Synthesis, Structural Elucidation, Spectral Studies and Antimicrobial Activity of Pyrano-Pyrazole Derivatives

Introduction

The Pyrano-pyrazole moiety is a fascinating template in the pharmaceutical industry, and it is responsible for the molecule’s vast range of biological activity.  Compound containing pyrano -pyrazole moiety can act as antimicrobial¹, analgesic², vasodilator³, anticancer⁴, anti-inflammatory⁵, inhibitors of human Chkl kinase⁶, antifungicidal⁷ and also as biodegradable agrochemicals⁸.  Different methods of one pot multicomponent reactions (MCRs) known for the preparation of pyrano [2,3-c] pyrazole structures, either in two-, three-, or four-component reactions. ^9-14.

In this study, we aimed to synthesis the substituted pyrano-pyrazole derivatives having biological activity as anti-bacterial and anti-fungal activities.

Experimental Section

Materials

All the compounds were synthesized using a high purity grade chemical, reagents, and solvents purchased from S. D. Fine chem and Merck Chemical Co. Aluminum precoated sheets (Merck Kieselgel 60 GF254) were used for TLC, which was observed using an ultraviolet lamp (254 and 365 nm). 

Instrumentation

M. P. observed by a Veergo M. P. Instrument sand uncorrected. ¹H NMR spectral data was obtained through Bruker (300 MHz) spectrophotometer at Sophisticated Analytical Instrumentation Facility (SAIF), Punjab. Mass spectra recorded with Waters Micromass and Fourier Transform Infrared spectroscopy was measured on Shimadzu Spectrophotometer.

Procedure

PART-A: [6-Amino-4-(4-chloro-phenyl)-5-cyano-3-methyl-4H-pyrano[2,3-c]pyrazol-1-yl]-pyridin-4-yl-methanone

A mixture of P-chloro aldehyde (0.01M), pyridine-4-carbohydrazide(0.01M), Ethyl 3-oxobutanoate (0.01M), Propanedinitrile(0.01M) and Sodium benzoate (5 mol%) was stirred in absolute Ethanol (10 ml) for 5-10 minutes at room temperature. After getting the solid, the crude product was filtered using whatman filter paper. Pure product was obtained by recrystallizing the crude product from ethanol.

PART-B: (substituted phenyl amino)-acetyl chloride

4-nitro aniline (0.01M) was dissolved in glacial acetic (15ml) containing 15ml of saturated solution of Sodium acetate. If substance not dissolved completely, the mixture was then warmed at 60 to 70 °C in water bath and subsequently it was cooled in ice-bath with continuous stirring. To avoid vigorous reaction Chloro acetyl chloride (1ml) was added drop wise. The white product was separated out and filtered after 30 minutes. The yield was washed with cooled H₂O and was purify by recrystallization from ethanol.

PART-C:N-[4-(4-Chloro-phenyl)-5-cyano-3-methyl-1-(pyridine-4-carbonyl)-1,4-dihydro-pyrano [2,3-c]pyrazol-6-yl]-2-(substituted phenylamino)-acetamide

A mixture of products from Part-A (0.04M) and Part-B (0.04M) was taken in 25ml of absolute alcohol and refluxed for 4 hours at 65 to 70 °C. TLC was used to monitor the reaction’s conclusion. The reaction mass was put into pieces of ice and filtered when it was finished. To get the pure yield, the product was purify with recrystallization from hot ethanol.

Scheme 1 Click here to View scheme

Scheme 2 Click here to View scheme

Table 1: Structure of all final synthesized compounds. Click here to View table

Compound B1

Yield B1 got as solid (69%). Melting Point 125.6.  Ft-IR spectra, v, cm^-1 : 1426, 1623 (C=C str. Aromatic), 3034 (C-H Aromatic), 1684,1726 (Ar-C=O, C=O), 2242 (CN), 3379 (N-H), 1495(-CH₂), 3034 (-CH₃), 1523 (-NO₂), 742 (-Cl).        

