Design, synthesis and preliminary bioactivity studies of 1,3,4-thiadiazole hydroxamic acid derivatives as novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors have emerged as a new class of anticancer agents, targeting the biological processes including cell cycle, apoptosis and differentiation. In the present study, a series of 1,3,4-thiadiazole based hydroxamic acids were developed as potent HDAC inhibitors. Some of them showed good inhibitory activity in HDAC enzyme assay and potent growth inhibition in some tumor cell lines. Among them, compound 6i (IC(50) = 0.089 μM), exhibited better inhibitory effect compared with SAHA (IC(50) = 0.15 μM).     

The mevalonate/isoprenoid pathway inhibitor apomine (SR-45023A) is antiproliferative and induces apoptosis similar to farnesol

Apomine (SR-45023A) is a new antineoplastic compound which is currently in clinical trials and representative of the family of cholesterol synthesis inhibitors 1,1-bisphosphonate esters. Apomine inhibits growth of a wide variety of tumor cell lines with IC(50) values ranging from 5 to 14 microM. The antiproliferative activity of apomine was studied in comparison with that of other inhibitors of the mevalonate/isoprenoid pathway of cholesterol synthesis, simvastatin, farnesol, and 25-hydroxycholesterol. All these compounds inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. Apomine (IC(50) = 14 microM), simvastatin (IC(50) = 3 microM), farnesol (IC(50) = 60 microM), and 25-hydroxycholesterol (IC(50) = 2 microM) inhibited HL60 cell growth. Growth inhibition due to simvastatin was reverted by mevalonate, whereas the antiproliferative activity of apomine, farnesol, and 25-hydroxycholesterol was not. Apomine triggered apoptosis in HL60 cells in less than 2 h. Apomine and farnesol induced caspase-3 activity at concentrations similar to their IC(50) values for cell proliferation, whereas a 10-fold excess of simvastatin was necessary to trigger apoptosis compared to its potency on proliferation. Caspase-3 activity was not induced by 25-hydroxycholesterol. The overall similar profile on mevalonate synthesis inhibition, cell growth inhibition, and apoptosis suggests that apomine acts as a synthetic mimetic of farnesol.     

Novel antiproliferative analogs of the Taq DNA polymerase inhibitor catalpol

The naturally occurring iridoid catalpol (1) is a Taq DNA polymerase inhibitor. However, its poor lipophilicity might account for the lack of biological activity against human solid tumor cell lines. The traditional prodrug approach by means of peracetylation of the free hydroxyl groups led to a compound, which showed a marginal growth inhibition against the most sensitive cell line A2780 (ovarian cancer). However, the formation of analogs bearing one to three silyl ether groups led to antiproliferative compounds against a panel of six human solid tumor cell lines, with GI50 values in the range 1.8-4.8 microM. Cell cycle studies revealed arrest in G0/G1 phase that is consistent with DNA polymerase inhibition.     

Synthesis and antitumor activity of 1,2,4-triazoles having 1,4-benzodioxan fragment as a novel class of potent methionine aminopeptidase type II inhibitors

A series of 1,2,4-triazole derivatives containing 1,4-benzodioxan (5a-5q) have been designed, synthesized, structurally determined, and their biological activities were evaluated as potential MetAP2 inhibitors. All the synthesized compounds were first reported. Among the compounds, compound 5k showed the most potent biological activity against HEPG2 cancer cell line (IC(50)=0.81 μM for HEPG2 and IC(50)=0.93 μM for MetAP2), which was comparable to the positive control. Docking simulation by positioning compound 5k into the MetAP2 structure active site was performed to explore the possible binding model. The results of apoptosis and Western-blot assay demonstrated that compound 5k possessed good antitumor activity against HEPG2 cancer cell line. Therefore, compound 5k with potent inhibitory activity in tumor growth inhibition may be a potential antitumor agent against HEPG2 cancer cell.     

