Phosphorus, Sulfur, and Silicon and the Related

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N-Phosphorylated Derivatives of 5-Nitroindazole as Antimicrobial and Antioxidant Agents and Docking Study against DNA GyraseA a

a

a

b

Sk Thaslim Basha , Devineni Subba Rao , Golla Madhava , Shaik Thahir Basha , Mundla c

d

d

Nagalakshmi Devamma , Saddala Madhu Sudhana , Asupatri Usha Rani & Chamarthi Naga Raju

a

a

Department of Chemistry, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India b

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Department of Virology, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India c

Department of Botany, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India

d

Department of Zoology, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India Accepted author version posted online: 16 Oct 2014.

To cite this article: Sk Thaslim Basha, Devineni Subba Rao, Golla Madhava, Shaik Thahir Basha, Mundla Nagalakshmi Devamma, Saddala Madhu Sudhana, Asupatri Usha Rani & Chamarthi Naga Raju (2014): N-Phosphorylated Derivatives of 5Nitroindazole as Antimicrobial and Antioxidant Agents and Docking Study against DNA GyraseA, Phosphorus, Sulfur, and Silicon and the Related Elements, DOI: 10.1080/10426507.2014.965818 To link to this article: http://dx.doi.org/10.1080/10426507.2014.965818

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ACCEPTED MANUSCRIPT Running Head: N-Phosphorylated Derivatives of 5-Nitroindazole and their Biological activity. N-Phosphorylated Derivatives of 5-Nitroindazole as Antimicrobial and Antioxidant Agents and Docking Study against DNA GyraseA

SK Thaslim Basha1, Devineni Subba Rao1, Golla Madhava1, Shaik Thahir Basha2, Mundla


Nagalakshmi Devamma3, Saddala Madhu Sudhana4, Asupatri Usha Rani4, Chamarthi Naga Raju1*

1

Department of Chemistry, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh,

India. 2

Department of Virology, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India.

3

4

Department of Botany, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India.

Department of Zoology, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India.

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Abstract A series of new phosphoramidate derivatives 7a-l were synthesized by reacting 5nitroindazole with 4-chlorophenyl dichlorophosphate (4), and then further with various biopotent aromatic/hetero/alkyl amines 6a-l. All the newly synthesized compounds were


characterized by spectroscopic data (IR, 1H,

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C,

31

P NMR and mass) and elemental analysis.

The products 7a-l were evaluated for their antioxidant activity (DPPH, H2O2 methods and IC50 values) and antimicrobial activity against the growth of Gram bacterial (positive and negative) and fungal pathogens. In the entire bio-screening data, we observed that 7j is the most active compound in terms of its antimicrobial and antioxidant activities. Further to know the binding interactions of title products with DNA GyraseA enzyme (E. coli), molecular docking studies were performed which revealed 7j and 7k exhibited high binding affinities towards the enzyme, DNA GyraseA (GyrA), and are higher than that of the standard antibiotic, streptomycin.

Antimicrobial Activity O2N

O2N N N H 3

N N O P R O

Cl

Docking

7(a-l)

Antioxidant activity

Keywords: 5-Nitroindazole, Phosphoramidates, Antimicrobial activity, Antioxidant activity, Molecular docking studies.

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INTRODUCTION Organophosphorus compounds have gained considerable intensive interest because of the wide range of applications in numerous fields such as medicine, agriculture and industry. 1-3 Among the various kinds of phosphorus compounds, phosphoramidates having a P-N


functionality play a vital role in biology. Particularly, these derivatives are often used as prodrugs to increase the water solubility and enhance the bio-availability of the parent drug and increase the penetration nature of the drug into the cell membrane. Many phosphoramidates have been synthesized as potential anticancer and antiviral agents.4-5 Therefore, extensive research has been focused on the synthesis of new bio-active phosphoramidate entities. Recently our research group has reported the synthesis of phophoramidate derivatives6-7 and evaluated their antimicrobial and antioxidant activity8 and found that phosphoramidate derivatives showed excellent activities. Further, nitrogen containing heterocyclic compounds such as 1H-indazole, pyrazole, pyrazoline, and pyrazolidine are the building blocks for a wide range of biologically active compounds.9 Among these indazole is an important pharmaceutical and emerging heterocyclic compound with a broad spectrum of activities such as antibacterial,10 antifungal,11 antiprotozoal,12 anticancer,13 anti-inflammatory,14 HIV protease inhibitor,15 antihypertensive agent,16 anti-obesity agent,17 thrombin receptor antagonist,18 lipase and phospholipase inhibitors,19 carbonyl reductase inhibitor,20 and 5-hydroxytryptamine agonist.21 Indazole containing a nitro group at the 5-position are considered as a very important moiety for biological applications. According to Aran et al.22 1-(2-((diethylamino)methyl)benzyl)-5-

