Removal of metabolic liabilities enables development of derivatives of procaspase-activating compound 1 (PAC-1) with improved pharmacokinetics

Article abstract of DOI:10.1021/acs.jmedchem.5b00413

Procaspase-activating compound 1 (PAC-1) is an o-hydroxy-N-acylhydrazone that induces apoptosis in cancer cells by chelation of labile inhibitory zinc from procaspase-3. PAC-1 has been assessed in a wide variety of cell culture experiments and in vivo models of cancer, with promising results, and a phase 1 clinical trial in cancer patients has been initiated (NCT02355535). For certain applications, however, the in vivo half-life of PAC-1 could be limiting. Thus, with the goal of developing a compound with enhanced metabolic stability, a series of PAC-1 analogues were designed containing modifications that systematically block sites of metabolic vulnerability. Evaluation of the library of compounds identified four potentially superior candidates with comparable anticancer activity in cell culture, enhanced metabolic stability in liver microsomes, and improved tolerability in mice. In head-to-head experiments with PAC-1, pharmacokinetic evaluation in mice demonstrated extended elimination half-lives and greater area under the curve values for each of the four compounds, suggesting them as promising candidates for further development.

Full text of DOI:10.1021/acs.jmedchem.5b00413

Article
pubs.acs.org/jmc
Removal of Metabolic Liabilities Enables Development of Derivatives
of Procaspase-Activating Compound 1 (PAC-1) with Improved
Pharmacokinetics
Howard S. Roth,† Rachel C. Botham,† Steven C. Schmid,† Timothy M. Fan,‡ Levent Dirikolu,§
and Paul J. Hergenrother*,†
†Department of Chemistry, ‡Department of Veterinary Clinical Medicine, and §Department of Veterinary Biosciences, University of
Illinois, Urbana, Illinois 61801, United States
S
* Supporting Information
ABSTRACT: Procaspase-activating compound 1 (PAC-1) is an o-hydroxy-N-acylhydrazone that induces apoptosis in cancer
cells by chelation of labile inhibitory zinc from procaspase-3. PAC-1 has been assessed in a wide variety of cell culture
experiments and in vivo models of cancer, with promising results, and a phase 1 clinical trial in cancer patients has been initiated
(NCT02355535). For certain applications, however, the in vivo half-life of PAC-1 could be limiting. Thus, with the goal of
developing a compound with enhanced metabolic stability, a series of PAC-1 analogues were designed containing modifications
that systematically block sites of metabolic vulnerability. Evaluation of the library of compounds identified four potentially
superior candidates with comparable anticancer activity in cell culture, enhanced metabolic stability in liver microsomes, and
improved tolerability in mice. In head-to-head experiments with PAC-1, pharmacokinetic evaluation in mice demonstrated
extended elimination half-lives and greater area under the curve values for each of the four compounds, suggesting them as
promising candidates for further development.
the overexpression of procaspase-3 in a number of cancer types,
including lymphoma,15,16 leukemia,17,18 melanoma,19,20 glio-
blastoma,21,22 pancreatic cancer,23 liver cancer,24 lung can-
cer,25−27 breast cancer,28−31 esophageal cancer,32 and colon
cancer,8,33−35 have made the small-molecule-mediated activa-
tion of procaspase-3 an attractive strategy for personalized
medicine.8−11
Procaspase-activating compound 1 (PAC-1, 1; Figure 1) was
identified via a high-throughput screen for compounds that
could enhance procaspase-3 enzymatic activity in vitro.8 PAC-1
is cytotoxic against a diverse array of cancer cells in culture,
including cell lines derived from hematopoietic tumors
(lymphoma,8,36−43 leukemia,8,38,42−47 and multiple myelo-
ma47), carcinomas of diverse origin (breast,8,38,42−46,48−50
renal,8 adrenal,8,51 colon,8,42,47,49,50,52 lung,8,42−50,52−55 cervi-
cal,38,47 gastric,42,43,47,49,50,52 ovarian,47 liver,42,43,47 prostate,42,43
INTRODUCTION
■
The development of personalized therapeutics has emerged as a
promising strategy in anticancer drug discovery. Translocation,
mutation, and abnormal expression of genes can produce
unique proteins that exist only in tumors, and the selective
modulation of these proteins can kill cancer cells with little
effect on healthy cells, minimizing adverse side effects.1 As
evasion of apoptosis is a hallmark of cancer,2,3 many recent
efforts to develop new anticancer drugs have focused on
inhibition of antiapoptotic proteins, including MDM2,4 Bcl-2,5,6
and XIAP.7 Similarly, small molecules capable of enhancing the
activity of proapoptotic proteins hold promise for the treatment
of cancer. One target that has received considerable attention is
procaspase-3,8−11 a member of the caspase family of proteases
critical to apoptosis. Both the intrinsic and extrinsic pathways of
apoptosis converge to activate executioner caspases-3, -6, and -7
from their proenzyme forms.12,13 The low frequency of
procaspase-3 mutations in cancer,14 its downstream location
relative to apoptotic proteins that are frequently mutated,13 and
Received: March 10, 2015
© XXXX American Chemical Society
A
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this direct procaspase-3 activation mechanism, cells treated with
PAC-1 or a derivative show cleaved procaspase-3 and poly-ADP
ribose polymerase-1 prior to release of cytochrome c from the
mitochondria or cleavage of initiator procaspases-8 and
-9,8,42,43,66 and PAC-1-mediated apoptosis occurs regardless
of the status of Bcl-2 family proteins.67,68 Because of this unique
mechanism, PAC-1 is increasingly being used as a tool to
directly activate procaspase-3 in a variety of biological
settings.66,67,69,70 In addition, PAC-1 and its derivatives have
shown synergy with experimental therapeutics18,48 and with the
anticancer drug paclitaxel.55
Despite the potential for promiscuity and/or instability with
certain o-hydroxy-N-acylhydrazones,71 PAC-1 shows minimal
inhibitory activity toward zinc-dependent enzymes, including
matrix metalloproteinases-9 and -14,72 and a derivative of PAC-
1 showed minimal inhibition toward carboxypeptidase A and
histone deacetylases.18 These results are consistent with the
known modest affinity of PAC-1 for zinc,36 allowing for a high
degree of selectivity for chelation of zinc ions from the labile
pool over essential zinc ions in canonical zinc-binding sites
within metalloproteins. In addition, PAC-1 is stable in aqueous
solution; degradation of PAC-1 is observed only when the
compound is subjected to extremes in temperature and pH
outside of relevant physiological ranges.73
Pharmacokinetic studies with PAC-1 in mice and dogs
revealed that serum concentrations of approximately 10 μM can
be achieved with few adverse events,39 with transient
neuroexcitation observed only at elevated doses when PAC-1
is administered via intravenous (iv) or intraperitoneal (ip)
injection.38 A phase 1 clinical trial of PAC-1 given orally to
cancer patients has been initiated (NCT02355535). A
sulfonamide-containing derivative of PAC-1, called S-PAC-1
(2; Figure 1), is well tolerated by mice at doses of 350 mg/kg
or higher via ip injection, with peak plasma concentrations of
3.5 mM at this dose.38 It is likely that the improved safety
profile is due in large part to the decreased ability of S-PAC-1
to cross the blood−brain barrier, as compared to PAC-1.41 In
addition to the ability of S-PAC-1 to induce cell death to a
variety of cancer cell lines in culture,38 recent efforts have
Figure 1. Structures of PAC-1 (1) and S-PAC-1 (2).
and gallbladder42,43), and other solid tumor histologies
(melanoma,8,38,42,43 osteosarcoma,47 neuroblastoma,8,47,49,50
and glioblastoma42,43). PAC-1 and its derivatives also induce
apoptosis in patient-derived samples from colon cancer8 and
chronic lymphocytic leukemia,18 and have anticancer efficacy in
multiple murine tumor models8,42,43,48,54−56 and in pet dogs
with cancer.38
The o-hydroxy-N-acylhydrazone was identified as the key
pharmacophore of PAC-1 through extensive studies of
structure−activity relationships (SARs).36,37 Several PAC-1
derivatives containing this motif have comparable activity in
vitro and in cell culture, but modification of the core results in a
loss of activity.37 The o-hydroxy-N-acylhydrazone is known to
chelate metals, including iron,57 copper,58 and zinc,59 and many
divalent metal cations are also known to inhibit procaspase36
and caspase60−63 enzymes. In particular, zinc from the labile
zinc pool, which is bound loosely to certain proteins, colocalizes
with procaspase-3 in cells64 and inhibits the enzymatic activity
of both procaspase-336 and caspase-3,61 and a putative binding
site on procaspase-3/caspase-3 for labile zinc ions has been
identified.65 The mechanism of action of PAC-1 most likely
involves the chelation of labile zinc from procaspase-3, relieving
the zinc-mediated inhibition and allowing procaspase-3 to
process itself to the active form.36,37,41 Using genetically
encoded zinc sensors, PAC-1 has been shown to mobilize the
labile zinc pool in cancer cells.41 Providing further support to
Figure 2. PAC-1 is susceptible to enzymatic oxidation in vitro and in vivo, giving metabolites that result from N-debenzylation, olefin oxidation, and
arene oxidation.75
B
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Figure 3. Nine hydrazides and five aldehydes were used to construct a library of 45 PAC-1 derivatives designed to display enhanced metabolic
stability by blocking oxidative N-debenzylation, olefin oxidation, and/or arene oxidation.
Scheme 1. Synthesis of PAC-1 Analogues: (a) Synthesis of Hydrazides 46a−i, (b) Synthesis of Aldehydes 47a−e, (c)
Condensation of Hydrazides and Aldehydes To Form PAC-1 Analogues 1−45
demonstrated the potential for S-PAC-1 to sensitize cancer cells
in culture to ionizing radiation.74 Encouragingly, S-PAC-1 was
effective in reducing or stabilizing tumor growth in four out of
six canine patients with spontaneously occurring lymphoma,
and the compound was well tolerated in all six dogs.38 These
results demonstrate the potential for procaspase-3 activation as
a safe and promising anticancer strategy.
While studies with PAC-1 and S-PAC-1 have been
encouraging, a challenge in using these compounds in animals
is the relatively short in vivo half-lives of both PAC-1 (2.1 0.3
h in dogs)39 and S-PAC-1 (1.09 0.02 h in dogs)38 following
iv administration. A study in rats identified three main pathways
of metabolism for PAC-1, including oxidative N-debenzylation,
olefin oxidation, and arene oxidation (Figure 2).75 While many
of these metabolites may be active based on the predicted
structure−activity relationships, the alcohols and secondary
amines resulting from these metabolites provide sites for
conjugation, including sulfation and glucuronidation; these
conjugates are then cleared from circulation. The metabolic
liabilities present in PAC-1 likely contribute to its pharmaco-
kinetic profile, necessitating relatively large doses to achieve
therapeutic levels in vivo. A PAC-1 analogue lacking some of
these liabilities may allow for lower dosing, which could
potentially reduce off-target toxicity. In this work, we describe
C
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a
b
c
Table 1. Cytotoxicity, Metabolic Stability, and Mouse Toxicity of PAC-1 Analogues
1−45
compd
R1
R2
R3
U-937 72 h IC50 (μM)
RLM 3 h stability (%)
mouse toxicity
1 (PAC-1)
Bn
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
H
10.2 0.3
8.9 0.6
12.1 1.3
13.7 0.9
13.1 3.7
11.1 2.1
10.2 1.7
15.3 6.7
6.6 1.9
9.6 2.1
4.9 0.4
9.4 1.3
9.0 1.2
12.8 2.7
10.0 1.7
7.3 0.9
4.1 0.4
4.8 1.2
17.0 1.4
−
38
84
89
48
90
31
86
16
85
30
61
71
30
61
24
69
15
64
64
−
2
1
4
2
4
1
2
1
6
1
2
3
2
3
2
4
2
1
4
severe
2 (S-PAC-1)
3
4-SO2NH2-Bn
Bz
none
−
−
−
−
4
4-CN-Bn
4-CN-Bz
4-F-Bn
5
6
7
4-F-Bz
moderate
8
4-CF3-Bn
4-CF3-Bz
Bn
−
lethal*
−
−
−
−
−
−
−
−
lethal*
−
−
−
−
−
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
4-SO2NH2-Bn
Bz
4-CN-Bn
4-CN-Bz
4-F-Bn
4-F-Bz
4-CF3-Bn
4-CF3-Bz
Bn
4-SO2NH2-Bn
Bz
F
H
F
H
15.7 2.6
88
1
4
4-CN-Bn
4-CN-Bz
4-F-Bn
F
H
−
−
88
−
F
H
15.3 1.3
−
F
H
−
−
−
4-F-Bz
F
H
15.3 0.8
4.7 0.3
8.7 0.5
9.5 0.9
9.8 1.3
8.6 2.0
12.7 2.0
10.1 2.0
10.3 4.1
8.5 1.4
3.4 0.6
6.5 0.6
8.9 1.2
8.7 0.4
12.3 1.0
11.2 0.9
9.4 1.2
7.5 0.7
7.5 1.4
3.9 0.6
5.2 0.6
86
30
87
56
89
93
65
95
57
92
49
90
49
62
86
49
66
48
67
40
64
2
5
3
1
3
7
2
4
1
3
3
2
6
3
5
5
3
1
3
1
5
4-CF3-Bn
4-CF3-Bz
Bn
F
H
F
H
moderate
F
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
−
lethal
4-SO2NH2-Bn
Bz
F
F
moderate
4-CN-Bn
4-CN-Bz
4-F-Bn
F
−
mild
F
F
−
severe
4-F-Bz
F
4-CF3-Bn
4-CF3-Bz
Bn
F
−
F
moderate
F
−
−
−
−
4-SO2NH2-Bn
Bz
F
F
4-CN-Bn
4-CN-Bz
4-F-Bn
F
F
mild
F
−
severe
−
4-F-Bz
F
4-CF3-Bn
4-CF3-Bz
F
F
lethal
a
Cells were treated with the compounds for 72 h. Biomass was quantified by sulforhodamine B assay. IC50 values shown are the mean SEM (n =
b
c
3). Rat liver microsomes were treated with the compounds (10 μM) for 3 h. The stability (%) values shown are the mean SEM (n = 3). Mice (n
= 3 per compound) were dosed with the compound via ip injection at 200 mg/kg (except where noted with an asterisk, in which case the dose was
100 mg/kg). A dash indicates that the compound was not evaluated.
the design, synthesis, and evaluation of a family of PAC-1
derivatives with the goal of enhancing metabolic stability, and
we report on promising compounds with enhanced metabolic
stability in vitro and in vivo.
RESULTS
■
Library Design. The structure−activity relationships of
PAC-1 indicate that modifications to the aryl rings can be
tolerated, as long as the core o-hydroxy-N-acylhydrazone
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Figure 4. The metabolic stability of PAC-1 and its derivatives was evaluated in rat liver microsomes at 10 μM for 3 h. LC/MS results of liver
microsome experiments for (A) PAC-1, (B) S-PAC-1, (C) 3, (D) 19, (E) 7, and (F) 32 are shown. Data shown are representative of three
independent experiments.
remains intact.8,36−38 The synthetic strategy that has been
adopted to access these active compounds involves the late-
stage condensation of a hydrazide and an aldehyde to form the
key o-hydroxy-N-acylhydrazone.8,37,38,40,42−46,49,51,53 This strat-
egy was useful for the generation of a large combinatorial
library of 837 diverse PAC-1 analogues.40 However, for this
study, we sought a more focused library design, with an
emphasis on the creation of derivatives with systematic removal
of the metabolic liabilities. The library (Figure 3) consists of 45
PAC-1 analogues (1−45), constructed from 9 hydrazides
(46a−i) and 5 aldehydes (47a−e). To avoid oxidative N-
debenzylation, the benzyl moiety was modified to a benzoyl (as
in 46c, 46e, 46g, and 46i), hypothesized to be more resistant to
oxidation.76 To avoid olefin oxidation, the allyl group was
changed to a propyl group (as in 47b and 47e) or removed
entirely (as in 47c). Finally, to block arene oxidation, building
blocks were introduced containing nitrile (46d,e), fluorine
(46f,g, 47c−e), and trifluoromethyl (46h,i) substituents.
Multiple derivatives were synthesized containing only one
modification to the PAC-1 core, so that the effect of individual
changes could be systematically evaluated.
the alkylation of piperazine (48) with ethyl chloroacetate (49)
to form monosubstituted piperazine 50. Compound 50 was
then reacted with substituted benzyl or benzoyl halides to give
disubstituted piperazines 51a−i in high yields. Reaction of the
esters with hydrazine then gave hydrazides 46a−i. Synthesis of
the aldehydes is shown in Scheme 1b. Both salicylaldehyde
(52) and 5-fluorosalicylaldehyde (47c) were alkylated with allyl
bromide to give (allyloxy)benzaldehydes 53a,b in high yields.
These compounds underwent Claisen rearrangements upon
heating at 200 °C, yielding aldehydes 47a and 47d in
approximately 50% yield. Finally, hydrogenation with diphenyl
sulfide as a catalyst poison allowed for chemoselective
reduction of the olefins,77 giving aldehydes 47b and 47e in
high yield. As shown in Scheme 1c, each of the hydrazides
46a−i was condensed with each of the aldehydes 47a−e in the
presence of a catalytic amount of HCl to give PAC-1 derivatives
1−45, the structures of which are given in Table 1.
