Identification of Purines and 7-Deazapurines as Potent and Selective Type i Inhibitors of Troponin I-Interacting Kinase (TNNI3K)

Article

pubs.acs.org/jmc

Identiﬁcation of Purines and 7‑Deazapurines as Potent and Selective

Type I Inhibitors of Troponin I‑Interacting Kinase (TNNI3K)

Brian G. Lawhorn,*^,†Joanne Philp,^†Yongdong Zhao,^†Christopher Louer,^†Marlys Hammond,^†

Mui Cheung,^†Harvey Fries,^†Alan P. Graves,^‡Lisa Shewchuk,^‡Liping Wang,^‡Joshua E. Cottom,^‡

Hongwei Qi,^‡Huizhen Zhao,^‡Rachel Totoritis,^‡Guofeng Zhang,^‡Benjamin Schwartz,^‡Hu Li,^‡

Sharon Sweitzer,^‡Dennis A. Holt,^†Gregory J. Gatto, Jr.,^†and Lara S. Kallander^†

^†Heart Failure Discovery Performance Unit and ^‡Platform Technology and Sciences, GlaxoSmithKline, 709 Swedeland Road, King of

Prussia, Pennsylvania 19406, United States

S

* Supporting Information

ABSTRACT: A series of cardiac troponin I-interacting kinase

(TNNI3K) inhibitors arising from 3-((9H-purin-6-yl)amino)-N-

methyl-benzenesulfonamide (1) is disclosed along with fundamental

structure−function relationships that delineate the role of each

element of 1 for TNNI3K recognition. An X-ray structure of 1

bound to TNNI3K conﬁrmed its Type I binding mode and is used

to rationalize the structure−activity relationship and employed to

design potent, selective, and orally bioavailable TNNI3K inhibitors.

Identiﬁcation of the 7-deazapurine heterocycle as a superior template (vs purine) and its elaboration by introduction of C4-

benzenesulfonamide and C7- and C8−7-deazapurine substituents produced compounds with substantial improvements in

potency (>1000-fold), general kinase selectivity (10-fold improvement), and pharmacokinetic properties (>10-fold increase in

poDNAUC). Optimal members of the series have properties suitable for use in in vitro and in vivo experiments aimed at

elucidating the role of TNNI3K in cardiac biology and serve as leads for developing novel heart failure medicines.

mentary to the Type II compounds derived from sorafenib

reported from our laboratories (Figure 1).^3,6Fundamental

structure−function relationships are presented along with a

cocrystal structure of 1 bound to the ATP binding site of

TNNI3K, and these results are rationalized and used to design

potent, selective, and orally bioavailable TNNI3K inhibitors.

INTRODUCTION

■

Cardiac troponin I-interacting kinase (TNNI3K or CARK) is a

member of the tyrosine-like kinase (TLK) family that is

selectively expressed in heart tissue, but its biological function is

poorly deﬁned.^1,2Although TNNI3K is a functional kinase

capable of autophosphorylation, interacts with components of

the sarcomere including cardiac troponin I, and has been linked

to activation of the p38 MAP kinase pathway, no genuine

substrates of TNNI3K have been identiﬁed to date.¹⁻³In

addition, the functional role of its autophosphorylation activity

and the details of how TNNI3K regulates p38 and other

unidentiﬁed signaling pathways are unclear. Overexpression of

Tnni3k triggers in vitro cardiac hypertrophy of neonatal rat

ventricular myocytes and exacerbates disease progression in

multiple in vivo settings including models of dilated

cardiomyopathy, pressure overload-induced heart failure, and

ischemia/reperfusion injury.³⁻⁵In complementary studies, a

Tnni3k knockout mouse exhibited reduced ischemic injury

demonstrating that deletion of TNNI3K is cardioprotective.³

These studies suggest that TNNI3K inhibition could serve as

a unique strategy for addressing acute ischemic injury and heart

failure. Consequently, we sought to identify selective inhibitors

that could be used to elucidate the cardiac biology of TNNI3K

and serve as templates for the development of novel heart

failure therapeutics. Herein, we provide the initial disclosure of

a series of TNNI3K inhibitors arising from 3-((9H-purin-6-

yl)amino)-N-methyl-benzenesulfonamide (1) that are comple-

RESULTS AND DISCUSSION

■

Hit Identiﬁcation. Evaluation of selected compounds from

the GSK kinase inhibitor collection against the kinase panel

assembled by Ambit, which includes TNNI3K, uncovered 1

(Figure 1) as a promising TNNI3K binder (K_d= 111 nM), and

further evaluation of 1 in our own TNNI3K assay conﬁrmed its

aﬃnity for TNNI3K (IC₅₀= 500 nM).⁷Of particular interest,

purine 1 exhibits excellent selectivity for TNNI3K against a

broad spectrum of kinases, binding signiﬁcantly to only 4% of

kinases (8 of 203 in Ambit panel) at 10 μM inhibitor

concentration. Compound 1 displays potent activity (IC₅₀= 32

nM for B-Raf V600E) against the closely related serine/

threonine-speciﬁc protein kinases B-Raf and c-Raf, which share

67% sequence identity (82% similarity) with TNNI3K among

residues comprising their ATP-binding sites. Raf kinase

inhibition has been linked to eﬀects in heart failure models,

and although this was not viewed as a prohibitive liability for 1,

Received: June 16, 2015

A

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Type I and II TNNI3K inhibitors.

the development of inhibitors selective against Raf kinases

would be desireable, especially in producing tools to character-

ize the biology of TNNI3K.⁸

Structure of 1 Bound to TNNI3K. A cocrystal structure of

1 bound to TNNI3K was solved to 2.7 Å resolution and

revealed that 1 resides in the ATP binding site (Figure 2). In

the presence of 1, the activation loop exists in the active

conformation rather than the alternative “DFG-out” orienta-

tion, indicating that 1 behaves as a Type I kinase inhibitor. The

purine ring of 1 contacts the kinase hinge recognition feature of

TNNI3K, with N3 serving as an H-bond acceptor for the

Ile542 backbone amide NH, while N9−H engages in a H-bond

donor interaction with the Ile542 backbone amide CO. The

benzenesulfonamide moiety projects toward the back pocket

and participates in three hydrogen bonding interactions. The

sulfonamide NH serves as a hydrogen bond donor to the side

chain OH of Thr539, which is the gatekeeper residue of

TNNI3K, while the sulfonamide oxygen atoms interact with the

backbone NH of Asp606 and an active site water that bridges

the Phe607 backbone NH and the carboxylate of Glu509. As a

consequence of this hydrogen bond network, the sulfonamide

N-Me group is directed upward where it is situated snugly into

a small, lipophilic pocket formed by Ala488, Ile489, Lys490,

and Ile537. In order to simultaneously accommodate the two

purine hydrogen bond contacts and the three sulfonamide

hydrogen bond interactions, the purine and benzene rings

adopt a coplanar binding conformation (dihedral angle ∼12°).

Eﬀect of Modiﬁcations of the Purine Moiety. An initial

set of derivatives of 1 were evaluated to deﬁne the role of each

of its features for TNNI3K recognition. Modiﬁcations of the

purine (Figure 3) included N9-methylation, which produced an

inactive compound (2, IC₅₀> 25 000 nM), suggesting that the

purine N9−H plays a key role, consistent with the cocrystal

structure of 1 bound to TNNI3K which reveals that the N9−H

Figure 3. Eﬀect of modifying the purine moiety of 1.

donates a hydrogen bond to the backbone carbonyl of Ile542.

Both electron-withdrawing (e.g., Cl, 5, IC₅₀= 16 000 nM) and

electron-donating (e.g., NH₂, 4, IC₅₀= 8000 nM) C2-

substituents displayed signiﬁcantly reduced TNNI3K activity

in a manner that correlates with substituent size (IC₅₀: H (500

nM) > F (3200 nM) > NH₂(8000 nM) > Cl (16 000 nM))

indicating a steric eﬀect where this portion of the molecule

contacts TNNI3K. Indeed, the structure shows C2 of 1 lying

within 3.5 Å of Gln540. Translocation of N7 to N8 (6, IC₅₀

=

50 nM) or removal of N7 (7, IC₅₀= 80 nM) improved

TNNI3K activity, and the apparent detrimental eﬀect of N7

was initially attributed to its desolvation upon binding to

TNNI3K, where the N7 atom projects into a hydrophobic

region and lacks a hydrogen bonding partner (Figure 2). The

tautomeric eﬀects unique to the purine ring system may also

disrupt TNNI3K recognition. In contrast to purine 1, the

pyrazolo[3,4-d]pyrimidine of 6 and pyrrolo[2,3-d]pyrimidine of

7 are predisposed for binding, as they lack the competing 7H-

purine tautomer (Figure 4) available to 1 that is unable to

engage the N9−H hydrogen bond donor interaction observed

as the 9H-purine tautomer of 1 binds to the TNNI3K active

Figure 2. Cocrystal structure of 1 (green) bound to TNNI3K (gray).

B

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 4. Structure-based rationalization of key results.

site (Figure 2).⁹Thus, the 7H-purine tautomer would introduce

a repulsive electrostatic interaction with the carbonyl of Ile542,

and the absence of this complicating factor in 6 and 7 would

enhance their interaction with TNNI3K.

Further alteration of 7 by elimination of the purine N1

nitrogen to give the pyrrolo[2,3-b]pyridine 9 resulted in a

substantial reduction in activity (100-fold) indicative of a

pivotal function for the N1 nitrogen atom. Structural

information revealed no binding contacts between N1 and

TNNI3K. However, N1 does appear to allow 1 to assume a

coplanar orientation that would be energetically disfavored by 9

(see Figure 4), but which is essential for TNNI3K binding by

this scaﬀold, as illustrated by the cocrystal structure (Figure 2).

Thus, N1 likely imparts a signiﬁcant conformational eﬀect on

binding of 1 to TNNI3K, and the consequences that arise from

this eﬀect along with other conformational considerations will

be further elaborated in a separate report. The 6-oxo-6,7-

dihydro-5H-pyrrolo[2,3-d]pyrimidine 10 exhibited activity

similar to purine 1, and was 5-fold less active than its parent

7. Notably, 10 shares a similar electronic arrangement with 1,

and also would suﬀer from the partial desolvation of its

carbonyl upon TNNI3K binding because one of the carbonyl

lone pairs is directed toward Gly544, preventing its interaction

with solvent water.

Eﬀect of Benzenesulfonamide Substitutions. An

assessment of substituted benzenesulfonamides demonstrated

that this moiety is also sensitive to modiﬁcation (Figure 5),

consistent with its intimate contact within the TNNI3K active

site (Figure 2). Extension of the N-Me group of 1 to the N-Et

reduced aﬃnity (11, IC₅₀= 2000 nM) implying a steric

limitation, whereas removal of the N-Me group to give 12 (IC₅₀

= 40 000 nM) resulted in an 80-fold reduction in activity.

Furthermore, the N-Me group could not be replaced with polar

atoms such as OH (13, IC₅₀= 20 000 nM). These results are

strong evidence that the N-Me group makes hydrophobic

contact within TNNI3K, consistent with the TNNI3K-

compound 1 costructure observations where this methyl

group is situated comfortably in a small, lipophilic pocket

formed by Ala488, Ile489, Lys490, and Ile537. The magnitude

of binding energy change (2.6 kcal/mol using ΔG₁− ΔG₂=

RT*ln(K₁/K₂) where K is approximated by IC₅₀values) arising

from this N-Me group is beyond the level usually observed for

ﬁlling a hydrophobic pocket (1.4 kcal/mol) and may reﬂect

Figure 5. Eﬀect of benzenesulfonamide substitutions.

additional energetic advantages of the N-Me functionality of

1.¹⁰Notably, 12 would presumably suﬀer a signiﬁcant penalty

for desolvation of one of its sulfonamide NH atoms which has

no apparent H-bonding partner upon binding to TNNI3K,

whereas the sulfonamide of 1 engages in a full complement of

H-bonding interactions upon binding.

Methylation (14, IC₅₀= 8000 nM) or removal (15, IC₅₀

=

6300 nM) of the sulfonamide NH produced similar reductions

in potency (∼15-fold) as anticipated from its role as a hydrogen

bond donor (Figure 2). The 6-Me substituted benzenesulfo-

namide 16 is substantially less active than 1, demonstrating that

this position of the benzenesulfonamide is near the wall of the

binding pocket, and crystallography results conﬁrm that C6

projects toward the catalytic side chains of Lys490 and Asp606.

In contrast, 4-Me-substituted benzenesulfonamide 17 is

equipotent to 1, consistent with the observation that the C4

position of the benzene points toward the opening of the

pocket and is thus available for further exploration.

The 7-deazapurine template 7 provided the opportunity to

expand the SAR generated from the purine scaﬀold by

examining two analogues modiﬁed at the nitrogen atom

which links the two aromatic groups (Figure 5). Both the N-

Me variant 18 (IC₅₀= 5000 nM) and the O-linked 19 (IC₅₀

=

16 000 nM) exhibited substantial reductions in TNNI3K

aﬃnity. In the case of 18, we hypothesize that this arises

from disruption of the coplanar binding conformation observed

in the crystal structure of 1 bound to TNNI3K (Figure 2). In a

coplanar orientation, the N-Me group of 18 would interpose

C

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

the neighboring benzene C4−H and deazapurine C7−H atoms,

forcing at least one severe H/H overlapping interaction (Figure

4), and thereby cause 18 to adopt an alternate, ineﬃcient

orientation upon binding to TNNI3K.¹¹The O-linked 19 is

also anticipated to favor an orthogonal alignment of its aryl

rings, as has been observed for similar diaryl ethers, whose

coplanar conformations are disfavored as a result of the

increased aryl−aryl repulsion arising from the smaller linking

oxygen atom.¹²Thus, an orthogonal conformation of 19 allows

optimal overlap between the oxygen p-orbital lone pair and the

π-system of the electron-deﬁcient pyrimidine ring while

minimizing steric repulsion between the two aryl rings (Figure

4). Finally, the 5-pyridyl analogue 20 was examined and

displayed activity (IC₅₀= 250 nM) only 3-fold less than 7,

consistent with structural evidence that shows the C5 position

approaches the opening of the pocket and points toward

solvent space (Figure 2).

34%) compared to 1, which displayed no measurable oral

exposure and could not be dosed intravenously due to its poor

solubility in the dosing vehicle. The heightened solubility of 7

may arise from its augmented basicity (calculated BH⁺pK_a=

5.2) versus 1 (calculated BH⁺pK_a= 3.2), and this is in contrast

to the potent pyrazolopyrimidine 6 (calculated BH⁺pK_a= 4.0),

which like 1, displays poor solubility and low oral bioavailability

(Cl = 72 mL/min/kg, F = 5%). Deazapurine 7 maintains the

excellent broad-spectrum kinase selectivity of 1, showing >100-

fold selectivity against 84% (155/185) and >10-fold selectivity

against 97% (180/185) of kinases tested in the Millipore

KinaseProﬁler panel (Millipore.com, see Supporting Informa-

tion for details). Like 1, compound 7 manifests potent activity

against the B-Raf oncogenic mutant (V600E), though, in the

case of 7, it is nearly equipotent against TNNI3K, whereas

compound 1 favors B-Raf inhibition by 15-fold, implying that 7

is a preferred scaﬀold for divergence into TNNI3K speciﬁc

binding. An additional beneﬁt of 7 is that it permits the

appendage of substituents from both the C7 and C8 ring

positions into the unﬁlled portion of the TNNI3K pocket.

These atoms along with the benzenesulfonamide C4 site were

targeted for further elaboration as the structural and

biochemical results signify that such modiﬁcations would be

permissible without disrupting TNNI3K recognition.

Selection of a Lead Compound. On the basis of these

structural and structure−function results, the 7-deazapurine 7

was selected as a suitable lead for further optimization (Figure

6). In addition to its enhanced aﬃnity for TNNI3K, 7 exhibits

Eﬀect of C7- and C8−7-Deazapurine Substitutions. An

evaluation of C7 and C8 7-deazapurine subsituents (Figure 7)

conﬁrmed that a variety of modiﬁcations are tolerated, as

anticipated from the crystal structure of 1 bound to TNNI3K.

The 7-Me variant (21, IC₅₀= 40 nM) displayed activity

comparable to 7 (IC₅₀= 80 nM), whereas larger alkyl

substitutions produced slightly weaker TNNI3K binders (22,

23, IC₅₀= 126 nM) than 21, suggesting that ﬁlling toward the

periphery of the binding site is of little energetic consequence.

The 7-Cl (25, IC₅₀= 32 nM) and 7-Br (26, IC₅₀= 32 nM)

variants exhibited potencies similar to the 7-Me bearing 21,

demonstrating little electronic eﬀect on TNNI3K binding.

Conversely, 25 and 26 show a marked improvement in activity

against B-Raf compared to 21, and this apparent electronic

eﬀect is indicative of divergent binding interactions for B-Raf vs

TNNI3K. Pertinent to this result is the observation that the

Figure 6. Comparison of hit 1 and lead 7. Kinase proﬁling details are

available in Supporting Information.

increased aqueous solubility (50 μM) and as a result oﬀers an

improved rat pharmacokinetic proﬁle (Cl = 68 mL/min/kg, F =

Figure 7. Eﬀect of C7- and C8-deazapurine substituents.

D

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

residue that lies below C7 of the deazapurine diﬀers in

TNNI3K (Leu595) and B-Raf (Phe583), and this disparity

presumably gives rise to their diﬀering responses to C7-

substituents (Figure 8). Importantly, C7-substitution had

or pharmacokinetics, and as such, 44 as well as the C8-

substitutions were not further pursued.

