Discovery of the first N -hydroxycinnamamide-based histone deacetylase 1/3 dual inhibitors with potent oral antitumor activity

Article

pubs.acs.org/jmc

Discovery of the First N‑Hydroxycinnamamide-Based Histone

Deacetylase 1/3 Dual Inhibitors with Potent Oral Antitumor Activity

Xiaoyang Li,^†Elizabeth S. Inks,^‡Xiaoguang Li,^†Jinning Hou,^†C. James Chou,^‡Jian Zhang,^†Yuqi Jiang,^†

Yingjie Zhang,*^,†and Wenfang Xu*^,†

^†Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, P. R. China

^‡Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of South Carolina,

Charleston, South Carolina 29425, United States

S

* Supporting Information

ABSTRACT: In our previous study, we designed and

synthesized a novel series of N-hydroxycinnamamide-based

HDAC inhibitors (HDACIs), among which the representative

compound 14a exhibited promising HDACs inhibition and

antitumor activity. In this current study, we report the

development of a more potent class of N-hydroxycinnama-

mide-based HDACIs, using 14a as lead, among which,

compound 11r gave IC₅₀values of 11.8, 498.1, 3.9, 2000.8,

5700.4, 308.2, and 900.4 nM for the inhibition of HDAC1,

HDAC2, HDAC3, HDAC8, HDAC4, HDAC6, and HDAC11, exhibiting dual HDAC1/3 selectivity. Compounds 11e, 11r, 11w,

and 11y showed excellent growth inhibition in multiple tumor cell lines. In vivo antitumor assay in U937 xenograft model

identiﬁed compound 11r as a potent, orally active HDACI. To the best of our knowledge, this work constitutes the ﬁrst report of

oral active N-hydroxycinnamamide-based HDACIs with dual HDAC1/3 selectivity.

p21 and p27 (cyclin-dependent Kinase inhibitors).⁶A recent

study showed that HDAC1 largely accumulated in mature areas

INTRODUCTION

■

Epigenetics are widely implicated in tumor initiation and

progression by diﬀerent modiﬁcations of DNA and histones.

There is a growing body of evidence demonstrating the

of diﬀerentiated tumor on the mice with teratoma, indicating

that HDAC1 is a possible biomarker of benign teratoma.⁷

HDAC3 is one of the most frequently upregulated genes in

importance of histone modiﬁcation in silencing tumor

human cancers⁸and is involved in each of the three major

suppressor genes.¹Among the various histone modiﬁers,

targets of cancer therapy: cell cycle control, diﬀerentiation, and

histone acetyltransferases (HATs) and histone deacetylases

(HDACs) are two reversible enzymes regulating histone

apoptosis.⁹There are many direct links between HDAC3 and

various tumor types. In colon cancer cells HCT116 and Caco-2,

acetylation status, attaching or removing acetyl groups from

the lysine residues in histone tails.²HATs prevent the positive

silencing of HDAC3 expression resulted in growth inhibition, a

decrease in cell survival, and increased apoptosis through

lysine residues from interacting more closely with the DNA

phosphate backbone, resulting in an “open” chromatin state,

whereas the HDACs remove acetyl groups, resulting in a

“closed” conﬁguration, which blocks the access of the

stimulating p21 promoter activity and expression.¹⁰In HeLa

cells, the majority of cellular HDAC3 is found to associate with

SMRT and N-CoR complexes, and knockdown of HDAC3

resulted in hyperacetylation of histone H3 and apoptosis.^9,11In

transcription machinery to DNA, suppressing gene expression.³

metastatic breast cancer cell MDA-MB-231, HDAC3 eﬃciently

To date, 18 mammalian HDAC isoforms have been

inhibited CREB3-enhanced NF-κB activation. Moreover, high

identiﬁed in humans; 11 of them (Class I, II, IV) are Zn²⁺-

HDAC3 expression is also associated with gastric cancer,¹²

dependent HDAC enzymes (classical HDACs).⁴Class I

Glioma,¹³renal cancer,¹⁴liver cancer,¹⁵and chronic lympho-

HDAC, comprising HDAC 1, 2, 3, and 8, are homologous to

cytic leukemia.¹⁶On the basis of the above evidence,

yeast RPD3 (reduced potassium dependency-3) protein; class II

members include HDAC 4, 5, 6, 7, 9, and 10 and are

structurally related to yeast Hda 1 (histone deacetylases 1).

developing HDACIs more speciﬁcally against HDAC1/3 may

prove to be a worthwhile goal.

In the past 10 years, over 490 clinical trials of more than 20

HDACIs candidates have been initiated, culminating in the

approval of two antitumor drugs vorinostat (SAHA) and

romidepsin (FK228). Recently, development of class or isoform

Class IV HDAC has the sole member, HDAC11. Class III

HDACs, called Sirtuins (SIRT1−7), require NAD⁺for their

activity.⁵

HDAC1 mainly localizes in the nucleus due to lack of the

nucleus export signal. The knockout of HDAC1 in mice caused

proliferation defects and development retardation because of an

arrest of cell growth that is associated with upregulation of the

Received: December 5, 2013

Published: April 2, 2014

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Figure 1. Structure of selective HDACIs.

selective HDACIs has drawn increased attention. Although

selective HDACIs were hypothesized to have fewer side eﬀects

than other pan-HDACIs, their therapeutic advantages have yet

to be conﬁrmed clinically. HDACIs are classiﬁed into diﬀerent

classes depending on their chemical structures, namely,

hydroxamates, benzamides, aliphatic acids, cyclic tetra peptides,

electrophillic ketones, and miscellaneous groups, among which

hydroxamates are considered as the most common HDACIs.

Although some class I selective,^17,18HDAC6 (class IIb)¹⁹

selective, and class IIa²⁰selective hydroxamates inhibitors have

been reported (Figure 1), hydroxamates are generally thought

to have limitation in selectivity against desired HDAC isoforms

due to their very strong chelating ability with zinc ion.²¹To the

best of our knowledge, most of the subclass I selective

inhibitors in research are benzamides HDACIs, such as MS-275

(30), MGCD0103 (31), CI994 (32), and so on (Figure 1).^22,23

Additionally, recent study demonstrated aryl substituents in the

o-phenylenediamine of benzamide HDACIs led to dramatic

improvements in potency and selectivity for HDAC1 and 2

versus HDAC3 (Figure 1).^24,25

KG1, HL60, K562, PC-3, A549, MDA-MB-231, HCT116, and

MCF-7 cell lines. In vivo antitumor assay in U937 xenograft

model showed compound 11r is a potent, orally active HDACI.

RESULTS AND DISCUSSION

■

Chemistry. We designed and synthesized a library of

cinnamamide derivatives based on the lead compound 41 (14a)

(Figure 2). The library was designed to fully explore the

inﬂuence of variation in the methoxy group of ferulic acid and

the cap group. The synthesis of compounds 10a and its

derivatives 11a−11x is described in Scheme 1. Synthesis of 3

was started with ^L-try by methyl ester protection and Boc-

protection. Methyl ester protection of 4-hydroxycinnamic acid

Recently, we have embarked on development of N-

hydroxycinnamamide-based HDACIs; in our previous study,

compound (S,E)-tert-butyl (1-(4-(3-(hydroxyamino)-3-oxo-

prop-1-en-1-yl)-2-methoxyphenoxy)-3-(1H-indol-3-yl)propan-

2-yl)carbamate (14a) exhibited modest HDAC inhibitory.²⁶

Here in this article, we report the design, synthesis, and

biological evaluation of a new series of selective hydroxymates

HDACIs describing their structure−activity relationship (SAR)

studies, isotype-selectivity, and anticancer activities. The

representative compound 11r shows dual selectivity and high

potency toward HDAC1/3. Several compounds show an

excellent eﬀect in proliferation inhibition against U937, HEL,

Figure 2. Modiﬁcation site and general structure of target compounds.

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a

Scheme 1. Synthesis of Compounds 10a−11y

a

Reagents and conditions: (a) CH₃COCl, CH₃OH, 95%; (b) (Boc)₂O, Et₃N, CH₂Cl₂, 90%; (c) LiAlH₄, anhydrous THF, 86%; (d) PPh₃, DEAD,

anhydrous THF, 55%; (e) AcOEt/HCl, 80%; (f) carboxylic acids or Boc-protected amino acids, TBTU, Et₃N, anhydrous CH₂Cl₂(60−65%); (g)

NH₂OH, KOH, anhydrous CH₃OH, (30−40%).

a

Scheme 2. Synthesis of Compound 14

a

Reagents and conditions: (a) CH₃I, KOH, TBABr, H₂O, THF, 50%; (b) PPh₃, DEAD, anhydrous THF, 55%; (c) NH₂OK, anhydrous CH₃OH,

30%.

5 gave 6. Reduction of 3 by LiAlH₄led to 4, which was further

subjected to Mitsunobu reaction with 6 and gave 7. Compound

7 was directly converted to the target hydroxamic acid 10a.

Intermediate 8, the N-deprotected product of 7, reacted with

diﬀerent carboxylic acids or Boc-protected amino acids by

TBTU-mediated amide formation to aﬀord 9b−9z. Com-

pounds 9b−9z were converted to hydroxamic acid compounds

11a−11z by NH₂OK in dry methanol.

Compound 4 was treated with CH₃I and phase transfer

catalyst TBABr in H₂O/THF containing KOH to get the

methyl protected 12 (Scheme 2). Subsequent reaction of 12

with 6 through Mitsunobu reaction in the presence of PPh₃and

DEAD aﬀorded compound 13. Finally, the hydroxamic acid

group was achieved in usual conditions to get compound 14.

Scheme 3 shows the preparation of secondary amine 21a−

21c. Intermediate 8 was mixed with benzaldehyde, 4-

chlorobenzaldehyde, or 4-ﬂuorobenzaldehyde in anhydrous

CH₃OH, respectively, then NaBH₄was added to give 18a−18c.

Boc-protected products 19a−19c were converted to hydroxa-

mic acids 20a−20c. Subsequent deprotection gave the end-

products 21a−21c.

Unlike Scheme 1, Scheme 4 used 3-hydroxycinnamic acid

instead of 4-hydroxycinnamic acid to give another new series of

compounds 27a−27c. The reagents and conditions of Scheme

4 are the same as those in Scheme 1.

HeLa Cell Extract Inhibition of the Target Com-

pounds. In our previous study we found the lead compound

41 displayed modest inhibition toward HDACs.²⁴At the

beginning of our present research, we made an attempt to ﬁnd a

new lead compound by modifying the R₁and R₂groups of 41

(Table 1). We changed the R₂group of 41 by replacing the

methoxy group with H to get compound 10a (Scheme 1) and

changed the R₁group of 10a with a methyl group to get

compound 14 (Scheme 2). We used HeLa cell extract as the

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a

Scheme 3. Synthesis of Compounds 21a−21c

a

Reagents and conditions: (a) benzaldehyde (4-chlorobenzaldehyde or 4-ﬂuorobenzal dehyde), Et₃N, NaBH₄, anhydrous CH₃OH, 85%; (b)

(Boc)₂O, Et₃N, CH₃OH, 65%; (c) NH₂OK, anhydrous CH₃OH, 30−40%; (d) AcOEt/HCl, 70%.

a

Scheme 4. Synthesis of Compounds 27a−27c

a

Reagents and conditions: (a) CH₃COCl, CH₃OH, 95%; (b) PPh₃, DEAD, anhydrous THF, 55%; (c) AcOEt/HCl, 80%; (d) carboxylic acids,

TBTU, Et₃N, anhydrous CH₂Cl₂(60−65%); (e) NH₂OH, KOH, anhydrous CH₃OH, (30−40%).

enzyme source to eﬃciently screen our target compounds. The

result in Table 1 showed that compound 10a exhibited slightly

increased HDAC inhibitory activity compared with 41 and 14.

This indicated that the methoxy group in R₂and methyl group

in R₁were not necessary, so we selected 10a as the lead

compound for further optimization and modiﬁcation.

Table 1. HDAC Inhibition Activity of Compounds 41, 10a,

and 13

In the following work, we designed an analogue to probe the

eﬀect of the N-substituent by replacing the Boc group of 10a

with other functional groups (Scheme 1). First, we synthesized

compounds 11a−11o with diﬀerent substituents including Boc-

protected amino acids, carboxylic acids, sulfo acids, and several

anti-inﬂammatory acids (Table 2). It was obvious that the

compounds with the structure of acid amide substituents were

more potent than those with sulfamide substituents (Table 2).

