Targeting Solid Tumors With BTK Inhibitors.

The repurposing of FDA-approved Bruton's tyrosine kinase (BTK) inhibitors as therapeutic agents for solid tumors may offer renewed hope for chemotherapy-resistant cancer patients. Here we review the emerging evidence regarding the clinical potential of BTK inhibitors in solid tumor therapy. The use of BTK inhibitors may through lead optimization and translational research lead to the development of new and effective combination regimens for metastatic and/or therapy-refractory solid tumor patients.

Targeted and modular architectural polymers employing bioorthogonal chemistry for quantitative therapeutic delivery.

There remain several key challenges to existing therapeutic systems for cancer therapy, such as quantitatively determining the true, tissue-specific drug release profile in vivo, as well as reducing side-effects for an increased standard of care. Hence, it is crucial to engineer new materials that allow for a better understanding of the in vivo pharmacokinetic/pharmacodynamic behaviours of therapeutics. We have expanded on recent "click-to-release" bioorthogonal pro-drug activation of antibody-drug conjugates (ADCs) to develop a modular and controlled theranostic system for quantitatively assessing site-specific drug activation and deposition from a nanocarrier molecule, by employing defined chemistries. The exploitation of quantitative imaging using positron emission tomography (PET) together with pre-targeted bioorthogonal chemistries in our system provided an effective means to assess in real-time the exact amount of active drug administered at precise sites in the animal; our methodology introduces flexibility in both the targeting and therapeutic components that is specific to nanomedicines and offers unique advantages over other technologies. In this approach, the in vivo click reaction facilitates pro-drug activation as well as provides a quantitative means to investigate the dynamic behaviour of the therapeutic agent.

Investigation on the impact of three different quaternary methyl ammonium cartridges on the radiosynthetic yields of [18 F]fluoromethyl tosylate.

Our recent investigations for the radiosynthesis of [18 F]fluoromethyl tosylate have highlighted that choice of quaternary methyl ammonium (QMA) cartridge used during the radiosynthesis can significantly impact the radiochemical yields. Often the details of the QMA cartridge used in fluourine-18 syntheses are not fully described. However, our studies demonstrate that the type, the size, and nature (method by which it has been conditioned) of the QMA cartridge used during the radiosynthesis can make a significant impact in the labelling efficiency. This paper investigates the use of three QMA cartridges and demonstrates that radiochemical yield (decay corrected) of [18 F]fluoromethyl tosylate can increase from 46% to 60% by simply changing the QMA cartridge (and leaving all other reagents and labelling conditions exactly the same). These learnings may be applied to improve the radiochemical yields of a number of [18 F]-fluorinated tracers (and synthons), where the labelling step is base-sensitive to increase the radiochemical yield, thereby significantly benefiting the radiochemistry and nuclear medicine community. This paper also highlights the necessity of the radiochemistry community to ensure the details of QMA cartridges used in fluorine-18 chemistry are fully and accurately described, since this will improve the translation of radiochemical methods from one laboratory to another.

Synthesis of 18 F-radiolabeled diphenyl gallium dithiosemicarbazone using a novel halogen exchange method and in vivo biodistribution.

18 F-radiolabeled diphenyl gallium thiosemicarbazone was prepared by [18 F] fluoride exchange of a nitrato anion under mild conditions. The diphenyl gallium thiosemicarbazone chloride is easily prepared in gram quantities and can be used at room temperature in the presence of oxygen. The corresponding nitrate complex is prepared using silver nitrate in methanol solvent and can be stored under nitrogen for weeks before radiolabeling. The biodistribution of this new tracer was studied in mice using positron emission tomography (PET).

N-(4-[18F]fluorobenzyl)cholylglycine, a novel tracer for PET of enterohepatic circulation of bile acids: Radiosynthesis and proof-of-concept studies in rats.

