Class i HDAC Inhibitors Display Different Antitumor Mechanism in Leukemia and Prostatic Cancer Cells Depending on Their p53 Status

Article abstract of DOI:10.1021/acs.jmedchem.8b00136

Previously, we designed and synthesized a series of o-aminobenzamide-based histone deacetylase (HDAC) inhibitors, among which the representative compound 11a exhibited potent inhibitory activity against class I HDACs. In this study, we report the development of more potent hydrazide-based class I selective HDAC inhibitors using 11a as a lead. Representative compound 13b showed a mixed, slow, and tight binding inhibition mechanism for HDAC1, 2, and 3. The most potent compound 13e exhibited low nanomolar IC₅₀s toward HDAC1, 2, and 3 and could down-regulate HDAC6 in acute myeloid leukemia MV4-11 cells. The EC₅₀ of 13e against MV4-11 cells was 34.7 nM, which is 26 times lower than its parent compound 11a. In vitro responses to 13e vary significantly and interestingly based on cell type: in p53 wild-type MV4-11 cells, 13e induced cell death via apoptosis and G1/S cell cycle arrest, which is likely mediated by a p53-dependent pathway, while in p53-null PC-3 cells, 13e caused G2/M arrest and inhibited cell proliferation without inducing caspase-3-dependent apoptosis.

Full text of DOI:10.1021/acs.jmedchem.8b00136

Subscriber access provided by UNIV OF SCIENCES PHILADELPHIA
Article
Class I HDAC inhibitors display different anti-tumor mechanism in
leukemia and prostatic cancer cells depended on their p53 status
Xiaoyang Li, Yuri K. Peterson, Elizabeth S. Inks, Richard Himes,
Jiaying Li, Yingjie Zhang, Xiujie Kong, and C. James Chou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00136 • Publication Date (Web): 02 Mar 2018
Downloaded from http://pubs.acs.org on March 3, 2018
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 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 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 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Class I HDAC inhibitors display different
antiꢀtumor mechanism in leukemia and prostatic
cancer cells depended on their p53 status
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
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
1
1
2
1
3
Xiaoyang Li , Yuri K. Peterson , Elizabeth S. Inks , Richard Himes , Jiaying Li , Yingjie Zhang ,
3
1,2*
Xiujie Kong , C. James Chou
1
Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy,
Medical University of South Carolina, Charleston, South Carolina 29425, United States
2
Lydex Pharmaceuticals, 330 Concord St. Unit 6A, Charleston, South Carolina 29401, United
States
3
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University,
Ji'nan, Shandong 250012, PR China
ABSTRACT: Previously, we designed and synthesized a series of oꢀaminobenzamideꢀbased
HDAC inhibitors, among which the representative compound 11a exhibited potent inhibitory
activity against class I HDACs. In this study, we report the development of more potent
hydrazideꢀbased class I selective HDAC inhibitors using 11a as a lead. Representative
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 59
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
compound 13b showed a mixed, slow, and tight binding inhibition mechanism for HDAC1, 2
and 3. The most potent compound 13e exhibited low nanoꢀmolar IC s toward HDAC1, 2 and 3,
5
0
and could downꢀregulate HDAC6 in acute myeloid leukemia MV4ꢀ11 cells. The EC of 13e
5
0
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
against MV4ꢀ11 cells was 34.7 nM, which is 26 times lower than its parent compound 11a. In
vitro responses to 13e vary significantly and interestingly based on cell type: in p53 wildꢀtype
MV4ꢀ11 cells, 13e induced cell death via apoptosis and G1/S cell cycle arrest which is likely
mediated by a p53ꢀdependent pathway, while in p53ꢀnull PCꢀ3 cells 13e caused G2/M arrest and
inhibited cell proliferation without inducing caspaseꢀ3 dependent apoptosis.
INTRODUCTION
Targeting histone modifying histone deacetylases (HDACs) to restore the expression of
1
tumor suppressor genes has shown clinical benefits especially for the treatment of cancer.
HDACs together with histone acetyltransferases (HATs) form two families of enzymes with
reversible actions for modifying the acetylation status of chromosomal histones and nonꢀhistone
proteins. HDACs remove acetyl groups from the lysine residues, resulting in a “closed”
configuration of chromatin blocking the access of the transcription machinery to DNA, and
finally suppresses gene expression. HATs on the other hand, perform antagonist actions which
2
ꢀ3
trigger an “open” state of chromatin and promote gene expression. Remarkably, a growing
number of nonꢀhistone proteins are also described as targets of HDACs and HATs, which
include transcription factors, DNA binding nuclear receptors, signal mediators, transcriptional
4ꢀ7
coꢀregulators and cytoskeletal proteins. Acetylation of nonꢀhistone targets impacts protein
2
ACS Paragon Plus Environment

Page 3 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
stability, protein cellular localization and proteinꢀprotein/proteinꢀnucleotide interactions, which
8
ꢀ9
can finally influence cell proliferation, survival and apoptosis. The tumor suppressor p53 was
1
0
the first reported nonꢀhistone target of HDACs and HATs. Acetylation of p53 activates its
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
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
sequenceꢀspecific DNA binding activity and consequently, increases activation of its target
11
genes. Furthermore, acetylated p53 induces cell apoptosis and basic autophagy by
transcriptionally upregulating tuberous sclerosis 2 (TSC2), AMPꢀactivated protein kinase
(AMPK), and damageꢀregulated autophagy modulator (DRAM) thereby suppressing
mammalian target of rapamycin (mTOR) and the uncꢀ51 like autophagy activating kinase 1
1
2
(ULK1) complex further downstream in MESꢀSA cells. p53ꢀfamily members p63 and p73
13ꢀ14
which can compensate for p53 could also mediate apoptosis.
To date, a total of eighteen HDACs isoforms have been identified in humans, eleven of
which contain a zincꢀbinding site; these can be divided into class I, II and IV, while class III
+
15
HDACs (SIRT1ꢀ7), require NAD for their activity. Class I HDACs, including HDAC 1, 2, 3
and 8, are homologous to yeast Rpd3 (reduced potassium dependencyꢀ3) protein. Class II
comprise HDAC 4, 5, 6, 7, 9, and 10, and are structurally related to yeast Hda1 (histone
1
6
deacetylases 1). Class III possesses only one member, HDAC11.
So far, four HDAC inhibitors (HDACIs) have been approved by the FDA: vorinostat
1
7
18
19
20
(
SAHA), romidepsin (FK228), belinostat (PXDꢀ101) and panobinostat (LBH589) for the
treatment of cutaneous tꢀcell lymphoma (CTCL), peripheral Tꢀcell lymphoma (PTCL) or
multiple myeloma (MM) (Figure 1). Understanding the structural regions of HDACIs is
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 59
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
essential to design potent and selective inhibitors. Pharmacophore models of most HDACIs
contain three structural parts: a cap group, a linker, and a zincꢀbinding group (ZBG). The cap
group interacts with the surface of the enzyme; the linker occupies the long hydrophobic tunnel
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
2
1ꢀ22
leading to the zinc site; and the ZBG functions in the bottom catalytic site.
The classification
of HDACIs primarily depends on their ZBG chemical structures: hydroxamates, benzamides,
aliphatic acids, electrophilic ketones and so on. To some extent, modification of the ZBG can
2
3ꢀ25
introduce a change in potency and in the selectivity profile.
For example, most
hydroxamates are panꢀHDACIs while the benzamides have increased class I selectivity.
Although hydroxamic acid is the most commonly used ZBG, it suffers from susceptibility to
26
hydrolysis leading to inactive carboxylic acid as well as glucuronidationꢀbased inactivation.
This structural instability contributes the main reason of poor pharmacokinetic profiles (eg. short
2
7ꢀ28
t1/2 and low Cmax) of clinical hydroxamate HDACIs.
(Figure 1 should be listed here)
Selective HDACIs have attracted increased attention as they are hypothesized to elicit fewer
side effects than the panꢀHDACIs. Class I HDACs are the most relevant with regard to the
2
+
29
initiation and development of cancer among all the Zn dependent HDACs, therefore
discovery of potent class I selective HDACIs is of great interest for the potential treatment of
cancer. Currently, most class I HDACIs, especially those in clinical research, are benzamides
HDACIs bearing oꢀaminobenzamide as the ZBG. Additionally, modifications to the
oꢀaminobenzamide moiety lead to alterations in inhibitor activity. Recent studies demonstrated
that aryl substituents on the oꢀaminobenzamide of benzamides HDACIs led to dramatic
4
ACS Paragon Plus Environment

