Sultam Thioureas: Synthesis and Antiviral Activity Against West Nile Virus

Abstract

The syntheses of eleven sultam thioureas, including nine new compounds, are described. These compounds were synthesized from thioureas and include the first sultam thioureas in which the two thiourea nitrogen groups are not identical. In addition, the first X-ray crystal structures of sultam thioureas and the antiviral activity of these compounds against West Nile virus (WNV) are reported.

Key words

medicinal chemistry - antiviral agents - West Nile virus - heterocycles - sultam thioureas - thioureas

References and Notes

• 1 Barklis E. Still A. Sabri MI. Hirsch AJ. Nikolich-Zugich J. Brien J. Dhenub TC. Scholz I. Alfadhli A. Antimicrob. Agents Chemother.  2007,  51:  2642 
• 2 Linden H. Goerdeler J. Tetrahedron Lett.  1977,  20:  1729 
• 3 Argyropoulos N. Science of Synthesis  2003,  13:  29 
• 4a Oliver JE. J. Org. Chem.  1971,  36:  3465 
• 4b Diveley WWR. inventors; Dithiazolium salts X;  US 3,166,564. 
• 5a Dixit SN. Verma VK. Indian Journal of Chemistry  1963,  1:  487 
• 5b Pandeya SN. Kumar A. Singh BN. Mishra DN. Pharmaceutical Research  1987,  4:  321 
• 5c Joshua C. Prasannan E. Thomas S. Australian Journal of Chemistry  1981,  34:  917 
• 6a Shibuya I, Tsucha T, Taguchi Y, and Ooishi A. inventors; Preparation of dithiazolium compounds;  JP 08027148. 
• 6b Epstein MG. Valente EJ. Wood WJL.
  J. Chem. Crystallogr.  2011,  41:  1060 
• 12 Georgiev AG, and Daskalov HP. inventors; 5-Substituted derivatives of 5H-dibenz(b,f)azepine and method for obtaining the same;  US 4,124,583. 
• 13a Sheldrick GM. Acta Crystallogr., Sect. A   2008.  64:  p.112 
• 13b Sheldrick GM. Program for crystal structure solution and refinement   University of Göttingen; Germany: 1998. 
• 18 Structure Correlation   Vol. 2:  Burgi H.-B. Dunitz JD. VCH Publishers; Weinheim: 1994. 

7

Typical procedure for the synthesis of sultam thioureas. To a flame-dried flask under nitrogen, were added thiourea (1 equiv) and THF to make a 0.4 M solution. Next NaH (60% in paraffin, 1.5 equiv) was added in portions. Phenylisothiocyanate (1 equiv) was added, the reaction mixture was heated in a 45 ˚C oil bath, and the reaction was monitored by TLC. The reaction mixture was cooled to room temperature and then cooled in an ice-water bath. To this reaction mixture was added a 0.5 M solution of HBr (5 equiv) in THF (made using 5.1 M HBr in acetic acid), followed by the addition of hydrogen peroxide (3.5 equiv, 30% H₂O₂ solution). The reaction was stirred overnight and filtered, after which, the solid was dissolved in CH₂Cl₂ (10 mL) and washed with sat. Na₂CO₃ (10 mL). The organic layer was removed and the aqueous layer was washed with a second portion of CH₂Cl₂ (10 mL). The combined CH₂Cl₂ solution was dried with anhydrous MgSO₄, filtered and concentrated in vacuo. The solid was dissolved in CH₂Cl₂ to make a 0.05 M solution, which was cooled in an ice bath under nitrogen. To this solution was added triethylamine (7.2 equiv based on the crude weight of the filtered solid), followed by dropwise addition of a 0.75 M solution of methanesulfonyl chloride (1.5 equiv based on the crude weight of the filtered solid) in CH₂Cl₂. After stirring the reaction for 1 h, the reaction was diluted with CH₂Cl₂ and washed twice with H₂O. The organic layer was dried with anhydrous MgSO₄, filtered and concentrated in vacuo.
The residue was purified by silica chromatography (ethyl acetate-n-hexane, 95% or CH₂Cl₂-n-hexane, 95%). The purity of the product was confirmed by HPLC analysis using two different conditions with a C18, 5 µm (250 × 4.6 mm) column at 40 ˚C, and monitored at 220 nm.

