U.S. patent application number 09/818201 was filed with the patent office on 2001-10-25 for amide derivatives for antiangiogenic and/or antitumorigenic use.
Invention is credited to Kalgutkar, Amit S., Marnett, Lawrence J..
Application Number | 20010034361 09/818201 |
Document ID | / |
Family ID | 22850011 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010034361 |
Kind Code |
A1 |
Kalgutkar, Amit S. ; et
al. |
October 25, 2001 |
Amide derivatives for antiangiogenic and/or antitumorigenic use
Abstract
A method of treating animals having cancer by administration of
secondary amide derivatives of various COOH-containing drugs, such
as COOH-containing NSAIDs, for instance, indomethacin.
Inventors: |
Kalgutkar, Amit S.;
(Nashville, TN) ; Marnett, Lawrence J.;
(Nashville, TN) |
Correspondence
Address: |
JENKINS & WILSON, PA
3100 TOWER BLVD
SUITE 1400
DURHAM
NC
27707
US
|
Family ID: |
22850011 |
Appl. No.: |
09/818201 |
Filed: |
March 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09818201 |
Mar 27, 2001 |
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09226693 |
Jan 7, 1999 |
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6207700 |
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Current U.S.
Class: |
514/420 ;
514/570 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/40 20130101; A61P 43/00 20180101; A61P 29/00 20180101 |
Class at
Publication: |
514/420 ;
514/570 |
International
Class: |
A61K 031/405; A61K
031/19 |
Goverment Interests
[0001] This research was funded by a research grant from the
National Institutes of Health (Research Grant No. CA47479). Thus,
the United States government has certain rights in the invention.
Claims
What is claimed is:
1. A method for cancer treatment in a warm blooded vertebrate
animal, comprising administering to the animal a treatment
effective amount sufficient to inhibit cancer of a carboxylic acid
secondary amide derivative of a compound, wherein: (1) the
derivative is selective for inhibition of cyclooxygenase-2, and (2)
the compound (a) is a cyclooxygenase inhibitor but is absent
selectivity for inhibition of cyclooxygenase-2 and (b) contains a
carboxylic acid moiety or a pharmaceutically acceptable salt
thereof.
2. The method of claim 1, wherein the cancer treatment is
antiangiogenic treatment and the amount sufficient to inhibit
cancer is sufficient to inhibit blood cells that feed cancer or
blood vessel cells that feed cancer.
3. The method of claim 1, wherein the cancer treatment is antitumor
treatment and the amount sufficient to inhibit cancer is sufficient
to inhibit tumor growth.
4. The method of claim 3, further including the derivative
possesses an analgesic, antiinflammatory, or antipyretic property
as possessed by the compound and the treatment provides an
analgesic, antiinflammatory, or antipyretic effect in the animal
absent concomitant administration of another compound for providing
such effect.
5. The method of claim 1, wherein the compound is a non-steroidal
antiinflammatory drug.
6. The method of claim 5, wherein the non-steroidal
antiinflammatory drug is selected from the group consisting of
fenamic acids, indoles, phenylalkanoic acids, phenylacetic acids,
pharmaceutically acceptable salts thereof, and combinations
thereof.
7. The method of claim 5, wherein the non-steroidal
antiinflammatory drug is selected from the group consisting of
indomethacin, 6-methoxy-.alpha.-methyl-2-naphthylacetic acid,
meclofenamic acid, diclofenac, flufenamic acid, niflumic acid,
mefenamic acid, sulindac, tolmetin, suprofen, ketorolac,
flurbiprofen, ibuprofen, aceloferac, alcofenac, amfenac,
benoxaprofen, bromfenac, carprofen, clidanac, diflunisal, efenamic
acid, etodolic acid, fenbufen, fenclofenac, fenclorac, fenoprofen,
fleclozic acid, indoprofen, isofezolac, ketoprofen, loxoprofen,
meclofenamate, naproxen, orpanoxin, pirprofen, pranoprofen,
tolfenamic acid, zaltoprofen, zomopirac, pharmaceutically
acceptable salts thereof, and combinations thereof.
8. The method of claim 1, wherein the secondary amide derivative is
a secondary amide derivative of a non-steroidal antiinflammatory
drug.
9. The method of claim 1, wherein the secondary amide derivative is
selected from the group consisting of secondary amide derivatives
of fenamic acids, indoles, phenylalkanoic acids, phenylacetic
acids, and combinations thereof.
10. The method of claim 1, wherein the secondary amide derivative
is selected from the group consisting of secondary amide
derivatives of indomethacin,
6-methoxy-.alpha.-methyl-2-naphthylacetic acid, meclofenamic acid,
diclofenac, flufenamic acid, niflumic acid, mefenamic acid,
sulindac, tolmetin, suprofen, ketorolac, flurbiprofen, ibuprofen,
aceloferac, alcofenac, amfenac, benoxaprofen, bromfenac, carprofen,
clidanac, diflunisal, efenamic acid, etodolic acid, fenbufen,
fenclofenac, fenclorac, fenoprofen, fleclozic acid, indoprofen,
isofezolac, ketoprofen, loxoprofen, meclofenamate, naproxen,
orpanoxin, pirprofen, pranoprofen, tolfenamic acid, zaltoprofen,
zomopirac, and combinations thereof.
11. The method of claim 1, wherein the secondary amide derivative
is selected from the group consisting of indomethacin-N-methyl
amide, indomethacin-N-ethan-2-ol amide, indomethacin-N-octyl amide,
indomethacin-N-nonyl amide, indomethacin-N-(2-methylbenzyl) amide,
indomethacin-N-(4-methylbenzyl) amide,
indomethacin-N-((R)-,4-dimethylben- zyl) amide,
indomethacin-N-((S)-,4-dimethylbenzyl) amide,
indomethacin-N-(2-phenethyl) amide, indomethacin-N-(4-fluorophenyl)
amide, indomethacin-N-(4-chlorophenyl) amide,
indomethacin-N-(4-acetamido- phenyl) amide,
indomethacin-N-(4-methylmercapto)phenyl amide,
indomethacin-N-(3-methylmercaptophenyl) amide,
indomethacin-N-(4-methoxyp- henyl) amide,
indomethacin-N-(3-ethoxyphenyl) amide,
indomethacin-N-(3,4,5-trimethoxyphenyl) amide,
indomethacin-N-(3-pyridy) amide,
indomethacin-N-5-((2-chloro)pyridyl) amide,
indomethacin-N-5-((1-ethyl)pyrazolo) amide,
indomethacin-N-(3-chloropropy- l) amide,
indomethacin-N-methoxycarbonylmethyl amide,
indomethacin-N-2-(2-L-methoxycarbonylethyl) amide,
indomethacin-N-2-(2-D-methoxycarbonylethyl) amide,
indomethacin-N-(4-methoxycarbonylbenzyl) amide,
indomethacin-N-(4-methoxy- carbonylmethylphenyl) amide,
indomethacin-N-(2-pyrazinyl) amide,
indomethacin-N-2-(4-methylthiazolyl) amide,
indomethacin-N-(4-biphenyl) amide, and combinations thereof.
12. The method of claim 1, wherein the treatment effective amount
sufficient to inhibit cancer ranges from about 0.5 milligram to
about 7.0 milligrams per kilogram of body weight of the animal per
day.
13. The method of claim 1, wherein the treatment effective amount
sufficient to inhibit cancer ranges from about 1.5 milligrams to
about 6.0 milligrams per kilogram of body weight of the animal per
day.
14. The method of claim 1, wherein the treatment effective amount
sufficient to inhibit cancer ranges from about 2.0 milligrams to
about 5.0 milligrams per kilogram of body weight of the animal per
day.
Description
TECHNICAL FIELD
[0002] The present invention, in general, relates to conversion of
the carboxylic acid moiety of various compounds into amide
derivatives of the compounds. More specifically, the present
invention relates to secondary amide derivatives of non-steroidal
antiinflammatory drugs (NSAIDs), particularly of indomethacin (an
NSAID), that exhibit inhibition of cyclooxygenase-2 (COX-2) far
exceeding inhibition of cyclooxygenase-1 (COX-1), and also, that
still exhibit the analgesic, antiinflammatory, and/or antipyretic
effect of the compound, i.e., of the NSAID, and moreover, also
exhibit cancer inhibition, i.e., an antiangiogenic and/or
antitumorigenic effect, in warm blooded vertebrate animals,
including humans.
TABLE OF ABBREVIATIONS
[0003]
1 Abbreviations Definitions NSAID non-steroidal antiinflammatory
drug COOH carboxylic acid moiety COX cyclooxygenase PGH.sub.2
prostaglandin H.sub.2 PGD.sub.2 prostaglandin D.sub.2 PGHS
prostaglandin endoperoxide synthase PER peroxidase SAR
structure-activity relationship GI gastrointestinal IC.sub.50
concentration in micromoles of indomethacin (or the indomethacin
derivative) at which there is 50% inhibition of COX activity-the
lower the IC.sub.50 is, then the more potent the drug is DMSO
dimethyl sulfoxide .sup.14C-AA [1-.sup.14C]-arachidonic acid HPLC
high performance liquid chromatography TLC thin layer
chromatography mg milligram kg kilogram mL milliliter .mu.M
micromole/liter .mu.L microliter N normal (when used in conjunction
with acid concentrations) NMR nuclear magnetic resonance Et.sub.2O
diethyl ether EtOAc ethyl acetate Et.sub.3N triethyl amine AcOH
acetic acid CDCl.sub.3 deuteriated chloroform rt room temperature
(about 72.degree. F., 22.degree. C.) BOP-Cl
bis(2-oxo-3-oxazolidinyl)phosphonic chloride (sold by Aldrich in
Wisconsin), and also see the journal article, Diago-Meseguer,
Palomo- Coll, Fernandez-Lizarbe, and Zugaza-Bilbao, "New Reagent
for Activating Carboxyl Groups; Preparation and Reactions of N,N-
Bis[2-oxo-3-oxazolidinyl] phosphorodiamidic Chloride", Synthesis
(1980) pp. 547-551 mp melting point FBS fetal bovine serum DMEM
Dulbecco's modified essential medium LPS lipopolysaccharide PBS
phosphate-buffered saline IFN-g interferon gamma
BACKGROUND OF THE INVENTION
[0004] As discussed in more detail below, the COX enzyme is really
two enzymes, COX-1 and COX-2, which serve different physiological
and pathophysiological functions. As is well known, at
antiinflammatory and/or analgesic doses, indomethacin, aspirin, and
other NSAIDs effect great inhibition of COX-1, which protects the
lining of the stomach from acid, along with relatively minimal
inhibition of COX-2, which provokes inflammation in response to
joint injury or a disease like arthritis. Also, certain NSAIDs
possess essentially the same inhibitory activity against both COX-1
and COX-2. Thus, zeroing in on inhibition of COX-2 alone has been
the goal of drug developers for several years in order to reduce or
eliminate the GI irritation caused by COX-1 inhibition.
[0005] More specifically, as discussed in Smith, Garavito, and
DeWitt, "D.L. Prostaglandin Endoperoxide H Synthases
(Cyclooxygenases)-1 and -2", J. Biol. Chem., (1996) Vol. 271, pp.
33157-33160, the pertinent step in prostaglandin and thromboxane
biosynthesis involves the conversion of arachidonic acid to
PGH.sub.2, which is catalyzed by the sequential action of the COX
and PER activities of PGHS, as set out in the following reaction
scheme: 1
[0006] That COX activity originates from two distinct and
independently regulated enzymes, termed COX-1 and COX-2, is
described in DeWitt and Smith, "Primary Structure of Prostaglandin
G/H Synthase from Sheep Vesicular Gland Determined from the
Complementary DNA Sequence", Proc. Natl. Acad. Sci. U.S.A. (1988)
Vol. 85, pp. 1412-1416; Yokoyama and Tanabe, "Cloning of Human Gene
Encoding Prostaglandin Endoperoxide Synthase and Primary Structure
of the Enzyme", Biochem. Biophys. Res. Commun. (1989) Vol. 165, pp.
