Design synthesis and antibacterial activity studies of new thiadiazoloquinolone compounds

Abstract

New 9-(alkyl/aryl)-4-fluoro-6-oxo[1,2,5]thiadiazolo[3,4-h]quinoline-5-carboxylic acids and their esters were designed and synthesized. A detailed discussion of the reactions utilized in the preparation of the intermediate and target compounds is reported. All the newly synthesized compounds were fully characterized using all the physico-chemical means needed. All the intermediates and the final esters and acids were tested against bacterial and fungal strains. The acids 25a and 25c proved to be very active against Gram positive and Gram negative bacteria with MIC 0.15–3 µg/mL. The structure–activity relationship of antibacterial thiadiazoloquinolones shows that compounds 25a and 25c are twice less potent than the corresponding cyclopropyl derivative 16. Therefore, the cyclopropyl moiety on N-9 seems to be the most suitable substituent.

• 6-Fluoroquinolone
• antibacterial activity
• synthesis
• thiadiazoloquinolone

Introduction

Norfloxacin (1) and ciprofloxacin (2) were among the first quinolones marketed as chemotherapeutic agents for the treatment of systemic bacterial infections1–3. Several new members of the quinolone family have emerged with enhanced activities such as grepafloxacin (3), levofloxacin (4), rufloxacin (5), and compounds 6–9 (Figure 1). Much has been learned about how molecular modifications of the core quinolone structure affect the antibacterial profile. The structure–activity relationship of the quinolones has been the subject of extensive reviews4–16.

Figure 1. Some fluoroquinolones of which 1–6 are in clinical use.

In our continuous effort to develop new chemotherapeutics with enhanced activities against resistant bacterial strains17,18, we introduced a new class of tricyclic compounds, namely thiadiazoloquinolones19. In our methodology, we took advantage of many known benzothiadiazoles (Figure 2) with diverse biopharmaceutical activities20–26 and we hybridized the thiadiazole moiety with the fluoroquinolone structure to introduce the new compound 9-cyclopropyl-4-fluoro-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline carboxylic acid (16). The latter compound was tested against several clinical isolate bacterial strains and did prove to be quite active compared to the drugs used in the market19.

Figure 2. Some compounds containing thiadiazolo moiety known in the literature.

Several drugs used in the market show a variation at N-1, e.g. cyclopropyl, ethyl, 2,4-difluorophenyl and many others. Our strategy for the synthesis of compound 16, banked on the introduction of the fluorine atom, for its crucial advantage, and also on the replacement of the ethyl with the cyclopropyl moiety, with respect to thiadiazoloquinolones 14 and 15.

The result of that study was very interesting, the strategy proved its success via enhancement of the activity compared to the previous thiadiazoloquinolones 14 and 15, and also the potency of compound 16 was comparable to the drug ciprofloxacin.19

Herein, we report on the synthesis and the antibacterial activities of new compounds based on the same strategy by fixing the fluorine atom and replacing the cyclopropyl with different moieties, namely ethyl, phenyl, p-fluorophenyl and p-methoxyphenyl, which are based on the structure of important fluoroquinolone agents.

Materials and methods

All the chemicals and solvents used in this study were purchased from Acros. Melting points were measured by Fischer-Johns Melting Point Apparatus and were uncorrected. ¹H NMR and ¹³C NMR spectra were measured on a Bruker DPX-300 instrument. Chemical shifts (δ) are expressed in ppm with reference to TMS as internal standard. High resolution mass spectra (HRMS) were measured in positive ion mode by electrospray ionization (ESI) on a Bruker instrument APEX IV 2008. The samples were dissolved in acetonitrile, diluted in spray solution (methanol/water 1:1 v/v + 0.1% formic acid) and injected using a syringe pump with a flow rate of 2 μL/min. External calibration was conducted using Arginine cluster in a mass range m/z 175–871. For all HRMS data, mass error: 0.00–0.50.

Chemistry

General procedure for the preparation of compounds

Ethyl 3-(N,N-dimethylamino)-2-(2,4-dichloro-5-fluoro-3-nitrobenzoyl)acrylate (18). It was prepared using reported methods starting from 2,4-dichloro-5-fluoro-3-nitrobenzoic acid 17 (Scheme 1).19

Preparation of ethyl 3-N-(aryl/alkyl)-2-(2,4-dichloro-5-fluoro-3-nitrobenzoyl)acrylates (19a–d). A stirred solution of (18) (∼38 mmol) in chloroform (50 mL) and methanol (10 mL), cooled to 8–10 °C, was treated dropwise with the appropriate aryl or alkylamine (1.5 eq) dissolved in MeOH (3 mL). The resulting mixture was further stirred for additional 10–15 min at 5–10 °C. Methanol (90 mL) was then added and the reaction mixture was stirred at rt for 1–2 h. The precipitated white solid product was filtered, washed with cold ethanol (10–20 mL) and air dried. The resultant product was used for the next step without further purification. For identification purpose, a small amount was purified on TLC plates eluting with 2% MeOH/CHCl₃ (50:1, v/v).

Ethyl 3-(N-ethylamino)-2-(2,4-dichloro-5-fluoro-3-nitrobenzoyl)acrylate (19a). Yield 95%; m.p. 154 °C; ¹H NMR δ ppm (CDCl₃): 1.01 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 1.35 (t, J = 7.1 Hz, 3H, NCH₂CH₃), 3.52 (q, J = 7.1 Hz, 2H, NCH₂CH₃), 3.98 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.11 (d, ³J_H–F = 8.0 Hz, 1H, H-6), 8.19 (d, J = 14.4 Hz, 1H, N–C(3″)-H), 10.99 (br d, J = 14.4 Hz, 1H, exchangeable N–H); ¹³C NMR δ ppm (CDCl₃): 13.9 (OCH₂CH₃), 15.6 (NCH₂CH₃), 40.5 (NCH₂CH₃), 60.1 (OCH₂CH₃), 99.8 (C-2″), 114.1 (d, ²J_C–F = 23.3 Hz, C-4), 115.8 (d, ²J_C–F = 23.3 Hz, C-6), 117.7 (d, ³J_C–F = 4.4 Hz, C-1), 144.0 (d, ³J_C–F = 6.2 Hz, C-3), 148.6 (C-2), 156.7 (d, ¹J_C–F = 253.4 Hz, C-5), 161.2 (N–C-3″), 165.9 (CO₂Et), 188.3 (C=O ketone); HRMS (ESI): calcd. for C₁₄H₁₃Cl₂FN₂O₅Na [M + Na]⁺: 401.00832, found: 401.00778.

Ethyl 3-(N-phenylamino)-2-(2,4-dichloro-5-fluoro-3-nitrobenzoyl)acrylate (19b). Yield 95%; m.p. 150 °C; ¹H NMR δ ppm (CDCl₃): 1.07 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 4.07 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.19 (d, ³J_H–F = 8.0 Hz, 1H, H-6), 7.24 (d, J = 7.6 Hz, 1H, H-4′), 7.28 (dd, J = 7.8, 7.6, Hz, 2H, H-3′/H-5′), 7.44 (d, J = 7.8 Hz, 2H, H-2′/H-6′), 8.67 (d, J = 13.8 Hz, 1H, N–C(3″)-H), 12.65 (br d, J = 13.8 Hz, 1H, exchangeable N–H); ¹³C NMR δ ppm (CDCl₃): 13.9 (OCH₂CH₃), 60.7 (OCH₂CH₃), 102.2 (C-2″), 114.3 (d, ²J_C–F = 23.3 Hz, C-4), 115.9 (d, ²J_C–F = 22.7 Hz, C-6), 118.0 (C-1), 118.5 (C-3′/C-5′), 127.0 (C-4′), 130.1 (C-2′/C-6′), 138.1 (C-1′), 143.5 (C-3), 148.6 (C-2), 154.2 (N–C-3″), 156.7 (d, ¹J_C–F = 254 Hz, C-5), 165.6 (CO₂Et), 189.3 (C=O ketone); HRMS (ESI): Calcd for C₁₈H₁₃Cl₂FN₂O₅Na [M + Na]⁺: 449.00832, found: 449.00778.