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ACCEPTED MANUSCRIPT nitro-1H-indazol-3-ol (1) exhibited remarkable trichomonacidal activity against Trichomonas vaginalis. Rodriguez et al.23 synthesized 1-(5-(butylamino)pentyl)-5-nitro-1H-indazol-3-ol (2), showed good anti-Trypanosoma cruzi activity. These nitro groups have the ability to produce radical species capable of inducing a cascade of reduced materials. These reduced intermediates can cause cellular damage directly by reacting with various biological macromolecules or by


generating highly reactive hydroxyl radicals.24 [Insert Figure 1 here] The above facts inspired us to synthesize phosphoramidate derivatives of 5-nitroindazole and to evaluate antimicrobial and antioxidant activities. Docking studies were performed to know the binding interaction of the target compounds. Some of the compounds exhibited potent antimicrobial and antioxidant activities and are known to possess good binding energies. RESULTS AND DISCUSSION Chemistry As a part of our research programme on designing the new bioactive phosphoramidate derivatives and their biological evaluation, we synthesized a series of N-Phosphorylated derivatives of 5-nitroindazole 7a-l which was outlined in Scheme 1. Initially, the intermediate, 4-chlorophenyl 5-nitro-1H-indazol-1-ylphosphonochloridate (5) was formed in good yield (65 %) by the reaction of 5-nitroindazole (3) and 4-chlorophenyl dichlorophosphate (4) in the presence of NaH. Subsequently, the target products, phosphoramidate derivatives of 5-nitroindazole 7a-l, were achieved by the substitution of various commercially available and bioactive substituted amines 6a-l (1o and 2o) in the place of ‘Cl’ present in the intermediate 5 in the presence of 1,4-dimethyl piperazine as a base. All the

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ACCEPTED MANUSCRIPT synthesized compounds were purified by column chromatography using chloroform:methanol (9:1) as an eluent and all the products obtained in good yields (68-79 %). The structures of the newly synthesized intermediate 5, and title compounds 7a-l were confirmed by spectroscopic data (IR, NMR [1H, 13C and

31

P] and mass) and elemental analysis.

The disappearance of the absorption band at 3267 cm-1 for –NH of 5-nitroindazole, appearance


of new band at 1205 cm-1 corresponding to the P=O functionality in the IR spectra and the disappearance of the signal at 9.80 ppm for –NH proton, and new signals in the aromatic region for the 4-chloro phenyl ring of 4-chlorophenyl dichlorophosphate in 1H NMR spectra of compound 5 confirmed that the –NH was involved in the substitution with 4-chlorophenyl dichlorophosphate (4). Similarly, stretching absorption bands were observed in the regions of 745-793, 1043-1079, 1212-1242, 3125-3334 and 3360-3391 cm-1 in the IR spectra of title compounds 7a-l which confirmed P-N, P-O, P=O, -NH and –OH bonds respectively. In

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P

NMR, the signal at δp from -4.4 to -14.4 confirmed the presence of P-atom in the synthesized compounds. In 1H NMR spectra, the signals in the regions of δ 6.24-8.91, 4.70-6.44 and 1.164.50 were assigned to the aromatic protons, NH proton and aliphatic protons respectively. The product obtained is a mixture of diastereomer and are non-resolvable. 13C NMR spectra and mass spectroscopy showed signals and molecular ions at their expected values and agreed well with the proposed structures respectively. Antimicrobial activity The in vitro antibacterial activity of the synthesized compounds 7a-l were tested against two Gram-positive bacteria, Bacillus subtilis (MTCC-441) and Staphylococcus aureus (MTCC-737) and Gram-negative bacteria, Escherichia coli (MTCC-443) and Pseudomonas aeruginosa

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ACCEPTED MANUSCRIPT (MTCC-741) at the concentration of 100 µg/mL using agar well diffusion method25 reveals that the compounds 7a, 7c, 7g and 7i exhibited good activity against B. subtilis. Similarly compounds 7b, 7d and 7k were more active against S. aureus, compounds 7a, 7g and 7i exhibited promising activity against E. coli and compounds 7e, 7h and 7j showed significant activity against P. aeruginosa. The results were tabulated in Table S 1.


The antifungal screening data (Figure S 1) against four fungal pathogens Viz., Aspergillus niger, Aspergillus flavus, Sclerotium rolfsii and Colletotrichum gloeosporioides by poisoned food technique26 revealed that all the title compounds exhibited good to moderate activity against all the fungal pathogens except C. gloeosporioides (foliar fungus). Whereas, compounds 7a, 7e, 7g and 7h showed good activity against A. niger, ranges from 53.3 to 56.6 % of inhibition over control, similarly the compounds 7e, 7h and 7k (51.1 % of inhibition over control) exhibited promising activity against A. flavus. Compounds 7c, 7h, 7j and 7k (50.0-55.5 % of inhibition over control) showed potential activity against S. rolfsii. Nonetheless the title compounds 7a-l showed less than 50 % of inhibition over control against C. gloeosporioides. From the results, the compounds 7a, 7h and 7j exhibited efficient activity against bacteria and fungi, when compared to other compounds. The structural units dopamine, furfural and 2piperazine pyridine respectively might be the reason to exhibit potency of activity. Hence, the molecules can be considered as the most biologically active compounds and can alter the structural components to enhance the activity in future studies. Further, the results indicate that the compounds 7a-l which suppressed the foliar pathogen, C. gloeosporioides is of high potent activity as it is highly difficult to manage the foliar pathogens in the field. Thus, these

