Acylurea connected straight chain hydroxamates as novel histone deacetylase inhibitors: Synthesis, SAR, and in vivo antitumor activity

Article abstract of DOI:10.1016/j.bmcl.2010.04.041

Thirty-six novel acylurea connected straight chain hydroxamates were designed and synthesized. Structure-activity relationships (SAR) were established for the length of linear chain linker and substitutions on the benzoylurea group. Compounds 5g, 5i, 5n, and 19 showed 10-20-fold enhanced HDAC1 potency compared to SAHA. In general, the cellular potency pIC₅₀ (COLO205) correlates with enzymatic potency pIC₅₀ (HDAC1). Compound 5b (SB207), a structurally simple and close analogue to SAHA, is more potent against HDAC1 and HDAC6 compared to the latter. As a representative example of this series, good in vitro enzymatic and cellular potency plus an excellent pharmacokinetic profile has translated into better efficacy than SAHA in both prostate cancer (PC3) and colon cancer (HCT116) xenograft models.

Full text of DOI:10.1016/j.bmcl.2010.04.041

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3314–3321
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acylurea connected straight chain hydroxamates as novel histone
deacetylase inhibitors: Synthesis, SAR, and in vivo antitumor activity
a
a
a
a
a
b
Haishan Wang a, , Ze-Yi Lim , Yan Zhou , Melvin Ng , Ting Lu , Ken Lee , Kanda Sangthongpitag ,
Kee Chuan Goh b, Xukun Wang b, Xiaofeng Wu b, Hwee Hoon Khng b, Siok Kun Goh b, Wai Chung Ong b,
Zahid Bonday b, Eric T. Sun a
*
a Department of Chemistry Discovery, S*BIO Pte Ltd, 1 Science Park Road, #05-09 The Capricorn, Singapore Science Park II, Singapore 117528, Singapore
b Department of Biology Discovery, S*BIO Pte Ltd, 1 Science Park Road, #05-09 The Capricorn, Singapore Science Park II, Singapore 117528, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 February 2010
Revised 1 April 2010
Accepted 10 April 2010
Available online 14 April 2010
Thirty-six novel acylurea connected straight chain hydroxamates were designed and synthesized.
Structure–activity relationships (SAR) were established for the length of linear chain linker and substitu-
tions on the benzoylurea group. Compounds 5g, 5i, 5n, and 19 showed 10–20-fold enhanced HDAC1
potency compared to SAHA. In general, the cellular potency pIC50 (COLO205) correlates with enzymatic
potency pIC50 (HDAC1). Compound 5b (SB207), a structurally simple and close analogue to SAHA, is more
potent against HDAC1 and HDAC6 compared to the latter. As a representative example of this series, good
in vitro enzymatic and cellular potency plus an excellent pharmacokinetic profile has translated into bet-
ter efficacy than SAHA in both prostate cancer (PC3) and colon cancer (HCT116) xenograft models.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HDAC inhibitor
Acylurea
Hydroxamic acid
SAR
Anticancer
Efficacy
Histone deacetylase (HDAC) enzymes are divided into four
different classes.1 Class I and II isozymes, specifically HDACs 1, 2,
3, 6 and 8, have been associated with uncontrolled tumor growth.
For example, selective knockdown studies on HDACs suggest that
the class I HDACs, particularly HDACs 1 and 3, are essential to
the proliferation and survival of mammalian carcinoma cells.2
HDAC inhibitors have been studied for their therapeutic effects
on cancer cells.3 Suberoylanilide hydroxamic acid (SAHA, vorino-
stat), the first HDAC inhibitor approved by the FDA in 2006 for
the treatment of cutaneous T-cell lymphoma, validates HDAC inhi-
bition as a strategy for cancer therapy.4
acid remains among the most potent and popular ZBGs reported
for inhibition of Class I HDACs. There is also a connection unit
(CU) or connector between the CAP and the linker. Examples of
CUs used for a variety of types of HDAC inhibitors are shown below
(Fig. 2).7
There are a number of HDAC inhibitors which are currently
undergoing clinical trials either as a single therapy or in combina-
tion for treatment of solid and hematologic malignancies (Fig. 1).
