Accepted Manuscript Synthesis of novel 4-nitropyrrole-based semicarbazide and thiosemicarbazide hybrids with antimicrobial and anti-tubercular activity Rajesh A. Rane, Shital S. Naphade, Pavan Kumar Bangalore, Mahesh B. Palkar, Mahamadhanif S. Shaikh, Rajshekhar Karpoormath PII: DOI: Reference:

S0960-894X(14)00505-8 http://dx.doi.org/10.1016/j.bmcl.2014.05.018 BMCL 21631

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

18 March 2014 30 April 2014 5 May 2014

Please cite this article as: Rane, R.A., Naphade, S.S., Bangalore, P.K., Palkar, M.B., Shaikh, M.S., Karpoormath, R., Synthesis of novel 4-nitropyrrole-based semicarbazide and thiosemicarbazide hybrids with antimicrobial and anti-tubercular activity, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl. 2014.05.018

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Synthesis of novel 4-nitropyrrole-based semicarbazide and thiosemicarbazide hybrids with antimicrobial and anti-tubercular activity Rajesh A. Ranea, Shital S. Naphadeb, Pavan Kumar Bangalorec, Mahesh B. Palkara, Mahamadhanif S. Shaikha and Rajshekhar Karpoormatha* a

Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-

Natal, Durban 4001, South Africa. b

Piramal Health Care Ltd, Goregaon, Mumbai, India.

c

S. P. P. School of Pharmacy and Technology Management, NMIMS University, Vile Parle,

Mumbai 400056, India. To receive all correspondence: Rajshekhar Karpoormath: Tel: +27 (0) 312607179, E-mail: [email protected] Abstract We report the synthesis and screening of forty novel 4-nitropyrrole-semicarbazide conjugates inspired from the reported bio-potential of bromopyrrole alkaloids and semicarbazide derivatives for antimicrobial activity. Herein, hybrids 5k-5o, 5r-5s and 5t displayed four-fold increased activity (MIC = 0.39 µg/mL) against E. coli compared to standard ciprofloxacin. Eight hybrids, 5k-5o and 5r-5t displayed equal antibacterial activity (MIC = 1.56 µg/mL) against K. pneumonia compared to standard Ciprofloxacin. Hybrid, 5k -5o (MIC = 0.195 µg/mL) displayed highly potent antibacterial activity against MSSA as compared to standard Ciprofloxacin. Eight-fold superior activity was observed for four hybrids 5k-5m and 5o (MIC = 0.39 µg/mL) against MRSA. Further, nine hybrids displayed four-fold superior antifungal activity (MIC = 0.78 µg/mL) compared to standard Amphotericin B. Encouraging MICs of these hybrids recognize them as promising leads for development of potential antimicrobial drugs. Keywords: 4-Nitropyrrole, semicarbazides, thiosemicarbazides, antimicrobial agent, antitubercular agent

Bacterial resistance to antibacterial agents or antibiotics is of grave concern in the medical community as many species of bacteria have evolved resistance to certain antibiotics and synthetic agents. Therefore, there is a rapidly growing global crisis in the clinical management of life-threatening infectious disease caused by multidrug-resistant strains of the Gram-positive pathogens Streptococcus, Enterococcus, and Staphylococcus as well as the Gram-negative pathogens Escherichia, Salmonella, and certain Pseudomonas strains. Especially, the emergence of multidrug-resistant strains of gram-positive bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermis and vancomycin-resistant Enterococcus is a primarily alarming problem of ever-increasing significance.This increased resistance demands for the discovery and development of novel antimicrobial leads with high efficacy.1-3 Over the years, molecular hybrid-based approachs4 had been exploited by researchers to discover some promising chemical architectures, displaying significant medicinal profiles. Using this approach, we have reported molecular hybridization assisted design, synthesis and evaluation of natural pyrrole alkaloids-inspired conjugates with antimicrobial and anticancer properties.5-7 Our recent exploration of this approach led to synthesis of 1,3,4-oxadiazole-4nitropyrrole hybrids7. The synthesized conjugates were tested for their antimicrobial and antitubercular activity. These hybrids showed promising antibacterial activity against Staphylococcus