Eﬀect of C4-Benzenesulfonamide Substitutions. An

evaluation of substituted-benzenesulfonamide analogues (Fig-

ure 9) demonstrated that TNNI3K binding aﬃnity can be

Figure 8. Crystal structure of 1 (green) bound to TNNI3K (gray)

overlaid with the B-Raf structure¹³(orange) (PDB code: 2FB8).

substantial eﬀects on pharmacokinetics with the halogen

bearing analogues showing up to 7-fold improvement in total

oral exposure (e.g., 26, poDNAUC = 0.55 h-kg/L) compared

to 7. The reduced clearance of 25 (Cl = 20 mL/min/kg) and

26 (Cl = 23 mL/min/kg) may arise from the halogen

substituents blocking oxidative metabolism of the pyrrole

ring, but the lower volumes of distribution of 25 (Vdss =0.45

L/kg) and 26 (Vdss = 0.63 L/kg) compared to 7 (Vdss = 1.4

L/kg) suggest that altered compound distribution may also

contribute to their elevated in vivo exposure (e.g., via protective

eﬀects from increased plasma protein binding).

Like the 7-Br-substituted 26, the 7-Ph analogue 27 exhibited

equipotent aﬃnity for TNNI3K (IC₅₀= 40 nM) but

substantially improved activity against B-Raf (IC₅₀= 4 nM)

compared to 21, perhaps reﬂecting the phenyl group’s greater

complementarity with the aromatic Phe583 side chain of B-Raf

than with the aliphatic Leu595 side chain of TNNI3K.

Substitutions on the 7-Ph ring produced only slight

modulations in TNNI3K and B-Raf activity, and these eﬀects

were not associated with any clear steric (cf. 27-30), electronic

(cf. 27, 30, 31), or electrostatic (cf. 32-34) trends.

Furthermore, the 7-Ph group displayed inferior pharmacoki-

netics to 26, and its high clearance (Cl = 93 mL/min/kg) could

only partially be restored through substitution (30, Cl = 65

mL/min/kg). Given these limitations and their preferential

binding to B-Raf over TNNI3K, 7-aryl analogues were not

pursued further.

Modiﬁcations of the C8 site included C8-bromination, which

had little eﬀect on TNNI3K binding (35, IC₅₀= 50 nM) but

did improve pharmacokinetics (poDNAUC = 0.26 h-kg/L).

Like the C7-phenyl compounds, the C8-phenyl variants (36-

41) displayed moderately enhanced (∼2−6 fold) potency

against TNNI3K, and substantially improved activity (∼10−50-

fold) against B-Raf. The C8-Ph substituent projects into the

front pocket, where residue diﬀerences between TNNI3K and

B-Raf (e.g., Gly545 vs Ser535, Ser549 vs His539) may give rise

to the divergent behavior of C8-substituents in binding to B-Raf

and TNNI3K (Figure 8). As with the C7-Ph compound (27),

the C8-Ph compound (36) displayed inferior pharmacokinetics

(poDNAUC = 0.01 h-kg/L) that could be partially rescued via

4-substitution (e.g., 40, poDNAUC = 0.14 h-kg/L). The

pyrimidoindole variant 44 was also evaluated, but it provided

no advantage over 7, as it had little eﬀect on binding, selectivity,

Figure 9. Eﬀect of C4-benzenesulfonamide substituents.

modulated by C4 groups in a manner that correlates with

substituent electronic properties (7, 45−47 IC₅₀: Cl (20 nM) >

H (80 nM) > Me (630 nM) > NHMe (6300 nM)), with

strongly electron-donating groups proving especially detrimen-

tal to activity (e.g., 47, 80-fold reduction vs 7). Given that the

C4-substituents are anticipated to project into a spacious

portion of the binding pocket (Figure 2), where they are

unlikely to introduce either beneﬁcial or repulsive interactions

with TNNI3K, the C4-substituents may elicit a remote

electronic eﬀect on key binding elements such as the H-

bonding network formed by the sulfonamide functionality or

the hydrophobic interactions of the benzene with nearby

residues (e.g., Ala605).

Of particular interest in light of the observed C4-substituent

electronic eﬀect, replacement of the N-methylamino substituent

47 (IC₅₀= 6300 nM) with the N,N-dimethylamino group (48,

IC₅₀= 50 nM) led to a substantial (∼100-fold) improvement in

binding aﬃnity for TNNI3K, and this result was conﬁrmed

through evaluation of the related morpholine-bearing com-

pound 49, which is also highly active (IC₅₀= 40 nM). This

dissimilarity in TNNI3K binding behavior highlights a key

stereoelectronic diﬀerence in ortho-substituted monoalkyl

anilines vs ortho-substituted dialkyl anilines (Figure 10).¹⁴

Monoalkyl anilines such as 47 adopt a coplanar orientation

wherein the nitrogen lone pair resides in a p-orbital parallel to

the π-system of the adjacent aryl ring to maximize resonance

stabilization. However, this conformation is unavailable to

dialkyl aniline 48 as its additional methyl group would

Figure 10. Conformations of anilines 47 and 48.

E

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

introduce a severe steric interaction with the ortho-NH group in

the coplanar orientation. As a result the dimethyl aniline group

of 48 must adopt a twisted conformation wherein its methyl

groups move out of the plane of the neighboring NH group and

its nitrogen lone pair orients perpendicular to the adjacent aryl

π-system, thereby disrupting the N-aryl resonance. Thus, in the

twisted conformation the strong resonance electron-donating

character of the aniline nitrogen is neutralized, resulting in a

signiﬁcant electronic divergence between the C4-substituents of

47 and 48 that is reﬂected in their TNNI3K binding aﬃnity.

Dialkyl anilines 48 (Cl = 30 mL/min/kg) and 49 (Cl = 39

mL/min/kg) exhibited improved in vivo clearance compared to

7 (Cl = 68 mL/min/kg, F = 34%) demonstrating the utility of

C4-substituents for modulating pharmacokinetics in the series,

presumably via blocking of oxidative metabolism in the aryl ring

system. The N,N-dimethylamino substituent was particularly

valuable, as it also led to increased aqueous solubility (330 μM

for 48 vs 50 μM for 7) and oral bioavailability (F = 100% for 48

vs 34% for 7), leading to an overall 7-fold improvement in oral

exposure (poDNAUC = 0.59 h-kg/L) compared to 7.

Deazapurine 48 also demonstrated good activity in a

TNNI3K cellular assay (IC₅₀= 50 nM) and oﬀers an enhanced

selectivity proﬁle, showing >100-fold selectivity against 95%

(175/185) and >10-fold selectivity against 99% (184/185) of

kinases tested in the Millipore KinaseProﬁler panel (Milli-

pore.com, see Supporting Information for details), with B-Raf

and c-Raf being the only kinases signiﬁcantly inhibited at 1 μM

compound concentration. Given its good pharmacokinetic

proﬁle and highly speciﬁc inhibition of the structurally related

TNNI3K, B-Raf, and c-Raf kinases, 48 represents an advanced

lead for further development (Figure 11).

interdependency of the C4 and C7 substituents was anticipated

from the structure of 1 bound to TNNI3K (Figure 2) as it

suggested that the C4 and C7 substituents would project

toward one another, and indeed, a subsequent crystal structure

of 53 bound to TNNI3K conﬁrmed the proximity (∼4 Å) of

the C4 and C7 substituents in the TNNI3K binding site

(Figure 13D). Analogues which bear a C4-morpholino group

show a signiﬁcant cellular activity enhancement (∼10-fold) in

combination with the C7-Me (52) and C7−Br (53)

modiﬁcations but to a lesser extent than the C4-NMe₂

compounds, suggesting a reduced complementarity between

the C4-morpholine moiety and the C7-substituents. Although

multiple energetic factors (e.g., electronic, conformational,

desolvation, hydrophobic eﬀect) may be in play, a comparison

of the molecular surface areas of 1, 26, 49, and 53 bound to

TNNI3K (Figure 13) reveals that improved hydrophobic

packing is a likely source of the synergistic eﬀect. By contrast to

the monosubstituted analogues, which are expected to make

only isolated hydrophobic interactions with TNNI3K through

either their C7−Br or C4-NR₂functional groups, the C7−Br

and C4-NC₄H₈O groups of the disubstituted 53 form a large,

contiguous lipophilic surface that forms multiple points of

contact with Leu595 that resides at the bottom of the pocket as

well as the Val477 and Ile469 side chains that lie above the

inhibitor. Moreover, the C7−Br and C4-NC₄H₈O groups

engage in an internal lipophilic interaction that may rigidify 53

into a molecular architecture that is highly complementary to

the shape of the TNNI3K pocket.

The C8−Br or C8−3-CF₃−Ph substitutions had only a small

eﬀect on binding aﬃnity (≤4-fold) when evaluated on either of

the C4-dialkylamine templates (54−57). These results are

similar to those found in the absence of a C4-substituent

(Figure 7, compds 35, 40) and consistent with the structural

evidence demonstrating the large distance between C4 and C8

groups upon TNNI3K binding. Overall, these results conﬁrm a

cooperative eﬀect between C4 and C7 substituents leads to the

exceptional activity of 50 and 51, which exhibit >1000-fold

increase in cellular activity compared to purine 1 (cellular IC₅₀

= 1300 nM).

Chemistry. Purine 1 and analogues were assembled via

substitution of chloro-heterocycles with appropriate anilines,

and this operation was accomplished through one of several

alternative methods (Scheme 1). In instances requiring the

purine scaﬀold, the desired products (1−5, 11−17) were

obtained by heating a mixture of the commercially available 6-

chloropurines and readily accessible anilines in isopropanol

(Method A: 58, 59, isopropanol, 80 °C, 16 h or μwave, 150 °C,

30 min, 13−91%).¹⁵In general, the 7-deazapurine analogues (7,

8, 18, 20, 21, 35, 43−45, 47−49, 54, 56) did not react well in

reﬂuxing isopropanol but could be accessed under AgOTf-

mediated coupling conditions (Method B: 59, 60, AgOTf (1

equiv), DMF, 80 °C, 16 h or AgOTf (1 equiv), μwave, 120 °C,

30 min, 3−86%).¹⁶However, the 7-halo-7-deazapurines (24−

26, 51, 53) proved to be more active substrates and were

successfully synthesized using Method A (8−53%), which also

was suﬃcient to access some 8-aryl variants (55, 57, 13−

23%).¹⁷Formation of pyrazolopyrimidine 6 and pyrrolopyr-

idine 9 required prolonged reaction times in reﬂuxing

isopropanol (5 days, 8% for 6) but the reactions could be

accelerated through acid catalysis (Method C: 59, 61, 1 M aq

HCl (1 equiv), isopropanol, μwave, 150 °C, 3 h, 45% for 9).¹⁵

Notably, 9 was also synthesized by way of the Hartwig−

Buchwald coupling method, thereby demonstrating that the 7-

Figure 11. Comparison of leads 7 and 48. Kinase proﬁling details are

available in Supporting Information.

Combined C4-Benzenesulfonamide and C7-/C8-Dea-

zapurine Substitutions. Given the desirable properties of the

advanced lead 48, we examined the eﬀect of C7- and C8-

deazapurine substitutions in combination with either the C4-

dimethylamino or C4-morpholino group (Figure 12). Of

interest, analogues bearing the C4-NMe₂functionality in

addition to either the C7-Me (50) or C7−Br group (51)

showed high aﬃnity for TNNI3K (IC₅₀< 10 nM), which

translated into exceptional cellular potency against TNNI3K

(IC₅₀∼ 1 nM). The ∼50-fold improvement in cellular activity

is indicative of a synergistic eﬀect between the C4 and C7

substituents as the C7−Br and C7-Me substitutions produced

only ∼2 to 3-fold improvement in TNNI3K binding in the

absence of the C4 substituent (Figure 7, compds 21, 26). The

F

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 12. Disubstituted 7-deazapurines. Compound IC₅₀values less than 10 nM could not be accurately obtained due to titration of TNNI3K in

the enzyme assay.

Scheme 1

Figure 13. Comparison of 1 (A), 26 (B), 49 (C), and 53 (D) bound

to TNNI3K.

azaindole does not rearrange under Method C, as might be

expected based on literature reports.¹⁸Method C also proved

useful as an alternative approach to prepare some substituted 7-

deazapurines (46, 50, 52, 10−20%). Finally, the synthesis of O-

linked 7-deazapurine 19 was accomplished through a base-

mediated substitution reaction (Method D: 60, 62, Cs₂CO₃,

DMF, μwave, 180 °C, 1 h, 4%) that was not useful for 3-amino-

benzenesulfonamide couplings as the sulfonamide preferentially

reacted instead of the aniline nitrogen under these conditions.

Postcoupling transformations of anilino-deazapurine com-

pounds provided additional analogues of interest (Scheme 2).

For instance, 10 was synthesized from 7 by way of oxidative

bromination (pyridinium bromide perbromide, t-BuOH, 25 °C,

1h) followed by a zinc reduction (Zn dust, AcOH, 25 °C, 15

min, 25%, 2 steps).¹⁶Suzuki couplings¹⁹employing bromide 26

were only partly successful, producing some of the targeted

biaryl compounds (28, 29, 31) in low yields (6−15%).

Protodebromination was the predominate pathway in these

reactions but could be avoided by masking the 7-deazapurine

N9−H atom with an electron-withdrawing functionality. Thus,

26 was converted to the di-Boc derivative 63 (Boc₂O, DMAP,

MeCN, 25 °C, 1 h, 53%) and Suzuki reactions (63, ArB(OH)₂,

Pd(dppf)Cl₂, aq Na₂CO₃, dioxane, 100 °C, 1 h) from this

template followed by prolonged heating of the reaction mixture

to achieve Boc removal (100 °C, 16 h) aﬀorded a wider range

of products (27, 30, 32−34) in modest yields (11−54%).

When using 63 as substrate, the conversions to product were

high under Suzuki conditions, but often separation of the

desired product from the reaction byproducts was challenging,

leading to low yields. Cross coupling reactions (35, ArB(OH)₂,

Pd(dppf)Cl₂or Pd(PPh₃)₄, aq Na₂CO₃, dioxane, μwave, 120

G

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 2

Scheme 4

The chloro-heterocycle and aniline building blocks required

to assemble each analogue were either commercially available,

synthesized according to literature reports,^16,20−25or prepared

by an original synthesis (Scheme 4, 5). Halogen metal exchange

(n-BuLi, THF, −78 °C, 1 h) on 67 followed by alkylation with

3-bromo-2-methylpropene (THF, −78 to 0 °C, 30 min, 17%)

produced 65 in a procedure analogous to that reported for the

synthesis of the 6-chloro-7-Me-7-deazapurine.²⁰The 6-chloro-

8-bromo-7-deazapurine 69 was prepared via a known three-step

sequence of benzenesulfonylation (NaH, PhSO₂Cl, 0 to 25 °C,

4 h, 98%), bromination (LDA, BrCl₂CCCl₂Br, −78 °C, 2 h,

61%), and hydrolysis (KOt-Bu, THF, 25 °C, 4 h, 34%).²⁶The

intermediate N-protected bromide 68 proved useful as a cross

coupling reagent and was subjected to Suzuki conditions (3-

CF₃−PhB(OH)₂, Pd(dppf)Cl₂, aq K₂CO₃, dioxane, 80 °C, 3 h,

47%) with subsequent sulfonamide hydrolysis (KOt-Bu, THF,

0 to 25 °C, 30 min, 99%) to give biaryl 70. Finally, the 7-CO₂t-

Bu derivative 72, which was utilized to access carboxylic acid

analogue 43 was synthesized via condensation of glycine tert-

butyl ester and 71 in a two-step procedure (Et₃N, EtOH, 25

°C, 24 h; NaH, DMF, 0 °C, 1 h, 25%) adapted from a similar

sequence developed for a related 7-deazapurine compound.²⁷

°C, 15 min) from 8-bromo-7-deazapurine 35 were also

successful using a variety of boronic acid substrates, though

variable yields were observed (3−51%).

7-Alkyl-7-deazapurines 22 and 23 were prepared via

reduction of their corresponding alkene functional groups,

which could be readily installed onto the 7-deazapurine core

(Scheme 3). In the case of 22, a cross coupling employing

isopropenylboronic acid pinacol ester under the previously

established conditions (63, Pd(dppf)Cl₂, aq Na₂CO₃, dioxane,

100 °C, 15 min, 17%) produced the mono-Boc, isopropenyl

derivative 64. In this instance, the long reaction times normally

used to eﬀect Boc removal produced complex mixtures.

However, following hydrogenation (H₂, Pd/C, MeOH, 25

°C, 16 h, 80%) of the alkene, Boc removal was easily

accomplished under acidic conditions (HCl, dioxane, MeCN,

25 °C, 16 h, 97%) to give 22. The alkene precursor of 23 was

installed on the 6-chloro-7-deazapurine core prior to aniline

coupling (Scheme 4), and with this building block (65) in

hand, 23 was assembled by way of a routine aniline substitution

(AgOTf, DMF, 80 °C, 10 h) followed by hydrogenation (H₂

(30 psi), Pd/C, MeOH, 25 °C, 16 h, 16%, 2 steps) (Scheme 3).

