Anti-nitric oxide production activity of isothiocyanates correlates with their polar surface area rather than their lipophilicity

Article abstract of DOI:10.1016/j.ejmech.2009.08.005

There is increasing demand for novel anti-inflammatory drugs with different mechanisms of action. We synthesized a series of isothiocyanates 2b-h based on 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) found in the pungent spice Wasabia japonica. Inhibit

Full text of DOI:10.1016/j.ejmech.2009.08.005

European Journal of Medicinal Chemistry 44 (2009) 4931–4936
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-nitric oxide production activity of isothiocyanates correlates with their polar
surface area rather than their lipophilicity
,
b
b
c
d
Toshiro Noshita a , Yumi Kidachi , Hirokazu Funayama , Hiromasa Kiyota , Hideaki Yamaguchi ,
*
Kazuo Ryoyama b
,
e
a Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, Hiroshima 727-0023, Japan
b Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Aomori University, 2-3-1 Kobata, Aomori 030-0943, Japan
c Department of Bioscience and Biotechnology for Future Bioindustry, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiya, Aoba-ku, Sendai 981-
8555, Japan
d Department of Pharmacy, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku, Nagoya 468-8503, Japan
e Graduate School of Environmental Sciences, Aomori University, 2-3-1 Kobata, Aomori 030-0943, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
There is increasing demand for novel anti-inflammatory drugs with different mechanisms of action. We
synthesized a series of isothiocyanates 2b–h based on 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC)
found in the pungent spice Wasabia japonica. Inhibitory activities against in-vitro growth of tumor cells
and production of nitric oxide (NO) using the mouse macrophage-like cell line J774.1 were noted. All
isothiocyanates were optimized by Hartree-Fock/3-21G model, and the log P values and the polar surface
area (PSA) values were calculated. Substitution of the methylsulfinyl group (CH3S(]O)–R) in 6-MITC
with a formyl (CHO–R), a methylsulfanyl (CH2S–R) or a methyl (CH3–R) group reduced the activities of
the parent isothiocyanate. Substitution with a formyl group resulted in lower lipophilicity (log P value)
whereas substitution with a methylsulfanyl or methyl group resulted in a lower PSA value. The inhibitory
activity of isothiocyanates showed better correlation with their PSA values rather than their partition
coefficient (log P) values. Isothiocyanates with higher PSA values and some degree of log P value may
have potent biological activity.
Received 22 July 2009
Accepted 12 August 2009
Available online 18 August 2009
Keywords:
Wasabi
Isothiocyanates
Nitric oxide
Structure–activity relationship
Polar surface area
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
antioxidant enzyme inducer potency of sulforaphane and its
analogs is influenced by the number of methylene (–CH2–) groups
Accumulating evidence from epidemiologic and clinical studies
indicates that chronic inflammatory disorders harbor an increased
risk of cancer, arthritis, heart attacks, and Alzheimer’s disease [1].
Sulforaphane [4-(methylsulfinyl)butyl isothiocyanate] is
a natural isothiocyanate present in cruciferous vegetables such as
broccoli and cabbage. It possesses potent anti-inflammatory and
chemopreventive activities [2]. The compound 6-(methyl-
sulfinyl)hexyl isothiocyanate (6-MITC) is found in the pungent
spice Wasabia japonica (wasabi) used in Japan [3,4] and is thought
to have anti-inflammatory and chemopreventive activities [5–7].
The relationship between structure and the biological activity of
alkyl isothiocyanates reveals that an increase in the length of the
carbon chain correlates with augmented induction of the activity of
NADPH:quinone oxidoreductase and its mRNA [8]. Morimitsu et al.
[5] and Prawan et al. [9] reported that the phase-II detoxifying/
in the bridge linking the functional group and isothiocyanate
moieties. Talalay et al. [10] suggested in structure–biological
activity studies of various alkyl and aromatic isothiocyanates that at
least one hydrogen on the carbon adjacent to the isothiocyanate
moiety (which leads to tautomerization of the methylene–iso-
thiocyanate moiety (–CH2–N]C]S) to a structure resembling an
a,
b-unsaturated thioketone) may be important for the inductive
ability of quinone reductase and glutathione S-transferases. The
oxidation state of sulfur in the methylsulfinyl functional group
appears to be important for the inducer activity of sulforaphane
and its analogs [5], and the type of the functional group may be
more important than the number of methylene groups [9]. Mor-
pholine and methylenedioxybenzene groups have been reported to
have superior inducer activity than other functional groups [9].
