U.S. patent application number 12/569639 was filed with the patent office on 2010-07-29 for lpaat-beta inhibitors and uses thereof.
This patent application is currently assigned to Cell Therapeutics, Inc.. Invention is credited to Lynn BONHAM, Robert E. Finney, Baoqing Gong, David M. Hollenback, J. Peter Klein, David W. Leung, Scott A. Shaffer, Norina M. Tang, John Tulinsky, Thayer H. White.
Application Number | 20100189726 12/569639 |
Document ID | / |
Family ID | 22921747 |
Filed Date | 2010-07-29 |
United States Patent
Application |
20100189726 |
Kind Code |
A1 |
BONHAM; Lynn ; et
al. |
July 29, 2010 |
LPAAT-BETA INHIBITORS AND USES THEREOF
Abstract
The invention relates to triazines and the use thereof to
inhibit lysophosphatidic acid acyltransferase .beta. (LPAAT-.beta.)
activity. The invention further relates to methods of treating
cancer using said triazines. The invention also relates to methods
for screening for LPAAT-.beta. activity.
Inventors: |
BONHAM; Lynn; (Seattle,
WA) ; Klein; J. Peter; (Vashon, WA) ; Finney;
Robert E.; (Shoreline, WA) ; Hollenback; David
M.; (Seattle, WA) ; Shaffer; Scott A.;
(Seattle, WA) ; Tang; Norina M.; (Ann Arbor,
MI) ; White; Thayer H.; (Bellevue, WA) ;
Leung; David W.; (Mercer Island, WA) ; Gong;
Baoqing; (Shoreline, WA) ; Tulinsky; John;
(Seattle, WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
Cell Therapeutics, Inc.
Seattle
WA
|
Family ID: |
22921747 |
Appl. No.: |
12/569639 |
Filed: |
September 29, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11601513 |
Nov 16, 2006 |
7608620 |
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12569639 |
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11224340 |
Sep 12, 2005 |
7199238 |
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11601513 |
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10712900 |
Nov 13, 2003 |
7064125 |
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11224340 |
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10236084 |
Sep 6, 2002 |
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10712900 |
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09984888 |
Oct 31, 2001 |
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10236084 |
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60244195 |
Oct 31, 2000 |
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Current U.S.
Class: |
424/178.1 ;
514/245; 544/208; 544/209 |
Current CPC
Class: |
C07D 251/54 20130101;
A61P 35/00 20180101; C07D 251/52 20130101; C07D 251/70 20130101;
C07D 251/50 20130101; C07D 251/58 20130101 |
Class at
Publication: |
424/178.1 ;
544/208; 544/209; 514/245 |
International
Class: |
A61K 31/53 20060101
A61K031/53; C07D 251/50 20060101 C07D251/50; C07D 405/12 20060101
C07D405/12; C07D 251/52 20060101 C07D251/52; A61K 39/00 20060101
A61K039/00; A61P 35/00 20060101 A61P035/00 |
Claims
1. A compound of the Formula: ##STR00015## wherein, R.sup.1 is
halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy or
substituted amino; R.sup.2, R.sup.3, R.sup.4 and R.sup.5, each of
which may be same or different, are hydrogen, alkyl, substituted
alkyl, alkenyl, alkynyl, aryl or substituted aryl; or R.sup.2 and
R.sup.3 or R.sup.4 and R.sup.5, together with the nitrogen to which
they are attached, form a piperidine, piperazine, or a morpholine
ring; or pharmaceutically acceptable salts thereof.
2. A compound of claim 1, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen and R.sup.3 and R.sup.5 are phenyl; or
pharmaceutically acceptable salts thereof.
3. A compound of claim 1, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is phenyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
4. A compound of claim 1, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is t-butyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
5. A compound selected from the group consisting of
6-chloro-N-(4-methoxy-phenyl)-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-butyl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,
6-chloro-N-isopropyl-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-tert-butyl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,
N-tert-butyl-6-chloro-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-cyclo-hexyl-N'-isopropyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,
6-chloro-N-isopropyl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
N-allyl-6-chloro-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,
N-tert-butyl-6-chloro-N'-cyclopentyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-methoxyphenyl)-N'-phenyl-[1,3,5]triazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4--
diamine,
6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N'-phenyl-[1,3,5]tr-
iazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-indan-5-yl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-propyl-[1,3,5]triazine-2,4-diamine,
N-(4-chloro-phenyl)-6-methoxy-N'-propyl-[1,3,5]triazine-2,4-diamine
and
N-(4-chloro-phenyl)-6-methylsulfanyl-N'-phenyl-[1,3,5]triazine-2,4-diamin-
e.
6. A compound of claim 1, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is 4-methoxyphenyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
7. A pharmaceutical composition comprising the compound of claim 1
and a pharmaceutically acceptable carrier.
8. A method for inhibiting LPAAT-.beta. (lysophosphatidic acid
acyltransferase .beta.) comprising contacting LPAAT-.beta. with an
effective amount of a compound of the Formula: ##STR00016##
wherein, R.sup.1 is halo, hydroxy, alkylmercapto, mercapto, alkoxy,
aryloxy or substituted amino; R.sup.2, R.sup.3, R.sup.4 and
R.sup.5, each of which may be same or different, are hydrogen,
alkyl, substituted alkyl, alkenyl, alkynyl, aryl or substituted
aryl; or R.sup.2 and R.sup.3 or R.sup.4 and R.sup.5, together with
the nitrogen to which they are attached, form a piperidine,
piperazine, or a morpholine ring; or pharmaceutically acceptable
salts thereof; thereby inhibiting LPAAT-.beta..
9. The method of claim 8, wherein said LPAAT-.beta. is found in an
animal.
10. The method of claim 9, wherein said animal is a mammal.
11. The method of claim 10, wherein said mammal is a human.
12. The method of claim 8, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen and R.sup.3 and R.sup.5 are phenyl; or
pharmaceutically acceptable salts thereof.
13. The method of claim 8, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is phenyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
14. The method of claim 8, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is t-butyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
15. The method of claim 8, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is 4-methoxyphenyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
16. The method of claim 8, wherein the compound is selected from
the group consisting of
6-chloro-N-(4-methoxy-phenyl)-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-butyl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,
6-chloro-N-isopropyl-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-tert-butyl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,
N-tert-butyl-6-chloro-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-cyclo-hexyl-N'-isopropyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,
6-chloro-N-isopropyl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
N-allyl-6-chloro-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,
N-tert-butyl-6-chloro-N'-cyclopentyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-methoxyphenyl)-N'-phenyl-[1,3,5]triazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4--
diamine,
6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N'-phenyl-[1,3,5]tr-
iazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-indan-5-yl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-propyl-[1,3,5]triazine-2,4-diamine,
N-(4-chloro-phenyl)-6-methoxy-N'-propyl-[1,3,5]triazine-2,4-diamine
and
N-(4-chloro-phenyl)-6-methylsulfanyl-N'-phenyl-[1,3,5]triazine-2,4-diamin-
e.
17. A method of inhibiting cell proliferation comprising contacting
a cell with an effective amount of a compound of the Formula:
##STR00017## wherein, R.sup.1 is halo, hydroxy, alkylmercapto,
mercapto, alkoxy, arylox or substituted amino; R.sup.2, R.sup.3,
R.sup.4 and R.sup.5, each of which may be same or different, are
hydrogen, alkyl, substituted alkyl, alkenyl, alkynyl, aryl or
substituted aryl; or R.sup.2 and R.sup.3 or R.sup.4 and R.sup.5,
together with the nitrogen to which they are attached, form a
piperidine, piperazine, or a morpholine ring; or pharmaceutically
acceptable salts thereof; thereby inhibiting the proliferation of
the cell.
18. The method of claim 17, wherein said cell is a cancer cell.
19. The method of claim 17, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen and R.sup.3 and R.sup.5 are phenyl; or
pharmaceutically acceptable salts thereof.
20. The method of claim 17, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is t-butyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
21. The method of claim 17, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is t-butyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
22. The method of claim 17, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is 4-methoxyphenyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
23. The method of claim 17, wherein the compound is selected from
the group consisting of
6-chloro-N-(4-methoxy-phenyl)-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-butyl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,
6-chloro-N-isopropyl-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-tert-butyl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,
N-tert-butyl-6-chloro-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-cyclo-hexyl-N'-isopropyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,
6-chloro-N-isopropyl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
N-allyl-6-chloro-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,
N-tert-butyl-6-chloro-N'-cyclopentyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-methoxyphenyl)-N'-phenyl-[1,3,5]triazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4--
diamine,
6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N'-phenyl-[1,3,5]tr-
iazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-indan-5-yl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-propyl-[1,3,5]triazine-2,4-diamine,
N-(4-chloro-phenyl)-6-methoxy-N'-propyl-[1,3,5]triazine-2,4-diamine
and
N-(4-chloro-phenyl)-6-methylsulfanyl-N'-phenyl-[1,3,5]triazine-2,4-diamin-
e.triazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4--
diamine,
6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N'-phenyl-[1,3,5]tr-
iazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine
and
6-chloro-N-indan-5-yl-N'-phenyl-[1,3,5]triazine-2,4-diamine.
24. A method for treating cancer, comprising administering to an
animal in need thereof, an effective amount of a compound of the
Formula: ##STR00018## wherein, R.sup.1 is halo, hydroxy,
alkylmercapto, mercapto, alkoxy, aryloxy or substituted amino;
R.sup.2, R.sup.3, R.sup.4 and R.sup.5, each of which may be same or
different, are hydrogen, alkyl, substituted alkyl, alkenyl,
alkynyl, aryl or substituted aryl; or R.sup.2 and R.sup.3 or
R.sup.4 and R.sup.5, together with the nitrogen to which they are
attached, form a piperidine, piperazine, or a morpholine ring; or
pharmaceutically acceptable salts thereof; wherein the cancer is
treated.
25. The method of claim 24, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen and R.sup.3 and R.sup.5 are phenyl; or
pharmaceutically acceptable salts thereof.
26. The method of claim 24, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is t-butyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
27. The method of claim 24, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is t-butyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
28. The method of claim 24, wherein R.sup.1 is chloro, R.sup.2 and
R.sup.4 are hydrogen, R.sup.3 is 4-methoxyphenyl and R.sup.5 is
4-chlorophenyl; or pharmaceutically acceptable salts thereof.
29. The method of claim 24, wherein said cancer is prostate,
breast, lung, ovarian, brain, cervical, colon or bladder
cancer.
30. The method of claim 24, where the compound is selected from the
group consisting of
6-chloro-N-(4-methoxy-phenyl)-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-butyl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,
6-chloro-N-isopropyl-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-tert-butyl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,
N-tert-butyl-6-chloro-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-cyclo-hexyl-N'-isopropyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,
6-chloro-N-isopropyl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
N-allyl-6-chloro-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,
N-tert-butyl-6-chloro-N'-cyclopentyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-methoxyphenyl)-N'-phenyl-[1,3,5]triazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4--
diamine,
6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N'-phenyl-[1,3,5]tr-
iazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-indan-5-yl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-propyl-[1,3,5]triazine-2,4-diamine,
N-(4-chloro-phenyl)-6-methoxy-N'-propyl-[1,3,5]triazine-2,4-diamine
and
N-(4-chloro-phenyl)-6-methylsulfanyl-N'-phenyl-[1,3,5]triazine-2,4-diamin-
e.