Page 5 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1, 30ꢀ31
32ꢀ33
improvements in potency and selectivity for HDAC1 and 2
or for HDAC3.
In our previous study, we designed and synthesized a series of HDAC class I selective
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
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
benzamide HDACIs bearing a tryptophan cap group. The most potent compound 11a,
(
S)ꢀ4ꢀ((3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(4ꢀmethoxybenzamido)propanamido)ꢀmethyl)ꢀNꢀ(2ꢀaminophenyl)be
nzamide exhibited potent inhibitory activity with IC against HDAC1, 2 and 3 of 20.6 nM, 157
50
3
4
nM, and 138 nM, respectively. However, as the ZBG, oꢀaminobenzamide reaches limits in its
26
inhibitory potency and is susceptible to glucuronidationꢀbased inactivation. In 2015, Wang et
3
5
al reported new HDAC class I selective inhibitors with a benzoylhydrazide scaffold. Our
group also generated a series of class I HDAC selective inhibitors that incorporated a hydrazide
as the ZBG, as shown for representative compound 3b (Figure 2). Furthermore, we
demonstrated that the hydrazide motif displayed allosteric inhibition kinetics and was
26
impervious to glucuronidation. As part of our current study, we modified our lead compound
11a by changing the linker and introducing the hydrazide motif as ZBG (Figure 2). Herein, we
discuss the structureꢀactivity relationships (SAR), isotype selectivity, enzymatic inhibition
kinetics, and p53 statusꢀdependency mechanism for this new series of compounds.
(
Figure 2 should be listed here)
CHEMISTRY
Scheme 1 shows the synthesis of intermediates 4a and 4b. Methyl protection of 1a and 1b
afforded 2a and 2b, respectively, which underwent amino acid condensation with compound 3 to
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 59
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
achieve 4a and 4b.
(
Scheme 1 should be listed here)
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
Compounds 4a, 4b, 5, 6 and 7 were treated with hydrazine monohydrate to achieve
hydrazide 8, 9, 10, 11 and 12, respectively. The hydrazides were reacted with different aliphatic
aldehydes, then reduced by NaBH CN to give the end products, 13aꢀ13h (Scheme 2).
3
Intermediate 14 was reacted with cyclopentylhydrazine or phenylhydrazine by a
TBTUꢀmediated amide formation to afford 15a and 15b, respectively (Scheme 3). Compound
1
6 (Scheme 4) was subjected to a similar reaction in order to produce 17, which was treated with
propionaldehyde and then NaBH CN to obtain end product 18.
3
(Scheme 2 should be listed here)
(Scheme 3 should be listed here)
(Scheme 4 should be listed here)
RESULTS AND DISCUSSION
In our previous study, we designed and synthesized a series of oꢀaninobenzamideꢀbased
HDACIs, among which, the representative compound 11a exhibited potent inhibitory activity
3
4
against class I HDACs (IC : HDAC1, 20.6 nM; HDAC2, 157 nM; HDAC3, 138 nM).
5
0
Although the oꢀaninobenzamide motif is the most commonly used ZBG for selective class I
HDACIs, it features several drawbacks: lower inhibitory activity, potential for heptatotoxicty
6
ACS Paragon Plus Environment

Page 7 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
due to the potential imine metabolites, and glucuronidationꢀbased inactivation. To explore new
HDACI motifs, Wang and colleagues demonstrated a novel hydrazideꢀbased class I HDACI,
based on which our lab generated a more potent series of class I HDAC selective inhibitors with
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
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
2
6, 35
hydrazide as a potential ZBG.
We have shown that hydrazide compounds are not
26
glucuronidated, and preliminary pharmacokinetics study in mice showed the C and AUC of
max
our hydrazide compound LPꢀ411 is at least 200 times higher than all FDA approved HDACIs.
Further, the t1/2 is much longer than approved hydroxamates HDACIs (data not shown), which
indicate hydrazide motifs have better in vivo stability. The side effect of hydrazideꢀbased
HDACIs is still unknown because they haven’t tested in clinic. However, compounds bearing
hydrazine/hydrazide motif are used clinically as antiꢀdepressants and antiꢀtuberculosis agent for
3
6
decades with great safety profiles.
Potential toxicity of the most recognizable
3
7
hydrazideꢀcontaining agent isoniazid is hepatotoxicity, but the rate was as low as 0.5%ꢀ2%,
3
8
and rarely was the hepatotoxicity lethal (1/25000). Due to the improved pharmacokinetic
profile and low toxicity of hydrazide, we optimize benzamides 11a by keeping its cap and linker
unchanged and introducing different hydrazide motifs to achieve compounds 13aꢀ13d, 15a and
15b (Table 1). More specifically, 13aꢀ13d possess linear alkyl chains 2 to 5 carbons in length on
the hydrazine motif, while 15a and 15b bear cyclic hydrazide substituents. HDAC1, 2, 3 and 6
enzymatic inhibition experiments and antiꢀproliferative assays based on the acute myeloid
leukemia MV4ꢀ11 cells were conducted to efficiently screen the newly designed compounds
(
Table 1). 13a-13d with linear alkyl motifs on hydrazide exhibited nanoꢀmolar IC s towards
50
HDAC1, 2 and 3. Further, unlike the parent compound 11a, 13b-13d exhibited a degree of
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 59
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
HDAC3 selective inhibition. Among the inhibitors tested, 13b bearing a propyl hydrazide
substituent exhibited the greatest selectivity toward HDAC1, 2 and 3: 13b’s IC50 against
HDAC3 was 5.63 nM, ~51ꢀfold and ~11ꢀfold lower than against HDAC2 and HDAC1,
respectively. However, compound 15a with cyclopentyl, and 15b with benzyl motifs showed no
inhibition against HDAC1, 2, or 3 up to 5000 nM. The compounds did not inhibit HDAC6 with
the exception of 13a, which has an ethyl hydrazide moiety. All of the hydrazide compounds
displayed subꢀmicromolar EC s against MV4ꢀ11 cells. The EC of the most potent compound
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
50
50
(13b) was 138.7 nM, 6.6 times more potent than parent benzamide 11a.
(Table 1 should be listed here)
In order to explore the inhibition mechanism of hydrazide versus benzamides HDAC
inhibitors, we conducted in vitro Vmax studies for hydrazide compound 13b and its parent
benzamides 11a using recombinant HDACs 1, 2 and 3. LineweaverꢀBurke analysis showed that
nd
1
3b clearly demonstrates a convergence in the 2 quadrant for HDAC1, 2 and 3, indicative of
mixed inhibition (Figure 3A, 3B and 3C). As indicated in our previous work, heterodimer
HDACs appear to contain an allosteric binding pocket created by the interface of the two
2
6
monomers in the heterodimer (Figure 4). Therefore, one way to interpret the results shown in
Figure 3A, 3B and 3C is that, in addition to being able to bind to the same site as the enzymatic
substrate (competitive), 13b may have an alternate binding site (i.e., a nonꢀcompetitive allosteric
site). Parent inhibitor 11a displayed entirely different binding kinetics against HDAC1 and
HDAC2 and HDAC3. For HDAC1, convergence of LineweaverꢀBurke plots for varied inhibitor
8
ACS Paragon Plus Environment

