International Journal of Medicinal ChemistryVolume 2011 (2011), Article ID 562421, 9 pagesdoi:10.1155/2011/562421

Research Article

Synthesis, Molecular Modeling, and Biological Evaluation of Novel Tetrahydro-β-Carboline Hydantoin and Tetrahydro-β-Carboline Thiohydantoin Derivatives as Phosphodiesterase 5 Inhibitors

Ashraf H. Abadi,¹ Jochen Lehmann,² Gary A. Piazza,³ Mohammad Abdel-Halim,¹ and Mohamed S. M. Ali¹

¹Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt²Institute of Pharmacy, Department of Pharmaceutical/Medicinal Chemistry, Friedrich-Schiller University, Philosophenweg 14, 07743 Jena, Germany³Division of Drug Discovery, Department of Biochemistry and Molecular Biology, Southern Research Institute, The University of Alabama at Birmingham, 2000 Ninth Avenue South, Birmingham, AL 35205, USA

Received 29 September 2010; Accepted 3 January 2011

Academic Editor: Qidong You

Copyright © 2011 Ashraf H. Abadi et al. Ashraf H. Abadi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Two series of fused tetrahydro-β-carboline hydantoin and tetrahydro-β-carboline thiohydantoin derivatives with a pendant 2,4-dimethoxyphenyl at position 5 were synthesized, and chiral carbons at positions 5 and 11a swing from R,R to R,S, S,R, and S,S. The prepared analogues were evaluated for their capacity to inhibit phosphodiesterase 5 (PDE5) isozyme. The R absolute configuration of C-5 in the β-carboline hydantoin derivatives was found to be essential for the PDE5 inhibition. Chiral carbon derived from amino acid even if of the S configuration (L-tryptophan) may lead to equiactive or more active isomers than those derived from amino acid with the R configuration (D-tryptophan). This expands the horizon from which efficient PDE5 inhibitors can be derived and may offer an economic advantage. The thiohydantoin derivatives were less active than their hydantoin congeners.

1. Introduction

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes responsible for the hydrolysis of cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) that are important intracellular second messengers playing a central role in regulating many relevant cell functions. These second messengers are converted to the biologically inactive monophosphates with subsequent termination of their physiological functions. Currently, the PDE system includes 11 families (PDE1-PDE11) comprising 21 different gene products [1–3].

The cGMP-specific phosphodiesterase 5 (PDE5) is abundant in the penile tissue, platelets, vascular, and smooth muscle tissues. This enzyme is the primary target for the development of small molecules, such as the well-known sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) to treat erectile dysfunction [4].

Nitric oxide activates guanylatecyclase to convert GTP to the second messenger cGMP which, in turn, results in smooth muscle relaxation and the erectile response. cGMP is hydrolysed by PDE5 to inactive GMP. PDE5 inhibitors such as sildenafil thus act to inhibit cGMP breakdown and thereby facilitate penile erection in patients suffering from MED [5]. Recently, sildenafil was approved for the treatment of pulmonary hypertension (Revatio), and there are numerous emerging reports relating the elevation of cGMP to anticancer apoptotic actions. This further illustrates the potential of PDE5 inhibitors as therapeutic agents.

Chemically speaking, the long-acting PDE5 inhibitor tadalafil is considered as a fused tetrahydro-β-carboline piperazinedione derivative with pendant 1,3-benzodioxol moiety at position 6 and a methyl substituent on the piperazinedione nitrogen, and it is of 2 chiral carbons both are of the absolute R configuration.

In the present work, we report the synthesis of novel tetrahydro-β-carboline analogues and evaluate the activity of these compounds as PDE5 inhibitors.

2. Chemistry

The general synthesis of the target β-carboline hydantoin and β-carboline thiohydantoin derivatives is illustrated in Schemes 1–4. Both D-tryptophan and L-tryptophan methyl ester were synthesized by a general synthetic procedure for amino acid esters. The ester and 2,4-dimethpxybenzaldehyde were subjected to a Pictet-Spengler reaction under nonstereospecific conditions. The diastereomeric nature of the produced cis- and transisomers of the 1,3-disubistituted THBC (1–4) allowed their separation by column chromatography using CH₂Cl₂ : CH₃OH (99.5 : 0.5) as an eluent.