31. A method for screening a patient for LPAAT-.beta. activity,
said method comprising detecting the presence or absence of an
increased amount of LPAAT-.beta. RNA, DNA or protein relative to a
predetermined control, whereby the presence of said increased
amount is indicative of cancer susceptibility in said patient.
32. The method of claim 31, comprising detecting the presence or
absence of an increased amount of LPAAT-.beta. RNA.
33. The method of claim 31, comprising detecting the presence or
absence of an increased amount of LPAAT-.beta. DNA.
34. The method of claim 31, comprising detecting the presence or
absence of an increased amount of LPAAT-.beta. protein.
35. A method of inhibiting cell proliferation comprising the
inhibition of LPAAT-.beta..
36. The method of claim 35, wherein said cell is a cancer cell.
37. A vaccine preparation capable of inducing an anti-tumor immune
response comprising a pharmaceutically acceptable carrier and an
anti-tumor immune response-inducing effective amount of
LPAAT-.beta. protein.
38. A method for screening a patient for LPAAT-.beta. activity,
said method comprising detecting the presence or absence of an
increased amount of a phospholipid of defined acyl-chain
composition relative to a predetermined control, whereby the
presence of said increased amount is indicative of cancer
susceptibility in said patient.
39. The method of claim 38, wherein said phospholipid is
phosphatidylinositol.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/601,513 filed Nov. 16, 2006, now allowed; which
application is a continuation of U.S. patent application Ser. No.
11/224,340 filed Sep. 12, 2005, which issued as U.S. Pat. No.
7,199,238; which application is a divisional of U.S. patent
application Ser. No. 10/712,900 filed Nov. 13, 2003, which issued
as U.S. Pat. No. 7,064,125; which application is a divisional of
U.S. patent application Ser. No. 10/236,084 filed Sep. 6, 2002,
abandoned; which application is a continuation of U.S. patent
application Ser. No. 09/984,888 filed Oct. 31, 2001, abandoned,
which claims priority to U.S. Provisional Application Ser. No.
60/244,195, filed Oct. 31, 2000, abandoned, the disclosures of
which are incorporated by reference herein in their entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
200144.sub.--405D3_SEQUENCE_LISTING.txt. The text file is 2 KB, was
created on Sep. 29, 2009 and is being submitted electronically via
EFS-Web.
BACKGROUND
[0003] 1. Technical Field
[0004] The invention is in the field of organic and medicinal
chemistry. In particular, the invention relates to triazines and
the use thereof to inhibit lysophosphatidic acid acyltransferase
.beta. (LPAAT-.beta.) activity. The invention further relates to
methods of treating cancer using said triazines. The invention also
relates to methods for screening for LPAAT-.beta. activity.
[0005] 2. Description of the Related Art
[0006] LPAAT catalyzes the acylation of lysophosphatidic acid (LPA)
to phosphatidic acid. LPA is the simplest glycerophospholipid,
consisting of a glycerol molecule, a phosphate group, and a fatty
acyl chain. LPAAT adds a second fatty acyl chain to LPA, producing
phosphatidic acid (PA). PA is the precursor molecule for certain
phosphoglycerides, such as phosphatidylinositol, and
diacylglycerols, which are necessary for the production of other
phosphoglycerides, such as phosphatidylcholine, and for
triacylglycerols, which are essential biological fuel
molecules.
[0007] In addition to being a crucial precursor molecule in
biosynthetic reactions, LPA has recently been added to the list of
intercellular lipid messenger molecules. LPA interacts with G
protein-coupled receptors, coupling to various independent effector
pathways including inhibition of adenylate cyclase, stimulation of
phospholipase C, activation of MAP kinases, and activation of the
small GTP-binding proteins Ras and Rho. Moolenaar, J. Biol. Chem.
28:1294 (1995). The physiological effects of LPA have not been
fully characterized as yet. However, one of the physiological
effects that is known is that LPA promotes the growth and invasion
of tumor cells. It has been shown that the addition of LPA to
ovarian or breast cancer cell lines induces cell proliferation,
increases intracellular calcium levels, and activates MAP kinase.
Xu et al., Biochem. J. 309:933 (1995). In addition, LPA has been
shown to induce MM1 tumor cells to invade cultured mesothelial cell
monolayers. Imamura et al. Biochem. Biophys. Res. Comm. 193:497
(1993).
[0008] Like LPA, PA is also a messenger molecule. PA is a key
messenger in a common signaling pathway activated by
proinflammatory mediators such as interleukin-1.beta., tumor
necrosis factor .alpha., platelet activating factor, and lipid A.
Bursten et al., Am. J. Physiol. 262:C328 (1992); Bursten et al., J.
Biol. Chem. 255:20732 (1991); Kester J. Cell Physiol. 156:317
(1993). PA has been implicated in mitogenesis of several cell lines
[English, Cell Signal 8: 341 (1996)]. PA level has been found to be
increased in either ras or fps transformed cell lines compared to
the parental Rat2 fibroblast cell line [Martin et al., Oncogene 14:
1571 (1997)]. Activation of Raf-1, an essential component of the
MAPK signaling cascade, by extracellular signals is initiated by
association with intracellular membranes. Recruitment of Raf-1 to
membranes has been reported to be mediated by direct association
with phosphatidic acid [Rizzo et al., J Biol Chem 275:23911-8
(2000)]. Thus, LPAAT, as an enzyme that regulate PA content in
cells, may play a role in cancer, and may also mediate inflammatory
responses to various proinflammatory agents.
BRIEF SUMMARY
[0009] The preferred embodiments of the present invention relate to
a compound of the Formula:
##STR00001##
wherein,
[0010] R.sup.1 is halo, hydroxy, alkylmercapto, mercapto, alkoxy,
aryloxy or substituted amino;
[0011] R.sup.2, R.sup.3, R.sup.4 and R.sup.5, each of which may be
same or different, are hydrogen, alkyl, substituted alkyl, alkenyl,
alkynyl, aryl or substituted aryl; or
[0012] R.sup.2 and R.sup.3 or R.sup.4 and R.sup.5, together with
the nitrogen to which they are attached, form a piperidine,
piperazine, or a morpholine ring; or
pharmaceutically acceptable salts thereof.
[0013] The preferred embodiments of the present invention further
relate to a method for inhibiting LPAAT-.beta. (lysophosphatidic
acid acyltransferase .beta.) comprising contacting LPAAT-.beta.
with an effective amount of a compound of the Formula:
##STR00002##
wherein,
[0014] R.sup.1 is halo, hydroxy, alkylmercapto, mercapto, alkoxy,
aryloxy or substituted amino;
[0015] R.sup.2, R.sup.3, R.sup.4 and R.sup.5, each of which may be
same or different, are hydrogen, alkyl, substituted alkyl, alkenyl,
alkynyl, aryl or substituted aryl; or
[0016] R.sup.2 and R.sup.3 or R.sup.4 and R.sup.5, together with
the nitrogen to which they are attached, form a piperidine,
piperazine, or a morpholine ring; or
[0017] pharmaceutically acceptable salts thereof;
thereby inhibiting LPAAT-.beta..
[0018] The preferred embodiments of the present invention further
relate to a method of inhibiting cell proliferation comprising
contacting a cell with an effective amount of a compound of the
Formula:
##STR00003##
wherein,
[0019] R.sup.1 is halo, hydroxy, alkylmercapto, mercapto, alkoxy,
arylox or substituted amino;
[0020] R.sup.2, R.sup.3, R.sup.4 and R.sup.5, each of which may be
same or different, are hydrogen, alkyl, substituted alkyl, alkenyl,
alkynyl, aryl or substituted aryl; or
[0021] R.sup.2 and R.sup.3 or R.sup.4 and R.sup.5, together with
the nitrogen to which they are attached, form a piperidine,
piperazine, or a morpholine ring; or
[0022] pharmaceutically acceptable salts thereof;
thereby inhibiting the proliferation of the cell.
[0023] The preferred embodiments of the present invention further
relate to a method for treating cancer, comprising administering to
an animal in need thereof, an effective amount of a compound of the
Formula:
##STR00004##
wherein,
[0024] R.sup.1 is halo, hydroxy, alkylmercapto, mercapto, alkoxy,
aryloxy or substituted amino;
[0025] R.sup.2, R.sup.3, R.sup.4 and R.sup.5, each of which may be
same or different, are hydrogen, alkyl, substituted alkyl, alkenyl,
alkynyl, aryl or substituted aryl; or
[0026] R.sup.2 and R.sup.3 or R.sup.4 and R.sup.5, together with
the nitrogen to which they are attached, form a piperidine,
piperazine, or a morpholine ring; or
[0027] pharmaceutically acceptable salts thereof;
wherein the cancer is treated.
[0028] The preferred embodiments of the present invention further
relate to a method for screening a patient for LPAAT-.beta.
activity, said method comprising detecting the presence or absence
of an increased amount of LPAAT-.beta. RNA, DNA or protein relative
to a predetermined control, whereby the presence of said increased
amount is indicative of cancer susceptibility in said patient.
[0029] The preferred embodiments of the present invention further
relate to a method of inhibiting cell proliferation comprising the
inhibition of LPAAT-.beta..
[0030] The preferred embodiments of the present invention further
relate to a vaccine preparation capable of inducing an anti-tumor
immune response comprising a pharmaceutically acceptable carrier
and an anti-tumor immune response-inducing effective amount of
LPAAT-.beta. protein.
[0031] The preferred embodiments of the present invention further
relate to a method for screening a patient for LPAAT-.beta.
activity, said method comprising detecting the presence or absence
of an increased amount of a phospholipid of defined acyl-chain
composition relative to a predetermined control, whereby the
presence of said increased amount is indicative of cancer
susceptibility in said patient.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0032] FIG. 1 shows the results on a breast intraductal
adenocarcinoma sample where there is moderate increase in
LAPPT-.beta. mRNA level in the tumor sample (top two panels).
[0033] FIG. 2 shows the results on a breast intraductal
adenocarcinoma sample where there is a large increase in
LPAAT-.beta. mRNA level in the tumor sample.
[0034] FIG. 3 shows three examples of ovarian cancer where the
LPAAT-.beta. mRNA levels are elevated and one example with
undetectable level of LPAAT-.beta. mRNA (lower right panel).
[0035] FIG. 4A shows the results on a prostate adenocarcinoma
sample where there is moderate increase in LPAAT-.beta. mRNA level
in the tumor sample.
[0036] FIG. 4B shows the results on immunohistochemical staining of
ovarian tissue with MoAb 4B12.
[0037] FIG. 4C shows the results on immunohistochemical staining of
cervical tissue with MoAb 4B12.
[0038] FIG. 4D shows the results on immunohistochemical staining of
lung tissue with MoAb 4B12.
[0039] FIG. 4E shows the summary of immunohistochemistry results of
the various tissue samples stained by MoAb 4B12.
[0040] FIG. 5A shows hemacytometer cell counts of ECV304 cell
lines.
[0041] FIG. 5B shows examples of cell morphology of NIH/3T3 cells
after exposure to specified agents.