Chromatographic purification of the library members yielded
the PAC-1 analogues in high purity (97% average purity). All
derivatives were at least 95% pure except compounds 20, 22,
and 24; because this standard of purity was not met for these
three compounds, they were excluded from further evaluation.
Evaluation of PAC-1 Analogues. Upon completion of the
synthesis of the 45 PAC-1 derivatives, the compounds were
evaluated in biological assays. First, the ability of the
compounds to induce cell death in U-937 (human lymphoma)
Compound Synthesis. Synthesis of the library involved
the construction of the nine hydrazides and five aldehydes,
followed by the condensation of each hydrazide with each
aldehyde to give the PAC-1 derivatives. The hydrazides were
synthesized according to Scheme 1a. The synthesis began with
E
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a
b
Table 2. Zinc Chelation and Caspase Activation by PAC-1 Derivatives
a
Increasing amounts of Zn(OTf)2 were added to a buffered solution of EGTA (7.3 mM) and PAC-1 derivative (100 μM). Kd was determined by
b
comparing the fluorescence intensity (excitation, 410 nm; emission, 475 nm) and free zinc concentration. U-937 cells were treated with the
compounds (30 μM) for 16 h and then lysed. Caspase-3/7 activity was assessed by cleavage of fluorogenic substrate Ac-DEVD-AFC.
cells in culture was determined (Table 1 and Supporting
Information). Each of the compounds induced dose-dependent
cell death under these conditions, and most of the compounds
were approximately as potent as PAC-1 and S-PAC-1,
confirming the previously determined SAR.8,37,38,40 The
metabolic stability of the compounds was then evaluated in
rat liver microsomes. The compounds were incubated with liver
microsomes for 3 h at 10 μM, and the metabolites were
observed by LC/MS, with ( )-propranolol hydrochloride as a
positive control;78 approximately 20% of the control remained.
The results of this assay are shown in Table 1. Compounds that
contained benzoyl substituents were significantly more stable
than analogous compounds containing benzyl groups; for
example, compound 3 was more stable than PAC-1, and
compound 32 was more stable than compound 31. The propyl-
containing compounds were less stable than the allyl-containing
compounds (e.g., PAC-1 was more stable than compound 10).
In addition, S-PAC-1 was relatively stable in the liver
microsomes, despite the short in vivo half-life of the
compound.38 This suggests that clearance mechanisms other
than oxidative metabolism are responsible for the elimination of
S-PAC-1 from treated animals.
The results of selected liver microsome experiments are
shown in Figure 4 (the full set of results is in the Supporting
Information). PAC-1 (Figure 4A) was 38% stable in the assay,
and several metabolites, including an N-dealkylated product, a
dihydroxylated product, and multiple monooxygenated prod-
ucts, were observed. S-PAC-1 (Figure 4B) was found to be
more stable than PAC-1, and fewer metabolites were observed.
One of the modifications that improved stability to the greatest
degree was the addition of the benzoyl in place of the benzyl
substituent, as demonstrated with compound 3 (Figure 4C).
This modification prevented the N-debenzylation completely
and increased stability to 89% during the 3 h incubation. In
addition, compounds with a benzoyl substituent had fewer
monooxygenated species than PAC-1, as the amide likely acted
to deactivate the aromatic ring toward oxidation. The addition
of fluorine to the benzylidene ring, as in compound 19 (Figure
4D), was also successful in reducing the number of
monooxygenated metabolites, and as expected, dihydroxylated
metabolites were not formed from compounds lacking the allyl
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group. Finally, combining multiple modifications, as in
compounds 7 (Figure 4E) and 32 (Figure 4F), led to highly
stable compounds that gave significantly fewer metabolites in
the liver microsome experiment.
Evaluation of Toxicity in Mice. On the basis of the
comparable cytotoxicity and improved in vitro metabolic
stability profile, several compounds were selected for further
in vivo evaluation. To assess the in vivo tolerability of the
compounds, they were administered to mice via intraperitoneal
injection at a dose of 200 mg/kg, the maximum tolerated dose
of PAC-1.48 Compounds were formulated at 5 mg/mL in a 200
mg/mL aqueous solution of (2-hydroxypropyl)-β-cyclodextrin.
The results of this experiment are shown in Table 1. Responses
are graded as mild, moderate, or severe; compounds that were
lethal to the mice at 200 mg/kg were also noted. Compounds 9
and 18 were lethal at a lower dose of 100 mg/kg, and
compound 36 induced hemolysis in the animals, so the
compounds containing the (trifluoromethyl)benzoyl substitu-
ent were not pursued further.
Secondary Assays. Because of their high stability,
comparable potency, and improved in vivo tolerability as
compared to PAC-1, compounds 7, 30, 32, and 41 were
selected for further investigation. To confirm that the hit
compounds act similarly to PAC-1, the compounds were
evaluated for their ability to chelate zinc in vitro, activate
executioner caspases in whole cells, and induce apoptosis in
cancer cells. Zinc binding was determined using an ethylene
glycol tetraacetic acid (EGTA) titration experiment.79 In this
experiment, varying concentrations of Zn(OTf)2 were added to
each well of a 96-well plate with a HEPES-buffered solution
containing EGTA and PAC-1 derivative, and the fluorescence
of the complex was analyzed, a slight variant of our previous
protocol for assessment of zinc binding.37 As shown in Table 2,
PAC-1 binds zinc with a Kd of 1.28 0.03 nM, while S-PAC-1
binds zinc with a Kd of 2.72 0.13 nM. Each of the four new
compounds displays affinity for zinc in the range of 1−2 nM.
PAC-1a, an inactive derivative of PAC-1 lacking the allyl and
hydroxyl substituents,8,36,37 does not bind zinc.
In addition, the ability of the compounds to activate
executioner caspases in whole cells was evaluated. Cells were
treated with the compounds for 0 or 16 h, then the cells were
lysed, and cleavage of the fluorescent caspase-3/7 substrate Ac-
DEVD-AFC was analyzed via kinetic reads. The activity (%) at
16 h was normalized to the slope of each compound at 0 h (0%
activity) and the slope of the positive control compound
staurosporine at 16 h (100% activity). As shown in Table 2 and
Figure 5, PAC-1 induces the highest degree of caspase
activation, while each of the other active compounds induces
greater than 60% activation of the executioner caspases. As
expected, treatment with DMSO alone or PAC-1a induces
minimal caspase activity in the cells.
To determine the mode of cell death induced by the
compounds, U-937 cells were treated with the compounds at
50 μM for 12 h, and viability was assessed by annexin V−
FITC/propidium iodide (FITC = fluorescein isothiocyanate)
staining (Figure 6). Each of these compounds induced
approximately 50% cell death under these conditions, and the
presence of large populations in the lower right quadrants of
the histograms (annexin V−FITC positive, propidium iodide
negative) confirms that the compounds induce apoptosis in
these cancer cells.
Figure 5. PAC-1 and its active derivatives activate executioner caspases
in cells. U-937 cells were treated with the compounds (30 μM for
PAC-1 derivatives, 1 μM for staurosporine) for 16 h and then lysed.
Caspase-3/7 activity was assessed by cleavage of the fluorogenic
substrate Ac-DEVD-AFC. Cells treated with vehicle alone or inactive
derivative PAC-1a show minimal caspase activity after 16 h. Values
shown are the mean SEM (n = 3).
lymphocytes18 in culture, the compounds were evaluated for
their ability to induce cell death in a panel of lymphoma and
leukemia cell lines, including Jurkat (human leukemia), GL-1
(dog lymphoma), OSW (dog lymphoma), and EL4 (mouse
lymphoma) cells, to complement the previously determined
IC50 values in U-937 (human lymphoma) cells. As shown in
Table 3, the compounds displayed comparable potency against
each given cell line. These results provide further support for
the previously determined structure−activity relationships, as
the modifications to improve metabolic stability had a minimal
effect on the activity of the new compounds, and further
suggest the potential of PAC-1 and derivatives for the treatment
of white blood cell cancers.
Pharmacokinetics. Because compounds 7, 30, 32, and 41
all chelate zinc, activate executioner caspases in whole cells, and
induce apoptosis, all four of the hit compounds were studied
further in vivo. The pharmacokinetics of the four compounds
plus PAC-1 and S-PAC-1 were evaluated in mice at a dose of 25
mg/kg (iv injection), and the results are shown in Figure 7 and
Table 4. PAC-1 and S-PAC-1 were cleared rapidly and were no
longer detectable after 5 and 6 h post-treatment, respectively. In
contrast, detectable levels of each of the four new derivatives
remained in circulation for at least 8 h post-treatment.
The elimination half-life of PAC-1 was 24.6 0.9 min, and
the half-life of S-PAC-1 was 38.1 3.3 min. Each of the four
new derivatives displayed half-lives of at least 88 min, with
compound 41 having the longest half-life at 122.3 1.4 min. In
addition, area under the curve (AUC) values from intravenous
administration for the four new derivatives were all significantly
higher than that of PAC-1. Compounds 7, 32, and 41 were also
found to display increased oral bioavailability as compared to
PAC-1 and S-PAC-1.
DISCUSSION
■
The introduction of substituents designed to block oxidative
metabolism is among the most attractive methods to achieve a
favorable pharmacokinetic profile, as the drug can pass through
the liver without being modified and remain in circulation for
longer periods of time. The knowledge of the metabolites
formed from PAC-1 in vivo facilitated the design of a library of
PAC-1 derivatives whose members lacked many of the
As many PAC-1 derivatives show activity against white blood
cell cancer lines8,37,38,40,42−47 and patient-derived leukemic
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Figure 6. PAC-1 and its derivatives induce apoptosis in U-937 cells. Cells were treated for 12 h at 50 μM, and viability was assessed by annexin V−
FITC/propidium iodide staining. Data shown are representative of three independent experiments.
a
Table 3. PAC-1 and Derivatives Are Cytotoxic to White Blood Cell Cancer Lines in Culture
72 h IC50 (μM)
cell line
species
origin
PAC-1
S-PAC-1
7
30
32
41
U-937
Jurkat
GL-1
OSW
EL4
human
human
dog
lymphoma
leukemia
10.2 0.3
4.4 0.6
3.0 0.1
10.0 0.8
6.5 0.5
8.9 0.6
4.5 1.2
3.2 0.2
9.8 0.1
7.9 0.5
10.2 1.7
4.0 0.5
3.0 0.1
9.3 0.2
6.5 0.8
8.6 2.0
4.1 0.7
3.4 0.2
10.0 0.6
7.3 1.2
10.1 2.0
3.5 0.2
2.4 0.4
9.5 0.7
5.1 0.4
9.4 1.2
3.4 0.6
2.2 0.3
8.5 0.7
4.7 0.7
lymphoma
lymphoma
lymphoma
dog
mouse
a
Cells were treated with the compounds for 72 h. Biomass was quantified by sulforhodamine B assay. IC50 values shown are the mean SEM (n =
3).
metabolic liabilities present on the parent compound. The
flexible, modular nature of the PAC-1 synthesis allowed for the
rapid generation of 45 derivatives from 9 hydrazides and 5
aldehydes.
Metal Binders in Medicine. Given the increased attention
paid to so-called “PAINS” (pan-assay interference compounds),
a further discussion of PAC-1 and its derivatives in relationship
to PAINS compounds is warranted. The metal-binding ability
of o-hydroxy-N-acylhydrazones can cause members of this class
of compounds to appear as hits in screening assays due to
interference with the assay screening system, rather than via
specific interactions with biological targets.71 In these cases,
attempts to validate such hits will fail because the apparent
activity of the screening hit is unrelated to the target. However,
rather than interfering with the in vitro procaspase-3 enzymatic
assay, the chelation of zinc from procaspase-3 in vitro by PAC-1
is highly biologically relevant: PAC-1 directly modulates zinc,
an endogenous inhibitor of procaspase-3, and the binding site
on procaspase-3/caspase-3 for this inhibitory zinc has been
identified.65 Through this “inhibiting the inhibitor” mechanism,
PAC-1 is akin to compounds that bind to other endogenous
Figure 7. Pharmacokinetic profiles of PAC-1 and selected derivatives
following a 25 mg/kg intravenous dose (n = 2). Detectable levels of
the novel derivatives are present in serum for at least 8 h post-
treatment, while PAC-1 and S-PAC-1 are no longer detectable after 5
and 6 h post-treatment, respectively.
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a
Table 4. Pharmacokinetic Parameters for PAC-1 and Selected Derivatives
compd
t1/2 (min)
AUC (iv) (min·μg/mL)
AUC (po) (min·μg/mL)
Foral (%)
PAC-1
24.6 0.9
38.1 3.3
89.5 19.3
120.5 16.3
88.7 3.3
122.3 1.4
210.3 9.3
446.0 114.1
362.3 55.8
291.0 40.6
364.9 5.6
313.4 5.5
31.6 1.6
54.0 12.4
92.2 6.0
15.1 1.4
12.9 6.1
25.6 2.3
8.5 5.1
S-PAC-1
7
30
32
41
25.7 18.3
105.2 24.2
113.0 3.7
28.8 6.2
36.1 0.5
a
A 25 mg/kg dose was administered via intravenous injection or oral gavage (per os, po). Values shown are the mean standard deviation (n = 2).
apoptotic inhibitors and induce apoptosis, such as those
binding MDM24 and XIAP.7
the role of the benzylpiperazine in PAC-1 activity merits further
study. Evaluation of the metabolic stability of the library
members in rat liver microsomes suggested that N-debenzyla-
tion was the main route of metabolism in vitro; the PAC-1
derivatives containing benzoyl substituents were more stable
than those containing benzyl substituents. These substitutions
also reduced the extent of arene oxidation, providing further
support for advancement of these compounds.
Four compounds (7, 30, 32, and 41) were identified with
favorable cell culture potency, in vitro metabolic stability, and in
vivo tolerability. Each of these compounds contains the benzoyl
substitution, as well as at least one arene substituent (fluorine
and/or nitrile) not present on PAC-1. The introduction of
fluorine is common in medicinal chemistry, especially the use of
aryl fluorides to block undesired metabolic arene oxidation, as
in the cholesterol-lowering drug ezetimibe.86 Aryl nitriles are
also commonly employed to accomplish this goal, as nitriles
typically pass through the body unmodified, and the electron-
withdrawing nature of the group deactivates the arene toward
oxidative metabolism at other sites.87 Trifluoromethyl groups
can deactivate arenes similarly in certain cases,88 and in vitro
results with the (trifluoromethyl)benzoyl-containing PAC-1
derivatives were encouraging. However, these compounds were
not evaluated further due to unacceptable levels of toxicity in
vivo.
Further evaluation of the four lead compounds demonstrates
that they chelate zinc, activate executioner caspases in whole
cells, and induce apoptosis similarly to PAC-1 and S-PAC-1.
The four derivatives display 3−5-fold higher elimination half-
lives and up to 2-fold higher overall compound exposure
compared to PAC-1. Results from the liver microsome
experiment are mostly consistent with the observed in vivo
pharmacokinetic profiles: fewer metabolites formed from the
new derivatives than from PAC-1 in vitro, and the compounds
remain in serum for longer periods of time than PAC-1. In
contrast, S-PAC-1 is stable in the liver microsome assay but has
a relatively short in vivo half-life. This suggests that the main
mode of clearance for S-PAC-1 may not be via oxidative
metabolism; instead, the compound may be excreted without
modification. A more thorough understanding of this
phenomenon may allow for the design of PAC-1 derivatives
that improve upon the pharmacokinetics even further than
those described in this paper. The rapid clearance of PAC-1 and
S-PAC-1 from circulation makes them challenging to evaluate
in certain efficacy models in vivo; these studies typically require
large doses of compound that increase the potential for toxicity.
The four novel derivatives remain in circulation for longer than
either PAC-1 or S-PAC-1, and thus offer promise as novel
therapeutic agents for the treatment of cancer.
PAC-1 also chelates labile zinc in cancer cells in culture, as
determined by detailed experiments with genetically encoded
fluorescent sensors specific for zinc.41 This removal of zinc
leads to the observed anticancer effect in cell culture. PAC-1
shows no activity toward several other enzymes as assessed by
in vitro assays,80 PAC-1 and its derivatives do not affect the
activity of proteins that rely on tightly bound zinc,18,72 and
PAC-1 derivatives that do not bind zinc in vitro are inactive in
cells.37 As shown by multiple investigators, treatment of cells
with PAC-1 or a derivative results in the cleavage of procaspase-
3 prior to the cleavage of initiator procaspases-8 and -9,42,43 and
cotreatment of PAC-1 with a covalent inhibitor of caspase-9
does not prevent cleavage of procaspase-3,66 further supporting
the hypothesis that the proapoptotic activity of PAC-1 is due to
chelation of labile inhibitory zinc from procaspase-3, leading to
the activation of procaspase-3 and apoptotic cell death.
While many metal chelators will interfere with in vitro
enzyme assays, it would be inappropriate to disregard all metal
chelators from consideration as drug candidates due to this in
vitro artifact. Indeed, metal chelators have a rich history in drug
discovery and have made a positive impact on many diseases
through a diverse range of mechanisms and targets.81 The many
examples of therapeutic metal-binding compounds include the
marketed drugs vorinostat (Zn2+)82 and penicillamine (Cu2+)81
and the entire class of bisphosphonates (Ca2+),83 as well as the
experimental therapeutics elesclomol (Cu2+),81 ML-133/Apto-
253 (Zn2+),84 and triapine (Fe3+/Fe2+),85 all of which rely on
metal chelation in vivo for their mechanism of action. It is safe
to say that many of these compounds would interfere with
certain in vitro assays that are contingent upon metal-bound
proteins. While it is reasonable for metal chelators and metal-
chelating motifs to be structural alerts when examining
screening hits, if the desired biological activity is metal
chelation, then obviously metal chelation is the precise trait
to look for in a screening hit.