Figure 1: Conformational representation of the energy minimized form of compound VIII by MMF94 followed by conformational search, showing a semiperpendicular disposition of the pendant dimethoxyphenyl relative to the tetracyclic part. Torsional angle of 77.2°.

A docking experiment was implemented to dock 8 to the human PDE5 using the MOE software [10]. For recognition between the protein and ligand, it is important that the two molecules form a stable complex. The factors contributing to the stabilization of the complex structure include complimentarily of shape, hydrogen bonding, electrostatic and hydrophobic properties, and internal strain when the complex is formed.

Detailed mode photo showed that compound 8 is able to dock to the active pocket of PDE5 and interact with the side chain of Gln 817 which forms a single, not bidentate, hydrogen bond with the indole NH group of the respective compound; interestingly, tadalafil interacts in the same fashion; however, unlike tadalafil the π-π stacking with Phe820 is missed. This may be the reason why this compound is less active than tadalafil, Figure 2.

Figure 2: detailed photo mode showing interaction of compound VIII with human PDE5 through H-bonding with Gln 817. The missing of π-π stacking with Phe820 may be the reason why this compound is less active than tadalafil.

4. Experimental

4.1. General

All starting materials were commercially available and used without further purification. All reactions were carried out with the use of standard techniques under an inert atmosphere (N₂). The analytical thin-layer chromatography (TLC) was carried out on E. Merck 60-F254 precoated silica gel plates, and components were usually visualized using UV light. Flash column chromatography was performed on silica gel 60 (E. Merck, 230–400 mesh). Melting points were determined on Buchi Melting Point apparatus and are uncorrected. Proton NMR (¹H NMR) and carbon NMR (¹³C NMR) spectra were recorded at ambient temperature on Varian Mercury VX-300 MHz spectrometer using tetramethylsilane as internal standard, and proton chemical shifts are expressed in ppm in the indicated solvent. The following abbreviations are used for multiplicity of NMR signals: (s) singlet, (d) doublet, (t) triplet, (q) quadruplet, (dd) double doublet, and (m) multiplet. The elemental analyses were performed by the Microanalytical Unit, Faculty of Science, Cairo University and are within 0.4% of the theoretical value, unless stated otherwise.

4.1.1. General Procedure for the Preparation of D- and L-Tryptophan Methyl Ester

A 250 mL round flask was charged with methanol (150 mL) and cooled with an ice water bath, then acetyl chloride (23 mL) was added dropwise using a dropping funnel over a period of 15 min. The solution was stirred for a further 10 min, then solid D- or L-tryptophan (12 g) was added in one portion, and the solution was heated to reflux for 5 hrs. The solution was allowed to cool to room temperature, and the solvent was removed under reduced pressure. The crude methyl tryptophan ester hydrochloride was extracted with ammonia solution (50 mL) and methylene chloride ( mL). The organic layer was dried over anhydrous Na₂SO₄ evaporated under reduced pressure to give yellowish white oil which solidifies on cooling in almost quantitative yield. It was used without further purification.

4.1.2. General Procedure for the Preparation of Methyl 1-(2,4-dimethoxyphenyl)-2,3,4,9-tetrahydro-1H-β-carboline-3-Carboxylate (1–4)

The appropriate tryptophan methyl ester (6.84 g, 31.4 mmol) and 2,4-dimethoxybenzaldehyde (5.73 g, 34.5 mmol) were dissolved in CH₂Cl₂ (25 mL) and cooled to 0°C in an ice bath. To this solution, trifluoroacetic acid (TFA) (1 mL) was added dropwise, and the mixture was stirred at room temperature for 4 days under N₂ atmosphere. The reaction mixture was then basified with dilute NH₄OH solution and extracted with CH₂Cl₂ (3 times 10 mL). The organic layer was washed with water, brine, dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The residue was purified, and the produced diastereomers were separated by column chromatography on silica gel eluting with CH₂Cl₂ : CH₃OH (99.5 : 0.5), giving first the appropriate cisisomer followed by the trans one.

