Discovery of a class of diheteroaromatic amines as orally bioavailable CDK1/4/6 inhibitors

Yan Fu a,#, Shuai Tang b,# Yi Su b, Yan Ye a, Chuantao Zha a, Lei Li a, Jianhua Cao a, Yi Chen b, Lei Jiang a,

Ying Huang a, Jian Ding b, Meiyu Geng b, Min Huang b,*, Huixin Wan a,*

,a Shanghai Haihepharmaceutial, Co.,Ltd, No. 421 Newton Road, Zhangjiang Hi-tech Park, Pudong New

Area, Shanghai, 201203,China b Shanghai Institute of Materia Medica, Chinese Academy of Science, No. 555 Zuchongzhi Road,

Zhangjiang Hi-tech Park, Pudong New Area, Shanghai, 201203, China#, these authors contributed equally to this work*, corresponding authors

Supplementary Data

Contents

General Computational Method S - 3

General Chemistry Method S - 4

Experimental Section (NMR, LC-MS) S - 11

In Vitro Biochemical and Cellular Assay S - 12

In vivo Mouse Pharmacokinetic and Efficacy Assay S- 13

1

General Computational Method

The crystal structure of CDK1 was downloaded from Protein Data Bank (http://www.rcsb.org/, PDB ID:

5hq0). The preparation of the protein was done using Protein preparation wizard tool in the Maestro

(Maestro, version 10.2, Schrödinger, LLC, New York, NY), with default parameters. This process fixed the

protein structure by verifying proper assignment of bonds and bond orders, adding hydrogens, and deleting

unwanted bound water molecules. The crystal structure of CDK1 in complex with an ATP-competitive

inhibitor (PDB code 5HQ0) was selected for the docking studies. In this structure, there are two crystal

waters which bridge the interaction between the inhibitor and CDK1. Further, the protein structure was

subjected to restrained minimization using the OPLS2005 force field. Before docking, every compound

was sketched and prepared using Ligprep module of maestro with default parameters. A 7.0 ± 0.2 pH value

was used to determine the ionization state. The docking of each compound to CDK1 was performed using

standard precision (SP) Induced Fit Docking (IFD) with default settings.

Figure 2. Putative binding mode of LY2835219 (PDB code 5HQ0). LY2835219 was shown as magenta

sticks. Hydrogen bonds (black dash) are labeled.

2

Figure3. Docking mode of Compound 5c and CDK1 (PDB code 5HQ0). Compound 5c was shown as

green sticks. Hydrogen bonds (black dash) are labeled.

3

General Chemistry Method

Chemicals and solvents were obtained from commercial suppliers and were used without further

purification. Solvents and reagents are abbreviated as follows: dichloromethane (DCM),

dimethylformamide (DMF), ethyl acetate (EtOAc), hydrochloric acid (HCl), sodium carbonate (Na2CO3),

N,N-diisopropylethylamine(DIPEA), potassium carbonate(K2CO3), 1,1’-bis(diphenylphosphino) ferrocene

dichloropalladium(II) (Pd(dppf)Cl2), tris(dibenzylideneacetone)dipalladium (Pd2(dba)3), sodium hydride

(NaH), tetrahydrofuran (THF), sodium iodide (NaI), acetic acid (HOAc), sodium triacetoborohydride

(NaBH(OAc)3), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-Phos), methanol (MeOH),

sodium cyanoborohydride(NaBH3CN), thionyl chloride (SOCl2). Flash chromatography was performed on

a Biotage Isolera One Flash Chromatography System using Silica Gel packed SNAP KP-Sil Cartridge.

Microwave initiated reaction was performed with Biotage Initiator+. 1H-NMR spectra were recorded at

room temperature on Bruker Advance spectrometers operating at 400 MHz. Chemical shifts are given in

ppm (δ) from tetramethylsilane as an internal standard. Significant 1H NMR data are tabulated in the

following order: multiplicity (s, singlet; d, doublet, dd double doublet; t, triplet; q, quartet; m, multiplet; br,

broad), number of protons, coupling constant(s) in hertz (Hz). Analytical, preparative HPLC and Electron

Spray Ionization condition (ESI) mass spectra were performed on a Waters HPLC 515 equipped with

Waters 2489 UV/Visible Detector System, an Agilent Quadrupolem LC-MS (6120) and an Agilent Prep-

HPLC (1260 Infinity II) equipped with a Diode Array Detector and a Quadrupole MSD using mixture

gradients of formic acid/water/acetonitrile as system solvent.