888-894; and Hla and Neilson, "Human Cyclooxygenase-2-cDNA", Proc.
Natl. Acad. Sci. U.S.A. (1992) Vol. 89, pp. 7384-7388.
[0007] COX-1 is the constitutive isoform and is mainly responsible
for the synthesis of cytoprotective prostaglandins in the GI tract
and for the synthesis of thromboxane, which triggers platelet
aggregation in blood platelets. See, Allison, Howatson, Torrence,
Lee, and Russell, "Gastrointestinal Damage Associated with the Use
of Nonsteroidal Antiinflammatory Drugs", N. Engl. J. Med. (1992)
Vol. 327, pp. 749-754.
[0008] On the other hand, COX-2 is inducible and short-lived. Its
expression is stimulated in response to endotoxins, cytokines, and
mitogens. See, Kujubu, Fletcher, Varnum, Lim, and Herschman,
"TIS10, A Phorbol Ester Tumor Promoter Inducible mRNA from Swiss
3T3 Cells, Encodes a Novel Prostaglandin Synthase/Cyclooxygenase
Homologue", J. Biol. Chem. (1991) Vol. 266, pp. 12866-12872; Lee,
Soyoola, Chanmugam, Hart, Sun, Zhong, Liou, Simmons, and Hwang,
"Selective Expression of Mitogen-Inducible Cyclooxygenase in
Macrophages Stimulated with Lipopolysaccharide", J. Biol. Chem.
(1992) Vol. 267, pp. 25934-25938; and O'Sullivan, Huggins, Jr., and
Mccall, "Lipopolysaccharide-Induced Expression of Prostaglandin H
Synthase-2 in Aveolar Macrophages is Inhibited by Dexamethasone by
not by Aspirin", Biochem. Biophys. Res. Commun. (1993) Vol. 191,
pp. 1294-1300.
[0009] Importantly, COX-2 plays a major role in prostaglandin
biosynthesis in inflammatory cells (monocytes/macrophages) and in
the central nervous system. See, Masferrer, Zweifel, Manning,
Hauser, Leahy, Smith, Isakson, and Seibert, "Selective Inhibition
of Inducible Cyclooxygenase-2 in vivo is Antiinflammatory and
Nonulcerogenic", Proc. Natl. Acad. Sci. U.S.A. (1994) Vol. 91, pp.
3228-3232; Vane, Mitchell, Appleton, Tomlinson, Bishop-Bailey,
Croxtall, and Willoughby, "Inducible Isoforms of Cyclooxygenase and
Nitric Oxide Synthase in Inflammation", Proc. Natl. Acad. Sci.
U.S.A. (1994) Vol. 91, pp. 2046-2050; Harada, Hatanaka, Saito,
Majima, Ogino, Kawamura, Ohno, Yang, Katori, and Yamamoto,
"Detection of Inducible Prostaglandin H Synthase-2 in Cells in the
Exudate of Rat Carrageenin-Induced Pleurisy", Biomed. Res. (1994)
Vol. 15, pp. 127-130; Katori, Harada, Hatanaka, Kawamura, Ohno,
Aizawa, and Yamamoto, "Induction of Prostaglandin H Synthase-2 in
Rat Carrageenin-Induced Pleurisy and Effect of a Selective COX-2
Inhibitor", Advances in Prostaglandin, Thromboxane, and Leukotriene
Research (1995) Vol. 23, pp. 345-347; and Kennedy, Chan, Culp, and
Cromlish, "Cloning and Expression of Rat Prostaglandin Endoperoxide
Synthase (Cyclooxygenase-2) cDNA", Biochem. Biophys. Res. Commun.
(1994) Vol. 197, pp. 494-500.
[0010] Hence, the differential tissue distribution of COX-1 and
COX-2 provides a basis for the development of drugs that are
selective COX-2 inhibitors (i.e., specificity for inhibition of
COX-2 far exceeds inhibition of COX-1) as antiinflammatory,
analgesic, and/or antipyretic agents with minimization of or
without the GI and hematologic liabilities from COX-1 inhibition
that plague most all currently marketed NSAIDs, most of which
inhibit both COX-1 and COX-2, with specificity for COX-1 inhibition
greatly exceeding that for COX-2 inhibition, although some have
essentially similar inhibitory activity against both COX-1 and
COX-2. See, for instance, Meade, Smith, and DeWitt, "Differential
Inhibition of Prostaglandin Indoperoxide Synthase (Cyclooxygenase)
Isozymes by Aspirin and Other Non-Steroidal Antiinflammatory
Drugs", J. Biol. Chem., (1993) Vol. 268, pp. 6610-6614.
[0011] Detailed SAR studies have been reported for two general
structural classes of selective COX-2 inhibitors (specificity for
COX-2 inhibition far exceeds COX-1 inhibition) including certain
acidic sulfonamides and diarylheterocyclics. The in vivo activities
of these selective COX-2 inhibitors validate the concept that
selective COX-2 inhibition is antiinflammatory and nonulcerogenic,
as discussed in the following journal articles. Gans, Galbraith,
Roman, Haber, Kerr, Schmidt, Smith, Hewes, and Ackerman,
"Anti-Inflammatory and Safety Profile of DuP 697, a Novel Orally
Effective Prostaglandin Synthesis Inhibitor", J. Pharmacol. Exp.
Ther. (1990) Vol. 254, pp. 180-187; Penning, Talley, Bertenshaw,
Carter, Collins, Docter, Graneto, Lee, Malecha, Miyashiro, Rogers,
Rogier, Yu, Anderson, Burton, Cogburn, Gregory, Koboldt, Perkins,
Seibert, Veenhuizen, Zhang, and Isakson, "Synthesis and Biological
Evaluation of the 1,5-Diarylpyrazole Class of Cyclooxygenase-2
Inhibitors: Identification of
4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1-
H-pyrazol-1-yl]benzenesulfonamide (SC-58635, Celecoxib)", J. Med.
Chem. (1997) Vol. 40, pp. 1347-1365; Khanna, Weier, Yu, Xu, Koszyk,
Collins, Koboldt, Veenhuizen, Perkins, Casler, Masferrer, Zhang,
Gregory, Seibert, and Isakson, "1,2-Diarylimidazoles as Potent
Cyclooxygenase-2 Selective, and Orally Active Antiinflammatory
Agents", J. Med. Chem. (1997) Vol. 40, pp. 1634-1647; Khanna,
Weier, Yu, Collins, Miyashiro, Koboldt, Veenhuizen, Curie, Siebert,
and Isakson, "1,2-Diarylpyrroles as Potent and Selective Inhibitors
of Cyclooxygenase-2", J. Med. Chem. (1997) Vol. 40, pp. 1619-1633;
Tsuji, Nakamura, Konishi, Tojo, Ochi, Senoh, and Matsuo, "Synthesis
and Pharmacological Properties of 1,5-Diarylyrazoles and Related
Derivatives", Chem. Pharm. Bull. (1997) Vol. 45, pp. 987-995;
Riendeau, Percival, Boyce, Brideau, Charleson, Cromlish, Ethier,
Evans, Falgueyret, Ford-Hutchinson, Gordon, Greig, Gresser, Guay,
Kargman, Lger, Mancini, O'Neill, Quellet, Rodger, Thrien, Wang,
Webb, Wong, Xu, Young, Zamboni, Prasit, and Chan, "Biochemical and
Pharmacological Profile of a Tetrasubstituted Furanone as a Highly
Selective COX-2 Inhibitor", Br. J. Pharmacol. (1997) Vol. 121, pp.
105-117; Roy, Leblanc, Ball, Brideau, Chan, Chauret, Cromlish,
Ethier, Gauthier, Gordon, Greig, Guay, Kargman, Lau, O'Neill,
Silva, Thrien, Van Staden, Wong, Xu, and Prasit, "A New Series of
Selective COX-2 Inhibitors: 5,6-Diarylthiazolo[3,2-b][1,2,4]-tr-
iazoles", Bioorg. Med. Chem. Lett. (1997) Vol. 7, pp. 57-62;
Thrien, Brideau, Chan, Cromlish, Gauthier, Gordon, Greig, Kargman,
Lau, Leblanc, Li, O'Neill, Riendeau, Roy, Wang, Xu, and Prasit,
"Synthesis and Biological Evaluation of
5,6-Diarylimidazo[2.1-b]thiazoles as Selective COX-2 Inhibitors",
Bioorg. Med. Chem. Lett. (1997) Vol. 7, pp. 47-52; Li, Norton,
Reinhard, Anderson, Gregory, Isakson, Koboldt, Masferrer, Perkins,
Seibert, Zhang, Zweifel, and Reitz, "Novel Terphenyls as Selective
Cyclooxygenase-2 Inhibitors and Orally Active Anti-Inflammatory
Agents", J. Med. Chem. (1996) Vol. 39, pp. 1846-1856; Li, Anderson,
Burton, Cogburn, Collins, Garland, Gregory, Huang, Isakson,
Koboldt, Logusch, Norton, Perkins, Reinhard, Seibert, Veenhuizen,
Zhang, and Reitz, "1,2-Diarylcyclopentenes as Selective
Cyclooxygenase-2 Inhibitors and Orally Active Anti-Inflammatory
Agents", J. Med. Chem. (1995) Vol. 38, pp. 4570-4578; Reitz, Li,
Norton, Reinhard, Huang, Penick, Collins, and Garland, "Novel
1,2-Diarylcyclopentenes are Selective Potent and Orally Active
Cyclooxygenase Inhibitors", Med. Chem. Res. (1995) Vol. 5, pp.
351-363; Futaki, Yoshikawa, Hamasaka, Arai, Higuchi, Iizuka, and
Otomo, "NS-398, A Novel Nonsteroidal Antiinflammatory Drug with
Potent Analgesic and Antipyretic Effects, which Causes Minimal
Stomach Lesions", Gen. Phamacol. (1993) Vol. 24, pp. 105-110;
Wiesenberg-Boetcher, Schweizer, Green, Muller, Maerki, and
Pfeilschifter, "The Pharmacological Profile of CGP 28238, A Novel
Highly Potent Anti-Inflammatory Compound", Drugs Exptl Clin Res.
(1989) Vol. XV, pp. 501-509; Futaki, Takahashi, Yokoyama, Arai,
Higuchi, and Otomo, "NS-398, A New Anti-Inflammatory Agent,
Selectively Inhibits Prostaglandin G/H Synthase/Cyclooxygenase
(COX-2) Activity in vitro", Prostaglandins (1994) Vol. 47, pp.
55-59; Klein, Nusing, Pfeilschifter, and Ullrich, "Selective
Inhibition of Cyclooxygenase-2", Biochem. Pharmacol. (1994) Vol.
48, pp. 1605-1610; Li, Black, Chan, Ford-Hutchinson, Gauthier,
Gordon, Guay, Kargman, Lau, Mancini, Quimet, Roy, Vickers, Wong,
Young, Zamboni, and Prasit, "Cyclooxygenase-2 Inhibitors. Synthesis
and Pharmacological Activities of 5-Methanesulfonamido-1-indanone
Derivatives", J. Med. Chem. (1995) Vol. 38, pp. 4897-8905; Prasit,
Black, Chan, Ford-Hutchinson, Gauthier, Gordon, Guay, Kargman, Lau,
Li, Mancini, Quimet, Roy, Tagari, Vickers, Wong, Young, and
Zamboni, "L-745,337: A Selective Cyclooxygenase-2 Inhibitor", Med.
