Synthesis, characterization, and evaluation of thiazolidine derivatives of cysteine for suppressing eumelanin production

Article abstract of DOI:10.1248/cpb.c16-00509

In the pathway of melanin biosynthesis, cysteine (Cys) is utilized for the synthesis of pheomelanin. Accordingly, Cys is considered to suppress the formation of brown-black eumelanin. Although attempts have been made to utilize Cys and its derivatives as skin-whitening agents, their instability and odor hinders their application as a cosmetic agent. Herein, N-acetyl-2-methylthiazolidine-2,4-dicarboxylic acid ethyl ester (AcCP2Et) was proposed as a candidate for a stable and prolonged-release derivative of Cys to inhibit dopachrome formation after its degradation in melanocytes. It was synthesized by acetylation of 2-methylthiazolidine-2,4-dicarboxylic acid 2-ethyl ester (CP2Et), the condensation derivative of Cys and ethyl pyruvate. AcCP2Et suppressed melanogenesis in melanocytes in vitro, was stable in phosphate buffer at 70°C for five days, and exhibited far less odor than CP2Et. Therefore, AcCP2Et was validated to be a useful deriative of Cys for application as a skin-whitening agent. AcCP2Et comprises four stereoisomers; thus characterization of each stereoisomer was required. The stereochemistry of AcCP2Et was confirmed via a single-crystal X-ray structure analysis of N-acetyl-2-methylthiazolidine-2,4-dicarboxylic acid (AcCP) derived from AcCP2Et. In the synthesis of AcCP2Et, the acetylation of CP2Et proceeded with epimerization at C4 to give trans-isomers when excess acetyl chloride and an organic amine was used, whereas it proceeded while retaining the original (R) configuration at C4 to give the cis- and trans-isomer when an equivalent of acetyl chloride with an inorganic base was used. These results indicate that the formation of an intermolecular mixed acid anhydride is responsible for the isomerization at the C4 asymmetric center.

Full text of DOI:10.1248/cpb.c16-00509

Vol. 64, No. 12
Chem. Pharm. Bull. 64, 1681–1691 (2016)
1681
Regular Article
Synthesis, Characterization, and Evaluation of Thiazolidine Derivatives
of Cysteine for Suppressing Eumelanin Production
,a
Yusuke Amino,* Yoshinobu Takino,b Megumi Kaneko,a Fumie Ookura,b Mai Yamamoto,a
Tatsuki Kashiwagi,a and Keiji Iwasakia
a Institute for Innovation, Ajinomoto Co., Inc.; 1–1 Suzuki-cho, Kawasaki-ku, Kawasaki 210–8681, Japan: and
b Research Institute for Bioscience Products & Fine Cheicals, Ajinomoto Co., Inc.; 1–1 Suzuki-cho, Kawasaki-ku,
Kawasaki 210–8681, Japan.
Received June 24, 2016; accepted September 2, 2016
In the pathway of melanin biosynthesis, cysteine (Cys) is utilized for the synthesis of pheomelanin. Ac-
cordingly, Cys is considered to suppress the formation of brown–black eumelanin. Although attempts have
been made to utilize Cys and its derivatives as skin-whitening agents, their instability and odor hinders
their application as a cosmetic agent. Herein, N-acetyl-2-methylthiazolidine-2,4-dicarboxylic acid ethyl ester
(AcCP2Et) was proposed as a candidate for a stable and prolonged-release derivative of Cys to inhibit dopa-
chrome formation after its degradation in melanocytes. It was synthesized by acetylation of 2-methylthia-
zolidine-2,4-dicarboxylic acid 2-ethyl ester (CP2Et), the condensation derivative of Cys and ethyl pyruvate.
