Highly selective MERTK inhibitors achieved by a single methyl group

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Highly selective MERTK inhibitors achieved by a single methyl group Jichen Zhao, Dehui Zhang, weihe zhang, Michael A. Stashko, Deborah DeRyckere, Eleana Vasileiadi, Rebecca E Parker, Debra Hunter, Qingyang Liu, Yuewei Zhang, Jacqueline L. Norris-Drouin, Bing Li, David Drewry, Dmitri B. Kireev, Douglas K. Graham, Henry Shelton Earp, Stephen V. Frye, and Xiaodong Wang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01229 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Journal of Medicinal Chemistry

Highly selective MERTK inhibitors achieved by a single methyl group Jichen Zhao,± Dehui Zhang,±, ₸ Weihe Zhang,± Michael A. Stashko,± Deborah DeRyckere,‖ Eleana Vasileiadi,‖ Rebecca E. Parker,‖ Debra Hunter,¶ Qingyang Liu,± Yuewei Zhang,± Jacqueline Norris-Drouin,± Bing Li,± David H. Drewry,₸ Dmitri Kireev,± Douglas K. Graham,‖₸ Henry Shelton Earp,§₸¶ Stephen V. Frye,±₸¶ Xiaodong Wang,±¶* ±Center

for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy ₸Meryx,

§ Department ‖Aflac

Inc., 450 West Dr., Chapel Hill, NC 27599, USA

of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

Cancer and Blood Disorders Center of Children’s Healthcare of Atlanta and Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA 30322, USA

¶Lineberger

Comprehensive Cancer Center, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

ABSTRACT. Although all kinases share the same ATP binding pocket, subtle differences in the residues that form the pocket differentiate individual kinases’ affinity for ATP competitive inhibitors. We have found that by introducing a single methyl group, the selectivity of our MERTK inhibitors over another target, FLT3, was increased up to 1000-fold (compound 31). Compound 19 was identified as an in vivo tool compound with subnanomolar activity against MERTK and 38-fold selectivity over FLT3 in vitro. The potency and selectivity of 19 for MERTK over FLT3

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was confirmed in cell-based assays using human cancer cell lines. Compound 19 had favorable pharmacokinetic properties in mice. Phosphorylation of MERTK was decreased by 75% in bone marrow leukemia cells from mice treated with 19 compared to vehicle-treated mice.

INTRODUCTION MERTK belongs to TAM (TYRO3, AXL and MERTK) family of tyrosine kinases and plays important roles in the innate immune system.1-3 TAM family kinases act as an anti-inflammatory, negative regulatory system to enable tolerance to self and thereby limit autoimmunity and damage to host tissues.4 However, TAM kinases in infiltrating myeloid cells in the tumor microenvironment can diminish or subvert an effective anti-tumoral immune response during cancer progression.5 The high levels of MERTK expression in tumor-infiltrating macrophages may increase efficiency at clearing apoptotic cancer cells, limiting the time during which antigens from those dying cells can trigger an innate immune response, and polarizing the tumor macrophage towards an M2-like, wound healing phenotype. Overexpression of one or more TAM receptors is often associated with cancer progression and metastasis as well as resistance to targeted therapies and commonly used cytotoxic chemotherapeutics.5-7 Conversely, genetic knockdown of MERTK in a spectrum of cancer cell types induces cancer cell death and synergizes with chemotherapeutic agents, suggesting a direct effect of MERTK inhibition on cancer cells.6, 8-10 In addition, a role for MERTK in the tumor microenvironment has been suggested by syngeneic mouse cancer studies in which tumors grew more slowly and were poorly metastatic in Mertk-/- mice background.5 Therefore, MERTK inhibition could also decrease macrophage-aided cell clearance, increase inflammatory cytokine release (IL-12) and stimulate anti-tumor immune responses, irrespective of whether the cancer cells express MERTK.6, 11-17


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Recently, durable tumor regression and prolonged survival was reported in a subset of patients with various solid tumors treated with anti-PD1 monoclonal antibodies.18-21 The success of these agents demonstrate the importance and effectiveness of cancer immunotherapy that targets T-cell checkpoint pathway. The regulatory role of TAM family kinases in the innate immune system portends the use of MERTK inhibitors in cancer immunotherapy. In 2014, we have reported UNC2025 (1) as a potent and highly orally bioavailable inhibitor of MERTK and FMSlike tyrosine kinase (FLT3) (Figure 1a).14, 22 The dual inhibitory activity of 1 may not be ideal for its use as a cancer immunotherapy drug due to hematopoietic suppression which has been associated with inhibition of FLT3.23-24 Thus, MERTK-selective inhibitors are needed.

Figure 1. a. MERTK/FLT3 dual inhibitor 1; b. the superposed ligand binding pockets of MERTK (magenta; PDB ID: 4M3Q) and FLT3 (cyan; PDB ID: 4XUF) and their differing residues; c. MERTK-selective inhibitor 2. The sequence identity and similarity between MERTK and FLT3 (53% identity and 68% similarity in their ligand binding pocket) suggest that inhibitor selectivity for one of these targets is achievable. However, since physical properties (size, shape and charge) of the four differing residue pairs in the ATP-binding site are very much alike between MERTK and FLT3 (see Figure 1b), attaining selectivity has been challenging.



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Our initial effort to develop MERTK-selective inhibitors led to generation of macrocyclic pyrimidines.25 However, because of its poor ADME properties, the lead compound in this series, UNC2541 (2), could only be used as an in vitro tool compound (Figure 1c). While optimization of the series is still ongoing, we have made significant progress on other chemical series, including pyrrolopyrimidines (compound 1). Here we report further modification of compound 1 to dramatically improve its selectivity against FLT3 while retaining potent MERTK activity and favorable pharmacokinetic (PK) properties. RESULTS AND DISCUSSION Based on information from our previous structure-activity relationship (SAR) study of compound 1, the trans-4-hydroxycyclohexyl group at the nitrogen of the pyrrole ring of 1 was important, forming a critical hydrogen bond with MERTK protein while avoiding the hERG liabilities of the corresponding primary amino-group.12,

14

Therefore, this functionality was

retained for further SAR studies. The synthesis of pyrrolopyrimidine analogues was straightforward (Scheme 1). Starting with the key intermediate, trans-4-(5-bromo-2-chloro-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (I), in path a, SNAr displacement of the 2-chloro substituent with an amine R1NH2 yielded intermediate II. Then, Suzuki coupling between II and boronic acid R2B(OH)2 or ester R2B(pin) provided final product IV. In path b, Suzuki coupling between I and boronic acid R2B(OH)2 or ester R2B(pin) followed by an SNAr displacement of the 2-chloro substituent in the newly formed intermediate III with an amine R1NH2 yielded the desired analogue IV. Path a was used to explore the SAR at the R2 position while path b was engaged for SAR exploration at the R1 position.


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Br N R1

R1NH2

path a

Br N Cl

N

N

I

OH

path b

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


N H

N

N

R2B(OH)2/R2B(pin) Pd(PPh3)4, K2CO3 THF, H2O, µW, 90oC

i-PrOH, µW II

N

OH

R1 R2

R2B(OH)2/R2B(pin) Pd(PPh3)4, K2CO3 THF, H2O, µW, 90oC

R2

N H

N

N

N Cl

R1NH2

N

N

i-PrOH, µW

III

IV

OH

OH

Scheme 1. Synthesis of pyrrolopyrimidines. Recognizing that there are four pairs of residues in the ATP-binding pocket that differ between MERTK and FLT3 (Figure 1b), we investigated the possibility of achieving selectivity over FLT3 by introducing steric demands or changing the electronic density at the R1 and/or R2 positions. A set of diversified R1 and R2 groups were chosen as shown in Table 1. The newly synthesized analogues were tested in our in-house microcapillary electrophoresis (MCE) MERTK, AXL, Tryo3, and FLT3 assays.25-26 Surprisingly, the addition of a methyl group at the α-position of the butyl side chain in analogue 3 provided over 30-fold selectivity for MERTK over FLT3. This change also increased the activity of 3 against TYRO3 (3-fold more active than 1) while decreasing the AXL inhibitory activity (9-fold less active than 1). When moving the methyl group to the β-position of the butyl side chain (4), the selectivity between MERTK and FLT3 was reduced to 7-fold. Unexpectedly, increasing the size of the group at the β-position of the butyl side chain from methyl to ethyl, the resulting analogue 5 became more selective against TYRO3 over MERTK (3-fold), AXL (31-fold), and FLT3 (30-fold). A cyclohexyl group at the R1 position (6) reduced the activity against the TAM family kinases and selectivity between MERTK and FLT3 (6-fold). The dramatic change in the selectivity profile driven by the steric effects at the R1 position



could be explained by the position of the R1 group within a constricted portion of the MERTK protein. Based on MERTK X-ray structures complexed with small molecules,11, 13, 27 the R1 group fits tightly in a hydrophobic pocket within the kinase; thus, any change in the shape of the R1 group could decrease affinity and induce a change in the inhibitory activity against target proteins. In particular, there is one key residue difference between MERTK and FLT3: Met730/Leu818 (MERTK/FLT3). This difference would be expected to make FLT3 more sensitive to the steric effect at the R1 position due to branching at Leu818 (FLT3) that is absent in Met730 (MERTK). In contrast to the differential SAR at the R1 position, changes explored at the R2 position were not fruitful. The addition of a cyclopropyl ring to the CH2 bridge at the R2 position (increasing the size of the R2 group) had no effect on either the activity or selectivity of the corresponding analogue 7 compared to 1. Similarly, removal of the CH2 bridge yielded a weaker analogue 8 compared to 1 with the same selectivity profile, although the shape of the R2 group was different. In addition, the replacement of the hydrophobic CH2 bridge in 1 with a hydrophilic NH group in 9 reduced the MERTK, AXL and FLT3 activity (over 3-fold) without affecting the selectivity for MERTK over FLT3. Changing the basic (4-methylpiperazin-1-yl)methyl group in 1 to a phenoxyl group in 10 introduced 3-fold selectivity for MERTK over FLT3; however, analogue 10 was over 100-fold less potent than 1. These observations could also be explained by the environment around the R2 group. The R2 group is located near the solvent front and has less interaction with the MERTK and FLT3 proteins and thus, TAM activity is less sensitive to changes in residue size or electronic density. Based on these data, the R1 position was targeted for further exploration. Table 1. SAR at the R1 and R2 positions.


