Thiosemicarbazones of formyl benzoic acids as novel potent inhibitors of estrone sulfatase

Article abstract of DOI:10.1021/jm0611657

Thiosemicarbazones of the microbial metabolite madurahydroxylactone, a polysubstituted benzo[a]-naphthacenequinone, have been previously reported by us as potent nonsteroidal inhibitors of the enzyme estrone sulfatase (cyclohexylthiosemicarbazone 1, IC

Full text of DOI:10.1021/jm0611657

J. Med. Chem. 2007, 50, 3661-3666
3661
Thiosemicarbazones of Formyl Benzoic Acids as Novel Potent Inhibitors of Estrone Sulfatase†
Peter Ju¨tten, Winfried Schumann, Albert Ha¨rtl, Hans-Martin Dahse,* and Udo Gra¨fe
Leibniz Institute for Natural Products Research and Infection Biology, HKI, Beutenbergstrasse 11a, 07745 Jena, Germany
ReceiVed October 7, 2006
Thiosemicarbazones of the microbial metabolite madurahydroxylactone, a polysubstituted benzo[a]-
naphthacenequinone, have been previously reported by us as potent nonsteroidal inhibitors of the enzyme
estrone sulfatase (cyclohexylthiosemicarbazone 1, IC50 0.46 µM). The active pharmacophore of 1 has now
been identified to be 2-formyl-6-hydroxybenzoic acid cyclohexylthiosemicarbazone (25, IC50 4.2 µM). The
active partial structure was derivatized in the search for novel agents against hormone-dependent breast
cancer. Further substantial increases in activity were achieved by reversal of functional groups leading to
the cyclohexylthiosemicarbazones of 5-formylsalicylic acid (35, IC50 0.05 µM) and 3-formylsalicylic acid
(34, IC50 0.15 µM) as the most potent analogues identified to date. Both compounds were shown to be
noncompetitive inhibitors of estrone sulfatase with Ki values of 0.13 µM and 0.12 µM, respectively. The
compounds showed low acute toxicity in the hen’s fertile egg screening test.
Introduction
We have recently reported a novel type of nonsteroidal
sulfatase inhibitor. Thiosemicarbazone derivatives of the natural
compound madurahydroxylactone (MHL), a polysubstituted
benzo[a]naphthacenequinone, are highly potent but not estro-
genic and show low acute toxicity.32 Their structure-activity
relationships have been disclosed, and the cyclohexylthiosemi-
carbazone (1, Figure 1) was shown to be the most potent
inhibitor (IC50 0.46 µM).
While we were pleased with our initial results, there were
significant limitations and we wondered how further improve-
ments could be achieved. Madurahydroxylactone is a highly
complex benzo[a]naphthacenequinone. A laborious total syn-
thesis was rejected, particularly as a promising route is far from
being obvious. Moreover, because of their high affinity for
protein binding and their molecular weight (≈650 g mol-1), it
seemed doubtful whether the madurahydroxylactone thiosemi-
carbazones could be attractive as drug candidates themselves.
Hence, we focused our further studies on the development of
smaller molecules as inhibitors. Our approach was based on
the hypothesis that A-ring analogues of 1 (see Figure 1) might
be a promising lead structure. The present paper describes our
studies into a series of thiosemicarbazones of formylbenzoic
acids aiming at compounds with improved in Vitro potency
which might be expected to possess enhanced in ViVo properties.
Structure-activity relationships are discussed in detail.
Chemistry. The synthetic approaches to the target molecules
are unexceptional and need no detailed description. All of the
thiosemicarbazones and related semicarbazones in this study
(Tables 1 and 2) were prepared by standard methods as outlined
in Scheme 1 for compound 13. With the exception of the di-
ortho-substituted compounds 29 and 30, no evidence for the
formation of E-Z isomers mixture was noted, only the E
isomers being obtained. The starting materials, for the synthesis
of the thiosemicarbazones shown in Tables 1 and 2, were
obtained from commercial sources, synthesized by literature
methods (see Experimental Section), or were made as described
below.
Breast cancer is the leading site of new cancer cases in
women, and the second leading cause of cancer death among
women. In the countries of the European Union, one in twelve
women will be at risk of having breast cancer during her life.
Over one-third of all breast cancers require stimulation by
estrogens for optimal growth. The estrogen 17â-estradiol has a
pivotal role in the growth and maintenance of mamma carci-
noma in postmenopausal women.1 The source of estrogens in
premenopausal women are predominantly the ovaries, but after
the menopause, enzymatic synthesis of estradiol via sulfatase,
aromatase, and 17â-hydroxysteroid dehydrogenase in the tumor
itself contributes to the high estrogen levels measured in breast
cancer tissue.
