Synthesis of New Pyrrolo[2, 3-b]pyridines as a Potent Inhibitor of Tumour Necrosis Factor Alpha

Article abstract of DOI:10.1002/ardp.200300773

The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cytokines tumor necrosis factor α (TNF-α) and 1L-1β. Accordingly, new pyrrolo[2,3]pyridine derivatives 5 a-d were prepared from 2-amino-3-cyanopyrroles 3 a-d via the intermediate

Full text of DOI:10.1002/ardp.200300773

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 15–19
New Pyrrolo [2,3-b] Pyridines as Inhibitor of TNF-α 15
Khalid Mohammed
Hassan Hilmy
Synthesis of New Pyrrolo[2, 3-b]pyridines as a
Potent Inhibitor of Tumour Necrosis Factor Alpha
Department of Chemistry,
Faculty of Science,
Munoufiya University,
Shebin El-Koom, Egypt
The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cy-
tokines tumor necrosis factor α (TNF-α) and 1L-1β. Accordingly, new pyrrolo[2,3-
]pyridine derivatives 5 a–d were prepared from 2-amino-3-cyanopyrroles 3 a–d via
the intermediate propenylaminopyrroles 4 a–d. Then the compounds 5 a–d were
tested for their ability to inhibit the production of TNF-α in vivo in rats.The most po-
tent compounds 5 a and 5 b possess enhanced ability to inhibit the production of
TNF-α stimulated with bacterial lipopolysaccharide.
Keywords: Pyrrolo[2,3-b]pyridines; TNF-α inhibitor
Received: February 5, 2003; Accepted: July 4, 2003 [FP773]
DOI 10.1002/ardp.200300773
press the release of these pro-inflammatory cytokines
from mononuclear phagocytes and block their actions in
Introduction
Pyrrolo[2,3-b]pyridine derivatives deserve great interest
because of their biological and physiological properties
[1–6].Tumor necrosis factor TNF-α and interleukin IL-1β
are pro-inflammatory cyctokines and play an important
role in inflammatory diseases such as rheumatoid arthri-
tis (RA) [7–10], inflammatory bowel disease (IBD) [11],
psoriasis [12], and Crohn’s disease [13]. The release of
and responses to, TNF-α and 1L-1β are regulated by a
mitogen-activated protein (MAP) kinase, known as p38
[14–19]. Indeed, inhibitors of this kinase potently sup-
a variety of inflammatory cells. This paper reports the
synthesis of some new pyrrolo[2,3-b]pyridines, which
were tested for their ability to inhibit the production of
TNF-α stimulated with bacterial lipopolysaccharide
(LPS).
Results and discussion
The reaction of phenacylmalononitrile derivatives 2 a–d
with aniline under reflux in absolute ethanol in the pres-
(i) Abs. Ethanol, NaOH.
(ii) Abs. Ethanol, Conc. HCl, reflux.
Scheme 1.
Correspondence: Khalid Mohammed Hassan Hilmy, P.O. Box
73, Shoubra, Misr, Cairo, Egypt 11231.Phone:+20 2 270-2517
or +20 10 683-2128, Fax: +20
khaledhilmy@hotmail.com
2 275-5785, e-mail:
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

16 Hilmy
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 15–19
(i) Dry tolune, p-TSO3H, reflux, 2 h
(ii) Abs. Ethanol, Na, reflux, 3 h
Scheme 2.
ence of catalytic amounts of conc. HCl afforded a single
product in each reaction; these compounds were identi-
fied as 1,5-disubtituted 2-amino-3-cyano-pyrroles 3 a–d
(Scheme 1). When 3 a–d were refluxed with ethyl ace-
toacetate in dry toluene and p-TSO3H as a catalyst the
products yielded were identified as propenylaminopyr-
roles 4 a–d (Scheme 2). The structure of compounds
4 a–d were based on the elemental and spectral data.
On the other hand, when compounds 4 a–d were reflux-
ed with sodium ethoxide in absolute ethanol intermo-
lecular cyclization took place to give compounds 5 a–d
(Scheme 2).