[0042] FIG. 5C shows the growth profiles of transduced populations
of NIH/3T3 cells.
[0043] FIG. 5D shows the growth profiles of transduced populations
of LNCaP cells.
[0044] FIG. 5E shows the effect of
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine on the
proliferation of MCF-7 cells.
[0045] FIG. 6A shows detection of tumor formation from LPAAT-.beta.
overexpressing cells.
[0046] FIG. 6B shows the effect of
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine on the volume of
tumors in mice.
[0047] FIG. 6C shows the effect of
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine on the growth of
B16 melanoma cells.
[0048] FIG. 6D shows the effect of
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine on the growth of
Lewis Lung tumor cells.
[0049] FIG. 6E shows the effect of
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine on the growth of
DU145 prostate tumor cells.
[0050] FIG. 7 shows a colorimetric assay whose time course of color
development is dependent on LPAAT enzyme.
[0051] FIG. 8 shows the result from assaying a plate of various
compounds at 16 mM.
[0052] FIG. 9 shows the results of the effects of a compound
selected from secondary screening on LPAAT-.beta. activity and
LPAAT-.alpha. activity.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. LPAAT-.alpha. and LPAAT-.beta.: An Overview
[0053] Northern blot analysis shows that LPAAT-.alpha. is expressed
in all human tissues tested with the highest expression level found
in skeletal muscle (West et al. DNA Cell Biol. 16:691 (1997)). The
uniformity of LPAAT-.alpha. expression has also been found in
additional tissues such as prostate, testis, ovary, small
intestine, and colon (Stamps et al., Biochem. J. 326: 455 (1997))
as well as in mouse tissues (Kume et al., Biochem. Biophys. Res.
Commun. 237: 663 (1997)). A 2 kb and a 1.3 kb forms, possibly due
to alternative utilization of polyadenylation signals at the
3'-UTR, have been found in murine LPAAT-.alpha.mRNA (Kume et al.,
Biochem. Biophys. Res. Commun 237: 663 (1997)), whereas only one
major human LPAAT-.alpha. mRNA of 2 kb in size has been detected by
Northern analysis. West et al., DNA Cell Biol. 16: 691 (1997);
Stamps et al., Biochem. J. 326: 455 (1997).
[0054] In contrast, LPAAT-.beta. demonstrates a distinct tissue
distribution of mRNA expression. West et al., DNA Cell Biol. 16:
691 (1997). LPAAT-.beta. is most highly expressed in liver and
heart tissues. LPAAT-.beta. is also expressed at moderate levels in
pancreas, lung, skeletal muscle, kidney, spleen, and bone marrow;
and at low levels in thymus, brain and placenta. This differential
pattern of LPAAT-.beta. expression has been confirmed independently
(Eberhardt et al., J. Biol. Chem. 272: 20299 (1997)) with the only
discrepancy being that high level, instead of moderate level, of
LPAAT-.beta. has been detected in pancreas, possibly due to slight
lot variations in commercial RNA blots (Clontech, Palo Alto,
Calif.). In addition, moderate LPAAT-.beta. expression has been
found in prostate, testis, ovary, small intestine, and colon with
the small intestine containing relatively higher amounts. Eberhardt
et al., J Biol Chem 272: 20299 (1997). Within various brain
sections, high expression has been found in the subthalamic nucleus
and spinal cord; and least in the cerebellum, caudate nucleus,
corpus callosum, and hippocampus. LPAAT-.beta. can also be detected
in myeloid cell lines THP-1, HL-60, and U937 with the mRNA levels
remaining the same with or without phorbal-ester treatment. The
size difference between human LPAAT-.alpha. and LPAAT-.beta. mRNA
is consistent with the sequence data, in which LPAAT-.alpha. has a
longer 3'-UTR. The differential tissue expression pattern
LPAAT-.alpha. and LPAAT-.beta. mRNA would suggest these two genes
are regulated differently and are likely to have independent
functions. Therefore, a desirable feature in compounds that inhibit
LPAAT activity is that they are specific in inhibiting one isoform
of the enzyme over the other (i.e., LPAAT-.beta. over
LPAAT-.alpha.).
II. LPAAT-.beta. and Cancer
[0055] PA has been implicated in mitogenesis of several cell lines.
English, Cell Signal 8: 341 (1996). PA level has been found to be
increased in either ras or fps transformed cell lines compared to
the parental Rat2 fibroblast cell line (Martin et al., Oncogene 14:
1571 (1997). To test whether LPAAT expression may be enhanced in
certain tumor cells, the expression of LPAAT-.alpha. and
LPAAT-.beta. mRNA in human tumor panel blots (Invitrogen, Carlsbab,
Calif.) that contained tumor RNAs, isolated from various malignant
tissues and RNAs from the normal tissues in the surgical margins,
were examined. Leung et al., DNA Cell Biol. 17: 377 (1998). The
same blots were also reprobed using cDNAs encoding phosphatidic
acid phosphatase isoform PAP2-.alpha.; an enzyme that degrades,
rather than generates, PA. Of a total of eight different tissues
examined, LPAAT-.beta. mRNA was found to be elevated in three
tumors tissues (uterus, fallopian tube, and ovary), as compared to
its expression in the corresponding normal tissues. However, no
significant difference was found in LPAAT-.alpha. mRNA level
between the various tumor tissues and the normal adjacent tissues.
In two of the tumor tissues (fallopian tube and ovary) where
LPAAT-.alpha. mRNA was elevated, PAP2-.alpha. mRNA expression was
found to be suppressed, as it was also in tumors of the colon,
rectum, and breast.
[0056] Since the finding of differential expression of LPAAT-.beta.
mRNA in certain tumor versus normal tissues is based on Northern
analysis of a single specimen from a given tissue, more studies
will be needed to determine whether the relative elevation of
LPAAT-.beta. expression in selected tumor tissues can be applied
and extended to similar tissues derived from a larger number of
donors. Leung et al., DNA Cell Biol. 17: 377 (1998). Accordingly,
in situ hybridization was used to compare LPAAT-.beta. mRNA levels
in breast, ovary, and prostate tumor samples obtained from multiple
independent donors (LifeSpan Biosciences, Seattle, Wash.).
Specifically, the coding region of human LPAAT-.beta. was amplified
by PCR from the plasmid pCE9. LPAAT-.beta. with primers
5'-GCATGAATTC AAAGGCCTAC GTCGACATGG AGCTGTGGCC GTG-3' (SEQ ID NO:1)
and 5'-GTCGACTCTA GACTACTGGG CCGGCTGCAC-3' (SEQ ID NO:2). The
resultant 870 by PCR product was then cut with EcoR I and XbaI for
insertion in between the EcoR I and XbaI sites of the in vitro
transcription vector pDP18-T7/T3 (Ambion, Austin, Tex.) to generate
the plasmid pDP.sub.--1ptB. Serial tissue sections from paraffin
archival samples were hybridized with digoxigenin labeled
riboprobes transcribed from either a T3 (sense) or T7 (antisense)
transcription initiation site present in the plasmid pDP.sub.--1ptB
linearized with either EcoR I (antisense) or Xba I (sense). The
tissue sections from paraffin blocks were digested with proteinase
K (20 .mu.g/ml) for 4 minutes, then hybridized with the antisense
probe (1 .mu.g/ml) at 60.degree. C. for 22 hours and subsequently
washed with 2.times.SSC and 0.1.times.SSC at 50.degree. C. The
hybridization signals were detected with NBT/BCIP substrates using
three cycles of an alkaline phosphatase TSA amplification system
(NEN Life Sciences, Boston, Mass.). The specimens were then
counterstained with methyl green. The signal was developed within
30 minutes at room temperature. The slides were then imaged using a
digital camera mounted onto a microscope.
[0057] Breast and ovary tissues were chosen for further in situ
hybridization study, as initial Northern analysis showed elevation
of LPAAT-.beta. mRNA levels in tumors derived from the female
reproductive tract. Prostate tissue was chosen, as it responds to
steroid hormones and contains ductal structures in a manner similar
to breast and ovary tissues. Using an anti-sense cDNA probe, it was
demonstrated that expression of the .beta. isoform of this enzyme
(LPAAT-.beta.) was augmented in human tumor tissue in 10/11
ovarian, 14/20 breast, and 7/16 prostate biopsies as compared to
normal adjacent tissues. FIG. 1 shows an example of the results on
a breast intraductal adenocarcinoma sample where there is moderate
increase in LPAAT-.beta. mRNA level in the tumor samples (top 2
panels) as evidenced by more dark-purple to brown spots compared to
adjacent hyperplasia (bottom-left panel) and normal tissue
(bottom-right panel). The slight increase in LPAAT-.beta. mRNA
staining in the hyperplasia sample (bottom-left panel) versus the
normal sample (bottom-right panel) suggests that elevation occurs
at an early stage of oncogenesis. FIG. 2 shows an example of the
results on another breast intraductal adenocarcinoma sample where
there is large increase in LPAAT-.beta. mRNA level in the tumor
sample (left panel) as evidenced by more dark-purple spots versus
the adjacent normal tissue (right panel). FIG. 3 shows three
examples of ovarian cancer samples where the LPAAT-.beta. mRNA
levels are elevated and one example with undetectable level of
LPAAT-.beta. mRNA (lower right panel). FIG. 4A shows an example of
the results on a prostate adenocarcinoma sample where there is
moderate increase in LPAAT-.beta. mRNA level in the tumor samples
(left panel) as evidenced by more dark-purple spots versus the
adjacent normal tissue (right panel). In no cases have elevated
levels of LPAAT-.beta. mRNA expression been found in the adjacent
normal region from the same donor even in those cases of breast,
ovarian, or prostate tumor where LPAAT-.beta. mRNA levels happen to
be low or undetectable. The augmented expression of LPAAT-.beta. in
a high percentage of tumor samples from breast (70%), ovary (91%),
and prostate tissues (44%) would suggest that LPAAT-.beta.
overexpression may be a contributing factor for the development of
these tumors.
[0058] To determine if increased transcription of LPAAT-.beta. mRNA
in selected tumor tissues can be extended to increased LPAAT-.beta.
protein expression in a wider range of tissues, a monoclonal
antibody specific for human LPAAT-.beta. protein (MoAb 4B12) was
generated based on the petide sequence, DLGERMVRENLKVW, derived
from amino acids 155-168 of LPAAT-.beta. protein (BAbCO, Berkeley,
Calif.). FIG. 4B shows an example of the results on
immunohistochemical staining (PhenoPath, Seattle, Wash.) with MoAb
4B12 at 1:4000 dilution of ovarian tissue where there is
substantial increase in LPAAT-.beta. protein expression in the
tumor samples (right panels) as evidenced by more intense brown
stainings versus the normal tissue (left panel). FIG. 4C shows an
example of the results on immunohistochemical staining (PhenoPath,
Seattle, Wash.) with MoAb 4B12 at 1:4000 dilution of cervical
tissue where there is substantial increase in LPAAT-.beta. protein
expression in the tumor samples (right panels) as evidenced by more
intense brown stainings versus the normal tissue (left panel).
