Synthesis and biological evaluation of N-hydroxyphenylacrylamides and N-hydroxypyridin-2-ylacrylamides as novel histone deacetylase inhibitors

822 J. Med. Chem. 2010, 53, 822–839

DOI: 10.1021/jm901502p

Synthesis and Biological Evaluation of N-Hydroxyphenylacrylamides and

N-Hydroxypyridin-2-ylacrylamides as Novel Histone Deacetylase Inhibitors

Florian Thaler,*^,†,¥Andrea Colombo,^‡Antonello Mai,^§Raffaella Amici,^†,¥Chiara Bigogno,^‡Roberto Boggio,

Anna Cappa, ^,¥Simone Carrara,^‡Tiziana Cataudella, Fulvia Fusar, Eleonora Gianti,^†,¥Samuele Joppolo di Ventimiglia,^‡

Maurizio Moroni,^†Davide Munari, Gilles Pain,^†,bNickolas Regalia,^‡Luca Sartori, ^,¥Stefania Vultaggio, Giulio Dondio,^‡

,¥

Stefania Gagliardi,^‡Saverio Minucci,^{^,#}Ciro Mercurio, and Mario Varasi

^†Genextra Group, Congenia s.r.l., Via Adamello 16, 20139 Milan, Italy, ^‡NiKem s.r.l., Via Zambeletti 25, 20021 Baranzate (MI), Italy,

^§Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Universitaꢀ degli Studi di Roma

;

“La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, Genextra Group, DAC s.r.l., Via Adamello 16, 20139 Milan, Italy, ^{^}European Institute of

Oncology, Via Adamello 16, 20139 Milan, Italy, and ^#Department of Biomolecular Sciences and Biotechnologies, University of Milan, Via Celoria,

26, 20133 Milan, Italy. ^¥Present address: European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy. ^bPresent address:

Sigma—Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina km 30,400, 00040 Pomezia (RM), Italy.

Received October 9, 2009

The histone deacetylases (HDACs) are able to regulate gene expression, and histone deacetylase

inhibitors (HDACi) emerged as a new class of agents in the treatment of cancer as well as other human

disorders such as neurodegenerative diseases. In the present investigation, we report on the synthesis and

biological evaluation of compounds derived from the expansion of a HDAC inhibitor scaffold having

N-hydroxy-3-phenyl-2-propenamide and N-hydroxy-3-(pyridin-2-yl)-2-propenamide as core structures

and containing a phenyloxopropenyl moiety, either unsubstituted or substituted by a 4-methylpiper-

azin-1-yl or 4-methylpiperazin-1-ylmethyl group. The compounds were evaluated for their ability to

inhibit nuclear HDACs, as well as for their in vitro antiproliferative activity. Moreover, their metabolic

stability in microsomes and aqueous solubility were studied and selected compounds were further

characterized by in vivo pharmacokinetic experiments. These compounds showed a remarkable stability

in vivo, compared to hydroxamic acid HDAC inhibitors that have already entered clinical trials.

The representative compound 30b showed in vivo antitumor activity in a human colon carcinoma

xenograft model.

Introduction

HDACs, while class III enzymes (also known as sirtuins) are

defined by their dependency on NAD^þ.⁴The acetylation

status of histones is crucial in modulating gene expression

and cell fate, and its dysregulation is involved in the develop-

ment of several cancers. Histone hyperacetylation leads to

transcriptional activation of suppressed genes, some of them

being associated with cell cycle arrest, differentiation, or

apoptosis in tumor cells. Recent studies have shown that

acetylation of non-histone proteins is also relevant for tumor-

igenesis, cancer cell proliferation, and immune functions.⁵

Thus, inhibition of HDACs has emerged as a novel therapeu-

tic strategy to reverse aberrant epigenetic changes associated

with cancer.^6-10After the discovery that exposure of cultured

cells to sodium n-butyrate caused a reversible accumulation of

highly acetylated histones,¹¹several classes of HDAC inhibi-

tors have been found demonstrating potent anticancer activ-

ities in preclinical studies. These include short chain fatty

acids, hydroxamic acids, cyclic tetrapeptides/depsipeptides,

ketones, and benzamides. In October 2006 SAHA (suberoyl-

anilide hydroxamic acid) 1^12-14gained approval by the FDA

as the first HDAC inhibitor for the treatment of cutaneous

T-cell lymphoma, and various agents are currently at different

stages of clinical development. Other examples of inhibitors

currently being evaluated in clinical trials are the hydroxamic

acids panobinostat or LBH589 2 (Novartis, phase III),¹⁵

PXD101 or belinostat 3 (Topotarget, phase III),¹⁶ITF-2357

Since the original observation that reversible post-transla-

tional modification of histones, such as acetylation or methy-

lation, is linked to transcriptional regulation, the biological

function of these modifications has aroused considerable

interest.¹Later it was found that hyperacetylated histone

cores are generally allocated in transcriptionally active genes,

whereas hypoacetylated histones are in transcriptionally silent

regions of the genome.²Acetylation occurs at lysine residues

on the amino-terminal tails of the histones, which leads to

neutralization of the positive charge of the histone tails and

thus to a decreased affinity for DNA.³The acetylation status

of the lysine residues is tightly controlled by two counteracting

enzyme families: the histone acetyltransferases (HAT^a) and

the histone deacetylases (HDAC). Up to now, there are 18

known human histone deacetylases, categorized into four

classes based on the structure. Classes I (HDACs 1-3 and

8), II (HDACs 4-7, 9, and 10), and IV (HDAC 11) enzymes

are Zn^2þ-dependent enzymes and are the so-called “classical”

*To whom correspondence should be addressed. Phone: þ390294375130.

Fax: þ390294375138. E-mail: florian.thaler@ifom-ieo-campus.it.

^aAbbreviations: HDAC, histone deacetylases; HAT, histone acetyl-

transferases; K562, chronic myelogenous leukemia cell line; HCT-116,

human colorectal carcinoma cell line; MTT, 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide; PK, pharmacokinetics.

r

pubs.acs.org/jmc

Published on Web 12/17/2009

2009 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 823

Figure 1. HDAC inhibitors in clinical development.

Scheme 1^a

4 (Italfarmaco, phase II),¹⁷SB-939 5 (S*BIO, phase I),¹⁸PCI-

24781 6 (Pharmacyclics, phase I),¹⁹and JNJ-16241199 7 (J&J,

phase I),²⁰the benzamides SNDX-275 or MS-275 8 (Syndax,

phase II)²¹and MGCD-0103 9 (Methylgene, phase II),²²the

cyclic depsipeptide romidepsin or FK228 10 (Gloucester,

phase II),²³and valproic acid 11 (Topotarget, phase II)²⁴

(see Figure 1).

So far, given their potency, hydroxamic acid derivatives are

among the most promising candidates for HDAC inhibition.

However, several compounds have been shown to have short

half-lives in vitro as well as in vivo. Nevertheless, the com-

pounds showed in vivo activity. As a consequence, a “hit-and-

run” mechanism has been suggested whereby the transient

and short exposure to relatively high concentrations of the

drug might be sufficient for an antitumor effect in vivo.^25,26

In the present study, we report the synthesis and biological

characterization of novel HDAC inhibitors using as starting

points N-hydroxy-3-phenyl-2-propenamide and N-hydroxy-

3-(pyridin-2-yl)-2-propenamide, selected among a small series

of aryl- and heteroarylhydroxyamide fragments. Their phe-

nyloxopropenyl derivatives, unsubstituted or substituted with

different 4-methylpiperazin-1-yl or 4-methylpiperazin-1-yl-

methyl moieties, were prepared with the goal of synthesizing

novel, highly potent compounds with improved pharmacoki-

netic properties.

^aReagents and conditions: (a) CH₂Cl₂, EDC, HOBT, NH₂OBn,

room temp; (b) MeOH, Pd/C, NH₄HCO₂; (c) CH₂Cl₂, EDC, HOBT,

TEA, NH₂OTHP, room temp; (d) CH₂Cl₂, Et₂O, HCl, room temp.

Chemistry

as coupling agents. Cleavage of the protecting group with

hydrogen chloride (HCl) afforded the desired hydroxamates

13d-g and 15.

N-Hydroxybenzamide (13a) is commercially available, and

N-hydroxy-2-phenylacetamide (13b)²⁷was prepared according

to the literature. As shown in Scheme 1, N-hydroxy-3-phenyl-

propionamide (13c) was prepared by reacting 3-phenylpropio-

nic acid (12c) with NH₂OBn (O-benzylhydroxylamine) in the

presence of EDC (N-(3-dimethylaminopropyl)-N⁰-ethylcarbo-

diimide hydrochloride) and HOBt (N-hydroxybenzotriazole).

The benzyl group was then cleaved by hydrogenation with 10%

Pd-C and ammonium formate.

Phenylacrylic acid (12d) is commercially available; the

pyridylacrylic acids 12e-g²⁸and the pyrrolylacrylic acid

14²⁹were prepared according to the literature. The acrylic

acids 12d-g and 14 reacted with NH₂OTHP (O-(tetrahydro-

pyran-2-yl)hydroxylamine) in the presence of EDC and HOBt

As shown in Scheme 2, condensation of (un)substituted

acetophenones 16a-g (see Supporting Information) with

commercially available (4-formylphenyl)acrylic acid (17) was

carried out in ethanol in the presence of aqueous KOH. The

acrylic acid 18a was then treated with ethyl chloroformate and

triethylamine, followed by the addition of O-(2-methoxy-

2-propyl)hydroxylamine.³⁰Acidic workup in the presence of

the Amberlyst 15 ion-exchange resin furnished the desired

hydroxamic acid 19a. The conversion of the acid intermediates

18b-g into the hydroxamic acids 19b-g was carried out

according to the procedures described in Scheme 1 for the

preparation of compounds 13d-g.

824 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Thaler et al.

Scheme 2^a

^aReagents and conditions: (a) EtOH, KOH, room temp; (b) THF, DMF, EDC, HOBT, TEA, NH₂OTHP, room temp, or (for 19a) ClCOOC₂H₅,

TEA, THF, 0 ꢀC, and then NH₂OC(CH₃)₂OCH₃; (c) CH₂Cl₂, Et₂O, HCl, room temp, or (for 19a) Amberlyst 15, MeOH, 45 ꢀC.

Scheme 3^a

^aReagents and conditions: (a) CH₂Cl₂, DMSO, TEA, (COCl)₂, -60 ꢀC; (b) MeOH, TMOF, PTSA, room temp; (c) THF, i-PrMgCl, then DMF, room

temp; (d) THF, NaH, (EtO)₂P(O)CH₂COO^tBu, room temp; (e) THF, HCl, room temp to 80 ꢀC; (f) 16a, THF, KOH, room temp; (g) CH₂Cl₂, TFA,

room temp; (h) CH₂Cl₂EDC, HOBT, TEA, NH₂OTHP, room temp; (i) CH₂Cl₂, Et₂O, HCl, room temp.

Commercially available (2-bromopyridin-4-yl)methanol

(20) was oxidized through Swern oxidation (Scheme 3). After

protection of the carbaldehyde 21 with trimethyl orthofor-

mate in the presence of p-toluenesulfonic acid, the compound

was first treated with isopropylmagnesium chloride (i-Pr-

MgCl), then quenched with DMF and finally treated with

tert-butyl diethyl phosphonacetate according to Horner-

Emmons reaction forming the intermediate 22. After cleavage

of the dimethoxy group with HCl in THF the carbaldehyde

was treated with acetophenone 16a. The reaction was carried

out in THF instead of ethanol, since Michael addition of

acetophenone on the double bond had been observed as side

reaction in ethanol (9:1 ratio between the bis-acetophenone

adduct and the desired tert-butyl ester 23 was found in the

reaction mixture). Removal of the tert-butyl group with

trifluoroacetic acid (TFA) in dichloromethane and introduc-

tion of the hydroxamic acid moiety according to the proce-

dure described above in Scheme 1 for compounds 13d-g

afforded compound 24.

Commercially available 6-bromopyridine-3-carbaldehyde

(25) was protected with trimethyl orthoformate, then treated

with i-PrMgCl followed by DMF to give 5-dimethoxymethyl-

pyridine-2-carbaldehyde (26) (Scheme 4). Horner-Emmons

olefination of 26 and cleavage of the dimethoxy group gave

the formylpyridinyl ester 27. Condensation of (un)substituted

acetophenones 16a-g with intermediate 27 was carried out in

MeOH, EtOH, or THF in the presence of aqueous KOH as

outlined in Schemes 2 and 3. Hydrolysis of the tert-butyl ester

followed by EDC coupling with NH₂OTHP and final clea-

vage of the tetrahydropyranyl protecting group furnished the

hydroxamic acid derivatives 30a-g.

palladium acetate [Pd(OAc)₂], PPh₃, triethylamine, and NaH-

CO₃. Different coupling conditions (1,4-diazabicyclo[2.2.2]-

octane (DABCO), K₂CO₃, tetrabutylammonium bromide

(n-Bu₄NBr), and Pd(OAc)₂under microwave irradiation)

were used to prepare the tert-butyl acrylate 32b from 6-bro-

mopyridine-2-carbaldehyde (31b) (Scheme 5). Subsequent

condensation of these intermediates with acetophenone deri-

vatives (16a-g), cleavage of the tert-butyl ester group, and

conversion to the hydroxamic acid afforded 35a-n.

Biological Results and Discussion

The hydroxamic acid derivatives were profiled using a

commercially available HDAC assay kit, with HeLa cell

nuclear extractsasenzyme source andafluorogenicacetylated

histone peptide fragment (Fluor de Lys) as a substrate. The

results are expressed either as the percentage of inhibition at a

fixed dose or as IC₅₀(50% inhibitory concentration) values.

The antiproliferative activity of the prepared compounds

against the chronic myelogenous leukemia cell line K562

was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-

nyltetrazolium bromide (MTT). Furthermore, the ability of

selected compounds to inhibit cellular HDACs was evaluated

following the modification of the basal level of acetylation in

the above-reported cell line.³¹A microsomal stability assay

was performed as further characterization. Compounds were

exposed to mouse and human microsomes at 37 ꢀC, and the

percentage of the unmetabolized compound after 30 min of

incubation was established.

Initially, a small series of aryl- and heteroarylhydroxamic

acid derivatives were synthesized and tested to identify the

best minimal scaffold endowed with HDAC inhibitory activ-

ity. As shown in Table 1, 13a and 13b were demonstrated to be

inactive under the experimental conditions used, whereas 13c

showed a moderate biochemical activity with 36% enzyme

The meta-substituted tert-butyl acrylate 32a was prepared

starting from the commercially available 3-bromobenzalde-

hyde 31a according to the Heck reaction in the presence of

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 825

Scheme 4^a

^aReagents and conditions: (a) MeOH, TMOF, PTSA, room temp; (b) THF, i-PrMgCl, then DMF, room temp; (c) THF, NaH, (EtO)₂P(O)-

CH₂COO^tBu, room temp; (d) THF, HCl, room temp; (e) EtOH or THF or MeOH, KOH, room temp or 0 ꢀC; (f) CH₂Cl₂, TFA, room temp; (g) CH₂Cl₂,

THF, DMF, EDC, HOBT, TEA, NH₂OTHP, room temp; (h) CH₂Cl₂, Et₂O, HCl, room temp.

Scheme 5^a

^aReagents and conditions: (a) for 32a DMF, TEA, PPh₃, NaHCO₃, Pd(OAc)₂, 100 ꢀC, or (for 32b) DMF, DABCO, K₂CO₃, Bu₄NBr, Pd(OAc)₂,

120 ꢀC, microwave; (b) EtOH or THF, KOH, room temp or 0 ꢀC; (c) CH₂Cl₂, TFA, room temp; (d) CH₂Cl₂, THF, DMF, EDC, HOBT, TEA,

NH₂OTHP, room temp, or (for 35a) ClCOOC₂H₅, TEA, THF, 0 ꢀC, and then NH₂OC(CH₃)₂OCH₃; (e) CH₂Cl₂, Et₂O, HCl, room temp, or (for 35a)

Amberlyst 15, MeOH, 45 ꢀC.

inhibition at 1 μM. Introduction of an acrylic group (13d) led

to a further increase in activity. Thus, pyrrolyl- and pyridiny-

lacryl hydroxamates were prepared and tested. The pyridine-

2-yl derivative 13e was demonstrated to be the most potent

compound among this series, whereas the electron-rich pyr-

rolyl derivative 15 was less active than the other acryl hydrox-

amates.

The heteraryl hydroxamate 13e and the aryl hydroxa-

mate 13d were selected for further expansion in the first step

by preparing scaffolds with a phenyloxopropenyl moiety,

which is present in previously reported structures.³²The

HDAC inhibitory activity of the meta phenyl analogue 35a,

with an IC₅₀value of 0.085 μM, was substantially higher

than that of the corresponding para derivative 19a

(1.48 μM). This trend did not emerge so clearly in the

pyridinyl series, as the 2,5 disubstituted derivative 30a

and the 2,6 disubstituted analogue 35h showed similar

IC₅₀values (∼0.02 μM) and are around 15 times more

active than the pyridinyl derivative 24, substituted at the 2

and 4 positions (see Table 2).

Generally, the pyridinyl derivatives showed a better anti-

proliferative activity than the corresponding phenyl analo-

gues. In detail, the cellular IC₅₀values for 30a, 35h, and 24

were between 0.5 and 1 μM, whereas 19a and 35a exhibited

IC₅₀values of 2.3 and 3.2 μM, respectively.

All five compounds showed a limited stability in mouse

microsomes. After 30 min of incubation at 37 ꢀC, the percen-

tage of unaltered compounds was below 20%. Similar data

were obtained in human microsomes with the exception of

35h, which was found to be more stable than the other

inhibitors. Furthermore, the compounds showed a limited

solubility in the screening buffer (Table 2).

826 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Table 1. HDAC Inhibitory Activity of the Minimal Scaffold^a

Thaler et al.

^aValues are the mean of three experiments. The standard derivations are <20% of the mean. The numbers represent the percentage of inhibition of

HDAC enzymes (HeLa nuclear extract) at 0.1, 1.0, and 10 μM concentration of the inhibitors.

On the basis of the observations reported above, subse-

quent optimization was directed to obtain compounds with an

improved metabolic stability and solubility. As previously

mentioned, 24 and the precursor 23 were shown to be reactive

and THF had to be used as solvent in place of ethanol in the

aldol reaction of 16a with 22. Electrophilic reactivity is often

associated with in vivo toxicity;^33,34therefore, no further

structures related to compound 24 were synthesized.

