Discovery of clinical candidate CEP-37440, a selective inhibitor of focal adhesion kinase (FAK) and anaplastic lymphoma kinase (ALK)

Article abstract of DOI:10.1021/acs.jmedchem.6b00487

Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.

Full text of DOI:10.1021/acs.jmedchem.6b00487

Article
pubs.acs.org/jmc
Discovery of Clinical Candidate CEP-37440, a Selective Inhibitor of
Focal Adhesion Kinase (FAK) and Anaplastic Lymphoma Kinase (ALK)
Gregory R. Ott,*,† Mangeng Cheng,† Keith S. Learn,† Jason Wagner,† Diane E. Gingrich,†
Joseph G. Lisko,† Matthew Curry,† Eugen F. Mesaros,† Arup K. Ghose,† Matthew R. Quail,†
Weihua Wan,† Lihui Lu,† Pawel Dobrzanski,† Mark S. Albom,† Thelma S. Angeles,† Kevin Wells-Knecht,†
Zeqi Huang,† Lisa D. Aimone,† Elizabeth Bruckheimer,‡ Nathan Anderson,‡ Jay Friedman,‡
Sandra V. Fernandez,§ Mark A. Ator,† Bruce A. Ruggeri,† and Bruce D. Dorsey†
†Teva Branded Pharmaceutical Products R&D, 145 Brandywine Parkway, West Chester, Pennsylvania 19380, United States
‡Champions Oncology, Inc., One University Plaza, Suite 307, Hackensack, New Jersey 07601, United States
§Thomas Jefferson University, 233 South 10th Street, 1002 BLSB, Philadelphia, Pennsylvania 19107, United States
S
* Supporting Information
ABSTRACT: Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic
stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro
activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase
(FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis.
The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo
pharmacology, and pharmacokinetics, is reported herein.
other genetic drivers/variants of this disease that have not been
clinically validated with small-molecule/biologic therapeutics
(e.g., KRas).10
INTRODUCTION
■
The identification of tumor-specific genetic aberrations, their
validation as druggable targets, and therapeutic interdiction
with small molecule drugs and/or biologics has led to a distinct
clinical benefit for specific, genetically defined patient
populations and has further advanced the field of personalized
medicine.1 In particular, non-small-cell lung cancer (NSCLC)
has seen the development of targeted small-molecule therapies
to oncogenic driver mutations in the epidermal growth factor
(EGFR) gene (gefitinib, erlotinib, afatinib),2,3 the oncogenic
fusion protein echinoderm microtubule-associated protein-like
4-anaplastic lymphoma kinase gene (EML4-ALK) (crizotinib,
certinib, alectinib),4 as well as other oncogenic proteins.5 The
emergence of tumor resistance through drug-induced activating
mutations, gene amplification, and/or induced-resistance/
alternative signaling pathways has led to extensive effort in
developing second and third generation therapies.6−9 Despite
the ability to genotype and stratify patients to tailored therapy,
significant medical need exists in NSCLC to address the above-
mentioned resistance to targeted therapy, metastasis, as well as
The biological rationale, therapeutic intervention, and clinical
proof of concept data with small−molecule ALK inhibitors
have been well documented.11−13 Current approaches toward
ALK+ cancers have focused on improving potency toward
drug-induced activating mutations as well as incorporation of
ancillary activity against other oncogenic signaling mecha-
nisms14,15 to combat resistance and the recurrence of local
disease and distal metastases. Our initial efforts in the ALK field
led to the discovery of 1 (Figure 1), a selective ALK inhibitor
which advanced to preclinical development.16,17 Several distinct
challenges for our next generation of inhibitors were readily
apparent which included not only addressing the limitations of
the lead molecule itself (e.g., metabolic stability, high plasma
protein binding) but the broader, changing landscape of the
Received: April 1, 2016
© XXXX American Chemical Society
A
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specifically NSCL, breast, ovarian, prostate, and HNSCC
carcinomas.21,22 Interest in small molecule FAK inhibitors has
grown and is currently being evaluated at the clinical level.23
Furthermore, a very recent report identified FAK as a key
mediator of the immune response in certain cancers and
provides strong evidence that adjunctive administration of
small-molecule FAK inhibitors would be beneficial with
inhibitors of T-cell immune checkpoint antibodies (i.e., anti-
PD-1, CTLA-4).24
Herein, we describe the SAR, in vitro and in vivo properties,
and the discovery of a clinical level compound, 27b (CEP-
37440). Compound 27b improved upon the pharmaceutic
properties of 1 and also demonstrated an enhanced
pharmacological and kinase selectivity profile with activity in
ALK-positive NSCL tumor xenografts, favorable brain pene-
tration, and both in vitro and in vivo biochemical and
pharmacodynamic activity/antitumor efficacy against focal
adhesion kinase (FAK) and FAK-dependent tumors. Further-
more, this activity is especially relevant to treating subsets of
inflammatory breast cancer (IBC), an aggressive and highly
metastatic subset of breast cancer that has recently been shown
to have amplification FAK in the preponderance of tumors
evaluated.25 This selective, dual-action inhibitor provides a
novel therapeutic opportunity to treat ALK- and FAK-driven
malignancies particularly in NSCLC, IBC, and potentially as an
adjunctive to immunotherapy.
Figure 1. Teva small molecule ALK inhibitors.
small-molecule ALK inhibitor field, which has seen the approval
of three distinct molecular entities to treat EML4-ALK defined
NSCLC as first or second line therapy, as well as specific drug-
induced resistance. Toward this end, a multipronged strategy
was developed which included new chemical architecture by
constraining the small-molecule ligands into the putative active
conformation,18−20 as well as fine-tuning the current lead series.
This strategy made ample use of our broad knowledge base of
structure−activity relationships (SARs) which engendered
cross-fertilization among the architecturally unique core
scaffolds (2−4). Ultimately, we addressed the pharmaceutical
liabilities, demonstrated in vitro activity against clinically
relevant ALK mutations, and also capitalized upon SAR
surrounding ancillary activity against focal adhesion kinase
(FAK). In tumors, FAK activation mediates anchorage-
independent cell survival, one of the hallmarks of cancer cells,
as well as migration, invasion, and angiogenesis, all critical
components in the process of metastatic progression. FAK
overexpression and activation are indicative of multiple solid
tumors, particularly those with a propensity for bone metastasis,
RESULTS AND DISCUSSION
■
Our lead inhibitor 1, as well as other known26−28 ALK
inhibitors, contains a diaminopyrimidine core motif. The
synthetic strategy employed a highly convergent approach
with fragment disconnections centered around the 5-chloro-
2,6-diaminopyrimidine designated as A−B−C shown in Figure
2. The first disconnection at the 2-position affords amines A
and advanced 6-amino-2,5-dichloro fragments B−C. Further
disconnection yields starting 2,5,6-tricholoropyrimidine B and
the amines C. The synthesis of the key amines A and the
elaboration to the final diaminopyrimidines are shown in
Schemes 1−4.
The intermediate aniline 8 (Scheme 1) was derived from the
known ketone 529 through reductive amination with morpho-
Figure 2. Retrosynthetic disconnections for 4-chloro-2,6-diaminopyrimidines.
B
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a
Scheme 1
a
Conditions: (a) morpholine, NaBH(OAc)3, AcOH, THF (99%); (b) KNO3, TFAA, MeCN (19%); (c) H2, Pd/C, EtOH (99%).
a
Scheme 2
a
Conditions: (a) KNO3, TFAA, MeCN (36%); (b) amine, NaBH(OAc)3, AcOH, THF (24−85%); (c) H2, Pd/C, EtOH (61−99%).
a
Scheme 3
a
Conditions: (a) methanesulfonic acid, 1-methoxy-2-propanol, Δ or microwave.
line to provide 6 in near quantitative yield. Nitration provided a
mixture of 2-nitro (7) and 4-nitro isomers (approximately 1:1)
which were separable by silica gel chromatography. Reduction
of the nitro group provided racemic 8.
The 6-amino derivatives (Scheme 2) were synthesized from
known ketone 9.30 A diverse set of substituents were desired;
thus nitration provided 10 and 4-nitro, which were separable on
silica gel. Reductive amination provided amines 11b−d; for
11a, the process was reversed. Reduction of the nitro group
gave 12a−d.
The intermediate 13 (Scheme 3) was independently reacted
with 8 and 12a−d to provide final compounds 14−18 via
methanesulfonic acid promoted addition.
Intermediates 21a−i31 (Scheme 4) were available from
anilines 19a−i via base-promoted addition to 2,4,5-trichlor-
opyrimidine. The final compounds 22−30 were then
synthesized in an analogous fashion to that shown in Scheme 3.
Separation of racemic 12d (Scheme 5) into individual
enantiomers was accomplished using SFC (Chiralcel OJ-H).
The individual enantiomers were then converted using
C
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a
Scheme 4
a
Conditions: (a) K2CO3, DMF; (b) 12a or 12d or (R)-12d or (S)-12d, methanesulfonic acid, 1-methoxy-2-propanol, Δ or microwave.
a
Scheme 5
a
Conditions: (a) Chiralcel OJ-H (3 cm × 15 cm), 15% MeOH, 0.2% diethylamine, CO2 (SFC); (b) p-Br-benzoyl chloride.
procedures outlined in Scheme 4 to 27a,b. The absolute
stereochemistry was determined using X-ray (anomalous
dispersion) of a single crystal of the bis-p-bromobenzoyl
derivative 31, which was available via reaction of the first eluting
isomer with p-bromobenzoyl chloride.32 Thus, the first eluting
enantiomer used in the preparation of 27a was determined to
be the (R)-configuration; by analogy 27b was assigned (S)-
configuration.
microsome incubations provided clarity and direction for this
approach. As shown in Figure 3, the two major locations for
oxidative metabolism proved to be the morpholine ring, which
was oxidized and degraded, and the olefin of the bicyclo[2.2.1]
system which was epoxidized. The degree and location of
metabolism in rat and human liver microsomes were quite
similar and suggested that in vitro and in vivo screening in rats
would be reasonably predictive.
Concurrent with our strategy to advance backup compounds
to the lead candidate by developing novel chemical
architecture, we also focused on fine-tuning 1 by making
minor structural modifications to address both pharmaceutical
limitations and broader, pharmacological aspects. Despite
moderate in vivo clearance values for 1 [CD-1 mouse, Cl =
29 mL min−1 kg−1; rat, Cl = 17 mL min−1 kg−1; cynomolgus
monkey, Cl = 28 mL min−1 kg−1], the in vitro liver microsome
stability values [liver microsome t1/2 (min): CD-1 mouse, 13;
rat, 7; human, 10] suggested room for improvement. Enhanced
metabolic stability would potentially lead to lower in vivo
clearance and thus increase overall exposure and bioavailability,
since permeability and solubility were not limiting factors.
Furthermore, identification of metabolic hotspots from liver
The in vitro potency and metabolic stability results for the
initial series of analogues are shown in Table 1. A clear
deleterious effect on potency was observed when the 6-position
morpholine was moved to the α-position (relative to the
morpholine of 1) on the benzocycloheptane ring (14). In
contrast, when the morpholine was moved to the α′-position,
providing the separable diastereomers 15a and 15b, potency
was within 2- to 4-fold relative to 1. Selectivity for the insulin
receptor (INSR) was maintained, though unfortunately so was
the short t1/2 in liver microsomes. We decided to further pursue
C6-substituted analogs based upon the maintenance of
potency/selectivity relative to 1. A significant improvement in
rat liver microsome stability was gained when the morpholine
was switched to the N-methylpiperazine (t1/2 = 5 min for 15a,b,
D
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Figure 3. Major sites of metabolism from in vitro liver microsome incubation (10 μM concentration for 120 min in the presence of NADPH and
analysis by LC/MS/MS.).
t1/2 = 39−40 min for 16a,b) without eroding the potency or
selectivity. A similar result was observed by “opening” the
morpholine to give methoxyethylamine derivative 17. However,
in each of these cases, the human liver microsome stability was
still unfavorable (t1/2 ≤ 15 min).
changing the N-methyl substituent on the piperazine to a
hydroxyethyl substituent. The resultant analogues 18a,b proved
to be slightly more potent in the enzyme assay and 4- to 5-fold
more potent in cells, and potency was now in a similar range as
1. Other properties remained similar including selectivity
(INSR and kinome), hERG (IC50 > 10 μM), metabolic
stability, and oral bioavailability (10%). As noted above, the
other main metabolic hot spot in 1 proved to be the olefin of
the bicyclo[2.2.1]heptene systems. Previous efforts28 and
literature reports36 had demonstrated that aromatic sulfona-
mides/sulfones as well as N-alkylsulfonamides and other
hydrogen bond acceptor motifs37 were acceptable substituents
on the aromatic group in the 2-position. Toward defining the
favored substituent on the piperazine, another direct com-
parator set was synthesized with the 2-(N-methyl-
methanesulfonamide)phenyl ring in place of the bicyco[2.2.1]-
heptene system (cf. 22 and 23). This set proved equipotent
against enzyme (ALK and FAK) and in ALK cells and
demonstrated similar selectivity against INSR and the kinome.
However, hydroxyethyl derivative 23 demonstrated roughly 3-
fold higher oral exposure (22, AUC0−6h = 1300 ng·h/mL, vs 23,
AUC0−6h = 3959 ng·h/mL). This higher exposure resulted in
about 4-fold enhanced oral bioavailability for 23 relative to 22.
In vitro liver microsome stability for 23 across multiple species,
however, continued to show issues with metabolic liabilities. In
rodent liver microsomes (mouse and rat) 23 was quite stable
(t1/2 = 37 min each), but in higher species (cynomolgus
monkey and human) half-lives were significantly lower (t1/2 = 8
and 13 min, respectively). Metabolite identification studies in
liver microsomes 23 (data not shown) revealed that loss of the
methyl group from the N-methylmethanesulfonamide was the
major product in both human and monkey liver microsomes for
23; this metabolite was minor in rodent liver microsomes.
Upon the basis of these results, we profiled the des-methyl
derivative 24, which proved to be 9-fold less potent against
ALK and outside our desired range (IC50 < 10 nM). Another
tactic to potentially circumvent this metabolism involved tying
The inhibitors 16a,b were put through an expanded in vitro
and in vivo assay regimen to benchmark against 1. Table 2
shows the in vitro and in vivo profiling of the two individual
distereomers. Along with ALK (enzyme, cell assays), kinase
selectivity was assessed against an internal panel (INSR, JAKs 1,
2, 3, PYK2, TYK2, FAK, and cMet)33 as well as an external
panel (1 μM concentration, DiscoveRx).34 In vitro ADME/
toxicology profiling was also completed (hERG, CYP
inhibition, expanded liver microsome stability) as well as iv/
po pharmacokinetics in rats. Relative to compound 1 (ALK
IC50 = 1.9 nM, cell IC50 = 25 nM), 16a,b were about 4-fold less
potent in enzyme and 9- to 10-fold less potent in cells. Overall
kinome selectivity (1 μM screening concentration) was
improved relative to 1 with S(90) < 0.10 for both. Surprisingly,
the modification of moving the heterocycle from the 7-position
to the α-position resulted in introduction of near equivalent
activity against focal adhesion kinase (FAK) compared to the
60-fold separation for ALK over FAK observed for compound
1. The structural basis of this activity will be discussed in the
context of further optimized analogs (vide infra). Importantly,
reasonable hERG selectivity and CYP inhibition profiles were
maintained as well as an improvement in liver microsome
stability in mouse and rat; however, monkey and human
stability was less than desired. Pharmacokinetics in rat35
demonstrated high iv AUCs and low Cl (data not shown)
and reasonable oral AUCs for both compounds and thus low
overall bioavailability (F = 11%).
Upon the basis of these promising results, we focused our
strategy on further improving the potency of these inhibitors
for ALK, maintaining the ancillary FAK potency and improving
upon the human liver microsome metabolic stability. A subtle,
yet noteworthy, SAR point was identified in the series by
E
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Table 1. Effects of Position and Nature of Substituent on Benzocycloheptane Ring System
a
b
IC50 values reported as the average ( SD if ≥3 determinations). LM = liver microsome.
a
Table 2. Expanded Profiling of 16a and 16b
b
c
f
g
h
ALK/cell,
INSR IC50
(nM)
FAK IC50
(nM)
S(90) (1
hERG IC50
(μM)
CYP IC50
LM t1/2 (min),
F (%),
rat
h
compd
IC50 (nM)
μM)
(μM)
M, R, Mo, H
oral AUC (ng·h/mL)
d
1
1.9 0.5/42
14/200
1257
25
5
0.136
>10
9.1
>10
>10
>10
8, 16, 17, 11
40, 40, 9, 15
40, 39, 7, 11
25
11
11
1203 221
4405 1704
5526 682
e
16a
16b
382
8.5
6.5
0.093
e
9
3/200
508 209
0.057
7.9
a
b
IC50 values are reported as the average ( SD if ≥3 determinations). Biochemical cell activity measuring inhibition of NPM-ALK phosphorylation.
c
d
e
f
S(90) = no. kinases with >90% inhibition/total number kinases tested. DiscoveRx 256 kinase panel. DiscoveRx 402 kinase panel. CYP isozymes:
g
h
1A2, 2C9, 2C19, 2D6, 3A4. LM (liver microsome): M = mouse, R = rat, Mo = monkey, H = human. 1 mg iv dose, 5 mg/kg po dose.
the methyl group back onto the sulfonamide to give a cyclic
sultam (25). This proved to be acceptable for potency against
ALK and FAK, as well as yielded acceptable oral bioavailability
in rats (48%). However, metabolic stability was similar to the
acylic variant (t1/2 = 16 min, hLM). Furthermore, hERG activity
for this analog was noted (IC50 = 2.7 μM), potentially as a
result of the increased lipophilicity. The 2-N,N-dimethyl-
sulfonamide derivative (26) and the N-methylamide (27)
demonstrated similar ALK enzyme and cell potency (IC50 = 4
and 40 nM, respectively), and both showed decreased INSR
F
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Table 3. Optimized Analogs at C2 and C7
a
b
IC50 values are the average of at least 2 determinations ( SD if ≥3 determinations). Biochemical cell activity measuring inhibition of NPM-ALK
c
phosphorylation. 1 mg iv dose, 5 mg/kg po dose.
selectivity in the enzymatic assay relative to the N-linked
sulfonamides. Overall kinome selectivity was better for the N-
methylamide 27 (S90 = 0.08 vs 0.16). Analogues 26 and 27
displayed acceptable oral bioavailability (28% and 47%,
respectively), though hERG activity was an issue for 26 (IC50
= 0.24 μM) relative to 27 (IC50 ≥ 10 μM). Importantly, 27
demonstrated acceptable in vitro liver microsome stability
across species (rat and human t1/2 > 40 min). The amide
G
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a
Table 4. Expanded Profiling of Individual Enantiomers 27a,b
ALK
cell
IC50
FAK
cell
IC50
b
b
f
INSR
IC50
INSR
hERG CYPs
IC50 IC50
b
c
g
h
ALK IC50
(nM)
cell IC50 FAK IC50
S(90)
LM (t1/2 min), F (%),
h
compd
(nM)
(nM)
(nM)
(nM)
(nM) (at 1 μM) (μM)
(μM)
M, R, Mo, H
rat
oral AUC0−∞ (ng·h/mL)
d
1
1.9 0.5
5.7
42
50
22
662
81
>10000
30
944
0.136
0.095
0.084
>10
>10
>10
>10
>5.6
>5.6
8, 16, 17, 11
40, 40, 24, 40
40, 40, 21, 40
25
18
42
1203
1196
e
e
(R)-27a
(S)-27b
1.0
3.1 0.5
65
9
2000
2.0 0.2
80
8360
a
b
c
IC50 values are reported as the average ( SD if ≥3 determinations). Biochemical cell activity measuring inhibition of phosphorylation. S(90) =
d
e
f
no. kinases with >90% inhibition/total number kinases tested. DiscoveRx 256 linase panel. DiscoveRx 442 kinase panel. CYP isosymes: 1A2, 2C9,
2C19, 2D6, 3A4. LM (liver microsome): M = mouse, R = rat, Mo = monkey, H = human. 1 mg iv dose and 5 mg/kg, po dose.
g
h
isostere derivatives pyrazole (28) and imidazole (29) provided
similar ALK and FAK potencies and improved INSR selectivity
to that of 27 but suffered from hERG activity for 28 (IC50 = 3.7
μM) and poor metabolic stability for 29 (hLM t1/2 = 18 min).
Both analogues, though orally bioavailable (F = 19% for each),
were inferior to 27. The 2-methoxy derivative 30 was tested but
proved to be significantly less active against ALK (IC50 = 44
nM) (Table 3).
potency within 10-fold of ALK (DiscoveRx Kd = 2.3 nM): FAK
(2×), FLT3(D835Y) (7×), LTK (4×), PLK4 (7×), PYK2
(3×), RSK1 (4×), RSK2 (1×), STK33 (4×), TNK1 (4×).
These data confirmed the activity against FAK and also
demonstrated that 27b displays high selectivity for ALK and
FAK with only RSK2 (kinase domain) having equipotency.
Given the clinically derived mutations that have been
reported for patients undergoing treatment with small-molecule
ALK inhibitors, compound 27b was screened for activity using
the DiscoveRx platform against known ALK activating
mutations. Shown in Table 5 are the activities with DiscoveRx
As noted above, analogues 22−30 were screened as racemic
mixtures to expedite discovery. Upon the basis of the overall
profile of 27, with the dual ALK and FAK activity, improved
metabolic stability and promising oral PK profile, individual
enantiomers 27a and 27b were synthesized from intermediates
(R)- and (S)-12d that were separated using SFC (Chiralcel OJ-
H). Expanded profiling of the two enantiomers is shown in
Table 4. The profiles for 27a and 27b are quite similar across
on-target potency, off-target selectivity, and in vitro ADME/
toxicity parameters. Given that the predicted binding mode of
these inhibitors places the piperazinyl moiety in a solvent
accessible region of the kinase, this similarity is not entirely
surprising. However, what was unexpected were the oral PK
profiles in which the (S)-enantiomer 27b displayed a 2- to 3-
fold improvement in oral biovailability and significantly higher
oral AUC despite similar in vitro metabolic stability values; this
result was also consistent with mouse PK/PD studies (data not
shown). For both enantiomers, Caco-2 permeability was
classified as high (Papp(A→B) > 1.0 × 10−6 cm/s) with similar
efflux ratios (<5) suggesting absorption was not limiting.
Relative to 1, 27b displayed improved potency, additional
activity against the oncogenic kinase FAK (in both enzyme and
cellular formats), and superior kinome selectivity as well as in
vitro/in vivo ADME properties. However, INSR enzyme
selectivity for 27b was only about 20-fold. In a cellular format,
27b displayed an IC50 of roughly 2 μM, representing about a
30-fold shift relative to INSR enzyme. By comparison of cellular
results, 25- to 50-fold margins for the INSR/FAK and INSR/
ALK cell ratios were evident, and we felt that only at very high
exposures would INSR exert a significant pharmacological
influence. The full DiscoveRx kinase panel (442 kinases) and
the Kd values for those kinases displaying >90% inhibition at
the screening concentration of 1 μM is in Supporting
Information. The following kinases in the panel demonstrated
Table 5. DiscoveRx Profiling of 27b against WT ALK and
ALK Mutations
a
target
Kd (nM)
b
ALK (WT)
3.6
ALK(1151Tins)
ALK(C1156Y)
ALK(F1174L)
2.6
1.6
1.1
3.3
ALK(L1196M)
a
b
Average value (n = 2). Data from independent experiment.
ALK (WT) as a benchmark (Kd = 3.6 nM); this result is in
good agreement with our internal isolated enzyme assay (IC50
= 3.1 nM) and that reported from previous independent
experiments at DiscoveRx (Kd = 2.6 nM). Of particular note,
27b displayed similar potencies for the ALK mutants tested,
ALK(1151Tins)38 Kd = 2.6 nM, ALK(C1156Y)39 Kd = 1.6 nM,
ALK(F1174L)40 Kd = 1.1 nM, as well as the gatekeeper
mutation ALK(L1196M)38 Kd = 3.3 nM.
The majority of analogues in Table 4 displayed similar ALK
and FAK potency in contrast to 1. Examination of the structural
basis for the dual ALK/FAK potency via docking into published
crystal structures found a potentially significant set of molecular
interactions to explain this activity. Shown below (Figure 4) are
both 1 and 27b docked (Glide XP) in crystal structures of ALK
(PDB code 3LZT) and FAK (PDB code 3BZ3). By
examination of the ALK structure, an acidic residue in the
solvent exposed region is within reasonable distance to the
basic heterocyclic amine of both 1 (yellow) and 27b (gray) to
pick up an interaction from Glu1210. This type of interaction is
H
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Figure 4. Glide docking of 27b (gray) and 1 (yellow): left panel, ALK (PDB code 3LZT); right panel, FAK (PDB code 3BZ3).
supported with other ALK ligand/protein structures.41 On the
right side of Figure 4 is the docking in a crystal structure of
FAK. Since the FAK activity was highly determinant on the
location of the heterocycle attached to the benzocycloheptane
and appeared to have little activity dependence on the amine
attached to the chloropyrimidine core, we were not surprised to
find an acidic residue, Glu506, in this region with which the
amine of the piperazine of 27b (gray) could potentially form a
favorable interaction. However, the morpholine of 1 (yellow) is
displayed in an alternative trajectory and would not be able to
form this same interaction. Furthermore, in FAK, the residue
corresponding to Glu1210 in ALK is Val513 which offers a
potential explanation as to the decreased activity of 1 in FAK.
There are reports that suggest that the high energetic penalty
due to desolvation could be overcome by free energy binding
gains with such favorable electrostatic interactions.42 In the
absence of other obvious ligand/protein structural features
impacting the observed selectivity, the modeling offers a
potential explanation.
monkeys. It was unclear why there was such a large shift in Vd
between rodents and non-human primates. Oral bioavailability
was acceptable across all three species with CD-1 mouse
roughly 2-fold that of rat and cynomolgus monkey. Overall
plasma exposure was lower in cynomolgus monkey (both iv
and oral) relative to the rodents, ostensibly due to the higher
clearance and very high distribution, since oral bioavailability
was similar to that of rat. Importantly, from tissue distribution
studies in rats at 30 mg/kg oral dose, total brain exposure
relative to plasma was roughly 1.5- to 2-fold [brain Cmax (3160
ng/g) vs plasma Cmax (2116 ng/g); brain AUC0−48h (61 584 ng·
h/mL) vs plasma AUC0−48h (31 539 ng·h/mL)] which is
significant, since one aspect of clinically derived resistance of
NSCLC to crizotinib is manifested in the emergence of brain
metastases.43,44 Compared to 1, a significant improvement in
multiple pharmacokinetic parameters was noted across
species.16 For example, 27b displays lower iv clearance values
in CD-1 mouse (10 mL min−1 kg−1 vs 29 mL min−1 kg−1) and
rat (3 mL min−1 kg−1 versus 17 mL min−1 kg−1) and similar
values to cyno (30 mL min−1 kg−1 vs 28 mL min−1 kg−1).
Furthermore, higher oral (dose normalized) AUC values45
across all three species were observed for 27b vs 1 in mouse
(1643 ng·h/mL vs 299 ng·h/mL), rat (1672 ng·h/mL vs 246
ng·h/mL) and cyno (276 ng·h/mL vs 95 ng·h/mL).
To support the advancement of 27b and position the clinical
program appropriately, it was profiled in a variety of in vitro
assays and in vivo pharmacological models pertaining to the
ALK component (NPM-ALK+ ALCL models, EML4-ALK+
NSCLC cancer models). Furthermore, 27b was evaluated in
FAK-activated in vivo tumor models (both xenograft and
patient derived xenografts46) and demonstrated robust activity.
27b was screened against both NPM-ALK+ ALCL cell lines
(Sup-M2 and Karpas-299) and NPM-ALK− hematologic cell
lines (Hut-102 and K562) and demonstrated selective
cytotoxicity in the oncogene-addicted cell lines (Figure 5).
The calculated cellular IC50 values for cell growth inhibition by
27b were 84 nM (n = 2) in Sup-M2 cells and 131 nM (n = 2)
in Karpas-299 cells. Furthermore, this concentration-dependent
induction of proapoptotic caspases was also observed for the
ALK+ cell lines but was absent in tumor cell lines lacking
oncogenic ALK fusion proteins. These data suggest that this
selectivity is driven through inhibition of ALK kinase activity.
Furthermore, 27b inhibited EML4-ALK tyrosine phosphor-
ylation in two different EML4-ALK+ NSCLC-derived cell lines,
NCI-H2228 and NCI-H3122, with mean IC50 values of 175 nM
(n = 2) and 85 nM (n = 2), respectively. Inhibition of EML4-
In vivo pharmacokinetic parameters for 27b in three species
(CD-1 mouse, Sprague-Dawley rat, and cynomolgus monkey)
are shown in Table 6. Following iv dosing, low clearance was
noted in rodents and moderate to high clearance was seen in
monkey which is consistent with the liver microsome results.
Volume of distribution (Vd) was also significantly higher in
Table 6. Pharmacokinetic Parameters of 27b in CD-1
Mouse, Sprague-Dawley (SD) Rats, and Cynomolgus
Monkeys
CD-1
cynomolgus
b
a
b
PK parameter
mouse
SD rat
monkey
c
iv
dose (mg/kg)
1
1
2
1
t1/2 (h)
3.0
0.4
5.4 0.6
554 11
13.2 1.9
30 0.5
10
AUC0−∞ (ng·h/mL)
Vd (L/kg)
1612
2.7
4005 237
0.8 0.2
CL (mL min−1 kg−1
)
10
4
5
0.2
c
po
dose (mg/kg)
10
Cmax (ng/mL)
tmax (h)
1533
2
1340 107
3.3 0.7
239
6
6
AUC0−∞ (ng·h/mL)
F (%)
16429
102
8360 540
2757 114
50
42
3
3
a
Values are the mean from three animals from each time point.
b
c
Values
SEM are the mean from three animals. Formulated as
solutions: iv, 3% DMSO, 30% Solutol, 67% PBS (mouse, rat), dH2O
(monkey); oi, dH2O (mouse, monkey), PEG400 (rat).
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Figure 5. In vitro cytotoxicity and caspase 3/7 activation results for 27b.
Figure 6. PK/PD of 27b (single 30 mg/kg, po dose) in NPM-ALK+ ALCL tumor xenograft.
ALK phosphorylation in H2228 and NCI-H3122 cells by 27b
resulted in concentration-related cytotoxicity and growth
inhibition, with a mean IC50 values (n = 2) of 2900 nM in
H2228 cells and 1779 nM in H3122 cells. The shift in IC50
values between biochemical inhibition and the cytotoxicity/
growth inhibition was more significant for the EML4-ALK+
NSCLC lines; however, tumor growth inhibition was still
robust (see Figure 7).
Prior PK/PD and efficacy studies in NPM-ALK ALCL
models demonstrated that sustained and significant (>90%)
target inhibition was required in vivo to elicit a robust
antitumor response.28 Furthermore, we were hoping to
improve upon the activity of compound 1, which demonstrated
target inhibition following a single, oral dose of 30 mg/kg out
to 12 h and thus required b.i.d. dosing of 30 mg/kg to achieve
robust responses. Upon the basis of the improvement in
pharmacokinetic parameters in mice, we predicted that
sustained and prolonged plasma levels would be achieved at
this dose. Indeed, a single oral dose (30 mg/kg) with 27b in an
NPM-ALK+ ALCL tumor xenograft (Sup-M2) (Figure 6)
demonstrated significant (>85%) knockdown of NPM-ALK
phosphorylation out to 24 h. Plasma and tumor levels remained
high to this time point.
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Figure 7. In vivo efficacy of 27b on ALK+ ALCL and NSCLC animal xenograft models.
Figure 8. PK/PD of 27b (single 55 mg/kg, po dose) in FAK+ CWR22 tumor xenograft.
Dose-dependent antitumor efficacy of 27b with oral dosing
was confirmed in the NPM-ALK+ ALCL Sup-M2 model
(Figure 7, left panel) with dosing at 3 mg/kg (b.i.d.), 10 mg/kg
(b.i.d), 30 mg/kg (b.i.d and q.d.), and 55 mg/kg (q.d.). As
expected, the once daily 30 mg/kg dose resulted in tumor
regressions during the 12-day dosing paradigm. Furthermore,
the higher 55 mg/kg once daily regimen effected complete
tumor ablation. No significant effect was observed with the 3
and 10 mg/kg b.i.d. regimens. In an EML4-ALK+ NSCLC
H2228 tumor xenograft (Figure 7, right panel), oral dosing at
30 and 55 mg/kg (b.i.d.) and 55 mg/kg (q.d.) demonstrated
significant tumor responses at all doses. On day 12, all animals
were switched to the 55 mg/kg, q.d. dosing regimen and
continued to day 40. The animals were followed out to day 80
with no tumor reemergence.
Upon the basis of the in vitro enzyme and cellular activity
against FAK, 27b was evaluated for single dose PK/PD effects
(Figure 8) in the CWR22 prostate CA xenograft which had
very high expression levels of both total and activated (pFAK)
FAK. Time-dependent inhibition of FAK phosphorylation at a
55 mg/kg oral dose out to 24 h was observed; this PD effect
was comparable but of slightly diminished magnitude to the
pALK inhibition profile in ALK-positive ALCL xenografts over
a similar time course, consistent with the differences in cellular
potency of 27b for ALK versus FAK.
clinically in NSCLC and inflammatory breast cancer, we
decided to explore efficacy in those preclinical models with the
reasonable assumption that 27b would display similar PK/PD
effects as those reported above. Toward this end 27b was
evaluated for biochemical inhibition of phospho-FAK in HCC-
827 cells that have high levels of constitutively activated FAK
(ELM4-ALK negative) and displayed an IC50 = 357 nM which
is reasonably consistent with the IC50 in our screening assay
(IC50 = 82 nM). Thus, in vivo antitumor efficacy in a HCC-827
NSCLC tumor xenograft (Figure 9) was evaluated in SCID
mice at 30 and 55 mg/kg (b.i.d) oral doses of 27b. Dose-related
antitumor efficacy was observed with 60% incidence of partial
tumor regressions at 55 mg/kg (b.i.d.) and significant tumor
growth inhibitory activity at 30 mg/kg (b.i.d.). The dosing
regimens were well-tolerated, with no overt toxicity or weight
loss. 27b does not inhibit EGFR phosphorylation in HCC-827
cells (activated EGF-R due to E746-A750 deletion), which
suggests the activity is related to FAK inhibition.
The tumor PD response observed in the FAK+ CWR22
tumor xenograft and the marked antitumor activity achieved in
the FAK activated HCC-827 NSCLC xenograft led us to
further explore the FAK inhibitory component of 27b in
patient-derived xenograft (PDX) models46 that had a defined
pFAK expression profile. Shown in Figure 10 are the screening
results at a single dose (55 mg/kg, b.i.d.) from two separate
PDX models, CTG-0142 squamous cell carcinoma head and
neck (SCCHN) and CTG-0785 sarcoma model. Relative to
The CWR22 PK/PD study validated the in vivo biochemical
FAK activity of 27b. Since 27b was going to be positioned
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Article
rational approach, liabilities associated with the advanced lead 1
were diminished, including specific metabolic liabilities of both
amino moieties attached to the pyrimidine core ring system.
Furthermore, we identified a small structural change on the
benzocycloheptyl ring that provided consistent, potent FAK
inhibition while maintaining high levels of kinome selectivity.
The structural basis for this potency was identified through
molecular docking experiments. Compound 27b displayed
favorable in vitro ADME properties and acceptable oral
bioavailability in three species. Furthermore, 27b imparts
equipotent in vitro activity against clinically-derived resistance
mutations and favorable brain exposure which may impact
physiologically relevant escape pathways such as brain
metastases. Dose-dependent antitumor efficacy was observed
in multiple animal models of ALK+ and FAK-driven cancers.
This dual FAK and ALK active compound is of unique interest
in light of the recent findings in inflammatory breast cancer
where subsets have ALK or FAK oncogenic components.
Additionally, exploration of the clinical significance of FAK
inhibition in conjunction with the recent findings in
immunotherapy is warranted. 27b is currently undergoing
human clinical trials.48
Figure 9. Activity of 27b in HCC-827 human NSCLC xenograft
(ALK−/FAK+).
EXPERIMENTAL SECTION
■
Unless otherwise indicated, all reagents and solvents were obtained
from commercial sources and used as received. H and 13C NMRs
1
undosed control mice, significant tumor growth inhibitory
activity (p < 0.05) was observed in both PDX models. In the
left-hand panel, over 28 days of dosing at of 55 mg/kg (b.i.d),
tumor stasis and a 75% TGI were observed. In the sarcoma
PDX, a 70% TGI was realized with only 13 days of dosing.
Finally, in regard to the pharmacology of 27b and FAK
inhibition, a potentially attractive therapeutic avenue for the
dual ALK and FAK activity is in inflammatory breast cancer
(IBC), a particularly aggressive subtype of breast cancer
refractory to conventional treatment regimens.25 Elevated levels
of pFAK have also been recently identified in a subset of IBC
(in the absence of ALK activity).47 In vitro testing of 27b
against a panel of FAK-activated IBC cell lines demonstrated
concentration dependent inhibition of cell proliferation as well
as robust in vivo antitumor activity against mammary gland-
implanted xenografts in SCID mice and importantly the
absence of brain metastases relative to control.47
were obtained on a Bruker Avance at 400 and 101 MHz, respectively,
in the solvent indicated with tetramethylsilane as an internal standard.
Analytical HPLC was run using a Zorbax RX-C8, 5 mm × 150 mm
column, eluting with a mixture of acetonitrile and water containing
0.1% trifluoroacetic acid with a gradient of 10−100% over 5 or 20 min,
wavelength of 254 and 290 nm to assess purity of final targets. LC/MS
results were obtained on either of two instruments. LC/MS analysis
was performed on a Waters Aquity Ultra Performance LC with a 2.1
mm × 50 mm Waters Aquity UPLC BEH C18 1.7 μm column. The
target column temperature was 45 °C, with a run time of 2 min, a flow
rate of 0.600 mL/min, and a solvent mixture of 5% (0.1% formic acid/
water):95% (acetonitrile/0.1% formic acid). The mass spectrometry
data were acquired on a Micromass LC-ZQ 2000 quadrupole mass
spectrometer. Alternatively, LCMS analysis was performed on a Bruker
Esquire 200 ion trap. Purity was determined for final compounds using
a combination of the analytical HPLC methods described above. All
final compounds were determined to have ≥95% purity as assessed by
area percent (A%) as noted in the tabular data. Automated column
chromatography (SiO2) was performed on a CombiFlash Companion
(ISCO, Inc.). Reverse phase HPLC was carried out on Gilson GX-281
series HPLC with Phenomenex Gemini-NX, C18 reverse phase 150
mm × 30 mm, 5 μm column, acetonitrile/water gradient, 0.1% TFA
CONCLUSIONS
In summary, we have advanced 27b, a selective inhibitor of
FAK and ALK, as a novel anticancer therapeutic. Following a
■
Figure 10. Activity of 27b in CTG-0977 human SCCHN PDX (ALK−/FAK+) and CTG-0785 sarcoma PDX (ALK−/FAK+).
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modifier gradient elution with automatic fraction collection. Melting
points were taken on a Mel-Temp apparatus and are uncorrected.
General Procedure for Reductive Amination. Ketone (1
equiv) and amine (1.1 equiv) were treated with sodium triacetox-
yborohydride (1.4 equiv) and acetic acid (1 equiv) in THF (10 mL/
mmol substrate) and stirred at room temperature until completion.
The reaction mixture was then concentrated and partitioned between
dichloromethane and saturated sodium bicarbonate solution. The
organic phase was washed with water, dried on MgSO4, and
concentrated. Automated flash chromatography purification (ISCO,
silica cartridge; gradient elution, 0−10% MeOH/CH2Cl2 or 10−100%
ethyl acetate−hexane) afforded amine products.
Hz, 1H), 2.87−2.70 (m, 4H), 2.61−2.47 (m, 3H), 2.30 (br t, J = 10.5
Hz, 1H), 2.15−2.03 (m, 2H), 1.93−1.78 (m, 1H), 1.47−1.32 (m, 1H).
(8) 4-Methoxy-6-morpholino-6,7,8,9-tetrahydro-5H-benzo-
[7]annulen-3-amine. Using the general procedure for hydro-
genation, 4-(4-methoxy-3-nitro-6,7,8,9-tetrahydro-5H-benzocyclohept-
en-6-yl)morpholine (7) (0.44 g, 1.42 mmol) provided 8 (0.39 g, 99%
yield) as a light tan solid which was used without further purification.
LC/MS (ESI+): 277.07 (M + H). 1H NMR (400 MHz, CDCl3) δ 6.67
(d, J = 7.8 Hz, 1H), 6.51 (d, J = 7.8 Hz, 1H), 3.80−3.66 (broad m,
9H), 3.44 (br d, J = 12.9 Hz, 1H), 2.76 (br s, 2H), 2.67−2.52 (m, 4H),
2.44−2.26 (m, 2H), 2.13−1.96 (m, 2H), 1.85−1.72 (m, 1H), 1.42−
1.29 (m, 1H).
General Procedure for Nitration.49 Aromatic substrate (1 equiv)
was treated with trifluoroacetic anhydride (30 equiv) and potassium
nitrate (1 equiv) in acetonitrile (3 mL/mmol) at 0 °C for 3 h. The
reaction mixture was partitioned between sodium bicarbonate solution
and dichloromethane and the aqueous phase extracted with additional
dichloromethane. The organics were dried (Na2SO4) and concen-
trated. Automated flash chromatography purification (ISCO, silica
cartridge; gradient elution, 0−10% MeOH/CH2Cl2 or 10−100% ethyl
acetate−hexane) afforded separated nitro regioisomers.
(10) 1-Methoxy-2-nitro-5,7,8,9-tetrahydrobenzo[7]annulen-
6-one. Using the general procedure for nitration, 1-methoxy-5,7,8,9-
tetrahydrobenzo[7]annulen-6-one (25 g, 0.131 mol) provided 10
1
(10.7 g, 34.6% yield). H NMR (400 MHz, CDCl3) 7.70 (d, J = 8.3
Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 3.92 (s, 3H), 3.80 (s, 2H), 3.13−
3.09 (m, 2H), 2.60 (t, J = 7.0 Hz, 2H), 2.10−2.03 (m, 2H).
(11a) 4-(1-Methoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzo[7]-
annulen-6-yl)morpholine. Using the general procedure for
reductive amination followed by the general procedure for nitration,
1-methoxy-5,7,8,9-tetrahydrobenzo[7]annulen-6-one (9) (0.51 g, 1.95
1
General Procedure for Hydrogenation. Nitro compound (1
equiv), 10% palladium on carbon (50% wet) (0.1 equiv), and ethanol
(20 mL/mmol substrate) were combined and subjected to hydro-
genation at 50 psi in a Parr apparatus until hydrogenation was
complete. The reaction mixture was filtered through Celite.
Evaporation of solvent under reduced pressure provided the amines
which were generally used without further purification.
General Procedure for SNAr Displacement on 2,4,5-
Trichloropyrimidine. 2,4,5-Trichloropyrimidine (1 equiv), 2-pyr-
azol-1-ylphenylamine (1 equiv), N,N-dimethylformamide (10 mL/
mmol substrate), and potassium carbonate (1.3 equiv) were combined
and stirred heated to 75 °C until completion. The mixture was poured
onto ice and stirred until all the ice had melted. The mixture was
filtered on a Buchner and air-dried and the solid used without further
purification or purified by automated flash chromatography
purification (ISCO, silica cartridge; gradient elution, 0−10%
MeOH/CH2Cl2 or 10−100% ethyl acetate−hexane).
General Procedure for SNAr Displacement on 2,5-Dichloro-
N-arylpyrimidin-4-amines. Amine (0.468 mmol), 2,5-dichloro-N-
arylpyrimidin-4-amine (0.312 mmol), 1-methoxy-2-propanol (1 mL/
mmol), and methanesulfonic acid (0.111 mL, 1.71 mmol) were
combined in a sealed tube and heated at 90 °C o/n. The reaction
mixture was diluted with DCM and washed with saturated aq
NaHCO3. The organic layer was dried, filtered, and concentrated.
Purification via reverse phase HPLC (Gilson GX-281 series HPLC
with Phenomenex Gemini-NX, C18 reverse phase 150 mm × 30 mm,
5 μm column, 5−50% acetonitrile/water gradient, 0.1% TFA
modifier). The desired fractions were either lyophilized to give the
desired product as a trifluoroacetic acid salt or free-based (partitioned
between dichloromethane and saturated aq NaHCO3, dried (Na2SO4),
filtered, and concentrated).
(6) 4-(4-Methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-
yl)morpholine. Using the general procedure for reductive amination,
4-methoxy-5,7,8,9-tetrahydrobenzocyclohepten-6-one (5) (2.25 g,
11.8 mmol) and morpholine (1.13 mL, 13.0 mmol) gave 6 as a
white solid (3.09 g, 99% yield). LC/MS (ESI+) m/z 262.0 (M + H)+.
1H NMR (400 MHz, CDCl3) δ 7.06 (t, J = 8.0 Hz, 1H), 6.72 (dd, J =
mmol) provided 11a as a yellow oil (0.18 g, 24%, 2 steps). H NMR
(400 MHz, CDCl3) δ 7.09 (t, J = 7.8 Hz, 1H), 6.81 (d, J = 7.6 Hz,
1H), 6.76 (d, J = 8.1 Hz, 1H), 3.81 (d, J = 1.3 Hz, 3H), 3.75 (t, J = 4.2
Hz, 4H), 3.40 (dd, J = 14.0, 7.2 Hz, 1H), 2.94−2.83 (m, 2H), 2.74−
2.66 (m, 2H), 2.61−2.54 (m, 2H), 2.44−2.36 (m, 1H), 2.35−2.26 (m,
1H), 2.14−2.00 (m, 1H), 1.87−1.75 (m, 1H), 1.38−1.25 (m, 1H).
(11b) 1-(1-Methoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzo[7]-
annulen-6-yl)-4-methylpiperazine. Following the general proce-
dure for reductive amination, 1-methoxy-2-nitro-5,7,8,9-tetrahydro-
benzo[7]annulen-6-one (10) (3.09 g, 13.1 mmol) and 1-methylpiper-
azine (13.1 g, 131 mmol) provided 11b (4.19 g, 99%). 1H NMR (400
MHz, CDCl3) δ 7.60 (d, J = 8.3 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H),
3.86 (s, 3H), 3.31 (br dd, J = 14.7, 7.6 Hz, 1H), 3.03−2.88 (m, 2H),
2.81−2.68 (m, 2H), 2.60 (br dd, J = 10.4, 4.8 Hz, 2H), 2.54−2.37 (m,
6H), 2.32 (s, 3H), 2.19−2.07 (m, 2H), 1.93−1.80 (m, 1H), 1.45−1.25
(m, 1H).
(11c) 1-Methoxy-N-(2-methoxyethyl)-2-nitro-6,7,8,9-tetra-
hydro-5H-benzo[7]annulen-6-amine. Following a general proce-
dure for reductive amination, 1-methoxy-2-nitro-5,7,8,9-tetrahydro-
benzo[7]annulen-6-one (10) (1.85 g, 7.87 mmol) and 2-methox-
yethylamine (5.91 g, 78.7 mmol) were converted to 11c (1.86 g, 80%
1
yield) as a brown oil. H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.3
Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 3.93−3.85 (m, 3H), 3.31 (br dd, J =
14.7, 7.6 Hz, 1H), 3.02−2.88 (m, 2H), 2.78−2.70 (m, 2H), 2.60 (br
dd, J = 10.4, 4.8 Hz, 2H), 2.54−2.38 (m, 4H), 2.32 (s, 4H), 2.17−2.05
(m, 2H), 1.95−1.79 (m, 1H), 1.44−1.26 (m, 1H).
(11d) 2-[4-(1-Methoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzo-
[7]annulen-6-yl)piperazin-1-yl]ethanol. Following the general
procedure for reductive amination, 1-methoxy-2-nitro-5,7,8,9-
tetrahydrobenzo[7]annulen-6-one (10) (15.1 g, 64.2 mmol) and 2-
piperazin-1-ylethanol provided 11d (19 g, 85% yield). LC/MS (ESI+)
1
m/z = 350 (M + H)+; H NMR (400 MHz, CDCl3) 7.56 (d, J = 8.2
Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 3.82 (s, 3H), 3.63−3.06 (m, 2H),
3.29−3.24 (m, 1H), 3.00−2.86 (m, 3H), 2.72−2.67 (m, 2H), 2.60−
2.51 (m, 8H), 2.46−2.37 (m, 2H), 2.12−2.07 (m, 2H), 1.87−1.78
(m,1H), 1.37−1.29 (m, 1H).
(12a) 1-Methoxy-6-morpholino-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-2-amine. Following the general procedure for
hydrogenation, 4-(1-4-(1-methoxy-2-nitro-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-6-yl)morpholine (11a) (1.5 g, 4.9 mmol) gave 12a
11.0, 8.0 Hz, 2H), 3.82 (s, 3H), 3.74 (dt, J = 5.7, 3.7 Hz, 4H), 3.61 (br
d, J = 12.9 Hz, 1H), 2.81−2.66 (m, 4H), 2.56−2.48 (m, 2H), 2.37−
2.29 (m, 1H), 2.27−2.19 (m, 1H), 2.08−1.98 (m, 2H), 1.89−1.76 (m,
1H), 1.48−1.34 (m, 1H).
(7) 4-(4-Methoxy-3-nitro-6,7,8,9-tetrahydro-5H-benzo[7]-
annulen-6-yl)morpholine. Using the general procedure for
nitration, 4-(4-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)-
morpholine (6) (2.00 g, 7.65 mmol) gave 7 (second eluting peak)
isolated as a yellow oil (0.43 g, 19%). LC/MS (ESI+) m/z 307.05 (M +
H)+. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.3 Hz, 1H), 6.95 (d,
J = 8.3 Hz, 1H), 3.89 (s, 3H), 3.78−3.67 (m, 4H), 3.50 (br d, J = 13.4
1
as a light brown solid (0.8 g, 61%). H NMR (400 MHz, CDCl3) δ
6.76 (d, J = 8.1 Hz, 1H), 6.54 (d, J = 8.1 Hz, 1H), 3.76−3.72 (m, 5H),
3.71 (d, J = 1.3 Hz, 3H), 3.23 (br dd, J = 14.5, 7.2 Hz, 1H), 2.92−2.64
(m, 4H), 2.63−2.53 (m, 2H), 2.44−2.30 (m, 2H), 2.16−2.03 (m, 2H),
1.84−1.71 (m, 1H), 1.42−1.29 (m, 1H).
(12b) 1-Methoxy-6-(4-methylpiperazin-1-yl)-6,7,8,9-tetrahy-
dro-5H-benzo[7]annulen-2-amine. Following the general proce-
dure for hydrogenation, 1-(1-methoxy-2-nitro-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-6-yl)-4-methylpiperazine (11b) (1.18 g, 3.68 mmol)
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1
was hydrogenated to give 12b (1.1 g, 99% yield). 1H NMR (400 MHz,
CDCl3) δ 6.76 (d, J = 7.8 Hz, 1H), 6.53 (d, J = 8.1 Hz, 1H), 3.67 (s,
3H), 3.23 (br dd, J = 14.1, 7.1 Hz, 1H), 2.76 (br d, J = 13.6 Hz, 4H),
2.67−2.59 (m, 2H), 2.57−2.46 (m, 3H), 2.45−2.34 (m, 3H), 2.32 (s,
3H), 2.16−2.03 (m, 2H), 1.85−1.72 (m, 2H), 1.42−1.30 (m, 2H).
(12c) 1-Methoxy-N6-(2-methoxyethyl)-6,7,8,9-tetrahydro-
5H-benzo[7]annulene-2,6-diamine. Following the general proce-
dure for hydrogenation, 1-methoxy-N-(2-methoxyethyl)-2-nitro-
6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-amine (11c) (1.86 g, 6.31
mmol) was hydrogenated to give 12c (1.61 g, 97% yield) as a tan solid.
1H NMR (400 MHz, CDCl3) δ 6.83 (d, J = 8.1 Hz, 1H), 6.52 (d, J =
LC/MS (ESI+) m/z 539 (M + H)+; H NMR (CDCl3, 400 MHz) δ
8.21 (d, J = 8 Hz, 1H), 7.90 (s, 1H), 7.42 (s, 1H), 7.03 (d, J = 8 Hz,
1H), 6.93 (d, J = 8 Hz, 1H), 6.39 (m, 1H), 6.34 (m, 1H), 4.37 (t, J = 8
Hz, 1H), 3.73 (m, 7H), 3.23 (m, 1H), 3.08 (s, H), 2.96 (s, 1H), 2.88
(m, 2H), 2.69 (m, 2H), 2.56 (m, 2H), 2.50 (d. J = 8 Hz, 1H), 2.37 (m,
2H), 2.25 (d, J = 9 Hz, 1H), 2.12 (m, 2H), 1.81 (m, 1H), 1.64 (d, J = 9
Hz, 1H), 1.34 (m, 1H). HPLC purity: 97 A%.
(16a and 16b) (1S,2S,3R,4R)-3-[[5-Chloro-2-[[(6S)-1-me-
thoxy-6-(4-methylpiperazin-1-yl)-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]bicyclo-
[2.2.1]hept-5-ene-2-carboxamide and (1S,2S,3R,4R)-3-[[5-
Chloro-2-[[(6S)-1-methoxy-6-(4-methylpiperazin-1-yl)-6,7,8,9-
tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]-
amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide. Following the
general procedure for SNAr displacement on 2,5-dichloro-N-arylpyr-
imidin-4-amine, 12b (3.82 g, 13.2 mmol) and 13 (3.95 g, 13.2 mmol)
gave two separable diastereomers. The stereochemistry at the 6-
position was not assigned. First eluting diasteromer from RP-HPLC,
8.1 Hz, 1H), 3.80 (br t, J = 5.1 Hz, 2H), 3.68 (s, 3H), 3.47 (s, 3H),
3.35 (s, 3H), 3.22 (t, J = 5.2 Hz, 2H), 3.16 (br s, 4H), 2.51 (br t, J =
12.9 Hz, 1H), 2.42 (br d, J = 10.6 Hz, 1H), 2.05 (br d, J = 7.6 Hz, 2H),
1.50−1.36 (m, 1H).
(12d) 2-[4-(2-Amino-1-methoxy-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-6-yl)piperazin-1-yl]ethanol. Following the gen-
eral procedure for hydrogenation, 2-[4-(1-methoxy-2-nitro-6,7,8,9-
tetrahydro-5H-benzo[7]annulen-6-yl)piperazin-1-yl]ethanol (11d)
(19.0 g, 54.4 mmol) gave 12d as a foamy white solid (17.25 g, 99%
16a (0.850 g, 12% yield): LC/MS (ESI+) m/z 552.22 (M + H)+; H
1
NMR (400 MHz, CDCl3) δ 8.20 (d, J = 8.1 Hz, 1H), 7.90 (s, 1H),
7.40 (s, 1H), 6.97 (br d, J = 7.3 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H),
6.41−6.37 (m, 1H), 6.35−6.31 (m, 1H), 5.60 (br s, 1H), 5.35 (br d, J
= 6.1 Hz, 1H), 5.31 (s, 3H), 4.37 (t, J = 7.3 Hz, 1H), 3.72 (s, 3H),
3.28−3.21 (m, 1H), 3.08 (br s, 1H), 2.95 (br s, 1H), 2.89−2.83 (m,
2H), 2.73 (br d, J = 5.1 Hz, 2H), 2.65 (br s, 2H), 2.55−2.39 (m, 5H),
2.32 (s, 3H), 2.25 (d, J = 9.3 Hz, 1H), 2.13 (br d, J = 12.1 Hz, 2H),
1.85−1.71 (m, 1H), 1.68−1.53 (m, 2H), 1.42−1.29 (m, 1H). HPLC
purity 95 A%. Second eluting diastereomer from RP-HPLC, 16b (0.52
g, 7% yield): LC/MS (ESI+) m/z 552.24; 1H NMR (400 MHz,
CDCl3) δ 8.20 (d, J = 8.3 Hz, 1H), 7.90 (s, 1H), 7.41 (s, 1H), 6.99 (br
d, J = 8.1 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.41−6.37 (m, 1H), 6.35−
6.32 (m, 1H), 5.68 (br s, 1H), 5.46 (br s, 1H), 4.40−4.34 (m, 1H),
3.73 (s, 3H), 3.25 (br dd, J = 13.8, 7.2 Hz, 1H), 3.09 (br s, 1H), 2.96
(br s, 1H), 2.90−2.85 (m, 2H), 2.73 (br d, J = 5.3 Hz, 2H), 2.68−2.64
(m, 2H), 2.63 (s, 2H), 2.56−2.37 (m, 5H), 2.32 (s, 3H), 2.26 (d, J =
9.3 Hz, 1H), 2.13 (br dd, J = 10.5, 4.7 Hz, 2H), 1.91−1.72 (m, 2H),
1.65 (br d, J = 9.1 Hz, 1H), 1.43−1.25 (m, 1H). HPLC purity: 96 A%.
(17) (1S,2S,3R,4R)-3-[[5-Chloro-2-[[1-methoxy-6-(2-methox-
yethylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]-
amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-car-
boxamide. Following the general procedure for SNAr displacement
on 2,5-dichloro-N-arylpyrimidin-4-amine, 12c (0.2 g, 0.8 mmol) and
13 (0.3 g, 0.8 mmol) gave 17 as a tan solid (30 mg, 8%). NMR and
HPLC appeared coincident for diastereomers. Mp 98−99 °C; LC/MS
1
yield). LC/MS (ESI+) m/z = 320 (M + H)+. H NMR (400 MHz,
CDCl3) 6.76 (d, J = 7.9 Hz, 1H), 6.53 (d, J = 7.9 Hz, 1H), 3.72 (broad
s, 3H), 3.71 (s, 3H),3.64 (t, J = 5.4 Hz, 2H), 3.26−3.20 (m, 1H),
2.84−2.72 (m, 5H), 2.62−2.56 (m, 8H), 2.42−2.35 (m, 2H), 2.40−
2.37 (m, 1H), 1.81−1.74 (m, 1H), 1.70 (broad s,1H), 1.41−1.33 (m,
1H).
(R)-12d and (S)-12d. An amount of 34 g of racemic 12d was
separated using SFC (supercritical fluid CO2) chromatography using a
Chiralcel OJ-H (3 cm × 15 cm) 808041 column with 15% methanol
(0.2% DEA)/CO2, 100 bar eluent at 80 mL/min flow rate, monitoring
the wavelength of 220 nm with an injection volume of 0.5 mL, 20 mg/
mL ethanol. 16.9 g of the first-enantiomer and 17 g of the second
eluting enantiomer were isolated with a chemical purity of >99% and
an enantiomeric excess (ee) of >99% (measured using a Chiralcel OJ-
H analytical column). NMR and mass spectrometry results were
equivalent to those of the racemic material.
(14) (1S,2S,3R,4R)-3-[[5-Chloro-2-[(4-methoxy-6-morpholi-
no-6,7,8,9-tetrahydro-5H-benzo[7]annulen-3-yl)amino]-
pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxa-
mide. Following the general procedure for SNAr displacement on 2,5-
dichloro-N-arylpyrimidin-4-amine, 8 (150 mg, 0.54 mmol) and
(1S,2S,3R,4R)-3-[(2,5-dichloropyrimidin-4-yl)amino]bicyclo[2.2.1]-
hept-5-ene-2-carboxamide (13) (162 mg, 0.54 mmol) gave 14 as a
light tan solid (155 mg, 53% yield) as an inseparable ∼1:1 mixture of
diastereomers. Mp: 139−148 °C; LC/MS (ESI+) m/z 539.16 (M +
H)+; 1H NMR (CDCl3) 8.17 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.37 (s,
1H), 6.97 (m, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.37 (m, 1H), 6.31 (m,
1H), 5.61 (br, 1H), 5.38 (br s, 1H), 4.36 (m, 1H), 3.76 (s, 3H), 3.74
(br s, 4H), 3.43 (d, J = 13.1 Hz, 1H), 3.07 (s, 1H), 2.94 (s, 1H), 2.72
(m, 4H), 2.58 (m, 2H), 2.49 (m, 2H), 2.33 (m, 1H), 2.23 (d, J = 9.2
Hz, 1H), 2.07 (m, 2H), 1.79 (m, 1H), 1.62 (m, 1H), 1.39 (m, 1H).
