Design, synthesis and pharmacological evaluation of some pyrazolopyrimidin-6(7H)-ones and tricyclic 8-oxo-dihydrooxazolopyrazolopyrimidin-9-ium chloride derivatives

2.1 General procedure for the synthesis of 5-amino-N-substituted-1H-pyrazole-4-carbonitrile (1a–1e)

10 mmol of hydrazine hydrate and substituted phenyl hydrazine (prepared by diazotization of substituted anilines followed by reduction with Na₂SO₃·7H₂O) (Das et al., 2008a) were dissolved in 25 mL of ethanol in a round bottom flask. 10 mmol of ethoxymethylenemalononitrile (EMMN) was added to it and the mixture was then refluxed for 5 h. It was then filtered and the filtrate evaporated in vacuo to yield the product followed by recrystallization from methanol.

2.2 General procedure for the synthesis of 4-amino-1-substituted-pyrazolo [3,4-d]pyrimidin-6(7H)-one (2a–2e)

0.01 mol of (1a–1e) was heated with 0.02 mol of urea at 180–200 °C until the clear melt becomes solid. Cooled solid was then dissolved in 2 N NaOH and the solution was boiled with charcoal for 10 min and filtered. The boiling filtrate was acidified with glacial acetic acid. This acidified solution was filtered hot to yield a tan colored product that was purified by dissolving small sample in boiling dilute NaOH and precipitating it from hot solution of acetic acid. The procedure was repeated to yield the pure compounds (Robins, 1956).

2.3 General procedure for the synthesis of 4-amino-8-oxo-1-substituted-7,8-dihydro-1H-oxazolo[3,2-a]pyrazolo[4,3-e]pyrimidin-9-ium chloride (3a–3e)

A solution of 2 mmol of (2a–2e), 2 mmol N-(4-bromophenyl)-2-chloroacetamide and 4 mmol triethylamine in 20 mL absolute toluene was refluxed for 3 h. Crystalline triethylamine was filtered off and washed with a small amount of benzene, which was combined with the filtrate. The mixture was treated with HCl (10%) with constant stirring and evaporated. The residue was washed with ice water, dried and recrystallized from propanol–hexane (1:2) to give the final dihydrooxazolo derivative of pyrazolopyrimidin-9-ium chloride (Kulakov, 2009).

2.4 Pharmacological evaluation

Healthy Charles Foster rats (150–200 g) and Swiss albino mice (20–25 g) of either sex were procured from animal house, Institute of Medical Science, Banaras Hindu University for the activity studies. The animals were housed in Perspex cages under standard conditions of temperature (24 ± 1 °C) and relative humidity (65 ± 10%) with free access to standard pellet diet and water ad libitum. All the animals were randomly distributed into groups of six each. Animals were allowed to acclimatize for seven days to their environment before experimentation. The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC) [Approval No. Dean/10–11/61], Institute of Medical Sciences, Banaras Hindu University. Experiments were conducted following the guidelines issued by the National Institute of Health (NIH) for the care and use of laboratory animals. All the synthesized derivatives (2a–2e) and (3a–3e) were evaluated for anti-inflammatory and analgesic activity along with an assessment of their ulcerogenic liability.

2.5 Determination of acute toxicity

The acute toxicity determination for the synthesized derivatives was carried out as per OECD guidelines (OECD 423-2002) on healthy albino mice of either sex.

2.6 Anti-inflammatory activity

2.6.2

2.6.2 Acute ulcerogenic activity

Ulcerogenic activity was evaluated after p.o. administration of test compounds or indomethacin at the dose of 10 mg/kg. Control rats received p.o. administration of vehicle (suspension of 1% carboxy methyl cellulose). Food but not water was removed 24 h before administration of the test compounds. Twenty-four hours after the dosing, rats under deep ether anesthesia were sacrificed so that the stomach could be removed, opened along the curvature, washed with distilled water and cleaned gently by dipping in saline. Stomach samples of all the groups obtained from ulcerated rats were fixed in 10% formalin which was embedded in paraffin blocks for sectioning. The obtained sections (1–3 mm) were stained with hematoxylin and eosin dye and finally monitored and photographed using a Nikon digital microscope (Eclipse 200) at 10× magnification (Figs. 1a–3) (Hafez et al., 2010).

