Inhibitors of histone deacetylase suppress the growth of MCF-7 breast cancer cells

Article abstract of DOI:10.1002/(SICI)1521-4184(199910)332:10<353::AID-ARDP353>3.0.CO;2-X

Inhibitors of histone deacetylase are attracting increasing interest due to their influence on transcription, differentiation, and apoptosis. We have investigated two synthetic inhibitors 3 and 4 of histone deacetylase and the natural product inhibitor tr

Full text of DOI:10.1002/(SICI)1521-4184(199910)332:10<353::AID-ARDP353>3.0.CO;2-X

Histone Deacetylase Inhibitors
353
Inhibitors of Histone Deacetylase Suppress the Growth of MCF-7
Breast Cancer Cells
a)
a)
b)
Kathrin Schmidt , Ronald Gust , and Manfred Jung*
a) Institut für Pharmazie der Freien Universität Berlin, Abteilung Pharmazeutische Chemie, Königin-Luise-Str. 2 + 4, 14195 Berlin, Germany
b) Institut für Pharmazeutische Chemie, Westfälische Wilhelms-Universität Münster, Hittorfstr. 58–62, 48149 Münster, Germany
Key Words: Histone deacetylase; trichostatin A; cancer; MCF-7
[2,3,5,]
acetyltransferases or deacetylases
. In several cases
Summary
aberrant chromatin acetylation could be linked to malignant
[5,7–9]
diseases
.
Inhibitors of histone deacetylase are attracting increasing interest
due to their influence on transcription, differentiation, and apop-
tosis. We have investigated two synthetic inhibitors 3 and 4 of
histone deacetylase and the natural product inhibitor trichostatin A
for their ability to suppress the growth of MCF-7 breast cancer
cells and here present complete and improved synthetic proce-
dures. The compounds show a dose dependent inhibition of growth
with activities in the low micromolar and nanomolar range.
Trichostatin shows cytocidal effects at 100 nM and still has activity
comparable to cisplatin (0.5 µM) at 10 nM. Whereas the synthetic
inhibitor 3 has cytocidal activity at 10 µM compound 4 shows a
maximum of 40% growth suppression at that concentration.
In the course of our studies to synthesize simple inhibitors
of histone deacetylase and the exploration of their potential
for cancer therapy and chemoprevention we have found
[10]
promising lead substances
. Among them were com-
pounds 3 and 4 constructed from structural elements of the
established natural product inhibitors trapoxin B (1) and
[11]
trichostatin A (2) (Scheme 1)
.
Whereas the impact of inhibitors of histone deacetylase on
leukaemic cells has been studied at length comparatively little
is known about their influence on cell lines derived from solid
tumors. Just recently it has been shown that the anticancer
drug cisplatin is crosslinking nuclear proteins to the DNA in
MCF-7 breast cancer cells and it was suggested that its
clinical activity is linked partly to that effect. Among those
nuclear proteins were the estrogen receptor and histone
Introduction
[12]
deacetylase HDAC-1 . So we studied the growth inhibition
Reversible acetylation of ε-amino groups of highly con-
served lysine residues in the N-terminal tails of histones has
a modulating impact on chromatin structure. This affects key
nuclear processes such as DNA replication, transcription,
by reference histone deacetylase inhibitor trichostatin A (2)
and of our synthetic lead structures 3 and 4 on that human
breast cancer cell line.
[1]
differentiation, and apoptosis . This modification is estab-
lished and maintained by histone acetyltransferases and his-
tone deacetylases, enzymes which have been identified as
Results and Discussion
homologs of regulators of transcription and nucleolar phos-
Chemistry
[2–4]
phoproteins
. Increasing evidence shows that transcrip-
tion factors involved in the regulation of proliferation and
As described previously 3 and 4 are accessible in four
differentiation exert their function by recruitment of histone synthetic steps using standard peptide coupling methodology.
Scheme 1. Inhibitors of histone deacetylase.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0365-6233/99/1010/0353 $17.50 +.50/0

354
Schmidt, Gust, and Jung
Scheme 2. Synthesis of Inhibitors 3 and 4.
