Enantiospecific, selective cyclooxygenase-2 inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00133-6

Cyclooxygenase inhibition studies with novel indomethacin alkanolamides demonstrate the potential for dramatic differences in inhibitor properties conferred by subtle structural modifications. The transformation of non-selective α-(S)-substituted indometh

Full text of DOI:10.1016/S0960-894X(02)00133-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1315–1318
Enantiospecific, Selective Cyclooxygenase-2 Inhibitors
Kevin R. Kozak, Jeffery J. Prusakiewicz, Scott W. Rowlinson
and Lawrence J. Marnett*
Departments of Biochemistry and Chemistry, Vanderbilt-Ingram Cancer Center and Center in Molecular Toxicology,
Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Received 14 November 2001; accepted 6 February 2002
Abstract—Cyclooxygenase inhibition studies with novel indomethacin alkanolamides demonstrate the potential for dramatic dif-
ferences in inhibitor properties conferred by subtle structural modifications. The transformation of non-selective a-(S)-substituted
indomethacin ethanolamides to potent, COX-2 selective inhibitors by simple stereocenter inversion highlights this property. # 2002
Elsevier Science Ltd. All rights reserved.
Cyclooxygenases (COXs) catalyze the bis-dioxygenation
of arachidonic acid to provide prostaglandin (PG) H2,
the precursor to the primary PGs and thromboxanes
(TXs).1 The therapeutic efficacy of nonsteroidal antiin-
flammatory drugs (NSAIDs) derives from their inhibi-
tion of COXs.2 Two COX isoforms exist. COX-1 is a
constitutive enzyme, whereas COX-2 is inducible and
highly regulated by a range of agonists. COX-1 activity
accounts for PG and TX production in gastric mucosa,
kidney, and platelets. COX-2 activity is thought to be
responsible for elevated PG levels associated with
inflammation.3 The link between COX-2 activity and
inflammation has been dramatically confirmed by the
clinical efficacy of two antiinflammatory, selective
COX-2 inhibitors, rofecoxib and celecoxib.
metabolic stability. This strategy is similar to that used
in the development of anandamide analogues with
enhanced metabolic stability (e.g., (R)-methanand-
amide).5 In the course of these investigations, we have
synthesized and evaluated several novel indomethacin
amides with substitutions near the amide linkage. The
results demonstrate that subtle structural modifications
of indomethacin amides can result in profound changes
in inhibitory properties and have identified an unique
enantioselective capacity of COX-1.
Indomethacin amides were prepared using standard
methods. Briefly, amides were generated by EDCI- or
BOP-Cl-mediated coupling of indomethacin with the
appropriate amine and purified by silica gel chromato-
graphy. Enantiomeric purity was determined by chiral,
high-performance liquid chromatography and estab-
lished that no racemization occurred during coupling
reactions (Chiralpak AD, 250Â4.6 mm, Chiral Tech-
nologies Inc., Exton, PA). (R)-2-Methoxy-1-methyl-
ethylamine was prepared by methylating (R)-2-tert-
butoxycarbonylamino-1-propanol with MeI in the pre-
sence of NaH followed by BOC deprotection with gas-
eous HCl. COX-1 was purified from ram seminal
vesicles and apoenzyme was prepared as described.6,7
Site-directed mutagenesis, expression, and purification of
COX-2 enzymes was performed as previously described.8
COX activity was quantified as reported previously.9
We recently demonstrated that modification of the
nonselective NSAID indomethacin by amidation pro-
vides COX-2-selective inhibitors. Initial results indi-
cated that sterically demanding amines or alcohols
could be coupled to indomethacin without significant
losses in potency or selectivity.4 This observation led to
an effort to develop similar compounds that possess
readily functionalizable moieties to allow for the
attachment of additional pharmacophores. In addition,
the potential for amidase/esterase metabolism of indo-
methacin derivatives providing the ulcerogenic parent
NSAID prompted related efforts to increase steric hin-
drance near the amide/ester bond, potentially enhancing
To examine the effects of amide substituents on COX-2
selectivity and potency of indomethacin amides, initial
modifications involved coupling the free acid with ami-
noalkanols of varied chain length. Neither COX-2
*Corresponding author. Tel.: +1-615-343-7329; e-mail: marnett@
toxicology.mc.vanderbilt.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00133-6

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K. R. Kozak et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1315–1318
selectivity nor potency was significantly affected by
increases in alkyl chain length (Table 1). As reference,
indomethacin displays IC50 values of 50 nM and 210
nM under these conditions for oCOX-1 and mCOX-2,
respectively.4
COX-2 inhibitory potency, but, the (S)-enantiomer, 7,
also proved to be a potent inhibitor of COX-1.
