Discovery of (R)-1-(3-(4-Amino-3-(4-phenoxyphenyl)-1 H-pyrazolo [3

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Discovery of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4d]pyrimidin-1-yl)piperidin-1-yl)-2-(dimethylamino)ethanone (CHMFL-FLT3-122) as a Potent and Orally Available FLT3 Kinase Inhibitor for FLT3-ITD Positive Acute Myeloid Leukemia Xixiang Li, Aoli Wang, Kailin Yu, Ziping Qi, Cheng Chen, Wenchao Wang, Chen Hu, Hong Wu, Jiaxin Wu, Zheng Zhao, Juan Liu, Fengming Zou, Li Wang, Beilei Wang, Wei Wang, Shanchun Zhang, Jing Liu, and Qingsong Liu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01611 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 5, 2015

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Journal of Medicinal Chemistry

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Discovery of (R)-1-(3-(4-amino-3-(4phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1yl)piperidin-1-yl)-2-(dimethylamino)ethanone (CHMFL-FLT3-122) as a Potent and Orally Available FLT3 Kinase Inhibitor for FLT3-ITD Positive Acute Myeloid Leukemia Xixiang Li1,2,6, Aoli Wang1,3,6, Kailin Yu1,3,6, Ziping Qi1,2,6, Cheng Chen1,2, Wenchao Wang1,2, Chen Hu1,2, Hong Wu1,3, Jiaxin Wu1,3, Zheng Zhao1,2, Juan Liu1,2, Fengming Zou1,2, Li Wang1,2, Beilei Wang1,2, Wei Wang1,2, Shanchun Zhang2,4, Jing Liu1,2*, Qingsong Liu1,2,3,5* 1. 2. 3. 4. 5. 6.

High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350 Shushanhu Road, Hefei 230031, Anhui, P. R. China CHMFL-HCMTC Target Therapy Joint Laboratory, Shushanhu Road, Hefei 230031, Anhui, P. R. China University of Science and Technology of China, P. R. China, Anhui, Hefei, 230036 Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, 230031, Anhui, P. R. China Hefei Science Center, Chinese Academy of Sciences, Shushanhu Road, Hefei 230031, Anhui, P. R. China These authors contribute equally


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ABSTRACT: FLT3-ITD mutant has been observed in about 30% AML patients and extensively studied as a drug discovery target. Based on the structure of PCI-32765 (Ibrutinib), a BTK kinase inhibitor that was recently reported to bear FLT3 kinase activity, through a structure-guided drug design approach, we have discovered compound 18 (CHMFL-FLT3-122), which displayed an IC50 of 40 nM against FLT3 kinase and achieved selectivity over BTK kinase (over 10-fold). It significantly inhibited the proliferation of FLT3-ITD positive AML cancer cell lines MV4-11 (GI50: 22 nM), MOLM13/14 (GI50: 21 nM/42 nM). More importantly, 18 demonstrated 170-fold selectivity between FLT3 kinase and c-KIT kinase (GI50: 11 nM versus 1900 nM) in the TELfusion isogenic BaF3 cells indicating a potential to avoid the FLT3/c-KIT dual inhibition induced myelosuppression toxicity. In the cellular context it strongly affected FLT3-ITD mediated signaling pathways and induced apoptosis by arresting the cell cycle into G0/G1 phase. In the in vivo studies 18 demonstrated a good bioavailability (30%) and significantly suppressed the tumor growth in MV4-11 cell inoculated xenograft model (50 mg/kg) without exhibiting obvious toxicity. Compound 18 might be a potential drug candidate for FLT3-ITD positive AML.

INTRODUCTION Acute Myeloid Leukemia (AML) accounts for approximately 80-90% of leukemia in adults. Over ten thousand new AML patients are diagnosed and lead to similar number of deaths per year in US alone.1,

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The current standard chemotherapy for AML usually lacks the durable

efficacy and is also poorly tolerated especially in the elder patients. FMS-Like tyrosine kinase (FLT3) is a type III receptor tyrosine kinase that plays important roles in the differentiation and survival of hematopoietic stem cells in bone marrow and has been observed over-expressed in