8

Compound 1: ^¹H NMR (400 MHz, CDCl₃): δ = 4.38 (s, 2 H), 6.90-7.44 (m, 15 H). ^¹³C NMR (100 MHz, CDCl₃):
δ = 46.46, 126.7, 126.8, 127.7, 127.9, 128.8, 128.9, 129.3, 129.4, 129.5, 131.9, 144.0, 144.8, 161.8, 188.4. HRMS (ESI+): m/z [M + H]⁺ calcd for C₂₁H₁₈N₃O₂S₃: 440.0556; found: 440.0545. HPLC conditions A: MeCN/H₂O with 0.1% AcOH and the following gradient: 5% MeCN for 2 min, 5-95% MeCN over 16 min, 95% MeCN for 2 min at 0.5 mL/min. HPLC conditions B: MeOH/H₂O with 0.1% AcOH and the following gradient: 5% MeOH for 2 min, 5-95% MeOH over 16 min, 95% MeOH for 2 min at 1.0 mL/min. The retention time of the product under conditions A was 16.9 min (>99% purity) and under conditions B it was 17.9 min (>99% purity).
Compound 6a: ^¹H NMR (400 MHz, CDCl₃): δ = 1.83 (d, J = 6.8 Hz, 3 H), 4.51 (q, J = 6.8 Hz, 1 H), 6.85-7.50 (m, 15 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 14.7, 55.1, 126.7, 126.8, 127.6, 127.9, 128.6, 128.6, 129.2, 129.4, 129.4, 132.2, 144.1, 144.8, 160.7, 188.4. HRMS (ESI+): m/z [M + H]⁺ calcd for C₂₂H₂₀N₃O₂S₃: 454.0712; found: 454.0701. HPLC conditions A: MeCN/H₂O with 0.1% AcOH and the following gradient: 45% MeCN for 3 min, 45-95% MeCN over 15 min, 95% MeCN for 2 min at 1.0 mL/min. HPLC conditions B: MeOH/H₂O with 0.1% AcOH and the following gradient: 55% MeOH for 3 min, 55-95% MeOH over 15 min, 95% MeOH for 2 min at 1.0 mL/min. The retention time of the product under conditions A was 17.4 min (>99% purity) and under conditions B was 18.0 min (>99% purity). Compound 6b: ^¹H NMR (400 MHz, CDCl₃): δ = 1.91 (s, 6 H), 6.90-7.02 (m, 4 H), 7.02-7.14 (m, 3 H), 7.14-7.22 (m, 2 H), 7.22-7.42 (m, 6 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 23.4, 63.7, 126.7, 126.8, 127.6, 127.9, 128.4, 128.7, 129.2, 129.3, 129.3, 132.5, 144.1, 144.9, 160.1, 188.5. HRMS (ESI+): m/z [M + H]⁺ calcd for C₂₃H₂₂N₃O₂S₃: 468.0869; found: 468.0860. HPLC conditions A: MeCN/H₂O with 0.1% AcOH and conditions B: MeOH/H₂O with 0.1% AcOH. The following gradient was used: 40% organic solvent for 3 min, 40-95% organic solvent over 15 min, 95% organic solvent for 2 min at 1.0 mL/min. The retention time of the product under conditions A was 13.6 min (>99% purity) and under conditions B it was 15.8 min (>99% purity).

9

For procedures see the Supporting Information.