Effect of resveratrol and beta-sitosterol in combination on reactive oxygen species and prostaglandin release by PC-3 cells

The objective of this project was to identify some possible mechanisms by which two common phytochemicals, resveratrol and beta-sitosterol, inhibit the growth of human prostate cancer PC-3 cells. These mechanisms include the effect of the phytochemicals on apoptosis, cell cycle progression, prostaglandin synthesis and the production of reactive oxygen species (ROS). Prostaglandins have been known to play a role in regulating cell growth and apoptosis. PC-3 cells were supplemented with 50 microM resveratrol or 16 microM beta-sitosterol alone or in combination for up to 5 days. Phytochemical supplementation resulted in inhibition in cell growth. beta-Sitosterol was more potent than resveratrol and the combination of the two resulted in greater inhibition than supplementation with either alone. Long-term supplementation with resveratrol or beta-sitosterol elevated basal prostaglandin release but beta-sitosterol was much more potent than resveratrol in this regard. beta-Sitosterol was more effective than resveratrol in inducing apoptosis and the combination had an intermediate effect after 1 day of supplementation. Cells supplemented with resveratrol were arrested at the G1 phase and at the G2/M phase in the case of beta-sitosterol while the combination resulted in cell arrest at the two phases of the cell cycle. beta-Sitosterol increased ROS production while resveratrol decreased ROS production. The combination of the two phytochemicals resulted in an intermediate level of ROS. The observed changes in prostaglandin levels and ROS production by these two phytochemicals may suggest their mediation in the growth inhibition. The reduction in ROS level and increase by resveratrol supplementation in PC-3 cells reflects the antioxidant properties of resveratrol. It was concluded that these phytochemicals may induce the inhibition of tumor growth by stimulating apoptosis and arresting cells at different locations in the cell cycle and the mechanism may involve alterations in ROS and prostaglandin production.     

The convenient synthesis and evaluation of the anticancer activities of new resveratrol derivatives

In the present study we report the simple synthesis and antitumour activity of novel stilbene derivatives 13-22. The key synthetic strategies involved Wadsworth-Horner-Emmons condensation and coupling reactions in high yields. All compounds showed significant growth inhibition on human tumour cell lines, with the most potent compound (19) exhibiting an IC(50) of 5.7 microM-11.4 microM in vitro.     

Heterocyclic substituted cantharidin and norcantharidin analogues--synthesis, protein phosphatase (1 and 2A) inhibition, and anti-cancer activity

Norcantharidin (3) is a potent PP1 (IC(50)=9.0+/-1.4 microM) and PP2A (IC(50)=3.0+/-0.4 microM) inhibitor with 3-fold PP2A selectivity and induces growth inhibition (GI(50) approximately 45 microM) across a range of human cancer cell lines including those of colorectal (HT29, SW480), breast (MCF-7), ovarian (A2780), lung (H460), skin (A431), prostate (DU145), neuroblastoma (BE2-C), and glioblastoma (SJ-G2) origin. Until now limited modifications to the parent compound have been tolerated. Surprisingly, simple heterocyclic half-acid norcantharidin analogues are more active than the original lead compound, with the morphilino-substituted (9) being a more potent (IC(50)=2.8+/-0.10 microM) and selective (4.6-fold) PP2A inhibitor with greater in vitro cytotoxicity (GI(50) approximately 9.6 microM) relative to norcantharidin. The analogous thiomorpholine-substituted (10) displays increased PP1 inhibition (IC(50)=3.2+/-0 microM) and reduced PP2A inhibition (IC(50)=5.1+/-0.41 microM), to norcantharidin. Synthesis of the analogous cantharidin analogue (19) with incorporation of the amine nitrogen into the heterocycle further increases PP1 (IC(50)=5.9+/-2.2 microM) and PP2A (IC(50)=0.79+/-0.1 microM) inhibition and cell cytotoxicity (GI(50) approximately 3.3 microM). These analogues represent the most potent cantharidin analogues thus reported.     