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R2 N R1

N

N

OH

Compound

R1

R2

MERTK

AXL

TYRO3

FLT3

IC50 (nM)a

IC50 (nM)a

IC50 (nM)a

IC50 (nM)a

3b

N

N H

4b

3.5 ± 0.7

125 ± 96

6.7 ± 2.3

109 ± 64

5.2 ± 5.1

100 ± 71

12 ± 0.6

37 ± 25

19 ± 8.8

206 ± 55

6.7 ± 2.7

202 ± 172

13 ± 3.3

340 ± 26

266 ± 42

73 ± 55

1.3 ± 0.4

15 ± 8.5

27 ± 10

0.9 ± 0.4

3.5 ± 0.5

26 ± 7.3

22 ± 4.5

1.5 ± 0.4

5.2 ± 4.2

42 ± 25

34 ± 12

3.4 ± 2.2

N

N H

N N

5

N H

N N

6

N

N H

7

N

N H

N N

8 9

N H

N

N

N H

NH NH

10 a

N H

O

78 ± 9.8

1690 ± 110 1662 ± 1331

Values are the mean of two or more independent assays ± SD. b racemic mixture.


214 ± 48


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During our initial SAR studies, analogue 3 was prepared as a racemic mixture. Its (S)enantiomer, analogue 11 (Table 2), proved to be more active against MERTK (5-fold), AXL (5fold) and FLT3 (8-fold) than its (R)-enantiomer 12, while activity against TYRO3 was the same for both enantiomers. The same trend was observed for analogues 13 and 14 which had a CF3 group at the α-position of the butyl side chain; however, the difference in activity between 13 and 14 was larger: 13 was 55-fold more active against MERTK than 14 and had 220-fold selectivity over FLT3 while 14 only had 8-fold selectivity over FLT3. The decreased MERTK activity and the increased FLT3 selectivity of 13 compared to 11 may be due to the polarity or increased bulk of the CF3 group. In addition, adding one or two more carbons at the end of the butyl side chain at the R1 position was well tolerated; analogues 15 and 16 had similar activity and a better selectivity profile compared to 11. However, a polar hydroxyl group at the α-methyl group (17) reduced the inhibitory activity against TAM family kinases and FLT3 and increased FLT3 selectivity to over 300-fold. Table 2. SAR at the R1 position.

N

N

N R

1

N

N

OH

Compound

R1

R2

MERTK

AXL

TYRO3

FLT3

IC50 (nM)

IC50 (nM)

IC50 (nM)

IC50


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(nM) 11

N

N H

N

N H

CF3

N

N

N H

17

N H

N

560 ± 397

13 ± 4.4

543 ± 369

20 ± 1.8

2543 ±

99 ± 99

4372 ±

1595

3112

1068 ±

11037 ±

151 ±

8299 ±

144

320

113

3806

2.6 ± 0.5

64 ± 3.8

4.3 ± 1.5

162 ± 117

3.6 ± 2.2

81 ± 38

6.9 ± 4.7

544 ± 247

24 ± 5.6

3015 ± 945

143 ± 73

7513 ±

N

N N

HO N

N H

a

22 ± 0.4

N

N H

16

66 ± 27

N

CF3

15

12 ± 4.9

N

N H

14

118 ± 57

N

12

13

4.3 ± 2.8

N

4770

Values are the mean of two or more independent assays ± SD. Next, the SAR at the R2 position was explored with the smallest and most active (S)-pentan-

2-yl group at the R1 position to maximize the ligand efficiency of newly synthesized analogues. Although, changes at the R2 position wouldn’t affect the selectivity profile dramatically based on initial SAR (Table 1), it provides an opportunity to tune physiochemical and PK properties in the corresponding analogues. As shown in Table 3, replacement of the N-methyl piperazine group with

morpholine,

1-methyl-1,4-diazepane,

(R)-N,N-dimethylpyrrolidine,

or

(1S,4S)-7-

azabicyclo[2.2.1]heptane yielded analogues 18-21 with similar activity and selectivity as 11, independent of the ring size. When the CH2 linker between phenyl and N-methylpiperazine in 11



was changed to sulfonyl, the corresponding analogue 22 was more selective against FLT3 compared to 11 (54-fold vs 15-fold); while the NH linker in analogue 23 resulted in similar activity and selectivity as 11. Surprisingly, the replacement of 4-piperidine with a hydrophobic cyclohexyl ring yielded a 19-fold weaker analogue 24 compared to 23. Furthermore, directly attaching a 4tetrahydropyran ring to the phenyl ring at the para-position also reduced the activity of the corresponding analogue 25 (MERTK: 14-fold; AXL: 28-fold; TYRO3: 37-fold; FLT3: 16-fold) while the selectivity for MERTK over FLT3 was retained compared to 11. In addition, ring-opened analogue 26 had similar TAM inhibitory activity and selectivity over FLT3 as 11. Interestingly, meta-substitution of the phenyl ring increased the FLT3 selectivity to 64-fold for 27 and 95-fold for 28 while retaining their TAM kinase activity. The R2 position also tolerated substituted heteroaryls such as 4-pyridine (29), 3-pyridine (30), pyrimidine (31), and pyrazole (32) with better (29) or comparable (30‒32) activity against MERTK and better FLT3 selectivity (36, 114, 1061, 20-fold respectively) compared to 11. In particular, analogue 31 achieved over 1000-fold selectivity of MERTK over FLT3. This result could not be explained by any specific proteininhibitor interactions because the R2 group is mostly exposed to the solvent. Hence, the most plausible explanation of the R2 effect on MERTK/FLT3 selectivity was that different R2 groups could differentially transfer the momenta resulting from their interactions with the solvent to the rest of the ligand, which in turn could differentially increase/decrease the number of ‘bad’ contacts with M730/L818 in MERTK or FLT3 respectively. For example, a bicycloaryl 2-methyl benzo[d]oxazole group at the R2 position in 33 decreased TAM kinase activities (MERTK: 5-fold; AXL, 28-fold; TYRO3: 22-fold) compared to 11, but dramatically increased FLT3 selectivity (170-fold). Table 3. SAR at the R2 position.


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R2 N N H

N

N

OH

R2

Compound

MERTK

AXL

TYRO3

FLT3

IC50 (nM)

IC50 (nM)

IC50 (nM)

IC50 (nM)

18

N

3.2 ± 1.4

218 ± 54

10 ± 7.3

61 ± 30

2.4 ± 1.6

80 ± 45

9.1 ± 5.3

39 ± 22

4.1 ± 0.4

152 ± 28

11 ± 5.1

51 ± 27

2.0 ± 1.3

84 ± 16

3.4 ± 2.2

19 ± 2.4

3.5 ± 1.5

191 ± 62

33 ± 24

189 ± 104

4.9 ± 0.5

206 ± 47

26 ± 0.4

107 ± 48

O

19

N N

20

N

21 22

N

N

F

O S O N N

23

NH NH

24

NH

25 26

O

72 ± 41

7356 ± 3745 850 ± 230

1296 ± 891

61 ± 17

3291 ± 1734 445 ± 235

1026 ± 583

2.5 ± 1.4

HN

80 ± 24

4.4 ± 2.8

N


49 ± 26


27

N

28

N

N

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4.9 ± 2.0

213 ± 53

29 ± 4.9

316 ± 180

3.1 ± 4.2

230 ± 156

21 ± 21

296 ± 140

1.2 ± 0.9

79 ± 55

9.0 ± 6.5

43 ± 26

4.9 ± 4.2

416 ± 143

50 ± 27

558 ± 97

13 ± 8.2

1084 ± 396

150 ± 60

13790 ± 6003

2.2 ± 0.2

161 ± 76

24 ± 6.4

45 ± 2.7

22 ± 5.0

3295 ± 2091

268 ± 21

3729 ± 1576

N

29

NH N N

30

COOMe N

31 32 33 a

N N

N N NH

O N

Values are the mean of two or more independent assays ± SD. A few structurally diversified analogues with good in vitro activity against MERTK (IC50