As the target compounds are very polar we chose a strategy Moreover, a patient with multi-resistent acute promyelocytic
where no chromatography is required after the last synthetic leukaemia was found to respond again to retinoid treatment
step. Thus a benzyl-protected hydroxylamine is employed by combination with the unspecific histone deacetylase in-
that allows for chromatographic purification of the protected hibitor phenylbutyrate. Six months after treatment RT-PCR
hydroxamate. After hydrogenation the compounds can be did not show any mRNA of the oncogenically transformed
purified by precipitation from methanolic solutions. The use retinoid receptors that recruit histone deacetylase to suppress
of BOP-Cl in all coupling steps in the synthesis of 3 led to transcription, thus indicating absence of residual disease.
[17]
increased yields compared to the water-soluble carbodiimide Again no severe side effects were observed . Thus this first
EDC. For 4 we had already proven that the use of BOP-Cl is report of the growth inhibition of MCF-7 breast cancer cells
crucial in the first coupling step as mixed anhydride or by potent inhibitors of histone deacetylase adds to the mount-
carbodiimide methods failed due to the inactivating effect of ing evidence that this class of compounds possesses great
[10]
the para-dimethylamino group
(Scheme 2).
potential for the treatment of cancer. Additionally it supports
the use of in-vitro assays of histone deacetylase activity as
[18–20]
screening tools in drug discovery
. Further investiga-
Biological Activities
tions that involve differences in response depending on estro-
gen receptor status of the cell and combination with other
antineoplastic agents are part of ongoing studies.
The impact of the different inhibitors of histone deacetylase
on MCF-7 cells was studied in our standard microtiter assay
using crystal violet staining to measure total biomass relative
to control over a prolonged period of exposure to the antineo-
Acknowledgements
[13]
plastic agent
(Figure 1).
. Cisplatin was used as a reference compound
M. Jung wishes to thank Prof. Unterhalt, Münster for his support and
H. Deguara for technical assistance. Financial support by the Fonds der
Chemischen Industrie, the DPhG-Stiftung für den wissenschaftlichen Nach-
wuchs im Stifterverband für die Deutsche Wissenschaft, the Förderergesell-
schaft der Westfälischen Wilhelms-Universität and the Deutsche
Forschungsgemeinschaft is gratefully acknowledged. We thank ASTA
Medica AG, Frankfurt, for gifts of 6-aminocaproic acid and 2.
Trichostatin A (2) and inhibitor 3 are able to reach cytocidal
effects at 100 nM (2) and 10 µM (3), respectively. In contrast,
compound 4 did not exceed 40% suppression of cell growth
even at 10 µM. Even at 10 nM, 2 still has an activity compa-
rable to cisplatin at 500 nM. This difference between 2 and
our inhibitors reflects the potency in the in-vitro enzyme
[14]
inhibition assay (IC : 2: 3 nM, 3: 500 nM, 4: 100 nM)
.
50
Experimental Part
After approximately 60 h there was an increase in cell growth
in some of the experiments (2 at 10 nM, 3 at 10 µM, 4 at
10 µM) which may reflect hydrolysis of the hydroxamates to
the inactive carboxylic acids.
Although the dose response curves are rather steep it is
encouraging that inhibitors of histone deacetylase can sup-
press the growth of breast cancer cells and even show cyto-
cidal effects in some cases. It has been shown recently that
this enzyme can indeed be inhibited safely in humans. The
depsipeptide FR-901228 which was chosen for clinical trials
Melting point: uncorrected.– Elemental analysis: Perkin Elmer.– IR: Shi-
madzu 470.– 1H-NMR: Varian Gemini 200 (200 MHz).– 13C-NMR: Varian
Gemini 200 (50.29 MHz).– MS: Varian MAT 44S, Finnigan MAT 312.–
Flash chromatography (FC): silica gel 60, 230–400 mesh (Merck).– Dichlo-
romethane was dried over molecular sieves (3Å).
Chemistry
General Procedure for Amide Formation
To a solution or suspension of the acid in dry CH2Cl2 (5 mL/mmol) was
added one equivalent of triethylamine (TEA) under nitrogen and the mixture
was stirred for 10 min. Then 1.1 equivalents of BOP-Cl, one equivalent of
the amine or hydroxylamine hydrochloride, and another three equivalents of
due to its good antitumor activity was discovered to be an
[15]
inhibitor of histone deacetylase
. First results from clinical
[16]
phase I studies showed encouragingly low toxicity
.
Arch. Pharm. Pharm. Med. Chem. 332, 353–357 (1999)

Histone Deacetylase Inhibitors
355
Figure 1. Growth inhibition of MCF-7 breast cancer cells by inhibitors of histone deacetylase.
Arch. Pharm. Pharm. Med. Chem. 332, 353–357 (1999)

356
Schmidt, Gust, and Jung
TEA were added. After stirring overnight most of the CH2Cl2 was removed
under reduced pressure and 100 mL ethyl acetate and 50 mL of 3% Na-
HCO3 solution were added. The organic phase was separated and washed
consecutively with 50 mL each water and sat. brine. The organic layer was
dried over Na2SO4 and the solvent was evaporated.
36.63, 34.89, 32.21, 28.30, 28.11 and 24.97 (CH2, one resonance is ob-
scured).– Anal. (C18H26N2O5) 350.46: C, H, N.
Methyl 6-(4-Dimethylaminobenzoylamino)caproate
General method for amide formation: 1.65 g (10 mmol) 4-dimethylamino
benzoic acid, 1.39 mL (1.01 g, 10 mmol) TEA, 2.80 g (11 mmol) BOP-Cl,
1.82 g (10 mmol) methyl 6-aminocaproate hydrochloride, 4.17 mL (3.03 g,
30 mmol) TEA. The crude was purified by FC using ethyl acetate/hexane
(2:1). Yield 1.70 g (58%) colorless crystals.– Mp 71 °C.– IR (KBr): ν =
3275s, 2935s, 1730s, 1590s.– MS (70 eV); m/z (%) = 292 (18) [M+], 219
(10), 148 (100).– 1H NMR (CDCl3): δ = 7.70–7.65 (m, 2 H, 2′-H, 6′-H),
6.70–6.64 (m, 2 H, 3′-H, 5′-H), 6.12 (bs, 1 H, NH), 3.66 (s, 3 H, OCH3),
3.48–3.38 m (m, 2 H, CONHCH2), 3.01 (s, 6 H, NMe2), 2.37–2.30 (m, 2 H,
CH2COOMe), 1.71–1.55 (m, 4 H, NHCH2CH2CH2CH2), 1.47–1.35 (m, 2
H, NHCH2CH2CH2).– 13C NMR (CDCl3): δ = 174.07 (COOMe), 167.49
(CONH), 152.47 (Me2NC), 128.36 (C-3′, C-5′), 121.76 (CCONH), 111.17
(C-2′, C-6′), 51.46 (OCH3), 40.14 (CONHCH2), 39.61 (NMe2), 33.94
(CH2COOMe), 29.53, 26.51 and 24.58 (CH2).
Methyl N-Benzyloxysuberoylamide
General method for amide formation: 1.99 g (10 mmol) monomethyl
suberate, 1.39 mL (1.01 g, 10 mmol) TEA, 2.80 g (11 mmol) BOP-Cl, 1.60 g
(10 mmol) O-benzylhydroxylamine hydrochloride, 4.17 mL (3.03 g,
30 mmol) TEA. The organic phase was also washed with 2 M HCl. The
resulting oil was purified by FC using ethyl acetate/hexane (1:1). Yield 5.28 g
(95%) colorless oil.– IR (KBr): ν = 3200s, 2940s, 1740s, 1660s.– MS (70
eV); m/z (%) = 170 (1), 106 (12), 91 (100).– (1H NMR (CDCl3): δ = 7.47
(bs, 1 H, NH), 7.38–7.34 (m, 5 H, Ar-H), 4.88 (s, 2 H, OCH2), 3.66 (s, 3 H,
OMe), 2.34–2.27 (m, 2 H, CH2), 1.99–1.88 (m, 2 H, CH2), 1.67–1.60 (m, 4
H, CH2), 1.38–1.26 (m, 4 H, CH2).– 13C NMR (CDCl3): δ = 174.20
(COOMe), 129.18 (Ar-CH), 128. 73 (Ar-1), 128.60 (Ar-CH), 78.26 (OCH2),
51.44 (OMe), 33.95, 32.99, 32.80, 28.71, 25.00 and 24.71 (CH2).