The morphology of the HDAC inhibitor pharmacophore, as exem-
plified by SAHA (Fig. 1), is characterized by three portions: a metal-
or zinc-binding group (ZBG), a hydrophobic group (CAP) for protein
surface recognition or interaction, and a linker to connect both ZBG
and CAP. The common linkers are aliphatic chain (e.g., six carbon
chain in SAHA), aromatic ring (e.g., phenylene in MS-275)5 and
vinyl-aromatic (e.g., styryl in PXD-101).6 The most common ZBGs
are hydroxamic acid and benzamide (e.g., MS-275). Hydroxamic
* Corresponding author. Tel.: +65 68275019; fax: +65 68275005.
E-mail address: haishan_wang@sbio.com (H. Wang).
Figure 1. Examples of clinically tested HDAC inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.041

H. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3314–3321
3315
CU for HDAC inhibitor. This CU can be used not only for generating
novel HDAC inhibitor from a patenting perspective, but also for
investigating interactions with the region between the binding
channel and the entrance area of the HDAC catalytic pocket.9,10
The optimal location for such an interaction is tunable by adjusting
the length of the linker. The acylurea moiety has more hydrogen
bond acceptors and donors available compared to either amide
or urea, hence the conformation or orientation of the linker
and/or the CAP may change, thus influencing potency and/or
isoform-selectivity. This unit will likely change the overall drug-
like properties as well.
In this report, we describe the synthesis and evaluation of
acylurea connected straight chain hydroxamates as novel HDAC
inhibitors, some of them have shown promising pharmacological
and pharmacokinetic properties.
Scheme 1, Route A, illustrates the general procedure used for
preparing acylurea connected hydroxamic acids 5 and 9. Amino
acid ester 2, if not commercially available, was prepared from ami-
Figure 2. Examples of connection units (CUs) used for HDAC inhibitors.
As part of our ongoing efforts to discover novel anticancer
agents, we have examined acylurea and sulfonylurea connected
hydroxamates as low molecular weight novel HDAC inhibitors.8
To the best of our knowledge, acylurea (–CONHCONH–), which
can be considered as a combination of amide and urea, is a new
Scheme 1. Reagents and conditions: (a) SOCl2 (1.6 equiv), MeOH, À78 °C to rt; (b) ArCON@C@O (3), Et3N, CH2Cl2, À78 °C to rt; (c) NH2OHÁHCl (10 equiv), NaOMe (12 equiv),
MeOH, 0 °C to rt; (d) NaBH(OAc)3, CH2Cl2; (e) Fmoc-Cl, Na2CO3, H2O/dioxane; (f) O-(2,4-dimethoxy-benzyl)-hydroxylamine, DCC, CH2Cl2, rt; (g) piperidine, MeOH, rt; (h) 5%
TFA in CH2Cl2, rt.

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H. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3314–3321
reductive alkylation of 2 with aldehyde 6. For compounds contain-
ing a functional group labile to strong basic conditions (NaOMe/
MeOH) or for easy workup during parallel synthesis, O-2,4-dime-
thoxy-benzyl11 protected hydroxamate precursor 11 was used to
prepare hydroxamates 5 and 9 (Scheme 1, Route B).
Benzoylisocyanate (3, Ar = phenyl) is commercially available,
more complex acylisocyanates 3 were prepared according to
Scheme 2. Method A is a traditional way of making acylisocyanates
by reaction of silver cyanate with acid chloride 14.12 Alternatively,
much cheaper sodium cyanate was used to react with acid chloride
14 under elevated temperature to give crude 3,13 which was used
without further purification. Acylisocyanates 3 with electron-with-
drawing groups, such as 3a–c and 3g–j, were prepared by method
B. 3d–f and 3k were made by method A.
TofurtherdiversifytheCAPgroup(theArmoiety)of5 (Scheme1),
a series of synthetic routes were developed to install Ar as a mono- or
di-substituted phenyl, or a (substituted) fused ring starting from
methylester4 (Scheme1) as depictedin Scheme3. Nitro compounds
4k was reduced to aniline 15, and then subsequently acylated or
Scheme 2. Reagents and conditions: Method (A) AgOCN (1.1 equiv), CH2Cl2, rt;
Method (B) NaOCN (1.3 equiv), SnCl4 (0.06 equiv), 1,2-dichlorobenzene, 180 °C.
no acid 1, and then reacted with acylisocyanate 3 to give acylurea
connected methyl ester 4, which was treated with excessive
hydroxylamine and afforded hydroxamate (5) in good yield.
