A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. RESULTS: A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 µM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 µM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. CONCLUSION: The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Discovery of novel anaplastic lymphoma kinase (ALK) and histone deacetylase (HDAC) dual inhibitors exhibiting antiproliferative activity against non-small cell lung cancer.

A series of novel benzimidazole derivatives were designed and synthesised based on the structures of reported oral available ALK inhibitor and HDAC inhibitor, pracinostat. In enzymatic assays, compound 3b, containing a 2-acyliminobenzimidazole moiety and hydroxamic acid side chain, could inhibit both ALK and HDAC6 (IC50 = 16 nM and 1.03 µM, respectively). Compound 3b also inhibited various ALK mutants known to be involved in crizotinib resistance, including mutant L1196M (IC50, 4.9 nM). Moreover, 3b inhibited the proliferation of several cancer cell lines, including ALK-addicted H2228 cells. To evaluate its potential for treating cancers in vivo, 3b was used in a human A549 xenograft model with BALB/c nude mice. At 20 mg/kg, 3b inhibited tumour growth by 85% yet had a negligible effect on mean body weight. These results suggest a attracting route for the further research and optimisation of dual ALK/HDAC inhibitors.

Antagonizing pathological α-synuclein-mediated neurodegeneration by J24335 via the activation of immunoproteasome.

The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.

Inhibitor development of MTH1 via high-throughput screening with fragment based library and MTH1 substrate binding cavity.

MutT Homolog 1 (MTH1) has been proven to hydrolyze oxidized nucleotide triphosphates during DNA repair. It can prevent the incorporation of wrong nucleotides during DNA replication and mitigate cell apoptosis. In a cancer cell, abundant reactive oxygen species can lead to substantial DNA damage and DNA mutations by base-pairing mismatch. MTH1 could eliminate oxidized dNTP and prevent cancer cells from entering cell death. Therefore, inhibition of MTH1 activity is considered to be an anti-cancer therapeutic target. In this study, high-throughput screening techniques were combined with a fragment-based library containing 2,313 compounds, which were used to screen for lead compounds with MTH1 inhibitor activity. Four compounds with MTH1 inhibitor ability were selected, and compound MI0639 was found to have the highest effective inhibition. To discover the selectivity and specificity of this action, several derivatives based on the MTH1 and MI0639 complex structure were synthesized. We compared 14 complex structures of MTH1 and the various compounds in combination with enzymatic inhibition and thermodynamic analysis. Nanomolar-range IC50 inhibition abilities by enzyme kinetics and Kd values by thermodynamic analysis were obtained for two compounds, named MI1020 and MI1024. Based on structural information and compound optimization, we aim to provide a strategy for the development of MTH1 inhibitors with high selectivity and specificity.

4-Substituted 2-amino-3,4-dihydroquinazolines with a 3-hairpin turn side chain as novel inhibitors of BACE-1.

Herein, we report the identification, design, and synthesis of a series of 4-substituted 2-amino-3,4-dihydroquinazolines with hairpin turn side chains as novel inhibitors of BACE-1. The dihydroquinazoline derivatives were rationally designed by modifying the amide group and relocating the α -hydrophobic substituent on the hairpin turn side chain of lead compound 2 to the C4-position on the 3,4-dihydroquinazoline scaffold to facilitate interactions with the S1, S2 and S1' subsites of BACE-1. Among these derivatives, two compounds exhibited potent BACE-1 inhibitory activity: 4-methyl-substituted (22a, BACE-1 CFA IC50 = 0.38 µM; BACE-1 WCA IC50 = 0.14 µM) and 4-cyclohexylmethyl-substituted (22b, BACE-1 CFA IC50 = 0.49 µM; BACE-1 WCA IC50 = 0.14 µM) 2-amino-3,4-dihydroquinazoline, each bearing a side chain of N-cyclohexyl-N-((1-methyl-1H-pyrazol-4-yl)methyl amide. The results suggest that the structural modifications maintain the hairpin turn topology similar to that of compound 2 and provide an additional interaction with the S2 subsite.

A highly HDAC6-selective inhibitor acts as a fluorescent probe.