Page 9 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
concentrations directly on the yꢀaxis indicates competitive inhibition (Figure 3D); however, the
compound displayed mixed type inhibition toward HDAC2 and HDAC3.
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
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
(Figure 3 should be listed here)
To better understand the potential binding modes and patterns between the two protein
heterodimers we tested 11a and 13b in four conditions each by probing the competitive site (CS)
and the allosteric site (AS) of HDAC1 and HDAC3 using computational docking. These results
were particularly useful in determining how much variability was in potential binding modes for
each of these eight total conditions (Figure 4), and the potential interaction maps of 11a and 13b
with both sites of HDAC1 and HDAC3 (Supporting Information S2ꢀS3). The overall size of
catalytic site (CS) and allosteric site (AS) for each isoform was reasonably similar (Figure 5). As
shown using scoring in the docking simulations (Figure 4), there was a very large discrimination
between the high affinity CS for 11a in HDAC1 and the very low affinity AS for 11a in HDAC1.
The interaction of 11a with the HDAC1 CS also indicated it could form hydrogen bonds with
Cys100, Tyr303, His140 and Gly149 as well as a πꢀπ interaction with Phe150, while interaction
of 11a with HDAC1 AS was weaker and appeared to only partially bury the indole moiety
(Supporting Information S2). Therefore, 11a has a higher preference with HDAC1 CS, which
might be the reason 11a exhibited competitive inhibition against HDAC1. For 13b interaction
with HDAC1 we see a modest preference for the CS over the AS (Figure 4), furthermore,
interaction of 13b with HDAC1 CS and AS also showed similar hydrogen bonding (Supporting
Information S3). Compound 11a and 13b binding to HDAC3 appeared to be almost equal in
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 59
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
preference for the two sites in the protein (Figure 4). More possible interactions were found with
the HDAC3 AS than HDAC1 AS (Supporting Information S2ꢀS3), which is in agreement with
the kinetics data where both of these compounds displayed mixed inhibition toward HDAC3.
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
(Figure 4 should be listed here)
(Figure 5 should be listed here)
To further examine whether the binding of inhibitor to the enzymes was timeꢀdependent, we
determined IC curves of 13b and 11a against HDAC1, 2 and 3 for several preꢀincubation times
5
0
(
15 min, 30 min, 60 min and 90 min; Figure 6). Doseꢀresponse curves and calculated IC values
50
for 13b and 11a all varied with enzymeꢀinhibitor preꢀincubation time. For example, after a 90
min preꢀincubation, the IC of 13b for HDAC3 was 5.7 nM, a 3ꢀfold decrease compared to the
50
IC measured after 15 min preꢀincubation; additionally, the IC did not reach a steadyꢀstate
5
0
50
value for 90 min (Figure 6C). Similar results were also observed for 13b with HDAC1 and 2 and
for 11a with HDAC1, 2 and 3 (Figure 6). These observations suggest that 13b and 11a are both
slow, tightꢀbinding inhibitors of class I HDACs.
(Figure 6 should be listed here)
After optimizing the ZBG, we explored the effect of varying the linker on compounds’
activity and selectivity. For this purpose, we kept the cap group and ZBG of the most selective,
potent compound (13b, Table 1) unchanged and introduced several different linker groups to
obtain compounds 13e-13h and 18 (Table 2). The modified compounds maintained 13b’s
10
ACS Paragon Plus Environment

Page 11 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
selectivity pattern in subclass I HDACs and showed no inhibition against HDAC6 up to 100 ꢁM.
Surprisingly, 13e, with a linker simply truncated by a single methylene spacer compared to 13b,
exhibited much higher activity against HDAC1, 2 and 3, and higher in vitro activity against
MV4ꢀ11 cells as well. IC s of 13e against HDAC1, 2 and 3 were 9.54, 28.04 and 1.41 nM,
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
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
50
which were 6.6ꢀfold, 10.2ꢀfold and 6.1ꢀfold lower than those for 13b, respectively. What’s more,
the antiproliferative activity of 13e (EC : 34.7 nM) in MV4ꢀ11 cells was 4.1 times higher than
5
0
1
3b (EC : 138.7 nM).
50
As 13e is the most potent compound, we then tested the IC50 of 13e for the other
2
+
Zn ꢀdependent HDACs (HDAC4, 5, 7, 8 and 9) in order to assess its subtype selectivity profile.
Inhibitory activity toward HDAC10 and 11 could not be determined because there are no
3
9
appropriate substrates. As expected, 13e did not show inhibition against any Class IIa HDACs
(
HDAC4, 5, 7 and 9) up to 50 ꢁM (Table 3 and Supporting Information Figure S4). IC for 13e
5
0
against Class I member HDAC8 was 626.12 nM, which was about 58.4ꢀfold, 20.0ꢀfold and
395.2ꢀfold higher than HDAC1, HDAC2 and HDAC3, respectively (Table 3 and Supporting
Information Figure S5). Therefore, 13e is a HDAC1, 2 and 3 selective inhibitor, or HDAC class I
selective inhibitor if we take the modest HDAC8 inhibition into consideration. To examine the
2
+
specificity of 13e we further tested the inhibitory activity of 13e against two different Zn
metalloenzymes, MMPꢀ2/MMPꢀ9 and APN/CD13 that have been suggested to play a role in
4
0ꢀ41
cancer survival.
Results indicated that 13e did not inhibit the two enzymes at concentrations
up to 50 ꢁM (Supporting Information Figure S6ꢀS7), suggesting that hydrazide HDAC inhibitors’
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 59
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
antiꢀcancer activity were not due to nonꢀspecific metalloprotease inhibition. We also predicted a
binding mode of 13e with catalytic site (CS) and allosteric site (AS) of HDAC1 and HDAC3.
2
+
The hydrazide functional group could form a bidentate interaction with Zn , which is similar to
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
hydroxamic acid in the CS (Supporting Information Figure S8).
(Table 2 should be listed here)
(Table 3 should be listed here)
To follow up on these experiments, we examined the HDAC inhibitory activity of our
HDACIs in cell culture, selecting the most potent compound (13e) for analysis in MV4ꢀ11 cells
with SAHA as a positive control. Results showed compound 13e can markedly increase the
level of acetylated histoneH3 and acetylated histoneH4, which was consistent with the HDAC1,
2 and 3 inhibition activities (Figure 7). Unexpectedly, the level of acetylꢀtubulin also increased,
although 13e does not inhibit HDAC6 which controls the deacetylation of tubulin. We
hypothesize that HDAC6 was downꢀregulated when treated with 13e; thus, we also
characterized the level of HDAC6. As expected, HDAC6 was nearly absent following treatment
with ≥ 0.5ꢁM 13e or with 5 ꢁM SAHA for 24 h. Further studies showed that 13e induced
HDAC6 repression require a more than of 12 h treatment. This suggested HDAC6
downꢀregulation is likely due to transcriptional repression, and not through protein degradation
(proteinase and lysosome, etc.), as the latter one would occur within hours. HDAC6 is required
42
for tumor cells to be resistant to anoikis then facilitated tumor cell invasion and metastasis.
12
ACS Paragon Plus Environment

