Discovery of a series of 2,5-diaminopyrimidine covalent irreversible inhibitors of Bruton's tyrosine kinase with in vivo antitumor activity

Article

pubs.acs.org/jmc

Discovery of a Series of 2,5-Diaminopyrimidine Covalent Irreversible

Inhibitors of Bruton’s Tyrosine Kinase with in Vivo Antitumor Activity

Xitao Li,^†Yingying Zuo,^†Guanghui Tang,^†Yan Wang,^†Yiqing Zhou,^†Xueying Wang,^†

Tianlin Guo,^†Mengying Xia,^†Ning Ding,^‡and Zhengying Pan*^,†

^†Key Laboratory of Chemical Genomics, Key Laboratory of Structural Biology, School of Chemical Biology and Biotechnology,

Shenzhen Graduate School, Peking University, Shenzhen, 518055, China

^‡Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Lymphoma,

Peking University Cancer Hospital and Institute, No. 52 Fucheng Road, Haidian District, Beijing, 100142, China

S

* Supporting Information

ABSTRACT: Bruton’s tyrosine kinase (Btk) is an attractive

drug target for treating several B-cell lineage cancers. Ibrutinib

is a ﬁrst-in-class covalent irreversible Btk inhibitor and has

demonstrated impressive eﬀects in multiple clinical trials.

Herein, we present a series of novel 2,5-diaminopyrimidine

covalent irreversible inhibitors of Btk. Compared with ibrutinib,

these inhibitors exhibited a diﬀerent selectivity proﬁle for the

analyzed kinases as well as a dual-action mode of inhibition of

both Btk activation and catalytic activity, which counteracts a

negative regulation loop for Btk. Two compounds from this

series, 31 and 38, showed potent antiproliferative activities

toward multiple B-cell lymphoma cell lines, including germinal

center B-cell-like diﬀuse large B cell lymphoma (GCB-DLBCL)

cells. In addition, compound 31 signiﬁcantly prevented tumor growth in a mouse xenograft model.

such as a long drug−target residence time and the decoupling of

pharmacokinetic and pharmacodynamic properties.¹⁸Btk inhib-

itors have been shown to be highly eﬃcacious in animal models of

rheumatoid arthritis, lupus, and lymphoma;^19,20multiple clinical

trials of ibrutinib and 3 have also shown encouraging results in

patients.^21,22Therefore, developing Btk inhibitors is a promising

strategy to treat overactive B-cell diseases such as autoimmune

disorders and lymphoma.

Targeted covalent drugs have recently garnered signiﬁcant

interest, particularly for kinase inhibitors.²³⁻²⁵Since the

pioneering work on EGFR/HER2 inhibitors,²⁶⁻²⁸in addition

to Btk, selective covalent inhibitors have been developed for

Bmx,²⁹FGFR,³⁰GSK3β,³¹Itk,³²JNK,³³Kit/PDGFR,³⁴Nek2,³⁵

PI3kα,³⁶RSK,³⁷and Src family kinases.^38,39This ﬁeld has also

been discussed in several excellent reviews.^40,41Covalent bond

formation between the inhibitors and nonconserved cysteine

residues proximal to the ATP binding pockets of the kinase

catalytic domain enhances the potency and selectivity of this type

of inhibitor. We use a two-step procedure to develop such

inhibitors. In the ﬁrst step, we optimize the template without

covalent reactive groups. In the second step, structural information

is used to guide the placement of a reactive group at a suitable

position on the parent template to enable an interaction with

the sulfhydryl group of the targeted cysteine. After the ﬁrst step,

INTRODUCTION

■

The B-cell receptor (BCR) signaling pathway plays an

important role in B-cell development and diﬀerentiation.^1,2

Bruton’s tyrosine kinase (Btk) is a crucial component of the

BCR pathway and is expressed only in hematopoietic cells except

natural killer or T cells.³Patients with X-linked agammaglobu-

linemia, which is a rare genetic disease resulting from mutations

in Btk, exhibit a reduced number of mature B-cells.^4,5Btk has

also been linked to chronic BCR activation in activated B-cell-like

diﬀuse large B cell lymphoma (ABC-DLBCL), which is a critical

factor in the promotion of lymphoma cell survival.^6,7

Since Btk was discovered in the 1990s, multiple Btk inhibitors

have been developed.^8,9These inhibitors can be divided into two

major classes, reversible and covalent irreversible ones (Figure 1).

LFM-A13,¹⁰dasatinib,¹¹1 (CGI-1746),¹²2 (GDC-0834),¹³and

RN-486^9,14are reversible Btk inhibitors. These inhibitors have

shown potent eﬀects for immune disorders in multiple animal

models, and 2 has been advanced into a phase I clinical trial.

Ibrutinib¹⁵from Celera/Pharmacyclics/Janssen and 3 (CC-292)¹⁶

from Celgene and the imidazoquinoxaline compounds¹⁷from

Pﬁzer are the primary examples of covalent irreversible Btk

inhibitors. Ibrutinib and 3 are currently in multiple clinical trials,

and ibrutinib was just approved to treat patients with mantle cell

lymphoma in November 2013. Both compounds interact with a

cysteine residue (Cys481) located at the rim of the ATP-binding

pocket in Btk. These types of irreversible Btk inhibitors have

unique characteristics compared with traditional reversible ones,

Received: November 16, 2013

A

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Figure 1. Examples of Btk inhibitors.

we select compounds reaching low nanomolar potency, which

often suggest that the template substantially interacts with the

target kinase, thus facilitating the selection of reactive groups in the

second step. In the second step, a mild reactive group is typically

preferred to limit the oﬀ-target eﬀects that would result from

indiscriminate reactions with other cellular nucleophiles.

phosphorylated and also inhibit Btk’s own catalytic ability.

Developing such a dual-action mode Btk inhibitor that inhibits

both Btk activation and catalysis could be a concise and eﬀective

strategy to further down-regulate BCR and other signal

transduction pathways in which Btk plays an important role and

to overcome potential resistance mechanisms due to increased Btk

activation.

In cells, Btk is ﬁrst activated by its upstream kinases through

phosphorylation of a key tyrosine residue (Tyr551), which

increases the catalytic activity of Btk by 10-fold.⁴²Following

autophosphorylation of another tyrosine residue (Tyr223), Btk

becomes fully activated and phosphorylates its substrates, such

as PLC-γ2 in the BCR pathway.⁴³Btk is an eﬃcient enzyme;

only a small fraction of existing Btk is activated, and approximately

30 min of Btk activity is required for full signal transduction

along the BCR pathway.⁴⁴In vivo studies also indicate that

more than 60% of Btk activity must be inhibited to achieve

observable eﬃcacy in animal models.⁴⁵Thus, an irreversible

inhibitor with a long drug−target residence time would be

preferable for sustained Btk inhibition. In addition, Btk is also

inﬂuenced by a negative feedback loop in the BCR pathway. The

kinase PKCβ is downstream of Btk in the BCR pathway, and it has

been suggested that PKCβ directly and negatively regulates Btk

activity by impeding Btk’s engagement with the signaling

complex.⁴⁶Treating Namalwa cells with ibrutinib increases

Tyr551 phosphorylation, the ﬁrst step in Btk activation, suggesting

the existence of Btk-mediated negative feedback loop.⁴⁷It has

been suggested that enhanced kinase activation is a potential

resistance mechanism that cells adopt to overcome the eﬀects of

inhibitors.⁴⁸⁻⁵⁰Multiple p38 kinase inhibitors have shown only

transient activity in clinical trials.⁵¹However, a dual-action p38

kinase inhibitor that prevents both the activation and kinase

activity of p38 in cells, 4-(5-(cyclopropylcarbamoyl)-2-methyl-

phenylamino)-5-methyl-N-propylpyrrolo[1,2-f ][1,2,4]triazine-6-

carboxamide (BMS-582949), has shown clinical eﬃcacy in a

phase IIa rheumatoid arthritis trial without tachyphylaxis.^52,53

Inspired by this observation, we sought scaﬀolds with a dual-

action mode that could prevent Btk Tyr551 from being

Herein, we present the ﬁrst series of dual-action mode Btk

covalent irreversible inhibitors based on a type II scaﬀold. These

compounds are potent Btk inhibitors with high selectivity and

have demonstrated signiﬁcant antiproliferation activities in a

mouse xenograft model of human B-cell lymphoma (DoHH2).

STRUCTURE-BASED DESIGN OF COVALENT BTK

INHIBITORS

■

Kinase inhibitors can be categorized based on the kinase

conformation that they recognize.⁵⁴Type I kinase inhibitors

(e.g., erlotinib and dasatinib) bind their target kinases in the

active conformation, whereas type II inhibitors (e.g., imatinib

and sorafenib) bind kinases in the inactive conformation.

Because one of our goals is to prevent Btk activation, type I

inhibitors that bind kinases in the active conformation may

not be suitable; thus, we focused our eﬀorts on type II

inhibitors. Btk shares high sequence similarity with the Src

family kinase Lck. We identiﬁed only one single amino acid

diﬀerence between Btk (Cys481) and Lck (Ser323) within the

direct contact range for analogues of ibrutinib.¹⁵In addition,

the literature has suggested the existence of cross-reactivity for

many Btk and Lck inhibitors.^11,15,55Thus, we hypothesized that

known type II Lck inhibitors would provide useful insights

regarding the design of novel Btk inhibitors; therefore, we used

the 2,5-diaminopyrimidine compounds developed by scientists

at Amgen as a starting point.⁵⁶

A Btk crystal structure that adopts a DFG-out conformation

(PDB code 3PJ3)⁵⁷was used as a template for our design. The

2,5-diaminopyrimidine compound 4 was easily docked into this

structure without obvious steric conﬂicts. As indicated in the

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Figure 2. (a) Structure of compound 4 and the “head”, “center”, and “tail” regions in the structure−activity relationship (SAR) study. (b)

Compound 4 (carbon atoms in yellow) was docked in a structure of Btk (PDB code 3PJ3). The red dots indicate potential hydrogen bonds between

compound 4 and Btk. Cys481 (carbon atoms in cyan) is highlighted.

a

model, compound 4 could form several important hydrogen

bonds with Btk, including with Met477 at the hinge region and

the gatekeeper residue Thr474, as well as hydrogen bonds with

Glu445 and Ser538. Compared to the conformation of its

parent compound in Lck (PDB code 3BYU), compound 4

adopts an almost identical conformation and forms similar

interactions with the protein. This model suggests that 2,5-

diaminopyrimidine compounds have potential as inhibitors

against Btk, but the selectivity between Btk and Lck might be

diﬃcult to achieve for this scaﬀold. However, because ibrutinib

has demonstrated remarkable eﬃcacy and safety in clinical

studies even though it is a potent inhibitor of Lck, Lyn, and

other Src family kinases, the selectivity between Btk and Lck

was not a critical factor in our program.

Table 1. Head Region SAR

RESULTS AND DISCUSSION

■

Structure−Activity Relationship. On the basis of our

docking model, we divided the 2,5-diaminopyrimidine scaﬀold

into head, center, and tail regions (Figure 2), which were then

explored sequentially.

The head area is proximal to the hinge region in Btk, and the

amino acid composition of this region is very similar between

Btk and Lck; thus, the IC₅₀values for the ﬁrst set of compounds

were surprising (Table 1). Compound 4 yielded an IC₅₀of 149 nM

against Lck but only 3.42 μM for Btk, a 23-fold diﬀerence.

Attaching an ethyl or cyclopropyl group at the R position (5 and 6)

did not substantially improve the potency. A phenyl group (7) at

this position improved the IC₅₀values for Btk and Lck to 133 and

7 nM, respectively. Our model suggested that Btk’s Tyr476 would

form π−π interactions with the newly added phenyl group in

inhibitor 7. Thus, we hypothesized that tuning the electron density

of the phenyl group might impact inhibitor potency. Decorating

the phenyl group with an m-CF₃group yielded a less potent

inhibitor (8). Substitution with the electron-donating group MeO

(9) improved potency for Btk. However, substitution with

electron-withdrawing F (10) yielded an inhibitor with a potency

equal to that of 9. Fortunately, substituting an amino group at

either the meta or para positions of the phenyl ring produced

inhibitors with comparable nanomolar potencies against both Btk

and Lck. In the absence of crystal structures for the inhibitor−

kinase complexes, it is diﬃcult to identify the exact basis for the

a

IC₅₀values are reported as means of duplicates. ND, not determined.

