Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4- d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N2-(2-Ethoxy-4-(4-methyl-4 H-1,2,4-triazol-3-yl)phenyl)-6-methyl- N8-neopentylpyrido[3,4- d]pyrimidine-2,8-diamine (BOS172722).

Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.

Introduction

MPS1 (monopolar spindle 1, also known as TTK) is a dual-specificity kinase that occupies a central role in mitosis. MPS1 is one of the main components of the spindle assembly checkpoint (SAC)[1−4] and ensures cells do not progress from metaphase to anaphase until the kinetochores are properly attached to the microtubules and under the appropriate tension at the metaphase plate.[2,3] Cancer cells heavily rely on MPS1 to cope with aneuploidy resulting from aberrant numbers of chromosomes.[5−8] The kinase has been found to be upregulated in a large number of tumor types[6,7,9−13] strongly suggesting MPS1 inhibition as a therapeutic approach for the treatment of cancer. As a result, MPS1 inhibitors have been pursued by a number of organizations, and accordingly, at least four compounds have reached Phase 1 clinical trials: BAY1161909 (1),[14] BAY1217389 (2),[14] CFI-402257 (3),[15] and S 81694 (structure undisclosed). We recently reported advanced inhibitors including CCT251455 (4)[16] and a series of pyrido[3,4-d]pyrimidines (34h (5))[17] (Figure ).

Figure 1

Published MPS1 inhibitors.

While the single agent efficacy of MPS1 inhibitors has been described to be limited,[18−21] a number of recent reports have documented that inhibition of MPS1 is particularly effective when used in combination with other drugs, for example, tubulin-targeting agents or CDK4/6 inhibitors.[22−26] We were particularly intrigued by the use of MPS1 inhibitors in combination with paclitaxel for triple negative breast cancer. Paclitaxel is often used for this aggressive and highly proliferative cancer but, on its own, does frequently not lead to durable responses, particularly in the metastatic setting.[27] Triple negative breast cancer thus remains a high medical need, and new effective therapeutic regimens are needed.

The work presented here builds upon a series of pyrido[3,4-d]pyrimidines that we recently disclosed.[17] Advanced compounds in this series showed excellent potency in biochemical and cellular assays, exemplified by 5 (Figure ); which was effective in inhibiting MPS1 in vivo.[17] However, this series in general, and 5 in particular, suffered from key liabilities that prevented further development, specifically high turnover in human microsomes as well as excessive lipophilicity.

Figure 2

MPS1 inhibitor 5. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells. Solubility conditions: HPLC, 1% DMSO, 10 mM PBS, pH 7.4.

Herein, we describe our optimization of the pyrido[3,4-d]pyrimidine series[17] culminating in the discovery of a Phase 1 clinical candidate compound.

Chemistry

Des methyl pyrido[3,4-d]pyrimidine compounds were made using the route shown in Scheme . Two complementary approaches could be used to gain access to the des methyl compounds. First, the amine was introduced into 7 by displacement of the chloride, followed by m-CPBA oxidation to give sulfone 9. Displacement of the sulfone with the appropriate formamide under NaH/THF conditions gave rise to the desired des methyl pyrido[3,4-d]pyrimidine compounds (Scheme ). Alternatively, the steps could be reversed, carrying out the m-CPBA oxidation as the first step to afford sulfone 18. Displacement with the appropriate formamide could then be carried out as previously, before introducing the amine at the final step, through reaction of the chloro-intermediate 20 with neopentylamine in NMP at elevated temperatures.

Scheme 1

Reagents and conditions: (i) amine, NMP, 100–130 °C; (ii) m-CPBA, CH2Cl2, 0 °C–r.t.; (iii) ArNHCHO 10–13 or 19, NaH, THF, 0 °C–r.t.

Compounds in the 6-methyl pyrido[3,4-d]pyrimidine series were prepared from the key intermediate 8-chloro-2-(methylthio)pyrido[3,4-d]pyrimidine 22, the synthesis of which we have previously reported.[28] Treatment of this intermediate with an amine at elevated temperature in NMP gave rise to sulfides 23–25. Oxidation of these compounds with m-CPBA afforded sulfones 26–28, which were ideally set up to undergo selective displacement. The aniline moiety was introduced using the corresponding formamide with either NaH in THF or cesium carbonate in DMSO, affording the desired 6-methyl pyrido[3,4-d]pyrimidines 34–42 (Scheme ).

Scheme 2

Reagents and conditions: (i) amine, NMP, 100–130 °C; (ii) m-CPBA, CH2Cl2, 0 °C–r.t.; (iii) ArNHCHO 12, 13, or 29–33, NaH, THF, 0 °C–r.t.; (iv) ArNHCHO, Cs2CO3, DMSO, 120 °C.

Again the order of steps could be reversed with the oxidation with m-CPBA being carried out first to give sulfone 43, which could then undergo the same coupling with the appropriate formamide as previously described using NaH in THF. Displacement of the chloro intermediate 44 was then carried out with amines at elevated temperatures in NMP (Scheme ).

Scheme 3

Reagents and conditions: (i) m-CPBA, CH2Cl2, 0 °C–r.t.; (ii) ArNHCHO 29, NaH, THF, 0 °C–r.t.; (iii) amine, NMP, 100–130 °C.

All amines used were commercially available, and the formamides were synthesized from the corresponding anilines by refluxing in formic acid. The anilines were prepared by standard transformations (see Supporting Information for procedures).

Results and Discussion

We routinely tested our compounds in a caliper-based MPS1 kinase assay at 1 mM ATP. As described in our preceding publication,[17] this relatively high ATP concentration was necessary due to the high potency of advanced compounds (Kis < 1 nM) that was beyond the dynamic range of the assay at lower ATP concentrations. Furthermore, we progressed compounds of sufficient potency to an MSD-based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells.[16] In addition, we routinely determined selectivity against CDK2, a cell cycle kinase with a high homology to MPS1 in terms of the ATP binding domain. Since the CDK2 assay was run at much lower ATP concentrations, we converted the IC50 values from the MPS1 assay (at 1 mM ATP) and those from the CDK2 assay (at 10 μM ATP) into Ki values and used these calculated Kis to assess the selectivity ratio.

Our key goal for the optimization of the pyrido[3,4-d]-pyrimidines series was to significantly improve the low human liver microsomal stability observed for compound 5 and other compounds in this series. Extensive attempts to identify major metabolites showed multiple oxidations and dealkylation products but failed to identify a consistent site of metabolism. Furthermore, we had not observed a correlation between stability in human microsomes and lipophilicity indicating that lowering LogP was not a promising approach. We thus suspected that rapid turnover was driven by recognition of a specific pharmacophore within our series and decided to systematically derivatize the molecule to discover modifications that would block this recognition and increase the metabolic stability.

