Discovery of Indolinone-Based Multikinase Inhibitors as Potential

Sep 30, 2017 - Idiopathic pulmonary fibrosis (IPF) is a serious and deadly disease for which treatment options are limited. The recent approval of ant...
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Letter

Discovery of Indolinone-based Multi-kinase Inhibitors as Potential Therapeutics for Idiopathic Pulmonary Fibrosis Zhenhua Huang, Heran Li, Qian Zhang, Fangzheng Lu, Mei Hong, Zhigang Zhang, Xiaocui Guo, Yuanju Zhu, Sanming Li, and Hongzhuo Liu ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00164 • Publication Date (Web): 30 Sep 2017 Downloaded from http://pubs.acs.org on October 1, 2017

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Discovery of Indolinone-based Multi-kinase Inhibitors as Potential Therapeutics for Idiopathic Pulmonary Fibrosis Zhenhua Huang† Heran Li†, Qian Zhang‡, Fangzheng Lu†, Mei Hong‡, Zhigang Zhang‡, Xiaocui Guo‡, Yuanju Zhu‡, Sanming Li† and Hongzhuo Liu†* †

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, P. R. China; KBP Biosciences, 401, Building 2, Jinan Pharm Valley, North Section of Gangxing Three Road, Jinan, Shandong, 250101, P. R. China KEYWORDS: idiopathic pulmonary fibrosis (IPF); multi-kinase inhibitors; SAR; PK properties; in vivo study. ‡

100

Sham Model Nintedanib 100mg/kg, q.d Nintedanib 30mg/kg, q.d

90

O N

O O

N 3 H

O N

NH IC50: c-KIT 10 nM RET 7.6 nM O PDGFR: 25 nM

Survival rate( % )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80

* ** 70

**

KBP-7018 100mg/kg, q.d KBP-7018 30mg/kg, q.d KBP-7018 10mg/kg, q.d

60

*

50

Log-rank test *P30

N

620

15

100

100

>30

N

4700

24

300

110

>30

O

9b, N

9c,

PDGFRβ 15

9

N H

O

FGFR1 140

R2

O

O

NR1R2/R

R1 N

O

N

9d, 9e,

N

N

1600 25 110 100 >30 3, N O 2, Nintedanib 41 1.8 3.1 85 2.6 NA: not available. The data were presented with two significant figures due to standard experimental error.

When evaluating compounds for their kinome selectivities, PDGFR was chosen as the key target due to its critical role in the initiation and progression of IPF. Therefore, the initial strategy was to improve the drug-like properties and potency of nintedanib (2) against PDGFR. Derivatives of compound 9 containing dihydroindole groups were designed and synthesized by introducing a variety of amines (Table 1). In all cases, the inhibitory activities of each derivative toward FGFR and VEGFR were weaker than nintedanib, indicating that the dihydroindole core may not be optimal for improving the efficacy against FGFR and VEGFR. Fortunately, the compounds showed a reserved affinity for PDGFR, with IC50 values ranging from 10-100 nM. A compound with a small Me2N group (compound 9a) exhibited a reasonable inhibitory potency (IC50, 15 nM), whereas a compound with a larger amino group (9b) exhibited a loss of activity. Surprisingly, neither the removal of the basicity of the nitrogen (9c) nor the aromatization of the amino group (9d and 9e) had an impact on potency. The morpholinyl-bearing compound 3 (KBP-7018) also inhibited PDGFR activity, despite the lack of affinity for FGFR and a decreased inhibition of VEGFR. Taken together, basicity was not a prerequisite for high inhibitory potency against PDGFR, and as various basic groups with different pKa values were all tolerated, the dihydroindol core may play a key role in affinity to the hinge region of the PDGFR. Because an X-ray structure of PDGFR is not available, a detailed explanation of its activity is beyond the scope of the present study. The designed compounds were further evaluated for their abilities to inhibit the h-PDGF-bb-stimulated proliferation of 3T3 cells. Compounds 9b-f and 3 were potent in the ~100 nM range, indicating sufficient cell permeability. Compounds with high potencies were further evaluated for their effects on the hERG K. To our surprise, a morpholino substituent at region 2 (compound 3, also known as KBP- 7018) significantly reduced the inhibitory effects

