Synthesis of [19, 35, 36-13C3]-labeled TAK779 as a molecular probe

Article abstract of DOI:10.1016/j.bmc.2009.07.026

N,N-Dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride (TAK779) is a potent and selective non-peptide CCR5 antagonist. To use a site-specifically labeled form as a molecula

Full text of DOI:10.1016/j.bmc.2009.07.026

Bioorganic & Medicinal Chemistry 17 (2009) 5769–5774
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Bioorganic & Medicinal Chemistry
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Synthesis of [19, 35, 36-13C3]-labeled TAK779 as a molecular probe
c
Hiroyuki Konno a,b, , Saburo Aimoto , Steven O. Smith c,d, Kazuto Nosaka a, Kenichi Akaji a,
*
*
a Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan
b Department of Chemistry and Chemical Engineering, Graduate School of Science and Technology, Yamagata University, Yonezawa, Yamagata 992-8510, Japan
c Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
d Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
N,N-Dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]ben-
zyl]tetrahydro-2H-pyran-4-aminium chloride (TAK779) is a potent and selective non-peptide CCR5
antagonist. To use a site-specifically labeled form as a molecular probe, TAK779 containing 13C at posi-
tions C19, 35, and 36 was produced. A commercially available [13C]-methyl iodide was employed for
the labeling. Starting from a known carboxylic acid segment containing no labeled carbon, the labeled
TAK779 was constructed by the successive coupling of [13C]-labeled tolyl boronic ester by the Suzuki–
Miyaura reaction and a [13C]-labeled aniline segment by amide bond formation.
Received 25 June 2009
Revised 11 July 2009
Accepted 14 July 2009
Available online 18 July 2009
Key words:
TAK779
Stable isotope
CCR5
Ó 2009 Elsevier Ltd. All rights reserved.
Chemokine receptor
1. Introduction
and/or 15N, accurate inter-nuclear distances between the isotopic
labels can be estimated. Thus, site-specifically labeled TAK779
The b-chemokine receptor CCR5, a G protein-coupled seven-
transmembrane domain receptor, acts as a major co-receptor for
the fusion and entry of macrophase-tropic (M-tropic or R5) HIV-1
into host cells.1 As a result, CCR5 is considered an attractive target
for the inhibition of M-tropic HIV replication.2 In 1999, N,N-di-
methyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclo-
hepten-8-yl] carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-amin-
ium chloride (1, TAK779) was reported as the first small molecule
that acts as a potent and selective non-peptide CCR5 antagonist with
a IC50 value of 1.4 nM.3 TAK779 (1) inhibited the entry of HIV-1 into
target cells by blocking the interaction between the gp120/CD4
complex and CCR5. Following the discovery of TAK779, other small
molecule CCR5 antagonists with improved potency and/or pharma-
ceutical properties were also been described.4,5
The structural and molecular interactions of CCR5 inhibitors,
including TAK779 with CCR5, however, are not clearly understood.
Combining site-directed mutagenesis of CCR5 and molecular mod-
eling, the interactions of small molecule inhibitors with CCR5 have
been examined.6–8 Although possible inhibitor-binding pockets of
CCR5 have been proposed, additional experimental data are re-
quired to clarify the exact mode of binding. High resolution so-
lid-state NMR spectroscopy can provide structural information
on complex solids. By combining solid-state NMR spectroscopy
and site-specific labeling with stable isotopes such as 13C, 2D,
would be a useful probe for examining the molecular interactions
between TAK779 and CCR5 by solid-state NMR techniques. Here,
we report the synthesis of 100% enriched [13C3]-labeled TAK779
(2), which contains 13C isotopes at C-19, -35, and -36 (Fig. 1).
2. Results and discussion
In an energy-minimized form, TAK779 (1) adopts a bent confor-
mation with the bend dividing a hydrophobic aromatic segment
from a positively charged aniline segment. Previously, it was sug-
gested that the two contrasting parts of TAK779 (1) interact with
complementary binding pockets of CCR5.7 To obtain experimental
data regarding the proposed model, TAK779 containing 13C iso-
topes at both ends, that is, positions C-19, -35, and -36, was se-
lected as a probe (2) (Fig. 1).
For the convenient introduction of 100% 13C-enriched isotope
carbon, a commercially available [13C]-methyl iodide was selected.
Thus, the scheme for the synthesis of TAK7793b was modified to
enable the introduction of methyl groups at C-19, -35, and -36
by [13C]-methyl iodide. The backbone of the 13C-labeled TAK779
(2) was constructed by successive coupling of the left-side boro-
nate segment (4) and right-side aniline segment (6) to the central
segment (5) (Scheme 1). The central carboxylic acid segment (5)
containing no labeled carbon was prepared according to a pub-
lished procedure3b starting with bromobenzene in eight steps
without difficulty. For the synthesis of the [13CH3]-labeled boronyl
toluene derivative (4), boronic acid (7) and [13C]-methyl iodide
* Corresponding authors. Tel./fax: +81 0 75 465 7659 (K.A.).
E-mail address: akaji@koto.kpu-m.ac.jp (K. Akaji).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.026

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H. Konno et al. / Bioorg. Med. Chem. 17 (2009) 5769–5774
H
H
N
35
36
13CH3
35 36
N
13CH3
H3C CH3
H313C
O
N
H3C
O
N
19
19
Cl
O
Cl
O
1
2
Figure 1. TAK779 (1) and [13C3]-labeled TAK779 (2).