For example, the IC₅₀value of 11g in HeLa extract was 1703.4

nM, much lower than compound 11h (>5000 nM). The same

a

cpd

R₁

R₂

IC₅₀of HeLa extract (nM)

41

H

CH₃O

H

610.5 20.8

530.7 18.6

600.1 30.7

10a

14

H

CH₃

H

a

Assays were performed in replicate (n ≥ 2); data are shown as mean

SD.

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Table 2. HDAC Inhibition Activity of Compounds 10a and Its Derivatives 11a−11o

a

Assays were performed in replicate (n ≥ 2); data are shown as mean SD.

result was also shown in compounds 11e and 11f. Additionally,

compound 11i was more potent than 11j, which indicated that

a short side alkyl chain might advance HDAC inhibitory

activity. Among compounds 11a−11o, the most potent

compounds, 11e and 11k, with IC₅₀values of 15.4 and 20.8

nM, respectively, were far more potent than SAHA (120.8 nM).

The common element they all share is a simple benzamide as

their N-substituent, so further modiﬁcation focused on

compound 11e.

Next, we synthesized 11p and 11q, which had diﬀerent alkyl

chain lengths relative to 11e. Activity data showed 11e with the

shortest side chain had the best activity, which also agreed with

the conclusion shown in 11i and 11j, that a short side alkyl

chain may promote their activity. Then we synthesized

compounds 11r−11y (Scheme 1) to probe the eﬀect of

substituent in benzene ring of benzamide (Table 3).

Surprisingly, the para-substituted compounds 11r, 11w, 11x,

and 11y showed a marked increase in the HDAC inhibition

potency compared with 11e, the IC₅₀values of 11r, 11w, 11x,

and 11y were 5.6, 6.7, 20.4, and 4.8 nM, respectively, however,

the ortho-substituted, metha-substituted, and disubstituted

compounds 11s, 11t, 11u, and 11v exhibited inferior activity

relative to 11e. The result showed para-substituents on a

benzene ring have an obvious promoting eﬀect on HDAC

inhibition.

Scheme 4 was designed to evaluate the activity of 3-

hydroxycinnamide-based series. We modiﬁed some of the most

potent compounds, 11e, 11r, and 11w, to achieve compounds

27a−27c. The HDAC inhibition result in Table 4 showed

compounds 27a−27c exhibited poor activity compared with

11e, 11r, and 11w, which revealed that 4-hydroxycinnamide-

based series were superior to the 3-hydroxycinnamide-based

series. Then 21a−21c were designed to improve water

solubility and chemical stability by replacing the amide of

11e, 11r, and 11w with a secondary amine. Compounds 21a−

21c showed mild inhibition, and the activity was 15 times lower

than 11e, 11r, and 11w. The possible explanation for why

compounds 11r, 21c, and 27c with similar structure exhibited

quite diﬀerent activity was described in the molecule docking

section.

In Vitro HDAC Isoform-Selectivity of the Compounds.

In order to explore the HDAC isoform selectivity proﬁle, we

chose the most potent compounds 11e, 11p, 11r, 11w, and 11y

described in the above discussion, as well as secondary amine

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Table 3. HDAC Inhibition Activity of Compounds 11e and Its Derivatives 11p−11x

a

Assays were performed in replicate (n ≥ 2); data are shown as mean SD.

analogue 21c and 3-hydroxycinnamide-based analogue 27c to

conduct enzyme inhibitory assays against HDAC1, HDAC2,

HDAC3, and HDAC6. Results presented in Table 5 reveal that

compounds 11e, 11r, 11w, and 11y exhibited dual selectivity

against HDAC1/3, among which compound 11r exhibited the

most obvious dual selectivity, particularly against HDAC3. IC₅₀

value of 11r against HDAC3 was 3.9 nM, which was ∼80-fold

and ∼130-fold lower than that of HDAC6 and HDAC2,

respectively. The positive control, SAHA, had almost no

selectivity toward these HDAC isoforms. To ascertain the

selectivities of our compounds across the broader family of

HDAC isoforms, we next proﬁled representative 11r against

HDAC8 (class I), HDAC4 (class IIa), and HDAC11 (class IV).

Compound 11r also displayed low micromolar activity against

HDAC8, HDAC4, and HDAC11 compared to the low

nanomolar activity against HDAC1 and HDAC3 (Table 6).

These data establish 11r to be a potent and HDAC1/3 dual

selective inhibitor.

In Vitro Antiproliferative Assay. Compounds 11e, 11p,

11r, 11w, and 11y with the most potent HDACs inhibitory

activity were selected to test their antiproliferative activity

against 10 types of solid or hematological tumor cell lines,

which were most frequently used in evaluating HDACIs (Table

7). Data showed cell lines HL60, MCF-7, and A549 had lower

sensitivity with our tested compounds than the other cell lines.

All the tested compounds exhibited much more superior

antiproliferative potency than SAHA against U937, K562, HEL,

KG1, MDA-MB-231, PC-3, and HCT116 cells. Compounds

11e, 11r, 11w, and 11y, which displayed the highest HDAC

enzyme inhibitory potency, also exhibited the best antiprolifer-

ative activity. Among these analogues, compound 11r exhibited

the highest potency, and human leukemic monocyte lymphoma

U937 seemed to be the most sensitive cell line to our HDACIs.

So cell line U937 was chosen for further evaluation.

Furthermore, cytotoxicity of 11r against Human Umbilical

Vein Endothelial Cells (HUVEC) was tested in our lab, and the

IC₅₀value was 61.48

6.17 μM, which revealed our

compounds’ selectivity over nontransformed cells when

compared with tumor cells.

Western Blot Analysis and Flow Cytometry Analysis.

Although the HDAC inhibitory activity of our HDACIs was

conﬁrmed in cell-free assays, the results were not as clear in cell

based assays. In order to investigate the HDAC inhibitory

activity of our HDACIs in cells further, we chose compounds

that were used in evaluating HDAC isoform-selectivity to

conduct Western blot assay in U937 cell. Intracellular levels of

acetylated histoneH3, acetylated histoneH4 (known substrates

for HDAC1, 2, and 3), and acetylated α-tubulin (a known

substrate for HDAC6)²⁷were compared with SAHA and

LBH589 (one of the most potent pan-HDACIs) as reference

compounds (Figure 3). Results show compounds 11e, 11p,

11r, 11w, and 11y can markedly increase the level of acetylated

histoneH3 and acetylated histoneH4, which were much

superior to SAHA, while comparable to LBH589. However,

the levels of acetylated α-tubulin of these compounds,

especially 11r, were much lower than LBH589. These data

demonstrated that some of our compounds showed lower

potency against HDAC6 than class I HDACs, which is in

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Table 4. HDAC Inhibition Activity of Compounds 11e, 11r, 11w, 21a−21c, and 27a−27c

a

Assays were performed in replicate (n ≥ 2); values are shown as mean SD.

a

Table 5. In Vitro Inhibition of HDACs Isoforms of Representative Compounds

IC₅₀of

HDAC1

(nM)

IC₅₀of

HDAC2

(nM)

IC₅₀of

HDAC3

(nM)

IC₅₀of

HDAC6

(nM)

HDAC2/ HDAC3

isoform selectivity

HDAC6/ HDAC3

isoform selectivity

HDAC2/ HDAC1

isoform selectivity

HDAC6/ HDAC1

isoform selectivity

cpd

11e

10.3

13.2

11.8

16.7

6.0

535.5

432.1

498.1

457.0

413.6

498.5

791.2

184.7

14.1

143.0

3.9

142.2

143.7

308.2

101.7

185.6

164.4

293.6

63.0

37.9

3.0

10.1

1.0

51.7

32.6

42.2

27.2

68.7

1.5

13.7

10.8

26.1

6.01

30.8

0.5

11p

11r

127.7

83.1

129.2

1.2

79.0

18.5

58

11w

11y

5.5

3.2

27c

329.9

355.4

34.6

403.2

47.0

90.1

0.4

21c

16.8

2.0

6.2

2.2

0.8

SAHA

0.7

5.3

1.8

a

Assays were performed in replicate (n ≥ 2); the SD values are <20% of the mean.

agreement with the isoform selectivity data observed in cell-free

assays.

an inhibitor of caspase-activated DNase (iCAD), which

liberates the DNase (CAD) to digest chromosomal DNA and

cause cell death.²⁸We can see from the result that our

compounds 11e, 11k, 11r, 11w, and 11y dramatically reduced

the level of procaspase3, which was most likely due to the fact

that procaspase3 was cleaved to the active form (caspase3),

which could promote apoptosis of U937 cells. At the same

time, we performed ﬂow cytometry analysis to conﬁrm the

eﬀect of these compounds in inducing apoptosis. In this assay,

we chose compounds 11r and 11w as representative

As our compounds displayed HDAC1/3 dual selectivity and

some studies reported that knockdown of HDAC1 and

HDAC3 could increase the percent of apoptotic cells in

carcinoma cells,¹¹we also evaluated the level of procaspase3

(inactive form of apoptotic eﬀector caspase3) in U937 cells

(Figure 4). Cleavage of pro-caspases3 results in the production

of an active eﬀector, caspase3, which can cleave essential

structural proteins such as cytokeratins, nuclear lamins, and also

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a

Table 6. In Vitro Inhibition of HDACs Isoforms of 11r

classes of

HDACs

fold selective for fold selective for

isoforms

IC₅₀

11.8

HDAC3

HDAC1

class II

HDAC1

HDAC2

HDAC3

HDAC8

HDAC4

HDAC6

HDAC11

498.1

3.9

127.7

42.2

2000.8

5700.7

308.2

900.4

513.0

1461.7

79.0

169.6

483.1

26.1

class IIa

class IIb

class IV

230.9

76.3

a

Assays were performed in replicate (n ≥ 2); the SD values are <20%

of the mean.

Figure 3. Western blot analysis of acetylated tubulin, acetylated

histone H3, acetylated histone H4, and pro-caspase3 in U937 cell lines

after 24 h treatment with compounds at 1 μM using SAHA (SA) and

LBH589 (LBH) as positive control. Hsp90 was used as a loading

control.

compounds and SAHA as the positive control. Results

demonstrated that 11r, 11w, and SAHA can signiﬁcantly

induce apoptosis in a time-dependent and dose-dependent

manner. At the same concentration (1 μM), 11r and 11w

induced 61.76% and 64.25% cell apoptosis, respectively, which

were much higher than that of SAHA (26.83%). Detailed

activity evaluation and apoptosis mechanism studies are

currently underway in our laboratory.

47%). In the mice group treated by 11r, no signiﬁcant body

weight loss and no evident toxic signs in liver and spleen were

detected (Figures 6 and 7).

Stability of Compounds in Artiﬁcial Gastric Juice,

Artiﬁcial Intestinal Juice, Rat Liver Homogenate, and

Human Plasma. Having demonstrated that 11r had ideal in

vitro activity, we next set out to assess its tumor growth

inhibitory eﬀects in subcutaneous cell lines and primary tumor

derived xenograft models. Before we conducted the in vivo

study, we developed HPLC methods to brieﬂy study the

stability of 11r in artiﬁcial gastric juice, artiﬁcial intestinal juice,

rat liver homogenate, and human plasma. Compound 11r was

incubated under each condition at 37 °C for 24 h, extracted,

and analyzed by HPLC. We observed that 11r was stable in

artiﬁcial gastric juice, artiﬁcial intestinal juice, rat liver

homogenate, and human plasma (Figure 5).

