Structure-based drug discovery of novel fused-pyrazolone carboxamide derivatives as potent and selective AXL inhibitors

As a novel and promising antitumor target, AXL plays an important role in tumor growth, metastasis, immunosuppression and drug resistance of various malignancies, which has attracted extensive research interest in recent years. In this study, by employing the structure-based drug design and bioisosterism strategies, we designed and synthesized in total 54 novel AXL inhibitors featuring a fused-pyrazolone carboxamide scaffold, of which up to 20 compounds exhibited excellent AXL kinase and BaF3/TEL-AXL cell viability inhibitions. Notably, compound 59 showed a desirable AXL kinase inhibitory activity (IC50: 3.5 nmol/L) as well as good kinase selectivity, and it effectively blocked the cellular AXL signaling. In turn, compound 59 could potently inhibit BaF3/TEL-AXL cell viability (IC50: 1.5 nmol/L) and significantly suppress GAS6/AXL-mediated cancer cell invasion, migration and wound healing at the nanomolar level. More importantly, compound 59 oral administration showed good pharmacokinetic profile and in vivo antitumor efficiency, in which we observed significant AXL phosphorylation suppression, and its antitumor efficacy at 20 mg/kg (qd) was comparable to that of BGB324 at 50 mg/kg (bid), the most advanced AXL inhibitor. Taken together, this work provided a valuable lead compound as a potential AXL inhibitor for the further antitumor drug development.

Synthesis and evaluation of antifungal activity studies of novel 2-(2-pyridyl)-2H-pyrazole-3-carboxamide derivatives

Background: Invasive fungal infections have negative impact on the health of immunocompromised individuals. With the development of fungal resistance to currently available antifungal drugs, there is a need to develop novel compounds with antifungal activity. Aims and Objectives: The objectives of the study are as follows: (1) To synthesize novel 2-(2-pyridyl)-2H-pyrazole-3-carboxamide derivatives and (2) to evaluate the antifungal activity of novel 2-(2-pyridyl)-2H-pyrazole-3-carboxamide derivatives. Materials and Methods: Novel 2-(2-pyridyl)-2H-pyrazole-3-carboxamide derivatives were prepared by multi step synthesis and characterized by LC-MS, 1HNMR and 13C NMR. The antifungal activity of these derivatives was assessed using Fusarium oxysporum, Aspergillus flavus, and Candida albicans by disc diffusion method. Results: We have synthesized fourteen derivatives of 2-(2-pyridyl)-2H-pyrazole-3-carboxamide. Most of the compounds possess good antifungal activity against F. oxysporum and C. albicans strains. Conclusion: We synthesized a series of novel 2-(2-pyridyl)-2H-pyrazole-3-carboxamide derivatives having good antifungal activity against F. oxysporus and C. albicans using a simple and low cost procedure.

Characterization of inthomycin biosynthetic gene cluster revealing new insights into carboxamide formation / 中国天然药物

Inthomycins are polyketide antibiotics which contain a terminal carboxamide group and a triene chain. Inthomycin B (1) and its two new analogues 2 and 3 were isolated from the crude extract of Streptomyces pactum L8. Identification of the gene cluster for inthomycin biosynthesis as well as the N-labeled glycine incorporation into inthomycins are described. Combined with the gene deletion of the rare P450 domain in the NRPS module, a formation mechanism of carboxamide moiety in inthomycins was proposed via an oxidative release of the assembly chain assisted by the P450 domain.

Synthesis and Antitumor Activities of N-hydroxy-5-(3-substituted ureido)-1H-indole-2-carboxamide Derivatives / 中国药学杂志

OBJECTIVE: To design and synthesize N-hydroxy-5-(3-substituted urea)-1H-indole-2-amide derivatives and investigate their anti-tumor activity in vitro. METHODS: A series of new N-hydroxy-5-(3-substituted ureido)-1H-indole-2-carboxamide derivatives (7a-7n)were designed and prepared from p-nitrophenylhydrazine and ethyl pyruvate. Their antitumor activities against Hela (breast cancer) lines, cervical cancer cell (MCF7) and human hepatoma cell lines (HepG2) were evaluated by methyl thiazolyl (MTT) method. RESULTS: Target compounds indicated certain antitumor activities, especially compound 7h showed the strongest cytotoxicity to Hela cells with a half inhibition concentration (IC50) of 7.31 μmol•L-1. CONCLUSION: The series of compounds show preferable antitumor activities, which are worthy of further study.

