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Design, Synthesis, and Structure–Activity Relationship Studies of 3-(Phenylethynyl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine Derivatives as a New Class of Src Inhibitors with Potent Activities in Models of Triple Negative Breast Cancer

April 2015
Journal of Medicinal Chemistry 58(9)

April 2015
58(9)

DOI:10.1021/acs.jmedchem.5b00270

Source
PubMed

Authors:

ming wu Zheng

Sichuan University

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A series of 3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine derivatives were designed and synthesized. Structure-activity relationship (SAR) analysis of these compounds led to the discovery of compound 1j, which showed the highest inhibitory potency against the Src kinase and the most potent anti-viability activity against the typical TNBC cell line MDA-MB-231 among all the synthesized compounds. Further kinase inhibition assays showed that compound 1j was a multikinase inhibitor and potently inhibited Src (IC50: 0.0009 μM) and MAPK signaling protein kinases B-RAF and C-RAF. In an MDA-MB-231 xenograft mouse model, a once-daily dose of compound 1j at 30mg/kg for 18 days completely suppressed the tumor growth with a tumor inhibition rate large than 100% without obvious toxicity. It also displayed good pharmacokinetic properties in a preliminary pharmacokinetic assay. Western blot and immunohistochemical assays revealed that compound 1j significantly inhibited Src and MAPK signaling, and markedly induced apoptosis in tumor tissues.

Chemical structures of representative Src inhibitors.

…

(A) Structures of ponatinib and compound 1a. (B) Schematic showing regions that are the focus of structural modifications.

…

Kinase Inhibition Profile of Compound 1j against Human Src and a Panel of Other Selected Protein Kinases a

…

(A) 1j inhibited colony formation of MDA-MB-231 cells. MDA-MB-231 cells were seeded in six-well plates and treated with 1j or dasatinib for 12 days. Colonies were stained with crystal violet and pictures taken. (B, C) 1j induced apoptosis and G 0 /G 1 cell cycle arrest of MDA-MB-231 cells. MDA-MB-231 cells were harvested after treatment with various concentrations of 1j and dasatinib for 24 and 48 h. Cells were stained with an annexin V-FITC apoptosis detection kit or cell cycle and apoptosis analysis kit, respectively.

…

(A) 1j inhibited MDA-MB-231 cell migration in wound healing assay. Cells were wounded by the pipet and then treated with various concentrations of compounds for 20 h. Scale bar, 100 μm. (B) 1j inhibited MDA-MB-231 cells invasion in transwell invasion assay. The bottom chambers of the transwells were filled with 600 μL of DMEM with 10% FBS, while the top chambers were seeded with 1 × 10 5 MDA-MB-231 cells in 200 μL of DMEM and treated with different concentrations of compounds for 24 h. Scale bar, 50 μm.

…

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Design, Synthesis, and Structure−Activity Relationship Studies of

3‑(Phenylethynyl)‑1H‑pyrazolo[3,4‑d]pyrimidin-4-amine Derivatives

as a New Class of Src Inhibitors with Potent Activities in Models

of Triple Negative Breast Cancer

Chun-Hui Zhang,

†,§

Ming-Wu Zheng,

†,§

Ya-Ping Li,

†,§

Xing-Dong Lin,

†

Mei Huang,

†

Lei Zhong,

†

Guo-Bo Li,

†

Rong-Jie Zhang,

†

Wan-Ting Lin,

†

Yan Jiao,

†

Xiao-Ai Wu,

†

Jiao Yang,

†

Rong Xiang,

‡

Li-Juan Chen,

†

Ying-Lan Zhao,

†

Wei Cheng,

†

Yu-Quan Wei,

†

and Sheng-Yong Yang*

,†

†

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and Collaborative Innovation Center for Biotherapy,

Sichuan University, Sichuan 610041, China

‡

Department of Clinical Medicine, School of Medicine, Nankai University, Tianjin 300071, China

SSupporting Information

ABSTRACT: A series of 3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-

amine derivatives were designed and synthesized. Structure−activity relationship

(SAR) analysis of these compounds led to the discovery of compound 1j, which

showed the highest inhibitory potency against the Src kinase and the most potent

antiviability activity against the typical TNBC cell line MDA-MB-231 among all

the synthesized compounds. Further kinase inhibition assays showed that com-

pound 1j was a multikinase inhibitor and potently inhibited Src (IC50 =0.0009μM)

and MAPK signaling protein kinases B-RAF and C-RAF. In an MDA-MB-231

xenograft mouse model, a once-daily dose of compound 1j at 30 mg/kg for

18 days completely suppressed the tumor growth with a tumor inhibition rate

larger than 100% without obvious toxicity. It also displayed good pharmacokinetic properties in a preliminary pharmacokinetic

assay. Western blot and immunohistochemical assays revealed that compound 1j signiﬁcantly inhibited Src and MAPK signaling

and markedly induced apoptosis in tumor tissues.

1. INTRODUCTION

Breast cancer is one of the most common cancer types in women

worldwide. Within breast cancer, the “triple-negative”subtype

(triple-negative breast cancer, TNBC), which lacks the expres-

sion of estrogen and progesterone receptor (ER/PR) and HER2,

is the most aggressive and also the most deadly.

1,2

TNBC has

been of great interest to oncologists because these cancers do not

beneﬁt from hormonal therapies or treatments targeted against

HER2.

3,4

Currently, chemotherapy is the only systemic therapy

and prognosis remains poor.

5,6

Compared with chemotherapy, targeted therapies have the

advantage of maximizing eﬃcacy while often reducing toxicity.

However, unlike other molecular subtypes of breast cancer,

there is no validated speciﬁc biomarker for TNBC.

7−9

Preclinical

studies have identiﬁed several potential targets, and Src is one

of the targets that have recently received increasing interest.

10,11

Src is a nonreceptor tyrosine kinase and has been revealed to

be a critical regulator of a large number of intracellular signaling

pathways.

12−14

Abnormal activation or ampliﬁcation of Src

has been detected in TNBC and demonstrated to play a role in

proliferation, migration, and invasion of breast cancer cell

lines.

10,15,16

Furthermore, a number of recent studies have shown

that dysregulation of Src is strongly associated with tumor

metastasis and a poor prognosis of TNBC.

17,18

Src therefore

represents a rational molecular target for TNBC.

10,19−22

Several

agents (see Figure 1) are currently available for inhibiting

Src,

23−28

and dasatinib is the only one that is in clinical trials for

treating TNBC. Dasatinib is an orally active tyrosine kinase

inhibitor that targets Src and BCR-ABL as well as several other

kinases and has been approved for the treatment of imatinib-

resistant BCR-ABL-positive leukemia.

Though dasatinib has a

good potency against Src kinase (IC50 = 0.0003 μM), it just

showed moderate anti-TNBC activity. For example, the IC50

value of dasatinib against the typical TNBC cell line MDA-MB-231

is just 0.178 μM. The updated reports from clinical trial studies of

dasatinib in the treatment of TNBC indicated that single-agent

dasatinib just showed limited eﬃcacy in TNBC.

Reasons

causing the limited eﬃcacy of the Src inhibitor in treating TNBC

could be complicated but should be mainly due to the refractory

of TNBC and certain unknown imperfect properties of this

compound. Therefore, discovery of new Src inhibitors with

potent anti-TNBC activity is now strongly demanded for drug

research and development (R&D) against TNBC.

In an eﬀort to discover Src inhibitors with high potency against

TNBC tumor, we recently performed a rational drug design in

Received: February 15, 2015

Article

pubs.acs.org/jmc

J. Med. Chem. XXXX, XXX, XXX−XXX

which scaﬀold hopping was applied on ponatinib (see

Figure 2A). Ponatinib was selected, since we are interested in

the 1,2-diarylethyne structure motif; in addition to that it can

potently inhibit Src (IC50 = 0.003 μM).

31,32

Before the scaﬀold

hopping, we collected a total of 209 potential “hinge-binder”

fragments derived from known kinase inhibitors; a hinge-binder

fragment indicates a moiety in a kinase inhibitor that can form

one to three hydrogen bonds with the hinge region of kinase

domain.

The collected hinge-binder fragments were used to

replace the imidazo[1,2-b]pyridazine moiety in ponatinib; the

imidazo[1,2-b]pyridazine moiety corresponds to the hinge-

binder of ponatinib. Molecular docking was then used to evaluate

the binding aﬃnities of constructed molecules with Src, and

GoldScore

was adopted to rank these compounds (for details,

see Supporting Information). Chemical structures of the top 100

compounds are given in Supporting Information (see Table S1).

Interestingly, the second ranked compound happened to be a

known potent Src inhibitor.

Encouraged by this result, we

decided to choose one from the top of these compounds to

synthesize; a compound was selected if it is a new compound and

synthetically accessible and can potentially form at least two

hydrogen bonds with the hinge region of kinase domain (for

details, see Supporting Information). The ﬁnally obtained com-

pound is 3-((4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-

3-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-

3-(triﬂuoromethyl)phenyl)benzamide (1a; Figure 2A), which

showed a slightly improved kinase inhibitory potency (IC50 =

0.002 μM) against Src compared with ponatinib. More importantly,

it exhibited an IC50 value of 0.069 μM against the TNBC MDA-

MB-231 cells in antiviability assays, which is approximately

2.5 times more potent than dasatinib and ponatinib (see Table 1).

This result indicated that improving the anti-TNBC potency of

Src inhibitors was feasible and encouraged us to carry out a

further structural optimization to compound 1a.

In this investigation, we shall perform structural modiﬁcations

to compound 1a. A series of 3-(phenylethynyl)-1H-pyrazolo-

[3,4-d]pyrimidin-4-amine derivatives are synthesized, and

the structure−activity relationship (SAR) of this series of com-

pounds is discussed. To the most active compound in enzymatic

and cellular assays, an in depth anti-TNBC activities both in vitro

and in vivo are evaluated. The mechanism of action of this

compound is also investigated.

Figure 1. Chemical structures of representative Src inhibitors.

Figure 2. (A) Structures of ponatinib and compound 1a. (B) Schematic showing regions that are the focus of structural modiﬁcations.

Journal of Medicinal Chemistry Article

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2. CHEMISTRY

All the target compounds in this investigation were readily

prepared using palladium-catalyzed Sonogashira coupling as a

key step (Schemes 1−3). Synthetic routes of compound 1a−h

are schematically illustrated in Scheme 1. First, intermediate

3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2), which was

prepared through NIS iodination of commercially available

1H-pyrazolo[3,4-d]pyrimidin-4-amine, reacted with commer-

cially available haloalkanes or self-prepared methanesulfonates

to produce the key intermediates 3a−i. Second, commercially

Table 1. Kinase Inhibitory Potency against Src and Antiviability Activities against MDA-MB-231 and HepG2 Cells of

Compounds 1a−h

IC50 values were determined from KinaseProﬁler of Euroﬁns. The data represent the mean values of two independent experiments.

Each

compound was tested in triplicate; the data are presented as the mean ±SD.

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Scheme 1. Synthetic Routes for Compounds 1a−h

Reagents and conditions: (a) NIS, DMF, 80 °C, N2; (b) R3-X(Br, I) or R3-OMs, K2CO3, DMF, 80 °C, N2; (c) MeOH, conc H2SO4 cat,reﬂux;

(d) ethynyltrimethylsilane, CuI, Pd(PPh3)4, triethylamine, THF, rt, N2; (e) (i) K2CO3, MeOH, rt, (ii) NaOH, H2O, rt; (f) diisobutylaluminum

hydride, anhydrous THF, N2, rt; (g) pyridinium p-toluenesulfonate, amine, sodium triacetoxyborohydride, EA, N2, 110−50 °C; (h) (i) SOCl2,

80 °C, (ii) DIPEA, EA, 0 °C to rt ((iii) this step only for 10h, LiOH, THF/H2O, rt); (j) (PPh3)2PdCl2, CuI, DIPEA, DMF, 80 °C, N2.

Scheme 2. Synthetic Routes for Compounds 1i−r

Reagents and conditions: (a) (PPh3)2PdCl2, CuI, DIPEA, DMF, 80 °C, N2; (b) TFA, DCM, 0 °C to rt; (c) acrylyl chloride, triethylamine,

DCM, −10 °C to rt.

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J. Med. Chem. XXXX, XXX, XXX−XXX

available 3-iodo-4-methylbenzoic acid was esteriﬁcated and

subsequently coupled with ethynyltrimethylsilane under

Sonogashira coupling conditions, followed by deprotection to

yield 3-ethynyl-4-methylbenzoic acid 6. Third, reduction of

benzonitrile 7a−cwith DIBAL cleanly aﬀorded aldehyde 8a−c

in excellent yield. 8a−cwere then coupled with amine through

a classical Borch reductive amination to produce 9a,c,d,f−h.

Fourthly, 9a,c,d,f−htogether with 4-((4-methylpiperazin-1-

yl)methyl)aniline (9b) and 3-(triﬂuoromethyl)aniline (9e),

which were purchased from market, reacted with 6under basic

conditions to give the key intermediates 10a−h. Finally, target

compounds 1a−hwere obtained through a palladium-catalyzed

Sonogashira coupling reaction between 10a−hand 3a. Similarly,

target compounds 1i−r, which contain various substituents at the

N-1 position of 1H-pyrazolo[3,4-d]pyrimidine (R3) (Scheme 2),

were prepared again through a Sonogashira coupling reaction

between 3b−iand 10a. Among them, 1q was produced by

deprotection of 1p, and amidation of 1q led to 1r.

The synthetic routes for compounds 14a−gare outlined in

Scheme 3. Terminal-alkyne-containing intermediates 11 was ﬁrst

prepared through a Sonogashira coupling reaction of 3b with

ethynyltrimethylsilane and a succeeding deprotection. Then com-

mercially available or self-prepared benzoic acids 12a−fwere

coupled with 4-((4-methylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)-

aniline (9a) to produce iodobenzamides 13a−f, which under-

went Sonogashira coupling to yield ﬁnal compounds 14a−f, and

hydrolysis of 14f with BBr3led to compound 14g. Self-prepared

iodobenzoic acids 12c,dwere synthesized by esteriﬁcation of

corresponding benzoic acids 15a,b, followed by iodination and

hydrolysis.

3. RESULTS AND DISCUSSIONS

3.1. SAR Analyses of 3-(Phenylethynyl)-1H-pyrazolo-

[3,4-d]pyrimidin-4-amine Derivatives. Since our goal here

is to discover Src inhibitors with high potency against TNBC,

both enzymatic and cellular assays were utilized to evaluate the

bioactivity of synthesized compounds in the SAR studies. For the

cellular assays, two tumor cell lines, MDA-MB-231 and HepG2,

were chosen. MDA-MB-231 is a typical human TNBC cell line

and expresses a high level of Src. HepG2 is a human liver cancer

cell line that expresses very low level of Src; HepG2 was used

here for ruling out the possible oﬀ-target eﬀects and cellular toxic

eﬀects.