(1R, 3R) Methyl-1-(2,4-dimethoxyphenyl)-2,3,4,9-tetrahydro-1-H-pyrido[3,4-b]indole-3-carboxylate (1)Yield: 21%; mp 86–88°C; IR (cm⁻¹) 3380 (NH), 1740(CO); 1H-NMR (CDCl₃), δ ppm, 2.90–3.10 (m, 1H, CHaHb), 3.10–3.30 (m, 1H, CHaHb), 3.81(s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.87 (s, 3H, COOCH₃), 3.9–4.0 (m, 1H, −CHCOOCH₃), 5.7 (s, 1H, CHPh), 6.40–6.60 (m, 2H, Ar), 7.10–7.60 (m, 5H, Ar); 13C-NMR (CDCl₃), δ ppm, 25.74, 51.44 (C₁), 52.12, 55.44, 55.70, 57.04 (C₃), 98.86, 104.88, 108.39, 110.78, 117.99, 119.40, 121.56, 125.99, 127.24, 129.92, 135.33, 135.89 160.72, 173.43. MS (m/z): 366 (M+, 100%). Anal. Calcd. for C₂₁H₂₂N₂O₄·0.5H₂O; C: 67.17, H: 6.17, N: 7.47; Found C: 67.44, H: 5.94, N: 7.23.

(1S,3R) Methyl-1-(2,4-dimethoxyphenyl)-2,3,4,9-tetrahydro-1-H-pyrido[3,4-b]indole-3-carboxylate (2)Yield: 57%; mp 146–147°C; IR (cm⁻¹) 3345 (NH), 1700 (CO); ¹H-NMR (CDCl₃), δ ppm, 2.70–2.75 (m, 1H, HaHb), 2.99–3.10 (m, 1H, HaHb), 3.43–3.49 (m, 6H, OCH₃), 3.58 (s, 3H, OCH₃), 3.78 (m, 1H, CHCOOCH₃), 5.42 (s, 1H, CHph), 5.95–7.74 (m, 7H, Ar); ¹³C-NMR (CDCl₃), δ ppm, 25.08, 48.86 (C ₁), 51.83, 52.01, 55.32, 55.46 (C ₃), 98.70, 103.38, 109.03, 110.84, 117.99, 119.24, 121.62, 122.34, 126.90, 129.63, 133.10, 136.12, 157.97, 160.84, 173.90. MS (m/z): 366 (M⁺, 100%). Anal. Calcd. for C₂₁H₂₂N₂O₄·0.5H₂O; C: 67.17, H: 6.17, N: 7.47; Found C: 67.52, H: 6.10, N: 7.35.

(1S,3S) Methyl-1-(2,4-dimethoxyphenyl)-2,3,4,9-tetrahydro-1-H-pyrido[3,4-b]indole-3-carboxylate (3)Yield: 19%; mp 88–90°C; IR (cm⁻¹) 3322 (NH), 1726 (CO); ¹H-NMR (CDCl₃), δ ppm, 3.02–3.07 (m, 1H, HaHb), 3.20–3.21 (m, 1H, HaHb), 3.80–3.82 (m, 9H, OCH₃), 3.88–3.98 (m, 1H, CHCOOCH₃), 5.6 (s,1H, CHph), 6.3–6.5 (m, 2H, Ar), 7.1–7.9 (m, 5H, Ar); ¹³C-NMR (CDCl₃), δ ppm, 25.49, 51.33 (C₁), 51.97, 55.23, 55.45, 56.70 (C3), 98.63, 104.65, 108.08, 110.73, 117.75, 119.16, 120.97, 121.36, 126.98, 129.83, 134.87, 135.79, 158.21, 160.55, 173.16. MS (m/z): 366 (M⁺, 100%). Anal. Calcd. for C₂₁H₂₂N₂O₄·0.25H₂O; C: 68.84, H: 6.05, N: 7.65; Found C: 68.62, H: 6.09, N: 7.54.

