Discovery of Novel Janus Kinase (JAK) and Histone Deacetylase

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Discovery of Novel Janus Kinase (JAK) and Histone Deacetylase (HDAC) Dual Inhibitors for the Treatment of Hematological Malignancies Xuewu Liang, Jie Zang, Xiaoyang Li, Shuai Tang, Min Huang, Mei-Yu Geng, C. James Chou, Chunpu Li, Yichun Cao, Wenfang Xu, Hong Liu, and Yingjie Zhang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01597 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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Journal of Medicinal Chemistry

Discovery of Novel Janus Kinase (JAK) and Histone Deacetylase (HDAC) Dual Inhibitors for the Treatment of Hematological Malignancies Xuewu Liang, †,‡ Jie Zang, † Xiaoyang Li, § Shuai Tang, ‡ Min Huang, ‡ Meiyu Geng, ‡ C. James Chou, § Chunpu

Li, ‡ Yichun Cao,

∥

Wenfang Xu, † Hong Liu, *,‡ and Yingjie Zhang. *,†

† Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, P. R. China ‡ State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai, 201203, China. § Department

of Drug Discovery and Biomedical Sciences, College of Pharmacy, Medical University of South Carolina,

Charleston, SC, 29425, United States ∥

School of Pharmacy, Fudan University, 826 Zhanghen Road, Shanghai, 201203, China.

ABSTRACT: Concurrent inhibition of JAK and HDAC could potentially improve the efficacy of the HDAC inhibitors in the treatment of cancers and resolve the problem of HDAC inhibitor resistance in some tumors. Here a novel series of pyrimidin-2-amino-pyrazol hydroxamate derivatives as JAK and HDAC dual inhibitors were designed, synthesized and evaluated, among which 8m possessed potent and balanced activities against both JAK2 and HDAC6 with IC50 at nanomolar level. 8m exhibited improved antiproliferative and proapoptotic activities over SAHA and ruxolitinib in several hematological cell lines. Remarkably, 8m exhibited more potent antiproliferation effect than the combination of SAHA and ruxolitinib in HEL cells bearing JAK2V617F mutation. Pharmacokinetic studies in mice showed that 8m possessed good bioavailability after intraperitoneal administration. Finally, 8m showed antitumor efficacy with no significant toxicity in a HEL xenograft model. Collectively, the results confirm the therapeutic potential of JAK and HDAC

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dual inhibitors in hematological malignancies and provide valuable leads for further structural optimization and antitumor mechanism study.

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INTRODUCTION HDAC inhibitors are widely applied in the treatment of various cancers.1-3 However, recent research showed that HDAC inhibitors promoted BRD4-mediated activation of leukemia inhibitory factor receptor (LIFR), which in turn activated JAK-STAT signaling and restrained the efficacy of HDAC inhibitors in some solid tumors. Concurrent inhibition of JAK or BRD4 sensitized some solid tumors to HDAC inhibitors.4 In addition, a recent study also found that the resistance to HDAC inhibitors in lymphoma cell lines and cutaneous T-cell lymphoma (CTCL) patients was associated with the persistent activation of STATs, a key module in JAK-STAT signaling pathway.5 Therefore, the development of JAK and HDAC dual inhibitors is of great value and significance in the treatment of cancers. Janus kinases (JAKs), a family of intracellular nonreceptor tyrosine kinases consisting of JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2), are critical in transducing cytokine mediated signals via the JAK-signal transducer and activator of transcription (STAT) pathway and are implicated in the pathogenesis of various hematological malignancies and autoimmune diseases.6,7 The discovery of the activating V617F mutation in JAK2 in the majority of patients with Philadelphia chromosome– negative myeloproliferative neoplasms (MPNs),8-11 including myelofibrosis (MF), polycythemia vera (PV), and essential thrombocythemia (ET), provided a strong impetus for the development of JAK2 inhibitors, culminating in the US Food and Drug Administration (FDA) approval of the JAK1/2 inhibitor ruxolitinib (1, Figure 1) for patients with MF or PV.12 Despite the success of ruxolitinib in ameliorating MPN-related symptoms, the ability of JAK inhibitors to reduce neoplastic allele burden in MPN patients seems limited and resistance/persistence upon chronic drug exposure has been

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seen.13-19 It has been revealed that JAK2 inhibitor persistence is associated with overexpression of JAK2 and heterodimerization between JAK2 and JAK1 or TYK2.19 Encouragingly, many preclinical researches indicated that histone deacetylase (HDAC) inhibitors could strongly inhibit the growth of tumor cells bearing the JAK2V617F mutation through downregulation of the JAK2V617F protein level and depletion of its downstream signaling.20-22 Therefore, it is reasonable to believe that the combination of JAK and HDAC inhibitors holds the promise of improving therapeutic efficacy and circumventing drug resistance/persistence in MPN patients.13-15 Preliminary results from a phase Ib trial of JAK inhibitor ruxolitinib and HDAC inhibitor panobinostat (2, Figure 1) in patients with MF have been published in abstract form, which showed that the combination therapy was well tolerated and resulted in reductions in JAK2V617F allele burden and improvements in bone marrow fibrosis in some patients.23 In addition, there are three active clinical trials of ruxolitinib in combination with HDAC inhibitors for MPN patients (NCT01433445, NCT01693601, NCT02267278).24 HDACs are a family of important epigenetic targets for the treatment of cancer, inflammation, neurological disorders, and infections.25,26 To date, four HDAC inhibitors, vorinostat (3, SAHA), romidepsin (4, FK228), belinostat (5, PXD101) and panobinostat (2, LBH589) were approved by the US Food and Drug Administration, and one HDAC inhibitor chidamide (6, CS005) was approved by the China Food and Drug Administration (CFDA) for the therapy of hematologic malignancies (Figure 1).27 To be specific, SAHA is for the treatment of CTCL, FK228 for the treatment of CTCL and peripheral T-cell lymphoma (PTCL), PXD101 and CS005 for the treatment of PTCL, and LBH589 for the treatment of multiple myeloma (MM). It has been revealed that constitutive activation of STAT is involved in resistance to SAHA across a variety of lymphoma cell lines,

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including CTCL.5 Combination of SAHA with a pan-inhibitor of JAK, the upstream activator of STAT, resulted in synergistic growth inhibition in SAHA-resistant CTCL cell lines. These results seem to be of clinical relevance, because high levels of phosphorylated STAT3 and nuclear localization of STAT1 in skin biopsies obtained from CTCL patients correlate with a lack of clinical response to SAHA.5 CN

N N

O

H N

HN N N

N H

O

O O S N H

OH

3 (Vorinostat, SAHA)

1 (Ruxolitinib) N H

O

OH

5 (Belinostat, PXD101)

H

O O

N H

O

OH

F

N H NH S S HN O

O

N H

N H

O O

H N

O

N H N

N H

NH2

O

H

2 (Panobinostat, LBH589)

4 (Romidepsin, FK228)

6 (Chidamide, CS005)

Figure 1. The structures of approved JAK inhibitor and HDAC inhibitors.

Based on this information, rational combination therapy with JAK and HDAC inhibitors is a potential therapeutic strategy for not only MPN, but also CTCL, and even other intractable hematological malignancies. However, the employment of drug combination is often concomitant with poor patient compliance, unpredictable pharmacokinetic (PK) profile and drug−drug interactions.28-30 An alternative strategy to circumvent these drawbacks is the development of JAK and HDAC dual inhibitors. Herein, we report the design, synthesis, activity and pharmacokinetic evaluation of pyrimidin-2-amino-pyrazol hydroxamate derivatives as JAK and HDAC dual inhibitors, which were derived from our previously reported 4-amino-(1H)-pyrazole-based JAK inhibitor.31 In comparison to the approved HDAC inhibitor SAHA and JAK inhibitor ruxolitinib, most of the

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compounds exhibited superior antiproliferative activities in several hematological cell lines due to their potent and balanced activities against both JAKs and HDACs. In the JAK2V617F-mutated erythroleukemia cell line HEL and the JAK2 wild type chronic myeloid leukemia cell line K562, combination of SAHA and ruxolitinib exhibited synergistic and additive effects, respectively. Strikingly, our dual inhibitors were significantly more potent than the drug combination. RESULTS AND DISCUSSION Rational Design of Novel JAK and HDAC Dual Inhibitors. Most HDAC inhibitors fit a common pharmacophore, which is mainly composed of a zinc binding group (ZBG) that chelates the catalytic zinc ion located at the bottom of the HDAC active site, a surface recognition cap (SRC) that interacts with the amino acid residues around the entrance of the active site, and a linker that connects the ZBG with SRC (Figure 2).32 Generally, potent HDAC inhibitors can be obtained through tethering a ZBG, such as hydroxamic acid, to various aromatic SRCs via a linker of five to seven carbons, like the approved HDAC inhibitor SAHA (Figure 2, 3A). JAK ATP active pocket contains hinge region and p-loop. Most JAK inhibitors possess specific functional groups which could form strong hydrogen bonds with JAK hinge region, like the approved JAK inhibitor ruxolitinib (Figure 3B). In our previous research, a potent 4-amino-(1H)-pyrazole-based JAK inhibitor 7 with notable antiproliferative potency was identified.31 Structural analysis indicated that the alkyl hydroxamate of SAHA could be readily tethered to compound 7 via the alkylation of N1 atom of pyrazole, leading to a potential JAK and HDAC dual inhibitor 8a (Figure 2). It is worth noting that during the preparation of our work, a series of ruxolitinib-based JAK and HDAC dual inhibitors designed by the similar strategy was reported, among which compound 9 exhibited potent in vitro antiproliferative activities

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against a range of hematological cell lines.33 Before synthesis, the proposed binding modes of compound 8a in the active sites of HDAC2 and JAK2 were analyzed using molecular docking study. Figure 3C showed the binding mode of 8a in the active site of HDAC2, which displayed the classical binding mode of hydroxamate-based HDAC inhibitors: the hydroxamic acid groups chelate the Zn2+ in a bidentate manner and form multiple hydrogen bonds, the aliphatic chains occupy the hydrophobic channel, and the terminal cap groups interact with the amino acid residues at the entrance of the active site. Figure 3D showed the binding mode of 8a in the ATP pocket of JAK2. Compound 8a formed dual hydrogen bonds between its aminopyrimidine moiety and the Leu932 residue. The phenyl group of 8a occupied the same hydrophobic pocket of JAK2 with the pyrrole ring of compound 1. It is worth noting that the alkyl hydroxamate part of 8a was projected towards the solvent region, minimally interfering the binding between 8a and JAK2. The above docking results strongly supported the chemical synthesis and biological evaluation of 8a and its analogs as JAK and HDAC dual inhibitors. Linker

SRC

O

H N

ZBG

N H

O

HDAC inhibitor pharmacophore

OH

3 (Vorinostat, SAHA) O

N

NH N

HN

N

N Cl

HN

N N

Cl

HN

HN

Cl

Cl

8a (designed JAK and HDAC dual inhibitor)

CN HN

O

HN

N

OH

N

7 (reported JAK inhibitor)

N

N H

N N

1 (Ruxolitinib)

N N

N N

N H

OH

9 (reported JAK and HDAC dual inhibitor)

7



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Figure 2. The design of JAK and HDAC dual inhibitors based on the HDAC inhibitor pharmacophore. The three parts of HDAC inhibitor pharmacophore are indicated in three colors.

Figure 3. (A). The binding mode of SAHA (3) in the active site of HDAC2 (PDB code: 5IWG). The hydroxamic acid group of SAHA chelates the Zn2+ in a bidentate manner and forms multiple hydrogen bonds with the Tyr308 residue and the His145 residue. The aliphatic chain occupies the hydrophobic channel, and the phenylamino group

8


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interacts with the Asp104 residue at the entrance of the active site. (B). The binding mode of ruxolitinib (1) in the active site of JAK2 (PDB code: 3FUP). The pyrrolopyrimidine ring of compound 1 forms dual hydrogen bonds with the backbone residues (Leu932 and Glu930) of the hinge region of JAK2. (C). The binding mode of 8a in the active site of HDAC2. (D). The binding mode of of 8a in the ATP pocket of JAK2. Yellow dashed lines represent the hydrogen bonds. Oxygen, nitrogen, chlorine and polar hydrogen atoms are shown in pink, blue, violet and white, respectively. The figures were generated using PyMol (http://www.pymol.org/).