HDAC6 receives great attention because of its therapeutic potential for the treatment of various diseases. Selective fluorescence imaging for HDAC6 is important for its pathological and biological studies. However, specific detection of HDAC6 by using a fluorescent small molecule probe remains a great challenge. Herein, a series of fluorescent HDAC6-selective inhibitors incorporating a naphthalimide skeleton were designed and synthesized. A structure-activity relationship study identified that compound JW-1 had the greatest inhibitory activity and superior specificity against HDAC6. JW-1 could substantially increase α-tubulin acetylation and was active against a panel of six cancer cell lines. Photophysical characterization and cellular imaging of MDA-MB-231 cells demonstrated that JW-1 is a highly fluorescent, cell penetrable, small-molecule inhibitor of HDAC6 that can be used for the detection of HDAC6 in complex cellular environments.

J24335 exerts neuroprotective effects against 6-hydroxydopamine-induced lesions in PC12 cells and mice.

Parkinson's disease is the second most prevalent age-related neurodegenerative disease and disrupts the lives of people aged >60 years. Meanwhile, single-target drugs becoming inapplicable as PD pathogenesis diversifies. Mitochondrial dysfunction and neurotoxicity have been shown to be relevant to the pathogenesis of PD. The novel synthetic compound J24335 (11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime), which has been researched similarly to J2326, has the potential to be a multi-targeted drug and alleviate these lesions. Therefore, we investigated the mechanism of action and potential neuroprotective function of J24335 against 6-OHDA-induced neurotoxicity in mice, and in PC12 cell models. The key target of action of J24335 was also screened. MTT assay, LDH assay, flow cytometry, RT-PCR, LC-MS, OCR and ECAR detection, and Western Blot analysis were performed to characterize the neuroprotective effects of J24335 on PC12 cells and its potential mechanism. Behavioral tests and immunohistochemistry were used to evaluate behavioral changes and brain lesions in mice. Moreover, bioinformatics was employed to assess the drug-likeness of J24335 and screen its potential targets. J24335 attenuated the degradation of mitochondrial membrane potential and enhanced glucose metabolism and mitochondrial biosynthesis to ameliorate 6-OHDA-induced mitochondrial dysfunction. Animal behavioral tests demonstrated that J24335 markedly improved motor function and loss of TH-positive neurons and dopaminergic nerve fibers, and contributed to an increase in the levels of dopamine and its metabolites in brain tissue. The activation of both the CREB/PGC-1α/NRF-1/TFAM and PKA/Akt/GSK-3ß pathways was a major contributor to the neuroprotective effects of J24335. Furthermore, bioinformatics predictions revealed that J24335 is a low toxicity and highly BBB permeable compound targeting 8 key genes (SRC, EGFR, ERBB2, SYK, MAPK14, LYN, NTRK1 and PTPN1). Molecular docking suggested a strong and stable binding between J24335 and the 8 core targets. Taken together, our results indicated that J24335, as a multi-targeted neuroprotective agent with promising therapeutic potential for PD, could protect against 6-OHDA-induced neurotoxicity via two potential pathways in mice and PC12 cells.

Discovery of HDAC6, HDAC8, and 6/8 Inhibitors and Development of Cell-Based Drug Screening Models for the Treatment of TGF-ß-Induced Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is incurable, and its progression is difficult to control and thus can lead to pulmonary deterioration. Pan-histone deacetylase inhibitors such as SAHA have shown potential for modulating pulmonary fibrosis yet with off-target effects. Therefore, selective HDAC inhibitors would be beneficial for reducing side effects. Toward this goal, we designed and synthesized 24 novel HDAC6, HDAC8, or dual HDAC6/8 inhibitors and established a two-stage screening platform to rapidly screen for HDAC inhibitors that effectively mitigate TGF-ß-induced pulmonary fibrosis. The first stage consisted of a mouse NIH-3T3 fibroblast prescreen and yielded five hits. In the second stage, human pulmonary fibroblasts (HPFs) were used, and four out of the five hits were tested for caco-2 permeability and liver microsome stability to give two potential leads: J27644 (15) and 20. This novel two-stage screen platform will accelerate the discovery and reduce the cost of developing HDAC inhibitors to mitigate TGF-ß-induced pulmonary fibrosis.

