Design and synthesis of novel small-molecule inhibitors of the hypoxia inducible factor pathway

Article abstract of DOI:10.1021/jm201018g

Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility, and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels but interfere with the ability of HIF-1α/HIF-1β to interact with cofactors p300/CBP to form an active transcriptional complex. (Figure presented)

Full text of DOI:10.1021/jm201018g

Article
pubs.acs.org/jmc
Design and Synthesis of Novel Small-Molecule Inhibitors of the
Hypoxia Inducible Factor Pathway
Suazette Reid Mooring,† Hui Jin,† Narra S. Devi,‡ Adnan A. Jabbar,§ Stefan Kaluz,∥,‡ Yuan Liu,∥
Erwin G. Van Meir,*,‡,§,∥ and Binghe Wang*,†
†Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30302-4098,
United States
§
‡Department of Neurosurgery and Department of Hematology and Medical Oncology, Emory University School of Medicine,
Atlanta, Georgia 30322, United States
∥Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, United States
ABSTRACT: Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors.
Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance
to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of
transcription factors, which induce target genes that regulate adaptive biological processes such as
anaerobic metabolism, cell motility, and angiogenesis. Clinical evidence has demonstrated that
expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1
contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an
important therapeutic target for inhibition by small molecules. Herein, we describe the design and
synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds
exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these
inhibitors do not alter HIF-1 levels but interfere with the ability of HIF-1α/HIF-1β to interact with
cofactors p300/CBP to form an active transcriptional complex.
elements (HRE) on the gene regulatory DNA sequences.13,14
The genes targeted for activation include those that encode
enzymes that carry out anaerobic glycolysis, erythropoietin, a
hormone that triggers red blood cell production, and vascular
INTRODUCTION
■
Hypoxia is a hallmark of solid tumors, largely due to inadequate
vascularization, and is characterized by a reduction in the partial
oxygen pressure in cells or tissue.1−3 Tumor hypoxia has been
endothelial cell growth factor (VEGF), a powerful activator of
shown to reduce the effectiveness of radiation and chemo-
new capillary formation and major driver of tumor angio-
therapy.4,5 The hypoxia inducible factor (HIF) family consists
genesis.15,16 Elevated levels of HIF-1α have been found in many
of the primary transcription factors activated by hypoxia and are
responsible for orchestrating a number of cellular responses
human cancers,17−19 and this is associated with poor response
to treatment and patient mortality.17,20−23 As a result, the HIF
such as angiogenesis and glycolysis that help tumor cells adapt
to hypoxic conditions.6 HIFs are basic helix−loop−helix
pathway has been exploited for the development of new cancer
therapies,24−27 including the development of small molecule
heterodimers composed of an oxygen sensitive HIF-α subunit
inhibitors targeting HIF-1.28−31
and a constitutively expressed HIF-1β.7 The levels of HIF-1α
To identify novel compounds for HIF-1 pathway inhibition,
are determined by intracellular oxygen concentration. Under
normoxic conditions, HIF-α is continually degraded by
ubiquitination and proteosomal degradation. Degradation
occurs when HIF-1α is hydroxylated at Pro 564 and Pro 402
located at its oxygen-dependent degradation domain (ODDD).
This process is facilitated by a family of prolyl hydroxylases
some 10 000 compounds from a 2,2-dimethylbenzopyran
combinatorial library were screened.32 The benzopyran moiety
was chosen because it appears in more than 4000 natural
products and is considered to be lipophilic enough to cross the
blood−brain barrier.33 The library was screened using a human
glioma cell line containing an HRE-alkaline phosphatase reporter
(PHDs) that require oxygen, iron, and 2-oxoglutarate as the
gene.33,34 1 (KCN-1) was identified as a potent inhibitor.35
Further tests in animal models for cancer have shown its ability
to inhibit cancer growth.36,37Separate mechanistic studies
indicate that 1 does not alter HIF-1 levels but interferes with
the ability of the HIF-1α/HIF-1β complex to associate with
transcriptional cofactors p300/CBP (CREB-binding protein).36
p300/CBP cofactors are transcription coactivating proteins. They
cosubstrate to hydroxylate the specific amino acid residues.8−10
After hydroxylation, HIF-1α binds to the von Hippel−Lindau
protein (pVHL) which is part of an E3-ubiquitin ligase complex
that marks HIF-1 for proteasomal degradation through
ubiquitination.11,12 Oxygen is required for the function of
PHDs; therefore, under hypoxic conditions, HIF-1α is
stabilized, accumulates, and translocates to the nucleus where
it interacts with HIF-1β to form the active transcription factor
HIF-1.11,12 HIF-1 then specifically activates the transcription of
over 100 selected genes by binding to hypoxia-responsive
Received: July 30, 2011
Published: October 27, 2011
© 2011 American Chemical Society
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interact with various transcription factors such as HIF-1 and
increase the expression of their target genes. Such a mechanism
is different from HIF-1 inhibitors and may lead to significant
insight into novel approaches to control solid tumor. With the
desire to improve potency, our goal for this study was to build an
SAR profile around the lead compound 1 in search of more
potent and soluble small-molecule inhibitors of HIF-1-mediated
transcription. This will help our effort in addressing the poor
solubility of 1 and its need for cremophor/ethanol in
formulation36 which is associated with toxicity.38
replaced by an amide group (class 7). Regions II and III were
modified with several alkyl and aryl substitutions, using known
synthetic procedures (class 2). The aldehyde derivative of the
core structure underwent reductive amination with a variety of
alkyl and aryl primary amines to provide modifications
to region II. Then sulfonylation of the resulting secondary
amines with various sulfonyl chlorides allowed modifications to
region III. The benzopyran ring of region IV was probed to
determine the influence of subtle and major modifications
of this region on activity. The first modification was the
replacement of the gem-dimethyl group of region IV with the
2-ethyl-2-methyl group (class 3). The benzopyran ring of
region IV was also replaced with a quinoline ring (class 4) and
benzofuran ring (class 5). Finally, the benzopyran ring of region
IV was replaced with a pyranopyridine fused ring to give classes
6a, 6b, and 6c.
RESULTS AND DISCUSSION
■
Design. The modification of compound 1 was approached
in a systematic manner in which the molecule was divided into
four regions as shown in Figure 1. In all, seven classes of
compounds were designed and synthesized (Figure 2). The
sulfonamide group of region I was either eliminated (class 1) or
Chemistry. Class 1: Benzopyran Analogues A. For the
synthesis of the class 1 analogues, the benzopyran moiety was
retained while the sulfonyl and 3,4-dimethoxyphenyl groups
were eliminated. To afford these analogues, the aldehyde
derivative of the benzopyran moiety was synthesized followed
by reductive amination and methylation of the resulting
secondary amine (in some cases).
The synthesis of class 1 analogues began with 2,2-dimethyl-
2H-chromene-6-carbaldehyde that was synthesized according
to literature procedures.39 Reductive amination of 2,2-dimethyl-
2H-chromene-6-carbaldehyde with several primary amines gave
analogues 2. Methylation of secondary amine 2 with MeI and
NaH generated analogues 3 (Scheme 1).
Figure 1. Four regions for modification of 1. IC50 = 0.65 ± 0.09 μM
(n = 42).
Figure 2. Analogues designed and synthesized.
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a
Scheme 1. Synthesis of Benzopyran Analogues A
a
R1 = 3,4-dimethoxyphenyl (2a, 3a), 2-pyridinyl (2b, 3b), 2,4-dimethylphenyl (2c, 3c), 4-carboxyphenyl (2d), 2-bromophenyl (2e), 2-fluorophenyl
(2f). Reagents and conditions: (a) R1NH2, ZnCl2, NaCNBH3, room temp, 11−70%; (b) MeI, NaH, THF, room temp, 50−81%.
a
Scheme 2. Synthesis of Benzopyran Analogues B
a
R2 = 3,4-dimethoxyphenyl and R1 = phenyl (1), isopropyl (5a), propargyl (5b), butyl (5c), t-butyl (5d), allyl (5e), isobutyl (5f), cyclopentyl (5g),
cyclopropyl (5h), cyclohexyl (5i). R1 = phenyl and R2 = 4-methoxyphenyl (5j), 3,5-dimethylphenyl (5k), 2,5-dichlorophenyl (5l), 2-trifluoromethoxy-
4-bromophenyl (5m). Reagents and conditions: (a) R1NH2, ZnCl2, NaCNBH3, room temp, 60−70%; (b) R2SO2Cl, Et3N, DCM, room temp,
30−95%.
a
Scheme 3. Synthesis of 2-Ethyl-2-Methylbenzopyran Analogue
a
Reagents and conditions: (a) 3-methyl-pent-1-yn-3-ol DBU, TFAA, CuCl, CH3CN, 0 °C to room temp, 30%; (b) xylene, microwave (220 W, 200
Torr, 120 °C, 100 min); (c) aniline, ZnCl2, NaCNBH3, room temp, overnight, 41%; (d) 3,4-dimethoxybenzylsulfonyl chloride, Et3N, DCM, room
temp, 24 h, 32%.
Class 2: Benzopyran Analogues B. Next, modifications
were separately made to region II (5a−i) and then to region III
(5j−m) of compound 1 with various alkyl and aryl substituents
in order to probe their effect on activity. Reductive amination
of 2,2-dimethyl-2H-chromene-6-carbaldehyde with various aryl-
or alkylamines afforded compound 4 that was subsequently
converted to sulfonamides with various sulfonyl chlorides to
give analogues 5 (Scheme 2).
Class 3: 2-Ethyl-2-methylbenzopyran Analogues. Next,
class 3 analogues were generated that involved a slight modi-
fication to the benzopyran portion (region IV) of 1. The ethyl
group replaced one of the gem-dimethyl groups on the benzopyran
ring (Scheme 3). For the synthesis of these analogues, O-alkylation
of 4-hydroxybenzophenone 6 with 3-methylpentyn-3-ol afforded
compound 7. Claisen rearrangement and rearomatization of 7 by
microwave irradiation yielded compound 8. Reductive amination of
aldehyde 8 gave the secondary amine 9 that was converted to the
corresponding sulfonamide 10 with 3, 4-dimethoxybenzenesulfonyl
chloride.
Class 4: Quinoline Analogues. The next modification to
region IV was the replacement of the benzopyran ring with
another fused ring system: quinoline (Scheme 4). Commer-
cially available quinoline aldehyde 11 was subjected to
reductive amination, followed by sulfonylation to afford
compound 13 in 45% yield.
Class 5: Benzofuran Analogues. Additionally, the 2,2-
dimethylbenzopyran ring (region IV) of 1 was replaced with
a 2,2-dimethylbenzofuran ring (Scheme 5). Commercially
available 2,2-dimethyl-2,3-dihydrobenzofuran-5-carbaldehyde
14 was subjected to reductive amination with various primary
amines to give compound 15 and then sulfonylation with 3,4-
dimethoxybenzenesulfonyl chloride to give analogues 16.
Class 6: Pyranopyridines. We also replaced one of the
carbons on the aromatic portion of the benzopyran ring with
nitrogen to afford pyranopyridine analogues. The pyridine
nitrogen was separately placed in each of the three available
positions on the benzopyran ring. It was envisioned that these
compounds would provide increased water solubility and
additional interaction points and therefore increased activity.
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a
Scheme 4. Synthesis of Quinoline Analogue
a
Reagents and conditions: (a) aniline, ZnCl2, NaCNBH3, room temp, 64%; (b) 3,4-dimethoxybenzylsulfonyl chloride, pyridine, room temp, 45%.
a
Scheme 5. Synthesis of Benzofuran Analogues
a
R1 = phenyl (16a), cycloheptyl (16b), isopropyl (16c), butyl (16d), cyclohexyl (16e), cyclopentyl (16f). Reagents and conditions: (a) R1NH2,
ZnCl2, NaCNBH3, room temp, 2 h; (b) 3,4-dimethoxybenzenesulfonyl chloride, Et3N, DCM, room temp, 14−42%.
a
Scheme 6. Synthesis of Pyrano[2,3-b]pyridine Analogues
a
R1 = phenyl (20a), cyclohexyl (20b). Reagents and conditions: (a) (i) BuLi, −78 °C; (ii) DMF, anhydrous ether, 31%; (b) R1NH2, ZnCl2,
NaCNBH3, MeOH, 49%; (c) 3,4-diethoxybenzenesulfonyl chloride, Et3N, DCM, room temp, 43−60%.
The incorporation of a nitrogen atom may also decrease the electron
density and make it more stable toward oxidative metabolism.
The first set of these compounds was the pyrano[2,3-
b]pyridines 20. The 2H-pyrano[2,3-b]pyridine core 17 was
synthesized as previously described.40 Formylation of 17 with
BuLi and DMF gave compound 18. Reductive amination with
aniline (19a) or cyclohexylamine (19b) followed by
sulfonylation with 3,4-dimethoxybenzesulfonyl chloride af-
forded compounds 20a and 20b (Scheme 6).
The second set of analogues in this class was the pyrano-
[3,2-b]pyridines that were prepared using the following
procedure: O-alkylation of commercially available 2-bromo-5-
hydroxypyridine 22 followed by Claisen rearrangement and
formylation gave compound 24 with a 23% overall yield for the
two steps. Subsequent reductive amination of 24 and then
reaction of secondary amine 25 with various sulfonyl chlorides
afforded analogues 26 (Scheme 7).
The final pyranopyridine derivative was the pyrano[2,3-c]-
pyridines (class 6c). To synthesize these analogues, 2-hydroxy-
5-methylpyridine 27 was brominated to afford compound
28.41N-Oxidation of 28 with m-CPBA gave product 29 in 70%
yield. Rearrangement of 29, facilitated by TFAA, afforded
compound 30. O-Alkylation of 30 with 3-chloro-3-methyl-1-
butene followed by Claisen rearrangement gave compound 32.
Nucelophilic substitution of the primary alcohol 32 with
bromine gave compound 33. Subsequent nucleophilic sub-
stitution of 33 with various primary amines followed by
removal of the bromine with BuLi afforded compound 35. Next
sulfonylation of 35 with arylsulfonyl chlorides resulted in
analogues 36 (Scheme 8).
Class 7: Amide Analogue. Finally, we replaced the
sulfonamide of compound 26a with an amide group. The
amide group is a common bioisostere for sulfonamide and may
enhance activity. In this case, the previously synthesized 25a was
reacted with 3,4-dimethoxybenzoyl chloride in the presence of
triethylamine to give the product 37 with a 98% yield (Scheme 9).
Biology. The synthesized analogues of 1 were evaluated for
their potential to inhibit HIF-1-mediated transcription under
hypoxia (1% O2) using a human glioma cell line LN229-HRE-
Lux,35 which stably expresses a hypoxia-responsive luciferase
reporter gene (Tables 1−9). The IC50 values of all compounds
were calculated based on a concentration curve testing of
compounds at 0, 1, 5, 10, and 25 μM. The compounds were
tested in single (n = 1) or multiple (n > 1) independent
experiments each carried out in quadruplicate. Compound 1 was
always tested along with the new analogues and has an IC50 of
0.65 ± 0.09 μM (n = 42)35 using this cell-based reporter assay
(Figure 1).
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a
Scheme 7. Synthesis of Pyrano[3,2-b]pyridine Analogues
a
R2 = 3,4-dimethoxyphenyl and R1 = phenyl (26a), butyl (26b), 3,4-dimethoxyphenyl (26c), cyclopentyl (26d), cyclohexyl (26e), tetrahydranapthyl
(26f), cycloheptyl (26g), cyclooctyl (26h), cyclobutyl (26i), p-fluorophenyl (26j). R1 = phenyl and R2 = cyclohexyl (26k), isopropyl (26l),
cyclopropyl (26m), butyl (26n), propyl (26o), isobutyl (26p), 4-biphenyl (26q), benzodioxolyl (26r), quinolin (26s), 2,3-dihydrobenzo(1,4)-
dioxinyl (26t). Reagents and conditions: (a) 2-methylbut-3-yn-2-ol, TFAA, DBU, CH3CN; (b) xylene, microwave heating, 120 °C, 30 min,
23% for two steps; (c) (1) BuLi, (2) DMF, anhydrous THF, 23%; (d) R1NH2, ZnCl2, NaCNBH3, MeOH; (d) R2SO2Cl, Et3N, DCM, 40−65% for
two steps.
a
Scheme 8. Synthesis of Pyrano[2,3-c]pyridine Analogues
a
R1 = phenyl and R2 = 4-methoxyphenyl (36a), 4-nitrophenyl (36b). R1 = cyclohexyl and R2 =4-isopropylphenyl (36c), 3,4-dimethoxyphenyl (36d).
Reagents and conditions: (a) Br2, pyridine, 0 °C, 74%; (b) m-CPBA, THF, 70%; (c) (1) TFAA, (2) MeOH, 30%; (d) 3-chloro-3-methyl-1-butene,
K2CO3, KI, CuCl2, acetone, 57%; (e) CuCl, toluene, microwave heating (200 W, 120 °C, 1 h), 70%; (f) CBr4, PPh3, DCM, 40%; (g) DIEA, DMF,
60 −78%; (h) BuLi, THF, −78 °C, 50−70%; (i) R2SO2Cl, pyridine, room temp, 70−89%.
Scheme 9. Synthesis of Compound 37
compounds 2a−f, only 2a and 2b had IC50 values below 10 μM;
the others were higher than 25 μM. The best compound in
that series was the 3,4-dimethoxyphenyl derivative 2a with an
IC50 of 3.0 μM. Analogues 3a−c showed similar IC50 values as
their secondary amine counterparts 2a−c, with the exception of
the 2,4-dimethoxyphenyl derivative 3c that had an IC50 of
2.6 μM. Therefore, methylation of the secondary amine had no
effect. As a result, it was concluded that the sulfonyl group was
essential to the activity of these compounds and was retained in
future modifications of compound 1.
Next, region II of the molecule was probed with various alkyl
and aryl substituents (5a−k). All the compounds were active to
some extent (Table 2). The best of this group was the propargyl
derivative 5b, isobutyl derivative 5f, and the cyclopropyl derivative
5h with IC50 values of 1.3, 1.6, and 1.5 μM, respectively.
Class I (benzopyran A) analogues were designed to probe
the importance of the sulfonyl group. In general, removal of the
sulfonyl group in compounds 2a−f and 3a−c resulted in a
marked decrease in activity (Table 1). For secondary amine
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Table 1. Structures and Activities of Analogues 2a to 3c
Table 2. Structures and Activities of Analogues 5a−i
In general, longer branched alkyl chains such as the isobutyl
group of 5f (1.6 μM) tended to do better than long unbranched
chains such as the butyl group of 5c (3.3 μM) or shorter
branched chains such as the tert-butyl group of 5d (3.5 μM).
Also, alkyl rings smaller than six carbons were better tolerated.
Compounds 5j−m were modified at region III of 1 with
various aryl substitutions (Table 3). The best compound in this
group was the 4-methoxyphenyl substituted 5j and 3,5-
dimethylphenyl substituted 5k with IC50 values of 0.6 and
0.5 μM, respectively. The 2-trifluoromethoxy-4-bromophenyl
substitution (5m) resulted in a significant decrease in activity
Compound 10 represented a subtle change to region IV of 1.
In this case, simply substituting one of the gem-dimethyls of the
benzopyran ring system of 1 with an ethyl group resulted in a
decrease in activity with an IC50 of 2.2 μM (Table 4). In the
case of compound 13, replacement of the benzopyran ring of 1
with a quinoline ring led to a reduction in HIF-1 inhibitory
activity with an IC50 of 3.5 μM (Table 4).
The benzofuran derivatives 16 afforded some potent
compounds (Table 5). A comparison of compound 16a
(IC50 = 0.5 μM) to 1 shows that the substitution of the
benzopyran ring with benzofuran did not necessarily result in a
much more potent compound than 1, but the benzofuran
analogue was comparable to that of 1. The foreseeable benefit
of the benzofuran structure of 16 is that it eliminates the double
bond on the pyran ring of 1. Since that double bond may be
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a
The next group of compounds in this class was the
pyrano[3,2-b]pyridine (class 6b). Compounds 26a−j were
modified at region I with various alkyl- and arylamines. This
class of compounds was among the best in the pyranopyridine
class (Table 7). All of these compounds inhibited HIF-
mediated transcription with the exception of the 2,4-dimethoxy
derivative 26c. Phenyl derivative 26a is similar in structure to 1
with the exception of the pyrano[3,2-b]pyridine core. This
compound had an IC50 of 1.3 μM, which is within the range
of activity observed for 1. Replacement of the phenyl group
of 26a with a cyclopentyl group (26d) and cyclohexyl group
(26e) was effective. The best compound in this group
(26a−t) was the cyclobutyl derivative 26i with an IC50 of
0.25 μM. It is noted that 26i was tested many times in
independent experiments (n = 30) side by side with 1 and
consistently showed ∼2.5-fold higher potency than 1. When
all the cylcoalkyl analogues were compared, the general trend
remained about the same as that of other series, in that
smaller rings (<6 carbons) tend to have better activity than
larger ring derivatives (>6 carbons). The tetrahydronaph-
thalene derivatives 26f had the lowest activity in this series
with IC50 of 6.15.
Table 3. Structures and Activities of Analogues 5j−m
a
Additionally, these pyrano[3,2-b]pyridines 26k−t were also
modified at region III with alkyl- and arylsulfonyl derivatives.
Generally, this group did not produce very active compounds
(Table 8). The cyclohexyl group was well tolerated in this
position, leading to derivative 26k, with an IC50 of 0.4 μM. In
this case, the cyclopropyl derivative (26m) showed almost no
activity within the experimental conditions. Compounds 26r
and 26t were an attempt to fix the conformation of the 2,4-
dimethoxybenzyl group to see if this modification would
enhance activity. Compound 26r that separated the oxygen
atoms by one carbon showed a decrease in activity (IC50 = 6.5
μM) compared to the 3,4-dimethoxy substituted compound
26a (1.3 μM). However, 26t, in which two carbons separate the
oxygen atoms, showed an IC50 of 0.9 μM. Also noted is the
quinoline derivative 26s that showed relatively good activity
with an IC50 of 0.9 μM.
Results were from single runs.
a
Table 4. Structures and Activities of Analogues 10 and 13
The third group of compounds in class 6 was the pyrano[2,3-c]-
pyridinyl derivatives (class 6c). These compounds also showed
good activities, which were comparable to 1 (Table 9). Derivatives
36a, 36b and 36d all showed similar IC50 values of 1.4, 1.8 and
1.1 μM, respectively. The isopropylphenyl derivative 36c showed a
5-fold decrease in activity when compared to the 3,4-dimethoxy
derivative 36d.
a
Results were from single runs.
Since the pyranopyridine analogues were among the most
potent compounds, we decided to replace the sulfonamide group
with an amide group to see what effect this modification would
have on activity. An amide is a commonly used bioisostere for
sulfonamide. The amide derivative showed a 2-fold increase in
activity over the sulfonamide derivative (Figure 3). This amide
group can be incorporated in future modifications of the
compound.
Following chemical optimization, the best compounds were
subsequently retested in the luciferase reporter assay in a dose−
response fashion to establish an IC50 of inhibitory activity on
HIF transcription (Figure 4 and data not shown). Four-
parameter logistic function was used to describe the dose−
response relationship, and the data were fitted into the
nonlinear mixed effects42model by nlme library in R.43 Each
compound was tested a minimum of 6 times on different days.
To address variations from experiment to experiment, we used
the nonlinear mixed effects model in which IC50 was set to have
susceptible to epoxidation in vivo and thereby introduce
toxicity, the benzofuran ring may be a better alternative. The
ring size of the cycloalkyl derivatives seems to have an effect on
activity. A comparison of the cycloheptyl ring of 16b (9.1 μM),
the cyclohexyl ring of 16e (8.2 μM), and the cyclopentyl ring of
16f (0.4 μM) seems to suggest that smaller rings (ring size 5 or
smaller) tend to be more favorable than large rings (six carbons
or more). This is similar to the trend seen with the benzopyran
analogues B (class 2).
The first of the pyranopyridine analogues was class 6a, the
pyrano[2,3-b]pyridines. Two compounds were synthesized in
this class. Compound 20a had the same substitutions as
compound 1 with the exception of the pyranopyridine core.
This compound showed modest activity with an IC50 of
2.5 μM. However, it was not as potent as 1. Replacing the
phenyl ring by the cyclohexyl ring (20b) resulted in a loss of
activity; that is, the IC50 was higher than 25 μM (Table 6).
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a
Table 5. Structures and Activities of Analogues 16a−f
a
Results were from single runs.
accumulation under hypoxia.33 It was found that some of the
compounds 5g and 16a reduced the level of expression of HIF-
1α at 20 μM, whereas the remaining compounds did not
(Figure 7). These data suggest that inhibition of HIF-1α
expression is not a general cause of the strong inhibition seen
against HIF-mediated transcription in the reporter assay. Such
results suggest that at least for some of the compounds, 16d,
16f, and 26i, the main biological activity is not mediated by
inhibiting HIF-1α gene expression or affecting HIF-1α turnover
through a blockage in translation of HIF-1α mRNA, or
accelerated protein degradation. Instead, these findings
imply that these inhibitors render the HIF transcriptional
complex functionally inactive. Potential mechanisms may
involve protein misfolding, incomplete protein modifica-
tions, and/or lack of HIF complex assembly. To dissect the
precise mechanism of action of this class of HIF pathway
inhibitors, additional work is needed. Ongoing mechanistic
studies indicate that 1 does not alter HIF-1 levels but
interferes with the ability of the HIF-1α/HIF-1β complex to
associate with transcriptional cofactors p300/CBP,36 and we
anticipate that this will be similar for the new analogues
identified here.
Table 6. Structures and Activities of Analogues 20a and 20b
an associated random effect. The results indicate that five
compounds displayed IC50 values in the submicromolar range
(Table 10), with 16a and 26i being the most promising, as they
both showed more than 2-fold improvement over 1.
We further confirmed inhibitory activity of these compounds
on the HIF pathway by conducting additional experiments with
the hypoxia-responsive promoter of the endogenous HIF
transcriptional target gene vascular endothelial growth factor
(VEGF). Using LN229 glioma cells stably transfected with a
VEGF promoter-luciferase reporter (LN229-VEGF-Luc), we
found that the tested compounds at 10 μM all significantly
inhibited hypoxia-induced transcription from the VEGF
promoter (Figure 5).
For further mechanistic studies, we picked the representative
compounds and previously characterized HIF pathway
inhibitors (1, 38 (Figure 6)33 and bortezomib) as controls to
evaluate their molecular basis of action using biochemical
techniques. As HIF regulation typically occurs at the protein
level, we probed by Western blotting whether the selected
compounds had a direct effect on HIF-1α protein accumulation
under hypoxia. HIF-1α levels were examined in cell extracts
from cells grown under hypoxia in the presence or absence of
inhibitor (20 μM). As expected, the results show that
bortezomib, a proteasome inhibitor, leads to the accumulation
of HIF-1α in an inactive form,44 whereas 38, a HIF-1α
translation inhibitor, leads to a blockage of HIF-1α
CONCLUSION
■
Several potent analogues of the lead compound 1 were
synthesized. These analogues were able to yield information on
the important functional groups at each of the four regions
identified in Figure 1. General conclusions about the SAR of
this molecule were determined (Figure 8). Analogues 2a−2f
demonstrated that the sulfonyl group was required for the
activity of these compounds. Also, alkyl rings of five carbons or
shorter, as well as longer branched chains, were well tolerated at
region II of 1. At region III, aryl substitutions seem to be better
than alkyl substitutions. The benzofuran analogues (16) were
also successful in that analogues in this series showed activity
comparable to that of 1. These benzofuran analogues may
be a good future alternative to the benzopyran derivatives, since
they do not contain electron-rich double bonds. To date, the
pyrano[3,2-b]pyridine analogues (26) provided the most
improvement in activity compared to 1. The best overall
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a
Table 7. Structures and Activities of Analogues 26a−j
a
Results were from single runs.
a
Table 8. Structure and Activities of Analogues 26k−t
a
Results were from single runs.
compound came from this group, the cyclobutyl derivative 26i,
which had an IC50 of 280 nM. The improvement in activity
of these analogues over 1 may be a result of increased
hydrophilicity and/or hydrogen bond interactions as a result
of the addition of the pyridine ring. Overall, these small
molecules show potential as effective HIF-1 inhibitors for
antitumor therapy. These compounds can be especially useful
in tumors that exhibit hypoxic resistance to chemotherapy and
radiotherapy.
EXPERIMENTAL SECTION
■
Biology. LN229-HRE-luciferase glioblastoma cells were used to
perform the assay. These cells contain a stably integrated reporter
construct (pBI-GL HRE V6R plasmid) made of six copies of the HIF
responsive element derived from the VEGF gene as previously
described.26The 48-well plates were seeded with 3 × 104 cells per well
and incubated under normoxic conditions for 24 h. Cells were then
pretreated with different concentrations of 1 or its analogues for 1 h
and then transferred to hypoxic conditions. After 24 h, the medium
was aspirated, cells were lysed, and reporter activity was measured in
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a
Table 9. Structures and Activities of Analogues 36
a
Results were from single runs.
Figure 3. Comparison of structures and activities of 26a and 37.
the lysate using the luciferase assay system (Promega, Madison, WI)
with a 20/20n luminometer (Promega).
Chemistry. General. All commercial chemicals and solvents were
reagent grade and were used without further purification unless
otherwise indicated. Microwave heating was performed in a single-
mode microwave cavity of a Discover Synthesis System (CEM Corp.),
and all microwave-irradiated reactions were conducted in heavy walled
1
glass vials sealed with Teflon septa. H NMR and 13C NMR were
recorded at 400 and 100 MHz, respectively, on a Bruker 400 NMR
spectrometer with TMS or deuterated solvent as the internal standard.
Coupling constants are in Hz. Mass spectral analysis was performed by
the Mass Spectrometry Facilities at Georgia State University. The
purities of tested compounds were assessed as being at least 95% with
analytical HPLC, which was performed using a C18 5 μm (250 mm ×
4.6 mm) column at 254 nm and with elution with a gradient of 70−
80% solvent B (methanol) in solvent A (water) at 0.8 mL/min.
General Procedure for Reductive Amination for Synthesis of
2a−f. To a solution of 2,2-dimethyl-2H-chromene-6-carbaldehyde
(1 equiv) in methanol was added the amine (2 equiv), sodium
cyanoborohydride (2 equiv), and zinc chloride (anhydrous) (2 equiv).
The mixture was stirred overnight. Then the solvent was removed by
rotary evaporation and 1 M NaOH added to the residue. The organic
layer was extracted with ethyl acetate or DCM (×2), dried over
magnesium sulfate, and concentrated in vacuo. The crude product was
purified by flash column chromatography (silica gel).
Figure 4. Dose response of a selected set of compounds using the
HRE-luciferase reporter system. Cells were pretreated with various
concentrations of inhibitors (control 1% DMSO) for 1 h in normoxia,
followed by 24 h of incubation in hypoxia with inhibitors present.
Luciferase activity was measured in cell extracts using a 20/20n
luminometer (Turner Biosystems). Data, expressed as percent of
control, are averages from at least four independent experiments
carried out in triplicate.
(3,4-Dimethoxyphenyl)-(2,2-dimethyl-2H-chromen-6-
1
ylmethyl)amine (2a). Yield: 60%. H NMR (CDCl3): δ 7.09 (dd,
J = 8.2, 2.1 Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H),
6.30 (s, 1H), 6.30−6.24 (m, 1H), 6.17 (dd, J = 8.5, 2.6 Hz, 1H), 5.61
(d, J = 9.8 Hz, 1H), 4.15 (s, 2H), 3.81 (t, J = 6.2 Hz, 6H), 1.43 ppm
(s, 6H). 13C NMR (CDCl3): δ 152.2, 150.0, 143.3, 141.6, 131.6, 131.1,
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(d, J = 9.6 Hz, 1H), 4.80 (s, br, 1H), 4.40 (s, 2H), 1.41 ppm (s, 6H).
HRMS (ESI) m/z calcd for C17H18N2O [(M + H)+] 267.149; found,
267.1505. HPLC: tR = 12.8 min, 99.6%.
Table 10. Average IC50 (from n Independent Experiments in
μM) of Selected Arylsulfonamide HIF-1 Pathway Inhibitors
As Established in the HRE-Luciferase Assay
( 2 , 2 - D i m e t h y l - 2 H - c h r o m e n - 6 - y l m e t h y l ) - ( 2 , 4 -
dimethylphenyl)amine (2c). Yield: 69%. 1H NMR (CDCl3): δ
7.10 (dd, J = 6.0 Hz, 2.4 Hz, 1H), 6.99 (d, J = 8.0 Hz), 6.92−6.90
(m, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.54 (d, J = 7.6 Hz, 1H), 6.30 (d,
J = 9.6 Hz, 1H) 4.21 (s, 2H), 2.23 (s, 3H), 2.12 (s, 3H), 1.43 ppm (s,
6H). MS (ESI) m/z 292 [(M + H)+]. HPLC: tR = 20.84 min, 97.9%
4-[(2,2-Dimethyl-2H-chromen-6-ylmethyl)amino]benzoic
Acid (2d). White powder. Yield: 58%. Mp 148 °C. 1H NMR
((CD3)2SO): δ7.71 ppm (s, 2H), 7.08 (s, 1H), 7.03 (s, 1H), 6.68 (d,
J = 8.2 Hz, 1H), 6.54 (d, J = 7.7 Hz, 2H), 6.37 (d, J = 9.8 Hz, 1H),5.72
(d, J = 9.8 Hz, 1H), 4.17 (d, J = 5.5 Hz, 2H), 1.35 (s, 6H). HRMS
(ESI) m/z calcd for C19H19NO3 [(M − H)+] 308.1287; found,
308.1276.
a
compd
no. of repeats
IC50
std error
95% CI
1
42
6
0. 648
0.306
0.813
0.478
0.378
0.280
0.044
0.06
0.562, 0.734
0.187, 0.425
0.533, 1.093
0.270, 0.686
0.255, 0.501
0.237, 0.323
16a
5g
6
0.141
0.105
0.062
0.022
16d
16f
26i
6
6
30
a
CI: confidence interval.
(2-Bromophenyl)-(2,2-dimethyl-2H-chromen-6-ylmethyl)-
1
amine (2e). Yield: 11%. H NMR (CDCl3): δ 7.43 (d, J = 7.8 Hz,
1H), 7.20−7.04 (m, 2H), 6.97 (s, 1H), 6.75 (d, J = 8.2 Hz, 1H), 6.62
(d, J = 8.0 Hz, 1H), 6.58 (d, J = 7.1 Hz, 1H), 6.30 (d, J = 9.8 Hz, 1H),
5.61 (d, J = 9.8 Hz, 1H), 4.63 (s, 1H), 4.26 (d, J = 5.3 Hz, 2H), 1.43
ppm (s, 6H). 13C NMR (CDCl3): δ 152.3, 144.9, 132.3, 131.1, 130.7,
128.5 128.1, 125.4, 122.2, 121.4, 117.9, 116.5, 111.6, 109.6, 76.3, 47.6,
28.0 ppm. HRMS (ESI) m/z calcd for C18H18NOBr [(M + H)+]
344.0650; found, 344.0663. HPLC: tR = 19.71 min, 97.9%.
(2,2-Dimethyl-2H-chromen-6-ylmethyl)-(2-fluorophenyl)-
1
amine (2f). Yield: 71%. H NMR (CDCl3): δ 7.10 (dd, J = 6.0, 2.0
Hz, 1H), 6.99−6.94 (m, 3H), 6.76−6.56 (m, 3H), 6.29 (d, J = 9.6 Hz,
1H), 5.61 (d, J = 9.6 Hz, 1H), 4.23 (s, 3H), 1.43 ppm (s, 6H). 13C
NMR (CDCl3): δ 152.4, 143.9, 138.1, 136., 130.9, 128.3, 128.2, 127.5,
127.1, 124.7, 123.5, 122.3, 121.2, 116.2, 77.4, 28.1, 21.5 ppm. HRMS
(ESI) m/z calcd for C18H18NOF [(M + H)+] 284.1451; found,
284.1442. HPLC: tR = 16.55 min, 97.3%.
General Procedure for Synthesis of 3a, 3b, and 3c by
Methylation of Secondary Amines 2a, 2b, and 2c, Respec-
tively. A solution of secondary amine 2 (1 equiv) in THF was added
to a flask containing NaH (2 equiv) in THF. After 5 min, MeI
(2 equiv) was added and the mixture stirred overnight. The reaction
mixture was quenched with water and diluted with ethyl acetate. The
organic layer was washed with water and brine, dried over MgSO4, and
concentrated in vacuo. The crude product was purified by flash
column chromatography (silica gel).
Figure 5. Luciferase reporter assays showing the effect of the selected
set of compounds in LN229-VEGF-luc cells. Cells were pretreated
with inhibitors (10 μM final concentration) for 1 h in normoxia,
followed by 24 h of incubation in normoxia (N) or hypoxia (H) and
luciferase measured as indicated in Figure 4. Each value represents an
average from triplicates ± standard deviation. RLU = relative light
units; C = vehicle control.
(3,4-Dimethoxyphenyl)-(2,2-dimethyl-2H-chromen-6-
ylmethyl)methylamine (3a). Yield: 60%. 1H NMR (CDCl3): δ
6.98 (dt, J = 7.2, 3.6 Hz, 1H), 6.87 (s, 1H), 6.78 (d, J = 8.7 Hz, 1H),
6.73 (s, 1H), 6.43 (s, 1H), 6.27 (d, J = 10.1 Hz, 2H), 5.59 (d, J = 9.8
Hz, 1H), 4.31 (s, 2H), 3.82 (d, J = 2.2 Hz, 6H), 2.89 (s, 3H), 1.42
ppm (s, 6H). 13C NMR (CDCl3): δ 149.7, 145.6, 131.2, 130.9, 127.9,
125.1, 122.3, 121.3, 116.3, 113.0, 104.8, 99.5, 77.4, 77.0, 76.7, 76.2,
57.6, 56.7, 55.8, 38.9, 28.0 ppm. HRMS (ESI) m/z calcd for
C21H25NO3 [(M + H)+] 340.1913; found, 340.1900. HPLC: tR = 21.7,
98.7%.
Figure 6. Structure of 38 (103D5).33
(2,2-Dimethyl-2H-chromen-6-ylmethyl)methylpyridin-2-yl-
1
amine (3b). Yield: 81%. H NMR (CDCl3): δ 8.21 (m, 1H), 7.45
(m, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.86 (s, 1H), 6.72 (d, J = 8.0 Hz,
1H), 6.52−6.59 (m, 2H), 6.28 (d, J = 10 Hz, 1H), 5.60 (d, J = 10 Hz,
1H), 4.70 (s, 2H), 3.06 (s, 3H), 1.44 ppm (s, 6H). 13C NMR
(CDCl3): δ 151.9, 148.0, 137.3, 130.9, 130.8, 127.8, 125.0, 122.4,
121.3, 116.3, 11.7, 105.8, 76.1, 52.6, 36.0, 28.0 ppm. HRMS (ESI) m/z
calcd for C18H20N2O [M + H)+] 281.1654; found, 281.1659. HPLC:
tR = 16.55, 97.3%.
( 2 , 2 - D i m e t h y l - 2 H - c h r o m e n - 6 - y l m e t h y l ) - ( 2 , 4 -
dimethylphenyl)methylamine (3c). Yield: 48%. 1H NMR
(CDCl3): δ 7.13 (dd, J = 2.0, 6.0 Hz, 1H), 7.05−7.00 (m, 4H),
6.75 (d, J = 8.0 Hz, 1H), 6.34 (d, J = 9.6 Hz, 1H), 5.63 (d, J = 9.2 Hz,
1H), 3.88 (s, 2H), 2.55 (s, 3H), 2,40 (s, 3H), 2.31 (s, 3H), 1.45 ppm
(s, 6H). MS (ESI) m/z 308 [(M + H)+]. HPLC: tR = 12.17, 96.8%.
General Procedure for Synthesis of 5a−m by Alkyl
Sulfonylation. To a solution of 2,2-dimethyl-2H-chromene-6-
carbaldehyde (1 equiv) in methanol was added the primary amine
(1 equiv), ZnCl2 (2 equiv), and the mixture was stirred at room
Figure 7. Western blots showing the effect of the selected set of
compounds on hypoxic accumulation of HIF-1α in LN229 cells. Cells
were pretreated with indicated inhibitors at 20 μM final concentration
(Btz, bortezomib, 100 nM) for 1 h before incubation in normoxia or
hypoxia for 24 h. Immunoblotting of HIF-1α and actin was as
described earlier. C = vehicle control.33
128.4, 125.7, 122.2, 121.4, 116.4, 113.3, 103.6, 99.0, 76.2, 56.7, 55.7,
48.8, 28.1 ppm. HRMS (ESI) m/z calcd for C20H23NO3 [(M + H)+]
326.1756; found, 326.1750. HPLC: tR = 9.41 min, 99.5%.
(2,2-Dimethyl-2H-chromen-6-ylmethyl)methylpyridin-2-yl-
1
amine (2b). Yield: 60%. H NMR (CDCl3): δ 8.10−8.08 (m, 1H),
7.77−7.35 (m, 1H), 7.08−7.07 (m, 1H), 7.00−6.96 (m, 1H), 6.77−
6.72 (m, 1H), 6.50−6.58 (m, 1H), 6.36 (d, J = 8.4 Hz, 1H), 6.28
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Figure 8. Structure−activity relationship of 1.
temperature for 2 h. Then NaCNBH3 (2 equiv) was added, and the
mixture was stirred at room temperature overnight. The solvent was
removed by rotary evaporation, and EtOAc was added to the residue.
The solid was filtered through Celite and the filtrate washed with 1 M
NaOH, water, and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The crude secondary amine
product 4 was used without further purification.
N-tert-Butyl-N-(2,2-Dimethyl-2H-chromen-6-ylmethyl)-3,4-
dimethoxybenzenesulfonamide (5d). Yield: 49%. 1H NMR
(CDCl3): δ 7.41 (dd, J = 8.5, 2.1 Hz, 1H), 7.22−7.12 (m, 2H),
7.06 (d, J = 2.1 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.74 (t, J = 9.7 Hz,
1H), 6.30 (t, J = 8.9 Hz, 1H), 5.62 (t, J = 9.5 Hz, 1H), 4.56 (s, 2H),
3.94 (d, J = 13.4 Hz, 3H), 3.85 (d, J = 14.6 Hz, 3H), 1.48−1.39
(m, 6H), 1.33 ppm (s, 9H). MS (ESI) m/z 468 [(M + Na)+]. HPLC:
tR = 13.2 min, 96.3%.
To a solution of the secondary amine 4 (1 equiv) in DCM was
added triethylamine (3 equiv) and the sulfonyl chloride (1.5 equiv).
The mixture was stirred for 24−48 h. Then water was added and the
organic layer extracted with DCM, dried over MgSO4, concentrated
under reduced pressure, and purified by flash column chromatography.
N-(2,2-Dimethyl-2H-chromen-6-ylmethyl)-N-isopropyl-3,4-
dimethoxybenzenesulfonamide (5a). Yield: 58%. Mp 132
N-Allyl-N-(2,2-dimethyl-2H-chromen-6-ylmethyl)-3,4-dime-
1
thoxybenzenesulfonamide (5e). Yield: 53%. Mp 94−98 °C. H
NMR (CDCl3): δ 7.48 (dd, J = 8.4, 2.2 Hz, 1H), 7.29 (t, J = 2.3 Hz,
1H), 7.01−6.91 (m, 2H), 6.88 (d, J = 2.1 Hz, 1H), 6.72 (t, J = 9.7 Hz,
1H), 6.29 (d, J = 6.4 Hz, 1H), 5.64 (t, J = 10.6 Hz, 1H), 5.53 (ddt, J =
16.7, 10.2, 6.5 Hz, 1H), 5.09 (ddd, J = 18.4, 13.6, 1.3 Hz, 2H), 4.28 (d,
J = 25.4 Hz, 2H), 4.03−3.95 (m, 3H), 3.95−3.88 (m, 3H), 3.84−3.72
(m, 2H), 1.44 ppm (s, 6H). 13C NMR (CDCl3): δ 152.6, 152.4, 149.1,
132.4, 132.4, 131.9, 129.4, 127.9, 126.6, 122.1, 121.3, 121.1, 199.2,
116.2, 110.6, 109.8, 76.4, 56.2, 56.18, 49.6, 49.2, 28.0 ppm. EI probe:
M+ 429. HPLC: tR = 11.9 min, 97.0%
1
−134 °C. H NMR (CDCl3): δ 7.39 (dd, J = 6.4, 2.0 Hz, 1H), 7.22
(d, J = 2.0 Hz, 1H), 7.07 (dd, J = 6.0, 2.0 Hz, 1H), 7.00 (d, J = 2.0 Hz,
1H), 6.90 (t, J = 8.6 Hz, 1H), 6.69 (d, J = 8.2 Hz, 1H), 6.28 (d, J = 9.8
Hz, 1H), 5.60 (d, J = 9.8 Hz, 1H), 4.39−4.19 (m, 2H), 4.23−4.02 (m,
1H), 4.04−3.73 (m, 6H), 1.41 (s, 6H), 1.05 ppm (d, J = 7.2 Hz, 6H).
13C NMR (CDCl3): δ 152.2, 152.2, 149.0, 133.2, 131.0, 130.8, 128.6,
N-(2,2-Dimethyl-2H-chromen-6-ylmethyl)-3,4-dimethoxy-N-
(3-methylbutyl)benzenesulfonamide (5f). Yield: 31%. Mp 103−
126.0, 122.3, 121.2, 120.8, 116.1, 110.5, 109.6, 76.3, 56.2, 56.1, 50.0,
46.0, 27.9, 21.3 ppm. HRMS (ESI) m/z calcd for C23H29NO5S [(M +
Na)+] 451.1664; found, 451.1651. HPLC: tR = 11.76 min, 97.2%.
N-(2,2-Dimethyl-2H-chromen-6-ylmethyl)-3,4-dimethoxy-N-
prop-2-ynylbenzenesulfonamide (5b). Yield: 95%. 1H NMR
(CDCl3): δ 7.53 (dd, J = 2.0 Hz, 1H), 7.38 (d, J = 2.0 Hz, 1H),
7.09−7.07 (m, 1H), 7.00−6.96 (m, 2H), 6.74 (d, J = 8.0 Hz, 1H), 6.31
(d, J = 9.6 Hz, 1H), 5.64 (d, J = 9.6 Hz, 1H), 4.24 (s, 1H), 4.01−3.96
(m, 7 H), 1.59 (s, 2H), 1.43 ppm (s, 6H). HRMS (ESI) m/z calcd for
1
105 °C. H NMR (CDCl3): δ 7.44 (dd, J = 6.4, 2.0 Hz, 1H), 7.29−
7.28 (m, 1H), 6.96−6.94 (m, 2H), 6.83 (s, 1H), 6.24 (d, J = 10.0 Hz,
1H), 5.62 (d, J = 10.0 Hz, 1H), 4.23 (s, 2H), 3.97(s, 3H), 3.92 (s, 3H),
2.90 (d, J = 7.6 Hz, 2H), 1.75 (sep, J = 6.8 Hz, 1H), 1.43 (s, 6H),1.28
(s, 2H), 0.792−0.779 ppm (m, 6H). 13C NMR (CDCl3): δ 152.5,
152.3, 149.0, 132.1, 131.1, 129.2, 128.5, 126.5, 122.1, 121.3 121.1,
116.2, 110.5, 109.9, 76.3, 56.2, 56.2, 55.8, 52.1, 27.9, 26.9, 20.0 ppm.
HRMS (ESI) m/z calcd for C24H31NO5S 468.1821 [(M + Na)+];
found, 468.1801. HPLC: tR = 13.92 min, 97.5%.
C23H25NO5S [(M + Na)+] 450.1351; found, 450.1352. HPLC: tR
=
N-Cyclopentyl-N-(2,2-dimethyl-2H-chromen-6-ylmethyl)-
3,4-dimethoxybenzenesulfonamide (5g). Yield: 58%. Mp 110
−112 °C. 1H NMR (CDCl3): δ 7.39 (d, J = 6.4 Hz, 1H), 7.21 (d, J =
2.1 Hz, 1H), 7.10−6.82 (m, 3H), 6.67 (d, J = 8.2 Hz, 1H), 6.26 (d, J =
9.8 Hz, 1H), 5.57 (d, J = 9.8 Hz, 1H), 4.22 (s, 2H), 3.88 (d, J = 20.2
Hz, 6H), 1.70−1.18 (m, 15H). 13C NMR (CDCl3): δ 152.2, 152.1,
150.0, 132.7, 131.0, 130.9, 127.9, 152.3, 122.3, 121.2, 121.0, 116.1,
110.5, 109.8, 76.2, 59.5, 56.2, 56.1, 46.8, 29.3, 28.0, 23.5 ppm. HRMS
(ESI) m/z calcd for C25H31NO5S 480.1842 [(M + Na)+]; found,
480.1822. HPLC: tR = 14.11 min, 98.1%.
10.65 min, 96.61%.
(N-Butyl-N-(2,2-dimethyl-2H-chromen-6-ylmethyl)-3,4-di-
methoxybenzenesulfonamide (5c). Yield: 55%. Mp 82 °C. 1H
NMR (CDCl3): δ 7.46 (dd, J = 6.4, 2.1 Hz, 1H), 7.28 (m, 1H), 7.00−
6.89 (m, 2H), 6.71 (d, J = 8.0 Hz, 1H), 6.27 (d, J = 10.0 Hz, 1H), 5.63
(d, J = 9.6 Hz, 1H), 4.23 (s, 1H), 3.97−3.92 (m, 6H), 3.10 (t, J = 7.6
Hz, 2H), 1.43(s, 6H), 1.38−1.16 (m, 6H), 0.79 (t, J = 7.6 Hz, 3H).
13C NMR (CDCl3): δ 152.3, 149.0, 132.2, 131.1, 129.1, 128.5, 126.4,
122.1, 121.3, 121.0, 116.2, 110.6, 109.8, 76.3, 56.2, 56.2, 51.1, 47.4,
30.0, 27.9, 19.9, 13.6 ppm. HRMS (ESI) m/z calcd for C24H31NO5S +
Na 468.1821; found, 468.1815. HPLC: tR = 14.0 min, 96.3%.
N-Cyclopropyl-N-(2,2-dimethyl-2H-chromen-6-ylmethyl)-
3,4-dimethoxybenzenesulfonamide (5h). Off-white semisolid.
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Yield: 47%. Mp 94 °C. 1H NMR (CDCl3): δ 7.46 (dd, J = 2.0, 6.4 Hz,
1H), 7.27 (d, J = 2.0 Hz, 1H), 7.06 (dd, J = 2.0, 6.4 Hz, 1H), 6.97−
6.93 (m, 2H), 6.70 (d, J = 8.0 Hz, 1H), 6.29 (d, J = 9.6 Hz, 1H), 4.27
(s, 2H), 3.94 (s, 3H), 3.90 (s, 3H), 5.62 (d, J = 9.6 Hz, 1H), 2.01
(quin, J = 4.0 Hz, 1H), 1.44 (s, 6H), 0.72 (q, J = 3.2 Hz, 2H), 0.59 (q,
J = 3.2 Hz, 2H). 13C NMR (CDCl3): δ 152.5, 148.9, 131.0, 130.5,
129.7, 129.0, 126.9, 122.2, 121.6, 121.1, 116.0, 110.4, 110.2, 76.3, 56.2,
56.2, 54.2, 30.6, 28.0, 27.3 ppm. HRMS (ESI) m/z calcd for
2-Ethyl-2-methyl-2H-chromene-6-carbaldehyde (8). A solu-
tion of 7 (170 mg) in xylene (3 mL) was subjected to microwave
irradiation for 100 min at 220 W, 200 Torr, 120 °C. The solvent was
removed in vacuum to give a quantitative yield of the product
1
(170 mg). H NMR (CDCl3): δ 9.81 (s, 1H), 7.63 (dd, J = 8.0 Hz,
2.0 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.42
(d, J = 10.0 Hz, 1H), 5.62 (d, J = 10.0 Hz, 1H), 1.81−1.66 (m, 3H),
1.43 (s, 3H), 0.97 ppm (t, J = 7.6 Hz, 3H).
C23H27NO5S 452.1508 [(M + Na)+]; found, 452.1489. HPLC: tR
=
N-((2-Ethyl-2-methyl-2H-chromen-6-yl)methyl)benzenamine
(9). Reaction was carried out following the same procedure as for 2a−
7.48 min, 99.5%.
1
f using 170 mg of 8 to give 90.9 mg (41%) of product. H NMR
N-Cyclohexyl-N-(2,2-dimethyl-2H-chromen-6-ylmethyl)-3,4-
dimethoxybenzenesulfonamide (5i). Yield: 79%. 1H NMR
(CDCl3): δ 7.42 (dd, J = 2.0 Hz, 6.4 Hz, 1H), 7.30−7.15 (m, 1H),
7.16 (d, J = 8.4 Hz, 1H), 7.10−7.08 (m, 1H), 7.02 (d, J = 2.0 Hz, 1H),
6.91 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.31 (d, J = 9.6 Hz,
1H), 5.63 (d, J = 9.6 Hz, 1H), 4.31 (s, 2H), 3.95 (s, 3H), 3.90 (s, 3H),
1.70−1.54 (m, 4H), 1.43 (s, 6H), 1.27−1.20 ppm (m, 6H). Yield:
79%. HPLC: tR = 14.4 min, 99.5%.
General Procedure for the Synthesis of 5j−m. To a solution
of secondary amine 4 (1 equiv) in DCM were added triethylamine
(3 equiv) and then the appropriate sulfonyl chloride (2 equiv). The
mixture was stirred at room temperature for 24 h. Then saturated
NH4Cl was added to the reaction mixture, which was extracted with
DCM (×2). After drying over MgSO4, the DCM solution was
concentrated under vacuum. The crude product was purified by flash
column chromatography (silica gel).
N-((2,2-Dimethyl-2H-chromen-6-yl)methyl)-4-methoxy-N-
phenylbenzenesulfonamide (5j). Yield: 26.4 mg (30%). 1H NMR
(CDCl3): δ 7.60 (d, J = 9.2 Hz, 2H), 7.28−7.22 (m, 3H), 7.00−6.93
(m, 4H), 6.91−6.88 (m, 2H), 6.62 (s, 1H), 6.24 (d, J = 10.0 Hz, 1H),
5.58 (d, J = 10 Hz, 1H), 4.62 (s, 2H), 3.90 (s, 3H), 1.40 ppm (s, 6H).
13C NMR (CDCl3): δ 162.9, 139.1, 130.9, 129.8, 129.3, 129.1, 128.8,
128.1, 127.8, 126.6, 122.3, 116.0, 114.0, 76.3, 55.6, 54.3, 28.0 ppm.
HRMS (ESI) m/z calcd for C23H27NO5S 452.1508 [(M + H)+];
found, 452.1489. HPLC: tR = 14.03 min, 96.1%.
(CDCl3): δ 7.22−7.18 (m, 5H), 7.11 (dd, J = 6.0 Hz, 2.4 Hz, 1H),
6.99 (d, J = 2.0 Hz, 2H), 6.77 −6.74 (m, 3H), 6.68−6.66 (m, 2H),
6.36 (d, J = 10.0 Hz, 1H), 5.58 (d, J = 10.0 Hz, 1H), 4.21 (s, 2H),
1.76−1.71 (m, 3H), 1.33(s, 3H), 0.96 ppm (t, J = 7.6 Hz, 3H).
N-((2-Ethyl-2-methyl-2H-chromen-6-yl)methyl)-3,4-dime-
thoxy-N-phenylbenzenesulfonamide (10). To a solution of 9 (80
mg, 0.29 mmol) in DCM (3 mL) were added Et3N (0.12 mL, 0.85
mmol) and 3,4-dimethoxybenzenesulfonyl chloride (135 mg, 0.573
mmol). After 24 h, saturated NH4Cl was added to the reaction mixture
and the aqueous layer was extracted with DCM (5 × 2 mL). The
organic layers was combined, dried over MgSO4, and concentrated
under vacuum. The crude product was purified by column (silica gel,
5:1 hexane/EtOAc) to give a white solid (42.6 mg). Yield: 32%. Mp
129−130 °C. 1H NMR (CDCl3): δ 7.35 (dd, J = 6.4 Hz, 2.0 Hz, 1H),
7.25−7.23 (m, 3H), 7.02−6.98 (m, 3H), 6.94 (d, J = 8.4 Hz, 1H),
6.91−6.88 (m, 2H), 6.59 (d, J = 8 Hz, 1H), 6.28 (d, J = 10 Hz, 1H),
5.53 (d, J = 10 Hz, 1H) 4.62 (s, 2H), 3.98 (s, 3H), 3.77 (s, 3H),
1.71−1.65 (m, 2H), 1.43 (s, 3H), 0.94 ppm (t, J = 7.6 Hz, 3H). 13C
NMR (CDCl3): δ 152.8, 152.5, 148.7, 139.2, 130.5, 129.8, 129.3,
129.2, 128.8, 127.9, 127.8, 126.6, 122.8, 121.4, 121.1, 115.8, 110.4,
79.0, 56.2, 56.1, 54.3, 34.0, 26.0 ppm. MS (ESI) m/z 502 [(M +
Na)+]. HPLC: tR = 5.9 min, 98.9%.
N-Phenylquinolin-3-amine (12). To a solution of quinoline-3-
carbaldehyde 11 (79 mg, 0.5 mmol) in MeOH (5 mL) were added
aniline (0.05 mL, 0.55 mmol) and ZnCl2 (136 mg, 2.0 mmol), and the
mixture was stirred at room temperature for 15 min. Then NaCNBH3
(62.8 mg, 2.0 mmol) was added and the mixture was stirred overnight
at room temperature. The solvent was removed by rotary evaporation
and the residue suspended in EtOAc. The organic layers were
combined and washed with NaHCO3, water, and brine and then dried
over MgSO4. Concentration in vacuo gave the crude product, which
was used in the next step without further purification. 1H NMR
(CDCl3): δ 9.05 (s, 1H), 8.23 (d, J = 7.6 Hz, 2H), 7.77 (d, J = 8.0 Hz,
1H), 7.71−7.66 (m, 1H), 7.54−7.51 (m, 1H), 7.21−7.16 (m, 2H),
6.76−6.72 (m, 1H), 6.68−6.60 (m, 2H), 4.54 (s, 2H), 4.16 (s, br, 1H).
3,4-Dimethoxy-N-phenyl-N-(quinolin-3-ylmethyl)-
benzenesulfonamide (13). To a solution of 12 (75 mg, 0.320
mmol) in DCM was added 3,4-dimethoxybenzylsulfonyl chloride
(83 mg, 0.35 mmol) and triethylamine (0.09 mL, 0.640 mmol). The
mixture was stirred overnight at room temperature, and the reaction
mixture was washed with water and brine. The organic layer was dried
over MgSO4, concentrated in vacuo, and purified by column
chromatography (silica gel, 2:1 hexane/EtOAc) to give a white
powder. Yield: 45%. Mp 173. 1H NMR (CDCl3): 8.86 (s, 1H), 8.14(d,
J = 8.0 Hz, 2H), 7.84 (dd, J = 7.2 Hz, 1.2 Hz, 1H), 7.80−7.76 (m, 1H),
7.64 −7.60 (m, 1H), 7.49 (dd, J = 6.0 Hz, 2.4 Hz, 1H), 7.38−7.30 (m,
3H), 7.17−7.14 (m, 2H), 7.08−7.05 (m, 2H), 5.03 (s, 2H), 4.08
(s, 3H), 3.85 ppm (s, 3H). 13C NMR (CDCl3): 152.8, 150.8, 148.8,
147.6, 138.0 135.7, 129.6, 129.2, 129.1, 129.0, 128.2, 127.7, 127.7,
126.9, 121.6, 110.5, 110.4, 56.2, 56.1, 52.4 ppm. MS (ESI) m/z 435
[(M + H)+]. HPLC: tR = 14.6 min, 95.2%.
N-((2,2-Dimethyl-2H-chromen-6-yl)methyl)-3,5-dimethyl-N-
phenylbenzenesulfonamide (5k). Yield: 65 mg, (40%). 1H NMR:
δ 7.54 (s, 2H), 7.30−7.23 (m, 3H), 7.00−6.97 (m, 1H), 6.90−6.88 (m,
2H), 6.60 (d, J = 8.8 Hz, 1H), 6.24 (d, J = 10 Hz, 1H), 5.58 (d, J = 9.6
Hz, 1H), 4.63 (s, 2H), 2.41 (s, 3H), 2.36 (s, 3H), 1.42(s, 6H). MS
(ESI) m/z 458 [(M + Na)+]. HPLC: tR = 23.7 min, 96.8%.
2,5-Dichloro-N-((2,2-dimethyl-2H-chromen-6-yl)methyl)-N-
1
phenylbenzenesulfonamide (5l). Yield: 69 mg (32%). H NMR
(CDCl3): δ 7.84 (d, J = 2.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.44−
7.40 (m, 1H), 7.23−7.21 (m, 3H), 7.06−7.04 (m, 2H), 6.92−6.90 (m,
2H), 6.64 (d, J = 7.6 Hz, 1H), 6.27 (d, J = 10.0 Hz, 1H), 5.60 (d, J =
9.6 Hz, 1H), 4.92 (s, 2H), 1.42 ppm (s, 6H). MS (ESI) m/z 471
[(M + Na)+].
4,4-Bromo-N-((2,2-dimethyl-2H-chromen-6-yl)methyl)-N-
phenyl-2-(trifluoromethoxy)benzenesulfonamide (5m). Yield:
1
23%. H NMR (CDCl3): δ 1.45 (s, 6H), 4.95 (s, 2H), 5.60 (d, 1H,
J = 9.6), 6.27 (d, 1H, J = 10), 6.64 (s, 1H, J = 7.6), 6.91 (m, 2H), 7.05
(m, 2H), 7.21 (m, 3H,) 7.43 (m, 1H) 7.48 (m, 1H), 7.84 ppm (d, 1H,
2.4). HPLC: tR = 19.6 min, 96.7%.
4-(3-Methylpent-1-yn-3-yloxy)benzaldehyde (7). To a solu-
tion of 3-methyl-1-pentyn-3-ol 6 (0.319 mL, 2.83 mmol) in
acetonitrile (3 mL) at 0 °C was added DBU (0.55 mL, 3.69 mmol).
Then TFAA (0.34 mL, 2.46 mmol) was added dropwise and the
solution was stirred at 0 °C for 30 min. To a solution of 4-
hydroxybenzaldehyde (300 mg, 2.46 mmol) in acetonitrile at 0 °C
were added DBU (0.55 mL 3.69 mmol) and CuCl2·2H2O (0.42 mg,
0.0025 mmol). The first mixture was added to the second mixture over
a period of 5 min. The mixture was stirred overnight. The solvent was
removed by rotary evaporation and the residue diluted with DCM.
Then the organic layer washed with 1 M HCl, 1 M NaOH, saturated
NaHCO3, and brine, dried over MgSO4, and concentrated in vacuum
N-((2,2-Dimethyl-2H-chromen-6-yl)methyl)benzenamine
(15a). To a solution of 2,3-dihydro-2,2-dimethylbenzofuran-5-
carboxaldehyde 14 (250 mg, 1.42 mmol) in MeOH (10 mL) were
added aniline (0.14 mL, 1.022 mmol), NaCNBH3 (178 mg, 2.84 mmol),
and ZnCl2 (dried in oven) (387 mg, 2.84 mmol). The mixture was
stirred at room temperature overnight, and then the solvent was removed
by rotary evaporation. Then 0.1 M NaOH was added to the resulting
residue, which was extracted with EtOAc (×2). The combined organic
1
to give 170 mg (30%) of product. H NMR (CDCl3): δ 9.91(s, 1H),
7.83−7.81 (m, 2H), 7.36−7.34 (m, 2H), 2.69 (s, 1H), 2.06−1.92 (m,
2H), 1.66 (s, 3H), 1.12 ppm (t, J = 7.2 Hz, 3H).
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Journal of Medicinal Chemistry
Article
layers were dried over MgSO4 and concentrated in vacuum to give
(m, 7H), 1.47(s, 6H), 1.27−1.22 ppm (m, 4H). HRMS (ESI) m/z
calcd for C25H33 NO5S [(M + Na)+] 482.1977; found, 482.1981.
HPLC: tR = 16.4 min, 95.6%.
1
301 mg of the product (84%). H NMR (CDCl3): δ 7.29−7.22 (m,
3H), 7.17 (d, J = 8.0 Hz, 1H), 6.95−6.70 (m, 4H), 4.28 (s, 2H), 3.06
(s, 2H), 1.55 ppm (s, 6H).
N-Cyclopentyl-N-((2,2-dimethyl-2,3-dihydrobenzofuran-5-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (16f). Yield:
N-((2,2-Dimethyl-2,3-dihydrobenzofuran-5-yl)methyl)-3,4-
dimethoxy-N-phenylbenzenesulfonamide (16a). To a solution
of 15a (100 mg, 0.395 mmol) in DCM (5 mL) were added triethylamine
(0.17 mL, 0.790 mmol) and 3,4-dimethoxybenzenesulfonyl chloride
(187 mg, 0.790 mmol) dissolved in 1 mL of DCM, and the mixture was
stirred for 72 h. Ammonium chloride was added to the reaction mixture,
which was then extracted with DCM (×2). After drying of the combined
organic layers over MgSO4 and concentration in vacuum, the crude
reaction mixture was purified by column (silica gel, 3:1 hexane/EtOAc)
1
31%. H NMR (CDCl3): δ 7.44 (d, J = 8.5, 1H), 7.27 (s, 2H), 7.04
(d, J = 8.10, 1H), 6.92 (d, J = 8.4, 1H), 6.65 (d, J = 8.1, 1H), 4.29
(s, 3H), 3.95 (s, 3H), 3.90 (s, 3H), 2.99 (s, 2H), 1.85−1.58 (m, 3H),
1.60−1.22 ppm (m, 12 H). HRMS (ESI) m/z calcd for C24H31NO5S
[(M + Na)+] 468.1821; found, 468.1817. HPLC: tR = 14.4 min, 95.0%
2,2-Dimethyl-2H-pyrano[2,3-b]pyridine-6-carbaldehyde
(18). To a solution of 17 (100 mg, 0.390 mmol) in dry ether (2 mL)
was added BuLi (0.25 mL, 2.5 M solution in THF) dropwise at
−65 °C, and the mixture was stirred for 15 min. Then DMF
(anhydrous) was added dropwise and the mixture was stirred at
−65 °C for 1.5 h. Water was added to quench the reaction, which was
extracted with EtOAc (×2). The organic layers were washed with
water (×1), brine (×1), dried over MgSO4, and concentrated in vacuo
to give a yellow oil. Purification by column chromatography (silica gel)
1
to give a white solid (76 mg, 42%). Mp 136 °C. H NMR (CDCl3):
δ 7.33 (dd, J = 2.13, 8.44, 1H), 7.26−7.21 (m, 3H), 7.09 (s, 1H) 7.00−
6.96 (m, 3H), 6.92 (d, J = 8.5, 1H), 6.83 (d, J = 8.1,1H), 6.52 (d, J = 8.1,
1H), 4.62 (s, 2H), 3.95 (s, 1H), 3.75 (s, 1H), 2.92 (s, 2H), 1.42 ppm (s,
1H). 13C NMR (CDCl3): δ 158.4, 152.5, 148.7, 139.3, 129.2, 127.5,
125.6, 121.4, 110.4, 108.9, 86.9, 56.2, 56.1, 54.6, 42.7, 28.2 ppm. MS
(ESI) m/z 435 [(M + H)]. HPLC: tR = 10.5 min, 96.1%.
1
with 6:1 hexane/EtOAc gave a white solid. Yield: 23 mg (31%). H
NMR (CDCl3): δ 1.58 (s, 1H), 5.79 (d, 1H, J = 8.0 Hz), 6.36 (d, 1H,
J = 9.6 Hz), 7.76 (s, 1H), 8.50 (s, 1H), 9.92 ppm (s, 1H).
General Procedure for the Synthesis of Compounds 16b−f. To
a solution of 14 in methanol was added amine (1 0.1 equiv) and zinc
chloride (2 equiv), and the reaction mixture was stirred for 2 h before
NaCNBH3 (2 equiv) was added. The mixture was then stirred at
room temperature overnight. The solvent was removed by rotary
evaporation and the residue diluted with EtOAc and washed with
Na2CO3 (saturated) and brine. The organic layers were dried over
MgSO4 and concentrated in vacuo. The product was used without
further purification in the next step. To the resulting secondary amine
15 (1 equiv) in DCM was added Et3N (2 equiv) and the appropriate
aryl- or alkylsulfonyl chloride (1.1 equiv), and the mixture was stirred
at room temperature overnight. The reaction mixture was diluted with
DCM and washed with water and brine. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The crude product was
purified by flash chromatography (silica gel).
N-((2,2-Dimethyl-2H-pyrano[2,3-b]pyridin-6-yl)methyl)-
benzenamine (19a). To a solution of 2,2-dimethyl-2H-pyrano[2,3-b]-
pyridine-6-carbaldehyde 18 (20 mg, 0.106 mg) in methanol (1 mL)
were added aniline (0.01 mL, 0.12 mmol), NaCNBH3 (13 mg, 0.212
mmol), and ZnCl2 (29 mg, 0.212 mmol). The mixture was stirred for
30 min, after which the solvent was removed by rotary evaporation and
the 1 M NaOH added to the residue, extracted with DCM, dried
over MgSO4, and concentrated in vacuo. Purification by column
chromatography (3:1 hexane/EtOAc) gave a white solid. Yield: 20 mg
1
(72%). H NMR (CDCl3) δ 8.01 (s, 1H), 7.29 (s, 1H), 7.26−7.16
(m, 2H), 6.74 (t, J = 7.2 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 6.26 (d, J =
9.6 Hz, 1H), 5.67 (d, J = 9.6 Hz, 1H), 4.21 (s, 2H), 1.51 ppm (s, 6H).
13C NMR (CDCl3) δ 159.6, 147.8, 146.4, 133.8, 132.2, 129.3, 128.4,
120.9, 118.0, 115.4, 113.0, 79.2, 45.4, 28.8 ppm. HRMS (ESI) m/z
calcd for C11H11NO2 [(M + H)+] 190.0868; found, 190.0870.
N-((2,2-Dimethyl-2H-pyrano[2,3-b]pyridin-6-yl)methyl)-
cyclohexanamine (19b). To a solution of 2,2-dimethyl-2H-pyrano-
[2,3-b]pyridine-6-carbaldehyde 18 (23 mg, 0.121 mmol) in MeOH
(1 mL) were added cyclohexylamine (0.014 mL, 0.121 mmol),
NaCNBH3 (15 mg, 0.242 mmol), and zinc chloride (33 mg, 0.242
mmol), and the mixture was stirred overnight . The solvent was
removed by rotary evaporation and the residue dissolved in EtOAc and
washed with 1 M NaOH, water, and brine, dried over MgSO4, and
concentrated in vacuo. The product was used in the next step without
further purification.
N-Cycloheptyl-N-((2,2-dimethyl-2,3-dihydrobenzofuran-5-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (16b). Yield:
14%. Mp 90 °C. 1H NMR (CDCl3): δ 7.43 (dd, J = 2.14, 8.44,
1H), 7.25−7.24 (m, 2H), 7.05 (d, J = 8.13, 1H), 6.92 (d, J = 8.5, 1H),
6.65 (d, J = 8.1, 1H), 4.28 (s, 2H), 3.96 (s, 3H), 3.90 (s, 3H), 3.00 (s,
2H), 1.63−1.51 (m, 8H), 1.48 (s, 6H), 1.45−1.27 ppm (m, 7H).
HRMS (ESI) calcd for C26H35NO5S m/z [(M + Na)+] 496.2134;
found, 496.2122. HPLC: tR = 18.6 min, 99.4%.
N-((2,2-Dimethyl-2,3-dihydrobenzofuran-5-yl)methyl)-N-iso-
propyl-3,4-dimethoxybenzenesulfonamide (16c). Yield: 18%. 1H
NMR (CDCl3): δ 7.45 (d, J = 8.2 Hz, 1H), 7.27 (s, 1H), 7.10 (d, J =
15.6 Hz, 1H), 6.94 (t, J = 7.5 Hz, 2H), 6.63 (d, J = 7.6 Hz, 1H), 4.24
(s, 2H), 3.94 (d, J = 19.3 Hz, 6H), 3.05−2.93 (m, 2H), 2.91 (d,
J = 6.9 Hz, 2H), 1.82−1.66 (m, 1H), 1.48 (s, 6H), 0.77 ppm (d, J =
5.7 Hz, 6H). 13C NMR (CDCl3): δ 158.5, 152.3, 149.0, 132.1, 128.4,
127.9, 127.7, 125.6, 121.1, 110.5, 109.9, 109.0, 87.1, 77.4, 77.1, 76.7,
56.2, 56.1, 55.8, 52.4, 42.7, 28.1, 26.9, 20.0 ppm. HRMS (ESI) m/z
calcd for C23H31NO5S [(M + Na)+] 456.1821; found, 456.1833.
HPLC: tR = 11.5 min, 97.6%.
N-((2,2-Dimethyl-2H-pyrano[2,3-b]pyridin-6-yl)methyl)-3,4-
dimethoxy-N-phenylbenzenesulfonamide (20a). To a solution
of 19a (20 mg, 0.075 mmol) in DCM (1 mL) were added 3,4-
dimethoxybenzenesulfonyl chloride (36 mg, 0.150 mmol) and
triethylamine (0.021 mL, 0.150 mmol). The mixture was stirred for
24 h at room temperature. The reaction mixture was washed with
water (×2) and the organic layer dried over MgSO4 and concentrated
in vacuo. Column chromatography (2:1 hexane/EtOAc) gave a white
N-Butyl-N-((2,2-dimethyl-2,3-dihydrobenzofuran-5-yl)-
methyl)-3,4-dimethoxybenzenesulfonamide (16d). Yield: 21%.
1H NMR (CDCl3): δ 7.47 (dd, J = 8.4, 1.5 Hz, 1H), 7.34−7.22 (m,
1
solid (15 mg). Yield: 43%. H NMR (CDCl3): δ 1.47(s, 6H), 3.76 (s,
3H), 3.97 (s, 3H), 4.60 (s, 2H), 5.66 (d,1H, J = 9.6), 6.26 (d, 1H, J =
10), 6.93−6.99 (m, 4H), 7.23−7.25 (m, 3H), 7.32−7.36 (m, 2H), 7.63
ppm (d, 1H, J = 2.4). HRMS (ESI) m/z calcd for C25H25N2O5S [M +
H)+] 467.1641; found, 467.1636. HPLC: tR = 7.5 min, 99.0%
N-Cyclohexyl-N-((2,2-dimethyl-2H-pyrano[2,3-b]pyridin-6-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (20b). Yield:
2H), 7.13 (s, 1H), 6.96 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.1 Hz, 1H),
4.25 (s, 2H), 3.95 (d, J = 16.7 Hz, 6H), 3.22−3.03 (m, 2H), 2.99 (s,
2H), 1.57 (d, J = 21.8 Hz, 1H), 1.49 (s, 6H), 1.41−1.24 (m, 3H),
1.23−1.08 (m, 2H), 0.79 ppm (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3):
δ 158.6, 152.2, 149.2, 143.0, 132.3, 128.3, 127.4, 125.5, 120.9, 110.5,
109.8, 109.0, 87.1, 77.4, 77.0, 76.7, 56.2, 56.1, 51.3, 47.3, 42.7, 29.9,
28.1, 19.9, 13.7 ppm. HRMS (ESI) m/z calcd for C23H31NO5S [(M +
Na)+] 456.1821; found, 456.1812. HPLC: tR = 11.2 min, 98.0%.
N-Cyclohexyl-N-((2,2-dimethyl-2,3-dihydrobenzofuran-5-yl)-
methyl)-3,4-dimethoxybenzenesulfonamide (16e). Yield: 36%.
1
60%. H NMR (CDCl3): δ 7.89 (s, 1H), 7.49 (s, 1H), 7.44 (d, J =
2.0 Hz, 1H), 7.28 (s, 1H), 6.93 (d, J = 8.8 Hz, 1H), 6.31 (d, J = 10 Hz,
1H), 5.69 (d, J = 10 Hz, 1H), 4.30 (s, 2H), 3.95 (s, 3H), 3.91 (s, 3H),
1.71−1.52 (m, 10 H), 1.29−1.21 ppm (m, 6H). HRMS (ESI) m/z
calcd for C25H32N2O5S [(M + H)+] 473.2110; found, 473.2127.
HPLC: tR = 11.4 min, 95.0%.
1
Mp 102 °C. H NMR (CDCl3) δ 7.41 (d, J = 8.4, 1H), 7.22 (s, 2H),
7.03 (d, J = 7.9, 1H), 6.90 (d, J = 8.3, 1H), 6.63 (d, J = 7.9, 1H), 4.31
(s, 2H), 3.70 (1H) 3.94 (s, 3H) 3.88 (s, 3H), 2.98 (s, 1H), 1.69−1.52
6-Bromo-2,2-dimethyl-2H-pyrano[3,2-b]pyridine (23). To a
solution 2-methyl-3-butyn-2-ol (21) in acetonitrile (6 mL) was added
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DBU (0.80 mL, 6.61 mmol) at 0 °C. Then TFAA was added dropwise,
also at 0 °C. The mixture was stirred for 30 min. In another round-
bottom flask, DBU (0.80 mL, 6.61 mmol) was added to a solution of
2-bromo-5-hydroxypyridine (1 g, 5.75 mmol) in 6 mL of acetonitrile at
0 °C. Then 2-methyl-3-butyn-2-ol was added dropwise into this
mixture, which was stirred for 30 additional min. The solvent was
removed by rotary evaporation, and the residue was diluted with
DCM. After separation, the organic layer was washed with 1 M HCl,
1 M NaOH, saturated NaHCO3, and brine, dried over MgSO4, and
concentrated in vacuum. The crude product was dissolved in 2 mL of
xylene and subjected to microwave irradiation (130 °C, 220 W) for
30 min. The solvent was removed by rotary evaporation and the
product concentrated in vacuum. The crude product was purified by
column chromatography (silica gel) (10:1 hexane/EtOAc) to give
300 mg of a yellow solid (23% over the two steps). 1H NMR (CDCl3):
1.45 (s, 6H), 5.86 (d, 1H, J = 10.4), 6.44 (d, 1H, J = 10), 6.90 (d, 1H,
J = 8.8), 7.14 ppm (s, 1H, J = 8.4). HRMS (ESI) m/z calcd for
C10H11NOBr [(M + H)+] 240.0024; found, 240.0026.
2,2-Dimethyl-2H-pyrano[3,2-b]pyridine-6-carbaldehyde
(24). To a solution of 23 (200 mg, 0.83 mmol) in anhydrous THF
(5 mL) at −78 °C was added BuLi (2.5M, 0.35 mL), and the mixture
was stirred for 35 min. Then DMF (0.08 mL, 0.1 mmol) was added
dropwise. The mixture was stirred at −78 °C for an 30 additional min.
Water (3 mL) was added to quench the reaction and was extracted
with EtOAc. The organic layer was washed with water, brine, dried
over MgSO4 and concentrated under reduced pressure. The crude
product was purified by flash column chromatography, using 20:1
hexane/EtOAc to give the product as a yellowish solid (23% yield). 1H
NMR: δ 1.53 (s, 6H), 6.01 (d 1H, J = 10.4), 6.58 (d, 1H, J = 10.4),
7.13 (d, 1H, J = 8.4), 7.77 (d, 1H, J = 8.4), 9.93 ppm (s, 1H). 13C
NMR δ 28.8, 78.7, 123.0, 123.3, 123.4, 136.3, 145.8, 153.7, 191.9 ppm
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-
aniline (25a). To a solution of 2,2-dimethyl-2H-pyrano[3,2-b]-
pyridine-6-carbaldehyde (434 mg, 2.28 mmol) in methanol (3 mL)
were added aniline (0.3 mL, 2.52 mmol) and zinc chloride (621 mg,
4.56 mmol), and the mixture was stirred at room temperature for 2 h.
Then NaCNBH3 (287 mg, 4.56 mmol) was added, and the mixture
was stirred overnight. Purification by column chromatography with 4:1
hexane/EtOAc gave an off-white solid. Yield: 48%. 1H NMR (CDCl3):
δ 1.49 (s, 6H), 4.361 (s, 2H), 5.91 (d, 1H, J = 10), 6.55 (d, 1H, J =
10), 6.68−6.71 (m, 3H), 7.01 (d, 1H, J = 8.4), 7.08 (d, 1H, J = 8.4),
7.18−7.38 ppm (m, 2H).
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-3,4-
dimethoxy-N-phenylbenzenesulfonamide (26a). To a solution
of the 25a (60 mg, 0.237 mmol) in dichloromethane (2.5 mL) were
added triethylamine (0.07 mL, 0.474 mmol) and the 3,4-dimethox-
ybenzenesulfonyl chloride (84 mg, 0.355 mmol). The mixture was
stirred for 24 h. The reaction mixture was diluted with DCM and the
organic layer washed with water and brine, dried over magnesium
sulfate, and concentrated in vacuo. The crude product was purified by
column chromatography (silica gel, 3:1 hexane/EtOAc to 1:1 hexane/
EtOAc) to give an off-white solid (56 mg). Yield: 50%. Mp 143 °C. 1H
NMR (CDCl3): δ 7.29 (dd, J = 8.4, 2.1 Hz, 2H), 7.25−7.17 (m, 3H),
7.14−7.09 (m, 2H), 6.96 (d, J = 8.4 Hz, 1H), 6.92−6.86 (m, 2H), 6.32
(d, J = 10.1 Hz, 1H), 5.80 (d, J = 10.1 Hz, 1H), 4.78 (s, 2H), 3.94 (s,
3H), 3.72 (s, 3H), 1.40 ppm (s, 6H). HRMS (ESI) m/z calcd for
C25H26N2O5S [(M + H)+] 467.1641; found, 467.1641. HPLC: tR = 9.7
min, 98.1%.
General Procedure for the Synthesis of 26b−j by Reaction
with Alkylsulfonyl Chloride. To a solution of 25 (1 equiv) in
methanol was added the primary amine (1 equiv) and ZnCl2
(2 equiv), and the mixture was stirred at room temperature for 2 h.
Then NaCNBH3 (2 equiv) was added and the mixture was stirred at
room temperature overnight. The solvent was removed by rotary
evaporation, and EtOAc was added to the residue. The solid was
filtered through Celite and the filtrate washed with 1 M NaOH, water,
and brine. The organic layer was dried over MgSO4 and concentrated
under reduced pressure. The crude secondary amine product was used
without further purification.
To a solution of the secondary amine (1 equiv) in DCM was added
triethylamine (3 equiv) and the sulfonyl chloride (1.5 equiv). The
mixture was stirred for 24−48 h. Then water was added and the
organic layer extracted with DCM, dried over MgSO4, concentrated
under reduced pressure, and purified by flash column chromatography.
N-Butyl-N-((2,2-dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)-
methyl)-3,4-dimethoxybenzenesulfonamide (26b). Yield: 42%.
1H NMR (CDCl3): δ 7.45 (d, J = 2.4 Hz, 1H), 7.29−7.26 (m, 2H),
7.04 (d, J = 8.4, 1H), 6.94 (d, J = 8.8, 1H), 6.40 (d, J = 8.0, 1H), 5.88
(d, J = 10, 1H), 4.37 (s, 2H), 3.96 (s, 3H), 3.93 (s, 3H), 3.18 (t, J =
7.6, 2H), 1.48 (s, 6H), 1.38 (m, 2H), 1.18 (sx, J = 7.2, 2H), 0.79 ppm
(t, J = 7.2, 3H). 13C NMR (CDCl3): δ 152.4, 149.0, 148.8, 148.7,
135.4, 131.6, 123.7, 123.7, 122.7, 121.0, 110.5, 109.8, 56.2, 56.2, 53.3,
48.8, 30.2, 28.2, 19.9, 13.6 ppm. HRMS (ESI) m/z calcd for
C23H31N2O5S [(M + H)+] 447.1954; observed, 447.1937. HPLC: tR =
11.4 min, 95.4%.
N-(3,4-Dimethoxyphenyl)-N-((2,2-dimethyl-2H-pyrano[3,2-
b]pyridin-6-yl)methyl)-3,4-dimethoxybenzenesulfonamide
(26c). Yield: 51%. Mp 117 °C. 1H NMR δ (CDCl3): δ 7.32−7.38 (m,
2H), 7.01−6.98 (m, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz.
1H), 6.68−6.62 (m, 2H), 6.34 (dd, J = 12.0, 0.4 Hz, 1H), 5.82 (d, J =
10.0 Hz, 1H), 4.76 (s, 2H), 3.96 (s, 3H), 3.83 (s, 3H), 3.79 (s, 3H),
3.72 (s, 3H), 1.43 ppm (s, 6H). 13C NMR (CDCl3): δ 152.6, 148.7,
148.7, 148.6, 148.5, 147.8, 140.2, 135.3, 132.2, 129.6, 123.6, 123.6,
122.6, 121.8, 121.0, 112.5, 110.5, 110.3, 56.2, 56.2, 56.1, 55.9, 28.2
ppm. HRMS (ESI) m/z calcd for C27H31N2O7S [(M + H)+] 527.1852;
found, 527.1866. HPLC: tR = 7.6 min, 98.4%.
N-Cyclopentyl-N-((2,2-dimethyl-2H-pyrano[3,2-b]pyridin-6-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (26d). Yield:
1
31%. H NMR (CDCl3): δ 7.55−7.35 (m, 2H), 7.35−7.22 (m, 1H),
7.07 (d, J = 8.4 Hz, 1H), 6.98−6.86 (m, 1H), 6.46 (dd, J = 14.7, 10.2
Hz, 1H), 5.89 (d, J = 10.1 Hz, 1H), 4.44−4.24 (m, 3H), 3.96 (s, 3H),
3.93 (s, 3H), 1.76−1.15 ppm (m, 15H). 13C NMR (CDCl3): δ 152.4,
150.7, 149.0 148.5,140.0 135.4, 132.2, 123.8, 123.7, 121.9, 121.2,
110.5, 109.8, 59.4, 56.3, 56.2, 48.6, 29.1, 28.2, 23.4 ppm. HRMS (ESI)
m/z calcd for C24H31N2O5 [(M + H)+] 459.1954; found, 459.1938.
HPLC: tR = 11.3 min, 96.9%.
