N-Phenyl-N'-(2-chloroethyl)ureas (CEU) as potential antineoplastic agents. Part 2: role of omega-hydroxyl group in the covalent binding to beta-tubulin

Tubulin is the target of many anticancer drugs, including N-phenyl-N'-(2-chloroethyl)urea (CEU). Unlike most anti-beta-tubulin agents, CEUs are protein monoalkylating agents binding through their N'-(2-chloroethyl)urea moiety to an amino acid nearby the colchicine-binding site on beta-tubulin isoform-2. Following the previously synthesized and attractive N-(3-omega-hydroxyalkylphenyl)-N'-(2-chloroethyl)urea that exhibited growth inhibitory activity at the nanomolar level, we investigated the importance of lower alkyl and alkoxy groups to evaluate the effect of hydroxylated group and chain length on both cell growth inhibition and the mechanism of action of CEU. Here, we describe the preparation of two new series of CEU and show that the most potent CEU derivatives beside the omega-hydroxylated 1f were 2f and 3e, respectively. We have confirmed that the pentyl substituted CEUs 1f, 2f, and 3e are still covalently binding to beta-tubulin and still arrest cell division in G(2)/M phase.     

N-Phenyl-N'-(2-chloroethyl)ureas (CEUs) as potential antineoplastic agents. Part 3: role of carbonyl groups in the covalent binding to the colchicine-binding site

In the course of the development of N-phenyl-N'-(2-chloroethyl)ureas (CEUs) as potential antineoplastic agents, we investigated the effect of carbonylated substituting chains of the aromatic ring of CEU on their covalent binding to the colchicine-binding site (C-BS). In this study, we found that CEU, 5e, 5f, 8e, and 8f substituted by either a methyl ester or a methyl ketyl group at the omega-position exhibited a significant antiproliferative activity on HT-29, M21, and MCF-7 tumor cells. SDS-PAGE assays and cell cycle analysis confirmed that 5e, 5f, 8e, and 8f covalently bind to the C-BS and arrest the cell division in G(2)/M phase. Surprisingly, the presence of omega-carboxyl, omega-ethyl esters or omega-amides decreased significantly both the antiproliferative activity and the specificity toward beta-tubulin.     

N-Phenyl-N′-(2-chloroethyl)urea analogues of combretastatin A-4: Is the N-phenyl-N′-(2-chloroethyl)urea pharmacophore mimicking the trimethoxy phenyl moiety?

A series of novel N-phenyl-N'-(2-chloroethyl)urea derivatives potentially mimicking the structure of combretastatin A-4 were synthesized and tested for their cell growth inhibition and their binding to the colchicine-binding site of beta-tubulin. Compounds 2a, 3a, and 3b were found to inhibit cell growth at the micromolar level on four human tumor cell lines. Flow cytometric analysis indicates that the new compounds act as antimitotics and arrest the cell cycle in G(2)/M phase. Covalent binding of 2a, 3a, and 3b to the colchicine-binding site of beta-tubulin was confirmed also using SDS-PAGE and competition assays.     

A comparative molecular field and comparative molecular similarity indices analyses (CoMFA and CoMSIA) of N-phenyl-N'-(2-chloroethyl)ureas targeting the colchicine-binding site as anticancer agents

To decipher the mechanism underlying the covalent binding of N-phenyl-N'-(2-chloroethyl)ureas (CEU) to the colchicine-binding site on beta(II)-tubulin and to design new and selective antimitotic drugs, we developed 3D quantitative structure-activity relationships (3D-QSAR) models using CoMFA and CoMSIA analyses. The present study correlates the cell growth inhibition activities of 56 structurally related CEU derivatives to several physicochemical parameters representing steric, electrostatic, and hydrophobic fields. Both CoMFA and CoMSIA models using two different optimum numbers of components (ONC) 10 and 4, respectively, gave good internal predictions and their cross-validated r2 values were between 0.639 and 0.743. These comprehensive CoMFA and CoMSIA models are useful in understanding the structure-activity relationships of CEU. The two models were compared to the X-ray crystal structure of the complex of tubulin-colchicine and analyzed for similarities between the two modes of analysis. These models will inspire the design of new CEU derivatives with enhanced inhibition of tumor cell growth and targeting specificity of beta(II)-tubulin and the cytoskeleton.     

