Gedatolisib

Bis(morpholino-1,3,5-triazine) Derivatives: Potent Adenosine 50-Triphosphate Competitive Phosphatidylinositol-3-kinase/Mammalian Target of Rapamycin Inhibitors: Discovery of Compound 26 (PKI-587), a Highly Efficacious Dual Inhibitor

Aranapakam M. Venkatesan,*,† Christoph M. Dehnhardt,† Efren Delos Santos,† Zecheng Chen,† Osvaldo Dos Santos,† Semiramis Ayral-Kaloustian,† Gulnaz Khafizova,† Natasja Brooijmans,† Robert Mallon,‡ Irwin Hollander,‡ Larry Feldberg,‡ Judy Lucas,‡ Ker Yu,‡ James Gibbons,‡ Robert T. Abraham,‡ Inder Chaudhary,§ and Tarek S. Mansour†

Abstract

The PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis, and glucose metabolism. It has been recognized recently that inhibiting this pathway might provide a viable therapy for cancer. A series of bis(morpholino-1,3,5-triazine) derivatives were prepared and optimized to provide the highly efficacious PI3K/mTOR inhibitor 1-(4-{[4-(dimethylamino)piperidin-1-yl]car- bonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea 26 (PKI-587). Compound 26 has shown excellent activity in vitro and in vivo, with antitumor efficacy in both subcutaneous and orthotopic xenograft tumor models when administered intravenously. The structure-activity relationships and the in vitro and in vivo activity of analogues in this series are described.

Introduction

Phosphatidylinositol-3-kinases (PI3Ks ) are lipid kinases that phosphorylate phosphatidylinositol diphosphate (PIP2) to its corresponding phosphatidylinositol triphosphate (PIP3). It has been recognized recently that the PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis, and glucose metabolism.1-6 The product of PI3K activity, namely, PIP3, acts as a second messenger responsible for the activation of the downstream kinase Akt.1 PI3Ks are categorized as class IA, class IB, class II, and class III enzymes, based on sequence homology and substrate prefer- ences. The PI3K related kinases (PIKKs) include mTOR, ATR, ATM, SMG1, and DNA-PK. PIKKs share a conserved kinase domain with PI3Ks and regulate cellular response to nutrient levels and environmental stress. The class IA subgroup consists of PI3KR, PI3Kβ, and PI3Kδ isoforms. Among these three isoforms, the gene encoding the PI3KR subunit, PIK3- CA, is mutated and/or overexpressed in breast, ovarian, color- ectal, brain (glioblastoma), and gastric cancers.6,9-11 Addi- tionally, the PIP3 lipid phosphatase, phosphatase and tensin homologue deleted on chromosome ten (PTEN), is frequently inactivated in numerous late stage tumors, causing elevated genesis.7,8,12 Therefore, inhibiting PI3KR is an attractive strat- egy for cancer therapy. Furthermore, it has been demonstrated that the PI3K pathway is paradoxically activated following selective mTOR inhibition, thus providing a compelling ratio- nale for developing dual PI3K/mTOR kinase inhibitors.13,14
Toward this end, several pharmaceutical companies and academic institutions have been concentrating their efforts on the development of small molecule PI3K/mTOR inhibitors.2,15-17 Among the various small molecule inhi- bitors, BEZ-235 1 (Figure 1) is the most advanced clini- cal candidate.19a,b It inhibits PI3K and mTOR in an ATP- competitive manner. This compound is also reported to inhibit cancer cell proliferation, causing cell cycle arrest at the G1 phase.19a,b Another compound that inhibits all class I PI3K isoforms, but with significantly lower potency against mTOR, is 2 GDC-0941.18 This compound was reported by Genentech and has entered phase I clinical studies. Another pan PI3K inhibitor that has gone into the clinic is Semafore compound, SF1126.20 Highly selective mTOR inhibitors based on a pyrazolopyrimidine scaffold such as 3a were reported by Zask et al.21a,b and recently entered clinical development. We also recently described a dual PI3K/mTOR inhibitor 3 (PKI-402) based on a triazolopyrimidine scaffold that has potent in vivo efficacy.21c
In the present article, we describe the design, synthesis, and identification of a highly potent bis(morpholino-1,3,5- triazine) derivative 26 (PKI-587) as a PI3K/mTOR inhibitor. Compound 26 decreases cell survival and proliferation and increases apoptosis in both in vitro and in vivo models. It shows compelling antitumor efficacy in subcutaneous and orthotopic human xenograft tumor models when admini- stered intravenously (iv) as a single agent.