Chemistry. The synthesis of key intermediates 15a-h and 17a-o is described in Schemes 1-2, respectively. In Scheme 1, reduction of the starting material 10, followed by Boc protection, led to compound 12, which reacted with commercially available bromoalkanes 13a-h under basic condition of cesium carbonate or DBU to produce intermediates 14a-h. Deprotection of 14a-h yielded intermediates 15a-h, respectively. In Scheme 2, the intermediates 17a-o were obtained by the reaction of 5-substituted-2,4-dichloropyrimidine 16a-d with various aromatic or aliphatic amines under different conditions. Scheme 1. Synthesis of intermediates 15a-h a

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O

n

() Br

()

n

O

N

13a-f

N H2N. HCl 15a-f

14a-f

13a,14a,15a, n=2, R0=CH3 13d,14d,15d, n=5, R0=C2H5

O R0

N d

BocHN

O

+

O R0

N c

R0

O

n

()

13b,14b,15b, n=3, R0=C2H5 13e,14e,15e, n=6, R0=C2H5

13c,14c,15c, n=4, R0=C2H5 13f,14f,15f, n=7, R0=C2H5

O H N N

H N

a

b

N

H 2N

O 2N

H N N

11

10

+

BocHN

O O

O O

c

Br

O d

N

N

N

12

N

BocHN 13g

14g

H2N. HCl

O

15g O

O

O

O O

+ Br

c

d

N

N

N BocHN

13h

a

N H 2N . HCl

14h

15h

Reagents and conditions: (a) Pd/C, H2, EtOH, rt, 1~2 days, 90%; (b) (Boc)2O, NaHCO3/H2O, THF,

rt, 1~2 days, 75%; (c) Cesium carbonate, CH3CN, 40-50 °C, 10 h, 51%-82%; (d) HCl/ EtOAc, rt, 6~10 h, 63%-93% Scheme 2. Synthesis of intermediates 17a-o a H N

17a-k, R1=Cl, R2=

H N Cl

H N

H N

H N

O

Cl

H N F

Cl

Cl N

17a

Cl e

N

N

R1 16a-d 16a, R1=Cl 16b, R1=H 16c, R1=F 16d, R1=CF3

O

N

R1 17a-o

N

N

17g

R2

Cl

17b

17h

17c

17d H N

NH

17i

17e

17j

17f

H N

17k

H N

17l,

1

R =H,

17m, R1=F, 1

2

R = R 2=

F

F

2

17n,o, R =CF3, R =

H N

H N

H N

F

a

Reagents and conditions: (e) for 17a and 17l, corresponding amines, Na2CO3, EtOH, rt, overnight,

40%-44%; for 17b, 17d-f, corresponding amines, DIPEA, DMF, r.t. overnight, 57%-82%; for 17c,

10


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corresponding amine, DIPEA, NMP, 100 °C, overnight, 40%; for 17g-k, corresponding amines, EtOH, 0 °C, 0.5~3 h, 63%-83%; for 17m, corresponding amine, H2O/CH3OH=3:1, 50 °C, 5 h, 68%; for 17n and 17o, corresponding amines, EtOH, -50 °C, 1 h, 25%-30%. The target compounds 8a-z were synthesized according to the procedures in Scheme 3. Intermediates 17a-o reacted with intermediates 15a-h using TFA as a catalyst at high temperature to give 18a-z, which were converted to hydroxamic acid compounds 8a-z by NH2OK in dry methanol, respectively.

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Scheme 3. Synthesis of target compounds 8a-z a R2 R1

( )n

N

N H

f

R

R =C2H5 (18t, R0=CH3)

18a-x

N H

N

1

H N

2

1

Cl 18b, 8b, n=6, R1=Cl, R2=

Cl

18c, 8c, n=6, R1=Cl, R2=

H N H N

H N H N

18e, 8e, n=6, R1=Cl, R2= O

H N

2

18f, 8f, n=6, R =Cl, R =

18i, 8i, n=6, R1=Cl, R2=

a

R1

1

HN OH

18j, 8j, n=6, R1=Cl, R2=

N H

1

H N

18p, 8p, n=6, R1=H, R2=

F

2

18q, 8q, n=6, R =F, R =

F

O

N

H N

18k, 8k, n=6, R1=Cl, R2=

18r, 8r, n=6, R1=CF3, R2=

F

H N

18l, 8l, n=5, R1=Cl, R2=

H N

Cl 1

2

18m, 8m, n=5, R =Cl, R = 1

2

18n, 8n, n=5, R =Cl, R =

18s, 8s, n=6, R1=CF3, R2= 1

Cl

2

18t, 8t, n=2, R =Cl, R = H N Cl

1

2

18u, 8u, n=3, R =Cl, R =

F

8z H N

H N

2

1

N N

N

18o, 8o, n=5, R =Cl, R =

H N

O

R2 g

N

HN OH

8y

O

H N

F 18g, 8g, n=6, R1=Cl, R2=

N H

N

2

N N

N

H N H N

Cl

18d, 8d, n=6, R1=Cl, R2=

1

O

1

18z

18h, 8h, n=6, R =Cl, R =

18a, 8a, n=6, R =Cl, R =

R

g

O

N H

8a-x

R2

N N

N

N HN OH N N H

N 18y

R2 R1

N

O

N N

N

O

( )n

N

O

1

N

f

R1 g

0

R2 17a-o + 15a-h

R2

0 O R

N N

N

f

O

18v, 8v, n=4, R =Cl, R =

18w, 8w, n=5, R1=Cl, R2=

18x, 8x, n=7, R1=Cl, R2=

H N

F

H N

F

H N

F H N

18y, 8y, R1=Cl, R2= H N

2

18z, 8z, R1=Cl, R2=

F

H N

F

H N

F

Reagents and conditions: (f) TFA, n-BuOH, 130 °C, 3 h, 20%-70%; (g) NH2OK, CH3OH (dry), rt,

0.5-1 h, 34%-80%. In Vitro JAK and HDAC Inhibition Assay. The JAK and HDAC inhibitory activities of 8a were evaluated by determining its IC50 values against JAK2 and HeLa nuclear extract, respectively. The JAK inhibitor 7 and the HDAC inhibitor 3 were used as the positive controls. The results in Table 1 showed that 8a exhibited comparable JAK2 inhibitory activity to 7 (IC50 value: 0.010 μM vs 0.002 μM) and more potent HDAC inhibitory activity than 3 (IC50 value: 0.020 μM vs 0.168 μM), validating our JAK and HDAC dual inhibitor design strategy shown in Figure 2. Compounds 8b-k with the

12


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same aliphatic linker length as 8a (n=6) were synthesized to investigate the effects of the terminal phenyl group on JAK2 and HDAC inhibition. All these analogs exhibited potent JAK2 inhibition with IC50 values lower than 50 nM. Among these analogs, compounds 8i, 8l, 8m and 8w exhibited the most potent JAK2 inhibition with IC50 values lower than 10 nM. Note that the aliphatic linker length of 5 or 6 methylenes had no dramatic influence on the JAK2 inhibition (8l vs 8a, 8m vs 8b, 8n vs 8c, 8o vs 8k, 8w vs 8f). With reference to their HDAC inhibitory activities, structure-activity relationships (SARs) were revealed: (1) Analogs with aromatic terminal groups (8a-8f) were generally more potent than analogs with aliphatic terminal groups (8g-8k). (2) Para-substitution on the terminal phenyl group was beneficial (The HDAC IC50 values of the para-substituted compounds 8b, 8e and 8f reached the single-digit nM range, over 50-fold more potent than the positive control 3. The above two SARs were also observed in compounds with shorter aliphatic chain (8l-8o and 8w, n=5), among which, 8l-8n and 8w with the aromatic terminal group of substituted phenyls exhibited more potent HDAC inhibition than 8o with the terminal methyl group, and the para-chlorine compound 8m was more potent than its meta- and ortho-chlorine analogs (8l and 8n). Taken together, the results in Table 1 indicate that the structural modification of the terminal phenyl group of 8a was generally tolerated for JAK2 and HDAC inhibition, leading to compound 8m with the most potent activities toward both JAK2 and total HDAC. Table 1. Structures and In Vitro Enzymes Inhibitory Activities Comparison of the Target Compounds 8a~o and 8w a

Compd.

JAK2

HDAC

IC50 (μM)

IC50 (μM)

Structure

13



Cl

8a

Cl

N

N H

Cl

N

Cl

8b

N

N H

N

Cl

8c

N

N H

Cl

8d

N

N H

N

O

N H

Cl

8e

N

N H F

N

Cl

8f

N

Cl N

N H

Cl

N

N

N

Cl

N H

N

8i N

N H Cl

8j

N

Cl

8l

8m

N H

N H N

N H

8k

N H

N

N

8h

N H N

N H

O

N H

N

N H

Cl

8g

N H

N H

N

N H

Cl Cl

N H

Cl

Cl N H

N N

N H

N N

O OH N NH N ()n=6

0.010±0.002

0.020±0.002

O OH N NH N ()n=6

0.014±0.004

0.002±0.001

O OH N NH N ()n=6

0.046±0.012

0.010±0.001

O OH N NH N ()n=6

0.012±0.003

0.019±0.003

O OH N NH N ()n=6

0.010±0.002

0.003±0.001

O OH N NH N ()n=6

0.015±0.005

0.003±0.002

O OH N N N ()n=6 H

0.020±0.004

0.037±0.006

O OH N N N ()n=6 H

0.050±0.008

0.040±0.004

O OH N NH N ()n=6

0.005±0.001

0.013±0.006

O OH N NH N ()n=6

0.030±0.011

0.026±0.009

0.036±0.010

0.015±0.003

O OH N NH N ()n=5

0.007±0.002

0.004±0.001

O OH N NH N ()n=5

0.004±0.001

0.002±0.001

O OH N NH N ()n=6

N

N H

Page 14 of 82

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Cl

8n

N H

Cl

N

Cl

8o

O OH N NH N ()n=5

N N H

O OH N NH N ()n=5

N

N H F

N

N H

Cl

8w

N H

N

N H

Cl

7

3

O OH N NH N ()n=5

N

Cl

N H

N NH

N N

N H

0.018±0.004

0.036±0.011

0.104±0.018

0.005±0.001

0.026±0.009

0.002±0.001

NAb

NAb

0.168±0.054

O

H N

N H

O

a

0.018±0.006

OH

Assays were performed in replicate (n>2), IC50 values are shown as mean ± SD.

b Not

active at 10 μM.

To investigate the effects of substituents of the pyrimidine group on JAK2 and HDAC inhibition, compounds 8p, 8q and 8r were synthesized and compared with 8f. The results in Table 2 showed that replacement of the chlorine on the pyrimidine of 8f with H, F, and CF3 led to decreased JAK2 inhibition (8p, 8q and 8r). In reference to their HDAC inhibitory activities, compounds 8q (IC50 = 0.003 μM) and 8f (IC50 = 0.003 μM) with halogen substituents exhibited the most potent activities, which were over 50-fold more potent than the positive control 3 (IC50 = 0.168 μM). Similar trends were observed in the comparison of 8k and 8s. Table 2. Structures and In Vitro Enzymes Inhibitory Activities Comparison of the Target Compounds 8p~s, 8f and 8k a

Compd.

JAK2

HDAC

IC50 (μM)

IC50 (μM)

Structure

15



F

H

8p F

N

N

F3C

N H

N

N H Cl

7

3

O OH N NH N ()n=6

0.015±0.005

0.003±0.001

O OH N NH N ()n=6

>0.1

0.041±0.007

O OH N NH N ()n=6

0.036±0.010

0.015±0.003

O OH N NH N ()n=6

0.085±0.025

0.037±0.005

0.002±0.001

NAb

NAb

0.168±0.054

N H

N

N H

0.003±0.001

N H

N

N H

>0.1

N

N H Cl

O OH N NH N ()n=6

N H

N

F3C

8r

0.018±0.008

N

N H F

8s

N

Cl

8f

>0.1

N H N

N H F

8k

N

F

8q

O OH N NH N ()n=6

N

N H

Cl

N H

N NH

N N

Page 16 of 82

N H O

H N O

N H

OH

a


b

Not active at 10 μM. To investigate the effects of linker on JAK2 and HDAC inhibition, compounds 8t-8z were

synthesized and evaluated (Table 3). For compounds with aliphatic linkers (8t-8x and 8f), HDAC inhibitory activities first increased and then decreased with the linker length, culminating in compound 8f (IC50 = 0.003 μM); however no obvious trend on JAK2 inhibition was observed from the aliphatic linker length. For compounds with aromatic linkers (8y and 8z), the Nhydroxybenzamide 8y possessed more potent HDAC and JAK2 inhibitory activities than the N-

16


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hydroxycinnamide 8z. Table 3. Structures and In Vitro Enzymes Inhibitory Activities Comparison of the Target Compounds 8t~z and 8f a

Compd.

JAK2

HDAC

IC50 (μM)

IC50 (μM)

O OH N NH N ()n=2

0.022±0.005

>10

O OH N NH N ()n=3

0.010±0.002

>10

O OH N NH N ()n=4

0.046±0.012

0.569±0.126

O OH N NH N ()n=5

0.005±0.001

0.026±0.009

O OH N NH N ()n=6

0.015±0.005

0.003±0.002

O OH N NH N ()n=7

0.061±0.018

0.038±0.001

0.021±0.008

0.062±0.017

0.043±0.013

0.144±0.006

0.002±0.001

NAb

NAb

0.168±0.054

Structure

F

Cl

8t

N

N H F

N

Cl

8u

N

N H F

N

Cl

8v

N

Cl

8w

N

Cl

8f

N

Cl

8x

N H N

N H F

N H N

N H F

N H N

N H F

N H

N H N

N H

N

N H

O

8y

N H F

Cl

N N

N

N H

N

N H

O

8z

F

Cl

N

N H

N

N H

N N

Cl

7

3

Cl

N H

OH

N NH

N N

N H

OH NH

O

H N O

N H

OH

17



a

Page 18 of 82


b Not

active at 10 μM.

