Synthesis of new 2-arylamino-6-trifluoromethylpyridine-3-carboxylic acid derivatives and investigation of their analgesic activity

Article abstract of DOI:10.1016/j.bmc.2004.05.025

A new series of 2-arylamino-6-trifluoromethyl-3-carboxylic acid derivatives was synthesized and assayed in vivo for their analgesic properties by means of writhing test in rats. When compared to aspirin, ibuprofen and flufenamic acid some of the new compo

Full text of DOI:10.1016/j.bmc.2004.05.025

Bioorganic & Medicinal Chemistry 12 (2004) 4169–4177
Synthesis of new 2-arylamino-6-trifluoromethylpyridine-3-
carboxylic acid derivatives and investigation
of their analgesic activity
Maria T. Cocco,a,* Cenzo Congiu,a Valentina Onnis,a Micaela Morelli,a
Vicente Felipob and Omar Caulia,b
aDipartimento di Tossicologia, Universitꢀa degli Studi di Cagliari, Via Ospedale 72, Cagliari I-09124, Italy
bFundaciꢁon Valenciana de Investigaciones Biomedicas, c/Amadeo de Saboya, 4, 46010 Valencia, Spain
Received 21 January 2004; accepted 17 May 2004
Available online 24 June 2004
Abstract—A new series of 2-arylamino-6-trifluoromethyl-3-carboxylic acid derivatives was synthesized and assayed in vivo for their
analgesic properties by means of writhing test in rats. When compared to aspirin, ibuprofen and flufenamic acid some of the new
compounds exhibited a comparable or improved analgesic activity and a lower ulcerogenic effect.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
After Vane first established a link between NSAID
pharmacological activity and prostaglandin production
in 1971,4 subsequent studies have highlighted the central
role of cyclo-oxygenase (COX) inhibition, with sub-
sequent reduction of prostaglandin biosynthesis, in
mediating both clinical efficacy and toxicity. In partic-
ular, NSAIDs inhibit the COX activity of prostaglandin
G/H synthase, the enzyme that mediates the biotrans-
formation of arachidonic acid into a series of prosta-
noids (prostaglandins and thromboxanes). These
compounds display complex biological functions in
several body systems and play a pivotal role in mediat-
ing inflammation and pain.5–7
Nonsteroidal anti-inflammatory drugs (NSAIDs) are a
class of compounds characterized by a similar pharma-
cological profile encompassing anti-inflammatory,
analgesic and anti-pyretic activities. They are indicated
for the treatment of musculo-skeletal disorders and
other syndromes involving pain.1 Despite their well-
known therapeutic efficacy, NSAIDs have the potential
to cause adverse reactions, especially with regard to the
digestive and the renal system.2 The most common ad-
verse events related to the use of NSAIDs involve the
gastrointestinal (GI) tract, and include a range of dis-
orders, from functional problems such as dyspepsia,
heartburn and abdominal discomfort to GI haemor-
rhage, peptic ulcer and perforation. Epidemiological
studies have shown that approximately 10–20% of
NSAID-treated patients have gastroduodenal erosions
or ulcers detected at endoscopy, and 60% of those with
serious GI complications are on long-term NSAID
therapy.3 Given the vast epidemiological proportions of
the problem of NSAID-induced GI damage, preclinical
and clinical researchers have spent substantial efforts
trying to identify the mechanisms responsible for the
therapeutic and toxic effects of these drugs in order to
devise strategies for effective prevention of GI toxicity.
In the early 1990s, molecular biology studies identified
two COX isoenzymes, named COX-1 and COX-2,
which have different molecular structures and are en-
coded by different genes.8 Subsequent studies showed
that COX-1 is constitutive and catalyses the production
of prostaglandins involved in GI mucosal protection
and other physiological activities, whereas COX-2,
mostly inducible, is responsible for the production of
prostaglandins that mediate inflammation, pain and
fever.6 The discovery of these two different COX
isoenzymes has allowed researchers a better under-
standing of the varying degrees of GI toxicity associated
with the use of different NSAIDs by hypothesizing that
COX-2 inhibition is responsible for the therapeutic
effects of these drugs, whereas the typical adverse reac-
tions are attributable to COX-1 inhibition.6;9 Recent
* Corresponding author. Tel.: +39-0706758645; fax: +39-0706758612;
e-mail: tcocco@unica.it
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.025

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M. T. Cocco et al. / Bioorg. Med. Chem. 12 (2004) 4169–4177
studies have further clarified the physiological role of
COX isoenzymes. COX-1 is expressed in most tissues
(mainly platelets, GI tract, central nervous system
(CNS) and kidney), where it mediates functions such as
platelet aggregation, neurotransmission, hyperaemia,
cell proliferation and differentiation.10–12 COX-1
expression has also been detected in synovial tissue of
patients with inflammatory joint disease.13 COX-2 is
induced by pro-inflammatory cytokines, mitogens and
growth factors.10 In platelet activation syndromes,
COX-2 appears to increase prostacyclin production. In
this respect, selective COX-2 inhibitors, which are
known to decrease urinary excretion of prostacyclin
metabolites, might increase the risk of thrombosis in
predisposed patients.14 Moreover, contrary to previous
beliefs, COX-2 appears to be constitutively expressed in
organs such as CNS, kidney, ovarian follicles and testes,
where it regulates important biological functions.6–15
However, the predominant physiological activity of
COX-2 appears to be the production of prostanoids that
mediate inflammation and pain. Based on the know-
ledge of the differential activities of the two COX
isoenzymes, new NSAIDs have been developed, such as
meloxicam, nimesulide, celecoxib and rofecoxib that are
capable of exerting a preferential or selective inhibition
of the COX-2 isoenzyme without interfering to the same
extent as nonselective NSAIDs with the normal func-
tions and mucosal protective activities mediated by
COX-1.