Propenylaminopyrroles 4 a–d showed characteristic
bands in their IR spectra at 1661–1654 cm–1 for ester
carbonyl, 2225–2227 cm–1 (CN), and at 3436–2970 cm–1
(NH) which confirmed their structures.The aromatic pro-
tons were resonated at δ 6.70–7.50 producing a multi-
plet together with a broad singlet at δ 9.90–9.97 (1 H)
due to NH proton, and a broad singlet at δ 4.57–5.70
(2 H) for the NH2 protons in the corresponding com-
pounds 3 a–d in the 1H-NMR (CDCl3-d6) spectra of 4 a–d
disappeared. The structures of compounds 5 a–d,
confirmed by the IR spectra exhibited vibrations near
3427–3458 cm–1 (NH2) and 1669–1672 cm–1 (CO) for
Table 1. TNF-α inhibitation data of pyrrolo[2,3-b]pyridine derivatives 5 a–d.
Compound
Range
ng/mL
Mean
ng/mL
I. S. D
Value
Significance
Control
5 a
54–50
4.5–5.2
0–3
70–77
30–70
47.30
4.80
0.13
73.00
50.00
2.25
0.35
0.15
3.60
20.00
–
<0.01
<0.01
>0.05
>0.05
H.S
H.S
N.S
N.S
5 b
5 c
5 d
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 15–19
New Pyrrolo [2,3-b] Pyridines as Inhibitor of TNF-α 17
Figure 1. Effect of compounds 5 a–d adminstrated on LPS (0.1 mg/kg) inducedTNF-α release in male albino rats as a
comparison.
ester carbonyl. A singlet at δ 6.85–6.95 (2 H) for (NH2)
and multiplet near δ 7.09–7.53 was found to be present
because of aromatic protons in the 1H-NMR (DMSO-d6)
spectra of compounds 5 a–d. Compounds 5 a–d were
tested for their ability to inhibit the production of TNF-α
(Table 1).The inhibitory effect of 5 a and 5 b may be due
to the binding of these compounds at the active site of
the transcription or translation processes of the TNF-α
formation. In case of compound 5 c, the methoxy group
may form hydrogen bonding at any location far from the
active site of the enzyme without any effect on the config-
uration and the activity of the active site, while compound
5 d with the fluorine atom may attack the terminal NH2 of
the enzyme similar to Sangers reagent.This may be the
reasons of inactivity of 5 c and 5 d.Figure 1 illustrates the
effect of compunds 5 a–d adminstrated on LPS
(0.1 mg/kg) induced TNF-α release in male albino rats
as a comparison.
recorded on KBr pellets on a Perkin Elmer 1720, Infrared Fou-
1
rier Transform Spectrometer. H-NMR and 13C-NMR spectra
were recorded on a Varian Gemini 2000 NMR spectrometer at
300 MHz for 1H- and 75.5 MHz for 13C-NMR with TMS as an in-
ternal standard. Chemical shifts were recorded in ppm (δ) from
an internal tetramethylsilane standard in deuterochloroform or
deuterodimethyl sulphoxide as specified below. EI spectra
were recorded on a Finnigan MAT SSQ 710. All the above ana-
lytical data were recorded at the University of Southern Den-
mark.The progress of the reaction was monitored by TLC ana-
lytical silica gel plates 60 F254. Merck silica gel (0.040–0.063
mm) was used for column chromatography (Merck, Darmstadt,
Germany). Sovents were reagent grade and, when necessary,
were purified and dried by standards methods. Sovents were
removed under reduced pressure.Melting points were taken on
an electro thermal melting point apparatus and are uncorrect-
ed. Elemental analyses were performed by Atlantic Microlab,
Inc., Norcross, GA, USA.
General Procedure for the preparation of Phenacylmalononi-
triles 2 a–d
To a stirred solution of equimolecular amounts (0.05 mol) of the
substituted phenacyl bromides 1 a–d and malononitrile (3.3 g)
in ethanol (50 mL), a solution of sodium hydroxide (2 g,
0.05 mol) in water (50 mL) was added dropwise.After complete
addition, the reaction mixture was diluted with water (50 mL)
and the precipitate was collected and recrystallized from etha-
nol to give the phenacylmalononitrile derivatives 2 a–d, respec-
tively [20].
Acknowledgement
The author would like to acknowledge Dr.Esmat.B.Sha-
hin for her help with the biological activity work.
Experimental
General Procedure for the preparation 3 a–d
Chemistry
A mixture of aniline (0.01 mol) and 3-5 drops of conc. hydro-
chloric acid was added to a solution of phenacylmalononitrile
derivatives 2 a–d (0.01 mol) in ethanol (50 mL). This mixture
Analytical data were recorded for the compounds described
below using the following general procedures. IR spectra were
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

18 Hilmy
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 15–19
121.84–135.37 (arom.), 157.98 (NHC-), 169.92 (CO); EI MS
m/z 449 (M+ – 1), 451 (M+ + 1).
was refluxed for 5, 7, 9 days to obtain 3 a, 3 b, 3 c, d, respective-
ly and examined byTLC (Silica gel) with CH2Cl2/CH3OH (39:1).