aureus,

Bacillus

subtilis,

Escherichia

coli,

Methicillin-resistant

Staphylococcus aureus and Methicillin-sensitive Staphylococcus aureus. Herein, three hybrids demonstrated promising anti-tubercular activity at MIC value less than 1.0 µg/mL against Mycobacterium tuberculosis H37RV. Likewise, equal antifungal activity (MIC = 1.56 µg/mL) compared to standard Amphotericin-B was displayed by most of the hybrids. The promising antibacterial, antifungal and anti-tubercular results along with their non-toxicity profile for mammalian cells indicates that integration of 4-nitropyrrole feature with 1,3,4oxadiazole was favourable for antimicrobial activity. Further from the literature reports it is known that 4-nitropyrrole-based natural products and their derivatives have shown potent antimicrobial activities8-12 (Figure 1). The bio-potential of semicarbazides and thiosemicarbazide derivatives as antibacterial, antifungal and anti-tubercular agents have been well documented in literature (Figure 1).13-19 Semicarbazides are biologically active and nontoxic compounds due to the presence of ureido unit (–NH-CO-NH–), which acts as pseudodipeptide motif. Considering the biological importance of substituted semicarbazides/thiosemicarbazides and 4-nitropyrrole templates, an attempt was made to design a series of novel antimicrobial conjugates incorporating these

features (Figure 2). In this communication we report the synthesis and in vitro screening of semicarbazide and thiosemicarbazide hybrids for their antibacterial, antifungal and antitubercular activities against Escherichia coli (ATCC- 25922), Klebsiella pneumoniae (recultured), Methicillin-resistant Staphylococcus aureus ((MRSA; ATCC 43866) , Methicillin-sensitive Staphylococcus aureus (MSSA; ATCC 35556) and Mycobacterium tuberculosis H37RV. We also reviewed the effect of N-methylation of pyrrole core on antimicrobial activity. These hybrids were further evaluated for their toxicity profile on mammalian cell using VERO cell lines. The target semicarbazide and thiosemicarbazide hybrids were synthesized as illustrated in Scheme 1. Initial key intermediates 1, 2 and 3 were synthesized as per previously reported reaction

scheme

by

our

group.7

Acetylation

of

pyrrole/N-methylpyrrole

using

trichloroacetylchloride gave 2-trichloroacetylpyrrole/N-methyl-2-trichloroacetylpyrrole 1a and 1b in good yield. The acetylated products 1a and 1b underwent reaction with fuming nitric acid in acetic anhydride resulting in to compounds 2a and 2b (4-nitro-1H-2trichloroacetylpyrrole). The obtained nitrated products were reacted with excess of hydrazine hydrate in absolute alcohol to give hydrazide derivatives 3a and 3b.7 Equivalent amounts of arylisocyanates/arylisothiocyanates were added to the hydrazide compounds 3a and 3b in absolute alcohol under reflux conditions to obtain the desired compounds 4 a-t and 5 a-t.20

The synthesized (thio)semicarbazides exhibited encouraging antibacterial, antifungal and anti-tubercular activities as shown in Table 1. Moderate to excellent antibacterial activity (MIC = 0.39 – 6.25 µg/mL) was exhibited by synthesized hybrids against Gram-negative bacterium E. coli. Among the thiosemicarbazides, eight hybrids (5k-5o and 5r-5t) displayed four-fold increased antibacterial activity (MIC = 0.39 µg/mL) followed by 5p (MIC = 0.78 µg/mL) with two-fold superior activity against E. coli compared to standard ciprofloxacin (MIC = 1.56 µg/mL). Presence of halogen on phenyl ring at ring B in hybrids 5m-5o is beneficial for antibacterial activity against Gram-negative bacterium E. coli. N-methylation of pyrrole core is more favored for antibacterial activity against E. coli than pyrrole-NH-free (5c-5e; MIC = 1.56 µg/mL). Hybrids 5l and 5t highlight the benefit of having methoxy group on phenyl ring at ring B for antibacterial activity against E. coli. Superior activity shown by hybrids with pyrrole-N-CH3 substitution (5b and 5j) compared to their NH-free analogs (5l and 5t) point out the favorable effect of N-methylation of pyrrole core for antibacterial activity against E. coli. All the pyrrole-NH-free analogs (5a-5i and 5j) demonstrated similar antibacterial activity (MIC 1.56 µg/mL) compared to standard ciprofloxacin against E. coli

