Discovery of a Potent, Selective, Orally Bioavailable, and Efficacious

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Discovery of a Potent, Selective, Orally Bioavailable and Efficacious Novel 2-(pyrazol-4-ylamino)-pyrimidine Inhibitor of the Insulin-like Growth Factor-1 Receptor (IGF-1R). Sébastien L. Degorce, Scott Boyd, Jon O Curwen, Richard Ducray, Christopher T Halsall, Clifford D. Jones, Franck Lach, Eva M. Lenz, Martin Pass, Sarah Pass, and Catherine B Trigwell J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00203 • Publication Date (Web): 14 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Discovery a Potent, Selective, Orally Bioavailable and Efficacious Novel 2-(pyrazol-4-ylamino)pyrimidine Inhibitor of the Insulin-like Growth Factor-1 Receptor (IGF-1R). Sébastien L. Degorce,*,†,‡ Scott Boyd,† Jon O. Curwen,† Richard Ducray,‡ Christopher T. Halsall,† Clifford D. Jones,†,‡ Franck Lach,‡ Eva M. Lenz,† Martin Pass,† Sarah Pass,† and Catherine Trigwell.† †

Oncology Innovative Medicines Unit, AstraZeneca, Mereside, Alderley Park, Macclesﬁeld,

Cheshire SK10 4TG, United Kingdom;

‡

Oncology Innovative Medicines Unit, AstraZeneca,

Centre de Recherches, Z.I. la Pompelle, BP1050, 51689 Reims Cedex 2, France. KEYWORDS.

IGF-1R,

ligand

lipophilic

efficiency,

2-(pyrazol-4-ylamino)-pyrimidine,

AZD9362.

ABSTRACT

Optimization of cellular lipophilic ligand efficiency (LLE) in a series of 2-anilino-pyrimidine IGF-1R kinase inhibitors led to the identification of novel 2-(pyrazol-4-ylamino)-pyrimidines with improved physico-chemical properties. Replacement of the imidazo[1,2-a]pyridine group of

ACS Paragon Plus Environment


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the previously reported inhibitor 3 with the related pyrazolo[1,5-a]pyridine improved IGF-1R cellular potency. Substitution of the amino-pyrazole group was key to obtaining excellent kinase selectivity and pharmacokinetic parameters suitable for oral dosing, which led to the discovery of (2R)-1-[4-(4-{[5-Chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)-2-pyrimidinyl]amino}-3,5-dimethyl1H-pyrazol-1-yl)-1-piperidinyl]-2-hydroxy-1-propanone (AZD9362, 28), a novel, efficacious inhibitor of IGF-1R.


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INTRODUCTION The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase that, upon ligand binding, engages with the PI3K-AKT and Ras-Raf-MEK signaling cascades which are key cellular pathways controlling proliferation and apoptosis. Increasingly the IGF-1R signalling axis has been linked with resistance to various therapeutic approaches.1 Deregulation of the IGF1R axis has been implicated in a variety of solid tumour types including lung, breast, colorectal and prostate. IGF-1R has been considered as a potential therapeutic target for solid cancers for many years and both monoclonal antibodies and small molecule kinase inhibitors have reached clinical trials with mixed success.2 Amongst the most studied IGF-1R inhibitors and currently still active in the clinic, linsitinib3 (1, Figure 1) and BMS-7548074 (2) are potent against both IGF-1R and the Insulin Receptor (IR), highlighting difficulties in obtaining selectivity against this close homolog. Structurally, 1 displays an aminopyrazine hinge binder (HB) motif and 2 an aminopyrazole HB. Our group has previously published the discovery of a series of 2-anilino-4(imidazo[1,2-a]pyridin-3-yl)-pyrimidines5, 6 such as compound 3, belonging to a HB class known internally as Mono-Anilino-Pyrimidines (MAPs). This structural motif is found in a number of clinical candidates such as the CDK inhibitor AZD55977 (4), the macrocyclic CDK2/Flt3/JAK2 inhibitor pacritinib8 (5) and the irreversible covalent EGFRm inhibitor osimertinib9 (6). Our

previously

reported

2-anilino-4-(imidazo[1,2-a]pyridin-3-yl)-pyrimidines

showed

excellent in vitro kinase inhibitory activity against IGF-1R, along with a good kinase selectivity profile (with the notable exception of the closely related Insulin Receptor) and adequate DMPK properties for oral dosing. Nevertheless further optimisation was required to improve the overall profile of 3, which has low aqueous solubility, high protein binding and potent inhibition of