reduced gastrointestinal side effects. So we undertook a
study on the influence of the introduction of a 6-CF3
group on 2-aminonicotinic acid derivatives. In this
paper we report the synthesis, the pharmacological and
toxicological properties of 2-arylamino-6-trifluorom-
ethylpyridine derivatives. We explored the consequence
on analgesic activity of the kind and the position of
substituents on arylamino moiety of 2-arylamino-6-tri-
fluoromethylpyridine-3-carboxylic acids 5. We also
explored the effects of substitution of the carboxylic acid
functionality with nitrile (compounds 4), ester (com-
pounds 7 and 9) or amide (compounds 6) moieties on
selected 2-arylamino-6-trifluoromethylpyridine-3-car-
boxylic acids that showed the best analgesic activity.
2. Results and discussion
2.1. Chemistry
The preparation of 2-anilinonicotinic acid derivatives is
generally achieved by condensation of a 2-halonicotinic
acid (or esters) and an aniline derivative at high tem-
perature22–24 or in the presence of anhydrous potassium
carbonate and Cu powder,25–29 pyridine and p-TsOH,30
or KI31 as catalysts. These reactions are well described
and applicable to various 6-H or 6-arylsubstituted 2-
halonicotinic acids. Whereas, to the best of our knowl-
edge, only an example of application of this reaction to
methyl 6-trifluoromethyl-2-bromonicotinate has been
reported.32 The introduction of a trifluoromethylated
function on a pyridine ring is not a trivial reaction, as
matter of fact few approaches for the preparation of
trifluoromethyl heterocycles have been developed.
Among these the use of easily available trifluorome-
thylated building blocks has often found to be effective
for introduction of a trifluoromethyl group into het-
erocyclic systems.33 We have previously reported an easy
and convenient method34 for the synthesis of pyridines
bearing trifluoromethyl group by reaction of trifluoro-
acetylvinyl ethers with 3-amino-3-dialkylaminopropen-
enitriles.
Pyridine derivatives form an important class of hetero-
cyclic compounds and have attracted the attention of
many scientists. Some pyridine compounds16 such as
derivatives of aminopyridynylmethanols and amino-
methylpyridinamines have been found useful as anal-
gesic as well as anti-inflammatory agents and for
treating Alzheimer’s disease.17 The pyridine nucleus is
present in niflumic acid and flunixin, two traditional
NSAIDs belonging to the class of fenamates. These
drugs are derivatives of N-phenyl (or hetero-
aryl)anthranilic acid that are employed for their analge-
sic, anti-inflammatory and anti-pyretic properties.
Unlike most other NSAIDs, the fenamates appear also
to compete with prostaglandins for binding at the
prostaglandin receptor site and thus potentially antag-
onize the physiopathological effects of prostaglandins
that have already been formed. Fenamates are used to
relieve mild to moderate pain when the duration of
therapy will not exceed one week. Fenamates are
endowed of most of the adverse effects induced by
NSAIDs, particularly gastrointestinal bleeding, ulcera-
tion and perforation. Unlike aspirin and most other
NSAIDs, the fenamates, do not affect platelet aggrega-
tion and bleeding time. Substitution of the carboxylic
acid functionality of several fenamates with heterocycles
containing an acidic functionality provided dual inhib-
itors of COX and 5-lypoxygenase.18 Furthermore recent
papers show that substitution at 2-position of anthra-
nilic acid by different substituted aryl or heteroaryl
moieties markedly modulate the biological activity.19–21
Prompted by the above observations we became inter-
ested into pyridine derivatives designed to obtain novel
molecules endowed with analgesic activity as well as
Schemes 1–4 show the synthetic approaches to obtain
6-trifluoromethylpyridine derivatives 4–7 and 9 (Table
1). According to Scheme 1, 2-arylamino-6-trifluoro-
methyl-3-pyridinecarbonitriles 4 were obtained through
an one-pot, two-step reaction. 3-Amino-3-ethoxypro-
penenitrile 1 was first treated with the appropriate
substituted arylamine in MeCN solution to give the no
isolable 3-amino-3-arylaminopropenenitriles 2 and then
with 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-one. The
temperature was gradually allowed to reach 60 °C. After
3 h pyridinecarbonitriles 4 are obtained in good yields
through the formation of the intermediate 1,1,1-triflu-
oro-3,5-hexadien-2-ones 3. Adducts 3 are the main
products of the reaction between propenenitriles 2b, 2c
and 2e, while in all other cases pyridines 4 are exclusively
obtained. The ratio of compounds 3 and 4 depended on
reaction time, in fact the prolongation of reaction time
up to 6 h resulted in formation of pyridines 4 as only
product.

M. T. Cocco et al. / Bioorg. Med. Chem. 12 (2004) 4169–4177
4171
CN
CN
CN
F3COC
CN
and / or
i)
ii)
F3C
N
NHAr
H2N
OEt
H2N
NHAr
H2N
NHAr
1
2
3
4
Scheme 1. Synthesis of pyridines 3. Reagents and conditions: (i) ArNH2, MeCN, rt; (ii) 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-one, 60 °C.
CN
COOH
NHAr
butoxy-3-buten-2-one in MeCN solution to give pyr-
idine derivatives 9.
i)
F3C
N
4
NHAr
F3C
N
5
2.2. Pharmacology
ii)
All compounds 4–7, 9 were screened for analgesic
activity using acetic acid writhing test35 in rats. Their
activity as compared with three clinically used NSAIDs
drugs namely aspirin (asa), ibuprofen (ibu) and flufen-
amic acid (flu) (Fig. 1).
