthene-3,6-diol (23b and
23c)
[0468] To a solution of 22a (134 mg, 0.48 mmol), KI (165.7 mg, 1.28
mmol) and KIO.sub.3 (135 mg, 0.63 mmol) in methanol (0.26 mL) and
water (1.54 mL) was slowly added 1M HCl (1.99 mL, 1.99 mmol) at
room temperature. The the reaction was stirred overnight before the
addition of ice water to stop the reaction. The reaction mixture
was extracted with ethyl acetate and the combined ethyl acetate
layers were washed with water and brine, and dried over
Na.sub.2SO.sub.4. After filtering off the solid, the solvent was
evaporated under reduced pressure. The crude product was purified
with silica gel column chromatography (hexane: acetate 20:1) to
afford 23b (156 mg, 42%) and 23c (53 mg, 17%). 23b: .sup.1H-NMR
(CDCl.sub.3): .delta. 7.53 (s, 2H), 5.92 (s, bro, 2H), 3.69-3.67
(d, J=6.8 Hz 1H), 1.90 (m, 1H), 1.61-1.15 (m, 8H); .sup.13C-NMR
(CDCl.sub.3): .delta. 153.3, 153.3, 137.8, 120.8, 74.4, 74.1, 50.0,
42.4, 29.4, 24.0; HRMS-ESI (-): Calcd for
C.sub.18H.sub.14I.sub.4O.sub.3: 785.9198. Found: 784.7060
[M-H].sup.-. ESI-MS: 784.8 [M-H].sup.-. 23c: .sup.1H-NMR
(CDCl.sub.3): .delta. 7.50 (s, 1H), 7.47 (s, 1H), 6.91 (s, 1H),
5.86 (s, bro, 2H), 3.68-3.66 (d, J=6.4 Hz, 1H), 1.87 (m, 1H),
1.54-1.44 (m, 6H), 1.13 (m, 2H); HRMS-ESI: Calcd for
C.sub.18H.sub.15I.sub.3O.sub.3: 660.0233. Found: 658.8079
[M-H].sup.-. ESI-MS: 659.1 [M-H].sup.-.
2,4,5,7-Tetraiodo-9-isopropyl-9H-xanthene-3,6-diol (23d)
[0469] The synthesis of 23d followed the same procedure as for 23b
in yield 43%. .sup.1H-NMR (CDCl.sub.3): .delta. 7.50 (s, 2H), 5.93
(s, 2H), 3.65-3.64 (d, J=4.0 Hz 1H), 1.88-1.83 (m, 1H), 0.98-0.74
(m, 6H); .sup.13C-NMR (CDCl.sub.3): .delta. 153.3, 153.3, 138.1,
119.1, 74.6, 74.0, 44.5, 38.0, 18.6, 14.2; ESI-MS: 758.8
[M-H].sup.-.
2-(3-Acetamidophenoxy)-4-nitrobenzoic acid (26,27)
[0470] To a solution of 24 (1.5 g, 7.44 mmol) in DMF (40 mL) was
added 25 (1.24 g, 8.19 mmol), K.sub.2CO.sub.3 (1.5 g, 10.9 mmol)
and copper powder (61 mg, 0.85 mmol). The reaction mixture was
heated at 130.degree. C. overnight. The reaction was cooled to room
temperature and poured slowly over an iced 1N HCl solution (150
mL). The mixture was stirred until a brown solid formed. The solid
was filtered and washed with cold water to give 26.
[0471] The crude solid was dissolved in concentrated sulfuric acid
(10 mL) and heated at 80.degree. C. for 1 hr. After cooling to room
temperature, the reaction mixture was poured into ice (150 mL) and
stirred for 1 hr. The precipitate was filtered and re-suspended in
2.5% aq. sodium carbonate. The solid was filtered and washed with
cold water and dried under vacuum overnight. Product 27 was used
for the next step directly without further purification.
.sup.1H-NMR (DMSO): .delta. 8.36-8.29 (m, 2H), 8.15-8.13 (m, 2H),
7.88-7.86 (d, J=8.8 Hz, 1H), 6.76-6.55 (m, 4H); ESI-MS: 279.0
[M+Na].sup.+.
3,6-Diamino-9H-xanthen-9-one (28)
[0472] To a solution of 27 (1.20 g, 4.22 mmol) in ethanol (100 mL)
was added SnCl.sub.2 (3.80 g, 16.88 mmol). The mixture was heated
at reflux overnight. The solvent was evaporated under reduced
pressure and residue was basified with 1N NaOH (80 mL) resulting in
brown precipitates, which was directly used for the next step.