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CYP3A4, whilst retaining a suitable level of IGF-1R cellular potency, good pan-kinase selectivity and adequate oral exposure for efficacy. Figure 1.


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SYNTHESIS Compounds reported herein were synthesized as shown in Schemes 1–2. Alkylation of 4-nitro-pyrazoles with N-Boc-4-(methylsulfonyloxy)-piperidine in DMF (Scheme 1), followed by hydrogenation provided 4-aminopyrazoles which were coupled to either 33 or 34, the syntheses of which were described previously.10 Under the coupling conditions, removal of the Boc protecting group occurred readily to give the free piperidines 10, 12 and 17 which were then acylated with acetic anhydride to afford compounds 7-9, 11, 13-16 and 18-21. In the case of 3-methoxy- and 3-ethoxy-4-nitropyrazoles, the alkylation was regioselective. In contrast, 3-methyl and 3-ethyl-4-nitropyrazoles afforded a mixture of regioisomers which were separated on silica gel at the nitro stage. Analogues 22-32 were obtained in a similar manner via piperidine intermediate 17 (Scheme 2), which was then alkylated using potassium carbonate in DMF (22-24) or coupled with carboxylic acids under standard HATU conditions (24-32). For examples 29-32 the appropriate Boc protected amino acid was utilized and the procedure was modified to effect an in situ TFA deprotection following the coupling.



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Scheme 1.a Synthesis of substituted pyrazoles.

a

Reagents and conditions: (a) tert-butyl 4-[(methylsulfonyl)oxy]piperidine-1-carboxylate, Cs2CO3, DMF, 120-140 °C, 2-3 hours; (b) H2, PtO2, EtOAc/EtOH, 50 PSI (10%-67% over 2 steps); (c): 33 or 34, aminopyrazole, p-TsOH, pentanol, 140 °C; (d) Ac2O (11%-68% over 2 steps).


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Scheme 2.a Synthesis of 3,5-dimethyl pyrazoles 22-32.

a

Reagents and conditions: (a) RCH2X, K2CO3, DMF, 16 hours (60%-61%); (b) RCO2H, HATU, DIPEA, DMF, 16 hours (25–28, 57%-66%); (c) RCO2H, HATU, DIPEA, DMF, 16 hours then TFA, 24 hours (29–32, 38%-62%).



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RESULTS AND DISCUSSION We planned to achieve better physicochemical properties combined with the required cell potency through optimization of ligand lipophilic efficiency11 calculated using cellular potency (thought to be a more relevant endpoint than enzyme potency as it requires a combination of good cellular penetration and inhibition of the target in a more physiological relevant environment) and experimental logD7.4 (cell LLE = cellular pIC50 – logD7.4). The kinase solvent channel region, which is occupied by the piperazine aniline in 3, is generally most tolerant of hydrophilic substitution.

However, previous attempts to improve potency and/or physical

properties by aniline substitution was only achieved at the expense of increased inhibition of the hERG ion channel.6 An alternative approach targeted replacements of the aniline based solvent channel groups with suitable heterocyclic replacements occupying a less lipophilic property space. We prepared amino-pyrazole 7 (Table 1) which incorporates a piperidin-4-yl group attached at pyrazole N1. Reasonable IGF-1R activity was maintained relative to 3, with a 5-fold decrease in activity in the biochemical assay and a larger 10-fold decrease in the cellular assay. However, the concurrent decrease in lipophilicity resulted in a more modest decrease in cell LLE (∆LLE=−0.4). Unfortunately, 7 suffered from a complete lack of selectivity against a number of CDKs as it was found to hit CDK1,2,7,8 and 9 with IC50’s of 0.066, 0.025, 0.021, 0.042 and 0.037 µM respectively. Pan-CDK activity is highly undesirable since it may result in a range of toxicities,12 and we decided to use our internal CDK2 assay as a surrogate for pan-CDK activity. Hitting CDK2 was a drawback that we anticipated based on the fact that similar compounds (e.g. 4) had been developed as CDK2 inhibitors, and the known importance of the ortho-methoxy group present in 3 to confer a good selectivity profile in the aniline series.5,