(1R,3S) Methyl-1-(2,4-dimethoxyphenyl)-2,3,4,9-tetrahydro-1-H-pyrido[3,4-b]indole-3-carboxylate (4)Yield: 11%; mp 148–150°C; IR (cm⁻¹) 3152 (NH), 1721 (CO); ¹H-NMR (CDCl₃), δ ppm,2.94–3.02 (m, 1H, HaHb), 3.20–3.27 (m, 1H, HaHb), 3.72 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 3.85–3.89 (m, 1H, CHCOOCH₃), 5.69 (s,1H, CHph), 6.24–7.58 (m, 7H, Ar), 8.46 (m, 1H, NH); ¹³C-NMR (CDCl₃), δ ppm, 24.94, 48.92 (C₁), 51.55, 51.83, 55.14, 55.23 (C ₃), 98.51, 103.23, 108.74, 110.84, 117.76, 118.97, 121.39, 122.06, 126,71, 129.58, 132.84, 136.11, 157.79, 160.32, 173.66. MS (m/z): 366 (M⁺, 100%). Anal. Calcd. for C₂₁H₂₂N₂O₄·0.25H₂O; C: 68.84, H: 6.05, N: 7.65, Found C: 67.79, H: 6.24, N: 7.15.

4.1.3. General Procedures for the Preparation of 2-Substituted-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione and 2-Substituted-1-oxo-5-(2,4-dimethoxyphenyl)-5,6,11,11a tetrahydro-1H-imidazo[5,1:6,1]pyrido[3,4-b]indole-3 (2H)-thione (5–16 and 17–20)

The appropriate isocyanate or isothiocyanate (1.6 mmol) was added to a well-stirred solution of the appropriate β-carboline (0.36 g, 1 mmol) in methyl ethyl ketone (10 mL), and the mixture was stirred at reflux for 16 hours under nitrogen atmosphere. The solvent was evaporated under reduced pressure; the residue was purified using preparative TLC and developed with CH₂Cl₂. The required band was stripped and dissolved in CH₂Cl₂. The solution was then filtered to remove the undesired silica; the filtrate was evaporated under reduced pressure to obtain the pure desired product.

(5R,11aR)-2-Ethyl-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (5)Yield: 50%; mp 216–218°C; IR (cm⁻¹): 3397 (NH), 1758, 1701 (CO); ¹H-NMR (CDCl₃), δ ppm: 1.22–1.28 (m, 3H,CH₂CH₃), 2.8–2.9 (m, 1H, HaHb), 3.49–3.55 (m, 1H, HaHb), 3.62–3.64 (m, 2H, CH₂CH₃), 3.79 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃), 4.55–4.57 (m,1H, CHCO), 6.45–6.56 (m, 3H, Ar, and CHph), 7.03–7.49 (m, 5H, Ar), 8.21 (s, 1H, NH). MS (m/z): 405 (M⁺, 100%). Anal. Calcd. for C₂₃H₂₃N₃O₄·0.8H₂O; C: 65.80, H: 5.91, N: 10.01. Found C: 66.47, H: 5.61, N: 9.40.

(5S,11aR)-2-Ethyl-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (6)Yield: 62%; mp 190–193°C; IR (cm⁻¹): 3395 (NH), 1759, 1701 (-CO-); ¹H-NMR (CDCl₃), δ ppm: 1.23–1.28 (m, 3H,CH₂CH₃), 2.86–2.90 (m, 1H, HaHb), 3.45–3.54 (m, 1H, HaHb), 3.62–3.65 (m, 2H, CH₂CH₃), 3.79 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃), 4.53–4.59 (m,1H, CHCO), 6.45–6.57 (m, 3H, Ar, and CHph), 7.03 (m, 5H, Ar), 8.21 (s, 1H, NH). MS (m/z): 428 (M⁺+23, 100%). Anal. Calcd. for C₂₃H₂₃N₃O₄·0.25H₂O; C: 67.39, H: 5.78, N: 10.25. Found C: 67.45, H: 5.48, N: 9.83.