3,6-Bis(dimethylamino)-9H-xanthen-9-one (29)
[0473] To a solution of 28 (1 g, 4.42 mmol) in 20 mL DMF was added
K.sub.2CO.sub.3 (3.66 g, 26.5 mmol) and iodomethane (1.65 mL, 26.5
mmol). The reaction mixture was heated at 100.degree. C. overnight
before being cooled down to room temperature and addition of 100 mL
DCM. The reaction mixture was washed with water and brine, dried
over Na.sub.2SO.sub.4, and filtered. Solvent evaporation under
reduced pressure gave a crude product, which was purified by column
chromatography (hexane: acetate 10:1 to 2:1) to afford 29 (975 mg,
78%). .sup.1H-NMR (CDCl.sub.3): .delta. 8.13-8.08 (d, J=5.2 Hz,
2H), 6.77-6.71 (m, 2H), 6.52-6.49 (M, 2H), 3.12 (S, 12H); ESI-MS:
283.1 [M+H].sup.+.
9-Cyclopentyl-N3,N3,N6,N6-tetramethyl-9H-xanthene-3,6-diamine
(30)
[0474] To a suspension of magnesium (64 mg, 2.67 mmol) in 10 ml
anhydrous THF was added cyclopropyl bromide (0.27 mL, 2.5 mmol).
The reaction was heated at reflux for 3 hr. At that point the
magnesium almost completely disappeared. The reaction was cooled
down to room temperature. A solution of 29 (100 mg, 0.35 mmol) in
10 mL anhydrous THF was added slowly to the reaction mixture. The
reaction was stirred at room temperature overnight. Saturated
NH.sub.4Cl was added before extraction of the reaction mixture with
ethyl acetate. The combined organic layers were washed with water
and brine, dried over Na.sub.2SO.sub.4, and filtered. Solvent
evaporation under reduced pressure resulted in a brown residue,
which was directly used for the next step.
[0475] To the crude product in 10 mL methanol was added a catalytic
amount of 10% Pd--C. The mixture was degassed under vacuum before
flushing with hydrogen 3 times. Hydrogenation was carried out at
room temperature with a balloon filled with hydrogen. The reaction
mixture was passed through silica gel in a small funnel followed by
washing 2 times with methanol. Solvent evaporation under reduced
pressure followed by purification by silica gel column
chromatography (hexane: acetate 15:1) afforded 30 (64 mg, 54%).
.sup.1H-NMR (CDCl.sub.3): .delta. 7.10-7.08 (d, J=8.0 Hz, 2H),
6.53-6.51 (m, 2H), 3.74 (m, 1H), 3.03 (s, 12H), 2.01 (m, 1H),
1.58-1.46 (m, 8H); .sup.13C-NMR (CDCl.sub.3): .delta. 153.7, 150.2,
129.4, 114.6, 107.6, 100.2, 50.8, 41.6, 40.7, 29.5, 24.3; ESI-MS:
337.1 [M+H].sup.+.
[0476] Results
[0477] To evaluate the inhibitory effect of synthesized Rose Bengal
("RB") analogs (Table 4), EcSecA N68 was used for the initial
enzymatic ATPase screening assay. EcSecA N68 is a truncated protein
of E. coli SecA that lacks the down regulatory C-terminus, which
allosterically inhibit the ATPase activity, and is the best SecA
protein for screening a large number of compounds as described
previously (Chen et al., Bioorg Med Chem 2010, 18(4), 1617-1625;
Huang et al., ChemMedChem 2012, 7(4), 571-577). The initial
screening was conducted at 100 .mu.M. As can be seen from FIG. 1,
two series of RB analogs, 22a-d and 23a-d showed significant
inhibition of enzyme activities. RB analogs containing the `D-ring`
(ring bearing the carbonyl group) and the chloro groups from ring A
removed, exhibited substantially reduced activity or essentially no
activity. Compounds with these showed no antimicrobial activity
against E. coli NR698 (MIC: >250 .mu.M) either. Masking the
hydroxyl group in 22a-d with a methyl group (21a-d, Table 4) or
replacing hydroxyl group with --N(CH.sub.3).sub.2 (30) also
resulted in compounds with weak or no activity (FIG. 1).
[0478] Analogs that showed substantial inhibition in the initial
screening were evaluated in the channel activity assay using both
EcSecA and BsSecA. This is a semi-physiological assay in the
oocytes (Hsieh et al., J. Biol. Chem. 2011, 286, 44702-44709; Lin
et al., J. Membr. Biol. 2006, 214, 103-113; Lin et al., J. Membr.
Biol. 2012, 245, 747-757) developed to measure SecA-mediated
protein-channel activity in a liposome environment, which closely
mimics the situation in bacteria. This method serves as an
excellent confirmative assay and is used for the generation of
quantitative data for SAR work. In the channel activity assay, many
compounds showed potent inhibitory activities (Table 5). The
potency is about the same against EcSecA and BsSecA with the
exception of 22d, which is more potent against EcSecA than BsSecA
by about 2-fold. The results suggest that the 9-position of
xanthene can tolerate a fairly large degree of modifications
including aryl groups and cycloaliphatic and linear aliphatic
substitutions. Further, the synthesized new analogs do not need to
have a carboxyl group on the group attached to the 9-position to
show potency. Such results suggest that the biologically active
form of RB is most like the lactone form, not the ring opening with
a free carboxylate group. Such cyclization resulting from a Michael
addition type of reaction of the quinoid moiety is well known for
this class of compounds including fluorescein. For example, the
lactone form is commercially available. Further studies with
decarboxylate RB also showed inhibition potency equal or better
than RB itself.