4

General Procedure for preparation of compound 5a-5k.

Step 1: To a suspension of 4-fluoro-1-isopropyl-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)-

1H-benzo[d]imidazole (2.3 g，7.22 mmol), 4,6-dichloropyrimidine or 4-bromo-2-chloropyridine (7.94

mmol) in 1,4-dioxane (40.0 mL) and water (10.0 mL) was added sodium carbonate (2.29 g，21.66 mmol).

The mixture was bubbled with Ar for 15 mins and then Pd (dppf) Cl2(528 mg，0.72 mmol) was added. The

mixture was then heated to 80 oC and stirred for 4-6 hours under Ar atmosphere. The reaction mixture was

cooled to room temperature and concentrated to remove the organic solvent after reaction completion. The

residue was diluted with water (100 mL) and extracted with EtOAc (150 mL×3). The organic layer was

separated and washed with saturated brine, then dried with anhydrous Na2SO4. The organic phase was then

concentrated and the residue was further purified with flash chromatography or preparative HPLC to afford

the desired intermediate 2.

LC-MS: 304.2(M+H).

LC-MS: 322.1(M+H); 1H-NMR (400MHz, CDCl3) δ 8.33 (d, J=2.4 Hz, 1H), 7.51 (s,

1H), 7.46 (d, J=5.6 Hz, 1H), 7.15 (d, J=11.2 Hz, 1H), 4.68-4.75 (m, 1H), 2.68 (s, 3H), 1.68 (d, J=6.8 Hz,

6H).

LC-MS: 305.4(M+H); 1H-NMR (400MHz, CDCl3) δ 9.016(s, 1H), 8.170(d, J=1.2 Hz,

1H), 7.733(s, 1H), 7.560-7.591(m, 1H), 4.739-4.773(m, 1H), 2.698(s, 3H), 1.649-1.726 (m, 6H).

LC-MS: 323.2(M+H).

Step 2: In a dried round-bottom flask was charged with heteroaromatic methyl bromide (10 mmol) and

acetonitrile (15mL), then N-ethyl piperazine (20 mmol) was added to the above mixture at room

5

temperature. The reaction mixture was then stirred for 2 hours and precipitate was formed. The precipitate

was collected by filtration, dried under vacuum and then de-protective group using 4N HCl in dioxane to

afford the desired intermediate 4, which was used directly for next step without further purification.

LC-MS: 222.1(M+H)

LC-MS: 222.3(M+H)

LC-MS: 222.1(M+H)

Step 3: To a microwave vial equipped with magnetic stir bar, intermediate 2 (0.155 mmol) and 4 (0.186

mmol) were dissolved in 1 mL of dioxane, then K2CO3 powder (0.31 mmol), Pd2(dba)3 (0.015 mmol) and

X-phos (0.029 mmol) were added and the mixture was bubbled with Ar for 15mins. Then the reaction

vessel was sealed and irradiated with Biotage Initiator + for 0.5-2 hours at a temperature of 120 °C. The

resulting reaction mixture was cooled to room temperature and diluted with dichloromethane and water.

The organic phase was washed with brine, dried over anhydrous Na2SO4 and then concentrated to dryness.

The residue was loaded on a column and flashed on silica gel to afford the desired compound (5a-5k) as

product.

