Trithiocarbonates as a novel class of HDAC inhibitors: SAR studies, isoenzyme selectivity, and pharmacolozgical profiles

J. Med. Chem. 2008, 51, 3985–4001

3985

Trithiocarbonates as a Novel Class of HDAC Inhibitors: SAR Studies, Isoenzyme Selectivity,

and Pharmacological Proﬁles

Florian Dehmel,^‡Steffen Weinbrenner,^†Heiko Julius,^†Thomas Ciossek,^§Thomas Maier,^†Thomas Stengel,^†Kamal Fettis,^†

Carmen Burkhardt,^†Heike Wieland,^†and Thomas Beckers*^,|

Nycomed GmbH, Therapeutic Area Oncology, Byk-Gulden-Strasse 2, D-78467 Konstanz, Germany

ReceiVed January 30, 2008

Inhibitors of histone deacetylases (HDAC) are currently developed for the treatment of cancer. These include

compounds with a sulfur containing head group like depsipeptide, alkylthiols, thiocarboxylates, and

trithiocarbonates with a carbonyl group in the R-position. In the present investigation, we report on the

synthesis and comprehensive SAR analysis of HDAC inhibitors bearing a tri- or dithiocarbonate motif.

Such trithiocarbonates are readily accessible from either preformed or in situ prepared R-halogenated

methylaryl ketones. A HDAC isotype selectivity and a substrate competitive mode-of-action is shown for

deﬁned analogues. Exploration of the head group showed the necessity of the dithio-R-carbonyl motif for

potent HDAC inhibition. Highly potent, substrate competitive HDAC6 selective inhibitors were identiﬁed

(12ac:IC₅₀) 65 nM and K_i) 110 nM). Trithiocarbonate analogues with an aminoquinoline-substituted

pyridinyl-thienoacetyl cap demonstrate a cytotoxicity proﬁle and potency comparable to that of suberoylanilide

hydroxamic acid (SAHA) as an approved cancer drug.

Scheme 1^a

Introduction

Posttranslational modiﬁcation of proteins by reversible lysine

acetylation is one important modiﬁcation modulating protein

function.¹The modiﬁcation of core histone proteins H3 and H4

by acetylation of N-terminal lysine residues is well described

and part of the so-called histone code important in epigenetic

processes.²Nevertheless, a raising number of nonhistone

proteins modiﬁed by lysine acetylation have been reported,

including the signal transducer and activator of transcription 1

and 3 (STAT1, STAT3),^aheat shock protein 90 (Hsp90), the

tumor suppressor protein p53, and R-tubulin.³The status of H3/

H4 core histone acetylation correlates with the transcriptional

activity of chromatin with histone acetyltransferases (HATs)

and histone deacetylases (HDACs) as key players regulating

reversible histone acetylation.^1bUp to now, 11 different HDAC

isoenzymes belonging to the class I (HDAC 1, 2, 3, 8), class II

(HDAC 4-7, 9, 10), and class IV (HDAC11) family have been

described.⁴There is clear evidence for a pathophysiological

relevance of HDAC mediated histone acetylation and cancer.⁵

These include (i) mutations of cAMP binding protein (CBP) as

a HAT are associated with Rubinstein-Taybi syndrome, a

cancer predisposition,⁶(ii) aberrant recruitment of HDAC1 by

^aReagents and conditions: (a) EtSNa, CS₂, THF, rt.

transcription factors in acute promyelocytic leukemia (APL)

mediated by the PML-retinoic acid receptor R fusion gene in

non-Hodgkin lymphoma by the overexpressed BCL6 protein

and in acute myelogenous leukemia (AML M2 subtype) by the

AML-ETO fusion protein,⁷(iii) overexpression of HDAC2 in

colon carcinoma upon constitutive activation of the wnt/ꢀ-

catenin/TCF signaling pathway,⁸and ﬁnally (iv) a high expres-

sion of HDAC1, 2, and 3 in gastric, prostate, and colorectal

cancer correlating with survival and staging.⁹As a consequence,

the identiﬁcation of HDAC inhibitors is currently one major

topic in oncology drug discovery programs. Inhibition of HDAC

class I/II enzymes is well established as an experimental clinical

approach for solid and hematological tumor therapy.^4b,10HDAC

inhibitors affect the transcriptional regulation and induce or

repress genes involved in differentiation, proliferation, cell cycle

regulation, protein turnover, and apoptosis.^4bHDAC inhibitors

of divergent chemical structure are currently in clinical develop-

ment.¹¹These include short chain fatty acids, hydroxamic acids,

cyclic tetrapeptides/peptolides, ketones, and benzamides.¹²

Various agents are currently in clinical development, namely

the hydroxamate analogues (E)-3-(4-(((2-(1H-indol-3-yl)ethyl)(2-

hydroxyethyl)amino)methyl)phenyl)-N-hydroxyacrylamide (LBH589),

N-hydroxy-3-(3-(N-phenylsulfamoyl)phenyl)acrylamide (PXD101,

Belinostat), N-(2-(4-(hydroxycarbamoyl)phenoxy)ethyl)-3-(isopropyl-

amino)benzofuran-2-carboxamide (PCI024781) and N¹-hydroxy-⁸--

phenyloctanediamide (suberoylanilide hydroxamic acid/SAHA,

Vorinostat), the benzamide analogues (4-(2-aminophenylcarbamoyl)-

benzylamino)methyl nicotinate (MS275 or SNDX275) and N-

(2-aminophenyl)-4-((4-(pyridin-3-yl)pyrimidin-2-ylamino)methyl)-

benzamide (MGCD0103), the cyclic peptolide depsi-

* To whom correspondence should be addressed. Phone: +761-5155916.

Fax: +761-5155595. E-mail: Thomas.Beckers@oncotest.de. Address: On-

cotest GmbH, Institute for Experimental Oncology, Am Flughafen 12-14,

D-79108 Freiburg, Germany.

†

Nycomed GmbH, Therapeutic Area Oncology.

Present address: Henkel KGaA, D-40589 Du¨sseldorf, Germany.

Present address: Boehringer Ingelheim Pharma.

Present address: Oncotest GmbH, Institute for Experimental Oncology.

‡

§

|

^aAbbreviations: APL, acute promyelocytic leukemia; AML, acute

myelogenous leukemia; AMC, 7-amino-4-methylcoumarin, ATCC,

American Tissue Type Collection; CBP, cAMP binding protein; CTCL,

cutaneous T-cell lymphoma; FACS, ﬂourescence activated cell sorting;

HDAC, histone deacetylase; HAT, histone acetyltransferase; HTS, high

throughput screening; NSCLC, nonsmall cell lung cancer; PVDF,

polyvinylidendiﬂouride;rHDAC,recombinantHDAC;SAR,structure-activity

relationship; SAHA, suberoylanilide hydroxamic acid; SDS, disodium-

dodecylsulfate; SDS-PAGE, SDS polyacrylamide gel electrophoresis;

STAT, signal transducers and activators of transcription; TSA, tricho-

statin A; VPA, valproic acid.

10.1021/jm800093c CCC: $40.75

2008 American Chemical Society

Published on Web 06/18/2008

3986 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13

Dehmel et al.

Scheme 2^a

^aReagents and conditions: (a) nPrMgCl, CS₂, CuI, THF, -20 °C; (b) EtOH, KOH, CS₂, H₂O, rt; (c) KSAc, THF, 50 °C, then NaOH, MeOH, rt; (d)

EtNCS, THF, rt; (e) EtO(CdO)Cl, DIPEA, THF, rt; (f) EtNCO, THF, rt; (g) RC₂H₄SH, NaH, THF, 0 °C, CS₂; (h) morpholine, NaOAc, CS₂, THF, rt; (i)

CS₂, NaOH, THF, then EtOC₂H₄Br, rt.

peptide/FK228 (rhomidepsin) and valproic acid (VPA). As

shown for SAHA and the natural product trichostatin A (TSA),

the hydroxamic acid head group complexes the Zn²⁺in the

active site, thereby inhibiting the enzyme in a substrate

competitive manner.¹³So far, HDAC class I isotype selectivity

has been shown only for benzamide analogues with MGCD0103

as a prominent example.¹⁴Selective HDAC6 inhibitors have

been rarely described with the hydroxamate analogue Tubacin

as one example.¹⁵Obviously, isotype selective inhibitors will

open new avenues for treating cancer and noncancer diseases

like rheumatoid arthritis, Crohn’s disease or muscle dystrophia,

displaying a different overall safety proﬁle.

The structural basis of HDAC inhibition is well established,

with the Zn²⁺complexing head group, a linker/spacer, and

the cap group of high structural variability.^13a,16Nevertheless,

the identiﬁcation of new head groups bearing druglike

properties proved to be difﬁcult.¹⁷Besides the well-studied

benzamides and hydroxamates,¹⁸compounds with a free thiol

head group were described as potent and occasionally isotype-

selective HDAC inhibitors,¹⁹but in general cellular activity

was very weak.²⁰Depsipeptide is a remarkable exception by

acting as a prodrug, liberating the reduced sulfhydryl group

only intracellular.²¹This concept has also been exploited at

similar structures bearing a disulﬁde moiety derived from

thio-pyridine.²²Different head groups containing sulfur

including thiocarboxylates,²³thioglycolamides,²⁴and R-thio-

substituted acetyl compounds²⁵have been described. Re-

cently, it was found that a trithiocarbonate group in R-position

to the carbonyl moiety represents a promising new head group

for HDAC inhibition.²⁶In the present study, we report on

the exploition of this novel structural motif for HDAC

inhibition and its biological activity in detail.

Chemistry. The HDAC inhibitors containing the acetyl

trithiocarbonate moiety are typically prepared from an R-halo-

acetophenone and conversion with sodium ethyl trithiocarbonate

(Scheme 1).²⁶The advanced hit compound 2 as well as its

methoxy analogue 3a were obtained in a clean reaction.

Analogues of the trithiocarbonate headgroup were acces-

sible from R-bromo-4-methoxy-acetophenone 1a in similar

conversions, as dithiobutyrate prepared from n-propyl Grig-

nard and carbondisulﬁde provided access to its dithiocar-

boxate analogue 3b (Scheme 2). The corresponding xantho-

genate 3c, the functionalized trithiocarbonates (3j and 3k),

and the dithiocarbamate 3m were prepared from the requisite

alcoholate, thiolate, or amine and carbondisulﬁde followed

by addition of the electrophilic bromo-acetophenone. The

synthesis of other trithiocarbonate analogues required 2-mer-

capto-acetophenone 4 as precursor, which was prepared in

two steps using potassium thioacteate followed by saponi-

ﬁcation. The thiol 4 was used to prepare the functionalized

Trithiocarbonates as a NoVel Class of HDAC Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3987

Scheme 3^a

^aReagents and conditions: (a) EtNCS, NEt₃, THF, rt; (b) EtBr, CS₂, 1 M NaOH, ethyl acetate/EtOH, rt.

Scheme 4^a

^aReagents and conditions: (a) HOC₂H₄OH, amberlyst 15, PhH, 80 °C; (b) NaH, imidazole, THF, 0 °C, then CS₂, then EtI; (c) cat. HCl, THF/H₂O, 60 °C.

Scheme 5^a

Scheme 6^a

^aReagents and conditions: (a) PTT, THF, rt; then EtS(CdS)SNa.

trithiocarbonate 3l, using commercially available methoxy-

ethyl bromide (Scheme 2), but the yield for this conversion

was found to be rather low. The O-ethyl thiocarbonate 3h

and the N-ethyl carbamates 3d and 3i were prepared in

moderate to good yield from 4 by simple addition of ethyl

chloroformiate, ethyl isothiocyanate, or ethyl isocyanate,

respectively.

^aReagents and conditions: (a) i. n-PrMgCl, CS₂, CuI, THF, -20 °C,

then HCl, H₂O, ii. 9, NEt₃, DMF, rt; (b) EtOH, KOH, CS₂, H₂O, rt; (c)

EtNH₂, aq NaOH, CS₂, DMF, 10 °C to rt.

Scheme 3 illustrates the preparation of nitrogen analogues

of the trithiocarbonate moiety. The addition of ethyl isothio-

cyanate to amino-acetophenone 5 resulted in thiourea 3f, while

conversion with carbondisulﬁde and ethyl bromide provided the

S-ethyl carbamate 3g in high yields. It is noteworthy that all

N-ethyl derivatives (3d, 3i, and 3f) exist in an equilibrium with

a cyclized species, the ratio being dependent on temperature

and solvent environment.

Scheme 7^a

The preparation of the S-ethyl dithiocarbonate 3e proved to

be more cumbersome (Scheme 4). 2-Hydroxy-acetophenone 6

was converted into the dioxolane to allow the installation of

the required functionality using sodium hydride, carbon sulﬁde,

and ethyl iodide before the protecting group was removed under

acid catalysis to yield 3e.

A one-pot procedure was elaborated to access substituted

acetophenone trithiocarbonates, which brominated precursors

are not commercially available (Scheme 5).

The acetophenone was brominated in situ with phenyltri-

methylammonium tribromide (PTT) in tetrahydrofuran. De-

colorization of the orange solution and formation of whitish

precipitate indicated the completion of the bromination

reaction and a solution sodium ethyl trithiocarbonate solution

(stock solution, stored under nitrogen for several weeks) was

added to yield 8a and 8e-g. Chlorides of type 9 or 10 instead

of bromo compounds also represented suitable electrophiles

for the preparation of trithiocarbonates (8h-j, Scheme 7) as

^aReagents and conditions: (a) EtS(CdS)SNa, THF, rt.

well as their CH₂-, O-, and NH-analogues (8b-d, Scheme

6), which were prepared using similar procedures as described

in Scheme 2.