Differences in physicochemical properties (e.g., solubility, acid–
base properties) may also influence the biological activity of iso-
thiocyanates. Jiao et al. [11] reported that inhibitory activities of
isothiocyanates against 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK)-induced lung tumorigenesis show better
* Corresponding author. Tel.: þ81 824 74 1000; fax: þ81 824 74 0191.
E-mail address: noshita@pu-hiroshima.ac.jp (T. Noshita).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.08.005

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T. Noshita et al. / European Journal of Medicinal Chemistry 44 (2009) 4931–4936
correlation with their high lipophilicity and low reactivity against
glutathione (making conjugations with thiol groups).
Clarification of the biological roles of the methyl sulfinyl group
of alkyl isothiocyanates is the subject of this contribution. A series
of new isothiocyanate derivatives replaced the methyl sulfinyl
group with other functional groups, though the six-carbon-chain
framework of 6-MITC was maintained. Structure–biological activity
relationships of the newly synthesized isothiocyanates were eval-
uated for their inhibitory activities against in-vitro growth of tumor
cells and production of nitric oxide (NO) using the mouse macro-
phage-like cell line J774.1.
2. Results and discussion
2.1. Chemistry
Chemical structures of 6-MITC and analogs are illustrated in
Fig. 1. Four isothiocyanates (2b, 2c, 2d and 2e) were prepared from
1,6-hexanediol via alkyl halide 1a [12] and their isothiocyanato
(–NCS) group formed by adopting the method of Ding et al [13]
(Scheme 1). Two isothiocyanates (2f and 2g) and thiocyanate 4
were prepared from 1,6-dichlorohexane (Scheme 2). The iso-
thiocyanato group of 2f and 2g was formed by adopting the method
of Ding et al [13], and the thiocyanato (–SCN) group of 4 was
synthesized by nucleophilic substitution of the thiocyanate anion
with alkyl halide 1c. The structures of all isothiocyanates (2b, 2c,
2d, 2e, 2f and 2g) and thiocyanate (4) were confirmed by 1H NMR
and IR spectral data, and all new compounds gave satisfactory
HRMS data. Isothiocyanates (R–N]C]S) showed strong and broad
absorption at about 2200 cmꢀ1 to 2100 cmꢀ1, whereas thiocyanate
(R–S–C^N) showed strong and sharp absorption at about
2150 cmꢀ1 in IR spectra. In addition, n-hexyl isothiocyanate (2h)
was purchased from Sigma–Aldrich (St. Louis, MO, USA).
Scheme 1. Reagents & Conditions: (a) i. NaN3, DMF, reflux, 3 h, 79%; ii. Ph3P, ether, 12 h,
rt., then CS2, reflux, 2 h, 82%; (b) TsOH, MeOH, 3 h, rt, 71%; (c) Ac2O, pyr., 16 h, rt, 56%;
(d) (CF3CO)2O, pyr., 18 h, rt, 32%; (e) Dess-Martin periodinane, CH2Cl2, 18 h, rt., 27%.
This indicates 6-MITC inhibits one of functions of normal macro-
phages, and suggests a possibility that 6-MITC would inhibit the
function of macrophages in vivo as well. According to this result, we
selected a mouse macrophage-like cell line J774.1 to test biological
activities of isothiocyanate compounds, instead of peritoneal
exudates macrophages because of their inconvenience in
preparation.
The newly synthesized isothiocyanates 2b–g, purchased 2h and
thiocyanate 4 were screened for their anti-proliferative and anti-
NO production activities in vitro using J774.1 cells. The half-
maximal inhibitory concentration (IC50) values of compounds 2b–h
and thiocyanate 4 are listed in Table 2. Thiocyanate 4 had no
activity, indicating that the inhibitory activities of 6-MITC were
entirely dependent on its isothiocyanato group. This observation is
in accordance with that of Morimitsu et al. [5]. That is, the meth-
ylthioalkyl isothiocyanate but not the methylthioalkylamine has
the induction potency of glutathione S-transferase (GST) activity,
indicating that the isothiocyanate moiety is essential for the
In addition, all isothiocyanates were optimized by Hartree-Fock/
3-21G model, and the log P values and the polar surface area (PSA)
values were calculated by using of Spartan ’06 program (Wave-
function, Incorporated, CA, USA).