In Vivo Antitumor Activity Assay. Because of the great

stability of 11r, it was feasible to evaluate its in vivo activity

orally. We established a subcutaneous U937 xenograft model

using SAHA as positive control. U937 cells (5 × 10⁶) were

subcutaneously implanted in the right ﬂanks of female nude

mice (BALB/c-nu). When tumor size reached about 100 mm³,

mice were randomized to ﬁve per group and were treated with

compound 11r or SAHA (100 mg/kg/day) by oral gavage for

16 days. Tumor growth inhibition (TGI) and relative

increment ratio (T/C) were calculated at the end of treatment

to reveal the antitumor eﬀects in tumor weight and tumor

volume, respectively. Compound 11r demonstrated potent in

vivo oral antitumor activity with higher TGI value (55.1%) and

lower T/C value (37%) than SAHA (TGI = 32.1%; T/C =

Docking Results. Figure 8a revealed the docked con-

formation of compounds 11r, 21c, and 27c. The docked

conformations of 11r and 21c were very similar in the linker

and ZBG, while they were quite diﬀerent in the aromatic ring

and indole ring of cap group. Moreover, conformation of 27c

was diﬀerent from 11r and 21c, leading to diverse binding

modes shown in Figure 8c−e. Figure 8c,d showed 11r and 21c

had similar docking mode and hydrogen bonds; the greater

potency of 11r might be amenable to the stiﬀening of the

scaﬀold introduced by the amide bond, which forces the

lipophilic p-chlorophenyl moiety to yield closer van der Waals

interactions with the protein rather than projecting itself toward

solvent as in 21c. Furthermore, in cap group, hydrogen bonds

formed between 21c and residues Asp92 and Asp93 had a

longer bond length (2.19 and 1.97 Å) than 11r (1.95 and 1.59

Å), which also might be factors that made 11r more eﬀective

against HDAC3 than 21c. Compared with 11r (Figure 8c),

compound 27c (Figure 8e) could form two diverse hydrogen

bonds with amide N−H of Phe200 and imidazole N−H of

His172 through carbonyl oxygen atom and etheric oxygen

atom, respectively, but at the same time, 27c lost two hydrogen

bonds with Asp92 in the cap group and Tyr298 around zinc

ion. In addition, the carbonyl oxygen of 27c had the same

distance with 11r to zinc ion (2.1 Å), but the hydroxyl oxygen

of 27c had a longer distance to the zinc ion (4.4 Å) than 11r

(1.9 Å), which indicated that 27c could only form a weak

monodentate interaction with zinc, while 11r could form a

Table 7. In Vitro Antiproliferative Activity of Representative Compounds

a

IC₅₀(μM)

cpd

11e

U937

K562

HEL

KG1

HL60

MDA-MB-231

PC-3

MCF-7

HCT116

A549

0.33

0.32

0.16

0.18

0.34

0.73

1.45

0.79

0.68

0.51

1.01

0.89

0.94

3.24

0.20

0.27

0.19

0.16

0.27

0.49

0.39

0.72

0.22

0.24

0.47

0.49

1.59

2.11

1.59

1.69

1.04

1.68

2.17

4.26

0.24

0.41

0.22

0.27

0.15

0.31

1.72

0.33

0.53

0.46

0.51

0.29

0.37

3.57

3.47

2.95

2.68

2.7

0.37

0.57

0.52

0.37

0.22

0.58

2.81

3.39

3.91

2.74

2.96

3.27

3.19

3.9

11p

11r

11w

11y

2.32

3.25

3.78

21c

SAHA

a

Assays were performed in replicate (n ≥ 2); the SD values are <20% of the mean.

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Figure 4. Induction of apoptosis at 12 and 24 h by compounds 11r, 11w, and SAHA in diﬀerent concentrations in U937 cells.

strong bidentate interaction with zinc. This might be the reason

why 27c was much less potent than 11r (Figure 8b).

(3.9 nM), with micromolar or submicromolar IC₅₀values

against HDAC2, HDAC8, HDAC4, HDAC6, and HDAC11. In

addition, a few of the examples of the new series in

antiproliferative study exhibited high potency against several

solid or hematological cells, even though antiproliferative

activity of the compounds seemed inferior to their HDAC

inhibition activity. For example, antiproliferative activity of 11r

was about 10 times higher than SAHA, while its HDAC

inhibition activity was about 100 times higher than SAHA. We

CONCLUSIONS

■

For the ﬁrst time, N-hydroxycinnamamide-based derivatives

have been shown to be potent dual HDAC1/3 selective

HDACIs. The representative compound 11r showed low

nanomolar IC₅₀values against HDAC1 (11.8 nM) and HDAC3

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Figure 5. Stability of compound 11r in artiﬁcial gastric juice, artiﬁcial intestinal juice, rat liver homogenate, and human plasma. Points were achieved

after 0, 1, 2, 4, 6, 8, and 24 h, respectively, and values are shown as mean SD.

potent hydroximates HDACIs in clinical) in U937 cell line. At

the same time, some of the compounds decreased the level of

pro-caspase3, which was consistent with the result of ﬂow

cytometry analysis in inducing apoptosis. Compound 11r was

evaluated for its in vivo antitumor study in a U937 xenograft

mice model and showed higher eﬃcacy compared to the

approved drug SAHA. More detailed studies of the antitumor

proﬁles of these promising compounds are underway in our lab.

Using 11r as lead, continued studies are underway to search for

more promising HDACIs with improved transcellular perme-

ability and isoform selectivity.

EXPERIMENTAL SECTION

Figure 6. Antitumor activity comparison of 11r and SAHA against

U937 human tumor xenografts implanted in mice, expressed as mean

tumor volume. Values are shown as mean SD (5 mice per group).

■

1

Materials and Methods. H NMR spectra were obtained on a

Bruker DRX spectrometer at 300 MHz with TMS as an internal

standard, δ in parts per million, and J in hertz. High-resolution mass

spectrometry was performed by Shandong Analysis and Test Center in

Ji’nan, China. ESI-MS spectra were recorded on an API 4000

spectrometer. All reactions were monitored by TLC using 0.25 mm

silica gel plates (60GF-254). UV light and ferric chloride were used to

visualize the spots. Silica gel was used for column chromatography

puriﬁcation. Flash chromatography was accomplished using the

automated CombiFlash Rf system from Teledyne ISCO and was

done using silica gel of 200−300 mesh. Melting points were

determined on an electrothermal melting point apparatus. All tested

compounds are >95% pure by HPLC analysis, performed on an

Agilent 1100 HPLC instrument using an ODS HYPERSIL column (5

μm, 4.6 mm × 250 mm) according to one of the following methods.

Method A: compounds 10a, 11a−11x, and 27a−27c were eluted with

35% acetonitrile/65% water (containing 0.4% formic acid)−65%

acetonitrile/35% water (containing 0.4% formic acid) over 20 min,

with detection at 290 nm and a ﬂow rate of 1 mL/min. Method B:

compounds 21a-21c were eluted with 30% acetonitrile−70% 0.05 M

potassium dihydrogen phosphate/phosphoric acid (PH = 3.0) over 20

min, with detection at 290 nm and a ﬂow rate of 1 mL/min. The

temperature of the column was 25 °C, and quantity of injection was 20

μM.

Figure 7. Picture of dissected U937 tumor tissues.

supposed 11r had poor transcellular permeability; this

speculation was conﬁrmed by parallel artiﬁcial membrane

permeation assay (PAMPA)²⁹conducted on 11r and SAHA

(data not shown). Future work to improve potency should

focus on enhancing permeability of 11r.

Compound 11e, 11r, 11w, and 11y increased histone H3 and

H4 acetylation in the same level of LBH589 (one of the most

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Figure 8. (a) Docked conformational alignment modeling of compounds 11r, 21c, and 27c. For clarity, HDAC3 (derived by modiﬁcation of PDB

code 4A69 using Tripos SYBYL 8.0) used for docking is not shown. (b) Proposed docked mode of 27c and 11r for binding zinc. (c−e) Proposed

binding preferences comparison of compounds 11r (c), 21c (d), and 27c (e) at HDAC3. The zinc ion is shown as a red sphere, and the dashed lines

stand for the hydrogen bonds (atom types: H, white; N, blue; O, red).

(S)-Methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride

(2), (S)-Methyl 2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)-

propanoate(3), and (S)-tert-butyl(1-hydroxy-3-(1H-indol-3-yl)-

propan-2-yl)carbamate (4) were synthesized as described previously.²⁴

General Procedure for the Preparation of 6 and 23. (E)-

Methyl 3-(4-hydroxyphenyl)acrylate. (6). p-Coumaric acid (16.4g,

100mmol) was dissolved in 200 mL of MeOH, then acetyl chloride

(24g, 300mmol) was added dropwise at 0 °C; the mixed solution was

reﬂuxed at 75 °C for 5 h. The solvent was evaporated under vacuum;

the product was dissolved with EtOAc, washed by saturated NaHCO₃

solution (2 × 100 mL), 1 M aqueous citric acid (2 × 100 mL), and

brine (2 × 100 mL), dried over MgSO₄overnight, and evaporated

under vacuum to obtained product compound 6, a white solid (15.6g,

87.5%). Mp: 140−141 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 3.69 (s,

3H), 6.43 (d, J = 16.2 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H), 7.54−7.59

(m, 3H), 10.01 (s, 1H). ESI-MS m/z: 179.2 [M + H]⁺.

(E)-Methyl 3-(4-Hydroxyphenyl)acrylate. (23). Mp: 90−91 °C. H

1

NMR (300 MHz, DMSO-d₆) δ 3.72 (s, 1H), 6.54 (d, J = 15.9 Hz,

1H), 6.83−6.86 (m, 1H), 7.04−7.05 (m, 1H), 7.15 (d, J = 7.5 Hz,

1H), 7.20−7.25 (m, 1H), 7.60 (d, J = 15.9 Hz, 1H), 7.62 (s, 1H). ESI-

MS m/z: 179.2 [M + H]⁺.

General Procedure for the Preparation of 12. tert-Butyl (1-

Hydroxy-3-(1-methyl-1H-indol-3-yl)propan-2-yl)carbamate (12).

Compound 4 (2.9g, 10mmol) was taken up in THF (50 mL), then

(CH₃CH₂CH₂CH₂)₄N(Br) (TBABr, 0.5g) was added followed by

CH₃I (3.5g, 25mmol). The reaction was stirred overnight, THF was

evaporated under vacuum, and the residue was extracted with EtOAc

(3 × 50 mL). The organic layer was washed with saturated NaHCO₃

solution (2 × 100 mL), 1 M aqueous citric acid (2 × 100 mL), and

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brine (2 × 100 mL) and dried over MgSO₄overnight. EtOAc was

evaporated under vacuum. The crude product was puriﬁed by

recrystallization with EtOAc and petroleum ether. The desired

compound 12 was white solid (1.6g, 54% yield). Mp: 122−124 °C.

¹H NMR (300 MHz, DMSO-d₆) δ 1.35 (s, 9H), 2.60−2.73 (m, 1H),

2.84−2.91 (m, 1H), 3.22−3.40 (m, 2H), 3.58−3.67 (m, 1H), 3.72 (s,

3H), 4.64 (t, J = 5.4 Hz, 1H), 6.53 (d, J = 8.1 Hz, 1H), 7.69−7.07 (m,

2H), 7.15 (t, J = 7.8 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 7.8

Hz, 1H). ESI-MS m/z: 305.5 [M + H]⁺.

(E)-Methyl 3-(4-((S)-2-((R)-2-((tert-Butoxycarbonyl)amino)-3-hy-

droxypropanamido)-3-(1H-indol-3-yl)propoxy)phenyl)acrylate (9c).

Colorless oil crystallized upon standing, 75% yield.

(E)-Methyl 3-(4-((2S)-2-((2R)-2-((tert-Butoxycarbonyl)amino)-3-

hydroxybutanamido)-3-(1H-indol-3-yl)propoxy)phenyl)acrylate

(9d). Colorless oil crystallized upon standing, 70% yield.

(S,E)-Methyl 3-(4-(2-Benzamido-3-(1H-indol-3-yl)propoxy)-

phenyl)acrylate (9e). Colorless oil crystallized upon standing, 78%

yield.

(S,E)-Methyl 3-(4-(2-([1,1′-Biphenyl]-4-ylcarboxamido)-3-(1H-

indol-3yl)propoxy)phenyl)acrylate (9g). Colorless oil crystallized

upon standing, 76% yield. ESI-MS m/z: 531.4 [M + H]⁺.

(S,E)-Methyl 3-(4-(2-([1,1′-Biphenyl]-4-ylsulfonamido)-3-(1H-

indol-3-yl)propoxy)phenyl)acrylate (9h). White solid, 76% yield.

Mp: 148−150 °C. ESI-MS m/z: 567.5 [M + H]⁺.

(S,E)-Methyl 3-(4-(2-(2-Ethylbutanamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (9i). Colorless oil crystallized upon standing,

70% yield. ESI-MS m/z: 449.5 [M + H]⁺.

General Procedure for the Preparation of 7, 13, and 24.

(S,E)-Methyl 3-(4-(2-((tert-Butoxycarbonyl)amino)-3-(1H-indol-3yl)-

propoxy)phenyl)acrylate (7). Compound 4 (5.8g, 20mmol), 6 (4.2g,

24mmol), and PPh₃(7.9g, 30mmol) were dissolved in anhydrous

THF, and then DEAD (5.2g, 30mmol) was added dropwise at 0 °C;

the mixture was stirred for 4 h, and then THF was evaporated. The

crude material was puriﬁed via ﬂash chromatography to aﬀord the

1

compound 7, a white solid (5.4g, 60%). Mp: 110−112 °C. H NMR

(300 MHz, DMSO-d₆) δ 1.36 (s, 9H), 2.85−3.00 (m, 2H), 3.70 (s,

3H), 3.95−4.07 (m, 3H), 6.51 (d, J = 15.9 Hz, 1H), 6.93−6.99 (m,

4H), 7.08 (t, J = 7.4 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H), 7.34 (d, J = 7.8

Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.66 (d, J

= 8.4 Hz, 2H), 10.82 (d, J = 1.5 Hz, 1H). ESI-MS m/z: 451.5 [M +

H]⁺.

