Anti-inflammatory potentials of some novel Murrayanine containing 1,3,4-Oxadiazole derivatives

 

Debarshi Kar Mahapatra 1*, Ruchi S. Shivhare 2, Vinod G. Ugale 3

1Department of Pharmaceutical Chemistry, Dadasaheb Balpande College of Pharmacy, Nagpur 440037, Maharashtra, India

2Department of Pharmaceutical Chemistry, Kamla Nehru College of Pharmacy, Nagpur 441108, Maharashtra, India

3Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur 425405, Dist. Dhule, Maharashtra, India

*Corresponding Author E-mail: dkmbsp@gmail.com

 

ABSTRACT

Based on the assumption that designing compounds with all the three features; a natural product, a synthetic component, and a functional Schiff’s base will surely lead to the development of more potent and safe analogs. The present research endeavors at designing a novel molecule from a previously reported starting material (E)-2-((1-methoxy-9H-carbazol-3-yl)methylene)hydrazinecarboxamide that comprised of a natural product (murrayanine, a carbazole alkaloid present in the Indian curry plant Murraya koenigii L.), a synthetic component (oxadiazole, a well-known versatile heterocycle which has potential for treating numerous ailments), and a functional Schiff’s base attaching a substituted aromatic portion. The produced derivatives were studied for exploration of their in vivo anti-inflammatory activity using carrageenan-induced paw edema method. The sophisticated analytical techniques established the structures of murrayanine-oxadiazole hybrids. The acute toxicity studies highlighted their certain degree of safety. The compound 4e containing 3,5-OCH3 and 4-OH expressed the highest biological activity after 3 hrs, which may be due to the interaction of the substituents with the active sites of inflammation targets like cyclooxygenase (COX) and lipoxygenase (LOX). A structural influence on the biological activity cannot be predicted very clear, however, it might be plausibly expressed that the hydrophilic groups or electron-donating substituents at 3 to 5 positions have a vital role. This research will be an emerging perspective towards the fabrication of natural and synthetic components in one hybridized form which results in synergistic pharmacological activity. The fabricated products have the potential of usage as future therapeutic agents with good safety profile.

 

KEYWORDS: Murrayanine; Murraya koenigii; Oxadiazole; Inflammation; Hybrid; Schiff’s Base.

 

INTRODUCTION:

The syntheses of natural product-based synthetic heterocyclic hybrids are an emerging area for drug discovery and development.1 Several natural alkaloids with a particular pharmacological potential are made hybrids with heterocyclic compounds with a standpoint of synergistic biological activity.2 The Schiff’s base play an imperative role in any compound owing to diverse applications in medicinal chemistry and biology.3 Based on the assumption that designing compounds with all the three features; a natural product, a synthetic component, and a functional Schiff’s base will surely lead to the development of more potent and safe analogs. Murrayanine, a carbazole alkaloid present in the Indian curry plant Murraya koenigii L. is a very simple molecule with high therapeutic potentials.4 The molecule facilitates semi-synthesis at several positions which differentiates it from other natural products.5 The semi-synthetic outcome may be an expression of multifarious biological activities by modulating molecular targets.6

Oxadiazole is a well-known versatile heterocycle which finds application in treating numerous ailments like inflammation, analgesia, tuberculosis, microbial infection, cancer, trypanosomal condition, retrovirus, helminthic, Parkinson, etc.7,8 At present, this heterocycle is a part of marketed formulations such as Raltegravir®, an antiretroviral drug; Zibotentan®, an anticancer agent; furamizole, an antibiotic; tiodazosin, an antihypertensive drug; and several other drugs.9,10

 

The present research endeavors at designing a novel molecule from a previously reported starting material (E)-2-((1-methoxy-9H-carbazol-3-yl)methylene)hydrazi necarboxamide that comprised of a natural product (murrayanine), a synthetic component (oxadiazole), and a functional Schiff’s base attaching a substituted aromatic portion. The produced derivatives were studied for exploration of their in vivo anti-inflammatory activity using carrageenan-induced paw edema method.