2.2. Inhibitory activities against in-vitro growth of tumor cells and
NO production
First of all, we examined inhibitory activity of 6-MITC on NO
production by mouse peritoneal macrophages. Table 1 shows
6-MITC inhibited the NO production in a dose-dependent manner.
Scheme 2. Reagents & Conditions: (a) i. NaN3, DMF, reflux, 3 h, 100%; ii. Ph3P, ether,
12 h, rt., then CS2, reflux, 2 h, 83%; (b) (CH3S)2CO, nBu4NBr, 30% aq. NaOH, 0.5 h, reflux,
67%; (c) i. NaN3, DMF, reflux, 3 h, 100%; ii. Ph3P, ether, 12 h, rt., then CS2, reflux, 2 h,
80%; (d) NaSCN, MeOH, reflux, 4 h, 39%; (e) 30% H2O2, AcOH, 0.5 h, rt., 64%.
Fig. 1. Chemical structures of 6-MITC and analogues used in this study.

T. Noshita et al. / European Journal of Medicinal Chemistry 44 (2009) 4931–4936
4933
Table 1
shifts are given in d unit (ppm). MS data were measured with a Jeol
Effect of 6-MITC on nitric oxide (NO) production by mouse peritoneal exudate
macrophages.
JMS-700 spectrometer in EI and FAB modes, and IR spectra were
recorded on a JASCO FT/IR-5300 spectrometer (Jasco, Tokyo, Japan).
6-MITC (
m
M)
NO (
m
M)a
Inhibition (%)
Data are given in
n
max (cmꢀ1). Preparative TLC work was carried
0
8.6 ꢁ 0.3
7.7 ꢁ 0.4
8.1 ꢁ 0.3
6.0 ꢁ 0.3
0.7 ꢁ 0.2
0
10
6
30
92
out using plates coated with Kieselgel 60 F254 (0.75-mm thick;
Merck KGaA, Darmstadt, Germany). All chemicals and solvents
were of reagent grade, and the latter were distilled and dried before
use. Jiao et al. [11] suggested that the application of log P (partition
coefficients between 1-octanol and aqueous phases) values is
suitable for measurement of the relative lipophilicity of iso-
thiocyanates, and that the log P values measured for iso-
thiocyanates are consistent with those predicted on the basis of
their chemical structures. We adopted the Spartan ’06 program
(Wavefunction, Incorporated, CA, USA) for calculation of log P from
Crippen model and polar surface area (PSA) values in Å2 of newly
synthesized isothiocyanates.
0.01
0.1
1
5
a
Mean ꢁ SE (n ¼ 6).
induction of GST activity. 6-MITC and N-acetyl cysteine have been
reported to spontaneously make conjugates in aqueous solution
[14]. Compounds 2b, 2c, 2d and 2f had almost identical anti-
proliferative and anti-NO production activities as those of 6-MITC.
Compounds 2e, 2g and 2h were less active than 6-MITC.
Hou et al. [8] reported that an increase in the length of the
carbon chain in alkyl isothiocyanates correlates with augmentation
of their biological activities. Increasing the carbon-chain length
appears to increase their log P values. The biological activity of
isothiocyanates may therefore increase with increment of their
log P values. An inverse correlation between log P and the activities
of each compound was noted, i.e., both activities tend to decrease
with increment in log P values, though the correlation was poor
(Fig. 2). A better correlation between PSA and the activities of each
compound were observed (Fig. 3). The low biological activities of
compounds 2h and 2g could therefore be due to their low PSA
values. Compound 2e showed weaker activity than compound 2f,
though the PSA value of compound 2e was larger than that of
compound 2f. This could be because the aldehyde group of
compound 2e tends to be readily attacked by nucleophiles and its
log P value is quite low. A certain degree of the log P value of iso-
thiocyanates is necessary for their effective activity. As mentioned
above, the biological activities of isothiocyanates should be through
binding of their isothiocyanato groups with the SH groups of target
molecules. It may be hypothesized that the PSA of isothiocyanates
facilitates formation of isothiocyanate–SH conjugations.
3.1.1. Synthesis of 2-(6-Isothiocyanatohexyloxy)-tetrahydro-2H-
pyran (2a)
A solution of 2-(6-iodohexyloxy)-tetrahydro-2H-pyran (1.10 g,
5 mmol) and sodium azide (400 mg, 6.0 mmol) in DMF (10 mL) was
stirred at reflux temperature for 2 h. The reaction mixture was
poured into water (100 mL) and extracted with n-hexane (50 mL, 3
times). Combined extracts were washed with water and brine,
dried over anhydrous CaCl2, and concentrated under reduced
pressure. A solution of dry residue (650 mg, containing 3.8 mmol of
2-(6-azidohexyloxy)-tetrahydro-2H-pyran) and PPh3 (1.23 g,
4.7 mmol) in Et2O (10 mL) was stirred at room temperature for 8 h.
After removing the solvent under diminished pressure, 1.5 mL of
CS2 was added to the reaction system. The resulting mixture was
refluxed for another 2 h. The reaction mixture was poured into
water (100 mL) and extracted with Et2O (30 mL,
3 times).
Combined extracts were washed with water and brine, dried over
anhydrous CaCl2, and concentrated. The residue was chromato-
graphed over silica gel and eluted with n-hexane/benzene (2:1 ¼ v/
v) to give 2-(6-isothiocyanatohexyloxy)-tetrahydro-2H-pyran (2a,
530 mg, 2.2 mmol, 44% yield from 2-(6-chlorohexyloxy)-tetrahy-
dro-2H-pyran) as a colorless oil. IR nmax (film) cmꢀ1: 2940, 2865,
2180, 2105, 1454, 1348, 1123, 1076, 1034. 1H NMR (CDCl3)
d: 1.35–
3. Experimental protocols
1.90 (14H, m), 3.40 (1H, dt, J ¼ 9.7, 6.4 Hz), 3.46–3.56 (1H, m), 3.52
(2H, t, J ¼ 6.6 Hz), 3.75 (1H, dt, J ¼ 9.5, 6.6 Hz), 3.82–3.92 (1H, m),
4.55–4.60 (1H, m). 13C NMR(CDCl3)
d: 19.8, 25.5, 25.6, 26.4, 29.5,
3.1. Chemistry
29.9, 30.8, 45.0, 62.5, 67.3, 99.0 (carbon of the isothiocyanato group
was not observed). HREI-MS: Found: 243.1282, calcd. for
C12H21NO2S (Mþ): 243.1293.
1H and 13C NMR data were measured by a JNM-AL300 spec-
trometer (Jeol, Tokyo, Japan) using CDCl3 as a solvent. Chemical
3.1.2. Synthesis of 6-Isothiocyanatohexan-1-ol (2b)
A
mixture of 2-(6-isothiocyanatohexyloxy)-tetrahydro-2H-
Table 2
In vitro NO production and tumor growth inhibitory activities of isothiocyanates 2b-
h and thiocyanate 4.
pyran (2a, 530 mg, 2.2 mmol) and a catalytic amount of p-TsOH in
dry MeOH (10 mL) was stirred for 3 h at room temperature under
an atmosphere of nitrogen. The reaction mixture was then
concentrated in vacuo. The residue was purified by preparative TLC
(n-hexane-AcOEt ¼ 4:1) to give pure 6-isothiocyanatohexan-1-ol
(2b, 250 mg, 1.7 mmol, 71% yield) as a colorless oil. IR nmax (film)
cmꢀ1: 3348, 2935, 2860, 2361, 2106, 1456, 1346, 1163, 1053, 945,
Compounds
Inhibition (IC50
,
m
M)a
Growth
PSA(Å2)b
log Pb
NO production
6-MITC
4 (thiocyanate)
5.7 ꢁ 0.5
>200
8.0 ꢁ 0.6
>200
19.9
–
1.24
–
Isothiocyanates
902, 729. 1H NMR (CDCl3)
d: 1.35–1.55 (4H, m), 1.55–1.65 (2H, m),
2b
2c
2d
2f
2e
2g
2h
6.0 ꢁ 1.2
6.6 ꢁ 1.2
4.4 ꢁ 0.3
4.1 ꢁ 0.2
23.1
23.8
23.2
10.2
17.5
2.7
1.41
1.64
2.78
1.36
0.94
2.08
2.61
1.70–1.80 (2H, m), 3.52(2H, t, J ¼ 6.4 Hz), 3.65 (2H, t, J ¼ 6.4 Hz). 13C
9.1 ꢁ 1.0
5.6 ꢁ 0.4
NMR(CDCl3) d: 25.0, 26.4, 29.9, 32.4, 45.0, 62.7 (carbon of the iso-
11.5 ꢁ 5.9
25.5 ꢁ 5.9
30.4 ꢁ 6.3
66.8 ꢁ 13.5
8.1 ꢁ 0.6
thiocyanato group was not observed). HREI-MS: Found: 159.0722,
19.7 ꢁ 1.1
14.8 ꢁ 0.9
47.9 ꢁ 10.8
calcd. for C7H13NOS (Mþ): 159.0718.