10-fold improvement (compounds 7−12) in potency toward Btk

but the largely ﬂat SAR for Lck. This diﬀerence may be not only

due to the identities of the amino acid residues that directly

contact the inhibitors but also due to the overall plasticity of the

C

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kinases in this region. Since the selectivity between Btk and Lck

was not considered critical for our program, we did not explore

this aspect of the inhibitors further.

by the movement of the phenyl group of the phenylalanine

residue in the “DFG” sequence. As expected, an aromatic group

was required at the R position (Table 3) because compounds

The docking structure of compound 7 in the Btk cocrystal

structure with a DFG-out conformation (PDB code 3PJ3)

indicated that Cys481 is approximately 6−7 Å away from the

phenyl ring at the head region of the inhibitors (Supporting

Information Figure 1). We anticipated that the addition of a

linker group extending from the meta position of this phenyl

ring would yield substituents that could directly interact with

the Cys481 sulfhydryl group. Acylating the m-amino group with

simple acids (13 and 14) largely maintained the potency even

though the bis-acylated compound 15 exhibited signiﬁcantly

reduced potency. These results indicate that it is possible to

accommodate a substitution at the meta position.

a

Table 3. Tail Region SAR

compd

R

Btk IC₅₀(nM)

Next, we probed the SAR at the central region (Table 2).

The angular methyl group at the R position might ﬁt snugly

12

21

22

23

24

25

26

27

28

29

30

3-CF₃-phenyl

Et

14

8563

4946

20

a

cyclopropyl

3-Cl-phenyl

3-NMe₂-phenyl

3-F-phenyl

Table 2. Center Region SAR

47

166

18

3-Cl-5-CF₃-phenyl

4-Cl-3-CF₃-phenyl

4-CF₃-phenyl

styryl

9

25

24

2-naphthyl

39

a

IC₅₀values are reported as means of duplicates.

with either ethyl (21) or cyclopropyl (22) groups exhibited

signiﬁcantly reduced potency, likely due to a signiﬁcant decrease

in hydrophobic interactions with the protein. However, various

aromatic ring substitutions were generally tolerated. By use of the

m-CF₃-substituted compound 12 as a benchmark, a chlorine

substitution yielded an inhibitor (23) with comparable potency, a

dimethylamino group (24) increased the IC₅₀value by approxi-

mately 3-fold, and a ﬂuorine atom (25) reduced the potency by

more than 10-fold. A secondary chlorine substitution either meta

or para to the amide did not signiﬁcantly change the IC₅₀values

(26 and 27). Moving the CF₃group to the para position (28)

was well-tolerated, and compound 29, which contained a styryl

group, also had comparable potency. Introducing a naphthyl

group at the R position (30) maintained the IC₅₀at 39 nM.

Collectively, these data suggest that Btk has a relatively large and

plastic pocket in the tail region.

The above SAR studies (Tables 1−3) indicated that

compound 12 was highly potent against Btk, and our model

indicated that the meta position (R¹) is the preferred site for

anchoring reactive groups (Table 4). Thus, 12 was used as the

template to probe the interaction between the inhibitors and

Cys481. A panel of potential covalent reactive groups was

attached to the free amino group at the R¹position. Because the

apparent potency of irreversible inhibitors varies with the

length of the preincubation time between the enzyme and

inhibitors, the preincubation time in our kinase activity assays

was kept constant at 10 min. Compound 31, which includes

the classic acrylamide group, yielded an IC₅₀of 5 nM for Btk.

Adding a methyl group to the terminal carbon of the acrylamide

(32) reduced inhibitor potency, which was partially recovered

through further substitution with a dimethylamino group at the

allylic position (33), consistent with the trend observed for other

covalent inhibitors of Btk and EGFR.^15,58The methyl group

reduced the electrophilicity and increased the steric hindrance of

a

IC₅₀values are reported as mean values of duplicates.

into in a small hydrophobic pocket formed with the gatekeeper

residue Thr474. Replacing this methyl group with a common bio-

isostere (Cl) yielded the equally potent compound 16. Decreasing

the sizes of substitution to F (17) and H (18) decreased the

potency by 3- and 16-fold, respectively. The amide at the L

position was expected to form hydrogen bonds with Glu445 and

Ser538, which may be important for type II inhibitors. Reversing

the amide group orientation (19) reduced the potency 3-fold for

Btk, while a urea linkage (20) reduced the potency 5-fold; thus, an

amide linkage was selected in the next SAR studies.

For type II inhibitors, we anticipated that tail region

substitutions would occupy the hydrophobic pocket vacated

D

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a

On the basis of their exceptional potency toward Btk,

compounds 31 and 38 were selected for further biological

evaluations.

Table 4. Reactive Group SAR

Compounds 31 and 38 Are Selective Covalent

Inhibitors of Btk. The selectivity of compound 31 was ﬁrst

proﬁled against a panel of kinases that included representatives

of major classes of kinases and those kinases that directly

participate in the BCR pathway (Figure 3). At 0.5 μM, compound

Figure 3. Selectivity of compound 31 against 18 kinases. The values

are the mean of duplicates.

31 was inactive against the majority of the kinases tested and

weakly active against Jak2. Compound 31 was moderately active

against CSK but potently inhibited ABL, HER4, Lck, p38α, and

VEGFR. Type II inhibitors for ABL, p38α, and VEGFR kinases

have been well-documented in the literature.⁵⁹⁻⁶³Notably, the

kinase selectivity of compound 31 was similar to that of ibrutinib

for kinases that are directly involved in the BCR pathway, i.e.,

potent activity against Btk and Lck, which are representative Tec

and Src family kinases, with no activity against Syk or PKC.

Reversible and irreversible inhibitors have diﬀerent require-

ments for sustained inhibitory eﬀects in cells.⁶⁴Reversible

inhibitors are in equilibrium with the biological target, whereas

irreversible inhibitors bind continuously to the targeted protein

until the protein is degraded through biological processes.

Thus, reversible inhibitors must be maintained at a high enough

concentration to suppress the target activity. However, irreversible

inhibitors require only a high-concentration pulse to occupy the

target binding sites and exert a sustained eﬀect. In fact, irreversi-

ble inhibitors with short half-lives may be preferred to mitigate

potential oﬀ-target eﬀects. Thus, for covalent irreversible inhibitors,

special attention should be paid to kinases that share a cysteine

residue at the structurally identical position. Btk is among a group

of 11 kinases out of more than 500 in the human kinome with a

cysteine at the identical position (Cys481 in Btk). These kinases

include Blk, Jak3, MAP2K7, Tec family (Bmx, Btk, Itk, Rlk, and

Tec), and EGFR family (EGFR, HER2, and HER4) kinases. We

measured the inhibitory eﬀects of compounds 31 and 38 against

10 of these kinases (Table 5). They are quite potent against Blk,

Bmx, Btk, Rlk, Tec, EGFR, HER2, and HER4. Notably, both

compounds had weak activity against Jak3 in the micromolar range

and no activity against Itk up to 30 μM. However, they were

almost as potent as ibrutinib (5 nM) against EGFR. It is unclear

whether this aﬃnity for EGFR would produce major side eﬀects in

clinical studies. Also, caution should be taken when interpreting

the selectivity of covalent inhibitors solely based on IC₅₀values, as

a recent study indicated that both reversible binding aﬃnity and

a

IC₅₀values are reported as mean values of duplicates.

the Michael acceptor, which reduced the potency of compound

32 by 7-fold, while the dimethylamino group in compound 33

was proposed to form a hydrogen bond with the cysteine residue

thiol group, thus enhancing its nucleophilicity.⁵⁸The vinyl

sulfonamide 34 and two additional compounds with good

leaving groups (35 and 36) were also potent, but the potency of

the bis-acylated compound (37) was reduced by more than

40-fold compared with compound 31. Interestingly, compound

38, which includes a glycyl spacer, exhibited excellent potency

that was below the detection limit of our Btk enzyme assay. With

acrylamide as the reactive group, variations at the R²position

yielded compounds 39−41, which were all potent inhibitors

of Btk.

One concern regarding covalent kinase inhibitors is that they

may react nonselectively with thiols other than the intended

target in a physiological environment. We used glutathione

(GSH), which is a tripeptide that is naturally abundant in cells,

as a surrogate to investigate acrylamide reactivity toward free

thiols. When compound 31 (100 μM) was incubated with 5 mM

GSH in PBS buﬀer at pH 7.4 at 37 °C, no adducts between

compound 31 and GSH were detected after 2 h, suggesting that

the acrylamide group does not react indiscriminately with thiols.

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Table 5. Selectivity against Kinases with a Cysteine Structurally Identical to Cys481 in Btk

IC₅₀(nM)

compd

Blk

Btk

Bmx

EGFR

HER2

HER4

Itk

Jak3

Rlk

Tec

31

38

2.7

4.2

5

1.9

11

7

19

7

5.5

3.3

>30 000

2680

6370

1.9

1.4

8.2

6.3

<4.4

0.71

a

IC₅₀values are reported as mean values of duplicates.

the chemical reactivity of these compounds can contribute

separately to the overall biochemical potency.⁶⁵

(PDB code 3OCS) indicates that this compound extends into a

deep pocket formed by Phe413, Leu542, Val546, and Tyr551,

resulting in the movement of Tyr551 away from the solvent

accessible area such that it may be unavailable for phos-

phorylation by upstream kinases.¹²

Mass spectrometry was employed to determine whether

compounds 31 and 38 formed covalent adducts with Btk.

Incubating the Btk kinase domain with 3 equiv of each

compound caused a complete mass peak shift, and the mass

numbers for the new peaks were consistent with 1-to-1

complexes between the compounds and protein (Figure 4).

To test the inhibition mode of action of compounds 31 and

38, we measured their abilities to inhibit the BCR pathway by

monitoring the phosphorylation levels of Tyr551 in Btk and

Tyr1217 in the Btk substrate PLC-γ2. Both compounds

strongly inhibited both phosphorylation events in live Ramos

cells. Compounds 31 and 38 exhibited IC₅₀values of 14 and

13 nM, respectively, for Btk Tyr551 phosphorylation and IC₅₀

values of 8.5 and 13 nM, respectively, for PLC-γ2 Tyr1217

phosphorylation. In comparison, racemic version of ibrutinib

(compound 4 in ref 15) inhibited PLC-γ2 phosphorylation

only, with an IC₅₀of 14 nM, and showed virtually no inhibition

(>7.5 μM) of Btk Tyr551 phosphorylation. Thus, compounds

31 and 38 clearly exhibit a dual-action mode of inhibition in

live cells.

Next, washout experiments were performed to conﬁrm

irreversible bond formation between Btk and the inhibitors in

live cells (Figure 5). The acrylamide-containing compounds 31

Figure 4. Mass spectra of the Btk kinase domain indicating that 1-to-1

adducts were formed between 31 or 38 and Btk. The molecular weight

of apo Btk (387−659) is 32136. After incubation with 31 (exact mass,

560.5) or 38 (exact mass, 617.6) at 4 °C for 1 h, the peaks were fully

shifted.

The adduct between compound 31 and Btk was digested with

trypsin. Mass spectrometric analysis of the digested peptide

fragments indicated that the modiﬁcation site was, as expected,

Cys481 (Supporting Information).

Compounds 31 and 38 Irreversibly Inhibit Btk

Phosphorylation in Live Cells. In cells, BCR signaling is a

graded process that is inﬂuenced by the convergence of both

activating and negative regulatory events.⁴⁶Upon BCR pathway

activation, Btk moves to the cell membrane and is ﬁrst

phosphorylated at Tyr551 by upstream kinases. After an

additional autophosphorylation step, Btk is fully activated and

phosphorylates its physiological substrate, PLC-γ2. Approx-

imately 30 min later, Btk activity returns to the basal level

through a negative feedback loop. PKCβ, which is activated

downstream of Btk, directly and negatively regulates Btk

through Ser180 phosphorylation, which impairs Btk migration

to the membrane and its overall phosphorylation/activation

level.⁴⁶Inhibiting Btk activity would interrupt downstream

events in the BCR pathway, including the negative feedback

loop, thus maintaining high Btk phosphorylation levels.

Ibrutinib has been shown to augment Btk Tyr551 phosphor-

ylation levels in Namalwa cells.⁴⁷Among known Btk inhibitors,

1 binds the inactive conformation of Btk, in which Tyr551

is not phosphorylated. The crystal structure of Btk with 1

Figure 5. Washout experiments with the reversible inhibitor 13 and

irreversible inhibitors 31 and 38. In Ramos cells, Btk Tyr551

phosphorylation was measured after the cells were treated with

compound for 1 h in a normal experiment or after treatment for 1 h

followed by washout with fresh media for 2 h in the washout

experiment.

and 38 maintained their inhibitory eﬀects in cells even after a

2 h washout with fresh media, whereas Btk phosphorylation

levels were largely restored in cells treated with the reversible

analogue 13, which contains a simple propylamide group, after

removal of the compound. These data showed that both

compounds 31 and 38 are irreversible inhibitors.

