Synthesis of highly deuterium-labeled (R)-K-13675, PPAR α agonist, for use as an internal standard for low-level quantification of drugs in plasma

Article abstract of DOI:10.1016/j.bmc.2009.01.049

Two highly deuterium-labeled compounds, (R)-K-13675-d₁₁ and (R)-K-13675-d₇, were prepared for use as internal standards for low-level quantification of plasma drugs by LC/MS/MS. We successfully demonstrated their utility in pharmacok

Full text of DOI:10.1016/j.bmc.2009.01.049

Bioorganic & Medicinal Chemistry 17 (2009) 1911–1917
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of highly deuterium-labeled (R)-K-13675, PPAR
a agonist, for use as an
internal standard for low-level quantification of drugs in plasma
*
Yukiyoshi Yamazaki, Shin-ichiro Ogawa, Kimiyuki Shibuya
Tokyo New Drug Research Laboratories, Pharmaceutical Division, Kowa Co., Ltd, 2-17-43, Noguchicho, Higashimurayama, Tokyo 189-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Two highly deuterium-labeled compounds, (R)-K-13675-d11 and (R)-K-13675-d7, were prepared for use
as internal standards for low-level quantification of plasma drugs by LC/MS/MS. We successfully demon-
strated their utility in pharmacokinetic studies for sensitive and precise drug quantification.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 4 December 2008
Revised 20 January 2009
Accepted 21 January 2009
Available online 13 February 2009
Keywords:
Deuterium-labeled
Pt/C catalyzed deuteration
Precise quantification
Internal standard
1. Introduction
O
N
O
O
O
N
OH
Peroxisome proliferator-activated receptor
a (PPARa) is a mem-
O
ber of one of the nuclear receptor superfamilies, activation of
which leads to a decrease in triglyceride levels and an increase in
HDL-cholesterol levels in humans.1 During the course of our drug
discovery program, we identified (R)-2-[3-[[benzoxazol-2-yl[3-(4-
(R)-K-13675 (1)
methoxyphenoxy)propyl]amino]methyl]phenoxy]butanoic
acid
Figure 1. Structure of (R)-K-13675.
((R)-K-13675 (1), Fig. 1) as a highly potent and selective PPAR
a
agonist, and its in vivo efficacy was shown to be more than 100
times higher than that of fenofibrate.2
dard for sensitive and precise quantification in pharmacokinetic
studies. Herein we report details of synthetic procedures and pre-
liminary pharmacokinetic studies.
As we might anticipate a clinical dose as low as one hundredth
that of fenofibrate, it was necessary to develop and validate a
method for quantifying trace amounts (less than ng/mL) of 1 in hu-
man plasma for pharmacokinetic studies. Deuterium-labeled com-
pounds are well known to be useful as internal standards for such
applications.3 However, for precise low-level quantifications, the
purities of deuterated compounds must be very high. Otherwise
residual non-deuterated compounds that can still be detected with
high sensitivity, might interfere. Therefore the efficient synthesis
of such compounds from appropriate reagents is a significant chal-
lenge in medicinal chemistry. Although a number of deuteration
methods have been reported,4 most reactions require harsh reac-
tion conditions or special equipment, are limited by substrate
availability, and result in unsatisfactory deuterium incorporation.
Despite these obstacles we succeeded in the synthesis of highly
pure deuterium-labeled (R)-K-13675 as an excellent internal stan-
2. Results and discussion
Our synthetic strategy towards deuterated (R)-K-13675 was
based on the construction of two major precursors, 2-aminophe-
nol-d4 (2) and 4-methoxyphenol-d7 (3) (Scheme 1). Envisaging that
post-synthetic deuteration would be feasible in this synthetic
scheme. We planned to synthesize 2 by hydrogen–deuterium (H–
D) exchange displacement of 2-aminophenol (5) in deuterium
oxide (D2O),5 while 3 was to be derived from a synthetic combina-
tion of the commercially available 1,4-hydroquinone-d6 (9) and
methanol-d4.
First, we focused on the H–D exchange reaction of Sajiki et al.,6
because it is applicable to a variety of aromatic compounds and its
implementation is straightforward. Thus we conducted the H–D
exchange reaction of 2-aminophenol (5) using 5% Pt/C (Aldrich,
Bach#03505ME) in D2O (99.9 atom % D; Aldrich) under a hydrogen
* Corresponding author. Tel.: +81 42 391 6211; fax: +81 42 395 0312.
E-mail address: k-sibuya@kowa.co.jp (K. Shibuya).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.049

1912
Y. Yamazaki et al. / Bioorg. Med. Chem. 17 (2009) 1911–1917
Next, we turned our attention to construction of the two key
D
OH
parts of (R)-K-13675-d11 (4) (Scheme 3).8 Treatment of 2 with
potassium xanthogenate in ethanol afforded the cyclized 2-mer-
captobenzoxazole-d4 (7), subsequent chlorination of which with
thionyl chloride furnished 2-chlorobenzoxazole-d4 (8) in moderate
yield. For the other key part, after several attempts9 we achieved
the synthesis of 4-methoxyphenol-d7 (3) by application of Bellas’
method.10 Exposure of 9 to benzoquinone-d4 and sulfuric acid in
methanol-d4 at room temperature proceeded smoothly to provide
the desired product 3 in 95% yield. Thus obtained, 3 was alkylated
by Michael reaction with acrylonitrile in the presence of Triton B at
80 °C to give the nitrile 10 (64%), followed by reduction with bor-
ane-THF at 80 °C to provide the amine 11 (73%). Reductive alkyl-
ation of 11 with 3-hydroxybenzaldehyde (12) and sodium
borohydride yielded the secondary amine 13 (90%). Interestingly,
the ratio of deuterium content was unaffected in a series of reduc-
tive reaction conditions. The seven-deuterium-labeled amine 13
was reacted with 8 to afford 14 in 83% yield. Etherification of the
labeled phenol 14 with n-butyl (S)-2-trifluoromethanesulfonyloxy-
butanoate in the presence of K2CO3 in acetonitrile at room temper-
ature gave the phenyl ether 15 in 97% yield. Finally, hydrolysis of
15 in aqueous NaOH provided (R)-K-13675-d11 (4) in 72% yield.