CONH2
NHAr
F3C
N
6
Scheme 2. Synthesis of pyridines 5 and 6. Reagents and conditions: (i)
H2SO4, H2O, 100 °C; (ii) 20% NaOH, 100 °C.
The writhing test is a model of inflammatory pain that it
has long been used as a screening tool for evaluation of
putative analgesic and anti-inflammatory agents.36–38
Moreover this model has been shown to be useful in
correlating the ED50 values of NSAID antinociception
in experimental animals with those in humans.39;40
COOH
NHAr
COOMe
NHAr
i)
F3C
N
F3C
N
After ip administration all compounds 4 and 5, except
for 4a, 4f and 5e, induced significant analgesic activity
(at least P < 0:05 as compared to vehicle injected rats
(Fig. 1). The percentage of inhibition of acetic acid-in-
duced writhing (Table 1) revealed that all pyridine-3-
carbonitriles 4, except for 4a and 4f, at the dose of 5 mg/
kg displayed a similar analgesic activity induced by
aspirin, ibuprofen and flufenamic acid (Fig. 1 and Table
1). Pyridine-3-carboxylic acids 5f, 5g and 5h induced a
significantly higher analgesic activity as compared to the
reference drugs (P < 0:05, Fig. 1). Acids 5a–5d show
analgesic activity comparable to that of reference com-
pounds (Fig. 1 and Table 1). Otherwise, administration
of compounds 5e, 6, 7 and 9 did not induced a signifi-
cant analgesic activity as compared to vehicle injected
rats (Fig. 1 and Table 1).
5g
7g
Scheme 3. Synthesis of pyridine 7g. Reagents and conditions: (i)
DMF–DMA, Toluene, 90 °C.
COOEt
OEt
COOEt
NHAr
i), ii)
H2N
F3C
N
9
8
Scheme 4. Synthesis of pyridines 8g–h. Reagents and conditions: (i)
ArNH2, MeCN, rt; (ii) 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-one,
60 °C.
Cyanopyridines 4 were the starting materials for the
preparation of pyridine-3-carboxylic acids 5 and pyr-
idine-3-carboxamides 6 (Scheme 2). High yields of
compounds 5 were achieved by hydrolysis of derivatives
4 in 50% aqueous sulfuric acid. Sodium hydroxide cat-
alyzed hydrolysis of selected compounds 4 afforded
pyridine-3-carboxamides 6.
In order to assess the possible nonspecific sedative or
motor effects of the investigated compounds and to
distinguish analgesia from drug-induced motor changes,
the motor activity after ip administration of the most
active compounds was evaluated (Fig. 2). None of the
tested compounds 4 and 5 induced significant motor
changes (Fig. 2) indicating that they give analgesia de-
void of nonspecific sedative or stimulating effects.
Reaction of pyridine-3-carboxylic acid 5g with dimeth-
ylformamide dimethyl acetal (DMF–DMA), in toluene
solution, afforded the corresponding methyl ester 7g in
80% yield. (Scheme 3).
Successively analgesic activity (Fig. 3) after po admin-
istration of the compounds that have shown analgesic
activity after ip administration (4b, 4c, 4d, 4e, 4g, 4h, 5a,
5b, 5c, 5d, 5f, 5g, 5h) was evaluated. After po adminis-
tration, compounds 4b–e, 4g, 4h, 5a–d and 5f–h at the
dose of 10 mg/kg demonstrated noteworthy analgesic
activity (at least P < 0:05 as compared to vehicle in-
jected rats, Fig. 3) similar to that induced by the refer-
ence drugs.
A chemistry similar to that described in the synthesis of
compounds 4 was used for the preparation ethyl esters 9.
As reported in Scheme 4, ethyl 3-amino-3-ethoxyprop-
enoate 8 was sequentially treated with the appropriate
substituted arylamine, then with 1,1,1-trifluoro-4-iso-

4172
M. T. Cocco et al. / Bioorg. Med. Chem. 12 (2004) 4169–4177
Table 1. Percentage of inhibition of acetic acid-induced writhing test (see Pharmacology section) in rats pretreated with one of the reference drugs
(aspirin, ibuprofen or flufenamic acid), one of the 2-arylamino-6-trifluoromethylpyridine derivatives or vehicle
X
F3C
N
NHAr
Compound
X
Ar
ip test (5 mg/kg)% inhibition
acetic acid-induced writhing
po test (10 mg/kg)% inhibition
acetic acid-induced writhing
4a
4b
4c
CN
CN
2-MeC6H4
3-CF3C6H4
4-CF3C6H4
16
61
63
59
59
53
62
72
65
68
72
61
40
91
97
92
31
18
49
50
49
63
74
66
0
––
75
78
73
72
––
83
97
70
57
74
75
––
90
94
90
––
––
––
––
––
60
70
60
0
CN
CN
4d
4e
2,6-Me2C6H3
2-Me,3-ClC6H3
2-Me,4-ClC6H3
2-Me,5-ClC6H3
2-Me,6-ClC6H3
2-MeC6H4
CN
CN
4f
4g
4h
5a
5b
5c
CN
CN
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
CONH2
CONH2
COOMe
COOEt
COOEt
3-CF3C6H4
4-CF3C6H4
5d
5e
2,6- Me2C6H3
2-Me,3-ClC6H3
2-Me,4-ClC6H3
2-Me,5-ClC6H3
2-Me,6-ClC6H3
2-Me,3-ClC6H3
2-Me,5-ClC6H3
2-Me,5-ClC6H3
2-Me,5-ClC6H3
2-Me,6-ClC6H3
5f
5g
5h
6e
6g
7g
9g
9h
ibu
asa
flu
vehicle
Only compounds, which displayed analgesic activity after ip administration were subsequently tested for their analgesic activity after po adminis-
tration.