INTRODUCTION: Enterohepatic circulation (EHC) of conjugated bile acids is an important physiological process crucial for regulation of intracellular concentrations of bile acids and their function as detergents and signal carriers. Only few bile acid-derived imaging agents have been synthesized and hitherto none have been evaluated for studies of EHC. We hypothesized that N-(4-[18F]fluorobenzyl)cholylglycine ([18F]FBCGly), a novel fluorine-18 labeled derivative of endogenous cholylglycine, would be a suitable tracer for PET of the EHC of conjugated bile acids, and we report here a radiosynthesis of [18F]FBCGly and a proof-of-concept study by PET/MR in rats. METHODS: A radiosynthesis of [18F]FBCGly was developed based on reductive alkylation of glycine with 4-[18F]fluorobenzaldehyde followed by coupling to cholic acid. [18F]FBCGly was investigated in vivo by dynamic PET/MR in anesthetized rats; untreated or treated with cholyltaurine or rifampicin. Possible in vivo metabolites of [18F]FBCGly were investigated by analysis of blood and bile samples, and the stability of [18F]FBCGly towards enzymatic de-conjugation by Cholylglycine Hydrolase was tested in vitro. RESULTS: [18F]FBCGly was produced with a radiochemical purity of 96% ±â€¯1% and a non-decay corrected radiochemical yield of 1.0% ±â€¯0.3% (mean ±â€¯SD; n = 12). PET/MR studies showed that i.v.-administrated [18F]FBCGly underwent EHC within 40-60 min with a rapid transhepatic transport from blood to bile. In untreated rats, the radioactivity concentration of [18F]FBCGly was approximately 15 times higher in bile than in liver tissue. Cholyltaurine and rifampicin inhibited the biliary secretion of [18F]FBCGly. No fluorine-18 metabolites of [18F]FBCGly were observed. CONCLUSION: We have developed a radiosynthesis of a novel fluorine-18 labeled bile acid derivative, [18F]FBCGly, and shown by PET/MR that [18F]FBCGly undergoes continuous EHC in rats without metabolizing. This novel tracer may prove useful in PET studies on the effect of drugs or diseases on the EHC of conjugated bile acids.

Investigation of two- and three-bond carbon-hydrogen coupling constants in cinnamic acid based compounds.

Two- and three-bond coupling constants (2 JHC and 3 JHC ) were determined for a series of 12 substituted cinnamic acids using a selective 2D inphase/antiphase (IPAP)-single quantum multiple bond correlation (HSQMBC) and 1D proton coupled 13 C NMR experiments. The coupling constants from two methods were compared and found to give very similar values. The results showed coupling constant values ranging from 1.7 to 9.7 Hz and 1.0 to 9.6 Hz for the IPAP-HSQMBC and the direct 13 C NMR experiments, respectively. The experimental values of the coupling constants were compared with discrete density functional theory (DFT) calculated values and were found to be in good agreement for the 3 JHC . However, the DFT method under estimated the 2 JHC coupling constants. Knowing the limitations of the measurement and calculation of these multibond coupling constants will add confidence to the assignment of conformation or stereochemical aspects of complex molecules like natural products. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis, characterization and biological activities of semicarbazones and their copper complexes.

Substituted semicarbazones/thiosemicarbazones and their copper complexes have been prepared and several single crystal structures examined. The copper complexes of these semicarbazone/thiosemicarbazones were prepared and several crystal structures examined. The single crystal X-ray structure of the pyridyl-substituted semicarbazone showed two types of copper complexes, a monomer and a dimer. We also found that the p-nitrophenyl semicarbazone formed a conventional 'magic lantern' acetate-bridged dimer. Electron Paramagnetic Resonance (EPR) of several of the copper complexes was consistent with the results of single crystal X-ray crystallography. The EPR spectra of the p-nitrophenyl semicarbazone copper complex in dimethylsulfoxide (DMSO) showed the presence of two species, confirming the structural information. Since thiosemicarbazones and semicarbazones have been reported to exhibit anticancer activity, we examined the anticancer activity of several of the derivatives reported in the present study and interestingly only the thiosemicarbazone showed activity while the semicarbazones were not active indicating that introduction of sulphur atom alters the biological profile of these thiosemicarbazones.