A wide range of trithiocarbonates of type 12 was accessible

from substituted acetophenones 7 using the in situ bromination

with PTT followed by addition of sodium ethyl trithiocarbonate

(Scheme 8, method a and Table 2). This methodolgy was also

successful with certain heterocyclic ethanones such as pyrroles

(13, Scheme 9).

The presence of six-membered nitrogen bearing heterocycles,

especially pyridine groups, however, prevented the application

of this methodology because no bromide formation with PTT

was observed for these substrates. To circumvent this obstacle,

alternative bromination methods were examined. NBS bromi-

3988 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13

Dehmel et al.

Scheme 8^a

blue proliferation/cytotoxicity assay.²⁸Recently, we reported

about HDAC inhibitors bearing ethyl trithiocarbonate as a

preferred head group²⁶found in compounds 2 or 3a. The lead

compound 2 now was more deeply studied for its biochemical

properties. Recombinant HDAC isoenzymes were inhibited with

IC₅₀) 6.99 µM for rHDAC1 and IC₅₀) 1.25 µM for rHDAC6

(Figure 1B/C). A substrate competitive mode-of-action was

demonstrated in enzyme kinetic studies using rHDAC1 with a

K_ivalue of 7 µM calculated from a Lineweaver-Burk plot

(Figure 2A). Cellular HDAC activity in HeLa cells was

concentration dependently inhibited, evident by an EC₅₀of 4.8

µM for induction of histone H3 hyperacetylation and an IC₅₀

of 81.2 µM for inhibition of cellular HDAC activity (Figure

3A/B). A broad cytotoxicity was observed for compound 2 with

a mean IC₅₀of 8.31 µM over a panel of 21 different tumor cell

lines representing 15 different cancer entities (Figure 5A) and

ﬁrst evidence for a proliferation-dependent cytotoxicity (Figure

5B). Compound 2 induced potent apoptosis in RKO colon

carcinoma cells, evident by a massive population of cells in a

subG1 cell cycle state (Figure 6A). These experiments with the

initial HTS hit compound 2 proved the HDAC inhibitory mode-

of-action with similarities to the hydroxamate analogue SAHA

in biochemical and cellular assays.

^aReagents and conditions: (a) PTT, THF, rt; then EtS(CdS)SNa (method

a); (b) TBDMSOTf, NEt₃, THF; then NBS, 0 °C, then EtS(CdS)SNa, rt

(method b); (c) NaHMDS, THF, -78 °C, then TMSCl, then Br₂, then

EtS(CdS)SNa, warm to rt (method c).

nation of silylenolethers in situ prepared from the acetophenone

and TBDMSOTf followed by addition of sodium ethyl trithio-

carbonate was successful for the preparation of 12ag (Scheme

8, method b, Table 2).

More general applicable was the preparation of silyleno-

lethers using NaHMDS as base and TMSCl in tetrahydrofuran

at low temperature. Addition of bromine to the reaction

mixture followed by the addition of solution of sodium ethyl

trithiocarbonate and warming to rt provided trithiocarbonates

bearing pyridine and quinoline substituents (Scheme 8,

method c and Table 2). Puriﬁcation of the resulting products

was sometimes difﬁcult and required puriﬁcation by prepara-

tive HPLC.

Several trithiocarbonates containing sulfonamide moieties

were prepared from 2-bromo-acetyl-benzenesulfonyl chlorides

14 by subsequent nucleophilic displacement with amines and

then sodium ethyl trithiocarbonate (Scheme 10, method d and

Table 2).

Outlined in Scheme 11 is the preparation of trithiocarbon-

ate possessing a thiophene core structure. These compounds

were derived from 5-acetyl-thiophene-2-carboxylic acid (15),

which was converted into the amide using iso-butyl chloro-

formiate and N-methyl morpholine and subsequent addition

of the requisite amine. The reaction mixture was directly

treated with PTT followed by addition of sodium ethyl

trithiocarbonate, therefore giving access to compounds 16a-e

in a four-step one-pot sequence (Table 2).

Its C1-homologues (18a-e) were prepared in similar

sequence from commercially available chloro-acetyl thiophene

carboxylic acid 17, thus the bromination step was omitted

in the sequence as shown in Scheme 12 (Table 2).

Finally, two related trithiocarbonates bearing a 2′-pyridinyl-

2,5-thieno-acetyl core unit were prepared from commercially

available 5-acetyl-2-thienyl-boronic acid (19) and respective

2-bromopyridine 20 using Suzuki-coupling conditions to yield

the coupling products 21 in acceptable yield (Scheme 13).

The conversion into the trithiocarbonates 22 and 23 required

again low temperature R-acetyl deprotonation using sodium

hexamethyldisilazide (NaHDMS) followed by silylenolether

formation, its subsequent bromination, and ﬁnally substitution

with sodium ethyl trithiocarbonate.

Biological Results and Discussion. The prepared trithiocar-

bonates and their analogues were examined for their HDAC

inhibiting properties. For this purpose, the compounds were

tested using a HDAC isoenzyme mixture puriﬁed from HeLa

cervical carcinoma nuclei, containing at least HDAC isoenzymes

1, 2, 3, 5, and 8, as well as the recombinant isoforms rHDAC1

and rHDAC6 puriﬁed from HEK293 cells.¹⁴Their cellular

activity in HeLa cervical carcinoma cells was evaluated by

different assays, namely (i) an enzymatic HDAC activity assay,²⁷

(ii) by quantiﬁcation of histone H3 hyperacetylation using a

single cell imaging assay,¹⁴and ﬁnally (iii) using the Alamar

On the basis of the published results for afﬁx variations on

ethyl trithiocarbonates,²⁶special emphasis was laid on the

relevance of the trithiocarbonate moiety itself acting as the

HDAC inhibitory head group. For the methoxy trithiocarbonate

3a, a submicromolar inhibition of the nuclear extract HDAC

isoenzyme mixture was determined (IC₅₀) 0.375 µM, Table

1). When the ethyl trithiocarbonate is exchanged for a dithiobu-

tyrate (Z ) CH₂, 3b), the HDAC inhibition is slightly enhanced

(IC₅₀) 0.123 µM). Similar HDAC inhibition is observed for

the dithiocarbamate 3d (Z ) NH, IC₅₀) 0.195 µM), while the

dithiocarbonate 3c is less active (Z ) O, IC₅₀) 0.811 µM).

Thus, the nuclear extract is inhibited by all four ethyl derivatives

3a-3d.

While the nuclear extract HDACs and rHDAC1 were almost

equipotently inhibited by all dithio analogues 3b-3d (1.1-3.4-

fold difference, Table 1), the trithiocarbonate 3a inhibited

rHDAC1 about 12-fold less potently (IC₅₀) 4.56 µM) as the

nuclear extract. This indicates the presence of another, not tested

HDAC isoform(s) or HDAC multiprotein complex distinct to

rHDAC1 and present in the nuclear extract, preferentially

inhibited by trithiocarbonate representative 3a compared to other

dithio analogues 3b-3d. In contrast, 3a exhibited a slightly

more potent rHDAC6 inhibition (IC₅₀) 1.22 µM), a cytosolic

isoenzyme not present in the nuclear extract. About 10 times

more potent rHDAC6 inhibition was found for all dithio

analogues 3b-3d, especially for dithiocarboxylate 3b and

dithiocarbamate 3d (Table 1).

For the trithiocarbonate 3a, its enzymatic inhibition of

rHDAC1 correlated well with cellular HDAC inhibition (IC₅₀

) 4.67 µM) and is also expressed by inhibition of proliferation/

cytotoxicity toward HeLa cells (IC₅₀) 1.96 µM). A direct

correlation between rHDAC1 inhibition and cellular activity,

however, is not found for dithiocarboxylate 3b or dithiocar-

bamate 3d and is less distinct for the dithiocarbonate 3c.

The replacement of the phenacyl bound sulfur in the

headgroup for oxygen (X ) O, 3e) or nitrogen (X ) NH, 3g or

3f) results in a complete loss of any HDAC inhibitory activity

(Table 1). Finally, substitution of the central sulfur for oxygen

was probed for the N- and O-analogues (Y ) O, 3h and 3i).

These compounds exhibited HDAC inhibition in a similar range

as the corresponding sulfur analogues (Y ) S, 3c and 3d) for

Trithiocarbonates as a NoVel Class of HDAC Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3989

Table 1. Enzymatic and Cellular Assay Results for Compounds 3^a

HDAC nuc IC₅₀

rHDAC1 IC₅₀

rHDAC6 IC₅₀

cell HDAC IC₅₀

HeLa cytotoxicity IC₅₀

compd

X

Y

Z

R

[µM]

3a

3b

3c

3d

3e

S

CH₂

O

NH

S

C₂H₅

0.375

0.123

0.811

0.195

16% @

32 µM

-6% @

30 µM

-3% @

32 µM

2.81

0.115

0.128

0.467

0.293

2% @

32 µM

4.56

0.422

1.38

0.215

6% @

32 µM

nd

1.22

4.67

3.41

4.72

nd

28% @

50 µM

nd

1.96

5.71

4.64

7.80

2.99

S

O

0.110

0.448

0.094

16% @

32 µM

nd

3f

NH

S

NH

S

C₂H₅

5.81

3g

-17% @

32 µM

21.2

0.368

9.72

7.51

8.94

0% @

32 µM

-8% @

32 µM

5.36

0.132

4.57

1.64

2.13

-2% @

32 µM

nd

26% @

50 µM

18.5

13.3

5.46

11.1

7.88

-8% @

50 µM

3 h

3i

3j

3k

3l

3m

S

O

S

O

NH

S

C₂H₅

C₂H₄OPh

C₂H₄NHAc

C₂H₄OCH₃

9.79

3.18

11.6

3.47

5.53

0% @

32 µM

bond -N-morpholinyl

^aBiochemical and cellular assays were done in replicate and mean values are shown. IC₅₀values were calculated from respective concentration-effect

curves. Inhibition of cellular HDAC enzymatic activity and cytotoxicity was quantiﬁed in HeLa cervical carcinoma cells. nd: not determined.

Scheme 9^a

In a next step, substitution of the ethyl group of the trithiocar-

bonates were investigated with the option to improve physico-

chemical properties of such compounds. Introduction of phenoxy-

(3j) and methoxy-groups (3l) were tolerated as the nuclear extract

(IC₅₀) 0.128 and 0.293 µM, respectively, Table 1) was inhibited

in a similar range as the unsubstituted ethyl derivative 3a (IC₅₀

)

0.375 µM). Similarily, the N-acetamide substitutent (3k) was found

to be an active HDAC inhibitor. All three substituted ethyl

derivatives 3j-3l showed slightly decreased rHDAC1 and rHDAC6

activity compared to 3a. The inhibition of cellular HDAC activity

was found to be similar to 3a and correlating well with rHDAC1

inhibition (Table 1). Interestingly, the most sterically demanding

phenoxy derivative 3j showed the lowest activity in both test

systems. In contrast to cellular HDAC inhibitory activity, the

cytotoxicity of the substituted derivatives 3j-3l was signiﬁcantly

weaker compared to their unsubstituted parent compound 3a. While

variations of the ethyl moiety of trithiocarbonates were compatible

with HDAC inhibition, a variation of dithiocarbamates toward more

bulky, secondary dithiocarbamates lead to completely inactive

compounds (3m).

^aReagents and conditions: (a) PTT, THF, rt; then EtS(CdS)SNa.

Scheme 10^a

^aReagentsandconditions:(a)RR’NH,DIPEA,THF,rt;thenEtS(CdS)SNa

(method d).

Scheme 11^a

These results for the variation of the head group were

reconﬁrmed in the structurally related N-acetamide series 8a-8d

(Table 2). Again, all four analogues inhibit the nuclear extract

in a similar range (IC₅₀values from 87 to 386 nM, Table 2)

but more potently than the methoxy compounds 3a-3d. Again,

the differences for the nontrithiocarbonates 8b-8d between

inhibition of nuclear extract and rHDAC1 are subtle (1.4-1.9

times), while 8a inhibits rHDAC1 about 5 times less potently.

Potent rHDAC6 inhibition is found for all N-acetamide ana-

logues 8a-8d, especially dithiocarboxylate 8b and dithiocar-

bamate 8d, showing low nanomolar inhibition (IC₅₀) 23 and

18 nM, respectively, Table 2).

^aReagents and conditions: (a) NMM, IBCF, THF, 0 °C, then RR’NH,

rt, then PTT, then EtS(CdS)SNa. NMM: N-methyl-morpholine; IBCF:

i-butylchloroformiate.

Scheme 12^a

^aReagents and conditions: (a) NMM, IBCF, THF, 0 °C, then RR’NH,

rt,thenEtS(CdS)SNa.NMM:N-methyl-morpholine;IBCF:i-butylchloroformiate.

The complete inhibition of nuclear extract HDACs, rHDAC1,

and rHDAC6 is exempliﬁed for 8b in Figure 1. As seen before, a

good correlation between enzymatic and cellular activity is found

only for the trithiocarbonate 8a, whereas for the dithio analogues

8b-8d, this correlation is less distinct (Table 2). Respective

concentration-effect curves for cellular HDAC inhibition and

histone H3 hyperacetylation exempliﬁed for 8c are shown in Figure

3 (IC₅₀values of 2.9 and 28 µM, respectively), veriﬁed by Western

the isoenzyme mixture, rHDAC1, and rHDAC6 (Table 1). The

cellular HDAC inhibition and cytotoxicity of derivatives 3h and

3i was about 2-fold weaker compared to the sulfur analogues,

resulting in a less pronounced correlation between rHDAC1

inhibition and cellular HDAC activity.

3990 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13

Dehmel et al.

