Design, synthesis, and anaplastic lymphoma kinase (ALK) inhibitory activity for a novel series of 2,4,8,22-tetraazatetracyclo[14.3.1.13,7.1 9,13]docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene macrocycles

Article abstract of DOI:10.1021/jm201333e

A novel set of 2,4,8,22-tetraazatetracyclo[14.3.1.1^3,7.1 ^9,13]docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene macrocycles were prepared as potential anaplastic lymphoma kinase (ALK) inhibitors, designed to rigidly lock an energy-minimized bioactive conformation of the diaminopyrimidine (DAP) scaffold, a well-documented kinase platform. From 13 analogues prepared, macrocycle 2m showed the most promising in vitro ALK enzymatic (IC₅₀ = 0.5 nM) and cellular (IC₅₀ = 10 nM) activities. In addition, macrocycle 2m exhibited a favorable kinase selectivity preference for inhibition of ALK relative to the highly homologous insulin receptor (IR) kinase (IR/ALK ratio of 173). The inclusive in vitro biological results for this set of macrocycles validate this scaffold as a viable kinase template and further corroborate recent DAP/ALK solid state studies indicating that the inverted "U" shaped conformation of the acyclic DAPs is a preferred bioactive conformation.

Full text of DOI:10.1021/jm201333e

Article
pubs.acs.org/jmc
Design, Synthesis, and Anaplastic Lymphoma Kinase (ALK) Inhibitory
Activity for a Novel Series of 2,4,8,22-Tetraazatetracyclo[14.3.1.13,7.19,13]-
docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene Macrocycles
Henry J. Breslin,* Brandon M. Lane, Gregory R. Ott, Arup K. Ghose, Thelma S. Angeles, Mark S. Albom,
Mangeng Cheng, Weihua Wan, R. Curtis Haltiwanger, Kevin J. Wells-Knecht, and Bruce D. Dorsey
Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380-4245, United States
ABSTRACT: A novel set of 2,4,8,22-tetraazatetracyclo-
[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9(21),10,12,16,18-
nonaene macrocycles were prepared as potential anaplastic
lymphoma kinase (ALK) inhibitors, designed to rigidly lock an
energy-minimized bioactive conformation of the diaminopy-
rimidine (DAP) scaffold, a well-documented kinase platform.
From 13 analogues prepared, macrocycle 2m showed the most
promising in vitro ALK enzymatic (IC50 = 0.5 nM) and cellular
(IC50 = 10 nM) activities. In addition, macrocycle 2m
exhibited a favorable kinase selectivity preference for inhibition
of ALK relative to the highly homologous insulin receptor (IR) kinase (IR/ALK ratio of 173). The inclusive in vitro biological
results for this set of macrocycles validate this scaffold as a viable kinase template and further corroborate recent DAP/ALK solid
state studies indicating that the inverted “U” shaped conformation of the acyclic DAPs is a preferred bioactive conformation.
myofibroblastic tumors (tropomyosin 3-anaplastic lymphoma
kinase, TPM3-ALK).8 Promisingly, a number of ATP-competitive
ALK inhibitors have demonstrated oncolytic activity against ALK-
positive cancer cells in vitro and in vivo. This has led to several
ALK inhibitors advancing to the level of clinical studies, including
crizotinib9 (Figure 1), which recently gained FDA approval for
ALK-positive cancers.
INTRODUCTION
■
Cancer, a group of diseases characterized by uncontrolled
growth and spread of abnormal cells, was the second leading
cause of deaths in the U.S. in 2010, accounting for one of every
four mortalities, as reported by the American Cancer Society.1
In recent years progress has been made in understanding the
biomolecular etiologies contributing to certain cancers,
resulting in successful targeted treatments for some of these
better characterized cancers.2 One such vein of targeted cancer
treatment is inhibition of various aberrant up-regulated and
unregulated kinases, which have been shown to be intimately
tied to certain cancers.3 Kinases are a group of over 500 different
proteins that are typically responsible for strategic internal
regulation of normal cellular signaling pathways, via phosphory-
lation of key cellular substrates.4 Kinases are subgrouped, depen-
dent on which amino acid of the key cellular substrate is phos-
phorylated, via catalytic transfer of a phosphate group from
adenosine triphosphate (ATP). One major subgroup of the kinase
family is the receptor tyrosine kinases. One particular tyrosine
kinase that has gained increasing attention as a therapeutic target is
analplastic lymphoma kinase (ALK), a member of the insulin
receptor (IR) superfamily.5 The chimeric protein nucleophosmin-
anaplastic lymphoma kinase (NPM-ALK) has been identified in
about 60−70% of cases of anaplastic large cell lymphoma (ALCL),
a non-Hodgkin’s lymphoma cancer arising most often in children
and young adults, with constitutively active ALK kinase activity
directly associated with the pathogenesis of disease.6 Other cancers
that have been linked with translocated ALK proteins that have led
to oncogenic signaling include non-small-cell lung cancer
(NSCLC) (echinoderm microtubule-associated protein-like 4-
anaplastic lymphoma kinase, EML4-ALK)7 and inflammatory
Figure 1.
2,4-Diaminopyrimidines (DAPs) have exhibited potent
enzymatic inhibition of ALK and have also shown significant
antiproliferative activity for overexpressed ALK-positive cancer
cells both in vitro and in vivo.10 Compound 1a, a representative
DAP ALK inhibitor, was evaluated via small-molecule X-ray
crystallography and found to possess an inverted “U” shaped
conformation for the three core aryl groups, as depicted in
Figure 2. We questioned if locking appropriately substituted
Received: October 6, 2011
Published: December 15, 2011
© 2011 American Chemical Society
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DAPs in this particular conformation (Figure 3) would yield
macrocycles with enhanced ALK inhibitory activities and/or
Figure 4. Calculated minimum energy conformation of 1c.
minimum energy conformations at 1.29 Å (Figure 5). These
encouraging modeling results added confidence that the proposed
macrocycles 2 would show promise as ALK inhibitors. Subsequent
to these modeling experiments, solid state crystal structures of apo
and ligand bound structures (including DAP bound) of ALK were
reported in the literature and showed similar conformational
arrangement of the DAP’s three aryl groups.13
Figure 2.
Scheme 1 comprehensively outlines the routes used to
prepare macrocycles 2a−m. A minimally substituted macro-
cycle (2a) was prepared to validate the synthetic sequence. 3-
Bromoaniline (3a) was reacted with 2,4,5-trichloropyrimidine
(4) to yield intermediate 5a, which was coupled with 3-
vinylaniline (6a) to give acyclic DAP 7a. This set the stage for
macrocyclization by employing the Heck reaction between the
vinyl (W) and bromo (Y) coupling partners, which yielded 8a
in 65% yield using microwave heating (120 °C for 30 min).
Pt-catalyzed hydrogenation of 8a gave the desired macrocycle
2a. In tandem with the reduction of the olefin, some dechlori-
nation of the substituted pyrimidine was observed, but this side
product was readily separated via chromatography. Note-
worthy, during the course of our work, an alternative synthesis
of the minimally substituted macrocycle 8a was reported in the
patent literature, via a ring closing olefin metathesis reaction,
albeit in a modest 9% yield.14
Encouraged by our preparation of core structure 2a, we
embarked on synthesizing macrocycles 2b−m (Table 1),
decorating the core macrocycle 2a with key appendages that
have been shown to enhance ALK activity. These synthetic
efforts were initially focused on introducing varied R groups
on 2a. Prior to the synthesis of R group substituted
macrocycles 2b−d, substituted anilines 6c and 6d were
prepared as outlined in Scheme 2. Intermediate 5b was
generated in a similar manner as 5a. Then anilines 6b−d were
reacted with 5b to form 7b−d under identical reaction conditions
used to construct DAP 7a. Comparing the cyclization step for
macrocycles 8b−d relative to macrocycle 8a demonstrated that
the Heck macrocyclization reaction proceeded equally well
regardless of whether the vinyl and bromide coupling partners
were interchanged as W or Y (Scheme 1). Final saturated targets
2b−d were generated cleanly by the selective diimide olefin
reductions of 8b−d. The diimide reduction circumvented
potential dechlorination issues, as had been observed in the
Pt catalyzed reduction of 2a. As the related chloro moiety in
acyclic DAPs has proven biologically favorable, we preferred to
maintain the 6-Cl substituent as a constant on key analogues 2;
therefore, the diimide reduction was employed for all subsequent
analogues.
Figure 3.
kinase selectivity profiles. A few recent overviews of varied
macrocyclic efforts directed at a panorama of biomolecular
targets share some other potential advantages, as well as
hurdles, that have been established for this type of strategic
endeavor.11 Prior to initiating our efforts, a literature search
revealed that a Janssen group had reported preparing DAP
macrocycles.12 However, their macrocycles had extended
linkers presenting flexible conformations that were not
necessarily focused specifically toward ALK. Following are
our experimental results appraising rigidly constrained, suitably
functionalized DAP macrocycles that were designed purposely
as ALK inhibitors.
RESULTS
■
To initially assess that locking the conformation of the DAPs
proposed in Figure 3 might prove biologically favorable, we
evaluated the potential overlap of an energy minimized
macrocyclic motif relative to its respective energy minimized
acyclic parent DAP via molecular modeling. Analogue 1c was
chosen as a preferable acyclic DAP for the modeling study
because it was a minimally substituted DAP that possessed
favorable ALK activity, as identified through routine screening.
Consistent with the X-ray conformation for the aryl groups of
DAP 1a (Figure 2), the computational evaluation of the energy
minimized structure of DAP 1c displayed the three aryl groups
aligned in a similar inverted “U” shaped conformation (Figure 4).
Tying the pendent phenyl groups of 1c with an ethylene linker
yielded macrocycle 2g, whose energy minimized conformation
overlapped nicely with DAP 1c, with the root-mean-square
deviation of all the common non-hydrogen atoms in their
Initial R″ substituted analogue 2e was prepared following the
general conditions used to generate 2a. It was envisioned that
the R,R″ disubstituted analogues 2f and 2g would be syn-
thesized following the general procedures of analogues 2b−d.
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Figure 5. Stereoview of superimposed, minimum-energy calculated conformations of 1c and 2g.
Scheme 1. Method A
Following this plan required the preparation of precursor
2-methoxy-5-vinylphenylamine (3d, method A), which was
converted to intermediate 5d as expected (Scheme 1).
However, initial attempts to prepare DAP 7f using the
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Table 1
a
b
ALK enzyme IC50
(nM)
NPM-ALK cell IC50
(nM)
synthesis
method
c
compd
X
R
R′
R″
IR/ALK
1b
1c
2a
2b
2c
4-Me-piperazinyl
4-Me-piperazinyl
H
H
H
H
H
H
H
67 13
NT
100
NT
NT
NT
1.3
F
-OMe
H
22
7
3.6
F
-CH2CH2-
-CH2CH2-
-CH2CH2-
>10000
ND
ND
ND
A
1-morpholinyl
H
>10000
A
-OCH2CH2-N-
pyrrolodine
H
283 121
B, A
2d
2e
2f
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CHCH-
-CHCH-
-CHCH-
4-Me-piperazinyl
H
H
H
H
H
H
H
H
H
92 10
360 80
>1000
NT
NT
NT
150
1200
70
2.8
>27
ND
67
B, A
-OMe
-OMe
-OMe
A
1-morpholinyl
4-Me-piperazinyl
4-Me-piperazinyl
4-Me-piperazinyl
4-Me-piperazinyl
4-Me-piperazinyl
4-Me-piperazinyl
4-Me-piperazinyl
4-Me-piperazinyl
4-Me-piperazinyl
4-Me-piperazinyl
A
2g
2h
2i
3.1 0.7
35 16
3.7 0.5
3.4 0.8
B, A
-CONHMe
-SO2-i-Pr
-N(Me)SO2Me
H
3.7
10
B, A, C
D, B, A
E, B, A
B, A
2j
120
800
30
75
2k
2l
-OMe
-OMe
-OMe
H
5
2
58
-SO2-i-Pr
-N(Me)SO2Me
H
1.5 0.1
19
D, B, A
E, B, A
B, A
2m
8d
8g
8m
0.51 0.02
392 149
259 74
10
173
4
NT
NT
120
H
-OMe
>11
>140
B, A
-OMe
-N(Me)SO2Me
7.0 0.8
E, B, A
a
b
Reported as the average the standard deviation of three or more determinations. Reported as the average of two determinations. NT = not
tested. (IR enzyme IC50)/(ALK enzyme IC50). ND = not determined.
c
Scheme 2. Method B
conditions to synthesize 7b−d were unsuccessful. With
2-methoxyethanol as the solvent for 7f, acid catalyzed attack
by the solvent’s hydroxyl moiety at the benzylic position of the
olefin yielded its respective ether (7n) as a major side product
(Scheme 3). Changing the protic solvent to the more
encumbered t-BuOH thwarted this undesirable reaction and
led to clean conversions for both 7f and 7g, which were carried
on to give 2f and 2g.
Toward the preparation of target 2h, it was found that the initial
intermediate 5e was prepared most efficiently by running the
reaction neat using 4 as both reactant and solvent. Subsequent
chemistries from 5e en route to 8h proceeded smoothly following
Scheme 1. Amide 2h was prepared over three steps from ester 8h
(Scheme 4). Intermediate 8n, generated by acid hydrolysis, was
subjected to an amine coupling reaction, and the resulting amide
8o was reduced to give 2h.
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Scheme 3
a
Scheme 4. Method C
a
(a) LiOH, H2O, MeOH; (b) EDAC, HOBt, H2NMe; (c) diimide.
Target 2k was also prepared following the general protocol
of Scheme 1. The syntheses of the last targeted group of macro-
cycles (2i, 2j, 2l, 2m) were initiated with the preparations of
anilines 3f−i (Schemes 5 and 6). Anilines 3f−i were treated
with 4 under basic conditions to yield respective pyrimidines
5f−i, all in reasonable yields. Subsequently, 5f−i provided 2i,
2j, 2l, and 2m following Scheme 1.
The acyclic DAP analogues 1b and 1c were prepared as
outlined in Scheme 7, following similar synthetic methods used
for intermediates of the macrocycles illustrated in Scheme 1.
Stepwise addition of the required anilines to 4 proceeded smoothly
over two steps to provide the regiocontrolled products.
2a did not demonstrate ALK inhibition at the highest testing
concentration (ALK IC50 > 10 μM). However, from subsequent
analogues prepared with singly R appended substitution on 2,
favorable ALK enzyme activity was observed; 2d (R = 4-Me-
piperazinyl; ALK IC50 = 92 nM) was more potent than 2c (R =
-OCH2CH2-N-pyrrolidine; ALK IC50 = 283 nM), with 2b (R =
1-morpholinyl; ALK IC50 > 10 μM) proving inactive. Note-
worthy, the activity of macrocycle 2d (ALK IC50 = 92 10 nM)
was comparable to the activity of its parent DAP 1b (ALK IC50 =
67 13 nM). This was the first supportive experimental evidence
that macrocycles 2 were constrained in a biologically desired
conformation.
The general protocol to evaluate biological promise for the
newly prepared compounds was to initially screen each for ALK
enzyme inhibitory activity and subsequently select only the
more potent enzyme inhibitors (ALK IC50 < 50 nM) for testing
in the secondary ALK cellular assay, measuring inhibition of
NPM-ALK phosphorylation. The compiled biological results
for all target structures are outlined in Table 1.
With ALK being a member of the IR tyrosine kinase
superfamily and recognizing that the IR has a vital role with a
normal physiological function, a preferred ALK inhibitor would
be devoid of IR kinase inhibition. This reasoning is supported
by preclinical in vivo studies where it has been shown that
inhibition of the IR resulted in variability of glucose homeo-
stasis.15 Therefore, an IR kinase enzyme assay was chosen as a
primary kinase selectivity counterscreen within the testing
paradigm for the targeted macrocycles. For simplicity of
viewing the relative IR/ALK kinase selectivity profile, the
related IR results listed in Table 1 are shown as a relative ratio
to the reported ALK activity.
SAR evaluation of the R″ appended groups on 2 began with
replacing hydrogen (2a) (ALK IC50 > 10 μM) with -OMe (2e)
(ALK IC50 = 360 nM). The significant increase in ALK potency
of 2e was further enhanced ∼100-fold when the R = H of 2e
was replaced with R = 4-Me-piperazinyl (2g) (ALK IC50
=
3.1 nM). Analogue 2g was the macrocycle that overlapped
nicely with its DAP acyclic parent 1c via molecular modeling
(Figure 5), so it was interesting to find that 2g proved greater
than 5-fold more potent than 1c in the ALK enzymatic assay.
Along with improved target potency, 2g showed an improved
IR/ALK selectivity relative to 1c (67× and 3.6×, respectively),
suggesting that the conformationally constrained macrocycle
avoids a preferred binding to IR. Unfortunately, the boost in
enzymatic inhibition of macrocycle 2g relative to DAP parent
1c did not translate in the cellular assay, with both compounds
displaying comparable activity (2g, cell IC50 = 150 nM; 1c, cell
IC50 = 100 nM). However, the fact that 2g exhibited cellular
activity was encouraging, as it was the first macrocycle screened in
the cellular secondary assay and further validated the macro-
cycles. The lack of any observed ALK activity for macrocycle 2f
was surprising (ALK IC50 > 1000 nM), based on the positive
results for analogues 2e and 2g, suggesting that the morpholine
interacts with ALK differently from the piperazine moiety.
DISCUSSION AND CONCLUSIONS
■
The initial synthetic efforts were focused on confirming the
feasibility of the planned synthetic route to generate macro-
cycles 2, which was validated by the successful preparation of
initial analogue 2a. Not surprisingly, the simplified macrocycle
Having found that the most potent macrocycles shared the
commonality of R as a 4-Me-piperazinyl moiety, additional SAR
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a
Scheme 5. Method D
a
(a) 2-Propanethiol, EtOH, 50 °C, 72 h; (b) mCPBA, CH2Cl2, rt, 16 h; (c) vinylboronic acid dibutyl ester, Pd(PPh3)4, Na2CO3, glyme, H2O, 80 °C;
(d) Fe, AcOH, THF, 35 °C, 16 h.
a
Scheme 6. Method E
a
(a) (CF3CO)2O, KNO3, CH3CN; (b) MeI, K2CO3, DMF; (c) vinylboronic acid dibutyl ester, Pd(PPh3)4, Na2CO3, glyme, H2O, 90 °C, 16 h;
(d) Fe, AcOH, THF, 35 °C, 16 h.
Scheme 7. Method F
studies on R′ and R″ were pursued holding R constant. Three
additional R″ substituted macrocycles prepared for comparison
to the methoxy-substituted analogue 2g (ALK IC50 = 3.1 nM;
cell IC50 = 150 nM) resulted in two comparably potent ALK
inhibitors, sulfone 2i (R″ = -SO2-i-Pr; ALK IC50 = 3.7 nM; cell
IC50 = 70 nM) and sulfonamide 2j (R″ = -N(Me)SO2Me; ALK
IC50 = 3.4 nM; cell IC50 = 120 nM), with amide 2h (R″=
-CONHMe; ALK IC50 = 35 nM; cell IC50 = 1200 nM) proving
about 10-fold less active. Also encouraging for sulfonamide 2j
was its favorable 75-fold IR/ALK selectivity ratio, while sulfone
2i proved less selective with only a 10-fold IR/ALK ratio.
Having identified R,R″ substituted macrocycles exhibiting
fairly robust ALK inhibition, we next turned our attention to
the R′ substituent. The R′ substituent as a methoxy moiety has
been recognized as a key ALK selectivity and potency deter-
minant for DAPs.10 This extrapolation to macrocycles 2 proved
fruitful, as exemplified by comparing the activities of 2k (ALK
IC50 = 5 nM) to 2d (ALK IC50 = 92 nM). The most promising
compound from this exercise, and for the series as a whole, was
sulfonamide 2m, with subnanomolar ALK enzyme inhibition
(IC50 = 0.51 nM) and low nanomolar ALK cellular activity (cell
IC50 = 10 nM). Compound 2m had concurrent improvements
in both enzymatic (7-fold) and cellular (12-fold) activities when
compared to its direct R′ desmethoxy analogue 2j (IC50 = 3.4 nM;
cell IC50 = 120 nM); these analogues proved the most definitive
comparators from this series in showing overall potency
improvement contributed by the R′ = methoxy group. Analogue
2m also displayed the best overall kinase selectivity for the series
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with an IR/ALK ratio of 173, again illustrating the effectiveness of
the R′ = methoxy.
speculate that macrocycles 2 might prove worthwhile as a
scaffold for other therapeutic targets where encouraging activi-
ties have been found for DAPs, be it in the kinase area or
otherwise.
Following the publication of apo and ligand bound crystal
structures of ALK, an additional modeling study demonstrated
a complementary fit of 2m in the ALK binding pocket,
supported by two favorable hydrogen bonding interactions at
the hinge (Met-1199) and one with the salt-bridge Lys-1150
(Figure 6). Noteworthy, the binding mode of 2m was also
EXPERIMENTAL SECTION
Chemistry. Table 1 lists all final products 2 and identifies the
respective synthetic method(s) followed for their preparations. All
■
1
final products 2 were characterized by 400 MHz H NMR (Bruker
Avance, in the solvent indicated, with tetramethylsilane as an internal
standard), LC/MS (Bruker Esquire 2000 mass spectrometer with
the Agilent 1100 HPLC equipped with an Agilent Eclipse XDB-C8,
2 mm × 30 mm 3.5 μm rt column, flow rate of 1.0 mL/min, and a
solvent mixture of 10% (0.1% formic acid/water)/90% (acetonitrile/0.1%
formic acid)), and HPLC (>98% purity at 215 and 254 nm by two
methods, (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% run over 5 min and (b) Agilent Eclipse
XD8-C8, 4.6 mm × 150 mm column, eluting with a mixture of
acetonitrile and water containing 0.1% trifluoroacetic acid with a
gradient of 10−100% run over 20 min). Also, final products 2d, 2f, 2g,
2i, 2j, 2l, and 2m were evaluated by high resolution mass spectrum
(HRMS) analyses (Waters Synapt G2 Q-TOF mass spectrometer
using leucine−enkephalin as a lock-mass standard). These HRMS
were carried out internally at Cephalon. Reported melting points were
taken on a Mel-Temp apparatus and are uncorrected. All spectral
analysis results are included in the Experimental Section.
Figure 6. Glide/XP generated binding mode of compound 2m docked
in the ALK binding pocket, with the ALK structure retrieved from the
PDB (PDB code 3LCT). Only the key residues of the ALK protein
were made visible for clarity.
1H NMR data of key intermediates were also obtained from the
400 MHz NMR instrument and are included in the Experimental
Section. When indicated, compounds assayed by thin-layer chroma-
tography were evaluated on Whatman MK6F (1 in. × 3 in. × 250 μm)
silica gel plates.
consistent with the DAP bound ALK structure (2XB7) in the
Protein Data Bank (PDB).
The last significant SAR observation was that olefin linked
analogues 8 proved considerably less potent when compared to
A CEM Discover unit was used for microwave assisted reactions.
For purifications, automated normal phase column chromatography
was performed on a CombiFlash Companion (ISCO, Inc.) using
eluent solvent systems as defined for specific compounds. Reverse
phase preparative HPLC was performed on a Gilson GX-281 equipped
with Gilson 333 and 334 pumps using a Phenomenex 00F-4454-00-AX
Gemini-NX 5 μm C18 column with mixtures of acetonitrile and
water, both containing 0.1% trifluoroacetic acid, as the eluent. All
reagents were commercially available unless otherwise specified, and all
reactions were run under an inert atmosphere of Ar or N2 unless
otherwise specified.
their respective ethylene linked macrocycles 2 [8d (ALK IC50
392 nM) relative to 2d (ALK IC50 = 92 nM), 8g (ALK IC50
259 nM) relative to 2g (ALK IC50 = 3 nM), 8m (ALK IC50
=
=
=
7 nM, cell IC50 = 120 nM) relative to 2m (ALK IC50 = 0.5 nM,
cell IC50 = 10 nM)]. A possible explanation for the loss of activity
for analogues 8 may pertain to the olefinic sp2 carbons adding
further constraint to the macrocycle, leading to a less desirable
conformer.
Molecular Modeling. Conformational Analysis. Most of the
computational work was done using the Schrodinger/Maestro
molecular modeling package (Maestro, version 9.1.107, Schrodinger,
LLC, New York, NY):
In conclusion, the synthetic and medicinal chemistry objec-
tives outlined for macrocycles 2 were accomplished success-
fully. We demonstrated that conformationally constrained
macrocycles 2 could be synthesized efficiently as inverted “U”
shaped conformer mimics of DAPs 1 by employing a Heck
reaction for the macrocyclization followed by selective diimide
olefin reductions (Scheme 1). Subsequent comparison of ALK
biological results for DAPs 1b and 1c relative to their respective
constrained macrocycles 2d and 2g showed ALK activities to be
fairly consistent, with a potential indication of improved kinase
selectivity for the macrocycles. The similarity of biological
activities between these directly related constrained macrocycles
with their acyclic DAP parents is also good complementary evi-
dence to the recently published DAP/ALK solid state cocrystal
findings that an inverted “U” shaped conformer of DAPs is a
preferred biologically active conformation. Finally, macrocycle 2m
was identified as an extremely potent ALK inhibitor with a
favorable kinase selectivity profile relative to IR and proved to
have the most promising overall profile from our initial set of 13
macrocycles prepared.
(1) Build 3D structures using LigPrep.
(2) Load all ligands in the Project Table, and select all.
(3) Set MacroModel Conformational Search Panel for
Project Table (selected entries, using the OPLS_2005
force field and water as the implicit solvent, minimization
method Polak−Ribiere conjugate gradient (PRCG),
maximum iteration of 500, convergence gradient of
0.05, and 1000 steps of mixed torsion low mode sampling).
Docking Study. The essential steps in the docking experiment are
summarized below:
(1) Prepare an ALK structure from the Protein Data Bank
(PDB) (PDB code 3LCT)16 using Maestro protein
preparation workflow.
(2) Use the lowest energy conformation of the ligands for
the docking study.
(3) Use Glide/XP flexible docking and keep the top 10 binding
poses.
In addition to these findings demonstrating that macrocycles
2 are a useful template as an ALK scaffold, one may further
(4) Select the binding mode using our knowledge based
approach.17
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Method A. 2-Methoxy-5-vinylphenylamine (3d). To a room
temperature (rt) mixture of 5-bromo-2-methoxyphenylamine (3c)
(610 mg, 3.0 mmol), potassium carbonate (K2CO3) (1.73 g, 12.5
mmol), and tetrakis(triphenylphsosphine)palladium(0) (Pd(PPh3)4)
(600 mg, 0.5 mmol) in 1,2-dimethoxyethane (18 mL) and water
(3 mL) was added vinylboronic acid dibutyl ester (2.37 mL,
10.8 mmol). The mixture was then warmed to 90 °C for 16 h. The
resulting mixture was concentrated under reduced pressure, and the
residue was acidified with aqueous 1 N HCl. Attempted extraction
with ethyl ether (Et2O) or ethyl acetate (EtOAc) yielded a solid
between the biphasic mix, which was filtered off. The aqueous phase of
the filtrate was neutralized while cooling with 30% NaOH, and the
resulting mixture was washed with ethyl acetate (EtOAc) (2×). The
latter EtOAc extracts were combined, dried over Na2SO4, filtered, and
concentrated under reduced pressure to yield 320 mg (71%) of 3d as
an oil, which was used for subsequent steps without further
manipulation. 1H NMR (DMSO-d6) δ 6.79−6.78 (d, 1H), 6.77−
6.75 (d, 1H), 6.64−6.61 (m, 1H), 6.58−6.50 (m, 1H), 5.53−5.49 (d, 1H),
5.05−5.02 (d, 1H), 4.91 (bs, 2H), 3.75 (s, 3H).
treated with 1:1 MeOH/water. The resulting solid was filtered,
retriturated with 1:1 MeOH/water, and refiltered, and the resulting
solid was triturated in MeOH at 50 °C. After the mixture was filtered,
the solid was rinsed liberally with MeOH. After air drying, 5e was
recovered in 52% yield as a brown solid. LC/MS: M + H = 377.9. 1H
NMR (DMSO-d6) δ 11.25 (s, 1H), 8.84−8.83 (d, J = 1.89 Hz, 1H),
8.60 (s, 1H), 7.97−7.95 (d, J = 8.52 Hz, 1H), 7.50−7.47 (m, 1H), 3.90
(s, 3H).
[5-Bromo-2-(propane-2-sulfonyl)phenyl]-(2,5-dichloropyrimidin-
4-yl)amine (5f). A mixture of 3f (310 mg, 1.11 mmol) and 4 (153 μL,
1.34 mmol) in DMF (2 mL) at 0 °C was treated with neat NaH
(89 mg, 2.2 mmol, 60% oil dispersion) over 30 s. After an hour at
0 °C, additional 4 (150 μL, 1.30 mmol) was added followed by neat
NaH (160 mg, 4 mmol, 60% oil dispersion). After another hour the
mixture was treated with saturated aqueous NH4Cl, then diluted with
water. The resulting brownish solid was filtered and washed liberally
with water. The solid was then rinsed with ice cold CH3CN (3 mL).
The resulting 415 mg (87%) of tannish solid 5f was used for
subsequent steps without further manipulation. LC: 100%. LC/MS:
M + H = 425.9. 1H NMR (DMSO-d6) δ 9.87 (s, 1H), 8.68 (bs, 1H), 8.60
(s, 1H), 7.82−7.79 (d, J = 8.49 Hz, 1H), 7.69−7.67 (d, J = 7.89 Hz, 1H),
3.59−3.56 (m, 1H), 1.18−1.17 (d, J = 7.76 Hz, 6 H).
(3-Bromophenyl)-(2,5-dichloropyrimidin-4-yl)amine (5a). 3-Bro-
moaniline (3a) (790 mg, 4.6 mmol), 2,4,5-trichloropyrimidine (4)
(840 mg, 4.58 mmol), and K2CO3 (1.90 g, 14 mmol) in N,N-dimethyl-
formamide (DMF) (20 mL) was warmed to 80 °C for 16 h. The
mixture was cooled to rt and then diluted with water (220 mL). The
resulting solid was filtered and rinsed with a small amount of water.
After air drying, the solid was triturated with ice cold acetonitrile
(CH3CN) (10 mL), filtered, and rinsed with a small amount of ice
cold CH3CN to yield 720 mg (49%) of desired 5a as a white solid, mp
N-[4-Bromo-2-(2,5-dichloropyrimidin-4-ylamino)phenyl]-N-
methylmethanesulfonamide (5g). Compounds 3g (550 mg, 2.0
mmol) and 4 (678 μL, 5.9 mmol) were combined with EtN(i-Pr)2
(940 μL, 5.4 mmol) in N-methylpyrrolidinone (5 mL) and warmed to
100 °C for 2 days. The resulting mixture was concentrated under high
vacuum and the residue treated with 1:1 H2O/CH3CN (8 mL). The
resulting solid was filtered and rinsed with ice cold 1:1 H2O/CH3CN
(2 mL), then with MeOH, yielding 587 mg (70%) of desired 5g as a
tannish solid, which was used for subsequent steps without further
1
146−148 °C. LC: 98%. LC/MS: M + H = 320.0. H NMR (DMSO-
d6) δ 9.60 (s, 1H), 8.43 (s, 1H), 7.89−7.88 (m, 1H), 7.68−7.64 (m,
1H), 7.37−7.36 (m, 2H).
1
manipulation. LC: 95%. LC/MS: M + H = 426.9. H NMR (DMSO-
(2,5-Dichloropyrimidin-4-yl)-3-vinylphenyl)amine (5b). Com-
pound 5b was prepared in a similar manner as 5a after substituting
3-vinylphenylamine (3b) for 3a, substituting N,N-diisopropylethyl-
amine (EtN(i-Pr)2) for K2CO3, and altering the reaction temperature
from 80 °C to rt. Workup varied in that after 16 h the mixture was
diluted with water, which yielded an oil that crystallized to a solid. The
solid was filtered, triturated with water, and refiltered to yield desired
5b in 87% yield as a white solid, mp 87.5−90 °C. LC: 97%. LC/MS:
M + H = 266.0. 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.69 (s, 1H), 7.59−
7.56 (m, 1H), 7.40−7.37 (m, 1H), 7.28−7.26 (m, 2H), 6.79−6.71
(m, 1H), 5.85−5.80 (d, J = 17.7 Hz, 1H), 5.35−5.33 (d, J = 10.9 Hz, 1H).
(5-Bromo-2-methoxyphenyl)-(2,5-dichloropyrimidin-4-yl)amine
(5c). Compound 5c was prepared in a similar manner as 5a after
substituting 5-bromo-2-methoxyphenylamine (3c) for 3a and altering
the reaction time from 16 to 3 h. Workup varied in that after 16 h the
resulting solid in the reaction mixture was filtered, rinsed with DMF,
then water. The resulting solid was triturated with CH3CN, filtered,
and air-dried to yield desired 5c in 54% yield as a white solid, mp
d6) δ 9.04 (s, 1H), 8.49 (s, 1H), 8.11 (s, 1H), 7.65−7.63 (d, J = 8.57
Hz, 1H), 7.56−7.54 (d, J = 8.50 Hz, 1H), 3.18 (s, 3H), 3.06 (s, 3H).
(2,5-Dichloropyrimidin-4-yl)-[2-(propane-2-sulfonyl)-5-
vinylphenyl]amine (5h). Compound 5h was prepared in a similar
manner as 5e after substituting 3h for 3e. The workup varied in that
after concentration of the reaction mixture the residue was partitioned
between CHCl3 (2×) and water. The organic phase was dried over
Na2SO4, filtered, and the crude product in the filtrate was adsorbed
directly onto silica gel. The crude material was purified via normal
phase chromatography (EtOAc/hexane eluent) to give desired 5h in
1
51% yield as a yellow solid. LC: 100%. LC/MS: M + H = 372.1. H
NMR (DMSO-d6) δ 9.79 (s, 1H), 8.55−8.32 (d, 1H), 7.85−7.83 (bs,
1H), 6.89−6.82 (m, 1H), 6.07−6.03 (d, 1H), 5.57−5.54 (d, 1H), 3.52
(bm, 1H), 1.17−1.15 (d, 6H).
N-[2-(2,5-Dichloropyrimidin-4-ylamino)-4-vinylphenyl]-N-methyl-
methanesulfonamide (5i). Compound 5i was prepared in a similar
manner as 5e after substituting 3i for 3e. The workup varied in that
after concentration of the reaction mixture the residue was partitioned
between EtOAc and water. The organic phase was dried over Na2SO4,
filtered, and the crude product in the filtrate was adsorbed directly
onto silica gel. The crude material was purified via normal phase
chromatography (EtOAc/hexane eluent) to give desired 5i in 50%
1
205−208 °C. LC: 99%. LC/MS: M + H = 349.9. H NMR (DMSO-
d6) δ 9.00 (s, 1H), 8.38 (s, 1H), 7.81−7.80 (d, J = 2.48 Hz, 1H), 7.43−
7.41 (m, 1H), 7.12−7.10 (d, J = 8.85 Hz, 1H), 3.82 (s, 3H).
(2,5-Dichloropyrimidin-4-yl)-(2-methoxy-5-vinylphenyl)amine
(5d). Compounds 3d (970 mg, 6.5 mmol), 4 (1.2 g, 6.5 mmol), and
EtN(i-Pr)2 (1.2 mL, 7.2 mmol) were combined in DMF (9 mL) at rt
and stirred for 72 h. The reaction mixture was then diluted with ice
cold water (100 mL). The resulting solid was filtered and rinsed
liberally with water. The solid was further purified by trituration with
methanol (2×) to yield desired 5d in 66% yield as an off white solid.
1
yield as a yellow solid. LC: 93%. LC/MS: M + H = 373.2. H NMR
(DMSO-d6) δ 9.04 (s, 1H, exchangeable), 8.45 (s, 1H), 7.93 (s, 1H),
7.64−7.62 (d, 1H), 7.47−7.45 (d, 1H), 6.82−6.75 (m, 1H), 5.91−5.87
(d, 1H), 5.40−5.37 (d, 1H), 3.17 (s, 3H), 3.04 (s, 3H).
N(4)-(3-Bromophenyl)-5-chloro-N(2)-(3-vinylphenyl)pyrimidine-
2,4-diamine (7a). 5a (318 mg, 1.0 mmol), 3-vinylphenylamine (6a)
(119 mg, 1.0 mmol), and 4.0 M HCl in dioxane (275 μL, 1.10 mmol)
were combined in 2-methoxyethanol (10 mL) and warmed to 110 °C
for 16 h. The resulting solution was concentrated under reduced pre-
ssure, and the residue was partitioned between CHCl3 and saturated
aqueous NaHCO3. The organic phase was dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue was triturated
with CH3CN, filtered, and rinsed with a small amount of CH3CN to
yield 297 mg (74%) of desired 7a as an off white solid, mp 142−
145 °C. LC: 96%. LC/MS: M + H = 402.9. 1H NMR (CDCl3) δ 8.12
(s, 1H), 7.84 (s, 1H), 7.57−7.55 (d, J = 8.33 Hz, 1H), 7.51−7.49
1
LC: 97%. LC/MS: M + H = 296.1. H NMR (DMSO-d6) δ 9.04
(s, 1H), 8.36 (s, 1H), 7.70 (s, 1H), 7.36−7.35 (d, 1H), 7.13−7.11
(d, 1H), 6.73−6.66 (m, 1H), 5.72−5.68 (d, 1H), 5.19−5.17 (d, 1H),
3.82 (s, 3H).
4-Bromo-2-(2,5-dichloropyrimidin-4-ylamino)benzoic acid meth-
yl ester (5e). Compound 5e was prepared in a similar manner as 5b
after substituting 2-amino-4-bromobenzoic acid methyl ester (3e) for
3b. In addition, rather than use of DMF as solvent, 4 (10 equiv) was
used as the solvent as well as reactant, and the mixture was warmed to
120 °C rather than 80 °C for 16 h. The workup also varied in that the
excess 4 was removed under high vacuum, and the residue was then
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Journal of Medicinal Chemistry
Article
(m, 2H), 7.34−7.28 (m, 2H), 7.24−7.20 (m, 1H), 7.15−7.13 (d, J =
7.60 Hz, 1H), 7.07 (bs, 1H), 7.01 (bs, 1H), 6.71−6.64 (m, 1H), 5.72−
5.67 (d, J = 17.69 Hz, 1H), 5.26−5.23 (d, J = 11.11 Hz, 1H).
N(2)-(3-Bromo-4-morpholin-4-ylphenyl)-5-chloro-N(4)-3-
vinylphenyl)pyrimidine-2,4-diamine (7b). Compound 7b was pre-
pared in a similar manner as 7a after substituting 5b for 5a and
3-bromo-4-morpholin-4-ylphenylamine (6b) for 6a. Also, EtOAc was
used for the extractive workup rather than CHCl3. The desired
product 7b was isolated in 71% as a white solid, mp 142−144 °C. LC:
methanesulfonic acid was used rather than 4.0 M HCl in dioxane.
The desired product 7h was isolated in 52% as a tan solid and was
used for subsequent cyclization to 8h without further manipulation.
1
LC: 98%. LC/MS: M + H = 559.0. H NMR (DMSO-d6) δ 11.04
(s, 1H), 9.49 (s, 1H), 8.98 (s, 1H), 8.30 (s, 1H), 7.95−7.93 (d, J =
8.56 Hz, 1H), 7.60−7.56 (m, 2H), 7.35−7.32 (d, J = 10.16 Hz, 1H),
7.07−7.05 (d, J = 8.57 Hz, 1H), 7.01−6.93 (m, 1H), 5.58−5.53 (d, J =
17.37 Hz, 1H), 5.21−5.18 (d, J = 11.81 Hz, 1H), 3.91 (s, 3H), 2.84 (s,
4H), 2.5 (bs, 4H + DMSO), 2.23 (s, 3H).
1
91%. LC/MS: M + H = 406.1. H NMR (DMSO-d6) δ 9.37 (s, 1H),
N(4)-[5-Bromo-2-(propane-2-sulfonyl)phenyl]-5-chloro-N(2)-[4-
(4-methylpiperazin-1-yl)-3-vinylphenyl]pyrimidine-2,4-diamine
(7i). Compounds 5f (150 mg, 0.35 mmol) and 6e (77 mg, 0.35 mmol)
and methanesulfonic acid (33 μL, 0.50 mmol) were combined in 2-
methoxyethanol (4 mL) and warmed to 110 °C for 3 h. The resulting
solution was concentrated under reduced pressure and the residue
partitioned between CHCl3 (2×) and saturated aqueous NaHCO3. The
combined organic phases were dried over Na2SO4, filtered, and con-
centrated under reduced pressure. The 270 mg of remaining brown oil
was dissolved in CH3CN (3 mL). The solid which crystallized was
filtered and rinsed with a small amount of ice cold CH3CN to yield
90 mg (40%) of desired 7i as a light brown solid, which was used
without further manipulation for its subsequent cyclization to 8i. LC:
92%. LC/MS: M + H = 607.0.
N-(4-Bromo-2-{5-chloro-2-[4-(4-methylpiperazin-1-yl)-3-
vinylphenylamino]pyrimidin-4-ylamino}phenyl)-N-methylmethane-
sulfonamide (7j). Compounds 5g (213 mg, 0.50 mmol) and 6e
(109 mg, 0.50 mmol) and methanesulfonic acid (46 μL, 0.71 mmol)
were combined in 2-methoxyethanol (6 mL) and warmed to 110 °C
for 4 h. The resulting solution was concentrated under reduced
pressure and the residue partitioned between EtOAc (2×) and
saturated aqueous NaHCO3. The combined organic phases were dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
remaining brown oil was dissolved in CH3CN (3 mL). The solid
which crystallized was filtered and rinsed with a small amount of ice
cold CH3CN to yield 118 mg (39%) of desired 7j as an off white solid,
which was used without further manipulation for its subsequent cycli-
zation to 8j. LC: 92%. LC/MS: M + H = 608.1. 1H NMR (DMSO-d6)
δ 9.40 (s, 1H), 8.45 (s, 1H), 8.39 (s, 1H), 8.23 (s, 1H), 7.60−7.58 (m,
3H), 7.41−7.39 (d, J = 8.60 Hz, 1H), 7.05−7.03 (d, J = 8.32 Hz, 1H),
7.00−6.92 (m, 1H), 5.55−5.51 (d, J = 17.81 Hz, 1H), 5.21−5.18 (d,
J = 11.24 Hz, 1H), 3.19 (s, 3H), 3.12 (s, 3H), 2.83 (bs, 4H), 2.50 (bs,
4H + DMSO), 2.24 (s, 3H).
N(2)-[5-Bromo-2-methoxy-4-(4-methylpiperazin-1-yl)phenyl]-5-
chloro-N(4)-(3-vinylphenyl)pyrimidine-2,4-diamine (7k). Com-
pound 7k was prepared in a similar manner as 7a after substituting
5b for 5a, substituting 6f for 6a, and heating the mixture for 8 h rather
than 16 h. The workup also varied in that after concentrating the reac-
tion mixture, the crude mix was purified straightaway via preparative
HPLC. The purest fractions were then partitioned between EtOAc and
saturated aqueous NaHCO3. The organic phase was dried over
Na2SO4, filtered, and concentrated under reduced pressure to give
desired 7k as an oil in 10% yield. This material was used without
further manipulation for its subsequent cyclization to 8k. LC: 95%.
LC/MS: M + H = 530.9.
N(2)-[5-Bromo-2-methoxy-4-(4-methylpiperazin-1-yl)phenyl]-5-
chloro-N(4)-[2-(propane-2-sulfonyl)-5-vinylphenyl]pyrimidine-2,4-
diamine (7l). Compounds 5h (200 mg, 0.54 mmol) and 6f (177 mg,
0.59 mmol) and methanesulfonic acid (45.3 μL, 0.70 mmol) were
combined in 2-methoxyethanol (3 mL) and warmed to 105 °C for 3 h.
The resulting solution was concentrated under reduced pressure, dis-
solved in DMSO, and purified via preparative HPLC. The purest
fractions of desired 7l were combined and partitioned between EtOAc
(2×) and saturated aqueous NaHCO3. The combined organic phases
were dried over Na2SO4, filtered, and concentrated under reduced
pressure. The 215 mg of yellow residue was triturated with ice cold
CH3CN, filtered, and rinsed with a small amount of ice cold CH3CN
to yield 70 mg (20%) of desired 7l as an off white solid, which was
used without further manipulation for its subsequent cyclization to 8l.
LC: 99%. LC/MS: M + H = 637.1. 1H NMR (DMSO-d6) δ 9.48 (s, 1H,
exchangeable), 8.53 (s, 1H), 8.46 (s, 1H, exchangeable), 8.32 (s, 1H),
7.78−7.76 (m, 2H), 7.49−7.47 (d, J = 7.64 Hz, 1H), 6.80 (s, 1H),
8.86 (s, 1H), 8.16 (s, 1H), 7.85 (s, 1H), 7.70 (s, 1H), 7.63−7.60 (m,
2H), 7.38−7.34 (t, 1H), 7.28−7.26 (d, 1H), 6.99−6.96 (d, 1H), 6.76−
6.68 (m, 1H), 5.81−5.77 (d, 1H), 5.27−5.24 (d, 1H), 3.73−3.71 (m,
4H), 2.88−2.86 (m, 4H).
N(2)-[3-Bromo-4-(2-pyrrolidin-1-ylethoxy)phenyl]-5-chloro-N(4)-
(3-vinylphenyl)pyrimidine-2,4-diamine (7c). Compound 7c was
prepared in a similar manner as 7b after substituting 6c for 6b and
substituting methanesulfonic acid for 4.0 M HCl in dioxane. Also,
CHCl3 was used for the extractive workup rather than EtOAc. The
desired product 7c was isolated in 52% as a white solid. LC/MS: M +
H = 515.4
N(2)-[3-Bromo-4-(4-methylpiperain-1-yl)phenyl]-5-chloro-N(4)-
(3-vinylphenyl)pyrimidine-2,4-diamine (7d). Compound 7d was
prepared in a similar manner as 7b after substituting 6d for 6b.
Also, CHCl3 was used for the extractive workup rather than EtOAc.
The desired product 7d was isolated in 62% as a tan solid, mp 149−
1
153 °C. LC: 93%. LC/MS: M + H = 501.0. H NMR (DMSO-d6) δ
9.35 (s, 1H), 8.85 (s, 1H), 8.16 (s, 1H), 7.83 (s, 1H), 7.70 (s, 1H),
7.61−7.58 (m, 2H), 7.37−7.33 (t, 1H), 7.28−7.26 (d, 1H), 6.97−6.95
(d, 1H), 6.75−6.68 (m, 1H), 5.81−5.77 (d, 1H), 5.27−5.24 (d, 1H),
2.87 (s, 4H), 2.46 (s, 4H), 2.23 (s, 3H).
N(4)-(5-Bromo-2-methoxyphenyl]-5-chloro-N(2)-(3-vinylphenyl)-
pyrimidine-2,4-diamine (7e). Compound 7e was prepared in a
similar manner as 7a after substituting 5c for 5a and heating the
mixture for 8 h rather than 16 h. The desired product 7e was isolated
in 76% as a white solid, mp 183−185 °C. LC: 97%. LC/MS: M + H =
432.9. 1H NMR (DMSO-d6) δ 9.44 (s, 1H), 8.26 (s, 1H), 8.19 (s, 1H),
8.09 (bs, 1H), 7.56−7.54 (m, 2H), 7.36−7.33 (m, 1H), 7.22−7.18 (m,
1H), 7.11−7.08 (d, J = 8.78 Hz, 1H), 7.06−7.04 (d, J = 7.71 Hz, 1H),
6.61−6.54 (m, 1H), 5.68−5.64 (d, J = 17.55 Hz, 1H), 5.22−5.19 (d, J =
10.96 Hz, 1H), 3.83 (s, 3H).
N(2)-[3-Bromo-4-morpholin-4-ylphenyl]-5-chloro-N(4)-(2-me-
thoxy-5-vinylphenyl)pyrimidine-2,4-diamine (7f). Compound 7f
was prepared in a similar manner as 7b after substituting 5d for 5b
and after altering the solvent system from 2-methoxyethanol to a 1:1
mixture of tert-butyl alcohol/1,2-dimethoxyethane (120 mL of solvent
for a mmol reaction). The reaction mixture was warmed for 6 days at
85 °C, followed by the standard workup using EtOAc for the extractive
workup. Some desired 7f crystallized relatively pure from EtOAc on
concentrating the filtrate, amd the remainder was purified via normal
phase chromatography to yield the desired product 7f in 55% overall
yield as a yellow tinted solid, mp 186−190 °C. LC: 100%. LC/MS:
1
M + H = 518.0. H NMR (DMSO-d6) δ 9.36 (s, 1H), 8.23 (s, 1H),
8.16 (s, 1H), 7.95 (s, 1H), 7.8 (s, 1H), 7.58−7.57 (m, 1H), 7.33−7.32
(m, 1H), 7.13−7.11 (d, 1H), 6.96−6.94 (d, 1H), 6.7−6.6 (m, 1H),
5.67−5.62 (d, 1H), 5.13−5.10 (d, 1H), 3.85 (s, 3H), 3.74−3.71 (m, 4H),
2.88−2.85 (m, 4H).
N(2)-[3-Bromo-4-(4-methylpiperazin-1-yl)phenyl]-5-chloro-N(4)-
(2-methoxy-5-vinylphenyl)pyrimidine-2,4-diamine (7g). Com-
pound 7g was prepared in a similar manner as 7f after substituting
6d for 6b. After the reaction mixture was concentrated, the crude mix
was purified straightaway via preparative HPLC. The purest fractions
were then partitioned between EtOAc and saturated aqueous
NaHCO3. The organic phase was dried over Na2SO4, filtered, and con-
centrated under reduced pressure to give desired 7g in 20% yield. This
material was used without further manipulation for subsequent
cyclization to 8g. LC: 93%. LC/MS: M + H = 531.0.
4-Bromo-2-(5-chloro-2-[4-(4-methylpiperazin-1-yl)-3-
vinylphenylamino]pyrimidin-4-ylamino)benzoic Acid Methyl Ester
(7h). Compound 7h was prepared in a similar manner as 7a after
substituting 5e for 5a and after substituting 6e for 6a. Also,
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(21),10,12,14,16,18-decaene (8d). Compound 8d was prepared in a
similar manner as 8a after substituting 7d for 7a. The desired product
8d was isolated in 75% yield as an off white solid. LC: 92%, LC/MS:
M + H = 419.1.
6.67−6.57 (m, 1H), 5.87−5.83 (d, J = 17.72 Hz, 1H), 5.40−5.37 (d, J =
10.85 Hz, 1H), 3.78 (s, 3H), 3.46−3.41 (m, 1H), 2.97 (s, 4H), 2.5 (s,
4H + DMSO), 2.24 (s, 3H), 1.18−1.16 (d, J = 6.68 Hz, 6H).
N-(2-{2-[5-Bromo-2-methoxy-4-(4-methylpiperazin-1-yl)-
phenylamino]-5-chloropyrimidin-4-ylamino}-4-vinylphenyl)-N-
methylmethanesulfonamide (7m). Compounds 5i (192 mg,
0.52 mmol) and 6f (170 mg, 0.57 mmol) and methanesulfonic acid
(43.4 μL, 0.67 mmol) were combined in 2-methoxyethanol (3 mL)
and warmed to 105 °C for 4 h. The resulting solution was concen-
trated under reduced pressure, dissolved in DMSO, and purified via
preparative HPLC. The purest fractions of desired 7m were combined
and partitioned between EtOAc (2×) and saturated aqueous
NaHCO3. The combined organic phases were dried over Na2SO4,
filtered, and concentrated under reduced pressure. The 270 mg of
yellow residue was triturated with ice cold CH3CN, filtered, and rinsed
with a small amount of ice cold CH3CN to yield 110 mg (34%) of
desired 7m as an off white solid, which was used without further
manipulation for its subsequent cyclization to 8m. LC: 98%. LC/MS:
A 25 mg sample of the 92% pure material was further purified via
preparative HPLC to return 17 mg of 8d as a TFA salt white
lyophylate, which was the sample used for biological testing. LC:
100%. LC/MS: M + H = 419.1. 1H NMR (DMSO-d6) δ 9.58 (bs, 1H),
9.29 (s, 1H), 9.12 (s, 1H), 8.61 (s, 1H), 8.45−8.44 (d, 1H), 8.12 (s, 1H),
7.29−7.25 (t, 1H), 7.20−7.18 (d, 1H), 7.02−6.97 (m, 3H), 6.83−6.80
(d, 1H), 6.75−6.72 (d, 1H), 3.74 (bs, H2O), 3.54−3.51 (d, 2H), 3.29−
3.22 (m, 4H), 2.93−2.89 (m, 5H).
(14Z)-10-Methoxy-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]-
docosa-1(20),3(22),4,6,9(21),10,12,14,16,18-decaene (8e). Com-
pound 8e was prepared in a similar manner as 8a after substituting
7e for 7a. The workup varied slightly in that the initial solid collected
was dissolved in tetrahydrofuran (THF), filtered, and the filtrate was
concentrated under reduced pressure. The resulting solid was
triturated in CH3CN, filtered, and rinsed with CH3CN followed by
Et2O. The desired product 8e was isolated in 51% yield as a tannish
1
M + H = 638.0. H NMR (DMSO-d6) δ 8.41 (s, 1H, exchangeable),
8.23 (s, 1H, exchangeable), 8.21 (s, 1H), 8.18 (s, 1H), 7.82 (s, 1H),
7.58−7.56 (d, J = 8.25 Hz, 1H), 7.36−7.34 (d, J = 8.04 Hz, 1H), 6.81
(s, 1H), 6.61−6.54 (m, 1H), 5.77−5.72 (d, J = 17.69 Hz, 1H), 5.27−
5.25 (d, J = 10.77 Hz, 1H), 3.79 (s, 3H), 3.18 (s, 3H), 3.10 (s, 3H),
2.98 (s, 4H), 2.5 (s, 4H + DMSO), 2.24 (s, 3H).
1
solid, mp 273−275 °C. LC: 99%. LC/MS: M + H = 351.1. H NMR
(DMSO-d6) δ 9.49 (s, 1H), 8.99 (s, 1H), 8.80 (bs, 1H), 8.21 (s, 1H),
7.83 (s, 1H), 7.25−7.21 (m, 1H), 7.08−7.04 (m, 3H), 6.94−6.92 (d,
J = 7.37 Hz, 1H), 6.55−6.44 (m, 2H), 3.91 (s, 3H).
N(2)-(3-Bromo-4-morpholin-4-ylphenyl)-5-chloro-N(4)-{2-me-
thoxy-5-[1-(2-methoxyethoxy)ethyl]phenyl}pyrimidine-2,4-diamine
(7n). 7n is the side product that was isolated from initial reaction
toward 7f when 2-methoxyethanol was used as solvent. LC: 95%. LC/
(14Z)-6-Chloro-10-methoxy-17-(morpholin-4-yl)-2,4,8,22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,14,16,18-decaene (8f). Compound 8f was prepared in a
similar manner as 8a after substituting 7f for 7a. The desired product
8f was isolated in 53% yield as a gray solid. LC: 87%. LC/MS: M +
H = 436.2.
(14Z)-6-Chloro-10-methoxy-17-(4-methylpiperazin-1-yl)-
2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3-
(22),4,6,9(21),10,12,14,16,18-decaene (8g). Compound 8g was
prepared in a similar manner as 8a after substituting 7g for 7a. The
desired product 8g was isolated in 94% yield as an off white solid. LC:
94%, M + H = 449.1.
1
MS: M + H = 594.1. H NMR (DMSO-d6) δ 9.39 (s, 1H), 8.49
(s, 1H), 8.15 (s, 1H), 7.70−7.68 (m, 2H), 7.52−7.49 (m, 1H), 7.19−
7.16 (m, 1H), 7.13−7.11 (d, J = 8.56 Hz, 1H), 6.98−6.95 (d, J = 8.80
Hz, 1H), 4.40−4.36 (m, 1H), 3.80 (s, 3H), 3.73−3.71 (m, 4H), 3.38−
3.30 (m, 4H), 3.18 (s, 3H), 2.87−2.85 (m, 4H), 1.31−1.30 (d, J = 6.41
Hz, 3H).
(14Z)-6-Chloro-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]-
docosa-1(20),3(22),4,6,9(21),10,12,14,16,18-decaene (8a). Palladi-
um acetate (Pd(OAc)2) (40 mg, 0.2 mmol), tri-o-tolylphosphine
(240 mg, 0.79 mmol), and DAP 7a (150 mg, 0.37 mmol) were
combined with CH3CN (4 mL) and triethylamine (Et3N) (300 μL,
2 mmol). The mixture was treated under microwave irradiation at
120 °C for 30 min. After the mixture was cooled, the resulting solid
was filtered and rinsed with ice cold CH3CN to yield 78 mg (65%) of
desired 8a as an off white solid, mp >250 °C. TLC: 25% EtOAc/
hexane Rf = 0.25; 5:1 CH2Cl2/MeOH Rf = 0.85. LC: 96%. LC/MS:
A small sample of the 94% pure material was further purified via
preparative HPLC to return pure 8g as a TFA salt white lyophylate,
which was the sample used for biological testing. LC: 99%. LC/MS:
1
M + H = 449.1. H NMR (DMSO-d6) δ 9.6 (bs, 1H), 9.38 (s, 1H),
9.04−9.03 (d, 1H), 8.41−8.40 (d, 1H), 8.19 (d, 1H), 7.84 (s, 1H),
7.11−7.01 (m, 4H), 6.68−6.60 (m, 2H), 3.90 (s, 3H), 3.5−3.2 (bm,
6H + H2O), 2.96−2.89 (m, 5H).
Methyl (14Z)-6-Chloro-17-(4-methylpiperazin-1-yl)-2,4,8,22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,14,16,18-decaene-10-carboxylate (8h). Compound 8h
was prepared in a similar manner as 8a after substituting 7h for 7a.
The desired product 8h was isolated in 54% yield as a bronze solid.
LC: 100%. LC/MS: M + H = 477.1. 1H NMR (DMSO-d6) δ 10.72 (s,
1H), 9.59 (s, 1H), 9.42 (s, 1H), 8.39 (s, 1H), 8.23 (s, 1H), 7.97−7.95
(d, 1H), 7.13−7.06 (m, 2H), 7.01−6.99 (d, 1H), 6.94−6.90 (d, 1H),
6.68−6.65 (d, 1H), 3.91 (s, 3H), 2.86 (s, 4H), 2.50 (s, 4H + DMSO),
2.25 (s, 3H).
1
M + H = 321.2. H NMR (DMSO-d6) δ 9.37 (s, 1H, exchangeable),
9.12 (s, 1H, exchangeable), 8.94 (s, 1H), 8.65 (s, 1H), 8.15 (s, 1H),
7.32−7.28 (m, 1H), 7.22−7.19 (m, 2H), 7.04 (bm, 2H), 6.93−6.91 (d,
J = 7.6 Hz, 1H), 6.64−6.55 (m, 2H).
(14Z)-6-Chloro-17-(morpholin-4-yl)-2,4,8,22-tetraazatetracyclo-
[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9(21),10,12,14,16,18-deca-
ene (8b). Compound 8b was prepared in a similar manner as 8a after
substituting 7b for 7a. In addition, after the CH3CN trituration, the
sample was also triturated with ethyl ether (Et2O). The desired
product 8b was isolated in 43% yield as an off white solid, mp 267−
(14Z)-6-Chloro-10-(propane-2-sulfonyl)-17-(4-methylpiperazin-
1-yl)-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3-
(22),4,6,9(21),10,12,14,16,18-decaene Trifluoroacetate (1:1) (8i).
Pd(OAc)2 (48 mg, 0.21 mmol), tri-o-tolylphosphine (275 mg, 0.90 mmol),
and DAP 7i (160 mg, 0.26 mmol) were combined with CH3CN (4 mL)
and Et3N (250 μL, 1.8 mmol). The mixture was treated under microwave
irradiation at 120 °C for 120 min. After cooling, the resulting mixture was
filtered and the filtrate was concentrated under reduced pressure. The
concentrated residue was partitioned between 1 N HCl and EtOAc (2×).
The aqueous phase was cooled and neutralized with KOH solution until
∼pH 7 and then diluted with saturated aqueous NaHCO3. The resulting
basic solution was extracted with CHCl3 (2×). The combined CHCl3
extracts were dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified via preparative HPLC and the purest
fractions were combined and lyophilized to yield desired 8i as its TFA salt
in 32% yield. This material was carried on for subsequent conversion to
2i without further manipulation. LC: 100%. LC/MS: M + H = 525.1.
1H NMR (DMSO-d6) δ 9.65 (bs, 1H), 9.55 (s, 1H), 9.48 (s, 1H),
1
270 °C. LC: 95%. LC/MS: M + H = 406.1. H NMR (DMSO-d6) δ
9.18 (s, 1H), 9.03 (s, 1H), 8.65 (s, 1H), 8.44−8.43 (d, 1H), 8.10
(s, 1H), 7.27−7.23 (t, 1H), 7.18−7.16 (d, 1H), 7.01−6.97 (m, 2H),
6.95−6.93 (d, 1H), 6.85−6.82 (d, 1H), 6.72−6.68 (d, 1H), 3.76−3.75
(m, 4H), 2.84 (m, 4H).
(14Z)-6-Chloro-17-[2-(pyrrolidin-1-yl)ethoxy]-2,4,8,22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,14,16,18-decaene (8c). Compound 8c was prepared in a
similar manner as 8a after substituting 7c for 7a. The desired product
8c was isolated in 62% yield as a copper colored solid. LC: 100%. LC/
1
MS: M + H = 434.1. H NMR (DMSO-d6) δ 9.7 (bs, 1H), 9.24 (s,
1H), 9.11 (s, 1H), 8.63 (s, 1H), 8.51 (d, 1H), 8.12 (s, 1H), 7.29−7.25
(t, 1H), 7.21−7.19 (d, 1H), 7.04−6.99 (m, 2H), 6.96−6.94 (d, 1H),
6.89−6.86 (d, 1H), 6.73−6.70 (d, 1H), 4.28−4.26 (m, 2H), 3.64−3.56
(bm, 2H + water), 3.18 (m, 2H), 2.07 (bm, 2H), 1.91−1.89 (m, 2H).
(14Z)-6-Chloro-17-(4-methylpiperazin-1-yl)-2,4,8,22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
458
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Journal of Medicinal Chemistry
Article
9.26−9.25 (d, 1H), 8.46−8.45 (d, 1H), 8.28 (s, 1H), 7.77−7.75 (d, 1H),
7.33−7.31 (m, 1H), 7.12−7.05 (m, 2H), 7.02−6.98 (d, 1H), 6.77−6.74
(d, 1H), 4.1 (bs, H2O), 3.54−3.51 (d, 2H), 3.42−3.38 (m, 1H), 3.30−
3.22 (m, 4H), 2.96−2.89 (m, 5H), 1.17−1.15 (d, 6H).
through a Phenomenex Strat-X-C cation catch and release resin, which
was then rinsed twice with CH3CN followed by MeOH. The resin
cartridge was transferred to a new filter flask, and the resin was rinsed
twice with 2 M NH3 in MeOH. The latter methanol solution was con-
centrated under reduced pressure to give a yellow residue. This
material was treated with CH3CN (2 mL) to yield a yellow solid,
which was filtered and rinsed with a small amount of ice cold CH3CN
to give 57 mg (72%) of desired 8m as a yellow solid, mp 278−280 °C.
(14Z)-6-Chloro-10-(2-methanesulfonylmethylamino)-17-(4-
methylpiperazin-1-yl)-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]-
docosa-1(20),3(22),4,6,9(21),10,12,14,16,18-decaene Trifluoroace-
tate (1:1) (8j). Pd(OAc)2 (24 mg, 0.10 mmol), tri-o-tolylphosphine
(140 mg, 0.45 mmol), and DAP 7j (80 mg, 0.13 mmol) were
combined with CH3CN (2 mL) and Et3N (120 μL, 0.9 mmol). The
mixture was treated under microwave irradiation at 120 °C for 45 min.
By LC analysis, the reaction had not proceeded to completion, so the
reaction mixture was filtered and the solution recharged with second
portions of Pd(OAc)2 (35 mg, 0.16 mmol), tri-o-tolylphosphine
(170 mg, 0.56 mmol), and Et3N (160 μL, 1.1 mmol). The mixture was
again treated under microwave irradiation at 120 °C for 120 min. After
cooling, the resulting mixture was filtered and the filtrate was con-
centrated under reduced pressure. The concentrated residue was
partitioned between 1 N HCl (1.4 mL) and EtOAc (2×). The aqueous
phase was purified directly via preparative HPLC and the purest
fractions were combined and lyophilized to yield desired 8j as its TFA
salt in 37% yield. This material was carried on for subsequent
conversion to 2j without further manipulation. LC: 100%. LC/MS:
1
LC: 97%. LC/MS: M + H = 556.1. H NMR (DMSO-d6) δ 8.90 (s,
1H), 8.37 (s, 1H), 8.21 (s, 1H), 8.18 (s, 1H, exchangeable), 8.02 (s,
1H, exchangeable), 7.57−7.55 (d, J = 8.25 Hz, 1H), 7.15−7.13 (d, J =
8.08 Hz, 1H), 6.78−6.75 (d, J = 13.0 Hz, 1H), 6.99 (s, 1H), 6.58−6.55
(d, J = 13.1 Hz, 1H), 3.85 (s, 3H), 3.21 (s, 3H), 3.08 (s, 3H), 2.92
(bm, 4H), 2.5 (4H + DMSO), 2.25 (s, 3H).
6-Chloro-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1-
(20),3(22),4,6,9(21),10,12,16,18-nonaene (2a). Compound 8a (50
mg, 0.2 mmol) was combined with platinum dioxide (PtO2) (10 mg,
0.04 mmol) in THF (6 mL), and the mixture was blanketed under H2
at atmospheric pressure. After being stirred at rt for 5 days the mixture
was carefully filtered, rinsed with a small amount of THF, and the
filtrate was subsequently concentrated. The residue was purified via
preparative HPLC to yield after lyophylization 16 mg (30%) of desired
2a as a white lyophylate. LC: 99%. LC/MS: M + H = 323.2. 1H NMR
(DMSO-d6) δ 9.27 (s, 1H), 9.03 (s, 1H), 8.15 (s, 1H), 7.98 (s, 1H),
7.81 (s, 1H), 7.27−7.23 (m, 1H), 7.14−7.10 (m, 2H), 7.02−7.00 (d,
J = 8.6 Hz, 1 H), 6.94−6.92 (d, J = 9.1 Hz, 1H), 6.82−6.80 (d, J = 7.4
Hz, 1H), 2.90 (s, 4H).
1
M + H = 526.1. H NMR (DMSO-d6) δ 9.80 (bs, 1H), 9.42 (s, 1H),
8.89 (s, 1H), 8.42 (s, 1H), 8.23 (s, 1H), 8.20 (s, 1H), 7.58−7.56 (d,
1H), 7.20−7.18 (d, 1H), 7.07−7.01 (m, 2H), 6.88−6.84 (d, 1H),
6.74−6.71 (d, 1H), 3.53−3.51 (d, 2H), 3.35−3.21 (m, 7H), 3.09 (s,
3H), 2.97−2.91 (m, 2H), 2.89 (s, 3H).
6-Chloro-17-(morpholin-4-yl)-2,4,8,22-tetraazatetracyclo-
[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene
(2b). At rt, dipotassium azodicarboxylate18 (784 mg, 4.0 mmol) was
added to a solution of 8b (70 mg, 0.172 mmol) in pyridine (10 mL).
The resulting mixture was treated with acetic acid (AcOH) (0.56 mL,
9.8 mmol). After 3 days the reaction mixture was treated with addi-
tional portions of dipotassium azodicarboxylate (330 mg, 1.7 mmol)
and AcOH (0.28 mL, 4.9 mmol) and warmed to 35 °C for 2 days (LC
supported reaction, >95% complete). The mixture was then combined
with water, and the resulting solid was collected. This solid was
dissolved in DMSO and purified via preparative HPLC to yield 28 mg
(31%) of desired 2b as a white lyophylate. LC: 100%. LC/MS: M +
H = 408.2. 1H NMR (DMSO-d6) δ 9.47 (bs, 2H), 8.16 (s, 1H), 7.95−
7.94 (d, 1H), 7.80 (s, 1H), 7.25−7.21 (t, 1H), 7.05−7.00 (m, 3H),
6.91−6.88 (m, 1H), 3.76−3.74 (m, 4H), 3.02−2.96 (m, 4H), 2.78−
2.77 (m, 4H).
(14Z)-6-Chloro-17-(4-methylpiperazin-1-yl)-19-methoxy-
2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3-
(22),4,6,9(21),10,12,14,16,18-decaene (8k). Compound 8k was
prepared in a similar manner as 8a after substituting 7k for 7a. The
workup varied slightly in that no product precipitated from the reac-
tion mixture; therefore, the reaction mixture was concentrated under
reduced pressure. The residue was partitioned between 1 N HCl and
EtOAc (2×). The aqueous phase was neutralized and extracted with
CHCl3 (3×). The CHCl3 extracts were combined, dried over Na2SO4,
filtered, and concentrated under reduced pressure to give crude 8k in
51% yield, which was carried on for subsequent reduction toward 2k
without further manipulation. LC/MS: M + H = 449.0.
