Practical synthesis of precursor of [N-methyl-11C]vorozole, an efficient PET tracer targeting aromatase in the brain

Article abstract of DOI:10.1016/j.bmc.2010.12.057

A practical method to prepare precursor of [N-methyl-¹¹C] vorozole ([¹¹C]vorozole), an efficient positron emission tomography (PET) tracer for imaging aromatase in the living body, was established. Sufficient amount of the racemate including norvorozole, a demethylated vorozole derivative used as a precursor of [¹¹C]vorozole, became available by means of high-yield eight-step synthesis. The enantiomers were separated by preparative HPLC using a chiral stationary phase column to give optically pure norvorozole and its enantiomer. From the latter, ent-[¹¹C]vorozole, an enantiomer of [¹¹C]vorozole, was prepared and used in the PET study for the first time, which was shown to bind very weakly to aromatase in rhesus monkey brain supporting the previous pharmacological results. The stable supply of norvorozole will facilitate further researches on aromatase in the living body including brain by the PET technique.

Full text of DOI:10.1016/j.bmc.2010.12.057

Bioorganic & Medicinal Chemistry 19 (2011) 1464–1470
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Practical synthesis of precursor of [N-methyl-11C]vorozole, an efficient PET
tracer targeting aromatase in the brain
Kayo Takahashi a, Gen Yamagishi b, Toshiyuki Hiramatsu b,c, Ayako Hosoya b,c, Kayo Onoe a, Hisashi Doi a,
Hiroko Nagata a, Yasuhiro Wada a, Hirotaka Onoe a, Yasuyoshi Watanabe a, Takamitsu Hosoya b,c,
⇑
a RIKEN Center for Molecular Imaging Science, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
b Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
c Laboratory of Chemical Biology, Graduate School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai,
Chiyoda-ku, Tokyo 101-0062, Japan
a r t i c l e i n f o
a b s t r a c t
A practical method to prepare precursor of [N-methyl-11C]vorozole ([11C]vorozole), an efficient positron
emission tomography (PET) tracer for imaging aromatase in the living body, was established. Sufficient
amount of the racemate including norvorozole, a demethylated vorozole derivative used as a precursor
of [11C]vorozole, became available by means of high-yield eight-step synthesis. The enantiomers were
separated by preparative HPLC using a chiral stationary phase column to give optically pure norvorozole
and its enantiomer. From the latter, ent-[11C]vorozole, an enantiomer of [11C]vorozole, was prepared and
used in the PET study for the first time, which was shown to bind very weakly to aromatase in rhesus
monkey brain supporting the previous pharmacological results. The stable supply of norvorozole will
facilitate further researches on aromatase in the living body including brain by the PET technique.
Ó 2010 Elsevier Ltd. All rights reserved.
Article history:
Received 10 December 2010
Revised 27 December 2010
Accepted 28 December 2010
Available online 1 January 2011
Keywords:
Aromatase inhibitor
Vorozole
Positron emission tomography (PET)
Carbon-11
1. Introduction
receptor, which is expressed in the tumor cells. Furthermore,
63–72% of breast cancer cells possess aromatase activity,9 which
Aromatase, one of the monooxygenase families of enzymes,1
catalyzes the biosynthetic conversion of androgens to estrogens.2,3
It consists of a specific aromatase cytochrome P450, the CYP19
gene product, and the flavoprotein NADPH-cytochrome P450
reductase. Aromatase has a single binding site for the substrates
in the highly conserved heme domain of the enzyme.4 The recent
X-ray structural study using native aromatase purified from hu-
man placenta clearly showed that the substrates fit tightly in the
androgen-specific cleft, in which the aromatization takes place.5
This makes a sharp contrast with many other microsomal P450s,
which can recognize and metabolize various structurally-diverse
drugs and xenobiotics owing to their flexible active sites.
In humans, aromatase is expressed in a wide variety of tissues
such as placenta, ovary, adipose tissue, skin, central nervous sys-
tem, hair follicles, testicle, liver, and muscle.2,4 Abundant amount
of aromatase is expressed in the ovary, which is the major source
of circulating estrogens in premenopausal women. Though, after
menopause, the ovary ceases to produce estrogens, aromatase
continues to synthesize estrogens in the non-ovarian tissues,6
including the breast tissue.7 Approximately two thirds of postmen-
opausal breast cancer patients have estrogen-dependent cancer,8
that is, estrogen can stimulate tumor growth by binding to its
triggers the local synthesis of estrogen promoting the tumor
growth. Hence, aromatase is one of the most common target
proteins in the treatment of hormone-dependent breast cancer.
Indeed, several aromatase inhibitors have been used clinically for
the treatment of breast cancer in postmenopausal patients.8,10
Aromatase inhibitors can be categorized into two general types
based on their structural feature, namely steroidal and non-
steroidal inhibitors (Fig. 1).8,10 Exemestane (1, Aromasin™),
structurally similar to endogenous androgens, is a representative
steroidal inhibitor, which binds irreversibly to the enzyme and
inactivates it.11 In contrast, the non-steroidal inhibitors such
as anastrozole (2, Arimidex™)12 and letrozole (3, Femara™),13
competitively inhibit the enzymatic activity of aromatase in a
⇑
Corresponding author. Tel./fax: +81 3 5280 8117.
E-mail address: thosoya.cb@tmd.ac.jp (T. Hosoya).