We started by altering the five-membered ring heterocycle of 5 maintaining a neopentyl substituent in the 8-position of the pyrido[3,4-d]pyrimidine core. These compounds are summarized in Table . Methylated and dimethylated pyrazole containing compounds (21, 14, and 15) showed only relatively modest activity in the high ATP assay, as well as in the cellular assay. The imidazole substituted compound (16) demonstrated excellent levels of biochemical activity, as well as showing promising levels of selectivity over CDK2 and cellular activity. Finally, triazole containing 17, while exhibiting nanomolar potency for CDK2, stood out in terms of its single digit nanomolar potency in the cellular assay and represents one of the most potent MPS1 inhibitors known to us. In fact, the potency of this compound was beyond the lower range of our biochemical assay even at 1 mM ATP. The reason why replacing the pyrazole moiety in 21 with imidazole (15) and particularly triazole (16) significantly boosted biochemical activity remained unclear, and crystal structures (vide infra) did not provide any additional insights.

Table 1

Biochemical and Cellular Data for Aniline Modifications on Neopentyl-Substituted Corea

Results are in nM unless otherwise stated and are mean for n ≥ 3, or mean values of two independent determinations or samples run n = 1. For SD (for n ≥ 3) and individual determinations (n = 2), see Table S1. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells. Kis were calculated from the IC50s using the Cheng–Prusoff equation.

We tested the human liver microsome (HLM) stability of compounds 16 and 17, but both still underwent extensive metabolism showing Clint values of 79 and 92 μL/min/mg protein, respectively. Nevertheless, the comparably low molecular weight (418) and lipophilicity (3.6) made 17 an excellent starting point for further investigation, and we decided to improve CDK2 selectivity and HLM stability.

In order to develop hypotheses on how to reduce CDK2 activity, we superimposed X-ray structures of our compounds[17] with published CDK2 structures (Figure ).[29,30]

Figure 3

Top: Superimposed crystal structure of MPS1 (green) bound to a pyrido[3,4-d]pyrimidine core (carbon atoms colored green), extracted from PDB code 5EH0, onto the structure of CDK2 (blue), extracted from PDB code 1H08 (ligand hidden for clarity), showing the different gatekeeper residues present in MPS1 and CDK2. Bottom: 6-position methyl group on pyrido[3,4-d]pyrimidine core.

Fourteen residues are different within the ATP binding pockets of MPS1 and CDK2, including the gatekeeper residue, which is Met602 in MPS1 and a bulkier phenylalanine (Phe80) in CDK2 (Figure ). We hypothesized that introducing a methyl group at the 6-position of the pyrido[3,4-d]pyrimidine core would be less tolerated in CDK2 than in MPS1, due to a clash with the CDK2 Phe80 gatekeeper residue.

We thus set out to prepare a series of methyl substituted compounds. The significant investigations required to access pyrido[3,4-d]pyrimidines with substitution in this position were disclosed by us recently.[28]

Table shows the biochemical and cellular results for two initial proof-of-concept compounds made as matched pairs to the corresponding unsubstituted compounds (16 and 17). Both compounds show potent biochemical inhibition of MPS1 and lower but still acceptable levels of inhibition in cells. As hypothesized, both 6-methylated compounds (34 and 35) demonstrated a significant improvement in selectivity for MPS1 over CDK2 (Ki ratio is between 500 and 7600). Even more importantly and somewhat unexpectedly, we observed a large improvement in HLM metabolism for the 6-methylated compounds. Compounds 34 and 35 represented by far the most stable compounds we had observed and thus a breakthrough in terms of optimizing the up to this point persisting HLM liability. We thus decided to focus on the methylated amino-pyrido[3,4-d]pyrimidine core.

Table 2

Biochemical and Cellular Data for Two Matched Pairs of Compounds Containing H or Me at the 6-Position of the Pyrido[3,4-d]pyrimidine Corea

Results are in nM unless otherwise stated and are mean for n ≥ 3, or mean values of two independent determinations or samples run n = 1. For SD (for n ≥ 3) and individual determinations (n = 2), see Table S2. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells. Kis were calculated from the IC50s using the Cheng–Prusoff equation.

To understand why these compounds showed greatly improved stability, we identified the metabolites for the matched pair 17 and 35. Interestingly, these experiments showed that the introduction of the methyl group completely changed the nature of the main metabolites in HLM (Figure ). Incubation of compound 17 with HLM primarily led to metabolites in which the triazole and aniline moieties were oxidized (Figure ). In sharp contrast, treatment of 35 with HLM led to oxidation on the neopentyl chain followed by loss of the entire chain (Figure ). Importantly, the metabolic hotspots are not only different for these compounds but, in both cases, also distant from the position of the newly introduced methyl group of 35. This observation thus suggests that the reduction of HLM metabolism is not due to blocking of a metabolically labile position (a commonly applied strategy, particularly using fluorine atoms) but instead to blocking of the pharmacophore through which 17 is recognized and bound. This hypothesis is consistent with the nature of P450 enzymes where substrate recognition and catalytic sites are spatially separated.

Figure 4

Results of a Met ID study showing oxidation products for the match pair of compounds 17 and 35 after treatment with HLM.

Compound 35 thus represented a significant step forward, and we explored whether HLM stability, CDK2 selectivity, cellular potency, and solubility could be further optimized. Table summarizes modifications of the methoxy group and the triazole ring substituents. Based on existing SAR, we hypothesized that introducing an ethoxy group in place of the methoxy group of 35 improves selectivity further. The corresponding ethoxy compound (36) showed similar levels of biochemical potency (IC50 11 vs 13 nM), albeit with a slight drop in cellular potency (P-MPS1 IC50 63 vs 30 nM). As hypothesized, this transformation resulted in an improved selectivity window over CDK2 (Ki ratio 500 (35) vs 46 (36)) and significantly improved stability in HLM.

Table 3

Biochemical and Cellular Data for Triazole Modifications Based on 35a

Results are in nM unless otherwise stated and are mean for n ≥ 3, or mean values of two independent determinations or samples run n = 1. For SD (for n ≥ 3) and individual determinations (n = 2), see Table S3. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells.

Introduction of a methyl group onto the triazole ring (37) resulted in very similar levels of potency to 35 both in the biochemical and cellular assays (Table ), albeit with an increase in lipophilicity (ALogP = 4.49 vs 3.88). The bicyclic triazole derivatives 38 and 39 were also potent biochemical inhibitors but showed significantly weaker inhibition in cells. Interestingly, this matched pair (38 and 39) also demonstrated a similar increase in selectivity between methoxy and ethoxy derivatives. Finally, adding a basic dimethylamino group to improve solubility (40) resulted in loss of cellular potency (P-MPS1 IC50 230 nM) possibly due to a decrease in cellular permeability of the more polar dimethylamine tail group, though the solubility (77.6 μM [HPLC method, 1% DMSO, 10 mM PBS, pH 7.4]) of this compound was greatly improved in comparison to 35.

From this investigation, 36 emerged as an attractive compound, and we tested if the overall properties could be further optimized by modification of the neopentyl amine.