(S,E)-Methyl 3-(4-(3-(1H-Indol-3-yl)-2-(2-propylpentanamido)-

propoxy)phenyl)acrylate (9j). White pure solid, 70% yield. Mp:

1

170−172 °C. H NMR (300 MHz, DMSO-d₆) δ 0.73−0.80 (m, 6H),

0.96−1.00 (m, 2H), 1.04−1.11 (m, 4H), 1.30−1.42 (m, 2H), 2.10−

2.17 (m, 1H), 2.86−3.03 (m, 2H), 3.70 (s, 3H), 3.98−4.07 (m, 2H),

4.34−4.42 (m, 1H), 6.51 (d, J = 15.9 Hz, 1H), 6.92−6.95 (m, 3H),

7.07 (t, J = 7.5 Hz, 1H), 7.14 (d, J = 2.4 Hz, 1H), 7.33 (d, J = 7.8 Hz,

1H), 7.58 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 17.0 Hz, 1H), 7.67 (d, J =

9.0 Hz, 2H), 7.94 (d, J = 7.8 Hz, 1H), 10.82 (d, J = 1.5 Hz, 1H). ESI-

MS m/z: 485.5 [M + H]⁺.

(S,E)-Methyl 3-(4-(2-((tert-Butoxycarbonyl)amino)-3-(1-methyl-

1H-indol-3yl)propoxy) phenyl)acrylate (13). Colorless oil crystallized

upon standing, 50% yield.

(S,E)-Methyl 3-(3-(2-((tert-Butoxycarbonyl)amino)-3-(1H-indol-3-

yl)propoxy)phenyl)acrylate (24). Colorless oil crystallized upon

standing, 50% yield. ESI-MS m/z: 451.4 [M + H]⁺.

(S,E)-Methyl 3-(4-(2-(2-Hydroxybenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (9k). Colorless oil crystallized upon stand-

ing, 74% yield.

General Procedure for the Preparation of 8 and 25. (S,E)-

Methyl 3-(4-(2-Amino-3-(1H-indol-3-yl)propoxy)phenyl)acrylate Hy-

drochloride (8). Compound 7 was dissolved in a solution of EtOAc (8

mL) saturated by dry HCl gas. The solution was stirred at room

temperature overnight. The ﬁltered precipitate was washed by diethyl

ether to give the compound 8, a white solid powder (3.8g, 83%). Mp:

(S,E)-Methyl 3-(4-(2-(2-((2,3-Dimethylphenyl)amino)benzamido)-

3-(1H-indol-3-yl)propoxy)phenyl)acrylate (9l). White pure solid, 76%

yield. Mp: 84−86 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 2.08 (s, 3H),

3.27 (s, 3H), 3.08−3.16 (m, 2H), 3.70 (s, 3H), 4.11−4.22 (m, 2H),

4.55−4.64 (m, 1H), 6.49 (d, J = 15.9 Hz, 1H), 6.74 (t, J = 7.8 Hz, 1H),

6.83 (d, J = 7.8 Hz, 1H), 6.91−6.99 (m, 4H), 7.03−7.09 (m, 3H),

7.20−7.26 (m, 2H), 7.34 (d, J = 8.1 Hz, 1H), 7.57−7.64 (m, 5H), 8.57

(d, J = 7.8 Hz, 1H), 9.39 (s, 1H), 10.84 (d, J = 1.5 Hz, 1H). ESI-MS

m/z: 574.5 [M + H]⁺.

(E)-Methyl 3-(4-((2S)-3-(1H-Indol-3-yl)-2-(2-(4-isobutylphenyl)-

propanamido)propoxy)phenyl)acrylate (9m). Colorless oil crystal-

lized upon standing, 74% yield. ESI-MS m/z: 539.5 [M + H]⁺.

(E)-Methyl 3-(4-((2S)-2-(2-(4-Benzoylphenyl)propanamido)-3-

(1H-indol-3yl)propoxy)phenyl)acrylate (9n). Colorless oil crystallized

upon standing, 73% yield. ESI-MS m/z: 563.4 [M + H]⁺.

(E)-Methyl 3-(4-((S)-3-(1H-Indol-3-yl)-2-((S)-2-(6-methoxynaph-

thalen-2-yl)propanamido)propoxy)phenyl)acrylate (9o). Colorless

oil crystallized upon standing, 70% yield. ESI-MS m/z: 587.3 [M +

H]⁺.

1

198−200 °C. H NMR (300 MHz, DMSO-d₆) δ 3.10−3.24 (m, 2H),

3.71 (s, 3H), 3.71−3.75 (m, 1H), 4.07 (dd, J = 5.7 Hz, J = 10.5 Hz,

1H), 4.21 (dd, J = 5.7 Hz, J = 10.5 Hz, 1H), 6.53 (d, J = 15.9 Hz, 1H),

6.97−7.01 (m, 3H), 7.11 (t, J = 6.9 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H),

7.38 (d, J = 8.1 Hz, 1H), 7.59−7.64 (m, 2H), 7.70 (d, J = 8.7 Hz, 2H),

8.40 (s, 2H), 11.05 (s, 1H). ESI-MS m/z: 351.5 [M + H]⁺.

(S,E)-Methyl 3-(3-(2-Amino-3-(1H-indol-3-yl)propoxy)phenyl)-

acrylate Hydrochloride (25). Pale yellow oil. ESI-MS m/z: 351.5

[M + H]⁺.

General Procedure for the Preparation of 9a−9y and 26a−

2 6 c . ( S , E ) - M e t h y l 3 - ( 4 - ( 3 - ( 1 H - I n d o l - 3 - y l ) - 2 - ( 4 -

methylphenylsulfonamido)propoxy)phenyl)acrylate (9f). To a sol-

ution of p-toluene sulfonic acid (0.34g, 2mmol) in anhydrous CH₂Cl₂

was added 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tet-

raﬂuoroborate (TBTU, 0.71g, 2.2mmol), followed by Et₃N (0.3g,

3mmol). After 30 min, compound 8 (0.85g, 2.2mmol) was added

followed by TEA (0.3g, 3mmol). Four hours later, the solution of

CH₂Cl₂was washed with 1 N HCl (2 × 30 mL), saturated Na₂CO₃(2

× 30 mL), and brine (2 × 30 mL), dried over MgSO₄overnight, and

the solvent was evaporated under vacuum. The crude product was

puriﬁed by recrystallization with EtOAc and petroleum ether to

(S,E)-Methyl 3-(4-(3-(1H-Indol-3-yl)-2-(2-phenylacetamido)-

propoxy)phenyl)acrylate (9p). White pure solid, 60% yield. Mp:

1

154−156 °C. H NMR (300 MHz, DMSO-d₆) δ 2.90−3.06 (m, 2H),

3.43 (s, 2H), 3.71 (s, 3H), 4.01 (d, J = 5.4 Hz, 2H), 4.27−4.35 (m,

1H), 6.52 (d, J = 15.9 Hz, 1H), 6.91−6.96 (m, 3H), 7.08 (t, J = 7.5 Hz,

1H), 7.10 (d, J = 2.1 Hz, 1H), 7.17−7.27 (m, 5H), 7.34 (d, J = 8.1 Hz,

1H), 7.57 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 16.2 Hz, 1H), 7.67 (d, J =

8.7 Hz, 2H), 8.30 (d, J = 7.8 Hz, 1H), 10.83 (d, J = 1.5 Hz, 1H). ESI-

MS m/z: 470.5 [M + H]⁺.

1

achieve a white pure solid (0.51g, 50% yield). Mp: 72−74 °C. H

NMR (300 MHz, DMSO-d₆) δ 2.30 (s, 3H), 2.85 (dd, J = 6.0 Hz, J =

14.4 Hz, 1H), 3.01 (dd, J = 8.1 Hz, J = 14.4 Hz, 1H), 3.60−3.65 (m,

1H), 3.70 (s, 3H), 3.86 (d, J = 4.8 Hz, 2H), 6.50 (d, J = 16.2 Hz, 1H),

6.76 (d, J = 8.7 Hz, 2H), 6.91 (t, J = 7.4 Hz, 1H), 7.00−7.06 (m, 2H),

7.20 (d, J = 8.1 Hz, 2H), 7.24−7.30 (m, 2H), 7.56−7.62 (m, 5H), 7.99

(d, J = 7.2 Hz,1H), 10.77 (d, J = 1.5 Hz, 1H). ESI-MS m/z: 505.4 [M

+ H]⁺.

(S,E)-Methyl 3-(4-(3-(1H-Indol-3-yl)-2-(3-phenylpropanamido)-

propoxy)phenyl)acrylate (9q). Colorless oil crystallized upon stand-

ing, 70% yield.

(S,E)-Methyl 3-(4-(2-(4-Chlorobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate(9r). Colorless oil crystallized upon standing,

75% yield. ESI-MS m/z: 489.4 [M + H]⁺.

(S,E)-Methyl 3-(4-(2-(2-Chlorobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (9s). Colorless oil crystallized upon stand-

ing, 78% yield.

(S,E)-Methyl 3-(4-(2-(2-((tert-Butoxycarbonyl)amino)acetamido)-

3-(1H-indol-3-yl)propoxy)phenyl)acrylate (9a). Colorless oil crystal-

lized upon standing, 70% yield.

(S,E)-Methyl 3-(4-(2-(3-Chlorobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (9t). Colorless oil crystallized upon stand-

ing, 77% yield.

(E)-Methyl 3-(4-((S)-2-((R)-2-((tert-Butoxycarbonyl)amino)-4-

(methylthio)butanamido)-3-(1H-indol-3-yl)propoxy)phenyl)-

acrylate (9b). Colorless oil crystallized upon standing, 73% yield.

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(S,E)-Methyl 3-(4-(2-(2,4-Dichlorobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (9u). Colorless oil crystallized upon stand-

ing, 70% yield.

(S,E)-Methyl 3-(4-(2-(3,4-Dichlorobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (9v). Colorless oil crystallized upon stand-

ing, 68% yield.

(S,E)-Methyl 3-(4-(2-(4-Fluorobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (9w). Colorless oil crystallized upon stand-

ing, 80% yield. ESI-MS m/z: 477.4 [M + H]⁺.

(S,E)-Methyl 3-(4-(2-(4-Bromobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (9x). Colorless oil crystallized upon stand-

ing, 70% yield.

(S,E)-Methyl 3-(3-(2-Benzamido-3-(1H-indol-3-yl)propoxy)-

phenyl)acrylate (26a). Colorless oil crystallized upon standing, 65%

yield.

(S,E)-Methyl 3-(3-(2-(4-Chlorobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (26b). Colorless oil crystallized upon

standing, 73% yield.

(S,E)-Methyl 3-(4-(2-(4-Fluorobenzamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (26c). Colorless oil crystallized upon

standing, 70% yield.

343mmol) were dissolved, respectively, in 70 and 120 mL of MeOH to

get solution A and solution B. Next, solution A was added dropwise to

solution B. After ﬁltering the precipitate (KCl), a NH₂OK solution was

obtained. Compound 7 (0.45g, 1 mmol) was dissolved in the NH₂OK

solution and stirred overnight. After the reaction was complete, it was

evaporated under vacuum. The residue was acidiﬁed with 1 N HCl to a

pH 3−4 and then extracted with EtOAc (3 × 20 mL). The organic

layer was washed with brine (2 × 30 mL) and dried over Na₂SO₄

overnight. The crude material was puriﬁed via ﬂash chromatography to

1

aﬀord the compound 10a (0.15g, 30% yield). Mp: 151−152 °C. H

NMR (300 MHz, DMSO-d₆) δ 1.37 (s, 9H), 2.79−3.00 (m, 2H),

3.90−4.04 (m, 3H), 6.33 (d, J = 15.6 Hz, 1H), 6.92−7.00 (m, 4H),

7.08 (td, J = 0.9 Hz, J = 7.6 Hz, 1H), 7.13 (d, J = 1.8 Hz, 1H), 7.34 (d,

J = 7.8 Hz, 1H), 7.42 (d, J = 15.6 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H),

7.56 (d, J = 7.8 Hz, 1H), 8.97 (s, 1H), 10.66 (s, 1H), 10.83 (d, J = 1.8

Hz, 1H). HRMS (AP-ESI) m/z calcd for C₂₅H₂₉N₃O₅[M + H]⁺

452.2180, found 452.2182. Retention time: 18.9 min, eluted with 40%

acetonitrile/60% water (containing 0.4% formic acid).