HPLC purity: 98 A%.
(15a and 15b) (1S,2S,3R,4R)-3-[[5-Chloro-2-[[(6S)-1-me-
thoxy-6-morpholino-6,7,8,9-tetrahydro-5H-benzo[7]annulen-
2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-
carboxamide and (1S,2S,3R,4R)-3-[[5-Chloro-2-[[(6R)-1-me-
thoxy-6-morpholino-6,7,8,9-tetrahydro-5H-benzo[7]annulen-
2-yl]amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-
carboxamide. Following the general procedure for SNAr displace-
ment on 2,5-dichloro-N-arylpyrimidin-4-amine, 112a (200 mg, 0.70
mmol) and 13 (190 mg, 0.65 mmol) gave 15a (48 mg, 14% yield) as a
mauve solid as the first eluting diastereomer from the RP-HPLC. The
stereochemistry at the 6-position was not assigned. Mp 146−148 °C.
LC/MS (ESI+) m/z 539 (M + 1); 1H NMR (CDCl3, 400 MHz) δ 8.21
(d, J = 8 Hz, 1H), 7.91 (s, 1H), 7.42 (s, 1H), 7.01 (br s, 1H), 6.93 (d, J
= 8 Hz, 1H), 6.40 (m, 1H), 6.34 (m, 1H), 4.37 (t, J = 8 Hz, 1H), 3.74
(m, 7H), 3.23 (m, 1H), 3.09 (s, H), 2.96 (s, 1H), 2.85 (m, 2H), 2.71
(m, 2H), 2.60 (m, 2H), 2.51 (d. J = 8 Hz, 1H), 2.42 (m, 2H), 2.26 (d,
J = 9 Hz, 1H), 2.14 (m, 2H), 1.81 (m, 1H), 1.64 (d, J = 9 Hz, 1H),
1.40 (m, 1H). HPLC purity: 99 A%. The second eluting diastereomer
15b (65 mg, 18% yield) was isolated as a white solid. Mp 148−51 °C.
(ESI+) m/z 527 (M + H)+; H NMR (CDCl3, 400 MHz) δ 8.20 (m,
1
1H), 7.92 (s, 1H), 7.43 (s, 1H), 7.00 (d, J = 8 Hz, 1H), 6.91 (d, J = 8
Hz, 1H), 6.40 (m, 1H), 6.35 (m, 1H), 5.70 (br s, 1H), 5.56 (br s, 1H),
4.37 (m, 1H), 3.73 (s, 3H), 3.52 (m, 2H), 3.37 (s, 3H), 3.10 (m, 2H),
2.96−2.82 (m, 5H), 2.62 (m, 2H), 2.51 (d, J = 8 Hz, 1H), 2.25 (d, J =
8 Hz, 1H), 2.10 (m, 1H), 1.96 (m, 1H), 1.77 (m, 2H), 1.64 (d, J = 8
Hz, 1H), 1.50 (m, 1H). HPLC purity: 95 A%
(18a and 18b) (1S,2S,3R,4R)-3-[[5-Chloro-2-[[(6S)-6-[4-(2-
hydroxyethyl)piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro-
5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]-
bicyclo[2.2.1]hept-5-ene-2-carboxamide and (1S,2S,3R,4R)-3-
[[5-Chloro-2-[[(6R)-6-[4-(2-hydroxyethyl)piperazin-1-yl]-1-me-
thoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]-
pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxa-
mide. Following the general procedure for SNAr displacement on 2,5-
dichloro-N-arylpyrimidin-4-amine, 12c (0.2 g, 0.8 mmol) and 13 (0.2
g, 0.8 mmol) gave a diastereomeric mixture which was not separable
using C18 RP-HPLC but could be separated employing SFC
(Chiralpak AD-H column and 40% ethanol, 0.1% DEA modifier).
The stereochemistry at the 6-position was not assigned. The first
eluting peak 18a (108 mg, 30% yield) was isolated as a tan foam. LC/
1
MS (ESI+) m/z 582.16 (M + H)+. H NMR (400 MHz, CDCl3) δ
8.22 (d, J = 8.1 Hz, 1H), 7.91 (s, 1H), 7.42 (s, 1H), 7.00 (br d, J = 9.9
Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.39 (br d, J = 3.3 Hz, 1H), 6.37−
6.32 (m, 1H), 5.62 (br s, 1H), 5.35 (br s, 1H), 4.42−4.35 (m, 1H),
3.74 (s, 3H), 3.65 (br s, 2H), 3.32−3.22 (m, 1H), 3.09 (br s, 1H), 2.96
(br s, 1H), 2.91−2.84 (m, 2H), 2.74 (br s, 3H), 2.59 (broad m, 8H),
N
DOI: 10.1021/acs.jmedchem.6b00487
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2.52 (br d, J = 9.1 Hz, 1H), 2.48−2.38 (m, 2H), 2.26 (br d, J = 9.9 Hz,
1H), 2.13 (br d, J = 10.6 Hz, 2H), 1.84−1.75 (m, 1H), 1.65 (br d, J =
9.6 Hz, 1H), 1.43−1.33 (m, 1H). HPLC purity: 99 A%. The second
eluting peak 18b (102 mg, 28% yield) was isolated as of a tan foam.
LC/MS (ESI+) m/z 582.15 (M + H)+. 1H NMR (400 MHz, CDCl3) δ
8.23 (d, J = 8.3 Hz, 1H), 7.91 (s, 1H), 7.42 (s, 1H), 7.05−6.98 (m,
1H), 6.94 (d, J = 8.1 Hz, 1H), 6.43−6.38 (m, 1H), 6.35 (br d, J = 2.8
Hz, 1H), 5.61 (br s, 1H), 5.34 (br s, 1H), 4.38 (t, J = 7.5 Hz, 1H), 3.73
(s, 3H), 3.69−3.63 (m, 2H), 3.30−3.22 (m, 1H), 3.09 (br s, 1H), 2.96
(br s, 1H), 2.91−2.82 (m, 2H), 2.79−2.71 (m, 2H), 2.60 (br s, 8H),
2.52 (d, J = 7.8 Hz, 2H), 2.44 (br d, J = 11.6 Hz, 2H), 2.27 (d, J = 9.3
Hz, 1H), 2.11 (m, 2H), 1.87−1.74 (m, 1H), 1.65 (br d, J = 8.3 Hz,
1H), 1.44−1.34 (m, 1H). HPLC purity 95 A%.
amino]pyrimidin-4-yl]amino]phenyl]methanesulfonamide.
Following the general procedure for SNAr displacement on 2,5-
dichloro-N-arylpyrimidin-4-amines, 12d (190 mg, 0.59 mmol) and
21c36 (132 mg, 0.40 mmol) gave 24 (65 mg, 26% yield) as an off-white
1
foam. LC/MS (ESI+) m/z 616.18 (M + H)+; H NMR (400 MHz,
CDCl3) δ 8.13 (s, 1H), 7.71 (s, 2H), 7.57−7.53 (m, 1H), 7.51 (s, 1H),
7.46 (s, 1H), 7.41−7.35 (m, 2H), 6.73 (d, J = 8.3 Hz, 1H), 3.70 (s,
3H), 3.65 (t, J = 5.4 Hz, 2H), 3.51 (s, 1H), 3.26−3.17 (m, 1H), 2.94
(s, 3H), 2.86−2.79 (m, 2H), 2.72 (br d, J = 5.1 Hz, 2H), 2.66−2.53
(m, 8H), 2.45−2.34 (m, 2H), 2.10 (br d, J = 10.9 Hz, 2H), 1.78 (q, J =
11.2 Hz, 1H), 1.40−1.26 (m, 1H). HPLC purity >95 A%.
(25) 2-[4-[2-[[5-Chloro-4-[2-(1,1-dioxo-1,2-thiazolidin-2-yl)-
anilino]pyrimidin-2-yl]amino]-1-methoxy-6,7,8,9-tetrahydro-
5H-benzo[7]annulen-6-yl]piperazin-1-yl]ethanol. Following the
general procedure for SNAr displacement on 2,5-dichloro-N-arylpyr-
imidin-4-amines, 12d (0.18 g, 0.57 mmol) and 21d (0.12 g, 0.33
mmol) gave 25 (73.6 mg, 34% yield) as a white foam. LC/MS (ESI+)
(21d) 2,5-Dichloro-N-[2-(1,1-dioxo-1,2-thiazolidin-2-yl)-
phenyl]pyrimidin-4-amine. Following the general procedure for
SNAr displacement on 2,4,5-trichloropyrimidine, 2-(1,1-dioxo-1,2-
thiazolidin-2-yl)aniline 19d (1.0 g, 5.4 mmol) and 2,4,5-trichloropyr-
imidine 20 (1.2 g, 5.6 mmol) gave 21d (1.15 g, 60% yield) as a yellow
1
m/z 642 (M + H)+; H NMR (400 MHz, CDCl3) δ 8.41−8.35 (m,
1
solid. LC/MS (ESI+) m/z 358.9 (M + H)+. H NMR (400 MHz,
2H), 8.11 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.46 (br s, 3H), 7.27−7.20
(m, 1H), 6.87 (d, J = 8.3 Hz, 1H), 3.76−3.69 (m, 5H), 3.65 (t, J = 5.3
Hz, 2H), 3.46 (t, J = 7.7 Hz, 2H), 3.25 (br dd, J = 14.3, 6.9 Hz, 1H),
2.93−2.80 (m, 2H), 2.74 (br d, J = 5.1 Hz, 2H), 2.67−2.55 (m, 10H),
2.48−2.37 (m, 2H), 2.18−2.07 (m, 2H), 1.87−1.73 (m, 2H), 1.36 (br
d, J = 11.9 Hz, 1H). HPLC purity >95 A%.
DMSO-d6) δ 9.04 (s, 1H), 8.44 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.54
(br d, J = 7.8 Hz, 1H), 7.47 (br t, J = 7.7 Hz, 1H), 7.37−7.28 (m, 1H),
3.71 (t, J = 6.3 Hz, 2H), 3.45 (br t, J = 7.3 Hz, 2H), 2.37 (quin, J = 6.9
Hz, 2H).
(21g) 2,5-Dichloro-N-(2-pyrazol-1-ylphenyl)pyrimidin-4-
amine. Following the general procedure for SNAr displacement on
2,4,5-trichloropyrimidine, 2-pyrazol-1-ylphenylamine 19g (2.00 g, 12.6
mmol) and 2,4,5-trichloropyrimidine 20 (2.30 g, 12.6 mmol) gave 21g
(26) 2-[[5-Chloro-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-1-
methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]-
pyrimidin-4-yl]amino]-N,N-dimethylbenzenesulfonamide. Fol-
lowing the general procedure for SNAr displacement on 2,5-dichloro-
N-arylpyrimidin-4-amines, 12d (92 mg, 0.3 mmol) and 21e29 (0.1 g,
0.3 mmol) gave 26 (76 mg, 24% yield) as a tan solid. Mp 196−197 °C;
LC/MS (ESI+) m/z 630.18 (M + H)+; 1H NMR (400 MHz, CDCl3) δ
9.42 (s, 1H), 8.57 (d, J = 8.3 Hz, 1H), 8.16 (s, 1H), 8.02 (d, J = 8.1
Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.52 (s,
1H), 7.31−7.25 (m, 2H), 6.92−6.87 (m, 1H), 3.74 (s, 3H), 3.69 (br t,
J = 5.2 Hz, 2H), 3.26 (br dd, J = 14.1, 6.8 Hz, 1H), 2.95−2.82 (br s,
4H), 2.76 (s, 6H), 2.75−2.61 (m, 8H), 2.54−2.37 (m, 2H), 2.20−2.10
(m, 2H), 1.82 (br s, 1H), 1.45−1.30 (m, 1H). HPLC purity 98 A%.
(27) 2-[[5-Chloro-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-1-
methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]-
pyrimidin-4-yl]amino]-N-methylbenzamide. Following the gen-
eral procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-
4-amines, 12d (0.13 g, 0.42 mmol) and 21f (0.10 g, 0.30 mmol) gave
27 (39 mg, 20% yield) as a pale yellow foam. LC/MS (ESI+) m/z
580.17 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 11.02 (s, 1H), 8.69
(d, J = 8.9 Hz, 1H), 8.13 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H),7.59−
7.50(m, 2H), 7.41 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 8.1 Hz,
1H), 6.21 (s, 1H), 3.74 (m, 3H), 3.66−3.63 (m, 2H), 3.29−3.23 (m,
1H), 3.06 (d, J = 4.3 Hz, 3H), 2.92−2.72 (m, 5H), 2.66−2.55 (m,
8H), 2.48−2.39 (m, 2H), 2.16−2.10 (m, 2H), 1.87−1.77 (m, 1H),
1.42−1.32 (m, 1H). HPLC purity 99 A%.
(27a) 2-[[5-Chloro-2-[[(6R)-6-[4-(2-hydroxyethyl)piperazin-1-
yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]-
amino]pyrimidin-4-yl]amino]-N-methylbenzamide. Following
the general procedure for SNAr displacement on 2,5-dichloro-N-
arylpyrimidin-4-amines, (R)-12d (91 mg, 0.28 mmol) and 21f (56 mg,
0.19 mmol gave 27a (56 mg, 50% yield). LC/MS (ESI+) m/z 580.1
(M + H)+; 1H NMR (400 MHz, CDCl3) δ 11.02 (s, 1H), 8.69 (d, J =
8.9 Hz, 1H), 8.13 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H),7.59−7.50(m, 2H),
7.41 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.21 (s,
1H), 3.74 (m, 3H), 3.66−3.63 (m, 2H), 3.29−3.23 (m, 1H), 3.06 (d, J
= 4.3 Hz, 3H), 2.92−2.72 (m, 5H), 2.66−2.55 (m, 8H), 2.48−2.39 (m,
2H), 2.16−2.10 (m, 2H), 1.87−1.77 (m, 1H), 1.42−1.32 (m, 1H).
HPLC purity: 95 A%.
1
as a tan solid (1.82 g, 47% yield). LC/MS (ESI+) m/z 306.03. H
NMR (CDCl3, 400 MHz): δ 10.88 (s, 1H), 8.57 (d, J = 8.3 Hz, 1H),
8.19 (s, 1H), 7.85 (dd, J = 1.0, 6.44 Hz, 2H); 7.46 (t, J = 8.3 Hz, 1H),
7.40 (d, J = 8.0 Hz, 1H), 7.28−7.235 (m, 1H), 6.54 (s, 1H).
(21h) 2,5-Dichloro-N-[2-(1-methylimidazol-2-yl)phenyl]-
pyrimidin-4-amine. Following the general procedure for SNAr
displacement on 2,4,5-trichloropyrimidine, 2-(1-methylimidazol-2-
yl)aniline, 19h (220 mg, 1.27 mmol) and 2,4,5-trichloropyrimidine
20 (233 mg, 1.27 mmol) gave 21h (140 mg, 33% yield). LC/MS
1
(ESI+) m/z 320.06 (M + H)+; H NMR (400 MHz, DMSO-d6) δ
11.69−11.37 (broad s, 1H), 8.40 (s, 1H), 8.33 (dd, J = 8.3, 1.0 Hz,
1H), 7.70 (dd, J = 7.8, 1.3 Hz, 1H), 7.52 (ddd, J = 8.5, 7.3, 1.5 Hz,
1H), 7.35 (d, J = 1.3 Hz, 1H), 7.31 (td, J = 7.6, 1.1 Hz, 1H), 7.14 (d, J
= 1.3 Hz, 1H), 3.78 (s, 3H).
(22) N-[2-[[5-Chloro-2-[[1-methoxy-6-(4-methylpiperazin-1-
yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]-
pyrimidin-4-yl]amino]phenyl]-N-methylmethanesulfonamide.
Following the general procedure for SNAr displacement on 2,5-
dichloro-N-arylpyrimidin-4-amines, 12b (63 mg, 0.22 mmol) and
21b24 (75 mg, 0.22 mmol) gave 22 (72 mg, 46% yield) isolated as a
1
tan lyophilate, TFA salt. LC/MS (ESI+) m/z 600.10 (M + H)+; H
NMR (400 MHz, CDCl3) δ 9.42 (s, 1H), 8.58 (d, J = 8.3 Hz, 1H),
8.17 (s, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.64
(br t, J = 8.8 Hz, 1H), 7.51 (s, 1H), 6.89 (d, J = 8.3 Hz, 1H), 4.14 (q, J
= 7.2 Hz, 1H), 3.75 (s, 3H), 3.65 (br s, 2H), 3.31−3.22 (m, 1H),
2.93−2.83 (m, 2H), 2.77 (s, 6H), 2.59 (m, 5H), 2.50−2.36 (m, 2H),
2.19−2.08 (m, 2H), 1.86−1.75 (m, 1H), 1.57 (br s, 3H), 1.43−1.33
(m, 1H), 1.28 (t, J = 7.1 Hz, 1H). HPLC purity >95 A%
(23) N-[2-[[5-Chloro-2-[[6-[4-(2-hydroxyethyl)piperazin-1-
yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]-
amino]pyrimidin-4-yl]amino]phenyl]-N -met hyl-
methanesulfonamide. Following the general procedure for SNAr
displacement on 2,5-dichloro-N-arylpyrimidin-4-amines, 12d (68 mg
0.22 mmol) and 21b (75 mg, 0.22 mmol) gave 23 (106 mg, 65%
yield) as a tan lyophilate, TFA salt. LC/MS (ESI+) m/z 630.16 (M +
1
H)+; H NMR (400 MHz, CDCl3) δ 10.44−10.37 (m, 1H), 9.15 (s,
1H), 8.06−7.99 (m, 1H), 7.97 (s, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.39
(d, J = 1.3 Hz, 3H), 6.81 (d, J = 8.3 Hz, 1H), 4.08−4.02 (m, 3H), 3.83
(br s, 4H), 3.73 (s, 4H), 3.39 (br dd, J = 14.4, 7.1 Hz, 1H), 3.31 (s,
6H), 3.19 (br s, 3H), 3.11−3.02 (m, 1H), 2.99 (s, 3H), 2.45−2.36 (m,
2H), 2.34−2.24 (m, 1H), 1.97−1.82 (m, 1H), 1.51−1.34 (m, 1H).
HPLC purity 99 A%
(27b) 2-[[5-Chloro-2-[[(6S)-6-[4-(2-hydroxyethyl)piperazin-1-
yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]-
amino]pyrimidin-4-yl]amino]-N-methylbenzamide. To a sealed
vessel (S)-12d (2.69 g, 8.41 mmol) and 21f (2.00 g, 6.73 mmol) were
combined in 1-methoxy-2-propanol (120 mL) followed by the
addition of methanesulfonic acid (2.44 mL, 37.7 mmol). The reaction
was then heated at 90 °C for 18 h. The reaction mixture was added to
a separatory funnel and diluted with sat. aq NaHCO3 until a cloudy
(24) N-[2-[[5-Chloro-2-[[6-[4-(2-hydroxyethyl)piperazin-1-
yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]-
O
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
precipitate formed. This was extracted 3× with dichloromethane. The
organic layer was then washed with brine, dried over MgSO4, filtered,
and concentrated. The residue was pumped dry and then purified by
automated flash chromatography (ISCO, gradient elution 0−10%
dichloromethane/10% NH4OH in MeOH). The desired product
eluted around 9−10%, and the 10% gradient was held until product
eluted completely. Mixed fractions were concentrated and were
purified by RP-HPLC (gradient elution, 0−40% CH3CN/water).
Chromatography was repeated using normal phase silica and reverse
phase HPLC to effect further purification as desired. Following
neutralization and concentration of all the material, the resulting solid
was obtained by taking the foam up into EtOAc and concentrating to
dryness several times to give 27b (1.1 g, 28%) as an off-white foamy
washed with water and brine, filtered, and concentrated. The solid was
recrystallized from methanol to afford 31 (404 mg, 80% yield). Several
of the crystals were suitable for X-ray. LC/MS (ESI+) m/z 686 (M +
1
H)+; H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 8.19 (br d, J = 8.1
Hz, 1H), 7.92 (br d, J = 7.3 Hz, 2H), 7.77 (br d, J = 7.3 Hz, 2H), 7.66
(br d, J = 7.8 Hz, 2H), 7.61 (br d, J = 8.1 Hz, 2H), 7.00 (br d, J = 7.8
Hz, 1H), 4.48 (br t, J = 5.3 Hz, 2H), 3.75 (s, 3H), 3.21 (br dd, J =
14.7, 6.1 Hz, 1H), 2.97−2.70 (m, 6H), 2.64 (br s, 6H), 2.51−2.37 (m,
2H), 2.12 (br d, J = 11.4 Hz, 2H), 1.88−1.75 (m, 1H), 1.37 (br d, J =
10.6 Hz, 1H).
In Vitro and in Vivo Screening Assays and in Silico
Modeling. Experimental details regarding enzymatic, cellular,
ADME screening assays, and in silico modeling have been reported
previously; see refs 16−20, 28, 33, 46 and references therein. All in
vivo experiments were conducted in accordance with Teva IUCUC
protocols.
1
solid. LC/MS (ESI+) m/z = 580.0 (M + H)+; H NMR (400 MHz,
CDCl3) δ 11.02 (s, 1H), 8.69 (d, J = 8.9 Hz, 1H), 8.13 (s, 1H), 8.08
(d, J = 8.4 Hz, 1H), 7.59−7.50 (m, 2H), 7.41 (s, 1H), 7.13 (t, J = 7.4
Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.21 (s, 1H), 3.74 (m, 3H), 3.66−
3.63 (m, 2H), 3.29−3.23 (m, 1H), 3.06 (d, J = 4.3 Hz, 3H), 2.92−2.72
(m, 5H), 2.66−2.55 (m, 8H), 2.48−2.39 (m, 2H), 2.16−2.10 (m, 2H),
1.87−1.77 (m, 1H), 1.42−1.32 (m, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 168.9, 158.5, 155.0, 154.7, 149.0, 139.3, 137.3, 135.6,
131.4, 130.3, 127.9, 124.4, 121.8, 121.3, 120.5, 104.8, 63.1, 61.0, 60.4,
58.5, 53.8, 47.7, 38.1, 33.9, 26.4, 26.3, 25.7, 25.5. High resolution mass
spectrum m/z 580.2807 [(M + H)+ calcd for C30H39ClN7O3
580.2803]. HPLC purity: 99 A%.
Representative Procedures for Measuring FAK Activity in Vitro and in
Vivo. the human prostate carcinoma cell lines, CWR22 and human
non-small-cell lung cancer cell line HCC-827 were obtained from
American Tissue Culture Collection (ATCC, Manassas, VA) and were
cultured in RPMI medium supplemented with 10% fetal bovine serum
(FBS). The rabbit phospho-FAK(Tyr397) (catalog no. 3283) and
FAK antibodies (catalog no. 3285) were purchased from Cell Signaling
Technology (Beverly, MA). Immunoblot analyses of phospho-FAK
and total FAK were carried out according to the protocols provided by
the antibody suppliers (Cell Signaling Technology). In brief, 2 mL of
cells (at a density of 0.75 × 106 cells/mL in RPMI medium containing
10% FBS (regular culture medium) or 75% murine or human plasma
diluted in regular culture medium were seeded in each well of 6-well
plates and an amount of 2 mL of test compound diluted in culture
medium at 2× indicated final concentrations was added to each well.
After incubation for 2−2.5 h, the cells were collected into 15 mL
centrifuge tubes. The cells were spun down by centrifugation at 233g
for 5 min, the medium was aspirated, the cells were resuspended in 1
mL of PBS, transferred to 1.5 mL microcentrifuge, and centrifuged at
10 621g for 2 min at 40 °C. The cells were then lysed in 150 μL of
Frak lysis buffer [10 mM Tris, pH 7.5, 1% Triton X-100, 50 mM
sodium chloride, 20 mM sodium fluoride, 2 mM sodium
pyrophosphate, 0.1% BSA, plus freshly prepared 1 mM activated
sodium vanadate, 1 mM DTT, and 1 mM PMSF and the protease
inhibitor cocktail III (1:100 dilution)] on ice for 10 min. After brief
sonication, lysates were cleared by centrifugation at 20 817g for 10 min
at 4 °C. The supernatants (90 μL) were transferred to fresh 1.5 mL
microcentrifuge tubes containing 30 μL of 4× LDS sample buffer. The
samples were heat-inactivated at 90 °C for 5 min; 20 μL of each
sample was resolved by NuPAGE 7% Tris-acetate gels at 150 V until
the dye front was out of the gels. The gels were transferred to
nitrocellulose membranes for 2 h at 30 V constant using a wet XCell II
blot module. The membranes were blocked in Tris-buffered saline
(TBS) containing 0.2% Tween-20 (TBST) and 3% Nestle Carnation
nonfat milk (Nestle USA Inc., Solon, OH) at room temperature (rt)
for 1 h. The membranes were incubated with antiphospho-FAK
(Tyr397) antibody (diluted 1:1000 in TBST containing 3% bovine
serum albumin) for 1.5 h at rt or overnight at 4 °C while rocking
gently. After washing 3 times with TBST for 10 min each time, the
membranes were incubated with goat-anti-rabbit antibody conjugated
with horseradish peroxidase (HRP) diluted in TBST containing 3%
nonfat milk for 1 h at rt while rocking gently. After washing 3 times
with TBST for 10 min each time and one time with TBS for 5 min, the
membranes were incubated with 5 mL of ECL-Western blotting
detection reagents for 5 min and exposed to Kodak chemilumines-
cence BioMax films for visualization. The membranes were then
stripped by incubating with stripping buffer (62.5 mM Tris HCl, pH
6.8, 2% SDS, and 100 mM 2-mercaptoethanol) for 30 min at 56 °C
and reblotted with anti-FAK antibody similarly as described above for
phospho-FAK antibody.
(28) 2-[4-[2-[[5-Chloro-4-(2-pyrazol-1-ylanilino)pyrimidin-2-
yl]amino]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-
6-yl]piperazin-1-yl]ethanol. Following the general procedure for
SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amines, 12d
(0.19 g, 0.59 mmol) and 21g (0.12 g, 0.40 mmol) gave 28 (81 mg,
1
35% yield) as a white foam. LC/MS (ESI+) m/z 589.1 (M + H)+; H
NMR (400 MHz, CDCl3) δ 10.19 (s, 1H), 8.53 (d, J = 8.1 Hz, 1H),
8.10−8.05 (m, 2H), 7.84 (dd, J = 17.2, 1.5 Hz, 2H), 7.47−7.38 (m,
3H), 7.27−7.21 (m, 1H), 6.90 (d, J = 8.1 Hz, 1H), 6.53 (s, 1H), 3.72
(s, 3H), 3.65 (t, J = 5.3 Hz, 2H), 3.25 (br dd, J = 14.1, 7.1 Hz, 1H),
2.95−2.82 (m, 2H), 2.75 (br d, J = 5.3 Hz, 2H), 2.69−2.54 (m, 8H),
2.49−2.37 (m, 2H), 2.12 (br d, J = 11.1 Hz, 2H), 1.87−1.75 (m, 1H),
1.44−1.32 (m, 1H). HPLC purity >95 A%.
(29) 2-[4-[2-[[5-Chloro-4-[2-(1-methylimidazol-2-yl)anilino]-
pyrimidin-2-yl]amino]-1-methoxy-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-6-yl]piperazin-1-yl]ethanol. Following the gen-
eral procedure for SNAr displacement on 2,5-dichloro-N-arylpyrimidin-
4-amines, 12d (0.15 g, 0.49) and 21h (0.10 g, 0.31 mmol) gave 29 (52
mg, 26% yield) as white crystals. Mp 95−96 °C. LC/MS (ESI+) m/z
603 (M + H)+; 1H NMR (400 MHz, CDCl3) δ 10.70 (s, 1H), 8.60 (d,
J = 8.3 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 8.06 (s, 1H), 7.51−7.41 (m,
3H), 7.25 (s, 1H), 7.24−7.19 (m, 1H), 7.02 (s, 1H), 6.92 (d, J = 8.3
Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.65 (t, J = 5.3 Hz, 2H), 3.50 (q, J
= 7.0 Hz, 1H), 3.25 (br dd, J = 14.1, 6.8 Hz, 1H), 2.94−2.70 (m, 4H),
2.67−2.55 (m, 8H), 2.49−2.37 (m, 2H), 2.17−2.08 (m, 2H), 1.82−
1.79 (m, 1H), 1.37 (q, J = 11.8 Hz, 1H). HPLC purity >95 A%.
(30) 2-[4-[2-[[5-Chloro-4-(2-methoxyanilino)pyrimidin-2-yl]-
amino]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-
yl]piperazin-1-yl]ethanol. Following the general procedure for
SNAr displacement on 2,5-dichloro-N-arylpyrimidin-4-amines, 12d
(0.14 g, 0.45 mmol) and 21i (0.08 g, 0.30 mmol) gave 30 (74 mg. 45%
1
yield) as a yellow solid. LC/MS (ESI+) m/z 553.17 (M + H)+; H
NMR (400 MHz, CDCl3) δ 8.47 (br d, J = 8.1 Hz, 1H), 8.10−8.06
(m, 2H), 7.82 (s, 1H), 7.43 (s, 1H), 7.16−7.07 (m, 1H), 7.06−6.99
(m, 1H), 6.95 (br dd, J = 14.0, 8.2 Hz, 2H), 3.96 (s, 3H), 3.74 (s, 3H),
3.65 (br t, J = 4.7 Hz, 2H), 3.26 (br dd, J = 14.0, 6.4 Hz, 1H), 2.97−
2.80 (m, 3H), 2.75 (br d, J = 4.5 Hz, 2H), 2.68−2.54 (m, 8H), 2.49−
2.37 (m, 2H), 2.12 (br d, J = 11.1 Hz, 2H), 1.81 (q, J = 11.6 Hz, 1H),
1.38 (br d, J = 11.6 Hz, 1H). HPLC purity 90 A%.
(31) 2-[4-[(6R)-2-[(4-Bromobenzoyl)amino]-1-methoxy-
6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl]piperazin-1-yl]-
ethyl 4-Bromobenzoate. 12d (0.38 g, 0.75 mmol) was treated with
4-bromobenzoyl chloride, (0.20 g, 0.90 mmol), triethylamine (0.16
mL, 1.13 mmol) in dichloromethane (40 mL) at 0 °C, warmed to
room temperature, and stirred overnight. The mixture was partitioned
between sat. aq NaHCO3 and dichloromethane. The organics were
Generation of Subcutaneous Human Tumor Xenografts in SCID/Beige
or Nu/Nu Mice Female. SCID/Beige (6−8 weeks, Taconic, Hudson,
NY) or Nu/Nu mice (6−8 weeks, Charles River Laboratory,
Wilmington, MA) were maintained 5/cage in microisolator units on
P
DOI: 10.1021/acs.jmedchem.6b00487
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a standard laboratory diet (Teklad Labchow, Harlan Teklad, Madison,
WI). Animals were housed under humidity- and temperature-
controlled conditions and the light/dark cycle was set at 12 h
intervals. Mice were quarantined at least 1 week prior to experimental
manipulation. Briefly, the cells were collected and resuspended in
RPMI medium at a density of 5 × 107/mL, and an aliquot (100 μL) of
the cell suspension (5 × 106 cells) was inoculated subcutaneously to
the left flank of each mouse with a 23 G needle. The mice were then
monitored daily. When the tumor xenograft volumes reached
approximately 300−500 mm3, the mice received a single oral
administration of either PEG-400 vehicle or test compound at100
μL/dose. At indicated time point(s) after dosing, the mice (3−4 mice
at each time point) were sacrificed and the blood was collected in 1.5
mL microcentrifuge tubes containing 20 μL of heparin sodium (10 000
unit/mL in H2O) and left on ice briefly. The tubes were centrifuged at
20 817g for 8 min at 4 °C, and the plasma was collected and
transferred to 1.5 mL microcentrifuge tubes, which were then stored at
−80 °C. The tumors were excised and weighed, cut into small pieces
with a scalpel, and placed into a round-bottom 14 mL tube on ice.
Two volumes of completed FRAK lysis buffer without detergent [10
mM Tris, pH 7.5, 50 mM sodium chloride, 20 mM sodium fluoride, 2
mM sodium pyrophosphate, 0.1% BSA, plus freshly prepared 1 mM
activated sodium vanadate, 4 mM DTT, 1 mM PMSF, and the
protease inhibitor cocktail III (1:100 dilution)] were added to 1
volume of tumor (e.g., an amount of 500 μL of FRAK lysis buffer was
added to 250 mg of tissue). The tissues were then disrupted with a
hand-held tissue blender 2−3 times, 10−15 s each time with 1−2 min
interval. The lysates were then sonicated twice, 4−5 strokes each time.
The tissue lysates were transferred to 1.5 mL microcentrifuge tubes
and centrifuged at 20 817g for 10 min at 4 °C. The supernatants (12
μL) were transferred to 1.5 mL microcentrifuge tubes containing 108
μL of FRAK lysis buffer and 40 μL of 4× LDS sample buffer with
freshly added 100 nM dithiotreitol. The samples were subjected for
phospho-FAK and total FAK detection with immunoblotting as
described above. The remaining tumor lysates were stored at −80 °C.
The compound levels in both plasma and tumor lysates were measured
by LC−MS/MS.
Antitumor Activity Study with Test Compound with Oral Admin-
istration. The tumor-bearing mice were randomized into different
treatment groups (8−10 mice/group) and were administered orally
with either vehicle (PEG-400) or test compound formulated in PEG-
400 at indicated doses (expressed as mg/kg equivalents of free base)
and with indicated dosing frequency, with 100 μL per dosing volume.
The length (L) and width (W) of each tumor was measured with a
vernier caliper, and the mouse body weight was determined every 2−3
days. The tumor volumes were then calculated with the formula
0.5236LW(L + W)/2. Statistical analyses of tumor volumes and mouse
body weight were carried out using the Mann−Whitney rank sum test.
The TGI values were calculated at the end of study by comparing the
tumor volumes (TV) of each treatment group with those of vehicle-
treated group with the following formula: [1 − (the last day TV of
compound-treated group/the last day TV of vehicle treated group)] ×
100.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mark Olsen and Emily Kordwitz (SFC separations),
Damaris Rolon-Steele (PK), Kelly Ziegler (PK), Maxime
Siegler (Johns Hopkins University, X-ray) for their assistance.
ABBREVIATIONS USED
■
ALCL, anaplastic large-cell lymphoma; ALK, anaplastic
lymphoma kinase; c-MET, mesenchymal epithelial transition
factor; CYP, cytochrome P450; EGFR, epidermal growth factor
receptor; EML4, echinoderm microtubule-associated protein-
like 4; EML4-ALK, echinoderm microtubule-associated pro-
tein-like 4-anaplastic lymphoma kinase; FAK, focal adhesion
kinase; hERG, human ether-a-go-go-related gene; IBC,
inflammatory breast cancer; INSR, insulin receptor kinase;
JAK, Janus kinase; KRas, Kirsten rat sarcoma virus; LM, liver
microsome; NPM, nucleophosmin; NSCLC, non-small-cell
lung cancer; PDX, patient-derived xenograft; PYK, pyruvate
kinase; TYK, tyrosine kinase; SAR, structure−activity relation-
ship
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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.6b00487.
Experimental conditions for X-ray data collection and
solution, PK for compound 1, DiscoveRx kinase
selectivity data for 27b (PDF)
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of Anaplastic Lymphoma Kinase in Lung Cancer: A Paradigm for
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9781118468791.ch35.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: 1-610-738-6861. E-mail: gregory.ott@tevapharm.
com.
Q
DOI: 10.1021/acs.jmedchem.6b00487
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Journal of Medicinal Chemistry
Article
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