Discovery of Novel Pyrrolo[2,3-d]pyrimidine-based Derivatives as Potent JAK/HDAC Dual Inhibitors for the Treatment of Refractory Solid Tumors

pubs.acs.org/jmc

Article

Discovery of Novel Pyrrolo[2,3‑d]pyrimidine-based Derivatives as

Potent JAK/HDAC Dual Inhibitors for the Treatment of Refractory

Solid Tumors

Xuewu Liang, Shuai Tang, Xuyi Liu, Yingluo Liu, Qifu Xu, Xiaomin Wang, Abdusaid Saidahmatov,

Chunpu Li, Jiang Wang, Yu Zhou, Yingjie Zhang, Meiyu Geng, Min Huang,* and Hong Liu *

Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c02111

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: It remains a big challenge to develop HDAC

inhibitors eﬀective for solid tumors. Previous studies have

suggested that the feedback activation of JAK-STAT3 pathway

represents a key mechanism leading to resistance to HDAC

inhibitors in breast cancer, suggesting the therapeutic promise of

JAK/HDAC dual inhibitors. In this work, we discovered a series of

pyrrolo[2,3-d]pyrimidine-based derivatives as potent JAK and

HDAC dual inhibitors. Especially, compounds 15d and 15h

potently inhibited JAK1/2/3 and HDAC1/6 and displayed

antiproliferative and proapoptotic activities in triple-negative breast

cancer cell lines. Besides, compounds 15d and 15h also diminished

the activation of LIFR-JAK-STAT signaling triggered by tumor-

associated ﬁbroblasts, which suggests that these compounds could

potentially overcome the drug resistance caused by the tumor microenvironment. More importantly, compound 15d eﬀectively

inhibited the tumor growth in MDA-MB-231 xenograft tumor model. Overall, this work provides valuable leads and novel antitumor

mechanisms for the treatment of the SAHA-resistant triple-negative breast cancers.

panobinostat, and chidamide (approved in China) have been

approved for the treatment of hematological malignancies,

including refractory cutaneous T cell lymphoma (CTCL),

peripheral T cell lymphoma, and multiple myeloma.^9,10

Despite the great success achieved in treatment of hemato-

logical malignancies, most of the known HDAC inhibitors used

as a single agent failed to show clinical beneﬁts in nearly all

types of solid tumors,¹¹including breast cancer, renal cancer,¹²

prostate cancer,¹³and head and neck cancer.¹⁴Simultaneously

inhibiting HDAC and other targets involved in the patho-

genesis of solid tumors may solve the dilemma.

Actually, chimeric HDAC inhibitors, which simultaneously

modulate HDAC and other targets in feedback or

compensatory pathways, could provide a potential treatment

for relapsing and drug-resistant cancers. Thus far, a dozen

diﬀerent types of chimeric HDAC inhibitors have been

developed,¹⁵including chimeric EGFR/HDAC inhibitors,¹⁶

INTRODUCTION

■

Simultaneous intervention of two or even multiple targets in

the complex pathogenesis may be beneﬁcial to the treatment of

patients suﬀered from complex diseases like malignant tumors

and central nervous system diseases.^1,2To tackle complex

diseases like cancer, modulating the activity of multiple targets

could possibly achieve better therapeutic beneﬁt, which

provides a reasonable scientiﬁc basis for multitargeted drug

design.³In clinical applications, multitargeted drugs possess

advantages such as optimal therapeutic eﬃcacy (like sorafenib

and dasatinib), slowing down drug resistance, as well as

predictable pharmacokinetic (PK) proﬁle and avoiding drug−

drug interactions compared with combinational therapies.⁴⁻⁶

However, it is undeniable that multitargeted eﬀects are also a

very important cause of drug side eﬀects. Nevertheless,

multitarget drug design strategies are widely used in antitumor

drugs.

Histone deacetylases (HDACs), a family of epigenetic

enzymes, regulate the deacetylation of a variety of their

substrates including histone and nonhistone proteins, then

further control the expression level of many oncogenes and

apoptotic genes and hence impact many cancer cellular

processes such as cell proliferation, cell migration, cell

apoptosis, and angiogenesis.^7,8Up to now, ﬁve HDAC

inhibitors, namely vorinostat (SAHA), romidepsin, belinostat,

Special Issue: New Horizons in Drug Discovery -

Understanding and Advancing Kinase Inhibitors

Received: December 7, 2020

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

A

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Chemical structures of representative JAK/HDAC dual inhibitors.

chimeric VEGFR/HDAC inhibitors,^17,18dual c-Src/HDAC

inhibitors,¹⁹dual Janus kinase (JAK)/HDAC inhibitors,¹⁰and

so on.

myelocytic leukemia (AML), multiple myeloma (MM), and

erythroleukemia cell lines (EL) showed that compound 1

could eﬀectively block both the JAK-STAT and HDAC

pathways.²⁷Compound 2 broadly inhibited JAK1 and

HDACs 1, 2, 3, 6, and 10, with IC₅₀values of less than 20

nM. Broad cellular antiproliferative potency of compound 2

was supported by demonstration of JAK-STAT and HDAC

pathway blockade in hematological cell lines.²⁸It is worth

mentioning that compound 3 with strong JAK2 potency (IC₅₀

= 3.1 nM) and HDAC6 potency (IC₅₀= 1.2 nM) showed 16−

25-fold selectivity against the other three JAK isoforms. It also

showed submicromolar potencies against a panel of four solid

tumor cell lines.²⁹In 2018, Sheng’s group reported a novel

JAK/HDAC dual inhibitor 4. Compound 4 possessed potent

inhibitory activities against JAK2 and HDAC6 with IC₅₀values

at 8 and 46 nM, respectively. Further studies in vivo showed

that compound 4 possessed excellent antitumor eﬃcacy in

several AML models and synergized with ﬂuconazole for the

treatment of resistant Candida albicans infections.³⁰In 2019,

our group reported a novel series of pyrimidin-2-amino-pyrazol

hydroxamate derivatives as JAK and HDAC dual inhibitors,

among which compound 5a possessed potent and balanced

activities against both JAK2 and HDAC6 with IC₅₀values at

4.3 and 14.4 nM, respectively. Compound 5a exhibited

improved antiproliferative and proapoptotic activities over

SAHA and ruxolitinib in several hematological cell lines.³¹It is

worth mentioning that the studies of these JAK/HDAC dual

inhibitors are more likely to focus on hematological

malignancies rather than solid tumors which are insensitive

to JAK inhibitors or HDAC inhibitors.

Invasive solid tumors, such as triple-negative breast cancer,

possess strong aggression, high mortality, and poor prognosis,

which lack eﬀective clinical treatment.^20,21Development of

more eﬀective therapy for treatment of such human

malignancies is very urgent. Interestingly, recent research

showed that HDAC inhibitors promoted BRD4-mediated

activation of leukemia inhibitory factor receptor (LIFR), which

in turn activated JAK1-STAT3 signaling and restrained the

eﬃcacy of HDAC inhibitors in breast cancer.²²Concurrent

inhibition of JAK sensitized breast cancer, including the triple-

negative subset, to HDAC inhibitors.²²Thus, the development

of JAK/HDAC dual inhibitors may be a reasonable and

eﬀective treatment for triple-negative breast cancer. Of note,

JAKs are a family of intracellular tyrosine kinases comprising

JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2) that play a

critical role in both tumor cells and in the tumor micro-

environment.²³JAKs, activated by diﬀerent cytokines, lead to

phosphorylation and dimerization of the signal transducers and

activators of transcription (STAT) family of proteins that

translocate to the nucleus and stimulate gene expression.²⁴⁻²⁶

Inhibition of JAKs is promising to block the biological crosstalk

between tumor cells and the surrounding cells in the tumor

microenvironment. As such, combining JAK and HDAC is

expected to overcome the limitations of HDAC inhibitors and

bring clinical beneﬁts to solid tumors. We have previously

discovered that LIFR-JAK1-STAT3 signaling limits response to

HDAC inhibition in the solid tumors.²²Given JAK1 and JAK2

share the same downstream molecules in cancer cells, dual

inhibition of JAK1 and JAK2 may avoid compensatory

activation and ensure the blockage of the feedback activation

of STAT3 signaling. Co-inhibition of HDACs with JAK1/

JAK2 may be more suitable for treating solid tumors.

It remains a big challenge to develop HDAC inhibitors

eﬀective for solid tumors. Combining JAK and HDAC is

expected to overcome the limitations of HDAC inhibitors and

bring clinical beneﬁts to solid tumors. Therefore, we developed

a novel series of pyrrolo[2,3-d]pyrimidine-based derivatives as

potent JAK/HDAC dual inhibitors and explore the advantages

of multitarget drugs for the treatment of relapsing and drug-

resistant cancers, in particular the triple-negative breast cancers

in this work.

So far, a few JAK/HDAC dual inhibitors have been

developed (Figure 1). Dymock’s group ﬁrst reported JAK/

HDAC dual inhibitors 1, 2, and 3 based on the structure of

JAK inhibitor templates pacritinib, ruxolitinib, and XL019,

respectively.²⁷⁻²⁹Compound 1 was selective for JAK2 and

HDAC6 against other JAK and HDAC isoforms with IC₅₀

values at 1.4 and 2.1 nM. Further detailed studies in acute

B

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. Rational design of novel JAK and HDAC dual inhibitors. (A) Predicted binding mode of compound 5b in the ATP pocket of JAK2 (PDB

3FUP). The unoccupied pocket is indicated by arrow. (B) Design strategy of JAK and HDAC dual inhibitors based on compound 5b. The three

parts of HDAC inhibitor pharmacophore are indicated in three colors (ZBG, zinc-binding group). In addition, the structure of compound 6a is

shown.

Figure 3. (A) The predicted binding mode of compound 6a in the ATP pocket of JAK2 (PDB 3FUP). (B) The predicted binding mode of

compound 6a in the active site of HDAC6 (PDB 5EEI). (C) Surface representation of compound 6a in the ATP pocket of JAK2 (PDB 3FUP).

(D) Surface representation of compound 6a in the active site of HDAC6 (PDB 5EEI). Yellow dashed lines represent the hydrogen bonds. In the

ligand, oxygen, nitrogen, carbon, and polar hydrogen atoms are shown in red, blue, green, and white, respectively. The ﬁgures were generated using

PyMol (http://www.pymol.org/).

on the solid tumors, which are not sensitive to JAK inhibitors

or HDAC inhibitors. Through analyzing the binding mode

between compound 5b and JAK2 protein using molecular

docking study, we found a hydrophobic pocket in the ATP

pocket of JAK2 protein that had not yet been occupied (Figure

2A). Thus, we tried to ﬁll this hydrophobic pocket with an

aromatic ring on the ligand molecule to improve the binding of

ligand to protein. In addition, considering the large cavity in

the surface recognition cap (SRC) of HDAC protein, the

introduction of an aromatic ring on the ligand molecule would

RESULTS AND DISCUSSION

■

Rational Design of Novel JAK and HDAC Dual

Inhibitors. In our previous research, a potent JAK/HDAC

dual inhibitor 5b with notable antiproliferative and proapop-

totic activities was identiﬁed.³¹However, this series of

compounds showed a very potent in vitro but a mild in vivo

anticancer activities against hematological malignancies. In this

work, we carried out further structural modiﬁcations based on

compound 5b in the hope of obtaining desired in vivo eﬃcacy

C

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 1. Synthesis of Intermediate Compounds 11a,b

a

Conditions and reagents: (a) Pd/C, H₂, EtOH, rt, 2 days, 90%; (b) (Boc)₂O, NaHCO₃/H₂O, THF, rt, 2 days, 75%; (c) Cs₂CO₃, CH₃CN, 40−50

°C, 10 h, 76−78%; (d) HCl/EtOAc, rt, 10 h, 63−75%.

a

Scheme 2. Synthesis of Intermediate Compounds 13a−s

a

Conditions and reagents: (e) for 13a−e. CuI, trans-1,2-cyclohexanediamine, K₃PO₄, dioxane, 130 °C, microwave 1 h, 20−50%; For 13f−s,

K₂CO₃, CH₃CN, 40−50 °C, 5 h, 67−96%.

not aﬀect the binding of the ligand to HDAC protein. To

improve the biological activity, fused ring strategy in the ligand

molecule was frequently applied in the design of JAK

inhibitors.^32,33On the basis of the above analysis, we

introduced diﬀerent aromatic rings to the pyrimidine ring on

the compound 5b, including phenyl ring, biphenyl ring, and

pyrrole ring (Figure 2B). By preliminary inhibitory activity

against JAK2, compounds containing quinazoline and phenyl-

pyrimidine scaﬀolds showed weaker JAK2 inhibition than the

compound containing pyrrolo[2,3-d]pyrimidine scaﬀold.

Therefore, we selected pyrrole ring as the optimal fused ring

to design the novel pyrrolo[2,3-d]pyrimidine-based derivatives

as novel and potent JAK/HDAC dual inhibitors.

Compound 6a, one of the representative pyrrolo[2,3-

d]pyrimidine derivatives which showed the most similar

structure with compound 5b, was analyzed for its proposed

binding modes in the active sites of JAK2 and HDAC6 using

molecular docking study. The docking results in Figures

3(A,C) showed the binding mode of compound 6a in the ATP

active pocket of JAK2. It can be seen that aminopyrimidine

moiety of compound 6a forms dual hydrogen bonds with

Leu932, which is the same as compound 5b. As we predicted,

the pyrrole ring introduced to the pyrimidine moiety perfectly

ﬁlls this unoccupied hydrophobic pocket and ﬁxes the

orientation of the phenyl group. In addition, alkyl hydroxamate

part of compound 6a is projected toward the solvent region

and forms hydrogen bonds with the amino acid residues

Arg980 and Lys857 at the entrance of the ATP active pocket in

JAK2. The docking results in Figure 3B,D showed the binding

mode of compound 6a in the active site of HDAC6. It can be

seen that the hydroxamic acid group of compound 6a chelates

the Zn²⁺in a bidentate manner and forms multiple hydrogen

bonds with Tyr745, His573, and His574 residues in the deep

active pocket of HDAC6 protein, which contribute to its high

inhibition potency against HDAC6. The aliphatic chain

occupies the hydrophobic channel, which perfectly connect

hydroxamic acid group with terminal cap group. The above

docking results strongly support the rationality of the design

strategy of the novel pyrrolo[2,3-d]pyrimidine-based deriva-

tives as potent JAK/HDAC dual inhibitors.

On the basis of the structure of compound 6a, we further

introduced some substituents on the phenyl group to

investigate the inﬂuence of substituents on the biological

activities and designed compounds 6b−e. Meanwhile, we

reduced the length of aliphatic linker from 6 to 5 and designed

compounds 6f,g. Considering the relatively strong rigidity of

the compounds 6a−g, we replaced the phenyl groups with

diﬀerent substituted benzyl groups, phenethyl groups, and

cycloalkyl groups to increase their structural ﬂexibility and

designed the other target compounds (15a−l, 16a−j, and

17a−c).

Chemistry. Chemical synthesis of JAK/HDAC dual

inhibitors are depicted in Schemes 1−3. 4-Nitro-1H-pyrazole

7, as the starting material is reduced by hydrogen to produce

1H-pyrazol-4-amine 8. Intemediate 8 is protected by Boc

group to obtain intemediate 9, which reacted with

commercially available bromoalkanes under basic condition

of cesium carbonate to produce intermediates 10a,b. Boc

deprotection of intermediates 10a,b under strong acid

condition gave intermediates 11a,b, respectively (Scheme

1).³¹Intermediates 13a−s were obtained by the reaction of 2-

chloro-7H-pyrrolo[2,3-d]pyrimidine 12 as the starting material

D

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 3. Synthesis of Target Compounds 6a−g, 15a−l, 16a−j, and 17a−c

a

Conditions and reagents: (f) EtOH, 130 °C, microwave 2 h, 18−41%; (g) NH₂OK, CH₃OH, rt, 0.5−1 h, 70−95%.

with diﬀerent aryl bromides under diﬀerent conditions

(Scheme 2). Intermediates 14a−z and 14A−F were prepared

by the reaction of the key intermediates 13a−s and the key

intermediates 11a,b under the conditions of high temperature

and acid catalyst in microwave apparatus. Intermediates 14a−z

and 14A−F were subsequently reacted with NH₂OK/

methanol solution to give target compounds 6a−g, 15a−l,

16a−j, and 17a−c, respectively (Scheme 3).

reduced the length of aliphatic linker from 6 to 5 and obtained

compounds 6f,g. Comparing their JAK2 and HDAC6

inhibitory activities data, we found that aliphatic linker length

of 5 or 6 methylenes had no dramatic inﬂuence on the JAK2

and HDAC6 inhibition. All the compounds (6a−g) containing

5 or 6 methylenes showed high potencies on the JAK2 and

HDAC6 inhibition. Linker length of 5 or 6 methylenes is both

accepted in the structural modiﬁcation of these JAK/HDAC

dual inhibitors.

