Anti-inflammatory, analgesic and antipyretic 4,6-disubstituted 3-cyano- 2-aminopyridines

Article abstract of DOI:10.1016/S0223-5234(99)80057-9

4,6-diaryl and 4,6-aryl-indolyl substituted 3-cyano-2-aminopyridines were synthesized and submitted to evaluation for their anti-inflammatory, analgesic and antipyretic activity. The electronegativity of the substituents and their displacement on the 4- o

Full text of DOI:10.1016/S0223-5234(99)80057-9

Eur. J. Med. Chem. 34 (1999) 245−254
© Elsevier, Paris
245
Original article
Anti-inflammatory, analgesic and antipyretic 4,6-disubstituted
3-cyano-2-aminopyridines
Fedele Mannaa*, Franco Chimentia, Adriana Bolascoa, Bruna Bizzarria, Walter Filippellib,
Amelia Filippellib, Luigi Gagliardic
aDipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università di Roma ‘La Sapienza’,
P. le Aldo Moro 5, 00185 Rome, Italy
bIstituto di Farmacologia e Tossicologia, Facoltà di Medicina e Chirurgia, II Università di Napoli,
Via Costantinopoli 16, 80138 Naples, Italy
cLaboratorio di Chimica del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
(Received 29 October 1997; revised 6 October 1998; accepted 8 October 1998)
Abstract – 4,6-diaryl and 4,6-aryl-indolyl substituted 3-cyano-2-aminopyridines were synthesized and submitted to evaluation for their
anti-inflammatory, analgesic and antipyretic activity. The electronegativity of the substituents and their displacement on the 4- or 6-aryl ring
of the 4,6-diaryl-3-cyano-2-aminopyridine nucleus (3a–q) influenced the anti-inflammatory activity which was higher in the presence of
electron-realising groups. The introduction of the indol-3-yl substituent in the 4-position of the 3-cyano-2-aminopyridine nucleus (6a–x)
increased the anti-inflammatory and analgesic power, but there was no evidence of the relationship among the electronic characteristic of the
substituents, their displacement on the 6-phenyl ring and the activity. Conversely, the displacement of the 2-hydroxyphenyl group in the
4-position (4a–e) and of the indol-3-yl group in the 6-position (8h–w) decreased the anti-inflammatory activity. All derivatives did not show
any significative antipyretic activity. © Elsevier, Paris
anti-inflammatory activity / analgesic activity / antipyretic activity / 4,6-diarylsubstituted 3-cyano-2-aminopyridine synthesis /
4,6-aryl-indolylsubstituted 3-cyano-2-aminopyridine synthesis
1. Introduction
relationship. The indolic nucleus was also inserted in the
3-cyano-2-aminopyridine structure on account of its pres-
ence in the most potent anti-inflammatory derivatives,
including indomethacin.
In this work we report the synthesis of 4,6-diaryl and
4,6-aryl-indolyl substituted 3-cyano-2-aminopyridines
and the evaluation of their anti-inflammatory, analgesic
and antipyretic activity.
4,6-disubstituted 3-cyano-2-aminopyridines were not
much studied: only some 4,6-diaryl or 4,6-aryl-hete-
rocyclyl-3-cyano-2-aminopyridines were tested for anti-
microbial activity and were found to be active [1, 2].
Furthermore, 6-indolyl-3-cyano-2-aminopyridines were
synthesized but not pharmacologically tested [3]. In a
previous paper [4], we reported the synthesis of 6-(2-
hydroxyphenyl)-4-(2,4-dimethoxyphenyl)-3-cyano-2-ami-
nopyridine 3w’ and of the corresponding 4-(2,4-
dichlorophenyl) derivative 3u’, which were ten times less
active than indomethacin.
2. Chemistry
The 6-(2-hydroxyphenyl)-4-(R,R’-phenyl)-3-cyano-2-
aminopyridines (3a–q) and 6-(R,R’-phenyl)-4-(2-
hydroxyphenyl)-3-cyano-2-aminopyridines (4a–e) were
synthesized as previously reported [4] by reaction of
substituted 1,3-diaryl-2-propen-1-ones with malononi-
trile. 1,3-diaryl-2-propen-1-ones were obtained by con-
densation of o-hydroxyacetophenone with substituted
These results led us to study a new series of 3-cyano-
2-aminopyridine derivatives and their structure–activity
*Correspondence and reprints

246
The 6-(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-amino-
pyridines (6a–x) were synthesized by reaction of
3-(indol-3yl)-1-(R,R’-phenyl)-2-propen-1-ones (5a–x)
with malononitrile in the presence of ammonium acetate
in EtOH (figure 2) (table III). The low yields were
probably due to the lone pair present on the indolic
nitrogen atom that decreased the electrophilic character
of the acceptor in the Michael addition. The indolic
chalcones (5a–x) were obtained by condensation of
indol-3-carboxyaldehyde with substituted acetophenones
in presence of piperidine as catalyst in EtOH.
