Synthesis of the β-Lactamase Indicators Cefesone and Nitrocefin

Abstract

The synthesis of the b-lactamase indicators Cefesone [(6R,7R)-3-(2,4-dinitrostyryl)-7-(phenylacetamido)ceph-3-em-4-carboxylic acid] and Nitrocefin [(6R,7R)-3-(2,4-dinitrostyryl)-7-(2thienylacetamido)ceph-3-em-4-carboxylic acid] from tert-butyl (1S,6R,7R)-3-bromomethyl-1-oxo-7-(phenylacetamido)ceph-3-em-4carboxylate is reported. Phosphonylation of the latter compound with triphenylphosphine gave the corresponding phosphonium derivative in 93% yield. Reduction followed by Wittig coupling with 2,4-dinitrobenzaldehyde gave a 1:12 mixture of E-and Z-isomers in 83% yield. The tert-butyl protecting group was removed with titanium tetrachloride to give Cefesone as the pure crystalline E-isomer in 63% yield. De-acylation was achieved by enzymatic hydrolysis in 77% yield. Finally the 2-thienylacetyl side chain was introduced to give crystalline Nitrocefin in 67% yield. Downloaded by: University of Pittsburgh. Copyrighted material. 1665 (CO, amide), 1525 cm -1 (NO 2 ). MS (DCI): m/z = 584.2 (MNH 4 + ). 1 H NMR (CDCl 3 ; 360 MHz): d = 1.53 (s, 9H, tert-butyl), 2.75 (d, 1H, J = 18.2 Hz, H 2 ), 3.25 (d, 1H, J = 18.2 Hz, H 2 ), 3.63 (ABq, 2H, J = 15.9 Hz, ArCH 2 ), 4.91 (d, 1H, J = 4.9 Hz, H 6 ), 5.84 (dd, 1H, J 6.7 = 4.9 Hz, J 7,NH = 9.1 Hz, H 7 ), 6.02 (d, 1H, J = 9.1 Hz, NH), 6.84 (ABq, 2H, J = 11.9 Hz, CH=CH), 7.3 (m, 5H, ArH), 7.57 (d, 1H, J = 8.6 Hz, ArH), 8.88 (d, 1H, J = 2.3 Hz, ArH).(6R,7R)-3-(2,4-Dinitrostyryl)-7-(phenylacetamido)ceph-3-em-4carboxylic Acid, E-Isomer (4): A stirred solution of 3 (67.41 g, purity 8% E-isomer, 81% Z-isomer, 105.9 mmol) in CH 2 Cl 2 (1685 mL) was cooled to -25 °C. TiCl 4 (53 mL, 482 mmol) was added in 10 min and the temperature was brought to 0°C. After 1.75 h, chilled 2 M HCl (1685 mL) was added at such a rate that the temperature remained under 10 °C. The organic phase was separated and extracted with 2 M HCI (2 ´1685 mL), H 2 O (1685 mL) and brine (1685 mL). The organic phase was concentrated under reduced pressure to give an orange foam. Crude 4 thus obtained was crystallized by dissolving in acetone (1350 mL) at 65 °C and adding H 2 O (675 mL). Crystallization was allowed to proceed for 16 h at 0°C and the crystals were collected by filtration. Recrystallization of the product was performed by dissolving the material in acetone/ HOAc (2:1) at 53 °C, removing the solvent (1700 mL) by evaporation under reduced pressure, and stirring for 16 h at 20 °C. Crystals were collected by filtration, washed with HOAc (300 mL) and Et 2 O (250 mL), and dried under vacuum at 45 °C to give 34.33 g (purity 99% E-isomer, 66.6 mmol; 63%) of 4 as yellow crystals. IR (KBr): n = 3300 (NH), 1780 (CO, b-lactam), 1715 (CO, amide), 1625 (CO, carboxylic acid), 1525 cm -1 (NO 2 ). MS (DCI): m/z = 528.0 (MNH 4 + ). 1 H NMR (CDCl 3 /DMSO-d 6, 1:2; 360 MHz): d = 3.50/3.58 (ABq, 2H, J = 14.1 Hz, ArCH 2 ), 3.62/3.77 (ABq, 2H, J = 17.5 Hz, H 2 ), 5.05 (d, 1H, J = 4.9 Hz, H 6 ), 5.72 (dd, 1H, J 6,7 = 4.9 Hz, J 7,NH = 8.3 Hz, H 7 ), 7.3 (m, 6H, ArH + CH=C), 7.63 (d, 1H, J = 16.1 Hz, C=CH), 7.82 (d, 1H, J = 8.8 Hz, ArH),...

A Practical Synthesis of Nitrocefin

“…The NMR spectra were identical to the reported values and to those of the authentic commercial samples. 4 The E and Z mixture of 4 before isomerization: 1 H NMR (500 MHz, 10% DMSO-d6 in CDCl3) δ 2.75, 3.13 (2d, 2H, J ) 18.3 Hz, Z-H2), 3.50 (1d, 1H, J ) 17.3 Hz, E-H2), 3.60-3.69 (m, 5H, E and Z-thiophene-CH2 and H2), 4.80 (d, 1H, J ) 4.5 Hz, Z-H6), 4.91 (d, 1H, J ) 4.9 Hz, E-H6), 5.62 (dd, 1H, J ) 4.9 Hz, J ) 8.9 Hz, Z-H7), 5.72 (dd, 1H, J ) 4.9 Hz, J ) 8.9 Hz, E-H7), 6.70…”

“…The second report also follows the same type of strategy (oxidation of the cephem sulfur and the requisite reduction at the end of the synthesis). 4 However, they had to incorporate an additional enzymic step for the removal of the C 7 -acyl moiety and the subsequent acylation by the 2-thienylacetyl group present in the structure of nitrocefin. Both synthetic strategies are multistep and low-yielding.…”

“…The yield was as good as typical Wittig reactions with cephalosporin derivatives (aqueous NaOH or NaHCO 3 in methylene chloride). 3,4,[8][9][10] Deprotection of the p-methoxybenzyl group of compound 10 was achieved by treatment with trifluoroacetic acid at icewater temperature for 15 min, resulting in nitrocefin as a mixture of Z and E isomers (the ratio became 6:5). The Z to E isomerization for some cephalosporin derivatives has been reported in the literature during the acidpromoted deprotection of 4-carboxylates with TFA 8 or with Lewis acid (TiCl 4 , SnCl 4 , etc) 4,11 A more prolonged treatment with TFA resulted in an increased ratio of E/Z but was accompanied by the decomposition of the desired product.…”

Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as β-lactamase substrates

“…The acid products were obtained as Z/E mixtures after TFA induced cleavage of either the benzhydryl ester (in the case of UW57 and UW58) or of the p-methoxybenzyl ester (in the case of UW154) groups. The Ghavami et al Assay for drug discovery 5 compounds could be isomerized slowly and completely in a solution of 10% DMSO in chloroform to the pure E stereoisomer as described in the synthesis of nitrocefin by the Mobashery group [13]. We discovered, however, that the Z to E isomerization occurred cleanly, completely and almost instantaneously if the sample of the acid was dissolved in neat DMSO or DMSO-d 6 , as indicated by proton NMR analysis.…”

“…A number of groups, including ours, have reported other chromogenic (CENTA [4], Cefesone (S1) [5], HMRZ-86 [6], PADAC [7], CLS405 [8], Chromacef [9]), and fluorogenic substrates, Ghavami et al Assay for drug discovery 4 (CA [10]; FC-3 to FC-5 [11]), and, recently, a near-infrared fluorescent probe [12]); some of which have advantages over nitrocefin in certain applications.…”