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The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110α with IC50 ≤ 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials. [Mol Cancer Ther 2009;8(7):1725–38] [Mol Cancer Ther 2009;8(7):1725–38]

Supplementary Figure 1 from Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

Supplementary Figure 3C from Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

Supplementary Table 1 from Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

Supplementary Figure 2 from Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

Starting from HTS hit 1a, X-ray co-crystallization and molecular modeling were used to design potent and selective inhibitors of PI3-kinase. Bioavailablity in this series was improved through careful modulation of physicochemical properties. Compound 12 displayed in vivo knockdown of PI3K pharmacodynamic markers such as pAKT, pPRAS40, and pS6RP in a PC3 prostate cancer xenograft model.

Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the discovery of the exceptionally potent 6-aryl morpholino thienopyrimidine 6. In an effort to reduce the melting point in analogs of 6, the thienopyrimidine was modified by the addition of a methyl group to disrupt planarity. This modification resulted in a general improvement in in vivo clearance. This discovery led to the identification of GNE-477 (8), a potent and efficacious dual PI3K/mTOR inhibitor.

The PI3K/AKT/mTOR pathway has been shown to play an important role in cancer. Starting with compounds 1 and 2 (GDC-0941) as templates, (thienopyrimidin-2-yl)aminopyrimidines were discovered as potent inhibitors of PI3K or both PI3K and mTOR. Structural information derived from PI3K gamma-ligand cocrystal structures of 1 and 2 were used to design inhibitors that maintained potency for PI3K yet improved metabolic stability and oral bioavailability relative to 1. The addition of a single methyl group to the optimized 5 resulted in 21, which had significantly reduced potency for mTOR. The lead compounds 5 (GNE-493) and 21 (GNE-490) have good pharmacokinetic (PK) parameters, are highly selective, demonstrate knock down of pathway markers in vivo, and are efficacious in xenograft models where the PI3K pathway is deregulated. Both compounds were compared in a PI3K alpha mutated MCF7.1 xenograft model and were found to have equivalent efficacy when normalized for exposure.

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110α with IC50 ≤ 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials. [Mol Cancer Ther 2009;8(7):1725–38] [Mol Cancer Ther 2009;8(7):1725–38]

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

In our continued effort to identify selective MRP1 modulators, we have developed two novel templates, 3 and 4, through rational drug design by identifying the key pharmacophore interaction at the 7-position of the pyrrolopyrimidine template 1. Further synthesis and SAR work on these novel templates gave a number of potent MRP1 modulators with great selectivity against Pgp. Additional studies to reduce the CYP3A4 inhibition are also reported. Several compounds of these classes were subjected to in vivo xenograft studies and in vivo efficacies were demonstrated.

Acid dissociation constants in water have been determined for proton loss from a series of N-aryl-N′-cyanoguanidines 1. Treatment of the same compounds with a strong base, butyllithium, followed by excess methyl iodide leads to successive N-methylation to yield mono-, di- and tri-methylated N-aryl-N′-cyanoguanidines. The mechanism of the methylation is discussed.

A series of N-aryl-N′-cyanoguanidines has been prepared and studied in [2H6]Me2SO by 1H and 15N NMR spectroscopy. The 15N NMR coupling patterns for natural abundance and 15N-enriched guanidines show unequivocally a structure with the CN conjugated with the cyano group. The invariance of δH for the nitrogen bound protons with guanidine concentration, the temperature variation, the lack of water-induced change in δH, the linear free energy relationship (LFER) analysis and coupling patterns, all show that no prototropic tautomerisation is occurring on the NMR timescale. The findings are considered in terms of possible dissociative and non-dissociative mechanisms.

Of the four class I phosphoinositide 3-kinase (PI3K) isoforms, PI3Kα has justly received the most attention for its potential in cancer therapy. Herein we report our successful approaches to achieve PI3Kα vs PI3Kβ selectivity for two chemical series. In the thienopyrimidine series of inhibitors, we propose that select ligands achieve selectivity derived from a hydrogen bonding interaction with Arg770 of PI3Kα that is not attained with the corresponding Lys777 of PI3Kβ. In the benzoxepin series of inhibitors, the selectivity observed can be rationalized by the difference in electrostatic potential between the two isoforms in a given region rather than any specific interaction.

The discovery of 2 (GDC-0980), a class I PI3K and mTOR kinase inhibitor for oncology indications, is described. mTOR inhibition was added to the class I PI3K inhibitor 1 (GDC-0941) scaffold primarily through the substitution of the indazole in 1 for a 2-aminopyrimidine. This substitution also increased the microsomal stability and the free fraction of compounds as evidenced through a pairwise comparison of molecules that were otherwise identical. Highlighted in detail are analogues of an advanced compound 4 that were designed to improve solubility, resulting in 2. This compound, is potent across PI3K class I isoforms with IC(50)s of 5, 27, 7, and 14 nM for PI3Kα, β, δ, and γ, respectively, inhibits mTOR with a K(i) of 17 nM yet is highly selective versus a large panel of kinases including others in the PIKK family. On the basis of the cell potency, low clearance in mouse, and high free fraction, 2 demonstrated significant efficacy in mouse xenografts when dosed as low as 1 mg/kg orally and is currently in phase I clinical trials for cancer.

Deracemization, the conversion of a racemic mixture into an enantiomerically enriched material, is achieved with the retro-aldol reaction of rac-1 in the presence of lithium (-)-ephedrinate [Eq. (1)]. Remarkably, two stereogenic centers are simultaneously deracemized. This reaction shows that efficient thermodynamic control of the enantioselectivity of aldol reactions is possible.

In HMPA-THF solution, lithiated 1-naphthylacetonitrile undergoes highly syn-selective addition to aromatic aldehydes, providing the first access to such syn-aldols. Syn-selectivity is also observed with two other arylacetonitriles. Aldolate equilibration and crossover experiments demonstrate that HMPA promotes retro-aldol reaction and that aldol diastereoselectivity under these conditions is thermodynamically controlled.

Deracemization, the conversion of a racemic mixture into an enantiomerically enriched material, is achieved with the retro-aldol reaction of rac-1 in the presence of lithium (-)-ephedrinate [Eq. (1)]. Remarkably, two stereogenic centers are simultaneously deracemized. This reaction shows that efficient thermodynamic control of the enantioselectivity of aldol reactions is possible.

Deracemisierung, d. h. Umwandlung einer racemischen in eine enantiomerenangereicherte Mischung, gelingt mit der Retro-Aldol-Reaktion von rac-1 in Gegenwart von Lithium-(−)-Ephedrinat [Gl. (1)]. Bemerkenswerterweise erfolgt die Deracemisierung dabei an zwei Stereozentren. Anhand dieser Reaktion last sich zeigen, das eine wirkungsvolle thermodynamische Kontrolle der Enantioselektivitat von Aldol-Reaktionen moglich ist.

In HMPA−THF solution, lithiated 1-naphthylacetonitrile undergoes highly syn-selective addition to aromatic aldehydes, providing the first access to such syn-aldols. Syn-selectivity is also observed with two other arylacetonitriles. Aldolate equilibration and crossover experiments demonstrate that HMPA promotes retro-aldol reaction and that aldol diastereoselectivity under these conditions is thermodynamically controlled.

An X-ray structural analysis of the title compound, C9H10N4O, shows a planar guanidine portion, the N-aryl group tilted ca 57° relative to the guanidine plane and E geometry about the formal `imino' bond.