Discovery of Oral Anticancer 1,2-Bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine Hybrids That Inhibit Angiogenesis and Induce DNA Cross-Links.

Designing hybrid molecules with dual functions is one approach to improve the therapeutic efficacy of combination treatment. We have previously conjugated phthalazine and bis(hydroxymethyl)pyrrole pharmacophores to form hybrids bearing antiangiogenesis and DNA interstrand cross-linking activities. To improve the bioavailability, we adopted a benzology approach to design and synthesize a new series of 1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazines. These new hybrids retained the dual functions and could be formulated into vehicles for intravenous and oral administration. Among them, we demonstrated that compound 19a with dimethylamine at the C6 position markedly suppressed the tumor growth of human small cell lung cancer cell line H526, squamous lung cancer cell line H520, and renal cancer cell line 786-O in nude mice, implying that compound 19a is a broad-spectrum anticancer agent. Our results implicated that the conjugation of antiangiogenic and DNA cross-linking is likely to be a helpful approach to improving the efficacy of combination therapy.

Antitumor Effects of the Novel Quinazolinone Holu-12: Induction of Mitotic Arrest and Apoptosis in Human Oral Squamous Cell Carcinoma CAL27 Cells.

BACKGROUND/AIM: Quinazolinone is a privileged chemical structure employed for targeting various types of cancer. This study aimed to demonstrate the antitumor activity of synthesized 6,7-disubstituted-2-(3-fluorophenyl) quinazolines (HoLu-11 to HoLu-14). MATERIALS AND METHODS: The cytotoxicity was assessed by the sulforhodamine B (SRB) assay. The cell cycle was examined by flow cytometry. The expression levels of cell cycle- and apoptosis-related proteins were estimated by western blotting. A xenograft animal model was used to explore the antitumor effects of HoLu-12. RESULTS: Among four synthetic quinazolinone derivatives, HoLu-12 significantly reduced the viability of oral squamous cell carcinoma (OSCC) cells. HoLu-12 induced G2/M arrest and increased the expression of cyclin B, histone H3 (Ser10) phosphorylation, and cleaved PARP, indicating that HoLu-12 could induce mitotic arrest and then apoptosis. Moreover, the combination of HoLu-12 and 5-fluorouracil (5-FU) displayed synergistic toxic effect on OSCC cells. HoLu-12 significantly inhibited tumor growth in vivo. CONCLUSION: HoLu-12 induces mitotic arrest and leads to apoptosis of OSCC cells. Furthermore, HoLu-12 alone or in combination with 5-FU is a potential therapeutic agent for OSCC.

[Electroacupuncture at "Zusanli"(ST36) and "Xuanzhong"(GB39) improves inflammatory reactions and reduces levels of serum MMP-3 and MMP-9 in adjuvant arthritis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli" (ST36) and "Xuanzhong" (GB39) on joint inflammatory reactions and serum matrix metalloproteinase-3 (MMP-3) and MMP-9 contents in rats with adjuvant arthritis (AA), so as to explore its mechanism underlying improvement of AA. METHODS: Forty male SD rats were randomly divided into control, model, acupoint and non-acupoint groups (n=10 in each group). The arthritis model was established by hypodermic injection of complete Freund's adjuvant (0.1 mL) into the bilateral footpads. EA (2 Hz, 3 V) was applied to bilateral ST36 and GB39 or two non-acupoints (5 mm left to ST36 and GB39) for 15 min, once every other day for a total of 8 times. The arthritis index score was evaluated according to the severity of local erythema and swelling of the ankle joint, plantar joint, toe joint and foot metacarpal joint (0-4 points). The inflammatory conditions of the ankle joint were observed by H.E. staining, and the contents of serum MMP-3 and MMP-9 were assayed by ELISA. RESULTS: The arthritis index score and serum concentrations of MMP-3 and MMP-9 were significantly increased in the model group relevant to the control group (P<0.01), and obviously decreased after EA intervention on the 18th day (P<0.01). The therapeutic effect of acupoint EA was notably superior to non-acupoint EA in down-regulating the arthritis index score and serum MMP-3 and MMP-9 concentrations (P<0.01). Under light microscope, marked proliferation of the synovial cells, inflammatory cell infiltration and increase of newly blood vessels were observed in the ankle joint of the model group, which was relatively milder in the acupoint group. CONCLUSION: acupoint EA intervention can significantly alleviate the inflammatory reaction of AA rats, which may be related to its effects in reducing the levels of serum MMP-3 and MMP-9.