Much effort has been devoted to the development of inhibitors
of the aromatase pathway which converts the androgen precursor
androstenedione to estrone. However, estrone production is not
completely abolished by aromatase inhibitors.2 Furthermore, at
physiological concentrations of estrone sulfate and androstene-
dione, productions of estrone from estrone sulfatase by hormon-
dependent breast tumor is ten times faster than the rate of estrone
production via the aromatase pathway.3 Consequently, the
control of estrone sulfatase inside the cancer cell with specific
inhibitors may have considerable advantage over single therapy
with aromatase inhibitors.4-7
Promising initial results in the blocking of this enzyme with
steroidal inhibitors like estrone 3-O-sulfamate (EMATEa),8
however, have been hampered by their strong estrogenic side
effect.9,10 There is currently increasing interest in developing a
potent nonsteroidal inhibitor for treatment of endocrine-
responsive breast cancer. A series of phenol sulfamate esters,
like 4-methylcoumarin 7-O-sulfamate (COUMATE), proved to
be inhibitors of the sulfatase,11-29 thus demonstrating that the
steroid nucleus as a whole is unnecessary for enzyme inhibi-
tion.30,31
† This paper is dedicated to the memory of Prof. Dr. U. Gra¨fe who
unexpectedly died during the course of this study.
The tetrazole 40 was prepared readily by reaction of 3-cy-
anobenzaldehyde, protected as the diethyl acetal, with tributyltin
azide (Scheme 1). The tributyltin group was removed employing
sodium hydroxide, and the product was isolated most conve-
niently as the N-trityl derivative 39. Subsequent deprotection
* Corresponding author. Tel: +49-3641-65-6878. Fax: +49-3641-65-
6879. E-mail: hans-martin.dahse@hki-jena.de.
a Abbreviations: MHL, madurahydroxylacton; EMATE, estrone 3-O-
sulfamate; COUMATE, 4-methylcoumarin 7-O-sulfamate.
10.1021/jm0611657 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/20/2007

3662 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Ju¨tten et al.
Figure 1. MHL cyclohexylthiosemicarbazone (1) and its partial structure (A-ring analogue).
Table 1. Estrone Sulfatase Inhibition Data for 2-24
compd
R1
R2
R3
R4
X
R5
IC50 [µM]
pKa
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
COOH
H
H
OH
H
H
COOH
H
H
OH
H
COOH
H
H
H
COOH
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
S
S
S
S
S
S
O
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
c-hexyl
n-butyl
n-hexyl
c-propyl
c-pentyl
c-heptyl
c-octyl
phenyl
4.0
1.3
24
8.0
4.0
6.4
5.4
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
9.8
10.2
H
H
2.3
>100
>100
13
COOH
COOCH3
SO3Na
B(OH)2
H
COOH
COOH
COOH
COOH
COOH
COOH
COOH
H
H
H
H
4.0
-
7.7
5.2
>100
0.81
NIa
NIa
>100
43
13
60
>100
tetrazol-5-yl
H
H
H
H
H
H
H
COOH
COOH
COOH
COOH
H
H
H
H
n-butyl
n-hexyl
c-pentyl
c-heptyl
58
12
20
4.0
H
H
H
a NI, no inhibition.
Table 2. Estrone Sulfatase Inhibition Data for Cyclohexylthiosemicarbazones 25-38 and Reference Compounds
compd
R1
R2
OH
OCH3
OH
OCH3
OH
OCH3
OH
R3
R4
R5
IC50 [µM]
pKa
25
26
27
28
29
30
31
32
33
34
35
36
37
38
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
OH
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
H
4.2
>100
0.73
>100
2.5
4.3
H
35
20
4.5
OCH3
OH
OCH3
H
OH
H
OH
OCH3
COOH
COOH
H
>100
0.15
0.05
0.25
5.0
3.8
4.2
5.8
H
OH
OH
COOH
COOEt
H
H
OH
H
H
SO3Na
H
4.6
COUMATE
EMATE
-
-
-
-
-
-
-
-
-
-
2.6
0.08
effected by hydrochloric acid hydrolysis gave the target
molecule 40.
The phthalaldehydic acids 45-48, which exist as an intramo-
lecular hemiacetal, were prepared from the corresponding methyl
ethers (41-44) by treatment with boron tribromide (Scheme
2). At -78 °C, boron tribromide regioselectively cleaved the
methyl ether ortho to the carboxyl group. The catechol 49 was
obtained from the guajacol 48 by dealkylation with aluminum
trichloride. The details of these cleavage procedures are
described in the Experimental Section.

Inhibitors of Estrone Sulfatase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3663
Table 3. Kinetic Parameters and Acute Toxicity of 34-36 and 1
than the 4-isomer 4. (4) The inhibitory activity of compounds
5, 6, and 7 was gradually increased as the position of the
hydroxyl group was moved from ortho to para, with IC50 values
of 8.0, 4.0, and 2.3 µM, respectively.
Having in this fashion identified the formylbenzoic acid
thiosemicarbazones 2 and 3 as potent estrone sulfatase inhibitors,
it was of interest to examine what structural features were
required for their activity. Esterification of the carboxyl function
caused a decrease in inhibitory activity. A similar result was
obtained later in a second series of compounds (cf. 10 with 2,
Table 1, and 37 with 36, Table 2). This suggests that the acid
group plays an important role in its interaction with the enzyme.
acute tox.
compd
IC50 [µM]
Km [µM]
Ki [µM]
[mg/kg]
34
35
36
1
0.15
0.05
0.25
0.46
29
31
0.12
0.13
>50
>50
>100
>50
NDa
27
NDa
0.46
a ND, not determined.
Scheme 1a
Replacement of the sulfur in 3 by oxygen led to the
corresponding semicarbazone 8. Compound 8 showed a dra-
matical drop in activity (IC50 >100 µM). This agrees with our
previously reported finding that the thiosemicarbazone is
mandatory for potent inhibition of estrone sulfatase.32 Substitu-
tion of the imine hydrogen with the more bulky methyl group
produced the ketone derivative 9 which was inactive as an
inhibitor at up to 100 µM. This argues for an interaction with
the enzyme which is sensitive to steric effects.
Having established the importance of the acid group and its
proper position, we concluded that further study of this key
substituent was well warranted. In an attempt to produce
analogues of greater potency, the carboxylic acid groups of 2
and 3 were replaced by some isosteres. The acid mimics would
allow us to vary pKa values as well as the geometry and charge
distribution about the acid center. pKa values of selected
compounds are listed in Tables 1 and 2. Since both carboxylic
acids (compounds 2 and 3) and phenols (5, 6, and 7) are
inhibitors of estrone sulfatase, there is obviously no correlation
between the acidity of the aromatic substituents and activity. It
is, however, known that differences in charge distribution and
geometry at the acid center can have profound effects.33,34
The most potent analogue arising from this effort was
tetrazole 13, which inhibited estrone sulfatase with an IC50 of
0.81 µM. This made the tetrazole the first compound with an
IC50 below 1 µM. Tetrazoles are of particular interest to the
medicinal chemist since they probably constitute the most
commonly used bioisostere of the carboxyl group. The tetrazole
is an “azole-type” carboxylic acid isostere and has a planar
geometry. Tetrazole 13 has a pKa value of 5.2 similar to that of
the parent carboxylic acid 3 (pKa 5.4).
a Reagents: (a) Bu3SnN3, toluene, reflux; (b) TrtCl, NaOH, H2O; (c)
HCl, H2O, THF; (d) NaOH, H2O; (e) cyclohexylthiosemicarbazide, EtOH,
reflux.
Biological Testing. Enzyme activity was determined using
a microsomal preparation from human placenta. The sulfatase
activities in the presence of increasing amounts of inhibitor
concentration were determined to evaluate the relative potency
of the inhibitors. A negative control (absence of inhibitors) was
run simultaneously. Some standard compounds (EMATE,
COUMATE) were tested in our assay system for comparison
with literature data. Our results were generally consistent with
data obtained by other groups. The IC50 data are shown in Tables
1 and 2.
We performed kinetic analyses of 34 and 35, some of the
most active compounds. The analysis of the results following
Lineweaver-Burk plot representations (data not shown) indi-
cates that the compounds are noncompetitive inhibitors of human
placental estrone sulfatase (Table 3). This behavior is in
agreement with that of MHL cyclohexylthiosemicarbazone 1.32
The sulfonic acid 11 had an IC50 value of 4.0 µM which
compared favorably with the parent carboxylic acid. On the other
hand, in the hydroxyl-substituted series (Table 2), the sulfonic
acid 38 reduced activity by a factor of 18 when compared to
carboxylic acid 36. These results indicate that, in certain
circumstances, an acid with tetrahedral geometry, in which the
charge of the anion is spread over three oxygen atoms, can be
tolerated by the sulfatase enzyme. The boronic acid 12 was
essentially inactive. Compound 12 was a weaker acid, but more
important its acidity is based on hydroxyl ion acceptance rather
than proton donation.