< 5 nM) and selectivity over FLT3 (> 10-fold) were chosen for short PK studies in mice (n = 2 with 6 time points). Compounds were administered at a dose of 3 mg/kg via an intravenous (iv) route to evaluate stability in mice (Table 4). Analogue 19 had the lowest clearance (CL: 29 mL/min/kg), good half-life (T1/2: 3.0 h), and optimal volume of distribution (Vss: 3.3). Analogue 18 had medium CL and shorter T1/2 (1.4 h) while the CL of 21 was very high (302 mL/min/kg, 3.4-fold higher than normal mouse blood flow rate). Analogue 22 had moderate CL and small Vss (0.98 L/kg), thus a very short T1/2 (0.65 h). Both 26 and 29 had moderate CL while 32 had a large maximum concentration (Cmax) (13.8 µM) which might induce undesired toxicity. Based on these data, analogue 19 was chosen for further characterization in an extended PK study. Mice were treated with 19 at a dose of 3 mg/kg administered via oral (po) or intraperitoneal (ip) routes. Under these conditions, 19 had 15% oral bioavailability with a 61 nM po Cmax and 600 nM ip Cmax (25


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and 250-fold above the MERTK IC50 for 19, respectively) and a 1.43 h T1/2 via the ip route. Thus, 19 is an useful MERTK-selective in vivo tool compound. Table 4. In Vivo PK Parameters of selected analogues (n = 2 mice per time point). Compound

a iv c

Dose

T1/2

Cmax

AUClast

Vss

CL

(mg/kg)

(h)

(μM)

(h*μM)

(L/kg)

(mL/min/kg)

18a

3

1.4

8.6

2.2

1.4

41

19b,c

3

3.0

5.2

2.7

3.3

29

21a

3

1.8

0.10

0.28

37.2

302

22b

3

0.65

6.5

1.6

0.98

50

26a

3

3.65

1.7

1.2

16.4

54

29b

3

2.6

3.5

1.5

4.2

55

32b

3

2.1

13.8

2.8

1.6

32

formulation: 10% DMSO, 10% Tween 80 in normal saline; biv formulation: normal saline (0.9% NaCl);

full PK (n = 3).

Morrison Kis of 19 against TAM family kinases and FLT3 were also determined. Analogue 19 was ATP competitive and was very active against MERTK (Ki (n = 5): MERTK, 0.43 ± 0.12 nM: AXL, 40.22 ± 10.32 nM; TYRO3 3.87 ± 3.77 nM; FLT3 16.14 ± 3.84 nM) and had good selectivity for MERTK over AXL (94-fold), TYRO3 (9-fold), and FLT3 (38-fold). Furthermore, the overall kinome profile of 19 was assessed in duplicate versus 56 kinases at Carna Biosciences using an MCE assay similar to our in-house assay. These 56 kinases were the kinases that were inhibited more than 50% in response to treatment with 100 nM 1 when tested against the Carna full kinome panel,14 and thus should provide an adequate view of the selectivity of 19 considering the structural similarity between 1 and 19. A concentration of 100 nM was used to make a direct comparison with 1 and is >200-fold above the MERTK Ki. Analogue 19 proved to be more selective than 1 as only 24 kinases were inhibited by greater than 50% at 100 nM (data not



Page 14 of 44

included). As expected, MERTK was 100% inhibited. We obtained IC50s from Carna for 19 versus the top 10 kinases inhibited by 1 to further confirm the improved selectivity of 19. As shown in table 5, 19 was equally-potent against MERTK, TRKA, and TYRO3, and weaker on FLT3 (7fold), AXL (7-fold) and SLK (8-fold) compared to 1. The IC50s for QIK, NUAK1 and KIT were above 100 nM. The Carna FLT3 IC50 value was much lower than our in-house data (16-fold), possibly because we used a different FLT3 construct from ThermoFisher which had linear kinetics while the Carna FLT3 construct had a non-linear initial rate in our in-house FLT3 assay. Table 5. Carna IC50 of 19 on the Top 10 kinases inhibited by 1.

Kinase

FLT3

MERTK

AXL

TRKA

TRKC

QIK

TYRO3

SLK

NuaK1

KIT

IC50 (nM) for 1

0.35

0.46

1.65

1.67

4.38

5.75

5.83

6.14

7.97

8.18

IC50 (nM) for 19

2.50

0.60

12.1

3.60

8.40

>100

3.10

47.0

>100

>100

The activity and selectivity of 19 were further evaluated in cell-based assays using human cancer cell lines. The impact of treatment with 19 on phosphorylation of MERTK, FLT3, and AXL proteins was assessed in 697 B-cell acute lymphoblastic leukemia (B-ALL), SEM B-ALL, and A549 non-small cell lung cancer (NSCLC) cell lines, respectively. IC50 values were determined and revealed potent activity against MERTK (IC50 = 14 nM; Figure 2a,b) and selectivity for MERTK over both FLT3 (5-fold; Figure 2c,d) and AXL (61-fold; Figure 2e,f). Of note, the selectivity for MERTK over FLT3 appears reduced in cell-based assays (5-fold) compared to the enzymatic assays described above (16-fold). The cell-based studies were conducted in 3 different cell lines to allow robust and quantitative detection of all 3 proteins. However, comparison of IC50 values across cell lines introduces the possibility of cell line specific differences that impact the activity of the compound across all targets, such that differences between targets are artificially


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increased or decreased when activity in multiple cell lines is compared. Thus, while the cell-based assays clearly demonstrate greater activity mediated by 19 against MERTK compared to FLT3, additional experiments to directly compare the effects of 19 against MERTK and FLT3 in the same cell line would be required to establish a more robust quantitation of the degree of selectivity in cells. 19 was also active in vivo. Immunocompromised mice were inoculated with 697 human BALL cells by iv injection and mice with advanced leukemia were treated with a single 30 mg/kg ip dose of 19. Bone marrow cells were isolated 10 min later when 19 was expected to be at or near to reach maximum serum concentration based on the PK studies (Tmax = 5 min; T1/2 3.0 h). Phosphorylation of human MERTK protein was reduced by 75 +/- 8.7% in leukemia cells isolated from the bone marrow of mice treated with 19 relative to vehicle (normal saline)-treated mice (Figure 3). These studies validate 19 as a MERTK-selective tyrosine kinase inhibitor suitable for translational application.



Figure 2. Compound 19 selectively inhibits MERTK phosphorylation in cell-based assays. B-ALL (697 and SEM) and NSCLC (A549) cell lines were treated with 19 or vehicle for 1 h and then pervanadate phosphatase inhibitor was added to the cultures for an additional 3 min. MERTK (A,B), FLT3 (C,D) and AXL (E,F) proteins were immunoprecipitated from cell lysates and phosphorylated and total proteins were detected by immunoblot. IC50 values and 95% confidence intervals were determined by non-linear regression.


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Figure 3. Compound 19 inhibits MERTK phosphorylation in vivo. Immunocompromised NOD/SCID IL2Rgamma (NSG) mice were inoculated with 697 B-ALL cells by injection into the tail vein. Mice with advanced leukemia were treated with a single dose of 30 mg/kg 19 or an equivalent volume of vehicle (normal saline) were administered by ip injection. Bone marrow was collected 10 min post-treatment and then incubated with pervanadate phosphatase inhibitor for an additional 10 min. MERTK protein was immunoprecipitated from cell lysates and phosphorylated and total MERTK were detected by immunoblot. (A) Representative immunoblots are shown. (B) Proteins were quantitated by densitometry. Mean fractions of phosphorylated MERTK and standard errors derived from 7 mice over 2 independent experiments are shown (p95%) was determined by LC-MS (additional analytical HPLC determination for key compounds in Table 4). General procedure A (Scheme 2): N Br

Br

Cl

O

N

N NH2 HCl

N

N

N H

N

K2CO3, DIPEA, iPrOH, 120 oC, sealed tube, 4 d 34

OH

35

B O

N

OH

N

N N

N

Pd(PPh3)4, K2CO3, dioxane/H2O (4:1), microwave, 90 oC, 1h

N H

11

N

N

OH

Scheme 2. Synthetic route for general procedure A. To a solution of trans-4-(5-bromo-2-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan1-ol (34) (3.00 g, 9.07 mmol) and (S)-pentan-2-amine hydrochloride (2.24 g, 18.15 mmol) in iPrOH

(6.0 mL) was added potassium carbonate (3.76 g, 27.2 mmol) (powder) and N,N-

diisopropylethylamine (DIPEA) (4.75 mL, 27.2 mmol). The mixture was heated in a sealed tube under nitrogen atmosphere at 120 oC for 4 d, then quenched by water. The mixture was extracted with EtOAc (3X). The combined organic solution was washed with water and brine, dried (Na2SO4), and concentrated. The residue was purified by an ISCO silica gel column to afford the desired product 35 as a white foam (3.09 g, 89%). 1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1H), 7.12 (s, 1H), 4.51–4.39 (m, 1H), 4.1–4.02 (m, 1H), 3.71–3.60 (m, 1H), 2.12–1.83 (m, 6H), 1.68– 1.37 (m, 6H), 1.21 (d, J = 6.9, 3H), 0.94 (d, J = 9.0Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 160.3,



Page 20 of 44

153.3, 150.1, 123.3, 112.2, 88.9, 70.2, 54.3, 47.7, 40.2, 35.3, 35.3, 31.5, 31.3, 21.0, 20.5, 14.4. MS (ESI) m/z: calcd for C17H26BrN4O: 381.12 [M+H]+; found 381.11.; LC-MS: >95% purity. A solution of 35 (38.1 mg, 0.10 mmol) in dioxane (0.80 mL) and water (0.20 mL) was added

potassium

carbonate

(41.1

mg,

0.30

mmol)