Structural investigation on phenyl- and pyridin-2-ylamino(methylene)naphthalen-2(3H)-one. Substituent effects on the NMR chemical shifts.

Schiff bases bearing phenyl and pyridyl groups were synthesized by condensation of appropriate amines with 2-hydroxynaphthaldehyde. These Schiff bases were obtained as colored crystalline solids. The proton NMR spectra of these compounds showed a doublet for the NH protons indicating a keto tautomer for these Schiff bases. The pyridyl-substituted Schiff bases containing hydroxyl moiety were found to show the most downfield shift for the NH protons in DMSO solvent, and this was rationalized due to the formation of a six- and five-membered ring using hydrogen bonds for these two compounds. Correspondingly, the olefinic proton of the Schiff bases is also found to be a doublet due to coupling to the amine proton. These Schiff bases exhibited thermochromic properties. Detailed NMR spectral analysis for both the phenyl- and pyridyl-substituted Schiff bases is presented.

Synthesis and characterization of benzothiazolyl-substituted anils.

New Schiff bases containing a hydroxynaphthyl ring and substituted benzothiazolyl groups have been synthesized. High-resolution NMR spectra confirmed that these anils exist as enol-keto tautomers in solution. The results from NMR data demonstrated that the proportion of enol tautomer exceeded 90% in these substituted anils. Some compounds exhibited thermochromism in solid state.

In vivo pharmacokinetics, metabolism, toxicity, and anti-HIV activity of N'-[2-(2-Thiophene)ethyl]-N'-[2-(5-bromopyridyl)]thiourea (HI-443), a potent non-nucleoside inhibitor of HIV reverse transcriptase.

N'-[2-(2-Thiophene)ethyl]-N'-[2-(5bromopyridyl)]thiourea (CAS 258340-15-7, HI-443) is a potent non-nucleoside inhibitor of HIV reverse transcriptase (NNRTI) that was rationally designed as a candidate anti-HIV agent. The purpose of the present study was to examine the in vivo pharmacokinetics, metabolism, toxicity, and anti-HIV activity of HI-443. HI-443 was very well tolerated in CD-1 mice and Lewis rats without any detectable toxicity at single parenteral bolus dose levels as high as 80 mg/kg. Intraperitoneally administered HI-443 exhibited anti-HIV activity in the Hu-PBL-SCID mouse surrogate model for hunnan AIDS at a non-toxic daily dose level of 10-20 mg/kg. These preclinical research studies provide the basis for future preclinical studies and clinical development of HI-443 as a new NNRTI candidate.

Effect of targeting janus kinase 3 on the development of intestinal tumors in the adenomatous polyposis coli(min) mouse model of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is associated with germ-line mutations in the tumor suppressor gene, adenomatous polyposis coli (APC) located on chromosome 5q21. Multiple intestinal neoplasia (Min) in mice resembles FAP in humans, resulting from a single point mutation in the murine homolog of the APC gene. The effects of the rationally-designed Janus kinase 3 (JAK3) inhibitor JANEX-1 (4-(4'-hydroxyphenyl) amino-6,7-dimethoxy-quinazoline, WHI-P131, CAS 202475-60-3) on the development of intestinal tumors in the APC (min/+) mouse model of FAP were examined. The Min mice were fed with rodent chow or chow supplemented with JANEX-1 once a week starting at 1.5 months of age. The cumulative proportions of mice remaining alive at 7 months were 13 +/- 6% for control mice versus 72 +/-12% for JANEX-1 treated mice (P<0.0002). In contrast, Compound DDE24, a synthetically activated genistein, an inhibitor of Epidermal Growth Factor receptor (EGF-R), cellular homologue of oncogene product from Raus Avian sarcoma virus (SRC) and Syk tyrosine kinases, which Thus, selective targeting of JAK3 was highly effective in preventing development of intestinal tumors in Min mice resulting in markedly improved survival outcomes. JAK3 inhibitors may therefore be useful in the prevention of colorectal cancer in individuals with FAP.