There is also more staining in the surrounding stromal cells
(indicated by arrows) in the tumor tissue vs the normal tissue,
suggesting that the tumor may also induce LPAAT-.beta. protein
expression in the surrounding cells. FIG. 4D shows another example
of the results on immunohistochemical staining (PhenoPath, Seattle,
Wash.) with MoAb 4B12 at 1:4000 dilution of lung tissue where there
is extensive increase in LPAAT-.beta. protein expression in the
tumor samples (right panels) as evidenced by more intense brown
stainings versus the normal tissue (left panel). FIG. 4E shows the
summary of immunohistochemistry (IHC) results of the various tissue
samples stained by MoAb 4B12. The augmented expression of
LPAAT-.beta. in a high percentage of tumor samples again suggest
that LPAAT-.beta. overexpression may be a contributing factor for
the development of these tumors and that LPAAT-.beta. may be a
useful target for the development of anti-cancer compounds.
[0059] The aforementioned antibody may also be used for diagnostic
and prognostic purposes when a tumor is present both on biopsies
and in serum or plasma. For example, ELISA may be performed on
serum to detect lung or ovarian cancer. It should be mentioned that
currently there are no useful early diagnostics for these types of
cancers.
[0060] The overexpression of LPAAT-.beta. in selected tumor tissues
would also suggest the LPAAT-.beta. protein may constitute a useful
antigen for the development of tumor vaccines against those tumors
where LPAAT-.beta. is overexpressed. Fong et al., Annu. Rev.
Immunol. 18: 245 (2000); Schreurs, et al., Crit. Rev. Oncol. 11: 1
(2000). One such approach may use autologous dendritic cells, a
type of potent antigen-presenting cells, to present LPAAT-.beta. as
a tumor-associated antigens for the generation of tumor-specific
immunity through the MHC class I and II processing pathways.
Administration of dendritic cells loaded ex vivo with LPAAT-.beta.
as a therapeutic vaccine to patients with tumors with augmented
LPAAT-.beta. expression may induce T cell-mediated tumor
destruction.
[0061] To assess whether LPAAT-.beta. overexpression in cells would
lead to certain phenotypic changes that are commonly observed in
transformed cells, the growth and adherence characteristics of
ECV304 cells (American Type Culture Collection, Richmond, Va.)
expressing LPAAT-.beta. (LPTb), expressing a catalytically inactive
form of LPAAT-.beta. (b-M8) whereby the arginine at position 175
was changed to alanine using the GeneEditor.TM. in vitro
site-directed mutagenesis system (Promega, Madison, Wis.), or
expressing green fluorescent protein (GFP) as a control were
compared. The aforementioned cells that express GFP may be
considered to be a non-limiting example of a "predetermined
control," according to the preferred embodiments of the present
invention. That is, such cells may be used to gauge whether a cell
is over- or under-expressing LPAAT-.beta. DNA, RNA or protein. FIG.
5A shows the growth curve of these three cell lines. Each cell line
was seeded at 200,000 cells per 60 mm plate. The cell numbers at
various times after seeding were determined by counting with a
hemacytometer. The growth rate of the three cell lines were similar
until they reached confluence at 100 hours after plating. After
confluence, the LPTb cells were able to continue to proliferate,
while the b-M8 and GFP cells' growth started to level off. This
demonstrated that ECV304 cells overexpressing LPAAT-.beta. could
continue to grow and could form a plurality of layers after they
had formed a confluent monolayer of cells. The proliferation of the
cells with the inactive mutant or the control cells slowed down
after confluence. The loss of contact inhibition and the propensity
for growth to an unusually high cell density are changes commonly
observed in tumorigenesis. The fact that the inactive LPAAT-.beta.
mutant (b-M8) expressing cells, like the vector control cells, are
constrained by density-dependent inhibition of cell division
strongly suggests that the capacity to overcome contact inhibition
may be due to increases in LPAAT-.beta. enzymatic activity. The
development of compounds that inhibit LPAAT-.beta. enzymatic
activity may reverse the growth pattern and hence tumorigenesis in
cells with abnormally high level of LPAAT-.beta. expression.
[0062] To determine if the observation from LPAAT-.beta. expressing
ECV304 cells can be extended to other cell types and to animal
models of tumorigenesis, LPAAT-.beta. cDNA was inserted into a
retroviral expression vector, pLOXSN, for the generation of
recombinant viral stocks in a packaging cell line, PT67 (Clontech,
Palo Alto, Calif.), for transduction into various cell lines. The
vector pLOXSN was derived from pLXSN with insertion of a 19 bp
oligonucleotide coding for the locus of recombination (lox) signal
sequence as well as a ClaI recognition site into the NheI site
within the 3'-LTR region of pLXSN. Miller and Rosman BioTechniques
7: 980 (1989); Hoess. and Abremski, Nucleic Acid and Mol. Biol. 4:
99 (1990). This lox sequence will be duplicated within the 5'-LTR
region during viral replication. Hence the sequence in between the
two lox sites located within the 5'- and the 3'-LTR can be excised
if required in the presence of the enzyme cre recombinase supplied
in trans from a separate retroviral vector with a different
selectable marker.
[0063] Over-expression of the normal cellular LPAAT-.beta. cDNA in
NIH/3T3 cells was associated with transformation in 3 out of 9
transduced populations. As is the case with normal cellular
proto-oncogenes, over-expression of LPAAT-.beta. is not sufficient,
but may contribute to transformation along with other, spontaneous
events. FIG. 5B shows examples of cell morphology of NIH/3T3 cells:
a bulk population transfected with a plasmid overexpressing the
Ki-ras oncogene (top left panel), a selected clone transduced with
a retroviral vector overexpressing LPAAT-.beta. (Hc2; lower left
panel) and cells with the LPAAT-.beta. cDNA excised using the
lox-cre recombination in the lower left and normal, untransduced
cells (top right panel). Sauer, Methods 14: 381 (1998). The control
untransduced cells exhibited normal fibroblast morphology and grew
as a contact-inhibited, adherent monolayer (top right panel). In
contrast, both the Ki-ras and LPAAT-.beta. overexpressing cells
were more elongated and spiked, were not contact-inhibited and
formed foci typical of transformation of these immortalized
fibroblasts. After removal LPAAT-.beta. transgene by lox-cre
recombination from the Hc2 clone (bottom right panel), this
transformed morphology was lost, suggesting that LPAAT-.beta.
overexpression is a contributing factor to this transformation
phenotype rather than being the result entirely of spontaneous
events during in vitro passage.
[0064] Another common parameter of cancer cells is a reduced
requirement for elements present in serum. FIG. 5C compares the
growth profiles of transduced populations of NIH/3T3 cells in low
(2%) serum. Two independent populations (LPT Hc2, LPT L bulk)
overexpressing LPAAT-.beta. have an increased ability to
proliferate compared to a control vector clone expressing alkaline
phosphatase (APc1) and those corresponding populations with
deletion of the LPAAT-.beta. transgene by lox-cre recombination
(LPT Hc2cre, LPT L bulkcre), suggesting that LPAAT-.beta.
overexpression is a contributing factor to this transformed
phenotype of proliferation with a reduced requirement for growth
factors.
[0065] Similarly, out of a total of 12 populations of human
prostate LNCaP cells (American Type Culture Collection, Manassas,
Va.) transduced with LPAAT-.beta. expressing vector, most of them
show augmented proliferation in low serum when compared to control
cells (FIG. 5D).
[0066] To determine whether administration of LPAAT-.beta.
inhibitor would have any effect on cell proliferation in tissue
culture, proliferation of human breast tumor MCF-7 cells in
microplates were measured by CyQUANT analysis using a
green-fluorescent nucleic acid stain optimized to produce a linear
detection range from 50 to 50,000 cells in 200 .mu.l volume
(Molecular Probes, Eugene, Oreg.) in the presence of various
concentrations of a LPAAT-.beta. inhibitor. FIG. 5E shows the
triazine compound shows
6-chloro-N,N-diphenyl-[1,3,5]triazine-2,4-diamine at .gtoreq.20
.mu.M is effective in blocking the proliferation of MCF-7
cells.
[0067] To determine if LPAAT-.beta. overexpression would contribute
to tumorigenesis in mice, 2.times.10.sup.6 NIH3T3 cells
overexpressing LPAAT-.beta. (LPAAT vector) and control cells were
injected subcutaneously into nude mice. FIG. 6A shows tumor could
be detected after 14 days from the LPAAT-.beta. overexpressing
cells, while no tumor formation was detected in vector control
cells after 28 days. The cells with the transgene removed by
lox-cre recombination showed delay of tumor formation compared to
LPAAT-.beta. overexpressing cells by .about.7 days. Recovery and
analysis of the lox-cre cells from mice showed that there had been
in vivo selection of a small sub-population that had not been
recombined to remove the LPAAT-.beta. transgene. This analysis
demonstrated that the only cells to form tumors retained the
original LPAAT vector and indeed had a high level of LPAAT activity
as well as G418 resistance (the neo gene is also removed along with
LPAAT-.beta. during the cre-lox procedure). These data show
LPAAT-.beta. overexpression is a contributing factor for
tumorigenesis in vivo.
[0068] To determine whether administration of LPAAT-.beta.
inhibitor would have any effect on tumor growth in mice,
5.times.10.sup.5 NIH/3T3 cells overexpressing the oncogene Ki-ras
were injected subcutaneously into nude mice. An LPAAT-.beta.
inhibitor 6-chloro-N,N-diphenyl-[1,3,5]triazine-2,4-diamine, at
concentrations that range from 10 mg/Kg to 100 mg/Kg of mouse body
weight was injected intra-peritoneally on day 1, 2, 3 and 4 after
injection of tumor cells. The size of tumors was then measured on
day 8. FIG. 6B shows the volume of the tumors in mice is decreased
as the concentration of the LPAAT-.beta. inhibitor increases,
suggesting that administration of this LPAAT-.beta. inhibitor is
efficacious in slowing down tumor growth in vivo.
[0069] In addition to slowing down tumor growth of NIH3T3/Ki-ras
cells in nude mice,
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine has also been
found to decrease the growth of B16 melanoma cells (FIG. 6C) and
Lewis Lung tumor cells (FIG. 6D) in syngeneic mice as well as the
growth of human DU145 prostate tumor cells in nude mice in a
xenograft study (FIG. 6E).
[0070] Analysis and characterization of phospholipids and other
complex lipids represent another strategy to measure effects of
small molecule inhibitors on phospholipid metabolizing enzymes
involved in tumor progression, including but not limited to,
LPAAT-.beta.. Measurements of phospholipids and other complex
lipids may be derived from cell lines cultured in vitro, from
tissue or plasma in vivo (e.g., murine or other animal studies), or
from human subjects (e.g., phlebotomy or biopsy). Phospholipids,
which are the primary constituents of a cellular bilayer, contain a
universal phosphoric acid residue connected to a glycerol backbone.
Phospholipid classes are defined by the chemical identity of the
"head group" on the phosphoric acid moiety. However, each
phospholipid class is often a complex mixture of discrete molecular
species due to the fact that the glycerol backbone has two
substituents residing at the Sn1 and Sn2 position of attachment.
