Synthesis and Antihypertensive Screening of New Derivatives of Quinazolines Linked with Isoxazole

A series of 7-substituted-3-(4-(3-(4-substitutedphenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-substituted quinazolin-4(3H)-one (1–30) have been synthesized by the cyclization of (E)-3-(4-(3-substitutedphenyl)acrylolyl)phenyl)-2-(substitutedphenyl)-7-substituted quinazolin-4-(3H)-one with hydroxylamine hydrochloride. The synthesized compounds were examined for their in vivo antihypertensive activity using albino rats. All the titled compounds exhibited good to moderate antihypertensive activity. Compounds 7-Chloro-3-(4-(3-(4-chlorophenyl)-4,5- dihydroisoxazol-5-yl)phenyl)-2-p-tolylquinazolin-4(3H)-one (23) and 7-Chloro-3-(4-(3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)phenyl)-2-(4-methoxyphenyl)quinazolin-4(3H)-one (24) exhibited potent antihypertensive activity through their anticipated α 1-adrenergic receptor blocking property similar to its clinically used analogue, prazosin, without affecting heart rate with prolonged duration of action when tested in adrenaline induced hypertension in anaesthetized rats.


Introduction
Among the major risk factors for arterial diseases, hypertension is one of the most life threatening health problems in the modern world [1,2]. In 2000, the figure of total number of adults with hypertension was 972 million and this may rise by about 60% to a total of 1.56 billion by 2025 [3]. Moreover, in India, about 70% of coronary heart diseaserelated deaths occur in people younger than 70 years [4]. 1 -Adrenergic receptor antagonist like prazosin [5,6], terazosin, and doxazosin are reputed class of antihypertensive agents derived from quinazoline. Prazosin has selectivity to blocks postsynaptic 1 -adrenergic receptors while having no effect on presynaptic 2 -adrenergic receptor and is responsible for the inhibition of norepinephrine release from sympathetic nerve, and the piperazine ring is also very sensitive towards enzymatic hydroxylation, whereas doxazosin and terazosin [7] were found to possess longer duration and less reflex tachycardia than prazosin. Since quinazoline derivatives, which belong to the -containing heterocyclic compounds, have universal concerns due to their widely and distinct biological activities such as diuretic [8][9][10], antihypertensive [11], antihistaminic [12,13] analgesic, and anti-inflammatory [14,15] anticancer [16] and anti-HIV [17] activities, it was therefore worthwhile to study the possibility of replacing or changing in position of the labile furoylpiperazine moiety in prazosin by a more stable isoxazole group so that the antihypertensive activity of the new derivatives remains unaltered but possesses longer duration of action due to the increased stability against enzymatic degradation. In present study we have reported 30 molecules containing quinazoline and isoxazole heterocyclic ring.

Materials
All the chemicals used were of laboratory grade and procured from E. Merck (Darmstadt, Germany) and S.D. Fine Chemicals (Mumbai, India). Melting points were determined by open capillary tubes in a Hicon melting point apparatus and are uncorrected. Purity of the compounds was checked by thin layer chromatography (TLC) plates (silica gel G). Iodine chamber and UV lamp were used for the visualization of TLC spots. The Fourier Transform Infrared (FT-IR) spectra were recorded on Bio-rad FTS-135 spectrophotometer using KBr pellets; ] max values are given in cm −1 . The 1 H NMR spectra were taken on a Bruker 400 Ultrashield (300/400 MHz) NMR spectrometers using dimethylsulfoxide (DMSO)-d6 solvent and tetramethylsilane (TMS) as an internal standard (chemical shift in , ppm). 13 C NMR spectra were recorded on a Bruker 400 Ultrashield (400 MHz) NMR spectrometers using dimethylsulfoxide (DMSO)-d6 solvent. Mass spectra recorded on UPLC-MS/MS (water, Q-TOF-ESI, and Mass Lyns v 4.1) mass serial no. JAA-272 (Synapt MS-G 1 ) were presented as / . Elemental analysis was carried out on CHN elemental (Perkin Elmer 240 analyser) using sulphanilic acid as a standard and tungsten (VI) oxide as a combusting agent. All the results of elemental analyses corresponded to the calculated values within experimental error. The reaction was monitored by thin-layer chromatography (TLC) and spots were visualized by iodine vapors or irradiation with UV light (254 nm).