Antiproliferative Activities. Initial testing of compound

31 at two concentrations (1 and 10 μM) against multiple non-

Hodgkin’s lymphoma and leukemia cell lines indicated that this

compound was active in preventing the growth of these cancer

cells (Figure 6). Cell viability assays further conﬁrmed the

initial results. Both compounds 31 and 38 displayed single-digit

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Table 6. Antiproliferation Activity against Several Cancer

a

Cell Lines

GI₅₀(μM)

compd

DoHH2

Pfeiﬀer

SU-DHL-6

WSU-NHL

ibrutinib

31

0.41

0.002

0.008

0.58

1.09

0.073

0.040

0.038

0.043

0.031

0.029

38

a

GI₅₀values are reported as mean values of two or three independent

experiments in triplicate.

direction is to examine the changes of proteins directly involved

in the BCR pathway, and the other one is to investigate potential

involvements of additional pathways because of the fact that

these compounds are also potent against quite a few of kinases

with important functions. Overall, these results are certainly

encouraging for potentially expanded applications of Btk inhibitors

in clinical studies.

Pharmacokinetic Properties of Compounds 31 and

38. Both compounds 31 and 38 displayed fair solubility and

excellent stability in fasted state simulated intestinal ﬂuid

(FaSSIF) (Table 7),⁶⁶which mimics the human intestinal

environment. We estimated that these compounds would have

a moderate hepatic clearance rate in mice based on a mouse

microsome stability study. However, in vivo pharmacokinetic

studies in mice showed that compound 31 had a fair half-life of

2.8 h, whereas compound 38 had a much shorter half-life of

0.6 h. Both compounds had small distribution volumes, and they

were expected to primarily remain in the circulatory system.

Figure 6. Compound 31 is a potent inhibitor of proliferation in

multiple malignant B cell lines. The values shown are the averages of

triplicates.

nanomolar potency in Pfeiﬀer cells and better eﬃcacy than

ibrutinib in DoHH2 cells with GI₅₀values of 73 nM (31) and

40 nM (38), respectively (Figure 7). SU-DHL-6 and WSU-NHL

are two germinal center B-cell-like diﬀuse large B cell lymphoma

(GCB-DLBCL) cell lines. It has been suggested that their

survival does not solely rely on the BCR pathway; thus, they

were not successfully targeted by ibrutinib in clinical studies.

However, compared with ibrutinib, the antiproliferative potency

of compounds 31 and 38 was approximately 13-fold stronger

against SU-DHL-6 cells and 35-fold stronger against WSU-NHL

cells (Table 6). We are actively investigating the biological basis

for these results and will report our ﬁndings in due course. One

Figure 7. Tumor cell proliferation assays. Representative dose−response curves of 31, 38, and ibrutinib treating DoHH2, Pfeiﬀer, SU-DHL-6, and

WSU-NHL cells. Cells were seeded in 96-well plates, incubated for 24 h, then treated with compounds for 72 h, and measured with a luminescent

cell viability assay.

G

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Condensation reactions with aromatic acids with variations at

the R position, followed by deprotection of the Boc group,

aﬀorded compounds 12 and 16−18. 12 was further acylated

with diﬀerent reactive groups or other acids to yield

compounds 13−15, 31−38. Compound 11 with an amino

group at the para position was prepared in a manner similar to

that for 12 (Supporting Information). Reverse amide and urea

compounds (19 and 20) were prepared using similar methods

based on Amgen’s procedures, which were also described in the

Supporting Information.

An alternative synthetic route is outlined in Scheme 3. Cbz-

protected compound 45 was coupled with 44 followed by

removal of the Cbz group to aﬀord intermediate 46.

Compounds 21−30 were prepared by amide coupling with

various carboxylic acids and the removal of Boc protection

group. Finally, compounds 39−41 were obtained by acylating

amine precursors with acryloyl chloride.

Table 7. Select PK Parameters for Compounds 31 and 38

compound

parameter

solubility (μM)

31

38

FaSSIF

7.02

94

2.23

99

stability (% remaining at 24 h)

Mouse Liver Microsome

t_1/2(h)

0.64

25.7

47.8

0.59

28.1

49.8

CL (μL min⁻¹mg⁻¹

)

CL^hep(mL min⁻¹kg⁻¹

)

a

In Vivo (iv)

t_1/2(h)

2.8

0.6

CL (mL min⁻¹kg⁻¹

)

6.85

0.20

11.49

0.13

V_ss(L/kg)

a

The in vivo pharmacokinetics of compounds 31 and 38 were

evaluated in ICR mice (n = 3) following intravenous (iv) injection at

1 mg/kg dose level. At various time points, blood samples were

collected and analyzed by LC−MS method.

CONCLUSIONS

■

We developed novel covalent irreversible inhibitors of Btk

based on a type II scaﬀold. These compounds exhibited potent

inhibitory activity in biochemical and cellular assays, presented

a dual-action mode of inhibition that counteracted the negative

regulation loop for Btk, and have a diﬀerent selectivity proﬁle

from ibrutinib. These inhibitors were up to 35-fold more potent

than the leading clinical drug, ibrutinib, in lymphoma cell

viability assays. In a mouse xenograft model, compound 31

eﬀectively prevented lymphoma tumor growth in a dose-

dependent manner. Because of the emerging importance of Btk

in a wide range of B-cell lineage hematological cancers, our

results support a new direction for the development of novel

Btk inhibitors. Further optimization of this series of compounds

will be reported in due course.

In Vivo Eﬃcacy of Compound 31. On the basis of its

potency and PK properties, compound 31 was further

examined in a mouse xenograft model inoculated with the

human lymphoma DoHH2 cells. This study comprised four

groups, with a vehicle control group and three treatment groups

that included one dose of ibrutinib (10 mg/kg) and two doses

of compound 31 (10 and 20 mg/kg). Ibrutinib was used as a

positive control because its in vivo antiproliferative eﬀects in

a xenograft model of DoHH2 have been reported in the

literature.⁶⁷The compounds were administered through the tail

vein once a day for 14 consecutive days, and the tumor sizes

were measured periodically. Plasma concentrations of com-

pounds were determined at 2 and 24 h after the last drug

administration. While all treatment groups achieved nice

exposure of compounds at 2 h, neither the 10 mg/kg ibrutinib

nor the 10 mg/kg compound 31 treatment groups had any

detectable levels of compounds at 24 h and the 20 mg/kg

compound 31 treatment group showed a dramatically reduced

drug concentration (Supporting Information). As shown in

Figure 8, at the end of the study, tumor sizes of the ibrutinib-

treated group were reduced by 32% (P = 0.145), and both

compound 31 dosage groups displayed more potent anti-

proliferative eﬀects. Compared with the vehicle control group,

the tumor size was reduced by 60% (P = 0.016) in the 10 mg/kg

compound 31 treatment group, whereas the tumor size was

reduced by 66% (P = 0.004) in the 20 mg/kg group. In every

group, the mice slightly gained weight after the 14-day

treatment, and no signiﬁcant weight ﬂuctuations were observed

during the treatment. Thus, compound 31 was well-tolerated

and exhibited a clear antiproliferative eﬀect in this mouse

xenograft model.

EXPERIMENTAL SECTION

■

Chemistry. All materials were obtained from commercial suppliers

and used without further puriﬁcation unless otherwise noted. Yields

refer to chromatographic yields unless otherwise stated. Anhydrous

THF was distilled from sodium. DCM and toluene were distilled from

calcium hydride. Reactions were monitored by thin-layer chromatog-

raphy (TLC) carried out on 0.25 mm Yantai silica gel plates (HSGF

254) using UV light as visualizing agents or an ethanolic solution of

phosphomolybdic acid or ninhydrin as developing agents. Yantai silica

gel (ZCX-II, particle size 0.048−0.075 mm) was used for ﬂash column

1

chromatography. H NMR and ¹³C NMR spectra were recorded on a

Bruker Avance 300 (¹H, 300 MHz; ¹³C, 75 MHz) or Bruker Advance

400 (¹H, 400 MHz; ¹³C, 100 MHz) or Bruker Advance 500 (¹H, 500

MHz; ¹³C, 125 MHz) spectrometer at ambient temperature. Chemical

shifts are reported in ppm from the solvent resonance. Data are

reported as follows: chemical shift, multiplicity (s = singlet, d =

doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling

constants, and number of protons. Mass spectrometric data were

obtained using an AB Q-Star mass spectrometer. All ﬁnal compounds

(4−41) were puriﬁed to >95% purity as determined by high

performance liquid chromatography (HPLC) with UV detection at

254 nm. HPLC experiments were performed on an Agilent 1200 series

LC system equipped with a quaternary pump (G1311A), a vacuum

degasser (G1322A), a diode array detector (G1315D), and an

autosampler (G1329A) using an Agilent Eclipse Plus C18 (5 μm,

4.6 mm × 250 mm) column. The mobile phase was constituted of

H₂O and CH₃CN (eluent A, 30−100% CH₃CN, 15 min; 100%

CH₃CN, 5 min; 1.00 mL/min ﬂow rate; eluent B, 30−100% 0.1% TFA

in CH₃CN/0.1% TFA in H₂O, 15 min; 100% 0.1% TFA in CH₃CN,

5 min; 1.00 mL/min ﬂow rate).

CHEMISTRY

■

The syntheses of this novel series of compounds were outlined

in Schemes 1, 2, and 3 where the core structure was prepared

using a method previously reported by Amgen scientists.⁵⁶In

general, reactions proceeded smoothly and provided desired

products in good yields. Compound 42 was coupled with

various amines, followed by a reduction step to form

intermediates 43, which underwent amide condensation to

yield compounds 4−10 (Scheme 1).

As shown in Scheme 2, aniline 44 was prepared from

compound 42 via S_NAr reaction followed by catalytic reduction.

Synthesis of tert-Butyl 3-(5-Aminopyrimidin-2-ylamino)-

phenylcarbamate (44). Step 1. Potassium carbonate (0.702 g,

H

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Figure 8. Btk inhibitor 31 in vivo eﬃcacy study. (a) Compound 31 signiﬁcantly prevented tumor growth in a xenograft mouse model. DoHH2

cells were implanted subcutaneously in female CB17/SCID mice. When tumors reached ∼167 mm³on average, mice were randomized into four

groups (n = 6/group). Compounds were administered via tail vein as a clear solution in polyoxyl 15 hydroxystearate/ethanol (1:1). Tumor size

and weight were monitored periodically for 14 days. Statistical analysis was conducted by one-way ANOVA. The Dunnett test was used to

analyze the statistical signiﬁcance between each treatment group and the vehicle group. (∗) P < 0.05 and (∗∗) P < 0.01 indicate statistical

signiﬁcance of tumor growth inhibition. (b) The body weights of the mice changed over time, and the mice in every group slightly gained weight

at the end of the study.

a

Scheme 1. Preparation of Compounds 4−10

a

Reagents and conditions: (a) ammonium hydroxide, THF, rt, 1 h (96%); (b) R-NH₂, K₂CO₃, CH₃CN, H₂O, rt, 3 h (81−96%); (c) 5% Pd/C, H₂,

methanol, rt, overnight (88−93%); (d) 2-methyl-5-(3-(triﬂuoromethyl)benzamido)benzoic acid, HATU, DIEA, DMF, rt, 8 h (71−98%).

I

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a

Scheme 2. Preparation of Compounds 12−18, 31−38

a

Reagents and conditions: (a) 3-(tert-butoxycarbonylamino)aniline or 4-(tert-butoxycarbonylamino)aniline, K₂CO₃, CH₃CN, H₂O, rt, 3 h (89%);

(b) 5% Pd/C, H₂, methanol, rt, overnight (88%); (c) various aromatic acids, HATU, DIEA, DMF, rt, 6 h (72−80%); (d) TFA, DCM, rt, 2 h (71−

98%); (e) various acyl chlorides, THF−H₂O, rt, 1 h (13, 91%; 31, 90%; 35, 87%); or various carboxylic acids, HATU, DIEA, DMF, rt, 5 h (14, 73%;

32, 90%; 33, 83%; 36, 81%; 38, 88%); or various acyl chlorides, THF, rt, 1 h (15, 91%; 37, 93%); or 2-chloroethanesulfonyl chloride, DIEA, DMAP,

THF, rt, 7 h (34, 84%).

a

Scheme 3. Alternative Route to 2,5-Diaminopyrimidines 21−30, 39−41

a

Reagents and conditions: (a) 44, HATU, DIEA, DMF, 5 h (71%); (b) 5% Pd/C, H₂, methanol, 60 °C, 10 h (53%); (c) R-COOH, HATU, DMF,

rt, 5 h (82−93%); (d) TFA, DCM, rt, 3 h (88−91%); (e) acryloyl chloride, THF, H₂O, rt, 1 h (84−98%).

