Synthesis, structure, antimycobacterial and anticancer evaluation of new pyrrolo-phenanthroline derivatives

Abstract

A study concerning design, synthesis, structure and in vitro antimycobacterial and anticancer evaluation of new fused derivatives with pyrrolo[2,1-c][4,7]phenanthroline skeleton is described. The strategy adopted for synthesis involves a [3 + 2] dipolar cycloaddition of several in situ generated 4,7-phenanthrolin-4-ium ylides to different substituted alkynes and alkenes. Stereo- and regiochemistry of cycloaddition reactions were discussed. The structure of the new compounds was proven unambiguously, single-crystal X-ray diffraction studies including. The antimycobacterial and anticancer activity of a selection of new synthesized compounds was evaluated against Mycobacterium tuberculosis H37Rv under aerobic conditions and 60 human tumour cell line panel, respectively. Five of the tested compounds possess a moderate antimycobacterial activity, while two of the compounds have a significant antitumor activity against renal cancer and breast cancer.

• 3 + 2 Cycloadditions
• N-ylides
• pyrrolo[2,1-c][4,7]phenanthrolines

Introduction

Phenanthroline derivatives attracted attention in the last years especially due to their biological effects1–3, materials science applications4, crystal engineering5^,6, their unique π-electrons delocalization7^,8 and complexation properties9.

Phenanthrolines polycyclic skeletons are also present in sterols, sex hormones, cardiac glycosides, bile acids and morphine alkaloids10. Recently, compounds with phenathroline skeleton have been synthesized and biologically evaluated11–15 as analogues of benzo[c]phenanthridine alkaloids (e.g. nitidine, fagaronine, sanguinarine) that attracted attention due to their interesting biological properties16–19.

Compared to 1,10-phenanthroline that has been widely studied both for synthesis and applications, 4,7-phenanthroline received much less interest due to difficulties in its synthesis. However, there are several reports regarding biological properties of 4,7-phenanthroline derivatives and their analogues as antibacterial activity20, amoebicide21 or antiviral properties22. Besides, 4,7-phenathroline derivatives were found to exhibit staining properties to HeLa cells and can be used as fluorophores which can bind with proteins23. Other derivatives were reported as high affinity triple-helix DNA stabilizing agents24.

As part of our ongoing research in the field of design and synthesis of new anti-TB25–28 and anticancer derivatives29–32 with azaheterocycles skeleton, we report here the design, synthesis, structure and in vitro antimycobacterial and anticancer evaluation of new class of compounds with pyrrolo[2,1-c][4,7]phenanthroline scaffold. To the best of our knowledge, there is no previous report, concerning the synthesis of pyrrolo[2,1-c][4,7]-phenanthroline derivatives.

Methods

General

Melting points were recorded on an A. Krüss Optronic Melting Point Meter KSPI and are uncorrected. Proton and carbon nuclear magnetic resonance (δ_H, δ_C) spectra were recorded on a DRX-500 Bruker (500 MHz). All chemical shifts are quoted on the δ-scale in ppm. Coupling constants are given in Hz. IR spectra were recorded on a FTIR Shimadzu or Jasco 660 plus FTIR spectrophotometer. Thin layer chromatography (TLC) was carried out on Merck silica gel 60F₂₅₄ plates. Column chromatography was carried out on silica gel (Roth 60, 0.04–0.063 mm). Visualization of the plates was achieved using a UV lamp (λ_max = 254 or 365 nm). All commercially available products were used without further purification unless otherwise specified.

X-ray crystallography

Crystallographic measurements were carried out with an Oxford-Diffraction XCALIBUR E CCD diffractometer equipped with graphite-monochromated Mo Kα radiation. Single crystals were positioned at 40 mm from the detector and 211 and 248 frames were measured each for 60 and 180 s over 1 ° scan width for 2259 and 2340, respectively. The unit cell determination and data integration were carried out using the CrysAlis package of Oxford Diffraction33. The structures were solved by direct methods using Olex234 software with the SHELXS structure solution program and refined by full-matrix least-squares on F² with SHELXL-9735. Atomic displacements for non-hydrogen atoms were refined using an anisotropic model. Hydrogen atoms were placed in fixed, idealized positions and refined as rigidly bonded to the corresponding atoms. The molecular plots were obtained using the Olex2 program. The main crystallographic data together with refinement details are summarized in Supplemental Table S1. CCDC-1048147 (for 7a) and 1048148 (for 11f) contain the supplementary crystallographic data for this contribution. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: [phone]; or [email protected]).

General procedure for synthesis of compounds 4, 6, 7, 8, 9, 10 and 11

The cycloimmonium salt 1a–e (1 mmol, 1 equiv.) and dipolarophile (DMAD, EP, N-phenylmaleimide or acrylonitrile, 1.1 mmol, 1.1 equiv.) were added to 5 mL of dichloromethane and the obtained suspension is stirred at room temperature (rt) under N₂ atmosphere. Triethylamine (TEA, 3 mmol, 3 equiv.) was added drop-wise over 1 h (magnetic stirring) and the resulting mixture was then stirred for 24 h at rt under nitrogen. Methanol (5 mL) was added and the resulting mixture is kept for 24 h without stirring. The formed precipitate was collected by filtration to give a powder which was washed with few mL of methanol. The product was crystallized from an appropriate solvent or purified by column chromatography.

Dimethyl 9-(4-methoxybenzoyl)pyrrolo[2,1-c][4,7]phenanthroline-7,8-dicarboxylate (4a)

Crystallized from ethanol–chloroform 1:1, (v/v). Yellow powder (51% yield), mp = 244–247 °C. IR [KBr, ν (cm⁻¹)]: 2953, 1739, 1702, 1596, 1252, 1211, 1161, 1086, 803. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.53 (s, 3H, 21-H), 3.90 (s, 3H, OMe), 3.91 (s, 3H, 19-H), 6.99 (d, J = 8.5 Hz, 2H, phenyl-H), 7.57 (dd, J = 8.0, 4.0 Hz, 1H, 3-H), 7.92 (d, J = 9.5 Hz, 1H, 11-H), 8.02 (d, J = 8.5 Hz, 2H, phenyl-H), 8.07 (d, J = 9.5 Hz, 1H, 12-H), 8.29 (d, J = 9.5 Hz, 1H, 5-H), 8.37 (d, J = 9.5 Hz, 1H, 6-H), 8.80 (d, J = 8.0 Hz, 1H, 4-H), 8.95 (d, J = 4.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 51.9 C-19, 52.6 C-21, 55.8 OMe, 105.0 C-7, 114.2 2 × CH-Ph, 118.9 C-6, 120.9 C-14, 121.7 C-5, 122.0 C-11, 122.5 C-3, 125.0 C-13, 126.3 C-9, 128.6 C-8, 130.4 Cq-Ph, 130.9 C-12, 131.0 C-16, 131.3 C-4, 132.6 2 × CH-Ph, 137.0 C-15, 146.0 C-17, 150.6 C-2, 163.6 C-18, 164.7 Cq-Ph, 165.4 C-20, 186.2 CO ppm. Anal. Calcd. for C₂₇H₂₀N₂O₆: C, 69.22; H, 4.30; N, 5.98. Found: C, 69.39, H, 4.28; N, 6.12.

