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Synthesis Acute Toxicity and Analgesic Activity of New Derivatives of Pyrrole.

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Arch. Pharm. Chem. Life Sci. 2006, 339, 670 – 674
Full Paper
Synthesis, Acute Toxicity, and Analgesic Activity of New
Derivatives of Pyrrole
Nikolai Danchev1, Atanas Bijev2, Diana Yaneva2, Stanislava Vladimirova2, and Irina Nikolova1
1
2
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Medicinal University, Sofia, Bulgaria
Department of Organic Synthesis and Fuels, University of Chemical Technology and Metallurgy, Sofia,
Bulgaria
Ten pyrrole derivatives (including six new compounds) were synthesized and evaluated as potential platform for analgesic agents’ development. Acute intraperitoneal toxicity and analgesic
activity studies (acetic acid writhing test) were performed on mice with acetylsalicylic acid used
as a reference substance. Products 3c, 3d, 3e, and 3h exhibited a dose-dependant activity demonstrating 1.5 to 2.5-fold better protections than the reference. The most prospective compounds
comprised salicylic acid moieties, whose 4-substituted derivatives were related to lower acute
toxicity and considerable activity. 4-[3-(Ethoxycarbonyl)-2-methyl-5-(3,4-dimethoxy-phenyl)-1Hpyrrol-1-yl]-2-hydroxy-benzoic acid 3c was pointed out as the most prospective substance due to
its lower acute toxicity (378 mg/kg body weight, intraperitoneally) and highest analgesic activity
(up to 89.3% protection) in a dose range of 1/10 to 1/40 parts of LD50.
Keywords: Acute toxicity / Analgesic activity / Nonsteroidal inhibitors / Pyrroles /
Received: July 24, 2006; Accepted: September 21, 2006
DOI 10.1002/ardp.200600116
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the
most widely prescribed and effective therapy for decreasing pain and inflammation. Unfortunately, most of the
classical NSAIDs are associated with gastrointestinal side
effects, whereas the popularity of the new generation of
selective cyclooxygenase-2 (COX-2) inhibitors is being
undermined by escalating doubts about possible cardiovascular risks [1, 2]. The development of new active entities with lower toxicity and fewer adverse effects is a
priority of the current global research.
Due to the favorable presence of a pyrrole moiety in
known active structures, pyrrole derivatives provoked a
special interest in the search for new active NSAIDs in
two generalized classes: carboxylic acids and analogs of
Correspondence: Assoc. Prof. Dr. Atanas Bijev, DSi Department of Organic Synthesis and Fuels, University of Chemical Technology and Metallurgy, 8 ”Kl. Ohridski” Blvd., 1756 Sofia, Bulgaria.
E-mail: a.bijev@uctm.edu
Fax: +359 2 8685488
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
effective COX-2 inhibitors [3–6]. Recently, the effects on
nociception of nine substituted N-pyrrolylcarboxylic
acids, previously reported as anti-inflammatory agents,
were examined in male Wistar rats by the Randall-Selitto
paw-pressure test [7]. In the current study we applied the
acetic acid writhing test in mice for evaluation of more
diverse pyrrole patterns as a possible structural platform
for NSAIDs development. This alternative pharmacological approach is a widely used model for visceral pain [8].
Results and discussion
Chemistry
The design of the target products was based on the architecture of the tricyclic COX-2 inhibitors with celecoxib
(CAS 169590-42-5) as a typical representative, relying on
the bioisosteric replacement of the five-membered central ring by pyrrole heterocycle. The pyrrole synthesis
was accomplished by Paal–Knorr cyclization [9, 10]
between intermediately prepared 1,4-dicarbonyl compounds 2 and amino partners according to Scheme 1.
Arch. Pharm. Chem. Life Sci. 2006, 339, 670 – 674
Synthesis, Toxicity, Analgesic Activity of Pyrroles
671
Reagents and conditions: (i) CH3COCH2COR3, Na, dry EtOH, 20–258C, 2 h; 2 – intermediate 1,4-dicarbonyl compounds were used in (ii) without isolation; (ii) R4NH2, glacial acetic acid (3a–c, 3f, 3g, 3i, 3j)
or DMSO (3d, 3e, 3h), 608C (3a–c, 3d, 3e, 3h) or boiling temperature (3f, 3g), 1–12 h.
Scheme 1. Synthesis of compounds 3a-j via Paal–Knorr cyclization.