N-Cyclohexyl-N-((2,2-dimethyl-2H-pyrano[3,2-b]pyridin-6-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (26e). Yield:
1
46%. H NMR (CDCl3): 7.47 (dd, J = 2.4, 6.4 Hz, 1H), 7.42 (d,
J = 8.4 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.06 (d, J = 8.4, 1H), 6.93
(d, J = 8.8 Hz, 1H), 6.45 (d, J = 10 Hz, 1H), 5.88 (d, J = 10 Hz, 1H),
4.34 (s, 2H), 3.96 (s, 3H), 3.92 (s, 3H), 3.80 (m, 1H), 1.64 (m, 3H),
1.48 (m, 9H), 1.25 −1.20 ppm (m, 4H). 13C NMR (CDCl3): δ 152.2,
150.7, 149.1, 148.5, 140.0, 135.2, 133.2, 123.7, 123.7, 122.5, 120.7,
110.6, 109.5, 58.4, 56.2, 56.1, 48.5, 31.3, 28.2, 26.1, 25.2 ppm. HRMS
(ESI) m/z calcd for C25H33N2O5S [(M + H)+] 473.2110; found,
473.2118. HPLC: tR = 13.1 min, 96.8%
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-3,4-
dimethoxy-N-(5,6,7,8-tetrahydronaphthalen-2-yl)-
benzenesulfonamide (26f). 1H NMR (CDCl3): δ 7.39−7.31 (m,
2H), 6.99 (dd, J = 13.4, 5.2 Hz, 2H), 6.92 (dd, J = 8.1, 2.8 Hz, 2H),
6.81 (d, J = 7.6 Hz, 2H), 6.35 (d, J = 10.1 Hz, 1H), 5.94−5.71 (m,
1H), 4.78 (d, J = 25.5 Hz, 2H), 3.97 (s, 3H), 3.79 (d, J = 5.3 Hz, 3H),
2.65 (dd, J = 25.6, 13.3 Hz, 4H), 1.83−1.66 (m, 4H), 1.41 ppm (d, J =
16.1 Hz, 6H). MS (ESI) [(M + H)+] 521. HPLC: tR = 15.6 min,
96.9%.
N-Cycloheptyl-N-((2,2-dimethyl-2H-pyrano[3,2-b]pyridin-6-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (26g). Yield:
1
18%. Mp 73 °C. H NMR (CDCl3): δ 7.47 (dt, J = 4.4, 2.2 Hz,
1H), 7.44 (d, J = 8.4 Hz, 1H), 7.31−7.27 (m, 1H), 7.07 (d, J = 8.4 Hz,
1H), 6.94 (dd, J = 8.5, 4.9 Hz, 1H), 6.45 (dd, J = 10.1, 0.5 Hz, 1H),
5.88 (d, J = 10.1 Hz, 1H), 4.44−4.29 (m, 2H), 3.96 (s, 3H), 3.94 (s,
1H), 3.93 (s, 3H), 1.62−1.29 ppm (m, 18H). HPLC: tR = 15.2 min,
98.8%.
N-Cyclooctyl-N-((2,2-dimethyl-2H-pyrano[3,2-b]pyridin-6-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (26h). Yield:
1
45%. Mp 117 °C. H NMR (CDCl3): δ 7.49 (d, J = 6.4, 1H), 7.46
(d, J = 10, 1H), 7.32−7.29 (m, 1H), 7.07 (d, J = 8.0, 1H), 6.94 (d, J =
8.4, 1H), 6.45 (d, J = 10, 1H), 5.89 (d, J = 10, 1H), 4.38 (s, 2H),
8485
dx.doi.org/10.1021/jm201018g|J. Med. Chem. 2011, 54, 8471−8489

Journal of Medicinal Chemistry
Article
3.97−3.93 (m, 7H), 1.61−1.42 ppm (m, 20H). MS (ESI) m/z [(M +
H)+] 501. HPLC: tR = 16.5 min, 99.0%.
6H), 1.03 ppm (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3): δ 148.78,
148.18, 139.59, 135.38, 129.28, 128.35, 127.62, 123.67, 123.63, 122.53,
77.34, 77.02, 76.70, 56.19, 53.17, 28.21, 17.08, 13.09 ppm. MS (ESI)
m/z [(M + H)+] 373. HPLC: tR = 9.23 min, 97.4%.
N-Cyclobutyl-N-((2,2-dimethyl-2H-pyrano[3,2-b]pyridin-6-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (26i). Yield:
1
40%. H NMR (CDCl3): δ 7.44 (dd, J = 8.4, 2.2 Hz, 1H), 7.36 (d,
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-2-
1
J = 8.4 Hz, 1H), 7.31−7.21 (m, 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.94 (t,
J = 6.9 Hz, 1H), 6.40 (dd, J = 32.8, 10.0 Hz, 1H), 5.88 (t, J = 10.6 Hz,
1H), 4.49−4.28 (m, 3H), 3.96 (s, 3H), 3.93 (d, J = 3.0 Hz, 3H), 2.08−
1.83 (m, 4H), 1.60−1.40 ppm (m, 8H). 13C NMR (CDCl3): δ 152.5,
150.3, 149.0, 148.6, 140.1, 135.4, 131.7, 123.8, 123.7, 121.8, 121.0,
110.5, 109.6, 77.4, 77.0, 76.7, 56.2, 56.2, 52.7, 49.3, 28.9, 28.2, 15.0
ppm. MS (ESI+) m/z [(M + H)+] 445. HPLC: tR = 8.7 min, 97%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-(4-
fluorophenyl)-3,4-dimethoxybenzenesulfonamide (26j). Yield:
methyl-N-phenylpropane-1-sulfonamide (26p). Yield: 28%. H
NMR (CDCl3): δ 7.40−7.30 (m, 4H), 7.27−7.21 (m, 1H), 7.19 (d, J =
8.3 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.42 (d, J = 10.1 Hz, 1H), 5.89
(dd, J = 21.3, 10.0 Hz, 1H), 4.93 (s, 2H), 3.01 (dd, J = 14.1, 6.5 Hz,
2H), 2.34 (dd J = 13.3, 6.7 Hz, 1H), 1.47 (d, J = 14.5 Hz, 6H), 1.10
ppm (d, J = 6.7 Hz, 6H). MS (ESI) m/z [(M + H)+] 387. HPLC: tR =
10.4 min, 96.3%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenylbiphenyl-4-sulfonamide (26q). Yield: 17%. Mp 170 °C.
1H NMR (CDCl3): δ 7.69 (s, 4H), 7.67−7.60 (m, 2H), 7.57−7.41 (m,
1
12%. H NMR (CDCl3): δ 7.55−7.40 (m, 1H), 7.38−7.23 (m, 3H),
7.16−7.01 (m, 4H), 7.00−6.87 (m, 5H), 6.40 (t, J = 11.7 Hz, 1H),
5.88 (t, J = 13.4 Hz, 1H), 4.83 (d, J = 14.0 Hz, 2H), 3.97 (s, 3H),
3.86−3.73 (m, 3H), 1.44 ppm (s, 6H). HRMS (ESI) m/z calcd for
C25H25FN2O5S [(M + H)+] 485.1546; found, 485.1531. HPLC: tR =
10.2 min, 97.6%.
3H), 7.36−7.22 (m, 5H), 7.21−7.13 (m, 2H), 7.00 (t, J = 7.3 Hz, 1H),
6.34 (d, J = 10.1 Hz, 1H), 5.83 (t, J = 9.5 Hz, 1H), 4.86 (d, J = 5.9 Hz,
2H), 1.43 ppm (s, 6H). HRMS (ESI) m/z calcd for C29H27N2O3S
[(M + H)+] 483.1742; found, 483.1723. HPLC: tR = 16.6 min, 96.1%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenylbenzo[d][1,3]dioxole-4-sulfonamide (26r). Yield: 65%.
General Procedure for 26k−t. To a solution of 25a (1 equiv) in
pyridine at 0 °C was added the appropriate sulfonyl chloride dropwise.
The mixture was allowed to warm to room temperature overnight. The
reaction mixture was then diluted with EtOAc, and the organic layer
was washed with 10% citric acid, saturated NaHCO3, water, and brine.
The organic layer was dried over MgSO4 and concentrated in vacuo.
The crude product was purified by flash column chromatography
(silica gel).
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenylcyclohexanesulfonamide (26k). Yield: 60%. 1H NMR
(CDCl3): δ 7.30−7.22 (m, 4H), 7.07−7.01 (m, 2H), 6.90 (dd, J =
24.2, 1.8 Hz, 1H), 6.46 (dd, J = 29.3, 2.1 Hz, 1H), 5.86 (d, J = 29.3 Hz,
1H), 5.05 (d, J = 48.2 Hz, 1H), 4.90 (d, J = 48.2 Hz, 1H), 2.13−1.96
(m, 4H), 1.78−1.70 (m, 5H), 1.44 (d, J = 6.3 Hz, 6H), 1.27 ppm (m,
2H). 13C NMR (CDCl3): δ 148.5, 148.3, 143.6, 140.4, 135.2, 129.1,
123.9, 123.8, 123.6, 122.0, 121.8, 92.4, 34.5, 31.8, 28.3, 28.2, 24.7, 21.7,
21.3 ppm. MS (ESI+) m/z [(M + Na)+] 435. HPLC: tR = 10.16 min,
97.6%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenylpropane-2-sulfonamide (26l). Yield: 58%. 1H NMR
(CDCl3): δ 7.37−7.19 (m, 4H), 7.16−6.99 (m, 2H), 6.93 (t, J = 9.4
Hz, 1H), 6.46 (dd, J = 10.1, 0.5 Hz, 1H), 5.87 (d, J = 10.1 Hz, 1H),
4.92 (dd, J = 36.5, 16.5 Hz, 2H), 1.81 (s, 3H), 1.75 (d, J = 11.8 Hz,
4H), 1.45 ppm (t, J = 5.8 Hz, 6H). 13C NMR (CDCl3): δ 148.5, 148.2,
143.1, 140.5, 135.3, 129.1, 124.2, 123.7, 123.6, 122.2, 122.1, 86.0, 28.3,
28.2, 27.4, 25.4 ppm. MS (ESI+) m/z [(M + H)+] 373. HPLC: tR =
10.0 min, 97.8%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenylcyclopropanesulfonamide (26m). Yield: 46%. 1H NMR
(CDCl3): δ 7.44−7.42 (m, 2H), 7.34−7.23 (m, 4H), 6.99 (d, J = 8 Hz,
1H), 6.40 (d, J = 10, 1H), 5.85 (dd, J = 10, 1H), 4.98 (s, 2H), 2.55 (m,
1H), 1.44 (d, J = 16.1 Hz, 6H), 1.13−1.11 (m, 2H), 0.98−0.95 ppm
(m, 2H). 13C NMR (CDCl3): δ 148.7, 148.1, 140.4, 139.7, 135.3,
129.1, 128.8, 127.7, 123.7, 123.6, 122.4, 56.2, 28.6, 28.2, 5.16 ppm.
HPLC: tR = 8.31 min, 95.8%. HRMS (ESI) m/z calcd for
C20H22N2O3S [(M + Na)+] 393.1212; found, 393.1231. HPLC: tR =
10.1 min, 98.3%.
1
Mp 187 °C. H NMR (CDCl3): δ 7.35−7.21 (m, 4H), 7.21−7.12
(m, 3H), 7.08 (t, J = 3.9 Hz, 1H), 7.02−6.93 (m, 1H), 6.88−6.78 (m,
1H), 6.36 (dd, J = 10.1, 0.5 Hz, 1H), 6.09 (s, 2H), 5.84 (dd, J = 14.0,
9.0 Hz, 1H), 4.82 (s, 2H), 1.68 (s, 2H), 1.43 ppm (s, 6H). HRMS
(ESI) m/z calcd for C24H23N2O5S [(M + H)+] 451.1328; found,
451.1316. HPLC: tR = 10.7 min, 99.7%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenylquinoline-8-sulfonamide (26s). Yield: 65%; mp 149 °C.
1H NMR (CDCl3): δ 9.19 (dd, J = 4.2, 1.8 Hz, 1H), 8.43−8.16 (m,
2H), 8.00 (dt, J = 10.5, 5.3 Hz, 1H), 7.66−7.56 (m, 2H), 7.54−7.47
(m, 1H), 7.14−6.94 (m, 6H), 6.40 (d, J = 10.1 Hz, 1H), 5.85 (t, J =
15.1 Hz, 1H), 5.58 (d, J = 35.1 Hz, 2H), 1.45 ppm (s, 6H). 13C NMR
(CDCl3): δ 151.3, 150,0, 148.5, 144.2, 140.1, 139.6, 137.1, 136.5,
135.1, 133.7, 133.5, 128.8, 128.8, 128.2, 127.2, 125.4, 123.9, 123.8,
122.4, 122.1, 77.4, 77.1, 76.9, 76.7, 58.8, 28.2 ppm. HRMS (ESI) m/z
calcd for C26H24N3O3S [(M + H)+] 485.1538; found, 485.1543.
HPLC: tR = 10.0 min, 96.9%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenyl-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide
1
(26t). Yield: 57%. Mp 131 °C. H NMR (CDCl3): δ 1.419 (s, 6H),
4.33−4.28 (m, 2H), 4.81 (s, 2H), 5.82 (d, 1H, J = 10), 6.35 (d, 1H, J =
10), 6.89 (d, 1H, J = 8.4), 6.97 (d, 1H, J = 8.4), 7.06 (dd, 1H), 7.12
−7.15 (m, 2H), 7.20−7.30 ppm (m, 5H). 13C NMR (CDCl3): δ 148.6,
147.7, 147.5, 143.4, 140.2, 139.4, 135.2, 130.4, 128.8, 128.5, 127.6, 123.7,
123.6, 122.4, 121.6, 117.4, 117.4, 77.4, 77.1, 77.0, 76.7, 64.6, 64.1, 60.4,
55.7, 28.2, 21.1, 14.2 ppm. HRMS (ESI) m/z calcd for C15H15N2O5S
[(M + H)+] 465.1484; found, 465.1489. HPLC: tR = 10.9 min, 98.2%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-3,4-
dimethoxy-N-phenylbenzamide (27). To a solution of 25a
(60 mg, 0.226 mmol) in DCM (3 mL) were added triethylamine
(0.06 mL, 0.452 mmol) and 3,4-dimethoxybenzoyl chloride (54 mg,
0.271 mmol). The mixture was stirred overnight at room temperature.
The reaction mixture was washed with H2O (×2) and saturated
NaHCO3 (×2), dried over MgSO4, and concentrated in vacuo.