Dimethyl 9-(4-nitrobenzoyl)pyrrolo[2,1-c][4,7]phenanthroline-7,8-dicarboxylate (4b)

Crystallized from ethanol–chloroform 1:1, (v/v). Orange powder (50% yield), mp = 237–238 °C. IR [KBr, ν(cm⁻¹)): 1736, 1718, 1699, 1488, 1340, 1235, 1203, 1091, 805. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.48 (s, 3H, 21-H), 3.94 (s, 3H, 19-H), 7.68 (dd, J = 8.5, 4.5 Hz, 1H, 3-H), 7.87 (d, J = 9.5 Hz, 1H, 11-H), 8.16 (d, J = 8.5 Hz, 3H, phenyl-H, 12-H), 8.36 (d, J = 8.5 Hz, 2H, phenyl-H), 8.51 (d, J = 9.5 Hz, 1H, 5-H), 8.57 (d, J = 9.5 Hz, 1H, 6-H), 8.91 (d, J = 8.5 Hz, 1H, 4-H), 9.05 (d, J = 4.5 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 52.2 C-19, 52.8 C-21, 105.9 C-7, 118.9 C-6, 121.4 C-14, 122.1 C-11, 122.8 C-3, 123.3 C-5, 123.9 2 × CH-Ph, 125.0 C-9, 125.1 C-13, 130.0 C-8, 130.9 2 × CH-Ph, 131.0 C-16, 131.0 C-4, 131.5 C-12, 137.7 C-15, 142.6 Cq-Ph, 146.2 C-17, 150.8 Cq-Ph, 151.0 C-2, 163.3 C-18, 165.2 C-20, 184.5 CO ppm. Anal. Calcd. for C₂₆H₁₇N₃O₇: C, 64.60; H, 3.54; N, 8.69. Found: C, 64.69, H, 3.48; N, 8.83.

Dimethyl 9-(4-chlorobenzoyl)pyrrolo[2,1-c][4,7]phenanthroline-7,8-dicarboxylate (4c)

Crystallized from ethanol–chloroform 1:1, (v/v). Yellow powder (55% yield), mp = 245–247 °C. IR [KBr, ν (cm⁻¹)]: 2947, 1726, 1710, 1644, 1223, 1212, 1086. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.51 (s, 3H, 21-H), 3.93 (s, 3H, 19-H), 7.50 (d, J = 8.0 Hz, 2H, phenyl-H), 7.65 (dd, J = 8.0, 4.0 Hz, 1H, 3-H), 7.89 (d, J = 9.5 Hz, 1H, 11-H), 7.96 (d, J = 8.0 Hz, 2H, phenyl-H), 8.12 (d, J = 9.5 Hz, 1H, 12-H), 8.44 (d, J = 9.5 Hz, 1H, 5-H), 8.55 (d, J = 9.5 Hz, 1H, 6-H), 8.88 (d, J = 8.0 Hz, 1H, 4-H), 9.03 (as, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 52.0 C-19, 52.7 C-21, 105.4 C-7, 118.9 C-6, 121.2 C-14, 122.1 C-11, 122.5 C-5, 122.7 C-3, 125.1 C-13, 125.6 C-9, 129.2 2 × CH-Ph, 130.1 C-8, 131.0 C-16, 131.2 C-4, 131.3 C-12, 131.4 2 × CH-Ph, 136.1 Cq-Ph, 137.7 C-15, 140.7 Cq-Ph, 146.2 C-17, 150.9 C-2, 163.5 C-18, 165.3 C-20, 185.8 CO ppm. Anal. Calcd. for C₂₆H₁₇ClN₂O₅: C, 66.04; H, 3.62; N, 5.92. Found: C, 66.20, H, 3.53; N, 5.99.

Dimethyl 9-cyanopyrrolo[2,1-c][4,7]phenanthroline-7,8-dicarboxylate (4d)

Crystallized from ethanol–chloroform 1:1, (v/v). Brown powder (50% yield), mp = 261–263 °C. IR [KBr, ν (cm⁻¹)]: 2955, 2216, 1740, 1694, 1225, 1099. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.97 (s, 3H, 19-H), 4.07 (s, 3H, 21-H), 7.71 (dd, J = 8.5, 4.0 Hz, 1H, 3-H), 8.42 (d, J = 10.0 Hz, 1H, 12-H), 8.46 (d, J = 10.0 Hz, 1H, 5-H), 8.51 (d, J = 10.0 Hz, 1H, 6-H), 8.92 (d, J = 8.5 Hz, 1H, 4-H), 9.11 (ad, J = 4.0 Hz, 1H, 2-H), 9.51 (d, J = 10.0 Hz, 1H, 11-H). ¹³C NMR (125 MHz, CDCl₃): δ 52.3 C-19, 53.4 C-21, 98.3 C-9, 106.9 C-7, 114.7 CN, 118.1 C-11, 118.9 C-6, 121.1 C-14, 122.5 C-5, 123.0 C-3, 125.1 C-13, 131.4 C-4, 132.0 C-16, 133.4 C-8, 132.7 C-12, 136.8 C-15, 146.4 C-17, 151.4 C-2, 162.7 C-18, 163.3 C-20 ppm. Anal. Calcd. for C₂₀H₁₃N₃O₄: C, 66.85; H, 3.65; N, 11.69. Found: C, 66.81, H, 3.58; N, 11.72.

Trimethyl pyrrolo[2,1-c][4,7]phenanthroline-7,8,9-tricarboxylate (4e)

Crystallized from ethanol–chloroform 1:1, (v/v). Pale pink powder (55% yield), mp = 215–218 °C. IR [KBr, ν (cm⁻¹)]: 2953, 1741, 1704, 1677, 1263, 1161, 1099, 802. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.94 (s, 3H, 19-H), 4.00 (s, 3H, OMe), 4.03 (s, 3H, 21-H), 7.76 (dd, J = 8.0, 4.0 Hz, 1H, 3-H), 8.36 (d, J = 10.0 Hz, 1H, 11-H), 8.38 (d, J = 9.5 Hz, 1H, 5-H), 8.43 (d, J = 10.0 Hz, 1H, 12-H), 8.49 (d, J = 9.5 Hz, 1H, 6-H), 9.01 (d, J = 8.5 Hz, 1H, 4-H), 9.05 (dd, J = 4.0, 1.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 52.1 C-19, 52.9 OMe, 53.2 C-21, 105.4 C-7, 117.6 C-9, 119.3 C-6, 121.1 C-14, 122.2 C-5, 122.5 C-3, 124.1 C-11, 125.2 C-13, 128.3 C-12, 131.6 C-16, 132.3 C-8, 133.6 C-4, 137.9 C-15, 144.1 C-17, 148.7 C-2, 161.1 COOMe, 163.2 C-18, 165.9 C-20 ppm. Anal. Calcd. for C₂₁H₁₆N₂O₆: C, 64.28; H, 4.11; N, 7.14. Found: C, 64.20, H, 4.03; N, 7.20.