(S,E)-tert-Butyl(2-((1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)amino)-2-oxoethyl)-

1

carbamate (11a). Pale yellow solid, 34% yield. Mp: 72−74 °C. H

General Procedure for the Preparation of 18a−18c. (S,E)-

Methyl 3-(4-(2-(Benzylamino)-3-(1H-indol-3-yl)propoxy)phenyl)-

acrylate (18a). The solution of compound 8 (0.76g, 2mmol) in

CH₃OH was added Et₃N (0.22g, 2.2mmol) for neutralization, followed

by the addition of methanol (0.21g, 2mmol); the resulting solution

was stirred for 2 h and the solvent evaporated. The residue was

dissolved in anhydrous CH₃OH, NaBH₄(0.15g, 4mmol) was added

slowly at 0 °C, the mixture was stirred at room temperature overnight,

and after the reaction ﬁnished, the reaction was quenched by adding

ice water slowly. CH₃OH was evaporated, and the residue was

extracted by EtOAc (3 × 30 mL). The organic layer was washed with

brine (2 × 30 mL) and saturated Na₂CO₃(2 × 30 mL) and dried over

MgSO₄overnight. The solvent was evaporated under vacuum to get

NMR (300 MHz, DMSO-d₆) δ 1.39 (s, 9H), 2.85−3.10 (m, 2H),

3.48−3.57 (m, 2H), 3.96 (m, 2H), 4.31−4.35 (m, 1H), 6.33 (d, J =

15.9, 1H), 6.99−6.90 (m, 4H), 7.08 (t, J = 6.9 Hz, 1H), 7.14 (d, J = 2.1

Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 15.9 Hz, 1H), 7.49 (d, J

= 8.4 Hz, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 8.98

(s, 1H), 10.66 (s, 1H), 10.85 (d, J = 1.8 Hz, 1H). HRMS (AP-ESI) m/

z calcd for C₂₇H₃₂N₄O₆[M + H]⁺509.2395, found 509.2392.

Retention time: 7.4 min, eluted with 40% acetonitrile/60% water

(containing 0.4% formic acid).

tert-Butyl((R)-1-(((S)-1-(4-((E)-3-(hydroxyamino)-3-oxoprop-1-en-

1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)amino)-4-(methylthio)-

1-oxobutan-2-yl)carbamate (11b). Pale yellow solid, 30% yield. Mp:

1

108−110 °C. H NMR (300 MHz, DMSO-d₆) δ 1.37 (s, 9H), 1.68−

1

crude product 11a, a colorless oil (0.79g, 90%). H NMR (300 MHz,

1.82 (m, 2H), 2.42 (t, J = 7.8 Hz, 2H), 2.97−3.04 (m, 2H), 3.36 (s,

3H), 3.90−4.05 (m, 3H), 4.25−4.42 (m, 1H), 6.32 (d, J = 15.6 Hz,

1H), 6.90−6.97 (m, 4H), 7.08 (t, J = 6.9 Hz, 1H), 7.20 (d, J = 2.1 Hz,

1H), 7.34 (d, J = 7.8 Hz, 1H), 7.41 (d, 15.9 Hz, 1H), 7.48 (d, 8.4 Hz,

2H), 7.60 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H), 8.97 (s, 1H),

10.66 (s, 1H), 10.85 (s, 1H). HRMS (AP-ESI) m/z calcd for

C₃₀H₃₈N₄O₆S [M + H]⁺583.2585, found 583.2486. Retention time:

10.55 min, eluted with 47% acetonitrile/53% water (containing 0.4%

formic acid).

DMSO-d₆) δ 2.95 (d, J = 6.3 Hz, 2H), 3.14−3.18 (m, 1H), 3.70 (s,

3H), 3.87 (s, 2H), 3.94 (d, J = 5.1 Hz, 2H), 6.50 (d, J = 15.9 Hz, 1H),

6.92 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 7.06 (t, J = 7.4 Hz,

1H), 7.14 (d, J = 2.4 Hz, 1H), 7.16−7.22 (m, 1H), 7.26−7.33 (m,

5H), 7.43 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.65 (d, J =

8.7 Hz, 2H), 8.99 (s, 1H), 10.83 (s, 1H). ESI-MS m/z: 441.5 [M +

H]⁺.

(S,E)-Methyl 3-(4-(2-((4-Fluorobenzyl)amino)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (18b). Colorless oil, 85% yield.

(S,E)-methyl 3-(4-(2-((4-Chlorobenzyl)amino)-3-(1H-indol-3-yl)-

propoxy)phenyl)acrylate (18c). Colorless oil, 88% yield.

tert-Butyl((R)-3-hydroxy-1-(((S)-1-(4-((E)-3-(hydroxyamino)-3-ox-

oprop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)amino)-1-

oxopropan-2-yl)carbamate (11c). Pale yellow solid, 34% yield. Mp:

General Procedure for the Preparation of 19a−19c. (S,E)-

Methyl 3-(4-(2-(Benzyl(tert-butoxycarbonyl)amino)-3-(1H-indol-3-

yl)propoxy)phenyl)acrylate (19a). Et₃N (0.61g, 6mmol) followed

by (Boc)₂O (0.78g, 3.6mmol) were added to the solution of

compound 11a (0.79g, 1.8mmol) in CH₂Cl₂. The reaction was stirred

at room temperature overnight. The resulting solution was washed by

saturated NaHCO₃solution (2 × 30 mL), 1 M aqueous citric acid (2

× 30 mL), and brine (2 × 30 mL) and dried over MgSO₄overnight.

The solvent was evaporated under vacuum to get crude product 12a, a

1

118−120 °C. H NMR (300 MHz, DMSO-d₆) δ 1.36 (s, 9H), 2.91−

3.05 (m, 2H), 3.51−3.54 (m, 2H), 3.93−4.02 (m, 3H), 4.31−4.38 (m,

1H), 4.84 (t, J = 6.3 Hz, 1H), 6.33 (d, J = 15.6 Hz, 1H), 6.65 (d, 8.1

Hz, 1H), 6.92−6.97 (m, 3H), 7.08 (t, J = 6.9 Hz, 1H), 7.16 (d, J = 2.1

Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 15.9 Hz, 1H), 7.49 (d, J

= 8.4 Hz, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 8.98

(s, 1H), 10.66 (s, 1H), 10.85 (d, J = 1.5 Hz, 1H). HRMS (AP-ESI) m/

z calcd for C₂₈H₃₄N₄O₇[M + H]⁺539.2500, found 539.2496.

Retention time: 5.3 min, eluted with 40% acetonitrile/60% water

(containing 0.4% formic acid).

1

colorless oil (0.92g, 95%). H NMR (300 MHz, DMSO-d₆) δ 1.43−

1.47 (m, 9H), 2.90−3.16 (m, 2H), 3.70 (s, 3H), 4.00−4.07 (m, 2H),

4.31−4.37 (m, 2H), 4.56 (m, 1H), 6.49 (d, J = 16.2 Hz, 1H), 6.77−

6.80 (m, 2H), 6.98 (t, J = 7.8 Hz, 1H), 7.09 (m, 2H), 7.12−7.17 (m,

1H), 7.20−7.24 (m, 4H), 7.35 (d, J = 7.8 Hz, 1H), 7.41−7.47 (m,

1H), 7.56−7.64 (m, 3H), 10.84 (s, 1H). ESI-MS m/z: 541.4 [M +

H]⁺.

tert-Butyl((2R)-3-hydroxy-1-(((S)-1-(4-((E)-3-(hydroxyamino)-3-

oxoprop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)amino)-

1-oxobutan-2-yl)carbamate (11d). Pale yellow solid, 29% yield. Mp:

1

102−104 °C. H NMR (300 MHz, DMSO-d₆) δ 1.01 (d, J = 5.7 Hz,

3H), 1.37 (s, 9H), 2.92−3.05 (m, 2H), 3.86−3.94 (m, 4H), 4.34−4.36

(m, 1H), 4.78 (d, J = 5.4 Hz, 1H), 6.27−6.35 (m, 2H), 6.91−6.97 (m,

3H), 7.08 (t, J = 7.2 Hz, 1H), 7.16 (d, J = 1.8 Hz, 1H), 7.31−7.48 (m,

4H), 7.60 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 8.96 (s, 1H),

10.65 (s, 1H), 10.83 (s, 1H). HRMS (AP-ESI) m/z calcd for

C₂₉H₃₆N₄O₇[M + H]⁺553.2657, found 553.2658. Retention time: 6.5

min, eluted with 40% acetonitrile/60% water (containing 0.4% formic

acid).

(S,E)-Methyl 3-(4-(2-((tert-Butoxycarbonyl)(4-ﬂuorobenzyl)-

amino)-3-(1H-indol-3-yl) propoxy)phenyl)acrylate (19b). Colorless

oil, 90% yield.

(S,E)-Methyl 3-(4-(2-((tert-Butoxycarbonyl)(4-chlorobenzyl)-

amino)-3-(1H-indol-3-yl) propoxy)phenyl)acrylate (19c). Colorless

oil, 91% yield.

General Procedure for the Preparation of 10a, 11a−11x, 14,

20a−20c, and 27a−27c. (S,E)-tert-Butyl(1-(4-(3-(hydroxyamino)-3-

oxoprop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)-

carbamate (10a). KOH (28g, 509mmol) and NH₂OH·HCl (23.35g,

(S,E)-N-(1-(4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)phenoxy)-

3-(1H-indol-3-yl)propan-2-yl)benzamide (11e). Pale yellow solid,

31% yield. Mp: 164−166 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 3.12

3336

dx.doi.org/10.1021/jm401877m | J. Med. Chem. 2014, 57, 3324−3341

Journal of Medicinal Chemistry

Article

(d, J = 6.6 Hz, 2H), 4.04−4.21 (m, 2H), 4.54−4.65 (m, 1H), 6.33 (d, J

= 15.9 Hz, 1H), 6.93−6.99 (m, 3H), 7.08 (td, J = 0.9 Hz, J = 7.4 Hz,

1H), 7.19 (d, J = 2.1 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.42 (d, J =

15.9 Hz, 1H), 7.43−7.55 (m, 5H), 7.64 (d, J = 7.8 Hz, 1H), 7.85 (d, J

= k7.5 Hz, 2H), 8.53 (d, J = 7.8 Hz, 1H), 8.98 (s, 1H), 10.66 (s, 1H),

10.83 (d, J = 1.8 Hz, 1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₅N₃O₄

[M + H]⁺456.1918, found 456.1916. Retention time: 12.6 min, eluted

with 40% acetonitrile/60% water (containing 0.4% formic acid).

(S,E)-3-(4-(3-(1H-Indol-3-yl)-2-(4-methylphenylsulfonamido)-

propoxy)phenyl)-N-hydroxyacrylamide (11f). Pale yellow solid, 34%

478.2699, found 478.2699. Retention time: 24.5 min, eluted with 40%

acetonitrile/60% water (containing 0.4% formic acid).

(S,E)-2-Hydroxy-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-

yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11k). Pale

yellow solid, 35% yield. Mp: 120−122 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 3.14 (d, J = 6.9, 2H), 4.08−4.22 (m, 2H), 4.59−4.70 (m,

1H), 6.33 (d, J = 15.9 Hz, 1H), 6.86−6.91 (m, 2H), 6.96−7.00 (m,

3H), 7.09 (t, J = 7.4 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H), 7.35 (d, J = 8.1

Hz, 1H), 7.37−7.42 (m, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 7.8

Hz, 1H), 7.92 (dd, J = 1.5 Hz, J = 8.1 Hz, 1H), 8.87 (d, J = 8.1 Hz,

1H), 8.98 (s, 1H), 10.66 (s, 1H), 10.85 (d, J = 1.8 Hz, 1H), 12.37 (s,

1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₅N₃O₅[M + H]⁺472.1867,

found 472.1868. Retention time: 17.2 min, eluted with 40%

acetonitrile/60% water (containing 0.4% formic acid).

1

yield. Mp: 118−120 °C. H NMR (300 MHz, DMSO-d₆) δ 2.31 (s,

3H), 2.85 (dd, J = 6.0 Hz, J = 14.4 Hz, 1H), 3.01 (dd, J = 8.1 Hz, J =

14.4 Hz, 1H), 3.60−3.62 (m, 1H), 3.84 (d, J = 4.8 Hz, 2H), 6.32 (d, J

= 15.9 Hz, 1H), 6.76 (d, J = 8.7 Hz, 2H), 6.92 (td, J = 0.9 Hz, J = 7.4

Hz, 1H), 7.00−7.06 (m, 2H), 7.21 (d, J = 8.1 Hz, 2H), 7.27 (d, J =

10.2 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.41 (d, J = 15.9 Hz, 1H), 7.44

(d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 6.9 Hz, 1H),

8.99 (s, 1H), 10.66 (s, 1H), 10.78 (d, J = 1,8 Hz, 1H). HRMS (AP-

ESI) m/z calcd for C₂₇H₂₇N₃O₅S [M + H]⁺506.1715, found

506.1744. Retention time: 22.9 min, eluted with 40% acetonitrile/

60% water (containing 0.4% formic acid).

(S,E)-2-((2,3-Dimethylphenyl)amino)-N-(1-(4-(3-(hydroxyamino)-

3-oxoprop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)-

1

benzamide (11l). Pale yellow solid, 33% yield. Mp: 126−128 °C. H

NMR (300 MHz, DMSO-d₆) δ 2.08 (s, 3H), 2.27 (s, 3H), 3.05−3.12

(m, 2H), 4.08−4.20 (m, 2H), 4.55−4.67 (m, 1H), 6.32 (d, J = 15.9

Hz, 1H), 6.74 (t, J = 7.5H, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.91−6.98

(m, 4H), 7.03−7.09 (m, 3H), 7.23−7.26 (m, 2H), 7.34 (d, J = 8.1 Hz,

1H), 7.41 (d, J = 15.9 Hz, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.64 (t, J =

7.2 Hz, 2H), 8.58 (d, J = 8.1 Hz, 1H), 8.97 (s, 1H), 9.40 (s, 1H), 10.65

(s, 1H), 10.84 (d, J = 1.5 Hz, 1H). HRMS (AP-ESI) m/z calcd for

C₃₅H₃₄N₄O₄[M + H]⁺534.1023, found 575.2653. Retention time:

10.2 min, eluted with 40% acetonitrile/60% water (containing 0.4%

formic acid).

(S,E)-N-(1-(4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)phenoxy)-

3-(1H-indol-3-yl)propan-2-yl)-[1,1′-biphenyl]-4-carboxamide (11g).

1

Pale yellow solid, 30% yield. Mp: 174−176 °C. H NMR (300 MHz,

DMSO-d₆) δ 3.14 (d, J = 6.9 Hz, 2H), 3.99−4.12 (m, 1H), 4.18−4.28

(m, 1H), 4.56−4.67 (m, 1H), 6.33 (d, J = 15.9 Hz, 1H), 6.94−7.00

(m, 3H), 7.09 (t, J = 7.2 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 7.35 (d, J =

8.1 Hz, 1H), 7.37−7.43 (m, 2H), 7.47−7.52 (m, 4H), 7.66 (d, J = 7.8

Hz, 1H), 7.72−7.78 (m, 4H), 7.96 (d, J = 8.4 Hz, 2H), 8.60 (d, J = 6.9

Hz, 1H), 10.67 (s, 1H), 10.84 (s, 1H). HRMS (AP-ESI) m/z calcd for

C₃₃H₂₉N₃O₄[M + H]⁺532.2192, found 532.2231. Retention time: 5.0

min, eluted with 40% acetonitrile/60% water (containing 0.4% formic

acid).

(E)-3-(4-((2S)-3-(1H-Indol-3-yl)-2-(2-(4-isobutylphenyl)-

propanamido)propoxy)phenyl)-N-hydroxyacrylamide (11m). Pale

yellow solid, 30% yield. Mp: 116−118 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 0.81−0.86 (m, 6H), 1.24−1.31 (m, 3H), 1.71−1.85 (m,

1H), 2.35−2.41 (m, 2H), 2.84−3.05 (m, 2H), 3.59−3.62 (m, 1H),

3.92 (m, 1H), 3.96−3.98 (m, 1H), 4.26−4.36 (m, 1H), 6.33 (dd, J =

4.5 Hz, J = 15.9 Hz, 1H), 6.84−7.08 (m, 6H), 7.13−7.19 (m, 3H),

7.42−7.61 (m, 5H), 8.14 (t, J = 6.4 Hz, 1H), 8.97 (s, 1H), 10.66 (s,

1H), 10.83 (s, 1H). HRMS (AP-ESI) m/z calcd for C₃₃H₃₇N₃O₄[M +

H]⁺540.2857, found 540.2856. Retention time: 7.7 min, eluted with

40% acetonitrile/60% water (containing 0.4% formic acid).

(E)-3-(4-((S)-3-(1H-Indol-3-yl)-2-((S)-2-(6-methoxynaphthalen-2-

yl)propanamido)propoxy)phenyl)-N-hydroxyacrylamide (11o). Pale

yellow solid, 29% yield. Mp: 134−136 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.35−1.42 (m, 3H), 2.89−3.08 (m, 2H), 3.74−3.80 (m,

1H), 3.85−3.86 (m, 3H), 3.92−4.00 (m, 2H), 4.29−4.39 (m, 1H),

6.31 (d, J = 15.9 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.94−7.09 (m,

3H), 7.11−7.51 (m, 8H), 7.62 (d, J = 7.8 Hz, 1H), 7.67−7.75 (m,

3H), 8.23 (d, J = 7.8 Hz, 1H), 8.98 (s, 1H), 10.66 (s, 1H), 10.85 (s,

1H). HRMS (AP-ESI) m/z calcd for C₃₆H₃₃N₃O₅[M + H]⁺564.2493,

found 564.2495.

(S,E)-3-(4-(2-([1,1′-Biphenyl]-4-ylsulfonamido)-3-(1H-indol-3-yl)-

propoxy)phenyl)-N-hydroxyacrylamide (11h). Pale yellow solid, 28%

yield. Mp: 167−169 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 2.85−2.91

(m, 1H), 2.98−3.05 (m, 1H), 3.64−3.75 (m, 1H), 3.82−3.91 (m, 1H),

6.29 (d, J = 15.9 Hz, 1H), 6.75 (d, J = 8.7 Hz, 2H), 6.91 (t, J = 7.8 Hz,

1H), 7.01 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 2.1 Hz, 1H), 7.24 (d, J = 8.1

Hz, 1H), 7.30−7.45 (m, 5H), 7.48−7.53 (m, 2H), 7.64−7.68 (m, 4H),

7.77 (d, J = 8.4 Hz, 2H), 8.17 (d, J = 7.2 Hz, 1H), 8.99 (s, 1H), 10.65

(s, 1H), 10.80 (d, J = 1.8 Hz, 1H). HRMS (AP-ESI) m/z calcd for

C₃₂H₂₉N₃O₅S [M + H]⁺568.1861, found 568.1901. Retention time:

5.1 min, eluted with 65% acetonitrile/35% water (containing 0.4%

formic acid).

(S,E)-2-Ethyl-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)butanamide (11i). Pale yel-

low solid, 31% yield. Mp: 170−172 °C. ¹H NMR (300 MHz, DMSO-

d₆) δ 0.69 (t, J = 7.2 Hz, 3H), 0.79 (t, J = 7.5 Hz, 3H), 1.23−1.47 (m,

4H), 1.91−2.02 (m, 1H), 2.90−3.04 (m, 2H), 3.99 (d, J = 5.4 Hz,

2H), 4.36−4.44 (m, 1H), 6.33 (d, J = 15.9 Hz, 1H), 6.95 (d, 8.4 Hz,

2H), 6.98 (td, J = 0.9 Hz, J = 7.1 Hz, 1H), 7.08 (td, J = 0.9 Hz, J = 7.6

Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.42 (d, J

= 15.6 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.97

(d, J = 8.1 Hz, 1H), 8.97 (s, 1H), 10.66 (s, 1H), 10.82 (d, J = 1.5 Hz,

1H). HRMS (AP-ESI) m/z calcd for C₂₆H₃₁N₃O₄[M + H]⁺450.2387,

found 450.2389. Retention time: 10.9 min, eluted with 40%

acetonitrile/60% water (containing 0.4% formic acid).

(S,E)-N-(1-(4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)phenoxy)-

3-(1H-indol-3-yl)propan-2-yl)-2-propylpentanamide (11j). Pale yel-

low solid, 27% yield. Mp: 171−172 °C. ¹H NMR (300 MHz, DMSO-

d₆) δ 0.73−0.80 (m, 6H), 1.97−1.20 (m, 6H), 1.30−1.46 (m, 2H),

2.12−2.18 (m, 1H), 2.86−3.03 (m, 2H), 3.98 (d, J = 5.1 Hz, 2H),

4.34−4.42 (m, 1H), 6.33 (d, J = 15.9 Hz, 1H), 6.95 (d, J = 8.7 Hz,

2H), 6.98 (td, J = 0.9 Hz, J = 6.8 Hz, 1H), 7.08 (td, J = 0.9 Hz, J = 7.2

Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.42 (d, J

= 15.6 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.63

(d, J = 9.0 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H), 10.66 (s, 1H), 10.83 (d, J

= 1.5 Hz, 1H). HRMS (AP-ESI) m/z calcd for C₂₈H₃₅N₃O₄[M + H]⁺

(S,E)-3-(4-(3-(1H-Indol-3-yl)-2-(2-phenylacetamido)propoxy)-

phenyl)-N-hydroxyacrylamide (11p). Pale yellow solid, 29% yield.

1

Mp: 154−156 °C. H NMR (300 MHz, DMSO-d₆) δ 2.90−3.06 (m,

2H), 3.43 (s, 2H), 3.99 (d, J = 5.1 Hz, 2H), 4.27−4.38 (m, 1H), 6.34

(d, J = 15.9 Hz, 1H), 6.91−6.97 (m, 3H), 7.08 (td, J = 0.9, J = 7.4 Hz,

1H), 7.11 (d, J = 2.1 Hz, 1H), 7.17−7.28 (m, 5H), 7.35 (d, J = 8.1 Hz,

1H), 7.42 (d, J = 15.9 Hz, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.58 (d, J =

7.8 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 8.99 (s, 1H), 10.66 (s, 1H),

10.84 (d, J = 1.5 Hz, 1H). HRMS (AP-ESI) m/z calcd for C₂₈H₂₇N₃O₄

[M + H]⁺470.2074, found 470.2074. Retention time: 12.8 min, eluted

with 40% acetonitrile/60% water (containing 0.4% formic acid).

(S,E)-3-(4-(3-(1H-Indol-3-yl)-2-(3-phenylpropanamido)propoxy)-

phenyl)-N-hydroxyacrylamide (11q). Pale yellow solid, 30% yield.

1

Mp: 166−168 °C. H NMR (300 MHz, DMSO-d₆) δ 2.41 (t, J = 8.1

Hz, 2H), 2.81 (t, J = 7.8 Hz, 2H), 2.85−3.02 (m, 2H), 3.94 (d, J = 5.1

Hz, 2H), 4.28−4.38 (m, 1H), 6.33 (d, J = 15.9 Hz, 1H), 6.96 (d, J =

8.7 Hz, 2H), 6.07 (t, J = 6.9 Hz, 1H), 7.03−7.09 (m, 2H), 7.13−7.35

(m, 5H), 7.34 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 15.9 Hz, 1H), 7.50 (d, J

= 8.7 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 8.99

(s, 1H), 10.67 (s, 1H), 10.83 (d, J = 1.8 Hz, 1H). HRMS (AP-ESI) m/

z calcd for C₂₉H₂₉N₃O₄[M + H]⁺484.2236, found 484.2231.

3337

dx.doi.org/10.1021/jm401877m | J. Med. Chem. 2014, 57, 3324−3341

Journal of Medicinal Chemistry

Article

Retention time: 17.4 min, eluted with 40% acetonitrile/60% water

(containing 0.4% formic acid).

+ H]⁺474.1824, found 474.1824. Retention time: 12.0 min, eluted

with 40% acetonitrile/60% water (containing 0.4% formic acid).

(S,E)-4-Bromo-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11x). Pale yel-

low solid, 32% yield. Mp: 182−184 °C. ¹H NMR (300 MHz, DMSO-

d₆) δ 3.11 (d, J = 6.9 Hz, 2H), 4.06−4.20 (m, 2H), 4.51−4.61 (m,

1H), 6.32 (d, J = 15.6 Hz, 1H), 6.93−6.98 (m, 3H), 7.08 (td, J = 0.9, J

= 7.8 Hz, 1H), 7.17 (d, J = 2.1 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.41

(d, J = 15.6 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 9.3 Hz,

1H), 7.69 (d, J = 1.8 Hz, 2H), 7.81 (d, J = 1.8 Hz, 2H), 8.63 (d, J = 7.8

Hz, 1H), 8.98 (s, 1H), 10.66 (s, 1H), 10.83 (d, J = 1.8 Hz, 1H).