Scheme 13^a

^aReagents and conditions: (a) Pd(dppf)Cl₂, Cs₂CO₃, dioxane/H₂O, rt; (b) NaHMDS, THF, -78 °C, then TMSCl, then Br₂, then EtS(CdS)SNa, warm to

rt; NaHMDS: sodium hexamethyldisilazide.

Having identiﬁed an amide substitutent at the phenacyl moiety

to be beneﬁcial for HDAC inhibition, several small analogues

of 8a were tested. Introduction of the N-acetyl substituent in

meta-position (8e) instead of para-position (8a) lead to a

decrease in enzymatic (about 2-fold) and cellular (about 5-fold)

activity. Even more prominent was this difference between the

meta- and para-position in the case of C1-homologues (8f and

8g). While para-analogue 8g exhibited slightly reduced HDAC

activity compared to 8a (about 2-4 times, Table 2), the meta-

derivative 8f was more than 10-fold less active in nuclear extract

and rHDAC1 inhibition (IC₅₀values of 6.59 and 38.2 µM,

respectively). Interestingly, rHDAC6 inhibition by 8f and the

cellular HDAC inhibition were found to be less reduced (about

2-3 times). Other para-N-acetyl derivatives were also studied.

The N-methylsulfonamide 8h proved to be a weaker HDAC

inhibitor in the overall proﬁle. N-acetyl-methylation of the C1-

homologue (8i) or C2-homologenation (8j) resulted in potent

rHDAC1 inhibitors in submicromolar range (IC₅₀values of

0.946 and 0.607 µM, respectively, Table 2), but their cellular

activity, especially cytotoxicity toward Hela cells, were surpris-

ingly modest.

Next, trithiocarbonates with a phenylacetyl moiety as the core

bearing larger amide substituents were studied in detail for their

HDAC inhibitory effects (Table 3). Simple phenyl amide

derivatives 12aa-12ad proved to be effective HDAC inhibitors

in regard to the HDAC isoenzyme mixture displaying submi-

cromolar activity. In general, those compounds possess a

remarkable selectivity for rHDAC6 over rHDAC1. The complete

inhibition of nuclear extract, rHDAC1, and rHDAC6 is shown

for 12ac in Figure 1. A substrate competitive mode-of-action

for inhibition of rHDAC6 was determined with a K_ivalue of

0.11 µM (Figure 2B). In A549 NSCLC cells, R-tubulin

hyperacetylation as a hallmark for cellular HDAC6 inhibition

Figure 1. Inhibition of HDAC enzymatic activity. Trithiocarbonate

was strongly induced by 12ac at 10 µM concentration, whereas

the effect on histone H3 hyperacetylation was weak (Figure 4B).

This nicely proved the HDAC6 selectivity also in a cellular

context.

The differences in activity between acetyl benzoic acid

derived anilides 12aa and 12ab and the inverse amides 12ac

and 12ad are marginal except for rHDAC6 inhibition, which

was more effectively inhibited by 12ac and 12ad. As seen

before for small amides, the position of ring substitution has

a strong inﬂuence on rHDAC1 inhibition: meta-substituted

derivatives 12aa and 12ac inhibit rHDAC1 with low micro-

analogues, namely 2 as initial HTS hit, 3b (dithiobutyrate analogue),

12ac (phenylamide analogue), 18a and 22 (thiophene analogues) were

tested for inhibition of HeLa nuclear extract HDAC activity (A),

rHDAC1 (B), and rHDAC6 (C) as class I and IIb representatives,

respectively. SAHA was included as a reference HDAC inhibitor with

the hydroxamate head group. IC₅₀values calculated from the

concentration-effect curves using Graphpad prism nonlinear curve

ﬁtting are shown.

blotting for histone H3 hyperacetylation and p21^wafinduction in

Figure 4 for 8b and 8c.

Trithiocarbonates as a NoVel Class of HDAC Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3991

Figure 2. Enzyme kinetic analysis of rHDAC1 and rHDAC6 inhibition. The initial hit 2 and the thiophene analogue 18d were tested for inhibition

of rHDAC1 enzymatic activity at various substrate concentrations up to 100 µM. Respective Lineweaver-Burk plots with calculated K_ivalues are

shown in (A). A similar analysis was done for isoenzyme selective analogues 12ac and 13 regarding inhibition of rHDAC6 at various substrate

concentrations up to 100 µM. Respective Lineweaver-Burk plots with calculated K_ivalues are shown in (B). Ac-GGK(Ac)-AMC was used as the

substrate for both, rHDAC1 and rHDAC6 assays.

Figure 3. Inhibition of HDAC activity in HeLa cells. The inhibition of cellular HDAC activity by selected analogues 2, 8c, 18a, 18c, 22, and

SAHA as reference was measured in two independent assays in HeLa cells. The induction of histone H3 hyperacetylation was quantiﬁed by single-

cell image analysis using the Cellomics Array Scan (A). The inhibition of HDAC enzymatic activity was quantiﬁed with the cell permeable HDAC

substrate Boc-K(Ac)-AMC. After cell lysis, only the deacetylated substrate Boc-K-AMC is cleaved by trypsin with subsequent release of the

ﬂuorophore AMC.

molar activity (IC₅₀values of 1.67 and 1.22 µM, respec-

tively). The corresponding para-substituted derivatives,

however, strongly loose their rHDAC1 inhibitory activity in

case of the aniline derivative 12ad (IC₅₀) 10.4 µM) or

almost completely in case of benzoic acid derivative 12ab

(IC₅₀) 27.9 µM). Structural rigidity of the phenyl amide

moiety might be an explanation for this remarkable isoen-

zyme selectivity, as the corresponding structurally more

ﬂexible para- and meta-substituted sulfonamide derivates

12ae and 12af do not discriminate in their rHDAC1 activity.

The conformationally restricted amino-quinazoline 12ag also

inhibited the HDAC nuclear extract and rHDAC6 in submi-

cromolar concentration, but rHDAC1 inhibition is also

signiﬁcantly reduced (IC₅₀) 25.8 µM). The amides of

3-pyridine-carboxylic acid and 3-quinoline-carboxylic acid,

12ah-12ak, were found to be strong HDAC inhibitors (IC₅₀

3992 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13

Dehmel et al.

Figure 4. Induction of p21^wafexpression, histone H3, and R-tubulin hyperacetylation. The induction of histone H3 K^1-20hyperacetylation and

p21^waf1as a HDAC inhibitor marker gene was analyzed in HeLa cells, treated with analogues 8b, 8c, 12az, 18a, 18c, 22, and 23 at concentrations

of 5 and 50 µM for 24 h (A). As a control, rehybridization was done with a ꢀ-actin speciﬁc antibody. The induction of histone H3 K^1-20and

R-tubulin hyperacetylation was studied in A549 NSCLC cells treated for 24 h with the HDAC6 selective analogues 12ac and 13 (B). The natural

product HC-toxin was included as HDAC inhibitor with weak, µM inhibition of HDAC6. As a control, rehybridization was done with antibodies

speciﬁc for histone H3 or R-tubulin.

Figure 5. Cytotoxicity proﬁle for selected trithiocarbonate analogues. The overall cytotoxicity proﬁle was studied for 2 as the initial HTS hit and

the potent thiophene analogues 18a and 22. A selection of 21 different solid and hematological tumor cell lines representing 15 different histotypes

was treated for 72 h with the HDAC inhibitors before quantiﬁcation of metabolic activity, correlating with cell viability/cell number. Mean graph

blots with the deviation from the mean IC₅₀expressed in log IC₅₀[M] are shown in (A). In this graphic representation, the mean IC₅₀value for a

given compound is deﬁned as “0” and deviations of individual IC₅₀values (log IC₅₀[M]) to higher and lower potency are expressed as negative

and positive values, respectively. Mean IC₅₀values were determined as 8.31 µM (-5.08) for 2, 1.41 µM (-5.85) for 18a, and 2.37 µM (-5.57)

for 22. Individual concentration-effect curves are shown for proliferating and arrested, nonproliferating RKO p21^waf1colon carcinoma cells in (B).

RKO p21^waf1cells were arrested by induction of the cdk inhibitory p21^waf1protein.

as low as 36 nM) and preferential inhibition of rHDAC6.

rHDAC6 selectivity, however, is less prominent for the para-

substituted derivatives 12ai and 12ak, as their rHDAC1

inhibition is in the micromolar range. For these compounds,

the rHDAC1 inhibition correlates well with cellular HDAC

inhibition resulting in submicromolar cellular activity, albeit

their cytotoxic activity is weak. More evidence for the

dependence of rHDAC1 inhibition on structural ﬂexibility

Trithiocarbonates as a NoVel Class of HDAC Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3993

Table 2. Enzymatic and Cellular Assay Results for Compounds 8^a

compd

X

R

HDAC nuc IC₅₀[µM] HDAC1 IC₅₀[µM] rHDAC6 IC₅₀[µM] cell HDAC IC₅₀[µM] HeLa cytotoxicity IC₅₀[µM]

8a

8b

8c

8d

8e

8f

8g

8h

8i

S

4-AcNH

CH₂4-AcNH

4-AcNH

NH 4-AcNH

0.318

0.087

0.386

0.094

0.506

6.59

0.719

1.26

0.384

0.587

1.68

0.164

0.536

0.141

2.93

38.24

7.12

6.11

0.180

0.023

0.090

0.018

0.329

0.816

0.346

1.09

0.857

1.37

26.2

1.12

3.28

6.82

2.07

8.95

6.33

1.48

1.46

6.41

3.61

11.0

9.38

11.3

17.9

45.8

21.5

12.8

O

S

3-AcNH

3-AcNHCH₂

4-AcNHCH₂

4-MeSO₂NH

4-AcN(Me)CH₂

4-AcNHCH₂CH₂

0.946

0.607

0.256

0.102

8j

^aBiochemical and cellular assays were done in replicate and mean values are shown. IC₅₀values were calculated from respective concentration-effect

curves. Inhibition of cellular HDAC enzymatic activity and cytotoxicity was quantiﬁed in HeLa cervical carcinoma cells.

Figure 6. Cell cycle analysis and induction of histone H3S¹⁰phosphorylation. The effects of selected analogues 2, 8c, 18a, and 22 on the cell

division cycle of RKO colon carcinoma cells was studied by ﬂow cytometry. RKO cells were treated for 24 h with the trithiocarbonate analogues

at 1 and 10 µM concentration. The percentage of cells in apoptosis (subG1), G1(2N), S, and G2/M (4N) was determined and is shown in a column

diagram in (A). 10 nM taxol as a tubulin interacting, antimitotic agent and 5 µM SAHA were included as control agents. The phosphorylation of

histone H3 at residue S¹⁰in RKO cells treated for 24 h with 2, 8c, 18a, and 22 at 1, 3, and 10 µM concentration was analyzed by Western blotting

(B). Hyperacetylation of histone H3 K^1-20as well as R-tubulin was analyzed in parallel. As a control, rehybridization was done with a ꢀ-actin

speciﬁc antibody.

of the core phenyl substitution is provided by compounds

12al-12an. A more than 8-fold difference in rHDAC1

inhibition between meta- and para-substituted benzylamides

12al and 12am was observed, but the constitutional para-

isomer 12an, which possess a less rigid connection to the

phenyl core, again retained rHDAC1 inhibition (IC₅₀) 0.523

µM). Another pair of meta- and para-substituted phenyl with

potent HDAC inhibitory properties are phenoxy-ethylamino

carboxamides 12ao and 12ap, which are low submicromolar

HDAC inhibitors in the enzymatic assays as well as potent

inhibitors of cellular HDAC activity, but again weakly

cytotoxic. A ﬁnal example for the loss of rHDAC1 inhibitory

efﬁcacy by the structural rigidity of para-substituted phenyl

analogues represents the bis-phenyl urea 12aq, which is 30-

fold more active toward rHDAC6 (IC₅₀) 0.606 µM) and

470-fold more active toward the nuclear extract isoenzyme

mixture (IC₅₀) 0.040 µM) compared to rHDAC1 (IC₅₀

18.8 µM).

)

The next series of trithiocarbonates that were probed for

HDAC inhibition were phenylsulfonamides (Table 3). Four

3994 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13

Table 3. Enzymatic and Cellular Assay Results for Compounds 12^a

Dehmel et al.

^aBiochemical and cellular assays were done in replicate and mean values are shown. IC50 values were calculated from respective concentration-effect

curves. Inhibition of cellular HDAC enzymatic activity and cytotoxicity was quantiﬁed in HeLa cervical carcinoma cells.

Trithiocarbonates as a NoVel Class of HDAC Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3995

Table 4. Enzymatic and Cellular Assay Results for Compounds 16 and 18^d

^aPreparation according to Scheme 11. ^bPreparation according to Scheme 12. ^cPreparation according to Scheme 13. ^dBiochemical and cellular assays

were done in replicate and mean values are shown. IC50 values were calculated from respective concentration-effect curves. Inhibition of cellular HDAC

enzymatic activity and cytotoxicity was quantiﬁed in HeLA cervical carcinoma cells.

4-chloro-phenyl derivatives were compared and, as expected,

the para-4-chloro-aniline carboxamide 12ar was inactive in the

rHDAC1 enzyme assay, while the corresponding sulfonamide

12as exhibited submicromolar rHDAC1 and cellular HDAC

inhibiton, besides a very potent inhibition of nuclear HDACs

(IC₅₀) 8 nM). The constitutional analogue 12at was consider-

ably less potent toward rHDAC1 (IC₅₀) 2.49 µM), and the

meta-substituted analogue 12au has even lower enzymatic

inhibitory activity that is also reﬂected in low cellular HDAC

inhibition. The biochemical discrimination between meta- and

para-substituted benzene sulfonamides was probed for the

4-methoxy-aniline sulfonamides 12av and 12aw. Both isomers

were found to be potent inhibitors of the nuclear extract, but in

contrast to the carboxamides (e.g., 12aa and 12ab), the para-

substituted sulfonamide showed slightly enhanced activity at

rHDAC1 and rHDAC6 (about 2-fold) and clearly superior

cellular HDAC inhibition. Interestingly, all activity was 10-fold

reduced by methylation of the sulfonamide moiety (12ax).

