Design, synthesis, and biological activity of folate receptor-targeted prodrugs of thiolate histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.05.040

Aiming to develop selective anticancer drugs, we designed and synthesized three disulfides bearing a folic acid moiety as candidate folate receptor (FR)-targeted prodrugs of thiolate histone deacetylase inhibitors. Among them, compound 1 displayed growth-inhibitory activity toward folate receptor-positive MCF-7 breast cancer cells. The activity of 1 was significantly reduced by free folic acid, suggesting that cellular uptake of 1 is mediated by FR.

Full text of DOI:10.1016/j.bmcl.2007.05.040

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4208–4212
Design, synthesis, and biological activity of folate receptor-targeted
prodrugs of thiolate histone deacetylase inhibitors
Takayoshi Suzuki,a,* Shinya Hisakawa,a Yukihiro Itoh,a Nobuaki Suzuki,a
Katsumasa Takahashi,a Masatoshi Kawahata,b Kentaro Yamaguchi,b
Hidehiko Nakagawaa and Naoki Miyataa,*
aGraduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
bEvaluation Group, Drug Research Department, R&D Division, Pharmaceuticals Group, Nippon Kayaku Co., Ltd, 31-12,
Shimo 3-chome, Kita-ku, Tokyo 115-8588, Japan
Received 22 April 2007; revised 10 May 2007; accepted 11 May 2007
Available online 17 May 2007
Abstract—Aiming to develop selective anticancer drugs, we designed and synthesized three disulfides bearing a folic acid moiety as
candidate folate receptor (FR)-targeted prodrugs of thiolate histone deacetylase inhibitors. Among them, compound 1 displayed
growth-inhibitory activity toward folate receptor-positive MCF-7 breast cancer cells. The activity of 1 was significantly reduced
by free folic acid, suggesting that cellular uptake of 1 is mediated by FR.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) have recently emerged as
a new target for the development of anticancer drugs,
and some small-molecular HDAC inhibitors, including
suberoylanilide hydroxamic acid (SAHA) (also known
as vorinostat) and MS-275, have been developed as anti-
cancer drugs (Fig. 1).1–6 Inhibition of HDACs causes
histone hyperacetylation which leads to the disruption
of the chromatin structure and the transcriptional acti-
vation of genes associated with cancer. Indeed, HDAC
inhibitors have shown anticancer activity in vitro, in ani-
mal models and in patients with solid tumors and hema-
tological malignancies.7 Nevertheless, they have been
reported to cause adverse events, such as nausea, vomit-
ing, anorexia, anemia, thrombocytopenia, and fatigue,
in the course of clinical trials.7–9 Therefore, it is neces-
sary to find HDAC inhibitors that show selective anti-
cancer activity.
O
O
H
O
N
NH2
N
OH
H
H
N
N
H
N
O
O
SAHA
MS-275
SH
H
N
N
S
O
NCH-31
Figure 1. Structures of SAHA, MS-275, and the thiolate HDAC
inhibitor NCH-31.
internalized via receptor-mediated endocytosis.10 Since
the FR is overexpressed on certain malignant cell types
and is undetectable or present only at low levels in most
normal tissues,11–13 targeting of the FR has been pro-
posed as a potential mechanism for delivery of drugs
to treat cancer.14 In addition, small molecules including
folate-drug conjugates may avoid the limitations associ-
ated with antibody-mediated targeting.15,16 Here, we
report on the design, synthesis, and biological activity
of FR-targeted prodrugs of HDAC inhibitors.
We have focused on folate receptor (FR)-targeted pro-
drugs for selectively targeting cancer cells. The vitamin
folic acid and its analogues display extremely high affin-
ity for the folate receptor on the cell surface, and are
We previously reported that thiol-based analogues,
including NCH-31 (Fig. 1), are potent HDAC inhibi-
tors.17–19 Thiols are thought to inhibit HDACs by coor-
dinating the zinc ion, which is required for deacetylation
Keywords: Histone deacetylase inhibitor; Selective anticancer activity;
Folate conjugate; Prodrug.