The substituents are acyl chains and typically consist of long
chain fatty acids but may also include a long chain ether, acetyl,
or hydroxyl group. Chemical measurements of phospholipids can
involve a variety of analytical methods including, but not limited
to, HPLC-MS (High Performance Liquid Chromatography-Mass
Spectrometry), HPLC-MS/MS (High Performance Liquid
Chromatography-Tandem Mass Spectrometry), one or two dimensional
TLC (Thin Layer Chromatography), and radiometry. While all the
stated methods can be used to quantitate bulk mass changes in a
particular phospholipid class of interest, mass spectrometry offers
the unique ability to measure all molecular species within a
phospholipid class in a single measurement with a high degree of
precision.
[0071] The above approach is demonstrated by performing HPLC-MS
analyses of phospholipid extracts from murine NIH/3T3 immortalized
fibroblasts, both normal wild type, .beta.Hc2 cells (i.e.,
overexpressing LPAAT-.beta., and Hc2cre cells (i.e., LPAAT-.beta.
gene removed by site-specific recombination). Analysis of
phosphatidylinositol in these cell populations clearly indicate a
combined effect of LPAAT-.beta. overexpression and cellular
transformation for the Hc2 population over that of the normal wild
type. This effect is characterized by an increase in unsaturated
(i.e., palmitate and stearate) and monounsaturated (i.e., oleate)
fatty acyl chains indicated by an increased molecular abundance of
ions at m/z 807, 833, 835, 861, and 863 which correspond most
likely to phosphatidylinositol species with acyl chains designated
as 16:0-16:1, 16:1-18:1 (and/or 16:0-18:2), 16:0-18:1, 18:1-18:1
(and/or 18:0-18:2), and 18:0-18:1, respectively. While multiple
molecular species may reside at the same nominal mass, these
species can be differentiated by tandem (MS/MS) mass spectrometry
methods. Additionally, note that actual determination of positional
location (i.e., Sn1 versus Sn2) requires other analytical methods
and only the most prevalent configuration is listed here. In
addition to the increase in unsaturated and monounsaturated acyl
chains in the LPAAT-.beta. overexpressing population (.beta.Hc2),
there is also a corresponding decrease in polyunsaturated (i.e.,
arachidonate) fatty acyl chains at m/z 857 (16:0-20:4) and m/z 885
(18:0-20:4). Removal of the LPAAT-.beta. transgene results in
phosphatidylinositol distributions similar to that of the normal
wild type 3T3 cells.
[0072] In summary, endogenous LPAAT-.beta. expression is detected
at high levels by both in situ hybridization and
immunohistochemistry in particular tumor tissues and often in
surrounding stroma and is associated with tumor progression.
LPAAT-.beta. overexpression appears to contribute reversibly to
transformation and tumorigenesis of immortalized rodent cells and
may also contribute to increased transformation of weakly
tumorigenic human cell lines. Compounds selected from screening of
LPAAT-.beta. inhibitors from different structural families can
inhibit proliferation of numerous tumor cell lines in vitro. Both
nude and immunocompetent mice can tolerate at least 100 mg/kg/day
for 4-5 days of the
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine maintaining body
weight and overall health with no discernable gross pathology. This
compound can inhibit the growth of numerous tumor models in mice
and may be a tumor-static compound.
III. LPAAT-.beta. Inhibitors
[0073] In one aspect, the compounds of the present invention relate
to triazines of the Formula:
##STR00005##
wherein,
[0074] R.sup.1 is halo, hydroxy, alkylmercapto, mercapto, alkoxy,
aryloxy or substituted amino;
[0075] R.sup.2, R.sup.3, R.sup.4 and R.sup.5, each of which may be
same or different, are hydrogen, alkyl, substituted alkyl, alkenyl,
alkynyl, aryl or substituted aryl; or
[0076] R.sup.2 and R.sup.3 or R.sup.4 and R.sup.5, together with
the nitrogen to which they are attached, form a piperidine,
piperazine, or a morpholine ring; or
pharmaceutically acceptable salts thereof.
[0077] As used herein, "alkyl" refers to straight- or
branched-chain hydrocarbons having from 1 to 10 carbon atoms and
more preferably 1 to 8 carbon atoms which includes, by way of
example, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl and
the like.
[0078] The term "alkyl" also refers to an "unsaturated alkyl"
moiety, which means that it contains at least one alkene or alkyne
moiety. "Alkene" or "alkenyl" refers to a group consisting of at
least two carbon atoms and at least one carbon-carbon double bond.
"Alkyne" or "alkynyl" refers to a group consisting of at least two
carbon atoms and at least one carbon-carbon triple bond. The alkyl
moiety, whether saturated or unsaturated, may be branched,
non-branched, or cyclic.
[0079] "Substituted alkyl" refers to an alkyl group, preferably
containing from 1 to 10 carbon atoms, having from 1 to 5
substituents including halogen, hydroxyl, alkyl, aryl or
substituted amino. A preferred substituted alkyl group is
trifluoromethyl.
[0080] "Alkoxy" refers to the group "alkyl-O--" which includes, by
way of example, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy,
t-butoxy and the like.
[0081] "Substituted amino" refers to the group --NRR, wherein each
R group is independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, cycloalkyl, aryl, substituted
aryl, or the R groups can be joined together with the nitrogen to
form a heterocyclic ring (e.g., piperidine, piperazine, or a
morpholine ring).
[0082] "Aryl" refers to an unsaturated aromatic carbocyclic group
of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or
multiple condensed rings (e.g., naphthyl or anthryl).
[0083] "Substituted aryl" refers to aryl group which are
substituted with 1 to 3 substituents selected from hydroxy, alkyl,
substituted alkyl, alkoxy, amino, aryl, --O--(CH.sub.2).sub.n--O--
(wherein n is an integer from 1 to 3), --(CH.sub.2).sub.m--
(wherein m is an integer from 3 to 5) or halogen.
[0084] "Cycloalkyl" refers to cyclic alkyl groups containing
between 3 and 8 carbon atoms having a single cyclic ring including,
by way of example, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl
and the like.
[0085] "Halogen" or "halo" refers to fluoro, chloro, bromo, iodo.
Most preferred halogens are chloro and fluoro.
[0086] "Mercapto" refers to the group --SR wherein the R group is
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, cycloalkyl, aryl or substituted aryl. The
term "alkylmerecapto" refers to the group --SR when R is alkyl,
substituted alkyl or cycloalkyl.
[0087] Compounds of the preferred embodiments of the present
invention include those compounds in Table 1.
TABLE-US-00001 LPAAT.beta. CT Colorimetric Assay Number Structure
IC.sub.50 (nM) 31867 ##STR00006## 750 31942 ##STR00007## 400 31978
##STR00008## 1,000 32028 ##STR00009## 200 32042 ##STR00010## 200
32099 ##STR00011## 650 116988 ##STR00012## 160 117147 ##STR00013##
3,800 31888 ##STR00014## 3,100
[0088] Preferred compounds include, but are not limited to,
6-chloro-N-(4-methoxy-phenyl)-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-butyl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,
6-chloro-N-isopropyl-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
N-tert-butyl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,
N-tert-butyl-6-chloro-N'-p-tolyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-cyclo-hexyl-N'-isopropyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,
6-chloro-N-isopropyl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
N-allyl-6-chloro-N'-cyclohexyl-[1,3,5]triazine-2,4-diamine,
2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,
N-tert-butyl-6-chloro-N'-cyclopentyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-methoxyphenyl)-N'-phenyl-[1,3,5]triazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4--
diamine,
6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N'-phenyl-[1,3,5]tr-
iazine-2,4-diamine,
N-benzo[1,3]dioxol-5-yl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-indan-5-yl-N'-phenyl-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chloro-phenyl)-N'-propyl-[1,3,5]triazine-2,4-diamine,
N-(4-chloro-phenyl)-6-methoxy-N'-propyl-[1,3,5]triazine-2,4-diamine
and
N-(4-chloro-phenyl)-6-methylsulfanyl-N'-phenyl-[1,3,5]triazine-2,4-diamin-
e.
[0089] Most preferred compounds include, but are not limited to,
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine,
N-tert-butyl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,
6-chloro-N-(4-chlorophenyl)-N'-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diam-
ine,
6-chloro-N-(4-chlorophenyl)-N'-phenyl-[1,3,5]-triazine-2,4-diamine.
[0090] The compounds of the preferred embodiments of the present
invention inhibit LPAAT-.beta. and thereby inhibit cell
proliferation. Therefore, the compounds of the preferred
embodiments of the present invention may be useful in the treatment
of cancer. The types of cancer that may be treated with the
compounds of the preferred embodiments of the present invention
include, but are not limited to, prostate, breast, lung, ovarian,
brain, cervical, colon or bladder cancer, and not limited to tumor
cells expressing high levels of LPAAT-.beta. as evidenced by the
decrease in NIH/3T3 Ki-ras tumor cell growth in vitro and in vivo
when treated with,
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine.
IV. Pharmacological Compositions, Therapeutic and Other
Applications
[0091] The compound of the present invention, or its
pharmaceutically acceptable salt, can be administered to a human
patient per se, or in pharmacological compositions where it is
mixed with pharmaceutically acceptable carriers or excipient(s).
Techniques for formulation and administration of drugs may be found
in "Remington's Pharmaceutical Sciences," Mack Publishing Co.,
Easton, Pa., latest edition.
[0092] A. Routes of Administration.
[0093] Suitable routes of administration may include, without
limitation, oral, rectal, transmucosal or intestinal administration
or intramuscular, subcutaneous, intramedullary, intrathecal, direct
intraventricular, intravenous, intraperitoneal or intranasal
injections.
[0094] Alternately, one may administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a solid tumor, often in a depot or sustained
release formulation.
[0095] Furthermore, one may administer the drug in a targeted drug
delivery system, for example, in a liposome coated with
tumor-specific antibody. The liposomes will be targeted to and
taken up selectively by the tumor.
[0096] B. Composition/Formulation.
[0097] Pharmacological compositions of the compounds and the
pharmaceutically acceptable salts thereof are preferred embodiments
of this invention. Pharmacological compositions of the present
invention may be manufactured by processes well known in the art;
e.g., by means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0098] Pharmacological compositions for use in accordance with the
present invention thus may be formulated in a conventional manner
using one or more pharmaceutically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0099] For injection, the compounds of the invention may be
formulated as sterile aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0100] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmacological preparations for oral use can be made with the use
of a solid excipient, optionally grinding the resulting mixture,
and processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0101] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0102] Pharmacological compositions which can be used orally
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules can contain the active ingredients
in admixture with a filler such as lactose, binders such as
starches, and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0103] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0104] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0105] The compounds may be formulated for parenteral
administration, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0106] Pharmacological compositions for parenteral administration
include sterile aqueous solutions of the active compounds in water
soluble form. Additionally, suspensions of the active compounds may
be prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0107] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0108] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0109] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation (see, for
example, U.S. Pat. No. 5,702,717 for a biodegradable depot for the
delivery of a drug). Such long acting formulations may be
administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compounds may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt. The pharmacological
compositions herein also may comprise suitable solid or gel phase
carriers or excipients. Examples of such carriers or excipients
include but are not limited to calcium carbonate, calcium
phosphate, various sugars, starches, cellulose derivatives,
gelatin, and polymers such as polyethylene glycols.