(14Z)-6-Chloro-10-(propane-2-sulfonyl)-17-(4-methylpiperazin-
1-yl)-19-methoxy-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]-
docosa-1(20),3(22),4,6,9(21),10,12,14,16,18-decaene (8l). Pd-
(OAc)2 (6.5 mg, 0.03 mmol) was dissolved in CH3CN (3 mL). Then
tri-o-tolylphosphine (55 mg, 0.18 mmol) was added and the mixture was
stirred at rt for 15 min under Ar. DAP 7l (60 mg, 0.094 mmol) and Et3N
(92 μL, 0.66 mmol) were added to the resulting reaction mixture. The
mixture was treated under microwave irradiation at 120 °C for 15 min.
After cooling, the resulting solution was filtered through a Phenomenex
Strat-X-C cation catch and release resin, which was then rinsed twice
with CH3CN followed by MeOH. The resin cartridge was transferred to
a new filter flask, and the resin was rinsed twice with 2 M NH3 in
MeOH. The latter methanol solution was concentrated under reduced
pressure to give a yellow residue. This material was treated with CH3CN
(2 mL) to yield a yellow solid, which was filtered and rinsed with a small
amount of ice cold CH3CN to give 33 mg (63%) of desired 8l as a
yellow solid. LC: 97%. LC/MS: M + H = 555.1. 1H NMR (DMSO-d6) δ
9.49 (s, 1H, exchangeable), 9.27 (s, 1H), 8.42 (s, 1H), 8.25 (s, 1H), 8.16
(s, 1H, exchangeable), 7.74−7.72 (d, J = 8.21 Hz, 1H), 7.28−7.26 (d,
8.00 Hz, 1H), 6.92−6.89 (d, J = 13.08 Hz, 1H), 6.72 (s, 1H), 6.62−6.58
(d, J = 13.20 Hz, 1H), 3.87 (s, 3H), 3.40−3.37 (m, 1H), 2.92 (s, 4H),
2.54 (s, 4H), 2.25 (s, 3H), 1.17−1.15 (d, J = 6.76 Hz, 6H).
6-Chloro-17-[2-(pyrrolidin-1-yl)ethoxy]-2, 4, 8, 22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,16,18-nonaene Trifluoroacetate (1:1) (2c). Compound 2c
was prepared in a similar manner as 2b after substituting 8c for 8b.
The workup varied slightly in that after the addition of water no solid
precipitated, so the aqueous solution was partitioned between saturated
aqueous NaHCO3 and EtOAc. The organic phase was dried over Na2-
SO4, filtered, and concentrated under reduced pressure. The residue was
purified via preparative HPLC. The desired product 2c was isolated as a
TFA salt, white lyophylate in 40% yield. LC: 100%. LC/MS: M + H =
436.1. 1H NMR (DMSO-d6) δ 9.7 (bs, 1H), 9.16 (s, 1H), 9.05 (s, 1H),
8.09 (s, 1H), 7.82 (s, 1H), 7.80 (s, 1H), 7.23−7.19 (t, 1H), 7.07−7.05
(d, 1H), 6.97−6.95 (d, 1H), 6.88 (m, 2H), 4.25−4.23 (m, 2H), 3.64 (bs,
2H + H2O), 3.20 (m, 2H), 2.91 (s, 4H), 2.08 (m, 2H), 1.91 (m, 2H).
6 - C h l o r o - 1 7 - ( 4 - m e t h y l p i p e r a z i n - 1 - y l ) - 2 , 4 , 8 , 2 2 -
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,16,18-nonaene Trifluoroacetate (1:1) (2d). Compound
2d was prepared in a similar manner as 2b after substituting 8d for 8b.
The workup followed was akin to that for 2c. The desired product 2d
was isolated in 63% yield as a TFA salt, white lyophylate. LC: 99%,
(14Z)-6-Chloro-10-(2-methanesulfonylmethylamino)-17-(4-
methylpiperazin-1-yl)-19-methoxy-2,4,8,22-tetraazatetracyclo-
[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9(21),10,12,14,16,18-deca-
ene (8m). Pd(OAc)2 (6.5 mg, 0.03 mmol) was dissolved in CH3CN
(3 mL). Then tri-o-tolylphosphine (55 mg, 0.18 mmol) was added and
the mixture was stirred at rt for 15 min under Ar. DAP 7m (90 mg,
0.14 mmol) and Et3N (300 μL, 2 mmol) were added to the resulting
reaction mixture. The mixture was treated under microwave irradiation
at 120 °C for 15 min. After cooling, the resulting solution was filtered
1
M + H = 421.0. H NMR (DMSO-d6) δ 9.7 (bs, 1H), 9.40 (s, 1H),
9.27 (s, 1H), 8.14 (s, 1H), 8.03−8.02 (d, 1H), 7.82 (s, 1H), 7.26−7.22
(t, 1H), 7.07−7.05 (d, 1H), 7.02−7.01 (d, 1H), 7.00 (s, 1H), 6.93−
6.90 (m, 1H), 3.52−3.49 (d, 2H), 3.26−3.2 (m, 2H), 3.09−3.05 (d,
2H), 2.99−2.90 (m, 9H). HRMS calcd for C23H25ClN6 (MH+):
421.1907. Found: 421.1913.
10-Methoxy-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-
1(20),3(22),4,6,9(21),10,12,16,18-nonaene (2e). Compound 2e was
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Journal of Medicinal Chemistry
Article
prepared in a similar manner as 2a after substituting 8e for 8a. The
crude product was dissolved in a small amount of warmed DMSO and
purified via preparative HPLC. The purest fractions were combined
and partitioned between CHCl3 and saturated aqueous NaHCO3. The
organic phase was dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was triturated with CH3CN, filtered,
and rinsed with ice cold CH3CN to give desired product 2e in 50%
yield as a white solid, mp 252−254 °C. LC: 98%. LC/MS: M + H =
353.2. 1H NMR (DMSO-d6) δ 9.33 (s, 1H), 8.15 (s, 1H), 7.95 (s, 1H),
7.86 (s, 1H), 7.81 (s, 1H), 7.17−7.13 (t, 3H), 7.00 (s, 2H), 6.95−6.93
(d, 1H), 6.87−6.84 (d, 1H), 3.85 (s, 3H), 2.82 (s, 4H).
6-Chloro-10-(propane-2-sulfonyl)-17-(4-methylpiperazin-1-yl)-
19-methoxy-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1-
(20),3(22),4,6,9(21),10,12,16,18-nonaene Trifluoroacetate (1:1)
(2l). Compound 2l was prepared in a similar manner as 2b after sub-
stituting 8l for 8b. The workup followed was akin to that for 2c. The
desired product 2l was isolated in 53% yield as a white lyophylate. LC:
1
99%. LC/MS: M + H = 557.1. H NMR (DMSO-d6) δ 9.71 (bs, 1H,
exchangeable), 9.30 (s, 1H, exchangeable), 8.38 (bs, 2H, 1 exchange-
able), 7.86 (s, 1H), 7.70−7.68 (d, J = 8.17 Hz, 1H), 7.22−7.20 (d, J =
8.16 Hz, 1H), 6.73 (s, 1H), 3.81 (s, 3H), 3.54−3.51 (d, 2H), 3.40−
3.33 (m, 1H), 3.29−3.21 (m, 2H), 3.19−3.15 (d, 2H), 3.05−2.91 (m,
9H), 1.13−1.11 (d, J = 6.77 Hz, 6H). HRMS calcd for C27H33-
ClN6O3S (MH+): 557.2102. Found: 557.2101.
6-Chloro-10-(2-methanesulfonylmethylamino)-17-(4-methyl-
piperazin-1-yl)-19-methoxy-2,4,8,22-tetraazatetracyclo-
[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9(21),10,12,16,18-non-
aene Trifluoroacetate (1:1) (2m). Compound 2m was prepared in
a similar manner as 2b after substituting 8m for 8b. The workup
followed was akin to that for 2c. The desired product 2m was isolated
in 74% yield as a white lyophylate. LC: 99%. LC/MS: M + H = 558.0.
1H NMR (DMSO-d6) δ 9.83 (bs, 1H, exchangeable), 8.32 (s, 1H,
6-Chloro-10-methoxy-17-(morpholin-4-yl)-2,4,8,22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,16,18-nonaene Trifluoroacetate (1:1) (2f). Compound 2f
was prepared in a similar manner as 2b after substituting 8f for 8b. The
desired product 2f was isolated in 19% yield as a TFA salt, white lyo-
1
phylate. LC: 99%. LC/MS: M + H = 438.2. H NMR (DMSO-d6) δ
9.38 (bs, 1H), 8.20 (bs, 1H), 8.16 (s, 1H), 7.93 (s, 1H), 7.87−7.86 (d,
1H), 7.05−6.90 (m, 4H), 3.84 (s, 3H), 3.76−3.74 (m, 4H), 3.5 (bs,
H2O), 2.96−2.89 (M, 4H), 2.80−2.78 (m, 4H). HRMS calcd for
C23H24ClN5O2 (MH+): 438.1697. Found: 438.1698.
exchangeable), 8.25 (s, 1H, exchangeable), 8.19 (s, 1H), 8.04 (s, 1H),
7.85 (s, 1H), 7.51−7.49 (d, J = 8.21 Hz, 1H), 7.11−7.09 (d, J = 8.20
Hz, 1H), 6.72 (s, 1H), 3.81 (s, 3H), 3.54−3.51 (d, 2H), 3.28−3.21 (m,
2H), 3.18−3.14 (m, 5H), 3.04−2.92 (m, 12H). HRMS calcd for
C26H32ClN7O3S (MH+): 558.2054. Found: 558.2050.
6-Chloro-10-methoxy-17-(4-methylpiperazin-1-yl)-2,4,8,22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,16,18-nonaene Trifluoroacetate (1:1) (2g). Compound
2g was prepared in a similar manner as 2b after substituting 8g for 8b.
The workup followed was akin to that for 2c. The desired product 2g
was isolated in 41% yield as a white lyophylate. LC: 99%. LC/MS: M +
Method B. 1-[3-(2-Bromo-4-nitrophenoxy)propyl]pyrrolidine
(10a). A mixture of 2-bromo-1-fluoro-4-nitrobenzene (1.10 g, 5.0 mmol),
N-β-hydroxyethylpyrrolidine (6.0 mL, 52 mmol), and K2CO3 (1.38 g,
10 mmol) was warmed to 100 °C for 24 h. The mixture was cooled to rt
and diluted with water. The resulting solid was filtered, rinsed with water,
and air-dried to yield 1.32 g (84%) of 10a as a copper colored solid. LC:
1
H = 451.2. H NMR (DMSO-d6) δ 9.6 (bs, 1H), 9.38 (s, 1H), 8.15
(s, 1H), 8.04 (bs, 1H), 7.95 (bs, 1H), 7.92−7.91 (d, 1H), 7.06−7.03
(d, 1H), 6.98−6.92 (m, 3H), 3.83 (s, 3H), 3.61 (bs, H2O), 3.53−3.50
(d, 2H), 3.25−3.22 (m, 2H), 3.10−3.07 (d, 2H), 2.99−2.96 (d, 2H),
2.92−2.87 (m, 7H). HRMS calcd for C24H27ClN6O (MH+): 451.2013.
Found: 451.2015.
1
93%. H NMR (DMSO-d6) δ 8.43−8.42 (d, 1H), 8.28−8.25 (m, 1H),
7.36−7.34 (d, 1H), 4.36−4.31 (t, 2H), 2.88−2.86 (t, 2H), 2.58−2.55 (m,
4H), 1.70−1.67 (m, 4H).
6-Chloro-10-(propane-2-sulfonyl)-17-(4-methylpiperazin-1-yl)-
2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3-
(22),4,6,9(21),10,12,16,18-nonaene (2i). Compound 2i was prepared
in a similar manner as 2b after substituting 8i for 8b. The workup
followed was akin to that for 2c except that after the extractive workup
the resulting crude material was not purified via preparative HPLC but
rather via crystallization from CH3CN. The desired product 2i was
isolated in 50% yield as a yellow tinted solid, mp >250 °C. LC: 98%. LC/
MS: M + H = 527.2. 1H NMR (DMSO-d6) δ 9.47 (s, 1H), 9.27 (s, 1H),
8.36−8.35 (d, 1H), 8.25 (s, 1H), 7.99−7.97 (d, 1H), 7.71−7.69 (d, 1H),
7.28−7.26 (m, 1H), 7.04−7.02 (d, 1H), 6.95−6.92 (m, 1H), 3.31 (bs,
1H + H2O), 3.04−2.98 (m, 4H), 2.81−2.79 (m, 4H), 2.5 (bs, 4H +
DMSO), 2.25 (s, 3H), 1.14−1.12 (d, 6H). HRMS calcd for
C26H31ClN6O2S (MH+): 527.1996. Found: 527.2007.
1-(2-Bromo-4-nitrophenyl)-4-methylpiperazine (10b). Com-
pound 10b was prepared in a similar manner as 10a after substituting
1-methylpiperazine for N-β-hydroxyethylpyrrolidine. In addition, no
K2CO3 was used, and the reaction was run at 90 °C rather than
100 °C. The desired product 10b was isolated in 97% yield as a yellow
solid. LC: 98%. 1H NMR (DMSO-d6) δ 8.38−8.37 (d, 1H), 8.21−8.18
(m, 1H), 7.29−7.27 (d, 1H), 3.18−3.16 (bm, 4H), 2.50 (s, 4H +
DMSO), 2.25 (s, 3H).
1-Bromo-2-fluoro-4-methoxy-5-nitrobenzene (10c) and 1-
Bromo-4-fluoro-2-methoxy-5-nitrobenzene (10d). 1-Bromo-2,4-di-
fluoro-5-nitrobenzene (960 mg, 4.0 mmol) was dissolved in MeOH
(20 mL). The mixture was cooled to 0 °C and treated with 0.5 M
sodium methoxide in MeOH solution (8.1 mL, 4 mmol). After 2 h the
reaction solution was concentrated under reduced pressure and the
residue was partitioned between EtOAc and water. The organic phase
was dried over Na2SO4, filtered, and concentrated under reduced
6-Chloro-10-(2-methanesulfonylmethylamino)-17-(4-methyl-
piperazin-1-yl)-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1-
(20),3(22),4,6,9(21),10,12,16,18-nonaene Trifluoroacetate (1:1) (2j).
Compound 2j was prepared in a similar manner as 2b after sub-
stituting 8j for 8b. The workup followed was akin to that for 2c. The
desired product 2j was isolated in 72% yield as a white lyophylate. LC:
1
pressure to give 833 mg (83%) of yellowish oil. H NMR spectral
analysis supported that the product was a mixture of 10c and 10d
(∼1:2 ratio). The assignments of the respective regioisomers were
determined based on further analysis of a purified 10e analogue,
generated from the subsequent step to this reaction. 1H NMR
(DMSO-d6) δ 8.41−8.39 (d, 0.63H), 8.36−8.34 (d, 0.26H), 7.57−7.54
(d, 0.30H), 7.45−7.42 (d, 0.70H), 4.01 (s, 2.2H), 3.95 (s, 0.9H).
1-(2-Bromo-5-methoxy-4-nitrophenyl)-4-methylpiperazine
(10e). The 833 mg mixture of 10c and 10d was treated at rt with 1-
methylpiperazine (10 mL, 100 mmol). After 3 h the mixture was
diluted with water and then extracted with EtOAc. The organic phase
was dried over MgSO4, filtered, and concentrated under reduced pressure
to yield 1.13 g of oil. This material was purified via preparative HPLC to
separate 10e from its respective regioisomer. The purifed fractions of 10e
from the HPLC were partitioned between EtOAc and saturated aqueous
NaHCO3. The organic phase was dried over Na2SO4, filtered, and con-
centrated under reduced pressure to give 0.26 g (24%) of desired 10e as
an oil, which crystallized upon sitting. Free basing of the purified regio-
isomer of 10e, i.e., 1-(4-bromo-5-methoxy-2-nitrophenyl)-4-methylpiperazine,
1
100%. LC/MS: M + H = 528.1. H NMR (DMSO-d6) δ 9.58 (bs,
1H), 9.38 (s, 1H), 8.21 (s, 1H), 8.18 (s, 1H), 8.10 (s, 1H), 8.02 (s,
1H), 7.53−7.51 (d, J = 8.09 Hz, 1H), 7.14−7.12 (d, J = 8.12 Hz, 1H),
7.06−7.04 (d, J = 8.43 Hz, 1H), 6.96−6.94 (d, J = 8.48 Hz, 1H), 3.53−
3.50 (d, 2H), 3.25−3.22 (m, 2H), 3.19 (s, 3H), 3.11−2.91 (m, 14H).
HRMS calcd for C25H30ClN7O2S (MH+): 528.1948. Found: 528.1951.
6-Chloro-17-(4-methylpiperazin-1-yl)-19-methoxy-2,4,8,22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,16,18-nonaene Trifluoroacetate (1:1) (2k). Compound
2k was prepared in a similar manner as 2b after substituting 8k for 8b.
The workup followed was akin to that for 2c. The desired product 2k
was isolated in 40% yield as a TFA salt, white lyophylate. LC: 99%.
1
LC/MS: M + H = 451.1. H NMR (DMSO-d6) δ 9.7 (bs, 1H), 9.36
(s, 1H), 8.14 (s, 1H), 8.12 (s, 1H), 7.90 (s, 1H), 7.76 (s, 1H), 7.25−
7.21 (t, 1H), 7.05−7.03 (d, 1H), 7.00−6.98 (d, 1H), 6.69 (s, 1H), 3.81
(s, 3H), 3.70 (bs, water), 3.53 (d, 2H), 3.27−3.24 (m, 2H), 3.14−3.11
(d, 2H), 3.01−2.98 (d, 2H), 2.94 (s, 3H), 2.92−2.91 (d, 4H).
460
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Journal of Medicinal Chemistry
Article
resulted in a return of 0.56 g (51%) of this side product. Preliminary analysis
of 10e: LC, 100%. LC/MS: M + H = 330.0.
propyl)-N′ethylcarbodiimide hydrochloride (150 mg, 0.8 mmol) was
added to a rt mixture of 8n (28 mg, 0.06 mmol), 1-hydroxybenzo-
triazole (11 mg, 0.084 mmol), methylammonium chloride (100 mg,
1 mmol), and triethylamine (100 μL, 0.7 mmol). After 7 days the
reaction mixture was partitioned between EtOAc (2×) and saturated
aqueous NaHCO3. The organic phases were combined, dried over
Na2SO4, filtered, and concentrated under reduced pressure to yield
28 mg (98%) of 8o, which was used toward 2h without further mani-
pulation. LC: 98%. LC/MS: M + H = 476.2.
A small sample of the 98% pure material was further purified via
preparative HPLC to return pure 8o as a TFA salt white lyophylate.
LC: 100%. LC/MS: M + H = 476.1. 1H NMR (DMSO-d6) δ 11.55 (s,
1H), 9.55 (bs, 1H), 9.40 (s, 1H), 9.39 (s, 1H), 8.76−8.74 (m, 1H),
8.46−8.45 (d, 1H), 8.19 (s, 1H), 7.77−7.75 (d, 1H), 7.13−7.04 (m,
3H), 6.91−6.87 (d, 1H), 6.70−6.66 (d, 1H), 3.5 (d, 2H), 3.35−3.2
(bm, 4H + H2O), 2.95−2.89 (m, 5H), 2.81−2.80 (d, 3H).
1-Methyl-4-(4-nitro-2-vinylphenyl)piperazine (11). Compound
11 was prepared in a similar manner as 3d after substituting 10b for
3c. In addition, the reaction was only heated for 5 h. The workup
varied in that after concentration of the reaction mixture under
reduced pressure, it was partitioned between EtOAc and 1 N HCl. The
aqueous phase was basified with 8 N KOH while cooling. The
resulting solid was filtered off, rinsed with ice cold water, and air-dried
yielding 11 in 70% yield as a yellow solid. LC: 98%. LC/MS M + H =
1
248.1, H NMR (DMSO-d6) δ 8.24−8.23 (d, 1H), 8.12−8.09 (m,
1H), 7.18−7.16 (d, 1H), 6.82−6.75 (m, 1H), 5.97−5.93 (d, 1H),
5.46−5.43 (d, 1H), 3.08−3.06 (m, 4H), 2.50 (s, 4H + DMSO), 2.25
(s, 3H).
3-Bromo-4-(2-pyrrolidin-1-ylethoxy)phenylamine (6c). Com-
pound 10a (0.74 g, 2.3 mmol) was combined with Raney nickel
(120 mg) in ethanol (EtOH) (50 mL), and the mixture was warmed
to reflux. Hydrazine hydrate (1.0 mL, 2 mmol) was added dropwise to
the refluxing solution, and the elevated temperature was maintained
for 1 h. After cooling, the reaction mixture was filtered and the filtrate
concentrated under reduced pressure to yield 0.58 g (87%) of 6c as a
brown oil, which was used without further manipulation. LC/MS: M +
6-Chloro-10-(N-methylcarboxamido)-17-(4-methylpiperazin-1-
yl)-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3-
(22),4,6,9(21),10,12,16,18-nonaene Trifluoroacetate (1:1) (2h). Com-
pound 2h was prepared in a similar manner as 2b after substituting 8o
for 8b. The workup followed was akin to that for 2c. The desired
product 2h was isolated in 20% yield as a TFA salt, white lyophylate.
1
1
H = 285.3. H NMR (CDCl3) δ 7.6.92−6.91 (d, 1H, J = 2.74 Hz),
LC: 100%. LC/MS: M + H = 478.2. H NMR (DMSO-d6) δ 11.2
6.79−6.77 (d, 1H, J = 8.64 Hz), 6.60−6.57 (m, 1H), 4.10−4.07 (t,
2H), 3.48 (bs, 2H), 2.94−2.91 (t, 2H), 2.68−2.65 (m, 4H), 1.84−1.80
(m, 4H).
(s, 1H), 9.61 (bs, 1H), 9.38 (s, 1H), 8.66−8.65 (m, 1H), 8.40 (s, 1H),
8.18 (s, 1H), 8.08 (s, 1H), 7.70−7.67 (d, 1H), 7.08−6.96 (m, 3H),
3.53 (d, 2H), 3.26−3.23 (m, 2H), 3.12−3.09 (d, 2H), 3.0−2.93 (m,
6H), 2.91 (d, 3H), 2.79 (d, 3H).
3-Bromo-4-(4-methylpiperazin-1-yl)phenylamine (6d). Com-
pound 6d was prepared in a similar manner as 6c after substituting
10b for 10a. The crude material was triturated with water to give 6d as
a light brown solid in 93% yield, which was used without further
Method D. 4-Bromo-1-isopropylsulfanyl-2-nitrobenzene (13).
2-Propanethiol (0.76 mL, 8 mmol) was added to a rt solution of 4-
bromo-1-fluoro-2-nitrobenzene (12) (1.10 g, 5.0 mmol) in EtOH
(5 mL), and the resulting solution was warmed at 50 °C for 4 days.
After cooling to rt, the solution was treated with water (15 mL). The
resulting yellow solid was filtered and rinsed liberally with water. After
air drying there remained 1.35 g (98%) of desired 13 as a yellow solid.
This material was used for the subsequent step without further
1
manipulation. H NMR (DMSO-d6) δ 6.90−6.88 (d, 1H), 6.82−6.81
(d, 1H), 6.54−6.51 (m, 1H), 5.03 (s, 2H), 2.79−2.77 (m, 4H), 2.43
(bm, 2H), 2.21 (s, 3H).
4-(4-Methylpiperazin-1-yl)-3-vinylphenylamine (6e). Iron (2.93 g,
52 mmol) was added at rt to a stirring solution of 11 (1.95 g, 7.9 mmol)
in THF (30 mL) and acetic acid (60 mL). The mixture was warmed to
35 °C and stirred for 16 h. The mixture was then cooled to rt, filtered,
and the resulting solution was concentrated under reduced pressure. The
residue was partitioned between CHCl3 (2×) and saturated aqueous
NaHCO3. The combined organic phases were dried over Na2SO4, filtered,
and concentrated under reduced pressure to yield 1.71 g (100%) of 6e as
a yellow tinted oil, which crystallized upon sitting. This material was used
for subsequent steps without further manipulation. LC/MS: M + H =
1
manipulation. LC: 96%. H NMR (DMSO-d6) δ 8.30−8.29 (d, 1H),
7.90−7.87 (m, 1H), 7.67−7.64 (d, 1H), 3.76−3.73 (m, 1H), 1.30−
1.28 (d, 6H).
4-Bromo-2-nitro-1-(propane-2-sulfonyl)benzene (14). m-Chloro-
perbenzoic acid (1.18 g, 6.84 mmol) was added neat to a 0 °C solution
of 13 (0.55 g, 2.0 mmol) in CH2Cl2 (15 mL). The reaction was
warmed to rt and stirred for 16 h. The reaction solution was con-
centrated under reduced pressure, and the residual white solid was
treated with saturated aqueous NaHCO3 (40 mL). After being stirred
for 10 min, the mixture was filtered and the resulting solid was washed
liberally with water. After air drying there remained 0.56 g (91%) of
desired 14 as a white solid. This material was used for the subsequent
1
218.1. H NMR (DMSO-d6) δ 7.04−6.96 (m, 1H), 6.82−6.80 (d, 1H),
6.75−6.74 (d, 1H), 6.50−6.48 (m, 1H), 5.59−5.54 (m, 1H), 5.17−5.14
(m, 1H), 4.77 (s, 2H), 2.27−2.70 (m, 4H), 2.43 (s, 4H), 2.20 (s, 3H).
5-Bromo-2-methoxy-4-(4-methylpiperazin-1-yl)phenylamine
(6f). Compound 6f was prepared in a similar manner as 6c after
substituting 10e for 10a. After workup a mass return of 100% was
returned, but 1H NMR and LC/MS confirmed the crude to be 80% 6f,
with a 20% impurity of the des-Br analogue of 6f. This crude material
was used for subsequent reactions without further manipulation. It was
also found that 6f could be prepared pure in 96% yield cleanly by
following the procedure akin to 6e after substituting 10e for 11. LC/
1
step without further manipulation. LC: 95%. H NMR (DMSO-d6) δ
8.49−8.48 (d, 1H), 8.19−8.16 (m, 1H), 7.97−7.95 (d, 1H), 3.77−3.73
(m, 1H), 1.27−1.26 (d, 6H).
5-Bromo-2(propane-2-sulfonyl)phenylamine (3f). Iron (710 mg,
13 mmol) was added at rt to a stirring solution of 14 (539 mg, 1.75
mmol) in THF (10 mL) and acetic acid (14 mL). The mixture was
warmed to 35 °C and stirred for 2 h. The mixture was then cooled to
rt, filtered, and the resulting solution was concentrated under reduced
pressure. The residue was partitioned between CHCl3 (2×) and
saturated aqueous NaHCO3. The combined organic phases were dried
over Na2SO4, filtered, and concentrated under reduced pressure to
yield 334 mg (69%) of 3f as a clear oil, which crystallized to a white
solid upon sitting. This material was used for its subsequent step
without further manipulation. LC: 97%. LC/MS: M + H = 279.8.
2-Nitro-1-(propane-2-sulfonyl)-4-vinylbenzene (15). Compound
15 was prepared in a similar manner as 3d after substituting 14 for
3a. The workup varied in that after concentration of the reaction
mixture, the residue was partitioned between EtOAc (2×) and water.