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ACCEPTED MANUSCRIPT compounds can be exploited for field trials and can be integrated in the Indian agriculture after toxicological studies. Antioxidant activity The in vitro antioxidant activity of the newly synthesized compounds was screened using DPPH radical scavenging27 and H2O2 scavenging methods28 (Table S 2). In addition, IC50 values


were also determined in DPPH method which is summarized in Figure S 3. All the target compounds exhibited good to potential antioxidant activity. Compounds 7d, 7g and 7j showed the highest activity among the title compounds, the reason might the presence of aliphatic cyclic tertiary amine directly linked to the phosphorus atom which involves in the abstraction of radicals. Compounds 7d (IC50, 18.53 µg/mL) and 7j (IC50, 18.21 µg/mL) possess almost equal IC50 values when compared to the standard ascorbic acid (IC50, 17.30 µg/mL). Docking studies To elucidate the binding mode of the lead compounds, in silico studies were carried out for phosphoramidate derivatives of 5-nitroindazole 7a-l endowed for DNA GyraseA (E.coli) inhibition. GyrA contains 889 amino acids; tyrosine is the active site that forms a covalent intermediate with the DNA. Docking studies were made to investigate the binding affinities between the compounds and active site of protein. Docking calculation were performed using Auto Dock Vina in PyRx Virtual Screening tool29 (runs-10, exhaustiveness-8, No. modes-5, energy range-25000, grid size- x-67, y-72, z-63) and X-ray crystal structure of GyrA was obtained from Protein Data Bank (PDB) code 1AB4. The docking scores and figures were summarized in Tables S 3 – S 5 and Figure S 4 respectively. (See Supplemental Materials)

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ACCEPTED MANUSCRIPT The results revealed that all the synthesized compounds 7a-l show high binding energies than 5-nitroindazole and standard antibiotic, streptomycin. Among the title compounds 7j, 7k, 7b and 7f exhibited the highest binding energies than the remaining molecules. The interaction between ligand and protein may be due to polar (hydrogen bonds) and non polar bonds (Van der Waal and electrostatic bonds).30-32 Compound 7j showed interaction with His45, Tyr266, and Gln267


having the highest binding affinity of -9.2 kcal/mol whereas 7k binds with only one amino acid, Arg517 and exhibited good binding energy of -8.3 kcal/mol. Interestingly, the docking studies coincide with in vitro antimicrobial activity. The docking images of the potent compounds 7j and 7k were summarised in Figure 2. [Insert Figure 2 here] EXPERIMENTAL Synthetic

procedure

for

the

synthesis

of

4-chlorophenyl-5-nitro-1H-indazol-1-

ylphosphonochloridate (5) 5-Nitroindazole (3) (2 mmol, 326 mg) was taken in a 50 mL flask containing THF (15 mL) and cooled to 0 oC using an ice-bath. Hexane washed sodium hydride (3 mmol, 72 mg) was added to the reaction mixture and stirred for 2h. After completion of the reaction, checked by TLC (H2 gas was evolved during the reaction), 4-chlorophenyl phosphorodichloridate (4) (2 mmol, 0.32 mL) in THF (5 mL) was added drop-wise at 0 oC through a dropping funnel. After addition was over, the reaction was maintained at 35 oC. The progress of the reaction was monitored by TLC (ethyl acetate:hexane 9:1). After completion of the reaction, the reaction mixture was filtered off to remove the NaCl salt and the filtrate was concentrated under reduced pressure to obtain the crude monochloride intermediate, 4-chlorophenyl-5-nitro-1H-indazol-1-

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ACCEPTED MANUSCRIPT ylphosphonochloridate (5). The product was purified by column chromatography using ethyl acetate: hexane (3:2) as an eluent. 4-Chlorophenyl-5-nitro-1H-indazol-1-ylphosphonochloridate (5): Pale yellow solid, Yield: 65 %, Mp: 121-124 oC. IR (νmax cm-1): 1205 (P=O str), 1053 (P-O str), 756 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.84 (1H, s, Ar-H), 8.41 (1H, s, Ar-H), 8.19 (1H, d, J = 2.0 Hz, Ar-H),


8.08 (1H, d, J = 2.0 Hz, Ar-H), 7.74 (2H, d, J = 5.0 Hz, Ar-H), 7.33 (2H, d, J = 5.0 Hz, Ar-H). 31

P NMR (161.9 MHz, DMSO-d6) δ: 4.1. MS (m/z): 371 (100%) (M+H+), 373 (34%).