N-Benzyl-3-sulfonamidopyrrolidines as novel inhibitors of cell division in E. coli

A new small molecule inhibitor of bacterial cell division has been discovered using a high-throughput screen in Escherichia coli. Although the lead screening hit (534F6) exhibited modest inhibition of the GTPase activity of FtsZ (20+/-5% at 100microM of compound), a primary target for bacterial cell division inhibitors, several analogs caused potent bacterial growth inhibition with negligible antagonism of FtsZ GTPase activity. A library of analogs has been prepared and several alkyne-tagged photoaffinity probes have been synthesized for use in experiments to elucidate the primary target of this compound.     

Synthesis, biological evaluation, and molecular docking studies of 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives as novel antitubulin agents

A series of 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives (1h-20h) have been designed and synthesized, and their biological activities were also evaluated as potential antiproliferation and tubulin polymerization inhibitors. Among all the compounds, 2h showed the most potent activity in vitro, which inhibited the growth of MCF-7, Hep-G2 and A549 cell lines with IC(50) values of 0.70 ± 0.05, 0.68 ± 0.02 and 0.86 ± 0.05 μM, respectively. Compound 2h also exhibited significant tubulin polymerization inhibitory activity (IC(50)=3.06 ± 0.05 μM). The result of flow cytometry (FCM) demonstrated that compound 2h induced cell apoptosis. Docking simulation was performed to insert compound 2h into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. Based on the preliminary results, compound 2h with potent inhibitory activity in tumor growth may be a potential anticancer agent.     

Indomethacin inhibits cell growth of medullary thyroid carcinoma by reducing cell cycle progression into S phase

Indomethacin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the growth of medullary thyroid carcinoma (MTC) cells in vitro. However, the mechanism of inhibition of MTC cell growth by indomethacin and its potency have yet to be revealed. We examined the effect of indomethacin on three different MTC cell lines (TT cells, DRO 81-1 cells and HRO 85-1 cells) and two non-MTC cells. The mechanism of indomethacin action in MTC cells was investigated by analyzing intracellular prostaglandin level, apoptosis, and cell cycle in TT cells. Indomethacin inhibited cell growth of all three MTC cell lines but not normal thyroid cells or anaplastic thyroid carcinoma cells. Indomethacin at 10 microM or greater showed a dose response inhibition of cell growth. Indomethacin at 25 muM, a putative therapeutic serum indomethacin level, showed potency similar to 100 to 200 nM sunitinib, a receptor tyrosine kinase inhibitor. To examine whether prostaglandin depletion might determine the inhibition of MTC cell growth, we created different prostaglandin E2 (PGE2) levels in TT cells using three different NSAIDs. A profound PGE2 depletion by indomethacin-ester, a potent cyclooxygenase (COX) II inhibitor, showed the least inhibition of cell growth. Indomethacin did not increase apoptosis of TT cells. Indomethacin, but not naproxen or indomethacin-ester, reduced cell cycle progression into S phase; this was unrelated to the degree of PGE2 depletion. The expression of phosphorylated retinoblastoma (pRb) protein that shifts cells from G(1) to S phase was reduced after exposure to indomethacin. In conclusion, indomethacin has specific anti-tumor effect on MTC cells, probably by reducing cell cycle progression into S phase rather than by prostaglandin depletion. Since no drug therapy is currently available for MTC, indomethacin may be one of the therapeutic candidates.     

3Beta-hydroxy-6-aza-cholestane and related analogues as phosphatidylinositol specific phospholipase C (PI-PLC) inhibitors with antitumor activity

6-Aza steroid analogues were synthesized as PI-PLC inhibitors. The most active compound, 3beta-hydroxy-6-aza-cholestane (1) showed potent PI-PLC inhibition (IC50 = 1.8 microM), similar to that of the commercially available steroid analogue U73122 (IC50 = 1-2.1 microM). Compound 1 exhibited significant growth inhibition effects (IC50 = 1.3 microM in each case) against MCF-7 and HT-29 cancer cells in in vitro cell culture. Compound 1 also inhibited the in vitro adhesion and transmigration of HT-1080 fibrosarcoma cells at 2.5 and 5.0 microM, respectively. In vivo, compound 1, at 1 mg/kg/day, reduced the volume of MCF-7 tumors in xenograft models, without weight loss in mice. Structure activity relationships of this series of compounds revealed that a hydrophobic cholesteryl side chain, 3beta-hydroxy group and a C-6 nitrogen containing a hydrogen atom at position-6 are crucial for activity. N-Maleic amidoacid derivative 11 also exhibited weak inhibition (IC50 = 16.2 microM).     

