Development of Allosteric Hydrazide-Containing Class i Histone Deacetylase Inhibitors for Use in Acute Myeloid Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.6b01385

One of the biggest hurdles yet to be overcome for the continued improvement of histone deacetylase (HDAC) inhibitors is finding alternative motifs equipotent to the classic and ubiquitously used hydroxamic acid. The N-hydroxyl group of this motif is highly subject to sulfation/glucoronidation-based inactivation in humans; compounds containing this motif require much higher dosing in clinic to achieve therapeutic concentrations. With the goal of developing a second generation of HDAC inhibitors lacking this hydroxamate, we designed a series of potent and selective class I HDAC inhibitors using a hydrazide motif. These inhibitors are impervious to glucuronidation and demonstrate allosteric inhibition. In vitro and ex vivo characterization of our lead analogues' efficacy, selectivity, and toxicity profiles demonstrate that they possess low nanomolar activity against models of acute myeloid leukemia (AML) and are at least 100-fold more selective for AML than solid immortalized cells such as HEK293 or human peripheral blood mononuclear cells.

Full text of DOI:10.1021/acs.jmedchem.6b01385

Subscriber access provided by United Arab Emirates University | Libraries Deanship
Article
Development of Allosteric Hydrazide-Containing Class I Histone
Deacetylase Inhibitors for Use in Acute Myeloid Leukemia
Jesse McClure, Cheng Zhang, Elizabeth Inks, Yuri K. Peterson, Jiaying Li, and C. James Chou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01385 • Publication Date (Web): 18 Oct 2016
Downloaded from http://pubs.acs.org on October 20, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Development of Allosteric HydrazideꢀContaining
Class I Histone Deacetylase Inhibitors for Use in
Acute Myeloid Leukemia
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jesse J. McClureǂ, Cheng Zhangǂ, Elizabeth S. Inks, Yuri Peterson, Jiaying Li, C. James Chou*
Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy,
Medical University of South Carolina, Charleston, South Carolina 29425, USA
Keywords: Histone Deacetylases, HDAC, Hydrazide, Allosteric, Acute Myeloid Leukemia,
Glucuronidation, MichaelisꢀMenten.
ABSTRACT
One of the biggest hurdles yet to be overcome for the continued improvement of Histone
Deacetylase (HDAC) inhibitors is finding alternative motifs equipotent to the classic and
ubiquitously used hydroxamic acid. The Nꢀhydroxyl group of this motif is highly subject to
sulfation/glucoronidationꢀbased inactivation in humans; compounds containing this motif require
much higher dosing in clinic to achieve therapeutic concentrations. With the goal of developing
a second generation of HDAC inhibitors, lacking this hydroxamate, we designed a series of
potent and selective class I HDAC inhibitors using a hydrazide motif. These inhibitors are
impervious to glucuronidation and demonstrate allosteric inhibition. In vitro and ex vivo
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
characterization of our lead analogs’ efficacy, selectivity, and toxicity profiles demonstrate they
possess low nanomolar activity against models of Acute Myeloid Leukemia (AML) and are at
least 100ꢀfold more selective for AML than solid immortalized cells such as HEK293 or human
peripheral blood mononuclear cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Histone Deacetylases (HDAC) are a collective of evolutionarily conserved enzymes that are
capable of removing acylꢀbased post translational modifications (PTMs) from εꢀnitrogens of
1
lysine residues of histones and other proteins.
The 11 identified human HDACs are
phylogenetically subdivided into four classes: class I, IIa, IIb, and IV, with class III being the
2
Sirtuin family of enzymes. Of these enzymes, only HDACs 1, 2, 3, and 6, members of the class
3
I and IIb families, have shown biologically relevant deacetylation ability. The canonical targets
of HDACs 1, 2, and 3 are acetylated histones H3 (HH3) and H4 (HH4), whereas HDAC6
4
ꢀ6
activity is the sole controller of tubulin acetylation.
Inhibition of HDACs 1, 2, 3, and 6 is a promising pathway for treating hematologic cancers in
clinic, with FDA approvals of HDAC inhibitors for the treatment of Tꢀcell lymphoma and
Multiple Myeloma (MM) (Chart 1). Additionally, selective HDAC inhibitors have shown great
promise in treating preꢀclinical models of cognitive dysfunction and inflammatoryꢀmediated
7ꢀ19
diseases.
However, despite their clinical successes and increasing promise as novel therapies
in a multitude of diseases, approved HDAC inhibitors are commonly associated with doseꢀ
20ꢀ34
limiting toxicities.
The hydroxamate group, until the FDA approval of vorinostat, was
considered to be an undesirable motif in medicinal chemistry. The nonꢀselective metal chelating
properties of the hydroxamate are what allow it to bind the catalytic zinc metal that HDACs rely
ACS Paragon Plus Environment

Page 3 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
on, but also cause compounds containing these groups to be associated with many undesirable
3
5
effects secondary to nonꢀtargeted active site metal chelation. While all FDA approved
inhibitors demonstrate very strong efficacy in the subꢀmicromolar range, the doses given in clinic
are relatively much higher. The reason for this increased required dose, and the subsequent side
effects due to offꢀtarget effects, could be in part to a metabolic inactivation of some of these
compounds. The hydroxamic acid that is present on three of the four FDA approved HDAC
inhibitors, vorinostat, panobinostat, and belinostat, is known to be sulfated or glucuronidated
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
6ꢀ39
extensively, specifically by UDP glucuronosyltransferase 1A1.
In the case of vorinostat, the
process of glucuronidation has been demonstrated to abrogate its HDAC inhibition activity, and
3
6
is further evidenced with its issues concerning achieving therapeutic concentrations in clinic.
As belinostat and panobinostat also share this hydroxamic acid motif, and have subsequently
been shown to be glucuronidated in vivo, a safe assumption can be made that this metabolism
causes inactivation for these compounds as well.
Attempts to generate novel, nonꢀhydroxamic acid metalꢀchelating motifs have resulted in the
use of orthoꢀaminoanilideꢀbased HDAC inhibitors, most notably MSꢀ275 (entinostat). However,
4
0
the benzylic primary amine of this motif is also subject to glucuronidationꢀbased inactivation.
4
1
Further, this inhibitor was shown to be hepatotoxic in mice.
In a more recent attempt to
generate nonꢀhydroxamic acid metalꢀchelating motifs, Wang and colleagues demonstrated a
4
2
novel, potent, class I HDACꢀspecific inhibitor series based around a hydrazide motif. Their
synthetically refined, lead analog from their initial publication possessed 60 nM HDAC3
inhibition, 500 nM and 100 nM for HDACs 1 and 2 respectively, and at least 20 fold selectivity
for class I HDACs compared to HDAC6. With slightly better potency than the similarly
selective orthoꢀaminoanilide ꢀbased inhibitor entinostat, and containing a novel moiety that
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
would not likely be subject to glucuronidation/sulfation, we postulated a more potent series of
class I selective inhibitors could be generated using hydrazide rather than hydroxamic acid or a
benzylic amine. Herein we report on a series of nanomolar to picomolar hydrazide containing
HDAC inhibitors, the most potent of which display nanomolar efficacy as single agents in
leukemiaꢀbased ex vivo models. Further, these compounds are impervious to glucuronidation,
inhibit class I HDACs via an allosteric mechanism, display superior, selective toxicity profiles
compared to panobinostat at equivalent concentrations in peripheral blood mononuclear cells
(PBMCs), and demonstrate greater than 100ꢀfold selectivity toward killing Acute Myeloid
Leukemia (AML) when compared to solid tumor cells such as HEK293. This combination of
potency, novel inhibition mechanism, and inability to be inactivated via glucuronidation and
likely sulfation, and selective toxicity toward leukemia cells make for promising candidates in
the treatment of AML.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RESULTS
The goal of our structureꢀactivity relationship studies for the hydrazideꢀbased compounds was
to develop a potent and selective inhibitor of class I HDACs, with particular emphasis on
HDAC3 inhibition. HDAC3 has shown increasing promise as a novel target to combat leukemia
4
3
as its inhibition controls hematopoiesis. As such, lead analogs from these studies were tested in
preꢀclinical leukemia models to determine efficacy and tested against solid tumor models and
PBMCs to demonstrate selectivity.
ACS Paragon Plus Environment