After the completion of evaluation for JAK and HDAC enzymatic activity of all compounds, SARs were developed and summarized in Figure 4. To sum up, (1) the hydroxamate group of the target compounds acts as the zinc binding group that chelates the catalytic zinc ion in HDAC and therefore achieves a potent HDAC inhibitory activity. (2) The aminopyrimidine moiety of the target compounds acts as the pharmacophore of JAKs and therefore achieves a potent JAK inhibitory activity. (3) Proper linker length is very important for HDAC inhibitory activity. The target compounds with the linker length 5 or 6 methylene groups possess the most potent inhibitory activities against HDACs. However, no obvious trend on JAK2 inhibition is observed from the aliphatic linker length and the linker types. (4) R1 group shows a decisive effect on JAK2 inhibitory activity. The target compounds with the chlorine atom on the R1 group possess the most potent inhibitory activity against JAK2. (5) Different substituents of R2 group are well tolerated in both JAK2 and HDACs. Pharmacophore of JAKs Aliphatic linker length n=2~7 Aromatic linkers

Modification of group R1

R1 Modification of the group R2 substituted aromatic amine or fatty amine

R2

N N

N N

SRC

N H

O Linker

Linker

N OH H

Zinc binding group (ZBG) that chelates the catalytic zinc ion

ZBG

Compounds 8a-z

Figure 4. SARs summary of JAK and HDAC dual inhibitors.

18


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JAK Isoform Selectivity Profile. The JAK2 IC50 values of five representative compounds 8a, 8b, 8h, 8i and 8m were determined. The results in Table 4 confirm the significant JAK2 inhibitory activity of these five compounds, among which, compound 8a, 8i and 8m possessed the IC50 values in the single-digit nM range. To profile their JAK isoform selectivity, these five compounds were further evaluated against the other three JAK isoforms (JAK1, JAK3 and TYK2). It was revealed that compounds with aromatic terminal groups (8a, 8b, 8m) were moderately selective to JAK1/2/3 over TYK2, while compounds with aliphatic terminal groups, especially compound 8h, showed JAK3 preference to some extent. Table 4. JAK Isoform Selectivity Profiles of Representative Compounds a IC50 (μM) Compd.

Structure

Cl

H N

8a 8b

8m

N

H N

H N

N N

Cl H N

0.0033

0.067

0.014

0.010

0.0076

0.094

N ()n=6 NH N OH O

0.050

0.030

0.0037

0.060

N ()n=6 NH N OH O

0.005

0.014

0.0020

0.012

0.004

0.0048

0.0074

0.049

0.002

0.0034

0.0035

ND b

N ()n=6 NH N OH O

N ()n=5 NH N OH O

N

Cl

Cl

0.015

H N

N

H N Cl

a

H N

N

Cl

7

0.010

N ()n=6 NH N OH O

N

N

Cl

TYK2

H N

N

Cl

8i

JAK3

N

H N

8h

JAK1

H N

N

Cl

Cl

JAK2

H N

N N

NH N

Assays were performed in replicate (n>2); the SD values are < 20% of the means.

b Not

determined. 19



Page 20 of 82

HDAC Isoform Selectivity Profile. Class I HDAC isoforms and Class IIb HDAC isoforms are involved in the tumorigenesis and development. To profile the HDAC isoform selectivity of these JAK and HDAC dual inhibitors, seven representative compounds 8a, 8k, 8m, 8v, 8x, 8y and 8z were tested against two class I isoforms (HDAC2/8) and one class IIb isoform (HDAC6) with compound 3 as the positive control. Based on the results in Table 5, we see that, except compound 8y being HDAC6/8 selective over HDAC2, all tested compounds showed HDAC2/6 preference over HDAC8, which was similar to the selectivity profile of 3. It is worth noting that compounds 8a, 8k and 8m exhibited more potent inhibitory activity against all the three HDAC isoforms tested than 3. Table 5. HDACs Isoform Selectivity Profiles of Representative Compounds a IC50 (μM) Compd.

Structure

Cl

H N

8a

H N

8m

8v

8x

H N

N

H N Cl

H N

F

N ()n=5 NH N OH O

N

H N

N

N ()n=4 NH N OH O

N

Cl

H N

H N

N

Cl

F

N ()n=6 NH N OH O

N

Cl

N ()n=6 NH N OH O

N

Cl

8k

H N

N

H N

N

N ()n=7 NH N OH O

N

Cl

O

8y

H N F

Cl

H N

N N

HDAC2

HDAC6

HDAC8

0.13

0.041

0.84

0.19

0.029

4.07

0.12

0.014

2.47

1.4

0.31

5.1

0.43

0.30

1.7

0.73

0.013

0.12

OH NH

N N

20


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O

8z

H N F

3 a

H N

N N

Cl

OH NH

1.0

0.063

14.1

0.22

0.090

5.6

N N

O

H N O

N H

OH

Assays were performed in replicate (n>2); the SD values are < 20% of the means. Western Blot Analysis. Western blot analysis was performed to evaluate the cellular potency of

the target compounds. The results in Figure 5 show that, similar to 3 (SAHA), our compounds 8a and 8k simultaneously increased the intracellular levels of the HDAC1/2/3 substrate acetyl-histone H4 (Ac-HH4) and the HDAC6 substrate acetyl-α-tubulin (Ac-Tubulin). Note that the HDAC6 selective inhibitor tubastatin A (Tub A, HDAC6 IC50: 15 nM, 1093-fold selective vs HDAC1) 34 only increased the intracellular levels of acetyl-α-tubulin. It was worth noting that at the same concentration of 80 nM and 400 nM, the effects of 8a and 8k on acetyl-histone H4 were comparable to that of 3, while at the same concentration of 80 nM, the effects of 8a and 8k on acetyl-α-tubulin were much superior to those of tubastatin A and 3.

21



Page 22 of 82

Figure 5. (A) A549 cells were treated with DMSO or compounds for 6 h. The levels of acetyl-α-tubulin (Ac-Tub) and acetyl-histone H4 (Ac-HH4) were determined by immunoblotting. β-Actin was used as a loading control. The result is the representative of three independent experiments. (B) Image J was used to quantify the western-blot data. At 80 nM, the effects of 8a and 8k on acetyl-α-tubulin were much superior to those of tubastatin A and 3.

It was recently revealed that HDAC inhibitor SAHA activates JAK-STAT pathway and attenuates the efficacy of HDAC inhibitors in some solid tumors, and concurrent inhibition of JAK and HDAC improves the efficacy of the HDAC inhibitors in the treatment of breast cancers.4 To validate the intracellular dual actions of 8m, another set of western blot analysis was performed. The results in Figure 6 validate that 3 (SAHA) upregulates p-STAT3-Tyr705 level via activating JAK-STAT pathway, and cotreatment with 1 (ruxolitinib) successfully suppresses the JAK-STAT pathway. To our delight, our dual inhibitor 8m led to decreased levels of p-STAT3-Tyr705 relative to 3, and

22


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increased levels of Ac-tubulin and Ac-HH3 relative to the control group, indicating the concurrent inhibition of intracellular JAK and HDAC (Figure 6).

Figure 6. MDA-MB-231 cells were treated with DMSO or compounds for 12 h. The levels of phosphorylated STAT3 (p-STAT3-Tyr705), total STAT3, acetyl-α-tubulin (Ac-Tub) and acetyl-histone H3 (Ac-HH3) were determined by immunoblotting. GAPDH was used as a loading control.

In Vitro Antiproliferation Assay. Given the promising cellular potencies validated by western blot analysis, most of our target compounds were progressed to in vitro antiproliferation assay. In addition to the human erythroleukemia cell line HEL bearing the JAK2V617F mutation, three other human hematological cancer cell lines including the chronic myelogenous leukemia cell line K562, the acute lymphoblastic leukemia cell line MOLT4, and the acute T cell leukemia cell line Jurkat were tested as both JAK inhibitors and HDAC inhibitors are of great potential for the therapy of hematological malignancies.35,36 From the results listed in Table 6, it was found that compared with the positive controls 1 and 3, almost all our tested compounds showed greater antiproliferative activity against the four hematological cancer cell lines. Note that K562 cell line was less sensitive to our

23



Page 24 of 82

compounds than HEL, MOLT4 and Jurkat cell lines. Moreover, the combinatorial cytotoxic effects of the JAK inhibitor 1 and the HDAC inhibitor 3 were determined, revealing the strong synergistic effect (CI = 0.48) in the JAK2V617F-mutated HEL cell line and the additive effect (CI = 0.97) in JAK2 wild type K562 cell line. Strikingly, most of our dual inhibitors were more potent than the combination of 1 and 3. Of particular note were compounds 8l and 8m,whose IC50 values against HEL, MOLT4 and Jurkat cell lines were all below 0.1 μM, reflecting their potent and balanced inhibition activity against both JAK and HDAC as shown in Table 1. Table 6. In Vitro Antiproliferative Activities of Representative Compounds a IC50 (μM) Compd. HEL

K562

MOLT4

Jurkat

8a

0.14±0.03

0.38±0.03

0.13±0.02

0.10±0.01

8b

0.12±0.02

0.32±0.03

0.12±0.01

0.10±0.02

8c

0.34±0.07

0.86±0.05

0.51±0.06

0.25±0.01

8d

0.18±0.04

0.42±0.16

0.16±0.02

0.09±0.04

8e

0.21±0.02

0.62±0.04

0.17 ±0.02

0.13±0.02

8f

0.21±0.03

0.50±0.13

0.22±0.05

0.12±0.02

8g

0.47±0.10

0.91±0.15

0.32±0.02

0.17±0.01

8i

0.08±0.01

0.38±0.08

0.15±0.01

0.05±0.01

24


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a


8k

0.10±0.02

0.59±0.01

0.11±0.02

0.07±0.01

8l

0.08±0.01

0.58±0.06

0.08±0.02

0.05±0.01

8m

0.09±0.01

0.49±0.08

0.08±0.02

0.06±0.01

8n

0.43±0.07

1.81±0.18

0.63±0.14

0.31±0.03

8r

0.42±0.08

0.48±0.11

0.24±0.07

NDb

8w

0.15±0.03

0.42±0.14

0.12±0.01

0.06±0.02

8z

0.60±0.15

1.03±0.07

0.33±0.06

0.15±0.01

1

18.6±6.2

23.2±2.0

15.8±1.4

>5

3

0.65±0.07

1.11±0.05

0.4±0.07

0.27±0.02

1 + 3c

0.30±0.02

1.03±0.03

NDb

NDb

(CI = 0.48)d

(CI = 0.97)d


b Not

determined.

c Combined

d

at a mole ratio of 1:1.

CI (combination index) was calculated based on the Chou−Talalay method.37 CI > 1 indicates

antagonism, CI = 1 indicates an additive effect, and CI < 1 indicates synergism. Apoptosis Assay. The apoptosis-inducing abilities of compounds 8a and 8m were evaluated by

25



Page 26 of 82

flow cytometry analysis. In this figure, there are four regions. The lower left corner and the upper left corner of the diagram show the number of living cancer cells and dead cancer cells, respectively. The upper right, combined with the lower right region of the diagram, represents the number of apoptotic cancer cells. It was demonstrated that 1.0 μM of 8m could induce almost complete HEL cell apoptosis (Figure 7) and over 70% K562 cell apoptosis (Figure 8), which was in line with its antiproliferative results that HEL was more sensitive to 8m than K562 (Table 6). Notably, at the same concentration of 1.0 μM, the apoptosis-inducing abilities of 8a and 8m in both HEL (Figure 7) and K562 (Figure 8) cells were much stronger than those of 1 (ruxolitinib) and 3 (SAHA), confirming the notable cellular potencies of our JAK and HDAC dual inhibitors.

26


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Figure 7 (A). Induction of apoptosis in HEL cells after 24 h of treatment. (B) The ability of compounds 3, 1, 8a and 8m to induce apoptosis in HEL cells after 24 h of treatment.

27



Page 28 of 82

Figure 8 (A). Induction of apoptosis in K562 cells after 24 h of treatment. (B) The ability of compounds 3, 1, 8a and 8m to induce apoptosis in K562 cells after 24 h of treatment.