 

MATERIALS AND METHODS:

Chemicals and Instrumentation

The chemical synthesis started with a previously reported chemical entity (E)-2-((1-methoxy-9H-carbazol-3-yl)methylene)hydrazinecarboxamide. The chemicals and solvents used for the synthesis were of analytical grade and procured from Sigma-Aldrich, HiMedia, and Merck. Sophisticated analytical techniques like FT-IR (Shimadzu® IRAffinity-1 instrument) using KBr system, 1H-NMR (Bruker Avance-II instrument) using tetramethylsilane (TMS), and mass spectra (MICROMASS Q-TOF instrument). The progress of the reaction process was examined by Merck pre-coated silica gel-G TLC plates. The Elemental Carbon-Hydrogen-Nitrogen (CHN) analyses were carried using PerkinElmer Elemental Analyzer 2400 instrument.

 

Animals

The exploration of the anti-inflammatory potentials of the fabricated hybridized oxadiazoles involved albino rats of 5-6 weeks age having the body weight in the range of 170-290 g. After receiving approval from the Department Ethical Committee (DEC) and CPCSEA (1389/a/10/CPCSEA), the experiment was commenced. The rats were kept under the hygienic controlled environment (24–25ºC temperature, humidity 50–60%, and 12 hr light and dark) in the animal house. Free access to water and feeding of standard rodent pellets were allowed.

 

Synthesis of target compounds

The synthesis of murrayanine based oxadiazole molecules (4a-e) involved cyclization of (E)-2-((1-methoxy-9H-carbazol-3-yl)methylene)hydrazinecarboxa mide (1) into amine-containing oxadiazole moiety (2) using sodium acetate and bromine. The produced oxadiazole moiety (2) was further reacted with a series of aldehydes such as benzaldehyde (3a), cuminaldehyde (3b), vanillin (3c), veratraldehyde (3d), and syringaldehyde (3e) to form Schiff’s base analogs in presence of few drops of glacial acetic acid. The reaction scheme of the murrayanine-oxadiazole derivatives is depicted in Scheme 1.

 


 

Scheme 1. The rational synthesis of novel murrayanine-1,3,4-oxadiazole derivatives.


 

Synthetic protocol for 5-(1-methoxy-9H-carbazol-3-yl)-1,3,4-oxadiazol-2-amine (2)

In a round bottom flask containing magnetic stirrer, the starting material (E)-2-((1-methoxy-9H-carbazol-3-yl)methylene)hydrazinecarboxamide (1) and (0.01 M) and sodium acetate (0.02 M) were solubilized in 50 mL of glacial acetic acid. 1 mL of bromine was added drop wise under constant stirring (1 mL of bromine in 5 mL of glacial acetic acid). After completion of reaction after few hours, crushed ice was added to the reaction mixture to obtain precipitated solid product. The obtained product was separated using Whatman filter paper, dried carefully, and recrystallized with ethanol.

 

45% yield; FTIR (KBr) υ (cm-1): 3354 (-NH2), 3268 (-NH, stretching), 3107 (C-H, aromatic), 1645 (C=C, aromatic), 1593 (-NH, bending), 1261 (C-N), 1219 (C-O); 1H NMR (δ, ppm, CDCl3): 10.12 (9, 1H), 8.15 (11, 2H), 7.2-8.2 (Aromatic, 6H), 3.87 (1, 3H). MS: M+ 280. Anal. Calcd. for C15H12N4O2: C, 64.28; H, 4.32; N, 19.99. Found: C, 64.06; H, 4.13; N, 19.47

 

Synthetic protocol for (E)-5-(1-methoxy-9H-carbazol-3-yl)-N-(substituted-benzylidene)-1,3,4-oxadiazol-2-amine (4a-e)

To the alcoholic solution of 5-(1-methoxy-9H-carbazol-3-yl)-1,3,4-oxadiazol-2-amine (2) (0.01 M) under continuous stirring, the various benzaldehyde derivatives (3a-e) were added and the reaction mixture was refluxed for 6-8 hrs in the presence of 8-9 drops of glacial acetic acid. The progress of the reaction was monitored by TLC technique. The hot reaction content was poured onto the crushed ice and the obtained precipitate was filtered off, washed thoroughly, and recrystallized using suitable solvents.