Large-scale synthesis of GMP grade N'- [2-(2-thiophene) ethyl]-N'- [2- (5-bromopyridyl)] -thiourea (HI-443), a new anti-HIV drug candidate.

The thiourea compound N'-[2-(2-thiophene)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (HI-443, CAS 258340-15-7), was found to be a potent anti-HIV agent with remarkable activity against nucleoside analog reverse transcriptase (NRT)-resistant, non-nucleoside analog reverse transcriptase (NNRT)-resistant, as well as multidrug-resistant HIV. Now the method of producing HI-443 under current Good Manufacturing Practice (cGMP) conditions on the scale of kilograms is reported. The availability of GMP-grade HI-443 will promote the preclinical and clinical development efforts aimed at making this new drug candidate available to HIV-infected persons.

N'-[2-(2-thiophene) ethyl]-N'-[2-(5-bromopyridyl)]thiourea (HI-443), a rationally designed non-nucleoside reverse transcriptase inhibitor compound with potent anti-HIV activity.

The in vitro activity profile of N'- [2-(2-thiophene)ethyl]-N'-[2-(5-bromopyridyl)] thiourea (CAS 258340-15-7, HI-443) was examined against 38 clinical isolates of HIV-1. HI-443 inhibited the replication and/or infectivity of each of the 7 HIV-1 isolates of non-B envelope subtype, each of the 22 isolates with genotypic nucleoside reverse transcriptase inhibitor (NRTI) resistance, each of the 6 multidrug-resistant HIV isolates with genotypic NRTI/non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance, each of the 3 isolates with no RT mutations, and each of the 3 laboratory strains of HIV-I with NNRTI or NNRTI/NRTI resistance. The potency of HI-443 against clinical HIV-1 isolates with NRTI- or NNRTI-resistance warrants the further development of this rationally designed NNRTI as a new anti-HIV agent.

In vivo pharmacokinetics and toxicity of a novel hydrophilic oral formulation of the potent non-nucleoside reverse transcriptase inhibitor compound N'-[2-(2-thiophene)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (HI-443).

The thiophene ethyl thiourea (TET) compound N'-[2-(2-thiophene)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (HI-443) is a potent non-nucleoside reverse transcriptase inhibitor (NNRTI). The pharmacokinetics of 17 different novel oral formulations of HI-443 were compared in an attempt to identify the most suitable dosage form for clinical use in HIV-infected persons. Plasma concentrations of HI-443 were monitored in mice after administration of the drug using these 17 different formulations at three time points. Two-way ANOVA showed highly significant formulation (p < 0.0001), time (p < 0.0001) and formulation*time interaction effects (p = 0.0003). Planned linear contrasts were performed to identify which formulations showed the highest bioavailability at 10, 30, 60 min and at all time points relative to DMSO alone. A significant positive regression was observed comparing bioavailibility of HI-443 at 10 min and hydrophilic-lipophilic balance (HLB) values of the formulations (R2 = 26%, p < 0.0001). The results showed that formulations that were hydrophilic, containing PEG400 and propylene glycol, gave the highest overall drug concentrations over the 60-min time period. The lead oral formulation of HI-443 exhibited a very favorable toxicity profile in BALB/c mice.

Large-scale synthesis of GMP grade alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl) propenamide (LFM-A13), a new anti-cancer drug candidate.

The leflunomide (CAS 75706-12-6) metabolite (LFM) analog alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13, DDE-28, CAS 244240-24-2), is a rationally designed inhibitor of the anti-apoptotic enzyme Bruton's tyrosine kinase (BTK). LFM-A13 is being developed as a novel dual-function anticancer drug with apoptosis-promoting and anti-thrombotic properties. LFM-A13 was prepared under current Good Manufacturing Practice (cGMP) conditions on the scale of kilograms.