Translational Potential of PAC-1 Derivatives. Cell
culture evaluation of the compounds reported herein confirm
previously determined structure−activity relationships, in that
substituents can be introduced to the aromatic rings without
abolishing activity if the core o-hydroxy-N-acylhydrazone
remains intact. Removal of the allyl group leads to a diminution
in cell culture potency, consistent with previous reports,8,37
although reduction to the fully saturated propyl group was
tolerated in the cell culture experiment. It is likely that the
increased hydrophobicity of the alkyl chain contributes to
increased cell permeability, as the allyl group does not affect the
ability of PAC-1 to bind zinc.37
The benzoyl-containing compounds display cell culture
activity similar to that of PAC-1. This substitution changes
the electronics at both the arene and the piperazine nitrogen;
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4-Benzoyl-1-piperazineacetohydrazide (46c). The title com-
pound was synthesized according to general procedure C: 51c (2.87 g,
10.4 mmol, 1.0 equiv), anhydrous hydrazine (1.31 mL, 41.6 mmol, 4.0
equiv), EtOH (20 mL, 0.5 M). 46c (1.41 g, 51.5%) was obtained as a
white solid after extraction without further purification. 1H NMR (500
MHz, CDCl3): δ 8.10 (s, 1H), 7.39−7.34 (m, 5H), 3.84 (br s, 2H),
3.77 (br s, 2H), 3.43 (br s, 2H), 3.08 (s, 2H), 2.56 (br s, 2H), 2.44 (br
s, 2H). 13C NMR (125 MHz, CDCl3): δ 170.5, 169.9, 135.5, 130.0,
128.7, 127.1, 60.6, 53.9 (br), 53.4 (br), 47.7 (br), 42.2 (br). HRMS
(ESI): m/z 263.1513 (M + H)+; calcd for C13H19N4O2, 263.1508.
Ethyl 4-[(4-Cyanophenyl)methyl]-1-piperazineacetate (51d).
The title compound was synthesized according to general procedure
A: 4-(bromomethyl)benzonitrile (54d; 2.0 g, 10.2 mmol, 1.0 equiv),
50 (2.64 g, 15.3 mmol, 1.5 equiv), K2CO3 (4.22 g, 30.6 mmol, 3.0
equiv), acetone (50 mL, 0.2 M). Purification by silica gel column
chromatography (50−100% EtOAc/hexanes) afforded 51d (2.71 g,
92.3%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 7.60 (d, 2H,
J = 8.0 Hz), 7.44 (d, 2H, J = 8.0 Hz), 4.18 (q, 2H, J = 7.0 Hz), 3.55 (s,
2H), 3.20 (s, 2H), 2.61 (br s, 4H), 2.51 (br s, 4H), 1.26 (t, 3H, J = 7.0
Hz). 13C NMR (125 MHz, CDCl3): δ 170.4, 144.4, 132.3, 129.7,
119.2, 111.0, 62.5, 60.8, 59.6, 53.1, 53.1, 14.4. HRMS (ESI): m/z
288.1718 (M + H)+; calcd for C16H22N3O2, 288.1712.
EXPERIMENTAL SECTION
■
General Methods. All reactions requiring anhydrous conditions
were conducted under a positive atmosphere of nitrogen or argon in
oven-dried glassware. Standard syringe techniques were used for
anhydrous addition of liquids. Unless otherwise noted, all starting
materials, solvents, and reagents were acquired from commercial
suppliers and used without further purification. Flash chromatography
was performed using 230−400 mesh silica gel. Compound syntheses
are discussed in the order in which they appear in the text. Syntheses
of 46a,8 46b,37 46h,40 47a,37 50,37 PAC-1 (1),8 S-PAC-1 (2),37 and
PAC-1a8 have been described previously.
All NMR experiments were recorded in CDCl3 (Sigma or
Cambridge), CD3OD (Sigma), or (CD3)2CO (Sigma or Cambridge)
on a Varian Unity 500 MHz spectrometer with residual undeuterated
solvent as the internal reference for 1H NMR and 13C NMR, and C6F6
as the internal reference for 19F NMR. The chemical shift [δ (ppm)],
coupling constants [J (Hz)], multiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, quint = quintet, sext = sextet, m = multiplet, br =
broad), and integration are reported. High-resolution mass spectral
data were recorded on a Micromass Q-Tof Ultima hybrid quadrupole/
time-of-flight ESI mass spectrometer or a Micromass 70-VSE at the
University of Illinois Mass Spectrometry Laboratory. Compound
purity was assessed by analytical HPLC (monitoring at 254 nm) on a
Waters Alliance e2695 HPLC system with a Waters XBridge C18
column, 4.6 × 150 mm. Mobile phase A was 0.1% F3CCO2H in H2O,
and solvent B was MeCN. A gradient was run with 0% B for 1 min,
then 0−100% B for 10 min, then constant 100% B for 5 min, then
100−0% B for 1 min, and then constant 0% B for 5 min. All
compounds evaluated in biological assays were ≥95% pure.
General Procedure A: Synthesis of Dialkylated Piperazines.
To a round-bottom flask were added benzyl halide (1.0 equiv), K2CO3
(3.0 equiv), and acetone (0.2 M). The mixture was stirred, and 50 (1.5
equiv) was added. The reaction mixture was stirred at reflux overnight.
The reaction mixture was cooled to room temperature. The solid was
filtered and washed with acetone. The filtrate was concentrated, and
the product was purified by silica gel column chromatography.
General Procedure B: Synthesis of Amides. To an oven-dried
round-bottom flask were added 50 (1.0 equiv), anhydrous
tetrahydrofuran (0.2 M), and freshly distilled Et3N (2.0 equiv). The
solution was stirred at 0 °C under N2, and benzoyl chloride (1.0 equiv)
was added. The reaction mixture was stirred overnight at room
temperature under N2. The reaction mixture was diluted with EtOAc
and washed with satd NaHCO3 (2×), H2O, and brine. The organic
layer was dried over MgSO4, filtered, and concentrated. The product
was purified by silica gel column chromatography.
4-[(4-Cyanophenyl)methyl]-1-piperazineacetohydrazide
(46d). The title compound was synthesized according to general
procedure C: 51d (2.71 g, 9.43 mmol, 1.0 equiv), anhydrous hydrazine
(1.18 mL, 37.7 mmol, 4.0 equiv), EtOH (19 mL, 0.5 M). 46d (1.73 g,
67.1%) was obtained as an off-white solid after extraction without
1
further purification. H NMR (500 MHz, CDCl3): δ 8.10 (br s, 1H),
7.60 (d, 2H, J = 8.0 Hz), 7.43 (d, 2H, J = 8.5 Hz), 3.84 (br d, 2H, J =
5.0 Hz), 3.55 (s, 2H), 3.08 (s, 2H), 2.55 (br s, 4H), 2.46 (br s, 4H).
13C NMR (125 MHz, CDCl3): δ 170.6, 144.1, 132.4, 129.6, 119.1,
111.2, 62.5, 60.8, 53.8, 53.3. HRMS (ESI): m/z 274.1673 (M + H)+;
calcd for C14H20N5O, 274.1668.
Ethyl 4-(4-Cyanobenzoyl)-1-piperazineacetate (51e). The
title compound was synthesized according to general procedure B:
50 (5.20 g, 30.2 mmol, 1.0 equiv), anhydrous tetrahydrofuran (150
mL, 0.2 M), freshly distilled Et3N (8.4 mL, 60.4 mmol, 2.0 equiv), 4-
cyanobenzoyl chloride (54e; 5.0 g, 30.2 mmol, 1.0 equiv). Purification
by silica gel column chromatography (0−10% MeOH/EtOAc)
1
afforded 51e (5.95 g, 65.4%) as a white solid. H NMR (500 MHz,
CDCl3): δ 7.68 (d, 2H, J = 8.0 Hz), 7.47 (d, 2H, J = 8.0 Hz), 4.14 (q,
2H, J = 7.0 Hz), 3.80 (br s, 2H), 3.37 (br s, 2H), 3.23 (s, 2H), 2.67 (br
s, 2H), 2.53 (br s, 2H), 1.23 (t, 3H, J = 7.0 Hz). 13C NMR (125 MHz,
CDCl3): δ 170.0, 168.3, 140.1, 132.5, 127.9, 118.2, 113.6, 60.9, 59.0,
52.9, 52.3, 47.5, 42.1, 14.3. HRMS (ESI): m/z 302.1501 (M + H)+;
calcd for C16H20N3O3, 302.1505.
4-(4-Cyanobenzoyl)-1-piperazineacetate (46e). The title
compound was synthesized according to general procedure C with
modification as noted: 51e (5.59 g, 18.6 mmol, 1.0 equiv), anhydrous
hydrazine (2.4 mL, 74.4 mmol, 4.0 equiv), EtOH (35 mL, 0.5 M).
After extraction with CH2Cl2, the aqueous layer was extracted with
EtOAc (3×). 46e (2.98 g, 55.8%) was obtained as an off-white solid
after extraction without further purification. 1H NMR (500 MHz,
CDCl3): δ 8.02 (br s, 1H), 7.70 (d, 2H, J = 8.5 Hz), 7.48 (d, 2H, J =
8.5 Hz), 3.86 (br d, 2H, J = 3.5 Hz), 3.78 (br s, 2H), 3.37 (br s, 2H),
3.11 (s, 2H), 2.60 (br s, 2H), 2.46 (br s, 2H). 13C NMR (125 MHz,
CDCl3): δ 169.8, 168.4, 139.8, 132.6, 127.9, 118.1, 113.8, 60.6, 53.8
(br), 53.3 (br), 47.6 (br), 42.2 (br). HRMS (ESI): m/z 288.1464 (M +
H)+; calcd for C14H18N5O2, 288.1461.
Ethyl 4-[(4-Fluorophenyl)methyl]-1-piperazineacetate (51f).
The title compound was synthesized according to general procedure
A: 4-fluorobenzyl chloride (54f; 2.5 g, 2.1 mL, 17.3 mmol, 1.0 equiv),
50 (4.48 g, 26.0 mmol, 1.5 equiv), K2CO3 (7.19 g, 52.0 mmol, 3.0
equiv), acetone (90 mL, 0.2 M). Purification by silica gel column
chromatography (gradient, 50−100% EtOAc/hexanes) afforded 51f
(3.66 g, 75.4%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.24−
7.20 (m, 2H), 6.95−6.91 (m, 2H), 4.13 (q, 2H, J = 7.0 Hz), 3.42 (s,
2H), 3.15 (s, 2H), 2.55 (br s, 4H), 2.46 (br s, 4H), 1.21 (t, 3H, J = 7.0
Hz). 13C NMR (125 MHz, CDCl3): δ 170.3, 162.0 (d, JC−F = 243.5
Hz), 133.9, 130.6 (d, JC−F = 8.0 Hz), 115.0 (d, JC−F = 21.0 Hz), 62.2,
General Procedure C: Synthesis of Hydrazides. To a round-
bottom flask were added ethyl ester (1.0 equiv) and EtOH or 2:1
EtOH/MeOH (0.5 M). The solution was stirred, and anhydrous
hydrazine (4.0 equiv) was added dropwise. The reaction mixture was
stirred at reflux overnight. The reaction mixture was cooled to room
temperature and concentrated. The resulting residue was partitioned
between CH2Cl2 and 1:1 brine/0.1 M KOH. The layers were
separated, and the aqueous layer was extracted with CH2Cl2 (2×). The
combined organic layers were dried over MgSO4, filtered, and
concentrated. Purification by silica gel column chromatography or
recrystallization yielded pure hydrazide.
Ethyl 4-Benzoyl-1-piperazineacetate (51c). The title com-
pound was synthesized according to general procedure B: 50 (2.45 g,
14.2 mmol, 1.0 equiv), anhydrous tetrahydrofuran (70 mL, 0.2 M),
freshly distilled Et3N (4.0 mL, 28.4 mmol, 2.0 equiv), benzoyl chloride
(54c; 2.0 g, 1.7 mL, 1.0 equiv). Purification by silica gel column
chromatography (50−100% EtOAc/hexanes) afforded 51c (2.87 g,
73.1%) as a pale yellow oil. 1H NMR (500 MHz, CDCl3): δ 7.41−7.38
(m, 5H), 4.19 (q, 2H, J = 7.0 Hz), 3.85 (br s, 2H), 3.48 (br s, 2H),
3.25 (s, 2H), 2.68 (br s), 2.54 (br s, 2H), 1.27 (t, 3H, J = 7.0 Hz). 13C
NMR (125 MHz, CDCl3): δ 170.5, 170.2, 135.9, 129.9, 128.7, 127.3,
61.0, 59.4, 53.3 (br), 52.8 (br), 47.8 (br), 42.1 (br), 14.4. HRMS
(ESI): m/z 277.1552 (M + H)+; calcd for C15H21N2O3, 277.1552.
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60.6, 59.6, 53.1, 52.8, 14.3. 19F NMR (470 MHz, CDCl3): δ −119.1.
HRMS (ESI): m/z 281.1659 (M + H)+; calcd for C15H22FN2O2,
281.1665.
CDCl3): δ −66.0. HRMS (ESI): m/z 331.1374 (M + H)+; calcd for
C14H18F3N4O2, 331.1382.
2-Hydroxy-3-propylbenzaldehyde (47b). To a round-bottom
flask were added aldehyde 47a (1.62 g, 10.0 mmol, 1.0 equiv), 5% Pd/
C (324 mg, 20 wt % relative to 47a), diphenyl sulfide (17 μL, 0.10
mmol, 0.010 equiv), and EtOAc (40 mL, 0.25 M). The reaction
mixture was stirred overnight at room temperature under an
atmosphere of H2 (balloon pressure). The reaction mixture was
filtered through Celite and washed thoroughly with EtOAc. The
filtrate was concentrated to afford aldehyde 47b (1.50 g, 91.7%) as a
4-[(4-Fluorophenyl)methyl]-1-piperazineacetohydrazide
(46f). The title compound was synthesized according to general
procedure C: 51f (3.0 g, 10.7 mmol, 1.0 equiv), anhydrous hydrazine
(1.4 mL, 42.8 mmol, 4.0 equiv), EtOH (20 mL, 0.5 M). 46f (2.59 g,
91.1%) was obtained as a white solid after extraction without further
1
purification. H NMR (500 MHz, CDCl3): δ 8.15 (br s, 1H), 7.22−
7.19 (m, 2H), 6.95−6.91 (m, 2H), 3.84 (br s, 2H), 3.40 (s, 2H), 3.01
(s, 2H), 2.47 (br s, 4H), 2.39 (br s, 4H). 13C NMR (125 MHz,
CDCl3): δ 170.5, 162.0 (d, JC−F = 243.6 Hz), 133.7 (d, JC−F = 2.8 Hz),
130.6 (d, JC−F = 8.3 Hz), 115.1 (d, JC−F = 21.1 Hz), 62.0, 60.6, 53.7,
53.0. 19F NMR (470 MHz, CDCl3): δ −118.9. HRMS (ESI): m/z
267.1630 (M + H)+; calcd for C13H20FN4O, 267.1621.
1
yellow oil. H NMR (500 MHz, CDCl3): δ 11.27 (s, 1H), 9.88 (s,
1H), 7.41−7.38 (m, 2H), 6.95 (t, 1H, J = 7.5 Hz), 2.64 (t, 2H, J = 7.5
Hz), 1.65 (sext, 2H, J = 7.5 Hz), 0.96 (t, 3H, J = 7.5 Hz). 13C NMR
(125 MHz, CDCl3): δ 197.0, 160.0, 137.4, 131.7, 131.4, 120.4, 119.6,
31.3, 22.7, 14.1. HRMS (EI): m/z 164.08383 (M+); calcd for
C10H12O2, 164.08373.
Ethyl 4-(4-Fluorobenzoyl)-1-piperazineacetate (51g). The
title compound was synthesized according to general procedure B:
50 (2.58 g, 15.0 mmol, 1.0 equiv), anhydrous tetrahydrofuran (30 mL,
0.5 M), freshly distilled Et3N (4.2 mL, 30.0 mmol, 2.0 equiv), 4-
fluorobenzoyl chloride (54g; 1.8 mL, 15.0 mmol, 1.0 equiv).
Purification by silica gel column chromatography (50−100%
EtOAc/hexanes) afforded 51g (3.74 g, 84.7%) as a pale yellow oil.