Design and Synthesis of 1,2-Bis(hydroxymethyl)pyrrolo[2,1- a]phthalazine Hybrids as Potent Anticancer Agents that Inhibit Angiogenesis and Induce DNA Interstrand Cross-links.

Hybrid molecules are composed of two pharmacophores with different biological activities. Here, we conjugated phthalazine moieties (antiangiogenetic pharmacophore) and bis(hydroxymethyl)pyrrole moieties (DNA cross-linking agent) to form a series of bis(hydroxymethyl)pyrrolo[2,1- a]phthalazine hybrids. These conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, arresting cell cycle progression at the G2/M phase, triggering apoptosis, and inhibiting vascular endothelial growth factor receptor 2 (VEGFR-2) in endothelial cells. Among them, compound 29d encapsulated in a liposomal formulation (e.g., 29dL) significantly suppressed the growth of small-cell lung cancer cell (H526) xenografts in mice. Based on immunohistochemical staining, the tumor xenografts in mice treated with 29dL showed time-dependent decreases in the intensity of CD31, a marker of blood vessels, whereas the intensity of γ-H2AX, a marker of DNA damage, increased. The present data revealed that the conjugation of antiangiogenic and DNA-damaging agents can generate potential hybrid agents for cancer treatment.

Disruption of IGF­1R signaling by a novel quinazoline derivative, HMJ­30, inhibits invasiveness and reverses epithelial-mesenchymal transition in osteosarcoma U­2 OS cells.

Osteosarcoma is the most common primary malignancy of the bone and is characterized by local invasion and distant metastasis. Over the past 20 years, long-term outcomes have reached a plateau even with aggressive therapy. Overexpression of insulin-like growth factor 1 receptor (IGF­1R) is associated with tumor proliferation, invasion and migration in osteosarcoma. In the present study, our group developed a novel quinazoline derivative, 6-fluoro­2-(3-fluorophenyl)-4-(cyanoanilino)quinazoline (HMJ­30), in order to disrupt IGF­1R signaling and tumor invasiveness in osteosarcoma U­2 OS cells. Molecular modeling, immune-precipitation, western blotting and phosphorylated protein kinase sandwich ELISA assays were used to confirm this hypothesis. The results demonstrated that HMJ­30 selectively targeted the ATP-binding site of IGF­1R and inhibited its downstream phosphoinositide 3-kinase/protein kinase B, Ras/mitogen-activated protein kinase, and IκK/nuclear factor-κB signaling pathways in U­2 OS cells. HMJ­30 inhibited U­2 OS cell invasion and migration and downregulated protein levels and activities of matrix metalloproteinase (MMP)­2 and MMP-9. An increase in protein levels of tissue inhibitor of metalloproteinase (TIMP)­1 and TIMP­2 was also observed. Furthermore, HMJ­30 caused U­2 OS cells to aggregate and form tight clusters, and these cells were flattened, less elongated and displayed cobblestone-like shapes. There was an increase in epithelial markers and a decrease in mesenchymal markers, indicating that the cells underwent the reverse epithelial-mesenchymal transition (EMT) process. Overall, these results demonstrated the potential molecular mechanisms underlying the effects of HMJ­30 on invasiveness and EMT in U­2 OS cells, suggesting that this compound deserves further investigation as a potential anti-osteosarcoma drug.

A Low-Toxicity DNA-Alkylating N-Mustard-Quinoline Conjugate with Preferential Sequence Specificity Exerts Potent Antitumor Activity Against Colorectal Cancer.

Efficacy and safety are fundamental prerequisites for anticancer drug development. In the present study, we explored the anti-colorectal cancer (CRC) activity of SL-1, a DNA-directed N-mustard-quinoline conjugate. The N-mustard moiety in SL-1 induced DNA strand breaks, interstrand cross-links (ICLs), G2/M arrest, and apoptosis, whereas its quinoline moiety preferentially directed SL-1 to target the selective guanine sequence 5'-G-G/C-N-G-C/T-3'. Notably, SL-1 was highly cytotoxic to various CRC cell lines. Experiments using xenograft models revealed that SL-1 was more potent than 5-fluorouracil (5-FU) and oxaliplatin for suppressing the growth of RKO and RKO-E6 (oxaliplatin-resistant subline) cells as well as metastatic SW620 cells. In addition, SL-1 combined with 5-FU was more effective than oxaliplatin and 5-FU for suppressing RKO or SW620 cell growth in mice. Significantly, compared with cisplatin, oxaliplatin, or 5-FU, SL-1 alone or in combination with 5-FU did not cause obvious kidney or liver toxicity in ICR mice. In summary, SL-1, a DNA-directed alkylating agent, is established as an anti-CRC agent with high efficacy and low toxicity and thus warrants further development for the treatment of CRC patients.