50
40
30
*
*
*
20
10
0
*
*
*
*
*
*
*
*
*
*
a
**
a
*
a
**
*
VEH IBU ASA FLU 4a 4b 4c 4d 4e 4f 4g 4h 5a 5b 5c 5d 5e 5f 5g 5h 6e 6g 7g 9g 9h
Figure 1. Stretches induced by ip acetic acid injection in rats pretreated ip (30 min before) with vehicle, one of the reference drugs or one of the
experimental new compounds (5 mg/kg). *P < 0:05; **P < 0:01 as compared to vehicle pretreated rats; aP < 0:05 as compared to ibuprofen (ibu),
aspirin (asa) and flufenamic acid (flu) pretreated rats.
3000
2000
1000
0
Particularly all tested compounds 4 display percentage
of inhibition of acetic acid-induced writhing higher after
po administration as compared to that of the same
compounds obtained after ip administration. Further-
more compounds 4h, 5f, 5g and 5h induced even a sig-
nificant higher analgesic activity as compared to that
induced by the reference drugs (P < 0:05).
veh IBU ASAFLU 4b 4c 4d 4e 4g 4h 5a 5b 5c 5d 5f 5g 5h
Since one of the common side effects of NSAID therapy
is the gastrointestinal toxicity, the compounds that have
shown analgesic activity (4b, 4c, 4d, 4e, 4g, 4h, 5a, 5b, 5c,
5d, 5f, 5g, 5h), were subsequently tested for their ability
Figure 2. Locomotor activity, recorded in 90 min, starting 30 min later
after ip administration of the vehicle, the reference drugs or the
experimental compounds (5 mg/kg).

M. T. Cocco et al. / Bioorg. Med. Chem. 12 (2004) 4169–4177
4173
50
40
30
20
10
0
tive compounds as 5e (5e: 40%, 6e: 31%). Similarly the
esterification of 3-carboxylic function leds to the almost
inactive compounds 7g and 9g. Independently from the
kind of ester, methylic or ethylic, their values of per-
centage of inhibition of acetic acid-induced writhing are
about the half of that showed by the parent acid 5g.
Generally substitution of 3-carboxylic group with a
cyano moiety led to slight reduction in analgesic activity,
except for compounds 5e and 4e, where this substitution
have an helpful effect on the activity. The introduction
of 2-methyl-5-chlorophenylamino or 2-methyl-6-chlor-
ophenylamino functions at 2-position of showed by
nitriles 4 and acids 5 is effective in inducing strong
analgesic activity. The displacement of chlorine atom
from 5- or 6-position to 4-position on the phenylamino
ring produces variable effects: in nitriles (compound 4f)
is detrimental, while in acids the activity is maintained
(compound 5f). On the contrary displacement of chlo-
rine atom from 5- or 6-position to 3-position does not
affect activity in nitriles (compound 4e), while it causes a
drop in activity in acids (compound 5e). When 2-methyl-
6-chlorophenylamino group is replaced by a 2,6-di-
methylphenylamino moiety as in 4d and 5d the activity is
reduced.
*
*
*
*
*
*
*
*
*
*
*
a
**
a
**
*
a
**
a
**
veh IBU ASA FLU 4b 4c 4d 4e 4g 4h 5a 5b 5c 5d 5f 5g 5h
Figure 3. Stretches induced by ip acetic acid injection in rats pretreated
po (60 min before) with vehicle, one of the reference drugs or one of the
experimental new compounds (10 mg/kg). * P < 0:05, ** P < 0:01 as
compared to vehicle pretreated rats; aP < 0:05 as compared to ibu-
profen (ibu), aspirin (asa) and flufenamic acid (flu) pretreated rats.
to produce, gastric ulcers compared to those induced by
the three reference drugs. The severity of gastric damage
was expressed as lesion index (Table 2).
Compounds 4b–e, 4g, 4h, 5a–d and 5f–h at the dose of
100 mg/kg induced a significantly lower (compounds 4b,
4c, 4e, 5b–d and 5f–h, lesion index between 0.1 and 0.7,
Table 2) or no (compounds 4d, 4h and 5a, lesion index:
0, Table 2) ulcerogenic effect as compared to reference
drugs.
3. Experimental
3.1. Chemistry
Analysis of all pharmacological data indicates that
analgesic activity appears to be related to some struc-
tural requirements, to the presence of a 3-carboxylic acid
moiety (compounds 5) and to 2-arylaminosubstituents.
In fact the amidation of the carboxyl on 3-position is
detrimental for analgesic activity. As matter of fact if we
consider the most active pyridine-3-carboxylic acid 5g
(percentage of inhibition of acetic acid-induced writhing
97%) the corresponding pyridine-3-carboxamide 6g
shows a 18% percentage of inhibition of acetic acid-in-
duced writhing. The same trend is displayed by less ac-
Melting points were determined on a Stuart Scientific
Melting point SMP1 and are uncorrected. Proton NMR
spectra were recorded on a Varian Unity 300 spec-
trometer. The chemical shift are reported in part per
million (d, ppm) downfield from tetramethylsilane
(TMS), which was used as internal standard. Infrared
spectra were obtained with a Bruker Vector 22 spec-
trophotometer. Elemental analyses were carried out
with a Carlo Erba model 1106 Elemental Analyzer and
the values found were within 0.4% of theoretical val-
ues.