Scheme 3

H

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

substantial reductions in activity (∼10−100-fold). These results

were corroborated by an X-ray structure of 1 bound to

TNNI3K, which revealed a novel coplanar binding mode

consisting of bidentate hinge-recognition by the purine coupled

with an elaborate hydrogen bonding network for the

sulfonamide group. Notably, the sulfonamide serves as a

hydrogen bond donor to the TNNI3K gatekeeper residue

(Thr539) and presumably contributes to the broad spectrum

kinase selectivity of the series.³¹The 7-deazapurine 7 was

identiﬁed as a superior lead compound vs purine 1 as it

demonstrated improved potency and pharmacokinetics, and

elaboration of this template by way of C4, C7, and C8-

substitutions produced potent (<10−100 nM) and selective

(>10-fold selectivity vs 97% of kinome) inhibitors, some of

which (e.g., 48) display good pharmacokinetics (poDNAUC =

0.59 h-kg/L) and excellent selectivity (>100-fold selectivity vs

95% of kinome), showing appreciable activity at only the

structurally related Raf kinases. Other derivatives (e.g., 51)

exhibited exceptional potency improvements (>1000-fold) over

1 and displayed subnanomolar activity against TNNI3K in a

cellular context (IC₅₀= 0.7 nM). Thus, anilino-7-deazapurine

TNNI3K inhibitors suitable as in vitro and in vivo biological

tools have been discovered, and these compounds along with

the structure−function results established herein serve as the

foundation for eﬀorts ultimately leading to the deﬁnition of the

fundamental cardiac biology of TNNI3K.³

Scheme 5

EXPERIMENTAL SECTION

Several benzenesulfonamide intermediates which lacked

literature precedent were prepared through standard methods

(Scheme 5). Methylation of 73 (H₂CO, AcOH, NaBH(OAc)₃,

CH₂Cl₂, 25 °C, 15 min, 40%)²⁸proceeded smoothly to give N-

Me aniline 74 under conditions that selectively produce the

monomethylated aniline vs dimethylated aniline species (4:1).

3-Hydroxy-N-Me-benzenesulfonamide 62 was synthesized from

the commercially available sulfonyl chloride 75 via sequential

sulfonamide formation (MeNH₂, DMAP, THF, 25 °C, 30 min,

79%) and demethylation (BBr₃, CH₂Cl₂, 0 to 25 °C, 2 h, 96%).

Amino-pyridyl intermediate 79 was obtained by modiﬁcation of

a reported synthesis of related amino-sulfonamide pyridines.²⁹

Thus, bromination of 76 (Br₂, 130 °C, 8 h) and subsequent

sulfonamide formation (CH₃NH₂, H₂O, 0 to 25 °C, 3 h, 17%, 2

steps) aﬀorded 78, which successfully underwent a copper-

catalyzed substitution reaction (NH₄OH, CuCl, sealed tube,

130 °C, 18 h, 61%) to install the aniline functionality.²⁹

Chlorosulfonylation of 2-ﬂuoronitrobenzene 80 (HSO₃Cl,

100 °C, 18 h, 65%) to provide 81 followed by sulfonamide

formation (MeNH₂.HCl, Et₃N, THF, −35 °C, 1 h, 90%) at low

temperatures gave 82, which was utilized to access a variety of

C4-substituted anilines 83.³⁰Addition of the appropriate

amines (MeNH₂, Me₂NH, morpholine) to 82 (R¹R²NH,

Et₃N, THF, 25 °C, 1 h, 90−98%)³⁰and subsequent reduction

of the nitro group (H₂, Pd/C, THF, 67−89%) delivered 3,4-

diaminobenzenesulfonamides 83 in good yields (Scheme 5).

■

General Experimental. The purity of each inhibitor was

determined to be >95% (except as noted) on an Agilent 1100

HPLC equipped with a Sunﬁre C18 5.0 μm column (3.0 mm × 50

mm) using a gradient of 10% to 100% MeCN/H₂O/0.05% TFA at 1

mL/min ﬂow rate with detection at 220 and 254 nm. Mass

determinations were conducted using an Agilent 6110 Quadrupole

MS with positive ESI. Preparative HPLC was conducted on a Waters

2525 system at a ﬂow rate of 50 mL/min with 254 nm detection using

either a Sunﬁre C18 OBD 5 μm column (30 × 150 mm) with a

gradient of MeCN/H₂O/0.1% TFA or an XBridge C18 OBD 5 μm

column (30 × 150 mm) with a gradient of MeCN/aq NH₄OH pH 10.

Flash column chromatography was conducted on silica gel eluting with

EtOAc-hexanes, EtOAc-petroleum ether or MeOH−CH₂Cl₂mixtures.

The following intermediates were synthesized following literature

procedures: 4-chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidine,¹⁶4-

chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine,²⁰4-chloro-5-ﬂuoro-

7H-pyrrolo[2,3-d]pyrimidine,²¹4,5-dichloro-7H-pyrrolo[2,3-d]-

pyrimidine,²²5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine,²³4-

chloro-9H-pyrimido[4,5-b]indole,²⁴6-bromo-4-chloro-7-(phenylsul-

fonyl)-7H-pyrrolo[2,3-d]pyrimidine (68),²⁶6-bromo-4-chloro-7H-

pyrrolo[2,3-d]pyrimidine (69),²⁶3-amino-N-hydroxy-benzenesulfona-

mide,²⁴and 3-amino-4-chloro-N-methylbenzenesulfonamide.²⁵Other

required reagents and building blocks were either purchased and used

as is or synthesized as described below.³²

Method A. General Coupling Method for Anilines and Chloro-

Purines or Chloro-7-Deazapurines. A mixture of a 6-chloropurine or

6-chloro-7-deazapurine (i.e., 4-chloro-7H-pyrrolo[2,3-d]pyrimidine)

and a 3-aminobenzenesulfonamide (1.5 equiv) in isopropanol (2

mL) was stirred at 80 °C for 16 h or subjected to microwave

irradiation (150 °C) for 30 min before being cooled to room

temperature. The solid was collected by ﬁltration, washed with MeOH,

and dried to aﬀord products that were analytically pure (1−5, 11, 14,

15, 25, 51, 53) or else were subjected to preparative HPLC to give

analytically pure solids (12, 13, 16, 17, 24, 26, 55, 57).

CONCLUSIONS

■

A series of anilino-purines and anilino-7-deazapurines arising

from 1 (Figure 1) were prepared and evaluated as inhibitors of

TNNI3K. Fundamental structure−activity relationships were

conducted for this initial series of Type I TNNI3K inhibitors

and used to deﬁne the role of each element of 1 for TNNI3K

recognition. The essential functions of the purine heterocycle,

the sulfonamide moiety, and their linking N atom were

established through multiple modiﬁcations that led to

Method B. General Coupling Method for Anilines and Chloro-7-

Deazapurines. The indicated 6-chloro-7-deazapurine, the required 3-

amino-benzenesulfonamide (1.3 equiv), and AgOTF (1 equiv) were

dissolved in DMF (2 mL) and stirred at 80 °C overnight or were

dissolved in i-PrOH (2 mL) and subjected to microwave irradiation

I

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(120−150 °C) for 60 min before being diluted with MeOH, and

ﬁltered. Analytically pure solids were obtained after puriﬁcation by

reverse-phase HPLC (7, 8, 18, 20, 21, 47, 48, 49), ﬂash

chromatography (35, 43, 54, 56), precipitation (44), or SCX cation

exchange (45).

Method C. General Coupling Method for Anilines and Chloro-

Heterocycles. A mixture of the indicated chloro-heterocycle and the

required 3-aminobenzenesulfonamide (1.5 equiv) in i-PrOH (2 mL)

was treated with 1 M aqueous HCl (1 equiv) and subjected to

microwave irradiation (150 °C) for 60 min before being ﬁltered and

subjected to reverse phase HPLC to give analytically pure products (6,

9, 46, 50, 52).

92% purity): ¹H NMR (400 MHz, CD₃OD) δ ppm 8.35 (s, 1 H), 8.18

(s, 1 H), 7.91 (d, J = 7.5 Hz, 1 H), 7.79 (d, J = 8.1 Hz, 1 H), 7.72 (dd,

J = 7.5, 8.1 Hz, 1 H), 7.39 (d, J = 3.51 Hz, 1 H), 6.77 (d, J = 3.51 Hz, 1

H), 3.91 (s, 3 H), 2.62 (s, 3 H). MS (m/z) 318.0 (M + H⁺).

3-((1H-Pyrrolo[2,3-b]pyridin-4-yl)amino)-N-methylbenzenesulfo-

namide (9). Compound 9 was prepared from 4-chloro-1H-pyrrolo-

[2,3-b]pyridine and N-methyl 3-aminobenzenesulfonamide using

1

Method C as a white solid in 19% yield (13 mg): H NMR (400

MHz, DMSO-d₆) δ ppm 12.39 (br. s., 1 H), 10.28 (s, 1 H), 8.11 (d, J =

7.03 Hz, 1 H), 7.76 (s, 1 H), 7.65−7.75 (m, 3 H), 7.61 (q, J = 5.02 Hz,

1 H), 7.43−7.47 (m, 1 H), 6.81−6.87 (m, 2 H), 2.48 (d, J = 5.02 Hz, 3

H). MS (m/z) 303.0 (M + H⁺).

N-Methyl-3-(1H-purin-6-ylamino)benzenesulfonamide (1). Com-

pound 1 was prepared from 6-chloropurine and N-methyl 3-

aminobenzenesulfonamide using Method A as a yellow solid in 81%

yield (80 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.34 (br. s, 1

H, aniline-NH), 8.53 (s, 1 H, purine C2-H), 8.48 (s, 1 H, purine C8-

H), 8.41 (s, 1 H, benzene C2-H), 8.17 (dd, J = 8.06, 1.26 Hz, 1 H,

benzene C4-H), 7.57 (t, J = 7.93 Hz, 1 H, benzene C5-H), 7.48 (m, 2

H, benzene C6-H, sulfonamide-NH)), 2.48 (d, J = 4.78 Hz, 3 H,

sulfonamide−CH₃). MS (m/z) 305.0 (M + H⁺).

N - M e t h y l - 3 - ( ( 9 - m e t h y l - 9 H - p u r i n - 6 - y l ) a m i n o ) -

benzenesulfonamide (2). Compound 2 was prepared from 6-chloro-

9-methyl-9H-purine and N-methyl 3-aminobenzenesulfonamide using

Method A as a white solid in 50% yield (76 mg): ¹H NMR (400 MHz,

CD₃OD) δ ppm 8.49 (m, 2 H), 8.22 (m, 1 H), 7.92 (m, 1 H), 7.82 (m,

1 H), 7.73 (m, 1 H), 3.97 (s, 3 H), 2.61 (s, 3 H). MS (m/z) 319.1 (M

+ H⁺).

N-Methyl-3-((6-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-

amino)benzenesulfonamide (10). A solution of 7 (100 mg, 0.330

mmol) in tert-butanol (20 mL) was treated with pyridine hydro-

bromide perbromide (211 mg, 0.659 mmol), and the mixture was

stirred at 25 °C for 1 h before being poured into water and extracted

with EtOAc. The organic extracts were washed (H₂O, saturated

aqueous NaCl), dried (Na₂SO₄), and concentrated to give a yellow-

brown solid. The residue was dissolved in AcOH (15 mL), treated

with zinc dust (300 mg, 4.59 mmol), and the mixture was stirred for

15 min before being ﬁltered, concentrated, and subjected to reverse

phase HPLC (10−50% MeCN/water/0.1%TFA) to give 10 (12 mg,

1

11%) as a white solid: H NMR (400 MHz, DMSO-d₆) δ ppm 11.13

(s, 1 H), 9.24 (s, 1 H), 8.36 (s, 1 H), 8.12 (t, J = 1.88 Hz, 1 H), 8.07

(dd, J = 8.03, 1.25 Hz, 1 H), 7.54 (t, J = 8.03 Hz, 1 H), 7.44 (q, J =

5.02 Hz, 1 H), 7.38 (d, J = 8.28 Hz, 1 H), 2.55 (s, 2 H), 2.45 (d, J =

5.02 Hz, 3 H). MS (m/z) 320.0 (M + H⁺).

3-((2-Fluoro-9H-purin-6-yl)amino)-N-methylbenzenesulfonamide

(3). Compound 3 was prepared from 6-chloro-2-ﬂuoro-9H-purine and

N-methyl 3-aminobenzenesulfonamide using Method A as an oﬀ-white

solid in 23% yield (27 mg, 90% purity): ¹H NMR (400 MHz, DMSO-

d₆) δ ppm 10.62 (s, 1H), 8.38 (s, 1H), 8.32 (s, 1H), 8.09 (dd, J = 1.3,

8.0 Hz, 1H), 7.60 (dd, J = 7.8, 8.0 Hz, 1H), 7.47 (m, 2H), 2.49 (d, J =

4.8 Hz, 3H). MS (m/z) 323.0 (M + H⁺).

3-((9H-Purin-6-yl)amino)-N-ethylbenzenesulfonamide (11). Com-

pound 11 was prepared from 6-chloropurine and 3-amino-N-

ethylbenzenesulfonamide using Method A as a pale yellow solid in

89% yield (94 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.10 (br.

s, 1H), 10.02 (br. s, 1H), 8.37 (d, J = 2.01 Hz, 1H), 8.27 (br. s, 1H),

8.20 (d, J = 6.78 Hz, 1H), 7.54−7.67 (m, 2H), 7.48 (d, J = 7.28 Hz,

1H), 7.29−7.40 (m, 1H), 6.83 (dd, J = 1.88, 3.39 Hz, 1H), 2.74−2.93

(m, 2H), 1.01 (t, J = 7.28 Hz, 3H). MS (m/z) 318.1 (M + H⁺).

3-((9H-Purin-6-yl)amino)benzenesulfonamide (12). Compound

12 was prepared from 6-chloropurine and 3-aminobenzenesulfona-

3-((2-Amino-9H-purin-6-yl)amino)-N-methylbenzenesulfona-

mide (4). Compound 4 was prepared from 6-chloro-9H-purin-2-amine

and N-methyl 3-aminobenzenesulfonamide using Method A as a white

1

solid in 15% yield (15 mg): H NMR (400 MHz, CD₃OD) δ ppm

1

mide using Method A as an oﬀ-white solid in 44% yield (42 mg): H

8.37 (m, 1H), 8.19 (s, 1H), 8.11 (m, 1H), 7.6−7.7 (m, 2H), 2.60 (s,

NMR (400 MHz, DMSO-d₆) δ ppm 13.25 (br. s., 1 H, purine N9-H),

10.13 (br. s., 1 H, aniline-NH), 8.61 (br. s., 1 H, purine C2-H), 8.43 (s,

1 H, purine C8-H), 8.34 (s, 1 H, benzene-C2-H), 8.09 (dt, J = 7.74,

1.79 Hz, 1 H, benzene C4-H), 7.44−7.56 (m, 2 H, benzene C5-H, C6-

H), 7.36 (s, 2 H, SO₂NH₂). MS (m/z) 291.1 (M + H⁺).

3H). MS (m/z) 320.1 (M + H⁺).

3-((2-Chloro-9H-purin-6-yl)amino)-N-methylbenzenesulfona-

mide (5). Compound 5 was prepared from 2,6-dichloro-9H-purine and

N-methyl 3-aminobenzenesulfonamide using Method A as a tan solid

in 91% yield (342 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.89

(s, 1H), 8.59 (s, 1H), 8.40 (m, 1H), 8.08 (m, 1H), 7.61 (dd, J = 8.0,

8.1 Hz, 1H), 7.49 (m, 2H), 2.51 (s, 3H). MS (m/z) 338.9 (M + H⁺).

3-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)amino)-N-methylbenzene-

sulfonamide (6). Compound 6 was prepared from 4-chloro-1H-

pyrazolo[3,4-d]pyrimidine and N-methyl 3-aminobenzenesulfonamide

using Method C as a white solid in 8% yield (112 mg): ¹H NMR (400

MHz, DMSO-d₆) δ ppm 13.75 (s, 1H), 10.33 (s, 1H), 8.47 (s, 1H),

8.36 (s, 1H), 8.32 (s, 1H), 8.29 (d, J = 8.3 Hz, 1H), 7.63 (dd, J = 7.8

Hz, 8.0 Hz, 1H), 7.50 (m, 2H), 2.48 (d, J = 5.0 Hz, 3H). MS (m/z)

305.0 (M + H⁺).

3-((9H-Purin-6-yl)amino)-N-hydroxybenzenesulfonamide (13).

Compound 13 was prepared from 6-chloropurine and 3-amino-N-

hydroxybenzenesulfonamide²⁴using Method A as a white solid in 24%

yield (35 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.22 (s, 1 H,

aniline-NH), 9.60 (s, 2 H, SO₂NHOH), 8.61 (br. s., 1 H, purine C2-

H), 8.44 (s, 1 H, purine C8-H), 8.36 (s, 1 H, benzene C2-H), 8.22 (d,

J = 8.0 Hz, 1 H, benzene C4-H), 7.58 (dd, J = 7.8, 8.0 Hz, 1 H,

benzene C5-H), 7.50 (d, J = 7.8 Hz, 1 H, benzene C6-H). MS (m/z)

307.0 (M+H⁺). The structure of 13 was further conﬁrmed by its

successful conversion to 3-((9H-purin-6-yl)amino)benzenesulﬁnic

acid. Thus, 13 (3 mg) was dissolved in 0.5 mL of 1N NaOH and

heated at 80 °C for 5 min to give a yellow solution which consisted of

3-((9H-purin-6-yl)amino)benzenesulﬁnic acid (100%) based on

LCMS analysis: MS (m/z) 276.0 (M + H⁺).

3-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzenesul-

fonamide (7). Compound 7 was prepared from 4-chloro-7H-

pyrrolo[2,3-d]pyrimidine and N-methyl 3-aminobenzenesulfonamide

1

using Method B as a white solid in 80% yield (330 mg): H NMR

3-((9H-Purin-6-yl)amino)-N,N-dimethylbenzenesulfonamide (14).