 

(E)-N-benzylidene-5-(1-methoxy-9H-carbazol-3-yl)-1,3,4-oxadiazol-2-amine (4a)

46% yield; FTIR (KBr) υ (cm-1): 3434 (-NH, stretching), 3176 (C-H, aromatic), 1673 (Azomethine, C=N), 1641 (C=C, aromatic), 1566 (-NH, bending), 1253 (C-N), 1231 (C-O); 1H NMR (δ, ppm, CDCl3): 10.22 (9, 1H), 8.63 (Azomethine, 1H), 7.1-7.8 (Aromatic, 11H), 3.89 (1, 3H). MS: M+ 368. Anal. Calcd. for C22H16N4O2: C, 71.73; H, 4.38; N, 15.21. Found: C, 71.42; H, 4.11; N, 14.96

 

(E)-N-(4-isopropylbenzylidene)-5-(1-methoxy-9H-carbazol-3-yl)-1,3,4-oxadiazol-2-amine (4b)

53% yield; FTIR (KBr) υ (cm-1): 3249 (-NH, stretching), 3131 (C-H, aromatic), 1696 (Azomethine, C=N), 1629 (C=C, aromatic), 1550 (-NH, bending), 1234 (C-N), 1212 (C-O); 1H NMR (δ, ppm, CDCl3): 10.14 (9, 1H), 8.55 (Azomethine, 1H), 7.2-7.9 (Aromatic, 10H), 3.82 (1, 3H), 1.19 (17, 6H). MS: M+ 410. Anal. Calcd. for C25H22N4O2: C, 73.15; H, 5.40; N, 13.65. Found: C, 72.83; H, 5.14; N, 13.26

(E)-2-methoxy-4-(((5-(1-methoxy-9H-carbazol-3-yl)-1,3,4-oxadiazol-2-yl)imino)methyl)phenol (4c)

58% yield; FTIR (KBr) υ (cm-1): 3379 (-OH), 3233 (-NH, stretching), 3164 (C-H, aromatic), 1692 (Azomethine, C=N), 1656 (C=C, aromatic), 1576 (-NH, bending), 1268 (C-N), 1225 (C-O); 1H NMR (δ, ppm, CDCl3): 10.18 (9, 1H), 8.59 (Azomethine, 1H), 7.1-8.2 (Aromatic, 9H), 5.24 (16, 1H), 3.82 (1, 6H). MS: M+ 414. Anal. Calcd. for C23H18N4O4: C, 66.66; H, 4.38; N, 13.52. Found: C, 66.21; H, 4.03; N, 13.15

 

(E)-N-(3,4-dimethoxybenzylidene)-5-(1-methoxy-9H-carbazol-3-yl)-1,3,4-oxadiazol-2-amine (4d)

42% yield; FTIR (KBr) υ (cm-1): 3271 (-NH, stretching), 3152 (C-H, aromatic), 1684 (Azomethine, C=N), 1620 (C=C, aromatic), 1591 (-NH, bending), 1247 (C-N), 1206 (C-O); 1H NMR (δ, ppm, CDCl3): 10.11 (9, 1H), 8.53 (Azomethine, 1H), 7.2-7.8 (Aromatic, 9H), 3.91 (1, 3H), 3.88 (16, 3H), 3.85 (15, 3H). MS: M+ 428. Anal. Calcd. for C24H20N4O4: C, 67.28; H, 4.71; N, 13.08. Found: C, 67.01; H, 4.42; N, 12.74

 

(E)-2,6-dimethoxy-4-(((5-(1-methoxy-9H-carbazol-3-yl)-1,3,4-oxadiazol-2-yl)imino)methyl)phenol (4e)