Table shows a representative set of amine substitutions at the 8-position of the pyrido[3,4-d]pyrimidine core. Compound 45 bears the same branched primary amine used in our previously reported MPS1 inhibitor 5.[17] Compound 45 demonstrated good activity against MPS1 in the biochemical and cellular assays (Table ) but exhibits poor solubility (2.2 μM), a possible consequence of the increased ALogP (4.61). The introduction of secondary amines including pyrrolidine (46) and substituted azetidines (47, 48, and 42) exhibited varied activity against MPS1. Pyrrolidine containing 46 displayed a significant decrease in the biochemical and cellular potency observed. Compound 47 exhibited excellent activity (P-MPS1 IC50 37 nM) and good levels of selectivity over CDK2 coupled with an increased solubility of 31.5 μM, possibly due to the decreased ALogP of 3.85. However, movement of the gem dimethyl group one carbon round the ring into the α-position (48) resulted in loss of all MPS1 activity (P-MPS1 IC50 3.8 μM). Cyano-substituted azetidine compound (42) also showed acceptable levels of potency (P-MPS1 IC50 110 nM) and selectivity over CDK2 (CDK2 IC50 2.70 μM), combined with a much lower AlogP of 3.25. However, this reduced ALogP did not translate into increased solubility, with 42 only showing a solubility of 6.8 μM. Introduction of a polar spirocyclic amine (41) resulted in a dramatic drop in lipophilicity (AlogP = 3.08), which, as expected, translated into an increase in solubility (56.2 μM).

Table 4

Biochemical and Cellular Data for Amine Modifications Based on 36a

Results are in nM unless otherwise stated and are mean for n ≥ 3, or mean values of two independent determinations or samples run n = 1. For SD (for n ≥ 3) and individual determinations (n = 2), see Table S4. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells.

To understand the observed MPS1 SAR (Table ), we solved the crystal structure of compound 36 bound to MPS1 (Figure A). As expected, the binding mode of 36 was nearly identical to that of the previously described pyrido[3,4-d]pyrimidine inhibitors.[17] The pyrido[3,4-d]pyrimidine scaffold of 36 binds to the hinge region, the 6-methyl group important for CDK2 selectivity and for the reduction in HLM metabolism, is located close to the side chain of the gatekeeper residue Met602, and the ethoxy moiety also important for selectivity binds in the selectivity pocket above the hinge. Somewhat surprisingly, the triazole moiety was not engaged in a hydrogen bond, and thus the X-ray structure did not explain why replacing the pyrazole of 21 with triazole (17, Table ) led to a significant increase in activity (vide supra). Together with previously reported structures of CCT251455 (4) and (5), the X-ray structure of 36 allowed us to rationalize the SAR summarized in Table . As previously described for compound 5, the neopentyl chain binds to a hydrophobic pocket that is created by a reordering of the MPS1 activation loop into an inactive conformation (Figure B).[16,17] The shape of the neopentyl chain represents an excellent match to this pocket resulting in several hydrophobic contacts explaining why this moiety is critical for activity. The two azetidine derivatives 47 and 48 exemplify the importance of the correct shape of the amine substituent for potent inhibition. The 3,3-dimethylazetidine substituent of derivative 47 can be regarded as a constrained mimetic of the neopentyl chain that can likely engage in similar hydrophobic contacts, and 47 maintains potent inhibition. The 2,2-dimethylazetidine moiety of 48, however, differs significantly in its overall shape from the neopentyl chain leading to less favorable interactions and a 40-fold higher IC50.

Figure 5

(A) Crystal structure of 36 bound to MPS1 (pdb code 6H3K). Compound 36 is shown with carbon atoms in yellow. Selected amino acids are shown in sea green and are labeled. Key hydrogen bonds are indicated as black dotted lines. (B) Close up of the neopentyl binding pocket. The neopentyl substituent is enveloped by residues Met671 and Pro673 from the activation loop. Protein surface is displayed as a transparent blue surface. The compound surface is shown in transparent yellow.

To investigate which of the potent and selective compounds in Table can be progressed further, we tested the stability in liver microsomes. Gratifyingly, all compounds tested showed satisfactory stability (Clint < 45 μL/min/mg protein in mouse and Clint < 26 μL/min/mg protein in human) (Table ). This represented a vast improvement over the human intrinsic clearance values seen for compound 5 (Clint 151.2 μL/min/mg protein), the starting point of our investigation.

With a number of active, selective, and soluble compounds in hand, we decided to investigate the mouse and rat pharmacokinetics (PK) of a selection of compounds (36, 45, 47, and 41) at 5 mg/kg administered both intravenous (iv) and orally (po) (Table ).[31] The resulting data showed moderate clearance for all compounds in both mouse (8.4–22.6 mL/min/kg) and rat (6.00–10.1 mL/min/kg). All compounds with the exception of 41 showed high oral bioavailability in both species (63–92%) with moderate volumes of distribution (Table ). The plasma protein binding for all four of these compounds was high (>98%), the lowest unsurprisingly shown by that presenting the lowest AlogP (41). This corresponded to a higher blood clearance and shorter half-life (0.66 h) for 41 compared to 36 (2.68 h).

Table 5

Mouse and Rat Blood Pharmacokinetics of 36, 41, 45, and 47 at 5 mg/kg iv and po, unless Otherwise Stateda,b,c

2.5 mg/kg (iv).

1 mg/kg (iv).

Compounds were administered iv and po (Mouse, 0.1 mL/10 g in 10% DMSO, 5% Tween 20 in saline; Rat, 0.05 mL/10 g in 10% DMSO, 5% Tween 20 in saline).

AUClast 6 h for mouse, 24 h for rat, unless otherwise stated.

AUClast 6 h.

We thus decided to progress both 36 and 41 to a single dose pharmacokinetic/pharmacodynamic (PK/PD) experiment in DLD1 xenografts to investigate if the in vitro and PK profiles translated into sustained inhibition of MPS1 in vivo (Figure ). We recently disclosed[32] a xenograft model to assess modulation of MPS1 activity in vivo. Briefly, this model (Dox-DLD1) measures the level of MPS1 autophosphorylation in DLD1 cancer cells and, importantly, overcomes the issue of low MPS1 levels through doxycycline inducible expression of the kinase. We tested both compounds at 25 mg/kg and in addition compound 36 at 50 mg/kg. The data are summarized in Figure . At 25 mg/kg, both compounds led to a pronounced reduction of MPS1 autophosphorylation after 6 h. Consistent with its longer half-life, only 36 showed significant inhibition at 24 h. As expected, the 50 mg/kg dose of 36 resulted in an increased suppression of MPS1 autophosphorylation at 6 and 24 h. The comparison of the plasma levels determined in the PK/PD study (Figure ) after blood to plasma correction with the compound levels determined in the PK study described above and performed at lower doses (Table ) was consistent with linear PK.

Figure 6

Bar charts show the ratio of phosphylated-MPS1/total-MPS1 levels (gray) in the Dox-DLD1 model after treatment with 36 (left) and 41 (right) at specified doses and time points. The plasma levels at the respective time points are given in the table below the bar chart.