The SAR analyses below will mainly focus on discussing the

inﬂuences of various substituents at the following three regions

(see Figure 2B) on the bioactivities: the phenyl ring of the amino

terminal of amide (Ring A; R1,R

2), the N-1 position of 1H-

pyrazolo[3,4-d]pyrimidine (R3), and the phenyl ring of the

carboxyl terminal of amide (Ring B; R4).

3.1.1. Substitution Eﬀects of the Phenyl Ring of the Amino

Terminal of Amide (Ring A; R1,R

2). In this section, we shall

explore the eﬀects of diﬀerent substituents at the meta-position

(R1) or para-position (R2) of ring A. In the ﬁrst step, we ﬁxed

R2as its original group, namely, (4-methylpiperazin-1-yl)methyl,

Scheme 3. Synthetic Routes for Compounds 14a−g

Reagents and conditions: (a) Pd(PPh3)4, CuI, ethynyltrimethylsilane, triethylamine, DMF, 80 °C, N2; (b) K2CO3, MeOH, rt; (c) (i) SOCl2,80°C,

(ii) 4-((4-methylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)aniline (7a), DIPEA, EA, 0 °C to rt; (d) CuI, (PPh3)2PdCl2, DIPEA, DMF, 80 °C, N2;

(e) BBr3, DCM, −78 °C to rt; (f) MeOH, conc H2SO4 cat,reﬂux; (g) NaIO4, I2, conc H2SO4, acetic anhydride, acetic acid, 5−40 °C; (h) EtOH/H2O,

NaOH, rt.

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and varied R1. Bioactivities of the resulting compounds (1a−d)

are shown in Table 1. Obviously, replacement of triﬂuoromethyl

group by hydrogen led to a slight drop in bioactivities in both

enzymatic and cellular assays (1b). Introduction of chloride or

bromide at R1did not improve the biochemical and cellular

potencies too much, rather likely increased the antiviability

activity against HepG2, implying a possible increase in toxicity. In

the second step, we thus ﬁxed R1as triﬂuoromethyl group and

changed R2with diﬀerent substituents. Compared with 1a,

substitution of R2by hydrogen (1e) or 4-methylmorpholine (1f)

decreased the enzymatic activity and the antiviability potency

against MDA-MB-231 cells. For replacement of R2with 1-ethyl-

4-methylpiperidine (1g), its enzymatic and cellular potencies

remained. However, introduction of 2-(4-methylpiperazin-1-

yl)ethanol (1h)atR

2slightly increased the antiviability against

HepG2, implying an increase in toxicity. Obviously, the results

indicated that modiﬁcations of 1a on R1and R2had almost no

eﬀect on the improvement of bioactivity. Therefore, in

subsequent structural modiﬁcation processes, R1and R2will be

kept as their original triﬂuoromethyl and (4-methylpiperazin-1-

yl)methyl, respectively.

3.1.2. Inﬂuences of Various Substituents at the N-1 Position

of 1H-Pyrazolo[3,4-d]pyrimidine Moiety (R3). To examine the

possible inﬂuences of substituents at the N-1 position of

1H-pyrazolo[3,4-d]pyrimidine moiety, various alkyl groups

with diﬀerent size were ﬁrst used to replace the original

isopropyl. Bioactivities of the synthesized derivatives (1a,1i−r)

are shown in Table 2. From Table 2, we can see that all the

derivatives have good bioactivities in both enzymatic and cellular

levels. The most active compound corresponds to 1j (Src, IC50 =

0.0009 μM; MDA-MB-231, IC50 = 0.011 μM), which contains an

ethyl group at R3. Compared with 1j, compounds with a smaller

size hydrophobic group (methyl, 1i), or a larger size hydrophobic

group (isopropyl (1a), cyclobutyl (1k), and cyclopentyl (1l)), at

the R3position aﬀorded a slightly lower bioactivity, implying that

ethyl represents the most suitable size of alkyl substituent. Then,

various polar substituents including methoxymethyl (1m),

(S)-tetrahydrofuran-3-yl (1n), (R)-tetrahydrofuran-3-yl (1o),

1-(Boc)piperidin-4-yl) (1p), piperidin-4-yl (1q), and 1-acryl-

oylpiperidin-4-yl (1r) were used to replace the ethyl group.

However, bioactivities of these compounds (Table 2) still did not

exceed that of compound 1j.

3.1.3. Possible Impacts of Various Substituents at the C-4

Position of Phenyl Ring B (R4). To explore the possible impact of

diﬀerent substituents at the C-4 position (R4) of phenyl ring B,

we synthesized compounds 14a−g, which contain varied

substituents at R4and ﬁxed groups at R1,R

2, and R3as their

optimal forms. Bioactivities of compounds 14a−gare shown in

Table 3. From Table 3, we can see that replacement of methyl

group by hydrogen slightly decreased the biochemical and

cellular anti-TNBC activities. A bulky alkyl substituent at R4, such

as ethyl or i-Pr, remarkably decreased its bioactivities. Further, a

halide (Cl, F) or a polar group (methoxyl and hydroxyl) also

considerably decreased the bioactivity. These results indicated

that methyl is the best substituent group at R4.

3.2. Preliminary in Vivo Antitumor Assays for Screening

Compounds with the Most Potent Anti-TNBC Activity. The

SAR analyses above led to the discovery of a number of Src

inhibitors that exhibited higher potency than dasatinib and

ponatinib in in vitro antiviability assays against human TNBC

MDA-MB-231 cells. To screen compounds with potent anti-

TNBC activity in vivo, we chose three compounds, namely, 1n,

1i, and 1j, to carry out a preliminary in vivo anti-TNBC study in a

MDA-MB-231 xenograft mouse model. The three compounds

were chosen because they are potent Src inhibitors with an IC50

value less than 10 nM and ranked as the top three most active

ones among all the compounds in the in vitro antiviability assay

against the TNBC MDA-MB-231 cells. In the preliminary in vivo

assays, compound 1j was still the most active one (see Figure 3).

Therefore, further in-depth studies including in vitro and in vivo

anti-TNBC activities, and the mechanism of action, were sub-

sequently carried out with compound 1j.

3.3. Biochemical Activities of Compound 1j against

Various Recombinant Human Protein Kinases. The kinase

inhibition proﬁle of compound 1j against a panel of selected

recombinant human protein kinases is presented in Table 4.

Compound 1j potently inhibited Src (IC50 = 0.0009 μM), Yes

(IC50 = 0.0008 μM), Fyn (IC50 = 0.005 μM), and Blk (IC50 =

0.019 μM), which all belong to the Src family kinases (SFKs).

Compound 1j also exhibited considerable potency against

several other kinases, including B-RAFV600E (IC50 = 0.015 μM),

B-RAF (IC50 = 0.092 μM), C-RAF (IC50 = 0.027 μM), BCR-ABL

(IC50 = 0.001 μM), EphA2 (IC50 = 0.016 μM), EphB2 (IC50 =

0.026 μM), TrkA (IC50 = 0.027 μM), DDR2 (IC50 = 0.128 μM),

TAK1 (IC50 = 0.061 μM), Btk (IC50 = 0.067 μM), IKKα(IC50 =

0.353 μM), IKKβ(IC50 = 0.164 μM), Axl (IC50 = 0.578 μM),

PDGFRα(IC50 = 0.890 μM), JAK2 (IC50 = 2.911 μM), and

EGFR(IC50 = 3.518 μM). Compound 1j displayed almost

no inhibitory activity against 18 other tested protein kinases

(IC50 >10μM). These data demonstrate that compound 1j is a

multikinase inhibitor with high potencies against Src family

kinases (SFKs) and several other kinases including MAPK

signaling protein kinases B-RAF and C-RAF.

3.4. Antiviability Activities of Compound 1j against

Various Cancer Cell Lines. The antiviability potencies of

compound 1j against various tumor cell lines including TNBC,

and other breast cancer subtypes, as well as other selected cancer

types, were measured using the MTT assay method. As shown in

Table 5, compound 1j displayed potent activities against TNBC

cell lines MDA-MB-231 and MDA-MB-435 with IC50 values of

0.011 and 0.005 μM, respectively. It also exhibited considerable

potencies against several other cell lines, including MDA-MB-

436 (TNBC, IC50 = 0.316 μM), MDA-MB-453 (TNBC, IC50 =

0.325 μM), MM.1S (myeloma, IC50 = 0.872 μM), U-87 MG

(glioblastoma, IC50 = 0.742 μM). For other 12 cell lines including

BT474 (breast cancer), MCF-7 (breast cancer), SKBR-3 (breast

cancer), HBL-1 (lymphadenoma), OCI-LY10 (lymphadenoma),

RAMOS (lymphadenoma), THP-1 (leukemia), HepG2 (hep-

atocarcinoma), Hela (cervical cancer), H358 (lung cancer),

A2058 (melanoma), PANC-1 (pancreatic cancer), compound 1j

just showed very weak activity or no activity (IC50 >10μM).

Colony-forming assays were then used to examine the anti-

proliferation activity of compound 1j. It was found that com-

pound 1j at concentrations larger than 0.01 μM signiﬁcantly

inhibited the colony formation of MDA-MB-231 cells (see

Figure 4A). Dasatinib also considerably decreased the formation

of colonies of MDA-MB-231 cells at concentrations of >0.01 μM.

Nevertheless, in terms of the potency in inhibiting the formation

of colonies, compound 1j is obviously more potent than dasatinib.

3.5. Eﬀects of Compound 1j on Cell Apoptosis and Cell

Cycle Progression. Aﬂow cytometry assay was performed to

examine cell apoptosis upon treatment with compound 1j.As

shown in Figure 4B, compound 1j induced apoptosis in MDA-

MB-231 cells in a concentration-dependent manner. At a con-

centration of 0.3 μM, an apoptosis rate of 34.4% was observed

after 48 h. We further examined the inﬂuence of compound 1j

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Table 2. Kinase Inhibitory PotencyagainstSrc and Antiviability Activities against MDA-MB-231and HepG2 Cells of Compounds 1i−rand1a

IC50 values were determined from KinaseProﬁler of Euroﬁns. The data represent the mean values of two independent experiments.

Each

compound was tested in triplicate; the data are presented as the mean ±SD.

nd means not determined.

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treatment on the cell cycle of MDA-MB-231 cells using ﬂow

cytometry. The results indicated that compound 1j could induce

cell cycle arrest in G0/G1phase (Figure 4C). As a positive

control, dasatinib also showed the same eﬀects in both ﬂow

cytometry assays but was relatively weaker than compound 1j in

terms of the potency. Taken together, these data demonstrated

that compound 1j could eﬀectively lead to cell apoptosis and

G0/G1phase arrest in MDA-MB-231.

3.6. Eﬀects of Compound 1j on Tumor Cell Migration

and Invasion. The eﬀects of compound 1j on tumor cell

migration and invasion were assessed using wound healing assays

and transwell assays, respectively. As displayed in Figure 5A, after

treatment of compound 1j in concentrations of >0.03 μM for

20 h, the migration of MDA-MB-231 cells was signiﬁcantly

inhibited. Results from the transwell assays showed that 0.1 μM

compound 1j potently blocked the invasion ability of MDA-MB-

231 (Figure 5B). Again, dasatinib also exhibited the same eﬀects

but was relatively weaker than compound 1j in terms of the

potency. These results demonstrated that 1j could eﬃciently

inhibit the migration and invasion of tumor cells.

3.7. Inactivation of Key Signaling Proteins in Intact

Cells. The ability of compound 1j to inhibit the activation of key

signaling proteins in intact cells was assessed using Western blot.

After a 5 h treatment with increasing concentrations of com-

pound 1j, MDA-MB-231 cells were harvested and lysed for an

IP/wt assay. As shown in Figure 6, compound 1j inhibited Src

phosphorylation in a dose-dependent manner with an estimated

IC50 value of 0.03 μM. Consistent with the inhibition of Src

activation, the phosphorylation of its downstream signaling

Figure 3. Preliminary in vivo anti-TNBC assays of compounds 1n,1i,

and 1j. Daily oral administration of compounds 1n,1i, and 1j at

concentrations of 40 mg kg−1d−1, respectively, was initiated when the

MDA-MB-231 tumors reached approximately 200 mm3in volume

(three mice per group). Points indicate mean tumor volume (mm3);

bars indicate SD.

Table 4. Kinase Inhibition Proﬁle of Compound 1j against

Human Src and a Panel of Other Selected Protein Kinases

kinase IC50

(μM) kinase IC50

(μM)

Src 0.0009 TAK1 0.061 CHK1 >10

Src (T341M) 0.030 Btk 0.067 FAK >10

Yes 0.0008 IKKα0.353 GSK3β>10

Fyn 0.005 IKKβ0.164 JNK1α1 >10

Blk 0.019 Axl 0.578 MAPK1 >10

B-RAF (V600E) 0.015 PDGFRα0.890 MEK1 >10

B-RAF 0.092 JAK2 2.911 mTOR >10

C-RAF 0.027 EGFR 3.518 PAK1 >10

BCR-ABL 0.001 Aurora A >10 Pim-1 >10

EphA2 0.016 Ark5 >10 PKBα>10

EphB2 0.026 CDK2 >10 PKBβ>10

TrkA 0.027 CDK7 >10 PKCα>10

DDR2 0.128 TBK1 >10 PI3Kα>10

IC50 values were determined from KinaseProﬁler of Euroﬁns. The

data represent the mean values of two independent experiments.

Table 5. Antiviability Activities of Compound 1j against

Various Cancer Cell Lines

cell line tumor type IC50 (μM)

MDA-MB-231 TNBC 0.011

MDA-MB-435 TNBC 0.005

MDA-MB-436 TNBC 0.316

MDA-MB-453 TNBC 0.325

BT474 breast cancer >10

MCF-7 breast cancer 7.25

SKBR-3 breast cancer 4.523

HBL-1 lymphadenoma ∼10

OCI-LY10 lymphadenoma 2.168

RAMOS lymphadenoma ∼10

MM.1S myeloma 0.872

THP-1 leukemia 5.11

U-87 MG glioblastoma 0.742

HepG2 hepatocarcinoma 8.672

Hela cervical cancer 5.410

H358 lung cancer 1.665

A2058 melanoma 1.908

PANC-1 pancreatic cancer 6.556

Each compound was tested in triplicate; the data are presented as the

mean ±SD.

Table 3. Kinase Inhibitory Potency against Src and

Antiviability Activities against MDA-MB-231 and HepG2

Cells of Compounds 14a−g and 1j

anti-cell viability (IC50,μM)

compd R4

kinase inhibition

(IC50,μM),

Src MDA-MB-231 HepG2

1j Me 0.0009 0.011 ±0.001 >5

14a H 0.005 0.024 ±0.002 >5

14b Cl 0.011 0.052 ±0.004 >5

14c Et 0.083 0.418 ±0.029 >5

14d i-Pr 1.409 1.225 ±0.090 >5

14e Fnd

1.743 ±0.222 >5

14f OMe 0.059 0.321 ±0.041 4.640 ±0.387

14g OH 3.191 >5 >5

IC50 values were determined from KinaseProﬁler of Euroﬁns. The

data represent the mean values of two independent experiments.

