Novel pirfenidone analogues: Synthesis of pyridin-2-ones for the treatment of pulmonary fibrosis

Article abstract of DOI:10.1002/ardp.200600017

A new series of polysubstituted 1-aryl-2-oxo-1,2-dihydropyridine-3- carbonitriles and pyrazolo[3,4-b]pyridine-5-carbonitriles and pyrido[2,3-d]pyrimidine-6-carbonitriles have been synthesized and tested for their antifibrotic activity. Among the tested co

Full text of DOI:10.1002/ardp.200600017

Arch. Pharm. Chem. Life Sci. 2006, 339, 429–436
Y. A. Ammar et al.
429
Full Paper
Novel Pirfenidone Analogues: Synthesis of Pyridin-2-ones for
the Treatment of Pulmonary Fibrosis
Yousry A. Ammar1, Magda M. F. Ismail2, Hend M. El-Sehrawi2, Eman Noaman3, Ashraf H. Bayomi4,
Taghreed Z. Shawer2
1 Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
2 Pharmaceutical Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr City, Cairo,
Egypt
3 Department of Radiation Biology, Natural Center for Radiation Research and Technology, Atomic Energy
Authority, Cairo, Egypt
4 Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt
A new series of polysubstituted 1-aryl-2-oxo-1,2-dihydropyridine-3-carbonitriles and pyrazolo[3,4-
b]pyridine-5-carbonitriles and pyrido[2,3-d]pyrimidine-6-carbonitriles have been synthesized and
tested for their antifibrotic activity. Among the tested compounds, compounds IIc and IVb exhi-
bited higher antifibrotic activity than the standard pirfenidone PD with a reduction of the
hydroxyproline level to 50 and 140 lmol/lung, respectively. However, bicyclic pyridone VIIIb dis-
played a high mortality rate. Detailed syntheses, spectroscopic and biological data are reported.
Keywords: Antifibrotic activity / Bicyclic pyridone / 2-Pyridone / Synthesis> /
Received: January 24, 2006; accepted: April 19, 2006
DOI 10.1002/ardp.200600017
Supporting information for this article is available on the WWW under
http://www.wiley-vch.de/contents/jc_2019/2006/200600017_s.pdf
an excess deposition of collagen in the lung interstitium
[4]. Pirfenidone (PD), 5-methyl-1-phenyl-2-(1H) pyridone, is
an investigational drug that, when administered at 0.5%
(w/w) of the diet, decreased both histological and bio-
chemical evidence of lung fibrosis in a variety of animal
models [5–9].
Herein, we report the preparation and antifibrotic
activity of new PD analogues (monocyclic and bicyclic
derivatives) with the aim of determining the electronic
and/or lipophilic influence of different 1- and 4-substitu-
ents of the pyridin-2-one nucleus on the biological activ-
ity.
Introduction
Interstitial pulmonary fibrosis (IPF) remains a challen-
ging and perplexing problem for both researchers and
clinicians. IPF is a crippling disease with a survival rate of
less than five years. There is no known effective therapy
for treating this disease. The current and most common
therapy involves treatment with corticosteroids. How-
ever, less than 20% of the patients respond to this line of
treatment. Moreover, corticosteroid therapy is associated
with severe side effects [1]. IPF is a result of a wide variety
of injuries to the lung [2]. These are caused by initial
inflammation including production of cytokines, infil-
tration of immune cells, cell-cell interaction, and fibro-
blast proliferation followed by scarring [3]. This results in
Results and discussion
Chemistry
The designed target compounds are depicted in
Schemes 1–3. Substituted 1-aryl-2-oxo-1,2-dihydropyri-
dine-3-carbonitriles IIa–g were obtained through con-
densation and cycloaddition of cyanoacetanilides Ia, b
Correspondence: Dr. Magda Ismail, Pharmaceutical Chemistry, Faculty
of Pharmacy (Girls), Al-Azhar University, Nasr City, Cairo 11754, Egypt.