Substituted 2-Benzamidobenzoic Acid (a1-a12).
To the substituted anthranilic acid (2 mmol) dissolved in 10% sodium hydroxide (10 mL), substituted benzoyl chloride (2.2 mmol) was added with stirring at room temperature for over 1 h. Upon completion, reaction mixture was quenched with cold water to obtain solid residue, which was washed with dilute HCl followed by water and recrystallized from ethanol. Yield: 90-96%.
To a magnetically stirred solution of substituted quinazolinone (c1-c12, 0.01 mmol) in ethanol, substituted aldehyde (0.01 mmol) was added and the reaction mixture was stirred for 2 h. Upon completion of reaction, the mixture was poured on crushed ice and then acidified with dilute HCl. A precipitate formed which was filtered, washed with water, and recrystallized with ethanol. . A mixture of (E)-3-(4-(3-substitutedphenyl)acrylolyl) phenyl)-2-(substitutedphenyl)-7-substituted quin-azolin-4-(3H)-one (d1-d30, 0.01 mol) in absolute ethanol (25 mL) and water; hydroxylamine hydrochloride (0.01 mol) was added. The reaction mixture was heated under reflux for 5 h in the presence of 30% KOH. The mixture was poured in ice water and then acidified with dilute HCl. The resulting solid was filtered, washed with water, dried, and crystallized with ethanol.

Pharmacology.
All the experimental protocols were carried out with the permission from the Institutional Animal Ethics committee (IAEC), project proposal no. 781 and the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments in Animal (CPC-SEA). Animals were obtained from the Central Animal House Facility, Hamdard University, New Delhi. Registration number and date of registration are 173/CPCSEA, 28th of January, 2000. All rats were housed in a temperature and humidity controlled room at an ambient temperature of 25 ± 2 ∘ C with a 12 h light/dark cycle. Animals were provided with pellets diet (Lipton, Calcutta, India) and water ad libitum.

Conditioning/Training of Animals.
For conducting the BP measurement studies, the animals were kept in restrainers for 10 min every day for one week. This exercise was done to avoid the fluctuation in blood pressure due to aggressive behaviour of the animal.

Induction of Hypertension in Albino Rats.
After recording the initial BP of rats, the animals were divided into groups of 6 animals each. One group was taken as control. Hypertension was induced in the remaining groups by subcutaneous injection of methyl prednisolone acetate (20 mg/kg body wt./wk) for 2 weeks as per method reported by Krakoff et al. [19].

Dose Establishment of Synthesized Compounds for
Screening of Antihypertensive Activity. Systolic blood pressure was measured in conscious rats using CODA noninvasive blood pressure recorder by Tail Cuff method (Kent Scientific Corporation, USA). The restrainers carrying the rat were placed in the BP instrument with the tail protruding out. The tail was gently placed in contact with a transducer membrane, which was connected to the digital BP display panel. The instrument was then turned on and allowed to stabilize until steady pulse rate was observed. Once the "pulse level ready" signal appeared, the BP recording button was pressed and the mean arterial BP was recorded. Albino rats (body weight 200-250 g) were used in the study. Rats were assigned to groups of six animals in each. Each compound (1 mg/day, 2 mg/day,. . .,10 mg/day) was administered orally after suspending in 1% carboxy methyl cellulose (CMC) solution. The blood pressure was recorded at various time intervals.