[0479] To study the antimicrobial effect of these compounds, the
active analogs against E. coli NR698, a leaky mutant, and B.
subtilis 168 were evaluated. In the antimicrobial assay, all the
non-halogenated analogs (22a-d) showed weak inhibitory activities
with MIC in the double-digit micromolar range (Table 5). However,
the halogenated analogs (23a-d), although with higher molecular
weights, showed potent antimicrobial activities against both E.
coli NR698 and B. subtilis 168. Against E. coli NR698, 23a-d showed
equal or more potent activities than RB with single digit
micromolar MIC values. Against B. subtilis 168, RB only showed very
weak activity with MIC value of 100 .mu.M. However, 23a had an MIC
of 22 .mu.M and the other halogenated analogs (23b-d) had MIC in
the single digit micromolar range. The non-halogenated analogs
(22a-d) with much lower molecular weight also showed more potent
activity than RB with MIC in the range of 13-75 .mu.M. Overall, the
synthetic analogs were more potent than RB in antimicrobial
assays.
[0480] The in vitro enzymatic activity and ion-channel activity
assays of these analogs do not always parallel that of
antimicrobial activities. On one hand, this is not surprising since
antimicrobial activities also depend on permeability and
solubility, among other factors. For example, the higher molecular
weight and the charged carboxylate group of RB could easily impede
its membrane permeability and thus lead to reduced antimicrobial
activity. Such phenomenon has been observed in other SecA analogs
(Chen et al., Bioorg Med Chem 2010, 18:1617-1625; Huang et al.,
ChemMedChem 2012, 7:571-577). In addition, the modified RB analogs
do not have the same planarity issues as RB and thus may not stack
and aggregate as much, which should help improve solubility and
consequently permeability.
[0481] Bactericidal studies were conducted and 20 .mu.M of 22a or
22c was found sufficient to kill 4-5 logs of B. subtilis 168 in one
hour while RB had little effect (FIG. 2). Thus although the
enzymatic inhibition potency of these analogs is not as good as RB,
the antimicrobial activity is much stronger. These results also
show the importance of using multiple assays in screening and
assessing SecA inhibitors.
TABLE-US-00004 TABLE 4 Structures of RB analogs ##STR00083##
##STR00084## Comp ID MW R R1 R2 R3 R4 R5 R6 RB 1017.6 chlorinated I
I I I --NaO .dbd.O benzoate 18a 228.2 .dbd.O H H H H OH OH 18b
543.8 .dbd.O Br Br Br Br OH OH 18c 480.0 .dbd.O H I I H OH OH 20a
308.4 cyclopentylidene H H H H OMe OMe 20b 282.3 propane-2-lidene H
H H H OMe OMe 21a 310.4 cyclopentane H H H H OMe OMe 21b 284.4
iso-propyl H H H H OMe OMe 21c 326.4 n-hexyl H H H H OMe OMe 21d
324.4 cyclohexane H H H H OMe OMe 22a 282.3 cyclopentyl H H H H OH
OH 22b 256.3 iso-propyl H H H H OH OH 22c 298.2 n-hexyl H H H H OH
OH 22d 296.2 cyclohexyl H H H H OH OH 23a 597.9 cyclopentyl Br Br
Br Br OH OH 23b 785.9 cyclopentyl I I I I OH OH 23c 660.0
cyclopentyl I I H I OH OH 23d 759.9 iso-propyl I I I I OH OH 30
336.1 cyclopentyl H H H H NMe.sub.2 NMe.sub.2
TABLE-US-00005 TABLE 5 Biological activities of RB analogs Ion
channel, Comp IC.sub.50 (.mu.M) MIC (.mu.M) 0 ID MW EcSecA BsSecA
E. coli NR698 B. subtilis 168 RB 1017.6 0.4 0.3 5 100 22a 282.3 3.4
3.0 45 25 22b 256.3 4.3 4.9 90 75 22c 298.2 2.3 2.5 19 13 22d 296.3
2.8 6.6 25 22 23a 597.9 2.3 2.4 2 22 23b 785.9 2.5 3.8 1 6 23c
660.0 2.2 2.8 6 6 23d 759.9 2.8 2.5 4 6
[0482] Summary
[0483] In summary, twenty three new RB analogs were successfully
synthesized and evaluated. The result of SAR studies indicated that
(1) the xanthene ring is important for activity; (2) the
chlorinated benzoate position can tolerate fairly substantial
modifications and an aryl ring is not essential; (3) a carboxyl
group is not important for activity; and (4) halogen substitution
of the xanthene ring is important.