N-alkylated hydroxamate 9 was made by using similar procedures
but starting from secondary amine 7 which was prepared by
Scheme 3. Reagents and conditions: (a) SnCl2Á2H2O, CH2Cl2–MeOH; (b) PhCH2COCl, CH2Cl2, Et3N; (c) PhSO2Cl, CH2Cl2, Et3N; (d) NH2OHÁHCl (10 equiv), NaOMe (12–20 equiv),
MeOH, 0 °C to rt; (e) R3B(OH)2, Pd(PPh3)4, PhMe–MeOH (9:1), NaHCO3 (sat), 80 °C; (f) Et3N, 1,4-dioxane, 80 °C; (g) SnCl2Á2H2O (5 equiv), HOAC–MeOH (1:9), 40 °C.

H. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3314–3321
3317
Table 1
SAR of linker length (m) for 5a–d and R2 for 9a–e
R2
HDAC1 IC50 (nM)
HDAC8 IC50 (nM)
COLO205 IC50
(lM)
a
a
b
Compound
m
5a
5b
5c
5d
9a
9b
9c
9d
9e
SAHA
3
5
6
7
5
5
5
6
6
—
H
H
H
H
>10,000
53 17
145 35
>10,000
608 113
270 14
405 106
328d
NTc
2.21 0.41
2.68 0.28
8.28 2.85
NTc
3.19 0.95
14.7 6.1
NTc
97
4
Ph(CH2)3–
PhCH2–
Pyridin-2-yl-CH2–
PhCH2–
Pyridin-2-yl-CH2–
—
392d
355 49
245 49
1600 749
555 191
121 37
NTc
NTc
NTc
NTc
9.06 0.38
2.20 0.14
272 127
a
b
c
Values are expressed as mean standard deviation of at least two independent duplicate experiments.
Values are expressed as mean standard deviation of at least two independent triplicate experiments.
NT: not tested.
d
Values obtained from only one duplicate or triplicate experiment.
sulfonylated to afford hydroxamates 16a or 16b after reacting with
excessive hydroxylamine. Halogenated 4g was attached with an aryl
orheteroarylgroupviaSuzukireaction, thenbiaryl17wasconverted
to hydroxamate 16c. Compound 19, a 3-amino analogue of 5f, was
made by reducing the nitro group of 4o to aniline 18 first before con-
verting it to hydroxamic acid. The chloro of 4o was displaced by an
amine R4R5NH (20) to afford 21, which was further converted to
hydroxamate 22. Compound 21 (when R4 = H) was also transformed
to benzimidazole 24 by reacting it with aldehyde R6CHO (23) or for-
mic acid in the presence of a nitro reducing reagent (e.g., SnCl2).14
Reacting the methyl ester 24 with hydroxylamine afforded 3-(1H-
benzimidazole-5-carbonyl)-ureido containing hydroxamate 25.
The CAP or Ar group for initially synthesized hydroxamates
(Table 1) was fixed as phenyl, for comparison with SAHA, derived
from readily available benzoyl isocyanate. Historically, we had
both HDAC8 and HDAC1 as respective target for our two in-house
HDAC programs, and human colon cancer cell line COLO205 was
used for routine anti-proliferation assay.15 Thus these compounds
were profiled against both HDAC1 and HDAC8 enzymes and
COLO205 cells (Table 1). Clearly m = 5 is optimal for both HDAC1
and COLO205 potency when comparing compounds (5a–d) with
an increasing number of methylenes in the linker. This SAR is in-
line with some similar studies on straight chain hydroxamates;
with the shorter amide group as the CU, a six-methylene linker is
optimal for HDAC enzymatic acitivity.16 As for urea as the CU, a
five-methylene is optimal for HDAC enzymatic activity.7a When
acylurea is CU, it is longer than both amide and urea, thus it is
not a surprise that the optimal length is five-methylene.
emerges (5g vs 5h; 5k vs 5l), the potency is related to the size
and lipophilicity of the substituent, in the order of I > Br > Cl > H,
NO2 > F. For example, the enzymatic potency was enhanced
4- and 7-fold respectively by simple 4-bromo or 4-iodo substitu-
tion. Neither amide 16a nor sulfonamide 16j is improved over 5b
in terms of HDAC1 potency. Their cellular activities are even worse,
probably due to their poor physicochemical properties. Lipophilic
tert-butyl group (5j) is well tolerated at position 4. Rigid pyrimi-
dine (16c) can maintain the enzymatic potency, but has weaker
cellular activity.