General procedure for the synthesis of compounds 7a-l The mixture of 4-chlorophenyl-5-nitro-1H-indazol-1-ylphosphonochloridate (5) (1 mmol, 372 mg), tryptamine (6b) (1 mmol, 160 mg) and 1,4-dimethyl piperazine (DMPipz) (1.2 mmol, 0.16 mL) as a base in THF (10 mL) was stirred for 4 h at 50 oC. The progress of the reaction was monitored by TLC using chloroform:methanol (4:1). After completion of the reaction, DMPipz.HCl salt was removed by filtration and the filtrate was evaporated to obtain the crude product, 4-chlorophenyl N-2-(1H-indol-3-yl)ethyl-P-(5-nitro-1H-indazol-1-yl) phosphonamidate (7b). The crude product was purified by column chromatography using chloroform:methanol (9:1) as an eluent and the same synthetic procedure was adopted for the synthesis of remaining title products as shown in Scheme 1. [Insert Scheme 1 here] Physical and spectral data of the newly synthesized compounds 4-Chlorophenyl

N-3,4-dihydroxyphenethyl-P-(5-nitro-1H-indazol-1-yl)phosphonamidate

(7a): Dark brown solid, Yield: 75 %, Mp: 154-157 oC. IR (νmax cm-1): 3380 (OH str), 3235 (NH str), 1212 (P=O str), 1079 (P-O str), 786 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 12.91 (2H,

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ACCEPTED MANUSCRIPT s, Ar-OH), 8.90 (1H, s, Ar-H), 8.47 (1H, s, Ar-H), 8.27 (1H, d, J = 2.0 Hz, Ar-H), 8.25 (1H, d, J = 2.0 Hz, Ar-H), 7.80-6.68 (7H, m, Ar-H), 5.81 (1H, s, NH), 2.70 (2H, t, J = 2.0 Hz), 2.57 (2H, t, J = 2.0 Hz). 13C NMR (125 MHz, DMSO-d6) δ: 153.2 (C7), 148.6 (C8), 143.4 (C4), 141.7 (C19), 141.5 (C18), 129.1 (C10, 12), 128.2 (C1), 128.1 (C11), 125.5 (C2), 122.4 (C9, 13), 122.1 (C16), 122.0 (C5), 121.3 (C3), 120.8 (C21), 118.8 (C17), 111.8 (C20), 110.9 (C6), 45.4 (C14), 39.3 (C15).

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P


NMR (161.9 MHz, DMSO-d6) δ: -12.4. MS (m/z): 489 (100%) (M+H +), 491 (35%). Anal. Calcd for C21H18ClN4O6P C, 51.60; H, 3.71; N, 11.46 Found: C, 51.52; H, 3.67; N, 11.55.

4-Chlorophenyl

N-2-(1H-indol-3-yl)ethyl-P-(5-nitro-1H-indazol-1-yl)phosphonamidate

(7b): Brown Solid, Yield: 79 %, Mp: 170-173 oC. IR (νmax cm-1): 3120 (NH str), 1236 (P=O str), 1043 (P-O str), 787 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 11.01 (1H, s, Ar-NH), 8.88 (1H, s, Ar-H), 8.41 (1H, s, Ar-H), 8.20 (1H, d, J = 2.0 Hz, Ar-H), 8.18 (1H, d, J = 2.0 Hz, Ar-H), 7.53-6.95 (9H, m, Ar-H), 5.74 (1H, s, NH), 3.63 (2H, t, J = 3.5 Hz, NCH2), 2.98 (2H, t, J = 3.5 Hz, NCH2CH2).

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C NMR (125 MHz, DMSO-d6) δ: 153.2 (C7), 146.2 (C8), 143.7 (C4), 136.0

(C22), 129.2 (C10, 12), 128.0 (C17), 127.3 (C1), 126.2 (C11), 125.0 (C2), 123.4 (C23), 122.2 (C9, 13), 122.0 (C5), 121.9 (C3), 121.5 (C20), 118.7 (C19), 118.2 (C18), 112.0 (C6), 110.1 (C16), 107.9 (C21), 45.5 (C14), 24.6 (C15).

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P NMR (161.9 MHz, DMSO-d6) δ: -11.7. MS (m/z): 496 (100%)

(M+H+), 162 (60%), 498 (33%). Anal. Calcd for C23H19ClN5O4P C, 55.71; H, 3.86; N, 14.12; Found: C, 55.66; H, 3.91; N, 14.07.

