Synthesis and anti-inflammatory activity of the major metabolites of imrecoxib

Article abstract of DOI:10.1016/j.bmcl.2009.02.090

We have developed a novel and moderately selective COX-2 inhibitor, imrecoxib, as a new anti-inflammatory drug. We describe herein the preparation of the major metabolites M2 and M4 of imrecoxib, as well as the in vitro and in vivo activities of the two c

Full text of DOI:10.1016/j.bmcl.2009.02.090

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2270–2272
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and anti-inflammatory activity of the major metabolites of imrecoxib
b
Zhiqiang Feng a, Fengming Chu a, Zongru Guo a, , Piaoyang Sun
*
a Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing 100050, China
b Hengrui Pharmaceutical Company, Lianyungang, Jiangsu 222002, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed a novel and moderately selective COX-2 inhibitor, imrecoxib, as a new anti-inflam-
matory drug. We describe herein the preparation of the major metabolites M2 and M4 of imrecoxib, as
well as the in vitro and in vivo activities of the two compounds. The results showed that both M2 and M4
are potential COXs inhibitors with a moderate COX-1/COX-2 selectivity, and their anti-inflammatory
activity in vivo was equal to or slightly higher than the clinical celecoxib.
Received 25 September 2008
Revised 6 February 2009
Accepted 24 February 2009
Available online 27 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Imrecoxib
Metabolites
Selective COX-2 inhibitor
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most
common medications for the treatment of pain, inflammation
and fever, and their anti-inflammatory effects are exhibited by
inhibiting cyclooxygenase (COX) which catalyzes the bioconver-
sion of arachidonic acid to prostaglandins.1 Nowadays, it is well
established that there are at least two COX isozymes, COX-1 and
COX-2.2,3 The isozyme COX-1 is constitutive and responsible for
the physiological production of prostaglandins, and is critical for
protection of gastric mucosa, platelet aggregation and renal blood
flow; whereas the COX-2 isozyme is inducible and responsible for
the elevated production of prostaglandins during inflammation.4,5
Traditional NSAIDs, such as naproxen, ibuprofen, diclofenac, flurbi-
profen, indomethacin and aspirin, inhibit both isoforms of COX,
furthermore COX-1 more strongly than COX-2, but harmful side ef-
fects in particular gastrointestinal (GI) irritation, ulcerogenicity
and renal toxicity, often limit their chronic use.6 Thus, several
new inhibitors were developed recently which selectively inhibit
the COX-2 enzyme without interfering with COX-1 enzymatic
activity. These molecules include celecoxib,7 rofecoxib8 and val-
decoxib,9 with fewer gastrointestinal side effects to traditional
NSAIDs. But an increased risk of myocardial infarction and cardio-
vascular thrombotic events associated with the use of highly selec-
tive COX-2 inhibitors were subsequently observed, which resulted
in the recent withdrawal of Rofecoxib and Valdecoxib from the
market.10,11 Hence an appropriately selective inhibition of COX-2
over COX-1 may be useful for the treatment of inflammation and
inflammation-associated disorders with reduced gastrointestinal
toxicities when compared with traditional NSAIDs and less cardio-
vascular thrombotic events than highly selective COX-2 inhibitors.
Imrecoxib, 4-(4-methylsulfonyl-phenyl)-1-propyl-3-(p-tolyl)-
3-pyrrolin-2-one, developed in our laboratory, is a novel and mod-
erately selective COX-2 inhibitor. It inhibits COX-1 and COX-2 with
IC50 values of 115 28 nmol/L and 18 4 nmol/L, respectively.12
Imrecoxib has been in the phase III of clinical trials in China for
the treatment of acute and chromic inflammatory diseases.
In a preclinical study, it was found that the half-life t1/2 of
imrecoxib is short, however, the time period of its anti-inflamma-
tory activity lasted longer. This observation is probably due to the
presence of active metabolites of imrecoxib. Further metabolic
study showed that imrecoxib was metabolized after iv administra-
tion with less than 2% of the dose recovered in urine and feces as
parent drug;13 seven metabolites (Scheme 1) of imrecoxib were
identified in vivo, including the 40-hydroxymethyl (M4), 40-car-
boxy-(M2), 40-hydroxymethyl-5-hydroxyl (M3), 40-hydroxy-
methyl-5-carbonyl (M5) metabolites, the glucuronide conjugate
of M4 (M1), the glucuronide conjugate of M2 (M6), and the glucu-
ronide conjugate of M3 (M7); approximately 75% of the dose was
excreted as the 40-carboxy metabolite (M2), and mainly in feces,
and ca. 15% of the dose as the 40-hydroxymethyl metabolite
(M4), mainly in urine; The major metabolic pathway was 40-
methyl group hydroxylation to form the 40-hydroxymethyl metab-
olite (M4) which was further oxidized to the 40-carboxy metabolite
(M2).14 The parent compound was primarily excreted as the car-
boxylic acid metabolite in feces after dosing. In order to further
confirm the structure and activity of the main metabolites M2
and M4, we performed the synthesis and the evaluation for anti-
inflammatory activity of the two metabolites.
It was found that the preparation of M2 and M4 by direct chem-
ical oxidation of Imrecoxib failed because of intricate products, for
example, the C5-methylene oxidation. A scheme of de novo syn-
thesis using 4-acetoxymethyl phenylacetic acid and 4-ethoxycar-
* Corresponding author. Tel.: +86 10 631 65249; fax: +86 10 83155752.
E-mail address: zrguo@imm.ac.cn (Z. Guo).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.090