To test if this chiral discrimination was a general prop-
erty of substituted ethanolamides of indomethacin, a
series of chiral derivatives was synthesized and eval-
uated against wild-type cyclooxygenases. As seen in
Table 2, the enantioselective inhibition of COX-1 by (S)
enantiomers of a-substituted indomethacin ethanolamides
holds for a wide range of substituents.
Indomethacin was then amidated with an array of
readily available and inexpensive 1,2-amino alcohols to
expand this class of inhibitors. Initially, indomethacin
was coupled to alaninol to provide enantiomers 6 and 7
(Table 2). Interestingly, both enantiomers demonstrated
An exception to this general pattern is seen with proli-
nol amides of indomethacin. Both enantiomers dis-
played attenuated hCOX-2 inhibitory potency
(IC50=1.7 and 1.8 mM for (R) and (S), respectively) and
neither enantiomer inhibited oCOX-1 (IC50>80 mM).
Table 1. Chain length SAR: n-amino-1-alkanol indomethacin amide
inhibition of wild-type cyclooxygenases
To determine if the regiochemistry of substitution was
critical in enantiospecific COX-2-selective inhibition,
the effects of b-substitution were examined (Table 3).
Similar to the a-substituted indomethacin ethanol-
amides, all b-substituted compounds retained COX-2
inhibitory potency. However, in contrast to a-sub-
stituted indomethacin ethanolamides, no tested b-sub-
stituted indomethacin ethanolamide possessed potency
against COX-1. Thus, substitution at the a position is
required for the observed enantiospecifity.
Compd
n
oCOX-1
inhibition
IC50 (mM)a
hCOX-2
inhibition
IC50 (mM)a
IC50 (COX-1)/
IC50 (COX-2)
1
2
3
4
5
2
3
4
5
6
>66
64
86
>83
>63
0.16
0.22
0.28
0.29
0.21
>410
290
310
>290
>300
An effort to establish the structural requirements for
enantiospecific COX-1 inhibition was undertaken by
systematically evaluating derivatives of 6 (Table 4). As
discussed above, a-substitution is required for enantio-
selective COX-1 inhibition (compare 6/7 with 21/22). In
addition, the presence of the hydroxyl moiety in 6/7 is
required for the enantioselective pattern of inhibition
observed in Table 2. Inhibitors that include methoxy
(25/26) or methyl (27/28) substitutions for the hydroxyl
moiety in 6/7 displayed altered chirality-dependent
inhibition of COX isoforms. In contrast to the related
alcohols, the (R)-enantiomers, 25 and 27, possessed
greater inhibitory potency toward COX-1 than the cor-
responding (S)-enantiomers, 26 and 28, and are thus less
aValues represent the average of at least two determinations.