Ethyl 3-(N-4-fluorophenylamino)-2-(2,4-dichloro-5-fluoro-3-nitrobenzoyl)acrylate (19c). Yield 91%; m.p. 130 °C; ¹H NMR δ ppm (CDCl₃): 1.05 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 4.05 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.09 (d, ³J_H–F = 8.0 Hz, 1H, H-6), 7.15 (d, J = 8 Hz, 2H, H-2′/C-6′), 7.25 (dd, J _H–H = 8 Hz, ³J _H–F = 8 Hz, 2H, H-3′/H-5′), 8.57 (d, J = 11.9 Hz, 1H, N–C(3″)-H), 12.66 (br d, J = 11.9 Hz, 1H, exchangeable N–H); ¹³C NMR δ ppm (CDCl₃): 13.8 (OCH₂CH₃), 60.6 (OCH₂CH₃), 102.2 (C-2″), 114.3 (d, ²J_C–F = 23.3 Hz, C-4), 115.9 (d, ²J_C–F = 23.6 Hz, C-6), 117.0 (d, ²J_C–F = 23.3 Hz, C-3′/C-5′), 117.8 (C-1), 120.3 (d, ³J_C–F = 8.3 Hz, C-2′/C-6′), 134.5 (C-1′), 143.4 (C-3), 148.6 (C-2), 154.6 (N–C-3″), 156.7 (d, ¹J_C–F = 254.1 Hz, C-5), 161.2 (d, ¹J_C–F = 245.8 Hz, C-4′), 165.5 (CO₂Et), 189.3 (C=O ketone); HRMS (ESI): Calcd for C₁₈H₁₂Cl₂F₂N₂O₅Na [M + Na]⁺: 466.99890, found: 466.99835.

Ethyl 3-(N-4-methoxyphenylamino)-2-(2,4-dichloro-5-fluoro-3-nitrobenzoyl)acrylate (19d). Yield 97%; m.p. 162 °C; ¹H NMR δ ppm (CDCl₃): 1.05 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 3.81 (s, 3H, OCH₃), 4.04 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 6.93 (d, J = 8.9 Hz, 2H, H-3′/H-5′), 7.15 (d, ³J_H–F = 8.0 Hz, 1H, H-6), 7.21 (d, J = 8.9 Hz, 2H, H-2′/H-6′), 8.56 (d, J = 13.9 Hz, 1H, N–C(3″)-H), 12.7 (br d, J = 13.9 Hz, 1H, exchangeable N–H); ¹³C NMR δ ppm (CDCl₃): 13.9 (OCH₂CH₃), 55.7 (OCH₃), 60.5 (OCH₂CH₃), 101.5 (C-2″), 114.3 (d, ²J_C–F = 23.3 Hz, C-4), 115.2 (C-3′/C-5′), 115.9 (d, ²J_C–F = 23.4 Hz, C-6), 117.8 (C-1), 120.0 (C-2′/C-6′), 131.4 (C-1′), 143.6 (d, ³J_C–F = 6.3 Hz, C-3), 148.6 (C-2), 154.3 (C-3″), 156.7 (d, ¹J_C–F = 253.7 Hz, C-5), 158.6 (C-4′), 165.6 (CO₂Et), 188.8 (C=O Ketone); HRMS (ESI): Calcd for [M + H]⁺: 457.03694, found: 457.03694.

Preparation of ethyl 7-chloro-1-(aryl/alkyl)-6 fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylates (20a–d). A solution of the particular precursor 19a–d (30 mmol) in DMF (50 mL) and potassium carbonate (90 mmol) was heated at 60 °C. The progress of the cyclization reaction was monitored by TLC (eluent: EtOAc: N–Hexane, 1:1/v:v). The reaction was complete within 80 to 90 min. The reaction mixture was then poured slowly onto crushed ice (100 g) under vigorous stirring, the precipitated pale yellow solid product was collected, washed with water, triturated with cold ethanol, and air dried to produce the pure product.

Ethyl 7-chloro-1-ethyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (20a).Yield 97%; m.p. 150 °C; ¹H NMR δ ppm (CDCl₃): 1.39 (t, J = 7.2 Hz, 3H, OCH₂CH₃), 1.43 (t, J = 7.2 Hz, 3H, NCH₂CH₃), 4.04 (q, J = 7.2 Hz, 2H, NCH₂CH₃), 4.40 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 8.43 (s, 1H, H-2), 8.47 (d, ³J_H–F = 8.34 Hz, 1H, H-5); ¹³C NMR δ ppm (CDCl₃): 14.7 (OCH₂CH₃), 16.1 (NCH₂CH₃), 50.3 (NCH₂Me), 60.9 (OCH₂Me), 112.1 (C-3), 115.8 (d, ²J_C–F = 22.8 Hz, C-5), 120.7 (d, ²J_C–F = 23.9 Hz, C-7), 128.8 (C-4a), 130.9 (d, ⁴J_C–F = 5.7 Hz, C-8a), 140.5 (C-8), 152.6 (C-2), 154.0 (d, ¹J_C–F = 249 Hz, C-6), 164.0 (CO₂Et), 170.4 (C-4) (C=O ketone); HRMS (ESI): calcd. for C₁₄H₁₂ClFN₂O₅ [M + H]⁺: 343.04970, found: 343.04915.

Ethyl 7-chloro-6-fluoro-8-nitro-4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylate (20b). Yield 97%; m.p. 194 °C; ¹H NMR δ ppm (CDCl₃): 1.36 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 4.36 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.33 (d, J = 7.7 Hz, 2H, H-2′/H-6′), 7.48 (dd, J = 7.7, 7.3 Hz, 2H, H-3′/H-5′), 7.56 (d, J = 7.3 Hz, 1H, H-4′), 8.37 (s, 1H, H-2), 8.44 (d, ³J_H–F = 8.3 Hz, 1H, H-5); ¹³C NMR δ ppm (CDCl₃): δ 14.4 (OCH₂CH₃), 61.6 (OCH₂Me), 112.0 (C-3), 115.4 (d, ²J_C–F = 22.9 Hz, C-5), 121.8 (d, ²J_C–F = 23.9 Hz, C-7), 127.6 (C-3′/C-5′), 128.8 (d, ³J_C–F = 3 Hz, C-4a), 129.8 (C-8a), 129.9 (C-2′/C-6′), 131.0 (C-4′), 139.5 (C-1′), 141.5 (C-8), 152.0 (C-2), 154.8 (d, ¹J_C–F = 253 Hz, C-6), 164.1 (CO₂Et), 171.0 (C-4) (C=O ketone); HRMS (ESI): Calcd for C₁₈H₁₂ClFN₂O₅Na [M + Na]⁺: 413.03165, found: 413.03110.