against hERG K compared to nintedanib. Furthermore, the substituents other than morpholine (eg. 9b-e) also reduced the hERG K inhibitory effect. Because of its high potency and minimal effects on hERG K, compound 3 (KBP-7018) is a promising kinase inhibitor. Based on the additional physicochemical properties of compound 3, such as its acceptable solubility and clean properties, we evaluated this compound in subsequent tests. The lactam 15 and 17 showed similar SAR trends in PDGFR activity. However, the affinities for FGFR1 and VEGFR were increased compared with compounds 9b-f (Table 2). Both Senantiomer (15a) and R-enantiomer (15b) of piperizinyl compounds showed a comparable potency to nintedanib against both VEGFR and PDGFR, whereas the activity in FGFR1 was decreased. On the other hand, a Me2N analog (15c) also showed decreased activities against PDGFR, FGFR1 and VEGFR compared with nintedanib. Furthermore, the isomeric piperazinyl analog (17a), a racemate, did not show activity against PDGFR, whereas individual enantiomers (17b and 17c) were also active in cells with potencies similar to compounds 9b-e and 3. Disappointingly, the presence of the piperazine nucleus elicited a significant inhibitory effect of hERG K, such as in compound 15a (IC50, 0.95 µM against hERG K). Therefore, these compounds were not suitable for further optimization. Because the ester group is regarded as a metabolic liability (Table 2), we next performed SAR studies by modifying the substituent in position 6 of the indolinone group with various isosteric groups. All isosteric heterocycles showed reasonable potencies against FGFR, PDGFR and VEGFR compared with nintedanib. The triazole derivative 21h provided the desired potency, whereas 5oxazole-bearing compound 21f was the least potent. Most of the other compounds, such as 21c and 21h, had nanomolar potencies against PDGFR and reasonable activities against FGFR1 and VEGFR.

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Table 2. The SAR of the indoline derivatives 15, 17 and 21.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O N

O O

15

R1 N R2

N

NH

NH

O

O

N H

O O

17

NR1R2/R 15a, (S),

N

N

15b, (R), 15c, NMe2 N

N

N

N

17a, 17b, (R or S) 17c, (R or S) 21b, OCF3 21c, 5-oxadiazolyl 21d, 3-oxadiazolyl 21e, 5-(2-Nmethyl)tetrazolyl 21f, 5-oxazolyl 21g, 2-furanyl 21h, 2-triazolyl 2, Nintedanib

O

R1 N R2

O N

NH

N

N

21

O

N H

R

N H

FGFR1 120

PDGFRβ 4.3

IC50 (nM) VEGFR2 1.6

100

3.2

8.3

97

1.4

3T3 (h-PDGFbb) 110

IC50 (µM) hERG K 0.95

94

14

51

NA

NA

140

45

12

NA

NA

NA NA 200 62 44 170

NA NA 27 4.5 5.3 13.0

NA NA 34 4.0 4.6 4.9

130 110 NA NA NA NA

NA NA NA NA NA NA

270 15 61 41

34 12 1.5 1.8

8.0 9.3 1.9 3.1

NA NA NA 85

NA NA NA 2.6

NA: not available. The data were presented with two significant figures due to standard experimental error.

However, all tested compounds in this subseries exhibited signifi-

Table 3. Results of the assessment of the activity (%) of 1 µM

cant toxicity, as evidenced by the lethality of an intravenous injec-

KBP-7018 toward 64 enzymes in a kinase panel compared with

tion of 5 mg/kg of either compound 21c or 21d. Analogs modi-

nintedanib (compounds with 80% inhibition of only 5 enzymes at 1 µM, whereas nintedanib (2) inhibited 19 enzymes, indicating the high selectivity of KBP-7018 compared with nintedanib (Table 3).