H
N
35
36
H
N
13CH3I
13CH3
13CH3
13CH3
N
H313C
O
N
H313C
O
19
Cl
O
13C3-labeled TAK779 (2)
O
3
H2N
O
B
13CH3
N
O
OH
Br
H313C
O
O
5
6
4
O2N
Br
H
N
13CH3I
HO
B
+
13CH3I
+
O
OH
7
8
Scheme 1. Synthetic plan for [13C3]-labeled TAK779 (2).
were selected as the coupling components. The [13CH3]-labeled
aniline derivative (6) was prepared via the N-methylation of amine
(8) by [13C]-methyl iodide.
First, p-bromophenyl boronic acid (7) was synthesized by the
reaction of trimethyl borate with the Grignard reagent prepared
from 1,4-dibromobenzene in 87% yield, according to the method
reported by Kabalka et al.10 Successive palladium (0)-catalyzed
cross coupling of 7 with [13C]-methyl iodide proceeded at 25 °C un-
der Gooben’s conditions.11 Subsequent purification by careful dis-
In the preparation of 13C-labeled p-tolylboronic acid ester (4),
[
13CH3]-labeled p-bromotoluene (9), instead of 4-methylphenylbo-
ronic acid employed in the original synthesis of non-labeled
TAK779 (1), is required. Although the synthesis of [13CH3]-labeled
p-bromotoluene (9) by zeolite-mediated bromination of [13CH3]–
toluene has been reported by Kesling et al.,9 separation of the prod-
uct mixture containing para/ortho derivatives was not effectively
achieved. In addition, with this route, very expensive [13CH3]-la-
beled toluene was necessary. To enable the use of readily available
tillation (bp = 58 °C, 20 mmHg) afforded
[
13CH3]-labeled p-
bromotoluene (9) in 30 % yield. The spectral data of the resulting
compound (9) was well consistent with those of the reported com-
pound.9 The labeled product (9) was then converted to the desired
[
13CH3]-labeled p-tolylboronate (4) in 68% yield by treatment with
bis(pinacorato)diboron12 in the presence of PdCl2(dppf).
[
13C]-methyl iodide, a route starting from 4-dibromobenzene was
selected (Scheme 2).
Previously in the synthesis of non-labeled TAK779 (1), the right-
segment, the aniline derivative (6), was prepared by successive
13CH3I, Pd(OAc)2
(Naph)3P, K3PO4
Br
Br
1) Mg, ether
Br
HO
2) B(OMe)3
then HNO3
THF, H2O
30%
H313C
B
OH
Br
9
7
87%
O
O
B
B
O
B
O
O
O
PdCl2(dppf), KOAc
DMSO, 80ºC
H313C
4
68%
Scheme 2. Synthesis of [13CH3]-p-tolylboronate (4).

H. Konno et al. / Bioorg. Med. Chem. 17 (2009) 5769–5774
5771
reductive amination of 4-nitro-benzylamine using tetrahydro-4H-
pyran-4-one and formaldehyde. In the present study, the stepwise
introduction of substitution groups on the nitrogen atom, which
enables the introduction of a labeled methyl group by [13C]-methyl
iodide, was employed. Thus, the desired [13CH3]-labeled aniline
derivative (6) was produced starting with N,N-Boc(o-Nos)NH (10)
(Scheme 3). The coupling of 10 with 4-tetrahydropyranyl alcohol
by Fukuyama’s protocol13 gave the corresponding amine, which
was then treated with TFA to provide nosyl amine (11) in 38% over-
all yield. The same procedure was repeated on 11 with p-nitro ben-
zyl alcohol to obtain the nitro derivative (12) in 52% yield. After
removal of the nosyl group of 12 in the presence of mercapto acetic
acid and LiOH (62% yield), methylation of amine (8) with [13C]-
methyl iodide in the presence of NaH afforded [13CH3]-labeled N-
methyl amine (13) in 72% yield. The coupling constant of the prod-
uct was 133 Hz (doublet, 3H) in the 1H NMR spectrum, which con-
firmed the successful 13C labeling in 13. Reduction of the nitro
group of 13 gave the desired aniline (6) in 76% yield.
was modified to enable the efficient use of [13C] methyl iodide.
Studies on molecular interactions between the synthesized la-
beled-TAK779 and CCR5 using solid-state NMR techniques are
now underway and will be reported in due course.
3. Experimental
3.1. General
[
13C]-Methyl iodide was purchased from ISOTEC. All manipula-
tions were conducted under an inert atmosphere (N2). All solvents
were of reagent grade. THF was distilled from sodium and benzo-
phenone ketyl. CH2Cl2 was distilled from CaH2. All commercial re-
agents were of the highest purity available. Analytical TLC was
performed on silica gel (60 F-254, Plates 0.25 mm). Column chro-
matography was carried out on Wakogel 60 (particle size, 0.063–
0.200 mm) or Dowex 1 ꢀ 8 (Clꢁ form: particle size, 100–200 mesh).
1H (300 MHz), and 13C (75 MHz) NMR spectra were recorded on a
Bruker AM-300. Chemical shifts are expressed in ppm relative to
TMS (0 ppm) or CHCl3 (7.28 ppm for 1H and 77.0 ppm for 13C) or
MeOH (3.30 ppm for 1H and 49.0 ppm for 13C). IR spectra were ob-
tained on a HORIBA FREEXACT-II FT-710 spectrometer. Low-reso-
lution mass spectra (LRMS) and high-resolution mass spectra
(HRMS) were obtained on either a JEOL JMS-HX-211A or a JMS-
HX-110A (EI or FAB) or a Bruker Autoflex-II (MALDI-TOF).