Page 13 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Another study showed oncogenic Kꢀras contributes to SAHA resistance by upregulating
4
3
HDAC6 and cꢀmyc expression. Therefore, downꢀregulation of HDAC6 may have benefits in
overcoming HDAC inhibition resistance. To the best of our knowledge, ours is the first report of
a hydrazide HDAC class I inhibitor that downꢀregulated HDAC6 instead of acting through direct
inhibition on that HDAC isoform.
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
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
(Figure 7 should be listed here)
We tested our HDACIs against the acute myeloid leukemia cell line MV4ꢀ11 and carried out
further evaluations of the antiꢀproliferative activity of 13e against a prostate p53 null cancer cell
line (PCꢀ3) and human embryonic kidney cells, HEK293 (a nonꢀtumor cell line). The EC50
curves are shown in Figure 8. The EC value (11020 nM) against the nonꢀtumor cell line
5
0
HEK293 was more than 300ꢀfold and 30ꢀfold higher than against the MV4ꢀ11 and PCꢀ3 cell
lines, respectively, revealing our compounds’ cytotoxic selectivity for tumor cells over
nonꢀtransformed cells. EC value of 13e towards PCꢀ3 was 351.0 nM, which was more than 10
50
times higher than toward MV4ꢀ11 cells. Interestingly, after 48 h treatment of 13e at high
concentration, almost all MV4ꢀ11 cells were dead, while ~20% PCꢀ3 remained viable compared
to control; therefore, we speculated that 13e actually causes cell death in MV4ꢀ11 cells while
merely preventing cell proliferation in PCꢀ3 cells without killing the cells.
(Figure 8 should be listed here)
In order to assess this hypothesis, we examined the MV4ꢀ11 and PCꢀ3 cell numbers after
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 59
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
treatment with 10 ꢁM and 2 ꢁM 13e for 24 h and 48 h, respectively. As with the EC assay, the
50
®
CellTiterꢀBlue (a buffered solution containing highly purified resazurin) was used as the assay
reagent for determining cell viability. Functional mitochondria in viable cells convert the reagent
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
4ꢀ45
to highly fluorescent resorufin.
Fluorescence intensity of resorufin is therefore directly
correlated to the number of viable cells in culture. In contrast to MV4ꢀ11 cells, for which the
viable cell population decreased with time (Figure 9), the viable PCꢀ3 cell population either
slightly increased (2 ꢁM) or remained unchanged (10 ꢁM) following 13e treatment. We further
examined the PCꢀ3 cell line with a phaseꢀcontrast light microscope for changes in cell
morphology in response to 48 h treatment with 10 ꢁM and 2 ꢁM 13e. Meeting our expectation,
1
3e did not obviously affect cell morphology at the concentration of 2 ꢁM. Even at 10 ꢁM,
cells were still alive and attached to the culture flask (Figure 10). These observations were
consistent with our hypothesis. Collectively, these data indicate that there are different, cellꢀtype
dependent in vitro responses to our HDAC inhibitor. It is of interest to note that these two cell
lines are distinct in their p53 genetic status: MV4ꢀ11 possesses wildꢀtype p53, while PCꢀ3 is p53
null. Activation and accumulation of p53 in the nucleus regulates the transcription of numerous
target genes using specific DNA response elements, which occurs along with apoptosis, cell
cycle arrest, DNA repair and differentiation. As the first tumor suppressor gene linked to
apoptosis, p53 can function as a “master regulator” of the apoptotic program not only by the
activation of proꢀapoptotic proteins such as Bax, Bak and PUMA and repression of
antiꢀapoptotic Bclꢀ2 proteins, but also by the activation of CD95 and TRAIL receptor 2
46ꢀ47
(TRAILꢀR2/DR5), which can sensitize cells to deathꢀreceptorꢀmediated apoptosis.
In human
14
ACS Paragon Plus Environment

Page 15 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
tumors, the p53 tumor suppressor gene is the most frequently mutated gene, and loss of wild
type p53 function can both disable apoptosis and accelerate tumor development in transgenic
4
8
mice. As p53 is a critical initiator of apoptosis, to further assess whether the different
responses of MV4ꢀ11 cells and PCꢀ3 cells to 13e was related to p53 status, we conducted
western blot analysis and determined the levels of proꢀcaspase3, cleaved caspase3, and cleaved
PARP (poly ADPꢀribose polymerase) in each cell line after 24 h of treatment with 1 ꢁM and 2
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
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
ꢁ
M of 13e (Figure 11). In MV4ꢀ11 cells, 13e caused the cleavage of inactive proꢀcaspase3,
resulting in the subsequent increase in the levels of cleaved active caspase3 and cleaved PARP,
indicating an activation of apoptosis. In PCꢀ3 cells, 13e treatment resulted in no observed
cleavage of proꢀcaspase3 or PARP. Also, in contrast to MV4ꢀ11 cells, 13e did not induce
downꢀregulation of HDAC6 nor increase the level of acetylated tubulin in PCꢀ3 cells. However,
unlike HDAC6 downꢀregulation, in both two cell lines 13e did not induce downꢀregulation of
class I HDAC1. Other studies have demonstrated that introduction of exogenous wildꢀtype p53
into cancer cells and its subsequent overexpression result in growth arrest, initially shown to
49ꢀ51
occur at the G1/S cell cycle checkpoint.
Thus, we examined the cell cycle of MV4ꢀ11 cells
and PCꢀ3 cells after treatment with 13e for 24 h. Consistent with previous reports, 13e induced
G1/S and subꢀG1 cell cycle arrest in MV4ꢀ11 cells (Figure 12). The population of G1 and
subꢀG1 cells increased after 24 h treatment with 0.5 ꢁM 13e, and subꢀG1 cells increased in a
doseꢀdependent manner, which indicated significant cellular apoptosis (Figure 12 and
52ꢀ53
Supporting Information Figure S10).
While different from MV4ꢀ11 cells, 13e triggered
G2/M arrest in PCꢀ3 cells (Figure 12 and Supporting Information Figure S11). Based on these
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 59
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
data, we hypothesized that the effects of 13e were mediated through a p53ꢀdependent
mechanism resulting in a G1/S and subꢀG1 cell cycle arrest and cell death.
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
We also noticed that 13e can cause p53 degradation in MV4ꢀ11 cells (Figure 11), as
degradation of p53 happened within 6 h with treatment of 0.5 ꢁM 13e (Supporting Information
54
Figure S9). p53 was degraded through the proteasome, and proteasome inhibitor Bortezomib
blocks this degradation upon hydrazide HDAC inhibitor treatment (Supporting Information
Figure S12). Degradation of p53 is linked to concurrent depletion of antiꢀapoptotic protein such
5
5
as cꢀFLIP and XIAP resulting in cellular apoptosis. In addition, inactivation of p53 by deletion,
depletion or inhibition in nonꢀtransformed or malignant human cell lines (such as HFFF2
fibroblasts, HCT116 colon cancer cells, SHꢀSY5Y neuroblastoma and HeLa cervical cancer
5
6ꢀ57
cells) can trigger autophagy,
both of apoptosis and autophagy can result in cell death.
(Figure 9 should be listed here)
(Figure 10 should be listed here)
(Figure 11 should be listed here)
(Figure 12 should be listed here)
CONCLUSION
A new series of hydrazideꢀbased HDACIs were designed, synthesized, and had SAR
interrogated. Their isotype selectivity, enzymatic binding kinetics, and in vitro anticancer
16
ACS Paragon Plus Environment

Page 17 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
activities were also characterized. Modifications to the hydrazide motifs as well as to the linker
fragments of the compounds affected both HDAC inhibitory activity and efficacy against
MV4ꢀ11 leukemia cells. Of the variations to the hydrazide motif, 13b with a 3ꢀcarbon linear
alkyl substituent exhibited the best selectivity and antiꢀproliferative activity. Enzymatic
inhibition kinetics studies showed that 13b was a slow and mixed type inhibitor toward HDAC1,
2 and 3. Subsequent linker modifications of 13b led to the optimized compoundꢀ13e, which
showed low nanoꢀmolar IC toward HDAC1, 2 and 3, subꢀmicro molar IC against HDAC8
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
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
50
and no inhibitory activity against class IIa (HDAC4, 5, 7, 9) and class IIb (HDAC6) up to 50 ꢁM.
The EC of 13e toward MV4ꢀ11 cells was 34.7 nM, which was about 26 times lower than that
5
0
of the parent compound 11a. 13e displayed no inhibition of HDAC6 up to 100 ꢁM, though the
compound did markedly increase the level of acetylated tubulin via a mechanism involving
downꢀregulation of HDAC6. Cell based studies showed that 13e exhibited completely different
mechanisms of antiꢀproliferation activity against two different cancer cell lines, MV4ꢀ11 and
PCꢀ3. These cell lines have distinct p53 genetic statuses: MV4ꢀ11 is p53 wildꢀtype while PCꢀ3 is
p53 null. Our studies indicated that 13eꢀinduced death of p53 wild type MV4ꢀ11 cells might be
mediated by a p53 dependent apoptotic pathway resulting in G1/S and subꢀG1 cell cycle arrest.
In p53 null PCꢀ3 cells, 13e could not induce apoptosis, instead producing cytostatic effects via
promotion of only G2/M arrest.
EXPERIMENTAL SECTION
General Chemistry. All solvents, reagents, and compound precursors were purchased from
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 59
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
SigmaꢀAldrich or other chemical vendor and used as received, unless otherwise noted. H NMR
1
3
and C NMR data were collected in deuterated solvent using a Bruker Nanobay 400 MHz
instrument with TMS as an internal standard. Chemical shifts (δ) are given in parts per million,
and coupling constants (J) reported in hertz (Hz). Mass spectral data were gathered on a Thermo
LCQ Fleet mass spectrometer using electrospray ionization. Purification was performed using a
Teledyne Isco Combiflash 200 on prepacked C18ꢀAq columns. All target compounds were at
least 95% pure as confirmed via UV detection of ESIꢀLCMS, performed on an Agilent 1100
HPLC instrument using an ODS HYPERSIL column (5ꢁm, 4.6 mm × 250 mm) using a gradient
of water/methanol with 0.1% formic acid added.
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
(4ꢀmethoxybenzoyl)ꢀLꢀtryptophan
(3)
methyl
(S)ꢀ4ꢀ((3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(4ꢀmethoxybenzamido)propanamido)methyl)benzoate (5), methyl
(
S)ꢀ4ꢀ(3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(4ꢀmethoxybenzamido)propanamido)benzoate
S)ꢀ6ꢀ(3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(4ꢀmethoxybenzamido)propanamido)hexanoate
(6),
methyl
(
(7),
(S)ꢀ4ꢀ((3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(4ꢀmethoxybenzamido)propanamido) methyl)benzoic acid (14) and
(
S,E)ꢀ3ꢀ(4ꢀ(3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(4ꢀmethoxybenzamido)propoxy)phenyl)acrylic acid (16) were
3
4
synthesized as described previously.
General Procedure for the Preparation of 2a and 2b.
7
-methoxy-7-oxoheptan-1-aminium chloride (2a)
7ꢀaminoheptanoic acid (1.45 g, 10 mmol) was dissolved in 200 mL methanol. To this solution,
18
ACS Paragon Plus Environment

Page 19 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
acetyl chloride (2.4 g, 30 mmol) was added dropwise at 0 °C. The mixed solution was refluxed
at 75 °C for 5 h. Volatiles were removed via vacuum and the residue was recrystallized from
1
ethyl acetate to afford the desire product 2a as a white powder, 95% yield. H NMR (400 MHz,
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
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
DMSOꢀd ) δ 8.05 (s, 2H), 3.58 (s, 3H), 2.78ꢀ2.68 (m, 2H), 2.31 (t, J = 7.4 Hz, 2H), 1.60ꢀ1.45 (m,
6
+
4
H), 1.341.23 (m, 5H). ESIꢀMS m/z: 160.25 [M + H] .
8-methoxy-8-oxooctan-1-aminium chloride (2b)
Using the synthetic method of 2a, compound 8ꢀaminooctanoic acid gave 2b as a white powder,
9
3% yield.
General Procedure for the Preparation of 4a and 4b.
Methyl (S)-7-(3-(1H-indol-3-yl)-2-(4-methoxybenzamido)propanamido)heptanoate (4a)
Compound 3 (1.02 g, 3 mmol) was dissolved in 50 mL dichloromethane, and to this solution
was added 2ꢀ(1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium tetrafluoroborate (TBTU, 1.05
g, 3.6 mmol) and trimethylamine (0.6 mL, 4.5 mmol). 30 min later, 2a (0.52 g, 3.3 mmol) was
added followed by 0.6 mL additional triethylamine. The reaction was allowed to stir overnight,
then was washed with 1 N HCl (2 × 30 mL), saturated Na CO (2 × 30 mL), and brine (2 × 30
2
3
mL), then dried over MgSO . Volatiles were removed under vacuum to get the crude product 4a,
4
which was used for the next step in the synthesis of 11 without further purification. ESIꢀMS m/z:
+
4
80.17 [M + H] .
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 59
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
Methyl (S)-8-(3-(1H-indol-3-yl)-2-(4-methoxybenzamido)propanamido)octanoate (4b)
1
Using the synthetic method of 4a, compounds 3 and 2b gave 4b as a white solid, 55% yield. H
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
NMR (400 MHz, DMSOꢀd ) δ 10.76 (s, 1H), 8.29 (d, J = 8.1 Hz, 1H), 8.00 (t, J = 5.6 Hz, 1H),
6
7.81 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 2.2 Hz,
1
H), 7.05 (t, J = 7.0 Hz, 1H), 6.95ꢀ6.99 (M, 3H), 4.69ꢀ4.63 (m, 1H), 3.80 (s, 3H), 3.58 (s, 3H),
3.22ꢀ2.99 (m, 4H), 2.28 (t, J = 7.4 Hz, 2H), 1.54ꢀ1.48 (m, 2H), 1.42ꢀ1.29 (m, 2H), 1.23 (s, 6H).
+
ESIꢀMS m/z: 494.17 [M + H] .
General Procedure for the Preparation of 8, 9, 10, 11 and 12.
(S)-N-(1-((4-(hydrazinecarbonyl)benzyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-4-met
hoxybenzamide (8)
To a solution of 5 (2.4 g, 5 mmol) in methanol was added hydrazine monohydrate (1.6 g, 25
mmol). The mixture was refluxed at 75 °C for 48 h. Volatiles were then removed to afford
1
compound 8, white solid, 95% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.78 (s, 1H), 9.71 (s,
6
1H), 8.65 (t, J = 6.0 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 7.87ꢀ7.81 (m, 2H), 7.75 (d, J = 8.3 Hz,
2
H), 7.70 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 2.2
Hz, 1H), 7.06 (dd, J = 11.1, 4.0 Hz, 1H), 6.98 (t, J = 8.0 Hz, 3H), 4.76 (td, J = 9.5, 5.0 Hz, 1H),
4
.47 (s, 2H), 4.36 (d, J = 5.9 Hz, 2H), 3.80 (s, 3H), 3.22 (ddd, J = 24.2, 14.5, 7.3 Hz, 2H).
+
ESIꢀMS m/z: 486.08 [M + H] .
(S)-N-(1-((4-(hydrazinecarbonyl)phenyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-4-met
20
ACS Paragon Plus Environment