Figure 1. Clinically-used aromatase inhibitors.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.057

K. Takahashi et al. / Bioorg. Med. Chem. 19 (2011) 1464–1470
1465
reversible manner. The triazole moiety of these drugs has been
considered to coordinate to the iron atom that resides in the heme
domain of the enzyme showing highly selective inhibitory activity
toward aromatase.14 These three drugs are included in the third-
generation aromatase inhibitors and have been evaluated
clinically.15
Expression of aromatase is detected not only in the peripheral
systems, but also in the brain, in which its functions have been
attracting much attention.16 It has been considered that aromatase
in the brain is related to reproductive behaviors and sexual differ-
entiation of the brain.16a Recently, it has been suggested to be
involved in neuroprotection16b,c as well as emotional behaviors.17
Although most of the studies of detecting aromatase proteins in
tissues had been performed by immunohistochemistry and
Western blot and Northern blot analyses, these techniques are
inadequate to survey the dynamic functions of aromatase in the
living body including the brain. In fact, the relationships between
the aromatase enzymatic activity and its diverse functions in the
brain are not fully understood.
the literatures including patents. With the aim of promoting the
PET study of aromatase in the brain, we examined to establish a
practical preparative method of norvorozole ((S)-5). Herein we de-
scribe the synthetic route of racemic mixture of norvorozole ((S)-5)
and its enantiomer as well as the conditions of their optical resolu-
tion. The results of PET studies comparing the binding of
[
11C]vorozole and its enantiomer toward aromatase in rhesus mon-
key brain are also noted.
2. Results and discussion
2.1. Synthesis and optical resolution of racemic mixture of
norvorozole and its enantiomer
At the early phase of the drug development, the biological activ-
ities of vorozole ((S)-4) were evaluated as a racemic mixture with
its enantiomer, which was referred to as R 76713 (rac-4) (Fig. 2).28a
Afterward, the (+)-(S)-isomer, R 83842, and its enantiomer, R
83839 (ent-vorozole, (R)-4), were prepared in an optically pure
form and the former isomer with more potent bioactivities was re-
ferred to as vorozole ((S)-4).20,23,28 Indeed, the IC50 values of voroz-
ole ((S)-4) and ent-vorozole ((R)-4) toward aromatase in rat
granulosa cells were reported as 0.44 and 240 nM, respectively,
showing more than 500-fold difference in potency between the
enantiomers.28c The Ki value of vorozole ((S)-4) against human pla-
cental aromatase was 0.7 nM, which was also 25-times more po-
tent than ent-vorozole.28c Therefore, the clinical studies were
focused on the more potent enantiomer, vorozole ((S)-4).
Vorozole ((S)-4), in an optically pure form, was prepared from
an optically pure synthetic intermediate, which was obtained by
selective crystallization of diasteromeric salts of hydrazine deriva-
tive and a chiral acid.29 This optical resolution procedure was opti-
mized to obtain vorozole ((S)-4) and preparation of no other
related compounds in optically pure form including norvorozole
((S)-5) were reported. In principle, norvorozole ((S)-5) can be ob-
tained directly from vorozole ((S)-4) by removing its N-methyl
group. In usual, however, it is not easy to remove N-methyl group
under mild condition without racemization. Moreover, preparing
optically pure norvorozole ((S)-5) by the method mentioned above
for vorozole ((S)-4) seemed unsuitable because N-methyl group
was introduced at the early stage of the synthesis.29 Thus, we
decided to establish a new synthetic route, which is appropriate
to obtain sufficient amount of norvorozole ((S)-5).
In order to study aromatase expressed in the living body, positron
emission tomography (PET) technique, a noninvasive in vivo molec-
ular imaging method, has been introduced.18 In the PET study, a PET
tracer labeled with a short-lived positron-emitting radionuclide
such as 11C and 18F with a half-life period of 20 and 110 min, respec-
tively, is employed. So far, some PET tracers with potent aromatase
inhibitory activity including [N-methyl-11C]vorozole ([11C]vorozole,
(S)-[11C]-4) (Fig. 2),19 11C-labeled sulfonanilide analogs,20 11C-
labeled sulfamate derivatives (dual aromatase–steroid sulfatase
inhibitors),21 and [cyano-11C]letrozole,22 have been developed.
Vorozole ((S)-4) is a potent and specific non-steroidal aroma-
tase inhibitor, which was developed to treat breast cancer.23 It
shares a common triazole substructure with anastrozole (2) and
letrozole (3) showing higher inhibitory activity toward aromatase
than these clinically-used drugs.24 Since vorozole has a methyl
group, which is covalently bound to one of the nitrogen atom on
the benzotriazole ring, [11C]vorozole has been prepared easily by
SN2-type N-[11C]methylation of norvorozole ((S)-5), a demethyl
derivative of vorozole, treating it with [11C]H3I under basic condi-
tions.19 Recently, it was pointed out that all of three possible N-
methylations proceed by this reaction and careful separation of
[
11C]vorozole ((S)-[11C]-4) from the inactive isomers should be ta-
ken into account to obtain reliable PET images.25 By using
11C]vorozole as a PET tracer, the localization of aromatase on the
[
amygdala and hypothalamus in living rhesus monkey brain was
discovered,26a and increased aromatase level in the hypothalamic
region of rats evoked by treatment of anabolic-androgenic steroids
was observed as well.26b,c Recent PET study using [11C]vorozole in
human brain has revealed unique distribution of aromatase in thal-
amus and medulla.27
In order to prepare norvorozole ((S)-5) in a practical manner, we
chose to synthesize the racemate first and separate the enantio-
mers by preparative HPLC using a chiral stationary phase column.
We also decided to construct the benzotriazole unit at the final
step to avoid the unnecessary difficulties for structural analysis
of intermediate compounds, which would arise from the formation
of benzotriazole isomers. Based on these ideas, we finally suc-
ceeded in developing a practical preparative method of norvoroz-
ole ((S)-5) and its enantiomer ent-norvorozole ((R)-5) as
summarized in Scheme 1.