Analogously (R)-K-13675-d7 (18) was prepared from the non-
deuterated 2-chlorobenzoxazole following the same synthetic
operation (Scheme 4).
D
D
NH2
OH
D
D
D
D
D
2
OCD3
3
D
D
D
D
N
O
O
O
O
N
OH
D
D
D
D
(R)-K-13675-d11 (4)
OCD3
Scheme 1. Synthetic strategy of (R)-K-13675-d11
.
atmosphere at 100 °C for 24 h. The deuterium content was deter-
mined on the basis of acetyl groups by 1H NMR after diacetylation
of 2-aminophenol-d4 (2) (Scheme 2, Table 1). Surprisingly, we
could achieve a deuterium content of only 86% D, significantly low-
er than the values reported in the literature (89% D at 80 °C, 97% D
at 180 °C).6d After these unsatisfying results, we decided on a dif-
ferent approach.
The deuterium-labeled compounds 4 and 18 were then ana-
lyzed by monitoring with LC/MS/MS.11 The multiple reaction mon-
itoring (MRM) ion chromatograms of (R)-K-13675-d7 (18) and (R)-
K-13675-d11 (4) are shown in Figure 2a and b. When both com-
pounds were measured after injection of 5 ng, 18 was observed
to contain a small amount (0.19%) of non-deuterated (R)-K-
13675, and 4 showed a trace amount (0.02%) of (R)-K-13675, as
summarized in Table 2.
We then devised a method for exploiting the kinetic isotope ef-
fect,7 in which almost full deuterium incorporation can be
achieved by multiple cycles of Pt/C-catalyzed deuteration. The ra-
tio of deuterium content was increased markedly in each cycle
and reached 97% D by the third cycle. A slight increase was ob-
served from the fourth cycle (fourth cycle, 97% D; fifth cycle, 98%
D). As detailed in Table 1, this efficient method let to easily repro-
ducible results even on a 10-g-scale (third cycle, 94% D; fifth cycle,
98% D). In summary, we have evidenced that it is possible to obtain
highly deuterated 2 by repeating the Pt/C-catalyzed reaction under
a hydrogen atmosphere, without requiring either extremely high
temperatures or specialized equipment.
For quantification of (R)-K-13675 (1) in rat plasma using isotope
dilution mass spectrometry, a calibration curve for 1 was con-
structed by spiking 100 lL of rat plasma with 0.25 ng of (R)-K-
13675-d11 (4) and 0.02, 0.04, 0.1, 0.2, 0.4, 1, 2, or 4 ng of 1, respec-
tively (Fig. 3 and Table 3).12 The calibration curve showed excellent
linearity (accuracy: 97.4–102%) and the correlation coefficient was
0.9999. Interestingly, compounds labeled with more deuterium
were shown to be superior to those with less deuterium for validat-
ing low-level quantifications of plasma drugs by LC/MS/MS. Indeed,
we carried out the quantifications of drugs in plasma using (R)-K-
13675-d11 (4) as an internal standard administered to rats and dogs
at a dose of 0.1 mg/kg. The results indicated that we have success-
fully established a method to determine low-level concentrations
(0.1 ng/mL) of drugs in plasma in each species. Details of these
pharmacokinetic studies will be reported elsewhere in due time.
D
H
2
NH
OH
D
D
NH
OH
2
2
5% Pt/C
D O, 100 ºC, 24 h
2
D
5
2
6
D
AcCl
D
NHAc
OAc
Et N
3
EtOAc, rt
D
D
Scheme 2. Synthesis of 2-aminophenol-d4 (2) and the diacetylated 2-aminophenol-
d4 6.
3. Conclusions
We have developed a useful method to obtain 2-aminophenol-
d4 with excellent deuterium incorporation by repetitive cycles of
H–D exchange, and have succeeded in the synthesis of highly deu-
terated (R)-K-13675-d11 and (R)-K-13675-d7. Furthermore, we
demonstrated that (R)-K-13675-d11 served as an excellent internal
standard for sensitive and precise quantification in pharmacoki-
netic studies.
Table 1
Deuterium incorporation of 2-aminophenol-d4 (2) after H–D exchange reactiona
Reaction cycle
Deuterium contentb (% D)
1
2
3
4
5
86
91
97
97
98
4. Experimental
4.1. General
a
The first reaction involved treatment of 5 (1.0 g) with 5% Pt/C (200 mg) in D2O
(50 mL, 99.9 atom% D) at 100 °C under a hydrogen atmosphere for 24 h. From the
second cycle, the reactions were performed with the same ratio of reagents under
the same conditions.
Commercially available reagents and solvents were used with-
out further purification. TLC analyses were carried out on silica
gel 60 F254 plates (Merck). 1H NMR and 13C NMR spectra were re-
b
Deuterium content (% D) of 6 was determined on the basis of the acetyl groups
by 1H NMR after diacetylation of 2.

Y. Yamazaki et al. / Bioorg. Med. Chem. 17 (2009) 1911–1917
1913
D
D
2
D
D
D
D
D
D
D
NH
OH
D
D
2
N
N
O
a
c
b
d
SH
Cl
O
D
D
7
3
8
D
D
D
D
DO
D
D
D CO
3
D
D CO
3
D
O
D CO
3
D
O
e
h
CN
OD
OH
D
OH
D
D
NH
2
D
9
D
D
10
11
D
D
D
D
D
OH
D
D
N
HN
N
O
OH
O
n
OHC
g
O
O
O
O
N
f
D
N
O Bu
O
D
D
D
14
D
12
15
13
D
D
D
D
D
3
OCD
D
3
D
D
3
D
D
OCD
OCD
D
N
O
O
O
O
N
D
OH
D
i
D
(R)-K-13675-d (4)
11
D
D
3
OCD
Scheme 3. Reagents and conditions: (a) potassium xanthogenate, EtOH, 55%; (b) SOCl2, DMF, 49%; (c) benzoquinone-d4, H2SO4, methanol-d4 , 95%; (d) acrylonitrile, Triton B,
64%; (e) BH3–THF, THF, 73%; (f) 11, NaBH4, MeOH, 90%; (g) 8, iPr2NEt, DMF, 83%; (h) n-butyl 2-(S)-trifluoromethanesulfonyloxybutanoate, K2CO3, MeCN, 97%; (i) 4 M aq NaOH,
EtOH, 72%.