Using the labeled segments above, [13C3]-labeled TAK779 (2)
was synthesized via two routes (routes (a) and (b), in Scheme 4).
The left segment (4) was introduced to the central segment (5) first
in route (a), whereas the right segment (6) was introduced first in
route (b). With route (a), Suzuki–Miyaura coupling of [13C]-labeled
boronate (4) and phenyl bromide (5) was conducted in the pres-
ence of potassium carbonate and a catalytic amount of PdCl2(dppf)
to obtain [13C]-labeled methyl ester (14) in 64% yield. After sapon-
ification of 14, the resulting [13C]-labeled carboxylic acid (15) was
treated with oxalyl chloride in the presence of a catalytic amount
of DMF. The addition of [13C]-labeled aniline (6) and triethylamine
to the mixture gave the desired [13C]-labeled amide (3) in 74%
yield. With route (b), coupling of carboxylic acid (16) and [13C]-la-
beled aniline (6) afforded [13C]-labeled amide (17) in 47% yield,
and a subsequent cross-coupling reaction gave [13C2]-labeled
amide (3) in 36% yield. Thus, the synthesis via route (a) resulted
in a better yield than that via route (b). Finally, treatment of
3.2. [13CH3]-p-Bromotoluene (9)
To a solution of palladium acetate (56 mg, 0.249 mmol), tri-
naphthylphosphine (205 mg, 0.498 mmol), and potassium phos-
phate (4.11 g, 19.9 mmol) in THF (20 ml) were added [13C]-methyl
iodide (2.00 g, 14.9 mmol), p-bromophenylboronic acid (7) (2.00 g,
9.96 mmol), and H2O (0.350 ml, 19.9 mmol). The reaction mixture
was stirred at room temperature for 12 h, poured into water
(10 ml), and extracted with hexane. The combined organic layers
were dried over MgSO4, and filtered through a plug of silica, and
the product was carefully distilled to give [13CH3]-p-bromotoluene
[
13C2]-labeled amide (3) with [13C]-methyl iodide in DMF and sub-
sequent counter ion exchange gave the desired [13C3]-labeled
TAK779 (2) in 70% yield. The product was confirmed to have the
expected molecular weight by MALDI TOF-MS. The expected large
coupling constants of the methyl protons of the tolyl group
(126 Hz, doublet, 3H) and quaternary ammonium group (144 Hz,
doublet, 6H) were observed in the 1H NMR spectrum of 2. In the
13C NMR spectrum, extremely strong signals from three methyl
carbons were also observed as expected. These spectral data clearly
support a successful 13C labeling in 2.
(9) (516 mg, 2.99 mmol, 30%) as
a white solid, bp = 58 °C
(20 mmHg), mp = 16 °C. 1H NMR (CDCl3) d: 2.32 (3H, d,
J = 126.6 Hz), 7.06 (2H, dd, J = 8.4, 4.5 Hz), 7.38 (2H, d, J = 8.4 Hz).
13C NMR (CDCl3) d: 21.0 (13C), 119.1, 130.9 (d, J = 3.1 Hz), 131.3,
136.7 (d, J = 44.2 Hz). The spectral data were well consistent with
those reported by Kesling et al.9
3.3. [13CH3]-p-(B-Pinacolate-boronyl)toluene (4)
In conclusion, [13C3]-labeled TAK779 (2) was produced by intro-
ducing 100% 13C-enriched isotope carbon at C-19, -35, and -36. For
convenience, the readily available [13C]-methyl iodide was em-
ployed, and the method used to synthesize non-labeled TAK7793b
To a solution of [1,10-bis(diphenylphosphino)-ferrocene]dichlo-
ropalladium (II) (22 mg, 0.030 mmol), KOAc (294 mg, 3.00 mmol),
and bis(pinacolate)diboron (279 mg, 1.10 mmol) in DMSO (6 ml)
HO
1)
O2N
mercaptoacetic
O
O2N
O2N
acid and LiOH
Nos
N
Nos
HN
OH
O
DIAD, PPh3, THF
2) TFA , CH2Cl2
38%
H
N
Boc
S
O
DMF
62%
DIAD, PPh3
THF
52%
O
O
12
o
-Nos
11
10
O2N
H2N
13CH3
N
O2N
13CH3I
13CH3
N
reduced Fe
H
N
NaH
O
AcOH
76%
O
THF
72%
O
6
8
13
Scheme 3. Synthesis of [13CH3]-aniline derivative (6).

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H. Konno et al. / Bioorg. Med. Chem. 17 (2009) 5769–5774
route (b)
1) (COCl)2, cat. DMF, CH2Cl2
Br
H
N
1M NaOH
Br
Br
H N
13CH3
N
2)
2
13CH3
N
MeOH/
THF
80%
CO2Me
CO2H
6
O
17
5
16
O
Et3N,THF
route (a)
O
O
B
47%
O
B
PdCl2(dppf) (cat.)
K2CO3, DME, reflux
PdCl2(dppf) (cat.)