The IR spectra of 6a–x showed multiple bands at
3500–3300 cm–1 (NH2), at 2220–2210 cm–1 (CN) and at
1640–1580 cm–1 (C=N–). The NMR spectra showed a
singlet at ppm 11.9–11.7 attributable to the NH proton
and multiplets for the aromatic, indolic and aminic
protons in the region ppm 9.2–6.7. In most cases, the
signals of the aminic protons were superimposed to that
of the aromatic protons.
The 6-(indol-3-yl)-4-(R,R’-phenyl)-3-cyano-2-amino-
pyridines (8h–w) were synthesized by reaction of
Figure 1.
6-(2-hydroxyphenyl)-4-(R,R’-phenyl)-3-cyano-2-
aminopyridines (3a–q) and 6-(R-phenyl)-4-(2-hydroxyphenyl)-
3-cyano-2-aminopyridines (4a–e).
benzaldehydes (1a–q) and of o-hydroxyphenylcar-
boxaldehyde with substituted acetophenones (2a–e)
(figure 1) (tables I and II).
The IR spectra of 3a–q and 4a–e showed bands at
3400–3200 cm–1 (NH2), 2220–2210 cm–1 (CN) and
1640–1580 cm–1 (C=N–). The NMR spectra showed a
broad singlet at ppm 13.6–13.4 attributed to the hydroxy-
lic proton and a multiplet of the aromatic and aminic
protons at ppm 8.1–6.7.
Figure 2.
pyridines (6a–x).
6-(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-amino-

247
Table I. Physical constants for the 6-(2-hydroxyphenyl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines (3a–q).
Compound
R
R1
Yield (%)
M.p. (°C)
Formula
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
3-NO2
4-NO2
4-N(CH3)2
3-OCH3
5-OCH3
28
26
34
15
28
38
33
35
47
29
37
39
49
51
11
22
43
235
257–260
270
243–245
265
285
C18H12N3OCl
C18H12N3OCl
C18H12N3OCl
C18H12N3OBr
C18H12N3OBr
C18H12N3OBr
C19H15N3O
260
275–277
253–256
203
209–210
263–265
225–227
220–221
177–179
265–268
228–230
C19H15N3O
C19H15N3O
C19H15N3O2
C19H15N3O2
C19H15N3O2
C18H12N4O3
C18H12N4O3
C18H17N4O
2-OCH3
2-COH3
C20H17N3O3
C20H17N3O3
3-(R,R’-phenyl)-1-(indol-3-yl)-2-propen-1-ones (7h–w)
with malononitrile in the presence of ammonium acetate
(figure 3) (table IV). The 7h–w compounds were obtained
by condensation of 3-acetylindole with substituted ben-
zaldehydes.
The IR spectra of 8h–w showed bands at
3500–3300 cm–1 (NH2), 2220–2210 cm–1 (CN) and
1640–1580 cm–1 (C=N–). The NMR spectra showed
signals of the NH proton at ppm 11.9 and of the aromatic,
indolic and aminic protons at ppm 8.0–6.7.
3. Pharmacology
All derivatives were investigated with respect to their
anti-inflammatory, analgesic and antipyretic activity, at
dose of 50 mg/kg corresponding to their ED50 values.
The (4-methyl)-, (3-methoxy)- and (2,4-dimethoxy)-4-
phenyl derivatives (3i, 3k and 3w’ respectively), charac-
terized by electron-releasing or alkyl substituents, were
the most potent anti-inflammatory compounds of this
series showing an activity approximately ten times lower
than that of indomethacin (table V). Strong electron-
withdrawing substituents (Cl or NO2) decreased the
Table II. Physical constants for the 6-(R-phenyl)-4-(2-
hydroxyphenyl)-3-cyano-2-aminopyridines (4a–e).