Mass spectra were obtained on a JEOL MS-BU20 mass spectrome-
N
OH
ter. Elemental analyses (C, H, N) were performed by Yanaco MT-
5. Melting points were determined in open glass capillaries on a
Buchi B-545 melting point apparatus.
HN
OH
O
O
N
j
k
O
D
D
D
D
4.2. Synthesis of 2-aminophenol-d4 (2) (large scale)
D
D
D
D
T o a suspension of 2-aminophenol (5) (10.0 g, 91.6 mmol) in
D2O (70 mL, 99.9 atom % D; Aldrich) was added 5% Pt/C (2.0 g, Al-
drich, Bach; #03505ME) under an argon atmosphere. The reaction
mixture was stirred at 100 °C under a hydrogen atmosphere for
24 h and diluted with EtOAc. The mixture was filtered through a
pad of Celite. The organic layer was washed with brine, dried over
Na2SO4 and concentrated in vacuo to give 2 as a brown solid
(8.77 g). This obtained product was used in the next reaction with-
out purification. The second reaction was performed with the same
ratio of reagents under the same conditions. Finally, the desired
OCD3
OCD3
13
16
N
O
N
O
O
O
O
O
N
OH
N
OBn
l
O
O
D
D
D
D
D
D
D
D
product 2 was obtained after the fifth reaction cycle (5.57 g,
13
*
*
*
54%): C NMR (68 MHz, DMSO-d6) d 113.71 , 114.20 , 118.63 ,
OCD3
(R)-K-13675-d7 (18)
OCD3
*
119.25 , 136.41, 143.90; IR (solid sample): 3376, 3351, 3328,
17
3305, 1578, 1438, 1300 cmꢀ1; HRMS (EI): m/z [M+] calcd for
Scheme 4. Reagents and conditions: (j) 2-chlorobenzoxazole, iPr2NEt, DMF, 87%;
(k) benzyl (S)-2-trifluoromethanesulfonyloxybutanoate, K2CO3, MeCN, 100%; (l) 4 M
aq NaOH, EtOH, 85%.
C6H3D4NO: 113.0775; found: 113.0773.
4.3. Synthesis of the diacetylated 2-aminophenol-d4 6.
Determination of deuterium content (% D)
corded on a JEOL JNM-LA 400 MHz and JEOL GSX-270. Tetrameth-
ylsilane was used as an internal standard. Chemical shifts (d) are
given in parts per million (ppm), coupling constants J values are gi-
To a solution of 2-aminophenol-d4 (2) (295 mg, 2.61 mmol) and
triethylamine (527 mg, 5.21 mmol) in EtOAc (10 mL) was added
acetyl chloride (409 mg, 5.21 mmol) at 0 °C. The reaction mixture
was stirred at room temperature for 1 h and quenched with water.
The organic layer was washed with water and brine, dried over
*
ven in Hertz (Hz) and are reported to the nearest 0.1 Hz. Starred ( )
values in the 13C NMR are for small peaks. Infrared (IR) spectra
were recorded on a Thermo Nicolet 370 FT-IR (ATR) spectrometer.

1914
Y. Yamazaki et al. / Bioorg. Med. Chem. 17 (2009) 1911–1917
(a) (R)-K-13675-d7 (18)
(b) (R)-K-13675-d11 (4)
5.5e6
5.0e6
4.5e6
4.0e6
3.5e6
3.0e6
2.5e6
2.0e6
1.5e6
1.0e6
5.0e5
0.0
4.8e6
4.4e6
(m/z 498/107)
(m/z 502/107)
x 1
x 1
4.0e6
3.6e6
3.2e6
2.8e6
2.4e6
2.0e6
1.6e6
1.2e6
8.0e5
4.0e5
0.0
(R)-K-13675-d7
(R)-K-13675-d11
(R)-K-13675-d0
(R)-K-13675-d0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
1
2
3
4
5
6
7
8
9
10 11 12 13 14
4.8e6
4.4e6
5.5e6
5.0e6
4.5e6
4.0e6
3.5e6
3.0e6
2.5e6
2.0e6
1.5e6
1.0e6
5.0e5
0.0
x 100
x 100
4.0e6
3.6e6
3.2e6
2.8e6
2.4e6
2.0e6
1.6e6
1.2e6
8.0e5
4.0e5
0.0
(R)-K-13675-d7
(R)-K-13675-d11
(R)-K-13675-d0
(R)-K-13675-d0
12.32
1
2
3
4
5
6
7
8
9
10 11 12 13 14
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Time, min
Time, min
Figure 2. MRM ion chromatograms of the deuterated and non-deuterated peaks.
Table 2
Table 3
Peak area and ratio in MRM ion chromatograms.11
Accuracy of (R)-K-13675 (1) for calibration curve samples in rat plasma
Compounds
Peak areaa
Ratiob (%)
1
2
3
4
5
6
7
8
Deuterated
Non-deuterated
Concentration (ng/mL)
Accuracya (%)
0.2
99.8
0.4
99.6
1
102
2
100
4
99.7
10
101
20
101
40
97.4
4 (d11
18 (d7)
)
71,200,000 1,100,000
88,800,000 7,000,000
16,900 800
167,000 7000
0.02 0.00
0.19 0.01
a
Accuracy (%) = (calculated concentration/nominal concentration) ꢁ 100.12.
a
Values are means S.D. (n = 10).