*Data for the following 45 kinases are not shown: AKT1, AMPKa1, AURA/B, BRAF, BRK, CDK2, CHK1, CK1d, CSK, DYRK1a, EGFR, EGFR (T790M), ERK2, GSK3, p70S6K, HER2/4, IGF1R, IKKβ, IRAK1/4, JAK1/2, JNK2/3, MAPKAPK2/5, c-MET, MSK1, MST2, NEK2, P38α/β, p70S6K, PDK1, PI3Kα/δ, PKCa, c-Raf, ROCK2, RSK1, SGK, SYK, ZAP70 (the kinase abbreviations shown in Supporting Information).

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Page 5 of 7 Table 4. PK properties of KBP-7018.

ed improved PK profiles compared with nintedanib, as supported by its acceptable oral bioavailability and half-life in all tested

Species

Dose (mg/kg) 50 10 20

Mouse Rat Dog

t1/2 (h) 4.3 4.8 3.3

AUC (h·ng/mL) 17,004 6406 2964

F (p.o.) 51% 68% 29%

species. In a previous study, KBP-7018 showed a significantly lower efflux ratio against P-gp than nintedanib in a Caco-2 cell monolayer transport system13, thereby increasing intestinal permeability and absorption in vivo. Second, KBP-7018 demonstrat-

KBP-7018 was chosen for further in vivo testing because of its

ed a highly selectively inhibition of tyrosine kinases compared

cellular potency, reduced effect on hERG K and attractive selec-

with nintedanib. Specifically, it exhibited a lower affinity for

tivity profiles. The PK properties of KBP-7018 were studied in

VEGFR2, a known anti-cancer target, resulting in a lower death

mice, rats and dogs. The oral bioavailability and the half-life of

rate in the mouse model of BLM-induced pulmonary fibrosis.

KBP-7018 were acceptable in all three species (Table 4), indicat-

Accordingly, KBP-7018 is superior to nintedanib and displays a

ing its drug-like profile.

wider therapeutic window (Figure 2). Finally, KBP-7018 had a less profound effect on hERG K, thus reducing the relating cardi-

Finally, KBP-7018 was evaluated in a C57 mouse model of bleomycin

(BLM)-induced

chronic

pulmonary

fibrosis

ac side effect.

using

nintedanib (2) as a positive control (Figure 2)11, 12. All three doses

Notably, KBP-7018 inhibited c-KIT and RET (which are impli-

(10, 30, 100 mg/kg, q.d.) significantly improved the 28-day sur-

cated in angiogenesis and fibrosis14, 15) with a potency in the na-

vival rate in a dose-dependent manner. At 30 mg/kg/day, KBP-

nomolar range (IC50 = 10 nM and 7.6 nM, respectively). Based on

7018 showed a higher in vivo efficacy compared to nintedanib.

the results from the present study, KBP-7018 shows promising

Notably, after the oral administration of 100 mg/kg/day

potency against IPF in a mouse model of BLM-induced pulmo-

nintedanib, the mice exhibited significantly reduced survival

nary fibrosis. The superior in vivo activities of KBP-7018 indicate

compared with the mice that received the oral administration of

that the combination of PDGFR, c-KIT and RET as potential ther-

the lower dose of 30 mg/kg/day, indicating a narrow therapeutic

apeutic targets for IPF.

window for nintedanib. In contrast, mice displayed sufficient tolerance to KBP-7018 at doses ranging from from 10 mg/kg to

In conclusion, a novel series of indolinone-based multi-kinase

100 mg/kg.

inhibitors targeting the angiogenesis and fibrosis pathways were established. KBP-7018 showed sufficient potencies against three angiogenic kinases (c-KIT, PDGFR, and RET). The PK properties

100

90

Survival rate( %)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80

* ** 70

**

Sham Model Nintedanib 100mg/kg, q.d Nintedanib 30mg/kg, q.d

of KBP-7018 in mice, rats and dogs were favorable. In a mouse

KBP-7018 100mg/kg, q.d

dependently improved the 28-day survival rate when administered

KBP-7018 30mg/kg, q.d KBP-7018 10mg/kg, q.d

60

*

Log-rank test *P