Synthesis and structure-activity relationship study of chemical probes as hypoxia induced factor-1α/malate dehydrogenase 2 inhibitors

Article abstract of DOI:10.1021/jm501241g

A structure-activity relationship study of hypoxia inducible factor-1α inhibitor 3-aminobenzoic acid-based chemical probes, which were previously identified to bind to mitochondrial malate dehydrogenase 2, was performed to provide a better understanding of the pharmacological effects of LW6 and its relation to hypoxia inducible factor-1α (HIF-1α) and malate dehydrogenase 2 (MDH2). A variety of multifunctional probes including the benzophenone or the trifluoromethyl diazirine for photoaffinity labeling and click reaction were prepared and evaluated for their biological activity using a cell-based HRE-luciferase assay as well as a MDH2 assay in human colorectal cancer HCT116 cells. Among them, the diazirine probe 4a showed strong inhibitory activity against both HIF-1α and MDH2. Significantly, the inhibitory effect of the probes on HIF-1α activity was consistent with that of the MDH2 enzyme assay, which was further confirmed by the effect on in vitro binding activity to recombinant human MDH2, oxygen consumption, ATP production, and AMP activated protein kinase (AMPK) activation. Competitive binding modes of LW6 and probe 4a to MDH2 were also demonstrated.

Full text of DOI:10.1021/jm501241g

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationship Study of Chemical
Probes as Hypoxia Induced Factor-1α/Malate Dehydrogenase 2
Inhibitors
Ravi Naik,†,∥ Misun Won,‡,∥ Hyun Seung Ban,‡,∥ Deepak Bhattarai,† Xuezhen Xu,† Yumi Eo,†
Ye Seul Hong,‡ Sarbjit Singh,§ Yongseok Choi,§ Hee-Chul Ahn,† and Kyeong Lee*,†
†BK21Plus R-FIND Team, College of Pharmacy, Dongguk University-Seoul, Koyang, 410-820, Korea
‡Korea Research Institute of Bioscience and Biotechnology, BioMedical Genomics Research Center, Daejeon 305-806, Korea
§College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
S
* Supporting Information
ABSTRACT: A structure−activity relationship study of
hypoxia inducible factor-1α inhibitor 3-aminobenzoic acid-
based chemical probes, which were previously identified to
bind to mitochondrial malate dehydrogenase 2, was performed
to provide a better understanding of the pharmacological
effects of LW6 and its relation to hypoxia inducible factor-1α
(HIF-1α) and malate dehydrogenase 2 (MDH2). A variety of
multifunctional probes including the benzophenone or the
trifluoromethyl diazirine for photoaffinity labeling and click
reaction were prepared and evaluated for their biological
activity using a cell-based HRE-luciferase assay as well as a
MDH2 assay in human colorectal cancer HCT116 cells. Among them, the diazirine probe 4a showed strong inhibitory activity
against both HIF-1α and MDH2. Significantly, the inhibitory effect of the probes on HIF-1α activity was consistent with that of
the MDH2 enzyme assay, which was further confirmed by the effect on in vitro binding activity to recombinant human MDH2,
oxygen consumption, ATP production, and AMP activated protein kinase (AMPK) activation. Competitive binding modes of
LW6 and probe 4a to MDH2 were also demonstrated.
Generally, affinity chromatography using biotinylated drugs
and the subsequent proteomic analysis of isolated proteins is
considered to be a common and powerful method. Although
the biotinylation of active compounds is relatively simple and
many drug targets have been successfully identified,1,2 this
method still has limitations due to the nonspecific protein
binding of hydrophobic molecules.
INTRODUCTION
■
Target identification of drugs is crucial for understanding their
molecular modes of action and to search for clinical biomarkers
in therapeutics. The target identification process of bioactive
small molecules has become a bottleneck in drug discovery,
since many novel therapeutic agents have been developed by
cell-based phenotypic screening, not by target-based in vitro
assay. Thus far, various chemical and biological technologies,
such as affinity chromatography, fluorescent imaging, proteo-
mic analysis, phase display biopanning, drug affinity responsive
target stability, and phenotype analysis using mutated or
overexpressed yeast and zebrafish, have been developed.1,2 On
the basis of recent significant developments in fluorescent
imaging of wide spectra and real-time observation of living cells,
chemical biology approach-driven probes have attracted much
attention in the field of drug discovery. Chemical probes of
small, bioactive molecules that can react rapidly with target
proteins are directly applicable for target identification in most
living cells without genetic manipulation. Especially, chemical
probes for biotin−streptavidin pull-down assay, activity-based
assay (ABPs), photoaffinity labeling, and fluorescence imaging
are the most frequently employed for target identification
studies of small molecules.3,4
Of note, photoaffinity labeling can be applicable for covalent
linkages between target proteins and small molecules, resulting
in the selective isolation of target proteins.5 Click chemistry for
fluorescent imaging is also useful when visualizing the
intracellular localization of the small molecules and to track
target proteins.5 On the basis of these techniques, a differential
fluorescence approach in two-dimensional gel electrophoresis
(2-DE) was employed for target identification using multifunc-
tional chemical probes containing functionalities for click
reaction and photolabeling.5−8 Figure 1 represents the
structures of the multifunctional chemical probes for target
identification, including 1a−b, 2, 3, and 4a−b.5−8
Hypoxia inducible factor 1 (HIF-1) is a heterodimeric
transcription factor that functions as a mast regulator in the
Received: August 13, 2014
Published: October 30, 2014
© 2014 American Chemical Society
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Figure 1. Chemical probes used for target identification.
a
Table 1. In Vitro HIF-1α and MDH2 Inhibitory Activity of LW6 and Acetylene Chemical Probes 4e and 4f
a
Values are the means of three experiments.
response of tumor growth to hypoxia.9 A high level of HIF-1α
subunit correlates with aggressive tumor growth, resistance to
radiation and chemotherapy, and poor clinical outcomes.10−13
We previously reported the synthesis and biological evaluation
of novel HIF-1α inhibitors.14−18 LW6, an aryloxyacetylamino
benzoic acid analogue, exhibited a potent inhibitory effect on
HIF-1α accumulation and target gene expression under hypoxia
(Table 1).14,19
Since the regulation pathway of HIF-1α expression is
complicated,20 it is challenging to understand the mechanism
of LW6 without identifying its direct target protein. Recently,
we have demonstrated that the target molecule of LW6 could
be mitochondrial malate dehydrogenase 2 (MDH2) in the
TCA cycle using chemical probes.7 Herein, we report the
synthesis and biological evaluation of a series of multifunctional
chemical probes containing functional groups including
acetylene, benzophenone, or trifluoromethyl diazirine, at
various sites of the parent compound LW6. The relevance of
HIF-1α inhibition to MDH2 inhibition and oxygen con-
sumption was also investigated using the probes to understand
their structure−activity relationships. In addition, ATP
production, AMPK signaling, and MDH2 kinetic assay were
carried out for the representative compounds.
RESULTS AND DISCUSSION
■
Chemistry. A series of chemical probes of LW6 were
synthesized as shown in Schemes 1−6. Chemical probes 4c and
4d carrying structural units, such as biotin reporter groups,
could be readily prepared from 6a or 6b, whose syntheses were
previously reported from 5a and 5b.14 Compound 6a was then
O-alkylated with (2-{2-[2-(2-chloro-acetylamino)-ethoxy]-
ethoxy}-ethyl)-carbamic acid tert-butyl ester under basic
conditions, to yield an adamantyl derivative with linker 7a.
This derivative was further subjected for Boc deprotection to
afford 8a. Biotinylation of 8a with (+)-biotin N-hydroxysucci-
nimide ester resulted in the desired biotin probe 4c. The
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a
Scheme 1. Synthesis of Biotin Chemical Probes 4c and 4d
a
Reagents and Conditions: (a) Methyl-3-amino-4-hydroxybenzoate, HBTU, DIPEA, DMF, rt, 12 h for 6a, 78.9%; 2-aminophenol, HBTU, DIPEA,
DMF, rt, 12 h for 6b, 76%; (b) K2CO3, Cs2CO3, KI, acetone/DMF, (2-{2-[2-(2-chloro-acetylamino)-ethoxy]-ethoxy}-ethyl)-carbamic acid tert-butyl
ester, 60 °C, 36 h, 43.8% for 7a and 58.4% for 7b; (c) TFA, DCM, rt, 1 h; (d) TEA, DMF, (+)-biotin N-hydroxysuccinimide ester, rt, 12 h, 33.3% for
4c and 66.6% for 4d.
a
Scheme 2. Synthesis of Acetylene Chemical Probes 4e and 4f
a
Reagents and Conditions: (a) Thionyl chloride, MeOH, 0 to 60 °C, 4 h, 93%; (b) p-methoxy benzyl chloride, K2CO3, KI, acetone, 0 to 60 °C, 12 h,
88.5%; (c) LiOH·H2O, THF/H2O, rt, 12 h, 92.1%; (d) propargyl bromide, K2CO3, DMF, 0 °C to rt, 12 h, 93.1%; (e) TFA, DCM, 0 °C to rt, 4 h,
90.2%; (f) NH4Cl, Zn, THF/H2O/MeOH, 70 °C, 3 h, 88.4%; (g) EDC·HCl, HOBt, DIPEA, DMF, 4-(1-adamantyl)-phenoxy acetic acid (5a) for
4e, rt, 12 h, 68.7%; and phenoxyacetic acid (5b) for 4f, rt, 12 h, 76.2%.
negative analogue 4d was prepared starting from 6b in a similar
manner (Scheme 1).
reduction of the ester 13 gave a key intermediate 15. Coupling
15 with 4-(1-adamantyl)-phenoxy acetic acid 5a, which was
readily prepared by our previously reported method, provided
probe 4e. Similarly, the corresponding negative control probe
4f was prepared by coupling 15 with a commercially available
phenoxyacetic acid (Scheme 2).14
As shown in Scheme 3, benzophenone-based multifunctional
probes were also prepared in four steps. O-Alkylation of phenol
with propargyl bromide afforded a phenyl propargyl ether
derivative 18. Friedel−Crafts acylation of 18 using m-
For the cellular localization study, chemical probes 4e and 4f,
in which a carboxylic acid functional group was replaced by an
acetylene moiety for click conjugation, were synthesized as
shown in Scheme 2. 3-Nitro-4-hydroxy benzoic acid was
esterified and protected with p-methoxybenzyl chloride to give
an ester derivative 11. Hydrolysis and O-alkylation with
propargyl bromide of 11 afforded a propargyl ester 13.
Subsequent p-methoxybenzyl deprotection and metal catalyzed
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a
Scheme 3. Synthesis of Benzophenone Chemical Probes 4g and 4h
a
Reagents and Conditions: (a) K2CO3, DMF, rt, 12 h, 71.4%; (b) AlCl3, m-nitrobenzoyl chloride, DCM, −10 °C to rt, 12 h, 46.7%; (c) NH4Cl, Fe,
THF/H2O/MeOH, 70 °C, 2 h, 75.4%; (d) EDC, HOBt, DIPEA, DMF, 4-(1-adamantyl)-phenoxy acetic acid for 4g, rt, 12 h, 61.1%; and 4-(1-
adamantyl)-phenoxyacrylic acid for 4h, rt, 12 h, 61.8%.
a
Scheme 4. Synthesis of Key Intermediate 30
a
Reagents and Conditions: (a) NH2OH·HCl, pyridine, 115 °C, 4 h, 80.3%; (b) p-TsCl, DMAP, Et3N, DCM, 0 °C to rt, 8 h, 84%; (c) NH3, Et2O,
−78 °C to rt, 12 h, 82.8%; (d) MnO2, CCl4, 0 °C to rt, 1 h, 50.5%; (e) NBS, benzoyl peroxide, CCl4, 77 °C, 2 h, 45.4%; (f) AlCl3, anisole, −10 °C to
rt, 24 h, 86%; (g) BBr3, DCM, 0 °C to rt, 2 h, 90%; (h) K2CO3, DMF, 40 °C, 4 h, 25.7%.
nitrobenzoyl chloride yielded (3-nitrophenyl)(4-(prop-2-
ynyloxy)phenyl)methanone 19.21 Selective reduction of the
nitro group in the presence of iron powder and acetic acid led
to the formation of a key intermediate 20. Coupling 20 with 4-
(1-adamantyl)-phenoxy acetic acid or 4-(1-adamantyl)-phenox-
yacrylic acid, which were readily prepared by our previously
reported method, gave benzophenone chemical probes 4g and
4h, respectively.14,18
For the synthesis of the trifluoromethyl diazirine probes 4i
and 4j, a key intermediate 30 was prepared by multistep
synthesis, as described in Scheme 4. Bromotrifluoromethyl
diazirine 26 was synthesized starting from commercially
available 2,2,2-trifluoro-1-p-tolylethanone by slightly modifying
the reported procedure.22−24 Compound 21 was converted to
an oxime derivative 22 by condensation with hydroxylamine
HCl in pyridine. O-Tosylation of 22 with p-toluenesulfonyl
chloride led to the formation of a tosylated oxime 23, which
later reacted with liquid ammonia in a sealed tube to yield a
diaziridine derivative 24 via elimination and subsequent
cyclization. Oxidation of 24 with MnO2 in CCl4 afforded a
diazirine 25. A key precursor 26 was then obtained by
bromination of 25 with N-bromosuccinimide (NBS) using
radical initiator benzoyl peroxide as a catalyst. Selective O-
alkylation of 26 with 3-(4-hydroxyphenyl)-adamantane-1-
carboxylic acid (29), which was readily prepared by our
previously reported method, furnished the key intermediate 30
(Scheme 4).25 The key intermediate 30 was subsequently
alkylated with ethyl chloroacetate to produce 31 in an excellent
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a
Scheme 5. Synthesis of Diazirine Chemical Probes 4i and 4j
a
Reagents and Conditions: (a) K2CO3, ethyl chloroacetate, DMF, rt, 12 h, 62.8%; (b) LiOH, THF/H2O, rt, 12 h, 80%; (c) HBTU, DIPEA, DMF, rt,
12 h, 19.8%; (d) K2CO3, acetone, propargyl bromide, 60 °C to rt, 12 h, 62.1%.
yield. Saponification of 31 under aq. LiOH conditions gave a
carboxylic acid derivative 32 and the subsequent coupling with
methyl prop-2-ynyl 3-amino-4-hydroxybenzoate (15) produced
the photoaffinity trimodular chemical probe 4i. The negative
control probe 4j was obtained by alkylation of 30 with
propargyl bromide under basic conditions, as shown in Scheme
5.
streptoavidin beads. In our initial study, the biotin probe of
LW6, 4c, was designed as the first choice to identify the target
protein for LW6 in human colorectal cancer HCT116 cells
under hypoxic conditions. The biotin probe 4c was prepared
along with the negative control 4d featuring an adamantyl-free
form. When the pull-down assay was performed, followed by
SDS-PAGE26 of copurified proteins with LW6, a number of
protein bands appeared with 4c, while few bands appeared with
the negative control 4d. This result was consistent with our
previous reports, which emphasized the importance of an
adamantyl group for HIF-1α inhibitory activity.14,18 Non-
specific protein or indirect bindings to 4c occurred due to a
hydrophobic group of LW6 under the experimental conditions
(Figure S1, Supporting Information). Even though target
proteins were purified by specific binding with LW6, they were
not selectable due to the limitations of the probes and
methodology. Therefore, we prepared multifunctional probes
by introducing a clickable group and photoreactive moiety at
different sites of LW6.