(5S,11aS)-2-Ethyl-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (7)Yield: 58%; mp 217–220°C; IR (cm⁻¹) 3397 (NH), 1758, 1701 (CO); ¹H-NMR (CDCl₃), δ ppm: 1.28–1.33 (m, 3H,CH₂CH₃), 2.75–2.86 (m, 1H, HaHb), 3.43–3.51 (m, 1H, HaHb), 3.57–3.67 (m, 2H, CH₂CH₃), 3.78 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 4.51–4.57 (m,1H, CHCO), 6.43–6.55 (m, 3H, Ar, and CHph), 7.02–7.49 (m, 5H, Ar), 8.3 (s, 1H, NH). MS (m/z): 405 (M⁺, 100%). Anal. Calcd. for C₂₃H₂₃N₃O₄0.8H₂O; C: 65.80, H: 5.91, N: 10.01. Found C: 66.10, H: 6.06, N: 9.47.

(5R,11aS)-2-Ethyl-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (8)Yield: 87%; mp 192–195°C; IR (cm⁻¹) 3397 (NH), 1759, 1701 (CO); ¹H-NMR (CDCl₃), δ ppm: 1.25–1.31 (m, 3H, CH₂CH₃), 2.77–2.88 (m, 1H, HaHb), 3.44–3.52 (m, 1H, HaHb), 3.58–3.67 (m, 2H, CH₂CH₃), 3.76 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 4.53–4.58 (m,1H, CHCO), 6.43–6.55 (m, 3H, Ar, and CHph), 7.02–7.52 (m, 5H, Ar), 8.28 (s, 1H, NH). MS (m/z): 405 (M⁺, 100%). Anal. Calcd. for C₂₃H₂₃N₃O₄0.5H₂O; C: 66.65, H: 5.84, N: 10.14. Found C: 66.83, H: 6.01, N: 10.39.

(5R,11aR)-2-Tertiarybutyl-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (9)Yield: 36%; mp 108–110°C; IR (cm⁻¹) 3405 (NH), 1759, 1704 (CO);¹H-NMR (CDCl₃),  ppm: 1.64 (s, 9H,C(CH₃)₃), 2.75–2.85 (m, 1H, HaHb), 3.43–3.49 (m, 1H, HaHb), 3.79 (s, 3H, OCH₃), 3.91(s, 3H, OCH₃), 4.43–4.45 (m,1H, CHCO), 6.55–7.26 (m, 8H, CHph, and Ar), 8.21 (s, 1H, NH). MS (m/z): 433 (M⁺, 100%), 305 (95%). Anal. Calcd. for C₂₅H₂₇N₃O₄H₂O; C: 66.50, H: 6.47, N: 9.31. Found C: 66:13, H: 6.51, N: 9.94.

(5S,11aR)-2-Tertiarybutyl-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (10)Yield: 44%; mp 191-193°C; IR (cm⁻¹) 3334 (NH), 1758, 1704 (CO);¹H-NMR (CDCl₃),  ppm: 1.65 (s, 9H,C(CH₃)₃, 3.41–3.59 (m, 1H, HaHb), 3.63–3.65 (m, 1H, HaHb), 3.79 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 4.40–4.47 (m,1H, CHCO), 7.04–7.52 (s, 8H, CHph, and Ar), 8.21 (s, 1H, NH). MS (m/z): 433 (M⁺, 91%), 305 (100%). Anal. Calcd. for C₂₅H₂₇N₃O₄H₂O; C: 66.50, H: 6.47, N: 9.31, Found C: 66.44, H: 6.12, N: 9.11.

