An efficient total synthesis of a sphingosine-1-phosphate receptor agonist KRP-203

Article abstract of DOI:10.1016/j.tet.2008.01.086

An efficient total synthesis of the S1P₁ agonist, KRP-203, is described. The key step involves the conjugate addition of diethyl acetamidomalonate to a styrene compound to give the core structure of KRP-203. Multigram quantities of the targeted KRP-203, sufficient for several biological studies, were obtained in six steps with an overall yield of 58%.

Full text of DOI:10.1016/j.tet.2008.01.086

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 3859e3866
www.elsevier.com/locate/tet
An efficient total synthesis of a sphingosine-1-phosphate
receptor agonist KRP-203
*
Masao Chino , Masatoshi Kiuchi, Kunitomo Adachi
Chemistry Laboratory, Pharmaceuticals Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshida,
Aoba-ku, Yokohama 227-0033, Japan
Received 28 November 2007; received in revised form 23 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract
An efficient total synthesis of the S1P1 agonist, KRP-203, is described. The key step involves the conjugate addition of diethyl acetamido-
malonate to a styrene compound to give the core structure of KRP-203. Multigram quantities of the targeted KRP-203, sufficient for several
biological studies, were obtained in six steps with an overall yield of 58%.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
attenuating the adverse events, multigram quantities of 2
were required along with an efficient synthesis process for
construction of its analogs. Recently we reported an easily ac-
cessible synthesis of 2 by a Pd-catalyzed cross-coupling reac-
tion;19 however, the synthetic methodology is not suitable for
the preparation of its related analogs having a variety of hydro-
philic parts. An extensive SAR study on the hydrophilic part of
1 has been conducted to lead to a discovery of some unique
compounds.5 In our continuing synthetic studies on 2 and its
analogs having a modified hydrophilic part, we developed
a new procedure which is characterized by an addition reaction
between carbonyl compounds having activated methylenes
and styrenes using cesium carbonate in dimethylsulfoxide
(DMSO). The method enables us not only to easily synthesize
various hydrophilic part modified analogs of 2 but also to
allow for an easy preparation of multigram quantities of 2.
In recent years, many immunosuppressants have been de-
veloped, some of which have been introduced in clinical organ
transplantation. FTY720 (1, Fig. 1),1e6 a novel immunomod-
ulator series, is the first sphingosine-1-phosphate (S1P) recep-
tor agonist7,8 that has proven effective in animal models of
transplantation and autoimmunity9,10 and has achieved prom-
ising results in phase 1 and phase 2 trials of renal transplanta-
tion in humans.11 FTY720 (1) exhibits immunosuppressive
€
activity by inhibiting the response of naıve and mature lym-
phocytes to S1P, and inducing the sequestration of circulating
€
naıve and mature lymphocytes from the blood and peripheral
tissues into secondary lymphoid organs.12,13 Its effect relates
to the modulation of lymphocyte trafficking. A novel immuno-
modulator KRP-203 (2, Fig. 1),14e16 which is an analog of 1,
is a S1P receptor agonist with increased selectivity on the S1P1
receptor over the S1P3 receptor. The S1P3 receptor may be as-
sociated with clinically observed adverse events of 1 including
a transient, asymptomatic bradycardia.17,18 As part of research
into structureeactivity relationships (SAR) focused on
* Corresponding author.
E-mail address: chino.masao@ma.mt-pharma.co.jp (M. Chino).
Figure 1. Structures of FTY720 (1) and KRP-203 (2).
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.086

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M. Chino et al. / Tetrahedron 64 (2008) 3859e3866
2. Results and discussion
2.1. Development of conjugate addition
bases such as potassium tert-butoxide20 and sodium tert-but-
oxide21 also gave compound 8a in moderate yields (entries 5
and 6).
We next set out to define the scope of the coupling reaction
with 9 by using methyl malonate 7b as a simple carbonyl com-
pound to optimize the reaction conditions (entries 10 and 11).
Addition of 2.4 equiv of methyl malonate 7b to 1 equiv of sty-
rene 9 and 1.2 equiv of cesium carbonate in DMSO suspension
gave a 96% yield of the coupling product 8b (entry 10). In
contrast, a reaction employing 1.2 equiv of styrene 9,
1.2 equiv of cesium carbonate, and 1 equiv of malonate af-
forded 8b in only 45% yield, along with 52% recovery of 9
(entry 11). These results suggest that the malonate may act
as a hydride donor. It is noteworthy that no regioisomer was
obtained in any of the styrene coupling reactions. The yield
of 8a and 8b was significantly high when DMSO was de-
gassed by purging with nitrogen for 15 min prior to use.
When cesium carbonate was used as catalyst (0.1 equiv),
only a trace amount of the desired product (<5%) was de-
tected. No reactions were observed when 5 equiv of galvi-
noxyl as radical scavenger was added to the reaction
mixture (entries 9 and 12). And the side-product 1122
(<30%) was obtained when DEAM (7a) was used (Scheme
3). The precise reaction mechanism remains unclear at pres-
ent, but the above-mentioned findings indicate that the addi-
tion of compound 7a to styrene 9 involves some kinds of
free radical intermediates (Scheme 3).23
While our first attempted route based on the reported route
for 214,16 was capable of supplying small amounts of the de-
sired product, it suffered from the following disadvantages:
(i) multistep synthesis with low overall yields; (ii) low yield
of diethyl acetamidomalonate (7a, DEAM) coupling reaction
(Scheme 1; 6/8a, <40%). In the synthesis of 1,2e5 the
DEAM coupling reaction with 1-(2-iodoethyl)-4-octylbenzene
proceeded smoothly to give the coupling compound. In the
synthesis of 2, however, the coupling reaction gave the styrene
9 predominantly and the coupling product 8a was produced in
only low yield (Scheme 2).
2.2. Synthesis of KRP-203
With the desired styrene reaction in hand, we turned our
attention to the synthesis of 2 (Scheme 4). The reaction be-
tween 2-chloro-4-fluorobenzaldehyde (3) and 3-mercaptophe-
nol (12) was selected because of their commercial availability.