6, 13

With this

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knowledge, we prepared various substituted pyrazole derivatives with the aim of restoring an acceptable kinase selectivity profile whilst retaining or improving cell LLE further. 3-Methoxypyrazole 8 was successful in that regard, as it proved almost as potent as, and isolipophilic to 7, with a 20-fold decrease in CDK2 activity. 3-Methyl analogue 9 showed a >4-fold improvement in cell potency for a slightly lower logD, which led to a significant cell LLE improvement over 7 (∆LLE=+1.0), whilst maintaining some CDK2 selectivity.

Interestingly, the 5-methyl

regioisomer 11 had similarly selectivity and potency to 9. Our previous work in the aniline series indicated that the imidazo[1,2-a]pyridine (imidazopyridine) ring can be replaced by the corresponding pyrazolo[1,5-a]pyridine (pyrazolopyridine) heterocycle without loss of IGF-1R activity,6 and thus the matching pyrazolopyridines 14-16 were made and evaluated. These compounds were representative of a trend which is shown in Figure 2a: pyrazolopyridines were typically slightly more potent against IGF-1R in our enzyme assay (∆pIC50=+0.32±0.27, N=39), whilst increasing CDK2 activity by a similar factor (∆pIC50=+0.24±0.18, N=40), resulting in retention of selectivity versus CDK2 (∆∆pIC50=+0.08±0.32, N=39). The similar cellular LLEs of 8/11 versus 14/16 suggested that the improved potency is due to the increased lipophilicity of pyrazolopyridines compared to imidazopyridines. This was again confirmed across a number of matched pairs (Figure 2b), which showed similar increases in lipophilicity (∆logD7.4=+0.28±0.19, N=30) and cell potency (∆pIC50=+0.32±0.34, N=36), resulting in a near neutral effect on cell LLE (∆LLE=+0.07±0.40, N=30). Fortunately, in this case, exchanging increased lipophilicity for cellular potency had no detrimental effect on other lipophilicity driven parameters such as hERG activity. In contrast, the bulkier ethoxypyrazole 19 improved cell LLE (∆LLE=+0.6 relative to methoxy analogue 14) but was accompanied by slightly increased hERG potency (IC50=6.4 µM).


The increased


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lipophilicity of the 3- and 5-ethylpyrazole isomers 20 and 21 was not balanced by an increase in cellular potency and so these were less attractive than their methyl counterparts 15 and 16.


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Figure 2. Imidazopyridine vs. pyrazolopyridine matched pairs: (a) IGF-1R and CDK2 enzyme potencies (∆pIC50) and CDK2 selectivity expressed as ∆∆pIC50(IGF-1R–CDK2); (b) IGF-1R cell potency (∆pIC50), ∆logD7.4 and ∆LLE. The number of matched pairs (Count), median change (Median), mean change (Avg) and standard deviation (StdDev) are indicated in the tables. N

N N N

R5

R5

N N

R2

imidazopyridines

N N

R2

pyrazolopyridines

(a)

(b)