Determination of -Adrenergic Receptors
Blocking Activity. Albino rats were classified into ten groups; each group comprises 6 animals each, to investigate the 1 -adrenergic receptor blocking effect of some synthesized compounds. In each experiment, the effect of adrenaline, at a dose of 3 g/Kg intravenously, on the arterial blood pressure was recorded alone 30 min before i.p. injection (5 mg/kg b.w. for compounds 1, 16, 19, 20, 23, 27, and 28, whereas 4 mg/kg b.w. for compound 24) of the test compound and then its effect was determined again after 30, 60, and 120 min from the injection of the test compounds [20].

Statistical Analysis of Data.
The statistical analysis was performed using GRAPHPAD INSTAT 3 software (Graph Pad Software Inc., San Diego, CA). Data obtained from animal experiments were expressed as arithmetic mean ± SEM and ±S.E. The comparison between various groups was performed by one-way analysis of variance (ANOVA), and the effects in treatment groups were compared with toxic control or control group by Dunnett multiple comparison test. < 0.05 was considered to be significant ( * < 0.05; * * < 0.01).

Dose Establishment for the Screening of Antihypertensive
Activity. The minimum effective dose of test compounds was established by performing the gradual enhanced dose administration (1 mg/day, 2 mg/day, 3 mg/day,. . ., 10 mg/day), and when there was no further decrease, that is, ceiling effect in the blood pressure, observed at a certain dose, that dose was assumed as the minimum dose required to elicit the optimum antihypertensive activity of the synthesised compound. All titled compounds were screened to study their effect on the arterial blood pressure by using noninvasive Tail Cuff method. The results are compared with standard drug prazosin [30], as shown in Table 1. Most of the synthetic compounds showed the ceiling dose between 5-6 mg. The title compounds 2, 18, 19, 23, and 27 were found to show substantial reduction in systolic blood pressure, whereas compound 24 showed significant antihypertensive activity as compared to standard drug prazosin (Figure 2). The ceiling dose of compound 24 was found to be 4 mg; however, prazosin has a ceiling dose of 5 mg. The rest of the compounds showed moderate decrease in hypertension.

Determination of -Adrenergic Receptor Blocking
Property. The injection of prazosin, 30 min prior to i.v injection of adrenaline at a dose of 3 g/Kg [31], produced a significant ( < 0.05) drop in arterial blood pressure due to selective blockade of 1 -adrenergic receptor by prazosin (adrenaline antagonist) [11]; however, injection of prazosin, 60 min prior to adrenaline injection returned the blood pressure to normal level; this effect may be due to rapid onset and short duration of action of drug ( Figures  3 and 4). Amongst the tested compounds, compound 1 showed the significant ( < 0.05) increase in SABP and DABP and without changing heart rate at 30 min prior  to administration of adrenaline. Therefore this compound could serve as lead molecule for the synthesis of a series of compounds to treat hypotension. Compounds 23 and 24 showed reverse action of vasopressor effect of adrenaline with depressor reflex after 30 min, as shown in Table 2. This effect was further prolonged at 60 min (Figures 3 and 4). The activity was continued up to 120 min to check the further change in activity. On the basis of these observations, it may be hypothesized that these compounds possess rapid and prolonged 1 -adrenergic blocking property without causing reflex tachycardia. Compounds 16, 27, and 28 also exhibited moderate antagonist activity of adrenaline after 30 min. It indicates that these compounds also possess 1 -adrenergic receptor blocking effect with shorter duration of action like prazosin.

Conclusion
In conclusion, we have synthesized new quinazoline derivatives associated with isoxazole. These compounds showed significant antihypertensive activity with minimal side effects. The most potent 1 -adrenergic blocking property was exhibited by the compound 24, having an SABP & DABP -11.6, −12.6 at 30 min. The replacement of piperazine moiety   of prazosin at position-2 with substituted phenyl ring and incorporation of substituted isoxazole ring at position-3 of quinazoline moiety proved beneficial for antihypertensive activity. Our study shows that these synthesized compounds act as lead and may be useful in antihypertensive therapy.

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