E-mail: magda_f_ismail@yahoo.com
Fax: +20 24 052-968
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430
Y. A. Ammar et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 429–436
Scheme 1. Synthesis route of compounds IIa–g and IIIa, b.
Scheme 3. Synthesis route of compounds VIIIa, b to X.
dine–3-carbonitriles IVa–c. 4-Styryl pyridone derivatives
Va–c were obtained by reaction of IVa–c with 4-anisalde-
hyde in ethanol/NaOEt, whereas condensation of IVa
with DMF-DMA in xylene [13] produced the 4-enamine
derivative VI. The isoquinoline derivatives VIIa, b were
obtained via Michael addition of IVa, b with (2-benzyl-
idene/4-methoxybenzylidene)-malononitrile in ethanol/
NaOEt with loss of a HCN molecule [14]. Reacting IIb, c
with N2H4.H2O, AC2O [11] or PhNCS [15] led to the addi-
tional fused pyridones e.g. pyrazolo[3,4-b]pyridines
VIIIa, b and pyrido[2,3-d]pyrimidines IXa, b and X, respec-
tively.
Biology
Antifibrotic evaluation
In this report, we aim to provide guidelines for the opti-
mal substitutions on the pyridin-2-one nucleus of the
active lead compound (PD), in order to obtain a highly
efficient drug with the least side effects. Lung content of
hydroxyproline (HP) was determined as a biochemical
index of fibrosis (Figure 1). Results are expressed as lmol
of HP/lung of pulmonary fibrotic models pre- and post-
treated with our synthesized PD analogues: IIb, IIc, IVb,
Vb, and VIIIb.
Scheme 2. Synthesis route of compounds IVa–c to VIIa,b.
with various aldehydes, malononitrile/ethyl cyanoace-
tate in ethanol/piperidine [10]. Refluxing pyridone deriv-
atives IIb, c with triethyl orthoformate in acetic anhy-
Compound IIc, substituted in the 1 position by a 4-
ethoxyphenyl group, displayed the most potent antifi-
brotic activity compared to PD and the control groups.
dride [11] afforded the corresponding ethyl pyridin-2-yl- This is shown in the histopathological study (Figure 2).
formimidates IIIa, b. On the other hand, cyclocondensa- Its counterparts IIb carrying the 4-fluorophenyl group
tion of Ia, b with acetyl acetone or benzoyl acetone in
demonstrated no significant antifibrotic activity.
ethanol using a catalytic amount of piperidine [12], furn-
Taking these results into consideration, we suggest
ished 1-aryl-4-methyl-2-oxo-6-substituted-1,2-dihydropyri- that the 4-ethoxyphenyl group, i.e. electron releasing
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Arch. Pharm. Chem. Life Sci. 2006, 339, 429–436
Novel Pirfenidone Analogues
431
Figure 1. Concentration of 5-hydroxyproline (lmol/lung) in different groups of animals. * Significant difference from control group at
p <0.05. ** Significant difference from control group at p <0.01. *** Significant difference from control group at p <0.001.
Figure 2. Hematoxylin and eosin stain rat lung sections. (A) Dietary control rats; (B) Rats treated with bleomycin; (C) Rats treated
with bleomycin and compound IIc; (D) Rats treated with bleomycin and compound IVb. Scale bars = 100 lm.
group, plays a significant role in the antifibrotic activity drawing property of the F-group might have increased
of IIc (HP = 48.55 lmol/lung) whereas for compound IIb the hydroxyproline level (HP = 216.88 lmol/lung).
having the 4-fluorophenyl moiety, the electron with- Another derivative showing an impressive antifibrotic
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Y. A. Ammar et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 429–436
Table 1. liver- and kidney-function parameters in rats under different treatment conditions.