AcCP2Et suppressed melanogenesis in melanocytes in vitro, was stable in phosphate buffer at 70°C for five
days, and exhibited far less odor than CP2Et. Therefore, AcCP2Et was validated to be a useful deriative of
Cys for application as a skin-whitening agent. AcCP2Et comprises four stereoisomers; thus characterization
of each stereoisomer was required. The stereochemistry of AcCP2Et was confirmed via a single-crystal X-ray
structure analysis of N-acetyl-2-methylthiazolidine-2,4-dicarboxylic acid (AcCP) derived from AcCP2Et. In
the synthesis of AcCP2Et, the acetylation of CP2Et proceeded with epimerization at C4 to give trans-isomers
when excess acetyl chloride and an organic amine was used, whereas it proceeded while retaining the origi-
nal (R) configuration at C4 to give the cis- and trans-isomer when an equivalent of acetyl chloride with an
inorganic base was used. These results indicate that the formation of an intermolecular mixed acid anhydride
is responsible for the isomerization at the C4 asymmetric center.
Key words thiazolidine derivative; cysteine; suppressing eumelanin production; acetylation; stereoisomer
Melanin plays an important role in determining hair, eye, pheomelanin, production of eumelanin is suppressed and the
and skin color. It also protects the skin from UV radiation and skin color turns closer to yellow. Therefore, it is considered
inhibits photocarcinogenesis.1) However, hyperpigmentation, that production of eumelanin is suppressed by supplying Cys
which is caused by melanin overproduction and accumula- during melanin synthesis.7,8)
tion, causes freckles, age spots, melisma, and melanoma.2)
Accordingly, attempts have been made to utilize thiol-con-
Medical cosmetics that contain skin-whitening components taining compounds such as Cys and N-acetyl-cysteine (NAC)
are used for treating hyperpigmentation. The inhibitory effect as cosmetics, especially as skin-whitening agents. However,
of conventional pharmaceutical skin-whitening agents such as Cys and NAC are easily oxidized and have problems, includ-
hydroquinone, arbutin,3) and kojic acid4) on melanogenesis is ing poor stability and/or unpleasant odor; this makes them
attributed to the inhibition of tyrosinase. Therefore, skin-whit- unsuitable for formulation as cosmetic agents or external skin
ening agents that have a mechanism distinct from tyrosinase preparations. To solve these problems, the development of a
inhibition should be useful.
Cys derivative with improved stability is required.9)
Melanin is produced in human pigment cells (melanocytes)
Several 2-monosubstituted thiazolidine-4-carboxylic acids,
in the skinutilizing L-tyrosine (Tyr) and L-cysteine (Cys). The which are cyclic Cys derivatives with a masked sulfhydryl
melanin biosynthesis pathway comprises two main parts: the group, have been used as protective agents to liberate Cys
production of eumelanin and the production of pheomelanin. non-enzymatically at physiological pH and temperature.10–13)
During eumelanogenesis, Tyr is converted to 3,4-dihydroxy- Several 2-(substituted phenyl)thiazolidine-4-carbxylic acid
phenylalanine (DOPA) by tyrosinase, which then further derivatives have been investigated as agents for inhibition of
catalyzes the conversion of DOPA to dopaquinone. Dopaqui- melanogenesis or as tyrosinase inhibitors.14–16) However, some
none is converted to 5,6-dihydroxyindole-2-carboxylic acid of these compounds have the disadvantage of liberating toxic
and 5,6-dihydroxyindole intermediates, eventually resulting aldehyde upon degradation.