Each compound was tested in triplicate; the data are presented as the

mean ±SD.

nd means not determined.

Journal of Medicinal Chemistry Article

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J. Med. Chem. XXXX, XXX, XXX−XXX

protein FAK was signiﬁcantly inhibited at concentrations of

>0.03 μM. Additionally, phosphorylation of the MAPK signaling

proteins, MEK and ERK, was also strongly inhibited, which could

be due to the inactivation of B-RAF and C-RAF (see Table 4),

upstream signaling proteins of MAPK, by compound 1j. It is also

noteworthy that compound 1j has no impact on the phos-

phorylation of AKT, indicating no eﬀect on the AKT signaling.

Similar to compound 1j, dasatinib also eﬃciently inhibited Src

and FAK and showed no eﬀect on AKT signaling. However,

diﬀerent from compound 1j, dasatinib just displayed very weak

inhibitory potency against the ERK phosphorylation, suggesting

less inﬂuence on the MAPK signal pathway. From here, it is

reasonable to conclude that one of the main causes that com-

pound 1j showed a higher antiproliferation activity against

TNBC cells than dasatinib could be due to it being able to inhibit

MAPK signaling more eﬃciently than dasatinib, in addition to

the potent inhibition of Src signaling.

3.8. In Vivo Eﬀects of Compound 1j. The in vivo anti-

TNBC activities of compound 1j were evaluated using an MDA-

MB-231 xenograft model. When the tumor grew to a volume of

150−200 mm3, the mice were grouped and treated orally once

daily with 7.5, 15, or 30 mg kg−1d−11j for 18 days. The tumor

Figure 4. (A) 1j inhibited colony formation of MDA-MB-231 cells. MDA-MB-231 cells were seeded in six-well plates and treated with 1j or dasatinib for

12 days. Colonies were stained with crystal violet and pictures taken. (B, C) 1j induced apoptosis and G0/G1cell cycle arrest of MDA-MB-231 cells.

MDA-MB-231 cells were harvested after treatment with various concentrations of 1j and dasatinib for 24 and 48 h. Cells were stained with an annexin

V-FITC apoptosis detection kit or cell cycle and apoptosis analysis kit, respectively.

Journal of Medicinal Chemistry Article

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J. Med. Chem. XXXX, XXX, XXX−XXX

volumes were measured every 3 days. At all doses given,

compound 1j markedly inhibited the tumor growth. Notably,

a30mgkg

−1d−1dose of compound 1j completely stopped

the tumor growth with a tumor inhibitory rate of >100%

(Figure 7A) without obvious toxicity. On the contrary, dasatinib

(40 mg kg−1d−1) and paclitaxel (10 mg kg−1week−1), a clinically

used TNBC drug, just showed moderate anti-TNBC eﬀects in

the same model (Figure 7A).

To better understand the mechanism of antitumor activities

in vivo, the abilities of compound 1j to inhibit Src and MAPK

signaling, and cell proliferation (Ki67), as well as its eﬀect on

apoptosis in tumor tissues were also evaluated using tumor

tissues isolated from MDA-MB-231 tumor xenograft models at

the end of the treatment with histological and immunohis-

tochemical techniques. A signiﬁcant decrease in the phosphor-

ylation of Src and MEK/ERK was observed in the compound 1j

treated groups compared with the control groups (Figure 7B),

indicating inhibition of Src and MAPK signal pathways. These

tumors exhibited reduced staining of Ki67, implying a reduction

in the number of proliferating cells in the tumor tissues. Further,

more TUNEL-positive cells with AP-Red staining were observed,

indicating a signiﬁcant increased apoptosis in the treatment

group when compared with the control group.

3.9. Pharmacokinetic Characteristics of Compound 1j.

A preliminary pharmacokinetic property assessment of com-

pound 1j was carried out on rats. The plasma concentration

versus time proﬁle is presented in Figure 7C, and the key

pharmacokinetic parameters of compound 1j are summarized in

Table 6. After per os administration of 10 mg/kg compound 1j,

the area under the concentration−time curve (AUC0−∞) was

about 4772.5 μgL

−1h and the half-life (t1/2) was about 12.76 h.

In addition, compound 1j achieved a maximum plasma

concentration (Cmax) of 214.63 μg/L within about 12 h and

showed a clearance rate (CL) of 2.1 L h−1kg−1and apparent

distribution volume (Vss) of 38.57 L/kg. All of these indicated

that compound 1j has good pharmacokinetic properties.

4. CONCLUDING REMARKS

In this study, a series of new Src inhibitors bearing a

3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine scaf-

fold were designed and synthesized. Structure−activity relation-

ships of these compounds were discussed based on enzymatic

and cellular activities. A number of compounds showed high

potencies in both biochemical and cell functional assays. Three

most active compounds were selected to conduct a preliminary in

vivo anti-TNBC assay, and 1j exhibited the most potent in vivo

anti-TNBC activity. Then, further in depth studies were carried

out on 1j for its in vitro and in vivo anti-TNBC activities.

Figure 5. (A) 1j inhibited MDA-MB-231 cell migration in wound healing assay. Cells were wounded by the pipet and then treated with various

concentrations of compounds for 20 h. Scale bar, 100 μm. (B) 1j inhibited MDA-MB-231 cells invasion in transwell invasion assay. The bottom

chambers of the transwells were ﬁlled with 600 μL of DMEM with 10% FBS, while the top chambers were seeded with 1 ×105MDA-MB-231 cells in

200 μL of DMEM and treated with diﬀerent concentrations of compounds for 24 h. Scale bar, 50 μm.

Figure 6. 1j inhibited the phosphorylation of Src and its downstream

signaling proteins and the activation of MAPK signaling proteins in cell

cultures. MDA-MB-231 cells were treated with 1j or dasatinib for 20 h.

Cells were lysed, and the proteins were analyzed by Western blot

analysis.

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J. Med. Chem. XXXX, XXX, XXX−XXX

The results showed that compound 1j was a multikinase inhib-

itor with high potencies against Src and several other kinases

including MAPK signaling protein kinases B-RAF and C-RAF.

In cellular assays, compound 1j signiﬁcantly inhibited the

proliferation of TNBC MDA-MB-231 and MDA-MB-435 cells

and displayed good selectivity for these TNBC cells against

other cancer cells. This compound could eﬀectively block the

migration and invasion of MDA-MB-231 cells and lead to

apoptosis and G0/G1phase arrest. In an MDA-MB-231 xenograft

mouse model, a once-daily dose of compound 1j at 30 mg/kg

for 18 days led to complete tumor regression without obvious

toxicity. Western blot and immunohistochemical analyses

indicated that compound 1j potently inhibited Src and MAPK

signal pathways. Finally, it is also important to mention that

although dasatinib has a comparable potency with compound 1j

in Src inhibition, it has a relatively weaker activity than com-

pound 1j in inhibiting MAPK signaling; dysregulation of MAPK

signaling has been demonstrated to be associated with tumor

development and drug resistance of TNBC.

21,36,37

This could be

one of the most important reasons that compound 1j showed

more potent anti-TNBC activity than dasatinib.

5. EXPERIMENTAL SECTION

Kinase Inhibition Assays. Kinase inhibition proﬁles were obtained

using KinaseProﬁler services provided by Euroﬁns, and ATP con-

centrations used are the ATP Kmof corresponding kinases.

Cell Lines and Cell Culture Conditions. All of the cell lines used

were obtained from the American Type Culture Collection (Manassas,

VA, USA). These cell lines were cultured in the designated medium

containing 10% fetal bovine serum (FBS) (v/v) at 37 °C in a humidiﬁed

5% CO2incubator according to ATCC guidelines.

Cell Viability Assays. The viability of cells was determined using the

MTT assay method. The cell lines were seeded (1500−30 000 cells per

well, depending on the cell type) in 96-well plates. After incubation for

24 h in serum-containing media, the cells were treated with inhibitors

(0−10 μg/mL) diluted with culture medium for 72 h at 37 °C under a

5% CO2atmosphere. Then 20 μL of the MTT reagent (5 mg/mL) was

added to each well, and the plates were incubated for 2−4 h at 37 °C. For

the adherent cells, the media and MTT were carefully aspirated from

each well, and the formazan crystals were dissolved in 150 μL of 100%

DMSO. For the suspended cells, 50 μL of 20% acidiﬁed SDS (w/v) was

used to dissolve the oxidative product, and the cells were incubated

overnight. Finally, the absorbance at 570 nm was read using a Multiskan

Figure 7. (A) In vivo antitumor eﬃcacy of 1j against MDA-MB-231 tumor xenograft models. Daily oral administration of 1j at concentrations of 7.5, 15,

30 mg kg−1d−1and paclitaxel at a dose of 10 mg kg−1week−1though tail vein injection were initiated when the MDA-MB-231 tumors reached

appoximately 200 mm3in volume (6 mice per group). Points indicate mean tumor volume (mm3); bars indicate SD; iv, intravenous. (B) Mechanism of

action of 1j in human tumor xenograft models. Mice bearing MDA-MB-231 tumor xenograft which was treated with 1j at 30 mg/kg were humanly

euthanized at the end of the experiment, and the tumor tissues were removed for further immunohistochemistry analysis and TUNEL detection. Scale

bar, 50 μm. (C) Plasma concentration−time curve of 1j in SD rats after a single oral dose of 10 mg/kg. Blood was collected at the indicated times, and the

plasma concentrations were determined by LC/MS. Points, mean; bars, SD; n=5.

Table 6. Pharmacokinetic Parameters of Compound 1j after

Oral Administration to SD Rats (n=5)

parameter po (10 mg/kg)

AUC0−∞

(μgL

−1h) 4772.50

t1/2

(h) 12.76

Cmax

(μg/L) 214.63

Tmax

(h) 12.00

(L h−1kg−1) 2.10

Vss

(L/kg) 38.57

Mean area under the plasma concentration−time curve.

Mean half-

life associated with the terminal slope.

Mean peak plasma con-

centration.

Mean time to reach maximum plasma concentration.

Mean body clearance.

Mean volume of distribution at steady state.

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J. Med. Chem. XXXX, XXX, XXX−XXX

MK3 ELISA photometer (Thermo Scientiﬁc). All experiments were

performed in triplicate. The IC50 values were calculated using GraphPad

Prism software.

Colony Formation Assay. Cells were seeded in six-well plates at a

density of 5000 per well and treated with vehicle, dasatinib, or 1j the next

day. The medium containing vehicle or 1j was replaced every 4 days.

Cells were ﬁxed with methanol and stained with crystal violet after

treatment for 12 days.

Wound Healing Assay. MDA-MB-231 cells were cultured to

conﬂuence in 24-well plates and wounded using a sterilized pipet tip to

make a straight scratch. Cells were rinsed with physiological saline

gently, and then PBS was replaced with DMEM medium containing

vehicle, dasatinib, or 1j. Pictures were taken by an OLYMPUS digital

camera and analyzed by AxioVision Rel 4.8 (Carl Zeiss) after 20 h.

Transwell Invasion Assay. The cell invasion assay was performed

as described previously.

The cells were photographed with Leica

DM2500.

Cell Cycle Progression and Apoptosis Assays. A total of 2 ×105

MDA-MB-231 cells were plated in a six-well plate and treated with 1j

and dasatinib for 24 and 48 h at 37 °C. After incubation, the cells were

harvested and washed with ice-cold PBS. The cell cycle progression was

analyzed using a cell cycle and apoptosis analysis kit (Beyotime), and the

apoptosis ratio was performed with an annexin V-FITC Apoptosis

Detection Kit (keygentec).

Western Blot Analysis. After treatment with a series of con-

centrations of 1j for 20 h at 37 °C, MDA-MB-231 cells were harvested,

washed with ice-cold physiological saline, and lysed with RIPA lysis

buﬀer (Beyotime) including 1% cocktail (Sigma-Aldrich). Whole-cell

protein lysates were prepared and centrifuged for 10 min at 12 000 rpm

and 4 °C to remove any insoluble material. The total proteins were

determined using the Bradford method, and an equivalent quantity of

protein was combined with an SDS−PAGE loading buﬀer (Beyotime)

in boiled water for 5 min. Cell lysates were separated by SDS−PAGE

and electrotransferred onto PVDF membranes (Millipore). The PVDF

membranes were incubated with each antibody and detected according

to the immunoblot analysis principle. The antibodies were purchased

from Cell Signaling Technology, and the dilutions of the antibodies were

according to the instruction from Cell Signaling Technology.

In Vivo Models. The animal studies were conducted under the

approval of the Experimental Animal Management Committee of

Sichuan University. MDA-MB-231 cells were harvested during the

exponential-growth phase, washed 3 times with serum-free medium,

followed by resuspension at a concentration of 5 ×107per mL. A total of

100 μL of cell suspension was injected into SCID mice (5−6 weeks)

subcutaneously. After the tumors had grown to 150−200 mm3, all the

mice were randomized into 6 groups (6 mice for each group) and dosed

with 1j (7.5, 15, or 30 mg kg−1d−1), dasatinib, paclitaxel (10 mg/kg/

week), or vehicle. The compounds were dissolved in sterilization

water with 25% (v/v) PEG400 plus 5% DMSO and administered orally.

Mice were monitored for side eﬀects every day, and tumor growth was

measured every 3 days. The volume was calculated as follows: tumor

size = ab2/2 (a, long diameter; b, short diameter).

Histopathology and IHC. SCID mice bearing tumors were treated

with control or 1j as described before. At the indicated time after dosing,

individual mice were humanely euthanized. The tumors were ﬁxed with

formalin and embedded in paraﬃn. Sections measuring 4−8μmin

thickness were prepared for histological and immunostaining with the

Ki67 antibody (Thermo Fisher Scientiﬁc, Fremont, CA). Apoptosis was

determined using transferase-mediated dUTP nick-end labeling

(TUNEL) and AP-Red staining (Roche Applied Science). The tissues

of heart, liver, spleen, lung, and kidney were stained with hematoxylin

and eosin (H&E). Finally, images were acquired on an Olympus digital

camera attached to a light microscope.

Pharmacokinetic Assessments. The pharmacokinetics analysis of

1j was conducted in male Sprague−Dawley rats (Chinese Academy of

Medical Science, Beijing, China). Brieﬂy, catheters were surgically

placed into the jugular veins of the rats to collect serial blood samples.

The animals were administered a single dose of 10 mg/kg 1j by oral

gavage after fasting overnight. Blood was collected at the indicated

time and centrifuged immediately to isolate plasma. The plasma

concentrations were determined using high performance liquid

chromatography with tandem mass spectrometric detection (3200

QTRAP system, Applied Biosystems). Noncompartmental pharmaco-

kinetic parameters were ﬁtted using DAS software (Enterprise, version

2.0, Mathematical Pharmacology Professional Committee of China).