First, the probes 4e and 4f, in which an acetylene moiety was
installed for click conjugation with the azide linked molecule
(e.g., fluorescent tag), were evaluated for their ability to inhibit
HIF-1α using a cell-based HRE (hypoxia responsive element)-
luciferase assay and Western blot analysis in HCT116 cells
under hypoxia (Tables 1−3, Figure 2). The probe 4e retained
HIF-1α inhibitory activity (IC50 = 4.3 μM) of LW6, whereas
the adamantyl-free probe 4f as a negative control (>30 μM)
completely lost the inhibitory activity, as shown in Table 1 and
Figure 2A. Accordingly, probe 4e was used for the intracellular
localization study of LW6 through fluorescent tagging.7
Another type of multifunctional diazirine probe 4a and its
negative analogue 4b, which can be photolabeled and click-
conjugated, were synthesized in 11 steps (Scheme 6).
Commercially available 4-(1-adamntyl) phenol was subjected
to iodination with potassium iodide to yield monoiodo
derivative 34, which, following methylation, gave 35a. Reaction
of 1-trifluoroacetylpiperidine with an anion derived from 35a
provided trifluoroacetyl derivative 36a. Further, treatment of
36a with hydroxylamine hydrochloride gave oxime derivative
37a, which was tosylated with p-toluenesulfonyl chloride in
order to provide a good leaving group to obtain 38a. Reaction
of 38a with liquid ammonia in a sealed tube led to the
formation of diaziridine derivative 39a via elimination and
subsequent cyclization. Diaziridine 39a was further oxidized in
the presence of iodine to give 40a. Demethylation of 40a by
Lewis acid BBr3 formed 41a, which was further alkylated with
ethyl chloroacetate and subjected to hydrolysis with LiOH to
provide 43a. Coupling 43a with key intermediate 15 led to the
desired multifunctional probe 4a. Meanwhile, the negative
analogue, 4b, was also prepared by the same method, starting
from commercially available 2-iodo anisole 35b, as shown in
Scheme 6.
Biological Evaluation. Biotinylated probes are often used
in the affinity-based isolation of target proteins using
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a
Scheme 6. Synthesis of Diazirine Chemical Probes 4a and 4b
a
Reagents and Conditions: (a) Con:H2SO4, KI, H2O2 (30 wt % solution in water), MeOH, 0 °C to rt, 12 h, 83.8%; (b) K2CO3, MeI, acetone, 0 °C
to rt, 8 h, 97.3%; (c) n-BuLi, 1-trifluoroacetyl piperidine, THF; −78 °C, 12 h, 75.2% for 36a and 72.4% for 36b; (d) NH2OH·HCl, pyridine, 115 °C,
4 h, 70.6% for 37a and 90% for 37b; (e) p-TsCl, DMAP, Et3N, DCM, 0 °C to rt, 8 h, 79.6% for 38a and 84.5% for 38b; (f) NH3, Et2O, −78 °C to rt,
16 h, 81.7% for 39a and 85.7% for 39b; (g) I2, Et3N, MeOH, 0 °C to rt, 3 h, 94.3% for 40a and 86.9% for 40b; (h) BBr3, DCM, −10 °C to rt, 2 h,
44.7% for 41a and 37.4% for 41b; (i) ethyl chloroacetate, K2CO3, DMF, rt, 12 h, 48.4% for 42a and 33.3% for 42b; (j) LiOH·H2O, THF/H2O, rt, 12
h, 55.5% for 43a and 74% for 43b; (k) EDC·HCl, HOBt, DIPEA, DMF, rt, 12 h, 18.2% for 4a and 22.2% for 4b.
Figure 2. Effects of probes on hypoxia-induced HIF-1α accumulation. HCT116 cells were incubated for 6 h in the presence of probes (10 μM). (A)
Acetylene chemical probes. (B) Benzophenone chemical probes. (C) Diazirine chemical probes. The protein levels of HIF-1α and β-actin were
detected by immunoblot analysis with specific antibodies.
In addition to an acetylene moiety, compounds 4g and 4h
bearing a benzophenone moiety for covalent attachment to the
target protein were synthesized as the multifunctional chemical
probes. Since benzophenone generates highly reactive radical
intermediates upon photolysis, it is often used for photoaffinity
labeling.5,6 However, inhibition of HIF-1α activities of 4g and
4h were barely detectable in both the HRE-luciferase assay
(Table 2) and the Western blot analysis (Figure 2B). The
introduction of a benzophenone group to LW6 (4g) resulted in
complete loss of HIF-1α inhibitory activity. Replacement of the
oxyacetylamide linker portion with an oxyacrylic amide linker
(4h) also failed to retain activity (Table 2). We observed that
elongation of the molecule or the addition of both steric
bulkiness and rigidity led to the loss of HIF-1α inhibitory
activity.
photoactivity at long wavelengths, good chemical stability, rapid
photolysis, and a smaller size in comparison with benzophe-
none. For example, a trifluoromethyldiazirine group was
introduced to the adamantyl group with a benzyl ester linkage
to provide 4i (Table 3). Although probe 4i showed moderate
HIF-1α inhibitory activity (IC50 = 11.4 μM) in comparison
with LW6, this result proved the efficiency of the diazirine
moiety over benzophenone for LW6. However, probe 4j, in
which the amide linkage was removed and keeps the acetylene
moiety on the phenoxy group, showed no effect on inhibitory
activity. This result led to the understanding that a NH-phenyl
group is crucial for the inhibition of HIF-1α (Table 3 and
Figure 2C). In contrast, compound 4a wherein trifluoromethyl
diazirine was introduced directly to the phenyl ring of LW6 was
found to maintain the potency (IC50 = 4.7 μM), as shown in
the HRE-luciferase assay (Table 3) and Western blot of HIF-1α
(Figure 2C). The negative control, adamantyl-free probe 4b,
did not show inhibitory activity.
In order to explore the feasibility of different photoaffinity
groups, we utilized a photoreactive diazirine moiety which
generates highly reactive carbene species upon photolysis.
Trifluoro diazirine has several valuable characteristics, including
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Next, we confirmed the relation between HIF-1α and MDH2
inhibitory activities of the chemical probes derived from LW6
in the study. The MDH2 inhibitory activities of the probes
were determined using isolated MDH2 from HCT116 cells
treated with the chemical probes, as shown in Tables 1−3. LW6
(IC50 = 6.3 μM) was used as a reference compound for a
Table 2. In Vitro HIF-1α and MDH2 Inhibitory Activity of
Benzophenone Chemical Probes 4g and 4h
a
comparison. As expected, 4e (IC50 = 7.1 μM) and 4a (IC50
=
5.5 μM) showed similar MDH2 inhibitory activities. Com-
pound 4i (IC50 = 10.2 μM) exhibited moderate MDH2
inhibition. However, the negative controls (4b, 4f, and 4j)
showed negligible MDH2 inhibition activities (IC50 >20 μM).
Of note, the probes containing benzophenone (4g and 4h) did
not show any significant effect on MDH2 activities.
We further examined in vitro binding activities of the probes
to recombinant human MDH2 (rhMDH2) through photo-
affinity labeling and click reaction (Figure 3). The fluorescent
a
Values are the means of three experiments.
Encouraged by these results, we performed a target
identification experiment using 4e and 4a.7 The compound
4e conjugated with a fluorescent tag, alexa 488, was localized in
mitochondria, suggesting that LW6 may bind to mitochondrial
protein. Finally, malate dehydrogenase 2 (MDH2) of the
mitochondrial TCA cycle was identified as a target of LW6
using multifunctional probe 4a through photoaffinity con-
jugation and separation of target proteins by 2D PAGE.7
Figure 3. In vitro binding of chemical probes to recombinant human
MDH2 (rhMDH2, 2 μg). The rhMDH2 was incubated with probes
(10 μM) and then irradiated with UV (360 nm) for photoaffinity
labeling of MDH2. Click reactions of probes with azide-Cy3 were
performed to visualize probe−MDH2 binding.
a
Table 3. In Vitro HIF-1α and MDH2 Inhibitory Activity for Diazirine Chemical Probes 4i, 4j, 4a, and 4b
a
Values are the means of three experiments.
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bands, complexes of rhMDH2−probe−Cy3, were detected in
the lane of the probes 4a and 4i. However, the probes devoid of
a photoreactive moiety (4e and 4f) and lacking MDH2
inhibitory activity (4b and 4j) did not bind to rhMDH2. These
results indicate that both probes 4a and 4i bind to MDH2 and
inhibit HIF-1α activation through suppression of MDH2
activity inside cells. In the previous report, we demonstrated
that a pull-down complex with biotin probe 4c contained
MDH2 protein, as demonstrated by Western blot analysis,
indicating that MDH2 is a binding protein of LW6 and that
biotin probe 4c requires specificity to be used as a probe.7
Previously, we have shown that the inhibition of MDH2
activity by LW6 caused the suppression of mitochondrial
respiration, which resulted in a decrease of oxygen
consumption.7 Therefore, the effects of chemical probes that
retain MDH2 activity on oxygen consumption in HCT116 cells
were investigated. In control HCT116 cells, the oxygen
concentration in the medium was rapidly decreased (Figure
4). However, the representative chemical probe 4a demon-
Figure 5. Effects of LW6 and chemical probe 4a on ATP content and
ATP-related signaling in HCT116 cells under hypoxic conditions. (A)
ATP content in LW6 (10 μM), 4a (10 μM)-treated cells was
determined using a luciferase-based assay system. Statistical signifi-
cance: **P < 0.01, compared with untreated control. (B) Each protein
level was detected by immunoblot analysis with specific antibodies.
μM) showed competitive inhibition of MDH2 activity with
NADH, suggesting LW6 and its chemical probe 4a act as direct
competitors of NADH in binding to MDH2.
Collectively, the introduction of a clickable acetylene moiety
and photoactivatable trifluoromethyl diazirine to LW6 facili-
tated fluorescence imaging and conjugation to target molecules.
The introduction of trifluoromethyl diazirine to LW6 did not
have any significant effect on the activities of either HIF-1α or
MDH2, as with LW6 alone. Accordingly, the application of
these chemical probes to identify target molecules of LW6
overcame limitations of conventional methods, requiring
availability of detectable amounts of target molecules and a
high affinity of target molecules without inactivation or
structural distortion during experiments. The structure−activity
relationship of the compounds synthesized in this study using
HRE-luciferase activity was consistent with that of the MDH2
enzyme assay, confirming that MDH2 is the direct target
protein of LW6. These results also provide more information
for better understanding of HIF-1α inhibition by targeting
MDH2.
Figure 4. Effects of probes on oxygen consumption. HCT116 cells
were incubated with compound LW6, diazirine chemical probe 4a at
20 μM for 3 h, and then, oxygen consumption was measured as
described in the Experimental Section.
strated apparent suppression in oxygen consumption by
HCT116 cells, as LW6 did. This result suggests that probes
inhibit HIF-1α accumulation through suppressing mitochon-
drial respiration via interfering with MDH2 activity.
CONCLUSION
■
The affinity-based pull-down assay using biotinylated LW6 was
not successful in identifying the target protein of LW6 due to
lack of selectivity. Therefore, a series of multifunctional
chemical probes derived from LW6 were synthesized and
evaluated for their HIF-1α inhibition activity. The chemical
probe 4e, designed for intracellular imaging through click
chemistry, retained the activity in HIF-1α inhibition. On the
other hand, addition of benzophenone (4g and 4h), which
reacts with the receptor through a radical intermediate for
photoaffinity labeling, did not retain HIF-1α inhibitory activity,
presumably due to a rigid conformation or steric bulkiness.