Ethyl 9-(4-methoxybenzoyl)pyrrolo[2,1-c][4,7]phenanthroline-7-carboxylate (6a)

Purified by column chromatography: CH₂Cl₂→ CH₂Cl₂: MeOH (98:2, v:v), then crystallized from ethanol–chloroform 1:1, (v/v). Orange powder (50% yield), mp = 252–253 °C. IR [KBr, ν (cm⁻¹)]: 2982, 1694, 1232, 1166, 1080, 800. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 1.41 (t, J = 7.0 Hz, 3H, 20-H), 3.95 (s, 3H, OMe), 4.40 (q, 2H, J = 7.0 Hz, 19-H), 7.07 (d, J = 8.0 Hz, 2H, phenyl-H), 7.60 (dd, J = 8.0, 4.0 Hz, 1H, 3-H), 7.73 (s, 1H, 8-H), 8.20 (m, 4H, 11-H, 12-H, phenyl-H), 8.40 (d, J = 9.5 Hz, 1H, 5-H), 8.51 (d, J = 9.5 Hz, 1H, 6-H), 8.85 (d, J = 8.0 Hz, 1H, 4-H), 8.99 (d, J = 4.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 14.7 C-20, 55.8 OMe, 60.4 C-19, 107.4 C-7, 114.1 2 × CH-Ph, 118.6 C-6, 120.6 C-14, 122.3 C-3, 122.4 C-5, 123.3 C-11, 125.0 C-13, 128.0 C-9, 129.1 C-8, 130.8 Cq-Ph, 130.6 C-12, 131.2 C-4, 131.7 C-16, 132.6 2 × CH-Ph, 139.8 C-15, 146.3 C-17, 150.4 C-2, 163.9 Cq-Ph, 164.2 C-18, 184.0 CO ppm. Anal. Calcd. for C₂₆H₂₀N₂O₄: C, 73.57; H, 4.57; N, 6.60. Found: C, 73.52, H, 4.49; N, 6.62.

Ethyl 9-(4-nitrobenzoyl)pyrrolo[2,1-c][4,7]phenanthroline-7-carboxylate (6b)

Crystallized from ethanol–chloroform 1:1, (v/v). Orange powder (49% yield), mp = 332–335 °C. IR [KBr, ν (cm⁻¹)]: 3070, 2968, 1694, 1638, 1516, 1495, 1344, 1237, 1081, 801. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 1.42 (t, J = 7.0 Hz, 3H, 20-H), 4.41 (q, 2H, J = 7.0 Hz, 19-H), 7.68 (dd, J = 8.5, 4.0 Hz, 1H, 3-H), 7.75 (s, 1H, 8-H), 8.29 (m, 4H, 11-H, 12-H, phenyl-H), 8.44 (d, J = 8.5 Hz, 2H, phenyl-H), 8.59 (d, J = 9.5 Hz, 1H, 5-H), 8.63 (d, J = 9.5 Hz, 1H, 6-H), 8.94 (d, J = 8.5 Hz, 1H, 4-H), 9.06 (d, J = 4.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 14.7 C-20, 60.7 C-19, 108.4 C-7, 118.6 C-6, 121.1 C-14, 122.6 C-3, 123.4 C-11, 124.0 2 × CH-Ph, 124.1 C-5, 125.0 C-13, 127.4 C-9, 131.0 2 × CH-Ph, 131.4 C-12, 131.5 C-4, 131.6 C-8, 131.9 C-16, 141.2 C-15, 143.8 Cq-Ph, 146.5 C-17, 150.4 Cq-Ph, 150.8 C-2, 163.7 C-18, 181.8 CO ppm. Anal. Calcd. for C₂₅H₁₇N₃O₅: C, 68.33; H, 3.90; N, 9.56. Found: C, 68.35, H, 3.80; N, 9.63.

Ethyl 9-(4-chlorobenzoyl)pyrrolo[2,1-c][4,7]phenanthroline-7-carboxylate (6c)

Crystallized from ethanol–chloroform 1:1, (v/v). Yellow powder (59% yield), mp = 278–280 °C. IR [KBr, ν (cm⁻¹)]: 3049, 2977, 1697, 1229, 1211, 1077, 803. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 1.42 (t, J = 7.0 Hz, 3H, 20-H), 4.40 (q, 2H, J = 7.0 Hz, 19-H), 7.57 (d, J = 8.0 Hz, 2H, phenyl-H), 7.62 (dd, J = 8.0, 4.0 Hz, 1H, 3-H), 7.73 (s, 1H, 8-H), 8.09 (d, J = 8.0 Hz, 2H, phenyl-H), 8.22 (as, 2H, 11-H, 12-H), 8.46 (d, J = 9.5 Hz, 1H, 5-H), 8.54 (d, J = 9.5 Hz, 1H, 6-H), 8.88 (d, J = 8.0 Hz, 1H, 4-H), 9.01 (d, J = 4.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 14.7 C-20, 60.5 C-19, 107.8 C-7, 118.5 C-6, 120.8 C-14, 122.4 C-3, 123.2 C-5, 123.4 C-11, 125.0 C-13, 127.6 C-9, 129.1 2 × CH-Ph, 130.4 C-8, 130.8 C-12, 131.3 C-4, 131.6 2 × CH-Ph, 131.8 C-16, 136.7 Cq-Ph, 139.6 Cq-Ph, 140.5 C-15, 146.3 C-17, 150.6 C-2, 164.0 C-18, 185.3 CO ppm. Anal. Calcd. for C₂₅H₁₇ClN₂O₃: C, 70.01; H, 4.00; N, 6.53. Found: C, 69.99, H, 3.93; N, 6.60.

Ethyl 9-cyanopyrrolo[2,1-c][4,7]phenanthroline-7-carboxylate (6d)

Crystallized from ethanol–chloroform 1:1, (v/v). Brown powder (55% yield), mp = 224–226 °C. IR [KBr, ν (cm⁻¹)]: 3075, 2992, 2213, 1698, 1248, 1080, 799. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 1.45 (t, J = 7.0 Hz, 3H, 20-H), 4.43 (q, 2H, J = 7.0 Hz, 19-H), 7.67 (dd, J = 8.5, 4.0 Hz, 1H, 3-H), 7.96 (s, 1H, 8-H), 8.37 (d, J = 9.5 Hz, 1H, 12-H), 8.40 (d, J = 10.0 Hz, 1H, 5-H), 8.58 (d, J = 10.0 Hz, 1H, 6-H), 8.90 (d, J = 8.5 Hz, 1H, 4-H), 9.07 (ad, J = 4.0 Hz, 1H, 2-H), 9.46 (d, J = 9.5 Hz, 1H, 11-H). ¹³C NMR (125 MHz, CDCl₃): δ 14.6 C-20, 60.7 C-19, 97.7 C-9, 108.3 C-7, 115.8 CN, 118.2 C-11, 118.9 C-6, 120.5 C-14, 121.6 C-5, 122.8 C-3, 125.1 C-13, 128.7 C-8, 131.3 C-4, 132.3 C-12, C-16, 137.5 C-15, 146.2 C-17, 151.1 C-2, 163.2 C-18 ppm. Anal. Calcd. for C₁₉H₁₃N₃O₂: C, 72.37; H, 4.16; N, 13.33. Found: C, 72.35, H, 4.09; N, 13.39.