Page 5 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Chemistry of butylhydrazide analogs. We began our structureꢀactivity relationship studies
by attempting to identify an ideal substituent for the carbonyl of the hydrazide motif. As shown
in Scheme 1, we began with either an aromatic carboxylic acid, acyl chloride, or ester. Using
commercially available starting materials, we generated the corresponding hydrazide molecule
through reactions a, b, and c, depending on the composition of the starting molecule. Target
hydrazide compounds were reacted with butylaldehyde to generate the compound of interest.
Carboxylic acids were reacted with oxalyl chloride in methylene chloride with catalytic amounts
of dimethylformamide (Scheme 1, reaction a). The resulting acyl chloride, or commercially
purchased acyl chlorides were stirred in methanol to give the corresponding methyl ester
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(Scheme 1, reaction b). The generated or commercially purchased methyl esters were refluxed
in methanol with a hydrazine water salt to generate a hydrazide of interest (Scheme 1, reaction
c). The resulting compounds were then refluxed in ethanol in the presence of magnesium sulfate
and butylaldehyde followed by a reduction with sodium cyanoborohydride in acidified methanol
to give the desired products 1aꢀ1l (Scheme 1, reactions d and e). A notable exception to this
scheme was the synthesis of 1d, which was unable to be esterified from the commercially
available corresponding acyl chloride until the addition of two equivalents of triethylamine.
Additionally, compound 1i spontaneously formed the corresponding pyrazolidinone when
reacted with the hydrazine salt due to the presence of an unsaturated bond alpha to the ester
carbonyl. Instead, the carboxylic acid starting material was reacted with HOBt and DCC in
acetonitrile. The resulting intermediate was reacted with the hydrazine water salt in acetonitrile
to yield the corresponding hydrazide. Lastly, compound 1l was the result of reacting methyl 4ꢀ
(aminomethyl)benzoate with benzoyl chloride to generate the methyl ester corresponding to 1l.
From here, the reaction carried on as shown in Scheme 1, reactions d and e.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HDAC inhibition of series 1 inhibitors. With the primary goal of designing a potent and
ideally selective series inhibitor of class I HDAC inhibitors, we screened compounds 1aꢀ1l
against HEK293 cell lysates using a fluorogenic acetylated lysine substrate. All compounds
were tested thrice against HEK293 lysate and recombinant HDAC3. If the compound was found
to possess an IC less than 100 nM against HDAC3 and less than 1000 nM against HEK293
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
lysate, it was tested thrice against recombinant HDACs 1 and 2 to determine selectivity for class
I. The summation of the corresponding findings are summarized in Table 1, with data for
HDACs 1 and 2 in Supporting Information Table S1.
The relatively inefficacious compounds 1b, 1d, 1f, and 1g demonstrated that heterocyclic
substitution was not favored compared to phenyl groups. Further, a para position oxygen
provided mild benefit as seen in compounds 1a and 1c. However, a carbon, whether aliphatic or
aromatic, was superior in this position, as seen in 1e and 1l. Interestingly, the creation of an
unsaturated bond at the alpha position to the carbonyl, as seen in compound 1i, provided ~6ꢀfold
increased HDAC3 inhibition when compared to its saturated counterpart 1h. This can be
explained as it creates an acrylamideꢀlike structure which is likely capable of forming covalent
bonds with cysteine residues on the HDAC enzyme; allowing for enhanced inhibition ability.
While neither compound was particularly potent, it was interesting to see the sharp difference in
activity between 1j and 1k. This possibly demonstrates that the binding pocket near this position
is relatively narrow and the wider surface area of the 1ꢀnaphthyl group of 1j is unable to bind
adequately, whereas the 2ꢀnaphthyl group of 1k is able to fit more reasonably or is less sterically
hindered. The compound 1l possessed the most potent carbonyl attachment, with an IC of less
5
0
than 10 nM and K of less than 2 nM for HDAC3 and 8ꢀ to 10ꢀfold selectivity for HDAC3
i
ACS Paragon Plus Environment

Page 7 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compared to HDACs 1 and 2. Using this finding, we next pursued structure activity
relationships on substitutions of the βꢀnitrogen relative to the hydrazide.
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chemistry of N-(4-(hydrazide)benzyl)benzamide analogs. We began our second refinement
synthetic scheme by generating the Nꢀ(4ꢀ(hydrazinecarbonyl)benzyl)benzamide which would be
used in all subsequent reactions for this group. This was achieved as shown in Scheme 2,
reactions aꢀc. Briefly, the starting material, 4ꢀ(aminomethyl)benzoic acid, was refluxed in
methanol and concentrated acid to generate the much more soluble corresponding methyl ester
hydrochloride salt (Scheme 2, reaction a). This intermediate was reacted with benzoyl chloride
in ethyl acetate and water in the presence of potassium carbonate to form an amide bond and
afford 4ꢀ(benzamidomethyl)benzoate (Scheme 2, reaction b). This was in turn refluxed with the
hydrazine water salt in methanol to generate the desired intermediate (Scheme 2, reaction c).
Lastly, this intermediate was Nꢀmethylated using various aldehydes in ethanol and magnesium
sulfate followed by reduction using sodium cyanoborohydride in acidified methanol to yield the
desired products, 2aꢀ2m (Scheme 2, reactions d and e).
HDAC inhibition of series 2 inhibitors. We screened compounds 2aꢀ2m thrice against
HEK293 lysates and recombinant human HDAC3; the resulting data is summarized in Table 2.
Compounds demonstrating an IC less than 100 nM against HDAC3 and less than 1000 nM
5
0
against HEK293 lysates were then tested thrice against recombinant HDACs 1 and 2 to
determine selectivity profiles. Data from HDACs 1 and 2 can be found in Supporting
Information Table S2. Generally, branched alkyl substitution, as seen in 2b, provided inferior
inhibition compared to nonꢀbranched analogs such as 2d. There was an overall decrease in
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
inhibition as the chain length increased past six carbons seen with compounds 2h, 2i, and 2k.
Cyclic additions at the end of alkyl chains, such as those seen in 2g and 2m, provided negligible
effects. Lastly, the addition of a trifluoro group for compound 2f provided worse HDAC3
inhibition compared to the nonꢀfluorinated 2d. Overall, very little potency was gained from this
refinement series, seeing the slight benefit of an Nꢀsubstituted propyl chain of 2d compared to
the corresponding butyl chain of 1l which dropped the IC to less than 5 nM and lowered the K
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
i
to the subꢀnanomolar range for HDAC3.
Chemistry N-(4-(hydrazide)benzyl)cinnamamide analogs. With the information from our
second structureꢀactivity relationship study providing insight that a nonꢀbranched propyl chain
substitution on the βꢀnitrogen of the hydrazide seemingly is ideal, we moved forward by further
modifying the carbonyl substituent. We noticed an increase in the use of Nꢀhydroxylated
acrylamide groups for HDAC inhibitors, present even in panobinostat and belinostat, as well as a
multitude of preꢀclinical inhibitors. This motif is a known Michael acceptor and likely forms
covalent bonds with the sulfhydryl group of cysteines on proteins in vivo. Curious to see the
effect on efficacy of incorporating an α, β unsaturated ketone, a similar Michael acceptor, we
developed a cinnamamide derivative to build off of. This was achieved by again refluxing 4ꢀ
(aminomethyl)benzoic acid in acidified methanol (Scheme 3, reaction a). Cinnamoyl chloride
was formed using cinnamic acid and performing an acyl chlorination using oxalyl chloride in
methylene chloride with catalytic amounts of dimethylformamide (Scheme 3, reaction b). The
product of this reaction was mixed with the benzoate from reaction a in a 1:1 (v/v) mixture of
ethyl acetate and water with potassium carbonate to form the corresponding amide bond
(Scheme 3, reaction c). Here, we reacted this compound with lithium hydroxide in a solution of
ACS Paragon Plus Environment

Page 9 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
methanol, water, and tetrahydrofuran (2:1:2 v/v) to generate the corresponding carboxylic acid
(Scheme 3, reaction d). While it was never attempted, reactions c and d may theoretically be
combined using lithium hydroxide in place of potassium carbonate in reaction c to cleave the
methyl ester while also simultaneously forming the amide bond. With the carboxylic acid
generated, we performed an amine coupling with HOBt and DCC in dimethylformamide
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(Scheme 3, reaction e). This intermediate was then reacted with a hydrazine water salt at 0°C.
Finally, the hydrazide was reacted with the aldehyde of interest in ethanol with addition of
magnesium sulfate and subsequently reduced using sodium cyanoborohydride in acidified
methanol to yield compounds 3aꢀ3e (Scheme 3, reactions f and g).
HDAC inhibition of series 3 inhibitors. We ended our in vitro screening by interrogating the
IC values of 3aꢀ3e against HEK293 lysates and recombinant human HDACs 1, 2, and 3. The
5
0
results from these studies are listed in Table 3. The most striking change when comparing this
cinnamamide derivative to the benzamide derivatives are their potencies. All compounds tested
display less than 40 nM IC50 values against HDAC3 with the exception of the hexyl chain
containing 3e. The lead candidate from this study was the propyl chain containing 3b,
possessing a subꢀnanomolar IC50 and K against HDAC3 with 10ꢀ and 100ꢀfold selectivity
i
toward HDACs 1 and 2, respectively. The butyl chain derivative, 3c, that mimicked the
substituent of the very potent 1l provided singleꢀdigit nanomolar potency against HDAC3, yet
was approximately 3ꢀ5 fold weaker when compared to 3b. These findings suggest that a βꢀ
nitrogen alkyl substituent chain length of 3ꢀ4 carbons provides ideal conditions, with the ethyl,
pentyl, and hexyl groups of 3a, 3d, and 3e, respectively, having diminished inhibitory prowess
when compared to 3b or 3c.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Mass spectrometric analysis of select hydrazide-based inhibitors in glucuronidating
environments. To demonstrate that these compounds were indeed highly unlikely to be
glucuronidated in vivo, we adapted and performed an ex vivo assay using a protocol derived
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
from Walsky and colleagues. Briefly, vorinostat, 1l, 2d, and 3b were incubated with human
liver microsomes, UDP Glucuronic acid, and alamethicin (a poreꢀforming antibiotic) for 12
hours at 37°C.
The reaction was quenched with a 47:50:3 (v/v) mixture of
water:acetonitrile:formic acid. The vessel was subjected to centrifugation and the supernatant
filtered and examined via electrospray ionization liquid chromatography mass spectrometry. A
parallel study that lacked UDP Glucuronic acid was performed with each inhibitor as a negative
control. Comparing Figure 1a (blue) and Figure 1b (red) demonstrates the presence of the oꢀ
+
glucuronidated metabolite of vorinostat with a (m+H )/z of 441.04 (Figure 1b, bottom right), in
4
5
line with previous findings.
This mass shift was not present in the corresponding negative
control lacking UDP Glucuronic acid (Figure 1a, middle right). There are no differences
between masses or ionization patterns in any of the hydrazideꢀcontaining compounds (Figures
1c-d, Supporting Information Figures S1a-d), suggesting these compounds are not readily
glucuronidated in environments where vorinostat is extensively glucuronidated. It was also
noted that 3b, possessing an unsaturated bond, was reduced in this model, despite no NADPH
being added to the reaction vessel (Supporting Information Figures S1c-d). This may
demonstrate a potential metabolite of this compound.
Interrogation of in vitro binding kinetics of hydrazide-containing HDAC inhibitors.
Generally speaking, the very reason why groups like orthoꢀaminoanilide or hydroxamates are
ACS Paragon Plus Environment

Page 11 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
able to chelate zinc and other dicationic metals is the same reason why they are prime
glucuonidation targets. Their possession of primary alcohols and primary amines not only allow
them to ionically complex with the positively charged zinc, but also allows them to be
extensively inactivated via glucuronidation. After demonstrating that the hydrazide motif was not
glucuronidated in ex vivo environments, we questioned whether it was inhibiting HDACs
through a direct active zinc chelation. We hypothesized that the compound should demonstrate
typical MichaelisꢀMenten competitive inhibition, similar to vorinostat, if it was truly chelating
zinc in the active site of HDACs. Thus, we performed in vitro Vmax studies using recombinant
HDACs 1 and 3 and compounds 2d and 3b. Applying a double reciprocal conversion of the data
yielded LineweaverꢀBurke plots. Surprisingly, and contrary to previous data, our hydrazideꢀ
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
nd
containing compounds clearly demonstrate a convergence in the 2 quadrant, indicative of
mixed inhibition (Figures 2a-b) and nonꢀcompetitive inhibition (Figures 2c-d). This is in direct
4
2
contrast to the initial findings published by Wang and colleagues. Their published data seem to
match a canonical competitive inhibitor, with their corresponding LineweaverꢀBurke plots
demonstrating all doses and control intersecting directly on the yꢀaxis, representative of 1/Vmax
.
However, the corresponding Vmax plots from which these graphs were derived display changes in
Vmax. This would mean the yꢀintercepts on the double reciprocal LineweaverꢀBurke plots should
be different as well. To further ensure our findings were valid, we used vorinostat as a positive
control, a known competitive inhibitor with extensive kinetic analyses published
46
independently.
Matching established data, our results demonstrated complete convergence
directly on the yꢀaxis, indicative of competitive inhibition (Figures 2e-f), which is in direct
agreement with the very similar Vmax values seen at each dose. This finding, coupled with the
lack of glucuronidation ex vivo, points to an allosteric binding site on class I HDACs that is
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
strongly inhibited by our hydrazideꢀcontaining inhibitors, and has little to do with catalytic site
zinc chelation. The Vmax plots from which these LineweaverꢀBurke plots were derived may be
seen in Supporting Information Figures S2a-f.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HDAC3 molecular docking study of HDAC inhibitors. With the results thus far suggesting
an allosteric binding site, we were curious if we could identify a potential site at which our
inhibitors were binding. Using the solution structure of HDAC3 bound to Ncor2, pdb: 4A69, we
probed the HDAC3 surface with 3b resulting in a heavy concentration of poses near the interface
with the Ncor2 coenzyme (Supporting Information Figure S3a). We then calculated the
propensity for ligand interaction which revealed a very positive binding region directly
overlapping with the high density 3b binding area (Supporting Information Figure S3b).
Using this pocket, we performed a flexible induced fit binding of 2d, 3b, and vorinostat and
calculated binding scores from the top 20 (lowest energy) poses from each set. Our lead
inhibitors 2d and 3b display nearly identical binding scores, which is line with their near
identical in vitro activity and electrospatial occupancies. The canonical binder vorinostat bound
less tightly to this pocket, as evidenced by its higher energy posing scores (Supporting
Information Figure S3c). Lastly, when we posed the lowest energy poses for 2d and 3b, we see
significant overlap in electrospatial occupancy, indicative of homologous modes of binding
(Figure 3a), whereas the lowest pose for vorinostat results in a completely different orientation,
with the hydroxamic acid exposed to solvent and its phenyl group serving as the main area of
interaction (Figure 3b). Additionally, a narrow tunnel can be seen where the propyl hydrazide of
both 2d and 3b fit nicely. This matches the in vitro and ex vivo data demonstrating that adding
branching alkyl chains, or extending the alkyl chains past a length of four carbons leads to
ACS Paragon Plus Environment

