Inhibitors of the Hydrolytic Enzyme Dimethylarginine Dimethylaminohydrolase (DDAH): Discovery, Synthesis and Development

Abstract

Dimethylarginine dimethylaminohydrolase (DDAH) is a highly conserved hydrolytic enzyme found in numerous species, including bacteria, rodents, and humans. In humans, the DDAH-1 isoform is known to metabolize endogenous asymmetric dimethylarginine (ADMA) and monomethyl arginine (l-NMMA), with ADMA proposed to be a putative marker of cardiovascular disease. Current literature reports identify the DDAH family of enzymes as a potential therapeutic target in the regulation of nitric oxide (NO) production, mediated via its biochemical interaction with the nitric oxide synthase (NOS) family of enzymes. Increased DDAH expression and NO production have been linked to multiple pathological conditions, specifically, cancer, neurodegenerative disorders, and septic shock. As such, the discovery, chemical synthesis, and development of DDAH inhibitors as potential drug candidates represent a growing field of interest. This review article summarizes the current knowledge on DDAH inhibition and the derived pharmacokinetic parameters of the main DDAH inhibitors reported in the literature. Furthermore, current methods of development and chemical synthetic pathways are discussed.

Keywords: arginine; asymmetric dimethylarginine; dimethylarginine dimethylaminohydrolase; enzyme inhibitors; monomethyl arginine; nitric oxide; organic synthesis.

Figure 1

The endogenous methylated arginines, asymmetric dimethylarginine (ADMA 1), monomethylarginine (l-NMMA 2), and symmetric dimethylarginine (SDMA 3) act as direct or indirect inhibitors of the NOS family of enzymes. ADMA (1) and l-NMMA (2) are substrates for human DDAH-1.

Scheme 1

ADMA and l-NMMA are substrates for DDAH-1 and are converted to l-citrulline (4) and an amine species 5 or 6.

Scheme 2

Synthetic scheme for generating HcyNO (9). Reagents and Conditions: (i) 5 M NaOH, 37 °C, 5 min, 96%; (ii) NaNO₂, 2 M HCl, 37 °C, 15 min, >99%.

Scheme 3

Schematic of the reaction mechanism proposed by Knipp et al. [102] for the covalent inhibition of DDAH-1 by HcyNO (9) at Cys273. Adapted with permission from [102]. Copyright 2005 American Chemical Society.

Scheme 4

Synthetic scheme for generating 2-chloroacetamidine (11) (the conditions described afford the hydrochloride salt). Reagents and Conditions: (i) NaOMe, dry MeOH, overnight; (ii) AcOH, NH₄Cl, reflux, 6 h, 89%.

Scheme 5

Synthetic scheme for generating the DDAH-1 biochemical probe N-but-3-ynyl-2-chloro-acetamidine (18). Reagents and Conditions: (i) DCM, TEA, DMAP, MsCl, 0 °C for 2 h then 25 °C for 3 h, 62%; (ii) anhydrous DMF, NaN₃, 70 °C, 3.5 h; (iii) Et₂O, Ph₃P, 0 °C for 2 h, addition of H₂O, then 25 °C for 20 h, 49% (two steps); (iv) dry HCl_(g) at 0 °C for 1 h, stop HCl_(g), then 0 °C for 4 h and RT overnight 48%–76%; (v) H₂O, pH = 10, 0 °C for 3 h, 25 °C for 2 h, neutralized, RT for 1.5 days, 62%. Adapted with permission from [109]. Copyright 2009 American Chemical Society.

Scheme 6

Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid (4124W), as reported by Ueda et al. [80] using the methods of Ogawa et al. [32] and Corbin et al. [111]. Reagents and Conditions: (i) (1) 2,4-diamino-n-butyric acid dihydrochloride (20), copper acetate, 1:1 NH₄OH/H₂O; (2) N,S-dimethylthiopseudouronium iodide (19) RT, 24 h; (3) Dowex 50WX4 (NH₄⁺ form), 4124W (21) was crystallized as the hydrochloride salt.