7-Ethyl 9-methyl pyrrolo[2,1-c][4,7]phenanthroline-7,9-dicarboxylate (6e)

Crystallized from ethanol–chloroform 1:1, (v/v). Pale pink powder (49% yield), mp = 173–174 °C. IR [KBr, ν (cm⁻¹)]: 3076, 2993, 2213, 1726, 1689, 1248, 1080, 799. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 1.44 (t, J = 7.0 Hz, 3H, 20-H), 4.00 (s, 3H, OMe), 4.41 (q, 2H, J = 7.0 Hz, 19-H), 7.57 (dd, J = 8.5, 4.0 Hz, 1H, 3-H), 8.05 (s, 1H, 8-H), 8.21 (d, J = 10.0 Hz, 1H, 12-H), 8.29 (d, J = 9.5 Hz, 1H, 5-H), 8.45 (d, J = 9.5 Hz, 1H, 6-H), 8.54 (d, J = 10.0 Hz, 1H, 11-H), 8.98 (d, J = 8.5 Hz, 1H, 4-H), 8.98 (ad, J = 4.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 14.7 C-20, 52.4 OMe, 60.4 C-19, 107.4 C-7, 118.6 C-6, 119.7 C-9, 120.7 C-14, 121.6 C-5, 122.2 C-3, 123.2 C-11, 124.8 C-13, 127.5 C-8, 129.9 C-12, 131.3 C-4, 131.8 C-16, 139.5 C-15, 146.0 C-17, 150.3 C-2, 162.2 COOMe, 164.0 C-18 ppm. Anal. Calcd. for C₂₀H₁₆N₂O₄: C, 68.96; H, 4.63; N, 8.04. Found: C, 68.90, H, 4.57; N, 8.10.

Ethyl 9-(4-methoxybenzoyl)-8,9-dihydropyrrolo[2,1-c][4,7]phenanthroline-7-carboxylate (7a)

Purified by column chromatography: CH₂Cl₂→ CH₂Cl₂: MeOH (98:2, v:v), then crystallized from ethanol–chloroform 1:1, (v/v). Red crystals (25% yield), mp = 243–246 °C. IR [KBr, ν (cm⁻¹)]: 1684, 1650, 1266, 1222, 1163, 1060, 1033, 803. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 1.28 (t, J = 7.0 Hz, 3H, 20-H), 3.09 (dd, J = 15.5, 5.5 Hz, 1H, 8-H), 3.64 (at, J = 9.5 Hz, 8-H), 3.93 (s, 3H, OMe), 4.18 (q, 2H, J = 7.0 Hz, 19-H), 6.88 (d, J = 9.5 Hz, 11-H), 6.99 (dd, J = 14.0, 5.0 Hz, 1H, 9-H), 7.04 (d, J = 8.5 Hz, 2H, phenyl-H), 7.48 (dd, J = 8.5, 4.0 Hz, 1H, 3-H), 7.73 (d, J = 10.0 Hz, 1H, 5-H), 7.92 (d, J = 10.0 Hz, 1H, 6-H), 7.97 (d, J = 9.5 Hz, 12-H), 8.04 (d, J = 8.5 Hz, 2H, phenyl-H), 8.49 (d, J = 8.5 Hz, 1H, 4-H), 8.77 (d, J = 4.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 14.9 C-20, 33.7 C-8, 55.8 OMe, 59.0 C-19, 64.3 C-9, 90.1 C-7, 114.6 2 × CH-Ph, 117.3 C-11, 119.2 C-5, 115.2 C-14, 122.6 C-3, 124.8 C-13, 125.8 Cq-Ph, 129.5 C-6, 130.0 C-4, 130.1 C-16, 131.5 2 × CH-Ph, 132.8 C-12, 138.0 C-15, 144.0 C-17, 148.2 C-2, 164.7 Cq-Ph, 166.1 C-18, 191.5 CO ppm. Anal. Calcd. for C₂₆H₂₂N₂O₄: C, 73.23; H, 5.20; N, 6.57. Found: C, 73.22, H, 5.09; N, 6.62.

12-(4-Nitrobenzoyl)-10-phenyl-11a,12-dihydro-8aH-pyrrolo[3′,4′:3,4]pyrrolo[2,1-c][4,7]phenanthroline-9,11(8bH,10H)-dione (8b)

Crystallized from ethanol-chloroform 1:1, (v/v). Yellow powder (58% yield), mp = 177–180 °C. IR [KBr, ν (cm⁻¹)]: 1718, 1596, 1523, 1497, 1382, 1343, 1195. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.72 (t, J = 8.0 Hz, 1H, 8-H), 3.84 (ad, J = 8.0 Hz, 1H, 18-H), 5.39 (d, J = 8.0, Hz, 1H, 7-H), 5.99 (s, 1H, 12-H), 6.28 (ad, J = 10.0 Hz, 1H, 6-H), 6.79 (d, J = 9.0 Hz, 1H, 13-H), 7.16 (ad, J = 8.0 Hz, 3H, 5-H, 2 × phenyl-CH), 7.35-7.43 (m, 4H, 3 × phenyl-CH, 3-H), 7.86 (d, J = 9.0 Hz, 1H, 14-H), 8.29 (ad, J = 9.0 Hz, 1H, 4-H), 8.45 (d, J = 8.5 Hz, 2H, 2 × CH-(p)-NO₂-Ph, 8.54 (d, J = 8.5 Hz, 2H, 2 × CH-(p)-NO₂-Ph, 8.69 (ad, J = 3.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 46.7 C-18, 48.1 C-8, 61.1 C-7, 65.5 C-12, 114.0 C-17, 116.9 C-13, 120.1 C-6, 121.7 C-5, 121.9 C-3, 124.6 2 × CH-(p)-NO₂-phenyl, 125.8 C-16, 126.3 2 × CH-Ph, 129.3 CH-Ph, 129.4 2 × CH-Ph, 129.7 C-4, 130.5 2 × CH-(p)-NO₂-phenyl, 130.9 C-14, 131.4 Cq-Ph, 137.6 Cq-(p)-NO₂-phenyl, 139.5 C-19, 142.0 C-15, 147.4 C-2, 151.3 C-NO₂, 174.4 C-9, 176.2 C-11, 194.1 C-20 ppm. Anal. Calcd. for C₃₀H₂₀N₄O₅: C, 69.76; H, 3.90; N, 10.85. Found: C, 69.77, H, 3.82; N, 10.95.

12-(3-Methoxybenzoyl)-10-phenyl-11a,12-dihydro-8aH-pyrrolo[3′,4′:3,4]pyrrolo[2,1-c][4,7]phenanthroline-9,11(8bH,10H)-dione (8f)

