4-Chloroisocoumarins as Chlamydial Protease Inhibitors and Anti-Chlamydial Agents
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design and Synthesis of 4-Chloroisocoumarin Compounds
2.2. Inhibitory Properties of 4-Chloroisocoumarins against CtHtrA and HLE
2.3. 4-Chloroisocoumarin Cytotoxic Effects in HEp-2 Cells and McCoy B Cells
2.4. 4-Chloroisocoumarin Compound Effects against C. trachomatis
3. Materials and Methods
3.1. Chemistry
3.1.1. General Procedure for the Synthesis of Unsubstituted Homophthalate Monoesters (1a–1g)
- 2-(2-iso-butoxy-2-oxo-ethyl)benzoic acid (1f). White solid (0.800 g, 49%) 1H NMR: (400 MHz, CDCl3) δ 11.33 (br s, 1H, COOH), 8.13 (dd, J = 1.2, 8.0 Hz, 1H, H-6), 7.54 (td, J = 1.2, 7.6 Hz, 1H, H-5), 7.40 (td, J = 1.2, 7.6 Hz, 1H, H-4), 7.29 (d, J = 7.6 Hz, 1H, H-3), 4.08 (s, 2H, CH2CO2), 3.88 (d, 2H, OCH2), 1.91 (sxt, J = 6.8 Hz, 1H, CH), 0.90 (d, J = 6.8 Hz, 6H, 2 × CH3). 13C NMR: (100 MHz, CDCl3) δ 172.2, 171.5, 137.0, 133.2, 132.4, 131.9, 128.6, 127.5, 70.9, 40.7, 27.7, 19.0. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H16O4Na 259.0946; Found 259.0951. m.p. 120–122 °C.
- (±)-2-(2-sec-butoxy-2-oxo-ethyl)benzoic acid (1g). White solid (1.22 g, 62%), 1H NMR: (400 MHz, CDCl3) δ 8.12 (dd, J = 7.6, 1.2 Hz, 1H, H-6), 7.53, (td, J = 1.2 Hz, 7.6 Hz, 1H, H-5), 7.40 (td, J = 1.2, 7.6 Hz, 1H, H-4), 7.28 (d, J = 7.6 Hz, 1H, H-3), 4.87 (sxt, J = 6.4 Hz, 1H, C-1′), 4.03 (d, J = 2.0 Hz, 2H, CH2CO2), 1.66–1.47, m, 2H, CH2), 1.21 (d, J = 6.4 Hz, 3H, CH3), 0.88 (t, J = 7.2 Hz, 3H, CH3). 13C NMR: (100 MHz, CDCl3) δ 172.5, 171.2, 137.1, 133.2, 132.4, 131.8, 128.7, 127.4, 72.8, 41.1, 28.7, 19.3, 9.6. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H16O4Na 259.0946; Found 259.0942. m.p. 101–103 °C.
3.1.2. General Procedure for the Synthesis of 3-alkoxy-4-chloroisocoumarins (2a–2g) (General Procedure A)
- 3-Methoxy-4-chloroisocoumarin (2a). Pale-yellow needles (0.272 g, 20%). 1H NMR (500 MHz, CDCl3) δ 8.20 (d, J = 7.5 Hz, H-8), 7.77–7.72 (m, 2H, H-6 and H-5), 7.39 (dt, J = 8.5, 1.0 Hz, 1H, H-7), 4.46 (q, J = 7.0 Hz, 2H, CH2), 1.47 (t, J = 7.0 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 159.7, 153.1, 137.9, 135.6, 130.1, 126.2, 122.23 117.3, 91.3, 66.8, 14.9. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C11H10ClO3Na 247.0138; Found 247.0127. 1H, 13C NMR and melting point was consistent with previous reported data [31].
- 3-Ethoxy-4-chloroisocoumarin (2b). Pale-yellow needles (0.250 g, 23%). 1H NMR (500 MHz, CDCl3) δ 8.20 (d, J = 7.5 Hz, H-8), 7.77–7.72 (m, 2H, H-6 and H-5), 7.39 (dt, J = 8.5, 1.0 Hz, 1H, H-7), 4.46 (q, J = 7.0 Hz, 2H, CH2), 1.47 (t, J = 7.0 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 159.7, 153.1, 137.9, 135.6, 130.1, 126.2, 122.23 117.3, 91.3, 66.8, 14.9. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C11H10ClO3Na 247.0138; Found 247.0127. m.p. 94–96 °C. Melting point was consistent with the reported value [31].