in the formation of brown–black eumelanin.5) Dopaquinone
As an example of the application of 2,2-disubstituted deriv-
also reacts with glutathione (GSH) or Cys to form glutathi- atives (Chart 1), Wlodek and Rommelspacher investigated the
onyldopa (GSH-dopa) or cysteineyldopa, and eventually forms effect of L-2-methyl-thiazolidine-2,4-dicarboxylic acid (abbre-
yellow–red pheomelanin.6) When Tyr, a starting material viated as CP (1), i.e., cysteinyl pyruvic acid), a condensation
for eumelanin, is utilized for the synthesis of melanin, pro- product of the biological substances Cys and pyruvic acid, on
duction of eumelanin is promoted and the skin color turns paracetamol-induced hepatotoxicity as a non-toxic precursor
darker. Conversely, when Cys is utilized for the synthesis of of Cys and GSH.17,18) Takizawa et al. reported that CP (1) is
*To whom correspondence should be addressed. e-mail: yusuke_amino@ajinomoto.com
© 2016 The Pharmaceutical Society of Japan

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1: 2-Methyl-2,4-thiazolidinedicarboxylic acid (cysteinyl pyruvic acid, CP); 2: 2-Methyl-2,4-thiazolidinedicarboxylic acid 2-ethyl ester (cysteinyl pyruvic acid 2-ethyl
ester, CP2Et); 3: N-Acetyl-2-methyl-2,4-thiazolidine-dicarboxylic acid (AcCP); 4: N-Acetyl-2-methyl-2,4-thiazolidine-dicarboxylic acid 2-ethyl ester (AcCP2Et); 5: N-
Acetyl-2-methyl-2,4-thiazolidine-dicarboxylic acid anhydride (AcCP anhydride).
Chart 1. Synthesis of N-Acetyl-2-methyl-2,4-thiazolidinedicarboxylic Acid and Its 2-Ethyl Ester
stable compared to conventional Cys derivatives, and may be selective inversion at C2 of the 2,4-cis- or 2,4-trans-epimers
used as an external skin preparation or a cosmetic.19) Suzuki to give 2(S)-p-tolyl-3-acetyl-1,3-thiazolidine-4(R)-carboxylic
et al. reported that L-2-methylthiazolidine-2,4-dicarboxylic acid or 2(R)-p-tolyl-3-acetyl-1,3-thiazolidine-4(R)-carboxylic
acid 2-ethyl ester (abbreviated as CP2Et (2), i.e., cysteinyl py- acid, respectively, through a ring-opening mechanism involv-
ruvic acid 2-ethyl ester), a product of Cys and ethyl pyruvate ing Schiff-base intermediates. Selective epimerization at C4
condensation, is useful as a skin-whitening agent since it has takes place when N-acetylated derivatives are treated with
a suppressive effect upon eumelanin production.20) However, acetic anhydride at 100°C via a mixed acid anhydride type
these compounds exist in equilibrium with Cys and pyruvic intermediate.21) Conversely, Cremonesi et al. reported that
acid or ethyl pyruvate in aqueous solution via non-enzymatic the acetylation of (2S,4R)-1,3-thiazolidine-2,4-dicarboxylic
ring opening and hydrolysis. Therefore, these compounds are acid 4-methyl ester with acetyl chloride in the presence of
unsuitable as stable prodrugs of Cys. It is expected that acety- trimethylamine (Et3N) in dichloromethane afforded (2S,4R)-
lation at the amino group of CP (1) or CP2Et (2) will help 3-acetyl-1,3-thiazolidine-2,4-dicarboxylic acid 4-methyl ester
prevent their non-enzymatic degradation.
with retention of configuration at C4 because their substrate
Thus, we propose N-acetyl-2-methylthiazolidine-2,4-dicar- could not generate an acid anhydride intermediate involving
boxylic acid and its 2-ethyl ester (abbreviated as AcCP (3), the 4-carboxyl group.24)
i.e., N-acetyl cysteinyl pyruvic acid, and AcCP2Et (4), i.e.,
Conversely, little information is available about the stereo-
N-acetyl cysteinyl pyruvic acid 2-ethyl ester, respectively) as selectivity of CP derivatives in N-acetylation, i.e., 2,2-disub-
stable and prolonged-release derivatives of Cys, which, after stituted thiazolidine-4-carboxylic acid. The CP derivative also
enzymatic cleavage and non-enzymatic ring opening to lib- comprises four stereoisomers. In addition, it has a tetra-sub-
erate the parent Cys in melanocytes, are expected to act as stituted asymmetric carbon center at C2. Schubert obtained
o-dopaquinone scavengers to inhibit dopachrome formation. AcCP (3) and its acid anhydride by the reaction of CP (1) and
Since all constituents of these compounds, i.e., Cys, pyruvic acetic anhydride/pyridine. However, the stereochemistry of
acid, acetic acid, and ethanol, are biological compounds, the the reaction product was not disclosed.25)
CP derivatives are expected to be non-toxic.