Compound R2
Yield (%) M.p. (°C) Formula
4a
4b
4c
4d
4e
2-OCH3
30
32
26
31
33
> 350
> 350
> 350
> 350
> 350
C19H15N3O2
C19H15N3O2
C19H15N3O2
C19H15N3O
C19H15N3O
3-OCH3
4-OCH3
2-CH3
4-CH3

248
Table III. Physical constants for the 6-(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-aminopyridines (6a–x).
Compound
R
R1
Yield (%)
M.p. (°C)
Formula
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6r
6m
6n
6s
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2-NO2
3-NO2
4-NO2
2-OH
4-OH
4-Cl
9
10
8
74
9
8
226
271
276
223–25
280
C20H13N4Cl
C20H13N4Cl
C20H13N4Cl
C20H13N4Br
C20H13N4Br
C20H13N4Br
C21H16N4
> 280
10
8
220–224
235–238
240–245
222–225
235
215–218
210
254
C21H16N4
C21H16N4
17
88
25
13
9
16
15
10
36
24
30
7
C21H16N4O
C21H16N4O
C21H16N4O
C20H13N5O2
C20H13N5O2
C20H13N5O2
C20H14N4O
C20H14N4O
C20H13N4Cl2
C20H13N4Cl2
C22H18N4O2
C22H18N4O2
280
270–273
206–209
216–218
208–210
220–223
263–266
6t
H
6u
6v
6w
6x
2-Cl
2-Cl
2-OCH3
3-OCH3
5-Cl
4-OCH3
4-OCH3
20
potency, (3-chloro) and (4-chloro)-4-phenyl derivatives
(3b, 3c) being almost inactive, the bromine derivatives
3d, 3e showed a transient activity, as 2,5-dimethoxy 3q.
The displacement of the 2-hydroxyphenyl substituent
in the 4-position of the 3-cyano-2-aminopyridine (4a–e)
produced a decrease of potency and a transient activity
(table VI). All compounds 3 and 4 did not show signifi-
cant analgesic and antipyretic activities.
The replacement of the aryl in the 4-position with a
â-indolic group gave rise to compounds 6, characterized
by a better anti-inflammatory activity as compared to that
of the corresponding 3 (table VII), however, the influence
of the 6-phenyl substituents on the structure/activity
relationship was not so clear. Thus, while their electronic
characteristic did not influence the anti-inflammatory
activity, their position significantly influenced the po-
tency, the most potent derivatives being the 6-(3-phenyl)
monosubstituted compounds 6b, 6h, 6m and the disub-
stituted ones 6t–u. Furthermore, many derivatives
showed a transient activity (6f, 6j–k, 6r, 6v). The
displacement of the indolic group from 4 to 6 position of
the 3-cyano-2-aminopyridine nucleus (8h, 8w) decreased
the anti-inflammatory activity (table VIII).
Compounds 6h, 6m and 6x also showed a good
analgesic activity which was ten times lower than in-
domethacin (table IX), while the 8 derivatives were
nearly inactive. Furthermore, the 6 and 8 compounds had
a very low antipyretic properties.
The new 3-cyano-2-aminopyridine derivatives (3, 6)
generally showed a more potent anti-inflammatory activ-
ity in comparison with the corresponding 2-(1H)-
pyridone derivatives synthesized in our previous

249
search [4]. Furthermore, the activity of 3-cyano-2-
aminopyridine derivatives (3, 6) was increased by
electron-releasing substituents on the 4-aryl group and on
the 6-aryl group respectively, while the activity of 4,6-
diaryl-3-cyano-2-(1H)-pyridones was markedly increased
by electron-withdrawing substituents.
4. Conclusions
The presence of amine group in the 2-position of the
pyridine ring gave rise to an increment of the anti-
inflammatory activity and affected also the pharmacologi-
cal answer to the influence of the substituents on 4 or
6-aryl rings in comparison with the corresponding 4,6-
diaryl-3-cyano-2-(1H)-pyridones.
The electronegativity of the substituents and their
displacement on the 4-aryl ring of the 4,6-diaryl-3-cyano-
2-aminopyridines 3a–q influenced the anti-inflammatory
activity which was higher in the presence of electron-
releasing groups. Furthermore, a decrease of potency was
observed when the 2-hydroxyphenyl group was inserted
in the 4-position of the 3-cyano-2-aminopyridine nucleus
4a–e. All 3 and 4 derivatives did not show any interesting
analgesic and antipyretic activities.