In Vitro JAK and HDAC Inhibition Assay. The inhibitory

activities of these novel pyrrolo[2,3-d]pyrimidine-based

derivatives against JAK2 and HDAC6 were evaluated by

determining their IC₅₀values, with the approved JAK inhibitor

ruxolitinib (INCB) and approved hydroxamic acid-based

HDAC inhibitor SAHA as positive controls. The results in

Table 1 showed that 6a exhibited potent JAK2 and HDAC6

inhibitory activities with IC₅₀values at 16.8 and 13.1 nM,

validating the rationality of our JAK and HDAC dual inhibitor

design strategy shown in Figure 2. It is worth noting that

compound 6a showed much more potent HDAC6 inhibitory

activity than that of SAHA (IC₅₀value: 75.9 nM) and slightly

weaker JAK2 inhibitory activity than that of ruxolitnib (IC₅₀

value: 2.9 nM). Simultaneously inhibiting JAK2 and HDAC6

with nanomolar level is acceptable. Next, diﬀerent para-

substituents were introduced to the phenyl moiety of

compound 6a, leading to the compounds 6b−e. Comparing

JAK2 and HDAC6 inhibitory activities data of compounds

6b−e with that of compound 6a, it is obvious to ﬁgure out that

the introduction of diﬀerent para-substituents to the phenyl

moiety will slightly reduce the JAK2 and HDAC6 inhibitory

activities, excluding compound 6e. Compound 6e exhibited

comparable JAK2 inhibitory activity to 6a (IC₅₀values: 15.7 vs

16.8 nM) and a little more potent HDAC6 inhibitory activity

than 6a (IC₅₀values: 5.9 vs 13.1 nM). To investigate the

eﬀects of the linker on JAK2 and HDAC6 inhibition, we

To reduce the rigidity of compounds 6a−g and considering

the aliphatic linker length of 5 or 6 methylenes both accepted,

compounds 15a−l and compounds 16a−j were synthesized

and evaluated. Compounds 15a−j exhibited potent JAK2 and

HDAC6 inhibition with IC₅₀values lower than 50 nM (Table

1), validating that ortho- and meta- halogen substituents on the

phenyl moiety were well tolerated both in the JAK2 and

HDAC6 inhibition. However, compounds 15k−l exhibited

poor JAK2 inhibition (IC₅₀> 100 nM) and potent HDAC6

inhibition (IC₅₀lower than 50 nM), validating that meta-

methoxyl substituent on the phenyl moiety was not tolerated in

the JAK2 inhibition. Note that through this round of

compound screening, we obtained many potent JAK/HDAC

dual inhibitors with IC₅₀at nanomolar level, including 15a,

15c−e, and 15g−i. Among these compounds, 15g potently

inhibited JAK2 and HDAC6 with the IC₅₀values at 4.1 and

13.7 nM, respectively.

Next, para-substituents were introduced to the phenyl

moiety on the structures of JAK/HDAC dual inhibitors 15a

and 15b. The enzyme inhibition results in Table 2 showed that

para-substituents (no matter halogen or methoxyl group) on

the phenyl moiety were not beneﬁcial for JAK2 inhibition but

tolerated in HDAC6 inhibition. All compounds 16a−h

exhibited very poor JAK2 inhibition (IC₅₀> 100 nM), which

is not what we expected. In addition, both compounds 16i,j

E

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1. Structures and in Vitro Enzymes Inhibitory

Table 2. Structures and in Vitro Enzymes Inhibitory

Activities Comparison of the Target Compounds 6a−g and

Activities Comparison of the Target Compounds 16a−j and

a

Compounds 15a−l

Compounds 17a−c

a

b

Assays were performed in replicate (n ≥ 3). @10 nM means

c

HDAC6 inhibition rate at 10 nM. The IC₅₀(nM) values of

compounds 17a, 17b, 17c, and SAHA against HDAC6. Not active at

d

10 μM.

exhibited very poor JAK2 inhibition (IC₅₀> 100 nM),

validating that phenethyl group in R substituent were also

not beneﬁcial for JAK2 inhibition but tolerated in HDAC6

inhibition.

Finally, we also introduced some cycloalkyl substituents

instead of aromatic substituents as R group to the pyrrolo

moiety of the JAK/HDAC dual inhibitors, leading to the

compounds 17a−c. The enzyme inhibition results in Table 2

showed that compounds 17a and 17b possessed very potent

inhibitory activities against HDAC6 (IC₅₀: 22 and 16 nM,

a

Assays were performed in replicate (n ≥ 3), IC₅₀values are shown as

b

mean SD. Not active at 10 μM.

F

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 4. (A) Predicted binding mode of compound 15d in the ATP pocket of JAK2 (PDB 3FUP). (B) Predicted binding mode of compound 16b

in the ATP pocket of JAK2 (PDB 3FUP). (C) Predicted binding mode of compound 15d in the active site of HDAC6 (PDB 5EEI). (D) Predicted

binding mode of compound 16b in the active site of HDAC6 (PDB 5EEI). Yellow dashed lines represent the hydrogen bonds. In the ligand,

oxygen, nitrogen, carbon, ﬂuoro, and in polar hydrogen atoms are shown in red, blue, green, pale cyan, and white, respectively. The repulsion force

between the ligand molecule with the Lys857 residue of JAK2 protein was shown in a red dashed line. The ﬁgures were generated using PyMol

(http://www.pymol.org/).

a

Table 3. JAK Isoform and HDAC Selectivity Proﬁles of Representative Compounds

JAK2

JAK1

JAK3

TYK2

HDAC1

HDAC6

compd

IC₅₀(nM)

6a

15c

15d

15g

15h

16.8 2.0

10.0 0.3

32.9 8.2

4.1 0.7

15.7 3.7

2.9 0.3

ND

19.6 3.2

32.3 8.6

33.4 4.0

8.7 1.0

19.9 2.4

1.1 0.1

ND

81.2 4.8

18.4 3.1

25.1 0.2

12.6 1.9

16.1 0.1

20.7 0.9

ND

23.5 0.2

13.3 4.3

40.9 3.3

15.0 3.3

21.7 1.8

10.3 5.1

ND

52.1 0.9

144.7 23.7

34.2 1.3

13.1 2.5

15.3 0.7

8.4 0.5

13.7 1.0

14.7 2.4

ND

172.9 11.0

92.1 19.1

b

Ruxolitinib

SAHA

ND

180.3 10.9

75.9 5.2

a

b

Assays were performed in replicate (n ≥ 3), IC₅₀values are shown as mean SD. Not determined.

respectively) but very low inhibitory activities against JAK2

(IC₅₀> 100 nM). However, compound 17c possessed a

balanced but mild JAK2 and HDAC6 inhibition with the IC₅₀

values at 69 and 88 nM, respectively.

The inhibitory activities of the corresponding compounds

16a−h against JAK2 were greatly decreased when the para-

substituents were present on the phenyl moiety. To explain the

phenomenon, we analyzed the binding modes of compound

15d and compound 16b in the ATP pocket of JAK2 (Figure

4). The docking results in Figure 4A showed that, like

compound 6a, compound 15d showed a similar binding mode

with JAK2 protein. Aminopyrimidine moiety of compound

15d forms dual hydrogen bonds with Leu932 and alkyl

hydroxamate moiety forms multiple hydrogen bonds with the

amino acid residues Ser936, Arg938, and Asp939. Therefore,

compound 15d exhibited comparable and potent JAK2

inhibitory activity to 6a (IC₅₀values: 32.9 vs 16.8 nM). It is

worth noting that the para-site of the phenyl moiety in the

ligand molecule 15d was very close (3.7 Å, indicated by a red

dashed line) to the Lys857 residue at the P-loop region. The

introduction of a substituent in the para- site of the phenyl

moiety may aggravate the repulsion force between the ligand

molecule with the Lys857 residue of JAK2 protein. Therefore,

in the binding mode of compound 16b with JAK2 protein

G

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 4. Further HDAC Selectivity Data for Representative Lead 15d

HDAC class I

HDAC class IIa

HDAC class IIb

HDAC1

HDAC3

HDAC8

HDAC4

HDAC7

HDAC6

HDAC10

compd

IC₅₀(nM)

15d

34.2 1.3

3.8 0.5

9.2 0.8

>10000

8.4 0.5

3.6 0.4

SAHA

180.3 10.9

28.1 1.6

191.5 24.7

63.2 7.7

75.2 10.9

75.9 5.2

47.5 3.5

a

Assays were performed in replicate (n ≥ 3), IC₅₀values are shown as mean SD.

Figure 5. (A) MDA-MB-231 cells were treated with DMSO or compounds at 10 μM for 12 h. (B) MDA-MB-231 cells were treated with DMSO or

compounds at 3 μM for 12 h. The levels of phosphorylated STAT3 (p-STAT3-Tyr705), STAT3, acetyl-α-tubulin (Ac-tubulin), acetyl-H3K9/14

(Ac-H3K9/14) were determined by immunoblotting. β-Actin was used as a loading control. Ruxolitinib was indicated by INCB in the combined

treatment group.

(Figure 4B), the dominant conformation of the phenyl moiety

in the ligand molecule 16b has undergone a huge change. The

disadvantageous conformation change led to a sharp decrease

IIa (HDACs 4, 7), with IC₅₀values of larger than 10 μM

(Table 4).

Concurrent Inhibition of Intracellular JAK and HDAC.

To conﬁrm the intracellular dual actions of this series of JAK/

HDAC dual inhibitors, Western blot analysis was performed.

As the previous literature reported that concurrent inhibition

of BRD4 or JAK sensitized breast cancer to HDAC inhibitors,

we selected triple-negative breast cancer cell line MDA-MB-

231 to perform the Western blot. Consistent with our previous

ﬁndings, HDAC inhibitor SAHA at the concentration of 10

and 3 μM could cause the upregulation of phosphorylated

STAT3, suggesting the restrained eﬃcacy in breast cancer.

Cotreatment of HDAC inhibitor SAHA with JAK inhibitor

ruxolitinib (INCB) successfully suppressed the JAK-STAT

pathway. To our delight, at the concentration of 10 μM, our

designed compounds 15c, 15d, 15g, and 15h obviously

increased the HDAC substrates acetyl-α-tubulin and acetyl-

histone H3, suggesting the eﬀective of HDACs activity and

meanwhile prevented the feedback activation of phosphory-

lated STAT3 in a dose-dependent manner compared to the

control group, which validated that this series of newly

designed compounds simultaneously inhibited JAKs and

HDACs in MDA-MB-231 cells (Figure 5A). At the low

concentration of 3 μM, compounds 15c, 15d, 15g, and 15h

also eﬀectively simultaneously inhibited JAK and HDAC

proteins in the MDA-MB-231 cell lines (Figure 5B). In this

celluar assay, the inhibitory activities against JAK and HDAC

proteins of compounds 15d and 15h were more potent than

that of compounds 15c and 15g.

in the JAK2 inhibitory activity of the ligand molecules (IC₅₀

>

100 nM). In addition, compound 15d showed a similar

binding mode with 16b in the active site of HDAC6 (Figure

4C,D), which rationally conﬁrmed the comparable and potent

HDAC6 inhibitory activity of compounds 15d (IC₅₀= 8.4

nM) and 16b (62% inhibition at 10 nM).

In summary, we did a relatively comprehensive structure−

activity relationship (SAR) study based on the linker length

and R group. Aliphatic linker length of 5 or 6 methylenes, para-

substituted phenyl, ortho-, and meta- halogen substituted

benzyl groups were accepted in this series of potent JAK/

HDAC dual inhibitors.

JAK and HDAC Selectivity Proﬁle. Because of their

signiﬁcant JAK2 and HDAC6 inhibitory activities, Compounds

6a, 15c, 15d, 15g, and 15h were evaluated against JAK

isoforms and HDAC isoforms to perform the JAK and HDAC

selectivity proﬁle. As depicted in Table 3, this series of

compounds exhibited potent but undiﬀerentiated inhibitory

activities against JAK1, JAK2, JAK3, and TYK2 with the IC₅₀

values between 4 and 40 nM (except that compound 6a

showed a mild inhibitory activity against JAK3 with the IC₅₀

value of 81.2 nM). Therefore, from the experimental results,

these compounds tend to be pan-JAKs inhibitors. A

comprehensive HDAC selectivity study on the representative

compounds was also conducted. Results in Tables 3 and 4

showed that compounds 6a, 15c, 15d, 15g, and 15h all

exhibited potent inhibitory activities against HDAC1 and

HDAC6. Especially, compound 15d broadly inhibited HDAC

class I (HDACs 1, 3, 8), and HDAC class IIb (HDACs 6, 10),

with IC₅₀values of less than 50 nM. However, compound 15d

possessed very poor inhibitory activities against HDAC class

Considering their potent inhibitory activities against JAK

and HDAC proteins in MDA-MB-231 cell lines, we further

veriﬁed the eﬀect of the compounds 15d and 15h on the

inhibition of the two target proteins JAK and HDAC in the

triple-negative breast cancer cell line BT-549. The results in

H

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 6 showed that similarly to results obtained in MDA-

MB-231 cells, compounds 15d and 15h increased the levels of

(IC₅₀values: 0.79 vs 1.04 μM), the antiproliferative activity of

compound 15d is slightly better than that of SAHA.

Apoptosis Assay. Because compound 15d exhibited

excellent antiproliferative activity in breast cancer cells, we

continue to select this compound for further studies on its pro-

apoptotic activity via ﬂow cytometry. Results in Figure 7

showed that at the concentration of 2.5 μM, SAHA induced

about 39% MDA-MB-231 cell apoptosis and possessed

synergistic pro-apoptotic activity in combination with

ruxolitinib (SAHA+ ruxolinib, about 72% MDA-MB-231 cell

apoptosis). To our delight, compound 15d induced MDA-MB-

231 cell apoptosis in a dose-dependent manner. At the

concentrations of 10 and 5 μM, compound 15d could induce

about 75% and 62% MDA-MB-231 cell apoptosis, suggesting a

potent pro-apoptotic activity in MDA-MB-231 cells.

Next, we further investigate the potency of compound 15d

in inducing cell apoptosis in the 4T1 cell lines. Results in

Figure 8 showed that compound 15d induced 4T1 cell

apoptosis in a dose-dependent manner. At the concentration of

5 μM, SAHA showed a very low pro-apoptotic potency on the

4T1 cell lines (about 20% 4T1 cell apoptosis). To our delight,

at the same concentration of 5 μM, compound 15d could

induce about 90% 4T1 cell apoptosis, which is higher than that

of SAHA and ruxolitinib combination group (about 35% 4T1

cell apoptosis). Therefore, it can be seen that for 4T1 solid

tumor, compound 15d as a JAK and HDAC dual inhibitor

exhibited higher pro-apoptotic activity than the drugs

combination group.

Blockage of LIFR-JAK-STAT Signaling Pathway

Stimulated by the Crosstalk between Tumor Cells and

Tumor Associated Fibroblasts. In addition to tumor cells,

other cell components in the tumor microenvironment could

also contribute to JAK-STAT signaling activation via secreting

of IL-6 family of cytokines, such as LIF, leading to the

activation of JAK-STAT signaling, which also contributes to

the limited eﬃcacy of HDAC inhibitors in breast cancer.

Tumor associated ﬁbroblast cells could secrete cytokines like

LIF to activate JAK-STAT pathway. To investigate the eﬀect of

tumor microenvironment on HDAC inhibitor resistance in

MDA-MB-231 cancer cells, we treated MDA-MB-231 cancer

cells with the conditioned medium derived from tumor-

associated Wl-38 ﬁbroblasts. The results in Figure 9A,B

showed that cytokines secreted by ﬁbroblasts Wl-38 could

upregulate the LIFR-JAK-STAT signaling pathway in MDA-

MB-231 cancer cells. In the represence of conditioned

medium, HDAC inhibitor SAHA further substantially

ampliﬁed LIFR-JAK-STAT signaling pathway, presumably

rendering tumors more resistant to drug. To our delight, at

3 μM, compounds 15d and 15h eﬀectively diminished the

LIFR-JAK-STAT signaling pathway, which demonstrated the

potential therapeutic beneﬁts of JAK and HDAC dual

inhibitors in triple-negative breast cancer and provided a

reasonable basis for the advantages in the in vivo experiment.

Preliminary in Vivo Antitumor Activity Evaluation.

Considering its potent antiproliferative and proapoptotic

capacities in MDA-MB-231 breast cancer cell lines, compound

15d was progressed into a preliminary in vivo antitumor

activity study in a MDA-MB-231 xenograft model. Mice were

randomly separated into six groups (n = 6/group) and treated

with compound 15d (30 or 100 mg/kg/day, IP), SAHA (100

mg/kg/day, PO), ruxolitinib (100 mg/kg/day, PO), combi-

nation of SAHA and ruxolitinib (100 + 100 mg/kg/day, PO)

and saline for 14 consecutive days. Tumor growth inhibition

Figure 6. BT-549 cells were treated with DMSO or compounds at 3

μM for 12 h. The levels of phosphorylated STAT3 (p-STAT3-

Tyr705), acetyl-α-tubulin (Ac-tubulin), and acetyl-H3K9/14 (Ac-

H3K9/14) were determined by immunoblotting. GAPDH was used

as a loading control. Ruxolitinib was indicated by INCB in the

combined treatment group.

HDAC substrates, Ac-tubulin, and Ac-H3K9/14 yet did not

cause the feedback activation of of p-STAT3 at the

concentration of 3 μM, which further conﬁrmed the JAK

and HDAC dual inhibitory activity of the newly designed

compounds at the cellular level.

In Vitro Antiproliferation Assay. JAK/HDAC dual

inhibitors reported before tended to focus on hematological

malignancies rather than solid tumors which were insensitive

to JAK inhibitors or HDAC inhibitors. To further investigate

the antiproliferative eﬀect of these JAK/HDAC dual inhibitors

on solid tumors, we selected hepatoma, renal cell adenocarci-

noma, nonsmall cell lung carcinomas, colon cancer, and other

three breast cancer cell lines as the research objects. The

results in Supporting Information, Table S1, showed that most

of our dual inhibitors, especially the compounds 6a, 15d, and

15h, exhibited better antiproliferative activities against 4T1

and MDA-MB-231 breast cancer cell lines than other solid

cancer cell lines. The antiproliferative results preliminarily

validated that our designed compounds may provide an

eﬀective treatment for intractable breast cancers.

Encouraged by the cell-based anticancer activity in breast

cancer cells, we continue to select several breast cancer cells for

further antiproliferative activity test. Results in Table 5 showed

Table 5. In Vitro Antiproliferative Activities of

Representative Compounds on the Breast Cancer Cells

a

antiproliferative eﬀect on breast cancer cells (IC₅₀, μM)

compd

4T1

MDA-MB −231 BT-549 MDA-MB-468 Cal-51

15d

15h

SAHA

2.87

3.72

1.59

3.83

3.94

1.09

2.41

3.02

2.96

2.08

2.45

2.75

0.79

1.28

1.04

a

Assays were performed in replicate (n ≥ 3).

that compounds 15d and 15h not only showed potent

antiproliferative activities against the breast cancers 4T1 and

MDA-MB-231, with the IC₅₀values at the low digit

micromolar level, but also inhibited the growth of BT-549,

MDA-MB-468, and Cal-51 breast cancer cells. Meanwhile, in

the BT-549 (IC₅₀values: 2.41 vs 2.96 μM), MDA-MB-468

(IC₅₀values: 2.08 vs 2.75 μM), and Cal-51 breast cancer cells

I

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 7. (A) Induction of apoptosis in MDA-MB-231 cells after 48 h of treatment. (B) Eﬃcacy of compound 15d at diﬀerent concentrations to

induce apoptosis in MDA-MB-231 cells after 48 h of treatment.