2.5
3.1.3. Synthesis of 6-Isothiocyanatohexyl acetate (2c)
Data represent means and standard errors of 5-11 independent experiments.
A solution of a mixture of 2b (122 mg, 0.8 mmol) in Ac2O
(1 mL) and pyridine (1 mL) was stirred at room temperature for
16 h. The reaction was quenched with methanol, and the reaction
a
50% inhibition.
b
PSA (polar surface area) and log P value were obtained with Spartan ’06
program.

4934
T. Noshita et al. / European Journal of Medicinal Chemistry 44 (2009) 4931–4936
NO production
Cell growth
80
60
40
20
0
60
40
20
0
y = 8.3282x - 0.5617
R = 0.377
y = 14.438x - 5.1741
R= 0.457
0
0.5
1
1.5
2
2.5
3
0
0.5
1
1.5
2
2.5
3
log P
log P
Fig. 2. Linear regression curves for the interactions of log P with 50% inhibition of NO production and cell growth.
mixture concentrated in vacuo. The dry residue was purified by
silica gel vacuum flash column chromatography eluting with
n-hexane/EtOAc (2:1 ¼ v/v) to give pure 6-isothiocyanatohexyl
acetate (2c, 86 mg, 0.43 mmol, 56% yield) as a colorless oil. IR nmax
(film) cmꢀ1: 2941, 2860, 2361, 2339, 2179, 2106, 1738, 1456, 1365,
preparative TLC to give pure 6-isothiocyanatohexanal (2e, 35 mg,
0.22 mmol, 27% yield) as a colorless oil. IR nmax (film) cmꢀ1: 2928,
2361, 2339, 2104, 1722, 1456, 1348. 1H NMR (CDCl3)
d: 1.45–1.55
(4H, m), 1.60–1.75 (4H, m), 2.49 (2H, dt, J ¼ 1.5, 7.3 Hz), 3.53 (2H, t,
J ¼ 6.6 Hz), 9.80 (1H, t, J ¼ 1.5 Hz). 13C NMR (CDCl3)
d: 21.2, 26.1,
1238, 1037, 889, 731, 669. 1H NMR (CDCl3)
d
: 1.35–1.55 (4H, m),
29.8, 43.6, 44.8, 201.9 (carbon of the isothiocyanato group was not
observed). HREI-MS: Found: 157.0566, calcd. for C7H11NOS (Mþ):
157.0561.
1.60–1.80 (4H, m), 2.05 (3H, s), 3.53 (2H, t, J ¼ 6.6 Hz), 4.08 (2H, t,
J ¼ 6.6 Hz). 13C NMR(CDCl3)
d: 21.0, 25.3, 26.3, 28.4, 29.8, 44.9,
64.21, 171.1 (carbon of the isothiocyanato group was not
observed). HREI-MS: Found: 201.0831, calcd. for C9H15NO2S (Mþ):
201.0823.
3.1.6. Synthesis of 1-Isothiocyanato-6-methoxyhexane (2f)
A solution of 1-chloro-6-methoxyhexane [15] (1b, 950 mg,
6.3 mmol) and sodium azide (600 mg, 9.2 mmol) in DMF (10 mL)
was stirred at reflux temperature for 2 h. The reaction mixture
was poured into water (100 mL) and extracted with n-hexane
(50 mL, 3 times). The combined extracts were washed with water
and brine, dried over anhydrous CaCl2, and concentrated under
diminished pressure. The residue was used for the next step
without purification. A solution of dry residue (1.0 g, containing
6.3 mmol of 1-azido-6-methoxyhexane) and PPh3 (2.07 g,
7.9 mmol) in Et2O (10 mL) was stirred at room temperature for
8 h. After removing the solvent under diminished pressure, 2 mL
of carbon disulfide (CS2) was added to the reaction system. The
resulting mixture was refluxed for another 2 h. The reaction
mixture was poured into water (100 mL) and extracted with Et2O
(50 mL, 3 times). Combined extracts were washed with water and
brine, dried over anhydrous CaCl2, and concentrated. The residue
was chromatographed over silica gel and eluted with n-hexane/
benzene (2:1 ¼ v/v) to give 1-isothiocyanato-6-methoxyhexane
(2f, 820 mg, 4.7 mmol, 75% yield from 1-chloro-6-methoxyhex-
ane) as a colorless oil. IR nmax (film) cmꢀ1: 2936, 2861, 2180, 2106,
3.1.4. Synthesis of 6-Isothiocyanatohexyl 2,2,2-trifluoroacetate (2d)
A solution of a mixture of 2b (107 mg, 0.67 mmol) in trifluoro-
acetic anhydride (1 mL) and pyridine (1 mL) was stirred at room
temperature for 18 h. The reaction was quenched with methanol,
and the reaction mixture concentrated in vacuo. The dry residue
was purified by preparative TLC (n-hexane/EtOAc ¼ 2:1) to give
pure 6-isothiocyanatohexyl 2,2,2-trifluoroacetate (2d, 55 mg,
0.22 mmol, 32% yield) as a colorless oil. IR nmax (film) cmꢀ1: 2943,
2864, 2361, 2339, 2183, 2108, 1786, 1556, 1456, 1404, 1350, 1221,
1161, 937, 891, 777, 731, 669. 1H NMR (CDCl3)
d: 1.35–1.55 (4H, m),
1.65–1.85 (4H, m), 3.53 (2H, t, J ¼ 6.4 Hz), 4.38 (2H, t, J ¼ 6.4 Hz). 13C
NMR(CDCl3) d: 24.9, 26.2, 28.0, 29.8, 44.9, 67.9, 116.4, 157.8 (carbon
of the isothiocyanato group was not observed). HREI-MS: Found:
255.0543, calcd. for C9H12F3NO2S (Mþ): 255.0541.
3.1.5. Synthesis of 6-Isothiocyanatohexanal (2e)
Dess–Martin periodinane (810 mg, 1.9 mmol) was added in one
portion to
a stirred solution of 2b (120 mg, 0.75 mmol) in
dichloromethane (8 mL) at room temperature. After 18 h, the
1454, 1348, 1120. 1H NMR (CDCl3)
d: 1.32–1.51 (4H, m), 1.54–1.64
reaction mixture was filtered. The filtrate was concentrated in
(2H, m), 1.65–1.77 (2H, m), 3.34 (3H, s), 3.38 (2H, t, J ¼ 6.4 Hz),
vacuo to give
a
colorless residue, which was purified by
3.53 (2H, t, J ¼ 6.6 Hz). 13C NMR (CDCl3)
d: 25.4, 26.4, 29.4, 29.8,
NO production
Cell growth
80
60
40
20
0
60
y = -1.8414x + 48.488
R = 0.790
y = -1.1479x + 31.71
R = 0.704
40
20
0
0
5
10
15
20
25
0
5
10
15
20
25
PSA
PSA
Fig. 3. Linear regression curves for the interactions of PSA with 50% inhibition of NO production and cell growth.

T. Noshita et al. / European Journal of Medicinal Chemistry 44 (2009) 4931–4936
4935
44.9, 58.6, 72.5 (carbon of the isothiocyanato group was not
observed). HREI-MS: Found: 173.0877, calcd. for C8H15NOS (Mþ):
173.0874.
room temperature. After 30 min, the reaction mixture was diluted
with dichloromethane and neutralized by adding potassium
carbonate. The mixture was filtered and purified by preparative TLC
to give pure 1-(methylsulfinyl)-6-thiocyanatohexane (4) with
quantitative yield. IR nmax (film) cmꢀ1: 2935, 2861, 2153, 1460, 1300,
3.1.7. Synthesis of (6-Chlorohexyl) (methyl) sulfane (1c)
A
solution of 1,6-dichlorohexane (1.55 g, 10 mmol), S,S0-
1028. 1H NMR (CDCl3)
d: 1.44–1.57 (4H, m), 1.75–1.94 (4H, m), 2.58
dimethyl dithiocarbonate (730 mg, 6.0 mmol) and 50 mg of tetra
n-butyl ammonium bromide in 30% aqueous KOH (10 mL) was
stirred vigorously at reflux temperature for 30 min. The reaction
mixture was poured into water (100 mL) and extracted with diethyl
ether (30 mL, 3 times). Combined extracts were washed with water
and brine, dried over anhydrous CaCl2, and concentrated under
diminished pressure. The residue was chromatographed over silica
gel and eluted with n-hexane/benzene (4:1 ¼ v/v) to give (6-
chlorohexyl) (methyl) sulfane (1c, 1.45 g, 8.7 mmol, 87% yield). IR
nmax (film) cmꢀ1: 2934, 2857, 1435, 1271, 1044, 723. 1H NMR (CDCl3)
(3H, s), 2.68 (1H, dt, J ¼ 12.8, 7.9 Hz), 2.73 (1H, dt, J ¼ 12.8, 7.3 Hz),
2.96 (2H, t, J ¼ 7.2 Hz). 13C NMR (CDCl3)
d: 22.4, 27.5, 28.1, 29.6, 33.8,
38.6, 54.4, 112.2. HRFAB-MS: Found: 206.0676, calcd. for C8H16NOS2
(Mþ1þ): 206.0673.