Prompted by the reactivity of compound 24, the electrophilic

behavior of compounds 19a, 30a, 35a, and 35h was also

examined in more detail. By adaptation of the protocol

described by Wienkers et al.,³⁴the compounds were incubated

ata test concentration of 100 μM with glutathione (100μM) in

a mixture of PBS (pH 7.4)/acetonitrile (65:35, v:v) for 1 h at

room temperature. Aliquots were taken at 0 and 1 h and

analyzed by LC-UV spectroscopy. 19a, 30a, and 35a were

recovered quantitatively after 1 h of incubation, whereas a

minor formation of a glutathione adduct of 35h was detected

by mass spectrometry. In this case 84% of 35h was recovered

after 1 h of incubation. Considering the literature benchmark

value of less than 20% residual glutathione after reaction with

the test compound for electrophilicity,³⁴compound 35h was

also considered, together with 19a, 30a, and 35a, for further

optimization. The goal toward more soluble compounds was

approached by synthesizing derivatives containing a phe-

nyloxopropenyl moiety, which was substituted either by a

4-methylpiperazin-1-yl or by a 4-methylpiperazin-1-ylmethyl

group.

Tables 3 and 4 summarize the biological data of the 4-methyl-

piperazin-1-yl and 4-methylpiperazin-1-ylmethyl derivatives

(entries 19b-g, 30b-g, 35b-g, and 35i-n). Overall, the

introduction of this moiety led to compounds with good

solubility while maintaining or increasing enzymatic inhibi-

tory and antiproliferative activities.

Large differences were found in the enzymatic assay for

compounds containing a para-substituted central phenyl or

2,5-disubstituted pyridinyl ring (see Table 3). IC₅₀values

ranged from 0.012 μM (30c) to 2 μM (19f). Side by side com-

parison of the compounds containing a central para substi-

tuted phenyl ring with those with a central 2,5-disubstituted

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 827

Table 2. HDAC Enzyme, Antiproliferative Activity, Microsomal Stability, and Solubility Data for Unsubstituted Phenyloxopropenyl Derivatives^a

microsomal stability

antiproliferative activity

K562 (μM)

compd

X

position

enzyme (μM)

mouse (%)

human (%)

solubility (μM)

19a

24

CH

N

5

4

5

6

1.48

2.28

1.06

0.69

3.21

0.51

2

14

6

5

16

14

9

3

15

27

12

16

0.323

0.022

0.085

0.02

30a

35a

35h

N

CH

N

6

11

38

^aAssays were done in replicates (n g 2). Mean values are shown, and the standard derivations are <30% of the mean.

Table 3. HDAC Enzyme, Antiproliferative Activity, Microsomal Stability, and Solubility Data for Para Substituted Compounds 19b-g and 30b-g^a

microsomal stability

antiproliferative activity

K562 (μM)

compd

R

X

enzyme (μM)

mouse (%)

human (%)

solubility (μM)

19b

19c

19d

19e

19f

19g

30b

30c

30d

30e

30f

30g

2-(4-methyl piperazin-1-yl)

2-(4-methyl piperazin-1-ylmethyl)

3-(4-methylpiperazin-1-yl)

CH

N

0.186

0.350

0.280

0.226

1.99

1.60

1.42

0.85

2.06

0.90

1.28

1.22

2.73

0.36

0.35

0.41

0.23

34

4.2

6.2

17

9.1

17

61

53

20

46

3.8

56

38

56

44

23

33

61

65

93

41

32

43

47

210

453

16

3-(4-methylpiperazin-1-ylmethyl)

4-(4-methyl piperazin-1-yl)

287

1

4-(4-methylpiperazin-1-ylmethyl)

2-(4-methylpiperazin-1-yl)

0.183

0.141

0.012

0.030

0.053

0.175

0.027

244

469

490

104

475

29

2-(4-methyl piperazin-1-ylmethyl)

3-(4-methylpiperazin-1-yl)

N

3-(4-methyl piperazin-1-ylmethyl)

4-(4-methylpiperazin-1-yl)

4-(4-methylpiperazin-1-ylmethyl)

N

467

^aAssays were done in replicates (n g 2). Mean values are shown, and the standard derivations are <30% of the mean.

pyridinyl ring showed that inhibitors containing the pyridinyl

ring were more potent in the assay. Moreover, a methylene

spacer between the piperazine group and the distal phenyl

group resulted in an increase in enzyme inhibitory activity in

some examples as seen when comparing 19g and 19f, 30c and

30b, 30g and 30f. No clear trends were observed regarding

ortho, meta, and para substitution on the distal phenyl ring.

For example, the para-substituted 4-methylpiperazin-1-yl

derivative 19f, with an IC₅₀value of 2.00 μM, was substan-

tially less active than the corresponding ortho (19b, 0.186 μM)

and meta (19d, 0.280 μM) analogues. On the contrary, the

para-substituted 30f, with an IC₅₀value of 0.175 μM, and the

ortho (30b) analogue (0.141 μM) showed comparable pot-

ency, whereas the meta (30d) derivative had an IC₅₀value of

0.030 μM.

On the other hand (see Table 4), the compounds containing

a meta-substituted central phenyl or a 2,6-disubstituted pyri-

dinyl ring displayed consistently good, double digit nano-

molar HDAC inhibitory activity with no major differences

within this subseries. The compounds 35b-n exhibited IC₅₀

values between 15 and 87 nM and were generally more potent

than the analogues with the para-substituted aromatic ring.

This trend was more evident within the phenyl than in the

pyridinyl series.

For a further characterization of their isoform selectivity

profile, some representative compounds were tested together

with 1 against HDACs 1, 3, 8, 4, and 6 (Amphora Corp., Resea-

rch Triangle, NC).³⁵All compounds showed activities compa-

rable or in some cases superior to 1, and the structures can be

classified as typical pan-HDAC inhibitors (data not shown).

As observed for the unsubstituted phenyloxopropenyl ser-

ies, the antiproliferative activity of the compounds containing

a 2,5-disubstituted pyridinyl ring was generally higher than

that of those containing a para-substituted central phenyl

(Table 3). Differences in cellular activity were observed be-

tween compounds, wherein the distal phenyl ring was sub-

stituted on different positions: the ortho-substituted 19b with

a cellular IC₅₀value of 1.6 μM was less active than the meta

(19d) and the para (19f) analogues withIC₅₀valuesof 0.85and

0.90 μM, respectively. The same trend was found within the

pyridinyl series: 30b exhibited an IC₅₀value of 1.2 μM,

whereas the meta (30d) and the para (30f) 4-methylpipera-

zin-1-yl analogues had IC₅₀values of 0.36 and 0.41 μM,

respectively.

828 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Thaler et al.

Table 4. HDAC Enzyme, Antiproliferative Activity, Microsomal Stability and Solubility Data for Meta Substituted Compounds 35a-n^a

microsomal stability

antiproliferative activity

K562 (μM)

compd

R

X

enzyme (μM)

mouse (%)

human (%)

solubility (μM)

35b

35c

35d

35e

35f

35g

35i

2-(4-methylpiperazin-1-yl)

2-(4-methylpiperazin-1-ylmethyl)

3-(4-methylpiperazin-1-yl)

CH

N

0.087

0.074

0.020

0.024

0.027

0.017

0.033

0.053

0.032

0.056

0.015

0.041

1.29

0.98

0.99

1.09

1.17

1.10

0.70

0.76

0.11

0.20

0.14

0.20

44

35

7.2

25

5.9

33

57

40

29

40

19

46

62

55

39

40

44

58

67

35

45

35

51

177

473

46

3-(4-methylpiperazin-1-ylmethyl)

4-(4-methylpiperazin-1-yl)

405

15

4-(4-methylpiperazin-1-ylmethyl)

2-(4-methylpiperazin-1-yl)

134

450

460

380

450

7

35j

2-(4-methylpiperazin-1-ylmethyl)

3-(4-methylpiperazin-1-yl)

N

35k

35l

N

3-(4-methylpiperazin-1-ylmethyl)

4-(4-methylpiperazin-1-yl)

4-(4-methylpiperazin-1-ylmethyl)

N

35m

35n

N

447

^aAssays were done in replicates (n g 2). Mean values are shown, and the standard derivations are <30% of the mean.

Similar trends were found for compounds containing a

meta-substituted central phenyl or 2,6-disubstituted pyridinyl

ring (Table 4). Pyridinyl derivatives were shown to be more

potent than their phenyl analogues. Four derivatives (35k-n),

wherein the distal phenyl rings were either meta- or para-

substituted, were found to be among the most potent com-

pounds of this series with cellular IC₅₀values between 0.1 and

0.2 μM.

By comparison of the antiproliferative activities of the

inhibitors, in which the central aromatic ring is meta-substi-

tuted, with the corresponding para analogues, the results

indicate that the meta analogues generally exhibited a higher

potency (Tables 3 and 4).

Table 5. Histone Acetylation Assay of Standard Compound 1 and Nine

Representative Derivatives^a

compd

acetylation increment in presence of 0.5 μM inhibitor

1

3.61

2.87

4.89

2.22

3.78

3.72

2.54

8.35

3.84

3.88

19b

30b

30c

30e

30g

35c

35i

35k

35n

^aValues refer to a single experiment. The numbers represent the ratio

of the acetylation level of treated cells over controls.

Tables3 and 4 alsoinclude the microsomal stabilitydata for

the 4-methylpiperazin-1-yl and 4-methylpiperazin-1-ylmethyl

derivatives. The results are expressed as percentage (%)

remaining of the parent compound after 30 min of incubation.

The inhibitorsofboth series, whereinthe centralaromatic ring

is meta- or para-substituted, were in general found to be more

stable in human than in mouse microsomes. An increased

stability of hydroxamic acid derivatives in human over mice

microsomes has been recently described by Venkatesh et al.³⁶

The authors observed that N-hydroxy-3-[2-phenethyl-1-(2-

pyrrolidin-1-ylethyl)-1H-benzimidazol-5-yl]acrylamide (SB639)

showed a limited stability in mice microsomes with a half-life

of 3 min, whereas the compound was significantly more stable

in human microsomes (t_1/2= 59 min). At the same experi-

mental conditions, the observed half-life of compound 1 was

28 min in mouse and 60 min in human microsomes. Compar-

ison of compounds containing a phenyl group as central

aromatic ring with those having a pyridinyl ring showed that

the latter ones generally demonstrated an increased stability.

Compounds 30b, 30c, 30g, and 35i were the most stable

compounds in this test (less than 50% of the product was

metabolized in both mouse and human microsomes), and

three of them (30b, 30c, and 35i) are ortho-substituted on the

distal phenyl ring. In contrast, compounds with a 4-methylpi-

perazin-1-yl-moietyinparaposition areamong the most labile

compounds in mouse microsomes. As expected, the introduc-

tion of the piperazine group led to structures having generally

a good solubility. As outlined in Tables 3 and 4, the solubility

of most of the inhibitors exceeded 200 μM at a pH value of 7.4.

Compounds with solubility lower than 200 μM as well as

derivatives that were metabolized more than 80% in mouse

and human microsomes were not considered for further tests.

Compounds 35j and 35l were not further characterized, since

both compounds were shown to be potent inhibitors of the

cytochromes P-450 CYP2D6 and CYP3A4, inhibiting the

enzymes at least 85% at 10 μM.

Nine compounds (19b, 30b, 30c, 30e, 30g, 35c, 35i, 35k, and

35n) that fulfilled the requirements discussed above were

selected for a more extensive characterization.

First, human cancer leukemia K562 cells were incubated

with the compounds in order to assess the ability of the

molecules to inhibit HDACs in cells measuring the histone

acetylation levels by flow cytometry. Consistent with an

ability to inhibit cellular HDACs, all compounds tested

induced a 2-8 times increase in the basal level of acetylation

in K562 cells at 0.5 μM (Table 5).

The same selectedcompounds weresubsequently submitted

to pharmacokinetic studies in mouse. The compounds were

administered in single intravenous (iv) and oral doses of 5 and

15 mg/kg, respectively. All the compounds were dissolved in

0.9% NaCl solution for the intravenous dose or in water for

the oral dose. The results were compared with SAHA (1),

which was dissolved in water containing 1% DMSO and 10%

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 829

Table 6. Pharmacokinetic Parameters of Standard Compounds 1-3 and Nine Representatives Derivatives in Mice

comp

1

1³⁷

2³⁷

3³⁷

19b

30b

30c

30e

30g

35c

35i

35k

35n

AUC_iv(ng h/mL)^a

t_1/2(h)

657

0.5

743

273

1.37

18.3

15.1

4.6

431

698

2

1397

2.3

1492

5.55

3.35

11.7

4.5

736

6.7

1052

9.7

849

8.27

5.88

20.1

3.1

1898

8.6

1506

8.5

2524^b

7.3

0-¥

0.38

6.73

0.81

8.33

1.21

11.6

5.03

6.66

Cl ((L/h)/kg)

V_ss(L/kg)

F (%)

7.62

1.2

7.14

5.4

18.1

3.58

2.1

6.78

28.4

3.9

4.75

31.9

5.5

2.64

13.9

2.9

3.32

18.3

2

1.75

16.07

2.6

12.1

6.3

^aLiterature AUC data are normalized to the doses used in house. ^bAUC_ivfrom 0 to 24 h (ng h/mL).

Figure 2. Caspase-3/7 induction in human cancer cells K562, exp-

ressed as relative light unit (RLU): untreated or treated with solvent

(DMSO), 30b, or 1 at 3 μM for 24 h.

Figure 3. Antitumor activity of 30b and 1 against HCT-116 human

tumor xenografts implanted in mice, expressed as mean tumor

volume (expressed as mm³) ( standard error of the mean (SEM):

untreated or treated with 30b in PBS or 1 in PEG/H₂O/DMSO

(45:45:10).

encapsin as iv formulation or in water containing 5% DMSO

and 1% PEG400 for the oral dose. The main plasma phar-

macokinetic parameters are presented in Table 6, which also

include data reported in the literature for 1, LBH-589 (2), and

PXD-101 (3).³⁷The pharmacokinetic profiles for all the

compounds tested were quite similar after the iv administra-

tion.

As reported in the table, the compounds exhibited in gene-

ral a medium/high clearance but still lower than the three

reference compounds, whose clearance was greater than the

hepatic blood flow of 5.4 (L/h)/kg in mice.³⁸Furthermore, the

estimated elimination half-life of the tested compounds is

between 2 h (19b) and 9.7 h (30g) and is generally higher than

that of all three reported reference compounds.³⁷19b and 30b

exhibited a medium-high estimated steady-state volume of

distribution V_ss(5.4 and 2.1 L/kg, respectively), whereas the

other tested compounds had a high V_sswith more than 10 L/

kg. In comparison, reference compounds 1 and 3 had a

medium-high V_sswith 1.2 and 5.0 L/kg, respectively, much

lower than 2 (15.1 L/kg). A poor oral bioavailability char-

acterizes the compounds as well as the three reference mole-

cules. 19b was the only exception with an oral bioavailability

higher than 10%.

Despite the poor pharmacokinetic parameters, the three

reference compounds had shown antitumor activity in vivo.²⁵

These results had triggered even more the search for active

compounds with a more favorable PK profile, and as des-

cribed above, compound 30b emerged as a compound with an

acceptable pharmacokinetic profile.

With respect to the apoptotic properties, 30b was profiled in

comparison with 1 in terms of ability to induce caspase

activation in human leukemia cancer cells (K562). As shown

in Figure 2, 30b, at the fixed concentration of 3 μM, induced a

relevant caspase-3/7activation after 24 h of treatment, slightly

higher than 1.

at a dose of 25 mg/kg iv once a day or with 1at a dose of 50 mg/kg

intraperitoneally once a day, which corresponds, to our best

knowledge, to the optimal condition for this derivative. As

outlined in Figure 3, compound 30b was demonstrated to have

a better in vivo efficacy than reference compound 1, characterized

by tumor stabilization and by a partial tumor regression in 5 out

of 7 mice. No significant body weight difference among the

groups of mice and no signs of overt toxicity were observed

during the treatment.

Conclusion

A novel series of N-hydroxy-3-phenyl-2-propenamide and

N-hydroxy-3-(pyridin-2-yl)-2-propenamide derivatives con-

taining a phenyloxopropenyl moiety have been described as

HDAC inhibitors. Unsubstituted derivatives exhibited low

micromolar to double digit nanomolar IC₅₀values in the in

vitro HDAC inhibition studies and low micromolar to sub-

micromolar activities in the antiproliferative assay, but their

further development was hampered by limited solubility and

stability in human and mouse microsomes. The goal toward

more soluble compounds was approached by synthesizing

derivatives containing a phenyloxopropenyl moiety, which

was substituted either by a 4-methylpiperazin-1-yl or by a

4-methylpiperazin-1-ylmethyl group. Overall, the introduc-

tion of these moieties led to compounds with good solubility

while maintaining or increasing enzymatic inhibitory and

antiproliferative activity. Furthermore, an improvement of

microsomial stability was observed for several compounds.

The compounds (19b, 30b, 30c, 30e, 30g, 35c, 35i, 35k, and

35n) with the best balance of in vitro inhibitory potency and

ADME properties were submitted for a pharmacokinetic

profiling in mice. Compared to HDAC inhibitors, which are

already in advanced clinical studies (1-3), the whole series

showedin generala lowerclearance rateandanincreased half-

life. A representativeexample, 30b, wasdemonstratedtocause

tumor growth inhibition in vivo in a human colon carcinoma

xenograft model.

Finally, 30b and reference compound 1 were tested in an in

vivo efficacy experiment. In detail, we established a subcuta-

neous human HCT-116 xenograft model to determine the

effect of 30b or 1 on tumor growth in vivo. Once tumors be-

came palpable (∼100 mm³), mice were treated either with 30b

830 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Thaler et al.

Experimental Section

(E)-N-Hydroxy-3-(1-methyl-1H-pyrrol-2-yl)acrylamide (15).

Yield: 86 mg of 15 (17%). H NMR (DMSO-d₆) δ: 10.5 (bs,

1

Chemistry. Reagents and solvents used, unless stated other-

wise, were of commercially available reagent grade quality and

were used without further purification. Flash chromatography

purifications were performed on Merck silica gel 60 (0.04-0.063

mm). Nuclear magnetic resonance spectra (¹H NMR) were

recorded on a Bruker 300 MHz spectrometer at 300 K and are

referenced in ppm (δ) relative to TMS. Coupling constants (J)

are expressed in hertz (Hz). HPLC-MS experiments were

performed on an Acquity UPLC apparatus equipped with a

diode array and a Micromass ZQ single quadruple (Waters)

using a BEH C18 (1.7 μm, 2.1 mm ꢀ 50 mm) column. The flow

rate was adjusted to 0.6 mL/min, and the splitting ratio between

the amount submitted to the mass spectrometer and to the waste

was 1:4. Mobile phase A was composed by a mixture of water

and acetonitrile (95:5) containing 0.1% TFA, and mobile phase

B was composed by a mixture of water and acetonitrile (5:95)

containing 0.1% TFA. Purity refers to UV detection at 220 nm

or base peak intensity (BPI), and the compounds used for

biological tests were at least 95% pure with the exception of

35a (92%) (see Supporting Information).