The robust modulation of the PD biomarkers observed for 36 prompted us to focus on this compound since a long duration of action is desirable for cell cycle targets. At 1 mg/kg iv and 5 mg/kg po, 36 showed complete bioavailability (100%), low clearance (1.2 mL/min/kg), a moderate volume of distribution (1.1 L/kg), and a 12 h half-life (Table ) in a dog PK study.

Table 6

Dog Blood Pharmacokinetics not Dogblood 36 at 1 mg/kg iv and 5 mg/kg poa

The compound was administered as the dichloride salt in saline containing 10% DMSO.

AUClast 48 h.

We were intrigued by the high bioavailability of 36, particularly given its poor solubility. Interestingly, pKa values for 36 were determined as 6.22 and 2.63. This suggests that, while the compound primarily exists as the free base at physiological pH, it is protonated in the acidic environment of the stomach, likely accelerating the dissolution and enabling high bioavailability despite modest solubility at physiological pH. This is an attractive feature of the pyrido [3,4-d]pyrimidine scaffold since it avoids the well-recognized risk associated with drugs carrying a positive charge at physiological pH, namely, hERG inhibition, efflux, and detrimental effects on passive permeation while still allowing high bioavailability and salt formation. To shed light on the question of which atom represents the weakly basic center, we performed a calculation using MoKa.[33] This calculation predicted that the nitrogen atom of the pyridine ring is by far the most basic atom.

To complete the in vitro profiling, 36 was tested in a wide panel of more than 400 kinases (Supporting Information, Tables S5–S8). As we had seen with our previous MPS1 inhibitor 5,[17] only a small number of other kinases were inhibited by 36, in particular JNK1, JNK2, JNK3, and LRRK2 at >80% at 1 μM. Follow up IC50 values were obtained (JNK1 IC50 = 92 nM, JNK2 IC50 = 76 nM, JNK3 IC50 = 242 nM, and LRRK2 IC50 = 48 nM) showing that 36 is selective for MPS1 over these other kinases. Furthermore, 36 also showed a clean CYP and hERG profile (Supporting Information, Tables S9 and S10).

Following extensive in vivo testing, particularly in combination with paclitaxel, the results of which will be published in due course (manuscript in preparation), we nominated 36 as our candidate. The synthesis of 36 has been scaled up into the kilogram range, and the drug is currently undergoing Phase 1 clinical trials.

Conclusions

We describe herein the discovery of our MPS1 inhibitor 36 (BOS172722). The starting point for the work described here was a series of previously reported pyrido[3,4-d]pyrimidine inhibitors. These earlier compounds already showed promising in vitro potency and selectivity but suffered from a number of liabilities, particularly high lipophilicity and rapid metabolism in HLM. Optimizing HLM metabolism proved challenging since commonly used approaches, such as identification of metabolites and lowering lipophilicity, did not help. Key to overcoming this problem was the serendipitous finding that introduction of a methyl group at the 6-position of the pyrido[3,4-d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. Compound 17 is thus an interesting example where metabolism is not primarily driven by hydrophobicity or the presence of a particular metabolic hotspot but by recognition of a specific pharmacophore distant from the site of metabolism. These results underscore the importance of systematic chemical modification to solve high metabolic turnover.

Further optimization led to a set of compounds with promising in vitro profile, and we progressed a number of selected compounds to PK and subsequently PK/PD experiments. Compound 36 emerged as our candidate showing excellent PK in mouse, rat, and dog. Data showing robust efficacy of 36 in combination with paclitaxel in in vivo models will be published shortly.

Interestingly, 36 showed very good bioavailability in all three species despite very modest solubility at physiological pH. We attribute this observation to the weakly basic character of 36 (pKa = 6.22) and other compounds in this series, which is likely accelerating their dissolution in the acidic environment of the stomach.

The synthesis of 36 has been scaled to the kilogram range, and the compound is currently in Phase 1 clinical trials.

Experimental Section

General Chemistry Information

Starting materials, reagents, and solvents for reactions were reagent grade and used as purchased. Chromatography solvents were HPLC grade and were used without further purification. Thin layer chromatography (TLC) analysis was performed using silica gel 60 F-254 thin layer plates. Flash column chromatography was carried out using columns prepacked with 40–63 μm silica. Microwave-assisted reactions were carried out using a Biotage Initiator microwave system. LCMS and HRMS analyses were performed on a HPLC system with diode array detector operating at 254 nm, fitted with a reverse-phase 50 × 4.6 mm column at a temperature of 22 °C, connected to a time of flight (ToF) mass spectrometer (ESI). The following solvent system, at a flow rate of 2 mL/min, was used: solvent A, methanol; solvent B, 0.1% formic acid in water. Gradient elution was as follows: 1:9 (A:B) to 9:1 (A:B) over 2.5 min, 9:1 (A:B) for 1 min then reversion back to 1:9 (A:B) over 0.3 min, 1:9 (A:B) for 0.2 min. 1H NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer using an internal deuterium lock. NMR data is given as follows: chemical shift (δ) in ppm, multiplicity, coupling constants (J) given in Hz, and integration. The purity of final compounds was determined by HPLC as described above and is ≥95% unless specified otherwise.

Compounds 8-chloro-2-(methylthio)pyrido[3,4-d]pyrimidine[28]7, 2-(methylthio)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine[28]8, 2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine[28]9, 8-chloro-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine[28]18, N-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)formamide[28]19, 8-chloro-N-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyrido[3,4-d]pyrimidin-2-amine[28]20, N2-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine[17,28]21, 8-chloro-6-methyl-2-(methythio)pyrido[3,4-d]pyrimidine[28]22, 6-methyl-2-(methylthio)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine[28]23, and 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine[28]26 were synthesized by previously reported methods.

Preparation of Compounds in Scheme

General Procedure for NaH Mediated Displacement on 9 (Compounds 14–17)

To a cooled (0 °C) solution of appropriate formamide (1 equiv) in THF (3 mL) was added sodium hydride (60% dispersion in oil, 1.6 equiv). The reaction mixture was stirred at r.t. for 10 min. The reaction mixture was cooled to 0 °C, and appropriate sulfone (1.2 equiv) was added. The reaction mixture was stirred for 18 h, while slowly warming to r.t. aq NaOH (1 M, 1 mL) and MeOH (1 mL) were added, and the reaction mixture was stirred at r.t. for 1 h. The reaction mixture was concentrated in vacuo, and the residue was diluted with EtOAc and water. The aqueous layer was re-extracted with EtOAc and CH2Cl2. The combined organic layers were washed with water and brine, dried (MgSO4), and concentrated in vacuo. The residue was purified by flash column chromatography (0–10% MeOH in EtOAc or CH2Cl2) and, if necessary, followed by SCX-2 cartridge (MeOH, 1 M NH3 in MeOH) to afford the title compounds.