Chemistry Methods. All reagents and solvents were obtained from

commercial suppliers and used without further puriﬁcation unless

otherwise indicated. Anhydrous solvents were dried and puriﬁed by

conventional methods prior use. Column chromatography was carried

out on silica gel (300−400 mesh). All reactions were monitored by thin-

layer chromatography (TLC), and silica gel plates with ﬂuorescence

F-254 were used and visualized with UV light. All of the ﬁnal compounds

were puriﬁed to >95% purity, as determined by high-performance liquid

chromatography (HPLC). HPLC analysis was performed on a Waters

2695 HPLC system with the use of a Kromasil C18 reversed-column

(4.6 mm ×250 mm, 5 μm). The binary solvent system (A/B) was as

follows: 10 mmol of ammonium acetate in water (pH 9) (A) and

acetonitrile (B), A/B = 32/68. The absorbance was detected at 254 nm,

and the ﬂow rate was 1 mL/min. 1H NMR and 13C NMR spectra were

recorded on a Bruker AV-400 spectrometer at 400 and 100 MHz,

respectively. Coupling constants (J) are expressed in hertz (Hz). Spin

multiplicities are described as s (singlet), br s (broad singlet), t (triplet),

q (quartet), and m (multiplet). Chemical shifts (δ) are listed in parts per

million (ppm) relative to tetramethylsilane (TMS) as an internal

standard. Mass spectral (MS) data were acquired on a Waters Q-TOF

Premier mass spectrometer (Micromass, Manchester, U.K.).

3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2). A solution

of 1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 g, 148 mmol) and

N-iodosuccinimide (50 g, 222 mmol) in DMF (150 mL) was stirred

at 80 °C for 10−12 h under an N2atmosphere. A second batch of

N-iodosuccinimide (3.92 g, 2 mmol) was added and the solution stirred

for additional 12 h. Upon standing at room temperature, a precipitate

was formed which was separated by ﬁltration and washed with

dimethylformamide and ethanol to aﬀord 15.1 g of the title compound.

The ﬁltrate was concentrated in vacuo to about one-half of the original

volume, and 500 mL of water was added. The precipitated product was

separated by ﬁltration and washed with ethanol to aﬀord a second batch

of the product (18.8 g, combined yield 33.9 g, 87.8% yield). 1H NMR

(400 MHz, DMSO-d6)δ11.06 (br s, 1H), 8.17 (s, 1H), 7.91 (br s, 1H),

6.76 (br s, 1H). MS m/z(ESI): 262.1 [M + H]+.

3-Iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

(3a). To a suspension of 2(5.0 g, 19.2 mmol) in anhydrous N,N-

dimethylformamide (40 mL) under an N2atmosphere, potassium car-

bonate (5.3 g, 38.4 mmol) and 2-bromopropane (1.9 mL, 20.1 mmol)

were added sequentially. The resulting mixture was stirred at 80 °C for

5 h and then was allowed to cool to room temperature. The mixture was

ﬁltered. The ﬁltrate was concentrated in vacuo and then partitioned

between water and DCM. The organic layer was dried with sodium

sulfate and concentrated in vacuo. The crude product was puriﬁed using

silica gel chromatography with a methanol/dichloromethane gradient to

aﬀord the desired product as a yellow solid (5.4 g, 92.8% yield). 1H

NMR (400 MHz, DMSO-d6)δ8.19 (s, 1H), 7.72 (br s, 1H), 6.72 (br s,

1H), 4.99−4.93 (m, 1H), 1.42 (d, J= 6.7 Hz, 6H). MS m/z(ESI): 304.0

[M + H]+. Compounds 3b−iwere synthesized by using a similar pro-

cedure; yield 51.1−92.0%.

1-Ethyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3b).

1H NMR (400 MHz, DMSO-d6)δ8.20 (s, 1H), 7.82 (br s, 1H), 6.67

(br s, 1H), 4.30 (q, J= 7.2 Hz, 2H), 1.35 (t, J= 7.2 Hz, 3H). MS m/z

(ESI): 290.0 [M + H]+.

3-Iodo-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3c).

1H NMR (400 MHz, DMSO-d6)δ8.21 (s, 1H), 7.87 (br s, 1H), 6.67

(br s, 1H), 3.88 (s, 3H). MS m/z(ESI): 275.9 [M + H]+.

1-Cyclobutyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine

(3d). 1H NMR (400 MHz, DMSO-d6)δ8.19 (s, 1H), 7.69 (br s, 1H),

6.64 (br s, 1H), 5.32−5.13 (m, 1H), 2.71−2.54 (m, 2H), 2.36 (d, J= 4.7

Hz, 2H), 1.93−1.76 (m, 2H). MS m/z(ESI): 316.1 [M + H]+.

1-Cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine

(3e). 1H NMR (400 MHz, DMSO-d6)δ8.19 (s, 1H), 7.78 (br s, 1H),

6.70 (br s, 1H), 5.28−4.97 (m, 1H), 2.05 (dt, J= 12.1, 7.9 Hz, 2H),

Journal of Medicinal Chemistry Article

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J. Med. Chem. XXXX, XXX, XXX−XXX

1.99−1.88 (m, 2H), 1.88−1.78 (m, 2H), 1.72−1.59 (m, 2H). MS m/z

(ESI): 330.1 [M + H]+.

3-Iodo-1-(methoxymethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-

amine (3f). 1H NMR (400 MHz, DMSO-d6)δ8.26 (s, 1H), 7.87 (br s,

1H), 6.70 (br s, 1H), 5.54 (s, 2H), 3.26 (s, 3H). MS m/z(ESI): 306.0

[M + H]+.

(S)-3-Iodo-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]-

pyrimidin-4-amine (3g). 1H NMR (400 MHz, DMSO-d6)δ8.21 (s,

1H), 7.89 (br s, 1H), 6.62 (br s, 1H), 5.50−5.28 (m, 1H), 4.10−3.96

(m, 2H), 3.92−3.80 (m, 2H), 2.43−2.23 (m, 2H). MS m/z(ESI): 332.1

[M + H]+.

(R)-3-Iodo-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]-

pyrimidin-4-amine (3h). 1H NMR (400 MHz, DMSO-d6)δ8.21 (s,

1H), 7.89 (br s, 1H), 6.63 (br s, 1H), 5.60−5.28 (m, 1H), 4.15−3.96

(m, 2H), 3.94−3.76 (m, 2H), 2.44−2.22 (m, 2H). MS m/z(ESI): 332.1

[M + H]+.

tert-Butyl 4-(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-

1-yl)piperidine-1-carboxylate (3i). 1H NMR (400 MHz, DMSO-

d6)δ8.20(s, 1H), 7.79 (br s, 1H), 6.72 (br s, 1H), 4.87−4.75 (m, 1H),

4.06 (d, J= 10.8 Hz, 2H), 2.95 (br s, 2H), 1.99−1.81 (m, 4H), 1.43

(s, 9H). MS m/z(ESI): 445.3 [M + H]+.

3-Ethynyl-4-methylbenzoic Acid (6). 3-Iodo-4-methylbenzoate

(4). A solution of 3-iodo-4-methylbenzoic acid (15 g, 57.14 mmol)

and conc H2SO4(cat., 3 mL) in MeOH (100 mL) was heated at

65 °C for 24 h. The reaction mixture was cooled, and the solvent was

evaporated. The residue was dissolved in Et2O, washed with saturated

aqueous NaHCO3(3×), H2O, brine, dried over MgSO4, and the solvent

was evaporated to give the methyl 3-iodo-4-methylbenzoate 4(14.8 g,

94.0%) as an orange liquid.

Methyl 4-Methyl-3-((trimethylsilyl)ethynyl)benzoate (5). 4(14.8 g,

53.6 mmol), Pd(PPh3)4(3.3 g, 2.86 mmol), and CuI (1.1 g, 5.74 mmol)

were in suspended absolute THF (100 mL). The mixture underwent

three cycles of vacuum/ﬁlling with N2.Triethylamine(5mL,35.6mmol)

and trimethylsilylacetylene (2.65 mL, 18.7 mmol) were then added.

The mixture was stirred at room temperature overnight. Then the

mixture was ﬁltered, ﬁltrate concentrated in vacuo, and the residue

was diluted with EA (3×) and 0.5 M ammonia solution for extraction.

The combined organic phases were dried over sodium sulfate, ﬁltered,

and concentrated in vacuo. The crude product was puriﬁed using silica

gel chromatography with a petroleum ether/ethyl acetate gradient to

aﬀord the methyl 4-methyl-3-((trimethylsilyl)ethynyl)benzoate (5)asa

yellow liquid.

5was dissolved in methanol (80 mL). K2CO3(7.4 g, 53.6 mmol) was

added and stirred for 10 min at rt. Then to the mixture were added

NaOH (2.1 g, 53.6 mmol) and water (20 mL), and the mixture was

stirred for another 3 h. The mixture was acidiﬁed with hydrochloric acid

(pH 4) and extracted with DCM (3×). The combined organic phases

were washed with saturated sodium chloride solution and concentrated

under reduced pressure. Chromatographic puriﬁcation through a short

silica gel frit with a petroleum ether/ethyl acetate gradient yields 6(6.8 g,

74.1% yield). 1H NMR (400 MHz, DMSO-d6)δ13.04 (br s, 1H), 7.93

(s, 1H), 7.85 (d, J= 8.0 Hz, 1H), 7.43 (d, J= 8.0 Hz, 1H), 4.48 (s, 1H),

2.45 (s, 3H). MS m/z(ESI): 159.0 [M −H]−.

4-((4-Methylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)-

aniline (9a). 4-Amino-2-trifluoromethylbenzaldehyde (8a). 4-Amino-2-

(triﬂuoromethyl)benzonitrile (7a) (11.2 g, 60 mmol) was charged into a

500 mL three-necked, round-bottomed ﬂask and underwent three cycles

of vacuum/ﬁlling with N2. Dry THF (100 mL) was then added and a

solution of diisobutylaluminum hydride (100 mL, 1.5 M in toluene) was

added at 23−30 °C over 20 min. Upon complete addition, the resulting

solution was stirred for an additional 30 min. After the reaction was

complete, the mixture was cooled to −10 °C and methanol (18 mL) was

carefully added. Then the mixture was stirred for an additional 2 h at rt

and an aqueous saturated Rochelle salt solution was added dropwise.

After the quench was complete, the mixture was stirred at 45−50 °C for

10 min. tert-Butyl methyl ether (150 mL) was added and stirred for

10 min. Organic layers were separated, and more tert-butyl methyl ether

was added for extraction. The combined organic phases were dried over

MgSO4and concentrated under vacuum. The residue was used directly

in the next step. MS m/z(ESI): 188.0 [M −H]−.

4-Amino-2-triﬂuoromethylbenzaldehyde (8a) (1.13 g, 6 mmol) was

dissolved in ethyl acetate (35 mL), and aqueous 1 M HCl solution

(20 mL) was added. The mixture was stirred rapidly at 20−35 °C for

5 min. A solution of aqueous 1 M NaOH (15 mL) was charged to the

mixture and stirred for an additional 5 min. The organic layer was

separated, washed with 10% (w/w) aqueous NaCl solution (20 mL),

and charged to a 100 mL reactor. 1-Methylpiperazine (3.0 g, 30 mmol),

pyridinium p-toluenesulfonate (150.8 mg, 0.6 mmol), and toluene

(15 mL) were charged. The mixture underwent azeotropic distillation

under Dean−Stark conditions with a ﬁnal vessel temperature of 102−

110 °C. After each 15 mL portion of distillate had been collected, fresh

ethyl acetate (15 mL) was charged to the reaction mixture. This

distillation−addition operation was repeated until a total of 45 mL of

ethyl acetate was collected. The mixture was cooled to 50 °C, and

sodium triacetoxyborohydride (2.54 g, 12 mmol) was added in portions

at 50−60 °C. The reaction mixture was cooled to 10−15 °C and

quenched with water (25 mL) over 10 min while maintaining the batch

temperature below 20 °C. The organic layer was separated, washed with

water (2 ×25 mL), dried over MgSO4, and concentrated in vacuo. The

residue was washed with petroleum ether/ethyl acetate (v/v = 5), and

the title compound 9a was obtained as a white solid (1.26 g, 76.1%

yield). 1H NMR (400 MHz, CDCl3): δ7.47 (d, J= 8.4 Hz, 1H), 6.91 (d,

J= 2.4 Hz, 1H), 6.79 (dd, J= 8.4, 2.4 Hz, 1H), 3.76 (br s, 2H), 3.52 (s,

2H), 2.70−2.35 (m, 8H), 2.28 (s, 3H); MS (ESI) m/z274.2 [M + H]+.

Compounds 9c−dand 9f−hwere synthesized by using a similar

procedure; yield 51.1−85.0%.

4-(Morpholinomethyl)-3-(triﬂuoromethyl)aniline (9f). 1H

NMR (400 MHz, DMSO-d6)δ7.30 (d, J= 8.4 Hz, 1H), 6.85 (d, J=

2.1 Hz, 1H), 6.72−6.75 (m, 1H), 5.45 (s, 2H), 3.60−3.50 (m, 4H), 3.39

(s, 2H), 2.40−2.30 (m, 4H). MS (ESI) m/z261.1 [M + H]+.

4-((4-Ethylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)aniline

(9g). 1H NMR (400 MHz, DMSO-d6)δ7.29 (d, J= 8.2 Hz, 1H), 6.87

(d, J= 2.2 Hz, 1H), 6.76 (dd, J= 8.2, 2.2 Hz, 1H,), 5.42 (s, 2H), 3.39 (s,

2H), 2.34−2.26 (m, 10H), 0.97 (t, J= 5 Hz, 3H). MS (ESI) m/z288.2

[M + H]+.

2-(4-(4-Amino-2-(triﬂuoromethyl)benzyl)piperazin-1-yl)-

ethan-1-ol (9h). 1H NMR (400 MHz, CDCl3)δ7.47 (d, J= 8.3 Hz,

1H), 6.92 (d, J= 2.0 Hz, 1H), 6.79 (dd, J= 8.2, 2.0 Hz, 1H), 3.77 (br s,

2H), 3.60 (t, J= 5.4 Hz, 2H), 3.53 (s, 2H), 2.76 (br s, 1H), 2.56−2.49

(m, 10H). MS (ESI) m/z304.2 [M + H]+.

3-Ethynyl-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-

3-(triﬂuoromethyl)phenyl)benzamide (10a). 3-Ethynyl-4-methyl-

benzoic acid 6(571 mg, 3.56 mmol) was reﬂuxed in SOCl2for 2 h,

concentrated in vacuo, and redissolved in ethyl acetate (8 mL) to aﬀord

3-ethynyl-4-methylbenzoyl chloride solution.