- 3-Propoxy-4-chloroisocoumarin (2c). 1H, 13C NMR and melting point were consistent with those previously reported [19].
- 3-Butoxy-4-chloroisocoumarin (2d). 1H, 13C NMR and melting point were consistent with those previously reported [19].
- 3-iso-Propoxy-4-chloroisocoumarin (2e). Pale yellow solid (0.235 g, 34%) 1H NMR: (500 MHz, CDCl3) δ 8.21 (d, J = 8.0 Hz, 1H, H-8), 7.78–7.71 (m, 2H, H-6 and H-5), 7.40 (dt, J = 7.0, 1.5 Hz, 1H, H-7), 5.06 (sep, J = 6.0 Hz, 1H, CH), (d, J = 6.0 Hz, 6H, 2 × CH3). 13C NMR: (125 MHz, CDCl3) δ 160.0, 152.7, 137.8, 135.6, 130.1, 126.3, 122.5, 117.6, 93.0, 75.6, 22.3. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C12H11ClO3Na 261.0289; Found 261.0284. m.p. 58–60 °C.
- 3-iso-Butoxy-4-chloroisocoumarin (2f). Yellow solid (0.255 g, 34%) 1H NMR: (500 MHz, CDCl3) δ 8.20 (dd, J = 7.5, 1.5 Hz, 1H, H-8), 7.77–7.70 (m, 2H, H-6 and H-5), 7.39 (dt, J = 7.0, 1.5 Hz, 1H, H-7), 4.15 (d, J = 6.5 Hz, 2H, CH2), (sep, J = 7.0 Hz, 1H, CH), 1.05 (d, J = 7.0 Hz, 6H, 2 × CH3).13C NMR: (125 MHz, CDCl3) δ 159.7, 153.3, 138.0, 135.6, 130.1, 126.1, 122.2, 117.3, 91.0, 76.6, 28.4, 18.8. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H13ClO3Na 275.0451; Found 275.0444. m.p. 48–51 °C. Melting point was consistent with the reported value [16].
- (±)-3-sec-Butoxy-4-chloroisocoumarin (2g). Yellow solid (0.215 g, 28%) 1H NMR: (500 MHz, CDCl3) δ 8.21 (br d, J = 7.5 Hz, 1H, H-8), 7.77–7.71 (m, 2H, H-6 and H-5), 7.40 (dt, J = 7.5, 1.5 Hz, 1H, H-7), 4.87 (sxt, J = 6.0 Hz, 1H, CH), 1.86–1.66 (m, 2H, CH2), 1.40 (d, J = 6.5 Hz, 3H, OCHCH3), 1.03 (t, J = 7.5 Hz, 3H, CH2CH3). 13C NMR: (125 MHz, CDCl3) δ 160.0, 152.9, 137.9, 135.6, 130.1, 126.2, 122.4, 117.6, 92.6, 80.2, 29.2, 19.8, 9.5. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H13O3ClNa 275.0451; Found 275.0439. m.p. 57–59 °C.
3.1.3. General Procedure for the Synthesis of 5-Substituted Homophthalate Monoesters (9 and 13) (General Procedure B)
- (±)-5-Chloro-2-(2-sec-butoxy-2-oxo-ethyl)benzoic acid (9). White solid (0.300 g, 48%). 1H NMR: (400 MHz, CDCl3) δ 10.66 (br s, 1H, COOH), 8.09 (d, J = 2.4 Hz, 1H, H-6), 7.50 (dd, J = 2.4, 8.0 Hz, 1H, H-4), 7.22 (d, J = 7.6 Hz, 1H, H-3), 4.86 (sxt, J = 6.4 Hz, 1H, CH), 3.99 (d, J = 2.0 Hz, 2H, CH2CO2), 1.65–1.47 (m, 2H, CH2), 1.20 (d, J = 6.4 Hz, 3H, OCHCH3), 0.88 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 171.3, 170.7, 135.5, 133.7, 133.4, 133.2, 131.7, 130.1, 73.1, 40.4, 28.7, 19.3, 9.6. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H15O4Na 293.0557; Found 293.0548. m.p. 100–103 °C.