Compound 14 was prepared from 6-chloropurine and 3-amino-N,N-

dimethylbenzenesulfonamide using Method A as a white solid in 77%

yield (160 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.06 (br. s.,

1 H), 8.63 (br. s., 2 H), 8.45 (t, J = 1.76 Hz, 1 H), 8.26 (dd, J = 8.16,

1.13 Hz, 1 H), 7.66 (t, J = 7.91 Hz, 1 H), 7.46 (d, J = 7.28 Hz, 1 H),

2.67 (s, 6 H). MS (m/z) 319.1 (M + H⁺).

N-(3-(Ethylsulfonyl)phenyl)-9H-purin-6-amine (15). Compound

15 was prepared from 6-chloropurine and 3-(ethylsulfonyl)aniline

using Method A as a pale yellow solid in 64% yield (66 mg): ¹H NMR

(400 MHz, DMSO-d₆) δ ppm 11.06 (br. s., 1 H), 8.65 (d, J = 2.52 Hz,

2 H), 8.59 (t, J = 1.76 Hz, 1 H), 8.23−8.28 (m, 1 H), 7.68 (dd, J = 7.8,

(400 MHz, DMSO-d₆) δ ppm 12.23 (s, 1H, pyrrolopyrimidine-NH),

10.21 (s, 1H, aniline-NH), 8.40 (s, 1H, pyrrolopyrimidine C2-H), 8.23

(s, 1H, benzene C2-H), 8.19 (d, J = 8.0 Hz, 1H, benzene C4-H), 7.64

(dd, J = 7.9, 8.0 Hz, 1H, benzene C5-H), 7.52 (d, J = 7.9 Hz, 1H,

benzene C6-H), 7.50 (m, 1H, sulfonamide-NH), 7.38 (dd, J = 2.5, 3.0

Hz, 1H, pyrrolopyrimidine-C6-H), 6.84 (dd, J = 1.5, 3.0 Hz, 1H,

pyrrolopyrimidine-C5-H), 2.48 (d, J = 4.8 Hz, 3H, sulfonamide−

CH₃). MS (m/z) 304.0 (M + H⁺).

N-Methyl-3-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-

benzenesulfonamide (8). Compound 8 was prepared from 4-chloro-

7-methyl-7H-pyrrolo[2,3-d]pyrimidine and N-methyl 3-aminobenze-

nesulfonamide using Method B as a white solid in 86% yield (111 mg,

J

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8.0 Hz, 1 H), 7.61 (d, J = 7.81 Hz, 1 H), 3.30 (q, J = 7.30 Hz, 2 H),

1.15 (t, J = 7.43 Hz, 3 H). MS (m/z) 304.0 (M + H⁺).

A mixture of 63 (500 mg, 0.86 mmol), Pd(dppf)Cl₂−CH₂Cl₂(250

mg, 0.34 mmol), and isopropenylboronic acid pinacol ester (430 mg,

2.6 mmol) in 1,4-dioxane (12 mL) was treated with 0.4 M aq Na₂CO₃

(8.6 mL, 3.4 mmol), purged with nitrogen, and stirred at 100 °C for 15

min before being treated with water and extracted with EtOAc. The

organic extract was washed with brine, dried over Na₂SO₄, ﬁltered,

concentrated, and subjected to ﬂash chromatography (50% EtOAc/

hexanes) to give 64 (65 mg, 17%) as a yellow solid following

trituration with MeOH: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.08

(br. s, 1H), 8.45 (s, 1H), 8.42 (t, J = 1.88 Hz, 1H), 8.37 (s, 1H), 8.01

(dd, J = 1.38, 8.16 Hz, 1H), 7.56−7.62 (m, 1H), 7.45−7.52 (m, 2H),

5.25 (s, 1H), 5.06 (s, 1H), 3.31 (s, 3H), 2.21 (s, 3H), 1.25 (s, 9H); MS

(m/z) 443.9 (M + H⁺).

5-((9H-Purin-6-yl)amino)-N,2-dimethylbenzenesulfonamide (16).

Compound 16 was prepared from 6-chloropurine and 5-amino-N,2-

dimethylbenzenesulfonamide using Method A as a yellow solid in 14%

yield (48 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.17 (s, 1 H),

8.40−8.47 (m, 2 H), 8.37 (s, 1 H), 8.08 (dd, J = 8.28, 2.26 Hz, 1 H),

7.49 (q, J = 4.77 Hz, 1 H), 7.37 (d, J = 8.53 Hz, 1 H), 2.53 (s, 3 H),

2.51 (s, 3H). MS (m/z) 319.0 (M + H⁺).

3-((9H-Purin-6-yl)amino)-N,4-dimethylbenzenesulfonamide (17).

Compound 17 was prepared from 6-chlorpurine and 3-amino-N,4-

dimethylbenzenesulfonamide using Method A as a yellow solid in 13%

1

yield (43 mg): H NMR (400 MHz, DMSO-d₆) δ ppm 9.82 (br. s., 1

H), 8.39 (s, 1 H), 8.35 (s, 1 H), 7.91 (d, J = 1.51 Hz, 1 H), 7.58 (dd, J

= 1.8, 8.0 Hz, 1 H), 7.53 (d, J = 8.0 Hz, 1 H), 7.43 (q, J = 4.94 Hz, 1

H), 2.46 (d, J = 4.77 Hz, 3 H), 2.33 (s, 3 H). MS (m/z) 319.0 (M +

H⁺).

N-Methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-

benzenesulfonamide (18). Compound 18 was prepared from 4-

chloro-7H-pyrrolo[2,3-d]pyrimidine and N-methyl-3-(methylamino)-

benzenesulfonamide (74) using Method B as a white solid in 18%

yield (26 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.09 (br. s, 1

H), 8.40 (s, 1 H), 7.83−7.72 (m, 4 H), 7.58 (q, J = 4.94 Hz, 1 H), 2.43

(d, J = 5.02 Hz, 3 H). MS (m/z) 318.0 (M + H⁺).

N-Methyl-3-{[5-(1-methylethyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]-

amino}benzenesulfonamide hydrochloride (22). A solution of 64

(65 mg, 0.15 mmol) in MeOH (25 mL) was treated with 10% Pd/C

(60 mg, 0.056 mmol) and stirred at 25 °C under a balloon of H₂

overnight. After 16 h, the mixture was ﬁltered, and the ﬁltrate was

concentrated to give tert-butyl (3-((5-isopropyl-7H-pyrrolo[2,3-d]-

pyrimidin-4-yl)amino)phenyl)sulfonyl(methyl)carbamate (52 mg,

1

80%) as a gray solid: H NMR (400 MHz, DMSO-d₆) δ ppm 11.67

(br. s, 1H), 8.45 (br. s, 1H), 8.32−8.36 (m, 1H), 8.27 (s, 1H), 8.08

(dd, J = 1.63, 7.65 Hz, 1H), 7.58 (t, J = 8.03 Hz, 1H), 7.46−7.52 (m,

1H), 7.08 (s, 1H), 3.31 (s, 3H), 1.27 (s, 3H), 1.26 (s, 3H), 1.24 (s,

9H); MS (m/z) 446 (M + H⁺).

Method D. Synthesis of 3-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-

N-methylbenzenesulfonamide (19). A mixture of 4-chloro-7H-

pyrrolo[2,3-d]pyrimidine (41.0 mg, 0.267 mmol), 3-hydroxy-N-

methylbenzenesulfonamide (62) (50 mg, 0.267 mmol), and Cs₂CO₃

(174 mg, 0.534 mmol) in DMF (2 mL) was subjected to microwave

irradiation (180 °C) for 60 min. The solid was removed by ﬁltration,

and the ﬁltrate was diluted with MeOH and subjected to reverse phase

HPLC (20−60% MeCN/water/0.1%TFA) to give 19 as an oﬀ-white

tert-Butyl (3-((5-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-

amino)phenyl)sulfonyl(methyl)carbamate (52 mg, 0.117 mmol) in

CH₃CN (5 mL) at 25 °C was treated with HCl (0.5 mL of a 4 M

solution in 1,4-dioxane, 2.00 mmol) and stirred overnight before being

concentrated to give an oil. The oil was dissolved in MeOH and

ﬁltered, and the ﬁltrate was concentrated to give 22 (48 mg, 97%) as a

pale yellow solid: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.26 (br. s,

1H), 9.18 (br. s, 1H), 8.31 (s, 1H), 8.05 (s, 1H), 7.91 (d, J = 7.78 Hz,

1H), 7.49−7.71 (m, 3H), 7.24 (br. s, 1H), 3.56−3.69 (m, 1H), 2.48 (s,

3H), 1.28 (d, J = 6.53 Hz, 6H). MS (m/z) 346.0 (M + H⁺).4-Chloro-

5-(2-methyl-2-propen-1-yl)-1H-pyrrolo[2,3-d]pyrimidine (65)

N-Methyl-3-{[5-(2-methylpropyl)-1H-pyrrolo[2,3-d]pyrimidin-4-

yl]amino}benzenesulfonamide Triﬂuoroacetate (23) A mixture of 5-

bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (67)²¹(500 mg, 2.2

mmol) in THF (15 mL) at −78 °C was treated dropwise with n-BuLi

(4.3 mL of 2.5 M in hexanes, 10.75 mmol). After 1 h, 3-bromo-2-

methyl-1-propene (2.89 g, 21.5 mmol) was added, and the mixture was

stirred and allowed to warm to room temperature. After 30 min, the

reaction was quenched with the addition of water (1 mL), and the

mixture was concentrated and subjected to reverse phase HPLC

(MeCN/H₂O/0.1% TFA) to give 65 (100 mg, 22%) as a brown solid:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.33 (br. s, 1 H), 8.49 (s, 1

H), 7.43 (s, 1 H), 4.75 (br. s, 1 H), 4.44 (s, 1 H), 3.47 (s, 2 H), 1.73

(s, 3 H). MS (m/z) 208.0 (M + H⁺).N-Methyl-3-{[5-(2-methyl-2-

propen-1-yl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}-

benzenesulfonamide (66)

A mixture of 65 (100 mg, 0.483 mmol) and 3-amino-N-

methylbenzenesulfonamide (135 mg, 0.726 mmol) in DMF (5 mL)

was treated with AgOTf (124 mg, 0.483 mmol) and heated at 80 °C

for 10 h before being cooled, diluted with water, and extracted with

CH₂Cl₂(3 × 10 mL). The combined organic extracts were dried

(Na₂SO₄) and concentrated to give 66 (150 mg, 87%) as a black oil:

MS (m/z) 358 (M + H⁺).

1

solid (4 mg, 4%): H NMR (400 MHz, DMSO-d₆) δ ppm 12.32 (br.

s., 1 H), 8.34 (s, 1 H), 7.68−7.74 (m, 2 H), 7.58−7.64 (m, 2 H),

7.52−7.58 (m, 2 H), 2.46 (d, J = 5.02 Hz, 3 H). MS (m/z) 305.0 (M +

H⁺).

5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylpyridine-3-

sulfonamide (20). Compound 20 was prepared from 4-chloro-7H-

pyrrolo[2,3-d]pyrimidine and 5-amino-N-methylpyridine-3-sulfona-

mide (79) using Method B as an orange solid in 6% yield (6 mg):

¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.96 (br. s, 1H), 9.87 (s,

1H), 9.35 (d, J = 2.26 Hz, 1H), 8.86 (t, J = 2.26 Hz, 1H), 8.54 (d, J =

2.01 Hz, 1H), 8.39 (s, 1H), 7.74 (q, J = 4.94 Hz, 1H), 7.30−7.44 (m,

1H), 6.83 (dd, J = 1.76, 3.26 Hz, 1H), 2.50−2.55 (m, 3H). MS (m/z)

304.9 (M + H⁺).

N-Methyl-3-[(5-methyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-

benzenesulfonamide (21). Compound 21 was prepared from 4-

chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine²⁰and 3-amino-N-meth-

ylbenzenesulfonamide using Method B as a white solid in 3% yield (25

1

mg): H NMR (400 MHz, DMSO-d₆) δ ppm 11.52 (br. s, 1H), 8.39

(s, 1H), 8.21 (s, 1H), 8.19 (t, J = 1.82 Hz, 1H), 7.98 (dd, J = 1.24, 8.13

Hz, 1H), 7.51 (t, J = 7.94 Hz, 1H), 7.35−7.45 (m, 2H), 6.98−7.06 (m,

1H), 2.46−2.54 (presumed 3H, obscured by DMSO peak), 2.44 (d, J

= 5.02 Hz, 3H). MS (m/z) 318.1 (M + H⁺).1,1-Dimethylethyl 5-

bromo-4-[(3-{[{[(1,1-dimethylethyl)oxy]carbonyl}(methyl)amino]-

sulfonyl}phenyl)amino]-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate

(63)

N-Methyl-3-{[5-(1-methylethyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]-

amino}benzenesulfonamide hydrochloride (22) A mixture of 26 (10.0

g, 23.88 mmol) in CH₃CN (200 mL) at 25 °C was treated with Boc₂O

(16.64 mL, 71.7 mmol) and DMAP (5.84 g, 47.8 mmol) and stirred

for 15 min before being concentrated. The residue was dissolved in

EtOAc and washed with water and brine, dried over Na₂SO₄, ﬁltered,

concentrated, and subjected to ﬂash chromatography (20% EtOAc/

hexanes) to aﬀord a yellow solid, which was triturated with EtOAc to

give 63 (7.5 g, 54%) as a white solid: ¹H NMR (400 MHz, DMSO-d₆)

δ ppm 9.23 (s, 1H), 8.94 (s, 1H), 8.75−8.79 (m, 1H), 8.45−8.51 (m,

1H), 8.34 (s, 1H), 7.99−8.10 (m, 2H), 3.74 (s, 3H), 2.04 (s, 9H), 1.69

(s, 9H).1,1-Dimethylethyl methyl[(3-{[5-(1-methylethenyl)-1H-

pyrrolo[2,3-d]pyrimidin-4-yl]amino}phenyl)sulfonyl]carbamate (64)

N-Methyl-3-{[5-(2-methylpropyl)-1H-pyrrolo[2,3-d]pyrimidin-4-

yl]amino}benzenesulfonamide Triﬂuoroacetate (23). A solution of

66 (150 mg, 0.42 mmol) in MeOH (20 mL) was treated with 10%

Pd/C (100 mg) and stirred a 25 °C under 30 psi of H₂for 16 h before

being ﬁltered, concentrated, and subjected to reverse phase HPLC

(MeCN/H₂O/0.1% TFA) to give 23 (24 mg, 16%) as a brown solid:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.83 (br. s, 1 H), 8.59 (br. s,

1 H), 8.26 (s, 1 H), 8.15 (s, 1 H), 7.91 (d, J = 9.30, 1 H), 7.57 (t, J =

7.91 Hz, 1 H), 7.45 (m, 2 H), 7.10 (s, 1 H), 2.82 (d, J = 7.03 Hz, 2 H),

2.47 (d, J = 5.02 Hz, 3 H), 1.80−1.87 (m, 1 H). MS (m/z) 360.2 (M +

H⁺).

K

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3-[(5-Fluoro-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methyl-

benzenesulfonamide Triﬂuoroacetate (24). Compound 24 was

prepared from 4-chloro-5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidine²¹and

3-amino-N-methylbenzenesulfonamide using Method A as an white

N-Methyl-3-({5-[4-(methyloxy)phenyl]-1H-pyrrolo[2,3-d]-

pyrimidin-4-yl}amino)benzenesulfonamide Triﬂuoroacetate (31).

Compound 31 was prepared from 26 and 4-methoxyphenylboronic

acid using Method E as a white solid in 11% (28 mg): ¹H NMR (400

MHz, DMSO-d₆) δ ppm 12.18 (br. s, 1H), 8.39 (s, 1H), 8.12 (s, 1H),

7.97 (br. s, 1H), 7.70−7.77 (m, 1H), 7.49−7.58 (m, 3H), 7.46 (q, J =

4.94 Hz, 1H), 7.35−7.43 (m, 2H), 7.07 (d, J = 8.53 Hz, 2H), 3.82 (s,

3H), 2.45 (d, J = 5.02 Hz, 3H). MS (m/z) 410.0 (M + H⁺).

N-Methyl-3-{[5-(4-pyridinyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]-

amino}benzenesulfonamide (32). Compound 32 was prepared from

63 and 4-pyridylboronic acid using Method E as a pale orange solid in

26% yield (26 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.42 (br.

s, 1H), 8.54−8.61 (m, 3H), 8.41 (s, 1H), 8.07 (s, 1H), 7.86 (d, J =

8.28 Hz, 1H), 7.76 (s, 1H), 7.56 (d, J = 6.02 Hz, 2H), 7.51 (t, J = 7.91

Hz, 1H), 7.43 (q, J = 4.77 Hz, 1H), 7.37 (d, J = 7.78 Hz, 1H), 2.46 (d,

J = 5.02 Hz, 3H). MS (m/z) 380.1 (M + H⁺).

1

solid in 53% yield (68 mg): H NMR (400 MHz, DMSO-d₆) δ ppm

11.81 (br. s, 1H), 9.25 (br. s, 1H), 8.30 (s, 1H), 8.26 (t, J = 1.76 Hz,

1H), 8.03−8.13 (m, 1H), 7.51−7.64 (m, 1H), 7.37−7.51 (m, 2H),

7.30 (t, J = 2.51 Hz, 1H), 2.47 (d, J = 4.77 Hz, 3H). MS (m/z) 322.0

(M + H⁺).