Example 3: Injection of Proteoliposomes in Oocytes as a Tool for
Monitoring Membrane Channel Activities
[0484] Liposomes Preparation
[0485] E. coli total lipids extracts or synthetic lipids (Avanti
Polar Lipid, Inc) were dried in a Thermo Savant vacuum and
resuspended in TAK buffer containing Tris-HCl 50 mM pH 7.6, 20 mM
NH.sub.4Cl and 25 mM KCl. The suspension was subjected to
sonication (Fisher Scientific Sonic Dismembrator Model 500) at an
amplitude of 70% for 8 to 10 minutes with a two minute pause in a
0.degree. C. ice-water bath. The particle sizes of opalescent
liposomes were measured by a Beckman Coulter N5 submicron particle
size analyzer and showed a normal distribution with a peak around
130 nm. The liposomes were aliquoted and stored at -80.degree. C.
until use. The PC/PS ratio was 2:1 and the PE/PG ratio was 3:1.
[0486] Protein Purification
[0487] E. coli SecA was purified from BL21(.lamda.DE3)/pT7-SecA.
SecA homologous from other bacteria were purified similarly from
BL21.19. Purified proOmpA were prepared, and SecYEG and SecDF YajC
were purified.
[0488] Two Electron Whole Cell Recording
[0489] When the channel on the cell membrane is open, ions pass
through the membrane and generate an ionic current. Thus, the
recording of ionic current could also mean the opening of the
protein conducting channel. A two-electrode voltage clamp,
connected to an amplifier (Geneclamp 500, Axon instruments Inc.,
Foster City, Calif.), was used to measure the current across the
plasma membranes of oocytes after the oocytes were injected with
the inhibitor.
[0490] The cells were placed in a recording chamber (BSC-HT,
Medical System, Greenvale, N.Y.) on a supporting nylon mesh, so
that the perfusion solution washed both the top and the bottom
surface of the oocytes. The cells were then impaled using
electrodes filled with 3 M KCl. One electrode (1.0-2.0 M.OMEGA.)
was used for voltage recording. This electrode was connected to the
HS-2.times.1 L headstage (inpot resistance, 10.sup.11.OMEGA.). The
second electrode (0.3-0.6 M.OMEGA.) was used for current recording,
which was connected to the HS-2.times.10 .mu.MG headstage (maximum
current, 130 .mu.A). The electrodes were connected to the headstage
via a silver wire that was freshly chloridized for each experiment.
Oocytes were reused for further experiments only if the difference
between the leak currents measured before and after the experiments
were less than 10% of the peak currents. The leak current was not
considered during data analysis. The generated currents were
low-pass filtered (Bessel, 4-pole filter, 3 db at 5 kHz), digitized
at 5 kHz (12 but resolution), and subsequently analyzed using a
pClamp6 (Axon Instruments). The highest and lowest currents
recorded were eliminated, and the remaining presented as mean
current.+-.S.E. (standard error; n, number of oocytes). The
expression rates for each injection sample were also recorded to
determine the channel activity efficiency.
[0491] Results
[0492] Inhibitors Effects
[0493] SecA is essential for bacteria growth and serves as an
ATPase for protein translocation across membranes. SecA also
possesses intrinsic ATPase activity that is increased upon
interaction with lipids, and further enhanced with protein
precursors. The effective inhibition of channel activity (Table 6)
by SecA inhibitor corresponds to inhibition of protein
translocation by SecA-dependent ATPase with E. coli SecA system.
With the proteoliposomes injection methods, the inhibitory effects
of various SecA inhibitors on the channel activities for other
bacterial systems can also be investigated.
[0494] Rose Bengal was used to test the sensitivity of the
SecA-dependent channel activity to inhibitors. SecA-liposomes or
liposomes containing SecA and SecYEG and various concentrations of
Rose Bengal were administered and the IC.sub.50 for the bacteria's
sensitivity to Rose Bengal was recorded.
[0495] Inhibition of the channel activity in oocytes injected with
BaSecA-, SaSecA-, and PaSecA-liposomes were similar (Table 6).
Injection of the various SecA homologs complexed with SecYEG showed
intermediate sensitivity to Rose Bengal compared injection with the
SecA-liposome alone (Table 6). The PaSecA complex was the only
exception. Addition of SecDF YajC increased the IC.sub.50 values
somewhat.
