Discovery of kinase spectrum selective macrocycle (16E)-14-methyl-20-oxa-5, 7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27) ,9,11,16,21,23-decaene (SB1317/TG02), a potent inhibitor of cyclin dependent kinases (CDKs), Janus kinase 2 (JAK2), and Fms-like tyrosine kinase-3 (FLT3) for the treatment of cancer

Article abstract of DOI:10.1021/jm201112g

Herein, we describe the design, synthesis, and SAR of a series of unique small molecule macrocycles that show spectrum selective kinase inhibition of CDKs, JAK2, and FLT3. The most promising leads were assessed in vitro for their inhibition of cancer cell proliferation, solubility, CYP450 inhibition, and microsomal stability. This screening cascade revealed 26h as a preferred compound with target IC₅₀ of 13, 73, and 56 nM for CDK2, JAK2 and FLT3, respectively. Pharmacokinetic (PK) studies of 26h in preclinical species showed good oral exposures. Oral efficacy was observed in colon (HCT-116) and lymphoma (Ramos) xenograft studies, in line with the observed PK/PD correlation. 26h (SB1317/TG02) was progressed into development in 2010 and is currently undergoing phase 1 clinical trials in advanced leukemias and multiple myeloma.

Full text of DOI:10.1021/jm201112g

Article
pubs.acs.org/jmc
Discovery of Kinase Spectrum Selective Macrocycle (16E)-14-Methyl-
20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-
1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (SB1317/TG02), a Potent
Inhibitor of Cyclin Dependent Kinases (CDKs), Janus Kinase 2 (JAK2),
and Fms-like Tyrosine Kinase-3 (FLT3) for the Treatment of Cancer
Anthony D. William,* Angeline C.-H. Lee, Kee Chuan Goh, Stephanie Blanchard, Anders Poulsen,
́
Ee Ling Teo, Harish Nagaraj, Chai Ping Lee, Haishan Wang, Meredith Williams, Eric T. Sun,
Changyong Hu, Ramesh Jayaraman, Mohammed Khalid Pasha, Kantharaj Ethirajulu, Jeanette M. Wood,
and Brian W. Dymock
S*BIO Pte. Ltd., 1 Science Park Road, No. 05-09, The Capricorn, Singapore Science Park II, 117528, Singapore
S
* Supporting Information
ABSTRACT: Herein, we describe the design, synthesis, and SAR of a series
of unique small molecule macrocycles that show spectrum selective kinase
inhibition of CDKs, JAK2, and FLT3. The most promising leads were
assessed in vitro for their inhibition of cancer cell proliferation, solubility,
CYP450 inhibition, and microsomal stability. This screening cascade
revealed 26h as a preferred compound with target IC50 of 13, 73, and 56
nM for CDK2, JAK2 and FLT3, respectively. Pharmacokinetic (PK) studies
of 26h in preclinical species showed good oral exposures. Oral efficacy was
observed in colon (HCT-116) and lymphoma (Ramos) xenograft studies, in line with the observed PK/PD correlation. 26h
(SB1317/TG02) was progressed into development in 2010 and is currently undergoing phase 1 clinical trials in advanced
leukemias and multiple myeloma.
simultaneous inhibition of CDKs 1, 2, and 9 has recently been
shown to enhance apoptotic killing of lung cancer (H1299) and
osteosarcoma cells (U2OS) compared with inhibition of a
single CDK alone.10 Small molecule CDK inhibitors have been
in clinical testing for a number of years for hematological
malignancies and solid tumors (for example, seliciclib/CYC202
(1)11 and AT7519 (2),12 Figure 1) but have shown limited
efficacy as single agents and often require intravenous
administration because of their poor oral pharmacokinetics.13
To address the deficiencies that have hampered development of
CDK inhibitors, we set out to identify an orally available
inhibitor of a new structural class meeting a specific target
product profile: inhibition of at least CDKs 1, 2, and 9, an
acceptable safety profile, and physicochemical properties
suitable for oral dosing.
JAK kinases (JAK1, JAK2, JAK3, and Tyk2) are a family of
intracellular nonreceptor tyrosine kinases that transduce
cytokine-mediated signals via the JAK-STAT pathway.14
Mutations in JAK2 have been associated with myeloprolifer-
ative neoplasms (MPNs) including polycythemia vera, essential
thrombocythemia, and myelofibrosis as well as a wide range of
leukemias and lymphomas.15−17 JAK2 inhibitors such as
INTRODUCTION
■
Recent advances in the understanding of the molecular
etiologies of cancer have engendered hopes of new targeted
therapies, but to date, these have yielded limited success with
only incremental and short-lived benefits being seen with single
agent targeted therapy.1 Blocking more than one aberrant
pathway in cancer cells is the mainstay of current standard of
care, usually with combinations of chemotherapies that
increasingly include targeted agents such as kinase inhibitors.2,3
Blockade of multiple pathways with multiple targeted therapies
is one of the most rapidly developing areas in cancer therapy.1,4
For example, multikinase inhibitors such as sorafenib,5
approved for renal cell (RCC) and hepatocellular carcinomas
(HCC), and sunitinib,6 approved for RCC and gastrointestinal
stromal tumors (GIST), are effective antiangiogenic and
antitumor agents and owe their efficacy to blocking multiple
signaling pathways. More recently, compounds combining
inhibition of two independent pathways, one of which is a non-
kinase, within a single molecular entity have entered the clinic.7
Cyclin-dependent kinases (CDKs) are serine−threonine
kinases that play important roles in cell cycle control (CDKs
1, 2, 4, and 6), transcription initiation (CDKs 7 and 9) and
neuronal function (CDK5).8 Aberrations in the cell cycle CDKs
and their cyclin partners have been observed in various tumor
types, including those of the breast, colon, liver, and brain.9 The
Received: August 18, 2011
Published: December 8, 2011
© 2011 American Chemical Society
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Figure 1. Advanced CDK, JAK2, and FLT3 kinase inhibitors in clinical trials.
Figure 2. Evolution of CDK inhibitor motif by modulating the macrocycle linking moiety.
ruxolitinib/INCB018424 (3)18 and pacritinib/SB1518 (4)19
are in advanced clinical development for the treatment of
myelofibrosis.20−22
potentially compelling profile for the treatment of advanced
cancers.
Complex natural products with macrocyclic features have
always served as a prime source of inspiration for the synthetic
organic chemist and continue to do so to this day. Almost
limitless structural variations provide diverse functionality and
stereochemical complexity in a conformationally preorganized
ring structure. From a medicinal chemist’s perspective, this can
result in high affinity and selectivity for biological targets while
preserving sufficient bioavailability to reach intracellular
locations. Despite these valuable characteristics and the proven
success of macrocycle drugs derived from natural products, this
structural class has been historically poorly explored in small
molecule drug discovery.29 This is in part due to concerns
about synthetic intractability and nondruglike properties, but
recently, there has been greater interest in small molecule
synthetic macrocycles as not just viable but attractive druglike
molecules against a wide variety of targets.30 We have
demonstrated success with small macrocycles in the area of
JAK2 inhibitors with the recent disclosure of the aforemen-
tioned pacritinib.28 Using computational modeling, we
developed CDK active macrocycles 9 bearing a basic center
that “switched on” activity against CDKs (Figure 2) while
maintaining JAK2 and FLT3 inhibition. Optimization of these
multiple target activities, aiming at IC50 in isolated ATPase
assays of less than 50 nM while achieving a balance of druglike
features, will thus be described.
Class III receptor tyrosine kinase (RTK), fms-like receptor
tyrosine kinase 3 (FLT3), plays an important role in the
maintenance, growth, and development of hematopoietic and
nonhemotopotietic cells.23 Overexpression and activating
mutations of the RTKs are known to be involved in the
pathophysiology of diverse human cancers. Mutations of the
FLT3 receptor can lead to the development of leukemia.24
Internal tandem duplications of FLT3 (FLT3-ITD) are the
most common mutations associated with acute myelogenous
leukemia (AML) and are a prognostic indicator associated with
adverse disease outcome.25 Examples of FLT3 kinase inhibitors,
which do not inhibit CDKs or JAKs (but do inhibit many other
kinases), are the marketed compounds sunitinib/SU11248
(5)26 and sorafenib/Bay 43-9006 (6).27
We have recently reported the discovery of pacritinib, a
unique macrocyclic JAK2/FLT3 dual inhibitor, as a therapy for
myelofibrosis and lymphoma.28 Herein we describe the work
carried out to identify and develop a separate series of
macrocycles targeting CDKs, JAK2, and FLT3. Although we
were aiming to develop compounds with this inhibition profile,
we did not know what level of inhibition of each enzyme would
ultimately be required; hence, this work describes efforts
toward a target profile of isolated enzyme IC50 of less than 50
nM for each target enzyme, CDK2, JAK2 and FLT3, a
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a
Scheme 1
a
Reactions and conditions: (a) tetrakis(triphenylphosphine)palladium(0), Na2CO3, 1,2-dimethoxyethane, water, 80 °C, 53−91%; (b) SnCl2·H2O,
CH2Cl2/MeOH (1:1), room temp, 56−98%; (c) 12a or 12b, allyl bromide, KOH, TBAHSO4, CH2Cl2, room temp, 55−59%; (d) 12d, vinylacetic
acid, HOBt, EDCI, CH2Cl2, room temp, 77%; (e) 12e, allylamine, HOBt, EDCI, CH2Cl2, room temp, 59%. (f) 12a, 4-bromobut-1-ene, Cs2CO3,
DMF, 40 °C, 75%; (g) 12f, N-allylmethylamine or allylamine, Na(OAc)3BH, CH2Cl2, room temp, 88−95%.
12c was reduced to free amine 12d with SnCl2·H2O and
reacted with vinylacetic acid to form amide 14a. The reverse
amide 14b was acquired from 12e using allylamine under
standard amide coupling conditions. Alkylation of phenol 12a
via cesium carbonate assisted displacement with 4-bromobut-1-
ene furnished homoallyl ether 15a. Last, reductive amination of
aldehyde 12f with N-allylmethylamine afforded 14c with
excellent yield. Left hand fragments 15b−f were obtained in
the same way as 15a, starting from the corresponding
commercially available starting materials.
CHEMISTRY
■
Synthesis of the macrocycles was achieved by coupling of the
two halves of the molecules via acidic displacement between
chloropyrimidines (left-hand fragment) and anilines (right-
hand fragment) followed by ring-closing metathesis of the
resulting dienes, employing either Grubbs second generation
catalyst or Zhan-1B catalyst31,32 with trifluoroacetic acid as an
additive. Scheme 1 illustrates the synthetic routes for
intermediates 14 and 15 and Scheme 2 describes compounds
26a−j.
Because of their diversity, right-hand fragments were
prepared using a wide variety of methods, as illustrated in
Schemes 2 and 3. Amidation proceeded with good yield
between commercially available aniline 16a and pentenoic acid
to give 18a (Scheme 2). Benzaldehyde 16b reacted with the
corresponding allylamines via reductive amination, affording
The left-hand fragments were constructed by Suzuki
coupling of commercially available 2,4-dichloropyrimidine
(10a) and boronic acids 11a−e, affording the corresponding
biaryls 12a−f (Scheme 1). Alkylation of phenol 12a and
primary alcohol 12b were achieved under phase transfer
conditions with allyl bromide, giving allyl ethers 13a,b. Nitro
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a
Scheme 2
a
Reactions and conditions: (a) 16a, 4-pentenoic acid, HOBt, EDCI, CH2Cl2, room temp, 58%; (b) SnCl2·H2O, CH2Cl2/MeOH (1:1), room temp,
56−98%; (c) 16b, N-allylmethylamine or allylamine, Na(OAc)3BH, CH2Cl2, room temp, 88−95%; (d) (CF3CO)2O, Et3N, THF, room temp, 99%;
(e) 16a, allyl bromide, Et3N, CH2Cl2, room temp, 66%; (f) (Boc)2O, NaOH, THF, room temp, 86%; (g)16c, allyl bromide, KOH, TBAHSO4,
CH2Cl2, room temp, 78%; (h) 4 M HCl, n-butanol, 80−100 °C, 59−92%; (i) Grubbs second generation catalyst, TFA, CH2Cl2, 40−45 °C, 64−89%;
(j) 4 M HCl, MeOH/CH2Cl2, 40 °C, 79%.
18b−c in excellent yields. Secondary amine 18c was capped
with trifluoroacetic anhydride to give 18d in quantitative yield.
Aniline 16a was alkylated with allyl bromide, producing 18e,
which was then capped with a Boc group to give 18f. Last, the
nitro groups of all the above-described intermediates were
reduced by the aforementioned SnCl2·H2O conditions, giving
key anilines 21a−e in moderate to excellent yields.
Couping of the left- and right-hand fragments proceeded
efficiently in hot butanol to afford 26a−j in moderate to good
yields. The resulting dienes were macrocyclized as described
above with Grubb’s second generation catalyst. RCM of
compounds with basic centers proceeds best in the presence
of an acid as an additive; hence, we attempted the reaction with
TFA (4−5 equiv) and were delighted to find that the desired
products had formed with good yield.33,34 Deprotection of 26d
was achieved with hydrochloric acid in methanol.
Schemes 3 and 4 illustrate the synthetic routes employed to
obtain the diverse right-hand fragments 21f−o and 22a−n.
Similar to the synthesis of aniline 21b, anilines 21f−h were
obtained via reductive amination and nitro group reduction,
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a
Scheme 3
a
Reactions and conditions: (a) N-allylmethylamine, Na(OAc)3BH, CH2Cl2, room temp, 75%, quantitative; (b) Fe powder, NH4Cl, EtOH, water, 80
°C, 81−93% or SnCl2·H2O, CH2Cl2/MeOH (1:1), room temp, 52−97%; (c) 16h or 16c, 1,2-dichloroethane, K2CO3, DMF, 100 °C, 64−72%; (d)
amine, Na(OAc)3BH3, CH2Cl2, room temp, 68−88%; (e) allyl bromide, K2CO3, DMF, 70 °C, 48−91%; (f) amine, DMA, 90 °C, 75−90%; (g)
morpholine, ACN, room temp, 83%; (h) NH4Cl, MeOH, 60 °C, 95%; (i) ethanethiol, K2CO3, DMF, 70 °C, 86−97%; (j) MCPBA, CH2Cl2, room
temp, 46%; (k) TFA/H2O (1:1), CH2Cl2, room temp, quantitative; (l) conc HNO3, conc H2SO4, 0 °C, 80%.
starting from commercially available 16d−f. Nitro 16g was
prepared from 16f via standard nitration conditions.
anilines 22f and 22g were derived from 16h as follows. The
alkylation of phenol 16h with 2-bromoethyl methyl ether
followed by reductive amination with N-methylamine and
subsequent reduction of the aromatic nitro function afforded
22f, while the aniline 22g was obtained by alkylation with
methyl bromoacetate to afford 16q, which on lithium hydroxide
hydrolysis furnished acid 16r. Finally, the amide formation was
accomplished with pyrrolidine to 16s under standard amide
coupling conditions. Scheme 4 describes the synthesis to obtain
a 1,3,5-trisubstituted right-hand aniline. Starting with trifluor-
oacetylation of aniline 16v to give 16w followed by ester
reduction using DIBAL gave the aldehyde which was
reductively aminated with N-allylmethylamine followed by
nitro group reduction, affording aniline 22n.
Coupling of the left- and right-hand sides followed by RCM
was carried out, as described above for Scheme 2, to give final
products 26k−q and 27a−q (Scheme 5). Following macro-
cyclization, some of the macrocycles were further modified.
Trifluoroacetyl, a group necessary to protect the linker amine
for efficient ring closing metathesis, was deprotected under
mild basic conditions to give the corresponding 26g and 27s.
Amidation of 26g with bromoacetyl bromide followed by
Diversity at the R1 position was installed by reductive
amination of 16b and 16i with the desired primary amines
giving the corresponding secondary amines 17a−i. Alkylation
with allyl bromide followed by nitro group reduction gave
anilines 21i−o. Trisubstituted anilines 22a,b, with a side chain
at the R2 position, were formed via a two-step procedure first
by displacement of the terminal chloride with amine followed
by the nitro reduction. The R2 substituent can be further
diversified from 16e to give morpholine 22c and ethyl-
sulfonylaniline 22d. More examples of anilines with group R12
were prepared as 22h−m. The nitrophenol aldehydes 16h and
16o were alkylated cleanly with 1,2-dichloroethane in the
presence of base to afford 16i and 16u in moderate to good
yields. The displacement of the chloride of 16i with ethanethiol
under basic conditions furnished 16t, which along with 16i and
16u was subjected to reductive amination followed by
displacement of the terminal chloride with secondary and
cyclic amines. Finally, the reduction of the aromatic nitro
functions afforded anilines 22h−m in good yields. The aniline
22e was obtained from 16t as described previously. The
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a
Scheme 4
a
Reactions and conditions: (a) N-allylmethylamine, Na(OAc)3BH, CH2Cl2, room temp, 75%, quantitative; (b) Fe powder, NH4Cl, EtOH, water, 80
°C, 81−93% or SnCl2·H2O, CH2Cl2/MeOH (1:1), room temp, 52−97%; (c) 16h or 16c, 1,2-dichloroethane, K2CO3, DMF, 100 °C, 64−72%; (d)
amine, DMA, 90 °C, 75−90%; (e) 2-bromoethyl methyl ether, K2CO3, DMF, 80 °C, 86%; (f) methyl bromoacetate, K2CO3, ACN, room temp, 87%;
(g) LiOH, THF/H2O (1:1), room temp, quantitative; (h) pyrrolidine, HOBt, EDCI, CH2Cl2, room temp, quantitative; (i) (CF3CO)2O, Et3N, THF,
room temp, quantitative; (j) DIBAL, CH2Cl2, −78 °C, 48%.
displacement with isopropylamine afforded 26r in a one-pot
reaction. Macrocycle 27s was further derivatized with
ethanesulfonyl chloride and 4-morpholinecarbonyl chloride in
separate reactions to produce corresponding 27t−u in
moderate yield. Macrocycles 28a−e were synthesized from
diversified left-hand fragments 15b−f using Grubbs second
generation catalyst as described previously.
conformational energy and/or had unfavorable electrostatic
interactions with the protein. Intriguingly, when the phenol
ether of 26h is modified to benzyl ether 26i, at least 1 order of
magnitude in potency is lost even though the overall length of
the linker is maintained. Some recovery against CDK2 and
JAK2 is seen when the linker of 26i is reversed to give 26j,
probably through a hydrogen bond with Asp145. Hence, if the
basic nitrogen is correctly orientated in the allylic/benzylic
position of the linker, it confers good potency most likely
through a salt bridge interaction with Asp86 (see Figure 3).
When docked into CDK2, 26i is also forming a salt bridge to
Asp86 but the benzylic ether oxygen is clashing with the
backbone carbonyl of Gln131, offering a possible explanation
for the 50-fold drop in potency compared to 26h. When
docked into FLT3, 26h adopts a slightly different preferred
conformation in order to achieve the key interaction with
Asp698. A few low energy conformations of 26h docked well
into all three proteins, and the differences in the putative
bioactive conformations seem to be due to subtle changes in
the conformation of the active site rather than homology.
However, JAK2 has a serine in place of aspartic acid; hence, the
basic nitrogen of 26h probably makes a hydrogen bond
interaction with Ser936 (Figure 4) when docked into JAK2. On
the basis of this SAR, we prioritized the promising 26h linker
for further studies.
RESULTS AND DISCUSSION
■
Selection of the Preferred Linker. In the early stages of
our kinase inhibitor research we prepared various macrocyclic
linking moieties to explore the optimal choice for both our
JAK2 program28 and CDK program described herein. In
conjunction with JAK2 and FLT3 we selected CDK2 as the
primary biochemical ATPase assay for evaluation of com-
pounds. Previously we have described dioxygen linkers with
only moderately polar character. Given the acidic residue
Asp86/Asp698 at the entrance to the CDK2/Flt3 active site, we
explored nitrogen and amide containing groups in a screen for
new interactions. The equivalent residue in JAK2 is Ser936. We
observed less active compounds with amide containing linkers;
for example, moderate activity against all enzymes was found
for phenol ether amide 26a but amides 26b and 26c with basic
centers were quite weak (IC50 > 10 μM) against the enzymes
described in Table 1. More flexible and shorter 26e was quite
active against CDK2 but not for JAK2 or FLT3. However, the
one carbon longer 26g and its N-methyl analogue 26h were
potent in the 100 nM range against all enzymes. When docked
into CDK2, the more constrained amides were higher in
Optimization of the Linker Nitrogen Substituent. We
next turned our attention to the linker nitrogen substituent.
Modeling showed that substituents of three or four atoms could
be accommodated in this part of the active site. However, only
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a
Scheme 5
a
Reactions and conditions: (a) 4 M HCl, n-butanol, 80−100 °C, 49−97%; (b) Grubbs second generation catalyst, TFA, CH2Cl2, 40−45 °C, 42−
89%; (c) K2CO3, H2O, MeOH, room temp, 48−58%; (d) (i) bromoacetyl bromide, CH2Cl2, room temp; (ii) isopropylamine, DIEA, CH2Cl2, room
temp, two steps 39%; (e) ethanesulfonyl chloride, Et3N, CH2Cl2, room temp, 40%; (f) 4-morpholinecarbonyl chloride, Et3N, CH2Cl2, room temp,
48%.
the smallest lipophilic substituents (26h, 26k and compare with
26g) were potent against all enzymes (Table 2). All enzymes
are similarly sensitive to steric effects (26l, 26m), with activity
being completely lost with the very bulky tert-butyl 26n (see
Figures 3 and 5 where docking of 26h against CDK2 shows
little available space particularly for bulky substituents).