10

Compound 6c: ^¹H NMR (400 MHz, CDCl₃): δ = 2.27 (s, 3 H), 2.32 (s, 3 H), 6.77-7.32 (m, 13 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 21.0, 21.2, 46.4, 126.3, 127.4, 128.9, 129.1, 129.1, 129.3, 130.0, 132.0, 136.3, 137.5, 141.7, 142.3, 161.5, 188.2. HRMS (ESI+): m/z [M + H]⁺ calcd for C₂₃H₂₂N₃O₂S₃: 468.0859; found: 468.0857. The purity of the product was confirmed by HPLC using the same two conditions used for 6b. The retention time of the product under conditions A was 14.1 min (98% purity) and under conditions B was 16.5 min (>99% purity).
Compound 6d: ^¹H NMR (400 MHz, CDCl₃): δ = 2.65-2.80 (m, 2 H), 3.12-3.33 (m, 2 H), 4.30 (d, J = 12.0 Hz, 1 H), 4.34 (d, J = 12.0 Hz, 1 H), 6.70-6.82 (m, 2 H), 6.96-7.03 (m, 4 H), 7.17-7.37 (m, 7 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 30.1, 30.2, 46.4, 126.4, 126.8, 127.3, 127.4, 128.0, 128.4, 128.8, 129.2, 129.4, 130.4, 131.9, 134.1, 135.3, 142.2, 143.1, 161.5, 188.5. HRMS (ESI+): m/z [M + H]⁺ calcd for C₂₃H₂₀N₃O₂S₃: 466.0712; found: 466.0700. The purity of the product was confirmed by HPLC using the same two conditions used for 6b. The retention time of the product under conditions A was 12.7 min (96% purity) and under conditions B was 15.1 min (98% purity).

11

Compound 5d: ^¹H NMR (400 MHz, CDCl₃): δ = 2.76-2.92 (m, 2 H), 3.34-3.49 (m, 2 H), 6.05 (br s, 2 H), 7.18-7.52 (m, 8 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 30.0, 30.5, 126.7, 127.8, 128.3, 129.1, 129.3, 130.0, 131.7, 135.7, 136.9, 140.1, 143.7, 183.3. HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₅H₁₅N₂S: 255.0951; found: 255.0951. HPLC conditions A: MeCN/H₂O with 0.1% AcOH and conditions B: MeOH/H₂O with 0.1% AcOH. The following gradient was used: 35% organic solvent for 3 min, 35-95% organic solvent over 15 min, 95% organic solvent for 2 min at 1.0 mL/min. The retention time of the product under conditions A was 6.6 min (>99% purity) and under conditions B was 10.5 min (>99% purity). Crystal Data: CCDC 838607.^²² C₁₅H₁₄N₃S; MW = 254.34; monoclinic; a = 7.3785 (10) Å, b = 15.847 (3) Å, c = 11.1495 (16) Å, β = 101.204 (14)˚; U = 1278.8 (3) Å^³; T = 298 (2) K; space group P 2₁/n (#14); λ = 0.71073 Å; Z = 4; D_c = 1.321 Mg/m^³; F(000) = 536; colorless; dimensions 0.20 × 0.20 × 0.50 mm; µ = 0.235 mm^-¹; 3.32 < Θ < 32.23˚, 7898 reflection measured, 4179 unique reflections, R _int = 0.0773. R1 = 0.2121, 0.3045, wR2 = 0.2746, 0.3530, 1.772 goodness of fit on F^². Crystals are invariably twinned. The structure was solved by direct methods with SHELXS-86 and refined by full-matrix least-squares with SHELXL-97. In each case, non-hydrogen atoms were found in the initial E-maps. All non-hydrogen atom positions and anisotropic vibrational parameters
were refined in the developed models, which included contributions from the hydrogen atoms placed in calculated positions and assigned U_iso values equal to 120% of the U_eq of the adjacent atom.

14

McGuire, J. R.; Zoellner, R. W. preliminary unpublished computational data.