TABLE-US-00006 TABLE 6 Rose Bengal IC.sub.50 (.mu.M) inhibition of
SecA channel activity in oocytes. Liposome + +SecYEG + SecAs
Liposomes BA13/Re-13 SecYEG SecDF C EcSecA 0.4 4.7/0.4 3.0 3.8
BsSecA1 0.3 5.8/0.5 3.1 4.5 PaSecA 0.3 5.1/0.3 1.1 2 SaSecA1 0.4
6.1/0.5 3.1 4.2 BaSecA1 0.3 6.1/0.5 3.3 4.0 MtbSecA1 0.5 -- MsSecA1
0.4 --
[0496] Methods for Assaying Channel Inhibitor Kinetics.
[0497] As mentioned, SecA ATPases activities respond differently
when interacting with lipids, protein precursors, and SecA
inhibitors. SecA-dependent ATPase showed non-competitive inhibition
at low ATP concentrations with RB, but competitive inhibition at
high ATP concentrations.
[0498] FIG. 3A shows non-competitive inhibition of the channel
activity of SecA-dependent ATPase. The channel activity on injected
EcSecA-liposomes in the oocytes also showed similar non-competitive
inhibition in regards to ATP (FIG. 3B). The inhibitor kinetics with
other bacterial SecA was also determined. Using the injected
SecA-liposomes in the oocytes, RB also showed non-competitive
inhibition with ATP for the channel activity for PaSecA and SaSecA1
(FIGS. 3C and 3D, respectively).
Example 4: Rose Bengal and Rose Bengal Analogs Inhibitors of SecA
Exhibit Antimicrobial Activity, Inhibit Toxins Secretion, and
Bypass Some Efflux Pumps Against Methicillin-Resistant
Staphylococcus aureus
[0499] Bacterial Strains and Culture Condition
[0500] S. aureus strains ATCC 35556 and ATCC 6538 were obtained
from the American Type Culture collection. S. aureus strains Mu50,
Mu3, and N315 were kindly provided by Dr. Chung-Dar Lu of Georgia
State University. Five efflux pump related S. aureus strains
8325-4, K1758 (NorA.sup.-), K2361 (NorA.sup.++), K2908
(MepA.sup.-), K2068 (MepA.sup.++) were kindly provided by Dr. G W
Kaatz at Wayne State University School of Medicine and Jon D.
Dingell VA Medical Center. All strains were grown on Luria-Bertani
(LB) agar plates or broth at 37.degree. C.
[0501] Chemical Compounds
[0502] Rose Bengal was purchased from SIGMA-ALDRICH. All RB analogs
were synthesized as described in Example 2.
[0503] Protein Preparation
[0504] The SaSecA1 and SaSecA2 genes were amplified from S. aureus
ATCC35556. The SaSecA1 gene was cloned into pET-21d and the SaSecA2
gene was cloned into pET-29a. Both genes were over-expressed in
BL21.lamda.DE3 at 20.degree. C. with 0.5 mM IPTG. SaSecA1 and
SaSecA2 were purified with His-trap column and Superdex-200
column.
[0505] In Vitro ATPase Activity Assay
[0506] The ATPase activity was determined by malachite green
colorimetric assay (described in Example 2). The ATPase assays were
carried out with different concentrations of inhibitor at
37.degree. C. for 40 min in the presence of 5% DMSO in room
light.
[0507] Bacteriostatic Effect
[0508] Bacteriostatic effects were tested according to the
guidelines of the Clinical and Laboratory Standards Institute
(described in Example 2).
[0509] Bactericidal Effect
[0510] Bactericidal effect was determined in presence of 2.5% DMSO
in room light (described in Example 2).
[0511] SecA-Lipsomes Ion-Channel Activity Assays
[0512] The liposomes were prepared as described in Example 3.
Oocytes were obtained from live frog Xenopus laevis (Xenopus
Express, Inc) and injected with sample mixtures. 50 nl sample
mixtures containing 120 ng liposomes, 120 ng SecA, 14 ng proOmpA, 2
mM ATP, 1 mM Mg.sup.2+, and different concentration of inhibitors
were injected into the dark pole site of oocytes using a Nanoject
II injector (Drummond Scientific Co., Broomall, Pa.). The ion
current was recorded for 1 min after three hours of incubation at
23.degree. C.
[0513] Toxin Secretion
[0514] S. aureus Mu50 was grown in LB broth at 37.degree. C.
Inhibitors were added to the mid-log phase of S. aureus Mu50.
Cultures were collected after treating with inhibitor for 0 h, 2
hrs (or 2.5 hrs), and 4 hrs. The supernatant and cell pellet were
separated by centrifugation followed by filtration through a 0.45
.mu.M filter. Western blots with specific toxin antibodies were
used to detect the amount of toxins in the supernatant. Antibodies
include .alpha.-hemolysin, enterotoxin B, and toxin shock syndrome
toxin-1 (TSST-1), which were purchased from Abcam (website
abcam.com).