(5S,11aS)-2-Tertiarybutyl-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (11)Yield: 32%; mp 103–106°C; IR (cm⁻¹) 3405 (NH), 1762, 1701 (CO); ¹H-NMR (CDCl₃), δ ppm: 1.58 (s, 9H,C(C H₃)₃), 2.76–2.87 (m, 1H, HaHb), 3.41–3.49 (m, 1H, HaHb), 3.79 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 4.40–4.47 (m,1H, CHCO), 6.45–7.51 (m, 8H, CHph, and Ar), 8.21 (s, 1H, NH). MS (m/z): 433 (M⁺, 100%), 305 (82%). Anal. Calcd. for C₂₅H₂₇N₃O₄·H₂O; C: 66.50, H: 6.47, N: 9.31. Found C: 66.23, H: 6.18, N: 9.22.

(5R,11aS)-2-Tertiarybutyl-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (12)Yield: 40%; mp 193–195°C; IR (cm⁻¹) 3405 (NH), 1759, 1704 (CO); ¹H-NMR (CDCl₃), δ ppm: 1.66 (s, 9H,C(CH₃)₃), 3.39–3.48 (m, 1H, HaHb), 3.61–3.65(m, 1H, HaHb), 3.77 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.39–4.46 (m,1H, CHCO), 6.45–7.52 (s, 8H, CHph, and Ar), 8.35 (s, 1H, NH). MS (m/z): 433 (M⁺, 100%), 305 (74%). Anal. Calcd. for C₂₅H₂₇N₃O₄·1.25H₂O; C: 65.85, H: 6.52, N: 9.21. Found C: 66.71, H: 6.47, N: 9.40.

(5R,11aR)-2-(P-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (13)Yield: 53%; mp 134–136°C; IR (cm⁻¹) 3373 (NH), 1773, 1714 (CO); ¹H-NMR (CDCl₃), δ ppm: 2.89–2.96 (m, 1H, HaHb), 3.52–3.61 (m, 1H, HaHb), 3.80 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 4.69–4.75 (m, 1H, CHCO), 6.45–7.49 (m, 12H, CHph, and Ar). MS (m/z): 487 (M⁺, 88%), 488 (M⁺+1, 25%), 305 (100%). Anal. Calcd. for C₂₇H₂₂ClN₃O₄0.25H₂O; C: 65.85, H: 4.61, N: 8.53. Found C: 65.56, H: 4.61, N: 8.21.

(5S,11aR)-2-(P-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (14)Yield: 46%; mp 214–216°C; IR (cm⁻¹) 3419 (NH), 1774, 1716 (CO); 1H-NMR (CDCl₃), δ ppm: 2.81–2.92 (m, 1H, HaHb), 3.49–3.65 (m, 1H, HaHb), 3.79 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.67–4.73 (m,1H, CHCO), 6.44–7.54 (m, 12H, CHph, and Ar). MS (m/z): 510 (M⁺+Na⁺¹, 100%). Anal. Calcd. for C₂₇H₂₂ClN₃O₄0.25H₂O; C: 65.85, H: 4.61, N: 8.53. Found C: 65.96, H: 4.76, N: 8.50.

(5S,11aS)-2-(P-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (15)Yield: 28%; mp 135–138°C; IR (cm⁻¹) 3363 (NH), 1769, 1712 (CO); ¹H-NMR (CDCl₃), δ ppm, 2.91–3.02 (m, 1H, HaHb), 3.53–3.64 (m, 1H, HaHb), 3.80 (s, 3H, OCH₃), 3.88(s, 3H, OCH₃), 4.69–4.75 (m, 1H, CHCO), 6.45–7.55 (m, 12H, CHph, and Ar). MS (m/z): 487 (M+, 77%), 488 (M+ +1, 22%), 305 (100%). Anal. Calcd. for C₂₇H₂₂ClN₃O₄·0.5H₂O; C: 65.26, H: 4.67, N: 8.46. Found C: 65.45, H: 4.78, N: 8.42.