Transamidación y transamidación-reducción de N-benciltiramina con DMF / Transamidation and transamidation-reduction of N-benzyltyramine with DMF / Transamidação e transamidação-redução de N-benziltiramina com DMF

Resumen La tiramina y la N-benciltiramina reaccionan con formaldehído para formar azaciclofanos por medio de condensaciones tipo Mannich aromáticas y reaccionan con aldehídos no enolizables para formar las respectivas bases de Schiff. En este artículo se presenta la síntesis inesperada de N-bencil-N-formiltiramina y N-bencil-N-metiltiramina por medio de reacciones de transamidación y de transamidación-reducción de N-benciltiramina con N,N-dimetilformamida. Para explicar el curso de la reacción se propuso un mecanismo que involucra la formilación de N-benciltiramina y posterior reducción de Leuckart-Wallach inducida por ácido fórmico generado in situ.

Abstract Tyramine and N-benzyltyramine react with formaldehyde to form azacyclophanes by means of aromatic Mannich reactions and react with non-enolizable aldehydes to form the respective Schiff bases. In this paper we present the unexpected synthesis of N-formyl-N-benzyltyramine and N-methyl-N-benzyltyramine by means of transamidation and transamidation-reduction of N-benzyltyramine with N,N-dimethylformamide. A reaction mechanism involving formylation of N-benzyltiramine followed by a Leuckart-Wallach reduction is proposed for rationalising such transformation.

Resumo A tiramina e a N-benziltiramina reagem com formaldeído para formar azaciclofanos por meio de reações de Mannich aromáticas e reagem com aldeídos não-enolizáveis para formar as respectivas bases de Schiff. Neste trabalho apresenta-se a síntese inesperada de N-formil-N-benziltiramina e N-metil-N-benziltiramina por meio de transamidação e transamidação-redução de N-benziltiramina promovida pela N,N-dimetilformamida. Propõe-se um mecanismo de reação que envolve a formilação de N-benziltiramina seguida por uma redução de Leuckart-Wallach induzida pelo ácido fórmico gerado in situ.

Design, synthesis and anti-tumor activity of novel 5-amino-2-(benzylthio) thia-zole-4-carboxamide derivatives / 中国药科大学学报

A series of 5-amino-2-( benzylthio ) thiazole-4-carboxamide derivatives were designed and synthesized to discover novel compounds with anti-tumor activity. Compounds DDO-5401-DDO-5416 were synthesized using 2-amino-2-cyanoacetamide as the start material. The structures of the synthesized compounds were confirmed by IR, 1 H NMR and ESI-MS. The in vitro anti-tumor activities of the synthesized compounds were determined by MTT assay in HCT116 , HepG2 , A549 , MDA-MB-231 and MCF-7 cell lines. Target compounds showed good anti-tumor activity especially in A549 cell line. SAR study showed that electron donating groups were more favorable than electron absorption ones. Compound DDO-5413 exhibited noteworthy activity in MDA-MB-231 and MCF-7 cell lines with IC50 value lower than the positive reference dasatinib. It suggested that DDO-5413 might be the candidate for further investigation.

Determination of 4-( 4-Amino-3-fluorophenoxy )-N-methylpyridine-2-carboxamide in Regorafenib by LC-MS/MS / 中国药师

Objective: To establish an LC-MS/MS method for the determination of 4-( 4-amino-3-fluorophenoxy )-N-methylpyri-dine-2-carboxamide ( AFP-PMA) as a genotoxic impurity in regorafenib. Methods: The content of AFP-PMA was determined by an LC-MS/MS method. A Waters XBridge Shield RP18 column was adopted to separate the samples and the column temperature was 50℃. The mobile phase consisted of 5 mmol·L-1ammonium acetate aqueous (A)-acetonitrile (B) with gradient elution (0~9 min, 5%B→90%B) at a flow rate of 1. 0 ml·min-1. An electrospray ionization source (ESI) was used in a positive-ion and multiple reactions monitoring mode. The ion channel was m/z 262. 2→244. 1. Results:The standard curve was linear within the range of 2. 41-980. 90 ng·ml-1(r=0. 9998) and the limit of quantification was 8. 02 ng·ml-1. The limit of detection was 2. 41 ng·ml-1, which was e-quivalent to 0.000241% for the concentration of regorafenib. The average recovery was 100.95% and RSD was 2.37% (n=9). Conclusion:The method has good specificity, promising accuracy and high sensitivity, which can be used for determining the trace genotoxic impurity AFP-PMA in regorafenib.