N2-(4-(1,3-Dimethyl-1H-pyrazol-4-yl)-2-methoxyphenyl)-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 14

Using N-(4-(1,3-dimethyl-1H-pyrazol-4-yl)-2-methoxyphenyl)formamide 10 and 2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 9.[28] Yield = 21%. HRMS (ESI) m/z calcd for C24H30N7O (M + H) 432.2506, found 432.2502; 1H NMR (500 MHz, CD3OD) δ 9.07 (s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.10 (d, J = 1.5 Hz, 1H), 7.03 (dd, J = 8.0, 1.5 Hz, 1H), 6.85 (d, J = 6.0 Hz, 1H), 4.00 (s, 3H), 3.88 (s, 3H), 3.40 (s, 2H), 2.40 (s, 3H), 1.09 (s, 9H).

N2-(4-(1,5-Dimethyl-1H-pyrazol-4-yl)-2-methoxyphenyl)-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 15

Using N-(4-(1,5-dimethyl-1H-pyrazol-4-yl)-2-methoxyphenyl)formamide 11 and 2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 9.[28] Yield = 8%. HRMS (ESI) m/z calcd for C24H30N7O (M + H) 432.2506, found 432.2504; 1H NMR (500 MHz, CDCl3) δ 9.02 (s, 1H), 8.51 (br s, 1H), 8.05 (m, 1H), 7.84 (m, 1H), 7.58 (s, 1H), 6.99 (dd, J = 8.2, 1.8 Hz, 1H), 6.96 (d, J = 1.8 Hz, 1H), 6.80 (br s, 1H), 3.99 (s, 3H), 3.88 (s, 3H), 3.64 (br s, 2H), 2.42 (s, 3H), 1.14 (s, 9H).

N2-(4-(1,2-Dimethyl-1H-imidazol-5-yl)-2-methoxyphenyl)-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 16

Using N-(4-(1,2-dimethyl-1H-imidazol-5-yl)-2-methoxyphenyl)formamide 12 and 2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 9.[28] Yield = 21%. HRMS (ESI) m/z calcd for C24H30N7O (M + H) 432.2506, found 432.2504; 1H NMR (500 MHz, CDCl3) δ 9.04 (s, 1H), 8.76 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 7.94 (d, J = 6.0 Hz, 1H), 7.21 (s, 1H), 6.99 (dd, J = 8.0, 2.0 Hz, 1H), 6.91 (d, J = 2.0 Hz, 1H), 6.79 (d, J = 6.0 Hz, 1H), 6.58 (br s, 1H), 4.02 (s, 3H), 3.67 (s, 3H), 3.50 (d, J = 6.0 Hz, 2H), 2.82 (s, 3H), 1.12 (s, 9H).

N2-(2-Methoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 17

Using N-(2-methoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 13 and 2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 9.[28] Yield = 12%. HRMS (ESI) m/z calcd for C22H27N8O (M + H) 419.2302, found 419.2288; 1H NMR (500 MHz, CD3OD) δ 9.15 (s, 1H), 8.74 (d, J = 8.5 Hz, 1H), 8.57 (s, 1H), 7.77 (d, J = 6.0 Hz, 1H), 7.43 (d, J = 1.5 Hz, 1H), 7.35 (dd, J = 8.5, 1.5 Hz, 1H), 6.90 (d, J = 6.0 Hz, 1H), 4.07 (s, 3H), 3.87 (s, 3H), 3.44 (s, 2H), 1.10 (s, 9H).

Preparation of Compounds in Scheme

General Procedure for Amine Displacement on 22

To a solution of 8-chloro-6-methyl-2-(methythio)pyrido[3,4-d]pyrimidine 22 (1 equiv) in NMP (20 mL) was added appropriate amine (2 equiv) and triethylamine (5 equiv). The reaction mixture was heated to 100 °C for 36 h. The reaction mixture was diluted with EtOAc and water, dried (MgSO4), and concentrated in vacuo. The residue was purified by flash column chromatography (0–50% EtOAc in cyclohexane) to afford the title compounds.

2-(6-Methyl-2-(methylthio)pyrido[3,4-d]pyrimidin-8-yl)-7-oxa-2-azaspiro[3.5]nonane 24

Using 7-oxa-2-azaspiro[3.5]nonane. Yield = 40%. HRMS (ESI) m/z calcd for C16H21N4OS (M + H) 317.1431, found 317.1422; 1H NMR (500 MHz, CD3OD) δ 9.00 (s, 1H), 6.76 (app s, 1H), 4.33 (br s, 4H), 3.71 (app t, J = 5.0 Hz, 4H), 2.65 (s, 3H), 2.44 (d, J = 0.5 Hz, 3H), 1.90 (app t, J = 5.0 Hz, 4H).

3-Methyl-1-(6-methyl-2-(methylthio)pyrido[3,4-d]pyrimidin-8-yl)azetidine-3-carbonitrile 25

Using 3-methylazetidine-3-carbonitrile hydrochloride. Yield = 66%. HRMS (ESI) m/z calcd for C14H16N5S (M + H) 268.1121, found 268.1119; 1H NMR (500 MHz, CD3OD) δ 9.05 (s, 1H), 6.89 (app s, 1H), 4.83 (br d, J = 7.5 Hz, 2H), 4.48 (br d, J = 7.5 Hz, 2H), 2.64 (s, 3H), 2.47 (d, J = 0.5 Hz, 3H), 1.78 (s, 3H).

General Procedure for m-CPBA Oxidation on 23–25

To a cooled (0 °C) solution of appropriate sulfide (1 equiv) in CH2Cl2 (10 mL) was added m-CPBA (3 equiv). The reaction mixture was stirred for 18 h, while slowly warming to r.t. In some instances, further m-CPBA was needed to achieve full conversion. The reaction mixture was quenched with water and extracted with CH2Cl2. The combined organic layers were washed with aq. sat. NaHCO3, dried (MgSO4), and concentrated in vacuo. The residue was purified by flash column chromatography (0–10% MeOH in CH2Cl2).

2-(6-Methyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidin-8-yl)-7-oxa-2-azaspiro[3.5]nonane 27

Using 2-(6-methyl-2-(methylthio)pyrido[3,4-d]pyrimidin-8-yl)-7-oxa-2-azaspiro[3.5]nonane 24. Yield = 28%. HRMS (ESI) m/z calcd for C16H21N4O3S (M + H) 349.1329, found 349.1318; 1H NMR (500 MHz, CD3OD) δ 9.38 (s, 1H), 6.94 (s, 1H), 4.29 (br s, 4H), 3.73 (app t, J = 5.5 Hz, 4H), 3.43 (s, 3H), 2.53 (s, 3H), 1.92 (app t, J = 5.5 Hz, 4H).

3-Methyl-1-(6-methyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidin-8-yl)azetidine-3-carbonitrile 28

Using 3-methyl-1-(6-methyl-2-(methylthio)pyrido[3,4-d]pyrimidin-8-yl)azetidine-3-carbonitrile 25. Yield = 77%. HRMS (ESI) m/z calcd for C14H16N5O2S (M + H) 318.1019, found 318.1009; 1H NMR (500 MHz, CD3OD) δ 9.46 (s, 1H), 7.08 (s, 1H), 4.54 (br s, 4H), 3.44 (s, 3H), 2.56 (s, 3H), 1.80 (s, 3H).