Chemosensitivity of human prostate cancer cells PC3 and LNCaP to genistein isoflavone and beta-lapachone

A wide spectrum of anti-cancer activity of genistein and beta-lapachone in various tumors has been reported in single treatments. In this study the combined effects of genistein and beta-lapachone on the chemosensitivity of LNCaP and PC3 human prostate cancer cells was determined in vitro, using 3-[4,5-dimethylthiazol-2-yl]-2-,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) to study treatment-induced growth inhibition and cytotoxicity and, annexin V-fluoresceine (FI) and terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-propidium iodide (PI) assays to determine potential treatment-induced apoptosis and/or necrosis. The results showed: i) that both PC3 and LNCaP are sensitive to single and combination treatments regardless of hormone sensitivity status, ii) that treatment induced dual death pathways (apoptosis and necrosis) in both cell types, iii) that growth inhibition in both cell types correlated positively with cell death via apoptosis at lower drug concentrations and necrosis at higher concentrations, iv) that combination of genistein and beta-lapachone had synergistic inhibitory effects on growth and proliferation in both cell types. The synergistic inhibitory effect was correlated positively with treatment-induced cell death via apoptosis and necrosis. The overall results indicate that combination treatments with beta-lapachone and genistein are more potent in killing both PC3 and LNCaP cancer cells than treatment with either genistein or beta-lapachone alone. beta-lapachone acts at the G1 and S phase checkpoints in the cell cycle, while genistein induces cell cycle arrest at the G2-M stage. The current results are therefore in agreement with the hypothesis that drug combinations that target cell cycles at different critical checkpoints would be more effective in causing cell death. This result provides a rationale for in vivo studies to determine whether beta-lapachone-genistein combination will provide effective chemotherapy for prostate cancer, regardless of the tumor sensitivity to hormone.     

Synthesis, biological evaluation and molecular docking studies of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives as inhibitors of HDAC activity

In present study, a series of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives (5a-8d) were designed, synthesized, and evaluated for HDAC inhibition and tumor cell antiproliferation. All of these compounds are reported for the first time, the chemical structures of these compounds were confirmed by means of (1)H NMR, ESI-MS and elemental analyzes. Among the compounds, compound 8c showed the most potent biological activity against HCT116 cancer cell line (IC(50) of 0.42 ± 0.02 μM for HDAC-1 and IC(50)=0.62 ± 0.02 for HCT116). Docking simulation was performed to position compound 8c into the HDAC active site to determine the probable binding model. The results of antiproliferative assay and western-blot demonstrated that compound 8c with potent inhibitory activity in tumor growth inhibition may be a potential anticancer agent against HCT116 cancer cell.     