(powder),

tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) (11.6 mg, 0.010 mmol), and 1-methyl-4-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)piperazine (38.0 mg, 0.12 mmol). The reaction mixture was heated under nitrogen atmosphere microwave irradiation at 90 oC for 1 h, then quenched by water. The combined organic solution was washed with water and brine, dried (Na2SO4), and concentrated. The residue was purified by HPLC (or reversed column ISCO) to afford the TFA salt of the title compound 11, which was converted to HCl salt by the treatment with a 4.0 N solution of HCl in dioxane and dried under lyophilization as a pale yellow solid (27 mg, 45%). 1H NMR (400 MHz, CD3OD) δ 8.84 (s, 1H), 7.97 (s, 1H), 7.85–7.70 (m, 4H), 4.64– 4.52 (m, 3H), 4.25–4.15 (m, 1H), 3.90–3.55 (m, 9H), 3.02 (s, 3H), 2.21–2.00 (m, 6H), 1.76–1.43 (m, 6H), 1.33 (d, J = 6.6 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 156.0, 151.9, 140.1, 135.4, 133.6, 128.6, 128.5, 128.4, 118.1, 111.2, 69.9, 60.8, 55.3, 51.3, 48.9, 43.3, 39.5, 35.1, 31.0, 30.8, 20.4, 20.4, 14.3. mp: 275.6-280.1 oC; MS (ESI) m/z: calcd for C29H43N6O: 491.35 [M+H]+; found 491.35.; LC-MS: >95% purity. General procedure B (Scheme 3): NH2 O

Br

B

N H

NH2

O

N N

N

OH

O

N

Pd(PPh3)4, K2CO3, dioxane/H2O, MW 90 oC

35

HN

N H

N

36

N

N

NaBH(OAc)3, HOAc, DCM OH


N H

N

N

24 OH

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Scheme 3. Synthetic route for general procedure B. A solution of 35 (1.14 g, 3.0 mmol) in 1,4-dioxane (24 mL) and water (6.0 mL) was added potassium carbonate (828 mg, 6.0 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (986 mg, 4.5 mmol), and Pd(PPh3)4 (347 mg, 0.30 mmol). The reaction mixture was heated under nitrogen atmosphere and microwave radiation at 90 oC for 1h, then quenched by water. The mixture was extracted with EtOAc (3X). The combined organic solution was washed with water and brine, dried (Na2SO4), and concentrated. The residue was purified by an ISCO silica gel column to afford the desired product 36 as a white foam (500 mg, 42%). 1H NMR (400 MHz, CD3OD) δ 8.58 (s, 1H), 7.33 (d, J = 8.3 Hz, 2H), 7.12 (s, 1H), 6.77 (d, J = 8.3 Hz, 2H), 4.50–4.37 (m, 1H), 4.12–4.02 (m, 1H), 3.70–3.60 (m, 1H), 2.12–1.83 (m, 6H), 1.66–1.33 (m, 6H), 1.21 (d, J = 6.5 Hz, 3H), 0.94 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 159.2, 154.2, 150.0, 147.3, 128.4, 125.6, 119.6, 117.7, 117.0, 111.3, 70.3, 53.8, 47.7, 40.3, 35.4, 35.4, 31.4, 31.3, 21.2, 20.5, 14.5. MS (ESI) m/z: calcd for C23H32N5O: 394.26 [M+H]+; found 394.25; LC-MS: >95% purity. To a solution of 36 (120 mg, 0.25 mmol) and cyclohexanone (25 mg, 0.25 mmol) in dichloromethane (1.0 mL) was added sodium triacetoxyborohydride (NaBH(OAc)3) (80 mg, 0.38 mmol) followed by one drop of acetic acid. The mixture was stirred at room temperature for 1.0 h and was quenched by acetone and methanol. The mixture was concentrated and purified by HPLC (or reversed column ISCO) to afford the trifluoroacetic acid (TFA) salt of the title compound 24 which was converted to HCl salt by the treatment with a 4.0 N solution of HCl in dioxane and dried under lyophilization as a light yellow solid (18.4 mg, 10%). 1H NMR (400 MHz, CD3OD) δ 8.79 (s, 1H), 7.94 (s, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 4.62–4.52 (m, 1H), 4.25–4.15 (m, 1H), 3.75–3.68 (m, 1H), 3.52–3.42 (m, 1H), 2.20–1.98 (m, 8H), 1.88 (d, J = 12.9 Hz, 2H), 1.77–1.18 (m, 15H), 1.00 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 155.9,



Page 22 of 44

151.9, 140.1, 135.0, 133.5, 129.2, 129.0, 125.8, 117.4, 111.0, 69.9, 63.2, 55.3, 48.8, 39.5, 35.1, 31.0, 30.8, 30.2, 26.0, 25.4, 20.5, 20.3, 14.3. MS (ESI) m/z: calcd for C29H42N5O: 476.34 [M+H]+; found 476.40; LC-MS: >95% purity. General procedure C (Scheme 4): N

N O

Br N Cl

N

34

N

N

N

B O

CF3

N

OH

N

Pd(PPh3)4, K2CO3, dioxane/water (4:1), microwave 90 oC, 0.5 h

NH2

N Cl

N

37

Pd(OAc)2, BINAP, Cs2CO3, THF, microwave 130 oC, 1 h

N

CF3 N N H

N

N

13 OH

OH

Scheme 4. Synthetic route for general procedure C. A solution of 34 (1.512 mmol, 500 mg) in 1,4-dioxane (10 mL) and water (2.5 mL) was added 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)piperazine (0.407 g, 1.286 mmol), potassium carbonate (0.418 g, 3.02 mmol), and Pd(PPh3)4 (0.151 mmol, 0.175 g). The reaction mixture was heated under nitrogen atmosphere microwave irradiation at 90 oC for 0.5 h. The mixture was concentrated and purified by an ISCO reversed column (methanol/water + 0.1% TFA, 10~100%) to afford a slight yellow oil as TFA salt, which was dissolved in iPrOH/dichloromethane

(1:4) and basified by washing with sodium hydroxide solution to yield 37

(270 mg, 41%) as light yellow foam. 1H NMR (400 MHz, CD3OD) δ 9.04 (s, 1H), 7.89 (s, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 4.73–4.66 (m, 1H), 3.76–3.68 (m, 1H), 3.56 (s, 2H), 2.80–2.30 (m, 8H), 2.28 (s, 3H), 2.18–1.96 (m, 6H), 1.64–1.50 (m, 2H). 13C NMR (100 MHz, CD3OD) δ 154.0, 153.2, 151.4, 137.2, 133.5, 131.5, 127.8, 126.1, 117.4, 117.3, 70.1, 63.4, 55.6, 54.6, 53.4, 45.9, 35.2, 31.6. MS (ESI) m/z: calcd for C24H31ClN5O: 440.22 [M+H]+; found 440.25; LC-MS: >95% purity.


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To a solution of 37 (85 mg, 0.19 mmol) and (R)-1,1,1-trifluoropentan-2-amine (0.290 mmol, 0.041 g) in THF (2 mL) was added palladium(II) acetate (9.66 µmol, 2.169 mg), BINAP (0.019 mmol, 0.012 g), and Cs2CO3 (0.290 mmol, 0.094 g). The reaction mixture was heated under nitrogen atmosphere and microwave radiation at 130 oC for 1.0 h, then quenched by water. The mixture was extracted with EtOAc (3X). The combined organic solution was washed with water and brine, dried (Na2SO4), and concentrated. The residue was purified by HPLC to afford the TFA salt of the title compound 13 which was converted to HCl salt by the treatment with a 4.0 N solution of HCl in dioxane and dried under lyophilization as a light yellow solid (45 mg, 36%).1H NMR (400 MHz, CD3OD) δ 8.89 (s, 1H), 8.01 (s, 1H), 7.85–7.60 (m, 4H), 5.06–4.98 (m, 1H), 4.67–4.58 (m, 1H), 4.32 (s, 2H), 3.78–3.66 (m, 3H), 3.67–3.60 (m, 3H), 3.59–3.53 (m, 2H), 2.97 (s, 3H), 2.17–2.09 (m, 2H), 2.10–1.97 (m, 4H), 1.95–1.88 (m, 1H), 1.86–1.78 (m, 1H), 1.68–1.39 (m, 5H), 1.01 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 155.2, 152.3, 140.8, 135.1, 133.6, 129.2, 128.9, 128.6, 126.8 (q, J = 280.0 Hz), 118.6, 112.4, 73.6, 72.4, 69.9, 62.2, 55.4, 53.6 (q, J = 30.3 Hz), 43.8, 35.0, 31.0, 31.0, 30.8, 19.4, 13.8. mp: 252.8-256.3 oC; MS (ESI) m/z: calcd for C29H40F3N6O: 545.32 [M+H]+; found 545.10; LC-MS: >95% purity. General procedure D (Scheme 5): CHO

Br

(HO)2B

N N H

N

35

N

OH

H N

CHO

Pd(PPh3)4, K2CO3, Dioxane/H2O, microwave 90 oC, 1h

N

N N H

N

38

O

N

NaBH(OAc)3, AcOH, DCM OH

Scheme 5. Synthetic route for general procedure D.