Several other amino-aryl sulfonamides were low submicromolar

HDAC inhibitors with cytotoxic activity of IC₅₀) 7.7 and 9.18

µM for 12ay-12az, respectively. The cellular activity of 12az

as HDAC inhibitor was proven by a strong induction of p21^waf1

and histone H3 hyperacetylation at 50 µM concentration in HeLa

cells (Figure 4A).

potent and selective HDAC6 inhibitor in biochemical (IC₅₀)

56 nM) and cellular (EC₅₀≈ 5 µM) assays (Figure 4B),

displaying a substrate competitive mode-of-action as determined

by Lineweaver-Burk enzyme kinetic analysis (K_i) 0.14 µM,

Figure 2B).

Trithiocarbonates bearing a 2,5-disubstituted thiophene core

instead of benzene as in compounds of type 16 were also potent

HDAC inhibitors (Table 4). The HDAC isoenzyme mixture was

inhibited with IC₅₀values from 19 to 63 nM, while the inhibition

of rHDAC6 was weaker, especially for the sterically more

demanding 3,5-dimethylbenzyl amine derivative 16d. Remark-

ably, rHDAC1 was inhibited by all benzyl amine derivatives

16a-16d with IC₅₀values between 0.34 and 0.73 µM (Table

4), but as seen for the phenyl compounds 12, the rigidiﬁed

aniline amide 16e was almost inactive (IC₅₀) 31.6 µM).

Noteworthy, the two enantiomers 16b and 16c had almost

identical inhibitory activity. The activity of the amides 16 in

biochemical assays was reﬂected in potent cellular enzymatic

HDAC inhibition (IC₅₀values from 0.41 to 1.45 µM), but very

weak cytotoxicity was determined.

If the structural rigidity in the thienyl carboxamide bond was

reduced by introduction of a methylene linker as in aryl-bearing

amides 18a-18d, potent HDAC inhibitors were obtained. For

all derivatives, also reasonable rHDAC1 inhibitory activity was

The HDAC inhibitory activity of the structurally related

pyrrole-N-sulfonamide 13 was also studied in more detail

(Scheme 9). Nuclear HDACs were very potently inhibited with

an IC₅₀of 9 nM, while rHDAC1 inhibition was about 200-fold

weaker (IC₅₀) 2.1 µM). Compound 13 was found to be a very

found, even for aniline derived carboxamides as 18c (IC₅₀)

0.25 µM, Table 4), which was more active as its benzylamine

analogue 18b (IC₅₀) 0.89 µM). rHDAC6 inhibition was also

observed for 18a-18d. Complete inhibition of nuclear extract,

rHDAC1, and rHDAC6 isoenzymes is shown for 18a in Figure

3996 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13

Dehmel et al.

1, and the substrate competitive mode-of-action for rHDAC1

was proven for 18d exemplarily (K_i) 0.46 µM, Figure 2A).

Compound 18a displays potent cytotoxicity toward HeLa cells

(IC₅₀) 0.71 µM) and was studied together with the close

analogue 18c in more detail. Histone H3 hyperacetylation is

induced by 18a and 18c with EC₅₀values of 2.4 and 0.76 µM,

respectively (Figure 3A), also seen in respective Western Blot

experiments for p21^waf1and acetylated histone H3 (Figure 4A).

Inhibition of cellular HDAC enzymatic activity was up to 10-

fold weaker for both analogues. Compound 18a was also

characterized using the 21 tumor cell line panel, depicting a

mean cytotoxicity of IC₅₀) 1.41 µM, quite comparable to

SAHA (mean IC₅₀) 3.3 µM)²⁹with some preferential activity

toward hematological tumors (Figure 5).

effect. All analogues induced R-tubulin hyperacetylation as

evidence for cellular HDAC6 inhibition. Interestingly, this

R-tubulin hyperacetylation was strongly reduced for compounds

2 and 18a at the 10 µM concentration, inducing strong histone

H3 S¹⁰phosphorylation and most likely mitotic arrest. From

these experiments, we conclude that selected trithiocarbonate

analogues show unique cellular properties with similarities to

antimitotic agents like taxol, arresting RKO colon carcinoma

cells transiently in mitosis before execution of apoptosis. We

hypothesize that a new lysine speciﬁc protein deacetylase or a

hypothetical mitotic HDAC complex are targeted by these

antimitotic trithiocarbonate analogues.

Experimental Section

Finally, the two pyridinyl-thienoacetyl derivatives 22 and 23

were studied. Both compounds exhibited potent HDAC inhibi-

tion in the enzymatic assays. While the amino-quinoline

substituted derivative 22 was a very potent inhibitor of the

nuclear extract (IC₅₀) 8 nM, Table 4), inhibition of rHDAC1

and rHDAC6 with IC₅₀values of 0.56 and 0.44 µM, respec-

tively, was signiﬁcantly weaker. The concentration-dependent

activity of derivative 22 in the biochemical HDAC assays is

shown in Figure 1, with a partial inhibition of rHDAC6 only.

The 5-amino-pyridinyl bearing trithiocarbonate 23 was also

active in the HeLa nuclear extract and rHDAC1 assays but with

diminished potency. Nevertheless, rHDAC6 was potently in-

hibited with IC₅₀) 43 nM. For both compounds, the activity

in the cellular HDAC activity assay and cytotoxicity were in

the same range. Respective cellular data for histone H3

hyperacetylation, p21^waf1induction, and HDAC enzymatic

activity are shown for analogues 22 and 23 in Figure 3A/B and

Figure 4A. Compound 22 was characterized using the 21 tumor

cell line panel, depicting a mean cytotoxicity of IC₅₀) 2.37

µM and a selectivity pattern similar to 18a (Figure 5).

HDAC inhibitors are well-known for affecting cell cycle

regulation, depleting cells in the S-phase while transiently

arresting in G1 and G2/M as shown for 5 µM SAHA treated

RKO colon carcinoma cells in Figure 6. For most cytotoxic

trithiocarbonate analogues, a partial proliferation-dependent

cytotoxicity was seen in the RKO p21^waf1cell system, high-

lighted for compounds 18a and 22 in Figure 5B. This was

unexpected, although a partial proliferation-dependent cytotox-

icity was seen for selected benzamide analogues in previous

studies as well (data not shown). We therefore investigated the

effects on RKO cell cycle distribution by compounds 2, 8c, 18a,

and 22 in more detail. All analogues induced depletion of

S-phase cells and apoptosis after 24 h at 10 µM concentration

(Figure 6A). At 1 µM concentration, a partial arrest in G1 is

evident for the thiophene analogues 18a and 22, a partial arrest

in G2/M seen for 22 at the higher concentration of 10 µM.

Recently, the interaction of HDAC3 with the mitotic kinase

Aurora B and a HDAC3 dependent phosphorylation of histone

H3 at residue S¹⁰by Aurora B was shown.³⁰In this study,

hydroxamate analogues induced a strong reduction of histone

H3 S¹⁰phosphorylation, a marker for mitotic arrest. This has

been explained by dissociation of inactive HDAC3 from the

Aurora B complex and subsequent hyperacetylation of histone

H3 at residue K⁹.³⁰We therefore investigated the effect of

selected trithiocarbonates on histone H3 S¹⁰phosphorylation.

In contrast to the study by Li et al. using hydroxamates like

TSA, compounds 8c and 18a induced histone H3 S¹⁰phospho-

rylation and in parallel H3 K^1-20hyperacetylation (Figure 6B).

Compound 22 behaved as expected, only inducing histone H3

hyperacetylation, whereas compound 2 showed the opposite

Biological Methods. Biochemical HDAC Assay. HDAC activity

was isolated from HeLa nuclear extracts according to a method

originally described by Dignam et al.³¹Mass cultures of HEK293

cell lines with stable expression of human rHDAC1 and rHDAC6

(kindly provided by E.Verdin, Gladstone Institute for Immunology,

San Francisco) were lysed and ﬂag-tagged proteins puriﬁed by M2-

agarose afﬁnity chromatography (Sigma art. no. A-2220). Fractions

from the puriﬁcation were analyzed by Western blotting and for

speciﬁc activity in the biochemical enzyme assay. The HDAC

enzyme activity assay was done essentially as described.^14,32About

4 ng/well HDAC1 or 3 ng/well HDAC6 were incubated with 6 or

10 µM Ac-NH-GGK(Ac)-AMC, respectively, as a substrate for 2

or 3 h at 30 °C. For enzyme kinetic studies, rHDAC1 (speciﬁc

activity 0.39 µmol/min/mg) and rHDAC6 (speciﬁc activity 0.78

µmol/min/mg) enzymes were incubated with different substrate and

inhibitor concentrations. For both enzymes, the substrate Ac-

GGK(Ac)-AMC was used up to 100 µM ﬁnal concentration (K_m

values of 32.3 and 7.3 µM for rHDAC1 and rHDAC6, respectively).

For trithiocarbonate analogues, the concentrations were selected

1

based on IC₅₀values, namely /₄, /₂, 1, 2, and 4 times the EC₅₀

concentration. Substrate concentration (mol/L) was plotted versus

enzyme velocity (µmol/min), and K_ivalues were calculated from

Lineweaver-Burk plots using GraphPad prism algorithms. A

substrate competitive bindung mode is evident by a point of

intersection on the y-axis.

Cellular Histone H3/Tubulin Hyperacetylation, p21, and

Phosphohistone H3 Induction Assays. Histone H3 hyperacety-

lation was quantiﬁed using a single-cell based high content assay

(HCS) format on the Cellomics “ArrayScan II” platform (Thermo

Fisher Scientiﬁc, Waltham, MA) as described.¹⁴Brieﬂy, HeLa cells

in 96-well microtiter plates were treated with test compound for

24 h, ﬁxed, permeabilized, and stained with a polyclonal rabbit

antibody speciﬁc for histone H3K(Ac)^1-20(Calbiochem, Darmstadt/

Germany). Data analysis was done with the Cellomics algorithm

“mitotic index”, calculating normalized nuclear ﬂuorescence in-

tensity, correlating with the level of histone H3 hyperacetylation.

In parallel to the histone H3 hyperacetylation assay, the cellular

efﬁcacy and mode-of-action of HDAC inhibitors was studied in

treated HeLa, RKO, or A549 cell lysates by Western blotting using

antibodies speciﬁc for acetylated or phosphorylated histone H3,

acetylated R-tubulin, and p21^waf1. After treatment with compound

for 24 h, cells were lysed (50 mM Tris HCl pH 8, 150 mM NaCl,

1 v/v NP-40, 0.5% w/v Na-desoxycholate, 0.2% w/v disodium-

dodecylsulfate (SDS), 0.02% NaN₃, 1 mM Na-vanadate, 20 mM

NaF, 100 µg/mL PMSF, protease inhibitor mix/Roche and 50U/

mL benzonase) and respective equal amounts of protein separated

by SDS-PAGE before transfer to polyvinylidendiﬂuoride/PVDF

membrane (Biorad) by semidry blotting. The following antibodies

were used for Western blot analysis: Acetylated histone H3 K^1-20

(Calbiochem no. 382158), histone H3 (Cell Signaling no. 9715),

phosphorylated histone H3 S¹⁰(Cell Signaling no. 9701), p21 (Santa

Cruz no. SC-397), acetylated R-tubulin (Sigma T-6793), and ꢀ-actin

(Sigma A-5441).

Cellular HDAC Enzymatic Activity Assay. A 96-well micro-

titer plate assay was established using Boc-K(Ac)-AMC as substrate

Trithiocarbonates as a NoVel Class of HDAC Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3997

as described recently.²⁷HeLa cells were seeded into white 96-well

tissue culture plates at a density of 5 × 10³cells/well and cultivated

for 24 h under standard cell culture conditions. After treatment with

trithiocarbonate analogues for 3 h, 10 µL/well of a 2 mM stock

solution of the substrate Boc-K(Ac)-AMC in culture medium were

added and incubation continued for additional 3 h before addition

of 100µL/well lysis/developer buffer mix (50 mM Tris HCl pH

8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 vol % Nonidet

P40, 2.0 mg/mL trypsin, 10 µM TSA). After ﬁnal incubation for

3 h under cell culture conditions, AMC ﬂuorescence was measured

at an excitation of λ ) 355 nm and emission of λ ) 460 nm on the

Perkin-Elmer Wallac Victor II V multilabel plate reader (Perkin-

Elmer, Wellesley, MA).

chromatography (silica, hexanes to hexanes/ethyl acetate 1:1)

providing the title compounds.

Ethyl 2-(4-Methylphenyl)-2-oxoethyl Trithiocarbonate (2). 2

was prepared from 2-bromo-1-p-tolyl-ethanone using general

procedure A; yellow solid (59%). ¹H NMR (DMSO-d₆, 400 MHz,

rt) δ (ppm) : 7.94 (d, J ) 8.2 Hz, 2H), 7.37 (d, J ) 8.2 Hz, 2H),

5.10 (s, 2H), 3.38 (q, J ) 7.3 Hz, 2H), 2.38 (s, 3H), 1.29 (t, J )

7.3 Hz, 3H). LC-MS (m/e): 271 [M + H]⁺.

Ethyl 2-(4-Methoxyphenyl)-2-oxoethyl Trithiocarbonate (3a).