except 5f and 5g. The semicarbazide analogs (4a-4t) exhibited less antibacterial potency (MIC = 1.56 - 6.25 µg/mL) compared to thiosemicarbazides against E.coli. The thiosemicarbazides and N-methylated semicarbazides displayed superior antibacterial activity than their NH-free analogs.

From the forty tested hybrids, most of the compounds displayed moderate to good antibacterial activity (MIC = 1.56 - 6.25 µg/mL) against K. pneumoniae compared to standard ciprofloxacin (MIC = 1.56 µg/mL). The semicarbazide analogs (4a-4t) demonstrated two to three-fold less activity against K. pneumoniae compared to standard ciprofloxacin. However, eight thiosemicarbazide hybrids 5k-5o and 5r-5t showed identical antibacterial activity (MIC = 1.56 µg/mL) against K. pneumoniae compared to standard Ciprofloxacin. This indicated that the thio-analogs, thiosemicarbazides are more favored than semicarbazides for activity against K. pneumoniae. Further, biological data also revealed that N-methylation of the pyrrole core is more favored than pyrrole-NH-free for antibacterial activity against K. pneumoniae. Synthesized semicarbazide and thiosemicarbazide hybrids exhibited moderate to excellent antibacterial activity against Methicillin Susceptible Staphylococcus aureus (MSSA) (MIC = 0.195 - 3.125 µg/mL). Seventeen of the twenty semicarbazide analogs 4a-4t displayed equal antibacterial activity against MSSA (MIC = 1.56 µg/mL) while the hybrids 4e, 4h and 4p exhibited two fold less antibacterial potency (MIC = 3.125 µg/mL) against MSSA compared to standard Ciprofloxacin. However thiosemicarbazide analogs showed moderate to excellent antibacterial activity against MSSA (MIC = 0.195 – 3.125 µg/mL). Among these, four hybrids 5k, 5l, 5m and 5o significantly displayed an eight-fold antibacterial activity (MIC = 0.195 µg/mL) against MSSA as compared to standard Ciprofloxacin. MICs of hybrids 5m (MIC = 0.195 µg/mL) and 5n (MIC = 0.39 µg/mL) indicated that presence of halogen atom at para-position on the phenyl ring is more favored than at ortho- for antibacterial activity against MSSA. Further, antibacterial activity of hybrid 5l (MIC = 0.195 µg/mL) and 5t (MIC = 0.78 µg/mL) revealed that the presence methoxy group at para-position on phenyl ring enhanced the activity. However, substituting more than one methoxy group on phenyl ring decreased activity against MSSA compared to para-methoxyphenyl- substituted analog. Four-fold superior activity was observed for hybrids 5n, 5r and 5s (MIC = 0.39 µg/mL) against MSSA compared to standard Ciprofloxacin. Eight semicarbazide hybrids 5a-5f, 5h and 5i displayed equal antibacterial activity (MIC = 1.56 µg/mL) against MSSA. Analysis of

the anti-MSSA activity revealed that pyrrole-NH-free thiosemicarbazides were less potent compared to pyrrole-N-methylated analogs against MSSA. Overall, thiosemicarbazide hybrids are more favored for anti-MSSA activity than semicarbazides.