[0110] The compounds of the invention that inhibit LPAAT-.beta. may
be provided as physiologically acceptable salts wherein the claimed
compound may form the negatively or the positively charged species.
Examples of salts in which the compound forms the positively
charged moiety include, without limitation, quaternary ammonium
(defined elsewhere herein), salts such as the hydrochloride,
sulfate, carbonate, lactate, tartrate, maleate, succinate, etc.
formed by the reaction of an amino group with the appropriate
acid.
[0111] C. Dosage.
[0112] Pharmacological compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an amount effective to achieve its intended
purpose.
[0113] More specifically, a therapeutically effective amount means
an amount of compound effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subject being
treated.
[0114] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0115] For any compound used in the methods of the invention, the
therapeutically effective amount or dose can be estimated initially
from cell culture assays. For example, a dose can be formulated in
animal models to achieve a circulating concentration range that
includes the IC.sub.50 as determined in cell culture (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of LPAAT-.beta. activity). Such information can be used
to more accurately determine useful doses in humans.
[0116] Toxicity and therapeutic efficacy of the compounds described
herein can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals for determining the LD.sub.50
(the dose lethal to 50% of the population) and the ED.sub.50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio between LD.sub.50 and
ED.sub.50. Compounds which exhibit high therapeutic indices are
preferred. The data obtained from these cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage of such compounds lies preferably within a
range of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (see e.g., Fingl, et al., in
"The Pharmacological Basis of Therapeutics," (1975), Chapter 1, pp.
1).
[0117] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain LPAAT-.beta. inhibitory effects, or minimal effective
concentration (MEC). The MEC will vary for each compound but can be
estimated from in vitro data; e.g., the concentration necessary to
achieve 50-90% inhibition of LPAAT-.beta. using the assays
described herein. Dosages necessary to achieve the MEC will depend
on individual characteristics and route of administration. However,
HPLC assays or bioassays can be used to determine plasma
concentrations.
[0118] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%.
[0119] In cases of local administration or selective uptake, the
effective local concentration of the drug may not be related to
plasma concentration.
[0120] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician. An exemplary systemic
daily dosage is about 5 to about 200 mg/kg of body weight.
Normally, from about 10 to about 100 mg/kg of body weight of the
compounds of the preferred embodiments of the present invention, in
one or more dosages per day, is effective to obtain the desired
results. One of ordinary skill in the art can determine the optimal
dosages and concentrations of the compounds of the preferred
embodiments of the present invention with only routine
experimentation.
[0121] The compounds of the preferred embodiments of the present
invention are substantially pure and preferably sterile. The phrase
"substantially pure" encompasses compounds created by chemical
synthesis and/or compounds substantially free of chemicals which
may accompany the compounds in the natural state, as evidenced by
thin layer chromatography (TLC) or high performance liquid
chromatography (HPLC).
[0122] D. Other Applications
[0123] The compounds of the preferred embodiments of the present
invention may be employed not only for therapeutic purposes, but
also as aids in performing research in vitro. For example, the
compounds of the preferred embodiments of the present invention may
be used to study biochemical pathways that would require the
inhibition of LPAAT-.beta. to elevated levels of LPA. Inhibition of
LPAAT-.beta. may result in the prolonged or limited activity of
biochemical pathways that depend on, or respond to, elevated levels
of LPA.
[0124] Additionally, a cell culture medium comprising the compounds
of the preferred embodiments of the present invention is within the
scope of the invention.
V. Assays for LPAAT-fl DNA, RNA and Protein
[0125] DNA molecules encoding the human LPAAT-.beta. gene, or
fragments thereof, can be used to detect the level of LPAAT-.beta.
gene expression in tissue samples. Such a detection method can be
used, for example, to compare the amount of LPAAT-.beta. RNA in a
sample obtained from normal tissue and in a sample isolated from
methotrexate-resistant tumor tissue. The presence of relatively low
levels of LPAAT-.beta. RNA in the tumor sample would indicate that
methotrexate resistance is due, at least in part, to
underexpression of the LPAAT-.beta. gene.
[0126] In testing a tissue sample for LPAAT-.beta. RNA using a
nucleic acid hybridization assay, RNA can be isolated from tissue
by sectioning on a cryostat and lysing the sections with a
detergent such as SDS and a chelating agent such as EDTA,
optionally with overnight digestion with proteinase K. Such tissue
may be obtained by biopsy. A preferred quantity of tissue is in the
range of 10-100 milligrams. Protein may be removed by phenol and
chloroform extractions, and nucleic acids are precipitated with
ethanol. RNA may be isolated by chromatography on an oligo dT
column and then eluted from the column. Further fractionation can
also be carried out according to methods well known to those of
ordinary skill in the art.
[0127] A number of techniques for molecular hybridization are used
for the detection of DNA or RNA sequences in tissues. When large
amounts of tissue are available, analysis of hybridization kinetics
provides the opportunity to accurately quantitate the amount of DNA
or RNA present, as well as to distinguish sequences that are
closely related but not identical to the probe. Reactions are run
under conditions of hybridization (T.sub.m-25.degree. C.) in which
the rate of re-association of the probe is optimal. Wetmur et al.,
J. Mol. Biol. 31:349 (1968). The kinetics of the reaction are
second order when the sequences in the tissue are identical to
those of the probe; however, the reaction exhibits complex kinetics
when probe sequences have partial homology to those in the tissue.
Sharp et al., J. Mol. Biol. 86:709 (1974).
[0128] The concentration of probe to cellular RNA is determined by
the sensitivity desired. To detect one transcript per cell would
require about 100 pg of probe per mg of total cellular DNA or RNA.
The nucleic acids are mixed, denatured, brought to the appropriate
salt concentration and temperature, and allowed to hybridize for
various periods of time. The rate of reassociation can be
determined by quantitating the amount of probe hybridized either by
hydroxyapatite chromatography (Britten et al., Science 161:529
(1968)) or by S1 nuclease digestion (Sutton, Biochim. Biophys. Acta
240:522 (1971)).
[0129] Another method of hybridization is the Northern Blot
technique. The particular hybridization technique is not essential
to the invention, and any technique commonly used in the art is
within the scope of the present invention. Typical probe technology
is described in U.S. Pat. No. 4,358,535, incorporated by reference
herein. For example, hybridization can be carried out in a solution
containing 6.times.SSC (10.times.SSC: 1.5 M sodium chloride, 0.15 M
sodium citrate, pH 7.0), 5.times.Denhardt's (1.times.Denhardt's:
0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone, 0.02% Ficoll
400), 10 mM EDTA, 0.5% SDS and about 10.sup.7 cpm of
nick-translated DNA for 16 hours at 65.degree. C.
[0130] The aforementioned hybridization assays are particularly
well suited for preparation and commercialization in kit form, the
kit comprising a carrier means compartmentalized to receive one or
more container means (vial, test tube, etc.) in close confinement,
with each container means comprising one of the separate elements
to be used in hybridization assay. For example, there may be a
container means containing LPAAT-.beta. DNA molecules suitable for
labeling by "nick translation," or containing labeled LPAAT-.beta.
DNA or labeled LPAAT-.beta. RNA molecules. Further container means
may contain standard solutions for nick translation of DNA
comprising DNA polymerase I/DNase I and unlabeled
deoxyribonucleotides.
[0131] Antibodies to human LPAAT-.beta. protein can be obtained
using the product of an LPAAT-.beta. expression vector as an
antigen. The preparation of polyclonal antibodies is well-known to
those of skill in the art. See, for example, Green et al.,
"Production of Polyclonal Antisera," in Immunochemical Protocols
(Manson, ed.), pp. 1-5 (Humana Press 1992). Alternatively, an
LPAAT-.beta. antibody of the present invention may be derived from
a rodent monoclonal antibody (MAb). Rodent monoclonal antibodies to
specific antigens may be obtained by methods known to those skilled
in the art. See, for example, Kohler and Milstein, Nature 256:495,
1975, and Coligan et al. (eds.), Current Protocols in Immunology,
1:2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter "Coligan"].
Briefly, monoclonal antibodies can be obtained by injecting mice
with a composition comprising an antigen, verifying the presence of
antibody production by removing a serum sample, removing the spleen
to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma
cells to produce hybridomas, cloning the hybridomas, selecting
positive clones which produce antibodies to the antigen, culturing
the clones that produce antibodies to the antigen, and isolating
the antibodies from the hybridoma cultures.
[0132] MAbs can be isolated and purified from hybridoma cultures by
a variety of techniques that are well known in the art. Such
isolation techniques include affinity chromatography with Protein-A
Sepharose, size-exclusion chromatography, and ion-exchange
chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and
pages 2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, 10:79
(Humana Press, Inc. 1992). A LPAAT-.beta. antibody may also be
derived from a subhuman primate antibody. General techniques for
raising therapeutically useful antibodies in baboons may be found,
for example, in Goldenberg et al., International Patent Publication
No. WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46:310
(1990).
[0133] Alternatively, a therapeutically useful LPAAT-.beta.
antibody may be derived from a "humanized" monoclonal antibody.
Humanized monoclonal antibodies are produced by transferring mouse
complementary determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
and then, substituting human residues in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by the publication of Orlandi
et al., Proc. Nat'l. Acad. Sci. USA 86:3833 (1989). Techniques for
producing humanized MAbs are described, for example, by Jones et
al., Nature 321:522 (1986); Riechmann et al., Nature 332:323
(1988); Verhoeyen et al., Science 239:1534 (1988); Carter et al.,
Proc. Nat'l Acad. Sci. USA 89:4285 (1992); Sandhu, Crit. Rev.
Biotech. 12: 437 (1992); and Singer et al., J. Immun. 150:2844
(1993), each of which is hereby incorporated by reference.
[0134] As an alternative, a LPAAT-.beta. antibody of the present
invention may be derived from human antibody fragments isolated
from a combinatorial immunoglobulin library. See, for example,
Barbas et al., METHODS: A Companion to Methods in Enzymology 2:119
(1991); and Winter et al., Ann. Rev. Immunol. 12:433 (1994) which
are incorporated herein by reference. Cloning and expression
vectors that are useful for producing a human immunoglobulin phage
library can be obtained, for example, from STRATAGENE Cloning
Systems (La Jolla, Calif.). In addition, a LPAAT-.beta. antibody of
the present invention may be derived from a human monoclonal
antibody. Such antibodies are obtained from transgenic mice that
have been "engineered" to produce specific human antibodies in
response to antigenic challenge. In this technique, elements of the
human heavy and light chain locus are introduced into strains of
mice derived from embryonic stem cell lines that contain targeted
disruptions of the endogenous heavy chain and light chain loci. The
transgenic mice can synthesize human antibodies specific for human
antigens, and the mice can be used to produce human
antibody-secreting hybridomas. Methods for obtaining human
antibodies from transgenic mice are described by Green et al.,
Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994);
and Taylor et al., Int. Immun. 6:579 (1994).
[0135] Having now generally described this invention, the same will
be understood by reference to the following examples which are
provided herein for purposes of illustration only and are not
intended to be limiting unless otherwise specified.