Page 13 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
markedly diminished inhibition activity. Taken together, these findings illustrate the potential for
a novel, allosteric binding site that the propyl hydrazide motif is particularly astute at fitting into.
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Efficacy of lead analog in ex vivo cell models. Satisfied with results of our three structure
activity studies and the potential for allosteric inhibitors that are impervious to glucuronidation,
we transitioned from recombinant enzyme preꢀscreening to cellular treatment. Given that HDAC
inhibitors are most successful in hematologic malignancies such as lymphomas and myelomas,
as shown by their FDA approvals, we screened our most potent inhibitor, 3b, against an array of
leukemia and Multiple Myeloma cell lines. Briefly, Molmꢀ14, an acute monocytic leukemia cell
4
7
48
49
line ; HLꢀ60, an acute promyelocitic cell line ; RS4ꢀ11, an acute lymphoblastic leukemia ;
5
0
51
K562, a chronic myelogenous leukemia ; MV4ꢀ11, an acute myeloid leukemia ; and RPMIꢀ
52
8
226, a Multiple Myeloma cell line , were all used to determine the efficacy of 3b against a
multitude of different hematologic cancers. Cells were treated for 48 hours before viability was
assessed with CellTiterꢀBlue Cell Viability Assay. The active fluorescent reagent in this assay is
resazurin. This molecule is converted by the functional mitochondria in the viable cells to
resorufin. Resorufin intensity is therefore directly related to cell viability. The resulting EC50
values from each cell line is summarized in Table 4. 3b demonstrates the highest level of
potency against the AML cell line MV4ꢀ11; possessing an EC for the cells at less than 50 nM.
5
0
Taken together with the efficacy toward Molm14 and RS4ꢀ11, both acute myeloid leukemia
subsets, we can see that 3b is particularly selective for acute types of leukemia when compared
to its lack of efficacy toward the chronic leukemia cell line, K562 or the chromosomal
translocation containing HLꢀ60 cell line. Further, when tested against the classical Multiple
Myeloma cell line, RPMIꢀ8226, 3b demonstrated incomplete kill curves even at mM
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
concentrations. Following the potency findings of 3b in MV4ꢀ11 cells, we assessed the efficacy
of 1l, 2d, and 3b in these cells as single agents. Panobinostat, vorinostat, entinostat, 1l, 2d, and
3b were tested against MV4ꢀ11 for 48 hours of treatment. Cells were treated with CellTiterꢀBlue
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to determine viability which was measured using a spectrophotometer as a function of resorufin
intensity. Results from these experiments are shown as an EC cell viability curve in Figure 4a.
5
0
As a negative control, we also performed this experiment using 2c, a compound whose structure
very closely matches 2d, differing only by a nonꢀreduced nitrogenꢀcarbon bond, while
possessing no appreciable inhibition against recombinant HDAC3. Matching its weak prowess
as an HDAC inhibitor from our recombinant HDAC assay, this compound demonstrated no
appreciable activity against MV4ꢀ11 cells even at doses as high as 250 µM (Supporting
Information Figure S4a).
As single agents, these compounds possess less than 300 nM EC values against MV4ꢀ11 with
5
0
complete kill curves. To demonstrate that our initial HDAC activity preꢀscreening assay was
correlative with efficacy against MV4ꢀ11 cells, we plotted the compounds’ respective potency
toward HEK293 lysates as a function of their IC50 with their respective EC50 values against
MV4ꢀ11 cells (Supporting Information Figure S4b). The results demonstrate a linear
relationship with considerable correlation, demonstrating the ability of our HDAC activity preꢀ
screen assay to translate to ex vivo potency in AML cells.
To further demonstrate the correlation between MV4ꢀ11 cell death with hyperacetylation of
histones H3 and H4, we performed Western blot analysis of MV4ꢀ11 cell lysates after 24 hour
treatment with 100 nM of inhibitor (Figure 4b). The data demonstrate that our lead inhibitors, 1l,
2d, and 3b are more potent at increasing the concentrations of acetylated histones H3 and H4,
markers of HDACs 1, 2, and 3 inhibition, than the FDA approved vorinostat or the orthoꢀ
ACS Paragon Plus Environment

Page 15 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
aminoanilideꢀbased entinostat (Figure 4b-c). Additionally, this upregulation is doseꢀdependent,
with increasing levels of hyperacetylation seen with increasing concentrations of inhibitor
(Figure 4d). As a negative control, we once again used 2c to further ensure the validity of our
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
preꢀcellular screening. At a concentration of 100 nM, 2c was also used in MV4ꢀ11 cells for
western blot analysis and demonstrated no appreciable ability to increase acetylation of histones
H3 and H4 (Supporting Information Figure S4c). Further, upregulation of histones H3 and H4
is selective, as our hydrazide based inhibitors do not demonstrate any effect on levels of
acetylated tubulin, a marker of HDAC6 inhibition (Figures 4b and 3d). This is an important
characteristic, as upregulation of acetylated histones H3 and H4 is inversely and logarithmically
correlated with EC against AML, that is, an increase in acetylated histones H3 and H4 leads to
50
an exponential decrease in EC against these cells (Supporting Information Figures S4d-e).
50
This is in sharp contrast to acetylated tubulin, where increasing levels of acetylation are not as
directly correlative with EC against MV4ꢀ11 cells (Figure 4b).
50
Selectivity and toxicity profiling of lead analogs. Concluding our experiments, we sought to
determine if the hydrazide based inhibition of HDACs was a nonꢀselective cell death mechanism,
or if it was selective toward nonꢀsolid tumor cells, particularly various forms of leukemia. We
examined the ex vivo effect of 1l, 2d, and 3b in HEK293 and HeLa cells, wellꢀstudied solid
tumor cell lines. Briefly, cells were preꢀincubated for 24 hours prior to treatment. After
treatment, cells were incubated for an additional 48 hours before measurement of cell viability as
a function of resorufin intensity was determined. The corresponding EC values demonstrate at
50
least 100 fold higher selectivity toward the MV4ꢀ11 cell line (compare Figures 4a and 5a-b).
Further, we wanted to examine our lead compounds’ toxicity profile against healthy human
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
peripheral blood mononuclear cells (PBMCs). PBMCs were treated with titrating doses of
panobinostat, 2d, or 3b for 24 hours followed by treatment with CellTiterꢀBlue. Cell viability
was determined as a function of resorufin intensity via spectrophotometer, normalized to control
treatment. The data demonstrate 2d and 3b’s superiority over panobinostat from a toxicity
standpoint. Further, the lack of a Michael acceptor group on 2d seems to reduce its toxicity
toward PBMC’s 10 fold compared to 3b (Figure 5b). This functional group (present as an
acrylamide) may also explain panobinostat’s much greater toxicity compared to 2d and 3b.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DISCUSSION
To date, all FDA approved HDAC inhibitors or those in clinical trials contain promiscuous
metalꢀchelating groups. The hydroxamic acid was the first metalꢀchelating group to be
introduced, and to this day remains the most common, but others such as the disulfide bond of
romidepsin, or the orthoꢀaminoanilide of entinostat have also been used. These groups are all
generally acidic in nature, and have been shown to be subject to glucuronidation. This addition
of steric bulk and charge neutralization removes the ability of these inhibitors to get into the
narrow active site and ionically bind to the positively charged zinc that is paramount to HDAC
deacylase activity. In this study we demonstrated that very potent and selective inhibition of
class I HDACs is possible with the novel utilization of the metal chelating group, hydrazide.
Further, we demonstrated that these compounds likely work through an allosteric mode of
inhibition. This possibly indicates the presence of a nonꢀcatalytic metalꢀcenter target of these
hydrazide compounds. Thus far, discoveries of allosteric, small molecule inhibitors of class I
HDACs have not been well established in the literature. Inhibitors that lack a known metalꢀ
ACS Paragon Plus Environment