Association of ATIC and GSTP1 Genetic Polymorphisms with HD-MTX Plasma Concentration and Adverse Reactions in Children with Acute Lymphoblastic Leukemia / 医药导报

Objective To investigate the association of genetic polymorphisms of ATIC and GSTP1 with plasma concentrations and adverse reactions of high-dose methotrexate( HD-MTX)in children with acute lymphoblastic leukemia (ALL). Methods A total of 70 peripheral blood samples were obtained from ALL children for extraction of genome DNA.The gene polymorphisms of ATIC T26293C and GSTP1 A313G locus were examined by using PCR and direct sequencing.Enzyme multiplied immunoassay technique(EMIT)was employed to determine the plasma concentration of MTX in 48 h.Clinical data of patients were collected during HD-MTX chemotherapy,and the adverse reactions were statistically analyzed.The associations of ATIC and GSTP1 genotypes with MTX plasma concentration and adverse reactions were investigated. Results There were genetic polymorphisms at the SNP of ATIC T26293C and GSTP1 A313G.At the SNP of ATIC T26293C,the percentages of TT, CT and CC genotypes in ALL children were 4.35%,39.13% and 56.52%,respectively,and the frequencies of T and C alleles were 23.91% and 76.09%.At the SNP of GSTP1 A313G,the percentages of AA,GA and GG genotype were 68.57%,28.57%and 2.86%,respectively,in ALL children. The frequencies of A and G alleles were 82. 86% and 17. 14%,respectively. No statistically significant difference was found in the ratio of blood MTX concentration to MTX dose at 48 h between children with different genotypes(P>0.05).In the GSTP1 A313G site,genotypes that induced the gastrointestinal reactions in the order from low to high were AA,GA,GG,and there was a significant association between gene polymorphism and gastrointestinal side effects(P<0.05).In the GSTP1 A313G site,genotypes that induced myelosuppression in the order of low to high were GG,AA, GA,and a significant association was noted between gene polymorphism and myelosuppression(P<0.05). Conclusion There are significant associations between GSTP1 A313G polymorphism and gastrointestinal side effects or myelosuppression after HD-MTX chemotherapy in ALL children.

Early Treatment of AICAR Protects Hypoxia-ischemia Brain Injury in Neonatal Rats / 医药导报

Objective To investigate the neuroprotective effects and mechanisms of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/ IMP cyclohydrolase(AICAR) supplement (AMPK activator) in different stages of neonatal rats sufferring from hypoxia-ischemia encephalopathy ( HIE). Methods Neonatal rat hypoxia-ischemia brain injury model was employed in this study. A total of 160 neonatal rats were distributed into five groups: sham, model control,AICAR30 min, AICAR24 h and AICAR72 h. The neuroprotective effects of AICAR supplement (30 min, 24 h, 72 h post operation) were compared by cresyl violet staining; Expressions of P-AMPK,AMPK in the brain tissue were measured by Western blotting.Foot-faults method was used to evaluate the long-term prognosis of the rats. Results Compared with the sham group, the survival of rats brain in model control group was significantly decreased [(100.0± 0.1)% and (45.3± 6.3)%, P0.05). Compared with the sham group, the expression of P-AMPK significantly increased about three times, while ATP level decreased close to the same. Conclusion Early AICAR treatment can protect hypoxia-ischemia brain injury by increasing AMPK-ATP level.

Neuroprotective and Antioxidant Effects of Novel Benzofuran-2-Carboxamide Derivatives

In the present study, we synthesized a series of novel 7-methoxy-N-(substituted phenyl)benzofuran-2-carboxamide derivatives in moderate to good yields and evaluated their neuroprotective and antioxidant activities using primary cultured rat cortical neuronal cells and in vitro cell-free bioassays. Based on our primary screening data with eighteen synthesized derivatives, nine compounds (1a, 1c, 1f, 1i, 1j, 1l, 1p, 1q and 1r) exhibiting considerable protection against the NMDA-induced excitotoxic neuronal cell damage at the concentration of 100 muM were selected for further evaluation. Among the selected derivatives, compound 1f (with -CH3 substitution at R2 position) exhibited the most potent and efficacious neuroprotective action against the NMDA-induced excitotoxicity. Its neuroprotective effect was almost comparable to that of memantine, a well-known NMDA antagonist, at 30 muM concentration. In addition to 1f, compound 1j (with -OH substitution at R3 position) also showed marked anti-excitotoxic effects at both 100 and 300 muM concentrations. These findings suggest that -CH3 substitution at R2 position and, to a lesser degree, -OH substitution at R3 position may be important for exhibiting neuroprotective action against excitotoxic damage. Compound 1j was also found to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals and inhibit in vitro lipid peroxidation in rat brain homogenate in moderate and appreciable degrees. Taken together, our structure-activity relationship studies suggest that the compound with -CH3 substitution at R2 and -OH substitution at R3 positions of the benzofuran moiety might serve as the lead exhibiting potent anti-excitotoxic, ROS scavenging, and antioxidant activities. Further synthesis and evaluation will be necessary to confirm this possibility.

