Anti-Tumor Activity of A Small Molecule Hypoxia Inducible Factor -2 Antagonist in Models of Renal Cell Carcinoma

Supplementary Methods, Data and Figures

Peloton Therapeutics, Inc.

2330 Inwood Road

Dallas, Texas 75235

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Supplementary Method, Data and Figures

Supplementary method includes detailed experimental procedure for the synthesis of

PT2385. Supplementary Figures 1-7 – Lack of cytotoxicity of PT2385 in cultured 786-O

and A498 cells (1); PT2385 pharmacokinetics in CD-1 mice (2); Inhibition of HIF-2

gene expression and circulating hVEGFa in A498 mouse xenograft model after treatment

with PT2385 (3); HIF-2 mRNA and protein levels in 786-O cells in vitro after treatment

with PT2385 (4); Levels of HIF-1 and HIF-2 in patient-derived xenograft tumors (5);

Body weight of mice in efficacy studies treated with either vehicle, PT2385 or sunitinib

(6); Binding of PT2385 to rat HIF-2 as determined by ITC and inhibition of mouse

kidney EPO gene expression with PT2385 treatment (7).

PT2385 Synthesis

( S )-3-((2,2-Difluoro-1-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1 H -inden-4-yl)oxy)-5-

fluorobenzonitrile (PT2385)

Step A: Preparation of 2-hydroxy-5-(methylthio)benzaldehyde: To a suspension of 4-

methylsulfanylphenol (50 g, 357 mmol), paraformaldehyde (72.3 g, 2407 mmol), and

anhydrous magnesium chloride (50.9 g, 535 mmol) in acetonitrile (500 mL) was added

triethylamine (186 mL, 1337 mmol) at ambient temperature. After the addition, the

reaction mixture was stirred at 60 °C for 5 hours. After cooling to 0 °C, 1 N HCl was

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added slowly until two phases separated (ca. 1.5 L). MTBE (700 mL) was added. The

organic layer was separated, washed with brine, dried (sodium sulfate), filtered and

concentrated under reduced pressure. The residue obtained was purified by flash

chromatography on silica gel eluting with 1:1 hexane/dichloromethane to give 2-

hydroxy-5-methylsulfanyl-benzaldehyde (50.5 g, 300 mmol, 84% yield) as semisolid.

Step B: Preparation of 3-(2-hydroxy-5-(methylthio)phenyl)propanoic acid: Triethylamine

(2.5 mL, 17.8 mmol) was added slowly to formic acid (1.55 mL, 41.0 mmol) at 0 °C.

Then 2,2-dimethyl-1,3-dioxane-4,6-dione (1.84 g, 12.9 mmol) was added, followed by a

solution of 2-hydroxy-5-methylsulfanyl-benzaldehyde (2.0 g, 11.9 mmol) in N,N-

dimethylacetamide (4 mL). The reaction mixture was stirred at ambient temperature for

1 hour and then it was stirred at 100 °C for 6 hours. After cooling to ambient temperature,

water (100 mL) was added and the pH was adjusted with 3N NaOH to pH~9. Ethyl

acetate (50 mL) was added. The aqueous layer was separated, then acidified with

saturated potassium hydrogen sulfate to pH~3. This aqueous layer was extracted

with MTBE (50 mL). The organic layer was separated, washed with brine, dried (sodium

sulfate), filtered and concentrated under reduced pressure. The residue obtained was

purified by flash chromatography on silica gel eluting with1:1 hexane/ethyl acetate to

give 3-(2-hydroxy-5-methylsulfanyl-phenyl)propanoic acid (1.67 g, 7.9 mmol, 66%

yield) as solid.

Step C: Preparation of 3-[2-(3-cyano-5-fluoro-phenoxy)-5-methylsulfanyl-

phenyl]propanoic acid: A suspension of 3-(2-hydroxy-5-methylsulfanyl-

phenyl)propanoic acid (2.14 g, 10 mmol), 3,5-difluorobenzonitrile (3.51 g, 25 mmol), and

cesium carbonate (9.85 g, 30 mmol) in sulfolane (36 mL) and sec-butanol (4 mL) was

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stirred at 105 °C for 4 hours. After coolingto ambient temperature, water (100 mL) and

MTBE (100 mL) were added. The aqueous layer was separated, acidified with saturated

potassium hydrogen sulfate to pH~3-4 and extracted with MTBE. The organic layer was

washed with brine, dried (sodium sulfate), filtered and concentrated under reduced

pressure. Water (50 mL) was added and the mixture was stirred at ambient temperature

for 30 minutes. The resulting solid was collected by filtration and dried under vacuum.