5-Fluoro-2-(2-propenyloxy)benzaldehyde (53b). To a round-
bottom flask were added 5-fluorosalicylaldehyde (47c; 4.0 g, 28.5
mmol, 1.0 equiv), potassium carbonate (4.92 g, 35.6 mmol, 1.25
equiv), and DMF (20 mL). Allyl bromide (3.7 mL, 42.8 mmol, 1.5
equiv) was added slowly to the mixture. The reaction mixture was
stirred overnight at room temperature. The reaction mixture was
diluted with water (50 mL) and extracted with ethyl acetate (3 × 50
mL). The combined organic layers were washed with water (2 × 25
mL), 0.1 M KOH (2 × 25 mL), water (2 × 25 mL), and brine (2 × 25
mL), dried over MgSO4, filtered, and concentrated to yield 53b (4.80
1
1H NMR (500 MHz, CDCl3): δ H NMR (500 MHz, CDCl3): δ
7.38−7.34 (m, 2H), 7.06−7.01 (m, 2H), 4.13 (q, 2H, J = 7.0 Hz), 3.77
(br s, 2H), 3.43 (br s, 2H), 3.21 (s, 2H), 2.61 (br s, 2H), 2.52 (br s,
2H), 1.22 (t, 3H, J = 7.0 Hz). 13C NMR (125 MHz, CDCl3): δ 170.0,
169.4, 163.5 (d, JC−F = 248.1 Hz), 131.8, 129.5 (d, JC−F = 8.3 Hz),
115.6 (d, JC−F = 22.0 Hz), 60.8, 59.1, 52.8 (br), 47.8 (br), 42.2 (br),
14.3. 19F NMR (470 MHz, CDCl3): δ −113.4. HRMS (ESI): m/z
295.1457 (M + H)+; calcd for C15H20FN2O3, 295.1458.
4-(4-Fluorobenzoyl)-1-piperazineacetohydrazide (46g). The
title compound was synthesized according to general procedure C:
51g (3.73 g, 12.7 mmol, 1.0 equiv), anhydrous hydrazine (1.6 mL, 50.8
mmol, 4.0 equiv), EtOH (25 mL, 0.5 M). 46g (2.28 g, 64.1%) was
obtained as a white solid after extraction without further purification.
1H NMR (500 MHz, CDCl3): δ 1H NMR (500 MHz, CDCl3): δ 8.09
(br s, 1H), 7.37−7.33 (m, 2H), 7.06−7.02 (m, 2H), 3.85 (br s, 2H),
3.70 (br s, 2H), 3.42 (br s, 2H), 3.06 (s, 2H), 2.48 (br s, 4H). 13C
NMR (125 MHz, CDCl3): δ 169.8, 169.5, 163.5 (d, JC−F = 249.1 Hz),
1
g, 93.3%) as a pale yellow liquid. H NMR (500 MHz, CDCl3): δ
10.47 (d, 1H, J = 3.0 Hz), 7.50 (dd, 1H, J = 3.0, 8.0 Hz), 7.23 (ddd,
1H, J = 3.0, 7.5, 11.0 Hz), 6.95 (dd, 1H, J = 4.0, 9.0 Hz), 6.06 (tdd,
1H, J = 5.0, 10.5, 17.5 Hz), 5.44 (qd, 1H, J = 1.5, 17.0 Hz), 5.34 (ddd,
1H, J = 1.5, 2.5, 10.5 Hz), 4.64 (td, 2H, J = 1.5, 5.0 Hz). 13C NMR
(125 MHz, CDCl3): δ 188.8, 157.4 (d, JC−F = 1.9 Hz), 157.2 (d, JC−F
=
240.5 Hz), 132.4, 126.1 (d, JC−F = 5.9 Hz), 122.6 (d, JC−F = 23.8 Hz),
118.5, 114.8 (d, JC−F = 7.1 Hz), 114.2 (d, JC−F = 23.1 Hz), 70.1. 19F
NMR (470 MHz, CDCl3): δ −125.5. HRMS (EI): m/z 180.05789
(M+); calcd for C10H9FO2, 180.05866.
5-Fluoro-2-hydroxy-3-(2-propenyl)benzaldehyde (47d). 53b
(4.64 g, 25.8 mmol) was heated neat overnight at 200 °C. The crude
product was purified by silica gel column chromatography (gradient,
0−10% CH2Cl2/hexanes) to yield 47d (2.24 g, 48.3%) as a bright
1
131.5 (d, JC−F = 2.8 Hz), 129.4 (d, JC−F = 9.1 Hz), 115.7 (d, JC−F
=
yellow oil. H NMR (500 MHz, CDCl3): δ 11.10 (s, 1H), 9.83 (s,
22.0 Hz), 60.6, 53.5 (br), 47.7 (br), 42.3 (br). 19F NMR (470 MHz,
CDCl3): δ −113.1. HRMS (ESI): m/z 281.1409 (M + H)+; calcd for
C13H18FN4O2, 281.1414.
1H), 7.17 (dd, 1H, J = 3.0, 9.0 Hz), 7.11 (dd, 1H, J = 3.0, 7.5 Hz) 5.96
(tdd, 1H, J = 6.5, 10.0, 17.0 Hz), 5.16−5.14 (m, 1H), 5.12 (qd, 1H, J =
1.5, 11.0 Hz), 3.42 (d, 2H, J = 6.5 Hz). 13C NMR (125 MHz, CDCl3):
Ethyl 4-[4-(Trifluoromethyl)benzoyl]-1-piperazineacetate
(51i). The title compound was synthesized according to general
procedure B: 50 (2.58 g, 15.0 mmol, 1.0 equiv), anhydrous
tetrahydrofuran (30 mL, 0.5 M), freshly distilled Et3N (4.2 mL, 30.0
mmol, 2.0 equiv), 4-(trifluoromethyl)benzoyl chloride (54i; 2.2 mL,
15.0 mmol, 1.0 equiv). Purification by silica gel column chromatog-
raphy (50−100% EtOAc/hexanes) afforded 51i (4.01 g, 77.5%) as a
δ 195.9 (d, JC−F = 2.5 Hz), 156.0 (d, JC−F = 1.0 Hz), 155.7 (d, JC−F
=
238.8 Hz), 135.1, 131.6 (d, JC−F = 6.4 Hz), 124.8 (d, JC−F = 23.6 Hz),
119.8 (d, JC−F = 6.4 Hz), 117.3, 116.0 (d, JC−F = 22.3 Hz), 33.2. 19F
NMR (470 MHz, CDCl3): δ −126.9. HRMS (EI): m/z 180.05761
(M+); calcd for C10H9FO2 180.05866.
5-Fluoro-2-hydroxy-3-propylbenzaldehyde (47e). To a
round-bottom flask were added aldehyde 47d (1.10 g, 6.11 mmol,
1.0 equiv), 5% Pd/C (220 mg, 20 wt % relative to 47d), diphenyl
sulfide (10 μL, 0.061 mmol, 0.010 equiv), and EtOAc (25 mL, 0.25
M). The reaction mixture was stirred overnight at room temperature
under an atmosphere of H2 (balloon pressure). The reaction mixture
was filtered through Celite and washed thoroughly with EtOAc. The
filtrate was concentrated to afford aldehyde 47e (991 mg, 89.3%) as a
1
yellow oil. H NMR (500 MHz, CDCl3): δ 7.65 (d, 2H, J = 8.0 Hz),
7.49 (d, 2H, J = 8.0 Hz), 4.16 (q̧ 2H, J = 7.0 Hz), 3.82 (br s, 2H), 3.41
(br s, 2H), 3.24 (s, 2H), 2.68 (br s, 2H), 2.54 (br s, 2H), 1.24 (t, 3H, J
= 7.0 Hz). 13C NMR (125 MHz, CDCl3)δ 170.1, 168.9, 139.4 131.8
(q, JC−F = 32.6 Hz), 127.6, 125.7 (q, JC−F = 3.8 Hz), 123.8 (q, JC−F
=
271.0 Hz), 60.9, 59.1, 53.0 (br), 52.5 (br), 47.6 (br), 42.2 (br), 14.3.
19F NMR (470 MHz, CDCl3): δ −66.0. HRMS (ESI): m/z 345.1430
1
yellow oil. H NMR (500 MHz, CDCl3): δ 11.06 (br s, 1H), 9.80 (s,
(M + H)+; calcd for C16H20F3N2O3, 345.1426.
1H), 7.12 (dd, 1H, J = 3.0, 9.0 Hz), 7.06 (dd, 1H, J = 3.0, 7.5 Hz), 2.62
(t, 2H, J = 7.5 Hz), 1.63 (sext, 2H, J = 7.5 Hz), 0.96 (t, 3H, J = 7.5
Hz). 13C NMR (125 MHz, CDCl3): δ 195.9 (d, JC−F = 2.5 Hz), 156.3
(d, JC−F = 1.0 Hz), 155.5 (d, JC−F = 238.1 Hz), 133.9 (d, JC−F = 6.3
Hz), 124.7 (d, JC−F = 23.1 Hz), 119.6 (d, JC−F = 6.5 Hz), 115.4 (d, JC−F
= 22.4 Hz), 31.2, 22.4, 14.0. 19F NMR (470 MHz, CDCl3): δ −127.3.
HRMS (EI): m/z 182.07392 (M+); calcd for C10H11FO2, 182.07431.
General Procedure D: Synthesis of PAC-1 Analogues. To a 16
× 150 mm test tube were added hydrazide (1.0 equiv), aldehyde (1.0
equiv), EtOH or 2:1 MeOH/MeCN (0.15 M), and 1.2 M HCl (7 mol
4-[4-(Trifluoromethyl)benzoyl]-1-piperazineacetohydrazide
(46i). The title compound was synthesized according to general
procedure C: 51i (4.00 g, 11.6 mmol, 1.0 equiv), anhydrous hydrazine
(1.5 mL, 46.4 mmol, 4.0 equiv), EtOH (25 mL, 0.5 M). 46i (2.35 g,
61.4%) was obtained as a white solid after extraction without further
purification. 1H NMR (500 MHz, CDCl3): δ 8.09 (br s, 1H), 7.62 (d,
2H, J = 8.0 Hz), 7.45 (d, 2H, J = 8.0 Hz), 3.88 (br s, 2H) 3.75 (br s,
2H), 3.35 (br s, 2H), 3.07 (s, 2H), 2.56 (br s, 2H), 2.42 (br s, 2H). 13C
NMR (125 MHz, CDCl3)δ 169.8, 168.9, 139.1 131.8 (q, JC−F = 32.1
Hz), 127.5, 125.7 (q, JC−F = 3.6 Hz), 123.7 (q, JC−F = 271.1 Hz), 60.5,
53.7 (br), 53.2 (br), 47.6 (br), 42.2 (br). 19F NMR (470 MHz,
%). The reaction mixture was shaken overnight at reflux on a Buchi
̈
Syncore parallel synthesizer. The reaction mixture was cooled to room
K
DOI: 10.1021/acs.jmedchem.5b00413
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
temperature, concentrated, and purified by silica gel column
chromatography or recrystallization to yield the pure PAC-1 analogue.
N′-[[2-hydroxy-3-(2-propenyl)phenyl]methylene]-4-benzo-
yl-1-piperazineacetohydrazide (3). The title compound was
synthesized according to general procedure D, but in a round-bottom
flask: 46c (262 mg, 1.0 mmol, 1.0 equiv), 47a (162 mg, 1.0 mmol, 1.0
equiv), 1.2 M HCl (58 μL, 0.070 mmol, 0.070 equiv), EtOH (7 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−10% MeOH/EtOAc) yielded 3 (284 mg, 69.8%) as a white solid.
1H NMR (500 MHz, CDCl3): δ 11.19 (s, 1H), 9.94 (br s, 1H), 8.45 (s,
1H), 7.46−7.41 (m, 5H), 7.20 (d, 1H, J = 6.5 Hz), 7.08 (dd, 1H, J =
1.5, 7.5 Hz), 6.85 (t, 1H, J = 7.0 Hz), 6.03 (tdd, 1H, J = 6.5, 10.0, 16.5
Hz), 5.10−5.05 (m, 2H), 3.88 (br s, 2H), 3.58 (s, 2H), 3.52 (br s, 2H),
3.45 (d, 2H, J = 6.5 Hz), 3.25 (s, 2H), 2.68 (br s, 2H), 2.61 (br s, 2H).
13C NMR (125 MHz, CDCl3): δ 170.6, 165.4, 156.4, 151.6, 136.5,
was synthesized according to general procedure D: 46g (140 mg, 0.50
mmol, 1.0 equiv), 47a (81 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29
μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−10% MeOH/EtOAc)
1
yielded 7 (171 mg, 80.5%) as a white solid. H NMR (500 MHz,
CDCl3): δ 11.19 (br s, 1H), 9.91 (br s, 1H), 8.43 (s, 1H), 7.43−7.40
(m, 2H), 7.19 (dd, 1H, J = 1.0, 7.5 Hz), 7.12−7.09 (m, 2H), 7.06 (dd,
1H, J = 1.5, 8.0 Hz), 6.85 (t, 1H, J = 7.5 Hz), 6.02 (tdd, 1H, J = 6.5,
10.0, 16.5 Hz), 5.10−5.04 (m, 2H), 3.69 (br s, 4H), 3.44 (d, 2H, J =
6.5 Hz), 3.24 (s, 2H), 2.64 (br s, 4H). 13C NMR (125 MHz, CDCl3):
δ 169.6, 165.4, 163.6 (d, JC−F = 249.1 Hz), 156.4, 151.6, 136.5, 132.5,
131.4 (d, JC−F = 3.4 Hz), 129.5 (d, JC−F = 8.4 Hz), 129.3, 128.2, 119.2,
116.8, 115.8, 115.8 (d, JC−F = 21.5 Hz), 60.9, 53.6 (br), 47.7 (br), 42.3
(br), 33.9. 19F NMR (470 MHz, CDCl3): δ −112.8. HRMS (ESI): m/
z 425.1989 (M + H)+; calcd for C23H26FN4O3, 425.1989. HPLC
purity: 97%.
N′-[[2-Hydroxy-3-(2-propenyl)phenyl]methylene]-4-[[4-
(trifluoromethyl)phenyl]methyl]-1-piperazineacetohydrazide
(8). The title compound was synthesized according to general
procedure D: 46h (158 mg, 0.50 mmol, 1.0 equiv), 47a (81 mg,
0.50 mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv),
EtOH (3 mL, 0.15 M). Purification by silica gel column
chromatography (gradient, 0−15% MeOH/EtOAc) yielded 8 (125
mg, 54.4%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 11.32 (br
s, 1H), 10.11 (br s, 1H), 8.33 (s, 1H), 7.56 (d, 2H, J = 8.5 Hz), 7.43
(d, 2H, J = 8.0 Hz), 7.17 (dd, 1H, J = 1.5, 7.5 Hz), 7.04 (dd, 1H, J =
1.5, 8.0 Hz), 6.83 (t, 1H, J = 7.5 Hz), 6.02 (tdd, 1H, J = 6.5, 10.0, 16.5
Hz), 5.10−5.04 (m, 2H), 3.57 (s, 2H), 3.44 (d, 2H, J = 7.0 Hz), 3.19
(s, 2H), 2.63 (br s, 4H), 2.53 (br s, 4H). 13C NMR (125 MHz,
135.4, 132.5, 130.2, 129.3, 128.8, 128.3, 127.1, 119.2, 116.9, 115.8,
61.0, 53.7, 53.1, 47.6, 42.1, 33.9. HRMS (ESI): m/z 407.2077 (M +
H)+; calcd for C23H27N4O3, 407.2083. HPLC purity: 95%.
N′-[[2-Hydroxy-3-(2-propenyl)phenyl]methylene]-4-[(4-
cyanophenyl)methyl]-1-piperazineacetohydrazide (4). The title
compound was synthesized according to general procedure D: 46d
(273 mg, 1.0 mmol, 1.0 equiv), 47a (162 mg, 1.0 mmol, 1.0 equiv), 1.2
M HCl (58 μL, 0.070 mmol, 0.070 equiv), EtOH (7 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−15%
MeOH/EtOAc) yielded 4 (367 mg, 87.7%) as a white solid. 1H NMR
(500 MHz, CDCl3): δ 11.25 (br s, 1H), 9.99 (br s, 1H), 8.40 (s, 1H),
7.61 (d, 2H, J = 8.0 Hz), 7.44 (d, 2H, J = 8.0 Hz), 7.18 (dd, 1H, J =
1.5, 7.5 Hz), 7.07 (dd, 1H, J = 1.5, 7.5 Hz), 6.84 (t, 1H, J = 7.5 Hz),
6.02 (tdd, 1H, J = 6.5, 10.0, 16.5 Hz), 5.11−5.04 (m, 2H), 3.58 (s,
2H), 3.44 (d, 2H, J = 7.0 Hz), 3.19 (s, 2H), 2.63 (br s, 4H), 2.53 (br s,
4H). 13C NMR (125 MHz, CDCl3): δ 165.9, 156.5, 151.5, 144.0,
136.6, 132.5, 132.4, 129.6, 129.3, 128.4, 119.2, 119.1, 117.0, 115.8,
111.2, 62.4, 61.1, 53.8, 53.2, 34.0. HRMS (ESI): m/z 418.2242 (M +
H)+; calcd for C24H28N5O2, 418.2243. HPLC purity: 96%.
CDCl3): δ 166.0, 156.4, 151.2, 142.4, 136.6, 132.4, 129.5 (q, JC−F
=
32.0 Hz), 129.3, 129.3, 128.3, 125.3 (q, JC−F = 3.8 Hz), 123.9 (q, JC−F
= 270.6 Hz), 119.2, 117.0, 115.8, 62.3, 61.0, 53.7, 53.1, 34.0. 19F NMR
(470 MHz, CDCl3): δ −65.4. HRMS (ESI): m/z 461.2160 (M + H)+;
calcd for C24H28F3N4O2, 461.2164. HPLC purity: 95%.