[0515] Results
[0516] Inhibition of S. aureus SecA Proteins
[0517] Two SecA homologues have been previously identified in S.
aureus (Siboo et al., J Bacteriol, 2008. 190:6188-6196). Two low
molecular weight RB analogs, SCA-41 and SCA-50 (see FIG. 4), were
analyzed for inhibition of SaSecA1 and SaSecA2. SCA-41 and SCA-50
was shown to inhibit the ATPase activities of SaSecA1 and SaSecA2
(Table 7). This is an indication that both compounds have at least
two targets in S. aureus.
[0518] The inhibitory effects of Rose Bengal (RB) and RB analogs
against SaSecA1 were further investigated using a SecA-liposome
ion-channel activity assay. To evaluate SecA's function in the
membrane, SaSecA1 was injected simultaneously with liposomes into
oocytes in the presence or absence of RB and RB analogs. The RB
analogs displayed potent inhibition of the ion-channel activity of
SaSecA1 (IC.sub.50 from 0.3 .mu.g/mL to 3.4 .mu.g/mL; Table 7). The
RB analog with the highest activity, SCA-50 inhibits SecA-dependent
ion channel activity better than that of RB (IC.sub.50: 0.4
.mu.g/mL).
TABLE-US-00007 TABLE 7 Inhibition against activities of SaSecA1
proteins, IC.sub.50 (.mu.M) ATPase activity Ion-channel activity
SaSecA1 SaSecA2 SaSecA1 RB 1.0 2.5 0.4 SCA-41 37.5 32.5 3.4 SCA-50
20 17.5 1.1
[0519] Inhibition on the Secretion of S. aureus Toxins
[0520] In S. aureus, Sec-system is responsible for secretion of
more than 20 toxins or virulence factors, which play important
roles in the pathogenesis of S. aureus infection. Therefore,
targeting S. aureus SecA1, an essential component of Sec-system
could reduce virulence of S. aureus. To determine whether the SecA
inhibitors can inhibit the secretion of S. aureus toxins, 10 .mu.M
SCA-41 or SCA-50 was added into the mid-log phase of S. aureus
Mu50. Results from western blot show that these compounds
significantly decreased the amount of .alpha.-hemolysin,
enterotoxin B, and toxin shock syndrome toxin-1 (TSST-1) in the
supernatant
[0521] The OD readings of the control and the supernatant (treated
with 10 .mu.M SCA-41 or SCA-50) did not change after 15 hours. This
is an indication that protein synthesis was not affected. All three
toxins contain Sec-dependent signal peptide. Therefore, it appears
that SCA-41 and SCA-50 inhibit the in vivo function of SecA1.
Inhibition of SecA could dramatically reduce the virulence of S.
aureus.
[0522] Antimicrobial Activities of Novel RB Analogs Against MRSA
Strains
[0523] To determine whether the RB analogs possess antimicrobial
effect against methicillin resistant Staphylococcus aureus (MRSA),
the bacteriostatic effects of these compounds against three MRSA
strains (N315, Mu3, and Mu50) and one clinical isolated strain of
S. aureus, ATCC 6538 was investigated. These inhibitors showed
bacteriostatic effects against all tested S. aureus strains with
MICs around 3.7 .mu.g/ml to 25.6 .mu.g/ml (Table 8). The
bacteriostatic effects of all tested RB analogs were better than
that of RB.
[0524] SCA-50 showed the best bacteriostatic effect and best
inhibitory effects against ATPase and ion-channel activities of
SaSecAs. Its ability to kill bacteria was tested. MRSA strain Mu50
and a clinical isolated strain S. aureus 6538 were employed in this
assay. SCA-50 showed a concentration-dependent manner of
bactericidal activity for both strains, killing 2 log numbers of S.
aureus 6538 and more than 3 log numbers of S. aureus Mu50 at 9
.mu.g/ml (FIG. 5).
TABLE-US-00008 TABLE 8 Bacteriostatic effect, MIC (.mu.g/ml) S.
aureus S. aureus 6538 S. aureus Mu50 S. aureus N315 Mu3 RB 38.2
50.8 19.1 38.2 SCA-41 10.6 8.8 14.1 14.1 SCA-46 16.0 25.6 25.6 25.6
SCA-50 3.7 3.7 3.7 3.7 SCA-57 7.4 7.4 7.4 7.4
[0525] The Effect of Photooxidation
[0526] Previous studies demonstrated that part of RB's
antimicrobial activities is due to photooxidation (Inbaraj et al.,
Photochem Photobiol, 2005. 81:81-8; Demidova et al., Antimicrob
Agents Chemother, 2005. 49:2329-35; Wang et al., Curr. Microbiol.,
2006. 52:1-5). To determine whether the antimicrobial activity of
the novel RB analogs were due to photooxidation, the bactericidal
effect of RB and SCA-41 were investigated in the dark and under
light. In the dark, RB 1 showed little bactericidal effect, and its
bactericidal effect was dramatically increased by light (FIG. 6).
These results confirmed that photooxidation contribute to part of
RB's antimicrobial activity. However the bactericidal effect of
SCA-41 was not affected by light. These results indicated that the
antimicrobial activity of SCA-41 is not due to a photooxidation
mechanism.