In case of Methicillin Resistant Staphylococcus aureus (MRSA) the synthesized semicarbazides and thiosemicarbazides displayed moderate to excellent antibacterial activity (MIC = 0.39 - 6.25 µg/mL) compared to standard ciprofloxacin (MIC = 3.125 µg/mL). The semicarbazides showed moderate antibacterial activity (MIC = 3.125 – 6.25 µg/mL) against MRSA. Unlike semicarbazide hybrids, thiosemicarbazides demonstrated moderate to excellent activity (MIC = 0.78 – 3.125 µg/mL) against MRSA strain. Eight of the synthesized hybrids 5a-5f, 5h and 5i (MIC = 3.125 µg/mL), displayed an equal antibacterial activity against MRSA compared to standard ciprofloxacin. Eight-fold superior potency was observed for hybrids 5k-5m and 5o (MIC = 0.39 µg/mL) against MRSA. Presence of halogen atom like fluorine or chlorine at para-position on phenyl ring at ring B was highly favored for the antibacterial activity against MRSA. Likewise, presence of methoxy group on phenyl ring was preferred for antibacterial activity as indicated by the MICs of hybrids 5l (MIC = 0.39 µg/mL) and 5t (MIC = 1.56 µg/mL). This also showed that presence of three methoxy groups on phenyl ring is less favored for anti-MRSA activity compared to one on para-substition. Four-fold greater activity was observed for four hybrids 5n, 5p, 5r and 5t (MIC = 0.78 µg/mL) against MRSA. Substitution of the fluorine atom at para-postion on phenyl ring (5m, MIC = 0.39 µg/mL) was more preferred for anti-MRSA activity than at ortho-position (5n, MIC = 0.78 µg/mL). Hybrid 5p (MIC = 0.78 µg/mL) with ethyl linker has shown four-fold higher anti-MRSA activity compared to standard Ciprofloxacin indicating beneficial effect of linker between the aryl ring and thiosemicarbazide group for anti-MRSA activity. Overall thiosemicarbazides are more active against MRSA than semicarbazides. The N-methylation of pyrrole core among the thiosemicarbazide hybrid was more beneficial for anti-MRSA activity. Analysis

of

the

biological

data

of

the

hybrids

4

a-t

and

5

a-t

revealed that N-methylation of pyrrole core was beneficial for anti-fungal activity against C. albicans. All the synthesized hybrids displayed moderate to excellent antifungal activity (MIC = 0.78 - 3.125 µg/mL) against C. albicans compared to standard Amphotericin-B (MIC = 3.125 µg/mL). From the semicarbazide analogs (4a-4t), all pyrrole-N-methylated hybrids exhibited either identical or to two-fold greater antifungal activity (MIC = 1.56 - 3.125 µg/mL) against C.albicans. All the pyrrole-NH-free semicarbazides displayed equal

antifungal activity (MIC = 3.125 µg/mL). For thiosemicarbazides (5a-5t), all pyrrole-Nmethylated hybrids significantly displayed four-fold superior antifungal activity (MIC = 0.78 µg/mL) except 5q (MIC = 1.56 µg/mL), which exhibited two-fold superior activity against C.albicans. Hence thiosemicarbazides motif and N-methylation on pyrrole core are more favored features for antifungal activity against C.albicans.

The synthesized hybrids showed moderate to good anti-tubercular activity (MIC = 0.5 - 59.2 µg/mL) against Mycobacterium tuberculosis H37RV using REMA method. Hybrid 5a displayed the highest anti-tubercular activity (MIC = 0.50 µg/mL), significantly very close to the activity of standard Isoniazid (MIC = 0.40 µg/mL). Substitution of nitro group on phenyl ring at para-position was highly favored for anti-tubercular activity against M. tuberculosis. Anti-tubercular activity of hybrid 5a also revealed that pyrrole-NH-free core is beneficial for activity against M. tuberculosis. Hybrid 5k demonstrated less anti-tubercular activity (MIC = 53.8 µg/mL) compared to its N-methylated analog 5a indicating that pyrrole-N-methylation is not favored for activity against M. tuberculosis. Similarly hybrid 5c (MIC = 0.56 µg/mL) exhibited potent anti-tubercular activity close to that of standard isoniazid. Presence of halogen atom at para-position on phenyl ring was also highly favored for anti-tubercular activity. This observation was supported by comparing the anti-tubercular activities of hybrids 5c (MIC = 0.56 µg/mL) and 5d (MIC = 0.82 µg/mL), where in para substitution on phenyl ring with fluorine (5d) decreased anti-tubercular activity compared to ortho-position (5c). Further chloro analog 5e (MIC = 1.3 µg/mL) of hybrid 5c showed decreased antitubercular activity than later indicating that substituting at para-position on phenyl ring with fluorine is more favored than chlorine for activity against M. tuberculosis. Hybrid compounds 5a (MIC = 0.50 µg/mL), 5c (MIC = 0.56 µg/mL) and 5i (MIC = 0.67 µg/mL) have exhibited promising anti-tubercular activity compared to standard Isoniazid. The presence of 4nitrophenyl substitution at ring B was favored for activity against M. tuberculosis as displayed by the good activity of hybrid 4c (MIC = 0.80 µg/mL). In comparison the activity of hybrid 4c (MIC = 0.80 µg/mL) and 5a (MIC = 0.50 µg/mL) revealed that thiosemicarbazide