Group
Control
Fibrotic
IIb
LDH
Urea
Creatinine
ALP
ALT
30.00 l 3.51
100%
21.00 l 3.06
70%
27.50 l 7.50
91.7%
34.00 l 6.00
113%
AST
929.60 l 67.63
100%
802.33 l 228.05
86.3%
885.50 l 10.50
95.3%
853.50 l 25.50
91.8%
40.75 l 1.89
100%
0.58 l 0.03
100%
1060.75 l 107.41
100%
971.00 l 98.88
91.5%
1110.50 l 10.50
104.7%
1046.00 l 59.00
98.6%
182.40 l 14.54
100%
128.67 l 19.37
70.5%
47.67 l 6.74
116.9%
0.43 l 0.03b)
74.1%
34.50 l 0.50a)
84.7%
0.55 l 0.05
94.8%
0.50 l 0.10
86.2%
178.00 l 46.00
97.6%
IIc
37.50 l 3.50
92%
114.00 l 19.00a)
62.5%
IVb
884.33 l 31.21
95.1%
33.33 l 3.33
81.8%
0.60 l 0.10
103.4%
851.00 l 84.70
80.2%
23.33 l 3.33
77.8%
172.67 l 38.32
94.7%
Data is representing six animals per group (mean l S.E.)
a)
Significant difference from at p a 0.05.
Significant difference from control group at, p a 0.01.
b)
Table 2. BALF measurements following intratracheal adminis-
tration of water or bleomycin to rats under different conditions.
compounds. The best results were obtained with com-
pound IVb, as it improved the level of LDH and normal-
ized the protein content. Compound IIc ameliorated the
LDH activity level and the total cell count, while protein
concentration recorded no significant change. Com-
pound IIb decreased the LDH activity level while the total
cell count and the protein content showed a significant
increase compared to the control group (Table 2).
Concerning the requirements for an antifibrotic activ-
ity of this class of compounds, the following conclusions
may be drawn:
Compound IIc is three times more potent than PD,
whereas IVb exhibited an antifibrotic activity ratio of 4:3
compared to the reference PD;
An electron donating group in position 1 is essential
for activity;
Groups
LDH activity Total cell count Protein
Levels (U/L) concentration
(log10)a)
(Macrophages (lg/ml)
+ Neutrophils
+ Eosinophils)
Control
Fibrotic
25.00 l 3.52 5.6 l 0.34
141.67 l 21.1b) 6.24 l 0.25
87 l 5.3
233 l 6.2b)
159 l 5.5b)
200 l 4.0b)
97 l 1.7
Pirfenidone 132.52 l 22.2b) 6.05 l 0.11
IIb
IIc
lVb
38.00 l 6.01 8.60 l 0.38b)
58.00 l 3.54b) 3.65 l 0.124b)
87.67 l 12.7b) 5.80 l 0.044
74 l 1.44
a)
When necessary, data under log10.
Significant difference from control group at p a 0.001.
b)
activity (HP = 119.55 lmol/lung), is the 1-(4-ethoxyphe-
nyl)-4,6-dimethyl-2-oxo-pyridine-3-carbonitrile IVb. Once
again, the electron releasing effect of 4-methyl group
confers a higher antifibrotic activity compared to that of
the PD group (HP = 158.39 lmol/lung). 4-Styryl pyridone
derivative Vb demonstrated a high mortality rate and
was excluded from the biochemical evaluation as well as
the bicyclic PD analogue, pyrazolo[3,4-b] pyridine-5-car-
bonitrile VIIIb.
All parameters of liver and kidney functions of the
fibrotic animals were improved after treatment with the
investigated compounds, however, a significant depres-
sion was observed in aspartate aminotransferase (AST)
activity with compound IIc (Table 1).
Analysis of recovered bronchoalveolar lavage fluid
(BALF) four days after bleomycin administration revealed
a high increase in both lactate dehydrogenase (LDH)
activity and protein content. Administration of PD
reduced such levels, but their values were still very high
compared with the control group. LDH activity levels and
protein contents were greatly decreased by the tested
The lipophilic effect of 4-phenyl group in IIc and IIb is
probably not the sole reason for activity;
Toxicity of compound VIIIb implies that the fusion of
the pyridone nucleus on 3- and 4-positions leads to nega-
tive impact on the activity.