We were gratified to see that the desired addition progressed
smoothly. However, this coupling reaction turned out to be
exothermic and therefore difficult to control on a large scale
(Table 2, entry 2). To resolve this issue, we developed a new
procedure. 3-Mercaptophenol (12) was charged slowly into
the preheated reaction mixture at 50 ꢀC. This process allows
good control of the exothermic behavior. The optimized proce-
dure can be used on a 30 mmol scale to consistently give com-
pound 13 in 98% isolated yield (entry 3).
Scheme 1. Our first attempted route based on the reported route for KRP-203.
Subsequent treatment with benzyl bromide in the presence
of potassium carbonate afforded the desired aldehyde 14 with
98% yield. On the basis of this study, we performed a one-pot
synthesis of compound 14 directly from 3 in 67% yield. This
product 14 was then converted to styrene 9 under Wittig or
Peterson olefination conditions. The Peterson olefination route
was more efficient because the intermediate alcohol can be
used without any purification. Subsequent addition of potas-
sium tert-butoxide afforded the styrene 9 in 88% yield. To
complete the synthesis of KRP-203 (2), compound 8a was re-
duced with calcium borohydride to afford acetamidopropane-
diol 15. Subsequently, deprotection with hydrochloric acid
provided 2 in six linear steps from commercially available
starting materials.
Scheme 2. Issue of DEAM coupling to iodoethylbenzene (6).
Based on the above-mentioned results, we hypothesized
that the styrene 9 may act as an intermediate in the DEAM
coupling reaction with 6. After screening several bases and
solvents in the DEAM coupling reaction with 9, we found ce-
sium carbonate in DMSO to be effective for this condensation
process, as summarized in Table 1. Other solvents such as
DMF and THF were ineffective (entries 2 and 3) and, in the
absence of base, no product was observed (entry 4). Other

M. Chino et al. / Tetrahedron 64 (2008) 3859e3866
3861
Table 1
Reaction condition screeninga
Entry
R
Base
Solventb
Product
Yieldc (%)
1
7a, NHAc
7a, NHAc
7a, NHAc
7a, NHAc
7a, NHAc
7a, NHAc
7a, NHAc
7a, NHAc
7a, NHAc
7b, Me
Cs2CO3
Cs2CO3
Cs2CO3
No base
KOtBu
DMSO
DMF
THF
8a
8a
8a
8a
8a
8a
8a
8a
8a
8b
8b
8b
83
2
5
3
NRf
NRf
49
4
DMSO
DMSO
DMSO
THF/EtOH (1:1)
DMSO
DMSO
DMSO
DMSO
DMSO
5
6
NaOtBu
KOtBu
36
7
NRf
NRf
NRf
96
8
CsOAc
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
9d
10
11e
12d
a
7b, Me
7b, Me
45g
NRf
Reaction conditions: to a solution of 2.4 equiv of malonate 7a or 7b and 1.2 equiv of base in DMSO was added a solution of 1 equiv of styrene 9 in DMSO at
room temperature. The reaction mixture was stirred at 35 ꢀC24 overnight under a nitrogen atmosphere in an oil bath (bath temperature).
b
All solvents were degassed by purging with nitrogen for 15 min prior to use.
Isolated yields after silica gel column chromatography.
c
d
Galvinoxyl of 5 equiv as radical scavenger was added to the reaction mixture.
Malonate/Cs2CO3/styrene molar ratio¼1:1.2:1.2.
e
f
NR¼no reaction, starting material remains unchanged.
g
Yield based on malonate 7b; styrene 9 (52%) recovered.
2.3. Scope of the addition of malonates to styrene
and several functional groups were well tolerated. For exam-
ple, 2-isopropylmalonate 7c (entry 1), 2-methoxymalonate
7d (entry 2), and 2-fluoromalonate 7e (entry 3) gave the cor-
responding adducts in good to moderate yields. And 2,2-di-
methyl-5-nitrodioxane 7f,25e27 which has an acidic proton
(pKa 18 in DMSO),28e30 also reacted with styrene 9 (entry
4). Finally, amino acid derivatives (7g and 7h) were well
tolerated (entries 5 and 6).
Our unique coupling reaction was broadly applicable to
a variety of carbonyl compounds with styrene 9 to prepare an-
alogs of 2. Six examples of styrene 9 coupling reaction with
carbonyl compounds are summarized in Table 3. We were
pleased to find that these substrates were generally effective
3. Summary
A highly efficient procedure for the synthesis of KRP-203
on a multigram scale was developed. Scheme 4 summarizes
the new route for KRP-203 synthesis from a commercially
available compound. This process features conjugate addition
of malonates, amino acid derivatives, or nitro compounds to
styrenes in DMSO in the presence of cesium carbonate.
4. Experimental
4.1. General
All reactions were carried out in oven-dried glassware un-
der a positive pressure of nitrogen. Anhydrous acetonitrile, di-
methylsulfoxide, and tetrahydrofuran were purchased from
Aldrich and used without further purification. Flash column
chromatography was performed using Biotage SP1Ô system.
1H NMR spectra were recorded on a Bruker Avance 400
Scheme 3. Plausible reaction mechanism.

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M. Chino et al. / Tetrahedron 64 (2008) 3859e3866
Table 3
Scope of the addition of malonates to styrene 9a
Entry
1
Nucleophile
Product
Yield (%)
43
2
3
79
55
4
5
37
58
68
6
a
Reaction conditions: to a solution of 2.4 equiv of malonate 7a or 7b and
1.2 equiv of cesium carbonate in DMSO was added a solution of 1 equiv of
styrene 9 in DMSO at room temperature. The reaction mixture was stirred
at 35 ꢀC24 overnight under an N2 atmosphere in an oil bath (bath temperature).
DMSO was degassed by purging with nitrogen for 15 min prior to use. Yields
(average of two runs) were given for products isolated after silica gel column
chromatography.
Scheme 4. Concise synthesis of KPR-203 (2).
(400 MHz) spectrometer. Chemical shifts are expressed in
parts per million relative to TMS as an internal standard. Coup-
ling constants are in units of hertz (Hz). Splitting patterns de-
scribe apparent multiplicities and are designated as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), and br
(broad). IR spectra were recorded on a PerkinElmer FT-IR
Spectrum One spectrometer. Melting points were obtained
Elemental analyses were performed on a Yanako CHN coder
MT-5 analyzer. High resolution mass spectra (HRMS) were
obtained on a Waters LCT Premier XE-ESI mass spectrum.