In Vivo Pharmacokinetic (PK) Profile. Due to its closely matched JAK/HDAC inhibitory

28


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activity and potent antiproliferative and pro-apoptotic capacities, compound 8m was progressed into in vivo pharmacokinetic study. Compound 8m was administered intravenously (IV) at 2 mg/kg or orally (PO) at 10 mg/kg in male Sprague−Dawley (SD) rats. The PK parameters and concentration– time curves of 8m indicated the poor PK properties of 8m (Table S1 and Figure S1, supporting information). To be specific, after intravenous administration, 8m was rapidly cleared with the clearance (CL) of 160.9 mL/min/kg and the half-life (t1/2) of 0.32 h. The oral bioavailability of 8m was lower than 1%. In order to further investigate the in vivo pharmacokinetic properties in mice, 8m was intraperitoneally and intravenously administrated in male ICR (CD-1) mice. The results in Table 7 showed that 8m possessed a good bioavailability after intraperitoneal administration, however the clearance of 8m in mice was also very rapid. Table 7. PK Parameters of 8m in ICR (CD-1) mice (N = 9, Male) Dose

t1/2

Tmax

(h)

(h)

Cmax

AUC0-t

MRT0-

Vz

CL

F

Administration (mg/kg) 1

IP

0.26 0.12

1

IV

0.32

/

(ng/mL) (ng*h/mL)

t (h)

(L/kg) (mL/min/kg)

118

33.0

0.36

/

/

/

28.4

0.27

18.4

643

(%) 116

In Vitro Liver Microsomal Stability Assay. We further determined the metabolic stability of compound 8m in the liver microsome. The reduced nicotinamide adenine dinucleotide phosphate (NADPH) and uridine diphosphate glucuronic acid (UDPGA) were used as cofactors for the phase I and phase II metabolic stability evaluation, respectively. The results showed that compound 8m was rapidly degraded by phase I metabolism, with t1/2 of 14.6 min and 25.8 min in rat and human microsomes, respectively (Figure S2, supporting information). The phase II metabolic stability 29



Page 30 of 82

indicated that 8m was also readily transformed by UDP-glucuronosyltransferase (UGT) in rat microsome (t1/2 = 41.6 min). Interestingly, the phase II metabolic stability of 8m in human microsome was very good (t1/2 = 361.1 min), demonstrating the species differences in drug metabolism (Figure S3, supporting information). Taken together, compound 8m showed poor phase I and phase II metabolic stability in rat microsome, which could partially contribute to its rapid in vivo clearance. In Vivo Antitumor Activity Evaluation and Determination of Drug Exposure. Considering its good intraperitoneal bioavailability, 8m was intraperitoneally administrated in a HEL xenograft model to preliminarily evaluate its in vivo antitumor potency. Another objective of this set of experiments was to investigate the in vivo effects of the combination of 1 and 3. Compounds 3 (100 mg/kg), 1 (100 mg/kg), 8m (100 mg/kg), and 3 plus 1 (60 mg/kg plus 70 mg/kg, the similar mole number as 8m) were dosed intraperitoneally for 16 consecutive days. Tumor growth inhibition (TGI) was calculated at the end of treatment. As shown in Table 8, a combination of 1 and 3 exhibited improved in vivo antitumor potency compared with each drug alone and possessed the best antitumor potency with TGI of 57%. 8m showed a moderate in vivo antitumor potency with TGI of 36%. The tumor growth curve and the final tumor tissue size are presented in Figure 9A and 9B, respectively, which explicitly demonstrates the superior in vivo potency of the combination group.Though 8m possessed much better in vitro activity than 3, the in vivo potency of 8m was a little inferior, which could be attributed to its short half-life and rapid clearance shown in Table 7. To further determine the compound exposure, the tumor and plasma concentrations of compounds 1, 3 and 8m in mice were measured after the last of five daily doses (100 mg/kg/day, IP). The results in Figure 10A reveal the lower tumor exposure of 8m compared with compounds 3 and 1, which may provide a basis for

30


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the modest antitumor activity of 8m in comparison to compounds 3 and 1. The low concentration of 8m in plasma (about 100 nM in vivo, Figure 10B) suggests a desirable in vivo toxic profile of 8m. Furthermore, both the mice treated with 8m alone and the mice cotreated with 1 and 3 showed good tolerance, indicated by no significant body weight loss and no observable toxic signs. Table 8. In Vivo Antitumor Efficacy in HEL Xenograft Model.

a Compared

Compd

Administration

TGI (%) a

3

100 mg/kg/d, IP

42

1

100 mg/kg/d, IP

27

3+1

60 mg/kg/d + 70 mg/kg/d, IP

57

8m

100 mg/kg/d, IP

36

with the control group, all treated groups showed statistically significant (P < 0.05) TGI

by Student’s two-tailed t test. The TGI values are based on the tumor weights.

Figure 9. (A) Growth curve of implanted HEL xenograft in nude mice. (B) Picture of dissected HEL tumor tissues.

31



Page 32 of 82

Figure 10. (A) The drug concentrations in tumor. (B) The drug concentrations in plasma. The tumor and plasma concentrations of compounds 1, 3 and 8m in mice were measured after the last of five daily doses (100 mg/kg/day, IP). Mice were sacrificed at 0.5 h, 1.0 h and 4.0 h respectively after administration, then blood and tumor samples were collected.

Protein Kinases Selectivity Profile. Based on the low drug concentrations in plasma in vivo (about 100 nM, Figure 10B), compound 8m was further tested against 85 kinds of kinases to characterize its protein kinase selective profiles at 20 nM and 200 nM, respectively. The results in Table 9 show that, at the concentration of 20 nM, 8m led to over 90% inhibition of Ret kinase, which was encoded by RET proto-oncogene. The mutations of Ret kinase are associated with the development of various types of human cancers.38 In addition, 20 nM of compound 8m also exhibited over 50% inhibition against FMS-like receptor tyrosine kinase 3 (FLT3) and vascular endothelial growth factor receptor 2 (VEGFR2). In fact, FLT3 also plays important roles in the progress of hematological malignancies, which is simultaneously targeted by the JAK inhibitor pacritinib in phase III clinical trials,39 while VEGFR2 is a validated antiangiogenesis target.40 Meanwhile, at the large concentration of 200 nM, 8m could lead to over 90% inhibition of Ret kinase, FLT3 kinase, and

32


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VEGFR2, which is consistent with the conclusion above. Overall, our kinase profiling, as shown in Table 9, indicated the relatively low but acceptable kinase selectivity of the compound 8m. Table 9. Kinome Selectivity Profiles for compound 8m a 8m Kinases

@

20

nM

8m @ 200

8m Kinases

nM

@

20

nM

8m @ 200

Kinases

nM

8m

8m

@ 20

@ 200

nM

nM

Abl

3

26

FLT-4

38

85

PDGFRα

47

81

ALK

-15

35

Fms/CSFR

2

37

PDGFRβ

20

55

Akt1

38

40

FRK

8

11

PDK1

5

15

ALK4

6

27

Fyn

18

42

PEK

8

13

AMPKα

6

38

GCK

-5

26

PhKγ2

10

15

Arg

-3

30

GSK3β

8

48

Pim2

-5

1

AurA/Aur2

37

89

HER2/ErbB2

0

0

PKA

-10

0

Axl

-14

32

HIPK2

-4

48

PKN1

11

33

Brk

19

14

IGF1R

44

70

PRKX

-1

0

BRSK1

3

19

IKKα

8

7

PYK2

1

26

CaMK1α

2

8

IRAK1

10

41

B-Raf

3

5

CDK6

10

34

IRR

11

41

RAF-1

19

43

CHK1

12

2

JAK2

94

100

Ret

92

99

c-kit

6

35

JNK1

8

13

ROCK2

-3

-5

CLK1

14

52

VEGFR2

65

96

RSK1

1

26

33



a

Page 34 of 82

c-Met

5

37

Lck

1

1

SGK1

5

0

CRIK

-8

0

LIMK1

-13

0

Src

15

24

DCAMKL1

-11

9

Lyn A

10

11

Syk

8

66

DDR2

11

11

MEK5

16

33

TBK1

0

47

EGFR

-5

21

MARK1

8

10

TSSK1

-3

19

EphA1

0

27

MLK1

30

89

TTK

0

11

ERK1

-10

0

MNK1

0

0

TXK

6

26

FAK

7

38

MSK2

-2

17

Tyk2

4

68

FGFR1

-3

32

MST1

-4

23

ULK1

3

13

FGFR2

9

79

MYT1

-20

0

Wee1

23

38

FGFR3

36

65

NDR1

7

0

WNK2

10

19

FGFR4

-6

34

NEK2

4

15

Yes

18

44

FLT-1

-12

61

p38α

7

0

FLT-3

60

95

PAK2

-3

7

% inhibition of phosphorylation of substrates at the indicated test compound concentration, using a

LANCE or HTRF detection method supplied by Eurofins Cerep corporation. In these heat maps, inhibition 90% inhibition is assigned as red and 50%< inhibition 250 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.44 (s, 1H), 9.10 (s, 1H), 8.75 (s, 1H), 8.08 (d, J = 3.4 Hz, 1H), 7.78 (s, 3H), 7.39 (d, J = 8.0 Hz, 3H), 3.98 (s, 2H), 1.96 (t, J = 7.2 Hz, 2H), 1.79 – 1.53 (m, 2H), 1.44 (dd, J = 14.7, 7.5 Hz, 2H). 13C

NMR (101 MHz, DMSO-d6) δ 169.25, 155.61, 150.24, 130.07, 128.80, 123.87, 119.67, 51.51,

32.21, 30.08, 22.80. HRMS (AP-ESI) m/z calcd for C18H19ClFN7O2 [M + H]+ 420.1346, found 420.1346. Retention time: 12.9 min, eluted with 18% acetonitrile/82% water (containing 0.4% formic acid). 6-(4-((5-chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-Nhydroxyhexanamide (8w) Ethyl 6-(4-((5-chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-

48


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1H-pyrazol-1-yl)hexanoate (18w) was reacted using a procedure similar to the synthesis of compound 8a, affording 8w as a yellow solid, 42% yield. Mp. 190-192 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.23 (s, 1H), 8.94 (s, 1H), 8.70 (s, 1H), 8.06 (s, 1H), 7.94 – 7.07 (m, 6H), 3.83 (s, 2H), 1.91 (t, J = 7.3 Hz, 2H), 1.60 (s, 2H), 1.55 – 1.35 (m, 2H), 1.13 (s, 2H). 13C NMR (101 MHz, DMSOd6) δ 169.39, 157.62, 135.52, 123.44, 51.71, 32.59, 30.20, 26.09, 25.11. HRMS (AP-ESI) m/z calcd for C19H21ClFN7O2 [M + H]+ 434.1502, found 434.1503. Retention time: 15.3 min, eluted with 18% acetonitrile/82% water (containing 0.4% formic acid). 8-(4-((5-Chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-Nhydroxyoctanamide (8x) Ethyl 8-(4-((5-chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)1H-pyrazol-1-yl)octanoate (18x) was reacted using a procedure similar to the synthesis of compound 8a, affording 8x as a brown solid, 50% yield. Mp. 178-180 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.24 (s, 1H), 8.95 (s, 1H), 8.06 (s, 1H), 7.76 (s, 1H), 7.48 (s, 2H), 7.25 (s, 4H), 3.83 (s, 2H), 1.93 (t, J = 7.3 Hz, 2H), 1.58 (s, 2H), 1.47 (dd, J = 14.0, 7.0 Hz, 2H), 1.21 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 169.56, 157.67, 135.27, 123.44, 119.62, 119.25, 115.70, 115.49, 51.81, 32.71, 30.46, 28.96, 28.70, 26.37, 25.51. HRMS (AP-ESI) m/z calcd for C21H25ClFN7O2 [M + H]+ 462.1815, found 462.1812. Retention time: 9.0 min, eluted with 25% acetonitrile/75% water (containing 0.4% formic acid). 4-((4-((5-Chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)methyl)N-hydroxybenzamide

(8y)

Methyl

4-((4-((5-chloro-4-((4-fluorophenyl)amino)pyrimidin-2-

yl)amino)-1H-pyrazol-1-yl)methyl)benzoate (18y) was reacted using a procedure similar to the synthesis of compound 8a, affording 8y as a light yellow solid, 30% yield. Mp. 202-204 oC. 1H NMR

49



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(400 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.27 (s, 1H), 9.03 (s, 1H), 8.89 (s, 1H), 8.07 (s, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.48 (s, 4H), 7.15 (t, J = 8.6 Hz, 4H), 5.17 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.34, 157.62, 132.46, 130.79, 129.99, 127.56, 127.46, 124.15, 120.13, 115.64, 115.42, 55.02. HRMS (AP-ESI) m/z calcd for C21H17ClFN7O2 [M + H] + 454.1189, found 454.1192. Retention time: 18.5 min, eluted with 18% acetonitrile/82% water (containing 0.4% formic acid). (E)-3-(4-((4-((5-chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1yl)methyl)phenyl)-N-hydroxyacrylamide

(8z)

Methyl

(E)-3-(4-((4-((5-chloro-4-((4-

fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)methyl)phenyl)acrylate

(18z)

was

reacted using a procedure similar to the synthesis of compound 8a, affording 8z as a light yellow solid, 30% yield. Mp. 192-194 oC.1H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.88 (s, 1H), 8.06 (s, 1H), 7.86 – 6.90 (m, 11H), 6.46 (d, J = 15.9 Hz, 1H), 5.13 (s, 2H). 13C NMR (101 MHz, DMSOd6) δ 157.64, 134.61, 128.06, 124.13, 120.06, 119.65, 115.63, 115.41, 55.11, HRMS (AP-ESI) m/z calcd for C23H19ClFN7O2 [M + H]+ 480.1346, found 480.1349. Retention time: 9.3 min, eluted with 25% acetonitrile/75% water (containing 0.4% formic acid). 1H-Pyrazol-4-amine (11) To a solution of 4-nitro-1H-pyrazole (10, 5.0 g, 44.2 mmol) in ethanol (160 mL) was added Pd/C (0.8 g,10 wt. %). The reaction was stirred under the hydrogen atmosphere for 1~2 days. Upon completion of the reaction, the mixture was filtered through Celite and concentrated under reduced pressure to give compound 11 (3.6 g, yield 90%) as red oil, which became a red solid in a cold place. 1H-NMR (400 MHz, DMSO-d6) δ 11.87 (brs, 1H), 6.97 (s, 2H), 3.76 (br s, 2H). ESI-MS, m/z: 84.1 [M+H]+. tert-Butyl (1H-pyrazol-4-yl)carbamate (12) To a solution of 1H-pyrazol-4-amine (11, 3.0 g,

50


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36.1 mmol) in THF (7.5 g/160 mL) was slowly added with a solution of di-tert-butyl dicarbonate (8.6 g, 39.7 mmol) in THF (50 mL) and a solution of NaHCO3 (aq, 7.5 g/50 mL water). The reaction was stirred at room temperature for 1~2 days. Upon completion of the reaction, the resulting mixture was added 100 mL water and extracted with ethyl acetate (3 × 100 mL). The organic layer was combined, dried over sodium sulfate, and concentrated. The residue was stirred in hexane (160 mL) to give compound 12 (5.0 g, yield 75%) as a light brown solid. 1H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 9.07 (s,1H), 7.58 (s, 1H), 7.34 (s, 1H), 1.44 (s, 9H). ESI-MS, m/z: 184.1 [M+H]+. General

Procedure

for

the

Preparation

of

14a-14h.