3-((4-Methylpiperazin-1-yl)methyl)-5-(triﬂuoromethyl)aniline 9a

(812 mg, 2.97 mmol) and DIPEA (806 mg, 6.24 mmol) were dissolved

in ethyl acetate (10 mL) and cooled to 0 °C. Then 3-ethynyl-4-

methylbenzoyl chloride ethyl acetate solution was dropwise added,

resulting in an oﬀ-white suspension. The suspension was allowed to stir

for 1 h at rt. To the reaction mixture was added 30 mL of water, and

the aqueous phase was extracted with ethyl acetate (3 ×20 mL). The

combined organic phase was separated, dried over anhydrous sodium

sulfate, ﬁltered, and the ﬁltrate was evaporated to dryness under reduced

pressure to give a solid residue. The solid was puriﬁed by ﬂash column

chromatography using MeOH in dichloromethane to give 10a as an oﬀ-

white solid (1.1 g, 88% yield). 1H NMR (400 MHz, CDCl3)δ8.33 (s,

1H), 7.91 (s, 1H), 7.86 (d, J= 13.7 Hz, 2H), 7.73 (t, J= 7.1 Hz, 2H), 7.28

(s, 1H), 3.60 (s, 2H), 3.32 (s, 1H), 2.48 (br s, 11H), 2.30 (s, 3H). MS

m/z(ESI): 416.3 [M + H]+. Compounds 10b−gwere synthesized by

using a similar procedure; yield 79.1−88.2%.

3-Ethynyl-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-

phenyl)benzamide (10b). 1H NMR (400 MHz, CDCl3)δ7.91 (d, J=

10.3 Hz, 2H), 7.76 (d, J= 7.8 Hz, 1H), 7.57 (d, J= 7.7 Hz, 2H), 7.31 (d,

J= 7.7 Hz, 3H), 3.49 (s, 2H), 3.35 (s, 1H), 2.51 (s, 3H), 2.48 (br s, 8H),

2.32 (s, 3H). MS m/z(ESI): 348.2 [M + H]+.

N-(3-Chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-

ethynyl-4-methylbenzamide (10c). 1H NMR (400 MHz, CDCl3)δ

8.86 (s, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.81 (d, J= 7.9 Hz, 1H), 7.49 (d,

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.5b00270

J. Med. Chem. XXXX, XXX, XXX−XXX

J= 8.3 Hz, 1H), 7.35−7.29 (m, 2H), 3.64 (s, 2H), 3.33 (s, 1H), 3.03−

2.78 (m, 11H), 2.47 (s, 3H). MS m/z(ESI): 382.2 [M + H]+.

N-(3-Bromo-4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-

ethynyl-4-methylbenzamide (10d). 1H NMR (400 MHz, CDCl3)δ

9.30 (s, 1H), 8.10 (s, 1H), 8.03 (s, 1H), 7.86 (d, J= 7.9 Hz, 1H), 7.63 (d,

J= 8.4 Hz, 1H), 7.32 (d, J= 8.4 Hz, 1H), 7.27 (d, J= 7.9 Hz, 1H), 3.63 (s,

2H), 3.32 (s, 1H), 3.04−2.94 (m, 8H), 2.82 (s, 3H), 2.48 (s, 3H). MS

m/z(ESI): 426.1 [M + H]+.

3-Ethynyl-4-methyl-N-(3-(triﬂuoromethyl)phenyl)-

benzamide (10e). 1H NMR (400 MHz, DMSO-d6)δ10.55 (s, 1H),

8.25 (s, 1H), 8.11 (s, 1H), 8.07 (d, J= 8.4 Hz, 1H), 7.92 (d, J= 8.0 Hz,

1H), 7.60 (d, J= 8.0 Hz, 1H), 7.47 (t, J= 8.8 Hz, 2H), 4.54 (s, 1H), 2.47

(s, 3H). MS m/z(ESI): 304.1 [M + H]+.

3-Ethynyl-4-methyl-N-(4-(morpholinomethyl)-3-

(triﬂuoromethyl)phenyl)benzamide (10f). 1H NMR (400 MHz,

CDCl3)δ7.98 (s, 1H), 7.95 (s, 1H), 7.88 (d, J= 8.0 Hz, 1H), 7.87 (s,

1H), 7.82−7.75 (m, 2H), 7.34 (d, J= 8.0 Hz, 1H), 3.81−3.66 (m, 4H),

3.63 (s, 2H), 3.36 (s, 1H), 2.52 (s, 3H), 2.51−2.41 (m, 4H). MS m/z

(ESI): 403.2 [M + H]+.

N-(4-((4-Ethylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)-

phenyl)-3-ethynyl-4-methylbenzamide Hydrochloride Salt

(10g). 1H NMR (400 MHz, DMSO-d6)δ10.57 (s, 1H), 10.24 (br s,

1H), 8.22 (s, 1H), 8.11 (d, J= 8.9 Hz, 2H), 7.92 (d, J= 8.2 Hz, 1H), 7.72

(d, J= 8.5 Hz, 1H), 7.48 (d, J= 8.2 Hz, 1H), 4.55 (s, 1H), 3.67 (s, 2H),

3.43 (d, J= 12.1 Hz, 2H), 3.18−3.04 (m, 2H), 3.03−2.81 (m, 6H), 2.47

(s, 3H), 1.24 (t, J= 7.2 Hz, 3H). MS m/z(ESI): 430.2 [M + H]+.

3-Ethynyl-N-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-

3-(triﬂuoromethyl)phenyl)-4-methylbenzamide (10h). 3-Ethynyl-

4-methylbenzoic acid 6(1.04 g, 6.5 mmol) was reﬂuxed in SOCl2for 2 h,

concentrated in vacuo, and redissolved in ethyl acetate (15 mL) to aﬀord

3-ethynyl-4-methylbenzoyl chloride solution.

2-(4-(4-Amino-2-(triﬂuoromethyl)benzyl)piperazin-1-yl)ethan-1-ol

9h (910 mg, 3 mmol) and DIPEA (840 mg, 6.5 mmol) were dissolved in

ethyl acetate (20 mL) and cooled to 0 °C. Then 3-ethynyl-4-

methylbenzoyl chloride−ethyl acetate solution was dropwise added,

resultinginanoﬀ-white suspension. The suspension was allowed to stir for

1 h at rt. To the reaction mixture was added 40 mL of water, and the

aqueous phase was extracted with ethyl acetate (3×30 mL). The combined

organic phase was separated, dried over anhydrous sodium sulfate, ﬁltered,

and the ﬁltrate was evaporated to dryness under reduced pressure to give a

solid residue. The solid combined with LiOH (500 mg) was dissolved

in THF/H2O (v/v = 3, 16 mL) and was stirred at 25 °C overnight. The

mixture was diluted with EA (3×30 mL) and brine (30 mL) for extraction.

Combined organic phases were dried over MgSO4, concentrated in vacuo.

The residue was puriﬁed by ﬂash column chromatography using MeOH in

dichloromethane to give 10h as a solid (1.65 g, 61.7% yield).

3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (1a). 3a (151.6 mg, 0.5 mmol),

10a (207.7 mg, 0.5 mmol), CuI (9.5 mg, 0.05 mmol), and (PPh3)2PdCl2

(17.5 mg, 0.025 mmol) were suspended in anhydrous DMF (3 mL). The

mixture underwent three cycles of vacuum/ﬁlling with N2. Then DIPEA

(165 μL, 1 mmol) was added with syringe. The mixture was stirred at

80 °C for 8 h and then quenched with water. DCM and 5% ammonia were

added for extraction. The combined organic layer was concentrated in

vacuo and the crude product was puriﬁed using silica gel chromatography

with a methanol/dichloromethane gradient to aﬀord the title compound

1a as a white solid (136 mg, 46.0% yield). 1H NMR (400 MHz, DMSO-

d6)δ10.58 (s, 1H), 8.29 (s, 1H), 8.18 (s, 2H), 8.05 (d, J= 8.3 Hz, 1H),

7.91 (d, J= 8.0 Hz, 1H), 7.64 (d, J= 8.3 Hz, 1H), 7.46 (d, J= 8.0 Hz, 1H),

5.01−4.95 (m, 1H), 3.58 (s, 2H), 3.04 (br s, 4H), 2.62 (s, 3H), 2.56 (br s,

4H), 2.43 (s, 3H), 1.40 (d, J= 6.6 Hz, 6H). 13C NMR (100 MHz, DMSO-

d6)δ164.72, 157.80, 156.29, 152.32, 144.14, 138.53, 132.03, 131.46,

131.37, 131.09, 129.97, 128.75, 127.55 (d, J= 29.5 Hz), 124.89, 123.57,

122.93, 121.57, 117.42, 100.77, 91.09, 85.55, 56.65, 52.79, 49.54, 48.89,

42.71, 42.36, 21.76, 20.54. HRMS m/z(ESI) calcd for C31H34F3N8O

[M + H]+591.2808; found, 591.2806. Compounds 1b−pwere synthesized

by using a similar procedure; yield 39.0−58.1%.

3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-

phenyl)benzamide (1b). 1H NMR (400 MHz, DMSO-d6)δ10.54 (s,

1H), 8.28−8.25 (m, 3H), 8.09 (s, 1H), 7.94 (d, J= 7.9 Hz, 1H), 7.87 (br

s, 1H), 7.75 (d, J= 8.2 Hz, 1H), 7.36 (d, J= 8.2 Hz, 1H), 5.09−5.03 (m,

1H), 3.70 (s, 2H), 2.49 (s, 3H), 2.46 (br s, 8H), 2.24 (s, 3H), 1.49 (d, J=

6.4 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)δ164.00, 157.87, 156.26,

152.28, 141.15, 136.75, 135.52, 133.62, 131.61, 130.74, 130.01, 127.29

(d, J= 30.1 Hz), 125.10, 125.02, 123.80, 121.76, 121.32, 100.80, 91.76,

84.76, 57.40, 54.47, 52.40, 48.87, 45.33, 21.74, 19.89. HRMS m/z(ESI)

calcd for C30H35N8O[M+H]

+523.2934; found, 523.2928.

3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-N-(3-chloro-4-((4-methylpiperazin-1-yl)methyl)-

phenyl)-4-methylbenzamide (1c). 1H NMR (400 MHz, DMSO-d6)

δ10.43 (s, 1H), 8.32 (s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.94 (d, J= 8.0

Hz, 1H), 7.72 (d, J= 8.4 Hz, 1H), 7.54 (d, J= 8.0 Hz, 1H), 7.44 (d, J=

8.4 Hz, 1H), 5.13−4.99 (m, 1H), 3.53 (s, 2H), 2.58 (s, 3H), 2.43 (br s,

8H), 2.20 (s, 3H), 1.49 (d, J= 6.6 Hz, 6H). HRMS m/z(ESI) calcd for

C30H34ClN8O[M+H]

+557.2544; found, 557.2538.

3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-N-(3-bromo-4-((4-methylpiperazin-1-yl)methyl)-

phenyl)-4-methylbenzamide (1d). 1H NMR (400 MHz, DMSO-d6)

δ10.44 (s, 1H), 8.33 (s, 1H), 8.27 (s, 1H), 8.16 (s, 1H), 7.95 (d, J= 8.0

Hz, 1H), 7.80 (d, J= 8.2 Hz, 1H), 7.54 (d, J= 8.2 Hz, 1H), 7.44 (d, J=

8.0 Hz, 1H), 5.19−4.94 (m, 1H), 3.56 (s, 2H), 2.73 (br s, 11H), 2.58 (s,

3H), 1.49 (d, J= 6.0 Hz, 6H). HRMS m/z(ESI) calcd for C30H34BrN8O

[M + H]+601.2039; found, 601.2040.

3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-methyl-N-(3-(triﬂuoromethyl)phenyl)benzamide

(1e). 1H NMR (400 MHz, DMSO-d6)δ10.63 (s, 1H), 8.36 (s, 1H),

8.26 (s, 2H), 8.09 (d, J= 7.9 Hz, 1H), 7.97 (d, J= 7.7 Hz, 1H), 7.88 (br s,

1H), 7.61 (t, J= 7.9 Hz, 1H), 7.54 (d, J= 7.9 Hz, 1H), 7.46 (d, J= 7.5 Hz,

1H), 6.74 (br s, 1H), 5.20−4.92 (m, 1H), 2.58 (s, 3H), 1.48 (d, J= 6.4

Hz, 6H). HRMS m/z(ESI) calcd for C25H22F3N6O[M+H]

+479.1807;

found, 479.1813.

3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-methyl-N-(4-(morpholinomethyl)-3-(triﬂuoromethyl)-

phenyl)benzamide (1f). 1H NMR (400 MHz, DMSO-d6)δ10.56 (s,

1H), 8.34 (s, 1H), 8.27 (s, 1H), 8.23 (s, 1H), 8.09 (d, J= 8.3 Hz, 1H),

7.97 (d, J= 7.9 Hz, 1H), 7.75 (d, J= 8.3 Hz, 1H), 7.55 (d, J= 7.9 Hz,

1H), 5.12−5.03 (m, 1H), 3.59 (s, 6H), 2.59 (s, 3H), 2.39 (s, 4H), 1.49

(d, J= 6.8 Hz, 6H). HRMS m/z(ESI) calcd for C30H31F3N7O2[M + H]+

578.2491; found, 578.2488.

3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)-4-methylbenzamide (1g). 1HNMR

(400 MHz, DMSO-d6)δ10.59 (s, 1H), 8.35 (s, 1H), 8.27 (s, 1H),

8.24 (s, 1H), 8.10 (d, J= 8.2 Hz, 1H), 7.98 (dd, J= 7.9, 1.1 Hz, 1H), 7.73

(d, J= 8.6 Hz, 1H), 7.55 (d, J= 8.2 Hz, 1H), 5.10−5.03 (m, 1H), 3.62 (s,

2H), 2.59 (s, 3H), 2.55 (br s, 10H), 1.49 (d, J= 6.7 Hz, 6H), 1.10 (t, J=

6.9 Hz, 3H). HRMS m/z(ESI) calcd for C32H36F3N8O[M+H]

605.2964; found, 605.2962.

3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-N-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)-4-methylbenzamide (1h). 1HNMR

(400 MHz, DMSO-d6)δ10.56 (s, 1H), 8.34 (s, 1H), 8.26 (s, 1H),

8.22 (s, 1H), 8.07 (d, J= 8.2 Hz, 1H), 7.96 (d, J= 8.0 Hz, 1H), 7.72 (d,

J= 8.2 Hz, 1H), 7.55 (d, J= 8.0 Hz, 1H), 5.19−4.94 (m, 1H), 4.37 (s,

1H), 3.56 (s, 2H), 3.48 (d, J= 5.4 Hz, 2H), 2.58 (s, 3H), 2.38 (br s, J=

6.1 Hz, 10H), 1.49 (d, J= 6.3 Hz, 6H). HRMS m/z(ESI) calcd for

C32H36F3N8O2[M + H]+621.2913; found, 621.2917.