Chemistry

The 2,4-bismorpholino-1,3,4-triazine derivatives 8-30, which are exemplified in the present article, were prepared starting from the commercially available cyanuric chloride 4, as outlined in Scheme 1. Initially the two chlorines in cyanuric chloride 4 were replaced with 2 equiv of morpholine 5 in the presence of triethylamine at 0 °C to yield 6 in almost quanti- tative yield. The third chlorine in compound 6 was displaced by a 4-aminophenyl moiety using Suzuki coupling reaction with 4-aminophenylboronic acid pinacol ester to yield 7. Compound 7 was reacted with various alkyl and aryl iso- cyanates to yield 8-14, 17, 18, and 21. Alternatively, urea
derivatives 15, 16, 19, and 20 were obtained by reacting the intermediate 7 with triphosgene/triethylamine and the corres- ponding amines. In order to introduce amides bearing water solubilizing amine functionality, intermediate 7 was reacted with methyl 4-isocyanatobenzoate in dichloromethane at room temperature to give 21 in good yield. The ester group was hydrolyzed with 5 N NaOH in tetrahydrofuran/methanol at reflux temperature to yield 22. The acid derivative 22 was reacted with different amines in the presence of HOBt/EDCI and triethylamine at room temperature. The final products were purified either by flash column silica gel column chro- matography or by preparative HPLC. All analogues 8-30 obtained by the above-mentioned route were analyzed for purity by analytical HPLC on a prodigy ODS3 column (150 mm 4.6 mm) using acetonitrile/water mixture. The purity of the newly synthesized compounds was analyzed at 210-370 nm.