3a was prepared from 2-bromo-1-(4-methoxy-phenyl)-ethanone

using general procedure A; yellow solid (79%). ¹H NMR (DMSO-

d₆, 200 MHz, rt) δ (ppm): 8.02 (d, J ) 8.9 Hz, 2H), 7.08 (d, J )

8.9 Hz, 2H), 5.08 (s, 2H), 3.86 (s, 3H), 3.38 (q, J ) 7.4 Hz, 2H),

1.29 (t, J ) 7.4 Hz, 3H). LC-MS (m/e): 287 [M + H]⁺.

Proliferation Assay and Tumor Cell Lines. The antiprolifera-

tive activity of trithiocarbonate analogues on diverse tumor cell

lines was evaluated by using the Alamar Blue (Resazurin) cell

viability assay.²⁸Cells were seeded into 96-well ﬂat bottom plates

at respective densities to allow proliferation during 72 h cultivation

with test compound. The assay was done essentially done as

described.²⁹The following human tumor cell lines were used: H460

(nonsmall cell lung cancer, ATCC HTB-177), A549 (nonsmall cell

lung cancer, ATCC CCL-185), MCF7 (breast carcinoma, ATCC

HTB-22), MDA-MB468 (breast carcinoma, ATCC HTB-132),

MDA-MB435 (HTB129), MDA-MB231 (breast carcinoma, ATCC

HTB-26), SK-BR-3 (breast carcinoma, ATCC HTB-30), SK-OV-3

(ovarian carcinoma, ATCC HTB-77), HCT-15 (colon carcinoma,

ATCC CCL-225), PC3 (prostate carcinoma, ATCC CRL-1435),

AsPC1 (pancreatic carcinoma, ATCC CRL-1682), HeLa (cervical

carcinoma, ATCC CCL-2), Cal27 (tongue carcinoma, ATCC CRL-

2095), A-431 (vulva carcinoma, ATCC CRL-2592), Hec1A (en-

dometrial carcinoma, ATCC HTB-112), Saos-2 (osteosarcoma,

ATCC HTB-85), U87MG (glioblastoma, ATCC HTB-14), K562

(chronic myeloid leukemia, DSMZ ACC 10), EOL1 (acute hyper-

eosinophilic myeloid leukemia, DSMZ ACC386), and CCRF-CEM

and CCRF-CEM VCR1000 (acute lymphoblastic T-cell leukemia

sensitive and resistant toward vincristine and DSMZ 240).³³The

human colon adenocarcinoma cell line RKO and transfectant

RKOp21^waf1were described recently.³⁴p21^waf1expression was

induced by treatment with 10 µM ponasterone A for 24 h and RKO

cells with/without p21^waf1expression (2 × 10⁴cells/well induced,

6 × 10³cells/well not induced) were treated with the compounds

as described.²⁹All cell lines were cultivated at 37 °C, 5% CO₂in

medium as stated on the ATCC, DSMZ, and ECACC information

sheets or as published.

2-(4-Methoxyphenyl)-2-oxoethyl Dithiobutyrate (3b). A solu-

tion of 1-propylmagnesium bromide (1.0 equiv) in diethyl ether (2

M) was diluted with tetrahydrofuran (0.2 M) and cooled to -20

°C under a nitrogen atmosphere. Copper iodide (0.1 equiv) and, 5

min later, carbon disulﬁde (1.0 equiv) was added to yield a dark

solution. After 20 min, 2-bromo-1-(4-methoxy-phenyl)-ethanone

was added and the mixture was stirred for 5 min. The reaction

mixture was then poured into brine and the aqueous phase was

extracted with ethyl acetate. The organic phases were dried over

anhydrous magnesium sulfate and evaporated. Chromatographical

puriﬁcation (silica, hexanes/ethyl acetate) provided 3b as pale-

yellow oil (13%). ¹H NMR (DMSO-d₆, 200 MHz, rt) δ (ppm):

8.01 (d, J ) 9.6 Hz, 2H), 6.97 (d, J ) 9.6 Hz, 2H), 4.76 (s, 2H),

3.89 (s, 3H), 3.06 (t, J ) 7.5 Hz, 2H), 1.92 (quin, J ) 7.2 Hz, 2H),

1.00 (t, J ) 7.4 Hz, 3H). LC-MS (m/e): 269 [M + H]⁺.

O-Ethyl S-2-(4-methoxy-phenyl)-2-oxoethyl Dithiocarbon-

ate (3c). A 0.5 M solution of sodium ethanolate (1.0 equiv) was

added carbon disulﬁde (1.15 equiv) in tetrahydrofuran at room

temperature, and the resulting solution was stirred for 30 min.

4-Methoxy-phenacylbromide (1.0 equiv) was added via syringe.

After 1 h, the reaction mixture was diluted with half-saturated brine

and the aqueous phase was extracted with dichloromethane three

times. The organic layer was dried via ﬁltration through an IST

phase separator cartridge and evaporated to leave a yellowish

residue, which was puriﬁed by chromatography (silica gel, hexanes

to hexanes/ethyl acetate 1:1), providing 3c as whitish solid (93%).

¹H NMR (DMSO-d₆, 200 MHz, rt) δ (ppm): 8.00 (d, J ) 8.9 Hz,

2H), 7.08 (d, J ) 8.9 Hz, 2H), 4.88 (s, 2H), 4.22 (q, J ) 7.1 Hz,

2H), 3.86 (s, 3H), 1.21 (t, J ) 7.3 Hz, 3H). GC-MS: 271 [M]⁺,

135 [M-119]⁺.

Flow Cytometric Analysis. RKO colon carcinoma cells were

seeded at 5 × 10⁵cells/10 cm cell culture dish 24 h before treatment

to allow logarithmic proliferation during the experiment. After about

24 h, cells were treated with test compound for 24 and 48 h. Further

procedure and ﬂow cytomteric analysis was done as described using

the Becton Dickinson FACS Canto device.²⁹

Chemical Procedures. Reagents and solvents were used as

purchased from the supplier. Usually, reactions were run under

nitrogen atmosphere (N₂-balloon). Unless otherwise stated, prepara-

tions were typically performed on a 0.5 mmol scale. NMR spectra

were recorded on a Bruker DPX200, a Bruker AV400, a Bruker

AVII300, or on a Bruker AV600 in the solvent given. Mass spectra

were recorded on a LCQ classic by Thermoﬁnnigan and GC-MS

spectra on a Trace MS (Thermoﬁnnigan). Reaction progress was

monitored by analytical LC-MS on an Agilent 1100.

Preparation of Ethyl Trithiocarbonates (Procedure A). A 0.5

M solution of sodium ethyl trithiocarbonate was prepared by adding

carbon disulﬁde (1.3 equiv) to a suspension of sodium thioethanolate

(1.2 equiv) in tetrahydrofuran at room temperature and stirring of

the resulting yellow solution for 30 min. Phenacyl bromide or

chloride (1.0 equiv) was dissolved in ethyl acetate at room

temperature and a 0.5 M solution of sodium ethyl trithiocarbonate

(1.2 equiv) was added via syringe. After 1-3 h, the reaction mixture

was diluted with half-saturated brine, and the aqueous phase was

extracted with dichloromethane three times. The organic layer was

dried via ﬁltration through IST phase separator cartridge and

evaporated to leave a yellowish residue, which was puriﬁed by

N-Ethyl S-2-(4-Methoxy-phenyl)-2-oxoethyl Dithiocarbam-

ate (3d). A solution of 2-mercapto-1-(4-methoxy-phenyl)-ethanone

4 (1.0 equiv) in tetrahydrofuran (0.2 M) was treated with ethylth-

ioisocyanate (1.2 equiv) at 0 °C. The reaction mixture was allowed

to warm to rt overnight, poured into saturated sodium bicarbonate

solution, and extracted with ethyl acetate. The organic phases were

dried over anhydrous magnesium sulfate and evaporated to yield

3i as pale-white powder (71%). 3d exists in solution in equilibrium

with a cyclized hemiaminal with the ratio being dependent on the

1

solvent. H NMR (DMSO-d₆, 400 MHz, rt) open chain δ (ppm):

10.05 (bs, 1H), 8.01 (d, J ) 8.9 Hz, 2H), 7.08 (d, J ) 8.9 Hz, 2H),

4.84 (s, 2H), 3.86 (s, 3H), 3.66 (m, 2H), 1.15 (t, J ) 7.2 Hz, 3H).

Cyclized hemiaminal δ (ppm): 7.57 (s, 1H), 7.36 (d, J ) 8.8 Hz,

2H), 6.99 (d, J ) 8.8 Hz, 2H), 3.69 (s, 3H), 3.57 (s, 2H), 3.57 (qd,

J ) 7.1, 6.5 Hz, 1H), 3.22 (qd, J ) 7.1, 6.5 Hz, 1H), 0.98 (t, J )

7.1 Hz, 2H). LC-MS (m/e): 270 [M + H]⁺.

S-Ethyl O-2-(4-Methoxy-phenyl)-2-oxoethyl Dithiocarbon-

ate (3e). A mixture of 2-hyxdroxy-1-(4′-methoxyphenyl)-ethanone

(6) (0.50 g, 3.0 mmol), ethyleneglycol (4 mL), and amberlyst 15

(0.80 g) in benzene was reﬂuxed in a Dean-Stark apparatus for

3 h. The mixture was extracted with saturated bicarbonate solution

and brine, and the solvents were removed under reduced pressure

to leave the product, which was used without further puriﬁcation.

A solution of the dioxolane (0.6 g, 2.9 mmol) in tetrahydrofuran

(19 mL) was added dropwise to a suspension of NaH (60% in

mineral oil, 0.28 g, 7.1 mmol) and imidazole (10 mg) in tetrahy-

drofuran (25 mL) at 0 °C. After stirring for 30 min at rt, carbon

3998 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13

Dehmel et al.

disulﬁde (430 µL, 7.1 mmol) was added. The mixture was again

stirred for 30 min, before ethyl iodide (575 µL, 7.1 mmol) was

added and the mixture was stirred for further 1 h. Then the reaction

mixture was poured onto ice and extracted with ethyl acetate. The

organic phases were dried over anhydrous magnesium sulfate and

evaporated. The residue was puriﬁed using chromatography (silica,

dichloromethane/hexanes 4:1) to yield the product as whitish solid

(0.65 g, 72%). The mixture of the resulting dioxolane (0.30 g, 0.95

mmol) in tetrahydrofuran/H₂O (3:1, 4 mL) was treated with a few

drops of conc HCl and stirred at 60 °C for 4 h. The reaction mixture

was poured into water and extracted with dichloromethane. The

organic phases were dried over anhydrous magnesium sulfate and

evaporated. The residue was puriﬁed using chromatography (silica,

dichloromethane/hexanes 3:2) to yield 3e as a colorless solid (0.20

g, 78%). ¹H NMR (CDCl₃, 200 MHz, rt) δ (ppm): 7.92 (d, J ) 9.2

Hz, 2H), 6.97 (d, J ) 9.2 Hz, 2H), 5.84 (s, 2H), 3.88 (s, 3H), 3.19

(q, J ) 7.1 Hz, 2H), 1.39 (t, J ) 7.1 Hz, 3H). LC-MS (m/e): 271

[M + H]⁺.

Preparation of Trithiocarbonates (Procedure B). A 0.5 M

solution of alkyl sodium trithiocarbonate is prepared by adding

carbon disulﬁde (1.15 equiv) to a suspension of the requisite sodium

thiolate (1.0 equiv) in tetrahydrofuran at room temperature and

stirring of the resulting yellow solution for 30 min. Phenacyl

bromide (1.0 equiv) was dissolved in ethyl acetate at room

temperature, and a 0.5 M solution of sodium alkyl trithiocarbonate

(1.2 equiv) was added via syringe. After 1-3 h, the reaction mixture

was diluted with half-saturated brine and the aqueous phase was

extracted with dichloromethane three times. The organic layer was

dried via ﬁltration through an IST phase separator cartridge and

evaporated to leave a yellowish residue, which was puriﬁed by

chromatography (silica gel, hexanes to hexanes/ethyl acetate 1:1),

providing the title compounds.

8.1 Hz, 1H), 7.77 (d, J ) 7.9 Hz, 1H), 7.50 (d, J ) 7.0 Hz, 1H),

5.10 (s, 2H), 3.39 (q, J ) 7.4 Hz, 2H), 2.07 (s, 3H), 1.29 (t, J )

7.3 Hz, 3H). LC-MS (m/e): 314 [M + H]⁺.

2-[4-(Acetylamino-methyl)-phenyl]-2-oxoethyl Ethyl Trithio-

carbonate (8g). 8g was prepared from N-(4-acetyl-benzyl)-aceta-

1

mide according to general procedure C; yellow solid (70%). H

NMR (DMSO-d₆, 200 MHz, rt) δ (ppm): 8.43 (t, J ) 6.0 Hz, 1H),

8.00 (d, J ) 8.3 Hz, 2H), 7.42 (d, J ) 8.3 Hz, 2H), 5.10 (s, 2H),

4.34 (d, J ) 6.0 Hz, 2H), 3.38 (q, J ) 7.4 Hz, 2H), 1.90 (s, 3H),

1.29 (t, J ) 7.4 Hz, 3H). LC-MS (m/e): 328 [M + H]⁺.

Ethyl 2-(4-Methanesulfonylamino-phenyl)-2-oxoethyl Trithio-

carbonate (8h). 8h was prepared from 2-chloro-1-(4-methane-

sulfonylamino-phenyl)-ethanone using general procedure A; yellow

1

oil (64%). H NMR (DMSO-d₆, 200 MHz, rt) δ (ppm): 10.35 (s,

1H), 8.04 (d, J ) 8.8 Hz, 2H), 7.32 (d, J ) 8.8 Hz, 2H), 5.08 (s,

2H), 3.38 (q, J ) 7.3 Hz, 2H), 3.14 (s, 3H), 1.29 (t, J ) 7.4 Hz,

3H). LC-MS (m/e): 350 [M + H]⁺.