3.2. Biological activities
3.2.1. Preparation of peritoneal exudates cells (PEC)
Five-7week-old BALB/c mice received an intraperitoneal (i.p.)
injection of 3 mL of 2.4% thioglycolate broth. After 4–5 d, BALB/c
mice were sacrificed by heart punctuation after chloroform anes-
thetization, and PEC were aseptically obtained from the mice by
injecting i.p. twice 5 mL cold phosphate-buffered saline solution
containing 0.1% fetal calf serum (FCS), heparin (10 U/mL) and
10 mM glucose, massaging and collecting the cells immediately
thereafter. The cells were washed and resuspended in 10% FCS-
RPMI 1640 medium containing 5 ꢂ10ꢀ5 M 2-mercaptoethanol and
d
: 1.36–1.54 (4H, m), 1.56–1.68 (2H, m), 1.72–1.86 (2H, m), 2.10 (3H,
s), 2.50 (2H, t, J ¼ 7.2 Hz), 3.54 (2H, t, J ¼ 6.6 Hz). 13C NMR(CDCl3)
d:
15.6, 26.5, 28.0, 29.0, 32.5, 34.2, 45.0. HREI-MS: Found: 166.0587,
calcd. for C7H15ClS (Mþ): 166.0583.
3.1.8. Synthesis of 1-Isothiocyanato-6-(methylthio)hexane (2g)
A solution of (6-chlorohexyl) (methyl) sulfane (1c, 740 mg,
4.4 mmol) and sodium azide (400 mg, 6.0 mmol) in DMF (10 mL)
was stirred at reflux temperature for 2 h. The reaction mixture was
poured into water (100 mL) and extracted with n-hexane (50 mL, 3
times). Combined extracts were washed with water and brine,
dried over anhydrous CaCl2, and concentrated under diminished
pressure. A solution of dry residue (650 mg, containing a 3.8 mmol)
of 1-azido-6-methoxyhexanel and PPh3 (1.23 g, 4.7 mmol) in Et2O
(10 mL) was stirred at room temperature for 8 h. After removing the
solvent under diminished pressure, 1.5 mL of CS2 was added to the
reaction system. The resulting mixture was refluxed for another
2 h. To the reaction mixture was poured into water (100 mL) and
extracted with Et2O (30 mL, 3 times). Combined extracts were
washed with water and brine, dried over anhydrous CaCl2, and
concentrated. The residue was chromatographed over silica gel and
eluted with n-hexane/benzene (2:1 ¼ v/v) to give 1-isothiocyanato-
6-(methylthio)hexane (2g, 530 mg, 2.8 mmol, 64% yield from 7) as
a colorless oil. IR nmax (film) cmꢀ1: 2936, 2857, 2183, 2102, 1453,
gentamicin sulfate (10 mg/mL). Next, 200 mL cell suspension
(5 ꢂ105 cells/mL) was added to flat-bottomed 96-well microplate
(Greiner). Non-adherent cells were removed with a Titer mixture
MB-1 (Japan Tirika Co.) after 2 h incubation at 37 ꢃC in a humidified
CO2 (5%) atmosphere. Adherent cells comprised 90–95% of the
plated PEC and consisted of over 95% macrophages, by morpho-
logical definition (Giemusa staining). Thus, the numbers of initial
plated PEC are represented as macrophage number in this study. All
animal experiments in this paper followed the Guidelines for
Animal Experimentation of Aomori University.