Z. Feng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2270–2272
2271
OH
CH3
S
CH3
S
OH
CH3
O
CH2OH
O
HO
O
CH3
S
COOH
O
O
O
O
M5
O
O
O
N
N
CH3
M1
CH3
O
N
imrecoxib
CH3
CH3
S
O
CH2OH
O
CH3
S
CH3
S
O
CH2OH
COOH
O
O
O
M4
O
N
M2
M3
HO
O
CH3
O
N
N
CH3
CH3
OH
OH
OH
COOH
OH
HO
HO
O
CH3
S
CH3
COOH
O
O
S
O
O
O
O
M7
M6
O
O
HO
N
N
CH3
CH3
Scheme 1. The metabolites and metabolic pathway of imrecoxib.
bonyl phenylacetic acid, respectively, instead of 4-methyl phenyl-
acetic acid based on the synthetic route of imrecoxib15 was per-
formed (Schemes 2 and 3).
O
CH3
O
CH3
OH
COOEt
S
COOEt
S
O
4-(Bromomethyl)phenylacetic acid, prepared by the reported
method,16 was mixed with excessive sodium acetate and acetic
acid, and heated for 12 h to give 4-(acetoxymethyl)phenylacetic
acid 1 as one intermediate of M4. The commercially available 4-
(methylsulfonyl)acetophenone was brominated at the position of
ketone carbonyl to produce bromoacetopenone, that was reduced
by sodium boronhydride and followed by cyclized to yield 4-
(methylsulfonyl)styrene oxide, then reacted with n-propyl amine
to give another intermediate n-propylamino alcohol 2, which in
turn was acylated by the acyl chloride of 1 to afford acylamide 3.
The key intermediate 4 was obtained from the oxidation of 3 with
DMSO. Finally by the intramolecular condensation, 4 was cyclized
to provide the target compound M4.
O
+
a
C3H7
N
C3H7
N
CH2COOH
5
OH
2
O
6
CH3
S
O
O
CH3
COOEt
S
COOH
O
O
b
c
C3H7
N
O
O
N
O
CH3
7
M2
O
CH3
Scheme 3. The synthesis of metabolite M2. Reagents and condition: a: SOCl2,
reflux, 2/pyridine; b: DMSO/Ac2O; c: LiOH /t-butanol.
OAc O CH3
S
OAc
S
O
O
a
+
C3H7
N
C3H7
N
The metabolite M2 was prepared using a similar sequence of
reactions illustrated Scheme 2. The ethyl 4-ethoxycarbonyl phen-
ylacetate (di-ester) was produced by the alcoholysis of cyanic
CH2COOH
1
OH
OH
O
3
2
CH3
S
OAc
O
CH3
S
CH2OH
O
Table 1
O
O
b
The inhibitory effect of M2 and M4 on activity of COX-1 and COX-2
c
a
C3H7
N
Compounds
IC50 (10ꢀ7 M)
COX-2 SIb
COX-1
COX-2
O
O
N
O
M2
M4
Imrecoib
27.8
0.87
1.15
4.10
0.14
0.18
6.8
6.2
6.4
CH3
4
M4
a
Concentration required for 50% inhibition.
In vitro COX-2 selectivity index (IC50ꢁCOX-1/IC50ꢁCOX-2).
Scheme 2. The synthesis of metabolite M4. Reagents and condition: a: (1) SOCl2,
reflux; (2) 2/pyridine, rt b: DMSO/Ac2O; c: potassium t-butoxide/t-butanol.
b