The reaction partners were selected to provide some
specific structural elements typical for a number of popular active molecules, such as salicylic, isonicotinamide,
and pyrazolinone moieties or amino acids residues. The
choice of these structural features was additionally motivated by the significant anti-inflammatory activity (up to
100% inhibition of the carrageenan-induced edema)
registered recently for closely related compounds synthesized in our laboratory according to a similar reaction
scheme [11]. In both studies, the intermediate 1,4-dicarbonyl compounds were prepared and used in the next
cyclization step without isolation. Four of the compounds of interest, 3a, 3b, 3d, 3e, have also been synthesized in our laboratory before [12], but had not been evaluated so far; six, 3c, 3f–3j, are new compounds.
The structure elucidation of the new compounds was
based on the relevant 1H-NMR and IR spectral characteristics interpreted in Experimental, and the purity of the
products was confirmed both by TLC and elemental analyses in an acceptable error range € 0.4%.
Pharmacological studies
Adult male albino mice were used for acute intraperitoneal toxicity and analgesic activity studies. Acetylsalicylic acid (popular as aspirin [CAS 50-78-2]) served as a
reference analgesic compound.
An acute toxicity test was performed as a first stage of
the pharmacological evaluations. The registered results
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Table 1. Acute toxicity (LD50) of the compounds 3a–j after intraperitoneal administration in mice.
Compounds
LD50 (mg/kg)
(range of values)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
Aspirin
192.1 (172 211)
742.9 (656 731)*
378.4 (325 421)*
185.6 (171 199)
181.7 (163 201)
182.6 (160 203)
195.1 (171 212)
201.4 (182 219)
508.2 (451 567)*
761.8 (682 837)*
197.1 (171 223)
n = 8–10 animals per tested compound; * p f 0.05 compared to
acetylsalicylic acid
(in the form of LD50 values after i.p. (intraperitoneally)
administration) are presented in Table 1.
Compounds 3b and 3j were proved to be the least toxic
within the series, whereas the rest of the tested pyrrole
derivatives showed moderate toxicity with about 3.5-fold
lower LD50 values, which were found to be comparable
with those of the reference.
Standard acetic acid induced writhing test in mice was
performed for assessment of analgesic activity [13]. Mice
were treated intraperitoneally at dose ranges of 1/10 to 1/
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Arch. Pharm. Chem. Life Sci. 2006, 339, 670 – 674
Figures 1. The most active compounds 3c, 3d, 3e, and 3h bear salicylic acid moieties.
80 parts of LD50. The number of writhing and stretching
within the observation period was counted. The percentage protection was calculated using the ratio: (control
mean – treated mean)6100/control mean. Aspirin was
applied as a reference in 20 and 50 mg/kg b.w. dose (1/4
and 1/10 parts of LD50, respectively).
All tested compounds except 3g were proved to exhibit
analgesic activity in doses 1/10 parts of LD50 (Table 2).
Discussion
The nociceptive response, which is brought about by chemical peritoneal irritation, and the related release of
prostaglandins [14] is reduced by cyclooxygenase (COX)
inhibitors, including a number of pyrrole derivatives.
The later are known to comprise both popular drugs,
such as tolmetin (CAS 64490-92-2), zomepirac (CAS
64092-48-4), clopirac (CAS 42779-82-8), ketorolac (CAS
74103-06-3), etc. and products of contemporary research
[3–6, 15, 16]. The compliance with the architecture of tricyclic COX-2 inhibitors together with their pyrrole affiliation defined the target structures as potential NSAIDs
with probable analgesic activity. The choice of aspirin as
a reference compound was motivated by the capability of
salicylic acid derivatives to alleviate pain by virtue of a
peripheral action whereat effects on the CNS may also be
involved [17].
Compounds 3c, 3d, 3e, and 3h (presented in Fig. 1)
decreased the number of writhings dose dependently
demonstrating 1.5 to 2.5-fold better protection than the
reference aspirin tested at the same experimental conditions. Comparative analysis of the complex pharmacological results disclosed some general relationships on the
background of the structural diversity. The most pronounced effects manifested by 3c and 3e justified the
inclusion of salicylic acid moieties R4 in the target molecules, whose 4-substituted derivatives of 2-hydroxybenzoic acid were related to lower acute toxicity in combination with considerable activity. The alternative derivatives bearing amino acids residues at the same position
were inactive.
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Table 2. Effects of compounds 3a–j on acetic acid induced writhing in mice.