Example 1
Production of Recombinant LPAAT-.beta. for Various Assays
[0136] For the construction of Baculovirus expression vectors, the
full-length human LPAAT-.beta. cDNA was amplified by PCR from the
DNA template pCE9.LPAAT-.beta. (West et al., DNA Cell Biol.
16:691-701 (1997)) using the primers 5'-TGATATCCGA AGAAGATCTT
ATGGAGCTGT GGCCGTGTC-3' (olpb1F; SEQ ID NO:3) and 5'-CAGGCTCTAG
ACTACTGGGC CGGCTGCAC-3' (olpb1R; SEQ ID NO:4). The .about.870 by
fragment generated was reamplified by PCR using the primers 5'
CCTACGTCG ACATGGAACA AAAATTGATA TCCGAAGAAG ATC-3' (olpb2F; SEQ ID
NO:5) and 5'-CAGGCTCTAG ACTACTGGGC CGGCTGCAC-3' (olpb1R; SEQ ID
NO:6). The .about.890 by fragment generated was then cleaved with
Sal I and Xba I for insertion into pFastBac.TM. HTc vector (Life
Technologies, Gaithersberg, Md.) between the Sal I and Xba I sites
for the generation of the plasmid pFB.LPAAT-.beta.. This plasmid
was then transformed into E. coli DH10Bac.TM. (Life Technologies,
Gaithersberg, Md.) for the generation of recombinant Bacmid DNA for
transfection into HighFive (Invitrogen, San Diego, Calif.) or SF9
insect cells for the production of recombinant Baculovirus stocks
using the protocol described in the Bac-to-Bac.RTM. Baculovirus
Expression System (Life Technologies, Gaithersberg, Md.), a
eukaryotic expression system for generating recombinant baculovirus
through site-specific transposition in E. coli. Viral stocks
harvested from the transfected cells can then be used to infect
fresh insect cells for the subsequent expression of LPAAT-.beta.
fusion protein with a poly-histidine tag and a myc-epitope near its
N-terminus. The membrane fraction from these Sf9 cells would be the
source of LPAAT enzyme.
Example 2
Preparation of Cell Membranes from Sf9 Cells
[0137] For the preparation of membranes From Sf9 Cells, all steps
are performed on ice or at 4.degree. C. Sf9 cell pellets
(.about.10.sup.8 cells) were thawed and resuspended in 1-2 ml of
buffer A (20 mM Hepes, pH 7.5, 1 mM DTT, 1 mM EDTA, 20% w/v
glycerol, 1 mM Benzamidine, 1 .mu.g/ml soybean trypsin inhibitor
(SBTI), 1 .mu.g/ml pepstatin A) w/o DTT but with 1 mM Pefabloc. The
cells were lysed by sonication using a Branson Sonifier at
output=2, duty cycle=2, 10 pulses each at 10 s. with the tip of
small sonicator probe submerged but not touching the walls. DTT was
then added to 1 mM from a 1 M stock. The samples were centrifuged
at 1500 rpm for 5 min. The low speed supernatant was saved and
centrifuged (TLA 100.3 rotor, polycarbonate tubes, 2 ml/tube or 1.5
ml/tube minimum) at 100000.times.g for 1 hr. The high speed pellet
was resuspend in Buffer A with a probe sonicator (10 pulses @
output #2 and duty cycle 20%) as a source of LPAAT enzyme.
Example 3
Assay of LPAAT-.beta. Activity
[0138] LPAAT-.beta. catalyzes the transfer of an acyl group from a
donor such as acyl-CoA to LPA. The transfer of the acyl group from
acyl-CoA to LPA leads to the release of free CoA, which can be
reacted with the thiol reagent, 5,5'-dithiobis(2-nitrobenzoic acid)
(DTNB). The reaction between DTNB and the free sulfhydryl group
from CoA generates a yellow-colored product,
3-carboxylato-4-nitrothiophenolate (CNP), that absorbs at 413 nm.
LPAAT-.beta. derived from Sf9 cell membrane overexpressing
LPAAT-.beta. were resuspended in HEPES saline buffer (20 mM HEPES
pH 7.5, 150 mM NaCl), 1 mg/ml BSA and 72 .mu.l aliquots were
distributed into 96-well microtiter plates. 8 .mu.l of compound of
interest at 200 .mu.M dissolved in 100% DMSO was added into each
well. 20 .mu.l of 1 mM 18:1-CoA and 1 mM sn-1-18:1 lysoPA was then
added to each well to initiate the reaction and allowed to run at
room temperature for 25 min. 100 .mu.l of 1 mM DTNB in 100% ethanol
was then added to each well to quench the reaction and for color
development. The absorbance at 405 nm, measured using a
spectrophotometer plate reader, is proportional to the activity of
LPAAT-.beta. in the sample. This colorimetric assay was used for
the high throughput screening of LPAAT inhibitors. Compounds that
showed >50% inhibition of the change in absorbance at 405 nm
compared to control were selected for a secondary assay. FIG. 7
shows an example of the colorimetric assay of which the time course
of color development is dependent on the amount of LPAAT enzyme
added. FIG. 8 shows an example of the results obtained from
assaying a plate of various compounds at 16 .mu.M. Compounds that
gave a reading of less than 0.06 arbitrary units (indicated by
arrow on right margin) were selected for further study.
[0139] A secondary assay for LPAAT activity in cell extracts based
on either the conversion of fluorescent NBD-LPA to NBD-PA (West, et
al., DNA Cell Biol. 6: 691-701, 1997) or [.sup.14C]LPA to
[.sup.14C]PA using TLC analysis was used to screen compounds that
showed >50% inhibition of LPAAT activity in the primary
colorimetric assay. The radiometric assay was carried out in Sf9
cell membrane overexpressing LPAAT-.beta. resuspended in
HEPES-saline buffer, pH 7.5, 1 mg/ml BSA, 1 mM EDTA and 200 .mu.M
[.sup.14C]18:1-CoA and 200 .mu.M sn-1-18:1 lysoPA. The samples were
incubated 7 min at 37.degree. C., extracted into organic solvent
(CHCl.sub.3/CH.sub.3OH/HCl at 33/66/1), before loading onto TLC
plates. A more detailed protocol for the radiometric assay is
described below:
[0140] Specifically, this LPAAT assay is a modification of the
acyltransferase assay published previously (Hollenback and Glomset,
Biochemistry 37: 363-376 (1999)).
[0141] 1. The basic assay, in a total vol of 50 .mu.l, employs a
solution of substrates and the protein sample. Total assay volume,
as well as the volume of each solution, can be changed to fit an
experiment. In addition, other compounds, ex inhibitors and
activators, can be included in the assay as well.
[0142] 2. To prepare the solution of substrates: [0143] a. Stocks
of Hepes (pH 7.5), NaCl, EDTA, BSA and acyl-CoA (from Serdery or
Sigma) are mixed with water to make the appropriate concentration
of each compound. This can be varied from assay-to-assay, but the
final reaction mix is about 50 mM Hepes, 100 mM NaCl, 1 mM EDTA, 1
mg/ml BSA and 0-400 .mu.M acyl-CoA. [0144] b. The lysoPA (from
Avanti) is typically stored in chloroform and the .sup.14C-labeled
acyl-CoA (from Amersham) is typically stored in water/ethanol=1:1.
Appropriate amounts of each solution are added the to a 12.times.75
mm borosilicate glass test tube and dry the solvent under N.sub.2
or Ar. An appropriate volume of the solution prepared in 2a is
added to the lysoPA and .sup.14C-labeled acyl-CoA. The lipids are
resuspend by sonication for 15 sec in a bath sonicator. The
resulting suspension is then incubated (with occasional gentle
vortexing) for about 10 minutes at room temp. The sn-1-16:0 lysoPA
may require brief warming of the solvent to solubilize it. The
concentration of lysoPA and .sup.14C-labeled acyl-CoA can vary, but
typically the final lysoPA concentration ranges between 0 and 400
.mu.M and the .sup.14C-labeled acyl-CoA specific activity ranges
between 0.5 and 2 Ci/mol.
[0145] 3. Protein sample: varies from experiment-to-experiment.
[0146] 4. The assay is performed by mixing the components in
12.times.75 mm borosilicate glass test tubes (the order of addition
does not matter unless indicated) and incubating at 37.degree. C.
for 5 to 10 minutes such that the assay within the linear range for
time and protein.
[0147] 5. The reaction is quenched by adding 1.3 ml of
chloroform/methanol/HCl=48/51/0.7 and vortexing. 10 .mu.l of
carrier solution is then added (3 mg/ml each PA, ex. 16:0-18:1, and
lysoPA, ex sn-1-18:1, in chloroform). Two phases are formed by
adding 0.3 ml of water to each tube and vortexing.
[0148] 6. The sample is centrifuged for 3 minutes at 1000.times.g,
the upper (aqueous/methanol) phase is aspirated and the lower phase
is dried under nitrogen.
[0149] 7. Thin layer chromatography: [0150] a. The dried samples
are resuspended in 50 .mu.l of chloroform and a 15 .mu.l aliquot is
immediately spotted on an Analtech silica gel 60 HP-TLC plate
(10.times.20 cm). [0151] b. Plates are developed in
chloroform/methanol/acetic acid/water=85/12.5/12.5/3 (takes about
15 min) and dried. [0152] c. To be able to convert pixel volume
(determined by the Storm phosphor imager, see step 8b) into cpm,
cpm standard curve must be generated on the plate. .sup.14C-labeled
oleate dilutions in chloroform are made for this purpose. Four
stocks (50 cpm/.mu.l to 800 cpm/.mu.l) are made and 2 .mu.l of a
different concentration are spotted in each corner of the plate
(where previously there was no radioactivity). [0153] d. For
quality control purposes, the plates are stained with primuline and
scanned with the Storm (blue chemilluminescence mode).
[0154] The PA and lysoPA bands are easily detected in this system
because of the carrier added in step 5. PA and lysoPA have
respective Rf's of about 0.63 and 0.21.
[0155] 8. Quantitating activity: [0156] a. The plates are then
wrapped in saran wrap and exposed to a freshly blanked phosphor
screen overnight (longer exposures can also be done to increase the
signal). [0157] b. The screens are scanned (Phosphorimager mode),
and LPAAT activity is determined by quantifying the pixels in the
band comigrating with PA standard versus the standard curve
generated from the cpm standards that were spotted in step 7c.
[0158] FIG. 9 shows examples of a compound, namely,
6-chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine, selected from
the secondary screening that exhibit concentration-dependent
inhibition of LPAAT-.beta. activity (O). Moreover, these compounds
have minimal effect on LPAAT-.alpha. activity (.quadrature.),
suggesting they are isoform-specific inhibitors.
Example 4
[0159] Triazines of the preferred embodiments of the present
invention were synthesized by one of two methods. Symmetrically
substituted triazines were synthesized by the addition of two
equivalents of the appropriate amino compound, in the presence of
diisopropylethylamine, to cyanuric chloride. Non-symmetrical
triazines were synthesized in a stepwise fashion by the sequential
addition of the amino compound in the presence of potassium
carbonate with isolation of the intermediate
mono-amino-dichlorotriazine.