3-[(5-Chloro-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methyl-

benzenesulfonamide Hydrochloride (25). Compound 25 was

prepared from 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine²²and 3-

amino-N-methylbenzenesulfonamide using Method A as an tan solid

1

in 36% yield (72 mg): H NMR (400 MHz, DMSO-d₆) δ ppm 12.25

(br. s, 1H), 8.62 (s, 1H), 8.33 (s, 1H), 8.27 (t, J = 1.76 Hz, 1H), 8.00−

8.06 (m, 1H), 7.55−7.61 (m, 1H), 7.54 (d, J = 2.76 Hz, 1H), 7.43−

7.49 (m, 2H), 2.47 (d, J = 5.02 Hz, 3H). MS (m/z) 322.0 (M + H⁺).

3-[(5-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methyl-

benzenesulfonamide Triﬂuoroacetate (26). Compound 26 was

prepared from 5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine²²and

3-amino-N-methylbenzenesulfonamide using Method A as yellow solid

in 17% yield (157 mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.37

(br. s, 1H), 8.55 (s, 1H), 8.35 (s, 1H), 8.27 (t, J = 1.76 Hz, 1H), 7.98−

8.04 (m, 1H), 7.55−7.61 (m, 2H), 7.43−7.50 (m, 2H), 2.47 (d, J =

5.02 Hz, 3H). MS (m/z) 382.0 (M + H⁺).

Method E. General Suzuki Coupling Method for Aryl Boronic

Acids and 7- or 8-Bromo-7-Deazapurines.¹⁹A mixture of the

appropriate bromide (26, 35, or 63), aryl boronic acid (3 equiv),

PdCl₂(dppf)−CH₂Cl₂(0.4 equiv) or Pd(PPh₃)₄(0.2 equiv), and 0.4

M aqueous Na₂CO₃(4 equiv) in 1,4-dioxane (5 mL) was heated at

100 °C for 16 h or subjected to microwave irradiation (160 °C) for 15

min before being diluted with water and extracted with EtOAc. The

organic extract was washed (saturated aqueous NaCl), dried

(Na₂SO₄), and concentrated to give pure products (34), or else the

solids were subjected to ﬂash chromatography (27, 32, 33, 36, 37, 39),

preparative HPLC (28, 29, 30, 31, 40, 41) or SCX cation exchange

(MeOH wash, NH₃/MeOH elution, 38, 42) to give analytically pure

solids.

3-({5-[4-(Aminomethyl)phenyl]-1H-pyrrolo[2,3-d]pyrimidin-4-yl}-

amino)-N-methylbenzenesulfonamide (33). Compound 33 was

prepared from 63 and 4-aminomethylphenylboronic acid using

Method E as a tan solid in 54% yield (57 mg): ¹H NMR (400

MHz, CD₃OD) δ ppm 8.40−8.43 (m, 1H), 8.15−8.19 (m, 1H), 7.69−

7.75 (m, 1H), 7.52−7.63 (m, 4H), 7.46−7.50 (m, 2H), 7.30−7.33 (m,

1H), 3.95 (s, 2H), 2.55−2.59 (m, 3H). MS (m/z) 409.1 (M + H⁺).

4-[4-({3-[(Methylamino)sulfonyl] phenyl}amino)-1H-pyrrolo[2,3-

d]pyrimidin-5-yl]benzoic acid (34). Compound 34 was prepared

from 63 and (4-methoxycarbonyl)phenylboronic acid using Method E

1

as a brown solid in 41% yield (45 mg, 93% purity): H NMR (400

MHz, DMSO-d₆) δ ppm 12.89 (br. s, 1H), 12.28−12.37 (m, 1H), 8.40

(s, 1H), 8.31 (s, 1H), 8.14 (s, 1H), 7.97−8.04 (m, J = 8.28 Hz, 2H),

7.78 (dd, J = 1.38, 7.91 Hz, 1H), 7.67−7.72 (m, J = 8.53 Hz, 2H), 7.64

(d, J = 2.26 Hz, 1H), 7.50 (t, J = 7.91 Hz, 1H), 7.43 (q, J = 4.85 Hz,

1H), 7.36 (d, J = 8.03 Hz, 1H), 2.46 (d, J = 5.02 Hz, 3H). MS (m/z)

424.0 (M + H⁺).

3-[(6-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methyl-

benzenesulfonamide (35). Compound 35 was prepared from 69²⁶

and 3-amino-N-methylbenzenesulfonamide using Method B as an oﬀ-

white solid in 53% yield (413 mg): ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 12.68 (br. s, 1H), 9.66 (s, 1H), 8.24−8.36 (m, 3H), 7.57 (t, J =

8.06 Hz, 1H), 7.45 (q, J = 4.95 Hz, 1H), 7.40 (d, J = 7.81 Hz, 1H),

6.92 (s, 1H), 2.46 (d, J = 5.04 Hz, 3H). MS (m/z) 382.0 (M + H⁺).

N-Methyl-3-[(6-phenyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-

benzenesulfonamide (36). Compound 36 was prepared from 35 and

phenylboronic acid using Method E as a yellow solid in 38% yield (19

N-Methyl-3-[(5-phenyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-

benzenesulfonamide Triﬂuoroacetate (27). Compound 27 was

prepared from 63 and phenylboronic acid using Method E as a

1

white solid in 45% yield (13 mg): H NMR (400 MHz, DMSO-d₆) δ

ppm 12.19 (br. s, 1H), 8.40 (s, 1H), 8.13 (t, J = 1.89 Hz, 1H), 7.95 (s,

1H), 7.72 (dd, J = 1.26, 8.06 Hz, 1H), 7.60 (d, J = 7.05 Hz, 2H), 7.46−

7.54 (m, 3H), 7.44 (br. s, 1H), 7.34−7.41 (m, 3H), 2.45 (s, 3H). MS

(m/z) 380.0 (M + H⁺).

1

mg): H NMR (400 MHz, DMSO-d₆) δ ppm 12.39 (s, 1H), 9.75 (s,

1H), 8.32−8.39 (m, 3H), 7.86 (d, J = 7.81 Hz, 2H), 7.54−7.61 (m,

1H), 7.32−7.54 (m, 5H), 7.25 (d, J = 1.76 Hz, 1H), 2.47−2.49 (m,

3H). MS (m/z) 380.0 (M + H⁺).

3-{[5-(2-Chlorophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}-

N-methylbenzenesulfonamide Triﬂuoroacetate (28). Compound 28

was prepared from 26 and 2-chlorophenylboronic acid using Method

E as a white solid in 15% yield (19 mg): ¹H NMR (400 MHz, DMSO-

d₆) δ ppm 12.30 (br. s, 1H), 8.40 (s, 1H), 8.03 (s, 1H), 7.69 (br. s,

1H), 7.57−7.65 (m, 2H), 7.52−7.57 (m, 1H), 7.42−7.52 (m, 5H),

7.36 (d, J = 7.53 Hz, 1H), 2.44 (d, J = 5.02 Hz, 3H). MS (m/z) 414.0

(M + H⁺).

N-Methyl-3-{[6-(2-methylphenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-

yl]amino}benzenesulfonamide (37). Compound 37 was prepared

from 35 and 2-methylphenylboronic acid using Method E as a tan

1

solid in 7% yield (8 mg): H NMR (400 MHz, DMSO-d₆) δ ppm

12.12 (br. s, 1H), 9.70 (s, 1H), 8.33−8.39 (m, 3H), 7.54−7.62 (m,

2H), 7.28−7.47 (m, 5H), 7.00 (s, 1H), 2.50 (2.47−2.54 presumed 3H,

obscured by DMSO peak), 2.47 (d, J = 4.78 Hz, 3H). MS (m/z) 394.1

(M + H⁺).

3-{[5-(3-Chlorophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}-

N-methylbenzenesulfonamide Triﬂuoroacetate (29). Compound 29

was prepared from 26 and 3-chlorophenylboronic acid using Method

E as a white solid in 6% yield (8 mg): ¹H NMR (400 MHz, DMSO-d₆)

δ ppm 12.33 (br. s, 1H), 8.41 (s, 1H), 8.39 (br. s, 1H), 8.07−8.12 (m,

1H), 7.77−7.84 (m, 1H), 7.61−7.66 (m, 2H), 7.45−7.55 (m, 3H),

7.41−7.45 (m, 1H), 7.35−7.41 (m, 2H), 2.46 (d, J = 5.02 Hz, 3H).

MS (m/z) 414.0 (M + H⁺).

3-{[5-(4-Chlorophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}-

N-methylbenzenesulfonamide Triﬂuoroacetate (30). Compound 30

was prepared from 63 and 4-chlorophenylboronic acid using Method

E as a white solid in 11% yield (15 mg): ¹H NMR (400 MHz, DMSO-

d₆) δ ppm 12.27 (br. s, 1H), 8.39 (s, 1H), 8.29 (br. s, 1H), 8.08−8.15

(m, 1H), 7.74−7.82 (m, 1H), 7.48−7.61 (m, 6H), 7.44 (q, J = 4.85

Hz, 1H), 7.38 (d, J = 7.28 Hz, 1H), 2.46 (d, J = 4.77 Hz, 3H). MS (m/

z) 414.0 (M + H⁺).

N-Methyl-3-{[6-(3-methylphenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-

yl]amino}benzenesulfonamide (38). Compound 38 was prepared

from 35 and 3-methylphenylboronic acid using Method E as a brown

1

solid in 51% yield (40 mg): H NMR (400 MHz, DMSO-d₆) δ ppm

12.35 (br. s, 1H), 9.74 (s, 1H), 8.27−8.44 (m, 3H), 7.62−7.75 (m,

1H), 7.58 (t, J = 7.81 Hz, 1H), 7.43−7.54 (m, 1H), 7.34−7.43 (m,

2H), 7.23−7.32 (m, 1H), 7.17 (d, J = 7.55 Hz, 1H), 7.03 (br. s, 1H),

2.48 (d, J = 4.78 Hz, 3H), 2.39 (s, 3H). MS (m/z) 394.1 (M + H⁺).

N-Methyl-3-{[6-(4-methylphenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-

yl]amino}benzenesulfonamide (39). Compound 39 was prepared

from 35 and 4-methylphenylboronic acid using Method E as a tan

1

solid in 17% yield (9 mg): H NMR (400 MHz, DMSO-d₆) δ ppm

12.32 (br. s, 1H), 9.71 (s, 1H), 8.29−8.41 (m, 3H), 7.74 (d, J = 8.06

Hz, 2H), 7.58 (t, J = 8.06 Hz, 1H), 7.45 (q, J = 4.78 Hz, 1H), 7.39 (d, J

= 7.81 Hz, 1H), 7.30 (d, J = 8.06 Hz, 2H), 7.18 (d, J = 2.01 Hz, 1H),

2.47 (d, J = 4.78 Hz, 3H), 2.35 (s, 3H). MS (m/z) 394.1 (M + H⁺).

L

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

N-Methyl-3-({6-[3-(triﬂuoromethyl)phenyl]-1H-pyrrolo[2,3-d]-

pyrimidin-4-yl}amino)benzenesulfonamide (40). Compound 40 was

prepared from 35 and 3-triﬂuoromethylphenylboronic acid using

Method E as a white solid in 38% yield (45 mg): ¹H NMR (400 MHz,

DMSO-d₆) δ ppm 8.46 (br. s, 3 H), 8.13−8.18(m, 3 H), 7.82 (d, J =

7.78 Hz, 2 H), 7.62−7.69 (m, 2 H), 7.48−7.55 (m, 2 H), 2.55 (d, 3 H

(obscured by solvent)). MS (m/z) 448.1 (M + H⁺).

3-{[6-(4-Hydroxyphenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}-

N-methylbenzenesulfonamide Triﬂuoroacetate (41). Compound 41

was prepared from 35 and 4-hydroxphenylboronic acid using Method

E as a white solid in 3% yield (4 mg): ¹H NMR (400 MHz, MeOD) δ

ppm 8.31 (s, 1 H), 8.12 (s, 1 H), 7.84−7.92 (m, 2 H), 7.74−7.80 (m, 1

H), 7.63−7.70 (m, 2 H), 6.88−6.95 (m, 3 H), 2.62 (s, 3 H). MS (m/

z) 396.1 (M + H⁺).

δ ppm 11.76 (br. s, 1H, pyrrolopyrimidine NH), 8.78 (s, 1H, aniline

NH), 8.20 (s, 1H, pyrrolopyrimidine C2-H), 8.11 (d, J = 2.26 Hz, 1H,

benzene C2-H)), 7.49 (dd, J = 2.26, 8.53 Hz, 1H, benzene C6-H), 7.29

(q, J = 4.85 Hz, 1H, SO₂NHMe), 7.18−7.23 (m, 2H, benzene C5-H,

pyrrolopyrimidine C6-H), 6.55 (dd, J = 1.63, 3.14 Hz, 1H,

pyrrolopyrimidine C5-H), 2.76 (s, 6H, N(CH₃)₂), 2.43 (d, J = 5.02

Hz, 3H, SO₂NHCH₃). ¹³C NMR (500 MHz, DMSO-d₆) δ ppm

154.86 (pyrrolopyrimidine C4), 151.92 (pyrrolopyrimidine C7a),

151.86 (pyrrolopyrimidine C2), 151.04 (benzenesulfonamide C4),

131.71 (benzene C3), 131.64 (benzene C1), 125.72 (benzene C2),

124.32 (benzene C6), 123.17 (pyrrolopyrimidine C6), 118.97

(benzene C5), 104.27 (pyrrolopyrimidine C4a), 99.40 (pyrrolopyr-

imidine C5), 43.31 (N(CH₃)₂), 29.59 (SO₂NHCH₃). For compound

48, the ¹H NMR and ¹³C NMR resonances were assigned on the basis

of gCOSY45, gHMQC, gHMBC, and ¹³C GASPE spectra, and all 2D

correlations are in agreement with the assigned structure. MS (m/z)

347.0 (M + H⁺).

N-Methyl-3-{[6-(4-pyridinyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]-

amino}benzenesulfonamide (42). Compound 42 was prepared from

35 and 4-pyridylboronic acid using Method E as a brown solid in 50%

1

yield (37 mg): H NMR (400 MHz, DMSO-d₆) δ ppm 12.35 (br. s,

N-Methyl-4-(4-morpholinyl)-3-(1H-pyrrolo[2,3-d]pyrimidin-4-

ylamino)benzenesulfonamide (49). Compound 49 was prepared

from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-4-(morpho-

lino)-N-methylbenzenesulfonamide (83c) using Method B as tan solid

1H), 9.74 (s, 1H), 8.30−8.40 (m, 3H), 7.34−7.72 (m, 5H), 7.23−7.32

(m, 1H), 7.17 (d, J = 7.55 Hz, 1H), 7.03 (br. s, 1H), 2.48 (d, J = 4.78

Hz, 3H). MS (m/z) 381.0 (M + H⁺).

4-({3-[(Methylamino)sulfonyl] phenyl}amino)-1H-pyrrolo[2,3-d]-

pyrimidine-6-carboxylic acid (43). Compound was prepared from

72 and 3-amino-N-methylbenzenesulfonamide using Method B as a

1

in 37% yield (47 mg): H NMR (400 MHz, DMSO-d₆) δ ppm 11.86

(br. s, 1H), 8.67 (s, 1H), 8.52 (d, J = 2.01 Hz, 1H), 8.29 (s, 1H), 7.50

(dd, J = 2.13, 8.41 Hz, 1H), 7.39 (q, J = 4.94 Hz, 1H), 7.33 (d, J = 8.53

Hz, 1H), 7.25−7.31 (m, 1H), 6.54−6.61 (m, 1H), 3.65−3.76 (m, 4H),

2.91−2.98 (m, 4H), 2.45 (d, J = 5.02 Hz, 3H). MS (m/z) 389.1 (M +

H⁺).

4-(Dimethylamino)-N-methyl-3-[(5-methyl-1H-pyrrolo[2,3-d]-

pyrimidin-4-yl)amino]benzenesulfonamide Triﬂuoroacetate (50).

Compound 50 was prepared from 4-chloro-5-methyl-7H-pyrrolo[2,3-

d]pyrimidine²⁰and 3-amino-4-(dimethylamino)-N-methylbenzenesul-

fonamide (83b) using Method C as an orange solid in 20% yield (28

mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.04 (br. s, 1 H), 9.17

(br. s, 1H), 8.60 (br. s, 1H), 8.29 (s, 1 H), 7.54 (m, 1 H), 7.38−7.46

(m, 2 H), 7.21 (br. s, 1 H), 2.76 (s, 6H), 2.53 (s, 3 H), 2.46 (d, J =

5.02 Hz, 3 H). MS (m/z) 361.1 (M + H⁺).

3-[(5-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-4-(dimethy-

lamino)-N-methylbenzenesulfonamide Hydrochloride (51). Com-

pound 51 was prepared from 5-bromo-4-chloro-7H-pyrrolo[2,3-

d]pyrimidine²²and 3-amino-4-(dimethylamino)-N-methylbenzenesul-

fonamide (83b) using Method A as a gray solid in 8% yield (25 mg):

¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.38 (s, 1H), 9.31 (s, 1H),

9.26 (d, J = 2.01 Hz, 1H), 8.43 (s, 1H), 7.61 (s, 1H), 7.38−7.51 (m,

3H), 2.74 (s, 6H), 2.47 (d, J = 5.02 Hz, 3H). MS (m/z) 424.9 (M +

H⁺).

1

pale purple 43 solid in 57% yield (38 mg): H NMR (400 MHz,

DMSO-d₆) δ ppm 12.59 (br. s, 1H), 9.99 (br. s, 1H), 8.43 (s, 1H),

8.33−8.39 (m, 1H), 8.30 (d, J = 7.78 Hz, 1H), 7.55−7.67 (m, 2H),

7.41−7.55 (m, 2H), 2.47 (d, J = 4.77 Hz, 3H). MS (m/z) 347.9 (M +

H⁺).