All tested compounds induced a decrease in LDH activ-
ity, total cell count (macrophages, neutrophils, and eosi-
nophils) and protein concentration, and their antifibro-
tic activity is supported by their acceptable results of liver
and kidney functions.
Experimental
Synthesis
Elemental analyses (C, H, N) were performed on Perkin-Elmer
2400 analyzer (Perkin-Elmer, Norwalk, CT, USA) at the Microana-
lytical Unit of Cairo University. All compounds were within
l0.4% of the theoretical values. Melting points were determined
in open capillaries on an Electrothermal LA 9000 Series (Electro-
thermal Engineering Ltd., Essex, UK) and are uncorrected. TLC
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Arch. Pharm. Chem. Life Sci. 2006, 339, 429–436
Novel Pirfenidone Analogues
433
chromatography was performed on pre-coated silica gel 60 F254
plates (Merck, Sofia, Bulgaria). Infrared spectra were recorded on
Pye Unicam SP 1000 IR spectrophotometer (Thermoelectron,
4-(2-(Dimethylamino)vinyl)-1-(4-fluorophenyl)-6-methyl-
2-oxo-1,2-dihydropyridine-3-carbonitrile VI
To a solution of IVa (0.01 mol) in xylene (20 mL), DMF-DMA
(0.01 mol) was added, the mixture was refluxed for 4 h, cooled,
and the resulting solid was filtered off and recrystallized
(Table 3).
1
Egelsbach, Germany). H-NMR spectra were recorded on Varian
Gemini EM-300 MHz NMR spectrophotometer (Varian, Fort Col-
lins, CO, USA). DMSO-d6 was used as solvent, TMS was used as
internal standard and chemical shifts were measured in d ppm.
Mass spectra were recorded on Varian MAT 311-A 70 eV (Varian).
PD was obtained from Marnac, Inc. (Dallas, TX, USA); trans-4-
hydroxy-L-proline (Sigma, Germany), and bleomycin sulphate
(Nippon Kayoku Ltd, Tokyo, Japan). Unless otherwise stated, all
other chemicals and reagents were of analytical grade and were
obtained from Sigma Chemical Co., Germany.
8-Amino-2-(4-fluorophenyl)-6-(4-methoxyphenyl)-3-
methyl-1-oxo-1,2-dihydroisoquinoline-7-carbonitrile VIIa,
8-amino-2-(4-ethoxyphenyl)-3-methyl-1-oxo-6-phenyl-
1,2-dihydroisoquinoline-7-carbonitrile VIIb
A mixture of IVa, b (0.01 mol), 2-(benzylidene/4-methoxybenzyli-
dene)-malononitrile (0.01 mol), and sodium ethoxide (1 g
sodium in 10 mL ethanol), was refluxed in ethanol (30 mL) for
3 h. The reaction mixture was cooled and poured into crushed
ice, acidified with HCl, the solid product was filtered off and
recrystallized (Table 3).
2-Cyano-N-(4-fluoro/ethoxyphenyl) acetamides
Ia, b
According to the reported method [16]
6-Amino-1-(4-fluorophenyl)-4-methyl-2-oxo-1,2-
3-Amino-7-(4-fluoro/ethoxyphenyl)-6-oxo-4-phenyl-6,7-
dihydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitriles
VIIIa, b
A mixture of IIb, c (0.01 mol) and hydrazine hydrate (0.01 mol)
in ethanol (20 mL) was refluxed for 6 h, the mixture was cooled,
and the solid was recrystallized (Table 3).