- (±)-5-Methoxy-2-(2-sec-butoxy-2-oxo-ethyl)benzoic acid (13). Brown solid (1.53 g, 62%) 1H NMR: (400 MHz, CDCl3) δ 7.64 (d, J = 2.8 Hz, 1H, H-6), 7.18 (d, J = 8.4 Hz, 1H, H-3), 7.07 (dd, J = 3.2 Hz, 8.4 Hz, 1H, H-4), 4.86 (sxt, J = 6.4 Hz, 1H, CH), 3.95 (d, J = 2.0 Hz, 2H, CH2CO2), 3.85 (s, 3H, OCH3), 1.65-1.43 (m, 2H, CH2), 1.20 (d, J = 6.0 Hz, 3H, OCHCH3), 0.88 (d, J = 7.6 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 172.4, 171.6, 158.5, 133.4, 129.5, 129.2, 119.3, 116.5, 72.7, 55.5, 40.2, 28.7, 19.3, 9.6. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H18O5Na 289.1052; Found 289.1048. m.p. 115–118 °C.
3.1.4. Synthesis of (±)-5-Nitro-2-(2-sec-butoxy-2-oxo-ethyl)benzoic acid (4)
- (±)-5-Nitro-2-(2-sec-butoxy-2-oxo-ethyl)benzoic acid (4) White solid (6.50 g, 76%) 1H NMR: (400 MHz, CDCl3) δ 8.95 (s, J = 2.4 Hz, 1H, H-6), 8.38 (dd, J = 2.4 Hz, 8.4 Hz, 1H, H-4), 7.50 (d, J = 8.4 Hz, 1H, H-3), 4.88 (sxt, J = 6.2 Hz, 1H, CH), 4.16 (d, J = 2.8 Hz, 2H, H-2′), 1.66–1.50 (m, 2H, CH2CH3), 1.22 (d, J = 6.2 Hz, 3H, OCHCH3), 0.88 (t, J = 7.4 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 170.3, 169.8, 147.1, 143.9, 133.7, 130.02, 127.4, 126.8, 73.6, 40.9, 28.7, 19.3, 9.6. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H15NO6Na 304.0797; Found 304.0790. m.p. 112–115 °C.
- (±)-3-sec-Butoxy-7-nitro-4-chloroisocoumarin (5). Synthesized using general procedure A with minor modifications. The reaction was performed at reflux and the product was triturated with methanol to afford yellow needles (2.49 g, 59%). 1H NMR: (400 MHz, CDCl3) δ 9.02 (d, J = 2.0 Hz, 1H, H-8), 8.51 (dd, J = 9.2, 2.4 Hz, 1H, H-6), 7.82 (d, J = 8.8 Hz, 1H, H-5), 5.02 (m, 1H, CH), 1.90–1.70 (m, 2H, CH2), 1.45 (d, J = 6.2 Hz, 3H, OCHCH3), 1.03 (t, J = 7.4 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 157.8, 155.7, 145.0, 143.2, 129.6, 126.3, 123.5, 116.8, 90.8, 81.2, 29.2, 20.0, 9.4. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H12NO5ClNa 320.0302; Found 320.0292. m.p. 108–111 °C.
3.1.5. Synthesis of (±)-7-Amino-3-sec-butoxy-4-chloroisocoumarin (6a)
- (±)-7-Amino-4-chloro-3-sec-butoxyisocoumarin (6a). Yellow solid (1.70 g, 95%) 1H NMR: (400 MHz, CDCl3) δ 7.53 (d, J = 8.8 Hz, 1H, H-5), 7.45 (d, J = 2.4 Hz, 1H, H-8), 7.10 (dd, J = 8.8, 2.4 Hz, 1H, H-6), 4.72 (sxt, J = 6.0 Hz, 1H, CH), 3.95 (br s, 2H, NH2), 1.84–1.63 (m, 2H, CH2), 1.35 (d, J = 6.0 Hz, 3H, OCHCH3), 1.01 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 160.4, 150.5, 145.4, 128.8, 123.9, 123.6, 119.2, 113.1, 94.2, 80.4, 29.2, 19.6, 9.5. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H14ClNO3Na 290.0560; Found 290.0566. m.p. 86–88 °C.