Preparation of Arylamide Trithiocarbonates (Procedure D).

A solution of the acetyl-aryl carboxylic acid (1.0 equiv) in

tetrahydrofuran (0.3 M) was cooled to 0 °C, then isobutyl

chloroformate (1.1 equiv) and N-methyl-morpholine (1.05 equiv)

were added and the mixture was stirred for 45 min. The corre-

sponding amine (1.2 equiv) was added, and the mixture was allowed

to warm to rt slowly. After 18 h, phenyltrimethylammonium

tribromide (PTT, 1.1 equiv) was added and the mixture was stirred

at rt (1-18 h) until the orange solution became colorless and a

white precipitate was observed. A solution of sodium ethyl

trithiocarbonate (0.5 M in tetrahydrofuran, 2.0 equiv) was added

and the mixture was stirred again for 2-18 h. After completion of

the reaction, the reaction mixture was diluted with half-saturated

brine, and the aqueous phase was extracted with dichloromethane.

The organic layer was evaporated to leave a yellowish residue,

which was puriﬁed either by chromatography (silica, hexanes/ethyl

acetate gradient) or by washing the solid with a little ethyl acetate,

providing the title compound as a pale-yellow solid.

Ethyl 2-oxo-2-(3-Phenylcarbamoyl-phenyl)-ethyl Trithiocar-

bonate (12aa). 12aa was prepared from aniline and 3-acetyl-

benzoic acid according to general procedure D; yellow solid (18%).

¹H NMR (DMSO-d₆, 400 MHz, rt) δ (ppm): 10.45 (s, 1H), 8.58

(s, 1H), 8.27 (t, J ) 6.3 Hz, 2H), 7.79 (d, J ) 7.7 Hz, 2H), 7.75

(t, J ) 7.8 Hz, 1H), 7.39 (t, J ) 7.6 Hz, 2H), 7.14 (t, J ) 7.4 Hz,

1H), 5.23 (s, 2H), 3.40 (q, J ) 7.4 Hz, 2H), 1.30 (t, J ) 7.4 Hz,

3H). LC-MS (m/e): 376 [M + H]⁺.

Ethyl 2-oxo-2-(4-Phenylcarbamoyl-phenyl)-ethyl Trithiocar-

bonate (12ab). 12ab was prepared from aniline and 4-acetyl-

benzoic acid according to general procedure D; yellow solid (30%).

¹H NMR (DMSO-d₆, 200 MHz, rt) δ (ppm): 10.45 (s, 1H), 8.19

(d, J ) 8.5 Hz, 2H), 8.10 (d, J ) 8.5 Hz, 2H), 7.80 (d, J ) 7.6 Hz,

2H), 7.38 (t, J ) 7.6 Hz, 2H), 7.13 (t, J ) 7.3 Hz, 1H), 5.18 (s,

2H), 3.39 (q, J ) 7.3 Hz, 2H), 1.30 (t, J ) 7.3 Hz, 3H). LC-MS

(m/e): 376 [M + H]⁺.

Preparation of Pyrimidylamide Trithiocarbonates, Method

c (Procedure E). A solution of the 1-(hetero)aryl-ethanone (1.0

equiv) in tetrahydrofuran (0.08 M) was treated with a solution of

sodium hexamethyldisilazide (1.1-2.2 equiv) at -78 °C and then

stirred for 0.5 h. Trimethylsilylchloride (1.1-2.2 equiv) was added,

and the mixture was stirred for 1 h at -78 °C. After the addition

of a solution of bromine (1.0 equiv) in tetrahydrofuran (0.5 M),

the mixture was stirred again for 1 h at -78 °C and then allowed

to warm to rt. A solution of sodium ethyl trithiocarbonate (0.5 M

in tetrahydrofuran, 3.0 equiv) was added, and the mixture was stirred

again for 2-18 h. The reaction mixture was poured into a 2:1

mixture of 5% sodium bicarbonate and 5% sodium bisulﬁte solution

and extracted with ethyl acetate. The organic phases were washed

with brine and dried with sodium sulfate. Evaporation in vacuo

gave an oily residue, which was puriﬁed by chromatography (silica,

hexanes/ethyl acetate gradient) or by preparative HPLC (acetonitrile/

water gradient), providing the title compounds.

Preparation of Trithiocarbonates Using PTT in Situ Bromi-

nation, Method a (Procedure C). A solution of phenylethanone

7 (1.0 equiv) in tetrahydrofuran (0.2-0.5 M) was treated with

phenyltrimethylammonium tribromide (PTT, 1.1 equiv) at rt. The

mixture was stirred at rt until the reaction was complete (TLC

control and appearance of a white precipitate, 3 min to 16 h). To

this reaction mixture, a 0.5 M solution of sodium ethyl trithiocar-

bonate (1.5-3 equiv) was added via syringe. After completion of

the reaction, the reaction mixture was diluted with half-saturated

brine and the aqueous phase was extracted with dichloromethane

three times. The organic layer was dried via ﬁltration through an

IST phase separator cartridge and evaporated to leave a yellowish

residue, which was puriﬁed by chromatography (silica, hexanes to

hexanes/ethyl acetate 1:1), providing the title compounds.

2-(4-Acetylamino-phenyl)-2-oxoethyl Ethyl Trithiocarbon-

ate (8a). 8a was prepared from N-(4-acetyl-phenyl)-acetamide

1

according to general procedure C; yellow solid (53%). H NMR

(DMSO-d₆, 200 MHz, rt) δ (ppm): 10.30 (s, 1H), 8.03 (d, J ) 8.7

Hz, 2H), 7.76 (d, J ) 8.7 Hz, 2H), 5.08 (s, 2H), 3.39 (q, J ) 7.4

Hz, 2H), 2.10 (s, 3H), 1.29 (t, J ) 7.4 Hz, 3H). LC-MS (m/e): 314

[M + H]⁺.

O-Ethyl S-2-(4-Acetamido-phenyl)-2-oxoethyl Dithiocarbon-

ate (8c). Ethyl chloroformiate (210 µL, 2.2 mmoL) was added a

solution of 2-mercapto-1-(4-methoxy-phenyl)-ethanone (365 mg,

2.0 mmol) in 4 mL of tetrahydrofuran at rt. Diisopropylethylamine

(346 µL, 2.5 mmol) was added, and the mixture was stirred

overnight. The reaction mixture was treated with half-saturated

brine, and the aqueous phase was extracted with ethyl acetate. The

organic phases were dried over anhydrous magnesium sulfate and

evaporated. The residue was puriﬁed using rotating preparative thin

layer chromatography (hexanes/ethyl acetate gradient) to yield 8c

as whitish solid (93%). ¹H NMR (DMSO-d₆, 200 MHz, rt) δ (ppm):

8.00 (d, J ) 8.9 Hz, 2H), 7.08 (d, J ) 8.9 Hz, 2H), 4.53 (s, 2H),

4.22 (q, J ) 7.1 Hz, 2H), 3.86 (s, 3H), 1.21 (t, J ) 7.3 Hz, 3H).

GC-MS (m/e): 254 [M]⁺, 135 [M-119]⁺.

2-(3-Acetylamino-phenyl)-2-oxoethyl Ethyl Trithiocarbon-

ate (8e). 8e was prepared from N-(3-acetyl-phenyl) acetamide

according to procedure C; yellow solid (70%). ¹H NMR (DMSO-

d₆, 400 MHz, rt) δ (ppm): 10.15 (s, 1H), 8.19 (s, 1H), 7.90 (d, J )

2-(3-Benzylcarbamoyl-phenyl)-2-oxoethyl Ethyl Trithiocar-

bonate (12al). 12al was prepared from benzylamine and 3-acetyl-

benzoic acid according to general procedure D; yellow solid (63%).

Trithiocarbonates as a NoVel Class of HDAC Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3999

¹H NMR (DMSO-d₆, 400 MHz, rt) δ (ppm): 9.30 (t, J ) 5.9 Hz,

1H), 8.56 (s, 1H), 8.21 (d, J ) 7.8 Hz, 2H), 7.70 (t, J ) 7.7 Hz,

1H), 7.36-7.33 (m, 4H), 7.29-7.23 (m, 1H), 5.19 (s, 2H), 4.53

(d, J ) 5.9 Hz, 2H), 3.38 (q, J ) 7.4 Hz, 2H), 1.29 (t, J ) 7.4 Hz,

3H). LC-MS (m/e): 390 [M + H]⁺.

2-(4-Benzylcarbamoyl-phenyl)-2-oxoethyl Ethyl Trithiocar-

bonate (12am). 12am was prepared from benzylamine and 4-acetyl-

benzoic acid according to general procedure D; yellow solid (49%).

¹H NMR (DMSO-d₆, 200 MHz, rt) δ (ppm): 9.23 (t, J ) 5.9 Hz,

1H), 8.16 (d, J ) 8.5 Hz, 2H), 8.04 (d, J ) 8.5 Hz, 2H), 7.36-7.23

(m, 5H), 5.16 (s, 2H), 4.50 (d, J ) 5.9 Hz, 2H), 3.38 (q, J ) 7.4

Hz, 2H), 1.29 (t, J ) 7.4 Hz, 3H). LC-MS (m/e): 390 [M + H]⁺.

2-[5-(Benzylcarbamoyl-methyl)-thiophen-2-yl]-2-oxoethyl Eth-

yl Trithiocarbonate (18a). 18a was prepared from benzylamine

1

according to general procedure G; yellow solid (13%). H NMR

(DMSO-d₆, 200 MHz, rt) δ (ppm): 8.67 (t, J ) 5.7 Hz, 1H), 8.00

(d, J ) 3.8 Hz, 1H), 7.37-7.24 (m, 5H), 7.09 (d, J ) 3.9 Hz, 1H),

5.01 (s, 2H), 4.20 (d, J ) 5.8 Hz, 2H), 3.83 (s, 2H), 3.38 (q, J )

7.4 Hz, 2H), 1.29 (t, J ) 7.3 Hz, 3H). LC-MS (m/e): 410 [M +

H]⁺.

Ethyl 2-oxo-2-{5-[(2-Phenoxy-ethylcarbamoyl)-methyl]-thio-

phen-2-yl}-ethyl Trithiocarbonate (18d). 18d was prepared from

2-phenoxy-ethylamine according to general procedure G; yellow

1

solid (17%). H NMR (DMSO-d₆, 200 MHz, rt) δ (ppm): 8.46 (t,

J ) 5.5 Hz, 1H), 7.98 (d, J ) 3.9 Hz, 1H), 7.29 (t, J ) 8.1 Hz,

2H), 7.07 (d, J ) 3.9 Hz, 1H), 6.97-6.91 (m, 3H), 5.00 (s, 2H),

4.00 (t, J ) 5.5 Hz, 2H), 3.73 (s, 2H), 3.46 (t, J ) 5.6 Hz, 2H),

3.38 (t, J ) 7.4 Hz, 2H), 1.29 (t, J ) 7.3 Hz, 3H). LC-MS (m/e):

440 [M + H]⁺.

Preparation of Pyridinyl Acetylthiophenes (Procedure H). A

solution of pyridinyl-bromide (1.0 equiv) and 5-acetyl-thienyl-2-

boronic acid (1.05 equiv) in dioxane/water (10:1, 0.2 M) was

charged with Pd(dppf)Cl₂(0.01 equiv) and cesium carbonate (1.4

equiv), then purged with nitrogen and the mixture was stirred at

15 rt for 18 h. The reaction mixture was diluted with dichlo-

romethane and ﬁltered through a silica pad. Evaporation in vacuo

and subsequent puriﬁcation by ﬂash chromatography (silica, hex-

anes/ethyl acetate gradient) yielded the desired product.

Preparation of Pyrimidylamide Trithiocarbonates, Method

d (Procedure F). A solution of 4-(2-bromo-acetyl)-benzenesulfonic

acid chloride (1.0 equiv) in tetrahydrofuran (0.08 M) was treated

with the requisite arylamine (1.1-2.2 equiv) and diisopropylethy-

lamine (1.1 equiv) at rt and then stirred for 0.5 h. A solution of

sodium ethyl trithiocarbonate (0.5 M in tetrahydrofuran, 3.0 equiv)

was added, and the mixture was stirred again for 18 h. The reaction

mixture was poured into a 2:1 mixture of 5% sodium bicarbonate

and extracted with ethyl acetate. The organic phases were washed

with brine and dried with sodium sulfate. Evaporation in vacuo

gave an oily residue, which was puriﬁed by chromatography (silica,

hexanes/ethyl acetate gradient) or by preparative HPLC (acetonitrile/

water gradient), providing the title compounds.

Ethyl 2-oxo-2-[5-((S)-1-phenyl-ethylcarbamoyl)-thiophen-2-

yl]-ethyl Trithiocarbonate (16b). 16b was prepared from (S)-1-

phenyl-ethylamine and 5-acetyl-thiophene-2-carboxylic acid ac-

cording to general procedure E; yellow solid (6%). ¹H NMR

(DMSO-d₆, 400 MHz, rt) δ (ppm): 9.15 (d, J ) 8.0 Hz, 1H), 8.18

(d, J ) 4.0 Hz, 1H), 8.01 (d, J ) 4.0 Hz, 1H), 7.40 (d, J ) 7.2 Hz,

2H), 7.35 (t, J ) 7.2 Hz, 2H), 7.25 (t, J ) 7.1 Hz, 1H), 5.13 (quint,

J ) 7.4 Hz, 1H), 5.07 (s, 2H), 4.48 (d, J ) 6.0 Hz, 2H), 3.38 (q,

J ) 7.3 Hz, 2H), 1.50 (d, J ) 7.1 Hz, 3H), 1.29 (t, J ) 7.4 Hz,

3H). LC-MS (m/e): 410 [M + H]⁺.