dihydropyridine-3,5-dicarbonitrile IIa, 6-amino-1-(4-fluoro/
ethoxyphenyl)-2-oxo-4-phenyl-1,2-dihydropyridine-3,5-
dicarbonitriles IIb, c, 6-amino-1-(4-fluorophenyl)-2-oxo-4-
(substituted-phenyl)-1,2-dihydropyridine-3,5-dicarbo
nitriles IId, g, ethyl-2-amino-5-cyano-1-(4-fluorophenyl)-6-
oxo-4-(4-substituted-phenyl)-1,6-dihydropyridine-3-
8-(4-Fluoro/ethoxyphenyl)-2-methyl-4,7-dioxo-5-phenyl-
3,4,7,8-tetrahydro-pyrido[2,3-d]pyrimidine-6-carbonitriles
IXa, b
A suspension of IIb, c (0.01 mol) in acetic anhydride (15 mL ) was
refluxed overnight, cooled, and the resulting solid was filtered
off and recrystallized (Table 3).
carboxylates IIe, f
Equimolar amounts of Ia, b (0.01 mol), appropriate aldehyde
(0.01 mol), and malononitrile or ethyl cyanoacetate (0.01 mol) in
ethanol (30 mL) were refluxed for 3 h using two drops of piperi-
dine as a reaction catalyst. The solid so obtained was filtered off
and crystallized (Table 3).
Ethyl-N-3,5-dicyano-1-(4-fluoro/ethoxyphenyl)-6-oxo-4-
phenyl-1,6-dihydropyridin-2-yl-formimidates IIIa, b
8-(4-Ethoxyphenyl)-4-imino-7-oxo-3,5-diphenyl-2-thioxo-
1,2,3,4,7,8-hexahydro-pyrido[2,3-d]pyrimidine-6-
carbonitrile X
To a solution of IIc (0.01 mol) in ethanol (20 mL), phenyl isothio-
cyanate (0.01 mol) and triethylamine (0.5 mL) were added, the
mixture was refluxed overnight, cooled, and the resulting solid
was filtered off and recrystallized (Table 3).
A
mixture of IIb, c (0.01 mol) and triethyl-ortho-formate
(0.01 mol) in acetic anhydride (15 mL) was refluxed for 4 h, the
mixture was cooled, and the solid product was recrystallized
(Table 3).
1-(4-Fluoro/ethoxyphenyl)-4,6-dimethyl-2-oxo-1,2-
dihydropyridine-3-carbonitriles IVa, b, 1-(4-fluorophenyl)-
4-methyl -2-oxo-6-phenyl-1,2-dihydropyridine-3-
Biological assays
Antifibrotic activity
carbonitrile IVc
Male Swiss albino rats (100–110 g) were obtained from the Egyp-
tian Organization for Biological Products and Vaccines at Giza,
Egypt and were used throughout the experiments. Animals were
housed in cages under good ventilation and illumination condi-
tions; they had unlimited access to water and standard rodent
chow or the same chow containing 0.5% PD (w/w). Animal main-
tenance and treatments were conducted in accordance with the
National Institute of Health Guide for Animal, as approved by
Institutional Animal Care and Use Committee (IACUC). To pro-
duce pulmonary fibrosis, animals received endotracheally (by
the transoral route) a single sublethal dose of bleomycin dis-
solved in 0.9% NaCl (0.9 unit per animal). Control animals were
subjected to the same protocol but received the same volume of
intratracheal saline instead of bleomycin. Tracheal instillation
was carried out under light anesthesia. Seven days after endotra-
To a solution of Ia, b (0.01 mol) in ethanol (20 mL), acetylacetone
or benzoylacetone (0.01 mol) and piperidine (0.5 mL) were added
and the mixture was refluxed for 3 h, cooled, the solid was fil-
tered off and recrystallized (Table 3).
1-(4-Fluoro/ethoxyphenyl)-4-(4-methoxystyryl)-6-methyl-
2-oxo-1,2-dihydropyridine-3-carbonitriles Va, b, 1-(4-
fluorophenyl)-4-(4-methoxystyryl)-2-oxo-6-phenyl-1,2-
dihydropyridine-3-carbonitrile Vc
To a solution of IVa–c (0.01 mol) in ethanol (20 mL), p-anisalde-
hyde (0.01 mol) and sodium ethoxide (1 g sodium in 10 mL etha-
nol) were added, the mixture was refluxed for 3 h, and the solid
so obtained was filtered off and recrystallized (Table 3).