Ethyl 7-chloro-6-fluoro-1-(4-fluorophenyl)-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (20c). Yield 97%; m.p. 226 °C; ¹H NMR δ ppm (CDCl₃): 1.36 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 4.36 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.18 (dd, J = 8.9 Hz, 2J_H–F = 7.9 Hz, 2H, H-3′/H-5′), 7.35(dd, J_H–H = 8.9 Hz, 3J_H–F = 4.5 Hz, 2H, H-2′/H-6′), 8.31 (s, 1H, H-2), 8.43 (d, 3J_H–F = 8.2 Hz, 1H, H-5); 13C NMR δ ppm (CDCl₃): 14.4 (OCH₂CH₃), 61.7 (OCH₂Me), 112.2 (C-3), 115.4 (d, 2J_C–F = 22.8 Hz, C-5), 117.1 = (d, 2J_C–F = 23.2 Hz, C-3′/C-5′), 122.0 (d, 2J_C–F = 23.7 Hz, C-7), 128.7 (C-4a), 129.7 (C-8a), 129.8 (d, 3J_C–F = 9.2 Hz, C-2′/C-6′), 135.2 (d, 3J_C–F = 3.4 Hz, C-8), 135.5 (C-1′), 151.9 (C-2), 154.8 (d, 1J_C–F = 253 Hz, C-6), 163.5 (d, 1J_C–F = 253 Hz, C-4′), 164 (CO₂Et), 170.9 (C-4)(C=O); HRMS (ESI): Calcd for C₁₈H₁₁Cl₂F₂N₂O₅Na [M + Na]⁺: 431.02223, found: 431.02168.

Ethyl 7-chloro-6-fluoro-1-(4-methoxyphenyl)-8-nitro-4-oxo-1,4-dihydroquinoline-3- carboxylate (20d). Yield 96%; m.p. 176 °C; ¹H NMR δ ppm (CDCl₃): 1.33 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 3.86 (s, 3H, OCH₃), 4.32 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 6.91 (d, J = 8.8 Hz, 2H, H-2′/H-6′), 7.24 (d, J = 8.8 Hz, 2H, H-3′/H-5′), 8.31 (s, 1H, H-2), 8.38 (d, 3J_H–F = 8.1 Hz, 1H, H-5).13C NMR δ ppm (CDCl₃): 14.4 (OCH₂CH₃), 55.8 (OCH₃), 61.5 (OCH₂CH₃), 111.7 (C-3), 114.7 (C-3′/C-5′), 115.2 (d, 2J_C–F = 23 Hz, C-5), 121.7 (d, 2J_C–F = 23.5 Hz, C-7),128.6 (C-4a), 129.1 (C-2′/C-6′), 129.7 (C-8a), 132.0 (d, 3J_C–F = 3.4 Hz, C-8), 141.2 (C-1′), 152.4 (C-2), 154.6 (d, 1J_C–F = 252.8 Hz, C-6), 161.1 (C-4′), 164.1 (CO₂Et), 171.0 (C-4)(C=O). HRMS (ESI): Calcd for C₁₉H₁₄ClFN₂O₆Na [M + Na]⁺: 421.06027, found: 421.05972.

Preparation of ethyl 7-azido-1-(aryl/alkyl)-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylates (21a–d). A warm (∼35 to 40 °C) solution of soduim azide (7.8 g, 120 mmol) in dimethylsulfoxide (DMSO, 50 mL) was added to a solution of the appropriate ethyl 7-chloro-1-aryl(or alkyl)-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoxaline-3-carboxylate (20 mmol) (20a–d) in DMSO (50 mL). The resulting mixture acquired yellow turbidity within few minutes, and was further stirred at (∼35 to 40 °C) for 16–20 h. Thereafter, the reaction mixture was diluted with cold water (250 mL), and the precipitated solid product was collected under suction, washed with cold water and air dried.

Ethyl 7-azido-1-ethyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (21a). Yield 90%; m.p. 150 °C; ¹H NMR δ ppm (CDCl₃): 1.39 (m, 6H, NCH₂CH₃, OCH₂CH₃), 3.98 (q, J = 7.2 Hz, 2H, NCH₂CH₃), 4.38 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 8.37 (s, 1H, H-2), 8.39 (d, 3J_H–F = 11.2 Hz, 1H, H-5);13C NMR δ ppm (CDCl₃): 14.4 (OCH₂CH₃), 16.2 (NCH₂CH₃), 50.5 (NCH₂CH₃), 61.5 (OCH₂CH₃), 112.3 (C-3), 115.9 (d, 2J_C–F = 20.9 Hz, C-5), 128.2 (C-4a), 128.8 (d, ²J_C–F = 11.1 Hz, C-7), 127.7 (d, ⁴J_C–F = 5.7 Hz, C-8a), 135.2 (C-8), 151.3 (C-2), 154.9 (d, 1J_C–F = 250.2 Hz, C-6), 164.5 (CO₂Et), 171.0 (C-4)(C=O); HRMS (ESI): Calcd for C₁₄H₁₂FN₅O₅Na [M + Na]⁺: 372.07202, found: 372.07147.

Ethyl 7-azido-6-fluoro-8-nitro-4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylate (21b). Yield 92%; m.p. 160 °C; ¹H NMR δ ppm (CDCl₃): 1.35 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 4.35 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.30 (d, J = 7.6 Hz, 2H, H-2′/H-6′), 7.45 (dd, J = 7.6, 7.3 Hz, 2H, H-3′/H-5′),7.55 (d, J = 7.3 Hz, 1H, H-4′), 8.31 (d, 3J_H–F = 9.4 Hz, 1H, H-5), 8.37 (s, 1H, H-2). 13C NMR δ ppm (CDCl₃): 14.4 (OCH₂CH₃), 61.5 (OCH₂CH₃), 112.2 (C-3), 115.4 (d, 2J_C–F = 21.2 Hz, C-5), 118.1 (C-4a), 127.5 = (C-3′/C-5′), 128.9 (C-8a), 129.8 (C-2′/C-6′), 130.5 (C-4′), 132.4 (d, 2JC–F = 23.9 Hz, C-7), 141.5 (C-8), 139.6 (C-1′), 151.7 (C-2), 153.1 (d, 1J_C–F = 251 Hz, C-6), 164.2 (CO₂Et), 171.0 (C-4)(C=O). HRMS (ESI): calcd. for C₁₈H₁₂FN₅O₅Na [M + Na]⁺: 420.07202, found: 420.07147.

Ethyl 7-azido-6-fluoro-1-(4-fluorophenyl)-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (21c). Yield 91%; m.p. 172 °C; ¹H NMR δ ppm (CDCl₃): 1.36(t, J = 7.1 Hz, 3H, OCH₂CH₃), 4.36 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.14 (dd, J = 8.9 Hz, ⁴J_H–F = 2.2 Hz, 2H, H-2′/H-6′), 7.31 (dd, J = 8.9 Hz, 3J_H–F = 4.5 Hz, 2H, H-3′/H-5′), 8.34 (d, 3J_H–F = 11.2 Hz, 1H, H-5), 8.38 (s, 1H, H-2); 13C NMR δ ppm (CDCl₃): 14.3 (OCH₂CH₃), 61.6 (OCH₂CH₃), 112.2 (C-3), 116.8 (d, 2J_C–F = 22.8 Hz, C-5), 117.1 = (d, 2J_C–F = 22.5 Hz, C-3′/C-5′), 126.5 (C-4a), 129.7 (d, 3J_C–F = 7.2 Hz, C-2′/C-6′), 129.9 (d, 2J_C–F = 23.7 Hz, C-7), 132.6 (C-8a), 135.2 (d, ³J_C–F = 3.2 Hz, C-8), 135.5 (C-1′), 151.7 (C-2), 154.7 (d, 1J_C–F = 247 Hz, C-6), 163.5 (d, 1J_C–F = 252.2 Hz, C-4′), 164 (CO₂Et), 171.0 (C-4)(C=O). HRMS (ESI): Calcd for C₁₈H₁₁F₂N₅O₅Na [M + Na]⁺: 438.06259, found: 438.06205.