Design, synthesis and biological evaluation of benzo[1.3.2]dithiazolium ylide 1,1-dioxide derivatives as potential dual cyclooxygenase-2/5-lipoxygenase inhibitors.

3-(4-Bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide (5) was discovered as a new prototype for dual inhibitors of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). Thus, the structure-activity relationships of benzo[1.3.2]dithiazolium ylide 1,1-dioxide skeleton were carried out. The 6-NO(2) group played an essential role in the inhibitory activity. In addition, moderate-sized lipophilic substituents at the para-position of the 3-aryl moiety were required for dual COX-2/5-LOX inhibitory activity. Among the identified potent dual inhibitors, 3-(4-tbutylphenyl) derivative 30c (IC(50) values of 0.27 µM and 0.30 µM against COX-2 and 5-LOX, respectively) and 3-(4-biphenyl) derivative 30f (IC(50) values of 0.50 µM and 0.15µM against COX-2 and 5-LOX, respectively) were the most potent dual COX-2/5-LOX inhibitors. Intraperitoneal administration of 30c at 100mg/kg demonstrated potent acute anti-inflammatory activity. As a result, benzo[1.3.2]dithiazolium ylide 1,1-dioxide represented a novel scaffold for the exploitation in developing dual COX-2/5-LOX inhibitors.

Synthesis of 1-substituted carbazolyl-1,2,3,4-tetrahydro- and carbazolyl-3,4-dihydro-ß-carboline analogs as potential antitumor agents.

A series of 1-substituted carbazolyl-1,2,3,4-tetrahydro- and carbazolyl-3,4-dihydro-ß-carboline analogs have been synthesized and evaluated for antitumor activity against human tumor cells including KB, DLD, NCI-H661, Hepa, and HepG2/A2 cell lines. Among these, compounds 2, 6, 7, and 9 exhibited the most potent and selective activity against the tested tumor cells. As for inhibition of topoisomerase II, compounds 1-14 and 18 showed better activity than etoposide. Among them, compounds 3, 4, 7, 9, and 10 exhibited potent activity. The structure and activity relationship (SAR) study revealed correlation between carbon numbers of the side chain and biological activities. The molecular complex with DNA for compound 2 was proposed.

Discovery of 3-(4-bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide as a novel dual cyclooxygenase/5-lipoxygenase inhibitor that also inhibits tumor necrosis factor-alpha production.

In the present study we have discovered compound 1, a benzo[1.3.2]dithiazolium ylide-based compound, as a new prototype dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (5-LOX). Compound 1 was initially discovered as a COX-2 inhibitor, resulting indirectly from the COX-2 structure-based virtual screening that identified compound 2 as a virtual hit. Compounds 1 and 2 inhibited COX-1 and COX-2 in mouse macrophages with IC(50) in the range of 1.5-18.1microM. Both compounds 1 and 2 were also found to be potent inhibitors of human 5-LOX (IC(50)=1.22 and 0.47microM, respectively). Interestingly, compound 1 also had an inhibitory effect on tumor necrosis factor-alpha (TNF-alpha) production (IC(50)=0.44microM), which was not observed with compound 2. Docking studies suggested the (S)-enantiomer of 1 as the biologically active isomer that binds to COX-2. Being a cytokine-suppressive dual COX/5-LOX inhibitor, compound 1 may represent a useful lead structure for the development of advantageous new anti-inflammatory agents.

Synthesis and structure-activity relationship of 6-arylureido-3-pyrrol-2-ylmethylideneindolin-2-one derivatives as potent receptor tyrosine kinase inhibitors.

A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure-activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.

Cytotoxicity and reversal of multidrug resistance by tryptanthrin-derived indoloquinazolines.

