Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.     

Synthesis and biological evaluation of 4'-(6,7-disubstituted-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol as potent Chk1 inhibitors

A new series of potent tricyclic pyrazole-based Chk1 inhibitors are described. Analogues disubstituted on the 6- and 7-positions show improved Chk1 inhibition potency compared with analogues with a single substituent on either the 6- or 7-position. Based on the lead compound 4'-(6,7-dimethoxy-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol (2), detailed SAR studies on the 6- and 7-positions were performed. 3'-morpholin-4'-yl-propoxy, pyridin-4'-ylmethoxy, pyridin-3'-ylmethoxy, 2'-(5'-ethyl-pyridin-2'-yl)-ethoxy, pyridin-2'-ylethoxy, (6'-methyl-pyridin-2'-yl)-propoxyethoxy, 2',3'-dihydroxyl-1'-yl-propoxy, and tetrahydro-furan-3'-yloxy have been identified as the best groups on the 6-position when the 7-position is substituted with methoxyl group. Pyridin-2'-ylmethoxy and pyridin-3'-ylmethoxy have been identified as the best substituents at the 7-position while the 6-position bearing methoxyl group. These compounds significantly potentiate the cytotoxicity of DNA-damaging antitumor agents in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and the camptothecin-induced S checkpoints, suggesting that their potent biological activities are mechanism-based through Chk1 inhibition.     

Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles as Rigid Analogues of Combretastatin A-4 with Potent Antiproliferative Activity

A series of novel 3-alkyl-1,5-diaryl-1H-pyrazoles were synthesized as combretastatin A-4 (CA-4) analogues and evaluated for antiproliferative activity against three human cancer cell lines (SGC-7901, A549 and HT-1080). Most of the target compounds displayed moderate to potent antiproliferative activity, and 7k was found to be the most potent compound. Structure-activity relationships indicated that compounds with a trimethoxyphenyl A-ring at the N-1 position of the pyrazole skeleton were more potent than those with the A-ring at the C-5 position. Tubulin polymerization and immunostaining experiments revealed that 7k potently inhibited tubulin polymerization and disrupted tubulin microtubule dynamics in a manner similar to CA-4. Computational modelling demonstrated that the binding of 7k to the colchicine binding site on microtubules may involve a similar mode as CA-4.     

Synthesis and preliminary biological evaluation of new anti-tubulin agents containing different benzoheterocycles

A new series of compounds, in which the 2-amino-4-methoxyphenyl ring of phenstatin analogue 5 was replaced with 2- or 3-amino-benzoheterocycles, was synthesized and evaluated for antiproliferative activity and inhibition of colchicine binding. The lack of activity of 3',4'-dimethoxy- and 4'-methoxy-benzoyl derivatives (8 and 9, respectively) indicates that the 3',4',5'-trimethoxybenzoyl moiety is critical for the activity. Two compounds, 7 and 11, displayed potent antiproliferative activity, with IC50 values ranging from 25 to 100 nM against a variety of cancer cell lines. Derivative 11 was more active than CA-4 as an inhibitor of tubulin polymerization. The results demonstrated that the antiproliferative activity was correlated with inhibition of tubulin polymerization.     

Identification of diaryl 5-amino-1,2,4-oxadiazoles as tubulin inhibitors: The special case of 3-(2-fluorophenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole

The combination of experimental (inhibition of colchicine binding) and computational (COMPARE, docking studies) data unequivocally identified diaryl 5-amino-1,2,4-oxadiazoles as potent tubulin inhibitors. Good correlation was observed between tubulin binding and cytostatic properties for all tested compounds with the notable exception of the lead candidate, 3-(3-methoxyphenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole (DCP 10500078). This compound was found to be substantially more active in our in vitro experiments than the monofluorinated title compound, 3-(2-fluorophenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole (DCP 10500067/NSC 757486), which in turn demonstrated slightly better tubulin binding activity. Comparative SAR analysis of 25 diaryl 5-amino-1,2,4-oxadiazoles with other known tubulin inhibitors, such as combretastatin A-4 (CA-4) and colchicine, provides further insight into the specifics of their binding as well as a plausible mechanism of action.     