Increases in log P and reduction in solubility were undesirable
trends in this series. Moderate activity was regained with the
more planar pyridyl 26o and longer methoxyethyl 26p, but
these groups were still over an order of magnitude less active
than 26h. Introduction of hydrogen-bonding functionality
regained some CDK2 potency as seen with 26q, but this was
not observed with 26r, the latter being less flexible and α-
branched. However, the activity of 26r could be considered
surprising when compared with electron-withdrawing trifluor-
oacyl 26f. All activity is abolished with 26f, presumably through
destruction of the salt bridge interaction with Asp86 coupled
with unfavorable steric clashes with the enzyme. This surprising
activity of 26r is likely due to the hydrogen bond/salt bridge
interaction with Asp86 being achieved through the basic center
of the isopropylaminoacyl group (see Figure S1, Supporting
Information). Despite the striking SAR, none of these
compounds were close to the activity of the highly potent
simpler analogues. Hence, we were persuaded by the data to
avoid adding unnecessary molecular weight to this part of the
molecule.
Other examples of electron-withdrawing N-substituents were
prepared but with an additional R2 substituent on the phenyl
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Table 1. SAR of Diverse Linkers
a
b
c
All compounds were isolated as 95:5 trans/cis mixtures. All IC50 values are the mean SD (of at least two independent experiments). cLogP was
calculated using QikProp.35
Figure 3. Macrocycles docked into CDK2. The CDK2 ATP-binding site is shown in thin tube with gray carbon. Inhibitors are shown in thick tube
with green carbon. (A) In addition to the hydrogen bonds to the hinge residue Leu83, 26h is forming a salt bridge to Asp86. (B) 26i is also forming
a salt bridge to Asp86, but the ether oxygen is clashing with the backbone carbonyl of Gln131, resulting in a lower IC50.
ring (Table 3, 27l−m, pKa of the basic nitrogen of
approximately 6 for both compounds). Nevertheless, a
comparison between these two compounds is instructive:
trifluoroethyl 27l suffers complete loss of all CDK2 activity but
a less significant impact on either JAK2 or FLT3, losing only 3-
to 4-fold; cyanomethyl 27m retains CDK2 potency (compared
to 27k) despite being similar electronically to the trifluoroethyl.
Cyano is a much smaller group than trifluoromethyl,
supporting the hypothesis that a small substituent is preferred
for CDK2 potency. For optimum CDK2 activity in this series it
is vital to preserve the strength of the Asp86 salt bridge by
retaining basicity of the nitrogen center of at least pKa = 9 (see
electrostatic surface in Figure 5).
Exploration of Aromatic Substituents at R2 and R3.
With the optimal N-methyl installed in the linker, various side
chains were studied at the R2 position. Lipophilic groups such
as methoxy 27a, chloro 27b, trifluoromethoxy 27c, and
methoxyethoxy 27d were slightly less active against all enzymes
when compared to 26h (Table 3). However, 27b does retain a
profile very similar to that of 26h, but the cLogP is higher. In an
effort to reduce cLogP and increase solubility while maintaining
potency, more polar substituents were installed such as sulfones
27e and 27g and amide 27h. Unfortunately these compounds
all lost activity against either CDK2 or FLT3. The branched
amine substituents, such as morpholine 27i, were tolerated
quite well against JAK2 and FLT3 but lost over 5-fold activity
against CDK2. Efforts then turned to basic side chains installed
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Figure 4. (A) 26h docked into FLT3. The predicted bioactive conformation of the macrocycle is different from that docked into CDK2. However,
the interactions with the protein are conserved with two hydrogen bonds formed to the hinge backbone and a salt bridge to Asp698. (B) 26h docked
into JAK2. The conformation of the macrocycle is different from that docked into CDK2 and FLT3. The two hydrogen bonds formed to the hinge
backbone are conserved, but instead of the salt bridge interaction there is a hydrogen bond to Ser936.
Table 2. SAR Study of the Amine Substituent
a
b
c
All compounds were isolated as 95:5 trans/cis mixtures. All IC50 values are the mean SD (of at least two independent experiments). cLogP was
calculated using QikProp.35 High throughput solubility in PBS buffered at pH 7.0. Denotes not tested.
d
e
on an ethoxy linker, which we had found to be productive in
our selective JAK2 program.28 Compounds 27j and 27k had
good JAK2 and FLT3 activity, but CDK2 activity was reduced
by over an order of magnitude when compared to 26h.
Unfortunately, this trend continued with analogues 27n, 27o,
and 27p. Furthermore, we determined the PK profile of 27j
which was only 5% bioavailable with a very high volume of
distribution (see Table S1 and Figure S4). It is most probable
that other very close derivatives with two basic centers and a
high cLogP (27j−q) would exhibit a similar undesirable profile.
Our attentions then turned to the neighboring position on the
aromatic ring, R3. Combination of R2 and R3 was explored with
27q, which retained potency for JAK2 and FLT3 but
dramatically lost activity against CDK2, probably as a result
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activity in earlier analogues. Although only a cursory survey
with a limited group of compounds, these data give little
justification for further studies of biaryl substitutents
Further in Vitro Profiling of Preferred Compounds.
Selected compounds with broad spectrum potency (IC50 < 100
nM) across the three target enzymes were profiled further to
determine their in vitro ADME properties and cell proliferation
inhibition in a range of solid and hematological cell lines (Table
5). HL-60 cells (acute myeloid leukemia), a cell line known to
be sensitive to CDK inhibitors, were used to initially screen
compounds. Most compounds were then assessed for their
human liver microsomal (HLM) stability. The most potent
CDK2 inhibitor, 27r, was also the most potent in HL-60 cells
with a IC50 for proliferation of 6 nM. Such high cellular activity
could reflect the combinatorial strategy of inhibiting several key
signaling pathways at the same time. This compound was only
moderately stable in HLM (t1/2 = 21 min) and did not meet
our target profile for a once per day oral therapy and so was not
profiled further. A similar set of data revealed 27i and 27t to be
potent in cells but not satisfactorily stable enough in HLM. HL-
60 is a FLT3-driven cell line; hence, the lower HL-60 IC50 for
27t compared to 27r is most likely related to its reduced FLT3
activity. The least active compound in the HL-60 cell
proliferation assay was the N-cyclopropyl derivative 26k with
an IC50 of 1.38 μM, but this compound did have good stability
in HLM and mouse liver microsomes (MLM); hence, it was
profiled in a range of other cell lines. Unfortunately its poor cell
potency was a general feature, perhaps because of its low
solubility, and it was not progressed further. Potent CDK2
inhibitor 27b was disappointing in terms of cell proliferation
activity in three cell lines despite its good enzyme activity. The
high cLogP of 4.5 indicates that there may be a solubility
problem, but this was not studied further. The remaining
compounds, 26g and 26h, were potent against all the cell lines
tested including solid tumor cell lines such as colon (HCT-116,
COLO205) and prostate (DU145). In general both com-
pounds had a broadly similar pharmacological profile but 26h
was always more active in cells, particularly the prostate cancer
cell line DU145 where there was a 5-fold difference between
the compounds. Higher cellular potency of 26h is probably due
to its generally higher potency against the enzymes but could
also be due to better solubility and permeability. Furthermore,
26g was significantly less soluble than 26h. Taken together,
these data support 26h as the preferred compound, and as such,
it was selected for advanced profiling.
Intracellular Pharmacodynamic Marker Studies and
Kinase Profiling of 26h. Intracellular pharmacodynamic
marker studies showed that 26h potently inhibited the CDK2
biomarker pRb (phospho-Rb, retinoblastoma tumor suppressor
protein) in HCT-116 (Figure 8). Effects could be detected at
the 40 nM with the protein phosphorylation being completely
inhibited at 200 nM. Similar studies in leukemic cell lines36
showed that 26h was also potent against pRb in MV4-11 cells
(IC50 = 0.13 μM) and also inhibited pFLT3 and pSTAT5 in the
same cell line.
Extensive biological characterization, including kinase profil-
ing, intracellular mechanistic studies, and antiproliferative
effects on a wide range of leukemic cell lines, was reported
elsewhere.36 These data show that 26h has a highly novel
kinase inhibitory spectrum inhibiting 17 kinases from a panel of
63, 11 of which are CDK/JAK/FLT family members. The
others, Lck, Fyn, Fms, TYRO3, ERK5, and p38δ, are implicated
in inflammatory and proliferative processes, and further
Figure 5. 26h docked into CDK2 which is shown with an electrostatic
surface. The methyl substituent on the basic nitrogen is just the right
size. A slightly larger substituent like cyclopropyl may be
accommodated but will clash slightly with the protein. Larger or
more bulky substituents cannot be docked without compromising the
salt bridge to Asp86.
of a repulsive interaction with the carbonyl of His84 (this
interaction is not seen in docking studies of JAK2 and FLT3).
Hence mono-R3 substitutions were explored in further detail.
Very active compounds were found when aniline substituents
were installed: 27r, 27s, and 27t were all slightly more active
than 26h with IC50 below 10 nM. Potencies for JAK2 and FLT3
were generally also good for these compounds. An additional
hydrogen bond to His84 at the end of the hinge region is most
likely responsible for the high CDK2 activity (Figure 6).
Compound 27u was the exception, being over 10-fold less
potent against CDK2 probably because of the inflexible urea
group not being able to adopt a suitable conformation to
maintain the hydrogen bond donor to His84. Despite these
data, we felt that these compounds did not offer enough gains
in potency for the increase in molecular weight; hence, the
lower molecular weight 26h was favored.
Exploration of Biaryl Substitutions (Table 4). A small
number of compounds confirmed that substitution at R4 was
not tolerated by any of the target enzymes (28a and 28b).
Methyl substitution at R5 (28c) was expected to boost JAK2
potency28 but surprisingly reduced potency slightly for JAK2
and by an order of magnitude for CDK2. One possible
explanation for this is the twist induced in the biaryl system,
predicted by conformational analysis, reducing interactions
between the phenyl ring and Phe80 (Figure 7). On the other
hand, fluoro substitution at R6 (28d) did produce a CDK2 and
FLT3 potent compound (28d CDK2 IC50 = 9 nM and FLT3
IC50 = 22 nM), with only a slight reduction in JAK2 potency.
When docked into CDK2/FLT3, the fluoro of 28d is pointing
toward the backbone NH of Asp145/Asp829. In JAK2 the
peptide bond involving the homologous backbone NH is
rotated 180° compared to CDK2. So when docked into JAK2,
the fluoro is pointing toward the backbone carbonyl of Gly993.
Substitution at R7 was explored with a methoxy group (28e),
but this resulted in dramatic loss of CDK2 potency and
considerable loss of JAK2 and FLT3 activity. This was not
surprising because of the likely close interactions between the
methoxy and Lys33 and Asp145 in that part of the binding site
(Figure 7). The phenyl ring ortho substituents were not studied
because of the anticipated twist imparted to the biaryl system,
which we envisioned to be unfavorable for our desired target
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Table 3. SAR Study of Amine and Aromatic Ring Substitutions
a
b
c
All compounds were isolated as 95:5 trans/cis mixtures. All IC50 values are the mean SD (of at least two independent experiments). cLogP was
calculated using QikProp.35 High throughput solubility in PBS buffered at pH 7.0. Denotes not tested.
d
e
biological studies are underway to better understand these
activities in an in vivo setting.
Extensive ADME Profiling of 26h. In the Caco-2
bidirectional permeability assays, the permeability (Papp) of
26h in the apical to basolateral (Papp,A→B) direction and in the
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plasma Cmax observed at the maximum tolerated dose.
Compound 26h was highly bound to plasma proteins in
human, mouse, and dog plasma with PPB ranging between
99.4% to 99.9%.
Pharmacokinetics of 26h in Mice. The PK properties of
26h in mice are summarized in Table 7. 26h showed high
systemic clearance relative to liver blood flow and high volume
of distribution at steady state, with a terminal half-life of ∼5.0 h.
It showed rapid absorption (tmax = 0.5 h) and a mean Cmax and
AUC of 1029 ng/mL and 2523 ng·h/mL, respectively, with a
mean terminal half-life of 6.1 h following a single oral dose of
75 mg/kg. It showed an acceptable oral bioavailability of 24%.
The exposures achieved in mice at the 75 mg/kg dose far
exceeded the enzyme inhibiton (CDK2 IC50 = 0.013 μM, JAK2
IC50 = 0.073 μM, and FLT3 IC50 = 0.056 μM) and cell
proliferation concentrations in HCT-116 (IC50 = 0.079 μM)
and HL-60 (IC50 = 0.059 μM), correlating with the observed
efficacy of 26h in preclinical pharmacology models at similar
doses.36
Efficacy of 26h in Xenograft Models. On the basis of its
efficacy on a broad spectrum of tumor cell lines and good oral
bioavailability, 26h was selected for evaluation in human tumor
xenograft mouse models. Two models were selected based on
their relevance in cancer: HCT-116 colon cancer and Ramos B-
cell lymphoma. Prior to conducting both experiments, dosing
regimes in each model were explored and optimal schedules
selected for each model that would be tolerated for the duration
of the experiment.36
Figure 6. 27s docked into CDK2. The aniline nitrogen is forming an
additional hydrogen bond to the backbone carbonyl of His84 at the
end of the kinase hinge region. This is probably not a strong hydrogen
bond as evident from the increase in activity of only 2-fold over 26h.
basolateral to apical (Papp,B→A) direction was 28.0 × 10−6 and
27.4 x10−6 cm/s, respectively. The efflux ratio, defined as the
ratio of Papp, B→A to Papp,A→B, was less than 3 (1.0), indicating
that 26h was not a substrate for efflux by intestinal P-gp
transporters, suggestive of high intestinal absorption in humans
(Table 6). In human liver microsomes (HLM) 26h was found
to be stable with a half-life of 45 min, was moderately stable in
DLM (t1/2 = 33 min), and was quite rapidly cleared in MLM
(t1/2 = 12 min) and in RLM (t1/2 = 11 min). Human CYP1A2,
3A4, 2C9, and 2C19 isoforms were not inhibited by 26h at the
highest tested concentration of 25 μM, but the compound
inhibited CYP2D6 with IC50 = 0.95 μM, approximately at the
In the colon cancer model, HCT-116 cells were injected
subcutaneously and tumors were established with mean group
sizes of approximately 100 mm3. Treatment with 26h at doses
of 50 and 75 mg/kg po 3 times per week on a Monday,
Wednesday, Friday schedule was started 8 days after cell
inoculation for 15 days. Treatment with 26h at 75 mg/kg po
q.d. 3×/week significantly inhibited the growth of tumors with
Table 4. SAR Study of Aromatic Ring Substitutions
a
b
c
All compounds were isolated as 95:5 trans/cis mixtures. All IC50 values are the mean SD of at least two independent experiments. cLogP was
calculated using QikProp.35
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Figure 7. (A) 28c docked into CDK2. In the putative bioactive conformation, the methyl group interacts favorably with Phe80. However, the methyl
group induces a twist in the A and C rings, so they are less coplanar, shifting the A-ring slightly away from Phe80 and thus preventing the A-ring
from interacting with Phe80. (B) 28e docked into CDK2. The orange-black dashed lines indicate clashes between protein and ligand atoms. The
methoxy substituent clashes with the side chains of Lys33 and Asp145. These are flexible residues and may move to accommodate the substituents.
However, this is energetically unfavorable, as it will break the salt bridge between the two residues.
Table 5. In Vitro Profiling of Preferred Compounds
a
a
IC50 (μM)
t1/2 (min)
IC50 (μM)
b
compd
CDK2
JAK2
FLT3
solubility (μg/mL)
HLM
MLM
HL-60
HCT-116
RAMOS
COLO205
DU145
c
26g
26h
26k
27b
27i
0.021
0.013
0.036
0.024
0.074
0.008
0.008
0.15
0.11
14.7
71.8
16.6
48
10
0.089
0.059
1.38
0.135
0.079
1.6
ND
0.12
0.072
3.6
0.71
0.14
3.0
0.073
0.090
0.082
0.049
0.018
0.050
0.056
0.042
0.077
0.071
0.14
45
12
0.033
c
>60
>60
ND
c
c
c
c
c
ND
ND
21
ND
0.34
0.49
0.41
ND
0.43
ND
c
c
c
c
147.0
82.4
ND
0.076
0.13
ND
ND
ND
c
c
c
c
27t
27r
30
7
ND
ND
ND
ND
c
c
c
c
c
c
0.019
ND
21
ND
0.006
ND
ND
ND
ND
a
b
c
All IC50 values are the mean of at least two independent experiments. High throughput solubility in PBS at pH 7.0. ND denotes not tested.
Table 6. Physicochemical Properties and in Vitro ADME of
26h
property
value
molecular wt
no. of HDB
no. of HBA
372.47
1
4
a
cLogP
PSA (Å2)
4.1
43
a
b
permeability (Papp,A→B, ×10−6 cm/s)
28 (efflux ratio of 1.0)
Figure 8. HCT-116 cells were treated separately with the indicated
concentrations of 26h for 24 h prior to denaturing lysis. An amount of
30 μg of lysate from each treatment was resolved on 10% SDS−PAGE,
transferred onto PVDF membrane, and probed with antibodies against
phospho-Rb and β-actin.
metabolic stability (t1/2, min)
HLM
45
12
11
33
MLM
RLM
DLM
c
d
a mean TGI of 82%, while the lower dose of 50 mg/kg po 3×/
week was marginally effective (Figure 9).
human CYP inhibition IC50 (μM)
>25, 0.95
e
plasma protein binding (%)
In the lymphoma model Ramos cells were injected
subcutaneously and tumors were established with mean group
sizes of approximately 200 mm3. Two different dosing regimens
of 26h were explored in this model: 75 mg/kg po q.d. on a 2
days on and 5 days off schedule and 15 mg/kg ip q.d. on a 5
days on 5 days off schedule were started 12 days after cell
inoculation for 15 days. There were two vehicle control groups
that received either MC/Tween or DMA/CRE (see Exper-
imental Section for details). The treatment groups were
compared with the corresponding vehicle control groups for
human
mouse
dog
99.9
99.4
99.9
cLogP and PSA were calculated using QikProp.35 Caco-2 bidirec-
tional permeability assay. CYP3A4, 1A2, 2C9, 2C19. CYP2D6.
Equilibrium dialysis assay in plasma at 1000 ng/mL.
a
b
c
d
e
assessment of percentage TGI. Treatment with 26h using
either regime significantly inhibited the growth of tumors with
mean TGIs of 42% and 63% for the oral and ip delivery
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Table 7. Pharmacokinetics of 26h in Mice
(A) Intravenous Administration
dose (mg/kg) Cl (L h−1 kg−1) Vss (L/kg) t1/2 (h) AUC0−∞ (ng·h/mL)
5.0
6.6
23
4.6
752
a
(B) Oral Administration
dose
(mg/kg)
tmax
(h)
Cmax
(ng/mL)
t1/2
(h)
AUC0−∞
(ng·h/mL)
F
(%)
75
0.5
1029
6.1
2523
24
a
In preliminary PK studies, 26h showed oral bioavailability (F) of 4%
and 37% in rats and dogs, respectively.
Figure 10. 26h is efficacious in a sc xenograft model of human B-cell
lymphoma (Ramos). Ramos tumors were established in female BALB/
c nude mice by sc implantation of 7 × 106 Ramos cells. Treatment
with 26h and vehicle (MC/Tween for po or DMA/CRE for ip) was
initiated on day 1 and continued until day 15. The tumor volumes
from the 15 mg/kg ip group was significantly different compared to
the ip vehicle control group on day 15. Sample size, n = 8−10 mice per
group.
Figure 9. 26h is efficacious in a murine sc xenograft model of human
colon cancer (HCT-116). Tumors were established in female BALB/c
nude mice by sc implantation of 5 × 106 HCT-116 tumor cells. Tumor
burden (volume, mm3) was measured twice a week by (w2l)/2, where
w is the width and l is the length in mm of an HCT-116 carcinoma.
Treatment started on day 1 when the group mean tumor volume
reached 105 mm3 and treatment ended on day 15.
spectrum selective profile not previously reported. Application
of a hypothesis of conformational constraint generated
macrocycles that were synthesized using a RCM strategy.
Screening of initial compounds in functional biochemical assays
against CDK2, JAK2, and FLT3 kinases allowed selection of a
preferred linker moiety containing a phenolic ether, trans
double bond, and allylic/benzylic N-methyl group. SAR and
broader in vitro profiling, particularly cellular assays, identified
26h, a small molecule kinase inhibitor with a distinct kinase
inhibitory spectrum, as the preferred lead candidate. Further
evaluation revealed excellent pharmacokinetic properties of 26h
and dose-dependent efficacy in mouse models of cancer
including a HCT-116 model of colon cancer and a Ramos
model of lymphoma. On the basis of its favorable
pharmaceutical and pharmacological profile, 26h (SB1317/
methods, respectively (Figure 10). Given the encouraging TGIs
observed with both oral and ip dosing schedules in these
challenging models, 26h was selected for further preclinical
development.36
CONCLUSIONS
■
We have described the discovery of a series of small molecule
macrocycles as potent inhibitors of CDKs, JAK2, and FLT3, a
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−20 °C was added pyrrolidine (0.83 mL, 10.0 mmol) dropwise over a
period of 30 min. The resulting mixture was allowed to warm to room
temperature over 1 h. The reaction mixture was concentrated under
reduced pressure, and CH2Cl2 (50 mL) was added. It was washed with
1 N NaOH, water, dried over Na2SO4, and concentrated under
reduced pressure to obtain the crude residue. The crude material was
purified by column chromatography (EtOAc/hexane, 1:8) to afford
10d (1.15 g, 53%). LC/MS (ESI positive mode) m/z 218 ([M + H]+)
C8H9Cl2N3.