Ratio of non-deuterated compound (%) = (peak area of non-deuterated com-
pound/ deuterated compound) ꢁ 100.
b
(131 mg, 26%): 1H NMR (400 MHz, CD3OD) d 2.13 (s, 3H), 2.30 (s,
3H), 7.12 (s, 0.02H), 7.18 (s, 0.02H), 7.21 (s, 0.02H), 7.74 (s,
0.02H); Deuterium content on aromatic ring = 98% D; Anal. Calcd
for C10H7D4NO3: C, 60.90; H, 3.58; D, 4.08; N, 7.10. Found: C,
60.92; H, 3.64; D, 4.04; N, 7.12; IR (solid sample) 3334, 1736,
1686, 1588, 1509, 1439, 1413 cmꢀ1; HRMS (EI): m/z [M+] calcd
for C10H7D4NO3: 197.09858; found: 197.09783; mp 123–124 °C.
20
y = 0.52 x + 0.0139 (r = 0.9999)
15
10
5
4.4. Synthesis of 2-mercaptobenzoxazole-d4 (7)
To a solution of 2-aminophenol-d4 (2) (5.15 g, 45.5 mmol) in
ethanol (100 mL) was added potassium xanthogenate (8.03 g,
50.1 mmol) at room temperature. The reaction mixture was stirred
at 100 °C for 5 h and cooled to room temperature. Solvent was
evaporated under reduced pressure. The residue was dissolved in
water. The aqueous solution was acidified with 4 M HCl and ex-
tracted with EtOAc. The organic layer was washed with water
and brine, dried over Na2SO4 and concentrated in vacuo. The resi-
due was recrystallized from EtOAc/n-hexane to give 7 as a red-
brown crystal (2.94 g, 42%). The filtrate was concentrated in vacuo.
The residue was purified by silica gel column chromatography (n-
hexane/EtOAc = 4:1) to give 7 as a red-brown crystal (933 mg, 13%.
Total yield; 3.87 g, 55%): 1H NMR (400 MHz, DMSO-d6) d 7.03 (s,
0
0
4
8
12
16
20
24
28
32
36
40
Plasma concentration of (R) -K-13675 in rat plasma (ng/mL)
Figure 3. Calibration curve for (R)-K-13675 (1) in rat plasma.
Na2SO4 and concentrated in vacuo. The residue was purified by
preparative TLC (n-hexane/EtOAc = 1:2) to give 6 as a yellow solid

Y. Yamazaki et al. / Bioorg. Med. Chem. 17 (2009) 1911–1917
1915
0.07H), 7.25 (s, 0.07H), 7.30 (s, 0.07H), 7.51 (s, 0.07H), 13.86 (br s,
(50 mL) was added slowly to the mixture at 0 °C. The mixture was
stirred at 80 °C for 0.5 h, acidified with 4 M HCl until pH 1 at 0 °C
and stirred at 80 °C for 14 h. Solvent was evaporated under re-
duced pressure. The residue was dissolved in water. The aqueous
solution was washed with Et2O. The aqueous layer was alkalinized
with 4 M aqueous NaOH and extracted with toluene. The organic
layer was washed with water and brine, dried over Na2SO4 and
concentrated in vacuo to give 11 as a colorless solid (2.87 g,
73%): 1H NMR (400 MHz, CDCl3) d 1.33 (br s, 2H), 1.90 (quintet,
J = 6.1 Hz, 2H), 2.90 (t, J = 6.1 Hz, 2H), 3.60-3.64 (m, 0.01H), 4.00
(t, J = 6.1 Hz, 2H), 6.82 (s, 0.03H), 6.83 (s, 0.03H); 13C NMR
13
*
*
*
1H); C NMR (100 MHz, DMSO-d6) d 109.88 , 110.33 , 123.56 ,
*
124.92 , 131.08, 148.07, 180.12; Anal. Calcd for C7HD4NOS: C,
54.17; H, 0.65; D, 5.19; N, 9.02. Found: C, 54.33; H, 0.68; D, 5.29;
N, 8.91; IR (solid sample): 3341, 1599, 1476, 1368, 1327, 1112,
932 cmꢀ1; MS (EI) m/z 155 [M+]; mp 186–189 °C.
4.5. Synthesis of 2-chlorobenzoxazole-d4 (8)
To
a solution of 2-mercaptobenzoxazole-d4 (7) (2.00 g,
12.9 mmol) in thionyl chloride (4 mL) was added N,N-dimethyl-
formamide (940 mg, 12.9 mmol) dropwise over 5 min at 0 °C under
an argon atmosphere. The reaction mixture was stirred at same
temperature for 2 h and concentrated in vacuo. The residue was
dissolved in EtOAc. Water was added. The organic layer was
washed with water and brine, dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
*
*
*
(100 MHz, CD3OD) d 33.48, 39.82, 55.24 , 67.64, 115.25 , 116.08 ,
154.37, 155.20; IR (solid sample): 2937, 2872, 2216, 2070, 1600,
1426, 1378 cmꢀ1; HRMS (EI): m/z [M+] calcd for C10H8D7NO2:
188.15349; found: 188.15294.
4.9. Synthesis of 3-[[[3-(4-methoxyphenoxy-d7)propyl]
raphy (n-hexane/EtOAc = 10:1) to give 8 as a yellow oil (999 mg,
amino]methyl]phenol (13)
13
* * *
49%):
C NMR (68 MHz, CDCl3) d 110.11 , 119.39 , 124.07 ,
*
125.11 , 143.73, 150.78, 151.39; Anal. Calcd for C7D4ClNO: C,
53.35; D, 5.11; N, 8.89. Found: C, 53.21; D, 5.39; N, 8.77; IR (neat):
1783, 1584, 1523, 1436, 1370, 1208, 1113 cmꢀ1; MS (EI) m/z 157
[M+], 159 [M++2].