5.08 mmol) was added to a stirred solution of 2-chloro-5-nitropyrimidine

(42) (0.352 g, 1.69 mmol) and tert-butyl 3-aminophenylcarbamate

(0.270 g, 1.69 mmol) in acetonitrile (12 mL), and the mixture was

stirred for 3 h at room temperature. The solution was diluted with water

and extracted twice with ethyl acetate. The combined organic layers

were dried, concentrated under vacuum, and puriﬁed via column

chromatography (gradient, 20−75% EtOAc in hexanes) to yield tert-

butyl 3-(5-nitropyrimidin-2-ylamino)phenylcarbamate (0.500 g, 89%) as

yellow solids.

Step 2. tert-Butyl 3-(5-nitropyrimidin-2-ylamino)phenylcarbamate

(0.500 g, 1.51 mmol) and 5% Pd/C (0.16 g) were dissolved in 10 mL

of methanol, and then the mixture was stirred under hydrogen

atmosphere at room temperature for 5 h. Once the reaction was

completed, the mixture was ﬁltered through Celite and the ﬁltrate was

concentrated and puriﬁed by column chromatography (gradient,

50−85% EtOAc in hexanes) to give the title compound (0.40 g, 88%)

as yellow solids. ¹H NMR (400 MHz, DMSO-d₆): δ 9.13 (s, 1H), 8.86

(s, 1H), 7.95 (s, 2H), 7.77 (s, 1H), 7.25 (d, 1H, J = 8.1 Hz), 7.05 (t,

1H, J = 8.1 Hz), 6.92 (d, 1H, J = 7.8 Hz), 4.77 (s, 2H), 1.47 (s, 9H).

¹³C NMR (125 MHz, DMSO-d₆): δ 152.8, 152.7, 143.6, 141.9,

139.4, 135.5, 128.0, 111.7, 110.5, 107.9, 78.5, 28.0. HRMS (ESI) m/z

+

calculated for C₁₅H₂₀N₅O₂[M + H]⁺: 302.1617. Found: 302.1602.

Synthesis of N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-2-

methyl-5-(3-(triﬂuoromethyl)benzamido)benzamide (12). Step 1.

44 (0.100 g, 0.230 mmol), 3-(triﬂuoromethyl)benzoic acid (0.048 g,

0.25 mmol), and HATU (0.131 g, 0.345 mmol) were dissolved in DMF

(2 mL), followed by the addition of DIEA (0.075 mL, 0.46 mmol). The

mixture was stirred at room temperature for 7 h. Then the solution was

concentrated under vacuum and the residue was diluted with saturated

aqueous sodium bicarbonate and ethyl acetate. The aqueous phase was

separated and extracted twice with ethyl acetate. The combined organic

phases were washed with brine, dried over anhydrous magnesium

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sulfate, concentrated, and puriﬁed by column chromatography (gradient,

30−100% EtOAc in hexanes) to yield tert-butyl 3-(5-(2-methyl-5-

(3-(triﬂuoromethyl)benzamido)benzamido)pyrimidin-2-ylamino)-

phenylcarbamate (0.12 g) as yellow solids.

135.4, 131.8, 130.8, 130.7, 129.8, 128.2, 124.5, 124.1, 123.6, 122.2,

121.6, 119.2, 23.9, 18.8, 6.4. HRMS (ESI) m/z calculated for

+

C₂₃H₂₁F₃N₅O₂[M + H]⁺: 456.1647. Found: 456.1642.

2-Methyl-N-(2-(phenylamino)pyrimidin-5-yl)-5-(3-

(triﬂuoromethyl)benzamido)benzamide (7). White solids. ¹H

NMR (400 MHz, DMSO-d₆): δ 10.57 (s, 1H), 10.41 (s, 1H), 9.63 (s,

1H), 8.80 (s, 2H), 8.33 (s, 1H), 8.28 (d, 1H, J = 8.0 Hz), 7.98 (d, 1H,

J = 7.6 Hz), 7.93 (d, 1H, J = 2.0 Hz), 7.84 (dd, 1H, J = 2.1 Hz, J = 8.3

Hz), 7.80 (t, 1H, J = 7.8 Hz), 7.74 (d, 2H, J = 7.8 Hz), 7.33 (d, 1H, J =

8.4 Hz), 7.27 (t, 2H, J = 8.1 Hz), 6.93 (t, 1H, J = 7.3 Hz), 2.39 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 167.5, 163.9, 156.5, 149.9, 140.6,

Step 2. 3-(5-(2-Methyl-5-(3-(triﬂuoromethyl)benzamido)-

benzamido)pyrimidin-2-ylamino)phenylcarbamate (0.120 g,

0.198 mmol) was treated with TFA (2 mL) and DCM (2 mL). The

mixture was stirred at room temperature for 2 h and concentrated

under vacuum. The residue was diluted with saturated aqueous sodium

bicarbonate and ethyl acetate. The organic phase was separated and

washed with brine, dried over anhydrous magnesium sulfate,

concentrated, and puriﬁed by column chromatography (gradient,

50−80% EtOAc in hexanes) to give the title compound (0.096 g, 82%

for two steps) as white solids. ¹H NMR (500 MHz, DMSO-d₆):

δ 10.56 (s, 1H), 10.36 (s, 1H), 9.30 (s, 1H), 8.76 (s, 2H), 8.33 (s, 1H),

8.28 (d, 1H, J = 7.9 Hz), 7.98 (d, 1H, J = 7.8 Hz), 7.93 (d, 1H, J = 2.0

Hz), 7.85 (dd, 1H, J = 2.0 Hz, J = 8.3 Hz), 7.80 (t, 1H, J = 7.9 Hz),

7.33 (d, 1H, J = 8.4 Hz), 7.07 (s, 1H), 6.89 (t, 1H, J = 7.8 Hz), 6.84 (d,

1H, J = 8.1 Hz), 6.17 (d, 1H, J = 7.6 Hz), 4.94 (s, 2H), 2.39 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 167.5, 163.9, 156.8, 149.8, 148.8,

136.5, 135.4, 131.8, 130.9, 130.8, 129.8, 129.3, 129.0, 128.4, 128.2,

126.2, 124.1, 121.7, 121.0, 119.2, 118.4, 118.3, 18.8. HRMS (ESI) m/z

calculated for C₂₆H₂₁F₃N₅O₂⁺[M + H]⁺: 492.1647. Found: 492.1650.

2-Methyl-5-(3-(triﬂuoromethyl)benzamido)-N-(2-(3-

(triﬂuoromethyl)phenylamino)pyrimidin-5-yl)benzamide (8).

White solids. ¹H NMR (400 MHz, DMSO-d₆): δ 10.59 (s, 1H),

10.50 (s, 1H), 10.03 (s, 1H), 8.87 (s, 2H), 8.33 (s, 1H), 8.28 (d, 1H,

J = 7.9 Hz), 8.22 (s, 1H), 7.99 (t, 2H, J = 9.6 Hz), 7.94 (d, 1H, J = 2.2

Hz), 7.86−7.78 (m, 2H), 7.51 (t, 1H, J = 8.0 Hz), 7.33 (d, 1H, J = 8.5

Hz), 7.25 (d, 1H, J = 7.7 Hz), 2.39 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 169.5, 165.8, 158.0, 151.7, 143.3, 138.4, 138.3, 137.3,

133.7, 132.9, 132.8, 131.7, 131.5, 130.2, 128.8, 127.6, 127.2, 126.0,

124.9, 124.5, 123.7, 123.5, 121.5, 118.9, 115.7, 20.7. HRMS (ESI) m/z

calculated for C₂₇H₁₈F₆N₅O₂⁻[M − H]⁻: 558.1365. Found: 558.1370.

N-(2-(3-Methoxyphenylamino)pyrimidin-5-yl)-2-methyl-5-

141.1, 136.5, 136.4, 135.5, 131.8, 130.9, 130.8, 129.8, 129.3, 129.0,

128.6, 128.2, 125.8, 124.1, 121.7, 119.2, 107.7, 107.0, 104.2, 18.8. HRMS

(ESI) m/z calculated for C₂₆H₂₀F₃N₆O₂⁻[M − H]⁻: 505.1600. Found:

505.1591.

Synthesis of N-(2-(3-Acrylamidophenylamino)pyrimidin-5-

yl)-2-methyl-5-(3-(triﬂuoromethyl)benzamido)benzamide (31).

To a stirred suspension of 12 (0.080 g, 0.16 mmol) and DIEA (0.027 mL,

0.16 mmol) in 1 mL of THF and 1 mL of H₂O was added acryloyl

chloride (0.013 mL, 0.16 mmol). Then the mixture was kept stirring at

room temperature for 1 h, then concentrated and diluted with ethyl

acetate and 10% aqueous citric acid. The organic phase was separated,

washed with brine, dried over anhydrous magnesium sulfate, and puriﬁed

by column chromatography (gradient, 50−70% EtOAc in hexanes) to

1

(3-(triﬂuoromethyl)benzamido)benzamide (9). White solids. H

NMR (400 MHz, DMSO-d₆): δ 10.59 (s, 1H), 10.44 (s, 1H), 9.63 (s,

1H), 8.81 (d, 2H, J = 2.2 Hz), 8.32 (s, 1H), 8.28 (d, 1H, J = 7.8 Hz),

7.98 (d, 1H, J = 7.4 Hz), 7.94 (s, 1H), 7.85−7.78 (m, 2H), 7.48 (d,

1H, J = 1.8 Hz), 7.32 (t, 2H, J = 8.8 Hz), 7.19−7.14 (m, 1H), 6.50 (d,

1H, J = 8.0 Hz), 3.73 (d, 3H, J = 2.2 Hz), 2.38 (s, 3H). ¹³C NMR (125

MHz, DMSO-d₆): δ 167.5, 163.8, 159.5, 156.4, 149.8, 141.7, 136.4,

135.4, 131.6, 130.8, 130.7, 129.6, 129.0, 128.1, 126.2, 124.0, 121.7,

119.2, 110.8, 106.2, 104.3, 54.8, 18.6. HRMS (ESI) m/z calculated for

1

yield the title compound (0.080 g, 90%) as a white powder. H NMR

(300 MHz, DMSO-d₆): δ 10.59 (s, 1H), 10.44 (s, 1H), 10.10 (s, 1H),

9.69 (s, 1H), 8.82 (s, 2H), 8.33 (s, 1H), 8.28 (d, 1H, J = 8.0 Hz), 8.07

(s, 1H), 7.99 (d, 1H, J = 7.8 Hz), 7.94 (d, 1H, 2.0 Hz), 7.87−7.78 (m,

2H), 7.39−7.32 (m, 3H), 7.23−7.17 (m, 1H), 6.48 (dd, 1H, J = 10.0

Hz, J = 17.0 Hz), 6.25 (dd, 1H, J = 2.0 Hz, J = 17.0 Hz), 5.73 (dd, 1H,

J = 1.9 Hz, J = 10.0 Hz), 2.39 (s, 3H). ¹³C NMR (100 MHz, MeOD):

δ 177.0, 173.4, 172.5, 166.0, 159.3, 150.4, 148.6, 146.0, 145.0, 141.5,

141.3, 140.4, 140.3, 139.3, 138.8, 138.5, 138.1, 137.7, 136.0, 135.8,

134.8, 133.6, 131.2, 128.7, 123.5, 122.2, 119.0, 28.3. HRMS (ESI) m/z

calculated for C₂₉H₂₄F₃N₆O₃⁺[M + H]⁺: 561.1862. Found: 561.1859.