N′-[[2-Hydroxy-3-(2-propenyl)phenyl]methylene]-4-[4-
(trifluoromethyl)benzoyl]-1-piperazineacetohydrazide (9). The
title compound was synthesized according to general procedure D: 46i
(165 mg, 0.50 mmol, 1.0 equiv), 47a (81 mg, 0.50 mmol, 1.0 equiv),
1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−15%
MeOH/EtOAc) yielded 9 (211 mg, 89.1%) as a white solid. 1H NMR
(500 MHz, CDCl3): δ 11.28 (br s, 1H), 10.13 (br s, 1H), 8.27 (s, 1H),
7.65 (d, 2H, J = 8.0 Hz), 7.48 (d, 2H, J = 8.0 Hz), 7.15 (d, 1H, J = 8.0
Hz), 6.94 (d, 2H, J = 7.0 Hz), 6.79 (t, 1H, J = 7.5 Hz), 5.97 (tdd, 1H, J
= 6.5, 10.0, 17.0 Hz), 5.05−5.00 (m, 2H), 3.86 (br s, 2H), 3.43 (br s,
2H), 3.39 (d, 2H, J = 6.5 Hz), 3.21 (s, 2H), 2.66 (br s, 2H), 2.58 (br s,
2H). 13C NMR (125 MHz, CDCl3): δ 169.0, 165.4, 156.3, 151.5,
139.0, 136.4, 132.5, 131.9 (q, JC−F = 32.6 Hz), 129.3, 128.2, 127.5,
125.8 (q, JC−F = 3.5 Hz), 123.7 (q, JC−F = 271.1 Hz), 119.3, 116.8,
115.8, 60.8, 53.5 (br), 47.5 (br), 42.0 (br), 33.8. 19F NMR (470 MHz,
CDCl3): δ −66.0. HRMS (ESI): m/z 475.1964 (M + H)+; calcd for
C24H26F3N4O3, 475.1957. HPLC purity: 97%.
N′-[(2-Hydroxy-3-propylphenyl)methylene]-4-(phenylmeth-
yl)-1-piperazineacetohydrazide (10). The title compound was
synthesized according to general procedure D, but in a round-bottom
flask: 46a (248 mg, 1.0 mmol, 1.0 equiv), 47b (164 mg, 1.0 mmol, 1.0
equiv), 1.2 M HCl (58 μL, 0.070 mmol, 0.070 equiv), EtOH (7 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−20% MeOH/EtOAc) yielded 10 (345 mg, 87.3%) as an off-white
solid. 1H NMR (500 MHz, CDCl3): δ 11.30 (s, 1H), 10.12 (br s, 1H),
8.31 (s, 1H), 7.35−7.30 (m, 4H), 7.30−7.25 (m, 1H), 7.17 (d, 1H, J =
7.5 Hz), 7.03 (d, 1H, J = 7.5 Hz), 6.82 (t, 1H, J = 7.5 Hz), 3.54 (s,
2H), 3.19 (s, 2H), 2.67 (t, 2H, J = 7.5 Hz), 2.62 (br s, 4H), 2.54 (br s,
4H), 1.67 (sext, 2H, J = 7.5 Hz), 0.97 (t, 3H, J = 7.5 Hz). 13C NMR
(125 MHz, CDCl3): δ 165.9, 156.7, 151.2, 137.9, 132.5, 130.7, 129.2,
128.8, 128.4, 127.3, 118.9, 116.8, 62.9, 61.0, 53.7, 53.0, 32.0, 22.7, 14.2.
HRMS (ESI): m/z 395.2436 (M + H)+; calcd for C23H31N4O2,
395.2447. HPLC purity: 98%.
N′-[[2-Hydroxy-3-(2-propenyl)phenyl]methylene]-4-(4-cya-
nobenzoyl)-1-piperazineacetohydrazide (5). The title compound
was synthesized according to general procedure D: 46e (287 mg, 1.0
mmol, 1.0 equiv), 47a (162 mg, 1.0 mmol, 1.0 equiv), 1.2 M HCl (58
μL, 0.070 mmol, 0.070 equiv), EtOH (7 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−10% MeOH/EtOAc)
yielded 5 (378 mg, 87.6%) as a light yellow solid. 1H NMR (500 MHz,
CDCl3): δ 11.23 (br s, 1H), 9.98 (br s, 1H), 8.32 (s, 1H), 7.69 (d, 2H,
J = 8.5 Hz), 7.48 (d, 2H, J = 8.0 Hz), 7.17 (d, 1H, J = 7.0 Hz), 6.99
(dd, 1H, J = 1.5, 8.0 Hz), 6.81 (t, 1H, J = 7.5 Hz), 5.98 (tdd, 1H, J =
6.5, 10.0, 17.0), 5.08−5.02 (m, 2H), 3.85 (br s, 2H), 3.42−3.39 (m,
4H), 3.23 (s, 2H), 2.68 (br s, 2H), 2.86 (br s, 4H). 13C NMR (125
MHz, CDCl3): δ 168.4, 165.3, 156.4, 151.7, 139.7, 136.5, 132.6, 132.6,
129.4, 128.2, 127.9, 119.3, 118.1, 116.8, 115.9, 113.8, 60.9, 53.7 (br),
53.3 (br), 47.5 (br), 42.1 (br), 33.9. HRMS (ESI): m/z 432.2034 (M
+ H)+; calcd for C24H26N5O3, 432.2036. HPLC purity: 97%.
N′-[[2-Hydroxy-3-(2-propenyl)phenyl]methylene]-4-[(4-
fluorophenyl)methyl]-1-piperazineacetohydrazide (6). The title
compound was synthesized according to general procedure D: 46f
(133 mg, 0.50 mmol, 1.0 equiv), 47a (81 mg, 0.50 mmol, 1.0 equiv),
1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−10%
MeOH/EtOAc) followed by precipitation from Et2O yielded 6 (182
mg, 89.0%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 11.26 (br
s, 1H), 10.02 (br s, 1H), 8.41 (s, 1H), 7.29−7.26 (m, 2H), 7.19 (dd,
1H, J = 1.5, 7.5 Hz), 7.08 (dd, 1H, J = 1.5, 8.0 Hz), 7.02−6.99 (m,
2H), 6.85 (t, 1H, J = 7.5 Hz), 6.03 (tdd, 1H, J = 6.5, 10.0, 16.5 Hz),
5.11−5.04 (m, 2H), 3.50 (s, 2H), 3.45 (d, 2H, J = 6.5 Hz), 3.19 (s,
2H), 2.62 (br s, 4H), 2.51 (br s, 4H). 13C NMR (125 MHz, CDCl3): δ
166.0, 162.2 (d, JC−F = 243.9 Hz), 156.6, 151.5, 136.7, 133.7 (d, JC−F
=
3.1 Hz), 132.5, 130.7 (d, JC−F = 7.8 Hz), 129.3, 128.4, 119.2, 117.0,
115.8, 115.3 (d, JC−F = 21.0 Hz), 62.2, 61.2, 53.9, 53.1, 34.0. 19F NMR
(470 MHz, CDCl3): δ −118.8. HRMS (ESI): m/z 411.2203 (M +
H)+; calcd for C23H28FN4O2, 411.2196. HPLC purity: 98%.
N′-[[2-Hydroxy-3-(2-propenyl)phenyl]methylene]-4-(4-fluo-
4-{[4-[[[N′-[(2-Hydroxy-3-propylphenyl)methylene]-
robenzoyl)-1-piperazineacetohydrazide (7). The title compound
hydrazine]carbonyl]methyl]-1-piperazinyl]methyl}-
L
DOI: 10.1021/acs.jmedchem.5b00413
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Journal of Medicinal Chemistry
Article
benzenesulfonamide (11). The title compound was synthesized
according to general procedure D: 46b (164 mg, 0.50 mmol, 1.0
equiv), 47b (82 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035
mmol, 0.070 equiv), 2:1 MeOH/MeCN (3 mL, 0.15 M). Purification
by silica gel column chromatography (gradient, 0−20% MeOH/
15 (137 mg, 66.4%) as a white solid. 1H NMR (500 MHz, CDCl3): δ
11.26 (br s, 1H), 10.09 (br s, 1H), 8.31 (s, 1H), 7.26 (dd, 2H, J = 6.0,
8.0 Hz), 7.16 (dd, 1H, J = 1.5, 6.5 Hz), 7.02−6.97 (m, 3H), 6.80 (t,
1H, J = 7.5 Hz), 3.48 (s, 2H), 3.18 (s, 2H), 2.65 (t, 2H, J = 7.5 Hz),
2.61 (br s, 4H), 2.50 (br s, 4H), 1.65 (sext, 2H, J = 7.5 Hz), 0.95 (t,
3H, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3): δ 165.9, 162.1 (d, JC−F
= 243.6 Hz), 156.7, 151.3, 133.7 (d, JC−F = 3.0 Hz), 132.5, 130.7, 130.6
(d, JC−F = 7.8 Hz), 128.9, 118.9, 116.8, 115.2 (d, JC−F = 21.0 Hz), 62.1,
61.0, 53.8, 53.0, 32.0, 22.8, 14.2. 19F NMR (470 MHz, CDCl3): δ
−118.8. HRMS (ESI): m/z 413.2361 (M + H)+; calcd for
C23H30FN4O2, 413.2353. HPLC purity: 97%.
1
EtOAc) yielded 11 (211 mg, 89.0%) as a white solid. H NMR (500
MHz, (CD3)2CO): δ 11.78 (s, 1H), 10.76 (br s, 1H), 8.48 (s, 1H),
7.84 (d, 2H, J = 8.5 Hz), 7.51 (d, 2H, J = 8.5 Hz), 7.17 (d, 1H, J = 7.0
Hz), 7.14 (dd, 1H, J = 1.5, 8.0 Hz), 6.82 (t, 1H, J = 7.5 Hz), 6.54 (br s,
2H), 3.59 (s, 2H), 3.17 (s, 2H), 2.64−2.59 (m, 6H), 2.52 (br s, 4H),
1.63 (sext, 2H, J = 7.5 Hz), 0.93 (t, 3H, J = 7.5 Hz). 13C NMR (125
MHz, (CD3)2CO): δ 166.3, 157.3, 150.9, 144.0, 143.8, 132.7, 130.8,
129.9, 129.6, 126.9, 119.6, 118.3, 62.6, 61.7, 54.3, 53.6, 32.5, 23.4, 14.2.
HRMS (ESI): m/z 474.2175 (M + H)+; calcd for C23H32N5O4S,
474.2175. HPLC purity: 95%.
N′-[(2-Hydroxy-3-propylphenyl)methylene]-4-(4-fluoroben-
zoyl)-1-piperazineacetohydrazide (16). The title compound was
synthesized according to general procedure D: 46g (140 mg, 0.50
mmol, 1.0 equiv), 47b (82 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29
μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−15% MeOH/EtOAc)
N′-[(2-Hydroxy-3-propylphenyl)methylene]-4-benzoyl-1-pi-
perazineacetohydrazide (12). The title compound was synthesized
according to general procedure D: 46c (131 mg, 0.50 mmol, 1.0
equiv), 47b (82 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035
mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by silica gel
column chromatography (gradient, 50−100% EtOAc/hexanes, then
1
yielded 16 (133 mg, 62.4%) as a white solid. H NMR (500 MHz,
CDCl3): δ 11.24 (br s, 1H), 10.17 (br s, 1H), 8.25 (s, 1H), 7.37 (dd,
2H, J = 5.5, 8.5 Hz), 7.13 (dd, 1H, J = 1.5, 7.5 Hz), 7.06 (t, 2H, J = 8.5
Hz), 6.91 (dd, 1H, J = 1.5, 7.5 Hz), 6.77 (t, 1H, J = 7.5 Hz), 3.83 (br s,
2H), 3.49 (br s, 2H), 3.20 (s, 2H), 2.62−2.58 (m, 6H), 1.60 (sext, 2H,
J = 7.5 Hz), 0.91 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3): δ
169.6, 165.4, 163.6 (d, JC−F = 249.3 Hz), 156.6, 151.7, 132.6, 131.4 (d,
JC−F = 3.4 Hz), 129.5 (d, JC−F = 8.5 Hz), 128.9, 119.0, 116.7, 115.8 (d,
JC−F = 21.8 Hz), 60.9, 53.5 (br), 47.7 (br), 42.2 (br), 31.9, 22.7, 14.1.
19F NMR (470 MHz, CDCl3): δ −112.8. HRMS (ESI): m/z 427.2141
1
5% MeOH/EtOAc) yielded 12 (174 mg, 85.5%) as a white solid. H
NMR (500 MHz, CDCl3): δ 11.29 (s, 1H), 10.29 (br s, 1H), 8.23 (s,
1H), 7.41−7.34 (m, 5H), 7.13 (dd, 1H, J = 1.5, 7.5 Hz), 6.90 (dd, 1H,
J = 1.5, 7.5 Hz), 6.76 (t, 1H, J = 7.5 Hz), 3.80 (br s, 2H), 3.47 (br s,
2H), 3.18 (s, 2H), 2.71−2.52 (m, 6H, Ar-CH2-CH2), 1.61 (sext, 2H, J
= 7.5 Hz), 0.91 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3): δ
170.5, 165.5, 156.6, 151.5, 135.3, 132.5, 130.6, 130.1, 128.8, 128.7,
127.0, 118.9, 116.7, 60.8, 53.6 (br), 53.0 (br), 47.5 (br), 42.0 (br),
31.9, 22.7, 14.1. HRMS (ESI): m/z 409.2238 (M + H)+; calcd for
C23H29N4O3, 409.2240. HPLC purity: 98%.
N′-[(2-Hydroxy-3-propylphenyl)methylene]-4-[(4-
cyanophenyl)methyl]-1-piperazineacetohydrazide (13). The
title compound was synthesized according to general procedure D:
46d (273 mg, 1.0 mmol, 1.0 equiv), 47b (164 mg, 1.0 mmol, 1.0
equiv), 1.2 M HCl (58 μL, 0.070 mmol, 0.070 equiv), EtOH (7 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−15% MeOH/EtOAc) yielded 13 (373 mg, 88.8%) as a white solid.
1H NMR (500 MHz, CDCl3): δ 11.19 (br s, 1H), 9.99 (br s, 1H), 8.37
(s, 1H), 7.60 (d, 2H, J = 8.0 Hz), 7.44 (d, 2H, J = 7.5 Hz), 7.16 (dd,
1H, J = 1.5, 7.5 Hz), 7.04 (dd, 1H, J = 1.5, 7.5 Hz), 6.82 (t, 1H, J = 7.5
Hz), 3.58 (s, 2H), 3.19 (s, 2H), 2.66−2.63 (m, 6H), 2.52 (br s, 4H),
1.64 (sext, 2H, J = 7.5 Hz), 0.95 (t, 3H, J = 7.5 Hz). 13C NMR (125
MHz, CDCl3): δ 165.8, 156.8, 151.6, 144.0, 132.7, 132.3, 130.8, 129.6,
128.9, 119.1, 119.0, 116.8, 111.2, 62.4, 61.1, 53.8, 53.2, 32.0, 22.8, 14.2.
HRMS (ESI): m/z 420.2396 (M + H)+; calcd for C24H30N5O2,
420.2400. HPLC purity: 97%.
(M + H)+; calcd for C23H28FN4O3, 427.2145. HPLC purity: 96%.
N′-[(2-Hydroxy-3-propylphenyl)methylene]-4-[[4-
(trifluoromethyl)phenyl]methyl]-1-piperazineacetohydrazide
(17). The title compound was synthesized according to general
procedure D: 46h (158 mg, 0.50 mmol, 1.0 equiv), 47b (82 mg, 0.50
mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv),
EtOH (3 mL, 0.15 M). Purification by silica gel column
chromatography (gradient, 0−15% MeOH/EtOAc) yielded 17 (93.9
1
mg, 40.6%) as a yellow solid. H NMR (500 MHz, CDCl3): δ 11.23
(br s, 1H), 10.05 (br s, 1H), 8.33 (s, 1H), 7.57 (d, 2H, J = 8.0 Hz),
7.44 (d, 2H, J = 8.0 Hz), 7.16 (dd, 1H, J = 1.5, 7.5 Hz), 7.02 (dd, 2H, J
= 1.5, 7.5 Hz), 6.81 (t, 1H, J = 7.5 Hz), 3.58 (s, 2H), 3.19 (s, 2H),
2.67−2.62 (m, 6H), 2.53 (br s, 4H), 1.65 (sext, 2H, J = 7.5 Hz), 0.95
(t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3): δ 165.9, 156.8,
151.5, 142.4, 132.6, 130.8, 129.6 (q, JC−F = 32.0 Hz), 129.3, 128.9,
125.4 (q, JC−F = 3.6 Hz), 124.4 (q, JC−F = 270.5 Hz), 119.0, 116.9,
62.4, 61.1, 53.8, 53.2, 32.0, 22.8, 14.2. 19F NMR (470 MHz, CDCl3): δ
−65.5. HRMS (ESI): m/z 463.2321 (M + H)+; calcd for
C24H30F3N4O2, 463.2321. HPLC purity: 98%.
N′-[(2-Hydroxy-3-propylphenyl)methylene]-4-[4-
(trifluoromethyl)benzoyl]-1-piperazineacetohydrazide (18).
The title compound was synthesized according to general procedure
D: 46i (165 mg, 0.50 mmol, 1.0 equiv), 47b (82 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−15% MeOH/EtOAc) yielded 18 (216 mg, 90.8%) as a white solid.