Selective alkylation of beta(II)-tubulin and thioredoxin-1 by structurally related subsets of aryl chloroethylureas leading to either anti-microtubules or redox modulating agents

Aryl chloroethylureas (CEUs) are potent anti-neoplastic agents alkylating specific intracellular proteins such as beta(II)-tubulin. Recently we have identified a new subset of CEU derived from compound 36 that alkylates thioredoxin isoform 1 (Trx-1), inhibits the nuclear translocation of Trx-1, and favors the accumulation of cells in G(0)/G(1) phase. We have evaluated the effects of various substituents and their position on the aromatic ring of a series of derivatives of 36 on (i) the anti-proliferative activity, (ii) the cell cycle progression, (iii) the nuclear translocation of Trx-1, and (iv) their covalent binding to beta-tubulin. The same experiments were performed on representative CEU derivatives where the 2-chloroethyl amino moiety is replaced by either an ethyl, a 2-aminooxazolinyl or a 2-chloroacetyl group. On one hand, our results suggest that CEUs substituted on the phenyl ring at position 3 or 4 by cycloalkyl and substituted cycloalkyl or cycloalkoxy groups inhibit the nuclear translocation of Trx-1 and arrest the cell cycle progression in G(0)/G(1). On the other hand, CEUs substituted by a fused aromatic ring, an aliphatic chain, or a fused aliphatic ring are alkylating beta(II)-tubulin but not Trx-1. Beside the expected inactivity of the ethylurea derivatives, none of the modification to the electrophilic moiety led to cross-selectivity of the drugs toward beta-tubulin but increased the anti-proliferative activity and resulted in mitigated effects on Trx-1 translocation.     

Aromatic 2-chloroethyl urea derivatives and bioisosteres. Part 2: Cytocidal activity and effects on the nuclear translocation of thioredoxin-1, and the cell cycle progression

Recently, a subset of N-phenyl-N'-(2-chloroethyl)ureas (CEU) was found abrogating the nuclear translocation of thioredoxin-1 and arresting the cell cycle in G(0)/G(1) phase. Several derivatives were prepared to assess their effect on cell cycle progression and on the intracellular location of Trx-1. Compounds 1-20, 21-40, and 41-60 exhibited GI(50) between 1 and 80 microM. Immunocytochemistry analysis showed compounds 4, 6, 8, 10, 11, 23, 24, 26-31, 34, 37, 41, 44, 46-51, 53, 56, and 57 inhibiting the nuclear translocation of Trx-1. Our results suggest that increasing the electrophilic character of these molecules might enhance the antiproliferative activity at the expense of the selectivity toward thioredoxin-1 and the G(0)/G(1) phase arrest.     

Optimized N-phenyl-N'-(2-chloroethyl)ureas as potential antineoplastic agents: synthesis and growth inhibition activity

In our ongoing research program aimed at the optimization of microtubule-self-assembly disrupting agents, we have prepared three series of phenylurea analogues (CEU), derived from N-(3-ω-hydroxyalkyl or 4-ω-hydroxyalkyl or 3-ω-hydroxyalkynyl)-phenyl-N′-(2-chloroethyl)ureas. Most compounds exhibit potent growth inhibitory activity on human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7 tumor cell lines, with a GI₅₀ ranging from 250 nM to 8 μM. Among these new molecules, three CEUs exhibit GI₅₀ in the nanomolar range. They are more potent by approximately an order of magnitude than previously described CEU analogues. As such, they are attractive hit compounds for the development of potent new alkylating antitubulin drugs.     

Mechanism of action of N-phenyl-N′-(2-chloroethyl)ureas in the colchicine-binding site at the interface between α- and β-tubulin

Computational tools such as CoMSIA and CoMFA models reported in a recent study revealed the structure–activity relationships ruling the interactions occurring between hydrophobic N-phenyl-N′-(2-chloroethyl)ureas (CEU) and the colchicine-binding site (C-BS) on β_ΙΙ-tubulin. Here, we describe the mechanisms involved in the covalent binding of three subsets of CEU derivatives to the C-BS. The FlexiDock experiments confirmed that the interaction of non-covalent portions of the CEU auxophore moiety of CEU is involved in the binding of the drug to the C-BS facilitate the nucleophilic attack of Glu-β198 rather than Cys-β239. In addition, these studies suggest that Cys-β239 together with Asn-α99, Ser-α176, Thr-α177, Leu-β246, Asn-β247, Ala-β248, Lys-β252 and Asn-β256 are implicated in the stabilization of a C-BS–CEU complex prior to the acylation of Glu-β198 by CEU. Our molecular models propose the formation of a stabilized C-BS–CEU complex before the completion of the Glu-β198 acylation; acylation triggering conformational changes of β-tubulin, microtubule depolymerization and anoikis. The computational models presented here might be useful to the design of selective and more potent C-BS inhibitors. Of interest, in vivo acylation of acidic amino acid residues by xenobiotics is an unusual reaction and may open new approaches for the design of irreversible protein inhibitors such as tubulin.     