Table 2. Inhibition of wild-type cyclooxygenases by a-substituted
indomethacin ethanolamides
Table 3. Inhibition of wild-type cyclooxygenases by b-substituted
indomethacin ethanolamides
Compd
R
oCOX-1 hCOX-2 IC50 (COX-1)/
inhibition inhibition IC50 (COX-2)
IC50 (mM)a IC50 (mM)a
6
7
8
9
(R)-CH3
(S)-CH3
(R)-CH2CH3
33
0.59
59
0.35
>63
0.40
>63
<3.9
51
2.3
47
<1.3
>50
<3.9
0.48
0.17
0.27
0.10
0.28
0.11
0.37
0.50
0.17
0.25
1.0
0.44
0.085
0.22
0.085
0.11
190
2.2
590
1.3
>570
1.1
>130
<23
200
2.3
110
<15
>230
<46
4.4
(S)-CH2CH3
10
11
12
13
14
15
16
17
18
19
20
(R)-CH(CH3)2
(S)-CH(CH3)2
(R)-CH2CH(CH3)2
(S)-CH2CH(CH3)2
(R)-CH2CH2SCH3
(S)-CH2CH2SCH3
(R)-C6H5
Compd
R
oCOX-1
inhibition
IC50 (mM)a
hCOX-2
inhibition
IC50 (mM)a
IC50 (COX-1)/
IC50 (COX-2)
21
22
23
24
(R)-CH3
(S)-CH3
(R)-C6H5
(S)-C6H5
52
32
>63
56
0.33
0.32
0.21
0.047
160
100
>300
1200
(S)-C6H5
(R)-CH2C6H5
(S)-CH2C6H5
(S)-CH(CH3)CH2CH3
aValues represent the average of at least two determinations.
aValues represent the average of at least two determinations.

K. R. Kozak et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1315–1318
1317
COX-2 specific. This contrasts with the greater COX-2
specificity observed for (R)-enantiomers in the a-sub-
stituted indomethacin ethanolamide series. Surprisingly,
the dimethyl substituted derivative, 29, maintained
COX-1 inhibitory potency suggesting that the inability
of (R)-enantiomers in this series to significantly inhibit
COX-1 is not simply attributable to an unfavorable
steric interaction between the alkyl substituent and the
enzyme.
sulfide (APHS). APHS fails to inhibit mCOX-2 con-
taining the S530A mutation.13 This mutation had
essentially no effect on the potency of 6 or 7 (Table 5).
As COX-2 regions known to be involved in interactions
with other COX-2 selective inhibitors appeared to play
no role in the COX-2 inhibitory potency or enantiospe-
cificity of indomethacin ethanolamides, additional
active site mutations were examined. R120, Y355, and
E524 form a hydrogen bonding network at the opening
of the substrate access channel within COX enzymes.
Mutations in R120 and E524 (R120Q, R120A, and
E524L) failed to render the enzyme resistant to inhibi-
tion by 6 or 7 (Table 5). In contrast, substitution of
Y355 with an alanine or phenylalanine provided mutant
enzymes which were not inhibited by either enantiomer
(<10% inhibition at 2.4 mM). The importance of Y355
In order to identify enzyme residues responsible for the
observed enantiospecific inhibitory characteristics of
these compounds, several site-directed mutant murine
COX-2 enzymes were evaluated with 6 and 7. We
anticipated identifying a mutant which retained sensi-
tivity to inhibition by 7 but was resistant to inhibition
by 6. Initially, protein regions previously identified as
critical in conferring isoform selectivity upon known
COX-2 inhibitors were evaluated.
Table 5. Inhibition of wild-type and mutant murine COX-2 by 6 and
7a
The COX-2 side-pocket, which accounts for the COX-2
selectivity of diarylheterocycles, was investigated with a
V523I single mutant as well as the V523I/R513H/V434I
triple mutant. In contrast to diarylheterocycles which do
not inhibit these mutants, both enzymes remained sen-
sitive to inhibition by 6 and 7 (Table 5).10,11 The sub-
Enzyme
6 Inhibition
IC50 (nM)a
7 Inhibition
IC50 (nM)a
Wild-type
V523I
VRV
L503F
S530A
R120A
R120Q
E524L
V89I
I92L
Y115L
S119V
Y115L/S119V
H122N690
F357L
540
690
340
300
570
50
170
220
110
160
150
60
75
220
85
200
390
200
210
stitution of
a
4-bromobenzyl moiety for the
4-chlorobenzoyl group of indomethacin provides a
COX-2 selective inhibitor. The selectivity of this indo-
methacin derivative depends on the additional COX-2
active site volume provided by the presence of a leucine
at position 503 in COX-2 instead of a phenylalanine as
in COX-1. Consequently, the L503F mutant is not
inhibited by the 4-bromobenzyl derivative.4,12 In contrast,
the L503F mutant enzyme was potently inhibited by both
6 and 7 (Table 5). Finally, the serine residue acetylated by
aspirin, Ser 530, is required for inhibition by the COX-2
selective covalent modifier, O-(acetoxyphenyl)hept-2-ynyl
90
560
250
200
325
240
290
300
250
250
aValues represent the average of at least two determinations.