N N

NH

N

N

N

F

N

N

5a

LC-MS: 489.2 (M+1); 1H-NMR: (400 MHz, CDCl3) δ 8.86(s, 1H), 8.27(s, 1H), 8.19(s, 1H), 8.11(s, 1H),

7.71(s, 2H), 7.60(d, J=10.8Hz, 1H), 4.73-4.80(m, 1H), 3.65(s, 2H), 3.48-3.51 (m, 2H), 3.05-3.10(m, 4H),

2.84-2.96(m, 4H), 2.70(s, 3H), 1.72(d, J=7.2Hz, 6H), 1.47-1.51(t, J=7.0Hz, 3H).

N

NH

N

N

N

F

N

N

5b

LC-MS: 488.3 (M+1); 1H-NMR: (400MHz, CDCl3) δ 8.31(d, J=4.4Hz, 1H), 8.19(s, 1H),7.84(s, 1H), 7.59-

7.63(m, 1H), 7.53(s, 2H), 7.48(s, 1H), 7.22(m, 1H), 7.07-7.10(m, 1H), 4.71-4.76 (m, 1H), 3.47(s, 2H),

2.68(s, 3H), 2.30-2.62(m, 10H), 1.70(d, J=6.8Hz, 6H), 1.07-1.10 (t, J=6.8Hz, 3H).

5c

6

LC-MS: 490.6 (M+1); 1H-NMR (400MHz, CDCl3) δ 8.890 (d, J=8.0 Hz, 1H), 8.361 (s, 1H), 8.208 (s, 1H),

8.113 (s, 1H), 7.738 (s, 1H), 7.567 (d, J=11.2 Hz, 1H), 4.727-4.797 (m, 1H), 3.715 (s, 2H), 2.452-2.782 (m,

13H), 1.719 (d, J=6.8 Hz, 6H), 1.128-1.162 (m, 3H).

5d

LC-MS:489.1(M+H); 1H-NMR(400MHz, CDCl3) δ 8.87 (s，1H), 8.34(d, J=5.6Hz, 1H), 8.23(s, 1H ),

7.93(s, 1Hz), 7.52(s, 1H), 7.16(d, J=1.2Hz, 1H), 4.73(q, J=7.2Hz ,1H), 3.67(s, 2H), 2.41-2.68(m, 13H),

1.69(s, 3H), 1.65(s, 3H), 1.14(s, 3H).

5e

LC-MS: 507.4 (M+H); 1H NMR (400MHz, MeOD-d4) δ 8.25 (d, J=2.4 Hz, 1H), 8.06 (d, J=9.2 Hz, 1H),

7.97 (d, J=5.6 Hz, 1H), 7.77 (s, 1H), 7.66 (d, J=9.6 Hz, 1H), 7.28 (d, J=11.2 Hz, 1H), 4.86-4.90 (m, 1H),

3.83 (s, 2H), 2.65-3.21 (m, 13H), 1.69 (d, J=6.8 Hz, 6H), 1.29-1.33 (t, J=7.2 Hz, 3H).

5f

LC-MS: 489.2 (M+ H); 1H-NMR (400MHz, CDCl3) δ 8.90-9.02 (m, 1H), 8.42 (d, J=7.2 Hz, 1H), 8.32 (d,

J=5.2 Hz, 1H), 7.72 (d, J=6.8 Hz, 1H), 7.61(d, J=8.8 Hz, 1H), 7.51 (s, 1H), 7.32 (s, 1H), 7.14 (d, J=5.2 Hz,

1H), 4.69-4.76 (m, 1H), 3.83 (s, 2H), 2.68 (s, 3H), 2.35-2.63 (m, 10H), 1.69 (d, J=6.8 Hz, 6H), 1.07 (t,

J=7.2Hz, 3H).

5g

LC-MS: 490.3 (M+H); 1H-NMR (400MHz, CDCl3) δ 11.46 (s, 1H), 8.97 (d, J=9.2 Hz, 1H), 8.88 (s, 1H),

8.33 (d, J=4.8 Hz, 2H), 7.77-7.84 (m, 2H), 4.75-4.79 (m, 1H), 4.06 (s, 2H), 2.70 (m, 3H), 2.41-2.68 (m,

10H), 1.73(d, J=6.8Hz, 6H), 1.08 (t, J=6.8 Hz, 3H).N N

NH

N

N

N

N

N

N

F 5h

LC-MS: 490.2 (M+H); 1H-NMR: (400 MHz, CD3OD) δ 8.79(d, J=3.2Hz, 2H), 8.57 (s, 2H), 8.19(s, 1H),

7.13(d, J=10.8Hz, 1H), 4.87-4.89(m, 1H), 3.49(s, 2H), 2.67(s, 3H), 2.63-2.67(m, 8H), 2.36 (q, J=7.2Hz,

2H), 1.71(d, J=6.8Hz, 6H), 1.12(t, J=7.2Hz, 3H).