Improved oral bioavailability of anti-HIV agent N'-[2-(2-thiophene)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (HI-443) in a novel lipophilic formulation.

N'-[2-(2-Thiophene)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (CAS 258340-15-7, HI-443) is a rationally designed non-nucleoside reverse transcriptase inhibitor (NNRTI) with potent anti-HIV activity at nanomolar concentrations but poor oral bioavailability. Here the identification of a novel oleic acid containing lead formulation of HI-443 is described which resulted in a approximately 10-fold improvement of its oral bioavailability yielding 10-fold higher systemic exposure levels in mice. Formulated HI-443 exhibited a favorable pharmacokinetics and toxicity profile in mice.

Anti-retroviral activity of GMP-grade stampidine against genotypically and phenotypically nucleoside reverse transcriptase inhibitor resistant recombinant human immunodeficiency virus. An in vitro study.

The in vitro potency of GMP-grade stampidine (CAS 217178-62-6) was examined against 3 clinical HIV-1 isolates and 6 recombinant HIV-1 clones with multi-NRTI 'resistance (NRTI: nucleoside reverse transcriptase inhibitors). GMP-grade stampidine active drug substance (Lot #'s MPR-M0008.00-01 and MPR-M0008.01-01) as well as GMP-grade stampidine extracted from the clinical stampidine capsules (GMP-Grade Clinical Batch, Pharmaceutical Service Lot Number 159I0601) were highly potent and exhibited nanomolar IC50 values against clinical HIV-1 isolates as well as recombinant HIV-1 clones with multi-NRTI resistance containing common patterns of reverse transcriptase mutations responsible for NRTI resistance.

Preclinical toxicity and pharmacokinetics of the Bruton's tyrosine kinase-targeting anti-leukemic drug candidate, alpha-cyano-beta-hydroxy-beta-methyl-N- (2,5-dibromophenyl) propenamide (LFM-A13).

The leflunomide (CAS 75706-12-6) metabolite (LFM) analog alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13, DDE-28, CAS 244240-24-2) is a rationally-designed specific inhibitor of the TEC family protein tyrosine kinase, Bruton's tyrosine kinase (BTK). LFM-A13 exhibited favorable pharmacokinetics in CD-1 mice, BALB/c mice, rats, and dogs. The intraperitoneal bioavailability was estimated to be -100%, while the oral bioavailability was -30%. LFM-A13 enters, but does not bind to multiple tissues followed by a rapid elimination from most of the tissues. Limited distribution of LFM-A13 to extravascular tissues and its corresponding low volume of distribution could be attributed to its plasma protein binding. LFM-A13 was not toxic to mice, rats, or dogs at daily dose levels as high as 100 mg/kg. LFM-A13 formulated as a suspension and hard gelatin capsules for oral administration showed a rapid absorption and favorable pharmacokinetic features. These preclinical research studies provide the basis for future pre-IND studies and clinicaldevelopment of LFM-A13 as an intravenously or orally administered new anti-leukemia agent targeting BTK.

Large-scale synthesis and formulation of GMP-grade stampidine, a new anti-HIV agent.

The arylphosphoramidate derivative of stavudine (STV, d4T, 2,3'-didehydro-3'-deoxythymidine, CAS 3056-17-5), stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6), is a novel anti-HIV agent. STAMP was prepared under current Good Manufacturing Practice (cGMP) conditions on the scale of kilograms. Solid STAMP was subsequently formulated as a capsule under GMP conditions for oral administration.

Synthesis, separation and anti-HIV activity of distereoisomers of N-[p-(4-bromophenyl) -2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester (stampidine).

The distereoisomers of stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2'3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6) were separated using two different procedures. The first method involved separation of the isomers by fractional crystallization, and the second method utilized a preparative HPLC. Both isomers were active against the HIV-1 strain HTLV(IIIB) and neither isomer was more or less active than distereoisomeric mixture of stampidine.