Another test of a photoaffinity moiety, the diazirine moiety, as a
precursor for reaction with proteins was performed through a
highly reactive carbene intermediate. When a small labile
photoreactive group, like the trifluoromethyl diazirine moiety,
was attached to an adamantyl moiety through a benzyl ester
linkage, probe 4i exhibited HIF-1α inhibition activity, though
not as high as that of LW6. It is likely that this bulkier group at
the adamantyl ring may hinder efficient binding to its target
protein. Finally, introduction of a trifluoromethyl diazirine
moiety directly into the phenyl ring of adamantyl derivative 4a
exhibited the high potency in HIF-1α inhibitory activity among
Furthermore, we also found the inhibition of respiration by
LW6 and the probe 4a resulted in significant reduction of ATP
production in HCT116 cells (Figure 5A). Then, we examined
the activation of AMPK in the presence of LW6 and chemical
probe 4a because the elevation in the ratio of AMP/ATP
increases phosphorylation of AMPK.27 As shown in Figure 5B,
LW6 and 4a induced phosphorylation of AMPK and
inactivation of the downstream target ACC (acetyl-CoA
carboxylase), indicating that inhibition of respiration by LW6
or 4a resulted in activation of the AMPK signaling pathway. Of
significance, the design of chemical probes is as important as
the structure−activity relationship study, since probes should
retain the biological activity of the parent compound.
Then, we performed MDH2 kinetic assays of LW6 and
chemical probes to further understand their inhibition modes of
MDH2 using the purified His-MDH2 at various concentrations
of NADH. LW6 inhibited MDH2 activity in a NADH-
dependent manner, showing the typical intersecting line pattern
for competitive inhibition with the inhibition constant (Ki)
values of 1.9 μM (Figure 6). Furthermore, probe 4a (Ki = 4.3
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Figure 6. Kinetic study of MDH2 inhibition by LW6 and chemical probe 4a. Double-reciprocal plot of the effect of LW6 (A) or chemical probe 4a
■
○
▲
●
(B) on NADH-dependent MDH2 activity. Concentrations of LW6 and chemical probe (μM): 0 ( ), 1.25 ( ), 2.5 ( ), and 5 ( ).
using silica gel 60 (230−400 mesh Kieselgel 60). Additionally, thin-
layer chromatography on 0.25 mm silica plates (E. Merck; silica gel 60
F254) was used to monitor reactions. The purity of the final products
was checked by reversed phase high-pressure liquid chromatography
(RP-HPLC), which was performed on a Waters Corp. HPLC system
equipped with an ultraviolet (UV) detector set at 254 nm. The mobile
phases used were (A) H2O containing 0.05% trifluoroacetic acid and
(B) CH3CN. HPLC employed a YMC Hydrosphere C18 (HS-302)
column (5 μm particle size, 12 nm pore size) that was 4.6 mm in
diameter × 150 mm in size with a flow rate of 1.0 mL/min. The
compound purity was assessed either using (method A) a gradient of
20% B to 100% B in 35 min or (method B) a gradient of 25% B to
100% B in 35 min. All biologically evaluated compounds’ purity were
>95% in both method A and method B.
the synthesized derivatives. Then, in vitro binding of the probe
4a to recombinant human MDH2 (rhMDH2) through
photoaffinity labeling and click reaction was confirmed. The
representative probe 4a showed an inhibitory effect on MDH2
activity, oxygen consumption, and AMPK activation similar to
that of LW6, indicating the structure−activity relationships of
the probes in HIF-1α inhibitory activity were in accordance
with that in the MDH2 assay. Furthermore, 4a also showed
competitive inhibition of MDH2 activity with NADH like LW6.
These results suggest that well designed chemical probes based
on a study of structure−activity relationships provide a reliable
platform for the identification of direct target protein in drug
discovery.
Methyl 3-(2-(4-Adamantan-1-yl-phenoxy)acetamido)-4-hy-
droxybenzoate (6a).14 To the mixture of 4-(1-adamantyl)-phenoxy
acetic acid (5a) (0.3 g, 1.05 mmol), methyl-3-amino-4-hydroxyben-
zoate (0.16 g, 0.94 mmol), and HBTU (0.48 g, 1.26 mmol) in DMF
(10 mL) was added DIPEA (0.27 mL, 1.57 mmol). The reaction
mixture was stirred at room temperature overnight and then
partitioned between EtOAc and brine. The organic layer was
separated, dried over anhydrous MgSO4, filtered, and concentrated
under a vacuum. The resulting residue was purified by silica gel
column chromatography (n-hexane:EtOAc = 3:7) to give methyl 3-(2-
(4-adamantan-1-yl-phenoxy)acetamido)-4-hydroxybenzoate as a white
EXPERIMENTAL SECTION
■
All the commercial chemicals were of reagent grade and were used
without further purification. Solvents were dried with standard
procedures. All the reactions were carried out under an atmosphere
of dried argon in flame-dried glassware. The proton nuclear magnetic
resonance (1H NMR) spectra were determined on a Varian (300, 400,
or 500 MHz) spectrometer (Varian Medical Systems, Inc., Palo Alto,
CA, USA). 13C NMR spectra were recorded on a Varian (100 MHz)
spectrometer. The chemical shifts are provided in parts per million
(ppm) downfield with coupling constants in hertz (Hz). The mass
spectra were recorded using high-resolution mass spectrometry
(HRMS) (electron ionization MS) obtained on a JMS-700 mass
spectrometer (Jeol, Japan) or using HRMS (electrospray ionization
MS) obtained on a G2 QTOF mass spectrometer. The products from
all of the reactions were purified by flash column chromatography
1
solid (0.36 g, 78.9% yield). H NMR (300 MHz, DMSO-d6) δ 11.10
(s, 1H), 9.24 (s, 1H), 8.69 (m, 1H), 7.60−7.64 (m, 1H), 7.30 (d, J =
8.4 Hz, 2H), 6.94−6.99 (m, 3H), 4.74 (s, 2H), 3.79 (s, 3H), 2.04 (m,
3H), 1.83 (m, 6H), 1.72 (m, 6H); MS (ESI) m/z 434 (M − H)−;
HRMS (ESI) m/z calcd for C26H29O5NNa [(M + Na)+], 458.1943;
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found, 458.1942; purity 100% (as determined by RP-HPLC, method
A, tR = 22.583 min; method B, tR = 25.333 min).
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The
crude product was purified by preparative TLC (n-hexane:EtOAc:-
MeOH = 6:3:1) to obtain tert-butyl 2-[2-(2-{2-[2-(2-phenoxyaceta-
mido) phenoxy] acetamido} ethoxy)ethoxy]ethylcarbamate as a
yellow oil (0.1 g, yield 58.4%). 1H NMR (500 MHz, CD3OD) δ
7.94 (d, J = 7.5 Hz, 1H), 7.32 (t, J = 8.0 Hz, 2H), 7.15 (t, J = 8.0 Hz,
1H), 7.07 (d, J = 8.5 Hz, 2H), 6.98−7.03 (m, 3H), 4.71 (s, 2H), 4.61
(s, 2H), 3.51−3.54 (m, 6H), 3.44 (q, J = 5.5 Hz, 4H), 3.17 (t, J = 5.5
Hz, 2H), 1.41 (s, 9H); MS (EI) m/z 531 (M+).
N-(2-Hydroxyphenyl)-2-phenoxyacetamide (6b). To the
mixture of phenoxyacetic acid (5b) (0.3 g, 1.97 mmol), 2-
aminophenol (0.32 g, 2.96 mmol), and HBTU (1.12 g, 2.96 mmol)
in DMF (10 mL) was added DIPEA (0.52 mL, 2.96 mmol). The
reaction mixture was stirred at room temperature overnight and then
partitioned between EtOAc and brine. The organic layer was
separated, dried over anhydrous MgSO4, filtered, and concentrated
under a vacuum. The resulting residue was purified by silica gel
column chromatography (n-hexane:EtOAc = 4:6) to form N-(2-
hydroxyphenyl)-2-phenoxyacetamide as a yellow solid (0.364 g, 76.0%
Methyl 3-[2-(4-Adamantan-1-ylphenoxy)acetamido]-4-
{2,13-dioxo-17-[(3aS,4R,6aR)-2-oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl]-6,9-dioxa-3,12-diazaheptadecyloxy}benzoate
(4c). The compound 7a (0.06 g, 0.08 mmol) was dissolved in the 2
mL mixture of TFA and CH2Cl2 (1:3), and the solution was stirred at
room temperature for 1 h. Then, the solvents were removed and
coevaporated with toluene three times to obtain crude free amine
product 8a. The amine product 8a without further purification was
dissolved in 1 mL of DMF, to which 0.5 mL of TEA was added. After
that, to the solution was added (+)-biotin N-hydroxysuccinimide ester
(0.034 g, 0.1 mmol), and it was stirred at room temperature overnight.
The reaction was quenched by water and extracted with ethyl acetate.
The organic layers were dried over anhydrous MgSO4 and filtered, and
then concentrated under reduced pressure. The crude product was
purified by preparative TLC (CH2Cl2:MeOH = 10:1) to give the
compound methyl 3-[2-(4-adamantan-1-ylphenoxy)acetamido]-4-
{2,13-dioxo-17-[(3aS,4R,6aR)-2-oxohexahydro-1H-thieno[3,4-d]-
imidazol-4-yl]-6,9-dioxa-3,12-diazaheptadecyloxy}benzoate as a white
solid (0.023 g, 33.3% yield). 1H NMR (400 MHz, CD3OD) δ 8.55 (d,
J = 1.8 Hz, 1H), 7.76−7.75 (m,1H), 7.23 (d, J =8.0 Hz, 2H), 6.98 (d, J
= 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 2H), 4.61 (s, 4H), 4.37−4.34 (m,
1H), 4.18−4.15 (m, 1H), 3.78 (s, 3H), 3.47−3.34 (m, 12H), 3.07−
3.05 (m, 1H), 2.82−2.78 (m, 1H), 2.57 (d, J = 12.0 Hz, 1H), 2.08 (t, J
= 8.0 Hz, 2H), 1.97 (brs, 3H), 1.82−1.81 (m, 6H), 1.75−1.67 (m,
6H), 1.57−1.41 (m, 4H), 1.33−1.29 (m, 2H); 13C NMR (100 MHz,
CD3OD) δ 174.7, 168.8, 168.3, 166.4, 155.4, 152.3, 145.0, 134.4,
127.4, 126.4, 125.7, 123.8, 123.4, 114.2, 111.6, 69.8, 69.2, 69.0, 67.3,
61.9, 60.2, 55.6, 43.1, 39.6, 38.8, 38.7, 36.4, 35.4, 35.3, 29.0, 28.3, 28.1,
25.4; MS (FAB) m/z 850 (MH+); HRMS (FAB) m/z calcd for
C44H60N5O10S [MH+], 850.4061; found, 850.4061; purity 100% (as
determined by RP-HPLC, method A, tR = 12.3 min; method B, tR =
18.4 min).
1
yield). H NMR (300 MHz, DMSO-d6) δ 10.02 (brs, 1H), 9.16 (s,
1H), 7.99 (d, J = 7.8 Hz, 1H), 7.31−7.36 (m, 2H), 6.86−7.04 (m,
5H), 6.78 (dt, J = 1.8 Hz, 7.8 Hz, 1H), 4.74 (s, 2H); MS (EI) m/z 243
(M+).
(2-{2-[2-(2-Chloro-acetylamino)-ethoxy]-ethoxy}-ethyl)-car-
bamic acid tert-butyl ester. To a solution of 1,2-bis(2-amino-
ethoxy) ethane (0.3 g, 2.03 mmol) and triethylamine (0.56 g, 4.06
mmol) in methanol (4.0 mL) was added di-tert-butyl dicarbonate
(0.55 mL, 2.43 mmol). The reaction mixture was stirred at room
temperature overnight, and the methanol and TEA were removed in
vacuo to yield oily residue, which was dissolved in CH2Cl2 and washed
with a solution of sodium carbonate. The combined extracts were
dried on anhydrous MgSO4, filtered, and concentrated in vacuo. The
resulting residue was purified by silica gel column chromatography (n-
hexane:EtOAc = 2:8) to form monoprotected diamine as yellow oil
(0.1 g, 66.7% yield). The TLC control showed almost no bis-protected
diamine. The yellow oil was used without further purification. A
solution of chloroacetyl chloride (0.17 g, 1.58 mmol) in CH2Cl2 (3
mL) was added dropwise over 20 min to a solution of monoprotected
diamine (0.32 g, 1.32 mmol) and TEA in CH2Cl2 (3 mL) at −20 °C.
The resulting brown solution was left to stir at room temperature for
24 h. The reaction solution was removed in vacuo, and the residue was
dissolved in CH2Cl2 and washed with a solution of sodium carbonate.
The combined extracts were dried on anhydrous MgSO4, filtered, and
1
concentrated to give a brown oil (0.3 g, 70.4% yield). H NMR (300
MHz, CDCl3) δ 7.03 (s, 1H), 5.06 (s, 1H), 3.95 (s, 2H), 3.38−3.51
(m, 10H), 3.20 (m, 2H), 1.33 (s, 9H).