N-Hydroxy-3-phenylpropionamide (13c). A mixture of 12c

(300 mg, 2.0 mmol), O-benzylhydroxylamine (NH₂OBn) (0.28 mL,

2.4 mmol), EDC (766 mg, 4.0 mol), and HOBt (540 mg,

4.0 mmol) in dry CH₂Cl₂(20 mL) was stirred for 3 h at room

temperature. The solution was diluted with CH₂Cl₂, washed

with saturated NaHCO₃and brine, dried over Na₂SO₄, and

evaporated in vacuo to dryness. The resulting benzyl protected

hydroxamic acid was purified by flash chromatography

(AcOEt/hexane from 3:7 to 4:6). NH₄HCO₂(813 mg, 12.9 mmol)

was added to a suspension of the intermediate and Pd/C (10%,

236 mg) in methanol (30 mL), and the mixture was stirred for

30 min at 40 ꢀC. After filtration of the catalyst, the solvent was

evaporated in vacuo to dryness, and the crude product was

purified by flash chromatography (CH₂Cl₂/MeOH/CH₃-

COOH, 97:3:0.3). The resulting product was triturated with

petroleum ether and diethyl ether, affording the requisite hydro-

xamic acid 13c (234 mg, 71%). ¹H NMR (DMSO-d₆) δ: 10.37 (s,

1H), 8.71 (s, 1H), 7.29-7.25 (m, 2H), 7.20-7.15 (m, 3H), 2.80

(t, J=7.8 Hz, 2H), 2.25 (t, J=7.8 Hz, 2H). LC-MS (m/e): no

signal.

Typical Procedure for the Synthesis N-Hydroxyacrylamides

13d-g and 15. Example: (E)-N-Hydroxy-3-pyridin-3-ylacryla-

mide (13f). A mixture of 12f (546 mg, 3.66 mmol), NH₂OTHP

(357 mg, 3.05 mmol), EDC (773 mg, 4.03 mol), and HOBt (540

mg, 4.0 mmol) in dry CH₂Cl₂(10 mL) and dry DMF (5 mL) was

stirred for 24 h at room temperature. The solution was diluted

with CH₂Cl₂, washed with saturated NaHCO₃and brine, dried

over Na₂SO₄, and evaporated in vacuo to dryness. The resul-

ting crude compound was purified by flash chromatography

(CH₂Cl₂/MeOH, 95:5). The resulting intermediate was dis-

solved in CH₂Cl₂and treated with 1 M HCl in Et₂O (3.4 mL)

for 1 h. The precipitating yellow solid was filtered off to afford

13f as its hydrochloride salt (422 mg, 69%). ¹H NMR (DMSO-

d₆) δ: 8.98 (s, 1H), 8.73 (d, J = 5.2 Hz, 1H), 8.42 (d, J = 8 Hz,

1H), 7.80 (dd, J = 5.2 Hz, 8 Hz, 1H), 7.57 (d, J = 16 Hz, 1H),

6.71 (d, J = 16 Hz, 1H). LC-MS (m/e): 165 [M - H]^þ.

(E)-N-Hydroxy-3-phenylacrylamide (13d). Yield: 273 mg of

13d (55%). ¹H NMR (DMSO-d₆) δ: 10.75 (bs, 1H), 7.56

(m, 2H), 7.45 (d, J = 16 Hz, 1H), 7.43-7.35 (m, 3H), 6.47 (d,

J = 16 Hz, 1H). LC-MS (m/e): no signal.

(E)-N-Hydroxy-3-pyridin-2-ylacrylamide (13e). Yield: 404 mg

of 13e (66%). ¹H NMR (DMSO-d₆) δ: 8.68-8.67 (m, 1H), 8.02

(t, J = 8.0, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.54-7.50 (m, 2H),

6.98 (d, J = 15.6 Hz, 1H). LC-MS (m/e): 165 [M - H]^þ.

(E)-N-Hydroxy-3-pyridin-4-ylacrylamide (13g). Yield: 257 mg

of 13g (42%). ¹H NMR (DMSO-d₆) δ: 8.85 (d, J = 6.4, 2H), 8.07

(d, J = 6.4, 2H), 7.59 (d, J = 16.0 Hz, 1H), 6.97 (d, J = 16.0 Hz,

1H). LC-MS (m/e): 165 [M - H]^þ.

1H), 7.35 (d, J = 15.6 Hz, 1H), 6.90 (bs, 1H), 6.48 (bs, 1H), 6.11

(d, J = 15.6 Hz, 1H), 6.06 (m, 1H), 3.67 (s, 3H). LC-MS (m/e):

189 [M þNa]^þ.

(E)-N-Hydroxy-3-[4-((E)-3-oxo-3-phenyl-1-propen-1-yl)phenyl]-

acrylamide (19a). A mixture of acetophenone (16a, 66 μL, 0.56

mmol), 4-formylcinnamic acid (17, 99 mg, 0.56 mmol), and 2 M

KOH (1 mL) in 10 mL of water/ethanol (1:1) was stirred at room

temperature for 24 h. The solution was then acidified with 10%

HCl and the formed yellow precipitate was filtered to afford 120

mg (77%) of the acrylic acid 18a. ¹H NMR (DMSO-d₆) δ (ppm):

8.13-8.20 (m, 2H), 8.00 (d, J = 15.73 Hz, 1H), 7.90-7.96 (m,

2H), 7.76-7.81 (m, 2H), 7.75 (d, J=15.73 Hz, 1H), 7.65-7.72

(m, 1H), 7.62 (d, J=16.05 Hz, 1H), 7.54-7.63 (m, 2H), 6.64 (d,

J =16.05 Hz, 1H).

Ethyl chloroformate (48 μL, 0.5 mmol) and triethylamine

(75 μL, 0.54 mmol) were added to a cooled solution of the car-

boxylic acid 18a (117 mg, 0.42 mmol) in dry THF (10 mL), and

the mixture was stirred for 10 min. The reaction mixture was

filtered, and O-(2-methoxy-2-propyl)hydroxylamine (35 μL,

0.47 mmol) was added to the filtrate. The solution was stirred

for 15 min at 0 ꢀC, then evaporated under reduced pressure, and

the residue was diluted with methanol (10 mL). The solution of

the protected hydroxamate was added to an Amberlyst 15 ion-

exchange resin (0.3 g), and the resulting mixture was stirred at

45 ꢀC for 1 h. Thereafter, the reaction mixture was filtered, and

the filtrate was concentrated in vacuo and then purified by

crystallization from acetonitrile affording the desired hydroxa-

mic acid 19a (76 mg, 62%). ¹H NMR (DMSO-d₆) δ: 8.09-8.22

(m, 2 H), 7.96 (d, J=15.42 Hz, 1 H), 7.87-7.95 (m, 2 H), 7.74 (d,

J=15.73 Hz, 1 H), 7.62-7.71 (m, 3 H), 7.55-7.62 (m, 2 H), 7.49

(d, J = 15.73 Hz, 1 H), 6.58 (d, J = 16.05 Hz, 1 H). LC-MS

(m/e): 294 [M - H]^þ.

(E)-N-Hydroxy-3-(4-{(E)-3-[2-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}phenyl)acrylamide (19b). A mixture of 1-[2-(4-

methylpiperazin-1-yl)phenyl]ethanone (16b, 542 mg, 2.48 mmol),

17 (438 mg, 2.48 mmol), and 1.7 M KOH (2.92 mL) in 10 mL of

water/ethanol (1:1) was stirred at room temperature for 24 h. The

solution was then acidified with 10% HCl and the formed yellow

precipitate was filtered to afford the acrylic acid hydrochloride 18b

(930 mg, 90%). ¹H NMR (DMSO-d₆) δ: 12.44 (bs, 1 H), 10.37 (bs,

1 H), 7.81 (m, 4 H), 7.62 (d, J=15.85 Hz, 1 H), 7.46-7.59 (m, 4 H),

7.28 (d, J=7.63 Hz, 1 H), 7.11-7.24 (m, 1 H), 6.62 (d, J=15.85

Hz, 1 H), 3.33-3.57 (m, 2 H), 3.10-3.27 (m, 4 H), 2.77-3.04 (m,

2 H), 2.66 (s, 3 H).

A mixture of 18b (250 mg, 0.608 mmol), NH₂OTHP (85 mg,

0.73 mmol), EDC (232 mg, 1.22 mol), HOBt (164 mg, 1.22

mmol), and TEA (253 μL, 1.82 mmol) in dry THF (5 mL) and

DMF (5 mL) was stirred for 24 h at room temperature. The

solution was then diluted with water and extracted with AcOEt.

The organic layer was washed with water and brine, dried

over Na₂SO₄, evaporated in vacuo to dryness, and purified by

flash chromatography (CH₂Cl₂/MeOH/NH₄OH, 98:2:0.2).

The intermediate was dissolved in CH₂Cl₂and treated with

HCl in Et₂O for 1 h. The resulting yellow solid was filtered

to give the desired hydroxamic acid 19b as its hydrochloride

1

salt (115 mg, 44%). H NMR (DMSO-d₆) δ: 10.89 (bs, 1 H),

7.83 (m, 2 H), 7.64 (m, 2 H), 7.43-7.60 (m, 5 H), 7.28 (d, J =

7.92 Hz, 1 H), 7.21 (td, J = 7.63, 0.88 Hz, 1 H), 6.57 (d, J =

15.85 Hz, 1 H), 3.37-3.52 (m, 2 H), 3.09-3.37 (m, 4 H),

2.77-3.00 (m, 2 H), 2.66 (d, J = 4.40 Hz, 3 H). LC-MS (m/e):

392 [M - H]^þ.

(E)-N-Hydroxy-3-(4-{(E)-3-[2-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}phenyl)acrylamide (19c). A mixture

of 17 (189 mg, 1.077 mmol), 1-[2-(4-methylpiperazin-1-yl-

methyl)phenyl]ethanone (16c, 250 mg, 1.077 mmol), and 1.7 M

KOH (1.26 mL) in EtOH (5 mL) and H₂O (5 mL) was stirred at

room temperature overnight and then acidified with 10% aqueous

HCl. The resulting precipitate was filtered to give the carboxylic

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 831

acid 18c (350 mg, 70%, bis-hydrochloride). LC-MS (m/e): 391

[M - H]^þ.

NH₄OH, 96:4:0.2). The resulting intermediate was dissolved in

CH₂Cl₂and treated with HCl in Et₂O for 6 h. The precipitating

yellow solid was filtered to give the requisite hydroxamic acid

19f as its hydrochloride salt (7.86 g, 83%). ¹H NMR (DMSO-d₆)

δ: 11.33 (bs, 1 H), 8.11 (m, 2 H), 7.97 (d, J = 15.55 Hz, 1 H), 7.91

(m, 2 H), 7.68 (d, J = 15.55 Hz, 1 H), 7.63 (m, 2 H), 7.49 (d, J =

15.85 Hz, 1 H), 7.11 (m, J = 9.10 Hz, 2 H), 6.60 (d, J = 15.85 Hz,

1 H), 4.01-4.19 (m, 2 H), 3.43-3.56 (m, 2 H), 3.23-3.43 (m,

2 H), 2.97-3.22 (m, 2 H), 2.80 (d, J = 4.69 Hz, 3 H). LC-MS

(m/e): 392 [M - H]^þ.

(E)-N-Hydroxy-3-(4-{(E)-3-[4-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}phenyl)acrylamide (19g). A mixture

of 1-[4-(4-methylpiperazin-1-ylmethyl)phenyl]ethanone (16g,

392 mg, 1.69 mmol), 17 (300 mg, 1.69 mmol), and 1.7 M KOH

(2.0 mL, 3.4 mmol) in H₂O (5 mL) and EtOH (5 mL) was stirred

at room temperature for 24 h. The mixture was then acidified

with 10% HCl and the resulting yellow precipitate was filtered

off to obtain the carboxylic acid 18g (542 mg, 69%, dihydro-

chloride). LC-MS (m/e): 391 [M - H]^þ.

A mixture of 18c (350 mg, 0.756 mmol), HOBt (204 mg, 1.51

mmol), EDC (288 mg, 1.51 mmol), TEA (0.210 mL, 1.51 mmol),

and NH₂OTHP (106 mg, 0.907 mmol) in dry DMF (8 mL) was

stirred overnight at room temperature and then partitioned

between water and AcOEt. The aqueous layer was brought to

basic conditions with NH₄OH and extracted with CH₂Cl₂. The

collected organic extracts were dried over Na₂SO₄and evapo-

rated in vacuo. The crude mixture was purified by silica gel

chromatography (CH₂Cl₂/MeOH/NH₄OH, 95:5:0.2), and the

resulting product was dissolved in CH₂Cl₂and treated with

HCl/Et₂O for 1 h. The hygroscopic precipitate was filtered and

freeze-dried to afford the requisite hydroxamic acid 19c (229 mg,

63%, bis-hydrochloride salt). ¹H NMR (DMSO-d₆, 353 K,

þTFA) δ: 7.77 (m, 2 H), 7.61 (m, 2 H), 7.47-7.59 (m, 5 H),

7.43 (d, J = 15.55 Hz, 1 H), 7.33 (d, J = 16.14 Hz, 1 H), 6.63 (d,

J = 16.14 Hz, 1 H), 3.91 (s, 2 H), 3.08-3.25 (m, 4 H), 2.79-2.93

(m, 4 H), 2.71 (s, 3 H). LC-MS (m/e): 406 [M - H]^þ.

NH₂OTHP (164 mg, 1.4 mmol), EDC (447 mg, 2.34 mol),

HOBt (316 mg, 2.34 mmol), and TEA (488 μL, 3.51 mmol) were

added to a solution of 18g (542 mg, 1.17 mmol) in dry THF

(5 mL) and dry DMF (5 mL), and the mixture was stirred for

24 h at room temperature. The mixture was then diluted with

water and extracted with AcOEt. The organic layer was rinsed

with water, then with brine, dried over Na₂SO₄, and evaporated

in vacuo to dryness. The crude product was purified by flash

chromatography (CH₂Cl₂/MeOH/NH₃, 98:2:0.2). The resulting

intermediate was dissolved in CH₂Cl₂and treated with HCl in

Et₂O for 1 h to obtain a yellow precipitate. The solid was then

filtered off to obtain the desired hydroxamic acid 19g (300 mg,

53%, dihydrochloride). ¹H NMR (DMSO-d₆) δ: 11.73 (bs, 1 H),

8.23 (m, 2 H), 8.00 (d, J=15.55 Hz, 1 H), 7.95 (m, 2 H), 7.86 (m,

2 H), 7.77 (d, J=15.55 Hz, 1 H), 7.66 (m, 2 H), 7.50 (d, J=15.85

Hz, 1 H), 6.60 (d, J=15.85 Hz, 1 H), 4.45 (bs, 2 H), 3.19-3.70

(m, 8 H), 2.82 (s, 3 H). LC-MS (m/e): 406 [M - H]^þ.

(E)-N-Hydroxy-3-[4-((E)-3-oxo-3-phenyl-1-propen-1-yl)pyridin-

2-yl]acrylamide (24). Dry DMSO (1.73 mL, 24.4 mmol) was

added dropwise under N₂atmosphere to a stirred solution of

oxalyl chloride (1.08 mL, 12.7 mmol) in CH₂Cl₂(2 mL) at -78 ꢀC.

After the mixture was stirred for 20 min (2-bromopyridin-4-

yl)methanol (20) (2.0 g, 11 mmol) in CH₂Cl₂(10 mL) was added,

and the mixture was stirred for an additional 10 min at -60 ꢀC.

TEA (7.37 mL, 53.0 mmol) was added dropwise, and the

resulting solution was allowed to reach room temperature. After

20 min water was added and the product was extracted with

CH₂Cl₂. The organic layer was dried over Na₂SO₄, concen-

trated, and purified by column chromatography (petroleum

ether/AcOEt, 8:2) to afford 2-bromopyridine-4-carbaldehyde

21 as a white solid (1.0 g, 62%). ¹H NMR (CDCl₃) δ: 10.04 (s,

1 H), 8.65 (d, J=4.99 Hz, 1 H), 7.91 (dd, J=1.32, 0.73 Hz, 1 H),

7.69 (dd, 1 H).

A solution of 21 (850 mg, 4.57 mmol), trimethyl orthoformate

(0.678 mL, 5.88 mmol), and p-toluenesulfonic acid (87 mg, 0.13

mmol) in MeOH (15 mL) was stirred overnight at room tem-

perature, then partitioned between 5% NaHCO₃and Et₂O. The

organic layer was dried over Na₂SO₄and evaporated in vacuo to

give 880 mg (83%) of 2-bromo-4-dimethoxymethylpyridine.

The intermediate was dissolved in dry THF (20 mL) and added

to a stirred solution of i-PrMgCl (2 M in Et₂O, 2.5 mL) under N₂

atmosphere. The resulting mixture was stirred at room tempera-

ture under N₂, and further i-PrMgCl (2.5 mL) was added over

3 h. DMF (1.0 mL, 13 mmol) was added, and the mixture was

stirred at room temperature for 2 h and then partitioned

between water and Et₂O. The organic layer was dried over

Na₂SO₄and evaporated to dryness. The crude solid was purified

by column chromatography (CH₂Cl₂/AcOEt, 9:1) to give 420 mg

(60%) of 4-dimethoxymethylpyridine-2-carbaldehyde, which was

then dissolved in dry THF (10 mL) and added dropwise to a

stirred solution of tert-butyl diethyl phosphonacetate (0.710 mL,

(E)-N-Hydroxy-3-(4-{(E)-3-[3-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}phenyl)acrylamide (19d). A mixture of 1-[3-(4-

methylpiperazin-1-yl)phenyl]ethanone (16d, 406 mg, 1.86 mmol),

17 (328 mg, 1.86 mmol), and 1.7 M KOH (2.19 mL) in 10 mL of

ethanol was stirred at room temperature overnight. The solution

was then acidified with 10% HCl and the resulting yellow pre-

cipitate was filtered to afford the acrylic acid 18d as its hydro-

chloride salt (460 mg, 60%). LC-MS (m/e): 377 [M - H]^þ.