[0527] The Possibility of Overcoming the Effect of Efflux Pump:
[0528] In Gram-positive bacteria, drugs targeting SecA might be
directly accessible from the extracellular matrix and exert their
effect without entering the cell. Therefore targeting SecA may
bypass the negative effect of efflux pumps in bacteria, which is a
major concern for the development of current drug-resistance (Zhang
et al., Bioorg Med Chem Lett, 2007, 17:707-11; Nikaido et al., Curr
Opin Infect Dis, 1999. 12:529-36; Van Bambeke et al., Biochem
Pharmacol, 2000, 60: 457-70; Markham et al., Curr Opin Microbiol,
2001, 4:509-14; Levy et al., Symp Ser Soc Appl Microbiol,
2002:65S-71S). S. aureus Mu50 and S. aureus N315 are resistant to
QacA efflux-mediated antibiotics. The SecA inhibitors showed
promising bacteriostatic effects against S. aureus Mu50 and S.
aureus N315, suggesting that these SecA inhibitors might be able to
overcome QacA mediated efflux.
[0529] NorA and MepA are two efflux pumps of S. aureus with 23% or
4% overexpression frequencies. To determine whether overexpression
of NorA or MepA could affect the antimicrobial effect of the SecA
inhibitors, microbial inhibition assay against NorA or MepA
deletion or overexpression mutants and the parent S. aureus 8325-4
was carried out with RB, SCA-41 and SCA-50. For RB, overexpression
NorA increased MIC to 1.5 fold that of NorA deletion mutant and 2.5
fold that of parental strains (Table 9). Overexpression of MepA
increased MIC to 1.5 fold that of MepA deletion mutant (Table 9).
These results indicate that NorA could pump out RB, though not very
efficient. However, for SCA-50 and SCA-41, overexpression or
deletion NorA or MepA did not significantly change the MIC (Table
9). Such results strongly suggest that the SecA inhibitors may have
the intrinsic ability to overcome the effect of the efflux pumps in
drug-resistance development.
TABLE-US-00009 TABLE 9 Bacteriostatic effects against S. aureus
efflux strains, MIC (.mu.g/ml) Strains WT NorA.sup.- NorA.sup.++
MepA.sup.- MepA.sup.++ compounds 8325-4 K1758 K2361 K2908 K2068 RB
13.2 22.3 34.6 10.6 17.5 SCA-41 11.8 14.1 11.8 14.1 11.8 SCA-50 3.7
3.7 3.7 3.7 3.7
[0530] RB and RB Analogs Exert Stronger Efficacy than First-Line
Antibiotics Against MRSA
[0531] S. aureus Mu50 is a MRSA strain with intermediate level
resistance to vancomycin (VISA). As reported in Table 10, the
selected SecA inhibitors were far more potent in their
antimicrobial activity against S. aureus Mu50 than the majority of
commonly used antibiotics. The MIC of SCA-50 is 4 .mu.g/ml, which
is 250 fold less than the MIC of ampicillin, kanamyin,
erythromycin, and rifampicin. MICs of norfloxacin, tetracycline,
and polymyxin B are 60 fold to 7 fold higher than that of SCA-50.
MIC of vancomycin is two-fold higher than that of SCA-50.
TABLE-US-00010 TABLE 10 Comparison of the antimicrobial activities
of SecA inhibitors with other antibiotics against S. aureus Mu50
Bacteriostatic effect Antibiotics MIC (.mu.g/ml) Bactericidal
effect RB 50.8 + SCA-41 8.8 + SCA-50 3.7 + Vancomycin 7.8 +
Ampicillin 1000 + Kanamycin 1000 + Polymxin B 31.3 + Tetracycline
62.5 - Erythromycin >1250 - Norfloxacin 250 + Rifampicin
>1000 +
[0532] Summary
[0533] SecA is important in the protein translocation machinery
present in all bacteria. In S. aureus, SecA is critical for both
bacterial survival and virulence, being responsible for secretion
of more than 20 toxins or virulence factors, which play important
roles in the pathogenesis of S. aureus infection. Therefore,
targeting S. aureus SecA might achieve dual effects-decreasing
bacterial survivability and reducing virulence. Two SecA homologues
(SecA1 and SecA2) exist in S. aureus, making them more attractive
targets for the development of novel antimicrobials. Dual target
inhibition could increase the chance of combating infection and
reducing the occurrence of drug resistance in this bacterium. SecA
has no counterpart in mammalian cells, thus providing an ideal
target for developing antimicrobial agents. FIG. 7 shows the
structures of compounds that were synthesized. Some of the
compounds were evaluated for in vitro inhibition activity and/or
toxicity.