K2CO3, DME, reflux
O
O
H313C
64%
36%
H313C
4
4
1) (COCl)2, cat. DMF, CH2Cl2
H
N
1M NaOH
13CH3
N
2)
H2N
13CH3
N
MeOH
6
CO2Me
CO2H
H313C
O
O
THF
82%
H313C
H313C
3
15
14
O
Et3N,THF
74%
1) 13CH3I, DMF 2) ion exchange
chromatography
70%
Cl- form (pH 3~4)
H
13CH3
N
N
13CH3
O
H313C
O
Cl
[
13C3]-labeled TAK779 (2)
Scheme 4. Synthesis of [13C3]-labeled TAK779 (2).
was added [13CH3]-p-bromotoluene (9) (202 mg, 1.18 mmol). After
being stirred at 80 °C for 12 h, the product was extracted with ben-
zene, washed with water, dried over MgSO4 and evaporated in va-
cuo. The residue was purified by column chromatography (hexane/
AcOEt = 20:1) to give 4 (150 mg, 0.684 mmol, 68%) as a colorless
oil. IR (film) mmax cmꢁ1: 2978, 1606, 1362, 1146, 1018, 962, 656.
1H NMR (CDCl3) d: 1.38 (12H, s), 2.39 (3H, d, J = 126.3 Hz), 7.21
(2H, dd, J = 7.5, 4.5 Hz), 7.73 (2H, d, J = 7.5 Hz). 13C NMR (CDCl3)
3.5. 4-[N-Nosyl-N-(tetrahydropyran-4-yl)aminomethyl]-
nitrobenzene (12)
To a solution of 11 (1.87 g, 6.53 mmol) and p-nitrobenzyl alco-
hol (1.00 g, 6.53 mmol) in THF (10 ml) were added triphenyl phos-
phine (2.57 g, 9.80 mmol) and diisopropyl azodicarboxylate
(1.90 g, 9.80 mmol) at room temperature. After being stirred for
2 h, the mixture was concentrated in vacuo and the residue was
purified by column chromatography (CHCl3/MeOH = 95:5) to give
12 (1.43 g, 3.39 mmol, 52%) as a colorless solid: mp = 128–130 °C.
IR (film) mmax cmꢁ1: 3084, 2964, 1545, 1522, 1346, 1161, 872,
742, 723. 1H NMR (CDCl3) d: 1.58–1.70 (4H, m), 3.39 (2H, td,
J = 11.4, 3.9 Hz), 3.91 (2H, dd, J = 10.2, 2.7 Hz), 4.12 (1H, m), 4.66
(2H, s), 7.53 (2H, d, J = 9.0 Hz), 7.58–7.74 (3H, m), 7.96 (1H, dd,
J = 7.8, 1.2 Hz), 8.13 (2H, d, J = 8.7 Hz). 13C NMR (CDCl3) d: 31.7,
47.0, 55.8, 67.2, 123.6, 124.4, 128.2, 131.1, 131.8, 133.6, 133.9,
145.6, 147.3, 147.7, HRFABMS (M+H)+ calcd for C18H20N3O3S1:
422.1022, found: 422.1018.
d: 21.7 (13C), 24.9, 83.6, 128.5 (d, J = 2.3 Hz), 134.8 (d, J = 3.8 Hz),
141.3 (d, J = 40.5 Hz). HRCIMS (M)+ calcd for
C
13C1H20O2B1:
12
12
220.1556, found: 220.1597.
3.4. N-Nosyl-N-(tetrahydropyran-4-yl)amine (11)
To a solution of Boc(Nos)NH (1.78 g, 5.90 mmol) and 4-tetrahy-
dropyranyl alcohol (402 mg, 3.94 mmol) in THF (10 ml) were
added triphenyl phosphine (1.55 g, 5.90 mmol) and diisopropyl
azodicarboxylate (1.14 ml, 5.90 mmol) at room temperature. After
being stirred for 2 h, the mixture was concentrated in vacuo. To a
solution of the crude product (937 mg) in CH2Cl2 (2 ml) was added
TFA (2 ml) at room temperature. After being stirred for 2 h, the
mixture was concentrated in vacuo and AcOEt and H2O were added
to the residue. The organic layer was washed successively with
water and brine, dried over MgSO4, and evaporated in vacuo. The
crude product was purified by column chromatography (hexane/
AcOEt = 2:1) to give 11 (430 mg, 1.50 mmol, 38%) as a white solid,
mp = 156–160 °C. IR (film) mmax cmꢁ1: 3265, 3097, 2956, 2858,
1541, 1362, 1340, 1167, 1086, 742. 1H NMR (CDCl3) d: 1.51–1.66
(2H, m), 1.78–1.83 (2H, m), 3.40 (2H, td, J = 11.7, 2.4 Hz), 3.58
(1H, m), 3.90 (2H, dt, J = 12.0, 3.9 Hz), 5.36 (1H, d, J = 7.5 Hz),
7.77 (2H, m), 7.88 (1H, m), 8.19 (1H, m). 13C NMR (CDCl3) d:
34.9, 51.5, 67.6, 125.7, 125.9, 130.6, 131.4, 133.5, 133.9, 134.6,
134.9, 135.8, 137.6, 149.2, 149.5. HRFABMS (M+H)+ calcd for
C11H15N2O5S: 287.0702, found: 287.0707.
3.6. 4-[N-(Tetrahydropyran-4-yl)aminomethyl]-nitrobenzene
(8)
To a solution of 12 (1.43 g, 3.39 mmol) in DMF (5 ml) were
added mercaptoacetic acid (0.707 ml, 10.2 mmol) and LiOH
(853 mg, 20.3 mmol) at room temperature. The mixture was stir-
red for 2 h, and AcOEt and water were added. The organic layer
was separated and the aqueous layer was extracted with AcOEt.
The combined organic layer was washed successively with water
and brine, dried over MgSO4, and evaporated in vacuo. The residue
was purified by column chromatography (CHCl3/MeOH = 95:5) to
give 8 (495 mg, 2.10 mmol, 62%) as a colorless oil. IR (film) mmax
cmꢁ1: 3317, 2943, 2844, 1602, 1516, 1342, 1090, 1012, 860, 739.
1H NMR (CDCl3) d: 1.38–1.51 (4H, m), 1.88 (2H, d, J = 12.6 Hz),
2.73 (1H, m), 3.39 (2H, td, J = 11.7, 2.1 Hz), 3.95 (3H, s), 4.00 (1H,

H. Konno et al. / Bioorg. Med. Chem. 17 (2009) 5769–5774
5773
br s), 7.53 (2H, d, J = 9.0 Hz), 8.17 (2H, dd, J = 9.0, 1.2 Hz). 13C NMR
(CDCl3) d: 33.7, 49.7, 53.4, 66.6, 123.5, 128.5, 146.9, 148.6. HREIMS
(M+H)+ calcd for C12H16N2O3: 236.1161, found: 236.1137.
ture for 5 h. The mixture was concentrated in vacuo and extracted
with AcOEt after being acidified using 1 M HCl. The organic layer
was washed successively with water and brine, dried over MgSO4,
and evaporated in vacuo. The residue was purified by column chro-
3.7. 4-[N-[13CH3]-Methyl-N-(tetrahydropyran-4-yl)aminomethyl]-
nitrobenzene (13)
matography (CHCl3/MeOH = 9:1) to give 15 (7 mg, 25.1 lmol, 82%)
as a colorless prism, mp 138–139 °C. IR (film) mmax cmꢁ1: 3022,
2925, 2854, 1680, 1421, 1290, 1273, 1254, 808. 1H NMR (CDCl3)
d: 2.08 (2H, m), 2.40 (3H, d, J = 126.3 Hz), 2.69 (2H, t, J = 6.6 Hz),
7.23 (2H, m), 7.42–7.50 (3H, m), 7.54–7.61 (2H, m), 7.90 (1H, br
s). 13C NMR (CDCl3) d: 20.9 (13C), 27.2, 30.1, 34.8, 126.6, 127.2,
To a solution of 8 (250 mg, 1.06 mmol) in THF (2 ml) was added
NaH (60% in oil; 80 mg, 2.03 mmol) at 0 °C. The mixture was stirred
for 10 min, and [13C]-methyl iodide (120
ll, 1.95 mmol) was added.
The mixture was stirred at room temperature for 12 h. It was then
poured into water (2 ml) and the product was extracted with AcOEt.
The organic layer was washed with saturated NH4Cl aq and brine,
dried over MgSO4, and evaporated in vacuo. The residue was purified
by column chromatography (CHCl3/MeOH = 98:2) to give 13
129.4, 129.8, 131.3, 132.2, 134.4, 137.0, 137.4, 139.0, 140.6,
141.9, 171.7. HREIMS (M)+ calcd for
found: 279.1356.
C
13C1H18O2: 279.1307,
18
12
3.11. 2-Bromo-6,7-dihydro-5H-benzocycloheptene-8-carboxylic
acid (16)
(190 mg, 763 l :
mol, 72%) as a colorless oil. IR (film) mmax cmꢁ1
2949, 2843, 1599, 1520, 1346, 1140, 1009, 741. 1H NMR (CDCl3) d:
1.60–1.78 (4H, m), 2.21 (3H, d, J = 133.2 Hz), 2.65 (1H, m), 3.38
(2H, t, J = 11.7 Hz), 3.68 (2H, d, J = 5.7 Hz), 4.04 (2H, dd, J = 11.1,
3.3 Hz), 7.51 (2H, d, J = 8.1 Hz), 8.16 (2H, d, J = 8.1 Hz). 13C NMR
A solution of 5 (1.00 g, 3.57 mmol) and 1 M NaOH (5 ml) in
MeOH (5 ml) and THF (10 ml) was stirred at room temperature
for 5 h. The mixture was concentrated in vacuo and extracted with
AcOEt after being acidified using 1 M HCl. The organic layer was
washed successively with water and brine, dried over MgSO4,
and evaporated in vacuo. The residue was purified by column chro-
matography (CHCl3/MeOH = 9:1) to give 16 (760 mg, 2.86 mmol,
(CDCl3) d: 29.3, 37.7 (13C), 57.1, 60.1, 67.5, 123.5, 129.0, 147.0,
148.2. HREIMS (M)+ calcd for
251.1345.
C
13C1H18N2O3: 251.1317, found:
12
12
3.8. 4-[N-[13CH3]-Methyl-N-(tetrahydropyran-4-yl)aminomethyl]-
aniline (6)
80%) as a colorless prism, mp = 213–214 °C. IR (KBr) mmax cmꢁ1
:
2931, 2634, 1668, 1421, 1292, 1201, 916, 872. 1H NMR (CD3OD)
d: 2.02 (2H, m), 2.58 (2H, td, J = 6.6, 0.9 Hz), 2.76 (2H, m), 7.08
(1H, d, J = 8.1 Hz), 7.34 (1H, dd, J = 8.1, 2.1 Hz), 7.44 (1H, d,
J = 1.8 Hz), 7.62 (1H, s). 13C NMR (CD3OD) d: 29.0, 30.1, 35.3,
120.6, 132.2, 132.5, 135.6, 135.7, 138.0, 139.0, 143.4, 171.4.
HRFABMS (MꢁH)+ calcd for C12H10O2Br: 264.9864, found:
264.9860.
To a solution of 13 (190 mg, 763 lmol) in acetic acid (1 ml) was
added reduced iron (100 mg, 1.79 mmol), and the mixture was stir-
red at room temperature for 10 h. The solvent was evaporated in va-
cuo, and AcOEt was added to the residue. The precipitate was
removed by filtration and the filtrate was washed successively with
1 M NaOH, water and brine, dried over MgSO4 and evaporated in va-
cuo to give 6 (156 mg, 720 lmol, 76%) as a colorless oil. IR (film) mmax
3.12. N-[4-[N-[13CH3]-Methyl-N-(4-tetrahydropyranyl)amino-
methyl]-phenyl]-2-bromo-6,7-dihydro-5H-benzocycloheptene-
8-carboxamide (17)
cmꢁ1: 3350, 3222, 2956, 2852, 1612, 1520, 1398, 1292, 1086, 1011,
752. 1H NMR (CDCl3) d: 1.65–1.85 (4H, m), 2.31 (3H, d, J = 129.3 Hz),
2.89 (1H, m), 3.34 (2H, t, J = 11.7 Hz), 3.68 (2H, d, J = 4.5 Hz), 4.02
(2H, dd, J = 11.1, 3.0 Hz), 5.68 (2H, br s), 6.63 (2H, dd, J = 8.1,
1.5 Hz), 7.11 (2H, dd, J = 8.1, 1.2 Hz). 13C NMR (CDCl3) d: 28.1, 35.7
To a solution of 16 (35 mg, 131
added oxalyl chloride (40 ml, 316
l
mol) in CH2Cl2 (0.50 ml) were
l
mol) and DMF (1 drop) on ice.
(
13C), 56.5, 58.7, 67.0, 114.9, 125.0, 130.8, 146.1. HRFABMS (M+H)+
The mixture was stirred for 2 h at room temperature. The solvent
was evaporated in vacuo. The solution of the residue in THF
calcd for 12C1213C1H21N2O1: 222.1654, found: 222.1680.
3.9. Methyl 2-(4-[13CH3]-methylphenyl)-6,7-dihydro-5H-benzo-
cycloheptene-8-carboxylate (14)
(0.50 ml) was added dropwise to a solution of 6 (30 mg, 135
lmol)
and triethylamine (55 l, 400 mol) in THF (0.50 ml) on ice and the
l
l
reaction mixture was stirred for 2 h at room temperature. The sol-
vent was evaporated in vacuo, and AcOEt and water were added.
The organic layer was washed successively with water and brine,
dried over MgSO4, and evaporated in vacuo. The residue was puri-
fied by column chromatography (CHCl3/MeOH = 19:1) to give 17
To a solution of bromo ester (5) (30 mg, 107
pinacolate-boronyl)–toluene (4) (40 mg, 183
(10 mg) in DME (2 ml) was added PdCl2(dppf) (10 mg, 8.65
The mixture was refluxed overnight under an argon atmosphere,
and AcOEt was added. The organic layer was washed successively
with water and brine, dried over MgSO4, and evaporated in vacuo.
The residue was purified by column chromatography (hexane/
l
l
mol), [13CH3]-p-(B-
mol), and, K2CO3
mol).
l
(29 mg, 61.8 l :
mol, 47%) as a colorless oil. IR (film) mmax cmꢁ1
3294, 2943, 2844, 1653, 1599, 1516, 1408, 1246, 1140, 844, 816,
756. 1H NMR (CDCl3) d: 1.63–1.81 (4H, m), 2.11 (2H, m), 2.23
(3H, d, J = 133.2 Hz), 2.68 (2H, t, J = 6.6 Hz), 2.79 (2H, t,
J = 5.7 Hz), 3.38 (2H, t, J = 11.1 Hz), 3.60 (2H, d, J = 5.1 Hz), 4.03
(2H, m), 7.04 (1H, d, J = 8.1 Hz), 7.22 (1H, s), 7.29 (1H, d,
J = 8.1 Hz), 7.32 (2H, d, J = 6.9 Hz), 7.41 (1H, s), 7.56 (2H, d,
J = 6.9 Hz), 7.76 (1H, s). 13C NMR (CDCl3) d: 27.9, 29.1, 30.2, 34.2,
37.3 (13C), 57.2, 59.5, 67.6, 119.6, 120.1, 129.4, 130.9, 131.0,
AcOEt = 2:1) to give 14 (20 mg, 68 lmol, 64%) as a colorless oil. IR
(film) mmax cmꢁ1: 2929, 2858, 1238, 1186, 810. 1H NMR (CDCl3) d:
2.10 (2H, m), 2.42 (3H, d, J = 126.3 Hz), 2.69 (2H, t, J = 6.9 Hz), 2.88
(2H, m), 3.85 (3H, s), 7.24 (2H, m), 7.44–7.62 (5H, m), 7.80 (1H, br
s). 13C NMR (CDCl3) d: 21.1 (13C), 27.6, 30.3, 34.9, 52.1, 55.4, 126.7,
126.8, 126.9, 127.2, 127.3, 127.4, 128.8, 129.5, 129.85, 129.89,
131.3, 131.5, 132.6, 132.7, 134.6, 136.8, 137.4, 137.5, 139.1, 139.2,
132.5, 134.2, 135.6, 136.5, 136.8, 139.3, 141.2, 167.7. HRFABMS
(M+H)+ calcd for
C
13C1H30N2O2Br: 470.1491, found: 470.1507.