The reaction mixture was then allowed to cool to room temper-
ature. The precipitate was collected by filtration, dried, and re-
crystallized from ethanol. Synthesis of compound 3 a has been
reported previously [21].
3-[3-Cyano-5-(4-methoxy-phenyl)-1-Phenyl-1H-pyrrol-2-ylami-
no]-but-2-enoic acid ethyl ester (4 c)
Yield (62.4 %); mp 160–161 °C; IR (cm–1) (KBr): 3434, 3249,
2979 (NH); 2225 (CN); 1661 (CO); 1H-NMR (CDCl3-d3): 1.20 (t,
3 H, CH2-CH3), 1.90 (S, 3 H, CH3), 3.69 (S, 3 H, CH3); 4.08 (q,
2 H, CH2), 4.71 (s, 1 H, CH), 6.50 (s, 1 H, 4-H); 6.70–7.37 (m,
9 H, Harom.), 9.97 (br s, 1 H, NH); 13C-NMR (CDCl3-d6): 14.36
(CH3), 19.45 (CH3), 55.14 (OCH3), 59.04 (CH2), 88.94 (CH),
90.97 (C3), 108.95 (C4), 115.71 (CN), 123.32–135.69 (arom.),
158.35 (NHC-), 169.90 (CO); EI MS m/z 401 (M+).
2-Amino-5-(4-bromo-phenyl)-1-phenyl-1H-pyrrole-3-carbonitrile
(3 b)
Yield (39 %), mp 200–201 °C; IR (cm–1) (KBr): 3454, 3330,
3217 (NH2), 2206 (CN);1H-NMR (CDCl3-d6):4.57 (s, 2 H, NH2),
6.52 (s, 1 H, 4-H), 6.90–7.70 (m, 9 H, Harom.); 13C-NMR (CDCl3-
d6): 73.60 (C3), 108.65 (C4), 117.25 (CN), 120.56–135.58
(arom.), 147.53 (C2); EI MS m/z 337 (M+). Anal. Calcd.
(C17H12BrN3): C, 60.37; H, 3.58; N, 12.42. Found: C, 60.15; H,
3.70; N, 12.21.
3-[3-Cyano-5-(4-fluoro-phenyl)-1-phenyl-1H-pyrrol-2-ylamino]-
but-2-enoic acid ethyl ester (4 d)
Yield (47.80 %); mp 137–138 °C; IR (cm–1) (KBr): 3435, 3240,
2986 (NH), 2227 (CN), 1655 (CO); 1H-NMR (CDCl3-d3) 1.24 (t,
3 H, CH2-CH3), 1.90 (s, 3 H, CH3), 4.14 (q, 2 H, CH2), 4.30 (S,
1 H, CH), 6.60 (s, 1 H, 4-H), 6.90–7.41 (m, 9 H, Harom.), 9.95 (br
s, 1 H, NH); 13C-NMR (CDCl3-d6): 14.35 (CH3), 19.46 (CH3),
59.12 (CH2), 89.23 (CH), 91.14 (C3), 109.56 (C4), 115.50 (CN),
126.98–135.87 (arom.), 158.12 (NHC-), 163.70 (C-F), 169.92
(CO); EI MS m/z 389 (M+).
2-Amino-5-(4-methoxy-phenyl)-1-phenyl-1H-pyrrole-3-carboni-
trile (3 c)
Yield (53 %), mp 206–207 °C; IR (cm–1) (KBr): 3377, 3250
(NH2), 2203 (CN); 1H-NMR (DMSO-d6):3.72 (s, 3 H, CH3), 4.66
(s, 2 H, NH2), 6.58 (s, 1 H, 4-H), 6.80–7.60 (m, 9 H, Harom.); 13C-
NMR (DMSO-d6): 55.05 (CH3), 70.38 (C3), 106.80 (C4), 117.79
(CN), 123.80–138.82 (arom.), 145.31 (C2);EI MS m/z 289 (M+).
Anal. Calcd. (C18H15N3O. 0.25 H2O): C, 73.51;H, 5.11; N,14.29.
Found: C, 73.88; H, 5.27; N, 14.41.