Chem. Res. (1995) Vol. 5, pp. 364-374; Tanaka, Shimotori, Makino,
Aikawa, Inaba, Yoshida, and Takano, "Pharmacological Studies of the
New Antiinflammatory Agent 3-Formylamino-7-methylsulfonyla-
mino-6-phenoxy-4H-1-benzopyran-4-one. 1st Communication:
Antiinflammatory, Analgesic and Other Related Properties",
Arzniem.-Forsch./Drug Res. (1992) Vol. 42, pp. 935-944; Nakamura,
Tsuji, Konishi, Okumura, and Matsuo, "Studies on Anti-Inflammatory
Agents. I. Synthesis and Pharmacological Properties of
2'-(phenylthio)methanesulfonamides and Related Derivatives", Chem.
Pharm. Bull. (1993) Vol. 41, pp. 894-906; Chan, Boyce, Brideau,
Ford-Hutchinson, Gordon, Guay, Hill, Li, Mancini, Penneton, Prasit,
Rasori, Riendeau, Roy, Tagari, Vickers, Wong, and Rodger,
"Pharmacology of a Selective Cyclooxygenase-2 Inhibitor, L-745,337:
A Novel Nonsteroidal Anti-Inflammatory Agent with an Ulcerogenic
Sparing Effect in Rat and Nonhuman Primate Stomach", J. Pharmacol.
Exp. Ther. (1995) Vol. 274, pp. 1531-1537; and Graedon and Graedon,
"Pills Promise Relief without Ulcers", The Raleigh, N.C. News and
Observer, p. 8D (Sep. 13, 1998) which addresses, in general terms,
the development of celecoxib, meloxicam, and vioxx as selective
COX-2 inhibitors.
[0012] Representative acidic sulfonamides and diarylheterocyclics
that have been reported as selective COX-2 inhibitors in the
journal articles mentioned in the above paragraph are: 2
[0013] Although acidic sulfonamides and diarylheterocyclics have
been extensively studied as selective COX-2 inhibitors, there are
very few reports on converting NSAIDs that are selective COX-1
inhibitors into selective COX-2 inhibitors. See, Black, Bayly,
Belley, Chan, Charleson, Denis, Gauthier, Gordon, Guay, Kargman,
Lau, Leblanc, Mancini, Quellet, Percival, Roy, Skorey, Tagari,
Vickers, Wong, Xu, and Prasit, "From Indomethacin to a Selective
COX-2 Inhibitor: Development of Indolalkanoic Acids as Potent and
Selective Cyclooxygenase-2 Inhibitors", Bioorg. Med. Chem. Lett.
(1996) Vol. 6, pp. 725-730; Luong, Miller, Barnett, Chow, Ramesha,
and Browner, "Flexibility of the NSAID Binding Site in the
Structure of Human Cyclooxygenase-2", Nature Structural Biol.
(1996) Vol. 3, pp. 927-933; and Kalgutkar, Crews, Rowlinson,
Garner, Seibert, and Marnett, "Aspirin-Like Molecules that
Covalently Inactivate Cyclooxygenase-2", Science (1998) Vol. 280,
pp. 1268-1270.
[0014] Also, interesting is U.S. Pat. No. 5,681,964 (issued in
1997) to Ashton et al., assignors to the University of Kentucky
Research Foundation, which shows conversion of indomethacin (an
NSAID) into certain ester derivatives with concomitant reduction of
GI irritation (see, FIG. 1 of U.S. Pat. No. 5,681,964 for the
structure of the ester derivatives). Additionally, U.S. Pat. Nos.
5,607,966 (Parent) (issued in 1997) and 5,811,438 (CIP) (issued in
1998), both to Hellberg et al., assignors to Alcon Laboratories,
show conversion of various NSAIDs (such as indomethacin) into
certain ester derivatives and amide derivatives (which are useful
as antioxidants and inhibitors of 5-lipoxygenase) but do not
address COX-2 selective inhibition.
[0015] Moreover, although U.S. Pat. Nos. 3,285,908 (issued in 1966)
and 3,336,194 (issued in 1967), both to Shen, assignor to Merck
& Co., Inc., describe various secondary and tertiary amide
derivatives of indomethacin, the patents fail to address COX
inhibition, probably because COX inhibition (both COX-1 and COX-2)
was undiscovered in the 1960's, and thus fail to recognize that
tertiary amide derivatives do not inhibit either COX-1 or COX-2.
(Also, see comparison compounds 9 and 10 in the Examples below.)
However, U.S. Pat. Nos. 5,436,265 (issued in 1995) to Black et al.
and 5,510,368 (issued in 1996) to Lau et al., both patents assigned
to Merck Frosst Canada, Inc., describe, respectively,
1-aroyl-3-indolyl alkanoic acids and N-benzyl-3-indoleacetic acids
as COX-2 selective inhibitors.
[0016] In the present investigation, the possibility has been
explored for designing selective COX-2 inhibitors using as
templates various compounds, such as NSAIDs, (1) that are selective
COX-1 inhibitors or (2) that have essentially the same inhibitory
activity for both COX-1 and COX-2. These two kinds of compounds are
collectively referred to as compounds that are not selective COX-2
inhibitors.
[0017] More particularly, analysis of the human COX-2 crystal
structure complexed with zomepirac-derived selective COX-2
inhibitors indicates that the structural basis for selectivity by
zomepirac-derived compounds is different from that of
diarylheterocyclics. See, Luong et al. mentioned above. Unlike
diarylheterocyclics, zomepirac analogs do not utilize the side
pocket; instead they breech the constriction at the mouth of the
COX active site occupied by Arg106 and Tyr341 and project down the
lobby region. The projection into this sterically uncongested
region in the COX-2 active site opens the possibility that making a
wide range of analogs of COOH-containing NSAIDs, each with a
different pendent functional group replacing the OH of the COOH,
would accomplish many purposes related to drug discovery or
development. For example, certain pendent groups could improve
water-solubility, bioavailability, or pharmacokinetics. Another
possibility would be to attach a pendent pharmacophore in order to
target a completely different protein leading to compounds with
dual pharmacological functions.
[0018] Abbott Laboratories and Parke-Davis have attempted the
pharmacophore approach. See, respectively, Kolasa, Brooks,
Rodriques, Summers, Dellaria, Hulkower, Bouska, Bell, and Carter,
"Nonsteroidal Anti-Inflammatory Drugs as Scaffolds for the Design
of 5-Lipoxygenase Inhibitors", J. Med. Chem. (1997) Vol. 40, pp.
819-824; and Flynn, Capiris, Cetenko, Connor, Dyer, Kostlan, Niese,
Schrier, and Sircar, "Nonsteroidal Antiinflammatory Drug Hydroxamic
Acids. Dual Inhibitors of Both Cyclooxygenase and 5-Lipoxygenase",
J. Med. Chem. (1990) Vol. 33, pp. 2070-2072. Both Kolasa et al. and
Flynn et al. reported that replacement of the carboxylic acid group
in NSAIDs with a hydroxamic acid moiety or a hydroxyurea moiety
provided dual inhibitors of COX and 5-lipoxygenase. Nevertheless,
none of the analogs displayed any significant selective COX-2
inhibition, and furthermore the hydroxamates underwent facile
hydrolysis.
[0019] However, nothing in the above-discussed literature suggests
that converting a COOH-containing drug, such as a COOH-containing
NSAID, that is not selective for COX-2 inhibition into a derivative
that is selective for COX-2 inhibition would also result in that
derivative being a cancer inhibitor. Nevertheless, it is
interesting to note that sulindac sulfide (an NSAID which contains
a COOH moiety as well as a methyl sulfide moiety) is a 40-fold more
potent inhibitor against COX-1 than COX-2, yet also exhibits
inhibition of tumors. On the other hand, a derivative, namely
sulindac sulfone (which contains a COOH moiety as well as a methyl
sulfone moiety) does not inhibit either COX-1 or COX-2, but still
exhibits inhibition of tumors.
[0020] Thus, it would be desirable to find certain COOH-containing
drugs, such as COOH-containing NSAIDs, which are not selective
COX-2 inhibitors (either display an inhibition for COX-1 far
exceeding inhibition of COX-2 or display essentially the same
inhibition for COX-1 and COX-2) that would, when converted into
certain derivatives, become selective COX-2 inhibitors (display an
inhibition for COX-2 far exceeding inhibition for COX-1), as well
as retain the analgesic, antiinflammatory, and/or antipyretic
effect of the drug, and yet exhibit other effects not exhibited by
the drug prior to derivatization, such as cancer inhibition.
SUMMARY AND OBJECTS OF THE INVENTION
[0021] Surprisingly with the present invention, it has been found
that derivatization of the carboxylic acid moiety of certain
compounds, such as certain NSAIDs, that are not selective COX-2
inhibitors, such as indomethacin, into secondary amide analogs
creates isozyme specificity for COX-2. Moreover, the resultant
secondary amide derivative is not only a selective COX-2 inhibitor,
but also is a cancer inhibitor, i.e., exhibits antiangiogenic
and/or antitumorigenic activity, and preferably, also retains the
analgesic, antiinflammatory, and/or antipyretic of the
compound.
[0022] Therefore, the present invention provides a method for
cancer treatment in a warm blooded vertebrate animal. The method
comprises administering to the animal a treatment effective amount
sufficient to inhibit cancer of a carboxylic acid secondary amide
derivative of a compound. The derivative is selective for
inhibition of cyclooxygenase-2. The compound (a) is a
cyclooxygenase inhibitor but is not selective for inhibition of
cyclooxygenase-2 and (b) contains a carboxylic acid moiety or a
pharmaceutically acceptable salt thereof. Preferably, the compound
is a non-steroidal antiinflammatory drug, or a pharmaceutically
acceptable salt thereof.
[0023] Hence, it is an object of the invention to provide a cancer
treatment that minimizes or obviates GI irritation.
[0024] Moreover, it is an advantage of the present invention that
the cancer treatment is also analgesic, antiinflammatory, and/or
antipyretic, absent the concomitant administration of an analgesic,
antiinflammatory, and/or antipyretic drug, such as an NSAID or a
pharmaceutically acceptable salt thereof.
[0025] Some of the objects of the invention having been stated
above, other objects will become evident as the description
proceeds, when taken in connection with the Laboratory Examples as
described below.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention involves a method for treating cancer
in an animal that is a warm-blooded vertebrate. Therefore, the
invention concerns mammals and birds.
[0027] Contemplated is the treatment of mammals such as humans, as
well as those mammals of importance due to being endangered (such
as Siberian tigers), of economical importance (animals raised on
farms for consumption by humans) and/or social importance (animals
kept as pets or in zoos) to humans, for instance, carnivores other
than humans (such as cats and dogs), swine (pigs, hogs, and wild
boars), ruminants (such as cattle, oxen, sheep, giraffes, deer,
goats, bison, and camels), and horses. Also contemplated is the
treatment of birds, including the treatment of those kinds of birds
that are endangered, kept in zoos, as well as fowl, and more
particularly domesticated fowl, i.e., poultry, such as turkeys,
chickens, ducks, geese, guinea fowl, and the like, as they are also
of economical importance to humans.
[0028] Thus, contemplated is the treatment of livestock, including,
but not limited to, domesticated swine (pigs and hogs), ruminants,
horses, poultry, and the like.
[0029] More particularly, a treatment effective amount of a
secondary amide derivative of a carboxylic acid-containing compound
is administered to the warm-blooded vertebrate animal. Thus, the
invention comprises administration of the secondary amide
derivative in concentrations calculated to provide the animal being
treated with the appropriate milieu to provide prevention, control,
or cessation of cancer. Moreover, in the preferred embodiment, the
secondary amide derivative possesses an analgesic,
antiinflammatory, and/or antipyretic property as possessed by the
carboxylic acid-containing compound prior to derivatization, and
thus, the cancer treatment provides an analgesic, antiinflammatory,
and/or antipyretic effect in the animal and is free of concomitant
administration of another drug for providing such effect.
[0030] By carboxylic-acid containing compound or COOH-containing
compound as used herein in connection with the present invention,
it is intended to include pharmaceutically acceptable acid salts of
the compound. Thus, for instance, the COOH moiety includes COOM,
where M is Na and the like.