Methyl 3-[2-(4-Adamantan-1-yl-phenoxy)-acetylamino]-4-
(2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazahexadecan-
16-yloxy)benzoate (7a). To the solution of (2-{2-[2-(2-chloro-
acetylamino)-ethoxy]-ethoxy}-ethyl)-carbamic acid tert-butyl ester
(0.12 g, 0.38 mmol) in anhydrous acetone (10 mL) and DMF (3
mL) was subsequently added compound 6a (0.082 g, 0.19 mmol),
K2CO3 (0.052 g, 0.38 mmol), Cs2CO3 (0.030 g, 0.09 mmol), and KI
(0.016 g, 0.09 mmol). The reaction mixture was heated at 60 °C for 36
h and cooled to room temperature. The mixture was evaporated under
reduced pressure, and the residue was washed with water. The solution
was extracted with ethyl acetate, and the combined organic layers were
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The
crude product was purified by preparative TLC (n-hexane:EtOAc =
1:1) to obtain methyl 3-[2-(4-adamantan-1-yl-phenoxy)-acetylamino]-
4-(2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazahexadecan-16-
5-[(3aS,4R,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-
4-yl]-N-{2-[2-(2-{2-[2-(2-phenoxyacetamido)phenoxy]-
acetamido}ethoxy)ethoxy]ethyl}pentanamide (4d). The com-
pound 7b (0.058 g, 0.11 mmol) was dissolved in the 2 mL mixture of
TFA and CH2Cl2 (1:3), and the solution was stirred at room
temperature for 1 h. Then, the solvents were removed and
coevaporated with toluene three times to obtain crude free amine
product 8b. The amine product 8b without further purification was
dissolved in 1 mL of DMF, to which 0.5 mL of TEA was added. After
that, to the solution was added (+)-biotin N-hydroxysuccinimide ester
(0.043 g, 0.13 mmol), and it was stirred at room temperature
overnight. The reaction was quenched by water and extracted with
10% MeOH/MC. The organic layers were dried over anhydrous
MgSO4 and filtered, and then concentrated under reduced pressure.
The crude product was purified by preparative TLC (CH2Cl2:MeOH
= 10:1) to give compound 5-[(3aS,4R,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl]-N-{2-[2-(2-{2-[2-(2-phenoxyacetamido)-
phenoxy]acetamido}ethoxy)ethoxy]ethyl}pentanamide as a pale yel-
1
yloxy)benzoate as a yellow oil (0.06 g, yield 43.8%). H NMR (300
MHz, CDCl3) δ 8.74 (brs, 1H), 7.83−7.86 (m, 1H), 7.32 (d, J = 9.0
Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.91 (d, J = 9.0 Hz, 1H), 4.65 (s,
2H), 4.64 (s, 2H), 3.89 (s, 3H), 3.43−3.51 (m, 10H), 3.23 (m, 2H),
2.09 (brs, 3H), 1.88−1.86 (m, 6H), 1.76−1.74 (m, 6H), 1.41 (s, 9H);
MS (EI) m/z 723 (M+).
1
low solid (0.048 g, 66.6% yield). H NMR (500 MHz, DMSO-d6) δ
9.61 (s, 1H), 8.25 (t, J = 5.5 Hz, 1H), 7.96 (d, J = 7.5 Hz, 1H), 7.80 (t,
J = 5.5 Hz, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.10 (t, J = 8.0 Hz, 1H), 7.06
(d, J = 8.0 Hz, 2H), 7.02−6.97 (m, 3H), 6.40 (s, 1H), 6.34 (s, 1H),
4.74 (s, 2H), 4.59 (s, 2H), 4.29 (t, J = 7.0 Hz, 1H), 4.11 (t, J = 6.0 Hz,
1H), 3.43 (t, J = 6.0 Hz, 4H), 3.36 (t, J = 5.5 Hz, 2H), 3.16 (q, J = 6.0
Hz, 2H), 3.10−3.06 (m, 1H), 2.81 (dd, J = 5.0 Hz, 12.5 Hz, 1H), 2.57
(d, J = 12.5 Hz, 1H), 2.05 (t, J = 7.5 Hz, 2H), 1.62−1.58 (m, 1H),
1.52−1.48 (m, 3H), 1.50−1.43 (m, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 172.6, 168.2, 166.9, 163.1, 157.9, 148.8, 130.0, 127.3,
125.4, 122.0, 121.9, 121.8, 115.3, 113.6, 70.0, 69.9, 69.6, 68.3, 67.5,
tert-Butyl 2-[2-(2-{2-[2-(2-Phenoxyacetamido) phenoxy]
acetamido} ethoxy)ethoxy]ethylcarbamate (7b). To the solution
of (2-{2-[2-(2-chloro-acetylamino)-ethoxy]-ethoxy}-ethyl)-carbamic
acid tert-butyl ester (0.13 g, 0.40 mmol) in anhydrous acetone (10
mL) was subsequently added compound 6b (0.048 g, 0.2 mmol),
K2CO3 (0.069 g, 0.50 mmol), Cs2CO3 (0.055 g, 0.17 mmol), and KI
(0.04 g, 0.02 mmol). The reaction mixture was heated at 60 °C for 36
h and cooled to room temperature. The mixture was evaporated under
reduced pressure, and the residue was washed with water. The solution
was extracted with ethyl acetate, and the combined organic layers were
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61.5, 59.6, 55.9, 49.0, 38.9, 35.6, 28.6, 25.7; MS (FAB) m/z 658
(MH+); HRMS (FAB) m/z calcd for C32H44N5O8S [MH+], 658.2911;
found, 658.2911; purity 100% (as determined by RP-HPLC, method
A, tR = 10.2 min; method B, tR = 8.5 min).
Prop-2-ynyl 3-Amino-4-hydroxybenzoate (15). To a mixture
of prop-2-ynyl 4-hydroxy-3-nitrobenzoate (14) (0.8 g, 3.6 mmol) and
NH4Cl (1.93 g, 36.0 mmol) in THF/MeOH/H2O (10:5:3) (20 mL)
was added Zn dust (2.36 g, 36.0 mmol) at 60 °C, and then, the
mixture was refluxed for 3 h. After completion of the reaction, the
reaction mixture was filtered through Celite and partitioned between
EtOAc and brine. The organic layer was separated, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography
(hexane:EtOAc = 4:6) to give prop-2-ynyl 3-amino-4-hydroxybenzoate
Methyl 4-Hydroxy-3-nitrobenzoate (10). To a solution of 4-
hydroxy-3-nitro-benzoic acid (9) (2.0 g, 10.9 mmol) in methyl alcohol
(25 mL) was added SOCl2 (1.18 mL, 16.3 mmol) dropwise at 0 °C,
and then, the mixture was refluxed for 4 h. After completion of the
reaction, the reaction mixture was concentrated under reduced
pressure. The resulting residue was purified by silica gel column
chromatography (hexane:EtOAc = 8:2) to give methyl 4-hydroxy-3-
1
as a yellow solid (0.61 g, 88.4% yield). H NMR (500 MHz, DMSO-
1
nitrobenzoate as a yellow solid (2.0 g, 93.0% yield). H NMR (400
d6) δ 7.24 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H),
4.84 (s, 2H), 3.53 (t, J = 4.0 Hz, 1H); MS (ESI) m/z 190 (M − H).
(4-Adamantan-1-yl-phenoxy)acetic Acid (5a). This synthetic
procedure followed our previous method.14 White solid (1.76 g, 92.1%
MHz, CDCl3) δ 10.88 (s, 1H), 8.82 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H),
7.22 (d, J = 8.0 Hz, 1H), 3.94 (s, 3H); MS (ESI) m/z 196 (M − H).
Methyl 4-(4-Methoxybenzyloxy)-3-nitrobenzoate (11). To a
mixture of methyl 4-hydroxy-3-nitrobenzoate (10) (1.8 g, 9.1 mmol),
K2CO3 (3.78 g, 27.3 mmol), and KI (3.03 g, 18.2 mmol) in acetone
(18 mL) was added p-methoxy benzyl chloride (1.49 mL, 10.9 mmol)
dropwise at 0 °C, and then, the mixture was refluxed for 12 h. After
completion of the reaction, the reaction mixture was concentrated
under reduced pressure and then partitioned between EtOAc and
brine. The organic layer was separated, washed with water, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography
(hexane:EtOAc = 9:1) to give methyl 4-(4-methoxybenzyloxy)-3-
1
yield). H NMR (400 MHz, MeOD) δ 7.26 (d, J = 7.2 Hz, 2H), 6.85
(d, J = 8.0 Hz, 2H), 4.60 (s, 2H), 2.08 (brs, 3H), 1.90−1.86 (m, 6H),
1.76 (m, 6H); MS (EI) m/z 286 (M+).
Prop-2-ynyl 3-(2-(4-Adamantan-1-yl-phenoxy)acetamido)-4-
hydroxybenzoate (4e). To a solution of (4-adamantan-1-yl-
phenoxy)acetic acid (5a) (0.1 g, 0.35 mmol) and prop-2-ynyl 3-
amino-4-hydroxybenzoate (15) (0.06 g, 0.35 mmol) in DMF (5.0 mL)
were added (EDC·HCl) (0.08 g, 0.42 mmol), HOBt (0.056 g, 0.42
mmol), and DIPEA (0.15 mL, 0.87 mmol). The reaction mixture was
stirred at room temperature overnight and then partitioned between
EtOAc and brine. The organic layer was separated, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography
(hexane:EtOAc = 6:4) to give prop-2-ynyl 3-(2-(4-adamantan-1-yl-
phenoxy)acetamido)-4-hydroxybenzoate as a white solid (0.11 g,
1
nitrobenzoate as a brown solid (2.56 g, 88.5% yield). H NMR (400
MHz, CDCl3) δ 8.49 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.36 (d, J =
12.0 Hz, 2H), 7.16 (d, J = 12.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 2H), 5.17
(s, 2H), 3.92 (s, 3H), 3.81 (s, 3H); MS (ESI) m/z 340 (M + Na).
4-(4-Methoxybenzyloxy)-3-nitrobenzoic Acid (12). A solution
of methyl 4-(4-methoxybenzyloxy)-3-nitrobenzoate (11) (2.0 g, 6.3
mmol) in THF/H2O (3:1 20 mL) was treated with lithium hydroxide
monohydrate (1.05 g, 25.2 mmol) and stirred at room temperature
until the reaction was complete as judged by TLC. The reaction
mixture was then acidified with 10% HCl to pH 4 and then partitioned
between EtOAc and brine. The organic layer was separated, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography
(CH2Cl2:MeOH = 9:1) to give 4-(4-methoxybenzyloxy)-3-nitro-
1
68.7% yield). H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 9.31
(s, 1H), 8.75 (s, 1H), 7.70 (d, J = 12.0 Hz, 1H), 7.35 (d, J = 8.0 Hz,
2H), 7.06 (d, J = 4.0 Hz, 1H), 7.03 (d, J = 8.0 Hz, 2H), 4.95 (s, 2H),
4.80 (s, 2H), 3.63 (t, J = 4.0 Hz, 1H), 2.10 (brs, 3H), 2.05−1.89 (m,
6H), 1.81−1.75 (m, 6H); 13C NMR (100 MHz, DMSO-d6) δ 167.3,
165.2, 155.7, 152.4, 144.6, 127.2, 126.3, 126.2, 122.3, 120.1, 115.3,
114.8, 79.1, 78.2, 67.6, 52.5, 43.2, 36.6, 35.6, 28.8; HRMS [M + H]
calcd [C28H30NO5], 460.2124; found, 460.2112; purity >99.9% (as
determined by RP-HPLC, method A, tR = 26.74 min; method B, tR =
26.14 min).
1
benzoic acid as a pale yellow solid (1.76 g, 92.1% yield). H NMR
(400 MHz, MeOD) δ 8.38 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.42 (d, J
= 8.0 Hz, 1H), 7.38 (d, J = 4.0 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 5.26
(s, 2H), 3.79 (s, 3H); MS (ESI) m/z 302 (M − H).
Prop-2-ynyl 4-Hydroxy-3-(2-phenoxyacetamido) Benzoate
(4f). To a solution of phenoxyacetic acid (5b) (0.1 g, 0.65 mmol) and
prop-2-ynyl 3-amino-4-hydroxybenzoate (15) (0.12 g, 0.65 mmol) in
DMF (5.0 mL) were added EDC·HCl (0.15 g, 0.78 mmol), HOBt
(0.11 g, 0.78 mmol), and DIPEA (0.29 mL, 1.64 mmol). The reaction
mixture was stirred at room temperature overnight and then
partitioned between EtOAc and brine. The organic layer was
separated, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The resulting residue was purified by silica gel column
chromatography (n-hexane:EtOAc = 7:3) to form prop-2-ynyl 4-
hydroxy-3-(2-phenoxyacetamido) benzoate as a white solid (0.16 g,
Prop-2-ynyl 4-(4-Methoxybenzyloxy)-3-nitrobenzoate (13).
To a mixture of 4-(4-methoxybenzyloxy)-3-nitrobenzoic acid (12) (1.7
g, 5.6 mmol) and K2CO3 (2.0 g, 16.8 mmol) in DMF (17 mL) was
added propargyl bromide (1.0 mL, 11.2 mmol) dropwise at 0 °C, and
then, the mixture was stirred at room temperature for 12 h. After
completion of the reaction, the reaction mixture was partitioned
between EtOAc and brine. The organic layer was separated, washed
with water, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The resulting residue was purified by silica gel column
chromatography (hexane:EtOAc = 3:7) to give prop-2-ynyl 4-(4-
methoxybenzyloxy)-3-nitrobenzoate as a yellow solid (1.78 g, 93.1%
1
76.2% yield). H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 9.33
(s, 1H), 8.74 (s, 1H), 7.69 (d, J = 12.0 Hz, 1H), 7.39 (t, J = 8.0 Hz,
2H), 7.10−7.04 (m, 4H), 4.95 (s, 2H), 4.82 (s, 2H), 3.63 (t, J = 4.0
Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 167.1, 165.2, 157.9,
152.5, 130.1, 127.2, 126.1, 122.5, 122.0, 120.0, 115.3, 115.2, 79.1, 78.2,
67.5, 52.5; HRMS [M + H] calcd [C18H16NO5], 326.1028; found,
326.1027; purity >99.9% (as determined by RP-HPLC, method A, tR =
16.12 min; method B, tR = 14.83 min).