In the following compound series, the alkyl group in the
thiosemicarbazono moiety was modified. The cycloalkylthi-
osemicarbazones (2, 3, 16-19, 23-24) inhibited the sulfatase
activity completely and concentration-dependent with IC50s in
the range of 1.3 (3) to 60 µM (19). These values were not as
low as those reported for the madurahydroxylactone cycloalkyl-
thiosemicarbazones32 but the relative differences agree. Aliphatic
chains (14, 15, 21, 22) and aromatic substituents (20) gave rise
to compounds of lower activity. This roughly parallels the
madurahydroxylactone thiosemicarbazoness described previ-
Results and Discussion
With the hypothesis in mind that A-ring analogues of 1 might
be potent nonsteroidal estrone sulfatase inhibitors, we started a
preliminary screening for a primitive definition of the minimal
structural prerequisites. A set of ortho-, meta-, and para-
substituted benzaldehyde cyclohexylthiosemicarbazones bearing
methyl, fluorine, chlorine, bromine, hydroxy, methoxy, amino,
acetamido, nitro, carboxy, and cyano groups were prepared and
screened as inhibitors. The results obtained from these SAR
studies are partly shown in Table 1 and can be summarized as
follows: (1) Benzaldehyde cyclohexylthiosemicarbazone was
a very poor inhibitor (IC50 >100 µM). Monosubstitution
generally increased estrone sulfatase inhibition leading to
compounds with moderate activity. (2) The introduction of
certain substituents led to potent inhibitors (IC50s in the range
from 1.3 to 24 µM), the most potent compounds bearing a
carboxy (2-4) or a hydroxy group (5-7). Both functional
groups are found in the “natural” parent compound 1! (3) The
cyclohexylthiosemicarbazone of phthalaldehydic acid (2) and
isophthalaldehydic acid (3) were about 10 times more active

3664 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Ju¨tten et al.
Scheme 2a
a Reagents: (a) BBr3, CH2Cl2, -78 °C; (b) AlCl3, CH2Cl2.
ously.32 Surprisingly, the butyl and hexyl derivatives 14 and
15 were essentially inactive. The data suggested a hydrophobic
interaction in which a cycloalkyl of the proper size fits snugly.
Therefore, the cyclohexylthiosemicarbazone was selected for
all our investigations.
C-N bonds in the thiourea moiety cause the thiosemicarbazones
1
to be conformationally rather rigid molecules.35 The H NMR
signals of the phthalaldehydic derivatives 25, 27, and 31, as
well as the isophthalaldehydic thiosemicarbazones 3, 34, 35,
and 36, could be assigned unequivocally by a combination of
COSY, HMBC, and HMQC experiments. We hoped that the
1H-1H-NOESY spectra might give some insight into conforma-
tion and configuration of these sulfatase inhibitors. While the
NOESY spectra of 3 and 27 supported strong evidence that these
molecules adopt an extended coplanar configuration, E relative
to the aldimine bond and Z,Z regarding the thiourea, the other
compounds, unfortunately, did not show all relevant cross-peaks.
Further modular variations of the thiosemicarbazones completing
the preliminary structure-activity study should form the basis
of a molecular approach toward more potent nonsteroidal estrone
sulfatase inhibitors.
The following studies were undertaken to investigate the
pharmacological characteristics of the most potent compounds
34, 35, and 36 as new prototypic estrone sulfatase inhibitors.