Table 4. Structure–activity relationship: methyl substituted indomethacin ethanolamides and derivatives
Compd
X
R1
R2
R3
R4
oCOX-1
inhibition
IC50 (mM)a
hCOX-2
inhibition
IC50 (mM)a
IC50 (COX-1)/
IC50 (COX-2)
6
7
OH
OH
OH
OH
OCH3
OCH3
CH3
CH3
OH
H
CH3
H
H
H
CH3
H
CH3
CH3
CH3
H
H
H
CH3
H
CH3
H
CH3
H
H
H
CH3
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
33
0.59
52
32
6.8
>60
0.18
4.7
0.17
0.27
0.33
0.32
0.14
0.24
0.13
0.11
0.19
190
2.2
160
100
49
>250
1.4
42
21
22
25
26
27
28
29
0.80
4.2
aValues represent the average of at least two determinations.

1318
K. R. Kozak et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1315–1318
in indomethacin ethanolamide inhibition agrees with
previous results with other indomethacin amides.
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Several additional protein residues were examined by
site-directed mutagenesis. Unfortunately, no residue
substitution resulted in a mutant enzyme sensitive to
inhibition by 7 but resistant to inhibition by 6 (Table 5).
The structural basis for the enantioselective inhibition
of COX-1 remains unclear.
It is well established that interactions between chiral
small molecules and enzymes can display marked ste-
reospecificity. The demonstration that a-substituted
ethanolamides of indomethacin can be transformed
from nonselective to highly COX-2-selective inhibitors
by the simple inversion of stereochemistry at the single
chiral center in these compounds dramatically affirms
this principle. Although COX enzyme structure has
been extensively studied, the combination of site-direc-
ted mutagenesis and SAR studies suggests that subtle and
pharmacologically exploitable COX isoform differences
remain uncharacterized.
8. Rowlinson, S. W.; Crews, B. C.; Lanzo, C. A.; Marnett,
L. J. J. Biol. Chem. 1999, 274, 23305.
9. Kalgutkar, A. S.; Kozak, K. R.; Crews, B. C.; Hochgesang,
G. P.; Marnett, L. J. J. Med. Chem. 1998, 41, 4800.
10. Gierse, J. K.; McDonald, J. J.; Hauser, S. D.; Rangwala,
S. H.; Koboldt, C. M.; Seibert, K. J. Biol. Chem. 1996, 271,
15810.
11. Guo, Q.; Wang, L.-H.; Ruan, K.-H.; Kulmacz, R. J. J.
Biol. Chem. 1996, 271, 19134.
12. Black, W. C.; Bayly, C.; Belley, M.; Chan, C.-C.; Charle-
son, S.; Denis, D.; Gauthier, J. Y.; Gordon, R.; Guay, D.;
Kargman, S.; Lau, C. K.; Leblanc, Y.; Mancini, J.; Ouellet,
M.; Percival, D.; Roy, P.; Skorey, K.; Tagari, P.; Vickers, P.;
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References and Notes
1. Hamberg, M.; Samuelsson, B. Proc. Natl. Acad. Sci. U.S.A.
1973, 70, 899.
13. Kalgutkar, A. S.; Crews, B. C.; Rowlinson, S. W.; Garner,
C.; Seibert, K.; Marnett, L. J. Science 1998, 280, 1268.