Ethyl 7-azido-6-fluoro-1-(4-methoxyphenyl)-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (21d). Yield 94 %; m.p. 170 °C; ¹H NMR δ ppm (CDCl₃): 1.35 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 3.86 (s, 3H, OCH₃), 4.34 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 6.90 (d, J = 8.9 Hz, 2H, H-2′/H-6′), 7.21 (d, J = 8.9 Hz, 2H, H-3′/H-5′), 8.28 (d, 3J_H–F = 6.5 Hz, 1H, H-5) 8.38 (s, 1H, H-2); 13C NMR δ ppm (CDCl₃): 14.3 (OCH₂CH₃), 55.7 (OCH₃), 61.4 (OCH₂CH₃), 112.0 (C-3), 114.6 (d, 2J_C–F = 21.5 Hz, C-5), 117.9 (C-3′/C-5′), 126.6 (d, 2J_C–F = 23.5 Hz, C-7),128.6 (C-4a), 129.0 (C-2′/C-6′), 129.3 (C-8a), 132.1 (d, 3J_C–F = 3.4 Hz, C-8), 132.3 (C-1′), 152.2 (C-2), 154.6 (d, 1J_C–F = 252.8 Hz, C-6), 161.0 (C-4′), 164.4 (CO₂Et), 171.1 (C-4)(C=O). HRMS (ESI): Calcd for C₁₉H₁₄FN₅O₆ [M + H]⁺: 428.10064, found: 428.10009.

Preparation of ethyl 7,8-diamino-1-(aryl/alkyl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylates (22a–d). Anhydrous stannous chloride (5 g, 26 mmol) was added portionwise to a stirred ice-cooled (4–8 °C) solution of the appropriate ethyl 7-azido-1-alkyl or 1-aryl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (21a--d) (Scheme 2) (6.3 mmol) in concentrated hydrochloric acid (36%, 50 mL). The reaction mixture was further stirred at room temperature (24 h), and the reaction progress was monitored by TLC. Upon completion of the reaction, the reaction mixture was diluted with ice-cold water (50 mL), basified with 40% cold aqueous sodium hydroxide solution to pH ∼ 13 and set aside for a few minutes. The precipitated solid product was collected by suction filtration and recrystallized from 90% ethanol.

Ethyl 7,8-diamino-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (22a). Yield 85%; m.p. (dec) 347 °C; ¹H NMR δ ppm (CDCl₃): 1.08 (t, J = 7.0 Hz, 3H, OCH₂CH₃), 1.23 (t, J = 7.0 Hz, 3H, NCH₂CH₃), 4.16 (q, J = 7.0 Hz, 2H, NCH₂CH₃), 4.63 (q, J = 7.0 Hz, 2H, OCH₂CH₃), 4.73 (br s, 2H, C(8)–NH₂), 5.53 (br s, 2H, C(7)–NH₂),7.32 (d, 3J_H–F = 11.3 Hz, 1H, H-5), 8.38 (s, 1H, H-2).13C NMR δ ppm (CDCl₃):14.8 (OCH₂CH₃), 16.0 (NCH₂CH₃), 50.8 (NCH₂CH₃), 60.0 (OCH₂CH₃), 101.9 (d, 2J_C–F = 20.6 Hz, C-5), 108.7 (C-3), 120.7 (C-4a), 124.7 (d, 3J_C–F = 5.1 Hz, C-8), 127.7 (C-8a), 131.2 (d, 3J_C–F = 15.1 Hz, C-7), 150.0 (d, 1J_C–F = 236.6 Hz, C-6), 151.8 (C-2), 165.4 (CO₂Et), 173.1 (C-4)(C=O); IR (KBr) v: br 3472.33, 3387.38, 1710.58, 1682.94, 1611.55, 1555.40, 1460.96, 1202.94 cm⁻¹; HRMS (ESI): calcd. for C₁₄H₁₆FN₃O₃ [M + H]⁺: 294.12539, found: 294.12485.

Ethyl 7,8-diamino-6-fluoro-1-phenyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (22b). Yield 80%; m.p. 240 °C; ¹H NMR, δ ppm (DMSO-d₆): 1.19 (t, J = 7.0 Hz, 3H, OCH₂CH₃), 4.14 (q, J = 7.0 Hz, 2H, OCH₂CH₃), 3.87 (br s, 2H, C(8)-NH₂), 5.39 (br s, 2H, C(7)-NH₂),7.32 (dd, J_H–H = 8.6 Hz, J_H–F = 2.3 Hz, 2H, H-2′/H-6′), 7.38 (dd, 3J_H–H = 8.6 Hz, J_H–F = 4.3 Hz, 2H, H-3′/H-5′), 7.56 (d, 3J_H–F = 8.3 Hz, 1H, H-5), 8.15 (s, 1H, H-2); 13C NMR, δ ppm (DMSO-d₆): 14.7 (OCH₂CH₃), 60.3 (OCH₂Me), 112.2 (C-3), 115.4 (d, 3J_C–F = 22.8 Hz, C-5), 117.1 = (d, 2J_C–F = 23.2 Hz, C-3′/C-5′), 122.0 (d, 2J_C–F = 23.7 Hz, C-7),128.6 (C-4a), 129.8 (d, 3J_C–F = 7.2 Hz, C-2′/C-6′), 129.9 (C-8a), 135.2 (d, 3J_C–F = 3.4 Hz, C-8), 135.5 (C-1′), 151.9 (C-2), 154.8 (d, 1JC–F = 253 Hz, C-6), 163.5 (d, 1J_C–F = 253 Hz, C-4′), 164 (CO2Et), 170.9 (C-4); HRMS (ESI): Calcd for [M + H]⁺: 306.12540, found: 306.12475.

Ethyl 7,8-diamino-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (22c). Yield 83%; m.p. 274 °C; ¹H NMR, δ ppm (DMSO-d₆): 1.19 (t, J = 7.0 Hz, 3H, OCH₂CH₃), 4.14 (q, J = 7.0 Hz, 2H, OCH₂CH₃), 3.87 (br s, 2H, C(8)-NH₂), 5.39 (br s, 2H, C(7)-NH₂),7.32 (dd, J = 8.6 Hz, 4J_H–F = 2.3 Hz, 2H, H-2′/H-6′), 7.38 (dd, J = 8.6 Hz, 3J_H–F = 4.3 Hz, 2H, H-3′/H-5′), 7.56 (d, 3J_H–F = 8.3 Hz, 1H, H-5), 8.15 (s, 1H, H-2); 13C NMR, δ ppm (DMSO-d₆): 14.7 (OCH₂CH₃), 60.3 (OCH₂CH₃), 101.1 (d, 2J_C–F = 21 Hz, C-5), 109.0 (C-3), 116.8 (d, 2J_C–F = 23.0 Hz, C-3′/C-5′), 120.9 (d, 3J_C–F = 8.3 Hz, C-4a), 124.4 (d, 3J_C–F = 6.1 Hz, C-8), 125.8 (C-8a), 128.6 (d, 3J_C–F = 8.9 Hz, C-2′/C-6′), 129.7 (d, 2J_C–F = 16.1 Hz, C-7), 140.2 (d, 4J_C–F = 2.9 Hz, C-1′), 150.0 (d, 1J_C–F = 236 Hz, C-6), 151.2 (C-2), 162 (d, 1J_C–F = 244 Hz, C-4′), 164.9 (CO₂Et), 172.7 (C-4)(C=O); HRMS (ESI): calcd. for C₁₈H₁₅F₂N₃O₃Na [M + Na]⁺: 382.09792, found: 382.09737.