Tricyclic Pyrazoles: Synthesis, Characterization, Antimicrobial, Antituberculosis, and Antitumor Activity of N,1-Diphenyl-1,4-dihydrothiochromeno[ 4,3-c]pyrazole-3-carboxamide-5,5-dioxide Derivatives.

Aims: A series of N,1-diphenyl-1,4-dihydrothiochromeno[4,3-c]pyrazole-3-carboxamide 5,5-dioxide derivatives (6a-m) were synthesized and evaluated for anticancer, antibacterial, and antifungal activity. Methodology: Reaction of 2,3-dihydro-4H-thiochromen-4-one 1,1-dioxide 2 with diethyl oxalate in ethanol in the presence of a base afforded the Claisen condensation product 3. Subsequent reaction of 3 with phenylhydrazine hydrochloride at reflux in ethanol afforded ethyl 1-phenyl-1,4-dihydrothiochromeno[4,3-c]pyrazole-3-carboxylate 5,5-dioxide (4). Alkaline hydrolysis of 4 furnished the corresponding 1-phenyl-1,4- dihydrothiochromeno[4,3-c]pyrazole-3-carboxylic acid 5,5-dioxide 5. The pyrazole acid 5 was converted into the corresponding acid chloride followed by treatment with an excess of the appropriate amine to give 6a-m. Results: Compound 6k showed better activity than chloroamphenicol against Klebsiella pneumoniae and Escherichia coli and equipotent to clotrimazole in inhibiting the growth of Candida albicans (MIC 3.125 μg/mL). All compounds were screened for their cytotoxic activity against two tumor cell lines, namely, human colon tumor cell line (HCT116) and human cervical cancer cell line (HeLa) using the colorimetric MTT assay. Most of the tested compounds exhibited potent antitumor activity. Particularly, compound 6k displayed the highest activity among the tested compounds with IC50 equal to 17 μM (HeLa) and 15 μM (HCT116) respectively. Among the tested compounds, 6k was found to be more active against M. tuberculosis, (H37Rv) with minimum inhibitory concentration (7.8 μM). Conclusion: The chloro- (6b and 6c), 2-aminobenzothiazole- (6l), and 4-aminoantipyrine- (6k) linkages exhibited better antimicrobial activity than their counterparts. Compound 6k was found to possess comparatively more antimicrobial, antituberculosis, and antitumor activity against the other derivatives.

Use of Hydrazine Carboxamide-2-[(2-Hydroxy-1- Naphthalenyl) Methylene] as an Analytical Reagent for the Extractive Spectrophotometric Determination of U(Vi).

Extraction and spectrophotometric determination of Uranium (VI) was carried out using Hydrazine Carboxamide-2-[(2-Hydroxy-1-Naphthalenyl) Methylene] as an analytical reagent. A reddish brown coloured complex is formed at pH 9.2 between U(VI):HCHNM which is extracted in n-butanol. An intense peak for the complex was observed at 385nm, well separated from that of ligand. Beer’s law is obeyed over the range of 2 to20 ppm. Composition of M:L in the complex was ascertained by Job’s method, mole ratio method and slope ratio method and found to be 1:2. Extraction and spectrophotometric determination of U(VI) was carried out using Hydrazine Carboxamide-2-[(2-Hydroxy-1- Naphthalenyl) Methylene] as an analytical reagent. A reddish brown coloured complex is formed at pH 9.2 between U(VI):HCHNM which is extracted in n-butanol. An intence peak for the complex was observed at 385nm, well separated from that of ligand. Beer’s law is obeyed over the range of 2 to 20 ppm. Composition of M:L in the complex was ascertained by Job’s method, mole ratio method and slope ratio method and found to be 1:2. Diverse ion effect was studied for various ions and the proposed method was found to be highly selective for the trace concentration of U(VI). Molar absorptivity and Sandell sensitivity values calculated are 0.1566 x104L mol-1cm-1 and 0.0585g/cm2 respectively. Proposed method has been successfully applied to analyse synthetic samples and the results were found to be in good agreement with standard gravimetric methods.