7

5i

LC-MS: 507.4 (M+H); 1H-NMR (400MHz, CDCl3) δ 8.69 (d, J=0.8 Hz, 1H), 8.22 (d, J=1.2 Hz, 1H), 8.18

(s, 1H), 7.99 (d, J=5.6 Hz, 1H), 7.58 (s, 1H), 7.39 (s, 1H), 7.19 (d, J=7.6 Hz, 1H), 4.70-4.74 (m, 1H), 3.68

(s, 2H), 2.56-2.84 (m, 13H), 1.69 (d, J=6.8 Hz, 6H), 1.22-1.25 (m, 3H).

5j

LC-MS: 490.3 (M+H); 1H-NMR(400MHz, CDCl3) δ 9.09 (s, 2H), 8.83 (d, J=4.4Hz, 1H), 8.16 (s, 1H),

7.44(d, J=11.2Hz, 1H), 7.12 (s, 1H), 4.73-4.77 (m, 1H), 3.88 (s, 2H), 2.42-2.84 (m, 15H), 1.70 (d, 6H,

J=6.8Hz), 1.20 (s, 3H).

5k

LC-MS: 508.2(M+H); 1H-NMR(400MHz, DMSO-d6) δ10.39 (s, 1H), 9.29(s, 1H), 8.63(s, 1H), 8.42(s, 1H),

8.15(s, 1H), 7.64(d, J=12.4Hz, 1H), 4.83-4.86(m, 1H), 3.62 (s, 2H), 2.64(s, 3H), 2.38-2.46(m, 8H), 2.27-

2.33(m, 2H), 1.60 (d, J= 7.2Hz, 6H), 0.97(t, J=7.2Hz, 3H).

General Procedure for preparation of compound 8a-8e.

Compound 6 was preppared using N-Boc piperazine as starting material in the general procedure of

compound 5a-5k, then it was treated with 4N HCl in dioxane to de-protective group to obtain compound 7.

Compoound 8a-8e were obtained using alkyl bromide substitution with compound 7 or alkyl aldehyde

reductive amination with compound 7.

8a

LC-MS: 504.7 (M+H); 1H-NMR(400MHz, CDCl3) δ 8.88 (m, 2H), 8.36 (s, 1H), 8.21 (s, 1H), 8.12(s, 1H),

7.83(s, 1H), 7.56 (d, J=11.2Hz, 1H), 4.73~4.80 (m, 1H), 3.70 (s, 2H), 2.52-2.86 (m, 12H), 1.72 (d, J=6.8

Hz, 6H), 1.07 (d, J=6.4 Hz, 6H).

8

8b

LC-MS: 544.4 (M+H); 1H-NMR(400MHz, CDCl3) δ 8.91(s, 1H), 8.88(s, 1H), 8.38(d, J=12.8Hz, 2H ), 8.21(s, 1H), 8.10(s, 1H), 7.85(s，1H), 7.56(d, J=11.2Hz, 2H), 4.76(t, J=6.8Hz, 1H), 3.72(s, 2H), 3.49(s,

2H), 2.99(q, J=9.6Hz, 2H), 2.63-2.77(m, 12H), 1.72(d, J=6.4Hz, 6H).