2272
Z. Feng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2270–2272
Table 2
The effect of metabolites M4 and M2 on rat carrageenan-induced paw edema
Group
Dose (mg/kg)
The degree of the paw swelling (%) X SD
3 h
1 h
2 h
4 h
Control
M4
M2
Imrecoxib
Celecoxib
–
34.1 5.3*
54.7 6.9*
55.0 8.5*
53.3 6.6*
10
10
10
10
25.05 10.4** (26.5)
18.6 10.0** (45.4)
23.4 7.8** (31.3)
26.1 7.2** (23.5)
29.52 12.8** (46.0)
26.3 15.4** (51.9)
33.1 10.8** (39.5)
34.7 16.9* (36.6)
24.14 12.3** (56.1)
24.5 10.1** (55.5)
27.3 4.1** (50.4)
30.4 5.2* (44.7)
19.23 13.9** (63.9)
18.2 5.6** (65.9)
26.4 9.6** (50.4)
27.0 1.8* (49.3)
*p < 0.05, **p < 0.01 versus control. The data in parentheses represent inhibitory rate (%).
groups from commercially available 4-cyano phenylacetonitrile.
The aliphatic acid ester in di-ester was selectively hydrolyzed by
sodium carbonate to afford 4-ethoxycarbonyl phenylacetic acid 5.
Then 5 was treated with SOCl2 to give the acyl chloride, that re-
acted with n-propylamino alcohol 2 to yield an amide 6, followed
by the treatment of oxidant to give the key intermediate 7. The
metabolite M2 was obtained from the intramolecular cyclization
of 7, when treated with LiOH.
good anti-inflammation of M2 in vivo may result from the high
bioavailability and highly stable metabolism of this structure.
In summary, the structures of M2 and M4, as major metabolites
of imrecoxib, were further confirmed by the preparation of two
authoritative samples 3-(40-carboxy-phenyl)-4-(40-methylsulfo-
nyl-phenyl)-1-propyl-3-pyrrolin-2-one and 3-(40-hydroxymethyl-
phenyl)-4-(40-methylsulfonyl-phenyl)-1-propyl-3-pyrrolin-2-one.
The potential anti-inflammatory activity of M2 and M4 were dem-
onstrated by in vitro and in vivo assay for the two compounds,
which showed that both M2 and M4 were moderately selective
COX-2 inhibitors. This not only explained the long action period
of the parent drug, but may afford new potent candidates with im-
proved metabolic profile and bioavailability for the development of
anti-inflammatory drug. It is matter-of-course that the above-de-
scribed data are preliminary and not enough to develop M2 or
M4 as drug candidates. More in vitro and in vivo experiments such
as human whole blood assay, blood bleeding/clotting tests, and
preliminary pharmacokinetic experiments have to be conducted
before considering M2 and M4 as new candidates.
The structures of synthetic M4 and M2 were confirmed with the
authentic metabolic M4 and M2, which were identified by the
analysis from LC–MS, NMR spectra.
Then, compounds M4 and M2 synthesized were evaluated for
their ability to inhibit COX-2 and COX-1 by cellular assay, using
freshly harvested mouse peritoneal macrophages as described in
the literature.17 The results showed that both M4 and M2 exhibited
potent inhibition against COX-2 and COX-1 as listed in Table 1. The
inhibitory effect of M2 on calcimycin-induced COX-1 activity was
dose dependent at the concentrations of 10–10,000 nM, with IC50
value of 2.78 ꢂ 10ꢀ6 M, which was evidently lower than the parent
compound; Nevertheless the inhibitory effect of M4, with IC50 va-
lue of 8.7 ꢂ 10ꢀ8 M, was slightly higher than the parent compound.
In addition, the inhibitory effect of M2 on LPS-induced COX-2
activity was dose dependent at the concentrations of 10–
1000 nM, with IC50 value of 4.1 ꢂ 10ꢀ7 M, that was evidently lower
than the parent compound; However the inhibitory effect of M4,
with IC50 value of 1.4 ꢂ 10ꢀ8 M, was slightly higher than the parent
compound. The selective ratio (IC50ꢁCOX-1/IC50ꢁCOX-2) of inhibition by
M2 and M4 were close to that of inhibition in the parent com-
pound. Hence both M2 and M4 have a moderate COX-1/COX-2
selectivity, and may be also selective COX-2 inhibitors with fewer
gastrointestinal side effects to traditional NSAIDs, and fewer car-
diovascular side effects to highly selective COX-2 inhibitors (see
Table 2).
The oral anti-inflammatory activities of M2 and M4 were eval-
uated at a 10 mg/kg single dose using the carrageenan-induced
paw edema method in rats, and compared with imrecoxib and
celecoxib as reference compounds in this model. The results indi-
cated that the inhibitory effect of M2 at investigated time points
on paw edema was slightly higher than M4 and the control com-
pounds. In addition, the solubility of M2 in water is higher com-
pared to that of M4 and imrecoxib, owing to the carboxylic acid
group in M2 structure. Moreover, M2 is metabolic stable because
it is major metabolite of imrecoxib, which may avoid some side ef-
fects from the metabolism of M2 as lead compound. Hence, the
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