PMR(300 MHz, DMSO) : 2.54 (s, 3H), 3.42(s, 2H), 4.21(s, 1H), 4.61(s, 1H), 7.39 to 8.44 (m, 13H), 8.51 (s, 1H). m/z=569.96, Anal. Calc. (%) for C₂₈H₂₀ClN₇O₅  C 59.00, H 3.54. N 17.20 Instrument Value C 58.99, H 3.51, N 17.19.

Compound B2

Yield B2 got as solid (66%). Melting Point 135.9.  Ft-IR spectra, v, cm^-1 : 1431, 1634 (C=C str. Aromatic), 3039 (C-H Aromatic), 1690,1728 (Ar-C=O, C=O), 2248 (CN), 3384 (N-H), 1499(-CH₂), 3029 (-CH₃), 1534 (-NO₂), 748 (-Cl).        

PMR (300 MHz, DMSO) : 2.35 (s, 3H), 3.63(s, 2H), 4.29 (s, 1H), 4.68(s, 1H), 7.44 to 8.99 (m, 13H), 8.45 (s, 1H). m/z=569.96, Anal. Calc. (%) for C₂₈H₂₀ClN₇O₅ C 59.00, H 3.92. N 17.27 Instrument Value C 58.99, H 3.90, N 17.26.

Compound B3

Yield B3 got as solid (66%). Melting Point139.8Ft-IR spectra, v, cm^-1 : 1419, 1633 (C=C str. Aromatic), 3038 (C-H Aromatic), 1674,1729 (Ar-C=O, C=O), 2252 (CN), 3383 (N-H), 1479(-CH₂), 3044 (-CH₃), 785 (-Cl).

PMR(300 MHz, DMSO) : 2.59 (s, 3H), 3.48(s, 2H), 4.29(s, 1H), 4.73(s, 1H), 7.45 to 8.88 (m, 13H), 8.65 (s, 1H). m/z=559.40, Analytical Calculation (%)  C₂₈H₂₂Cl₂N₆O₃ C 60.12, H 3.60. N 15.02Instrument value C 60.10, H 3.58, N 14.99.

Compound B4

Yield B4 was got as solid (71%). Melting Point 143.8.  Ft-IR spectra, v, cm^-1 : 1418, 1638 (C=C str. Aromatic), 3053 (C-H Aromatic), 1699,1739 (Ar-C=O, C=O), 2263 (CN), 3364 (N-H), 1488(-CH₂), 3039 (-CH₃), 1530 (-NO₂), 746 (-Cl).       

 PMR (300 MHz, DMSO) : 2.67 (s, 3H), 3.31 (s, 2H), 4.11(s, 1H), 4.55(s, 1H), 7.49 to 8.91 (m, 13H), 8.66 (s, 1H). m/z=569.96, Anal. Calc. (%) for C₂₈H₂₀ClN₇O₅  C 59.00, H 3.54. N 17.20 Found C 58.98, H 3.52, N 17.17.

Compound B5

Yield B5 was got as solid (63 %). Melting Point 110.5.  Ft-IR spectra, v, cm^-1 : 1446, 1635 (C=C str. Aromatic), 3039 (C-H Aromatic), 1689,1741 (Ar-C=O, C=O), 2235 (CN), 3366 (N-H), 1489(-CH₂), 3039 (-CH₃), 749 (-Cl).  ¹H NMR (300 MHz, DMSO) : 2.34 (s, 3H), 3.48(s, 2H), 4.23(s, 1H), 4.66(s, 1H), 7.35 to 8.49 (m, 13H), 8.55 (s, 1H). m/z=538.98, Analytical Calculation (%) for C₂₉H₂₃ClN₆O₃ C64.62, H 4.30. N 15.59.Instrument Value C 64.59, H 4.29, N 15.58.