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In order to confirm the origin of the observed selectivity improvements with substituted pyrazoles we obtained crystal structures of the 3-methyl and 5-methyl des-acetyl analogues 10 and 12 (showing similar potency to their N-Ac counterparts, and selected for their improved solubility due to their basicity, but were otherwise flawed with strong CYP inhibition) bound to an unphosphorylated construct of the kinase domain of IGF-1R. The overlay of these structures (Figure 3a) shows an excellent alignment of the ligands where the 2-aminopyrimidine interact with the hinge in a classical manner and where protein can accommodate a methyl group on either side of the pyrazole ring. Both methyl groups induced a similar 32° twist of the pyrazole and that close to the hinge region (and present in 10) was assumed to play a similar role as the omethoxy group mentioned earlier. These results suggested that 3,5-disubstituted pyrazoles in the solvent channel could potentially improve both potency and selectivity. This was observed with both dimethyl pyrazoles 13 and 18, where the selectivity ratio against CDK2 was improved further to 34- and 48-fold respectively, which was a significant improvement over the single methyl isomers. The changes in selectivity profiles observed versus CDK2 were mirrored in broader kinase selectivity panels: mono-methyl pyrazole 16 had a sub-optimal profile with 7 (excluding IGF-1R and IR) of the 60 kinases tested showing an inhibition greater than 75% at 1 µM in external enzyme assays (Figure 4).

However, dimethyl pyrazole 18 gave a much

improved kinase inhibition profile compared to both 16 and 3 with only 3 kinase hits above 75% inhibition (excluding IGF-1R and IR): CDK2 (82% – which translated into a modest IC50=1.38 µM in our internal enzyme assay), Flt1 (80%) and JNK1 (76%). The lack of selectivity with the mono-methyl pyrazole was hypothesized to be related to the accessibility of an alternative binding mode where the methyl group is orientated away from the kinase hinge region. This would place the substituted piperidine into a different orientation, which is presumably less


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favorable for IGF-1R, but is accommodated in a range of other kinases. With the more selective dimethyl pyrazole, one methyl group is necessarily directed towards the hinge in either orientation, which is acceptable in IGF-1R but presumably not the majority of other kinases. Figure 3. X-ray crystal structures of 10 (cyan – PDB code 5FXR), 12 (purple – PDB code 5FXQ), and 24 (green – PDB code 5FXS) bound to IGF-1R: (a) overlay of 10 and 12; (b) overlay of 10 and 24.



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An additional unexpected benefit of the disubstituted pyrazole 18 compared to the monosubstituted analogues 15 and 16 was an improvement in rat PK parameters (Table 2). Both methyl pyrazole isomers 15 and 16 showed poor bioavailability (33 µM and hERG:enzyme ratios of at least >3,800), which was unexpected as similar basic analogues in the aniline series resulted in significant hERG


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inhibition.5 Disappointingly, 29-32 proved potent inhibitors of cytochrome CYP 3A4 (IC50=0.53.8 µM) and the potential increased risk of drug-drug interactions prevented further progression of these compounds. It is noteworthy that our original lead compound 3, is also a potent CYP 3A4 inhibitor (IC5010

23

0.012

0.025

12

3.2

4.4

15

>10

24

0.009

0.025

7.8

2.9

4.7

510

>10

25

0.012

0.029

22

2.6

4.9

2

>10

26

0.012

0.049

17

2.8

4.5

31

>10

27

0.010

0.045

23

2.8

4.6

17

>10

28

0.014

0.048

29

2.9

4.4

17

21

29

0.006

0.023

>33

1.3

6.3

260

0.5

30

0.008

0.018

>33

1.7

6.0

670

0.3

31

0.005

0.025

>33

1.7

5.9

620

1.0

32

0.006

0.019

>33

1.4

6.3

>1,000

3.8

a

Solu bilityb

Data expressed as in Table 1 unless otherwise specified. b Thermodynamic solubility of solid samples in a 0.1 M aqueous phosphate buﬀer at pH 7.4 after 24 h at 25 °C.14 e Inhibition of CYP3A4, n=1.