N-Methyl-3-(1H-pyrimido[4,5-b]indol-4-ylamino)-

benzenesulfonamide (44). Compound 44 was prepared from 4-

chloro-9H-pyrimido[4,5-b]indole²³and 3-amino-N-methylbenzenesul-

fonamide using Method B as a pale lavender solid in 90% yield (156

mg): ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.27 (s, 1H), 9.27 (br.

s., 1H), 8.50 (s, 1H), 8.46 (d, J = 7.81 Hz, 1H), 8.23 (t, J = 1.76 Hz,

1H), 8.07 (dd, J = 1.01, 8.06 Hz, 1H), 7.61 (t, J = 7.93 Hz), 7.53−7.58

(m, 1H), 7.44−7.53 (m, 3H), 7.29−7.36 (m, 1H), 2.48 (d, J = 4.78

Hz, 3H). MS (m/z) 354.1 (M + H⁺).

N,4-Dimethyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-

benzenesulfonamide (45). Compound 45 was synthesized from 4-

chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-N,4-dimethylbenze-

nesulfonamide using Method B as a pale green solid in 20% yield (75

1

mg): H NMR (400 MHz, DMSO-d₆) δ ppm 11.75 (br. s, 1H), 9.10

(s, 1H), 8.14 (s, 1H), 7.84 (d, J = 1.51 Hz, 1H), 7.48−7.57 (m, 2H),

7.42 (q, J = 4.85 Hz, 1H), 7.18−7.24 (m, 1H), 6.49 (dd, J = 1.76, 3.26

Hz, 1H), 2.45 (d, J = 5.02 Hz, 3H), 2.31 (s, 3H). MS (m/z) 318.0 (M

+ H⁺).

N-Methyl-3-[(5-methyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-

4-(4-morpholinyl)benzenesulfonamide (52). Compound 52 was

prepared from 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine²⁰and

3-amino-4-(morpholino)-N-methylbenzenesulfonamide (83c) using

4-Chloro-N-methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-

benzenesulfonamide Triﬂuoroacetate (46). Compound 46 was

prepared from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-4-

chloro-N-methylbenzenesulfonamide²⁵using Method C as a tan solid

1

Method C as gray solid in 10% yield (12 mg): H NMR (400 MHz,

1

in 12% yield (24 mg): H NMR (400 MHz, DMSO-d₆) δ ppm 12.09

DMSO-d₆) δ ppm 11.65 (br. s, 1 H), 9.26 (d, J = 2.01 Hz, 1 H), 8.75

(s, 1 H), 8.34 (s, 1 H), 7.57 (d, J = 8.28 Hz, 1 H), 7.43 (m, 2 H), 7.12

(s, 1 H), 3.77−3.84 (m, 4 H), 2.90−2.97 (m, 4 H), 2.66 (s, 3 H), 2.47

(s, 3 H). MS (m/z) 403.1 (M + H⁺).

(br. s., 1 H), 9.76 (br. s., 1 H), 8.25 (s, 1 H), 8.13 (d, J = 2.26 Hz, 1

H), 7.85 (d, J = 8.53 Hz, 1 H), 7.55−7.71 (m, 2 H), 7.32−7.37 (m, 1

H), 6.67 (br. s., 1 H), 2.49 (d, J = 5.02 Hz, 3 H). MS (m/z) 338.0 (M

+ H⁺).

3-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methyl-4-

(methylamino)benzenesulfonamide (47). Compound 47 was pre-

pared from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-N-

methyl-4-(methylamino)benzenesulfonamide (83a) using Method B

as a tan solid in 15% yield (16 mg): ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 11.64 (br. s., 1 H), 8.72 (s, 1 H), 8.10 (s, 1 H), 7.52 (dd, J = 8.66,

2.13 Hz, 1 H), 7.48−7.50 (m, 1 H), 7.12−7.15 (m, 1 H), 7.05 (q, J =

5.27 Hz, 1 H), 6.73 (d, J = 8.53 Hz, 1 H), 6.31 (br. s., 1 H), 5.91−6.02

(m, 1 H), 2.77 (d, J = 4.77 Hz, 3 H), 2.37 (d, J = 5.27 Hz, 3 H). MS

(m/z) 333.0 (M + H⁺).

3-[(5-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methyl-

4-(4-morpholinyl)benzenesulfonamide Hydrochloride (53). Com-

pound 53 was prepared from 5-bromo-4-chloro-7H-pyrrolo[2,3-

d]pyrimidine²²and 3-amino-4-(morpholino)-N-methylbenzenesulfo-

namide (83c) using Method A as a pale gray solid in 32% yield (35

1

mg): H NMR (400 MHz, DMSO-d₆) δ ppm 12.41 (br. s, 1H), 9.13

(d, J = 2.01 Hz, 1H), 9.03 (s, 1H), 8.41 (s, 1H), 7.64 (d, J = 2.51 Hz,

1H), 7.39−7.55 (m, 3H), 3.84−3.91 (m, 4H), 2.91−2.98 (m, 4H),

2.47 (d, J = 5.02 Hz, 3H). MS (m/z) 466.9 (M + H⁺).

3-[(6-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-4-(dimethy-

lamino)-N-methylbenzenesulfonamide (54). Compound 54 was

prepared from 68 and 3-amino-4-(dimethylamino)-N-methylbenzene-

sulfonamide (83b) using Method B as a white solid in 36% yield (162

4-(Dimethylamino)-N-methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-

ylamino)benzenesulfonamide (48). Compound 48 was prepared

from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-4-(dimethy-

lamino)-N-methylbenzenesulfonamide (83b) using Method B as an

oﬀ-white solid in 22% yield (51 mg): ¹H NMR (400 MHz, DMSO-d₆)

1

mg): H NMR (400 MHz, DMSO-d₆) δ ppm 12.56 (br. s, 1H), 8.89

(s, 1H), 8.17 (s, 1H), 8.00 (d, J = 2.01 Hz, 1H), 7.46−7.54 (m, 1H),

M

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.28 (d, J = 5.04 Hz, 1H), 7.19 (d, J = 8.56 Hz, 1H), 6.67 (s, 1H), 2.75

(s, 6H), 2.42 (d, J = 5.04 Hz, 3H). MS (m/z) 425.0 (M + H⁺).

4-(Dimethylamino)-N-methyl-3-({6-[3-(triﬂuoromethyl)phenyl]-

1H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)benzenesulfonamide Tri-

ﬂuoroacetate (55). Compound 55 was prepared from 70 and 3-

amino-4-(dimethylamino)-N-methylbenzenesulfonamide (83b) using

Method A as a white solid in 13% yield (18 mg): ¹H NMR (400 MHz,

DMSO-d₆) δ ppm 12.52 (br. s, 1 H), 8.87 (s, 1 H), 8.28 (s, 1 H), 8.23

(m, 1 H), 8.12 (d, J = 7.53 Hz, 1 H), 7.67−7.74 (m, 2 H), 7.50 (dd, J =

8.53, 2.26 Hz, 1 H), 7.31 (q, J = 4.94 Hz, 1 H), 7.22−7.29 (m, 2 H),

2.79 (s, 6 H), 2.44 (d, J = 5.02 Hz, 3 H). MS (m/z) 491.1 (M + H⁺).

3-[(6-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methyl-

4-(4-morpholinyl)benzenesulfonamide (56). Compound 56 was

prepared from 68 and 3-amino-4-(morpholino)-N-methylbenzenesul-

fonamide (83c) using Method B as a pale yellow powder in 65% yield

The resulting yellowish solid was suspended in 10 mL of hexanes and

ﬁltered to give 72 (718 mg, 56%) as an oﬀ-white solid: ¹H NMR (400

MHz, DMSO-d₆) δ ppm 13.33 (br. s, 1H), 8.74 (s, 1H), 7.14 (s, 1H),

1.58 (s, 9H). MS (m/z) 254.1 (M + H⁺).

N-Methyl-3-(methylamino)benzenesulfonamide (74). A mixture

of N-methyl-3-aminobenzenesulfonamide (1 g, 5.37 mmol) and

formaldehyde (0.440 mL, 5.91 mmol) in CH₂Cl₂at 25 °C was

treated with acetic acid (0.338 mL, 5.91 mmol) and stirred for 5 min

before sodium triacetoxyborohydride (1.707 g, 8.05 mmol) was added.

The mixture was stirred for 10 min before being treated with saturated

aqueous NaHCO₃and extracted with CH₂Cl₂. The combined organic

extracts were washed with brine, dried (Na₂SO₄), concentrated, and

subjected to ﬂash chromatography (35−40% EtOAc-hexanes) to give

74 (438 mg, 40%) as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 7.30−7.26 (m, 2H), 6.91−6.89 (m, 2H), 6.75 (d, J = 7.3 Hz, 1H),

6.18 (q, J = 5.0 Hz, 1H), 2.70 (d, J = 5 0.0 Hz, 3H), 2.40 (d, J = 3.8

Hz, 3H). MS (m/z) 201.0 (M + H⁺).

3-Hydroxy-N-methylbenzenesulfonamide (62). A mixture of 3-

(methyloxy)benzenesulfonyl chloride (250 mg, 1.21 mmol) and

DMAP (29.6 mg, 0.24 mmol) in a solution of methylamine (2 M in

THF, 4.8 mL, 9.6 mmol) was stirred at 25 °C for 30 min before being

ﬁltered and concentrated to give N-methyl-3-(methyloxy)-

benzenesulfonamide (267 mg, 85% purity, 93% yield) as a yellow

oil: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.50−7.57 (m, 1 H), 7.47

(br. s., 1 H), 7.35 (d, J = 7.78 Hz, 1 H), 7.28 (t, J = 2.01 Hz, 1 H), 7.23

(dd, J = 8.28, 2.26 Hz, 1 H), 3.83 (s, 3 H), 2.41 (s, 3 H). MS (m/z)

202.0 (M + H⁺).

1

(68 mg): H NMR (400 MHz, DMSO-d₆) δ ppm 12.64 (br. s, 1 H),

8.73 (s, 1 H), 8.34 (d, J = 2.01 Hz, 1 H), 8.23 (s, 1 H), 7.51 (dd, J =

8.31, 2.01 Hz, 1 H), 7.37 (q, J = 5.04 Hz, 1 H), 7.30 (d, J = 8.56 Hz, 1

H), 6.69 (s, 1 H), 3.64−3.72 (m, 4 H), 2.91−2.98 (m, 4 H), 2.44 (d, J

= 5.04 Hz, 3 H). MS (m/z) 467.0 (M + H⁺).

N-Methyl-4-(4-morpholinyl)-3-({6-[3-(triﬂuoromethyl)phenyl]-1H-

pyrrolo[2,3-d]pyrimidin-4-yl}amino)benzenesulfonamide Triﬂuor-

oacetate (57). Compound 57 was prepared from 70 and 3-amino-

4-(morpholino)-N-methylbenzenesulfonamide (83c) using Method A

1

as a white solid in 23% yield (35 mg, 93% purity): H NMR (400

MHz, DMSO-d₆) δ ppm 12.88 (br. s, 1 H), 8.36 (s, 1 H), 8.24 (br. s, 1

H), 8.17 (m, 2 H), 7.71−7.78 (m, 2 H), 7.59−7.66 (m, 2 H), 7.45 (m,

1 H), 7.38 (m, 1 H), 7.26 (br. s, 1 H), 2.96−3.04 (m, 4 H), 2.46 (d, J =

5.02 Hz, 3 H). MS (m/z) 533.2 (M + H⁺).

A mixture of N-methyl-3-(methyloxy)benzenesulfonamide (267 mg,

1.13 mmol) in CH₂Cl₂(2.8 mL) at 0 °C was treated with boron

tribromide (2.26 mL of 1 M in CH₂Cl₂, 2.26 mmol) and allowed to

warm to 25 °C. After 2 h the reaction was carefully quenched by the

cautious addition of dry methanol (∼1 mL) and then concentrated

and the residue partitioned between DCM and water. The organic

layer was then collected via hydrophobic frit and concentrated to give

62 (56 mg, 27%) as a brown oil. The aqueous layer was passed

through an OASIS cartridge (Waters) eluting with MeOH to give

4-Chloro-6-[3-(triﬂuoromethyl)phenyl]-1H-pyrrolo[2,3-d]-

pyrimidine (70). A mixture of 68²⁵(1.97 g, 4.34 mmol), 3-

(triﬂuoromethyl)phenylboronic acid (0.823 g, 4.34 mmol) and

K₂CO₃(0.959 g, 6.94 mmol) in 1,4-dioxane (36 mL) and water (12

mL) was purged with nitrogen and treated with PdCl₂(dppf)−CH₂Cl₂

(0.5 g, 0.612 mmol). The mixture was stirred at 82 °C for 3 h before

being cooled and partitioned between 200 mL EtOAc and 30 mL

brine. The organic layer was washed with brine, dried over MgSO₄,

concentrated, and subjected to ﬂash column chromatography (0−35%

EtOAc-hexanes) to give 4-chloro-7-(phenylsulfonyl)-6-[3-

(triﬂuoromethyl)phenyl]-7H-pyrrolo[2,3-d]pyrimidine (0.893 g,

47.0%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm

8.90 (s, 1 H), 7.89−7.96 (m, 5 H), 7.78 (t, J = 7.53 Hz, 2 H), 7.61−

7.69 (m, 2 H), 7.16 (s, 1 H). MS (m/z) 438.0 (M + H⁺).

A solution of 4-chloro-7-(phenylsulfonyl)-6-[3-(triﬂuoromethyl)-

phenyl]-7H-pyrrolo[2,3-d]pyrimidine (500 mg, 1.14 mmol) in THF

(20 mL) was treated with KOt-Bu (641 mg, 5.71 mmol) at 0 °C

before being warmed to 25 °C. After 30 min, the mixture was diluted

with 50 mL of sat. aqueous Na₂CO₃and extracted with EtOAc (100

mL × 2). The combined organic extracts were washed with brine,

dried over MgSO₄, and concentrated to give 70 (345 mg, 100%) as a

light brown solid: MS (m/z) 298.0 (M + H⁺).

1,1-Dimethylethyl 4-chloro-1H-pyrrolo[2,3-d]pyrimidine-6-car-

boxylate (72). To a suspension of 4,6-dichloro-pyrimidine-5-

carbaldehyde (71) (2 g, 11 mmol) in EtOH (50 mL) at 25 °C was

added glycine tert-butyl ester (1.894 g, 11 mmol) followed by Et₃N

(3.9 mL, 28 mmol). The reaction mixture was stirred for 24 h before

being concentrated, and the residue was taken up in water and

extracted with CH₂Cl₂. The organic layer extract was concentrated and

subjected to ﬂash chromatography (0−90% EtOAc-hexanes) to give

1,1-dimethylethyl 4-chloro-5-hydroxy-5,6-dihydro-1H-pyrrolo[2,3-d]-

pyrimidine-6-carboxylate (1.38 g, 44.9%) as a mix of two isomers, a

light yellow solid: MS (m/z) 272.1 (M + H⁺).

1

additional 62 (146 mg, 69%) as a yellow oil: H NMR (400 MHz,

DMSO-d₆) δ ppm 9.89−10.19 (m, 1 H), 7.33−7.42 (m, 2 H), 7.12−

7.21 (m, 2 H), 6.97−7.03 (m, 1 H), 2.39 (d, J = 4.77 Hz, 3 H). MS

(m/z) 188.0 (M + H⁺).

5-Bromo-3-pyridinesulfonyl Chloride (77). A mixture of 3-

pyridinesulfonyl chloride hydrochloride (8.9 g, 44 mmol) and bromine

(14 g, 88 mmol) was heated to 130 °C for 8 h. The resulting mixture

of 77 was cooled and used immediately in the subsequent step.

5-Bromo-N-methyl-3-pyridinesulfonamide (78). A solution of

CH₃NH₂(50 mL of a 23−30% in H₂O) at 0 °C was treated with

77 (44 mmol), warmed to 25 °C, and stirred for 3 h before being

extracted with EtOAc. The organic extract was concentrated washed

with 10:1 hot petroleum ether-EtOAc to give 78 (2.4 g, 18% yield, 2

steps) as a brown solid that was used immediately in the subsequent

step.

5-Amino-N-methyl-3-pyridinesulfonamide (79). A mixture of 78

(2.4 g, 9.6 mmol), CuCl (0.100 g, 1.01 mmol), and concentrated

aqueous NH₄OH (5 mL) was heated to 130 °C for 18 h in a sealed

tube. The reaction mixture was then treated with sodium sulﬁde and

extracted with EtOAc. The combined organic extracts were

concentrated and the residue was washed with 20:5:3 hot petroleum

1

ether-EtOAc-MeOH to give 79 (1.1 g, 61%) as a brown solid: H

NMR (400 MHz, DMSO-d₆) δ ppm 8.11 (d, J = 2.51 Hz, 1H), 8.04

(d, J = 1.76 Hz, 1H), 7.47 (br. s, 1H), 7.24 (t, J = 2.13 Hz, 1H), 5.83

(br. s, 2H), 2.44 (s, 3H); MS (m/z) 188.1 (M + H⁺).

4-Fluoro-3-nitrobenzenesulfonyl Chloride (81). 1-Fluoro-2-nitro-

benzene (80) (50.0 g, 0.354 mol) was added to chlorosulfonic acid (91

g, 0.778 mol) at 65 °C. The resulting mixture was then heated to 100

°C for 18 h. The mixture was cooled to 25 °C, poured over ice and

extracted with CH₂Cl₂. The combined organic layers were then

washed with sat. aqueous NaHCO₃, then brine, dried over MgSO₄,

ﬁltered, and concentrated to give 81 (55.3 g, 65%) as a brown oil: ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 8.24 (dd, J = 7.40, 2.13 Hz, 1 H),

To a solution of 1,1-dimethylethyl 4-chloro-5-hydroxy-5,6-dihydro-

1H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (1.38 g, 5.06 mmol) in

DMF (20 mL) at 0 °C was added NaH (0.186 g of a 60% dispersion in

mineral oil, 4.64 mmol). The resulting mixture was stirred for 1 h

before being quenched slowly with the addition of 2 mL of water and

partitioned between 30 mL sat. aqueous NH₄Cl solution and 250 mL

EtOAc. The organic layer was washed with sat. aqueous NH₄Cl (2 ×

20 mL) then brine (30 mL), dried over MgSO₄, and concentrated.