N2-(4-(1,2-Dimethyl-1H-imidazol-5-yl)-2-methoxyphenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 34

To a cooled (0 °C) solution of N-(4-(1,2-dimethyl-1H-imidazol-5-yl)-2-methoxyphenyl)formamide 12 (17 mg, 0.069 mmol) in THF (3 mL) was added sodium hydride (2.7 mg, 0.11 mmol, 60% dispersion in oil). The reaction mixture was stirred at r.t. for 10 min. The reaction mixture was cooled to 0 °C, and 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 26(28) (25.6 mg, 0.083 mmol) was added. The reaction mixture was stirred for 18 h, while slowly warming to r.t. aq NaOH (1 M, 1 mL) and MeOH (1 mL) were added, and the reaction mixture was stirred at r.t. for 1 h. The reaction mixture was concentrated in vacuo, and the residue diluted with EtOAc and water. The aqueous layer was re-extracted with EtOAc and CH2Cl2. The combined organic layers were washed with water and brine, dried (MgSO4), and concentrated in vacuo. The residue was purified by flash column chromatography (0–10% MeOH in EtOAc) and followed by SCX-2 cartridge (MeOH, 1 M NH3 in MeOH) to afford the title compound (3.3 mg, 11%). HRMS (ESI) m/z calcd for C25H32N7O (M + H) 446.2663, found 446.2648; 1H NMR (500 MHz, CD3OD) δ 9.02 (s, 1H), 8.57 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 7.02 (dd, J = 8.5, 2.0 Hz, 1H), 6.88 (s, 1H), 6.70 (d, J = 1.0 Hz, 1H), 4.02 (s, 3H), 3.61 (s, 3H), 3.45 (s, 2H), 2.45 (s, 3H), 2.44 (d, J = 1.0 Hz, 3H), 1.09 (s, 9H).

General Procedure for Cesium Carbonate Mediated Substitution on 26, 27, or 28

To a solution of the appropriate sulfone (1 equiv) in DMSO (20 mg sulfone/mL) was added appropriate formamide (1.2 equiv) and cesium carbonate (2 equiv). The reaction mixture was heated to 120 °C in a closed cap vial for 18 h. The reaction mixture was diluted with EtOAc and water. The aqueous layer was re-extracted with EtOAc, and the combined organic layers were dried (MgSO4) and concentrated in vacuo. The residue was purified by flash column chromatography (0–10% MeOH in CH2Cl2) and if necessary followed by SCX-2 cartridge (MeOH, 1 M NH3 in MeOH) to afford the title compounds.

N2-(2-Methoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 35

Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 26 and N-(2-methoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 13. Yield = 48%. HRMS (ESI) m/z calcd for C23H29N8O (M + H) 433.2459, found 433.2447; 1H NMR (500 MHz, CD3OD) δ 9.05 (s, 1H), 8.75 (d, J = 8.5 Hz, 1H), 8.56 (s, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.34 (dd, J = 8.5, 2.0 Hz, 1H), 6.71 (d, J = 1.0 Hz, 1H), 4.07 (s, 3H), 3.87 (s, 3H), 3.48 (s, 2H), 2.44 (app s, 3H), 1.10 (s, 9H).

N2-(2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 36

Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 26 and N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 29. Yield = 70%. HRMS (ESI) m/z calcd for C24H31N8O (M + H) 447.2615, found 447.2629; 1H NMR (500 MHz, acetone-d6) δ 9.11 (s, 1H), 8.80 (d, J = 8.5 Hz, 1H), 8.37 (s, 1H), 8.21 (br s, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.5, 2.0 Hz, 1H), 6.74 (d, J = 0.5 Hz, 1H), 4.35 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.51 (s, 2H), 2.41 (d, J = 0.5 Hz, 3H), 1.55 (t, J = 7.0 Hz, 3H), 1.08 (s, 9H).

N2-(4-(4,5-Dimethyl-4H-1,2,4-triazol-3-yl)-2-ethoxyphenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 37

Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 26(28) and N-(2-ethoxy-4-(4,5-dimethyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 30. Yield = 56%. HRMS (ESI) m/z calcd for C25H33N8O (M + H) 461.2772, found 461.2756; 1H NMR (500 MHz, CD3OD) δ 9.06 (s, 1H), 8.75 (d, J = 8.5 Hz, 1H), 7.34 (d, J = 1.9 Hz, 1H), 7.28 (dd, J = 8.5, 1.9 Hz, 1H), 6.72 (s, 1H), 4.30 (q, J = 7.2 Hz, 2H), 3.71 (s, 3H), 3.48 (s, 2H), 2.53 (s, 3H), 2.45 (s, 3H), 1.56 (t, J = 7.2 Hz, 3H), 1.10 (s, 9H).

N2-(4-(6,7-Dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)-2-methoxyphenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 38

Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 26(28) and N-(4-(6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)-2-methoxyphenyl)formamide 31. Purified by SCX-2 cartridge (MeOH, 1 M NH3 in MeOH) followed by purification by HPLC. Yield = 31%. HRMS (ESI) m/z calcd for C25H31N8O (M + H) 459.2615, found 459.2571; 1H NMR (500 MHz, CDCl3) δ 8.92 (s, 1H), 8.71 (d, J = 8.3 Hz, 1H), 8.07 (s, 1H), 7.66 (d, J = 1.9 Hz, 1H), 7.26 (br d, J = 1.9 Hz, 1H), 6.59 (d, J = 0.9 Hz, 1H), 6.53 (br t, J = 5.9 Hz, 1H), 4.24 (t, J = 6.9 Hz, 2H), 4.04 (s, 3H), 3.49 (d, J = 5.9 Hz, 2H), 3.06 (dd, J = 8.3, 6.9 Hz, 2H), 2.95–2.80 (m, 2H), 2.48 (d, J = 0.9 Hz, 3H), 1.11 (s, 9H).

N2-(4-(6,7-Dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)-2-ethoxyphenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 39

Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 26(28) and N-(4-(6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)-2-ethoxyphenyl)formamide 32. Yield = 35%. HRMS (ESI) m/z calcd for C26H33N8O (M+H) 473.2777, found 473.2769; 1H NMR (500 MHz, CD3OD) δ 9.00 (s, 1H), 8.67 (d, J = 8.5 Hz, 1H), 7.51 (d, J = 1.8 Hz, 1H), 7.39 (dd, J = 8.5, 1.8 Hz, 1H), 6.67 (s, 1H), 4.35–4.28 (m, 4H), 3.47 (s, 2H), 3.04–3.01 (m, 2H), 2.91–2.87 (m, 2H), 2.43 (s, 3H), 1.57 (t, J = 6.9 Hz, 3H), 1.10 (s, 9H).

