INTRODUCTION:

Inflammation is a very complex process that participates in the aggravation of several diseases.¹ The arachidonic acid derived chemical mediators play crucial role in the mediating of the inflammatory processes. At present, studies are being conducted to understand the role of cyclooxygenase-1/2 (COX-1/2) and related enzyme in various ailments and their possible treating approaches.² At present, there are several inhibitors available in the market for the inhibition of COX-1/2 which leads to the inability to convert arachidonic acid (AA) to prostaglandin H₂ (PGH₂). PGH₂ is an active component that mediates inflammation and pain sensitivity to the afferent nerves.³ There are several NSAIDs in market with similar attribute in inhibiting COX-1/2, thromboxane A₂ (TXA₂) production and inhibition of platelet aggregation, etc.⁴ However, in the majority of the therapeutic regimen, gastrointestinal complications like ulceration and bleeding are often reported.⁵ Therefore, it is a fundamental need to discover some new molecules for treating inflammatory conditions with more potency alongside least possible adverse effects.

Compounds from natural origin are one of the best options for finding alternatives.⁶ The natural products have reduced adverse effects and superior safety profile which makes them fruitful for the long-term prophylactic use, however, the therapeutic efficacy often suffers as compared to synthetic molecules.⁷ With that intension to discover new compounds with pronounced anti-inflammatory activity; a natural precursor known as murrayanine, a carbazole alkaloid present in the extract of Murraya koenigii L. (Rutaceae), and well known for anti-inflammatory action was principally used.⁸ Murrayanine-chalcone, a new compound (semi)-synthesized by our research group in which a chalcone is tagged was employed as the template for designing the inhibitors.⁹ In order to develop compounds with pronounced and elevated biological activity, heterocycles were produced from the parent chalcone. The pyrimidines are the well famed candidates for treating the inflammatory states.¹⁰ Several experimentally produced compounds, molecules in clinical trials, and products in market with anti-inflammatory potentials have pyrimidine ring.¹¹

In the present research, murrayanine-chalcone based pyrimidine derivatives were designed and synthesized using urea in alkaline ethanolic media with an intention to exploit their anti-inflammatory activity. Therefore, these novel compounds comprising of hybridized components of murrayanine, chalcone, and pyrimidine was believed to express noteworthy anti-inflammatory activity.

MATERIALS AND METHODS:

Chemical and Instrumentation:

The chemicals and solvents were of analytical grade and purchased from Sigma-Aldrich, Germany. The spectroscopic analyses were performed using IRAffinity-1 infrared spectrometer (IR spectroscopy), Bruker Avance-II instrument (¹H-NMR), and MICROMASS Q-TOF instrument (mass spectra). The tetramethylsilane (TMS) was used as an internal standard and the chemical shifts are expressed in ppm. The pre-coated silica gel-G TLC plate (Merck) was employed to monitor the progress of the chemical reaction. The CHN analysis was performed using Elemental Analyzer of PerkinElmer 2400 model.

Animals:

The anti-inflammatory activity of the fabricated pyrimidine compounds was screened after obtaining ethical permission from the institution. As per the screening protocol, albino rats of the same sex of weight 180-250 g were employed. The animals were kept polypropylene cages under hygienic condition at controlled temperature and humidity of 25–26ºC / 50–55%. A 12/12 hrs light/dark cycles was also maintained.

Synthesis of target compounds:

The novel murrayanine based pyrimidine derivatives (3a-h) were rationally designed from the previously reported starting material by our research group, known as the “murrayanine-chalcone” (1). In this proposed design, the heterocyclic form was fabricated utilizing the chalcone portion by simultaneously converting the carbonyl part into closed ring pyrimidine form, using the urea (2). The Scheme 1 depicts the reaction scheme of the novel analogs.

Scheme 1. The synthetic outline for the fabrication of pyrimidine derivatives from the murrayanine-chalcone.