Ethyl 7,8-diamino-6-fluoro-1-(4-methoxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (22d). Yield 89%; m.p. 274 °C; ¹H NMR, δ ppm (DMSO-d₆): 1.20 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 3.75 (br s, 2H, C(8)-NH₂), 3.81 (s, 3H, OCH₃), 4.14 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 5.29 (br s, 2H, C(7)-NH₂), 7.09 (d, J = 8.9 Hz, 2H, H-2′/H-6′), 7.32 (d, 3J_H–F = 11.3 Hz, 1H, H-5), 7.48 (d, J = 8.9 Hz, 2H, H-3′/H-5′), 8.11 (s, 1H, H-2). 13C NMR, δ ppm (DMSO-d₆): 14.8 (OCH₂CH₃), 56.1 (OCH₃), 60.1 (OCH₂CH₃), 100.9 (d, 2J_C–F = 20.9 Hz, C-5), 108.5 (C-3), 115.4 (C-3′/C-5′), 120.1 (d, 3J_C–F = 7.2 Hz, C-4a), 124.5 (d, 3J_C–F = 6.1 Hz, C-8), 125.7 (C-8a), 127.8 (C-2′/C-6′), 129.3 (d, 2J_C–F = 16.2 Hz, C-7), 136.6 (C-1′), 150.0 (d, 1J_C–F = 235.5 Hz, C-6), 151.2 (C-2), 159.8 (C-4′), 164.9 (CO₂Et), 172.7 (C-4). IR (cm⁻¹, KBr): br 3294.77, 2980.30, 1716.58, 1674.64, 1616.90, 1508.57, 1461.32 cm⁻¹; HRMS (ESI): Calcd for C₁₉H₁₈FN₃O₄ [M + H]⁺: 372.13596, found: 372.13541.

Preparation of ethyl(methyl) 9-(aryl/alkyl)-4 fluoro-6-oxo-6,9-dihydro-[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylates (23a,b,d/24b–d). To a stirred suspension of the particular diamine compound (22a–d) (Scheme 3) (3 mmol) in dry benzene (25 mL), was added purified thionylchloride (6 mL) and the resulting mixture was heated at 80–85 °C for 8 h. Benzene and excess thionyl chloride were distilled off in vacuum and the residue was cooled, dissolved in CHCl₃ and washed with water (2 × 30 mL). The organic layer was separated, dried (anhydrous MgSO₄) and the solvent CHCl₃ was then evaporated to dryness under reduced pressure. The residual product was recrystallized from dichloromethane/methanol. In some cases, methanol was used to destroy the excess thionyl chloride within the reaction. Upon using this workup process transesterfication occurs and the resultant product was either methyl ester instead of ethyl ester or a mixture of both esters.

Methyl 9-ethyl-4-fluoro-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylate (23a). Yield 80%, m.p. 325 °C. ¹H NMR, δ ppm (D₂O): 1.42 (t, J = 6.9 Hz, 3H, NCH₂CH₃), 3.76 (s, 3H, CO₂CH₃), 5.05 (q, J = 6.9 Hz, 2H, NCH₂CH₃), 8.49 (d, 3J_H–F = 10.2 Hz, 1H, H-5), 8.86 (s, 1H, H-2); 13C NMR, δ ppm (D₂O): 16.5 (NCH₂CH₃), 52.1 (OCH₃), 52.1 (NCH₂CH₃), 109.1 (d, 2J_C–F = 19.7 Hz, C-5), 114.1 (C-7), 127.5 (d, 3J_C–F = 5.8 Hz, C-5a), 130.4 (Hz, C-9a), 148.4 (d, 2J_C–F = 10.5 Hz, C-3a), 148.8 (d, 3J_C–F = 1.6 Hz, C-9b), 149.5 (C-8), 150.0 (d, 1J_C–F = 256 Hz, C-4), 165.1 (CO₂Me), 171.3 (C-6)(C=O); HRMS (ESI): Calcd for C₁₄H₁₂FN₃O₃S [M + H]⁺: 308.05052, found: 308.04982.

Methyl 4-fluoro-6-oxo-9-phenyl-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinol-ine-7-carboxylate (23b). Yield 40% m.p. 300 °C; ¹H NMR, δ ppm (DMSO-d₆): 3.74 (s, 3H, CO₂CH₃), 7.61 (m, 5H, H-2′/H-3′/H-4′/H-5′/H-6′), 8.09 (d, 3J_H–F = 10.8 Hz, H-5), 8.43 (s, 1H, H-8); 13C NMR, δ ppm (DMSO-d₆): 52.2 (CO₂CH₃), 108.9 (d, 2J_C–F = 18.7 Hz, C-5), 113.9 (C-7), 127.6 (C-5a), 127.7 (C-3′/C-5′), 130.1 (C-2′/C-6′), 130.2 (C-4′), 130.9 (C-9a), 143.6 (C-1′), 148.5 (C-3a), 148.9 (C-9b), 149.3 (C-8), 150.0 (d, 1J_C–F = 214 Hz, C-4), 164.7 (CO₂Et), 171.7 (C-6); HRMS (ESI): Calcd for C₁₇H₁₀FN₃O₃SNa [M + Na]⁺: 378.03246, found: 378.03209.

Methyl 4-fluoro-9-(4-methoxyphenyl)-6-oxo-6,9-dihydro[1,2,5]thiadiazolo [3, 4-h]quinoline-7-carboxylate (23d). Yield 40%; m.p. 281 °C; ¹H NMR, δ ppm (CDCl₃):δ 3.93 (s, 6H, OCH₃, CO₂CH₃), 7.04 (d, J = 8.9 Hz, 2H, H-2′/H-6′), 7.29 (d, J = 8.9 Hz, 2H, H-3′/H-5′), 8.32 (d, 3J_H–F = 10.3 Hz, 1H, H-5), 8.52 (s, 1H, H-8); 13C NMR, δ ppm (CDCl₃): 52.5 (CO₂CH₃), 55.8 (OCH₃), 109.7 (d, 2J_C–F = 19.6 Hz, C-5), 113.8 (C-7), 114.9 (C-3′/C-5′), 128.2 (C-2′/C-6′), 128.7 (d, 3J_C–F = 5.7 Hz, C-5a), 130.5 (C-9a), 135.9 (C-1′), 148.5 (C-3a), 148.8 (C-9b), 149.6 (C-8), 150.5 (d, 1J_C–F = 261 Hz, C-4), 160.5 (C-4′), 165.5 (CO₂Et), 172.3 (C-6); HRMS (ESI): Calcd for C₁₈H₁₂FN₃O₄SNa [M + Na]⁺: 408.04302, found: 408.04225.

Ethyl 4-fluoro-6-oxo-9-phenyl-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylate (24b). Yield 40%, m.p. 285 °C; ¹H NMR, δ ppm (DMSO-d₆): 1.25 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 4.22 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.60 (m, 5H, H-2′/H-3′/H-4′/H-5′/H-6′), 8.11 (d, 3J_H–F = 10.8 Hz, H-5), 8.41 (s, 1H, H-2); 13C NMR, δ ppm (DMSO-d₆): 14.7 (OCH₂CH₃), 60.9 (OCH₂CH₃), 108.9 (d, 2J_C–F = 19.2 Hz, C-5), 114.4 (C-7), 127.8 (C-3′/C-5′),128.7 (d, 3J_C–F = 5.8 Hz, C-5a), 130.1 (C-2′/C-6′), 130.2 (C-4′), 130.9 (d, 4J_C–F = 3 Hz, C-9a), 139.0 (C-1′), 148.4 (d, 2J_C–F = 10.5 Hz, C-3a), 148.7 (d, 3J_C–F = 1.6 Hz, C-9b), 148.9 (C-8), 150.5 (d, 1J_C–F = 214 Hz, C-4), 164.2 (CO₂Et), 171.7 (C-6)(C=O); HRMS (ESI): Calcd for C₁₈H₁₂FN₃O₃SNa [M + Na]⁺: 392.04811, found: 392.04934.