Disubstituted phenyls (5m, 22a–f, Table 3) were also investi-
gated. Substitutions at both 2 and 6 positions (5m) are not good
for enzymatic potency. 4-Amino substituted compounds (22a–f)
can maintain or slightly enhance enzymatic potency, but less bulky
(22a) or flexible basic chains (22e–f) are optimal for both enzy-
matic and cellular activities. Compound 19 is the most potent
disubstituted analogue of 5b in Table 3. Compared to 5f, the 3-ami-
no group of 19 slightly improves both the enzymatic and cellular
potency. Compounds 25a–g are cyclic analogs of 22e–f, but there
are no significant changes in their HDAC1 potencies. The benzimid-
azole ring is basic, and not particularly lipophilic (as in an all-car-
bon ring like 5n) hence solubility is good. Despite lower solubility,
compound 5n is the most potent fused ring analog of 5b. This con-
firms that lipophilicity is important for the CAP group.
The above studies have demonstrated that the potency of these
acylurea connected straight chain hydroxamates can be improved
by modifying the CAP region of 5b, compounds 5g, 5i, 5n, and 19
are representative examples with low nanomolar HDAC1 IC50 val-
ues. We also assessed the relationship between enzymatic and cel-
lular potency. In general, the cellular potency pIC50 (COLO205)
correlates significantly (p = 0.017, n = 32) with the enzymatic po-
tency pIC50 (HDAC1) although linear regression is not excellent
(Fig. S1, Supplementary data). This is not surprising due to the piv-
otal role HDAC1 plays in cell proliferation and survival.
Compounds with N-alkylated side chains showed much
reduced HDAC potency (9a–c vs 5b; 9d–e vs 5c). It seems that
these branched groups are not well tolerated probably due to unfa-
vorable interactions at the binding pocket rim region. Remiszewski
et al. reported that potency of urea connected straight chain
hydroxamates also reduced about threefold after N-methylation
of the urea moiety.16a
These compounds in Table 1 are much weaker inhibitors of
HDAC8 enzyme activity compared to HDAC1. As HDAC1 plays an
important role in cell proliferation,2 the HDAC1 assay was selected
as the only HDAC isoform for SAR studies.
HDAC isoform profiling15 work confirmed that compounds 5b
and 5g are pan-HDAC inhibitors (Table 3). Compound 5b shares a
similar profile as SAHA,17 but it is more potent against HDAC1
and HDAC618 than the latter. Compound 5g is much more potent
against all the isoforms except HDAC8. Compounds 5b and 5g
are relatively poor HDAC8 inhibitors, because they share the simi-
lar straight alkyl chain linker as SAHA. The linker region is more in-
volved in direct interactions with the hydrophobic pocket of
HDAC8, trichostatin A has a bulkier linker and exhibits better
Compound 5b with m = 5 is more potent than SAHA in the
HDAC1 assay and was selected for further modification. The
mono-substituted benzoylureas (5e–l, 16a–c, Table 2) were made
according to Scheme 3. A preference for 4-substituted phenyls

3318
H. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3314–3321
Table 2
Biological activities of compounds with Ar being mono-substituted phenyl (5e–l, 16a–c), disubstituted phenyl (5m, 19, 22a–f), or fused aromatic ring (5n, 25a–g)
a
b
Compound
Ar
HDAC1 IC50 (nM)
COLO205 IC50 (lM)
5b
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
16a
16b
16c
19
Ph
4-F-Ph
4-Cl-Ph
4-Br-Ph
3-Br-Ph
4-I-Ph
4-tert-Bu-Ph
4-NO2-Ph
3-NO2-Ph
2,6-Dichloro-Ph
Naphthalene-2-yl
4-(PhCH2CONH)-Ph
4-(PhSO2NH)-Ph
4-(Pyrimidin-5-yl)-Ph
3-NH2–4-Cl-Ph
53 17
2.21 0.41
2.55 0.21
1.60 0.14
0.87 0.49
3.15 0.21
0.87 0.19
2.35 0.07
2.31 0.09
2.80 0.00
2.95c
2.15 0.21
13.2 5.3
>100
10.0 2.9
1.25 0.21
115
26
13
60
8
24
45
92
7
1
5
4
1
1
2
1
185 21
6
44
97
26
11
1
6
5
2
1
22a
22b
20
46
1
6
3.45 1.48
3.25 0.64
22c
22d
43 10
11.5 2.2
12.0 1.4
55
2
22e
22f
22
20
3
1
2.42 0.43
1.70 0.28
25a
25
3
9.70 0.14
25b
25c
25d
20
49
21
2
2
1
3.75 1.77
19.5 0.7
5.10 0.71
25e
20
1
3.70 0.71

H. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3314–3321
3319
M)
Table 2 (continued)
a
b
Compound
Ar
HDAC1 IC50 (nM)
COLO205 IC50
(l
25f
34
22
1
0
5.38 0.10
25g
2.91 1.26
a,b,c
See footnote of Table 1.