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EXPERIMENTAL SECTION General Procedures. All experiments were carried out at ambient temperature under an inert atmosphere. Evaporations were carried out by rotary evaporation or utilizing Genevac equipment in vacuo and work up procedures were carried out after removal of residual solids by filtration. Flash chromatography purifications were performed on an automated Armen Glider Flash: Spot II Ultimate (Armen Instrument, Saint-Ave, France) using prepacked Merck normal phase Si60 silica cartridges (granulometry: 15-40 or 40-63 µm) obtained from Merck, Darmstadt, Germany. Preparative chromatography was performed on a Waters instrument (600/2700 or 2525) fitted with a ZMD or ZQ ESCi mass spectrometers and a Waters X-Terra, a Waters X-Bridge or a Waters SunFire reverse-phase column (C-18, 5 µm silica, 19 mm diameter, 100 mm length, flow rate of 40 mL/min) using decreasingly polar mixtures of water (containing 0.2% ammonium carbonate) and acetonitrile as eluent. Intermediates were not fully purified but their structure and purity were assessed by TLC, NMR, HPLC and mass techniques and are consistent with the proposed structures. The purities of the compounds for biological testing were assessed by NMR, HPLC and mass spectral techniques and are consistent with the proposed structures characterized; purity was at least 95%. Unless stated otherwise proton magnetic resonance spectra were determined using a Bruker Avance 500 (500 MHz) instrument. Measurements were taken at ambient temperature unless otherwise specified. 1H and 13C NMR spectra are reported as chemical shifts in parts per million (ppm) relative to an internal solvent reference. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublet; dt, doublet of triplets; bs, broad signal. Analytical HPLC was carried out using a Waters Alliance HT (2790 & 2795) fitted with a


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Waters ZQ ESCi or ZMD ESCi mass spectrometer and an X Bridge 5 µm C-18 column (2.1 x 50 mm) at a flow rate of 2.4 mL/min, using a solvent system of 95% A + 5% C to 95% B + 5% C over 4 minutes, where A = water, B = methanol, C = 1:1 methanol:water (containing 0.2% ammonium carbonate). Detection was by Electrospray Mass Spectrometry and by UV absorbance at a wave length of 254 nm. Accurate mass spectra were recorded on a Waters Xevo Qtof 2 in +ve ion mode with an Acquity UPLC Binary Solvent Manager, Sample manager and PDA as the sample inlet. All IC50 data are quoted as geometric mean values, and statistical analysis is available in the Supporting Information. All experimental activities involving animals were carried out in accordance with AstraZeneca animal welfare protocols which are consistent with The American Chemical Society Publications rules and ethical guidelines and were approved by the AstraZeneca animal welfare ethics committee. Chemical synthesis of representative example compound 28. The synthesis and characterisation of all other novel molecules reported therein are available in the Supplementary Information. tert-Butyl 4-(3,5-dimethyl-4-nitro-1H-pyrazol-1-yl)piperidine-1-carboxylate 3,5-Dimethyl-4-nitro-1H-pyrazole

(334

g,

2.4

mol),

tert-butyl

4-

(methylsulfonyloxy)piperidine-1-carboxylate (956 g, 3.1 mol) and cesium carbonate (926 g, 2.8 mol) were suspended in DMF (3 L). The reaction was purged with nitrogen and heated to 120 °C over a period of 3 hours, after which the reaction mixture was quenched with water. The resulting precipitate was filtered, washed with water and dried under vacuum at 50 °C over P2O5. The process was repeated on the same scale and the combined crudes were recrystallised from



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EtOAc / i-Hexane to afford tert-butyl 4-(3,5-dimethyl-4-nitro-1H-pyrazol-1-yl)piperidine-1carboxylate (915 g, 61 %) as a cream-coloured solid. 1

H NMR (400 MHz, DMSO-d6, 303K) 1.42 (9H, s), 1.74 – 1.87 (4H, m), 2.39 (3H, s), 2.63

(3H, s), 2.90 (2H, s), 4.05 (2H, d, J 13), 4.51 (1H, p, J 7.8). m/z (ES+) 269 (M-tBu)+. 2-Methyl-2-propanyl 4-(4-amino-3,5-dimethyl-1H-pyrazol-1-yl)-1-piperidinecarboxylate tert-Butyl 4-(3,5-dimethyl-4-nitro-1H-pyrazol-1-yl)piperidine-1-carboxylate (338 g, 1.0 mol) and 5% w/w Palladium on carbon (JM Type87L) (68 g, 16 mmol) in MeOH (3.3 L) were stirred under an atmosphere of hydrogen at 5 bar and 50 °C for 16 hours. A total charge of 638g was processed in batches. The reaction mixtures were filtered, the filtrates combined and evaporated to dryness. The resulting crude mixtures were triturated with Et2O, filtered and dried to give tertbutyl 4-(4-amino-3,5-dimethyl-1H-pyrazol-1-yl)piperidine-1-carboxylate as a pale pink solid (459 g, 80 %). 1