[0534] The tested RB analogs showed promising inhibition against
the activities of both SaSecA1 and SaSecA2, and exert better
antimicrobial activities than RB. The most active compound, SCA-50
showed potent concentration-dependent bactericidal activity. The
MIC of SCA-50 is 4 .mu.g/ml, better than that of vancomycin, which
is the last sort against MRSA. Moreover, vancomycin only decreases
bacterial survivability, while the SCA-50 decreases bacterial
survivability and inhibited toxin secretion simultaneously.
[0535] The data showed that the over-productions of NorA and MepA
in S. aureus strains have no effect on the MIC SCA-41 and SCA-50.
Such results strongly suggest that SecA inhibitors may have the
intrinsic ability to overcome the effect of the efflux-pumps in
drug-resistance development. In such a case, the drug-efflux pump
would have less negative effects on the inhibitor's ability to
exert antimicrobial activity. This is the first approach, to our
best knowledge, of the development of new antimicrobials that have
the intrinsic ability to overcome the effects of efflux that
bacteria use in developing multi-drug resistance. Given the
wide-spread nature of efflux in bacteria and its importance in
drug-resistance, such a finding by itself would be of extraordinary
novelty and significance.
[0536] In the treatment against bacterial infection, the
traditional thinking has been almost solely on achieving
bactericidal and/or bacteriostatic effects. Such approaches
continue to be very effective and play an important role. However,
combination approaches might yield a more effective outcome. These
combinatorial approaches may include the regulation and/or
inhibition of virulence factor production, inhibition of bacterial
quorum sensing, and inhibition or bypassing efflux, which is a key
mechanism of multi-drug resistance in bacteria. Some of the
additional approaches do not exert the same kind of evolutionary
pressure as bactericidal and bacteriostatic agents do and thus are
less likely to quickly induce drug resistance. Along this line,
targeting SaSecA proteins ia a very attractive antimicrobial
strategy, because inhibition SecA could decrease bacterial
survivability, reducing virulence, and by-passing efflux at the
same time.
Example 5: Compounds of Formula I-X as SecA Inhibitors
[0537] Bacterial Strain and Growth Conditions
[0538] An outer membrane leaky mutant strain, E. coli NR698 (Ruiz
et al., Cell, 2005, 121:307-317; provided by Thomas J Silhavy of
Princeton University) and B. subtilis 168 (lab stock) were grown in
Luria-Bertani (LB) medium at 37.degree. C. S. aureus strains Mu50
were kindly provided by Dr. Chung-Dar Lu of Georgia State
University. B. anthracis Sterne and S. aureus 6538 were obtained
from American Type Culture Center. All strains were grown on
Luria-Bertani (LB) agar plates or broth at 37.degree. C.
[0539] Protein Preparation
[0540] EcSecAN68, a truncated mutant of EcSecA containing the
N-terminal catalytic domain, EcSecA, and BsSecA were used to study
the in vitro inhibition effect of RB analogs. These proteins were
purified as previously described (Chen et al., J. Biol. Chem. 1996,
271:29698-29706; Chen et al., J. Bacteriol. 1998, 180:527-537).
[0541] In Vitro ATPase Activity Assay
[0542] The malachite green colorimetric assay was used to determine
the inhibition effect of RB analogs against the ATPase activity of
SecA proteins. In this assay, ATPase assays were carried out at
different concentrations of the inhibitor, and IC50 was defined as
the concentration of the compound, which could inhibit 50% ATPase
activity of the enzyme. Because RB analogs were dissolved in 100%
DMSO, there was 5% DMSO in the final assay.
[0543] Bacteriostatic Effect
[0544] Bacteriostatic effects were tested by a liquid microdilution
method according to the guidelines of the Clinical and Laboratory
Standards Institute (Performance standards for antimicrobial
susceptibility testing. M100-S21; 21st informational supplement.
Clinical and Laboratory Standards Institute, Wayne, Pa. 2011). This
assay was performed in a 96-well microtiter tray under normal room
light condition. All bacteria were grown in LB broth, and when the
OD.sub.600 reach 0.5, the culture was diluted to
OD.sub.600.apprxeq.0.05. 97.5 .mu.l diluted culture and 2.5 .mu.l
of compound were added to each well. Cells were incubated at
37.degree. C. with shaking (250 rpm) for 24 hr. MIC is the lowest
concentration of inhibitors at which cells were not able to
grow.
[0545] Results:
[0546] A series of compounds from the genus described by Formula
I-X were screened against EcSecA using the intrinsic ATPase of the
truncated N-terminal catalytic domain EcN68 (unregulated ATPase).
Those compounds with significant IC50 values are shown in FIG.
8.
[0547] The compounds were also screened for their inhibitory
activities against the bacterial strains B. anthracis, S. aureus
6538, S. aureus Mu50, E. coli NR698, and B. subtilis 168. The
inhibitory activities of those compounds with significant IC.sub.50
values are shown in FIG. 8.
[0548] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0549] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *