Discovery of selective and noncovalent diaminopyrimidine-based inhibitors of epidermal growth factor receptor containing the T790M resistance mutation

Article

pubs.acs.org/jmc

Discovery of Selective and Noncovalent Diaminopyrimidine-Based

Inhibitors of Epidermal Growth Factor Receptor Containing the

T790M Resistance Mutation

Emily J. Hanan,*^,†Charles Eigenbrot,^‡Marian C. Bryan,^†Daniel J. Burdick,^†Bryan K. Chan,^†

Yuan Chen,^§Jennafer Dotson,^†Robert A. Heald,^∇Philip S. Jackson,^∇Hank La,^§Michael D. Lainchbury,^∇

Shiva Malek,^∥Hans E. Purkey,^†Gabriele Schaefer,^⊥Stephen Schmidt,^∥Eileen M. Seward,^∇Steve Sideris,^∥

Christine Tam,^#Shumei Wang,^†Siew Kuen Yeap,^∇Ivana Yen,^∥Jianping Yin,^‡Christine Yu,^‡

Inna Zilberleyb,^#and Timothy P. Heﬀron^†

†

‡

§

∥

Departments of Discovery Chemistry, Structural Biology, Drug Metabolism and Pharmacokinetics, Biochemical and Cellular

Pharmacology, ^⊥Molecular Oncology, and ^#Protein Expression, Genentech Inc., 1 DNA Way, South San Francisco, California 94080,

United States

^∇Argenta, Early Discovery Charles River, 7/8 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, United Kingdom

S

* Supporting Information

ABSTRACT: Activating mutations within the epidermal

growth factor receptor (EGFR) kinase domain, commonly

L858R or deletions within exon 19, increase EGFR-driven cell

proliferation and survival and are correlated with impressive

responses to the EGFR inhibitors erlotinib and geﬁtinib in

nonsmall cell lung cancer patients. Approximately 60% of

acquired resistance to these agents is driven by a single

secondary mutation within the EGFR kinase domain,

speciﬁcally substitution of the gatekeeper residue threonine-

790 with methionine (T790M). Due to dose-limiting toxicities

associated with inhibition of wild-type EGFR (wtEGFR), we

sought inhibitors of T790M-containing EGFR mutants with

selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR

inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel

diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over

wtEGFR, broad kinase selectivity, and desirable physicochemical properties.

both quinazoline-based inhibitors and reduces their binding

aﬃnity. This is similar to the resistance mechanism observed

for Abl tyrosine-kinase inhibitors (TKIs) in CML, which is also

the result of a gatekeeper residue substitution

(T315I).^{12,13,17−20}A 2008 report proposed a second

contributing mechanism, in which the T790M-containing

mutants have an increased aﬃnity for ATP, resulting in

reduced cellular potency for the ATP-competitive inhibitors.²¹

Several second-generation EGFR inhibitors form a covalent

bond with Cys-797 within the EGFR active site and have

shown preclinical activity against T790M-containing mutants of

EGFR. However, their clinical eﬃcacy has been limited by

associated skin rash and gastrointestinal toxicity, possibly

because of their potency against wild-type EGFR

(wtEGFR).^22,23Additionally, there have been reports of

acquired resistance to one such covalent inhibitor via the

INTRODUCTION

■

Nonsmall cell lung cancers (NSCLC) harboring mutations in

the tyrosine kinase domain of the epidermal growth factor

receptor (EGFR) are well-studied examples of oncogene

addiction.¹Activating mutations, most commonly the point

mutation L858R or deletions within exon 19 (e.g., residues

746−750), increase EGFR-driven cell proliferation and

survival.²⁻⁵The ﬁrst-generation EGFR inhibitors erlotinib

and geﬁtinib have had remarkable success for the treatment of

EGFR-mutated NSCLC.⁶⁻¹⁰However, the dramatic initial

clinical responses to these agents are always followed by an

acquired resistance.¹¹⁻¹³Approximately 60% of this acquired

resistance arises from a particular secondary mutation within

the EGFR kinase domain, leading to the substitution of the

gatekeeper residue threonine-790 with methionine

(T790M).¹²⁻¹⁶This mutation maintains the catalytic function

of the enzyme but reduces the activity of geﬁtinib and erlotinib

through two mechanisms. The bulkier side chain of the

methionine residue occludes part of the binding site utilized by

Received: October 13, 2014

A

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Table 1. Initial Hits

a

b

c

LLE is calculated using cLogD at pH 7.4. K_idata are an average of at least two independent experiments. Ratio of wtEGFR K_iappover

d

EGFR(TMLR) K_iapp

.

Ratio of wtEGFR K_iappover EGFR(TMdel) K_iapp

.

T790M mutation, and it is questionable if drug levels can be

achieved to suﬃciently inhibit T790M mutant forms of

EGFR.^24,25It is therefore desirable to develop a potent

inhibitor of T790M-containing EGFR mutants with reduced

activity against wtEGFR. Recently, third-generation covalent

inhibitors including AZD9291 and CO-1686 have been

generated that demonstrate selectivity for T790M-containing

EGFR mutants over wtEGFR, and early phase I data indicate

promising eﬃcacy and tolerability with this approach.²⁶⁻³⁰

The compelling nature of T790M EGFR mutants as a drug

target and an understanding of the relationship between

wtEGFR inhibition and dose-limiting toxicities led us to initiate

an eﬀort to identify inhibitors of the major resistance mutations

of EGFR, the T790M/L858R mutation (TMLR), and the

T790M/del746−750 mutation (TMdel), with selectivity over

wtEGFR. It is worth noting that second- and third-generation

EGFR inhibitors described to date have been almost exclusively

covalent in nature. Due to the low TMLR and TMdel K_mfor

ATP (∼1−2 μM),³¹it was expected that exquisite biochemical

potency (K_i< 1 nM) against both T790M-containing mutants

of EGFR would be required for a robust cellular eﬀect. While

covalent inhibitors cannot be displaced by ATP and are able to

circumvent this issue, concerns about possible toxicity with

covalent inhibitors, both oﬀ-target and resulting from time-

dependent inhibition of wtEGFR, led us to pursue a

noncovalent strategy.³²⁻³⁵Herein we describe our eﬀorts to

identify reversible inhibitors of T790M mutant forms of EGFR

beginning with a potent inhibitor of Jak2/Tyk2. X-ray crystal

structures revealed two distinct binding modes of these early

inhibitors that enabled design of subsequent T790M EGFR

inhibitors with high levels of selectivity over wtEGFR, broad

kinase selectivity, and with desirable physicochemical properties

that render them appealing for further optimization.

RESULTS AND DISCUSSION

■

A high-throughput biochemical screen identiﬁed compounds

1−3 as moderately potent inhibitors of both T790M-containing

mutant forms of EGFR (Table 1). Interestingly, the acyclic

amine 1 has only marginal selectivity for TMLR and TMdel

over wtEGFR (5−10 times), whereas piperidine-containing 2

and 3 have a higher level of selectivity (11−53 times). In order

to advance these initial hits into a viable lead series, a number of

parameters required optimization. First, these compounds were

originally identiﬁed as potent inhibitors of Jak2 and Tyk2 with

K_ivalues between 1 and 16 nM, and it was desirable to

minimize the inhibition of Jak family kinases (see Table S1 of

the Supporting Information for the Jak family activity of 1−

3).³⁶Broader kinase selectivity was promising: when tested at 1

μM against a panel of 296 kinases, 3 inhibited only 5 other

kinases at >80%.³⁷Second, compounds 1−3 are highly

lipophilic, resulting in both poor solubility and rapid turnover

in liver microsomes (Cl_hep> 15 mL/min/kg determined in

human liver microsomes). With the dual goals of increasing

potency and decreasing lipophilicity, the lipophilic ligand

eﬃciency (LLE) was a useful parameter for measuring progress

during compound optimization.³⁸⁻⁴⁰A low lipophilicity

starting point was desired to enhance the likelihood of

identifying molecules with high LLE during lead optimization.

For advancement of the series, we sought a combined target

B

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proﬁle with TMLR and TMdel biochemical potency <10 nM,

wtEGFR selectivity >20 times, LogD < 2.5, and kinetic

solubility >50 μM.

An X-ray crystal structure of compound 1 with the TMLR

kinase domain revealed a binding mode analogous with those

seen for similar compounds in the active site of Tyk2 (Figure

1a).³⁶The aminopyridine functionality forms hydrogen bonds

corresponding increase in population of non-Thr790-interact-

ing conformations.⁴⁴

We attempted to modify the dihaloaryl motif found in

compounds 1−3, as this group is a signiﬁcant contributor to the

high lipophilicity and poor solubility of these molecules.

However, modiﬁcation of the dihalophenyl group was not

productive (see the Supporting Information, Table S2).

Nonaromatic amides do improve solubility but lack the

potency required for further consideration. Polar substituents

on the phenyl ring, as well as heteroaryl replacements, provide

no improvement in solubility or potency compared with

compounds 1−3.

We next turned our attention to replacement of the amide

functionality common to compounds 1−3. Conversion of the

amide into a fused heterocyclic ring reduces the degrees of

rotational freedom and could therefore improve potency.

Furthermore, the crystal structure of 1 suggests additional space

in the binding site adjacent to the gatekeeper residue that could

accommodate a fused ﬁve-membered heterocyclic ring. A series

of bicyclic compounds were therefore designed with the general

substructure shown in Figure 2. Depending on the choice of

Figure 2. Design of the bicyclic motif.

heterocycle, this additional ring provides alternative substitu-

tion vectors: into the ribose pocket (R¹), toward the catalytic

lysine (R²), and toward the gatekeeper side chain (R³).

Compounds 4 and 5 (Table 2) were synthesized as proof of

concept molecules for this design, with the inclusion of a

pyrazole substituent at R²(see Figure 2) designed to interact

with catalytic Lys745. We were gratiﬁed to see that while there

is a slight decrease in potency for 4 and 5 compared to 3,

replacement of the dihalophenyl amide results in a signiﬁcant

reduction in lipophilicity and a net increase in LLE. X-ray

crystal structures of both compounds 4 and 5 with the TMLR

kinase domain revealed that the compounds bind in the active

site as designed, with the pendant pyrazole ring interacting with

the side chains of both Lys745 and Glu762 (Figure 3).

However, the complete loss of selectivity for both compounds

against wtEGFR is diﬃcult to rationalize. While the imidazo

compound 4 could engage in a water-mediated hydrogen bond

with the wtEGFR gatekeeper Thr790 side chain, as described

above, and might be expected to have similar TMLR and

wtEGFR activity, the pyrrolo compound 5 positions a

hydrophobic face toward the gatekeeper residue and could

make no such hydrogen-bonding interaction. However, the

alternative rotamer of the Thr790 side chain could provide

positive lipophilic interactions similar to the lipophilic Met790

side chain.

Figure 1. (a) X-ray structure of compound 1 complexed with TMLR

and (b) overlay of 1 (green) and erlotinib (magenta) complexed with

wtEGFR (PDB ID: 1M17)⁴³

with the backbone carbonyl and NH of the hinge residue

Met793. The hydroxyethyl group extends into the ribose

pocket and the oxygen makes an intramolecular hydrogen bond

with the amide NH. The carbonyl of the amide group is

directed toward the gatekeeper residue Met790, in close

proximity (3.2 Å) to the terminal methyl of the methionine side

chain. When modeled into the structure of the wtEGFR kinase

domain complexed with erlotinib,⁴¹the pyridyl ring of 1

overlays with the quinazoline portion of erlotinib (Figure 1b).

The quinazoline N3 of erlotinib is observed to make a water-

mediated hydrogen bond with the gatekeeper Thr790 side

chain, and we anticipated that the carbonyl of 1 could make a

similar water-bridged interaction with the Thr790 of

wtEGFR.^42,43Compounds 2 and 3 would not be conforma-

tionally restricted by the same intramolecular hydrogen bond,

and the observed reduction in potency for these compounds

against wtEGFR may be a result of the additional rotational

degrees of freedom around the amide bond, with a

While selectivity was not optimized with bicyclic compounds

4 and 5, the structures with TMLR suggest that the

unsubstituted N1 of both compounds could provide a useful

vector toward the ribose pocket in the center of the binding site

(vector R¹in Figure 2). We hypothesized that additional

potency for EGFR could be gained by occupying the ribose

C

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Table 2. Bicyclic Amide Replacements

a

b

c

LLE is calculated using LogD measured at pH 7.4. K_idata are an average of at least two independent experiments. Ratio of wtEGFR K_iappover

EGFR(TMLR) K_iapp

.

with compound 6 in order to rationalize this selectivity and

were surprised to discover that the ligand adopts a diﬀerent

binding mode than previous inhibitors.

As shown in Figure 4a, relative to compounds 4 and 5

(Figure 3) or 1 (Figure 1a), 6 has ﬂipped 180° horizontally and

is positioned deeper within the binding site, with the pyridine

nitrogen still maintaining a hydrogen bond with the backbone

NH of Met793. However, the NH of the aminopyrimidine in

the ligand now forms a hydrogen bond with the backbone

carbonyl of Gln791. The pyrimidine ring is positioned

underneath the gatekeeper Met790 side chain, providing

substantial hydrophobic contact, and the pyrimidine N1

nitrogen achieves a hydrogen bond with the side chain

hydroxyl of Thr854. Adding to the back pocket interactions,

the oxygen of the methoxypiperidine group is within hydrogen-

bonding distance of Lys745. The N-isopropyl group does

occupy the ribose pocket as designed, although it approaches

from the opposite direction than originally anticipated. In light

of what appear to be favorable hydrophobic contacts between

the ligand and the Met790 side chain, and the lack of possible

hydrogen bonding interactions with Thr790, the enhanced

potency of 6 against T790M mutants relative to wtEGFR is

understandable.⁴⁵While it appears that the original binding

mode should be accessible by 6, the isopropyl group may

achieve more favorable hydrophobic contacts in the ﬂipped

binding mode, serving as the driving force for that

conformation. An overlay of the bound conformations of 5

Figure 3. X-ray structures of 4 (green) and 5 (magenta) complexed

with TMLR.

pocket with lipophilic groups, as this pocket is particularly

lipophilic in EGFR with Cys797 forming the ﬂoor of the

binding site. The addition of an isopropyl substituent to the N1

position (compound 6) does provide an improvement in

biochemical potency when compared to the N1-unsubstituted 5

(Table 2). Interestingly, 6 also regains selectivity (22-fold) for

TMLR over wtEGFR. We sought a TMLR crystal structure

D

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Figure 4. a) X-ray structure of 6 complexed with TMLR and (b) overlay of ligands 5 (magenta) and 6 (blue).

and 6 highlights the dramatic diﬀerence in binding mode that is

possible with a very minor structural change (NH vs N-iPr)

(Figure 4b).

Methionine−aryl interactions are frequently found in protein

structures, and the coplanar interaction of Met790 with the

pyrimidine of 6 is similar to the arrangement often observed in

the binding sites of adenine rings.⁴⁶However, in both the CSD

and the PDB, Bissantz et al. have determined that thioether

sulfur atoms are preferentially found in the plane of a phenyl

ring and not in a coplanar arrangement (see Figure S-8 of ref

46). The π system···thioether interaction observed in the 6/

TMLR structure, as depicted in Figure 5a, is outside of that

preferred geometry. To better understand the kinase selectivity

of these inhibitors, we were interested in applying a distribution

analysis more speciﬁcally to kinases. Within the PDB, we found

58 unique examples of methionine-gatekeeper kinases where

the sulfur atom of the gatekeeper side chain is within 7 Å of an

aromatic ring in the ligand.⁴⁷Following the methodology of

Bissantz, we plotted the distribution of interaction geometries

between ligand aromatic rings and methionine sulfur atoms

within these structures (Figure 5b). Interestingly, the

methionine interaction geometry observed in the 6/TMLR

complex lies in a relatively unpopulated area of the plot.

Additional reﬁnement of this analysis to examine phenyl vs

heteroaryl ring systems does not change the distribution; the

small number of structures in the PDB with a ligand heteroaryl

ring in close proximity to a kinase methionine gatekeeper side

chain (only 10 structures) further supports the uncommon

nature of this binding mode in a kinase active site. Finally, while

methionine-gatekeeper residues are common, the combination

with a threonine residue (or other hydrogen-bond donor) in a

comparable position to Thr854 is relatively unusual.⁴⁸There-

fore, we believe that the combination of a coplanar pyrimidine

interaction with the Met790 side chain and a hydrogen-bonding

interaction with Thr854 might provide an opportunity for

achieving broader kinase selectivity in this binding mode.

The appealing LLE and selectivity over wtEGFR led us to

Figure 5. The centroid shift describes the distance within the plane of

a sulfur atom from the center of the aromatic ring. (a) Pyrimidine···

Met790 geometry observed in the 6/TMLR complex: centroid shift ≈

1.375 Å, height ≈ 3.34 Å measured roughly perpendicular to the plane

at 92.9°. (b) Distribution of gatekeeper methionine sulfur in the

proximity of a ligand aromatic ring for methionine-gatekeeper kinases

in the PDB.

use this new, T790M-selective binding mode as a starting point

for structure-based design, and we constructed a series of

bicyclic inhibitors, as shown in Table 3. The crystal structure of

6 suggests that the pendant pyrazole ring is pointed toward

solvent and is likely not providing additional potency (Figure

4a). Compound 7 (Table 3) seemingly conﬁrms this

hypothesis, in which a hydrogen replaces the pyrazole ring

E

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Table 3. Heterocycle Modiﬁcations

a

b

c

LLE is calculated using LogD measured at pH 7.4. K_idata are an average of at least two independent experiments. Ratio of wtEGFR K_iappover

d

EGFR(TMLR) K_iapp. Ratio of wtEGFR K_iappover EGFR(TMdel) K_iapp.

and provides a compound of comparable potency for both

TMLR and TMdel, as well as similar LLE and selectivity over

wtEGFR. Furthermore, a crystal structure of 7 with TMLR

recapitulates the binding mode observed with 6, with ligands

closely overlapping (data not shown).

A signiﬁcant beneﬁt in removing this pyrazole was realized

with respect to general selectivity over other kinases. Pyrazole-

containing compounds 4−6 exhibit undesirable activity against

CDK2, with 6 also showing decreased selectivity against Aurora

B and KDR as compared to 3−5 (Table 4). Interestingly, while

the imidazopyridine compound 4 ablates the Jak2 and Tyk2

activity exhibited by original hits 1−3 (<50% inhibition of Jak2

or Tyk2 activity observed at 1 μM), both pyrrolopyridine

compounds 5 and 6 retain signiﬁcant Jak family activity. NH-

pyrrolopyridine 5 is equipotent against TMLR and Jak2/Tyk2,

while N-isopropyl 6 exhibits approximately 10-fold selectivity

F

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and Jak2 (Table 4). This improvement in broader kinase

selectivity is perhaps unsurprising given the alternative hinge-

binding motif provided by the unsubstituted pyrazole.

Table 4. Kinase Selectivity Proﬁles

IC₅₀(nM)

a

b

compd

Aurora B

KDR

CDK2

Jak2

44

Tyk2 K_i(nM)

While 7 has >20-fold selectivity over Aurora B, KDR, and

Jak2, we sought to improve its selectivity over CDK2 in order

to drive an EGFR-dependent antiproliferative eﬀect. Mod-

iﬁcation of the ribose-pocket group from an isopropyl to the

slightly larger cyclopentyl (compound 8) to ﬁll a greater

portion of the lipophilic ribose pocket does improve potency

against TMLR and TMdel slightly (∼2-fold). However, this

change from isopropyl to cyclopentyl increases lipophilicity and

does not substantially aﬀect kinase selectivity. A comparison of

X-ray crystal structures of 8 with TMLR and CDK2 (Figures

7a,b) suggested an opportunity to enhance selectivity in

subsequent designs. The binding mode of 8 in TMLR is

consistent with that observed for 6 (Figure 7a), while in CDK2,

the binding mode corresponds to the original binding mode

observed for compounds 2, 4, and 5.⁴⁹The gatekeeper residue

in CDK2 is a phenylalanine (Phe80), and this bulky side chain

precludes binding of the ligand in the orientation observed in

TMLR. Noting that the phenyl group of the Phe80 side chain

provides hydrophobic contacts with the aromatic methines of

the pyrrole ring, we hypothesized that introducing polarity in

this region would disfavor interactions with CDK2 Phe80 and

improve selectivity, similar to the selectivity beneﬁt seen with 4

as compared with 5. Indeed, the addition of an extra ring

nitrogen in imidazopyridine compound 9 provides an improve-

ment in selectivity over all key oﬀ-targets as compared to 7 and

8 (Table 4), with a 24-fold reduction in activity against CDK2.

Aurora B, KDR, and Jak2 also have lipophilic gatekeeper

residues (Aurora B = Leu170, KDR = Val914, Jak2 = Met929),

and the concurrent reduction in all of these oﬀ-target activities

suggests that the binding mode observed for 8 in CDK2 may be

shared among these other oﬀ-targets, consistent with the Jak2

model described in Figure 6. When tested at 1 μM in a broader

panel of 219 kinases, 9 inhibits only 5 kinases at >80%.^50,51

Compared with 7, compound 9 also has reduced lipophilicity

while potency is maintained and thus provides an increased

LLE. The improvement in physicochemical properties also

results in a signiﬁcant increase in kinetic aqueous solubility

(Table 3).

3

>10000

3260

3290

1360

219

503

90

16

4

>1000

96

3100

36

5

1770

5

6

107

12

10

154

7

6190

1990

2270

80

718

8

4030

93

225

c

9

>10000

1660

2220

530

476

>1000

c

10

11

>1000

2240

a

Aurora B, KDR, and CDK2 IC₅₀values determined at Invitrogen.

b

Jak2 IC₅₀and Tyk2 K_ivalues determined using an assay method

previously described.^36,53<20% inhibition observed at 1000 nM.

c

for TMLR over Jak2. The diﬀerential activity against Jak2 for 4

relative to 5 and 6 may be understood using a model of all three

compounds bound in the Jak2 active site in the orientation

observed for 4 and 5 in TMLR (Figure 6). In this orientation,

Figure 6. Models of 4 (a, green), 5 (b, magenta) and 6 (b, blue) in the

Jak2 active site (PBD ID: 4hge), with the protein surface colored

according to lipophilicity.

the ﬁve-membered heterocycle is adjacent to the gatekeeper

residue and the hydrophobic side chain of Jak2’s Met929

gatekeeper can be expected to preferentially interact with the

pyrrolo C3 methines of 5 and 6, while the imidazo N2 of 4

introduces unfavorable polarity in this hydrophobic region. The

selectivity of compound 6 can be improved with removal of the

terminal pyrazole moiety; as compared to 6, compound 7

exhibits minimal activity against the oﬀ-targets Aurora B, KDR,

Both pyrazolopyridine compounds 10 and 11 were expected

to result in a similar polarity mismatch with the CDK2 binding

site, and as shown in Table 4, both of these compounds have

good selectivity against all tested oﬀ-targets. Additionally, the 3-

aminopyrazolopyridine compound 10 is 3−8-fold more potent

than 9 with a TMLR K_iof 12 nM and TMdel K_iof 37 nM. This

Figure 7. Structures of 8 complexed with (a) TMLR and (b) CDK2.

G

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a

Scheme 1. Preparation of Compounds 4−6

a

Reagents and conditions: (a) polyphosphoric acid, 200 °C, 18 h, quant.; (b) sodium hydride, SEM-Cl, THF, DMF, rt, 2 h, 81%; (c) iodine

monochloride, NaOAc, HOAc, 70 °C, 18 h, 34%; (d) methanesulfonyl chloride, triethylamine, DCM, 0 °C to rt, 2 h, 46%; (e) 4-ethynyl-1H-

pyrazole, Pd(PPh₃)₂Cl₂, CuI, triethylamine, DMF, 50−100 °C, 33%; (f) 4-ethynyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole, Pd(PPh₃)₂Cl₂,

CuI, triethylamine, DMF, 50−100 °C, 34%; (g) NaH, SEM-Cl, THF, 0 °C to rt, 5 h, 36%; (h) 2-iodopropane, Cs₂CO₃, DMF, 90 °C, 48 h, 40%; (i)

4-methoxypiperidine hydrochloride, Cs₂CO₃, DMF, 120 °C, 18 h, 87%; (j) 16, 20a, or 20b, Pd₂dba₃, X-Phos or Xantphos, Cs₂CO₃, 1,4-dioxane, 120

°C, 28−81%; (k) HCl, MeOH, rt to 50 °C, 22−76%.

compound reached our initial objective of simultaneously

achieving good potency with selectivity against wtEGFR (8−26

times) and with acceptable physicochemical properties that are

substantially improved over those of compounds 1−3 (LogD <

2.5, kinetic solubility >50 μM). Critically, 10 has good

selectivity over a broad panel of kinases, inhibiting only 5 out

of 219 at >80% when tested at 1 μM.^51,52

determined in human liver microsomes ranging from 11 to

19 mL/min/kg, suggesting directions for further optimization.

CONCLUSION

■

In conclusion, beginning with potent inhibitors of Jak2/Tyk2,

we identiﬁed a novel series of noncovalent diaminopyrimidine-

based T790M EGFR inhibitors. Replacement of the biaryla-

mide motif with bicyclic substructures provided a signiﬁcant

reduction in lipophilicity. X-ray crystal structures revealed two

distinct binding modes that enabled design of T790M EGFR

inhibitors with high levels of selectivity over wtEGFR and with

favorable physicochemical properties that provide a desirable

starting point for further optimization. The unusual binding

mode utilizing interactions with both the gatekeeper Met790

and Thr854 side chains provides a framework for broad kinase

selectivity. The bicyclic scaﬀolds identiﬁed in Table 3 (7, 9, and

10) enable a substantial improvement in LLE compared to the

initial HTS hits (1−3) and are promising leads in the search for

selective and noncovalent inhibitors of T790M-containing

EGFR mutants. Further optimization of this scaﬀold to achieve

improved cellular potency and metabolic stability will be the

focus of future publications.

Other bicyclic systems were tested but did not result in

further improvement over 10 (see Table 3). 2-Oxoimidazole 12

was similarly anticipated to insult the CDK2 and Jak2 active

sites and provide good kinase selectivity; however, compared

with 10, this compound has decreased activity against TMLR

and was therefore not pursued. The dihydropyrrolo 13 was

designed to increase the percentage of sp³centers in an attempt

to improve the solubility of pyrrolopyridine 7. This compound

does exhibit an improvement in kinetic aqueous solubility

(increased from 8 to 69 μM), but it has signiﬁcantly reduced

TMLR potency. While the structure of 8 with TMLR suggests

that the sp³carbons of 13 should be accommodated sterically, a

greater energetic beneﬁt may be provided by the aliphatic−

aromatic contacts of pyrroles 7 and 8 sandwiched between

Leu718 and Gly796 as compared to the aliphatic−aliphatic

contacts aﬀorded by the dihydropyrrole of 13.⁴⁶Finally,

pyridone 14 was synthesized to evaluate 6−6 bicyclic systems,

but the lack of potency and poor solubility led to no additional

pursuit.

CHEMISTRY

■

Compounds 1−3 were prepared as generally described by

Liang et al.^36,54Compounds 4−6 were prepared as described in

Scheme 1. 2-Bromo-4,5-diaminopyridine and pyrazole 4-

carboxylate were converted to imidazopyridine 15 via a

polyphosphoric acid-mediated cyclization. Subsequent bis-

SEM protection aﬀorded intermediate 16. The corresponding

pyrrolopyridine intermediates were prepared by starting with 2-

bromo-4-aminopyridine 17, which was iodinated and N-

Further characterization of compounds in Table 3 indicated

only modest cellular activity in a pEGFR assay in H1975 cells,

an EGFR(TMLR)-driven cell line (8 IC₅₀= 1.2 μM, 10 IC₅₀

=

1.3 μM). Additionally, the in vitro metabolic stability of all

compounds in Table 3 is less than desirable with Cl_hep

H

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a

Scheme 2. Preparation of Compounds 7 and 8

a

Reagents and conditions: (a) 2-iodopropane or iodocyclopentane, Cs₂CO₃, DMF, 90 °C, 18−20 h, 56−90%; (b) 22, chloro[[BrettPhos][2-(2-

aminoethylphenyl)palladium(II)]]/[BrettPhos], NaOtBu, tBuOH, 90 °C, 5 h, 33−49%.

a

Scheme 3. Preparation of Compound 9

a

Reagents and conditions: (a) isopropylamine, triethylamine, THF, rt, 3 h, quant.; (b) 22, Pd₂dba₃, X-Phos, Cs₂CO₃, 1,4-dioxane, reﬂux, 3 h, 72%;

(c) Pd/C, EtOAc, MeOH, H₂, rt, 18 h, 92%; (d) trimethyl orthoformate, formic acid, 100 °C, 1 h, 27%.

a

Scheme 4. Preparation of Compound 10

a

Reagents and conditions: (a) CuCl, tert-butyl nitrite, acetonitrile, 50 °C to reﬂux, 1 h, 72%; (b) palladium(II) acetate, triethylamine, MeOH, CO,

60 °C, 18 h, 47%; (c) diisobutylaluminum hydride, DCM, −78 °C, 1 h, 55%; (d) hydrazine hydrate, triethylamine, dioxane, reﬂux, 3 d, 34%; (e)

iodine, potassium hydroxide, DMF, 40 C, 16 h; (f) 2-iodopropane, sodium hydride, DMF, rt, 16 h, 44% over two steps; (g) diphenylmethanimine,

Pd₂dba₃, Xantphos, Cs₂CO₃, 1,4-dioxane, 90 °C, 3 h, 58%; (h) 22, Pd₂dba₃, X-Phos or Xantphos, Cs₂CO₃, 1,4-dioxane, 100 °C, 2 h, 57%; (i) HCl,

dioxane, rt, 0.5 h, 60%.

sulfonylated to provide 18. Annulation with optionally SEM-

protected 4-alkynylpyrazoles provided the pyrrolopyridines 19a

and 19b. N1 alkylation or SEM protection of 19a,b provided

the key intermediates 20a,b. S_NAr reaction of 4-methoxypiper-

idine with 2-chloro-4-aminopyrimidine 21 aﬀorded intermedi-

ate 22. Finally, palladium-catalyzed coupling of intermediates

16 or 20a,b with 22 yielded compounds 4−6.

Schemes 2−4 describe preparation of additional bicyclic

pyridine analogs. Pyrrolopyridines 7 and 8 were readily

prepared as shown in Scheme 2. Alkylation of commercially

available 6-chloro-1H-pyrrolo[3,2-c]pyridine to give 23a,b

followed by palladium-catalyzed coupling to aminopyrimidine

22 provided compounds 7 and 8. As described in Scheme 3, a

nucleophilic displacement reaction with isopropylamine and

2,4-dichloro-5-nitropyridine provided chloropyridine 24, which

was coupled with the aminopyrimidine fragment 22 followed

by reduction of the nitro group to provide 25. Closure of the

imidazo ring with trimethyl orthoformate yielded 9. Scheme 4

describes the synthesis of 3-aminopyrazolopyridine 10,

beginning with compound 2-bromo-5-iodopyridin-4-amine. A

Sandmeyer reaction converted the 4-amino functionality to a 4-

chloro group (26). Conversion of the 3-iodo group to a methyl

ester proceeded via palladium-catalyzed carbonylation. Sub-

sequent reduction to the aldehyde with diisobutylaluminum

hydride aﬀorded compound 27. Treatment with hydrazine

eﬀected the ring closure to pyrazolopyridine 28. Iodination at

the 3-position to 29 followed by N-alkylation provided key

intermediate 30. Sequential palladium-catalyzed couplings ﬁrst

at the iodo position and then at the bromo position, followed

by deprotection, provided compound 10.

The preparation of compounds 11−14 is described in the

Supporting Information.

I

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ﬁltered and dried to aﬀord the title compound (0.950 g, 46%). LCMS

(ESI): [M + H] = 379.0. H NMR (400 MHz, DMSO-d₆): δ 8.62 (s,

1H), 7.52 (s, 1H), 3.26 (s, 3H).

EXPERIMENTAL SECTION

General. Unless otherwise indicated, all reagents and solvents were

purchased from commercial sources and used without further

puriﬁcation. Moisture or oxygen sensitive reactions were conducted

■

1

6-Bromo-2-(1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine

(19a). To a solution of 4-iodo-1H-pyrazole (11.0 g, 56.7 mmol) and

ethynyl(trimethyl)silane (22.3 g, 227 mmol) in THF (80 mL) were

added diethylamine (80 mL, 758 mmol), bis(triphenylphosphine)-

palladium(II) dichloride (6.03 g, 8.51 mmol), and copper(I) iodide

(1.62 g, 8.51 mmol), and the reaction mixture was stirred at room

temperature for 18 h. The solvent was removed in vacuo and the

resulting residue was dissolved in Et₂O (400 mL) and ﬁltered. The

ﬁltrate was concentrated and the residue was puriﬁed by silica gel

chromatography (solvent gradient of 0−100% Et₂O in heptane) to

aﬀord 4-((trimethylsilyl)ethynyl)-1H-pyrazole (5.1 g, 55%) as a brown

oil. LCMS (ESI): [M + H]⁺= 165.2. ¹H NMR (400 MHz, DMSO-d₆):

δ 12.92 (s, 1H), 7.87 (s, 1H), 7.46 (s, 1H), 0.01 (d, J = 6.4 Hz, 9H).

To a solution of 4-((trimethylsilyl)ethynyl)-1H-pyrazole (5.0 g, 30

mmol) in THF (50 mL) was added a solution of lithium hydroxide

hydrate (1.9 g, 46 mmol) in water (10 mL). After stirring for 18 h at

room temperature, the reaction mixture was neutralized with acetic

acid and concentrated. The residue was partitioned between 1-butanol

and water, and the combined organic layers were concentrated to yield

4-ethynyl-1H-pyrazole (2.8 g, quantitative). ¹H NMR (400 MHz,

DMSO-d₆): δ 13.08 (s, 1H), 7.84 (s, 2H), 3.93 (s, 1H).

1

under an atmosphere nitrogen gas. Unless otherwise stated, H NMR

spectra were recorded at room temperature using Varian Unity Inova

Bruker AVANCE III UltraShield-Plus Digital NMR spectrometer at

indicated frequencies. Chemical shifts are expressed in ppm relative to

an internal standard, tetramethylsilane (=0.00 ppm). The following

abbreviations are used: br = broad signal, s = singlet, d = doublet, dd =

doublet of doublets, t = triplet, q = quartet, m = multiplet. Puriﬁcation

by silica gel chromatography was carried out using a CombiFlash by

Teledyne ISCO system with prepacked cartridges. Puriﬁcation by

reverse-phase high-performance liquid chromatography (HPLC) and

supercritical ﬂuid chromatography (SFC) was also used. All ﬁnal

compounds were puriﬁed to ≥95% chemical purity as determined by

HPLC with UV detection at 254 nm. Further details on the analytical

conditions used for individual compounds may be found in the

Supporting Information.

6-Bromo-2-(1H-pyrazol-4-yl)-3H-imidazo[4,5-c]pyridine (15).

A mixture of 6-bromopyridine-3,4-diamine (100 mg, 0.53 mmol) and

1H-pyrazole-4-carboxylic acid (60 mg, 0.53 mmol) in polyphosphoric

acid (1 g) was heated in a sealed vial at 200 °C for 18 h. The reaction

mixture was diluted with water and made basic by addition of NaOH

(50% aq) and a white precipitate formed. The solid thus formed was

collected by ﬁltration and washed with water to aﬀord the title

compound (150 mg, quant.). LCMS (ESI): [M + H]⁺264.1.

6-Bromo-3-(2-trimethylsilanylethoxymethyl)-2-[1-(2-trime-

thylsilanylethoxymethyl)-1H-pyrazol-4-yl]-3H-imidazo[4,5-c]-

pyridine (16). To a suspension of 6-bromo-2-(1H-pyrazol-4-yl)-3H-

imidazo[4,5-c]pyridine (150 mg, 0.53 mmol) in THF (2 mL) under a

nitrogen atmosphere was added NaH (60% dispersion in mineral oil,

63 mg, 1.6 mmol). DMF (2 mL) was added to facilitate dissolution.

After eﬀervescence had ceased, 2-(trimethylsilyl)ethoxymethyl chlor-

ide (265 mg, 1.6 mmol) was added and stirring was continued for 2 h.

The reaction mixture was partitioned between water and EtOAc. The

aqueous phase was further extracted with EtOAc (3×), and the

combined organic phases were dried (MgSO₄) and concentrated in

vacuo. The resulting residue was puriﬁed by chromatography on silica

(solvent gradient of 0−40% EtOAc in cyclohexane) to aﬀord the title

compound (a mixture of two regioisomers) as a yellow oil that

solidiﬁed on standing (224 mg 81%). LCMS (ESI): [M + H]⁺524.2.

2-Bromo-5-iodopyridin-4-amine (18a). To a solution of 2-

bromopyridin-4-amine (15.0 g, 86.7 mmol) and sodium acetate (14.2

g, 173.4 mmol) in glacial acetic acid (150 mL) was added a solution of

iodine monochloride (4.9 mL, 95.4 mmol) in glacial acetic acid (70

mL). After heating for 18 h at 70 °C, the reaction was cooled to room

temperature and poured into water (800 mL). The aqueous solution

was partitioned twice with EtOAc. The combined organic layers were

washed with saturated aqueous Na₂CO₃, saturated aqueous Na₂S₂O₃,

and brine and dried over MgSO₄. The organic layer was ﬁltered and

concentrated in vacuo, and the resulting residue was puriﬁed by silica

gel chromatography (solvent gradient of 10−60% EtOAc in heptane)

to aﬀord the title compound (8.9 g, 34%). LCMS (ESI): [M + H]⁺=

300.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.16 (s, 1H), 6.77 (s, 1H),

6.50 (s, 2H).

A mixture of N-(2-bromo-5-iodopyridin-4-yl)methanesulfonamide

(1500 mg, 4.0 mmol), 4-ethynyl-1H-pyrazole (440 mg, 4.8 mmol),

bis(triphenylphosphine)palladium(II) dichloride (140 mg, 0.20

mmol), copper(I) iodide (38 mg, 0.20 mmol), and triethylamine

(2.5 mL, 18 mmol) in DMF (35 mL) was heated at 100 °C for 3 h and

then cooled to 50 °C. DBU (1.8 mL) was added and stirring

continued at 50 °C for 30 min. After cooling to room temperature, the

solution was diluted with saturated NH₄Cl and extracted twice with

EtOAc. The combined organic layers were dried over MgSO₄, ﬁltered,

and concentrated in vacuo. The resulting residue was puriﬁed by silica

gel chromatography (solvent gradient of 5−100% EtOAc in heptane)

to aﬀord the title compound (350 mg, 33%). LCMS (ESI): [M + H]⁺

1

= 263.0. H NMR (400 MHz, DMSO-d₆): δ 13.13 (s, 1H), 11.92 (s,

1H), 8.51 (s, 1H), 8.26 (s, 1H), 8.01 (s, 1H), 7.48 (s, 1H), 6.72 (s,

1H).

6-Bromo-2-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-

4-yl)-1H-pyrrolo[3,2-c]pyridine (19b). Into a 1 L three-necked

round-bottom ﬂask purged and maintained with nitrogen were added

4-iodo-1H-pyrazole (45.0 g, 232 mmol, 1.00 equiv) and N,N-

dimethylformamide (500 mL). This was followed by addition of

sodium hydride (60% dispersion in mineral oil, 10.2 g, 2.55 mmol) in

several batches at 0 °C. The reaction mixture was stirred for 30 min at

0 °C in an ice/salt water bath. To the reaction mixture was added [2-

(chloromethoxy)ethyl]trimethylsilane (42.3 g, 254 mmol) dropwise

with stirring. The resulting solution was stirred for an additional 3 h at

room temperature. The reaction mixture was quenched by addition of

water/ice (250 mL) and extracted with EtOAc (2 × 500 mL), and the

organic layers were separated and combined. The combined organic

layer was washed with brine, dried over anhydrous sodium sulfate, and

concentrated under vacuum to aﬀord 4-iodo-1-[[2-(trimethylsilyl)-

ethoxy]methyl]-1H-pyrazole (80.0 g) as light yellow oil without

further puriﬁcation.

Into a 1 L three-necked round-bottom ﬂask purged and maintained

with an inert atmosphere of nitrogen were added 4-iodo-1-[[2-

(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (75.0 g, 231 mmol), CuI

(880 mg, 4.62 mmol), dichlorobis(triphenylphosphine)palladium(II)

(3.25 g, 4.63 mmol), triethylamine (93.5 g, 926 mmol),

(trimethylsilyl)acetylene (56.7 g, 578 mmol), and THF (500 mL).

After being stirred for 5 h at room temperature, the reaction mixture

was diluted with water (300 mL) and EtOAc (1000 mL), ﬁltered,

washed with brine, dried over anhydrous sodium sulfate, and

concentrated under vacuum to aﬀord 1-[[2-(trimethylsilyl)ethoxy]-

methyl]-4-[2-(trimethylsilyl)ethynyl]-1H-pyrazole (80.0 g) as brown

oil. LCMS (ESI): [M + H]⁺= 295.

N-(2-Bromo-5-iodopyridin-4-yl)methanesulfonamide (18).

Methanesulfonyl chloride (2.15 mL, 27.3 mmol) in DCM (8 mL)

was added dropwise to a cold (0 °C) solution of 2-bromo-5-

iodopyridin-4-amine (1.63 g, 5.45 mmol) and triethylamine (3.84 mL,

27.3 mmol) in DCM (20 mL). The reaction mixture was allowed to

warm to room temperature and stirred for 2 h. The solvent was

removed in vacuo and the resulting residue was puriﬁed by silica gel

chromatography (solvent gradient of 5−100% EtOAc in heptane) to

aﬀord N-(2-bromo-5-iodopyridin-4-yl)-N-(methylsulfonyl)-

methanesulfonamide (1.1 g, 44% yield) as an oﬀ-white solid that

was dissolved in aqueous NaOH solution (10%, 15 mL) in THF (15

mL) and stirred at room temperature for 16 h. The reaction mixture

was concentrated. Water was added and then the mixture was acidiﬁed

to pH 4 using an aqueous citric acid solution. The resulting solid was

Into a 100 mL three-necked round-bottom ﬂask purged and

maintained with nitrogen were added 1-[[2-(trimethylsilyl)ethoxy]-

J

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methyl]-4-[2-(trimethylsilyl)ethynyl]-1H-pyrazole (80.0 g, 272 mmol,

1.00 equiv) and THF (100 mL). This was followed by addition of

tetrabutylammonium ﬂuoride (1 N solution in THF, 300 mL)

dropwise with stirring at room temperature. After being stirred for 4 h

at room temperature, the reaction mixture was diluted with EtOAc

(300 mL), washed with brine, dried over anhydrous sodium sulfate,

and concentrated under vacuum. The residue was puriﬁed by

chromatography with EtOAc/petroleum ether (1:50) to aﬀord 4-

ethynyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (30.0 g,

50.0%) as yellow oil. LCMS (ESI): [M + H]⁺= 223. ¹H NMR

(300 MHz, CDCl₃): 7.68 (s, 1H), 7.59 (s, 1H), 5.34 (s, 2H), 3.50 (m,

2H), 2.99 (s, 1H), 0.85 (m, 2H), 0.03 (s, 9H).

gradient of 0−100% EtOAc in cyclohexane) and then triturated with

cyclohexane, aﬀording a second batch of the title compound (2.38 g,

87% combining the two batches). H NMR (400 MHz, CDCl₃): δ

7.94 (1H, d, J = 5.60 Hz), 5.74 (1H, d, J = 5.60 Hz), 4.53 (2H s),

4.33−4.24 (2H, m), 3.47−3.37 (4H, m), 3.33−3.24 (2H, m), 1.98−

1.87 (2H, m), 1.60−1.47 (2H, m).

1

[2-(4-Methoxypiperidin-1-yl)pyrimidin-4-yl]-[2-(1H-pyrazol-

4-yl)-3H-imidazo[4,5-c]pyridin-6-yl]amine (4). A mixture of 6-

bromo-3-(2-trimethylsilanylethoxymethyl)-2-[1-(2-trimethylsilanyle-

thoxymethyl)-1H-pyrazol-4-yl]-3H-imidazo[4,5-c]pyridine (120 mg,

0.23 mmol), 22 (53 mg, 0.25 mmol), Pd₂dba₃(10 mg, 0.01 mmol),

XPhos (22 mg, 0.046 mmol), and Cs₂CO₃(273 mg, 0.84 mmol) in

dioxane (2 mL) was purged with argon (3×) and heated at 120 °C for

18 h. The cooled reaction mixture was diluted with DCM and ﬁltered

through Celite. The ﬁltrate was concentrated in vacuo and the

resulting residue was puriﬁed by chromatography on silica (solvent

gradient of 0−10% MeOH in DCM, followed by 0−15% MeOH in

EtOAc and by 0−10% 2 M NH₃/MeOH in DCM) to aﬀord [2-(4-

methoxypiperidin-1-yl)pyrimidin-4-yl]-[3-(2-trimethylsilanylethoxy-

methyl)-2-[1-(2-trimethylsilanylethoxymethyl)-1H-pyrazol-4-yl]-3H-

imidazo[4,5-c]pyridin-6-yl]amine (120 mg, 81%). LCMS (ESI): [M +

H]⁺652.4.

A mixture of N-(2-bromo-5-iodopyridin-4-yl)methanesulfonamide

(5.9 g, 16.0 mmol), 4-ethynyl-1-((2-(trimethylsilyl)ethoxy)methyl)-

1H-pyrazole (4.5 g, 20.0 mmol), bis(triphenylphosphine)palladium(II)

dichloride (550 mg, 0.78 mmol), copper(I) iodide (150 mg, 0.78

mmol), and triethylamine (6.6 mL, 47.0 mmol) in DMF (50 mL) was

heated at 100 °C for 3 h and then cooled to 50 °C. DBU (7.1 mL) was

added and stirring continued for an additional 30 min at 50 °C. After

stirring at room temperature for 18 h, the mixture was diluted with

NH₄Cl aqueous solution and extracted twice with EtOAc. The

combined organic layers were dried over MgSO₄and concentrated in

vacuo. The resulting residue was puriﬁed by silica gel chromatography

(gradient of 0−100% EtOAc in heptane) to aﬀord the title compound

[2-(4-Methoxypiperidin-1-yl)pyrimidin-4-yl]-[3-(2-trimethylsilany-

lethoxymethyl)-2-[1-(2-trimethylsilanylethoxymethyl)-1H-pyrazol-4-

yl]-3H-imidazo[4,5-c]pyridin-6-yl]amine (120 mg, 0.18 mmol) was

suspended in MeOH (5 mL) and HCl (6 M aqueous solution, 0.5

mL). The reaction mixture was stirred at room temperature for 3 h

and then at 50 °C for a further 3 h. A solid precipitated on standing.

The suspension thus obtained was dissolved in MeOH by the use of

heat and sonication and the resulting solution was loaded onto an SCX

cartridge. The cartridge was washed with MeOH while the product

was eluted with 2 M NH₃in MeOH. The product containing fractions

were combined and concentrated in vacuo, and the resulting residue

was puriﬁed by chromatography on silica (solvent gradient of 0−10%

2 M NH₃/MeOH in DCM) to aﬀord the title compound as a white

1

(2.1 g, 34%). LCMS (ESI): [M + H]⁺= 395.2. H NMR (400 MHz,

DMSO-d₆): δ 8.57 (s, 1H), 8.41 (s, 1H), 8.09 (s, 1H), 7.53 (s, 1H),

6.80 (s, 1H), 5.51 (s, 2H), 3.67−3.55 (m, 2H), 0.97−0.74 (m, 2H),

0.00 (s, 9H).

6-Bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-2-(1-((2-

(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)-1H-pyrrolo-

[3,2-c]pyridine (20a). Sodium hydride (60% dispersion in mineral

oil, 270 mg, 6.7 mmol) was added slowly to a solution of 6-bromo-2-

(1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine (350 mg, 1.3 mmol) in

THF (10 mL) at 0 °C and stirred for 30 min followed by addition of

2-(chloromethoxy)ethyltrimethylsilane (0.94 mL, 5.3 mmol). The

reaction mixture was allowed to warm to room temperature and stirred

for an additional 5 h. The reaction was quenched with water (50 mL),

and the aqueous layer was extracted twice with EtOAc. The combined

organic layers were concentrated in vacuo, and the resulting residue

was puriﬁed by silica gel chromatography (solvent gradient of 0−50%

EtOAc in heptane) to aﬀord the title compound (250 mg, 36%).

LCMS (ESI): [M + H]⁺= 524.0. ¹H NMR (500 MHz, DMSO-d₆): δ

8.60 (d, J = 4.1 Hz, 1H), 8.36−8.33 (m, 1H), 8.01 (d, J = 2.9 Hz, 2H),

6.85 (s, 1H), 5.64 (s, 2H), 5.48 (d, J = 8.3 Hz, 2H), 3.61−3.48 (m,

4H), 0.94−0.76 (m, 4H), 0.01−0.00 (m, 18H).

6-Bromo-1-isopropyl-2-(1-((2-(trimethylsilyl)ethoxy)-

methyl)-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine (20b). 2-

Iodopropane (0.4 mL, 4.0 mmol) was added to a mixture of 6-

bromo-2-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)-1H-

pyrrolo[3,2-c]pyridine (500 mg, 1.0 mmol) and cesium carbonate

(1000 mg, 4.0 mmol) in DMF (4 mL). After stirring at 90 °C for 48 h,

the mixture was diluted with EtOAc (80 mL) and washed with water.

The organic layer was concentrated in vacuo and the residue was

puriﬁed by silica gel chromatography (solvent gradient of 0−40%

EtOAc in heptane) to aﬀord the title compound (240 mg, 40%).

LCMS (ESI): [M + H]⁺= 437.4. ¹H NMR (400 MHz, DMSO-d₆): δ

8.62 (s, 1H), 8.31 (s, 1H), 7.87 (d, J = 9.9 Hz, 2H), 6.65 (s, 1H), 5.79

(s, 1H), 5.52 (s, 2H), 4.84−4.71 (m, 1H), 3.72−3.59 (m, 2H), 1.58 (d,

J = 6.9 Hz, 6H), 0.99−0.85 (m, 2H), 0.00 (s, 9H).

1

solid (55 mg, 76%). LCMS (ESI): [M + H]⁺391.9. H NMR (400

MHz, DMSO-d₆): δ 13.29 (1H, s), 12.67 (1H, s), 9.61 (1H, s), 8.51

(1H, s), 8.43−8.09 (2H, br s), 8.03 (1H, s), 7.93 (1H, d, J = 5.9 Hz),

6.58−6.50 (1H, m), 4.25−4.14 (2H, m), 3.48−3.30 (3H, m), 3.28

(3H, s), 1.93−1.84 (2H, m), 1.47−1.35 (2H, m).

N-(2-(4-Methoxypiperidin-1-yl)pyrimidin-4-yl)-2-(1H-pyra-

zol-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-amine (5). A mixture of 6-

bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-2-(1-((2-(trimethylsilyl)-

ethoxy)methyl)-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine (125 mg,

0.239 mmol), 22 (64.6 mg, 0.310 mmol), tris(dibenzylideneacetone)-

dipalladium(0) (10.9 mg, 0.012 mmol), Xantphos (14.2 mg, 0.024

mmol), and cesium carbonate (233 mg, 0.716 mmol) in 1,4-dioxane

(1.5 mL) and 1,2-dimethoxyethane (1.5 mL) was added to a

microwave tube. After purging with nitrogen for 5 min, the sealed

tube was heated at 120 °C in a CEM microwave for 60 min. The

reaction mixture was ﬁltered through a pad of Celite and the ﬁltrate

was concentrated in vacuo. The resulting residue was puriﬁed by silica

gel chromatography (solvent gradient of 0−100% EtOAc in heptane)

to aﬀord N-(2-(4-methoxypiperidin-1-yl)pyrimidin-4-yl)-1-((2-

(trimethylsilyl)ethoxy)methyl)-2-(1-((2-(trimethylsilyl)ethoxy)-

methyl)-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-amine (43 mg,

28%; LCMS (ESI): [M + H]⁺= 652.0) as an oﬀ-white solid which was

dissolved in MeOH (1 mL) and 10% aqueous NaOH (0.1 mL) and

stirred at room temperature for 30 min. The mixture was evaporated

and the residue was puriﬁed by HPLC (C18 silica on a 20 min

gradient of 5−50% acetonitrile/0.1% NH₄OH in water) to aﬀord the

2-(4-Methoxypiperidin-1-yl)pyrimidin-4-ylamine (22). 2-

Chloropyrimidin-4-ylamine (3.5 g, 27.0 mmol), 4-methoxypiperidine

hydrochloride (4.09 g, 27.0 mmol), and Cs₂CO₃(26.4 g, 81.0 mmol)

were suspended in DMF (60 mL) and heated at 120 °C for 18 h. The

reaction mixture was partitioned between water and EtOAc. The

aqueous phase was washed with EtOAc (2×), and the combined

organic phases were washed with brine, dried over MgSO₄, and

concentrated in vacuo aﬀording the title compound as a solid (2.5 g).

The aqueous phase was concentrated in vacuo and the slurry was

extracted with EtOAc. The volatiles were removed in vacuo, and the

resulting residue was puriﬁed by silica gel chromatography (solvent

1

title compound (5.0 mg, 20%). LCMS (ESI): [M + H]⁺= 391.2. H

NMR (400 MHz, DMSO-d₆): δ 9.46 (s, 1H), 8.44 (s, 1H), 8.07 (s,

2H), 7.96−7.87 (m, 2H), 6.63−6.53 (m, 2H), 4.24 (dt, J = 13.0, 4.6

Hz, 2H), 3.52−3.35 (m, 2H), 3.30 (s, 3H), 1.97−1.85 (m, 2H), 1.51−

1.36 (m, 2H).

1-Isopropyl-N-(2-(4-methoxypiperidin-1-yl)pyrimidin-4-yl)-

2-(1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-amine (6). 6-

Bromo-1-isopropyl-2-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyra-

zol-4-yl)-1H-pyrrolo[3,2-c]pyridine (236 mg, 0.542 mmol), 22 (169

K

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mg, 0.813 mmol), tris(dibenzylideneacetone)dipalladium(0) (24.8 mg,

0.0271 mmol), Xantphos (32.3 mg, 0.054 mmol), cesium carbonate

(442 mg, 1.36 mmol), and 1,4-dioxane (4 mL) were added to a

pressure tube. After purging with nitrogen for 5 min, the sealed tube

was heated at 120 °C for 20 h. The reaction mixture was ﬁltered

through a pad of Celite and washed with EtOAc. The ﬁltrate was

concentrated in vacuo and the residue was puriﬁed by silica gel

chromatography (solvent gradient of 0−100% EtOAc in heptane) to

aﬀord 1-isopropyl-N-(2-(4-methoxypiperidin-1-yl)pyrimidin-4-yl)-2-

(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)-1H-pyrrolo-

mL, 16 mmol) was heated under nitrogen at 90 °C for 5 h. The

reaction mixture was ﬁltered through Celite, rinsing with 2-propanol,

and concentrated. The crude product was puriﬁed via ﬂash

chromatography on silica gel (4 g of silica, solvent gradient of 0−

10% MeOH in DCM) followed by achiral SFC to yield 48.3 mg (33%)

1

of the title compound. LCMS (ESI): [M + H]⁺= 393.2. H NMR

(400 MHz, DMSO-d₆): δ 9.60 (s, 1H), 8.52 (d, J = 0.9 Hz, 1H), 8.29

(s, 1H), 7.93 (d, J = 5.6 Hz, 1H), 7.43 (d, J = 3.3 Hz, 1H), 6.52 (dd, J

= 3.4, 0.8 Hz, 1H), 6.36 (d, J = 5.7 Hz, 1H), 4.76−4.67 (m, 1H),

4.28−4.19 (m, 2H), 3.51−3.43 (m, 1H), 3.43−3.33 (m, 2H), 3.29 (s,

3H), 2.19−2.08 (m, 2H), 1.98−1.79 (m, 6H), 1.78−1.65 (m, 2H),

1.51−1.34 (m, 2H).

(2-Chloro-5-nitropyridin-4-yl)isopropylamine (24). Isopropyl-

amine (1.43 mL, 16.7 mmol) was added dropwise to a solution of 2,4-

dichloro-5-nitropyridine (3 g, 13.9 mmol) and triethylamine (3.9 mL,

27.8 mmol) in THF (50 mL) at room temperature, resulting in a mild

exotherm. The reaction mixture was stirred at room temperature for 3

h and then partitioned between water and EtOAc. The aqueous phase

was washed with EtOAc, and the combined organic phases were

washed with brine, dried (MgSO₄), and concentrated in vacuo,

aﬀording the title compound as a yellow solid (quantitative). ¹H NMR

(400 MHz, CDCl₃): δ 9.02 (1H, s), 8.08 (1H, s), 6.74 (1H, s), 3.88−

3.72 (1H, m), 1.37 (6H, d, J = 6.4 Hz).

[3,2-c]pyridin-6-amine (110 mg, 36%; LCMS (ESI): [M + H]⁺

=

563.6) as an oﬀ-white solid. This material was dissolved in 4 N HCl in

1,4-dioxane (4 mL) and stirred at room temperature for 1 h. The

mixture was concentrated and the residue was puriﬁed by preparatory

HPLC (C18 silica on a 20 min gradient of 5−50% acetonitrile/0.1%

NH₄OH in water) to aﬀord the title compound (19 mg, 22%). LCMS

1

(ESI): [M + H]⁺= 433.3. H NMR (400 MHz, DMSO-d₆): δ 13.21

(s, 1H), 9.56 (s, 1H), 8.50 (s, 1H), 8.33 (s, 1H), 7.93 (d, J = 5.7 Hz,

1H), 6.45 (s, 2H), 4.72 (p, J = 7.1 Hz, 1H), 4.33−4.24 (m, 2H), 3.50−

3.34 (m, 2H), 3.30 (s, 3H), 1.91 (d, J = 12.2 Hz, 2H), 1.57 (d, J = 7.0

Hz, 6H), 1.43 (dq, J = 8.2, 4.5, 3.9 Hz, 2H).

6-Bromo-1-isopropyl-1H-pyrrolo[3,2-c]pyridine (23a). A mix-

ture of 6-bromo-1H-pyrrolo[3,2-c]pyridine (250.4 mg, 1.271 mmol),

2-iodopropane (0.50 mL, 5.0 mmol), cesium carbonate (878 mg, 2.67

mmol), and DMF (5.0 mL, 64 mmol) was heated at 90 °C for 20 h.

The reaction mixture was cooled to room temperature, diluted with

EtOAc, washed with water (2×) and brine, dried over magnesium

sulfate, ﬁltered, and evaporated in vacuo. The crude product was

puriﬁed via ﬂash chromatography on silica gel (12 g silica, solvent

gradient of 0−100% EtOAc in DCM) to yield 272.4 mg (90%) of the

title compound. LCMS (ESI): [M + H]⁺= 239. ¹H NMR (400 MHz,

DMSO-d₆): 8.59 (d, J = 1.3 Hz, 1H), 7.83 (s, 1H), 7.65 (d, J = 3.5 Hz,

1H), 6.65 (dd, J = 3.5, 0.8 Hz, 1H), 4.82 (p, J = 6.6 Hz, 1H), 1.44 (d, J

= 6.7 Hz, 6H).

N⁴-Isopropyl-N²-[2-(4-methoxypiperidin-1-yl)pyrimidin-4-

yl]pyridine-2,4,5-triamine (25). (2-Chloro-5-nitropyridin-4-yl)-

isopropylamine (517 mg, 2.4 mmol), 11 (500 mg, 2.4 mmol),

XPhos (229 mg, 48 μmol), Pd₂(dba)₃(109 mg, 12 μmol), and

Cs₂CO₃(1.56 g, 4.8 mmol) were suspended in 1,4-dioxane (10 mL).

The reaction mixture was degassed with argon, sonicated, and then

heated under reﬂux for 3 h. The cooled reaction mixture was

partitioned between water and EtOAc. The aqueous phase was washed

with EtOAc (2×), and the combined organic phases were washed with

brine, dried over MgSO₄, and concentrated in vacuo. The resulting

dark brown solid was triturated with diethyl ether, aﬀording N⁴-

isopropyl-N²-[2-(4-methoxypiperidin-1-yl)pyrimidin-4-yl]-5-nitropyri-

6-Chloro-1-cyclopentyl-1H-pyrrolo[3,2-c]pyridine (23b). A

mixture of 6-chloro-1H-pyrrolo[3,2-c]pyridine (100 mg, 0.65 mmol),

iodocyclopentane (254 mg, 1.3 mmol), and Cs₂CO₃(422 mg, 1.3

mmol) in DMF (1 mL) was heated at 80 °C for 18 h. The cooled

reaction mixture was partitioned between water and EtOAc. The

aqueous phase was further extracted with EtOAc, and the combined

organic phases were dried (MgSO₄) and concentrated in vacuo. The

resulting residue was puriﬁed by chromatography on silica (solvent

gradient of 0−50% EtOAc in cyclohexane) to aﬀord the title

1

dine-2,4-diamine as a bright yellow solid (667 mg, 72%). H NMR

(400 MHz, CDCl₃): δ 9.02 (1H, s), 8.25 (1H, m), 8.11 (1H, d, J = 5.5

Hz), 7.66 (1H, s), 7.30 (1H, s), 6.06 (1H, d, J = 5.5 Hz), 4.33−4.20

(2H, m), 3.94−3.82 (1H, m), 3.56−3.44 (3H, m), 3.41 (3H, s), 2.00−

1.89 (2H, m), 1.68−1.57 (2H, m), 1.37 (6H, d, J = 6.38 Hz).

N⁴-Isopropyl-N²-[2-(4-methoxypiperidin-1-yl)pyrimidin-4-yl]-5-ni-

tropyridine-2,4-diamine (2.18 g, 5.6 mmol) and Pd/C (10% by

weight) (300 mg) were suspended in a mixture of EtOAc/MeOH

(100 mL/10 mL) and stirred under a hydrogen atmosphere for 18 h.

The reaction mixture was ﬁltered and the ﬁltrate was concentrated in

vacuo. The resulting residue was puriﬁed by silica gel chromatography

(solvent gradient of 0−10% 2 M NH₃/MeOH in DCM) to aﬀord the

1

compound as a colorless oil (80 mg, 56%). H NMR (400 MHz,

CDCl₃): δ 8.64 (1H, s), 7.31 (1H, s), 7.21 (1H, d, J = 3.3 Hz), 6.57

(1H, d, J = 3.2 Hz), 4.76−4.65 (1H, m), 2.30−2.17 (2H, m), 1.97−

1.72 (6H, m).

1

title compound (1.84 g, 92%). H NMR (400 MHz, CDCl₃): δ 7.99

(1-Isopropyl-1H-pyrrolo[3,2-c]pyridin-6-yl)-[2-(4-methoxypi-

peridin-1-yl)pyrimidin-4-yl]amine (7). A mixture of 6-bromo-1-

isopropylpyrrolo[3,2-c]pyridine (95.7 mg, 0.400 mmol), 22 (112.4 mg,

0.5397 mmol), chloro[[BrettPhos][2-(2-aminoethylphenyl)palladium-

(II)]]/[BrettPhos] admixture (31.0 mg, 0.0232 mmol), sodium tert-

butoxide (118.1 mg, 1.23 mmol), and tert-butanol (1.5 mL, 16 mmol)

was heated under nitrogen at 90 °C for 5 h. The reaction mixture was

ﬁltered through Celite, rinsing with 2-propanol, and concentrated. The

crude product was puriﬁed via ﬂash chromatography on silica gel (4 g

of silica, solvent gradient of 0−10% MeOH in DCM) followed by

achiral SFC to yield 71.9 mg (49%) of the title compound. LCMS

(ESI): [M + H]⁺= 361.2. ¹H NMR (400 MHz, DMSO-d₆): δ 9.60 (s,

1H), 8.52 (d, J = 1.2 Hz, 1H), 8.29 (s, 1H), 7.93 (d, J = 5.6 Hz, 1H),

7.45 (d, J = 3.3 Hz, 1H), 6.52 (dd, J = 3.3, 0.8 Hz, 1H), 6.36 (d, J = 5.7

Hz, 1H), 4.59 (p, J = 6.7 Hz, 1H), 4.31−4.17 (m, 2H), 3.51−3.42 (m,

1H), 3.42−3.33 (m, 2H), 1.98−1.84 (m, 2H), 1.49 (d, J = 6.7 Hz,

6H), 1.46−1.36 (m, 2H).

(1H, d, J = 5.7 Hz), 7.61 (1H, s), 7.27 (1H, s), 7.18 (1H, s), 6.14 (1H,

d, J = 5.7 Hz), 4.37−4.2 (3H, m), 3.76−3.64 (1H, m), 3.50−3.33 (6H,

m), 2.82 (2H, s), 1.99−1.89 (2H, m), 1.65−1.52 (2H, m), 1.28 (6H, d,

J = 6.3 Hz).

(1-Isopropyl-1H-imidazo[4,5-c]pyridin-6-yl)-[2-(4-methoxy-

piperidin-1-yl)pyrimidin-4-yl]amine (9). To a solution of N⁴-

isopropyl-N²-[2-(4-methoxypiperidin-1-yl)pyrimidin-4-yl]pyridine-

2,4,5-triamine (70 mg, 0.19 mmol) in trimethyl orthoformate (2 mL)

was added formic acid (5 drops) and the reaction mixture was heated

at 100 °C for 1 h. The volatiles were removed in vacuo and the

resulting residue was puriﬁed by HPLC (C18 silica on a 20 min

gradient of 20−60% acetonitrile/0.1% NH₄OH in water followed by a

20 min gradient of 5−50% acetonitrile/0.1% HCO₂H in water) to

aﬀord the title compound as a formate salt (19 mg, 27%). LCMS

(ESI): [M + H]⁺367.9. ¹H NMR (400 MHz, DMSO-d₆): δ 9.74 (1H,

s), 8.60 (1H, d, J = 0.9 Hz), 8.35 (1H, s), 8.28 (1H, s), 8.14 (0.4H, s),

7.92 (1H,d, J = 5.7 Hz), 6.33 (1H, d, J = 5.7 Hz), 4.65−4.54 (1H, m),

4.24−4.12 (2H, m), 3.24 (3H, s), 2.46−2.25 (3H, m), 1.91−1.77 (2H,

m), 1.51 (6H, d, J = 6.8 Hz), 1.44−1.31 (2H, m).

(1-Cyclopentyl-1H-pyrrolo[3,2-c]pyridin-6-yl)-[2-(4-methox-

ypiperidin-1-yl)pyrimidin-4-yl]amine (8). A mixture of 6-bromo-

1-cyclopentylpyrrolo[3,2-c]pyridine (98.5 mg, 0.371 mmol), 22 (105.4

mg, 0.5061 mmol), chloro[[BrettPhos][2-(2-aminoethylphenyl)-

palladium(II)]]/[BrettPhos] admixture (26.1 mg, 0.0195 mmol),

sodium tert-butoxide (119.0 mg, 1.24 mmol), and tert-butanol (1.5

2-Bromo-4-chloro-5-iodopyridine (26). To a three-necked

round-bottom ﬂask containing acetonitrile (1.5 L), 2-bromo-5-

iodopyridin-4-amine (125 g, 418 mmol), and copper(I) chloride

L

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(61.0 g, 622 mmol) was added tert-butyl nitrite (86.0 g, 835 mmol)

dropwise with stirring at 50 °C. The resulting solution was heated to

reﬂux for 1 h. The reaction mixture was cooled to room temperature

and concentrated in vacuo. The crude product was puriﬁed via ﬂash

chromatography on silica gel (solvent gradient of 1−10% EtOAc in

petroleum ether) to aﬀord the title compound (96 g, 72%) as a yellow

solid.

dipalladium(0) (75 mg, 0.08 mmol), 4,5-bis(diphenylphosphino)-9,9-

dimethylxanthene (36 mg, 0.08 mmol), and cesium carbonate (540

mg, 1.66 mmol) in 1,4-dioxane (6 mL) was stirred for 3 h at 90 °C in

an inert atmosphere of nitrogen. The solution was cooled to room

temperature, diluted with water (20 mL), and extracted with EtOAc

(2×). The combined organic layers were washed with brine, dried over

anhydrous sodium sulfate, ﬁltered, and concentrated in vacuo. The

resulting residue was puriﬁed by silica gel chromatography (solvent

gradient of 0−60% EtOAc in petroleum ether) to aﬀord the title

compound (200 mg, 58%) as a yellow solid. LCMS (ESI): [M + H]⁺=

419.

6-Bromo-4-chloropyridine-3-carbaldehyde (27). Into a 2 L

pressure tank reactor were placed 2-bromo-4-chloro-5-iodopyridine

(30.0 g, 94.2 mmol), MeOH (500 L), triethylamine (28.6 g, 282

mmol), and palladium(II) acetate (2.10 g, 9.35 mmol). The resulting

solution was stirred at 60 °C under an atmosphere of CO (1−2 atm).

After 18 h, the reaction was cooled to room temperature, ﬁltered, and

concentrated in vacuo. The crude product was puriﬁed via ﬂash

chromatography on silica gel (solvent gradient of 2−20% EtOAc in

petroleum ether) to aﬀord methyl 6-bromo-4-chloropyridine-3-

carboxylate (11 g, 47%) as a white solid. LCMS (ESI): [M + H]⁺=

1-Isopropyl-N⁶-(2-(4-methoxypiperidin-1-yl)pyrimidin-4-yl)-

1H-pyrazolo[4,3-c]pyridine-3,6-diamine (10). A mixture of 6-

bromo-N-(diphenylmethylene)-1-isopropyl-1H-pyrazolo[4,3-c]-

pyridin-3-amine (80 mg, 0.19 mmol), tris(dibenzylideneacetone)-

dipalladium(0) (17.5 mg, 0.02 mmol), 4,5-bis(diphenylphosphino)-

9,9-dimethylxanthene (9.1 mg, 0.02 mmol), 22 (48 mg, 0.23 mmol),

and cesium carbonate (125 mg, 0.39 mmol) in 1,4-dioxane (2 mL) was

stirred for 2 h at 100 °C in an inert atmosphere of nitrogen. The

resulting mixture was cooled to room temperature and concentrated in

vacuo. The resulting residue was puriﬁed by silica gel chromatography

(solvent gradient 0−65% EtOAc in petroleum) to aﬀord N³-

(diphenylmethylene)-1-isopropyl-N⁶-(2-(4-methoxypiperidin-1-yl)-

pyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridine-3,6-diamine (60 mg, 57%)

as a yellow solid. LCMS (ESI): [M + H]⁺= 547.

1

250. H NMR (300 MHz, DMSO-d₆): δ 8.14 (s, 1H), 7.64 (s, 1H),

3.98 (s, 3H).

To a three-necked round-bottom ﬂask under nitrogen containing

methyl 6-bromo-4-chloropyridine-3-carboxylate (35.0 g, 139 mmol) in

DCM (700 mL) was added diisobutylaluminum hydride (1 M in

hexanes) (153 mL) dropwise with stirring at −78 °C. The resulting

solution was maintained at −78 °C for 1 h and then warmed to room

temperature. After 1 h, the reaction was quenched by addition of HCl

(2 N in water, 100 mL) followed by water (500 mL). The resulting

solution was extracted with DCM (3 × 500 mL), and the combined

organic layers were washed with brine, dried over anhydrous sodium

sulfate, and concentrated in vacuo. The crude product was puriﬁed via

ﬂash chromatography on silica gel (solvent gradient of 1−10% EtOAc

in petroleum ether) to aﬀord the title compound (17 g, 55%) as a

white solid. LCMS (ESI): [M + H]⁺= 220.

To a reaction vessel were added N³-(diphenylmethylene)-1-

isopropyl-N⁶-(2-(4-methoxypiperidin-1-yl) pyrimidin-4-yl)-1H-

pyrazolo[4,3-c]pyridine-3,6-diamine (60 mg, 0.11 mmol) and HCl in

1,4-dioxane (4 M, 8 mL). The resulting solution was stirred for 0.5 h at

room temperature. The pH value of the solution was adjusted to 8

with sodium bicarbonate (aq) and the mixture was extracted with of

EtOAc (2×). The resulting mixture was concentrated in vacuo. The

resulting residue was puriﬁed via reverse-phase HPLC and lyophilized

to aﬀord the title compound (25.3 mg, 60%) as a light yellow solid.

LCMS (ESI): [M + H]⁺= 383.15. ¹H NMR (400 MHz, DMSO-d₆): δ

9.76 (s, 1H), 8.63 (s, 1H), 8.05 (s, 1H), 7.97 (d, J = 5.6 Hz, 1H), 6.35

(d, J = 5.6 Hz, 1H), 5.77 (s, 2H), 4.50−4.44 (m, 1H), 4.25−4.20 (m,

2H), 3.49−3.40 (m, 1H), 3.38−3.34 (m, 2H), 3.32 (s, 3H), 1.92−1.90

(m, 2H), 1.50−1.30 (m, 8H).

6-Bromo-1H-pyrazolo[4,3-c]pyridine (28). To a reaction vessel

were added 6-bromo-4-chloropyridine-3-carbaldehyde (24.0 g, 109

mmol), 1,4-dioxane (600 mL), triethylamine (22.0 g, 217 mmol), and

hydrazine hydrate (9.50 g, 131 mmol), and the reaction mixture was

heated to reﬂux for 3 days. The reaction was cooled to room

temperature and concentrated in vacuo. The crude product was

puriﬁed via ﬂash chromatography on silica gel (solvent gradient of 1−

5% MeOH in DCM) to aﬀord the title compound (7.4 g, 34%) as a

Enzymatic assays. Experiments were carried out as previously

described.³¹

1

yellow solid. LCMS (ESI): [M + H]⁺= 198. H NMR (400 MHz,

In Vitro Microsome Metabolic Stability. Experiments were

carried out as previously described.⁵⁵

DMSO-d₆): δ 13.62 (br, 1H), 8.93 (s, 1H), 8.35 (s, 1H), 7.81 (s, 1H).

6-Bromo-3-iodo-1H-pyrazolo[4,3-c]pyridine (29). To a reac-

tion vessel were added a solution of 6-bromo-1H-pyrazolo[4,3-

c]pyridine (5.50 g, 27.8 mmol) in DMF (100 mL), potassium

hydroxide (3.60 g, 64.2 mmol), and iodine (14.2 g, 55.9 mmol), and

the resulting mixture was stirred for 16 h at 40 °C. The reaction was

cooled to room temperature and diluted with water (500 mL). The

resulting suspension was ﬁltered, and the solids were collected and

dried in an oven under reduced pressure to aﬀord the title compound

ASSOCIATED CONTENT

* Supporting Information

■

S

Tabulated biochemical Jak family activity for compounds 1−3;

tabulated structures and data for amide modiﬁcations; PDB

codes and measurements used for the generation of Figure 5b;

experimental procedures for kinetic solubility experiments;

preparation and characterization of compounds 1−3 and 11−

14; protein expression, puriﬁcation, and crystallographic

methods; data collection and reﬁnement statistics; and HPLC

methods used to determine chemical purity. This material is

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

1

(10.5 g) as a yellow solid. LCMS (ESI): [M + H]⁺= 324. H NMR

(400 MHz, DMSO-d₆): δ 14.00 (s, 1H), 8.61 (s, 1H), 7.88 (s, 1H).

6-Bromo-3-iodo-1-isopropyl-1H-pyrazolo[4,3-c]pyridine

(30). To a solution of 6-bromo-3-iodo-1H-pyrazolo[4,3-c]pyridine

(10.4 g, 32.1 mmol) in DMF (100 mL) under nitrogen were added

sodium hydride (2.10 g, 52.5 mmol) and 2-iodopropane (21.0 g, 123

mmol), and the reaction mixture was stirred at room temperature.

After 16 h, the reaction was quenched with water (400 mL) and

extracted with EtOAc (2 × 300 mL). The combined organic layers

were washed with brine, dried over anhydrous sodium sulfate, and

concentrated in vacuo. The crude product was puriﬁed via ﬂash

chromatography on silica gel (eluent:of 10% EtOAc in petroleum

ether) to aﬀord the title compound (5.2 g, 44%) as a white solid.

Accession Codes

The PDB codes are as follows: 4RJ7 for 1 complexed with

TMLR, 4RJ6 for 4 complexed with TMLR, 4RJ5 for 5

complexed with TMLR, 4RJ4 for 6 complexed with TMLR,

4RJ8 for 8 complexed with TMLR, and 4RJ3 for 8 complexed

with CDK2.

1

LCMS (ESI): [M + H]⁺= 366. H NMR (400 MHz, DMSO-d₆): δ

8.57 (s, 1H), 8.13 (s, 1H), 5.07−5.00 (m, 1H), 1.46−1.45 (d, J = 6.8

AUTHOR INFORMATION

Corresponding Author

*Phone: 650-225-4577. E-mail: hanan.emily@gene.com.

Notes

■

Hz, 6 H).

6-Bromo-N-(diphenylmethylene)-1-isopropyl-1H-pyrazolo-

[4,3-c]pyridin-3-amine (31). A mixture of 6-bromo-3-iodo-1-

isopropyl-1H-pyrazolo[4,3-c]pyridine (300 mg, 0.82 mmol), diphe-

nylmethanimine (150 mg, 0.83 mmol), tris(dibenzylideneacetone)-

The authors declare no competing ﬁnancial interest.

M

dx.doi.org/10.1021/jm501578n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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(8) Pao, W.; Miller, V.; Zakowski, M.; Doherty, J.; Politi, K.; Sarkaria,

I.; Singh, B.; Heelan, R.; Rusch, V.; Fulton, L.; Mardis, E.; Kupfer, D.;

Wilson, R.; Kris, M.; Varmus, H. EGF receptor gene mutations are

common in lung cancers from “never smokers” and are associated with

sensitivity of tumors to gefitinib and erlotinib. Proc. Natl. Acad. Sci. U.

S. A. 2004, 101, 13306−13311.

(9) Jackman, D. M.; Miller, V. A.; Cioffredi, L. A.; Yeap, B. Y.; Janne,

P. A.; Riely, G. J.; Ruiz, M. G.; Giaccone, G.; Sequist, L. V.; Johnson, B.

E. Impact of epidermal growth factor receptor and KRAS mutations on

clinical outcomes in previously untreated non-small cell lung cancer

patients: Results of an online tumor registry of clinical trials. Clin.

Cancer Res. 2009, 15, 5267−5273.

(10) Rosell, R.; Moran, T.; Queralt, C.; Porta, R.; Cardenal, F.;

Camps, C.; Majem, M.; Lopez-Vivanco, G.; Isla, D.; Provencio, M.;

Insa, A.; Massuti, B.; Gonzalez-Larriba, J. L.; Paz-Ares, L.; Bover, I.;

Garcia-Campelo, R.; Moreno, M. A.; Catot, S.; Rolfo, C.; Reguart, N.;

Palmero, R.; Sanchez, J. M.; Bastus, R.; Mayo, C.; Bertran-Alamillo, J.;

Molina, M. A.; Sanchez, J. J.; Taron, M. Screening for epidermal

growth factor receptor mutations in lung cancer. N. Engl. J. Med. 2009,

361, 958−967.

(11) Pao, W.; Chmielecki, J. Rational, biologically based treatment of

EGFR-mutant non-small-cell lung cancer. Nat. Rev. Cancer 2010, 10,

760−774.

(12) Kobayashi, S.; Boggon, T. J.; Dayaram, T.; Janne, P. A.; Kocher,

O.; Meyerson, M.; Johnson, B. E.; Eck, M. J.; Tenen, D. G.; Halmos, B.

EGFR mutation and resistance of non-small-cell lung cancer to

gefitinib. N. Engl. J. Med. 2005, 352, 786−792.

(13) Pao, W.; Miller, V. A.; Politi, K. A.; Riely, G. J.; Somwar, R.;

Zakowski, M. F.; Kris, M. G.; Varmus, H. Acquired resistance of lung

adenocarcinomas to gefitinib or erlotinib is associated with a second

mutation in the EGFR kinase domain. PLoS Med. 2005, 2 (e73), 225−

235.

(14) Balak, M. N.; Gong, Y.; Riely, G. J.; Somwar, R.; Li, A. R.;

Zakowski, M. F.; Chiang, A.; Yang, G.; Ouerfelli, O.; Kris, M. G.;

Ladanyi, M.; Miller, V. A.; Pao, W. Novel D761Y and common

secondary T790M mutations in epidermal growth factor receptor-

mutant lung adenocarcinomas with acquired resistance to kinase

inhibitors. Clin. Cancer Res. 2006, 12, 6494−6501.

(15) Kosaka, T.; Yatabe, Y.; Endoh, H.; Yoshida, K.; Hida, T.;

Tsuboi, M.; Tada, H.; Kuwano, H.; Mitsudomi, T. Analysis of

epidermal growth factor receptor gene mutation in patients with non-

small cell lung cancer and acquired resistance to gefitinib. Clin. Cancer

Res. 2006, 12, 5764−5769.

(16) Yu, H. A.; Arcila, M. E.; Rekhtman, N.; Sima, C. S.; Zakowski,

M. F.; Pao, W.; Kris, M. G.; Miller, V. A.; Ladanyi, M.; Riely, G. J.

Analysis of tumor specimens at the time of acquired resistance to

EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers.

Clin. Cancer Res. 2013, 19, 2240−2247.

(17) Shah, N. P.; Nicoll, J. M.; Nagar, B.; Gorre, M. E.; Paquette, R.

L.; Kuriyan, J.; Sawyers, C. L. Multiple BCR-ABL kinase domain

mutations confer polyclonal resistance to the tyrosine kinase inhibitor

imatinib (STI571) in chronic phase and blast crisis chronic myeloid

leukemia. Cancer Cell 2002, 2, 117−125.

(18) Kwak, E. L.; Sordella, R.; Bell, D. W.; Godin-Heymann, N.;

Okimoto, R. A.; Brannigan, B. W.; Harris, P. L.; Driscoll, D. R.; Fidias,

P.; Lynch, T. J.; Rabindran, S. K.; McGinnis, J. P.; Wissner, A.; Sharma,

S. V.; Isselbacher, K. J.; Settleman, J.; Haber, D. A. Irreversible

inhibitors of the EGF receptor may circumvent acquired resistance to

gefitinib. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 7665−7670.

(19) Quintas-Cardama, A.; Kantarjian, H.; Cortes, J. Flying under the

radar: The new wave of BCR-ABL inhibitors. Nat. Rev. Drug Discovery

2007, 6, 834−848.

ACKNOWLEDGMENTS

■

We thank Christopher Hamman, Michael Hayes, Yutao Jiang,

Baiwei Lin, Deven Wang, and Mengling Wong for puriﬁcation

and analytical support, Baiwei Lin for conducting kinetic

solubility and LogD experiments, and Ning Liu for conducting

liver microsome stability experiments. We thank the CCDC⁵⁶

for their help in the use of Relibase to query the PDB and, in

particular, Tjelvar Olsson, who provided scripts for the radial

distribution plots. We thank Kristina Grabowski of the

Chemical Computing Group⁵⁷for her svl script to calculate

the geometries speciﬁc to gatekeeper methionines. The

Advanced Light Source is supported by the Director, Oﬃce

of Science, Oﬃce of Basic Energy Sciences, of the U.S.

Department of Energy under Contract No. DE-AC02-

05CH11231. The Berkeley Center for Structural Biology is

supported in part by the National Institutes of Health, National

Institute of General Medical Sciences, and the Howard Hughes

Medical Institute. Use of the LS-CAT Sector 21 of the

Advanced Photon Source (APS) was supported by the

Michigan Economic Development Corp. and the Michigan

Technology Tri-Corridor (Grant 085P1000817). APS, an

Oﬃce of Science User Facility operated for the U.S.

Department of Energy Oﬃce of Science by Argonne National

Laboratory, was supported by the U.S. DOE under Contract

No. DE-AC02-06CH11357.

ABBREVIATIONS USED

■

CSD, Cambridge Structural Database; EtOAc, ethyl acetate;

HOAc, acetic acid; Jak, Janus kinase; KDR, kinase insert

domain receptor; LLE, lipophilic ligand eﬃciency; MeOH,

methanol; NaOAc, sodium acetate; NaOtBu, sodium tert-

butoxide; SEM, 2-(trimethylsilyl)ethoxymethyl; TKI, tyrosine

kinase inhibitor; TMdel, epidermal growth factor receptor

(T790M/del746−750); TMLR, epidermal growth factor

receptor (T790M/L858R); Tyk2, tyrosine kinase 2; wtEGFR,

wild-type epidermal growth factor receptor.

REFERENCES

■

(1) Sharma, S. V.; Settleman, J. Oncogene addiction: Setting the

stage for molecularly targeted cancer therapy. Genes Dev. 2007, 21,

3214−3231.

(2) Sordella, R.; Bell, D. W.; Haber, D. A.; Settleman, J. Gefitinib-

sensitizing EGFR mutations in lung cancer activate anti-apoptotic

pathways. Science 2004, 305, 1163−1167.

(3) Tracy, S.; Mukohara, T.; Hansen, M.; Meyerson, M.; Johnson, B.

E.; Janne, P. A. Gefitinib induces apoptosis in the EGFRL858R non-

small-cell lung cancer cell line H3255. Cancer Res. 2004, 64, 7241−

7244.

(4) Gazdar, A. F.; Minna, J. D. Inhibition of EGFR signaling: All

mutations are not created equal. PLoS Med. 2005, 2 (e377), 1085−

1087.

(5) Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Epidermal

growth factor receptor mutations in lung cancer. Nat. Rev. Cancer

2007, 7, 169−181.

(6) Lynch, T. J.; Bell, D. W.; Sordella, R.; Gurubhagavatula, S.;

Okimoto, R. A.; Brannigan, B. W.; Harris, P. L.; Haserlat, S. M.; Supko,

J. G.; Haluska, F. G.; Louis, D. N.; Christiani, D. C.; Settleman, J.;

Haber, D. A. Activating mutations in the epidermal growth factor

receptor underlying responsiveness of non-small-cell lung cancer to

gefitinib. N. Engl. J. Med. 2004, 350, 2129−2139.

(7) Paez, J. G.; Janne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.;

Gabriel, S.; Herman, P.; Kaye, F. J.; Lindeman, N.; Boggon, T. J.;

Naoki, K.; Sasaki, H.; Fujii, Y.; Eck, M. J.; Sellers, W. R.; Johnson, B.

E.; Meyerson, M. EGFR mutations in lung cancer: Correlation with

clinical response to gefitinib therapy. Science 2004, 304, 1497−1500.

(20) Erlotinib activity in biochemical assays: wtEGFR K_i= 0.1 nM,

EGFR(L858R) K_i= 0.3 nM, EGFR(d746−750) K_i= 0.1 nM,

EGFR(T790M/L858R) K_i= 123 nM, and EGFR(T790M/d746−750)

K_i= 95 nM.

(21) Yun, C. H.; Mengwasser, K. E.; Toms, A. V.; Woo, M. S.;

Greulich, H.; Wong, K. K.; Meyerson, M.; Eck, M. J. The T790M

N

dx.doi.org/10.1021/jm501578n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

mutation in EGFR kinase causes drug resistance by increasing the

affinity for ATP. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 2070−2075.

(22) Janne, P. A.; Schellens, J. H.; Engelman, J. A.; Eckhardt, S. G.;

Millham, R.; Denis, L. J.; Britten, C. D.; Wong, S. G.; Boss, D. S.;

Camidge, D. R. Preliminary activity and safety results from a phase I

clinical trial of PF-00299804, an irreversible pan-HER inhibitor, in

patients (pts) with NSCLC. J. Clin. Oncol. 2008, 26, (May 20 suppl;

abstr 8027).

(23) Sequist, L. V.; Besse, B.; Lynch, T. J.; Miller, V. A.; Wong, K. K.;

Gitlitz, B.; Eaton, K.; Zacharchuk, C.; Freyman, A.; Powell, C.;

Ananthakrishnan, R.; Quinn, S.; Soria, J. C. Neratinib, an irreversible

pan-ErbB receptor tyrosine kinase inhibitor: Results of a phase II trial

in patients with advanced non-small-cell lung cancer. J. Clin. Oncol.

2010, 28, 3076−3083.

(34) Potashman, M. H.; Duggan, M. E. Covalent modifiers: An

orthogonal approach to drug design. J. Med. Chem. 2009, 52, 1231−

1246.

(35) Kalgutkar, A. S.; Dalvie, D. K. Drug discovery for a new

generation of covalent drugs. Expert Opin. Drug Discovery 2012, 7,

561−581.

(36) Liang, J.; Tsui, V.; Van Abbema, A.; Bao, L.; Barrett, K.;

Beresini, M.; Berezhkovskiy, L.; Blair, W. S.; Chang, C.; Driscoll, J.;

Eigenbrot, C.; Ghilardi, N.; Gibbons, P.; Halladay, J.; Johnson, A.;

Kohli, P. B.; Lai, Y.; Liimatta, M.; Mantik, P.; Menghrajani, K.; Murray,

J.; Sambrone, A.; Xiao, Y.; Shia, S.; Shin, Y.; Smith, J.; Sohn, S.;

Stanley, M.; Ultsch, M.; Zhang, B.; Wu, L. C.; Magnuson, S. Lead

identification of novel and selective TYK2 inhibitors. Eur. J. Med.

Chem. 2013, 67, 175−187.

(24) Sos, M. L.; Rode, H. B.; Heynck, S.; Peifer, M.; Fischer, F.;

Kluter, S.; Pawar, V. G.; Reuter, C.; Heuckmann, J. M.; Weiss, J.;

Ruddigkeit, L.; Rabiller, M.; Koker, M.; Simard, J. R.; Getlik, M.; Yuza,

Y.; Chen, T. H.; Greulich, H.; Thomas, R. K.; Rauh, D. Chemo-

genomic profiling provides insights into the limited activity of

irreversible EGFR Inhibitors in tumor cells expressing the T790M

EGFR resistance mutation. Cancer Res. 2010, 70, 868−874.

(25) Kim, Y.; Ko, J.; Cui, Z.; Abolhoda, A.; Ahn, J. S.; Ou, S. H.; Ahn,

M. J.; Park, K. The EGFR T790M mutation in acquired resistance to

an irreversible second-generation EGFR inhibitor. Mol. Cancer Ther.

2012, 11, 784−791.

(26) Ranson, M.; Pao, W.; Kim, D. W.; Kim, S. W.; Ohe, Y.; Felip, E.;

Planchard, D.; Ghiorghiu, S.; Cantarini, M.; Janne, P. A. Preliminary

results from a phase I study with AZD9291: An irreversible inhibitor of

epidermal growth factor receptor (EGFR) activating and resistance

mutations in non-small-cell lung cancer (NSCLC). Eur. J. Cancer

2013, 49 (Suppl 3), S15.

(27) Sequist, L. V.; Soria, J.-C.; Gadgeel, S.; Wakelee, H.; Camidge,

D. R.; Varga, A.; Fidias, P.; Wozniak, A. J.; Neal, J. W.; Doebele, R. C.;

Garon, E. B.; Jaw-Tsai, S.; Stern, J. C.; Allen, A.; Goldman, J. W. First-

in-human evaluation of CO-1686, an irreversible, selective, and potent

tyrosine kinase inhibitor of EGFR T790M. J. Clin. Oncol. 2013, 31,

2524.

(28) Cross, D. A.; Ashton, S. E.; Ghiorghiu, S.; Eberlein, C.; Nebhan,

C. A.; Spitzler, P. J.; Orme, J. P.; Finlay, M. R.; Ward, R. A.; Mellor, M.

J.; Hughes, G.; Rahi, A.; Jacobs, V. N.; Red Brewer, M.; Ichihara, E.;

Sun, J.; Jin, H.; Ballard, P.; Al-Kadhimi, K.; Rowlinson, R.; Klinowska,

T.; Richmond, G. H.; Cantarini, M.; Kim, D. W.; Ranson, M. R.; Pao,

W. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated

resistance to EGFR inhibitors in lung cancer. Cancer Discovery 2014, 4,

1046−1061.

(29) Tjin Tham Sjin, R.; Lee, K.; Walter, A. O.; Dubrovskiy, A.;

Sheets, M.; Martin, T. S.; Labenski, M. T.; Zhu, Z.; Tester, R.; Karp,

R.; Medikonda, A.; Chaturvedi, P.; Ren, Y.; Haringsma, H.; Etter, J.;

Raponi, M.; Simmons, A. D.; Harding, T. C.; Niu, D.; Nacht, M.;

Westlin, W. F.; Petter, R. C.; Allen, A.; Singh, J. In vitro and In vivo

characterization of irreversible mutant-selective EGFR inhibitors that

are wild-type sparing. Mol. Cancer Ther. 2014, 13, 1468−1479.

(30) Janne, P. A.; Ramaligam, S. S.; Yang, J. C. H.; Ahn, M.-J.; Kim,

D.-W.; Kim, S.-W.; Planchard, D.; Ohe, Y.; Felip, E.; Watkins, C.;

Cantarini, M.; Ghiorghiu, S.; Ranson, M. Clinical activity of the

mutant-selective EGFR inhibitor AZD9291 in patients (pts) with

EGFR inhibitor-resistant non-small-cell lung cancer (NSCLC). J. Clin.

Oncol. 2014, 32 (Suppl. 5), 8009.

(31) Lee, H. J.; Schaefer, G.; Heffron, T. P.; Shao, L.; Ye, X.; Sideris,

S.; Malek, S.; Chan, E.; Merchant, M.; La, H.; Ubhayakar, S.; Yauch, R.

L.; Pirazzoli, V.; Politi, K.; Settleman, J. Noncovalent wild-type-sparing

inhibitors of EGFR T790M. Cancer Discovery 2013, 3, 168−181.

(32) Preliminary phase I data suggest that irreversible inhibitors

AZD9291 and CO-1686 do not appear to have the toxicities seen with

previous irreversible inhibitors of EGFR.

(37) Percent inhibition of 296 kinases determined at Invitrogen for

compound 3; inhibition of >80% at 1 μM observed for

EGFR(T790M/L858R), EGFR(T790M), Jak2, Jak2 JH1/JH2,

MAP4K4, Mink1, and Tyk2.

(38) Leeson, P. D.; Springthorpe, B. The influence of drug-like

concepts on decision-making in medicinal chemistry. Nat. Rev. Drug

Discovery 2007, 6, 881−890.

(39) Shultz, M. D. Setting expectations in molecular optimizations:

Strengths and limitations of commonly used composite parameters.

Bioorg. Med. Chem. Lett. 2013, 23, 5980−5991.

(40) Hopkins, A. L.; Keseru, G. M.; Leeson, P. D.; Rees, D. C.;

Reynolds, C. H. The role of ligand efficiency metrics in drug discovery.

Nat. Rev. Drug Discovery 2014, 13, 105−121.

(41) PBD accession code 1M17.

(42) When compound 1 is modeled into the 1M17 wtEGFR

structure, if the water molecule adjacent to the gatekeeper side chain is

allowed to rotate, the carbonyl oxygen of compound 1 is within

hydrogen-bonding distance (<3.0 Å) of that water.

(43) Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. Structure of the

epidermal growth factor receptor kinase domain alone and in complex

with a 4-anilinoquinazoline inhibitor. J. Biol. Chem. 2002, 277, 46265−

46272.

(44) Conformational searches were carried out in MacroModel 10.0,

using the OPLS_2005 forceﬁeld and mixed torsional/low-mode

sampling for 1000 steps. Low-energy conformations within 1 kcal/

mol of the global minimum for each molecule were compared, and the

number of conformers around the amide bond was used as a proxy for

the degrees of freedom.

(45) A structure of compound 6 complexed with wtEGFR was not

obtained. Neither of the two binding modes oﬀers a hydrogen-bonding

partner for Thr790; if recapitulated in wtEGFR, the binding mode for

6 observed in TMLR would displace the water molecule typically

observed to solvate Thr854, while failing to provide a compensating

interaction.

(46) Bissantz, C.; Kuhn, B.; Stahl, M. A medicinal chemist’s guide to

molecular interactions. J. Med. Chem. 2010, 53, 5061−5084.

(47) See Table S3 of the Supporting Information for PDB codes and

measurements.

(48) A total of 34 out of 531 (6%) kinases have equivalent Met + Thr

residues and 27 out of 531 (5%) kinases have equivalent Met + Ser

residues.

(49) Electron density suggests that the catalytic Lys of CDK2 is

acetylated in the complex with compound 8; however, we believe that

this artifact of the crystallization system should not impact the binding

mode of this compound.

(50) Percent inhibition of 219 kinases at 1 μM determined at

Invitrogen; kinases inhibited at > 80%: MAP4K4, MELK, Mink1,

MST1, Rse.

(51) While pyrrolopyridine-based inhibitors have been reported to

inhibit MPS1, compounds 9 and 10 inhibit MPS1 at only 24−27%

when tested at 1 μM (Invitrogen). Naud, S.; Westwood, I. M.; Faisal,

A.; Sheldrake, P.; Bavetsias, V.; Atrash, B.; Cheung, K. M.; Liu, M.;

Hayes, A.; Schmitt, J.; Wood, A.; Choi, V.; Boxall, K.; Mak, G.;

Gurden, M.; Valenti, M.; de Haven Brandon, A.; Henley, A.; Baker, R.;

McAndrew, C.; Matijssen, B.; Burke, R.; Hoelder, S.; Eccles, S. A.;

(33) Evans, D. C.; Watt, A. P.; Nicoll-Griffith, D. A.; Baillie, T. A.

Drug-protein adducts: An industry perspective on minimizing the

potential for drug bioactivation in drug discovery and development.

Chem. Res. Toxicol. 2004, 17, 3−16.

O

dx.doi.org/10.1021/jm501578n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Raynaud, F. I.; Linardopoulos, S.; van Montfort, R. L.; Blagg, J.

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine

inhibitors of mitotic kinase monopolar spindle 1 (MPS1). J. Med.

Chem. 2013, 56, 10045−10065.

(52) Percent inhibition of 219 kinases at 1 μM determined at

Invitrogen; kinases inhibited at >80%: MAP4K4, Mink1, MST1,

MST2, Rse.

(53) Zak, M.; Hurley, C. A.; Ward, S. I.; Bergeron, P.; Barrett, K.;

Balazs, M.; Blair, W. S.; Bull, R.; Chakravarty, P.; Chang, C.; Crackett,

P.; Deshmukh, G.; DeVoss, J.; Dragovich, P. S.; Eigenbrot, C.;

Ellwood, C.; Gaines, S.; Ghilardi, N.; Gibbons, P.; Gradl, S.; Gribling,

P.; Hamman, C.; Harstad, E.; Hewitt, P.; Johnson, A.; Johnson, T.;

Kenny, J. R.; Koehler, M. F.; Bir Kohli, P.; Labadie, S.; Lee, W. P.;

Liao, J.; Liimatta, M.; Mendonca, R.; Narukulla, R.; Pulk, R.; Reeve, A.;

Savage, S.; Shia, S.; Steffek, M.; Ubhayakar, S.; van Abbema, A.;

Aliagas, I.; Avitabile-Woo, B.; Xiao, Y.; Yang, J.; Kulagowski, J. J.

Identification of C-2 hydroxyethyl imidazopyrrolopyridines as potent

JAK1 inhibitors with favorable physicochemical properties and high

selectivity over JAK2. J. Med. Chem. 2013, 56, 4764−4785.

(54) See Supporting Information for experimental procedures.

(55) Halladay, J. S.; Wong, S.; Jaffer, S. M.; Sinhababu, A. K.;

Khojasteh-Bakht, S. C. Metabolic stability screen for drug discovery

using cassette analysis and column switching. Drug Metab. Lett. 2007,

1, 67−72.

(56) Cambridge Crystallographic Database, Cambridge Crystallo-

graphic Data Centre, 12 Union Road, Cambridge CB2 1EZ, United

Kingdom.

(57) Chemical Computing Group, Kaiser-Wilhelm-Ring 11, 50672

Koln, Germany.

P

dx.doi.org/10.1021/jm501578n | J. Med. Chem. XXXX, XXX, XXX−XXX

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Discovery of selective and noncovalent diaminopyrimidine-based inhibitors of epidermal growth factor receptor containing the T790M resistance mutation

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