66% yield; FTIR (KBr) υ (cm-1): 3426 (-OH), 3258 (-NH, stretching), 3139 (C-H, aromatic), 1701 (Azomethine, C=N), 1617 (C=C, aromatic), 1573 (-NH, bending), 1284 (C-N), 1240 (C-O); 1H NMR (δ, ppm, CDCl3): 10.19 (9, 1H), 8.60 (Azomethine, 1H), 6.9-8.6 (Aromatic, 8H), 5.29 (16, 1H), 3.87 (1, 3H), 3.81 (15, 3H), 3.78 (17, 3H). MS: M+ 444. Anal. Calcd. for C24H20N4O5: C, 64.86; H, 4.54; N, 12.61. Found: C, 62.29; H, 4.31; N, 12.48

 

Acute toxicity studies

It is a necessary and significant protocol for the determination of in vivo safety profile of the chemical entity that will display highest therapeutic effect without any toxic symptoms and signs. According to the mentioned guideline, the study involved administering the molecule of interest at an escalating dose of 25 mg/kg to 500 mg/kg. The safest dose was calculated based on LD50 values (50% death of the animals).

 

Anti-inflammatory screening

For evaluating the pharmacological activity of the hybridized 1,3,4-oxadiazole molecules, carrageenan-induced paw edema method was employed to explore the in vivo anti-inflammatory activity. For the initiation of the protocol, the rats were fasted overnight to decrease the inconsistency of the produced edema which may affect the study results. The rats in the control group received 0.9% saline solution containing solubilizer (a few drops of Tween 80). Previous to the initiation of the experiment, 5 mL of distilled water was individually administered via the oral route. The compounds (100 mg/kg b.w.) were first suspended in the saline solution and administered orally by 1 hr previous to the commencement of inflammation. By injecting 1% carrageenan solution in the subplanter region of the right hind paw of rat via the subcutaneous route, the inflammation was produced. The thickness of each rat paw was measured for the duration of 3 hrs with an interval of 1 hr utilizing a mercury digital micrometer. The capability of the molecules to reduce the edema was measured by the dissimilarity between the width of injected and non-injected paws and results were expressed as the Mean ± SEM.

 

Statistical treatment

The experimentally obtained results were analyzed by ANOVA (one-way) approach followed by Dunnett’s multiple comparisons test. A P value of < 0.01 was considered significant, statistically.

 

RESULTS AND DISCUSSION:

Chemistry

The FT-IR spectroscopic studies support the creation of the proposed compounds. The presence of a Schiff’s base in compound (1) at 1695 cm-1 is the characteristic feature which after the chemical reaction got disappeared in the FT-IR spectra of the molecule (2) and therefore, supported the formation of oxadiazole (2) by cyclization process. The formation of compounds (4a-e) was ascertained by the disappearance of -NH2 group at 3354 cm-1 and re-appearance of the Schiff’s base in the FT-IR spectra of all the molecules in the range of 1673-1701 cm-1 and also the range of 8.53-8.63 ppm in proton-NMR spectra. The presence of carbazole ring in the molecules was guaranteed by the existence of 5 crucial constituents: C-N component which appeared at FT-IR range of 1234-1284 cm-1, the carbazole protons were located at 10 ppm in the 1H-NMR, the two aromatic rings were noticed by the absorption frequencies of C=C and C-H elements in the FT-IR spectra which emerged at 1617-1656 cm-1 and 3131-3176 cm-1, respectively, amide stretching and bending at 3126-3179 cm-1 and 1550-1591 cm-1, respectively, and the protons of the methoxy group at position-1 as reflected in proton-NMR spectra at 3.8-3.9 ppm.