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Figure 1 (a) Histopathology: Rat’s stomach of the control group showing intact epithelium. (b) Indomethacin group showing development of ulcers as indicated by disruption of epithelium.

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Figure 2 (a) Histopathology: Rat’s stomach of test compound (3e) indicating minimal ulcer (33.3%). (b) Intact mucosa of control group.

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Figure 3 Indomethacin showing mucosal bleeding.

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2.7 Analgesic activity

2.8 Statistical analysis

All values are expressed as mean ± standard error of the mean (SEM) for ‘n’ number of observations, where ‘n’ represents number of animals being studied. The results were analyzed by one way analysis of variance (ANOVA) followed by Dunnett’s test. ‘p’ values more than 0.05 was considered as non-significant, less than 0.05 as significant and less than 0.01 as extremely significant as compared with the control group.

3.1 Chemistry

Proposed derivatives have been synthesized according to Scheme 1 starting from EMMN. The structures of the compounds obtained were supported by spectroscopic measurements and elemental analysis. The FT-IR spectra of 2a–2e and 3a–3e showed two characteristic stretchings of primary amine at around 3158–3349 cm⁻¹ and 3158–3387 cm⁻¹ respectively. A peak at around 1620–1668 cm⁻¹ and 1720–1732 cm⁻¹ is attributed to the presence of –C⚌O in pyrimidine nucleus and dihydrooxazolo moiety of 2a–2e and 3a–3e respectively. Further, all the derivatives showed diagnostic IR absorptions at 1528–1594 cm⁻¹ stretching for –C⚌N in the pyrazole nucleus. Additional peak at 1070–1116 cm⁻¹ confirmed C–O–C stretching in dihydrooxazolo ring system of 3a–3e.

¹H-NMR spectra of all the derivatives showed a peak within the range of δ 7.79–8.55 corresponding to –CH (Ha) proton of pyrazole nucleus. The free –NH₂ protons appear as broad singlet with a corresponding chemical shift value in the range of δ 6.53–6.90. The –NH proton (Hc) of pyrimidine nucleus (2a–2e) displayed singlet signal in the range of δ 7.78–8.07 while the secondary N1 proton of unsubstituted derivatives (2a and 3a) showed singlet at 8.10 and 7.91 respectively. Derivatives 3a–3e displayed an additional signal due to the presence of Hc methylene protons which appears as singlet at around δ 4.34–4.74. All the other protons belonging to the N1 substituted phenyl ring were seen according to the expected chemical shift. The elemental analysis results were within ±0.4% of the theoretical values.

3.2 Pharmacology

In post-acute toxicity studies, the synthesized derivatives (2a–2e; 3a–3e) were evaluated by the carrageenan induced rat paw edema method as described by Winter et al. (1962) followed by the evaluation of their ulcerogenic liability (Table 1).

Carrageenan induced paw edema model was used to evaluate the effects of acute inflammation. Carrageenan is a mixture of polysaccharides composed of sulfated galactose units and it is derived from Irish Sea moss, Chondrous crispus. The time course of edema development in carrageenan-induced paw edema model in rats is generally represented by a biphasic curve. The early or the first phase of inflammation occurs after an hour of carrageenan injection and is partly attributed to trauma of injection and also to histamine, and serotonin components. The late or the second phase (after 3 h of carrageenan injection) is associated with the production of bradykinin, protease, prostaglandin, and lysosome (Vinegar et al., 1969).

The anti-inflammatory activity of synthesized derivatives was assessed from their ability to inhibit the paw edema induced by carrageenan in rats. The tested compounds exhibited anti-inflammatory activity spanning over a range from 53.46% to 89.06% as compared to 90.15% inhibition by standard drug indomethacin after 6 h.