Most of the reactions were monitored by thin layer chroma-
tography on 0.25 mm E. Merck silica gel plates (60 F254),
visualized with UV light, and stained with 5% ethanolic
phosphomolybdic acid or p-anisaldehyde solution.
The following compounds in this paper are known com-
pounds: 2-amino-2-(2-{4-[3-(benzyloxy)phenylthio]-2-chloro-
phenyl}ethyl)propane-1,3-diol hydrochloride (KRP-203, 2),14,16
2,2-dimethyl-5-nitro[1,3]dioxane (7f),25e27 2-acetylamido-
3-ethoxycarbonyl-but-2-enedioic acid diethyl ester (11).22
on a Buchi 535 melting point apparatus and are uncorrected.
¨
Table 2
Reaction condition screening for the synthesis of 13a
4.2. General procedure for malonate coupling with styrene
Entry Base
Solvent
Temp (ꢀC) Yield (%) Reaction scale
A 50-mL, two-necked, round-bottomed flask equipped with
a Teflon-coated magnetic stir bar, a rubber septum, and an in-
ert gas inlet tube that was attached to a nitrogen manifold. The
flask was charged with ethyl acetamidomalonate 7a (444 mg,
2.05 mmol, 2.4 equiv) and 6 mL of anhydrous DMSO. To the
1
2
3
NaH
NaH
DMSO
DMSO
45
45
73
33
98
6 mmol in 60 mL
110 mmol in 260 mL
32 mmol in 110 mL
K2CO3 DMSO/THF 50
a
Yields given for products isolated after silica gel column chromatography.

M. Chino et al. / Tetrahedron 64 (2008) 3859e3866
3863
solution was added cesium carbonate (333 mg, 1.02 mmol,
4.2.4. 2-(2-{4-[3-(Benzyloxy)phenylthio]-2-chlorophenyl}-
1.2 equiv) at room temperature and the resulting mixture
was stirred at room temperature for 30 min. To the reaction
mixture was slowly added styrene 9 (300 mg, 0.85 mmol,
1 equiv, in 2.5 mL of anhydrous DMSO) at room temperature,
and was then stirred at 35 ꢀC in an oil bath (bath temperature)
overnight. The reaction mixture was cooled to 0 ꢀC in an ice-
water bath, and 10 wt % aqueous citric acid was added to the
reaction mixture. The aqueous suspension was extracted with
dichloromethane (3ꢁ20 mL). The combined organic phases
were washed with brine, dried over MgSO4, filtered, and con-
centrated by rotary evaporation. The residue was purified by
flash silica gel chromatography (gradient from EtOAc/hexane,
13:87 to EtOAc only) to give 403 mg (83%) of 8a as a color-
less oil.
ethyl)-2-methoxlmalonic acid dimethyl ester (8d)
Colorless oil; 1H NMR (CDCl3, 400 MHz) d 2.34e2.38 (m,
2H), 2.67e2.71 (m, 2H), 3.44 (s, 3H), 3.81 (s, 6H), 5.02 (s,
2H), 6.87e6.95 (m, 3H), 7.14 (s, 2H), 7.23 (t, 1H,
J¼7.9 Hz), 7.31e7.39 (m, 6H); 13C NMR (CDCl3,
100 MHz) d 27.1, 32.5, 52.9, 53.6, 70.1, 84.6, 114.1, 117.4,
123.7, 127.5, 128.1, 128.6, 129.6, 130.2, 131.1, 131.5,
134.5, 135.2, 136.2, 136.5, 137.4, 159.3, 168.8; IR (ATR):
1739, 1586, 1474, 1454, 1225 cmꢂ1. Anal. Calcd for
C27H27ClO6S$1/4H2O: C, 62.42; H, 5.34. Found: C, 62.46;
H, 5.32.
4.2.5. 2-(2-{4-[3-(Benzyloxy)phenylthio]-2-chlorophenyl}-
ethyl)-2-fluoromalonic acid diethyl ester (8e)
1
Colorless oil; H NMR (CDCl3, 400 MHz) d 1.31 (t, 6H,
4.2.1. 2-Acetamido-2-(2-{4-[3-(benzyloxy)phenylthio]-2-
chlorophenyl}ethyl)malonic acid diethyl ester (8a)
J¼7.2 Hz), 2.41e2.51 (m, 2H), 2.80e2.85 (m, 2H), 4.29 (q,
4H, J¼7.2 Hz), 5.02 (s, 2H), 6.88e6.95 (m, 3H), 7.14 (s,
2H), 7.24 (t, 1H, J¼7.9 Hz), 7.31e7.40 (m, 6H); 13C NMR
(CDCl3, 100 MHz) d 13.9, 14.0, 26.8, 26.9, 33.9, 34.1, 59.0,
62.4, 62.7, 70.1, 93.2, 95.2, 114.3, 117.6, 123.9, 127.5,
128.1, 128.6, 129.4, 130.2, 131.1, 131.4, 134.6, 135.6,
135.9, 136.5, 136.6, 159.3, 164.0, 165.8, 166.1; IR (ATR):
1748, 1586, 1474, 1185, 1016 cmꢂ1. Anal. Calcd for
C28H28ClFO5S$1/2H2O: C, 62.27; H, 5.41. Found: C, 62.44;
H, 5.55.
1H NMR (CDCl3, 600 MHz) d 1.26 (t, 6H, J¼7.1 Hz), 2.05
(s, 3H), 2.53e2.58 (m, 2H), 2.61e2.67 (m, 2H), 4.19e4.28
(m, 4H), 5.02 (s, 2H), 6.84 (s, 1H), 6.86e6.94 (m, 3H),
7.09e7.15 (m, 2H), 7.23 (t, 1H, J¼8.0 Hz), 7.29e7.41 (m,
6H); 13C NMR (CDCl3, 150 MHz) d 14.0, 23.0, 27.5, 32.0,
62.7, 66.2, 70.1, 114.2, 117.3, 123.6, 127.5, 128.1, 128.6,
129.6, 130.2, 130.9, 131.5, 134.5, 135.1, 136.2, 136.5,
137.2, 159.3, 167.9, 169.1; IR (ATR): 1736, 1678, 1474,
1192, 1016 cmꢂ1. Anal. Calcd for C30H32ClNO6S$1/3H2O:
C, 62.54; H, 5.72; N, 2.43. Found: C, 62.46; H, 5.63; N,
2.73.