H NMR (400 MHz, CDCl3, 303 K) 1.46 (9H, s), 1.79-1.82 (2H, m), 2.11 (2H, m), 2.20 (2H,

m), 2.16 (6H, s), 2.81 (2H, br s), 3.98 (1H, m), 4.27 (2H, m). m/z (ES+) 295 (M+H)+. 5-Chloro-N-[3,5-dimethyl-1-(piperidin-4-yl)-1H-pyrazol-4-yl]-4-(pyrazolo[1,5-a]pyridin-3yl)pyrimidin-2-amine (17) 3-(2,5-Dichloropyrimidin-4-yl)pyrazolo[1,5-a]pyridine (3.98 g, 15.0 mmol), tert-butyl 4-(4amino-3,5-dimethyl-1H-pyrazol-1-yl)piperidine-1-carboxylate (4.86 g, 16.5 mmol) and 4methylbenzenesulfonic acid hydrate (5.70 g, 30.0 mmol) were suspended in 2-pentanol (50 mL). The reaction was degased, purged with nitrogen and heated to 140 °C for 4 hours in an autoclave. The crude residues were purified by flash chromatography on silica, eluting a gradient of 0 – 10% 7M NH3/MeOH in DCM. Fractions containing the desired product were combined, evaporated and triturated with Et2O to afford 17, 5-chloro-N-(3,5-dimethyl-1-(piperidin-4-yl)-


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1H-pyrazol-4-yl)-4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2-amine as an off-white solid (3.27 g, 52 %). 1

H NMR (500 MHz, DMSO-d6, 323K) 1.79 (2H, d), 1.94 – 2.06 (5H, m), 2.11 (3H, s), 2.29

(2H, t), 3.12 (2H, d), 4.09 – 4.20 (1H, m), 7.08 – 7.17 (1H, m), 7.28 (1H, s), 8.00 (1H, s), 8.32 (1H, s), 8.41 (1H, s), 8.79 (1H, d), 8.95 (1H, s). HRMS (ESI+): Anal cald for C21H24ClN8 (M+H)+: 423.1812; Found: 423.1777. (2R)-1-[4-(4-{[5-Chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)-2-pyrimidinyl]amino}-3,5-dimethyl1H-pyrazol-1-yl)-1-piperidinyl]-2-hydroxy-1-propanone (28) HATU (847 mg, 2.2 mmol) was added portionwise to a stirred solution of 5-chloro-N-(3,5dimethyl-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2-amine (725 mg, 1.7 mmol), (R)-2-hydroxypropanoic acid (170 mg, 1.9 mmol) and DIPEA (0.45 mL, 2.6 mmol) in DMF (7.5 mL) at room temperature. The resulting solution was stirred at room temperature for 2 hours. MeOH (2 mL) and NaOH (aq) 2N (2 mL) were added and stirring continued for 16 hours. The resulting mixture was diluted with DCM and washed with water. The organic layer was dried over MgSO4, filtered and evaporated. The crudes were purified by flash silica chromatography, elution gradient 0 – 5% EtOH in DCM. Fractions containing the desired product were evaporated to dryness and triturated with EtOAc to afford (2R)-1-(4-(4-(5chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2-ylamino)-3,5-dimethyl-1H-pyrazol-1yl)piperidin-1-yl)-2-hydroxypropan-1-one as a colourless solid (540 mg, 64 %). 1

H NMR (500 MHz, DMSO-d6, 373 K) 1.29 (3H, d, J 5.9), 1.89 – 1.97 (2H, m), 1.97 – 2.11

(5H, m), 2.16 (3H, s), 3.08 (2H, s), 4.27 – 4.44 (3H, m), 4.46 – 4.56 (2H, m), 7.09 (1H, td, J 1.4, 6.8), 7.24 – 7.31 (1H m), 8.13 (1H, s), 8.19 (1H, d, J 8.7), 8.32 (1H, s), 8.74 (1H, dt, J 1.0, 6.9), 8.92 (1H, s).