A. Method 1: Synthesis of Symmetrical Triazines
Synthesis of
6-Chloro-N,N'-diphenyl-[1,3,5]triazine-2,4-diamine(CT-113020)
[0160] To a mixture of cyanuric chloride (5.07 g, 27.5 mmol) and
acetonitrile (60 ml), cooled in an ice bath, was added a solution
of aniline (5.3 ml, 58.2 mmol) and diisopropylethylamine (10.5 ml,
60.3 mmol) in acetonitrile (15 ml) over 30 minutes. After stirring
at room temperature for 20 hours, the mixture was filtered. The
solid was washed with ethyl acetate (4.times.25 ml), suspended in
water (50 ml), stirred for 1 hour, filtered, washed with water, and
dried under vacuum to give
6-chloro-N,N-diphenyl-[1,3,5]triazine-2,4-diamine (3.97 g, 48%
yield) as a white solid. .sup.1H NMR (d.sub.6-DMSO) .delta. 7.61
(2H, br s), 7.28-7.37 (4H, m), 7.05-7.11 (4H, m). .sup.13C NMR
(d.sub.6-DMSO) .delta. 167.7, 164.0, 138.5, 128.4, 124.5,
121.3.
B. Method 2: Synthesis of Unsymmetrical Triazines
Synthesis of
6-Chloro-N-(4-chlorophenyl)-N'-phenyl-[1,3,5]triazine-2,4-diamine(CT-1164-
33)
[0161] To a mixture of cyanuric chloride (5.15 g, 27.9 mmol),
potassium carbonate (3.98 g, 28.8 mmol) and 18-crown-6 (158 mg,
0.60 mmol) in toluene (40 ml), cooled in an ice bath, was added a
solution of 4-chloroaniline (3.61 g, 28.3 mmol) in toluene (20 ml)
over 15 minutes. After stirring at room temperature for 20 hours,
the mixture was treated with ethyl acetate (60 ml) and filtered
through a pad of celite under suction. The filtrate was
concentrated under vacuum and recrystallized (chloroform) to give
(4-chloro-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (4.06 g,
53% yield) as a white solid. .sup.1H NMR (d.sub.6-DMSO) .delta.
11.23 (1H, s), 7.62 (2H, d, J=9 Hz), 7.47 (2H, d, J=9 Hz). .sup.13C
NMR (d.sub.6-DMSO) .delta. 169.5, 168.5, 163.6, 135.7, 128.6,
122.5, ESMS m/z 273 (M-H).sup.-.
[0162] To a mixture of
(4-chloro-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (3.97 g,
14.4 mmol), potassium carbonate (2.20 g, 15.9 mmol) and 18-crown-6
(46 mg, 0.17 mmol) in toluene (25 ml), cooled in an ice bath, was
added a solution of aniline (1.4 ml, 15.4 mmol) in toluene (10 ml)
over 15 minutes. After stirring at room temperature for 24 hours,
the mixture was treated with ethyl acetate (35 ml) and filtered
through a pad of celite under suction. The filtrate was
concentrated under vacuum and the residue was recrystallized
(chloroform) to give
6-Chloro-N-(4-chlorophenyl)-N-phenyl-[1,3,5]triazine-2,4-diamine
(2.20 g, 46% yield) as a white solid. .sup.1H NMR (d.sub.6-DMSO)
.delta. 10.1-10.4 (2H, br s), 7.5-7.8 (4H, br s), 7.3-7.5 (4H, m),
7.15-7.05 (1H, m), ESMS m/z 330 (M-H).sup.-.
Example 5
Synthesis of
6-Chloro-N-(4-chlorophenyl)-N'-(4-methoxy-phenyl)-[1,3,5]triazine-2,4-dia-
mine(CT-31867)
[0163] The reaction of
(4-chloro-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine with
p-anisidine, according to method 2, gave
6-chloro-N-(4-chlorophenyl)-N'-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diam-
ine (51 mg, 62%): .sup.1H NMR (CDCl.sub.3) 7.21-7.50 (6H, m), 6.91
(2H, d, J=11 Hz), 3.85 (3H, s).
Example 6
Synthesis of
6-Chloro-N-(4-methoxyphenyl)-N'-phenyl-[1,3,5]triazine-2,4-diamine(CT-319-
42)
[0164] To a solution of
2-(4-methoxyphenyl)amino-4,6-dichloro-1,3,5-triazine (57 mg, 0.21
mmoles) in acetonitrile (0.5 ml) was added a solution of aniline
(0.021 ml, 0.23 mmoles) and diisopropylethylamine (0.05 ml, 0.29
mmoles) in acetonitrile (0.5 ml). After stirring for 24 hours, the
mixture was concentrated under vacuum and the residue was dissolved
in ethyl acetate (10 ml). The solution was washed with a solution
composed of saturated aqueous sodium chloride solution and 1 M
hydrochloric acid (1:1, 2.times.10 ml), dried over sodium sulfate,
filtered, and concentrated under vacuum. The residue was purified
by flash chromatography on silica gel eluting with 10% ethyl
acetate-hexane to provide CT-31942 (38 mg, 57% yield). .sup.1H NMR
(d.sub.6-DMSO) .delta. 10.00-10.22 (m, 2H), 7.26-7.82 (m, 6H),
7.01-7.10 (m, 1H), 6.91 (d, J=9 Hz, 2H), 3.74 (s, 3H).
Example 7
Synthesis of
N-Benzo[1,3]dioxol-5-yl-6-chloro-N'-(4-chlorophenyl)-[1,3,5]triazine-2,4--
diamine(CT-31978)
[0165] The reaction of
4,6-dichloro-N-(4-chlorophenyl)-[1,3,5]triazine-2-amine with
3,4-methylenedioxyaniline using the method described for the
synthesis of CT-116433 provided CT-31978 (56 mg, 65% yield).
.sup.1H NMR (d.sub.6-DMSO) .delta. 10.08-10.37 (m, 2H), 7.58-7.87
(m, 2H), 7.21-7.40 (m, 3H), 6.85-7.03 (m, 2H), 6.00 (s, 2H).
Example 8
Synthesis of
6-Chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N'-phenyl-[1,3,5]triazine-2-
,4-diamine(CT-32028)
[0166] To a solution of
4,6-dichloro-N-phenyl-[1,3,5]triazine-2-amine (95 mg, 0.39 mmoles)
in tetrahydrofuran (2 ml) was added a solution of
1,4-benzodioxan-6-amine (64 mg, 0.42 mmoles) and triethylamine
(0.07 ml, 0.50 mmoles) in tetrahydrofuran (0.5 ml). After stirring
for 24 hours, the mixture was concentrated under vacuum and the
residue was dissolved in ethyl acetate (10 ml). The solution was
washed with a solution composed of saturated aqueous sodium
chloride solution and 1 M hydrochloric acid (1:1, 2.times.10 ml),
dried over sodium sulfate, filtered, and concentrated under vacuum.
The residue was purified by flash chromatography on silica gel
eluting with 25% ethyl acetate-hexanes to provide CT-32028 (97 mg,
69% yield). .sup.1H NMR (d.sub.6-DMSO) .delta. 9.88-10.28 (m, 2H),
7.60-7.85 (m, 2H), 7.20-7.40 (m, 3H), 6.96-7.10 (m, 2H), 6.77-6.81
(m, 1H), 4.21 (s, 4H).
Example 9
Synthesis of
N-Benzo[1,3]dioxol-5-yl-6-chloro-N'-phenyl-[1,3,5]triazine-2,4-diamine(CT-
-32042)
[0167] The reaction of
4,6-dichloro-N-phenyl-[1,3,5]triazine-2-amine with
3,4-methylenedioxyaniline using the method described for the
synthesis of CT-32028 provided CT-32042 (51 mg, 42% yield). .sup.1H
NMR (d.sub.6-DMSO) .delta. 10.05-10.29 (m, 2H), 7.57-7.80 (m, 2H),
7.21-7.42 (m, 3H), 6.80-7.13 (m, 3H), 6.03 (s, 2H).
Example 10
Synthesis of
6-Chloro-N-indan-5-yl-N'-phenyl-[1,3,5]triazine-2,4-diamine(CT-32099)
[0168] The reaction of
4,6-dichloro-N-phenyl-[1,3,5]triazine-2-amine with 5-aminoindan
using the method described for the synthesis of CT-32028 provided
CT-32099 (36 mg, 37% yield). .sup.1H NMR (d.sub.6-DMSO) .delta.
10.15-10.28 (m, 2H), 7.56-7.72 (m, 3H), 7.05-7.39 (m, 5H), 2.75-90
(m, 4H), 1.94-2.09 (m, 2H).
Example 11
Synthesis of
6-Chloro-N-2-(4-chlorophenyl)-N-4-propyl-[1,3,5]triazine-2,4-diamine(CT-1-
16988)
[0169] The reaction of
4,6-dichloro-N-(4-chlorophenyl)-[1,3,5]triazine-2-amine with
propylamine using the method described for the synthesis of
CT-116433 provides CT-116988.
Example 12
Synthesis of
N-(4-Chlorophenyl)-6-methoxy-N'-propyl-[1,3,5]triazine-2,4-diamine(CT-117-
147)
[0170] A mixture of CT-116988 and sodium methoxide (3.0 molar
equivalents) in methanol is heated at reflux for 18 hours. After
cooling to room temperature, the reaction mixture is concentrated
under vacuum. The residue is suspended in ethyl acetate and washed
with water. The ethyl acetate phase is dried over sodium sulfate,
filtered and concentrated under vacuum. The residue is purified by
flash chromatography on silica gel to provide CT-117147.
Example 13
Synthesis of
N-(4-chlorophenyl)-6-methylsulfanyl-N'-phenyl-[1,3,5]triazine-2,4-diamine-
(CT-31888)
[0171] A mixture of CT-116433 (108 mg, 0.32 mmol) and sodium
methanethiolate (79 mg, 1.13 mmol) in dimethyl sulfoxide (3 ml) was
heated at 70.degree. C. for 18 hours. After cooling to room
temperature the mixture was diluted with ethyl acetate (25 ml) and
was washed with a 1:1 solution of water and saturated aqueous
sodium chloride solution (4.times.25 ml). The organic phase was
dried over sodium sulfate, filtered, and concentrated under vacuum.
The residue was purified by flash chromatography on silica gel
eluting with 5% ethyl acetate-hexane to provide CT-31888 (76 mg,
69% yield). .sup.1H NMR (d.sub.6-DMSO) .delta. 7.51-7.60 (m, 4H),
7.26-7.39 (m, 5H), 7.00-7.17 (m, 2H), 2.56 (s, 3H).
[0172] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention,
and without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions without undue experimentation. All
patents, patent applications and publications cited herein are
incorporated by reference in their entirety.
Sequence CWU 1
1
5143DNAArtificial SequencePrimer 1gcatgaattc aaaggcctac gtcgacatgg
agctgtggcc gtg 43230DNAArtificial SequencePrimer 2gtcgactcta
gactactggg ccggctgcac 30339DNAArtificial SequencePrimer 3tgatatccga
agaagatctt atggagctgt ggccgtgtc 39429DNAArtificial SequencePrimer
4caggctctag actactgggc cggctgcac 29542DNAArtificial SequencePrimer
5cctacgtcga catggaacaa aaattgatat ccgaagaaga tc 42
* * * * *