3.1.6. Synthesis of (±)-7-(dimethylamino)-3-sec-butoxy-4-chloroisocoumarin (6b)
- (±)-7-(Dimethylamino)-3-sec-butoxy-4-chloroisocoumarin (6b). 1H NMR: (400 MHz, CDCl3) δ 7.58 (d, J = 8.8 Hz, 1H, H-8), 7.39 (d, J = 2.8 Hz, 1H, H-5), 7.18 (dd, J = 2.8, 9.2 Hz, 1H, H-6), 4.72 (sxt, 6.0 Hz, 1H, CH), 3.03 (s, 6H, N(CH3)2), 1.85–1.63 (m, 2H, CH2), 1.36 (d, J = 6.4 Hz, 3H, OCHCH3), 1.02 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 160.9, 150.1, 149.1, 126.6, 123.6, 120.8, 119.1, 110.3, 94.5, 80.3, 40.5, 29.2, 19.6, 9.5. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C15H18ClNO3Na 318.0867; Found 318.0870.
3.1.7. Synthesis of (±)-N-(4-Chloro-1-oxo-3-sec-butoxy-isochromen-7-yl)acetamide (6c)
- (±)-N-(4-Chloro-1-oxo-3-sec-butoxy-isochromen-7-yl)acetamide (6c). 1H NMR: (400 MHz, CDCl3) δ 8.21 (dd, J = 2.1 Hz, 8.8 Hz, 1H, H-6), 8.11 (d, J = 2.2 Hz, 1H, H-8), 7.69 (d, J = 8.8 Hz, 1H, H-5), 7.61 (br s, 1H, NH), 4.85–4.78 (m, 1H, CH), 2.23 (s, 3H, NHCOCH3), 1.85-1.67 (m, 2H, CH2), 1.38 (d, J = 6.2 Hz, 3H, OCHCH3), 1.02 (t, J = 7.4, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 168.9, 160.0, 152.1, 136.9, 133.8, 128.0, 123.5, 119.4, 117.9, 93.2, 80.6, 29.2, 24.4, 19.8, 9.5. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C15H16ClNO4Na 332.0660; Found 332.0660,. m.p. 161 °C (decomposed with gas evolution).
3.1.8. (±)-N-(4-chloro-1-oxo-3-sec-butoxy-isochromen-7-yl)-2,2,2-trifluoroacetamide (6d)
3.1.9. General Procedure for the Synthesis of 6e and 6f
- (±)-3-sec-Butoxy-4-chloro-7-iodoisocoumarin (6e). The mixture was poured into an aqueous solution (100 mL) of 5% sodium bicarbonate containing sodium thiosulfate (7.5 g). The product was extracted into hexane (75 mL) and then washed with water (50 mL) and brine (50 mL) and then dried using anhydrous magnesium sulfate. The solvent was loaded onto a silica gel column and purified with a gradient of 0–20% dichloromethane in hexanes, affording a yellow solid (1.38 g, 65%).1H NMR: (400 MHz, CDCl3) δ 8.49 (d, J = 1.6 Hz, 1H, H-8), 7.99 (dd, J = 2.0, 8.4 Hz, 1H, H-6), 7.43 (d, J = 8.4 Hz, 1H, H-5), 4.87 (sxt, 6.0 Hz, 1H, CH), 1.86–1.59 (m, 2H, CH2), 1.39 (d, J = 6.0 Hz, 3H, CHCH3), 1.02 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 158.4, 153.2, 144.1, 138.4, 137.3, 124.1, 118.9, 91.9, 89.6, 80.5, 29.2, 19.9, 9.5. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H12ClIO3Na 400.9417; Found 400.9408. m.p. 69–72 °C
- (±)-7-Bromo-3-sec-butoxy-4-chloro-isocoumarin (6f). The solvent was removed with reduced pressure at room temperature, and the crude solid was sonicated in hexanes for 15 min. The slurry was loaded onto a silica gel column and purified with a gradient of 0–20% dichloromethane in hexanes, affording a yellow solid (0.170 g, 17%). 1H NMR: (400 MHz, CDCl3) δ 8.32 (d, J = 2.0 Hz, 1H, H-8), 7.82 (dd, J = 2.0, 8.4 Hz, 1H, H-6), 7.58 (d, J = 8.8 Hz, 1H, H-5), 4.87 (sxt, 6.0 Hz, 1H, CH), 1.87–1.66 (m, 2H, CH2), 1.39 (t, J = 6.4 Hz, 3H, CH2CH3), 1.01 (d, J = 6.4 Hz, 3H, CHCH3). 13C NMR: δ 159.6, 153.1, 138.6, 136.9, 132.3, 124.2, 119.4, 118.8, 92.0, 80.6, 29.2, 19.8, 9.5. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H12BrClO3Na 352.9556; Found 352.9542. m.p. 78–80 °C.
