Synthesis and biological screening of diethyl [ N -(thiazol-2-yl)carbamoyl]methylphosphonates

A three-step synthesis, involving condensation of bromomethyl aryl ketones with urea to afford 2-aminothiazoles, their chloroacetylation and subsequent solvent-free Arbuzov phosphonation has afforded a series of novel diethyl [ N -(thiazol-2-yl)carbamoyl]methylphosphonates 3a - 3f in good overall yields; the 4-carboxythiazole analogue 3g was obtained by selective hydrolysis of the corresponding ethyl ester 3f . The phosphonate esters exhibited significant anti-cancer activity (nM - low µM IC 50 values) against SH-SY5Y cells and, in one case, 7.6 µM MIC90 anti-TB activity against the virulent M. tuberculosis H 37 Rv strain; the chloroacetamido precursors all exhibited some antimalarial ( Pf LDH) activity, three with IC 50 values in the range 1.0 - 8.9 µΜ.

The thiazole scaffold is well represented in medicinal systems.Mjambili et al. have prepared a library of Naryl substituted 2-(pyridin-2-yl)thiazol-4-amines and explored their anti-TB and antimalarial activity, and compounds containing electronegative para substituents were shown to inhibit P. falciparum with submicromolar IC 50 values 11 .The anti-cancer potential of certain thiazole-based compounds has been investigated, the most active of which, ethyl 2- [3-(diethylamino)propanamido]thiazole-4-carboxylate, exhibited a GI 50 value of 0.08 μM against the RPMI-8226 leukemia cell line and broad-spectrum activity against 60 tumour cell lines with a GI 50 value of 38.3 µΜ 12,13 .Jaishree et al. showed that 2-methyl-4trifluoromethylthiazole-5-carboxamides exhibited promising anti-parasitic and insecticidal properties, but without herbicidal effect. 14n this communication, we discuss: (i) the synthesis of a series of [N-(thiazol-2yl)carbamoylmethylphosphonate esters 3 as fosmidomycin analogues which satisfy the Lipinski "Rule of Five"; and (ii) screening of these compounds and their synthetic precursors for antimalarial, anti-cancer and antituberculosis activity.

Result and Discussion
The aminothiazole scaffolds 6a-e were obtained using conventional Hantzsch condensation of the α-bromo ketones 4a-f with thiourea (Scheme 1).On completion of each of the reactions, the desired product was precipitated out by pouring the reaction mixture into ice-cold water.The known thiazole derivatives 3a-f [15][16][17] were thus isolated in excellent yields (93-100%; Table 1) and subsequently treated with chloroacetyl chloride in the presence of triethylamine in dichloromethane, using a modification of the method reported by Xu et al. 18 to afford the 2-(2-chloroacetamido)thiazole analogues 7a-f in yields ranging from 54 to 100%.Solventfree Arbuzov phosphonation of the 2-(2-chloroacetamido)thiazoles 7a-f was effected by boiling with triethyl phosphite at 110 ⁰C for 9 h.Excess triethyl phosphite was removed by stirring the crude products with hexane.Column chromatography afforded the desired phophonate esters 3a-e in 68-98% yield, while treatment of the carbethoxy analogue 3f with methanolic potassium hydroxide, followed by acidification, permitted selective hydrolysis of the carboxlic ester moiety to give diethyl [(4-carboxythiazol-2-yl)carbamoyl]methylphosphonate 3g in 59% yield.The phosphonate esters 3a-g are all new and were fully characterised using 1-and 2-D NMR, IR and HRMS methods.
Reagents and conditions: i) EtOH, 70 ᵒ C, 1 h; ii) Chloroacetyl chloride, 0 ᵒ C -rt, 2 h; iii) (EtO) 3 P, 110 ᵒ C, 9h; and iv) KOH, MeOH, rt, 2h, then 20% HCl.It is apparent from the data summarised in Table 2 that, at low concentrations, the phosphonate esters 3ac,e,g exhibit encouragingly effective inhibition of the SH-SY5Y cell line with IC 50 values in the nanomolar to very low micromolar range but little, if any, activity against the HeLa cell line 19,20 (≥ 95% viability at 20 μM), thus reflecting clear selectivity against the former cell line.Compounds 7a-f, however, exhibit 15-40% inhibition of the HeLa cells at 20 μM.The resazurin-based whole-cell PfLDH bioassay was conducted to explore the antimalarial activities of the crucial intermediates and final compounds using 20 µM as the cut-off concentration before determining IC 50 values for compounds with significant levels of inhibition.The results (Table 1) show that the unsubstituted aminothiazole intermediates 6a-f exhibit low levels of inhibition (66-88% PfLDH viability), whereas the 2-(2chloroacetamido)thiazole intermediates 7a-e exhibit significant activity at 20 μM, with IC 50 values of 8.87, 1.86 and 1.04 μM for compounds 7d, 7a and 7e , respectively.The phosphonate esters 3a-g, which were designed Apart from the para-bromophenyl product 3c, which exhibited an MIC90 value of 7.62 µM, all other compounds in this series (3) showed little if any inhibitory effect (MIC90 and MIC99 values > 20 µM) on the growth of M. tuberculosis H 37 Rv 19 .The relatively high predicted Log P value of 3.87 for compound 3c may contribute to its absorption across the lipophilic membrane of M. tuberculosis H 37 Rv, whereas the Log P values for the other compounds 3a, 3b, 3d, 3e, 3f and 3g (2.98, 3.62, 3.18, 1.54 and 1.10, respectively) are all lower.