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ACCEPTED MANUSCRIPT 4-Chlorophenyl 5-nitro-1H-indazol-1-yl (pyrrolidin-1-yl)phosphinate (7c): Semi solid, Yield: 73 %. IR (νmax cm-1): 1233 (P=O str), 1043 (P-O str), 785 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.85 (1H, s, Ar-H), 8.44 (1H, s, Ar-H), 8.20 (1H, d, J = 2.5 Hz, Ar-H), 8.17 (1H, d, J = 2.5 Hz, Ar-H), 7.75 (2H, d, J = 9.0 Hz, Ar-H), 7.60 (2H, d, J = 9.0 Hz, Ar-H), 2.92 (4H, t, J = 3.5 Hz, NCH2CH2), 2.16 (4H, t, J = 3.5 Hz NCH2CH2).

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C NMR (125 MHz, DMSO-d6) δ:


153.4 (C7), 148.4 (C8), 143.3 (C4), 129.4 (C10, 12), 128.1 (C1), 127.7 (C11), 125.4 (C2), 122.6 (C9, 13),

122.0 (C5), 121.2 (C3), 111.9 (C6), 48.6 (C14,

17),

27.6 (C15,

16).

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P NMR (161.9 MHz,

DMSO-d6) δ: -6.8. MS (m/z): 407 (75%) (M+H+), 335 (100%), 409 (25%). Anal. Calcd for C17H16ClN4O4P C, 50.20; H, 3.96; N, 13.77; Found: C, 50.11, H, 3.90; N, 13.85.

4-Chlorophenyl morpholino(5-nitro-1H-indazol-1-yl)phosphinate (7d): Yellow solid, Yield: 72 %, Mp: 230-233 oC. IR (νmax cm-1): 1220 (P=O str), 1067 (P-O str), 790 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.84 (1H, s, Ar-H), 8.42 (1H, s, Ar-H), 8.20 (1H, d, J = 2.0 Hz, Ar-H), 8.17 (1H, d, J = 2.0 Hz, Ar-H), 7.76 (2H, d, J = 8.5 Hz, Ar-H), 7.59 (2H, d, J = 8.5 Hz, Ar-H), 3.60 (4H, t, J = 3.5 Hz, OCH2), 3.01 (4H, t, J = 3.5 Hz, NCH2). 13C NMR (125 MHz, DMSO-d6) δ: 153.9 (C7), 148.5 (C8), 143.4 (C4), 129.3 (C10, 12), 128.5 (C1), 128.2 (C11), 125.6 (C2), 122.5 (C9, 13), 122.1 (C5), 121.5 (C3), 112.0 (C6), 64.4 (C14, 17), 42.8 (C15, 16).

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P NMR (161.9 MHz,

DMSO-d6) δ: -7.1. MS (m/z): 423 (100%) (M+H+), 425 (35%). Anal. Calcd. for C17H16ClN4O5P C, 48.30; H, 3.81; N, 13.25; Found: C, 48.22; H, 3.90; N, 13.33.

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ACCEPTED MANUSCRIPT 4-Chlorophenyl P-5-nitro-1H-indazol-1-yl-N-(thiophen-2-ylmethyl) phosphonamidate (7e): Grey solid, Yield: 77 %, Mp: 190-193 oC. IR (νmax cm-1): 3210 (NH str), 1242 (P=O str), 1068 (P-O str), 765 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.81 (1H, s, Ar-H), 8.41 (1H, s, ArH), 8.06-7.26 (9H, m, Ar-H), 4.76 (1H, s, NH), 3.63 (2H, s, NCH2).

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C NMR (125 MHz,

DMSO-d6) δ: 153.0 (C7), 148.1 (C8), 143.5 (C4), 139.0 (C15), 137.2 (C16), 129.9 (C10, 12), 128.5


(C1), 128.1 (C11), 124.3 (C2), 122.0 (C9, 12), 121.9 (C5), 121.2 (C3), 112.7 (C17), 112.1 (C6), 108.4 (C18), 36.6 (C14). 31P NMR (161.9 MHz, DMSO-d6) δ: -5.7. MS (m/z): 449 (100%) (M+H+), 286 (62%) 451 (33%). Anal. Calcd for C18H14ClN4O4PS C, 48.17; H, 3.14; N, 12.48; Found: C, 48.26; H, 3.16; N, 12.41.

4-Chlorophenyl4-methylpiperazin-1-yl(5-nitro-1H-indazol-1-yl) phosphinate (7f): Semi solid, Yield: 71 %, IR (νmax cm-1): 1238 (P=O str), 1067 (P-O str), 788 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.81 (1H, s, Ar-H), 8.40 (1H, s, Ar-H), 8.19 (1H, d, J = 2.5 Hz, Ar-H), 8.17 (1H, d, J = 2.5 Hz, Ar-H), 7.74 (2H, d, J = 9.0 Hz, Ar-H), 7.60 (2H, d, J = 9.0 Hz, Ar-H), 2.852.50 (11H, m, N-methyl piperazine). 13C NMR (125 MHz, DMSO-d6) δ: 153.1 (C7), 147.9 (C8), 143.5 (C4), 129.5 (C10, 12), 127.8 (C1), 126.2 (C11), 125.4 (C2), 122.5 (C9, 13), 122.1 (C5), 121.4 (C3), 111.9 (C6), 57.1 (C15, 16), 48.6 (C14, 17), 46.4 (C18).