Despite the utility of [11C]vorozole in PET studies, its precursor,
norvorozole ((S)-5), is not easily available. At present, it almost en-
tirely depends on donation from the pharmaceutical company and
its detailed synthetic procedure has not been appeared in any of
We selected commercially available 4,40-dichloro-3-nitrobenz-
ophenone (6) as the starting material. At first, one of the two chloro
groups attached on the more electron deficient benzene ring was
selectively displaced by an azido group to give 7. Subsequent cat-
alytic hydrogenation of 7 led to reduction of azido and nitro groups
at once to give o-phenylenediamine derivative 8. The protection of
the both amino groups of 8 by Boc group followed by sodium boro-
hydride reduction gave benzhydrol derivative 10. Treatment of
alcohol 10 with mesyl chloride in the presence of triethylamine
afforded a mixture of corresponding mesylate 11 and chloride 12,
which was produced by chlorination of 11. Since both compounds
could be used in the next step equally, we did not optimize the
Figure 2. Structures of vorozole, [N-methyl-11C]vorozole, norvorozole, and their
enantiomers.

1466
K. Takahashi et al. / Bioorg. Med. Chem. 19 (2011) 1464–1470
anesthetized rhesus monkeys and their brain was scanned by a
O
O
PET camera. The distribution volume ratio (DVR) images obtained
for horizontal and coronal sections are shown in Figure 3, in which
the binding potential of each tracer to brain aromatase are repre-
sented. Consequently, a high DVR of [11C]vorozole ((S)-[11C]-4)
was observed in the amygdala and hypothalamus which was con-
sistent with the previous study.26a On the contrary, relatively low
DVR of ent-[11C]vorozole ((R)-[11C]-4) was detected throughout
the brain.
NO2
R
R
R
b
d
Cl
Cl
6: R = Cl
7: R = N3
8: R = NH2
9: R = NHBoc
a
c
N
N
N
X
Based on the results of PET images, the time–activity curves of
PET tracers were obtained. As employed in the previous study,26a
the cerebellum region was selected as a reference region, which
was regarded not to contain aromatase enzyme. Thus, the normal-
ized time–activity curves of [11C]vorozole ((S)-[11C]-4) and ent-
NHBoc
NHBoc
R
R
f
h
Cl
Cl
10: X = OH
13: R = NHBoc
15: R = NH2
e
g
11: X = OMs
12: X = Cl
[
11C]vorozole ((R)-[11C]-4) in the aromatase-rich regions, the
amygdala and hypothalamus, were generated by dividing by that
of the cerebellum region (Fig. 4). As a result, the normalized SUVs
(standardized uptake values), the ratios of aromatase-rich regions
to the reference region, of [11C]vorozole ((S)-[11C]-4), were higher
than 1.0 throughout the monitoring time, while that of ent-
N
N
N
N
N
N
H
NHBoc
NHBoc
N
N
[
[
11C]vorozole ((R)-[11C]-4) were close to 1.0, indicating that
11C]vorozole ((S)-[11C]-4) binds to aromatase more specifically,
N
Cl
Cl
rac-5
14
but ent-[11C]vorozole ((R)-[11C]-4) does not.
Scheme 1. Synthesis of racemic mixture of norvorozole and its enantiomer.
Reagents and conditions: (a) NaN3, DMF, 0 °C to room temperature, 2 h, 98%; (b) H2,
10% Pd–C, THF, room temperature, 15 h, 82%; (c) Boc2O, iPr2NEt, reflux, 24 h, 44%;
(d) NaBH4, THF–EtOH (1:1, v/v), 0 °C to room temperature, 2 h, quant.; (e) CH3SO2Cl,
Et3N, THF–CH2Cl2 (1:1, v/v), 0 °C to room temperature, 4 h; (f) 1,2,4-triazole, K2CO3,
acetone, room temperature, 16 h, 69% of 13 (two-steps) and 15% of 14 (two-steps);
(g) TFA, CH2Cl2, 0 °C, 4 h, 85%; (h) NaNO2, aq HCl, 0 °C, 1 h, 93%.
DVR images and normalized time–activity curves indicated that
the binding of ent-[11C]vorozole ((R)-[11C]-4) in the amygdala and
hypothalamus is less specific than that of [11C]vorozole ((S)-[11C]-
4). Our present result showing the large difference of aromatase
binding between [11C]vorozole ((S)-[11C]-4) and ent-[11C]vorozole
((R)-[11C]-4) was consistent with previously reported IC50 and Ki val-
ues for these enantiomers.28 Furthermore, it was demonstrated that
nosignificant epimerizationof vorozoleoccursin invivo atleastdur-
ingtheperiodof PETscanning. Thisinformation, in general, wouldbe
important to achieve for optically active drug candidates.
reaction conditions any more and the mixture was used without
further purification. Thus, the mixture of 11 and 12 was reacted
with 1,2,4-triazole under basic conditions to form approximately
5:1 mixture of desired triazole derivative 13 and its regioisomer
14, which were easily separated by chromatography. After depro-
tection of Boc groups of 13 by TFA, the resulting diamine 15 was
treated with sodium nitrite under acidic conditions to construct
the benzotriazole unit affording rac-5, a racemic mixture of norv-
orozole ((S)-5) and its enantiomer, ent-norvorozole ((R)-5). The
TLC behavior and 1H NMR spectrum of the synthetic sample
matched exactly with those of the authentic sample. It took only
eight simple reaction steps in approximately 20% overall yield to
prepare rac-5 from the starting material, showing the practicality
of the synthetic route.