(5R,11aS)-2-(P-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-6,1]pyrido[3,4-b]indole-1,3-dione (16)Yield: 67%; mp 212–215°C; IR (cm⁻¹) 3363 (NH), 1769, 1709 (CO); ¹H-NMR (CDCl₃), δ ppm, 2.81–2.92 (m, 1H, HaHb), 3.49–3.57 (m, 1H, HaHb), 3.79 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.67–4.73 (m,1H, CHCO), 6.44–7.54 (m, 12H, CHph, and Ar). MS (m/z): 487 (M⁺, 77%), 488 (M⁺+1, 22%), 305 (100%). Anal. Calcd. for C₂₇H₂₂ClN₃O₄0.8H₂O; C: 64.55, H: 4.74, N: 8.36. Found. C: 65.43, H: 4.88, N: 8.39

(5R,11aS)-2-Methyl-1-oxo-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1H-imidazo[5,1-6,1]pyrido[3,4-b]indole-3(2H)-thione (17)Yield: 51%; mp 188–191°C; IR (cm⁻¹) 3407 (NH), 1744 (CO), 1640 (CS); ¹H-NMR (CDCl₃), δ ppm, 2.8–2.9 (m, 1H, CHaHb), 3.53 (m, 1H, CHaHb), 3.54 (s, 3H, CH₃), 3.79 (s, 3H,OCH₃), 3.96 (s, 3H, OCH₃), 4.73–4.78 (m, 1H, CHCO), 6.46–7.50 (m, 8H, CHph, and Ar), 8.19 (s, 1H, NH). MS (m/z): 407 (M⁺, 100%). Anal. Calcd. for C₂₂H₂₁N₃O₃S0.3H₂O; C: 57.25, H: 5.90, N: 9.10. Found C: 56.92, H: 5.52, N: 8.99.

(5S,11aR)-2-Methyl-1-oxo-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1-H-imidazo[5,1-1,6]pyrido[3,4-b]indole-3(2H)-thione (18)Yield: 65%; mp 188–190°C; IR (cm⁻¹) 3407 (NH), 1744 (CO), 1640 (CS); ¹H-NMR (CDCl₃), δ ppm, 2.8–2.9 (m, 1H, CHaHb), 3.5–3.6 (m, 1H, CHaHb), 3.54 (s, 3H, CH₃), 3.79 (s, 3H, OCH₃), 3.96 (s, 3H, OCH₃), 4.73–4.78 (m, 1H, CHCO), 6.46–7.50 (m, 8H, CHph, and Ar), 8.19 (s, 1H, NH). MS (m/z): 407 (M⁺, 100%). Anal. Calcd. for C₂₂H₂₁N₃O₃S0.3H₂O; C: 57.25, H: 5.90, N: 9.10. Found C: 56.88, H: 5.92, N: 9.09.

(5R,11aS)-2-Allyl-1-oxo-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1H-imidazo[5,1-1,6]pyrido[3,4-b]indole-3(2H)-thione (19)Yield: 58%; mp 224–227°C; IR (cm⁻¹) 3415 (NH), 1725 (CO), 1613 (CS); ¹H-NMR (CDCl₃), δ ppm, 2.82–2.92 (m, 1H, CHaHb), 3.40–3.49 (m, 1H, CHaHb), 3.75 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 4.36 (m, 2H,–CH₂–), 5.01–5.13 (m, 3H, CHCO, and CH = CH₂), 5.76–5.87 (m, 1H, CH = CH₂), 6.47–7.48 (m, 8H, CHph, and Ar). MS (m/z): 433 (M⁺+1, 100%). Anal. Calcd. for C₂₄H₂₃N₃O₃S0.5H₂O; C: 65.14, H: 5.47, N: 9.50. Found C: 65.25, H: 5.38, N: 9.15.