8c

LC-MS: 520.3 (M+H); 1H-NMR (400MHz, CDCl3) δ 8.91 (s, 1H), 8.88 (s, 1H), 8.35 (s, 1H), 8.20 (s, 1H),

8.10 (s, 1H), 7.68-7.76 (m, 1H), 7.57 (d, J=11.6 Hz, 1H), 4.73-4.80 (m, 1H), 3.72 (s, 2H), 3.49-3.67 (m,

2H), 3.35 (s, 3H), 2.52-2.88 (m, 13 H), 1.72 (d, J=7.2 Hz, 6H).

8d

LC-MS: 516.4 (M+H); 1H-NMR(400MHz, CDCl3) δ 8.88 (s, 1H), 8.37 (s, 1H), 8.21 (s, 1H), 8.10(s, 1H),

7.55-7.58(m, 2H), 4.74~4.78 (m, 1H), 3.71 (s, 2H), 2.54-2.70 (m, 11H), 2.28 (d, J=6.4 Hz, 2H), 1.72 (d,

J=7.2 Hz, 6H), 0.85-0.87 (m, 1H), 0.49-0.53 (m, 2H), 0.10-0.11 (m, 2H).

8e

LC-MS: 530.3(M+H); 1H-NMR(400MHz, CDCl3) δ 9.22(s, 1H), 8.90(s, 1H), 8.47(s, 1H), 8.24(s, 1H),

8.16(s, 1H), 7.96(d, 1H) 4.89(q, 1H), 4.03(s, 2H), 3.54(d, J=6.8Hz, 2H), 2.76 (s,1H), 2.50(s, 3H ), 2.01(d,

J=7.2Hz, 2H), 1.66(m, 14H).

Preparation of compound 8f

Step 1: To a dried flask was added 2-bromo-5-(bromomethyl) pyrazine (100 mg，0.40 mmol) and ethyl

((5-bromopyrazin-2-yl)methyl)phosphonate (1.0 mL) in acetonitrile (1.0 mL). The mixture was stirred at

100 oC for 4 hours and cooled to room temperature. The mixture was concentrated to dryness and purified

with flash chromatography to afford compound 9 (100 mg，white solid), LC-MS: 311 (M+H).

Step 2: 1-ethyl-piperidin-4-one (49.3 mg，0.388 mmol) was dissolved in THF (10 mL) and added

dropwise to a stirred mixture of phosphoric acid diethyl ester 9 (100 mg，0.324 mmol) and NaH (60% oil

dispersion, 25.9 mg，0.648 mmol) in THF (10 mL) under N2. The solution was stirred at room temperature

9

for 6 h, and then partitioned between water (30 mL) and AcOEt (50 mL×3). The organic phase was dried

over Na2SO4 and evaporated in vacuo. The crude mixture was purified by silica gel chromatography

(EtOAc:PE=1:1). The collected fractions gave 60 mg of desired compound 10 as a white solid. LC-MS：

284 (M+H)

Step 3: Compound 11 was prepared according to the procedure of compound 5a-5k using 6-(4-fluro-1-

isopropyl-2-methyl-1H-benzo[d]imdazole) pyrimidyl-4-amine (50 mg，0.175 mmol) and 5-bromo-2-((1-

ethylpiperidinyl-4-ene) methylene pyrimidine (60 mg，0.213 mmol) as starting materials. LC-MS: 487.4

(M+H);1H-NMR:(400MHz,CDCl3) δ 8.91 (s, 1H), 8.87 (s, 1H), 8.20 (s, 1H), 8.18 (s, 1H), 8.04 (s, 1H),

7.59 (s, 1H), 7.57 (d, J=11.6Hz, 1H), 6.28 (s, 1H),4.72~4.78 (m, 1H), 2.98~3.02 (m, 2H), 2.69 (s, 3H),

2.52~2.63(m, 8H), 1.72 (d, J=6.8Hz, 6H), 1.16 (t, J=7.0Hz, 3H).

Step 4: Compound 8f was prepared using Pd/C catalytic hydrogenation to afford the desired compound.