13

C NMR (176 MHz, DMSO, 303 K) 8.8, 11.6, 20.6, 20.8, 31.4, 31.5, 32.0, 32.2,

40.7, 40.8, 43.4, 43.6, 54.2, 64.0, 64.4, 107.2, 113.4, 114.4, 116.7, 120.4, 126.8, 129.5, 133.7,



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139.5, 143.2, 143.3, 155.3, 158.1, 160.3, 172.0 (Observed complexity is due to rotameric effects at the experimental temperature).

HRMS (ESI+): Anal cald for C24H28ClN8O2 (M+H)+:

495.2024; Found: 495.2023. Chiral Analysis: 5µm Chiralpak AD-H (250mm x 4.6mm) column, iso-Hexane/IPA/TEA 80/20/0.1, 25°C, 1mL/min, retention time 60.2 mins, >98% ee. Efficacy In the in vivo studies using the P12 allograft described herein, 5x106 cells were inoculated subcutaneously into female nude (nu/nu) mice and allowed to grow for 7 days before animals were randomized into groups. One group was dosed orally once daily with vehicle (1% ploysorbate 80), while 3 other groups were dosed with compound 28 at 25, 12.5 or 6.25 mg/kg. In the experiment using the Colo205 human line, 3x106 cells were inoculated subcutaneously into female nude mice in 50% Matrigel and allowed to grow for 8 days before animals were randomized into 5 groups. Three of these groups were dosed with vehicle, compound 28 orally at 50 mg/kg for 2 days per week or Gemcitabine alone twice weekly via the intraperitoneal route at 100 mg/kg. Two more groups were dosed with the agents in combination using 50 or 25 mg/kg compound 28. The group receiving 50 mg/kg compound 28 as part of the combination were dosed for one week only while animals dosed with 25 mg/kg compound 28 were dosed for 2 weekly cycles. SUPPLEMENTARY MATERIAL Complete experimental details for the syntheses of intermediates and all final compounds are described together with assay statistical analyses. Crystallographic data: authors will release the atomic coordinates and experimental data upon article publication under the following PDB accession codes: 5FXR (10), 5FXQ (12), 5FXS (24).


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ACKNOWLEDGMENTS The authors wish to acknowledge Calum Cook, Steven Glossop, Maryannick Lamorlette, Antoine Le Griffon, Arshed Mahmood, Mickael Maudet and Fabrice Renaud for the synthesis of some of the compounds described herein. We would also like to thank Paul R Davey, Marta Wylot and Steven Glossop for assistance with accurate mass and NMR spectral data, Patrice Koza for purification of some final compounds and Dawn Brison for technical input to the mouse allograft studies. Christopher Phillips is thanked for assistance with the deposition of crystal structures and Caroline Truman for the generation of enzyme data. Finally, we would like to thank Jamie Scott for his valuable suggestions to the manuscript. ABBREVIATIONS ADME, absorption, distribution, metabolism and excretion ; CYP, cytochrome P450; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; HATU, O-(7-Azabenzotriazol-1yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; hERG, human Ether-à-go-go-Related Gene; LLE, ligand lipophilic efficiency; PPB, Plasma Protein Binding; RT, Room Temperature; AUTHOR INFORMATION *Tel:

+44

(0)1625

514382.

Fax:

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[email protected]


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TABLE OF CONTENTS GRAPHIC O N N Cl

N

N N

N N

N N

O

N H

Cl N

O

3 cell IC50 = 20 nM solubility = 1 µM CYP3A4 IC50 < 1 µM

N N

N N H

28 cell IC50 = 48 nM solubility = 17 µM (crystalline) CYP3A4 IC50 = 21 µM


OH

Discovery of a Potent, Selective, Orally Bioavailable, and Efficacious

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