motif

was

more

preferential

for

anti-tubercular

activity than

semicarbazide. The anti-tubercular activity of hybrid 5d (MIC = 0.82 µg/mL) was almost identical to that of 4c. It was also observed that heterocyclic furfuryl- substitution at ring B in hybrid 4i (MIC = 0.92 µg/mL) favored for anti-tubercular activity against M. tuberculosis. Likewise, potent anti-tubercular activity was observed for hybrids 4d (MIC = 1.1 µg/mL), 5b MIC = 1.3 µg/mL) and 5e (MIC = 1.3 µg/mL). In summary thiosemicarbazide analogs are

more potent than semicarbazides and N-methylation of the pyrrole core has negative effect on the anti-tubercular activity.

The synthesized analogs were further studied for their mammalian toxicity (IC50) using VERO cell lines at concentrations of 62.0 and 62.5 µg/mL. Viability was assessed, after 72 hours, by the conversion of MTT into formazan product using Promega Cell Titre 96 nonradioactive cell proliferation assay22. All the hybrids were found to be non-toxic and the data is tabulated in Table 1. The standard INH toxicity has been taken as >454.7 which was reported previously.23 It can be safely deduced that these synthetic compounds have shown toxicity profiles on-par with standard INH cell toxicity (>454) and are safe to use for further studies.

In conclusion, we have synthesized forty novel 4-nitroopyrrole-semicarbazide conjugates based on the reported bio-prospective of bromopyrrole alkaloids and semicarbazide derivatives, with the objective of evaluating their antimicrobial properties against Escherichia coli, Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus, Methicillinsensitive Staphylococcus aureus and Mycobacterium tuberculosis. The structure activity relational studies give us glimpse that N-alkylation of pyrrole core has shown superior antibacterial and antifungal activity. On the contrary, hybrids with pyrrole-NH-free displayed superior activity against Mycobacterium tuberculosis compared to its N-methylated analogs. All the tested microbes showed more sensitivity towards thiosemicabazide analogs than semicarbazides. In general, presence of substitutions like halogen atom on phenyl ring at ring B is beneficial for antimicrobial activity. Hybrids 5k, 5l, 5m, 5n, 5o, 5r, 5s and 5t displayed four-fold increased activity (MIC = 0.39 µg/mL) against E. coli compared to standard ciprofloxacin. Eight hybrids 5k-5o and 5r-5t displayed equal antibacterial activity (MIC = 1.56 µg/mL) against K. pneumonia compared to standard Ciprofloxacin. Further five thiosemicarbazide 5k-5o (MIC = 0.195 µg/mL) displayed highly potent antibacterial activity against MSSA compared to standard Ciprofloxacin. It was observed that hybrids 5k-5m and 5o (MIC = 0.39 µg/mL) displayed eight-fold increase in potency against MRSA. Nine pyrrole-N-methylated hybrids significantly displayed four-fold superior antifungal activity (MIC = 0.78 µg/mL) than standard Amphotericin B. Excellent MICs shown by these hybrids identify them as promising leads for development of novel antibacterial and antifungal drugs. Further potent activity displayed by five hybrids 5a, 5c, 5i, 5d, and 4c with MICs of 0.50,

0.56, 0.67, 0.82 and 0.80 µg/mL respectively; against Mycobacterium tuberculosis H37RV recognize them as future anti-tubercular lead candidates.