Synthetic protocol for 6-(substituted)-4-(1-methoxy-9H-carbazol-3-yl)-1,6-dihydropyrimidin-2-ol (3a-h)

An equmolar mixture of 0.05 M of the murrayanine-chalcone derivatives (1a-h) and urea (2) were refluxed for 6 hours under alkaline ethanolic media, produced by 1 g of KOH with 25 mL of ethanol. The progress of the reaction was monitored by TLC plates. After the completion of the reaction, the content was cooled and poured onto crushed ice with vigorous stirring. The solid product (3a-h) was suitably filtered under vacuum, comprehensively washed with cold water, and recrystallized from ethanol.¹²

6-(2-fluorophenyl)-4-(1-methoxy-9H-carbazol-3-yl)-1,6-dihydropyrimidin-2-ol (3a)

48% yield; FTIR (KBr) υ (cm^-1): 3494 (-OH), 3295 (-NH), 3123 (C-H, aromatic), 1649 (C=N, aromatic), 1616 (C=C, aromatic), 1574 (-NH, bending), 1312 (C-N), 1282 (C-O), 1129 (C-F). ¹H NMR (δ, ppm, CDCl₃): 10.26 (9, 1H), 7.2-8.3 (Aromatic, 10H), 4.66 (12, 1H), 3.89 (1, 3H), 2.27 (11, 1H). MS: M⁺ 387. Anal. Calcd. for C₂₃H₁₈FN₃O₂: C, 71.31; H, 4.68; N, 10.85. Found: C, 71.01; H, 4.38; N, 10.54.

6-(4-fluorophenyl)-4-(1-methoxy-9H-carbazol-3-yl)-1,6-dihydropyrimidin-2-ol (3b)

35% yield; FTIR (KBr) υ (cm^-1): 3477 (-OH), 3233 (-NH), 3159 (C-H, aromatic), 1684 (C=N, aromatic), 1607 (C=C, aromatic), 1598 (-NH, bending), 1339 (C-N), 1264 (C-O), 1112 (C-F). ¹H NMR (δ, ppm, CDCl₃): 10.19 (9, 1H), 7.1-8.2 (Aromatic, 10H), 4.58 (12, 1H), 3.93 (1, 3H), 2.21 (11, 1H). MS: M⁺ 387. Anal. Calcd. for C₂₃H₁₈FN₃O₂: C, 71.31; H, 4.68; N, 10.85. Found: C, 70.96; H, 4.41; N, 10.53.

6-(2-iodophenyl)-4-(1-methoxy-9H-carbazol-3-yl)-1,6-dihydropyrimidin-2-ol (3c)

71% yield; FTIR (KBr) υ (cm^-1): 3467 (-OH), 3246 (-NH), 3163 (C-H, aromatic), 1664 (C=N, aromatic), 1624 (C=C, aromatic), 1602 (-NH, bending), 1318 (C-N), 1273 (C-O), 706 (C-I). ¹H NMR (δ, ppm, CDCl₃): 10.27 (9, 1H), 7.2-8.4 (Aromatic, 10H), 4.62 (12, 1H), 3.84 (1, 3H), 2.19 (11, 1H). MS: M⁺ 495. Anal. Calcd. for C₂₃H₁₈IN₃O₂: C, 55.77; H, 3.66; N, 8.48. Found: C, 55.37; H, 3.49; N, 8.34.

6-(4-iodophenyl)-4-(1-methoxy-9H-carbazol-3-yl)-1,6-dihydropyrimidin-2-ol (3d)

67% yield; FTIR (KBr) υ (cm^-1): 3490 (-OH), 3269 (-NH), 3144 (C-H, aromatic), 1653 (C=N, aromatic), 1633 (C=C, aromatic), 1595 (-NH, bending), 1337 (C-N), 1286 (C-O), 681 (C-I). ¹H NMR (δ, ppm, CDCl₃): 10.22 (9, 1H), 7.1-8.3 (Aromatic, 10H), 4.64 (12, 1H), 3.81 (1, 3H), 2.23 (11, 1H). MS: M⁺ 495. Anal. Calcd. for C₂₂H₁₈IN₃O: C, 55.77; H, 3.66; N, 8.48. Found: C, 54.97; H, 3.51; N, 8.36.