The combined organic phases were dried over MgSO4, filtered, and the
filtrate was adsorbed directly onto silica gel under reduced pressure. The
residue was purified via normal phase chromatography (EtOAc/hexane
eluent) to give desired 15 in 55% yield. LC: 99%. 1H NMR (DMSO-d6)
1
MS: M + H = 302.0. H NMR (CDCl3) δ 6.93 (s, 1H), 6.62 (s, 1H),
3.85 (s, 3H), 3.66 (s, 2H), 2.99 (bs, 4H), 2.61−2.59 (bm, 4H), 2.38
(s, 3H).
Method C. (14Z)-6-Chloro-17-(4-methylpiperazin-1-yl)-2,4,8,22-
tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3(22),4,6,9-
(21),10,12,14,16,18-decaene-10-carboxylic Acid (8n). A solution of
LiOH (11 mg, 0.46 mmol) in water (0.6 mL) was added to a solution
of 8 h (29 mg, 0.06 mmol) in MeOH (6 mL), and the resulting
mixture was warmed to 65 °C for 16 h. After cooling to 0 °C, the
mixture was treated with 1 N HCl (0.46 mL, 0.46 mmol), then con-
centrated under reduced pressure and used directly for the subsequent
reaction toward 8o without further manipulation. LC: 100%. LC/MS:
M + H = 463.1.
(14Z)-6-Chloro-10-(N-methylcarboxamido)-17-(4-methylpipera-
zin-1-yl)-2,4,8,22-tetraazatetracyclo[14.3.1.13,7.19,13]docosa-1(20),3-
(22),4,6,9(21),10,12,14,16,18-decaene (8o). N-(3-Dimethylamino-
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δ 8.26 (s, 1H), 8.02−7.98 (m, 2H), 6.95−6.87 (m, 1H), 6.27−6.23 (d,
1H), 5.67−5.64 (d, 1H), 3.78−3.70 (m, 1H), 1.28−1.26 (d, 6H).
2-(Propane-2-sulfonyl)-5-vinylphenylamine (3h). Compound 3h
was prepared in a similar manner as 6e after substituting 15 for 11.
The workup was similar except that EtOAc was used for extractions
rather than CHCl3. Compound 3h was recovered in 99% yield as a
brown oil and used directly after workup for subsequent steps without
The solid was filtered, then triturated with MeOH, refiltered, and
rinsed with a small amount of MeOH to yield desired 21b in 31% yield
as a white solid. LC: 100%. LC/MS: M + H = 270.0. 1H NMR (CDCl3)
δ 8.52−8.49 (m, 1H), 8.19 (s, 1H), 8.14 (bs, 1H), 7.15−7.03 (m, 2H),
6.96−6.94 (m, 1H), 3.96 (s, 3H).
5-Chloro-N(2)-[4-(4-methylpiperazin-1-yl)phenyl]-N(4)-phenyl-
pyrimidine-2,4-diamine Trifluoroacetate (1:1) (1b). Compound 1b
was prepared in a similar manner as 7a after substituting 21a for 5a
and 4-(4-methylpiperazin-1-yl)phenylamine (22) for 6a. Also, the
mixture was heated for 48 h rather than 16 h. After workup the crude
product was isolated as a purple tinted solid. This material was purified
via preparative reverse phase HPLC to yield the desired product 1b as
a TFA salt in 71% as a white lyophylate. LC: 100%. LC/MS: M + H =
1
further manipulation. LC/MS: M + H = 226.0 . H NMR (CDCl3) δ
7.62−7.60 (d, 1H), 6.89−6.87 (d, 1H), 6.72 (s, 1H), 6.68−6.61 (m,
1H), 5.86−5.82 (d, 1H), 5.42−5.39 (d, 1H), 5.08 (bs, 2H), 3.37−3.30
(m, 1H), 1.34−1.32 (d, 6H).
Method E. N-(4-Bromo-2-nitrophenyl)methanesulfonamide
(17). Potassium nitrate (0.34 g, 3.36 mmol) was added neat to a
0 °C solution of N-(4-bromophenyl)methanesulfonamide (16) (0.75 g,
3.0 mmol) in CH3CN (20 mL) and trifluoroacetic anhydride (2.6 mL).
After 2 h the 0 °C reaction mixture was slowly treated with saturated
aqueous NaHCO3 until the reaction mixture was basic. The resulting
solid was filtered and rinsed liberally with ice cold water. After air drying
there remained 0.75 g (85%) of desired 17 as a yellow solid, which was
used for the subsequent step without further manipulation. LC: 98%. 1H
NMR (DMSO-d6) δ 9.88 (s, 1H), 8.23 (s, 1H), 7.95−7.93 (d, 1H),
7.59−7.57 (d, 1H), 3.152 (s, 3H).
1
395.0. H NMR (DMSO-d6) δ 9.79 (bs, 1H), 9.37 (s, 1H), 8.98 (s,
1H), 8.15 (s, 1H), 7.68−7.66 (d, J = 7.90 Hz, 2H), 7.48−7.46 (d, J =
8.92 Hz, 2H), 7.38−7.34 (m, 2H), 7.17−7.13 (m, 1H), 6.88−6.86 (d,
J = 9.00 Hz, 2H), 3.74−3.71 (m, 2H), 3.54−3.51 (m, 2H), 3.18 (bm,
2H), 2.92−2.87 (m, 5H).
5-Chloro-N(4)-(2-methoxyphenyl)-N(2)-[4-(4-methylpiperazin-1-
yl)phenyl]pyrimidine-2,4-diamine Fumarate (1:1) (1c). Compounds
21b (81 mg, 0.30 mmol) and 22 (57 mg, 0.30 mmol) and 10-
camphorsulfonic acid (77 mg, 0.33 mmol) were combined in
isopropanol (3 mL) with two drops of water, and the entire mixture
was warmed under microwave conditions at 110 °C for 1 h. An
attempt to partition the reaction mixture between EtOAc and
saturated aqueous NaHCO3 led to a solid precipitating. This solid
was filtered and rinsed with a small amount of water to yield 40 mg of
crude product after drying. The crude product was dissolved in a small
amount of warm methanol and combined with a warm ethanol (1 mL)
solution of fumaric acid (12 mg, 0.10 mmol). The resulting warm
solution was filtered, and on cooling a solid precipitated. The solid was
filtered, rinsed with a small amount of ice cold ethanol, and air-dried to
yield 17 mg (13%) of 1c as a slightly purple tinted solid, mp 242−244
°C. LC: 100%. LC/MS: M + H = 425.1. 1H NMR (DMSO-d6) δ 9.13
(s, 1H), 8.10−8.07 (m, 3H), 7.42−7.40 (d, J = 8.59 Hz, 2H), 7.15−
7.13 (m, 2H), 6.96 (m, 1H), 6.82−6.79 (d, J = 8.85 Hz, 2H), 6.60 (s,
2H), 3.86 (s, 3H), 3.32 (bs, 4H + H2O), 3.06 (bs, 4H), 2.26 (s, 3H).
Biology. Kinase Enzyme and Cellular Assays and Related
Determinations of Binding and Inhibitory Activity. ALK
Assay. Compounds were tested for their ability to inhibit the kinase
activity of baculovirus-expressed human ALK cytoplasmic domain
using a modification of the assay protocol reported for trkA.19
Phosphorylation of the substrate, phospholipase C-γ (PLC-γ)
generated as a fusion protein with glutathione S-transferase
(GST),20 was detected with a europium-labeled anti-phosphotyrosine
antibody and measured by time-resolved fluorescence (TRF). Briefly,
each 96-well plate was coated with 100 μL/well of 10 μg/mL substrate
solution (recombinant GST-PLCγ) in Tris-buffered saline (TBS). The
ALK assay mixture (total volume of 100 μL/well) consisting of 20 mM
HEPES (pH 7.2), 1 μM ATP, 5 mM MnCl2, 0.1% BSA, and test
compound (diluted in DMSO, 2.5% DMSO final in assay) was then
added to the assay plate. Enzyme (50 ng/mL ALK) was added, and the
reaction was allowed to proceed at 37 °C for 15 min. Detection of the
phosphorylated product was performed by adding 100 μL/well Eu-N1
labeled PT66 antibody diluted 1:5000 in 0.25% BSA in TBS
containing 0.1% Tween-20 (TBS-T). Incubation at 37 °C then
proceeded for 1 h, followed by addition of 100 μL of enhancement
solution. The plate was gently agitated, and after 30 min, the
fluorescence of the resulting solution was measured using the
PerkinElmer EnVision 2102 (or 2104) multilabel plate reader
(PerkinElmer, Waltham, MA). Data analysis was performed using
ActivityBase (IDBS, Guilford, U.K.). IC50 values were calculated by
plotting percent inhibition versus log10 of the concentration of
compound and fitting to the nonlinear regression sigmoidal dose−
response (variable slope) equation in XLFit (IDBS, Guilford, U.K.).
IR Kinase Assay. The IR kinase assay was performed using the TRF
assay as described above for ALK. The nucleotide substrate ATP was
used at 20 μM, and recombinant human baculovirus-expressed IR
cytoplasmic domain was added to the assay at a final concentration of
20 ng/mL. In place of Eu-N1 labeled PT66 antibody, Eu-N1 labeled
N-(4-Bromo-2-nitrophenyl)-N-methylmethanesulfonamide
(18). Methyl iodide (430 μL, 6.9 mmol) was added to a mixture of 17
(0.68 g, 2.3 mmol) and K2CO3 (1.3 g, 9.2 mmol) in DMF (10 mL).
After 16 h the reaction mixture was treated with water (20 mL). The
resulting solid was filtered, rinsed liberally with water, and air-dried to
yield 0.68 g (96%) of desired 18 as a white solid, which was used for
the subsequent step without further manipulation. LC: 100%. 1H
NMR (DMSO-d6) δ 8.23 (s, 1H), 8.02−8.00 (d, 1H), 7.80−7.77 (d,
1H), 3.26 (s, 3H), 3.06 (s, 3H).
N-(2-Amino-4-bromophenyl)-N-methylmethanesulfonamide
(3g). Compound 3g was prepared in a similar manner as 3f after
substituting 18 for 11. The workup was similar except that EtOAc was
used for extractions rather than CHCl3. Compound 3g was recovered
in 98% yield as a clear oil and used directly for subsequent steps
without further manipulation. LC: 93%. 1H NMR (DMSO-d6) δ
7.14−7.12 (d, 1H), 6.92 (s, 1H), 6.69−6.67 (d, 1H), 5.43 (s, 2H), 3.05
(s, 6H).
N-Methyl-N-(2-nitro-4-vinylphenyl)methanesulfonamide (19). Com-
pound 19 was prepared in a similar manner as 3d after substituting 18
for 3a. The workup varied in that after concentration of the reaction
mixture, the residue was partitioned between EtOAc (2×) and water.
The combined organic phases were dried over MgSO4, filtered, and
the crude product in the filtrate was adsorbed directly onto silica gel
under reduced pressure. The residue was purified via normal phase
chromatography (EtOAc/hexane eluent) to give desired 19 in 69%
yield. LC: 98%. LC/MS: M + 23 = 279.0.
N-(2-Amino-4-vinylphenyl)-N-methylmethanesulfonamide (3i). Com-
pound 3i was prepared in a similar manner as 6e after substituting
19 for 11. The workup was similar except that EtOAc was used for
extractions rather than CHCl3. Compound 3i was recovered in 99%
yield as a brown oil and used directly after workup for subsequent
steps without further manipulation. LC: 97%. LC/MS: M + 23 =
1
249.0. H NMR (CDCl3) δ 7.11−7.09 (d, 1H), 6.85−6.83 (m, 2H),
6.66−6.59 (m, 1H), 5.74−5.70 (d, 1H), 5.29−5.27 (d, 1H), 4.24 (bs,
2H), 3.25 (s, 3H), 2.98 (s, 3H).
Method F. (2,5-Dichloropyrimidin-4-yl)phenylamine (21a). Com-
pound 21a was prepared in a similar manner as 5a after substituting
aniline (20a) for 3a, substituting EtN(i-Pr)2 for K2CO3, and altering
the reaction temperature from 80 °C to rt. Workup varied in that after
16 h the reaction was diluted with water, which yielded an oil that
crystallized to a solid. The solid was filtered and rinsed liberally with
water to yield desired 21a in 81% yield as a white solid. LC: 99%.
LC/MS: M + H = 240.0. 1H NMR (CDCl3) δ 8.20 (s, 1H), 7.64−7.61
(m, 2H), 7.43−7.38 (m, 2H), 7.25 (bs, 1H), 7.23−7.16 (m, 1H).
(2,5-Dichloropyrimidin-4-yl)-(2-methoxyphenyl)amine (21b). Com-
pound 21b was prepared in a similar manner as 5a after substituting
o-anisidine (20b) for 3a. Workup varied in that after 16 h the reaction
was diluted with water, which yielded an oil that crystallized to a solid.
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Journal of Medicinal Chemistry
Article
PY100 antibody (diluted 1:10000 in antibody dilution buffer) was
utilized for detection.
REFERENCES
■
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Data analysis was performed using ActivityBase (IDBS, Guilford,
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 610-738-6695. E-mail: HBreslin60@gmail.com.
ACKNOWLEDGMENTS
■
We appreciate the encouraging and helpful discussions with
Amos B. Smith III during the course of this work and the
assistance of Craig Zificsak in nomenclature assignments.
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; ALK, analplastic lymphoma kinase;
IR, insulin receptor; NPM-ALK, nucleophosmin-anaplastic
lymphoma kinase; NSCLC, non-small-cell lung cancer; EML4-
ALK, echinoderm microtubule-associated protein-like 4-anaplastic
lymphoma kinase; TPM3-ALK, tropomyosin 3-anaplastic lym-
phoma kinase; DAP, 2,4-diaminopyrimidine; PPZ, piperazine;
PDB, Protein Data Bank; HRMS, high resolution mass spectrum;
PRCG, Polak−Ribiere conjugate gradient; PLC-γ, phospholipase
C-γ; GST, glutathione S-transferase; TRF, time-resolved fluo-
rescence; TBS-T, Tween-20; rt, room temperature
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