Crystallized from ethanol–chloroform 1:1, (v/v). Orange powder (82% yield), mp = 173–176 °C. IR [KBr, ν (cm⁻¹)]: 1706, 1582, 1490, 1377, 1263, 1169. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.69 (t, J = 8.0 Hz, 1H, 8-H), 3.92 (s, 3H, OMe), 3.87 (ad, J = 8.0 Hz, 1H, 18-H), 5.45 (d, J = 8.0, Hz, 1H, 7-H), 5.96 (s, 1H, 12-H), 6.26 (ad, J = 10.0 Hz, 1H, 6-H), 6.80 (d, J = 9.0 Hz, 1H, 13-H), 7.13 (ad, J = 8.5 Hz, 1H, 5-H), 7.20 (ad, J = 7.5 Hz, 2H, 2 × phenyl-CH), 7.25 [overlapped signal, 1H, (m)-OMe-phenyl-H], 7.36–7.43 (m, 3H, 3 × phenyl-CH), 7.32 (dd, J = 4.0, 9,0 Hz, 1H, 3-H), 7.51 (t, J = 8.0 Hz, 1H, (m)-OMe-phenyl-H), 7.82 (d, J = 9.0 Hz, 1H, 14-H), 7.84 (s, 1H, (m)-OMe-phenyl-H), 7.98 (d, J = 7.5 Hz, 1H, (m)-OMe-phenyl-H), 8.26 (ad, J = 9.0 Hz, 1H, 4-H), 8.66 (ad, J = 3.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 47.3 C-18, 48.1 C-8, 55.8 OMe, 61.1 C-7, 65.0 C-12, 112.8 CH-(m)-OMe-phenyl, 113.8 C-17, 117.2 C-13, 120.0 C-6, 121.7 C-5, 121.8 C-3, 122.0 CH-(m)-OMe-phenyl, 125.6 C-16, 126.5 2 × CH-Ph, 129.2 CH-Ph, 129.3 2 × CH-Ph, 129.4 C-4, 130.5 CH-(m)-OMe-phenyl, 130.8 CH-(m)-OMe-phenyl, 130.8 C-14, 131.5 Cq-Ph, 134.4 Cq-(m)-OMe-phenyl, 140.0 C-19, 143.1 C-15, 147.2 C-2, 160.4 C-OMe, 174.9 C-9, 176.5 C-11, 195.1 C-20 ppm. Anal. Calcd. for C₃₁H₂₃N₃O₄: C, 74.24; H, 4.62; N, 8.38. Found: C, 74.27, H, 4.52; N, 8.45.

Dimethyl 8-(phenylcarbamoyl)-8,9-dihydropyrrolo[2,1-c][4,7]phenanthroline-7,9-dicarboxylate (9e)

Purified by column chromatography: CH₂Cl₂: MeOH (99.6:0.4, v:v). Orange red powder (30% yield), mp = 166–168 °C. IR [KBr, ν (cm⁻¹)]: 2951, 1751, 1721, 1678, 1615, 1597, 1549, 1211, 1186. ¹H NMR (500MHz, CDCl₃, 25 °C): δ 3.81 (s, 3H, OMe), 3.89 (s, 3H, OMe), 3.36 (d, J = 3.0 Hz, 1H, 8-H), 6.28 (dd, J = 3.0 Hz, 1H, 9-H), 7.09 (t, J = 7.5 Hz, 1H, Ph-H), 7.32 (t, J = 7.5 Hz, 2H, Ph-H), 7.41 (d, J = 9.0 Hz, 1H, 11-H), 7.52 (dd, J = 8.5, 4.0 Hz, 1H, 3-H), 7.60 (ad, J = 8.0 Hz, 3H, 6-H, Ph-H), 8.09 (d, J = 10.0 Hz, 1H, 5-H), 8.15 (d, J = 9.0 Hz, 1H, 12-H), 8.50 (d, J = 8.5 Hz, 1H, 4-H), 8.86 (dd, J = 4.0, 1.0 Hz, 1H, 2-H), 10.19 (s, 1H, NH). ¹³C NMR (125 MHz, CDCl₃): δ 51.3 OCH₃, 51.4 C-8, 53.4 OCH₃, 62.2 C-9, 115.9 C-14, 117.5 C-11, 118.5 C-7, 119.7 2 × CH-Ph, 6-H, 122.8 C-3, 124.1 CH-Ph, 125.0 C-13, 129.1 2 × CH-Ph, 129.9 C-4, 131.3 C-5, 133.8 C-12, 137.5 C-16, 151.1 C-15, 144.3 C-17, 149.0 C-2, 168.32 CO, 170.1 CO. Anal. Calcd. for C₂₆H₂₁N₃O5: C, 68.56; H, 4.65; N, 9.23. Found: C, 68.50, H, 4.58; N, 8.28.

9-(4-Nitrobenzoyl)-6a,7,8,9-tetrahydropyrrolo[2,1-c][4,7]phenanthroline-7-carbonitrile (10b)

Crystallized from ethanol–chloroform 1:1, (v/v). Dark red powder (45% yield), mp = 265–268 °C. IR [KBr, ν (cm⁻¹)]: 2911, 2176, 1708, 1630, 1499, 1472, 1344, 792. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 2.20–2.26 (m, 1H, 8a-H), 2.88–2.93 (m, 1H, 8b-H), 3.50 (at, J = 5.5 Hz,1H, 7-H), 5.31 (at, J = 2.5 Hz, 1H, 15-H), 5.57 (dd, J = 9.0, 6.5 Hz, 1H, 9-H), 5.82 (ad, J = 10.5 Hz, 1H, 6-H), 6.53 (d, J = 9.0 Hz, 1H, 11-H), 7.26 (overlapped signal, 1H, 5-H), 7.33 (dd, J = 4.0, 9.0 Hz, 1H, 3-H), 7.77 (d, J = 9.0 Hz, 1H, 12-H), 8.25 (ad, J = 9.0 Hz, 3H, 2 × phenyl-H, 4-H), 8.44 (d, J = 8.5 Hz, 2H, 2 × phenyl-H), 8.65 (ad, J = 3.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 32.3 C-8, 35.9 C-7, 61.9 C-9, 62.2 C-15, 112.0 C-14, 116.6 C-11, 117.9 C-6, 122.0 C-3, 119.1 CN, 123.6 C-5, 124.7 2 × CH-Ph, 125.8 C-13, 129.1 C-4, 129.9 2 × CH-Ph, 131.4 C-12, 138.3 Cq-Ph, 139.6 C-16, 143.2 C-17, 147.3 C-2, 151.2 Cq-Ph, 196.2 C-18 ppm. Anal. Calcd. for C₂₃H₁₆N₄O₃: C, 69.69; H, 4.07; N, 14.13. Found: C, 69.78, H, 4.03; N, 14.19.

Methyl 7-cyano-8,9-dihydropyrrolo[2,1-c][4,7]phenanthroline-9-carboxylate (11e)

Crystallized from ethanol–chloroform 1:1, (v/v). Dark red powder (45% yield), mp = 230–232 °C. IR [KBr, ν (cm⁻¹)]: 2948, 2175, 1723, 1630, 1276, 833, 792. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.15 (dd, J = 16.0, 4.0 Hz, 1H, 8b-H), 3.48 (t, J = 15.0 Hz, 1H, 8a-H), 3.84 (s, 3H, OMe), 5.22 (dd, J = 13.0, 4.5 Hz, 1H, 9-H), 6.96 (d, J = 10.0 Hz, 1H, 6-H), 7.28 (d, J = 9.5 Hz, 1H, 11-H), 7.70 (d, J = 10.0 Hz, 1H, 5-H), 7.90 (dd, J = 8.5, 2.5 Hz, 1H, 3-H), 8.82 (ad, J = 4.0 Hz, 1H, 2-H), 8.89 (d, J = 9.0 Hz, 1H, 12-H), 8.95 (d, J = 8.5 Hz, 1H, 4-H). ¹³C NMR (125 MHz, CDCl₃): δ 33.3 C-8, 53.1 OCH₃, 61.2 C-9, 72.1 C-7, 119.7 C-6, 121.2 C-11, 114.7 C-14, 119.1 CN, 122.7 C-3, 125.4 C-12, 127.6 C-5, 126.4 C-13, 138.3 C-4, 135.0 C-17, 140.2 C-16, 140.4 C-2, 153.0 C-15, 169.3 CO. Anal. Calcd. for C₁₈H₁₃N₃O₂: C, 71.28; H, 4.32; N, 13.85. Found: C, 71.30, H, 4.29; N, 13.88.