[0031] The derivatives useful in the method of the present
invention are secondary amide derivatives of drugs having a
carboxylic acid moiety or a pharmaceutically acceptable salt
thereof, for instance, secondary amide derivatives of non-steroidal
anti-inflammatory drugs having a carboxylic acid moiety or a
pharmaceutically acceptable salt thereof. A number of chemical
classes of non-steroidal anti-inflammatory drugs have been
identified, as described in CRC Handbook of Eicosanoids:
Prostaglandins, and Related Lipids, Volume II, Drugs Acting Via the
Eicosanoids, pages 59-133, CRC Press, Boca Raton, Fla. (1989).
[0032] The NSAID may be selected, therefore, from a variety of
chemical classes including, but not limited to, fenamic acids, such
as flufenamic acid, niflumic acid, and mefenamic acid; indoles,
such as indomethacin, sulindac, and tolmetin; phenylalkanoic acids,
such as suprofen, ketorolac, flurbiprofen, and ibuprofen; and
phenylacetic acids, such as diclofenac. Further examples of NSAIDs
are listed below:
2 aceloferac etodolic acid loxoprofen alcofenac fenbufen
meclofenamate amfenac fenclofenac naproxen benoxaprofen fenclorac
orpanoxin bromfenac fenoprofen pirprofen carprofen fleclozic acid
pranoprofen clidanac indoprofen tolfenamic acid diflunisal
isofezolac zaltoprofen efenamic acid ketoprofen zomopirac
[0033] More specifically, preferred secondary amide derivatives
useful in the present invention include, but are not limited to,
secondary amide derivatives of the following COOH-containing
NSAIDs: 6-methoxy-.alpha.-methyl-2-naphthylacetic acid (and its Na
acid salt form known as naproxen), meclofenamic acid, and
diclofenac, with secondary amide derivatives of indomethacin being
preferred, and that indomethacin derivative described below as
compound 11 being especially preferred. Also, the secondary amide
derivatives of indomethacin, where the Cl at the 4-position of the
benzoyl moiety is replaced with Br or F, should work in the present
invention.
[0034] Even more preferred are the secondary amide derivatives of
indomethacin including, but are not limited to,
indomethacin-N-methyl amide, indomethacin-N-ethan-2-ol amide,
indomethacin-N-octyl amide, indomethacin-N-nonyl amide,
indomethacin-N-(2-methylbenzyl) amide,
indomethacin-N-(4-methylbenzyl) amide,
indomethacin-N-((R)-,4-dimethylben- zyl) amide,
indomethacin-N-((S)-,4-dimethylbenzyl) amide,
indomethacin-N-(2-phenethyl) amide, indomethacin-N-(4-fluorophenyl)
amide, indomethacin-N-(4-chlorophenyl) amide,
indomethacin-N-(4-acetamido- phenyl) amide,
indomethacin-N-(4-methylmercapto)phenyl amide,
indomethacin-N-(3-methylmercaptophenyl) amide,
indomethacin-N-(4-methoxyp- henyl) amide,
indomethacin-N-(3-ethoxyphenyl) amide,
indomethacin-N-(3,4,5-trimethoxyphenyl)amide,
indomethacin-N-(3-pyridyl) amide,
indomethacin-N-5-((2-chloro)pyridyl) amide,
indomethacin-N-5-((1-ethyl)pyrazolo) amide,
indomethacin-N-(3-chloropropy- l) amide,
indomethacin-N-methoxycarbonylmethyl amide,
indomethacin-N-2-(2-L-methoxycarbonylethyl) amide,
indomethacin-N-2-(2-D-methoxycarbonylethyl) amide,
indomethacin-N-(4-methoxycarbonylbenzyl) amide,
indomethacin-N-(4-methoxy- carbonylmethylphenyl) amide,
indomethacin-N-(2-pyrazinyl) amide,
indomethacin-N-2-(4-methylthiazolyl) amide,
indomethacin-N-(4-biphenyl) amide, and combinations thereof.
[0035] The secondary amide derivative may be administered to the
animal as a suppository or as a supplement to fluids that are
administered internally or parenterally, for instance nutriment
fluids such as intervenous sucrose solutions. Furthermore,
intraoral (such as buccal or sublingual) administration or
transdermal (such as with a skin patch) administration to the
animal is also contemplated. A good discussion of intraoral
administration can be seen in U.S. Pat. No. 4,229,447 issued Oct.
21, 1980 to Porter and U.S. Pat. No. 5,504,086 issued Apr. 2, 1996
to Ellinwood and Gupta. A good discussion of transdermal
administration can be seen in U.S. Pat. No. 5,016,652 issued May
21, 1991 to Rose and Jarvik.
[0036] Additionally, administration to the animal may be by various
oral methods, for instance as a tablet, capsule, or powder
(crystalline form) that is swallowed. Also, oral administration may
include that the secondary amide derivative is admixed in a carrier
fluid appropriate therefor so that it is administered as a liquid
(solution or suspension) that is drunk. When the secondary amide
derivative is admixed in a carrier fluid, appropriate fluids
include, but are not limited to, water, rehydration solutions
(i.e., water with electrolytes such as potassium citrate and sodium
chloride, for instance the solution available under the trade name
RESOL.RTM. from Wyeth Laboratories), nutritional fluids (i.e.,
milk, fruit juice), and combinations thereof. Thus, the oral
administration may be as a component of the diet, such as human
food, animal feed, and combinations thereof.
[0037] In addition to oral administration such as by way of the
mouth, contemplated also is administration of a solution or
suspension to the esophagus, stomach, and/or duodenum, such as by
gavage, i.e., by way of a feeding tube. Gavage type of
administration is useful for when the cancer has progressed and the
animal can no longer swallow food, medicine, et cetera, by
mouth.
[0038] Hence, it is also contemplated that additional ingredients,
such as various excipients, carriers, surfactants, nutriments, and
the like, as well as various medicaments other than a secondary
amide derivative, or combinations thereof, may be present together
with the secondary amide derivative, whatever the form that the
derivative is in. Medicaments other than a secondary amide
derivative may include, but are not limited to, osmolytic polyols
and osmolytic amino acids (i.e., myo-inositol, sorbitol, glycine,
alanine, glutamine, glutamate, aspartate, proline, and taurine),
cardiotonics (i.e., glycocyamine), analgesics, antibiotics,
electrolytes (i.e., organic or mineral electrolytes such as salts),
and combinations thereof.
[0039] A suitable dosing amount of secondary amide derivative for
administration to the animal should range from about 0.5 mg to
about 7.0 mg per kg of body weight of the animal per day, more
preferably from about 1.5 mg to about 6.0 mg per kg of body weight
of the animal per day, and even more preferably from about 2.0 mg
to about 5.0 mg per kg of body weight of the animal per day.
Administration may be one or more times per day to achieve the
total desired daily dose. Of course, the amount can vary depending
on the severity of the cancer and/or the age of the animal.
[0040] The present invention should be useful in the treatment of
cancer in animals, wherein the cancer is caused by pathogens (i.e.,
parasites, bacteria, protozoa, and viruses, including toxic agents
in food), nutritional factors (i.e., excess mineral salts, excess
protein, allergic agents in food, undigestible food components, or
poor quality ingredients in food), environmental factors that act
as stressors or pollutants (i.e., heat, chilling, shipment of
animals, or toxins such as from air and/or water pollution), and/or
physiological disorders such as those of the digestive tract,
pulmonary/circulatory system, liver, kidneys, colon, and/or
pancreas.
[0041] The present invention indicates that COOH-containing drugs
that are not COX-2 selective inhibitors, such as the NSAID known as
indomethacin, when converted into secondary amides, results in
isozyme specificity for COX-2 and thus presents an efficient
strategy for the generation of potent and selective COX-2
inhibitors. The below-discussed extensive SAR study conducted with
indomethacin suggests that a variety of secondary amide
substituents are tolerated for replacing the OH in the COOH moiety
of indomethacin, and these resultant derivatives are as potent and
selective as COX-2 inhibitors as are the diarylheterocyclics
discussed above. Thus, this strategy has great potential in the
development of nonulcerogenic antiinflammatory agents. Moreover, as
illustrated in the Examples below, the secondary amide derivatives
of the invention also exhibit an anticancer effect.
LABORATORY EXAMPLES
[0042] The following is noted in connection with the materials and
procedures below.
[0043] The amides that were made and their selective COX-2
inhibition properties are listed in the Table below. A total of 31
analogs (31 amide derivatives) of indomethacin were prepared.
Various nitrogen-containing substituents (i.e., amines) that
replaced the OH of the COOH, in order to create an amide, included
aminoalkyl, aminoaryl, aminoarylalkyl, aminoethers, or
aminopyridinyl moieties as part of the nitrogen-containing
substituent. The most potent amide analogs in the indomethacin
derivative series displayed IC.sub.50 values for inhibition of
purified human COX-2 in the low nanomolar range with COX-2
selectivity ratios ranging from >1000 to 4000.
[0044] Well established methodology was utilized in the synthesis
of amide derivatives of indomethacin by treatment of the
indomethacin with an appropriate amine (designated as R) utilizing
BOP-Cl as the carboxylic acid activator to replace the OH of the
COOH with R and create an amide. If R was a primary amine, the
resultant derivative was a secondary amide, and if R was a
secondary amine, the resultant derivative was a tertiary amide.
[0045] More specifically, a reaction mixture containing
indomethacin (300 mg, 0.84 mmol) and BOP-Cl (218 mg, 0.84 mmol) in
5 mL of anhydrous CH.sub.2Cl.sub.2 was treated with Et.sub.3N (167
mg, 0.84 mmol) and allowed to stir at rt for 10 minutes. The
mixture was then treated with the appropriate amine (0.94 mmol)
designated as R and stirred overnight at rt. Following dilution
with CH.sub.2Cl.sub.2 (30 mL), the resultant solution was washed
with water (2.times.25 mL), 3 N NaOH (2.times.25 mL), water
(2.times.30 mL), dried (in the presence of MgSO.sub.4), filtered,
and the solvent concentrated in vacuo. The crude amide was purified
by chromatography on silica gel or recrystallization in the
appropriate solvent. The reaction scheme was as follows: 3
[0046] IC.sub.50 values for the inhibition of purified human COX-2
or ovine COX-1 by test compounds were determined by the TLC assay
discussed below. Ovine COX-1 was used because it is very easy to
isolate and to purify the enzyme from sheep seminal vesicles,
whereas human COX-1 is normally obtained by over expression in an
insect cell system and is very difficult to handle and especially
to purify. COX-1 from sheep is >90% similar to COX-1 from
humans. Finally, inhibition of COX-1 from these two sources by
NSAIDs has been reported in the published literature and the
IC.sub.50 values are similar, suggesting no dramatic active site
differences. COX-1 was purified from ram seminal vesicles obtained
from Oxford Biomedical Research, Inc. (Oxford, Mich.). The specific
activity of the protein was 20 (.mu.MO.sub.2/minute)/mg, and the
percentage of holoprotein was 13.5%. Samples of human COX-2 (1.62
.mu.g/.mu.l) were obtained by expression of insect cell cloned
human COX-2 carried on baculovirus vectors, followed by
purification.
[0047] The enzymes obtained after purification were apo (i.e., they
lacked the hemprosthetic group). They were reconstituted with
hematin purchased from Sigma Chemical Co. (St. Louis, Mo.) in the
assays to render them to their natural states which is holo (i.e.,
natural COX-1 and natural COX-2 contain the hemprosthetic group) so
that the inhibition by test compounds had physiological
relevance.