AIM: To evaluate the effects and elucidate the mechanisms of a series of indoloquinazolines as novel anticancer agents. METHODS: Condensation of the substituted isatoic anhydride with the substituted isatin was performed to prepare compounds 1-4, followed by adding malononitrile to prepare compounds 5-7. Cytotoxicity was measured by MTT assays. Apoptosis induction was evaluated using DNA fragmentation, cell cycle assay, caspase 3/7 activity and Western blot. RESULTS: Compounds 3, 4, and 5 display cytotoxicity against MCF-7, HeLa, SKOV3, and A498 cancer cells. DNA ladders appear in cells treated with compounds 3, 4, and 5. Within those, compound 4 exhibits the greatest activity in regards to sub-G(1) accumulations in the cell cycle and the activation of caspase-3/7. Furthermore, Fas and Fas ligand levels are elevated by compound 4, implying that the apoptosis is in part mediated through the signals. On the other hand, compounds 1 and 7 display chemosensitizing activity since cytotoxicity of doxorubicine and etoposide is enhanced in combination with compound 1 and 7, respectively, in MCF-7/adr (doxorubicin-resistant) and MCF-7/vp (etoposide-resistant). CONCLUSION: The cytotoxicity of indoloquinazolines is structure-dependent rather than cell type-dependent due to the similar degree of cytotoxicity induced by the individual compounds in all four cell lines. Further modification of the tryptanthrin skeleton is important to develop novel anticancer agents bearing either cytotoxicity against MCF-7 cells or drug resistance reversal in MCF-7/adr and MCF-7/vp.

Structure of Human Phosphodiesterase 5A1 Complexed with Avanafil Reveals Molecular Basis of Isoform Selectivity and Guidelines for Targeting α-Helix Backbone Oxygen by Halogen Bonding.

Phosphodiesterase 5A1 (PDE5) is a key target for treating cardiovascular diseases and erectile dysfunction. Here, we report the crystal structure of PDE5 complexed with the sole second generation drug avanafil. Analysis of protein-drug interactions revealed the structural basis of avanafil's superior isoform selectivity. Moreover, a halogen bonding was observed between avanafil and a backbone carbonyl oxygen of an adjacent α-helix, whose contribution to inhibitory potency illustrates the feasibility of exploiting α-helix backbone in structure-based drug design.

An improved screening model to identify inhibitors targeting zinc-enhanced amyloid aggregation.

Zinc, which is abundant in senile plaques consisting mainly of fibrillar beta-amyloid (Abeta), plays a critical role in the pathogenesis of Alzheimer's disease. Treatment with zinc chelators such as clioquinol has been used to prevent Abeta aggregation in Alzheimer's patients; however, clioquinol produces severe side effects. A simple, easy, inexpensive, and versatile screen to identify zinc chelators for inhibition of Abeta aggregation is currently unavailable. We thus developed a high-throughput screen that identifies zinc chelators with anti-Abeta aggregation activity. The recombinant Abeta peptides, aggregated on solid-phase microplates, formed Abeta-immunopositive beta-sheet-containing structures in the presence of zinc. Formation of these Abeta fibrils was specifically blocked by metal ion chelators. This screening model improves identification of zinc-enhanced Abeta fibrils and anti-Abeta aggregation mediated by zinc chelating. The convenient system could qualitatively and quantitatively assay a large sample pool for Abeta aggregation inhibition and dissolution of Abeta aggregates. This screen is practical, reliable, and versatile for comprehensive detection of amyloid fibrillation and identification of inhibitors of Abeta aggregation.

Downregulation of GSTpi expression by tryptanthrin contributing to sensitization of doxorubicin-resistant MCF-7 cells through c-jun NH2-terminal kinase-mediated apoptosis.

Overexpression of GSTpi and underexpression of Topo II expression are associated with multidrug resistance (MDR) phenotype through nontransporter pathway. Tryptanthrin, a quinazoline derivative, was reported to sensitize resistant cells to doxorubicin by downregulation of MDR1 expression. This study aims to extendedly investigate the effect of tryptanthrin on the role of nontransporter-based genes in determining the MDR response in doxorubicin-resistant MCF-7 cells (MCF-7/adr). Results show that tryptanthrin downregulates GSTpi expression and reduces glutathione S-transferase (GST) activity, but has no effect on Topo II expression. Less production of GSTpi decomposes the protein-protein interactions of GSTpi and c-jun NH2-terminal kinase (JNK). The resulting free-form JNK undergoes phosphorylation upon elevated intracellular doxorubicin accumulation and subsequently activates JNK-mediated apoptosis. In conclusion, in addition to transporter pathway, tryptanthrin reverses MDR partly by modulating GSTpi-related pathway, a nontransporter pathway, in MCF-7/adr cells. It indicates that tryptanthrin may act as a potential chemoadjuvant agent through multiple targets.