3-((4-Amino-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (1i). 1H NMR (400 MHz,

DMSO-d6)δ10.60 (s, 1H), 8.33 (s, 1H), 8.26 (s, 1H), 8.22 (s, 1H), 8.09

(d, J= 8.5 Hz, 1H), 7.95 (d, J= 8.1 Hz, 1H), 7.69 (d, J= 8.5 Hz, 1H),

7.52 (d, J= 8.1 Hz, 1H), 3.93 (s, 3H), 3.62 (s, 2H), 2.92 (br s, 4H), 2.58

(br s, 7H), 2.48 (s, 3H). 13C NMR (100 MHz, DMSO-d6)δ164.79,

157.79, 156.57, 153.26, 144.19, 138.48, 132.12, 131.46, 131.38, 131.30,

129.96, 128.69, 127.53 (d, J= 31.0 Hz), 125.04, 123.52, 121.61, 117.36,

117.30, 99.55, 91.12, 85.41, 56.82, 53.27, 50.27, 33.97, 20.50, 17.33.

HRMS m/z(ESI) calcd for C29H30F3N8O[M+H]

+563.2495; found,

563.2497.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.5b00270

J. Med. Chem. XXXX, XXX, XXX−XXX

3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (1j). 1H NMR (400 MHz,

DMSO-d6)δ10.56 (s, 1H), 8.34 (s, 1H), 8.27 (s, 1H), 8.22 (d, J= 1.7

Hz, 1H), 8.07 (d, J= 8.5 Hz, 1H), 7.97 (d, J= 8.1 Hz, 1H), 7.72 (d, J=

8.5 Hz, 1H), 7.55 (d, J= 8.1 Hz, 1H), 4.38 (q, J= 7.2 Hz, 2H), 3.57 (s,

2H), 2.58 (s, 3H), 2.39 (br s, 8H), 2.16 (s, 3H), 1.42 (t, J= 7.2 Hz, 3H).

13C NMR (100 MHz, DMSO-d6)δ164.77, 157.86, 156.52, 152.75,

144.17, 138.19, 132.23, 132.20, 131.38, 131.32, 129.99, 128.72, 127.75

(d, J= 30.0 Hz), 125.11, 123.56, 123.02, 121.62, 117.31, 100.19, 91.18,

85.47, 57.50, 54.78, 52.75, 45.78, 41.99, 20.55, 14.68. HRMS m/z(ESI)

calcd for C30H32F3N8O[M+H]

+577.2651; found, 577.2652.

3-((4-Amino-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (1k). 1H NMR (400 MHz,

DMSO-d6)δ10.56 (s, 1H), 8.35 (s, 1H), 8.26 (s, 1H), 8.22 (s, 1H), 8.08

(d, J= 8.5 Hz, 1H), 7.97 (d, J= 8.0 Hz, 1H), 7.72 (d, J= 8.5 Hz, 1H),

7.55 (d, J= 8.0 Hz, 1H), 5.38−5.29 (m, 1H), 3.57 (s, 2H), 2.76−2.62

(m, 2H), 2.59 (s, 3H), 2.41 (br s, 10H), 2.18 (s, 3H), 1.95−1.80 (m,

2H). 13C NMR (100 MHz, DMSO-d6)δ164.66, 157.82, 156.43, 152.71,

144.14, 138.18, 132.13, 132.10, 131.33, 131.26, 129.95, 128.74, 127.42

(d, J= 30.0 Hz), 125.30, 123.51, 122.98, 121.54, 117.27, 100.77, 91.25,

85.45, 57.43, 54.68, 52.61, 50.39, 45.62, 29.36, 20.53, 14.41. HRMS m/z

(ESI) calcd for C32H34F3N8O[M+H]

+603.2808; found, 603.2808.

3-((4-Amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-

yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (1l). 1H NMR (400 MHz,

DMSO-d6)δ10.59 (s, 1H), 8.33 (s, 1H), 8.25 (s, 1H), 8.22 (s, 1H), 8.11

(d, J= 8.5 Hz, 1H), 7.95 (d, J= 7.9 Hz, 1H), 7.70 (d, J= 8.5 Hz, 1H),

7.54 (d, J= 7.9 Hz, 1H), 5.29−5.14 (m, 1H), 3.65 (s, 2H), 3.03−2.87

(m, 4H), 2.75 (s, 3H), 2.57 (s, 3H), 2.45−2.26 (m, 4H), 2.14−2.07 (m,

2H), 2.05−1.93 (m, 2H), 1.93−1.78 (m, 2H), 1.74−1.61 (m, 2H).

HRMS m/z(ESI) calcd for C33H36F3N8O[M+H]

+617.2964; found,

617.2968.

3-((4-Amino-1-(methoxymethyl)-1H-pyrazolo[3,4-d]-

pyrimidin-3-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-

yl)methyl)-3-(triﬂuoromethyl)phenyl)benzamide (1m). 1H NMR

(400 MHz, DMSO-d6)δ10.57 (s, 1H), 8.36 (s, 1H), 8.32 (s, 1H), 8.22

(s, 1H), 8.07 (d, J= 8.3 Hz, 1H), 7.98 (d, J= 7.9 Hz, 1H), 7.72 (d, J= 8.3

Hz, 1H), 7.55 (d, J= 7.9 Hz, 1H), 5.63 (s, 2H), 3.58 (s, 2H), 3.31 (s,

3H), 2.59 (s, 3H), 2.40 (br s, 8H), 2.19 (s, 3H). HRMS m/z(ESI) calcd

for C30H32F3N8O2[M + H]+593.2600; found, 593.2597.

(S)-3-((4-Amino-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]-

pyrimidin-3-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-

yl)methyl)-3-(triﬂuoromethyl)phenyl)benzamide (1n). 1H NMR

(400 MHz, DMSO-d6)δ10.59 (s, 1H), 8.36 (s, 1H), 8.28 (s, 1H), 8.23

(s, 1H), 8.10 (d, J= 8.0 Hz, 1H), 7.97 (d, J= 7.6 Hz, 1H), 7.72 (d, J= 8.0

Hz, 1H), 7.55 (d, J= 7.6 Hz, 1H), 5.58−5.41 (m, 1H), 4.18−3.98 (m,

2H), 3.91 (dd, J= 13.4, 9.1 Hz, 2H), 3.62 (s, 2H), 2.59 (s, 3H), 2.54

(s, 3H), 2.44 (br s, 8H), 2.38−2.29 (m, 2H). 13C NMR (100 MHz,

DMSO-d6)δ164.63, 157.85, 156.51, 153.17, 144.19, 138.18, 132.12,

131.34, 131.25, 129.98, 129.94, 128.80, 127.42 (d, J= 29.3 Hz), 125.45,

123.52, 123.01, 121.52, 117.27, 100.83, 91.37, 85.36, 71.57, 67.39, 57.48,

54.76, 52.73, 45.75, 31.82, 20.56, 14.66. HRMS m/z(ESI) calcd for

C32H34F3N8O2[M + H]+619.2757; found, 619.2756.

(R)-3-((4-Amino-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-

d]pyrimidin-3-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-

1-yl)methyl)-3-(triﬂuoromethyl)phenyl)benzamide (1o). 1H

NMR (400 MHz, DMSO-d6)δ10.60 (s, 1H), 9.98 (br s, 1H), 9.44

(br s, 1H), 8.35 (s, 1H), 8.28 (s, 1H), 8.24 (s, 1H), 8.12 (d, J= 8.2 Hz,

1H), 7.98 (d, J= 8.0 Hz, 1H), 7.72 (d, J= 8.2 Hz, 1H), 7.55 (d, J= 8.0

Hz, 1H), 5.61−5.36 (m, 1H), 4.19−3.98 (m, 2H), 3.91 (dd, J= 12.3, 7.6

Hz, 2H), 3.67 (s, 2H), 3.08 (s, 6H), 2.91 (br s, 2H), 2.74 (s, 3H), 2.59 (s,

3H), 2.43−2.33 (m, 2H). HRMS m/z(ESI) calcd for C32H34F3N8O2

[M + H]+619.2757; found, 619.2761.

tert-Butyl 4-(4-Amino-3-((2-methyl-5-((4-((4-methylpipera-

zin-1-yl)methyl)-3-(triﬂuoromethyl)phenyl)carbamoyl)-

phenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-

1-carboxylate (1p). 1H NMR (400 MHz, DMSO-d6)δ10.60 (s, 1H),

8.33 (s, 1H), 8.24 (s, 1H), 8.22 (s, 1H), 8.09 (d, J= 8.0 Hz, 1H), 7.95 (d,

J= 7.7 Hz, 1H), 7.69 (d, J= 8.0 Hz, 1H), 7.52 (d, J= 7.7 Hz, 1H),

4.98−4.78 (m, 1H), 4.10−4.01 (m, 2H), 3.61 (s, 2H), 3.03−2.81 (m,

6H), 2.55 (br s, 7H), 2.48 (s, 3H), 1.99−1.90 (m, 4H), 1.41 (s, 9H).

HRMS m/z(ESI) calcd for C38H45F3N8O[M+H]

+732.3597; found,

732.3598.

3-((4-Amino-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-

3-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-

3-(triﬂuoromethyl)phenyl)benzamide (1q). 1p (1.46 g, 2 mmol)

was dissolved in DCM (20 mL) and cooled to 0 °C. Then 10 mL of

CF3COOH was slowly added and stirred at rt for 2 h. The mixture was

concentrated and suspended in water. Aqueous sodium hydrogen

carbonate was added and adjusted to pH 8. Then the mixture was

ﬁltered and washed with water. The solid was dried in vacuo and

recrystallized in acetone. Then the mixture was ﬁltered to aﬀord 1q as a

light yellow solid (1.07 g, 84.9% yield). 1H NMR (400 MHz, DMSO-d6)

δ10.56 (s, 1H), 8.35 (s, 1H), 8.28 (s, 1H), 8.22 (s, 1H), 8.08 (d, J= 7.9

Hz, 1H), 7.98 (d, J= 7.9 Hz, 1H), 7.72 (d, J= 8.2 Hz, 1H), 7.55 (d, J=

8.2 Hz, 1H), 5.09−4.98 (m, 1H), 4.36−4.32 (m, 1H), 3.58 (s, 2H),

3.21−3.08 (m, 2H), 2.99 (d, J= 7.0 Hz, 2H), 2.58 (s, 3H), 2.42 (br s,

8H), 2.31 (d, J= 11.7 Hz, 2H), 2.22 (s, 3H), 2.12 (d, J= 11.6 Hz, 2H).

HRMS m/z(ESI) calcd for C33H37F3N9O[M+H]

+632.3073; found,

632.3071.

3-((1-(1-Acryloylpiperidin-4-yl)-4-amino-1H-pyrazolo[3,4-d]-

pyrimidin-3-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-

yl)methyl)-3-(triﬂuoromethyl)phenyl)benzamide (1r). 1q (510 mg,

0.807 mmol) and triethylamine (223 μL, 1.6 mmol) were dissolved in

10 mL of DCM. Then the mixture was cooled to −10 °C, and to it was

dropped a solution of acrylyl chloride (72 μL, 0.888 mmol) in

dichloromethane (2 mL) within 0.5 h. Then the mixture was stirred at rt

for an additional 0.5 h and quenched with water. DCM and more water

were added for extraction. The combined organic layer was dried with

sodium sulfate, ﬁltered, concentrated, and the crude product was puriﬁed

using silica gel chromatography with a methanol/dichloromethane gradient

to aﬀord the 1r as a yellow solid (440 mg, 72.3%). 1H NMR (400 MHz,

DMSO-d6)δ10.54 (s, 1H), 8.33 (s, 1H), 8.27 (s, 1H), 8.21 (s, 1H), 8.06 (d,

J=7.7Hz,1H),7.96(d,J= 7.7 Hz, 1H), 7.71 (d, J= 7.8 Hz, 1H), 7.54 (d,

J= 7.8 Hz, 1H), 6.88 (dd, J= 17.7, 10.2 Hz, 1H), 6.14 (d, J= 17.7 Hz, 1H),

5.71 (d, J= 10.2 Hz, 1H), 5.08−4.94 (m, 1H), 4.56 (d, J= 10.5 Hz, 1H),

4.21 (d, J= 10.9 Hz, 1H), 3.57 (s, 2H), 2.97−2.84(m,1H),2.73−2.62 (m,

1H),2.57(s,3H),2.39(brs,8H),2.19(s,3H),2.01(brs,4H).HRMSm/z

(ESI) calcd for C36H39F3N9O2[M + H]+686.3179; found, 686.3181.

1-Ethyl-3-ethynyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

(11). 1-Ethyl-3-((trimethylsilyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-

amine. 3b (5.15 g, 17.8 mmol), CuI (339 mg, 1.78 mmol), and Pd(PPh3)4

(1 g, 0.89 mmol) were suspended in anhydrous DMF (25 mL). The

mixture underwent three cycles of vacuum/ﬁlling with N2.Triethylamine

(5 mL, 35.6 mmol) and trimethylsilylacetylene (2.65 mL, 18.7 mmol)

were then added. The mixture was stirred at 80 °C for 5 h and then

quenched with water. EA and more water were added for extraction. The

combined organic layer was dried with sodium sulfate, ﬁltered, con-

centrated, and the crude product was puriﬁed using silica gel chroma-

tography with a methanol/dichloromethane gradient to aﬀord the

1-ethyl-3-((trimethylsilyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-

amine as a yellow solid.

1-Ethyl-3-((trimethylsilyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-

amine was dissolved in methanol. Then K2CO3(4.9 g, 35.6 mmol) was

added, and the mixture was stirred for 10 min at room temperature. The

mixture was ﬁltered, and the ﬁltrate was concentrated in vacuo and

then partitioned between water and DCM. The organic layer was dried

with sodium sulfate and concentrated in vacuo. The crude product

was puriﬁed using silica gel chromatography with a methanol/

dichloromethane gradient to aﬀord the title compound 11 as a white

solid (1.49 g, 44.7% yield). 1H NMR (400 MHz, DMSO-d6)δ8.20 (s,

1H), 7.82 (br s, 1H), 6.67 (br s, 1H), 4.30 (q, J= 7.2 Hz, 2H), 1.35 (t, J=

7.2 Hz, 3H). MS m/z(ESI): 188.2 [M + H]+.

3-Iodo-4-isopropylbenzoic Acid (12d). Methyl 4-isopropylben-

zoate (16b). A solution of 4-isopropylbenzoic acid 15b (1 g, 6.1 mmol),

and conc H2SO4(cat., 1 mL) in MeOH (10 mL) was heated at 65 °C for

20 h. The reaction mixture was cooled, and the solvent was evaporated.