LC-MS: 489.4 (M+H); 1H-NMR(400MHz, CDCl3) δ 8.90 (s, 1H), 8.87 (s, 1H), 8.20 (s, 1H), 8.17 (s, 1H),

8.04 (s, 1H), 7.60 (s, 1H), 7.56 (d, J=12.0Hz, 1H), 6.28 (s, 1H),4.72~4.78 (m, 1H), 2.98~3.01 (m, 2H),

2.72~2.74 (m, 2H), 2.69(s, 3H), 2.43~2.45 (m, 2H), 1.79~2.00 (m, 7H), 1.72 (d, J=7.2Hz, 6H), 1.11 (t,

J=7.2Hz, 3H).

Preparation of compound 8h

Step 1: N-ethyl piperidin-4-ol(200 mg，1.55 mmol) was added to dry THF (10 mL) and the mixture was

cooled to 0oC, then NaH (60%, 124 mg，3.1 mmol) was added slowly. The mixture was stirred at 0oC for

0.5 h and then 2,5-dichloropyrodazine(231 mg，1.55 mmol) was added and stirred overnight. The reaction

was quenched with iced water and extracted with DCM, the organic layer was washed with brine, dried and

concentrated to afford compound 12(330 mg, white solid).LC-MS: 242.1(M+H).

Step 2: Compound 8h was prepared according to the procedure of compound 5a-5k using compound 12 as

starting material. LC-MS：491 (M+H);1H-NMR: (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.70 (s, 1H), 8.15 (s,

1H), 8.98 (s, 1H), 7.56 (s, 1H), 7.50-7.52 (d, J=11.2Hz, 1H), 7.41 (s, 1H), 5.05 (s, 1H), 4.73-7.76 (m,1H) ,

2.83 (s, 2H), 2.68 (s, 3H), 2.49 (s, 2H), 2.13 (s, 2H), 1.90 (s, 2H), 1.66-1.71 (m, 8H), 1.15 (t, J=7.2Hz, 3H).

Preparation of compound 8g

Step 1: Compound 13 was prepared according to the procedure of compound 5a-5k using 6-(4-fluro-1-

10

isopropyl-2-methyl-1H-benzo[d]imidazole-6-yl) pyrimidinyl-4-amine (57.0 mg，0.2 mmol) and 1-(5-

chloro-2-pyradazinyl)-ethynone (37.44 mg，0.24 mmol) as starting materials. Compound 13 (51

mg，0.126 mmol) was added to THF (3 mL) and the mixture was cooled to 0oC. NaBH4 (10 mg) was

slowly added to the mixture and stirred for 1 h. The mixture was diluted with water (10 mL) and EtOAc (20

mL×3). The organic layer was collected and washed with brine, dried and concentrated to afford compound

14(48 mg ), which was used directly for next step without further purification，LCMS: 408.0 (M+H).

Step 2: Compound 14 (48 mg，0.118 mmol) was dissolved in dried toluene (5 mL), then SOCl2(1.5 mL)

and a drop of DMF were added to the mixture. The reaction mixture was stirred at reflux for 3 hours and

then cooled to room temperature, concentrated to dryness to afford crude compound 15(41 mg). LCMS:

425.3 (M+H).

Step 3: To a 25 mL flask was added crude compound 15 (41 mg，0.097 mmol) and DIPEA (0.5 mL) in

acetonitrile (5 mL) , then N-ethyl piperazine (33.1 mg，0.291 mmol) was added to the mixture and stirred

at reflux for 2 hours. The mixture was cooled to room temperature and diluted with water (10 mL) and

EtOAc (20 mL×3), the organic phase was washed with brine and concentrated to dryness. The residue was

purified with preparative HPLC to afford the desired compound (5 mg，white solid). LC-MS: 504.3

(M+H)；1H-NMR: (400 MHz, CDCl3) δ 8.87 (d, J=7.6Hz, 1H), 8.35 (s, 1H), 8.21 (s, 1H)，8.10 (s, 1H),

7.51 (s, 1H), 7.56 (d，J=7.6Hz, 1H), 4.76-4.78 (m, 1H), 3.71-3.73 (m, 1H), 2.51-2.69 (m, 11H), 2.11(s,