The introduction of the indol-3-yl group in the
4-position of the 3-cyano-2-aminopyridine nucleus gave
rise to derivatives 6a–x which exhibited better anti-
inflammatory and analgesic properties, but there was no
evidence of the relationship between the electronic nature
of the substituents, their displacement on the 6-phenyl
ring and the activity. In addition, the introduction of the
Figure 3. 6-(indol-3-yl)-4-(R,R’-phenyl)-3-cyano-2-aminopy-
ridines (8h–w).
Table IV. Physical constants of the 6-(indol-3-yl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines (8h–w).
Compound
R
R1
Yield (%)
M.p. (°C)
Formula
8h
8w
H
3-CH3
4-OCH3
7
6
203–205
208–210
C21H16N4
C22H19N4O2
2-OCH3

250
Table V. Anti-inflammatory activity of the 6-(2-hydroxyphenyl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines (3a–q).
Compound
mg/kg/os
Volume of edema (mL ± S.E.)
a
0
1 h
2 h
4 h
Controls
Indomethacin
Carrag. 1 % Sol.
5
50
50
50
50
50
50
50
50
50
50
50
50
50
50
0.8 ± 0.1
1.0 ± 0.1
0.9 ± 0.1
0.9 ± 0.1
1.1 ± 0.1
1.0 ± 0.1
0.7 ± 0.1
0.8 ± 0.1
1.0 ± 0.1
1.1 ± 0.1
1.1 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
1.1 ± 0.1
0.8 ± 0.1
1.1 ± 0.1
0.9 ± 0.1
0.9 ± 0.1
1.2 ± 0.1
0.7 ± 0.1
1.2 ± 0.1
0.9 ± 0.1
0.8 ± 0.1
1.2 ± 0.1 (+50)
1.2 ± 0.1 (–50)
1.2 ± 0.1 (–25)
1.2 ± 0.1 (–25)
1.5 ± 0.1 (0)
1.2 ± 0.1 (+50)
1.2 ± 0.1 (–50)
1.2 ± 0.1 (–25)
1.3 ± 0.1 (0)
1.5 ± 0.1 (+87)
1.2 ± 0.1 (–71)
1.4 ± 0.1 (–28)
1.6 ± 0.1 (0)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
1.5 ± 0.1 (0)
1.7 ± 0.1 (–14)
1.6 ± 0.1 (–14)
1.3 ± 0.1 (–28)
1.3 ± 0.1 (–28)
1.5 ± 0.1 (–28)
1.5 ± 0.1 (–43)
1.4 ± 0.1 (–57)
1.3 ± 0.1 (–43)
1.3 ± 0.1 (–57)
1.6 ± 0.1 (–28)
1.4 ± 0.1 (–14)
1.6 ± 0.1 (–28)
1.5 ± 0.1 (–14)
1.5 ± 0.1 (–14)
1.8 ± 0.1 (–14)
1.8 ± 0.1 (+157.1)
1.5 ± 0.1 (–132.1)
1.4 ± 0.1 (–101.6)
1.3 ± 0.1 (–94.6)
1.2 ± 0.1 (–50)
0.9 ± 0.1 (–50)
1.1 ± 0.1 (–25)
1.3 ± 0.1 (–25)
1.3 ± 0.1 (–50)
1.3 ± 0.1 (–50)
1.2 ± 0.1 (–25)
1.2 ± 0.1 (–50)
1.4 ± 0.1 (–25)
1.1 ± 0.1 (–25)
1.4 ± 0.1 (–25)
1.2 ± 0.1 (–25)
1.2 ± 0.1 (–25)
1.4 ± 0.1 (–50)
1.2 ± 0.1 (+ 71.4)
1.3 ± 0.1 (–63.1)
1.2 ± 0.1 (–38.1)
1.0 ± 0.2 (–46.0)
1.3 ± 0.1 (–25)
1.0 ± 0.1 (–25)
1.2 ± 0.1 (0)
1.4 ± 0.1 (0)
1.4 ± 0.1 (–25)
1.3 ± 0.1 (–50)
1.2 ± 0.1 (–25)
1.2 ± 0.1 (–50)
1.5 ± 0.1 (0)
1.2 ± 0.1 (0)
1.5 ± 0.1 (0)
1.3 ± 0.1 (0)
1.2 ± 0.1 (–25)
1.5 ± 0.1 (–25)
1.4 ± 0.1 (+100.0)
1.5 ± 0.2 (–75.0)
1.3 ± 0.1 (–55.6)
1.2 ± 0.1 (–50.0)
50
50
50
b
Controls
Carrag. 1 % sol.
