Synthesis and biological evaluation of carbamates d erived from aminocombretastatin A-4 as vascular disrupting agents

A series of twenty-six carbamates derived from amin ocombretastatin A-4 (AmCA-4) were synthesized and evaluated for their capacity to aff ect cell proliferation, tubulin polymerization, mit otic cell arrest, microtubule network organization, apop tosis and endothelial tubular structures in vitro. The anti-proliferative activity of the synthetic carbam tes was measured on several human tumor cell lines (i.e. HT-29, MCF-7, HeLa, A-549, MDA-MB-231, HL-60) as well as on the endothelial cell line HMEC-1 and the non-tumor cell line HEK-293. The com pounds showed anti-proliferative activity in the nanomolar range thereby exceeding by far the ac tivity of combretastatin A-4 (CA-4) and, in some cases, the activity of AmCA-4. The most active comp unds proved to be the carbamates bearing chloro, bromo or methoxy groups in the meta position of the phenyl ring. Moreover, all carbama tes inhibited in vitro tubulin polymerization, in a sim lar manner to that of CA-4 and AmCA-4 by interacting with the colchicine binding site in tub ulin. The synthetic carbamates proved as active as AmCA-4 in causing mitotic arrest, as assessed in A5 49 human lung cancer cells, and disruption of the microtubule cellular network. Some selected carbama tes induced apoptosis at concentrations as low as 10 nM, being more active than AmCA-4. Finally, thes e elected carbamates displayed a vascular disrupting activity on endothelial cells in a dosedependent manner. In conclusion, our data indicate

The poor water solubility of CA-4 has prevented its use as an anticancer drug which explains the development of aminocombretastatin (AmCA-4), a non-natural combretastatin, and Ombrabuline, a serine-derivative from AmC4-4 (see figure 2). These compounds also show strong cytotoxicity, inhibition of tubulin polymerization and antivascular activity [8].
Several drugs exhibit a carbamate group in their structures as in rivastigmine (used for the treatment of dementia), retigabine (prescribed for the treatment of epilepsies), morizicine (used for the treatment of arrhythmias), zafirlukast (used for the treatment of asthma), and as in antiretroviral drugs used for the treatment of HIV/AIDS, like efavirenz, ritonavir, darunavir, fosamprenavir, atazanavir and cobicistat. Carbamate functionality confers chemical and proteolytic stability and a great capacity to pass through cell membranes which explains the increasing use of carbamates in medicinal chemistry [9]. The delocalization of nonbonded electrons of nitrogen over the carboxyl moiety causes a conformational rigidity. In addition, the NH and the carboxyl group exhibit excellent capacities for the formation of hydrogen bonds. Thus, substitution on the N-and O-termini of a carbamate allows to finetune the biological and pharmacokinetic properties of carbamate-bearing molecules.
In particular, the cis-stilbene structural fragment of CA-4 can be considered as a privileged motif with regard to VDA activity [10].

Research purpose
During the past few years we have been investigating a range of analogues of natural products for their potential value in anticancer therapy [11]. We have published several reports on the biological properties of combretastatin A-4 derivatives [12]. Therefore, on the basis of the aspects commented above and in continuation of our research on novel natural product analogues with potential utility in cancer therapy, we wanted to study whether carbamoylation of the amino group in aminocombretastatin A-4 improves its biological activity [13]. The synthesis of this new family of combretastatin-derived carbamates is described in scheme 1. The structures of the synthesized carbamates are indicated in figure 3.