The residue was dissolved in Et2O, washed with saturated aqueous

NaHCO3(3×), brine, dried over MgSO4, and the solvent was evaporated

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.5b00270

J. Med. Chem. XXXX, XXX, XXX−XXX

to give the 16b (998 mg, 92.0%) as an orange liquid. 1H NMR (400

MHz, DMSO-d6)δ7.89 (d, J= 8.3 Hz, 2H), 7.40 (d, J= 8.3 Hz, 2H),

3.84 (s, 3H), 3.07−2.86 (m, 1H), 1.22 (d, J= 6.9 Hz, 6H). MS m/z

(ESI): 179.1 [M + H]+.

Methyl 3-Iodo-4-isopropylbenzoate (17b). To 8 mL of acetic

acid and 4 mL of acetic anhydride in a 50 mL round-bottom ﬂask were

added NaIO4(599 mg, 2.8 mmol) and powdered I2(507.62 mg,

2 mmol) at 5−10 °C with stirring. Then conc H2SO4(1 mL) was slowly

added dropwise while keeping the temperature at 5−10 °C followed

with methyl 4-isopropylbenzoate (998 mg, 5.6 mmol) at the same

temperature. A brown suspension was stirred for 1 h at room tem-

perature and 4 h at 40 °C (during this time a brown-violet solution

almost decolorized). After cooling, water (30 mL) was added at 0 °C and

product was extracted with CH2Cl2(3×20 mL). Extracts were washed

with 20% NaOH (approximately 3 mL; water layer was strongly alkaline

after extraction), dried over MgSO4, and evaporated to aﬀord a yellow

oil. Crude product was puriﬁed using silica gel chromatography with a

petroleum ether gradient to aﬀord the desired product as a light yellow

solid (1.56 g, 91.5% yield). 1H NMR (400 MHz, DMSO-d6)δ8.34 (d,

J= 1.7 Hz, 1H), 7.94 (dd, J= 8.1, 1.7 Hz, 1H), 7.49 (d, J= 8.1 Hz, 1H),

3.85 (s, 3H), 3.24−3.06 (m, 1H), 1.21 (d, J= 6.8 Hz, 6H). MS m/z

(ESI): 305.0 [M + H]+.

3-Iodo-4-isopropylbenzoic Acid (12d). To a solution of 17b

(1.56 g, 5.1 mmol) in ethanol (20 mL) was added 1 M NaOH (6 mL).

After stirring for 2 h at room temperature a clear pale yellow solution was

obtained. The mixture was concentrated in vacuo and suspended in

water. Then the solution was adjusted to pH 2 by addition of 1 M HCl

and the solvent was stripped oﬀ. The residue was washed with water and

taken up three times in toluene (30 mL) and evaporated to aﬀord 12d as

a beige powder (1.33 g, 89.9% yield). 1H NMR (400 MHz, DMSO-d6)δ

13.10 (s, 1H), 8.32 (d, J= 1.6 Hz, 1H), 7.92 (dd, J= 8.1, 1.6 Hz, 1H),

7.46 (d, J= 8.1 Hz, 1H), 3.22−3.07 (m, 1H), 1.21 (d, J= 6.8 Hz, 6H).

MS m/z(ESI): 289.0 [M −H]−. Compound 12c was prepared by using

a similar procedure; yield 78.1%.

4-Ethyl-3-iodobenzoic Acid (12c). 1H NMR (400 MHz, DMSO-

d6)δ8.31 (s, 1H), 7.89 (d, J= 7.8 Hz, 1H), 7.44 (d, J= 7.8 Hz, 1H), 2.73

(q, J= 7.2 Hz, 2H), 1.16 (t, J=7.2Hz,3H).MSm/z(ESI): 275.0 [M −H]−.

Compounds 13a−fwere prepared in a similar manner to that

described for 10a from 4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)aniline and diﬀerent benzoic acid; yield 67.9−85.0%.

3-Iodo-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (13a). 1H NMR (400 MHz,

DMSO-d6)δ10.57 (s, 1H), 8.32 (s, 1H), 8.18 (s, 1H), 8.04 (d, J= 8.5 Hz,

1H), 7.98 (d, J= 7.8 Hz, 2H), 7.71 (d, J= 8.5 Hz, 1H), 7.36 (t, J= 7.9 Hz,

1H), 3.57 (s, 2H), 2.40 (br s, 8H), 2.17 (s, 3H). MS m/z(ESI): 504.1

[M + H]+.

4-Chloro-3-iodo-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide Hydrochloride Salt (13b).

1H NMR (400 MHz, DMSO-d6)δ10.76 (s, 1H), 8.54 (s, 1H), 8.24 (s,

1H), 8.13 (d, J= 8.3 Hz, 1H), 8.01 (d, J= 8.3 Hz, 1H), 7.89 (br s, 1H),

7.77 (d, J= 8.4 Hz, 1H), 3.90 (br s, 2H), 3.47 (br s, 2H), 3.16 (br s, 4H),

2.78 (s, 3H), 2.71 (br s, 2H). MS m/z(ESI): 538.1 [M + H]+.

4-Ethyl-3-iodo-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (13c). 1H NMR (400 MHz,

CDCl3), δ8.29(s, 1H), 8.16 (s, 1H), 7.88−7.85 (m, 2H), 7.78 (d, J= 8.0

Hz, 1H), 7.74 (d, J= 8.4 Hz, 1H), 7.29 (d, J= 8.0 Hz, 1H), 3.62 (s, 2H),

2.77 (q, J= 7.6 Hz, 2H), 2.51 (br s, 8H), 2.41 (s, 3H), 1.22 (t, J= 7.6 Hz,

3H). MS m/z(ESI): 532.1 [M + H]+.

3-Iodo-4-isopropyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (13d). 1H NMR (400 MHz,

CDCl3)δ8.37 (s, 1H), 8.31 (s, 1H), 7.95 (d, J = 10.1 Hz, 2H), 7.90 (d,

J= 7.6 Hz, 1H), 7.57 (d, J= 6.4 Hz, 1H), 7.36 (d, J= 8.1 Hz, 1H), 3.73 (s,

2H), 3.27−3.23 (m, 1H), 3.04 (br s, 4H), 2.87 (s, 4H), 2.74 (s, 3H), 1.26

(d, J= 6.8 Hz, 6H). MS m/z(ESI): 546.1 [M + H]+.

4-Fluoro-3-iodo-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (13e). 1H NMR (400 MHz,

DMSO-d6)δ10.56 (s, 1H), 8.46 (d, J= 5.7 Hz, 1H), 8.17 (s, 1H),

8.03 (d, J= 8.3 Hz, 2H), 7.71 (d, J= 8.6 Hz, 1H), 7.45 (t, J= 8.2 Hz, 1H),

3.58 (s, 2H), 2.43 (br s, 8H), 2.24 (s, 3H). MS m/z(ESI): 522.1

[M + H]+.

3-Iodo-4-methoxy-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide Hydrochloride Salt (13f).

1H NMR (400 MHz, DMSO-d6)δ10.43 (s, 1H), 9.62 (br s, 1H), 8.43

(d, J= 1.9 Hz, 1H), 8.19 (s, 1H), 8.14−7.97 (m, 2H), 7.69 (d, J= 8.5 Hz,

1H), 7.16 (d, J= 8.7 Hz, 1H), 3.92 (s, 3H), 3.65 (s, 2H), 3.15 (br s, 4H),

2.75 (s, 3H), 2.67 (br s, 4H). MS m/z(ESI): 534.1 [M + H]+.

Compounds 14a−fwere prepared in a similar manner to that

described for 1a from 1-ethyl-3-ethynyl-1H-pyrazolo[3,4-d]pyrimidin-

4-amine 11 and diﬀerent iodobenzamide (13a−f); yield 37.9−51.6%.

3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (14a). 1H NMR (400 MHz,

DMSO-d6)δ10.65 (s, 1H), 8.36 (s, 1H), 8.27 (s, 1H), 8.23 (s, 1H),

8.13−8.01 (m, 2H), 7.97 (s, 1H), 7.73 (d, J= 7.6 Hz, 1H),7.65 (s, 1H),

4.37 (q, J= 7.2 Hz, 2H), 3.58 (s, 2H), 2.43 (br s, 8H), 2.23 (s, 3H), 1.42

(t, J= 7.2 Hz, 3H). HRMS m/z(ESI) calcd for C29H30F3N8O[M+H]

563.2495; found, 563.2499.

3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-chloro-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (14b). 1H NMR (400 MHz,

DMSO-d6)δ10.67 (s, 1H), 8.48 (s, 1H), 8.29 (s, 1H), 8.20 (s, 1H), 8.05

(s, 2H), 7.83 (d, J= 8.2 Hz, 1H), 7.73 (d, J= 8.2 Hz, 1H), 4.39 (d, J= 6.9

Hz, 2H), 3.57 (s, 2H), 2.40 (br s, 8H), 2.19 (s, 3H), 1.42 (t, J= 6.9 Hz,

3H). HRMS m/z(ESI) calcd for C29H29ClF3N8O[M+H]

+597.2105;

found, 597.2107.

3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-ethyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (14c). 1H NMR (400 MHz,

DMSO-d6)δ10.62 (s, 1H), 8.35 (s, 1H), 8.28 (s, 1H), 8.24 (s, 1H), 8.09

(d, J= 8.3 Hz, 1H), 8.00 (d, J= 8.0 Hz, 1H), 7.72 (d, J= 8.3 Hz, 1H),

7.56 (d, J= 8.0 Hz, 1H), 4.38 (q, J= 7.6 Hz, 2H), 3.60 (s, 2H), 2.95 (q,

J= 7.2 Hz, 2H), 2.51 (br s, 8H), 2.34 (s, 3H), 1.42 (t, J= 7.6 Hz, 3H),

1.29 (t, J= 7.2 Hz, 3H). HRMS m/z(ESI) calcd for C31H34F3N8O

[M + H]+591.2808; found, 591.2805.

3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-isopropyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (14d). 1H NMR (400 MHz,

DMSO-d6)δ10.58 (s, 1H), 8.33 (s, 1H), 8.28 (s, 1H), 8.23 (s, 1H), 8.08

(d, J= 8.1 Hz, 1H), 8.02 (d, J= 8.1 Hz, 1H), 7.72 (d, J= 8.4 Hz, 1H),

7.62 (d, J= 8.4 Hz, 1H), 4.39 (q, J= 7.2 Hz, 2H), 3.61 (s, 2H), 3.59−3.50

(m, 1H), 2.39 (s, 8H), 2.33 (s, 3H), 1.42 (t, J= 7.2 Hz, 3H), 1.32 (d, J=

6.9 Hz, 6H). HRMS m/z(ESI) calcd for C32H36F3N8O[M+H]

605.2964; found, 605.2970.

3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-ﬂuoro-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (14e). 1H NMR (400 MHz,

DMSO-d6)δ10.64 (s, 1H), 8.47 (d, J= 8.1 Hz, 1H), 8.28 (s, 1H), 8.20

(s, 1H), 8.15−8.08 (m, 1H), 8.05 (d, J= 8.1 Hz, 1H), 7.73 (d, J= 8.6 Hz,

1H), 7.59 (t, J= 9.0 Hz, 1H), 4.38 (q, J= 7.2 Hz, 2H), 3.57 (s, 2H), 2.39

(br s, 8H), 2.16 (s, 3H), 1.42 (t, J= 7.2 Hz, 3H). HRMS m/z(ESI) calcd

for C29H29F4N8O[M+H]

+581.2400; found, 581.2405.

3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-methoxy-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (14f). 1H NMR (400 MHz,

DMSO-d6)δ10.50 (s, 1H), 8.40 (s, 1H), 8.29 (s, 2H), 8.22 (s, 1H),

8.16−8.02 (m, 2H), 7.71 (d, J= 7.9 Hz, 1H), 7.34 (d, J= 8.8 Hz, 1H),

6.73 (br s, 1H), 4.36 (d, J= 6.7 Hz, 2H), 4.04 (s, 3H), 3.60 (s, 2H), 2.39

(br s, 8H), 2.34 (s, 3H), 1.42 (t, J= 6.7 Hz, 3H). HRMS m/z(ESI) calcd

for C30H32F3N8O2[M + H]+593.2600; found, 593.2598.

3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-

ethynyl)-4-hydroxy-N-(4-((4-methylpiperazin-1-yl)methyl)-3-

(triﬂuoromethyl)phenyl)benzamide (14g). To a stirred suspension

of 3-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-

methoxy-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(triﬂuoromethyl)-

phenyl)benzamide 14f (296 mg, 0.5 mmol) in dry DCM (10 mL)

at −78 °C was added dropwise BBr3(1.0 M solution in DCM, 6 mL,

6 mmol) under an N2atmosphere. Then the reaction mixture was stirred

at this temperature for 20 min and moved to rt for another 8 h. The

reaction was then quenched by the dropwise addition of water. The

reaction mixture was poured into ammonia solution (30 mL, pH 8), and

more DCM was added for extraction. The combined organic extracts

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.5b00270

J. Med. Chem. XXXX, XXX, XXX−XXX

were dried (Na2SO4) and the solvent was removed under reduced

pressure to give a solid which was puriﬁed by ﬂash chromatography to

aﬀord the title compound as a yellow solid (173.5 mg, 60.1% yield). 1H

NMR (400 MHz, DMSO-d6)δ10.68 (s, 1H), 8.41 (s, 1H), 8.31 (s, 1H),

8.28 (s, 1H), 8.14 (d, J= 8.6 Hz, 1H), 8.04 (d, J= 8.7 Hz, 1H), 7.98 (d,

J= 8.7 Hz, 1H), 7.73 (d, J= 8.6 Hz, 1H), 7.58 (s, 1H), 4.44 (q, J= 7.2 Hz,

2H), 3.67 (s, 2H), 3.11 (br s, 4H), 2.70 (s, 3H), 2.65 (br s, 4H), 1.46 (t,

J= 7.2 Hz, 3H). HRMS m/z(ESI) calcd for C29H30F3N8O2[M + H]+

579.2444; found, 579.2446.

■ASSOCIATED CONTENT

SSupporting Information

Chemical structures of top 100 compounds, a full description of

the scaﬀold hopping and molecular docking, and a csv ﬁle of

molecular formula strings. This material is available free of charge

via the Internet at http://pubs.acs.org.

■AUTHOR INFORMATION

Corresponding Author

*Phone: +86-28-85164063. Fax: +86-28-85164060. E-mail:

yangsy@scu.edu.cn.

Author Contributions

C.-H.Z., M.-W.Z., and Y.-P.L. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

■ACKNOWLEDGMENTS

This work was supported by the 973 Program (Grant

2013CB967204), the National Natural Science Foundation

(Grant 81325021, 81172987, and 81473140), and the Program

for Changjiang Scholars and Innovative Research Team in

University (Grant IRT13031).