N-(2-Aminopyrimidin-5-yl)-2-methyl-5-(3-(triﬂuoromethyl)-

benzamido)benzamide (4). White solids. ¹H NMR (300 MHz,

DMSO-d₆): δ 10.57 (s, 1H), 10.17 (s, 1H), 8.51 (s, 2H), 8.32 (s, 1H),

8.27 (d, 1H, J = 7.7 Hz), 7.98 (d, 1H, J = 7.4 Hz), 7.89 (d, 1H, J = 2.2

Hz), 7.84−7.77 (m, 2H), 7.30 (d, 1H, J = 8.4 Hz), 6.55 (s, 2H), 2.36

(s, 3H). ¹³C NMR (125 MHz, DMSO-d₆): δ 167.3, 163.8, 160.5,

150.7, 136.7, 136.3, 135.4, 131.6, 130.7, 130.6, 129.6, 128.04, 128.01,

124.2, 124.0, 123.9, 121.5, 119.1, 18.6. HRMS (ESI) m/z calculated for

−

C₂₇H₂₁F₃N₅O₃[M − H]⁻: 520.1596. Found: 520.1600.

N-(2-(3-Fluorophenylamino)pyrimidin-5-yl)-2-methyl-5-(3-

(triﬂuoromethyl)benzamido)benzamide (10). White solids. ¹H

NMR (400 MHz, DMSO-d₆): δ 10.59 (s, 1H), 10.49 (s, 1H), 9.92 (s,

1H), 8.86 (s, 2H), 8.33 (s, 1H), 8.28 (d, 1H, J = 7.8 Hz), 7.98 (d, 1H,

J = 7.5 Hz), 7.95 (s, 1H), 7.86−7.78 (m, 3H), 7.47 (d, 1H, J = 8.0 Hz),

7.35−7.26 (m, 2H), 6.72 (td, 1H, J = 2.1 Hz, J = 8.4 Hz), 2.39 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 169.5, 165.8, 165.4, 163.1, 158.0,

151.7, 144.3, 138.4, 137.3, 133.7, 132.9, 131.9, 131.7, 131.2, 130.1,

128.7, 127.2, 126.0, 124.5, 123.7, 121.1, 115.9, 109.1, 106.5, 20.7.

HRMS (ESI) m/z calculated for C₂₆H₁₈F₄N₅O₂⁻[M − H]⁻: 508.1397.

Found: 508.1402.

N-(2-(4-Aminophenylamino)pyrimidin-5-yl)-2-methyl-5-(3-

(triﬂuoromethyl)benzamido)benzamide (11). White solids. ¹H

NMR (300 MHz, MeOD): δ 8.64 (s, 2H), 8.27 (s, 1H), 8.20 (d, 1H,

J = 8.1 Hz), 7.93−7.88 (m, 2H), 7.74 (d, 1H, J = 7.9 Hz), 7.67 (dd,

1H, J = 2.2 Hz, J = 8.2 Hz), 7.34−7.31 (m, 3H), 6.74 (d, 2H, J = 8.7

Hz), 4.63 (s, 3H), 2.45 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ

167.4, 163.9, 157.5, 157.3, 151.3, 150.2, 143.6, 143.4, 136.6, 136.4,

135.4, 134.9, 131.8, 130.9, 129.8, 128.2, 125.0, 124.1, 121.1, 120.9,

+

C₂₀H₁₇F₃N₅O₂[M + H]⁺: 416.1334. Found: 416.1338.

N-(2-(Ethylamino)pyrimidin-5-yl)-2-methyl-5-(3-(triﬂuoromethyl)-

benzamido)benzamide (5). White solids. ¹H NMR (400 MHz,

DMSO-d₆): δ 10.56 (s, 1H), 10.15 (s, 1H), 8.55 (s, 1H), 8.32 (s,

1H), 8.28 (d, 1H, J = 7.9 Hz), 7.98 (d, 1H, J = 7.8 Hz), 7.89 (d, 1H,

J = 2.2 Hz), 7.85−7.78 (m, 2H), 7.30 (d, 1H, J = 8.4 Hz), 7.07 (t, 1H, J =

5.6 Hz), 3.30−3.26 (m, 2H), 2.37 (s, 3H), 1.12 (t, 3H, J = 7.1 Hz). ¹³C

NMR (100 MHz, DMSO-d₆): δ 168.9, 165.4, 160.9, 152.3, 138.2, 137.9,

137.0, 133.3, 132.7, 132.4, 132.2, 131.3, 130.8, 129.7, 125.6, 125.4, 123.1,

120.7, 37.1, 20.3, 16.2. HRMS (ESI) m/z calculated for C₂₂H₁₉F₃N₅O₂⁻

[M − H]⁻: 442.1491. Found: 442.1496.

N-(2-(Cyclopropylamino)pyrimidin-5-yl)-2-methyl-5-(3-

(triﬂuoromethyl)benzamido)benzamide (6). White solids. ¹H

NMR (500 MHz, DMSO-d₆): δ 10.55 (s, 1H), 10.17 (s, 1H), 8.58 (s,

1H), 8.32−8.27 (m, 3H), 7.97 (d, 1H, J = 7.6 Hz), 7.89 (d, 1H, J = 2.0

Hz), 7.84−7.78 (m, 2H), 7.32−7.29 (m, 2H), 2.68−2.66 (m, 1H),

2.36 (s, 3H), 0.67−0.63 (m, 2H), 0.47−0.43 (m, 2H). ¹³C NMR

(100 MHz, DMSO-d₆): δ 167.4, 163.9, 160.3, 150.6, 136.7, 136.4,

+

119.2, 114.0, 18.8. HRMS (ESI) m/z calculated for C₂₆H₂₂F₃N₆O₂

[M + H]⁺: 507.1756. Found: 507.1741.

2-Methyl-N-(2-(3-propionamidophenylamino)pyrimidin-5-

yl)-5-(3-(triﬂuoromethyl)benzamido)benzamide (13). White

1

solids. H NMR (300 MHz, DMSO-d₆): δ 10.59 (s, 1H), 10.43 (s,

1H), 9.81 (s, 1H), 9.63 (s, 1H), 8.80 (s, 2H), 8.32 (s, 1H), 8.28 (d,

1H, J = 7.8 Hz), 7.98−7.93 (m, 3H), 7.86−7.77 (m, 2H), 7.33 (d, 2H,

J = 8.3 Hz), 7.28 (d, 1H, J = 7.7 Hz), 7.15 (t, 1H, J = 8.1 Hz), 2.38 (s,

3H), 2.30 (q, 2H, J = 7.5 Hz), 1.07 (t, 3H, J = 7.5 Hz). ¹³C NMR (100

MHz, DMSO-d₆): δ 173.7, 169.4, 165.8, 158.5, 151.7, 142.7, 141.4,

138.4, 138.3, 137.4, 133.7, 132.9, 132.7, 131.7, 131.2, 130.9, 130.3,

130.1, 128.2, 126.0, 123.7, 121.1, 115.5, 114.4, 111.3, 31.3, 20.7, 11.6.

HRMS (ESI) m/z calculated for C₂₉H₂₆F₃N₆O₃⁺[M + H]⁺: 563.2018.

Found: 563.2012.

K

dx.doi.org/10.1021/jm4017762 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

N-(2-(3-(3-(Dimethylamino)propanamido)phenylamino)-

pyrimidin-5-yl)-2-methyl-5-(3-(triﬂuoromethyl)benzamido)-

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-2-methyl-5-(3-

(3-(triﬂuoromethyl)phenyl)ureido)benzamide (20). White solids.

¹H NMR (500 MHz, DMSO-d₆): δ 10.31 (s, 1H), 9.29 (s, 1H), 9.10

1

benzamide (14). White solids. H NMR (400 MHz, DMSO-d₆): δ

10.59 (s, 1H), 10.43 (s, 1H), 10.13 (s, 1H), 9.67 (s, 1H), 8.81 (s, 2H),

8.32 (s, 1H), 8.28 (d, 1H, J = 7.9 Hz), 8.03 (s, 1H), 7.99−7.96 (m,

2H), 7.84−7.78 (m, 2H), 7.36−7.28 (m, 3H), 7.21−7.17 (m, 1H),

3.28−3.06 (m, 4H), 2.94−2.90 (m, 1H), 2.79 (t, 1H, J = 7.0 Hz), 2.74

(s, 4H), 2.39 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.0,

167.5, 163.9, 156.4, 149.8, 140.9, 139.0, 136.5, 136.4, 135.4, 131.8,

130.9, 130.8, 129.8, 128.6, 128.2, 126.3, 125.3, 124.1, 122.6, 121.8,

119.3, 113.9, 112.4, 109.4, 52.9, 42.6, 31.2, 18.8. HRMS (ESI) m/z

calculated for C₃₁H₃₁F₃N₇O₃⁺[M + H]⁺: 606.2440. Found: 606.2433.

2-Methyl-N-(2-(3-(N-propionylpropionamido)phenylamino)-

pyrimidin-5-yl)-5-(3-(triﬂuoromethyl)benzamido)benzamide

(15). White solids. ¹H NMR (500 MHz, DMSO-d₆): δ 10.88 (s, 1H),

10.59 (s, 1H), 9.93 (s, 1H), 9.11 (s, 2H), 8.31 (s, 1H), 8.27 (d, 1H, J =

7.8 Hz), 7.98−7.97 (m, 2H), 7.83 (dd, 1H, J = 2.1 Hz, J = 8.3 Hz),

7.78 (d, 1H, J = 7.8 Hz), 7.53 (s, 1H), 7.49 (d, 1H, J = 8.2 Hz), 7.34

(d, 1H, J = 8.4 Hz), 7.30 (t, 1H, J = 8.0 Hz), 6.89 (d, 1H, J = 8.8 Hz),

2.44 (q, 2H, J = 7.4 Hz), 2.38 (s, 3H), 2.29 (q, 2H, J = 7.6 Hz), 1.07−

1.04 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ 173.7, 172.1, 168.1,

164.0, 155.9, 149.3, 141.5, 140.0, 136.5, 135.8, 135.4, 132.0, 131.8, 131.1,

131.0, 129.8, 129.1, 128.2, 124.1, 122.2, 119.3, 118.1, 117.4, 29.5, 28.5,

18.8, 9.5, 9.4. HRMS (ESI) m/z calculated for C₃₂H₃₀F₃N₆O₄⁺[M + H]⁺:

619.2281. Found: 619.2274.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-2-chloro-5-(3-

(triﬂuoromethyl)benzamido)benzamide (16). White solids. ¹H

NMR (400 MHz, DMSO-d₆): δ 10.74 (s, 1H), 10.62 (s, 1H), 9.38 (s,

1H), 8.76 (s, 2H), 8.34 (s, 1H), 8.29 (d, 1H, J = 7.9 Hz), 8.06 (d, 1H,

J = 2.4 Hz), 8.00 (d, 1H, J = 7.7 Hz), 7.97 (dd, 1H, J = 2.5 Hz, J = 8.8

Hz), 7.82 (t, 1H, J = 7.6 Hz), 7.61 (d, 1H, J = 8.8 Hz), 7.09 (s, 1H),

6.91 (t, 1H, J = 7.7 Hz), 6.86 (d, 1H, J = 8.1 Hz), 6.20 (d, 1H, J = 7.5

Hz), 5.08 (s, 2H). ¹³C NMR (125 MHz, DMSO-d₆): δ 164.5, 164.1,

156.9, 150.9, 149.7, 148.6, 140.9, 137.8, 136.2, 135.1, 131.7, 131.6,

130.0, 129.7, 128.5, 128.3, 125.4, 124.3, 124.1, 122.8, 120.4, 107.8, 107.1,

104.4. HRMS (ESI) m/z calculated for C₂₅H₁₉ClF₃N₆O₂⁺[M + H]⁺:

527.1210. Found: 527.1209.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-2-ﬂuoro-5-(3-

(triﬂuoromethyl)benzamido)benzamide (17). White solids. ¹H

NMR (500 MHz, MeOD): δ 8.70 (s, 2H), 8.27 (s, 1H), 8.20 (d, 1H,

J = 7.9 Hz), 8.13 (dd, 1H, J = 2.7 Hz, J = 6.3 Hz), 7.94−7.91 (m, 1H),

7.88 (d, 1H, J = 7.8 Hz), 7.72 (t, 1H, J = 7.9 Hz), 7.27 (t, 1H, J = 9.3

Hz), 7.20 (t, 1H, J = 2.0 Hz), 7.02 (t, 1H, J = 7.9 Hz), 6.92 (dd, 1H,

J = 1.0 Hz, J = 8.1 Hz), 6.40 (dd, 1H, J = 1.3 Hz, J = 7.8 Hz), 4.56 (s,

1H). ¹³C NMR (125 MHz, MeOD): δ 167.1, 165.0, 164.9, 159.0, 158.9,

156.5, 152.4, 149.1, 142.1, 136.8, 136.4, 136.3, 132.4, 130.7, 130.3, 129.5,

127.1, 126.5, 125.6, 124.4, 123.9, 117.8, 110.9, 107.8. HRMS (ESI) m/z

calculated for C₂₅H₁₇F₄N₆O₂⁻[M − H]⁻: 509.1349. Found: 509.1355.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-3-(3-

(triﬂuoromethyl)benzamido)benzamide (18). White solids. ¹H

NMR (400 MHz, DMSO-d₆): δ 10.70 (s, 1H), 10.36 (s, 1H), 9.32 (s,

1H), 8.78 (s, 2H), 8.36 (s, 2H), 8.31 (d, 1H, J = 8.0 Hz), 8.06 (d, 1H,

J = 8.2 Hz), 8.00 (d, 1H, J = 7.8 Hz), 7.82 (t, 1H, J = 7.8 Hz), 7.77 (d,

1H, J = 7.9 Hz), 7.57 (t, 1H, J = 7.8 Hz), 7.09 (s, 1H), 6.91 (t, 1H, J =

7.7 Hz), 6.86 (d, 1H, J = 8.0 Hz), 6.19 (d, 1H, J = 7.5 Hz), 4.98 (s,

2H). ¹³C NMR (125 MHz, DMSO-d₆): δ 165.2, 164.0, 156.8, 150.7,

148.6, 141.0, 138.9, 135.4, 134.7, 131.7, 129.6, 129.0, 128.6, 128.5,

128.1, 125.6, 124.9, 124.1, 123.6, 122.7, 120.0, 107.7, 107.1, 104.3.