9-(3-Methoxybenzoyl)-8,9-dihydropyrrolo[2,1-c][4,7]phenanthroline-7-carbonitrile (11f)

Crystallized from ethanol–chloroform 1:1, (v/v). Dark red powder (40% yield), mp = 221–224 °C. IR [KBr, ν (cm⁻¹)]: 3072, 2916, 2168, 1689, 1633, 1268, 1136, 794. ¹H NMR (500 MHz, CDCl₃, 25 °C): δ 3.02 (dd, J = 15.0, 5.0 Hz, 1H, 8b-H), 3.63 (t, J = 14.0 Hz, 1H, 8a-H), 3.88 (s, 3H, OMe), 6.03 (dd, J = 14.0, 5.0 Hz, 1H, 9-H), 6.81 (d, J = 9.5 Hz, 1H, 11-H), 6.89 (d, J = 10.5 Hz, 1H, 5-H), 7.25 (overlapped signal, 1H, 3-H), 7.48–7.52 (m, 2H, 2 × phenyl-H), 7.54–7.58 (m, 2H, 2 × phenyl-H), 7.87 (d, J = 10.0 Hz, 1H, 6-H), 7.98 (d, J = 9.0 Hz, 1H, 12-H), 8.45 (d, J = 8.5 Hz, 1H, 4-H), 8.80 (dd, J = 4.0, 1.0 Hz, 1H, 2-H). ¹³C NMR (125 MHz, CDCl₃): δ 33.6 C-8, 55.7 OCH₃, 64.6 C-9, 66.9 C-7, 113.6 CH-Ph, 116.8 C-5, C-11, 115.1 C-14, 119.6 CN, 121.3 C-3, CH-Ph, 122.8 CH-Ph, 129.7 C-4, 129.9 C-6, 130.5 CH-Ph, 133.3 C-12, 133.9 Cq-Ph, 138.1 C-16, 125.3 C-13, 148.5 C-2, 155.1 C-15, 143.7 C-17, 160.5 Cq-Ph, 191.9 CO. Anal. Calcd. for C₂₄H₁₇N₃O₂: C, 75.97; H, 4.52; N, 11.08. Found: C, 76.02, H, 4.49; N, 11.13.

Microbiology

Antimycobacterial activities of the compounds were performed by Center of Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) at Southern Research Institute36–38.

The Primary Cycle High Throughput Screening (HTS). Determination of 90% inhibitory concentration (IC₉₀), 50% inhibitory concentration (IC₅₀) and Minimum Inhibitory Concentration (MIC)

The MIC of compound was determined by measuring bacterial growth after 5 days in the presence of test compounds. Compounds were prepared as 10-point two-fold serial dilutions in DMSO and diluted into 7H9-Tw-OADC medium in 96-well plates with a final DMSO concentration of 2%. The highest concentration of compound was 200 µM where compounds were soluble in DMSO at 10 mM. For compounds with limited solubility, the highest concentration was 50 × less than the stock concentration, e.g. 100 µM for 5 mM DMSO stock, 20 µM for 1 mM DMSO stock. For potent compounds, assays were repeated at lower starting concentrations. Each plate included assay controls for background (medium/DMSO only, no bacterial cells), zero growth (100 µM Rifampicin) and maximum growth (DMSO only), as well as a rifampicin dose response curve. Plates were inoculated with M. tuberculosis and incubated for 5 days: growth was measured by OD₅₉₀ and fluorescence (Ex 560/Em 590) using a BioTek™ Synergy 4 plate reader. Growth was calculated separately for OD₅₉₀ and RFU. To calculate the MIC, the 10-point dose response curve was plotted as % growth and fitted to the Gompertz model using GraphPad Prism 5. Data points obtained from a dose response growth inhibition assay are curve-fitted using the Gompertz model to calculate MIC (Figure 1A) and the Levenberg–Marquardt algorithm to calculate IC₅₀ and IC₉₀ (Figure 1B). (A) The MIC is the concentration at which complete inhibition growth is seen and is derived from the point of inflection at which the curve meets the lower asymptote (zero growth). (B) IC₅₀ and IC₉₀ are points at which growth is inhibited by 50% and 90% respectively. Orange line = MIC; Green line = IC₅₀; Blue line = IC₉₀.

Figure 1. Dose response curves used to calculate MIC, IC₅₀ and IC₉₀.

MIC values were reported when the following quality control criteria were satisfied:

• For each plate

  • No growth in the background (un-inoculated) control wells.

  • OD₅₉₀ > 0.3 in maximum growth wells.

  • Rifampicin MIC within 3-fold of the expected value.

• For each compound curve. MICs were reported if

  • There were two points with growth >75%.

  • There were two points with growth <75%.

• If only one point was >75% inhibition then the MIC value was reported as the maximum concentration tested.

• If no point reached 75% inhibition, the MIC was reported as > maximum concentration tested.

Results and discussion

Chemistry

All monoquaternary 4,7-phenanthrolin-4-ium bromides 1 were prepared following our previously reported procedures39. Then, a variety of functionalized alkynes and alkenes were used as dipolarophiles for 3 + 2 cycloaddion of the in situ generated N-ylides 2 in order to synthesize a series of different substituted pyrrolo[2,1-c][4,7]phenanthroline derivatives. Thus, the dipolar cycloaddition reactions of the ylides 2(a–e) [generated under triethylamine treatment from the corresponding salts 1] to dimethylacetylene dicarboxylate (DMAD) and ethyl propiolate (EP) gave moderate yields of the expected aromatic pyrrolo[2,1-c][4,7]phenanthroline derivatives 4(a–e) and 6(a–e) (Scheme 1). From the reactions of ylides 2 with EP, only a single regioisomer has been isolated in accordance with the electronic effects in both reactants. The reaction of ylide 2a and EP gave a mixture consisting of 8,9-dihydropyrrolo[2,1-c][4,7]phenanthroline derivative 7a and aromatized compound 6a. We assume that all cycloadditions in Scheme 1, result initially in formation of the unisolable 9,15-dihydropyrrolo[2,1-c][4,7]phenanthroline intermediates 3 and 5 that undergo an oxidative aromatization, converting into more stable aromatic compounds 4 and 6. The formation of dihydroderivative 7a takes place probably via a regioselective prototropic rearrangement.

Scheme 1. Synthesis of fused pyrrolo derivatives 4, 6 and 7.

The structures of the newly compounds were proven by elemental and spectral (IR, NMR, X-ray) methods.

The ¹H and ¹³C NMR spectra of compound 4 and 6 prove the pyrrolo ring formation. Interestingly, the signal of proton 11 that appear in the region of 7.87–8.54 ppm in the spectra of cycloadducts 4a–c, 4e, 6a–c and 6e shifts to the lower fields at 9.51 ppm in the spectrum of 4d, and 9.46 ppm in the spectrum of 6d, compounds in which the position 9 is substituted with a cyano group. We suppose that shifting is due to the positioning of the proton 11 in a deshielding anisotropy region of cyano group rather than a possible H-bond involving.