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Arch. Pharm. Chem. Life Sci. 2006, 339, 429–436
Table 3. Physicochemical data of synthesized compounds.
Compound Spectral data
No.
Formulae
C9H7FN2O
C11H12N2O2
Yield
(%)
M.p.
(C8)
Solvent
R
Y
X
Ia
HNMR (DMSO-d6) d 3.85 (s, 2H,
75
158–160
Methanol
–
–
F
CH2CN), 7.38 (m, 4H, Ar-H), 9.5
(s, 1H, NH)
Ib
IR (KBr, cm– 1): 3200 (NH), 2250
(CN), 1700 (CO)
80
50
165–168
340–342
Methanol
Ethanol
–
–
OC2H5
F
IIa
1HNMR (CD3COCD3) d 2.05 (s, 3H, C14H9FN4O
CH3), 7.00–7.70 (m, 6H, 4Ar-H +
2NH2)
CH3
CN
IIb
IIc
1HNMR (DMSO-d6) d 7.38–7.60
(m, 9H, Ar-H), 7.92 (br.s, 2H, NH2,
D2O exchangeable)
C19H11FN4O
70
76
329–331
305–307
Ethanol
Ethanol
C6H5
C6H5
CN
CN
F
1HNMR (DMSO-d6) d 1.38 (t, 3H,
CH3), 4.10 (q, 2H, CH2), 7.11, 7.29
(2d, AB system J = 9Hz, 4H), 7.57
(m, 5H, ArH), 7.80 (br.s, 2H, NH2
D2O exchangeable).
C21H16N4O2
OC2H5
IId
IIe
1HNMR (DMSO-d6) d 7.39–7.66
(m, 8H, Ar-H), 7.83 (br.s, 2H, NH2
D2O exchangeable)
C19H10ClFN4O 60
C21H15ClFN3O3 65
330–332
233–235
Ethanol
Ethanol
C6H4-CL-4
C6H4-CL-4
CN
F
F
1HNMR ( DMSO-d6) d 0.70 (t, 3H,
CH3), 3.77 (q, 2H, CH2), 6.96–7.63
(m, 8H, Ar-H), 9.23 (s, 2H, NH2-D2O
exchangeable)
COOC2H5
IIf
1HNMR ( DMSO-d6 ) d 0.70 (t, 3H, C22H18FN3O4 70
CH3), 3.71 (q, 2H, CH2), 3.87 (s, 3H,
OCH3), 6.88–8.22 (m, 8H, Ar-H),
210–213
320–322
238–240
200–202
Ethanol
Ethanol
Ethanol
Ethanol
C6H4-OCH3-4
COOC2H5
F
8.90 (s, 2H, NH2-D2O exchangeable)
IIg
1HNMR ( DMSO-d6) d 3.77 (s, 3H
OCH3), 3.84 (s, 6H, 2OCH3), 6.87 (s,
2H, Ar-H), 7.41–7.44 (m, 4H, Ar-H),
7.85 (s, 2H, NH2-D2O exchangeable)
IR (KBr, cm– 1: 2222 (CN), 1692
(CO), 1641 (CH=N-), M.S (m/z):
386(M+, 92.3%), 357 (24.7%), 330
(65.1%), 329 (100%)
C22H17FN4O4 75
C22H15FN4O2 65
C6H2-(OCH3)3- CN
3,4,5
F
IIIa
IIIb
C6H5
C6H5
CN
CN
F
1HNMR ( DMSO-d6) d 0.99 (t, 3H,
CH3, OC2H5-CH=), 1.23 (t, 3H, CH3),
3.99–4.14 (m, 4H, 2CH2), 7.07, 7.22
(2d, AB system, J = 8Hz, 4H), 7.54–
7.63 (m, 5H, ArH), 8.29 (s, 1H,
CH=N)
C24H20N4O3
60
OC2H5
IVa
IVb
1HNMR ( DMSO-d6) d 1.95 (s, 3H, C14H11FN2O
CH3) 2.36 (s, 3H, CH3), 6.40 (s, lH,
95
70
180–183
209–211
Ethanol
Ethanol
CH3
CH3
–
–
F
C(5) H ), 7.32–7.35 (m, 4H, Ar-H)
1HNMR (DMSO-d6 ) d 1.35 (t, 3H,
CH3), 1.97 (s, 3H, CH3), 2.37 (s, 3H,
CH3), 4.07 (q, 2H, CH2), 6.42 (s, 1H,
C(5)H), 7.03, 7.18 (2d, AB system,
J = 9Hz, 4H)
C16H16N2O2
OC2H5
IVc
Va
1HNMR (DMSO-d6) d 2.06 (s, 3H,
CH3), 6.51 (s, 1H, C (5) H), 7.12–
7.58 (m, 9H, Ar-H)
C19H13FN2O
50
190–193
230–232
Ethanol
Benzene
C6H5
CH3
–
–
F
F
1HNMR(DMSO-d6) : d 2.03 (s, 3H, C22H17FN2O2 70
CH3), 3.83 (s, 3H, OCH3), 6.43 (s,
1H, C(5)H), 6.80–7.69 (m, 10H,
8Ar-H+ 2CH=)
Vb
IR (KBr, cm– 1): 2217 (CN), 1660
(CO), MS (m/z): 386 (M+, 41.8),