Group
(n)
Dose
(mg/kg) (i.p.)
Vehicle
3a
3a
3b
3b
3c
3c
3c
3c
3d
3d
3d
3e
3e
3e
3f
3f
3g
3h
3h
3h
3i
3i
3j
3j
Aspirin
Aspirin
Aspirin
19.2
9.6
20.0
10.0
38.0
19.0
8.5
4.2
18.6
9.3
4.6
18.7
9.1
4.5
18.3
9.2
19.5
20.2
10.0
5.0
51.0
25.5
76.1
38.0
10.0
20.0
50.0
Number
of writhings
(mean € SEM)
29.1 € 2.8
15.6 € 2.3*
27.9 € 3.7
16.3 € 3.1*
25.3 € 4.2
3.1 € 1.1*
8.2 € 1.6*,a)
21.2 € 2.8*,b)
27.9 € 3.6
14.9 € 4.8*
19.6 € 3.1*,a)
26.5 € 3.9
6.2 € 2.1*
22.4 € 3.2*,a)
33.3 € 4.1
21.8 € 3.1*
30.4 € 4.3
29.5 € 4.9
12.5 € 2.1*
20.3 € 3.6*,a)
29.8 € 4.2
7.8 € 1.6*,a)
24.3 € 4.1
22.6 € 3.2*
31.1 € 3.7
29.2 € 3.7
23.1 € 2.6
18 € 2.1
Protection
(%)
46.4
4.1
43.9
1.3
89.3
71.8
27.1
4.1
48.8
32.6
8.9
78.7
23.0
–14.4
25.1
–3.1
–1.3
57.0
30.2
–2.4
73.2
16.5
22.3
–6.8
–0.3
20.6
38.1
n = 7–8 animals; * p f 0.05 compared to vehicle treated group;
a)
p f 0.05 compared to 1/10 LD50; b) p f 0.05 compared to 1/20
LD50.
The introduction of a CH3O group into the 5-aryl ring
proved to be favorable, since it decreased the acute toxicity 3afi3b, whereas the presence of the second methoxy
group increased the analgesic activity 3bfi3c.
Isonicotinamide derivative 3i showed also notable
dose-dependently analgesic effects (up to 73% protection)
in compliance with the high anti-inflammatory activity
recently registered for four analogous compounds in our
previous study [11].
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Arch. Pharm. Chem. Life Sci. 2006, 339, 670 – 674
Conclusion
The complex findings define a platform for further developments of analgesic agents based on the general formula 3, favoring structures with salicylic acid or isonicotinamide moieties in R4. Compound 3c could serve as the
most prospective target for further optimizations due to
its highest activity (up to 89.3% protection in a dose
range of 1/10 to 1/40 parts of LD50) on the background of
acceptable acute i.p. toxicity (LD50 378.4 mg/kg).
Experimental
Synthesis, Toxicity, Analgesic Activity of Pyrroles
673
acute intraperitoneal toxicity and analgesic activity studies. The
animals were housed individually, water and food being supplied ad libitum; animal room temperature 228C € 38C; humidity
30%; lighting schedule 12 h light/dark cycle. Prior to administration, animals fasted for one day. All the experimental procedures were conducted according to the NIH Guidelines of the
Care and Use of Laboratory animals (USA) and approved by Ethical Committee for Care and Management of Laboratory Animals
at Faculty of Pharmacy – MU, Sofia, Bulgaria.
Acute intraperitoneal toxicity
The compounds were suspended using Tween 80. Acute intraperitoneal toxicity studies were performed according to OECD
Guideline 425 “Up and Down procedure” with computer program calculations [18] (n = 8–10 animals per tested compound).
General
Analgesic activity – Acetic acid- induced writhing test
All commercial chemicals used as starting materials and
reagents in this study were purchased from Merck (Darmstadt,
Germany) and were of reagent grade. Yields refer to purified products and were not optimized. The synthesis progress and the
purity of the products were controlled by TLC (Silica gel 60 F254,
on aluminium sheets; Merck). Melting points were determined
in open capillaries on a AZ9003MK4 apparatus, (Electrothermal,
Southend-on-Sea, England). IR spectra were registered on Specord 71 IR (Carl-Zeiss, Jena, Germany) and Ehinox Bruker (Faenlanden, Switzerland) (for compounds 3i) and are expressed in
cm–1. 1H-NMR spectra were recorded with a Bruker-Spectrospin
spectrometer WM250 (250 MHz, Faenlanden, Switzerland) in
the indicated solvent (TMS as internal standard). The values of
the chemical shifts are expressed in ppm, and the coupling constants (J) are expressed in Hertz (Hz). All COOH and NH protons
were D2O exchangeable. The chemical IUPAC names were created with ACD/ Name Version 2.51.