Table 3
Table 5
Profiling of selected compounds
Compounds 5b, 5g, and SAHA are pan-HDAC inhibitors
HDACa
Kib (nM)
5g
Compound
Solubilitya
M)
Solubilityb
g/mL)
Log Dc
c log Pd
PSAe
(
l
(l
5b
16
44
23
20
SAHA
5b
5g
177
51
28
526
15
0.75
1.72
NT
NT
NT
NT
NT
1.04
1.07
1.86
2.11
0.65
1.09
3.45
2.97
1.84
108
108
137
133
169
129
129
78
1
2
3
4
5
6
7
8
9
2
2
5
4
8
12
8
6
13
8
2
1
3
1
2
0
5
63 13
40
29
16
33 14
18
126 60
205 35
65 26
50
36
4
9
1
16a
16c
22e
25f
25g
SAHA
NT
NT
NT
NT
NT
166
17
>205
>250
>211
>250
46 14
7
2
5
NTc
31
252 25
315 48
a
b
c
55
40
39
9
9
6
6
9
9
2
2
2
Kinetic solubility (pH 7).
Thermodynamic solubility (pH 7.4).
Determined in n-octanol/sodium phosphate buffer (pH 7.4).
c log P (octanol/water) and
10
11
7
6
d
e
a
b
c
HDAC isoenzymes.
PSA (polar surface area) were calculated using MOE 2008.10, Chemical Com-
Dissociation constant.15
NT: not tested.
puting Group.
dependent manner (Fig. S2, Supplementary data). In one of our
preliminary studies on histone acetylation pharmacodynamics,
compound 5b and SAHA were dosed to COLO205 tumor-bearing
mice at 160 mg/kg via ip injection, and both compounds demon-
strated hyperacetylation of histone H3 and H4 in tumor tissues
at 1.5 h time point, but compound 5b seemed more efficient
(Fig. S3, Supplementary data).
Kinetic solubility of selected compounds (Table 5) was assessed.
Compounds 5b and compounds with protonable basic centers at
physiological pH (22e, 25f–g) showed relatively high solubility.
Compound 5b was further assessed on its thermodynamic solubil-
inhibitory effect on HDAC8 compared to SAHA.19 Compounds 16a–
c, 19, 22a–f, and 25a–g have structurally diverse CAP groups, hence
their HDAC isoform inhibitory profiles may be expected to be dif-
ferent from those of 5b and 5g.
Compounds with representative Ar groups were selected for fur-
ther evaluation in cellular assays. Broad activity against human tu-
mor cell lines, such as HCT116 (colon cancer), A2780 (ovarian
cancer) and PC3 (prostate cancer) was encouraging (Table 4). Com-
pounds 5b was also further profiled against more human tumor cell
lines, for example, NCI-H522 (non-small cell lung cancer,
IC50 = 0.50 0.31
IC50 = 0.46 M). As comparison, SAHA showed IC50 = 1.28 0.35
and 0.65 0.36 M for these two cell lines, respectively.
lM), Ramos (human Burkitt’s lymphoma,
ity in pH 7.4 buffer, the value (526
lg/mL or 1.8 mM) is higher than
l
lM
that of SAHA (166 g/mL, 0.63 mM). SAHA has a reported value of
l
l
0.1 mg/mL.4 Compound 5b has a measured log D = 0.75, which is
more polar than SAHA (log D = 1.04), thus more soluble.