2H), 1.72 (d, J=6.8Hz, 6H), 1.47 (d, J=6.4Hz, 3H), 1.22 (s, 3H)。

11

In Vitro Determination of CDK1/4/6 Inhibitory Activity

The inhibition of compounds activity of CDK1, 4, 6 was characterized by LANCE Ultra kinase assays kit

(PerkinElmer, USA) according to the manufacturer’s instructions. The kinase reaction was performed by

mixing the kinase、ULight-substrate, ATP and inhibitors being tested in a suitable buffer. The reaction was

incubated for an hour at room temperature. The detection reaction was performed by adding EDTA to stop

the kinase reaction, then the LANCE Europium-anti-phospho specific antibody was added to the reaction in

LANCE Detection buffer. The mixture was incubated for an hour at room temperature. Reading the plate

by the PerkinElmer EnVision® Reader.

In Vitro Determination of Cell Proliferation Inhibitory Activity in Colo-205

Human colon cancer cell lines Colo-205 were purchased from American Type Culture Collection (ATCC)

and cultured in media according to recommendations with 10% FBS (Gibco). Cells were seeded in 96-well

plates. After attachment, cells were treated with various concentrations of compounds and further cultured

for 96 h. Cell proliferation was then determined using CCK8 assay (Dojindo, Japan). The plates were read

by SPECTRA max 384 PLUS (Molecular Devices, USA). IC50 values were calculated by concentration-

response curve fitting using four-parameter method.

12

In vivo Determination of Mouse Pharmacokinetic Parameters

The purpose of this study was to determine the pharmacokinetic parameters of compounds in male ICR

mice following intravenous (iv) and oral (po) administration. IV formulation: compound was dissolved in

5%DMSO/40%PEG400/55%Saline to yield a final concentration of 0.2 mg/mL for each compound for

intravenous administration. PO formulation: compound was dissolved in 0.4%MC in deionized water to

yield a final concentration of 0.5 mg/mL for each compound for oral administration. Blood samples (~ 200

µL/ sample) were collected via the retro-orbital puncture after anesthetized by isoflurane or via cardiac

puncture (terminal collection) at appropriate time points. Samples were placed in tubes containing heparin

sodium and stored on ice until centrifuged. The PK parameters were determined for the test article from

mean concentration-time data in the test species. A non-compartmental module of WinNonlin®

Professional was used to calculate parameters. Any concentrations that were BLQ were omitted from the

calculation of PK parameters in individual animals. The bioavailability was calculated as F (%) = (Doseiv ×

AUCoral(0-∞)) / (Doseoral × AUCiv(0-∞)) × 100%

In vivo Determination of Efficacy in Colo-205 Cell Based Xenograft Model

Female BALB/c nude mice (4−6 weeks) were housed at six mice per cage in a specific pathogen free room

with a 12 h light/dark schedule at 25 ± 1 °C; the animals were fed an autoclaved chow diet and water ad

libitum. All the animal experiments were performed according to the institutional ethical guidelines of

animal care.

Colo-205 cells at a density of 5×106 were first implanted subcutaneously into the right flank of each mouse

and then allowed to grow to 700–800 mm3, which was defined as a well-developed tumor. The well-

developed tumors were cut into 1.5 mm3 fragments and transplanted s.c. into the right flank of nude mice

using a tracer. When the tumor volume reached 160 mm3, the mice were randomly assigned into control

and treatment groups. The control groups were given vehicle alone and the treatment groups received

compounds at the indicated doses via oral administration once daily for 21 days. The sizes of the tumors

were measured twice per week using micro calipers. The tumor volume (TV) was calculated as follows: TV

= (length × width2)/2. The tumor volume shown was obtained on the indicated days as the median tumor

volume ± SEM for indicated groups of mice. The relative tumor volume (RTV) values were measured on

the final day of the study for the drug-treated mice compared with the vehicle-treated mice and were

calculated as RTV = Vt/V0, while V0 is the tumor volume at day 0, Vt is the tumor volume measured each

time point. The percentage of tumor growth inhibition (TGI) values were also measured. Significant

differences between the treated versus the control groups (P ≤0.05) were determined using Student’s t test.

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