[0048] HoloCOX-2 (66 nM) or holoCOX-1 (44 nM) in 100 mM Tris-HCl,
pH 8.0 containing 500 .mu.M phenol was treated with several
concentrations of indomethacin or an amide derivative of
indomethacin at 25.degree. C. for 20 minutes. Since the recombinant
COX-2 had a lower specific activity than the ovine COX-1, the
protein concentrations were adjusted such that the percentages of
total products obtained following catalysis of arachidonic acid
(purchased from Nu Chek Prep, Elysian, Minn.) by the two isoforms
were comparable.
[0049] More specifically, time- and concentration-dependent
inhibition of cyclooxygenase activity for ovine COX-1 (44 nM) and
human COX-2 (66 nM) using the TLC assay was determined as follows.
Reaction mixtures of 200 .mu.L contained hematin-reconstituted
protein in 100 mM Tris-HCl, pH 8.0, 500 .mu.M phenol, and
[1-.sup.14C]-arachidonic acid (50 .mu.M, .about.55-57 mCi/mmol).
For the time-dependent inhibition assay, hematin-reconstituted
COX-1 (44 nM) or COX-2 (66 nM) was preincubated at rt for 20
minutes with varying inhibitor concentrations in DMSO followed by
the addition of [1-.sup.14C]-arachidonic acid (50 .mu.M) for 30
seconds at 37.degree. C. [1-.sup.14C]-arachidonic acid
(.about.55-57 mCi/mmol) was purchased from New England Nuclear,
Dupont, or American Radiolabeled Chemicals (St. Louis, Mo.).
[0050] Reactions were terminated by solvent extraction in
Et.sub.2O/CH.sub.3OH/1 M citrate, pH 4.0 (30:4:1). The phases were
separated by centrifugation at 2000 g-force for 2 minutes and the
organic phase was spotted on a TLC plate (obtained from J. T.
Baker, Phillipsburg, N.J.). The plate was developed in
EtOAc/CH.sub.2Cl.sub.2/gl- acial AcOH (75:25:1) at 4.degree. C.
Radiolabeled prostanoid products were quantitatively determined
with a radioactivity scanner (obtained from Bioscan, Inc.,
Washington, D.C.). The percentage of total products observed at
different inhibitor concentrations was divided by the percentage of
products observed for protein samples preincubated for the same
time with DMSO.
[0051] Control experiments in the absence of indomethacin indicated
.about.25-30% conversion of fatty acid substrate to products, which
was sufficient for assessing the inhibitory properties of all test
compounds. Under these conditions, indomethacin displayed selective
time- and concentration-dependent inhibition of COX-1 (i.e.,
IC.sub.50 (COX-1).about.0.050 .mu.M; IC.sub.50 (COX-2).about.0.75
.mu.M), whereas the secondary amide derivatives displayed selective
COX-2 inhibition and the tertiary amide derivatives did not inhibit
either COX-1 or COX-2 (i.e., measurement of COX-2 was stopped at an
extremely high IC.sub.50 and still >80% COX-2 activity
remained). Also, the following is noted for NS-398 and
2-methyl-4-phenyl-5-sulfoamidophenyl oxazole, which are two of the
above-mentioned acidic sulfonamides: namely, NS-398: IC.sub.50
(COX-2).about.0.12 .mu.M; IC50 (COX-1)>66 .mu.M; and
2-methyl-4-phenyl-5-sulfoamidophenyl oxazole: IC.sub.50
(COX-2).about.0.06 .mu.M; IC.sub.50 (COX-1)>66 .mu.M.
[0052] For certain comparison tests, inhibition of COX-2 activity
in activated murine RAW264.7 cells was determined as follows. Low
passage number murine RAW264.7 cells were grown in DMEM containing
10% heat-inactivated FBS. Cells (6.2.times.10.sup.6 cells /T25
flask) were activated with 500 ng/mL LPS and 10 units/mL IFN-g in
serum-free DMEM for 7 hours. Vehicle (DMSO) or inhibitor in DMSO (0
to 1 .mu.M) was added for 30 minutes at 37.degree. C. Inhibition of
exogenous arachidonic acid metabolism or inhibition of PGD.sub.2
synthesis was determined by incubating the respective cells with 20
.mu.M .sup.14C-AA for 15 minutes at 25.degree. C. Aliquots (200
.mu.L) were removed into termination solution and total products
were quantitatively determined by the TLC assay as described
earlier.
[0053] Melting points were determined using a Gallenkamp melting
point apparatus and were uncorrected. Chemical yields were
unoptimized specific examples of one preparation. NSAIDs (i.e.,
indomethacin) were purchased from Sigma (St. Louis, Mo.). All other
chemicals were purchased from Aldrich (Milwaukee, Wis.). Methylene
chloride was purchased as anhydrous from Aldrich and was used as
received. All other solvents were HPLC grade. Analytical TLC
(Analtech uniplates.TM.) was used to follow the course of
reactions. Silica gel (Fisher, 60-100 mesh) was used for column
chromatography. .sup.1H NMR and .sup.13C NMR spectra in CDCl.sub.3
were recorded on a Bruker WP-360 spectrometer or an AM-400
spectrometer. Chemical shifts were expressed in parts per million
(ppm) relative to tetramethylsilane as an internal standard. Spin
multiplicities were reported as s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quartet), and m (multiplet).
Coupling constants (J) were reported in hertz (Hz).
Example I
[0054] The following carboxylic acid amide derivatives of
indomethacin, designated as compounds 1 through 31, were made.
(Note: compounds 1, 2, and 9 through 13 are also disclosed in the
above-discussed U.S. Pat. Nos. 3,285,908 and 3,336,194, both to
Shen, assignor to Merck & Co., Inc.)
[0055] Indomethacin-N-methyl amide (compound 1) was obtained upon
chromatography on silica gel (EtOAc:hexanes; 10:90 then 50:50) as a
bright yellow solid (271 mg, 79%). mp=187-189.degree. C.; .sup.1H
NMR (CDCl.sub.3) .delta. 7.64-7.67 (dd, 2H, J=6.6 Hz and 1.9 Hz,
ArH), 7.47-7.50 (dd, 2H, J=6.7 Hz and 1.9 Hz, ArH), 6.88-6.89 (dd,
1H, J=9.1 Hz and 2.5 Hz, ArH), 6.84-6.87 (d, 1H, J=9.0 Hz, ArH),
6.68-6.72 (dd, 1H, J=9.1 Hz and 2.5 Hz, ArH), 5.22 (bs, 1H, NH),
3.83 (s, 3H, CH.sub.3), 3.65 (s, 2H, CH.sub.2), 2.75-2.76 (d, 3H,
J=4.8 Hz, CH.sub.3), 2.39 (s, 3H, CH.sub.3).
[0056] Indomethacin-N-ethan-2-ol amide (compound 2) was obtained
upon chromatography on silica gel (EtOAc) as a pale yellow solid
(143 mg, 39%). mp=162-164.degree. C.; .sup.1H NMR (CDCl.sub.3)
.delta. 7.66-7.68 (dd, 2H, J=6.7 Hz and 1.7 Hz, ArH), 7.47-7.50
(dd, 2H, J=6.9 Hz and 1.9 Hz, ArH), 6.85-6.89 (d and s, 2H, J=9.2
Hz, ArH), 6.68-6.72 (dd, 1H, J=9.0 Hz and 2.5 Hz, ArH), 6.03 (bs,
1H, NH), 3.82 (s, 3H, CH.sub.3), 3.67 (bs, 4H, 2CH.sub.2),
3.35-3.40 (q, 2H, J=4.8 Hz, CH.sub.2), 2.44 (bs, 1H, OH), 2.39 (s,
3H, CH.sub.3).
[0057] Indomethacin-N-octyl amide (compound 3) was obtained upon
chromatography on silica gel (EtOAc:hexanes; 30:70) as a yellow
solid (164 mg, 42%). mp=109-111.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.62-7.65 (d, 2H, J=8.2 Hz, ArH), 7.46-7.49
(d, 2H, J=8.2 Hz, ArH), 6.85-6.89 (m, 2H, ArH), 6.68-6.71 (d, 1H,
J=8.9 Hz, ArH), 5.67 (s, 1H, NH), 3.82 (s, 3H, CH.sub.3), 3.64 (s,
2H, CH.sub.2), 3.16-3.22 (m, 2H, CH.sub.2), 2.38 (s, 3H, CH.sub.3),
1.39 (m, 2H, CH.sub.2), 1.19 (m, 10H, 5CH.sub.2), 0.83-0.88 (t,
J=6.2 Hz, CH.sub.3).
[0058] Indomethacin-N-nonyl amide (compound 4) was obtained upon
chromatography on silica gel (EtOAc:hexanes; 30:70) as a yellow
solid (191 mg, 47%).%). mp=128-130.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.64-7.67 (d, 2H, J=8.4 Hz, ArH), 7.47-7.50
(d, 2H, J=8.4 Hz, ArH), 6.89 (s, 1H, ArH), 6.85-6.88 (d, J=8.9 Hz,
ArH), 6.68-6.72 (dd, 1H, J=9.0 Hz and 2.4 Hz, ArH), 5.60-5.63 (bt,
J=5.3 Hz, NH), 3.82 (s, 3H, CH.sub.3), 3.64 (s, 2H, CH.sub.2),
3.16-3.22 (m, 2H, CH.sub.2), 2.38 (s, 3H, CH.sub.3), 1.36-1.41 (m,
2H, CH.sub.2), 1.19-1.28 (m, 12H, 6CH.sub.2), 0.84-0.89 (t, J=6.5
Hz, CH.sub.3).
[0059] Indomethacin-N-(2-methylbenzyl)amide (compound 5) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 50:50)
as a yellow solid (218 mg, 56%). mp=177-179.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.60-7.61 (d, 2H, J=8.1 Hz, ArH), 7.44-7.46
(d, 2H, J=8.1 Hz, ArH), 7.06-7.15 (m, 4H, ArH), 6.83-6.89 (m, 2H,
ArH), 6.67-6.70 (d, 1H, J=8.1 Hz, ArH), 5.84 (s, 1H, NH), 4.40-4.41
(d, 2H, J=5.3 Hz, CH.sub.2), 3.79 (s, 3H, CH.sub.3), 3.70 (s, 2H,
CH.sub.2), 2.37 (s, 3H, CH.sub.3), 2.19 (s, 3H, CH.sub.3).
[0060] Indomethacin-N-(4-methylbenzyl)amide (compound 6) was
obtained upon recrystallization from methanol as a yellow solid
(142 mg, 37%). mp=191-192.degree. C.; .sup.1H NMR (CDCl.sub.3)
.delta. 7.63-7.60 (d, 2H, J=8.5 Hz, ArH), 7.46-7.44 (d, 2H, J=8.4
Hz, ArH), 7.08-7.01 (m, 4H, ArH), 6.88 (s, 1H, ArH), 6.87-6.85 (d,
1H, J=6.3 Hz, ArH), 6.71-6.67 (dd, 1H, J=9.0 Hz and 2.4 Hz, ArH),
5.89 (bt, 1H, NH), 4.38-4.36 (d, 2H, J=5.9 Hz, CH.sub.2), 3.78 (s,
3H, CH.sub.3), 3.69 (s, 2H, CH.sub.2), 2.35 (s, 3H, CH.sub.3), 2.30
(s, 3H, CH.sub.3).
[0061] Indomethacin-N-((R)-4-dimethylbenzyl) amide (compound 7) was
obtained upon recrystallization from methanol to yield a pale
yellow solid (124 mg, 31 %). mp=201-202.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.62-7.64 (d, 2H, J=8.4 Hz, ArH), 7.45-7.48
(d, 2H, J=8.6 Hz, ArH), 7.01-7.08 (m, 4H, ArH), 6.87-6.90 (d, 1H,
J=9.0 Hz, ArH), 6.83-6.84 (d, 1H, J=2.3 Hz, ArH), 6.68-6.72 (dd,
1H, J=9.0 Hz and 2.4 Hz, ArH), 5.76-5.78 (bd, 1H, J=8.0 Hz, NH),
5.09-5.14 (m, 1H, CH), 3.76 (s, 3H, CH.sub.3), 3.63-3.64 (d, 2H,
J=2.8 Hz, CH.sub.2), 2.34 (s, 3H, CH.sub.3), 2.30 (s, 3H,
CH.sub.3), 1.35-1.38 (d, 3H, J=6.8 Hz, CH.sub.3).