Synthetic work
Aminocombretastatin was prepared by total synthesis following a published procedure [14]. For the synthesis of carbamates two procedures were applied. In the indicated Method a in scheme 1 carbamates were directly achieved upon reaction of AmCA-4 with commercially available aryl chloroformates in THF in the presence of pyridine [15]. The low commercial availability of aryl chloroformates allowed only the synthesis of three carbamates with the application of Method a (compounds 1, 4 and 7, see figure 3). The remaining carbamates were prepared by one-pot Method b in which AmCA-4 was converted into the corresponding trichloromethylcarbamate, upon reaction with triphosgene and Et3N, which in turn was transformed into carbamates by reaction with a range of phenols (see scheme 1) [16].   1). These latter compounds are mainly substituted in meta and para positions with halogen atoms or their bioisosters. The results achieved with the carbamates are shown in table 2 and are compared with those attained in the absence of any ligand (control) and in the presence of CA-4 or AmCA-4. All carbamates increased the CrC value relative to the value measured in the control (absence of ligand) with CrC values similar to those achieved in the presence of CA-4 or AmCA-4. Figure 4 shows the effects of some selected ligands on the in vitro tubulin polymerization process studied by turbidimetry time-course measurements at 350 nm and at 37ºC. In order to compare, paclitaxel, a microtubule stabilizer, was also evaluated.

Tubulin interaction at the colchicine binding site
In order to check whether the carbamates interact with tubulin in the colchicine-binding site the EBI assay was undertaken. This method is based on the property of N,N'-ethylene-bis(iodoacetamide) (EBI), a homobifunctional thioalkylating agent, to crosslink the Cys-239 and the Cys-354 residues present in the colchicine-binding site of β-tubulin. The covalent binding of EBI to β-tubulin forms an adduct that is easily detected by Western Blot as a second immunoreacting band of β-tubulin that migrates faster than the native β-tubulin band. As a consequence, treatment of the cells with a compound that binds to this colchicine-binding site will impair the binding of EBI, resulting in the absence of the second band [17]. The carbamates tested were selected according to their IC50   As shown in figure 5, there is a dose-response effect for all compounds tested. Concretely, compounds 10, 12 and 25 were able to inhibit the formation of EBI adduct at 3 µM, showing a behaviour similar to that of colchicine at this concentration. However, compounds 11 and 13 were less effective since they required higher concentrations (100 µM for 11 and 10 µM for 13) to avoid being displaced by EBI from tubulin. Thus, this experiment demonstrates that the compounds bind tubulin at the colchicine-binding site and that their antiproliferative capacity correlates with tubulin binding.

Mitotic arrest and inhibition of interphase microtubules of cultured
The effects of carbamates on cell cycle distribution were evaluated in A549 cells. Thus, cells were incubated for 20 h in the presence of CA-4, AmCA-4 and compounds 1-26 and then, DNA content was measured by flow cytometry (see experimental section). All the carbamates extensively arrested cells in the G2/M phase at a concentration half of their IC50 value. Next, we studied the effects of compounds 1-26 on the microtubule cytoskeleton. Therefore, A549 cells were incubated for 16 h in the presence of AmCA-4 and compounds 1-26 at concentrations twice their IC50 value. Figure 6 depicts some selected results. It can be appreciated that in the presence of AmCA-4 and carbamates, tubulin appears agregated and nuclei are compressed and fragmented, which is characteristic of cells that have been disturbed during their division process.

Induction of apoptosis
Cell cycle distribution ( Table 3) clearly shows that the percentage of subG0 cells is increased in cells treated with CA-4, AmCA-4 and carbamates. This exhibition of a sub-diploid DNA content, together with the high percentage of cells arrested in mitotic phase, could be characteristic of apoptosis.
Consequently, induction of apoptosis was studied by measuring the translocation of phosphatidylserine from the cytoplasmic to the extracellular side of the plasma membrane. Thus, A549 cells were incubated for 20 h in the presence of AmCA-4 and some representative carbamates, after which annexin content was measured by flow cytometry. Carbamates which displayed the best antiproliferative activity were selected for this assay.

Tube destruction
Vascular-disrupting agents do not only affect tumor cells but also endothelial cells. Thus, the capacity of synthetic carbamates to destroy a preexisting vasculature network formed by endothelial cells was evaluated. Therefore, cells were seeded on top of Matrigel, which induces the formation of a network of endothelial tubes. Then, the cultures were treated with different concentrations of AmCA-4 and carbamates and pictures were taken 4 h later in order to evaluate the tube destruction effect. selected for this assay. Additionally, two less active carbamates (11 and 13) were tested in order to establish a correlation between antiproliferative capacity and tube destruction. As shown in figure 8, all compounds tested displayed a vascular disrupting activity in a dose-dependent manner. Concretely, compounds 10, 12 and 25 exhibited this property at concentrations higher than 3 nM, improving the effect manifested by AmCA-4 and being about 10-fold more active than 13 and 100-fold more active than 11. Therefore, the higher the antiproliferative activity, the greater the capacity for tube destruction cycle distribution.