Table 2. Gastric lesion index induced by an acute administration of
the tested compounds (100 mg/kg po)
3.1.1. General procedure for the synthesis of 2-arylamino-
6-trifluoromethylpyridin-3-carbonitriles 4. The appropri-
ate arylamine (10 mmol) was added to a solution of
3-amino-3-ethoxypropenenitrile 1 (1.10 g, 10 mmol) in
anhydrous acetonitrile (20 mL). The resulting solution
was kept at room temperature for 4 days and then 1,1,1-
trifluoro-4-iso-butoxy-3-buten-2-one (1.96 g, 10 mmol)
was added. The resulting mixture was stirred at room
temperature for 0.5 h and then refluxed for 6 h. Then
solvent was removed to dryness. The resulting residue
was tritured with isopropyl ether, the obtained solid was
separated by filtration and recrystallized from a suitable
solvent.
Compound
Ulcer index
ibu
asa
flu
4b
37 5.5
58 6.2
4.83
3
0.5 0.2ꢀ
0.33 0.2ꢀ
0ꢀ
4c
4d
4e
4g
0.7 0.2ꢀ
0.6 0.2ꢀ
0ꢀ
4h
5a
0ꢀ
5b
5c
0.1 0.1ꢀ
0.17 0.1ꢀ
0.5 0.2ꢀ
0.3 0.15ꢀ
0.17 0.1ꢀ
0.5 0.1ꢀ
0ꢀ
5d
5f
5g
5h
vehicle
3.1.1.1. 2-((2-Methylphenyl)amino)-6-trifluoromethyl-
pyridine-3-carbonitrile 4a. Yield 50%. Mp 138–140 °C
1
* P < 0:01 as compared to ibuprofen (ibu), aspirin (asa) and flufenamic
acid (flu).
(cyclohexane). H NMR (CDCl3): d 2.34 (s, 3H, CH3),
7.08–7.95 (m, 7H, aryl and NH). IR (Nujol) 3321, 2233,

4174
M. T. Cocco et al. / Bioorg. Med. Chem. 12 (2004) 4169–4177
1609, 1585 cmꢁ1. Anal. Calcd for C14H10F3N3: C, 60.65;
H, 3.64; N, 15.16. Found: C, 60.60; H, 3.66; N, 15.20.
The resulting solution was heated in boiling water bath
for 6 h. After cooling the reaction mixture was diluted
with water (50 mL) and the formed precipitate was
separated by filtration, dried and recrystallized from a
suitable solvent.
3.1.1.2. 2-((3-Trifluoromethylphenyl)amino)-6-trifluo-
romethylpyridine-3-carbonitrile 4b. Yield 24%. Mp 138–
1
140 °C (cyclohexane). H NMR (CDCl3): d 7.29–8.17
(m, 7H, aryl and NH). IR (Nujol) 3346, 2232, 1620,
1592 cmꢁ1. Anal. Calcd for C14H7F6N3: C, 50.77; H,
2.13; N, 12.69. Found: C, 50.70; H, 2.12; N, 12.71.
3.1.2.1. 2-((2-Methylphenyl)amino)-6-trifluoromethyl-
pyridine-3-carboxylic acid 5a. Yield 88%. Mp 234–
235 °C (toluene). 1H NMR (CDCl3): d 2.20 (s, 3H, CH3),
4.10 (br s, 1H, OH), 6.80-8.27 (m, 6H, aryl), 10.25 (s,
1H, NH). IR (Nujol) 3347, 1672, 1598, 1527 cmꢁ1. Anal.
Calcd for C14H11F3N2O2: C, 56.76; H, 3.74; N, 9.46.
Found: C, 56.81; H, 3.76; N, 9.40.
3.1.1.3. 2-((4-Trifluoromethylphenyl)amino)-6-trifluo-
romethylpyridine-3-carbonitrile 4c. Yield 58%. Mp 184–
1
185 °C (cyclohexane). H NMR (CDCl3): d 7.23–8.03
(m, 6H, aryl), 7.36 (s, 1H, NH). IR (Nujol) 3357, 2232,
1620, 1597, 1581 cmꢁ1. Anal. Calcd for C14H7F6N3: C,
50.77; H, 2.13; N, 12.69. Found: C, 50.72; H, 2.14; N,
12.66.
3.1.2.2. 2-((3-Trifluoromethylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxylic acid 5b. Yield 85%. Mp
1
224–225 °C (toluene). H NMR (DMSO-d6): d 4.20 (br
3.1.1.4. 2-((2,6-Dimethylphenyl)amino)-6-trifluoro-
methylpyridine-3-carbonitrile 4d. Yield 33%. Mp 184–
s, 1H, OH), 7.31–8.47 (m, 6H, aryl), 10.25 (s, 1H, NH).
IR (Nujol) 3321, 1686, 1612, 1592, 1550 cmꢁ1. Anal.
Calcd for C14H8F6N2O2: C, 48.01; H, 2.30; N, 8.00.
Found: C, 48.07; H, 2.32; N, 7.97.
1
185 °C (cyclohexane). H NMR (CDCl3): d 2.22 (s, 6H,
CH3), 6.70 (s, 1H, NH), 7.03–7.92 (m, 5H, aryl). IR
(Nujol) 3321, 2235, 1601, 1581 cmꢁ1. Anal. Calcd for
C15H12F3N3: C, 61.85; H, 4.15; N, 14.43. Found: C,
61.80; H, 4.16; N, 14.40.