4.2.6. 5-(2-{4-[3-(Benzyloxy)phenylthio]-2-chlorophenyl}-
ethyl)-2,2-dimethyl-5-nitro[1,3]dioxane (8f)
4.2.2. 2-(2-{4-[3-(Benzyloxy)phenylthio]-2-chlorophenyl}-
ethyl)-2-methylmalonic acid diethyl ester (8b)
White solid; mp 101 ꢀC; 1H NMR (CDCl3, 400 MHz)
d 1.39 (s, 3H), 1.45 (s, 3H), 2.03e2.08 (m, 2H), 2.60e2.64
(m, 2H), 3.97 (d, 2H, J¼13.0 Hz), 4.55 (d, 2H, J¼13.0 Hz),
5.01 (s, 2H), 6.89e6.96 (m, 3H), 7.04e7.13 (m, 2H), 7.22e
7.40 (m, 7H); 13C NMR (CDCl3, 100 MHz) d 20.5, 26.1,
26.9, 33.9, 63.9, 70.1, 86.1, 99.2, 114.3, 117.8, 124.1, 127.5,
128.1, 128.6, 129.3, 130.2, 130.9, 131.2, 134.3, 135.6,
135.7, 136.3, 136.5, 159.3; IR (ATR): 1590, 1537,
822 cmꢂ1. Anal. Calcd for C27H28ClNO5S: C, 63.09; H,
5.49; N, 2.72. Found: C, 62.79; H, 5.66; N, 2.58.
1
Colorless oil; H NMR (CDCl3, 400 MHz) d 1.26 (t, 6H,
J¼7.1 Hz), 1.52 (s, 3H), 2.09e2.14 (m, 2H), 2.65e2.69 (m,
2H), 4.20 (q, 4H, J¼7.1 Hz), 5.02 (s, 2H), 6.86e6.94 (m, 3H),
7.15 (s, 2H), 7.23 (t, 1H, J¼8.0 Hz), 7.31e7.40 (m, 6H); 13C
NMR (CDCl3, 100 MHz) d 14.1, 19.9, 28.4, 35.6, 53.5, 61.3,
70.1, 114.1, 117.3, 123.6, 127.5, 128.1, 128.6, 129.7, 130.1,
130.9, 131.6, 134.4, 134.8, 136.3, 136.6, 138.2, 159.3,
172.0; IR (ATR): 1727, 1586, 1474, 1225 cmꢂ1. Anal. Calcd
for C29H31ClO5S$1/3H2O: C, 65.34; H, 5.99. Found: C,
65.44; H, 5.85.
4.2.3. 2-(2-{4-[3-(Benzyloxy)phenylthio]-2-chlorophenyl}-
4.2.7. 4-{4-[3-(Benzyloxy)phenylthio]-2-chlorophenyl}-2-
ethyl)-2-isopropylmalonic acid diethyl ester (8c)
(diphenylmethyleneamino)butyric acid ethyl ester (8g)
1
Colorless oil; H NMR (CDCl3, 400 MHz) d 1.03 (d, 6H,
1
Colorless oil; H NMR (CDCl3, 400 MHz) d 1.27 (t, 3H,
J¼6.9 Hz), 1.30 (t, 6H, J¼7.1 Hz), 2.11e2.15 (m, 2H), 2.41
(q, 1H, J¼7.1 Hz), 2.64e2.68 (m, 2H), 4.24 (q, 4H,
J¼6.9 Hz), 5.01 (s, 2H), 6.86e6.94 (m, 3H), 7.14 (s, 2H),
7.22 (t, 1H, J¼7.9 Hz), 7.31e7.38 (m, 6H); 13C NMR
(CDCl3, 100 MHz) d 14.2, 18.6, 28.8, 32.2, 33.9, 60.9, 61.6,
70.1, 114.1, 117.2, 123.5, 127.5, 128.1, 128.6, 129.7, 130.1,
131.0, 131.6, 134.4, 134.7, 136.4, 136.5, 138.6, 159.3,
J¼7.2 Hz), 2.16e2.24 (m, 2H), 2.61e2.76 (m, 2H), 4.09e
4.23 (m, 3H), 5.01 (s, 2H), 6.86e6.94 (m, 3H), 7.06e7.11
(m, 2H), 7.15e7.43 (m, 15H), 7.68 (d, 2H, J¼7.2 Hz); 13C
NMR (CDCl3, 100 MHz) d 14.2, 29.7, 33.3, 61.0, 64.9,
70.1, 114.0, 117.2, 123.5, 127.5, 127.8, 128.1, 128.5, 128.6,
128.7, 128.9, 129.6, 130.1, 130.4, 130.8, 131.6, 134.4,
134.6, 136.3, 136.4, 136.6, 138.3, 139.5, 159.3, 170.9,
170.9; IR (ATR): 1721, 1586, 1474, 1242, 1221, 1020 cmꢂ1
.
172.0; IR (ATR): 1732, 1586, 1474, 1223, 1024, 694 cmꢂ1
.
Anal. Calcd for C31H35ClO5S: C, 67.07; H, 6.35. Found: C,
66.91; H, 6.18.
Anal. Calcd for C38H34ClNO3S: C, 73.59; H, 5.53; N, 2.26.
Found: C, 73.24; H, 5.67; N, 2.29.