[0062] Indomethacin-N-((S)-4-dimethylbenzyl) amide (compound 8) was
obtained upon recrystallization from methanol as a pale yellow
solid (163 mg, 41 %). mp=200-201.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.53-7.55 (d, 2H, J=8.3 Hz, ArH), 7.37-7.40
(d, 2H, J=8.4 Hz, ArH), 6.94-7.01 (m, 4H, ArH), 6.76-6.82 (m, 2H,
ArH), 6.61-6.64 (dd, 1H, J=9.0 Hz and 2.5 Hz, ArH), 5.77-5.79 (bd,
1H, J=7.8 Hz, NH), 5.02-5.07 (m, 1H, CH), 3.69 (s, 3H, CH.sub.3),
3.58-3.59 (d, 2H, J=2.9 Hz, CH.sub.2), 2.27 (s, 3H, CH.sub.3), 2.23
(s, 3H, CH.sub.3), 1.28-1.30 (d, 3H, J=6.9 Hz, CH.sub.3).
[0063] Comparison
[0064] Indomethacin-N-methylphenethyl amide (compound 9) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 50:50)
as a yellow solid (288 mg, 72%). mp=61-63.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.64-7.67 (d, 2H, J=8.4 Hz, ArH), 7.45-7.48
(d, 2H, J=8.5 Hz, ArH), 7.02 (d, 1H, J=2.4 Hz, ArH), 6.81-6.84 (d,
1H, J=9.0 Hz, ArH), 6.63-6.66 (dd, 1H, J=9.0 Hz and 2.5 Hz, ArH),
3.82 (s, 3H, CH.sub.3), 3.71 (s, 2H, CH.sub.2), 3.57-3.60 (t, 2H,
J=5.4 Hz, CH.sub.2), 3.43-3.46 (t, 2H, J=5.3 Hz, CH.sub.2), 2.38
(s, 3H, CH.sub.3), 1.59-1.61 (m, 2H, CH.sub.2), 1.52-1.53 (m, 2H,
CH.sub.2), 1.42-1.43 (m, 2H, CH.sub.2).
[0065] Comparison
[0066] Indomethacin-N-piperidinyl amide (compound 10) was obtained
upon chromatography on silica gel (EtOAc:hexanes; 40:60) as a pale
yellow solid (146 mg, 41%). mp=161-163.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.64-7.67 (d, 2H, J=8.4 Hz, ArH), 7.45-7.48
(d, 2H, J=8.5 Hz, ArH), 7.02 (d, 1H, J=2.4 Hz, ArH), 6.81-6.84 (d,
1H, J=9.0 Hz, ArH), 6.63-6.66 (dd, 1H, J=9.0 Hz and 2.5 Hz, ArH),
3.82 (s, 3H, CH.sub.3), 3.71 (s, 2H, CH.sub.2), 3.57-3.60 (t, 2H,
J=5.4 Hz, CH.sub.2), 3.43-3.46 (t, 2H, J=5.3 Hz, CH.sub.2), 2.38
(s, 3H, CH.sub.3), 1.59-1.61 (m, 2H, CH.sub.2), 1.52-1.53 (m, 2H,
CH.sub.2), 1.42-1.43 (m, 2H, CH.sub.2).
[0067] Indomethacin-N-(2-phenethyl) amide (compound 11) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 30:70)
as a bright yellow solid (169 mg, 44%). mp=148-150.degree. C.;
.sup.1H NMR (CDCl.sub.3) .delta. 7.58-7.60 (d, J=8.4 Hz, ArH),
7.46-7.48 (d, 2H, J=8.5 Hz, ArH), 7.12-7.14 (m, 3H, ArH), 6.85-6.95
(m, 4H, ArH), 6.69-6.73 (dd, 1H, J=8.9 Hz and 2.4 Hz, ArH), 5.61
(s, 1H, NH), 3.81 (s, 3H, CH.sub.3), 3.59 (s, 2H, CH.sub.2),
3.43-3.49 (m, 2H, CH.sub.2), 2.68-2.72 (t, 2H, J=6.7 Hz, CH.sub.2),
2.04 (s, 3H, CH.sub.3).
[0068] Indomethacin-N-(4-fluorophenyl) amide (compound 12) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 5:95 to
20:80) as an orange solid (217 mg, 57%). mp=200-202.degree. C.;
.sup.1H NMR (CDCl.sub.3) .delta. 7.65-7.67 (d, 2H, J=8.3 Hz, ArH),
7.47-7.50 (d, 2H, J=8.3 Hz, ArH), 7.32-7.35 (m, 3H, ArH), 6.94-6.99
(m, 3H, ArH, NH), 6.85-6.88 (d, 1H, J=9.0 Hz, ArH), 6.70-6.73 (dd,
1H, J=9.0 Hz and 2.0 Hz, ArH), 3.81 (s, 3H, CH.sub.3), 3.79 (s, 2H,
CH.sub.2), 2.45 (s, 3H, CH.sub.3).
[0069] Indomethacin-N-(4-chlorophenyl) amide (compound 13) was
obtained upon recrystallization from methanol as a pale yellow
solid (234 mg, 56%). mp=209-210.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.58-7.61 (d, 2H, J=8.2 Hz, ArH), 7.40-7.42
(d, 2H, J=8.2 Hz, ArH), 7.13-7.27 (m, 5H, ArH), 6.84 (s, 1H, NH),
6.77-6.80 (d, 1H, J=9.0 Hz, ArH), 6.62-6.65 (d, 1H, J=9.0 Hz, ArH),
3.72 (s, 2H, CH.sub.2), 3.72 (s, 3H, CH.sub.3), 2.37 (s, 3H,
CH.sub.3).
[0070] Indomethacin-N-(4-acetamidophenyl) amide (compound 14) was
obtained upon recrystallization from methanol as a pale yellow
solid (221 mg, 54%). mp=256-257.degree. C.; .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.14 (s, 1H, NH), 9.86 (s, 1H, NH),
7.62-7.70 (m, 4H, ArH), 7.48 (s, 4H, ArH), 7.18 (d, 1H, J=2.3 Hz,
ArH), 6.90-6.93 (d, 1H, J=9.0 Hz, ArH), 6.68-6.72 (dd, 1H, J=9.1 Hz
and 2.5 Hz, ArH), 3.73 (s, 3H, CH.sub.3), 3.71 (s, 2H, CH.sub.2),
2.27 (s, 3H, CH.sub.3), 1.99 (s, 3H, CH.sub.3).
[0071] Indomethacin-N-(4-methylmercapto)phenyl amide (compound 15)
was obtained upon chromatography on silica gel (EtOAc:hexanes;
50:50) as a bright yellow solid (162 mg, 40%). mp=195-196.degree.
C.; .sup.1H NMR (CDCl.sub.3) .delta. 7.67-7.70 (d, 2H, J=8.4 Hz,
ArH), 7.48-7.50 (d, 2H, J=8.4 Hz, ArH), 7.30-7.33 (d, 2H, J=8.6 Hz,
ArH), 7.17-7.22 (m, 3H, 2ArH and NH), 6.92-6.93 (d, 1H, J=2.3 Hz,
ArH), 6.85-6.88 (d, 1H, J=9.0 Hz, ArH), 6.69-6.73 (dd, 1H, J=9.0 Hz
and 2.4 Hz, ArH), 3.80 (s, 3H, CH.sub.3), 3.79 (s, 2H, CH.sub.2),
2.45 (s, 3H, C.sub.3), 2.44 (s, 3H, CH.sub.3).
[0072] Indomethacin-N-(3-methylmercaptophenyl) amide (compound 16)
was obtained upon chromatography on silica gel (EtOAc:hexanes;
15:85) as a yellow solid (218 mg, 54%). mp=129-131.degree. C.;
.sup.1H NMR (CDCl.sub.3) .delta. 7.62-7.64 (d, 2H, J=8.2 Hz, ArH),
7.45-7.48 (d, 2H, J=8.4 Hz, ArH), 7.39 (s, 1H, NH), 7.09-7.18 (m,
2H, ArH), 6.94-6.96 (m, 3H, ArH), 6.86-6.89 (d, 1H, J=9.0 Hz),
6.69-6.72 (d, 1H, J=8.9 Hz, ArH), 3.80 (s, 3H, CH.sub.3), 3.78 (s,
2H, CH.sub.2), 2.42 (s, 3H, CH.sub.3).
[0073] Indomethacin-N-(4-methoxyphenyl) amide (compound 17) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 10:90 to
25:75) as an orange solid (239 mg, 61%). mp=201-202.degree. C.;
.sup.1H NMR (CDCl.sub.3) .delta. 7.67-7.70 (dd, 2H, J=6.8 Hz and
1.8 Hz, ArH), 7.48-7.51 (d, 2H, J=7.1 Hz, ArH), 7.28-7.29 (d, 1H,
J=2.0 Hz, ArH), 7.20 (s, 1H, NH), 6.94-6.95 (d, 1H, J=2.4 Hz, ArH),
6.86-6.89 (d, 1H, J=9.0 Hz, ArH), 6.78-6.84 (m, 2H, ArH), 6.69-6.73
(dd, 1H, J=9.0 Hz and 2.4 Hz, ArH), 3.81 (s, 3H, CH.sub.3, 3.79 (s,
2H, CH.sub.2), 3.76 (s, 3H, CH.sub.3), 2.45 (s, 3H, CH.sub.3).
[0074] Indomethacin-N-(3-ethoxyphenyl) amide (compound 18) was
obtained upon recrystallization from methanol as a bright yellow
solid (297 mg, 74%). mp=152-154.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.68-7.70 (d, 2H, J=8.4 Hz, ArH), 7.48-7.51
(d, 2H, J=8.4 Hz, ArH), 7.24 (s, 1H, NH), 7.13-7.18 (m, 2H, ArH),
6.94-6.82 (m, 3H, ArH), 6.70-6.73 (dd, 1H, J=9.0 Hz and 2.4 Hz),
6.61-6.65 (dd, 1H, J=8.2 Hz and 1.7 Hz, ArH), 3.96-4.03 (q, 2H,
J=7.0 Hz, CH.sub.2), 3.81 (s, 3H, CH.sub.3), 3.80 (s, 2H,
CH.sub.2), 2.45 (s, 3H, CH.sub.3), 1.36-1.40 (t, 3H, J=7.0 Hz,
CH.sub.3).
[0075] Indomethacin-N-(3,4,5-trimethoxyphenyl) amide (compound 19)
was obtained upon chromatography on silica gel (EtOAc:hexanes;
10:90 to 30:70) as a light orange solid (191 mg, 44%).
mp=239-241.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 7.67-7.69
(d, 2H, J=8.5 Hz, ArH), 7.48-7.51 (d, 2H, J=8.5 Hz, ArH), 7.20 (s,
1H, NH), 6.94 (d, 1H, J=8.9 Hz, ArH), 6.70-6.74 (m, 3H, ArH),
3.78-3.81 (m, 14H, 3CH.sub.3 & CH.sub.2), 2.45 (s, 3H,
CH.sub.3).