(TGI) was calculated at the end of treatment. As shown in

Table 6 and Figure 10, compound 15d showed signiﬁcant

antitumor potency with TGI of 37% at the low dose of 30 mg/

kg/day and 49% at the large dose of 100 mg/kg/day. The

antitumor activity of compound 15d is comparable to or

slightly better than that of the combination group SAHA and

Ruxolitinib. In addition, the body weight of mice in the low-

dose administration (30 mg/kg/d, IP) group of compound

15d did not change signiﬁcantly, but the mice in the high-dose

administration group (100 mg/kg/d, IP) showed some weight

loss (about 15%). Nevertheless, the result indicated that

compound 15d as a potent JAK and HDAC dual inhibitor

exhibited eﬀective antitumor activity in vivo against triple-

negative breast cancer.

Protein Kinase Selectivity Proﬁle. Because of its potent

JAK/HDAC dual inhibitory activities and anticancer capacities,

compound 15d was further tested against 134 kinases to

characterize its protein kinase selective proﬁles at 50 and 500

nM, respectively. As depicted in Supporting Information,

Table S2 and Figure 11, at the concentration of 50 nM,

compound 15d exhibited over 75% inhibition of JAK1, JAK2,

JAK3, and TYK2, which further conﬁrmed that compound 15d

is a pan-JAK inhibitor. In addition, compound 15d exhibited

65% inhibition of FMS-like receptor tyrosine kinase 3 (Flt3),

which plays important roles in the progress of tumors.

Although compound 15d possessed strong inhibitory activities

against other kinases, such as VEGFR-2, Met, FGFR at 500

nM, overall, compound 15d was relatively selective to the

inhibition of JAK.

CONCLUSION

■

It is urgent to develop a more eﬀective therapy for the

treatment of invasive solid tumors, especially triple-negative

breast cancer. In the present work, a novel series of

pyrrolo[2,3-d]pyrimidine-based derivatives simultaneously in-

hibiting JAKs and HDACs were rationally designed, synthe-

sized, and evaluated. Most of these compounds possessed

JAK/HDAC dual inhibitory activities with IC₅₀values at

nanomolar levels. Lead 15d, as the representative compound,

showed the potent antiproliferative and proapoptotic activities

in several breast cancer cell lines. Western blot analysis

conﬁrmed that the newly designed compounds eﬀectively and

simultaneously inhibited JAK and HDAC proteins in the breast

cancer cell lines. Besides, compounds 15d and 15h eﬀectively

downregulated the LIFR-JAK-STAT signaling pathway via the

inhibition of phosphorylated STAT3, which implies that JAK/

J

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 8. (A) Induction of apoptosis in 4T1 cells after 48 h of treatment. (B) Eﬃcacy of compound 15d at diﬀerent concentrations to induce

apoptosis in 4T1 cells after 48 h of treatment.

Q-TOF spectrometer. Melting points were determined on an

electrothermal melting point apparatus and were uncorrected. All

the ﬁnal compounds achieved a minimum of 95% purity determined

by HPLC analysis, which was carried out on an Agilent 1200 coupled

with a diode array detector (DAD) and an extended C18 column (4.6

mm × 250 mm, 5 μm).

HDAC dual inhibitors can eﬀectively overcome the drug

resistance caused by the tumor microenvironment. More

importantly, 15d eﬀectively inhibits tumor growth in MDA-

MB-231-bearing mice. In summary, this work conﬁrms the

therapeutic potency of JAK and HDAC dual inhibitors and

proposes a novel constructive antitumor mechanism for solid

tumors.

General Procedure for the Preparation of 6a−6g. N-Hydroxy-7-

(4-((7-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-

1-yl)heptanamide (6a). A mixture of ethyl 7-(4-((7-phenyl-7H-

pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)heptanoate

(14a, 0.20 g, 0.46 mmol) in NH₂OK/CH₃OH (20 mL) was stirred at

room temperature for 0.5 h. When TLC showed the reaction was

completed, evaporation of the solvent under reduced pressure gave a

residue, which was dissolved in water (20 mL). the mixture was

neutralized by adding a 1 M HCl solution. The white precipitate was

ﬁltered and dried in cabinet drier to give the crude material. MeOH

(3−5 mL) was added and the slurry was sonicated and then stirred at

40 °C for 4 h. The solids were collected by suction ﬁltration and

allowed to air-dry overnight to give the pure compound 6a (0.15 g,

EXPERIMENTAL SECTION

■

Chemistry. Unless otherwise noted, all solvents and reagents were

commercially available (Sinopharm, Bide, Adamas-beta, SigmaAl-

drich, TCI, J&K, etc.) and used without further puriﬁcation. All

reactions were monitored by thin-layer chromatography on 0.25 mm

silica gel plates (60 GF-254) and visualized with UV light, or iodine

1

vapor. H NMR and ¹³C NMR were generated in CDCl₃, DMSO-d₆,

or CD₃OD on Varian Mercury 300, 400, or 500 NMR spectrometers.

Chemical shifts were reported in parts per million (ppm). Multiplicity

of ¹H NMR signals was reported as single (s), double (d), triplet (t),

quarter (q), and multiplet (m). ESI (Low resolution mass spectra)

were recorded on a Thermo Fisher LCQ-DECA spectrometer; ESI

(high resolution mass spectra) were determined on an Agilent G6520

1

78% yield) as a white solid, mp 148−150 °C. H NMR (400 MHz,

DMSO-d₆) δ 10.33 (s, 1H), 9.35 (s, 1H), 8.72 (s, 1H), 8.65 (s, 1H),

7.91 (s, 1H), 7.89−7.83 (m, 2H), 7.58 (dd, J = 9.7, 5.7 Hz, 3H), 7.47

K

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 9. (A) Compounds 15d and 15h inhibited LIFR-JAK-STAT signaling in MDA-MB-231 stimulated by the conditioned medium (CM) of

Wl-38 cells. The concentrations of compounds 15d, 15h, and SAHA were all 3 μM in this Western blot. (B) Compounds 15d and 15h could

diminish the activation of LIFR-JAK-STAT signaling triggered by tumor-associated ﬁbroblasts, which suggests that JAK/HDAC dual inhibitors

could potentially overcome the drug resistance caused by the tumor microenvironment and improve the in vivo anticancer activity.

((7-(4-bromophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14b, 0.10 g, 0.19 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 6b (0.08

g, 83% yield) as a white solid, mp 182−184 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.31 (s, 1H), 9.39 (s, 1H), 8.73 (d, J = 3.2 Hz, 1H),

8.65 (s, 1H), 7.96−7.69 (m, 5H), 7.58 (dd, J = 7.6, 3.8 Hz, 1H), 7.47

(d, J = 2.9 Hz, 1H), 6.65 (t, J = 3.5 Hz, 1H), 4.02 (t, J = 7.2 Hz, 2H),

2.03−1.87 (m, 2H), 1.72 (q, J = 7.2 Hz, 2H), 1.45 (p, J = 7.3 Hz,

2H), 1.39−1.12 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.5,

156.6, 138.3, 137.2, 132.5, 129.6, 125.8, 125.6, 123.9, 119.2, 102.7,

51.9, 32.6, 30.3, 28.6, 26.3, 25.5. HRMS (AP-ESI) m/z calcd for

C₂₂H₂₄BrN₇O₂[M + H]⁺498.1248, found 498.1248. Retention time:

28.7 min, eluted with 23% acetonitrile/77% water (containing 0.4%

formic acid).

Table 6. In Vivo Antitumor Eﬃcacy in MDA-MB-231

Xenograft Model

compd

administration

TGI (%)

SAHA

ruxolitinib

SAHA + ruxolitinib

15d

100 mg/kg/d, po

100 + 100 mg/kg/d, po

30 mg/kg/d, ip

26

12

33

37

a

100 mg/kg/d, ip

49

a

Compared with the control group, the treated group showed

statistically signiﬁcant (P < 0.05) TGI by Student’s two-tailed t test.

7-(4-((7-(4-Chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (6c). Ethyl 7-(4-

((7-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14c, 0.08 g, 0.17 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 6c (0.06

g, 77% yield) as a white solid, mp 185−187 °C. ¹H NMR (600 MHz,

DMSO-d₆) δ 10.30 (s, 1H), 9.37 (s, 1H), 8.72 (s, 1H), 8.63 (s, 1H),

7.95−7.84 (m, 3H), 7.66−7.61 (m, 2H), 7.57 (d, J = 3.7 Hz, 1H),

7.46 (s, 1H), 6.64 (d, J = 3.7 Hz, 1H), 4.00 (t, J = 7.1 Hz, 2H), 1.90

(t, J = 7.4 Hz, 2H), 1.70 (p, J = 7.2 Hz, 2H), 1.44 (p, J = 7.4 Hz, 2H),

1.22 (p, J = 8.6 Hz, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.4,

156.4, 152.2, 151.8, 136.8, 130.9, 129.7, 129.6, 125.6, 125.3, 123.8,

119.2, 112.2, 102.7, 51.9, 32.6, 30.3, 28.6, 26.3, 25.5. HRMS (AP-ESI)

m/z calcd for C₂₂H₂₅ClN₇O₂[M + H]⁺454.1753 found 454.1751.

Retention time: 22.5 min, eluted with 23% acetonitrile/77% water

(containing 0.4% formic acid).

7-(4-((7-(4-Fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (6d). Ethyl 7-(4-

((7-(4-ﬂuorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14d, 0.08 g, 0.17 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 6d (0.06

g, 77% yield) as a white solid, mp 173−175 °C. ¹H NMR (600 MHz,

DMSO-d₆) δ 10.30 (s, 1H), 9.35 (s, 1H), 8.72 (s, 1H), 8.63 (d, J =

1.8 Hz, 1H), 7.96−7.81 (m, 3H), 7.53 (d, J = 3.7 Hz, 1H), 7.49−7.38

(m, 3H), 6.62 (d, J = 3.7 Hz, 1H), 3.99 (t, J = 7.1 Hz, 2H), 1.89 (t, J

= 7.4 Hz, 2H), 1.69 (t, J = 7.2 Hz, 2H), 1.43 (t, J = 7.3 Hz, 2H),

1.27−1.09 (m, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.5, 161.6,

159.7, 152.3, 134.2, 129.8, 126.0, 123.7, 119.3, 116.5, 116.3, 102.3,

51.8, 32.6, 30.2, 28.6, 26.2, 25.4. HRMS (AP-ESI) m/z calcd for

Figure 10. Growth curve of the implanted MDA-MB-231 xenograft in

mice (RTV SEM). Ruxolitinib was indicated by INCB.

(s, 1H), 7.40 (t, J = 7.4 Hz, 1H), 6.62 (d, J = 3.7 Hz, 1H), 3.98 (t, J =

7.0 Hz, 2H), 1.91 (t, J = 7.3 Hz, 2H), 1.70 (p, J = 7.0 Hz, 2H), 1.44

(p, J = 7.3 Hz, 2H), 1.22 (h, J = 6.9, 6.4 Hz, 4H). ¹³C NMR (101

MHz, DMSO-d₆) δ 169.5, 156.2, 152.2, 151.4, 137.9, 129.7, 126.8,

126.0, 123.8, 119.2, 112.2, 102.4, 51.9, 32.6, 30.2, 28.6, 26.2, 25.4.

HRMS (AP-ESI) m/z calcd for C₂₂H₂₆N₇O₂[M + H]⁺420.2142,

found 420.2151. Retention time: 7.6 min, eluted with 23%

acetonitrile/77% water (containing 0.4% formic acid).

7-(4-((7-(4-Bromophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (6b). Ethyl 7-(4-

L

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 11. Kinome selectivity proﬁles for compound 15d. Percent 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 <50% is

assigned as green, inhibition >90% inhibition is assigned as red, and 50% < inhibition < 90% is assigned as yellow. In the kinome selectivity proﬁles,

ATP concentration is close to the K_mof each tested kinase.

C₂₂H₂₅FN₇O₂[M + H]⁺438.2048, found 438.2055. Retention time:

9.4 min, eluted with 23% acetonitrile/77% water (containing 0.4%

formic acid).

7-(4-((7-(4-(Methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-

2-yl)amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (6e). Ethyl 7-

(4-((7-(4-(methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14e, 0.10 g, 0.19 mmol) was

reacted using a procedure similar to the synthesis of 6a, aﬀording

compound 6e (0.08 g, 83% yield) as a white solid, mp 198−200 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 10.32 (s, 1H), 9.44 (s, 1H), 8.77

(s, 1H), 8.65 (s, 1H), 8.26 (d, J = 8.5 Hz, 2H), 8.12 (d, J = 8.5 Hz,

2H), 7.92 (s, 1H), 7.73 (d, J = 3.8 Hz, 1H), 7.53 (s, 1H), 6.72 (d, J =

3.8 Hz, 1H), 4.04 (t, J = 7.1 Hz, 2H), 3.30 (s, 3H), 1.91 (t, J = 7.4 Hz,

2H), 1.79−1.67 (m, 2H), 1.52−1.38 (m, 2H), 1.24 (d, J = 6.1 Hz,

4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.5, 156.7, 142.1, 129.9,

128.7, 123.7, 123.5, 51.9, 44.1, 32.6, 30.4, 28.6, 26.2, 25.5. HRMS

(AP-ESI) m/z calcd for C₂₃H₂₇N₇O₄S [M + H]⁺498.1918, found

498.1917. Retention time: 8.5 min, eluted with 23% acetonitrile/77%

water (containing 0.4% formic acid).

6-(4-((7-(4-(Methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-

2-yl)amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (6f). Ethyl 6-

(4-((7-(4-(methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (16f, 0.10 g, 0.20 mmol) was

reacted using a procedure similar to the synthesis of 6a, aﬀording

compound 6f (0.08 g, 83% yield) as a white solid, mp 208−210 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 10.36 (s, 1H), 9.46 (s, 1H), 8.77

((7-(4-bromophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl) amino)-1H-

pyrazol-1-yl)hexanoate (14g, 0.10 g, 0.20 mmol) was reacted using

a procedure similar to the synthesis of 6a, aﬀording compound 6g

(0.09 g, 93% yield) as a white solid, mp 174−176 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 10.30 (s, 1H), 9.37 (s, 1H), 8.72 (d, J = 3.0 Hz,

1H), 8.64 (s, 1H), 7.94−7.68 (m, 5H), 7.57 (dd, J = 7.6, 3.7 Hz, 1H),

7.47 (s, 1H), 6.63 (t, J = 3.5 Hz, 1H), 4.00 (t, J = 7.1 Hz, 2H), 1.91

(td, J = 7.5, 5.2 Hz, 2H), 1.71 (p, J = 7.2 Hz, 2H), 1.50 (h, J = 7.2 Hz,

2H), 1.25−1.13 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.3,

156.6, 152.0, 138.3, 137.3, 132.5, 129.7, 125.8, 125.7, 123.9, 119.2,

119.1, 102.7, 51.8, 32.6, 30.2, 26.2, 25.2. HRMS (AP-ESI) m/z calcd

for C₂₁H₂₂BrN₇O₂[M + H]⁺484.1091, found 484.1112. Retention

time: 17.9 min, eluted with 23% acetonitrile/77% water (containing

0.4% formic acid).

The Procedures for the Preparation of Compounds 8, 9, 10a,b,

and 11a,b Were Described in Our Previous Paper.³¹General

Procedure for the Preparation of 13a−13e; 2-Chloro-7-phenyl-7H-

pyrrolo[2,3-d]pyrimidine (13a). To a solution of 2-chloro-7H-

pyrrolo[2,3-d]pyrimidine (1.0 g, 6.51 mmol) and iodobenzene

(2.66 g, 13.02 mmol) in dioxane (20 mL) was added CuI (0.5 g,

7.24 mmol), K₃PO₄(2.0 g, 9.46 mmol), and trans-1,2-cyclohexanedi-

amine (0.20 g, 1.94 mmol). The resulting mixture was irradiated in a

microwave (150 W) at 130 °C for 1 h. The resulting mixture was

ﬁltered through Celite and concentrated under reduced pressure to

give a residue, which was dissolved in DCM (300 mL). The organic

layer was washed with 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 (CH₂Cl₂/

EtOAc, 20:1 V/V) to give compound 13a (0.3 g, 20% yield) as a

white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.08 (s, 1H), 8.08 (d, J

= 3.7 Hz, 1H), 7.87−7.79 (m, 2H), 7.69−7.59 (m, 2H), 7.49 (ddt, J =

7.9, 6.9, 1.1 Hz, 1H), 6.95 (d, J = 3.7 Hz, 1H). ESI-MS m/z = 230.1

[M + H]⁺.

(s, 2H), 8.26 (d, J = 8.7 Hz, 2H), 8.13 (d, J = 8.6 Hz, 2H), 7.91 (s,

1H), 7.73 (d, J = 3.7 Hz, 1H), 7.54 (s, 1H), 6.73 (d, J = 3.8 Hz, 1H),

4.04 (t, J = 6.9 Hz, 2H), 3.37 (s, 3H), 1.92 (t, J = 7.3 Hz, 2H), 1.80−

1.67 (m, 2H), 1.60−1.43 (m, 2H), 1.21 (dt, J = 15.1, 7.6 Hz, 2H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 169.4, 156.7, 152.6, 152.2, 142.1,

138.1, 129.9, 128.7, 125.1, 123.8, 123.5, 119.5, 103.6, 51.8, 44.1, 32.5,

30.2, 26.1, 25.1. HRMS (AP-ESI) m/z calcd for C₂₂H₂₅N₇O₄S [M +

H]⁺484.1761, found 484.1762. Retention time: 3.8 min, eluted with

23% acetonitrile/77% water (containing 0.4% formic acid).