Synthesis and biological evaluation of enantiomerically pure cyclopropyl analogues of combretastatin A4

To evaluate the influence of stereochemistry on biological activities of cis-cyclopropyl combretastatin A4 (CA4) analogues, we have prepared several cyclopropyl compounds in their pure enantiomeric forms. The key reactions in our synthesis are the cyclopropanation of a (Z)-alkenylboron compound bearing a chiral auxiliary, and the cross-coupling of both enantiomeric cyclopropyl trifluoroborate salts with aryl and olefinic halides. Three pairs of cis-cyclopropyl CA4 analogues were evaluated for their potential antivascular activities. The diarylcyclopropyl compounds with SR-configuration (?)-1b, (?)-2b and the cyclopropylvinyl enantiomer (+)-3a with RR-configuration were the most potent tubulin polymerization inhibitors. A correlation was noted between anti-tubulin activity and rounding up activity of endothelial cells. The cytotoxic activity on B16 melanoma cells was in the submicromolar range for most compounds, but unlike the anti-tubulin activity, there was no difference in cytotoxic activity between racemic and enantiomerically pure forms for the three series of compounds. Molecular docking studies within the colchicine binding site of tubulin were in good agreement with the tubulin polymerization inhibitory data and confirmed the importance of the configuration of the synthesized cis-cyclopropyl CA4 analogues for potential antivascular activities.     

Substitution at the 8-position of 3'-deoxy-cyclic ADP-carbocyclic-ribose, a highly potent Ca2+-mobilizing agent, provides partial agonists

We previously showed that 3'-deoxy-cyclic ADP-carbocyclic-ribose (3'-deoxy-cADPcR, 4) is a stable and highly potent analogue of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. From these results, we designed and synthesized other 3'-modified analogues of cADPcR having a substituent at the 8-position and found that this modification at the 8-position made them partial agonists. Among these compounds, 8-NH(2)-3'-deoxy-cADPcR (10) was identified as a potent partial agonist with an EC(50) value of 17 nM.     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Carbonylbisphosphonate and (diazomethylene)bisphosphonate analogues of AZT 5(')-diphosphate

A novel nucleotide analogue is described, in which the alpha,beta-phosphoric anhydride oxygen of a nucleoside 5(')-diphosphate is replaced by a carbonyl group: the carbonylbisphosphonate analogue 5 of 2('),3(')-dideoxy-3(')-azidothymidine 5(')-diphosphate (AZT 5(')-diphosphate). 5 was synthesized from tetramethyl (diazomethylene)bisphosphonate 1 via the trimethyl ester 4 of the corresponding AZT 5(')-(diazomethylene)bisphosphonate 6, which is also a new type of nucleotide analogue. The ultimate product 5 was isolated by reverse-phase HPLC, and characterized by 31P, 13C, and 1H NMR; and by high-resolution mass spectrometry. The ketone group of 5 is a visible chromophore (yellow) and reversibly forms a colorless hydrate. The ketone hydrate 'pK' is about 4.2 when excess of magnesium ion is present. The potential of such analogues as novel inhibitors of enzymes mediating nucleotide-dependent biochemical processes is discussed.     

Synthesis and evaluation of diaryl sulfides and diaryl selenide compounds for antitubulin and cytotoxic activity

We have devised a procedure for the synthesis of analogs of combretastatin A-4 (CA-4) containing sulfur and selenium atoms as spacer groups between the aromatic rings. CA-4 is well known for its potent activity as an inhibitor of tubulin polymerization, and its prodrugs combretastatin A-4 phosphate (CA-4P) and combretastatin A-1 phosphate (CA-1P) are being investigated as antitumor agents that cause tumor vascular collapse in addition to their activity as cytotoxic compounds. Here we report the preparation of two sulfur analogs and one selenium analog of CA-4. All synthesized compounds, as well as several synthetic intermediates, were evaluated for inhibition of tubulin polymerization and for cytotoxic activity in human cancer cells. Compounds 3 and 4 were active at nM concentration against MCF-7 breast cancer cells. As inhibitors of tubulin polymerization, both 3 and 4 were more active than CA-4 itself. In addition, 4 was the most active of these agents against 786, HT-29 and PC-3 cancer cells. Molecular modeling binding studies are also reported for compounds 1, 3, 4 and CA-4 to tubulin within the colchicine site.     

Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. Part 1: Structure-activity relationships of the 1- and 3-positions

1-Benzoyl-3-cyanopyrrolo[1,2-a]quinoline (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a had good activity against several breast cancer cell lines but was much less active against several other cancer cell lines. SAR studies of 2a found that substitution at the 4-position of the 1-benzoyl group was important for activity. Replacing the 3-cyano group by an ester or ketone group led to inactive compounds. Interestingly, 4-substituted analogs such as 1-(4-(1H-imidazol-1-yl)benzoyl)-3-cyanopyrrolo[1,2-a]quinoline (2k) were found to be broadly and highly active in the caspase activation assay as well as in the cell growth inhibition assay with low nM EC(50) and GI(50) values in human breast cancer cells T47D, human colon cancer cells HCT116, and hepatocellular carcinoma cancer cells SNU398. Compound 2a was found not to inhibit tubulin polymerization up to 50 microM, while 2k was found to inhibit tubulin polymerization with an IC(50) value of 5 microM, indicating that certain substituents at the 4-position of the 1-benzoyl group can change the mechanism of action.     

Synthesis and anticonvulsant activity of new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones

Thirteen new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones were synthesized and their pharmacological activity determined with the objective to better understand their SAR for anticonvulsant activity. The anticonvulsant effects of these compounds were evaluated by standard pentylenetetrazol (scPTZ test) and maximum electroshock seizure (MES test) models in mice. Most of the compounds showed ability to protect against the pentylenetetrazol-induced convulsions. Compound 3o (the N-1'-p-nitrophenyl, N-3'-ethyl derivative) in the N-1'-aryl, N-3'-alkyl disubstituted series exhibited maximum activity with ED(50) of 41.8 mg/kg in scPTZ convulsion model.     

Synthesis and molecular docking of N′-arylidene-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbohydrazides as novel hypoglycemic and antioxidant dual agents

Herein, the design and synthesis of 10 novel N′-arylidene pyrazole-3-carbohydrazides are described. Compounds were pretended to act as dual agents against diabetes and oxidative stress, two correlated pathologies involved in metabolic syndrome development and progression. The antioxidant capacity was evaluated by means of DPPH and FRAP in vitro assays. It was found that compounds bearing a hydroxyl group at 4-position of the hydrazone moiety are potent antioxidant entities, being compound 3g (a syringaldehyde derivative) the most active compound. In addition, the in vivo hypoglycemic effect of the analogues was determined. With regard to the above, the cinnamaldehyde derivatives showed a scarce biological activity, while the 4-hydroxy analogues showed the higher glycemia reduction at 7 h after administration. Interestingly, the most potent antioxidants 3b and 3g also were of the most active compounds in reducing the plasma glucose, reaching 80% of reduction in the case of 3g. Molecular docking binding poses conducted to a plausible interpretation of the biological outcomes and a possible interaction between a hydroxy group and Asn287 of CB1R was proposed as an important feature for enhancing the observed activity.     

Synthesis and biological evaluation of 1,2,3-triazole linked aminocombretastatin conjugates as mitochondrial mediated apoptosis inducers

A series of 1,2,3-triazole linked aminocombretastatin conjugates were synthesized and evaluated for cytotoxicity, inhibition of tubulin polymerization and apoptosis inducing ability. Most of the conjugates exhibited significant anticancer activity against some representative human cancer cell lines and two of the conjugates 6d and 7c displayed potent cytotoxicity with IC₅₀ values of 53 nM and 44 nM against A549 human lung cancer respectively, and were comparable to combretastatin A-4 (CA-4). SAR studies revealed that 1-benzyl substituted triazole moiety with an amide linkage at 3-position of B-ring of the combretastatin subunit are more active compared to 2-position. G2/M cell cycle arrest was induced by these conjugates 6d and 7c and the tubulin polymerization assay (IC₅₀ of 1.16 μM and 0.95 μM for 6d and 7c, respectively) as well as immunofluorescence analysis showed that these conjugates effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Colchicine competitive binding assay suggested that these conjugates bind at the colchicine binding site of tubulin as also observed from the docking studies. Further, mitochondrial membrane potential, ROS generation, caspase-3 activation assay, Hoechst staining and DNA fragmentation analysis revealed that these conjugates induce cell death by apoptosis.     