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M. Chino et al. / Tetrahedron 64 (2008) 3859e3866
4.2.8. 3-(2-{4-[3-(Benzyloxy)phenylthio]-2-chlorophenyl}-
ethyl)-3-(diphenylmethyleneamino)dihydrofuran-
2(3H)-one (8h)
manifold. The flask was charged with compound 13 (4.7 g,
17.7 mmol, 1 equiv), potassium carbonate (5.9 g, 42.6 mmol,
2.4 equiv), and 80 mL of anhydrous MeCN. The resulting
mixture was stirred at 0 ꢀC in an ice-water bath, then benzyl
bromide (3.6 g, 21.3 mmol, 1.4 equiv, in 20 mL of anhydrous
MeCN) was added via syringe over 30 min. And the reaction
mixture was stirred at 45 ꢀC for 1.5 h, then the mixture was
cooled to 0 ꢀC in an ice-water bath, and 10 wt % aqueous citric
acid was added to the reaction mixture. The aqueous suspen-
sion was extracted with EtOAc (3ꢁ50 mL). The combined or-
ganic phases were washed with brine, dried over MgSO4,
filtered, and concentrated by rotary evaporation. The residue
was purified by flash silica gel chromatography (gradient
from EtOAc/hexane, 15:85e30:70) to give 6.2 g (98%) of
14 as a white solid: mp 48e49 ꢀC; 1H NMR (CDCl3,
400 MHz) d 5.07 (s, 2H), 7.05e7.07 (m, 2H), 7.11e7.13 (m,
3H), 7.33e7.42 (m, 6H), 7.74 (d, 1H, J¼8.0 Hz), 10.35 (s,
1H); 13C NMR (CDCl3, 100 MHz) d 70.2, 116.6, 120.6,
125.4, 127.0, 127.5, 127.7, 128.2, 128.7, 129.5, 129.6,
130.8, 131.2, 136.3, 138.5, 148.5, 159.6, 188.8; IR (ATR):
1684, 1575, 1242, 1019 cmꢂ1. Anal. Calcd for C20H15ClO2S:
C, 67.69; H, 4.26. Found: C, 67.64; H, 4.41.
1
White solid; mp 80e81 ꢀC; H NMR (CDCl3, 400 MHz)
d 1.73e1.78 (m, 1H), 2.05e2.16 (m, 1H), 2.48e2.60 (m,
2H), 4.06e4.12 (m, 1H), 4.28e4.33 (m, 1H), 4.58e4.62 (m,
1H), 5.00 (s, 2H), 5.22e5.25 (m, 1H), 6.71 (d, 1H,
J¼7.9 Hz), 6.82e6.86 (m, 2H), 6.95e7.05 (m, 4H), 7.11e
7.21 (m, 4H), 7.28e7.43 (m, 9H), 7.64 (d, 2H, J¼7.7 Hz);
13C NMR (CDCl3, 100 MHz) d 39.2, 44.2, 46.9, 64.2, 65.5,
70.1, 113.6, 117.0, 123.1, 126.7, 127.2 (ꢁ2), 127.46, 127.52,
127.6, 128.1, 128.58, 128.62, 129.9, 130.0, 131.0, 134.5,
135.0, 136.4, 136.5, 138.4, 142.5, 146.5, 159.3, 179.3; IR
(ATR): 1766, 1584, 1445, 1212, 1019, 697 cmꢂ1; HRMS (ESI):
m/zcalcdforC38H33ClNO3S(MþH)þ 618.1870,found618.1874.
Anal. Calcd for C38H32ClNO3S$1/2H2O$3/2HCl: C, 66.93; H,
5.10; N, 2.05. Found: C, 66.62; H, 4.91; N, 2.04.
4.3. 2-Chloro-4-[3-(hydroxy)phenylthio]benzaldehyde (13)
A 500-mL, three-necked, round-bottomed flask equipped
with an overhead mechanical stirrer with a large Teflon-coated
blade, a rubber septum, and a Liebig reflux condenser fitted
with an inert gas inlet tube that was attached to a nitrogen
manifold. The flask was charged with 2-chloro-4-fluoro-benz-
aldehyde (5.0 g, 31.7 mmol, 1 equiv), potassium carbonate
(8.7 g, 63.3 mmol, 2.0 equiv), and 80 mL of anhydrous
DMSO. The resulting mixture was stirred in an oil bath at
50 ꢀC (internal temperature), then 3-hydroxybenzenthiol
(4.0 g, 31.7 mmol, 1.0 equiv, in 30 mL of anhydrous THF)
was added via syringe over 30 min. The addition proceeded
at a rate that kept the internal temperature below 50 ꢀC, usu-
ally taking 30 min. And the reaction mixture was stirred at
50 ꢀC for 1.5 h, then the mixture was cooled to 0 ꢀC in an
ice-water bath, and 1 N HCl was added to the reaction mix-
ture. The aqueous suspension was extracted with EtOAc
(3ꢁ50 mL). The combined organic phases were washed with
brine, dried over MgSO4, filtered, and concentrated by rotary
evaporation. The residue was purified by flash silica gel chro-
matography (gradient from EtOAc/hexane, 15:85e30:70) to
4.4.1. One-pot approach to synthesis of 14
A 500-mL, three-necked, round-bottomed flask equipped
with an overhead mechanical stirrer with a large Teflon-coated
blade, a rubber septum, and a Liebig reflux condenser fitted
with an inert gas inlet tube that was attached to a nitrogen
manifold. The flask was charged with 2-chloro-4-fluoro-benz-
aldehyde (5.3 g, 33.6 mmol, 1 equiv), potassium carbonate
(9.3 g, 67.2 mmol, 2.0 equiv), and 70 mL of anhydrous
DMSO. The resulting mixture was stirred in an oil bath at
50 ꢀC (internal temperature), then 3-hydroxybenzenthiol
(4.2 g, 33.6 mmol, 1.0 equiv, in 30 mL of anhydrous THF)
was added via syringe over 30 min. The addition proceeded
at a rate that kept the internal temperature below 50 ꢀC, usu-
ally taking 30 min. And the reaction mixture was stirred at
50 ꢀC for 1.5 h. After complete conversion was confirmed
by TLC, the reaction mixture was stirred at 0 ꢀC in an ice-
water bath, then benzyl bromide (6.9 g, 40.3 mmol, 1.2 equiv,
in 30 mL of anhydrous THF) was added via syringe over
30 min. And the reaction mixture was stirred at 45 ꢀC for fur-
ther 1.5 h, then the mixture was cooled to 0 ꢀC in an ice-water
bath, and 10 wt % aqueous citric acid was added to the reac-
tion mixture. The aqueous suspension was extracted with
EtOAc (3ꢁ50 mL). The combined organic phases were
washed with brine, dried over MgSO4, filtered, and concen-
trated by rotary evaporation. The residue was purified by flash
silica gel chromatography (gradient from EtOAc/hexane,
15:85e30:70) to give 8.0 g (67%) of 14 as a white solid.