(5S,11aR)-2-Allyl-1-oxo-5-(2,4-dimethoxyphenyl)-5,6,11,11a-tetrahydro-1H-imidazo[5,1-6,6,1]pyrido[3,4-b]indole-3(2H)-thione (20)Yield: 55%; mp 224–227°C; IR (cm⁻¹) 3415 (NH), 1725 (CO), 1613 (CS); ¹H-NMR (CDCl₃), δ ppm, 2.75–2.85 (m, 1H, CHaHb), 3.45–3.54 (m, 1H, CHaHb), 3.80 (s, 3H, OCH₃), δ 3.87 ppm (s, 3H, OCH₃), 4.52–4.54 (m, 2H, –CH₂–) 4.73–4.80 (m, 1H, CHCO), 5.21–5.33 (m, 2H, CH = CH₂), 5.85–6.98 (m, 1H, CH = CH₂), 6.48–7.50 (m, 8H, CHph, and Ar), 8.23 (s, 1H, NH). MS (m/z): 433 (M⁺+1, 100%). Anal. Calcd. for C₂₄H₂₃N₃O₃S0.5H₂O; C: 65.14, H: 5.47, N: 9.50. Found C: 65.71, H: 5.64, N: 9.18.

4.2. Biological Testing

4.2.1. Phosphodiesterase Inhibitory Activity

PDE activity was measured using a modification of the IMAP fluorescence polarization phosphodiesterase assay from Molecular Devices. The assay was modified to use tetramethylrhodamine- (TAMRA-) cGMP as substrate. PDE hydrolysis of the fluorescent labeled substrate allows it to bind the IMAP binding reagent, which results in increased FP. The excitation and emission spectrum of the (TAMRA-) cGMP were at 485 and 530 nm, respectively. The assays were performed in 96-well microtiter plates using a reaction buffer containing 10 mM Tris-HCl (pH 7.2), 10 mM MgCl₂, 0.05% NaN₃, and 0.1% phosphate-free BSA as the carrier. Each well contained 20 L of recombinant enzyme (5 units//mL, BPS Biosciences, San Diego, CA) and 10 L test agent. The reaction was initiated by the addition of 10 μL of a substrate solution containing 50 nM TAMRA-cGMP. After incubating at room temperature for 60 minutes, the reaction was terminated by adding 120 L of binding solution. FP was measured by a Perkin Elmer Envision plate reader.

Data AnalysisDrug effects on PDE activity were measured, and potency was expressed by an IC₅₀ value (50% inhibitory concentration). The IC₅₀ value was determined by testing a range of 10 concentrations with at least two replicates per concentration. Dose response curves were analyzed using Prism 4 software (GraphPad) to calculate IC₅₀ values using a four-parameter logistic equation. All in vitro experiments involved dose-response analysis were repeated at least twice to confirm reproducibility of IC₅₀ values.

4.3. Molecular Modeling

4.3.1. Energy Minimization Procedure

The compounds with the correct stereochemistry were drawn on ChemSketch 11 and stored in mol format. The structure was recalled in molecular operating environment (MOE) [10], and all hydrogen atoms were added. The compound was energy minimized using Hamiltonian-Force Field-MMFF94x, followed by systematic conformational search (RMS gradient 0.01); the best 30 conformers were stored in an mdb database format.

4.3.2. Docking

The crystal structure of human phosphodiesterase 5 complexed with tadalafil was downloaded from the protein data bank (PDB ID code 1UDU) and opened with MOE software. Only one chain out of the 2 was left for the docking experiment. Also, the old ligand was removed. The molecular operating environment of docking was used to calculate the docking energies between ligand as its conformationally searched database and the enzyme pocket as given in the software manual. The lowest energy conformation was selected as the best.

Acknowledgments

The authors are grateful to the Faculty of Postgraduate Studies, German University in Cairo for partial finance of the Project and to the Alexander von Humboldt Foundation for sponsoring a postdoctoral fellowship to the first author during which a part of the work has been carried out.

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“Molecular Operating Environment (MOE 2007.09),” Chemical Computing Group, Inc. Montreal, Quebec, Canada.