Results and Discussion

Final compounds 8-30 were tested in vitro against PI3KR, PI3Kγ, and mTOR. The IC50 values against PI3KR and PI3Kγ enzymes were determined using a fluorescence polari- zation format assay.22 The corresponding mTOR inhibition for the newly synthesized compounds was determined using a DELFIA format ELISA.23 PI3KR, PI3Kγ, and mTORas well as the cell proliferation (3 days growth inhibition) assay24 IC50 values in MDA-361 (breast, PI3K mutant [E545K]/Her2 ) and PC3MM2 (prostate, PTEN deletion) human tumor cell lines are shown in Table 1. The solubility (at pH 7.4), PAMPA permeability, and clogP data for analogues 8-30 are also listed in Table 1.
Several morpholine bearing fused pyrimidines such as imidazolopyrimidines,25 pyrrolopyrimidines,26 and triazolo- pyrimidines21c have been reported by us previously as PI3K/ mTOR dual inhibitors. In all these scaffolds, the morpholine oxygen formed a pivotal hinge region hydrogen bond inter- action with Val851 of the PI3KR catalytic domain18,21a-c but also presented a metabolic liability via oxidation R to the morpholine ring oxygen, causing loss of potency. Many potent PI3K enzyme inhibitors based on the above-mentioned scaffolds failed to show antitumor efficacy in in vivo models. The most advanced compound 3 showed good efficacy in MDA-361 xenograft model. However, advanced studies with 3 were halted because of its poor solubility.
Bearing these facts in mind, we decided to probe the other scaffolds that resemble a pyrimidine core but have lower clogP values than the triazolopyrimidine core in PKI-402. Among the various heterocycles we synthesized, the 1,3,5-triazine scaffold17 was very attractive for the following reasons:
(1) 1,3,5-Triazine is a monocyclic, symmetrical molecule similar to the pyrimidine core with two nitrogens in the 1,3- positions, and it has been used as a kinase inhibitor scaffold.17 Hence, we envisaged that 1,3,5-triazine could mimic the pyrimidine scaffold and allow us to position the critical substituents observed in our earlier series for optimum inter- action with the binding site of PI3KR.
(2) The presence of three nitrogens in the 1,3,5-triazine core can inherently impart polarity to the whole molecule, and the clogP values of the designed compounds can be about 2 or lower.
(3) We also envisioned also that a bismorpholino-1,3,5- triazine scaffold would have an advantage under conditions where one morpholine gets metabolically oxidized, still leav- ing another morpholine to make the vital hinge region H bond interaction in the PI3K catalytic domain.
However, we were aware that these modifications can solve the clogP problem, and to an extent, the morpholine meta- bolic liability might not address permeability and the micro- somal stability problems. In order to explore the above- mentioned points, compound 8 was designed and modeled in the PI3Kγ homology model (Figure 2). The structural similarity of the ATP-binding sites of PI3KR and PI3Kγ isoforms enabled a homology model to be built based on an in-house X-ray crystal structure of PI3Kγ in complex with a related compound (3IBE.pdb).21a Overall sequence similarity between PI3KR and PI3Kγ is 43% in the kinase catalytic domain; however, the ATP-binding site is significantly more conserved with 81% of the residues being identical. As indicated in Figure 2, compound 8 docked in the homology model forms the critical hinge region hydrogen-bond to Val851 through a morpholine oxygen. The urea forms two hydrogen-bonding interactions to Asp810 through both urea-NH groups and one to the catalytic lysine (Lys802) through the urea carbonyl group. Hence, compound 8 was prepared as depicted in Scheme 1. As can be seen from Table 1, compound 8 was found to have potent PI3KR, PI3Kγ, and mTOR inhibitory activity but only moderate potency in MDA-361 and PC3MM2 cell proliferation assays.
This was attributed to the poor solubility and permeability of compound 8.
However, this initial enzyme potency result encouraged us to probe the structure-activity relationship more system- atically to further optimize the cellular potency. There was an observed drop in the potency against PI3KR and PI3Kγ when the phenyl group in compound 8 was replaced with alkyl groups such as methyl 10, ethyl 9, or saturated heterocyclyl 29. However, the corresponding mTOR activity was not affected. The structural basis for the observed difference in PI3KR/γ and mTOR activity for compounds 9, 10, and 29 is not entirely clear because of a lack of detailed structural information on the mTOR enzyme (Figure 3). However, three residue differ- ences are observed in the loop that forms the top of the binding site around the urea substituents. Asp805 in PI3KR is a glutamic acid in mTOR, and this residue plays a critical role in stabilizing the catalytic lysine (Lys802 in PI3KR, Lys2187 in mTOR) through the formation of a salt bridge. Substituted ureas of this type result in a concerted motion of the catalytic lysine and the aspartic/glutamic acid,21a,b and it is possible that mTOR with the longer side chain in the glutamic acid, compared to the aspartic acid in PI3KR, undergoes this motion more easily. The second noted residue difference in this area is the different positioning of glycine. In PI3KR, a glycine (Gly804) is directly adjacent to the aspartic acid involved in the salt bridge, whereas in mTOR, a histidine (His2189) separates the glutamic acid from the glycine (Gly2188). Again, it is possible that the different position of the glycine allows the mTOR loop to accommodate the hydro- phobic groups coming off the urea more easily. Substitution of the phenyl group in compound 8 with 4-fluoro 11, 4-methyl 12, 4-chloro 13, and 2,4-difluorophenyl 14 led to compounds with moderate PI3KR, PI3Kγ, and mTOR potency. It can also be noted from Table 1 that substitution of the phenyl group with a lipophilic group (examples 11, 12, 13, and 14) led to a decrease in the potency against PI3KR. However, all these compounds, with the exception of 29, had excellent potency against mTOR. It appears from the different examples that PI3KR potency is favored if the R group is an aryl substituent. Molecular model- ing using the PI3Kγ homology model revealed that the sub- stituents on the R groups are solvent exposed (Figure 4). Hence, compounds 15 and 16, bearing polar entities such as -CH2OH and -CH2CH2OH were prepared.