Ethyl 2-oxo-2-[5-((R)-1-Phenyl-ethylcarbamoyl)-thiophen-2-

yl]-ethyl Trithiocarbonate (16c). 16c was prepared from (R)-1-

phenyl-ethylamine and 5-acetyl-thiophene-2-carboxylic acid ac-

cording to general procedure D; yellow solid (44%). ¹H NMR

(DMSO-d₆, 400 MHz, rt) δ (ppm): 9.15 (d, J ) 8.0 Hz, 1H), 8.18

(d, J ) 4.0 Hz, 1H), 8.01 (d, J ) 4.0 Hz, 1H), 7.40 (d, J ) 7.2 Hz,

2H), 7.35 (t, J ) 7.2 Hz, 2H), 7.25 (t, J ) 7.1 Hz, 1H), 5.13 (quint,

J ) 7.4 Hz, 1H), 5.07 (s, 2H), 4.48 (d, J ) 6.0 Hz, 2H), 3.38 (q,

J ) 7.3 Hz, 2H), 1.50 (d, J ) 7.1 Hz, 3H), 1.29 (t, J ) 7.4 Hz,

3H). LC-MS (m/e): 410 [M + H]⁺.

2-(5-{5-[(3-Amino-quinolinyl)-methyl]-pyridin-2-yl}-thiophen-

2-yl)-2-oxoethyl Ethyl Trithiocarbonate (22). 2-(5-{5-[(3-amino-

quinolinyl)-methyl]-pyridin-2-yl}-thiophen-2-yl)-ethanone (21a) was

prepared from (4-bromo-benzyl)-quinolin-3-yl-amine according to

procedure H; yellow solid (51%). ¹H NMR (DMSO-d₆, 400 MHz,

rt) δ (ppm): 8.68 (d, J ) 1.6 Hz, 1H), 8.56 (d, J ) 2.8 Hz, 1H),

8.04 (d, J ) 8.2 Hz, 1H), 7.95-7.91 (m, 2H), 7.87 (d, J ) 4.1 Hz,

1H), 7.79 (d, J ) 8.0 Hz, 1H), 7.62 (d, J ) 7.8 Hz, 1H), 7.38 (t,

J ) 6.7 Hz, 1H), 7.34 (t, J ) 6.7 Hz, 1H), 7.08 (d, J ) 1.6 Hz,

1H), 6.99 (t, J ) 4.0 Hz, 1H), 5.07 (s, 2H), 4.48 (t, J ) 4.0 Hz,

2H), 2.55 (s, 3H). LC-MS (m/e): 360 [M + H]⁺.

22 was prepared from 21a according to general procedure E:

1

yellow solid (9%). H NMR (DMSO-d₆, 400 MHz, rt) δ (ppm):

8.69 (s, 1H), 8.56 (d, J ) 2.7 Hz, 1H), 8.18 (d, J ) 4.1 Hz, 1H),

8.07 (d, J ) 8.2 Hz, 1H), 7.95-7.91 (m, 2H), 7.79 (d, J ) 7.9 Hz,

1H), 7.62 (d, J ) 7.8 Hz, 1H), 7.38 (t, J ) 6.7 Hz, 1H), 7.34 (t, J

) 6.7 Hz, 1H), 7.09 (s, 1H), 6.98 (t, J ) 6.0 Hz, 1H), 5.07 (s, 2H),

4.48 (t, J ) 6.0 Hz, 2H), 3.39 (q, J ) 7.4 Hz, 2H), 1.29 (t, J ) 7.4

Hz, 3H). LC-MS (m/e): 496 [M + H]⁺.

2-[5-(3,5-Dimethyl-benzylcarbamoyl)-thiophen-2-yl]-2-oxoet-

hyl Ethyl Trithiocarbonate (16d). 16d was prepared from 3,5-

dimethyl-benzylanime and 5-acetyl-thiophene-2-carboxylic acid

2-(5-(5-Amino-pyrid-2-yl)-thieno-2-yl)-2-oxoethyl Ethyl Trithio-

carbonate (23). 2-(5-(5-Amino-pyrid-2-yl)-thieno-2-yl)-ethanone

(21b) was prepared from 5-amino-2-bromo-pyridine according to

general procedure H; yellowish solid (44%). ¹

1

according to general procedure D; yellow solid (21%). H NMR

H NMR (DMSO-d₆,

(DMSO-d₆, 400 MHz, rt) δ (ppm): 9.27 (t, J ) 5.9 Hz, 1H), 8.16

(d, J ) 4.1 Hz, 1H), 7.90 (d, J ) 4.1 Hz, 1H), 6.92 (s, 2H), 6.90

(s, 1H), 5.07 (s, 2H), 4.40 (d, J ) 5.9 Hz, 2H), 3.39 (q, J ) 7.4

Hz, 2H), 2.25 (s, 6H), 1.29 (t, J ) 7.4 Hz, 3H). LC-MS (m/e): 424

[M + H]⁺.

400 MHz, rt) δ (ppm): 7.94 (d, J ) 2.6 Hz, 1H), 7.85 (d, J ) 4.0

Hz, 1H), 7.69 (d, J ) 8.6 Hz, 1H), 7.53 (d, J ) 4.1 Hz, 1H), 6.97

(dd, J ) 8.6, 2.7 Hz, 1H), 5.78 (s, 2H), 2.56 (s, 3H). LC-MS (m/

e): 219 [M + H]⁺

.

23 was prepared from 21b according to general procedure E;

1

Preparation of Thiophenylmethylamide Trithiocarbonates

(Procedure G). A solution of [5-(2-chloro-ethanoyl)-thiophen-2-

yl]-acetic acid (1.0 equiv) in tetrahydrofuran (0.2 M) was cooled

to 0 °C, then isobutyl chloroformate (1.2 equiv) and N-methyl-

morpholine (1.2 equiv) were added and the mixture was stirred for

45 min. The corresponding amine (1.2 equiv) was added, and the

mixture was allowed to warm to rt slowly. After 1 h, a solution of

sodium ethyl trithiocarbonate (0.5 M in tetrahydrofuran, 1.5 equiv)

was added and the mixture was stirred again for 18 h. After

completion of the reaction, the reaction mixture was diluted with

half-saturated brine and the aqueous phase was extracted with

dichloromethane. The organic layer was evaporated to leave a

yellowish residue, which was puriﬁed by chromatography (silica,

hexanes/ethyl acetate gradient) followed by preparative HPLC

(acetonitrile/water gradient), providing the title compound as a pale-

yellow solid.

yellow solid (10%). H NMR (DMSO-d₆, 400 MHz, rt) δ (ppm):

8.09 (d, J ) 4.0 Hz, 1H), 7.95 (d, J ) 2.6 Hz, 1H), 7.73 (d, J )

8.6 Hz, 1H), 7.59 (d, J ) 4.1 Hz, 1H), 6.98 (dd, J ) 8.5, 2.7 Hz,

1H), 5.77 (s, 2H), 5.03 (s, 2H), 3.39 (q, J ) 7.4 Hz, 2H), 1.29 (t,

J ) 7.4 Hz, 3H). LC-MS (m/e): 355 [M + H]⁺.

HPLC Purity. Purity of all compounds was determined by

analytical HPLC using a HPLC equipment of the LiChroGraph

series by Hitachi (pressure gradient pump L-7100), a reversed phase

column by Waters (Xterra, 100 mm × 2.1 mm inner diameter),

and as eluent a 10 mM ammoniumacetate/acetonitrile gradient

(buffer to pH 7,4). The purity of the compounds was determined

using a diode array detector (Hitachi L-7455, cell: 10 mm, 8 µL)

at λ ) 230-330 nm. All compounds possessed a purity of greater

than 95% as determined as average value of two independent

injections (see Table 5 for key compounds). Tracings of key

compounds are given as Supporting Information.

4000 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13

Table 5. Purity of Key Compounds as Determined by Analytical HPLC

Dehmel et al.

(4) (a) de Ruijter, A. J.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van

Kuilenburg, A. B. Histone deacetylases (HDACs): characterization

of the classical HDAC family. J. Biochem. 2003, 370, 737–749. (b)

Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Anticancer activities of

histone deacetylase inhibitors. Nat. ReV. Drug. DiscoVery 2006, 5,

769–784.

(5) (a) Marks, P.; Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller, T.;

Kelly, W. K. Histone deacetylases and cancer: causes and therapies.

Nat. ReV. Cancer 2001, 1, 194–202. (b) 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. (c) Glozak,

M. A.; Seto, E. Histone deacetylases and cancer. Oncogene 2007, 26,

5420–5432.

(6) Murata, T.; Kurokawa, R.; Krones, A.; Tatsumi, K.; Ishii, M.; Taki,

T.; Masuno, M.; Ohashi, H.; Yanagisawa, M.; Rosenfeld, M. G.; Glass,

C. K.; Hayashi, Y. Defect of histone acetyltransferase activity of the

nuclear transcriptional coactivator CBP in Rubinstein-Taybi syn-

drome. Hum. Mol. Genet. 2001, 10, 1071–1076.

(7) Wang, J.; Hoshino, T.; Redner, R. L.; Kajigaya, S.; Liu, J. M. ETO,

fusion partner in t(8;21) acute myeloid leukemia, represses transcription

by interaction with the human N-CoR/mSin3/HDAC1 complex. Proc.

Natl. Acad. Sci. U.S.A. 1998, 95, 10860–10865.

purity

(%)

no.

2

ethyl 2-(4-methylphenyl)-2-

oxoethyl trithiocarbonate

ethyl 2-(4-methoxyphenyl)-2-

oxoethyl trithiocarbonate

2-(4-methoxyphenyl)-2-

100/100

3a

3b

3c

99.1/98.9

96.0/95.9

97.8/98.1

oxoethyl dithiobutyrate

O-ethyl S-2-

(4-methoxy-phenyl)-2-

oxoethyl dithiocarbonate

3d

3e

N-ethyl S-2-

(4-methoxy-phenyl)-2-

oxoethyl dithiocarbamate

S-ethyl O-2-

96.8/96.8

98.3/98.1

95.2/95.7

(4-methoxy-phenyl)-2-

oxoethyl dithiocarbonate

2-(4-acetylamino-phenyl)-2-

oxoethyl ethyl trithiocarbonate

O-ethyl S-2-

8a

8c

(8) Zhu, P.; Martin, E.; Mengwasser, J.; Schlag, P.; Janssen, K. P.;

Go¨ttlicher, M. Induction of HDAC2 expression upon loss of APC in

colorectal tumorigenesis. Cancer Cell 2004, 5, 455–463.

100/100

(4-acetamido-phenyl)-2-

oxoethyl dithiocarbonate

2-(3-acetylamino-phenyl)-2-

oxoethyl ethyl trithiocarbonate

2-[4-(acetylamino-methyl)-phenyl]-2-

oxoethyl ethyl trithiocarbonate

ethyl 2-(4-methanesulfonylamino-phenyl)-2-

oxoethyl trithiocarbonate

ethyl 2-oxo-2-(3-phenylcarbamoyl-phenyl)-

ethyl trithiocarbonate

(9) (a) Weichert, W.; Ro¨ske, A.; Gekeler, V.; Beckers, T.; Ebert, M.; Pross,

M.; Dietel, M.; Denkert, C.; Ro¨cken, C. Class I HDAC expression

patterns are highly prognostic in human gastric cancer. Lancet

Oncology 2008, 9, 139–148. (b) Weichert, W.; Ro¨ske, A.; Niesporek,

S.; Noske, A.; Buckendahl, A. C.; Dietel, M.; Gekeler, V.; Boehm,

M.; Beckers, T.; Denkert, C. Class I histone deacetylase expression

has independent prognostic impact in human colorectal cancer: speciﬁc

role of class I HDACs in vitro and in vivo. Clin. Cancer. Res. 2008,

14, 1669–1677. (c) Weichert, W.; Ro¨ske, A.; Gekeler, G.; Beckers,

T.; Stephan, C.; Jung, K.; Niesporek, S.; Denkert, C.; Dietel, M.;

Kristiansen, G. Expression, function and prognostic role of class I

histone deacetylase isoforms in prostate cancer in vitro and in vivo.

Br. J. Cancer 2008, 98, 604–610.

(10) (a) Yoo, C. B.; Jones, P. A. Epigenetic therapy of cancer: past, present

and future. Nat. ReV. Drug DiscoVery 2006, 5, 37–50. (b) Riester, D.;

Hildmann, C.; Schwienhorst, A. Histone deacetylase inhibitors-turning

epigenic mechanisms of gene regulation into tools of therapeutic

intervention in malignant and other diseases. Appl. Microbiol. Bio-

technol. 2007, 75, 499–514.

8e

96.7/95.9

99.7/99.7

98.6/98.9

97.8/98.1

98.0/97.5

99.5/99.8

97.4/97.9

95.2/95.3

8g

8h

12aa

12ab

12al

ethyl 2-oxo-2-(4-phenylcarbamoyl-phenyl)-

ethyl trithiocarbonate

2-(3-benzylcarbamoyl-phenyl)-2-

oxoethyl ethyl trithiocarbonate

12am 2-(4-benzylcarbamoyl-phenyl)-2-

oxoethyl ethyl trithiocarbonate

ethyl 2-oxo-2-[5-((S)-1-

phenyl-ethylcarbamoyl)-thiophen-

2-yl]-ethyl trithiocarbonate

ethyl 2-oxo-2-[5-((R)-1-

phenyl-ethylcarbamoyl)-thiophen-

2-yl]-ethyl trithiocarbonate

2-[5-(3,5-dimethyl-benzylcarbamoyl)-

thiophen-2-yl]-2-oxoethyl

ethyl trithiocarbonate

2-[5-(benzylcarbamoyl-methyl)-

thiophen-2-yl]-2-oxoethyl

16b

16c

16d

18a

18d

22

99.4/99.3

96.5/97.0

99.5/99.5

98.7/98.7

95.7/95.7

98.5/98.6

(11) Sorbera, L. A. Epigenetic targets as an approach to cancer therapy

and chemoprevention. Drugs Future 2006, 31, 335–344.