N2-(4-(5-(2-(Dimethylamino)ethyl)-4-methyl-4H-1,2,4-triazol-3-yl)-2-ethoxyphenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 40

Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 26(28) and N-(4-(5-(2-(dimethylamino)ethyl)-4-methyl-4H-1,2,4-triazol-3-yl)-2-ethoxyphenyl)formamide 33. Purified by reverse phase flash column chromatography (0–100% water in MeOH, followed by 0–30% 2 M methanolic ammonia in CH2Cl2). Yield = 18%. HRMS (ESI) m/z calcd for C28H40N9O (M + H) 518.3356, found 518.3370; 1H NMR (500 MHz, CD3OD) δ 9.05 (s, 1H), 8.75 (d, J = 8.5 Hz, 1H), 7.34 (d, J = 1.8 Hz, 1H), 7.28 (dd, J = 8.5, 1.8 Hz, 1H), 6.71 (s, 1H), 4.30 (q, J = 7.0 Hz, 2H), 3.73 (s, 3H), 3.47 (s, 2H), 3.09 (app t, J = 7.5 Hz, 2H), 2.91 (app t, J = 7.5 Hz, 2H), 2.44 (s, 3H), 2.42 (s, 6H), 1.56 (t, J = 7.0 Hz, 3H), 1.09 (s, 9H).

N-(2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methyl-8-(7-oxa-2-azaspiro[3.5]nonan-2-yl)pyrido[3,4-d]pyrimidin-2-amine 41

Using 2-(6-methyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidin-8-yl)-7-oxa-2-azaspiro[3.5]nonane 27 and N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 29. Yield = 46%. HRMS (ESI) m/z calcd for C26H31N8O2 (M + H) 487.2564, found 487.2511; 1H NMR (500 MHz, CD3OD) δ 9.06 (s, 1H), 8.57 (d, J = 8.5 Hz, 1H), 8.56 (s, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.36 (dd, J = 8.5, 2.0 Hz, 1H), 6.78 (s, 1H), 4.31–4.27 (m, 6H), 3.87 (s, 3H), 3.69 (app t, J = 5.5 Hz, 4H), 2.44 (s, 3H), 1.89 (app t, J = 5.5 Hz, 4H), 1.54 (t, J = 7.0 Hz, 3H).

1-(2-((2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)amino)-6-methylpyrido[3,4-d]pyrimidin-8-yl)-3-methylazetidine-3-carbonitrile 42

Using 3-methyl-1-(6-methyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidin-8-yl)azetidine-3-carbonitrile 28 and N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 29. Yield = 38%. HRMS (ESI) m/z calcd for C24H26N9O (M + H) 456.2255, found 456.2180; 1H NMR (500 MHz, CD3OD) δ 9.10 (s, 1H), 8.56 (s, 1H), 8.46 (d, J = 8.5 Hz, 1H), 7.40 (dd, J = 8.5, 2.0 Hz, 1H), 7.39 (d, J = 2.0 Hz, 1H), 6.91 (s, 1H), 4.75 (d, J = 9.0 Hz, 2H), 4.39 (d, J = 9.0 Hz, 2H), 4.28 (q, J = 7.0 Hz, 2H), 3.88 (s, 3H), 2.47 (s, 3H), 1.75 (s, 3H), 1.53 (t, J = 7.0 Hz, 3H).

8-Chloro-6-methyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine 43

A suspension of 8-chloro-6-methyl-2-(methylthio)pyrido[3,4-d]pyrimidine 22 (1.13 g, 5.01 mmol) in CH2Cl2 (50 mL) was treated with m-CPBA (77% w/w, 2.60 g, 11.57 mmol) at 0 °C. The reaction mixture was stirred for 18 h, while slowly warming to r.t. The reaction was quenched with water and extracted with CH2Cl2. The combined organic layers were washed with aq. sat. NaHCO3, dried (MgSO4), and concentrated in vacuo. The residue was purified by flash column chromatography (0–70% EtOAc in cyclohexane) to afford the title compound (972 mg, 75%). HRMS (ESI) m/z calcd for C9H9ClN3O2S (M + H) 258.0099, found 258.0092; 1H NMR (500 MHz, CD3OD) δ 9.82 (s, 1H), 7.96 (d, J = 0.5 Hz, 1H), 3.54 (s, 3H), 2.78 (d, J = 0.5 Hz, 3H).

8-Chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 44

To a solution of N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 29 (1.88 g, 7.63 mmol) in THF (70 mL) was added NaH (60% w/w, 500 mg, 12.50 mmol) at 0 °C. The reaction mixture was stirred at r.t. for 30 min. The mixture was cooled to 0 °C, and 8-chloro-6-methyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine 43 (2.50 g, 9.70 mmol) was added. The reaction mixture was stirred for 18 h, while slowly warming to r.t. aq. NaOH (2 M, 25 mL) and MeOH (25 mL) were added, and the resulting mixture stirred at r.t. for 1 h. The reaction mixture was concentrated in vacuo, and the residue was diluted with CH2Cl2 and water. The aqueous layer was extracted with CH2Cl2, and the combined organic layers were dried (MgSO4) and concentrated in vacuo. The residue was purified by flash column chromatography (0–6% MeOH in CH2Cl2) to afford the title compound (3.24 g, quant). HRMS (ESI) m/z calcd for C19H19ClN7O (M + H) 396.1339, found 396.1335; 1H NMR (500 MHz, (CD3)2SO) δ 9.46 (s, 1H), 8.85 (d, J = 8.3 Hz, 1H), 8.79 (s, 1H), 8.56 (s, 1H), 7.74 (d, J = 1.0 Hz, 1H), 7.49–7.36 (m, 2H), 4.25 (q, J = 7.0 Hz, 2H), 3.80 (s, 3H), 2.58 (s, 3H), 1.43 (t, J = 7.0 Hz, 3H).

General Procedure for Amine Displacement on 44 (Compounds 45–48)

To a solution of the appropriate chloro compound (1 equiv) in NMP (3 mL) was added the appropriate amine or salt thereof (2–5 equiv) and triethylamine (5 equiv). The reaction mixture was heated to 100 °C in a closed cap vial for 18 h. The reaction mixture was diluted with EtOAc and water. The organic layer was washed with brine, dried (MgSO4), and concentrated in vacuo. The residue was purified by flash column chromatography (eluting with the appropriate solvent system) and, if necessary, followed by SCX-2 cartridge (MeOH, 1 M NH3 in MeOH).