Methyl

3-(4-((tert-

butoxycarbonyl)amino)-1H-pyrazol-1-yl)propanoate (14a) Cesium carbonate (3.5 g, 10.8 mmol) was added to a solution of methyl 3-bromopropanoate (13a, 1.07 g, 6.5 mmol) and tert-butyl (1Hpyrazol-4-yl) carbamate (12, 1.0 g, 5.43 mmol) in acetonitrile (150 mL). The resulting mixture was heated at 40-50 oC for 10 h. Upon completion of the reaction, evaporation of the solvent under reduced pressure gave a residue, which was dissolved in ethyl acetate (300 mL). The organic layer was washed with citric acid (100 mL), water (100 mL) and brine (100 mL), dried over sodium sulfate, filtered, and concentrated to give the crude product, which was purified by silica gel chromatography (petroleum ether/ethyl acetate, 5:1) to give compound 14a (1.2 g, yield 82%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 7.63 (s, 1H), 7.27 (s, 1H), 4.25 (t, J = 6.6 Hz, 2H), 3.59 (s, 3H), 2.82 (t, J = 6.6 Hz, 2H), 1.44 (s, 9H). ESI-MS m/z: 270.4 [M + H]+. Ethyl 4-(4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)butanoate (14b) Commercially available ethyl 4-bromobutanoate (13b) and tert-butyl (1H-pyrazol-4-yl) carbamate (12) were reacted using a procedure similar to the synthesis of 14a, affording compound 14b as colorless oil, yield 75%.

51



1H

Page 52 of 82

NMR (400 MHz, CDCl3) δ 7.66 (s, 1H), 7.32 (s, 1H), 6.41 (s, 1H), 4.13 (q, J = 7.1 Hz, 4H), 2.29

(t, J = 7.2 Hz, 2H), 2.15 (p, J = 6.9 Hz, 2H), 1.50 (s, 9H), 1.25 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 298.2 [M + H]+. Ethyl 5-(4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)pentanoate (14c) Commercially available ethyl 5-bromopentanoate (13c) and tert-butyl (1H-pyrazol-4-yl) carbamate (12) were reacted using a procedure similar to the synthesis of 14a, affording compound 14c as colorless oil, yield 71%. 1H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1H), 7.67 (s, 1H), 7.31 (s, 1H), 4.07 – 4.02 (m, 2H), 4.00 (d, J = 6.6 Hz, 2H), 2.38 – 2.22 (m, 2H), 1.77 – 1.63 (m, 2H), 1.44 (s, 9H), 1.40 (d, J = 7.5 Hz, 2H), 1.16 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 312.2 [M + H]+. Ethyl 6-(4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl) hexanoate (14d) Commercially available ethyl 6-bromohexanoate (13d) and tert-butyl (1H-pyrazol-4-yl) carbamate (12) were reacted using a procedure similar to the synthesis of 14a, affording compound 14d as colorless oil, 76% yield. 1H

NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1H), 7.62 (s, 1H), 7.25 (s, 1H), 4.06 – 4.01 (m, 2H), 4.01

– 3.95 (m, 2H), 2.25 (t, J = 7.4 Hz, 2H), 1.76 – 1.64 (m, 2H), 1.54 – 1.48 (m, 2H), 1.44 (s, 9H), 1.22 – 1.13 (m, 5H). ESI-MS m/z: 326.2 [M + H]+. Ethyl 7-(4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)heptanoate (14e) Commercially available ethyl 7-bromoheptanoate (13e) and tert-butyl (1H-pyrazol-4-yl) carbamate (12) were reacted using a procedure similar to the synthesis of 14a, affording compound 14e as colorless oil, 78% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 7.60 (s, 1H), 7.23 (s, 1H), 4.05 – 4.00 (m, 2H), 3.99 – 3.94 (m, 2H), 2.23 (t, J = 7.4 Hz, 2H), 1.67 (p, J = 7.1 Hz, 2H), 1.49 – 1.44 (m, 2H), 1.42 (s, 9H), 1.27 – 1.21 (m, 2H), 1.15 (td, J = 7.2, 3.6 Hz, 5H). ESI-MS m/z: 340.3 [M + H]+.

52


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Ethyl 8-(4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)octanoate (14f) Commercially available ethyl 8-bromooctanoate (13f) and tert-butyl (1H-pyrazol-4-yl) carbamate (12) were reacted using a procedure similar to the synthesis of 14a, affording compound 14f as colorless oil, 84% yield. ESI-MS m/z: 354.3 [M + H]+. Methyl

4-((4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)methyl)benzoate

(14g)

Commercially available methyl 4-(bromomethyl)benzoate (13g) and tert-butyl (1H-pyrazol-4-yl) carbamate (12) were reacted using a procedure similar to the synthesis of 14a, affording compound 14g as a white solid, 55% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.78 (s, 1H), 7.36 – 7.25 (m, 3H), 5.34 (s, 2H), 3.84 (s, 3H), 1.43 (s, 9H). ESI-MS m/z: 332.2 [M + H]+. Methyl

(E)-3-(4-((4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)methyl)

phenyl)acrylate (14h) Commercially available (E)-3-(4-(bromomethyl)phenyl)acrylate (13h) and tert-butyl (1H-pyrazol-4-yl) carbamate (12) were reacted using a procedure similar to the synthesis of 14a, affording compound 14h as a white solid, 51% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 7.74 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 16.1 Hz, 1H), 7.31 (s, 1H), 7.22 (d, J = 8.1 Hz, 2H), 6.62 (d, J = 16.0 Hz, 1H), 5.26 (s, 2H), 3.72 (s, 3H), 1.43 (s, 9H). ESI-MS m/z: 358.2 [M + H]+. General Procedure for the Preparation of 15a-15h. Methyl 3-(4-amino-1H-pyrazol-1yl)propanoate hydrochloride (15a) A mixture of methyl 3-(4-((tert-butoxycarbonyl)amino)-1Hpyrazol-1-yl)propanoate (14a, 0.6 g, 2.23 mmol) in HCl/ethyl acetate (25 mL) was stirred at room temperature for 6~10 h. The white precipitate was filtered to give the pure compound 15a (0.4 g,

53



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yield 87%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 3H), 7.96 (s, 1H), 7.53 (s, 1H), 4.36 (t, J = 6.6 Hz, 2H), 3.59 (s, 3H), 2.88 (t, J = 6.6 Hz, 2H). ESI-MS m/z: 170.1 [M + H]+. Ethyl 4-(4-amino-1H-pyrazol-1-yl)butanoate hydrochloride (15b) Ethyl 4-(4-((tertbutoxycarbonyl)amino)-1H-pyrazol-1-yl)butanoate (14b) was reacted using a procedure similar to the synthesis of 15a, affording compound 15b as a white solid, 85% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 3H), 7.97 (s, 1H), 7.54 (s, 1H), 4.15 (t, J = 6.9 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H), 1.99 (p, J = 7.1 Hz, 2H), 1.17 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 198.2 [M + H]+. Ethyl 5-(4-amino-1H-pyrazol-1-yl)pentanoate hydrochloride (15c) Ethyl 5-(4-((tertbutoxycarbonyl)amino)-1H-pyrazol-1-yl)pentanoate (14c) was reacted using a procedure similar to the synthesis of 15a, affording compound 15c as a white solid, 80% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 3H), 7.97 (s, 1H), 7.53 (s, 1H), 4.12 (t, J = 6.9 Hz, 2H), 4.04 (q, J = 7.1 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.87 – 1.68 (m, 2H), 1.44 (dt, J = 15.1, 7.6 Hz, 2H), 1.17 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 212.2 [M + H]+. Ethyl 6-(4-amino-1H-pyrazol-1-yl)hexanoate hydrochloride (15d) Ethyl 6-(4-((tertbutoxycarbonyl)amino)-1H-pyrazol-1-yl) hexanoate (14d) was reacted using a procedure similar to the synthesis of 15a, affording compound 15d as a white solid, 75% yield.1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 3H), 7.97 (s, 1H), 7.52 (s, 1H), 4.10 (t, J = 7.0 Hz, 2H), 4.04 (q, J = 7.1 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 1.82 – 1.65 (m, 2H), 1.61 – 1.44 (m, 2H), 1.19 (dt, J =14.2, 7.6 Hz, 5H). ESI-MS m/z: 226.3 [M + H]+. Ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate hydrochloride (15e) Ethyl 7-(4-((tert-

54


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butoxycarbonyl)amino)-1H-pyrazol-1-yl)heptanoate (14e) was reacted using a procedure similar to the synthesis of 15a, affording compound 15e as a white solid, 63% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 3H), 7.95 (s, 1H), 7.52 (s, 1H), 4.10 (t, J = 7.0 Hz, 2H), 4.04 (q, J = 7.1 Hz, 2H), 2.22 (s, 2H), 1.82 – 1.65 (m, 2H), 1.57 – 1.40 (m, 2H), 1.28 (dd, J = 15.2, 7.3 Hz, 2H), 1.24 – 1.19 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 240.1 [M + H]+. Ethyl

8-(4-amino-1H-pyrazol-1-yl)octanoate

hydrochloride

(15f)

Ethyl

8-(4-((tert-

butoxycarbonyl)amino)-1H-pyrazol-1-yl)octanoate (14f) was reacted using a procedure similar to the synthesis of 15a, affording compound 15f as a white solid, 93% yield. ESI-MS m/z: 254.1 [M + H]+. Methyl 4-((4-amino-1H-pyrazol-1-yl)methyl)benzoate hydrochloride (15g) Methyl 4-((4((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)methyl)benzoate (14g) was reacted using a procedure similar to the synthesis of 15a, affording compound 15g as a white solid, 75% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 3H), 8.13 (s, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.60 (s, 1H), 7.35 (d, J = 8.3 Hz, 2H), 5.46 (s, 2H), 3.84 (s, 3H). ESI-MS m/z: 232.1 [M + H]+. Methyl (E)-3-(4-((4-amino-1H-pyrazol-1-yl)methyl)phenyl)acrylate hydrochloride (15h) Methyl (E)-3-(4-((4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)methyl) phenyl)acrylate (14h) was reacted using a procedure similar to the synthesis of 15a, affording compound 15h as a white solid, 63% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.12 (d, J = 39.7 Hz, 3H), 8.09 (s, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 16.1 Hz, 1H), 7.58 (s, 1H), 7.27 (d, J = 8.2 Hz, 2H), 6.63 (d, J = 16.1 Hz, 1H), 5.38 (s, 2H). ESI-MS m/z: 258.3 [M + H]+. 2,5-Dichloro-N-(3-chlorophenyl)pyrimidin-4-amine

(17a)

To

a

solution

of

2,4,5-

trichloropyrimidine (16a, 0.6 g, 3.3 mmol) and 3-chloroaniline (0.48 g, 3.8 mmol) in ethanol (30 mL)

55



Page 56 of 82

was added Na2CO3 (1.4 g, 13.2 mmol). The mixture was stirred at room temperature overnight. When the reaction was complete as determined by TLC, the resulting mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (120 mL), and washed with water (50 mL), 1 M citric acid (50 mL), and brine (100 mL), and then dried over sodium sulfate. Filtration and solvent evaporation gave the crude product, which was purified by silica gel chromatography (petroleum ether/ethyl acetate, 5:1) to afford 17a (0.4 g, yield 44%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.43 (s, 1H), 7.76 (s, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H). ESI-MS m/z: 274.5 [M + H]+. 2,5-Dichloro-N-(4-chlorophenyl)pyrimidin-4-amine

(17b)