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the AML and ALL.3-5 A variety of gain-of-function mutations have been identified in the AML patients such as FLT3-ITD (internal tandem duplication), FLT3-D835Y/E/V/H, and FLT3K663Q etc, among which FLT3-ITD accounts for about 30% and is associated with poor prognosis.6 The mutated FLT3-ITD kinase promotes the AML blast survival and proliferation through the downstream signaling mediators including Stat5, ERK and AKT.7 Therefore, FLT3ITD kinase has been considered as a validated drug discovery target for FLT3-ITD positive AML. A number of small molecule inhibitors have been reported to exhibit potent FLT3 kinase inhibitory activities such as sunitinib8, sorafenib,9 PKC412,10, CEP-701,11 UNC2025,12 MLN518,13 KW-244914 and AMG-92515 etc. In addition, the relatively more selective second generation FLT3 inhibitors such as AC2206, crenolanib16 and PLX339717 are being tested in the clinic now which have shown initial transient responses but usually followed by quick development of resistance. Besides, the c-KIT kinase and FLT3 kinase dual inhibition induced synthetic lethal myelosuppression toxicity observed from the most advanced clinical trial compounds such as PKC412 and AC220 is also a big safety concern.18 Currently there is no FLT3 targeted therapies get approved for clinical application yet.19 Hence, there is still a critical unmet medical need remaining for FLT3-ITD positive AML. Recently we discovered that PCI-32765 (1), a clinically used BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), also displayed submicromolar GI50 against FLT3-ITD positive AML cancer cell lines.20, 21 In addition, compound 1 did not exhibit apparent c-KIT kinase inhibition which indicated a better safety profile for this class of compounds. In order to further improve the FLT3 kinase potency and selectivity of this new class of compounds, starting from 1, through a detailed SAR study, we discovered a potent,


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selective and orally available FLT3 kinase inhibitor 18 (CHMFL-FLT3-122) that displayed impressive in vitro and in vivo activities against FLT3-ITD positive AML (Figure 1).

Figure 1. Chemcial structure of PCI-32765 (1) and CHMFL-FLT3-122 (18). RESULTS AND DISCUSSTION We first examined the binding mode of PCI-32765 to BTK and FLT3 kinase in order to direct the chemical modification (Figure 2). In BTK kinase, PCI-32765 forms three hydrogen bonds with Met477, Glu475 and Thr474 respectively in the hinge binding area. The signature of this binding is that the acrylamide of PCI-32765 forms a covalent bond with Cys481 to achieve the irreversible binding. The O-bridged diphenyl moiety directs into the hydrophobic pocket formed by DFG motif and α-cHelix (Figure 2A). In comparison, PCI-32765 forms two similar hydrogen bonds with Cys694 and Glu692 of FLT3 kinase in the hinge binding area. However, the Cys481 in BTK is replaced by Asp698 in FLT3 which makes the irreversible binding impossible (Figure 2B). Superimposition of BTK and FLT3 kinase reveals that the Phe830 in the DFG motif of FLT3 kinase (white in Figure 2C) flips more flat than Phe540 in BTK kinase (purple in Figure 2C), which might provide more space for this hydrophobic pocket. However, the α-cHelix of FLT3 kinase shifts closer to the hinge binding area than the α-cHelix of BTK kinase hence this


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will shrink the hydrophobic pocket (Figure 2C). Combining all of the analyses, we proposed that there are two directions we can take to improve the binding affinity for new lead compounds against FLT3 kinase. First, we can modify the O-bridged diphenyl group (R2, Figure 2D) to investigate if there is a larger pocket created by α-cHelix-in and Phe830-out. Second, since in FLT3 kinase there is no approachable cysteine residues required by irreversible binding, we can modify the piperidine acrylamide moiety (R1 and R3, Figure 2D) to achieve the better selectivity and stronger binding affinity against FLT3 kinase.

Figure 2. Binding mode analysis of PCI-32765 (1) to BTK kinase and FLT3 kinase. (A) PCI32765 docked into BTK kinase (PDB ID: 3GEN). (B) PCI-32765 docked into FLT3 kinase (PDB ID: 1RJB). (C) Superimposition of BTK and FLT3 kinase in complex with PCI-32765 (purple, BTK kinase; white, FLT3 kinase). (D) Illustration of the chemical modification strategy of compound 1. We first made compound 2 which bears a double bond linked diphenyl moiety to explore the possibility of FLT3 kinase occupying a larger inner hydrophobic pocket formed by DFG motif and α-cHelix. However, compared with 1, it significantly lost the activity against intact AML cancer cell line MV4-11 (GI50: 10 µM versus 0.33 µM) (Table 1), which expresses FLT3-ITD