In this report, we present a detailed study of the stereo-
There have been many reports on the synthesis of N-acyl- specificity in the N-acetylation of CP (1) and CP2Et (2), and
ated 2-monosubstituted thiazolidine-4-carboxylic acid. These explore the stability of AcCP2Et (4) and its suppression of
derivatives comprise four stereoisomers because of their two eumelanin production in vitro.
asymmetric centers at C2 and C4; thus, characterization of
each stereoisomer has received considerable attention.21–23)
Results and Discussion
Szilágyi and Györgydeák studied the stereoselectivity in the
The synthesis of N-acetyl-2-methylthiazolidine-2,4-dicar-
acetylation of thiazoline-4-carboxylic acids. They obtained boxylic acid (AcCP) derivatives is outlined in Chart 1. Deriva-
2-p-tolyl-1,3-thiazolidine-4(R)-carboxylic acid as a 1:1 mix- tives of 2-methyl-tiazolidine-2,4-dicarboxylic acid (CP: 1a, b;
ture of C2 epimers by the condensation of L-(R)-Cys with p- CP2Et: 2a, b) were prepared by condensation of pyruvic acid
tolualdehyde. N-Acetylation using acetic anhydride/pyridine at or ethyl pyruvate with L-(R)-Cys. When these compounds
25°C or acetic anhydride/water at 100°C was accompanied by are reacted in ethanol or water at room temperature (r.t.), a

Vol. 64, No. 12 (2016)
Chem. Pharm. Bull.
1683
Table 1. Reaction of Thiazolidine Derivatives under Various Conditions
Entry
Substrate (ratio)
Acetylation
Products (ratio)
Yield (%)
Hydrolysis
3: AcCP (ratio)
Yield (%)
1
2
3
4
5
1a, b (1:1)
2a, b (1:1)
2a, b (1:1)
2a, b (1:1)
2c, d (1:1)
(CH3CO)2O/—a)
CH3COCl/Et3Nb)
(CH3CO)2O/—c)
5a, c (—)
4b, d (1:1)
4b, d (1:1)
4a, b (1:1)
4c, d (1:1)
53.1
58.8
46.5
93.6
93.2
HCl (aq)e)
NaOH (aq)f)
—
3a, c (1:1)
21.7g)
66.8
—
3b, d (1:1)
—
3a, b, d (45:50:5)
—
d)
CH3COCl/K2CO3
CH3COCl/K2CO3
NaOH (aq)f)
93.1g)
d)
—
—
Reagents and conditions: a) (CH3CO)2O (4eq.), 100°C, 2h; b) CH3COCl (1.5eq.), Et3N (2eq.), AcOEt, rt, overnight; c) (CH3CO)2O (2eq.), AcOEt, 90°C, 3h; d) CH3COCl
(1.2eq.), K2CO3 (1.2eq.), AcOEt, r.t., overnight; e) 5% citric acid, r.t., overnight, then added conc. HCl, 70°C, 1h; f) 4N NaOH (2.5eq.), EtOH, 60°C, overnight; g) total yield.
Fig. 1. ORTEP Drawing of the Crystal Structure of AcCP (3)
(A): Racemic cis-(2R,4R)- and cis-(2S,4S)-isomers of AcCP (3a, c) (Table 1, entry 1) with thermal ellipsoids at the 50% probability level; atom names of cis-(2S,4S)-
isomer of AcCP (3c) are labeled. (B): Racemic trans-(2S,4R)- and trans-(2R,4S)-isomers of AcCP (3b, d) (Table 1, entry 2) with thermal ellipsoids at the 50% probability
level; atom names of trans-(2S,4R)-isomer of AcCP (3b) are labeled.