TG02) was advanced into development and is currently being
evaluated in phase 1 clinical trials in leukemia patients.
EXPERIMENTAL SECTION
■
Chemistry. Solvents and reagents were used directly from the
suppliers without further purification and were of the highest generally
available quality. Workup for chemical reactions was typically done by
diluting the reaction mixture or residue with the reaction solvent or
extraction solvent and then washing with the indicated aqueous
solution(s). Product solutions were dried over anhydrous sodium
sulfate prior to filtration, and the solvents were removed under
reduced pressure using a rotary evaporator. All compounds were
purified by either flash or reverse phase chromatography. Flash column
chromatography was conducted using silica gel 60 (Merck KGaA,
0.040−0.063 mm, 230−400 mesh ASTM). Reverse-phase preparative
high performance liquid chromatography (rpHPLC) was operated on
a Phenomenex column (Luna 5 μm C18 (2) 100A 150 mm × 21.2
mm) with adjustable solvent gradients, usually 5−95% acetonitrile in
water + 0.1% trifluoroacetic acid (TFA) with a run time of 18 min at a
flow rate of 20 mL/min, and was used for routine purification.
Fractions containing the desired product were lyophilized or
evaporated to dryness under vacuum to provide the dry compound
or evaporated to remove volatile organic solvents and then extracted
with a suitable organic solvent (ethyl acetate or dichloromethane were
commonly used; if necessary, the pH of the aqueous solution was
adjusted in order to get free base, acid, or the neutral compound).
The preliminary purity and identity of all compounds were assessed
after purification by tandem HPLC/mass spectrometry (LC/MS)
analyses on a Waters Micromass ZQ mass spectrometer in
electrospray ionization (ESI) positive mode after separation on a
Waters 2795 separations module. The HPLC separations were
performed on a Phenomenex column (Luna 5 μm C18 (2) 100A 50
mm × 2.00 mm) with a flow rate of 0.8 mL/min and a 4 min gradient
of x − 100% acetonitrile in water + 0.05% TFA (x = 5 or 30 or 50),
using a Waters 2996 photodiode array detector. Purity and identity
were assessed on the integrated UV chromatogram (220−400 nm)
and the mass spectrum (homogeneity of the product peak and its
fragmentation(s)).
3-(2-Chloropyrimidine-4-yl)phenol (12a). To a degassed
(argon) solution of 2,4-dichloropyrimidine (10 g, 67.1 mmol) and
3-hydroxyphenylboronic acid (11.1 g, 80.5 mmol) in 1,2-dimethoxy
ethane (150 mL) were added aqueous NaHCO3 (10.6 g in 15 mL
H2O) and freshly prepared Pd(PPh3)4 (7.7 g, 6.6 mmol). The reaction
mixture was heated to 80 °C for 7 h. It was cooled to room
temperature and quenched with saturated NH4Cl solution. The
reaction mass was extracted with CH2Cl2 (3 × 50 mL). The combined
organic extracts were washed with brine, dried over Na2SO4, and
concentrated under reduced pressure to obtain the crude residue. The
crude material was purified by column chromatography (EtOAc/
hexane, 1:4) to afford 12a (12.20 g, 88%) as an off white solid. LC/MS
1
(ESI positive mode) m/z 207 ([M + H]+) C10H7ClN2O; H NMR
(CDCl3) δ 9.71 (br, 1H), 8.71 (d, 1H), 7.98 d, 1H), 7.49−7.54 (m,
2H), 7.30 (t, 1H), 6.94 (dd, 1H); 13C NMR (CDCl3) δ 166.7, 161.58,
160.95, 158.54, 136.30, 130.77, 119.6, 118.6, 117.5, 116.5, 114.2. Anal.
Calcd for C10H7ClN2O: C, 58.13; H, 3.14; N, 13.56; Cl, 17.16. Found:
C, 58.22; H, 3.33; N, 13.32; Cl, 13.45. IR (KBr pellet): 3173, 1536,
1360, 789, 625, 1082, 791 cm−1.
[3-(2-Chloropyrimidine-4-yl)phenyl]methanol (12b). The
synthesis for this intermediate was prepared using a different Suzuki
coupling procedure compared to 12a. To a degassed (nitrogen)
solution of 10a (50 g, 335 mmol) and 3-(hydroxymethyl)-
phenylboronic acid 11b (48.5 g, 318 mmol) in THF (500 mL, 10
vol) were added saturated Na2CO3 (88.6 g, 838 mmol), palladium
acetate [Pd(OAc)2] (0.15 g, 0.67 mmol), and triphenylphosphine
(0.35 g, 1.34 mmol). The resultant mixture was stirred at 70 °C for 6 h
and filtered. Water was added to the filtrate, and it was extracted with
EtOAc (3 × 250 mL). The combined organic extracts were washed
with brine, dried (Na2SO4), and filtered. The filtrate was treated with
activated charcoal, and the mixture was filtered. The filtrate was
concentrated under reduced pressure, and the crude product was
slurried in isopropyl alcohol (300 mL). The mixture was then filtered
and the solid was slurried in cold n-heptane and was again filtered to
furnish 12b (42.8 g, 61%). This material was taken up for the next step
without any purification. LC/MS (ESI positive mode) m/z 221 ([M +
The final purity was determined using a Waters 2695 separations
module on a Waters Xterra RP18 3.5 μm, 4.6 mm × 20 mm IS column
with a flow rate of 2.0 mL/min, gradient 5−65% B over 4 min, then
65−95% B over 1 min, and 95% B for an additional 0.1 min (solvent
A, H2O with 0.1% TFA; solvent B, acetonitrile with 0.1% TFA), and a
Waters 2996 photodiode array detector. For compounds not suitable
for the above HPLC methods either because of polarity or poor peak
separation, a longer column (Phenomenex Gemini 5 μm C18, 110A,
4.6 mm × 150 mm) together with a flow rate of 1.0−1.2 mL/min and
a 15 min gradient of 5−95% acetonitrile in water + 0.1% TFA was
used for purity determination. Purity was >95% for all test compounds
except those indicated in their respective synthesis descriptions.
1
H]+) C11H9ClN2O; H NMR (DMSO-d6) δ 8.81 (d, 1H), 8.15 (br,
1H), 8.10 (d, 1H), 8.04 (dt, 1H), 7.57−7.54 (m, 1H), 4.63 (s, 2H);
13C NMR (DMSO-d6) δ 166.7, 161.4, 160.8, 143.9, 134.7, 130.5,
129.4, 126.1, 125.4, 116.4, 62.9.
Following a procedure similar to that for 12a, the following
intermediates were synthesized.
1
All the 1D and 2D NMR experiments for H (400.13 MHz) and
13C (100.61 MHz) nuclei were performed on a Bruker AVANCE-400
2-Chloro-4-(3-nitrophenyl)pyrimidine (12c). The title com-
pound was synthesized from 10a and 3-nitrophenylboronic acid 11c
(91%). LC/MS (ESI positive mode) m/z 236 ([M + H]+)
C10H6ClN3O2; 1H NMR (CDCl3) δ 8.94 (t, 1H), 8.76 (d, 1H),
8.48 (qd, 1H), 8.40 (m), 7.77−7.70 (m, 2H).
1
1
digital NMR spectrometer. H−1H and H−13C experiments (COSY,
HMQC, HSQC, and HMBC) were run with Z-gradient selection.
NMR spectra are reported in ppm with reference to an internal
tetramethylsilane standard (0.00 ppm for 1H and 13C) or solvent
peak(s) of CDCl3 (7.26 and 77.1 ppm) or CD3OD (3.31 and 49.0
ppm) or DMSO-d6 (2.50 and 39.5 ppm). Other NMR solvents were
used as needed. When peak multiplicities are reported, the following
abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, br = broadened, dd = doublet of doublets, dt = doublet
of triplets, and bs = broadened singlet. Coupling constants, when
given, are reported in hertz.
3-(2-Chloropyrimidin-4-yl)benzoic Acid (12e). The title
compound was synthesized from 10a and 3-carboxyphenylboronic
acid 11d (63%). LC/MS (ESI positive mode) m/z 235 ([M + H]+)
1
C11H7ClN2O2; H NMR (MeOD-d4) δ 8.66 (s, 1H), 8.62 (d, 1H),
8.29 (d, 1H), 8.17 (d, 1H), 7.69 (d, 1H), 7.55 (t, 1H).
3-(2-Chloropyrimidin-4-yl)benzaldehyde (12f). The title com-
pound was synthesized from 10a and 3-carbonylphenylboronic acid
11e (60%). LC/MS (ESI positive mode) m/z 219 ([M + H]+)
C11H7ClN2O.
3-(2-Chloro-6-methylpyrimidin-4-yl)phenol (12g). The title
compound was synthesized from 2,4-dichloro-6-methylpyrimidine 10b
and 3-hydroxyphenylboronic acid 11a (81%). LC/MS (ESI positive
mode) m/z 221 ([M + H]+) C11H9ClN2O.
All melting points are uncorrected. Elemental analyses of CHN
were performed on a Perkin-Elmer 2400 CHN/CHNS elemental
analyzer. HRMS data were obtained from a Bruker microTOF-Q II
with direct injection.
2,4-Dichloro-6-pyrrolidin-1-ylpyrimidine (10d). To a mixture
of 2,4,6-trichloropyrimidine (1.83 g, 10.0 mmol) in THF (50 mL) at
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3-(2-Chloro-6-pyrrolidin-1-ylpyrimidin-4-yl)phenol (12h).
The title compound was synthesized from 10d and 3-hydroxyphe-
nylboronic acid 11a (56%). LC/MS (ESI positive mode) m/z 276
([M + H]+) C14H14ClN3O.
3-(2-Chloro-5-methylpyrimidin-4-yl)phenol (12i). The title
compound was synthesized from 2,4-dichloro-5-methylpyrimidine
10e and 3-hydroxyphenylboronic acid 11a (66%). LC/MS (ESI
positive mode) m/z 221 ([M + H]+) C11H9ClN2O.
added HOBt (0.184 g, 1.36 mmol) and EDCI (0.26 g, 1.36 mmol).
The resulting mixture was stirred for 4 h. The reaction mixture was
cooled to 0 °C and quenched with H2O. The aqueous layer was
extracted with CH2Cl2 thrice, and the combined organic extracts were
washed with saturated NaHCO3 followed by brine, dried over Na2SO4,
and concentrated under reduced pressure to give 14a (0.18 g, 77%)
which was taken for the next step without further purification. LC/MS
(ESI positive mode) m/z 274 ([M + H]+) C14H12ClN3O.
3-(2-Chloropyrimidin-4-yl)-5-fluorophenol (12j). The title
compound was synthesized from 10a and 3-fluoro-5-hydroxyphenyl-
boronic acid 11f (53%). LC/MS (ESI positive mode) m/z 225 ([M +
H]+) C10H6ClFN2O.
Following a procedure similar to that of 14a, the following
intermediates were synthesized.
N-Allyl-3-(2-chloropyrimidin-4-yl)benzamide (14b). The title
compound was synthesized from 12e and allylamine (59%). LC/MS
1
(ESI positive mode) m/z 274 ([M + H]+) C14H12ClN3O; H NMR
Acetic Acid 5-(2-Chloropyrimidin-4-yl)-2-methoxyphenyl
Ester (12k). The title compound was synthesized from 10a and 3-
acetoxy-4-methoxyphenylboronic acid pinacol ester 11g (74%). LC/
MS (ESI positive mode) m/z 279 ([M + H]+) C13H11ClN2O3.
3-(2-Chloropyrimidin-4-yl)phenylamine (12d). To a solution
of 12c (0.3 g, 1.07 mmol) in CH2Cl2/MeOH (1:1, 10 mL), cooled to
0 °C, SnCl2·H2O (1.15 g, 5.00 mmol) was added. The reaction mixture
was gradually raised to room temperature and stirred for 7 h. After
completion of the reaction, the solvent from the reaction mixture was
removed under reduced pressure and the residue was taken into
saturated Na2CO3 and CH2Cl2 (20 mL). The reaction mixture was
filtered through filter paper. The organic layer was separated from the
filtrate, and the aqueous layer was extracted with CH2Cl2 (3 × 10 mL).
The combined organic extracts were dried (Na2SO4) and concentrated
under reduced pressure to afford 12d (0.2 g, 91%) as a thick syrup
which was used for the next step without further purification. LC/MS
(ESI positive mode) m/z 206 ([M + H]+) C10H8ClN3.
5-(2-Chloropyrimidin-4-yl)-2-methoxyphenol (12l). To a
solution of 12k (0.27 g, 0.96 mmol) in THF (2.5 mL) was added
LiOH (0.07 g, 2.88 mmol, dissolved in 2.5 mL of H2O). The reaction
mixture was stirred at room temperature for 2 h. Upon completion of
the reaction, 1 M HCl added to acidify the mixture and the aqueous
layer was extracted with CH2Cl2 (3 × 10 mL). The combined organic
extracts were dried (Na2SO4) and concentrated under reduced
pressure to afford 12l (0.22 g, 97%) as a pale orange solid which
was used for the next step without further purification. LC/MS (ESI
positive mode) m/z 237 ([M + H]+) C11H9ClN2O2; 1H NMR
(CDCl3) δ 8.56 (d, 1H), 7.71 (dd, 1H), 7.54 (d, 1H), 6.95 (d, 1H),
3.98 (s, 3H).
4-(3-Allyloxymethylphenyl)-2-chloropyrimidine (13b). A
mixture of 12b (20 g, 90.6 mmol), KOH (23.8 g, 172 mmol), and
tetrabutylammonium hydrogen sulfate (1.5 g, 4.53 mmol) in allyl
bromide (80 mL, 4 vol) was stirred at room temperature for 12 h. The
reaction mixture was quenched with water and extracted with CH2Cl2
(3 × 25 mL). The combined organic extracts were washed with water,
brine, dried (Na2SO4), and concentrated under reduced pressure to
obtain the crude residue which was purified by column chromatog-
raphy (using 15% EtOAc in hexane) to furnish 13b (13 g, 55%) as a
pale yellow oil. LC/MS (ESI positive mode) m/z 261 ([M + H]+)
C14H13ClN2O; 1H NMR (CDCl3) δ 8.62 (d, 1H), 8.06 (br, 1H), 8.00
(dt, 1H), 7.66 (d, 1H), 7.54−7.46 (m, 2H), 6.02−5.93 (m, 1H), 5.27
(ddq, 2H), 4.60 (s, 2H), 4.08 (dq, 2H); 13C NMR (CDCl3) δ 167.1,
161.9, 159.9, 139.7, 135.3, 134.6, 131.2, 129.7, 129.3, 126.6, 117.5,
115.4, 71.7, 71.6; IR (KBr pellet): 1568, 1535, 1344, 1186, 1079 cm−1.
Following a procedure similar to that of 13b, the following
intermediates were synthesized.
(CDCl3) δ 8.69 (d, 1H), 8.51 (t, 1H), 8.24 (m, 2H), 7.96 (m, 1H),
7.72 (d, 1H), 7.61 (t, 1H), 6.02−5.89 (m, 1H), 5.37−5.21 (m, 2H),
4.14 (m, 2H).
Pent-4-enoic Acid (3-Nitrophenyl)amide (18a). The title
compound was synthesized from 3-nitroaniline 16a and pent-4-enoic
acid (yield, 58%). LC/MS (ESI positive mode) m/z 221 ([M + H]+)
C11H12N2O3.
4-(3-Allyloxyphenyl)-2-chloropyrimidine (15a). To a solution
of 12a (8 g, 38.8 mmol) in anhydrous DMF (40 mL), 4-bromo-1-
butene (23.6 mL, 233 mmol) was added slowly followed by cesium
carbonate (57 g, 174.7 mmol). The reaction mixture was heated to 40
°C for 12 h. The mixture was brought to room temperature and
quenched with water (140 mL). The reaction mixture was extracted
with CH2Cl2 (3 × 75 mL), and the combined organic extracts were
dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by column chromatography (EtOAc/hexane,
1:19) to afford 15a (7.5 g, 75%) as a white solid. LC/MS (ESI positive
mode) m/z 261 ([M + H]+) C13H11ClN2O; 1H NMR (CDCl3) δ 8.63
(d, 1H), 7.64−7.67 (m, 2H), 7.62 (d, 1H), 7.41 (dd, 1H), 5.90−5.94
(m, 1H), 5.15−5.22 (m, 1H), 4.12 (t, 1H), 2.59 (p, 1H); 13C NMR
(CDCl3) δ 167.0, 161.8, 159.8, 159.6,136.5, 134.3, 130.2, 119.8, 118.3,
117.2, 115.3, 113.4, 67.5,33.6. Anal. Calcd for C14H13ClN2O: C, 64.49;
H, 5.03; N, 10.74; Cl, 13.60. Found: C, 64.73; H, 5.07; N, 10.54; Cl,
13.34. IR (KBr pellet): 1573, 1455, 1351, 1223, 1183, 1037, 784 cm−1.
Following a procedure similar to that of 15a, the following
intermediates were synthesized.
4-(3-But-3-enyloxyphenyl)-2-chloro-6-methylpyrimidine
(15b). The title compound was synthesized from 12g and 4-bromo-1-
butene (70%). LC/MS (ESI positive mode) m/z 275 ([M + H]+)
C15H15ClN2O.
4-(3-But-3-enyloxyphenyl)-2-chloro-6-pyrrolidin-1-ylpyrimi-
dine (15c). The title compound was synthesized from 12h and 4-
bromo-1-butene (41%). LC/MS (ESI positive mode) m/z 330 ([M +
H]+) C18H20ClN3O.
4-(3-But-3-enyloxyphenyl)-2-chloro-5-methylpyrimidine
(15d). The title compound was synthesized from 12i and 4-bromo-1-
butene (56%). LC/MS (ESI positive mode) m/z 275 ([M + H]+)
C15H15ClN2O.
4-(3-But-3-enyloxy-5-fluorophenyl)-2-chloropyrimidine
(15e). The title compound was synthesized from 12j and 4-bromo-1-
butene (65%). LC/MS (ESI positive mode) m/z 279 ([M + H]+)
C14H12ClFN2O.
4-(3-But-3-enyloxy-4-methoxyphenyl)-2-chloropyrimidine
(15f). The title compound was synthesized from 12l and 4-bromo-1-
butene (45%). LC/MS (ESI positive mode) m/z 291 ([M + H]+)
C15H15ClN2O2; 1H NMR (CDCl3) δ 8.55 (d, 1H), 8.13 (m, 2H), 7.60
(d, 1H), 6.97 (d, 1H), 6.04−5.97 (m, 1H), 5.38−5.22 (m, 2H), 4.60
(s, 2H), 4.14−4.12 (m, 2H), 3.91 (s, 3H).
5-Nitro-2-trifluoromethoxybenzaldehyde (16g). To a cooled
solution of 2-trifluoromethoxybenzaldehyde 16f (2.58 g, 13.6 mmol)
in concentrated H2SO4 (8.1 mL) at 0 °C was added concentrated
HNO3 (1.7 mL) dropwise, and reaction mixture was stirred for 2 h.
Cold water was added, and resulting mixture was filtered. The product
16g was obtained as an off-white solid (2.55 g, 80%) and was taken to
the next step without further purification. LC/MS (ESI positive mode)
m/z 236 ([M + H]+) C8H4F3NO4.
4-(3-Allyloxyphenyl)-2-chloropyrimidine (13a). The title com-
pound was synthesized from 12a and allyl bromide (59%). LC/MS
(ESI positive mode) m/z 247 ([M + H]+) C13H11ClN2O.
1-Allyloxymethyl-3-nitrobenzene (18g). The title compound
was synthesized from 3-nitrobenzyl alcohol 16c and allyl bromide
(78%). LC/MS (ESI positive mode) m/z 194 ([M + H]+)
1
C10H11NO3; H NMR (CDCl3) δ 8.27 (s, 1H), 8.18 (dd, 1H), 7.73
(dd, 1H), 7.57 (t, 1H), 6.01 (m, 1H), 5.38 (m, 1H), 5.29 (m, 1H),
4.65 (s, 2H), 4.13 (dt, 2H).
But-3-enoic Acid [3-(2-Chloropyrimidin-4-yl)phenyl]amide
(14a). To a mixture of 12e (0.2 g, 0.852 mmol) and vinylacetic acid
(81 μL, 0.942 mmol) in CH2Cl2 (4 mL) at room temperature were
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Journal of Medicinal Chemistry
Article
Allylmethyl-(3 nitrobenzyl)amine (18b). To a solution of 3-
nitrobenzaldehyde 16b (20 g, 132.4 mmol) in CH2Cl2 (500 mL) was
added N-allylmethylamine (18.8 mL, 198.6 mmol), and the resulting
mixture was stirred at room temperature for 12 h after which
Na(OAc)3BH (56.13 g, 265 mmol) was added portionwise and the
mixture stirred for an additional 4 h. The reaction mixture was then
quenched with saturated NH4Cl solution and extracted with CH2Cl2
(3 × 150 mL). The combined organic extracts were dried over Na2SO4
and concentrated under reduced pressure to give the crude material
which was purified by column chromatography (EtOAc/hexane, 1:19)
to afford 18b (25.9 g, 95%) as a pale yellow syrup. LC/MS (ESI
positive mode) m/z 207 ([M + H]+) C11H14N2O2; 1H NMR (CDCl3)
δ 8.20 (s, 1H), 8.11 (dd, 1H), 7.68 (dd, 1H), 7.48 (t, 1H), 5.85−5.94
(m, 1H), 5.20 (t, 1H), 3.60 (s, 2H), 3.07 (dd, 1H), 2.22 (s, 3H); 13C
NMR (CDCl3) δ 148.4, 141.5, 135.20, 134.9, 129.1, 123.7, 122.1,
118.1, 60.52, 60.50. Anal. Calcd for C11H14N2O2: C, 64.06; H, 6.84; N,
13.58. Found: C, 62.31; H, 6.69; N, 13.25. IR (KBr pellet): 2787,
1528, 1349, 924, 733 cm−1.
and the resulting mixture was refluxed for 7 h. Upon completion, the
reaction mixture was concentrated under reduced pressure to give an
oil, which was dissolved in CH2Cl2. It was washed with water once,
dried over Na2SO4, and concentrated under reduced pressure to
furnish colorless oil 16j (2.2 g, 95%). LC/MS (ESI positive mode) m/
z 232 ([M + H]+) C9H10ClNO4.
2-Dimethoxymethyl-1-ethylsulfanyl-4-nitrobenzene (16k).
To a solution of 16j (2.0 g, 8.63 mmol), K2CO3 (2.39 g, 17.3
mmol) in DMF (50 mL) was added ethanethiol (0.64 mL, 8.63
mmol), and the resulting mixture was refluxed for 7 h. Upon
completion, the reaction mixture was concentrated under reduced
pressure to give thick syrup, which was dissolved in CH2Cl2. It was
washed with water once, dried over Na2SO4, and concentrated under
reduced pressure to furnish colorless oil 16k (1.9 g, 86%). LC/MS
(ESI positive mode) m/z 258 ([M + H]+) C11H15NO4S.
2-Dimethoxymethyl-1-ethanesulfonyl-4-nitrobenzene (16l).
To a cooled, degassed (N2) solution of 16k (1 g, 3.88 mmol) in
CH2Cl2 (20 mL) at 0 °C was added 3-chloroperbenzoic acid (1.34 g,
7.76 mmol), and the resulting mixture was stirred at room temperature
overnight. The reaction mixture was cooled to 0 °C and quenched
with water. The product was extracted with CH2Cl2 thrice, and the
combined organic extracts were dried over Na2SO4 and concentrated
under reduced pressure to furnish an oil, which was purified by column
chromatography (EtOAc/hexane) to obtain 0.52 g of 16l (46%). LC/
MS (ESI positive mode) m/z 290 ([M + H]+) C11H15NO6S.
2-Ethanesulfonyl-5-nitrobenzaldehyde (16m). To a cooled
solution of 16l (0.5 g, 1.73 mmol) in water (10 mL) at 0 °C was added
50% TFA/CH2Cl2 (10 mL), and the resulting mixture was stirred at
room temperature for 1 day. The organic layer was separated, and the
aqueous layer was basified using saturated NaHCO3 solution and
extracted with CH2Cl2 twice. The combined organic extracts were
dried over Na2SO4 and concentrated under reduced pressure to
furnish 16m (0.42 g, quantitative) as an off-white solid. LC/MS (ESI
positive mode) m/z 244 ([M + H]+) C9H9NO5S.
2-Morpholin-4-yl-5-nitrobenzaldehyde (16n). To a mixture of
2-chloro-5-nitrobenzaldehyde 16e (2 g, 10.8 mmol) in ACN (40 mL,
20 vol) was added morpholine (3.5 mL, 32.4 mmol), and the resulting
mixture was stirred at room temperature overnight. The reaction
mixture was cooled to room temperature, and water (60 mL) was
added to quench the reaction. The aqueous layer was extracted thrice
with EtOAc (30 mL each), and the combined organic extract was
washed with brine, dried (Na2SO4), and concentrated under reduced
pressure. Product 16n (2.11 g, 83%) was taken forward for the next
step without any purification. LC/MS (ESI positive mode) m/z 237
([M + H]+) C11H12N2O4.
Following a procedure similar to that of 18b, the following
intermediates were synthesized.
N-Allyl-3-(2-chloropyrimidin-4-yl)benzamide (14c). The title
compound was synthesized from 12f and N-allylmethylamine (60%).
LC/MS (ESI positive mode) m/z 274 ([M + H]+) C11H14N2O2.
Allyl-(3-nitrobenzyl)amine (18c). The title compound was
synthesized from 10b and allylamine (88%). LC/MS (ESI positive
1
mode) m/z 193 ([M + H]+) C10H12N2O2; H NMR (CDCl3) δ 8.20
(s, 1H), 8.07 (d, 1H), 7.68 (d, 1H), 7.47 (t, 1H), 5.93−5.87 (m, 1H),
5.22−5.11 (m, 2H), 3.88 (s, 2H), 3.28−3.26 (m, 2H).
N-Allyl-2,2,2-trifluoro-N-(3-nitrobenzyl)acetamide (18d). To
a solution of 18c (0.673 g, 3.51 mmol) and trifluoroacetic anhydride
(0.98 mL, 7.00 mmol) in THF (10 mL) was added triethylamine (0.98
mL, 7.00 mmol) dropwise, and the resulting mixture was stirred at
room temperature for 1 h. Aqueous NaHCO3 (20 mL) was added and
extracted with CH2Cl2 (3 × 15 mL). The combined organic extracts
were dried over Na2SO4 and concentrated under reduced pressure to
give 18d (1 g, 99%) as an orange liquid. This material was taken up for
the next step without further purification. LC/MS (ESI positive mode)
1
m/z 289 ([M + H]+) C12H11F3N2O3; H NMR (CDCl3) δ 8.19 (m,
1H), 8.09 (s, 1H), 7.62−7.55 (m, 2H), 5.81−5.75 (m, 1H), 5.38−5.26
(m, 2H), 4.70 (s, 2H), 4.01−3.96 (m, 2H).
3-Nitro-5-(2,2,2-trifluoroacetylamino)benzoic Acid Methyl
Ester (16w). Following a procedure similar to that of 18d, the title
compound was synthesized from 3-amino-5-nitrobenzoic acid methyl
ester 16v (yield, quantitative). LC/MS (ESI positive mode) m/z 293
([M + H]+) C10H7F3N2O5.
Allyl-(3-nitrophenyl)amine (18e). To a solution of 3-nitroaniline
16a (1 g, 7.24 mmol) and allyl bromide (0.86 mL, 7.60 mmol) in
CH2Cl2 (20 mL) was added triethylamine (2.0 mL, 14.5 mmol), and
the resulting mixture was stirred at room temperature for 3 h. The
reaction mixture was cooled to 0 °C and quenched with water. The
product was extracted with CH2Cl2 thrice, and the combined organic
extracts were dried over Na2SO4 and concentrated under reduced
pressure to furnish an oil, which was purified by column (EtOAc/
hexane) to obtain 0.89 g of 18e (66%). LC/MS (ESI positive mode)
m/z 179 ([M + H]+) C9H10N2O2.
Allyl-(3-nitrophenyl)carbamic Acid tert-Butyl Ester (18f). To
a solution of 18e (0.3 g, 1.68 mmol), di-tert-butyl dicarbonate (0.55 g,
2.52 mmol), and 4-(dimethylamino)pyridine (10 mg, 0.084 mmol) in
THF (5 mL) was added triethylamine (0.33 mL, 2.36 mmol), and the
resulting mixture was stirred at room temperature for 4 h. Upon
completion, THF was removed under reduced pressure and water (20
mL) was added. The product was extracted with CH2Cl2 (3 × 15 mL)
and the combined organic extracts were dried over Na2SO4 and
concentrated under reduced pressure to give 18f (0.4 g, 86%) as a
colorless liquid. This material was taken up for the next step without
further purification. LC/MS (ESI positive mode) m/z 279 ([M + H]+)
C14H18N2O4.
2-(2-Chloroethoxy)-5-nitrobenzaldehyde (16i). To a mixture
of 2-hydroxy-4-nitrobenzaldehyde 16h (50 g, 299 mmol) and 1,2-
dichloroethane (300 mL, 6 vol) in DMF (600 mL, 12 vol)) was added
K2CO3 (62.5 g, 450 mmol), and the resulting mixture was stirred at
100−105 °C for 6 h. The reaction mixture was quenched with water
(500 mL), and the product was extracted four times with CH2Cl2 (200
mL each). The combined organic extracts were washed with water
(500 mL), brine, dried (Na2SO4), and concentrated under reduced
pressure, and hexane (500 mL) was added to the crude product. The
resulting mixture was stirred for 30 min and evaporated to dryness
under reduced pressure followed by hexane slurry. The slurry with
cold hexane (500 mL) procedure was repeated, and the yellow solid
was collected by filtration. Product 15e (49.3 g, 72%) was taken
forward to the next step without any purification. LC/MS (ESI
positive mode) m/z 230 ([M + H]+) C9H8ClNO4; 1H NMR (CDCl3)
δ 10.52 (s, 1H), 8.71 (d, 1H), 8.45 (dd, 1H), 7.14 (d, 1H), 4.51 (t,
2H), 3.96 (t, 2H); 13C NMR (CDCl3) δ 187.3, 164.2, 142.1, 130.6,
125.0, 124.6, 113.2, 69.4, 41.3.
2-(2-Chloroethoxy)-3-methoxy-5-nitrobenzaldehyde (16u).
Following a procedure similar to that of 16i, the title compound
was synthesized from 2-hydroxy-3-methoxy-5-nitro-benzaldehyde 16o
and 1,2-dichloroethane (64%). LC/MS (ESI positive mode) m/z 260
([M + H]+) C10H10ClNO5.
2-(2-Methoxyethoxy)-5-nitrobenzaldehyde (16p). To a mix-
ture of 2-hydroxy-5-nitrobenzaldehyde 16h (5 g, 29.9 mmol) and 1-
1-Chloro-2-dimethoxymethyl-4-nitrobenzene (16j). To a
solution of 2-chloro-5-nitrobenzaldehyde 16e (1.85 g, 1.00 mmol) in
MeOH (50 mL) was added ammonium chloride (0.2 g, 3.67 mmol),
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Article
bromo-2-methoxyethane (5.6 mL, 59.8 mmol) in DMF (50 mL, 10
vol) was added K2CO3 (6.25 g, 45.0 mmol), and the resulting mixture
was stirred at 100−105 °C for 6 h. The reaction mixture was cooled to
0 °C and quenched with water. The product was extracted with
CH2Cl2 thrice and the combined organic extracts were dried over
Na2SO4 and concentrated under reduced pressure to furnish an oil,
which was purified by column (EtOAc/hexane) to obtain 5.8 g of 16p
(86%). LC/MS (ESI positive mode) m/z 226 ([M + H]+)
C10H11NO5.
(2-Formyl-4-nitrophenoxy)acetic Acid Methyl Ester (16q).
To a mixture of 2-hydroxy-5-nitrobenzaldehyde 16h (0.5 g, 2.99
mmol) and methyl bromoacetate (1.4 mL, 14.9 mmol) in DMF (15
mL) was added K2CO3 (0.83 g, 5.98 mmol), and the resulting mixture
was stirred at 40 °C for 20 h. The reaction mixture was cooled to 0 °C
and quenched with water. The product was extracted with CH2Cl2
thrice and the combined organic extracts were dried over Na2SO4 and
concentrated under reduced pressure to furnish an oil, which was
purified by column (EtOAc/hexane) to obtain 0.62 g of 16q (87%).
LC/MS (ESI positive mode) m/z 240 ([M + H]+) C10H9NO6.
(2-Formyl-4-nitrophenoxy)acetic Acid (16r). Following a
procedure similar to that of 12l, the title compound was synthesized
from 16q (yield, quantitative). LC/MS (ESI positive mode) m/z 226
([M + H]+) C9H7NO6.
(83%). LC/MS (ESI positive mode) m/z 244 ([M + H]+)
C13H13N3O2.
(2-Methoxyethyl)-(3-nitrobenzyl)amine (17f). The title com-
pound was synthesized from 16b and 2-methoxyethylamine (88%).
LC/MS (ESI positive mode) m/z 211 ([M + H]+) C10H14N2O3.
2-(3-Nitrobenzylamino)ethanol (17g). The title compound was
synthesized from 16b and 2-aminoethanol (75%). LC/MS (ESI
positive mode) m/z 197 ([M + H]+) C9H12N2O3; 1H NMR (CDCl3)
δ 8.19 (s, 1H), 8.10 (d, 1H), 7.64 (dd, 1H), 7.51−7.44 (m, 1H), 3.90
(s, 2H), 3.67 (t, 2H), 2.76 (t, 2H).
[2-(2-Chloroethoxy)-5-nitrobenzyl]-(2,2,2-trifluoroethyl)-
amine (17h). The title compound was synthesized from 16b and
2,2,2-trifluoroethylamine (79%). LC/MS (ESI positive mode) m/z
313 ([M + H]+) C11H12ClF3N2O3.
[2-(2-Chloroethoxy)-5-nitrobenzylamino]acetonitrile (17i).
The title compound was synthesized from 16b and 2-amino-
acetonitrile (75%). LC/MS (ESI positive mode) m/z 270 ([M +
H]+) C11H12ClN3O3.
Following a procedure similar to that of 18b, the following
intermediates were synthesized.
Allyl-(2-methoxy-5-nitrobenzyl)methylamine (18h). The title
compound was synthesized from 2-methoxy-5-nitrobenzaldehyde 16d
and N-allylmethylamine (95%). LC/MS (ESI positive mode) m/z 237
([M + H]+) C12H16N2O3.
Allyl-(2-chloro-5-nitrobenzyl)methylamine (18i). The title
compound was synthesized from 2-chloro-5-nitrobenzaldehyde 16e
and N-allylmethylamine (86%). LC/MS (ESI positive mode) m/z 241
([M + H]+) C11H13ClN2O2.
Allylmethyl-(5-nitro-2-trifluoromethoxybenzyl)amine (18j).
The title compound was synthesized from 16g and N-allylmethyl-
amine (91%). LC/MS (ESI positive mode) m/z 291 ([M + H]+)
C12H13F3N2O3.
Allyl-[2-(2-methoxyethoxy)-5-nitrobenzyl]methylamine
(18k). The title compound was synthesized from 16p and N-
allylmethylamine (92%). LC/MS (ESI positive mode) m/z 281 ([M +
H]+) C14H20N2O4.
Allyl-(2-ethanesulfonyl-5-nitrobenzyl)methylamine (18l).
The title compound was synthesized from 16m and N-allylmethyl-
amine (78%). LC/MS (ESI positive mode) m/z 299 ([M + H]+)
C13H18N2O4S.
Allylmethyl-(2-morpholin-4-yl-5-nitrobenzyl)amine (18m).
The title compound was synthesized from 16n and N-allylmethyl-
amine (yield, quantitative). LC/MS (ESI positive mode) m/z 292 ([M
+ H]+) C15H21N3O3.
Allyl-[2-(2-ethylsulfanylethoxy)-5-nitrobenzyl]methylamine
(18n). The title compound was synthesized from 16t and N-
allylmethylamine (93%). LC/MS (ESI positive mode) m/z 311 ([M +
H]+) C15H22N2O3S.
2-{2-[(Allylmethylamino)methyl]-4-nitrophenoxy}-1-pyrroli-
din-1-ylethanone (18o). The title compound was synthesized from
16s and N-allylmethylamine (92%). LC/MS (ESI positive mode) m/z
335 ([M + H]+) C17H23N3O4.
5-Nitro-2-(2-oxo-2-pyrrolidin-1-ylethoxy)benzaldehyde
(16s). Following a procedure similar to that of 14a, the title
compound was synthesized from 16r (yield, quantitative). LC/MS
(ESI positive mode) m/z 279 ([M + H]+) C13H14N2O5.
2-(2-Ethylsulfanylethoxy)-5-nitrobenzaldehyde (16t). Follow-
ing a procedure similar to that of 16k, the title compound was
synthesized from 16i and ethanethiol (97%). LC/MS (ESI positive
mode) m/z 256 ([M + H]+) C11H13NO4S.
2,2,2-Trifluoro-N-(3-formyl-5-nitrophenyl)acetamide (16x).
To a cooled mixture of 16u (1.4 g, 4.73 mmol) in CH2Cl2 (6 mL,
10 vol) at 0 °C was added 1.0 M DIBAL solution (4 mL, 4.00 mmol)
dropwise. The resulting mixture was stirred for 1 h and allowed to
warm to room temperature. Water was added, and product was
extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts
were washed with brine, dried (Na2SO4), and concentrated under
reduced pressure to obtain the crude residue which was purified by
column chromatography (using 35% EtOAc in hexane) to furnish 11g
(230 mg, 48%) as a colorless oil. LC/MS (ESI positive mode) m/z
263 ([M + H]+) C9H5F3N2O4.
Cyclopropylmethyl-(3-nitrobenzyl)amine (17c). To a mixture
of 3-nitrobenzaldehyde 16b (1.2 g, 7.94 mmol) in CH2Cl2 (12 mL)
was added aminomethylcyclopropane (1.35 mL, 15.6 mmol), and the
resulting mixture was stirred at room temperature for 4 h after which
Na(OAc)3BH (3.3 g, 15.6 mmol) was added portionwise and the
mixture stirred for additional 4 h. The reaction mixture was then
quenched with water (20 mL) and extracted with CH2Cl2 (3 × 15
mL). The combined organic extracts were dried over Na2SO4 and
concentrated under reduced pressure to afford 17c (1.4 g, 86%) as a
pale yellow syrup. LC/MS (ESI positive mode) m/z 207 ([M + H]+)
C11H14N2O2.
Allyl-[2-(2-chloroethoxy)-5-nitrobenzyl]methylamine (18p).
The title compound was synthesized from 16i and N-allylmethylamine
(99%). LC/MS (ESI positive mode) m/z 285 ([M + H]+)
C13H17ClN2O3.
Following a procedure similar to that of 17c, the following
intermediates were synthesized.
Cyclopropyl-(3-nitrobenzyl)amine (17a). The title compound
was synthesized from 16b and cyclopropylamine (72%). LC/MS (ESI
positive mode) m/z 193 ([M + H]+) C10H12N2O2; 1H NMR (CDCl3)
δ 10.20 (br, 1H), 8.32 (s, 1H), 8.11 (d, 1H), 8.01 (d, 1H), 7.50 (t,
1H), 4.20 (s, 2H), 2.40−2.37 (m, 1H), 1.07 (m, 2H), 0.66 (m, 2H).
Isobutyl-(3-nitrobenzyl)amine (17b). The title compound was
synthesized from 16b and isobutylamine (79%). LC/MS (ESI positive
mode) m/z 209 ([M + H]+) C11H16N2O2.
Allyl-[2-(2-chloroethoxy)-3-methoxy-5-nitrobenzyl]-
methylamine (18q). The title compound was synthesized from 16u
and N-allylmethylamine (84%). LC/MS (ESI positive mode) m/z 315
([M + H]+) C14H19ClN2O4.
N-{3-[(Allylmethylamino)methyl]-5-nitrophenyl}-2,2,2-tri-
fluoroacetamide (18r). The title compound was synthesized from
16x and N-allylmethylamine (96%). LC/MS (ESI positive mode) m/z
318 ([M + H]+) C13H14F3N3O3.
(2,2-Dimethylpropyl)-(3-nitrobenzyl)amine (17d). The title
compound was synthesized from 16b and 2,2-dimethylpropan-1-amine
(68%). LC/MS (ESI positive mode) m/z 223 ([M + H]+)
C12H18N2O2.
(3-Nitrobenzyl)pyridin-2-ylmethylamine (17e). The title com-
pound was synthesized from 16b and 2-(aminomethyl)pyridine
Allylcyclopropyl-(3-nitrobenzyl)amine (19a). To a stirring
solution of 17a (0.18 g, 0.938 mmol) and K2CO3 (0.39 g, 2.81
mmol) in DMF (2 mL) was added allyl bromide (0.2 mL, 2.25 mmol)
dropwise, and the resulting mixture was stirred at 70 °C for 3 h. The
reaction mixture was then brought to room temperature, quenched
with water (15 mL), and extracted with CH2Cl2 (3 × 20 mL). The
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combined organic extracts were washed with water, brine, dried
(Na2SO4), and concentrated under reduced pressure to afford the
crude 19a (0.12 g, 86%) as a yellow syrup. This intermediate was
taken forward to the next step without any purification. LC/MS (ESI
positive mode) m/z 233 ([M + H]+) C13H16N2O2.
Following a procedure similar to that of 19a, the following
intermediates were synthesized.
Allylisobutyl-(3-nitrobenzyl)amine (19b). The title compound
was synthesized from 17b and allyl bromide (83%). LC/MS (ESI
positive mode) m/z 249 ([M + H]+) C14H20N2O2.
Allylcyclopropylmethyl-(3-nitrobenzyl)amine (19c). The title
compound was synthesized from 17c and allyl bromide (80%). LC/
MS (ESI positive mode) m/z 245 ([M + H]+) C14H18N2O2; 1H NMR
(CDCl3) δ 8.23 (s, 1H), 8.08 (dd, 1H), 7.72 (d, 1H), 7.47 (t, 1H),
5.94−5.84 (m, 1H), 5.22−5.15 (m, 2H), 3.76 (s, 2H), 3.22 (d, 2H),
2.37 (d, 2H), 0.88−0.85 (m, 1H), 0.51−0.47 (m, 2H), 0.09−0.06 (m,
2H).