HRMS (ESI) m/z calculated for C₂₅H₂₀F₃N₆O₂⁺[M + H]⁺: 493.1600.

Found: 493.1648.

(s, 1H), 8.90 (s, 1H), 8.75 (s, 2H), 8.03 (s, 1H), 7.65 (s, 1H), 7.57 (d,

1H, J = 8.0 Hz), 7.51 (t, 1H, J = 7.9 Hz), 7.45 (d, 1H, J = 8.4 Hz), 7.31

(d, 1H, J = 7.4 Hz), 7.23 (d, 1H, J = 8.2 Hz), 7.07 (s, 1H), 6.89 (t, 1H,

J = 7.8 Hz), 6.84 (d, 1H, J = 8.0 Hz), 6.17 (d, 1H, J = 7.5 Hz), 4.96 (br

s, 2H), 2.34 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆): δ 167.5, 156.7,

152.4, 149.8, 148.2, 141.1, 140.4, 136.9, 136.6, 130.8, 129.7, 129.3,

128.7, 128.5, 125.8, 125.2, 121.8, 119.8, 118.0, 117.2, 114.1, 107.8,

+

107.3, 104.5, 18.5. HRMS (ESI) m/z calculated for C₂₆H₂₃F₃N₇O₂

[M + H]⁺: 522.1865. Found: 522.1852.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-2-methyl-5-

propionamidobenzamide (21). White solids. ¹H NMR (500 MHz,

DMSO-d₆): δ 10.29 (s, 1H), 9.95 (s, 1H), 9.26 (s, 1H), 8.74 (s, 2H),

7.76 (d, 1H, J = 1.6 Hz), 7.59 (dd, 1H, J = 1.8 Hz, J = 8.2 Hz), 7.22 (d,

1H, J = 8.4 Hz), 7.07 (s, 1H), 6.89 (t, 1H, J = 7.8 Hz), 6.84 (d, 1H, J =

8.2 Hz), 6.18 (d, 1H, J = 1.9 Hz), 4.92 (s, 2H), 2.35−2.30 (m, 5H),

1.09 (t, 3H, J = 7.6 Hz). ¹³C NMR (125 MHz, DMSO-d₆): δ 172.0,

167.6, 156.7, 149.8, 148.6, 141.0, 136.9, 136.4, 130.7, 129.5, 128.5, 125.8,

120.3, 117.8, 107.7, 107.1, 104.3, 29.4, 18.5, 9.5. HRMS (ESI) m/z

calculated for C₂₁H₂₃N₆O₂⁺[M + H]⁺: 391.1882. Found: 391.1913.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-5-

(cyclopropanecarboxamido)-2-methylbenzamide (22). White

solids. ¹H NMR (400 MHz, DMSO-d₆): δ 10.32 (d, 2H, J = 5.4

Hz), 9.33 (s, 1H), 8.76 (s, 2H), 7.78 (s, 1H), 7.60 (d, 1H, J = 8.2 Hz),

7.23 (d, 1H, J = 8.3 Hz), 7.08 (s, 1H), 6.90 (t, 1H, J = 7.7 Hz), 6.85 (d,

1H, J = 8.1 Hz), 6.18 (d, 1H, J = 7.6 Hz), 4.97 (s, 2H), 2.34 (s, 3H),

1.81−1.76 (m, 1H), 0.82−0.80 (m, 4H). ¹³C NMR (100 MHz, DMSO-

d₆): δ 171.7, 167.6, 156.8, 149.8, 148.8, 141.1, 137.1, 136.5, 130.9, 129.6,

128.7, 125.9, 120.2, 117.7, 107.7, 106.9, 104.2, 18.7, 14.5, 7.2. HRMS (ESI)

m/z calculated for C₂₂H₂₃N₆O₂⁺[M + H]⁺: 403.1882. Found: 403.2030.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-5-(3-chloroben-

zamido)-2-methylbenzamide (23). White solids. ¹H NMR

(500 MHz, DMSO-d₆): δ 10.43 (s, 1H), 10.34 (s, 1H), 9.27 (s,

1H), 8.77 (s, 2H), 8.04 (s, 1H), 7.95−7.94 (m, 2H), 7.82 (d, 1H, J =

8.3 Hz), 7.67 (d, 1H, J = 8.0 Hz), 7.58 (t, 1H, J = 7.9 Hz), 7.31 (d, 1H,

J = 8.4 Hz), 7.08 (s, 1H), 6.90 (t, 1H, J = 7.8 Hz), 6.86 (d, 1H, J = 8.1

Hz), 6.19 (d, 1H, J = 7.6 Hz), 4.92 (s, 2H), 2.39 (s, 3H). ¹³C NMR

(100 MHz, DMSO-d₆): δ 167.5, 164.0, 156.8, 149.9, 148.8, 141.1,

136.60, 136.57, 136.54, 133.3, 131.5, 130.9, 130.8, 130.5, 128.7, 127.3,

126.5, 125.9, 121.7, 119.2, 107.7, 107.0, 104.2, 18.8. HRMS (ESI) m/z

calculated for C₂₅H₂₂ClN₆O₂⁺[M + H]⁺: 473.1493. Found: 473.1519.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-5-(3-

(dimethylamino)benzamido)-2-methylbenzamide (24). White

1

solids. H NMR (400 MHz, DMSO-d₆): δ 10.53 (s, 1H), 10.38 (s,

1H), 9.33 (s, 1H), 8.80 (s, 2H), 8.00 (s, 1H), 7.86 (d, 1H, J = 6.7 Hz),

7.34−7.26 (m, 4H), 7.09 (s, 1H), 6.94−6.84 (m, 3H), 6.18 (d, 1H, J =

7.5 Hz), 4.97 (br s, 2H), 2.98 (s, 6H), 2.39 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 169.5, 168.0, 158.6, 152.2, 151.7, 150.7, 143.0, 138.9, 138.1,

137.3, 132.7, 132.3, 130.8, 130.6, 127.9, 123.6, 121.2, 117.2, 117.1, 113.0,

109.5, 108.8, 106.0, 26.9, 20.8. HRMS (ESI) m/z calculated for

C₂₇H₂₈N₇O₂⁺[M + H]⁺: 482.2304. Found: 482.2508.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-5-(3-ﬂuoroben-

1

zamido)-2-methylbenzamide (25). White solids. H NMR (400

MHz, DMSO-d₆): δ 10.44 (s, 1H), 10.38 (s, 1H), 9.32 (s, 1H), 8.77 (s,

2H), 7.95 (d, 1H, J = 2.1 Hz), 7.85−7.78 (m, 3H), 7.64−7.58 (m,

1H), 7.46 (td, 1H, J = 2.5 Hz, J = 8.8 Hz), 7.32 (d, 1H, J = 8.4 Hz),

7.08 (s, 1H), 6.90 (t, 1H, J = 7.9 Hz), 6.85 (d, 1H, J = 8.2 Hz), 6.18 (d,

1H, J = 7.6 Hz), 4.96 (s, 2H), 2.39 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 167.6, 164.1, 163.2, 160.8, 156.8, 149.9, 148.8, 141.2,

137.0, 136.6, 131.0, 130.7, 130.6, 128.7, 125.9, 123.9, 121.7, 119.2,

118.6, 114.4, 107.8, 107.0, 104.3, 18.8. HRMS (ESI) m/z calculated for

N³-(2-(3-Aminophenylamino)pyrimidin-5-yl)-4-methyl-N¹-

(3-(triﬂuoromethyl)phenyl)isophthalamide (19). White solids.

¹H NMR (300 MHz, MeOD): δ 8.73 (s, 2H), 8.18 (s, 1H), 8.15 (d,

1H, J = 1.8 Hz), 8.00 (dd, 1H, J = 1.9 Hz, J = 8.0 Hz), 7.94 (d, 1H, J =

8.4 Hz), 7.58−7.42 (m, 3H), 7.21 (t, 1H, J = 2.0 Hz), 7.06−6.92 (m,

2H), 6.40 (dt, 1H, J = 1.1 Hz, J = 7.8 Hz), 2.56 (s, 3H). ¹³C NMR (75

MHz, MeOD): δ 168.8, 166.3, 157.4, 150.7, 147.6, 140.8, 140.6, 139.4,

136.2, 132.0, 131.0, 130.9, 130.5, 129.3, 129.0, 128.8, 126.3, 125.4,

123.7, 120.4, 117.0, 109.7, 109.5, 106.4, 18.5. HRMS (ESI) m/z

calculated for C₂₆H₂₂F₃N₆O₂⁺[M + H]⁺: 507.1756. Found: 507.1743.

+

C₂₅H₂₂FN₆O₂[M + H]⁺: 457.1788. Found: 457.1844.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-5-(3-chloro-5-

(triﬂuoromethyl)benzamido)-2-methylbenzamide (26). White

solids. ¹H NMR (300 MHz, MeOD): δ 8.73 (s, 2H), 8.24 (d, 2H, J =

11.4 Hz), 7.95 (d, 2H, J = 2.3 Hz), 7.69 (dd, 1H, J = 2.3 Hz, J = 8.3

Hz), 7.33 (d, 1H, J = 8.4 Hz), 7.23 (t, 1H, J = 2.0 Hz), 7.05 (t, 1H, J =

7.9 Hz), 6.94 (d, 1H, J = 8.1 Hz), 6.43 (d, 1H, J = 9.0 Hz), 4.63

L

dx.doi.org/10.1021/jm4017762 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(s, 3H), 2.47 (s, 3H). ¹³C NMR (75 MHz, MeOD): δ 170.8, 165.6,

158.8, 152.0, 148.9, 142.2, 139.0, 137.6, 137.3, 136.7, 133.9, 133.7, 133.4,

132.6, 132.5, 130.3, 126.9, 124.1, 124.0, 121.0, 111.1, 111.0, 107.9, 19.3.

HRMS (ESI) m/z calculated for C₂₆H₁₉ClF₃N₆O₂⁻[M − H]⁻: 539.1210.

Found: 539.1218.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-5-(4-chloro-3-

(triﬂuoromethyl)benzamido)-2-methylbenzamide (27). White

solids. ¹H NMR (400 MHz, DMSO-d₆): δ 10.63 (s, 1H), 10.37 (s, 1H),

9.31 (s, 1H), 8.75 (s, 2H), 8.41 (d, 1H, J = 1.5 Hz), 8.28 (dd, 1H, J = 1.7

Hz, J = 8.4 Hz), 7.94 (d, 1H, J = 8.4 Hz), 7.90 (d, 1H, J = 2.0 Hz), 7.82

(dd, 1H, J = 2.0 Hz, J = 8.3 Hz), 7.33 (d, 1H, J = 8.4 Hz), 7.07 (s, 1H),

6.89 (t, 1H, J = 7.9 Hz), 6.84 (d, 1H, J = 8.2 Hz), 6.17 (d, 1H, J = 7.6

Hz), 4.96 (s, 2H), 2.38 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆): δ

167.4, 162.9, 156.8, 149.8, 148.6, 141.0, 136.5, 136.2, 133.9, 133.8, 133.2,

131.9, 130.8, 128.5, 126.8, 126.5, 125.7, 123.6, 121.7, 121.4, 119.2, 107.7,

107.0, 104.3, 18.6. HRMS (ESI) m/z calculated for C₂₆H₁₉ClF₃N₆O₂⁻

[M − H]⁻: 539.1210. Found: 539.1212.