Page 21 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
hoxybenzamide (9)
Using the synthetic method of 8, compound 6 and hydrazine monohydrate gave 9 as a white
1
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
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
solid, 95% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.81 (s, 1H), 10.45 (s, 1H), 9.66 (s, 1H),
6
8.55 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 8.9 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 7.8 Hz,
1
H), 7.70 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 2.2 Hz, 1H), 7.06 (t, J = 7.5
Hz, 1H), 7.01ꢀ6.97 (m, 3H), 4.92ꢀ4.83 (m, 1H), 4.45 (s, 2H), 3.81 (s, 3H), 3.30ꢀ3.22 (m, 2H).
+
ESIꢀMS m/z: 472.00 [M + H] .
(S)-N-(1-((6-hydrazinyl-6-oxohexyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-4-methoxy
benzamide (10)
Using the synthetic method of 8, compound 7 and hydrazine monohydrate gave 10 as a white
1
solid, 95% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.76 (s, 1H), 8.92 (s, 1H), 8.28 (d, J = 8.0
6
Hz, 1H), 8.02 (t, J = 5.3 Hz, 1H), 7.81 (d, J = 8.7 Hz, 2H), 7.68 (d, J = 7.8 Hz, 1H), 7.30 (d, J =
8
.0 Hz, 1H), 7.18 (s, 1H), 7.05 (t, J = 7.4 Hz, 1H), 7.00ꢀ6.95 (m, 3H), 4.69ꢀ4.63 (m, 1H), 4.17 (s,
2H), 3.80 (s, 3H), 3.23ꢀ2.97 (m, 4H), 1.99 (t, J = 7.4 Hz, 2H), 1.52ꢀ1.44 (m, 2H), 1.39ꢀ1.34 (m,
+
2
H), 1.30ꢀ1.14 (m, 2H). ESIꢀMS m/z: 466.17 [M + H] .
(S)-N-(1-((7-hydrazinyl-7-oxoheptyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-4-methox
ybenzamide (11)
Using the synthetic method of 8, compound 4a and hydrazine monohydrate gave 11 as a white
1
solid, 95% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.76 (s, 1H), 8.93 (s, 1H), 8.29 (d, J = 8.1
6
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 59
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
Hz, 1H), 8.01 (t, J = 5.6 Hz, 1H), 7.81 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 7.8 Hz, 1H), 7.30 (d, J =
8
1
.0 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H), 7.05 (t, J = 7.0 Hz, 1H), 6.95ꢀ6.99 (m, 3H), 4.69ꢀ4.63 (m,
H), 4.23 (s, 2H), 3.80 (s, 3H), 3.21ꢀ3.00 (m, 4H), 2.00 (t, J = 7.4 Hz, 2H), 1.53ꢀ1.42 (m, 2H),
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
+
1.38ꢀ1.35 (m, 2H), 1.21 (s, 4H). ESIꢀMS m/z: 480.25 [M + H] .
(S)-N-(1-((8-hydrazinyl-8-oxooctyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-4-methoxy
benzamide (12)
Using the synthetic method of 8, compound 4b and hydrazine monohydrate gave 12 as a white
1
solid, 95% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.76 (s, 1H), 8.91 (s, 1H), 8.29 (d, J = 8.1
6
Hz, 1H), 8.00 (t, J = 5.6 Hz, 1H), 7.81 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 7.8 Hz, 1H), 7.30 (d, J =
8
.0 Hz, 1H), 7.18 (d, J = 2.2 Hz, 1H), 7.05 (t, J = 7.0 Hz, 1H), 6.99ꢀ6.95 (m, 3H), 4.69ꢀ4.63 (m,
1H), 4.14 (s, 2H), 3.80 (s, 3H), 3.23ꢀ2.98 (m, 4H), 1.99 (t, J = 7.4 Hz, 2H), 1.51ꢀ1.45 (m, 2H),
+
1
.41ꢀ1.31 (m, 2H), 1.22 (s, 6H). ESIꢀMS m/z: 494.25[M + H] .
General Procedure for the Preparation of 13a-13h.
(S)-N-(1-((4-(2-ethylhydrazine-1-carbonyl)benzyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-
yl)-4-methoxybenzamide (13a)
Compound 8 (0.97 g, 2 mmol) was dissolved in 50 mL ethanol, then acetaldehyde was added
followed by 20 mmol MgSO . Upon completion of the reaction, MgSO was filtered off and the
4
4
volatiles were removed from the filtrate under vacuum. The residue was dissolved in 50 mL
methanol, followed by the addition of NaBH CN and 2 drops of concentrated HCl. The mixture
3
22
ACS Paragon Plus Environment

Page 23 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was allowed to stir overnight, volatiles were removed under vacuum, and the crude product was
purified on combiꢀflash system C18 reverse phase columns eluted with acetonitrile and water to
1
give 13a, a white powder, 60% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.80 (d, J = 1.6 Hz,
6
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
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
1H), 9.98 (s, 1H), 8.67 (t, J = 6.0 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.9 Hz, 2H),
7
.77 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 8.3 Hz,
2H), 7.22 (d, J = 2.2 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H),7.01ꢀ6.97 (m, 3H), 5.10 (s, 1H), 4.83ꢀ4.72
m, 1H), 4.37 (d, J = 5.9 Hz, 2H), 3.81 (d, J = 7.5 Hz, 3H), 3.31ꢀ3.15 (m, 2H), 2.82 (q, J = 7.2
(
13
Hz, 2H), 1.05 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, DMSOꢀd ) δ 172.57, 166.22, 165.65,
6
162.08, 143.33, 136.55, 132.08, 129.82, 127.71, 127.47, 127.24, 126.76, 124.20, 121.36, 118.99,
118.69, 113.79, 111.79, 111.02, 55.80, 54.90, 45.98, 42.33, 28.02, 13.57. ESIꢀMS m/z:
+
514.08[M + H] . (λ ) purity 97.9%. Retention time: 7.74 min.
254
(S)-N-(3-(1H-indol-3-yl)-1-oxo-1-((4-(2-propylhydrazine-1-carbonyl)benzyl)amino)propan-
2
-yl)-4-methoxybenzamide (13b)
Using the synthetic method of 13a, compound 8 and propionaldehyde gave 13b as a white solid,
1
5
8% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.80 (s, 1H), 9.97 (s, 1H), 8.67 (t, J = 5.9 Hz,
6
1H), 8.42 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.2 Hz, 2H), 7.71 (d, J = 7.8
Hz, 1H), 7.32 (s, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 1.8 Hz, 1H), 7.07 (t, J = 7.3 Hz, 1H),
7
.01ꢀ6.97 (m, 3H), 5.10 (s, 1H), 4.80ꢀ4.75 (m, 1H), 4.37 (d, J = 5.8 Hz, 2H), 3.80 (s, 3H),
1
3
3.30ꢀ3.15 (m, 2H), 2.76 (t, J = 7.0 Hz, 2H), 1.55ꢀ1.40 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H). C
NMR (100 MHz, DMSOꢀd ) δ 172.57, 166.22, 165.61, 162.08, 143.31, 136.55, 132.09, 129.82,
6
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 59
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
127.72, 127.47, 127.24, 126.77, 124.20, 121.35, 118.99, 118.69, 113.79, 111.79, 111.02, 55.80,
+
5
4.90, 53.58, 42.33, 28.02, 21.33, 12.15. ESIꢀMS m/z: 528.17[M + H] . (λ ) purity 97.0%.
2
54
Retention time: 8.30 min.
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
(S)-N-(1-((4-(2-butylhydrazine-1-carbonyl)benzyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2
yl)-4-methoxybenzamide (13c)
-
Using the synthetic method of 13a, compound 8 and butyraldehyde gave 13c as a white solid, 60%
1
yield. H NMR (400 MHz, DMSOꢀd ) δ 10.79 (s, 1H), 9.97 (s, 1H), 8.67 (t, J = 6.0 Hz, 1H),
6
8
.41 (d, J = 8.0 Hz, 1H), 7.89ꢀ7.82 (m, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 7.8 Hz, 1H),
7.33 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 2.2 Hz, 1H), 7.10ꢀ7.03 (m, 1H),
.02ꢀ6.94 (m, 3H), 5.08 (s, 1H), 4.82ꢀ4.70 (m, 1H), 4.37 (d, J = 5.9 Hz, 2H), 3.80 (s, 3H),
7
1
3
3.33ꢀ3.15 (m, 2H), 2.79 (t, J = 7.0 Hz, 2H), 1.51ꢀ1.30 (m, 4H), 0.90 (t, J = 7.2 Hz, 3H). C
NMR (100 MHz, DMSOꢀd ) δ 172.56, 166.22, 165.60, 162.07, 143.31, 136.55, 132.08, 129.81,
6
1
27.71, 127.46, 127.23, 126.76, 124.19, 121.35, 118.98, 118.69, 113.79, 111.79, 111.02, 55.79,
+
54.89, 51.38, 42.33, 30.27, 28.01, 20.31, 14.40. ESIꢀMS m/z: 542.08[M + H] . (λ ) purity
254
9
9.9%. Retention time: 8.71 min.
(S)-N-(3-(1H-indol-3-yl)-1-oxo-1-((4-(2-pentylhydrazine-1-carbonyl)benzyl)amino)propan-
-yl)-4-methoxybenzamide (13d)
2
Using the synthetic method of 13a, compound 8 and valeraldehyde gave 13d as a white solid, 60%
1
yield. H NMR (400 MHz, DMSOꢀd ) δ 10.80 (s, 1H), 9.98 (s, 1H), 8.68 (t, J = 5.8 Hz, 1H),
6
24
ACS Paragon Plus Environment