6-(4-bromophenyl)-4-(1-methoxy-9H-carbazol-3-yl)-1,6-dihydropyrimidin-2-ol (3e)

44% yield; FTIR (KBr) υ (cm^-1): 3482 (-OH), 3254 (-NH), 3132 (C-H, aromatic), 1677 (C=N, aromatic), 1614 (C=C, aromatic), 1589 (-NH, bending), 1346 (C-N), 1275 (C-O), 663 (C-Br). ¹H NMR (δ, ppm, CDCl₃): 10.18 (9, 1H), 7.2-8.4 (Aromatic, 10H), 4.56 (12, 1H), 3.86 (1, 3H), 2.26 (11, 1H). MS: M⁺ 447; M+2 449. Anal. Calcd. for C₂₃H₁₈BrN₃O₂: C, 61.62; H, 4.05; N, 9.37. Found: C, 61.39; H, 3.92; N, 9.15.

4-(1-methoxy-9H-carbazol-3-yl)-6-(2-(trifluoromethyl)phenyl)-1,6-dihydropyrimidin-2-ol (3f)

49% yield; FTIR (KBr) υ (cm^-1): 3473 (-OH), 3278 (-NH), 3151 (C-H, aromatic), 1681 (C=N, aromatic), 1601 (C=C, aromatic), 1551 (-NH, bending), 1326 (C-N), 1262 (C-O), 1074 (C-F). ¹H NMR (δ, ppm, CDCl₃): 10.25 (9, 1H), 7.2-8.3 (Aromatic, 10H), 4.67 (12, 1H), 3.83 (1, 3H), 2.22 (11, 1H). MS: M⁺ 437. Anal. Calcd. for C₂₄H₁₈F₃N₃O₂: C, 65.90; H, 4.15; N, 9.61. Found: C, 65.13; H, 4.02; N, 9.46.

6-(3,5-bis(trifluoromethyl)phenyl)-4-(1-methoxy-9H-carbazol-3-yl)-1,6-dihydropyrimidin-2-ol (3g)

62% yield; FTIR (KBr) υ (cm^-1): 3486 (-OH), 3237 (-NH), 3120 (C-H, aromatic), 1692 (C=N, aromatic), 1639 (C=C, aromatic), 1564 (-NH, bending), 1324 (C-N), 1269 (C-O), 1206 (C-F). ¹H NMR (δ, ppm, CDCl₃): 10.21 (9, 1H), 7.2-8.4 (Aromatic, 10H), 4.61 (12, 1H), 3.85 (1, 3H), 2.18 (11, 1H). MS: M⁺ 505. Anal. Calcd. for C₂₅H₁₇F₆N₃O₂: C, 59.41; H, 3.39; N, 8.31. Found: C, 59.27; H, 3.23; N, 8.18.

6-(2,4-dichloro-5-fluorophenyl)-4-(1-methoxy-9H-carbazol-3-yl)-1,6-dihydropyrimidin-2-ol (3h)

57% yield; FTIR (KBr) υ (cm^-1): 3461 (-OH), 3255 (-NH), 3166 (C-H, aromatic), 1656 (C=N, aromatic), 1621 (C=C, aromatic), 1569 (-NH, bending), 1301 (C-N), 1258 (C-O), 1195 (C-F), 788 (C-Cl). ¹H NMR (δ, ppm, CDCl₃): 10.23 (9, 1H), 7.1-8.4 (Aromatic, 10H), 4.59 (12, 1H), 3.92 (1, 3H), 2.24 (11, 1H). MS: M⁺ 455; M+2 457. Anal. Calcd. for C₂₃H₁₆Cl₂FN₃O₂: C, 60.54; H, 3.53; N, 9.21. Found: C, 59.14; H, 3.33; N, 8.87.