Synthesis and structure-activity relationship studies on tryprostatin A, an inhibitor of breast cancer resistance protein

Tryprostatin A is an inhibitor of breast cancer resistance protein, consequently a series of structure-activity studies on the cell cycle inhibitory effects of tryprostatin A analogues as potential antitumor antimitotic agents have been carried out. These analogues were assayed for their growth inhibition properties and their ability to perturb the cell cycle in tsFT210 cells. SAR studies resulted in the identification of the essential structural features required for cytotoxic activity. The absolute configuration L-Tyr-L-pro in the diketopiperazine ring along with the presence of the 6-methoxy substituent on the indole moiety of 1 was shown to be essential for dual inhibition of topoisomerase II and tubulin polymerization. Biological evaluation also indicated the presence of the 2-isoprenyl moiety on the indole scaffold of 1 was essential for potent inhibition of cell proliferation. Substitution of the indole N(a)-H in 1 with various alkyl or aryl groups, incorporation of various L-amino acids into the diketopiperazine ring in place of L-proline, and substitution of the 6-methoxy group in 1 with other functionality provided active analogues. The nature of the substituents present on the indole N(a)-H or the indole C-2 position influenced the mechanism of action of these analogues. Analogues 68 (IC(50)=10 microM) and 67 (IC(50)=19 microM) were 7-fold and 3.5-fold more potent, respectively, than 1 (IC(50)=68 microM) in the inhibition of the growth of tsFT210 cells. Diastereomer-2 of tryprostatin B 8 was a potent inhibitor of the growth of three human carcinoma cell lines: H520 (IC(50)=11.9 microM), MCF-7 (IC(50)=17.0 microM) and PC-3 (IC(50)=11.1 microM) and was equipotent with etoposide, a clinically used anticancer agent. Isothiocyanate analogue 71 and 6-azido analogue 72 were as potent as 1 in the tsFT210 cell proliferation and may be useful tools in labeling BCRP.     

Synthesis and SAR of C12–C13-oxazoline derivatives of epothilone A

The SAR of a series of new epothilone A derivatives with a 2-substituted-1,3-oxazoline moiety trans-fused to the C12–C13 bond of the deoxy macrocycle have been investigated with regard to tubulin polymerization induction and cancer cell growth inhibition. Significant differences in antiproliferative activity were observed between different analogs, depending on the nature of the substituent at the 2-position of the oxazoline ring. The most potent compounds showed comparable activity with the natural product epothilone A. Modeling studies provide a preliminary rationale for the observed SAR.     

Novel [1,2,4]triazolo[1,5-a]pyrimidine derivatives as potent antitubulin agents: Design,multicomponent synthesis and antiproliferative activities

As restricted CA-4 analogues, a novel series of [1,2,4]triazolo[1,5-a]pyrimidines possessing 3,4,5-trimethoxylphenyl groups has been achieved successfully via an efficient one-pot three-component reaction of 3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine, 1,3-dicarbonyl compounds and aldehydes. Initial biological evaluation demonstrated some of target compounds displayed potent antitumor activity in vitro against three cancer cell lines. Among them, the most highly active analogue 26 inhibited the growth of HeLa, and A549 cell lines with IC₅₀ values at 0.75, and 1.02 μM, respectively, indicating excellent selectivity over non-tumoural cell line HEK-293 (IC₅₀ = 29.94 μM) which suggested that the target compounds might possess a high safety index. Moreover, cell cycle analysis illustrated that the analogue 26 significantly induced HeLa cells arrest in G2/M phase, meanwhile the compound could dramatically affect cell morphology and microtubule networks. In addition, compound 28 exhibited potent anti-tubulin activity with IC₅₀ values of 9.90 μM, and molecular docking studies revealed the analogue occupied the colchicine-binding site of tubulin. These observations suggest that [1,2,4]triazolo[1,5-a]pyrimidines represent a new class of tubulin polymerization inhibitors and well worth further investigation aiming to generate potential anticancer agents.     

Conformationally restricted macrocyclic analogues of combretastatins

New analogues of combretastatins have been evaluated as inhibitors of tubulin polymerization. These compounds present a macrocyclic structure, in which the para positions of the aromatic moieties have been linked by a 5- or 6-atoms chain, in order to produce a conformational restriction. This could contribute to determine the active conformation for these ligands. Such a conformational restriction and/or the steric hindrance makes them less potent inhibitors than the model compound CA-4.     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 5: Exploration of pyridazinones containing 6-amino-substituents

The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.     

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)