The position of the two protons on the dihydropyrrolo ring of 7a were established on the basis of NMR and confirmed by single-crystal X-ray study. Thus, the ¹H-NMR spectrum of 7a shows in the aliphatic region two signals at 3.09 and 3.64 ppm corresponding to the two geminal protons 8 coupled with the deshielded proton 9 that furnishes a signal at 6.99 ppm. As well, the chemical shift of the carbonyl ester groups shifts in ¹³C-NMR spectrum shows an attachment to a Csp2 (166.1 ppm). The result of single crystal X-ray study of compound 7a (crystallized from CH₂Cl₂:MeOH 1:1 (v:v)) is shown in Figure 2, while the bond lengths and angles are summarized in Supplemental Table S2. The X-ray data show that dihydropyrrolo[2,1-c][4,7]phenanthroline core is almost planar, the maximal deviation of the atoms from the mean plane being 0.082 Å.

Figure 2. X-ray molecular structure of compound 7a with thermal ellipsoids at 50% probability level.

In the crystal structure, molecules 7a are stocked through the CH···O hydrogen bonding into a columnar array aligned parallel to the c crystallographic axis, as shown in Figure 3. Significant π–π stacking interactions are also present within the column. The ring-centroid separation between inversion molecules are of 3.732 Å, and shift distance of 1.711 Å. The crystal structure of compound 7a clearly demonstrated that it crystallizes without any solvent molecules.

Figure 3. View of supramolecular columnar architecture in the crystal structure of 7a. Only H atoms involved in hydrogen bonding are shown. H-bonds parameters: C10–H⋯O3 [C10–H 0.93 Å, H⋯O3 2.38 Å, C10⋯O3(2 − x, 1 − y, −z) 3.308(4) Å, C10–H⋯O3 175.8 °]; C3–H⋯O3 [C3–H 0.93 Å, H⋯O3 2.49 Å, C3⋯O3(2 − x, 1 − y, −z) 3.419(4) Å, C10–H⋯O3 173.0°]; C14–H⋯O3 [C14–H 0.97 Å, H⋯O3 2.44 Å, C14⋯O3(−1 + x, y, z) 3.299(4) Å, C10–H⋯O3 148.0°].

Next step in our research plan was the study of 3 + 2 cycloaddition of ylides 2b, e, f to symmetrical (N-phenylmaleimide) and unsymmetrical (acrylonitrile) substituted alkenes. Thus, N-ylides 2 were in situ generated and reacted with N-phenylmaleimide giving dihydropyrrolo[3′,4′:3,4]pyrrolo[2,1-c][4,7]phenanthroline derivatives 8 (Scheme 2).

Scheme 2. Synthesis of fused pyrrolo derivatives 8, 9, 10 and 11.

The cycloaddition reactions with N-phenylmaleimide occur highly stereoselective, a single isomer (8) being obtained. Interestingly, from the reaction of 1e in the same conditions, we isolated compound 9e formed by hydrolysis of unstable intermediate 8 during column chromatography separation process. Thus, using CH₂Cl₂/MeOH (99.6:0.4; v/v) as eluent under acidic conditions on silica gel, the imidic cycle opened forming compound 9e. As far, for the cycloaddition with acrylonitrile, the reactions are as expected highly regioselective. The reaction pathway with acrylonitrile occurs differently according the starting ylide we used: a single compound with tetrahydropyrrolo structure 10b was obtained in the case of ylide type b, while for ylides type e and f, a dihydroderivative (11e and 11f, respectively) have been obtained. The formation of dihydroderivatives 11e and 11f could be explained by an oxidative dehydrogenation of tetrahydro intermediates 10, leading to thermodynamically more stable product 11.

In ¹H NMR of compounds 8, there are four distinct signals in the aliphatic region corresponding to the four hydrogen atoms on the pyrrolo ring. The signals delivered by H12 appear as singlets, the lack of coupling for H12 proton being a proof for a trans position relative to proton 18. On the other hand, the big values for the coupling constants J_18,8 (8.0 Hz) and J_8,7 (8.0 Hz) show us that hydrogen atoms H18, H8 and H7 lay on the same side of the tetrahydropyrrolic ring, but on the opposite side of H12.

The structure of compound 9e is proved by the existence in the ¹H NMR spectrum, of two singlets in the aliphatic region (3.81 and 3.89 ppm) corresponding to the methyl ester groups, two coupled doublets (at 4.36 and 6.28 ppm) corresponding to the dihydropyrrolo hydrogens and the singlet at low field (10.19 ppm) furnished by the amidic proton.

The presence of five signals in the aliphatic region of the ¹H NMR of compound 10b, corresponds to the expected tetrahydropyrrolo ring hydrogen atoms, the most deshielded being H9 (dd, 5.57 ppm).

The similarity of ¹H NMR spectra of compound 11e, 11f and 7a diminished the work for elucidation of structure 11, these compounds being probably obtained by an oxidative process in which the intermediate compounds 10 lose two hydrogen atoms. The single crystal X-ray diffraction investigation has demonstrated that the structure of compound 11f [crystallized from CH₂Cl₂:MeOH 1:1 (v:v)] is built up from molecular entities depicted in Figure 4, which confirmed the conclusions made on the base of above-mentioned methods.

Figure 4. The X-ray molecular structure of compound 11f with the thermal ellipsoids at 50% probability level.

The crystal structure of compound 11f resembles that of compound 7a, since the core is planar (the maximal deviation of the atoms from the mean plane is 0.079 Å) and similar columnar supramolecular architecture represents the main packing motif. The molecules 11f are interacting within the column through the CH···O hydrogen-bonding donation to the oxygen atom O1 and π–π stacking interactions, as shown in Figure 5. The ring-centroid distance between parallel packed aromatic systems exhibits the value of 3.757 Å and shift distance of 1.740 Å. No molecules of solvent are present in the crystal structure of compound 11f.

Figure 5. View of supramolecular columnar architecture in the crystal structure of 11f. Only H atoms involved in hydrogen bonding are shown. H-bonds parameters: C7–H⋯O1 [C7–H 0.93 Å, H…O1 2.48 Å, C7⋯O1(1 + x, y, z) 3.158(5) Å, C10–H⋯O3 129.8 °].

Design and biological activity

We have recently discovered that 1-[2-(4-methoxyphenyl)-2-oxoethyl]-4-[[1-(ethoxycarbonyl)-3-benzoyl]-indolizin-7-yl]pyridinium bromide (Scheme 3) is a potent compound against both replicating and non-replicating M. tuberculosis25. On the other hand, phenanthroline, azaindolizine and phenanthridine derivatives were successful identified as DNA intercalating agents or possessing antimycobacterial activity26^,30^,40–43. In the same time, benzo[c]phenanthridinium alkaloids as fagaronine and nitidine which showed antileukemic activity on rodents44 and act as topoisomerase I and II inhibitors45–47, played the role of model compound for the development of new anticancer agents14. Having all these above consideration in mind and encouraged by our previous promising results in the field of anti-TB25–28 and anticancer29^,30^,32 derivatives with indolizine and/or phenanthroline skeleton, we have focused on the design of novel structures that contain four or five fused (hetero)cycles with pyrrolo[2,1-c][4,7]phenanthroline skeleton (Scheme 3). In equal measure, we were interested to see the influence of the substituents at pyrrolo ring (especially the pharmacophoric moiety p-chloro-benzoyl26) on the pharmacological properties, and if the increasing of number of fused rings from four to five will affect these properties (Scheme 3).

