Three component Kabachnik-Fields condensation leading to substituted aminomethane-P -hydroxymethylphosphonic acids as a tool for screening of bacterial urease inhibitors

Condensation of hydroxyalkane-H -phosphinic acids, formaldehyde and secondary amines has given entry to the synthesis of variously substituted aminomethane-P -hydroxymethylphosphinic acids. The proposed strategy allowed to perform a feasible synthesis of several molecules with designed biological activity towards bacterial urease – an enzyme which is a medicinally relevant molecular target. The inhibitory potency of compounds was validated using enzyme purified from Bacillus pasteurii


Introduction
2][3][4] This approach offers the possibility of producing large number of compounds with the same scaffold and diverse side chains, thus allowing structure optimization in respect of activity, selectivity and other important pharmacological features of the active molecule.Although the most widely studied MCR is Ugitype condensation, other reactions are also used.Among them, Mannich-type three component condensation leading to aminophosphonic acids was applied for construction of compounds with interesting biological activities. 5,6reasethe enzyme catalyzing hydrolysis of urea, is a molecular target for potential therapeutics. 7,8Ureolytic activity of human pathogens Helicobacter pylori and Proteus mirabilis is the main causative factor of severe clinical conditions concerning the gastric and urinary tract.Ammonia and carbon dioxide released in urea hydrolysis are crucial for H. pylori infections pathogenesis.High ammonia concentration results in local pH increase in stomach.Urease activity may have serious consequences including development of gastric ulcer and/or duodenal ulcers, MALT lymphoma, Ménétrier disease and gastric cancer. 9Moreover, infection of the human and animal urinary tract by urease producing species results in urolithiasis.Ammonia elevates urine pH causing a precipitation of magnesium and/or calcium salts as struvite and carbonate apatite.To confront disadvantages of antibiotic treatment in urease-producing bacterial infections, substances that inhibit urease activity have been intensively studied. 10Several classes of urease inhibitors were investigated including urea derivatives, phosphoramidates, hydroxamic acids, quinones, and polyphenols.Amides of phosphoric acid, being classical transition state analogs, represent the group with the highest activity, but unfortunately their utility is strongly limited by low hydrolytic stability (the reported half-life of N-(diaminophosphinyl)-4fluorobenzamide, flurofamide at pH 2 is around 5 min.). 11Interestingly, a moderately active urease inhibitoracetohydroxamic acid was approved by FDA in 1983 for urinary tract infections treatment (trademarks Lithostat, Uronefrex), although it exhibits severe side effects including teratogenicity, psychoneurologic and musculo-integumentary symptoms. 12,13n this paper we apply a multi-component reaction strategy for screening urease's phosphinate inhibitors.The use of the Kabachnik-Fields three-component condensation of a hydroxyalkane-H-phosphinic acid, formaldehyde and a secondary amine to produce substituted aminomethane-P-hydroxymethylphosphinic acids is studied.The optimization of a straightforward method for the synthesis of compounds with potential anti-urease activity gives the possibility to assay a variety of analogues.

Design
5][16] N-Methylaminomethane-P-hydroxymethylphosphinic acid 1 was one of the most effective analogues.Using the structure of 1 as a scaffold, two types of modifications were envisaged (Scheme 1), namely the substitution of hydroxymethyl fragment (compounds 2-6), or amine moiety (structures 7-12).In order to test the synthetic methodology as well as the complementarity with the enzyme active site, substituents of various size and character (aliphatic/aromatic) were proposed.Scheme 1.The outline of urease inhibitors screening procedure.

Synthesis
Inhibitors construction started with the preparation of α-hydroxyalkanephosphinic acids.Known methods of preparation of these compounds include a variation of the Pudovik addition reaction of commercially available 50 wt % aqueous hypophosphorous acid to aldehydes under acidic conditions. 17Mono-and bis(α-hydroxyalkyl)phosphinic acids are obtained with prolonged heating.In a previous paper 15 we reported the optimized reaction conditions for the synthesis of hydroxymethane-H-phosphinic acid 16, which was used in the next step without being isolated (Scheme 2).The same reaction conditions e.g.50 wt % aqueous hypophosphorous acid with 1.2 equiv. of aldehyde (acetaldehyde for 13, benzaldehyde for 14, 2-phenylacetaldehyde for 15) in the presence of 1.2 equiv.6M hydrochloric acid in ethanol refluxed for 48h, afforded the αhydroxyalkane-H-phosphinic acids 13-15 as main products (60-89%), accompanied by the corresponding bis-adducts.The latter compounds give signals around 40 ppm in 31 P NMR while the former around 33 ppm.The chemical shift of the carbon atom in the α-position of the phosphorus (δ ( 13 C) ~69 ppm) and its coupling constant with the phosphorus atom ( 1 JPC ~ 110 Hz) are consistent with the presence of an α-hydroxyalkanephosphinic moiety.The CH proton resonates in most cases as a multiplet at about 3.6 ppm.
Since α-hydroxyalkane-H-phosphinic acids were obtained, the one pot synthesis of substituted α1-aminomethane-α2-hydroxyalkanephosphinic acids was available by the classical Kabachnik-Fields three-component reaction. 18Several N,N-disubstituted phosphinates 3, 5, 6, 8, 10, 12, 17-21 were obtained in around 65% yield by combining the α-hydroxyalkane-Hphosphinic acid, formaldehyde, and the secondary amine.When the phosphinate planned to be N-monosubstituted, the N-benzyl substituent was easily removed by catalytic hydrogenolysis after the three-component condensation.Compounds 3, 5, 6, 8, 10, 12, 17-21 were purified by dowex cation exchange resin followed, if necessary, by reversed phase silica gel column chromatography.Most of the secondary amines used were commercially available while the others were synthesized in a stepwise procedure of reductive amination.This involves imine formation in methanol followed by reduction with NaBH4, a procedure which is known to avoid the usual dialkylation problem. 19 H O (a)