Acknowledgements The authors sincerely thank College of Health Science, University of KwaZulu-Natal, Durban, South Africa for funding this project and Professor R.S. Gaud for providing facility at SPP SPTM, NMIMS, Mumbai to carry out these experiments.

References 1. (a) Suresha, G. P.; Suhas, R.; Kapfo, W.; Gowda, D. C. Eur. J. Med. Chem. 2011, 46, 2530; (b) Lohray, B. B.; Lohray, V. B.; Srivastava, B. K.; Kapadnis, P. B.; Pandya, P. Bioorg. Med. Chem. 2004, 12, 4557. 2. Fu, L.; Liu, Xin.; Ling, C.; Cheng, J.; Guo, X.; He, H.; Ding, S.; Yang, Y. Bioorg. Med. Chem. Lett. 2012, 22, 814. 3. Siwek, A.; Staczek, P.; Stefanska, J. Eur. J. Med. Chem. 2011, 46, 5717. 4. (a) Sashidhara, K. V.; Kumar, M.; Modukuri, R. K.; Srivastava, R. K.; Soni, A.; Srivastava, K.; Singh, S. V.; Saxena, J. K.; Gauniyal, H. M.; Puri, S. K. Bioorg. Med. Chem. 2012, 20, 2971; (b) Barbosa, T. P.; Sousa, S. C. O.; Amorim, F. M.; Rodrigues, Y. K. S.; de Assis, P. A. C.; Caldas, J. P. A.; Oliveira, M. R.; Vasconcellos, M. L. A. A. Bioorg. Med. Chem. Lett. 2011, 19, 4250; (c) Viegas-Junior, C.; Danuello, A.; Bolzani, V. S.; Barreiro, E. J.; Fraga, C. A. Curr. Med. Chem. 2007, 14, 1829. 5. (a) Rane, R. A.; Sahu, N. U.; Shah, C. P. Bioorg. Med. Chem. let. 2012, 22 (23), 7131. (b) Rane, R. A.; Gutte, S. D.; Sahu, N. U. Bioorg. Med. Chem. Lett. 2012, 22, 6429. (c) Rane, R. A.; Sahu, N. U.; Shah, C. P.; Shah, N. K. J. enz. Inh. Med. Chem. 2013, 1. 6. Rane, R. A.; Sahu, N. U.; Gutte, S. D.; Mahajan, A. A.; Shah, C. P.; Bangalore. P. Eur. J. Med. Chem. 2013, 63, 793. 7. Rane, R. A.; Bangalore, P.; Borhade, S. D.; Khandare, P. K. Eur. J. Med. Chem. 2013, 70, 49. 8. Raju, R.; Andrew, M. P.; Diaz, L. X. B.; Zeinab, K.; Robert, J. C. Org. Lett. 2010, 12, 5158. 9. Santo, R. D.; Costi, R.; Artico, M.; Massa, S.; Lampis, G.; Deidda, D.; Pompei, R. Bioorg. Med. Chem. Lett. 1998, 8, 2931.