1H NMR (500 MHz, CDCl3): δ 11.24 (br s, 1H), 10.14 (br s, 1H),
N′-[(2-Hydroxy-3-propylphenyl)methylene]-4-(4-cyanoben-
zoyl)-1-piperazineacetohydrazide (14). The title compound was
synthesized according to general procedure D: 46e (287 mg, 1.0
mmol, 1.0 equiv), 47b (164 mg, 1.0 mmol, 1.0 equiv), 1.2 M HCl (58
μL, 0.070 mmol, 0.070 equiv), EtOH (7 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−15% MeOH/EtOAc)
1
yielded 14 (377 mg, 86.9%) as a light yellow solid. H NMR (500
MHz, CDCl3): δ 11.15 (br s, 1H), 9.92 (br s, 1H), 8.32 (s, 1H), 7.71
(d, 2H, J = 8.0 Hz), 7.49 (d, 2H, J = 7.5 Hz), 7.16 (d, 1H, J = 7.0 Hz),
6.98 (dd, 1H, J = 1.5, 7.5 Hz), 6.80 (t, 1H, J = 7.5 Hz), 3.86 (br s, 2H),
3.44 (br s, 2H), 3.24 (s, 2H), 2.70 (br s, 2H), 2.64−2.57 (m, 4H), 1.62
(sext, 2H, J = 7.5 Hz), 0.93 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz,
CDCl3): δ 168.5, 165.2, 156.7, 152.0, 139.8, 132.9, 132.7, 130.8, 129.0,
127.9, 119.1, 118.1, 116.7, 113.9, 61.0, 53.5 (br), 47.5 (br), 42.1 (br),
32.0, 22.8, 14.2. HRMS (ESI): m/z 434.2188 (M + H)+; calcd for
C24H28N5O3, 434.2192. HPLC purity: 98%.
N′-[(2-Hydroxy-3-propylphenyl)methylene]-4-[(4-
fluorophenyl)methyl]-1-piperazineacetohydrazide (15). The
title compound was synthesized according to general procedure D:
46f (133 mg, 0.50 mmol, 1.0 equiv), 47b (82 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−20% MeOH/EtOAc) followed by precipitation from Et2O yielded
8.23 (s, 1H), 7.64 (d, 2H, J = 8.0 Hz), 7.47 (d, 2H, J = 8.0 Hz), 7.13
(d, 1H, J = 8.0 Hz), 6.89 (d, 1H, J = 7.5 Hz), 6.76 (t, 1H, J = 7.5 Hz),
3.85 (br s, 2H), 3.43 (br s, 2H), 3.21 (s, 2H), 2.73−2.58 (m, 6H), 1.60
(sext, 2H, J = 7.5 Hz), 0.90 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz,
CDCl3): δ 169.0, 165.4, 156.5, 151.6, 139.0, 132.7, 131.9 (q, JC−F
=
32.5 Hz), 130.6, 128.8, 127.5, 125.8 (q, JC−F = 3.5 Hz), 123.7 (q, JC−F
= 271.3 Hz), 119.0, 116.7, 60.8, 53.5 (br), 47.5 (br), 42.0 (br), 31.9,
22.7, 14.1. 19F NMR (470 MHz, CDCl3): δ −66.0. HRMS (ESI): m/z
477.2108 (M + H)+; calcd for C24H28F3N4O3, 477.2114. HPLC purity:
99%.
N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-4-(phenylmeth-
yl)-1-piperazineacetohydrazide (19). The title compound was
synthesized according to general procedure D: 46a (124 mg, 0.50
mmol, 1.0 equiv), 47c (70 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29
μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−20% MeOH/EtOAc)
M
DOI: 10.1021/acs.jmedchem.5b00413
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
yielded 19 (173 mg, 93.7%) as a white solid. H NMR (500 MHz,
CDCl3): δ 10.81 (br s, 1H), 10.13 (br s, 1H), 8.39 (s, 1H), 7.33−7.30
(m, 4H), 7.28−7.25 (m, 1H), 7.00 (dt, 1H, J = 3.0, 9.0 Hz), 6.94−6.89
(m, 2H), 3.55 (s, 2H), 3.19 (s, 2H), 2.63 (br s, 4H), 2.53 (br s, 4H).
13C NMR (125 MHz, CDCl3): δ 166.3, 155.9 (d, JC−F = 235.8 Hz),
154.8, 150.0, 137.9, 129.3, 128.5, 127.4, 118.9 (d, JC−F = 23.1 Hz),
Hz), 6.85 (dd, 1H, J = 4.5, 9.0 Hz), 6.79 (dd, 1H, J = 3.0, 8.5 Hz), 3.82
(br s, 2H), 3.41 (br s, 2H), 3.22 (s, 2H), 2.67 (br s, 2H), 2.54 (br s,
2H). 13C NMR (125 MHz, CDCl3): δ 168.4, 165.5, 155.7 (d, JC−F
=
236.1 Hz), 154.5, 149.9, 139.7, 132.6, 127.8, 119.0 (d, JC−F = 23.1 Hz),
118.2 (d, JC−F = 7.6 Hz), 118.1, 117.4 (d, JC−F = 7.5 Hz), 116.0 (d,
JC−F = 23.6 Hz), 113.7, 60.8, 53.4 (br), 52.7 (br), 47.4 (br), 42.0 (br).
19F NMR (470 MHz, CDCl3): δ −128.1. HRMS (ESI): m/z 410.1623
118.4 (d, JC−F = 7.6 Hz), 117.6 (d, JC−F = 7.5 Hz), 116.1 (d, JC−F
=
23.8 Hz), 63.0, 61.1, 53.9, 53.1. 19F NMR (470 MHz, CDCl3): δ
−128.5. HRMS (ESI): m/z 371.1877 (M + H)+; calcd for
C20H24FN4O2, 371.1883. HPLC purity: 95%.
(M + H)+; calcd for C21H21FN5O3, 410.1628. HPLC purity: 96%.
N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-4-[(4-
fluorophenyl)methyl]-1-piperazineacetohydrazide (24). The
title compound was synthesized according to general procedure D:
46f (133 mg, 0.50 mmol, 1.0 equiv), 47c (70 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−20% MeOH/EtOAc) yielded 24 (152 mg, 78.2%) as a pale yellow
4-{[4-[[[N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-
hydrazine]carbonyl]methyl]-1-piperazinyl]methyl}-
benzenesulfonamide (20). The title compound was synthesized
according to general procedure D: 46b (164 mg, 0.50 mmol, 1.0
equiv), 47c (70 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035
mmol, 0.070 equiv), 2:1 MeOH/MeCN (3 mL, 0.15 M). Purification
by silica gel column chromatography (gradient, 0−20% MeOH/
1
solid. H NMR (500 MHz, CDCl3): δ 10.83 (br s, 1H), 10.19 (br s,
1H), 8.33 (s, 1H), 7.25 (dd, 2H, J = 5.5, 8.5 Hz), 6.99−6.95 (m, 3H),
1
EtOAc) yielded 20 (172 mg, 82.1%) as a yellow solid. H NMR (500
6.90−6.86 (m, 2H), 3.47 (s, 2H), 3.18 (s, 2H), 2.60 (br s, 4H), 2.49
(br s, 4H). 13C NMR (125 MHz, CDCl3): δ 166.3, 162.1 (d, JC−F
=
MHz, (CD3)2CO): δ 11.33 (br s, 1H), 10.95 (br s, 1H), 8.49 (s, 1H),
7.85 (d, 2H, J = 8.0 Hz), 7.49 (d, 2H, J = 8.0 Hz), 7.12 (dd, 1H, J =
3.0, 9.0 Hz), 7.07 (dt, 1H, J = 3.0, 8.5 Hz), 6.91 (dd, 1H, J = 5.0, 9.0
Hz), 6.62 (br s, 2H), 3.55 (s, 2H), 3.19 (s, 2H), 2.59 (br s, 4H), 2.49
(br s, 4H). 13C NMR (125 MHz, (CD3)2CO): δ 166.8, 156.4 (d, JC−F
= 233.5 Hz), 155.4, 155.1 (d, JC−F = 2.8 Hz), 143.9, 143.6, 129.9,
243.9 Hz), 155.8 (d, JC−F = 236.0 Hz), 154.7 (d, JC−F = 1.4 Hz), 149.7
(d, JC−F = 2.6 Hz), 133.7 (d, JC−F = 3.0 Hz), 130.6 (d, JC−F = 7.9 Hz),
118.8 (d, JC−F = 23.3 Hz), 118.3 (d, JC−F = 7.6 Hz), 117.6 (d, JC−F
=
7.5 Hz), 116.0 (d, JC−F = 23.8 Hz), 115.2 (d, JC−F = 21.1 Hz), 62.1,
61.0, 53.8, 52.9. 19F NMR (470 MHz, CDCl3): δ −118.8, −128.4.
HRMS (ESI): m/z 389.1787 (M + H)+; calcd for C20H23F2N4O2,
389.1789. HPLC purity: 94%.
126.7, 119.2 (d, J
= 7.6 Hz), 118.7 (d, JC−F = 17.8 Hz), 118.6,
C−F
116.6 (d, JC−F = 23.9 Hz), 62.5, 61.5, 54.1, 53.4. 19F NMR (470 MHz,
(CD3)2CO): δ −127.3. HRMS (ESI): m/z 450.1609 (M + H)+; calcd
for C20H25FN5O4S, 450.1611. HPLC purity: 92%.
N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-4-(4-fluoroben-
zoyl)-1-piperazineacetohydrazide (25). The title compound was
synthesized according to general procedure D: 46g (140 mg, 0.50
mmol, 1.0 equiv), 47c (70 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29
μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−20% MeOH/EtOAc)
N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-4-benzoyl-1-pi-
perazineacetohydrazide (21). The title compound was synthesized
according to general procedure D: 46c (131 mg, 0.50 mmol, 1.0
equiv), 47c (70 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035
mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by silica gel
column chromatography (gradient, 0−20% MeOH/EtOAc) yielded
21 (157 mg, 81.6%) as a white solid. 1H NMR (500 MHz, CDCl3): δ
10.89 (br s, 1H), 10.50 (br s, 1H), 8.21 (s, 1H), 7.40−7.33 (m, 5H),
6.93 (dt, 1H, J = 2.5, 8.5 Hz), 6.84 (dd, 1H, J = 4.5, 9.0 Hz), 6.74 (dd,
1H, J = 2.5, 8.5 Hz), 3.79 (br s, 2H), 3.48 (br s, 2H), 3.17 (s, 2H), 2.60
(br s, 2H), 2.53 (br s, 2H). 13C NMR (125 MHz, CDCl3): δ 170.5,
165.8, 155.7 (d, JC−F = 235.8 Hz), 154.5, 149.7, 135.3, 130.1, 128.7,
127.0, 118.8 (d, JC−F = 23.1 Hz), 118.2 (d, JC−F = 7.5 Hz), 117.6 (d,
JC−F = 7.4 Hz), 116.0 (d, JC−F = 23.6 Hz), 60.7, 53.6 (br), 53.4 (br),
47.6 (br), 42.0 (br). 19F NMR (470 MHz, CDCl3): δ −128.3. HRMS
(ESI): m/z 385.1674 (M + H)+; calcd for C20H22FN4O3, 385.1676.
HPLC purity: 97%.
N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-4-[(4-
cyanophenyl)methyl]-1-piperazineacetohydrazide (22). The
title compound was synthesized according to general procedure D:
46d (137 mg, 0.50 mmol, 1.0 equiv), 47c (70 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−20% MeOH/EtOAc) yielded 22 (169 mg, 85.5%) as a white solid.
1H NMR (500 MHz, CDCl3): δ 10.84 (br s, 1H), 10.20 (br s, 1H),
1
yielded 25 (101 mg, 50.1%) as a white solid. H NMR (500 MHz,
CDCl3): δ 10.81 (br s, 1H), 10.25 (br s, 1H), 8.29 (s, 1H), 7.38 (dd,
2H, J = 5.5, 8.5 Hz), 7.07 (t, 2H, J = 8.5 Hz), 6.98−6.94 (m, 1H), 6.86
(dd, 1H, J = 4.0, 9.0 Hz), 6.79 (dd, 1H, J = 2.0, 8.0 Hz), 3.63 (br s,
4H), 3.21 (s, 2H), 2.59 (br s, 4H). 13C NMR (125 MHz, CDCl3): δ
169.7, 165.7, 163.6 (d, JC−F = 249.4 Hz), 155.8 (d, JC−F = 236.3 Hz),
154.6 (d, JC−F = 0.9 Hz), 150.0 (d, JC−F = 2.3 Hz), 131.3 (d, JC−F = 3.4
Hz), 129.5 (d, JC−F = 8.4 Hz), 119.0 (d, JC−F = 23.1 Hz), 118.3 (d, JC−F
= 7.5 Hz), 117.5 (d, JC−F = 7.3 Hz), 116.0 (d, JC−F = 24.6 Hz), 115.8
(d, JC−F = 21.9 Hz), 60.9, 53.5, 47.7, 42.2. 19F NMR (470 MHz,
CDCl3): δ −112.6, −128.2. HRMS (ESI): m/z 403.1573 (M + H)+;
calcd for C20H21F2N4O3, 403.1582. HPLC purity: 96%.
N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-4-[[4-
(trifluoromethyl)phenyl]methyl]-1-piperazineacetohydrazide
(26). The title compound was synthesized according to general
procedure D: 46h (158 mg, 0.50 mmol, 1.0 equiv), 47c (70 mg, 0.50
mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv),
EtOH (3 mL, 0.15 M). Purification by silica gel column
chromatography (gradient, 0−20% MeOH/EtOAc) yielded 26 (194
1
mg, 88.6%) as a pale yellow solid. H NMR (500 MHz, CDCl3): δ
10.83 (br s, 1H), 10.17 (br s, 1H), 8.34 (s, 1H), 7.56 (d, 2H, J = 8.0
Hz), 7.43 (d, 2H, J = 8.0 Hz), 6.98 (dt, 1H, J = 3.0, 8.0 Hz), 6.91−6.86
(m, 2H), 3.57 (s, 2H), 3.19 (s, 2H), 2.62 (br s, 4H), 2.52 (br s, 4H).
13C NMR (125 MHz, CDCl3): δ 166.3, 155.8 (d, JC−F = 236.0 Hz),
154.7 (d, JC−F = 1.5 Hz), 149.8 (d, JC−F = 2.4 Hz), 142.4 (d, JC−F = 0.8
Hz), 129.5 (q, JC−F = 32.1 Hz), 129.3, 125.3 (q, JC−F = 3.8 Hz), 124.4
(q, JC−F = 270.6 Hz), 118.9 (d, JC−F = 23.0 Hz), 118.3 (d, JC−F = 7.6
Hz), 117.6 (d, JC−F = 7.5 Hz), 116.1 (d, JC−F = 23.6 Hz), 62.3, 61.0,
53.8, 53.1. 19F NMR (470 MHz, CDCl3): δ −65.4, −128.4. HRMS
(ESI): m/z 439.1765 (M + H)+; calcd for C21H23F4N4O2, 439.1757.
HPLC purity: 96%.
N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-4-[4-
(trifluoromethyl)benzoyl]-1-piperazineacetohydrazide (27).
The title compound was synthesized according to general procedure
D: 46i (165 mg, 0.50 mmol, 1.0 equiv), 47c (70 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−20% MeOH/EtOAc) yielded 27 (173 mg, 76.5%) as a white solid.
8.31 (s, 1H), 7.56 (d, 2H, J = 8.5 Hz), 7.41 (d, 1H, J = 8.0 Hz), 6.95
(dt, 1H, J = 3.0, 9.0 Hz), 6.88−6.85 (m, 2H), 3.54 (s, 2H), 3.18 (s,
2H), 2.60 (br s, 4H), 2.50 (br s, 4H). 13C NMR (125 MHz, CDCl3): δ
166.2, 155.7 (d, JC−F = 235.9 Hz), 154.6, 149.6, 144.0, 132.2, 129.5,
118.9 (d, JC−F = 22.6 Hz), 118.6, 118.2 (d, JC−F = 7.6 Hz), 117.5 (d,
JC−F = 7.5 Hz), 116.0 (d, JC−F = 23.8 Hz), 110.9, 62.2, 61.0, 53.6, 53.0.
19F NMR (470 MHz, CDCl3): δ −128.4. HRMS (ESI): m/z 396.1838
(M + H)+; calcd for C21H23FN5O2, 396.1836. HPLC purity: 94%.
N′-[(5-Fluoro-2-hydroxyphenyl)methylene]-4-(4-cyanoben-
zoyl)-1-piperazineacetohydrazide (23). The title compound was
synthesized according to general procedure D: 46e (144 mg, 0.50
mmol, 1.0 equiv), 47c (70 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29
μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−20% MeOH/EtOAc)
1
yielded 23 (144 mg, 70.1%) as a white solid. H NMR (500 MHz,
CDCl3): δ 10.79 (br s, 1H), 10.16 (br s, 1H), 8.28 (s, 1H), 7.67 (d,
2H, J = 8.0 Hz), 7.47 (d, 2H, J = 8.5 Hz), 6.95 (dt, 1H, J = 3.0, 8.0
N
DOI: 10.1021/acs.jmedchem.5b00413
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H NMR (500 MHz, CDCl3): δ 10.80 (br s, 1H), 10.20 (br s, 1H),
8.28 (s, 1H), 7.65 (d, 2H, J = 8.0 Hz), 7.48 (d, 2H, J = 7.5 Hz), 6.96
(dt, 1H, J = 2.5, 8.0 Hz), 6.87 (dd, 1H, J = 4.5, 8.5 Hz), 6.79 (dd, 1H, J
= 2.5, 8.0 Hz), 3.84 (br s, 2H), 3.44 (br s, 2H), 3.22 (s, 2H), 2.70 (br
s, 2H), 2.55 (br s, 2H). 13C NMR (125 MHz, CDCl3): δ 169.1, 165.6,
D: 46d (137 mg, 0.50 mmol, 1.0 equiv), 47d (90 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−15% MeOH/EtOAc) yielded 31 (164 mg, 75.3%) as a white solid.