Structure-activity relationship of aza-steroids as PI-PLC inhibitors

A number of aza-steroids were synthesized as potent phosphatidylinositol phospholipase C (PI-PLC) inhibitors. The epimeric mixtures 22,25-diazacholesterol (8a) and 3beta-hydroxy-22,25-diazacholestane (8b) were among the most active of these inhibitors, with IC(50) values of 7.4 and 7.5 microM, respectively. The 20alpha epimer, 8a2 (IC(50)=0.64 microM), whose stereochemistry at C-20 coincides with that of cholesterol, was found 50 times more potent than the 20beta epimer, 8a1 (IC(50)=32.2 microM). In diaza-estrone derivatives, the 3-methoxy group on the aromatic A-ring of 23 exhibited moderate PI-PLC inhibitory activity (IC(50)=19.7 microM), while compound with a free hydroxyl group (21) was inactive. However, in diaza-pregnane derivatives, epimers with a 3-hydroxyl group (8a, IC(50)=7.4 microM) exhibited more potent PI-PLC inhibitory activity than their counterparts with 3-methoxyl group on the non-aromatic A-ring (26, IC(50)=17.4 microM). We have illustrated in our previous publication that 3-hydroxyl-6-aza steroids are potent PI-PLC inhibitors.(3) However, simultaneous presence of the 6-aza and 22,25-diaza moieties in one molecule as in 13, led to loss of activity. Epimeric mixture 8a showed selective growth inhibition effects in the NCI in vitro tumor cell screen with a mean GI(50) value (MG-MID) of 5.75 microM for 54 tumors.     

The lethal effect of bis-type azridinylnaphthoquinone derivative on oral cancer cells (OEC-M1) associated with anti-apoptotic protein bcl-2

Several drugs of aziridinylbenzoquinone analogs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill tumor cells preferentially within the hypoxic microenvironment. From our previous reported data, it was found that the synthesized 2-aziridin-1-yl-3-[(2-[2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy]ethyl)thio]naphthoquinone (AZ-1) is a bioreductive compound with potent lethal effect on oral cancer cell, OEC-M1. It was found in this study that the lethal effect of the oral cancer cell lines OEC-M1 induced by AZ-1 was mediated through the cell cycle arrest and apoptosis pathway. The LC50 values of OEC-M1 and KB cells induced by AZ-1 compound were 0.72 and 1.02 microM, respectively, which were much lower than that of normal fibroblast cells (SF with LC50 = 5.6 microM) with more than 90% of normal fibroblasts surviving as compared to control at a concentration of AZ-1 as high as 2 microM. It was interesting to note that the LC50 of monotype diaziridinylbenzoquinone compound, diaziquone (AZQ), was 50 microM on OEC-M1 cells. Comparing the cytotoxicity of AZ-1 and AZQ on OEC-M1 cells, AZ-1 is approximately 70 times more potent than AZQ. By using Western blot, both G2/M phase cell cycle arresting protein, cyclin B, and anti-apoptotic protein, bcl-2, were expressed in OEC-M1 cell when the concentrations of AZ-1 were increased from 0.125 to 0.5 microM and then decreased from 1 to 2 microM of AZ-1 treatment as compared with control for 24 h. Both proteins were expressed most abundantly at 0.5 microM AZ-1. However, the expression of bcl-2 protein in OEC-M1 was significantly decreasing in a dose-dependent manner and was only about 50% protein level at 2 microM AZ-1 for 48h as compared with control. The cell survival check protein p53 increased from 1.72- to 2.8-fold and 1.36- to 2.16-fold at concentrations of AZ-1 from 0.125 to 2.0 microM in a dose-dependently increasing manner on OEC-M1 as compared with control for 24 and48 h treatments, respectively. The apoptotic-related phenomena were observed, which included apoptotic body formation and the enzyme activity change of caspase-3. The apoptotic bodies and caspase-3 activity of OEC-M1 were induced only at 2 microM AZ-1 for a 24h treatment, yet apoptotic body formation was observed at as low as 0.5 microM AZ-1 and in a dose-dependently increasing manner for a 48 h treatment. The caspase-3 activity was increased 20.6%, 26.8%, and 84.2%, respectively, at 0.5, 1, and 2muM concentrations of AZ-1 for a 48 h treatment as compared with control. These results indicate that AZ-1 induced the cell death of OEC-M1 through the G2/M phase arrest of cell cycle and anti-apoptosis first and then apoptosis following a 48 h treatment. All of the pathway might be associated with bcl-2 and p53 protein expression. We propose that the AZ-1 could be used as anti-oral cancer drug for future studies with animal models.     