10. Ching, Y. W.; Chung, W. C.; Keiji, M.; Preston, D. M. T.B. George, Antimicrob. Agents Chemother. 1975, 8, 216. 11. Roberto, D. S.; Roberta, C.; Marino, A.; Silvio, M.; Giorgio, L.; Delia, D.; Raffaello, P. Bioorg. Med. Chem. Lett. 1998, 8, 2931. 12. Pourmorad, F.; Shafiee, A. J. Sci. I. R. Iran. 1998, 9, 30. 13. Asghar, S. F.; Yasin, K. A.; Rehman, H.; Aziz, S. Nat. Prod. Res. 2010, 24, 315 (and the references cited therein). 14. Ismail, I. I.; Mandour, A. H.; Azeem, F. A. Commun. Fac. Sci. Univ. Ank. Series B. 1993, 39, 19 (and the references cited therein). 15. Mohareb, R. M.; Mohamed, A. A. Int. J. Pure. Appl. Chem. 2012, 2, 144 (and the references cited there in). 16. El-Subbagh, H. I.; El-Naggar, W. A.; Badria, F. A.; Med. Chem. Res. 1994, 3, 503. 17. El-Subbagh, H. I.; Al-Obaid, A. M. Eur. J. Med. Chem. 1996, 31, 1017. 18. Shehata, I. A.; El-Subbagh, H. I.; Abdelal, A. M.; El-Sher-beny, M. A. Med. Chem. Res. 1996, 6, 148. 19. Dogan, H. N.; Rollas, S.; Erdeniz, H. Farmaco, 1998, 53, 462. 20. Salgin-Goksen, U.; Gokhan-Kelecki, N.; Goktas, O.; Koysal, Y.; Kilic, E.; Isik, S.; Aktay, G.; Ozalp, M. Bioorg. Med. Chem. 2007, 15, 5738. 21. Palomino, J. C.; Martin, A.; Camacho, M.; Guerra, H.; Swings, J.; Portaels, F. Antimicrob. Agents Chemother. 2002, 46, 2720. 22. Gundersen, L. L.; Nissen-Meyer, J.; Spilsberg, B. J. Med. Chem. 2002, 45, 1383. 23. Sriram. D.; Yogeeswari, P.; Dhakla, P.; Senthilkumar, P.; Banerjee, D. Bioorg. Med. Chem. Lett. 2007, 17, 1888.

Scheme 1: Synthesis of 2-(N-(arylaminocarbonyl)-hydrazinocarbonyl)-4-nitro-1H-pyrrol (4) and 2-(N-(arylaminothiocarbonyl)-hydrazinocarbonyl)-4-nitro-1H-pyrrol (5) O 2N ClCOCCl3 N R R=H/CH3

Anh. ether K 2CO3

N R

CCl3 Fuming HNO3 Acetic anhy. O -10 0C, 2h

1 1a-R=H 1b-R=CH3

4a - phenyl 4b - 4-methoxyphenyl 4c - 4-nitrophenyl 4d - 4-bromophenyl 4e - 4-chlorophenyl 4f - 2,5-dimethoxyphenyl 4g - benzyl 4h - pheneth-2-yl 4i - furfuryl 4j - cyclopentyl 4k - phenyl 4l - 4-methoxyphenyl 4m - 4-nitrophenyl 4n - 4-bromophenyl 4o - 4-chlorophenyl 4p - 2,5-dimethoxyphenyl 4q - benzyl 4r - pheneth-2-yl 4s - furfuryl 4t - cyclopentyl

O2N N R

CCl3 O

N2H4, RT N R

Abs. alc, 2h

2 2a-R=H 2b-R=CH3 5a - 4-nitrophenyl 5b - 4-methoxyphenyl 5c - 4-fluorophenyl 5d - 2-fluorophenyl 5e - 4-chlorophenyl 5f - pheneth-2-yl 5g - benzyl 5h - cyclohexyl 5i - o-tolyl 5j - 3,4,5-trimethoxyphenyl 5k - 4-nitrophenyl 5l - 4-methoxyphenyl 5m - 4-fluorophenyl 5n - 2-fluorophenyl 5o - 4-chlorophenyl 5p - pheneth-2-yl 5q - benzyl 5r - cyclohexyl 5s - o-tolyl 5t - 3,4,5-trimethoxyphenyl

H N

NH2

O

3 3a-R=H 3b-R=CH3

Abs. alc Reflux

Ar-NCO or Ar-NCS

TEA, 4-5h O2N

N R

H N O

X N H

N H

4/5 Ar = aryl/heteroaryl 4(a-j) - R=H, X=O 4(k-t) - R=CH3, X=O 5(a-j) - R=H, X=S 5(k-t) - R=CH3, X=S

Ar

Fig 1: Literature reports for semicarbazide and thiosemicarbazide analogs with their biological activities

Fig 2. Design of (thio)-semicarbazides of 4-nitropyrrole using molecular hybridization method.