N N H

N

18

N

OH

O


A solution of 35 in water (2.0 mL) and 1,4-dioxane (8.0 mL) was added (4-formylphenyl) boronic acid (224 mg, 1.5 mmol), potassium carbonate (276 mg, 2.0 mmol), and Pd(PPh3)4 (116 mg, 0.10 mmol). The reaction mixture was heated under nitrogen atmosphere and microwave radiation at 90 oC for 0.5 h, then quenched by water. The mixture was extracted with EtOAc (3X). The combined organic solution was washed with water and brine, dried (Na2SO4), and concentrated. The residue was purified by an ISCO silica gel column (EtOAc/Hexane 0~100%) to afford the desired product 38 as a white foam (307 mg, 76%). 1H NMR (400 MHz, CD3OD) δ 9.90 (s, 1H), 8.75 (s, 1H), 7.92–7.82 (m, 2H), 7.78–7.70 (m, 2H), 7.58 (s, 1H), 4.56–4.45 (m, 1H), 4.12– 4.05 (m, 1H), 3.76–3.66 (m, 1H), 2.13–1.92 (m, 6H), 1.70–1.40 (m, 6H), 1.22 (d, J = 6.5 Hz, 3H), 0.96 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 193.4, 159.8, 155.0, 150.9, 142.4, 135.4, 131.5, 127.3, 123.4, 115.7, 110.5, 70.3, 54.2, 47.7, 40.26, 35.4, 31.5, 31.3, 21.1, 20.6, 14.5. MS (ESI) m/z: calcd for C24H31N4O2: 407.24 [M+H]+; found 407.25; LC-MS: >95% purity. To a solution of 38 (94 mg, 0.23 mmol) and morpholine (100 mg, 1.15 mmol) in dichloromethane (5.0 mL) was added NaBH(OAc)3 (244 mg, 1.15 mmol) followed by acetic acid (0.10 mL). The reaction mixture was stirred at room temperature for 1.0 h, quenched by acetone and methanol, and concentrated. The residue was purified by HPLC (or reversed column) to afford to title compound as TFA salt, which was converted to HCl salt with a 4.0 N solution of HCl in dioxane and lyophilized to afford 18 (85 mg, 67% yield) as a light yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.79 (s, 1H), 7.95 (s, 1H), 7.82–7.76 (m, 2H), 7.66 (d, J = 8.3 Hz, 2H), 4.63–4.55 (m, 1H), 4.41 (s, 2H), 4.24–4.17 (m, 1H), 4.09–4.02 (m, 2H), 3.86–3.65 (m, 3H), 3.43–3.36 (m, 2H), 3.27–3.19 (m, 2H), 2.20–2.01 (m, 6H), 1.76–1.44 (m, 6H), 1.33 (d, J = 6.6 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 156.0, 152.0, 140.1, 135.3, 133.5, 128.7, 128.6, 128.4, 118.1, 111.3, 69.9, 64.9, 61.5, 55.3, 52.8, 48.9, 39.5, 35.1, 31.0, 30.9, 20.5, 20.4, 14.3. mp:


Page 24 of 44

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234.1-237.7 oC; MS (ESI) m/z: calcd for C28H40N5O2: 478.32[M+H]+; found 478.40; HPLC: 97% purity. rac-trans-4-(5-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-2-(pentan-2-ylamino)-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (3) The title compound 3 (76.5 mg, 28%) was prepared according to general procedure A from 34 (148 mg, 0.45 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD); δ 8.80 (s, 1H), 7.94 (s, 1H), 7.70–7.65 (m, 4H), 4.64–4.55 (m, 1H), 4.44 (s, 2H), 4.24–4.16 (m, 1H), 3.77–3.68 (m, 3H), 3.69–3.61 (m, 3H), 3.61–3.53 (m, 2H), 3.00 (s, 3H), 2.19–2.11 (m, 2H), 2.11–1.97 (m, 4H), 1.76–1.61 (m, 2H), 1.61–1.40 (m, 5H), 1.33 (d, J = 6.6 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H). mp: 279.2-283.3 oC; MS (ESI) m/z: calcd for C29H43N6O: 491.34 [M+H]+; found 491.15; LC-MS: >95% purity. trans-4-(2-((2-Methylbutyl)amino)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (4) The title compound 4 (76.5 mg, 35%) was prepared according to general procedure A from 34 (120 mg, 0.36 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.80 (s, 1H), 7.93 (s, 1H), 7.76 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 8.2 Hz, 2H), 4.63–4.56 (m, 1H), 4.34 (s, 2H), 3.77–3.44 (m, 9H), 3.34–3.31 (m, 2H), 2.98 (s, 3H), 2.19–2.01 (m, 6H), 1.87–1.77 (m, 1H), 1.59–1.47 (m, 3H), 1.35–1.26 (m, 1H), 1.06-0.98 (m, 6H). mp: 267.3-271.4 oC; MS (ESI) m/z: calcd for C29H43N6O: 491.32 [M+H]+; found 491.15; LC-MS: >98% purity. trans-4-(2-((2-Ethylbutyl)amino)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-7H-pyrrolo [2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (5) The title compound 5 (22.0 mg, 54.3%) was prepared according to general procedure A from 34 (33.0 mg, 0.10 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.85 (s, 1H), 7.97 (s, 1H), 7.82–7.72 (m, 4H), 4.67–4.58 (m, 1H), 4.55 (s, 2H),



3.91–3.59 (m, 9H), 3.51 (d, J = 6.3 Hz, 2H), 3.02 (s, 3H), 2.19–2.01 (m, 6H), 1.70–1.60 (m, 1H), 1.59–1.40 (m, 6H), 1.01 (t, J = 7.4 Hz, 6H). mp: 280.6-285.1 oC; MS (ESI) m/z: calcd for C30H45N6O: 505.36 [M+H]+; found 505.40; LC-MS: >95% purity. trans-4-(2-(Cyclohexylamino)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-7H-pyrrolo [2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (6) The title compound 6 (78 mg, 42%) was prepared according to general procedure A from 34 (69 mg, 0.2 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.79 (s, 1H), 7.92 (s, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.2 Hz, 2H), 4.60–4.50 (m, 1H), 4.36 (s, 2H), 4.00–3.88 (m, 1H), 3.76–3.41 (m, 9H), 2.98 (s, 3H), 2.20–2.00 (m, 7H), 1.90–1.78 (m, 2H), 1.75–1.69 (m, 1H), 1.59–1.26 (m, 8H). mp: >300 oC; MS (ESI) m/z: calcd for C30H43N6O: 503.34 [M+H]+; found 503.40; LC-MS: >95% purity. trans-4-(2-(Butylamino)-5-(4-(1-(4-methylpiperazin-1-yl)cyclopropyl)phenyl)-7H-pyrrolo [2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (7) The title compound 7 (42 mg, 34%) was prepared according to general procedure A from 34 (66 mg, 0.20 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.77 (s, 1H), 7.88 (s, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 4.64–4.54 (m, 1H), 3.76–3.63 (m, 3H), 3.60–3.44 (m, 6H), 3.28–3.22 (m, 1H), 2.88–2.78 (m, 4H), 2.20–2.02 (m, 6H), 1.75–1.68 (m, 2H), 1.59–1.45 (m, 4H), 1.37–1.29 (m, 2H), 1.10–0.98 (m, 5H). mp: 219.9-223.5 oC; MS (ESI) m/z: calcd for C30H43N6O: 503.35[M+H]+; found 503.32; LCMS: >95% purity. trans-4-(2-(Butylamino)-5-(4-(4-methylpiperazin-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)cyclohexan-1-ol (8) The title compound 8 (24.5 mg, 17 %) was prepared according to general procedure A from 34 (79 mg, 0.25 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.69 (s, 1H), 7.73 (s, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 4.63–4.53 (m, 1H), 3.96–


Page 26 of 44

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3.87 (m, 2H), 3.76–3.69 (m, 1H), 3.67–3.60 (m, 2H), 3.54 (t, J = 7.1 Hz, 2H), 3.35–3.25 (m, 2H), 3.15–3.06 (m, 2H), 2.98 (s, 3H), 2.17–2.01 (m, 6H), 1.76–1.66 (m, 2H), 1.60–1.41 (m, 4H), 1.02 (t, J = 7.4 Hz, 3H). MS (ESI) m/z: calcd for C27H39N6O: 463.32 [M+H]+; found 463.30; LCMS: >95% purity. trans-4-(2-(Butylamino)-5-(4-(piperidin-4-ylamino)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)cyclohexan-1-ol (9) The title compound 9 (35.6 mg, 25%) was prepared according to general procedure B from 35 (95 mg, 0.25 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.67 (s, 1H), 7.69 (s, 1H), 7.53 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 8.1 Hz, 2H), 4.63–4.51 (m, 1H), 3.78– 3.69 (m, 2H), 3.56–3.44 (m, 4H), 3.19–3.10 (m, 2H), 2.32–2.22 (m, 2H), 2.17–2.00 (m, 6H), 1.84– 1.65 (m, 4H), 1.58–1.41 (m, 4H), 1.02 (t, J = 7.4 Hz, 3H). MS (ESI) MS (ESI) m/z: calcd for C27H39N6O: 463.32 [M+H]+; found 463.35; LC-MS: >95% purity. trans-4-(2-(Butylamino)-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan1-ol (10) The title compound 10 (40.5 mg, 29 %) was prepared according to general procedure A from 34 (79 mg, 0.25 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.71 (s, 1H), 7.76 (s, 1H), 7.65–7.59 (m, 2H), 7.41–7.34 (m, 2H), 7.14 (t, J = 7.4 Hz, 1H), 7.11–7.05 (m, 2H), 7.05– 7.00 (m, 2H), 4.66–4.54 (m, 1H), 3.76–3.67 (m, 1H), 3.54 (t, J = 7.1 Hz, 2H), 2.18–2.02 (m, 6H), 1.75–1.67 (m, 2H), 1.58–1.43 (m, 4H), 1.08–0.98 (m, 3H). MS (ESI) m/z: calcd for C28H33N4O2: 457.26 [M+H]+; found 457.30; LC-MS: >95% purity. trans-4-(5-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-2-(((S)-pentan-2-yl)amino)-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (11) The preparation of title compound 11 was general procedure A.