A mixture of 18d (224 mg, 0.543 mmol), NH₂OTHP (76 mg,

0.65 mmol), EDC (207 mg, 1.09 mol), HOBt (147 mg, 1.09

mmol), and TEA (151 μL, 1.09 mmol) in dry THF (5 mL) and

dry DMF (5 mL) was stirred for 60 h at room temperature. The

solution was then diluted with water and extracted with AcOEt.

The organic layer was washed with water and brine, dried over

Na₂SO₄, evaporated in vacuo to dryness, and purified by flash

chromatography (CH₂Cl₂/MeOH/NH₄OH, 96:4:0.2). The re-

sulting intermediate was dissolved in CH₂Cl₂and treated with

HCl in Et₂O for 1 h. The precipitating yellow solid was filtered to

give the desired hydroxamic acid 19d (187 mg, 80%, hydro-

chloride salt). ¹H NMR (DMSO-d₆) δ: 11.06 (bs, 1 H),

7.86-8.03 (m, 3 H), 7.74 (d, J = 15.55 Hz, 1 H), 7.61-7.70

(m, 4 H), 7.49 (d, J = 15.55 Hz, 1 H), 7.47 (dd, J = 8.22, 7.63 Hz,

1 H), 7.33 (dd, J = 8.22, 2.05 Hz, 1 H), 6.60 (d, J = 15.85 Hz,

1 H), 3.81-4.09 (m, 2 H), 3.39-3.64 (m, 2 H), 3.03-3.32 (m,

4 H), 2.82 (d, J = 4.69 Hz, 3 H). LC-MS (m/e): 392 [M - H]^þ.

(E)-N-Hydroxy-3-(4-{(E)-3-[3-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}phenyl)acrylamide (19e). The hy-

droxamic acid 19e was prepared according to the procedure

described for compound 19c. The hydroxamic acid 19e was

purified by preparative HPLC (200 mg, 30% from 16d, bis-

trifluoroacetate). ¹H NMR (DMSO-d₆, 353 K, þTFA) δ:

7.98-8.16 (m, 2 H), 7.80-7.91 (m, 2 H), 7.82 (d, J = 15.85

Hz, 1 H), 7.77 (d, J = 13.50 Hz, 1 H), 7.61-7.72 (m, 3 H), 7.58 (t,

J = 7.48 Hz, 1 H), 7.50 (d, J = 15.85 Hz, 1 H), 6.63 (d, J = 15.55

Hz, 1 H), 3.86 (s, 2 H), 3.25 (t, J = 4.99 Hz, 4 H), 2.81-2.89 (m,

4 H), 2.80 (s, 3 H). LC-MS (m/e): 406 [M - H]^þ.

(E)-N-Hydroxy-3-(4-{(E)-3-[4-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}phenyl)acrylamide(19f). A mixture of 1-[4-(4-

methylpiperazin-1-yl)phenyl]ethanone (16f, 6.59 g, 30.2 mmol), 17

(5.32 g, 30.2 mmol), and 1.7 M KOH (35.5 mL) in ethanol (90 mL)

was stirred at room temperature overnight. The solution was then

filtered to afford 9.2 g (73%) of the potassium acrylate 18f. LC-

MS (m/e): 377 [M - H]^þ.

18f (9.2 g, 22 mmol) was then dissolved in dry THF (60 mL)

and dry DMF (60 mL), and TEA (6.2 mL, 44 mmol),

NH₂OTHP (3.1 g, 27 mmol), EDC (8.5 g, 44 mmol), and HOBt

(6.0 g, 44 mmol) were added to the resulting slurry. The mixture

was stirred overnight at room temperature, then diluted with

water and extracted with CH₂Cl₂. The organic layer was washed

with water and brine, dried over Na₂SO₄, evaporated in vacuo to

dryness, and purified by flash chromatography (CH₂Cl₂/MeOH/

832 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Thaler et al.

3.02 mmol) and NaH (60% oil dispersion, 140 mg, 3.48 mmol) in

dry THF (10 mL). The solution was stirred for 1 h at room

temperature under N₂and was then partitioned between water

and Et₂O. The organic layer was washed with brine, dried over

Na₂SO₄, and evaporated to dryness. The crude mixture was

purified by column chromatography (petroleum ether/AcOEt,

9:1) to give the (E)-3-(4-dimethoxymethylpyridin-2-yl)acrylic

(CDCl₃) δ: 10.00 (d, J = 0.59 Hz, 1 H), 8.80 (ddd, J = 1.76,

0.59 Hz, 1 H), 8.01 (dddd, J=7.92, 2.05, 0.59 Hz, 1 H), 7.95 (dd,

J =7.92, 0.88 Hz, 1 H), 5.59 (s, 1 H), 3.31 (s, 6 H).

tert-Butyl diethylphosphonoacetate (5.00 g, 19.8 mmol) was

dissolved in dry THF (20 mL) and added dropwise to a stirred

suspension of NaH (60% oil dispersion, 936 mg, 23.4 mmol) in

dry THF (20 mL). After 10 min at room temperature, a solution

of 26 (3.26 g, 18.0 mmol) in dry THF (30 mL) was added

dropwise under N₂, keeping the temperature below 25 ꢀC. The

resulting mixture was stirred for 1 h and then partitioned

between water and AcOEt. The organic layer was dried over

Na₂SO₄and evaporated to dryness. The crude product was

purified by flash chromatography (petroleum ether/AcOEt,

95:5) to obtain 3.86 g (77%) of (E)-3-(5-dimethoxymethylpyr-

idin-2-yl)acrylic acid tert-butyl ester as a pale-yellow oil. The

compound was then stirred for 4 h at room temperature in THF

(27 mL) and 1 M HCl (27 mL), brought to basic pH with 10%

aqueous K₂CO₃, and extracted twice with AcOEt. The com-

bined organic layers were dried over Na₂SO₄and evaporated in

vacuo to give 27 as a white solid (2.97 g, 92%). ¹H NMR

(CDCl₃) δ: 10.13 (s, 1 H), 9.08 (d, J = 1.47 Hz, 1 H), 8.18 (dd,

J=8.07, 2.20 Hz, 1 H), 7.63 (d, J=15.85 Hz, 1 H), 7.57 (d, J=

7.92 Hz, 1 H), 6.99 (d, J =15.55 Hz, 1 H), 1.56 (s, 9 H).

1

acid tert-butyl ester 22 (550 mg, 86%). H NMR (CDCl₃) δ:

8.65 (d, J=4.99 Hz, 1 H), 7.62 (d, J=15.85 Hz, 1 H), 7.46-7.56

(m, 1 H), 7.34 (dd, J=4.99, 1.47 Hz, 1 H), 6.86 (d, J=15.85 Hz,

1 H), 5.41 (s, 1 H), 3.35 (s, 6 H), 1.55 (s, 9 H).

The tert-butyl ester 22 (550 mg, 1.99 mmol) was dissolved in

THF (10 mL) and 1 M HCl (10 mL), stirred overnight at room

temperature, and then heated to 80 ꢀC for 4 h. The mixture was

partitioned between 5% NaHCO₃and AcOEt, and the organic

layer was dried over Na₂SO₄and evaporated in vacuo to afford

186 mg (40%) of (E)-3-(4-formylpyridin-2-yl)acrylic acid tert-

butyl ester. A mixture of the resulting intermediate, acetophe-

none (16a) (96 mg, 0.80 mmol), and 1.7 M KOH (0.52 mL) was

dissolved in THF (8 mL) and stirred at room temperature

overnight. The solution was then partitioned between water

and AcOEt, and the organic layer was dried over Na₂SO₄and

evaporated to dryness. The crude solid was purified by column

chromatography (petroleum ether/AcOEt, 9:1) to afford 168 mg

of tert-butyl (E)-3-[4-((E)-3-oxo-3-phenyl-1-propen-1-yl)pyridin-

2-yl]acrylate 23 (62%). ¹H NMR (DMSO-d₆) δ (ppm): 8.72 (d,

J=4.99 Hz, 1H), 8.28-8.33 (m, 1H), 8.24 (d, J=15.85 Hz, 1H),

8.13-8.22 (m, 2H), 7.81 (dd, J=4.99, 1.47 Hz, 1H), 7.68-7.76

(m, 1H), 7.70 (d, J=15.55 Hz, 1H), 7.57-7.66 (m, 2H), 7.59 (d,

J =15.85 Hz, 1H), 6.91 (d, J= 15.85 Hz, 1H), 1.51 (s, 9H).

A mixture of 23 (168 mg, 0.5 mmol) and TFA (3 mL) in

CH₂Cl₂(9 mL) was stirred for 2 h at room temperature, and then

the solvent was removed in vacuo. The acrylic acid trifluo-

roacetate salt was then dissolved in CH₂Cl₂(5 mL), and TEA

(0.49 mL, 3.5 mmol), EDC (191 mg, 1.0 mmol), HOBt (135 mg,

1.0 mmol), and NH₂OTHP (70 mg, 0.6 mmol) were added. The

solution was stirred overnight at room temperature. The mix-

ture was then partitioned between water and AcOEt, and the

organic layer was dried over Na₂SO₄and evaporated to dryness.

The crude product was purified by column chromatography

(CH₂Cl₂/MeOH/NH₄OH, 97:3:0.1). The resulting intermediate

was then dissolved in CH₂Cl₂and treated with HCl/Et₂O for 2 h

at room temperature. The precipitate was filtered and crystal-

lized from EtOH to give 24 as its hydrochloride salt (32 mg,

20%). ¹H NMR (DMSO-d₆) δ: 11.00 (bs, 1 H), 8.72 (d, J = 4.99

Hz, 1 H), 8.23 (d, J = 15.55 Hz, 1 H), 8.14-8.23 (m, 3 H), 7.85

(dd, J = 4.99, 1.17 Hz, 1 H), 7.71 (d, J = 15.85 Hz, 1 H),

7.66-7.78 (m, 1 H), 7.58-7.66 (m, 2 H), 7.55 (d, J = 15.26 Hz, 1

H), 7.02 (d, J = 15.26 Hz, 1 H). LC-MS (m/e): 295 [M - H]^þ.

(E)-3-(5-Formylpyridin-2-yl)acrylic Acid tert-Butyl Ester (27).

A solution of 6-bromopyridine-3-carbaldehyde (25) (8.01 g, 43.1

mmol), p-toluenesulfonic acid (819 mg, 4.31 mmol), and tri-

methyl orthoformate (TMOF, 9.42 mL, 86.1 mmol) in MeOH

(80 mL) was stirred at room temperature for 3 h. The mixture

was brought to basic pH with 5% aqueous K₂CO₃and extracted

with diethyl ether. The organic layer was dried over Na₂SO₄and

then evaporated to dryness. The crude solid was purified by

silica gel chromatography (petroleum ether/AcOEt, 9:1) to give

7.18 g (72%) of 2-bromo-5-dimethoxymethylpyridine as a pale-

yellow oil. The intermediate (7.16 g, 30.9 mmol) was dissolved in

THF (20 mL) and added dropwise to a stirred solution of

i-PrMgCl (2 M in diethyl ether, 20.1 mL) in THF (20 mL) under

N₂, keeping the temperature below 25 ꢀC. The resulting mixture

was stirred for 4 h, DMF (3.09 mL, 40.1 mmol) was added

dropwise, and the solution was stirred for additional 2 h. The

mixture was then partitioned between water and AcOEt, and the

organic layer was dried over Na₂SO₄and evaporated in vacuo.

The crude product was purified by column chromatography

(petroleum ether/AcOEt, 9:1) to afford 5-dimethoxymethylpyr-

idine-2-carbaldehyde 26 as a yellow oil (3.27 g, 60%). ¹H NMR

(E)-N-Hydroxy-3-[5-((E)-3-oxo-3-phenyl-1-propen-1-yl)pyridin-

2-yl]acrylamide (30a). A mixture of 27 (364 mg, 1.56 mmol), 16a

(188 mg, 1.56 mmol), and 1.7 M KOH (1.8 mL) in MeOH (10 mL)

was stirred at 0 ꢀC for 3 h. The precipitating product was filtered

off to afford the tert-butyl ester 28a as a yellow powder (130 mg,

1

25%). H NMR (CDCl₃) δ: 8.88 (d, 1H), 8.02-8.09 (m, 2H),

7.97 (dd, J=8.22, 2.35 Hz, 1H), 7.81 (d, J=15.85 Hz, 1H), 7.63

(d, J=15.55 Hz, 1H), 7.62 (d, J=15.85 Hz, 1H), 7.59-7.64 (m,

1H), 7.51-7.58 (m, 2H), 7.49 (d, J = 8.22 Hz, 1H), 6.91 (d,

J =15.55 Hz, 1H), 1.56 (s, 9H).

A solution of 28a (130 mg, 0.38 mmol) in CH₂Cl₂(4 mL) and

TFA (1 mL) was stirred for 4 h at room temperature. Then the

solvent was removed in vacuo and the resulting oil was crystal-

lized from Et₂O to give the acrylic acid 29a (165 mg, quantita-

tive, trifluoroacetate salt). ¹H NMR (DMSO-d₆) δ: 9.09 (d, J=

2.05 Hz, 1H), 8.45 (dd, J = 8.22, 2.05 Hz, 1H), 8.14-8.23 (m,

2H), 8.13 (d, J=15.85 Hz, 1H), 7.86 (d, J=8.22 Hz, 1H), 7.79

(d, J=15.55 Hz, 1H), 7.66-7.74 (m, 1H), 7.64 (d, J=15.55 Hz,

1H), 7.54-7.64 (m, 2H), 6.92 (d, J=15.85 Hz, 1H).

HOBt (113 mg, 0.83 mmol), EDC (160 mg, 0.83 mmol), TEA

(126 mg, 1.26 mmol), and NH₂OTHP (59 mg, 0.50 mmol) were

added to a solution of 29a (165 mg, 0.41 mmol) in THF/DMF

(1:1, 10 mL). The mixture was stirred for 6 h at room tempera-

ture and then partitioned between water and Et₂O. The com-

bined organic layers were washed with brine, dried over

Na₂SO₄, evaporated in vacuo, and purified by silica gel chro-

matography (petroleum ether/AcOEt, 4:6). The resulting oil was

dissolved in CH₂Cl₂and treated with HCl/Et₂O for 1.5 h. The

precipitate was filtered to give the desired hydroxamic acid 30a

(47 mg, 33%, hydrochloride salt). ¹H NMR (DMSO-d₆) δ: 9.06

(d, J = 1.89 Hz, 1 H), 8.45 (dd, J = 8.34, 2.04 Hz, 1 H),

8.15-8.23 (m, 2 H), 8.11 (d, J = 15.73 Hz, 1 H), 7.79 (d, J =

15.73 Hz, 1 H), 7.73 (d, J = 7.87 Hz, 1 H), 7.66-7.72 (m, 1 H),

7.57-7.64 (m, 2 H), 7.53 (d, J = 15.73 Hz, 1 H), 7.04 (d, J =

15.42 Hz, 1 H). LC-MS (m/e): 295 [M - H]^þ.

(E)-N-Hydroxy-3-(5-{(E)-3-[2-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (30b). A mixture of

27 (7.0 g, 30 mmol), 1.7 M KOH (14.7 mL), and 16b (5.5 g, 25

mmol) in THF (200 mL) was stirred at room temperature

overnight. Further 27 (0.35 g, 1.5 mmol) was added, and stirring

continued overnight. The mixture was partitioned between

water and AcOEt, and the aqueous layer was washed three

times with AcOEt. The combined collected organic layers were

dried over Na₂SO₄and evaporated in vacuo. The crude solid

was purified by column chromatography (petroleum ether/

AcOEt, 1:1, and then CH₂Cl₂/MeOH/NH₄OH, 485:15:1) to

afford the tert-butyl ester 28b as a yellow powder (9.25 g,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 833

85%). ¹H NMR (CDCl₃) δ: 8.88 (d, J=2.05 Hz, 1 H), 7.92 (dd,

J=8.07, 2.20 Hz, 1 H), 7.71 (d, J=16.14 Hz, 1 H), 7.64 (d, J=

15.85 Hz, 1 H), 7.60-7.64 (m, 1 H), 7.62 (d, J=15.85 Hz, 1 H),

7.46-7.54 (m, 2 H), 7.08-7.18 (m, 2 H), 6.90 (d, J=15.85 Hz,

1 H), 2.98-3.21 (m, 4 H), 2.38-2.54 (m, 4 H), 2.22 (s, 3 H), 1.56

(s, 9 H).

A mixture of 28b (9.25 g, 20.1 mmol) and TFA (2 mL) in

CH₂Cl₂(8 mL) was stirred for 6 h at room temperature. The

solvent was removed in vacuo and the residue triturated in Et₂O

to give the acrylic acid 29b (13.6 g, quantitative, trifluoroacetate

salt). ¹H NMR (CDCl₃) δ: 8.96 (d, J=2.35 Hz, 1 H), 7.97 (dd,

J= 8.22, 2.05 Hz, 1 H), 7.67 (d, J=15.85 Hz, 1 H), 7.47-7.62

(m, 4 H), 7.42 (d, J=16.14 Hz, 1 H), 7.08-7.27 (m, 2 H), 6.91 (d,

J = 15.85 Hz, 1 H), 3.50-3.71 (m, 2 H), 3.21-3.48 (m, 4 H),

2.89-3.13 (m, 2 H), 2.80 (s, 3 H).