3. Conclusion
In summary, we established a practical preparative method of
optically pure norvorozole ((S)-5) by means of high-yield eight-
step synthesis of the racemate followed by its separation by pre-
parative HPLC using a chiral stationary phase column. The stable
supply of norvorozole ((S)-5) will facilitate further researches on
aromatase in the living body by the aromatase specific PET tracer,
[
11C]vorozole ((S)-[11C]-4).
Next, we examined to separate the enantiomers of rac-5 by pre-
parative HPLC using a chiral stationary phase column. After some
efforts to screen separating conditions including column types,
combination and composition of solvents for mobile phase, we
found the practical conditions. Consequently, using CHIRALPAK
AD-H column (Daicel Chemical) eluting with EtOH/MeOH (7:3,
v/v) was effective to obtain both norvorozole ((S)-5) and its enan-
tiomer ent-norvorozole ((R)-5) in optically pure forms (Supple-
mentary Fig. 1). The isomer that eluted later was unambiguously
determined as norvorozole ((S)-5) by comparing it with the
authentic sample.
2.2. PET imaging using [11C]vorozole and ent-[11C]vorozole in
the rhesus monkey brain
With both norvorozole ((S)-5) and ent-norvorozole ((R)-5) in
hand, we were interested in comparing the brain distribution of
[
11C]vorozole ((S)-[11C]-4) and its enantiomer, ent-[11C]vorozole
Figure 3. The distribution volume ratio (DVR) images of [11C]vorozole ((S)-[11C]-4)
(upper panels) and ent-[11C]vorozole ((R)-[11C]-4) (lower panels) in the rhesus
monkey brain. Left panels: horizontal sectional view; Right panels: coronal
sectional view. White and yellow arrows indicate amygdala and hypothalamus,
respectively.
((R)-[11C]-4). Thus, both PET tracers were synthesized and carefully
purified according to the modified method reported recently.25
The prepared PET tracer, [11C]vorozole ((S)-[11C]-4) or ent-
[
11C]vorozole ((R)-[11C]-4), were intravenously administered to

K. Takahashi et al. / Bioorg. Med. Chem. 19 (2011) 1464–1470
1467
room temperature and stirred for 2 h. To this was added water
(300 mL) and the mixture was extracted with EtOAc
(150 mL ꢀ 3). The combined organic extracts were successively
washed with water (100 mL ꢀ 3) and brine (100 mL ꢀ 1), dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography (silica-gel
220 g, n-hexane/EtOAc = 9:1) to give 4-azido-40-chloro-3-nitro-
benzophenone (7) (17.6 g, 98.4%) as a pale yellow solid; TLC
Rf = 0.41 (n-hexane/EtOAc = 4:1); 1H NMR (300 MHz, CDCl3) d
7.47 (d, 1H, J = 8.5 Hz, aromatic), 7.49–7.57 (AA0BB0, 2H, aromatic),
7.69–7.78 (AA0BB0, 2H, aromatic), 8.08 (dd, 1H, J = 2.0, 8.5 Hz, aro-
matic), 8.34 (d, 1H, J = 2.0 Hz, aromatic); 13C NMR (75.5 MHz,
CDCl3) d 120.8, 127.6, 129.0 (2C), 131.1 (2C), 133.4, 134.4, 134.7,
138.6, 139.7, 140.1, 191.6; IR (KBr, cmꢁ1) 687, 714, 754, 847,
876, 914, 964, 980, 1015, 1072, 1090, 1175, 1256, 1294, 1352,
1400, 1487, 1533, 1566, 1587, 1607, 1661, 2131; HRMS (EI) m/z
302.0205 (M+, C13H735ClN4O3 required 302.0207).
Figure 4. The normalized time–activity curves of [11C]vorozole ((S)-[11C]-4) (solid
line) and ent-[11C]vorozole ((R)-[11C]-4) (dashed line) at aromatase-rich regions, the
amygdala (filled rhombus) and hypothalamus (filled square), in the rhesus monkey
brain. SUV (standardized uptake value) represents as (radioactivity in tissue/
volume of tissue)/(injected radioactivity/body weight).
4.1.2. 3,4-Diamino-40-chlorobenzophenone (8)
Under argon atmosphere, to a solution of 4-azido-40-chloro-3-
nitrobenzophenone (7) (5.00 g, 16.5 mmol) in THF (80 mL) was
added palladium/charcoal activated (10%) (500 mg) at room tem-
perature. The gas in the reaction flask was evacuated and refilled
with hydrogen gas. After stirring for 15 h, the internal gas was re-
turned to argon and to the mixture was added CH2Cl2 (300 mL) and
stirred for 30 min under air. The catalyst was removed by filtration
and the filtrate was concentrated under reduced pressure. The res-
idue was purified by column chromatography (silica-gel 220 g,
CH2Cl2/CH3OH = 40:1) to give 3,4-diamino-40-chlorobenzophenone
4. Experimental
4.1. Chemistry
General: All chemical reagents used were commercial grade and
used as received. Analytical thin-layer chromatography (TLC) was
performed on precoated (0.25 mm) silica-gel plates (Merck Chem-
icals, Silica Gel 60 F254). Column chromatography was conducted
using silica-gel (Kanto Chemical Co., Inc., Silica Gel 60N, spherical
(8) (3.36 g, 82.4%) as
a brown solid; TLC Rf = 0.35 (CH2Cl2/
CH3OH = 15:1); 1H NMR (300 MHz, DMSO-d6) d 4.73 (s, 2H, NH2),
5.54 (s, 2H, NH2), 6.52 (dd, 1H, J = 1.4, 1.8 Hz, aromatic), 6.87 (dd,
1H, J = 1.8, 8.1 Hz, aromatic), 7.03 (dd, 1H, J = 1.4, 8.1 Hz, aromatic),
7.50–7.56 (AA0BB0, 2H, aromatic), 7.56–7.62 (AA0BB0, 2H, aromatic);
13C NMR (75.5 MHz, DMSO-d6) d 112.3, 115.4, 122.9, 124.9, 128.2
neutral, particle size 40–50
resolution of racemic compound was performed by preparative
HPLC using chiral stationary phase column CHIRALPAK
guard column;
lm or 63–210 lm). The optical
a
(2C), 130.8 (2C), 134.0, 135.8, 138.3, 141.6, 192.9; IR (KBr, cmꢁ1
)
AD-H (20 mm i.d. ꢀ 250 mm, attached with
a
758, 818, 845, 870, 905, 1090, 1142, 1161, 1292, 1325, 1396,
1449, 1518, 1558, 1587, 1612, 3370, 3435; HRMS (FAB+/NBA+KCl)
m/z 285.0204 ([M+K]+, C13H1135ClN2OK required 285.0197).
10 mm i.d. ꢀ 20 mm, Daicel Chemical Ind., Ltd, Japan). The optical
purity of separated compounds was determined by analytical
HPLC using a chiral stationary phase column CHIRALPAK AD-H
(4.6 mm i.d. ꢀ 250 mm, attached with
4.0 mm i.d. ꢀ 10 mm, Daicel Chemical Ind., Ltd, Japan). Specific
optical rotation ([ ]D) was measured on a JASCO DIP-370 digital
a
guard column;
4.1.3. 40-Chloro-3,4-di(tert-butoxycarbonylamino)benzo-
phenone (9)
a
Under argon atmosphere, to a solution of 3,4-diamino-40-chloro-
benzophenone (8) (2.86 g, 11.6 mmol) in THF (100 mL) was succes-
sively added N,N-diisopropylethylamine (4.85 mL, 27.8 mmol) and
di-tert-butyl dicarbonate (6.40 mL, 27.9 mmol) at room tempera-
ture, and the mixture was refluxed (oil bath temperature 70 °C)
for 24 h. After cooling to room temperature, to this was added 1 M
aqueous HCl solution (300 mL) and the mixture was extracted with
EtOAc (100 mL ꢀ 3). The combined organic extracts were succes-
sively washed with water (100 mL ꢀ 1) and brine (100 mL ꢀ 1),
dried over Na2SO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography (silica-
gel 230 g, n-hexane/EtOAc = 6:1) to give 40-chloro-3,4-di(tert-but-
oxycarbonylamino)benzophenone (9) (2.28 g, 44.0%) as a colorless
solid; TLC Rf = 0.57 (n-hexane/EtOAc = 2:1); 1H NMR (300 MHz,
CDCl3) d 1.52 (s, 9H, 3CH3), 1.54 (s, 9H, 3CH3), 6.42–6.60 (br, 1H,
NH), 7.15–7.24 (br, 1H, NH), 7.42–7.51 (AA0BB0, 2H, aromatic),
7.59 (dd, 1H, J = 1.9, 8.5 Hz, aromatic), 7.71–7.77 (AA0BB0, 2H, aro-
matic), 7.80 (d, 1H, J = 1.9 Hz, aromatic), 7.90 (d, 1H, J = 8.5 Hz, aro-
matic); 13C NMR (75.5 MHz, CDCl3) d 28.11 (3C), 28.13 (3C), 81.3,
81.4, 121.4, 127.1, 127.9, 128.2, 128.5 (2C), 131.3 (2C), 132.3,
135.7, 136.3, 138.6, 152.9, 154.1, 194.0; IR (KBr, cmꢁ1) 737, 758,
1049, 1090, 1157, 1242, 1314, 1368, 1393, 1420, 1526, 1587,
1647, 1701, 1730, 2978, 3310; HRMS (FAB+/NBA+NaI) m/z
469.1508 ([M+Na]+, C23H2735ClN2O5Na required 469.1506).
polarimeter. 1H and 13C NMR spectra were obtained with a Varian
MERCURY 300 spectrometer at 300 and 75.5 MHz, respectively.
CDCl3 (CIL) and DMSO-d6 (CIL) were used as solvents for obtaining
NMR spectra. Chemical shifts (d) are given in parts per million
(ppm) downfield from (CH3)4Si (d 0.00 for 1H NMR in CDCl3) or
the solvent peak (d 2.49 for 1H NMR and d 39.5 for 13C NMR in
DMSO-d6, and d 77.0 for 13C NMR in CDCl3) as an internal reference
with coupling constants (J) in hertz (Hz). The abbreviations s, d, m,
and br signify singlet, doublet, multiplet, and broad, respectively.
IR spectra were measured by diffuse reflectance method on a SHI-
MADZU IRPrestige-21 spectrometer attached with DRS-8000A
with the absorption band given in cmꢁ1. High-resolution mass
spectra (HRMS) were measured on a JEOL JMS-700 mass spectrom-
eter under or electron impact ionization (EI) condition or positive
fast atom bombardment (FAB+) condition using m-nitrobenzyl
alcohol (NBA) as a matrix at the Center for Advanced Materials
Analysis (Suzukakedai), Technical Department, Tokyo Institute of
Technology.