Novel 3-phenylpropane-1,2-diamine derivates as inhibitors of aminopeptidase N (APN)

Aminopeptidase N (APN) is an essential peptidase involved in the process of tumor invasion and metastasis. Here we describe a novel class of inhibitor with 3-phenylpropane-1,2-diamine as scaffold to APN. Preliminary activity evaluation with enzyme inhibition studies showed that compound 12i exhibited potent and selective inhibitory activity towards APN with the IC(50) value 15.5+/-1.2microM.     

Discovery of 18β-glycyrrhetinic acid conjugated aminobenzothiazole derivatives as Hsp90-Cdc37 interaction disruptors that inhibit cell migration and reverse drug resistance

A series of 18β-glycyrrhetinic acid (GA) conjugated aminobenzothiazole derivatives were designed, synthesized and evaluated for disruption activity of Hsp90-Cdc37 as well as the effects of in vitro cell migration. These compounds exhibited relatively good disruption activity against Hsp90-Cdc37 with IC₅₀ values in low micromolar range. A docking study of the most active compound 11g revealed key interactions between 11g and Hsp90-Cdc37 complex in which the benzothiazole moiety and the amine chain group were important for improving activity. It is noteworthy that further antitumor activity screening revealed that some compounds exhibited better inhibitory activity than the commercial anticancer drug 5-FU and showed potent suppression activity against drug-resistant cancer cells. In particular, compound 11?g appeared to be the most potent compound against the A549 cell line, at least partly, by inhibition of the activity of Hsp90 and apoptosis induction. The treatment of A549 cells with compound 11g resulted in inhibition of in vitro cell migration through wound healing assay and S phase of cell cycle arrested. In addition, 11g-induced apoptosis was significantly facilitated in A549 cells. Thus, we conclude that GA aminobenzothiazole derivatives may be the potential Hsp90-Cdc37 disruptors with the ability to suppress cells migration and reversed drug-resistant.     

Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted aminoethanol amides

The acid ceramidase (AC) inhibitory activity of octanoylamides, p-tert-butylbenzamides and pivaloylamides of several 2-substituted aminoethanols is reported. All the aminoethanol amides bearing a hexadecyl substituent (C16), as well as (S)-N-(1-(hexadecylthio)-3-hydroxypropan-2-yl)pivaloylamide (SC16-tb) were inhibitory in cell lysates overexpressing AC, while all other compounds were not inhibitors. Kinetic experiments with (R,E)-N-(1-hydroxyoctadec-3-en-2-yl)pivaloylamide (E-tb) and SC16-tb showed that inhibition was competitive, with K(i) values of 34 and 94.0 microM, respectively. None of the compounds inhibited neutral ceramidase. Compounds E-tb and E-c7 (the octanoylamide of the unsaturated base E), which elicited a dose-response inhibition with IC(50) values around 15 microM, were the only AC inhibitors in intact cells. Both compounds were toxic to A549 cells with LD(50) values nearly 40 microM. Flow cytometry studies with E-tb evidenced that this compound induced a concentration-dependent cell cycle arrest at G(1) and a 20-25% apoptosis/late apoptosis/necrosis after a 24-h incubation at 50 microM. In agreement with its activity as acidic ceramidase inhibitor, this effect was accompanied with an increase in the amounts of C14, C16 and C18 ceramides (LC-MS analyses), which suggested that these lipids may be responsible for the cytotoxic activity of E-tb.     

Suzuki coupling based synthesis and in vitro cytotoxic evaluation of 7-heteroaryl-substituted camptothecin analogs

In an effort to discover potent antitumor agents, a series of novel C-7-heteroaryl-substituted camptothecin derivatives were designed and synthesized via microwave-promoted Suzuki coupling reaction. These analogs were then assessed for cytotoxicity against three human tumor cell lines, A549, HCT116, HT-29, and inhibitory effects on topoisomerase I. All of the new compounds showed potent inhibition of human tumor cell growth, among which compound 10a showed higher cytotoxic activity than that of SN-38. Furthermore, this series of compounds retained or enhanced Topo I inhibition.     

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.