*
Corresponding authors. Tel./fax: +81 52 836 3407; e-mail addresses:
suzuki@phar.nagoya-cu.ac.jp; miyata-n@phar.nagoya-cu.ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.040

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4208–4212
4209
of the acetylated lysine substrate. Further, thiolate ana-
H2N
S
N
a - c
S
H2N
logues showed potent cancer cell growth-inhibitory
activities.20,21 Based on these findings, we designed
FR-targeted prodrugs of HDAC inhibitors. Unlike
hydroxamates and o-aminoanilides, such as SAHA
and MS-275, thiolate HDAC inhibitors can be conju-
gated with a folic acid moiety via a disulfide bond, which
would be reduced under reductive conditions, releasing
the free thiol as an active species. We designed the folic
acid-NCH-31 conjugates 1 and 2 (Fig. 2), which are ex-
pected to be recognized by the FR located on the cell
surface, to enter cells via receptor-mediated endocytosis,
and then to release the HDAC inhibitor NCH-31 upon
cleavage of the disulfide bond in the cellular environ-
ment. We also designed the symmetrical disulfide 3 bear-
ing a folic acid moiety. The reduced form of compound
3 itself could behave as an HDAC inhibitor.
SH
4
5
O
d
BocHN
S
N
H
S
N
COOt-Bu
6
O
H
BocHN
S
S
N
N
e
N
H
6
COOt-Bu
O
S
7
O
H
N
H2N
S
N
f
N
H
S
6
COOH
O
S
The synthesis of the folic acid-NCH-31 conjugate 1 is
outlined in Scheme 1. Mercaptoethylamine 4 was con-
verted into 2-(2-(2-pyridinyl)disulfanyl)ethylamine 5 by
the Boc protection of 4, followed by treatment with
2,20-dithiopyridine and Boc deprotection. Compound 5
was then coupled with N-Boc-L-glutamic acid a-tert-bu-
tyl ester to give the amide 6. Treatment of compound 6
with NCH-3117,20 in DMF afforded the sulfur-ex-
changed product 7. Universal deprotection of 7 using
hydrochloric acid yielded the NCH-31-glutamic acid
linked compound 8. The folic acid-NCH-31 conjugate
1 was obtained in 92% yield by the reaction of 8 with
pteroyl azide 922 in DMSO in the presence of
tetramethylguanidine.
8
g
1
Scheme 1. Reagents and conditions: (a) (Boc)2O, Et3N, CH2Cl2, rt; (b)
2,20-dithiopyridine MeOH, rt; (c) HCl, AcOEt, rt, 39% (three steps);
(d) Boc-Glu-O-t-Bu, EDCI, HOBt, Et3N, CH2Cl2, rt, 76%; (e) NCH-
31, DMF, rt, 86%; (f) HCl, AcOEt, rt, quant; (g) pteroyl azide (9),
tetramethylguanidine, DMSO, rt, 92%.
a
H2N
NH2
BocHN
SH
NH2
O
O
2
2
10
11
c - h
H
b
BocHN
N
Scheme 2 shows the preparation of the other folic acid-
NCH-31 conjugate 2. Compound 2 was synthesized from
2,20-(ethylenedioxy)diethylamine 10. Reaction of the dia-
mine 10 with an equivalent amount of (Boc)2O gave the
mono-Boc-protected compound 11. Coupling between
amine 11 and 3-mercaptopropanoic acid in the presence
of EDCI and HOBt afforded 12. The folic acid-NCH-31
conjugate 2 was prepared from the thiol 12 in the same
manner as described for the synthesis of 1.
O
2
2
O
12
Scheme 2. Reagents and conditions: (a) (Boc)2O, CH2Cl2, 0 ꢁC to rt,
quant; (b) 3-mercaptopropanoic acid, EDCI, HOBt, CH2Cl2, rt, 53%;
(c) 2,20-dithiopyridine MeOH, rt; (d) HCl, AcOEt, rt; (e) Boc-Glu-O-t-
Bu, EDCI, HOBt, Et3N, CH2Cl2, rt; (f) NCH-31, DMF, rt; (g) HCl,
AcOEt, rt; (h) 9, tetramethylguanidine, DMSO, rt, 59% (six steps).