Novel N-mustard-benzimidazoles/benzothiazoles Hybrids, Synthesis and Anticancer Evaluation.

BACKGROUND: Bendamustine, an N-mustard-benzoimidazole hybrid conjugate, was recently approved for the treatment of chronic lymphocytic leukemia. However, the short half-life of bendamustine may limit its clinical applications. OBJECTIVE: The purpose of this study is to design and synthesize compounds with a more favorable pharmacokinetic profile. METHODS: We synthesized a series of hybrid molecules comprising a phenyl N-mustard moiety and benzothiazole or benzimidazole scaffold linked via a urea linker and evaluated their antitumor activity and plasma stability. RESULTS: We revealed that these agents exhibited significant cytotoxicity against a panel of human lymphoblastic leukemia and human solid tumor cells in culture. Human lymphoblastic leukemia CCRM-CEM cells were the most sensitive to the tested compounds. In general, the new hybrids were as potent as cisplatin, but significantly more cytotoxic than bendamustine. Phenyl N-mustard-benzothiazole compound 27d and phenyl N-mustardbenzimidaloe compound 32b possessed significant cytotoxicity and led to apoptotic death in the treated tumor cells. These two agents were able to induce DNA interstrand cross-linking and arrested cell cycle progression at the G2/M phase. Furthermore, we showed that these new hybrids were more chemically stable than bendamustine in rat plasma. CONCLUSION: Our results suggest that conjugation of phenyl N-mustard pharmacophore at C6 of benzimidazole or at C8 of the benzothiazole ring via a urea linker is likely an approach to increase the chemical stability and bioavailability. Highlights ⇒ Series of benzimidazoles and benzothiazoles linked to N-mustard were synthesized. ⇒ The newly synthesized derivatives induced DNA interstrand cross-links. ⇒ These derivatives induced cell cycle arrest in the G2/M phase and triggered apoptosis in H460 cells. ⇒ The new compounds are more cytotoxic than bendamustine. ⇒ The new compounds were chemically more stable than bendamustine in rat plasma.

Novel indolizino[8,7-b]indole hybrids as anti-small cell lung cancer agents: Regioselective modulation of topoisomerase II inhibitory and DNA crosslinking activities.

A novel series of bis(hydroxymethyl)indolizino[8,7-b]indole hybrids composed of ß-carboline (topoisomerase I/II inhibition) and bis(hydroxymethyl)pyrrole (DNA cross-linking) are synthesized for antitumor evaluation. Of tumor cell lines tested, small cell lung cancer (SCLC) cell lines are the most sensitive to the newly synthesized compounds. These hybrids induce cell cycle arrest at the G2/M phase, trigger tumor cell apoptotic death, and display diverse mechanisms of action involving topoisomerase II (Topo II) inhibition and induction of DNA cross-linking. Intriguingly, the substituent at N11 (H or Me) plays a critical role in modulating Topo II inhibition and DNA cross-linking activities. N11-Me derivatives predispose to induce DNA crosslinks, whereas N11-H derivatives potently inhibit Topo II. Computational analysis implicates that N11-Me restrict the torsion angles of the two adjacent OH on pyrrole resulting in a favorable of DNA cross-linking. Among these hybrids, compound 17a with N11-H is more effective than cisplatin and etoposide, but as potent as irinotecan, against the growth of SCLC H526 cells in xenograft model.

Lifeact and Utr230 induce distinct actin assemblies in cell nuclei.

Nuclear actin assembly in somatic cells has been an enigma for a long time. Recently, with the advancement of novel F-actin probes, researchers have started to uncover this mystery. In this study, we investigated the actin dynamics in somatic cell nuclei using two probes: Lifeact and Utr230. Surprisingly, we observed that both Lifeact and Utr230 significantly interfered with actin dynamics in cell nuclei. Moreover, these two probes induced distinct patterns of nuclear actin assembly. While Lifeact induced filamentous actin assembly in cell nuclei, Utr230 led to various patterns of actin aggregates, including fibers, small puncta, and large patches. Moreover, the interference of actin dynamics by Lifeact was limited to nuclear actin, while Utr230 induced actin aggregation in both the nucleus and cytoplasm. Using time-lapse microscopy, we found that Lifeact-induced actin fibers remained steady over hours of observation, indicating a deficiency of nuclear F-actin reorganization. These results suggest that Lifeact and Utr230 both interfere with nuclear actin dynamics but with distinct mechanisms. This is an important finding for research on nuclear actin assembly and highlights the potential value of these two probes for exploring the native mechanisms underlying nuclear actin dynamics, which appear to be altered in the presence of these probes.