Allyl-(2,2-dimethylpropyl)-(3-nitrobenzyl)amine (19d). The
title compound was synthesized from 17d and allyl bromide (91%).
LC/MS (ESI positive mode) m/z 263 ([M + H]+) C15H22N2O2.
Allyl-(3-nitrobenzyl)pyridin-2-ylmethylamine (19e). The title
compound was synthesized from 17e and allyl bromide (48%). LC/
MS (ESI positive mode) m/z 284 ([M + H]+) C16H17N3O2.
Allyl-(2-methoxyethyl)-(3-nitrobenzyl)amine (19f). The title
compound was synthesized from 17f and allyl bromide (77%). LC/MS
(ESI positive mode) m/z 251 ([M + H]+) C13H18N2O3.
2-[Allyl-(3-nitrobenzyl)amino]ethanol (19g). The title com-
pound was synthesized from 17g and allyl bromide (72%). LC/MS
(ESI positive mode) m/z 237 ([M + H]+) C12H16N2O3.
Allyl-[2-(2-chloroethoxy)-5-nitrobenzyl]-(2,2,2-
trifluoroethyl)amine (19h). The title compound was synthesized
from 17h and allyl bromide (57%). LC/MS (ESI positive mode) m/z
353 ([M + H]+) C14H16ClF3N2O3.
Allylmethyl-[5-nitro-2-(2-piperidin-1-ylethoxy)benzyl]amine
(19p). The title compound was synthesized from 18p and piperidine
(78%). LC/MS (ESI positive mode) m/z 334 ([M + H]+)
C18H27N3O3.
Allyl-[3-methoxy-5-nitro-2-(2-pyrrolidin-1-ylethoxy)benzyl]-
methylamine (19q). The title compound was synthesized from 18q
and pyrrolidine (78%). LC/MS (ESI positive mode) m/z 350 ([M +
H]+) C18H27N3O4.
Following a procedure similar to that of 12d, the following
intermediates were synthesized.
Pent-4-enoic Acid (3-Aminophenyl)amide (21a). The title
compound was synthesized from 18a (96%). LC/MS (ESI positive
mode) m/z 191 ([M + H]+) C11H14N2O.
3-[(Allylmethylamino)methyl]phenylamine (21b). The title
compound was synthesized from 18b (98%). LC/MS (ESI positive
1
mode) m/z 177 ([M + H]+) C11H16N2. H NMR (CDCl3) δ 7.09 (t,
1H), 6.57−6.69 (m, 2H), 6.55 (dd, 1H), 5.85−5.95 (m, 1H), 5.12−
5.17 (m, 2H), 3.67 (br, 1H), 3.39 (s, 2H), 3.02 (d, 2H), 2.18 (s, 3H);
13C NMR (CDCl3) δ 146.4, 140.3, 136.0, 129.1, 119.4, 117.4, 115.7,
113.8, 61.7, 60.6, 42.2. Anal. Calcd for C11H16N2: C, 74.96; H, 9.15; N,
15.89. Found: C, 74.34; H, 9.04; N, 15.68. IR (KBr pellet): 3346,
3210, 2782, 1605, 1460, 1294, 921, 785, 696 cm−1.
Allyl-(3-aminophenyl)carbamic Acid tert-Butyl Ester (21c).
The title compound was synthesized from 18f (56%). LC/MS (ESI
positive mode) m/z 249 ([M + H]+) C14H20N2O2.
N-Allyl-N-(3-aminobenzyl)-2,2,2-trifluoroacetamide (21d).
The title compound was synthesized from 18d (78%). LC/MS (ESI
positive mode) m/z 259 ([M + H]+) C12H13F3N2O.
3-[(Allylmethylamino)methyl]-4-methoxyphenylamine (21f).
The title compound was synthesized from 18h (63%). LC/MS (ESI
positive mode) m/z 207 ([M + H]+) C12H18N2O.
3-[(Allylmethylamino)methyl]-4-chlorophenylamine (21g).
The title compound was synthesized from 18i (95%). LC/MS (ESI
positive mode) m/z 211 ([M + H]+) C11H15ClN2.
3-[(Allylmethylamino)methyl]-4-trifluoromethoxyphenyl-
amine (21h). The title compound was synthesized from 18j (77%).
LC/MS (ESI positive mode) m/z 261 ([M + H]+) C12H15F3N2O.
3-[(Allylcyclopropylamino)methyl]phenylamine (21i). The
title compound was synthesized from 19a (88%). LC/MS (ESI
positive mode) m/z 203 ([M + H]+) C13H18N2; 1H NMR (CDCl3) δ
7.09 (t, 1H), 6.69 (m, 1H), 6.59 (dd, 1H), 6.04−5.87 (m, 1H), 5.22−
5.13 (m, 2H), 3.73 (s, 2H), 3.25 (d, 2H), 1.90 (br s, 1H), 0.59−0.40
(m, 4H).
3-[(Allylisobutylamino)methyl]phenylamine (21j). The title
compound was synthesized from 19b (65%). LC/MS (ESI positive
mode) m/z 219 ([M + H]+) C14H22N2.
3-[(Allylcyclopropylmethylamino)methyl]phenylamine
(21k). The title compound was synthesized from 19c (90%). LC/MS
(ESI positive mode) m/z 217 ([M + H]+) C14H20N2.
3-{[Allyl-(2,2-dimethylpropyl)amino]methyl}phenylamine
(21l). The title compound was synthesized from 19d (61%). LC/MS
(ESI positive mode) m/z 233 ([M + H]+) C15H24N2.
3-[(Allylpyridin-2-ylmethylamino)methyl]phenylamine
(21m). The title compound was synthesized from 19e (97%). LC/MS
(ESI positive mode) m/z 254 ([M + H]+) C16H19N3.
{Allyl-[2-(2-chloroethoxy)-5-nitrobenzyl]amino}acetonitrile
(19i). The title compound was synthesized from 17i and allyl bromide
(64%). LC/MS (ESI positive mode) m/z 310 ([M + H]+)
C14H16ClN3O3.
Allyl-[2-(2-diethylaminoethoxy)-5-nitrobenzyl]methylamine
(19j). To a solution of 18p (2 g, 7.02 mmol) in N,N-
dimethylacetamide (10 mL) was added dimethylamine (4 mL, 2
vol), and the resulting mixture was stirred at 90 °C for 20 h. The
reaction mixture was brought to room temperature and quenched with
water (20 mL) and extracted in ethyl acetate (3 × 20 mL). The
combined organic extracts were washed with water, brine, dried
(Na2SO4), and concentrated under reduced pressure to afford the
crude 19j (1.85 g, 82%) as a thick syrup. This intermediate was taken
forward to the next step without any purification. LC/MS (ESI
positive mode) m/z 322 ([M + H]+) C17H27N3O3.
Following a procedure similar to that of 19j, the following
intermediates were synthesized.
Allylmethyl-[5-nitro-2-(2-pyrrolidin-1-ylethoxy)benzyl]-
amine (19k). The title compound was synthesized from 18p and
pyrrolidine (75%). LC/MS (ESI positive mode) m/z 320 ([M + H]+)
C17H25N3O3.
Allyl-[5-nitro-2-(2-pyrrolidin-1-ylethoxy)benzyl]-(2,2,2-
trifluoroethyl)amine (19l). The title compound was synthesized
from 19h and pyrrolidine (79%). LC/MS (ESI positive mode) m/z
388 ([M + H]+) C18H24F3N3O3.
3-{[Allyl-(2-methoxyethyl)amino]methyl}phenylamine (21n).
The title compound was synthesized from 19f (64%). LC/MS (ESI
positive mode) m/z 221 ([M + H]+) C13H20N2O.
2-[Allyl-(3-aminobenzyl)amino]ethanol (21o). The title com-
pound was synthesized from 19g (76%). LC/MS (ESI positive mode)
m/z 207 ([M + H]+) C12H18N2O.
{Allyl-[5-nitro-2-(2-pyrrolidin-1-ylethoxy)benzyl]amino}-
acetonitrile (19m). The title compound was synthesized from 19i
and pyrrolidine (90%). LC/MS (ESI positive mode) m/z 345 ([M +
H]+) C18H24N4O3.
Allylmethyl-[2-(2-morpholin-4-ylethoxy)-5-nitrobenzyl]-
amine (19n). The title compound was synthesized from 18p and
morpholine (84%). LC/MS (ESI positive mode) m/z 336 ([M + H]+)
C17H25N3O4.
Allylmethyl-{2-[2-(4-methylpiperazin-1-yl)ethoxy]-5-
nitrobenzyl}amine (19o). The title compound was synthesized from
18p and N-methylpiperazine (88%). LC/MS (ESI positive mode) m/z
349 ([M + H]+) C18H28N4O3.
3-[(Allylmethylamino)methyl]-4-(2-ethylsulfanylethoxy)-
phenylamine (22e). The title compound was synthesized from 18n
(91%). LC/MS (ESI positive mode) m/z 281 ([M + H]+)
1
C15H24N2OS; H NMR (CDCl3) δ 6.77 (d, 1H), 6.69 (d, 1H), 6.54
(dd, 1H), 5.98−5.88 (m, 1H), 5.24−5.12 (m, 2H), 4.05 (t, 2H), 3.48
(s, 2H), 3.07 (d, 2H), 2.87 (t, 2H), 2.62 (q, 2H), 2.22 (s, 3H), 1.25 (t,
3H).
3-[(Allylmethylamino)methyl]-4-(2-methoxyethoxy)-
phenylamine (22f). The title compound was synthesized from 18k
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(64%). LC/MS (ESI positive mode) m/z 251 ([M + H]+)
C14H22N2O2.
3-[(Allylmethylamino)methyl]-4-(2-diethylaminoethoxy)-
phenylamine (22h). The title compound was synthesized from 19j
(59%). LC/MS (ESI positive mode) m/z 292 ([M + H]+)
C17H29N3O.
NaHCO3 was added, and pH was adjusted to 7−8. The organic layer
was separated, and the aqueous layer was extracted with CH2Cl2 (3 ×
50 mL). The combined organic extracts were dried (Na2SO4),
concentrated under reduced pressure, and purified by column
chromatography (1:3 EtOAc/hexane) to furnish 23f as a white solid
1
(7.4 g, 92%). LC/MS (ESI positive mode) m/z 401 ([M + H]+); H
3-[(Allylmethylamino)methyl]-4-(2-pyrrolidin-1-ylethoxy)-
phenylamine (22i). The title compound was synthesized from 19k
(88%). LC/MS (ESI positive mode) m/z 290 ([M + H]+)
C17H27N3O.
3-[(Allylmethylamino)methyl]-4-(2-morpholin-4-ylethoxy)-
phenylamine (22j). The title compound was synthesized from 19i
(54%). LC/MS (ESI positive mode) m/z 306 ([M + H]+)
C17H27N3O2.
3-[(Allylmethylamino)methyl]-4-[2-(4-methylpiperazin-1-yl)-
ethoxy]phenylamine (22k). The title compound was synthesized
from 19i (77%). LC/MS (ESI positive mode) m/z 319 ([M + H]+)
C18H30N4O.
3-[(Allylmethylamino)methyl]-4-(2-piperidin-1-ylethoxy)-
phenylamine (22l). The title compound was synthesized from 19p
(85%). LC/MS (ESI positive mode) m/z 304 ([M + H]+)
C18H29N3O.
3-[(Allylmethylamino)methyl]-5-methoxy-4-(2-pyrrolidin-1-
ylethoxy)phenylamine (22m). The title compound was synthesized
from 19q (63%). LC/MS (ESI positive mode) m/z 288 ([M + H]+)
C18H29N3O2.
N-{3-[(Allylmethylamino)methyl]-5-aminophenyl}-2,2,2-tri-
fluoroacetamide (22n). The title compound was synthesized from
18r (52%). LC/MS (ESI positive mode) m/z 320 ([M + H]+)
C13H16F3N3O; 1H NMR (CDCl3) δ 7.73 (br s, 1H), 7.10 (t, 1H), 6.67
(s, 1H), 6.53 (s, 1H), 5.93−5.86 (m, 1H), 5.30−5.15 (m, 2H), 3.86
(br s, 2H), 3.38 (s, 2H), 3.03 (d, 2H), 2.18 (s, 3H).
3-[(Allylmethylamino)methyl]-4-morpholin-4-ylphenyl-
amine (22c). Compound 18m (2.0 g, 6.86 mmol) was taken in EtOH
(20 mL, 10 vol), and fine Fe powder (1.15 g, 20.6 mmol) was added at
50−55 °C followed by NH4Cl solution (1.76 g, 32 mmol in 2 mL of
water). The reaction mixture was refluxed for 4 h, and EtOH was
removed from the reaction mixture under reduced pressure. The
residue was basified with NaHCO3 solution (pH 7−8) and extracted
with CH2Cl2 (3 × 25 mL). The combined organic extracts were
washed with brine, dried (Na2SO4), and concentrated under reduced
pressure to furnish 22c (1.67 g, 93%) as a brown oil. This material was
taken up for the next step without any purification. LC/MS (ESI
positive mode) m/z 262 ([M + H]+) C15H23N3O.
NMR (CDCl3) δ 8.46 (d, 1H), 7.69−7.60 (m, 4H), 7.49 (s, 1H), 7.37
(t, 1H), 7.29 (t, 1H), 7.13 (d, 1H), 7.04−6.99 (m, 2H), 5.95−5.87 (m,
2H), 5.22−5.13 (m, 4H), 4.10 (t, 2H), 3.51 (s, 2H), 3.05 (d, 2H), 2.57
(m, 2H), 2.22 (s, 3H); 13C NMR (CDCl3) δ 164.7, 160.3, 159.4,
158.6, 140.0, 139.7, 138.6, 136.0, 134.4, 129.8, 128.7, 123.2, 119.9,
119.6, 118.0, 117.5, 117.3, 117.2,113.1, 108.4, 67.4, 61.8, 60.6, 42.2,
33.7. Anal. Calcd for C25H28N4O: C, 74.97; H, 7.05; N, 13.99. Found:
C, 74.80; H, 7.15; N, 13.86. IR (KBr pellet): 3271, 3070, 2776, 1572,
1433, 1295, 913, 780, 627 cm−1.
Following a procedure similar to that of 23f, the following
intermediates were synthesized.
Pent-4-enoic Acid {3-[4-(3-Allyloxyphenyl)pyrimidin-2-
ylamino]phenyl}amide (23a). The title compound was synthesized
from 13a and 21a (76%). LC/MS (ESI positive mode) m/z 401 ([M
+ H]+) C24H24N4O2.
But-3-enoic Acid [3-(2-{3-[(Allylmethylamino)methyl]-
phenylamino}pyrimidin-4-yl)phenyl]amide (23b). The title
compound was synthesized from 14a and 21b (83%). LC/MS (ESI
positive mode) m/z 414 ([M + H]+) C25H27N5O.
N-Allyl-3-(2-{3-[(allylmethylamino)methyl]phenylamino}-
pyrimidin-4-yl)benzamide (23c). The title compound was
synthesized from 14b and 21b (91%). LC/MS (ESI positive mode)
m/z 414 ([M + H]+) C25H27N5O.
Allyl-{3-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-ylamino]-
phenyl}carbamic Acid tert-Butyl Ester (23d). The title compound
was synthesized from 15a and 21c (59%). LC/MS (ESI positive
mode) m/z 473 ([M + H]+) C28H32N4O3.
N-Allyl-N-{3-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-
ylamino]benzyl}-2,2,2-trifluoroacetamide (23e). The title com-
pound was synthesized from 15a and 21d (87%). LC/MS (ESI
positive mode) m/z 483 ([M + H]+) C26H25F3N4O2; 1H NMR
(CDCl3) δ 8.41 (t, 1H), 7.71−7.59 (m, 4H), 7.44 (m, 1H), 7.36 (m,
1H), 7.22 (m, 1H), 7.11 (m, 1H), 6.98 (dd, 1H), 5.96−5.89 (m, 1H),
5.78−5.73 (m, 1H), 5.33−5.07 (m, 4H), 4.66 (s, 2H), 4.13 (t, 2H),
3.94 (br, 2H), 2.59 (q, 2H).
{ 3 - [ ( A l l y l m e t h y l a m i n o ) m e t h y l ] p h e n y l } - [ 4 - ( 3 -
allyloxymethylphenyl)pyrimidin-2-yl]amine (23g). The title
compound was synthesized from 13b and 21b (87%). LC/MS (ESI
positive mode) m/z 401 ([M + H]+) C25H28N4O.
(4-{3-[(Allylmethylamino)methyl]phenyl}pyrimidin-2-yl)-(3-
allyloxymethylphenyl)amine (23h). The title compound was
synthesized from 14c and 21e (71%). LC/MS (ESI positive mode)
m/z 401 ([M + H]+) C25H28N4O.
{3-[(Allylcyclopropylamino)methyl]phenyl}-[4-(3-but-3-
enyloxyphenyl)pyrimidin-2-yl]amine (18a). The title compound
was synthesized from 15a and 21i (57%). LC/MS (ESI positive
mode) m/z 427 ([M + H]+) C27H30N4O; 1H NMR (CDCl3) δ 11.73
(br, 1H), 8.29 (d, 1H), 8.02 (d, 1H), 7.80 (s, 1H), 7.64 (m, 2H),
7.58−7.46 (m, 2H), 7.38 (m, 1H), 7.32 (d, 1H), 7.18 (d, 1H), 6.20−
6.10 (m, 1H), 5.97−5.83 (m, 1H), 5.57−5.53 (m, 2H), 5.29−5.13 (m,
2H), 4.33 (s, 2H), 4.10 (t, 2H), 2.62−2.57 (m, 2H), 2.34−2.29 (m,
1H), 0.83 (m, 4H).
{3-[(Allylisobutylamino)methyl]phenyl}-[4-(3-but-3-
enyloxyphenyl)pyrimidin-2-yl]amine (24b). The title compound
was synthesized from 15a and 21j (64%). LC/MS (ESI positive
mode) m/z 443 ([M + H]+) C28H34N4O.
Following a procedure similar to that of 22c, the following
intermediates were synthesized.
3-Allyloxymethylphenylamine (21e). The title compound was
synthesized from 18g (91%). LC/MS (ESI positive mode) m/z 164
([M + H]+) C10H13NO.
3-{[Allyl-(2,2,2-trifluoroethyl)amino]methyl}-4-(2-pyrrolidin-
1-ylethoxy)phenylamine (22a). The title compound was synthe-
sized from 19h (87%). LC/MS (ESI positive mode) m/z 358 ([M +
H]+) C18H26F3N3O.
{Allyl-[5-amino-2-(2-pyrrolidin-1-ylethoxy)benzyl]amino}-
acetonitrile (22b). The title compound was synthesized from 19i
(92%). LC/MS (ESI positive mode) m/z 315 ([M + H]+)
C18H26N4O.
3-[(Allylmethylamino)methyl]-4-ethanesulfonylphenyl-
amine (22d). The title compound was synthesized from 18l (81%).
LC/MS (ESI positive mode) m/z 267 ([M + H]+) C13H20N2O2S.
2-{2-[(Allylmethylamino)methyl]-4-aminophenoxy}-1-pyrro-
lidin-1-ylethanone (22g). The title compound was synthesized from
18o (86%). LC/MS (ESI positive mode) m/z 262 ([M + H]+)
C17H25N3O2.
{3-[(Allylcyclopropylmethylamino)methyl]phenyl}-[4-(3-but-
3-enyloxyphenyl)pyrimidin-2-yl]amine (24c). The title com-
pound was synthesized from 15a and 21k (56%). LC/MS (ESI
positive mode) m/z 441 ([M + H]+) C28H32N4O.
(3-{[Allyl-(2,2-dimethylpropyl)amino]methyl}phenyl)-[4-(3-
but-3-enyloxyphenyl)pyrimidin-2-yl]amine (24d). The title
compound was synthesized from 15a and 21l (96%). LC/MS (ESI
positive mode) m/z 457 ([M + H]+) C29H36N4O.
{ 3 - [ ( A l k y l m e t h y l a m i n o ) m e t h y l ] p h e n y l } - [ 4 - ( 3 -
allyloxyphenyl)pyrimidine-2-yl]amine (23f). To a solution of 15a
(4.22 g, 24 mmol) and 18b (5.2 g, 20 mmol) in n-butanol (70 mL)
was added concentrated HCl (3 mL), and the resulting mixture was
heated to 90−100 °C for 12 h. The reaction mixture was brought to
room temperature, and the solvent was removed under reduced
pressure. The residue was dissolved in CH2Cl2 (50 mL). Saturated
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{3-[(Allylpyridin-2-ylmethylamino)methyl]phenyl}-[4-(3-but-
3-enyloxyphenyl)pyrimidin-2-yl]amine (24e). The title com-
pound was synthesized from 15a and 21m (49%). LC/MS (ESI
positive mode) m/z 478 ([M + H]+) C30H31N5O.
(3-{[Allyl-(2-methoxyethyl)amino]methyl}phenyl)-[4-(3-but-
3-enyloxyphenyl)pyrimidin-2-yl]amine (24f). The title com-
pound was synthesized from 15a and 21n (77%). LC/MS (ESI
positive mode) m/z 445 ([M + H]+) C27H32N4O2.
2-(Allyl-{3-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-ylamino]-
benzyl}amino)ethanol (24g). The title compound was synthesized
from 15a and 21o (85%). LC/MS (ESI positive mode) m/z 431 ([M
+ H]+) C26H30N4O2.