7-(4-Bromophenyl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidine (13b).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.26 mmol) and 1-

bromo-4-iodobenzene (0.65 g, 2.30 mmol) were reacted using a

procedure similar to the synthesis of 13a, aﬀording compound 13b

(0.5 g, 50% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ

6-(4-((7-(4-Bromophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (6g). Ethyl 6-(4-

M

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

9.10 (s, 1H), 8.20 (d, J = 3.8 Hz, 1H), 8.16 (s, 4H), 7.01 (d, J = 3.8

Hz, 1H), 3.31 (s, 3H). ESI-MS m/z = 308.2 [M + H]⁺.

13j (0.78 g, 86% yield) as a white solid. ¹H NMR (400 MHz, DMSO-

d₆) δ 8.96 (s, 1H), 7.77 (d, J = 3.6 Hz, 1H), 7.41−7.31 (m, 3H), 7.16

(ddd, J = 5.5, 3.1, 1.8 Hz, 1H), 6.74 (d, J = 3.6 Hz, 1H), 5.46 (s, 2H).

ESI-MS m/z = 279.2 [M + H]⁺.

2-Chloro-7-(3-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidine

(13k). 2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.26 mmol) and

1-(chloromethyl)-3-methoxybenzene (0.56 g, 3.58 mmol) were

reacted using a procedure similar to the synthesis of 13f, aﬀording

compound 13k (0.70 g, 78% yield) as a white solid. ESI-MS m/z =

273.9 [M + H]⁺.

2-Chloro-7-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidine (13c).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.68 g, 4.43 mmol) and 1-

chloro-4-iodobenzene (2.11 g, 8.86 mmol) were reacted using a

procedure similar to the synthesis of 13a, aﬀording compound 13c

(0.45 g, 39% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆)

δ 9.04 (s, 1H), 8.05 (d, J = 3.7 Hz, 1H), 7.91−7.78 (m, 2H), 7.74−

7.61 (m, 2H), 6.92 (d, J = 3.7 Hz, 1H). ESI-MS m/z = 265.1 [M +

H]⁺.

2-Chloro-7-(4-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13l).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.26 mmol) and 1-

(bromomethyl)-4-ﬂuorobenzene (0.64 g, 2.78 mmol) were reacted

using a procedure similar to the synthesis of 13f, aﬀording compound

13l (0.70 g, 82% yield) as a white solid. ¹H NMR (400 MHz, DMSO-

d₆) δ 8.95 (s, 1H), 7.74 (d, J = 3.6 Hz, 1H), 7.37−7.28 (m, 2H),

7.22−7.13 (m, 2H), 6.73 (d, J = 3.6 Hz, 1H), 5.44 (s, 2H). ESI-MS

m/z = 262.1 [M + H]⁺.

2-Chloro-7-(4-ﬂuorophenyl)-7H-pyrrolo[2,3-d]pyrimidine (13d).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.50 g, 3.26 mmol) and 1-

ﬂuoro-4-iodobenzene (1.45 g, 6.51 mmol) were reacted using a

procedure similar to the synthesis of 13a, aﬀording compound 13d

(0.20 g, 25% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆)

δ 9.04 (s, 1H), 8.02 (d, J = 3.7 Hz, 1H), 7.87−7.77 (m, 2H), 7.50−

7.39 (m, 2H), 6.91 (dd, J = 3.7, 0.7 Hz, 1H). ESI-MS m/z = 248.1 [M

+ H]⁺.

2-Chloro-7-(4-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13m).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.50 g, 3.26 mmol) and 1-

(bromomethyl)-4-chlorobenzene (0.75 g, 3.27 mmol) were reacted

using a procedure similar to the synthesis of 13f, aﬀording compound

13m (0.70 g, 78% yield) as a white solid. ¹H NMR (400 MHz,

DMSO-d₆) δ 8.96 (s, 1H), 7.75 (d, J = 3.6 Hz, 1H), 7.45−7.37 (m,

2H), 7.30−7.22 (m, 2H), 6.74 (d, J = 3.6 Hz, 1H), 5.45 (s, 2H). ESI-

MS m/z = 278.2 [M + H]⁺.

2-Chloro-7-(4-methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13n).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.50 g, 3.26 mmol) and 1-

(bromomethyl)-4-methylbenzene (0.60 g, 3.27 mmol) were reacted

using a procedure similar to the synthesis of 13f, aﬀording compound

13n (0.70 g, 83% yield) as a white solid. ¹H NMR (400 MHz,

DMSO-d₆) δ 8.94 (s, 1H), 7.71 (d, J = 3.6 Hz, 1H), 7.15 (s, 4H), 6.71

(d, J = 3.6 Hz, 1H), 5.40 (s, 2H), 2.26 (s, 3H). ESI-MS m/z = 258.1

[M + H]⁺.

2-Chloro-7-(4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidine

(13o). 2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.50 g, 3.26 mmol)

and 1-(bromomethyl)-4-methoxybenzene (0.65 g, 3.27 mmol) were

reacted using a procedure similar to the synthesis of 13f, aﬀording

compound 13o (0.70 g, 79% yield) as a white solid. ESI-MS m/z =

274.3 [M + H]⁺.

2-Chloro-7-phenethyl-7H-pyrrolo[2,3-d]pyrimidine (13p). 2-

Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.50 g, 3.26 mmol) and (2-

bromoethyl)benzene (0.60 g, 3.27 mmol) were reacted using a

procedure similar to the synthesis of 13f, aﬀording compound 13p

(0.70 g, 83% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.88 (s, 1H), 7.58 (d, J = 3.6 Hz, 1H), 7.28−7.08 (m, 5H), 6.62 (d,

J = 3.6 Hz, 1H), 4.46 (dd, J = 7.7, 6.7 Hz, 2H), 3.11 (t, J = 7.2 Hz,

2H). ESI-MS m/z = 258.1 [M + H]⁺.

2-Chloro-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidine (13q). 2-

Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.7 g, 4.56 mmol) and

bromocyclopentane (1.36 g, 9.12 mmol) were reacted using a

procedure similar to the synthesis of 13f, aﬀording compound 13q

(0.70 g, 69% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.91 (s, 1H), 7.79 (d, J = 3.7 Hz, 1H), 6.71 (d, J = 3.7 Hz, 1H),

5.12−5.04 (m, 1H), 2.14 (ddt, J = 10.9, 5.3, 2.8 Hz, 2H), 1.93−1.83

(m, 4H), 1.76−1.67 (m, 2H). ESI-MS m/z = 222.3 [M + H]⁺.

2-Chloro-7-(cyclopropylmethyl)-7H-pyrrolo[2,3-d]pyrimidine

(13r). 2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (1.0 g, 6.51 mmol) and

(bromomethyl)cyclopropane (1.05 g, 7.81 mmol) were reacted using

a procedure similar to the synthesis of 13f, aﬀording compound 13r

(0.90 g, 67% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.92 (s, 1H), 7.76 (d, J = 3.6 Hz, 1H), 6.69 (d, J = 3.6 Hz, 1H), 4.08

(d, J = 7.2 Hz, 2H), 1.31−1.22 (m, 1H), 0.51 (dt, J = 8.0, 3.0 Hz,

2H), 0.47−0.38 (m, 2H). ESI-MS m/z = 208.2 [M + H]⁺.

2-Chloro-7-(cyclohexylmethyl)-7H-pyrrolo[2,3-d]pyrimidine

(13s). 2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (1.0 g, 6.51 mmol) and

(bromomethyl)cyclohexane (1.38 g, 7.81 mmol) were reacted using a

procedure similar to the synthesis of 13f, aﬀording compound 13s

(1.20 g, 74% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.92 (s, 1H), 7.67 (d, J = 3.5 Hz, 1H), 6.69 (d, J = 3.5 Hz, 1H), 4.06

2-Chloro-7-(4-(methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]-

pyrimidine (13e). 2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.26

mmol) and 1-bromo-4-(methylsulfonyl)benzene (0.65 g, 2.66 mmol)

were reacted using a procedure similar to the synthesis of 13a,

aﬀording compound 13e (0.5 g, 50% yield) as a white solid. ¹H NMR

(400 MHz, DMSO-d₆) δ 9.10 (s, 1H), 8.20 (d, J = 3.8 Hz, 1H), 8.16

(s, 4H), 7.01 (d, J = 3.8 Hz, 1H), 3.31 (s, 3H). ESI-MS m/z = 308.2

[M + H]⁺.

General Procedure for the Preparation of 13f−13s. 7-Benzyl-2-

chloro-7H-pyrrolo[2,3-d]pyrimidine (13f). To a solution of 2-chloro-

7H-pyrrolo[2,3-d]pyrimidine (0.8 g, 5.22 mmol) and (bromomethyl)-

benzene (0.97 g, 5.75 mmol) in acetonitrile (80 mL) was added

K₂CO₃(1.4 g, 10.44 mmol). The resulting mixture was heated at 40−

50 °C for 5 h. The resulting mixture was ﬁltered through Celite and

concentrated under reduced pressure to give a residue, which was

dissolved in DCM (150 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/

EtOAc, 8:1 v/v) to give compound 13f (1.0 g, 78% yield) as a white

solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (s, 1H), 7.75 (d, J = 3.6

Hz, 1H), 7.39−7.19 (m, 5H), 6.73 (d, J = 3.6 Hz, 1H), 5.45 (s, 2H).

ESI-MS m/z = 244.1 [M + H]⁺.

2-Chloro-7-(2-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13g).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.26 mmol) and 1-

(bromomethyl)-2-ﬂuorobenzene (0.70 g, 3.74 mmol) were reacted

using a procedure similar to the synthesis of 13f, aﬀording compound

13g (0.78 g, 92% yield) as a white solid. ¹H NMR (400 MHz,

DMSO-d₆) δ 8.96 (s, 1H), 7.77−7.61 (m, 1H), 7.37 (td, J = 7.6, 1.8

Hz, 1H), 7.28−7.20 (m, 1H), 7.20−7.08 (m, 2H), 6.74 (d, J = 3.6 Hz,

1H), 5.49 (d, J = 13.4 Hz, 2H). ESI-MS m/z = 262.1 [M + H]⁺.

2-Chloro-7-(2-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13h).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.6 g, 3.91 mmol) and 1-

(bromomethyl)-2-chlorobenzene (0.88 g, 4.30 mmol) were reacted

using a procedure similar to the synthesis of 13f, aﬀording compound

13h (0.87 g, 80% yield) as a white solid. ¹H NMR (400 MHz,

DMSO-d₆) δ 8.96 (s, 1H), 7.65 (d, J = 3.6 Hz, 1H), 7.51 (dd, J = 7.9,

1.3 Hz, 1H), 7.30 (dtd, J = 29.3, 7.5, 1.5 Hz, 2H), 6.82 (dd, J = 7.7,

1.7 Hz, 1H), 6.75 (d, J = 3.7 Hz, 1H), 5.52 (s, 2H). ESI-MS m/z =

279.1 [M + H]⁺.

2-Chloro-7-(3-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13i).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.26 mmol) and 1-

(bromomethyl)-3-ﬂuorobenzene (0.64 g, 2.78 mmol) were reacted

using a procedure similar to the synthesis of 13f, aﬀording compound

13i (0.80 g, 96% yield) as a white solid. ¹H NMR (400 MHz, DMSO-

d₆) δ 8.96 (s, 1H), 7.77 (d, J = 3.6 Hz, 1H), 7.39 (td, J = 7.9, 6.0 Hz,

1H), 7.25−6.96 (m, 3H), 6.75 (d, J = 3.6 Hz, 1H), 5.48 (s, 2H). ESI-

MS m/z = 262.2 [M + H]⁺.

2-Chloro-7-(3-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13j).

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.26 mmol) and 1-

(bromomethyl)-3-chlorobenzene (0.73 g, 3.58 mmol) were reacted

using a procedure similar to the synthesis of 13f, aﬀording compound

N

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(d, J = 7.4 Hz, 2H), 1.85 (dqd, J = 11.1, 7.5, 3.8 Hz, 1H), 1.65 (h, J =

4.2, 3.4 Hz, 2H), 1.59 (s, 1H), 1.48−1.43 (m, 2H), 1.20−1.10 (m,

3H), 0.97 (qd, J = 11.5, 3.5 Hz, 2H). ESI-MS m/z = 250.1 [M + H]⁺.

General Procedure for the Preparation of 14a−14z and 14A−

14F. Ethyl 7-(4-((7-Phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-

1H-pyrazol-1-yl)heptanoate (14a). 2-Chloro-7-phenyl-7H-pyrrolo-

[2,3-d]pyrimidine (13a, 0.23 g, 1.00 mmol) and ethyl 7-(4-amino-1H-

pyrazol-1-yl)heptanoate (hydrochloride) (11b, 0.20 g, 0.83 mmol)

were suspended in ethanol (16 mL). The mixture was irradiated in a

microwave (150 W) at 125 °C for 2−3 h. Evaporation of the solvent

under reduced pressure gave a residue, which was preliminarily

puriﬁed by silica gel chromatography (CH₂Cl₂/CH₃OH/Et₃N,

100:1:0.25 v/v/v) to give the product 14a (0.10 g, yield 27%) as a

white solid. ESI-MS m/z = 433.2 [M + H]⁺.

Ethyl 7-(4-((7-(4-Bromophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14b). 7-(4-Bromophenyl)-2-

chloro-7H-pyrrolo[2,3-d]pyrimidine (13b, 0.3 g, 0.97 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14b (0.10 g, yield 22%) as a

white solid. ESI-MS m/z = 511.2 [M + H]⁺.

Ethyl 7-(4-((7-(4-Chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14c). 2-Chloro-7-(4-chloro-

phenyl)-7H-pyrrolo[2,3-d]pyrimidine (13c, 0.26 g, 1.00 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.20 g, 0.83 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14c (0.08 g, yield 20%) as a

white solid. ESI-MS m/z = 467.1 [M + H]⁺.

Ethyl 7-(4-((7-(4-Fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14d). 2-Chloro-7-(4-ﬂuoro-

phenyl)-7H-pyrrolo[2,3-d]pyrimidine (13d, 0.22 g, 0.90 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.18 g, 0.75 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14d (0.08 g, yield 20%) as a

white solid. ESI-MS m/z = 451.2 [M + H]⁺.

6H), 6.43 (d, J = 3.5 Hz, 1H), 5.38 (s, 2H), 4.02 (tt, J = 7.1, 3.8 Hz,

4H), 2.23 (dt, J = 14.6, 7.3 Hz, 2H), 1.71 (h, J = 8.3, 7.7 Hz, 2H),

1.60−1.43 (m, 2H), 1.28−1.17 (m, 2H), 1.15 (d, J = 6.9 Hz, 3H).

ESI-MS m/z = 433.2 [M + H]⁺.

Ethyl 7-(4-((7-Benzyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-

1H-pyrazol-1-yl)heptanoate (14i). 7-Benzyl-2-chloro-7H-pyrrolo-

[2,3-d]pyrimidine (13f, 0.35 g, 1.44 mmol) and ethyl 7-(4-amino-

1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b, 0.25 g, 0.91

mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14i (0.15 g, yield 38%) as a white solid. ¹H

NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.63 (s, 1H), 7.93 (s,

1H), 7.48 (s, 1H), 7.34−7.22 (m, 6H), 6.43 (d, J = 3.5 Hz, 1H), 5.38

(s, 2H), 4.10−3.95 (m, 4H), 2.29−2.16 (m, 2H), 1.71 (p, J = 7.0 Hz,

2H), 1.48 (pd, J = 7.2, 2.4 Hz, 2H), 1.26 (ddd, J = 23.4, 11.8, 7.5 Hz,

4H), 1.18−1.05 (m, 3H). ESI-MS m/z = 447.1 [M + H]⁺.

Ethyl 6-(4-((7-(2-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14j). 2-Chloro-7-(2-ﬂuoroben-

zyl)-7H-pyrrolo[2,3-d]pyrimidine (13g, 0.35 g, 1.34 mmol) and ethyl

6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a, 0.25 g,

0.95 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14j (0.15 g, 35% yield) as a white solid. ESI-

MS m/z = 451.1 [M + H]⁺.

Ethyl 7-(4-((7-(2-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14k). 2-Chloro-7-(2-ﬂuoro-

benzyl)-7H-pyrrolo[2,3-d]pyrimidine (13g, 0.35 g, 1.34 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14k (0.16 g, yield 38%) as a

white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 9.73 (s, 1H), 8.73 (s,

1H), 7.98 (s, 1H), 7.54 (s, 1H), 7.42 (d, J = 3.7 Hz, 1H), 7.39−7.34

(m, 1H), 7.25 (dd, J = 10.4, 8.2 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H),

7.14 (t, J = 7.4 Hz, 1H), 6.58 (d, J = 3.7 Hz, 1H), 5.46 (s, 2H), 4.07−

4.00 (m, 4H), 2.24 (t, J = 7.4 Hz, 2H), 1.78−1.69 (m, 2H), 1.48 (q, J

= 7.5 Hz, 2H), 1.25 (dq, J = 26.2, 7.8 Hz, 4H), 1.15 (t, J = 7.1 Hz,

3H). ESI-MS m/z = 465.1 [M + H]⁺.

Ethyl 7-(4-((7-(4-(Methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]-

pyrimidin-2-yl) amino)-1H-pyrazol-1-yl)heptanoate (14e). 2-

Chloro-7-(4-(methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine

(13e, 0.30 g, 0.97 mmol) and ethyl 7-(4-amino-1H-pyrazol-1-

yl)heptanoate (hydrochloride) (11b, 0.25 g, 0.91 mmol) were reacted

using a procedure similar to the synthesis of 14a, aﬀording compound

14e (0.10 g, yield 20%) as a white solid. ESI-MS m/z = 511.1 [M +

H]⁺.

Ethyl 6-(4-((7-(2-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14l). 2-Chloro-7-(2-chloroben-

zyl)-7H-pyrrolo[2,3-d]pyrimidine (13h, 0.37 g, 1.33 mmol) and ethyl

6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a, 0.25 g,

0.95 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14l (0.15 g, 30% yield) as a white solid. ESI-

MS m/z = 467.2 [M + H]⁺.

Ethyl 7-(4-((7-(2-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14m). 2-Chloro-7-(2-chloro-

benzyl)-7H-pyrrolo[2,3-d]pyrimidine (13h, 0.35 g, 1.25 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14m (0.10 g, yield 20%) as a

white solid. ESI-MS m/z = 481.2 [M + H]⁺.