Page 17 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
chelating group that have been shown to inhibit HDACs were shown to display canonical
5
3
competitive MichaelisꢀMenten enzyme kinetics when further interrogated.
The results in this manuscript suggest our compounds are also highly resistant to
glucuronidation, indicating they possess little anionic charge, and display mixed/nonꢀcompetitive
inhibition, giving further credibility to the possibility that they may bind to a nonꢀcatalytic site on
HDACs irrespective of whether substrate is bound. Further, this allosteric site is present ex vivo,
as their recombinant enzyme inhibition prowess translated directly to cellular efficacy as well.
Discovery of a potential allosteric binding site benefits more than just medicinal chemistry
surrounding HDAC inhibitors. By having a combination of agents that work both at the active
site, and allosterically on HDACs, the possibility of dual mechanism therapies for acute myeloid
leukemia arises.
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Lastly, while this paper established a >60 fold improvement in potency over the initial lead
inhibitor generated by Wang and colleagues, our most potent of which breaching the picomolar
range for recombinant human HDAC3 inhibition, further refinement of these inhibitors is also
possible. Further alterations surrounding Nꢀbenzylbenzamide group that provided so much of
these molecules’ potencies would be the first place to start. Stepwise methylation at key sites, or
the introduction of electron donating or withdrawing groups around either of the phenyl rings
could provide additional potency.
Our lead inhibitors 1l, 2d, and 3b, were used ubiquitously throughout the manuscript. All of
these agents display higher levels of ex vivo potency than vorinostat or entinostat. The most
potent, 3b, contains a Michaelꢀacceptor group in the form of an α, βꢀunsaturated ketone. While
once a forbidden group in medicinal chemistry, recently approved effective therapies such as
dimethyl fumarate demonstrate that not all Michaelꢀacceptors are detrimental to patient health,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
even in long term use. However, when comparing 3b’s potency to that of 2d, we see an
approximate 2.5 fold decrease in EC in MV4ꢀ11 cells at the cost of a 10 fold increase in PBMC
50
based toxicity. Further, this was the only one of our compounds tested in human liver
microsomes to demonstrate a metabolite, being reduced about its unsaturated bond. This was
surprising, as no external reducing substrates such as NADH or NADPH were added to the
reaction vessel. This indicates that even the residual concentrations of these reducing agents
leftover from extraction were enough to metabolize this compound. As such, the superior
metabolic stability, and better toxicity profiles of 2d make it a more beneficial candidate worth
pursuing.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL PROCEDURES
General Chemistry. All reagents and chemical solvents were used from the respective
1
13
commercially available sources without further purification. H NMR and C NMR data were
collected in deuterated solvent with a Bruker 400 MHz with Trimethylsilane as a standard
reference. Chemical shifts are given in parts per million. NMR descriptions use the following
abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad peak.
Coupling constants (J) reported in Hz. Mass spectra data were gathered using a Thermo LCQ
Fleet mass spectrometer using electrospray ionization. Purification was performed using a
Teledyne Isco Combiflash 200 on prepacked C18 columns. All target compounds were at least
95% pure confirmed via UV detection (λ = 254 nm) on a Thermo LCQ Fleet HPLCꢀMS using an
Accucore RPꢀMS HPLC Column, 2.6 µm particle size, 30x4.6 mm. These runs used water and
methanol with 0.1% (v/v) formic acid were used as mobile phase. A gradient of 100% water was
run isocratically for 0.5 minutes at 500 µL/min. The gradient then increased to 100% methanol
ACS Paragon Plus Environment

Page 19 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
over 13.5 minutes, and run isocratically for one minute before returning to 100% water over the
next two minutes. Capillary temperature was 350°C, with a spray voltage of 5 kV. Purity was
calculated using the automatic peak detection function followed by dividing the area under the
curve of the peak by the total area of all peaks in the chromatograph.
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure Creation. Figures were generated using Adobe Illustrator CC, Adobe Photoshop CC,
Image Studio Lite 4.0, GraphPad Prism 6.0, ChemDraw 13.0, and Microsoft PowerPoint 2013.
Synthesis of butylhydrazide derivatives (1a-1l).
Series 1 General Procedure. Once the corresponding hydrazide was generated, 1.1 equivalents
of butaldehyde and 10 equivalents of magnesium sulfate were stirred in 10 mL of ethanol with
the hydrazide. The reaction was stirred at room temperature and monitored via TLC. After
disappearance of starting material, the excess magnesium sulfate was removed via vacuum
filtration, and the collected solution condensed under vacuum. The intermediate was
resuspended in 4 mL of methanol followed by addition of 1.2 equivalents of sodium
cyanoborohydride and a pinch of methyl orange. Argon was bubbled through the resulting
yellow solution for 5 minutes. At this time a solution of concentrated HCl in methanol (1:1 v/v)
was added dropwise until the solution turned and stayed red. The mixture was allowed to stir
overnight under argon. Volatiles were removed under vacuum and purified on C18 reverse
phase columns eluted with acetonitrile and water to yield the desired product.
N’ꢀbutylꢀ4ꢀmethoxybenzohydrazide (1a). 498.54 mg (3 mmol) of 4ꢀmethoxybenzohydrazide was
reacted as described in Series 1 General Procedure to yield 317.9 mg of dry product (48%
1
yield). H NMR (400 MHz, DMSO): δ 9.89 (s, 1H), 7.82 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
H), 4.99 (s, 1H), 3.81 (s, 3H), 2.79ꢀ2.76 (m, 2H), 1.48ꢀ1.40 (m, 2H), 1.38ꢀ1.32 (m, 2H), 0.89 (t,
1
3
J = 7.2 Hz, 3H). C NMR (100 MHz, DMSO): δ 165.4, 162.0, 129.3, 125.9, 114.0, 55.8, 51.5,
+
30.3, 20.3, 14.3. [(m+H )/z = 223.25]. (λ ) purity 95.4%, t 9.28 mins.
254
R
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N’ꢀbutylbenzofuranꢀ2ꢀcarbohydrazide (1b). 324.0 mg (2 mmol) of benzofuranꢀ2ꢀcarboxylic acid
was suspended in 5 mL of methylene chloride. The flask was flushed with argon for 10 minutes
before injection of 0.4 mL (4 mmol) oxalyl chloride. Two drops of dimethylformamide were
injected and furious bubbling began. The sealed vessel was continuously flushed with argon and
vented for 2 hours at room temperature. 10 mL of sieve dried ethanol was slowly injected and
allowed to stir for an additional hour. Volatiles were removed under vacuum and the crude
intermediate was resuspended in 5 mL ethanol. To this solution was added 250 mg (5 mmol) of
hydrazine water salt. The reaction was refluxed for 3 hours to give the corresponding hydrazide.
Volatiles were removed under vacuum and the product suspended in 10 mL of ethanol. From
here the reaction proceeded as described in Series 1 General Procedure to yield 369.1 mg of
1
dry product (72% yield). H NMR (400 MHz, DMSO): δ 10.26 (d, J = 6.0 Hz, 1H), 7.78 (d, J =
8
.0 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.54 (s, 1H), 7.49ꢀ7.45 (m, 1H), 7.36ꢀ7.32 (m, 1H), 5.16ꢀ
.13 (m, 1H), 2.84ꢀ2.79 (m, 2H), 1.48ꢀ1.41 (m, 2H), 1.39ꢀ1.33 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H);
C NMR (100 MHz, DMSO): δ 157.7, 154.7, 148.7, 127.5, 127.2, 124.2, 123.1, 112.2, 109.6,
5
1
3
+
51.2, 30.2, 20.3, 14.4. [(m+H )/z = 233.25]. (λ ) purity 98.8%, t 11.53 mins.
2
54
R
N’ꢀbutylꢀ4ꢀphenoxybenzohydrazide (1c). 685 mg (3 mmol) of 4ꢀphenoxybenzohydrazide was
suspended in 10 mL of ethanol. From here the reaction proceeded as described in Series 1
1
General Procedure to yield 596.7 mg of dry product (70% yield). H NMR (400 MHz, DMSO):
δ 9.99 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.46ꢀ7.42 (m, 2H), 7.23ꢀ7.20 (m, 1H), 7.09 (d, J = 8.0
Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 5.07 (s, 1H), 2.81ꢀ2.77 (m, 2H), 1.47ꢀ1.41 (m, 2H), 1.38ꢀ1.32
ACS Paragon Plus Environment