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mutant. In the isogenic BaF3 cells whose proliferation is dependent on FLT3-ITD, compound 2 also lost about 15-fold activity (GI50: 1.8 µM versus 0.12 µM). This indicated that FLT3 might not tolerate the larger hydrophobic moiety. We then turned our attention to the hinge binding area and made a series of modifications at R1 position to explore the SAR. Since there is no approachable cysteine residue which could be utilized to form the covalent bond as in BTK kinase, we first saturated the double bond in 1 to make 3 and found that it exhibited better inhibitory activities against MV4-11 (GI50: 0.13 µM) and FLT3-ITD-BaF3 isogenic cell line (GI50: 0.028 µM). But it also displayed a GI50 of 3.8 µM against parental BaF3 cell line which indicated there was still selectivity window could be explored (ideally, the compound that does not show apparent inhibitory activity against the parental BaF3 cells i.e. >10 µM is preferred). Variation of terminal ethyl group, e.g. methyl group (4) or bulky ones such as propyl (5), tertbutyl (6), 2-methyl-butyl (7), dimethyl propyl (8) and cyclopropyl (9) all led to significant lose of inhibitory activity. However, introduction of the methyl cyclopropyl group (10) started to gain back the activity against MV4-11 (GI50: 0.41 µM) and FLT3-ITD-BaF3 cells (GI50: 0.082 µM), though still weaker than 3. Methyl piperidine (11) and 1,4-dimethyl piperazine (12) exhibited comparably moderate activities to 3. Compounds that switching the linking carbonyl group into sulfonamide and bearing different sizes of substitutes (13-15) did not improve the antiproliferation efficacy either, despite that all of them still remained the moderate potencies. Table 1. Compounds (1-15) with different substituents at R1/R2 position and their antiproliferation efficacies against intact and isogenic cancer cell linesa MV4-11 Compd.

1

R1

BaF3

(GI50: µM)

BaF3-FLT3-ITD (GI50: µM)

(GI50: µM)

0.33

0.12

>10

R2


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2

>10

1.8

>10

3

0.13

0.028

3.8

4

0.35

0.07

5.5

5

1.2

0.45

>10

6

>10

1.8

>10

7

2.4

0.26

4.5

8

2.4

0.46

4.6

9

6.5

1.4

>10

10

0.41

0.082

3.6

11

0.16

0.077

5.0

12

0.23

0.093

2.3

13

0.17

0.048

>10


7

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a

14

0.57

0.14

4.0

15

0.76

0.16

8.6

All GI50s were obtained by triplet testing.

We then went back to analyze the binding mode again and envisioned that the Asp698 might serve as a hydrogen bond acceptor. If the substitutes at proper R1 position could provide a hydrogen bond donor then it might increase the potency. Therefore we replaced the acrylamide group with a series of amino acids and derivatives for SAR study. The glycine moiety (16) remarkably improved the anti-proliferation efficacy against MV4-11 (GI50: 0.022 µM) and BaF3-FLT3-ITD (GI50: 0.007 µM) meanwhile still remained proper selectivity against parental BaF3 cell (GI50: 3.8 µM) (Table 2), while the beta-alanine (17) lost over 30-fold activity against MV4-11. The N, N-dimethyl glycine (18) kept the activity against MV4-11 (GI50: 0.022 µM) and BaF3-FLT3-ITD (GI50: 0.011 µM) and showed a better selectivity against parental BaF3 (GI50: 4.9 µM). Introduction of other amino acids, e.g., alanine (19), serine (20), valine (21), threonine (22) and leucine (24) led to similar activities except that isoleucine (23) lost around 6-fold activities. These results indicated that the hydrogen bond formed between Asp698 and the nitrogen atom in R1 was critical for the binding (Figure 3). Table 2. Compounds (16-24) with amino acids and derivatives at R1 position and their antiproliferation efficaciesa Compd.

16

MV4-11

BaF3-FLT3-ITD

BaF3

(GI50: µM)

(GI50: µM)

(GI50: µM)

0.022

0.007

3.8

R1


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a

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17

0.75

0.39

>10

18

0.022

0.011

4.9

19

0.054

0.022

3.8

20

0.061

0.046

6.5

21

0.027

0.017

1.4

22

0.064

0.022

3.9

23

0.12

0.088

1.7

24

0.032

0.005

1.3


Figure 3. Compound 18 docked into FLT3 kinase (PDB ID: 1RJB).