Ethyl 6-(4-((7-(3-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14n). 2-Chloro-7-(3-ﬂuoroben-

zyl)-7H-pyrrolo[2,3-d]pyrimidine (13i, 0.35 g, 1.34 mmol) and ethyl

6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a, 0.25 g,

0.95 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14n (0.15 g, yield 35%) as a white solid.

ESI-MS m/z = 451.1 [M + H]⁺.

Ethyl 7-(4-((7-(3-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14o). 2-Chloro-7-(3-ﬂuoro-

benzyl)-7H-pyrrolo[2,3-d]pyrimidine (13i, 0.35 g, 1.34 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14o (0.16 g, yield 38%) as a

white solid. ESI-MS m/z = 465.1 [M + H]⁺.

Ethyl 6-(4-((7-(3-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14p). 2-Chloro-7-(3-chloroben-

zyl)-7H-pyrrolo[2,3-d]pyrimidine (13j, 0.37 g, 1.34 mmol) and ethyl

6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a, 0.25 g,

0.95 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14p (0.10 g, yield 20%) as a white solid.

ESI-MS m/z = 467.2 [M + H]⁺.

Ethyl 6-(4-((7-(4-(Methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]-

pyrimidin-2-yl) amino)-1H-pyrazol-1-yl)hexanoate (14f). 2-

Chloro-7-(4-(methylsulfonyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine

(13e, 0.30 g, 0.97 mmol) and ethyl 6-(4-amino-1H-pyrazol-1-

yl)hexanoate (hydrochloride) (11a, 0.25 g, 0.95 mmol) were reacted

using a procedure similar to the synthesis of 14a, aﬀording compound

14f (0.10 g, 22% yield) as a white solid. ¹H NMR (400 MHz, DMSO-

d₆) δ 9.45 (s, 1H), 8.77 (s, 1H), 8.25 (d, J = 8.8 Hz, 2H), 8.12 (d, J =

8.6 Hz, 2H), 7.92 (s, 1H), 7.73 (d, J = 3.8 Hz, 1H), 7.52 (s, 1H), 6.72

(d, J = 3.8 Hz, 1H), 4.04 (q, J = 6.3, 5.7 Hz, 2H), 3.95 (t, J = 6.6 Hz,

2H), 3.30 (s, 3H), 2.25 (t, J = 7.3 Hz, 2H), 1.80−1.68 (m, 2H),

1.52−1.47 (m, 2H), 1.26−1.21 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H).

ESI-MS m/z = 497.2 [M + H]⁺.

Ethyl 6-(4-((7-(4-Bromophenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14g). 7-(4-Bromophenyl)-2-

chloro-7H-pyrrolo[2,3-d]pyrimidine (13b, 0.3 g, 0.97 mmol) and

ethyl 6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a,

0.25 g, 0.95 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14g (0.15 g, 32% yield) as a

white solid. ESI-MS m/z = 497.1 [M + H]⁺.

Ethyl 6-(4-((7-Benzyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-

1H-pyrazol-1-yl)hexanoate (14h). 7-Benzyl-2-chloro-7H-pyrrolo-

[2,3-d]pyrimidine (13f, 0.35 g, 1.44 mmol) and ethyl 6-(4-amino-

1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a, 0.25 g, 0.95 mmol)

were reacted using a procedure similar to the synthesis of 14a,

1

aﬀording compound 14h (0.18 g, 45% yield) as a white solid. H

NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.63 (s, 1H), 8.02−7.84

(m, 1H), 7.49 (d, J = 0.9 Hz, 1H), 7.30 (dtd, J = 12.0, 8.3, 7.7, 5.8 Hz,

O

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Ethyl 7-(4-((7-(3-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14q). 2-Chloro-7-(3-chloro-

benzyl)-7H-pyrrolo[2,3-d]pyrimidine (13j, 0.37 g, 1.34 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14q (0.10 g, yield 20%) as a

white solid. ESI-MS m/z = 481.1 [M + H]⁺.

Ethyl 6-(4-((7-(3-Methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-

yl)amino)-1H-pyrazol-1-yl)hexanoate (14r). 2-Chloro-7-(3-methox-

ybenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13k, 0.37 g, 1.34 mmol) and

ethyl 6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a,

0.25 g, 0.95 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14r (0.12 g, yield 27%) as a

white solid. ESI-MS m/z = 463.2 [M + H]⁺.

Ethyl 7-(4-((7-(3-Methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-

yl)amino)-1H-pyrazol-1-yl)heptanoate (14s). 2-Chloro-7-(3-me-

thoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13k, 0.29 g, 1.09 mmol)

and ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride)

(11b, 0.25 g, 0.91 mmol) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 14s (0.10 g, yield 20%) as a

white solid. ESI-MS m/z = 477.3 [M + H]⁺.

Ethyl 6-(4-((7-(4-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14t). 2-Chloro-7-(4-ﬂuoroben-

zyl)-7H-pyrrolo[2,3-d]pyrimidine (13l, 0.35 g, 1.34 mmol) and ethyl

6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a, 0.25 g,

0.95 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14t (0.15 g, 35% yield) as a white solid.

ESI-MS m/z = 451.2 [M + H]⁺.

Ethyl 7-(4-((7-(4-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14u). 2-Chloro-7-(4-ﬂuoro-

benzyl)-7H-pyrrolo[2,3-d]pyrimidine (13l, 0.35 g, 1.34 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14u (0.16 g, yield 38%) as a

white solid. ESI-MS m/z = 465.2 [M + H]⁺.

Ethyl 6-(4-((7-(4-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14v). 2-Chloro-7-(4-chloroben-

zyl)-7H-pyrrolo[2,3-d]pyrimidine (13m, 0.35 g, 1.25 mmol) and ethyl

6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a, 0.25 g,

0.95 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14v (0.15 g, 34% yield) as a white solid.

ESI-MS m/z = 467.1 [M + H]⁺.

Ethyl 7-(4-((7-(4-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14w). 2-Chloro-7-(4-chloro-

benzyl)-7H-pyrrolo[2,3-d]pyrimidine (13m, 0.35 g, 1.25 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14w (0.15 g, yield 34%) as a

white solid. ESI-MS m/z = 481.1 [M + H]⁺.

Ethyl 6-(4-((7-(4-Methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14x). 2-Chloro-7-(4-methyl-

benzyl)-7H-pyrrolo[2,3-d]pyrimidine (13n, 0.35 g, 1.36 mmol) and

ethyl 6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a,

0.25 g, 0.95 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14x (0.20 g, 47% yield) as a

white solid. ESI-MS m/z = 447.2 [M + H]⁺.

3H), 6.98−6.82 (m, 2H), 6.40 (d, J = 3.5 Hz, 1H), 5.30 (s, 2H),

4.09−3.95 (m, 4H), 3.70 (s, 3H), 2.23 (dt, J = 14.5, 7.4 Hz, 2H),

1.80−1.68 (m, 2H), 1.53 (td, J = 7.8, 3.0 Hz, 2H), 1.30−1.18 (m,

2H), 1.13 (d, J = 6.3 Hz, 3H). ESI-MS m/z = 463.1 [M + H]⁺.

Ethyl 7-(4-((7-(4-Methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-

yl)amino)-1H-pyrazol-1-yl)heptanoate (14A). 2-Chloro-7-(4-me-

thoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13o, 0.35 g, 1.28 mmol)

and ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride)

(11b, 0.25 g, 0.91 mmol) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 14A (0.18 g, yield 41%) as a

white solid. ESI-MS m/z = 477.1 [M + H]⁺.

Ethyl 6-(4-((7-Phenethyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)hexanoate (14B). 2-Chloro-7-phenethyl-

7H-pyrrolo[2,3-d]pyrimidine (13p, 0.35 g, 1.36 mmol) and ethyl 6-

(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a, 0.25 g,

0.95 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14B (0.16 g, yield 38%) as a white solid.

ESI-MS m/z = 447.2 [M + H]⁺.

Ethyl 7-(4-((7-Phenethyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14C). 2-Chloro-7-phenethyl-

7H-pyrrolo[2,3-d]pyrimidine (13p, 0.35 g, 1.36 mmol) and ethyl 7-

(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b, 0.25 g,

0.91 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14C (0.16 g, yield 38%) as a white solid.

ESI-MS m/z = 461.2 [M + H]⁺.

Ethyl 7-(4-((7-Cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14D). 2-Chloro-7-cyclopentyl-

7H-pyrrolo[2,3-d]pyrimidine (13q, 0.30 g, 1.35 mmol) and ethyl 7-

(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b, 0.25 g,

0.91 mmol) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14D (0.12 g, yield 21%) as a colorless oil.

ESI-MS m/z = 425.2 [M + H]⁺.

Ethyl 7-(4-((7-(Cyclopropylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-

2-yl)amino)-1H-pyrazol-1-yl)heptanoate (14E). 2-Chloro-7-(cyclo-

propylmethyl)-7H-pyrrolo[2,3-d]pyrimidine (13r, 0.28 g, 1.35 mmol)

and ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride)

(11b, 0.25 g, 0.91 mmol) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 14E (0.10 g, 18% yield) as a

colorless oil. ESI-MS m/z = 425.2 [M + H]⁺.

Ethyl 7-(4-((7-(Cyclohexylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-

yl)amino)-1H-pyrazol-1-yl)heptanoate (14F). 2-Chloro-7-(cyclohex-

ylmethyl)-7H-pyrrolo[2,3-d]pyrimidine (13s, 0.32 g, 1.35 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14F (0.15 g, 25% yield) as a

white solid. ESI-MS m/z = 453.3 [M + H]⁺.

General Procedure for the Preparation of 15a−l, 16a−j, and

17a−c. 6-(4-((7-Benzyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-

1H-pyrazol-1-yl)-N-hydroxyhexanamide (15a). Ethyl 6-(4-((7-ben-

zyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-1-yl) hexa-

noate (14h, 0.16 g, 0.37 mmol) was reacted using a procedure

similar to the synthesis of 6a, aﬀording compound 15a (0.12 g, 80%

yield) as a white solid, mp 160−162 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.39 (s, 1H), 9.24 (s, 1H), 8.70 (s, 1H), 8.63 (s, 1H),

7.92 (s, 1H), 7.50 (s, 1H), 7.29 (td, J = 12.0, 9.8, 5.7 Hz, 6H), 6.43

(d, J = 3.6 Hz, 1H), 5.39 (s, 2H), 4.02 (t, J = 6.9 Hz, 2H), 1.93 (t, J =

7.3 Hz, 2H), 1.72 (p, J = 7.2 Hz, 2H), 1.50 (p, J = 7.5 Hz, 2H), 1.20

(p, J = 7.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.4, 156.3,

152.3, 151.1, 138.6, 129.6, 129.0, 127.8, 127.5, 126.8, 124.2, 119.1,

111.5, 100.1, 51.6, 47.5, 32.6, 30.2, 26.1, 25.2. HRMS (AP-ESI) m/z

calcd for C₂₂H₂₅N₇O₂[M + H]⁺420.2142, found 420.2147.

Retention time: 3.7 min, eluted with 23% acetonitrile/77% water

(containing 0.4% formic acid).

Ethyl 7-(4-((7-(4-Methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)heptanoate (14y). 2-Chloro-7-(4-methyl-

benzyl)-7H-pyrrolo[2,3-d]pyrimidine (13n, 0.35 g, 1.36 mmol) and

ethyl 7-(4-amino-1H-pyrazol-1-yl)heptanoate (hydrochloride) (11b,

0.25 g, 0.91 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14y (0.20 g, yield 47%) as a

white solid. ESI-MS m/z = 461.1 [M + H]⁺.

Ethyl 6-(4-((7-(4-Methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-

yl)amino)-1H-pyrazol-1-yl)hexanoate (14z). 2-Chloro-7-(4-methox-

ybenzyl)-7H-pyrrolo[2,3-d]pyrimidine (13o, 0.35 g, 1.28 mmol) and

ethyl 6-(4-amino-1H-pyrazol-1-yl)hexanoate (hydrochloride) (11a,

0.25 g, 0.95 mmol) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14z (0.18 g, 41% yield) as a

white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.61 (s,

1H), 8.04−7.95 (m, 1H), 7.51 (t, J = 0.7 Hz, 1H), 7.31−7.20 (m,

7-(4-((7-Benzyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyra-

zol-1-yl)-N-hydroxyheptanamide (15b). Ethyl 7-(4-((7-benzyl-7H-

pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-1-yl) heptanoate

(14i, 0.12 g, 0.26 mmol) was reacted using a procedure similar to

the synthesis of 6a, aﬀording compound 15b (0.12 g, 95% yield) as a

white solid, mp 150−152 °C. H NMR (400 MHz, DMSO-d₆) δ

10.35 (s, 1H) 9.23 (s, 1H), 8.67 (d, J = 1.8 Hz, 1H), 8.63 (s, 1H),

7.97−7.88 (m, 1H), 7.48 (d, J = 0.7 Hz, 1H), 7.36−7.22 (m, 6H),

1

P

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

6.43 (d, J = 3.6 Hz, 1H), 5.38 (s, 2H), 4.02 (t, J = 7.0 Hz, 2H), 1.92

(t, J = 7.3 Hz, 2H), 1.71 (q, J = 7.1 Hz, 2H), 1.44 (q, J = 7.2 Hz, 2H),

1.22 (d, J = 6.8 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.5,

156.3, 151.1, 138.6, 129.6, 129.0, 127.8, 127.5, 124.2, 119.1, 100.2,

51.7, 47.4, 32.6, 30.4, 28.6, 26.2, 25.4. ESI-MS m/z = 434 [M + H]⁺.

HRMS (AP-ESI) m/z calcd for C₂₃H₂₇N₇O₂[M + H]⁺434.2299,

found 434.2304. Retention time: 5.5 min, eluted with 23%

acetonitrile/77% water (containing 0.4% formic acid).

6-(4-((7-(2-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (15c). Ethyl 6-(4-

((7-(2-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl) amino)-1H-

pyrazol-1-yl)hexanoate (14j, 0.12 g, 0.26 mmol) was reacted using

a procedure similar to the synthesis of 6a, aﬀording compound 15c

(0.08 g, 73% yield) as a white solid, mp 156−158 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.36 (s, 1H), 9.23 (s, 1H), 8.76 (s, 1H), 8.63 (s,

1H), 7.94 (s, 1H), 7.48 (s, 1H), 7.34 (dtd, J = 8.7, 5.6, 2.7 Hz, 1H),

7.29−7.21 (m, 2H), 7.12 (d, J = 6.2 Hz, 2H), 6.45 (d, J = 3.5 Hz,

1H), 5.44 (s, 2H), 4.02 (t, J = 7.0 Hz, 2H), 1.93 (t, J = 7.4 Hz, 2H),

1.80−1.63 (m, 2H), 1.59−1.41 (m, 2H), 1.21 (tt, J = 9.6, 6.3 Hz,

2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.4, 161.4, 159.0, 156.3,

152.3, 151.2, 130.1, 130.1, 129.8, 129.6, 126.7, 125.2, 125.1, 125.1,

124.2, 119.1, 115.9, 115.7, 111.4, 100.4, 51.7, 41.3, 32.6, 30.2, 26.1,

25.2. HRMS (AP-ESI) m/z calcd for C₂₂H₂₄FN₇O₂[M + H]⁺

438.2048, found 438.2053. Retention time: 4.6 min, eluted with

23% acetonitrile/77% water (containing 0.4% formic acid).

7-(4-((7-(2-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (15d). Ethyl 7-(4-

((7-(2-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl) amino)-1H-

pyrazol-1-yl)heptanoate (14k, 0.15 g, 0.32 mmol) was reacted using

a procedure similar to the synthesis of 6a, aﬀording compound 15d

(0.10 g, 70% yield) as a white solid, mp 158−160 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 10.31 (s, 1H), 9.22 (s, 1H), 8.63 (d, J = 16.2 Hz,

2H), 7.92 (s, 1H), 7.45 (s, 1H), 7.35−7.29 (m, 1H), 7.26−7.19 (m,

2H), 7.13−7.04 (m, 2H), 6.43 (d, J = 3.6 Hz, 1H), 5.42 (s, 2H), 4.00

(t, J = 7.0 Hz, 2H), 1.90 (t, J = 7.4 Hz, 2H), 1.70 (t, J = 7.2 Hz, 2H),

1.49−1.37 (m, 2H), 1.21 (q, J = 12.7, 8.3 Hz, 4H). ¹³C NMR (101

MHz, DMSO-d₆) δ 169.5, 161.4, 159.0, 156.3, 152.3, 151.2, 130.1,

130.1, 129.8, 129.6, 126.7, 125.2, 125.1, 125.0, 124.2, 119.1, 115.9,

115.7, 111.4, 100.4, 51.7, 41.3, 32.6, 30.4, 28.6, 26.2, 25.5. HRMS

(AP-ESI) m/z calcd for C₂₃H₂₆FN₇O₂[M + H]⁺452.2205, found

452.2209. Retention time: 6.2 min, eluted with 25% acetonitrile/75%

water (containing 0.4% formic acid).

6-(4-((7-(2-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (15e). Ethyl 6-(4-

((7-(2-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)hexanoate (14l, 0.12 g, 0.25 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 15e

(0.08 g, 68% yield) as a white solid, mp 172−174 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 10.35 (s, 1H), 9.25 (s, 1H), 8.67 (s, 2H), 7.82 (s,

1H), 7.54 (dd, J = 8.0, 1.3 Hz, 1H), 7.43 (s, 1H), 7.33 (td, J = 7.7, 1.8

Hz, 1H), 7.27−7.23 (m, 2H), 6.79 (s, 1H), 6.49 (d, J = 3.6 Hz, 1H),

5.48 (s, 2H), 3.97 (t, J = 7.0 Hz, 2H), 1.92 (t, J = 7.4 Hz, 2H), 1.69

(p, J = 7.4 Hz, 2H), 1.49 (p, J = 7.4 Hz, 2H), 1.18 (p, J = 7.7 Hz, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 169.4, 156.3, 151.2, 135.7, 131.9,

156.3, 152.5, 151.2, 131.9, 129.8, 129.6, 128.7, 128.0, 126.8, 124.2,

118.9, 100.5, 51.8, 45.2, 32.6, 30.4, 28.6, 26.2, 25.4. HRMS (AP-ESI)

m/z calcd for C₂₃H₂₇ClN₇O₂[M + H]⁺468.1909, found 468.1900.