3.2.2. Inhibition of in vitro production of NO
Production of NO in peritoneal macrophages and J774.1 cells
(measured as NO2 concentration) was determined by Griess
reagent as described previously [17] except for FBS concentration;
the culture medium for J774.1 cells was RPMI-1640 containing 10%
FBS and gentamicin sulfate (10 mg/mL). In brief, peritoneal macro-
1346. 1H NMR (CDCl3)
d
: 1.40–1.50 (4H, m), 1.50–1.78 (4H, m), 2.10
phages were prepared as described above, and J774.1 cells (4 ꢂ104/
0.2 mL) in each well of a 96-well flat-bottomed microplate were
cultured at 37 ꢃC for 1 d. The culture medium was replaced with
freshly prepared warm (37 ꢃC) 10% FCS-RPMI 1640 medium. After
(3H, s), 2.51 (2H, t, J ¼ 7.5 Hz), 3.52 (2H, t, J ¼ 6.4 Hz). 13C NMR
(CDCl3) d: 15.5, 26.2, 27.9, 28.8, 29.8, 34.1, 45.0 (carbon of the iso-
thiocyanato group was not observed). HREI-MS: Found: 189.0651,
calcd. for C8H15NS2 (Mþ): 189.0646.
incubation for 30 min, 10
compounds and 10
m
L serial twofold diluted solution of test
mL
interferon gamma (2 U/mL) þ lipopoly-
3.1.9. Synthesis of 1-(Methylthio)-6-thiocyanatohexane (3)
saccharide (10 ng/mL) solution were added to each well. The
microplates were further incubated at 37 ꢃC. After 20–24 h, 0.1 mL
culture fluid from each well was withdrawn and mixed with 0.1 mL
Griess reagent. Mixtures were incubated for 10 min at room
temperature. Absorbance of each well, 540–655 nm, was measured
with a Bio-RAD model 550 microplate reader. NO2 concentrations
were measured using sodium nitrite as a standard.
Sodium thiocyanate (NaSCN, 810 mg, 10 mmol) was added to
a stirred solution of (6-chlorohexyl)(methyl)sulfane (1c, 1.1 g,
6.6 mmol) in methanol. The reaction mixture was heated at reflux
temperature for 4 h. After dilution with chloroform, the mixture
was filtered with silica gel, and the filtrate concentrated. The dry
residue was chromatographed over silica gel and eluted with
n-hexane/benzene (2:1 ¼ v/v) to give 1-(methylthio)-6-thio-
cyanatohexane (3, 490 mg, 3.2 mmol, 39% yield). 1H NMR (CDCl3)
d:
3.2.3. Inhibition activity of in-vitro cell growth
1.40–1.50 (4H, m), 1.50–1.70 (2H, m), 1.75–1.90 (2H, m), 2.10 (3H, s),
J774.1 cells were maintained in a humidified atmosphere of 5%
CO2 at 37 ꢃC and passaged every 4 d. The culture medium was
RPMI-1640 containing 5% fetal bovine serum (FBS) and genta-
2.50 (2H, t, J ¼ 7.2 Hz), 2.95 (2H, t, J ¼ 7.1 Hz). 13C NMR (CDCl3)
d
:
15.5, 27.5, 27.9, 28.8, 29.8, 33.9, 34.0, 112.3. IR nmax (film) cmꢀ1
:
2934, 2857, 2153, 1460, 1435. HREI-MS: Found: 189.0642, calcd. for
micin sulfate (10
m
g/mL). J774.1 cells (1ꢂ104/0.2 mL) in each well
C8H15NS2 (Mþ): 189.0646.
of a 96-well flat-bottomed microplate (Greiner) were cultured at
37 ꢃC for 1 d. After addition of 10
mL serial twofold diluted solu-
3.1.10. Synthesis of 1-(Methylsulfinyl)-6-thiocyanatohexane (4)
An aqueous solution (30%) of hydrogen peroxide (50 mL,
0.45 mmol) was added to a stirred solution of 1-(methylthio)-6-
tion of test compounds, microplates were further incubated at
37 ꢃC for 1 d. Cell viability was determined by a [3-(4,5-dime-
thylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide] (MTT) assay
(Sigma–Aldrich) [16].
thiocyanatohexane (3, 57 mg, 0.3 mmol) in acetic acid (1.0 mL) at

4936
T. Noshita et al. / European Journal of Medicinal Chemistry 44 (2009) 4931–4936
[9] A. Prawan, Y.-S. Keum, T.O. Khor, S.Yu.S. Nair, W. Li, L. Hu, Ah-Ng T. Kong, Structural
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