Compound B6

Yield B6 was got as solid (70 %). Melting Point 125.7.  Ft-IR spectra, v, cm^-1 : 1429, 1625 (C=C str. Aromatic), 3033 (C-H Aromatic), 1682,1728 (Ar-C=O, C=O), 2245 (CN), 3381 (N-H), 1498(-CH₂), 2968 (-CH₃), 745 (-Cl).  

PMR(300 MHz, DMSO) : 2.52 (s, 3H), 3.44(s, 2H), 4.23(s, 1H), 4.65(s, 1H), 7.40 to 8.43 (m, 13H), 8.49 (s, 1H). m/z=538.98, Analytical Calculation (%) for C₂₉H₂₃ClN₆O₃  C 64.62, H 4.30. N 15.59 Instrument Value C 64.60, H 4.28, N 15.56.

Compound B7

Yield, B7 was got as solid (64 %). Melting Point 113.2.  Ft-IR spectrum, v, cm^-1 : 1427, 1638 (C=C str. Aromatic), 3038 (C-H Aromatic), 1685,1729 (Ar-C=O, C=O), 2249 (CN), 3372 (N-H), 1499(-CH₂), 2925 (-CH₃), 745 (-Cl).  

PMR(300 MHz, DMSO) : 2.58 (s, 3H), 3.45(s, 2H), 4.25(s, 1H), 4.66(s, 1H), 7.44 to 8.49 (m, 13H), 8.55 (s, 1H). m/z=538.98, Analytical Calculation (%) for C₂₉H₂₃ClN₆O₃  C 64.62, H 4.30. N 15.59 Instrument Value C 64.61, H 4.28, N 15.57.

Compound B8

Yield B8 got as solid (65 %). Melting Point 161.8.  Ft-IR spectrav, cm^-1 : 1427, 1622 (C=C str. Aromatic), 3033 (C-H Aromatic), 1685,1725 (Ar-C=O, C=O), 2244 (CN), 3384 (N-H), 1497(-CH₂), 3038 (-CH₃),  747 (-Cl).PMR (300 MHz, DMSO) : 2.59 (s, 3H), 3.46(s, 2H), 4.25(s, 1H), 4.66(s, 1H), 7.44 to 8.54 (m, 13H), 8.55 (s, 1H). m/z=524.96, Analytical Calculation (%) for C₂₈H₂₁ClN₆O₃  C 64.06, H 4.03. N 16.01 Instrument Value C 64.02, H 4.01, N 16.00.

Results and Discussion

First synthesis of [2,3-c]pyrazole type derivatives were prepared by Gein et al. via one-pot  reaction manners¹⁵.  In Present work, one-pot design of pyrano[2,3-c]pyrazole derivatives through MCR of Ethyl 3-oxobutanoate, pyridine-4-carbohydrazide, Propanedinitrile, and substituted benzaldehydes. Here, we performed antimicrobial study of synthesized pyrano[2,3-c]pyrazoleheterocyclic derivatives.

Antibacterial activity

Figure 1: Antibacterial activity. Click here to View figure

Table 2: Antibacterial activity of synthesized compounds.

Compound	Gram +		Gram –
	MTCC 96	MTCC 442	MTCC 433	MTCC 1688
	S. Aureus	S. Pyogenus	E. Coli	P. Aeruginosa
B-1	100	62.5	125	125
B-2	250	500	250	200
B-3	250	250	250	250
B-4	125	200	125	100
B-5	62.5	200	50	100
B-6	125	250	250	500
B-7	250	100	100	125
B-8	50	100	62.5	25

 

Antibacterial activity shown that compound B-1 is good active against S.Pyogenus. B-5 is good active against S.Aures and E.coli and B-8 is good active against S. Aureus, E. Coli and very good activity on P. Aeruginosa.

Antifungal activity

Figure 2: Antifungal activity Click here to View figure

Table 3: Antifungal activity of synthesized compounds.