1
give 8.2 g (98%) of 13 as a yellow solid: mp 97e98 ꢀC; H
NMR (CDCl3, 400 MHz) d 5.77 (s, 1H), 6.95 (dd, 1H,
J¼8.2, 2.0 Hz), 7.03 (dd, 1H, J¼2.0, 2.0 Hz), 7.07e7.09 (m,
2H), 7.13 (d, 1H, J¼2.0 Hz), 7.32 (dd, 1H, J¼7.8, 7.8 Hz),
7.75 (d, 1H, J¼8.2 Hz), 10.3 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 117.0, 121.2, 125.4, 126.7, 127.6, 129.4, 129.5,
131.1, 131.2, 138.6, 148.8, 156.8, 189.3; IR (ATR): 3269,
1656, 1573, 1437, 1250 cmꢂ1. Anal. Calcd for C13H9ClO2S:
C, 58.98; H, 3.43. Found: C, 59.11; H, 3.63.
4.4. 4-[3-(Benzyloxy)phenylthio]-2-chlorobenzaldehyde (14)
4.5. 3-[3-(Benzyloxy)phenylthio]-6-vinyl-1-chlorobenzene (9)
A 500-mL, three-necked, round-bottomed flask equipped
with an overhead mechanical stirrer with a large Teflon-coated
blade, a rubber septum, and a Liebig reflux condenser fitted
with an inert gas inlet tube that was attached to a nitrogen
A 500-mL, three-necked, round-bottomed flask equipped
with a Teflon-coated magnetic stir bar, a rubber septum, and
an inert gas inlet tube that was attached to a nitrogen manifold.
The flask was charged with compound 14 (5.0 g, 14.1 mmol,

M. Chino et al. / Tetrahedron 64 (2008) 3859e3866
3865
1 equiv) and 140 mL of anhydrous THF. The resulting mixture
was stirred at 0 ꢀC in an ice-water bath, then 1 M
TMSCH2MgCl (22 mL, 22 mmol, 1.5 equiv) was added via
syringe over 30 min. And the reaction mixture was stirred at
room temperature for 30 min, then the mixture was cooled
to 0 ꢀC in an ice-water bath, and 1 N HCl was added to the
reaction mixture. The aqueous suspension was extracted
with EtOAc (3ꢁ20 mL). The combined organic phases were
washed with brine, dried over MgSO4, filtered, and concen-
trated by rotary evaporation. The crude residue was dissolved
in 140 mL of anhydrous THF. To the mixture was slowly
added 1 M KOtBu in THF solution (31 mL, 31 mmol,
2.2 equiv) at 0 ꢀC in an ice-water bath, and the reaction mix-
ture was stirred at room temperature for 30 min, then the mix-
ture was cooled to 0 ꢀC in an ice-water bath, and 1 N HCl was
added to the reaction mixture. The aqueous suspension was
extracted with EtOAc (3ꢁ30 mL). The combined organic
phases were washed with brine, dried over MgSO4, filtered,
and concentrated by rotary evaporation. The residue was puri-
fied by flash silica gel chromatography (gradient from hexane
only to EtOAc/hexane 30:70) to give 4.4 g (88%) of 9 as a yel-
an inert gas inlet tube that was attached to a nitrogen manifold.
The flask was charged with calcium chloride (2.9 g,
26.4 mmol, 2.5 equiv) and 20 mL of distilled water. To the re-
sulting mixture was added compound 8a (6.0 g, 10.5 mmol) in
100 mL of EtOH at 0 ꢀC in an ice-water bath, then sodium
borohydride (2.0 g, 52.7 mmol, 5 equiv) was added by
potion-wise. And the reaction mixture was stirred at room
temperature overnight. The mixture was cooled to 0 ꢀC in an
ice-water bath, and 1 N HCl was carefully added to the reac-
tion mixture. The resulting mixture was directly concentrated
by rotary evaporation. The residue was dissolved in CHCl3,
then washed with brine, dried over MgSO4, filtered, and con-
centrated by rotary evaporation to give 4.9 g of 15 (95%) as
a colorless oil which was sufficiently pure for use in the
1
next reaction without further purification: H NMR (CDCl3,
400 MHz) d 1.88e1.93 (m, 2H), 2.04 (s, 3H), 2.70e2.74 (m,
2H), 3.64 (dd, 2H, J¼11.3, 6.3 Hz), 3.73 (t, 2H, J¼6.0 Hz),
3.89 (dd, 2H, J¼11.3, 5.6 Hz), 5.02 (s, 2H), 6.02 (s, 1H),
6.87e6.94 (m, 3H), 7.13e7.18 (m, 2H), 7.23 (t, 1H,
J¼7.7 Hz), 7.31e7.41 (m, 6H); 13C NMR (CDCl3, 100 MHz)
d 24.1, 27.2, 33.3, 61.3, 65.7, 70.1, 114.1, 117.5, 123.7, 127.6,
128.1, 128.6, 129.8, 130.2, 131.0, 131.5, 134.1, 135.2, 136.1,
136.5, 138.0, 159.3, 171.7; IR (ATR): 3295, 1651, 1585, 1474,
1223, 1044, 1020, 684 cmꢂ1; HRMS (ESI): m/z calcd for
C26H29ClNO4S (MþH)þ 486.1506, found 486.1502.
1
low oil: H NMR (DMSO-d6, 400 MHz) d 5.11 (s, 2H), 5.46
(d, 1H, J¼11.2 Hz), 5.91 (d, 1H, J¼17.4 Hz), 6.94e7.05 (m,
4H), 7.20e7.22 (m, 1H), 7.30e7.42 (m, 7H), 7.69 (d, 1H,
J¼8.2 Hz); 13C NMR (DMSO-d6, 100 MHz) d 69.3, 114.9,
117.7, 118.0, 124.0, 127.4, 127.6, 127.8, 128.3, 128.6,
129.8, 130.7, 131.5, 132.5, 133.4, 134.1, 136.6, 136.8,
159.0; IR (ATR): 1583, 1472, 1222, 1024, 684 cmꢂ1. Anal.
Calcd for C21H17ClOS$1/8H2O: C, 71.02; H, 4.90. Found:
C, 71.03; H, 4.96.
4.7. 2-Amino-2-(2-{4-[3-(benzyloxy)phenylthio]-2-
chlorophenyl}ethyl)propane-1,3-diol hydrochloride
(KRP-203, 2)
A 500-mL, three-necked, round-bottomed flask equipped
with a Teflon-coated magnetic stir bar, a rubber septum, and
an inert gas inlet tube that was attached to a nitrogen manifold.