4.1.1. 4-Azido-40-chloro-3-nitrobenzophenone (7)
To a solution of 4,40-dichloro-3-nitrobenzophenone (6, Sigma–
Aldrich) (17.5 g, 59.1 mmol) in DMF (60 mL) was added sodium
azide (4.61 g, 70.9 mmol) at 0 °C. The mixture was warmed to

1468
K. Takahashi et al. / Bioorg. Med. Chem. 19 (2011) 1464–1470
4.1.4. 40-Chloro-3,4-di(tert-butoxycarbonylamino)benzhydrol
(10)
ylamino)benzene (14) (248 mg, 14.8%, two-step yield based on 10)
as a colorless solid; TLC Rf = 0.50 (EtOAc only), Rf = 0.12 (n-hexane/
EtOAc = 1:1); 1H NMR (300 MHz, CDCl3) d 1.49 (s, 9H, 3CH3), 1.52
(s, 9H, 3CH3), 6.50 (s, 1H, CH), 6.75–6.88 (m, 2H, aromatic), 7.00–
7.10 (AA0BB0, 2H, aromatic), 7.30–7.43 (m, 3H, aromatic and
2NH), 7.55 (d, 1H, J = 8.0 Hz, aromatic), 8.04 (s, 2H, aromatic); 13C
NMR (75.5 MHz, CDCl3) d 28.1 (6C), 62.6, 81.15, 81.20, 123.4,
124.0, 124.8, 129.1 (2C), 129.4 (2C), 131.0, 133.4, 135.0, 135.9,
142.6 (2C), 146.6, 153.7 (2C); IR (KBr, cmꢁ1) 650, 737, 783, 1015,
1024, 1051, 1091, 1157, 1246, 1308, 1368, 1491, 1522, 1708,
2978, 3294; HRMS (FAB+/NBA+NaI) m/z 522.1878 ([M+Na]+,
Under argon atmosphere, to a solution of 40-chloro-3,4-di(tert-
butoxycarbonylamino)benzophenone (9) (3.00 g, 6.71 mmol) in a
1:1 mixture of THF–EtOH (60 mL) was added sodium borohydride
(254 mg, 6.71 mmol) at 0 °C. The mixture was allowed to warm to
room temperature and stirred for 2 h. To this was added water
(200 mL) and the mixture was extracted with EtOAc
(150 mL ꢀ 3). The combined organic extracts were successively
washed with water (100 mL ꢀ 1) and brine (100 mL ꢀ 1), dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography (silica-gel
120 g, n-hexane/EtOAc = 2:1) to give 40-chloro-3,4-di(tert-butoxy-
carbonylamino)benzhydrol (10) (3.22 g, quant.) as a colorless so-
lid; TLC Rf = 0.36 (n-hexane/EtOAc = 2:1); 1H NMR (300 MHz,
CDCl3) d 1.499 (s, 9H, 3CH3), 1.504 (s, 9H, 3CH3), 2.57 (d, 1H,
J = 3.4 Hz, OH), 5.75 (d, 1H, J = 3.4 Hz, CH), 6.65–6.84 (br, 2H,
2NH), 7.06 (dd, 1H, J = 1.9, 8.2 Hz, aromatic), 7.27–7.31 (AA0BB0,
4H, aromatic), 7.43 (d, 1H, J = 8.2 Hz, aromatic), 7.47 (d, 1H,
J = 1.9 Hz, aromatic); 13C NMR (75.5 MHz, CDCl3) d 28.2 (6C),
74.6, 80.8, 80.9, 122.4, 123.5, 124.2, 127.8 (2C), 128.3 (2C), 129.4,
130.0, 132.8, 140.8, 142.0, 154.0 (2C); IR (KBr, cmꢁ1) 737, 770,
818, 1015, 1026, 1049, 1090, 1157, 1248, 1368, 1393, 1491,
1524, 1597, 1697, 2978, 3329; HRMS (FAB+/NBA+NaI) m/z
471.1674 ([M+Na]+, C23H2935ClN2O5Na required 471.1663).
C
25H3035ClN5O4Na required 522.1884).
4.1.6. 1,2-Diamino-4-{(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)-
methyl}benzene (15)
Under argon atmosphere, to
a solution of 4-{(4-chloro-
phenyl)(1H-1,2,4-triazol-1-yl)methyl}-1,2-di(tert-butoxycarbonyl-
amino)benzene (13) (1.00 g, 2.00 mmol) in CH2Cl2 (2.0 mL) was
added trifluoroacetic acid (2.0 mL) at 0 °C. After stirring at the
same temperature for 4 h, to this was added saturated aqueous
NaHCO3 solution (100 mL) and the mixture was extracted with
CH2Cl2 (50 mL ꢀ 3). The combined organic extracts were dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography (silica-
gel 120 g, EtOAc only to EtOAc/CH3OH = 15:1) to give 1,2-diami-
no-4-{(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl}benzene (15)
(511 mg, 85.2%) as a colorless solid; TLC Rf = 0.41 (EtOAc only);
1H NMR (300 MHz, DMSO-d6) d 4.48–4.55 (br, 4H, 2NH2), 6.24
(dd, 1H, J = 1.9, 8.0 Hz, aromatic), 6.41 (d, 1H, J = 1.9 Hz, aromatic),
6.47 (d, 1H, J = 8.0 Hz, aromatic), 6.75 (s, 1H, CH), 7.13–7.16
(AA0BB0, 2H, aromatic), 7.39–7.41 (AA0BB0, 2H, aromatic), 8.03 (s,
1H, aromatic), 8.42 (s, 1H, aromatic); 13C NMR (75.5 MHz, DMSO-
d6) d 65.4, 113.9, 114.0, 117.2, 126.7, 128.3 (2C), 129.5 (2C),
132.2, 135.0, 135.1, 139.1, 144.0, 151.7; IR (KBr, cmꢁ1) 611, 658,
679, 741, 789, 856, 961, 1015, 1090, 1136, 1202, 1275, 1296,
1406, 1445, 1491, 1518, 1593, 1626, 3345; HRMS (FAB+/NBA) m/z
299.0945 (M+, C15H1435ClN5 required 299.0938).