Page 25 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
8.42 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 7.8 Hz,
1
H), 7.33 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.1 Hz, 2H), 7.22 (s, 1H), 7.06 (t, J = 7.4 Hz, 1H),
.01ꢀ6.97 (m, 3H), 5.08 (s, 1H), 4.76 (dd, J = 13.0, 9.3 Hz, 1H), 4.36 (d, J = 5.7 Hz, 2H), 3.80 (s,
7
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
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
3H), 3.36ꢀ3.14 (m, 2H), 2.77 (t, J = 7.0 Hz, 2H), 1.44ꢀ1.46 (m, 2H), 1.39ꢀ1.23 (m, 4H), 0.88 (t, J
13
=
6.6 Hz, 3H). C NMR (100 MHz, DMSOꢀd ) 172.58, 166.22, 165.59, 162.07, 143.31, 136.54,
6
132.06, 129.82, 127.69, 127.46, 127.23, 126.74, 124.21, 121.35, 118.99, 118.69, 113.78, 111.79,
11.01, 55.79, 54.89, 51.66, 42.31, 29.35, 27.98, 27.77, 22.53, 14.42. ESIꢀMS m/z: 556.25[M +
1
+
H] . (λ ) purity 98.5%. Retention time: 9.03 min.
254
(S)-N-(3-(1H-indol-3-yl)-1-oxo-1-((4-(2-propylhydrazine-1-carbonyl)phenyl)amino)
propan-2-yl)-4-methoxybenzamide (13e)
Using the synthetic method of 13a, compound 9 and propionaldehyde gave 13e as a white solid,
1
6
2% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.82 (s, 1H), 10.50 (s, 1H), 9.92 (s, 1H), 8.59 (d,
6
J = 7.7 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 7.7 Hz, 1H), 7.72
(d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 2.1 Hz, 1H), 7.06 (t, J = 7.1 Hz, 1H),
7
.01ꢀ6.97 (m, 3H), 5.06 (s, 1H), 4.91ꢀ4.85 (m, 1H), 3.81 (s, 3H), 3.27ꢀ3.25 (m, 2H), 2.75 (t, J =
1
3
7.1 Hz, 2H), 1.54ꢀ1.41 (m, 2H), 0.94ꢀ0.88 (m, 3H). C NMR (100 MHz, DMSOꢀd ) δ 171.99,
6
1
66.37, 165.32, 162.15, 142.12, 136.49, 129.85, 128.30, 128.21, 127.68, 126.53, 124.32, 121.39,
119.08, 118.68, 113.85, 111.78, 110.66, 55.81, 53.63, 31.16, 21.34, 12.14. ESIꢀMS m/z:
+
514.08[M + H] . (λ ) purity 95.9%. Retention time: 8.36 min.
254
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 59
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
(S)-N-(3-(1H-indol-3-yl)-1-oxo-1-((6-oxo-6-(2-propylhydrazinyl)hexyl)amino)propan-2-yl)-
4
-methoxybenzamide (13f)
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
Using the synthetic method of 13a, compound 10 and propionaldehyde gave 13f as a white solid,
1
61% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.77 (d, J = 1.5 Hz, 1H), 9.22 (d, J = 5.7 Hz, 1H),
6
8
.29 (d, J = 8.1 Hz, 1H), 8.03 (t, J = 5.6 Hz, 1H), 7.82 (d, J = 8.9 Hz, 2H), 7.68 (d, J = 7.8 Hz,
1H), 7.31 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H), 7.09ꢀ7.02 (m, 1H), 7.01ꢀ6.92 (m, 3H),
4
.76 (d, J = 5.5 Hz, 1H), 4.70ꢀ4.64 (m, 1H), 3.80 (s, 3H), 3.23ꢀ2.99 (m, 4H), 2.68ꢀ2.57 (m, 2H),
.00 (t, J = 7.4 Hz, 2H), 1.57ꢀ1.43 (m, 2H), 1.44ꢀ1.30 (m, 4H), 1.29ꢀ1.15 (m, 2H), 0.86 (t, J =
2
1
3
7.4 Hz, 3H). C NMR (100 MHz, DMSOꢀ d ) δ 172.12, 171.34, 166.00, 162.03, 136.50, 129.75,
6
1
27.76, 126.82, 124.08, 121.29, 119.01, 118.61, 113.77, 111.73, 111.09, 55.78, 54.70, 53.50,
+
39.01, 33.93, 29.25, 28.20, 26.44, 25.44, 21.22, 12.06. ESIꢀMS m/z: 508.25[M + H] . (λ )
254
purity 95.9%. Retention time: 7.98 min.
(S)-N-(3-(1H-indol-3-yl)-1-oxo-1-((7-oxo-7-(2-propylhydrazinyl)heptyl)amino)propan-2-yl)-
-methoxybenzamide (13g)
4
Using the synthetic method of 13a, compound 11 and propionaldehyde gave 13g as a white solid,
1
6
1% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.77 (d, J = 1.4 Hz, 1H), 9.22 (s, 1H), 8.29 (d, J
6
=
8.1 Hz, 1H), 8.01 (t, J = 5.6 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 7.8 Hz, 1H), 7.31
(d, J = 8.0 Hz, 1H), 7.19 (d, J = 2.1 Hz, 1H), 7.05 (dd, J = 11.1, 3.9 Hz, 1H), 7.00ꢀ6.95 (m, 3H),
26
ACS Paragon Plus Environment