Acute toxicity studies:

The acute toxicity studies signifies the dose, where the maximum therapeutic effect with no such mortality was observed. This is an imperative parameter as far as in vivo safety is concerned. The lethal dose (LD₅₀) was established for the synthesized compounds in adult male albino rats. The protocol involved injection of a range of steadily rising (from 25 mg/kg to 500 mg/kg) doses of the compounds. The dose was computed based on 50% animal death.

Anti-inflammatory screening:

According to the protocol, the standard carrageenan-induced paw edema method was employed for the screening of in vivo anti-inflammatory potentials of the prepared molecules. With an intention to reduce the inconsistency of edema, the rats were fasted overnight. Before the commencement of the experiment, 5 mL of distilled water was individually administered by oral route. The test compound at a dose of 100 mg/kg b.w. was initially suspended in the saline solution and orally administered an hour before the induction of inflammation.

The inflammation was generated in the subplanter region of the right hind paw of rats by injecting 1% carrageenan solution via the subcutaneous route. Employing the mercury digital micrometer, the thickness of each rat paw was measured for the duration of 3 hrs at an interval of 1 hr to determine the edema. The difference between the width of injected and non-injected paws, reflects the potential of compounds in reducing the edema. The acquire data were expressed as mean ± standard error. The control group received the saline solution containing a few drops of Tween 80.¹³

RESULT AND DISCUSSION:

Chemistry:

The data obtained from the spectroscopy were thoroughly studied in order to elucidate the proposed structure. The formation of the pyrimidine structure from the chalcone precursor was confirmed by the disappearance of the IR peak in the range of 1690-1750 cm^-1, a characteristic location for ketonic C=O group in the spectra. Another distinct element in the transformation of the parent chalcone into pyrimidine was the appearance of –OH group. The hydroxyl moiety was detected primarily in the range of 3461-3490 cm^-1. Few prime features of the three aromatic rings were distinctly noticed in the spectra. The C-H stretching was found in the range of 3120-3166 cm^-1and the C=C stretching was seen chiefly in the range 1601-1639 cm^-1. The amide stretching and bending of the compounds were perceived in the characteristic ranges of 3233-3295 cm^-1and 1551-1602 cm^-1, respectively. The ¹H-NMR (proton NMR) disclosed several discrete aspects of the prepared derivatives. The carbazole nitrogen protons were predominantly observed at 10 ppm. The spectral region of 7.1-8.4 ppm largely represented the aromatic rings. The mass spectra revealed that the base peaks of the fabricated molecules were found quite similar or nearly exact to the molecular weight (theoretical). The isotopic forms (viz. chlorine and bromine) were identified in the spectra as molecular mass + 2. These above traits additional confirmed the transformation into the heterocyclic forms. Several fragment peaks of m/z 100-200 were also spotted in the spectra. Eventually, the elemental composition of the derivatives confirmed the formation of the derivatives as indicated by the ratio(s) of elements. Therefore, the sophisticated analytical tools confirmed the transformation of chalcone into the heterocyclic form.

Determination of LD₅₀ value:

The acute toxicity screening of the fabricated murrayanine-chalcone derivatives revealed that all the experimental compounds were safe over the dose range of 25-500 mg/kg b.w. as indicated by no signs of toxic effects or mortality. The anti-inflammatory prospective of the molecules was determined at the dose of 100 mg/kg b.w.

Anti-inflammatory activity:

The produced derivatives displayed noteworthy anti-inflammatory activity in carrageenan-induced paw edema model. The compound 3b, having para-fluorine substituent exhibited the highest anti-inflammatory activity with 58.33% reduction after 3 hrs. Next to the key candidate, molecule 3a with ortho-fluorine substituent and 3e with para-bromo demonstrated significant reduction of edema by 51.81% and 52.66% after 3 hrs, respectively. The compounds 3d and 3f showed moderate reduction in edema by 42.28% and 46.54%, respectively. The derivatives 3c, 3g, and 3h showed lowest activity ranging from 24.37-38.97% edema reduction in the above mentioned duration. The results illustrated that the fabricated molecules have the potential to be exploited as future inflammatory agents and may be applied for treating inflammatory conditions and arthritis after further clinical studies. The anti-inflammatory activity of the murrayanine-chalcone based pyrimidine compounds are described in Table 1.