Page 21 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
(
m, 2H), 0.89 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO): δ 165.1, 159.9, 156.1, 130.7,
+
1
29.7, 128.4, 124.7, 119.9, 117.9, 51.4, 30.3, 20.3, 14.4. [(m+H )/z = 285.25]. (λ ) purity
254
9
8.2%, t 12.60 mins.
R
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N’ꢀbutylnicotinohydrazide (1d). 1068 mg (6 mmol) of nicotinoyl chloride was suspended in 20
mL of methanol. To this solution was added 1.7 mL (12 mmol) of triethylamine. The reaction
was stirred at room temperature for 1 hour. 750 mg (15 mmol) hydrazine water salt was added
and the solution was refluxed for 3 hours yielding the corresponding hydrazide. Volatiles were
removed under vacuum and the intermediate was resuspended in 30 mL of methanol. From here
the reaction proceeded as described in Series 1 General Procedure to yield 721.7 mg of dry
1
product (62% yield). H NMR (400 MHz, DMSO): δ 10.21 (s, 1H), 8.99 (s, 1H), 8.72ꢀ8.70 (m,
1H), 8.19ꢀ8.10 (m, 1H), 7.52ꢀ7.49 (m, 1H), 5.15 (s, 1H), 2.83ꢀ2.79 (m, 2H), 1.48ꢀ1.42 (m, 2H),
1
3
1
.38ꢀ1.32 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO): δ 164.2, 152.3, 148.6,
+
1
35.2, 129.3, 124.0, 51.3, 30.2, 20.3, 14.4. [(m+H )/z = 194.25]. (λ ) purity 95.6%, t 6.17
2
54
R
mins.
N’ꢀbutylꢀ[1,1’ꢀbiphenyl]ꢀ4ꢀcarbohydrazide (1e). 890 mg (3.8 mmol) of methyl [1,1’]ꢀbiphenylꢀ4ꢀ
carboxylate was suspended in 20 mL of methanol. To this solution, 945 mg (18.9 mmol) of
hydrazine water salt was added. The solution was brought to reflux and reacted for 3 hours. The
solution was cooled and volatiles evaporated under vacuum. From here the reaction proceeded
1
as described in Series 1 General Procedure to yield 555.1 mg of dry product (54% yield). H
NMR (400 MHz, DMSO): δ 10.08 (d, J = 6 Hz, 1H), 7.94ꢀ7.92 (m, 2H), 7.78ꢀ7.73 (m, 4H),
7.52ꢀ7.48 (m, 2H), 7.44ꢀ7.40 (m, 1H), 5.13ꢀ5.09 (m, 1H), 2.84ꢀ2.79 (m, 2H), 1.49ꢀ1.42 (m, 2H),
1
3
1
.40ꢀ1.35 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO): δ 165.4, 143.2, 139.6,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
32.5, 129.5, 128.5, 128.2, 127.3, 127.0, 51.4, 30.3, 20.3, 14.4. [(m+H )/z = 269.25]. (λ )
254
purity 96.7%, t 12.82 mins.
R
N’ꢀbutylthiopheneꢀ2ꢀcarbohydrazide (1f). 1500 mg (10 mmol) of thiopheneꢀ2ꢀcarbonyl chloride
was injected into 10 mL of sieve dried methanol and bubbled with argon for 10 minutes. To this
mixture was injected two drops of dimethylformamide. The reaction proceeded at room
temperature for 1 hour before addition of 600 mg (12 mmol) hydrazine water salt. The solution
was refluxed for 3 hours before being cooled and condensed in vacuo. The reaction then
proceeded as described in Series 1 General Procedure to yield 1235 mg of dry product (62%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
yield). H NMR (400 MHz, DMSO): δ 10.02 (s, 1H), 7.76ꢀ7.74 (m, 2H), 7.15ꢀ7.13 (m, 1H),
5
.03 (s, 1H), 2.80ꢀ2.77 (m, 2H), 1.45ꢀ1.40 (m, 2H), 1.37ꢀ1.32 (m, 2H), 0.89 (m, J = 7.2 Hz, 3H);
1
3
C NMR (100 MHz, DMSO): δ 161.0, 138.8, 131.0, 128.4, 128.2, 51.3, 30.3, 20.3, 14.4.
+
[
(m+H )/z = 199.17]. (λ ) purity 97.5%, t 8.47 mins.
254
R
N’ꢀbutylfuranꢀ2ꢀcarbohydrazide (1g). 1100 mg (8.4 mmol) of furanꢀ2ꢀcarbonyl chloride was
injected into 10 mL of sieve dried methanol that was bubbled with argon for 10 minutes prior to
addition. To this mixture was injected 1780 mg (17.6 mmol) of triethylamine. After 60 minutes,
1
000 mg (20 mmol) of hydrazine water salt was added, and heated to reflux for 3 hours. The
reaction was cooled and condensed under vacuum. From here the reaction proceeded as
1
described in Series 1 General Procedure to yield 861 mg of dry product (56% yield). H NMR
(
400 MHz, DMSO): δ 9.90 (s, 1H), 7.83ꢀ7.82 (m, 1), 7.11ꢀ7.10 (m, 1H), 6.62ꢀ6.60 (m, 1H), 5.00
1
3
(s, 1H), 2.79ꢀ2.74 (m, 2H), 1.44ꢀ1.40 (m, 2H), 1.38ꢀ1.30 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H); C
+
NMR (100 MHz, DMSO): δ 157.6, 147.3, 145.4, 113.6, 112.1, 51.3, 30.2, 20.3, 14.4. [(m+H )/z
=
183.17]. (λ ) purity 98.4%, t 7.43 mins.
254 R
ACS Paragon Plus Environment

Page 23 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
N’ꢀbutylꢀ3ꢀphenylpropanehydrazide (1h). 1012 mg (6 mmol) of 3ꢀphenylpropanoyl chloride was
injected into 10 mL of argon bubbled, sieve dried methanol. The reaction was allowed to stir for
1
hour before addition of 900 mg (18 mmol) hydrazine water salt. The mixture was brought to
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
reflux for 3 hours before being cooled to room temperature and condensed under vacuum. From
here the reaction proceeded as described in Series 1 General Procedure to yield 911 mg of dry
1
product (68% yield). H NMR (400 MHz, DMSO): δ 9.25 (s, 1H), 7.29ꢀ7.25 (m, 2H), 7.21ꢀ7.26
(m, 3H), 4.77 (s, 1H), 2.82 (t, J = 7.6 Hz, 2H), 2.63ꢀ2.59 (m, 2H), 2.33 (t, J = 7.6 Hz, 2H), 1.31ꢀ
1
3
1.25 (m, 4H), 0.85 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO): δ 170.5, 141.5, 128.7 (d),
+
1
26.3, 51.2, 35.6, 31.5, 30.1, 20.2, 14.4. [(m+H )/z = 221.25]. (λ ) purity 98.9%, t 8.56 mins.
2
54
R
N’ꢀbutylcinnamohydrazide (1i). 1480 mg (10 mmol) of transꢀcinnamic acid was suspended in 50
mL of acetonitrile. To this solution was added 1620 mg (12 mmol) of 1ꢀhydroxybenzotriazole
and 2478 mg (12 mmol) N, N' –dicyclohexylcarbodiimide. The solution stirred overnight at
room temperature before addition of 600 mg (12 mmol) hydrazine water salt, which was refluxed
for 3 hours. The solution was brought to room temperature and condensed under vacuum. From
here the reaction proceeded as described in Series 1 General Procedure to yield 885 mg of dry
1
product (40% yield). H NMR (400 MHz, DMSO): δ 9.62 (s, 1H), 7.58ꢀ7.56 (m, 2H), 7.45ꢀ7.38
(m, 4H), 6.57 (d, J = 16.0 Hz, 1H), 5.05 (s, 1H), 2.76ꢀ2.72 (m, 2H), 1.44ꢀ1.28 (m, 4H), 0.89 (t, J
13
=
7.2 Hz, 3H); C NMR (100 MHz, DMSO): δ 164.3, 139.0, 135.4, 129.9, 129.4, 127.9, 120.8,
+
51.4, 30.2, 20.2, 14.4. [(m+H )/z = 219.25]. (λ ) purity 97.5%, t 10.95 mins.
2
54
R
N’ꢀbutylꢀ1ꢀnapthohydrazide (1j). 1033 mg (6 mmol) of 1ꢀnapthoic acid was suspended in 20 mL
of argon flushed methylene chloride. To this solution was injected 1143 mg (9 mmol) of oxalyl
chloride followed by injection of 2 drops of dimethylformamide. Furious bubbling was seen,
with gas being exhausted and argon flushing continually throughout the room temperature
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reaction. After one hour, 20 mL of sieve dried methanol was injected slowly. After reacting for
an additional hour at room temperature, the solution was condensed under vacuum and
resuspended in 30 mL of methanol. To this was added 1500 mg (30 mmol) of hydrazine water
salt. The reaction was brought to reflux for 3 hours before being cooled and condensed under
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
vacuum. From here the reaction proceeded as described in Series 1 General Procedure to yield
1
1
050 mg of dry product (72% yield). H NMR (400 MHz, DMSO): δ 9.97 (s, 1H), 8.22ꢀ8.19 (m,
H), 8.04ꢀ7.98 (m, 2H), 7.61ꢀ7.53 (m, 4H), 5.23 (s, 1H), 2.92ꢀ2.87 (m, 2H), 1.53ꢀ1.42 (m, 2H),
1
1
3
1.40ꢀ1.38 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO): δ 167.8, 133.7, 133.6,
+
1
30.4, 130.4, 128.7, 127.2, 126.7, 125.8, 125.7, 125.5, 51.3, 30.3, 20.3, 14.4. [(m+H )/z =
2
43.25]. (λ ) purity >99%, t 11.00 mins.
254
R
N’ꢀbutylꢀ2ꢀnapthohydrazide (1k). 1120 mg (6 mmol) of 2ꢀnapthohydrazide was reacted as
1
described in Series 1 General Procedure to yield 1016 mg of dry product (70% yield).
H
NMR (400 MHz, DMSO): δ 10.2 (s, 1H), 8.46 (s, 1H), 8.04 ꢀ7.93 (m, 4H), 7.64ꢀ7.58 (m, 2H),
5.17 (s, 1H), 2.87ꢀ2.83 (m, 2H), 1.51ꢀ1.46 (m, 2H), 1.41ꢀ1.35 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H);
13
C NMR (100 MHz, DMSO): δ 165.8, 134.6, 132.6, 131.1, 129.3, 128.4, 128.1, 128.0, 127.8,
+
1
27.2, 124.4, 51.4, 30.3, 20.3, 14.4. [(m+H )/z = 243.25]. (λ ) purity >99%, t 11.42 mins.
254
R
Nꢀ(4ꢀ(2ꢀbutylhydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (1l). To a mixture of sieve dried
methylene chloride, was added 711 mg (4 mmol) of methyl 4ꢀ(aminoethyl)benzoate. This vessel
was flushed with argon for 30 minutes before injection of 560 mg (4 mmol) of benzoyl chloride
and 607 mg (6 mmol) of trimethylamine. The reaction was stirred at room temperature for 2
hours before being condensed under vacuum. The crude intermediate was resuspended in 30 mL
of methanol and 1000 mg (20 mmol) of hydrazine water salt was added as one portion. The
solution was refluxed for 3 hours before being cooled and condensed under vacuum. From here
ACS Paragon Plus Environment