Conclusions
The novel diethyl [N-(thiazol-2-yl)carbamoyl]methylphosphonates (3) were successfully obtained in good overall yields.Although designed as potential antimalarial agents, these compounds failed to exhibit any activity against PfLDH, whereas their chloroacetamido precursors (7) all exhibited antimalarial (PfLDH) activity, three with IC 50 values in the range 1.0 -8.9 μM.The title compounds did, however, exhibit significant and selective anti-cancer activity (nM -low μM IC 50 values) against SH-SY5Y cells and, in one case, 7.6 μM MIC90 anti-TB activity against the virulent M. tuberculosis H 37 Rv strain.

Experimental Section
General.Reagents were supplied by Sigma-Aldrich and used without further purification.Tetrahydrofuran (THF) and methylene chloride were stored over 4 Å molecular sieves.The reaction progress and purity of the compounds were checked by thin layer chromatography (TLC) on pre-coated Merck ® silica gel G60 F 254 plates, and viewed under UV light at 254 and 365 nm.Melting points were recorded, uncorrected, using a Reichert hot-plate microscope.Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance II 600 MHz, Bruker Avance III HD 400 MHz and Bruker Fourier 300 MHz spectrometers.The NMR chemical shifts are reported in ppm downfield from tetramethylsilane (TMS), and the coupling constants are given in Hertz (Hz).NMR analyses were carried out in deuterated solvents, such as DMSO-d 6 , CDCl 3 , acetone-d 6 and methanol-d 4 for standard NMR experiments, and the spectra were calibrated using solvent signals [δ H : 7.26 ppm for residual CHCl 3 , 2.50 ppm for residual DMSO, 2.05 ppm for residual acetone and 3.31 for residual MeOH; δ C : 77.2 ppm (CDCl 3 ), 39.5 ppm (DMSO-d 6 ), 29.8 ppm (acetone-d 6 ) and 49.0 ppm (MeOH-d 4 )].Infrared (IR) spectra were obtained using a Perkin Elmer (R) Spectrum 400 Frontier / FT-IR spectrometer.High-resolution mass spectra (HRMS) were recorded on a Waters API Q-TOF Ultima spectrometer (University of Stellenbosch, Stellenbosch, South Africa).NMR spectra for all compounds and the bioassay procedures are provided in the Supporting Information.
The general procedure for the preparation of the known 2-(2-chloroacetamido)thiazoles (7) involved a modification of the procedure reported by Xu and collegues 18 .A solution of 2-amino-4-phenylthiazole 6a (0.530 g, 3 mmol) and Et 3 N (560 µL, 4 mmol) in dichloromethane (15 mL) was cooled to 0-5 ⁰C in an ice-bath and stirred for 30 min.2-Chloroacetyl chloride (578 µL, 6.6 mmol) in dry dichloromethane (1.5 mL) was then added slowly, and the reaction mixture was allowed to warm to room temperature and stirred until the amine was completely consumed (ca. 1 h, as monitored by TLC).The reaction mixture was diluted with dichloromethane and washed successively with water and saturated brine.The organic layer was dried over anhydrous Na 2 SO 4 , the solvent was removed under reduced pressure and the residue was recrystallised from ethanol to give compound 7a (0.413 g, 54%) as light-grey crystals, mp 170-171 o C (Lit. 18,20,26171-173 o C).The remaining analogues were obtained similarly [7b (100%) as a brown solid, mp 194-195 o C (Lit. 27mp not cited); 7c (64%) as a brown solid, mp 241-243 o C (Lit. 27

Table 2 .
Bioassay results for compounds 6a