3.1.10. Synthesis of Compounds 6g and 6h
- (±)-3-sec-Butoxy-4,7-dichloro-isocoumarin (6g). Synthesized using general procedure A to afford a yellow solid (0.328 g, 63%) 1H NMR: (400 MHz, CDCl3) δ 8.16 (d, J = 1.6 Hz, 1H, H-8), 7.67–7.64 (m, 2H, H-5 and H-6) 4.87 (sxt, J = 6.4 Hz, 1H, CH), 1.87–1.66 (m, 2H, CH2), 1.40 (d, J = 6.0 Hz, 3H, OCHCH3), 1.02 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 158.8, 153.1, 136.5, 135.8, 132.0, 129.3, 124.1, 118.6, 92.0, 80.6, 29.2, 19.8, 9.5. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C13H12Cl2O3H+ 309.0061; Found 309.0051. m.p. 86–88 °C.
- (±)-7-Methoxy-3-sec-butoxy-4-chloroisocoumarin (6h). Synthesized using general procedure A to afford a yellow solid (0.410 g, 77%) 1H NMR: (400 MHz, CDCl3) δ 7.62 (d, J = 8.8 Hz, 1H, H-5), 7.59 (d, J = 2.8 Hz, 1H, H-8), 7.32 (dd, J = 8.8, 2.8 Hz, 1H, H-6), 4.78 (sxt, J = 6.0 Hz, 1H, CH), 3.88 (s, 3H, OCH3), 1.85-1.64 (m, 2H, CH2), 1.37 (d, J = 6.0 Hz, 3H, OCHCH3), 1.01 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR: (100 MHz, CDCl3) δ 160.0, 158.2, 151.4, 131.4, 125.1, 124.1, 118.7, 110.4, 93.4, 80.4, 55.7, 29.2, 19.7, 9.4. HRMS (ESI/Q-TOF) m/z: [M + Na]+ Calcd for C14H15O4ClNa 305.0549; Found 305.0557. m.p. 55–56 °C.
3.2. Biological Studies
3.2.1. Recombinant Expression and Purification of CtHtrA
3.2.2. CtHtrA Protease Inhibition Assay
3.2.3. HLE Protease Inhibition Assay
3.2.4. MTS Cell Viability Assay
3.2.5. Chlamydia trachomatis Growth Inhibition Assay
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Compound | Relative Potency against CtHtrA a | Relative Potency against HLE b | Relative Selectivity c |
---|---|---|---|
JO146 | 1.00 | 1.00 | 1.00 |
2a | 2.33 | 0.0207 | 113.0 |
2b | 3.73 | 0.0664 | 56.4 |
2c | 2.23 | 0.323 | 6.92 |
2d | 1.57 | 0.310 | 5.08 |
2e | 5.32 | 0.419 | 12.7 |
2f | 1.50 | 0.368 | 4.08 |
2g | 6.58 | 0.451 | 14.6 |
6a | 0.326 | n.d. | n.d. |
6b | 0.113 | n.d. | n.d. |
6c | 0.158 | n.d. | n.d. |
6d | <0.001 | n.d. | n.d. |
6e | <0.001 | n.d. | n.d. |
6f | 0.057 | n.d. | n.d. |
6g | 0.390 | n.d. | n.d. |
6h | 0.184 | n.d. | n.d. |
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Phillips, M.J.A.; Huston, W.M.; McDonagh, A.M.; Rawling, T. 4-Chloroisocoumarins as Chlamydial Protease Inhibitors and Anti-Chlamydial Agents. Molecules 2024, 29, 1519. https://doi.org/10.3390/molecules29071519
Phillips MJA, Huston WM, McDonagh AM, Rawling T. 4-Chloroisocoumarins as Chlamydial Protease Inhibitors and Anti-Chlamydial Agents. Molecules. 2024; 29(7):1519. https://doi.org/10.3390/molecules29071519
Chicago/Turabian StylePhillips, Matthew J. A., Wilhelmina M. Huston, Andrew M. McDonagh, and Tristan Rawling. 2024. "4-Chloroisocoumarins as Chlamydial Protease Inhibitors and Anti-Chlamydial Agents" Molecules 29, no. 7: 1519. https://doi.org/10.3390/molecules29071519