Ethyl 4-fluoro-9-(4-fluorophenyl)-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylate (24c). Yield 79%, m.p. 274 °C; ¹H NMR, δ ppm (DMSO-d₆): 1.38 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 4.38 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.26 (d, J = 8.3 Hz, 2H, H-2′/H-6′), 7.41 (dd, J = 8.3 Hz, ⁴J_H–F = 4.6 Hz, 2H, H-3′/H-5′), 8.28 (d, 3J_H–F = 10.2 Hz, 1H, H-5), 8.46 (s, 1H, H-2); 13C NMR, δ ppm (DMSO-d₆): 14.4 (OCH₂CH₃), 61.5 (OCH₂Me), 109.6 (d, 2J_C–F = 19.6 Hz, C-5), 114.3 (C-7), 117.0 (d, 2J_C–F = 23.0 Hz, C-3′/C-5′),128.7 (d, 3J_C–F = 5.8; Hz, C-5a), 129.2 (d, 3J_C–F = 9.0 Hz, C-2′/C-6′), 130.1 (d, 4J_C–F = 3 Hz, C-9a), 139.0 (C-1′), 148.4 (d, 2J_C–F = 10.5 Hz, C-3a), 148.7 (d, 3J_C–F = 1.6 Hz, C-9b), 148.9 (C-8), 150.5 (d, 1J_C–F = 261 Hz, C-4), 163.3 (d, 1J_C–F = 249 Hz, C-4′), 164.7 (CO₂Et), 172.3 (C-6); HRMS (ESI): Calcd for C₁₈H₁₁F₂N₃O₃SNa [M + Na]⁺: 410.03869, found: 410.04116.

Ethyl 4-fluoro-9-(4-methoxyphenyl)-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylate (24d). Yield 40%; m.p. 237 °C; ¹H NMR, δ ppm (DMSO-d₆): 1.24 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 3.85 (s, 3H, OCH₃), 4.20 (q, J = 7.1 Hz, 2H, OCH₂CH₃), 7.09 (d, J = 8.8 Hz, 2H, H-2′/H-6′), 7.54 (d, J = 8.8 Hz, 2H, H-3′/H-5′), 8.04 (d, 3J_H–F = 10.8 Hz, 1H, H-5), 8.37 (s, 1H, H-2); 13C NMR, δ ppm (DMSO-d₆): 14.7 (OCH₂CH₃), 56.1 (OCH₃), 61.5 (OCH₂CH₃), 108.9 (d, 2J_C–F = 19.1 Hz, C-5), 114.1 (C-7), 115.1 (C-3′/C-5′),127.5 (d, 3J_C–F = 5.4 Hz, C-5a), 129.0 (C-2′/C-6′), 131.3 (C-9a), 136.6 (C-1′), 148.3 (C-3a), 148.9 (C-9b), 149.4 (C-8), 150.1 (d, 1J_C–F = 249 Hz, C-4), 160.4 (C-4′), 164.2 (CO₂Et), 171.7 (C-6); HRMS (ESI): Calcd for C₁₉H₁₄FN₃O₄SNa [M + Na]⁺: 422.05867, found: 422.05809.

Preparation of 9-(aryl/alkyl)-4-fluoro-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylic acids (25a–d). A vigorously stirred suspension of the ester (23a,b,d/24b–d). (1.5 mmol) in 6 N HCl (15 mL) and ethanol (6 mL) was heated at 80–85 °C under reflux conditions. Progress of the ester hydrolysis was monitored by TLC and was completed within 20–24 h. Thereafter, the reaction mixture was cooled, poured onto crushed ice (30 g) and the resulting heavy pale yellow precipitate was collected, washed with cold water, dried and recrystallized from N,N-dimethylformamide (DMF) to produce a pure compound.

9-Ethyl-4-fluoro-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylic acid (25a). Yield 95%; m.p. 315 °C; ¹H NMR δ ppm (D₂O): 1.48 (t, J = 7.0 Hz, 3H, NCH₂CH₃), 5.26 (q, J = 7.0 Hz, 2H, NCH₂CH₃), 8.23 (d, 3J_H–F = 10.4 Hz, 1H, H-5), 8.68 (s, 1H, H-8), 15.14 (br s, 1H, CO₂H); 13C NMR δ ppm (D₂O): 16.4 (NCH₂CH₃), 53.5 (NCH₂CH₃), 108.1 (d, 2J_C–F = 19.6 Hz, C-5), 111.9 (C-7), 125.7 (d, 3J_C–F = 6.5 Hz, C-5a), 132.2 (C-9a), 148.7 (d, 2J_C–F = 10.5 Hz, C-3a), 149.1 (d, 3J_C–F = 3 Hz, C-9b), 149.7 (C-8), 154.4 (d, 1J_C–F = 259 Hz, C-4), 165.7 (CO₂H), 176.3 (C-6); HRMS (ESI): Calcd for C₁₂H₈FN₃O₃S [M − H]⁻: 292.01921, found: 292.01976.

4-Fluoro-6-oxo-9-phenyl-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylic acid (25b). Yield 90%; m.p. 301 °C; ¹H NMR δ ppm (D₂O): 7.64 (m, 5H, H-1′/H-2′/H-3′/H-4′/H-5′), 8.23 (d, 3J_H–F = 10.7 Hz, 1H, H-5), 8.68 (s, 1H, H-8), 14.91 (br s,1H, CO₂H); 13C NMR δ ppm (D₂O): 107.9 (d, 2J_C–F = 19.0 Hz, C-5), 111.4 (C-7), 125.7 (Hz, C-5a), 127.5 (C-3′/C-5′), 130.2 (C-2′/C-6′), 130.7 (C-4′), 131.2 (C-9a), 143.4 (C-1′), 148.6 (d, 2J_C–F = 10.5 Hz, C-3a), 149.0 (C-9b), 149.7 (C-8), 154.3 (d, 1J_C–F = 259 Hz, C-4), 165.5 (CO₂H), 176.8 (C-6); HRMS (ESI): Calcd for C₁₆H₈FN₃O₃S [M  −  H]⁻: 340.01921, found: 340.01976.

4-Fluoro-9-(4-fluorophenyl)-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylic acid (25c). Yield 93%; m.p. 278 °C; ¹H NMR δ ppm (DMSO-d₆); 7.44 (d, J = 8.4 Hz, 2H, H-2′/H-6′), 7.72 (dd, J = 8.4 Hz, 3J_H–F = 4.4 Hz, 2H, H-3′/H-5′), 8.19 (d, 3J_H–F = 10.4 Hz, 1H, H-5), 8.71 (s, 1H, H-8), 14.9 (br s,1H, CO₂H); 13C NMR δ ppm (DMSO-d₆); 107.9 (d, 2J_C–F = 19.3 Hz, C-5), 114.3 (C-7), 117.0 (d, 2J_C–F = 23.0 Hz, C-3′/C-5′),125.7 (d, 3J_C–F = 6.5 Hz, C-5a), 130.1 (d, 3J_C–F = 9.0 Hz, C-2′/C-6′), 132.2 (C-9a), 139.6 (C-1′), 148.6 (d, 2J_C–F = 10.5 Hz, C-3a), 149.0 (d, 3J_C–F = 3 Hz, C-9b), 149.7 (C-8), 151.3 (d, 1J_C–F = 259 Hz, C-4), 163.1 (d, 1J_C–F = 245 Hz, C-4′), 165.6 (CO₂H), 176.6 (C-6); HRMS (ESI): Calcd for C₁₆H₇F₂N₃O₃SNa [M + Na]⁺: 382.00739, found: 382.00684.