1
yield). H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H), 8.19 (d, J = 8.0
Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 1H), 6.92 (d, J
= 8.0 Hz, 2H), 5.24 (s, 2H), 5.05 (s, 2H), 3.81 (s, 3H), 2.52 (t, J = 4.0
Hz, 1H); MS (ESI) m/z 340 (M − H).
Prop-2-ynyl 4-Hydroxy-3-nitrobenzoate (14). To a solution of
prop-2-ynyl 4-(4-methoxybenzyloxy)-3-nitrobenzoate (13) (1.6 g, 4.6
mmol) in CH2Cl2 (16 mL) was added TFA (1.43 mL, 18.7 mmol)
dropwise at 0 °C, and then, the mixture was stirred at room
temperature for 4 h. After completion of the reaction, the reaction
mixture was concentrated and coevaporated with toluene three times
under reduced pressure. The resulting residue was purified by silica gel
column chromatography (hexane:EtOAc = 1:1) to give prop-2-ynyl 4-
Phenyl Propargyl Ether (18). A suspension of phenol (16) (1.0
g, 10.6 mmol), anhydrous potassium carbonate (4.4 g, 31.8 mmol),
and propargyl bromide (17) (1.6 mL, 21.2 mmol) in DMF (10 mL)
was stirred overnight at room temperature. The reaction mixture was
diluted with ethyl acetate and subsequently washed with aqueous
sodium bicarbonate, brine, and water. The organic layer was dried over
anhydrous MgSO4. The solvent was filtered and evaporated under
reduced pressure to afford a crude product, which was purified by silica
gel column chromatography (hexane:EtOAc = 9:1) to give phenyl
1
hydroxy-3-nitrobenzoate as a yellow solid (0.93 g, 90.2% yield). H
NMR (400 MHz, CD3OD) δ 8.58 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H),
7.14 (d, J = 8.0 Hz, 1H), 4.83 (s, 2H), 2.89 (t, J = 4.0 Hz, 1H); MS
(ESI) m/z 220 (M − H).
1
propargyl ether as a colorless oil (1.0 g, 71.4% yield). H NMR (500
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MHz, CDCl3) δ 7.32−7.28 (m, 2H), 7.01−6.97 (m, 2H), 4.69 (s, 2H),
2.51 (t, J = 5.0 Hz, 1H); MS (ESI) m/z 133 (M + H).
11.4 Hz, 2H), 4.77 (s, 2H), 2.55 (t, J = 2.5 Hz, 1H), 2.10 (brs, 3H),
1.90−1.89 (m, 6H), 1.78−1.73 (m, 6H); 13C NMR (100 MHz,
CDCl3) δ 195.2, 164.8, 161.1, 158.3, 153.7, 148.1, 138.7, 138.3, 132.5,
130.6, 129.0, 126.4, 125.4, 123.6, 120.9, 117.3, 114.5, 103.9, 77.8, 76.2,
55.9, 43.2, 36.7, 35.9, 28.9; MS (EI) m/z 532 (M+); HRMS (EI) m/z
calcd for C35H34NO4 [M + H], 532.2410; found, 532.2488; purity
100% (as determined by RP-HPLC, method A, tR = 29.61 min;
method B, tR = 29.7 min).
2,2,2-Trifluoro-1-(4-methylphenyl)-1-ethanone Oxime (22).
To a stirred solution of 2,2,2-trifluoro-1-(2-methoxyphenyl)ethanone
(21) (3.0 g, 15.94 mmol) in pyridine (30.0 mL) was added
hydroxylamine hydrochloride (3.3 g, 47.83 mmol). Then, the mixture
was refluxed for 4 h. The pyridine was evaporated, 50 mL of an
aqueous solution of citric acid (10%) and CH2Cl2 were added to the
residue, and the organic layer was extracted. The organic layer was
separated, washed with brine, dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. The resulting residue was purified by silica
gel column chromatography (hexane:EtOAc = 2:8) to give 2,2,2-
trifluoro-1-(4-methylphenyl)-1-ethanone oxime as a white solid (2.6 g,
80.3% yield). 1H NMR (500 MHz, CDCl3) δ 8.08 (s, 1H), 7.40 (d, J =
10.0 Hz, 2H), 7.28 (d, J = 10.0 Hz, 2H), 2.40 (s, 3H); MS (EI) m/z
203 (M+).
2,2,2-Trifluoro-1-(4-methylphenyl)-1-ethanone O-(p-Tolue-
nesulfonyl) Oxime (23). To a mixture of 2,2,2-trifluoro-1-(4-
methylphenyl)-1-ethanone oxime (22) (2.5 g, 12.30 mmol), 4-
dimethylaminopyridine (0.75 g, 6.15 mmol), and triethylamine (2.57
mL, 18.45 mmol) in CH2Cl2 (25.0 mL) was added p-toluene sulfonyl
chloride (2.81 g, 14.76 mmol) at 0 °C, and then, the mixture was
stirred at room temperature for 8 h. After completion of the reaction,
the reaction mixture was partitioned between CH2Cl2 and brine. The
organic layer was separated, washed with water, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (hexane:EtOAc = 1:9) to
give 2,2,2-trifluoro-1-(4-methylphenyl)-1-ethanone O-(p-toluenesul-
fonyl) oxime as a white solid (3.6 g, 84% yield). 1H NMR (400
MHz, CDCl3) δ 7.88 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H),
7.31 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 2.47 (s, 3H), 2.40
(s, 3H); MS (EI) m/z 357 (M+).
(3-Nitrophenyl)(4-(prop-2-ynyloxy)phenyl)methanone
(19).21 The m-nitrobenzoyl chloride (1.0 g, 5.38 mmol) was gradually
added to a mixture of phenyl propargyl ether (18) (0.71 g, 5.38 mmol)
and anhydrous aluminum chloride (1.07 g, 8.08 mmol) in dichloro-
methane at −10 °C with continuous stirring. After addition, the
mixture was stirred overnight at room temperature and quenched with
10% hydrochloric acid. The mixture was diluted with dichloromethane
and dried over anhydrous magnesium sulfate. The filtrate was
concentrated under reduced pressure and purified by column
chromatography on silica gel to give (3-nitrophenyl)(4-(prop-2-
ynyloxy)phenyl)methanone as a pale yellow solid (0.7 g, 46.7%
1
yield). H NMR (500 MHz, CDCl3) δ 8.58 (s, 1H), 8.43−8.41 (m,
1H), 8.09 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 10.0 Hz, 2H), 7.69 (t, J =
10.0 Hz, 1H), 7.09 (d, J = 10.0 Hz, 2H), 4.80 (s, 2H), 2.57 (t, J = 5.0
Hz, 1H); MS (EI) m/z 282 (M + H).
(3-Aminophenyl)(4-(prop-2-ynyloxy)phenyl)methanone
(20). To a mixture of (3-nitrophenyl)(4-(prop-2-ynyloxy)phenyl)-
methanone (19) (0.6 g, 2.13 mmol) and NH4Cl (1.14 g, 21.3 mmol)
in THF/MeOH/H2O (10:5:3) (20 mL) was added iron powder (1.19
g, 21.3 mmol) at 60 °C, and then, the mixture was refluxed for 2 h.
After completion of the reaction, the reaction mixture was filtered
through Celite and partitioned between EtOAc and brine. The organic
layer was separated, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The resulting residue was purified by silica gel
column chromatography (hexane:EtOAc = 4:6) to give (3-
aminophenyl)(4-(prop-2-ynyloxy)phenyl)methanone as a yellow
1
semisolid (0.4 g, 75.4% yield). H NMR (400 MHz, CDCl3) δ 7.84
(d, J = 8.0 Hz, 2H), 7.24−7.22 (m, 1H), 7.10−7.07 (m, 2H), 7.04 (d, J
= 8.0 Hz, 2H), 6.88−6.86 (m, 1H), 4.78 (s, 2H), 3.80 (brs, 2H), 2.56
(t, J = 4.0 Hz, 1H); MS (ESI) m/z 252 (M + H).
2-(4-Adamantan-1-yl-phenoxy)-N-[3-(4-prop-2-
ynyloxybenzoyl)phenyl] Acetamide (4g). To a solution of (4-
adamantan-1-yl-phenoxy)acetic acid (5a) (0.1 g, 0.35 mmol) and (3-
aminophenyl)(4-(prop-2-ynyloxy)phenyl)methanone (20) (0.08 g,
0.35 mmol) in DMF (5.0 mL) were added (EDC·HCl) (0.08 g,
0.42 mmol), HOBt (0.056 g, 0.42 mmol), and DIPEA (0.15 mL, 0.87
mmol). The reaction mixture was stirred at room temperature
overnight and then partitioned between EtOAc and brine. The organic
layer was separated, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The resulting residue was purified by silica gel
column chromatography (hexane:EtOAc = 6:4) to give 2-(4-
adamantan-1-yl-phenoxy)-N-[3-(4-prop-2-ynyloxybenzoyl)phenyl]
acetamide as a white solid (0.11 g, 61.1% yield). 1H NMR (400 MHz,
CDCl3) δ 8.44 (brs, 1H), 8.00−7.98 (m, 1H), 7.85 (d, J = 8.0 Hz,
2H), 7.83 (t, J = 4.0 Hz, 1H) 7.53−7.45 (m, 2H), 7.33 (d, J = 8.0 Hz,
2H), 7.05 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 4.78 (s, 2H),
4.61 (s, 2H), 2.57 (t, J = 4.0 Hz, 1H), 2.09 (brs, 3H), 1.88−1.80 (m,
6H), 1.75−1.72 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 194.8,
166.8, 161.2, 154.7, 145.7, 138.9, 137.0, 132.5, 130.6, 129.1, 126.3,
126.1, 123.6, 121.0, 114.5, 114.4, 77.8, 76.2, 67.7, 55.9, 43.3, 36.7, 35.7,
28.9; HRMS [M + H] calcd [C34H34NO4], 520.2463; found,
520.2461; purity >99.9% (as determined by RP-HPLC, method A,
tR = 28.65 min; method B, tR = 28.35 min).
(E)-3-(4-Adamantan-1-ylphenoxy)-N-(3-(4-(prop-2-ynyloxy)-
benzoyl)phenyl)acrylamide (4h). To a solution of 4-(1-adaman-
tyl)-phenoxyacrylic acid (0.1 g, 0.33 mmol) and (3-aminophenyl)(4-
(prop-2-ynyloxy)phenyl)methanone (20) (0.08 g, 0.33 mmol) in
DMF (5.0 mL) were added (EDC·HCl) (0.076 g, 0.40 mmol), HOBt
(0.054 g, 0.40 mmol), and DIPEA (0.14 mL, 0.82 mmol). The
reaction mixture was stirred at room temperature overnight and then
partitioned between EtOAc and brine. The organic layer was
separated, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The resulting residue was purified by silica gel column
chromatography (hexane:EtOAc = 6:4) to give (E)-3-(4-adamantan-1-
ylphenoxy)-N-(3-(4-(prop-2-ynyloxy)benzoyl)phenyl)acrylamide as a
white solid (0.11 g, 61.8% yield). 1H NMR (500 MHz, CDCl3) δ 7.90
(d, J = 10.0 Hz, 2H), 7.84 (d, J = 5.0 Hz, 2H), 7.77 (s, 1H), 7.46−7.42
(m, 2H), 7.36 (d, J = 10.0 Hz, 2H), 7.06−7.03 (m, 5H), 5.63 (d, J =
3-(4-Methylphenyl)-3-trifluoromethyldiaziridine (24). Liquid
ammonia (6.0 mL) was added at −78 °C to an ether (30.0 mL)
solution of 2,2,2-trifluoro-1-(4-methylphenyl)-1-ethanone O-(p-tolue-
nesulfonyl) oxime (23) (3.0 g, 8.39 mmol) in a sealed tube. The
solution was stirred at room temperature for 16 h. The mixture was
carefully cooled to −78 °C, and the sealed tube was opened. The
ammonia was evaporated, and white precipitate was filtered and
washed with ether. The filtrate was concentrated in vacuo to give 3-(4-
methylphenyl)-3-trifluoromethyldiaziridine as a white solid (1.4 g,
1
82.8% yield). H NMR (500 MHz, CDCl3) δ 7.49 (d, J = 10.0 Hz,
2H), 7.22 (d, J = 10.0 Hz, 2H), 2.75 (d, J = 10.0 Hz, 1H), 2.37 (s, 3H),
2.17 (d, J = 10.0 Hz, 1H); MS (EI) m/z 202 (M+).
3-(4-Methylphenyl)-3-trifluoromethyldiazirine (25). To the
compound 3-(4-methylphenyl)-3-trifluoromethyldiaziridine (24) (1.0
g, 4.94 mmol) in CCl4 (10 mL) was added MnO2 (1.29 g, 14.83
mmol) at 0 °C, and then, the mixture was stirred at room temperature
for 1 h. After completion of the reaction, CCl4 was concentrated under
reduced pressure at low temperature due to volatility of the product.
Flash chromatography of the residue over silica gel (hexane:EtOAc =
9.5:0.5) afforded 3-(4-methylphenyl)-3-trifluoromethyldiazirine as a
1
colorless solid (0.5 g, 50.5% yield). H NMR (500 MHz, CDCl3) δ
7.19 (d, J = 10.0 Hz, 2H), 7.08 (d, J = 10.0 Hz, 2H), 2.35 (s, 3H); MS
(EI) m/z 200 (M+).
3-(4-Bromomethylphenyl)-3-trifluoromethyldiazirine (26). A
solution of 3-(4-methylphenyl)-3-trifluoromethyldiazirine (25) (0.4 g,
2.0 mmol) in CCl4 (4 mL) was heated to 70 °C, powdered NBS (0.66
g, 3.75 mmol) was added and stirred for 10 min and then benzoyl
peroxide (15 mg) was added, and the reaction was refluxed for 2 h.