4.1.5. 4-{(4-Chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl}-1,2-
di(tert-butoxycarbonylamino)benzene (13)
Under argon atmosphere, to a solution of 40-chloro-3,4-di(tert-
butoxycarbonylamino)benzhydrol (10) (1.50 g, 3.34 mmol) in a
1:1 mixture of THF–CH2Cl2 (28 mL) was successively added trieth-
ylamine (700
lL, 5.02 mmol) and methanesulfonyl chloride
(310 L, 4.01 mmol) at 0 °C. The mixture was allowed to warm to
l
room temperature and stirred for 4 h. This was poured into water
(100 mL) and the mixture was extracted with CH2Cl2 (100 mL ꢀ 3).
The combined organic extracts were washed with brine
(100 mL ꢀ 1), dried over Na2SO4, filtered, and concentrated under
reduced pressure to give a crude mixture of 4-{chloro(4-chloro-
phenyl)methyl}-1,2-di(tert-butoxycarbonylamino)benzene
and {3,4-bis(tert-butoxycarbonylamino)phenyl}(4-chloro-
(11)
4.1.7. 6-{(4-Chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl}-1H-
benzo[d][1,2,3]triazole (rac-5)
phenyl)methyl methanesulfonate (12) (2.02 g) as a colorless oil.
Under argon atmosphere, to a solution of the crude product ob-
tained as above in acetone (30 mL) was successively added potas-
sium carbonate (1.38 g, 10.0 mmol) and 1,2,4-triazole (461 mg,
6.68 mmol) at room temperature. After stirring for 16 h at the
same temperature, the mixture was filtered and the filtrate was
concentrated under reduced pressure. The residue was purified
by column chromatography (silica-gel 120 g, n-hexane/
EtOAc = 3:2–2:3) to give 4-{(4-chlorophenyl)(1H-1,2,4-triazol-1-
yl)methyl}-1,2-di(tert-butoxycarbonylamino)benzene (13) (1.16 g,
68.8%, two-step yield based on 10) as a colorless solid; TLC
Rf = 0.50 (n-hexane/EtOAc = 1:1); 1H NMR (300 MHz, CDCl3) d
1.49 (s, 9H, 3CH3), 1.51 (s, 9H, 3CH3), 6.67 (s, 1H, CH), 6.72 (br s,
1H, NH), 6.83 (br s, 1H, NH), 6.90 (dd, 1H, J = 2.0, 8.7 Hz, aromatic),
7.00–7.10 (AA0BB0, 2H, aromatic), 7.27–7.40 (m, 3H, aromatic), 7.56
(d, 1H, J = 8.7 Hz, aromatic), 7.97 (s, 1H, aromatic), 8.02 (s, 1H, aro-
matic); 13C NMR (75.5 MHz, CDCl3) d 27.9 (6C), 66.1, 80.4, 80.5,
123.3, 123.7, 124.1, 128.6 (2C), 129.0 (2C), 130.1, 130.4, 133.1,
134.0, 136.2, 143.2, 151.7, 153.4 (2C); IR (KBr, cmꢁ1) 660, 679,
737, 787, 1015, 1049, 1091, 1157, 1244, 1368, 1493, 1526, 1597,
1709, 2978, 3308; HRMS (FAB+/NBA+NaI) m/z 522.1900 ([M+Na]+,
C25H3035ClN5O4Na required 522.1884).
To a solution of 1,2-diamino-4-{(4-chlorophenyl)(1H-1,2,4-tria-
zol-1-yl)methyl}benzene (14) (460 mg, 1.53 mmol) in 0.2 M aque-
ous HCl (30 mL) was added sodium nitrite (111 mg, 1.61 mmol) at
0 °C. After stirring at the same temperature for 1 h, to this was
added saturated aqueous NaHCO3 solution (100 mL) and the mix-
ture was extracted with EtOAc (50 mL ꢀ 3). The combined organic
extracts were successively washed water (50 mL ꢀ 1) and brine
(50 mL ꢀ 1), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy (silica-gel 50 g, EtOAc only) to give 6-{(4-chlorophenyl)(1H-
1,2,4-triazol-1-yl)methyl}-1H-benzo[d][1,2,3]triazole
(rac-5)
(444 mg, 93.1%) as a colorless solid; TLC Rf = 0.48 (EtOAc only);
1H NMR (300 MHz, DMSO-d6) d 7.20–7.30 (AA0BB0, 2H, aromatic),
7.334 (s, 1H, aromatic or CH), 7.340 (d, 1H, J = 8.7 Hz, aromatic),
7.40–7.53 (AA0BB0, 2H, aromatic), 7.67 (s, 1H, aromatic or CH),
7.92 (d, 1H, J = 8.7 Hz, aromatic), 8.11 (d, 1H, J = 1.7 Hz, aromatic),
8.67 (d, 1H, J = 1.7 Hz, aromatic), 15.6–16.0 (br, 1H, NH); 13C NMR
(75.5 MHz, DMSO-d6) d 64.1, 114.3, 115.6, 125.8, 128.7 (2C), 130.0
(2C), 132.9, 136.3, 137.9, 138.6, 144.8, 152.2 (One carbon was not
observed or overlapped with other peak. See 13C NMR spectrum
in Supplementary Fig. 2.); IR (KBr, cmꢁ1) 677, 739, 777, 856, 959,
995, 1015, 1092, 1136, 1206, 1275, 1410, 1437, 1491, 1636,
3372; HRMS (FAB+/NBA) m/z 311.0809 ([M+H]+, C15H1235ClN6 re-
quired 311.0812).