We expected that the compounds are not active-site directed
inhibitors. In fact, in Vitro kinetic studies revealed 34 and 35 to
be noncompetitive inhibitors of estrone sulfatase. The kinetic
parameters obtained from Lineweaver-Burk plots are outlined
in Table 3. Thus, the mechanism of inhibition is different from
that of the phenol sulfamates, which compete with estrone
sulfate, but is similar to that of MHL cyclohexylthiosemicar-
bazone (1, see Table 3).32
MHL cyclohexylthiosemicarbazone 1 (IC50 0.46 µM), having
at least one hydroxy group in the A-ring, was 8.7 times stronger
than 2. Therefore, a further study was subsequently undertaken
to evaluate effects of introducing hydroxyl groups and other
substituents in the compounds 2 and 3. The results of this study
are shown in Table 2. Salicylic acid derivative 25 represents a
synthetic partial structure closer to 1 than 2. It was disappointing
that hydroxy analogue 25 was only equipotent with compound
2. Increasing the hydrophobicity by introduction of a methyl
group, compounds 27 and 29, led to an increase in estrone
sulfatase inhibition. The methyl group in 6-formyl-2-hydroxy-
3-methylbenzoic acid cyclohexylthiosemicarbazone 27 (IC50
0.73 µM) enhances activity more than threefold compared to
the regioisomer 29. Replacement of the methyl group in 27 by
a methoxy subsitutent (31) reduced activity whereas the catechol
32 reestablished the inhibitory power. The IC50 values of the
methyl ether derivatives 26, 28, 30, and 33 were about 1 order
of magnitude higher than those of the phenolic analogues (25,
27, 29, and 31). Thus, the potency of all these compounds were
clearly dependent on the presence of a free hydroxy group.
By analogy to the relationship between ortho-carboxylic acid
2 and the meta-compound 3, we hypothesized that hydroxylated
isophthalaldehydic acid cyclohexylthiosemicarbazones might
well be more effective than 25 and 27. Indeed, this was
effectively the case. Salicylic acid derivative 34 enhanced the
sulfatase inhibition by 1 order of magnitude (IC50 0.15 µM). It
is interesting to note that the position of the hydroxyl group
plays an important role since the regioisomeric salicylate 35
(IC50 0.05 µM) is three times more active than 34. Moving the
hydroxy group meta to both the carboxylic acid and the
thiosemicarbazone (36) slightly reduces the potency. However,
compound 36 still inhibits fairly well compared to 3, which is
much less active. The data clearly indicate the great importance
of the hydroxy group in these compounds with respect to
sulfatase inhibition. This represents a major difference in SAR
between the isophthalaldehydic and phthalaldehydic acid series.
Compounds 34, 35, and 36 all possess much greater inhibitory
activity than COUMATE. 5-Formylsalicylic acid cyclohexy-
lthiosemicarbazone 35 was the most potent inhibitor of all the
compounds presented and inhibited estrone sulfatase even more
stronger than EMATE.
The acute toxicities of 34 and 36 were studied in the hen’s
fertile egg screening test (HEST). Fifteen-day old chick embryos
receive the test compound through the air cell and deaths were
measured at 72-96 h after the treatment. The LD50 of HEST is
strongly positively correlated to i.v. LD50 obtained in mice and
rodents.36 The acute toxicity of 34-36 determined in the hen’s
fertile egg screening test were >50 mg kg-1 and >100 mg kg-1
,
respectively (Table 3). The compounds showed no antibacterial
activity. They inhibited the growth of K-562 leukemia cells and
HeLa cervix carcinoma cells with IC50 of >50 µg mL-1
.
The compounds 34, 35, and 36 are relatively small molecules
capable of forming sodium salts of the carboxylic acid. The
sodium salts obtained by careful treament with 1 equiv of
sodium hydroxide are sufficiently water soluble (>0.1 g mL-1
)
to permit easy formulation for in ViVo tests of estrone sulfatase
inhibition.
Conclusions
The active pharmacophore of the nonsteroidal sulfatase
inhibitor MHL cyclohexylthiosemicarbazone (1) has now been
identified to be 2-formylbenzoic acid cyclohexylthiosemicar-
bazone (2). The active partial structure was derivatized to
improve the inhibitory power. Thiosemicarbazones of formyl-
salicylic acids exemplified by 25 and 5-formyl-2-hydroxyben-
zoic acid cyclohexylthiosemicarbazone (35) are a new class of
In an attempt to rationalize the structure-activity data
discussed above and presented in Tables 1 and 2, we assume
that multiple hydrogen bonding and dipolar interactions of the
aromatic acid unit and the alkylthiosemicarbazone region are
important features for binding with the enzyme estrone sulfatase.
The aldimine double bond and the restricted rotation about the

Inhibitors of Estrone Sulfatase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3665
Hz, 2 H), 8.09 (m, J ) 8.5 Hz, 1 H), 8.09 (s, 1 H), 11.50 (s, 1 H).
Anal. (C15H19N3O2S) C, H, N, S.
nonsteroidal estrone sulfatase inhibitors. The thiosemicarbazone
35 was characterized by an IC50 of 50 nM and possesses greater
inhibitory activity than EMATE and COUMATE. Compounds
25, 35, and analogues such as 34 and 36 can be easily
synthesized. Our studies revealed that the salicylic acid moiety,
an isophthalaldehydic acid motif, and the thiosemicarbazone
were all critical for high sulfatase inhibition. The carboxylic
acid moiety can be replaced by selected acid mimics, for
example, a tetrazol-5-yl, to give compounds of similar activity.