5
50
50
b
Indomethacin
b
3w’
3u’
b
a
b
Times from the administration; published data [4]; percentages (in parantheses) indicate a variation of edema volume calculated in
comparison with control values.
indol-3-yl group in the 6-position of the 3-cyano-2-
aminopyridine nucleus markedly decreased potency
(8h–w). Furthermore, compounds 6 and 8 did not display
a significant antipyretic activity.
Table VI. Anti-inflammatory activity of the 6-(R-phenyl)-4-(2-hydroxyphenyl)-3-cyano-2-aminopyridines (4a–e).
Compound
mg/kg/os
Volume of edema (mL ± S.E.)
a
0
1 h
2 h
4 h
Controls
Indomethacin
Carrag. 1 % Sol.
5
0.8 ± 0.1
1.0 ± 0.1
0.8 ± 0.1
0.9 ± 0.1
0.7 ± 0.1
1.1 ± 0.1
1.0 ± 0.1
1.3 ± 0.1 (+62)
1.2 ± 0.1 (–60)
1.1 ± 0.1 (–60)
1.1 ± 0.1 (–60)
0.9 ± 0.1 (–60)
1.4 ± 0.1 (–40)
1.2 ± 0.1 (–60)
1.5 ± 0.1 (+87)
1.2 ± 0.1 (–71)
1.2 ± 0.1 (–43)
1.2 ± 0.1 (–57)
1.0 ± 0.1 (–57)
1.4 ± 0.1 (–57)
1.3 ± 0.1 (–57)
1.7 ± 0.1 (+112)
1.2 ± 0.1 (–78)
1.4 ± 0.1 (–44)
1.3 ± 0.1 (–55)
1.2 + 0.1 (–44)
1.6 ± 0.1 (–44)
1.5 ± (0.1) (–44)
4a
4b
4c
4d
4e
50
50
50
50
50
a
Times from the administration; percentages (in parantheses) indicate a variation of edema volume calculated in comparison with control
values.

251
Table VII. Anti-inflammatory activity of the 6-(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-aminopyridines (6a–x).
Compound
mg/kg/os
Volume of edema (mL ± S.E.)
a
0
1 h
2 h
4 h
Controls
Indomethacin
Carrag. 1 % Sol.
5
0.9 ± 0.1
0.9 ± 0.1
0.8 ± 0.1
0.8 ± 0.1
0.9 ± 0.1
0.9 ± 0.1
0.9 ± 0.1
0.7 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
0.8 ± 0.1
1.1 ± 0.1
0.7 ± 0.1
0.8 ± 0.1
0.7 ± 0.1
0.8 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
1.0 ± 0.1
0.8 ± 0.1
1.1 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
1.3 ± 0.1 (+44)
1.1 ± 0.1 (–50)
1.0 ± 0.1 (–25)
1.0 ± 0.1 (–50)
1.1 ± 0.1 (–50)
1.2 ± 0.1 (–25)
1.2 ± 0.1 (–25)
0.9 ± 0.1 (–50)
1.2 ± 0.1 (–25)
1.3 ± 0.1 (–25)
1.1 ± 0.1 (–25)
1.3 ± 0.1 (–50)
0.9 ± 0.1 (–50)
1.1 ± 0.1 (–25)
0.9 ± 0.1 (–50)
1.0 ± 0.1 (–50)
1.1 ± 0.1 (–25)
1.4 ± 0.1 (–25)
1.2 ± 0.1 (–50)
1.1 ± 0.1 (–25)
1.4 ± 0.1 (–50)
1.1 ± 0.1 (–25)
1.3 ± 0.1 (–25)
1.5 ± 0.2 (+66)
1.1 ± 0.1 (–67)
1.2 ± 0.1 (–30)
1.0 ± 0.1 (–50)
1.2 ± 0.1 (–50)
1.2 ± 0.1 (–50)
1.3 ± 0.1 (–33)
1.0 ± 0.1 (–50)
1.3 ± 0.1 (–33)
1.3 ± 0.1 (–50)
1.1 ± 0.1 (–50)
1.4 ± 0.1 (–33)
1.0 ± 0.1 (–50)
1.2 ± 0.1 (–33)
1.0 ± 0.1 (–50)
1.1 ± 0.1 (–50)
1.2 ± 0.1 (–50)
1.5 ± 0.1 (–33)
1.3 ± 0.1 (–50)
1.2 ± 0.1 (–33)
1.5 ± 0.1 (–50)
1.1 ± 0.1 (–50)
1.3 ± 0.1 (–50)
1.6 ± 0.2 (+78)
1.1 ± 0.1 (–71)
1.4 ± 0.1 (–14)
1.1 ± 0.1 (–57)
1.4 ± 0.1 (–29)
1.4 ± 0.1 (–28)
1.6 ± 0.1 (0)
1.2 ± 0.1 (–28)
1.5 ± 0.1 (–14)
1.3 ± 0.1 (–57)
1.3 ± 0.1 (–28)
1.5 ± 0.1 (–43)
1.2 ± 0.1 (–28)
1.4 ± 0.1 (–14)
1.2 ± 0.1 (–28)
1.1 ± 0.1 (–57)
1.3 ± 0.1 (–43)
1.5 ± 0.1 (–43)
1.4 ± 0.1 (–43)
1.3 ± 0.1 (–28)
1.7 ± 0.1 (–14)
1.1 ± 0.1 (–57)
1.3 ± 0.1 (–57)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6r
6m
6n
6s
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
6t
6u
6v
6w
6x
a
Times from the administration; percentages (in parantheses) indicate a variation of edema volume calculated in comparison with control
values.