The flask was charged with compound 15 (5.7 g, 11.7 mmol),
20 mL of EtOH, and 20 mL of 6 M HCl. The reaction mixture
was stirred at 80 ꢀC for 60 min. The precipitate formed was
collected by filtration to give 4.5 g of 2 (87%) as a white solid:
mp 148e149 ꢀC; 1H NMR (DMSO-d6, 400 MHz) d 1.76e1.80
(m, 2H), 2.71e2.76 (m, 2H), 3.55 (d, 4H, J¼4.8 Hz), 5.10 (s,
2H), 5.43 (t, 2H, J¼4.8 Hz), 6.90e7.01 (m, 3H), 7.25 (dd, 1H,
J¼7.9, 1.2 Hz), 7.30e7.42 (m, 8H), 7.91 (br s, 3H); 13C NMR
(DMSO-d6, 100 MHz) d 25.9, 31.1, 60.0, 60.8, 69.2, 114.3,
116.9, 123.1, 127.6, 127.8, 128.3, 129.7, 130.6, 131.3,
133.4, 134.2, 135.1, 136.6, 138.3, 158.8; IR (ATR): 3243,
3033, 2940, 1587, 1521, 1243, 1072, 695 cmꢂ1. Anal. Calcd
for C24H26ClNO3S$HCl: C, 60.00; H, 5.66; N, 2.92. Found:
C, 59.72; H, 5.74; N, 2.90.
4.5.1. Wittig approach to synthesis of 9
A 1-L, three-necked, round-bottomed flask equipped with
a Teflon-coated magnetic stir bar, a rubber septum, and an in-
ert gas inlet tube that was attached to a nitrogen manifold. The
flask was charged with methyltriphenylphosphonium bromide
(15.6 g, 43.7 mmol) and 200 mL of anhydrous THF. The re-
sulting mixture was stirred at 0 ꢀC in an ice-water bath, then
n-butyllithium (27 mL, 1.6 M in hexanes, 43.7 mmol) was
added via syringe over 30 min. The orange mixture was stirred
at room temperature for 1 h. To the reaction mixture was
slowly added compound 14 (12.9 g, 36.4 mmol) in 100 mL
of anhydrous THF at 0 ꢀC in an ice-water bath. And the reac-
tion mixture was stirred at room temperature for 3 h. The
reaction mixture was treated with saturated NH4Cl solution
(200 mL) and extracted with ethyl acetate (3ꢁ50 mL). The
combined organic phases were washed with brine, dried over
MgSO4, filtered, and concentrated by rotary evaporation.
The residue was purified by flash silica gel chromatography
(gradient from hexane only to EtOAc/hexane 30:70) to give
11.3 g (88%) of 9 as a yellow oil.
4.8. Triethyl-2-(acetylamino)ethylene-1,1,2-tricarboxylate (11)
1
Colorless oil; H NMR (CDCl3, 400 MHz) d 1.29 (t, 3H,
4.6. N-(3-{4-[3-(Benzyloxy)phenylthio]-2-chlorophenyl}-1,1-
bis(hydroxymethyl)propyl)acetamide (15)
J¼7.1 Hz), 1.31 (t, 3H, J¼7.1 Hz), 1.34 (t, 3H, J¼7.1 Hz),
2.20 (s, 3H), 4.23 (q, 2H, J¼7.1 Hz), 4.28 (q, 2H,
J¼7.1 Hz), 4.33 (q, 2H, J¼7.1 Hz), 10.94 (s, 1H); 13C NMR
(CDCl3, 100 MHz) d 11.8, 12.0, 12.1, 22.0, 59.9, 60.0, 60.7,
103.6, 144.4, 160.4, 162.1, 164.8, 165.9; IR (ATR): 1721, 1602,
A 500-mL, three-necked, round-bottomed flask equipped
with a Teflon-coated magnetic stir bar, a rubber septum, and

3866
M. Chino et al. / Tetrahedron 64 (2008) 3859e3866
1236, 1196 cmꢂ1. Anal. Calcd for C13H19NO7$1/10H2O: C,
51.20; H, 6.30; N, 4.59. Found: C, 51.15; H, 6.33; N, 4.79.
10. Budde, K.; Schmouder, R. L.; Nashan, B.; Brunkhorst, R.; Lucker, P. W.;
Mayer, T.; Brookman, L.; Nedelman, J.; Skerjanec, A.; Bohler, T.;
Neumayer, H.-H. Am. J. Transplant. 2003, 3, 846e854.
11. Tedesco-Silva, H.; Mourad, G.; Kahan, B. D.; Boira, J. G.; Weimar, W.;
Mulgaonkar, S.; Nashan, B.; Madsen, S.; Charpentier, B.; Pellet, P.;
Vanrenterghem, Y. Transplantation 2004, 77, 1826e1833.
12. Chiba, K.; Yanagawa, Y.; Masubuchi, Y.; Kataoka, H.; Kawaguchi, T.;
Ohtsuki, M.; Hoshino, Y. J. Immunol. 1998, 160, 5037e5044.
13. Yanagawa, Y.; Sugahara, K.; Kataoka, H.; Kawaguchi, T.; Masubuchi, Y.;
Chiba, K. J. Immunol. 1998, 160, 5493e5499.
4.9. 2,2-Dimethyl-5-nitro[1,3]dioxane (7f)
A50-mLround-bottomedflaskequippedwitha Teflon-coated
magnetic stir bar and a nitrogen inlet. The flask was charged
with 2,2-dimethyl-5-nitro[1,3]dioxan-5-ylmethanol25e27 (1.14 g,
6.0 mmol) and 15 mL 10 wt % aqueous sodium hydroxide. The re-
actionmixturewas heated to60 ꢀCfor 1 h. Thesolutionwascooled
to 5 ꢀC and at this temperature it was acidified to pH 5 with concen-
trated acetic acid. The precipitated solid was collected by filtration
and dried to give 170 mg (18%) of 7f as a pale brown solid: mp 60e
61 ꢀC; 1H NMR (DMSO-d6, 400 MHz)d1.23 (s, 3H), 1.42 (s, 3H),
4.28 (dd, 2H, J¼13.0, 1.8 Hz), 4.38 (dd, 2H, J¼13.0, 1.8 Hz),
4.63e4.64 (m, 1H); 13C NMR (DMSO-d6, 100 MHz) d 18.9,
28.0, 59.2, 77.4, 98.0. Anal. Calcd for C6H11NO4$1/10AcOH: C,
43.11; H, 6.63; N, 8.38. Found: C, 43.41; H, 6.52; N, 8.29.