The attempted route to 3 is shown in Scheme 3. In this
scheme, we anticipated that disulfide dimer 3 would be
obtained from the corresponding thiol monomer by
reaction with I2. The 7-bromoheptanoic acid ethyl ester
13 was hydrolyzed to give the carboxylic acid 14, after
which Curtius rearrangement of the acyl azide prepared
from 14 using diphenylphosphoryl azide (DPPA) pro-
vided the isocyanate. This, on treatment with tert-buta-
nol, gave the N-Boc compound 15. Treatment of 15 with
triphenylmethanethiol in the presence of NaOMe affor-
ded compound 16. Deprotection of the Boc group of 16
and coupling with N-Fmoc-L-glutamic acid a-tert-butyl
ester gave the amide 17. The Fmoc group of 17 was re-
moved using piperidine and coupling with pteroyl azide
9 afforded compound 18. Removal of the tert-butyl
group and the triphenylmethyl group of 18 under acidic
conditions gave the thiol 19. Although 19 was success-
fully obtained from 13 in eight steps, it was poorly sol-
uble. We examined a variety of solvents for the
dimerization of 19 using I2, but a suspension of 19 with
I2 failed to provide the disulfide dimer 3.
H2N
HN
N
O
N
N
H
N
O
H
H
N
S
N
N
S
X
6
O
COOH
S
O
Folic Acid
NCH-31
1: X = -NHCH2CH2-
2: X = -NHCH2CH2OCH2CH2OCH2CH2-
H2N
HN
N
O
N
H
N
N
O
H
N
S
N
H
O
COOH
2
3
Figure 2. Candidate FR-targeted prodrugs of thiolate HDAC
inhibitors.

4210
T. Suzuki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4208–4212
Table 1. HDAC inhibition data for NCH-31, 1–3, and 19a
EtO
HO
a
b
Br
6
Br
6
BocHN
15
Br
6
Entry
Compound
IC50 (lM)
O
O
14
0.17b
0.27
3.0
13
1
2
3
4
5
NCH-31
1c
2c
3c
19
O
Trt
c
d, e
FmocHN
Trt
BocHN
S
N
H
S
9.0
6
6
COOt-Bu
16
21
17
a Values are means of at least three experiments.
b Data taken from the literature (Ref. 20).
c Incubated with DTT (250 lM).
H2N
HN
N
N
N
H
N
f, g
O
H
O
N
Trt
N
S
H
6
O
COOt-Bu
disulfide bond of compound 1 was reduced to release
NCH-31 under the reductive conditions. On the other
hand, the HDAC-inhibitory activities of compounds 2
and 3 were weaker than that of 1. The reason for the
weaker activity of 2 is unclear, but it may be because
compound 2 is resistant to reduction as compared with
compound 1.
18
H2N
N
O
N
H
HN
N
h
N
O
H
N
N
H
SH
6
O
COOH
19
i
To confirm the effectiveness of the folic acid-based pro-
drugs of HDAC inhibitors, compounds 1 and 2 were
tested in a cancer cell growth inhibition assay24 using
FR-positive human breast cancer MCF-7 cells.25 Con-
sistent with the results in the enzyme assay, compound
1 displayed dose-dependent cell growth-inhibitory activ-
ity (Fig. 3). Further, a competition experiment with
100 lM free folic acid significantly reduced the cell
growth-inhibitory activity of 1, demonstrating that the
FR is responsible for the cellular entry of 1 (Fig. 4). In
addition, treatment of MCF-7 cells with compound 1
produced an increase in the accumulation of acetylated
histone H4 (Fig. 5),26 which indicated that the cell
growth-inhibitory activity of compound 1 significantly
correlates with the inhibition of HDACs. Furthermore,
the activity of 1 to cause histone hyperacetylation was
significantly reduced by 100 lM free folic acid (Fig. 5).
These results also suggested that the uptake of com-
pound 1 is FR mediated.