29b (13.6 g, 20.1 mmol) was suspended in DMF (100 mL) and

CH₂Cl₂(50 mL). TEA (8.91 mL, 64.1 mmol), HOBt (5.77 g, 42.7

mmol), EDC (8.16 g, 42.7 mmol), and NH₂OTHP (2.25 g, 19.2

mmol) were added, and the resulting mixture was stirred at room

temperature for 4 h. The solution was then partitioned between

water and AcOEt, and the organic layer was washed with water,

dried over Na₂SO₄, and evaporated in vacuo. The crude solid

was purified by column chromatography (CH₂Cl₂/MeOH/

NH₄OH from 98:2:0.2 to 96:4:0.4). The resulting intermediate

was dissolved in CH₂Cl₂and treated with HCl/Et₂O for 8 h. The

solvent was evaporated, the residue was dissolved in 2 M HCl

and washed with AcOEt. The aqueous layer was freeze-dried

to give the tris-hydrochloride salt of the hydroxamic acid 30b

(5.5 g, 55%). ¹H NMR (DMSO-d₆) δ: 11.34 (bs, 1 H), 9.01 (d,

J = 1.76 Hz, 1 H), 8.39 (dd, J = 8.22, 2.05 Hz, 1 H), 7.76 (d, J =

8.22 Hz, 1 H), 7.68 (d, J = 16.14 Hz, 1 H), 7.61 (d, J = 16.14 Hz,

1 H), 7.47-7.61 (m, 3 H), 7.29 (d, J = 7.92 Hz, 1 H), 7.21 (t, J =

7.34 Hz, 1 H), 7.06 (d, J = 15.55 Hz, 1 H), 3.35-3.50 (m, 2 H),

3.22-3.34 (m, 4 H), 2.77-3.01 (m, 2 H), 2.66 (d, J = 4.40 Hz,

3 H). LC-MS (m/e): 393 [M - H]^þ.

(E)-N-Hydroxy-3-(5-{(E)-3-[2-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (30c). A mix-

ture of 16c (829 mg, 3.57 mmol), the tert-butyl ester 27 (1.00 g,

4.29 mmol), and 1.7 M KOH (2.09 mL) in THF (40 mL) was

stirred overnight at room temperature. The resulting solution was

diluted with water and extracted with AcOEt. The organic layer

was dried over Na₂SO₄and evaporated in vacuo. The crude reac-

tion mixture was purified by column chromatography (eluent,

from petroleum ether/AcOEt, 1:1, to CH₂Cl₂/MeOH/NH₄OH,

93:7:0.2) to give the tert-butyl ester 28c (1.31 g, 82%). ¹H NMR

(CDCl₃) δ: 8.74 (d, J=2.05 Hz, 1 H), 7.85 (dd, J=8.22, 2.35 Hz,

1 H), 7.59 (d, J = 15.85 Hz, 1 H), 7.45 (d, J = 8.22 Hz, 1 H),

7.31-7.42 (m, 4 H), 7.22 (d, J = 16.43 Hz, 1 H), 7.11 (d, J =

16.43 Hz, 1 H), 6.88 (d, J=15.55 Hz, 1 H), 3.60 (s, 2 H), 2.21-2.53

(m, 8 H), 2.18 (s, 3 H), 1.54 (s, 9 H).

J = 15.55 Hz, 1 H), 3.58 (s, 2 H), 2.27-2.33 (m, 4 H), 2.11-2.21

(m, 4 H), 2.05 (s, 3 H). LC-MS (m/e): 407 [M - H]^þ.

(E)-N-Hydroxy-3-(5-{(E)-3-[3-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (30d). A mixture of

27 (610 mg, 2.62 mmol), 1.7 M KOH (1.54 mL), and 16d (521 mg,

2.38 mmol) in THF (3 mL) was stirred at 0 ꢀC for 2 h. The resulting

solution was partitioned between water and AcOEt, and the

organic layer was dried over Na₂SO₄and evaporated in vacuo.

The crude mixture was used in the next step without any further

purification (1.00 g, 97%). ¹H NMR (DMSO-d₆) δ: 9.07 (d, J =

2.05 Hz, 1 H), 8.42 (dd, J=8.22, 2.35 Hz, 1 H), 8.08 (d, J=15.85

Hz, 1 H), 7.85 (d, J=8.22 Hz, 1 H), 7.76 (d, J=15.85 Hz, 1 H),

7.53-7.65 (m, 2 H), 7.59 (d, J=15.55 Hz, 1 H), 7.41 (t, J=8.07

Hz, 1 H), 7.15-7.31 (m, 1 H), 6.87 (d, J = 15.85 Hz, 1 H),

3.09-3.27 (m, 4 H), 2.42-2.50 (m, 4 H), 2.24 (s, 3 H), 1.51 (s, 9 H).

The resulting tert-butyl ester 28d (1.00 g, 2.31 mmol) was

dissolved in CH₂Cl₂(5 mL) and TFA (2.5 mL). The solution was

stirred at room temperature for 18 h. The solvent was then

removed and the residue was triturated in MeOH to afford 29d

(0.90 g, 64%, bis-trifluoroacetate salt). 29d (900 mg, 1.49 mmol)

was dissolved in DMF (9 mL), and HOBt (372 mg, 2.75 mmol),

EDC (527 mg, 2.75 mmol), TEA (0.254 mL, 1.83 mmol), and

NH₂OTHP (322 mg, 2.75 mmol) were added. The mixture was

stirred at room temperature for 7 h and then partitioned

between water and AcOEt. The organic layer was dried over

Na₂SO₄and evaporated in vacuo, and the crude product was

purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH,

98:2:0.2). The resulting product was dissolved in CH₂Cl₂and

treated with HCl/Et₂O for 1 h. The precipitate was filtered off to

provide the desired hydroxamic acid 30d (290 mg, 42%, bis-

1

hydrochloride salt). H NMR (DMSO-d₆) δ: 11.09 (bs, 1 H),

9.08 (d, J = 1.76 Hz, 1 H), 8.45 (dd, J = 8.22, 2.05 Hz, 1 H), 8.09

(d, J = 15.55 Hz, 1 H), 7.78 (d, J = 15.85 Hz, 1 H), 7.61-7.78

(m, 3 H), 7.54 (d, J = 15.26 Hz, 1 H), 7.48 (dd, 1 H), 7.34 (dd,

J = 8.22, 1.76 Hz, 1 H), 7.04 (d, J = 15.55 Hz, 1 H), 3.80-4.16

(m, 2 H), 3.42-3.64 (m, 2 H), 3.02-3.32 (m, 4 H), 2.82 (d, J =

4.70 Hz, 3 H). LC-MS (m/e): 393 [M - H]^þ.

(E)-N-Hydroxy-3-(5-{(E)-3-[3-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (30e). KOH

(1.7 M, 0.634 mL) was added dropwise to a stirred mixture of 27

(250 mg, 1.08 mmol) and 16e (250 mg, 1.08 mmol) in EtOH

(15 mL). The resulting solution was stirred at 0 ꢀC for 7 h and then

diluted with water and extracted with AcOEt. The organic layer

was dried over Na₂SO₄and evaporated in vacuo. The crude pro-

duct was purified by chromatographic column (CH₂Cl₂/MeOH/

NH₄OH from 97:3:0.1 to 95:5:0.2), and the desired tert-butyl ester

was dissolved in CH₂Cl₂(4 mL) and TFA (1 mL). The mixture was

stirred at room temperature for 6 h and then the solvent was

removed in vacuo to give the desired carboxylic acid 29e (200 mg,

tris-trifluoroacetate salt). LC-MS (m/e): 392 [M - H]^þ.

The resulting tert-butyl ester 28c (1.31 g, 2.93 mmol) was

dissolved in CH₂Cl₂(15 mL) and TFA (4 mL) and stirred at

room temperature for 6 h. The solvent was evaporated to give

the carboxylic acid 29c (2.12 g, 99%, tris-trifluoroacetate salt).

A mixture of the carboxylic acid 29c (2.12 g, 2.90 mmol), HOBt

(783 mg, 5.8 mmol), EDC (1.1 g, 5.8 mmol), TEA (1.2 mL, 8.7

mmol), and NH₂OTHP (306 mg, 2.61 mmol) in DMF (15 mL)

was then stirred overnight at room temperature. The solution

was diluted with water, brought to basic conditions with

NH₄OH, and extracted with AcOEt and CH₂Cl₂. The combined

organic layers were dried over Na₂SO₄and evaporated in vacuo.

The crude mixture was purified by silica gel chromatography

(CH₂Cl₂/MeOH/NH₄OH, 96:4:0.2), and the resulting product

was dissolved in CH₂Cl₂and treated with HCl/Et₂O for 2 h. The

hygroscopic precipitate was filtered, crystallized from isopro-

panol, and freeze-dried to give the requisite hydroxamic acid 30c

(73.3 mg, 5%, tris-hydrochloride). ¹H NMR (DMSO-d₆, 353 K,

þNa₂CO₃) δ: 8.79 (d, J = 2.05 Hz, 1 H), 8.09 (dd, J = 8.22,

2.35 Hz, 1 H), 7.52 (d, J = 8.22 Hz, 1 H), 7.31-7.48 (m,

4 H), 7.25-7.30 (m, 2 H), 7.23 (d, J = 15.55 Hz, 1 H), 6.92 (d,

A mixture of the unpurified 29e (194 mg), HOBt (83 mg, 0.616

mmol), EDC (118 mg, 0.616 mmol), TEA (0.127 mL, 0.920

mmol), and NH₂OTHP (44 mg, 0.38 mmol) in DMF (10 mL)

was stirred at room temperature for 5 h. The resulting solution

was diluted with water and extracted with AcOEt and CH₂Cl₂.

The combined organic layers were washed with brine, dried over

Na₂SO₄, and evaporated in vacuo. The crude mixture was

purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH

from 97:3:0.1 to 95:5:0.2), and the resulting product was dis-

solved in CH₂Cl₂and treated with HCl/Et₂O for 3 h. The

precipitate was filtered and purified by preparative HPLC to

give the desired hydroxamic acid 30e (9 mg, 1% from 16e, tris-

trifluoroacetate salt). ¹H NMR (DMSO-d₆þTFA) δ: 11.20 (bs,

1 H), 9.09 (d, J = 2.05 Hz, 1 H), 8.50-8.60 (m, 1 H), 8.47 (dd,

J = 8.07, 2.20 Hz, 1 H), 8.15-8.22 (m, 1 H), 8.21 (d, J =

15.85 Hz, 1 H), 7.86-7.93 (m, 1 H), 7.83 (d, J = 15.85 Hz, 1 H),

7.70 (d, J = 7.92 Hz, 1 H), 7.67 (t, J = 7.63 Hz, 1 H), 7.54 (d,

J = 15.26 Hz, 1 H), 7.03 (d, J = 15.55 Hz, 1 H), 4.44 (bs,

2 H), 3.09-3.91 (m, 8 H), 2.84 (s, 3 H). LC-MS (m/e): 407

[M - H]^þ.

834 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Thaler et al.

(E)-N-Hydroxy-3-(5-{(E)-3-[4-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (30f). A mixture of

27 (261 mg, 1.12 mmol), 16f (246 mg, 1.12 mmol), and 1.7 M

KOH (125 mg, 2.24 mmol) in EtOH (10 mL) was stirred at room

temperature for 16 h. The formation of a precipitate could be

observed, and the solid was then filtered off to obtain the tert-

butyl ester 28f (222 mg, 45%). 28f (222 mg, 0.513 mmol) was

then treated with TFA (2 mL) in CH₂Cl₂(5 mL) at room

temperature for 5 h. The solvent was evaporated in vacuo to

dryness to give the desired carboxylic acid 29f (310 mg, quanti-

tative, bis-trifluoroacetate). A mixture of 29f (310 mg, 0.51

mmol), NH₂OTHP (78 mg, 0.67 mmol), EDC (155 mg, 0.81

mmol), HOBt (109 mg, 0.80 mmol), and TEA (280 μL, 2 mmol)

in THF (5 mL) and DMF (5 mL) was stirred for 72 h at room

temperature. The mixture was diluted with water and extracted

with AcOEt. The organic layer was washed with water, then

with brine, dried over Na₂SO₄, and evaporated in vacuo to

dryness. The crude product was purified by flash chromatogra-

phy (CH₂Cl₂/MeOH/NH₃, 96:4:0.2). The obtained intermediate

was dissolved in CH₂Cl₂and treated with HCl in Et₂O for 3 h to

obtain a dark-brown solid. The solid was then filtered off and

washed with CH₂Cl₂to obtain the requisite hydroxamic acid 30f

and purified by column chromatography (petroleum ether/

AcOEt, 95:5) to give the desired intermediate 32a as a pale-

yellow oil (3.5 g, 56%). ¹H NMR (DMSO-d₆) δ: 10.04 (s, 1 H),

8.24 (t, J=1.76 Hz, 1 H), 8.04 (dt, J=7.70, 1.28 Hz, 1 H), 7.92

(dt, J=7.63, 1.32 Hz, 1 H), 7.65 (d, J=16.14 Hz, 1 H), 7.64 (t,

J =7.63 Hz, 1 H), 6.65 (d, J =16.14 Hz, 1 H), 1.50 (s, 9 H).

(E)-N-Hydroxy-3-[3-((E)-3-oxo-3-phenyl-1-propen-1-yl)phenyl]-

acrylamide (35a). The hydroxamic acid 35a was prepared ac-

cording to the procedure described for compound 19a. Yield:

105 mg (64%). ¹H NMR (DMSO-d₆) δ: 10.77 (s, 1H), 9.03

(bs, 1H), 8.15-8.22 (m, 2H), 8.11-8.18 (m, 1H), 8.01 (d, J =

15.55 Hz, 1H), 7.87 (d, J=7.63 Hz, 1H), 7.77 (d, J=15.85 Hz,

1H), 7.66-7.73 (m, 1H), 7.44-7.67 (m, 5H), 6.58 (d, J = 15.85

Hz, 1H). LC-MS (m/e): 294 [M - H]^þ.

(E)-N-Hydroxy-3-(3-{(E)-3-[2-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}phenyl)acrylamide (35b). A mixture of 32a

(220 mg, 0.95 mmol), 16b (200 mg, 0.92 mmol), and 1.7 M KOH

(102 mg, 1.82 mmol) in EtOH (5 mL) and H₂O (1 mL) was

stirred at room temperature for 12 h. The mixture was then

partitioned between water and AcOEt, and the organic layer

was dried over Na₂SO₄and evaporated in vacuo. The crude

product was dissolved in CH₂Cl₂(2 mL) and TFA (1 mL), and

the resulting solution was stirred for 6 h at room temperature.

The solvent was then removed in vacuo to give the carboxylic

acid 34b (0.220 g, 49% from 32a, trifluoroacetate salt). 34b (220mg,

0.45 mmol), HOBt (95 mg, 0.702 mmol), EDC (134 mg, 0.70

mmol), TEA (0.245 mL, 1.75 mmol), and NH₂OTHP (68 mg, 0.58

mmol) were dissolved in THF (5 mL) and DMF (1 mL), stirred for

12 h at room temperature, and then partitioned between water and

AcOEt. The organic layer was dried over Na₂SO₄and evaporated

in vacuo, and the crude product was purified by column chroma-

tography (AcOEt to AcOEt/MeOH, 9:1). The resulting compound

was dissolved in CH₂Cl₂and treated with HCl/Et₂O for 2 h. The

precipitate was filtered and purified by preparative HPLC to obtain

the hydroxamic acid 35b as its trifluoroacetate salt (53 mg, 23%).

¹HNMR(DMSO-d₆) δ:10.75(bs, 1H), 9.69(bs, 1H),9.05(s, 1H),

7.98 (s, 1 H), 7.79 (d, J = 7.34 Hz, 1 H), 7.65 (d, J = 7.63 Hz, 1 H),

7.43-7.60 (m, 6 H), 7.29 (d, J = 7.63 Hz, 1 H), 7.21 (td, J = 7.50,

0.88 Hz, 1 H), 6.55 (d, J = 15.85 Hz, 1 H), 3.23-3.53 (m, 4 H),

3.02-3.22 (m, 2 H), 2.84-3.00 (m, 2 H), 2.71 (s, 3 H). LC-MS

(m/e): 392 [M - H]^þ.

(E)-N-Hydroxy-3-(3-{(E)-3-[2-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}phenyl)acrylamide (35c). KOH (1.7 M,

0.634 mL) was added dropwise to a stirred mixture of 16c (250 mg,

1.08 mmol) and 32a (250 mg, 1.08 mmol) in EtOH (15 mL) at 0 ꢀC.

The resulting mixture was stirred at 0 ꢀC for 4 h and then parti-

tioned between water and AcOEt. The organic layer was dried over

Na₂SO₄and evaporated in vacuo. The crude product was purified

by column chromatography (petroleum ether/AcOEt, 7:3, and then

CH₂Cl₂/MeOH/NH₄OH, 95:5:0.1) to give the tert-butyl acrylate

33c (384 mg, 80%). LC-MS (m/e): 447 [M - H]^þ.

The tert-butyl acrylate 33c (384 mg, 0.861 mmol) was dis-

solved in a mixture of TFA (1.5 mL) and CH₂Cl₂(6 mL) and

stirred for 6 h at room temperature. The solvent was removed in

vacuo, and the residue was dissolved in DMF (15 mL). HOBt

(232 mg, 1.72 mmol), EDC (328 mg, 1.71 mmol), TEA (0.239 mL,

1.72 mmol), and NH₂OTHP (121 mg, 1.03 mmol) were added,

and the resulting mixture was stirred at room temperature for

5 h. Water was added, and the product was extracted with AcOEt

followed by CH₂Cl₂. The combined organic layers were dried

over Na₂SO₄and evaporated in vacuo. The crude product was

purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH,

97:3:0.1), and the resulting product was dissolved in CH₂Cl₂and

treated with HCl/Et₂O for 2 h. The precipitate was filtered,

crystallized from i-PrOH/MeOH/diisopropyl ether and then

purified by preparative HPLC to give the desired hydroxa-

mic acid 35c (124 mg, 23% from 33c, bis-trifluoroacetate salt).

¹H NMR (DMSO-d₆, 353 K) δ: 7.91 (s, 1 H), 7.72 (d, J = 7.63

Hz, 1 H), 7.59-7.68 (m, 3 H), 7.56 (td, J = 7.34, 1.47 Hz, 1 H),

7.41-7.53 (m, 4 H), 7.37 (d, J=16.14 Hz, 1 H), 6.65 (d, J=15.55 Hz,

1

(156 mg, 66%, dihydrochloride salt). H NMR (DMSO-d₆) δ:

11.37 (bs, 1 H), 9.09 (d, J = 1.76 Hz, 1 H), 8.53 (dd, J = 8.22,

2.05 Hz, 1 H), 8.15 (d, J = 15.85 Hz, 1 H), 8.13 (m, 2 H), 7.81 (d,

J = 8.22 Hz, 1 H), 7.73 (d, J = 15.55 Hz, 1 H), 7.56 (d, J = 15.55

Hz, 1 H), 7.12 (m, 2 H), 7.08 (d, J = 15.55 Hz, 1 H), 4.03-4.19

(m, 2 H), 3.44-3.55 (m, 2 H), 3.26-3.42 (m, 2 H), 3.01-3.22 (m,

2 H), 2.80 (d, J = 4.69 Hz, 3 H). LC-MS (m/e): 393 [M - H]^þ.

(E)-N-Hydroxy-3-(5-{(E)-3-[4-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (30g). KOH

(1.7 M, 1.54 mL) was added dropwise to a stirred mixture of 27

(610 mg, 2.62 mmol) and 16g (608 mg, 2.62 mmol) in THF (20 mL).