MJ-66 induces malignant glioma cells G2/M phase arrest and mitotic catastrophe through regulation of cyclin B1/Cdk1 complex.

Malignant gliomas are among the most devastating cancers as they are resistant to many kinds of treatment. Despite recent advances in the diagnosis and treatment, the prognosis of patients remains very poor and the development of new drug is urgently needed. Here, we report that a synthetic quinazolinone analog 2-(naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone (MJ-66) induced glioma cell death. Immunofluorescence staining showed that MJ-66-induced cell death was associated with multinucleated phenotype and multipolar spindles that were typical characteristics of mitotic catastrophe. Flow cytometry analysis revealed that MJ-66 caused glioma cell cycle arrest at G2/M phase and increased the proportion of polyploidy cells. Western blotting indicated that the expression of cyclin B1, Cdk1 pY15 and Cdk1 increased after treatment with MJ-66. MJ-66 effectively inhibited tumor growth and induced apoptosis in the xenograft animal model of U87 human glioma cells. Together, these results suggest that MJ-66 inhibited malignant gliomas growth through inducing mitotic catastrophe by interference with G2/M cell cycle checkpoint which may open a new avenue for the treatment of malignant gliomas.

Combined versus monotherapy or concurrent therapy for treatment of thalassaemia.

A combined deferasirox (DFX) and deferiprone (DFP) treatment protocol for relieving thalassemia patients' iron-overload was designed and the pharmacokinetic study was performed by LC-MS/MS. For this open-label, randomized trial, eight patients were recruited and randomly allocated to different treatment regimens: (A) monotherapy with single oral dose of DFX 30 mg/kg, (B) monotherapy with DFP 80 mg/kg/day, twice daily, (C) combined therapy with DFX and DFP (DFX 30 mg/kg for first dose, DFP 40 mg/kg 7 hours later, and DFP 40 mg/kg after another 7 h) and (D) concurrent therapy with DFX 30 mg/kg and DFP 80 mg/kg. Descriptive statistics evaluated pharmacokinetic parameters, AUC0-t, AUC0-inf, Cmax, Tmax, T1/2 and MRT. A positive pharmacokinetic drug interaction was observed in combined therapy. In case of DFX, combined therapy tallied about 2-fold larger than monotherapy in AUC, 1.5-fold larger in Cmax, 1 h longer in Tmax, but 1 h shorter in T1/2. Regarding DFP, most such parameters of combined therapy concurred with monotherapy. Conversely, negative drug interaction was observed in concurrent therapy. With DFX, concurrent therapy attained 1.2- to 2.2-fold lower than monotherapy in AUC0-t and Cmax, 0.6-h shorter in Tmax, and 3-fold longer in T1/2. With DFP, concurrent therapy proved approximately 2-fold larger than monotherapy in AUC and Cmax, 2.5-fold longer in T1/2, and 1.4-fold longer in MRT. Follow-up of subjects' clinical examinations and subjective symptoms showed no adverse events. Our findings showed the combined therapy had advantages, safe, convenient and painless for patients, over the existing concurrent therapy with deferoxamine (DFO) and DFX.

The synthesis and biological evaluation of new DNA-directed alkylating agents, phenyl N-mustard-4-anilinoquinoline conjugates containing a urea linker.

We synthesized a series of phenyl N-mustard-4-anilinoquinoline conjugates to study their antitumorigenic effects. These agents were prepared by the condensation of 4-[N,N-bis(2-chloroethyl)amino]phenyl isocyanate with 6-amino-4-methylamino or 4-anilinoquinolines. The structure-activity relationship (SAR) studies revealed that the C2-methylquinoline derivatives (18a-o) were generally more cytotoxic than the C2-phenylquinoline conjugates (23a-d) in inhibiting the cell growth of various human tumor cell lines in vitro. However, the methylamino or aniline substituents at C4 of quinoline did not influence the cytotoxic effects. The title conjugates were capable of inducing DNA cross-linking and promoting cell-cycle arrest at the G2/M phase. This study demonstrates that phenyl N-mustard-4-anilinoquinoline conjugates are generally more potent than phenyl N-mustard-4-anilinoquinazoline conjugates against the cell growth of various tumor cell-lines.