3
Scheme 3. Reagents and conditions: (a) LiOH, THF, EtOH, H2O, rt,
quant; (b) 1—DPPA, Et3N, reflux, 2—t-BuOH, toluene, reflux, 44%;
(c) NaOMe, HS-Trt, toluene, EtOH, 60 ꢁC, 94%; (d) TFA, CH2Cl2, rt;
(e) Fmoc-Glu-O-t-Bu, EDCI, HOBt, CH2Cl2, rt, 76% (two steps); (f)
piperidine, DMF, rt, 87%; (g) 9, tetramethylguanidine, DMSO, rt,
21%; (h) TFA, CH2Cl2, rt, 94%; (i) I2.
We succeeded in obtaining 3 through the route outlined
in Scheme 4. In this route, a disulfide bond was formed
in the early stage. Initially, 6-aminohexanol 20 was con-
verted to compound 21 by N-Boc protection, O-tosyla-
tion, and treatment with potassium thioacetate. The
acetyl group of 21 was then removed to give the thiol
22, and the disulfide dimer 23 was obtained by the reac-
tion of the thiol monomer 22 with I2. The desired disul-
fide 3 was successfully obtained from 23 in 74% yield
using the same procedure as described for the synthesis
of 1.
In summary, we have designed and synthesized FR-tar-
geted prodrugs of thiolate HDAC inhibitors that possess
a cleavable disulfide bond. The folic acid-NCH-31 con-
jugate 1 showed potent HDAC-inhibitory activity under
reductive conditions. Furthermore, compound 1 exerted
growth-inhibitory activity against FR-positive breast
cancer MCF-7 cells, and the cellular uptake of 1 was
considered to be FR mediated, based on a competition
experiment with free folic acid. Our strategy of utilizing
Compounds 1–3 were initially tested in an in vitro
HDAC inhibition assay under reductive conditions
(Table 1).23 Among these compounds, compound 1
showed the most potent activity inhibiting HDACs with
an IC50 of 0.27 lM, and the activity was comparable
with that of NCH-31. This result suggested that the
d
a - c
BocHN
21
SAc
H2N
OH
6
6
20
140
120
100
80
e
S
BocHN
22
SH
BocHN
6
2
23
60
40
20
f - i
3
0
Scheme 4. Reagents and conditions: (a) (Boc)2O, THF, rt; (b) TsCl,
Et3N, THF, rt; (c) KSAc, acetone, rt, 75% (three steps); (d) aq NaOH,
MeOH, THF, rt, 71%; (e) I2, MeOH, rt, 95%; (f) HCl, AcOEt, rt; (g)
Boc-Glu-O-t-Bu, EDCI, HOBt, Et3N, CH2Cl2, rt; (h) HCl, AcOEt, rt;
(i) 9, tetramethylguanidine, DMSO, rt, 74% (four steps).
-8
-7
-6
-5
-4
-3
Log concentration (M)
Figure 3. Growth inhibition of FR-positive MCF-7 cells by com-
pounds 1 (d) and 2 (m).

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4208–4212
4211
8. Kelly, W. K.; O’Connor, O. A.; Krug, L. M.; Chiao, J. H.;
Heaney, M.; Curley, T.; MacGregore-Cortelli, B.; Tong,
W.; Secrist, J. P.; Schwartz, L.; Richardson, S.; Chu, E.;
Olgac, S.; Marks, P. A.; Scher, H.; Richon, V. M. J. Clin.
Oncol. 2005, 23, 3923.
9. Ryan, Q. C.; Headlee, D.; Acharya, M.; Sparreboom, A.;
Trepel, J. B.; Ye, J.; Figg, W. D.; Hwang, K.; Chung, E. J.;
Murgo, A.; Melillo, G.; Elsayed, Y.; Monga, M.; Kalnit-
skiy, M.; Zwiebel, J.; Sausville, E. A. J. Clin. Oncol. 2005,
23, 3912.
10. Antony, A. C. Blood 1992, 79, 2807.
11. Ross, J. F.; Chaudhuri, P. K.; Ratnam, M. Cancer 1994,
73, 2432.