Further elution with EtOAc also gave the isomer 4-{(4-chloro-
phenyl)(4H-1,2,4-triazol-4-yl)methyl}-1,2-di(tert-butoxycarbon-

K. Takahashi et al. / Bioorg. Med. Chem. 19 (2011) 1464–1470
1469
4.1.8. (S)-6-((4-Chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl)-1H-
benzo[d][1,2,3]triazole (norvorozole, (S)-5) and (R)-6-((4-chlo-
rophenyl)(1H-1,2,4-triazol-1-yl)methyl)-1H-benzo[d][1,2,3]-
triazole (ent-norvorozole, (R)-5)
the emission scans, transmission scans were performed for 30 min
for attenuation correction.
[
11C]Vorozole ((S)-[11C]-4) (102–
195 MBq) or ent-[11C]vorozole ((R)-[11C]-4) (185–205 MBq) was
administered intravenously as a bolus. The monkeys were scanned
for 90 min (4 ꢀ 30 s, 3 ꢀ 60 s, 2 ꢀ 150 s, 2 ꢀ 300 s, and 7 ꢀ 600 s)
using a microPET Focus 220 (Siemens, Knoxville, TN, USA).
The racemic mixture rac-5 was separated to optically pure (S)-5
and (R)-5 by preparative HPLC using CHIRALPAK AD-H: mobile
phase, EtOH/MeOH = 70:30 (v/v); flow rate, 3.0 mL/min; detection,
UV at 254 nm. After concentration of each collected fraction under
reduced pressure, the residue was redissolved in H2O/MeOH and
lyophilized to give a colorless solid. The enantiomeric excess (ee)
of both of separated compounds was determined to be >99% by
analytical HPLC using CHIRALPAK AD-H: mobile phase, EtOH/
MeOH = 70:30 (v/v); flow rate, 1.0 mL/min; detection, UV at
254 nm; retention time, (S)-5: 6.8 min, (R)-5: 4.5 min (see analyti-
cal HPLC charts for rac-5, (S)-5, and (R)-5 in Supplementary Fig. 1);
4.3.3. Analysis of PET data
For quantitative analysis of PET images, kinetic modeling,
PMOD software (PMOD Technologies Ltd, Zurich, Switzerland)
was employed. Volumes of interest (VOIs) were delineated in the
amygdala, hypothalamus, and cerebellum. For each of these re-
gions, decay-corrected time–activity curves were generated. The
data were analyzed with a Logan noninvasive model31 using the
time–activity curve of the cerebellum as an input function, and
the slope of regression (=distribution volume ratio (DVR) = associ-
ation rate constant/dissociation rate constant + 1) and binding po-
tential (BP = DVR ꢁ 1 = association rate constant/dissociation rate
constant) were calculated. Parametric images of DVR were also
generated based on pixel-wise kinetic modeling.
specific optical rotation, (S)-5: ½a D26
ꢂ
+24.0° (c 1.0 in CHCl3), (R)-5:
½
a 2D6
ꢂ
ꢁ24.0° (c 1.0 in CHCl3).
4.2. PET chemistry
a)
General: Carbon-11 was produced by a 14N(p, 11C nuclear reac-
tion by using a CYPRISHM-12S Cyclotron (Sumitomo Heavy Indus-
try, Tokyo, Japan). An original automated radiolabeling system
consisting of the heating of the reaction mixture, dilution, HPLC
injection, fractional collection, evaporation, and sterile filtration
was used for the production of [11C]H3I and the 11C-labeling. Puri-
fication with semi-preparative HPLC was performed on a JASCO
system (Tokyo, Japan). Radioactivity was quantified with an
ATOMLAB™300 dose calibrator (Aloka, Tokyo, Japan). Analytical
Acknowledgments
The authors thank Ms. Masayo Ishikawa at the Center for Ad-
vanced Materials Analysis (Suzukakedai), Technical Department,
Tokyo Institute of Technology for HRMS analysis, Mr. Masahiro
Kurahashi at Sumitomo Heavy Industry Accelerator Service Ltd
for operating the cyclotrons, Ms. Emi Hayashinaka and Ms. Chiho
Takeda at RIKEN Center for Molecular Imaging Science for support-
ing the PET scan operation and supporting monkey handling,
respectively, and Daicel Chemical Ind., Ltd, Japan for preliminary
screening of conditions for optical resolution. This work was par-
tially supported by the Molecular Imaging Program of Research
Base for Exploring New Drugs, from the Ministry of Education, Cul-
ture, Sports, Science, and Technology (MEXT), Japan.
HPLC was performed on
a Shimadzu system (Kyoto, Japan)
equipped with pumps and a UV detector, and the effluent radioac-
tivity was determined by using a RLC700 radio analyzer (Aloka).
The columns used for the analytical and semi-preparative HPLC
were COSMOSIL C18 AR-II and cholester (Nacalai Tesque). [11C]H3I
was prepared as previously described.30
4.2.1. Synthesis of [N-methyl-11C]vorozole ((S)-[11C]-4) and ent-
[N-methyl-11C]vorozole ((R)-[11C]-4)
Supplementary data
[N-Methyl-11C]vorozole ([11C]vorozole, (S)-[11C]-4) was synthe-
sized by N-[11C]methylation of norvorozole ((S)-5) using [11C]H3I
according to the previously reported procedure.25 ent-[N-
Methyl-11C]vorozole (ent-[11C]vorozole, (R)-[11C]-4) was also syn-
thesized in the same manner using ent-norvorozole ((R)-5). The
Supplementary data (analytical HPLC charts for rac-5, (S)-5, and
(R)-5 and 13C NMR spectra of rac-5) associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2010.12.057.
References and notes
specific radioactivities of
11C]vorozole ((R)-[11C]-4) at the end of synthesis were in the range
of 13.5–90.7 and 35–42.9 GBq/ mol, respectively, and their radio-
[
11C]vorozole ((S)-[11C]-4) and ent-
[
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