Table 1. In vivo anti-inflammatory potential of murrayanine-chalcone based pyrimidine compounds (3a–h) in carrageenan-induced paw edema rat models.

Group	-R	Percentage (%) inhibition of edema
		1 hr	2 hr	3 hr
3a	2-F	28.12 ± 2.64	39.84 ± 3.23	51.81 ± 3.12
3b	4-F	31.97 ± 3.29	42.72 ± 1.91	58.33 ± 2.47
3c	2-I	18.44 ± 2.32	30.13 ± 2.74	38.97 ± 2.39
3d	4-I	22.59 ± 2.98	31.52 ± 2.26	42.28 ± 2.73
3e	4-Br	27.94 ± 3.11	39.19 ± 2.56	52.66 ± 4.19
3f	2-CF₃	24.81 ± 2.35	35.21 ± 3.31	46.54 ± 2.52
3g	3,5-CF₃	11.38 ± 2.67	20.34 ± 3.17	31.48 ± 1.95
3h	2,4-Cl; 5-F	9.72 ± 2.49	17.29 ± 2.92	24.37 ± 2.82
Indo methacin	-	39.87 ± 2.41	48.53 ± 2.87	65.12 ± 3.33

n = 6; ED₅₀ of 100 mg/kg b.w. in male adult albino mice; P < 0.005

From this study, it was observed that the electron-withdrawing substituent have played a profound role in arbitrating the anti-inflammatory activity. The lipophilicity might be predicted as the driving force which permitted the compounds to cross the biological barriers and mediating the activity. In addition, it was scrutinized that the number and the position of substituents played imperative role in mediating the activity. Here, it was detected that fluorine, the highest lipophilic substituent (also the electronegative) has exhibited the best activity, followed by bromine, and iodine substituent. However, it was seen that while substituting the chlorine along with fluorine in the derivative, the activity fallen drastically. The reason may be the formation of micelles of the compounds or binding to the amino acid residues which may produce hindrance in crossing the biological barrier. Therefore, it led to considerably reduced anti-inflammatory activity. Furthermore, the introduction of bulky group -CF₃ did not show an impressive activity as compared to –F group. By increasing the number of substituent to two –CF₃, it was found that the activity reduces significantly. The reason might be due to steric hindrance which prevented access to the active sites of chemical mediators of inflammation like COX, resulting in diminished activity.

CONCLUSION:

The present research work revealed that the newly synthesized heterocyclic compounds from the chalcone parent have tremendous potential as anti-inflammatory agents. The compound 3b, having para-fluorine substituent exhibited the highest anti-inflammatory activity with 58.33% reduction after 3 hrs. The anti-inflammatory activity was observed to be reasonably analogous with that of indomethacin, the standard drug. A very clear structure-activity relationship was not established due to mixed results. It was detected that fluorine substituted compound exhibited the best activity, followed by bromine, and iodine substituent. It was also seen that while substituting the chlorine along with fluorine in the derivative, the activity fallen drastically. The introduction of bulky group did not show an impressive activity as compared to the single electron-withdrawing group. On increasing the number of substituent to double, it was found that the activity reduces significantly. This research will undoubtedly encourage scientists across the globe in the rational designing and synthesis of molecules with distinct pharmacological action(s).

ACKNOWLEDGEMENT:

Authors are highly thankful to Savitribai Phule Pune University, Pune, Maharashtra, India for providing research grants (Grant No. 13PHM000126).

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Received on 27.10.2017 Accepted on 11.12.2017

Asian J. Pharm. Res. 2018; 8(1): 06-10.

DOI: 10.5958/2231-5691.2018.00002.3