Acrylamide Functional Group Incorporation Improves Drug-like Properties: An Example with EGFR Inhibitors.

We demonstrate that the acrylamide group can be used to improve the drug-like properties of potential drug candidates. In the EGFR inhibitor development, both the solubility and membrane permeability properties of compounds 6a and 7, each containing an acrylamide group, were substantially better than those of gefitinib (1) and AZD3759 (2), respectively. We demonstrated that incorporation of an acrylamide moiety could serve as a good strategy for improving drug-like properties.

High-selective HDAC6 inhibitor promotes HDAC6 degradation following autophagy modulation and enhanced antitumor immunity in glioblastoma.

Glioblastoma is the most fatal type of primary brain cancer, and current treatments for glioblastoma are insufficient. HDAC6 is overexpressed in glioblastoma, and siRNA-mediated knockdown of HDAC6 inhibits glioma cell proliferation. Herein, we report a high-selective HDAC6 inhibitor, J22352, which has PROTAC (proteolysis-targeting chimeras)-like property resulted in both p62 accumulation and proteasomal degradation, leading to proteolysis of aberrantly overexpressed HDAC6 in glioblastoma. The consequences of decreased HDAC6 expression in response to J22352 decreased cell migration, increased autophagic cancer cell death and significant tumor growth inhibition. Notably, J22352 reduced the immunosuppressive activity of PD-L1, leading to the restoration of host anti-tumor activity. These results demonstrate that J22352 promotes HDAC6 degradation and induces anticancer effects by inhibiting autophagy and eliciting the antitumor immune response in glioblastoma. Therefore, this highly selective HDAC6 inhibitor can be considered a potential therapeutic for the treatment of glioblastoma and other cancers.

Quinazolin-2,4-dione-Based Hydroxamic Acids as Selective Histone Deacetylase-6 Inhibitors for Treatment of Non-Small Cell Lung Cancer.

We designed and synthesized quinazolin-2,4-dione-based hydroxamic acids to serve as selective competitive inhibitors of histone deacetylase-6 (HDAC6). The most potent and selective compound, 3d (IC50, 4 nM, HDAC6; IC50 > 10 µM, HDAC1), substantially increased acetylation of α-tubulin instead of histones in the lung cancer cell line, LL2. Paclitaxel in combination with 3d had a synergistic anticancer effect on reduction of programmed death-ligand 1 expression in LL/2 cells. When given orally, 3d was mainly found to locate in the liver and lungs, at a concentration 18- to 70-fold greater, respectively, than in plasma. As an orally active HDAC6 inhibitor, 3d (20 mg/kg) potentiated paclitaxel antitumor activity (percentage tumor growth inhibition, 67.5%) in a xenograft syngeneic non-small cell lung cancer mouse model.

Benzyl ether-linked glucuronide derivative of 10-hydroxycamptothecin designed for selective camptothecin-based anticancer therapy.

A beta-glucuronidase-activated prodrug approach was applied to 10-hydroxycamptothecin, a Camptotheca alkaloid with promising antitumor activity but poor water solubility. We synthesized a glucuronide prodrug of 10-hydroxycamptothecin ( 7) in which glucuronic acid is connected via a self-immolative 3-nitrobenzyl ether linker to the 10-OH group of 10-hydroxycamptothecin. Compound 7 was 80 times more soluble than 10-hydroxycamptothecin in aqueous solution at pH 4.0 and was stable in human plasma. Prodrug 7 was 10- to 15-fold less toxic than the parent drug to four human tumor cell lines. In the presence of beta-glucuronidase, prodrug 7 could be activated to elicit similar cytotoxicity to the parent drug in tumor cells. Enzyme kinetic studies showed that Escherichia coli beta-glucuronidase had a quite low K m of 0.18 microM for compound 7 and exhibited 520 times higher catalytic efficiency for 7 than for 6 (a glucuronide prodrug of 9-aminocamptothecin). Molecular modeling studies predicted that compound 7 would have a higher binding affinity to human beta-glucuronidase than compound 6. Prodrug 7 may be useful for selective cancer chemotherapy by a prodrug monotherapy (PMT) or antibody-directed enzyme prodrug therapy (ADEPT) strategy.