The precipitate was filtered, the solvent was removed in vacuo at 20
°C, and the crude product was purified by column chromatography
eluting with hexane/CH2Cl2 (20:1) to give 3-(4-bromomethylphenyl)-
1
3-trifluoromethyldiazirine as a yellow oil (0.25 g, 45.4% yield). H
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NMR (500 MHz, CDCl3) δ 7.19 (d, J = 10.0 Hz, 2H), 7.08 (d, J =
10.0 Hz, 2H), 4.50 (s, 3H); MS (EI) m/z 277 (M+).
35.5, 31.9, 29.7, 29.4, 28.6; HRMS [M + H] calcd [C38H35F3N3O7],
702.2378; found, 702.2378; purity >98% (as determined by RP-HPLC,
method A, tR = 28.8 min; method B, tR = 29.3 min).
3-(4-Hydroxyphenyl)-adamantane-1-carboxylic Acid (29).
The synthetic procedure was followed as per our previous method.25
4-[3-({4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzyloxy}-
carbonyl)adamantan-1-y]phenol (30).23 The mixture of 3-(4-
hydroxyphenyl)-adamantane-1-carboxylic acid (29) (0.34 g, 1.25
mmol) and 3-(4-bromomethylphenyl)-3-trifluoromethyl-3H-diazirine
(26) (0.52 g, 1.91 mmol) was dissolved in 10 mL of anhydrous DMF,
and KHCO3 (0.19 g, 1.91 mmol) was added. The reaction mixture was
heated at 40 °C for 4 h and quenched by aqueous NaHCO3. The
solution was extracted with EA, and the organic layers were dried over
MgSO4. The filtrate was concentrated and purified by column
chromatography on silica gel to form 4-[3-({4-[3-(trifluoromethyl)-
3H-diazirin-3-yl]benzyloxy}carbonyl)adamantan-1-y]phenol as a white
solid (0.15 g, 25.7% yield). 1H NMR (400 MHz, CDCl3) δ 7.36 (d, J =
8.4 Hz, 2H), 7.17−7.23 (m, 4H), 6.79 (d, J = 8.0 Hz, 2H), 5.15 (s,
2H), 2.23 (m, 2H), 2.04 (m, 2H), 1.96 (m, 4H), 1.89 (m, 4H), 1.72
(m, 2H); MS (EI) m/z 470 (M+).
Ethyl-2-{4-[3-({4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-
benzyloxy}carbonyl)adamantan-1-y]phenoxy}acetate (31).
The 4-[3-({4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyloxy}-
carbonyl)adamantan-1-y]phenol (30) (0.3 g, 0.63 mmol) and ethyl
chloroacetate (0.15 g, 1.27 mmol) were dissolved in 5 mL of
anhydrous DMF. K2CO3 (0.26 g, 1.91 mmol) was added and stirred at
room temperature overnight. The excess solvent was evaporated and
washed with water. The mixture was extracted with ethyl acetate and
dried over MgSO4. The filtrate was concentrated under reduced
pressure and the residue was purified by MPLC, which afforded ethyl-
2-{4-[3-({4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyloxy}-
carbonyl)adamantan-1-y]phenoxy}acetate as a colorless oil (0.22 g,
62.8% yield). 1H NMR (500 MHz, CDCl3) δ 7.35 (d, J = 8.0 Hz, 2H),
7.26 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 8.5 Hz,
2H), 5.10 (s, 2H), 4.60 (s, 2H), 4.27 (q, J = 7.0 Hz, 2H), 2.22 (m,
2H), 2.01 (m, 2H), 1.93 (m, 4H), 1.86 (m, 4H), 1.72 (m, 2H), 1.30 (t,
J = 7.0 Hz, 3H); MS (FAB+) m/z 556 (M+).
4-[3-({4-[3-(Trifluoromethyl)-3H-diazirin-3-yl] benzyloxy}
carbonyl) adamantan-1-y]-(prop-2-ynyloxy) Benzene (4j). The
compound 4-[3-({4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyloxy}-
carbonyl)adamantan-1-y]phenol (30) (0.15 g, 0.32 mmol) was
dissolved in 8 mL of anhydrous DMF, and K2CO3 (0.09 g, 0.64
mmol) was added. The mixture was heated at 60 °C for 2 h and cooled
to room temperature, and then, propargyl bromide (0.054 g, 0.48
mmol) was added. The reaction mixture was stirred overnight and
then washed with water. The mixture was extracted with dichloro-
methane and then washed with brine. The organic phase was dried
over MgSO4 and filtered. The filtrate was evaporated under reduced
pressure, and the residue was purified by column chromatography on
silica gel to afford 4-[3-({4-[3-(trifluoromethyl)-3H-diazirin-3-yl]
benzyloxy} carbonyl) adamantan-1-y]-(prop-2-ynyloxy) benzene as a
colorless oil (0.1 g, 62.1% yield). 1H NMR (400 MHz, CDCl3) δ 7.36
(d, J = 8.4 Hz, 2H), 7.28 (dd, J = 3.2 Hz, 5.2 Hz, 2H), 7.18 (d, J = 8.4
Hz, 2H), 6.94 (dd, J = 2.4 Hz, 6.8 Hz, 2H), 5.11 (s, 2H), 4.67 (d, J =
2.8 Hz, 2H), 2.51 (t, J = 2.4 Hz, 1H), 2.23 (m, 2H), 2.02 (m, 2H),
1.93 (m, 4H), 1.87 (m, 4H), 1.72 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 176.9, 155.6, 143.1, 138.2, 128.8, 128.0, 127.9, 126.7, 125.9,
114.5, 78.8, 75.4, 65.0, 55.8, 44.3, 42.2, 42.1, 41.9, 38.1, 35.9, 35.6,
28.7; HRMS [M + H] calcd [C29H28F3N2O3], 509.2020; found,
509.2023; purity >98% (as determined by RP-HPLC, method A, tR =
30.45 min; method B, tR = 30.11 min).
4-Adamantan-1-yl-2-iodophenol (34). A solution of 4-adaman-
tyl-phenol (33) (4.00 g, 17.5 mmol), potassium iodide (2.9 g, 17.5
mmol), and sulfuric acid (1.40 mL, 26.2 mmol) in methanol (400 mL)
was stirred at 0 °C while hydrogen peroxide (30%) (4.0 mL, 35.0
mmol) was added dropwise. Stirring was continued for 12 h at
ambient temperature. Then, the solvent was removed under reduced
pressure. The residue was diluted with concentrated sodium bisulfite
(100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined
organic layers were washed with brine, dried over MgSO4, filtered, and
concentrated under reduced pressure. Flash chromatography of the
residue over silica gel (hexane:EtOAc = 2:8) afforded 4-adamantan-1-
2-{4-[3-({4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzyloxy}-
carbonyl)adamantan-1-y]phenoxy}acetic Acid (32). The ethyl-2-
{4-[3-({4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyloxy}carbonyl)-
adamantan-1-y]phenoxy}acetate (31) (0.2 mg, 0.36 mmol) was
dissolved in 20 mL of THF, and LiOH·H2O was added and stirred
at room temperature, which was monitored by TLC. The excess
solvent was evaporated and washed with 10% HCl. The mixture was
extracted with EA and dried over MgSO4. The filtrate was
concentrated to give crude product 2-{4-[3-({4-[3-(trifluoromethyl)-
3H-diazirin-3-yl]benzyloxy}carbonyl)adamantan-1-y]phenoxy}acetic
acid without further purification.
1
yl-2-iodophenol as a white solid (5.2 g, 83.8% yield). H NMR (400
MHz, CDCl3) δ 7.61 (s, 1H), 7.23 (d, J = 8.0 Hz, 1H), 6.93 (d, J =
12.0 Hz, 1H), 5.10 (s, 1H), 2.08 (brs, 3H), 1.85−1.82 (m, 6H), 1.76−
1.70 (m, 6H); MS (ESI) m/z 353 (M + H).
4-Adamantan-1-yl-2-iodo-1-methoxybenzene (35a). To a
mixture of 4-adamantan-1-yl-2-iodophenol (34) (5.00 g, 14.1 mmol)
and K2CO3 (5.8 g, 42.3 mmol) in acetone (50 mL) was added methyl
iodide (1.75 mL, 28.2 mmol) dropwise at 0 °C, and then, the mixture
was stirred at room temperature for 8 h. After completion of the
reaction, evaporated acetone and the reaction mixture were partitioned
between CH2Cl2 and brine. The organic layer was separated, washed
with water, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The resulting residue was purified by silica gel column
chromatography (hexane:EtOAc = 1:9) to give 4-adamantan-1-yl-2-
iodo-1-methoxybenzene as a white solid (5.05 g, 97.3% yield). 1H
NMR (400 MHz, CDCl3) δ 7.73 (s, 1H), 7.28 (d, J = 8.0 Hz, 1H),
6.77 (d, J = 8.0 Hz, 1H), 3.85 (s, 3H), 2.08 (brs, 3H), 1.85−1.82 (m,
6H), 1.76−1.70 (m, 6H); MS (ESI) m/z 369 (M + H).
1-(5-Adamantan-1-yl-2-methoxyphenyl)-2,2,2-trifluoroetha-
none (36a). To a stirred solution of 4-adamantan-1-yl-2-iodo-1-
methoxybenzene (35a) (5.0 g, 13.57 mmol) in THF (50 mL) at −78
°C was added dropwise 1.6 M solution of n-butyllithium in hexane
(12.7 mL, 20.35 mmol) over a 1 h period. After stirring the mixture at
the same temperature for 8 h, a solution of 1-trifluoroacetyl piperidine
(2.4 mL, 16.25 mmol) in THF (10 mL) was added dropwise over 1 h.
Following the addition, the mixture stirred at −78 °C for a further 3 h
before the reaction was quenched with an aqueous solution of NH4Cl.
Ether was added, and the organic layer was extracted three times with
ether (3 × 50 mL). The combined organic layers were washed with
brine, dried over MgSO4, filtered, and concentrated under reduced
pressure. Flash chromatography of the residue over silica gel
(hexane:EtOAc = 2:8) afforded 1-(5-adamantan-1-yl-2-methoxyphen-
Prop-2-ynyl-4-hydroxy-3-(2-{4-[3-({4-[3-(trifluoromethyl)-
3H-diazirin-3-yl]benzyloxy}carbonyl)adamantan-1-yl]-
phenoxy}acetamido)benzoate (4i). The mixture of 2-{4-[3-({4-[3-
(trifluoromethyl)-3H-diazirin-3-yl]benzyloxy}carbonyl)adamantan-1-
y]phenoxy}acetic acid (32) (96.6 mg, 0.18 mmol), HBTU (104 mg,
0.27 mmol), and DIPEA (35 mg, 0.25 mmol) was dissolved in 4 mL of
DMF and MC. The solution was stirred at room temperature for 30
min, prop-2-ynyl 3-amino-4-hydroxybenzoate (15) (52 mg, 0.27
mmol) was added, and the mixture was stirred overnight. The excess
solvent was removed under reduced pressure, and the residue was
washed with water and 10% HCl. The mixture was extracted with EA
and dried over MgSO4. The filtrate was concentrated and purified by
column chromatography, which gave prop-2-ynyl-4-hydroxy-3-(2-{4-
[3-({4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyloxy}carbonyl)-
adamantan-1-yl]phenoxy}acetamido)benzoate as a pale yellow solid
1
(25 mg, 19.8% yield). H NMR (500 MHz, CDCl3) δ 9.80 (s, 1H),
8.58 (s, 1H), 7.88 (dd, J = 2.0 Hz, 8.5 Hz, 1H), 7.76 (d, J = 2.0 Hz,
1H), 7.34−7.37 (m, 3H), 7.19 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.5 Hz,
2H), 6.97 (d, J = 8.5 Hz, 2H), 5.12 (s, 2H), 4.90 (d, J = 2.5 Hz, 2H),
4.69 (s, 2H), 2.50 (t, J = 2.5 Hz, 1H), 2.03 (m, 2H), 1.94 (m, 6H),
1.88 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 176.8, 168.8, 164.9,
154.6, 153.7, 144.7, 138.2, 129.6, 128.0, 126.7, 126.5, 124.6, 124.5,
121.6, 120.2, 114.6, 75.0, 67.2, 65.1, 52.5, 44.2, 42.2, 41.9, 38.1, 36.1,
9534
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Journal of Medicinal Chemistry
Article
yl)-2,2,2-trifluoroethanone as a pale yellow semisolid (3.45 g, 75.2%
yield). H NMR (400 MHz, CDCl3) δ 7.65 (s, 1H), 7.61 (d, J = 8.0
CH2Cl2 (25.0 mL) was added p-toluene sulfonyl chloride (2.60 g,
13.68 mmol) at 0 °C, and then, the mixture was stirred at room
temperature for 8 h. After completion of the reaction, the reaction
mixture was partitioned between CH2Cl2 and brine. The organic layer
was separated, washed with water, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The resulting residue was purified
by silica gel column chromatography (hexane:EtOAc = 1:9) to give
2,2,2-trifluoro-1-(2-methoxyphenyl)ethanone O-tosyl oxime as a white
solid (3.6 g, 84.5% yield). 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J =
8.0 Hz, 2H), 7.49 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.09 (d,
J = 8.0 Hz, 1H), 7.02−6.92 (m, 2H), 3.79 (s, 3H), 2.48 (s, 3H); MS
(ESI) m/z 374 (M + H).