1H NMR (500 MHz, CDCl3): δ 11.12 (br s, 1H), 10.16 (br s, 1H),
8.28 (s, 1H), 7.57 (d, 2H, J = 8.0 Hz), 7.42 (d, 2H, J = 8.0 Hz), 6.88
(dd, 1H, J = 3.0, 9.0 Hz), 6.72 (dd, 1H, J = 3.0, 8.0 Hz), 5.94 (tdd, 1H,
J = 6.5, 10.0, 17.0 Hz), 5.09−5.05 (m, 2H), 3.55 (s, 2H), 3.38 (d, 2H, J
= 7.0 Hz), 3.19 (s, 2H), 2.62 (br s, 4H), 2.51 (br s, 4H). 13C NMR
(125 MHz, CDCl3): δ 166.0, 155.5 (d, JC−F = 235.5 Hz), 152.5 (d,
JC−F = 1.4 Hz), 149.9, 144.0, 135.7, 132.2, 130.1 (d, JC−F = 6.8 Hz),
129.5, 119.0 (d, JC−F = 23.0 Hz), 119.0, 116.8 (d, JC−F = 7.8 Hz),
155.8 (d, JC−F = 236.3 Hz), 154.6 (d, JC−F = 1.3 Hz), 150.0 (d, JC−F
=
1.9 Hz), 138.9, 132.0 (q, JC−F = 32.6 Hz), 127.5, 125.8 (q, JC−F = 3.6
Hz), 123.7 (q, JC−F = 271.3 Hz), 119.0 (d, JC−F = 23.1 Hz), 118.3 (d,
JC−F = 7.6 Hz), 117.4 (d, JC−F = 7.5 Hz), 116.0 (d, JC−F = 23.8 Hz),
60.8, 53.5 (br), 47.5 (br), 42.0 (br). 19F NMR (470 MHz, CDCl3): δ
−66.0, −128.1. HRMS (ESI): m/z 453.1552 (M + H)+; calcd for
C21H21F4N4O3, 453.1550. HPLC purity: 97%.
116.5, 113.9 (d, JC−F = 23.5 Hz), 111.0, 62.3, 60.9, 53.8, 53.1, 33.8. 19
F
N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]methylene]-
4-(phenylmethyl)-1-piperazineacetohydrazide (28). The title
compound was synthesized according to general procedure D: 46a
(124 mg, 0.50 mmol, 1.0 equiv), 47d (90 mg, 0.50 mmol, 1.0 equiv),
1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−20%
MeOH/EtOAc) yielded 28 (186 mg, 90.8%) as a white solid. 1H
NMR (500 MHz, CDCl3): δ 11.16 (s, 1H), 10.20 (br s, 1H), 8.28 (s,
1H), 7.34−7.30 (m, 4H), 7.28−7.25 (m, 1H), 6.91 (dd, 1H, J = 3.0,
9.0 Hz), 6.74 (dd, 1H, J = 3.0, 8.0 Hz), 5.98 (tdd, 1H, J = 6.5, 10.0,
16.5 Hz), 5.13−5.08 (m, 2H), 3.54 (s, 2H), 3.42 (d, 2H, J = 7.0 Hz),
3.20 (s, 2H), 2.63 (br s, 4H), 2.53 (br s, 4H). 13C NMR (125 MHz,
CDCl3): δ 166.2, 155.5 (d, JC−F = 235.8 Hz), 152.5 (d, JC−F = 1.4 Hz),
149.8 (d, JC−F = 2.5 Hz), 137.9, 135.7, 130.2 (d, JC−F = 6.8 Hz), 129.2,
128.4, 127.3, 119.0 (d, JC−F = 23.1 Hz), 116.8 (d, JC−F = 7.9 Hz),
116.6, 113.9 (d, JC−F = 23.5 Hz), 62.9, 61.0, 53.8, 53.0, 33.8. 19F NMR
(470 MHz, CDCl3): δ −128.6. HRMS (ESI): m/z 411.2191 (M +
H)+; calcd for C23H28FN4O2, 411.2196. HPLC purity: 99%.
4-{[4-[[[N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]-
methylene]hydrazine]carbonyl]methyl]-1-piperazinyl]methyl}-
benzensulfonamide (29). The title compound was synthesized
according to general procedure D: 46b (164 mg, 0.50 mmol, 1.0
equiv), 47d (90 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035
mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by silica gel
column chromatography (gradient, 0−20% MeOH/EtOAc) yielded
29 (214 mg, 87.2%) as a white solid. 1H NMR (500 MHz,
(CD3)2CO): δ 11.73 (br s, 1H), 10.94 (br s, 1H), 8.46 (s, 1H),
7.85 (d, 2H, J = 8.5 Hz), 7.48 (d, 2H, J = 8.5 Hz), 6.96 (d, 2H, J = 9.0
Hz), 6.62 (br s, 2H), 5.99 (tdd, 1H, J = 6.5, 10.0, 16.5 Hz), 5.10 (qd,
1H, J = 1.5, 17.0 Hz), 5.04 (qd, 2H, J = 1.5, 10.0 Hz), 3.55 (s, 2H),
3.40 (d, 2H, J = 7.0 Hz), 3.19 (s, 2H), 2.59 (br s, 4H), 2.49 (br s, 4H).
13C NMR (125 MHz, (CD3)2CO): δ 166.8, 156.1 (d, JC−F = 233.6
NMR (470 MHz, CDCl3): δ −128.6. HRMS (ESI): m/z 436.2144 (M
+ H)+; calcd for C24H27FN5O2: 436.2149. HPLC purity: 95%.
N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]methylene]-
4-(4-cyanobenzoyl)-1-piperazineacetohydrazide (32). The title
compound was synthesized according to general procedure D: 46e
(144 mg, 0.50 mmol, 1.0 equiv), 47d (90 mg, 0.50 mmol, 1.0 equiv),
1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−15%
MeOH/EtOAc) yielded 32 (196 mg, 87.2%) as a white solid. 1H
NMR (500 MHz, CDCl3): δ 11.10 (br s, 1H), 10.21 (br s, 1H), 8.20
(s, 1H), 7.65 (d, 2H, J = 8.0 Hz), 7.45 (d, 2H, J = 8.5 Hz), 6.84 (dd,
1H, J = 3.0, 9.0 Hz), 6.61 (dd, 1H, J = 3.0, 8.0 Hz), 5.88 (tdd, 1H, J =
7.0, 10.0, 16.5 Hz), 5.03−5.00 (m, 2H), 3.81 (br s, 2H), 3.40 (br s,
2H), 3.31 (d, 2H, J = 6.5 Hz), 3.21 (s, 2H), 2.66 (br s, 2H), 2.54 (br s,
2H). 13C NMR (125 MHz, CDCl3): δ 168.3, 165.4, 155.4 (d, JC−F
=
235.9 Hz), 152.3, 150.1, 139.7, 135.4, 132.5, 130.0 (d, JC−F = 6.8 Hz),
127.7, 119.1 (d, JC−F = 23.1 Hz), 118.0, 116.6 (d, JC−F = 7.9 Hz),
116.5, 113.8 (d, JC−F = 23.5 Hz), 113.6, 60.7, 53.3 (br), 47.3 (br), 42.0
(br), 33.6. 19F NMR (470 MHz, CDCl3): δ −128.3. HRMS (ESI): m/
z 450.1931 (M + H)+; calcd for C24H25FN5O3: 450.1941. HPLC
purity: 97%.
N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]methylene]-
4-[(4-fluorophenyl)methyl]-1-piperazineacetohydrazide (33).
The title compound was synthesized according to general procedure
D: 46f (133 mg, 0.50 mmol, 1.0 equiv), 47d (90 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−20% MeOH/EtOAc) yielded 33 (176 mg, 82.0%) as a yellow solid.
1H NMR (500 MHz, CDCl3): δ 11.15 (br s, 1H), 10.22 (br s, 1H),
8.26 (s, 1H), 7.25 (dd, 2H, J = 5.5, 8.5 Hz), 6.98 (t, 2H, J = 8.5 Hz),
6.89 (dd, 1H, J = 3.0, 9.0 Hz), 6.72 (dd, 1H, J = 3.0, 8.0 Hz), 5.95 (tdd,
1H, J = 6.5, 10.0, 17.0 Hz), 5.10−5.06 (m, 2H), 3.47 (s, 2H), 3.39 (d,
2H, J = 6.5 Hz), 3.18 (s, 2H), 2.61 (br s, 4H), 2.49 (br s, 4H). 13C
NMR (125 MHz, CDCl3): δ 166.2, 162.1 (d, JC−F = 243.8 Hz), 155.5
(d, JC−F = 235.5 Hz), 152.5 (d, JC−F = 0.9 Hz), 149.8, 135.7, 133.6 (d,
JC−F = 3.0 Hz), 130.6 (d, JC−F = 7.8 Hz), 130.1 (d, JC−F = 6.8 Hz),
119.0 (d, JC−F = 23.0 Hz), 116.8 (d, JC−F = 7.8 Hz), 116.5, 115.1 (d,
JC−F = 21.0 Hz), 113.9 (d, JC−F = 23.5 Hz), 62.1, 61.0, 53.7, 52.9, 33.8.
19F NMR (470 MHz, CDCl3): δ −118.8, −128.6. HRMS (ESI): m/z
Hz), 153.1 (d, JC−F = 1.3 Hz), 149.6, 143.9, 143.5, 136.6, 130.5 (d, JC−F
= 7.0 Hz), 129.8, 126.7, 118.7 (d, JC−F = 23.1 Hz), 118.4 (d, JC−F = 8.0
Hz), 116.5, 114.6 (d, JC−F = 23.6 Hz), 62.5, 61.5, 54.1, 53.4, 34.2. 19F
NMR (470 MHz, (CD3)2CO): δ −127.4. HRMS (ESI): m/z 490.1930
(M + H)+; calcd for C23H29FN5O4S 490.1924. HPLC purity: 98%.
N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]methylene]-
4-benzoyl-1-piperazineacetohydrazide (30). The title compound
was synthesized according to general procedure D: 46c (131 mg, 0.50
mmol, 1.0 equiv), 47d (90 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29
μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−15% MeOH/EtOAc)
429.2095 (M + H)+; calcd for C23H27F2N4O2, 429.2102. HPLC purity:
98%.
N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]methylene]-
4-(4-fluorobenzoyl)-1-piperazineacetohydrazide (34). The title
compound was synthesized according to general procedure D: 46g
(140 mg, 0.50 mmol, 1.0 equiv), 47d (90 mg, 0.50 mmol, 1.0 equiv),
1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−15%
MeOH/EtOAc) yielded 34 (163 mg, 73.8%) as a white solid. 1H
NMR (500 MHz, CDCl3): δ 11.10 (br s, 1H), 10.23 (br s, 1H), 8.25
(s, 1H), 7.38 (dd, 2H, J = 5.5, 8.5 Hz), 7.07 (t, 2H, J = 8.5 Hz), 6.88
(dd, 1H, J = 3.0, 9.0 Hz), 6.65 (dd, 1H, J = 3.0, 8.5 Hz), 5.92 (tdd, 1H,
J = 6.5, 9.5, 17.0 Hz), 5.10−5.04 (m, 2H), 3.82 (br s, 2H), 3.50 (br s,
2H), 3.36 (d, 2H, J = 6.5 Hz), 3.21 (s, 2H), 2.59 (br s, 4H). 13C NMR
(125 MHz, CDCl3): δ 169.7, 165.6, 163.6 (d, JC−F = 249.8 Hz), 155.5
(d, JC−F = 235.6 Hz), 152.5 (d, JC−F = 1.4 Hz), 150.3 (d, JC−F = 2.5
Hz), 135.6, 131.3 (d, JC−F = 3.5 Hz), 130.2 (d, JC−F = 6.9 Hz), 129.5
(d, JC−F = 8.4 Hz), 119.2 (d, JC−F = 23.1 Hz), 116.7 (d, JC−F = 7.8 Hz),
1
yielded 30 (186 mg, 87.8%) as a white solid. H NMR (500 MHz,
CDCl3): δ 11.17 (s, 1H), 10.45 (br s, 1H), 8.19 (s, 1H), 7.40−7.33
(m, 5H), 6.86 (dd, 1H, J = 3.0, 9.0 Hz), 6.60 (dd, 1H, J = 3.0, 8.5 Hz),
5.91 (tdd, 1H, J = 6.5, 9.5, 18.0 Hz), 5.09−5.03 (m, 2H), 3.80 (br s,
2H), 3.47 (br s, 2H), 3.35 (d, 2H, J = 6.5 Hz), 3.19 (s, 2H), 2.56 (br s,
4H). 13C NMR (125 MHz, CDCl3): δ 170.5, 165.6, 155.4 (d, JC−F
=
235.5 Hz), 152.4 (d, JC−F = 1.4 Hz), 150.1 (d, JC−F = 1.8 Hz), 135.6,
135.3, 130.1, 130.1, 128.7, 127.0, 119.0 (d, JC−F = 23.0 Hz), 116.8 (d,
JC−F = 7.9 Hz), 116.5, 113.9 (d, JC−F = 23.5 Hz), 60.7, 53.6 (br), 47.6
(br), 42.0 (br), 33.7. 19F NMR (470 MHz, CDCl3): δ −128.5. HRMS
(ESI): m/z 425.1991 (M + H)+; calcd for C23H26FN4O3, 425.1989.
HPLC purity: 97%.
N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]methylene]-
4-[(4-cyanophenyl)methyl]-1-piperazineacetohydrazide (31).
The title compound was synthesized according to general procedure
O
DOI: 10.1021/acs.jmedchem.5b00413
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
116.6, 115.9 (d, JC−F = 21.8 Hz), 113.9 (d, JC−F = 23.5 Hz), 60.9, 53.6
(br), 47.7 (br), 42.2 (br), 33.8. 19F NMR (470 MHz, CDCl3): δ
−112.6, −128.4. HRMS (ESI): m/z 443.1886 (M + H)+; calcd for
C23H25F2N4O3, 443.1895. HPLC purity: 98%.
(d, 2H, J = 8.0 Hz), 7.49 (d, 2H, J = 8.0 Hz), 6.98 (dd, 1H, J = 3.0, 9.5
Hz), 6.93 (dd, 1H, J = 3.0, 8.5 Hz), 6.59 (br s, 2H), 3.57 (s, 2H), 3.18
(s, 2H), 2.64−2.59 (m, 6H), 2.50 (br s, 4H), 1.63 (sext, 2H, J = 7.5
Hz), 0.93 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, (CD3)2CO): δ
166.6, 156.1 (d, JC−F = 233.1 Hz), 153.4 (d, JC−F = 1.4 Hz), 149.7 (d,
JC−F = 2.9 Hz), 143.9, 143.6, 132.8 (d, JC−F = 6.9 Hz), 129.9, 126.8,
N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]methylene]-
4-[[4-(trifluoromethyl)phenyl]methyl]-1-piperazineacetohy-
drazide (35). The title compound was synthesized according to
general procedure D: 46h (158 mg, 0.50 mmol, 1.0 equiv), 47d (90
mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070
equiv), EtOH (3 mL, 0.15 M). Purification by silica gel column
chromatography (gradient, 0−20% MeOH/EtOAc) yielded 35 (176
mg, 73.7%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 11.14 (br
s, 1H), 10.19 (br s, 1H), 8.28 (s, 1H), 7.56 (d, 2H, J = 8.0 Hz), 7.43
(d, 2H, J = 8.0 Hz), 6.90 (dd, 1H, J = 3.0, 9.0 Hz), 6.72 (dd, 2H, J =
3.0, 8.0 Hz), 5.96 (tdd, 1H, J = 6.5, 10.0, 17.0 Hz), 5.11−5.08 (m, 2H),
3.57 (s, 2H), 3.40 (d, 2H, J = 6.5 Hz), 3.20 (s, 2H), 2.63 (br s, 4H),
2.53 (br s, 4H). 13C NMR (125 MHz, CDCl3): δ 166.1, 155.6 (d, JC−F
= 235.5 Hz), 152.5 (d, JC−F = 1.3 Hz), 149.9 (d, JC−F = 2.4 Hz), 142.4,
135.7, 130.2 (d, JC−F = 6.8 Hz), 129.5 (q, JC−F = 32.0 Hz), 129.3, 125.3
(q, JC−F = 3.8 Hz), 124.4 (q, JC−F = 270.5 Hz), 119.1 (d, JC−F = 23.1
Hz), 116.9 (d, JC−F = 7.8 Hz), 116.6, 114.0 (d, JC−F = 23.5 Hz), 62.3,
61.0, 53.8, 53.1, 33.8. 19F NMR (470 MHz, CDCl3): δ −65.4, −128.5.