The resulting solution was stirred at room temperature overnight

and then diluted with water and extracted with AcOEt. The organic

layer was dried over Na₂SO₄and evaporated in vacuo. The crude

product was purified by chromatographic column (petroleum

ether/AcOEt, then CH₂Cl₂/MeOH/NH₄OH, 485:15:1) to give the

tert-butyl ester 28g (620 mg, 53%). LC-MS (m/e): 448 [M - H]^þ.

28g (620 mg, 1.39 mmol) was dissolved in CH₂Cl₂(4.5 mL)

and TFA (1.5 mL), and the mixture was stirred at room tem-

perature for 6 h. Then the solvent was removed in vacuo to give

the desired carboxylic acid 29g as its tris-trifluoroacetate salt

(1.02 g). A mixture of 29g (1.02 g), HOBt (375 mg, 2.78 mmol),

EDC (531 mg, 2.78 mmol), TEA (0.576 mL, 4.17 mmol), and

NH₂OTHP (146 mg, 1.28 mmol) in DMF (18 mL) was then

stirred at room temperature for 5 h. The resulting solution was

diluted with water and extracted with AcOEt and CH₂Cl₂. The

combined organic layers were washed with brine, dried over

Na₂SO₄, and evaporated in vacuo. The crude mixture was

purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH

from 485:15:1 to 480:20:1), and the resulting product was

dissolved in CH₂Cl₂and treated with HCl/Et₂O for 3 h. The

precipitate was filtered, washed with CH₂Cl₂, and freeze-dried

to give the desired hydroxamic acid 30g (85.3 mg, 12% from 28g,

tris-hydrochloride salt). ¹H NMR (DMSO-d₆þ TFA) δ: 11.20

(bs, 1 H), 9.06 (d, J = 2.18 Hz, 1 H), 8.42 (dd, J = 8.21, 2.25 Hz,

1 H), 8.23 (m, 2 H), 8.12 (d, J = 15.70 Hz, 1 H), 7.80 (d, J =

15.85 Hz, 1 H), 7.78 (m, 2 H), 7.71 (d, J = 8.16 Hz, 1 H), 7.53 (d,

J = 15.36 Hz, 1 H), 7.02 (d, J = 15.42 Hz, 1 H), 4.31 (bs, 2 H),

2.95-3.76 (m, 8 H), 2.82 (s, 3 H). LC-MS (m/e): 407 [M - H]^þ.

tert-Butyl (E)-3-(3-Formylphenyl)acrylate (32a). 3-Bromo-

benzaldehyde (31a, 5.0 g, 27 mmol) was dissolved in DMF (70

mL) and TEA (8.5 mL, 61 mmol). PPh₃(354 mg, 1.35 mmol),

Pd(OAc)₂(121 mg, 0.54 mmol), NaHCO₃(4.5 g, 54 mmol), and

tert-butyl acrylate (4.0 mL, 27 mmol) were added under N₂

atmosphere, and the solution was heated to 100 ꢀC for 3 h.

Additional Pd(OAc)₂(60 mg, 0.26 mmol) was added, and after

1 h at 100 ꢀC the slurry was partitioned between water and Et₂O.

The organic layer was dried over Na₂SO₄, evaporated in vacuo,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 835

1 H), 3.97 (s, 2 H), 3.08-3.36 (m, 4 H), 2.93 (bs, 4 H), 2.69 (s,

3 H). LC-MS (m/e): 406 [M - H]^þ.

stirred at room temperature for 6 h. The mixture was diluted

with water, and the resulting precipitate was filtered off and

dried. The solid was dissolved in CH₂Cl₂(4 mL) and TFA (2 mL),

and the solution was stirred at room temperature overnight. The

solvent was then removed in vacuo and the product was

triturated with diisopropyl ether to give the carboxylic acid

34f (0.360 g, trifluoroacetate salt). 34f (150 mg) was then

dissolved in THF (5 mL) and DMF (1 mL). HOBt (65 mg,

0.47 mmol), EDC (90 mg, 0.47 mmol), TEA (0.166 mL, 1.19

mmol), and NH₂OTHP (46 mg, 0.40 mmol) were added, and the

mixture was stirred at room temperature overnight. The mixture

was partitioned between water and AcOEt. The organic layer

was dried over Na₂SO₄and evaporated in vacuo, and the crude

product was purified by column chromatography (AcOEt to

AcOEt/MeOH, 9:1). The resulting intermediate (70 mg, yellow

powder) was dissolved in CH₂Cl₂and treated with HCl/Et₂O for

2 h. The precipitate was filtered and triturated with MeOH/H₂O

to give the hydroxamic acid 35f (35 mg, 11% from 32a, hydro-

chloride salt). ¹H NMR (DMSO-d₆þ TFA) δ: 10.36 (bs, 1 H),

8.07-8.17 (m, 3 H), 8.00 (d, J = 15.55 Hz, 1 H), 7.85 (d, J =

7.63 Hz, 1 H), 7.69 (d, J = 15.55 Hz, 1 H), 7.63 (d, J = 7.63 Hz,

1 H), 7.52 (d, J = 15.85 Hz, 1 H), 7.49 (t, J = 7.63 Hz, 1 H), 7.12

(m, 2 H), 6.58 (d, J = 15.85 Hz, 1 H), 4.01-4.22 (m, 2 H),

3.38-3.69 (m, 2 H), 3.04-3.32 (m, 4 H), 2.85 (s, 3 H). LC-MS

(m/e): 392 [M - H]^þ.

(E)-N-Hydroxy-3-(3-{(E)-3-[3-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}phenyl)acrylamide (35d). A solution of 16d

(350 mg, 1.6 mmol) in EtOH (5 mL) was added within 2 h to a

stirred mixture of 32a (372 mg, 1.6 mmol) and 1.7 M KOH

(89 mg, 1.6 mmol) in EtOH (10 mL) at -20 ꢀC. The resulting

mixture was stirred at room temperature overnight and then

partitioned between water and AcOEt. The organic layer was

dried over Na₂SO₄and evaporated in vacuo. The crude product

was dissolved in CH₂Cl₂(10 mL) and TFA (2 mL), and the

resulting solution was stirred at room temperature for 6 h. The

solvent was then removed in vacuo and the residue was tritu-

rated with Et₂O to give 308 mg of the carboxylic acid 34d as its

trifluoroacetate salt. The crude 34d (308 mg) was then dissolved

in DMF (7 mL), and HOBt (170 mg, 1.25 mmol), EDC (240 mg,

1.25 mmol), TEA (262 μL, 1.88 mmol), and NH₂OTHP (88 mg,

0.75 mmol) were added. The mixture was stirred overnight at

room temperature and then partitioned between water and

AcOEt. The organic layer was dried over Na₂SO₄and evapo-

rated in vacuo, and the crude reaction mixture was purified by

column chromatography (CH₂Cl₂/MeOH/NH₄OH, 98:2:0.2).

The resulting product was dissolved in CH₂Cl₂and treated with

HCl/Et₂O for 1 h. The precipitate was filtered and purified by

preparative HPLC, providing the requisite hydroxamic acid 35d

(24.7 mg, 3% from 32a, trifluoroacetate salt). ¹H NMR

(DMSO-d₆þ TFA) δ: 9.74 (bs, 1 H), 8.10 (s, 1 H), 7.96 (d,

J=15.55Hz,1H),7.87(d,J=7.34 Hz, 1 H), 7.75 (d, J=15.85 Hz,

1 H), 7.59-7.71 (m, 3 H), 7.40-7.58 (m, 3 H), 7.34 (dd, J = 7.92,

2.35 Hz, 1 H), 6.57 (d, J = 15.85 Hz, 1 H), 3.87-4.08 (m, 2 H),

3.40-3.70 (m, 2 H), 2.97-3.30 (m, 4 H), 2.89 (s, 3 H). LC-MS

(m/e): 392 [M - H]^þ.

(E)-N-Hydroxy-3-(3-{(E)-3-[3-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}phenyl)acrylamide (35e). KOH (1.7 M,

0.634 mL) was added dropwise to a stirred mixture of 16e (250 mg,

1.08 mmol) and 32a (250 mg, 1.08 mmol) in EtOH (15 mL) at 0 ꢀC.

The resulting mixture was stirred at 0 ꢀC for 4 h and then

partitioned between water and AcOEt. The organic layer was dried

over Na₂SO₄and evaporated to dryness. The crude product was

purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH

from 485:15:1 to 465:35:1) to give the tert-butyl acrylate 33e

(330 mg, 69%). LC-MS (m/e): 447 [M - H]^þ.

33e (330 mg, 0.740 mmol) was dissolved in a mixture of TFA

(1 mL) and CH₂Cl₂(4 mL) and stirred for 6 h at room

temperature. The solvent was removed in vacuo, and the residue

was dissolved in DMF (15 mL). HOBt (200 mg, 1.48 mmol),

EDC (283 mg, 1.48 mmol), TEA (0.206 mL, 1.48 mmol) and

NH₂OTHP (104 mg, 0.888 mmol) were added, and the resulting

mixture was stirred at room temperature overnight. Then water

was added, and the product was extracted with AcOEt followed

by CH₂Cl₂. The combined organic layers were dried over

Na₂SO₄and evaporated in vacuo. The crude product was

purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH

from 485:15:1 to 475:25.1), then dissolved in CH₂Cl₂and treated

with HCl/Et₂O for 2 h. The precipitate was filtered, crystallized

from i-PrOH/MeOH/diisopropyl ether and purified by prepara-

tive HPLC to give the desired hydroxamic acid 35e (83.7 mg,

18% from 33e, bis-trifluoroacetate salt). ¹H NMR (DMSO-d₆)

δ: 8.12-8.18 (m, 1 H), 8.09 (dt, J = 7.34, 1.76 Hz, 1 H),

7.98-8.05 (m, 1 H), 7.87 (d, J = 15.55 Hz, 1 H), 7.82 (dt, J =

8.22, 1.47 Hz, 1 H), 7.74 (d, J = 15.55 Hz, 1 H), 7.71 (dt, J =

7.34, 1.47 Hz, 1 H), 7.64 (dt, J = 7.70, 1.28 Hz, 1 H), 7.59 (t, J =

7.78 Hz, 1 H), 7.53 (d, J = 15.55 Hz, 1 H), 7.50 (t, J = 7.63 Hz,

1 H), 6.67 (d, J = 15.55 Hz, 1 H), 3.96 (s, 2 H), 3.22-3.41 (m,

4 H), 2.88-3.05 (m, 4 H), 2.79 (s, 3 H). LC-MS (m/e): 406

[M - H]^þ.

(E)-N-Hydroxy-3-(3-{(E)-3-[4-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}phenyl)acrylamide (35g). A mixture

of 32a (209 mg, 0.90 mmol), 16g (194 mg, 0.83 mmol), and 1.7 M

KOH (0.512 mL) in EtOH (10 mL) was stirred at 0-4 ꢀC

overnight and then partitioned between water and AcOEt.

The organic layer was dried over Na₂SO₄and evaporated in

vacuo. The crude mixture was purified by chromatographic

column (CH₂Cl₂/MeOH/NH₄OH, 97:3:0.1) to give the tert-

1

butyl acrylic ester 33g (148 mg, 40%). H NMR (DMSO-d₆)

δ: 8.16 (s, 1 H), 8.07 (m, 2 H), 7.89 (d, J = 15.85 Hz, 1 H),

7.78-7.84 (m, 1 H), 7.72 (d, J=15.85 Hz, 1 H), 7.66-7.73 (m,

1 H), 7.60 (d, J=15.85 Hz, 1 H), 7.41-7.53 (m, 3 H), 6.61 (d, J=

15.85 Hz, 1 H), 3.59 (s, 2 H), 2.42-2.48 (m, 4 H), 2.33-2.41 (m,

4 H), 2.20 (s, 3 H), 1.53 (s, 9 H).

33g (148 mg, 0.33 mmol) was dissolved in a mixture of TFA

(0.50 mL) and CH₂Cl₂(1 mL) and stirred at room temperature

for 4 h. The solvent was removed in vacuo, and the residue was

dissolved in CH₂Cl₂(3 mL) and DMF (1 mL). HOBt (81 mg, 0.6

mmol), EDC (115 mg, 0.59 mmol), TEA (0.167 mL, 1.2 mmol),

and NH₂OTHP (70 mg, 0.6 mmol) were added, and the resulting

mixture was stirred at room temperature overnight. CH₂Cl₂was

removed and the residue partitioned between water and AcOEt.

The organic layer was washed with water, dried over Na₂SO₄,

and evaporated in vacuo. The crude mixture was purified by

column chromatography (CH₂Cl₂/MeOH/NH₄OH, 95:5:0.1),

and the resulting product was dissolved in CH₂Cl₂and treated

with HCl/Et₂O for 3 h. The precipitate was filtered, crystallized

from i-PrOH, and then triturated with MeOH to give hydro-

1

xamic acid 35g (54 mg, 34% from 33g, bis-hydrochloride). H

NMR (DMSO-d₆, 353 K) δ: 8.13 (m, 2 H), 8.03 (t, J = 1.17 Hz,

1 H), 7.87 (d, J = 15.85 Hz, 1 H), 7.81 (dt, J = 7.63, 1.17 Hz,

1 H), 7.74 (d, J = 15.85 Hz, 1 H), 7.69 (m, 2 H), 7.60-7.66 (m,

1 H), 7.53 (d, J = 15.85 Hz, 1 H), 7.50 (t, J = 7.63 Hz, 1 H), 6.67

(d, J = 16.14 Hz, 1 H), 4.05 (s, 2 H), 3.22-3.49 (m, 4 H),

2.96-3.21 (m, 4 H), 2.78 (s, 3 H). LC-MS (m/e): 406 [M - H]^þ.

tert-Butyl (E)-3-(6-Formylpyridin-2-yl)acrylate (32b). Three

batches of 6-bromopyridine-2-carbaldehyde (31b, 1.0 g, 5.4 mmol),

tert-butyl acrylate (2.1 g, 16 mmol), 1,4-diazabicyclo[2.2.2]octane

(DABCO, 25 mg, 0.22 mmol), K₂CO₃(734 mg, 5.31 mmol),

Bu₄NBr (1.73 g, 5.38 mmol), and Pd(OAc)₂(25 mg, 0.11 mmol)

in DMF (10 mL) were heated by microwave irradiation at 120 ꢀC

for 5 h. The mixtures were combined and partitioned between

water and Et₂O. The organic layer was washed with water, dried

over Na₂SO₄, evaporated to dryness, and purified by column

chromatography (petroleum ether/AcOEt, 95:5) to afford 32b

(E)-N-Hydroxy-3-(3-{(E)-3-[4-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}phenyl)acrylamide (35f). A mixture of 32a

(425 mg, 1.83 mmol), 16f (400 mg, 1.83 mmol), and 1.7 M KOH

(202 mg, 3.60 mmol) in EtOH (5 mL) and H₂O (1 mL) was

836 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Thaler et al.

(2.8 g, 57%). ¹H NMR (CDCl₃) δ: 10.08 (d, J = 0.61 Hz, 1 H),

7.80-7.97 (m, 2 H), 7.65 (d, J=15.55 Hz, 1 H), 7.62 (dd, 1 H), 6.98

(d, J= 15.69 Hz, 1 H), 1.56 (s, 9 H).

(E)-N-Hydroxy-3-(6-{(E)-3-[2-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (35j). A so-

lution of 16c (110 mg, 0.47 mmol) in THF (4 mL) was added

dropwise within 30 min to a stirred solution of 32b (110 mg, 0.47

mmol) and 1.7 M KOH (0.276 mL) in THF (10 mL) at 0 ꢀC. The

mixture was stirred at 0 ꢀC for 1 h and then at room temperature

overnight. The solution was partitioned between water and

AcOEt, and the organic layer was dried over Na₂SO₄and

evaporated in vacuo. The crude mixture was purified by column

chromatography (CH₂Cl₂/MeOH/NH₄OH, 98:2:0.1) to give the

tert-butyl ester 33j (135 mg, 64%). LC-MS (m/e): 448 [M - H]^þ.

33j (135 mg, 0.30 mmol) was dissolved in a mixture of TFA

(0.456 mL) and CH₂Cl₂(1 mL) and stirred at room temperature

for 4 h. The solvents were evaporated to dryness and the residue

was dissolved in CH₂Cl₂(4 mL) and TEA (0.581 mL, 4.18 mmol).

EDC (145 mg, 0.756 mmol), HOBt (103 mg, 0.76 mmol), and

NH₂OTHP (66.7 mg, 0.570 mmol) were added, and the resulting

mixture was stirred at room temperature overnight. The solvent

was evaporated, and the residue was partitioned between water

and AcOEt. The organic layer was dried over Na₂SO₄and

evaporated in vacuo. The crude mixture was purified by column

chromatography (CH₂Cl₂/MeOH/NH₄OH, 93:7:0.1) and the

resulting product was dissolved in CH₂Cl₂and treated with

HCl/Et₂O for 1.5 h. The precipitate was filtered off, triturated

with i-PrOH, and purified by preparative HPLC to afford the

requisite hydroxamic acid 35j (36 mg, 16% from 33j, tris-

trifluoroacetate). ¹H NMR (DMSO-d₆þ TFA) δ: 10.92 (bs,

1 H), 9.47 (bs, 1 H), 7.92 (t, J= 7.63 Hz, 1 H), 7.79 (d, J = 7.34 Hz,

1 H), 7.41-7.68 (m, 7 H), 7.30 (d, J = 15.85 Hz, 1 H), 7.00 (d,

J = 15.55 Hz, 1 H), 3.75 (bs, 2 H), 3.24-3.40 (m, 2 H), 2.73-

3.00 (m, 4 H), 2.71 (s, 3 H), 2.30-2.45 (m, 2 H). LC-MS (m/e):

407 [M - H]^þ.

(E)-N-Hydroxy-3-[6-((E)-3-oxo-3-phenyl-1-propen-1-yl)pyridin-

2-yl]acrylamide (35h). 16a (116 μL, 1.0 mmol) was added drop-

wise to a solution of 32b (240 mg, 1.0 mmol) and 1.7 M KOH

(0.90 mL) in EtOH (5 mL), cooled to -15 ꢀC. The mixture was

kept at -15 ꢀC overnight and then at 0 ꢀC for a further 3 h. The

solution was then diluted with water and extracted with AcOEt.