Scheme 3. Design in the class of pyrrolo[2,1-c][4,7]phenanthroline derivatives.

The pyrrolo[2,1-c][4,7]phenanthroline derivatives 4 and 6 have been evaluated for in vitro antimycobacterial activity against M. tuberculosis H37Rv (grown under aerobic conditions), as a part of the TAACF TB screening program under direction of the US National Institute of Health, the NIAID division. Initially, the relative solubility of compounds in microbiological medium was measured using turbidity as a measure (Table 1). A serial dilution of compounds was prepared in DMSO and then transferred to microbiological medium at pH 6.8. Turbidity was measured and compared to a control; the lowest concentration at which compounds are insoluble (defined as turbidity > 300% of control) was recorded48. The data from Table 1 illustrate that all tested indolizine with phenanthroline skeleton have a good solubility in microbiological medium, which is quite promising from the pharmacological properties point of view. Then, the IC₅₀, IC₉₀ and MIC of compound were determined by measuring bacterial growth after 5 days in the presence of test compounds36–38^,49. The obtained results are listed in Table 1.

Table 1. Solubility in microbiological medium and antimycobacterial activity of compounds 4a–d and 6a–d against M. tuberculosis H37Rv under aerobic conditions.

				Compound solubility
Compounds	IC50 (µM)	IC90 (µM)	MIC (µM)	Starting concentration (µM)	Lowest insoluble concentration (µM)
4a	>200	>200	>200	200	12.5
4b	>50	>50	>50	100	12.5
4c	>100	>100	>100	20	10
4d	>50	>50	>50	50	12.5
6a	>50	>50	>50	20	20
6b	>20	>20	>20	200	N/A
6c	>50	>50	>50	200	100
6d	>200	>200	>200	200	N/A
Control*
Rifampicin	0.0036	0.0061	0.0055	0.04	N/A

N/A means the compound was soluble at the highest concentration.

*Each experiment runs included and other control compounds: metoprolol tartrate, phenytoin, haloperidol, simvastatin, diethylstilbestrol and tamoxifen.

Bold values indicate moderate active compounds.

As can be seen, five from the eight tested compounds (4a–d and 6a–d) had activity against M. tuberculosis H37Rv under aerobic conditions, with an IC₅₀ in a range of 20–50 μM. We may also notice that the presence of p-chloro-benzoyl moiety at position 9 did not affect the antimycobacterial activity of compounds 4c and 6c comparing with the other derivatives type 4 and 6. Unfortunately, we are not able at the moment to comment the influence of the number of rings on the biological activity because the antimycobacterial evaluation of compounds 8, 9, 10 and 11 is underway.

Four indolizines with phenanthroline skeleton, 4a, 4c, 6c and 6d, were selected and tested in vitro for anticancer activity by the National Cancer Institute (NCI, USA), under the Developmental Therapeutics Program (DTP), at a single high dose (10⁻⁵ M) cell assay. This assay was performed in a 60 human tumor cell line panel, representing leukemia, melanoma and cancers of lung, colon, brain, breast, ovary, kidney and prostate, in accordance with the protocol of the NCI [US National Cancer Institute (NCI), Bethesda; http://dtp.nci.nih.gov/]50–52. The results are expressed as “percentage growth inhibition” (PGI) term, and represent growth relative to the no-drug control, and relative to the time zero number of cells (Table 2). This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0)51. Supplementary information concerning the in vitro anticancer assay could be found in a research overview by Michael R. Boyd52 and in the ESI of this article.

Table 2. Percentage growth inhibition (PGI, μM) data of compounds 4a, 4c, 6c and 6d against an NCI 60 human tumour cell lines (selection).

Panel/Cell line	Compound 4a, PGI	Compound 4c, PGI	Compound 6c, PGI	Compound 6d, PGI
CNS Cancer
SF-SNB-19	84.77	95.43	74.19	86.66
Melanoma
M14	77.14	88.38	95.18	99.07
Renal Cancer
UO-31	81.49	84.21	58.02	76.07
Breast Cancer
MCF7	84.73	92.59	93.10	56.64
T-47D	80.69	81.09	86.01	70.76

The number reported for the one-dose assay, percentage growth inhibition (PGI), is growth relative to the no-drug control and relative to the time zero number of cells. Full results are presented in the SM.

Bold and underlined values indicate active compounds. Underlined values indicates moderate active compounds.

The results from Table 2 indicate that pyrrolo [2,1-c][4,7]phenanthroline derivatives 6c and 6d exhibit a significant antitumor growth inhibitory activity (around 50%) against renal cancer (UO-31) and breast cancer (MCF7), respectively. We may also notice a weaker antitumor growth inhibitory activity (around 25%) of 6c against CNS Cancer (SF-SNB-19), 4a against Melanoma (M14) and 6d against Breast Cancer (T-47D).

As to the mechanism, taking into account, the above consideration concerning biological activity, as well as the fact that the X-ray structure of the new indolizines with phenanthroline skeleton show coplanar cores of these compounds, we assert that an interaction with DNA via an intercalation mechanism would be reasonable.

Conclusions

A new class of compounds with pyrrolo[2,1-c][4,7]phenanthroline skeleton has been synthesized by [3 + 2] dipolar cycloaddition of 4,7-phenanthrolin-4-ium ylides generated in situ from the corresponding monoquaternary salts, to activated symmetrical and unsymmetrical substituted alkynes or alkenes. The cycloaddition reactions to symmetrically substituted alkenes (N-phenylmaleimide) occur highly stereoselective (a single isomer being obtained), whereas the cycloadditions to unsymmetrically substituted dipolarophiles (acrylonitrile and ethyl propiolate) occur highly regioselective (a single regioisomer being formed), under charge control. The formation of aromatized and dihydro-fused polycyclic indolizines (in some cases) was explained by an oxidative dehydrogenation of intermediate tetrahydro-fused derivatives, process that leads to thermodynamically more stable compounds. The structure of the new compounds was proved by elemental and spectral analysis, including X-ray diffraction. The antimycobacterial activity of eight compounds was investigated against Mycobacterium tuberculosis H37Rv under aerobic conditions, five of the tested compounds showing moderate activity. Four new compounds have been evaluated by NCI for anticancer activity. Two of the compounds exhibiting a significant antitumor growth inhibitory activity (around 50%) against renal cancer (UO-31) and breast cancer (MCF7), respectively.

Supplementary material available online

Supplemental Tables S1 and S2.

Acknowledgements

The authors gratefully acknowledge the National Institutes of Health and the National Institute of Allergy and Infectious Diseases (Contract No. HHSN272201100009I / HHSN27200001A08) for antimycobacterial tests and, to National Cancer Institute (NCI) for biological evaluation of compounds on their 60-cell panel: the testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (the URL to the Program's website: http://dtp.cancer.gov/). We also thank to the POSCCE-O 2.2.1, SMIS-CSNR 13984-901, Project no. 257/28.09.2010, CERNESIM, for the NMR experiments.

Declaration of interest

The authors report no conflict of interest. C. M. Al Matarneh is thankful for the financial support of grant POSDRU/159/1.5/S/137750, Project “Doctoral and Postdoctoral programs support for increased competitiveness in Exact Sciences research” co-financed by the European Social Found within the Sectorial Operational Program Human Resources Development 2007–2013.