■ABBREVIATIONS USED

TNBC, triple-negative breast cancer; ER, estrogen receptor; PR,

progesterone receptor; HER2, human epidermal growth factor

receptor 2; Src, sarcoma; B-RAF, serine/threonine protein kinase

B-RAF; C-RAF, RAF proto-oncogene serine/threonine protein

kinase; FAK, focal adhesion kinase; ERK, extracellular regulated

protein kinases; AKT, protein kinase B; MAPK, mitogen-

activated protein kinase; SFK, Src family kinase; SAR, structure−

activity relationship; R&D, research and development; i-Pr,

isopropyl; NIS, 1-iodopyrrolidine-2,5-dione; DIBAL, diisobuty-

laluminum hydride; EA, ethyl acetate

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May 2023
J BIOMOL STRUCT DYN

The pyrimidine and fused pyrimidine ring systems play vital roles to inhibit the c-Src kinase. The Src kinase is made of different domains but the kinase domain is responsible for inhibition of Src kinase. In which the kinase domain is the main domain that is made of several amino acids. The Src kinase is inhibited by its inhibitors when it is activated by phosphorylation. Although dysregulation of Src kinase caused cancer in the late nineteenth century, medicinal chemists have not explored it extensively; therefore it is still regarded as a cult pathway. There are numerous FDA-approved drugs on the market, yet novel anticancer drugs are still in demand. Existing medications have adverse effects and drug resistance owing to rapid protein mutation. In this review, we discussed the activation process of Src kinase, chemistry of pyrimidine ring and its different synthetic routes, as well as the recent development in c-Src kinase inhibitors containing pyrimidine and their biological activity, SAR, and selectivity. The c-Src binding pocket has been predicted in detail to discover the vital amino acids which will interact with inhibitors. The potent derivatives were docked to discover the binding pattern. The derivative 2 established three hydrogen bonds with the amino acid residues Thr341 and Gln278 and had the greatest binding energy of -13.0 kcal/mol. The top docked molecules were further studied for ADMET studies. The derivative 1, 2, and 43 did not show any violation of Lipinski's rule. All derivatives used for the prediction of toxicity showed toxicity.

4-Aminopyrazolopyrimidine scaffold and its deformation in the design of tyrosine and serine/threonine kinase inhibitors in medicinal chemistry

Article

Jul 2022

The 4-Aminopyrazolopyrimidine scaffold has been an interesting pharmacophore since the disclosure of the intimate connection between small-molecule inhibitors and the treatment of diseases. Modification of the 4-aminopyrazolopyrimidine scaffold according to different targets, especially tyrosine kinase and serine/threonine kinase, has resulted in a variety of small-molecule inhibitors. Kinase inhibitors with 4-aminopyrazolopyrimidine derivatives as scaffolds have been widely applied in the treatment of diseases. In this article, we summarized the reports on 4-aminopyrazolopyrimidine as well as its deformation and the application of its derivatives in designing small-molecule inhibitors and the treatment of diseases.

Potential Synthetic Lethality for Breast Cancer: A Selective Sirtuin 2 Inhibitor Combined with a Multiple Kinase Inhibitor Sorafenib

Article

Dec 2021
PHARMACOL RES

Sorafenib is a clinically useful multiple kinase inhibitor for the treatment of kidney cancer, liver cancer and acute myelocytic leukemia, while it has shown weak efficacy in suppressing breast cancer. Since sirtuin2 (SIRT2) is an important epigenetic regulator and associated with several cancer types including breast cancer, development and evaluation of new SIRT2 inhibitors to probe their therapeutic potentials is currently desirable. A highly selective SIRT2 inhibitor named I was previously developed by us, which showed activity to inhibit non-small cell lung cancer cell lines in vitro. We herein report expanded screening of I and its structurally similar inactive compound II against other cancer cell lines, and found that I had a wide spectrum of anticancer activity while II had no such effects. The I-sorafenib combination treatment exerted obvious synergistic reduction on cell viability of MCF-7 cells. We observed that the combination treatment could suppress cell proliferation, survival and migration, arrest cell cycle at G0/G1 phase, and induce apoptosis in MCF-7 cells, when compared with the single treatment. In vivo studies revealed that the combination treatment showed stronger tumor growth inhibition (87%), comparing with I-(42.8%) or sorafenib-solely-treated groups (61.1%) in MCF-7 xenograft model. In conclusion, this work clearly revealed a potential synthetic lethality effect for I combined with sorafenib, and will probably offer a new strategy at least for breast cancer treatment.

Combined SFK/MEK inhibition prevents metastatic outgrowth of dormant tumor cells

Article

Full-text available

Dec 2013
J CLIN INVEST

Breast cancer (BC) can recur as metastatic disease many years after primary tumor removal, suggesting that disseminated tumor cells survive for extended periods in a dormant state that is refractory to conventional therapies. We have previously shown that altering the tumor microenvironment through fibrosis with collagen and fibronectin deposition can trigger tumor cells to switch from a dormant to a proliferative state. Here, we used an in vivo preclinical model and a 3D in vitro model of dormancy to evaluate the role of Src family kinase (SFK) in regulating this dormant-to-proliferative switch. We found that pharmacological inhibition of SFK signaling or Src knockdown results in the nuclear localization of cyclin-dependent kinase inhibitor p27 and prevents the proliferative outbreak of dormant BC cells and metastatic lesion formation; however, SFK inhibition did not kill dormant cells. Dormant cell proliferation also required ERK1/2 activation. Combination treatment of cells undergoing the dormant-to-proliferative switch with the Src inhibitor (AZD0530) and MEK1/2 inhibitor (AZD6244) induced apoptosis in a large fraction of the dormant cells and delayed metastatic outgrowth, neither of which was observed with either inhibitor alone. Thus, targeting Src prevents the proliferative response of dormant cells to external stimuli, but requires MEK1/2 inhibition to suppress their survival. These data indicate that treatments targeting Src in combination with MEK1/2 may prevent BC recurrence.

Zhang S, Huang WC, Zhang L, Cancer Brain Metastases Src Family Kinases as Novel Therapeutic Targets to Treat Breast. Cancer Res. 73:5764-5774

Article

Full-text available

Aug 2013
CANCER RES

Despite better control of early stage disease and improved overall survival of patients with breast cancer, the incidence of life-threatening brain metastases continues to increase in some of these patients. Unfortunately, other than palliative treatments there is no effective therapy for this condition. In this study, we reveal a critical role for Src activation in promoting brain metastasis in a preclinical model of breast cancer, and we show how a Src-targeting combinatorial regimen can treat HER2+ brain metastases in this model. We found that Src was hyperactivated in brain-seeking breast cancer cells derived from human cell lines or from patients' brain metastases. Mechanistically, Src activation promoted tumor cell extravasation into the brain parenchyma via permeabilization of the blood-brain barrier. When combined with the EGFR/HER2 dual-targeting drug lapatinib, a Src-targeting combinatorial regimen prevented outgrowth of disseminated breast cancer cells through the induction of cell cycle arrest. More importantly, this combinatorial regimen inhibited the outgrowth of established experimental brain metastases, prolonging the survival of metastases-bearing mice. Our results provide a rationale for clinical evaluation of Src-targeting regimens to treat breast cancer patients suffering from brain metastasis.

Genome and Transcriptome Sequencing in Prospective Metastatic Triple-Negative Breast Cancer Uncovers Therapeutic Vulnerabilities

Article

Full-text available

Nov 2012
MOL CANCER THER

Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective metastatic mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors; particularly those that might inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene, when tumor gene expression was compared to nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer to all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/MTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer.

TCPTP regulates SFK and STAT3 signaling and is lost in triple-Negative breast cancers

Article

Full-text available

Nov 2012
MOL CELL BIOL

Tyrosine phosphorylation-dependent signaling, as mediated by members of the epidermal growth factor receptor (EGFR) family (ErbB1 to -4) of protein tyrosine kinases (PTKs), Src family PTKs (SFKs), and cytokines such as interleukin-6 (IL-6) that signal via signal transducer and activator of transcription 3 (STAT3), is critical to the development and progression of many human breast cancers. EGFR, SFKs, and STAT3 can serve as substrates for the protein tyrosine phosphatase TCPTP (PTPN2). Here we report that TCPTP protein levels are decreased in a subset of breast cancer cell lines in vitro and that TCPTP protein is absent in a large proportion of “triple-negative” primary human breast cancers. Homozygous TCPTP deficiency in murine mammary fat pads in vivo is associated with elevated SFK and STAT3 signaling, whereas TCPTP deficiency in human breast cancer cell lines enhances SFK and STAT3 signaling. On the other hand, TCPTP reconstitution in human breast cancer cell lines severely impaired cell proliferation and suppressed anchorage-independent growth in vitro and xenograft growth in vivo. These studies establish TCPTP's potential to serve as a tumor suppressor in human breast cancer.

TRIPLE-NEGATIVE BREAST CANCER: A SEARCH FOR NEW THERAPEUTIC TARGETS

Article

Sep 2010

Rationale for Targeting the Ras/MAPK Pathway in Triple-Negative Breast Cancer

Article

May 2014

"Triple negative" breast cancer (TNBC) is the most aggressive and least common clinical subtype of breast cancer. As its nomenclature implies, TNBC lacks specific biomarker expression marking response to an effective targeted therapy. The incidence of TNBC is higher in young minority women who suffer from high rates of early recurrence and death from their disease. Mounting preclinical evidence supports targeting the Ras/MAPK cell signaling pathway in the TNBC subtype, despite large genomic surveys such as The Cancer Genome Atlas demonstrating infrequent canonical mutations in this pathway. Due to the early spread of TNBC, targeted treatment in the neoadjuvant setting may offer the effective therapeutic punch needed to eliminate micro-metastatic disease and reduce mortality. Herein, we will review the evidence supporting clinical trials of targeted inhibitors of the Ras/MAPK pathway in TNBC, and discuss the obstacles and opportunities of this approach.

New Strategies for Triple-Negative Breast Cancer-Deciphering the Heterogeneity

Article

Feb 2014

Triple-negative breast cancer (TNBC) is a heterogeneous disease; gene expression analyses recently identified six distinct TNBC subtypes, each displaying a unique biology. Exploring novel approaches to treatment of these subtypes is critical because less than 30% of women with metastatic breast cancer survive five years and virtually all women with metastatic TNBC will ultimately die of their disease despite systemic therapy. To date, not a single targeted therapy has been approved for the treatment of TNBC and cytotoxic chemotherapy remains the standard treatment. We discuss the current and upcoming therapeutic strategies being explored in an attempt to "target" TNBC. Clin Cancer Res; 20(4); 782-90. ©2014 AACR.

Tackling the Diversity of Triple-Negative Breast Cancer

Article

Dec 2013

Triple-negative breast cancer (TNBC) comprises a highly diverse collection of cancers. Here, we review this diversity both in terms of gene expression subtypes and the repertoire of genetic events. Transcriptomic analyses of TNBC have revealed at least six subtypes, with the luminal androgen receptor (luminal AR) or molecular apocrine cancers forming a distinct group within triple-negative disease. Distinct from the gene expression subtypes, a diverse set of genetic events have been described in TNBC, with a number of potentially targetable genetic events found although all at relatively low frequency. Clinical trials to define the clinical utility of therapies targeting these low-frequency events will require substantial screening efforts to identify sufficient patients. Set against the diversity of TNBC, clinical studies of patients with triple-negative disease will need to be either focused on molecularly defined subsets with upfront molecular stratification, or powered for a secondary endpoint analysis of a molecularly defined subset. Such approaches will be crucial to realize the potential of precision medicine for patients with TNBCs. Clin Cancer Res; 19(23); 6380-8. ©2013 AACR.

Identification of GZD824 as an Orally Bioavailable Inhibitor That Targets Phosphorylated and Nonphosphorylated Breakpoint Cluster Region-Abelson (Bcr-Abl) Kinase and Overcomes Clinically Acquired Mutation-Induced Resistance against Imatinib

Article

Jan 2013
J MED CHEM

Various imatinib resistance-related mutations have been identified in different functional domains of Bcr-Abl kinase, among which the gatekeeper T315I point mutation poses a major challenge in the clinical management of chronic myelogenous leukemia (CML). Novel inhibitors are urgently needed to overcome drug resistance related to Bcr-Abl T315I and other mutations. Herein, we report GZD824 (10a) as a novel orally bioavailable inhibitor that targets a broad spectrum of Bcr-Abl mutants, including the gatekeeper T315I and p-loop mutations, which are associated with poor prognosis in CML. Compound 10a tightly bound phosphorylated and non-phosphorylated Bcr-Abl and inhibited their kinase activity with IC50 values in the low nM range. Compound 10a also strongly suppressed the proliferation of Bcr-Abl-positive CML cells and murine Ba/F3 cells ectopically expressing Bcr-Abl WT or resistance-related mutants with low-nM IC50 values. Compound 10a displayed good oral bioavailability (48.7%), a reasonable half-life (10.6 hrs) and promising in vivo anti-tumor efficacy. This compound induced the regression of tumors driven by wild-type Bcr-Abl or resistance-related mutants, such as T315I, G250E, Q252H, E255K and F317L, in mouse xenograft tumor models. The survival of mice bearing an allograft leukemia model that utilizes Ba/F3 cells harboring Bcr-Abl T315I was significantly improved by administration of 10a. Thus, compound 10a represents a promising lead candidate for the further development of Bcr-Abl inhibitors to overcome clinically acquired resistance against imatinib.

Selective Targeting of Distinct Active Site Nucleophiles by Irreversible Src-Family Kinase Inhibitors

Article

Nov 2012
J AM CHEM SOC

Src-family tyrosine kinases play pivotal roles in human physiology and disease, and several drugs that target members of this family are in clinical use. None of these drugs appear to discriminate among closely related kinases. However, assessing their selectivity toward endogenous kinases in living cells remains a significant challenge. Here, we report the design of two Src-directed chemical probes, each consisting of a nucleoside scaffold with a 5'-electrophile. A 5'-fluorosulfonylbenzoate (1) reacts with the conserved catalytic lysine (Lys295) and shows little discrimination among related kinases. By contrast, a 5'-vinylsulfonate (2) reacts with a poorly conserved, proximal cysteine (Cys277) found in three Src-family and six unrelated kinases. Both 1 and 2 bear an alkyne tag and efficiently label their respective endogenous kinase targets in intact cells. Using 1 as a competitive probe, we determined the extent to which ponatinib, a clinical Bcr-Abl inhibitor, targets Src-family kinases. Remarkably, while ponatinib had little effect on en-dogenous Fyn or Src, it potently blocked the critical T-cell kinase, Lck. Probes 1 and 2 thus enable competitive profiling vs. distinct kinase subsets in living cells.

Design, Synthesis, and Structure–Activity Relationship Studies of 3-(Phenylethynyl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine Derivatives as a New Class of Src Inhibitors with Potent Activities in Models of Triple Negative Breast Cancer

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