24
12
139.6, 139.7, 140.5, 141.8, 142.1, 169.1. HREIMS (M)+ calcd for
12
C
19
13C1H20O2: 293.1463, found: 293.1501.
3.13. N-[4-[N-[13CH3]-Methyl-N-(4-tetrahydropyranyl)amino-
methyl]-phenyl]-2-(4-[13CH3]-methylphenyl)-6,7-dihydro-5H-
benzocycloheptene-8-carboxamide (3)
3.10. 2-(4-[13CH3]-Methylphenyl)-6,7-dihydro-5H-benzocyclo-
heptene-8-carboxylic acid (15)
A solution of 14 (9 mg, 30.7
MeOH (0.20 ml) and THF (0.20 ml) was stirred at room tempera-
lmol) and 1 M NaOH (0.20 ml) in
To a solution of 15 (110 mg, 394
lmol) in CH2Cl2 (3 ml) were
added oxalyl chloride (300 l, 2.37 mmol) and DMF (1 drop) on
l

5774
H. Konno et al. / Bioorg. Med. Chem. 17 (2009) 5769–5774
ice. The mixture was stirred for 2 h at room temperature. The sol-
vent was evaporated in vacuo. A solution of the residue in THF
d, J = 8.7 Hz), 8.00 (1H, br s), 13C NMR (CD3OD) d: 19.1 (13CH3Ph),
26.1, 27.7, 29.1, 33.4, 45.54 (13CH3N), 45.59 (13CH3N), 45.64
(1.5 ml) was added dropwise to a solution of 6 (156 mg, 702
l
mol)
(
13CH3N), 57.1, 63.9, 65.5, 68.5, 70.2, 71.2, 120.2, 121.9, 125.5,
and triethylamine (300 l, 2.00 mmol) in THF (3 ml) on ice and the
l
125.7, 125.8, 126.6, 126.7, 127.4, 128.7, 128.5, 129.3, 132.8,
reaction mixture was stirred for 2 h at room temperature. The sol-
vent was evaporated in vacuo, and then AcOEt and H2O were
added. The organic layer was washed successively with water
and brine, dried over MgSO4, and evaporated in vacuo. The residue
was purified by column chromatography (CHCl3/MeOH = 19:1) to
134.1, 134.3, 136.3, 136.9, 137.1, 138.5, 140.6, 140.7, 169.7, MAL-
DI-TOFMS (MꢁCl)+ calcd for
C
13C3H40N2O2: 498.301, found:
30
12
498.164.
Acknowledgements
give 3 (141 mg, 292 lmol, 74%) as a colorless oil. IR (film) mmax
cmꢁ1: 3294, 3020, 2925, 2845, 2781, 1651, 1597, 1516, 1406,
1315, 1244, 1138, 810, 754, 1H NMR (CDCl3) d: 1.60–1.77 (4H,
m), 2.17–2.30 (2H, m), 2.26 (3H, d, J = 133 Hz, 13CH3N), 2.42 (3H,
d, J = 126 Hz, 13CH3Ph), 2.45 (1H, m), 2.75 (2H, t, J = 6.3 Hz), 2.91
(2H, m), 3.39 (2H, td, J = 11.7, 2.7 Hz), 3.62 (2H, d, J = 4.2 Hz),
4.06 (2H, dd, J = 10.2, 3.0 Hz), 7.24–7.36 (6H, m), 7.44–7.63 (6H,
m), 13C NMR (CDCl3) d: 21.1 (13CH3Ph), 28.0, 29.2, 30.4, 34.6, 37.5
We thank Dr. Nobutaka Fujii and Dr. Shinya Oishi (Kyoto Uni-
versity) for the measurements of mass spectra. This work was sup-
ported in part by a Grant-in-aid for Scientific research from the
Japan Society for the Promotion of Science (Grant 19790095 to
H.K.), and by NIH Grant GM-41412 to S.O.S.
(
13CH3N), 57.3, 59.5, 67.7, 120.0, 126.7, 126.8, 129.3, 129.5, 129.8,
References and notes
130.6, 134.3, 134.7, 135.9, 136.9, 137.1, 137.5, 138.0, 139.1,
141.2, 168.1, HRFABMS (M+H)+ calcd for
483.2855, found: 483.2924.
C
13C2H37N2O2:
30
12
1. (a) Choe, H.; Farzan, M.; Sun, Y.; Sullivan, N.; Rollins, B.; Ponath, P. D.; Wu, L.;
MacKay, C. R.; LaRosa, G.; Newman, G.; Gerard, W.; Gerard, N.; Sodroski, J. Cell
1996, 85, 1135–1148; (b) Doranz, B. J.; Rucker, J.; Smyth, Y.; Samson, R. J.;
Peiper, S. C.; Parmentier, M.; Collman, R. G.; Doms, R. W. Cell 1996, 85, 1149–
1158.