The most potent inhibitors show low acute toxicity and are not
cytotoxic. Compounds 34, 35, and 36 are therefore good lead
compounds for the development of potent novel agents against
hormone-dependent breast cancer.
3-(1H-Tetrazol-5-yl)-benzaldehyde Cyclohexylthiosemicar-
bazone (13). mp 228-229 °C; 1H NMR (DMSO-d6) δ 1.07-1.50
(m, 5 H), 1.60 (m, 1 H), 1.66-1.79 (m, 2 H), 1.82-1.95 (m, 2 H),
4.19 (m, 1 H), 7.65 (t, J ) 7.8 Hz, 1 H), 7.99-8.07 (m, 2 H), 8.11
(d, J ) 7.7 Hz, 1 H), 8.15 (s, 1 H, CHdN), 8.25 (br s, 1 H, H-2),
11.53 (s, 1 H, )N-NH). Anal. (C15H19N7S) C, H, N, S.
2-(4-Cyclohexylthiosemicarbazono)methyl-6-hydroxybenzo-
ic Acid (25). mp 197-199 °C; 1H NMR (DMSO-d6) δ 1.05-1.47
(m, 5 H), 1.53-1.78 (m, 3 H), 1.82-1.96 (m, 2 H), 4.10 (m, 1 H),
6.91 (m, 1 H, H-5), 7.24-7.31 (m, 2 H, H-3,4), 7.74 (d, J ) 8.4
Hz, 1 H, NH-cHex), 8.12 (s, 1 H, CHdN), 11.51 (s, 1 H, )N-
NH). Anal. (C15H19N3O3S) C, H, N, S.
6-(4-Cyclohexylthiosemicarbazono)methyl-2-hydroxy-3-me-
1
thylbenzoic Acid (27). mp 197-198 °C; H NMR (DMSO-d6) δ
Experimental Section
1.13 (m, 1 H), 1.27 (m, 2 H), 1.40 (m, 2 H), 1.59 (m, 1 H), 1.72
(m, 2 H), 1.87 (m, 2 H), 2.19 (s, 3 H, 3-CH3), 4.15 (m, 1 H), 7.29
(d, J ) 7.8 Hz, 1 H, H-4), 7.35 (d, J ) 7.8 Hz, 1 H, H-5), 7.80 (d,
J ) 8.5 Hz, 1 H, NH-cHex), 8.45 (s, 1 H, CHdN), 11.46 (s, 1 H,
dN-NH). Anal. (C16H21N3O3S) C, H, N, S.
Chemical Compounds and Synthetic Procedures. General
Information. The following substituted benzoic acids are available
commercially: phthalaldehydic acid, isophthalaldehydic acid, 2-acetyl-
benzoic acid, 2-formylbenzenesulfonic acid, 2-formylboronic acid,
3-formylsalicylic acid, and 5-formylsalicylic acid, as well as the
2-, 3-, and 4-hydroxybenzaldehydes. Minor modifications of
literature procedures furnished: 3-hydroxy-7-methoxy-3H-isoben-
zofuran-1-one (41),37 3-hydroxy-7-methoxy-6-methyl-3H-isoben-
zofuran-1-one (42),38 3-hydroxy-7-methoxy-4-methyl-3H-isoben-
zofuran-1-one (43),38 3-formyl-4-hydroxybenzoic acid,39 and 3-formyl-
4-hydroxybenzenesulfonic acid.40 The thiosemicarbazides and
semicarbazides are commercially available or were prepared by
literature procedures.41
Analytical thin layer chromatography (TLC) was performed on
precoated plates (Merck TLC aluminum sheets silica gel 60 F254).
Products and starting material were detected by viewing under UV
light (254 or 366 nm). Column chromatography was performed on
silica gel 60 (0.063-0.1 mm; Merck). Nuclear magnetic resonance
spectra (NMR) were obtained on a Bruker DPX 300 (1H, 300 MHz;
13C 75 MHz) and Bruker DRX 500 (1H, 500 MHz; 13C, 125 MHz)
instruments; chemical shifts (δ) are expressed in parts per million
(ppm) relative to the residual solvent peak. Electrospray mass
spectra (ESMS) were obtained with a VG Quattro mass spectrom-
eter. Melting points were taken on a Bu¨chi B-540 melting point
apparatus and are uncorrected. pKa measurements are obtained from
the titration curve applying the Kolthoff method. A 5-10 mg
amount of the test compound was dissolved in 20% aqueous DMF.