The filtered solid was suspended in 3:1 hexane/MTBE (~20 mL) and stirred at ambient

temperature for 30 minutes. The solid was collected by filtration, washed with hexane

and dried to give 3-[2-(3-cyano-5-fluoro-phenoxy)-5-methylsulfanyl-phenyl]propanoic

acid (2.9 g, 8.8 mmol, 87% yield) as solid.

Step D: Preparation of 3-fluoro-5-(7-methylsulfanyl-1-oxo-indan-4-yl)oxy-benzonitrile:

To a solution of 3-[2-(3-cyano-5-fluoro-phenoxy)-5-methylsulfanyl-phenyl]propanoic

acid (8.44 g, 25.5 mmol) in dichloromethane (50 mL) was added a drop of DMF, then

followed by addition of oxalyl chloride (2.62 mL, 30.6 mmol). The reaction mixture was

stirred at ambient temperature for 1 hour. Volatile solvents were removed under reduced

pressure. Dichloromethane (20 mL) was added. The resulting mixture was added slowly

to a suspension of aluminum trichloride (6.79 g, 50.0 mmol) in dichloromethane (50 mL).

The mixture was stirred at ambient temperature for 1 hour. The reaction mixture was

cooled to 0 °C. Aqueous 1 N HCl (20 mL) was added slowly, followed by water (50 mL)

and dichloromethane (100 mL). The organic layer was separated, washed with saturated

sodium bicarbonate, dried (sodium sulfate), filtered and concentrated under reduced

pressure to give 3-fluoro-5-(7-methylsulfanyl-1-oxo-indan-4-yl)oxy-benzonitrile (7.98 g,

25.5 mmol, 100% yield) as solid.

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Step E: Preparation of 3-fluoro-5-(7-methylsulfonyl-1-oxo-indan-4-yl)oxy-benzonitrile:

A suspension of 3-fluoro-5-(7-methylsulfanyl-1-oxo-indan-4-yl)oxy-benzonitrile (7.98 g,

25.5 mmol), and Oxone® (53.6 g, 87 mmol) in acetonitrile (40 mL) and water (20 mL)

was stirred at ambient temperature for 18 hours. Solid was removed by filtration and

washed with dichloromethane (40 mL). The organics were removed under reduced

pressure. Acetone (20 mL) and water (40 mL) were added. The resulting suspension was

stirred at ambient temperature for 30 minutes. The solid was collected by filtration and

dried to give 3-fluoro-5-(7-methylsulfonyl-1-oxo-indan-4-yl)oxy-benzonitrile (7.3 g, 21

mmol, 83% yield) as solid.

Step F: Preparation of 3-[( E, Z )-1-butylimino-7-methylsulfonyl-indan-4-yl]oxy-5-fluoro-

benzonitrile: Butan-1-amine (5.15 mL, 52 mmol) was added to 3-fluoro-5-(7-

methylsulfonyl-1-oxo-indan-4-yl)oxy-benzonitrile (450 mg, 1.3 mmol) and

trifluoroacetic acid (20 μL, 0.26 mmol) in benzene (10 mL) at room temperature then

warmed to reflux with the azeotropic removal of water by a Dean-Stark apparatus.

Progress of the reaction was monitored by 1H–NMR. When complete, the reaction was

cooled to room temperature then concentrated in vacuo. The residue was diluted with

water (10 mL), extracted with MTBE (3 x 10 mL), washed with brine and dried over

Na2SO4, filtered and concentrated. Crude 3-[(E, Z)-1-butylimino-7-methylsulfonyl-indan-

4-yl]oxy-5-fluoro-benzonitrile was used immediately without purification in the next

step.

Step G: Preparation of 3-(2,2-difluoro-7-methylsulfonyl-1-oxo-indan-4-yl)oxy-5-fluoro-

benzonitrile: Selectfluor® (1.15 g, 3.25 mmol) was added to crude 3-[(E, Z)-1-butylimino-

7-methylsulfonyl-indan-4-yl]oxy-5-fluoro-benzonitrile (520 mg, 1.3 mmol) and sodium

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sulfate (369 mg, 2.6 mmol) in acetonitrile (10 mL) then warmed to reflux for 6 hours.