In vitro metabolic stability (t1/2) of selected compounds assessed
in different species liver microsomes is presented in Table 6. In gen-
eral, these compounds showed good stability (t1/2 >30 min) in
human liver microsomal assay except compound 25f which has in-
creased liability due to its lipophilicity (c log P). The metabolic sta-
bility of 5b was comparable with that of SAHA in human, rat and
mouse liver microsomes.
A hallmark of HDAC inhibition is the increase in the acetylation
level of histones.3,20 Histone acetylation, including H3, H4 and H2A
can be detected by Western immuno-blotting. The mechanism of
action of compounds 5b–c, 5g, 5k, 9b, 16a, 16c, 22e, and 25f–g
was confirmed in vitro at 10 lM as evidenced by hyperacetylation
of histone H3 in COLO205 cells, other compounds were not tested.
Compound 5b also increased the level of both acylated histone
33,20 and
a
-tubulin,18 and up-regulated cyclin-dependant kinase
inhibitor p2121 protein expression in HCT116 cells in a dose-
Table 6
Liver microsomal stability (t1/2, min) of selected compounds
Table 4
Cellular IC50
Compound
Human
Rat
Mouse
>30
(l
M) of selected compounds
5b
5g
>60
>60
>30
>30
>60
53 10
NT
NT
NT
Compound
HCT116
A2780
PC3
31
3
5b
5g
22e
25g
SAHA
1.58a
0.70 0.06
0.45 0.05
0.99 0.16
1.35 0.07
1.62 0.47
0.74 0.21
0.66 0.07
0.92 0.20
1.25 0.12
1.21 0.85
16c
22e
25f
25g
SAHA
NT
NT
NT
NT
>30
0.60a
1.33a
21
2
3.52a
>30
>60
NT
>60
1.43 0.29
a
Values obtained from only one triplicate experiment.
NT: not tested.

3320
H. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3314–3321
The permeability of 5b was also assessed in Caco-2 cells. The
In summary, we have designed and synthesized a series of acylu-
rea connected straight chain hydroxamates, established structure–
activity relationships for the length of linear chain linker and substi-
tutions on the benzoylurea group. Compounds 5g, 5i, 5n, and 19
showed 10–20-fold enhanced HDAC1 potency compared to SAHA. A
structurally simple and representative compound, 5b (SB207), has
shown very good drug-like properties, and has demonstrated good
antitumor efficacy in both PC3 and HCT116 xenograft models. These
results encourage us to further evaluate and develop this series as
well as to better understand the HDAC isoform inhibitory profiles of
those with complex CAP groups such as substituted benzimidazole,
naphthalene, indole, and other polycyclic ring systems.
rate of transport (PappAtoB) of 5b was 3.12 Â 10À6 cm/s, which
was higher than that reported for SAHA (1.70 Â 10À6 cm/s).4
The results of PK studies on compound 5b, SAHA, LBH-589 and
PXD-101 in nude mice have been published.22 After a single iv dose
of 10 mg/kg injection, the systemic plasma clearance of 5b (5.03
L/h/kg) was lower than that of SAHA (6.73 L/h/kg) as well as the
hepatic blood flow of 5.4 L/h/kg in mice. The estimated steady-
state volume of distribution (Vss) of 5b (2.16 L/kg) was greater
than that of SAHA (0.81 L/kg). The estimated elimination half-life
for 5b was 1.54 h, which was longer than that of SAHA (0.38 h).
After an oral single dose of 50 mg/kg, the AUC and Cmax of 5b were
9 and 12 times greater than those of SAHA, respectively. Com-
pound 5b has an oral bioavailability in mouse of 62%, which is
much higher than that of SAHA (8.3%).