This was then titrated against 10 mM NaOH solution via a 5-mL
burette. Elemental analyses indicated by the symbols of the elements
were within (0.4% of the theoretical values. Solvents were dried
according to the standard procedures. Chemical yields were not
optimized.
3-(4-Cyclohexylthiosemicarbazono)methyl-2-hydroxybenzo-
ic Acid (34). mp 227-229 °C; 1H NMR (DMSO-d6) δ 1.02-1.52
(m, 5 H), 1.60 (m, 1 H), 1.65-1.79 (m, 2 H), 1.81-1.97 (m, 2 H),
4.18 (m, 1 H), 6.97 (t, J ) 7.8 Hz, 1 H, H-5), 7.84 (dd, J ) 7.8/1.7
Hz, 1 H, H-6), 8.00 (d, J ) 8.4 Hz, 1 H, NH-cHex), 8.30 (dd, J )
7.7/1.7 Hz, 1 H, H-4), 8.42 (s, 1 H, CHdN), 11.47 (s, 1 H, dN-
NH). Anal. (C15H19N3O3S) C, H, N, S.
5-(4-Cyclohexylthiosemicarbazono)methyl-2-hydroxybenzo-
ic Acid (35). mp 240-242 °C; 1H NMR (DMSO-d6) δ 1.14 (m, 1
H), 1.20-1.33 (m, 2 H), 1.34-1.48 (m, 2 H), 1.59 (m, 1 H), 1.65-
1.76 (m, 2 H), 1.80-1.91 (m, 2 H), 4.17 (m, 1 H), 7.01 (d, J ) 8.7
Hz, 1 H, H-3), 7.93 (d, J ) 2.1 Hz, 1 H, H-6), 7.05 (d, J ) 8.7 Hz,
1 H, HN-cHex), 8.01 (s, 1 H, CHdN), 8.12 (dd, J ) 8.7/2.0 Hz,
1 H, H-4), 11.28 (s, 1 H, )N-NH) Anal. (C15H19N3O3S) C, H, N,
S.
3-(4-Cyclohexylthiosemicarbazono)methyl-4-hydroxybenzo-
ic Acid (36). mp >350 °C; 1H NMR (DMSO-d6) δ 1.08-1.49 (m,
5 H), 1.51-1.76 (m, 3 H), 1.82-1.95 (m, 2 H), 4.16 (m, 1 H),
6.94 (d, J ) 8.6 Hz, 1 H, H-5), 7.80 (dd, J ) 8.6/2.2 Hz, 1 H,
H-6), 7.94 (d, J ) 8.4 Hz, 1 H, HN-cHex), 8.32 (d, J ) 2.2 Hz,
1 H, H-2), 8.38 (s, 1 H, CHdN), 10.78 (br s, 1 H), 11.38 (s, 1 H,
dN-NH), 12.59 (br, 1 H). Anal. (C15H19N3O3S) C, H, N, S.
Biology. Materials. Estrone sulfate was purchased from Sigma
Chemical Co. (Germany). [6,7-3H]Estrone sulfate (specific activity,
53 Ci/mmol) was purchased from New England Nuclear (Boston,
MA). Radioactive samples were analyzed with a Packard Tri-Carb
1600 Liquid scintillation counter. Liquid scintillation cocktail was
Ultima Gold from Packard (Meriden, CT).
Estrone Sulfatase Assay. 3H-Estrone sulfate (500,000 dpm/tube)
adjusted to 20 µM with unlabeled estrone sulfate in Tris-HCl buffer
(0.2 M, pH 8.0, 0.1 mL) was added to a test tube. An inhibitor at
various concentrations in Tris-HCl buffer (0.1 mL) was then added
to each tube. The assay began by the addition of placental
microsomes diluted with Tris-HCl buffer containing 2.5 mM
dithiothreitol (0.3 mL). The protein concentration was measured
using the Bradford method. The final volume of the assay was 0.5
mL. After 20 min of incubation at 37 °C, 1.5 mL of toluene was
added to quench the assay. Control samples with no inhibitor were
incubated simultaneously. The quenched samples were vortexed
for 1 min, and 50 µL of toluene was removed from the organic
phase and diluted with 3 mL of scintillation cocktail. The aliquots
were counted for 3 min to determine the amount of product
formation. All samples were run twice in duplicate. Product
formation for samples containing an inhibitor was compared to that
of the control sample.