To a solution of 11 (1.88 g, 10.0 mmol) in MeOH (25 mL) was
added 3-hydroxybenzaldehyde (1.22 g, 10 mmol) at room temper-
ature. The reaction mixture was stirred at 60 °C for 3 h. A solution
of NaBH4 (567 mg, 15.0 mmol) in water (10 mL) was added slowly
to this mixture at 0 °C. The mixture was stirred at room tempera-
ture for 14 h and filtered off. The isolated solid was rinsed with
water and dried in vacuo to give 13 as a colorless crystal (2.66 g,
90%): 1H NMR (400 MHz, DMSO-d6) d 1.82 (quintet, J = 6.8 Hz,
2H), 2.06 (br s, 1H), 2.60 (t, J = 6.8 Hz, 2H), 3.60 (s, 2H), 3.94 (t,
J = 6.8 Hz, 2H), 6.59 (dd, J = 7.8, 2.2 Hz, 1H), 6.72 (d, J = 7.8 Hz,
1H), 6.74 (s, 1H), 7.07 (t, J = 7.8 Hz, 1H), 9.24 (s, 1H); 13C NMR
4.6. Synthesis of 4-methoxyphenol-d7 (3)
To a solution of 1,4-hydroquinone-d6 (9) (98 atom% D, 4.48 g,
38.6 mmol) and benzoquinone-d4 (98 atom% D, 325 mg,
2.90 mmol) in methanol-d4 (99.8 atom % D, 25 mL) was added sul-
furic acid (4.80 g) over 5 min at room temperature. The reaction
mixture was stirred for 14 h, poured into ice-water and neutralized
with 4 M aqueous NaOH. The aqueous solution was extracted with
Et2O. The organic layer was washed with brine, dried over Na2SO4
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (n-hexane/EtOAc = 5:1) to give 3 as a pale
yellow solid (4.83 g, 95%): 1H NMR (400 MHz, CDCl3) d 3.72–3.76
(m, 0.06H), 4.87 (s, 1H), 6.76 (s, 0.10H), 6.79 (s, 0.10H); 13C NMR
*
(100 MHz, DMSO-d6) d 29.33, 45.50, 53.08, 54.43 , 66.37, 113.38,
*
*
114.72, 114.15 , 114.89 , 118.44, 128.92, 142.51, 152.57, 153.10,
157.26; Anal. Calcd for C17H14D7NO3: C, 69.36; H, 4.79; D, 4.79;
N, 4.76. Found: C, 69.34; H, 4.90; D, 4.76; N, 4.75; IR (solid sample):
3275, 1580, 1480, 1431, 1390, 1156, 1109 cmꢀ1; MS (EI) m/z 294
[M+]; mp 142–143 °C.
*
*
*
(100 MHz, CDCl3) d 55.06 , 114.59 , 115.80 , 149.41, 153.48; IR
4.10. Synthesis of 3-[[benzoxazol-2-yl-d4[3-(4-methoxy
phenoxy-d7)propyl]amino]methyl]phenol (14)
(solid sample): 3381, 2521, 2262, 2069, 1419, 1141, 1112 cmꢀ1
;
HRMS (EI): m/z [M+] calcd for C7HD7O2: 131.09564; found:
131.09642; mp 56–57 °C.
To a solution of 13 (1.87 g, 6.34 mmol) in DMF (15 mL) and
N,N-diisopropylethylamine (983 mg, 7.61 mmol) was added
8
4.7. Synthesis of 3-(4-methoxyphenoxy-d7)propionitrile (10)
(999 mg, 6.34 mmol) at room temperature. The reaction mixture
was stirred at 80 °C for 15 h and diluted with EtOAc and water.
The organic layer was washed with water and brine, dried over
Na2SO4 and concentrated in vacuo. The residue was purified by
silica gel column chromatography (n-hexane/EtOAc = 2:1) to give
14 as an amorphous brown solid (2.19 g, 83%): 1H NMR
To acrylonitrile (3.50 g, 65.3 mmol) were added 4-methoxyphe-
nol-d7 (3) (4.28 g, 32.7 mmol) and Triton B (0.3 mL) at room tem-
perature. The reaction mixture was stirred at 80 °C under an
argon atmosphere for 22 h and diluted with EtOAc and water.
The organic layer was washed with 1 M aqueous NaOH, water
and brine, dried over Na2SO4 and concentrated in vacuo. The resi-
due was recrystallized from EtOAc/n-heptane to give 10 as color-
less needles (3.87 g, 64%): 1H NMR (400 MHz, CDCl3) d 2.79 (t,
J = 6.4 Hz, 2H), 3.72–3.75 (m, 0.01H), 4.13 (t, J = 6.4 Hz, 2H), 6.84
(s, 0.03H), 6.86 (s, 0.03H); deuterium content = 99% D; 13C NMR
(400 MHz, CDCl3)
d 2.03 (quintet, J = 6.8 Hz, 2H), 3.45 (t,
J = 6.8 Hz, 2H), 3.89 (t, J = 6.8 Hz, 2H), 4.65 (s, 2H), 6.70–6.73 (m,
2H), 6.75 (s, 1H), 7.14 (t, J = 7.8 Hz, 1H), 8.75 (s, 1H); 13C NMR
*
*
*
(68 MHz, CDCl3) d 27.62, 45.01, 52.07, 54.89 , 65.32, 108.84 ,
*
*
*
113.12, 114.25 , 114.81 , 115.08 , 115.33, 118.77, 120.22 ,
123.53 , 129.79, 137.60, 141.40, 148.07, 152.61, 153.77, 157.82,
*
*
*
*
(100 MHz, CDCl3) d 18.63, 54.87 , 63.55, 114.38 , 115.66 , 117.35,
151.66, 154.55; Anal. Calcd for C10H4D7NO2: C, 65.19; H, 2.19; D,
7.65; N, 7.60. Found: C, 65.09; H, 2.22; D, 7.54; N, 7.54; IR (solid
sample): 2930, 2886, 2255, 2219, 2069, 1446, 1420 cmꢀ1; MS (EI)
m/z 184 [M+]; mp 65–66 °C.