This test was accomplished according to the modified method of
Koster et al. [13]. The writhes were induced by intraperitoneal
administration of 0.8% (v/v) acetic acid solution (10 mL/kg).
Thirty minutes prior to the administration of acetic acid, the
animals were treated intraperitoneally with the test substances,
vehicle or reference acetylsalicylic acid. Writhing and stretching
responses, defined as constriction of the abdomen with stretching of the hind limbs, were counted for 30 min after the i.p.
injection. The data were calculated using Student-Fischer t test.
Product characteristics
(Presented: chemical name; 1H-NMR (solvent); IR in KBr [cm-1]; m.
p. (8C); yield (%); Rf at 208C)
4-[3-(Ethoxycarbonyl)-2-methyl-5-(3,4-dimethoxyphenyl)-1H-1-pyrrolyl]-2-hydroxybenzoic acid 3c
1
General synthetic procedures for the preparation of
compounds 3c, 3f–j
To 11 mmol of relevantly substituted 1,4-dicarbonyl compound
2 (prepared according to [11]) dissolved in 10 mL glacial acetic
acid (for 3c, 3f, 3g, 3i, and 3j) or in 5 mL DMSO (for 3h) was added
the corresponding amino partner R4NH2: 13 mmol (2.15 g) L-bphenyl-alanine in the synthesis of 3f, 13 mmol (1.71 g) DL-leucine
in the synthesis of 3g; 10 mmol (1.53 g) 4-amino-2-hydroxy-benzoic acid in the syntheses of 3c and 3h, 10 mmol (1.37 g) isonicotinohydrazide in the synthesis of 3i; 10 mmol (2.03 g) 4-amino1,5-dimethyl-2-phenyl-1,2-dihydropyrazole-3-one in the synthesis of 3j. The mixture was stirred with the aid of a magnetic stirrer for 2-6 h at 608C (for 3c and 3h) or for 1-5 h at reflux (for 3f,
3g, 3i, and 3j) (TLC control). The products separated as crystal
solids after dilution with water. Compounds 3c and 3h were purified by dissolving into 2% aqueous NaOH and precipitation
with 5% HCl followed by a second precipitation with hexane
from ether. Compounds 3f, 3g, 3i, and 3j were purified by recrystallization from ethanol. 3g was additionally purified with ether
and hexane.
Pharmacological tests
Animals
Male albino mice, line H, 25–30g b.w. (Institute of Physiology,
Bulgarian Academy of Sciences, Sofia, Bulgaria) were used for
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
H-NMR (CDCl3): 1.38 (t, J = 7.1 Hz, 3H, OCH2CH3); 2.45 [s, 3H, 2CH3]; 3.66 (s, 3H, OCH3); 3.83 (s, 3H, OCH3); 4.33 (q, J = 7.1 Hz, 2H,
OCH2CH3); 6.72 [s, 1H, 4-H]; 6.52 – 6.73 (m, 4H, 59-H and 3H, 299-H,
599-H and 699-H); 6.84, 6.85 (d, 1H, 69-H, J = 1.9 Hz); 7.89, 7.92 (d, 1H,
39-H, J = 8.4 Hz); 10.66 (s, 1H, COOH); IR: 3350 (OH), weak bands by
2500, 2300 (COOH); 1680 (CO); 870, 810 (Ar); 66–688C; 51%; 0.41
(chloroform : ethanol = 10 : 1.5).
1-(1-Carboxy-2-phenyl-ethyl)-5-(3,4-dimethoxy-phenyl)2-methyl-1H-pyrrole-3-carboxylic acid ethyl ester 3f
1
H-NMR (CDCl3): 1.39 (t, J = 7.1 Hz, 3H, OCH2CH3); 2.70 [s, 3H, 2CH3]; 3.24, 3.39 (26dd, J = 14.1 Hz, 2H, CH2–CH); 3.71 (s, 3H,
OCH3); 3.88 (s, 3H, OCH3); 4.31 (q, J = 7.1 Hz, 2H, OCH2CH3); 5.01 –
5.14 (m, 1H, CH2–CH); 5.10 – 5.14 (brs, 1H, COOH); 6.29, 6.40 (d,
2H, 29-H and 69-H); 6.37 [s, 1H, 4-H]; 6.67 – 6.71 (m, 3H, 39-H, 49-H
and 59-H); 7.13 – 7.18 (m, 3H, 299-H, 599-H and 699-H); IR: 3400 (OH),
weak band by 2500, 2300 (COOH); 1710 (CO); 880, 830, 770, 710
(Ar); 112–1148C; 70%; 0.49 (chloroform : ethanol = 10 : 0.3).