Acknowledgments
Compound 5b was selected for evaluation in vivo to demon-
strate whether the acylurea connected hydroxamate is efficacious
and has any advantages over SAHA. As SAHA had demonstrated
efficacy in CWR22 human prostate xenograft in nude mice,3 our
in vivo xenograft studies were also initiated in a human prostate
cancer PC3 tumor-bearing nude mice.23 Compound 5b and SAHA
were given daily via intraperitoneal (ip) injection for 21 days at
doses of 160, 200, and 250 mg/kg (5b only) (Fig. S4, Supplementary
data). Compound 5b demonstrated significant antitumor activity
compared to vehicle control. Tumor growth inhibition (TGI) on
day 22 was 73% (200 mg/kg, maximum tolerated dose (MTD),
p <0.01) and 57% (160 mg/kg, p <0.05), but 250 mg/kg dose was
toxic with 2/9 treatment-related deaths on day 4. SAHA was used
as a positive control and showed moderate activity at 160 mg/kg
(MTD) with TGI = 35% (p > 0.05), but the 200 mg/kg dose was toxic
in this experiment, with 2/9 treatment-related deaths on days 16
and 22, respectively.
Compound 5b was also given orally once per day to HCT116
tumor-bearing mice (Fig. 3), with TGI on day 21 of 80% (100 mg/
kg, p < 0.01), but it was not tolerated at 200 mg/kg, with 2/10 treat-
ment-related deaths on days 6 and 10, respectively. SAHA showed
moderate activity at 200 mg/kg with TGI = 50% (p < 0.01) in this
experiment. Compared to SAHA, compound 5b can achieve better
efficacies at the same or lower dose level.
We thank the S*BIO sample management team for preparing
sample solutions for assays, the PKDM department for DMPK stud-
ies on 5b, Dr Ignacio Segarra for early animal work on 5b, and Dr.
Brian W. Dymock for critical review of the manuscript.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.041.
References and notes
1. (a) De Ruijter, A. J. M.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van Kuilenburg, A.
B. P. Biochem. J. 2003, 370, 737; (b) Michan, S.; Sinclair, D. Biochem. J. 2007, 404, 1.
2. (a) Curtin, M.; Glaser, K. Curr. Med. Chem. 2003, 10, 2373; (b) Glaser, K. B.; Li, J.;
Staver, M. J.; Wei, R.-Q.; Albert, D. H.; Davidsen, S. K. Biochem. Biophys. Res.
Commun. 2003, 310, 529.
3. Butler, L. M.; Agus, D. B.; Scher, H. I.; Higgins, B.; Rose, A.; Cordon-Cardo, C.;
Thaler, H. T.; Rifkind, R. A.; Marks, P. A.; Richon, V. M. Cancer Res. 2000, 60,
5165.
4. FDA approval and New Drug Application (NDA) documents for Zolinza™
(SAHA,
vorinostat):
http://www.accessdata.fda.gov/drugsatfda_docs/nda/
2006/021991s000_ZolinzaTOC.cfm.
5. (a) Saito, A.; Yamashita, T.; Mariko, Y.; Nosaka, Y.; Tsuchiya, K.; Ando, T.;
Suzuki, T.; Tsuruo, T.; Nakanishi, O. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4592;
(b) Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukasawa, N.; Saito, A.; Mariko, Y.;
Yamashita, T.; Nakanishi, O. J. Med. Chem. 1999, 42, 3001.
6. Plumb, J. A.; Finn, P. W.; Williams, R. J.; Bandara, M. J.; Romero, M. R.; Watkins,
C. J.; La Thangue, N. B.; Brown, R. Mol. Cancer Ther. 2003, 2, 721.
7. For review of chemical types and SAR of HDAC inhibitors, see: (a) Paris, M.;
Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505; (b) Miller,
T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem. 2003, 46, 5097; (c) Xie, A.; Liao, C.;
Li, Z.; Ning, Z.; Hu, W.; Lu, X.; Shi, L.; Zhou, J. Curr. Med. Chem. – Anti Cancer
Agents 2004, 4, 273.
8. Lim, Z.-Y.; Wang, H.; Zhou, Y. WO 2005040101A1, 2005.
9. Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.; Rifkind, R. A.; Marks, P. A.;
Breslow, R.; Pavletich, N. P. Nature 1999, 401, 188.
10. Wang, D.-F.; Helquist, P.; Wiech, N. L.; Wiest, O. J. Med. Chem. 2005, 48, 6936.
11. Barlaam, B.; Hamon, A.; Maudet, M. Tetrahedron Lett. 1998, 39, 7865.
12. Hill, A. J.; Degnan, W. M. J. Am. Chem. Soc. 1940, 62, 1595.
13. Caubere, P.; Den, M.-Z.; Lecolier, S.; Senet, J. P. Eur. Pat. Appl. EP 334720 B1,
1992.