General procedure for the preparation 5 a–d
To propenylaminopyrroles 4 a–d (5 mmol) in absolute ethanol
(50 mL) metallic sodium (5 mmol) was added.Then the reaction
mixtured was refluxed for 3 h. The reaction was monitored by
TLC with CH2Cl2/CH3OH (39:1).After cooling to room tempera-
ture, white precipitate of compounds 5 a–d were obtained and
then purified by column chromatography CH2Cl2/CH3OH
(39:1).
2-Amino-5-(4-Fluoro-phenyl)-1-phenyl-1H-pyrrole-3-carbonitrile
(3 d)
Yield (46 %); mp 169–170 °C; IR (cm–1) (KBr): 3455, 3339,
3223 (NH2), 2209 (CN);1H-NMR (CDCl3-d3):4.62 (s, 2 H, NH2),
6.57 (s, 1 H, 4-H), 6.80–7.70 (m, 9 H, Harom.); 13C-NMR (CDCl3-
d6): 73.27 (C3); 107.90 (C4), 117.26 (CN), 127.98–135.50
(arom.), 146.72 (C2); EI MS m/z 277 (M+). Anal. Calcd.
(C17H12FN3): C, 73.63; H, 4.36; N,15.15. Found: C, 73.48; H,
4.46; N, 15.21.
4-Amino-6-methyl-1,2-diphenyl-1H-pyrrolo[2,3-b]pyridine-5-car-
boxylic acid ethyl ester (5 a)
Yield (60.1 %); mp 207–208 °C; IR (cm–1) (KBr): 3441 (NH2),
1670 (CO); 1H-NMR (DMSO-d6): 1.31 (t, 3 H, OCH2-CH3), 2.55
(s, 3 H, CH3), 4.29 (q, 2 H, CH2), 6.95 (s, 2 H, NH2), 7.22–7.53
(m, 11 H, Harom. and pyrrole-3-CH); 13C-NMR (DMSO-d6): 14.11
(CH3), 26.88 (CH3), 60.02 (CH2), 100.93, 101.43 105.80,
127.20–136.80 (arom.) 149.93;155.20, 161.81, 168.71;EI-MS
m/z 371 (M+). Anal. Calcd. (C23H21N3O2 1.5 H2O): C, 69.32; H,
5.32; N, 10.54. Found: C, 69.28; H, 5.51; N, 10.37.
General procedure for the preparation 4 a–d
A mixture of 3 a–d (5 mmol) and MeCOCH2CO2Et (5 mmol) in
dry benzene (50 mL) in the presence of P-Me C6H4SO3H
(0.05 g) was azeotropically refluxed for 2 h. The reaction was
monitored by TLC with CH2Cl2/CH3OH (39:1). After cooling to
room temperature, the solvent was evaporated in vacuo, and
the solid product formed, which was then purified by column
chromatography CH2Cl2/CH3OH (39:1) to obtain compounds
4 a–d.
4-Amino-2-(4-bromo-phenyl)-6-methyl-1-phenyl-1H-
pyrrolo[2,3-b]pyridine-5-carboxylic acid ethyl ester (5 b)
3-(3-Cyano-1,5-diphenyl-1H-Pyrrol-2-ylamino)-but-2-enoic acid
ethyl ester (4 a)
Yield (85.80 %); mp 215–216 °C; IR (cm–1) (KBr): 3427, (NH2),
1670 (CO); 1H-NMR (DMSO-d6): 1.33 (t, 3 H, OCH2-CH3), 2.58
(s, 3 H, CH3), 4.31 (q, 2 H, CH2), 6.92 (s, 2 H, NH2), 7.09–7.43
(m, 10 H, Harom. and pyrrole-3-CH); 13C-NMR (DMSO-d6): 14.05
(CH3), 26.84 (CH3), 60.06 (CH2), 100.91, 101.51, 106.40,
122.34–136.72 (arom.) 149.61, 155.31, 161.85, 168.75; EI-MS
m/z 449 (M+ – 1), 451 (M+ + 1) Anal. Calcd. (C23H20BrN3O2 0.75
H2O): C, 59.55; H, 4.35;N, 9.06. Found:C, 59.65;H, 4.4; N, 8.94.
