A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose-and timedependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

The canonical Wnt pathway plays an important role in embryonic development, adult tissue homeostasis, and cancer. Germline mutations of several Wnt pathway components, such as Axin, APC, and ß-catenin, can lead to oncogenesis. Inhibition of the poly(ADP-ribose) polymerase (PARP) catalytic domain of the tankyrases (TNKS1 and TNKS2) is known to inhibit the Wnt pathway via increased stabilization of Axin. In order to explore the consequences of tankyrase and Wnt pathway inhibition in preclinical models of cancer and its impact on normal tissue, we sought a small molecule inhibitor of TNKS1/2 with suitable physicochemical properties and pharmacokinetics for hypothesis testing in vivo. Starting from a 2-phenyl quinazolinone hit (compound 1), we discovered the pyrrolopyrimidinone compound 25 (AZ6102), which is a potent TNKS1/2 inhibitor that has 100-fold selectivity against other PARP family enzymes and shows 5 nM Wnt pathway inhibition in DLD-1 cells. Moreover, compound 25 can be formulated well in a clinically relevant intravenous solution at 20 mg/mL, has demonstrated good pharmacokinetics in preclinical species, and shows low Caco2 efflux to avoid possible tumor resistance mechanisms.

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A powder X-ray diffraction method was developed and validated to measure the crystalline impurity 4-(5-cyclopentyloxy-carbonylamino-1-methyl-indol-3-ylmethyl)-3-methoxy-N-o-tolylsulfonylbenzamide hydrate in a pharmaceutical tablet ranging from 0.6 to 3% (w/w). The calibration plot was found to be linear with a correlation coefficient (r2) of 0.996, and was reproducible among operators. The detection limit was determined to be 0.6% with a signal-to-noise ratio of 3:1. The quantitation limit was determined to be 1% with a signal-to-noise ratio of 5:1. Instrument precision at the quantitation limit was 5.8%. Method precision was 6.1% at the quantitation limit and 7.4% at the detection limit. Intermediate precision at the quantitation limit was 7.3% during a 6-month study. Accuracy measurements using crystalline impurity standards prepared in an excipient mixture ranged from 89.3 to 105.5%. Accuracy measurements using tablets containing spiked quantities of crystalline impurity ranged from 72.0 to 92.7%. Accuracy measurements using spiked tablets were complicated because the crystalline impurity was lost during the manufacturing process and a correction factor was used. Ruggedness was assessed by evaluating repetitive assay, repetitive packing, sample packing, and sample stability. Repetitive assays show the exposure of standards to a relative humidity in excess of 57% caused displacement error because of an increase in sample volume and a peak-position shift. Repetitive-packing studies show the analyte was extracted from the sample at a low relative humidity because of a static-charge induction. Sample-packing studies show that two subjective packing techniques were equivalent, and that under- and over-packing samples cause changes in sample density which would not affect results within ±16%. Sample-stability studies show that the quantitation-limit standard was stable as long as the sample was exposed to a relative humidity below 57%.

A series of four beta-cyclodextrin complexes (called products) was formed by neutralizing an acidic drug to study the effect of drug solubility on complex formation and the dissolution performance from direct compression tablets. Four solid products were prepared by neutralizing the drug in 0.05, 0.10, 0.20, and 0.30 M tromethamine solutions with a constant 0.09 M beta-cyclodextrin concentration, filtering the solutions, and removing the water through evaporation with heat and vacuum. The four products contained drug and water in a distinct relationship, thus suggesting a complex formation that was dependent on the tromethamine concentration. Infrared, powder X-ray diffraction, differential scanning calorimetry (DSC), phase solubility, and scanning electron microscopy (SEM) techniques revealed distinct differences among the four products, suggesting three of the four products were complexes, and one product was either a weak complex or a physical mixture. Ultraviolet (UV) analysis showed no evidence of complex formation. Phase solubility results showed one product had a slight increase in drug solubility, and three products had no increase in drug solubility with increasing beta-cyclodextrin concentration. The lack of a solubility increase suggests insoluble complex formation. Drug dissolution in water was improved significantly in all tablets containing either a product or a physical mixture when compared to the pure drug. The products prepared with the two highest concentrations of tromethamine showed a dissolution performance that was superior to all other formulations. Enthalpy measurements by DSC were a good indicator of dissolution performance for tablets containing the four products. Drug dissolution through salt formation in the absence of beta-cyclodextrin showed the drug-salt dissolution varied from better to worse when compared to the dissolution profiles of the four products. The varying dissolution performance was attributed to the formation of distinct beta-cyclodextrin complexes with varing solubilities.

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