Compound	Minimal Inhibition Concentration
	MTCC 227	MTCC 282	MTCC 1323
	C. Albicans	A. Niger	A. Clavatus
B-1	500	500	>1000
B-2	500	500	1000
B-3	500	500	500
B-4	200	250	250
B-5	250	500	100
B-6	100	100	500
B-7	200	500	500
B-8	500	250	250

 

Antifungal activity shown that Product B-4 and B-7 are moderately reactive against  C.Albicans.  Product B-6 is better reactive against C. Albicans, A. Niger and compound B-5 good active against A. Clavatus.

Acknowledgment

The authors would like to acknowledge The HNSB. Ltd. Science College, Himmatnagar and P. T. Sarvajanik Science College, Surat for providing  lab. Facility.

Conflicts of Interest

The Authors declare that there are no conflict of interest regarding this research paper.

References

1. Mistry, P.T.; Kamdar, N.R.; Haveliwala, D.D.; Patel, S. K. J. Heterocycl. Chem. 2012, 49 (2), 349–357
   CrossRef
2. Kuo, S.C.; Huang, L.J.; Nakamura, H. J. Med. Chem. 1984, 27 (4), 539–544
   CrossRef
3. Ahluwalia, V.K.; Dahiya, A.; Garg, V. Indian J. Chem.,Sect B, 1997,36 (1), 88–90
4. Wang, J. L.; Liu, D.; Zheng, Z.J.; Shan, S.; Han, X.; Srinivasula, S.M.; Croce, C.M.; Alnemri, E.S.; Huang, Z. Proc. Natl. Acad. Sci. U.S.A.,2009, 97 (13), 7124–7129
5. Mandha, S. R.; Siliveri, S.; Alla, M.; Bommena, V. R.; Bommineni, M.R.; Balasubramanian, S. Bioorg. Med. Chem. Lett., 2012, 22 (16), 5272–5278
   CrossRef
6. Foloppe, N.; Fisher, L.M.; Howes, R.; Potter, A.; Robertson, A.G.S.; Surgenor, A.E. Bioorg. Med. Chem. 2006, 14(14), 4792–4802
   CrossRef
7. Ramiz, M.M.M.; Hafiz, I.S.A.; Rahim, M.A.M.A.; Gaber, H.M. J. Chin. Chem. Soc.2012,59 (1), 72–80
8. Kiyani, H.; Samimi, H.A.; Ghorbani, F.;Esmaieli, S.;Curr. Chem. Lett.2013,2 (4), 197–206
9. Otto, S.; Engberts, J.B.F.N. Pure Appl. Chem. 2000, 72 (7), 1365– 1372
   CrossRef
10. Shestopalov, A. M.; Emeliyanova, Y. M.; Shestopalov, A. A.; Rodinovskaya, L. A.; Niazimbetova, Z. I.; Evans, D.H. Tetrahedron, 2003, 59 (38), 7491–7496
    CrossRef
11. Zonouz, A. M.; Eskandari, I.; Khavasi, H.R. Tetrahedron Lett., 2012, 53 (41), 5519–5522
    CrossRef
12. Aslam, N.; White, J. M.; Zafar, A. M.; Abdul, M. J.; Ghafoor, N.; Sajid; Noreen, S; Khan, M.A. Arkivoc, 2018, part vi, 139-203
    CrossRef
13. Khalid M.; Shireen M.  Research Journal of Chemistry and Environment, 2019,    23(10). 139-156
14. Ganta, R. K.; Kerru, N.; Maddila S.; Jonnalagadda, S. B. Molecules, 2021, 26, 3270
    CrossRef
15. Gein, V.L.; Zamaraeva, T.M.; Kozulina, I.V. Russ. J. Org. Chem., 2014, 50 (5), 691–693
    CrossRef

---

This work is licensed under a Creative Commons Attribution 4.0 International License.