HRMS (ESI): m/z 479.2066 (M + H)+; calcd for C24H27F4N4O2,
479.2070. HPLC purity: 96%.
N′-[[5-Fluoro-2-hydroxy-3-(2-propenyl)phenyl]methylene]-
4-[4-(trifluoromethyl)benzoyl]-1-piperazineacetohydrazide
(36). The title compound was synthesized according to general
procedure D: 46i (165 mg, 0.50 mmol, 1.0 equiv), 47d (90 mg, 0.50
mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv),
EtOH (3 mL, 0.15 M). Purification by silica gel column
chromatography (gradient, 0−15% MeOH/EtOAc) yielded 36 (139
mg, 56.3%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 11.07 (br
s, 1H), 10.14 (br s, 1H), 8.26 (s, 1H), 7.66 (d, 2H, J = 8.5 Hz), 7.50
(d, 2H, J = 8.0 Hz), 6.89 (dd, 1H, J = 3.0, 9.0 Hz), 6.66 (dd, 1H, J =
3.0, 8.5 Hz), 5.93 (tdd, 1H, J = 7.0, 10.0, 16.5 Hz), 5.10−5.04 (m, 2H),
3.86 (br s, 2H), 3.45 (br s, 2H), 3.37 (d, 2H, J = 6.5 Hz), 3.23 (s, 2H),
2.69 (br s, 2H), 2.57 (br s, 2H). 13C NMR (125 MHz, CDCl3): δ
169.1, 165.5, 155.6 (d, JC−F = 235.9 Hz), 152.5, 150.4 (d, JC−F = 2.1
Hz), 138.9, 135.6, 132.0 (q, JC−F = 32.6 Hz), 130.3 (d, JC−F = 6.8 Hz),
127.5, 125.9 (q, JC−F = 3.8 Hz), 123.7 (q, JC−F = 271.1 Hz), 119.3 (d,
JC−F = 23.0 Hz), 116.7 (d, JC−F = 4.6 Hz), 116.7, 114.0 (d, JC−F = 23.5
Hz), 60.9, 53.6 (br), 47.5 (br), 42.2 (br), 33.8. 19F NMR (470 MHz,
CDCl3): δ −66.0, −128.4. HRMS (ESI): m/z 493.1868 (M + H)+;
calcd for C24H25F4N4O3, 493.1863. HPLC purity: 97%.
N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)methylene]-4-
(phenylmethyl)-1-piperazineacetohydrazide (37). The title
compound was synthesized according to general procedure D: 46a
(124 mg, 0.50 mmol, 1.0 equiv), 47e (91 mg, 0.50 mmol, 1.0 equiv),
1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−15%
MeOH/EtOAc) yielded 37 (174 mg, 84.6%) as an off-white solid. 1H
NMR (500 MHz, CDCl3): δ 11.10 (s, 1H), 10.19 (br s, 1H), 8.26 (s,
1H), 7.32−7.30 (m, 4H), 7.28−7.25 (m, 1H), 6.89 (dd, 1H, J = 3.0,
9.0 Hz), 6.71 (dd, 1H, J = 3.0, 8.5 Hz), 3.54 (s, 2H), 3.20 (s, 2H),
2.66−2.59 (m, 6H), 2.53 (br s, 4H), 1.64 (sext, 2H, J = 7.5 Hz), 0.95
(t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3): δ 166.1, 155.4 (d,
JC−F = 235.1 Hz), 152.8, 150.0, 137.9, 132.7 (d, JC−F = 6.6 Hz), 129.2,
128.4, 127.3, 119.1 (d, JC−F = 22.5 Hz), 116.7 (d, JC−F = 7.9 Hz), 113.4
(d, JC−F = 23.4 Hz), 63.0, 61.0, 53.8, 53.1, 31.9, 22.5, 14.0. 19F NMR
(470 MHz, CDCl3): δ −129.1. HRMS (ESI): m/z 413.2345 (M +
H)+; calcd for C23H30FN4O2, 413.2353. HPLC purity: 99%.
119.0 (d, JC−F = 22.8 Hz), 118.3 (d, JC−F = 8.1 Hz), 114.2 (d, JC−F
=
23.6 Hz), 62.5, 61.6, 54.2, 53.4, 32.3, 23.1, 14.1. 19F NMR (470 MHz,
(CD3)2CO): δ −127.7. HRMS (ESI): m/z 492.2074 (M + H)+; calcd
for C23H31FN5O4S, 492.2081. HPLC purity: 96%.
N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)methylene]-4-ben-
zoyl-1-piperazineacetohydrazide (39). The title compound was
synthesized according to general procedure D: 46c (131 mg, 0.50
mmol, 1.0 equiv), 47e (91 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29
μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−10% MeOH/EtOAc)
1
yielded 39 (189 mg, 88.9%) as a white solid. H NMR (500 MHz,
CDCl3): δ 11.13 (s, 1H), 10.48 (br s, 1H), 8.15 (s, 1H), 7.38−7.32
(m, 5H), 6.83 (dd, 1H, J = 3.0, 9.0 Hz), 6.55 (dd, 1H, J = 3.0, 8.5 Hz),
3.80 (br s, 2H), 3.46 (br s, 2H), 3.18 (s, 2H), 2.59−2.54 (m, 6H), 1.57
(sext, 2H, J = 7.5 Hz), 0.88 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz,
CDCl3): δ 170.5, 165.6, 155.3 (d, JC−F = 235.1 Hz), 152.6 (d, JC−F
=
1.4 Hz), 150.2 (d, JC−F = 2.5 Hz), 135.3, 132.5 (d, JC−F = 6.8 Hz),
130.1, 128.7, 126.9, 119.1 (d, JC−F = 22.6 Hz), 116.6 (d, JC−F = 7.9
Hz), 113.4 (d, JC−F = 23.4 Hz), 60.7, 53.5 (br), 47.5 (br), 42.0 (br),
31.8, 22.4, 13.9. 19F NMR (470 MHz, CDCl3): δ −129.0. HRMS
(ESI): m/z 427.2144 (M + H)+; calcd for C23H28FN4O3, 427.2145.
HPLC purity: 99%.
N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)methylene]-4-[(4-
cyanophenyl)methyl]-1-piperazineacetohydrazide (40). The
title compound was synthesized according to general procedure D:
46d (137 mg, 0.50 mmol, 1.0 equiv), 47e (91 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−15% MeOH/EtOAc) yielded 40 (180 mg, 82.1%) as a white solid.
1H NMR (500 MHz, CDCl3): δ 11.07 (br s, 1H), 10.16 (br s, 1H),
8.25 (s, 1H), 7.57 (d, 2H, J = 8.5 Hz), 7.42 (d, 2H, J = 8.0 Hz), 6.86
(dd, 1H, J = 3.0, 9.0 Hz), 6.68 (dd, 1H, J = 3.0, 8.0 Hz), 3.55 (s, 2H),
3.18 (s, 2H), 2.70−2.57 (m, 6H), 2.51 (br s, 4H). 13C NMR (125
MHz, CDCl3): δ 166.0, 155.4 (d, JC−F = 235.1 Hz), 152.7 (d, JC−F
=
1.4 Hz), 150.0 (d, JC−F = 2.5 Hz), 144.0, 132.6 (d, JC−F = 6.8 Hz),
132.2, 129.5, 119.1 (d, JC−F = 22.6 Hz), 119.0, 116.6 (d, JC−F = 8.0
Hz), 113.4 (d, JC−F = 23.5 Hz), 110.9, 62.3, 60.9, 53.7, 53.1, 31.9, 22.4,
14.0. 19F NMR (470 MHz, CDCl3): δ −129.0. HRMS (ESI): m/z
438.2301 (M + H)+; calcd for C24H29FN5O2, 438.2305. HPLC purity:
96%.
N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)methylene]-4-(4-
cyanobenzoyl)-1-piperazineacetohydrazide (41). The title com-
pound was synthesized according to general procedure D: 46e (144
mg, 0.50 mmol, 1.0 equiv), 47e (91 mg, 0.50 mmol, 1.0 equiv), 1.2 M
HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−10%
MeOH/EtOAc) yielded 41 (196 mg, 86.5%) as a white solid. 1H
NMR (500 MHz, CDCl3): δ 11.03 (br s, 1H), 10.17 (br s, 1H), 8.19
(s, 1H), 7.66 (d, 2H, J = 8.0 Hz), 7.46 (d, 2H, J = 8.0 Hz), 6.84 (dd,
1H, J = 3.0, 9.0 Hz), 6.59 (dd, 1H, J = 3.0, 8.5 Hz), 3.82 (br s, 2H),
3.41 (br s, 2H), 3.22 (s, 2H), 2.66 (br s, 2H), 2.57−2.52 (m, 2H), 1.55
(sext, 2H, J = 7.5 Hz), 0.87 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz,
CDCl3): δ 168.3, 165.4, 155.3 (d, JC−F = 235.5 Hz), 152.6 (d, JC−F
=
1.1 Hz), 150.3 (d, JC−F = 2.0 Hz), 139.7, 132.6 (d, JC−F = 6.8 Hz),
132.5, 127.7, 119.2 (d, JC−F = 22.6 Hz), 118.0, 116.4 (d, JC−F = 7.9
Hz), 113.6, 113.4 (d, JC−F = 23.3 Hz), 60.7, 53.4 (br), 47.4 (br), 42.0
(br), 31.7, 22.3, 13.9. 19F NMR (470 MHz, CDCl3): δ −128.7. HRMS
(ESI): m/z 452.2098 (M + H)+; calcd for C24H27FN5O3, 452.2098.
HPLC purity: 99%.
N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)methylene]-4-[(4-
fluorophenyl]methyl)-1-piperazineacetohydrazide (42). The
title compound was synthesized according to general procedure D:
46f (133 mg, 0.50 mmol, 1.0 equiv), 47e (91 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
4-{[4-[[[N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)-
methylene]hydrazine]carbonyl]methyl]-1- piperazinyl]-
methyl}benzenesulfonamide (38). The title compound was
synthesized according to general procedure D: 46b (164 mg, 0.50
mmol, 1.0 equiv), 47e (91 mg, 0.50 mmol, 1.0 equiv), 1.2 M HCl (29
μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M). Purification by
silica gel column chromatography (gradient, 0−20% MeOH/EtOAc)
1
yielded 38 (221 mg, 89.9%) as a white solid. H NMR (500 MHz,
(CD3)2CO): δ 11.68 (br s, 1H), 10.92 (br s, 1H), 8.46 (s, 1H), 7.84
P
DOI: 10.1021/acs.jmedchem.5b00413
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
0.15 M). Purification by silica gel column chromatography (gradient,
0−20% MeOH/EtOAc) yielded 42 (202 mg, 93.8%) as a yellow solid.
1H NMR (500 MHz, CDCl3): δ 11.07 (br s, 1H), 10.16 (br s, 1H),
Hz), 113.4 (d, JC−F = 23.4 Hz), 60.8, 53.5 (br), 47.5 (br), 42.0 (br),
31.8, 22.4, 14.0. 19F NMR (470 MHz, CDCl3): δ −66.0, −128.7.
HRMS (ESI): m/z 495.2008 (M + H)+; calcd for C24H27F4N4O3,
495.2019. HPLC purity: 98%.
8.26 (s, 1H), 7.26 (dd, 2H, J = 5.5, 8.5 Hz), 6.99 (t, 2H, J = 8.5 Hz),
6.88 (dd, 1H, J = 3.0, 9.0 Hz), 6.70 (dd, 1H, J = 3.0, 8.0 Hz), 3.48 (s,
2H), 3.18 (s, 2H), 2.66−2.58 (m, 6H), 2.50 (br s, 4H), 1.62 (sext, 2H,
J = 7.5 Hz), 0.94 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3): δ
166.1, 162.1 (d, JC−F = 243.8 Hz), 155.5 (d, JC−F = 235.0 Hz), 152.8
(d, JC−F = 0.9 Hz), 150.1 (d, JC−F = 2.0 Hz), 133.7, 132.7 (d, JC−F = 6.8
Hz), 130.7 (d, JC−F = 7.9 Hz), 119.2 (d, JC−F = 22.5 Hz), 116.7 (d, JC−F
= 7.9 Hz), 115.2 (d, JC−F = 21.0 Hz), 113.5 (d, JC−F = 23.4 Hz), 62.1,
61.0, 53.8, 53.0, 31.9, 22.5, 14.1. 19F NMR (470 MHz, CDCl3): δ
−118.8, −129.0. HRMS (ESI): m/z 431.2250 (M + H)+; calcd for
C23H29F2N4O2, 431.2259. HPLC purity: 98%.
N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)methylene]-4-(4-
fluorobenzoyl)-1- piperazineacetohydrazide (43). The title
compound was synthesized according to general procedure D: 46g
(140 mg, 0.50 mmol, 1.0 equiv), 47e (91 mg, 0.50 mmol, 1.0 equiv),
1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL, 0.15 M).
Purification by silica gel column chromatography (gradient, 0−10%
MeOH/EtOAc) yielded 43 (195 mg, 87.7%) as a white solid. 1H
NMR (500 MHz, CDCl3): δ 11.07 (br s, 1H), 10.32 (br s, 1H), 8.18
(s, 1H), 7.36 (dd, 2H, J = 5.0, 8.5 Hz), 7.05 (t, 2H, J = 8.5 Hz), 6.84
(dd, 1H, J = 3.0, 9.0 Hz), 6.57 (dd, 1H, J = 3.0, 8.5 Hz), 3.81 (br s,
2H), 3.48 (br s, 2H), 3.20 (s, 2H), 2.62−2.50 (m, 6H), 1.56 (sext, 2H,
J = 7.5 Hz), 0.88 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3): δ
169.6, 165.6, 163.5 (d, JC−F = 249.3 Hz), 155.4 (d, JC−F = 235.4 Hz),
152.7 (d, JC−F = 0.9 Hz), 150.3, 132.6 (d, JC−F = 6.8 Hz), 131.3 (d,
JC−F = 3.4 Hz), 129.4 (d, JC−F = 8.4 Hz), 119.2 (d, JC−F = 22.6 Hz),
ASSOCIATED CONTENT
■
S
* Supporting Information
Full biological protocols, NMR spectra, LC traces for liver
microsome experiments, and formation curves for IC50
determination and zinc-binding determination. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 217-333-0363. Fax: 217-244-8024. E-mail: hergenro@
illinois.edu.
Notes
The authors declare the following competing financial
interest(s): The University of Illinois has filed patents on
these compounds.
ACKNOWLEDGMENTS
■
We are grateful to the National Institutes of Health (Grant
R01-CA120439) and University of Illinois for support of this
work. H.S.R. was partially supported by the Richard B.
Silverman Predoctoral Fellowship from the American Chemical
Society Division of Medicinal Chemistry. R.C.B. is a National
Science Foundation predoctoral fellow, a Robert C. and
Carolyn J. Springborn graduate fellow, and a member of the
NIH Chemistry-Biology Interface Training Program (Grant
NRSA 1-T32-GM070421).
116.5 (d, JC−F = 7.9 Hz), 115.8 (d, JC−F = 21.6 Hz), 113.4 (d, JC−F
=
23.4 Hz), 60.7, 53.5 (br), 47.7 (br), 42.1 (br), 31.8, 22.4, 13.9. 19F
NMR (470 MHz, CDCl3): δ −112.6, −128.8. HRMS (ESI): m/z
445.2049 (M + H)+; calcd for C23H27F2N4O3, 445.2051. HPLC purity:
98%.
N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)methylene]-4-[[4-
(trifluoromethyl)phenyl]methyl]-1-piperazineacetohydrazide
(44). The title compound was synthesized according to general
procedure D: 46h (158 mg, 0.50 mmol, 1.0 equiv), 47e (91 mg, 0.50
mmol, 1.0 equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv),
EtOH (3 mL, 0.15 M). Purification by silica gel column
chromatography (gradient, 0−20% MeOH/EtOAc) yielded 44 (184
ABBREVIATION USED
■
PAC-1, procaspase-activating compound 1
REFERENCES
1
mg, 76.5%) as a light yellow solid. H NMR (500 MHz, CDCl3): δ
■
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11.08 (br s, 1H), 10.18 (br s, 1H), 8.25 (s, 1H), 7.56 (d, 2H, J = 8.0
Hz), 7.43 (d, 2H, J = 8.0 Hz), 6.88 (dd, 1H, J = 3.0, 9.0 Hz), 6.69 (dd,
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2.52 (br s, 4H), 1.62 (sext, 2H, J = 7.5 Hz), 0.93 (t, 3H, J = 7.5 Hz).
13C NMR (125 MHz, CDCl3): δ 166.1, 155.5 (d, JC−F = 235.1 Hz),
152.8 (d, JC−F = 1.2 Hz), 150.0 (d, JC−F = 2.6 Hz), 142.4, 132.7 (d,
JC−F = 6.8 Hz), 129.5 (q, JC−F = 32.1 Hz), 129.3, 125.3 (q, JC−F = 3.8
Hz), 124.4 (q, JC−F = 270.5 Hz), 119.2 (d, JC−F = 22.6 Hz), 116.7 (d,
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N′-[(5-Fluoro-2-hydroxy-3-propylphenyl)methylene]-4-[4-
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The title compound was synthesized according to general procedure
D: 46i (165 mg, 0.50 mmol, 1.0 equiv), 47e (91 mg, 0.50 mmol, 1.0
equiv), 1.2 M HCl (29 μL, 0.035 mmol, 0.070 equiv), EtOH (3 mL,
0.15 M). Purification by silica gel column chromatography (gradient,
0−10% MeOH/EtOAc) yielded 45 (140 mg, 56.7%) as a white solid.
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(q, JC−F = 271.0 Hz), 119.3 (d, JC−F = 22.6 Hz), 116.5 (d, JC−F = 7.9
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