N-ethyl-N-isopropylpropan-2-

amine (1.3 g, 10.0 mmol) was added to a solution of 2,4,5-trichloropyrimidine (16a, 1.0 g, 5.5 mmol) and 4-chloroaniline (1.27 g, 10 mmol) in DMF (30 mL). The mixture was stirred at room temperature overnight. When the reaction was completed as detected by TLC, the solution was poured into ethyl acetate (150 mL) and washed with water (50 mL×3),1 M citric acid (50 mL), and brine (100 mL), and then dried over sodium sulfate. Filtration and solvent evaporation gave the crude product, which was purified by silica gel chromatography (petroleum ether/ethyl acetate, 5:1) to afford 17b (0.9 g, yield 60%) as a white solid. 1H-NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.41 (s, 1H), 7.72 – 7.56 (m, 2H), 7.51 – 7.39 (m, 2H). ESI-MS, m/z: 274.7 [M + H]+. 2,5-Dichloro-N-(2-chlorophenyl)pyrimidin-4-amine

(17c)

N-ethyl-N-isopropylpropan-2-

amine (1.0 g, 7.7 mmol) was added to a solution of 2,4,5-trichloropyrimidine (16a, 1.0 g, 5.5 mmol) and 2-chloroaniline (0.74 g, 5.5 mmol) in NMP (20 mL). The mixture was stirred at 100 oC overnight. When the reaction was complete as determined by TLC, the solution was poured into ethyl acetate

56


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(150 mL) and washed with water (50 mL×3), 1 M citric acid (50 mL), and brine (100 mL) and then dried over sodium sulfate. Filtration and solvent evaporation gave the crude product, which was purified by silica gel chromatography (petroleum ether/ethyl acetate, 50:1) to afford 17c (0.6 g, yield 40%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.40 (s, 1H), 7.60 (dd, J = 7.8, 1.1 Hz, 1H), 7.52 (dd, J = 7.8, 1.4 Hz, 1H), 7.40 (m, 2H). ESI-MS m/z: 274.6 [M + H]+. 2,5-Dichloro-N-phenylpyrimidin-4-amine (17d) 2,4,5-Trichloropyrimidine (16a) and aniline were reacted using a procedure similar to the synthesis of compound 17b, affording 17d as a white solid, 77% yield. 1H-NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.86 – 8.36 (s, 1H), 8.09 – 7.15 (m, 5H). ESI-MS, m/z: 240.5 [M + H]+. 2,5-Dichloro-N-(4-methoxyphenyl)pyrimidin-4-amine (17e) 2,4,5-Trichloropyrimidine (16a) and 4-methoxyaniline were reacted using a procedure similar to the synthesis of compound 17b, affording 17e as a gold yellow solid, 82% yield. 1H-NMR (400 MHz, DMSO-d6) δ 9.44 (s, 1H), 8.32 (s, 1H), 7.43 (d, J = 8.9 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 3.77 (s, 3H). ESI-MS, m/z: 270.6 [M + H]+. 2,5-Dichloro-N-(4-fluorophenyl)pyrimidin-4-amine (17f) 2,4,5-Trichloropyrimidine (16a) and 4-fluoroaniline were reacted using a procedure similar to the synthesis of compound 17b, affording 17f as an off-white solid, 57% yield. 1H-NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 8.37 (d, J = 3.7 Hz, 1H), 7.66 – 7.53 (m, 2H), 7.31 – 7.18 (m, 2H). ESI-MS, m/z: 258.3 [M + H]+. 4-(2,5-Dichloropyrimidin-4-yl)morpholine (17g) To a solution of 2,4,5-trichloropyrimidine (16a, 1.0 g, 5.5 mmol) in ethanol (30 mL) was slowly added morpholine (0.87 g, 10 mmol) in ice bath. The mixture was stirred for about 3h, when the reaction was complete as determined by TLC,

57



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the resulting mixture was concentrated under reduced pressure. The residue was washed with 20 mL petroleum ether to obtain 17g (0.9 g, yield 70 %) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 3.84 – 3.75 (m, 2H), 3.10 – 3.02 (m, 2H). ESI-MS m/z: 234.5 [M + H]+. 2,5-Dichloro-4-(piperidin-1-yl)pyrimidine

(17h)

2,4,5-Trichloropyrimidine

(16a)

and

piperidine were reacted using a procedure similar to the synthesis of 17g, affording compound 17h as a white solid, 78% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.27 (s, 1H), 3.69 (d, J = 5.7 Hz, 4H), 1.61 (d, J = 7.9 Hz, 6H). ESI-MS m/z: 232.3 [M + H]+. 2,5-Dichloro-N-cyclopropylpyrimidin-4-amine (17i) 2,4,5-Trichloropyrimidine (16a) and cyclopropanamine were reacted using a procedure similar to the synthesis of 17g, affording compound 17i as a white solid, 63% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 3H), 2.53 (dd, J = 7.6, 4.2 Hz, 1H), 0.78 – 0.72 (m, 2H), 0.71 – 0.63 (m, 2H). ESI-MS m/z: 204.2 [M + H]+. 2,5-Dichloro-N-propylpyrimidin-4-amine (17j) 2,4,5-Trichloropyrimidine (16a) and propan1-amine were reacted using a procedure similar to the synthesis of 17g, affording compound 17j as a white solid, 73% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.19 (s, 3H), 2.82 – 2.60 (m, 2H), 1.74 – 1.47 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H). ESI-MS m/z: 206.3 [M + H]+. 2,5-Dichloro-N-methylpyrimidin-4-amine

(17k)

2,4,5-Trichloropyrimidine

(16a)

and

CH3NH2/Ethanol were reacted using a procedure similar to the synthesis of 17g, affording compound 17k as a white sold, 83% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.13 (s, 1H), 7.92 (br, 1H), 2.86 (d, J = 4.8 Hz, 3H). ESI-MS m/z: 177.6 [M + H]+. 2-Chloro-N-(4-fluorophenyl)pyrimidin-4-amine (17l) 2,4-Dichloropyrimidine (16b) and 4fluoroaniline were reacted using a procedure similar to the synthesis of compound 17a, affording 17l

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as a white solid, 40% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 8.15 (d, J = 5.9 Hz, 1H), 7.59 (dd, J = 8.8, 5.0 Hz, 2H), 7.29 – 7.16 (m, 2H), 6.71 (d, J = 5.9 Hz, 1H). ESI-MS m/z: 224.4 [M + H]+. 2-Chloro-5-fluoro-N-(4-fluorophenyl)pyrimidin-4-amine (17m) The solution of 2,4-dichloro5-fluoropyrimidine (16c, 1.0 g, 6.1 mmol) and 4-fluoroaniline (0.75 g, 6.7 mmol) in the mixed reagents (30 mL water/10 mL methanol) was stirred at 50 oC for 5 h until a lot of white precipitate appeared. The white precipitate was filtered and washed with 20 mL petroleum ether. The precipitate was collected and dried in air to afford 17m (1.0 g, yield 68%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.32 (d, J = 3.5 Hz, 1H), 7.82 – 7.60 (m, 2H), 7.32 – 7.18 (m, 2H). ESIMS m/z: 242.2 [M + H]+. 2-Chloro-N-(4-fluorophenyl)-5-(trifluoromethyl)pyrimidin-4-amine (17n) The solution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine (16d, 1.2 g, 5.5 mmol) and 4-fluoroaniline (0.74 g, 6.6 mmol) in 50 mL ethanol was stirred at -50 oC for 1h. When the reaction was complete as determined by TLC, the resulting mixture was concentrated under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether/ethyl acetate, 10:1) to afford 17n (0.4 g, 25%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.58 (s, 1H), 7.45 (dd, J = 8.7, 5.0 Hz, 2H), 7.25 (t, J = 8.7 Hz, 2H). ESI-MS m/z: 292.3 [M + H]+. 2-Chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine

(17o)

2,4-Dichloro-5-

(trifluoromethyl)pyrimidine (16d) and methylamine in ethanol were reacted using a procedure similar to the synthesis of compound 17n, affording 17o as a white solid, 30% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 8.04 (s, 1H), 3.00 (d, J = 4.5 Hz, 3H). ESI-MS m/z: 212.1 [M + H]+.

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General Procedure for the Preparation of 18a-18z. Ethyl 7-(4-((5-chloro-4-((3chlorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)heptanoate (18a) To a solution of 2,5-dichloro-N-(3-chlorophenyl)pyrimidin-4-amine (17a, 0.25 g, 0.91 mmol) and ethyl 7-(4-amino1H-pyrazol-1-yl)heptanoate hydrochloride (15e, 0.16 g, 0.59 mmol) in butanol (12 mL) was added trifluoroacetic acid (0.5 mL). The mixture was irradiated in a microwave (150 W) at 130 oC for 3 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane / methanol/ TEA, 100: 1: 0.25) to afford 18a (0.15 g, yield 53%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.96 (s, 1H), 8.10 (s, 1H), 7.50 – 7.29(m, 6H), 4.02 (q, J = 7.1 Hz, 2H), 3.89 (d, J = 6.8 Hz, 2H), 2.20 (dt, J = 21.5, 7.4 Hz, 2H), 1.60 (s, 2H), 1.45 (ddd, J = 12.3, 7.1, 3.4 Hz, 2H), 1.22 (t, J = 7.7 Hz, 2H), 1.15 (dd, J = 6.7, 5.3 Hz, 5H). ESI-MS m/z: 477.3 [M + H]+. Ethyl

7-(4-((5-chloro-4-((4-chlorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-

yl)heptanoate (18b) 2,5-Dichloro-N-(4-chlorophenyl)pyrimidin-4-amine (17b) and ethyl 7-(4amino-1H-pyrazol-1-yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18b as a white solid, 60% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 8.94 (s, 1H), 8.09 (s, 1H), 7.99 – 7.14 (m, 6H), 3.99 (t, J = 6.6 Hz, 2H), 3.89 (s, 2H), 2.24 (t, J = 7.3 Hz, 2H), 1.60 – 1.41 (m, 4H), 1.38 – 1.21 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS m/z: 477.6 [M + H]+. Ethyl


yl)heptanoate (18c) 2,5-Dichloro-N-(2-chlorophenyl)pyrimidin-4-amine (17c) and ethyl 7-(4amino-1H-pyrazol-1-yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the

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synthesis of compound 18a, affording 18c as a white solid, 38% yield. 1H NMR (400 MHz, DMSOd6) δ 9.21 (s, 1H), 8.91 (s, 1H), 8.04 (s, 1H), 7.95 – 7.23 (m, 4H), 7.09 (s, 1H), 6.87 (s, 1H), 4.02 (q, J = 7.1 Hz, 2H), 3.69 (s, 2H), 2.22 (dt, J = 14.2, 7.3 Hz, 2H), 1.74 – 1.34 (m, 4H), 1.22 (t, J = 7.8 Hz, 2H), 1.15 (dt, J = 7.1, 4.0 Hz, 5H). ESI-MS m/z: 477.5 [M + H]+. Ethyl 7-(4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)heptanoate (18d) 2,5-Dichloro-N-phenylpyrimidin-4-amine (17d) and ethyl 7-(4-amino-1H-pyrazol-1yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18d as a white solid, 60% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.84 (s, 1H), 8.06 (s, 1H), 7.50 (d, J = 19.1 Hz, 2H), 7.40 (t, J = 7.4 Hz, 3H), 7.23 – 7.13 (m, 2H), 4.06 – 3.95 (m, 2H), 3.85 (s, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.56 – 1.47 (m, 4H), 1.28 – 1.14 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS m/z: 443.3 [M + H] +. Ethyl

7-(4-((5-chloro-4-((4-methoxyphenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-

yl)heptanoate (18e) 2,5-Dichloro-N-(4-methoxyphenyl)pyrimidin-4-amine (17e) and ethyl 7-(4amino-1H-pyrazol-1-yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18e as a white solid, 53% yield. 1H NMR (400 MHz, DMSOd6) δ 9.16 (s, 1H), 8.76 (s, 1H), 8.01 (s, 1H), 7.28 – 7.18 (m, 4H), 6.99 (d, J = 7.2 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 3.79 (s, 5H), 2.25 (t, J = 7.3 Hz, 2H), 1.56 – 1.47 (m, 4H), 1.31 (dq, J = 14.6, 7.4 Hz, 4H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 473.5 [M + H]+. Ethyl

7-(4-((5-chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-

yl)heptanoate (18f) 2,5-Dichloro-N-(4-fluorophenyl)pyrimidin-4-amine (17f) and ethyl 7-(4-amino1H-pyrazol-1-yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the

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synthesis of compound 18a, affording 18f as a white solid, 63% yield. 1H NMR (400 MHz, DMSOd6) δ 10.46 (s, 1H), 10.16 (s, 1H), 8.30 (s, 1H), 7.67 – 7.21 (m, 6H), 4.07 – 3.94 (m, 2H), 3.81 (s, 2H), 2.35 – 2.14 (m, 2H), 1.58 – 1.44 (m, 4H), 1.37 – 1.19 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H). ESI-MS m/z: 461.3 [M + H]+. Ethyl 7-(4-((5-chloro-4-morpholinopyrimidin-2-yl)amino)-1H-pyrazol-1-yl) heptanoate (18g) 4-(2,5-Dichloropyrimidin-4-yl)morpholine (17g) and ethyl 7-(4-amino-1H-pyrazol-1yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18g as a white solid, 23% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H), 8.05 (s, 1H), 7.76 (s, 1H), 7.45 (s, 1H), 4.01 (dt, J = 13.3, 6.7 Hz, 4H), 3.79 – 3.67 (m, 4H), 3.63 – 3.50 (m, 4H), 2.25 (t, J = 7.3 Hz, 2H), 1.77 – 1.66 (m, 2H), 1.53 – 1.49 (m, 2H), 1.36 – 1.23 (m, 4H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 437.2 [M + H]+. Ethyl 7-(4-((5-chloro-4-(piperidin-1-yl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)heptanoate (18h) 2,5-Dichloro-4-(piperidin-1-yl)pyrimidine (17h) and ethyl 7-(4-amino-1H-pyrazol-1yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18h as a white solid, 60% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.99 (s, 1H), 7.77 (s, 1H), 7.45 (s, 1H), 4.10 – 3.93 (m, 4H), 3.55 (s, 4H), 2.25 (t, J = 7.3 Hz, 2H), 1.77 – 1.66 (m, 2H), 1.63 (s, 6H), 1.53 – 1.48 (m, 2H), 1.29 (ddd, J = 16.3, 12.1, 6.9 Hz, 4H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 435.4 [M + H]+. Ethyl