[0076] Indomethacin-N-(3-pyridyl) amide (compound 20) was obtained
upon chromatography on silica gel (EtOAc:hexanes; 50:50 to 75:25)
as a yellow solid (190 mg, 52%). mp=204-205.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 8.39-8.40 (d, 1H, J=2.1 Hz, ArH), 8.32-8.34
(d, 1H, J=4.4 Hz, ArH), 8.04-8.08 (m, 1H, ArH), 7.66-7.70 (m, 2H,
ArH), 7.48-7.52 (m, 2H, ArH), 7.38 (s, 1H, NH), 7.22-7.25 (m, 1H,
ArH), 6.93-6.94 (d, 1H, J=2.4 Hz, ArH), 6.85-6.88 (d, 1H, J=9.1 Hz,
ArH), 6.70-6.74 (dd, 1H, J=9.1 Hz and 2.5 Hz, ArH), 3.84 (s, 2H,
CH.sub.2), 3.81 (s, 3H, CH.sub.3), 2.47 (s, 3H, CH.sub.3).
[0077] Indomethacin-N-5-((2-chloro)pyridyl) amide (compound 21) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 5:95 to
50:50) as a pale yellow solid (221 mg, 56%). mp=196-198.degree. C.;
.sup.1H NMR (CDCl.sub.3) .delta. 8.19-8.20 (d, 1H, J=2.8 Hz, ArH),
8.03-8.06 (dd, 1H, J=8.7 Hz and 2.9 Hz, ArH), 7.59-7.63 (m, 2H,
ArH), 7.46-7.51 (m, 3H, ArH), 7.24 (s, 1H, NH), 6.92-6.93 (d, 1H,
J=2.4 Hz, ArH), 6.84-6.87 (d, 1H, J=9.0 Hz, ArH), 6.70-6.74 (dd,
1H, J=9.1 Hz and 2.5 Hz, ArH), 3.84 (s, 2H, CH.sub.2), 3.82 (s, 3H,
CH.sub.3), 2.46 (s, 3H, CH.sub.3).
[0078] Indomethacin-N-5-((1-ethyl)pyrazolo) amide (compound 22) was
obtained upon recrystallization from methanol as a pale yellow
solid (153 mg, 40%). mp=193-194.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 7.99 (bs, 1H, NH), 7.66-7.68 (d, 2H, J=8.2 Hz,
ArH), 7.47-7.50 (m, 3H, ArH), 7.00 (s, 1H, ArH), 6.83-6.86 (d, 1H,
J=9.0 Hz, ArH), 6.69-6.72 (d, 1H, J=8.9 Hz, ArH), 6.35 (s, 1H,
ArH), 4.01-4.04 (bd, 2H, J=6.8 Hz, CH.sub.2), 3.90 (s, 2H,
CH.sub.2), 3.82 (s, 3H, CH.sub.3), 2.47 (s, 3H, CH.sub.3),
1.24-1.29 (t, 3H, J=7.1 Hz, CH.sub.3).
[0079] Indomethacin-N-(3-chloropropyl) amide (compound 23) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 30:70)
as a off-white solid (153 mg, 40%). .sup.1H NMR (DMSO-d.sub.6)
.delta. 8.11 (bs, 1H, NH), 7.62-7.69 (m, 4H, ArH), 7.09 (s, 1H,
ArH), 6.92-6.95 (d, 1H, J=8.9 Hz, ArH), 6.68-6.71 (d, 1H, J=8.8 Hz,
ArH), 3.80 (s, 3H, CH.sub.3), 3.58-3.67 (t, 2H, J=6.3 Hz,
CH.sub.2), 3.52 (s, 2H, CH.sub.2), 3.15-3.17 (m, 2H, CH.sub.2),
2.20 (s, 3H, CH.sub.3), 1.81-1.85 (t, 2H, J=6.5 Hz, CH.sub.2).
[0080] Indomethacin-N-methoxycarbonylmethyl amide (compound 24) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 30:70)
as a yellow solid (265 mg, 76%). .sup.1H NMR (CDCl.sub.3) .delta.
7.66-7.68 (dd, 2H, J=6.7 Hz and 1.7 Hz, ArH), 7.47-7.50 (dd, 2H,
J=6.9 Hz and 1.9 Hz, ArH), 6.92-6.95 (m, 2H, ArH), 6.70-6.73 (m,
1H, ArH), 6.03 (bs, 1H, NH), 3.98-4.00 (d, 2H, J=5.5 Hz, CH.sub.2),
3.84 (s, 3H, CH.sub.3), 3.71 (s, 3H, CH.sub.3), 3.69 (s, 2H,
CH.sub.2), 2.38 (s, 3H, CH.sub.3).
[0081] Indomethacin-N-2-(2-L-methoxycarbonylethyl) amide (compound
25) was obtained upon chromatography on silica gel (EtOAc:hexanes;
30:70 and then 50:50) as a yellow solid (300 mg, 84%). .sup.1H NMR
(CDCl.sub.3) .delta. 7.67-7.70 (dd, 2H, J=8.5 Hz and 1.85 Hz, ArH),
7.47-7.50 (dd, 2H, J=8.4 Hz and 1.9 Hz, ArH), 6.91-6.96 (m, 2H,
ArH), 6.69-6.73 (m, 1H, ArH), 6.16-6.18 (d, 1H, J=7.4 Hz, NH),
4.57-4.62 (m, 1H, CH), 3.83 (s, 3H, CH.sub.3), 3.70 (s, 3H,
CH.sub.3), 3.65 (s, 2H, CH.sub.2), 2.37 (s, 3H, CH.sub.3),
1.32-1.34 (d, 3H, J=7.2 Hz, CH.sub.3).
[0082] Indomethacin-N-2-(2-D-methoxycarbonylethyl) amide (compound
26) was obtained upon chromatography on silica gel (EtOAc:hexanes;
40:60) as a yellow solid (803 mg, 67%). .sup.1H NMR (CDCl.sub.3)
.delta. 7.67-7.70 (dd, 2H, J=8.5 Hz and 1.85 Hz, ArH), 7.47-7.50
(dd, 2H, J=8.4 Hz and 1.9 Hz, ArH), 6.91-6.96 (m, 2H, ArH),
6.69-6.73 (dd, 1H, ArH), 6.16-6.18 (d, 1H, J=7.4 Hz, NH), 4.57-4.62
(m, 1H, CH), 3.83 (s, 3H, CH.sub.3), 3.70 (s, 3H, CH.sub.3), 3.65
(s, 2H, CH.sub.2), 2.36 (s, 3H, CH.sub.3), 1.32-1.34 (d, 3H, J=7.2
Hz, CH.sub.3).
[0083] Indomethacin-N-(4-methoxycarbonylbenzyl) amide (compound 27)
was obtained upon chromatography on silica gel (EtOAc:hexanes;
40:60) as a yellow solid (198 mg, 47%). .sup.1H NMR (CDCl.sub.3)
.delta. 7.91-7.94 (d, 2H, J=6.8 Hz, ArH), 7.61-7.65 (d, 2H, J=8.7
Hz, ArH), 7.45-7.48 (d, 2H, J=9.0 Hz, ArH), 7.19-7.21 (d, 2H, J=8.3
Hz, ArH), 6.83-6.88 (m, 2H, ArH), 6.68-6.72 (dd, 1H, J=9.0 Hz and
2.4 Hz, ArH), 5.97-5.99 (bt, 1H, J=5.9 Hz, NH), 4.45-4.47 (d, 2H,
J=6.1 Hz, CH.sub.2), 3.90 (s, 3H, CH.sub.3), 3.83 (s, 3H,
CH.sub.3), 3.72 (s, 2H, CH.sub.2), 2.38 (s, 3H, CH.sub.3).
[0084] Indomethacin-N-(4-methoxycarbonylmethylphenyl) amide
(compound 28) was obtained upon chromatography on silica gel
(EtOAc:hexanes; 20:80) as an yellow solid (100 mg, 23%). .sup.1H
NMR (CDCl.sub.3) .delta. 7.67-7.70 (d, 2H, J=8.5 Hz, ArH),
7.48-7.51 (d, 2H, J=8.5 Hz, ArH), 7.33-7.36 (d, 2H, J=8.4 Hz, ArH),
7.18-7.23 (d and bs, 3H, ArH and NH), 6.92-6.93 (d, 1H, J=2.3 Hz,
ArH), 6.85-6.88 (d, 1H, J=9.0 Hz, ArH), 6.70-6.73 (dd, 1H, J=9.0 Hz
and 2.0 Hz, ArH), 3.81 (s, 5H, CH.sub.2 and CH.sub.3), 3.67 (s, 3H,
CH.sub.3), 3.56 (s, 3H, CH.sub.2), 2.45 (s, 3H, CH.sub.3).
[0085] Indomethacin-N-(2-pyrazinyl) amide (compound 29) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 30:70 to
50:50) as a bright yellow solid (251 mg, 69%). .sup.1H NMR
(CDCl.sub.3) .delta. 9.58 (d, 1H, J=1.4 Hz, ArH), 8.33-8.34 (d, 1H,
J=2.5 Hz, ArH), 8.16-8.17 (m, 1H, ArH), 7.86 (bs, 1H, NH),
7.69-7.71 (d, 2H, J=8.5 Hz, ArH), 7.49-7.51 (d, 2H, J=8.5 Hz, ArH),
6.92-6.93 (d, 1H, J=2.4 Hz, ArH), 6.84-6.87 (d, 1H, J=8.9 Hz, ArH),
6.70-6.72 (dd, 1H, J=9.0 Hz and 2.5 Hz, ArH), 3.86 (s, 2H,
CH.sub.2), 3.81 (s, 3H, CH.sub.3), 2.47 (s, 3H, CH.sub.3).
[0086] Indomethacin-N-2-(4-methylthiazolyl) amide (compound 30) was
obtained upon chromatography on silica gel (EtOAc:hexanes; 30:70
and then 70:30) to afford the pure product as a pale yellow solid
which was recrystallized from ethyl ether (241 mg, 63%). .sup.1H
NMR (CDCl.sub.3) .delta. 8.68 (bs, 1H, NH), 7.70-7.74 (d, 2H, J=9.0
Hz, ArH), 7.48-7.52 (d, 2H, J=9.0 Hz, ArH), 6.79-6.85 (m, 2H, ArH),
6.67-6.71 (dd, 1H, J=9.0 Hz and 2.4 Hz, ArH), 6.52 (s, 1H,
Thiazole-H), 3.88 (s, 2H, CH.sub.2), 3.79 (s, 3H, CH.sub.3), 2.45
(s, 3H, CH.sub.3), 2.27 (s, 3H, CH.sub.3).
[0087] Indomethacin-N-(4-biphenyl) amide (compound 31) was obtained
upon chromatography on silica gel (EtOAc:hexanes; 30:70) to afford
the pure product as a pale yellow solid (421 mg, 59%). .sup.1H NMR
(CDCl.sub.3) .delta. 7.68-7.71 (d, 2H, J=8.4 Hz, ArH), 7.32-7.55
(m, 11H, ArH), 6.95-6.96 (d, 1H, J=2.0 Hz, ArH), 6.86-6.89 (d, 1H,
J=9.0 Hz, ArH), 6.73-6.74 (dd, 1H, J=1.7 Hz, ArH), 3.83 (s, 2H,
CH.sub.2), 3.81 (s, 3H, CH.sub.3), 2.47 (s, 3H, CH.sub.3).
[0088] The structures and IC.sub.50 values for indomethacin and
Compounds 1 through 31 are set out in the Table below.