The anti-inflammatory effects of the 4-amino-8-oxo-1-substituted-7,8-dihydro-1H-oxazolo[3,2-a]pyrazolo[4,3-e]pyrimidin-9-ium chloride derivatives (3a–3e) were found to be greater than those of 4-amino-1-substituted-pyrazolo [3,4-d]pyrimidin-6(7H)-one intermediate (2a–2e). After 6 h of carrageenan administration, compound 3e exhibited maximum anti-inflammatory effect (89.06%), followed by 3c and 3d with 85.66% and 82.26% inhibition respectively among the derivatives (3a–3e). Similarly, among derivatives (2a–2e), 2e exerted maximum anti-inflammatory effect (77.15%) closely followed by derivative 2d (76.54%) and derivative 2c (72.90% respectively). Rest of the derivatives exhibited moderate to weak anti-inflammatory activity. The above results reaffirm our belief that the dihydroxazolo moiety which has been inserted in a lateral position (3a–3e) is responsible for further improving the anti-inflammatory effect as compared to its corresponding 4-amino pyrazolopyrimidinone intermediates (2a–2e). Also, it might be stated that the synthesized derivatives exhibit their anti-inflammatory effect through the prostaglandin mediated mechanism as they seem to be more effective in the second phase of carrageenan-induced edema as compared to the early phase. The ulcerogenic liabilities of compounds (2a–2e; 3a–3e) were significantly less than that of indomethacin at the observed equimolar dose level. The 2,4-dichlorophenyl substitution at N1 position of pyrazolopyrimidine has considerably enhanced the anti-inflammatory activity profile of both 2e and 3e. From the obtained results as reported in Table 1, it has been noticed that all the synthesized derivatives exhibited reduced ulcerogenicity (16.6–66.6%) (Fig. 2b) and are considered to be safer than indomethacin which exhibited an ulcerogenic liability of 83.3% (Fig. 1b).

Derivatives (2c–2e) and (3c–3e) which displayed comparable anti-inflammatory activity with reduced ulcerogenicity were further evaluated for analgesic activity employing acetic acid induced writhing in mice and formalin induced paw edema in mice. Acetic acid is postulated to induce pain by increasing fluids comprising of PGE2 and PGF2α (Deraedt et al., 1980) at the peritoneal receptors. Acetic acid acts indirectly by inducing the release of endogenous mediators, which is responsible for the stimulation of the nociceptive neurons that are sensitive to non-steroidal anti-inflammatory drugs and narcotics (Bentley et al., 1983; Collier et al., 1968). In the acetic acid induced writhing test the analgesic activity of the derivatives is expressed as “mean increase in latency after drug administration ± SEM” relative to control and percentage inhibition in writhing reflex. Among the evaluated derivatives at an equimolar oral dose of 10 mg/kg indomethacin, derivative 3e exhibited maximum inhibition of writhings (70.48%) followed by derivatives 3d, 3c, 2e, 2d and 2c as compared to 78.22% inhibition of writhing by indomethacin. Surprisingly, the overall results suggest that probably the dihydroxazolo moiety (3c–3e) accentuates the analgesic profile of the derivatives as compared to their corresponding 4-amino pyrazolopyrimidinone intermediates (2c–2e).

The formalin-induced pain test consists of two distinct periods of response, i.e. early (tonic pain) and late phase (inflammatory pain) response. The response is evaluated by counting the time spent with licking and elevation (lifting) of the injected paw, flinching, and also protection of the paw from full pressure when walking or resting (Porro and Cavazzuti, 1993) Drugs that act primarily on the CNS inhibit both phases equally while peripherally acting drugs inhibit the late phase (Tjolsen et al., 1992). The formalin test assesses the behavioral response to injection of dilute formation into the paw of an animal. In the formalin test, pretreatment of mice with the derivatives (2c–2e; 3c–3e) at equimolar doses of 10 mg/kg of standard drugs indomethacin and pentazocin had a significant effect during the second phase (15–30 min) as can be seen from the data reported in Table 2 except that derivative 2c did not have any significant activity at both the phases. The results obtained clearly indicate that the active analgesic principal of the evaluated derivatives is acting peripherally and not through mechanisms involving the central nervous system.