Retention time: 11.7 min, eluted with 23% acetonitrile/77% water

(containing 0.4% formic acid).

6-(4-((7-(3-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (15g). Ethyl 6-(4-

((7-(3-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)hexanoate (14n, 0.16 g, 0.35 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 15g

(0.10 g, 67% yield) as a white solid, mp 156−158 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.35 (s, 1H), 9.23 (s, 1H), 8.64 (s, 2H), 7.91 (s,

1H), 7.48 (s, 1H), 7.41−7.33 (m, 1H), 7.31 (d, J = 3.6 Hz, 1H),

7.14−7.04 (m, 3H), 6.45 (d, J = 3.6 Hz, 1H), 5.41 (s, 2H), 4.02 (t, J =

7.0 Hz, 2H), 1.92 (t, J = 7.4 Hz, 2H), 1.72 (p, J = 7.2 Hz, 2H), 1.49

(h, J = 7.2 Hz, 2H), 1.29−1.12 (m, 2H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 169.4, 163.8, 161.4, 156.3, 152.3, 151.2, 141.6, 141.5,

131.1, 131.0, 129.6, 126.7, 124.2, 123.5, 123.5, 119.1, 114.7, 114.5,

114.4, 114.2, 100.3, 51.6, 46.9, 32.6, 30.2, 26.1, 25.1. HRMS (AP-ESI)

m/z calcd for C₂₂H₂₄FN₇O₂[M + H]⁺438.2048, found 438.2049.

Retention time: 4.7 min, eluted with 23% acetonitrile/77% water

(containing 0.4% formic acid).

7-(4-((7-(3-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (15h). Ethyl 7-(4-

((7-(3-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl) amino)-1H-

pyrazol-1-yl)heptanoate (14o, 0.15 g, 0.32 mmol) was reacted using

a procedure similar to the synthesis of 6a, aﬀording compound 15h

(0.10 g, 70% yield) as a white solid, mp 158−160 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.33 (s, 1H), 9.23 (s, 1H), 8.63 (s, 2H), 7.91 (s,

1H), 7.47 (s, 1H), 7.41−7.32 (m, 1H), 7.30 (d, J = 3.6 Hz, 1H),

7.13−7.06 (m, 3H), 6.44 (d, J = 3.6 Hz, 1H), 5.40 (s, 2H), 4.02 (t, J =

7.0 Hz, 2H), 1.92 (t, J = 7.4 Hz, 2H), 1.71 (t, J = 7.1 Hz, 2H), 1.51−

1.40 (m, 2H), 1.21 (dd, J = 8.1, 4.5 Hz, 4H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 169.5, 163.8, 161.4, 156.3, 152.3, 151.2, 141.6, 141.5,

131.1, 131.0, 129.6, 126.7, 124.1, 123.5, 123.5, 119.1, 114.7, 114.5,

114.4, 114.2, 100.3, 51.7, 46.9, 32.6, 30.4, 28.6, 26.2, 25.4. HRMS

(AP-ESI) m/z calcd for C₂₃H₂₆FN₇O₂[M + H]⁺452.2205, found

452.2205. Retention time:6.7 min, eluted with 23% acetonitrile/77%

water (containing 0.4% formic acid).

6-(4-((7-(3-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (15i). Ethyl 6-(4-

((7-(3-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)hexanoate (14p, 0.06 g, 0.13 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 15i

(0.05 g, 86% yield) as a white solid, mp 160−162 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.32 (s, 1H), 9.46 (s, 1H), 9.23 (s, 1H), 8.64 (s,

1H), 8.62 (s, 1H), 7.89 (s, 1H), 7.46 (s, 1H), 7.36−7.29 (m, 3H),

7.20 (dd, J = 6.8, 2.1 Hz, 1H), 6.43 (d, J = 3.6 Hz, 1H), 5.38 (s, 2H),

4.00 (t, J = 7.0 Hz, 2H), 1.90 (t, J = 7.4 Hz, 2H), 1.76−1.63 (m, 2H),

1.54−1.43 (m, 2H), 1.19 (q, J = 7.8 Hz, 2H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 169.4, 141.0, 133.6, 131.0, 129.8, 127.8, 127.4, 126.2,

123.8, 119.4, 111.5, 100.6, 51.7, 46.9, 32.6, 30.2, 26.1, 25.1. HRMS

(AP-ESI) m/z calcd for C₂₂H₂₅ClN₇O₂[M + H]⁺454.1753, found

454.1755. Retention time: 8.2 min, eluted with 23% acetonitrile/77%

water (containing 0.4% formic acid).

7-(4-((7-(3-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (15j). Ethyl 7-(4-

((7-(3-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14q, 0.09 g, 0.18 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 15j

(0.08 g, 91% yield) as a white solid, mp 150−152 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 10.33 (s, 1H), 9.24 (s, 1H), 8.63 (d, J = 12.3 Hz,

2H), 7.90 (s, 1H), 7.46 (s, 1H), 7.35−7.27 (m, 4H), 7.19 (d, J = 7.0

Hz, 1H), 6.43 (d, J = 3.5 Hz, 1H), 5.38 (s, 2H), 4.00 (t, J = 7.0 Hz,

2H), 1.89 (t, J = 7.4 Hz, 2H), 1.69 (t, J = 7.1 Hz, 2H), 1.43 (t, J = 7.3

Hz, 2H), 1.20 (h, J = 7.3 Hz, 4H).¹³C NMR (126 MHz, DMSO-d₆) δ

169.4, 140.8, 133.6, 131.0, 127.9, 127.5, 126.3, 51.8, 47.0, 32.6, 30.3,

28.6, 26.2, 25.4. HRMS (AP-ESI) m/z calcd for C₂₃H₂₇ClN₇O₂[M +

H]⁺468.1909, found 468.1917. Retention time: 11.3 min, eluted with

23% acetonitrile/77% water (containing 0.4% formic acid).

129.8, 129.6, 128.7, 128.0, 126.8, 124.2, 100.4, 51.7, 45.2, 32.6, 30.2,

26.1, 25.1. HRMS (AP-ESI) m/z calcd for C₂₂H₂₅ClN₇O₂[M + H]⁺

454.1753, found 454.1749. Retention time: 8.7 min, eluted with 23%

acetonitrile/77% water (containing 0.4% formic acid).

7-(4-((7-(2-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (15f). Ethyl 7-(4-

((7-(2-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14m, 0.10 g, 0.20 mmol) was reacted using

a procedure similar to the synthesis of 6a, aﬀording compound 15f

(0.08 g, 82% yield) as a white solid, mp 168−170 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 10.35 (s, 1H), 9.25 (s, 1H), 8.71−8.64 (m, 2H),

7.83 (s, 1H), 7.54 (dd, J = 8.0, 1.2 Hz, 1H), 7.42 (s, 1H), 7.33 (td, J =

7.7, 1.7 Hz, 1H), 7.27−7.22 (m, 2H), 6.78 (d, J = 7.7 Hz, 1H), 6.49

(d, J = 3.6 Hz, 1H), 5.48 (s, 2H), 3.98 (t, J = 7.0 Hz, 2H), 1.92 (t, J =

7.4 Hz, 2H), 1.68 (p, J = 7.2 Hz, 2H), 1.45 (p, J = 7.4 Hz, 2H), 1.22

(dp, J = 22.3, 7.4 Hz, 4H). ¹³C NMR (124 MHz, DMSO-d₆) δ 169.5,

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

N-Hydroxy-6-(4-((7-(3-methoxybenzyl)-7H-pyrrolo[2,3-d]-

pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)hexanamide (15k). Ethyl 6-

(4-((7-(3-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-

1H-pyrazol-1-yl)hexanoate (14r, 0.10 g, 0.21 mmol) was reacted

using a procedure similar to the synthesis of 6a, aﬀording compound

procedure similar to the synthesis of 6a, aﬀording compound 16c

(0.10 g, 70% yield) as a white solid, mp 158−160 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.34 (s, 1H), 9.23 (s, 1H), 8.65 (d, J = 10.2 Hz,

2H), 7.88 (s, 1H), 7.49 (s, 1H), 7.44−7.35 (m, 2H), 7.28 (dd, J = 6.1,

2.5 Hz, 3H), 6.44 (d, J = 3.6 Hz, 1H), 5.39 (s, 2H), 4.02 (t, J = 7.0

Hz, 2H), 1.93 (t, J = 7.4 Hz, 2H), 1.72 (p, J = 7.2 Hz, 2H), 1.51 (p, J

= 7.5 Hz, 2H), 1.20 (qd, J = 9.3, 8.7, 6.1 Hz, 2H). ¹³C NMR (101

MHz, DMSO-d₆) δ 169.4, 156.3, 152.3, 151.2, 137.6, 132.4, 129.6,

129.4, 129.0, 126.7, 124.2, 119.1, 100.3, 51.6, 46.8, 32.6, 30.2, 26.1,

25.2. HRMS (AP-ESI) m/z calcd for C₂₂H₂₄ClN₇O₂[M + H]⁺

454.1753, found 454.1757. Retention time: 9.6 min, eluted with 23%

acetonitrile/77% water (containing 0.4% formic acid).

7-(4-((7-(4-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (16d). Ethyl 7-(4-

((7-(4-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14w, 0.15 g, 0.31 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 16d

(0.10 g, 70% yield) as a white solid, mp 174−176 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 10.33 (s, 1H), 9.27 (s, 1H), 8.63 (s, 2H), 7.87 (s,

1H), 7.47 (d, J = 3.0 Hz, 1H), 7.39−7.24 (m, 5H), 6.44 (d, J = 3.6

Hz, 1H), 5.37 (s, 2H), 4.01 (t, J = 7.0 Hz, 2H), 1.91 (t, J = 7.4 Hz,

2H), 1.69 (p, J = 7.1 Hz, 2H), 1.45 (p, J = 7.3 Hz, 2H), 1.31−1.18

(m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.5, 156.3, 152.3,

151.2, 137.6, 132.4, 129.6, 129.3, 129.0, 126.7, 124.2, 119.1, 111.5,

100.3, 51.7, 46.8, 32.6, 30.4, 28.6, 26.2, 25.4. ESI-MS m/z = 468 [M +

H]⁺. HRMS (AP-ESI) m/z calcd for C₂₃H₂₆ClN₇O₂[M + H]⁺

468.1909, found 468.1905. Retention time: 15.6 min, eluted with

23% acetonitrile/77% water (containing 0.4% formic acid).

6-(4-((7-(4-Methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (16e). Ethyl 6-(4-

((7-(4-methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)hexanoate (14x, 0.15 g, 0.33 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 16e

(0.10 g, 70% yield) as a white solid, mp 140−142 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.34 (s, 1H), 9.21 (s, 1H), 8.64 (d, J = 17.4 Hz,

2H), 7.93 (s, 1H), 7.50 (s, 1H), 7.26 (d, J = 3.5 Hz, 1H), 7.22−7.09

(m, 4H), 6.41 (d, J = 3.6 Hz, 1H), 5.33 (s, 2H), 4.03 (t, J = 7.0 Hz,

2H), 2.25 (s, 3H), 1.93 (t, J = 7.4 Hz, 2H), 1.80−1.67 (m, 2H), 1.51

(p, J = 7.5 Hz, 2H), 1.21 (tt, J = 9.8, 6.4 Hz, 2H). ¹³C NMR (101

MHz, DMSO-d₆) δ 169.4, 156.2, 152.2, 151.0, 137.0, 135.6, 129.6,

129.5, 127.6, 126.7, 124.2, 119.1, 111.5, 100.1, 51.7, 47.2, 32.6, 30.3,

26.1, 25.2, 21.1. HRMS (AP-ESI) m/z calcd for C₂₃H₂₇N₇O₂[M +

H]⁺434.2299, found 434,2294. Retention time: 7.6 min, eluted with

23% acetonitrile/77% water (containing 0.4% formic acid).

7-(4-((7-(4-Methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (16f). Ethyl 7-(4-

((7-(4-methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14y, 0.15 g, 0.32 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 16f

(0.11 g, 70% yield) as a white solid, mp 166−168 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.35 (s, 1H), 9.20 (s, 1H), 8.62 (s, 2H), 7.94 (s,

1H), 7.50 (s, 1H), 7.25 (d, J = 3.5 Hz, 1H), 7.21−7.09 (m, 4H), 6.41

(d, J = 3.5 Hz, 1H), 5.33 (s, 2H), 4.03 (t, J = 7.0 Hz, 2H), 2.25 (s,

3H), 1.92 (t, J = 7.4 Hz, 2H), 1.72 (t, J = 7.0 Hz, 2H), 1.46 (t, J = 7.2

Hz, 2H), 1.31−1.17 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ

169.5, 156.2, 152.2, 151.1, 137.0, 135.6, 129.6, 129.5, 127.6, 126.7,

124.2, 119.1, 111.5, 100.1, 51.7, 47.2, 32.6, 30.4, 28.6, 26.2, 25.5, 21.1.

ESI-MS m/z = 448 [M + H]⁺. HRMS (AP-ESI) m/z calcd for

C₂₄H₂₉N₇O₂[M + H]⁺448.2455, found 448.2457. Retention time:

10.4 min, eluted with 23% acetonitrile/77% water (containing 0.4%

formic acid).

1

15k (0.07 g, 72% yield) as a white solid, mp 148−150 °C. H NMR

(600 MHz, DMSO-d₆) δ 10.34 (s, 1H), 9.27 (s, 1H), 8.64 (s, 2H),

7.93 (s, 1H), 7.51 (s, 1H), 7.30 (d, J = 3.6 Hz, 1H), 7.24 (t, J = 7.9

Hz, 1H), 6.88 (s, 1H), 6.83 (dt, J = 8.3, 2.5 Hz, 2H), 6.44 (d, J = 3.6

Hz, 1H), 5.35 (s, 2H), 4.02 (t, J = 7.0 Hz, 2H), 3.68 (s, 3H), 1.92 (t, J

= 7.4 Hz, 2H), 1.78−1.68 (m, 2H), 1.55−1.46 (m, 2H), 1.29−1.14

(m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.4, 159.8, 140.1,

130.1, 129.7, 119.7, 119.2, 113.6, 112.9, 100.2, 55.4, 51.6, 47.4, 32.6,

30.2, 26.1, 25.1. HRMS (AP-ESI) m/z calcd for C₂₃H₂₈N₇O₃[M +

H]⁺450.2248, found 450.2259. Retention time: 4.4 min, eluted with

23% acetonitrile/77% water (containing 0.4% formic acid).

N-Hydroxy-7-(4-((7-(3-methoxybenzyl)-7H-pyrrolo[2,3-d]-

pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)heptanamide (15l). Ethyl 7-

(4-((7-(3-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-

1H-pyrazol-1-yl)heptanoate (14s, 0.09 g, 0.18 mmol) was reacted

using a procedure similar to the synthesis of 6a, aﬀording compound

1

15l (0.07 g, 80% yield) as a white solid, mp 145−147 °C. H NMR

(400 MHz, DMSO-d₆) δ 10.31 (s, 1H), 9.22 (s, 1H), 8.62 (d, J = 20.4

Hz, 2H), 7.91 (s, 1H), 7.48 (s, 1H), 7.31−7.15 (m, 2H), 6.93−6.70

(m, 3H), 6.52−6.31 (m, 1H), 5.32 (s, 2H), 3.99 (d, J = 7.2 Hz, 2H),

3.66 (s, 3H), 1.90 (t, J = 7.3 Hz, 2H), 1.69 (t, J = 7.7 Hz, 2H), 1.53−

1.38 (m, 2H), 1.20 (s, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.5,

159.8, 152.3, 140.1, 130.1, 129.7, 119.7, 119.2, 113.6, 112.9, 100.3,

55.4, 51.7, 47.4, 32.6, 30.4, 28.6, 26.2, 25.4. HRMS (AP-ESI) m/z

calcd for C₂₄H₃₀N₇O₃[M + H]⁺464.2405, found 464.2405.

Retention time: 5.8 min, eluted with 23% acetonitrile/77% water

(containing 0.4% formic acid).

6-(4-((7-(4-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (16a). Ethyl 6-(4-

((7-(4-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl) amino)-1H-

pyrazol-1-yl)hexanoate (14t, 0.12 g, 0.26 mmol) was reacted using

a procedure similar to the synthesis of 6a, aﬀording compound 16a

(0.08 g, 73% yield) as a white solid, mp 176−178 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.34 (s, 1H), 9.22 (s, 1H), 8.64 (d, J = 12.3 Hz,

2H), 7.92 (s, 1H), 7.50 (s, 1H), 7.40−7.29 (m, 2H), 7.27 (d, J = 3.6

Hz, 1H), 7.20−7.09 (m, 2H), 6.42 (d, J = 3.6 Hz, 1H), 5.37 (s, 2H),

4.02 (t, J = 7.0 Hz, 2H), 1.92 (t, J = 7.4 Hz, 2H), 1.72 (t, J = 7.5 Hz,

2H), 1.49 (q, J = 7.5 Hz, 2H), 1.30−1.14 (m, 2H). ¹³C NMR (101

MHz, DMSO-d₆) δ 169.4, 163.1, 160.6, 156.3, 152.2, 151.1, 134.8,

134.8, 129.7, 129.7, 129.6, 126.6, 124.2, 119.1, 115.9, 115.7, 111.5,

100.3, 51.6, 46.7, 32.6, 30.2, 26.1, 25.1. HRMS (AP-ESI) m/z calcd

for C₂₂H₂₄FN₇O₂[M + H]⁺438.2048, found 438.2043. Retention

time: 5.1 min, eluted with 23% acetonitrile/77% water (containing

0.4% formic acid).