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Since compound 18 displayed the best activities so far, we then placed more efforts to investigate the R3 part. Inversion of the R stereo center on the piperidine ring of 18 to S (25) resulted in over 10-fold loss of activities indicating that the R conformation was preferred at this position (Table 3). Shifting the amide moiety from 3- position of piperidine to 4- position (26-28) or replacing the piperidine ring by the pyrrolidine ring (29-32) all led to the activity loss. Table 3. Anti-proliferation efficacies of compounds (25-32) bearing different R1 and R3 moietiesa MV4-11

BaF3-FLT3-ITD

BaF3

(GI50: µM)

(GI50: µM)

(GI50: µM)

25

0.28

0.12

5.8

26

0.11

0.075

3.65

27

0.63

0.19

>10

28

0.21

0.075

0.59

29

0.18

0.08

7.59

30

0.083

0.041

8.42

31

0.12

0.041

2.15

32

0.42

0.13

>10

Compd.

a

R1

R3



10


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Considering the potent anti-proliferation activities against FLT3-ITD positive cell line and the selectivity window between the FLT3-ITD-BaF3 cell and parental BaF3 cell, we decided to choose 18 for further characterization. We first examined compound 18’s selectivity with DiscoveRx’s KinomeScanTM technology. The results showed that compound 18 was very selective (S score (1)=0.03 at 1µM). Besides FLT3wt and FLT3/ITD, it also displayed strong binding against BLK, BTK, ERBB3, HCK, LCK, MEK5, PDGFRα, RET and SRMS kinases (percent activity remaining less than 1% at 1µM). (Fig. 4A and B and Supplemental Table 1) Given the fact that KinomeScanTM is a binding assay and may not fully reflect the inhibitory activity, we then used ADP-Glo based biochemical activity assay with the purified kinase proteins to confirm the compound 18’s inhibition activity against FLT3, BTK and c-KIT. The results demonstrated that compound 18 inhibited FLT3 kinase with an IC50 of 40 nM (Figure 4C). It also demonstrated a good selectivity over BTK kinase (IC50: 421 nM) and c-KIT kinase (IC50: 559 nM). We also used TEL transformed BaF3 system to further confirm the on-target activity and selectivity against other potential off-targets revealed by KinomeScanTM binding assay (Table 4). Interestingly, compound 18 was also very potent against FLT3-D835Y/V/H mutants, which are other important oncogenic mutations in the AML, in the BaF3 isogenic cell lines. While for the drug resistance mutations such as FLT3-ITD-D835Y, FLT3-ITD-F691L, it lost 1620 fold activities comparing to FLT3-ITD itself. In addition, 18 also exhibited good selectivity over c-Kit kinase (GI50: 1.9 µM versus 0.011 µM for FLT3-ITD, 170 fold selectivity) and other protein kinases such as MET, EGFR, JAK1/2/3, RET, PDGFRα and BMX (around 25-200 fold) (Table 4). In the SRC kinase family, compound 18 did not potently inhibits HCK (GI50: 0.88 µM), and LCK (GI50: 0.31 µM) and BLK (GI50: 0.94 µM). Combining the results from all of these different assay formats, we concluded that compound 18 is a highly potent FLT3 kinase


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inhibitor. Compound 18 displayed similar potencies in the FLT3-ITD dependent AML cancer cell line MOLM13 (GI50: 0.021 µM) and MOLM14 (0.042 µM) as in MV4-11 (Table 5 and 2). As expected, it did not exhibit potent inhibitory activities against FLT3 wt or negative intact cancer cell lines such as U937, SU-DHL-2, U2932, JVM-2, Namalwa and NB4 cell lines.

Figure 4. Selectivity profiling of compound 18 (A) KinomeScanTM profiling of compound 18 at a concentration of 1 µM against 468 kinases. (B) Kinases showed strong binding to compound 18 (less than 1% activity remaining comparing to DMSO control in the assay format with 1 µM of 18). (C) ADP-Glo biochemical characterization of compound 18 against FLT3 kinase, BTK kinase and c-KIT kinase.