12. Weitman, S. D.; Lark, R. H.; Coney, L. R.; Fort, D. W.;
Frasca, V.; Zurawski, V. R.; Kamen, B. A. Cancer Res.
1992, 52, 3396.
120
100
80
60
40
20
0
*
**
1 (25 M)
1 (25 M) 1 (50 M) 1 (50 M)
μ
μ
μ
μ
+ FA (100 μM)
+ FA (100 μM)
Figure 4. Growth inhibition of FR-positive MCF-7 cells by 1 and 1
plus 100 lM free folic acid (FA). *p < 0.05; **p < 0.01 by Student’s t
test.
13. Weitman, S. D.; Weinberg, A. G.; Coney, L. R.; Zuraw-
ski, V. R.; Jennings, D. S.; Kamen, B. A. Cancer Res.
1992, 52, 6708.
14. Leamon, C. P.; Reddy, J. A. Adv. Drug Deliv. Rev. 2004,
56, 1143.
1 (25 μM)
1 (50 μM)
control
1 (25 μM)
1 (50 μM) + FA (100 μM) + FA (100 μM)
15. Canevari, S.; Mezzanzanica, D.; Menard, S.; Ferrini, S.;
Moretta, L.; Colnaghi, M. I. Int. J. Cancer 1992, 7, 42.
16. Yamaguchi, T.; Tsurumi, H.; Kotani, T.; Yamaoka, N.;
Otsuji, E.; Kitamura, K.; Takahashi, T. Jpn. J. Cancer
Res. 1994, 85, 167.
Ac histone H4
β-actin
17. Suzuki, T.; Kouketsu, A.; Matsuura, A.; Kohara, A.;
Ninomiya, S.; Kohda, K.; Miyata, N. Bioorg. Med. Chem.
Lett. 2004, 14, 3313.
Figure 5. Western blot analysis of histone hyperacetylation in MCF-7
cells produced by 1 and 1 plus 100 lM free folic acid (FA).
18. Suzuki, T.; Matsuura, A.; Kouketsu, A.; Nakagawa, H.;
Miyata, N. Bioorg. Med. Chem. Lett. 2005, 15, 331.
19. Suzuki, T.; Kouketsu, A.; Itoh, Y.; Hisakawa, S.; Maeda,
S.; Yoshida, M.; Nakagawa, H.; Miyata, N. J. Med.
Chem. 2006, 49, 4809.
20. Suzuki, T.; Nagano, Y.; Kouketsu, A.; Matsuura, A.;
Maruyama, S.; Kurotaki, M.; Nakagawa, H.; Miyata, N.
J. Med. Chem. 2005, 48, 1019.
a disulfide bond to connect a thiolate HDAC inhibitor
with folic acid should be applicable to other anticancer
agents bearing a thiol group, such as thiolate matrix
metalloproteinase inhibitors27 and thiolate farnesyl-
transferase inhibitors.28 Our findings in this study pro-
vide the basis for a new approach to developing
candidate antitumor agents with potentially fewer side
effects.
21. Suzuki, T.; Hisakawa, S.; Itoh, Y.; Maruyama, S.;
Kurotaki, M.; Nakagawa, H.; Miyata, N. Bioorg. Med.
Chem. Lett. 2007, 17, 1558.
22. Luo, J.; Smith, M. D.; Lantrip, D. A.; Wang, S.; Fuchs, P.
L. J. Am. Chem. Soc. 1997, 119, 10004.
Acknowledgments
23. The HDAC activity assay was performed using an HDAC
fluorescent activity assay/drug discovery kit (AK-500,
BIOMOL Research Laboratories). HeLa Nuclear Extracts
(0.5 lL/well) were incubated at 37 ꢁC with 25 lM of Fluor
de LysTM substrate and various concentrations of samples.
Reactions were stopped after 30 min by adding Fluor de
LysTM Developer with trichostatin A which stops further
deacetylation. Then, 15 min after addition of this devel-
oper, the fluorescence of the wells was measured on a
fluorometric reader with excitation set at 360 nm and
emission detection set at 460 nm, and the % inhibition was
calculated from the fluorescence readings of inhibited
wells relative to those of control wells. The concentration
of compound which results in 50% inhibition was deter-
mined by plotting the log[Inh] versus the logit function of
the % inhibition. IC50 values are determined using a
regression analysis of the concentration/inhibition data.