Table 1: Antimicrobial results (MIC in µg/mL) of synthesized semicarbazide (4) and Thiosemicarabazide (5) derivatives Compound

E.colia

K.pneumoniaea

MSSA

MRSA

C.albicansb

4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 4s 4t 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t Ciproflixacin AmphoterecinB Isoniazide

3.125 3.125 3.125 3.125 3.125 3.125 6.25 6.25 3.125 3.125 1.56 1.56 1.56 1.56 1.56 1.56 3.125 3.125 1.56 1.56 1.56 1.56 1.56 1.56 1.56 3.125 3.125 1.56 1.56 1.56 0.39 0.39 0.39 0.39 0.39 0.78 1.56 0.39 0.39 0.39 1.56 NT

3.125 3.125 3.125 3.125 3.125 3.125 6.25 6.25 3.125 3.125 3.125 3.125 3.125 3.125 3.125 6.25 3.125 6.25 3.125 3.125 3.125 3.125 3.125 3.125 3.125 6.25 6.25 3.125 3.125 3.125 1.56 1.56 1.56 1.56 1.56 3.125 3.125 1.56 1.56 1.56 1.56 NT

1.56 1.56 1.56 1.56 3.125 1.56 1.56 3.125 1.56 1.56 1.56 1.56 1.56 1.56 1.56 3.125 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 3.125 1.56 1.56 3.125 0.195 0.195 0.195 0.39 0.195 0.39 0.78 0.39 0.39 0.78 1.56 NT

3.125 3.125 3.125 3.125 6.25 3.125 3.125 6.25 3.125 3.125 3.125 3.125 3.125 3.125 3.125 6.25 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 6.25 3.125 3.125 6.25 0.39 0.39 0.39 0.78 0.39 0.78 1.56 0.78 0.78 1.56 3.125 NT

NT

NT

NT

NT

3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 0.78 0.78 0.78 0.78 0.78 0.78 1.56 0.78 0.78 0.78 NT 3.125

Antitubercular activityc 40.6 2.5 0.80 1.1 2.0 14.5 15.2 10.3 0.92 12.5 59.2 26.1 11.9 7.8 30.5 42.4 51.9 42.1 17.0 38.7 0.50 1.3 0.56 0.82 1.3 3.9 7.3 2.8 0.67 2.7 53.8 19.4 6.7 3.2 16.3 23.0 31.9 27.5 11.9 21.5 NT NT

IC50 (µM)d 357.0 400.6 211.3 377.9 218.2 332.8 348.0 220.3 442.8 231.0 305.8 328.0 401.7 602.3 289.5 429.2 304.1 633.0 481.5 271.4 528.9 331.0 331.3 471.2 190.6 281.0 396.7 204.1 461.9 328.1 395.0 183.5 662.4 293.1 210.3 403.5 239.0 352.4 470.6 392.1 NT NT

NT

0.40

NT

a - MIC values are determined by broth dilution method (two fold dilution). b - MIC values are determined by agar dilution method (two fold dilution). c - MIC values are determined by Resazurin micro titer assay (REMA). d – Mammalian toxicity testing using VERO cell lines.

Graphical Abstract Synthesis of novel 4-nitropyrrole-based semicarbazide and thiosemicarbazide hybrids with antimicrobial and anti-tubercular activity Rajesh A. Ranea*, Shital S. Naphadeb, Pavan Kumar Bangalorec, Mahesh B. Palkara, Mahamadhanif S. Shaikha and Rajshekhar Karpoormatha* A series of forty novel semicarbazide and thiosemicarbazide hybrids with 4-nitropyrrole were synthesized and evaluated for antibacterial, antifungal and anti-tubercular activities. Pyrrole core R=H/CH3

O2N N R

H N O

X N H

N H

Ar

(thio)semicarbazide; X = O/S

Ring B Ar = aryl/heteroaryl