5-aminoimidazole-4-carboxamide Riboside Induces Apoptosis Through AMP-activated Protein Kinase-independent and NADPH Oxidase-dependent Pathways

It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-alpha1 phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-alpha1-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation.

Antidepressant and anxiolytic-like effects of 4n, a novel 5-HT3 receptor antagonist using behaviour based rodent models.

The present study was designed to investigate the putative antidepressant and anxiolytic-like effects of N-n-Butylquinoxalin-2-carboxamide (4n), a novel 5-HT3 receptor antagonist, with an optimal log P (2.01) and pA2 value (7.3) greater than ondansetron (6.9) using rodent behavioural models of depression and anxiety. Acute treatment of 4n (1-4 mg/kg, ip) in mice produced antidepressant-like effect in forced swim test (FST) without affecting the baseline locomotion in actophotometer test in mice. 4n (2-4 mg/kg, ip) treatment also potentiated the 5-hydroxytryptophan (5-HTP) induced head twitch response in mice. Further, 4n (1-4 mg/kg, ip) treatment antagonized reserpine induced hypothermia in rats. Chronic treatment (14 days) with 4n (1-4 mg/kg) and paroxetine (10 mg/kg) significantly attenuated the behavioural anomalies induced by bilateral olfactory bulbectomy in rats in modified open field paradigm. An anxiogenic-like behaviour was induced by light alone as the stimulus using light-dark aversion test. 4n (2-4 mg/kg, ip) treatment significantly increased no. of transitions between dark and lit area and the time spent in the lit area. In conclusion, these preliminary investigations confirm that 4n exhibited antidepressant and anxiolytic-like effects in rodent models of depression and anxiety.

Anti-depressant-like activity of a novel serotonin type-3 (5-HT3) receptor antagonist in rodent models of depression.

N-Cyclohexyl-3-methoxyquinoxalin-2-carboxamide (QCM-13), a novel 5-HT3 antagonist identified from a series of compounds with higher pA2 (7.6) and good log P (2.91) value was screened in rodent models of depression such as forced swim test (FST), tail suspension test (TST), interaction studies with standard anti-depressants and confirmatory studies such as reversal of parthenolide induced depression and reserpine induced hypothermia. In FST (2 and 4 mg/kg) and TST (2 and 4 mg/kg), QCM-13 significantly reduced the duration of immobility in mice without affecting the base line locomotion. QCM-13 (2 and 4 mg/kg) was also found to have significant interaction with standard anti-depressants (fluoxetine and bupropion in FST and TST respectively). Further, reversal of parthenolide induced depression in mice and reserpine induced hypothermia in rat models indicate the serotonergic influence of QCM-13 for anti-depressant potential.

Role of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside in the Growth Regulation of Anaplastic Thyroid Cancer Cells Lines / 대한내분비학회지

BACKGROUND: Anaplastic thyroid carcinoma is one of the most aggressive human cancers with a median survival of only 6 months. Local surgical tumor debulking combined with radio-chemotherapy is generally used to treat this malady, but the low success rate has prompted the search for new therapeutic targets. We used 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) as an AMP-activated protein kinase (AMPK) activator to induce growth suppression and apoptosis in the anaplastic thyroid carcinoma cells. METHODS: We investigated the effect of AICAR on the proliferation of thyroid cancer cell lines (ARO, WRO and FRO) by performing methyl-thiazoletetrazolium bromide assay. We wanted to see the effect of AICAR on the apoptosis and cell cycle of the thyroid cancer cells, and we wanted to determine the mechanism of these changes. RESULTS: The proliferation of all thyroid cancer cell lines was significantly inhibited by administration of AICAR. FRO was the most susceptible cell line to AICAR treatment and so further studies were then performed with this cell line. The suppressive effect of AICAR on cell proliferation was related with phosphorylation of AMPK and the increased apoptosis. Also, cell cycle analysis revealed that progression to the G2-M phase was arrested (S-phase arrest) by AICAR treatment. S-phase arrest was associated with the increased protein expression of p21. CONCLUSION: In the anaplastic thyroid cancer cell lines, AICAR inhibited proliferation due to the arrest in the S-phase; this was accompanied with the increased expression of p21. Overall, AMPK activation by AICAR or any other pharmacological agent could be a tempting potential target for thyroid cancer therapy.