5. Experimental protocols
5.1.1. General procedure for compounds 3a–q: 6-(2-
hydroxyphenyl)-4-(R,R’-phenyl)-3-cyano-2-
aminopyridines
5.1. Chemical synthesis
The compounds 3a–q were synthesized according to
Melting points were determined in open glass capillar-
ies with a Büchi 510 apparatus and are uncorrected. The
IR spectra were recorded on a Perkin Elmer 218 B
instrument, NMR spectra on a Varian EM-390 instru-
ment, using TMS as internal standard, and were listed in
table X. Results of elemental analysis were within ±0.4%
of the theoretical values.
the method described in [4]. IR (KBr) cm–1: 3400–3200
1
(NH2), 2200–2210 (CN), 1640–1580 (C=N–). H-NMR
(DMSO-d6) ppm: 13.6–13.4 (s, 1H, OH); 8.0–6.7 (m, 10
or 11H, aromatic and aminic protons).
Table VIII. Anti-inflammatory activity of the 6-(indol-3-yl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines (8h–w).
Compound
mg/kg/os
Volume of edema (mL ± S.E.)
a
0
1 h
2 h
4 h
Controls
Indomethacin
8h
Carrag. 1 % Sol.
5
50
50
0.9 ± 0.1
1.0 ± 0.1
1.2 ± 0.1
0.8 ± 0.1
1.3 ± 0.1 (+44)
1.2 ± 0.1 (–50)
1.5 ± 0.1 (–25)
1.1 ± 0.1 (–25)
1.5 ± 0.1 (+67)
1.2 ± 0.1 (–67)
1.6 ± 0.1 (–33)
1.2 ± 0.1 (–33)
1.7 ± 0.1 (+89)
1.2 ± 0.1 (–75)
1.8 ± 0.1 (–25)
1.4 ± 0.1 (–25)
8w
a
Times from the administration; percentages (in parantheses) indicate a variation of edema volume calculated in comparison with control
values.

252
Table IX. Analgesic activity of the 6-(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-aminopyridines (6a–x).