Cycloalkyl-substituted aryl chloroethylureas inhibiting cell cycle progression in G0/G1 phase and thioredoxin-1 nuclear translocation

1-(2-Chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) has been shown to abrogate the presence of thioredoxin-1 into the nucleus through its selective covalent alkylation. In the present letter we have evaluated the structure-activity relationships of the substituents at positions 3 and 4 of the phenyl ring of cHCEU derivatives on cell cycle progression and thioredoxin-1 nuclear translocation. Active CEU derivatives exhibited GI(50) ranging from 1.9 to 49muM on breast carcinoma MCF-7, skin melanoma M21, and colon carcinoma HT-29 cells. On one hand, compounds 1, 2, 9c, 10c, 13, and 14 arrested the cell cycle in G(2)/M phase while CEUs 3, 4, 5c, 6c, 11c, and 12c blocked the cell division in G(0)/G(1) phase. On the other hand, CEUs 2-4, 5c, 7c, 8c, 11c, and 12c abrogated the translocation of thioredoxin-1 while the other CEU derivatives were inactive in that respect. Our results suggest that CEU substituted on the phenyl ring at position 3 or 4 by lower cycloalkyl or cycloalkoxy groups arrest cell progression in G(0)/G(1) phase through mechanism of action different from their antimicrotubule counterparts, presumably via thioredoxin-1 alkylation and modulation of its activity. The mechanism of action of these new molecules is still undetermined. However, the significant accumulation of cells in G(0)/G(1) phase suggests that these molecules may act similarly to known chemopreventive agents against cancers. In addition, the inhibition of Trx-1 nuclear localization also suggests the abrogation of an important chemoresistance mechanism towards a variety of chemotherapeutic agents.     

Chloroethyl urea derivatives block tumour growth and thioredoxin-1 nuclear translocation

Aryl chloroethyl ureas (CEUs) are new protein alkylating agents exhibiting anticancer activity both in vitro and in vivo. We report herein that 14C-labeled CEU derivatives, designated CEU-025 and CEU-027, covalently bind to thioredoxin-1 (TRX1). Covalent binding of these molecules slightly decreases the disulfide-reducing activity of recombinant TRX1, when compared with the effect of strong thioalkylating agents such as N-ethylmaleimide. Moreover, site-directed mutagenesis and diamide competition assays demonstrated that TRX1 cysteinyl residues are not the prime targets of CEUs. CEU-025 abrogates the nuclear translocation of TRX1 in human cancer cells. In addition, we show that CEU-025 can block TRX1 nuclear translocation induced by cisplatin. Unexpectedly, pretreatment with sublethal CEU-025 concentrations that block TRX1 nuclear translocation protected the cells against cisplatin cytotoxicity. Overexpression of TRX1 in HT1080 fibrosarcoma cells attenuated CEU-025 cytotoxicity, while its suppression using TRX1-specific siRNA increased the effects of CEU-025, suggesting that loss of function of TRX1 is involved, at least in part, in the cytotoxic activity of CEU-025. These results suggest that CEU-025 and CEU-027 exhibit anticancer activity through a novel, unique mechanism of action. The importance of TRX1 and the dependence of the cytotoxicity of CEU-025 and CEU-027 on TRX1 intracellular localization are also discussed.     

Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the A ring of bound colchicine molecule

Isotypes of vertebrate tubulin have variable amino acid sequences, which are clustered at their C-terminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first-order conditions. Experiments with individual isotypes established that the presence of beta(III) in the purified tubulin is responsible for the biphasic kinetics. Because the isotypes mainly differ at the C termini, the colchicine-binding kinetics of unfractionated tubulin and the beta(III) isotype, cleaved at the C termini, have been tested under pseudo-first-order conditions. Removal of the C termini made no difference to the nature of the kinetics. Sequence alignment of different beta isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. To establish whether these differences lie at the colchicine-binding site or not, homology modeling of all beta-tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A ring of colchicine at the colchicine-binding site on beta tubulin. While the beta(III) isotype has two hydrophilic residues (serine(242) and threonine(317)), both beta(II) and beta(IV) have two hydrophobic residues (leucine(242) and alanine(317)). beta(II) has isoleucine at position 318, while beta(III) and beta(IV) have valine at that position. Thus, these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine-binding kinetics of the different isotypes of tubulin.     

Analysis of the colchicine-binding site of beta-tubulin.

Comparison of the beta-tubulin sequences with the equilibrium colchicine Ka and the Ki for inhibition by podophyllotoxin suggests that residue beta:316 is directly involved in binding the common trimethoxyphenyl-(or A-) ring. By contrast, the analysis indicates that the local hydrophobicity affects the rate of one of the two conformational changes associated with colchicine binding but does not determine the affinity of the colchicine-binding site.     