N

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.98 (ddd, J = 8.60, 4.45, 2.26 Hz, 1 H), 7.55 (dd, J = 11.29, 8.53 Hz, 1

H).

incubated at room temperature for 60 min. Fluorescence polarization

was measured on an Analyst plate reader (Ex/Em: 485/530 nm,

Dichroic: 505). For dose-response experiments, normalized data were

ﬁt by ABASE/XC₅₀to the following equation:

4-Fluoro-N-methyl-3-nitrobenzenesulfonamide (82). A solution

of 81 (43 g, 179.5 mmol) in THF (500 mL) was treated with Et₃N

(150 mL, 1.08 mol) and then cooled to −35 °C. Then a solution of

CH₃NH₂−HCl (14.5 g, 215.4 mmol) in water was added dropwise

and the mixture was stirred for 1 h before being warmed to 25 °C,

diluted with water and extracted with EtOAc. The organic extract was

washed with saturated aqueous NaHCO₃, then brine, dried over

MgSO₄, ﬁltered, concentrated, and subjected to ﬂash chromatography

(20% EtOAc-petroleum ether) to give 82 (38 g, 90%) as a yellow

solid: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.47 (dd, J = 7.03, 2.51

Hz, 1 H), 8.17 (ddd, J = 8.72, 3.95, 2.38 Hz, 1 H), 7.81−7.89 (m, 2

H), 2.47 (d, J = 5.02 Hz, 3 H). MS (m/z) 235.0 (M + H⁺).

3-Amino-4-(dimethylamino)-N-methylbenzenesulfonamide

(83b). A mixture of 82 (1.8 g, 7.7 mmol) and Me₂NH-HCl (0.63 g, 7.7

mmol) in THF (100 mL) at −40 °C was treated with Et₃N (1.8 mL,

12.8 mmol). The mixture was warmed to room temperature and

stirred for 30 min before being concentrated and subjected to ﬂash

chromatography (20% EtOAc-petroleum ether) to give 4-(dimethy-

lamino)-N-methyl-3-nitrobenzenesulfonamide (1.8 g, 90%) as a yellow

solid: MS (m/z) 260.0 (M+H⁺).

pIC₅₀= (log((b − y)/(y − a)))/d − log(x)

where x is the compound concentration, y is the % activity at speciﬁed

compound concentration, a is the minimum % activity, b is the

maximum % activity, and d is the Hill slope. The pIC₅₀values were

averaged to determine a mean value, for a minimum of 2 experiments,

but typically for ≥4 experiments. On average, pIC₅₀values fell within

0.3 units for individual experiments with a given compound.

Compound IC₅₀values less than 10 nM could not be accurately

obtained due to titration of TNNI3K in the assay.

TNNI3K Cellular Assay. The ability of compounds to inhibit

TNNI3K autophosphorylation was measured in HEKMSRII cells

overexpressing human myc-TNNI3K. After cell lysis, myc-TNNI3K is

captured using an antimyc tag antibody, and phosphorylation is

detected using europium-tagged antiphosphotyrosine antibody (Eu-

PY20, PerkinElmer Catalog No: AD0038) and measured by time-

resolved ﬂuorescence of europium on an EnVision plate reader

(PerkinElmer). This technique is known as dissociation-induced

lanthanide ﬂuorescence intensity assay (DELFIA). HEKMSRII cells

transduced with human myc-TNNI3K-expressing Bacmam were

cultured in DMEM/F12 with 0.1% FBS and 1% penicillin-

streptomycin at 37 °C in a 5% CO₂incubator overnight. These

myc-TNNI3K overexpressing cells were treated with test compounds

and incubated for 30 min. Pervanadate solution (0.8 mM vanadate,

0.01% H₂O₂, DMEM/F12 with 0.1% FBS and 1% penicillin-

streptomycin) was then added to cells. After 20 min of incubation

at room temperature, media was aspirated, and cells were lysed with

Cell Extraction Buﬀer (Invitrogen cat. No FNN0011) containing

Complete Protease Inhibitor Cocktail (Roche cat. no. 11 697 498

001). Fifteen microliters of cell lysate was transferred to a black

Maxisorp plate (Nunc cat no. 460518) coated with anti-Myc Tag

(clone 4A6, Millipore, cat. no. 05-724), which had been blocked and

washed with Superblock-TBS and TBS-T, respectively. After the plates

were incubated for 2 h at room temperature, they were washed with

TBS-T and treated with Superblock T20 (TBS) containing 100 μg/

mL Eu-PY20 antibody, and the plates were incubated for 1 h at room

temperature. The cells were then washed extensively with TBS-T,

DELFIA enhancement solution (PerkinElmer) was added and the cells

incubated for 20 min at room temperature, and ﬂuorescence was

quantiﬁed on an EnVision plate reader using an Europium-615 nm

ﬁlter. For dose response experiments, normalized data were ﬁt by

ABASE/XC₅₀to the following equation:

A solution of 4-(dimethylamino)-N-methyl-3-nitrobenzenesulfona-

mide (1.8 g, 6.9 mmol) in THF (100 mL) was treated with 10% Pd/C

(0.7 g) and stirred at 50 °C under an atmosphere of hydrogen for 16 h

before being ﬁltered and concentrated to give 83b (1.5 g, 94%) as a

1

white solid: H NMR (400 MHz, DMSO-d₆) δ ppm 7.03−7.10 (m,

2H), 7.00 (d, J = 8.28 Hz, 1H), 6.93 (dd, J = 2.13, 8.16 Hz, 1H), 5.13

(s, 2H), 2.62 (s, 6H), 2.38 (d, J = 5.02 Hz, 3H). MS (m/z) 230.2 (M

+H⁺).

3-Amino-N-methyl-4-(methylamino)benzenesulfonamide (83a).

Compound 83a was prepared from 82 and MeNH₂−HCl by a

procedure analogous to that for 83b to give 83a (1.3 g, 66%) as a

white solid: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 6.95 (dd, J = 8.16,

2.13 Hz, 1 H), 6.91 (d, J = 2.01 Hz, 1 H), 6.82 (q, J = 5.10 Hz, 1 H),

6.43 (d, J = 8.28 Hz, 1 H), 5.32 (q, J = 4.52 Hz, 1 H), 4.86 (s, 2 H),

2.77 (d, J = 5.02 Hz, 3 H), 2.32 (d, J = 5.02 Hz, 3 H). MS (m/z) 216.0

(M+H⁺).

3-Amino-N-methyl-4-(4-morpholinyl)benzenesulfonamide (83c).

Compound 83c was prepared from 82 and morpholine by a procedure

analogous to that for 83b to give 83c (1.98 g, 85%) as a white solid:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.07−7.17 (m, 2H), 7.01 (d, J

= 8.28 Hz, 1H), 6.94 (dd, J = 1.88, 8.16 Hz, 1H), 5.20 (s, 2H), 3.72−

3.81 (m, 4H), 2.80−2.89 (m, 4H), 2.38 (d, J = 4.77 Hz, 3H). MS (m/

z) 272.2 (M+H⁺).

TNNI3K Assay Reagents.³²His6-MBP-TEV-Full length human

TNNI3K (hTNNI3K) was expressed in a Baculovirus system and

puriﬁed from amylase aﬃnity chromatography followed by Super-

dex200. The ﬂuorescent ligand 5-({[2-({[3-({4-[(5-hydroxy-2-

methylphenyl)amino]-2-pyrimidinyl}amino)phenyl]carbonyl}amino)-

ethyl]amino}carbonyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic

acid (84) was prepared as described.³³Buﬀer components, including

MgCl₂, Bis-Tris, DTT and CHAPS were purchased from Sigma-

Aldrich.

pIC₅₀= (log((b − y)/(y − a)))/d − log(x)

where x is the compound concentration, y is the % activity at speciﬁed

compound concentration, a is the minimum % activity, b is the

maximum % activity, and d is the Hill slope. The pIC₅₀values were

averaged to determine a mean value, for a minimum of 2 experiments.

On average, pIC₅₀values fell within 0.3 units for individual

experiments with a given compound.

TNNI3K Enzyme Assay.³²A ﬂuorescence polarization assay was

used to determine the concentration-dependent binding of com-

pounds to the ATP binding pocket of hTNNI3K. The binding of 84 to

the hTNNI3K ATP binding pocket results in an increase in

ﬂuorescence polarization. Addition of compound displaces the

ﬂuorescent probe, which leads to a decrease in the polarization signal.

Ten milliliters of a 5 nM solution of 84 (Solution 1) was prepared by

mixing 5 μL of 1 M DTT, 80 μL of 10% (w/v) CHAPS and 5 μL of a

stock solution of 84 (10 μM in DMSO) into 9910 μL of buﬀer (20

mM Tris, 15 mM MgCl₂, pH 7.5). Solution 2 was formed by mixing

53.8 μL of 2.6 μM hTNNI3K with a 6946.2 μL aliquot of Solution 1 to

make up a 7 mL mixture of hTNNI3K and 84 (Solution 2). Fifty (50)

nL of inhibitors in DMSO (or DMSO controls) were stamped into a

384well low volume Greiner black plate, followed by addition of 5 μL

of Solution 1 to column 18 and 5 μL Solution 2 to columns 1−17 and

19−24 of the plate. The plate was then spun at 500 rpm for 30 s and

B-Raf Enzyme Assay.^32,34Selected compounds were tested for B-

Raf protein serine kinase inhibitory activity in a B-Raf Accelerated

MEK ATPase assay (BRAMA). Baculovirus-expressed His6-tagged

BRAFV600E full-length (amino acids 2−766) was used in the BRAMA

assay. The BRAMA assay is a high-sensitivity assay which measures an

intrinsic MEK-mediated ATP hydrolysis uncoupled from downstream

ERK phosphorylation by coupling the formation of ADP to NADH

oxidation through the enzymes pyruvate kinase and lactate

dehydrogenase. When ADP production is initiated by addition of

catalytic amounts of an activated Raf enzyme and nonphosphorylated

MEK, one observes robust ADP production concomitant with Raf-

mediated phosphorylation of MEK. The assay was conducted as

disclosed³⁴but with the following changes: (1) the assay was

performed with a ﬁnal MEK concentration of 150 nM, and (2) the

assay was read at a single end point instead of a kinetic read.

O

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Acceleration of MEK ATPase activity was determined from the data

and plotted as a function of inhibitor concentration to aﬀord

concentration response curves, from which the pIC₅₀values were

generated following a standard pIC₅₀ﬁtting protocol. For key

analogues, the concentration−response experiments were repeated

and, on average, the pIC₅₀values fell within 0.2 units from individual

experiments with a given compound.

Notes

The authors declare the following competing ﬁnancial

interest(s): Authors aﬃlliated with GlaxoSmithKline have

received compensation in the form of salary and stock.

ACKNOWLEDGMENTS

■

We gratefully acknowledge Nathan Gaul, Peter Caprioli,

Evanson Agyeman, Michael Shaber, Brian Dombroski, and

Laurie Carson for conducting the TNNI3K and B-Raf assays

and Will Burkhart for supplying recombinant TNNI3K protein

for crystallization studies.

Pharmacokinetics. Rat pharmacokinetics were determined in male

Sprague−Dawley rats in a noncrossover fashion. Two rats (fed)

received the designated compound as a 30 min intravenous infusion (1

mg/kg target dose; 4 mL/kg dose volume) formulated as a solution in

20% aqueous cavitron with 3−5% DMSO at pH 4−4.5. Two

additional rats (fed) received the designated compound as a gastric

bolus (2 mg/kg target dose, 16 mL/kg dose volume) formulated as a

solution in 6% cavitron with 3−5% DMSO at pH 4. Blood samples

were collected from the femoral artery at various times following

dosing and centrifuged to obtain plasma. Plasma concentrations of the

test compound were quantiﬁed by LC/MS/MS (LOQ = 1 ng/mL).

Noncompartmental methods were used for analysis of concentration

versus time data. Cl = iv plasma clearance; F = oral bioavailability;

poDNAUC = dose-normalized area under the oral concentration−

time curve. All studies were conducted after review by the GSK

Institutional Animal Care and Use Committee and in accordance with

the GSK Policy on the Care, Welfare and Treatment of Laboratory

Animals.

Protein X-ray Crystal Structures. TNNI3K protein (residues 402−

730 for complex 1 and residues 421−730 for complex 53) was

complexed with 5-fold molar excess of inhibitor for 1 h on ice. The

complex was concentrated to 8−10 mg/mL in 25 mM HEPES, 400

mM NaCl pH 8, 5 mM DTT, and 1 mM CHAPS. Crystals were grown

by sitting-drop vapor diﬀusion at 4 °C. Drops (600 nL) were set with a

Mosquito instrument (TTP LabTech; Melbourne, United Kingdom)

in MRC 2 Well Crystallization Plates (Swissci; Zug, Switzerland) by

combining 300 nL TNNI3K−inhibitor complex and 300 nL of well

solution. For complex 1, the well solution contained 0.1 M HEPES 7.0

and 0.3 M sodium potassium tartrate. For complex 53, the well

solution contained 0.1 M HEPES pH 7.0, 0.1 M ammonium sulfate,

and 8% PEG3350. TNNI3K−inhibitor complexed crystals were

harvested and cryoprotected with 25% glycerol (complex 1) or 35%

ethylene glycol (complex 53) prior to data collection. The initial

structure of TNNI3K was solved by molecular replacement using

MOLREP³⁵in the CCP4 program suite and Tak1 as a search model

(RCSB 2EVA). These crystals belonged to the P2₁space group with 4

mol/asu. Data for the 1 and 53 complexes were collected on a Saturn

944+/FRE+ Super Bright and reduced and scaled with HKL2000.³⁶

The structure was built using COOT³⁷and reﬁned with REFMAC.³⁸

Statistics for the data can be found in Supplementary Table 1. Atomic

coordinates and structure factors have been deposited into the Protein

Data Bank (PDB: 4YFI and PDB: 4YFF). Images of the X-ray crystal

structures (Figures 2, 8, 13) were generated using the PyMol

ABBREVIATIONS USED

■

TNNI3K, troponin I-interacting kinase; CARK, cardiac ankyrin

repeat kinase; DNAUC, dose-normalized area under the curve;

TLK, tyrosine-like kinase; GSK, GlaxoSmithKline; kcal, kilo-

calories; Cl, iv clearance; Vdss, volume of distribution; F, oral

bioavailability; μwave, microwave; i-Bu, isobutyl; dppf, 1,1′-

Bis(diphenylphosphino)ferrocene; SCX, strong cation ex-

change; Bis-Tris, Bis(2-hydroxyethyl)-amino-tris-

(hydroxymethyl)-methane; CHAPS, 3-[(3-Cholamidopropyl)-

dimethylammonio]-1-propanesulfonate; DELFIA, dissociation-

induced lanthanide ﬂuorescence intensity assay; DMEM/F12,

Dulbecco’s modiﬁed eagle medium−nutrient mixture F12; FBS,

fetal bovine serum; TBS, tris buﬀered saline; BRAMA, B-Raf

Accelerated MEK ATPase Assay; MEK, mitogen-activated

protein kinase kinase; HEPES, 4-(2-hydroxyethyl)-1-piperazi-

neethanesulfonic acid

REFERENCES

■

(1) Zhao, Y.; Meng, X. M.; Wei, Y. J.; Zhao, X. W.; Liu, D. Q.; Cao,

H. Q.; Liew, C. C.; Ding, J. F. Cloning and characterization of a novel

cardiac-specific kinase that interacts specifically with cardiac troponin I.

J. Mol. Med. (Berl) 2003, 81, 297−304.

(2) Lal, H.; Ahmad, F.; Parikh, S.; Force, T. Troponin I-interacting

protein kinase: a novel cardiac-specific kinase, emerging as a molecular

target for the treatment of cardiac disease. Circ. J. 2014, 78, 1514−

1519.

(3) Vagnozzi, R. J.; Gatto, G. J., Jr.; Kallander, L. S.; Hoffman, N. E.;

Mallilankaraman, K.; Ballard, V. L. T.; Lawhorn, B. G.; Stoy, P.; Philp,

J.; Graves, A. P.; Naito, Y.; Lepore, J. J.; Gao, E.; Madesh, M.; Force, T.

Inhibition of the cardiomyocyte-specific TNNI3K limits oxidative

stress, injury, and adverse remodeling in the ischemic heart. Sci. Transl.

Med. 2013, 5, 207ra141.

(4) Wang, L.; Wang, H.; Ye, J.; Xu, R. X.; Song, L.; Shi, N.; Zhang, Y.

W.; Chen, X.; Meng, X. M. Adenovirus-mediated overexpression of

cardiac troponin I-interacting kinase promotes cardiomyocyte hyper-

trophy. Clin. Exp. Pharmacol. Physiol. 2011, 38, 278−284.

(5) Wheeler, F. C.; Tang, H.; Marks, O. A.; Hadnott, T. N.; Chu, P.

L.; Mao, L.; Rockman, H. A.; Marchuk, D. A. Tnni3k modifies disease

progression in murine models of cardiomyopathy. PLoS Genet. 2009,

5, e1000647.

Molecular Graphics System, Version 1.7.2.1 Schrodinger, LLC.