Summary and conclusions
The effect of introduction of a carbamate group in the aminocombretastatin structure was studied.
As shown in table 1, carbamate derivatives offer IC50 values in the nanomolar range, improving by far the activity of CA-4 and, in some cases, the activity of AmCA-4. For instance, derivatives 6 (m-Cl), 9 (m-Br), 10 (p-Br), 12 (m-OMe) exceeded the activity of AmCA-4 in HT-29, MCF-7 and HMEC-1 cell lines and also offered a good selectivity over non-tumor cell lines. From the structures of these compounds it can be concluded that the most active carbamates are the ones bearing chloro, bromo or methoxy groups in the meta position of the phenyl ring.
In addition, all the carbamates proved as potent as CA-4 and AmCA-4 in inhibiting in vitro tubulin polymerization. We also demonstrated that carbamates interact with tubulin at the colchicine-binding site and that the compounds with higher antiproliferative activity (compounds 10 (p-Br), 12 (m-OMe) and 25 (m-Me-p-Cl) also show greater tubulin binding capacity. From cell cycle analysis and immunofluorescence studies it can be deduced that carbamates cause a mitotic arrest in A549 cells causing nuclei fragmentation and tubulin aggregation. Moreover, the carbamates induced apoptosis in a dose-dependent manner, with more than 90% of apoptotic cells at 100 nM. Compounds 9 (m-Br) and 12 (m-OMe) proved as active as AmCA-4 even at a concentration of 10 nM.
Finally, some selected carbamates displayed a vascular disrupting activity of endothelial cells in a dose-dependent manner which correlated with their antiproliferative activity. Thus, highly antiproliferative carbamates 10 (p-Br), 12 (m-OMe) and 25 (m-Me-p-Cl) were able to disrupt tubular network at concentrations higher than 3 nM, improving the effect shown by AmCA-4.

General procedures
NMR spectra were measured at 25°C. The signals of the deuterated solvent (CDCl3) were taken as the reference. Multiplicity assignments of 13 C signals were made by means of the DEPT pulse sequence. Complete signal assignments in 1 H and 13 C NMR spectra were made with the aid of 2D homo-and heteronuclear pulse sequences (COSY, HSQC, HMBC). High resolution mass spectra were run by the electrospray mode (ESMS). IR data were measured with oily films on NaCl plates (oils) and are given only for relevant functional groups (C=O, NH). Experiments which required an inert atmosphere were carried out under dry N2 in flame-dried glassware. Commercially available reagents were used as received.

Experimental procedure for the synthesis of carbamates by method a.
A solution of AmCA-4 (0.7 mmol) in THF (5 mL/mmol) was cooled at 0ºC and anhydrous pyridine (1.7 mmol) and the corresponding phenyl chloroformate (1.0 mmol) were added under inert atmosphere. The resulting mixture was stirred in the dark for 20 min at 0ºC and for 1 h at rt. After this time, H2O (3.4 mL) and HCl 1M (1.7 mL) were added to the reaction mixture, which was then extracted with CH2Cl2 (3 x 20 mL). The organic layer was washed with brine, and then dried on anhydrous Na2SO4. Removal of volatiles under reduced pressure afforded an oily residue which was subjected to column chromatography on silica-gel (Hexanes-EtOAc mixtures as eluant) obtaining the desired products with the yields indicated below. (1) ( ( 9, 149.1, 146.9, 137.2, 132.6, 130.8, 130.3, 126

3-Chloro-4-methylphenyl (Z)-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl)carbamate
In the case of MDA-MB-231 cells, 1 × 10 4 cells per well were seeded in 48-well plates in 1 mL of growth medium. One day later, 5-fold dilutions of the compounds were added. After 3 days of incubation, cells were trypsinized and counted in a Coulter counter (Rega Institute for Medical Research, KU Leuven). The IC50 value was determined as the compound concentration required to reduce cell proliferation by 50%.