spontaneous condensation–cyclization reaction leads to the AcCP (3a, c) based on chiral HPLC analysis and the single
formation of a mixture of the two diastereoisomers of the CP crystal X-ray structure analysis described below (Fig. 1A). As
derivatives (1a, b or 2a, b) in a ratio of ca. 1:1, as determined reported by Szilágyi and Györgydeák,21) the N-acetylation of
1
by H-NMR spectroscopy. These derivatives were converted cis-(2R,4R)-CP (1a) proceeded with retention of configuration
to N-acetyl-2-methylthiazolidine-2,4-dicarboxylic acid deriva- at C2 and C4 to give intramolecular acid anhydride 5a, where-
tives (AcCP: 3, AcCP2Et: 4) under various reaction conditions as N-acetylation of trans-(2S,4R)-CP (1b) proceeded with
as shown in Table 1. Since AcCP2Et (4) exists as four stereo- epimerization at C4 via intermolecular mixed acid anhydride
isomers due to its two asymmetric centers at C2 and C4, char- such as 6b and c followed by formation of intramolecular acid
acterization of each stereoisomer is complicated. Therefore, anhydride 5c as shown in (A) in Chart 2.
their stereochemistry was assigned on the basis of their ana-
Acetylation of 2-Methylthiazolidine-2,4-dicarboxylic Acid
lytical and spectral data. A reversed-phase HPLC (RP-HPLC) 2-Ethyl Ester (CP2Et: 2a, b) with Acetyl Chloride and
analysis and a chiral HPLC analysis were performed to evalu- Organic Amine Entry 2 in Table 1 shows the reaction of
ate the stereoisomer ratios of AcCP2Et (4a–d), and typical a ca. 1:1 mixture of CP2Et (2a, b), derived from L-(R)-Cys
chromatograms are shown in Supplementary Fig. S1.
and ethyl pyruvate, with acetyl chloride and Et3N. Contrary
Acetylation of 2-Methylthiazolidine-2,4-dicarboxylic Acid to CP (1a, b), CP2Et (2a, b) is highly soluble in ethyl acetate
(CP: 1a, b) with Acetic Anhydride Entry 1 in Table 1 (AcOEt), and was acetylated by acetyl chloride in the presence
shows the reaction of a ca. 1:1 mixture of CP (1a, b) and of Et3N at r.t. When 1.5 equiv. of acetyl chloride and 2.0 equiv.
acetic anhydride. When CP (1a, b) and acetic anhydride was of Et3N were used, acetylation completed to give AcCP2Et (4)
1
heated at 100°C according to Schubert’s method,25) a dark as a single stereoisomer, as indicated by H-NMR spectros-
brown homogeneous solution formed in 2h. The acid anhy- copy, in satisfactory isolated yield. However, the existence
dride of AcCP (5) was found to be stable to silica gel column of two stereoisomers was indicated by chiral HPLC. Similar
chromatography and isolated in modest yield. Acidic hydro- results were obtained when other organic amines such as
lysis of 5 gave a single stereoisomer, i.e., the cis-isomer of pyridine, N-methylmorpholine, or diisopropylethylamine were
1
AcCP (3), as indicated by H-NMR spectroscopy. In addition, used instead of Et3N.
this compound proved to be a mixture of cis-enantiomers of
Then, AcCP2Et (4) was subjected to alkaline saponification

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(A): Epimerization at C4 of 6b; Table 1, entry 1; (B): Epimerization at C4 of 7a; Table 1, entries 2 and 3.
Chart 2. Possible Epimerization Mechanisms at C4 of Thiazolidine Intermediates
at 60°C for 16h to give a single stereoisomer of AcCP (3) by cis-(2R,4R)-AcCP2Et (4a) and trans-(2S,4R)-AcCP2Et (4b).