14. Kohno, Y.; Ando, N.; Kuriyama, K.; Iwanami, S.; Kudou, S. WO 2003/
029205 A1, 2003; Chem. Abstr. 2003, 138, 304049.
15. Shimizu, H.; Takahashi, M.; Kaneko, T.; Murakami, T.; Hakamata, Y.;
Kudou, S.; Kishi, T.; Fukuchi, K.; Iwanami, S.; Kuriyama, K.; Yasue,
T.; Enosawa, S.; Matsumoto, K.; Takeyoshi, I.; Morishita, Y.; Kobayashi,
E. Circulation 2005, 111, 222e229.
16. Tsubuki, T.; Kobayashi, K.; Komatsu, H. WO 2006/041019 A1, 2006;
Chem. Abstr. 2006, 144, 412416.
17. Sanna, M. G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.-Y.; Peterson, M. S.;
Webb, B.; Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H. J. Biol. Chem. 2004,
279, 13839e13848.
18. Fujishiro, J.; Kudou, S.; Iwai, S.; Takahashi, M.; Hakamata, Y.; Kinoshita,
M.; Iwanami, S.; Izawa, S.; Yasue, T.; Hashizume, K.; Murakami, T.;
Kobayashi, E. Transplantation 2006, 82, 804e812.
19. Hamada, M.; Kiuchi, M.; Adachi, K. Synthesis 2007, 13, 1927e1929.
20. Rodriguez, A. L.; Bunlaksananusorn, T.; Knochel, P. Org. Lett. 2000, 2,
3285e3287.
21. Siperko, L. G.; Smith, F. X. Synth. Commun. 1979, 9, 383e389.
22. Zimmermann, D.; Janin, Y. L.; Brehm, L.; Brauner-Osborne, H.; Ebert,
¨
Acknowledgements
We are grateful to Prof. Tohru Fukuyama (University of
Tokyo) and Prof. Jonathan A. Ellman (University of Califor-
nia, Berkeley) for helpful discussions. We thank Mrs. Megumi
Furui for careful NMR studies.
B.; Johansen, T. N.; Madsen, U.; Krogsgaard-Larsen, P. Eur. J. Med.
Chem. 1999, 34, 967e976.
23. For leading references of malonyl radical annulation, see: (a) Curran,
D. P.; Chen, M.-H.; Spletzer, E.; Seong, C. M.; Chang, C.-T. J. Am.
Chem. Soc. 1989, 111, 8872e8878; (b) Wetter, C.; Jantos, K.; Woithe,
K.; Studer, A. Org. Lett. 2003, 5, 2899e2902; (c) For recent radical
addition to styrene, see: Byers, J. H.; Janson, N. J. Org. Lett. 2006, 8,
3453e3455.
References and notes
1. For recent review, see: Adachi, K.; Chiba, K. Perspect. Med. Chem. 2007,
1, 11e23.
24. Also evaluated was the reaction temperature, which is crucial for limiting
the formation of decarboxylation product. At above 100 ꢀC or using the
water content of DMSO, the major product was the corresponding decarb-
oxylated compound.
2. Adachi, K.; Kohara, T.; Nakao, N.; Arita, M.; Chiba, K.; Mishina, T.;
Sasaki, S.; Fujita, T. Bioorg. Med. Chem. Lett. 1995, 5, 853e856.
3. Fujita, T.; Hirose, R.; Yoneta, M.; Sasaki, S.; Inoue, K.; Kiuchi, M.; Hirase, S.;
Chiba, K.; Sakamoto, H.; Arita, M. J. Med. Chem. 1996, 39, 4451e4459.
4. Kiuchi, M.; Adachi, K.; Kohara, T.; Teshima, K.; Masubuchi, Y.; Mishina,
T.; Fujita, T. Bioorg. Med. Chem. Lett. 1998, 8, 101e106.
5. Kiuchi, M.; Adachi, K.; Kohara, T.; Minoguchi, M.; Hanano, T.; Aoki, Y.;
Mishina, T.; Arita, M.; Nakao, N.; Ohtsuki, M.; Hoshino, Y.; Teshima, K.;
Chiba, K.; Sasaki, S.; Fujita, T. J. Med. Chem. 2000, 43, 2946e2961.
6. Kiuchi, M.; Adachi, K.; Tomatsu, A.; Chino, M.; Takeda, S.; Tanaka, Y.;
Maeda, Y.; Sato, N.; Mitsutomi, N.; Sugahara, K.; Chiba, K. Bioorg. Med.
Chem. 2005, 13, 425e432.
25. Quirk, J. M.; Harsy, S. G.; Hakansson, C. L. Eur. Patent 348223 B1, 1989.
26. Koszytkowska-Stawinska, M.; Sas, W.; Sowinska, A. J. Chem. Res.,
Synop. 1996, 3, 162e163.
27. Uno, T.; Beausoleil, E.; Goldsmith, R. A.; Levine, B. H.; Zuckermann,
R. N. Tetrahedron Lett. 1999, 40, 1475e1478.
7. Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.; Xie, J.; Milligan,
J.; Thornton, R.; Shei, G.-J.; Card, D.; Keohane, C.; Rosenbach, M.; Hale,
J.; Lynch, C. L.; Rupprecht, K.; Parsons, W.; Rosen, H. Science 2002, 296,
346e349.
28. Bordwell, F. G.; Vanier, N. R.; Matthews, W. S.; Hendrickson, J. B.;
Skipper, P. L. J. Am. Chem. Soc. 1975, 97, 7160e7162.
29. Bordwell, F. G.; Bartmess, J. E.; Hautala, J. A. J. Org. Chem. 1978, 43,
3113e3116.
8. Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.; Cottens, S.; Hof,
R.; Bruns, C.; Prieschl, E.; Baumruker, T.; Hiestand, P.; Foster, C. A.;
Zollinger, M.; Lynch, K. R. J. Biol. Chem. 2002, 277, 21453e21457.
9. Chiba, K.; Adachi, K. Drugs Future 1997, 22, 18e22.
30. Bordwell, F. G. Acc. Chem. Res. 1988, 21, 456e463.