4-Fluoro-9-(4-methoxyphenyl)-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylic acid (25d). Yield 92%; m.p. 300 °C; ¹H NMR δ ppm (DMSO-d₆); 3.86 (OCH₃), 7.12 (d, J = 8.9 Hz, 2H, H-2′/H-6′), 7.57 (d, J = 8.9 Hz, H-3′/H-5′), 8.20 (d, 3J_H–F = 10.4 Hz, 1H, H-5), 8.64 (s, 1H, H-8), 14.86 (br s,1H, CO₂H); 13C NMR δ ppm (DMSO-d₆); 56.1 (OCH₃), 107.9 (d, 2J_C–F = 19.5 Hz, C-5), 111.4 (C-7), 115.2 (C-3′/C-5′), 125.7 (d, 3J_C–F = 6.5 Hz, C-5a), 128.8 (C-2′/C-6′), 132.2 (C-9a), 136.4 (C-1′), 148.6 (d, 2J_C–F = 10.5 Hz, C-3a), 149.0 (d, 3J_C–F = 3 Hz, C-9b), 150.0 (C-8), 150.9 (d, 1J_C–F = 259 Hz, C-4), 160.7 (C-4′), 165.5 (CO₂H), 176.8 (C-6).; IR (KBr) v: 3061.36, 1732.53, 1611.62, 1504.53, 1474.49, 1253.78 cm⁻¹; HRMS (ESI): Calcd for C₁₇H₁₀FN₃O₄S [M  −  H]⁻: 370.02978, found: 370.03033.

Antimicrobial activity

The biological screening of compounds 19–22a–d, 23a, 24b–d and 25a–d was performed in vitro by the two fold broth dilution method27. The antibacterial activity was tested against Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538 Gram positive strains, Escherichia coli ATCC 8739 and Haemophilus influenzae ATCC 19418 Gram negative ones using Mueller Hinton medium. The antifungal properties were evaluated against Candida tropicalis ATCC 1369 and Saccharomyces cerevisiae ATCC 9763 yeasts and Aspergillus niger ATCC 6275 mould in Sabouraud dextrose broth. The compounds at the concentration range of 0.0015–400 μg/mL in 1% DMSO were used in this study, with ciprofloxacin as reference antibacterial substance and miconazole as reference antifungal drug. DMSO was loaded as negative control. Suspensions of the microorganisms were prepared to inoculate 5 × 10⁵ bacteria/mL and 1× 10³ fungi/mL. The minimum inhibitory concentration (MIC, μg/mL) value was taken as the lowest concentration of compound that showed inhibition of microbial growth after 24 h of incubation at 37 °C for bacteria and after 48 h of incubation at 30 °C for fungi. Each experiment has been done in triplicate and repeated at least three times.

Results and discussion

Chemistry

Our synthesis banks on the reported strategy used by us and others in building the quinolone skeleton. Scheme 1 shows the synthesis of our N-aryl/alkyl substituted quinolones, in which the benzoic acid derivative 17 was converted to the benzoyl chloride via interaction with thionyl chloride under reflux conditions. The resulting benzoyl chloride was reacted with ethyl 3-(N,N-dimethylamine)acrylate in benzene to produce the intermediate 18 which was reacted with the desired amino compound to get the respective 19a–d as the predominant Z isomers.

Scheme 1. synthesis of ethyl 7-chloro-1-(ethyl/aryl)-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylates (20a–d).

The structures of compounds 19a–d are supported by spectral (NMR and MS) data. The various ¹H and 13C NMR signals were assigned based on DEPT and 2D NMR (HMQC) experiments. The ¹H NMR spectra show the methylene protons (O–CH₂CH₃) as quartet in the range δ 3.98–4.07 ppm, while the methyl protons (O–CH₂CH₃) resonate as triplet in the range δ 1.01–1.07 ppm. The H-3″ proton resonates as a doublet signal in the range δ 8.19–8.67 ppm (with J = 11–14 Hz) indicating that the products are the Z-isomers. The H-6 proton of this series is well characterized as a sharp doublet in the range δ 7.09–7.19 ppm, (with ³J_H–F = 8.0–8.2 Hz) due to spin–spin coupling interaction with the vicinal fluorine atom.

The 13C NMR spectra of compounds 19a–d reveal clearly the presence of the fluorine atom and its effect on the adjacent carbons; thus the following carbons showed through-bonds coupling with the fluorine atom and appear as doublets (C-5, ¹J = 253–254 Hz), (C-6, ²J = 22.5–22.7 Hz). The 13C NMR spectra also indicate the presence of the keto group at 189.3–189.8 ppm, while the carbonyl carbon of the ester group resonates in the range 164.1–165.9 ppm. The C-3″ carbon resonates in the range 154.2–161.2 ppm, while C-2″ in the range 99.8–102.2 ppm.

The quinolone ring in 20a–d was constructed through intramolecular cyclization of (19a–d) in which the chlorine atom at position 2 was replaced by the amino group via nucleophilic aromatic substitution (S_NAr) assisted by the neighboring nitro group and fluorine atom28,29.

In this group of compounds, ¹H NMR showed the disappearance of N–H broad peak at 11–12.7 ppm present in their acyclic precursors 19a–d; the spectrum showed a change in the doublet peak of CH to a singlet one. Both H-5 and H-2 in 20a–d are more deshielded than the corresponding protons in the 19a–d precursors. These changes constitute diagnostic criteria for ring closure (19a–d → 20a–d).

The 13C NMR spectra of compounds 20a–d reveal that most of the carbons are deshielded except for C-2, C-4 and C-8a.

In the 13C NMR spectrum, the carbon of the keto group [=O on C-4] for 20a–d resonates in the range 170.4–170.9 ppm, which is more shielded than the corresponding C-4 in compounds 19a–d (188–189 ppm); similarly, C-2 resonates around 152 ppm and C-8a resonates around 130 ppm and are more shielded than the corresponding carbons in 19a–d (154 ppm) and (148 ppm), respectively.

Having the essential quinolone compounds in hand, we could easily convert them to the diamino compounds 22a–d as shown in Scheme 2, in which the chlorine atom was replaced with the azide moiety via the standard nucleophilic aromatic substitution. The structure of compounds 21a–d is supported by spectral (NMR and MS) data which are in agreement with the literature data. In the ¹H NMR spectra, there is not any difference in the chemical shift compared to the compounds 20a–d. In the 13C NMR spectra there is a small difference for C-7, which appears downfield in compounds 21a–d (∼128 ppm) compared to the corresponding C-7 in 20a–d due to the replacement of –Cl by the azide group.

Scheme 2. Synthesis of ethyl 7,8-diamino-1-(aryl/alkyl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylates (22a–d).

Reduction of the particular ethyl 7-azido-1-(alkyl/aryl)-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (21a–d) with anhydrous stannous chloride in concentrated hydrochloric acid afforded the respective 7,8-diamino compounds (22a–d) in good yield (78–85%). The effective reduction of 21a–d to afford 22a–d was deduced from the presence of NH band at 3462–3295 cm⁻¹ in the IR spectra. The IR spectra of 22a–d also show absorption bands (1672, 1632 cm⁻¹), which indicate the presence of a conjugated keto group, while the carboxy group absorbs at 1718 cm⁻¹. The structure of 22a–d is further supported by NMR spectral data. The ¹H NMR spectra of 22a–d indicate the appearance of the amine protons signals at 5.05 (NH₂ on C-8) and 5.43 (NH₂ on C-7), while H-5 appears as a doublet at 7.19 ppm, and is thus shielded compared to its position in 21a–d. The 13C NMR spectra show the C-8 and C-5 resonating around 125.7 and 100.6 ppm, respectively, which are shielded compared to their positions in 21a–d (133.1 and 115.8 ppm, respectively).

