PHA-739358 (Danusertib), which was discovered and developed by Nerviano Medical Sciences, is currently in Phase II clinical studies. This inhibitor features a pyrrolopyrazole scaffold which had previously been identified as an ATP-mimetic pharmacophore suited for kinase binding [
27]. The SAR (structure activity relationship) analysis of several pyrrolopyrazole subclasses resulted in the synthesis of PHA-680632 which showed high anti-cancer activity in vitro and in vivo and have thus become a preclinical candidate [
27]. The X-ray crystallographic structure of PHA-680632 in complex with Aurora A guided further design. Through combinatorial expansion of a related 1,4,5,6-tetrahydropyrrolo[3,4-
c]pyrazole core and SAR refinements of the 5-amido-pyrrolopyrazole series a potent Aurora kinase inhibitor PHA-739358 was identified [
28]. It has been shown to inhibit Aurora A, B, and C in biochemical competitive assays with IC50 values of 13, 79, and 61 nM, respectively [
28]. However, this study also demonstrated that PHA-739358 predominantly has an Aurora B inhibition phenotype in cell cultures. At high concentrations, it has been reported to cross-react with Abl (Abelson), Ret (rearranged during transfection), Trk-A, and FGFR1 (fibroblast growth factor receptor 1) kinases [
32]. In this latter study, cell lines exposed to PHA-739358 were found to be sensitive to concentrations in the range 28 to 300 nM and the mode of action of PHA-739358 corresponded to Aurora B inhibition as assessed by phospho-histone H3(Ser10) inhibition. In addition, cells with tetraploid (4n) and polyploid (>4n) DNA content were observed to accumulate upon treatment with PHA-739358 [
32]. Preclinical efficacy and toxicity studies were also performed in nude mice transplanted with several human xenografts, employing maximum tolerated doses (MTD) of 60 mg/kg/day for 5 days, 30 mg/kg/day for 10 days, or 45 mg/kg/day for 10 days. Tumor growth inhibition (TGI) was observed to be 66% to 98%; the compound was fairly well tolerated with only mild weight loss and myelosuppression. PHA-739358 has also been tested in a rat model having DMBA (9,10-Dimethylbenz-A-Anthracene) induced mammary carcinomas. At 25 mg/kg, TGI was measured as 75% and a complete cure was achieved in one rat [
32]. Recently a Phase I study results were reported. Pharmacokinetic profiles were linear, and dose and time dependent. Of 80 patients assessed, stable disease was observed in 28, and in seven cases, this lasted for six months [
33]. In another Phase I study, 56 patients divided into two parts (part 1 has 40 patients and part 2 has 16 patients) received escalating doses (45, 90, 180, 360, 500, 580, 650 mg/m
2: 24 h infusion every 14 days) of PHA-739358 [
34]. In part 1, patients received escalating doses of PHA-739358 without the co-administration of G-CSF (granulocyte stimulating factor). Doses were further escalated in part 2 in the presence of G-CSF. The MTD established in part 1 was 500 mg/m
2. DLTs (dose limiting toxicity) were reported in 6 patients, which include neutropenia, grade 4 mucositis, and neutropenic infection. In part 2, 16 patients received the escalating doses of 500, 750, and 1000 mg/m
2 along with G-CSF. No severe DLTs in the presence of G-CSF were reported even at maximum dose administered (1000 mg/m
2). The dose 1000 mg/m
2 of PHA-739358 along with G-CSF induced objective response in one refractory small cell lung cancer patient. This is the first time that the partial responses have been reported for an AKI with minimum toxicities. Several prolonged disease stabilizations were also reported in part 1 schedule. Phase II and Phase III single agent studies without G-CSF are underway in 7 types of solid tumors [
34]. However, G-CSF is also being considered in further clinical studies. In addition to AKs, PHA-739358 has been also shown to inhibit BCR-ABL kinase (breakpoint cluster region-abelson) [
35]. Many chronic myeloid leukemia (CML) patients acquire resistance to the BCR-ABL inhibitor imatinib by specific BCR-ABL mutations, particularly the T315I gate-keeper mutation. Interestingly, PHA-739358 inhibited both wild type BCR-ABL (25 nM) and T315I mutated protein in kinase assays. Moreover, PHA-739358 reportedly has a higher affinity for the T315I form (5 nM) than the Abl wild type (21 nM) [
35], which may prove advantageous for clinical treatment. This compound is currently in Phase II studies, being investigated in imatinib-resistant CML patients [
33]. Twelve CML patients were enrolled and received doses from 250 to 400 mg/m
2/day (3 consecutive weeks every 4 weeks). Two patients with T315I BCR-ABL achieved complete hematological response. One patient displayed complete cytogenetic and molecular response after 3 months. PHA-739358 was well tolerated and only grade 3/4 neutropenia has been reported. As part of the pharmacodynamic study, CRKL phosphorylation was decreased in majority of treated patients. Additional studies in CML resistant patients are underway [
33].