{3-[(Allylmethylamino)methyl]-4-methoxyphenyl}-[4-(3-but-
3-enyloxyphenyl)pyrimidin-2-yl]amine (25a). The title com-
pound was synthesized from 15a and 21f (76%). LC/MS (ESI
positive mode) m/z 431 ([M + H]+) C26H30N4O2.
{3-[(Allylmethylamino)methyl]-4-chlorophenyl}-[4-(3-but-3-
enyloxyphenyl)pyrimidin-2-yl]amine (25b). The title compound
was synthesized from 15a and 21g (86%). LC/MS (ESI positive
mode) m/z 435 ([M + H]+) C25H27ClN4O.
{3-[(Allylmethylamino)methyl]-4-trifluoromethoxyphenyl}-
[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine (25c). The title
compound was synthesized from 15a and 21h (71%). LC/MS (ESI
positive mode) m/z 485 ([M + H]+) C26H27F3N4O2.
[3-[(Allylmethylamino)methyl]-4-(2-methoxyethoxy)-
phenyl]-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine
(25d). The title compound was synthesized from 15a and 22f (73%).
LC/MS (ESI positive mode) m/z 475 ([M + H]+) C28H34N4O3.
{3-[(Allylmethylamino)methyl]-4-ethanesulfonylphenyl}-[4-
(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine (25e). The title
compound was synthesized from 15a and 22d (88%). LC/MS (ESI
positive mode) m/z 492 ([M + H]+) C27H32N4O3S.
{3-[(Allylmethylamino)methyl]-4-[2-(4-methylpiperazin-1-
yl)ethoxy]phenyl}-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]-
amine (25n). The title compound was synthesized from 15a and 22k
(90%). LC/MS (ESI positive mode) m/z 543 ([M + H]+)
C32H42N6O2.
[3-[(Allylmethylamino)methyl]-4-(2-piperidin-1-ylethoxy)-
phenyl]-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine
(25o). The title compound was synthesized from 15a and 22l (93%).
LC/MS (ESI positive mode) m/z 528 ([M + H]+) C32H41N5O2.
[3-[(Allylmethylamino)methyl]-5-methoxy-4-(2-pyrrolidin-1-
ylethoxy)phenyl]-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]-
amine (25p). The title compound was synthesized from 15a and 22m
(68%). LC/MS (ESI positive mode) m/z 544 ([M + H]+)
C32H41N5O3.
N-{3-[(Allylmethylamino)methyl]-5-[4-(3-but-3-
enyloxyphenyl)pyrimidin-2-ylamino]phenyl}-2,2,2-trifluoroa-
cetamide (25q). The title compound was synthesized from 15a and
22n (89%). LC/MS (ESI positive mode) m/z 512 ([M + H]+)
C27H28F3N5O2.
{3-[(Allylmethylamino)methyl]phenyl}-[4-(3-but-3-enyloxy-
phenyl)-6-methylpyrimidin-2-yl]amine (25r). The title com-
pound was synthesized from 15b and 21b (92%). LC/MS (ESI
positive mode) m/z 415 ([M + H]+) C26H30N4O.
{3-[(Allylmethylamino)methyl]phenyl}-[4-(3-but-3-enyloxy-
phenyl)-6-pyrrolidin-1-ylpyrimidin-2-yl]amine (25s). The title
compound was synthesized from 15c and 21b (90%). LC/MS (ESI
positive mode) m/z 470 ([M + H]+) C29H35N5O.
{3-[(Allylmethylamino)methyl]phenyl}-[4-(3-but-3-enyloxy-
phenyl)-5-methylpyrimidin-2-yl]amine (25t). The title com-
pound was synthesized from 15d and 21b (68%). LC/MS (ESI
positive mode) m/z 415 ([M + H]+) C26H30N4O.
{3-[(Allylmethylamino)methyl]phenyl}-[4-(3-but-3-enyloxy-
5-fluorophenyl)pyrimidin-2-yl]amine (25u). The title compound
was synthesized from 15e and 21b (66%). LC/MS (ESI positive
mode) m/z 419 ([M + H]+) C25H27FN4O.
{3-[(Allylmethylamino)methyl]phenyl}-[4-(3-but-3-enyloxy-
4-methoxyphenyl)pyrimidin-2-yl]amine (25v). The title com-
pound was synthesized from 15f and 21b (51%). LC/MS (ESI
positive mode) m/z 431 ([M + H]+) C26H30N4O2.
[3-[(Allylmethylamino)methyl]-4-(2-ethylsulfanylethoxy)-
phenyl]-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine (25f).
The title compound was synthesized from 15a and 22e (67%). LC/
1
MS (ESI positive mode) m/z 505 ([M + H]+) C29H36N4O2S; H
NMR (CDCl3) δ 8.42 (d, 1H), 7.64−7.62 (m, 3H), 7.54 (d, 1H), 7.38
(t, 1H), 7.10−7.08 (m, 2H), 7.06−7.04 (m, 1H), 6.87 (d, 1H), 6.04−
5.88 (m, 2H), 5.22−5.12 (m, 4H), 4.15 (t, 2H), 4.08 (t, 2H), 3.57 (s,
2H), 3.11 (d, 2H), 2.93 (t, 2H), 2.66 (t, 2H), 2.62−2.58 (m, 2H), 2.26
(s, 3H), 1.31 (t, 3H).
2-{2-[(Allylmethylamino)methyl]-4-[4-(3-but-3-
enyloxyphenyl)pyrimidin-2-ylamino]phenoxy}-1-pyrrolidin-1-
ylethanone (25g). The title compound was synthesized from 15a
and 2g (82%). LC/MS (ESI positive mode) m/z 528 ([M + H]+)
C31H37N5O3.
{3-[(Allylmethylamino)methyl]-4-morpholin-4-ylphenyl}-[4-
(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine (25h). The title
compound was synthesized from 15a and 22c (63%). LC/MS (ESI
positive mode) m/z 586 ([M + H]+) C29H35N5O2.
[3-[(Allylmethylamino)methyl]-4-(2-diethylaminoethoxy)-
phenyl]-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine (25i).
The title compound was synthesized from 15a and 22h (97%). LC/
MS (ESI positive mode) m/z 516 ([M + H]+) C31H41N5O2.
[3-[(Allylmethylamino)methyl]-4-(2-pyrrolidin-1-ylethoxy)-
phenyl]-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine (25j).
The title compound was synthesized from 15a and 22i (77%). LC/MS
(ESI positive mode) m/z 514 ([M + H]+) C31H39N5O2.
[3-{[Allyl-(2,2,2-trifluoroethyl)amino]methyl}-4-(2-pyrroli-
din-1-ylethoxy)phenyl]-[4-(3-but-3-enyloxyphenyl)pyrimidin-
2-yl]amine (25k). The title compound was synthesized from 15a and
22a (67%). LC/MS (ESI positive mode) m/z 582 ([M + H]+)
C32H38F3N5O2.
{Allyl-[5-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-ylamino]-2-
(2-pyrrolidin-1-ylethoxy)benzyl]amino}acetonitrile (25l). The
title compound was synthesized from 15a and 22b (71%). LC/MS
(ESI positive mode) m/z 539 ([M + H]+) C32H38N6O2.
[3-[(Allylmethylamino)methyl]-4-(2-morpholin-4-ylethoxy)-
phenyl]-[4-(3-but-3-enyloxyphenyl)pyrimidin-2-yl]amine
(25m). The title compound was synthesized from 15a and 22j (88%).
LC/MS (ESI positive mode) m/z 530 ([M + H]+) C31H39N5O3.
(16E)-14-Methyl-20-oxa-5,7,14,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (26h). Diene 23f (1.0 g, 2.5 mmol) was
dissolved in CH2Cl2 (1 L). The reaction appartus was flushed with N2
vigorously. The reaction mixture was heated to 45 °C for 1 h. The
apparatus was flushed with N2, and TFA (1 mL) was added
maintaining the temperature at 45 °C. The mixture was then flushed
with nitrogen gas for an additional 15 min, and Grubbs second
generation catalyst (0.21 g, 0.25 mmol), predissolved in CH2Cl2 (10
mL), was added to the reaction mixture. The mixture was stirred at 45
°C for 5 h. Solvent was removed from the reaction mixture under
reduced pressure. The crude residue was purified by column
chromatography (EtOAc/MeOH) to furnish 1.2 g of the crude
product which was recrystallized with EtOAc/hexane to give 950 mg
of the desired product. This material was dissolved in CH2Cl2 (20 mL)
and washed with saturated NaHCO3. The organic layer was separated,
dried (Na2SO4), and concentrated under reduced pressure to obtain
(827 mg, 86%) of 26h as free base. The free amine was dissolved in 1
N HCl (20 mL). The resulting mixture was filtered through a filter
paper and lyophilized to afford 26h as 2HCl monohydrate salt (900
mg, 78%) as a light yellow solid. LC/MS (ESI positive mode) m/z 373
1
([M + H]+); H NMR (CDCl3) δ 10.6 (br, 1H), 9.9 (s,1H), 8.80 (s,
1H), 8.51 (d, 1H), 7.81 (s, 1H), 7.57 (d, 1H),7.40 (m, 2H), 7.29 (d,
1H), 7.23 (m, 2H), 7.16 (d, 1H), 6.08 (m, 1H), 5.74 (m, 1H), 4.50
(m, 1H), 4.17−4.07 (m, 2H), 3.93−3.67 (m, 3H), 2.51 (m, 2H), 2.42
(s, 3H); 13C NMR (CDCl3) δ 163.1, 160.2, 159.2, 141.6, 140.8, 138.6,
131.1, 130.7, 129.6, 124.0, 121.0,120.4, 119.7, 116.5, 115.1,108.5, 65.3,
57.7,57.5, 37.4, 32.4. Anal. Calcd for C23H28Cl2N4O2 (2HCl·H2O
salt): C, 59.61; H, 6.09; N, 12.09; Cl, 15.30. Found: C, 59.87; H, 5.82;
N, 12.28; Cl, 15.50. IR (KBr pellet): 3474, 3058, 1633, 1591, 1459,
1281, 1213, 1801, 1043, 793, 692 cm−1.
189
dx.doi.org/10.1021/jm201112g | J. Med. Chem. 2012, 55, 169−196

Journal of Medicinal Chemistry
Article
Following a procedure similar to that of 26h, the following products
were synthesized but isolated as free base.
3.07 (m, 1H), 2.60−2.73 (m, 3H), 1.78−1.98 (m, 1H), 0.60 (d, 3H),
0.51 (d, 3H).
(17E)-20-Oxa-5,7,13,26-tetraazatetracyclo[19.3.1.1(2,6).1-
(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,17,21,23-decaen-
14-one (26a). The title compound was synthesized from 23a (73%,
>95% trans by 1H NMR). LC/MS (ESI positive mode) m/z 373 ([M
(16E)-14-(Cyclopropylmethyl)-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (26m). The title compound
1
was synthesized from 24c (52%, >95% trans by H NMR). LC/MS
1
1
+ H]+) C22H20N4O2; H NMR (CDCl3) δ 11.23 (s, 1H), 8.34 (m,
(ESI positive mode) m/z 413 ([M + H]+) C26H28N4O; H NMR
1H), 8.13 (d, 1H), 7.71 (m, 1H), 7.50 (d, 1H), 7.41 (t, 1H), 7.24−
7.34 (m, 4H), 7.14 (s, 1H), 7.02 (m, 1H), 5.75−5.87 (m, 2H), 4.61 (d,
2H), 2.45 (s, 4H).
(CDCl3) δ 12.00 (s, 1H), 8.80 (s, 1H), 8.30 (br s, 1H), 7.93 (d, 1H),
7.38−7.59 (m, 6H), 7.17−7.21 (m, 1H), 6.12 (dt, 1H), 5.89 (dt, 1H),
4.79 (d, 1H), 4.22−4.27 (m, 3H), 3.94−4.00 (m, 1H), 3.86 (d, 1H),
2.68−2.86 (m, 4H), 1.05 (m, 1H), 0.64−0.72 (m, 2H), 0.19−0.23 (m,
2H).
(16E)-14-Methyl-5,7,14,20,26-pentaazatetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-dec-
aene-19-one (26b). The title compound was synthesized from 23b
(16E)-14-(2,2-Dimethylpropyl)-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (26n). The title compound
1
(81%, >95% trans by H NMR). LC/MS (ESI positive mode) m/z
1
386 ([M + H]+) C23H23N5O; H NMR (CDCl3) δ 8.55−8.54 (m,
1
1H), 8.27 (s, 1H), 8.03−7.97 (m, 2H), 7.54−7.52 (m, 1H), 7.38−7.35
(m, 1H), 7.31 (s, 1H), 7.25−7.10 (m, 3H), 6.77−6.75 (m, 1H), 5.85−
5.69 (m, 2H), 5.08 (s, 2H), 4.03−3.95 (m, 4H), 3.48 (s, 3H).
(16E)-14-Methyl-5,7,14,19,26-pentaazatetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-dec-
aene-20-one (26c). The title compound was synthesized from 23c
was synthesized from 24d (75%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 429 ([M + H]+) C27H32N4O; H NMR
(CDCl3) δ 8.79 (s, 1H), 8.39 (d, 1H), 7.86 (d, 1H), 7.68 (d, 1H),
7.45−7.47 (m, 2H), 7.42 (t, 1H), 7.27 (d, 1H), 7.14 (d, 1H), 7.05−
7.08 (m, 1H), 6.02 (dt, 1H), 5.87 (dt, 1H), 4.87−4.91 (m, 1H), 4.50−
4.51 (m, 1H), 4.27−4.31 (m, 1H), 4.04−4.12 (m, 1H), 3.80−3.85 (m,
2H), 2.97 (d, 2H), 2.65−2.70 (m, 2H), 0.66 (s, 9H).
1
(82%, >95% trans by H NMR). LC/MS (ESI positive mode) m/z
1
386 ([M + H]+) C23H23N5O; H NMR (CDCl3) δ 8.55−8.54 (m,
14-(Pyridin-2-ylmethyl)-20-oxa-5,7,14,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (26o). The title compound was
1H), 8.27 (s, 1H), 8.03−7.97 (m, 2H), 7.54−7.52 (m, 1H), 7.38−7.35
(m, 1H), 7.31 (s, 1H), 7.25−7.10 (m, 3H), 6.77−6.75 (m, 1H), 5.85−
5.69 (m, 2H), 5.08 (s, 2H), 4.03−3.95 (m, 4H), 3.48 (s, 3H).
1-(20-Oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1-
(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaen-
14-yl)-2,2,2-trifluoroethanone (26f). The title compound was
1
synthesized from 24e (42%, >95% trans by H NMR). LC/MS (ESI
positive mode) m/z 450 ([M + H]+) C28H27N5O; 1H NMR (CDCl3)
δ 11.9 (s, 1H), 8.90 (s, 1H), 8.59 (d, 1H), 8.27 (d, 1H), 7.99 (s, 1H),
7.67 (t, 1H), 7.66 (d, 1H), 7.36−7.57 (m, 7H, m), 7.19−7.21 (m, 1H),
6.07 (dt, 1H), 5.92 (dt, 1H), 4.55 (s, 2H), 4.31 (s, 2H), 3.88−3.89 (m,
2H), 2.64 (d, 2H), 2.64 (t, 2H).
1
synthesized from 23e (73%, 90% trans by H NMR). LC/MS (ESI
positive mode) m/z 455 ([M + H]+) C24H21F3N4O2; 1H NMR
(CDCl3) δ 8.85 (d, 1H), 8.38 (d, 1H), 7.99−7.94 (m, 1H), 7.52−7.34
(m, 4H), 7.27 (d, 1H), 7.03−6.92 (m, 2H), 5.79−5.46 (m, 2H), 4.80
(s, 2H), 4.20 (t, 2H), 4.09−4.02 (m, 2H), 2.54−2.50 (m, 2H).
14-Methyl-19-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-dec-
aene (26i). The title compound was synthesized from 23g (74%,
( 1 6 E ) - 1 4 - ( 2 - M e t h o x y e t h y l ) - 2 0 - o x a - 5 , 7 , 1 4 , 2 6 -
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (26p). The title compound
1
was synthesized from 24f (77%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 417 ([M + H]+) C25H28N4O2; H NMR
(MeOD-d4) δ 8.71 (s, 1H), 8.36 (d, 1H), 7.88 (s, 1H), 7.43 (d, 1H),
7.31 (t, 1H), 7.19−7.15 (m, 2H), 7.02 (dd, 1H), 6.96 (t, 2H), 5.76−
5.80 (m, 1H), 5.59−5.66 (m, 1H), 4.09 (t, 2H), 3.85 (s, 2H), 3.42 (d,
2H), 3.30−3.28 (m, 2H), 3.25, (s, 3H), 2.44, (br s, 2H), 2.43 (d, 2H).
2-(20-Oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1-
(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaen-
14-yl)ethanol (26q). The title compound was synthesized from 24g
1
15:85 cis/trans ratio by H NMR). LC/MS (ESI positive mode) m/z
1
373 ([M + H]+) C23H24N4O; H NMR (MeOD-d4) δ 8.66−8.85 (m,
1H), 8.46 (d, 1H), 8.31 (s, 1H), 7.92 (d, 1H), 7.55−7.52 (m, 1H),
7.51−7.50 (m, 1H), 7.39−7.34 (m, 2H), 7.21−7.18 (m, 1H), 7.09−
7.08 (m, 1H), 6.22 (td, 1H), 5.91−5.84 (m, 1H, CH), 4.61−4.58 (m,
2H), 4.15 (s, 2H), 3.94−3.86 (m, 2H), 2.73 (s, 2H), 2.59 (s, 3H).
(16E)-19-Methyl-14-oxa-5,7,19,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (26j). The title compound was
synthesized from 23h (64%, >95% trans by 1H NMR). LC/MS
1
(63%, >95% trans by H NMR). LC/MS (ESI positive mode) m/z
1
403 ([M + H]+) C24H26N4O2; H NMR (MeOD-d4) δ 9.09 (s, 1H),
8.41 (d, 1H), 7.91 (s, 1H), 7.48−7.53 (m, 1H), 7.28−7.40 (m, 3H),
7.08−7.15 (m, 3H), 6.13−6.20 (m, 1H), 5.77−5.82 (m, 1H), 4.12−
4.00 (m, 2H), 4.00−3.98 (m, 2H), 2.59−2.61 (m, 2H), 1.20−1.28 (m,
4H), 0.80−0.87 (m, 2H).
1
(ESI positive mode) m/z 373 ([M + H]+) C23H24N4O; H NMR
(MeOD-d4) δ 8.57−8.43 (m, 3H), 8.22 (d, 1H), 7.76−7.74 (m, 1H),
7.68 (t, 1H), 7.48 (d, 1H), 7.34 (t, 1 H), 7.16 (d, 1H), 7.08 (dt, 1H),
6.25 (dt, 1H), 6.03 (dt, 1H), 4.81 (s, 2H), 4.29−4.26 (m, 2H), 3.82−
3.79 (m, 2H), 2.91 (s, 3H).
(16E)-11-Methoxy-14-methyl-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (27a). The title compound
1
(16E)-14-Cyclopropyl-20-oxa-5,7,14,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (26k). The title compound was
was synthesized from 25a (71%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 403 ([M + H]+) C24H26N4O2; H NMR
(CDCl3) δ 12.19 (s, 1H), 8.72 (d, 1H), 8.33 (d, 1H), 7.93 (s, 1H),
7.56−7.60 (m, 2H), 7.51 (dd, 1H), 7.38 (d, 1H), 7.22−7.24 (m, 1H),
7.03 (d, 1H), 4.18−4.33 (m, 6H), 2.74 (q, 2H), 2.68 (s, 3H), 2.14 (s,
3H).
1
synthesized from 24a (49%, >95% trans by H NMR). LC/MS (ESI
positive mode) m/z 399 ([M + H]+) C25H26N4O; 1H NMR (MeOD-
d4) δ 9.05 (s, 1H), 8.44 (br s, 1H), 7.90 (br s, 1H), 7.52 (d, 1H), 7.43
(t, 1H), 7.38 (t, 1H), 7.31 (d, 1H), 7.22 (dd, 1H), 7.10−7.16 (m, 2H),
6.21 (dt, 1H), 5.80 (dt, 1H), 4.60 (br s, 1H), 3.90−4.19 (5H, m), 2.80
(m, 1H), 2.65−2.66 (m, 2H), 0.51−0.60 (m, 3H), 0.58−0.55 (br s,
1H).
( 1 6 E ) - 1 1 - C h l o r o - 1 4 - m e t h y l - 2 0 - o x a - 5 , 7 , 1 4 , 2 6 -
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (27b). The title compound
1
was synthesized from 25b (48%, >95% trans by H NMR). LC/MS
1
(16E)-14-Isobutyl-20-oxa-5,7,14,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (26l). The title compound was
synthesized from 24b (53%, >95% trans by 1H NMR). LC/MS
(ESI positive mode) m/z 407 ([M + H]+) C23H23ClN4O; H NMR
(DMSO-d6) δ 10.15 (s, 1H), 9.73 (br s, 1H), 9.10 (d, 1H), 8.63 (d,
1H), 7.90 (s, 1H), 7.69 (d, 1H), 7.50−7.55 (m, 2H), 7.39 (dd, 1H),
7.27 (dd, 1H), 6.12−6.23 (m, 1H), 5.67−5.78 (m, 1H), 4.59 (s, 2H),
4.08−4.27 (m, 4H), 3.85−3.89 (m, 2H), 2.59 (s, 3H).