Page 25 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the reaction proceeded as described in Series 1 General Procedure to yield 601 mg of dry
1
product (46% yield). H NMR (400 MHz, DMSO): δ 9.99 (s, 1H), 9.13ꢀ9.10 (t, J = 6.0 Hz, 1H),
7.93ꢀ7.91 (m, 2H), 7.81ꢀ7.79 (m, 2H), 7.57ꢀ7.47 (m, 3H), 7.41ꢀ7.39 (m, 2H), 5.07 (s, 1H), 4.54
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
(
d, J = 6.0 Hz, 2H); C NMR (100 MHz, DMSO): δ 166.7, 165.6, 143.5, 134.7, 132.2, 131.8,
+
128.8, 12.7, 127.6, 127.5, 51.4, 42.9, 30.3, 20.3, 14.4. HRMSꢀESI [(m+H )/z] calculated for
C H N O : 326.18697; found, 326.18622. (λ ) purity 96.3%, t 11.92 mins.
19
23
3
2
254
R
Synthesis of N-(4-(hydrazide)benzyl)benzamide derivatives (2a-2m).
Intermediate formation. 6050 mg (40 mmol) of 4ꢀ(aminomethyl)benzoic acid was dissolved in
200 mL of methanol, to which was added 6 mL of concentrated HCl in one portion. The mixture
was refluxed overnight and the volatiles condensed under vacuum. The resulting white solid was
suspended in ethyl ether and separated via vacuum filtration to yield the benzoate HCl salt. This
compound was dissolved in a 1:2 mixture of ethyl acetate and water and chilled to 0°C to which
1
1040 mg (80 mmol) was added followed by addition of 5623 mg benzoyl chloride (40 mmol).
The vessel was warmed to room temperature and stirred for 2 additional hours. The mixture was
separated via acid/base extraction, washing the water phase twice with ethyl acetate. All organic
phases were combined and condensed under vacuum to yield a white solid. This was suspended
in 200 mL of methanol and 10000 mg (200 mmol) of hydrazine water salt was added. The
solution was refluxed for 48 hours, cooled to room temperature, and volatiles were removed
under vacuum. This intermediate (2sm) was used as the starting material for all further reactions
for this family.
Series 2 General Procedure. The mixture of 2sm and aldehyde of interest were stirred at room
temperature overnight; vacuum filtration afforded the desired intermediate, which was dissolved
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in 30 mL of methanol. To this solution was added a pinch of methyl orange, and the solution’s
color turned yellow. The solution was bubbled under argon for 5 minutes, and 2.2 mmol of
sodium cyanoborohydride was then added to it. A 1:1 mixture of methanol and concentrated HCl
was added dropwise until the solution turned red. After addition, the mixture was stirred at room
temperature for 6 hours. The reaction was quenched with sodium hydroxide, and organic
solvents were removed under vacuum. The residues were extracted twice with ethyl acetate, and
organic phases were combined and dried over magnesium sulfate. After filtration, organic
solvents were removed and the residues were purified by flash chromatography.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(E)ꢀNꢀ(4ꢀ(2ꢀ(butꢀ2ꢀenꢀ1ꢀyl)hydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2a). 2 mmol of 2sm was
dissolved in 100 mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol of
magnesium sulfate and 2 mmol of (E)ꢀbutꢀ2ꢀenal. From here the reaction proceeded as described
1
in Series 2 General Procedure to yield 208 mg (32% yield). H NMR (400 MHz, DMSO): δ
9.97 (d, J = 5.6 Hz, 1H), 9.12 (t, J = 6.0 Hz, 1H), 7.93ꢀ7.91 (m, 2H), 7.81ꢀ7.78 (m, 2H), 7.58ꢀ
7.48 (m, 3H), 7.41ꢀ7.39 (m, 2H), 5.63ꢀ5.50 (m, 2H), 5.11ꢀ5.07 (m, 1H), 4.54 (d, J = 6.0 Hz, 2H),
13
3
.37ꢀ3.35 (m, 2H), 1.65 (d, J = 5.6 Hz, 3H); C NMR (100 MHz, DMSO): δ 166.8, 165.7,
+
1
43.5, 134.7, 132.2, 131.8, 128.8, 128.4, 128.2, 127.7, 127.6, 127.5, 53.6, 42.9, 18.2. [(m+H )/z
=
324.17]. (λ ) purity >99%, t 10.00 mins.
254 R
Nꢀ(4ꢀ(2ꢀisopropylhydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2b). 2 mmol of 2sm was dissolved in
00 mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol of magnesium
1
sulfate and 2 mmol of isopropanal. From here the reaction proceeded as described in Series 2
1
General Procedure to yield yielded 324 mg (52% yield). H NMR (400 MHz, DMSO): δ 9.95
(d, J = 6.8 Hz, 1H), 9.11 (t, J = 6.0 Hz, 1H), 7.92ꢀ7.90 (m, 2H), 7.82ꢀ7.80 (m, 2H), 7.58ꢀ7.48 (m,
3H), 7.41ꢀ7.39 (m, 2H), 4.95ꢀ4.92 (m, 1H), 4.53 (d, J = 6.0 Hz, 2H), 3.11ꢀ3.03 (m, 1H), 1.02 (d,
ACS Paragon Plus Environment