360 (37.9), 279 (29.9)
C24H22N2O3
65
245–248
Benzene
CH3
–
OC2H5
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Arch. Pharm. Chem. Life Sci. 2006, 339, 429–436
Table 3. Continued ...
Novel Pirfenidone Analogues
435
Compound Spectral data
No.
Formulae
Yield
(%)
M.p.
(C8)
Solvent
Benzene
Ethanol
R
Y
–
–
X
F
Vc
VI
1HNMR(DMSO-d6) : d 3.75 (s, 3H, C27H19FN2O2 70
OCH3), 6.20 (s, 1H, C(5)H), 7.00–
240–243
C6H5
–
7.98 (m, 15H, 13Ar-H + 2CH=)
1HNMR(DMSO-d6) : d 2.26 (s, 3H, C17H16FN3O
CH3), 2.99 (s, 6H, N(CH3)2), 3.91
(d, 1H, CH-N), 5.02 (d, 1H, CH=CH),
6.51 (s,1H, C(5)H), 7.16–7.75 (m,
4H, Ar-H)
75
130–132
F
VIIa
1HNMR (DMSO-d6) : d 1.93 (s, 3H, C24H18FN3O2 64
CH3), 3.84 (s, 3H, OCH3), 6.56 (s, 1H,
170–173
Benzene
C6H4-OCH3 –4
–
F
C(7)H), 6.69 (s, 1H, C(6)H), 6.77–
7.55 (m, 8H, Ar-H), 8.30 (s, 2H, NH2)
VIIb
IR (KBr, cm– 1): 3466, 3389 (NH2), C25H21N3O2
2207 (CN), 1654 (CO)
80
25
130–133
300–302
Benzene
Ethanol
C6H5
–
–
–
OC2H5
F
VIIIa
IR (KBr, cm– 1): 3460, 3308, 3227 C19H12FN5O
(NH, NH2), 2213 (CN), 1626 (CO),
MS (m/z): 345 (M+, 12.79), 302
(10.49), 237 (62.95), 111 (100)
VIIIb
IXa
1HNMR (DMSO-d6) : d 1.36 (t, 3H, C21H17N5O2
CH3), 4.05–4.12 (q, 2H, CH2), 5.35
(s, 1H, C(5)H), 7.02–7.63 (m, 9H,
Ar-H), 8.42 (s, 2H, NH2)
30
310–313
280–282
279–281
Ethanol
Ethanol
Ethanol
–
–
–
–
–
–
OC2H5
IR (KBr, cm– 1): 3444, 3337 (NH2), C21H13FN4O2 50
2219 (CN), 1660(CO), MS (m/z): 372
(M+, 6.40), 330 (100), 137 (20.19),
95 (94.13), 75 (50.99)
F
IXb
1HNMR (DMSO-d6) : d 1.36 (t, 3H, C23H18N4O3
CH3), 2.16 (s, 3H, CH3), 4.08 (q, 2H,
CH2), 7.02, 7.18 (2d, AB system, J =
9Hz, 4H), 7.32–7.47 (m, 5H, Ar-H),
12.62 (s, 1H, NH)
30
OC2H5
X
1HNMR (DMSO-d6): 1.05 (t, 3H,
CH3), 4.08 (q, 2H, CH2), 6.95–7.67
(m, 14H, Ar-H), 8.78 (s, 2H, 2NH)
C28H21SN5O2 50
100–103
Benzene
–
–
OC2H5
cheal bleomycin and/or compounds administration, the ani-
mals were scarified after light anesthesia with diethyl ether to
collect blood by heart puncture. Bronchoalveolar lavage was per-
formed and lungs were weighed and processed separately for
biochemical and histological studies as indicated below. The
seventh day after bleomycin administration was selected as the
approximate time for maximal rate of collagen synthesis. The
animals were randomly divided into eight groups: group 1: vehi-
cle; group 2: vehicle + bleomycin; group 3: PD + bleomycin;
group 4: IIb + bleomycin; group 5: IIc + bleomycin; group 6:
IVb + bleomycin; group 7: Vb + bleomycin; group 8: VIIIb + ble-
omycin. Treatments (vehicle, PD, or tested compounds) were
administered orally by gavage on a daily basis (at 9:00 a.m.)
started three days prior to intratracheal instillation of bleomy-