Compound
mg/kg/os
Mean no. of writhes ± S.E. within % decrease compared with the
25 min after treatment
control group
Controls
Indomethacin
Acetic acid
5
51.2 ± 10.8
26.3 ± 7.6
37.1 ± 1
–48.6
–27.7
–16.9
–19.7
–10.9
–1.95
– 9.3
–34.5
–46.4
–30.8
–30.1
–48.2
–27.5
–26.7
–56.8
–38.8
–26.9
–11.3
–32.4
–26.7
–22.6
–47.6
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6r
6m
6n
6s
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
42.5 ± 10.6
41.09 ± 9.3
45.6 ± 3.5
50.2 ± 3.5
46.4 ± 9.1
33.5 ± 9.2
27.6 ± 9.9
35.4 ± 6.8
35.8 ± 6.5
26.5 ± 11.2
37.1 ± 9.4
37.5 ± 8.4
22.1 ± 12
31.3 ± 9.8
37.4 ± 9.8
45.4 ± 7.6
34.6 ± 7.6
37.5 ± 8.1
39.6 + 5.4
26.2 ± 8.1
6t
6u
6v
6w
6x
5.1.2. General procedure for compounds 2a–e: 3-
(R,R’-phenyl)-1-(2-hydroxyphenyl)-2-propen-1-ones
0.1 mol of suitable acetophenone, dissolved in 30 mL
of EtOH, in the presence of 30% NaOH (15 mL), were
added dropwise to ice-cooled salicylic aldehyde
(0.1 mol). The reaction mixture was allowed to stand for
24 h at room temperature, then neutralized with 15%
HCl. The resulting solid was washed with petroleum
Table X. (a) 1H-NMR ppm data of the 6-(2-hydroxyphenyl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines (3a–q), 6-(R,R’-phenyl)-4-(2-
hydroxyphenyl)-3-cyano-2-aminopyridines (4a–e), 6-(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-aminopyridines (6a–x), 6-(indol-3-yl)-4-(R,R’-
phenyl)-3-cyano-2-aminopyridines (8h–w). (b) IR (KBr) cm–1 data of the 6-(2-hydroxyphenyl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines
(3a–q), 6-(R,R’-phenyl)-4-(2-hydroxyphenyl)-3-cyano-2-aminopyridines (4a–e), 6-(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-aminopyridines
(6a–x), 6-(indol-3-yl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines (8h–w).
OH
Ar, Ar’, NH2
NH indolic
Ar, Indolic, NH2
3a–q
4a–e
6a–x
8h–w
13.6–13.4 singlet
13.6–13.4 singlet
8.1–6.7 multiplet
8.1–6.7 multiplet
11.9–11.7 singlet
11.9 singlet
9.2–6.7 multiplet
8.0–6.7 multiplet
Table X. (b) IR (KBr) cm–1 data of the 6-(2-hydroxyphenyl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines (3a–q), 6-(R,R’-phenyl)-4-(2-
hydroxyphenyl)-3-cyano-2-aminopyridines (4a–e), 6-(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-aminopyridines (6a–x), 6-(indol-3-yl)-4-(R,R’-
phenyl)-3-cyano-2-aminopyridines (8h–w).
NH2
CN
C=N–
3a–q
4a–e
6a–x
8h–w
3400–3200
3400–3200
3500–3300
3500–3300
2220–2210
2220–2210
2220–2210
2220–2210
1640–1580
1640–1580
1640–1580
1640–1580

253
ether, then crystallized from 1:1 toluene/cyclohexane.
Physical and spectroscopic characteristics of these com-
pounds were comparable with known data [5, 6, 7, 8, 9,
10, 11, 12].
cm–1: 3100–3000 (NH), 1645–1630 (C=O) and
1590–1560 (CH=CH). H-NMR (DMSO-d6) ppm: 11.9
(s, 1H, NH), 8.4–8.2 (m, 4 or 5H, aromatic protons),
8–7.6 (2d, 2H, CH=CH, J = 18 Hz) and 7.8–7.1 (m, 5H,
indolic protons).
1
5.1.3. General procedure for compounds 4a–e: 6-
(R,R’-phenyl)-4-(2-hydroxyphenyl)-3-cyano-2-
aminopyridines
5.1.7. General procedure for compounds 8h–w: 6-
(indol-3-yl)-4-(R,R’-phenyl)-3-cyano-2-aminopyridines
0.011 mol of chalcone 5, 0.022 mol of malononitrile
and 0.088 mol of ammonium acetate were dissolved in
80 mL of EtOH and refluxed at 110 °C, with stirring, for
24 h. The resulting solid was washed with warm EtOH
and acetone. IR (KBr) cm–1: 3500–3300 (NH2),
2220–2210 (CN) and 1640–1580 (C=N). 1H-NMR
(DMSO-d6) ppm: 11.9 (s, 1H, NH) and 8.0–6.7 (m, 11 or
12H, aromatic, aminic and indolic protons).
A solution of chalcone 2 (0.022 mol), malononitrile
(0.044 mol) and ammonium acetate (0.0176 mol) in
80 mL of EtOH was refluxed under stirring, at 110 °C for
24 h. The solid separated was collected by filtration and
washed with warm EtOH and acetone. IR, (KBr) cm–1:
3400–3200 (NH2), 2220–2210 (CN) and 1640–1580
1
(C=N–). H-NMR (DMSO-d6) ppm: 13.6–13.4 (s, 1H,
OH); 8.1–6.8 (m, 10 or 11H, aromatic and aminic
protons).