7-(4-((5-chloro-4-(cyclopropylamino)pyrimidin-2-yl)amino)-1H-pyrazol-1-

yl)heptanoate (18i) 2,5-Dichloro-N-cyclopropylpyrimidin-4-amine (17i) and ethyl 7-(4-amino-1Hpyrazol-1-yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis

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of compound 18a, affording 18i as a white solid, 36% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 7.96 (s, 1H), 7.85 (s, 1H), 7.49 (s, 1H), 7.15 (s, 1H), 3.99 (t, J = 6.6 Hz, 4H), 2.90 – 2.78 (m, 1H), 2.25 (t, J = 7.3 Hz, 2H), 1.81 – 1.64 (m, 2H), 1.53 – 1.48 (m, 2H), 1.29 (ddd, J = 24.5, 16.1, 8.7 Hz, 4H), 0.87 (t, J = 7.4 Hz, 3H), 0.76 (d, J = 5.2 Hz, 2H), 0.71 – 0.58 (m, 2H). ESI-MS m/z: 407.3 [M + H]+. Ethyl 7-(4-((5-chloro-4-(propylamino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)heptanoate (18j)

2,5-Dichloro-N-propylpyrimidin-4-amine

(17j)

and

ethyl

7-(4-amino-1H-pyrazol-1-

yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18j as a white solid, 48% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 1H), 7.84 (s, 1H), 7.77 (s, 1H), 7.43 (s, 1H), 7.10 (s, 1H), 3.99 (dd, J = 13.1, 6.6 Hz, 4H), 3.36 (dd, J = 9.0, 4.8 Hz, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.79 – 1.66 (m, 2H), 1.60 (td, J = 14.8, 7.5 Hz, 2H), 1.53 – 1.48 (m, 2H), 1.36 – 1.24 (m, 4H), 0.89 (dt, J = 14.6, 7.4 Hz, 6H). ESI-MS m/z: 409.2 [M + H]+. Ethyl 7-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)heptanoate (18k) 2,5-Dichloro-N-methylpyrimidin-4-amine (17k) and ethyl 7-(4-amino-1H-pyrazol-1yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18k as a white solid, 64% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 7.84 (s, 1H), 7.81 (s, 1H), 7.46 (s, 1H), 7.08 (s, 1H), 4.18 – 3.89 (m, 4H), 2.91 (s, 3H), 2.25 (t, J = 7.3 Hz, 2H), 1.81 – 1.64 (m, 2H), 1.50 (td, J = 14.5, 7.2 Hz, 2H), 1.38 – 1.20 (m, 4H), 1.16 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 381.4 [M + H]+. Ethyl


yl)hexanoate (18l) 2,5-Dichloro-N-(3-chlorophenyl)pyrimidin-4-amine (17a) and ethyl 6-(4-amino-

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1H-pyrazol-1-yl)hexanoate hydrochloride (15d) were reacted using a procedure similar to the synthesis of compound 18a, affording 18l as a white solid, 43% yield. 1H NMR (400 MHz, DMSOd6) δ 9.26 (s, 1H), 8.95 (s, 1H), 8.11 (s, 1H), 7.95 – 7.01 (m, 6H), 3.98 (t, J = 6.6 Hz, 2H), 3.92 (s, 2H), 2.24 (t, J = 7.3 Hz, 2H), 1.64 (s, 2H), 1.30 (dq, J = 14.5, 7.3 Hz, 2H), 1.18 (d, J = 6.6 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 463.3 [M + H]+. Ethyl


yl)hexanoate (18m) 2,5-Dichloro-N-(4-chlorophenyl)pyrimidin-4-amine (17b) and ethyl 6-(4amino-1H-pyrazol-1-yl)hexanoate hydrochloride (15d) were reacted using a procedure similar to the synthesis of compound 18a, affording 18m as a white solid, 37% yield. 1H NMR (400 MHz, DMSOd6) δ 9.22 (s, 1H), 8.93 (s, 1H), 8.09 (s, 1H), 7.94 – 7.18 (m, 6H), 4.08 – 3.96 (m, 2H), 3.84 (s, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.65 (s, 2H), 1.38 – 1.23 (m, 2H), 1.20 (dd, J = 13.6, 6.6 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 463.2 [M + H]+. Ethyl


yl)hexanoate (18n) 2,5-Dichloro-N-(2-chlorophenyl)pyrimidin-4-amine (17c) and ethyl 6-(4-amino1H-pyrazol-1-yl)hexanoate hydrochloride (15d) were reacted using a procedure similar to the synthesis of compound 18a, affording 18n as a white solid, 50% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 8.93 (s, 1H), 8.09 (s, 1H), 7.94 – 7.18 (m, 6H), 4.08 – 3.96 (m, 2H), 3.84 (s, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.65 (s, 2H), 1.38 – 1.23 (m, 2H), 1.20 (dd, J = 13.6, 6.6 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 463.3 [M + H]+. Ethyl

6-(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)hexanoate

(18o) 2,5-Dichloro-N-methylpyrimidin-4-amine (17k) and ethyl 6-(4-amino-1H-pyrazol-1-

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yl)hexanoate hydrochloride (15d) were reacted using a procedure similar to the synthesis of compound 18a, affording 18o as a white solid, 57% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 7.83 (d, J = 10.7 Hz, 2H), 7.46 (s, 1H), 7.03 (s, 1H), 4.00 (dt, J = 13.2, 6.8 Hz, 4H), 2.91 (d, J = 4.2 Hz, 3H), 2.26 (t, J = 7.3 Hz, 2H), 1.80 – 1.65 (m, 2H), 1.52 (dd, J = 6.9, 2.2 Hz, 2H), 1.27 – 1.17 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 367.2 [M + H]+. Ethyl 7-(4-((4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)heptanoate (18p) 2-Chloro-N-(4-fluorophenyl)pyrimidin-4-amine (17l) and ethyl 7-(4-amino-1H-pyrazol-1yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18p as a white solid, 53% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.27 (s, 1H), 8.98 (s, 1H), 7.96 (d, J = 5.7 Hz, 1H), 7.71-7.69 (m, 3H), 7.41 (s, 1H), 7.15 (t, J = 8.6 Hz, 2H), 6.08 (d, J = 5.7 Hz, 1H), 4.86 (q, J = 6.3 Hz, 2H), 3.97 (t, J = 7.2 Hz, 2H), 2.20 (t, J = 7.3 Hz, 2H), 1.69 (t, J = 7.3 Hz, 2H), 1.48 (t, J = 7.4 Hz, 2H), 1.37 – 1.18 (m, 4H), 1.15 (d, J = 6.3 Hz, 3H). ESIMS m/z: 427.2 [M + H]+. Ethyl

7-(4-((5-fluoro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-

yl)heptanoate (18q) 2-Chloro-5-fluoro-N-(4-fluorophenyl)pyrimidin-4-amine (17m) and ethyl 7-(4amino-1H-pyrazol-1-yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18q as a white solid, 32% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H), 9.00 (s, 1H), 8.03 (d, J = 3.8 Hz, 1H), 7.70 (s, 3H), 7.33 (s, 1H), 7.19 (t, J = 8.8 Hz, 2H), 4.05 – 3.91 (m, 4H), 2.25 (t, J = 7.3 Hz, 2H), 1.73 – 1.60 (m, 2H), 1.53 – 1.48 (m, 2H), 1.29 (ddd, J = 24.6, 16.1, 8.4 Hz, 4H), 0.88 (dd, J = 7.3, 4.2 Hz, 3H). ESI-MS m/z: 445.4 [M + H]+. Ethyl

7-(4-((4-((4-fluorophenyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-

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pyrazol-1-yl)heptanoate (18r) 2-Chloro-N-(4-fluorophenyl)-5-(trifluoromethyl)pyrimidin-4-amine (17n) and ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18r as a white solid, 20% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.81 (s, 1H), 8.26 (s, 1H), 7.44 – 7.35 (m, 2H), 7.29 (t, J = 8.7 Hz, 2H), 7.18 (s, 1H), 7.07 (s, 1H), 4.00 (t, J = 6.5 Hz, 2H), 3.76 (t, J = 6.9 Hz, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.52 (dt, J = 6.5, 5.3 Hz, 4H), 1.29-1.19 (m, 4H), 0.93 – 0.82 (m, 3H). ESI-MS m/z: 495.2 [M + H]+. Ethyl

7-(4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-

yl)heptanoate (18s) 2-Chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (17o) and ethyl 7-(4amino-1H-pyrazol-1-yl)heptanoate hydrochloride (15e) were reacted using a procedure similar to the synthesis of compound 18a, affording 18s as a white solid, 54% yield. 1H NMR (400 MHz, DMSOd6) δ 9.55 (s, 1H), 8.08 (s, 1H), 7.85 (s, 1H), 7.53 (s, 1H), 7.07 (s, 1H), 4.01 (dt, J = 13.2, 6.4 Hz, 4H), 2.95 (s, 3H), 2.25 (t, J = 7.3 Hz, 2H), 1.83 – 1.57 (m, 2H), 1.53 – 1.48 (m, 2H), 1.37 – 1.23 (m, 4H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 415.2 [M + H]+. Methyl


yl)propanoate (18t) 2,5-dichloro-N-(4-fluorophenyl)pyrimidin-4-amine (17f) and methyl 3-(4amino-1H-pyrazol-1-yl)propanoate hydrochloride (15a) were reacted using a procedure similar to the synthesis of compound 18a, affording 18t as a white solid, 22% yield. 1H NMR (400 MHz, DMSOd6) δ 9.22 (s, 1H), 8.91 (s, 1H), 8.07 (s, 1H), 7.90 – 7.10 (m, 6H), 4.13 (s, 3H), 3.99 (t, J = 6.5 Hz, 2H), 2.76 (s, 2H). ESI-MS m/z: 391.1 [M + H]+. Ethyl


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yl)butanoate (18u) 2,5-Dichloro-N-(4-fluorophenyl)pyrimidin-4-amine (17f) and ethyl 4-(4-amino1H-pyrazol-1-yl)butanoate hydrochloride (15b) were reacted using a procedure similar to the synthesis of compound 18a, affording 18u as a white solid, 58% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.93 (s, 1H), 8.06 (s, 1H) 7.90 – 7.10 (m, 6H), 4.86 (p, J = 6.3 Hz, 2H), 3.83 (s, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.80 (s, 2H), 1.17 (d, J = 6.4 Hz, 3H). ESI-MS m/z: 419.1 [M + H]+. Ethyl


yl)pentanoate (18v) 2,5-Dichloro-N-(4-fluorophenyl)pyrimidin-4-amine (17f) and ethyl 5-(4-amino1H-pyrazol-1-yl)pentanoate hydrochloride (15c) were reacted using a procedure similar to the synthesis of compound 18a, affording 18v as a white solid, 49% yield. 1H NMR (400 MHz, DMSOd6) δ 10.57 (s, 1H), 9.46 (s, 1H), 9.12 (s, 1H), 8.08 (d, J = 3.6 Hz, 1H), 7.77 (s, 2H), 7.39 (d, J = 8.3 Hz, 3H), 4.01 (t, J =6.8 Hz, 4H), 2.29 (t, J = 7.3 Hz, 2H), 1.87 – 1.61 (m, 2H), 1.49 (dd, J = 15.9, 7.9 Hz, 2H), 1.18 – 1.10 (m, 3H). ESI-MS m/z: 433.3 [M + H]+. Ethyl


yl)hexanoate (18w) 2,5-Dichloro-N-(4-fluorophenyl)pyrimidin-4-amine (17f) and ethyl 6-(4-amino1H-pyrazol-1-yl)hexanoate hydrochloride (15d) were reacted using a procedure similar to the synthesis of compound 18a, affording 18w as a white solid, yield 38%. 1H NMR (400 MHz, DMSOd6) δ 9.23 (s, 1H), 8.93 (s, 1H), 8.06 (s, 1H), 7.77 (s, 1H), 7.48 (s, 2H), 7.25 (s, 3H), 4.86 (p, J = 6.2 Hz, 2H), 3.83 (s, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.60 (s, 2H), 1.50 (t, J = 7.7 Hz, 2H), 1.15 (d, J = 6.2 Hz, 5H). ESI-MS m/z: 447.2 [M + H]+. Ethyl


yl)octanoate (18x) 2,5-Dichloro-N-(4-fluorophenyl)pyrimidin-4-amine

(17f) and ethyl 8-(4-

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amino-1H-pyrazol-1-yl)octanoate hydrochloride (15f) were reacted using a procedure similar to the synthesis of compound 18a, affording 18x as a white solid, 38% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.93 (s, 1H), 8.06 (s, 1H), 7.61 – 7.50 (m, 2H), 7.24 (s, 4H), 3.99 (t, J = 6.6 Hz, 2H), 3.83 (s, 2H), 2.26 (t, J = 7.3 Hz, 2H), 1.55 – 1.46 (m, 4H), 1.31 (dt, J = 14.8, 7.4 Hz, 2H), 1.24 (d, J = 14.0 Hz, 4H), 0.87 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 478.2 [M + H]+. Methyl