̈

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b00931.

(6) Stoy, P. unpublished results.

(7) (a) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.;

Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.;

Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.;

Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.;

Wodicka, L. M.; Zarrinkar, P. P. A quantitiative analysis of kinase

inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127−131. (b) Fabian,

M. A.; Biggs, W. H., III; Treiber, D. K.; Atteridge, C. E.; Azimioara, M.

D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.;

Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.;

Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J.-M.; Mehta, S. A.;

Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar,

P. P.; Lockhart, D. J. A small molecule-kinase interaction map for

clinical kinase inhibitors. Nat. Biotechnol. 2005, 23, 329−335.

Table of statistics for X-ray crystal structures and kinase

selectivity results for 1, 7, and 48 (PDF)

Molecular Formula Strings (CSV)

Accession Codes

4YFI (TNNI3K-1), 4YFF (TNNI3K-53).

AUTHOR INFORMATION

■

Corresponding Author

*E-mail: brian.2.lawhorn@gsk.com. Phone: (610) 270-5347.

P

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(8) (a) Harris, I. S.; Zhang, S.; Treskov, I.; Kovacs, A.; Weinheimer,

C.; Muslin, A. J. Raf-1 kinase is required for cardiac hypertrophy and

cardiomyocyte survival in response to pressure overload. Circulation

2004, 110, 718−723. (b) Muslin, A. J. Role of Raf proteins in cardiac

hypertrophy and cardiomyocyte survival. Trends Cardiovasc. Med.

2005, 15, 225−229.

(9) Gonnella, N. C.; Roberts, J. D. Studies of the tautomerism of

purine and the protonation of purine and its 7- and 9-methyl

derivatives by nitrogen-15 nuclear magnetic resonance spectroscopy. J.

Am. Chem. Soc. 1982, 104, 3162−3164.

(10) Leung, C. S.; Leung, S. S. F.; Tirado-Rives, J.; Jorgensen, W. L.

Methyl effects on protein-ligand binding. J. Med. Chem. 2012, 55,

4489−4500.

(11) Schmid, S.; Rottgen, M.; Thewalt, U.; Austel, V. Synthesis and

conformational properties of 2,6-bis-anilino-3-nitropyridines. Org.

Biomol. Chem. 2005, 3, 3408−3421.

(12) Uno, B.; Kawakita, T.; Kano, K.; Ezumi, K.; Kubota, T.

Spectroscopic analysis and geometry assignment of the minimum

energy conformations of 2-phenoxypyridines and diphenyl ethers. Bull.

Chem. Soc. Jpn. 1992, 65, 2697−2703.

(13) King, A. J.; Patrick, D. R.; Batorsky, R. S.; Ho, M. L.; Do, H. T.;

Zhang, S. Y.; Kumar, R.; Rusnak, D. W.; Takle, A. K.; Wilson, D. M.;

Hugger, E.; Wang, L.; Karreth, F.; Lougheed, J. C.; Lee, J.; Chau, D.;

Stout, T. J.; May, E. W.; Rominger, C. M.; Schaber, M. D.; Luo, L.;

Lakdawala, A. S.; Adams, J. L.; Contractor, R. G.; Smalley, K. S. M.;

Herlyn, M.; Morrissey, M. M.; Tuveson, D. A.; Huang, P. S.

Demonstration of a genetic therapeutic index for tumors expressing

oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res. 2006,

66, 11100−11105.

(19) (a) Axten, J. M.; Romeril, S. P.; Shu, A.; Ralph, J.; Medina, J. R.;

Feng, Y.; Li, W. H. H.; Grant, S. W.; Heerding, D. A.; Minthorn, E.;

Mencken, T.; Gaul, N.; Goetz, A.; Stanley, T.; Hassell, A. M.; Gampe,

R. T.; Atkins, C.; Kumar, R. Discovery of GSK2656157: An optimized

PERK inhibitor selected for preclinical development. ACS Med. Chem.

Lett. 2013, 4, 964−968. (b) Devine, S. M.; Lim, S. S.;

Chandrashekaran, I. R.; Macraild, C. A.; Drew, D. R.; Debono, C.

O.; Lam, R.; Anders, R. F.; Beeson, J. G.; Scanlon, M. J.; Scammells, P.

J.; Norton, R. S. A critical evaluation of pyrrolo[2,3-d]pyrimidine-4-

amines as Plasmodium falciparum apical membrane antigen 1 (AMA1)

inhibitors. MedChemComm 2014, 5, 1500−1506. (c) Reader, J. C.;

Matthews, R. P.; Klair, S.; Cheung, K.-M. J.; Scanlon, J.; Proisy, N.;

Addison, G.; Ellard, J.; Piton, N.; Taylor, S.; Cherry, M.; Fisher, M.;

Boxall, K.; Burns, S.; Walton, M. I.; Westwood, I. M.; Hayes, A.; Eve,

P.; Valenti, M.; De Haven Brandon, A.; Box, G.; Van Montfort, R. L.

M.; Williams, D. H.; Aherne, G. W.; Raynaud, F. I.; Eccles, S. A.;

Garrett, M. D.; Collins, I. Structure-guided evolution of potent and

selective CHK1 inhibitors through scaffold morphing. J. Med. Chem.

2011, 54, 8328−8342. (d) Khalaf, A. I.; Huggan, J. K.; Suckling, C. J.;

Gibson, C. L.; Stewart, K.; Giordani, F.; Barrett, M. P.; Wong, P. E.;

Barrack, K. L.; Hunter, W. N. Structure-based design and synthesis of

antiparasitic pyrrolopyrimidines targeting pteridine reductase 1. J. Med.

Chem. 2014, 57, 6479.

(20) Pudlo, J. S.; Nassiri, M. R.; Kern, E. R.; Wotring, L. L.; Drach, J.

C.; Townsend, L. B. Synthesis, antiproliferative, and antiviral activity of

certain 4-substituted and 4,5-disubstituted 7-[(1,3-dihydroxy-2-

propoxy)methyl]pyrrolo[2,3-d]pyrimidines. J. Med. Chem. 1990, 33,

1984−1992.

(21) Wang, X.; Seth, P. P.; Ranken, R.; Swayze, E. E.; Migawa, M. T.

Synthesis and biological activity of 5-fluorotubercidin. Nucleosides,

Nucleotides Nucleic Acids 2004, 23, 161−170.

(22) Pudlo, J. S.; Saxena, N. K.; Nassiri, M. R.; Turk, S. R.; Drach, J.

C.; Townsend, L. B. Synthesis and antiviral activity of certain 4- and

4,5-disubstituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]-

pyrimidines. J. Med. Chem. 1988, 31, 2086−2092.

(23) (a) Showalter, H. D. H.; Bridges, A. J.; Zhou, H.; Sercel, A. D.;

McMichael, A.; Fry, D. W. Tyrosine kinase inhibitors. 16. 6,5,6-

tricyclic benzothieno[3,2-d]pyrimidines and pyrimido[5,4-b]- and

[4,5-b]indoles as potent inhibitors of the epidermal growth factor

receptor tyrosine kinase. J. Med. Chem. 1999, 42, 5464−5474.

(b) Reader, J. C.; Matthews, T. P.; Klair, S.; Cheung, K.-M. J.;

Scanlon, J.; Proisy, N.; Addison, G.; Ellard, J.; Piton, N.; Taylor, S.;

Cherry, M.; Fisher, M.; Boxall, K.; Burns, S.; Walton, M. I.; Westwood,

I. M.; Hayes, A.; Eve, P.; Valenti, M.; Brandon, A. d. H.; Box, G.; van

Montfort, R. L. M.; Williams, D. H.; Aherne, G. W.; Raynaud, F. I.;

Eccles, S. A.; Garrett, M. D.; Collins, I. Structure guided evolution of

potent and selective CHK1 inhibitors through scaffold morphing. J.

Med. Chem. 2011, 54, 8328−8342.

(24) Mincione, F.; Menabuoni, L.; Briganti, F.; Mincione, G.;

Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors:

inhibition of isozymes I, II, and IV with N-hydroxysulfonamides − a

novel class of intraocular pressure lowering agents. J. Enzyme Inhib.

1998, 13, 267−284.

(25) Petrow, V.; Stephenson, O.; Wild, A. M. Studies in the field of

diuretic agents part V. A new route to disulphamyl derivatives of

benzene. J. Pharm. Pharmacol. 1960, 12, 705−719.

(26) Mayasundari, A.; Fujii, N. Efficient formation of 4,6-

disubstituted pyrollo[2,3-d]pyrimidines: a novel route to TWS119, a

glycogen synthase kinase-3β inhibitor. Tetrahedron Lett. 2010, 51,

3597−3598.

(27) Clark, M. P.; George, K. M.; Bookland, R. G.; Chen, J.;

Laughlin, S. K.; Thakur, K. D.; Lee, W.; Davis, J. R.; Cabrera, E. J.;

Brugel, T. A.; VanRens, J. C.; Laufersweiler, M. J.; Maier, J. A.; Sabat,

M. P.; Golebiowski, A.; Easwaran, V.; Webster, M. E.; De, B.; Zhang,

G. Development of new pyrrolopyrimidine-based inhibitors of Janus

kinase 3 (JAK3). Bioorg. Med. Chem. Lett. 2007, 17, 1250.

(14) Ahlbrecht, H.; Duber, E. O.; Epsztajn, J.; Marcinkowski, R. M.

K. Delocalisation, conformation and basicity of anilines. Tetrahedron

1984, 40, 1157−1165.

(15) (a) Zhang, C.; Shokat, K. M. Enhanced selectivity for inhibition

of analog sensitive kinases through scaffold optimization. Tetrahedron

2007, 63, 5832−5838. (b) Tasler, S.; Muller, O.; Wieber, T.; Herz, T.;

Krauss, R.; Totzke, F.; Kubbutat, M. H. G.; Schachtele, C. N-

substituted 2′-(aminoaryl)benzothiazoles as kinase inhibitors: Hit

identification and scaffold hopping. Bioorg. Med. Chem. Lett. 2009, 19,

1349−1356. (c) Liu, X.-P.; Narla, R. K.; Uckun, F. M. Organic phenyl

arsonic acid compounds with potent antileukemic activity. Bioorg. Med.

Chem. Lett. 2003, 13, 581−583. (d) Bridges, A. J.; Denny, W. A.; Fry,

D.; Kraker, A.; Meyer, R. F.; Rewcastle, G. W.; Thompson, A. M.

Bicyclic compounds capable of inhibiting tyrosine kinases of the

epidermal growth factor receptor family. U.S. Patent 5,654,307, 1997.

(e) Gangjee, A.; Namjoshi, O. A.; Ihnat, M. A.; Buchanan, A. The

contribution of a 2-amino group on receptor tyrosine kinase inhibition

and antiangiogenic activity in 4-anilinosubstituted pyrrolo[2,3-d]-

pyrimidines. Bioorg. Med. Chem. Lett. 2010, 20, 3177−3181. (f) Deng,

X.; Okram, B.; Ding, Q.; Zhang, J.; Choi, Y.; Adrian, F. J.;

Wojciechowski, A.; Zhang, G.; Che, J.; Bursulaya, B.; Cowan-Jacob,

S. W.; Rummel, G.; Sim, R.; Gray, N. S. Expanding the diversity of

allosteric Bcr-Abl inhibitors. J. Med. Chem. 2010, 53, 6934−6946.

(16) Sun, L.; Cui, J.; Liang, C.; Zhou, Y.; Nematalla, A.; Wang, X.;

Chen, H.; Tang, C.; Wei, J. Rational design of 4,5-disubstituted-5,7-

dihydro-pyrrolo[2,3-d]pyrimidine-6-ones as a novel class of inhibitors

of epidermal growth factor receptor (EGF-R) and Her2(p18^5erbB

)

tyrosine kinases. Bioorg. Med. Chem. Lett. 2002, 12, 2153−2157.

(17) Voronkov, M. V.; Gu, K.; Baugh, S. D. P.; Becker, M. R. A

modular approach to 4,5-diaminopyrrolo[2,3-d]pyrimidines and 2,4,5-

triaminopyrrolo[2,3-d]pyrimidines. Tetrahedron Lett. 2006, 47, 4149−

4151.

(18) (a) Guillard, J.; Decrop, M.; Gallay, N.; Espanel, C.; Boissier, E.;

Herault, O.; Viaud-Massuard, M.-C. Synthesis and biological

evaluation of 7-azaindole derivatives, synthetic cytokinin analogues.

Bioorg. Med. Chem. Lett. 2007, 17, 1934−1937. (b) Girgis, N. S.;

Larson, S. B.; Robins, R. K.; Cottam, H. B. The synthesis of 5-

azaindoles by substitution-rearrangement of 7-azaindoles upon treat-

ment with certain primary amines. J. Heterocycl. Chem. 1989, 26, 317−

325.

(28) (a) Abdel-Magid, A. F.; Carson, K. C.; Harris, B. D.; Maryanoff,

C. A.; Shah, R. D. Reductive amination of aldehydes and ketones with

sodium triacetoxyborohoydride. Studies on direct and indirect

Q

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

reductive amination procedures. J. Org. Chem. 1996, 61, 3849−3862.

(b) Sabatucci, J. P.; Ashwell, M. A.; Trybulski, E.; O’Donnell, M.-M.;

Moore, W. J.; Harnish, D. C.; Chadwick, C. C. Substituted 4-

hydroxyphenyl sulfonamides as pathway-selective estrogen receptor

ligands. Bioorg. Med. Chem. Lett. 2006, 16, 854−858.

(29) Morisawa, Y.; Kataoka, M.; Nagahori, H.; Sakamoto, T.; Kitano,

N.; Kusano, K.; Sato, K. Studies on anticoccidial agents. 13. Synthesis

and anticoccidial activity of nitropyridine-2- and −3-sulfonamides and

derivatives. J. Med. Chem. 1980, 23, 1376−1380.

(30) Wendt, M. D.; Shen, W.; Kunzer, A.; McClellan, W. J.; Bruncko,

M.; Oost, T. K.; Ding, H.; Joseph, M. K.; Zhang, H.; Nimmer, P. N.;

Ng, S.-C.; Shoemaker, A. R.; Petros, A. M.; Oleksijew, A.; Marsh, K.;

Bauch, J.; Oltersdorf, T.; Belli, B. A.; Martineau, D.; Fesik, S. W.;

Rosenberg, S. H.; Elmore, S. W. Discovery and structure-activity

relationship of antagonists of B-cell lymphoma 2 family proteins with

chemopotentiation activity in vitro and in vivo. J. Med. Chem. 2006, 49,

1165−1181.

(31) For an example of using the H-bond donating ability of a

gatekeeper Thr to introduce kinase selectivity, see: Martin, M. W.;

Newcomb, J.; Nunes, J. J.; Boucher, C.; Chai, L.; Epstein, L. F.; Faust,

T.; Flores, S.; Gallant, P.; Gore, A.; Gu, Y.; Hsieh, F.; Huang, X.; Kim,

J. L.; Middleton, S.; Morgenstern, K.; Oliveira-dos-Santos, A.; Patel, V.

F.; Powers, D.; Rose, P.; Tudor, Y.; Turci, S. M.; Welcher, A. A.; Zack,

D.; Zhao, H.; Zhu, L.; Zhu, X.; Ghiron, C.; Ermann, M.; Johnston, D.;

Saluste, C.-G. P. Structure-based design of novel 2-amino-6-phenyl-

pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-ones as po-

tent and orally active inhibitors of lymphocyte specific kinase (Lck):

Synthesis, SAR, and in vivo anti-inflammatory activity. J. Med. Chem.

2008, 51, 1637−1648.

(32) Hammond, M.; Kallander, L. S.; Lawhorn, B. G.; Philp, J.;

Sarpong, M. A.; Seefeld, M. A. Azolopyrimidine compounds as kinase

inhibitors and their preparation and use in the treatment of disesases

and methods. PCT Int. Appl. WO2011149827, 2011.

(33) Casillas, L. N.; Chakravorty, S. J.; Charnley, A. K.; Eidam, P.;

Haile, P. A.; Hughes, T. V.; Jeong, J. U.; Kang, J.; Lakdawala, S. A.;

Leister, L. K.; Marquis, R. W.; Miller, N. A.; Price, D. J.; Sehon, C. L.;

Wang, G. Z.; Zhang, D. Preparation of indazolyl-substituted

pyrimidinediamine derivatives as RIP2 kinase inhibitors. PCT Int.

Appl. WO2011120025, 2011.

(34) May, E. W.; Rominger, C. M.; Schaber, M. D. Assay for B-Raf

activity based on intrinsic MEK ATPase activity. PCT Int. Appl. US

20060211073, 2006.

(35) Vagin, A.; Teplyakov, A. MOLREP: an automated program for

molecular replacement. J. Appl. Crystallogr. 1997, 30, 1022−1025.

(36) Otwinowski, Z.; Minor, W. Processing of X-ray diﬀraction data

collected in oscillation mode. In Methods in Enzymology; Carter, C. W.,

Jr., Sweet, R. M., Eds.; Academic Press: New York, 1997; pp 307−326.

(37) Emsley, P.; Cowtan, K. Coot: Model-building tools for

molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,

60, 2126−2132.

(38) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of

macromolecular structures by the maximum-likelihood method. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240−255.

R

DOI: 10.1021/acs.jmedchem.5b00931

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Identification of Purines and 7-Deazapurines as Potent and Selective Type i Inhibitors of Troponin I-Interacting Kinase (TNNI3K)

DOI: 10.1021/acs.jmedchem.5b00931

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.5b00931

Full text of DOI:10.1021/acs.jmedchem.5b00931

Products guided by the article

R&D Labs maybe for 1351091-88-7

Relevant to this article

Hot Product