1H-NMR, which was characteristically different from that of Therefore, it was concluded that the formation of a mixed
the above mentioned cis-enantiomers of AcCP (3a, c). The acid anhydride and the epimerization at C4 are avoided under
trans relative stereochemistry of the obtained AcCP (3) was these conditions using an inorganic base as in the case of the
estimated by comparing its 1H-NMR signals with those of 4-methyl ester derivative reported by Cremonesi et al.,24) and
cis-enantiomers of AcCP (3a, c). In addition, the single crystal that N-acetylation proceeded with retention of the C4 configu-
X-ray structure analysis described below revealed that this ration. Similarly, another set of stereoisomers of CP2Et (2c, d)
compound is a mixture of enantiomers of trans-isomers, i.e., obtained by the reaction of D-(S)-Cys and ethyl pyruvate gave
trans-(2S,4R)-AcCP (3b) and trans-(2R,4S)-AcCP (3d) (Fig. a mixture of cis-(2S,4S)- and trans-(2R,4S)-AcCP2Et (4c, d)
1B). It is concluded that the trans-enantiomers of AcCP2Et as shown in entry 5 in Table 1.
(4b, d) were obtained under these acetylation conditions.
Hydrolysis of cis-(2R,4R)- and trans-(2S,4R)-AcCP2Et (4a,
Therefore, it is proposed that the configuration at C4 of cis- b) under alkaline conditions at 60°C for 16h gave cis-(2R,4R)-
(2R,4R)-AcCP2Et (4a) is inverted to give the thermodynami- and trans-(2S,4R)-AcCP (3a, b). In addition, formation of ca.
cally stable trans-(2R,4S)-AcCP2Et (4d) via an intermolecular 5% of the trans-(2R,4S)-AcCP (3d) was observed (Table 1,
mixed acid anhydrides (7a, d) (Chart 2B).
entry 4). Therefore, the C4 asymmetric center of cis-(2R,4R)-
In addition, acetylation of CP2Et (2a, b) with more than AcCP2Et (4a) and/or cis-(2R,4R)-AcCP (3a) are liable to
2 equiv. acetic anhydride without amine in refluxing ethyl isomerize under basic conditions to generate the stable trans-
acetate also gave an enantiomeric mixture of trans-enantio- (2R,4S)-AcCP (3d).
mers of AcCP2Et (4b, d) (Table 1, entry 3). The presence of
Synthesis of Various Derivatives of 2-Methylthiazoli-
their anhydrides with acetic acid such as 7d (Chart 2B) were dine-2,4-dicarboxylic Acid Chart 3 shows examples of the
confirmed by 1H-NMR spectroscopy of the crude reaction derivatives of 2-methylthiazolidine-2,4-dicarboxylic acid pro-
mixture. This observation strongly suggests the involvement duced using CP (1a, b), CP2Et (2a, b), and 2-methylthiazoli-
of a mixed acid anhydride in the epimerization. Such a mech- dine-2,4-dicarboxylic acid 2-methyl ester (CP2Mt, 8). These
anism is consistent with that proposed in a study by Szilágyi derivatives were expected to possess different lipophilicities
and Györgydeák, in which selective epimerization at C4 of depending on the introduced functional groups. Reaction of
thiazolidine-4-carboxylic acids occurred via an intermediate CP2Et (2a, b) with a long-chain fatty acid chloride yielded
formation of thiazolidine-4-carboxylic acid-acetic acid mixed surfactant-like prodrugs such as N-hexadecanoyl derivatives
anhydrides.21)
11 and 12. The stereo-selectivity of N-acylation with a long-
Acetylation of 2-Methylthiazolidine-2,4-dicarboxylic Acid chain fatty acid chloride is similar to that of N-acetylation
2-Ethyl Ester (CP2Et: 2a, b) with Acetyl Chloride and with acetyl chloride, and depends on the amount of acylation
Inorganic Base Entry 4 in Table 1 shows the reaction of a reagent and the kind of base used. Esterification of the 2-car-
ca. 1:1 mixture of CP2Et (2a, b) with acetyl chloride in the boxylic acid group in cis-(2R,4R)- and trans-(2S,4R)-AcCP2Et
presence of potassium carbonate (K2CO3) in ethyl acetate at (4a, b) with an alcohol afforded more lipophilic prodrug forms
r.t. Under these reaction conditions, more than 1.2 equiv. of of Cys, such as 10a and b. Reaction of AcCP2Et (4a, b) with
acetyl chloride and 1.2 equiv. of K2CO3 were required to com- glycine (Gly) methyl ester or alanine (Ala) methyl ester afford-
plete the reaction in satisfactory yield. Tetrahydrofuran (THF) ed peptide-like derivatives, such as 13a and b. Of the synthe-
or CH2Cl2 were found to be suitable solvents for this reaction, sized compounds, AcCP (3), AcCP2Et (4), and AcCP2Me (9)
and potassium hydrogen carbonate, sodium carbonate, and show excellent water solubility and are suitable for assessing
sodium hydrogen carbonate were assessed as the inorganic melanogenesis inhibition activity.