HRMS (AP-ESI) m/z calcd for C₂₇H₂₄BrN₃O₄[M + H]⁺534.1023,

found 534.1021.

(S,E)-4-Chloro-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11r). Pale yel-

low solid, 32% yield. Mp: 182−184 °C. ¹H NMR (300 MHz, DMSO-

d₆) δ 3.11 (d, J = 6.6 Hz, 2H), 4.06−4.11 (m, 1H), 4.14−4.20 (m,

1H), 4.54−4.61 (m, 1H), 6.32 (d, J = 15.6 Hz, 1H), 6.93−6.98 (m,

3H), 7.08 (t, J = 6.9 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H), 7.34 (d, J = 7.8

Hz, 1H), 7.42 (d, J = 15.9 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.55 (d, J

= 8.7 Hz, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 8.63

(d, J = 7.8 Hz, 1H), 8.98 (s, 1H), 10.66 (s, 1H), 10.83 (d, J = 1.5 Hz,

1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₄ClN₃O₄[M + H]⁺

490.1526, found 490.1528. Retention time: 6.0 min, eluted with

40% acetonitrile/60% water (containing 0.4% formic acid).

(S,E)-2-Chloro-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11s). Pale yel-

low solid, 35% yield. Mp: 113−115 °C. ¹H NMR (300 MHz, DMSO-

d₆) δ 3.00−3.13 (m, 2H), 4.10 (d, J = 5.4 Hz, 2H), 4.49−4.57 (m,

1H), 6.33 (d, J = 15.9 Hz, 1H), 6.95−7.00 (m, 3H), 7.10 (t, J = 7.4 Hz,

1H), 7.22 (d, J = 2.1 Hz, 1H), 7.29 (dd, J = 1.8 Hz, J = 7.5 Hz, 1H),

7.32−7.40 (m, 3H), 7.42−7.50 (m, 4H), 7.63 (d, J = 7.8 Hz, 1H), 8.66

(d, J = 8.1 Hz, 1H), 8.98 (s, 1H), 10.67 (s, 1H), 10.87 (d, J = 1.8 Hz,

1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₄ClN₃O₄[M + H]⁺

490.1528, found 490.1528. Retention time: 10.8 min, eluted with

40% acetonitrile/60% water (containing 0.4% formic acid).

(S,E)-3-Chloro-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11t). Pale yel-

low solid, 33% yield. Mp: 148−149 °C. ¹H NMR (300 MHz, DMSO-

d₆) δ 3.13 (d, J = 6.9 Hz, 2H), 4.06−4.11 (m, 1H), 4.14−4.21 (m,

1H), 4.54−4.65 (m, 1H), 6.48 (d, J = 15.9 Hz, 1H), 6.94−6.99 (m,

2H), 7.03−7.08 (m, 1H), 7.12−7.19 (m, 2H), 7.27−7.32 (m, 2H),

7.34−7.55 (m, 4H), 7.65 (d, J = 7.8 Hz, 1H), 7.83−7.85 (m, 2H), 8.53

(d, J = 8.1 Hz, 1H), 9.06 (s, 1H), 10.72 (s, 1H), 10.83 (s, 1H). HRMS

(AP-ESI) m/z calcd for C₂₇H₂₄ClN₃O₄[M + H]⁺490.1528, found

490.1528. Retention time: 18.8 min, eluted with 40% acetonitrile/60%

water (containing 0.4% formic acid).

(S,E)-2,4-Dichloro-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-

yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11u). Pale

yellow solid, 29% yield. Mp: 159−160 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 3.03−3.16 (m, 2H), 4.10−4.19 (m, 2H), 4.51−4.60 (m,

1H), 6.33 (d, J = 15.6 Hz, 1H), 6.93−6.99 (m, 3H), 7.08 (t, J = 6.9 Hz,

1H), 7.18 (d, J = 2.1 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.42 (d, J =

15.9 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.81

(d, J = 1.8 Hz, 2H), 7.83 (d, J = 1.8 Hz, 1H), 8.77 (d, J = 7.8 Hz, 1H),

8.97 (s, 1H), 10.66 (s, 1H), 10.85 (d, J = 1.5 Hz, 1H). HRMS (AP-

ESI) m/z calcd for C₂₇H₂₃Cl₂N₃O₄[M + H]⁺534.1023, found

524.1138. Retention time: 10.1 min, eluted with 51% acetonitrile/49%

water (containing 0.4% formic acid).

(S,E)-3,4-Dichloro-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-

yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11v). Pale

yellow solid, 30% yield. Mp: 162−164 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 3.00−3.13 (m, 2H), 4.08−4.10 (m, 2H), 4.47−4.58 (m,

1H), 6.33 (d, J = 15.9 Hz, 1H), 6.95−6.99 (m, 3H), 7.10 (t, J = 7.2 Hz,

1H), 7.21 (d, J = 2.1 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.1

Hz, 1H), 7.42 (d, J = 15.9 Hz, 1H), 7.48 (dd, J = 2.1 Hz, J = 8.1 Hz,

1H), 7.50 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 2.1

Hz, 1H), 8.72 (d, J = 8.1 Hz, 1H), 8.99 (s, 1H), 10.66 (s, 1H), 10.86

(s, 1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₃Cl₂N₃O₄[M + H]⁺

524.1138, found 524.1138. Retention time: 14.2 min, eluted with 43%

acetonitrile/57% water (containing 0.4% formic acid).

(S,E)-4-Fluoro-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11w). Pale yel-

low solid, 27% yield. Mp: 180−182 °C. ¹H NMR (300 MHz, DMSO-

d₆) δ 3.11 (d, J = 6.9 Hz, 2H), 4.05−4.20 (m, 2H), 4.52−4.60 (m,

1H), 6.32 (d, J = 15.9 Hz, 1H), 6.93−6.99 (m, 3H), 7.08 (t, J = 6.9 Hz,

1H), 7.18 (d, J = 2.1 Hz, 1H), 7.26−7.33 (m, 3H), 7.41 (d, J = 15.9

Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.89−7.94

(m, 2H), 8.56 (d, J = 7.8 Hz, 1H), 8.98 (s, 1H), 10.66 (s, 1H), 10.83

(d, J = 1.5 Hz, 1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₄FN₃O₄[M

(S,E)-4-Methoxy-N-(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-

yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (11y). Mp:

1

180−182 °C. H NMR (300 MHz, DMSO-d₆) δ 3.10 (d, J = 6.9

Hz, 2H), 3.80 (s, 3H), 4.02−4.20 (m, 2H), 4.52−4.63 (m, 1H), 6.33

(d, J = 15.9 Hz, 1H), 6.93−7.00 (m, 5H), 7.08 (td, J = 0.9 Hz, J = 7.4

Hz, 1H), 7.17 (d, J = 2.1 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.42 (d, J

= 15.9 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 7.8 Hz, 1H), 7.85

(d, J = 8.7 Hz, 2H), 8.38 (d, J = 8.1 Hz, 1H), 8.98 (s, 1H), 10.66 (s,

1H), 10.82 (d, J = 1.5 Hz, 1H). HRMS (AP-ESI) m/z calcd for

C₂₈H₂₇N₃O₅[M + H]⁺486.2023, found 486.2023. Retention time:

10.1 min, eluted with 47% acetonitrile/53% water (containing 0.4%

formic acid).

(S,E)-tert-Butyl(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1-methyl-1H-indol-3-yl)propan-2-yl)carbamate (14).

Pale yellow solid, 33% yield. Mp: 146−148 °C. ¹H NMR (300

MHz, DMSO-d₆) δ 1.36 (s, 9H), 2.83−2.99 (m, 2H), 3.72 (s, 3H),

3.93−4.04 (m, 3H), 6.33 (dd, J = 4.5 Hz, J = 15.9 Hz, 1H), 6.95 (d, J =

8.4 Hz, 2H), 6.97−7.02 (m, 2H), 7.10−7.15 (m, 2H), 7.36−7.42 (m,

2H), 7.49 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 8.97 (s, 1H),

10.66 (s, 1H). HRMS (AP-ESI) m/z calcd for C₂₆H₃₁N₃O₅[M + H]⁺

466.2336, found 466.2332. Retention time: 13.8 min, eluted with 47%

acetonitrile/53% water (containing 0.4% formic acid).

(S,E)-tert-Butylbenzyl(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-

yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)carbamate (20a). A pale

yellow solid, 90% yield. ¹H NMR (300 MHz, DMSO-d₆) δ 1.15−1.25

(m, 9H), 2.90−3.06 (m, 2H), 3.97−4.06 (m, 2H), 4.31−4.40 (m, 2H),

4.56 (s, 1H), 6.31 (d, J = 15.6 Hz, 1H), 6.76−6.79 (m, 2H), 6.98 (t, J

= 7.2 Hz, 1H), 7.01−7.07 (m, 2H), 7.09−7.25 (m, 5H), 7.32−7.43 (m,

5H), 8.96 (s, 1H), 10.64 (s, 1H), 10.84 (s, 1H). ESI-MS m/z: 541.4

[M + H]⁺.

(S,E)-tert-Butyl 4-Chlorobenzyl(1-(4-(3-(hydroxyamino)-3-oxo-

prop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)carbamate

(20b). A pale yellow oil, 80% yield.

(S,E)-tert-Butyl 4-Fluorobenzyl(1-(4-(3-(hydroxyamino)-3-oxo-

prop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)carbamate

(20c). A pale yellow oil, 85% yield.

(S,E)-N-(1-(3-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)phenoxy)-

3-(1H-indol-3-yl)propan-2-yl)benzamide (27a). A pale yellow

1

powder, 30% yield. Mp: 104−106 °C. H NMR (300 MHz, DMSO-

d₆) δ 3.15 (d, J = 8.4 Hz, 2H), 4.06−4.11 (m, 1H), 4.16−4.22 (m,

1H), 4.54−4.64 (m, 1H), 6.48 (d, J = 15.6 Hz, 1H), 6.94−6.99 (m,

2H), 7.08 (t, J = 7.6 Hz, 1H), 7.12−7.14 (m, 2H), 7.19 (d, J = 2.4 Hz,

1H), 7.30 (d, J = 8.1 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.38 (d, J =

15.9 Hz, 1H), 7.42−7.54 (m, 3H), 7.65 (d, J = 7.8 Hz, 1H), 7.82−7.85

(m, 2H), 8.52 (d, J = 8.1 Hz, 1H), 9.05 (s, 1H), 10.71 (s, 1H), 10.82

(d, J = 1.5 Hz, 1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₅N₃O₄[M +

H]⁺456.1918, found 456.1919. Retention time: 11.5 min, eluted with

46% acetonitrile/54% water (containing 0.4% formic acid).

(S,E)-4-Flouro-N-(1-(3-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (27b). A pale

yellow powder, 28% yield. Mp: 96−98 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 3.05−3.19 (m, 2H), 4.05−4.11 (m, 1H), 4.13−4.20 (m,

1H), 4.52−4.61 (m, 1H), 6.48 (d, J = 15.6 Hz, 1H), 6.94−6.98 (m,

2H), 7.08 (t, J = 7.2 Hz, 1H), 7.11−7.18 (m, 3H), 7.26−7.34 (m, 4H),

7.42 (d, J = 15.9 Hz, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.89−7.94 (m,

2H), 8.54 (d, J = 7.8 Hz, 1H), 9.04 (s, 1H), 10.70 (s, 1H), 10.80 (s,

1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₄FN₃O₄[M + H]⁺

474.1824, found 474.1825.

3338

dx.doi.org/10.1021/jm401877m | J. Med. Chem. 2014, 57, 3324−3341

Journal of Medicinal Chemistry

Article

(S,E)-4-Chloro-N-(1-(3-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

Western Blot Analysis. Brieﬂy, U937 cells with diﬀerent

treatment were collected and lysed with lysis buﬀer (20 mM Tris-

HCl [pH 7.5], 1% NP-40, 1 mM sodium vanadate, 1 mM EDTA, 1

mM EGTA, 50 mM NaF, and 1 mM phenylmethyl sulfonylﬂuoride

[PMSF]) for 30 min, then centrifuged for 15 min at 14 000 rpm at 4

°C, and the supernatant was the whole-cell extracts. Total protein

extracts (30 μg per lane) were separated by 12% SDS polyacrylamide

gel electrophoresis and transferred onto PVDF membranes (Cat.