162.28; Anal. Calcd for C24H13D11N2O4: C, 69.37; H, 3.15; D,
5.33; N, 6.74. Found: C, 69.47; H, 3.24; D, 5.32; N, 6.75; IR (solid
sample): 2941, 1633, 1567, 1424, 1393, 1153, 1111 cmꢀ1; HRMS
(EI): m/z [M+] calcd for C24H13D11N2O4: 415.24150; found:
415.24342.
4.8. Synthesis of 3-(4-methoxyphenoxy-d7)propylamine (11)
4.11. Synthesis of n-butyl (R)-2-[3-[[benzoxazol-2-yl-d4 [3-(4-
methoxyphenoxy-d7)propyl]amino]methyl]phenoxy]
butanoate (15)
To a solution of 10 (3.82 g, 20.7 mol) in THF (20 mL) was added
BH3-THF (1.17 mol/L, 21.3 mL, 24.9 mmol) dropwise at room tem-
perature under an argon atmosphere. The reaction mixture was
stirred at 80 °C for 3 h and cooled to room temperature. Methanol
To a solution of 14 (2.16 g, 5.20 mmol) and K2CO3 (1.08 g,
7.79 mmol) in MeCN (30 mL) was added a solution of n-butyl

1916
Y. Yamazaki et al. / Bioorg. Med. Chem. 17 (2009) 1911–1917
*
*
(S)-2-trifluoromethanesulfonyloxybutanoate
(1.97 mg,
27.13, 45.22, 51.31, 54.45 , 65.50, 108.81, 113.92, 114.16 ,
*
6.76 mmol) in MeCN (10 mL) under an argon atmosphere. The
reaction mixture was stirred at room temperature for 19 h and
filtered off. The filtrate was concentrated in vacuo. The residue
was dissolved in EtOAc. Water was added. The organic layer
was washed with water and brine, dried over Na2SO4 and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane/EtOAc = 5:1) to give 15 as a brown
oil (2.82 g, 97%): 1H NMR (400 MHz, CDCl3) d 0.85 (t, J = 7.3 Hz,
3H), 1.05 (t, J = 7.1 Hz, 3H), 1.22–1.28 (m, 2H), 1.49–1.55 (m,
2H), 1.96 (quintet, J = 6.7 Hz, 2H), 2.14 (quintet, J = 6.8 Hz, 2H),
3.70 (t, J = 6.7 Hz, 2H), 3.96 (t, J = 6.7 Hz, 2H), 4.00-4.14 (m,
2H), 4.53 (t, J = 6.5 Hz, 1H), 4.72 (d, J = 15.6 Hz, 1H), 4.77 (d,
J = 15.6 Hz, 1H), 6.76 (dd, J = 7.8, 2.2 Hz,1H), 6.86 (s, 1H), 6.89
(d, J = 7.8 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H); 13C NMR (68 MHz,
CDCl3) d 9.67, 13.59, 18.92, 26.17, 27.51, 30.48, 45.11, 52.06,
114.42, 115.04 , 115.59, 117.90, 120.13, 123.88, 129.64, 138.73,
143.31, 148.46, 152.30, 153.27, 157.63, 162.26; Anal. Calcd for
24H17D7N2O4: C, 70.05; H, 4.16; D, 3.43; N, 6.81. Found: C,
70.05; H, 4.27; D, 3.42; N, 6.77; IR (solid sample): 2937, 1638,
1600, 1582, 1459, 1424, 1394 cmꢀ1; MS (EI) m/z 411 [M+]; mp
100–101 °C.
C
4.14. Synthesis of benzyl (R)-2-[3-[[benzoxazol-2-yl-d4 [3-(4-
methoxyphenoxy-d7)propyl]amino]methyl]phenoxy]
butanoate (17)
To a solution of 16 (4.59 g, 11.2 mmol) and K2CO3 (2.31 g,
16.7 mmol) in MeCN (60 mL) was added a solution of benzyl (S)-
2-trifluoromethanesulfonyloxybutanoate (4.00 g, 12.3 mmol) un-
der an argon atmosphere. The reaction mixture was stirred at room
temperature for 11 h and filtered off. The filtrate was concentrated
in vacuo. The residue was dissolved in EtOAc. Water was added.
The organic layer was washed with water and brine, dried over
Na2SO4 and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (n-hexane/EtOAc = 5:1) to give 17
as a colorless oil (6.97 g, 100%): 1H NMR (400 MHz, CDCl3) d 1.02 (t,
J = 7.2 Hz, 3H), 1.96 (quintet, J = 7.1 Hz, 2H), 2.13 (quintet,
J = 6.5 Hz, 2H), 3.68 (t, J = 6.5 Hz, 2H), 3.95 (t, J = 6.5 Hz, 2H), 4.57
(t, J = 6.7 Hz, 1H), 4.69 (d, J = 15.9 Hz, 1H), 4.74 (d, J = 15.9 Hz,
1H), 5.06 (d, J = 12.2 Hz, 1H), 5.15 (d, J = 12.2 Hz, 1H), 6.74 (dd,
J = 7.8, 2.4 Hz, 1H), 6.84 (s, 1H), 6.89 (d, J = 7.8 Hz, 1H), 7.00 (d,
J = 7.8 Hz, 1H), 7.14-7.30 (m, 8H), 7.37 (d, J = 7.8 Hz, 1H); 13C
*
*
*
55.19 , 64.96, 65.50, 77.60, 108.64 , 113.73, 113.86 , 114.66,
*
*
*
*
115.32 , 115.78 , 120.15 , 120.68, 123.44 , 129.82, 138.67,
143.41, 148.56, 152.69, 153.76, 158.31, 162.15, 171.67; Anal.
Calcd for C32H27D11N2O6: C, 68.91; H, 4.88; D, 3.97; N, 5.02.