base. Following these reaction conditions, a ca. 1:1 mixture
Single Crystal X-Ray Structure Analysis of the N-Ace-
of the cis- and trans-isomers of AcCP2Et (4) was indicated by tyl-2-methylthiazolidine-2,4-dicarboxylic Acid (AcCP, 3)
1H-NMR. Chiral HPLC analysis revealed that they were the
Crystal structures of the mixture of cis-enantiomers of AcCP

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Chart 3. Synthesis of N-Acyl-2-methyl-2,4-thiazolidinedicarboxylic Acid Derivatives
(3a, c) (Table 1, entry 1) and the mixture of trans-enantiomers AcCP2Et (4b, d) under the same conditions for 3d, and the
of AcCP (3b, d) (Table 1, entry 3) were determined by single melanin contents were measured to determine the melanogen-
crystal X-ray structure analysis. A summary of the crystallo- esis inhibition of the test molecules. Cys, CP2Et (2a, b), and
graphic analyses is shown in Supplementary Table S1.
AcCP2Et (4b, d) were found to reduce the melanin content in
Figure 1A illustrates the crystal packing in the racemic a concentration-dependent manner with IC50 values of 1108,
crystal of cis-(2R,4R)- and cis-(2S,4S)-AcCP (3a, c) as an 3120, and 4544µM, respectively. The activity of AcCP2Et (4b,
ORTEP drawing. This crystal belongs to the space group P-1 d) is estimated to be approximately one fourth of that of Cys
and its unit cell contains one molecule of cis-(2R,4R)-AcCP under these experimental conditions. As the stability of the
(3a), one molecule of cis-(2S,4S)-AcCP (3c), and two water compounds increases, their inhibition activity decreases. Cys
molecules. The cis-enantiomers of AcCP (3a, c) molecule is considered to be a key compound in the control of melano-
forms an intramolecular hydrogen bond between oxygen genesis, as it regulates tyrosinase activity as well as the ability
atoms O2 and O3, and adopts a relatively globular confor- of L-dopaquinone to generate eumelanin or pheomelanine.7,14)
mation. The hydrogen bonding network between a water We believe that the Cys liberated by degradation of CP2Et (2a,
molecule and oxygen atoms O1, O4, and O5 of neighboring b) or AcCP2Et (4b, d) is responsible for the melanogenesis in-
cis-enantiomers of AcCP (3a, c) molecules plays an important hibition observed. In the case of CP2Et (2a, b), it is thought
role in the crystal packing.
that Cys released by extracellular non-enzymatic degradation
Figure 1B demonstrates the crystal packing in the racemic is taken up into cells. Conversely, it is expected that AcCP2Et
crystal of trans-(2S,4R)- and trans-(2R,4S)-AcCP (3b, d) as (4b, d) is first taken up into the cells; Cys is then liberated
an ORTEP drawing. This crystal belongs to the space group inside the cells by enzymatic cleavage of its amide bond and/
P21/c and its unit cell contains two molecules of trans-(2S,4R)- or ester bond and followed by non-enzymatic ring opening.