Biphenyl derivatives incorporating urea unit as novel VEGFR-2 inhibitors: Design,synthesis and biological evaluation

A series of novel biphenyl urea derivates were synthesized and investigated for their potential to inhibit vascular endothelial growth factor receptor-2 (VEGFR-2). In particular, A7, B3 and B4 displayed significant enzymatic inhibitory activities, with IC₅₀ values of 4.06, 4.55 and 5.26 nM. Compound A7 exhibited potent antiproliferative activity on several cell lines. SAR study suggested that the introduction of methyl at ortho-position of the biphenyl urea and tertiary amine moiety could improve VEGFR-2 inhibitory activity and antitumor effects. Molecular docking indicated that the urea moiety formed four hydrogen bonds with DFG residue. These biphenyl ureas could serve as promising lead compounds for further optimization.     

Membrane interactions of a new class of anticancer agents derived from arylchloroethylurea: a FTIR spectroscopic study.

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and different lipids such as dimyristoylphosphatidylcholine (DMPC) in the absence and presence of 30 mol% of cholesterol, dimyristoylphosphatidylglycerol (DMPG) and a mixture made of 1-palmitoyl-2-oleylphosphatidylcholine (POPC) and DMPC by Fourier transform infrared (FTIR) spectroscopy. The results indicate that the drugs incorporate in the bilayer and cause a decrease of the phase transition temperature and an increase of the conformational disorder of the lipid acyl chains. These effects are dependent on the nature (degree of branching, length of the alkyl chain and presence of a sulfur atom), as well as on the position of the R substituent and are related to the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky branched substituent and those for which the disordering effect on the lipid bilayer is the greatest. On the other hand, the disordering effect is small for the long chain CEUs, such as 4-n-hexadecyl CEU, which have been shown to have weak cytotoxic activity.     

Detection and identification of transient intermediates in the reactions of tryptophan synthase with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Evidence for a tetrahedral (gem-diamine) intermediate

The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy. The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively). Rate constants determined by SWSF were either well resolved (i.e., k1[DHT], k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1[OXA], k-1 greater than or equal to k2, k-2 greater than k3, k-3). The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species. Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system. The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates.     

Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC(50) of 2.0 microM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26 Angstroms resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.     

In vitro inhibition of translation initiation by N,N'-diarylureas--potential anti-cancer agents

Symmetrical N,N'-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP·tRNA(i)(Met) ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N'-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.     

Microtuberization of layered shoots and nodal cuttings of potato: The influence of growth regulators and incubation periods

A protocol is presented for the rapid induction of microtubers on micropropagated, layered potato shoots of Kennebec, Russet Burbank and Superior in medium devoid of growth regulators. Layered shoots microtuberized more rapidly and produced significantly larger microtubers compared with nodal cuttings. The addition of coumarin or (2-chloroethyl)-trimethylammonium chloride and benzyladenine to microtuberization medium, either had no effect or significantly reduced microtuber weight per shoots compared with medium containing only 80 g × 1^-1 sucrose and minimally affected the number of microtubers per shoot. Increasing the incubation period from 28 to 56 days did not affect the number but significantly increased the weight of microtubers per shoot and substantially increased the proportion, up to 20%, of microtubers heavier than 1 gram.Abbreviations Ba benzyladenine - ccc (2-chloroethyl) trimethylammonium chloride - coumarin 2h-1-benzopyran-2-one - ga₃ gibberellic acid     

Synthesis, cytostatic activity and ADME properties of C-5 substituted and N-acyclic pyrimidine derivatives

The synthesis of the novel 5-alkyl pyrimidine derivatives, 5,6-dihydrofuro[2,3-d]pyrimidines and 5-alkyl N-methoxymethyl pyrimidine derivatives and evaluation of their cytostatic activities are described. The mechanism of antiproliferative effect of 5-(2-chloroethyl)-substituted pyrimidine 3 that exerted the pronounced cytostatic activity was studied in further details on colon carcinoma (HCT116) cells. The cell cycle perturbation analysis demonstrated severe DNA damage (G2/M arrest) pointing to a potential DNA alkylating ability of 3. Preliminary ADME data of 3 and its 6-methylated structural congener (6-Me-3) showed their high permeability and good metabolic stability.     

Spectroscopic characterization of DMPC/DOTAP cationic liposomes and their interactions with DNA and drugs

Gene and synthetic drug-delivery vectors have been developed and characterized to treat several genetic diseases and cancers. Our study aims at characterizing cationic liposomes containing the zwitterionic phospholipid DMPC and the cationic lipid DOTAP as well as their interactions with two types of DNA and a new class of antineoplastic agents derived from arylchloroethylureas (CEU). Results obtained using FTIR spectroscopy as well as ³¹P and ²H NMR indicate that DMPC and DOTAP form cationic liposomes in a highly disordered fluid phase at a molar ratio of 1:1. In addition, the FTIR results indicate that the presence of DNA or CEUs within the liposomes does not significantly affect the conformational order of both the DMPC and DOTAP acyl chains. Our results therefore provide a detailed characterization of complexes between cationic liposomes and both DNA and drugs and indicate that these complexes are stable and fluid assemblies.