Found: C, 68.80; H, 4.97; D, 3.94; N, 4.94; IR (neat): 2960,
2067, 1751, 1633, 1563, 1425, 1154 cmꢀ1; HRMS (EI): m/z [M+]
calcd for C32H27D11N2O6: 557.34087; found: 557.34196; ½a D20
ꢂ
+19.1 (c 0.89, CHCl3).
4.12. Synthesis of (R)-2-[3-[[benzoxazol-2-yl-d4 [3-(4-
methoxyphenoxy-d7)propyl]amino]methyl]phenoxy] butanoic
acid; (R)-K-13675-d11 (4)
*
*
NMR (100 MHz, CDCl3) d 9.52, 26.03, 27.27, 45.12, 52.00, 54.48 ,
*
To a solution of 15 (2.70 g, 4.84 mmol) in EtOH (20 mL) was
added 4 M aqueous NaOH (2.4 mL) at 0 °C. The reaction mixture
was stirred at room temperature for 1 h, diluted with water, acid-
ified with 4 M HCl and extracted with EtOAc. The organic layer was
washed with water and brine, dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (CHCl3/MeOH = 10:1) and recrystallized from EtOAc/n-hep-
tane to give (R)-K-13675 d11 as a colorless crystal (1.74 g, 72%):
1H NMR (400 MHz, DMSO-d6) d 0.96 (t, J = 7.1 Hz, 3H), 1.79-1.89
(m, 2H), 2.06 (quintet, J = 6.8 Hz, 2H), 3.65 (t, J = 6.8 Hz, 2H), 3.94
(t, J = 6.8 Hz, 2H), 4.60 (t, J = 6.4 Hz, 1H), 4.72 (s, 2H), 6.78 (dd,
J = 7.8, 2.4 Hz, 1H), 6.86 (s, 1H), 6.89 (d, J = 7.8 Hz, 1H), 7.25 (t,
J = 7.8 Hz, 1H), 13.1 (br s, 1H); 13C NMR (100 MHz, DMSO-d6) d
9.53, 25.42, 27.12, 45.29, 51.27, 65.47, 76.43, 113.30, 114.13,
119.77, 129.76, 139.02, 143.19, 148.43, 152.31, 153.25, 158.04,
162.24, 172.46; Anal. Calcd for C28H19D11N2O6: C, 67.04; H, 3.82;
D, 4.42; N, 5.58. Found: C, 66.91; H, 3.86; D, 4.34; N, 5.50; IR (solid
sample): 2959, 2939, 2887, 1716, 1628, 1426, 1377 cmꢀ1; MS
65.47, 66.64, 77.48, 108.69, 113.83, 114.19 , 114.61, 115.23 ,
116.10, 120.32, 120.66, 123.88, 128.15, 128.27, 128.45, 129.78,
135.30, 138.65, 143.43, 148.85, 152.63, 153.72, 158.17, 162.56,
171.30; Anal. Calcd for C35H29D7N2O6: C, 71.53; H, 4.97; D, 2.40;
N, 4.77. Found: C, 71.42; H, 5.11; D, 2.40; N, 4.75; IR (neat):
2938, 1752, 1638, 1459, 1578, 1425, 1153 cmꢀ1; HRMS (EI): m/z
[M+] calcd for C35H29D7N2O6: 587.30052; found: 587.30184; ½a 2D0
ꢂ
+16.4 (c 1.00, CHCl3).
4.15. Synthesis of (R)-2-[3-[[benzoxazol-2-yl [3-(4-methoxy
phenoxy-d7)propyl]amino]methyl]phenoxy]butanoic acid; (R)-
K-13675-d7 (18)
To a solution of 17 (6.30 g, 10.7 mmol) in EtOH (60 mL) was
added 4 M aqueous NaOH (5.4 mL) at 0 °C. The reaction mixture
was stirred at room temperature for 2 h and diluted with water.
The aqueous solution was washed with Et2O, acidified with cHCl
at 0 °C and extracted with EtOAc. The organic layer was washed
with water and brine, dried over Na2SO4 and concentrated in va-
cuo. The residue was purified by silica gel column chromatogra-
phy (CHCl3/MeOH = 10:1) and recrystallized from EtOAc–n-
heptane to give (R)-K-13675-d7 as colorless needles (4.55 g,
85%): 1H NMR (400 MHz, DMSO-d6) d 0.98 (t, J = 7.0 Hz, 3H),
1.83-1.92 (m, 2H), 2.07 (quintet, J = 6.5 Hz, 2H), 3.67 (t,
J = 6.5 Hz, 2H), 3.95 (t, J = 6.5 Hz, 2H), 4.64 (t, J = 6.3 Hz, 1H),
4.74 (s, 2H), 6.80 (dd, J = 7.8, 2.0 Hz, 1H), 6.90 (s, 1H), 6.91 (d,
J = 7.8 Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H),
7.26 (t, J = 7.8 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.36 (d,
J = 7.8 Hz, 1H), 13.1 (br s, 1H); 13C-NMR (100 MHz, DMSO-d6) d
(FAB) m/z 502 [M++1]; mp 95–96 °C; ½a 2D4
ꢂ
+17.1 (c 0.67, MeOH).