cin or saline till the end of the experiment (seven days post instil-
lation). The tested compounds were administered by stomach
intubation tube. The oral dose was 0.04 g/day for seven doses for
the tested compounds and 0.5% (w/w) for PD, given as repeated
doses in a final volume of 1 mL of 1% carboxymethyl cellulose
suspension (vehicle).
ments. The separated plasma from heparinized blood was used
for determination of liver function parameters including, ala-
nine aminotransferase (ALT) [17], AST [18], alkaline phosphatase
(ALP) [19], and LDH [20]. Creatinine and urea concentrations
were estimated according to the methods of Lustgasten et al. [21]
and Kaplan et al. [22], respectively.
Preparation of lung tissue
For the determination of the indicators of pulmonary fibrosis,
each animal was anesthetized with light anesthesia, the chest
was opened, and blood was collected by heart puncture at the
seventh day post tested-compounds treatment, a thoracotomy
was performed, and the trachea was exposed and cannulated.
The right bronchus was ligated, and the right lung was removed,
weighed, frozen in liquid nitrogen, and stored at –808C until
the determination of hydroxyproline content. The left lung was
inflated with 10% neutral-buffered formalin to a pressure of
20 cm H2O for 1 h. The trachea was then ligated, and the lung
was removed and placed in formalin. Sections from the upper,
middle, and lower portions of the lung were dehydrated and
embedded in paraffin, and 5 lm sections were cut and stained
with hematoxylin and eosin for histological evaluation.
Measurement of the biochemical parameters
Animals were fasted overnight prior to samples collection. Sam-
ples were collected after seven days from beginning of treat-
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the addition of 0.6 mL of chloramines T, 0.6 mL of Ehrlich’s
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barbital (300 mg/kg, i. p.), the lungs and trachea were removed
en bloc, and two successive 5.0 mL aliquots of warm (378C) phos-
phate-buffered saline (PBS, pH 7.4) were infused and slowly with-
drawn from the lungs through a cannula inserted into the tra-
chea. Bronchoalveolar lavage fluid (BALF) volumes routinely
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onto microscope slides using a Shandon Cytospin 2 centrifuge
(Shandon Southern Products Limited, Runcorn, Cheshire, Eng-
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cells/animal. Absolute cell numbers were log10 transformed for
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kit (Sigma LDH procedure No. DG1340-UV), and the BALF protein
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