Page 27 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4.79 (s, 1H), 4.70ꢀ4.64 (m, 1H), 3.80 (s, 3H), 3.23ꢀ2.99 (m, 4H), 2.61 (t, J = 7.1 Hz, 2H), 2.00 (t,
13
J = 7.4 Hz, 2H), 1.49ꢀ1.45 (m, 2H), 1.43ꢀ1.34 (m, 4H), 1.22 (s, 4H), 0.86 (t, J = 7.4 Hz, 3H). C
NMR (100 MHz, DMSOꢀd ) δ 172.10, 171.39, 166.03, 162.03, 136.50, 129.75, 127.76, 126.81,
6
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
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
124.06, 121.29, 119.00, 118.61, 113.78, 111.73, 111.08, 55.78, 54.72, 53.50, 39.03, 33.91, 29.38,
+
2
8.73, 28.17, 26.50, 25.64, 21.22, 12.06. ESIꢀMS m/z: 522.25[M + H] . (λ ) purity 98.0%.
2
54
Retention time: 8.29 min.
(
S)-N-(3-(1H-indol-3-yl)-1-oxo-1-((8-oxo-8-(2-propylhydrazinyl)octyl)amino)propan-2-yl)-4
methoxybenzamide (13h)
-
Using the synthetic method of 13a, compound 12 and propionaldehyde gave 13h as a white
1
solid, 61% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.76 (d, J = 1.7 Hz, 1H), 9.29 (s, 1H), 8.25
6
(d, J = 8.1 Hz, 1H), 8.00 (t, J = 5.6 Hz, 1H), 7.78 (d, J = 8.9 Hz, 2H), 7.65 (d, J = 7.8 Hz, 1H),
7
.31 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H), 7.08ꢀ7.02 (m, 1H), 7.01ꢀ6.92 (m, 3H), 4.76 (s,
H), 4.69ꢀ4.63 (m, 1H), 3.78 (s, 3H), 3.20ꢀ2.98 (m, 4H), 2.59 (t, J = 7.0 Hz, 2H), 1.99 (t, J = 7.3
1
1
3
Hz, 2H), 1.50ꢀ1.44 (m, 2H), 1.42ꢀ1.29 (m, 4H), 1.19 (s, 6H), 0.83 (t, J = 7.4 Hz, 3H). C NMR
(
100 MHz, DMSOꢀd ) δ 172.23, 171.71, 166.24, 162.08, 136.48, 129.71, 127.68, 126.61, 124.03,
6
121.38, 118.93, 118.72, 113.84, 111.77, 110.89, 55.77, 54.77, 53.45, 33.89, 29.30, 28.84, 28.13,
+
2
6.60, 25.58, 21.10, 11.98. ESIꢀMS m/z: 536.33[M + H] . (λ ) purity 99.0%. Retention time:
2
54
8
.54 min.
General Procedure for the Preparation of 15a-15c and 17.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 59
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
(S)-N-(1-((4-(2-cyclopentylhydrazine-1-carbonyl)benzyl)amino)-3-(1H-indol-3-yl)-1-oxopro
pan-2-yl)-4-methoxybenzamide (15a)
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
Compound 14 (0.47 g, 1 mmol) was dissolved in 50 mL dichloromethane and to this solution
was added 2ꢀ(1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium tetrafluoroborate (TBTU, 0.35
g, 1.2 mmol) and trimethylamine (0.2 mL, 1.5 mmol). 30 min later, cyclopentylhydrazine (0.12
g, 1.2 mmol) was added. The reaction was allowed to stir overnight, then was washed with brine
(
2 × 30 mL) and dried over MgSO . Volatiles were removed under vacuum and the crude residue
4
purified on combiꢀflash system C18 reverse phase columns eluted with acetonitrile and water to
1
yield the desired product. H NMR (400 MHz, DMSOꢀd ) δ 11.89 (s, 1H), 10.86 (d, J = 1.7 Hz,
6
1
H), 8.76 (t, J = 6.0 Hz, 1H), 8.47 (d, J = 8.0 Hz, 1H), 7.91ꢀ7.84 (m, 4H), 7.70 (d, J = 7.8 Hz,
1H), 7.37ꢀ7.31 (m, 3H), 7.22 (d, J = 2.1 Hz, 1H), 7.08ꢀ7.04 (m, 1H), 7.00ꢀ6.97 (m, 3H),
4
.82ꢀ4.70 (m, 1H), 4.39 (d, J = 5.9 Hz, 2H), 3.96 ꢀ3.85 (m, 1H), 3.80 (s, 3H), 3.33ꢀ3.14 (m, 3H),
1
3
1
.97ꢀ1.64 (m, 6H), 1.62ꢀ1.48 (m, 2H). C NMR (100 MHz, DMSOꢀ d ) δ 172.65, 166.21,
6
165.79, 162.08, 145.32, 136.55, 129.84, 129.18, 128.38, 127.70, 127.48, 126.73, 124.20, 121.33,
18.97, 118.68, 113.79, 111.80, 110.96, 62.23, 55.81, 55.01, 42.33, 28.59, 28.00, 24.40. ESIꢀMS
1
+
m/z: 554.17[M + H] . (λ ) purity 98.4%, Retention time: 8.67 min.
254
(S)-N-(3-(1H-indol-3-yl)-1-oxo-1-((4-(2-phenylhydrazine-1-carbonyl)benzyl)amino)
propan-2-yl)-4-methoxybenzamide (15b)
Using the synthetic method of 15a, compound 14 and phenylhydrazine gave 15b as a white solid,
28
ACS Paragon Plus Environment

Page 29 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
55% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.81 (d, J = 1.8 Hz, 1H), 10.33 (d, J = 3.0 Hz,
6
1
H), 8.71 (t, J = 6.0 Hz, 1H), 8.44 (d, J = 7.9 Hz, 1H), 7.91 (d, J = 2.9 Hz, 1H), 7.85 (d, J = 8.6
Hz, 4H), 7.71 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 3H), 7.23 (d, J = 2.2 Hz, 1H), 7.18ꢀ7.16
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
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
(m, 2H), 7.11ꢀ7.04 (m, 1H), 7.03ꢀ6.95 (m, 3H), 6.79 (d, J = 7.6 Hz, 2H), 6.73 (t, J = 7.3 Hz, 1H),
13
4
.83ꢀ4.72 (m, 1H), 4.39 (d, J = 5.8 Hz, 2H), 3.80 (s, 3H), 3.37ꢀ3.16 (m 2H). C NMR (100
MHz, DMSOꢀd ) δ 172.61, 166.65, 166.23, 162.08, 150.03, 143.81, 136.54, 131.86, 129.83,
6
1
29.22, 127.72, 127.38, 126.74, 124.22, 121.36, 119.04, 118.70, 113.79, 112.78, 111.80, 111.02,
+
5
5.80, 54.90, 42.36, 28.02. ESIꢀMS m/z: 562.17[M + H] . (λ ) purity 96.8%, Retention time:
254
9.75 min.
(
S,E)-N-(1-(4-(3-hydrazinyl-3-oxoprop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)-4
-methoxybenzamide (17)
Using the synthetic method of 15a, compound 16 and hydrazine monohydrate gave 17 as a
+
colorless oil, 55% yield. ESIꢀMS m/z: 485.17[M + H] .
General Procedure for the Preparation of 18.
(
S,E)-N-(1-(1H-indol-3-yl)-3-(4-(3-oxo-3-(2-propylhydrazinyl)prop-1-en-1-yl)
phenoxy)propan-2-yl)-4-methoxybenzamide (18)
Using the synthetic method of 13a, compound 17 and propionaldehyde gave 18 as a white solid,
1
6
5% yield. H NMR (400 MHz, DMSOꢀd ) δ 10.82 (s, 1H), 9.51 (s, 1H), 8.36 (d, J = 7.9 Hz,
6
1H), 7.85 (d, J = 8.9 Hz, 2H), 7.63 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.39 (d, J =
29
ACS Paragon Plus Environment