Biological activity
The obtained compounds were tested against urease purified from Bacillus pasteurii (Table 1) using color indophenol assay.Inhibition constants for compounds 2-4, 7, 9, 11 were in micromolar range.The highest activities were exhibited by compounds which were mono Nsubstituted analogues 2, 4, 7, 9 and 11.Within this group N-cyclohexyl-aminomethane-Phydroxymethylphosphinic acid (11) was the most potent with Ki = 92 M.With the exception of structure 3, N,N-disubstituted compounds were found to be inactive.In comparison to the starting structure 1, the tested modifications did not lead to improvement of inhibitory activity.

Conclusions
The present work describes the application of the three component Kabachnik-Fields reaction for the synthesis of a library of urease inhibitors.The development of a simple synthetic method to obtain compounds with the designed scaffold was achieved.The proposed synthetic approach is convenient for further optimization of aminophosphinic inhibitors urease.
1 H, 31 P, and 13 C NMR spectra were recorded on a 200 MHz spectrometer.Proton and carbon chemical shifts are referenced to residual solvent. 31P chemical shifts are reported on δ scale (in ppm) downfield from 85% H3PO4.The following abbreviations were used to explain the NMR multiplicities: s ) singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet, br ) broad.ESI mass spectral analyses were performed on a mass spectrometer MSQ Surveyor, Finnigan using direct sample injection.Negative or positive ion ESI spectra were acquired by adjusting the needle and cone voltages accordingly.

General procedure for the synthesis of α-hydroxyalkane-H-phosphinic acids
Hypophosphorous acid (50% aq.) (10 mmol) was added to the solution of the aldehyde (12 mmol) in ethanol (4 mL) and 6M aqueous hydrochloric acid (12 mmol).The resulting mixture was refluxed for 48 h.The reaction mixture was concentrated in vacuo and the residue was purified with column chromatography using CH2Cl2-MeOH-AcOH: 9-1-0.5 as eluent in the case of 13-15 or used without further purification in the case of 16.

General procedure for the synthesis of secondary amines
To the solution of the aldehyde (10 mmol) in methanol (10 mL) the amine (13 mmol) was added.
After stirring at room temperature for 1 h, the solution was cooled to 0 o C prior to adding sodium borohydride (5 mmol) portionwise.The resulting solution was stirred at room temperature for 1 h and then evaporated under vacuum.After addition of water and Et2O, the organic phase was washed with NaHCO3, water, dried over Na2SO4 and concentrated under vacuum.Column chromatography purification, using EtOAc-MeOH: 9-1 as eluent, provided secondary amines as colorless oils.General procedure for the synthesis of α1-aminoalkane-α2-hydroxyalkanephosphinates 1-Hydroxyalkane-H-phosphinic acid (1 mmol) was dissolved in water (2 mL), followed by the addition of the corresponding secondary amine (1 mmol) and 6N HCl (0.5 mL).The solution was warmed to 90 o C, p-formaldehyde (2 mmol) was added, and the mixture was kept at 90 o C for additional 5 h.After removing the volatiles, the crude product was dissolved in water and purified using Dowex AG50x4 cation exchange resin with water as eluent.Fractions that gave slightly positive color reaction with ninhydrin and appropriate NMR spectral data were combined and evaporated under vacuum to give the phosphinates as hygroscopic solids.In certain cases an extra purification step was added using reverse phase silica gel and watermethanol (9.5-0.5) as eluent.N,N-Dimethylaminomethane(P-1-hydroxyethyl)phosphinic acid (3).Yield 77%. 1 H NMR (200 MHz, D2O) δ 0.93-1.05(2  d, 3H, J = 7.4 Hz, CH3), 2.64 (s, 6H, (CH3)2), 3.07 (d, J = 8.6 Hz 2H, CH2P), 3.57 (q, 1H, J = 7.3 Hz CH). 13

Table 1 .
Inhibitory activity (IC50 and Ki) of analyzed compounds against Bacillus pasteurii urease