3-(5-Adamantan-1-yl-2-methoxyphenyl)-3-(trifluoromethyl)
Diaziridine (39a). Liquid ammonia (6.0 mL) was added at −78 °C to
an ether (30.0 mL) solution of 1-(5-adamantan-1y-l-2-methoxyphen-
yl)-2,2,2-trifluoroethanone O-tosyl oxime (38a) (3.0 g, 5.91 mmol) in
a sealed tube. The solution was stirred at room temperature for 16 h.
The mixture was carefully cooled to −78 °C, and the sealed tube was
opened. The ammonia was evaporated, and white precipitate was
filtered and washed with ether. The filtrate was concentrated in vacuo
to give 3-(5-adamantan-1-yl-2-methoxyphenyl)-3-(trifluoromethyl)
diaziridine as a pale yellow solid (1.7 g, 81.7% yield). 1H NMR
(500 MHz, CDCl3) δ 7.44 (s, 1H), 7.38 (d, J = 10.0 Hz, 1H), 6.88 (d,
J = 10.0 Hz, 1H), 3.85 (s, 3H), 2.67 (d, J = 10.0 Hz, 1H), 2.47 (d, J =
10.0 Hz, 1H), 2.09 (brs, 3H), 1.88−1.86 (m, 6H), 1.77−1.72 (m, 6H);
MS (ESI) m/z 353 (M + H).
1
Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 3.89 (s, 3H), 2.17 (brs, 3H), 1.85−
1.82 (m, 6H), 1.78−1.72 (m, 6H); MS (ESI) m/z 339 (M + H).
2,2,2-Trifluoro-1-(2-methoxyphenyl)ethanone (36b). To a
stirred solution of 1-iodo-2-methoxybenzene (35b) (5.0 g, 21.35
mmol) in THF (50 mL) at −78 °C was added dropwise 1.6 M
solution of n-butyllithium in hexane (24.8 mL, 32.0 mmol) over a 1 h
period. After stirring the mixture at the same temperature for 8 h, a
solution of 1-trifluoroacetyl piperidine (3.75 mL, 25.6 mmol) in THF
(11 mL) was added dropwise over 1 h. Following the addition, the
mixture was stirred at −78 °C for a further 3 h before the reaction was
quenched with an aqueous solution of NH4Cl. Ether was added, and
the organic layer was extracted three times with ether (3 × 50 mL).
The combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. Flash
chromatography of the residue over silica gel (hexane:EtOAc = 2:8)
afforded 2,2,2-trifluoro-1-(2-methoxyphenyl)ethanone as a pale yellow
1
semisolid (3.15 g, 72.4% yield). H NMR (400 MHz, CDCl3) δ 7.67
(d, J = 8.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.07−7.03 (m, 2H), 3.98
(s, 3H); MS (ESI) m/z 205 (M + H).
1-(5-Adamantan-1-yl-2-methoxyphenyl)-2,2,2-trifluoroetha-
none Oxime (37a). To a stirred solution of 1-(5-adamantan-1-yl-2-
methoxyphenyl)-2,2,2-trifluoroethanone (36a) (3.2 g, 9.45 mmol) in
pyridine (32 mL) was added hydroxylamine hydrochloride (1.31 g,
18.91 mmol). Then, the mixture was refluxed for 4 h. The pyridine was
evaporated, 25 mL of an aqueous solution of citric acid (10%) and
CH2Cl2 was added to the residue, and the organic layer was extracted.
The organic layer was separated, washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography
(hexane:EtOAc = 7:3) to give 1-(5-adamantan-1-yl-2-methoxyphen-
yl)-2,2,2-trifluoroethanone oxime as a white solid (2.4 g, 70.6% yield).
1H NMR (500 MHz, CDCl3) δ 8.33 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H),
3-(2-Methoxyphenyl)-3-(trifluoromethyl)diaziridine (39b).
Liquid ammonia (6.0 mL) was added at −78 °C to an ether (30.0
mL) solution of 2,2,2-trifluoro-1-(2-methoxyphenyl)ethanone O-tosyl
oxime (38b) (3.0 g, 8.03 mmol) in a sealed tube. The solution was
stirred at room temperature for 16 h. The mixture was carefully cooled
to −78 °C, and the sealed tube was opened. The ammonia was
evaporated, and white precipitate was filtered and washed with ether.
The filtrate was concentrated in vacuo to give 3-(2-methoxyphenyl)-3-
(trifluoromethyl)diaziridine as a pale yellow solid (1.5 g, 85.7% yield).
1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 8.0 Hz, 1H), 7.41 (t, J =
7.22 (s, 1H), 6.88 (d, J = 8.0 Hz, 1H), 3.82 (s, 3H), 2.09 (brs, 3H),
1.89−1.88 (m, 6H), 1.79−1.74 (m, 6H); MS (ESI) m/z 354 (M + H).
2,2,2-Trifluoro-1-(2-methoxyphenyl)ethanone Oxime (37b).
To a stirred solution of 2,2,2-trifluoro-1-(2-methoxyphenyl)ethanone
(36b) (3.0 g, 14.69 mmol) in pyridine (30.0 mL) was added
hydroxylamine hydrochloride (3.06 g, 44.08 mmol). Then, the mixture
was refluxed for 4 h. The pyridine was evaporated, 50 mL of an
aqueous solution of citric acid (10%) and CH2Cl2 was added to the
residue, and the organic layer was extracted. The organic layer was
separated, washed with brine, dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. The resulting residue was purified by silica
gel column chromatography (hexane:EtOAc = 1:9) to give 2,2,2-
trifluoro-1-(2-methoxyphenyl)ethanone oxime as a white solid (2.9 g,
90.0% yield). 1H NMR (400 MHz, CDCl3) δ 8.85 (s, 1H), 7.44 (d, J =
8.0 Hz, 1H), 7.01 (m, 2H), 3.84 (s, 3H); MS (ESI) m/z 220 (M + H).
1-(5-Adamantan-1y-l-2-methoxyphenyl)-2,2,2-trifluoroetha-
none O-Tosyl Oxime (38a). To a mixture of 1-(5-adamantan-1-yl-2-
methoxyphenyl)-2,2,2-trifluoroethanone oxime (37a) (2.8 g, 7.92
mmol), 4-dimethylaminopyridine (0.48 g, 3.96 mmol), and triethyl-
amine (1.65 mL, 11.88 mmol) in CH2Cl2 (28 mL) was added p-
toluene sulfonyl chloride (1.81 g, 9.50 mmol) at 0 °C, and then, the
mixture was stirred at room temperature for 8 h. After completion of
the reaction, the reaction mixture was partitioned between CH2Cl2 and
brine. The organic layer was separated, washed with water, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography
(hexane:EtOAc = 1:9) to give 1-(5-adamantan-1y-l-2-methoxyphen-
yl)-2,2,2-trifluoroethanone O-tosyl oxime as a white solid (3.2 g, 79.6%
8.0 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 3.79 (s,
3H), 2.69(d, J = 10.0 Hz, 1H), 2.47(d, J = 10.0 Hz,1H); MS (ESI) m/
z 219 (M + H).
3-(5-Adamantan-1-yl-2-methoxyphenyl)-3-(trifluorometh-
yl)-3H-diazirine (40a). To a mixture of 3-(5-adamantan-1-yl-2-
methoxyphenyl)-3-(trifluoromethyl)diaziridine (39a) (1.6 g, 4.54
mmol) and triethylamine (1.90 mL, 13.62 mmol) in methanol (16
mL) was added iodine (1.37 g, 4.99 mmol) at 0 °C, and then, the
mixture was stirred at room temperature for 3 h. After completion of
the reaction, aqueous citric acid 10% (10 mL) and Na2S2O3 were
added. The organic layer was extracted three times with ether. The
combined organic layers were washed with brine, dried over MgSO4,
filtered, and concentrated under reduced pressure. Flash chromatog-
raphy of the residue over silica gel (hexane:EtOAc = 9.5:0.5) afforded
3-(5-adamantan-1-yl-2-methoxyphenyl)-3-(trifluoromethyl)-3H-diazir-
1
ine as a pale yellow solid (1.5 g, 94.3% yield). H NMR (400 MHz,
CDCl3) δ 7.70 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 8.0 Hz,
1H), 3.85 (s, 3H), 2.09 (brs, 3H), 1.88−1.86 (m, 6H), 1.76−1.72 (m,
6H); MS (ESI) m/z 351 (M + H).
3-(2-Methoxyphenyl)-3-(trifluoromethyl)-3H-diazirine (40b).
To a mixture of 3-(2-methoxyphenyl)-3-(trifluoromethyl)diaziridine
(39b) (1.4 g, 6.41 mmol) and triethylamine (2.68 mL, 19.25 mmol) in
methanol (14 mL) was added iodine (1.79 g, 7.05 mmol) at 0 °C, and
then, the mixture was stirred at room temperature for 3 h. After
completion of the reaction, aqueous citric acid 10% (10 mL) and
Na2S2O3 were added. The organic layer was extracted three times with
ether. The combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. Flash
chromatography of the residue over silica gel (hexane:EtOAc =
9.5:0.5) afforded 3-(2-methoxyphenyl)-3-(trifluoromethyl)-3H-diazir-
1
yield). H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.0 Hz, 2H), 7.44
(d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.03 (s, 1H), 6.88 (d, J =
8.0 Hz, 1H), 3.74 (s, 3H), 2.48 (s, 3H), 2.09 (brs, 3H), 1.85−1.82 (m,
6H), 1.76−1.70 (m, 6H); MS (ESI) m/z 508 (M + H).
2,2,2-Trifluoro-1-(2-methoxyphenyl)ethanone O-Tosyl
Oxime (38b). To a mixture of 2,2,2-trifluoro-1-(2-methoxyphenyl)-
ethanone oxime (37b) (2.5 g, 11.40 mmol), 4-dimethylaminopyridine
(0.69 g, 5.7 mmol), and triethylamine (2.38 mL, 17.11 mmol) in
1
ine as a pale yellow oil (1.2 g, 86.9% yield). H NMR (400 MHz,
CDCl3) δ 7.47 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.00 (t, J =
8.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 3.93 (s, 3H); MS (ESI) m/z 217
(M + H).
9535
dx.doi.org/10.1021/jm501241g | J. Med. Chem. 2014, 57, 9522−9538

Journal of Medicinal Chemistry
Article
4-Adamantan-1-yl-2-(3-(trifluoromethyl)-3H-diazirin-3-yl)-
phenol (41a). To a solution of 3-(5-adamantan-1-yl-2-methoxyphen-
yl)-3-(trifluoromethyl)-3H-diazirine (40a) (1.4 g, 3.99 mmol) in
CH2Cl2 (14 mL) was added BBr3 (0.45 mL, 4.79 mmol) in CH2Cl2
(10 mL) through a septum using a syringe at −10 °C. After stirring for
30 min at the same temperature, the cold bath was removed, and the
mixture was stirred for 1.5 h. After completion of the reaction, the
mixture was partitioned between CH2Cl2 and water, and the organic
layer was washed with brine, dried, and concentrated. The resulting
residue was purified by silica gel column chromatography
(hexane:EtOAc = 9.5:0.5), which afforded 4-adamantan-1-yl-2-(3-
(trifluoromethyl)-3H-diazirin-3-yl)phenol as a pale yellow semisolid
vacuo to obtain 2-(4-adamantan-1-yl-2-(3-(trifluoromethyl)-3H-diazir-
in-3-yl)phenoxy)acetic acid as a white solid (0.1 g, 55.5% yield). H
NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H),
6.94 (d, J = 8.0 Hz, 1H), 4.75 (s, 2H), 2.12 (brs, 3H), 1.91−1.88 (m,
6H), 1.79−1.76 (m, 6H); MS (ESI) m/z 395 (M+).
1
2-(2-(3-(Trifluoromethyl)-3H-diazirin-3-yl)phenoxy)acetic
Acid (43b). A solution of ethyl 2-(2-(3-(trifluoromethyl)-3H-diazirin-
3-yl)phenoxy)acetate (42b) (0.12 g, 0.41 mmol) in THF/H2O (3:1, 2
mL) was treated with lithium hydroxide monohydrate (0.07 g, 1.66
mmol) and stirred at room temperature until the reaction was
complete as judged by TLC. The reaction mixture was then acidified
with 10% HCl to pH 4 and then partitioned between EtOAc and
brine. The organic layer was separated, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo to obtain 2-(2-(3-(trifluoromethyl)-
3H-diazirin-3-yl)phenoxy)acetic acid as a white solid (0.08 g, 74.0%
1
(0.6 g, 44.7% yield). H NMR (400 MHz, CDCl3) δ 10.93 (s, 1H),
7.71 (m, 2H), 7.10 (d, J = 8.0 Hz, 1H), 2.08 (brs, 3H), 1.85−1.82 (m,
6H), 1.76−1.70 (m, 6H); MS (ESI) m/z 337 (M + H).
1
yield). H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.0 Hz, 1H), 7.35
2-(3-(Trifluoromethyl)-3H-diazirin-3-yl)phenol (41b). To a
solution of 3-(2-methoxyphenyl)-3-(trifluoromethyl)-3H-diazirine
(40b) (1.0 g, 4.62 mmol) in CH2Cl2 (10 mL) was added BBr3
(0.52 mL, 5.55 mmol) in CH2Cl2 (10 mL) through a septum using a
syringe at −10 °C. After stirring for 30 min at the same temperature,
the cold bath was removed, and the mixture was stirred for 1.5 h. After
completion of the reaction, the mixture was partitioned between
CH2Cl2 and water, and the organic layer was washed with brine, dried,
and concentrated. The resulting residue was purified by silica gel
column chromatography (hexane:EtOAc = 9.5:0.5), which afforded 2-
(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol as a pale yellow oil (0.35
g, 37.4% yield). 1H NMR (400 MHz, CDCl3) δ 11.06 (s, 1H), 7.62 (d,
J = 8.0 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.93
(d, J = 8.0 Hz, 1H); MS (ESI) m/z 203 (M + H).