15

Compound 6e: ^¹H NMR (400 MHz, CDCl₃): δ = 3.11 (s, 0.5 H), 3.60 (s, 2.5 H), 4.33 (s, 1.7 H), 4.35 (s, 0.3 H), 6.79-6.83 (m, 2 H), 6.89-6.94 (m, 2 H), 7.05-7.56 (m, 6 H). HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₆H₁₆N₃O₂S₃: 378.0399; found: 378.0391. The purity of the product was confirmed by HPLC using the same two conditions used for 5d. The retention time of the product under conditions A was 11.4 min (95% purity) and under conditions B was 13.3 min (98% purity).
Compound 6f: ^¹H NMR (400 MHz, CDCl₃): δ = 0.80 (t, J = 7.1 Hz, 0.93 H), 1.19 (t, J = 7.1 Hz, 2.07 H), 3.52 (q, J = 7.1 Hz, 0.62 H), 4.13 (q, J = 7.1 Hz, 1.38 H), 4.32 (s, 1.35 H), 4.36 (s, 0.65 H), 6.76-6.82 (m, 1 H), 6.83-6.88 (m, 1 H), 7.08-7.18 (m, 4 H), 7.22-7.58 (m, 4 H). HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₇H₁₈N₃O₂S₃: 392.0556; found: 392.0546. The purity of the product was confirmed by HPLC using the same two conditions used for 5d. The retention time of the product under conditions A was 16.5 min (98% purity) and under conditions B was 14.2 min (98% purity). Compound 6g: ^¹H NMR (400 MHz, CDCl₃): δ = 4.31 (s, 1.25 H), 4.33 (s, 0.75 H), 4.74 (s, 0.76 H), 5.36 (s, 1.24 H), 6.61-6.66 (m, 1 H), 6.68-6.72 (m, 1 H), 6.82-6.88 (m, 1 H), 6.97-7.41 (m, 12 H). HRMS (ESI+): m/z [M + H]⁺ calcd for C₂₂H₂₀N₃O₂S₃: 454.0712; found: 454.0703. The purity of the product was confirmed by HPLC using the same two conditions used for 5d. The retention time of the product under conditions A was 17.9 min (99% purity) and under conditions B was 15.7 min (95% purity). Compound 6h: ^¹H NMR (400 MHz, CDCl₃): δ = 0.64-0.81 (m, 1 H), 0.81-1.15 (m, 3 H), 1.27-1.46 (m, 2 H), 1.46-1.63 (m, 2 H), 1.64-1.78 (m, 1 H), 1.87-2.02 (m, 1 H), 4.22 (tt, J = 3.5, 11.9 Hz, 0.5 H), 4.29 (s, 1.3 H), 4.33 (s, 0.7 H), 5.07 (tt, J = 3.5, 11.9 Hz, 0.5 H), 6.64-6.78 (m, 2 H), 6.98-7.05 (m, 1 H), 7.05-7.28 (m, 4 H), 7.28-7.59 (m, 3 H). HRMS (ESI+): m/z [M + H]⁺ calcd for C₂₁H₂₄N₃O₂S₃: 466.1025; found: 446.1015. The purity of the product was confirmed by HPLC using the same two conditions used for 6b. The retention time of the product under conditions A was 15.0 min (>99% purity) and under conditions B was 16.9 min (97% purity).