Scheme 7

Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid analogues. Reagents and Conditions: (i) DEAD, PPh₃, THF, 0 °C-RT, 3–16 h; (ii) DIPEA, CH₃CN, RT, 24 h; (iii) 4M HCl/1,4-dioxane, RT, 24–72 h, 12%–67% overall; (iv) SOCl₂, 0 °C for 30 min, reflux for 1 h, RT overnight, 44%–84%. Adapted with permission from [112]. Copyright 2005 American Chemical Society.

Scheme 8

Synthetic scheme for generating N,N-disubstituted alkylguanidinobutanoic acid and N-arylguanidine analogues. Reagents and Conditions: (i) DEAD, Ph₃P, THF, 0 °C-RT, 3–16 h; (ii) DIPEA, CH₃CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 0.29%–41% overall. Adapted with permission from [112]. Copyright 2005 American Chemical Society.

Scheme 9

Synthetic scheme for generating L-257 (39) and L-291 (40). Reagents and Conditions: (i) DEAD, Ph₃P, THF, 0 °C-RT, 3–16 h; (ii) DIPEA, CH₃CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 44% overall; (iv) SOCl₂, 0 °C for 30 min, reflux for 1 h, RT overnight, 80%. Adapted with permission from [112]. Copyright 2005 American Chemical Society.

Scheme 10

Synthetic scheme for generating N^G-(2-methoxyethyl)-l-arginine carboxylate bioisosteres. Reagents and Conditions: (i) CDI, DBU, methanesulfonamide, THF, RT, 7 h, 51%; (ii) HATU, DIPEA, O-methylhydroxylamine hydrochloride, THF, 0 °C-RT, 16 h, 86%; (iii) HATU, DIPEA, O-benzylhydroxylamine, THF, 0 °C-RT, 16 h, 57%; (iv) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%; (v) DIPEA, DCM, RT, 24 h, 43a 42%, 43b 22%, 43c 56%; (vi) TFMSA/TFA, RT, 1 h, 99%. Reproduced in part from [117] with permission of the Royal Society of Chemistry.

Scheme 11

Synthetic scheme for generating N^G-(2-methoxyethyl)-l-arginine carboxylate bioisosteres. Reagents and Conditions: (i) i-BuCOCl, N-methylmorpholine, NH₄OH (30%), THF, −10 °C-RT, 3 h, 99%; (ii) TFAA, TEA, THF, 0 °C-RT, 16 h, 47%; (iii) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%; (iv) DIPEA, DCM, RT, 24 h, 40%; (v) NaN₃, ZnBr₂, H₂O/2-propanol, reflux, 16 h, 30%; (vi) (1) NH₂OH·HCl, NaHCO₃, DMSO, reflux, 16 h; (2) CDI, DBU, THF, reflux, 30 min, 73%; (vii) TFMSA/TFA, DCM, RT, 30 min, 99%. Reproduced in part from [117] with permission of the Royal Society of Chemistry.

Scheme 12

Synthetic scheme for generating N⁵-(1-iminoalk(en)yl)-l-ornithine derivatives. Reagents and Conditions: (i) dry HCl_(g) at 0 °C for 1 h, stop HCl_(g), then 0 °C for 4 h and RT overnight; (ii) H₂O, 5 °C, pH = 10, 1.5 h, adjust pH = 7, overnight, Dowex 50WX8-400 (H₂O then 10% aqueous pyridine); (iii) 6 M HCl, EtOAc, 84%–94% from N^α-Boc-l-ornithine (52). Adapted with permission from [127]. Copyright 1999 American Chemical Society.

Figure 2

N⁵-(1-Iminoalk(en)yl)-l-ornithine derivatives, L-VNIO (54a), L-IPO (54b), and Cl-NIO (54c), as synthesized by Kotthaus et al. [114] and Wang et al. [128,129].

Scheme 13

Synthetic scheme for generating PFP sulfonate esters 61. Reagents and Conditions: (i) DCM, TEA, 0 °C, 59%; (ii) Method A: NaHCO₃, anhydrous MgSO₄, DCM, reflux, 24 h, 73%–90% [133]; Method B: NaOAc, EtOH, RT, 90% [134]; (iii) dry toluene, reflux, 1–24 h, 44%–75%. Reproduced in part from [130,131,132,133] with permission of the Royal Society of Chemistry and the American Chemical Society.