IPVH00010, Millipore). Membrane was blocked with 5% S36 milk in

TBS-T (10 mM Tris [pH 7.4], 150 mM NaCl, and 0.1% Tween 20)

for 1 h at room temperature, then incubated with a 1:1000 or 1:2000

dilution of primary antibody overnight at 4 °C. Then the membrane

was washed three times and incubated at 1:2000 dilution of antimouse

or antirabbit goat-HRP-conjugated secondary antibodies for 2 h at

room temperature. Finally the membrane was washed another three

times and developed by enhanced chemiluminescence (ECL, Cat.

WBKLS0050, Millipore).

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)benzamide (27c). A pale

1

yellow powder, 33% yield. Mp: 126−127 °C. H NMR (300 MHz,

DMSO-d₆) δ 3.09−3.12 (m, 2H), 4.06−4.20 (m, 2H), 4.54−4.61 (m,

1H), 6.48 (d, J = 15.9 Hz, 1H), 6.94−6.99 (m, 2H), 7.08 (t, J = 7.4 Hz,

1H), 7.12−7.14 (m, 2H), 7.18 (d, J = 2.1 Hz, 1H), 7.27−7.34 (m,

2H), 7.42 (d, J = 15.6 Hz, 1H), 7.55 (d, J = 8.7 Hz, 2H), 7.64 (d, J =

7.5 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 8.62 (d, J = 7.8 Hz, 1H), 9.05 (s,

1H), 10.71 (s, 1H), 10.82 (d, J = 1.5 Hz, 1H). HRMS (AP-ESI) m/z

calcd for C₂₇H₂₄ClN₃O₄[M + H]⁺456.1918, found 456.1919.

Retention time: 17.2 min, eluted with 40% acetonitrile/60% water

(containing 0.4% formic acid).

General Procedure for the Preparation of 21a−21c. (S,E)-tert-

Butylbenzyl(1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-

phenoxy)-3-(1H-indol-3-yl)propan-2-yl)carbamate (21a). This

method was the same as the general procedure for the preparation

of 8 and 25.

1

Apoptosis Assay. U937 cells (1.5 × 10⁵) were treated with

diﬀerent concentrations of 11r, 11w, or SAHA for 12 or 24 h. Cells

were harvested following incubation, washed twice in cold PBS,

centrifuged, and resuspended in 1× annexin-binding buﬀer. Cells were

diluted in 1× annexin-binding buﬀer to 1 × 10⁶cells/mL, preparing a

suﬃcient volume to have 100 μL per assay. Five microliters of Alexa

Fluor 488 annexin V and 1 μL of 100 μg/mL PI were added to each

100 μL of cell suspension. Cells were incubated at room temperature

for 15 min. After the incubation period, 150 μL of 1× annexin-binding

buﬀer was added, mixed gently, and kept on ice. The stained cells were

analyzed by ﬂow cytometry, measuring the ﬂuorescence emission at

530 and 575 nm (or equivalent) using 488 nm excitation. Alexa Fluor

488 annexin V/Dead Cell Apoptosis Kit with Alexa Fluor 488 annexin

V and PI for Flow Cytometry was used for this assay (Invitrogen).

Stability of Representitive Compound in Artiﬁcial Gastric

Juice, Artiﬁcial Intestinal Juice, Rat Liver Homogenate, and

Human Plasma. Preparation of artiﬁcial gastric juice: 160 mL of

water and 2 g of pepsin were added to 3.28 mL of diluted hydrochloric

acid. After mixing, the mixture was diluted to 200 mL with water.

Preparation of artiﬁcial intestinal juice: 1.36 g of sodium dihydrogen

phosphate was dissolved in 100 mL of water, and the pH was adjusted

to 6.8 using 0.4% NaOH. Then 2 g of pancreatin was added, and the

mixture was diluted to 200 mL with water. Preparation of rat liver

homogenate: Wistar rats (Experimental Animal Center, Shandong

University, female, 200 20 g) were sacriﬁced after fasting 12 h. Liver

was homogenized in quadruple phosphate butter (pH 7.4) and

centrifuged (4000 rpm), 10 min. The liver homogenate was stored at

−20 °C. Human plasma was obtained from Institution of Clinical

Pharmacology, Qilu Hospital. Artiﬁcial gastric juice, artiﬁcial intestinal

juice, rat liver homogenate, and human plasma were preincubated at

37 °C prior to the addition of 50 μL of stock solution of mutual

prodrugs (1 mg/mL in CH₃OH). Sample aliquots were taken for

processing and analysis after 24 h incubation at 37 °C. All of the

samples underwent extraction using 600 μL acetonitrile and were

ﬁltered (0.22 μm) after shocking 30 s and centrifugation at 12 000

rpm, 10 min. Chromatographic analysis was achieved by HPLC

method on an Agilent 1100 HPLC instrument using a ODS

HYPERSIL column (5 μm, 4.6 mm × 100 mm) according the

following method: compound was eluted with 42% acetonitrile/58%

water (containing 0.4% formic acid) over 20 min, with detection at

290 nm and a ﬂow rate of 1 mL/min. The temperature of the column

was 25 °C, and the quantity of injection was 20 μM.

A pale yellow oil, 65% yield. Mp: 136−138 °C. H NMR (300

MHz, DMSO-d₆) δ 3.17−3.27 (m, 1H), 3.41−3.47 (m, 1H), 3.67 (s,

1H), 4.12−4.17 (m, 1H), 4.25−4.27 (m, 1H), 4.36 (s, 2H), 6.38 (d, J

= 15.6 Hz, 1H), 6.91−6.99 (m, 3H), 7.10 (t, J = 7.2 Hz, 1H), 7.22 (d, J

= 2.4 Hz, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.42−7.50 (m, 6H), 7.64−

7.68 (m, 2H), 9.72 (s, 1H), 9.92 (s, 1H), 10.31 (s, 1H), 11.05 (d, J =

1.8 Hz, 1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₇N₃O₃[M + H]⁺

442.2125, found 442.2125. Retention time: 12.5 min.

(S,E)-tert-Butyl 4-Fluorobenzyl(1-(4-(3-(hydroxyamino)-3-oxo-

prop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)carbamate

1

(21b). A pale yellow oil, 68% yield. Mp: 138−140 °C. H NMR (300

MHz, DMSO-d₆) δ 3.17−3.26 (m, 1H), 3.41−3.48 (m, 1H), 3.70 (s,

1H), 4.12−4.17 (m, 1H), 4.25−4.28 (m, 1H), 4.36 (s, 2H), 6.39 (d, J

= 15.9 Hz, 1H), 6.92−6.99 (m, 3H), 7.10 (t, J = 6.9 Hz, 1H), 7.23 (d, J

= 2.1 Hz, 1H), 7.29 (d, J = 8.7 Hz, 2H), 7.35−7.42 (m, 2H), 7.48−

7.53 (m, 3H), 7.69−7.74 (m, 2H), 9.74 (s, 1H), 9.94 (s, 1H), 11.05 (d,

J = 1.8 Hz, 1H). HRMS (AP-ESI) m/z calcd for C₂₇H₂₆FN₃O₃[M +

H]⁺460.2031, found 460.2033. Retention time: 14.2 min.

(S,E)-tert-Butyl 4-Chlorobenzyl(1-(4-(3-(hydroxyamino)-3-oxo-

prop-1-en-1-yl)phenoxy)-3-(1H-indol-3-yl)propan-2-yl)carbamate

1

(21c). A pale yellow oil, 60% yield. Mp: 136−138 °C. H NMR (300

MHz, DMSO-d₆) δ 3.17−3.26 (m, 1H), 3.41−3.47 (m, 2H), 4.11−

4.16 (m, 1H), 4.24−4.27 (m, 1H), 4.37 (s, 2H), 6.38 (d, J = 15.9 Hz,

1H), 6.92−6.99 (m, 3H), 7.11 (t, J = 7.2 Hz, 1H), 7.23 (d, J = 2.1 Hz,

1H), 7.37 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 15.6 Hz, 1H), 7.48−7.53

(m, 5H), 7.70 (d, J = 8.4 Hz, 2H), 9.75 (s, 1H), 9.69 (s, 1H), 10.30 (s,

1H), 11.05 (d, J = 1.8 Hz, 1H). HRMS (AP-ESI) m/z calcd for

C₂₇H₂₆ClN₃O₃[M + H]⁺476.1735, found 476.1735. Retention time:

21.3 min.

In Vitro HDACs Inhibition Fluorescence Assay. In vitro

HDACs inhibition assays were conducted as previously described.²²

In brief, 10 μL of enzyme solution (HeLa nuclear extract, HDAC1,

HDAC2, HDAC3. or HDAC6) was mixed with various concentrations

of tested compound (50 μL). The mixture was incubated at 37 °C for

5 min, then luorogenic substrate Boc-Lys(acetyl)-AMC (40 μL) was

added. After incubation at 37 °C for 30 min, the mixture was stopped

by the addition of 100 μL of developer containing trypsin and TSA.

Twenty minutes later, ﬂuorescence intensity was measured using a

microplate reader at excitation and emission wavelengths of 390 and

460 nm, respectively. The inhibition ratios were calculated from the

ﬂuorescence intensity readings of tested wells relative to those of

control wells, and the IC₅₀values were calculated using a regression

analysis of the concentration/inhibition data.

In Vitro Antiproliferative Assay. All cell lines were maintained in

RPMI1640 medium containing 10% FBS at 37 °C in a 5% CO₂

humidiﬁed incubator. Cell proliferation assay was determined by the

MTT method. Brieﬂy, cells were passaged the day before dosing into a

96-well cell plate, allowed to grow for 12 h and then treated with

diﬀerent concentrations of compound sample for 48 h. A 0.5% MTT

solution was added to each well. After incubation for another 4 h,

formazan formed from MTT was extracted by adding 150 μL of

DMSO rocking for 15 min. Absorbance was then determined using an

ELISA reader at 570 nm.

In Vivo Antitumor Assay against U937 Xenograft. For in vivo

antitumor eﬃcacy research, 1 × 10⁷human histiocytic lymphoma cells

(U937) were inoculated subcutaneously in the right ﬂank of male

athymic nude mice (5−6 weeks old, SLAC LABORATORY ANIMAL,

Shanghai). Ten days after injection, tumors were palpable, and mice

were randomized into treatment and control groups (5 mice per

group). The treatment groups received compound 11r (100 mg/kg/d)

or SAHA (100 mg/kg/d) by oral administration, and the blank control

group received equal volume of PBS solution containing 40% DMSO.

During treatment, subcutaneous tumors were measured with a vernier

caliper every three days, and body weight was monitored regularly.

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After treatment, mice were sacriﬁced and dissected to weigh the tumor

tissues and to examine the internal organ injury. Tumor growth

inhibition (TGI) and relative increment ratio (T/C) were used as the

evaluation indicators to reveal the antitumor eﬀects in tumor weight

and tumor volume, respectively. Data were analyzed by Student’s two-

tailed t test. A P level < 0.05 was considered statistically signiﬁcant.

National Cancer Institute of the National Institutes of Health

(Award no. R01CA163452) and National High-tech R&D

Program of China, 863 Program (Grant No.2014AA020523).

ABBREVIATIONS USED

■

HDAC, histone deacetylase; HDACi, histone deacetylase

inhibitors; SAR, structure−activity relationships; SAHA,

suberoylanilide hydroxamic acid; ZBG, zinc-binding group;

Boc, tertbutoxycarbonyl

TGI = (the mean tumor weight of control group

− the mean tumor weight of treated group)

/the mean tumor weight of control group

Tumor volumes (V) were estimated using the equation (V = ab²/2,

where a and b stand for the longest and shortest diameter,

respectively). T/C was calculated according to the following formula:

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ASSOCIATED CONTENT

* Supporting Information

■

S

¹H NMR spectral information of the key intermediates 6, 7,

and 8 and representative target compounds 10a, 11k, 11e, 11p,

11r, 11w, 11y, and 27c. HPLC analysis chromatograms of

representative target compounds 10a, 11k, 11e, 11p, 11r, 11w,

11y, and 27c. This material is available free of charge via the

Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors

*(W.X.) Phone: 86-531-88382264. Fax: 86-531-88382264. E-

mail: wenfxu@gmail.com.

■

*(Y.Z.) E-mail: zhangyingjie@sdu.edu.cn

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The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

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Discovery of the first N -hydroxycinnamamide-based histone deacetylase 1/3 dual inhibitors with potent oral antitumor activity

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