3.1.2.3. 2-((4-Trifluoromethylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxylic acid 5c. Yield 78%. Mp
234–235 °C (2-PrOH). 1H NMR (DMSO-d6): d 6.89–
8.75 (m, 6H, aryl), 10.75 (s, 1H, NH), 12.83 (br s, 1H,
OH). IR (Nujol) 3212, 3170, 3136, 3106, 1678, 1637,
1623, 1604 cmꢁ1. Anal. Calcd for C14H8F6N2O2: C,
48.01; H, 2.30; N, 8.00. Found: C, 47.97; H, 2.29; N,
8.04.
3.1.1.5. 2-((3-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carbonitrile 4e. Yield 50%. Mp 152–
1
154 °C (cyclohexane). H NMR (CDCl3): d 2.37 (s, 3H,
CH3), 7.08 (s, 1H, NH), 7.13–7.97 (m, 5H, aryl). IR
(Nujol) 3302, 2234, 1600, 1571 cmꢁ1. Anal. Calcd for
C14H9ClF3N3: C, 53.95; H, 2.91; N, 13.48. Found: C,
53.90; H, 2.92; N, 13.50.
3.1.2.4. 2-((2,6-Dimethylphenyl)amino)-6-trifluoro-
methylpyridine-3-carboxylic acid 5d. Yield 65%. Mp
3.1.1.6. 2-((4-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carbonitrile 4f. Yield 46%. Mp 150–
1
260 dec °C (toluene). H NMR (DMSO-d6): d 2.04 (s,
6H, CH3), 7.04–8.33 (m, 5H, aryl), 9.64 (s, 1H, NH). IR
1
152 °C (cyclohexane). H NMR (CDCl3): d 2.31 (s, 3H,
(Nujol) 3339, 1672, 1588 cmꢁ1
. Anal. Calcd for
CH3), 7.01 (s, 1H, NH), 7.05–7.97 (m, 5H, aryl). IR
(Nujol) 3425, 2226, 1619, 1590, 1541 cmꢁ1. Anal. Calcd
for C14H9ClF3N3: C, 53.95; H, 2.91; N, 13.48. Found: C,
53.99; H, 2.90; N, 13.45.
C15H13F3N2O2: C, 58.07; H, 4.22; N, 9.03. Found: C,
61.80; H, 4.16; N, 14.40.
3.1.2.5. 2-((3-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxylic acid 5e. Yield 75%. Mp
3.1.1.7. 2-((5-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carbonitrile 4g. Yield 70%. Mp 148–
150 °C (toluene). 1H NMR (CDCl3): d 2.31 (s, 3H, CH3),
7.01 (s, 1H, NH), 7.13–7.97 (m, 5H, aryl). IR (Nujol)
3426, 2226, 1619, 1590, 1541 cmꢁ1. Anal. Calcd for
C14H9ClF3N3: C, 53.95; H, 2.91; N, 13.48. Found: C,
54.00; H, 2.90; N, 13.44.
1
234–235 °C (toluene). H NMR (DMSO-d6): d 2.27(s,
3H, CH3), 7.08–8.46 (m, 6H, aryl and NH), 11.10 (s, 1H,
OH). IR (Nujol) 3337, 1668, 1617, 1580, 1541 cmꢁ1
.
Anal. Calcd for C14H10ClF3N2O2: C, 50.85; H, 3.05; N,
8.47. Found: C, 50.90; H, 3.03; N, 8.50.
3.1.2.6. 2-((4-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxylic acid 5f. Yield 90%. Mp
3.1.1.8. 2-((6-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carbonitrile 4h. Yield 83%. Mp 144–
1
224–225 °C (toluene). H NMR (DMSO-d6): d 2.24 (s,
3H, CH3), 7.08–8.49 (m, 5H, aryl), 10.05 (s, 1H, NH).
1
145 °C (cyclohexane). H NMR (CDCl3): d 2.37 (s, 3H,
CH3), 7.08 (s, 1H, NH), 7.13–7.97 (m, 5H, aryl). IR
IR (Nujol) 3183, 3134, 1681, 1615, 1598, 1542 cmꢁ1
.
(Nujol) 3303, 2234, 1600 cmꢁ1
. Anal. Calcd for
Anal. Calcd for C14H10ClF3N2O2: C, 50.85; H, 3.05; N,
8.47. Found: C, 50.81; H, 3.06; N, 8.43.
C14H9ClF3N3: C, 53.95; H, 2.91; N, 13.48. Found: C,
54.00; H, 2.90; N, 13.44.
3.1.2.7. 2-((5-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxylic acid 5g. Yield 97%. Mp
218–220 °C (toluene). H NMR (CDCl3): d 2.35 (s, 3H,
CH3), 7.19–8.43 (m, 5H, aryl), 11.17 (s, 1H, NH). IR
(Nujol) 3333, 1678, 1614, 1587, 1537 cmꢁ1. Anal. Calcd
3.1.2. General procedure for the synthesis of 2-arylamino-
6-trifluoromethylpyridine-3-carboxylic acid 5. The
appropriate pyridine-3-carbonitrile 4 (10 mmol) was
added of 50% aqueous sulfuric acid solution (10 mL).
1

M. T. Cocco et al. / Bioorg. Med. Chem. 12 (2004) 4169–4177
4175
for C14H10ClF3N2O2: C, 50.85; H, 3.05; N, 8.47. Found:
C, 50.80; H, 3.07; N, 8.44.
separated by filtration and recrystallized from a suitable
solvent.
3.1.2.8. 2-((6-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxylic acid 5h. Yield 92%. Mp
214–215 °C (toluene). H NMR (DMSO-d6): d 2.26 (s,
3H, CH3), 7.15–8.43 (m, 6H, aryl and NH), 10.38 (s, 1H,
OH). IR (Nujol) 3384, 3338, 1668, 1618, 1580,
1541 cmꢁ1. Anal. Calcd for C14H10ClF3N2O2: C, 50.85;
H, 3.05; N, 8.47. Found: C, 50.79; H, 3.04; N, 8.51.