The organic layer was dried over Na₂SO₄and evaporated to

dryness, and the crude product was purified by column chro-

matography (petroleum ether/AcOEt, 8:2). LC-MS (m/e): 336

[M - H]^þ.

The tert-butyl ester 33h was dissolved in CH₂Cl₂(4 mL) and

TFA (1 mL) and stirred for 6 h at room temperature. Then the

solvent was removed in vacuo and the resulting white powder

was triturated with diisopropyl ether to give the acrylic acid 34h

(96 mg, trifluoroacetate salt). HOBt (37 mg, 0.27 mmol), EDC

(56 mg, 0.29 mmol), TEA (103 μL, 0.74 mmol), and NH₂OTHP

(29 mg, 0.25 mmol) were added to 93 mg of 34h dissolved in

THF/DMF (1:1, 10 mL). The mixture was stirred for 12 h at

room temperature and then partitioned between water and

AcOEt. The organic layer was washed with brine, dried over

Na₂SO₄, evaporated in vacuo, and purified by silica gel chro-

matography (CH₂Cl₂/MeOH/NH₄OH, 98:2:0.2). The resulting

oil was dissolved in CH₂Cl₂and treated with HCl/Et₂O for 4 h.

The precipitate was filtered to give the desired hydroxamic acid

35h (40 mg, 12% from 32b, hydrochloric salt). ¹H NMR

(DMSO-d₆) δ: 8.17 (d, J = 15.55 Hz, 1 H), 8.07-8.15 (m,

2 H), 7.96 (t, J = 7.34 Hz, 1 H), 7.90 (d, J = 7.04 Hz, 1 H),

7.55-7.79 (m, 5 H), 7.54 (d, J = 15.55 Hz, 1 H), 7.09 (d, J =

15.26 Hz, 1 H). LC-MS (m/e): 295 [M - H]^þ.

(E)-N-Hydroxy-3-(6-{(E)-3-[2-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (35i). A solution of

32b (200 mg, 0.86 mmol), 16b (187 mg, 0.86 mmol), and 1.7 M

KOH (0.504 mL) in THF (10 mL) was stirred at room tempera-

ture overnight and then partitioned between water and AcOEt.

The aqueous layer was extracted with AcOEt, and the combined

organic layers were dried over Na₂SO₄and evaporated in vacuo.

The crude product was purified by column chromatography

(CH₂Cl₂/MeOH/NH₄OH, 97:3:0.2 to 96:4:0.2) to give the tert-

butyl ester 33i (240 mg, 64%). ¹H NMR (DMSO-d₆) δ: 8.01 (d,

J=15.85 Hz, 1 H), 7.91 (t, J=7.92 Hz, 1 H), 7.66-7.73 (m, 2 H),

7.59 (d, J = 15.55 Hz, 1 H), 7.58 (d, J = 15.55 Hz, 1 H),

7.48-7.55 (m, 2 H), 7.22 (d, J = 9.10 Hz, 1 H), 7.09-7.15 (m,

1 H), 6.91 (d, J=15.55 Hz, 1 H), 3.00-3.05 (m, 2 H), 2.89-2.98

(m, 2 H), 2.35-2.46 (m, 4 H), 2.08 (s, 3 H), 1.53 (s, 9 H).

33i (240 mg, 0.55 mmol) was dissolved in a mixture of TFA

(1 mL) and CH₂Cl₂(4 mL) and stirred at room temperature for

8 h. The solvents were removed in vacuo, and the residue was

dissolved in DMF (15 mL). HOBt (150 mg, 1.11 mmol), EDC

(212 mg, 1.11 mmol), and TEA (0.231 mL, 1.66 mmol) were

added, and the resulting solution was stirred for 10 min.

NH₂OTHP (77.8 mg, 0.665 mmol) was added, and the mixture

was stirred at room temperature for 7 h. Further NH₂OTHP

(7.78 mg, 0.067 mmol) was added, and the solution was stirred at

room temperature overnight and then partitioned between water

and CH₂Cl₂. The aqueous phase was washed twice with CH₂Cl₂,

and the combined organic layers were dried over Na₂SO₄and

evaporated in vacuo. The crude mixture was purified by column

chromatography (CH₂Cl₂/MeOH/NH₄OH, 97:3:0.2 to 95:5:0.2).

The resulting product was dissolved in CH₂Cl₂and treated with

Et₂O/HCl for 4 h. The precipitate was filtered and freeze-dried to

give the desired hydroxamic acid 35i (40 mg, 16% from 33i, bis-

(E)-N-Hydroxy-3-(6-{(E)-3-[3-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (35k). A solution

of 16d (137 mg, 0.63 mmol) in THF (10 mL) was added dropwise

within 30 min to a stirred solution of 32b (146 mg, 0.63 mmol)

and 1.7 M KOH (370 μL) in THF (10 mL) at 0 ꢀC. The mixture

was stirred at 0 ꢀC for 1 h and then at room temperature

overnight. The solution was partitioned between water and

AcOEt, and the organic layer was dried over Na₂SO₄and

evaporated in vacuo. The crude product was purified by column

chromatography (CH₂Cl₂/MeOH/NH₄OH, 98:2:0.1) to give

the tert-butyl ester 33k as an orange solid (188 mg, 69%).

LC-MS (m/e): 434 [M - H]^þ.

33k (188 mg, 0.434 mmol) was then dissolved in a mixture of

TFA (1.5 mL) and CH₂Cl₂(3 mL) and stirred at room tem-

perature for 2 h. The solvents were evaporated to dryness, and

the residue was dissolved in CH₂Cl₂(4 mL) and TEA (0.800 mL,

5.80 mmol). EDC (198 mg, 1.03 mmol), HOBt (140 mg, 1.04

mmol), and NH₂OTHP (100 mg, 0.854 mmol) were added, and

the resulting mixture was stirred at room temperature overnight.

The solvent was evaporated, and the residue was partitioned

between water and AcOEt. The organic layer was dried over

Na₂SO₄and evaporated in vacuo. The crude product was

purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH,

97:3:0.1), and the resulting product was dissolved in CH₂Cl₂and

treated with HCl/Et₂O for 2 h. The precipitate was filtered,

triturated with i-PrOH, and purified by preparative HPLC to

give the desired hydroxamic acid 35k (27 mg, 10% from 33k, bis-

trifluoroacetate salt). ¹H NMR (DMSO-d₆) δ: 10.94 (bs, 1 H),

9.80 (bs, 1 H), 8.12 (d, J = 15.55 Hz, 1 H), 7.96 (t, J = 7.34 Hz,

1 H), 7.91 (dd, J = 7.63, 1.17 Hz, 1 H), 7.71 (d, J = 15.55 Hz,

1 H), 7.55-7.67 (m, 3 H), 7.51 (d, J = 2.93 Hz, 1 H), 7.51 (d, J =

15.85 Hz, 1 H), 7.37 (dd, J = 8.36, 1.91 Hz, 1 H), 7.06 (d, J =

15.26 Hz, 1 H), 3.90-4.12 (m, 2 H), 3.43-3.64 (m, 2 H), 3.14-3.33

(m, 2 H), 2.98-3.12 (m, 2 H), 2.89 (s, 3 H). LC-MS (m/e): 393

[M - H]^þ.

1

hydrochloride). H NMR (DMSO-d₆) δ: 10.92 (bs, 1 H), 10.58

(bs, 1 H), 7.96 (d, J = 15.55 Hz, 1 H), 7.95 (t, J = 7.63 Hz, 1 H),

7.74-7.81 (m, 1 H), 7.47-7.70 (m, 5 H), 7.29-7.37 (m, 1 H), 7.24

(t, J = 7.48 Hz, 1 H), 7.04 (d, J = 15.26 Hz, 1 H), 3.30-3.55 (m,

4 H), 3.12-3.30(m, 2 H), 2.82-3.01 (m, 2 H), 2.58 (d, J= 4.40 Hz,

3 H). LC-MS (m/e): 393 [M - H]^þ.

(E)-N-Hydroxy-3-(6-{(E)-3-[3-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (35l). A mix-

ture of 16e (200 mg, 0.85 mmol), 32b (200 mg, 0.85 mmol), and 1.7 M

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 837

KOH (0.504 mL) in THF (10 mL) was stirred at room temperature

overnight and then partitioned between water and AcOEt. The

organic layer was dried over Na₂SO₄and evaporated in vacuo.

The crude mixture was purified by column chromatography

(CH₂Cl₂/MeOH/NH₄OH, 97:3:0.2) to give the tert-butyl ester 33l

(300 mg, 78%). ¹H NMR (DMSO-d₆) δ:8.06(d, J=15.55 Hz, 1 H),

7.89-8.00(m, 3H), 7.81-7.88 (m, 1 H), 7.71-7.77 (m, 1 H), 7.67 (d,

J=15.55 Hz, 1 H), 7.60 (d, J=15.85 Hz, 1 H), 7.49-7.62 (m, 2 H),

6.89 (d, J=15.85 Hz, 1 H), 3.61 (s, 2 H), 2.35-2.47 (m, 8 H), 2.22 (s,

3 H), 1.54 (s, 9 H).

(E)-N-Hydroxy-3-(6-{(E)-3-[4-(4-methylpiperazin-1-ylmethyl)-

phenyl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (35n). A solu-

tion of 16g (189 mg, 0.815 mmol) in THF (8 mL) was added

dropwise within 30 min to a stirred solution of 32b (190 mg, 0.815

mmol) and 1.7 M KOH (0.48 mL) in THF (20 mL) at 0 ꢀC. The

mixture was stirred at 0 ꢀC for 1 h and then at room temperature

overnight. The solution was partitioned between water and

AcOEt, and the organic layer was dried over Na₂SO₄and evapo-

rated in vacuo. The crude product was purified by column

chromatography (CH₂Cl₂/MeOH/NH₄OH, 96:4:0.1) to give the

tert-butyl ester 33n (245 mg, 67%). ¹H NMR (CDCl₃) δ: 8.16 (d,

J=15.26 Hz, 1 H), 8.06 (m, 2 H), 7.77 (t, J=7.63 Hz, 1 H), 7.76 (d,

J = 15.26 Hz, 1 H), 7.64 (d, J = 15.55 Hz, 1 H), 7.51 (m, 2 H),

7.39-7.47 (m, 2 H), 6.97 (d, J=15.55 Hz, 1 H), 3.62 (s, 2 H), 2.55

(bs, 8 H), 2.35 (s, 3 H), 1.59 (s, 9 H).

33n (245 mg, 0.548 mmol) was dissolved in a mixture of TFA

(3 mL) and CH₂Cl₂(10 mL) and stirred at room temperature for

2 h. The solvent was evaporated, and the residue was dissolved

in CH₂Cl₂(6 mL) and TEA (1.1 mL, 8.14 mmol). EDC (283 mg,

1.47 mmol), HOBt (200 mg, 1.48 mmol), and NH₂OTHP (86.6 mg,

0.74 mmol) were added, and the resulting mixture was stirred at

room temperature overnight. The solvent was evaporated, and

the residue was partitioned between water and AcOEt. The

organic layer was dried over Na₂SO₄and evaporated in vacuo.

The crude product was purified by column chromatography

(CH₂Cl₂/MeOH/NH₄OH, 96:4:0.1), and the resulting inter-

mediate was dissolved in CH₂Cl₂and treated with HCl/Et₂O

for 3 h. The precipitate was filtered, rinsed with CH₂Cl₂, and

purified by preparative HPLC to give the hydroxamic acid 35n

(14 mg, 4% from 33n, tris-trifluoroacetate salt). ¹H NMR

(DMSO-d₆þ TFA) δ: 8.16 (d, J = 15.26 Hz, 1 H), 8.13 (m,

2 H), 7.96 (t, J = 7.63 Hz, 1 H), 7.89 (d, J = 7.04 Hz, 1 H), 7.72 (d,

J = 15.26 Hz, 1 H), 7.65 (d, J = 7.63 Hz, 1 H), 7.58 (m, 2 H), 7.54

(d, J = 15.55 Hz, 1 H), 7.06 (d, J = 15.26 Hz, 1 H), 3.86 (s, 2 H),

2.88-3.55 (m, 8 H), 2.81 (s, 3 H). LC-MS (m/e): 407 [M - H]^þ.

HDAC Enzyme Assay. The in vitro activity of HDAC in-

hibitors was assayed using the HDAC fluorescent histone

deacetylase activity assay kit (Biomol Research Laboratories,

Plymouth Meeting, PA). Briefly, 15 μL of a nuclear extract from

HeLa cells was diluted to 50 μL with the assay buffer containing

the HDAC inhibitor and the substrate (peptide containing

acetylated lysine) at 200 μM. The samples were incubated for

15 min at room temperature and then exposed to a developer for

a further 10 min. Substrate fluorescence was measured using an

excitation at 355 nm and emission at 460 nm, and the %

inhibition was calculated relative to untreated samples. IC₅₀

values were determined using a regression analysis of the con-

centration/inhibition data.

Cell Growth Assay. The effect of the HDAC inhibitors on cell

proliferation was measured using an adaptation of published

procedures.³⁹K562 cells (derived from human lymphoma) were

incubated for 72 h with different concentrations of the inhibi-

tors. Then 5 mg/mL of MTT in PBS was added at different time

points and the solution was incubated for 3-4 h. All incubations

were carried out at 37 ꢀC. The supernatant was then removed,

the formazan crystals were dissolved in a mixture of DMSO and

absolute EtOH (1:1, v:v), and the absorption of the solution was

measured at a wavelength between 550 and 570 nm. IC₅₀values

are determined using a regression analysis of the concentration/

inhibition data.

Metabolic Stability in Hepatic Microsomes. By adaptation of

the protocols described by Di et al.,⁴⁰the compounds at 1 μM

were preincubated for 10 min at 37 ꢀC in potassium phosphate

buffer (pH 7.4) together with 0.5 mg/mL mouse or human

hepatic microsomes (Xenotech, Kansas City). The cofactor

mixture comprising NADP, G6P, and G6P-DH was added,

and aliquots were taken after 0 and 30 min. Samples were

analyzed on an Acquity UPLC, coupled with a sample organi-

zer, and interfaced with a triple quadrupole Premiere XE

(Waters, Milford, MA). Mobile phases consisted of a phase A

33l (300 mg, 0.671 mmol) was then dissolved in a mixture of

TFA (1 mL) and CH₂Cl₂(4 mL) and stirred at room tempera-

ture for 5 h. Additional TFA (1 mL) was added, and the mixture

was stirred for further 4 h. The solvents were removed in vacuo,

and the residue was dissolved in DMF (15 mL). EDC (256 mg,

1.33 mmol), HOBt (181 mg, 1.34 mmol), and TEA (0.280 mL,

2.01 mmol) were added. After 10 min NH₂OTHP (94.1 mg,

0.804 mmol) was added, and the resulting mixture was stirred

overnight at room temperature. Further NH₂OTHP (63 mg,

0.54 mmol) was added in two portions over 24 h, and then the

resulting solution was partitioned between water and CH₂Cl₂.

The aqueous layer was extracted three times with CH₂Cl₂, and

the combined organic layers were dried over Na₂SO₄and

evaporated in vacuo. The crude mixture was purified by column

chromatography (CH₂Cl₂/MeOH/NH₄OH from 97:3:0.2 to

96:4:0.2), and the resulting product was dissolved in CH₂Cl₂

and treated with HCl/Et₂O for 4 h. The precipitate was filtered,

rinsed with CH₂Cl₂, and purified by preparative HPLC to give

the hydroxamic acid 35l (30 mg, 6% from 33l, tris-trifluoroace-

tate salt). ¹H NMR (DMSO-d₆þTFA) δ: 8.15 (d, J = 15.55 Hz,

1 H), 8.06-8.14 (m, 2 H), 7.96 (t, J = 7.78 Hz, 1 H), 7.89 (d, J =

7.63 Hz, 1 H), 7.74 (d, J = 15.55 Hz, 1 H), 7.60-7.74 (m, 3 H),

7.55 (d, J = 15.85 Hz, 1 H), 7.05 (d, J = 15.26 Hz, 1 H), 3.95

(s, 2 H), 3.05-3.99 (m, 8 H), 2.81 (s, 3 H). LC-MS (m/e): 407 [M

- H]^þ.

(E)-N-Hydroxy-3-(6-{(E)-3-[4-(4-methylpiperazin-1-yl)phenyl]-

3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide (35m). KOH (1.7 M,

0.580 mL) was added dropwise to a stirred mixture of 16f (215 mg,

0.987 mmol) and 32b (229 mg, 0.987 mmol) in EtOH (15 mL) at

0 ꢀC. The resulting solution was stirred at 0 ꢀC for 8 h, diluted with

water, and extracted with AcOEt. The organic layer was dried over

Na₂SO₄and evaporated to dryness. The crude product was

purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH,

10:4:0.2) to give a mixture (0.34 g) of tert-butyl (E)-3-(6-{1-

hydroxy-3-[4-(4-methylpiperazin-1-yl)phenyl]-3-oxopropyl}pyridin-

2-yl)acrylate and 33m. The mixture was dissolved in CH₂Cl₂(10 mL)

and TFA (2 mL), and the resulting solution was stirred at room

temperature overnight. The solvent was removed in vacuo, and the

residue was dissolved in THF (10 mL) and 1.7 M KOH (2.64 mL).

The solution was stirred at room temperature for 1 h and then

acidified with HCl/Et₂O and evaporated in vacuo. The resulting solid

was washed with water and filtered to give the carboxylic acid 34m

(0.14 g, bis-hydrochloride).

EDC (118 mg, 0.62 mmol), HOBt (100 mg, 0.62 mmol), TEA

(0.258 mL, 1.86 mmol), and NH₂OTHP (44 mg, 0.37 mmol)

were added to a solution of 34m (140 mg) in DMF (5 mL). The

mixture was stirred at room temperature for 6 h, then diluted

with water and extracted with AcOEt and CH₂Cl₂. The com-

bined organic layers were dried over Na₂SO₄and evaporated in

vacuo. The resulting crude product was purified by column

chromatography (CH₂Cl₂/MeOH/NH₄OH, 96:4:0.2). The pro-

duct was dissolved in CH₂Cl₂and treated with HCl/Et₂O for 2 h.