According to the published pharmacokinetic study results of
5b,22 when dosed orally at 50 mg/kg, the Cmax (22.4
l
M) was well
M), HCT116 (1.58 M),
M). Plasma concentration was maintained above
above the IC50 levels for HDAC1 (0.053
and PC3 (0.74
l
l
l
the HDAC1 IC50 for about 22 h, above HCT116 and PC3 IC50s for
2.2 and 4.3 h, respectively. Given that the doses in the PC3 and
HCT116 xenograft experiments were much higher than this PK
study, it is not surprising that 5b can achieve such good efficacy
as compared to SAHA in both xenograft studies.
14. Wang, H.; Yu, N.; Song, H.; Chen, D.; Zou, Y.; Deng, W.; Lye, P. L.; Chang, J.; Ng,
M.; Blanchard, S.; Sun, E. T.; Sangthongpitag, K.; Wang, X.; Goh, K. C.; Wu, X.;
Khng, H. H.; Fang, L.; Goh, S. K.; Ong, W. C.; Bonday, Z.; Stunkel, W.; Poulsen, A.;
Entzeroth, M. Bioorg. Med. Chem. Lett. 2009, 19, 1403.
15. For protocols used for HDAC enzymatic assays, see Ref. 14. Fluor de Lys-HDAC8
substrate (KI-178) was used for HDAC8 IC50 determination. Fluor de Lys™
substrate (KI-104) was used for HDAC1 IC50 and HDAC1-11 isoform Ki
determination: Ki = IC50/(1 + ([substrate]/Km)). Anti-proliferations assays:
similar protocols used as published in Ref. 14.
16. (a) Remiszewski, S. W.; Sambucetti, L. C.; Atadja, P.; Bair, K. W.; Cornell, W. D.;
Green, M. A.; Howell, K. L.; Jung, M.; Kwon, P.; Trogani, N.; Walker, H. J. Med.
Chem. 2002, 45, 753; (b) Jung, M.; Brosch, G.; Kölle, D.; Scherf, H.; Gerhäuser, C.;
Loid, P. J. Med. Chem. 1999, 42, 4669.
17. Khan, N.; Jeffers, M.; Kumar, S.; Hackett, C.; Boldog, F.; Khramtsov, N.; Qian, X.;
Mills, E.; Berghs, S. C.; Carey, N.; Finn, P. W.; Collins, L. S.; Tumber, A.; Ritchie, J.
W.; Jensen, P. B.; Lichenstein, H. S.; Sehested, M. Biochem. J. 2008, 409, 581.
18. Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. M.; Schreiber, S. L. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 4389.
19. Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E. C.; Brunetti, M.; Renzoni, D.;
Chakravarty, P.; Paolini, C.; De Francesco, R.; Gallinari, P.; Steinkühler, C.; Di
Marco, S. Proc. Natl. Acad. Sci. U.S.A. 2004, 19, 15064.
Figure 3. Antitumor activity of 5b and SAHA in HCT116 xenograft model. n = 10
female BALB/c nude mice (Animal Resources Centre, Australia) per dose group, po,
qd  21 days.

H. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3314–3321
3321
20. 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.
21. (a) Richon, V. M.; Sandhoff, T. W.; Rifkind, R. A.; Marks, P. A. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 10014; (b) Gui, C. Y.; Ngo, L.; Xu, W. S.; Richon, V. M.; Marks, P.
A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1241.
22. Yeo, P.; Liu, X.; Goh, E.; New, L. S.; Zeng, P.; Wu, X.; Venkatesh, P.; Kantharaj, E.
Biomed. Chromatogr. 2007, 21, 184.
23. Xenograft studies. Similar protocols used as published in Ref. 14. Compounds
were dissolved in 10% N,N-dimethylacetamide (DMA)/10% Cremophore/80%
water for ip route or 0.5% methylcellulose/0.1%Tween 80 (MC/TW) for oral
administration. Statistic analysis was done with one-way ANOVA followed by
Dunnett’s Multiple Comparison test (Prism 4.0).