5.2. Pharmacological evaluation
5.1.4. General procedure for compounds 5a–x: 3-
(indol-3-yl)-1-(R,R’-phenyl)-2-propen-1-ones
5.2.1. Anti-inflammatory activity
0.11 mol of indol-3-carboxyaldehyde and 0.11 mol of
suitable acetophenone, in the presence of 0.6 mL of
piperidine, were dissolved in 80 mL of anhydrous EtOH
and refluxed for 24 h at 110 °C under N2, with stirring.
The reaction mixture was poured into ice and neutralized
with diluted acetic acid; the solid was filtered and
crystallized from EtOH. IR (KBr) cm–1: 3100–3000
(NH), 1645–1630 (C=O) and 1590–1560 (CH=CH).
1H-NMR (DMF-d7) ppm: 12–11 (s, 1H, NH), 9–8.2 (m,
4 or 5H, aromatic protons), 8–7.5 (2d, 2H, CH=CH, J =
18 Hz) and 7.9–7.2 (m, 5H, indolic protons).
Anti-inflammatory activity was evaluated by the paw
edema test, using carrageenin (1%) on albino rats of both
sexes (pregnant rats excluded) weighing 180–250 g; each
group comprised five animals. The compounds were
administered by oral route (gavage) 30 min before the
carrageenin, at a dose of 50 mg/kg. The volume of the
rat’s paw was measured 1, 2 and 4 h after the adminis-
tration of carrageenin, and indomethacin was used as
reference compound (5 mg/kg).
The reported values were the average of five determi-
nations ± S.E. and the percentage of activity was calcu-
lated in comparison with controls. The significance was
calculated by Student’s t-test for coupled values (tables V,
VI, VII and VIII).
5.1.5. General procedure for compounds 6a–x: 6-
(R,R’-phenyl)-4-(indol-3-yl)-3-cyano-2-aminopyridines
A solution of indolic chalcone 5 (0.056 mol), malono-
nitrile (0.056 mol) and ammonium acetate (0.38mol) in
80 mL of anhydrous EtOH was refluxed at 110 °C, with
stirring, for 24 h. The resulting solid was collected by
filtration, washed with water and purified by repeated
washing with warm EtOH. IR (KBr) cm–1: 3500–3300
5.2.2. Analgesic activity
Analgesic activity was determined by means of acetic
acid test (writhing test) [13] carried out on Mus musculus.
Each group was composed of five animals of both sexes,
pregnant females excluded (body weight 18–25 g). The
compounds were administered orally at a dose of
50 mg/kg and indomethacin was used as reference com-
pound (5 mg/kg).
1
(NH2), 2220–2210 (CN) and 1640–1580 (C=N–). H-
NMR (DMSO-d6) ppm: 11.9–11.7 (s, 1H, NH) and
9.2–6.7 (m, 11 or 12H, aromatic, indolic and aminic
protons).
The animals were injected intraperitoneally with
0.25 mL /mouse of 0.5% aqueous acetic acid solution and
writhes were counted during the subsequent 25 min. An
oral dose of a test compound was administered 30 min
before the injection. The mean number of writhes for
each experimental group ± S.E. and percent decrease
compared with the control group (five mice not treated
with test compounds) were calculated. Results are given
in table IX.
5.1.6. General procedure for compounds 7h–w: 3-
(R,R’-phenyl)-1-(indol-3-yl)-2-propen-1-ones
0.07 mol of substituted benzaldehyde in 80 mL of
EtOH were added to 0.07 mol of acetylindole, in the
presence of 10% NaOH and were allowed to stand at
room temperature for 24 h, with stirring. The reaction
mixture was neutralized with 15% HCl; the resulting
solid was filtered and crystallized from EtOH. IR (KBr)

254
5.2.3. Antipyretic activity
Antipyretic activity was determined by measuring the
variation in the body temperature in albino rats of both
sexes (pregnant rats excluded) weighing 180–200 g. Fe-
ver was induced by a 10% solution of brewer’s yeast
(10 mL /kg) subcutaneously administered. Each group
was composed of five animals.
The reported values were the average of five determi-
nations ± S.E. and the percentage of activity was calcu-
lated in comparison with basal values. The significance
was calculated by the Student’s t-test for coupled values.
The compounds tested were administered by oral route
at a dose of 50 mg/kg and indomethacin was used as
reference compound (5 mg/kg).
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