General Procedure for the Preparation of Cyclohexylthi-
osemicarbazones 2-13 and 25-38. 4-Cyclohexylthiosemicarba-
zide (10 mmol) in one portion was added to a boiling solution of
the aldehyde (10 mmol) in ethanol (3 mL). The reaction mixture
was stirred under reflux until all the starting material was consumed
(TLC). After cooling, the solid was filtered off and recrystallized
from ethanol.
2-(4-Cyclohexylthiosemicarbazono)methylbenzoic Acid (2).
1
mp 232-234 °C; H NMR (DMSO-d6) δ 1.13-1.93 (m, 10 H),
4.17 (m, 1 H), 7.44-7.51 (m, 1H), 7.54-7.62 (m, 1 H), 7.78-
7.84 (m, 1 H), 8.13 (d, J ) 7.4 Hz, 1 H), 7.94 (d, J ) 8.5 Hz, 1
H), 8.74 (s, 1 H), 11.53 (s, 1 H). Anal. (C15H19N3O2S) C, H, N, S.
3-(4-Cyclohexylthiosemicarbazono)methylbenzoic Acid (3).
1
mp 239-241 °C; H NMR (DMSO-d6) δ 1.05-1.52 (m, 5 H),
1.54-1.79 (m, 3 H), 1.80-1.94 (m, 2 H), 4.07-4.27 (m, 1 H),
7.55 (t, J ) 8.0 Hz, 1 H, H-5), 7.94 (m, 1 H, H-6), 8.03-8.19 (m,
3 H, CHdN, H-2,4), 11.46 (s, 1 H, dN-NH), 13.14 (br, 1 H).
Anal. (C15H19N3O2S) C, H, N, S.
Kinetic Analysis of the Inhibition of Estrone Sulfatase
Activity. Estrone sulfatase assays were carried out as described
above using various concentrations of the substrate (2-20 µM).
The Km, Ki, and type inhibition for 34 and 35 at pH 8.0 were
determined by Lineweaver-Burk plot analysis and secondary
4-(4-Cyclohexylthiosemicarbazono)methylbenzoic Acid (4).
1
mp 255-260 °C; H NMR (DMSO-d6) δ 1.11-1.35 (m, 3 H),
1.37-1.53 (m, 2 H), 1.55-1.65 (m, 1 H), 1.67-1.78 (m, 2 H),
1.81-1.92 (m, 2 H), 4.12-4.26 (m, 1 H), 7.87, 7.94 (2 d, J ) 8.5

3666 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Ju¨tten et al.
(20) Ahmed, S.; James, K.; Owen, C. P. The design, synthesis, and
biochemical evaluation of derivatives of biphenyl sulfamate-based
compounds as novel inhibitors of estrone sulfatase. Biochem. Biophys.
Res. Commun. 2002, 294, 180-183.
(21) Ahmed, S.; James, K.; Owen, C. Inhibition of estrone sulfatase (ES)
by derivatives of 4-[(aminosulfonyl)oxy] benzoic acid. Bioorg. Med.
Chem. Lett. 2002, 12, 2391-2394.
(22) Ciobanu, L. C.; Luu-The, V.; Poirier, D. Nonsteroidal compounds
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Biochem. Mol. Biol. 2002, 80, 339-353.
(23) Nussbaumer, P.; Bilban, M.; Billich, A. 4,4′-Benzophenone-O,O′-
disulfamate: a potent inhibitor of steroid sulfatase. Bioorg. Med.
Chem. Lett. 2002, 12, 2093-2095.
replots of the slopes of Lineweaver-Burk plot versus the corre-
sponding inhibitor concentration (0, 0.5, 1, 2.5, 5 µM).
Acknowledgment. The Thu¨ringer Ministerium fu¨r Wissen-
schaft und Kunst supported this research. We wish to thank
Mrs Ursula Zscherpe and Mrs Gudrun Seide for their excellent
technical assistance.
Supporting Information Available: Full experimental details
and analytical data for compounds 2-40 and 45-49. This material
is available free of charge via the Internet at http://pubs.acs.org.
(24) Nussbaumer, P.; Lehr, P.; Billich, A. 2-Substituted 4-(thio)chrome-
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(26) Billich, A.; Meingassner, J. G.; Nussbaumer, P.; Desrayaud, S.; Lam,
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(27) Hejaz, H. A.; Woo, L. W.; Purohit, A.; Reed, M. J.; Potter, B. V.
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