Page 27 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
J = 10.4 Hz, 6H); C NMR (100 MHz, DMSO): δ 166.8, 166.0, 143.5, 134.7, 132.2, 131.8,
+
1
28.8, 127.7, 127.6, 127.4, 50.8, 42.9, 21.4. [(m+H )/z = 312.25]. (λ ) purity 98.8%, t 8.68
254
R
mins.
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
E)ꢀNꢀ(4ꢀ(2ꢀpropylidenehydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2c). 2 mmol of 2sm was
dissolved in 100 mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol of
magnesium sulfate and 2 mmol of propanal. From here the reaction proceeded as described in
1
Series 2 General Procedure to yield 347 mg (56% yield). H NMR (400 MHz, DMSO): δ
1
4
1
1.39 (s, 1H), 9.13 (t, J = 6.0 Hz, 1H), 7.93ꢀ7.90 (m, 2H), 7.84ꢀ7.75 (m, 3H), 7.58ꢀ7.38 (m, 5H),
1
3
.56ꢀ4.53 (m, 2H), 2.30ꢀ2.25 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO): δ
66.8, 163.1, 153.5, 143.9, 134.7, 131.8, 128.8, 128.1, 127.7, 127.5(d), 42.9, 25.9, 11.1. HRMSꢀ
+
ESI [(m+H )/z] calculated for C H N O : 310.15567; found, 310.15529. (λ ) purity 97.5%, t
1
8
19
3
2
254
R
1
1.60 mins.
Nꢀ(4ꢀ(2ꢀpropylhydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2d). 2 mmol of 2sm was dissolved in
100 mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol of magnesium
sulfate and 2 mmol of propanal. From here the reaction proceeded as described in Series 2
1
General Procedure to yield 318 mg (51% yield). H NMR (400 MHz, DMSO): δ 9.98 (d, J =
6.0 Hz, 1H), 9.11 (t, J = 6.0 Hz, 1H), 7.92ꢀ7.91 (m, 2H), 7.81ꢀ7.79 (m, 2H), 7.56ꢀ7.48 (m, 3H),
7
.41ꢀ7.39 (m, 2H), 5.11ꢀ5.07 (m, 1H), 4.53 (d, J = 6.0 Hz, 2H), 2.78ꢀ2.73 (m, 2H), 1.49ꢀ1.44 (m,
1
3
2
H), 0.92 (t, J = 7.6 Hz, 3H); C NMR (100 MHz, DMSO): δ 166.7, 165.6, 143.5, 134.7, 132.2,
+
131.8, 128.8, 127.7, 127.6, 127.4, 53.6, 42.9, 21.3, 12.1. HRMSꢀESI [(m+H )/z] calculated for
C H N O : 312.17132; found, 312.17127. (λ ) purity >99%, t 12.28 mins.
18
21
3
2
254
R
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Nꢀ(4ꢀ(2ꢀpentylhydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2e). 2 mmol of 2sm was dissolved in
100 mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol of magnesium
sulfate and 2 mmol of pentanal. From here the reaction proceeded as described in Series 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
General Procedure to yield 312 mg (46% yield). H NMR (400 MHz, DMSO): δ 9.99 (s, 1H),
9.11 (t, J = 6.0 Hz, 1H), 7.93ꢀ7.91 (m, 2H), 7.81ꢀ7.79 (m, 2H), 7.57ꢀ7.47 (m, 3H), 7.41ꢀ7.39 (m,
2H), 5.07 (s, 1H), 4.53 (d, J = 6.0 Hz, 2H), 2.78 (s, 2H), 1.48ꢀ1.44 (m, 2H), 1,33ꢀ1.29 (m, 4H),
1
3
0
.88 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, DMSO): δ 166.8, 165.6, 143.5, 134.7, 132.2,
+
131.8, 128.8, 127.7, 127.6, 127.5, 51.7, 42.9, 29.4, 27.8, 22.5, 14.4. [(m+H )/z = 340.25]. (λ )
254
purity >99%, t 11.30 mins.
R
Nꢀ(4ꢀ(2ꢀ(3,3,3ꢀtrifluoropropyl)hydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2f). 2 mmol of 2sm
was dissolved in 100 mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol
of magnesium sulfate and 2 mmol of 3,3,3ꢀtrifluoropropanal. From here the reaction proceeded
1
as described in Series 2 General Procedure to yield 445 mg (61% yield). H NMR (400 MHz,
DMSO): δ 10.03 (d, J = 6.0 Hz, 1H), 9.11 (t, J = 6.0 Hz, 1H), 7.92ꢀ7.90 (m, 2H), 7.82ꢀ7.80 (m,
2
3
1
H), 7.58ꢀ7.48 (m, 3H), 7.42ꢀ7.40 (m, 2H), 5.43ꢀ5.39 (m, 1H), 4.54 (d, J = 6.0 Hz, 2H), 3.05ꢀ
13
.00 (m, 2H), 2.54ꢀ2.44 (m, 2H); CꢀHSQC (100MHz, 400 MHz, DMSO) δ 131.8, 128.8, 127.7,
+
27.6, 127.5, 44.55, 42.8, 32.1. [(m+H )/z = 366.25]. (λ ) purity 97.7%, t 10.85 mins.
2
54
R
Nꢀ(4ꢀ(2ꢀ(cyclopropylmethyl)hydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2g). 2 mmol of 2sm was
dissolved in 100 mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol of
magnesium sulfate and 2 mmol of cyclopropanecarbaldehyde. From here the reaction proceeded
1
as described in Series 2 General Procedure to yield 415 mg (64% yield). H NMR (400 MHz,
DMSO): δ 10.06 (s, 1H), 9.13 (s, 1H), 7.93ꢀ7.92 (m, 2H), 7.85ꢀ7.80 (m, 2H), 7.57ꢀ7.47 (m, 3H),
7.41ꢀ7.39 (m, 2H), 5.12 (s, 1H), 4.55 (s, 2H), 2.66 (s, 2H), 0.93ꢀ0.91 (m, 1H), 0.46ꢀ0.44 (m, 2H),
ACS Paragon Plus Environment

Page 29 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
0
1
9
.16ꢀ0.15 (m, 2H); C NMR (100 MHz, DMSO): δ 166.8, 165.5, 143.5, 134.7, 132.2, 131.8,
+
28.8, 127.7, 127.6, 127.5, 56.6, 42.9, 9.95, 3.61. [(m+H )/z = 324.17]. (λ ) purity 97.4%, t
254
R
.27 mins.
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nꢀ(4ꢀ(2ꢀheptylhydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2h). 2 mmol of 2sm was dissolved in
00 mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol of magnesium
1
sulfate and 2 mmol of heptanal. From here the reaction proceeded as described in Series 2
1
General Procedure to yield 345 mg (47% yield). H NMR (400 MHz, DMSO): δ 10.00 (s, 1H),
9
2
1
.12 (t, J = 6.0 Hz, 1H), 7.93ꢀ7.91 (m, 2H), 7.82ꢀ7.80 (m, 2H), 7.57ꢀ7.47 (m, 3H), 7.41ꢀ7.39 (m,
H), 5.08ꢀ5.05 (m, 1H), 4.54 (d, J = 6.0 Hz, 2H), 2.81ꢀ2.76 (m, 2H), 1,48ꢀ1.44 (m, 2H), 1.34ꢀ
13
.26 (m, 8H), 0.86 (t, J = 6.4 Hz, 3H); C NMR (100 MHz, DMSO): δ 166.7, 165.6, 143.5,
134.7, 132.2, 131.8, 128.8, 127.7, 127.6, 127.4, 51.7, 42.9, 31.8, 29.1, 28.1, 27.1, 22.6, 14.4.
+
[
(m+H )/z = 368.33]. (λ ) purity >99%, t 13.25 mins.
254
R
Nꢀ(4ꢀ(2ꢀoctylhydrazineꢀ1ꢀcarbonyl)benzyl)benzamide (2i). 2 mmol of 2sm was dissolved in 100
mL ethanol with ultraꢀsonication aid. To this solution was added 40 mmol of magnesium sulfate
and 2 mmol of octanal. From here the reaction proceeded as described in Series 2 General
1
Procedure to yield 305 mg (40% yield). H NMR (400 MHz, DMSO): δ 10.00 (s, 1H), 9.12 (t,
J = 6.0 Hz, 1H), 7.93ꢀ7.91 (m, 2H), 7.82ꢀ7.80 (m, 2H), 7.55ꢀ7.46 (m, 3H), 7.41ꢀ7.39 (m, 2H),
5.07ꢀ5.06 (m, 1H), 4.54 (d, J = 5.6 Hz, 2H), 2.80ꢀ2.76 (m, 2H), 1.47ꢀ1.42 (m, 2H), 1.34ꢀ1.25 (m,
1
3
1
0H), 0.86 (t, J = 6.4 Hz, 3H); C NMR (100 MHz, DMSO): δ 166.7, 165.6, 143.5, 134.7,
132.2, 131.8, 128.8, 127.7, 127.6, 127.4, 51.7, 42.9, 31.7, 29.4, 29.2, 28.1, 27.2, 22.6, 14.4.
+
[
(m+H )/z = 382.33]. (λ ) purity 95.8%, t 13.97 mins.
254
R
ACS Paragon Plus Environment