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P NMR (161.9 MHz, DMSO-d6), δ

ppm: -8.2. MS (m/z): 436 (100%) (M+H+), 438 (33%). Anal. Calcd for C18H19ClN5O4P C, 49.61; H, 4.39; N, 16.07; Found: C, 49.53; H, 4.50; N, 16.13.

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ACCEPTED MANUSCRIPT 4-Chlorophenyl 5-nitro-1H-indazol-1-yl(piperidin-1-yl)phosphinate (7g): Yellow solid, Yield: 70 %, Mp: 233-236 oC. IR (νmax cm-1): 1224 (P=O str), 1067 (P-O str), 793 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.89 (1H, s, Ar-H), 8.40 (1H, s, Ar-H), 8.24 (1H, d, J = 2.5 Hz, Ar-H), 8.08 (1H, d, J = 2.5 Hz, Ar-H), 7.78 (2H, d, J = 9.0 Hz, Ar-H), 7.61 (2H, d, J = 9.0 Hz, Ar-H), 2.13 (4H, t, J = 2 Hz, piperidine), 1.59-1.53 (6H, m, piperidine).

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C NMR (125 MHz,


DMSO-d6) δ: 153.0 (C7), 148.5 (C8), 143.6 (C4), 129.7 (C10, 12), 128.4 (C1), 128.2 (C11), 124.9 (C2), 122.9 (C9, 13), 122.5 (C5), 121.5 (C3), 112.0 (C6), 48.4 (C14, 18), 23.5 (C15, 17), 19.2 (C16). 31P NMR (161.9 MHz, DMSO-d6) δ: -4.4. MS (m/z): 421 (100%) (M+H+), 315 (81%), 271 (50%), 423 (34%). Anal. Calcd for C18H18ClN4O4P C, 51.38; H, 4.31; N, 13.31; Found: C, 51.19; H, 4.42; N, 13.21.

4-Chlorophenyl

N-furan-2-ylmethyl-P-(5-nitro-1H-indazol-1-yl)phosphonamidate

(7h):

Semi solid, Yield: 69 %. IR (νmax cm-1): 3190 (NH str), 1216 (P=O str), 1078 (P-O str), 790 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.80 (1H, s, Ar-H), 8.40 (1H, s, Ar-H), 8.18 (1H, d, J = 2.1 Hz, Ar-H), 8.15 (1H, d, J = 2.1 Hz, Ar-H), 7.76 (2H, d, J = 9.0 Hz, Ar-H), 7.62 (2H, d, J = 9.0 Hz, Ar-H), 6.54-6.24 (3H, m, Ar-H), 4.67 (1H, s, NH), 3.24 (2H, s, NCH2). 13C NMR (125 MHz, DMSO-d6) δ: 152.9 (C7), 147.5 (C8), 143.9 (C4), 137.8 (C15), 137.2 (C19), 129.4 (C10, 12), 128.8 (C1), 128.6 (C11), 124.5 (C2), 122.5 (C9, 13), 122.3 (C5), 121.1 (C3), 113.1 (C17), 111.7 (C6), 107.4 (C16), 45.0 (C14). 31P NMR (161.9 MHz, DMSO-d6) δ: -6.2. MS (m/z): 433 (84%) (M+H+), 270 (100%), 435 (28%). Anal. Calcd for C18H14ClN4O5P C, 49.96; H, 3.26; N, 12.95; Found: C, 49.85; H, 3.32; N, 12.91.

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ACCEPTED MANUSCRIPT 4-Chlorophenyl-N-2-(3,4-dihydroxyphenyl)-2-hydroxyethyl-N-isopropyl-P-(5-nitro-1Hindazol-1-yl) phosphonamidate (7i): Grey solid, Yield: 68 %, Mp: 245-248 oC. IR (νmax cm-1): 3391 (OH str), 1233 (P=O str), 1065 (P-O str), 784 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 13.77 (2H, s, Ar-OH), 8.84 (1H, s, Ar-H), 8.41 (1H, s, Ar-H), 8.19 (1H, d, J = 2.5 Hz, Ar-H), 8.16 (1H, d, J = 2.5 Hz, Ar-H), 7.74-7.14 (7H, m, Ar-H), 6.77 (1H, br s, CH-OH), 4.51-4.50 (1H,


m, -NCH2CH-OH), 3.59-3.58 (2H, m, -NCH2), 2.37-2.30 (1H, m, -NCH(CH3)2), 1.21 (6H, d, J = 3.5 Hz, -NCH(CH3)2).