16

Crystal Data for 6e: CCDC 838606.^²² C₁₆H₁₅N₃O₂S₃; MW = 377.49; orthorhombic; a = 16.708 (4) Å, b = 18.397 (3) Å, c = 5.6729 (8) Å, β = 90˚; U = 1743.7 (6) Å^³; T = 300(2) K; space group P n a 21 (#33); λ = 0.71073 Å; Z = 4; D _c = 1.438 Mg/m^³; F(000) = 784; yellow; dimensions 0.69 × 0.06 × 0.03 mm; µ = 0.439 mm^-¹; 3.54 < Θ < 29.19˚, 9073 reflection measured, 4001 unique reflections, R _int = 1163. R1 = 0.0625, 0.1818, wR2 = 0.0656, 0.0781, 0.877 goodness of fit on F^². The structure was solved by direct methods and refined as described above. Compound 6f: CCDC 838608.^²² C₁₇H₁₇N₃O₂S₃; MW = 391.54; triclinic; a = 10.2747 (5) Å, b = 11.2638 (5) Å, c = 18.1559 (8) Å, β = 80.766 (4)˚; U = 1866.99 (15) Å^³; T = 299 (2) K; space group P-1; λ = 0.71073 Å; Z = 4; D _c = 1.393 Mg/m^³; F(000) = 816; yellow; dimensions 0.46 × 0.32 × 0.05 mm; µ = 0.413 mm^-¹; 3.35 < Θ < 30.59˚, 21500 reflection measured, 11234 unique reflections, R _int = 0.0342. R1 = 0.0407, 0.1264, wR2 = 0.0482, 0.0515, 1.005 goodness of fit on F^². The structure was solved by direct methods and refined as described above.

17

Compound 6i: ^¹H NMR (400 MHz, CDCl₃): δ = 0.78 (t, J = 7.2 Hz, 1.8 H), 1.17 (t, J = 7.1 Hz, 1.2 H), 2.78 (s, 1 H), 3.21-2.28 (m, 3.3 H), 3.81 (q, J = 7.2 Hz, 0.7 H), 4.35 (s, 0.8 H), 4.36 (s, 1.2 H), 7.36-7.42 (m, 2 H), 7.45-7.53 (m, 3 H). HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₂H₁₆N₃O₂S₃: 330.0399; found: 330.0399. The purity of the product was confirmed by HPLC using the same two conditions used for 6b. The retention time of the product under conditions A was 8.6 min (97% purity) and under conditions B was 10.3 min (96% purity). Crystal Data: CCDC 838605.^²² C₁₂H₁₅N₃O₂S₃; MW = 329.47; monoclinic; a = 5.2548 (4) Å, b = 15.8241 (13) Å, c = 17.885 (2) Å, β = 97.681 (9)˚; U = 1473.8 (2) Å^³; T = 150 (2) K; space group P 21/n (#14); λ = 0.71073 Å; Z = 4; D _c = 1.485 Mg/m^³; F(000) = 688; colorless; dimensions 0.62 × 0.04 × 0.01 mm; µ = 0.507 mm^-¹; 3.45 < Θ < 27.59˚, 10662 reflection measured, 3285 unique reflections, R _int = 0.0831. R1 = 0.0637, 0.1217, wR2 = 0.1185, 0.1248, 1.008 goodness of fit on F^². The structure was solved by direct methods and refined as described above.

19

Compound 6j: ^¹H NMR (400 MHz, CDCl₃): δ = 2.87 (s, 3 H), 3.31 (s, 3 H), 4.37 (s, 2 H), 7.37-7.43 (m, 2 H), 7.47-7.54 (m, 3 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 39.6, 42.0, 46.5, 129.5, 129.6, 130.1, 132.7, 161.3, 185.2. HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₁H₁₄N₃O₂S₃: 316.0243; found: 316.0243. The purity of the product was confirmed by HPLC using the same two conditions used for 5d. The retention time of the product under conditions A was 8.7 min (>99% purity) and under conditions B was 10.1 min (99% purity).

20

Cytotoxicity analyses were performed by using the assay developed for determining dehydrogenase activities in metabolically active cells as described in ref. 1.

21

Fifty percent effective (EC₅₀) concentrations of compounds against WNV were determined by evaluation of cytopathic effects (CPE) in Vero cells by limiting dilution, as described in ref. 1.

22

Crystallographic data for structures 5d, 6e, 6f, and 6i have been deposited with the Cambridge Crystallographic Data Centre (CCDC 838607, 838606, 838608, and 838605, respectively). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, United Kingdom [Fax: [phone] or e-mail: [e-mail]].