Tubulin self-assembly assay
Purified tubulin was used for these measurements. Tubulin polymerization was carried out in a 96 well-plate. In each well 50 µL of a solution of 25 µM of tubulin in GAB buffer was added to 50 µL of 27.5 µM solution of the corresponding compounds in GAB buffer (20 mM sodium phosphate, 10 mM MgCl2, 1 mM EGTA, 30% glycerol) and 0.1 mM GTP at pH = 6.5. Then, the plate was incubated at 37ºC in Multiskan® and absorbance at 340 nM was registered every 30 seconds during 2 hours.
After 1.5 h, cells were harvested and cell extracts were prepared for Western Blot analysis. Thirty μg of proteins were subjected to gel electrophoresis using 0.1% SDS (85% purity) and 10% polyacrylamide gels. After electrophoresis, proteins were transferred to pretreated Hybond-P polyvinylidene difluoride (PVDF) membranes, which were incubated overnight at 4ºC in blocking buffer (2.5% non-fat dry milk in PBS containing 0.1% Tween) and subsequently for 1 h at rt in blocking buffer primary antibody raised against β-tubulin. After washing, membranes were incubated with the corresponding HRPconjugated secondary antibody in blocking buffer for 30 min at rt. Next, membranes were washed extensively and immunoreactive proteins were detected by chemiluminiscence (ECLplus, Bio-Rad).

Cell cycle analysis
Progression of the cell cycle was analysed by means of flow cytometry with propidium iodide. After incubation with compounds for 24 h, A549 cells were fixed, treated with RNase and stained with propidium iodide following instructions of BD Cycletest TM DNA Kit. Analysis was performed with a BD Accuri TM C6 flow cytometer.

Immunofluorescence assay
Immunofluorescent analysis of the microtubule network was performed on the A-549 cell line. In this assay, 1,5x10 5 cells were plated on a coverglass and incubated with the different concentrations of selected compounds for 16 h. Cells were then washed with PEMP, permeabilized with PEM-Triton X-100 0.5% for 90 seconds at room temperature and fixed in 3.7% formaldehyde (in PEM pH 7.4) for 30 min at rt. Direct immunostaining was carried out for 2.5 h at 37°C in darkness with primary FITCconjugated anti-α-tubulin antibody (dilution 1:400 in PBS-BSA 1% from a 1 mg/mL solution; monoclonal antibody, clone DM1A, Sigma-Aldrich). Next, cells were washed with PBS and incubated for 30 min at room temperature in darkness with Hoechst 2 mM in water. Then, cells were washed in PBS and coverglasses were mounted with 10 μL of Glycine/Glycerol buffer. The cytoskeleton was imaged by a confocal laser scanning microscope (CLSM) Leica SP5 with a Leica inverted microscope, equipped with a Plan-Apochromat 63× oil immersion objective (NA=1.4). Each image was recorded with the CLSM's spectral mode selecting specific domains of the emission spectrum. The FITC fluorophore was excited at 488 nm with an argon laser and its fluorescence emission was collected between 496 nm and 535 nm.

Apoptosis assay
Apoptosis was determined by quantifying FITC-Annexin V translocation by means of flow cytometry. A549 cells were incubated with compounds for 24 h and then stained following instructions of BD Apoptosis Detection TM Kit. Analysis was performed with a BD Accuri TM C6 flow cytometer.

Tube destruction assay
Wells of a 96-well μ-plate for angiogenesis were coated with 12 μL of Matrigel (10 mg/mL, BD Biosciences) at 4ºC. After gelatinization at 37ºC for 30 min, HMEC-1 cells were seeded at 2 x 10 4 cells/well in 35 μL of culture medium on top of the Matrigel. After 20 h of incubation at 37ºC, when tube-like structures were detectable, compounds were added at different concentrations. Next, 4 h later, tube destruction was evaluated by giving a score from 0 to 3 (3: intact tubular network as seen in the control, 2: missing connections and/or dead ends; 1: many separate small tubes that are not connected; 0: no tubes).

Proteins and ligands
Calf brain tubulin was purified as described previously [19].