24. MCF-7 human breast cancer cells were purchased from
American Type Culture Collection (ATCC, Manassas,
VA, USA) and cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing penicillin and streptomycin,
which was supplemented with fetal bovine serum as
described in the ATCC instructions. MCF-7 cells were
plated in 96-well plates at initial densities of 5000 cells/well
(50 lL/well) and incubated at 37 ꢁC. After 24 h, cells were
exposed to a solution of test compounds in DMEM
(50 lL) at various concentrations in DMEM at 37 ꢁC in
This work was supported in part by Grants-in-Aid for
Young Scientists (B) from the Ministry of Education,
Science, Culture, Sports, Science, and Technology,
Japan, and a grant from Takeda Science Foundation.
References and notes
1. Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem.
2003, 46, 5097.
2. Miller, T. A. Expert Opin. Ther. Pat. 2004, 14, 791.
3. Biel, M.; Wascholowski, V.; Giannis, A. Angew. Chem.
Int. Ed. Engl. 2005, 44, 3186.
4. Suzuki, T.; Miyata, N. Curr. Med. Chem. 2006, 13, 935.
5. Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.;
Breslow, R.; Rifkind, R. A.; Marks, P. A. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 3003.
6. Saito, A.; Yamashita, T.; Mariko, Y.; Nosaka, Y.;
Tsuchiya, K.; Ando, T.; Suzuki, T.; Tsuruno, T.; Nak-
anishi, O. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4592.
7. Kelly, W. K.; Richon, V. M.; O’Connor, O.; Curley, T.;
MacGregor-Curtelli, B.; Tong, W.; Klang, M.; Schwartz,
L.; Richardson, S.; Rosa, E.; Drobnjak, M.; Cordon-
Cordo, C.; Chiao, J. H.; Rifkind, R.; Marks, P. A.; Scher,
H. Clin. Cancer Res. 2003, 9, 3578.

4212
T. Suzuki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4208–4212
5% CO2 for 72 h. Then, 10 lL of alamarBlueTM was added,
lysate were resolved in 15% SDS–polyacrylamide gel and
then transferred onto nitrocellulose membranes (Bio-Rad
Laboratories). After blocking for 30 min with Tris-buf-
fered saline (TBS) containing 3% skim milk, the transb-
lotted membrane was incubated overnight at 4 ꢁC with
hyperacetylated histone H4 antibody (Upstate Biotech-
nology) (1:2000 dilution) or b-actin antibody (Abcam)
(1:1000 dilution) in TBS containing 3% skim milk. After
probing with the primary antibody, the membrane was
washed twice with water, then incubated with goat anti-
rabbit or anti-mouse IgG-horseradish peroxidase conju-
gates (diluted 1:5000) for 2 h at room temperature, and
further washed twice with water. The immunoblots were
visualized by enhanced chemiluminescence.
and cells were incubated at 37 ꢁC for 3 h. The fluorescence
of the wells was measured on a fluorometric reader with
excitation set at 530 nm and emission detection set at
590 nm, and the percentage of cell growth was calculated
from the fluorescence readings.
25. Lee, E. S.; Na, K.; Bae, Y. H. J. Control. Release 2003, 91,
103.
26. MCF-7 cells were cultured in DME culture medium
containing penicillin and streptomycin, which was supple-
mented with fetal bovine serum as described in the ATCC
instructions. MCF-7 cells (5 · 105) were treated for 14 h
with samples at the indicated concentrations in 10% FBS-
supplemented DMEM then collected and extracted with
SDS buffer. Protein concentrations of the lysates were
determined using a Bradford protein assay kit (Bio-Rad
Laboratories); equivalent amounts of proteins from each
27. Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H.
Chem. Rev. 1999, 99, 2735.
28. Bell, I. M. J. Med. Chem. 2004, 47, 1869.