7-(4-((7-(4-Fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (16b). Ethyl 7-(4-

((7-(4-ﬂuorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl) amino)-1H-

pyrazol-1-yl)heptanoate (14u, 0.15 g, 0.32 mmol) was reacted using

a procedure similar to the synthesis of 6a, aﬀording compound 16b

(0.12 g, 83% yield) as a white solid, mp 160−162 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.34 (s, 1H), 9.22 (s, 1H), 8.63 (s, 2H), 7.91 (s,

1H), 7.50 (s, 1H), 7.37−7.30 (m, 2H), 7.26 (d, J = 3.6 Hz, 1H), 7.15

(t, J = 8.9 Hz, 2H), 6.42 (d, J = 3.5 Hz, 1H), 5.37 (s, 2H), 4.02 (t, J =

7.0 Hz, 2H), 1.92 (t, J = 7.4 Hz, 2H), 1.71 (t, J = 7.1 Hz, 2H), 1.50−

1.41 (m, 2H), 1.28−1.19 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 169.5, 160.6, 156.3, 152.2, 151.1, 134.8, 134.8, 129.7, 129.6, 126.6,

124.2, 119.1, 115.9, 115.7, 100.3, 51.7, 46.7, 32.6, 30.4, 28.6, 26.2,

25.4. ESI-MS m/z = 452 [M + H]⁺. HRMS (AP-ESI) m/z calcd for

C₂₃H₂₆FN₇O₂[M + H]⁺452.2202, found 452.2201. Retention time:

7.5 min, eluted with 23% acetonitrile/77% water (containing 0.4%

formic acid).

6-(4-((7-(4-Methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (16g). Ethyl 6-(4-

((7-(4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)hexanoate (14z, 0.10 g, 0.21 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 16g

(0.08 g, 95% yield) as a white solid, mp 118−120 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.36 (s, 1H), 9.23 (s, 1H), 8.70 (s, 1H), 8.62 (s,

1H), 7.97 (s, 1H), 7.52 (s, 1H), 7.29−7.22 (m, 3H), 6.91−6.84 (m,

2H), 6.40 (d, J = 3.5 Hz, 1H), 5.30 (s, 2H), 4.04 (t, J = 6.9 Hz, 2H),

6-(4-((7-(4-Chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyhexanamide (16c). Ethyl 6-(4-

((7-(4-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)hexanoate (14v, 0.15 g, 0.32 mmol) was reacted using a

R

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

3.70 (s, 3H), 1.93 (t, J = 7.4 Hz, 2H), 1.74 (t, J = 7.5 Hz, 2H), 1.51 (t,

J = 7.5 Hz, 2H), 1.28−1.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-

d₆) δ 169.4, 159.0, 156.2, 152.2, 151.0, 130.5, 129.6, 129.1, 126.6,

124.3, 119.1, 114.4, 111.6, 100.1, 55.5, 51.7, 46.9, 32.6, 30.3, 26.1,

25.2. HRMS (AP-ESI) m/z calcd for C₂₃H₂₇N₇O₃[M + H]⁺

450.2248, found 450.2249. Retention time: 4.0 min, eluted with

23% acetonitrile/77% water (containing 0.4% formic acid).

7-(4-((7-(4-Methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (16h). Ethyl 7-(4-

((7-(4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14A, 0.10 g, 0.21 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 16h

(0.08 g, 95% yield) as a white solid, mp 138−140 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 10.34 (s, 1H), 9.38 (s, 1H), 8.64 (s, 2H), 7.97 (s,

1H), 7.53 (s, 1H), 7.29 (d, J = 3.5 Hz, 1H), 7.25 (d, J = 8.6 Hz, 2H),

6.89−6.85 (m, 2H), 6.43 (d, J = 3.6 Hz, 1H), 5.30 (s, 2H), 4.04 (t, J =

7.0 Hz, 2H), 3.69 (s, 3H), 1.91 (t, J = 7.4 Hz, 2H), 1.72 (t, J = 7.1 Hz,

2H), 1.44 (q, J = 7.2 Hz, 2H), 1.22 (dq, J = 13.5, 6.9 Hz, 4H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 169.5, 159.0, 156.2, 152.2, 151.0,

130.5, 129.6, 129.1, 126.5, 124.2, 119.1, 114.3, 114.2, 111.5, 100.1,

55.5, 51.7, 46.9, 32.6, 30.4, 28.6, 26.2, 25.5. HRMS (AP-ESI) m/z

calcd for C₂₄H₂₉N₇O₃[M + H]⁺464.2405, found 464.2408.

Retention time: 5.9 min, eluted with 23% acetonitrile/77% water

(containing 0.4% formic acid).

6-(4-((7-Phenethyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)-N-hydroxyhexanamide (16i). Ethyl 6-(4-((7-pheneth-

yl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-1-yl) hexa-

noate (14B, 0.10 g, 0.22 mmol) was reacted using a procedure

similar to the synthesis of 6a, aﬀording compound 16i (0.08 g, 95%

yield) as a white solid, mp 168−170 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.35 (s, 1H), 9.19 (s, 1H), 8.67 (s, 1H), 8.59 (s, 1H),

7.98 (s, 1H), 7.55 (s, 1H), 7.32−7.19 (m, 5H), 7.19−7.10 (m, 1H),

6.34 (d, J = 3.5 Hz, 1H), 4.38 (dd, J = 8.4, 6.6 Hz, 2H), 4.05 (t, J =

7.0 Hz, 2H), 3.12 (t, J = 7.5 Hz, 2H), 1.93 (t, J = 7.4 Hz, 2H), 1.80−

1.71 (m, 2H), 1.55−1.46 (m, 2H), 1.29−1.16 (m, 2H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 169.4, 156.1, 152.2, 150.9, 139.0, 129.7,

129.0, 128.8, 126.8, 126.5, 124.4, 119.0, 99.6, 51.7, 45.4, 35.9, 32.6,

30.2, 26.1, 25.1. HRMS (AP-ESI) m/z calcd for C₂₃H₂₇N₇O₂[M +

H]⁺434.2299, found 434.2302. Retention time: 5.9 min, eluted with

25% acetonitrile/75% water (containing 0.4% formic acid).

7-(4-((7-Phenethyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)-N-hydroxyheptanamide (16j). Ethyl 7-(4-((7-pheneth-

yl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-1-yl) hepta-

noate (14C, 0.15 g, 0.32 mmol) was reacted using a procedure

similar to the synthesis of 6a, aﬀording compound 16j (0.10 g, 70%

yield) as a white solid, mp 158−160 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.36 (s, 1H), 9.41 (s, 1H), 9.18 (s, 1H), 8.59 (s, 1H),

7.99 (s, 1H), 7.55 (s, 1H), 7.32−7.18 (m, 5H), 7.13 (d, J = 3.6 Hz,

1H), 6.34 (d, J = 3.5 Hz, 1H), 4.38 (dd, J = 8.4, 6.5 Hz, 2H), 4.05 (t, J

= 7.0 Hz, 2H), 3.12 (dd, J = 8.3, 6.6 Hz, 2H), 1.92 (t, J = 7.4 Hz, 2H),

1.74 (s, 2H), 1.46 (t, J = 7.3 Hz, 2H), 1.24 (p, J = 3.6 Hz, 4H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 169.5, 156.1, 152.2, 150.9, 139.0,

129.7, 129.0, 128.8, 126.8, 126.5, 124.4, 119.1, 111.5, 99.6, 51.8, 45.4,

35.9, 32.6, 30.4, 28.6, 26.2, 25.5. HRMS (AP-ESI) m/z calcd for

C₂₄H₂₉N₇O₂[M + H]⁺448.2455, found 448.2454. Retention time:

7.0 min, eluted with 23% acetonitrile/77% water (containing 0.4%

formic acid).

calcd for C₂₁H₃₀N₇O₂[M + H]⁺412.2455, found 412.2444.

Retention time: 4.8 min, eluted with 23% acetonitrile/77% water

(containing 0.4% formic acid).

7-(4-((7-(Cyclopropylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (17b). Ethyl 7-(4-

((7-(cyclopropylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-

1H-pyrazol-1-yl)heptanoate (14E, 0.08 g, 0.19 mmol) was reacted

using a procedure similar to the synthesis of 6a, aﬀording compound

1

17b (0.06 g, 77% yield) as a white solid, mp 135−137 °C. H NMR

(400 MHz, DMSO-d₆) δ 10.30 (s, 1H), 9.14 (s, 1H), 8.64 (d, J = 1.7

Hz, 1H), 8.59 (s, 1H), 7.95 (s, 1H), 7.53 (s, 1H), 7.23 (d, J = 3.6 Hz,

1H), 6.36 (d, J = 3.6 Hz, 1H), 4.08−3.95 (m, 4H), 1.91 (t, J = 7.4 Hz,

2H), 1.79−1.67 (m, 2H), 1.44 (q, J = 7.2 Hz, 2H), 1.23 (q, J = 8.1,

4.7 Hz, 5H), 0.48 (dtd, J = 7.8, 6.1, 5.5, 2.7 Hz, 2H), 0.42 (tt, J = 5.1,

2.6 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.5, 156.2, 152.2,

150.9, 129.7, 126.5, 124.4, 119.1, 99.6, 51.7, 48.2, 32.6, 30.3, 28.6,

26.2, 25.4. 11.9. HRMS (AP-ESI) m/z calcd for C₂₀H₂₈N₇O₂[M +

H]⁺398.2299, found 398.2308. Retention time: 2.7 min, eluted with

23% acetonitrile/77% water (containing 0.4% formic acid).

7-(4-((7-(Cyclohexylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-

amino)-1H-pyrazol-1-yl)-N-hydroxyheptanamide (17c). Ethyl 7-(4-

((7-(cyclohexylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)heptanoate (14F, 0.10 g, 0.22 mmol) was reacted using a

procedure similar to the synthesis of 6a, aﬀording compound 17c

(0.08 g, 82% yield) as a white solid, mp 142−144 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 10.32 (s, 1H), 9.17 (s, 1H), 8.63 (s, 1H), 8.58 (s,

1H), 7.99 (s, 1H), 7.50 (s, 1H), 7.14 (d, J = 3.6 Hz, 1H), 6.35 (d, J =

3.5 Hz, 1H), 4.03 (t, J = 7.0 Hz, 2H), 3.97 (d, J = 7.1 Hz, 2H), 1.90

(t, J = 7.4 Hz, 3H), 1.73 (s, 2H), 1.64 (s, 2H), 1.61−1.51 (m, 3H),

1.49−1.41 (m, 2H), 1.23 (s, 4H), 1.17−1.07 (m, 3H), 0.99 (d, J =

12.1 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.4, 156.1, 152.5,

150.9, 129.5, 127.1, 124.5, 118.9, 111.4, 99.4, 51.8, 50.1, 38.4, 32.6,

30.8, 30.4, 28.6, 26.4, 26.3, 25.6, 25.4. HRMS (AP-ESI) m/z calcd for

C₂₃H₃₄N₇O₂[M + H]⁺440.2768, found 440.2766. Retention time:

16.7 min, eluted with 23% acetonitrile/77% water (containing 0.4%

formic acid).

Molecular Docking Study. The crystal structures of JAK2 (PDB

3FUP) and HDAC6 (PDB 5EEI) were obtained from the Protein

Data Bank. Before the docking process, the structure of protein was

treated by adding hydrogen atoms, deleting water molecules, assigning

AMBER7 FF99 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−Huckel method. Other docking

̈

parameters were kept to the default values. Molecular docking was

carried out via the Sybyl/Surﬂex-Dock module.

Janus Kinase Inhibition Assay. (1) Janus kinase 1/2/3

inhibition assay. Target compounds were dissolved in DMSO and

centrifuged for 5 min to obtain 10⁻²M solution. The 10⁻²M target

compound solutions were kept at 4 °C. The ﬁnal concentration of

DMSO in the assay was no more than 1%. In 384-well plates, a 5 μL

enzyme system (50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM

MgCl₂, 0.01% brij-35, 4 μM substrate, 0.4−5 ng/μL enzymes) was

added with 2.5 μL of target compound solution, 2.5 μL of 200 μM

ATP. The mixture was incubated at room temperature and away from

light for 60 min. Subsequently, 5 μL of reagent A diluted with

development buﬀer was added to each well and incubated at room

temperature and away from light for 60 min. Then, 5 μL of stop

solution was added to each well to terminate the reaction and

incubated at room temperature and away from light for 10 min.

Fluorescence intensity was measured using a microplate reader at

excitation and emission wavelengths of 400 and 445/520 nm,

respectively. Origin data analysis software were used to calculate the

IC₅₀data by the nonlinear curve ﬁtting method (allowed to ﬂoat and

ﬁtted as a parameter). (2) TYK2 kinase inhibition assay. Target

compounds were dissolved in DMSO and centrifuged for 5 min to

obtain 10⁻²M solution. The 10⁻²M target compound solutions were

kept at 4 °C. The ﬁnal concentration of DMSO in the assay was no

more than 1%. In 384-well plates, 3 μL of enzyme system (5 mM

7-(4-((7-Cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-

pyrazol-1-yl)-N-hydroxyheptanamide (17a). Ethyl 7-(4-((7-cyclo-

pentyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-

heptanoate (14D, 0.12 g, 0.28 mmol) was reacted using a procedure

similar to the synthesis of 6a, aﬀording compound 17a (0.10 g, 86%

yield) as a white solid, mp 159−161 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.30 (s, 1H), 9.13 (s, 1H), 8.63 (s, 1H), 8.57 (s, 1H),

7.92 (s, 1H), 7.54 (s, 1H), 7.24 (d, J = 3.7 Hz, 1H), 6.37 (d, J = 3.6

Hz, 1H), 4.96 (q, J = 7.5 Hz, 1H), 4.03 (t, J = 7.0 Hz, 2H), 2.11 (d, J

= 7.9 Hz, 2H), 1.89 (q, J = 6.8, 6.4 Hz, 6H), 1.72 (q, J = 8.4, 7.3 Hz,

4H), 1.50−1.40 (m, 2H), 1.23 (s, 4H). ¹³C NMR (101 MHz, DMSO-

d₆) δ 169.4, 156.0, 150.9, 129.8, 124.4, 124.0, 119.1, 112.0, 99.9, 55.2,

51.7, 32.6, 32.1, 30.3, 28.6, 26.2, 25.4, 24.1. HRMS (AP-ESI) m/z

S

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

MgCl₂, 1 mM MnCl₂, 1 mM DTT, 12.5 nM SEB, 0.8 ng/μL enzyme)

was added with 4 μL of target compound solution, 3 μL of substrate

mixture (33.3 μM ATP and 3.3 μM substrate) and incubated at room

temperature for 40 min. Subsequently, 10 μL of premix of SA-XL665

and TK Ab was added to stop the reaction and incubated the plate at

room temperature for 1 h and then read in a microplate reader using

HTRF settings. GraphPad software were used to calculate the IC₅₀

data by the nonlinear curve ﬁtting method (allowed to ﬂoat and ﬁtted

as a parameter).

240 min. Then stop solution containing trypsin was added. The

coupled reaction was incubated for another 90 min at 37 °C.

Fluorescent AMC released from substrate was measured in Synergy2

using ﬁlter sets as excitation = 355 nm and emission = 460 nm. IC₅₀

values were calculated by GraphPad Prism software (California, US).

Western Blot Analysis. Cells in the logarithmic growth stage

were inoculated into 6-well culture plates at an appropriate density

overnight before exposed to compounds of diﬀerent concentrations.

After treatment, cells were collected and lysed using 1× SDS sample

buﬀer (50 mM Tris pH 6.8, 100 mM DTT, 2% SDS, 0.1%

bromophenol blue, 10% glycerol). The cell lysate was collected,

heated in a boiling water bath for 10 min, and centrifuged at 10 000

rpm for 10 min. The supernatant was taken for SDS-page

electrophoresis. After the electrophoresis, the proteins were trans-

ferred to the cellulose nitrate membrane. The membrane was

incubated in sealing solution containing 5% skim milk powder (5%

skim milk, 20 mM tris-HCl PH 7.2−7.4, 150 mM NaCl, 0.1% Tween-

20) for 60 min at room temperature. and then placed in a primary

antibody diluted in 5% skim milk at 4 °C overnight. After washing

with 100 mM tris-HCl (PH. 7.2−7.4, 0.9% NaCl, 0.2% Tween-20) at

room temperature for 3 times, 10 min each, horseradish peroxidase-

labeled secondary antibody was added and incubated at room

temperature for 1 h. After three-time washing, and the blots were

visualized with an enhanced chemiluminescence assay.

In Vitro Antiproliferative Assay. SRB assay was used to

determine the growth inhibition of cancer cells. The speciﬁc steps

were as follows: 100 μL cells in the logarithmic growth stage were

seeded to the 96-well culture plate according to the appropriate

density and incubated overnight. Diﬀerent concentrations of

compounds were added and coincubated for 72 h. After the

coincubation, pour the culture solution and add 10% (w/v)

trichloroacetic acid (100 μL/well) at 4 °C for 1 h. Then the adherent

cells were washed with distilled water ﬁve times. After drying by air, to

each well was added 100 μL of SRB solution (Sigma, st. Louis, MO,

U.S.A) (4 mg/mL, soluble in 1% acetic acid). After incubation for 15

min, it was washed with 1% acetic acid for ﬁve times. After drying by

air, 10 mM Tris solution (100 μL) was added to each well, and the

optical density (OD value) at the wavelength of 560 nm was

determined by SPECTRA max PLUS.

Protein Kinase Selectivity Proﬁle. 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 kinases

was quantiﬁed by measuring the phosphorylation of their substrates

using human recombinant enzymes and the LANCE detection

method. For example, evaluation of the eﬀects of compounds on the

activity of the human ALK quantiﬁed by measuring the phosphor-

ylation of the substrate Ulight-CKKSRGDYMTMQIG (IRS-1) using

a human recombinant enzyme and the LANCE detection method.

The test compound, reference compound or water (control) were

mixed with the enzyme (about 0.25 ng) in a buﬀer containing 40 mM

Hepes/Tris (pH 7.4), 0.8 mM EGTA/Tris, 8 mM MgCl₂, 1.6 mM

DTT, and 0.008% Tween 20. Thereafter, the reaction is initiated by

adding 250 nM of the substrate Ulight-CKKSRGDYMTMQIG (IRS-

1) and 30 μM ATP, and the mixture is incubated for 60 min at room

temperature. For control basal measurements, the enzyme is omitted

from the reaction mixture. Following incubation, the reaction is

stopped by adding 13 mM EDTA. After 5 min, the antiphopho-PT66

antibody labeled with europium chelate is added. After 60 more min,

the ﬂuorescence transfer is measured at excitation and emission

wavelengths of 337 and 620/665 nm, using a microplate reader

(Envision, PerkinElmer). The enzyme activity is determined by

dividing the signal measured at 665 nm by that measured at 620 nm

(ratio). The results are expressed as a percent inhibition of the control

enzyme activity. Other kinase inhibition assays were performed as

above method.