trans-4-(5-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-2-(((R)-pentan-2-yl)amino)-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (12) The title compound 12 (21 mg, 40%) was prepared according to general procedure A from 34 (29 mg, 0.088 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD); δ 8.74 (d, J = 5.6 Hz, 1H), 7.85 (d, J = 7.3 Hz, 1H), 7.68 (dd, J = 8.3, 2.4 Hz, 2H), 7.52 (dd, J = 8.3, 2.5 Hz, 2H), 4.54–4.48 (m, 1H), 4.23–4.14 (m, 1H), 4.00 (s, 2H), 3.76–3.64 (m, 1H), 3.50–3.35 (m, 4H), 3.25–3.00 (m, 4H), 2.90 (s, 3H), 2.33–2.02 (m, 6H), 1.86– 1.42 (m, 6H), 1.31 (d, J = 6.5 Hz, 3H), 0.99 (t, J = 7.3 Hz, 3H). mp: 266.1-270.2 oC; MS (ESI) m/z: calcd for C29H43N6O: 491.35; found 491.15 [M+H]+; LC-MS: >95% purity. trans-4-(5-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-2-(((R)-1,1,1-trifluoropentan-2yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (13) The preparation of title compound 13 was general procedure C. trans-4-(5-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-2-(((S)-1,1,1-trifluoropentan-2yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (14) The title compound 14 (50 mg, 40%) was prepared according to general procedure C from 37 (85 mg, 0.19 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.90 (s, 1H), 8.03 (s, 1H), 7.83–7.62 (m, 4H), 5.07–4.96 (m, 1H), 4.68–4.60 (m, 1H), 4.47–4.37 (m, 2H), 3.79–3.67 (m, 3H), 3.68–3.61 (m, 3H), 3.59–3.53 (m, 2H), 2.99 (s, 3H), 2.18–2.09 (m, 2H), 2.10–1.98 (m, 4H), 1.95–1.88 (m, 1H), 1.87–1.77 (m, 1H), 1.67–1.39 (m, 5H), 1.01 (t, J = 7.4 Hz, 3H). mp: 269.1-273.2 oC; MS (ESI) m/z: calcd for C29H40F3N6O: 545.32 [M+H]+; found 545.10; LC-MS: >95% purity. rac-trans-4-(2-(((S)-Hexan-2-yl)amino)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (15) The title compound 15 (63 mg, 36%) was prepared according to general procedure A from 34 (100 mg, 0.285 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.82 (s, 1H), 7.95 (s, 1H), 7.85–7.69 (m, 4H), 4.64–4.54 (m, 1H),


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4.52 (s, 2H), 4.23–4.13 (m, 1H), 3.80–3.54 (m, 9H), 3.01 (s, 3H), 2.21–2.10 (m, 2H), 2.10–2.01 (m, 4H), 1.77–1.69 (m, 1H), 1.67–1.60 (m, 1H), 1.58–1.48 (m, 2H), 1.48–1.37 (m, 4H), 1.37–1.28 (m, 3H), 0.95 (t, J = 7.0 Hz, 3H). mp: 283.2-285.6 oC; MS (ESI) m/z: calcd for (C30H45N6O+): 505.36 [M+H]+; found 505.25; LC-MS: >95% purity. rac-trans-4-(2-(((S)-Heptan-2-yl)amino)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (16) The title compound 16 (9.4 mg, 15%) was prepared according to general procedure A from 34 (33 mg, 0.1 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD); δ 8.74 (s, 1H), 7.85 (s, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 4.63–4.54 (m, 1H), 4.22–4.14 (m, 1H), 3.93 (s, 2H), 3.75–3.69 (m, 1H), 3.44–3.33 (m, 4H), 3.12–2.94 (m, 4H), 2.90 (s, 3H), 2.19–2.10 (m, 3H), 2.10–2.01 (m, 3H), 1.76–1.68 (m, 1H), 1.68– 1.59 (m, 1H), 1.59–1.49 (m, 2H), 1.49–1.41 (m, 2H), 1.41–1.27 (m, 7H), 0.92 (t, J = 7.0 Hz, 3H); MS (ESI) m/z: calcd for C31H47N6O: 519.38 [M+H]+; found 519.20; LC-MS: >95% purity. trans-4-(2-((1-Hydroxyhexan-2-yl)amino)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-7H -pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (17) The title compound 17 (29 mg, 46%) was prepared according to general procedure A from 34 (33 mg, 0.1 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.79 (s, 1H), 7.87 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 4.65–4.54 (m, 1H), 4.27–4.17 (m, 1H), 4.12 (s, 2H), 3.78–3.63 (m, 3H), 3.54–3.40 (m, 4H), 3.28– 3.16 (m, 4H), 2.93 (s, 3H), 2.18–2.10 (m, 2H), 2.10–1.96 (m, 4H), 1.78–1.62 (m, 2H), 1.60–1.49 (m, 2H), 1.49–1.37 (m, 4H), 0.94 (t, J = 6.9 Hz, 3H); MS (ESI) m/z: calcd for C30H45N6O2: 521.36[M+H]+; found 521.20; LC-MS: >95% purity. trans-4-(5-(4-(Morpholinomethyl)phenyl)-2-(((S)-pentan-2-yl)amino)-7H-pyrrolo[2,3d]pyrimidin-7-yl)cyclohexan-1-ol (18) The preparation of title compound 18 was general procedure D.



trans-4-(5-(4-((4-Methyl-1,4-diazepan-1-yl)methyl)phenyl)-2-(((S)-pentan-2-yl)amino)-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (19) The title compound 19 (4.10 g, 56%) was prepared according to general procedure D from 38 (6.82 g, 13.1 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.85 (s, 1H), 7.97 (s, 1H), 7.83–7.74 (m, 4H), 4.66–4.51 (m, 3H), 4.28–4.15 (m, 1H), 4.06–3.81 (m, 4H), 3.79–3.40 (m, 5H), 3.00 (s, 3H), 2.40 (s, 2H), 2.21–2.03 (m, 6H), 1.80–1.43 (m, 6H), 1.34 (d, J = 6.6 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 156.0, 151.9, 140.1, 135.3, 133.4, 129.4, 128.6, 128.5, 118.1, 111.3, 69.9, 61.6, 56.5, 55.3, 53.9, 48.9, 44.6, 39.5, 35.1, 31.0, 30.8, 20.5, 20.4, 14.3. mp: 231.2-234.1 oC; MS (ESI) m/z: calcd for (C30H45N6O+): 505.36 [M+H]+; found 505.40; HPLC: 96% purity. trans-4-(5-(4-(((S)-3-(Dimethylamino)pyrrolidin-1-yl)methyl)phenyl)-2-(((S)-pentan-2yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (20) The title compound 20 (81 mg, 57%) was prepared according to general procedure D from 38 (94 mg, 0.23 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.81 (s, 1H), 7.94 (s, 1H), 7.81–7.70 (m, 4H), 4.65–4.55 (m, 3H), 4.31–4.17 (m, 2H), 3.95–3.48 (m, 5H), 2.97 (s, 6H), 2.70–2.60 (m, 1H), 2.55–2.40 (m, 1H), 2.20–2.01 (m, 6H), 1.76–1.43 (m, 6H), 1.32 (t, J = 8.2 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H). mp: 195.0199.5 oC; MS (ESI) m/z: calcd for C30H45N6O: 505.36 [M+H]+; found 505.40; LC-MS: >95% purity. trans-4-(5-(4-((7-Azabicyclo[2.2.1]heptan-7-yl)methyl)phenyl)-2-(((S)-pentan-2-yl)amino)7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (21) (Scheme 6)


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O

HO

H

N 1. MsCl, DIPEA, DCM

NaBH4, MeOH N

N N

N H

N

N

N H

39

2.