The precipitate was filtered and rinsed with CH₂Cl₂and Et₂O to

give the desired hydroxamic acid 35m (68 mg, 15% from 32b,

bis-hydrochloride). ¹H NMR (DMSO-d₆, 353 K, þTFA) δ: 8.08

(d, J = 15.85 Hz, 1 H), 8.00-8.05 (m, 2 H), 7.90 (t, J = 7.78 Hz,

1 H), 7.78 (d, J = 7.63 Hz, 1 H), 7.63 (d, J = 15.55 Hz, 1 H), 7.58

(dd, J = 7.78, 0.73 Hz, 1 H), 7.52 (d, J = 15.55 Hz, 1 H), 7.02-

7.18 (m, 3 H), 3.75 (bs, 4 H), 3.34 (bs, 4 H), 2.84 (s, 3 H). LC-MS

(m/e): 393 [M - H]^þ.

838 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Thaler et al.

[0.1% formic acid in a mixture of water and acetonitrile (95:5, v:

v)] and phase B [0.1% formic acid in a mixture of water and

acetonitrile (5:95, v:v)]. Separations were achieved at 40 ꢀC on

Acquity BEH C18 columns (50 mm ꢀ 2.1 mm ꢀ 1.7 μm with a

flow rate of 0.45 mL/min or 50 mm ꢀ 1 mm ꢀ 1.7 μm with a flow

rate of 0.2 mL/min). The column was conditioned with 2% of

phase B for 0.2 min, then brought to 100% of phase B within

0.01 min and maintained at these conditions for 1.3 min. The

operating parameters of the mass spectrometer were set as

follows: capillary voltage 3.4 kV, source termperature 115 ꢀC,

desolvation temperature 450 ꢀC, desolvation gas flow 900 L/h,

cell pressure 3.3 ꢀ 10^-3mbar. Cone voltage and collision energy

were optimized for each compound. LC-MS/MS analyses were

carried out using a positive electrospray ionization (ESI(þ))

interface in MRM (multiple reaction monitoring) mode with

verapamil as internal standard. The percentage of the com-

pound remaining after a 30 min incubation period was calcu-

lated according the following equation: [(area at time 30 min)/

(area at time 0 min)] ꢀ 100%.

K562 cells, plated at 10⁶/well in 96-multiwell plates, were

incubated for 24 h with the compounds at a final concentration

of 3 μM, or solvent (DMSO) prior to direct lysis by caspase-Glo

3/7 reagent. The luminescence produced, determined by the

cleavage of the substrate by the activated caspases, is expressed

as relative light unit (RLU), and results are expressed as the

mean and the standard deviation of triplicate data.

In Vivo Efficacy Experiments. For in vivo antitumor efficacy

studies, 5 million human cancer cells (HCT-116) were injected

subcutaneously in the flank of female CD-1 nude mice (aged

8-10 weeks, Charles River Laboratories, Wilmington, MA).

Tumors were allowed to grow until a volume of around 100 mm³

was reached. Mice bearing a tumor xenograft were randomized

into treated and control groups of 7 animals per group. HDAC

inhibitors were dissolved in vehicles (compound 1 in a mixture of

45% PEG400, 45% H₂O, and 10% DMSO and compound 30b

in PBS) and administered daily for 5 days per week for 2 weeks.

Tumor volumes were measured regularly by vernier caliper

during the experiments, and body weight was monitored three

times a week. Dimensions of the tumors were calculated using

the equation V = 0.5ab², where a and b are the longest and

shortest diameter, respectively,⁴¹and are expressed as mm³. The

reported tumor volumes are expressed as the mean ( SEM.

Histone Acetylation Assay. K562 cells were incubated with the

compounds at a final concentration of 0.5 μM for 3 h, then fixed

with 1% formaldehyde in PBS and permeabilized with a solu-

tion containing 0.1% Triton X-100 in PBS. After being washed,

the cells were preincubated with 10% goat serum in PBS for

30 min at 4 ꢀC, exposed to a monoclonal antibody against acetyl-

ated histones for 1 h at room temperature, and then incubated

for 1 h with a secondary antibody conjugated with FITC.

Histone acetylation levels were measured by cytofluorometry

(FACS) and expressed as ratio of the acetylation level of treated

versus untreated cells.³¹

Acknowledgment. The authors are grateful to Dr. Agnese

Abate, Dr. Giacomo Carenzi, Dr. Daniele Fancelli,

Dr. Simon Plyte, Dr. Manuela Villa, Dr. Teresa Mercurio,

Dr. Daniela Rapetti, Dr. Marco Giulio Rozio, and

Dr. Michele Zandi for their helpful discussions and excellent

technical support in the execution of the present work.

In Vivo Drug Pharmacokinetic Studies. Pharmacokinetic ex-

periments were performed using 4 week old male nude CD-1

mice (Charles River Laboratories, Calco, Italy). Animals were

quarantined for approximately 1 week prior to the study. They

were housed under standard conditions and had free access to

water and standard laboratory rodent diet.

Supporting Information Available: Synthesis and character-

ization of compounds 16b, 16c, 16e, and 16g; purity of key

compounds as determined by HPLC. This material is available

free of charge via the Internet at http://pubs.acs.org.

Compound 1 was dissolved in a mixture of 1% DMSO and

10% Encapsin in water at a concentration of 1 mg/mL for the iv

(rapid bolus) administration or 5% DMSO and 1% PEG400 in

water for the oral (gavage) dose at a concentration of 3 mg/mL.

Compounds 19b, 30b, 30c, 30e, 30g, 35c, 35i, 35k, and 35n were

dissolved in normal saline solution at a concentration of 1 mg/mL

for the iv formulation and in water for the oral administration at

3 mg/mL of the base. Each experimental group contained 27-

30 animals. The compounds were administered to mice either by

iv or oral route, and blood samples were collected after different

time points after dosing. Plasma was separated immediately after

blood sampling by centrifugation, plasma proteins were precipi-

tated using Sirocco filtration plates or Oasis HLB elution plates

according to the distributor instructions, and the plasma samples

were kept frozen (-80 ꢀC) until submission to LC/MS/MS ana-

lysis. Sample analysis was performed on an Acquity UPLC using

eithera Acquity BEHC18 column (50mmꢀ 2.1 mm ꢀ 1.7 μm) or

a Acquity HSS T3 column (50 mm ꢀ 2.1 mm ꢀ 1.8 μm), coupled

with a sample organizer and interfaced to a triple quadrupole

Premiere XE (Waters, Milford, MA). The mass spectrometer was

operated using electrospray interface (ESI) with a capillary voltage

of 3-4 kV, cone voltage of 25-52 V, source temperature of

115-120 ꢀC, desolvation gas flow of 800 L/h, and desolvation

temperature of 450-480 ꢀC. Collision energy was optimized for

each compound. LC-MS/MS analyses were carried out using a

positive electrospray ionization (ESI(þ)) interface in MRM

(multiple reaction monitoring) mode.

References

(1) Allfrey, V. G.; Faulkner, R.; Mirsky, A. E. Acetylation and

methylation of histones and their possible role in the regulation

of RNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 1964, 51, 786–794.

(2) Kouzarides, T. Histone acetylases and deacetylases in cell prolif-

eration. Curr. Opin. Genet. Dev. 1999, 9, 40–48.

(3) Hong, L.; Schroth, G. P.; Matthews, H. R.; Yau, P.; Bradbury,

E. M. Studies of the DNA binding properties of histone H4 amino

terminus. Thermal denaturation studies reveal that acetylation

markedly reduces the binding constant of the H4 “tail” to DNA.

J. Biol. Chem. 1993, 268, 305–314.

(4) Gregoretti, I. V.; Lee, Y. M.; Goodson, H. V. Molecular evolution

of the histone deacetylase family: functional implications of phy-

logenetic analysis. J. Mol. Biol. 2004, 338, 17–31.

(5) Spange, S.; Wagner, T.; Heinzel, T.; Kramer, O. H. Acetylation of

non-histone proteins modulates cellular signalling at multiple

levels. Int. J. Biochem. Cell Biol. 2009, 41, 185–198.

(6) Vidali, G.; Boffa, L. C.; Bradbury, E. M.; Allfrey, V. G. Butyrate

suppression of histone deacetylation leads to accumulation of

multiacetylated forms of histones H3 and H4 and increased DNase

I sensitivity of the associated DNA sequences. Proc. Natl. Acad.

Sci. U.S.A. 1978, 75, 2239–2243.

(7) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and the

promise of epigenetic (and more) treatments for cancer. Nat. Rev.

Cancer 2006, 6, 38–51.

(8) Glozak, M. A.; Seto, E. Histone deacetylases and cancer. Oncogene

2007, 26, 5420–5432.

(9) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone

deacetylase inhibitors: from bench to clinic. J. Med. Chem. 2008,

51, 1505–1529.

Pharmacokinetic parameters were calculated by a noncom-

partmental method using WinNolin 5.1 software (Pharsight,

Mountain View, CA).

Induction of apoptosis. Induction of apoptosis was examined

by measuring the activities of caspase-3 and caspase-7 in K562

cells, using the caspase-GLO3/7 assay (Promega, Madison, WI)

according to the manufacturer’s instructions.

(10) Mai, A.; Altucci, L. Epi-drugs to fight cancer: from chemistry to

cancer treatment, the road ahead. Int. J. Biochem. Cell Biol. 2009,

41, 199–213.

(11) Riggs, M. G.; Whittaker, R. G.; Neumann, J. R.; Ingram, V. M.

n-Butyrate causes histone modification in HeLa and Friend ery-

throleukaemia cells. Nature 1977, 268, 462–464.

(12) Richon, V. M.; Webb, Y.; Merger, R.; Sheppard, T.; Jursic, B.;

Ngo, L.; Civoli, F.; Breslow, R.; Rifkind, R. A.; Marks, P. A.

Article

Second generation hybrid polar compounds are potent inducers of

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 839

(26) McLaughlin, F.; La Thangue, N. B. Histone deacetylase inhibitors

open new doors in cancer therapy. Biochem. Pharmacol. 2004, 68,

1139–1144.

transformedcell differentiation. Proc. Natl. Acad. Sci. U.S.A. 1996,

93, 5705–5708.

(13) Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.; Breslow, R.;

Rifkind, R. A.; Marks, P. A. A class of hybrid polar inducers of

transformed cell differentiation inhibits histone deacetylases. Proc.

Natl. Acad. Sci. U.S.A. 1998, 95, 3003–3007.

(27) Massaro, A.; Mordini, A.; Reginato, G.; Russo, F.; Taddei, M.

Microwave-assisted transformation of esters into hydroxamic

acids. Synthesis 2007, 20, 3201–3204.

(28) Marvel, C. S.; Coleman, L. E., Jr.; Scott, G. P. Pyridine analogs of

chalcone and their polymerization reactions. J. Org. Chem. 1955,

20, 1785–1792.

(14) Marks, P. A. Discovery and development of SAHA as an antic-

ancer agent. Oncogene 2007, 26, 1351–1356.

(15) Maiso, P.; Carvajal-Vergara, X.; Ocio, E. M.; Lopez-Perez, R.; Mateo,

G.; Gutierrez, N.; Atadja, P.; Pandiella, A.; San Miguel, J. F. The

histone deacetylase inhibitor LBH589 is a potent antimyeloma agent

that overcomes drug resistance. Cancer Res. 2006, 66, 5781–5789.

(16) Plumb, J. A.; Finn, P. W.; Williams, R. J.; Bandara, M. J.; Romero,

M. R.; Watkins, C. J.; La Thangue, N. B.; Brown, R. Pharmaco-

dynamic response and inhibition of growth of human tumor

xenografts by the novel histone deacetylase inhibitor PXD101.

Mol. Cancer Ther. 2003, 2, 721–728.

(17) Golay, J.; Cuppini, L.; Leoni, F.; Mico, C.; Barbui, V.; Domenghini,

M.; Lombardi, L.; Neri, A.; Barbui, A. M.; Salvi, A.; Pozzi, P.;

Porro, G.; Pagani, P.; Fossati, G.; Mascagni, P.; Introna, M.;

Rambaldi, A. The histone deacetylase inhibitor ITF2357 has anti-

leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF

production by stromal cells. Leukemia 2007, 21, 1892–1900.

(18) Sangthongpitag, K.; Wang, H.; Yeo, P.; Liu, X.; Goh, E.; New, L.;

Zeng, P.; Wu, X.; Hu, C.; Kantharaj, E. ADME and PK/PD

attributes of SB939, a potent orally active HDAC inhibitor. Eur.

J. Cancer Suppl. 2006, 4, 53.

(29) New, J. S.; Christopher, W. L.; Yevich, J. P.; Butler, R.; Schlem-

mer, R. F., Jr.; VanderMaelen, C. P.; Cipollina, J. A. The thieno-

[3,2-c]pyridine and furo[3,2-c]pyridine rings: new pharmacophores

with potential antipsychotic activity. J. Med. Chem. 1989, 32,

1147–1156.

(30) Mori, K.; Koseki, K. Synthesis of trichostatin a, a potent differ-

entiation inducer of friend leukemic cells, and its antipode. Tetra-

hedron 1988, 6013–6020.

(31) Ronzoni, S.; Faretta, M.; Ballarini, M.; Pelicci, P.; Minucci, S. New

method to detect histone acetylation levels by flow cytometry.

Cytometry, Part A 2005, 66, 52–61.

(32) Mai, A.; Massa, S.; Pezzi, R.; Simeoni, S.; Rotili, D.; Nebbioso, A.;

Scognamiglio, A.; Altucci, L.; Loidl, P.; Brosch, G. Class II (IIa)-

selective histone deacetylase inhibitors. 1. Synthesis and biological

evaluation of novel (aryloxopropenyl)pyrrolyl hydroxyamides.

J. Med. Chem. 2005, 48, 3344–3353.

(33) Roy, D. R.;Parthasarathi, R.; Maiti, B.; Subramanian, V.; Chattaraj,

P. K. Electrophilicity as a possible descriptor for toxicity prediction.

Bioorg. Med. Chem. 2005, 13, 3405–3412.

(19) Buggy, J. J.; Cao, Z. A.; Bass, K. E.; Verner, E.; Balasubramanian, S.;

Liu, L.; Schultz, B. E.; Young, P. R.; Dalrymple, S. A. CRA-024781: a

novel synthetic inhibitor of histone deacetylase enzymes with antitu-

mor activity in vitro and in vivo. Mol. Cancer Ther 2006, 5, 1309–1317.

(20) Arts, J.; Angibaud, P.; Marien, A.; Floren, W.; Janssens, B.; King,

P.; van Dun, J.; Janssen, L.; Geerts, T.; Tuman, R. W.; Johnson,

D. L.; Andries, L.; Jung, M.; Janicot, M.; van Emelen, K. R306465

is a novel potent inhibitor of class I histone deacetylases with

broad-spectrum antitumoral activity against solid and haematolo-

gical malignancies. Br. J. Cancer 2007, 97, 1344–1353.

(21) Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukazawa, N.; Saito, A.;

Mariko, Y.; Yamashita, T.; Nakanishi, O. Synthesis and histone

deacetylase inhibitory activity of new benzamide derivatives.

J. Med. Chem. 1999, 42, 3001–3003.

(22) Zhou, N.; Moradei, O.; Raeppel, S.; Leit, S.; Frechette, S.; Gaudette,

F.; Paquin, I.; Bernstein, N.; Bouchain, G.; Vaisburg, A.; Jin, Z.;

Gillespie, J.; Wang, J.; Fournel, M.; Yan, P. T.; Trachy-Bourget,

M. C.; Kalita, A.; Lu, A.; Rahil, J.; MacLeod, A. R.; Li, Z.;

Besterman, J. M.; Delorme, D. Discovery of N-(2-aminophenyl)-

4-[(4-pyridin-3-ylpyrimidin-2-ylamino)methyl]benzamide (MGCD0103),

an orally active histone deacetylase inhibitor. J. Med. Chem. 2008, 51,

4072–4075.

(23) Nakajima, H.; Kim, Y. B.; Terano, H.; Yoshida, M.; Horinouchi,

S. FR901228, a potent antitumor antibiotic, is a novel histone

deacetylase inhibitor. Exp. Cell Res. 1998, 241, 126–133.

(24) Gottlicher, M. Valproic acid: an old drug newly discovered as inhibitor

of histone deacetylases. Ann. Hematol. 2004, 83 (Suppl. 1), S91–S92.

(25) Elaut, G.; Rogiers, V.; Vanhaecke, T. The pharmaceutical poten-

tial of histone deacetylase inhibitors. Curr. Pharm. Des. 2007, 13,

2584–2620.

(34) Wienkers, L. C.; Hauer, M. J.; Epps, D. E. Method for Determin-

ing Chemical Reactivity. US6979545, 2001.

(35) Blackwell, L.; Norris, J.; Suto, C. M.; Janzen, W. P. The use of

diversity profiling to characterize chemical modulators of the

histone deacetylases. Life Sci. 2008, 82, 1050–1058.

(36) Venkatesh, P. R.; Goh, E.; Zeng, P.; New, L. S.; Xin, L.; Pasha,

M. K.; Sangthongpitag, K.; Yeo, P.; Kantharaj, E. In vitro phase I

cytochrome P450 metabolism, permeability and pharmacokinetics

of SB639, a novel histone deacetylase inhibitor in preclinical

species. Biol. Pharm. Bull. 2007, 30, 1021–1024.

(37) Yeo, P.; Xin, L.; Goh, E.; New, L. S.; Zeng, P.; Wu, X.; Venkatesh,

P.; Kantharaj, E. Development and validation of high-perfor-

mance liquid chromatography-tandem mass spectrometry assay

for 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide, a novel

HDAC inhibitor, in mouse plasma for pharmacokinetic studies.

Biomed. Chromatogr. 2007, 21, 184–189.

(38) Davies, B.; Morris, T. Physiological parameters in laboratory

animals and humans. Pharm. Res. 1993, 10, 1093–1095.

(39) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czer-

winski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker,

R. H.; Boyd, M. R. Feasibility of drug screening with panels of

human tumor cell lines using a microculture tetrazolium assay.

Cancer Res. 1988, 48, 589–601.

(40) Di, L.; Kerns, E. H.; Gao, N.; Li, S. Q.; Huang, Y.; Bourassa, J. L.;

Huryn, D. M. Experimental design on single-time-point high-

throughput microsomal stability assay. J. Pharm. Sci. 2004, 93,

1537–1544.

(41) Carlsson, G.; Gullberg, B.; Hafstrom, L. Estimation of liver tumor

volume using different formulas: an experimental study in rats.

J. Cancer Res. Clin. Oncol. 1983, 105, 20–23.

Article Doi

Synthesis and biological evaluation of N-hydroxyphenylacrylamides and N-hydroxypyridin-2-ylacrylamides as novel histone deacetylase inhibitors

DOI: 10.1021/jm901502p

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Article abstract of DOI:10.1021/jm901502p

Full text of DOI:10.1021/jm901502p

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