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C NMR (125 MHz, DMSO-d6) δ: 153.4 (C7), 148.7 (C8), 143.0 (C4),

141.7 (C19), 141.4 (C18), 136.7 (C16), 129.4 (C10, 12), 127.5 (C1), 127.0 (C11), 125.8 (C2), 122.7 (C9, 13), 122.4 (C21), 122.0 (C5), 121.6 (C3), 120.8 (C20), 112.4 (C6), 110.5 (C17), 68.4 (C15), 45.5 (C14), 43.5 (C22), 19.8 (C23,

24).

31

P NMR (161.9 MHz, DMSO-d6) δ: -14.4. MS (m/z): 547

(100%) (M+H+), 261 (65%), 549 (33%). Anal. Calcd for C24H24ClN4O7P C, 52.71; H, 4.42; N, 10.24; Found: C, 52.66; H, 4.35; N, 10.30.

4-Chlorophenyl 5-nitro-1H-indazol-1-yl(4-(pyridin-2-yl)piperazin-1-yl)phosphinate (7j): Pale brown solid, Yield: 79 %, Mp: 124-127 oC. IR (νmax cm-1): 1237 (P=O str), 1066 (P-O str), 772 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.84 (1H, s, Ar-H), 8.40 (1H, s, Ar-H), 8.19 (1H, d, J = 2.0 Hz, Ar-H), 8.17 (1H, d, J = 2.0 Hz, Ar-H), 8.09 (1H, d, J = 5.0 Hz, Ar-H), 7.756.59 (7H, m, Ar-H), 3.02-3.01 (4H, t, J = 4.5 Hz), 2.76-2.74 (4H, t, J = 4.5 Hz). 13C NMR (125 MHz, DMSO-d6) δ: 165.0 (C20), 161.4 (C19), 153.2 (C7), 149.1 (C8), 143.6 (C4), 141.5 (C17), 129.1 (C10, 12), 127.0 (C1), 126.0 (C11), 125.8 (C2), 122.2 (C9, 13), 122.0 (C5), 121.9 (C3), 121.1 (C18), 111.6 (C6), 107.3 (C16), 53.9 (C15, 21), 45.7 (C14, 22). 31P NMR (161.9 MHz, DMSO-d6) δ: -

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ACCEPTED MANUSCRIPT 5.8. MS (m/z): 499 (100%) (M+H+), 382 (80%), 501 (33%). Anal. Calcd for C22H20ClN6O4P C, 52.97; H, 4.04; N, 16.85; Found: C, 52.85; H, 4.12; N, 16.86.

4-Chlorophenyl

N-4-chloro-2-fluorophenyl-P-(5-nitro-1H-indazol-1-yl)phosphonamidate


(7k): Pale yellow solid, Yield: 76 %, Mp: 237-240 oC. IR (νmax cm-1): 3334 (NH str), 1240 (P=O str), 1066 (P-O str), 745 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.88 (1H, s, Ar-H), 8.45 (1H, s, Ar-H), 8.23 (1H, d, J = 2.0 Hz, Ar-H), 8.21 (1H, d, J = 2.0 Hz, Ar-H), 7.80-6.80 (7H, m, Ar-H), 6.44 (1H, s, NH). 13C NMR (125 MHz, DMSO-d6) δ: 156.8 (C19), 153.5 (C7), 148.2 (C8), 143.2 (C4), 129.8 (C14), 129.3 (C10, 12), 127.9 (C1), 127.7 (C11), 127.4 (C17), 126.6 (C16), 125.4 (C2), 122.5 (C9, 13), 122.1 (C5), 121.4 (C3), 116.4 (C15), 112.5 (C6), 111.0 (C18). 31P NMR (161.9 MHz, DMSO-d6) δ: -8.2. MS (m/z): 481 (85%) (M+H+), 483 (28%), 317 (100%). Anal. Calcd for C19H12Cl2 FN4O4P C, 47.42; H, 2.51; N, 11.64; Found: C, 47.30; H, 2.45; N, 11.70.

(R)-

4-Chlorophenyl

N-1-hydroxybutan-2-yl-P-(5-nitro-1H-indazol-1-

yl)phosphornamidate (7l): Pale brown solid, Yield: 73 %, Mp: 180-183 oC. IR (νmax cm-1): 3360 (OH str), 3164 (NH str), 1234 (P=O str), 1064 (P-O str), 785 (P-N str). 1H NMR (500 MHz, DMSO-d6) δ: 8.87 (1H, s, Ar-H), 8.42 (1H, s, Ar-H), 8.20 (1H, d, J = 2.0 Hz, Ar-H), 8.18 (1H, d, J = 2.0 Hz, Ar-H), 7.72-7.60 (4H, m, Ar-H), 5.22 (1H, br s, OH), 4.70 (1H, s, NH), 3.833.60 (3H, m, CHCH2OH), 1.78-1.75 (2H, m, CH3CH2CH), 1.16 (3H, t, J = 3.0 Hz, CH3).