6-(4-Dimethylaminobenzoylamino)caproic Acid (6)
N-Benzyloxymonosuberoylamide (5)
1.17 g (4 mmol) of methyl 6-(4-dimethylaminobenzoylamino)caproate
were dissolved in 10 mL of THF and 4 mL (8 mmol) of a 1 M LiOH solution
were added. The mixture was stirred for 4 h at room temperature. 10 mL of
0.5 M NaOH and 50 mL of ethyl acetate were added. The aqueous phase was
treated with 2 M HCl to pH 6–7 and some of the acidprecipitated. The mother
liquor was evaporated to dryness and dried over P2O5 in a desiccator
overnight. The resulting solid was sufficiently pure for the next coupling step.
The spectral data was taken form the precipitated pure acid.– Mp 165 °C.–
IR (KBr): ν = 3380s, 2910s, 1700s, 1600s.– MS (70 eV); m/z (%) = 278 (36)
[M+], 219 (19), 148 (100).– 1H NMR (CD3OD): δ = 7.71–7.66 (m, 2 H, 2′-H,
6′-H,), 6.75–6.70 (m, 2 H, 3′-H, 5′-H), 3.37–3.28 (m, 2 H, CONHCH2), 3.01
(s, 6 H, NMe2), 2.34–2.27 (m, 2 H, CH2COOH), 1.80–1.55 (m, 4 H,
NHCH2CH2CH2CH2), 1.48–1.35 (m, 2 H, NHCH2CH2CH2).– 13C NMR
(DMSO-d6): δ = 174.32 (COOH), 165.97 (CONH), 151.94 (Me2NC), 128.34
(C-3′, C-5′), 121.48 (CCONH), 110.71 (C-2′, C-6′), 39.65 (NMe2), 38.81
(CONHCH2), 33.59 (CH2COOMe), 29.02, 26.02, 24.23 (CH2).– Anal.
(C15H18N2O3) 278.32: C, H, N.
2.77 g (10 mmol) of methyl N-benzyloxysuberoylamide were dissolved in
10 mL of THF and 10 mL (20 mmol) of a 1 M LiOH solution were added.
The mixture was stirred for 4 h at room temperature. 10 mL of 0.5 M NaOH
and 50 mL of ethyl acetate were added. The aqueous phase was acidified
with 2 M HCl (pH 3) and extracted three times with 50 mL portions of ethyl
acetate. The organic phase was dried over Na2SO4 and evaporated. The
resulting solid was sufficiently pure for the next coupling step. Yield 1.78 g
(68%) colorless crystals.– Mp 73 °C.– IR (KBr): ν = 3240s, 2940s, 1695s,
1655s.– MS (70 eV); m/z (%) = 105 (26), 91 (100), 77 (28).– 1H NMR
(CDCl3): δ = 8.29 (bs, 1 H, NH), 7.37 (s, 5 H, Ar-H), 4.89 (bs, 2 H, OCH2),
2.35–2.27 (m, 3 H, CH2), 2.03 (bs, 1 H, CH2), 1.60–1.57 (m, 4 H, CH2),
1.32–1.27 (m, 4 H, CH2).– 13C NMR (CDCl3): δ = 178.74 (COOH), 129.23
(Ar-CH), 128. 82 (Ar-1), 128.65 (Ar-CH), 78.39 (OCH2), 33.94, 32.71,
28.70, 28.62, 24.72 and 24.51 (CH2).