(t, J = 8.0 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H),
4.66 (s, 2H); MS (ESI) m/z 261 (M + H).
Prop-2-ynyl 3-(2-(4-Adamantan-1-yl-2-(3-(trifluoromethyl)-
3H-diazirin-3-yl)phenoxy)acetamido)-4-hydroxybenzoate (4a).
To a solution of 2-(4-adamantan-1-yl-2-(3-(trifluoromethyl)-3H-
diazirin-3-yl)phenoxy)acetic acid (43a) (0.08 g, 0.20 mmol) and
prop-2-ynyl 3-amino-4-hydroxybenzoate (15) (0.04 g, 0.20 mmol) in
DMF (2.0 mL) were added EDC·HCl (0.046 g, 0.24 mmol), HOBt
(0.032 g, 0.24 mmol), and DIPEA (0.09 mL, 0.5 mmol). The reaction
mixture was stirred overnight at room temperature and then
partitioned between EtOAc and brine. The organic layer was
separated, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The resulting residue was purified by silica gel column
chromatography (hexane:EtOAc = 6:4) to give prop-2-ynyl 3-(2-(4-
adamantan-1-yl-2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenoxy)-
acetamido)-4-hydroxybenzoate as a pale yellow solid (0.02 g, 18.2%
Ethyl 2-(4-Adamantan-1-yl-2-(3-(trifluoromethyl)-3H-diazir-
in-3-yl)phenoxy)acetate (42a). A suspension of 4-adamantan-1-yl-
2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol (41a) (0.5 g, 1.48
mmol), anhydrous potassium carbonate (0.61 g, 4.45 mmol), and
ethyl chloroacetate (0.32 mL, 2.97 mmol) in DMF (5 mL) was stirred
overnight at room temperature. The reaction mixture was diluted with
ethyl acetate and subsequently washed with aqueous sodium
bicarbonate, brine, and water. The organic layer was dried over
anhydrous MgSO4. The solvent was filtered and evaporated under
reduced pressure to afford a crude solid, which was purified by silica
gel column chromatography (hexane:EtOAc = 3:1) to give ethyl 2-(4-
adamantan-1-yl-2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenoxy)-
1
yield). H NMR (400 MHz, CDCl3) δ 10.10 (s, 1H), 10.00 (s, 1H),
8.08 (s, 1H), 7.94 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0
Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.91 (t, J =
4.0 Hz, 2H), 4.79 (s, 2H), 2.51 (t, J = 4.0 Hz, 1H), 2.12 (brs, 3H),
1.91−1.88 (m, 6H), 1.79−1.76 (m, 6H); 13C NMR (100 MHz,
CDCl3) δ 167.8, 164.9, 156.1, 154.2, 145.7, 134.4, 129.6, 129.1, 125.0,
124.9, 121.8, 120.1, 118.7, 115.0, 113.6, 77.7, 74.2, 67.3, 60.4, 52.4,
43.0, 36.5, 35.9, 29.7; HRMS [M + H] calcd [C30H29F3N3O5],
567.1915; found, 567.1909; purity >99.9% (as determined by RP-
HPLC, method A, tR = 23.83 min; method B, tR = 22.95 min).
Prop-2-ynyl-4-hydroxy-3-(2-(2-(3-(trifluoromethyl)-3H-dia-
zirin-3-yl)phenoxy)acetamido)benzoate (4b). To a solution of 2-
(2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenoxy)acetic acid (43b)
(0.06 g, 0.23 mmol) and prop-2-ynyl 3-amino-4-hydroxybenzoate
(15) (0.04 g, 0.23 mmol) in DMF (2.0 mL) were added EDC·HCl
(0.052 g, 0.27 mmol), HOBt (0.037 g, 0.27 mmol), and DIPEA (0.1
mL, 0.57 mmol). The reaction mixture was stirred at room
temperature overnight and then partitioned between EtOAc and
brine. The organic layer was separated, dried over anhydrous MgSO4,
filtered, and concentrated in a vacuum. The resulting residue was
purified by silica gel column chromatography (hexane:EtOAc = 7:3) to
give prop-2-ynyl 4-hydroxy-3-(2-(2-(3-(trifluoromethyl)-3H-diazirin-
3-yl)phenoxy)acetamido)benzoate as a white solid (0.02 g, 22.2%
yield). 1H NMR (400 MHz, CDCl3) δ 9.80 (s, 1H), 8.62 (s, 1H), 7.89
(d, J = 8.0 Hz, 1H), 7.78 (s, 1H), 7.39 (t, J = 8.0 Hz, 2H), 7.10 (t, J =
8.0 Hz, 1H), 7.02 (d, J = 8.0 Hz, 2H), 4.90 (s, 2H), 4.71 (s, 2H), 2.58
(t, J = 4.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 167.8, 164.9,
156.1, 154.2, 129.6, 129.1, 125.0, 124.9, 121.8, 120.1, 115.0, 113.6,
77.7, 74.2, 67.3, 60.4, 52.4; HRMS [M + H]+ calcd [C20H14F3N3O5],
434.2331; found, 434.2321; purity >99.9% (as determined by RP-
HPLC, method A, tR = 26.16 min; method B, tR = 25.38 min).
Cell Culture. Human colorectal carcinoma HCT116 cells were
cultured in a 5% CO2 atmosphere at 37 °C in Dulbecco’s modified
Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 5%
fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL
streptomycin (Gibco). Cells were seeded at a density of 5 × 105 cells/
mL/well in a 12-well tissue culture plate at 37 °C for 20 h for
subsequent experiments. Hypoxic conditions were achieved by
1
acetate as a white solid (0.3 g, 48.4% yield). H NMR (400 MHz,
CDCl3) δ 7.70 (s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz,
1H), 4.69 (s, 2H), 4.27 (q, J = 7.2 Hz, 2H), 2.10 (brs, 3H), 1.87−1.84
(m, 6H), 1.81−1.78 (m, 6H), 1.30 (t, J = 8.0 Hz, 3H); MS (ESI) m/z
423 (M + H).
Ethyl 2-(2-(3-(Trifluoromethyl)-3H-diazirin-3-yl)phenoxy)-
acetate (42b). A suspension of 2-(3-(trifluoromethyl)-3H-diazirin-
3-yl)phenol (41b) (0.3 g, 1.48 mmol), anhydrous potassium carbonate
(0.61 g, 4.45 mmol), and ethyl chloroacetate (0.32 mL, 2.97 mmol) in
DMF (3 mL) was stirred overnight at room temperature. The reaction
mixture was diluted with ethyl acetate and subsequently washed with
aqueous sodium bicarbonate, brine, and water. The organic layer was
dried over anhydrous MgSO4. The solvent was filtered and evaporated
under reduced pressure to afford a crude solid, which was purified by
silica gel column chromatography (hexane:EtOAc = 3:1) to give ethyl
2-(2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenoxy)acetate as a white
solid (0.14 g, 33.33% yield). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J
= 8.0 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.92
(d, J = 8.0 Hz, 1H), 4.60 (s, 2H), 4.24 (q, J = 7.2 Hz, 2H), 1.30 (t, J =
8.0 Hz, 3H); MS (ESI) m/z 289 (M + H).
2-(4-Adamantan-1-yl-2-(3-(trifluoromethyl)-3H-diazirin-3-
yl)phenoxy)acetic Acid (43a). A solution of ethyl 2-(4-adamantan-
1-yl-2-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenoxy)acetate (42a)
(0.2 g, 0.47 mmol) in THF/H2O (3:1, 2 mL) was treated with
lithium hydroxide monohydrate (0.07 g, 1.89 mmol) and stirred at
room temperature until the reaction was complete as judged by TLC.
The reaction mixture was then acidified with 10% HCl to pH 4 and
then partitioned between EtOAc and brine. The organic layer was
separated, dried over anhydrous MgSO4, filtered, and concentrated in
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replacing cells with 1% O2, 94% N2, and 5% CO2 in a multigas
incubator (Sanyo, Osaka, Japan).
Kinetic Assay of MDH2. The enzyme activity of MDH2 was
measured by oxaloacetate-dependent NADH oxidation assays, where
the NADH concentration was determined by measuring absorbance at
340 nm. The reaction was performed in 100 mM potassium phosphate
buffer (pH 7.4) with 0.25 nM His-MDH2, 600 μM oxaloacetic acid,
and various concentrations of NADH (60, 75, 100, 150, and 300 μM).
The Vmax and Km were determined from double-reciprocal
Lineweaver−Burk plots using Sigmaplot 13.0, and a graph was plotted
for the velocity against the concentration of NADH.
HRE-Luciferase Reporter Assay. HCT116 cells expressing both a
hypoxia response element (HRE)-dependent firefly luciferase reporter
and a CMV-renilla luciferase reporter were cultured in DMEM (Gibco,
Carlsbad, CA) supplemented with 5% fetal bovine serum (Gibco,
Carlsbad, CA), 100 U/mL penicillin, and 100 μg/mL streptomycin
(Gibco).18 The cells (2 × 104 cells/0.1 mL/well) were seeded in a 96-
well tissue culture plate for 20 h for subsequent experiments. The cells
were incubated for 12 h with or without drugs in normoxic or hypoxic
conditions (1% O2, 94% N2, and 5% CO2). The luciferase activities of
cells were measured using a Dual-Luciferase Assay System (Promega,
Madison, WI) with a Victor X Light luminescence reader
(PerkinElmer, Boston, MA).
Immunoblot Analysis. Cells were washed with PBS, lysed with
RIPA buffer (20 mM HEPES (pH 7.4), 1% Triton X-100, 10%
glycerol, 1 mM EDTA, 5 mM sodium fluoride, 10 μg/mL
phenylmethylsulfonylfluoride (PMSF), and 1 mM sodium vanadate)
for 15 min at 4 °C, and centrifuged at 13 000 rpm for 15 min. Lysates
were then boiled for 5 min in 5× sample buffer (50 mM Tris (pH 7.4),
4% sodium dodecyl sulfate (SDS), 10% glycerol, 4% 2-thioethanol, and
50 μg/mL bromophenol blue) at a ratio of 4:1. Protein samples were
subjected to SDS-polyacrylamide gel electrophoresis (PAGE), trans-
ferred to PVDF membranes (Millipore, Billerica, MA), and
immunoblotted with anti-HIF-1α (BD Transduction Laboratories,
San Diego, CA) or anti-β-actin (Abcam, Cambridge, U.K.). Protein
expression was visualized on Kodak Biomax X-ray film (Kodak,
Rochester, NY).
MDH2 Activity Assay. The activity of MDH2 was determined
using a MDH2 activity assay kit (Abcam) according to the
manufacturer’s instructions. Cells were lysed with RIPA buffer
containing protease and phosphatase inhibitors. A 100 μL portion of
proteins (100 μg/mL) was incubated for 3 h at room temperature in
each well. After washing three times with washing buffer, 50 μL of drug
diluted in base buffer was added to the well. This was preincubated for
1 h at room temperature and then for 3 h at 4 °C. The assay reagents
(the final concentrations of malate and NAD+ were 5 and 1 mM,
respectively) were added to the well, and the absorbance at 450 nm
was measured at 30 and 60 min.
In Vitro MDH2 Binding Assay. The recombinant human MDH2
(BioVision, Mountainview, CA, USA, 2 μg) was incubated with
probes, and then photoaffinity labeling was performed by irradiation
with 360 nm UV radiation (UVP, Upland, CA, USA) for 30 min on
ice. Click reactions of the probe and Cy3 azide (Click Chemistry
Tools, Scottsdale, AZ, USA) were established with the Click-iT
Protein Reaction Buffer Kit (Invitrogen, Carlsbad, CA, USA)
according to the manufacturer’s instructions. After the click reaction,
proteins were precipitated with methanol/chloroform/water (60/15/
40, v/v) and denatured by boiling for 5 min in a sample buffer.
Proteins were separated by SDS-PAGE, and fluorescence was detected
with a Typhoon 9410 imaging system (GE Healthcare, Piscataway, NJ,
USA).
ASSOCIATED CONTENT
* Supporting Information
Pull-down assay with biotin probes. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
■
*Address: BK21Plus R-FIND Team, College of Pharmacy,
Dongguk University-Seoul, 35, Dongguk-ro, Ilsandong-gu,
Goyang 410-820, Republic of Korea. Phone: +82-31-961-
5214. Fax: +82-31-961-5206. E-mail: kaylee@dongguk.edu.
Notes
The authors declare no competing financial interest.
∥R.N., M.W., H.S.B.: Co-first authors.
ACKNOWLEDGMENTS
■
This study was supported by National Research Foundation
(NRF) grants (2012M3A9C1053532, 2012M3A9B4028787,
and 2014R1A2A2A01005455) funded by Ministry of Science,
ICT and Future Planning, Korea, and KRIBB Initiative of the
Korea Research Council of Fundamental Science and
Technology. This work was inspired by the international and
interdisciplinary environments of the JSPS Asian CORE
Program, “Asian Chemical Biology Initiative”.
ABBREVIATIONS USED
■
HIF, hypoxia inducible factor; MDH2, malate dehydrogenase 2;
2D, two-dimensional; HRE, hypoxia-response element; AMPK,
AMP activated protein kinase; ACC, acetyl-CoA carboxylase;
ABPs, activity-based assay; SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis; NBS, N-bromosuccinimide;
br s, broad signal; n-BuLi, n-butyllithium; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; rt, room
temperature; THF, tetrahydrofuran
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