3.1.4.1. Ethyl 2-((5-chloro-2-methylphenyl)amino)-6-
trifluoromethylpyridine-3-carboxylate 9g. Yield 80%.
1
1
Mp 116–117 °C (hexane). H NMR (CDCl3): d 1.44 (t,
J ¼ 7:1 Hz, 3H, CH3), 2.37 (s, 3H, CH3), 4.43 (q,
J ¼ 7:1 Hz, 2H, CH2), 7.04–8.42 (m, 5H, aryl), 10.23 (s,
1H, NH). IR (Nujol) 3278, 1696, 1619, 1600, 1590 cmꢁ1
.
Anal. Calcd for C16H14ClF3N2O2: C, 53.57; H, 3.93; N,
7.81. Found: C, 53.62; H, 3.91; N, 7.77.
3.1.3. General procedure for the synthesis of 2-arylamino-
6-trifluoromethylpyridine-3-carboxamides 6. The appro-
priate pyridine-3-carbonitrile 4 (10 mmol) was added of
20% aqueous sodium hydroxide solution (10 mL). The
resulting mixture was heated in boiling water bath for
4 h. After cooling the reaction mixture was diluted with
water (50 mL) and the formed precipitate was separated
by filtration, dried and recrystallized from a suitable
solvent.
3.1.4.2. Ethyl 2-((6-chloro-2-methylphenyl)amino)-6-
trifluoromethylpyridine-3-carboxylate 9h. Yield 80%.
1
Mp 90–91 °C (hexane). H NMR (CDCl3): d 1.44 (t,
J ¼ 7:1 Hz, 3H, CH3), 2.37 (s, 3H, CH3), 4.43 (q,
J ¼ 7:1 Hz, 2H, CH2), 7.04–8.42 (m, 5H, aryl), 10.23 (s,
1H, NH). IR (Nujol) 3318, 3262, 1693, 1616, 1601,
1587 cmꢁ1. Anal. Calcd for C16H14ClF3N2O2: C, 53.57;
H, 3.93; N, 7.81. Found: C, 53.52; H, 3.94; N, 7.84.
3.1.5. General procedure for the synthesis of 6-amino-5-
cyano-6-arylamino-1,1,1-trifluoro-3,5-hexadien-2-ones 3.
The appropriate arylamine (10 mmol) was added to a
solution of 3-amino-3-ethoxypropenenitrile 1 (1.10 g,
10 mmol) in anhydrous acetonitrile (20 mL). The
resulting solution was kept at room temperature for
4 days and then 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-
one (1.96 g, 10 mmol) was added. The resulting mixture
was stirred at room temperature for 0.5 h and then ref-
luxed for 3 h. Then solvent was removed to dryness. The
resulting residue was constituted by a mixture of pyri-
dine-3-carbonitriles 4 and 3,5-hexadienones 3. These
compounds were separated each other by treating the
crude residue with EtOH, 3,5-hexadienones 3 precipi-
tated and were separated by filtration; while pyridine-3-
nitriles 4 were recovered from the ethanolic solution by
removal of solvent.
3.1.3.1. 2-((3-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxamide 6e. Yield 86%. Mp 234–
235 °C (toluene). H NMR (DMSO-d6): d 2.27 (s, 3H,
CH3), 7.11–8.44 (m, 7H, aryl and NH2), 10.43 (s, 1H,
NH). IR (Nujol) 3488, 3165, 3125, 1663, 1619,
1592 cmꢁ1. Anal. Calcd for C14H11ClF3N3O: C, 51.00;
H, 3.36; N, 12.74. Found: C, 50.96; H, 3.37; N, 12.76.
1
3.1.3.2. 2-((5-Chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxamide 6g. Yield 98%. Mp
1
214–215 °C (toluene). H NMR (CDCl3) 2.28 (s, 3H,
CH3), 6.98 (s, 2H, NH2), 7.00–7.93 (m, 5H, aryl), 10.51
(s, 1H, NH). IR (Nujol) 3462, 3184, 1681, 1615 cmꢁ1
.
Anal. Calcd for C14H11ClF3N3O: C, 51.00; H, 3.36; N,
12.74. Found: C, 51.06; H, 3.35; N, 12.70.
3.1.3.3. Methyl 2-((5-chloro-2-methylphenyl)amino)-6-
trifluoromethylpyridine-3-carboxylate 7g. To a solution
of acid 5g (0.83 g, 2.5 mmol) in dry toluene (5 mL),
DMF–DMA (0.66 mL, 5 mmol) was added. The result-
ing solution was heated at 90 °C for 2 h. The solvent was
removed at reduced pressure obtaining ester. Yield 80%.
3.1.5.1. 6-Amino-5-cyano-6-(3-trifluoromethylphenyl-
amino)-1,1,1-trifluoro-3,5-hexadien-2-one 3b. Yield 40%.
Mp 109–110 °C (cyclohexane). 1H NMR (CDCl3): d
5.44 (s, 2H, NH2), 7.05–8.09 (m, 6H, aryl, H-3 and H-4),
7.30 (s, 1H, NH). IR (Nujol) 3482, 3374, 3346, 2230,
1649, 1621, 1592 cmꢁ1. Anal. Calcd for C14H9F6N3O: C,
48.15; H, 2.60; N, 12.03. Found: C, 48.21; H, 2.59; N,
12.05.