N-Benzyloxy-N′-[1-(S)-methoxycarbonyl-2-phenylethyl]suberoylbisamide
General method for amide formation; 1.11 g (4 mmol) 5, 0.5 mL (0.41 g,
4 mmol) TEA, 0.86 g (4 mmol) L-phenylalanine methyl ester hydrochloride,
1.12 g (4.4 mmol) BOP-Cl, 1.68 mL (1.21 g, 12 mmol) TEA. The organic
phase was also washed with 2 M HCl. The crude was chromatographed using
ethyl acetate/hexane (5:1). Yield 1.25 g (71%) colorless crystals.– Mp
86 °C.– IR (KBr): ν = 3050s, 1730s, 1640s.– MS (70 eV); m/z (%) = 440 (3)
[M+], 274 (14), 162 (100).– 1H NMR (CDCl3): δ = 8.79 (bs, 1 H, NHOCH2),
7.37 (s, 5 H, OCH2Ph), 7.33–7.22 (m, 3 H, Phe-H), 7.11–7.06 (m, 2 H,
Phe-H), 6.02 (d, 3J = 7.53 Hz, 1 H, Phe-NH), 4.89–4.81 (m, 3 H, OCH2 and
CHCOOMe), 3.71 (s, 3 H, OMe), 3.19–3.02 (m, 2 H, CHCH2Ph), 2.17–2.09
(m, 2 H, CH2), 2.02 (bs, 2 H, CH2), 1.63–1.49 (m, 4 H, CH2), 1.27–1.24 (m,
4 H, CH2).– 13C NMR (CDCl3): δ = 172.70 (COOMe), 172.31 (CONH),
135.99 (Phe-1), 135.00 (OCH2C), 129.22, 129.18, 128.70, 128.60 and 127.14
(Ar-CH), 78.33 (OCH2), 53.06 (OMe), 52.27 (CHCOOMe), 37.94, 36.18,
32.70, 28.48, 25.20 and 24.82 (CH2). Anal. (C25H32N2O5) 440.59: C, H, N.
N-Benzyloxy-6-(4-dimethylaminobenzoylamino)capramide
General method for amide formation; combined 6 and crude saponification
product, 0.56 mL (0.41 g, 4 mmol) TEA, 0.64 g (4 mmol) O-benzylhydroxy-
lamine hydrochloride, 1.12 g (4.4 mmol) BOP-Cl, 1.68 mL (1.21 g,
12 mmol), TEA. The crude was chromatographed using ethyl acetate with
1% diethylamine. Yield (from the ester in two steps): 1.06 g (69%) colorless
crystals.– Mp 128 °C.– IR (KBr): ν = 3350s, 3150s, 2925s, 1660s, 1605s.–
MS (70 eV); m/z(%) = 383 (7) [M+], 212 (49), 148 (100).– 1H NMR (CDCl3):
δ = 7.62–7.57 (m, 2 H, 2′-H, 6′-H), 7.30 (s, 5 H, Ph), 6.60–6.55 (m, 2 H, 3′-H,
5′-H), 4.81 (s, 2 H, OCH2), 3.38–3.29 (m, 2 H, CONHCH2), 2.93 (s, 6 H,
NMe2), 2.01 (bs, 2 H, CH2CO), 1.68–1.45 (m, 4 H, NHCH2CH2CH2CH2),
1.36–1.25 (m, 2 H, NHCH2CH2CH2).– 13C NMR (CDCl3): δ = 167.61
(CONHCH2), 152.50 (Me2NC), 129.16 (Ar-CH), 128.67 (OCH2C), 128.58
(Ar-CH), 128.37 (Ar-CH), 121.58 (CCONHCH2), 111.18 (C-2′, C-6′), 78.35
(OCH2), 40.11 (CONHCH2), 39.50 (NMe2), 32.81 (CH2CO), 29.40, 26.27
and 24.51 (CH2).
N-Hydroxy-N′-[1-(S)-methoxycarbonyl-2-phenylethyl]suberoylbisamide (3)
1.00
g (2.27 mmol) N-benzyloxy-N′-[1-(S)-methoxycarbonyl-2-
N-Hydroxy-6-(4-dimethylaminobenzoylamino)capramide (4)
phenylethyl]-suberoylbisamide was dissolved in 15 mL of methanol and
100 mg 10% palladium on charcoal were added. The mixture was treated
with hydrogen under atmospheric pressure for 6 h and subsequently filtered.