(12) (a) Miller, T. A.; Witter, D. J.; Belvedere, S. Histone Deacetylase

Inhibitors. J. Med. Chem. 2003, 46, 5097–5116. (b) Hildmann, C.;

Wegener, D.; Riester, D.; Hempel, R.; Schober, A.; Merena, J.;

Guirato, L.; Guccione, S.; Nielsen, T. K.; Ficner, R.; Schwienhorst,

A. Substrate and inhibitor speciﬁcity of class 1 and class 2 histone

deacetylases. J. Biotechnol. 2006, 124, 258–270.

(13) (a) Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.; Rifkind,

R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P. Structures of a

histone deacetylase homologue bound to the TSA and SAHA

inhibitors. Nature 1999, 401, 188–193. (b) Somoza, J. R.; Skene, R. J.;

Katz, B. A.; Mol, C.; Ho, J. D.; Jennings, A. J.; Luong, C.; Arvai, A.;

Buggy, J. J.; Chi, El.; Tang, J.; Sang, B.-C.; Verner, E.; Wynands,

R.; Leahy, E. M.; Dougan, D. R.; Snell, G.; Navre, M.; Knuth, M. W.;

Swanson, R. V.; McRee, D. E.; Tari, L. W. Structural Snapshots of

Human HDAC8 Provide Insights into the Class I Histone Deacetylases.

Structure 2004, 12, 1325–1334.

ethyl trithiocarbonate

ethyl 2-oxo-2-{5-[(2-phenoxy-

ethylcarbamoyl)-methyl]-thiophen-

2-yl}-ethyl trithiocarbonate

2-(5-{5-[(3-amino-quinolinyl)-methyl]-

pyridin-2-yl}-thiophen-2-yl)-2-

oxoethyl ethyl trithiocarbonate

2-(5-(5-amino-pyrid-2-yl)-thieno-2-yl)-2-

oxoethyl ethyl trithiocarbonate

23

(14) Beckers, T.; Burkhardt, C.; Wieland, H.; Gimmnich, P.; Ciossek, T.;

Maier, T.; Sanders, K. Distinct pharmacological properties of 2nd

generation HDAC inhibitors with the benzamide or hydroxamate head

group. Int. J. Cancer 2007, 121, 1138–1148.

(15) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. M.;

Schreiber, S. L. Domain-selective small-molecule inhibitor of histone

deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc. Natl.

Acad. Sci. U.S.A. 2003, 100, 4389–4394.

Supporting Information Available: All information about

chemical synthesis and analytics of additional compounds as

presented in the manuscript but not shown in the Experimental

Section. This material is available free of charge via the Internet at

http://pubs.acs.org.

(16) Hodawadekar, S. C.; Marmorstein, R. Chemistry of acetyl transfer by

histone modifying enzymes: structure, mechanismand implications for

effector design. Oncogene 2007, 26, 5528–5540.

(17) (a) Suzuki, T.; Miyata, N. Rational Design of Non-Hydroxamate

Histone Deacetylase Inhibitors. Mini-ReV. Med. Chem. 2006, 6, 515–

526. (b) Suzuki, T.; Miyata, N. Non-hydroxamate Histone Deacetylase

Inhibitors. Curr. Med. Chem. 2005, 12, 2867–2880.

(18) (a) For recent studies on hydroxamtes and benzamides, see for instance

Anandan, S.-K.; Ward, J. S.; Brokx, R. D.; Denny, T.; Bray, M. R.;

Patel, D. V.; Xiao, X.-Y. Design and synthesis of thiazole-5-

hydroxamic acids as novel histone deacetylase inhibitors. Bioorg. Med.

Chem. Lett. 2007, 17, 5995–5999. (b) Moradei, O. M.; Mallais, T. C.;

Frechette, S.; Paquin, I.; Tessier, P. E.; Leit, S. M.; Fournel, M.;

References

(1) (a) Cohen, T.; Yao, T. P. AcK-Knowledge reversible acetylation. Sci.

STKE 3 2004, pe42. (b) Yang, X. J.; Seto, E. HATs and HDACs:

from structure, function and regulation to novel strategies for therapy

and prevention. Oncogene 2007, 26, 5310–5318.

(2) Strahl, B. D.; Allis, C. D. The language of covalent histone modiﬁca-

tions. Nature 2000, 403, 41–45.

(3) (a) Zhang, K.; Dent, S. Y. J. Histone modifying enzymes and cancer:

going beyond histones. Cell. Biochem. 2005, 96, 1137–1148. (b)

Johnstone, R. W.; Licht, J. D. Histone deacetylase inhibitors in cancer

therapy: is transcription the primary target? Cancer Cell 2003, 4, 13–

18.

Trithiocarbonates as a NoVel Class of HDAC Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 4001

Bonﬁls, C.; Trachy-Bourget, M.-C.; Liu, J.; Yan, T. P.; Lu, A. H.;

Rahil, J.; Wand, J.; Lefebvre, S.; Li, Z.; Vaisburg, A. F.; Bestermann,

J. M. Novel Aminophenyl Benzamide-Type Histone Deacetylase

Inhibitors with Enhanced Potency and Selectivity. J. Med. Chem. 2007,

50, 5543–5546. (c) Hamblett, C. L.; Methot, J. L.; Mampreian, D. M.;

Sloman, D. L.; Stanton, M. G.; Kral, A. M.; Fleming, J. C.; Cruz,

J. C.; Chenard, M.; Ozerova, N.; Hitz, A. M.; Wang, H.; Deshmukh,

S. V.; Nazef, N.; Harsch, A.; Hughes, B.; Dahlberg, W. K.; Szewczak,

A. A.; Middleton, R. E.; Mosley, R. T.; Secrist, J. P.; Miller, T. A.

The discovery of 6-amino nicotinamides as potent and selective histone

deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 5300–5309.

(19) (a) Suzuki, T.; Kouketsu, A.; Matsuura, A.; Kohara, A.; Ninomiya,

S.-i.; Kohda, K.; Miyata, N. Thiol-based SAHA analogues as potent

histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2004, 14,

3313–3317. (b) Nishino, N.; Jose, B.; Okamura, S.; Ebisusaki, S.; Kato,

T.; Sumida, Y.; Yoshida, M. Cyclic Tetrapeptides Bearing a Sulfhydryl

Group Potently Inhibit Histone Deacetylases. Org. Lett. 2003, 5, 5079–

5082. (c) Itoh, Y.; Suzuki, T.; Kouketsu, A.; Suzuki, N.; Maeda, S.;

Yoshida, M.; Nakagawa, H.; Miyata, N. Design, Synthesis, Structure-

Selectivity Relationship, and Effect on Human Cancer Cells of a Novel

Series of Histone Deacetylase 6-Selective Inhibitors. J. Med. Chem.

2007, 50, 5425–5438.

(20) Suzuki, T.; Nagano, Y.; Kouketsu, A.; Matsuura, A.; Maruyama, S.;

Kurotaki, M.; Nakagawa, H.; Miyata, N. Novel Inhibitors of Human

Histone Deacetylases: Design, Synthesis, Enzyme Inhibition, and

Cancer Cell Growth Inhibition of SAHA-Based Non-hydroxamates.

J. Med. Chem. 2005, 48, 1019–1032.

(21) Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K-H.; Nishiyama, M.;

Nakajima, H.; Tanaka, A.; Komatsu, Y.; Nishino, N.; Yoshida, M.;

Horinouchi, S. FK228 (Depsipeptide) as a Natural Prodrug That

Inhibits Class I Histone Deacetylases. Cancer Res. 2002, 62, 4916–

4921.

(22) (a) Yurek-George, A.; Cecil, A. R. L.; Mo, A. H. K.; Shijun, W.;

Rogers, H.; Habens, F.; Maeda, S.; Yoshida, M.; Packham, G.;

Ganesan, A. The First Biologically Active Synthetic Analogues of

FK228, the Depsipeptide Histone Deacetylase Inhibitor. J. Med. Chem.

2007, 50, 5720–5726. (b) Shivashimpi, G. M.; Amagai, S.; Kato, T.;

Nishino, N.; Maeda, S.; Nishino, T. G.; Yoshida, M. Molecular design

of histone deacetylase inhibitors by aromatic ring shifting in chlamy-

docin framework. Bioorg. Med. Chem. 2007, 15, 7830–7839.

(23) (a) Suzuki, T.; Hisakawa, S.; Itoh, Y.; Maruyama, S.; Kurotaki, M.;

Nakagawa, H.; Miyata, N. Identiﬁcation of a potent and stable

antiproliferative agent by the prodrug formation of a thiolate histone

deacetylase inhibitor. Bioorg. Med. Chem. Lett. 2007, 17, 1558–1561.

(b) Suzuki, T.; Kouketsu, A.; Itoh, Y.; Hisakawa, S.; Maeda, S.;

Yoshida, M.; Nakagawa, H.; Miyata, N. Highly Potent and Selective

Histone Deacetylase 6 Inhibitors Designed Based on a Small-Molecular

Substrate. J. Med. Chem. 2006, 49, 4809–4812.

(24) (a) Anandan, S.-K.; Ward, J. S.; Brokx, R. D.; Bray, M. R.; Patel,

D. V.; Xiao, X.-X. Mercaptoamide-based non-hydroxamic acid type

histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2005, 15,

1969–1972. (b) Chen, B.; Petukhov, P. A.; Jung, M.; Velena, A.;

Eliseeva, E.; Dritschilo, A.; Kozikowski, A. P. Chemistry and biology

of mercaptoacetamides as novel histone deacetylase inhibitors. Bioorg.

Med. Chem. Lett. 2005, 15, 1389–1392. (c) Suzuki, T.; Matsuura, A.;

Kouketsu, A.; Nakagawa, H.; Miyata, N. Identiﬁcation of a potent

non-hydroxamate histone deacetylase inhibitor by mechanism-based

drug design. Bioorg. Med. Chem. Lett. 2005, 15, 331–335.

(25) Gu, W.; Nusinzon, I.; Smith, R. D., Jr.; Horvarth, C. M.; Silverman,

R. B. Carbonyl- and sulfur-containing analogs of suberoylanilide

hydroxamic acid: Potent inhibition of histone deacetylases. Bioorg.

Med. Chem. 2006, 14, 3320–3329.

(26) Dehmel, F.; Ciossek, T.; Maier, T.; Weinbrenner, S.; Schmidt, B.;

Zoche, M.; Beckers, T. TrithiocarbonatessExploration of a new head

group for HDAC inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 4746–

4752.

(27) Ciossek, T.; Julius, H.; Wieland, H.; Maier, T.; Beckers, T. A

homogeneous cellular histone deacetylase assay suitable for compound

proﬁling and robotic screening. Anal. Biochem. 2008, 372, 72–81.

(28) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the

Alamar Blue (resazurin) ﬂuorescent dye for the assessment of

mammalian cell cytotoxicity. Eur. J. Biochem. 2000, 267, 5421–5426.

(29) Mahboobi, S.; Sellmer, A.; Hocher, H.; Garhammer, C.; Pongratz,

H.; Maier, T.; Ciossek, T.; Beckers, T. 2-Aroylindoles and 2-aroyl-

benzofurans with N-hydroxyacrylamide substructures as a novel series

of rationally designed histone deacetylase inhibitors. J. Med. Chem.

2007, 50, 4405–4418.

(30) Li, Y.; Kao, G. D.; Garcia, B. A.; Shabanowitz, J.; Hunt, D. F.; Qin,

J.; Phelan, C.; Lazar, M. A. A novel histone deacetylase pathway

regulates mitosis by modulating Aurora B kinase activity. Genes DeV.

2006, 20, 2566–2579.

(31) Dignam, J. D.; Lebovitz, R. M.; Roeder, R. G. Accurate transcription

initiation by RNA polymerase II in a soluble extract from isolated

mammalian nuclei. Nucleic Acids Res. 1983, 11, 1475–1489.

(32) Wegener, D.; Wirsching, F.; Riester, D.; Schwienhorst, A. A ﬂuoro-

genic histone deacetylase assay well suited for high-throughput activity

screening. Chem. Biol. 2003, 10, 61–68.

(33) Ise, W.; Heuser, M.; Sanders, K.; et al. P-Glycoprotein-associated

resistance to taxol and taxotere and its reversal by dexniguldipine-

HCl, dexverapamil-HCl, or cyclosporin A. Int. J. Oncol. 1996, 8, 951–

956.

(34) Schmidt, M.; Lu, Y.; Liu, B.; Fang, M.; Mendelsohn, J.; Fan, Z.

Differential modulation of paclitaxel-mediated apoptosis by p21waf1

and p27kip1. Oncogene 2000, 19, 2423–2429.

JM800093C

Article Doi

Trithiocarbonates as a novel class of HDAC inhibitors: SAR studies, isoenzyme selectivity, and pharmacolozgical profiles

DOI: 10.1021/jm800093c

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm800093c

Full text of DOI:10.1021/jm800093c

Products guided by the article

R&D Labs maybe for 1039453-78-5

Relevant to this article

Hot Product