1
Mp 99–100 °C (cyclohexane). H NMR (CDCl3): d 2.34
(s, 3H, CH3), 3.95 (s, 3H, OCH3), 7.01–8.37 (m, 5H,
aryl), 10.14 (s, 1H, NH). IR (Nujol) 1693, 1617, 1590,
1542 cmꢁ1. Anal. Calcd for C15H12ClF3N2O2: C, 52.26;
H, 3.51; N, 8.13. Found: C, 52.31; H, 3.50; N, 8.14.
3.1.5.2. 6-Amino-5-cyano-6-(4-trifluoromethylphenyl-
amino)-1,1,1-trifluoro-3,5-hexadien-2-one 3c. Yield 32%.
Mp 84–85 °C (cyclohexane). H NMR (CDCl3): d 5.40
3.1.4. General procedure for the synthesis of ethyl 2-
arylamino-6-trifluoromethylpyridine-3-carboxylates
1
9.
(s, 2H, NH2), 5.74–7.71 (m, 6H, aryl, H-3 and H-4),
11.73 (s, 1H, NH). IR (Nujol) 3485, 3368, 2229, 1646,
1619, 1605, 1575 cmꢁ1. Anal. Calcd for C14H9F6N3O: C,
48.15; H, 2.60; N, 12.03. Found: C, 48.10; H, 2.61; N,
12.00.
The appropriate arylamine (10 mmol) was added to a
solution of ethyl 3-amino-3-ethoxypropenoate 8 (1.60 g,
10 mmol) in anhydrous acetonitrile (20 mL). The
resulting solution was kept at room temperature for
6 days and then 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-
one (1.96 g, 10 mmol) was added. The resulting mixture
was stirred at room temperature for 0.5 h and then ref-
luxed for 3 h. Then solvent was removed to dryness and
the resulting residue was treated with isopropyl ether,
3.1.5.3. 6-Amino-5-cyano-6-(3-chloro-2-methylphen-
ylamino)-1,1,1-trifluoro-3,5-hexadien-2-one 3e. Yield
45%. Mp 114–115 °C (hexane). 1H NMR (CDCl3): d

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M. T. Cocco et al. / Bioorg. Med. Chem. 12 (2004) 4169–4177
2.37, 2.46 (s, 3H, CH3), 5.73–7.97 (m, 5H, aryl, H-3 and
H-4). IR (Nujol) 3302, 2234, 1601, 1571 cmꢁ1. Anal.
Calcd for C14H11ClF3N3O: C, 51.00; H, 3.36; N, 12.74.
Found: C, 51.06; H, 3.35; N, 12.70.
along the long axis of each cage (Opto-Varimex,
Columbus Instruments, Columbus, Ohio). Interruption
of a photocell beam was detected by a counter that
recorded the total number of interruptions caused by
rat’s movements during the test. Motor activity was
recorded for 90 min. Each experimental group consisted
of six rats.
3.2. Pharmacology
Pharmacological tests were conducted in adult male
Sprague–Dawley rats of 250–300 g body/weight. Animal
room was maintained on a 12 light/dark cycle. Food and
water were freely available except for the acute ulcero-
genesis test. All test compounds were suspended in a
solution of methylcellulose (0.5%) and administered ip
or po in a volume of 10 mL/kg body weight. All animal
experimentations have been conducted in accordance
with the guidelines for care and use of experimental
animals of the European Communities Directive (86/
609/EEC; D.L., 27.01.1992, number 116).
3.5. Acute ulcerogenesis
The ability to produce gastric damage was evaluated
according to previously reported method.41–43 Ulcero-
genic activity was evaluated after po administration of
the test compound or one of the three reference drugs at
the dose of 100 mg/kg. Control rats received po admin-
istration of vehicle (suspension of 0.5% methylcellulose).
Food but not water was removed 24 h before adminis-
tration of the test compound. Eight hours after po
administration of the test compound, rats were anaes-
thetized with chloral hydrate and the stomach was ex-
tracted and dipped in 1% formaldehyde solution for
about 15 min and then cut out along its great curvature.
The number and the length of ulcers were detected using
a microscope. The severity of the gastric lesion was
measured along its greatest length (1 mm ¼ rating of 1,
1–2 mm ¼ rating of 2, >2 mm ¼ rating according to their
length in mm). The overall total of length was desig-
nated as the ‘ulcer index’. Each experimental group
consisted of six rats.
3.3. Writhing test
The Writhing test was performed by an ip injection of
1% aqueous acetic acid solution in a volume of 2 mL/kg
body weight. Before acetic acid injection, rats were
placed individually in the test cage (33 w, 56 l, 20 h, cm)
and habituated for 30 min. Stretching movements con-
sisting of arching of the back, development of tension in
the abdominal muscles, elongation of the body and
extension of forelimbs were counted by an experimenter
unaware to the drug treatment for 15 min after the acetic
acid injection. Rats received acetic acid injection 30 or
60 min after ip or po administration of the test com-
pound, respectively. Screening of analgesic activity was
performed after ip administration of compounds at the
dose of 5 mg/kg. Compounds that showed comparable
or greater analgesic activity than the three reference
drugs (aspirin, ibuprofen and flufenamic acid) were
screened at the dose of 10 mg/kg after po administration.
Control rats received ip or po administration of vehicle
(suspension of 0.5% methylcellulose). Ibuprofen, aspirin
and flufenamic acid were used as reference drug at the
same dose of the investigated compounds. The analgesic
activity was expressed also, in terms of percentage of
inhibition:
3.6. Statistics
Mean and standard error of the mean (SEM) of writhes,
locomotor activity values, ulcers index were calculated
for each experimental group. Significant differences were
evaluated by one-way analysis of variance followed
Dunnett’s multiple comparison test.
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