1
(ESI positive mode) m/z 415 ([M + H]+) C26H30N4O; H NMR
(MeOD-d4) δ 8.95 (br s, 1H), 8.42 (d, 1H), 7.99 (t, 1H), 7.47 (t, 1H),
7.38 (t, 1H), 7.35 (t, 1H), 7.28 (d, 1H), 7.16 (dd, 1H), 7.11 (d, 1H),
7.08 (dd, 2H), 6.15 (dt, 1H), 5.85 (dt, 1H), 4.58 (d, 1H), 4.22−4.26
(m, 1H), 4.11 (d, 1H), 3.86−4.05 (m, 3H), 3.86−4.05 (m, 3H), 3.02−
(16E)-14-Methyl-11-trifluoromethoxy-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (27c). The title compound
1
was synthesized from 25c (53%, >95% trans by H NMR). LC/MS
190
dx.doi.org/10.1021/jm201112g | J. Med. Chem. 2012, 55, 169−196

Journal of Medicinal Chemistry
Article
1
(ESI positive mode) m/z 457 ([M + H]+) C24H23F3N4O2; H NMR
(DMSO-d6) δ 10.18 (s, 1H), 9.85 (br s, 1H), 9.13 (m, 1H), 8.63 (d,
1H), 7.90 (s, 1H), 7.69 (d, 1H), 7.55 (d, 1H), 7.52 (t, 1H), 7.46 (br s,
1H), 7.29 (dd, 1H), 6.12−6.23 (m, 1H), 5.67−5.78 (m, 1H), 3.95−
4.59 (m, 6H), 3.85−3.89 (m, 2H), 2.59 (s, 3H).
1H), 6.03−5.89 (m, 1H), 5.83−5.74 (m, 1H), 4.43 (m, 2H), 4.28−
4.25 (m, 4H), 4.01 (m, 2H), 3.77 (d, 2H), 3.70 (m, 2H), 3.38 (m,
2H), 3.15−3.07 (m, 2H), 2.63−2.61 (m, 2H), 2.24−2.20 (m, 4H).
11-(2-(Pyrrolidin-1-yl)ethoxy)-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaen-14-acetonitrile (27m). The
(16E)-11-(2-Methoxyethoxy)-14-methyl-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (27d). The title compound
1
title compound was synthesized from 25l (62%, >95% trans by H
NMR). LC/MS (ESI positive mode) m/z 511 ([M + H]+)
1
1
was synthesized from 25d (56%, >95% trans by H NMR). LC/MS
C30H34N6O2; H NMR (CDCl3) δ 8.44 (d, 1H), 8.30 (d, 1H), 7.84
1
(ESI positive mode) m/z 447 ([M + H]+) C26H30N4O3; H NMR
(s, 1H), 7.50 (d, 1H), 7.45 (t, 1H), 7.27 (d, 1H), 7.16 (d, 1H), 7.11
(d, 1H), 5.81 (dt, 1H), 5.52 (dt, 1H), 4.36 (t, 2H), 4.27 (t, 2H), 3.85−
3.62 (m, 8H), 3.33 (s, 2H), 3.24 (d, 2H), 2.11−2.15 (m, 4H).
14-Methyl-11-(2-(morpholin-4-yl)ethoxy)-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (27n). The title compound
(DMSO-d6) δ 9.48 (s, 1H), 8.52 (d, 1H), 8.50 (d, 1H), 7.87 (t, 1H),
7.64 (dd, 1H), 7.47 (t, 1H), 7.35 (d, 1H), 7.24 (dd, 1H), 7.04 (dd,
1H), 6.93 (d, 1H), 5.64−5.71 (m, 1H), 5.44−5.51 (m, 1H), 4.25 (t,
2H), 4.05−4.08 (m, 2H), 3.65−3.68 (m, 2H), 3.48 (s, 2H), 3.34 (s,
3H), 3.00 (d, 2H), 2.37−2.40 (m, 2H), 1.98 (s, 3H).
(16E)-11-Ethylsulfonyl-14-methyl-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (27e). The title compound
1
was synthesized from 25m (66%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 502 ([M + H]+) C29H35N5O3; H NMR
(CDCl3) δ 12.05 (s, 1H), 8.66 (d, 1H), 8.22 (d, 1H), 7.78 (s, 1H),
7.45−7.47 (m, 2H), 6.38 (dd, 1H), 7.29 (d, 1H), 7.08−7.11 (m, 1H),
6.83 (d, 1H), 5.98−6.05 (m, 1H), 5.72−5.78 (m, 1H), 4.33−4.39 (m,
2H), 4.19−4.25 (m, 2H), 3.96−4.00 (m, 6H,), 3.54−3.81 (m, 6H),
2.99−3.01 (m, 2H), 2.63−2.68 (m, 2H), 2.53 (s, 3H).
1
was synthesized from 25e (68%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 465 ([M + H]+) C25H28N4O3S; H NMR
(MeOD-d4) δ 9.51 (d, 1H), 8.62 (d, 1H), 8.05−7.97 (m, 2H), 7.65−
7.63 (m, 1H), 7.54−7.52 (m, 2H), 7.52−7.49 (m, 2H), 7.23−7.21 (m,
1H), 6.31−6.24 (m, 1H), 6.02−5.94 (m, 1H), 4.43−4.04 (m, 8H),
3.38−3.32 (m, 2H), 2.74 (s, 3H), 1.33 (t, 3H).
14-Methyl-11-(2-(4-methylpiperazin-1-yl)ethoxy)-20-oxa-
5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1-
(25),2(26),3,5,8(27),9,11,16,21,23-decaene (27o). The title com-
2-(14-Methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-dec-
aen-11-yl)oxy-1-(pyrrolidin-1-yl)ethanone (27h). The title com-
pound was synthesized from 25g (59%, >95% trans by 1H NMR). LC/
MS (ESI positive mode) m/z 500 ([M + H]+) C29H33N5O3; 1H NMR
(MeOD-d4) δ 8.82 (d, 1H), 8.39 (d, 1H), 7.87−7.86 (m, 1H), 7.51−
7.47 (m, 1H), 7.39 (t, 1H), 7.33 (d, 1H), 7.18 (dd, 1H), 7.14−7.09
(m, 2H), 6.18−6.10 (m 1H), 6.18−6.10 (m, 1H), 5.91−5.82 (m, 1H),
4.24−4.20 (m, 1H), 4.09−3.92 (m, 3H), 3.84−3.78 (m, 1H), 2.55 (s,
3H), 1.98−1.91 (m, 4H), 1.84−1.79 (m, 3H).
1
pound was synthesized from 25n (50%, >95% trans by H NMR).
1
LC/MS (ESI positive mode) m/z 515 ([M + H]+) C30H38N6O2; H
NMR (CDCl3) δ 11.99 (s, 1H), 8.73 (d, 1H), 8.27 (d, 1H), 7.86−7.87
(br s, 1H), 7.52−7.56 (m, 2H), 7.47 (dd, 1H), 7.35 (d, 1H), 7.16−
7.19 (m, 1H), 6.96 (d, 1H), 6.05−6.11 (m, 1H), 5.82−5.88 (m, 1H),
4.28−4.36 (m, 4H), 3.71−4.03. (m, 12H), 3.05 (br s, 2H), 2.91 (s,
3H), 2.60 (s, 3H), 2.05 (s, 2H).
14-Methyl-11-(2-(piperidin-1-yl)ethoxy)-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (27p). The title compound
(16E)-11-(4-Morpholino)-14-methyl-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (27i). The title compound
1
was synthesized from 25o (62%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 500 ([M + H]+) C30H37N5O2; H NMR
1
was synthesized from 25h (88%, >95% trans by H NMR). LC/MS
(CDCl3) δ 12.04 (s, 1H), 8.72 (d, 1H), 8.25 (d, 1H), 7.82 (br s, 1H),
7.51−7.52 (m, 2H), 7.41 (dd, 1H), 7.34 (d, 1H), 7.14−7.18 (m, 1H),
6.94 (d, 1H), 6.03−6.10 (m, 1H), 5.80−5.87 (m, 1H), 4.95 (d, 1H),
4.22−4.33 (m, 4H), 4.02−4.06 (m, 1H), 3.59−4.06 (m, 6H), 2.93 (br
s, 2H), 2.65−2.77 (m, 2H, CH2), 2.59 (s, 3H), 1.83−2.01 (m, 6H).
10-Methoxy-14-methyl-11-(2-(pyrrolidin-1-yl)ethoxy)-20-
oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]-
heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (27q).
The title compound was synthesized from 25p (44%, >95% trans by
1H NMR). LC/MS (ESI positive mode) m/z 516 ([M + H]+)
1
(ESI positive mode) m/z 458 ([M + H]+) C27H31N5O2; H NMR
(CDCl3) δ 10.83 (s, 1H), 9.04 (br s, 1H), 8.34 (d, 1H), 8.06 (t, 1H),
7.56−7.59 (m, 1H), 7.53 (t, 1H), 7.35−7.38 (m, 2H), 7.25 (s, 1H),
7.19 (dd, 1H), 6.10−6.17 (m, 1H), 5.91−5.96 (m, 1H), 4.27−4.30 (m,
2H), 4.03−4.04 (m, 2H), 3.84−3.94 (m, 6H), 3.00 (m, 2H), 2.90 (m,
2H), 2.69 (m, 2H), 2.64 (s, 3H).
(16E)-11-(2-(Diethylamino)ethoxy)-14-methyl-20-oxa-
5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1-
(25),2(26),3,5,8(27),9,11,16,21,23-decaene (27j). The title com-
pound was synthesized from 25i (50%, >95% trans by 1H NMR). LC/
MS (ESI positive mode) m/z 488 ([M + H]+) C29H37N5O2; 1H NMR
(CDCl3) δ 12.03 (s, 1H), 8.79 (d, 1H), 8.29 (d, 1H), 7.92−7.93 (br s,
1H), 7.56−7.60 (m, 2H), 7.49 (dd, 1H), 7.39 (d, 1H), 7.19−7.22 (m,
1H), 6.98 (d, 1H), 6.07−6.14 (m, 1H), 5.88−5.95 (m, 1H), 4.42 (br s,
2H), 4.34−4.38 (m, 2H), 3.90−4.09 (m, 2H), 3.28−3.58 (m, 6H),
2.74−2.85 (m, 2H), 2.66 (s, 3H), 1.47 (t, 3H), 1.42 (t, 3H).
(16E)-14-Methyl-11-(2-(pyrrolidin-1-yl)ethoxy)-20-oxa-
5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1-
(25),2(26),3,5,8(27),9,11,16,21,23-decaene (27k). The title com-
pound was synthesized from 25j (67%, >95% trans by 1H NMR). LC/
MS (ESI positive mode) m/z 486 ([M + H]+) C29H35N5O2; 1H NMR
(CDCl3) δ 11.94 (s, 1H), 8.73 (d, 1H), 8.26 (d, 1H), 7.85−7.86 (br s,
1H), 7.50−7.52 (m, 2H), 7.41 (dd, 1H), 7.34 (d, 1H), 7.13−7.16 (m,
1H), 6.92 (d, 1H), 6.04−6.09 (m, 1H), 5.82−5.89 (m, 1H), 4.28−4.37
(m, 4H), 3.92−4.06. (m, 4H), 3.05 (br s, 2H), 2.65−2.74 (m, 2H),
2.60 (s, 3H), 2.20−2.24 (m, 4H), 2.12−2.14 (m, 4H).
C30H37N5O3; 1H NMR (CDCl3) δ 8.59 (d, 1H), 8.49 (br s, 1H), 7.93
(s, 1H), 7.52 (d, 1H), 7.41 (t, 1H), 7.23 (d, 1H), 7.13 (dd, 1H), 6.97
(d, 1H), 6.18 (dt, 1H), 5.77 (m, 1H), 4.32−4.24 (m, 4H), 4.05 (d,
2H), 3.90 (s, 2H), 3.85−3.81 (m, 4H), 3.65 (t, 2H), 3.26−3.20 (m,
2H), 3.25 (s, 3H), 2.61 (s, 3H), 2.16−2.05 (m, 4H).
N-(14-Methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-dec-
aen-10-yl)-2,2,2-trifluoroacetamide (27r). The title compound
1
was synthesized from 25q (68%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 483 ([M + H]+) C25H24F3N5O2; H NMR
(MeOD-d4) δ 8.98 (s, 1H), 8.51 (d, 1H), 7.98 (s, 1H), 7.59−7.57 (m,
1H), 7.51−7.47 (m, 3H), 7.41−7.39 (m, 1H), 7.20−7.18 (m, 1H),
6.22 (dt, 1H), 5.83 (dt, 1H), 4.85−4.35 (m, 2H), 4.34−4.09 (m, 2H),
4.07−3.79 (m, 2H), 2.74−2.64 (m, 2H), 2.69 (s, 3H)
(16E)-4,14-Dimethyl-20-oxa-5,7,14,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (28a). The title compound was
(16E)-11-(2-(Pyrrolidin-1-yl)ethoxy)-14-(2,2,2-trifluoroethyl)-
20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]-
heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (27l).
The title compound was synthesized from 25k (51%, >95% trans by
1H NMR). LC/MS (ESI positive mode) m/z 554 ([M + H]+)
C30H34F3N5O2; H NMR (CDCl3) δ 8.61 (s, 1H), 8.30 (d, 1H), 7.92
(s, 1H), 7.57−7.54 (m, 2H), 7.40−7.36 (m, 2H), 7.24(d, 1H), 6.95 (d,
1
synthesized from 25r (56%, >95% trans by H NMR). LC/MS (ESI
positive mode) m/z 387 ([M + H]+) C24H26N4O; 1H NMR (DMSO-
d6) δ 9.62 (s, 1H), 8.60 (s, 1H), 8.42 (s, 1H), 7.92 (t, 1H), 7.64 (d,
1H), 7.46 (t, 1H), 7.34 (s, 1H), 7.16−7.22 (m, 2H), 7.05 (d, 1H), 6.86
(d, 1H, 5.48−5.74 (m, 2H), 4.20 (t, 2H), 3.48 (s, 2H), 3.06 (d, 2H),
2.45−2.46 (m, 2H), 2.42 (s, 3H), 2.01 (s, 3H).
1
191
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Journal of Medicinal Chemistry
Article
(16E)-14-Methyl-4-(pyrrolidin-1-yl)-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (28b). The title compound
2-(Isopropylamino)-1-(20-oxa-5,7,14,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaen-14-yl)ethanone (26r). To a solution of
26g (5 mg, 0.0168 mmol) and bromoacetyl bromide (1.5 μL, 0.0168
mmol) in CH2Cl2 (1 mL) was added diisopropylethylamine (3 μL,
0.0168 mmol), and the resulting solution was stirred at room
temperature for 4 h. Isopropylamine (5 μL) was added, and resulting
mixture was stirred for another 4 h. It was concentrated under reduced
pressure and purified by preparative HPLC to furnish 26r (3 mg, 39%,
>95% trans by 1H NMR) as a pale yellow solid. LC/MS (ESI positive
1
was synthesized from 25s (49%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 442 ([M + H]+) C27H31N5O; H NMR
(CDCl3) δ 12.6 (s, 1H), 8.73 (s, 1H), 7.86−7.91 (m, 2H), 7.41−7.48
(m, 2H), 7.34−7.35 (m, 1H), 7.10 (d, 1H), 6.45 (s, 1H), 6.03−6.18
(m, 1H), 5.86−5.92 (m, 1H), 4.13−4.30 (m, 4H), 3.83−3.84 (m, 2H),
3.68 (s, 2H), 2.74 (m, 2H), 2.56 (s, 2H), 2.21 (s, 3H), 1.63 (m, 4H).
(16E)-3,14-Dimethyl-20-oxa-5,7,14,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (28c). The title compound was
1
mode) m/z 458 ([M + H]+) C27H31N5O2; H NMR (MeOD-d4) δ
8.60 (s, 1H), 8.48 (d, 1H), 7.97 (d, 1H), 7.62 (m, 1H), 7.46 (t, 1H),
7.35 (m, 1H), 7.28 (t, 1H), 7.24 (dd, 1H), 7.06 (t, 1H), 6.86 (d, 1H),
5.78−5.71 (m, 1H), 5.52−5.42 (m, 1H), 4.23 (t, 2H), 4.18−4.10 (m,
5H), 3.92 (s, 2H), 2.52 (br, 2H), 1.39 (d, 6H).
1
synthesized from 25t (54%, >95% trans by H NMR). LC/MS (ESI
positive mode) m/z 387 ([M + H]+) C24H26N4O; 1H NMR (DMSO-
d6) δ 9.52 (s, 1H), 8.63 (br s, 1H), 8.42 (s, 1H), 7.51 (t, 1H), 7.44 (t,
1H), 7.32−7.37 (m, 1H), 7.13−7.19 (m, 2H), 7.02−7.08 (m, 1H),
6.82 (d, 1H), 5.64−5.72 (m, 1H), 5.62−5.71 (m, 1H), 5.51−5.55 (m,
1H), 4.20 (t, 2H), 3.41 (s, 2H), 3.07 (d, 2H), 3.34 (s, 3H), 1.98 (s,
3H).
(16E)-11-(2-(Ethylsulfonyl)ethoxy)-14-methyl-20-oxa-
5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1-
(25),2(26),3,5,8(27),9,11,16,21,23-decaene (27g). To a solution
of 27f (20 mg, 0.042 mmol) in acetic acid (2 mL) at 0 °C was added
50% hydrogen peroxide solution (0.1 mL), and the resulting mixture
was stirred at room temperature for 7 h. It was concentrated under
reduced pressure and purified by preparative HPLC to furnish 27g (4
( 1 6 E ) - 2 3 - F l u o r o - 1 4 - m e t h y l - 2 0 - o x a - 5 , 7 , 1 4 , 2 6 -
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (28d). The title compound
1
1
was synthesized from 25u (51%, >95% trans by H NMR). LC/MS
mg, 19%, >95% trans by H NMR) as a yellow solid. LC/MS (ESI
1
(ESI positive mode) m/z 391 ([M + H]+) C23H23FN4O; H NMR
positive mode) m/z 509 ([M + H]+) C27H32N4O4S; 1H NMR
(MeOD-d4) δ 8.66 (d, 1H), 8.41 (d, 1H), 7.97 (t, 1H), 7.52 (d, 1H),
7.42 (t, 1H), 7.26 (d, 1H,), 7.09−7.14 (m, 2H), 7.03 (d, 1H), 5.88 (dt,
1H), 5.72 (dt, 1H, CH), 4.49 (t, 2H), 4.20 (t, 2H), 3.91 (brs, 2H),
3.64 (t, 2H), 3.46−3.48 (m, 2H), 3.25−3.28 (m, 2H), 2.53−2.57 (m,
2H), 2.28 (s, 3H), 1.42 (t, 3H).
(CDCl3) δ 9.78 (s, 1H), 8.85 (s, 1H), 8.45 (m, 1H), 7.82 (s, 1H),
7.48−7.50 (m, 2H), 7.21−7.29 (m, 3H), 6.87 (td, 1H), 6.06−6.13 (m,
1H), 5.85−5.93 (m, 1H), 4.16−4.27 (m, 4H), 3.75−3.85 (m, 2H),
2.74−2.75 (m, 2H), 2.62 (s, 3H).
(16E)-22-Methoxy-14-methyl-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (28e). The title compound
N-(Ethylsulfonylmethyl)(14-methyl-20-oxa-5,7,14,26-
tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaen-10-yl)amine (27t). To a
solution of 27s (9 mg, 0.021 mmol) and ethanesulfonyl chloride (10
μL) in CH2Cl2 (1 mL) was added triethylamine (10 μL), and the
resulting solution was stirred at room temperature for 4 h. It was
concentrated under reduced pressure and purified by preparative
HPLC to furnish 27t (4 mg, 40%, >95% trans by 1H NMR) as a pale
yellow solid. LC/MS (ESI positive mode) m/z 480 ([M + H]+)
1
was synthesized from 25v (44%, >95% trans by H NMR). LC/MS
1
(ESI positive mode) m/z 403 ([M + H]+) C24H26N4O2; H NMR
(CDCl3) δ 11.8 (s, 1H), 8.41−8.44 (m, 2H), 8.26 (br s, 1H), 7.95 (d,
1H), 7.65 (br s, 1H), 7.52−7.54 (m, 1H), 7.45 (d, 1H), 7.45 (d, 1H),
7.31−7.34 (m, 1H), 7.07 (d, 1H), 6.18−6.24 (m, 2H), 4.67 (s, 2H),
4.08−4.27 (m, 4H), 3.99 (s, 3H), 3.93−4.04 (m, 1H), 3.65 (br s, 1H),
2.84 (s, 3H).
1
C26H31N5O3S; H NMR (MeOD-d4) δ 8.47 (s, 1H), 8.39 (d, 1H),
19-Oxa-5,7,13,25-tetraazatetracyclo[18.3.1.1(2,6).1(8,12)]-
hexacosa-1(24),2(25),3,5,8(26),9,11,15,20,22-decaene (26e). To
a solution of 26d (12 mg, 0.027 mmol) in MeOH/CH2Cl2 (0.2:1, 2
mL) was added 4 M HCl (0.5 mL), and the resulting mixture was
heated at 40 °C for 4 h. It was concentrated under reduced pressure
and purified by preparative HPLC to furnish 26e (7 mg, 79%, >95%
trans by 1H NMR) as a yellow solid. LC/MS (ESI positive mode) m/z
8.01 (s, 1H), 7.41−7.39 (m, 1H), 7.34 (t, 1H), 7.13 (d, 1H), 7.01−
6.98 (m, 1H), 7.86−7.83 (m, 2H), 5.68−5.65 (m, 2H), 4.14 (t, 2H),
3.60−3.55 (m, 2H), 3.39 (s, 2H), 3.17−3.13 (m, 2H), 3.07 (q, 2H),
2.54−2.52 (m, 2H), 2.28 (s, 3H), 1.28 (t, 3H).
