Sydnone Cycloaddition Route to Pyrazole-Based Analogs of Combretastatin A4

Abstract

The combretastatins are an important class of tubulin-binding agents. Of this family, a number of compounds are potent tumor Vascular Disrupting Agents (VDAs) and have shown promise in the clinic for cancer therapy. We have developed a modular synthetic route to combretastatin analogs based on a pyrazole core through highly-regioselective alkyne cycloaddition reactions of sydnones. These compounds show modest to high potency against human umbilical vein endothelial cell proliferation. Moreover, evidence is presented that these novel VDAs have the same mode of action as CA4P and bind reversibly to -tubulinbelieved to be a key feature in avoiding toxicity. The most active compound from in vitro studies was taken forward to an in vivo model and instigated an increase in tumor cell necrosis.

Evaluation of Sydnone‐Based Analogues of Combretastatin A‐4 Phosphate (CA4P) as Vascular Disrupting Agents for Use in Cancer Therapy

“…Instead, VDA effects on the microtubule cytoskeleton of endothelial cells (the primary target of VDAs) and subsequent changes in cell morphology are thought to be integral to their therapeutic efficacy . Indeed, recently, we have shown that assays using endothelial cells provided a good insight into the activity CA4 analogues that translated into in vivo efficacy …”

“…All compounds submitted for biological testing were recrystallised to consistent melting point and judged as >95 % pure by 1 H and 13 C NMR spectroscopy unless otherwise specified. Sydnones were prepared by standard methods (cyclodehydration of N ‐nitroso amino acid with trifluoroacetic anhydride) …”

“…4‐(3,4,5‐Trimethoxyphenyl)‐ N ‐(3‐hydroxy‐4‐methoxyphenyl)sydnone (11) : N ‐(3‐Hydroxy‐4‐methoxyphenyl)sydnone (473 mg, 2.27 mmol) and 5‐bromo‐1,2,3‐trimethoxybenzene (842 mg, 3.41 mmol) were subjected to the general conditions affording 11 as a colourless solid (402 mg, 47 %); mp: 196–197 °C (Lit . 196–197 °C); 1 H NMR (400 MHz, CDCl 3 ): δ =3.67 (6 H, s), 3.83 (3 H, s), 4.00 (3 H, s), 5.94 (1 H, s), 6.59 (2 H, s), 6.92–7.05 (2 H, m), 7.09 ppm (1 H, s); 13 C NMR (101 MHz, CDCl 3 ): δ =56.1, 56.5, 61.0, 104.7, 108.0, 111.0, 111.7, 117.2, 119.9, 127.8, 138.5, 147.0, 149.5, 153.3, 167.2 ppm; FTIR: max 3295 (br), 3082 (w), 2940 (w), 2836 (w), 1710 (s), 1600 (w), 1581 (m), 1509 (s), 1229 (s), 1125 (s) 1014 (m), 998 cm −1 (m); HRMS calculated for C 18 H 19 N 2 O 7 (ES + ) [ M +H] + : 375.1192, found: 375.1181.…”

“…Endothelial cell culture : Human Umbilical Vein Endothelial Cells (HUVECs) from pooled donors were purchased from Promocell GmbH (Promocell, C‐12203) and cultured as described before . Briefly, cells were maintained for up to a maximum of six passages in endothelial cell basal medium (EBM) (Promocell, C‐22010) supplemented with Endothelial Cell Growth Supplement Mix (Promocell, C‐39215) and an extra 8 % ( v / v ) low‐endotoxin foetal calf serum (Invitrogen).…”

“…Immunofluorescence staining of the endothelial cytoskeleton : Changes in cell morphology and the cytoskeleton were visualised by dual staining of microtubules and actin filaments as we described previously . Briefly, HUVECs were plated in Permanox Lab‐Tek chamber slides (Life Technologies) coated with 5 μg mL −1 fibronectin and were allowed to reach confluence before they were exposed to drug or vehicle for 30 min.…”