4-((4-((5-chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-

yl)methyl)benzoate (18y) 2,5-Dichloro-N-(4-fluorophenyl)pyrimidin-4-amine (17f) and methyl 4((4-amino-1H-pyrazol-1-yl)methyl)benzoate hydrochloride (15g) were reacted using a procedure similar to the synthesis of compound 18a, affording 18y as a white solid, 77% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.87 (s, 1H), 8.07 (s, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.46 (s, 4H), 7.20 (s, 2H), 7.11 (s, 2H), 5.23 (s, 2H), 3.84 (s, 3H). ESI-MS m/z: 453.3 [M + H]+. Methyl

(E)-3-(4-((4-((5-chloro-4-((4-fluorophenyl)amino)pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)methyl)phenyl)acrylate (18z) 2,5-Dichloro-N-(4-fluorophenyl)pyrimidin-4-amine (17f) and methyl (E)-3-(4-((4-amino-1H-pyrazol-1-yl)methyl)phenyl)acrylate hydrochloride (15h) were reacted using a procedure similar to the synthesis of compound 18a, affording 18z as a white solid, 74% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.85 (s, 1H), 8.06 (s, 1H), 7.74-7.57 (m, 4H), 7.43 (m, 3H), 7.17 (dt, J = 16.7, 8.1 Hz, 4H), 6.61 (dd, J = 16.0, 3.4 Hz, 1H), 5.17 (s, 2H), 3.32 (s, 3H). ESI-MS m/z: 479.1 [M + H]+. Molecular Docking Study. The crystal structures of JAK2 (PDB code: 3FUP) and HDAC2 (PDB code: 5IWG) were obtained from Protein Data Bank. Before the docking process, the structure of protein was treated by adding hydrogen atoms, deleting water molecules, assigning AMBER7 FF99

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charges and a 100-step minimization process using Sybyl X_2.1. The molecular structure was generated with the Sybyl/Sketch module and optimized using Powell’s method with the Tripos force field with convergence criterion set at 0.005 kcal/(Å mol) and assigned charges with the Gasteiger– Hückel method. Other docking parameters were kept to the default values. Molecular docking was carried out via the Sybyl/Surflex-Dock module. Kinase Inhibition Assay. The kinase inhibition assays were performed by eurofins cerep corporation in France. In brief, Evaluation of the effects of compounds on the activity of the human JAKs

was

quantified

by

measuring

the

phosphorylation

of

the

substrate

Ulight-

CAGAGAIETDKEYYTVKD using human recombinant enzymes and the LANCE detection method. Other kinase inhibition assays were performed as above method. Origin data analysis software were used to calculate the IC50 data by the non-linear curve fitting method (allowed to float and fitted as a parameter). In Vitro HDACs Inhibition Fluorescence Assay. In vitro HDACs inhibition assays were performed as previously described.43 In brief, 10 μL of enzyme solution (HeLa cell nuclear extract, HDAC2, HDAC6 or HDAC8) was mixed with different concentrations of tested compound (50 μL). The mixture was incubated at 37℃ for 5 mins, followed by adding 40 μL fluorogenic substrate (BocLys(acetyl)-AMC for HeLa cell nuclear extracts, HDAC2 and HDAC6, Boc-Lys(triflouroacetyl)AMC for HDAC8). After incubation at 37℃ for 30 mins, the mixture was quenched by addition of 100 μL of developer containing trypsin and Trichostatin A (TSA). Over another incubation at 37 ºC for 20 min, fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 390 and 460 nm, respectively. The inhibition ratios were calculated from the

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fluorescence intensity readout of tested wells relative to those of control wells, and the IC50 values were calculated using Prism non-linear curve fitting method (allowed to float and fitted as a parameter). Western Blot Analysis. The A549 cells were treated with compounds or DMSO for a specified period of time. Then the cells were washed twice with cold PBS and lysed in ice-cold RIPA buffer. Lysates were cleared by centrifugation. Protein concentrations were determined using the BCA assay. Equal amounts of cell extracts were then resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with ac-histone H4 antibody, ac-tubulin antibody and β-actin antibody, respectively. Blots were detected using an enhanced chemiluminescence system. The MDA-MB-231 cells were treated with compounds or DMSO for a specified period of time. The cells were collected and lysed in SDS buffer. Lysates were cleared by centrifugation. The supernate was then resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with p-STAT3 antibody, STAT3 antibody, ac-tubulin antibody, ac-histone H3 antibody and GAPDH antibody, respectively. Blots were detected using an enhanced chemiluminescence system. In Vitro Antiproliferative Assay. Cell proliferation assay was determined by the MTT (3-[4,5dimethyl-2-thiazolyl]-2,5-diphenyl-2h-tetrazolium bromide) method. Briefly, all cell lines were maintained in RPMI1640 medium containing 10% FBS at 37 °C in a 5% CO2 humidified incubator. Cells were passaged into a 96-well cell plate, allowed to grow for 12 h and then treated with different concentrations of compounds for 48 h. Then 0.5% MTT solution was added to each well. After incubation for another 4 h, formazan formed from MTT was extracted by adding 200 μL DMSO. Absorbance was then determined using an ELISA reader at 570 nm.

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Apoptosis Assay. HEL and K562 cells (2 × 105) were treated with different concentrations of test compounds for 24 h. Cells were collected after incubation, washed twice in cold PBS, centrifuged, and resuspended in 1 × annexin-binding buffer. Cells were diluted in 1 × annexin-binding buffer to about 1 × 106 cells/mL, preparing a sufficient volume to have 100 μL per assay. The cell suspension was added 5 μL annexin V and 10 μL PI. After gently mixed, the flow tube should be incubated at room temperature for 15 min. After the incubation period, the stained cells were analyzed by flow cytometry. Pharmacokinetics. All experiments involving laboratory animals were performed with the approval of the Shandong University Laboratory Animal Center ethics committee. Compound 8m was subjected to PK studies in SD rats and ICR (CD-1) mice. In SD rats, compound 8m was dissolved in the solution of 10% DMSO, 40% PEG400 and 50% pure water, then administrated via the oral route at 10 mg/kg (6 rats) or administrated via the intravenous route at 2 mg/kg (6 rats). Blood samples were collected from each animal via jugular vein and stored in ice (0-4 °C) at the specific time points. Plasma was separated from the blood by centrifugation and stored in a freezer at -80 °C. All samples for the tested compounds were analyzed by LC-MS/MS. The procedure in ICR (CD-1) mice is similar to above. The difference between the PK studies in ICR (CD-1) mice and in SD rats is that the former one used IP, not PO dosing (as was employed in SD rats). In Vitro Stability Evaluation in SD Rats and Human Liver Microsomes. For phase I and phase II metabolic stability evaluation, liver microsomes containing compound 8m were incubated with NADPH and UDPGA at 37 °C. At the specific time points, samples were added to acetonitrile to terminate the reaction, then subjected to vortex mixing for 5 min and stored in a freezer at -80 °C.

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Before analysis, the samples were centrifuged at 4000 rpm for 15 min. The remaining of 8m in the supernatants were analyzed by LC-MS/MS. The t1/2 values were calculated using the equation t1/2 = -0.693/k, where k is the slope found in the linear fit of the natural logarithm of the fraction remaining of 8m vs. incubation time. In Vivo Antitumor Activity Assay in HEL Xenograft Model. In vivo human tumor xenograft models were established as previously described.43 1 × 107 HEL cells were inoculated subcutaneously in the right flank of male BALB/c-nu mice (5-6 weeks old, Beijing HFK Bioscience Co., Ltd.). Ten days after injection, tumors were palpable and mice were randomized into treatment and control groups (6 mice per group). The treatment groups received specified concentrations of compounds by intraperitoneal administration, and the blank control group received intraperitoneal administration of equal volume of PBS (5% DMSO). During treatment, subcutaneous tumors were measured with vernier caliper every three days, and body weight was monitored regularly. Tumor volume (V) was estimated using the equation (V = ab2 / 2, where a and b stand for the longest and shortest diameter, respectively). After treatment, mice were sacrificed and dissected to weigh the tumor tissues and to examine the internal organ injury by macroscopic analysis. TGI was calculated according to the following formula: TGI = (the mean tumor weight of control group – the mean tumor weight of treated group) / the mean tumor weight of control group. All the obtained data were used to evaluate the antitumor potency and toxicity of compounds. Data were analyzed by Student’s two-tailed t test. A P level < 0.05 was considered statistically significant. Determination of Drug Concentrations in Plasma and Tumor. When tumors were palpable,

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the male BALB/c-nu mice were randomized into treatment and control groups. The treatment groups received specified concentrations of compounds by intraperitoneal administration, and the blank control group received intraperitoneal administration of equal volume of PBS (5% DMSO). After 5 days treatment with 3, 1 and 8m, mice were sacrificed at 0.5 h, 1.0 h and 4.0 h respectively after administration and dissected to collect the blood and weigh the tumor tissues at the specific time points. Plasma was separated from the blood by centrifugation and stored in a freezer at -80 °C. The tumor tissues were homogenized with normal saline (5:1 v/w) before ultrasound treatment. The plasma samples and tumor samples were deproteinized with methanol containing an internal standard. After centrifugation, the supernant were diluted with methanol and acetonitrile and then centrifugated again. The compound concentrations in the supernant were analyzed by LC-MS/MS.

ASSOCIATED CONTENT Supporting information 1H

NMR, 13C NMR and HPLC spectra of all target compounds and supplemental figures and tables;

Molecular formula stings (CSV) AUTHOR INFORMATION Corresponding Author *Y. Z.: phone, +86 531 88382009; E-mail: [email protected] *H. L.: phone, +86 21 50807042; E-mail: [email protected] ORCID Yingjie Zhang: 0000-0001-6118-6695

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Hong Liu: 0000-00033685-6268 ACKNOWLEGEMENTS This work was supported by National High-Tech R&D Program of China (863 Program) (2014AA020523), Natural Science Foundation of Shandong Province (Grant No. ZR2018QH007), Major Project of Science and Technology of Shandong Province (2017CXGC1401), Young Scholars Program of Shandong University (YSPSDU, 2016WLJH33). ABBREVIATIONS USED CTCL, cutaneous T-cell lymphoma; ET, essential thrombocythemia; FLT3, FMS-like receptor tyrosine kinase 3; HDAC, histone deacetylase; JAK, Janus kinase; LIFR, leukemia inhibitory factor receptor; MF, myelofibrosis; MM, multiple myeloma; MPNs, Philadelphia chromosome–negative myeloproliferative neoplasms; NADPH, nicotinamide adenine dinucleotide phosphate; PBS, Phosphate buffer solution; PTCL, peripheral T-cell lymphoma; PV, polycythemia vera; STAT, Signal transducers and activators of transcription; TGI, Tumor growth inhibition; TYK2, tyrosine kinase 2; UDPGA, uridine diphosphate glucuronic acid; UGT, UDP-glucuronosyltransferase; VEGFR2, vascular endothelial growth factor receptor 2. REFERENCES 1. Lane, A.A.; Chabner, B.A. Histone Deacetylase Inhibitors in Cancer Therapy. J. Clin. Oncol.

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Date, T.; Takasaki, M.; Terada, D.; Hirai, A.; Akashi, A.; Chen, F.; Adachi, Y.; Ishikawa, Y.; Hayakawa, F.; Hagiwara, S.; Naoe, T.; Kiyoi, H. A Novel Irreversible FLT3 Inhibitor, FF-10101, Shows Excellent Efficacy against AML cells with FLT3 Mutations. Blood. 2018, 131, 426-438. 43. Li, X.; Inks, E. S.; Li, X.; Hou, J.; Chou, C. J.; Zhang, J.; Jiang, Y.; Zhang, Y.; Xu, W. Discovery

of the First N-Hydroxycinnamamide-Based Histone Deacetylase 1/3 Dual Inhibitors with Potent Oral Antitumor Activity. J. Med. Chem. 2014, 57, 3324-3341.

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Table of Contents Graphic Cl Cl HN N

N HN

N N

H N

OH

O

8m Enzymatic assays:

Celluar assays:

JAK1: 4.8 nM JAK2: 4.3 nM JAK3: 7.4 nM Tyk2: 49 nM HDAC2: 120 nM HDAC6: 14.4 nM

HEL: 90 nM K562: 490 nM MOLT4: 80 nM Jurkat: 60 nM

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Discovery of Novel Janus Kinase (JAK) and Histone Deacetylase

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