N

N H

N

N

DIPEA ,iPrOH

40 OH

N

H N

OH

21

OH

Scheme 6. Synthesis of analogue 21. A solution of 39 (406 mg, 1.0 mmol) in methanol (10 mL) was added sodium borohydride (76 mg, 2.0 mmol) potion-wisely. The reaction mixture stirred at room temperature for 30 min, then quenched with water. The mixture was extracted with EtOAc (3X). The combined organic solution was washed with water and brine, dried (Na2SO4), and concentrated to provide 40 which used without further purification. A solution of 40 (139 mg, 0.34 mmol) in dichloromethane (6.0 mL) was added DIPEA (0.20 mL, 1.10 mmol) and MsCl (40 µL, 0.51 mmol) at 0 oC. The reaction mixture was stirred at 0 oC for 1 h and quenched with water. The mixture was extracted with EtOAc (3X). The combined organic solution was washed with water and brine, dried (Na2SO4), and concentrated to provide the crude material. A solution of this material in isopropyl alcohol (1.5 mL) was added 7-azabicyclo[2.2.1]heptane (98 mg, 1.0 mmol) and DIPEA (0.2 mL, 1.1 mmol). The reaction mixture was heated at 110 oC in sealed tube overnight and quenched with water. The mixture was extracted with EtOAc (3X). The combined organic solution was washed with water and brine, dried (Na2SO4), and concentrated. The residue was purified by HPLC to afford the TFA salt of the title compound 21 (21 mg, 11%). 1H NMR (400 MHz, CD3OD) δ 8.79 (s, 1H), 7.91 (s, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.2 Hz, 2H), 4.62–4.55 (m, 1H), 4.30 (s, 2H), 4.23–4.17 (m, 1H), 4.07–4.03 (m, 2H), 3.77–3.69 (m, 1H), 2.42–2.34 (m, 2H), 2.20–2.01 (m, 7H), 1.96–1.89 (m, 2H), 1.87–1.79 (m, 2H), 1.76–1.43 (m, 7H), 1.33 (d, J = 6.6 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H).

13C

NMR (100 MHz, CD3OD) δ 155.9, 152.1, 140.3, 134.9, 132.4, 131.3,



128.4, 128.3, 118.2, 111.3, 70.0, 64.8, 55.3, 50.7, 48.8, 39.6, 35.1, 31.0, 30.9, 28.4, 26.4, 20.5, 20.4, 14.3. mp: 225.9-229.5 oC; MS (ESI) m/z: calcd for C30H42N5O: 488.34 [M+H]+; found 488.35; HPLC: 96% purity. trans-4-(5-(2-Fluoro-4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-2-(((S)-pentan-2-yl)amino) -7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (22) The title compound 22 (14 mg, 10%) was prepared according to general procedure A from 34 (66 mg, 0.20 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.74 (d, J = 1.4 Hz, 1H), 8.02 (s, 1H), 7.97 (dd, J = 8.2, 7.4 Hz, 1H), 7.78–7.72 (m, 2H), 4.66–4.56 (m, 1H), 4.26–4.14 (m, 1H), 4.06–3.92 (m, 2H), 3.77–3.67 (m, 1H), 3.65–3.45 (m, 2H), 3.29–3.17 (m, 2H), 2.90 (s, 3H), 2.95–2.75 (m, 2H), 2.18–1.99 (m, 6H), 1.76– 1.41 (m, 6H), 1.33 (d, J = 6.6 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H). mp: 199.2-203.3 oC; MS (ESI) m/z: calcd for C28H40FN6O3S: 559.29 [M+H]+; found 559.30; HPLC: 96% purity. trans-4-(2-(((S)-Pentan-2-yl)amino)-5-(4-(piperidin-4-ylamino)phenyl)-7H-pyrrolo[2,3d]pyrimidin-7-yl)cyclohexan-1-ol (23) The title compound 23 (33 mg, 35 %) was prepared according to general procedure B from 35 (61 mg, 0.16 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.72 (s, 1H), 7.77 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.6 Hz, 2H), 4.604.53 (m, 1H), 4.23–4.15 (m, 1H), 3.87–3.78 (m, 1H), 3.76–3.68 (m, 1H), 3.55–3.45 (m, 2H), 3.21– 3.09 (m, 2H), 2.31–2.21 (m, 2H), 2.17–1.98 (m, 5H), 1.95–1.82 (m, 2H), 1.75–1.41 (m, 7H), 1.32 (d, J = 6.6 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H). MS (ESI) m/z: calcd for C28H41N6O: 477.33[M+H]+; found 477.35; LC-MS: >95% purity. trans-4-(5-(4-(Cyclohexylamino)phenyl)-2-(((S)-pentan-2-yl)amino)-7H-pyrrolo[2,3d]pyrimidin-7-yl)cyclohexan-1-ol (24) The preparation of title compound 24 was general procedure B.


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trans-4-(2-(((S)-Pentan-2-yl)amino)-5-(4-(tetrahydro-2H-pyran-4-yl)phenyl)-7H-pyrrolo [2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (25) The title compound 25 (44 mg, 48%) was prepared according to general procedure B from 35 (70 mg, 0.18 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.69 (s, 1H), 7.76 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.35 (t, J = 8.0 Hz, 2H), 4.61– 4.52 (m, 1H), 4.22–4.12 (m, 1H), 4.06–4.00 (m, 2H), 3.74–3.66 (m, 1H), 3.61–3.50 (m, 2H), 2.88– 2.77 (m, 1H), 2.16–1.98 (m, 6H), 2.85–1.72 (m, 4H), 1.72–1.40 (m, 6H), 1.31 (d, J = 8.0 Hz, 3H), 0.99 (t, J = 8.0 Hz, 3H). mp: 158.5-162.7 oC; MS (ESI) m/z: calcd for C28H39N4O2: 463.31[M+H]+; found 463.30; LC-MS: >95% purity. trans-4-(5-(4-(((2-(Diethylamino)ethyl)amino)methyl)phenyl)-2-(((S)-pentan-2-yl)amino)7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (26) The title compound 26 (66 mg, 29%) was prepared according to general procedure D from 37 (105 mg, 0.37 mmol) as a yellow solid. 1H

NMR (400 MHz, CD3OD) δ 8.82 (s, 1H), 7.94 (s, 1H), 7.80–7.70 (m, 4H), 4.64–4.53 (m, 1H),

4.37 (s, 2H), 4.25–4.16 (m, 1H), 3.77–3.69 (m, 1H), 3.67–3.59 (m, 4H), 3.38–3.32 (m, 4H), 2.20– 2.01 (m, 6H), 1.77–1.65 (m, 2H), 1.58–1.45 (m, 4H), 1.40 (t, J = 7.2 Hz, 6H), 1.34 (d, J = 6.5 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H). mp: 182.0-185.6 oC; MS (ESI) m/z: calcd for C30H47N6O: 507.38 [M+H]+; found 507.40; HPLC: 96% purity. trans-4-(5-(3-((4-Methylpiperazin-1-yl)methyl)phenyl)-2-(((S)-pentan-2-yl)amino)-7Hpyrrolo[2,3-d]pyrimidin-7-yl)cyclohexan-1-ol (27) The title compound 27 (36 mg, 24%) was prepared according to general procedure A from 34 (82.5 mg, 0.25 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 9.05 (s, 1H), 7.96–7.91 (m, 2H), 7.74 (d, J = 8.0 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 4.63–4.55 (m, 1H), 4.34–4.16 (d, J = 32.1 Hz, 3H), 3.80– 3.37 (m, 9H), 2.97 (s, 3H), 2.21–1.94 (m, 6H), 1.75–1.43 (m, 6H), 1.33 (d, J = 6.6 Hz, 3H), 1.00



(t, J = 7.3 Hz, 3H). MS (ESI) m/z: calcd for C29H43N6O: 491.35 [M+H]+; found 491.40; LC-MS: >95% purity. trans-4-(5-(3-(4-Methylpiperazin-1-yl)phenyl)-2-(((S)-pentan-2-yl)amino)-7H-pyrrolo[2,3d]pyrimidin-7-yl)cyclohexan-1-ol (28) The title compound 28 (34 mg, 36%) was prepared according to general procedure A from 34 (53 mg, 0.16 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.73 (s, 1H), 7.84 (s, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.25 (s, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.02 (dd, J = 8.2, 1.9 Hz, 1H), 4.61–4.53 (m, 1H), 4.24–4.15 (m, 1H), 3.99–3.90 (m, 2H), 3.74–3.66 (m, 1H), 3.65–3.57 (m, 2H), 3.35–3.25 (m, 2H), 3.19–3.06 (m, 2H), 2.97 (s, 3H), 2.19– 1.97 (m, 6H), 1.73–1.40 (m, 6H), 1.32 (d, J = 8.0 Hz, 3H), 0.99 (t, J = 8.0 Hz, 3H). mp: 193.3196.9 oC; MS (ESI) m/z: calcd for C28H41N6O: 477.33 [M+H]+; found 477.30; LC-MS: >95% purity. trans-4-(2-(((S)-Pentan-2-yl)amino)-5-(2-(piperazin-1-yl)pyridin-4-yl)-7H-pyrrolo[2,3d]pyrimidin-7-yl)cyclohexan-1-ol (29) The title compound 29 (38 mg, 41%) was prepared according to general procedure A from 34 (53 mg, 0.16 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 9.13 (s, 1H), 8.60 (s, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 4.68–4.55 (m, 1H), 4.28–4.16 (m, 1H), 4.16–4.04 (m, 4H), 3.77–3.65 (m, 1H), 3.58–3.39 (m, 4H), 2.22–1.92 (m, 6H), 1.76–1.41 (m, 6H), 1.33 (d, J = 8.0 Hz, 3H), 0.99 (t, J = 8.0 Hz, 3H). mp: 242.1-246.5 oC; MS (ESI) m/z: calcd for C26H38N7O: 463.31[M+H]+; found 463.30; HPLC: 98% purity. Methyl