6.73 (dt, 1H, J = 6.1 Hz, J = 15.4 Hz), 6.33 (d, 1H, J = 15.4 Hz), 3.17

(s, 2H), 2.39 (s, 3H), 2.26 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆):

δ 167.4, 163.8, 162.8, 156.4, 149.7, 140.8, 139.1, 136.4, 135.3, 131.7,

130.8, 130.7, 129.7, 129.2, 128.9, 128.4, 128.1, 127.0, 126.2, 125.2,

124.0, 122.5, 121.7, 119.1, 113.9, 112.5, 109.5, 59.2, 44.5, 18.7. HRMS

(ESI) m/z calculated for C₃₂H₃₁F₃N₇O₃⁺[M + H]⁺: 618.2440. Found:

618.2432.

2-Methyl-5-(3-(triﬂuoromethyl)benzamido)-N-(2-(3-(vinyl-

sulfonamido)phenylmino)pyrimidin-5-yl)benzamide (34).

White solids. ¹H NMR (400 MHz, DMSO-d₆): δ 10.57 (s, 1H),

10.42 (s, 1H), 9.92 (s, 1H), 9.68 (s, 1H), 8.81 (s, 2H), 8.33 (s, 1H),

8.28 (d, 1H, J = 7.8 Hz), 7.98 (d, 1H, J = 7.9 Hz), 7.94 (d, 1H, J = 1.7

Hz), 7.84 (dd, 1H, J = 1.7 Hz, J = 8.2 Hz), 7.80 (t, 1H, J = 7.7 Hz),

7.76 (s, 1H), 7.39 (d, 1H, J = 8.4 Hz), 7.34 (d, 1H, J = 8.4 Hz), 7.17 (t,

1H, J = 8.2 Hz), 6.77 (dd, 1H, J = 9.9 Hz, J = 16.4 Hz), 6.71 (d, 1H,

J = 7.9 Hz), 6.18 (d, 1H, J = 16.4 Hz), 6.06 (d, 1H, J = 9.9 Hz), 2.39 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 167.5, 163.9, 156.4, 149.8,

141.3, 138.0, 136.5, 136.3, 135.5, 131.8, 131.0, 130.9, 129.8, 129.4, 129.0,

128.9, 128.2, 127.6, 126.4, 124.1, 122.6, 121.8, 119.3, 113.9, 112.5, 109.6,

18.8. HRMS (ESI) m/z calculated for C₂₈H₂₄F₃N₆O₄S⁺[M + H]⁺:

597.1532. Found: 597.1516.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-2-methyl-5-(4-

(triﬂuoromethyl)benzamido)benzamide (28). White solids. ¹H

NMR (400 MHz, DMSO-d₆): δ 10.60 (s, 1H), 10.39 (s, 1H), 9.36 (s,

1H), 8.78 (s, 2H), 8.18 (d, 2H, J = 8.2 Hz), 7.97 (d, 1H, J = 1.8 Hz),

7.94 (d, 2H, J = 8.3 Hz), 7.83 (dd, 1H, J = 1.8 Hz, J = 8.3 Hz), 7.33 (d,

1H, J = 8.4 Hz), 7.12 (s, 1H), 6.94−6.87 (m, 2H), 6.21 (d, 1H, J = 7.3

Hz), 5.22 (br s, 2H), 2.40 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

169.4, 166.2, 158.7, 151.7, 150.1, 143.0, 140.3, 138.4, 138.3, 132.8,

132.7, 130.6, 130.4, 127.8, 127.3, 127.2, 124.4, 123.6, 121.1, 109.8,

N-(2-(3-(2-Chloroacetamido)phenylamino)pyrimidin-5-yl)-2-

methyl-5-(3-(triﬂuoromethyl)benzamido)benzamide (35).

White solids. ¹H NMR (400 MHz, DMSO-d₆): δ 10.58 (s, 1H),

10.43 (s, 1H), 10.25 (s, 1H), 9.70 (s, 1H), 8.82 (s, 2H), 8.33 (s, 1H),

8.28 (d, 1H, J = 7.9 Hz), 8.01−7.94 (m, 3H), 7.86−7.78 (m, 2H), 7.41

(d, 1H, J = 8.1 Hz), 7.35−7.28 (m, 2H), 7.21 (t, 1H, J = 8.1 Hz), 4.26

(s, 2H), 2.39 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 167.5,

164.5, 163.9, 156.5, 149.8, 141.0, 138.6, 136.5, 135.5, 131.8, 130.9,

130.8, 129.8, 129.4, 129.0, 128.7, 128.2, 126.4, 124.2, 124.1, 121.8,

119.3, 114.3, 112.5, 109.5, 43.6, 18.8. HRMS (ESI) m/z calculated for

+

109.2, 106.4, 20.7. HRMS (ESI) m/z calculated for C₂₆H₂₂F₃N₆O₂

[M + H]⁺: 507.1756. Found: 507.1754.

N-(2-(3-Aminophenylamino)pyrimidin-5-yl)-5-cinnamamido-

2-methylbenzamide (29). White solids. ¹H NMR (300 MHz,

DMSO-d₆): δ 10.37 (d, 2H, J = 3.0 Hz), 9.41 (s, 1H), 8.77 (s, 2H),

7.87 (d, 1H, J = 2.2 Hz), 7.70 (dd, 1H, J = 2.1 Hz, J = 8.3 Hz), 7.65−

7.56 (m, 3H), 7.47−7.40 (m, 3H), 7.27 (d, 1H, J = 8.7 Hz), 7.20 (s,

1H), 6.96 (d, 2H, J = 5.0 Hz), 6.81 (d, 1H, J = 15.8 Hz), 6.28 (s, 1H),

2.35 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 168.0, 164.0, 157.1,

153.8, 150.4, 150.1, 146.0, 141.7, 140.8, 137.4, 137.0, 135.0, 131.5,

130.6, 130.3, 129.5, 129.3, 128.2, 127.8, 126.5, 122.5, 121.0, 118.4,

−

C₂₈H₂₁ClF₃N₆O₃[M − H]⁻: 581.1316. Found: 581.1323.

2-Methyl-N-(2-(3-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-

acetamido)phenylamino)pyrimidin-5-yl)-5-(3-(triﬂuoromethyl)-

1

benzamido)benzamide (36). White solids. H NMR (400 MHz,

DMSO-d₆): δ 10.59 (s, 1H), 10.44 (s, 1H), 10.32 (s, 1H), 9.70 (s,

1H), 8.83 (s, 2H), 8.34 (s, 1H), 8.29 (d, 1H, J = 7.8 Hz), 8.05 (s, 1H),

8.01−7.98 (m, 2H), 7.94 (d, 1H, J = 1.8 Hz), 7.91 (d, 1H, J = 7.8 Hz),

7.86 (dd, 1H, J = 4.0 Hz, J = 8.4 Hz), 7.81 (t, 1H, J = 7.8 Hz), 7.72 (t,

1H, J = 7.2 Hz), 7.46 (t, 1H, J = 7.3 Hz), 7.39 (d, 1H, J = 8.2 Hz), 7.34

(d, 1H, J = 8.4 Hz), 7.29 (d, 1H, J = 8.2 Hz), 7.21 (t, 1H, J = 8.0 Hz),

4.71 (s, 2H), 2.40 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 167.5,

165.2, 164.9, 163.9, 156.5, 149.7, 141.4, 140.9, 138.7, 136.5, 135.4,

131.9, 131.8, 130.9, 130.8, 129.8, 129.3, 129.0, 128.7, 128.2, 126.4,

125.6, 125.3, 124.2, 124.1, 123.4, 122.6, 121.7, 119.2, 114.0, 112.3,

109.3, 46.1, 18.8. HRMS (ESI) m/z calculated for C₃₅H₂₇F₃N₇O₄S⁺

[M + H]⁺: 698.1797. Found: 698.1786.

N-(2-(3-(N-Acryloylacrylamido)phenylamino)pyrimidin-5-

yl)-2-methyl-5-(3-(triﬂuoromethyl)benzamido)benzamide (37).

White solids. ¹H NMR (500 MHz, DMSO-d₆): δ 10.89 (s, 1H), 10.60

(s, 1H), 10.24 (s, 1H), 9.13 (s, 2H), 8.32 (s, 1H), 8.28 (d, 1H, J = 7.9

Hz), 7.98 (d, 2H, J = 7.0 Hz), 7.86−7.79 (m, 2H), 7.61−7.59 (d, 2H,

J = 8.6 Hz), 7.39−7.35 (m, 2H), 6.92 (d, 1H, J = 8.4 Hz), 6.44−6.24

(m, 4H), 5.78−5.75 (m, 2H), 2.40 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 168.0, 165.6, 163.9, 163.2, 155.6, 149.3, 141.1, 139.7, 136.5,

135.8, 135.4, 132.1, 131.8, 131.6, 131.0, 130.9, 130.3, 129.7, 129.3, 128.2,

127.1, 125.2, 124.1, 124.0, 122.5, 122.1, 119.3, 18.7. HRMS (ESI) m/z

calculated for C₃₂H₂₆F₃N₆O₄⁺[M + H]⁺: 615.1968. Found: 615.2026.

N-(2-(3-(2-Acrylamidoacetamido)phenylamino)pyrimidin-5-

yl)-2-methyl-5-(3-(triﬂuoromethyl)benzamido)benzamide (38).

White solids. ¹H NMR (500 MHz, DMSO-d₆): δ 10.57 (s, 1H), 10.41

(s, 1H), 9.99 (s, 1H), 9.64 (s, 1H), 8.81 (s, 2H), 8.43 (t, 1H, J = 5.7

Hz), 8.33 (s, 1H), 8.28 (d, 1H, J = 7.8 Hz), 8.03 (s, 1H), 7.98 (d, 1H,

J = 7.8 Hz), 7.93 (s, 1H), 7.85 (d, 1H, J = 8.5 Hz), 7.81 (t, 1H, J = 7.8

Hz), 7.37−7.33 (m, 2H), 7.27 (d, 1H, J = 8.0 Hz), 7.19 (t, 1H, J = 8.1

Hz), 6.36 (dd, 1H, J = 10.3 Hz, J = 17.1 Hz), 6.12 (d, 1H, J = 17.0

Hz), 5.63 (d, 1H, 10.4 Hz), 4.00 (d, 2H, J = 5.8 Hz), 2.39 (s, 3H). ¹³C

NMR (125 MHz, DMSO-d₆): δ 167.5, 167.3, 164.9, 163.9, 156.5,

149.8, 140.8, 138.9, 136.4, 136.3, 135.4, 131.6, 131.4, 130.8, 130.7,

129.6, 128.4, 128.1, 126.2, 125.3, 124.9, 124.0, 122.7, 121.7, 119.2,

+

19.2. HRMS (ESI) m/z calculated for C₂₇H₂₅N₆O₂[M + H]⁺:

465.2039. Found: 465.2037.

N-(3-(2-(3-Aminophenylamino)pyrimidin-5-ylcarbamoyl)-4-

methylphenyl)-2-naphthamide (30). White solids. ¹H NMR

(400 MHz, DMSO-d₆): δ 10.56 (s, 1H), 10.40 (s, 1H), 9.34 (s,

1H), 8.79 (s, 1H), 8.62 (s, 1H), 8.11−7.89 (m, 6H), 7.88 (d, 1H, J =

7.83 Hz), 7.65 (s, 2H), 7.33 (d, 1H, J = 8.4 Hz), 7.11 (s, 1H), 6.92−

6.88 (m, 2H), 6.20 (d, 1H, J = 7.4 Hz), 5.16 (br s, 2H), 2.40 (s, 3H).

¹³C NMR (125 MHz, DMSO-d₆): δ 169.5, 167.4, 158.7, 151.7, 150.5,

143.0, 138.8, 138.4, 136.2, 133.9, 133.8, 132.8, 132.3, 130.8, 130.6,

129.9, 129.8, 129.7, 129.6, 128.8, 127.8, 126.2, 123.5, 120.9, 109.7,

+

109.0, 106.2, 20.7. HRMS (ESI) m/z calculated for C₂₉H₂₅N₆O₂[M

+ H]⁺: 489.2039. Found: 489.2048.