Yield (58.3 %); mp 152–153 °C; IR (cm–1) (KBr): 3436, 3239,
3061, 2970 (NH); 2227 (CN); 1654 (CO); 1H-NMR (CDCl3-d3):
1.25 (t, 3 H, CH2-CH3), 1.80 (s, 3 H, CH3), 4.10 (q, 2 H, CH2),
4.70 (s, 1 H, CH), 6.60 (s, 1 H, 4-H), 7.00–7.50 (m, 10 H, Harom.),
9.99 (br s, 1 H, NH); 13C-NMR (CDCl3-d6): 14.37 (CH3), 19.48
(CH3), 59.10 (CH2), 89.12 (CH), 91.17 (C3), 109.64 (C4), 115.62
(CN), 127.60–135.88 (arom.), 158.24 (NHC-), 169.93 (CO); EI
MS m/z 371 (M+).
4-Amino-2-(4-methoxy-phenyl)-6-methyl-1-phenyl-1H-
pyrrolo[2,3-b]pyridine-5-carboxylic acid ethyl ester (5 c)
3-[5-(4-Bromo-phenyl)-3-cyano-1-phenyl-1H-Pyrrol-2-ylamino]-
but-2-enoic acid ethyl ester (4 b)
Yield (55.30 %); mp 199–200 °C; IR (cm–1) (KBr): 3458, (NH2),
1669 (CO); 1H-NMR (DMSO-d6): 1.32 (t, 3 H, OCH2 CH3), 2.57
(s, 3 H, CH3), 3.76 (s, 3 H, OCH3), 4.34 (q, 2 H, CH2), 6.85 (s,
2 H, NH2), 7.09–7.45 (m, 10 H, Harom. and pyrrole-3-CH); 13C-
NMR (DMSO-d6) :14.08 (CH3), 26.86 (CH3), 59.95 (CH2),
99.79, 101.33, 105.74, 113.75-136.81 (arom.) 149.67, 154.72,
161.95, 168.70; EI-MS m/z 401 (M+). Anal. Calcd. (C24H23N3O3
H2O):C, 68.66;H, 5.48;N, 10.10.Found:C, 68.67;H, 5.59;N, 9.95.
Yield (78 %); mp 145–146 °C; IR (cm–1) (KBr): 3435, 3249,
2982 (NH); 2226 (CN); 1661 (CO); 1H-NMR (CDCl3-d3): 1.25 (t,
3 H, CH2-CH3), 1.90 (s, 3 H, CH3), 4.10 (q, 2 H, CH2), 4.75 (s,
1 H, CH), 6.60 (s, 1 H, 4-H), 6.90–7.40 (m, 9 H, Harom.), 9.90 (br
s, 1 H, NH); 13C-NMR (CDCl3-d6): 14.35 (CH3), 19.45 (CH3),
59.14 (CH2), 89.36 (CH), 91.22 (C3), 109.95 (C4), 115.39 (CN),
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 15–19
New Pyrrolo [2,3-b] Pyridines as Inhibitor of TNF-α 19
wood, S. Patel, C. I. Ragan, P. D. Lesson, J. Med. Chem.,
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4-Amino-2-(4-fluoro-phenyl)-6-methyl-1-phenyl-1H-pyrrolo[2,3-
b] pyridine-5-carboxylic acid ethyl ester (5 d)
Yield (48.60 %); mp 210–211 °C; IR (cm–1) (KBr): 3437 (NH2),
1672 (CO); 1H-NMR (DMSO-d6): 1.30 (t, 3 H, OCH2CH3), 2.58
(s, 3 H, CH3), 4.26 (q, 2 H, CH2), 6.90 (s, 2 H, NH2), 7.10–7.48
(m, 10 H, Harom. and pyrrole-3-CH); 13C-NMR (DMSO-d6): 14.03
(CH3), 26.82 (CH3), 59.89 (CH2), 100.84, 101.43, 105.70,
127.22-136.55 (arom.) 149.87, 155.20, 161.92, 168.63; EI-MS
m/z 389 (M+). Anal. Calcd. (C23H20FN3O2 H2O): C, 67.74; H,
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Lipopolysaccharides from Escherichia coli sero-type 026:B6
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Pre-coated goat anti-rabbit antibodies are used to capture a
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TNF-α conjugate (competitive ligand) and sample or standard
form a competition reaction forTNF-α specific antibody binding
sites. Therefore, as the concentration of TNF-α in the sample
increases, the amount of biotinylated TNF-α captured by the
antibody decreases.With the addition of streptavidin conjugat-
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TNF-α) followed by the addition of the color reagent solution,
the amount of biotinylated TNF-α is detected.This results in an
inverse relationship between Optical Density (OD) and concen-
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