5-(7-(trans-4-hydroxycyclohexyl)-2-(((S)-pentan-2-yl)amino)-7H-pyrrolo[2,3-

d]pyrimidin-5-yl)picolinate (30) The title compound 30 (20 mg, 9%) was prepared according to general procedure A from 34 (142 mg, 0.43 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.92 (s, 1H), 8.81 (s, 1H), 8.29-8.21 (m, 1H), 8.20–8.15 (m, 1H), 8.12 (s, 1H), 4.57–4.47 (m,


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1H), 4.19–4.05 (m, 1H), 3.92 (s, 3H), 3.59–3.53 (m, 1H), 2.12–1.90 (m, 6H), 1.69–1.33 (m, 6H), 1.25 (d, J = 6.5 Hz, 3H), 0.91 (q, J = 7.3 Hz, 3H). mp: 170.8-175.2 oC; MS (ESI) m/z: calcd for (C24H32N5O3+): 438.25 [M+H]+; found 438.20; LC-MS: >95% purity. trans-4-(5-(2-Cyclopropylpyrimidin-5-yl)-2-(((S)-pentan-2-yl)amino)-7H-pyrrolo[2,3d]pyrimidin-7-yl)cyclohexan-1-ol (31) The title compound 31 (65 mg, 30 %) was prepared according to general procedure A from 34 (142 mg, 0.43 mmol) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.92 (s, 2H), 8.76 (s, 1H), 7.99 (s, 1H), 4.57–4.43 (m, 1H), 4.19–4.01 (m, 1H), 3.70–3.53 (m, 1H), 2.26–2.18 (m, 1H), 2.12–1.87 (m, 6H), 1.68–1.32 (m, 6H), 1.24 (d, J = 6.5 Hz, 3H), 1.16–1.09 (m, 4H), 0.91 (t, J = 7.3 Hz, 3H). mp: 85.7-89.3 oC; MS (ESI) m/z: calcd for C24H33N6O: calcd. m/z 421.27 [M+H]+; found 421.30; LC-MS: >95% purity. trans-4-(2-(((S)-Pentan-2-yl)amino)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3d]pyrimidin-7-yl)cyclohexan-1-ol (32) The Boc protected compound was prepared according to general procedure A from coupling of 34 (990 mg, 3.0 mmol) and tert-butyl 4-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate.

The

Boc

protected product was added a 3.0 M solution of HCl in MeOH (5.0 mL). The resulting solution was stirred at room temperature for 1.0 h. The solvent was removed under reduced pressure to provide the title compound 32 (44 mg, 3%). 1H NMR (400 MHz, CD3OD) δ 8.77 (s, 1H), 8.20 (s, 1H), 7.89 (d, J = 4.0 Hz, 1H), 7.71 (s, 1H), 4.65–4.47 (m, 2H), 4.24–4.12 (m, 1H), 3.73–3.64 (m, 1H), 3.61–3.51 (m, 2H), 3.27–3.19 (m, 2H), 2.37–2.26 (m, 4H), 2.16–2.06 (m, 2H), 2.06–1.91 (m, 4H), 1.70–1.41 (m, 6H), 1.31 (d, J = 4.0 Hz, 3H), 0.98 (t, J = 8.0 Hz, 3H). mp: 152.3-155.2 oC; MS (ESI) m/z: calcd for C25H38N7O: 452.31 [M+H]+; found 452.30; HPLC: 97% purity. trans-4-(5-(2-Methylbenzo[d]oxazol-5-yl)-2-(((S)-pentan-2-yl)amino)-7H-pyrrolo[2,3d]pyrimidin-7-yl)cyclohexan-1-ol (33) The title compound 33 (76 mg, 35%) was prepared



according to general procedure A from 34 (142 mg, 0.43 mmol) as a light brown solid. 1H NMR (400 MHz, CD3OD) δ 8.60 (s, 1H), 7.73–7.69 (m, 1H), 7.53–7.49 (m, 2H), 7.31 (s, 1H), 4.51–4.36 (m, 1H), 4.08–3.95 (m, 1H), 3.77–3.55 (m, 1H), 2.56 (s, 3H), 2.09–1.87 (m, 6H), 1.57–1.32 (m, 6H), 1.15 (d, J = 6.5 Hz, 3H), 0.88 (t, J = 7.2 Hz, 3H). MS (ESI) m/z: calcd for (C25H32N5O2+): 434.26 [M+H]+; found 434.30; LC-MS: >95% purity. Cell Based Kinase Inhibition Assays 697 B-ALL, SEM B-ALL, and A549 NSCLC cells were cultured in the presence of 19 or vehicle only for 1.0 h. Pervanadate solution was prepared fresh by combining 20 mM sodium orthovanadate in 0.9x PBS in a 1:1 ratio with 0.3% (w/w) hydrogen peroxide in PBS for 15-20 min at room temperature. Cultures were treated with 120 M pervanadate for 3 min prior to collection and cell lysates were prepared in 50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM EDTA, 10% glycerol, and 1% Triton X-100, supplemented with protease inhibitors (Roche Molecular Biochemicals, #11836153001). MERTK, FLT3, and AXL proteins were immunoprecipitated with anti-MERTK (R&D Systems, #MAB8912), anti-FLT3 (Cell Signaling, #3462), or anti-AXL (R&D Systems, AF154) antibodies and Protein G agarose beads (InVitrogen). Phosphorylated proteins were detected by western blot using anti-phospho-MERTK antibody raised against a peptide derived from the tri-phosphorylated activation loop of MERTK8 (Phopshosolutions, Inc), antibody specific for phosphorylated FLT3 (Cell Signaling Technology, #3461), or antiphosphotyrosine antibody (Millipore, 4G10). Nitrocellulose membranes were stripped and total proteins were detected using anti-MERTK antibody (Abcam, ab52968), anti-FLT3 antibody (Cell Signaling, #3462) or anti-AXL antibody (R&D Systems, AF154). Relative phosphorylated and total protein levels were determined by densitometry using Image J software and IC50 values and


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95% confidence intervals were calculated by non-linear regression using Prism v7 software (Graphpad Software Inc.). Pharmacodynamic Assay All research involving animals was conducted in accordance with a protocol approved by the Emory University Institutional Animal Care and Use Committee. Mice were bred in house. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (23-35 weeks old) were transplanted with 2 x 106 697 B-ALL cells by intravenous injection into the tail vein and leukemia was established for 17 or 20 days prior to treatment with a single 30 mg/kg dose of 19 (n=7, 2 females and 4 males) or an equivalent volume (10 mL/kg) of saline vehicle (n=7, 2 females and 4 males). Pervanadate solution was prepared fresh as described above. Mice were euthanized and femurs were collected 10 mins after treatment and bone marrow cells were flushed with 1 mL of room temperature RPMI medium + 20% FBS + 1 µM MgCl2 + 100 U/ml DNase + 240 µM pervanadate and incubated at room temperature in the dark for 10 mins. Lysates were prepared, MERTK protein was immunoprecipitated, and total and phospho-MERTK proteins were detected and quantitated by western blot as described above. ABBREVIATIONS TAM, TYRO3, AXL and MERTK; FLT3, FMS-like tyrosine kinase; ADME, absorption, distribution, metabolism, and excretion; PK, pharmacokinetic; SAR, structure-activity relationship; MCE, microcapillary electrophoresis; iv, intravenous; CL, clearance; T1/2, half-life; Vss, volume of distribution; Cmax, maximum concentration; iv, intravenous; po, oral; ip, intraperitoneal; B-ALL, B-cell acute lymphoblastic leukemia; NSCLC, non-small

cell

lung

cancer;

NSG,

NOD/SCID

IL2Rgamma;

Pd(PPh3)4,

tetrakis(triphenylphosphine)palladium(0); NaBH(OAc)3, sodium triacetoxyborohydride; DIPEA, N,N-diisopropylethylamine.



ACKNOWLEDGMENT This work was supported by the University Cancer Research Fund and a philanthropic gift to the CICBDD. We thank Dr. Zibo Li and Dr. Li Wang for their help in the HPLC data collection. SUPPORTING INFORMATION AVAILABLE Biological methods (MCE assay, selectivity profiling, and PK study), 1H and 13C NMR spectra for compound 19, and analytical HPLC spectra for compounds 18, 19, 21, 22, 26, 29, 32. This material and Molecular Formula Strings is available free of charge via the internet at http://pubs.acs.org. Corresponding Author Information Xiaodong Wang: Tel: 919-843-8456. E-mail: [email protected]. Present Addresses † Jichen Zhang: China Novartis Institutes for Biomedical Research, 4218 Jinke Road, Shanghai, China; Dehui Zhang: RTI international, Room 115, Little BLDG, 3040 E Cornwallis Rd, Durham, NC 27709, USA; Weihe Zhang: BioCryst Pharmaceuticals, Inc., 2100 Riverchase Center, Bldg. 200, Suite 200, Birmingham, AL 35244, USA; Qingyang Liu: Department of chemistry, Montgomery College, 51 Mannakee Street, Rockville, MD 20850, USA; David H. Drewry: SGC Center for Chemical Biology, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 . References 1.

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Table of Contents Graphic

N

N N H

N

N

N

19 (UNC4203) IC50 MerTK 2.4 nM (Ki 0.43 nM) Axl 80 nM (Ki 40.22 nM) Tyro3 9.1 nM (Ki 3.87 nM) FLT3 39 nM (Ki 16.14 nM) T1/2: 3.0 h %F: 15 OH


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Highly selective MERTK inhibitors achieved by a single methyl group

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