N-(14-Methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-dec-
aen-10-yl)morpholine-4-carboxamide (27u). Following a proce-
dure similar to that of 27t, the title compound was synthesized from
27s and Grubbs second generation catalyst. LC/MS (ESI positive
1
346 ([M + H]+) C21H20N4O; H NMR (MeOD-d4) δ 8.58−8.57 (m,
2H), 8.52 (d, 1H), 8.02 (t, 1H), 7.74−7.72 (m, 1H), 7.56−7.41 (m,
3H), 7.30−7.27 (m, 2H), 7.13 (dd, 1H), 6.18 (td, 1H), 5.86 (td, 1H),
4.32−4.30 (m, 2H), 4.12 (d, 2H), 2.39−2.34 (m, 2H).
1
mode) m/z 501 ([M + H]+) C28H32N6O3; H NMR (MeOD-d4) δ
8.81 (s, 1H), 8.50 (d, 1H), 8.02 (s, 1H), 7.58−7.56 (m, 1H), 7.48 (t,
1H), 7.38 (d, 1H), 7.22−7.16 (m, 1H), 6.92 (s, 1H), 6.89−6.85 (m,
1H), 6.26−6.18 (m, 1H), 5.90−5.83 (m, 1H), 4.56 (t, 2H), 4.38−4.30
(m, 2H), 4.19−4.05 (m, 2H), 3.73−3.70 (m, 2H), 3.56−3.53 (m, 2H),
2.70 (s, 3H), 2.24−2.02 (m, 4H).
20-Oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]-
heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (26g).
To a mixture of 26f (45 mg, 0.100 mmol) in 5.5% H2O/MeOH (3
mL) was added K2CO3 (69 mg, 0.500 mmol), and the resulting
mixture was stirred at 90 °C for 6 h. It was concentrated under
reduced pressure and purified by preparative HPLC to furnish 26g (17
mg, 48%, >95% trans by 1H NMR) as a pale yellow solid. LC/MS (ESI
positive mode) m/z 360 ([M + H]+) C22H22N4O; 1H NMR (MeOD-
d4) δ 8.99 (br s, 1H), 8.44 (d, 1H), 8.44 (d, 1H), 8.01 (t, 1H), 7.51
(dd, 1H), 7.42 (t, 1H), 7.24−7.31 (m, 2H), 7.24−7.31 (m, 2H), 7.13
(dd, 1H), 7.02−7.06 (m, 2H), 5.85 (dt, 1H), 5.67 (dt, 1H), 4.19 (t,
2H), 3.93 (s, 2H), 3.48 (d, 2H), 2.53−2.57 (m, 2H).
Enzyme Assays. The recombinant enzymes (CDK2/cyclin A,
JAK2, and FLT3) were purchased from Invitrogen (catalog numbers
PV 3267, 4210, and 3182, respectively). All assays were carried out in
384-well white microtiter plates using the PKLight assay system from
Cambrex. This assay platform is a luminometric assay for the detection
of ATP in the reaction using a luciferase-coupled reaction. The
compounds were tested at eight concentrations prepared from 3- or 4-
fold serial dilution starting at 10 μM. For CDK2/cyclin A assay, the
reaction mixture consisted of the following components in 25 μL of
assay buffer (50 mM Hepes, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 5
mM BGP, 1 mM DTT, 0.1 mM sodium orthovanadate), 1.4 μg/mL of
CDK2/cyclin A complex, 0.5 μM RbING substrate (Invitrogen,
catalog number PV2939), and 0.5 μM ATP. The mixture was
incubated at room temperature for 2 h. Then 13 μL of PKLight ATP
detection reagent was added and the mixture was incubated for 10
min. Luminescence signals were detected on a multilabel plate reader
(Victor2 V 1420, Perkin-Elmer). The other kinase assays were similar,
(16E)-14-Methyl-20-oxa-5,7,14,26-tetraazatetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaen-10-amine (27s). Following a procedure
similar to that of 26g, the title compound was synthesized from 27r
1
(58%, >95% trans by H NMR). LC/MS (ESI positive mode) m/z
388 ([M + H]+) C23H25N5O; 1H NMR (CDCl3) δ 8.41 (d, 1H), 8.17
(s, 1H), 8.10 (t, 1H), 7.48−7.43 (m, 1H), 7.38 (t, 1H), 7.16 (s, 1H),
7.04−7.01 (m, 1H), 6.50 (s, 1H), 6.16 (t, 1H), 5.78− 5.69 (m, 2H),
4.19 (t, 2H), 3.62−3.59 (m, 2H), 3.29−3.27 (m, 2H), 2.91 (q, 2H),
2.53 (q, 2H), 2.24 (s, 3H).
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Journal of Medicinal Chemistry
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with the following differences in reagents: For FLT3 assays, the
mixture contained 2.0 μg/mL FLT3 enzyme, 5 μM poly(Glu,Tyr)
substrate (Sigma, catalog number P0275), and 4 μM ATP. For JAK2
assays, the reaction contained 0.35 μg/mL JAK2 enzyme, 10 μM
poly(Glu,Ala,Tyr) substrate (Sigma, catalog number P3899), and 0.15
μM ATP. The analytical software Prism 5.0 (GraphPad Software Pte
Ltd.) was used to generate IC50 values from the data.
Cell Proliferation Assays. All cell lines were obtained from the
American Type Culture Collection (ATCC, Manassas, VA) and
cultured according to the recommended guidelines. For proliferation
assays in 96-well plates, 20 000 cells were seeded in 100 μL of medium
and treated the following day with compounds (in triplicate) at
concentrations up to 10 μM for 48 h. Cell viability was monitored
using the CellTiter-96 Aqueous One solution cell proliferation assay
(Promega, Madison, WI). Dose−response curves were plotted to
determine IC50 values for the compounds using the XL-fit software
(IDBS Ltd., Alameda, CA).
(25 μM) for 60 min (CYP2C19), dextromethorphan (5 μM) for 30
min (CYP2D6), and midazolam (2.5 μM) for 5 min (CYP3A4) at 37
°C. The selective inhibitors α-naphthoflavone, sulfaphenazole,
tranylcypromine, quinidine, and ketoconazole were used as positive
controls for CYP1A, CYP2C9, CYP2C19, CYP2D6, and CYP3A4
inhibitor, respectively. For CYP1A, the reactions were terminated by
the addition of methanol, and the formation of the metabolite,
resorufin, was monitored by fluorescence (excitation wavelength of
535 nm, emission wavelength of 595 nm). For the CYP2C9,
CYP2C19, CYP2D6, and CYP3A4 incubations, the reactions were
terminated by the addition of methanol containing an internal
standard. The samples were centrifuged, and the supernatants were
combined for the simultaneous analysis of 4-hydroxytolbutamide, 4-
hydroxymephenytoin, dextrorphan, 1-hydroxymidazolam, and the
internal standard by LC−MS/MS. Formic acid in deionized water
(final concentration of 0.1%) was added to the final sample prior to
analysis. A decrease in the formation of the metabolites compared to
vehicle control was used to calculate an IC50 value (test compound
concentration that produces 50% inhibition).
Cell Pharmacodynamic Assay. HCT-116 cells (2 × 105 in 5 mL
of McCoy’s medium supplemented with 2 mM L-glutamine and 10%
fetal bovine serum) were seeded in 60 mm dishes 16−24 h before drug
treatment. Each dish was treated separately with different concen-
trations of 26h or DMSO for 24 h prior to lysis using a modified
radioimmunoprecipitation buffer (50 mM Tris-HCl, 150 mM NaCl,
1% sodium deoxycholate, 0.25 mM EDTA (pH 8.0), 1% Triton X-100,
0.2% NaF, and protease inhibitor cocktail (Sigma catalog number
P8340). Proteins were measured using the Bradford assay, and 30 μg
of lysate from each treatment was resolved on 10% SDS−PAGE and
transferred onto PVDF membrane. Western blot analyses using
antibodies against phospho-Rb (catalog number 9308, Cell Signaling
Technology, Danvers, MA) and β-actin (catalog number A2066,
Sigma-Aldrich, St. Louis, MO) were performed using dilutions
recommended by suppliers. Signals were detected using auto-
radiography with Pierce ECL Western blotting substrate (Thermo
Fisher Scientific, Rockford, IL).
Caco-2 Bidirectional Permeability Assay. 26h at 5 μM in
Hank’s balanced salt solution (HBSS), final DMSO concentration less
than 1%, was placed in 21−28 day confluent monolayer cells in
Transwell assay plates. Both apical and basolateral sides were
maintained at pH 7.4. When dosed on the apical side, the permeability
in the A → B direction was assessed, and when dosed on the
basolateral side, the B → A direction was assessed. Both apical and
basolateral sides were sampled at 2 h. The concentration of 26h was
determined by LC/MS using a four-point calibration curve. Atenolol
(Papp < 0.5 × 10−6 cm/s), propranolol (15 × 10−6 cm/s < Papp < 25 ×
10−6 cm/s), Lucifer yellow (Papp > 0.4 × 10−6 cm/s), and digoxin
(efflux ratio of >3) were used in the quality control of the monolayer
batch. The integrity of the monolayer was determined by measuring
the pre-experiment TEER (between 450 and 650 Ωcm2) and using
Lucifer yellow (efflux of ≤0.5%). The efflux ratio was defined as the
High Throughput Solubility Assay. This assay measures the
solubility of a compound in PBS in a high throughput mode. The assay
was done using 96-well semitransparent PP microplates with V-shaped
bottom (Greiner Bio-One) and 96-well UV transparent microplates
(Greiner Bio-One). Compound solutions (250 μM) were prepared in
10 mM phosphate buffer (pH 7.0) containing 20% DMSO in a total
volume of 0.2 mL. Plates were placed on a shaker set at 600 rpm for
1.5 h, following which the plates were allowed to stand for 2 h at room
temperature. The plates were centrifuged at 1500g for 15 min. The
supernatants were transferred to a UV transparent microplate and
analyzed by UV spectrophotometry at the appropriate absorption
maxima. The concentration of the compound in the supernatant was
quantified using a calibration curve. For calculated solubilities of 250
30 μM, solubilities are reported as >250 μM (>150 μg/mL).
Metabolic Stability in Liver Microsomes. Compounds (5 μM)
were incubated with MLM (mouse liver microsomes), RLM (rat liver
microsomes), DLM (dog liver microsomes), and HLM (human liver
microsomes) (final microsomal concentration of ∼0.87 mg/mL) in a
reaction mix containing 50 mM potassium phosphate buffer (pH 7.4)
and NADPH regeneration system, at 37 °C, in a total reaction volume
of 1 mL. Reactions were terminated at 0, 15, 30, 45, and 60 min of
incubation with a chilled mixture of acetonitrile and DMSO (80:20).
The mixture was vortexed for 5 min, centrifuged at 13 200 rpm for 15
min at 4 °C, and the supernatants were analyzed by LC−MS/MS.
Stability was assessed by plotting the percent of parent compound
remaining against time on a log−linear scale, and half-life was
estimated from the linear portion of the log−linear curve using the first
order equation t1/2 = 0.693/k, where k is the slope of the curve (equal
to the first order elimination rate constant).
ratio of Papp,B→A to Papp,A→B.
In Vitro Plasma Protein Binding. Equilibrium dialysis was
performed in a Micro-Equilibrium Dialyzer (Harvard Apparatus) with
a chamber volume of 500 μL (each compartment with a volume of 250
μL). The semipermeable membrane used was rinsed with Milli-Q
water and soaked for 10 min in PBS. 26h was added to plasma (from
mouse, dog, and humans) to obtain a final concentration of 1000 ng/
mL. The spiked plasma was vortexed, and 250 μL was aliquoted into
one chamber of the dialyzer cell. The other chamber was filled with
250 μL of PBS buffer. The assembled cell was placed into a water bath
at 37 °C, and dialysis was performed for 4 h. Following dialysis, 50 μL
of PBS dialyzed samples containing free 26h was transferred into 2 mL
Eppendorf tubes in triplicate for extraction. Samples were extracted
with 1500 μL of MTBE (methyl tert-butyl ether) for 30 min using a
mixer at motor speed setting 60 with pulsing. After 30 min, the sample
tubes were centrifuged at 4 °C for 10 min at 13 000 rpm in a
microcentrifuge. The supernatant (1400 μL) was transferred into fresh
2 mL Eppendorf tubes and dried in a SpeedVac at 43 °C for 35 min.
The dried samples were reconstituted with 100 μL of methanol/Milli-
Q H2O (60:40) and analyzed by LC−MS/MS.
Pharmacokinetics. Male BALB/c mice (aged ∼10−12 weeks and
weighing 17−22 g), male Beagle dogs (∼6−7 months of age, weighing
10−14 kg), and male Wistar rats (aged 6−8 weeks, weighing 239−249
g) were used in this study. All the animal studies were performed as
per approved internal protocols for animal care and use. The oral
doses for mice, dogs, and rats were 75, 10, and 10 mg/kg, respectively.
The doses were administered by gavage as suspensions (0.5%
methylcellulose and 0.1% Tween 80) to mice and rats, and as gelatin
capsules (12 Torpac) to dogs. Following oral dosing serial blood
samples were collected (cardiac puncture in mice, jugular vein in dogs,
and superior vena cava in rats) at different time points (0−24 h) in
tubes containing K3EDTA as anticoagulant, centrifuged, and plasma
was separated and stored at −70 °C until analysis. Plasma samples
were processed and analyzed by LC−MS/MS. Pharmacokinetic
parameters were estimated by noncompartmental methods using
WinNonlin (version 5.1, Pharsight, CA).
Human in Vitro CYP450 Inhibition Assay. 26h was incubated
(at concentrations of 0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final
DMSO concentration of 0.35%) with human liver microsomes (0.25
mg/mL for CYP1A and CYP3A4, 0.5 mg/mL for CYP2C19 and
CYP2D6, 1 mg/mL for CYP2C9) and NADPH (1 mM) in the
presence of the probe substrate ethoxyresorufin (0.5 μM) for 5 min
(CYP1A), tolbutamide (120 μM) for 60 min (CYP2C9), mephenytoin
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Materials and Methods for HCT-116 and Ramos Studies. 1.
Mice/Husbandry. Female BALB/c nude mice (ARC, West Australia),
10−12 weeks of age, were fed with sterilized tap water (ad libitum
water) and irradiated standard rodent diet consisting of 19% protein,
5% fat, and 5% fiber. Mice were housed in individual ventilated cages
on a 12 h light cycle at 21−22 °C and 40−60% humidity. The use of
animals in the Biological Resource Centre (BRC), Biopolis, Singapore,
is compliant with the recommendations of the Guide for Care and Use
of Laboratory Animals with respect to restraint, husbandry, surgical
procedures, feed and fluid regulation, and veterinary care. The animal
care and use program, 050076 at BRC, is Institutional Animal Care
and Use Committee (IACUC) accredited.
2. Tumor Implantation. 2.1. For HCT-116 Study. Mice were
implanted subcutaneously in the right flank with 5 × 106 cells of HCT-
116 human colon carcinoma. Each tumor was monitored twice per
week and subsequently daily as the neoplasms reached the desired size
of approximately 100 mm3. At day 8, when the tumors attained a
calculated tumor volume between 75 to 144 mm3, the animals were
pair-matched and distributed randomly into various treatment groups
(the mean tumor volume in each group was 105 mm3). Estimated
tumor volume was calculated using the formula
6. Statistics. 6.1. For HCT-116 study. The two-way ANOVA
followed by Bonferroni posttests was used to determine the statistical
significance of median tumor volume between groups. Statistical
analyses were conducted at a p level of 0.05. Prism (GraphPad),
version 3, was used for all statistical analyses and graphic presentations.
6.2. For Ramos study. The Mann−Whitney U test was used to
determine the statistical significance of median tumor volume between
a treatment group and the vehicle control group. Statistical analyses
were conducted at a p level of 0.05. Prism (GraphPad), version 3, was
used for all statistical analyses and graphic presentations.
Computational. The molecules were built using Maestro, version
8.0.308, or converted to 3D structures from the 2D structure using
LigPrep, version 2.1.207.35 Basic amines were protonated as in
aqueous solution at physiological pH. The conformational space was
searched using the Monte Carlo multiple minima (MCMM) method
as implemented in MacroModel, version 9.5.207.35 All heavy atoms
and hydrogens on heteroatoms were included in the test for duplicate
conformations. All rotatable single bonds were included in the
conformational search. All aliphatic rings were ring-opened, and
quaternary atoms were allowed to invert. Each search was continued
until the global energy minima were found at least 3 times. The energy
minimizations were carried out using the truncated Newton conjugate
gradient algorithm (TNCG) and the OPLS-AA force field as
implemented in MacroModel.37 Default parameters were used. The
conformational searches were done for aqueous solution using the
generalized Born/solvent accessible surface (GB/SA) continuum
solvation model.38
3
2
tumor volume(mm ) = (w l)/2
where w is the width and l is the length in mm of an HCT-116
carcinoma.
2.2. For Ramos Study. Mice were implanted subcutaneously in the
right flank with 7 × 106 cells of Ramos (ATCC No. CRL-9591) cells
(100 μL). The tumor size was monitored twice per week and
subsequently daily as the neoplasms reached the desired size,
approximately 200 mm3. On day 12, when the tumors attained a
volume of between 75 and 405 mm3, the animal were pair-matched
and distributed randomly into various treatment groups (the mean
tumor volume in each group was 216 mm3). Estimated tumor volume
was calculated using the formula
The CDK2 (PDB entry 1AQ1),39 and Flt3 (PDB entry 1RJB)40 X-
ray structures were downloaded from the Protein Data Bank (PDB41).
The protein structures were prepared using the protein preparation
wizard in Maestro with standard settings. Grids were generated using
Glide, version 4.5.208, following the standard procedure recom-
mended by Schrodinger.35 The conformational ensembles were
̈
docked flexibly using Glide with standard settings in both standard
and extra precision mode. Only poses with low energy conformations
and good hydrogen bond geometries were considered. cLogP values
were calculated using QikProp, version 3.0.207, with standard
settings.35
3
2
tumor volume(mm ) = (w l)/2
where w is the width and l is the length in mm of a Ramos tumor.
3. Drug. 26h hydrochloride was synthesized at S*BIO PTE LTD
and dissolved in 0.5% methyl cellulose/0.1% Tween 80 (MC/Tween)
for oral (po) dosing or in 10% dimethylacetamide (DMA) and 10%
Cremophor (DMA/CRE) for ip dosing. Dosing solutions were
prepared weekly in a feeding volume of 10 mL per kilogram body
weight and stored at 4 °C.
4. Treatment Plan. 4.1. For HCT-116 Study. On day 1, HCT-
116-bearing nude mice were pair-matched and placed into 3 groups of
9−10 animals each. Treatment with all drugs was initiated on day 1.
The test compound, 26h, was administered po at the following dosing
schedules: 50 and 75 mg/kg × 3 times a week (Monday, Wednesday,
and Friday; 3/w). There was a vehicle control group that received
vehicle (MC/Tween) on the same schedule. The study was terminated
on day 15.
ASSOCIATED CONTENT
* Supporting Information
■
S
Explanation for the potency of 26r against CDK2, structures of
the RCM catalysts employed and the iv/po PK of 27j in mice.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +65 62195443. E-mail: wanthony11@yahoo.com.
■
4.2. For Ramos Study. 26h was administered once daily at doses of
75 mg/kg po q.d. 2d_on−5d_off or 15 mg/kg ip q.d. 5d_on−5d_off.
There were two vehicle control groups that received either MC/
Tween or DMA/CRE. The treatment groups were compared with the
corresponding vehicle control group for the percentage of the tumor
growth inhibition (% TGI). The treatment was terminated after 14
days of dosing.
5. End Points.Tumor Growth Inhibition. Activity was assessed by
the tumor growth inhibition (TGI) method in which treatment-
effected decreases in tumor volume on day 15 of various treatment
groups were compared to the vehicle group. The percent tumor
growth inhibition (% TGI) was calculated as follows:
ACKNOWLEDGMENTS
■
We thank Bee Kheng Ng and Wai Chung Ong for the
biological screening data, Venkatesh Reddy for PK sample
analysis, Sam Ramanujulu Murugappan for NMR related work,
Zahid Bonday and Miah Kiat for protein preparation work,
Veronica Diermayr, Stefan Hart, Tracy Lawhon, and Rob
Mansfield for critical review of the manuscript, and Simon
Campbell for helpful discussions.
ABBREVIATIONS USED
■
AML, acute myeloid leukemia; CDK, cyclin-dependent kinase;
CYP, cytochrome P450; ERK5, extracellular signal-regulated
kinase-5; ET, essential thrombocythemia; FLT3, fms-like
receptor tyrosine kinase 3; JAK2, Janus kinase 2; MF,
myelofibrosis; MPN, myeloproliferative neoplasms; PV, poly-
cythemia vera; RCM, ring closing metathesis; RTKs, receptor
% TGI = (Cday_a − Tday_a)/(Cday_a − Cday_1) × 100
where Cday_1 is the median tumor volume for control group
(vehicle) on day 1, Cday_a is the median tumor volume for control
group (vehicle) on day a, and Tday_a is median tumor volume for
treatment group on day a.
194
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