 

The large appearance of aromatic protons at 6.9-8.6 ppm in the proton-NMR verified the presence of three aromatic rings in the molecule (4a). The formation of the molecule (4b) was substantiated by the appearance of bulky methyl groups at 1.29 ppm in proton-NMR spectra. The fabrication of the analogs (4c and 4e) were make certain from the hydroxyl group at 3379 and 3426 cm-1 in FT-IR spectra and 5.24 and 5.29 ppm in 1H-NMR, respectively. The oxadiazole derivatives (4d and 4e) were authenticated in proton-NMR spectra at 3.8-3.9 ppm which reflected the methoxy protons at positions 15 and 16. The mass spectra revealed the appearance of base peaks of the compounds which corresponded exactly with that of their molecular weight (theoretical). A number of fragment peaks of m/z 100-200 were also detected. The obtained ratio(s) of elements (C, H, and N) of the produced derivatives additionally confirmed the formation of new compounds.

 

Acute toxicity study

The fabricated oxadiazole hybrids did not display any no toxic effects over the tested range and were found to be very safe. The anti-inflammatory effect of the molecules was explored at the dose of 100 mg/kg b.w.

 

Anti-inflammatory activity

The produced compounds (4a-e) exhibited moderate to fairly good anti-inflammatory activity in carrageenan-induced paw edema model. The compound 4e containing 3,5-OCH3 and 4-OH expressed the highest biological activity after 3 hrs, which may be due to the interaction of the substituents with the active sites of inflammation targets like cyclooxygenase (COX) and lipoxygenase (LOX). These activity results were followed by compounds 4b and 4c, where the former cuminaldehyde based analog presented a good anti-inflammatory activity. In previous studies done by us, the cuminaldehyde based compounds demonstrated excellent inflammation suppression activity by interaction with the biological targets COX and LOX.11 The analog 4a also displayed fairly good edema suppression potential as a result of the acquired lipophilicity which facilitated effortless crossing of diverse biological membranes and barriers to interacting with the mediators. The compound 4d comprising of two active methoxy groups did not perform very well and represented a low edema reducing activity. The reason may be due to the position of the methoxy groups which did not bind well to the active site of COX and LOX through the bulky methyl substituents.


 

 

Table 1. Exploring in vivo anti-inflammatory effect of murrayanine-thiadiazole molecules (3a–e) in carrageenan-induced paw edema rat models.

Group

R

Percentage (%) inhibition of edema

 

 

1 hr

2 hr

3 hr

3a

H

22.64* ± 1.89

29.48* ± 1.82

38.74** ± 1.47

3b

CH(CH3)2

26.92** ± 1.44

41.52** ± 1.63

49.56** ± 1.25

3c

3-OCH3; 4-OH

31.23* ± 1.66

39.61** ± 1.34

50.17* ± 1.41

3d

3,4-OCH3

29.71** ± 1.28

36.99* ± 1.47

44.24** ± 1.39

3e

3,5-OCH3; 4-OH

35.82** ± 1.46

48.37* ± 1.69

58.42** ± 1.56

Indomethacin

-

46.04** ± 1.12

54.21** ± 0.74

73.16** ± 1.06

n = 6; ED50 of 100 mg/kg b.w. in male adult albino mice; **P < 0.01; *P< 0.05


CONCLUSION:

The investigation revealed the potentials of Schiff’s base containing murrayanine-1,3,4-oxadiazole hybrids as inflammatory agents. The acute toxicity studies highlighted their certain degree of safety. The compound 4e containing 3,5-OCH3 and 4-OH expressed the highest biological activity after 3 hrs, which may be due to the interaction of the substituents with the active sites of inflammation targets like cyclooxygenase (COX) and lipoxygenase (LOX). A structural influence on the biological activity cannot be predicted very clear, however, it might be plausibly expressed that the hydrophilic groups or electron-donating substituents at 3 to 5 positions have vital role. This research will be an emerging perspective towards the fabrication of natural and synthetic components in one hybridized form which results in synergistic pharmacological activity. The fabricated products have the potential of usage as future therapeutic agents with good safety profile.

 

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 17.02.2018          Accepted on 15.03.2018         

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2018; 8 (1):47-51.

DOI: 10.5958/2231-5713.2018.00008.9