(S)-N8-(3,3-Dimethylbutan-2-yl)-N2-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidine-2,8-diamine 45

Using 8-chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 44 and (S)-3,3-dimethylbutan-2-amine at 130 °C for 2 days. Yield = 52%. HRMS (ESI) m/z calcd for C25H33N8O (M + H) 461.2777, found 461.2777; 1H NMR (500 MHz, (CD3)2SO) δ 9.15 (s, 1H), 8.57 (s, 1H), 8.43 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 1.9 Hz, 1H), 7.31 (dd, J = 8.2, 1.9 Hz, 1H), 6.72 (d, J = 1.0 Hz, 1H), 6.43 (d, J = 9.5 Hz, 1H), 4.23 (q, J = 6.9 Hz, 2H), 4.13 (dq, J = 9.4, 6.6 Hz, 1H), 3.78 (s, 3H), 2.38 (s, 3H), 1.41 (t, J = 6.9 Hz, 3H), 1.16 (d, J = 6.6 Hz, 3H), 1.00 (s, 9H).

N-(2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methyl-8-(pyrrolidin-1-yl)pyrido[3,4-d]pyrimidin-2-amine 46

Using 8-chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 44 and pyrrolidine at 130 °C. Yield = 70%. HRMS (ESI) m/z calcd for C23H27N8O (M + H) 431.2308, found 431.2315; 1H NMR (500 MHz, (CD3)2SO) δ 9.13 (s, 1H), 8.56 (s, 1H), 8.34 (s, 1H), 8.23 (d, J = 8.2 Hz, 1H), 7.39–7.29 (m, 2H), 6.76 (d, J = 0.8 Hz, 1H), 4.21 (q, J = 6.9 Hz, 2H), 3.88 (s, 4H), 3.78 (s, 3H), 2.37 (s, 3H), 1.94–1.83 (m, 4H), 1.41 (t, J = 6.9 Hz, 3H).

8-(3,3-Dimethylazetidin-1-yl)-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 47

Using 8-chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 44 and 3,3-dimethylazetidine hydrochloride. Yield = 70%. HRMS (ESI) m/z calcd for C24H30N8O (M + 2H)/2 223.1266, found 223.1261; 1H NMR (500 MHz, CD3OD) δ 9.05 (s, 1H), 8.56 (d, J = 8.5 Hz, 1H), 8.54 (s, 1H), 7.37 (d, J = 1.5 Hz, 1H), 7.34 (dd, J = 8.5, 1.5 Hz, 1H), 6.77 (s, 1H), 4.29 (q, J = 7.0 Hz, 2H), 4.21 (br s, 4H), 3.87 (s, 3H), 2.44 (s, 3H), 1.54 (t, J = 7.0 Hz, 3H), 1.38 (s, 6H).

8-(2,2-Dimethylazetidin-1-yl)-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 48

Using 8-chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 44 and 2,2-dimethylazetidine. Yield = 89%. HRMS (ESI) m/z calcd for C24H29N8O (M + H) 445.2459, found 445.2461; 1H NMR (500 MHz, CD3OD) δ 8.98 (s, 1H), 8.54 (s, 1H), 8.52 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.33 (dd, J = 8.0, 2.0 Hz, 1H), 6.65 (s, 1H), 4.70–4.66 (m, 2H), 4.27 (d, J = 7.0 Hz, 2H), 3.85 (s, 3H), 2.39 (s, 3H), 2.22 (dd, J = 9.0, 7.5 Hz, 2H), 1.71 (s, 6H), 1.54 (t, J = 7.0 Hz, 3H).

Biochemical Assays

MPS1 and CDK2 counterscreen assay were performed as reported previously.[16]

MSD Assay

An electrochemiluminescence assay (Meso Scale Discovery, MSD) for detection of autophosphorylation of MPS1 at pTpS33/37 sites in cell lysates has been described previously.[16] Autophosphorylation of MPS1 at pTpS33/37 and total MPS1-GFP levels in MPS1-doxycycline inducible DLD-1 xenografts were determined by MSD assays as described previously.[32] Results were presented as the ratio of Phospho-MPS1/Total MPS1.

Microsomal Metabolism

Microsomal clearance was determined in female CD1 mice, female Sprague–Dawley rats, and mixed gender human liver microsomes obtained from Tebu-Bio (Peterborough, U.K.) following incubation of 1 μM compound at 37 °C in 1 mg/mL microsomal protein, 3 mmol/L MgCl2, 1 mmol/L NADPH, 2.5 mmol/L, UDP-glucuronic acid, and 10 mmol/L phosphate buffer (pH 7.4) (all purchased from Sigma-Aldrich, Gillingham, U.K). Reactions were started by addition of the cofactors following 10 min preincubation of microsomes with test compound and were terminated at −1, 0, 5, 10, 15, and 30 min with three volumes of ice-cold methanol containing internal standard. Samples were centrifuged at 2800g for 30 min at 4 °C and the supernatants analyzed. Control incubations were prepared as above with omission of cofactors. Compound measurements were performed by LCMS on an Agilent quadrupole time-of-flight instrument (Agilent 6510) following separation with a 6 min gradient of 0.1% formic acid in methanol on a 50 × 2.1 mm 2.6 μm C18 column (Kinetex Phenomenex). For metabolite identification, the gradient was extended to 20 min and MS/MS carried out with fragment elucidation for ions of interest.

Pharmacokinetic Studies

Animals were adapted to laboratory conditions for at least 1 week prior to dosing and were allowed food and water ad libitum. Compounds were administered iv or po (mouse, 0.1 mL/10 g in 10% DMSO, 5% tween 20 in saline; rat, 0.05 mL/10 g in 10% DMSO, 5% tween 20 in saline; dog, 10% in DMSO in saline). Blood samples were collected from the tail vein (20 μL) at 8 time points over the 24 h post dose and spotted on Whatman B cards (VWR) together with a standard curve and quality controls spiked in control blood. Cards were allowed to dry at r.t. for at least 6 h. Cards were punched and 6 mm discs were extracted with 200 μL of methanol containing 500 nM olomoucine as an internal standard. Following centrifugation, extracts were analyzed by multiple reaction monitoring of precursor and product ions by ESI-LCMS/MS on a QTRAP 4000 (ABSciex) following gradient separation with 0.1% formic acid in methanol on a Phenomenex Kinetex C18 UPLC column (50 × 2.1 mm, 2.6 μM). Quantitation was carried out with an external calibration (typically 8 points ranging from 1 nM to 25 μM). Quality controls were included (three concentrations) at the beginning and the end of the analytical run and were within 20% of nominal concentrations.

Pharmacokinetic parameters were derived from noncompartmental analysis using Phoenix Pharsight WinNonlin (model 200 and 201) version 6.1/6.3.

All experiments using animals were performed in accordance with the local Animal Welfare and Ethical Review Board, the UK Home Office Animals Scientific Procedures Act 1986, and with the United Kingdom National Cancer Research Institute Guidelines for the Welfare of Animals in Cancer Research.[31] The ICR does not undertake research in nonrodent species and requires internal ethical review when such studies are sponsored by organizations with whom we collaborate. Collaborator-sponsored nonrodent pharmacology studies of compound 36 necessary for the prediction of therapeutic window and application to the clinic were approved by the ICR Animal Welfare and Ethics Review Board and were conducted in full compliance with national regulations at AAALAC accredited R&D sites.

PK/PD Experiments

These experiments were conducted as previously described.[32]