Purification by column chromatography with silica gel and 3:1 DCM/
EtOAc gave a quantitative yield of a light yellow oil. 1H NMR
(CDCl3): δ 7.26−7.17 (m, 3H), 7.17−7.08 (m, 3H), 7.06−6.97 (m,
2H), 6.95 (t, J = 5.0 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H), 6.53−6.40 (m,
1H), 5.92−5.79 (m, 1H), 5.16 (s, 2H), 3.83 (s, 3H), 3.66 (s, 3H), 1.46
ppm (s, 6H). 13C NMR (CDCl3): δ 169.9, 150.3, 149.0, 148.6, 147.9,
144.7, 140.6, 135.1, 129.0, 127.8, 127.2, 126.3, 124.0, 123.6, 123.0,
122.3, 112.6, 109.9, 77.4, 77.0, 77.0, 76.7, 55.8, 55.7, 55.6, 28.2 ppm.
HRMS (ESI) m/z calcd for C26H27N2O4 [(M + H)+] 431.1971; found,
431.1951.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenylbutane-1-sulfonamide (26n). Yield: 48%. 1H NMR
(CDCl3): δ 7.41−7.29 (m, 4H), 7.27−7.22 (m, 1H), 7.18 (d, J =
8.3 Hz, 1H), 7.00 (dd, J = 12.4, 5.5 Hz, 1H), 6.41 (d, J = 10.1 Hz, 1H),
5.86 (d, J = 10.1 Hz, 1H), 4.94 (s, 2H), 3.38−2.79 (m, 2H), 1.96−1.77
(m, 2H), 1.54−1.38 (m, 8H), 1.12−0.77 ppm (m, 3H). 13C NMR
(CDCl3): δ 170.85, 148.78, 148.19, 139.60, 135.37, 129.28, 128.34,
127.62, 123.68, 123.62, 122.54, 56.25, 51.28, 28.22, 25.29, 21.68, 13.61
ppm. MS (ESI) m/z [(M + H)+] 387. HPLC: tR = 8.3 min, 95.8%.
N-((2,2-Dimethyl-2H-pyrano[3,2-b]pyridin-6-yl)methyl)-N-
phenylpropane-1-sulfonamide (26o). Yield: 20%. Mp 120 °C. 1H
NMR (CDCl3): δ 7.40−7.21 (m, 6H), 7.17 (d, J = 8.3 Hz, 1H), 6.98
(t, J = 7.2 Hz, 1H), 6.40 (d, J = 10.1 Hz, 1H), 5.85 (d, J = 10.1 Hz,
1H), 4.93 (s, 2H), 3.20−2.99 (m, 2H), 1.98−1.79 (m, 2H), 1.43 (s,
2-Bromo-6-(hydroxymethyl)pyridin-3-ol (30). A solution of
2-bromo-3-hydroxy-6-methylpyridine 1-oxide 29 (15 g, 0.075 mol) in
TFAA (50 mL, 0.375 mol) was stirred at 40 °C for 24 h. The solvent
was removed under vacuum. The residue was purified by column
chromatography (silica gel, EtOAc/hexane, 2:1). Yield: 4.5 g, 30%.
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1H NMR (CDCl3): δ 7.32 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 8.5 Hz,
1H), 4.56 (s, 2H).
1.27−1.16 ppm (m, 5H). 13C NMR (CD3OD): δ 151.5, 148.3, 136.7,
136.5, 128.7, 119.9, 119.0, 77.00, 56.0, 50.3, 32.4, 26.8, 25.8, 24.7 ppm.
General Procedure for Synthesis of 36. A mixture of
compound 35 (1 equiv) and the appropriate sulfonyl chloride
(2 equiv) in pyridine was stirred overnight at room temperature.
Then 1 M HCl was added to the reaction mixture and the solution
extracted with ethyl acetate (3 × 15 mL). The combined organic layers
were washed with water (3 × 20 mL), brine and dried over Na2SO4.
The solvent was removed under vacuum and the residue purified by
column chromatography (silica gel, EtOAc/hexane, 1:4).
N-((2,2-Dimethyl-2H-pyrano[2,3-c]pyridin-6-yl)methyl)-4-
methoxy-N-phenylbenzenesulfonamide (36a). Yield: 50%. 1H
NMR (CD3OD): δ 7.76 (s, 1H), 7.59−7.56 (m, 2H), 7.25−7.18 (m,
3H), 7.09−7.04 (m, 4H), 6.37 (d, J = 10 Hz, 1H), 5.97 (d, J = 10 Hz,
1H), 4.88 (s, 3H), 4.78 (s, 2H), 3.084 (s, 3H), 1.40 ppm (s, 6H). 13C
NMR (CD3OD): 163.5, 148.6, 139.3, 136.7, 136.1, 129.7, 129.3, 128.8,
128.5, 128.5, 127.6, 119.7, 119.4, 113.9, 77.1, 55.0, 54.9, 26.8 ppm. MS
(ESI) m/z [(M + H)+] 437. HPLC: tR = 9.6 min, 97.8%.
N-((2,2-Dimethyl-2H-pyrano[2,3-c]pyridin-6-yl)methyl)-4-
nitro-N-phenylbenzenesulfonamide (36b). Yield: 59%. 1H NMR
(CD3OD): δ 8.41 (dd, J = 2.0 Hz, 4.8 Hz, 2H), 7.89 (dd, J = 2.0 Hz,
4.8 Hz), 7.78 (s, 1H), 7.29−7.28 (m, 4H), 7.10−7.08 (m, 2H), 6.39
(d, J = 10 Hz, 1H), 6.00 (d, J = 10 Hz, 1H), 1.42 ppm (s, 6H). MS
(ESI) m/z [(M + H)+] 452. HPLC: tR = 9.03 min, 95.2%.
N-Cyclohexyl-N-((2,2-dimethyl-2H-pyrano[2,3-c]pyridin-6-
yl)methyl)-4-isopropylbenzenesulfonamide (36c). Yield: 28%.
1H NMR (CDCl3): δ 7.98 (s, 1H), 7.77−7.75 (m, 2H), 7.37−7.34 (m,
(6-Bromo-5-(2-methylbut-3-yn-2-yloxy)pyridin-2-yl)-
methanol (31). Compound 30 (1.02 g, 5 mmol) was dissolved in
acetone (20 mL. Then K2CO3 (166 mg, 7 mmol), KI (33.2 mg,
0.2 mmol), and CuCl2.2H2O (33.2 mg, 0.02 mmol) were added. The
suspension was stirred at 60 °C for 10 min. The solution of 3-chloro-3-
methyl-2-butyne (1.02 g, 5 mmol) in acetone (5 mL) was added
dropwise to the solution of 30. The reaction mixture was cooled to
room temperature and the suspension filtered. The solid residue was
washed with MeOH. The filtrate was concentrated under vacuum and
purified with column chromatography (silica gel, EtOAc/hexane, 1:1).
1
Yield: 700 mg, 57%. H NMR (CDCl3): δ 7.88 (d, J = 8.0 Hz, 1H),
7.23 (d, J = 8.0 Hz, 1H), 4.72 (s, 2H), 1.73 ppm (s, 6H). 13C NMR
(CDCl3): δ 154.7, 149.6, 137.03, 129.5, 120.4, 85.7, 75.8, 75.7, 64.6,
30.1 ppm.
(8-Bromo-2,2-dimethyl-2H-pyrano[2,3-c]pyridin-6-yl)-
methanol (32). A solution of 31 (700 mg, 2.5 mmol) in toluene
(10 mL) was subjected to microwave irradiation (200 W, 120 °C) for
1 h. The reaction mixture was cooled to room temperature and
filtered. The filtrate was concentrated under vacuum and the residue
purified by column chromatography (silica gel, EtOAc/hexane, 1:3 to
1:2). Yield: 500 mg, 70%. 1H NMR (CDCl3): δ 6.88 (s, 1H), 6.28 (d,
J = 10 Hz, 1H), 5.90 (d, J = 9.6 Hz, 1H), 4.63 (s, 2H), 1.51 ppm (s,
6H).
8-Bromo-6-(bromomethyl)-2,2-dimethyl-2H-pyrano[2,3-c]-
pyridine (33). To a solution of 32 in DCM (2 mL) was added CBr4
(66 mg, 0.2 mmol) and PPh3 (264 mg, 0.2 mmol). The reaction
mixture was stirred at room temperature for 1 h. The solvent was
removed under vacuum and the residue purified by column
chromatography (silica gel, EtOAc/hexane, 1:4). Yield: 260 mg,
40%. 1H NMR (CDCl3): δ 6.28 (d, J = 10 Hz, 1H), 5.91 (d, J = 10 Hz,
1H), 4.47 (s, 2H), 1.53 ppm (s, 6H).
General Procedure for the Synthesis of 34. To a degassed
flask with 33 (1 equiv) were added DMF, aniline (1.5 equiv), and
DIEA (1.5 equiv). The mixture was stirred at room temperature
overnight. Water (50 mL) was added to the reaction mixture, and the
resulting solution was extracted with ethyl acetate (3 × 25 mL). The
combined organic layers were washed with 0.5 N HCl (50 mL), 40%
NaHCO3 (50 mL), water (50 mL), and brine, dried over Na2SO4, and
concentrated under vacuum. The residue was purified by column
chromatography (silica gel).
N-((8-Bromo-2,2-dimethyl-2H-pyrano[2,3-c]pyridin-6-yl)-
methyl)benzenamine 34a. Yield: 78%. 1H NMR (CDCl3): δ
7.26−7.21 (m, 3H), 6.97−6.95 (m, 3H), 6.23 (d, J = 9.6 Hz, 1H), 5.85
(d, J = 9.6 Hz, 1H), 4.40 (s, 2H), 1.48 ppm (s, 6 H).
1H), 7.28 (d, J = 8.8 Hz, 1H), 6.34 (d, J = 9.6 Hz, 1H), 5.84 (d, J = 10
Hz, 1H), 4.42 (s, 2H), 3.80 (m, 1H), 2.99 (m, 1H), 1.75−1.59 (m,
3H), 1.55−1.39 (m, 9 H), 1.29−1.27 (m, 6H), 1.23 −1.19 ppm (m,
4H). 13C NMR (CDCl3): δ 153.8, 151.8, 148.1, 138.7, 136.7, 135.8,
128.1, 127.1, 127.3, 120.9, 118.9, 58.5, 48.7, 34.1, 28.0, 26.1, 25.1, 23.7
ppm. MS (ESI) m/z [(M + H)+] 455. HPLC: tR = 18.6 min, 98.2%.
N-Cyclohexyl-N-((2,2-dimethyl-2H-pyrano[2,3-c]pyridin-6-
yl)methyl)-3,4-dimethoxybenzenesulfonamide (36d). Yield:
1
28%. H NMR (CDCl3) δ 8.00 (s, 1H), 7.50−7.47 (m, 1H), 7.33−
7.29 (m, 2H), 6.94 (d, J = 8.4 Hz, 1H), 6.36 (d, J = 9.6 Hz, 1H), 5.86
(d, J = 9.6 Hz, 1H), 5.32 (s, 2H), 3.967 (s, 3H), 3.94 (s, 3H), 3.97 (m,
1H), 1.68 −1.52 (m, 4H), 1,48 (s, 6H), 1.28−1.20 ppm (m, 4H). 13C
NMR (CDCl3): δ 151.7, 149.1, 136.7, 135.9, 133.2, 128.1, 120.8,
120.7, 118.9, 110.6, 109.5, 83.1, 58.5, 56.2, 56.2, 48.6, 31.4, 28.0, 26.1,
25.1 ppm. MS (ESI) m/z [(M + H)+] 473. HPLC: tR = 10.3 min,
97.3%.
AUTHOR INFORMATION
■
N-((8-Bromo-2,2-dimethyl-2H-pyrano[2,3-c]pyridin-6-yl)-
methyl)cyclohexanamine (34b). Yield: 60%. 13C NMR (CD3OD):
δ 152.0, 145.2, 136.9, 129.7, 129.1, 119.8, 118.7, 78.5, 56.0, 49.8, 32.4,
26.8, 25.8, 24.7 ppm.
Corresponding Author
*For E.G.V.M.: telephone, 1-404-778-5563; e-mail, evanmei@
emory.edu. For B.W.: telephone, 1-404-413-5544; e-mail,
wang@gsu.edu.
General Procedure for the Synthesis of Compound 35. A
flask of secondary amine 34 (1 equiv) was degassed, and THF
(anhydrous) was added under nitrogen. The solution was cooled to
−78 °C and stirred for 1 h. BuLi (2.5 equiv) was the added to the
solution dropwise at −78 °C. The resulting solution was stirred for
1 h. Water (10 mL) was added to the solution, which was diluted with
ethyl acetate (25 mL). After separation, the aqueous layer was
extracted with ethyl acetate and washed with water (25 mL × 3) and
brine (25 mL), dried over Na2SO4, and concentrated under vacuum.
The residue was purified by column chromatography (silica gel,
EtOAc/hexane, 1:4)
ACKNOWLEDGMENTS
■
Financial support from the National Institutes of Health (Grant
R01CA116804 to E.V.G.M.), V Foundation (to E.V.G.M), and
an associated minority supplement (Grant R01CA116804-S1)
and a fellowship from the Georgia State University Molecular
Basis of Diseases Program (to S.R.M.) is gratefully acknowl-
edged. The authors thank Sarah Burroughs for her help with
the submission of this manuscript
N-((2,2-Dimethyl-2H-pyrano[2,3-c]pyridin-6-yl)methyl)-
1
benzenamine (35a). Yield: 70%. H NMR (CD3OD): δ 7.91 (s,
1H), 7.08−7.04 (m, 3H), 6.59−6.57 (m, 3H), 6.28 (d, J = 9.6 Hz,
1H), 5.92 (s, J = 10 Hz, 1H), 4.88 (s, 2H), 1.41 ppm (s, 6H). 13C
NMR (CD3OD): δ 152.3, 148.3, 148.2, 136.7, 135.2, 128.9, 128.7,
119.9, 117.8, 116.8, 112.6, 76.9, 26.8 ppm.
ABBREVIATIONS USED
■
AP, alkaline phosphatase; CBP, CREB-binding protein; HIF,
hypoxia inducible factor; HRE, hypoxia-responsive element;
ODDD, oxygen-dependent degradation domain; PHD, prolyl
hydroxylase; VEGF, vascular endothelial growth factor; pVHL,
von Hippel−Lindau protein
N-((2,2-Dimethyl-2H-pyrano[2,3-c]pyridin-6-yl)methyl)-
1
cyclohexanamine (35b). Yield: 50%. H NMR (CD3OD): δ 7.95
(s, 1H), 7.08 (s, 1H), 6.44 (d, J = 9.6 Hz, 1H), 6.03 (d, J = 10 Hz,
1H), 3.78 (s, 2H), 2.46 (m, 1H), 1.97−1.75 (m, 5 H), 1.46 (s, 6H,
8487
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