13

C

NMR (125 MHz, DMSO-d6) δ: 152.9 (C7), 148.6 (C8), 143.5 (C4), 129.1 (C10, 12), 128.0 (C1), 127.9 (C11), 125.3 (C2), 122.3 (C9, 13), 122.0 (C5), 121.8 (C3), 111.8 (C6), 63.4 (C17), 47.8 (C14),

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ACCEPTED MANUSCRIPT 23.4 (C15), 12.2 (C16).

31

P NMR (161.9 MHz, DMSO-d6) δ: -9.81. MS (m/z): 425 (100%)

(M+H+), 262 (76%), 427 (35%). Anal. Calcd for C17H18ClN4O5P C, 48.07; H, 4.27; N, 13.19; Found: C, 48.15; H, 4.22; N, 13.26. CONCLUSION In summary, we designed and synthesized twelve phosphorylated derivatives of 5-


nitroindazole in good yields. All the title compounds were screened for their antimicrobial activity and docking studies. The results of docking studies revealed that compound 7j exhibited the highest binding affinity (-9.2 kcal/mol) and compounds 7k showed good binding affinity (8.3 kcal/mol). In silico studies were also supported by in vitro antimicrobial studies. Further, in vitro antioxidant activity was evaluated for the title compounds 7a-l. Compounds 7d and 7j showed good radical scavenging activity at lower IC50 values than the other compounds. Finally, we concluded that 7j is the most active compound in terms of antimicrobial and antioxidant activities. Therefore, we would like to investigate its activity in vivo. ACKNOWLEDGMENT The author, SK. Thaslim Basha expresses thanks to UGC for financial support. (See the supplemental material for the additional information)

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ACCEPTED MANUSCRIPT References: 1. Engel, R. Chem. Rev. 1977, 77, 349-367. 2. Hiratake, J.; Oda, J. Biosci. Biotechnol. Biochem. 1997, 61, 211-218. 3. Schug, K.A.; Lindner, W. Chem. Rev. 2005, 105, 67-114. 4. Venkatachalam, T.K.; Samuel, P.; Qazi, S.; Uckun, F.M. Eur. J. Med. Chem. 2005, 40,


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ACCEPTED MANUSCRIPT 25. Barry, A.L. The Antimicrobial Susceptibility Test: Principle and Practices, Edited by Illus lea and Febiger, Philadelphia, PA USA 1976, p. 180; [Biol. Abstr. 1977, 64, 2518 3]. 26. Nene, Y.L.; Thapliyal, P.N. Fungicides in plant disease control, 3rd ed., Oxford and IBH Publishing Co.pvt. Ltd, New Delhi, 1993, 526. 27. (a) Burits, M.; Bucar, F. Phytother Res. 2000, 14, 323-328; (b) Cuendet, M.; Hostettmann,


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ACCEPTED MANUSCRIPT O O2 N

O 2N

P

N + Cl

Cl

O

THF 4h

N H

Cl

3

4

Cl + R

H 6

3

O2 N

1

2

4

1,4-dimethyl piperazine THF, 30-50 C

N 5

N

7 6

R

10

O

P


N N O P Cl O 5

NaH, 0-35 °C

9

Cl 8

11

O 12

13

7

Compd

Compd

R 17

HO

15 16

18

14

16

S

20 18

15

17

16

19

14

21

15

22

N H

H3 C

N

19

21 17

20 19

14

7k

N

7d

O

N 16

21

14

17

Cl

17

17

7h

16

15

H N

15

18

O

14

7l

16

14

22

H N

19

18 14

N

15

18

17

14

N

N

17 15

15

16

18

17

16

24

23

20

16

16

22

7j

N

7g

N

16 14

23 16

15

18

14

18

15

N

HO

14

15

14

7c

7i

17

HO

H N 7f

20

H N

15

18

21

HO

7b

17

7e 19

R OH

H N

7a

Compd

R

H N

F

17

OH

Scheme 1 Synthesis of N-phosphorylated derivatives of 5-nitroindazole 7a-l.

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ACCEPTED MANUSCRIPT OH OH

O 2N O2N

N

N

N N N

N H

1-(5-(butylamino)pentyl)-5-nitro1H-indazol-3-ol (2)

1-(2-((diethylamino)methyl)benzyl) -5-nitro-1H-indazol-3-ol (1)

O2N

N O

N

Cl


P O R

Phosphoramidate derivatives of 5-nitroindazole 7a-l

Figure 1 Biologically active 5-nitroindazole derivatives.

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3D Structure

Docking image

3D Structure

Docking image

5-Nitroindazole 7j

Streptomycin Figure 2 Docking images of the synthesized

7k potent compounds against DNA GyraseA.

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