4.13. Synthesis of 3-[[benzoxazol-2-yl[3-(4-methoxyphenoxy-
d7)propyl]amino]methyl]phenol (16)
To a solution of 13 (6.22 g, 21.1 mmol) in DMF (60 mL) and N,N-
diisopropylethylamine (2.73 g, 21.1 mmol) was added 2-chloro-
benzoxazole (3.24 g, 21.1 mmol) at room temperature. The reac-
tion mixture was stirred at 80 °C for 14 h and diluted with EtOAc
and water. The organic layer was washed with water and brine,
dried over Na2SO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography (n-hexane/
EtOAc = 2:1) to give a yellow oil. The oil was recrystallized from
tert-butyl methyl ether to give 16 as a colorless crystal (7.56 g,
87%): 1H NMR (400 MHz, DMSO-d6) d 2.06 (quintet, J = 6.8 Hz,
2H), 3.65 (t, J = 6.8 Hz, 2H), 3.94 (t, J = 6.8 Hz, 2H), 4.70 (s, 2H),
6.70 (dd, J = 7.8, 1.7 Hz, 1H), 6.69-6.76 (m, 2H), 7.00 (td, J = 7.8,
1.2 Hz, 1H), 7.15 (t, J = 7.8 Hz, 2H), 7.30 (d, J = 7.8 Hz, 1H), 7.36
(d, J = 7.8 Hz, 1H), 9.43 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d
*
9.46, 25.41, 27.14, 45.29, 51.30, 54.45 , 65.49, 76.41, 108.85,
*
*
113.36, 114.17, 114.18 , 115.05 , 115.67, 119.85, 120.18,
123.89, 129.74, 139.01, 143.29, 148.51, 152.34, 153.29, 158.04,
162.25, 172.39; Anal. Calcd for C28H23D7N2O6: C, 67.59; H,
4.66; D, 2.83; N, 5.63. Found: C, 67.66; H, 4.77; D, 2.82; N,
5.54; IR (solid sample): 2967, 2935, 1725, 1635, 1584, 1426,
1382 cmꢀ1
+16.5 (c 0.62, MeOH).
;
MS (FAB) m/z 498 [M++1]; mp 91–93 °C;
½ ꢂ
a 2D40

Y. Yamazaki et al. / Bioorg. Med. Chem. 17 (2009) 1911–1917
1917
D2O.4o We attempted to apply this method for preparation of 2-aminophenol-
d4 (2) under the same reaction conditions except using a Biotage microwave
oven. Unfortunately, satisfactory results could not be obtained due to the
mechanistic differences in the microwave oven. The maximum pressure of the
Biotage microwave oven was set as 22 bar. When the deuteration reaction was
carried out at 140 W and 175 °C, the reaction stopped within 2 min because the
pressure exceeded 22 bar.
Acknowledgments
We are grateful to Professor H. Sajiki of Gifu Pharmaceutical
University for helpful advice. We also wish to thank Dr. David Rick-
etts (Discovery Business Development) and Dr. Karsten Marx
(Polyphor) for their help in preparing the manuscript.
6. (a) Sajiki, H.; Hattori, K.; Aoki, F.; Yasunaga, K.; Hirota, K. Synlett 2002, 1149–
1151; (b) Sajiki, H.; Aoki, F.; Esaki, H.; Maegawa, T.; Hirota, K. Org. Lett. 2004, 6,
1485–1487; (c) Sajiki, H.; Esaki, H.; Aoki, F.; Maegawa, T.; Hirota, K. Synlett
2005, 1385–1388; (d) Sajiki, H.; Ito, N.; Esaki, H.; Maesawa, T.; Maegawa, T.;
Hirota, K. Tetrahedron Lett. 2005, 46, 6995–6998; (e) Maegawa, T.; Akashi, A.;
Esaki, H.; Aoki, F.; Sajiki, H.; Hirota, K. Synlett 2005, 845–847; (f) Ito, N.;
Watahiki, T.; Maesawa, T.; Maegawa, T.; Sajiki, H. Adv. Synth. Catal. 2006, 348,
1025–1028; (g) Ito, N.; Watahiki, T.; Maesawa, T.; Maegawa, T.; Sajiki, H.
Synthesis 2008, 9, 1467–1478; (h) Esaki, H.; Aoki, F.; Umemura, M.; Kato, M.;
Maegawa, T.; Monguchi, Y.; Sajiki, H. Chem. Eur. J. 2007, 13, 4052–4063; (i)
Kurita, T.; Hattori, K.; Seki, S.; Mizumoto, T.; Aoki, F.; Yamada, Y.; Ikawa, K.;
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Zimmermann, J. Angew. Chem., Int. Ed. 2007, 46, 7744–7765.
10. Bellas, M.; Cahill, R.; Hayes, L. G.B. Patent 1557237.
11. Liquid chromatography: Autosampler; 3133 HTS autosampler Z (Shiseido),
degasser/binary pump/column compartment; Agilent 1100 Series (Agilent
Technologies), Column; Symmetry C18 2.1 ꢁ 50 mm id, 5
lm (Waters). Mass
spectrometer: API4000 triple-quadrupole mass spectrometer (Applied
Biosystems) was equipped with an electrospray source, operating in the
positive ion mode. Quantification was performed in multiple reaction
monitoring (MRM) mode. The MS/MS was set to monitor at a transition of
m/z 491/107 for (R)-K-13675, m/z 498/107 for (R)-K-13675-d7 and m/z 502/107
for (R)-K-13675-d11, respectively. MRM data were acquired and integrated
using Analyst software (Ver.1.4.1, Applied Biosystems).
12. Methanol solution containing different amounts of 1 (0.02, 0.04, 0.1, 0.2, 0.4, 1,
2, and 4 ng/tube) was added to 250
evaporation under nitrogen at 40 °C, rat plasma (100
dried residue, and then 100 L of aqueous solution of 4 (2.5 ng/mL) and 900 lL
l
L of 2% propylene glycol in ethanol. After
lL) was added to the
l
of 0.1% aqueous acetic acid were added to the sample. Each sample was
vortexed, extracted with tert-butylmethyl ether (5 mL) with shaking for 10 min
and centrifuged at 3000 rpm for 10 min. The organic layer (3 mL) was
transferred into a glass tube and 250
was added to the solution. After evaporation under nitrogen at 40 °C, the dried
residue was dissolved in 150 L of 50% aqueous methanol. The samples thus
L) were analyzed using LC/MS/MS and monitored at m/z 491 and
lL of 2% propylene glycol in ethanol
l
5. Vaidyanathan et al. reported a simple and efficient method for post-synthesis
of 4-aminophenol-d4 by microwave irradiation in a CEM microwave oven at
140 W and 175 °C for 20 min in the presence of 35% deuterium chloride in
obtained (20
502.11
l