The reaction was cooled to ambient temperature, treated with concentrated HCl (1.0 mL,

12 mmol) and stirred for 15 minutes. The mixture was diluted with water (10 mL),

extracted with EtOAc (3 x 10 mL), washed with brine (10 mL), dried over MgSO4,

filtered and concentrated in vacuo. The residue was purified on silica gel eluting with a

gradient of 20–100% EtOAc/hexane which afforded 3-(2,2-difluoro-7-methylsulfonyl-1-

oxo-indan-4-yl)oxy-5-fluoro-benzonitrile (437 mg, 1.2 mmol, 88% yield).

Step H: Preparation of ( S )-3-((2,2-difluoro-1-hydroxy-7-(methylsulfonyl)-2,3-dihydro-

1 H -inden-4-yl)oxy)-5-fluorobenzonitrile ( PT2385 ): An ice cold solution of RuCl(p-

cymene)[(R,R)-Ts-DPEN] (40.7 mg, 0.06 mmol) in CH2Cl2 (30 mL) was added by

syringe under nitrogen to an ice cold solution of 3-(2,2-difluoro-7-methylsulfonyl-1-oxo-

indan-4-yl)oxy-5-fluoro-benzonitrile (2.44 g, 6.4 mmol), triethylamine (1.78 mL, 12.8

mmol) and formic acid (724 μL, 19.2 mmol) in CH2Cl2 (30 mL). The reaction was placed

in a refrigerator at 4 C for 16 hours. The mixture was concentrated to 10 mL then

purified directly on silica gel eluting with 10–50% EtOAc/hexane which afforded

PT2385 (2.15 g, 5.6 mmol, 87% yield). Enantiomeric excess (98%) was determined by

chiral HPLC. LCMS ESI (-) 428 (M+HCO2-). 1HNMR (400 MHz, CDCl3): δ 7.93 (d, 1

H), 7.27–7.24 (m, 1 H), 7.15–7.14 (m, 1 H), 7.07–7.03 (m, 1 H), 7.00 (d, 1 H), 5.63–5.58

(m, 1 H), 3.56–3.35 (m, 3 H), 3.24 (s, 3 H).

Supplementary Figures:

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Supplementary Figure 1. Lack of cytotoxicity of PT2385 on cultured cells. 786-O or

A498 cells were treated with indicated concentration of PT2385 for 72 hours. Cell

numbers were determined using MTT assay.

Supplementary Figure 2. Pharmacokinetics of PT2385 in CD-1 mice. Plasma

concentration of PT2385 was determined after a single dose of 10 mg/kg by oral gavage.

Supplementary Figure 3. A498 tumor-bearing mice received three oral doses of 10

mg/kg PT2385 on b.i.d. schedule. 12 hours after the last dose, tumors were excised. The

levels of mRNA for HIF-2-regulated genes VEGF-A, CCND1 and CXCR4 were

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decreased. The mRNA levels of HIF-2 were also reduced. Circulating human VEGF-A

levels were reduced. * p < 0.05; **p < 0.01 (unpaired T test comparing treated group

with vehicle control group).

Supplementary Figure 4. RT-qPCR detection of HIF-2 mRNA and immunoblotting

detection of HIF-2 protein in 786-O cells treated with 1 M of PT2385 for indicated

time. There were no significant changes in mRNA and protein levels.

Supplementary Figure 5. Immunoblotting detection of HIF-1 and HIF-2 protein in

the patient-derived xenograft model (Fig. 5c). Samples from three individual tumor

samples were analyzed.

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Supplementary Figure 6. PT2385 was well tolerated in mouse xenograft models.

Graphs of body weight measurements in three xenograft models. a, 786-O xenograft

model (n=8). b, A498 xenograft model (n=8). c, patient-derived ccRCC xenograft model

(n=8).

Supplementary Figure 7. PT2385 inhibits rodent HIF-2 activity. a, Determination of

PT2385 binding affinity to the PAS-B domain of rat HIF-2b, Reduction of mouse

EPO mRNA in the kidney of CD-1 mice treated with 2 doses of 3, 10, or 30 mg/kg at 12-

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hour interval. Kidney samples were collected 12 hours after the last dose. * p < 0.05;

(unpaired T test comparing treated group with vehicle control group)

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