The product was evaporated, redissolved in 5 mL of methanol, and precipi-
tated with diethyl ether. Yield 510 mg (64%) colorless crystals.– Mp
105 °C.– IR (KBr): ν = 3200m, 1730s, 1620s.– MS (70 eV); m/z (%) = 350
(9) [M+], 162 (75), 120 (100).– 1H NMR (CDCl3): δ = 7.34–7.23 (m, 3 H,
Ar-H), 7.14–7.10 (m, 2 H, Ar-H), 6.33 (d, 3J = 7.59 Hz, Phe-NH), 4.89–4.83
(m, 1 H, CHCOOMe), 3.74 (s, 3 H, OMe), 3.22–3.00 (m, CHCH2Ph),
2.20–2.07(m, 4H, CH2), 1.71–1.48 (m, 4 H, CH2), 1.36–1.21 (m, 4 H, CH2).–
13C NMR (CD3OD): δ = 172.19 (CO), 169.06 (CO), 137.28 (Phe-1), 128.95
and 128.11 (Phe-2,3,5,6), 126.41 (Phe-4), 53.31 (OMe), 51.70 (CHCOOMe),
383 mg (1 mmol) of N-benzyloxy-6-(4-dimethylaminobenzoylamino)-
capramide were dissolved in 15 mL of methanol and 40 mg 10% palladium
on charcoal were added. The mixture was treated with hydrogen under
atmospheric pressure for 6 h and was subsequently filtered. The product was
evaporated, redissolved in 5 mL of methanol, and precipitated with diethyl
ether. The last step was repeated once. Yield 0.16 g (56%) white powder.–
Mp 170 °C.– IR (KBr): ν = 3365s, 3160s, 1650m, 1600s.– MS (70 eV); m/z
(%) = 293 (5) [M+], 164 (19), 148 (100).– 1H NMR (DMSO-D6): δ = 10.33
3
(s, 1 H, CONHOH), 8.67 (s, 1 H, CONHOH), 8.07 (t, 1 H, J = 5.45 Hz,
CONHCH2), 7.71–7.65 (m, 2 H, 2′-H, 6′-H,), 6.69–6.65 (m, 2 H, 3′-H, 5′-H),
3.23–3.13 (m, 2 H, CONHCH2), 2.94 (s, 6 H, NMe2), 1.97–1.90 (m, 2 H,
Arch. Pharm. Pharm. Med. Chem. 332, 353–357 (1999)

Histone Deacetylase Inhibitors
357
CH2CO), 1.53–1.42 (m, 4 H, NHCH2CH2CH2CH2), 1.30–1.22 (m, 2 H,
NHCH2CH2CH2).– 13C NMR (DMSO-D6): δ = 169.08 (CONHOH), 166.01
(CONHCH2), 151.96 (Me2NC), 128.39 (C-3′, C-5′), 121.42 (CCONHCH2),
110.74 (C-2′, C-6′), 39.72 (NMe2), 38.66 (CONHCH2), 32.24 (CH2CO),
29.11, 26.14 and 24.92 (CH2).– Anal. (C15H23N3O3) 293.41: C, H, N.
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Cell Culture
The human MCF-7 breast cancer cell line was obtained from the American
Type Culture Collection (ATCC, Rockville, Md.; USA). Cell line banking
and quality control were performed according to the seed stock concept
reviewed by Hay [21]. The MCF-7 cells were maintained as a monolayer
culture at 37 °C in a humidified 95% air, 5% CO2 atmosphere in T-75 flasks
using L-glutamine containing Eagle’s MEM supplemented with BCS
(100mL/L) as growth medium. The cell line was weekly passaged after
treatment with trypsin/EDTA.
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In Vitro Chemosensitivity Assay
The in vitro testing of the inhibitors of histone deacetylase for antitumor
activity was carried out on MCF-7 cells according to a previously published
microtiter assay [13]. Briefly, 100 µL of a cell suspension at 500 cells/mL
culture medium were plated into each well of a 96-well microtiter plate and
incubated at growing conditions (see above) for three days. Then each
substance was added in three concentrations. After theproper incubation time
the medium was removed, the cells were fixed with a glutardialdehyde
solution and stored at 4 °C. Cell biomass was determined by a crystal violet
staining technique as described earlier [13,22]. The effectiveness (cytostatic
effect) of the compounds is expressed as:
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T/Ccorr [%] = [(T – Co) / (C – Co)] × 100
where T (test) and C (control) are the optical densities at 590 nm of the crystal
violet extract of the cells in the wells (i.e. the chromatin-bound crystal violet
extracted with ethanol 70%), and Co is the optical density of the cell extract
immediately before treatment. Cytocidal effect:
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For the automatic estimation of the optical density of the crystal violet extract
in the wells the Microplate PhotometerLabsystemMultiscan® Plus was used.
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27, 2057–2058.
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Received: May 6, 1999 [FP394]
Arch. Pharm. Pharm. Med. Chem. 332, 353–357 (1999)