2. (a) De Clercq, E. Biochim. Biophys. Acta 2002, 1587, 258–275; (b) LaBranche, C.
C.; Galasso, G.; Moore, J. P.; Bolognesi, D. P.; Hirsch, M. S.; Hammer, S. M.
Antiviral. Res. 2001, 50, 95–115.
3. (a) Baba, M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.; Sawada, H.; Iizawa, Y.;
Shiraishi, M.; Aramaki, Y.; Okonogi, K.; Ogawa, Y.; Meguro, K.; Fujino, M. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 5698–5703; (b) Shiraishi, M.; Aramaki, Y.; Seto,
M.; Imoto, H.; Nishikawa, Y.; Kanzaki, N.; Okamoto, M.; Sawada, H.; Nishimura,
O.; Baba, M.; Fujino, M. J. Med. Chem. 2000, 43, 2049–2063; (c) Ikemoto, T.;
Nishiguchi, A.; Mitsudera, H.; Wakimasu, M.; Tomimatsu, K. Tetrahedron 2001,
57, 1525–1529.
4. Seibert, C.; Sakmar, T. P. Curr. Pharm. Des. 2004, 10, 2041–2062.
5. (a) Kazmierski, W.; Bifulco, N.; Yang, H.; Boone, L.; DeAnda, F.; Watson, C.;
Kenakin, T. Bioorg. Med. Chem. 2003, 11, 2663–2676; (b) Zhao, Q.; Ma, L.; Jiang,
S.; Lu, H.; Liu, S.; He, Y.; Strick, N.; Neamati, N.; Debnath, K. Virology 2005, 339,
213–225.
6. Dragic, T.; Trkola, A.; Thompson, D. A. D.; Cormier, E. G.; Kajumo, F. A.; Maxwell,
E.; Lin, S. W.; Ying, W.; Smith, S. O.; Sakmar, T. P.; Moore, J. P. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 5639–5644.
7. Seibert, C.; Ying, W.; Gavrilov, S.; Tsamis, F.; Kuhmann, S. E.; Palani, A.; Tagat, J.
R.; Clader, J. W.; McCombie, S. W.; Baroudy, B. M.; Smith, S. O.; Dragic, T.;
Moore, J. P.; Sakmar, T. P. Virology 2006, 349, 41–54.
8. Maeda, K.; Das, D.; Ogata-Aoki, H.; Nakata, H.; Miyakawa, T.; Tojo, Y.; Norman,
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43, 1059–1068.
The coupling reaction of 17 with 4 using the same procedure as
above afforded 3 (36% yield; the spectroscopic data (IR, 1H NMR,
13C NMR, and MS) were identical as above).
3.14. N,N-[13CH3,13CH3]-Dimethyl-N-[4[[[2-(4-[13CH3]-methyl-
phenyl)-6,7-dihydro-5H-benzocycloheptan-8-yl]carbonyl]
amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride,
TAK779 (2)
A solution of 3 (9 mg, 18.6
(22 mg, 350 mol) in DMF (1 ml) was stirred for 12 h at room tem-
l
mol) and [13C]-methyl iodide
l
perature. The solvent was evaporated in vacuo, and AcOEt was
added to the residue. The precipitate was filtered, washed with
AcOEt and MeOH, and recrystallized from EtOH and AcOEt to give
an iodide salt (9 mg, 14.4
lmol) as a red prism. A solution of the
iodide salt (9 mg, 14.4 mol) in MeOH and water was subjected
l
to ion-exchange column chromatography (Dowex, 100–200 mesh,
Clꢁ form) to give [19, 35, 36-13C3]-labeled TAK779 (2) (7 mg,
13.1 l
mol, 70%) as a colorless prism. IR (film) mmax cmꢁ1: 3394,
3014, 2964, 2931, 2860, 2767, 2445, 1655, 1595, 1520, 1319,
1248, 1084, 847, 812, 756, 1H NMR (CD3OD) d: 2.01 (2H, m), 2.16
(2H, m), 2.27 (2H, d, J = 12.0 Hz), 2.39 (3H, d, J = 126 Hz, 13CH3Ph),
2.68 (2H, t, J = 6.0 Hz), 2.92 (2H, m), 2.99 (6H, dd, J = 144, 3.6 Hz,
10. Kabalka, G. W.; Sastry, U.; Sastry, K. A. R. J. Organometal. Chem. 1983, 259, 269–
274.
13CH3)2N), 3.52 (2H, t, J = 13.8 Hz), 3.76 (1H, m), 4.17 (2H, dd,
11. Gooben, L. J. Appl. Organometal. Chem. 2004, 18, 602–604.
12. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508–7510.
13. (a) Kan, T.; Fukuyama, T. J. Syn. Org. Chem. Jpn. 2001, 59, 779; (b) Kan, T.;
Fukuyama, T. Chem. Commun. 2004, 35; (c) Fukuyama, T.; Jow, C.-K.; Cheung,
M. Tetrahedron Lett. 1995, 36, 6373–6376.
(
J = 11.1, 3.6 Hz), 4.56 (2H, br s), 7.26 (2H, dd, J = 7.8, 4.5 Hz), 7.28
(1H, d, J = 8.1 Hz), 7.45 (1H, s), 7.48 (1H, dd, J = 5.7, 2.1 Hz), 7.53
(2H, d, J = 8.1 Hz), 7.59 (2H, d, J = 6.9 Hz), 7.61 (1H, s), 7.88 (2H,