1-(1-Carboxy-3-methyl-butyl)-5-(4-chloro-phenyl)-2methyl-1H-pyrrole-3-carboxylic acid ethyl ester 3g
1
H-NMR (CDCl3): 0.56 [d, 3H, CH3 (i-Pr)]; 0.71 [d, 3H, CH3 (i-Pr)]; 1.09
[s, 1H, CH(i-pr)]; 1.31 (t, J = 7.1 Hz, 3H, OCH2CH3); 1.87 (d, 2H, CHCH2-CH3); 2.57 [s, 3H, 2-CH3]; 4.27 (q, J = 7.1, 2H, OCH2CH3); 4.92 (q,
1H, CHCOOH); 5.40 (s, 1H, COOH); 6.56 [s, 1H, 4-H]; 7.31 (q, 4H,
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674
N. Danchev et al.
C6H4); IR: 3250, 3150 (OH), 2400 (COOH), 1750,1710 (CO), 820
(Ar); 157-1588C; 64%, 0.57 (chloroform : ethanol = 10 : 0.3).
5-(3-Acetyl-2-methyl-5-phenyl-1H-1-pyrrolyl)-2hydroxybenzoic acid 3h
1
H-NMR (CDCl3): 2.40 [s, 3H, 2-CH3]; 2.48 (s, 3H, CH3CO); 6.70 [s,
1H, 4-H]; 6.93, 6.96 [d, J = 8.7 Hz, 1H, 39-H]; 7.07 – 7.14 (m, 5H,
C6H5); 7.17, 7.19 (dd, 1H, 49-H, J = 2.2, 7.2 Hz); 7.66, 7.68 (d, 1H, 69H, J = 2.6 Hz); 11.48 (s, 1H, COOH). IR: 3350 (OH), 2550, 2300
(COOH), 1680, 1610 (CO), 860, 810, 760, 700 (Ar); 244–2468C
(dec.); 48%; 0.23 (chloroform : ethanol = 10 : 1.5).
Ethyl 2-methyl-5-phenyl-1-[(4-pyridylcarbonyl)amino]-1H3-pyrrolecarboxylate 3i
1
H-NMR (CDCl3): 1.31 (t, J = 7.1 Hz, 3H, OCH2CH3), 2.40 [s, 3H, 2CH3], 4.18 (q, J = 7.1 Hz, 2H, OCH2CH3), 6.56 [s, 1H, 4-H], 7.30 – 7.36
(m, 5H, C6H5), 7.65, 7.68 (d, 2H, 39-H, 59-H, J = 5.9 Hz); 8.60, 8.62 (d,
2H, 29-H, 6'-H, J = 5.8 Hz), 10.25 (s, 1H, NH); IR: 3214 (NH), 1707,
1662 (CO); 1533 (NH); 761, 654 (Ar); 182–1848C; 86%; 0.42 (chloroform : ethanol = 10 : 1.5).
Ethyl 1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-4pyrazolyl)-5-(3,4-dimethoxy-phenyl)-2-methyl-1H-3pyrrolecarboxylate 3j
1
H-NMR (CDCl3): 1.36 (t, J = 7.1 Hz, 3H, OCH2CH3), 1.71 [s, 3H, CH3],
2.50 [s, 3H, 2-CH3], 3.05 [s, 3H, 29-CH3], 3.66 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 4.30 (q, J = 7.1 Hz, 2H, OCH2CH3), 6.67 [s, 1H, 4-H], 6.856.86 (m, 3H, 299-H, 599-H and 699-H), 7.35-7.49 [m, 5H, C6H5(R4)]. IR:
1690, 1660 (CO); 860, 800, 760, 700 (Ar); 144-1468C; 72%; 0.58
(chloroform : ethanol = 10 : 1.5).
Arch. Pharm. Chem. Life Sci. 2006, 339, 670 – 674
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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