be due to their marginally improved permeability. The PAMPA permeability for compound 15 is 0.18 10-6 cm/s, and the Caco2 data for compound 16 is 2.9 (A > B) and 7.2 (B > A). Compounds 15 and 16 had poor human and nude mouse microsomal stabilities, which precluded them from further investigation. Heterocycles 18, 19, and 20 containing polar nitrogen also gave potent compounds with slightly increased human and nude mouse microsomal stabilities. However, these analogues did not have a good solubility profile at pH 7.4. Hence, compounds such as 24-30 bearing a basic amine moiety were prepared. As can be seen from Table 1, except for compound 29, compounds bearing a basic amine moiety with an amide linkage exhibited excellent enzyme and cell potencies. These analogues also had good microsomal stability in all three species and exhibited good to moderate solubility. This dramatic boost in the PI3KR enzyme potency by the amide substituent is not very well understood and may be due to an increased hydrogen bond accepting ability of the overall molecule. On the basis of enhanced potency, solubility, microsomal stability, and lack of Cyp inhibition, compound 26 was chosen for further in vitro and in vivo evaluations. Compound 26 was evaluated for its potency against class I PI3Ks (i.e., the β, γ, and δ isoforms) and the most frequently occurring mutant forms of PI3KR, notably the H1047R and E545K. The IC50 values are shown in Table 2. These results show that compound 26 is a pan- PI3K/mTOR inhibitor, with equivalent potency against mTOR (IC50 = 1.4 nM, Table 1). This compound was also evalu- ated against a panel of 236 other human protein kinases at 10 μM, and it was found to be highly selective for PI3K and mTOR.
In vitro, compound 26 showed good potency in cell growth inhibition assays using MDA-361 and PC3-MM2 cell lines. Tumor cell growth inhibition by 26 correlated with suppres- sion of phosphorylation of PI3K/mTOR signaling pathway proteins in MDA-361 tumor cells (Figure 5). Compound 26 prevented the phosphorylation of Akt (pAkt) at threonine 308 (T308; IC50 = 8 nM) (phosphoblot IC50 values were deter- mined by densitometric scans of Western blots) and induced cleaved PARP at 30 nM. Cleaved PARP is a marker for cells undergoing apoptosis. Full activation of Akt kinase occurs when the mTOR containing mammalian target of rapamycin 2 (TORC2) protein complex phosphorylates Akt at serine 473 (S473). Figure 5 also shows potent (IC50 < 10 nM) suppression of Akt phosphorylation at S473 by 26. Phophory- lation of Akt kinase effector proteins GSK3 kinase, endothe- lial nitric oxide synthase (ENOS), and prolinerich Akt sub- strate (PRAS 40) was also suppressed by 26 at concentrations less than 30 nM. The inhibition of mTOR containing TORC1 kinase activity by 26 was demonstrated by suppression of p70S6K and 4EBP1 phosphorylation at concentrations less than 30 nM by 26. Compound 26 did not affect the overall Akt content in MDA-361 cells at concentrations tested (Figure 5), indicating pAkt suppression was not an artifact of compound cytotoxicity. Initial blood level studies in female nude mice were done with compounds 24, 25, and 26 via both oral (po) and iv routes of administration. None of these three analogues had oral exposure in nude mice. However, when 26 was administered to nude mice at 25 mg/kg iv (in 5% dextrose [D5/W], 0.3% lactic acid, pH 3.5), low plasma clearance (7 (mL/min)/kg) compared to liver blood flow (90 (mL/min)/kg), high volume of distribution (7.2 L/kg) compared to plasma volume (0.7 L/kg), and long half-life, (14.4 h) were observed (Table 3). Low clearance indicated minimal metabolism, and high volume of distribution was suggestive of extensive tissue distribution in nude mice. When compound 26 was administered iv (single dose) at 25 mg/kg, it suppressed pAkt (T308 and S473) for up to 36 h and induced cleaved PARP up to 18 h in MDA-361 tumors staged in nude mice (Figure 6). Compound 26 exhibiting potent antitumor efficacy against MDA-361 tumors is also demonstrated in Figure 7. Com- pound 26 administered iv at 20 mg/kg on an intermittent regimen (days 1, 5, 9) caused regression of large staged ( 900 mm3) tumors. This effect was more pronounced than that observed for paclitaxel (Taxol) given at 60 mg/kg (single dose, ip). In subsequent in vivo studies the minimum effi- cacious dose (MED) of 26 was determined to be 3 mg/kg against MDA-361 tumors and maximum tolerated single dose (MTD) was determined to be 30 mg/kg. Potent antitumor activity of compound 26 was also demon- strated in an orthotopic version of the H1975 (non-small-cell lung carcinoma, mutant EGFR [L858R, T790M]) xenograft model (Figure 8). H1975 cells were injected into the pleural cavity of nude mice, and the animals were treated with 26 once weekly at 25 mg/kg for 7 weeks. As can be seen from Figure 8, all of the untreated control animals were dead by day 50, compared to 90% survival in the group treated with 26. Conclusion We have shown that bis(morpholinotriazine) compounds bearing bisarylureas Gedatolisib are potent dual PI3K/mTOR inhibitors.

References

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