Cyclocondensation of the synthesized diamine 22a–d using thionyl chloride produces the targeted bicyclic benzo[c][1,2,5]thiadiazoles system 23/24 (Scheme 3). The driving force of formation is due to: (i) strong electrophilicity of thionyl chloride, and (ii) stability of the tricyclic product due to aromaticity. It is worth mentioning that upon the workup procedure, where methanol was used to destroy the excess thionyl chloride, trans-esterification of the ethyl ester produced completely the methyl ester 23a, while a 1:1 ratio of methyl and ethyl esters were isolated for the product 23b/24b and 23d/24d. Compound 24c was isolated as the ethyl ester only.

Scheme 3. Synthesis of 9-(aryl/alkyl)-4-fluoro-6-oxo-6,9-dihydro[1,2,5]thiadiazolo[3,4-h]quinoline-7-carboxylic acids 25a--d.

The following changes constitute diagnostic criteria for ring closure: the ¹H NMR spectra of compounds (23/24) indicate the disappearance of the amine protons signals at 3.75–4.80 (NH₂ on C-8) and 5.29–5.61 (NH₂ on C-7), while H-5 appears as a doublet at 8.09–8.32 ppm, and is thus deshielded compared to its position in (22a–d). The 13C-NMR spectra show the C-3a and C-9b around 148.4 and 148.9 ppm, respectively, which are deshielded compared to their positions in 22a–d.

The hydrolysis of ethyl(methyl) 9-(aryl/alkyl)-4-fluoro-6-oxo-6,9-dihydro[1,2,5]thiadiazolo- [3,4-h]quinoline-7-carboxylate (23/24a–d) gave the corresponding acid products (25a–d) in ∼90 % yield by using 12% aqueous HCl under reflux conditions.

The structures of 25a–d are supported by spectral (¹H NMR and MS) data. The ¹H NMR spectra show the disappearance of ethyl or the methyl protons signals (in previous esters 23/24b–d) from the spectra and the appearance of a broad signal at 14.9 (exchangeable CO₂H proton). 13C-NMR spectra show the disappearance of ethyl or the methyl carbons signals (in previous esters 23/24b–d) from the spectra.

Antimicrobial activity

The in vitro antimicrobial properties of the acids 25a–d, their esters 23a and 24b–d and of the starting compounds 19/22a–d were detected against Gram positive and Gram negative bacteria, yeasts and mould.

Table 1 summarizes the results obtained when compounds 23a, 24b–d and 25a–d were assayed against B. subtilis and S. aureus Gram positive bacteria and E. coli and H. influenzae Gram negative ones. The antibacterial activity of desfluorinated N-ethylquinolone acid 14 and of the 6-fluoro-N–Cyclopropyl analogous 16 together with its ethyl ester is also reported for an useful comparison.

Table 1. In vitro antibacterial activity of thiadiazoloquinolones (minimum inhibitory concentration in μg/mL).

	Microorganism
Compound	Bacillus subtilis ATCC 6633	Staphylococcus aureus ATCC 6538	Escherichia coli ATCC 8739	Haemophilus influenzae ATCC 19418
23a	12	>400	>400	>400
24b	100	>400	>400	>400
24c	12	400	>400	200
24d	>400	>400	>400	>400
25a	0.15	1.5	3	1.5
25b	1.5	12	50	6
25c	0.15	1.5	3	0.7
25d	100	200	>400	>400
14*	nt†	6	6	nt
16 ethyl ester*	3	50	100	25
16*	0.07	0.7	0.7	0.3
Ciprofloxacin	0.03	0.3	0.015	0.15

*Results reported in previous article.19

†Not tested.

The thiadiazoloquinolone acids 25a–d are the most active compounds. N-ethyl derivative 25a and N-4-fluorophenyl derivative 25c had strong inhibitory effect on the growth of both Gram positive and Gram negative tested strains (MIC 0.15–3 μg/mL). Significant inhibition was also exhibited by phenyl derivative 25b with MIC values in the 1.5–50 μg/mL range. A lower activity was observed for 4-methoxyphenyl derivative 25d against B. subtilis and S. aureus (MIC 100–200 μg/mL). Gram positive B. subtilis was the most sensitive microorganism. Among thiadiazoloquinolone esters, N-ethyl derivative 23a and N-4-fluorophenyl derivative 24c showed good antimicrobial properties against B. subtilis at 12 μg/mL, whereas phenyl derivative 24b had some activity against the same strain at the concentration of 100 μg/mL. Compound 24c was the only ester that inhibited the growth of H. influenzae and S. aureus (MIC 200–400 μg/mL, respectively).

All the newly synthesized compounds exhibited antibacterial activity lower than that of ciprofloxacin used as reference substance. Furthermore, no inhibition was exerted against S. cerevisiae, C. tropicalis and A. niger fungal strains up to the concentration of 400 μg/mL (data not shown; MICs of miconazole 12 μg/mL, 6 μg/mL and 3 μg/mL, respectively).

Structure--activity relationship shows that, among the newly synthesized thiadiazoloquinolone acids and among their corresponding esters, the N-4-fluorophenyl derivatives 24c and 25c exhibited the highest activity, followed, in the order, by N-ethyl- (23a and 25a), N-phenyl- (24–25b) and N-4-methoxyphenyl- (24–25d) quinolones. However, these new thiadiazoloquinolones exhibited MIC values higher than those of the N–Cyclopropyl acid 16 and its ethyl ester, confirming the current knowledge that the replacement of the cyclopropyl at the N-1 position of the quinolone ring with ethyl or substituted and unsubstituted phenyl moiety plays a negative role on the antibacterial properties. Likewise, the introduction of fluorine at position C-6, in compound 25a, enhanced, by a factor four or two, the activity of 14 against S. aureus and E. coli, respectively.

As concerns the intermediates 19/22a–d, only B. subtilis, the most sensitive microorganism, was inhibited by 22c at 100 μg/mL and by 7-chloro-8-nitroquinolines 20a and 20c at 200 μg/mL. A low antibacterial activity appeared when compounds 19 cyclize to quinolone structure 20. This inhibition disappeared in the derivatives 21, probably owing to the loss of chlorine substituent. Replacing the azido fragment with the amino group for substances 22 led to an increase in antibacterial properties of 4-fluorophenyl derivative 22c against B. subtilis. The subsequent cyclization of the two amino groups at 7 and 8 positions on the fused thiadiazole ring (compounds 23a and 24b-d) enhanced the activity. Compound 24c showed a MIC value of 12 μg/mL towards B. subtilis and broadened the range of its inhibition against S. aureus and H. influenzae. This change in the structure resulted also in increased activity of 23a and 24b towards B. subtilis (MIC 12 and 100 μg/mL, respectively).

As expected, the sizeable increase of activity was observed in the target compounds 25a--d, whose activity is discussed above, by replacing the C-3 ester group with the carboxylate that is considered critical and not yet improved by any change.

Conclusion

The aim of this study is to investigate whether the thiadiazolo fused ring, as a bridge between the critical 7 and 8 positions of the fluoroquinolone scaffold, offers new insight into the structural–activity relationship of the class of quinolones antibacterials. In this study, 26 new compounds were successfully synthesized and fully characterized. The synthesized thiadiazolofluoroquinolones show broad spectrum of activity. The fluoroquinolone 25c was the most active compound in this study. The activities of this class of compounds were expected as a consequence of the modifications at the positions 7 and 8 of the fluoroquinolone ring, and are in agreement with the analogous previous generations of fluoroquinolones. This modification is expected to increase the intracellular targets on the type II topoisomerase and to impair the action of efflux pump, thus lowering the development of quinolone resistance in Gram positive pathogens. Extensive mechanism of action for this class of compounds will be investigated.

Declaration of interest

The authors report no conflicts of interests. The authors alone are responsible for the content and writing of this article.

Acknowledgements

We wish to thank the Deanship of Scientific Research (The University of Jordan, Amman, Jordan) for financial support.