In Vitro HDAC1/6 Inhibition Fluorescence Assay. In 384-well

plates, 5 μL of enzyme system (15 mM Tris-HCl pH 8.1, 250 μM

EDTA, 250 mM NaCl, 10% glycerol, 0.7−12.5 ng/μL enzymes) was

added with 2.5 μL of target compound solution and 2.5 μL of

substrate. The mixture was incubated at room temperature protected

from light for 60 min. Subsequently, 10 μL of trypsin diluted with

trypsin buﬀer was added to each well and incubated at room

temperature and away from light for 10 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 ﬂuorescence intensity readout of tested wells

relative to those of control wells, and the IC₅₀values were calculated

using Prism nonlinear curve ﬁtting method (allowed to ﬂoat and ﬁtted

as a parameter).

Apoptosis Assay. For ﬂow cytometry assay, annexin V-FITC/

propidium iodide (PI) staining assay (A211-02, Vazyme) was

employed to detect cells in early apoptotic and late apoptotic stages.

Cells were seeded in 6-well plates and incubated overnight, then

treated with DMSO or compounds for 48 h, and harvested and

washed in cold PBS. Cells were resuspended in 100 μL of annexin V-

FITC/PI staining solution and incubated for 10−15 min at room

temperature. After incubation, the cells were collected and analyzed

by FACSCalibur (BD, USA), and data were analyzed using FlowJo 7.6

Software.

In Vivo Antitumor Activity Assay. The female BALB/c nude

mice (IACUC no. 2019-04-GMY-12) were purchased from Beijing

Vital River Laboratory Animal Technology Co. Ltd. MDA-MB-231

cells were suspended in serum-free medium and injected with 0.2 mL

containing 2 × 10⁶cells into the right ﬂank of BALB/c-nude mice.

When the tumor reached a mean volume of 100−150 mm³, the

tumor-bearing nude mice were randomly divided into control and

treatment groups (6 mice/group) and received the vehicle, SAHA

(100 mg/kg/d, PO), ruxolintinib (100 mg/kg/d, PO), and 15d (30

and 100 mg/kg/d, IP) at indicated doses. The size of tumors was

measured individually twice per week. Tumor volume (TV) was

calculated as V = (length × width²)/2. All experiments were

performed according to the institutional ethical guidelines on animal

care and approved by the Institute Animal Care and Use Committee

at Shanghai Institute of Materia Medica.

In Vitro HDAC3 Enzymic Assay. A 10 μL reactive system (50

mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂,

0.01% Tween20) containing HDAC3 enzyme, H3(1−21)K9Ac

substrate, and the tested compounds was added in 384-well plates

and incubated at room temperature for 1 h. Subsequently, 10 μL of

premix of SA-XL665 and anti-H3K9me0-Eu(K) was added to stop

the reaction and incubated the plate at room temperature for 1 h and

then read in a microplate reader using HTRF settings. GraphPad

software were used to calculate the IC₅₀data by the nonlinear curve

ﬁtting method (allowed to ﬂoat and ﬁtted as a parameter).

In Vitro HDAC4, HDAC7, HDAC8, and HDAC10 Enzymic

Assay. The assays were carried out by Sundia MediTech Company,

Ltd. Brieﬂy, diﬀerent concentrations of compounds were incubated

with recombinant HDAC4, HDAC7, HDAC8 (Active Motif, USA),

and HDAC10 (BPS Biosciences, USA) at room temperature for 15

min, which was followed by adding mixture of Ac-peptide-AMC

substrates to initiate the coupled reaction in Tris-based assay buﬀer.

For HDAC4 and HDAC7, ﬂuorescent AMC released from substrate

was measured in Synergy2 (BioTek, US) using ﬁlter sets as excitation

= 355 nm and emission = 460 nm kinetically. For HDAC8 and

HDAC10, reaction mixtures were incubated at room temperature for

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02111.

T

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

Supporting tables; H NMR, and ¹³C NMR of all target

Yingjie Zhang − Department of Medicinal Chemistry, School

of Pharmacy, Shandong University, Ji’nan, Shandong

250012, P. R. China; orcid.org/0000-0001-6118-6695

Meiyu Geng − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

compounds; HPLC spectra and HRMS of the

representative compounds (PDF)

Molecular formula stings (CSV) (CSV)

Predicted binding mode of 6a in the ATP pocket of

JAK2; surface representation of 6a in the ATP pocket of

JAK2 (3FUP) (PDB)

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02111

Predicted binding mode of 15d in the ATP pocket of

JAK2 (3FUP) (PDB)

Predicted binding mode of 16b in the ATP pocket of

JAK2 (3FUP) (PDB)

Predicted binding mode of 6a in the active site of

HDAC6; surface representation of 6a in the active site of

HDAC6 (5EEI) (PDB)

Author Contributions

X.W. Liang and S. Tang, contributed equally to this work. All

authors have given approval to the ﬁnal version of the

manuscript.

Notes

Predicted binding mode of 15d in the active site of

HDAC6 (5EEI) (PDB)

The authors declare no competing ﬁnancial interest.

Predicted binding mode of 16b in the active site of

HDAC6 (5EEI) (PDB)

ACKNOWLEDGMENTS

■

This study was supported by National Natural Science

Foundation of China (21907102, 21632008, 91229204,

81473245, and 81772695).

AUTHOR INFORMATION

Corresponding Authors

■

Hong Liu − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China; orcid.org/0000-0003-3685-

6268; Phone: +86 21 50807042; Email: hliu@

mail.shcnc.ac.cn

Min Huang − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; Email: mhuang@

simm.ac.cn

ABBREVIATIONS USED

■

AML, acute myelocytic leukemia; CTCL, cutaneous T cell

lymphoma; EL, erythroleukemia cell lines; HDACs, histone

deacetylases; JAK, Janus kinase; LIFR, leukemia inhibitory

factor receptor; MM, multiple myeloma; INCB, ruxolitinib;

SAR, structure−activity relationship; SRC, surface recognition

cap; STAT, signal transducers and activators of transcription;

ZBG, zinc-binding group

Authors

Xuewu Liang − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

Shuai Tang − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

Xuyi Liu − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China

Yingluo Liu − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

Qifu Xu − Department of Medicinal Chemistry, School of

Pharmacy, Shandong University, Ji’nan, Shandong 250012,

P. R. China

Xiaomin Wang − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

Abdusaid Saidahmatov − State Key Laboratory of Drug

Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, Shanghai 201203, China

Chunpu Li − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

Jiang Wang − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

REFERENCES

■

(1) Tan, Z.; Zhang, S. Past, present, and future of targeting Ras for

cancer therapies. Mini-Rev. Med. Chem. 2016, 16, 345−357.

(2) Tan, Z.; Chaudhai, R.; Zhang, S. Polypharmacology in drug

development: A minireview of current technologies. ChemMedChem

2016, 11, 1211−1218.

(3) Zimmermann, G. R.; Lehar, J.; Keith, C. T. Multi-target

therapeutics: when the whole is greater than the sum of the parts.

Drug Discovery Today 2007, 12, 34−42.

(4) Morphy, R.; Rankovic, Z. Designed multiple ligands. An

emerging drug discovery paradigm. J. Med. Chem. 2005, 48, 6523−

6543.

(5) Csermely, P.; Agoston, V.; Pongor, S. The efficiency of multi-

target drugs: the network approach might help drug design. Trends

Pharmacol. Sci. 2005, 26, 178−182.

(6) Hartman, J. L. t.; Garvik, B.; Hartwell, L. Principles for the

buffering of genetic variation. Science 2001, 291, 1001−1004.

(7) Hesham, H. M.; Lasheen, D. S.; Abouzid, K. A. M. Chimeric

HDAC inhibitors: Comprehensive review on the HDAC-based

strategies developed to combat cancer. Med. Res. Rev. 2018, 38,

2058−2109.

(8) Xu, W. S.; Parmigiani, R. B.; Marks, P. A. Histone deacetylase

inhibitors: molecular mechanisms of action. Oncogene 2007, 26,

5541−5552.

(9) He, S.; Dong, G.; Li, Y.; Wu, S.; Wang, W.; Sheng, C. Potent

dual BET/HDAC inhibitors for efficient treatment of pancreatic

cancer. Angew. Chem., Int. Ed. 2020, 59, 3028−3032.

(10) Liang, X.; Liu, H.; Zhang, Y. Novel-targeted therapy for

hematological malignancies with JAK and HDAC dual inhibitors.

Future Med. Chem. 2019, 11, 1849−1852.

Yu Zhou − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China; orcid.org/0000-0002-7684-

2939

(11) Slingerland, M.; Guchelaar, H. J.; Gelderblom, H. Histone

deacetylase inhibitors: an overview of the clinical studies in solid

tumors. Anti-Cancer Drugs 2014, 25, 140−149.

U

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(12) Stadler, W. M.; Margolin, K.; Ferber, S.; McCulloch, W.;

Thompson, J. A. A phase II study of depsipeptide in refractory

metastatic renal cell cancer. Clin. Genitourin. Cancer 2006, 5, 57−60.

(13) Molife, L. R.; Attard, G.; Fong, P. C.; Karavasilis, V.; Reid, A.

H.; Patterson, S.; Riggs, C. E., Jr.; Higano, C.; Stadler, W. M.;

McCulloch, W.; Dearnaley, D.; Parker, C.; de Bono, J. S. Phase II,

two-stage, single-arm trial of the histone deacetylase inhibitor

(HDACi) romidepsin in metastatic castration-resistant prostate

cancer (CRPC). Ann. Oncol 2010, 21, 109−113.

(14) Haigentz, M., Jr.; Kim, M.; Sarta, C.; Lin, J.; Keresztes, R. S.;

Culliney, B.; Gaba, A. G.; Smith, R. V.; Shapiro, G. I.; Chirieac, L. R.;

Mariadason, J. M.; Belbin, T. J.; Greally, J. M.; Wright, J. J.; Haddad,

R. I. Phase II trial of the histone deacetylase inhibitor romidepsin in

patients with recurrent/metastatic head and neck cancer. Oral Oncol.

2012, 48, 1281−1288.

(15) Liu, T.; Wan, Y.; Xiao, Y.; Xia, C.; Duan, G. Dual-target

inhibitors based on HDACs: novel antitumor agents for cancer

therapy. J. Med. Chem. 2020, 63, 8977−9002.

dual inhibitors of Janus kinase (JAK) and histone deacetylase

(HDAC) based on ruxolitinib and vorinostat. J. Med. Chem. 2017,

60, 8336−8357.

(29) Chu-Farseeva, Y. Y.; Mustafa, N.; Poulsen, A.; Tan, E. C.; Yen,

J. J. Y.; Chng, W. J.; Dymock, B. W. Design and synthesis of potent

dual inhibitors of JAK2 and HDAC based on fusing the

pharmacophores of XL019 and vorinostat. Eur. J. Med. Chem. 2018,

158, 593−619.

(30) Huang, Y.; Dong, G.; Li, H.; Liu, N.; Zhang, W.; Sheng, C.

Discovery of janus kinase 2 (JAK2) and histone deacetylase (HDAC)

dual inhibitors as a novel strategy for the combinational treatment of

leukemia and invasive fungal infections. J. Med. Chem. 2018, 61,

6056−6074.

(31) Liang, X.; Zang, J.; Li, X.; Tang, S.; Huang, M.; Geng, M.;

Chou, C. J.; Li, C.; Cao, Y.; Xu, W.; Liu, H.; Zhang, Y. Discovery of

novel janus kinase (JAK) and histone deacetylase (HDAC) dual

inhibitors for the treatment of hematological malignancies. J. Med.

Chem. 2019, 62, 3898−3923.

(32) Kulagowski, J. J.; Blair, W.; Bull, R. J.; Chang, C.; Deshmukh,

G.; Dyke, H. J.; Eigenbrot, C.; Ghilardi, N.; Gibbons, P.; Harrison, T.

K.; Hewitt, P. R.; Liimatta, M.; Hurley, C. A.; Johnson, A.; Johnson,

T.; Kenny, J. R.; Bir Kohli, P.; Maxey, R. J.; Mendonca, R.; Mortara,

K.; Murray, J.; Narukulla, R.; Shia, S.; Steffek, M.; Ubhayakar, S.;

Ultsch, M.; van Abbema, A.; Ward, S. I.; Waszkowycz, B.; Zak, M.

Identification of imidazo-pyrrolopyridines as novel and potent JAK1

inhibitors. J. Med. Chem. 2012, 55, 5901−5921.

(33) Su, Q.; Ioannidis, S.; Chuaqui, C.; Almeida, L.; Alimzhanov,

M.; Bebernitz, G.; Bell, K.; Block, M.; Howard, T.; Huang, S.; Huszar,

D.; Read, J. A.; Rivard Costa, C.; Shi, J.; Su, M.; Ye, M.; Zinda, M.

Discovery of 1-methyl-1H-imidazole derivatives as potent Jak2

inhibitors. J. Med. Chem. 2014, 57, 144−158.

(16) Mahboobi, S.; Sellmer, A.; Winkler, M.; Eichhorn, E.; Pongratz,

H.; Ciossek, T.; Baer, T.; Maier, T.; Beckers, T. Novel chimeric

histone deacetylase inhibitors: a series of lapatinib hybrides as potent

inhibitors of epidermal growth factor receptor (EGFR), human

epidermal growth factor receptor 2 (HER2), and histone deacetylase

activity. J. Med. Chem. 2010, 53, 8546−8555.

(17) Peng, F. W.; Xuan, J.; Wu, T. T.; Xue, J. Y.; Ren, Z. W.; Liu, D.

K.; Wang, X. Q.; Chen, X. H.; Zhang, J. W.; Xu, Y. G.; Shi, L. Design,

synthesis and biological evaluation of N-phenylquinazolin-4-amine

hybrids as dual inhibitors of VEGFR-2 and HDAC. Eur. J. Med. Chem.

2016, 109, 1−12.

(18) Zang, J.; Liang, X.; Huang, Y.; Jia, Y.; Li, X.; Xu, W.; Chou, C.

J.; Zhang, Y. Discovery of novel Pazopanib-based HDAC and VEGFR

dual inhibitors targeting cancer epigenetics and angiogenesis

simultaneously. J. Med. Chem. 2018, 61, 5304−5322.

(19) Ko, K. S.; Steffey, M. E.; Brandvold, K. R.; Soellner, M. B.

Development of a chimeric c-Src kinase and HDAC inhibitor. ACS

Med. Chem. Lett. 2013, 4, 779−783.

(20) Makarevic, J.; Rutz, J.; Juengel, E.; Maxeiner, S.; Mani, J.; Vallo,

S.; Tsaur, I.; Roos, F.; Chun, F. K.; Blaheta, R. A. HDAC inhibition

counteracts metastatic re-activation of prostate cancer cells induced

by chronic mTOR suppression. Cells 2018, 7, 129−146.

(21) Terranova-Barberio, M.; Thomas, S.; Ali, N.; Pawlowska, N.;

Park, J.; Krings, G.; Rosenblum, M. D.; Budillon, A.; Munster, P. N.

HDAC inhibition potentiates immunotherapy in triple negative breast

cancer. Oncotarget 2017, 8, 114156−114172.

(22) Zeng, H.; Qu, J.; Jin, N.; Xu, J.; Lin, C.; Chen, Y.; Yang, X.; He,

X.; Tang, S.; Lan, X.; Yang, X.; Chen, Z.; Huang, M.; Ding, J.; Geng,

M. Feedback activation of leukemia inhibitory factor receptor limits

response to histone deacetylase inhibitors in breast cancer. Cancer Cell

2016, 30, 459−473.

(23) Clark, J. D.; Flanagan, M. E.; Telliez, J. B. Discovery and

development of janus kinase (JAK) inhibitors for inflammatory

diseases. J. Med. Chem. 2014, 57, 5023−5038.

(24) Pellegrini, S.; Dusanter-Fourt, I. The structure, regulation and

function of the janus kinases (JAKs) and the signal transducers and

activators of transcription (STATs). Eur. J. Biochem. 1997, 248, 615−

633.

(25) Nosaka, T.; Kitamura, T. Janus kinases (JAKs) and signal

transducers and activators of transcription (STATs) in hematopoietic

cells. Int. J. Hematol. 2000, 71, 309−319.

(26) Yamaoka, K.; Saharinen, P.; Pesu, M.; Holt, V. E.; Silvennoinen,

O.; O’Shea, J. J. The janus kinases (Jaks). Genome Biol. 2004, 5, 253.

(27) Yang, E. G.; Mustafa, N.; Tan, E. C.; Poulsen, A.; Ramanujulu,

P. M.; Chng, W. J.; Yen, J. J.; Dymock, B. W. Design and synthesis of

Janus kinase 2 (JAK2) and histone deacetlyase (HDAC) bispecific

inhibitors based on Pacritinib and evidence of dual pathway inhibition

in hematological cell lines. J. Med. Chem. 2016, 59, 8233−8262.

(28) Yao, L.; Mustafa, N.; Tan, E. C.; Poulsen, A.; Singh, P.; Duong-

Thi, M. D.; Lee, J. X. T.; Ramanujulu, P. M.; Chng, W. J.; Yen, J. J. Y.;

Ohlson, S.; Dymock, B. W. Design and synthesis of ligand efficient

V

https://dx.doi.org/10.1021/acs.jmedchem.0c02111

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of Novel Pyrrolo[2,3-d]pyrimidine-based Derivatives as Potent JAK/HDAC Dual Inhibitors for the Treatment of Refractory Solid Tumors

DOI: 10.1021/acs.jmedchem.0c02111

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c02111

Full text of DOI:10.1021/acs.jmedchem.0c02111

Products guided by the article

R&D Labs maybe for 909563-33-3

Relevant to this article

Hot Product