3TABLE Selective COX-2 Inhibition by Amide Derivatives of
Indomethacin 4 IC.sub.50 (.mu.M).sup.a Compound R COX-2 COX-1
Selectivity.sup.b Indomethacin OH 0.75 0.05 0.066 1 NHCH.sub.3 0.70
>66.sup.c >94 2 5 0.25 >66 287 3 6 0.0375 66 1760 4 7 0.04
16.5 412.5 5 8 0.15 >66.sup.c >440 6 9 0.06 8.0 133 7 10
0.0625 4.0 64 8 11 0.20 4.0 20 9 12 >33.sup.c >66 --
(Comparison) 10 13 >33.sup.c >66 -- (Comparison) 11 14 0.06
>66 >1100 12 15 0.06 >66.sup.c >1100 13 16 0.062
>66.sup.c >1064 14 17 0.12 >66.sup.c >550 15 18 0.12
>66.sup.c >550 16 19 0.22 >66.sup.c >300 17 20 0.056
>66.sup.c >1178 18 21 0.65 52.5 81 19 22 >1.0 >66
>66 20 23 0.052 >66 >1269 21 24 0.047 >66.sup.c
>1404 22 25 0.70 >66.sup.c >94 23 26 0.050 45 900 24 27
4.0 >66 >16.5 25 28 0.4 >66 >165 26 29 0.19 >66
>347 27 30 0.080 >66 >825 28 31 0.058 >66 >1138 29
32 4.0 >66 >16.5 30 33 4.0 >66 >16.5 31 34 0.5 >66
>132 .sup.aIC.sub.50 values were determined by incubating
several concn of inhibitor in DMSO with human COX-2 (66 nl) or
ovine COX-1 (44 nM) for 20 min followed by treatment with
1-.sup.14C-AA (50 .mu.M) at 37.degree. C. for 30 sec. Assay were
run in duplicate. .sup.bRatio of IC.sub.50 (COX-1): IC.sub.50
(COX-2). .sup.c>80% remaining COX-1 activity at this concn
DISCUSSION OF SECONDARY AMIDE DERIVATIVES OF INDOMETHACIN
[0089] Carboxylic Acid Aliphatic Secondary Amides Derivatives of
Indomethacin
[0090] The N-methyl amide derivative (compound 1) displayed
selective COX-2 inhibition (IC.sub.50 (COX-2).about.0.70 .mu.M;
IC.sub.50 (COX-1)>66 .mu.M). Increments in COX-2 inhibitory
potency and selectivity was observed with the higher octyl homolog
(compound 3); however, further increase in chain length to the
nonyl derivative (compound 4) led to some loss of COX-2 selectivity
(compound 3: IC.sub.50 (COX-2).about.37.5 nM; IC.sub.50 (COX-1) 66
.mu.M; compound 4: IC.sub.50 (COX-2).about.40 nM; IC.sub.50
(COX-1).about.16.5 .mu.M).
[0091] Carboxylic Acid Aromatic Secondary Amides Derivatives of
Indomethacin
[0092] Incorporation of methylene spacer units (compound 11)
between the amide nitrogen and the phenyl ring also generated
potent and selective COX-2 inhibitors.
[0093] For instance, the 4-methylbenzyl amide derivative (compound
6) was 133-fold selective for COX-2, whereas the corresponding
2-methylbenzyl isomer (compound 5) was >440 times more selective
as a COX-2 inhibitor. Furthermore, the R--methyl-(4-methylbenzyl)
enantiomer (compound 7) was a better inhibitor of COX-2 than the
corresponding S-methyl enantiomer (compound 8).
[0094] Additionally, the aromatic amides containing the 4-fluoro
(compound 12), 4-methylmercapto (compound 15), or the 3-pyridyl
substituent (compound 20), displayed potent and selective COX-2
inhibition, as noted below. 35
[0095] Tertiary Amides (Comparison Compounds 9 and 10)
[0096] Another interesting aspect in the SAR studies with the
indomethacin amides was that N,N-methyl-2-phenethyl (compound 9)
and the piperidinyl (compound 10) amide derivatives, both of which
are tertiary amides, were inactive against COX-2. In other words,
only the secondary amides were selective COX-2 inhibitors, whereas
the tertiary amides were devoid of any inhibitory effect towards
either isozyme i.e., measurement of COX-2 inhibition for the
tertiary amides was stopped at an extremely high IC value (see the
value of 33 for both compounds 9 and 10) and still >80% COX-1
activity remained.
Example II
[0097] Comparison with Sulfonamides of Another Study
[0098] A similar SAR study was previously reported in the
above-noted journal article by Li et al. for acidic sulfonamides.
(See, the structures drawn above for compounds L-745,337 and
NS-398.) Specifically, Li et al. found that replacement of the N--H
proton in the NHSO.sub.2CH.sub.3 moiety of L-745,337 or NS-398 with
a methyl group led to complete loss of inhibitory potency towards
either the COX-1 or COX-2 isozyme.
[0099] This behaviour may be explained from the recently solved
crystal structure of murine COX-2 complexed with NS-398. See,
Kurumbail et al., Abstract 197, Eicosanoids and Other Bioactive
Lipids in Cancer, Inflammation and Related Diseases, Fifth
International Conference, La Jolla, Calif. (Sep. 17-20, 1997).
Unlike the diarylheterocyclics, NS-398 does not utilize the side
pocket even though it contains a sulfonamide group. Instead the
sulfonamide binds to Arg106 in a fashion similar to the carboxylic
acid-containing NSAIDs.
[0100] Although the carboxylic acid secondary amide derivatives of
indomethacin in the present invention do not contain any
electron-withdrawing substituents, the above-discussed SAR
observations on the lack of inhibition by the carboxylic acid
tertiary amide derivatives suggest that the --CONH--R.sub.1 group
probably binds to a group on the enzyme (see below). This can be
seen from contrasting the data immediately below for the inventive
secondary amide derivative (compound 11) with the comparison
tertiary amide derivatives (compounds 9 and 10) and the comparison
derivative of prior art compound NS-398 in which the N--H proton in
the NHSO.sub.2CH.sub.3 moiety was replaced with methyl. 36
Example III
[0101] Additional Inhibitory Activity Testing with Mouse COX
[0102] Compound 11
[0103] The structural basis for COX-2 selectivity by compound 11
also was probed by site directed mutagenesis. More particularly,
the inhibitory potency of indomethacin as compared to that of
indomethacin-N-phenethyl amide (compound 11) was evaluated against
site directed murine COX-2 mutants (Arg106Gln and Tyr341Ala) which
represent key residues involved in the binding of the carboxylic
acid-containing NSAIDs. Arg106 is the only positively charged
residue in the fatty acid binding site and is important for binding
the carboxylic acid moiety of an NSAID with Tyr341Ala, which is
juxtaposed to Arg106 at the constriction site and is responsible
for the selective binding of the S-enantiomers but not the
R-enantiomers in the 2-phenylpropionate class of NSAIDs including
flurbiprofen. In addition to these mutants, also analyzed was the
inhibition profile of the Val509IleArg499HisVal420Ile mutant (also
known as the VRV mutant) which incorporates the major amino acid
changes between COX-2 and COX-1 in the side pocket region and is
responsible for binding the diarylheterocycles. The results were
that indomethacin displayed a slightly better potency against
wild-type mouse COX-2 than compound 11 (indomethacin: IC.sub.50
(mouse COX-2).about.25 nM; compound 11: IC.sub.50 (mouse
COX-2).about.35 nM). Furthermore, the Tyr341Ala and the triple
mutant VRV was resistant to inhibition by each of indomethacin and
compound 11, whereas the Arg106Gln mutant was resistant to
inhibition by indomethacin but was effectively inhibited by
compound 11 (IC.sub.50.about.25 nM).
[0104] Compound 17
[0105] Inhibition of COX-2 activity in intact mouse cells by
Compound 17 was assayed in murine RAW264.7 macrophages in which
COX-2 activity was induced by pathologic stimuli. The macrophages
were treated with LPS (500 ng/mL) and interferon-g (10 U/mL) for
7.5 hours to induce COX-2 and then treated with several
concentrations of the 4-methoxyphenyl amide derivative of
indomethacin (compound 17) for 30 minutes at 37.degree. C. The
IC.sub.50 value for PGD.sub.2 by compound 17 was 62.5 nM. Under
these conditions, indomethacin was a better inhibitor of COX-2
activity in intact mouse cells (IC.sub.50.about.10 nM) than
compound 17.
[0106] Indeed, comparison of the potency of indomethacin as a
inhibitor of purified mouse COX-2 versus purified human COX-2
revealed that indomethacin displayed greater inhibition of the
mouse enzyme than of the human isoform (IC.sub.50 (mouse
COX-2).about.350 nM; IC.sub.50 (human COX-2).about.1 .mu.M). On the
other hand, an indomethacin-amide derivative (compound 11) was a
better inhibitor of human COX-2 than of murine COX-2 (compound 11:
IC.sub.50 (mouse COX-2).about.120 nM; IC.sub.50 (human
COX-2).about.75 nM).
[0107] These results also strengthen another researcher's previous
observations which suggest that COX enzymes from the rat are
pharmacologically different from those from humans, as reported in
Ramesha, "Human and Rat Cyclooxygenases are Pharmacologically
Distinct", Adv. Exp. Med. Biol. (1997) Vol. 407, pp. 67-71.
Example IV
[0108] Testing for Reduction in Inflammation
[0109] Compound 14 was tested in a standard in vivo assay of
inflammation--the rat foot pad edema model. This assay is widely
used in the pharmaceutical industry to evaluate antiinflammatory
compounds. Rats were injected with carrageenan, which triggers a
rapid edema (swelling) within 3 hours that can be quantitatively
measured by volume displacement. A single dose of compound 14 (2
mg/kg) given orally 1 hour after carrageenan injection caused a
dramatic decrease in swelling.
[0110] In these experiments, the carrageenan that was injected was
in 0.1 mL of aqueous saline so that 0.1 mL volume increase was due
to the injection alone. Taking this into consideration,
approximately an 80-85% reduction in inflammation following
treatment with compound 14 was found. For comparison, indomethacin
was also tested in this assay at a dose of 2 mg/kg orally, and
comparable reduction in inflammation was found.
[0111] More specifically, male Sprague-Dawley rats (150 g) received
a subplantar injection of carrageenan (0.1 mL of a 1% suspension of
carrageenan in sterile aqueous saline) into the right hind footpad
while mildly anesthetized with methoxyflurane. At 1 hour
post-injection, the rats were gavaged with 0.5 mL corn oil
containing either 90 .mu.L DMSO or 90 .mu.L compound 14 for the
doses specified below. The ipsilateral footpad volume (mL) was
measured with a water displacement plethysmometer at time=3 hours
post-injection and compared to the time=0 pre-injection paw volume
for edema calculations.
[0112] For each dose, 6 rats were injected, and the results are
summarized below.
4 Compound 14 Concentration (mg/mL) 3 hour edema (mL) standard
deviation 0 0.87 0.1 0.2 0.55 0.04 0.5 0.47 0.07 1.0 0.39 0.03 2.0
0.38 0.07
Example V
[0113] Testing for Tumor Inhibition
[0114] The ability of compound 11 to inhibit the growth of a human
colon cancer cell line in nude mice (human tumor xenograft assay)
was determined. Following subcutaneous inoculation of the mice with
the human tumor cells, the animals were injected intraperitoneally
3 times a week with vehicle alone or vehicle containing 5 mg/kg of
compound 11.
[0115] More specifically, male athymic Nude-nu (nu/nu) mice (25 g,
6-8 weeks old, Harlan Sprague-Dawley)were injected
intraperitoneally with 100 .mu.L vehicle (5% ethanol, 5% Tween 80
in sterile aqueous PBS) or 100 .mu.L compound 11 in vehicle
immediately prior to subcutaneous implantation of 5.times.10.sup.6
HCA-7 (human colon adenocarcinoma, colony 29) cells in PBS into the
dorsal surface. The mice, 5 animals per group, received injections
of vehicle or vehicle containing compound 11 (5 mg/kg) 3 times per
week. Tumor volume was determined by external measurement with an
electronic caliper.
[0116] From comparison of the growth of the tumors as summarized
below, it can be seen that compound 11 significantly inhibited
tumor growth.
5 Control tumor compd 11 Day of Explant (vol. cm.sup.3) (tumor vol.
cm.sup.3) 22 0.34 0.10 25 0.47 0.11 27 0.62 0.13 29 0.70 0.14 31
0.76 0.17
[0117] It will be understood that various details of the invention
may be changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation--the
invention being defined by the claims.
* * * * *