Table 4. Anti-proliferation effects of compound 18 against a variety of isogenic BaF3 cell linesa


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Cell lines

GI50: µM

Cell lines

GI50: µM

BaF3-TEL-FLT3 wt

0.016

BaF3-TEL-c-KIT

1.9

BaF3-FLT3-ITDD835Y

0.17

BaF3-TEL-MET

2.2

BaF3-FLT3-ITDF691L

0.22

BaF3-TEL-EGFR

1.8

BaF3-FLT3-D835Y

0.033

BaF3-TEL-JAK1

3.8

BaF3-FLT3-D835V

0.003

BaF3-TEL-JAK2

0.3

BaF3-FLT3-D835H

0.004

BaF3-TEL-JAK3

2.5

BaF3-TEL-BLK

0.94

BaF3-TEL-BMX

0.28

BaF3-TEL-LCK

0.31

BaF3-TEL-HCK

0.88

BaF3-TEL-RET

0.65

BaF3-TEL-PDGFRb

0.68

a


Table 5. Anti-proliferation effects of compound 18 against a variety of intact cancer cell lines a

a

Cell lines

GI50: µM

Cell lines

GI50: µM

MOLM13

0.021

U2932

1.6

MOLM14

0.042

JVM-2

2.6

U937

9.7

Namalwa

6.4

SU-DHL-2

5.5

NB4

3.8

All GI50s were obtained by triplet testing


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We then investigated the inhibitory effect of 18 on FLT3 mediated signaling pathway. It significantly affected FLT3 auto-phosphorylation at Tyr589/591 site in MV4-11 and MOLM14 cell line (EC50 less than 30 nM) as well as MOLM13 cell line (EC50 about 100 nM) (Figure 5). FLT3 kinase downstream mediator Stat5’s phosphorylation was also remarkably inhibited in these cell lines (EC50 less than 30 nM). In addition, the phosphorylation of ERK, AKT and expression of cMYC were also significantly inhibited, which exhibited a similar trend to the well-established FLT3 inhibitor AC220. Compound 18 could also arrest cell cycle progression of these cell lines into the G0/G1 phase and induction of apoptosis (Figure 6).

Figure 5. Compound 18 inhibited FLT3 kinase mediated signaling pathway in MV4-11, MOLM13 and MOLM14 cancer cell lines.


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Figure 6. Compound 18 arrested cell cycle progression and induction of apoptosis in MV4-11, MOML14 and MOLM13 cells. We also evaluated the compound 18’s PK properties in rats following intravenous and oral administration (Table 6). The half-life of the 18 was about 1.4 h with IV and 1.2 h with PO and the bioavailability was about 30%. Overall it had a good volume distribution but relatively quick clearance. Table 6. PK properties of compound 18. MRT Compd.

T1/2

Tmax

Cmax

AUC(0-t)

AUC(0-∞)

Vz

CLz

18

(hr)

(hr)

(ng/mL)

ng/mL*hr

Ng/mL*hr

mL/kg

mL/hr/kg

(0-∞)

F (%)

(hr) IV (1mg/kg) Mean

1.38

0.02

1503.83

606.89

619.33

3233.38

1616.20

1.15

NA

SD

0.31

0.00

121.81

20.78

23.28

779.55

62.03

0.12

NA


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PO (10 mg/kg) Mean

1.24

1.67

458.24

1834.84

1883.80

NA

NA

2.75

30.42

SD

0.24

0.58

103.77

360.99

378.29

NA

NA

0.32

NA

We next tested the antitumor efficacy of compound 18 in MV4-11cell inoculated xenograft mouse model. With oral gavage, the 12.5, 25 and 50 mg/Kg/day administration did not significantly affect the mice body weight (Figure 7A). However, the 25mg/kg/day and 50 mg/kg/day dosage could almost completely suppress the tumor progression (Figure 7 B, C, D). It is worth to mention that the PK profile obtained from rat may not be directly transformed into the mice due to the species difference.

Figure 7. Compound 18’s anti-tumor efficacy in MV4-11 xenograft model. Female nu/nu mice bearing established MV4-11 tumor xenografts were treated with 18 at 12.5, 25.0, 50.0 mg/kg/d, or vehicle. Daily oral administration was initiated when MV4-11 tumors had reached a size of 200 to 400 mm3. Each group contained 5 animals. Data, mean ± SEM. (A) Body weight and (B) tumor size measurements from MV4-11 xenograft mice after 18 administration. Initial body


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weight and tumor size were set as 100%. (C) Representative photographs of tumors in each group after 12.5, 25.0 or 50.0 mg/kg/d 18 or vehicle treatment. (D) Comparison of the final tumor weight in each group after 22-day treatment period. Numbers in columns indicate the mean tumor weight in each group. ns, p>0.05, *p

Discovery of (R)-1-(3-(4-Amino-3-(4-phenoxyphenyl)-1 H-pyrazolo [3

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