(E)-N-(2-(3-But-2-enamidophenylamino)pyrimidin-5-yl)-2-

methyl-5-(3-(triﬂuoromethyl)benzamido)benzamide (32).

1

White solids. H NMR (400 MHz, MeOD): δ 8.76 (s, 2H), 8.27 (s,

1H), 8.21 (d, 1H, J = 7.7 Hz), 8.11 (s, 1H), 7.94 (s, 1H), 7.89 (d, 1H,

J = 7.7 Hz), 7.73 (t, 1H, J = 7.8 Hz), 7.68 (dd, 1H, J = 1.9 Hz, J = 8.3

Hz), 7.35−7.32 (m, 2H), 7.27 (d, 1H, J = 7.8 Hz), 7.22 (t, 1H, J = 8.0

Hz), 6.91 (dt, 1H, J = 7.0 Hz, J = 15.2 Hz), 6.15 (dd, 1H, J = 1.3 Hz,

J = 15.2 Hz), 2.46 (s, 3H), 1.91 (d, 3H, J = 6.8 Hz). ¹³C NMR

(100 MHz, MeOD): δ 170.8, 167.1, 166.7, 158.6, 152.0, 142.1, 140.3,

137.6, 137.5, 137.1, 133.6, 132.5, 132.3, 131.9, 130.7, 129.9, 129.5,

129.4, 127.3, 126.7, 125.6, 125.5, 124.1, 121.1, 116.2, 115.2, 112.2,

19.3, 17.9. HRMS (ESI) m/z calculated for C₃₀H₂₆F₃N₆O₃⁺[M + H]⁺:

575.2018. Found: 575.2015.

(E)-N-(2-(3-(4-(Dimethylamino)but-2-enamido)phenylamino)-

pyrimidin-5-yl)-2-methyl-5-(3-(triﬂuoromethyl)benzamido)-

1

benzamide (33). White solids. H NMR (400 MHz, DMSO-d₆): δ

10.57 (s, 1H), 10.41 (s, 1H), 10.02 (s, 1H), 9.62 (s, 1H), 8.81 (s, 2H),

8.32 (s, 1H), 8.28 (d, 1H, J = 7.9 Hz), 8.06 (s, 1H), 7.98 (d, 1H, J =

7.8 Hz), 7.93 (d, 1H, J = 1.9 Hz), 7.83 (dd, 1H, J = 2.0 Hz, J = 8.3 Hz),

7.80 (t, 1H, J = 7.8 Hz), 7.38−7.33 (m, 3H), 7.19 (t, 1H, J = 8.1 Hz),

M

dx.doi.org/10.1021/jm4017762 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

113.8, 112.5, 109.5, 42.6, 18.6. HRMS (ESI) m/z calculated for

In Vivo Pharmacodynamic Study. Female CB17/SCID mice

were injected subcutaneously with 5 × 10⁶DoHH2 cells. When

tumors reached ∼167 mm³on average, mice were randomized into

four groups (n = 6/group) and daily dosing began. Compounds were

administered via tail vein as a clear solution in polyoxyl 15 hydro-

xystearate (Solutol)/ethanol (1:1). Tumor size and body weight were

monitored periodically for 14 days. Statistical analysis was conducted

using one-way ANOVA. The Dunnett test was used to analyze the

statistical signiﬁcance between each treatment group and the vehicle

group. P < 0.05 is considered statistically signiﬁcant. All animal studies

were conducted under approved animal care protocols and AAALAC

standards.

+

C₃₁H₂₇F₃N₇O₄[M + H]⁺: 618.2077. Found: 618.2086.

N-(2-(3-Acrylamidophenylamino)pyrimidin-5-yl)-5-(3-ﬂuoro-

benzamido)-2-methylbenzamide (39). White solids. ¹H NMR

(300 MHz, DMSO-d₆): δ 10.45 (s, 2H), 10.12 (s, 1H), 9.69 (s, 1H),

8.82 (s, 2H), 8.08 (s, 1H), 7.96 (s, 1H), 7.85−7.77 (m, 3H), 7.60 (dd,

1H, J = 7.7 Hz, J = 13.6 Hz), 7.49−7.31 (m, 4H), 7.21 (t, 1H, J = 7.9 Hz),

6.48 (dd, 1H, J = 10.0 Hz, J = 16.9 Hz), 6.25 (d, 1H, J = 17.3 Hz), 5.74

(d, 1H, J = 10.8 Hz), 2.38 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆): δ

167.5, 164.0, 163.0, 162.8, 160.9, 156.5, 149.8, 140.8, 139.0, 136.8, 136.5,

132.0, 130.8, 130.6, 130.5, 128.5, 126.3, 123.7, 121.7, 119.1, 118.5, 118.3,

114.4, 114.2, 112.8, 109.7, 18.6. HRMS (ESI) m/z calculated for

C₂₈H₂₃FN₆O₃Na⁺[M + Na]⁺: 533.1713. Found: 533.1705.

N-(2-(3-Acrylamidophenylamino)pyrimidin-5-yl)-5-(4-

chloro-3-(triﬂuoromethyl)benzamido)-2-methylbenzamide

(40). White solids. ¹H NMR (500 MHz, DMSO-d₆): δ 10.62 (s, 1H),

10.41 (s, 1H), 10.08 (s, 1H), 9.65 (s, 1H), 8.82 (s, 2H), 8.43 (s, 1H), 8.29

(d, 1H, J = 8.4 Hz), 8.08 (s, 1H), 7.94 (d, 1H, J = 8.4 Hz), 7.92 (s, 1H),

7.84 (d, 1H, J = 8.3 Hz), 7.39 (t, 2H, J = 6.9 Hz), 7.34 (d, 1H, J = 8.3

Hz), 7.21 (t, 1H, J = 8.1 Hz), 6.48 (dd, 1H, J = 10.2 Hz, J = 16.9 Hz),

6.26 (d, 1H, J = 17.0 Hz), 5.74 (d, 1H, J = 10.2 Hz), 2.40 (s, 3H). ¹³C

NMR (125 MHz, DMSO-d₆): δ 168.0, 165.5, 163.9, 163.1, 155.5, 149.2,

141.1, 139.7, 136.4, 135.8, 135.4, 132.0, 131.6, 131.5, 130.9, 130.8, 130.2,

129.6, 129.2, 128.1, 126.9, 124.0, 122.5, 122.1, 119.3, 118.2, 117.8, 18.6.

HRMS (ESI) m/z calculated for C₂₉H₂₁ClF₃N₆O₃⁻[M − H]⁻: 593.1316.

Found: 593.1338.

ASSOCIATED CONTENT

* Supporting Information

Syntheses of compounds 11, 19, and 20, modeling study, and

detailed procedures and data for biological assays. This material

is available free of charge via the Internet at http://pubs.acs.org.

■

S

AUTHOR INFORMATION

Corresponding Author

*Phone: 86-755-26033072. E-mail: panzy@pkusz.edu.cn.

Notes

■

The authors declare no competing ﬁnancial interest.

N-(3-(2-(3-Acrylamidophenylamino)pyrimidin-5-ylcarbamo-

1

yl)-4-methylphenyl)-2-naphthamide (41). White solids. H NMR

ACKNOWLEDGMENTS

(400 MHz, DMSO-d₆): δ 10.56 (s, 1H), 10.45 (s, 1H), 10.11 (s, 1H),

9.68 (s, 1H), 8.85 (s, 2H), 8.62 (s, 1H), 8.11−8.02 (m, 6H), 7.88 (dd,

1H, J = 1.9 Hz, J = 8.3 Hz), 7.66−7.64 (m, 2H), 7.39 (d, 2H, J = 8.0

Hz), 7.34 (d, 1H, J = 8.4 Hz), 7.22 (t, 1H, J = 8.0 Hz), 6.50 (dd, 1H,

J = 10.1 Hz, J = 16.9 Hz), 6.25 (dd, 1H, J = 1.7 Hz, J = 17.0 Hz), 5.75

(dd, 1H, J = 1.8 Hz, J = 10.2 Hz), 2.41 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 169.5, 167.4, 164.9, 158.4, 151.7, 142.8, 141.0, 138.8,

138.4, 136.2, 133.9, 133.8, 133.7, 132.8, 132.4, 130.8, 130.5, 129.9, 129.8,

129.7, 129.5, 128.8, 128.5, 128.3, 126.2, 123.5, 121.0, 116.0, 114.6, 111.4,

20.7. HRMS (ESI) m/z calculated for C₃₂H₂₇N₆O₃⁺[M + H]⁺: 543.2145.

Found: 543.2126.

■

Authors thank the scientists at Crown Biosciences at Beijing

and Taicang, China, for their great eﬀorts in biological assays,

and Professor Junmin Quan at Peking University, China, for

helpful discussions. The following ﬁnancial support is gratefully

acknowledged: to Z.P., Grants 2013CB910700 (973 Program),

81373270 and 21142005 (National Natural Science Foundation

of China), KQTD201103, CXB201005260059A and JC201-

005270281A (Shenzhen Municipal Science and Technology

Innovation Council), and 20100001120030 (Ministry of Educa-

tion); to N.D., Grants 81201873 (National Natural Science

Foundation of China) and 7132050 (Beijing Natural Science

Foundation).

Biological Assays. Kinase Enzymology Assays. Kinases were

purchased from Carna Biosciences. Kinase enzymology assays were

performed according to the protocols speciﬁed in HTRF KinEase

assays sold by Cisbio Bioassays.

Cellular Phosphorylation Assays. The assays were performed

using a similar procedure as described in literature.¹⁵5 × 10⁵Ramos

cells were seeded in a 12-well plate overnight. After being treated with

compounds for 1 h, cells were stimulated with goat anti-human IgM

antibody for 3−5 min. After centrifugation, cells were lysed by boiling

in SDS buﬀer. Proteins were analyzed by Western blot. Data were

analyzed with ImageJ and Excel.

Cell Viability Assay. Lymphoma cells were seeded in 96-well plates

(5000 cells/well), incubated for 24 h, then treated with compounds for

72 h, and measured with CellTiter-Glo luminescent cell viability assay by

Promega. Data analysis was performed with GraphPad Prism 5.0.

Stability Test in FaSSIF. FaSSIF solutions of compounds in a

96-well plate were incubated on a minishaker at 350 rpm at 37 °C. At

each deﬁned time point (2, 6, 10, and 24 h), samples were collected

and measured with LC−MS method.

ABBREVIATIONS USED

■

ABL, asbelson murine leukemia viral oncogene; Bmx, bone

marrow tyrosine kinase gene in chromosome X protein; Blk, B

lymphoid tyrosine kinase; DIEA, ethyldiisopropylamine;

GI₅₀, the concentration for 50% of maximal inhibition of cell

proliferation; GSK3β, glycogen synthase kinase 3β; HATU,

(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexaﬂuorophosphate); HER, human epidermal growth factor

receptor; HTRF, homogeneous time-resolved ﬂuorescence; Itk,

IL2-inducible T-cell kinase; Jak, Janus kinase; JNK, c-Jun N-terminal

kinase; Lck, lymphocyte-speciﬁc protein tyrosine kinase;

MAP2K7, dual speciﬁcity mitogen-activated protein kinase kinase

7; Nek2, “never in mitosis gene a”-related kinase 2; NIMA, never

in mitosis gene a; PDGFR, platelet-derived growth factor;

PLC-γ2, 1-phosphatidylinositol 4,5-bisphosphate phosphodiester-

ase γ2; Rlk, resting lymphocyte kinase; RSK, ribosomal s6 kinase;

Syk, spleen tyrosine kinase; S_NAr, nucleophilic aromatic

substitution

Mouse Liver Microsome Stability Study. Compounds were

incubated with liver microsomes and NADPH at 37 °C. Samples were

collected at diﬀerent time points and centrifuged, and the supernatants

were analyzed with LC−MS/MS. First-order kinetics was used to

calculate t_1/2and CL. CL^hepwas derived from the equation CL^hep

=

(CL)(Q)/(CL + Q), where Q is the estimated liver blood ﬂow of mice

at 90 mL min⁻¹kg⁻¹. Testosterone, propranolol, and clozapine were

used as positive controls.

In Vivo Pharmacokinetic Study. The pharmacokinetics of

compounds 31 and 38 were evaluated in ICR mice (n = 3) following

intravenous (iv) injection at the 1 mg/kg dose level. At various time

points, blood samples were taken and analyzed by the LC−MS method.

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Discovery of a series of 2,5-diaminopyrimidine covalent irreversible inhibitors of Bruton's tyrosine kinase with in vivo antitumor activity

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