Scheme 14

Synthetic scheme for generating indolylthiobarbituric acid derivatives 68. Reagents and Conditions: (i) NaOMe, MeOH, 60 °C, 6 h, yields not reported; (ii) NaH, DMF, RT, overnight, 83%; (iii) 6 M HCl, EtOH, RT, 2–3 h, yields not reported.

Scheme 15

Synthetic scheme for generating ebselen (73). Reagents and Conditions: (i) (1) DCM, pyridine, SOCl₂, reflux, 2 h; (2) toluene, aniline, reflux, 3 h, 80%; (ii) dry THF, n-BuLi, nitrogen atmosphere, 0 °C, 30 min, 76% (determined via methylation of 72); (iii) same solution as step (ii), Se, 0 °C, 30 min; (iv) same solution as step (iii), CuBr₂, 30 min at −78 °C, then 2 h at RT, 63%. Adapted with permission from [147], copyright 1989 American Chemical Society, and [148], copyright 2003 The Pharmaceutical Society of Japan.

Scheme 16

Synthetic scheme for generating 4-hydroxy-2-nonenal (4-HNE, 76). Reagents and Conditions: (i) dry oxygen-free DCM, RT, 25 h, 75%, Hoveyda-Grubbs 2nd generation (0.025 eq.).

Scheme 17

Synthetic scheme for generating PD 404182 (79). Reagents and Conditions: Route A: (i) DMF, NaH, CS₂, argon atmosphere, 80 °C, 12 h, 88%; (ii) (1) 0.1 M NaOH, MeOH/H₂O (9:1), reflux, 12 h; (2) anhydrous EtOH, BrCN, argon atmosphere, reflux, 2 h, 61% (two steps). Route B: (iii) DMF, NaH, argon atmosphere, 80 °C, 2 h, 62%; (iv) TFA, CHCl₃, molecular sieves 4 Å, reflux, 1 h, 85%. Adapted with permission from [172,173]. Copyright 1975, 2010 American Chemical Society.

Figure 3

Chemical structures of commonly prescribed proton pump inhibitors (PPIs) 82a–f.

Scheme 18

Synthetic scheme for generating omeprazole (82a). Reagents and Conditions: Route A: (i) 1 M NaOH (2 eq.), EtOH, RT, 8 h; (ii) catalyst: V₂O₅, NaVO₃, NH₄VO₃, [(CH₃COCH₂COCH₃)₂VO] (0.0001–0.1 eq.), H₂O₂: 10%–70% aqueous or organic solution (1–3 eq.), solvent: halogenated hydrocarbons, ethers, alcohols, ketones, nitriles, or H₂O, temperature: 0 °C to boiling point of the solvent, 0.5–24 h, 89%–93%. Route B: (iii) condensation, conditions not specified in patent; (iv) [(CH₃COCH₂COCH₃)₂VO] (0.006 eq.), 30% H₂O₂, acetone, 0–5 °C for 1 h then 20–22 °C for 1 h, 90%; (v) 10% NaOH, nitrogen atmosphere, 50 °C, 3 h; (vi) CO₂, extraction into 1:1 v/v isopropanol/toluene, reflux, 20–30 min, 42% (across steps (v) and (vi)).

Scheme 19

Synthetic scheme for generating 1,2,3-triazoles 95. Reagents and Conditions: (i) 92a (Ph₃P)₂PdCl₂, CuI, TEA, TIPS-acetylene, DMF, 80 °C, 72 h, 30%, 92b (Ph₃P)₂PdCl₂, CuI, TEA, ethynyl-TMS, 80 °C, 20 h, 48%; (ii) DPPA, DBU, toluene, RT, 16 h, 94a 55%, 94b 83%; (iii) CuI, sodium ascorbate, TEA, DCM, RT, 24 h, 95a 65%, 95b 30%. Reproduced in part from [117] with permission of the Royal Society of Chemistry.