A novel antitubulin agent, DPQZ, induces cell apoptosis in human oral cancer cells through Ras/Raf inhibition and MAP kinases activation.

6-(N,N-Dimethylamino)-2-(naphthalene-1-yl)-4-quinazolinone (DPQZ)-induced apoptosis was accompanied by the characteristics of DNA fragmentation and phosphatidylserine externalization in human oral cancer HSC-3 cells. The IC50 (half maximal inhibitory concentration) value of DPQZ is about 0.25 µM at 24 h. The interference in the dynamics of tubulin and cell division of DPQZ, like vinblastine (0.01 µM), has been proven in this study. Treatment of HSC-3 cells with DPQZ resulted in many of mitotic cells with multipolar spindles. Up-regulation of MAP kinases, such as ERK, JNK, and p38, mediated by DPQZ appears to be involved in DPQZ-induced apoptosis in HSC-3 cells. It is worthy of note that the expression of Ras and c-Raf that lie upstream of ERK were inhibited by DPQZ. In addition, the DPQZ-induced cell death was attenuated by JNK inhibitor SP600125 (3 or 10 µM), not by the ERK or p38 inhibitors. JNK inhibitor abolished the DPQZ-induced increase in the phosphorylation of Bcl-2 and the protein levels of proform caspase-3, caspase-8, and caspase-9, indicating that JNK is an upstream activator of Bcl-2 and caspase family members and plays a key role in DPQZ-induced HSC-3 cell apoptosis. We also attempted to develop an anticancer drug that is designed to kill rapidly dividing cancer cells while causing less damage to normal cells. The DPQZ-induced cytotoxicity against human gingival fibroblasts was less than that against HSC-3 cells. Our work provides a new strategy and mechanism for developing anticancer drug and may contribute to clinical anticancer drug discovery and application.

Development of a fast LC-MS/MS assay for the determination of deferiprone in human plasma and application to pharmacokinetics.

A fast and accurate liquid chromatography/tandem mass spectrometric (LC-MS/MS) assay was first developed and validated for the determination of deferiprone in human plasma. The analytes were extracted with acetonitrile from only 50 µL aliquots of human plasma to achieve the protein precipitation. After extraction, chromatographic separation of analytes in human plasma was performed using a Synergi Fusion-RP 80A column at 30 °C. The mobile phase consisted of methanol and 0.2% formic acid containing 0.2 mM EDTA (60:40, v/v). The flow rate of the mobile phase was 0.8 mL/min. The total run time for each sample analysis was 4 min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor-to-parent ion transitions m/z 140.1 → 53.1 for deferiprone and m/z 143.1 → 98.1 for internal standard. A linear range was established from 0.1 to 20 µg/mL. The limit of detection was determined as 0.05 µg/mL. The validated method was estimated for linearity, recovery, stability, precision and accuracy. Intraday and interday precisions were 4.3-5.5 and 4.6-7.3%, respectively. The recovery of deferiprone was in the range of 80.1-86.8%. The method was successfully applied to a pharmacokinetic study of deferiprone in six thalassemia patients.

The synthesized novel fluorinated compound (LJJ-10) induces death receptor- and mitochondria-dependent apoptotic cell death in the human osteogenic sarcoma U-2 OS cells.

We designed the 6-fluoro-2-(3-fluorophenyl)-4-substituted anilinoquinazoline derivatives as less toxic anti-cancer candidates. Our result demonstrated that LJJ-10 has greater cytotoxicity than that of the other compounds in human osteogenic sarcoma U-2 OS cells. LJJ-10-induced apoptosis was associated with enhancing ROS generation, DNA damage, and an increase of the protein levels of Fas, FasL, FADD, caspase-8, cytochrome c, Apaf-1, AIF, Endo G, caspase-9 and caspase-3 in U-2 OS cells. LJJ-10-triggered growth inhibition was significantly attenuated by N-acetylcysteine, cyclosporine A, anti-FasL monoclonal antibody, and caspase-8, -9 and -3 specific inhibitors in U-2 OS cells. We suggest that LJJ-10-induced apoptotic cell death in U-2 OS cells through death receptor- and mitochondria-dependent apoptotic signaling pathways.