AcCP (3b) and two molecules of trans-(2R,4S)-AcCP (3d). Since AcCP2Et (4b, d) is more active than trans-enantiomers
Contrary to the cis-enantiomers of AcCP (3a, c) structure, of AcCP (3b, d) (data not shown), AcCP2Et (4b, d) may be
the trans-enantiomers of AcCP (3b, d) molecule adopts a more easily taken up into cells, which may be responsible for
relatively extended conformation. Two kinds of intermolecular its higher activity relative to AcCP (3b, d). These data suggest
hydrogen bond, i.e., the hydrogen bond between O1 atom and that AcCP2Et (4b, d) may have the potential as a stable de-
O2 atom of neighboring enantiomer, and the hydrogen bond rivative of Cys for skin whitening. Research on the detection
between O3 atom and O5 atom of neighboring enantiomer, of Cys released from AcCP2Et (4b, d) in a skin model incor-
play an important role in the crystal packing.
porating melanocytes is currently under investigation in our
Suppression of Eumelanin Production Figure 2 shows group, and the results will be published in due course.
the cell viabilities and the melanogenesis inhibition activity of
the examined compounds.
Stability of N-Acetyl-2-methylthiazolidine-2,4-dicarbox-
ylic Acid 2-Ethyl Ester (AcCP2Et, 4a, c) As mentioned
We treated B16 melanoma cells with L-Cys, CP2Et (2a, b), above, alkaline hydrolysis of the ethyl ester group at the qua-
and trans-enantiomers of AcCP2Et (4b, d) (Table 1, entry 2) ternary C2 in AcCP2Et (4) is slow. Therefore, it was assumed
to determine whether the compounds have cytotoxic effects. that AcCP2Et (4) would be stable in neutral or weakly acidic
Cell viabilities were determined using Neutral Red (NR) as- conditions. The stability of a ca. 1:1 mixture (20mg/20mL)
says. The results show that Cys is not cytotoxic at 2mM, and of the cis-(2R,4R)- and trans-(2S,4R)-isomers of AcCP2Et (4a,
that CP2Et (2a, b) and AcCP2Et (4b, d) are not cytotoxic at b) (Table 1, entry 4) over a pH range of 4 to 7 was investigat-
concentrations of 2, 4, 8, and 10mM to B16 melanoma over ed, and the results are shown in Table 2. No degradation was
3d.
observed at 70°C after 5d for the AcCP2Et (4b) at pH 5 to 7
B16 melanoma cells were incubated with various concentra- or for the AcCP2Et (4a) at pH 7. Three to 7% degradation was
tions of kojic acid (a positive control), Cys, CP2Et (2a, b), and observed for the AcCP2Et (4b) at pH 4 and for the AcCP2Et

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Vol. 64, No. 12 (2016)
Fig. 2. Effects of Thiazolidine Derivatives on Cell Viability and Melanogenesis in B16 Melanoma Cells
B16 melanoma cells were treated with kojic acid, cysteine, and two thiazolidine derivatives for 3d, and cell viability was determined in phenol red free DMEM. Cell
viabilities were determined using NR assays. Melanin contents were measured at 450nm. Kojic acid was used as a positive control. Each value is reported as a percentage
relative to a sample-free control. Data represents the mean S.D. of at least three independent experiments.
detected at pH 7. It was concluded that AcCP2Et (4b, d) shows
less sulfur odor due to decomposition, and exhibits superior
Table 2. Time–Course Stability Test of AcCP2Et (4a, b) at 70°C
Residual values (%)
preservation stability to CP2Et (2a, b) on storage. Therefore,
AcCP2Et is a useful compound with potential as a cosmetic
ingredient.
pH
4a (cis-Isomer)
4b (trans-Isomer)
4
5
6
7
69.6
93.5
95.8
99.8
Conclusion
96.9
>99.9
>99.9
AcCP2Et (4) was designed as a stable and prolonged-release
derivative of Cys for the suppression of eumelanin production,
and was synthesized by acetylation of CP2Et (2a, b), a simple
condensation product of Cys and ethyl pyruvate. trans-Enan-
tiomers of AcCP2Et (4b, d) exhibited eumelanin suppression
>99.9
The residual values (%) after five days are shown. Sample concentration: 20mg
sample in 20mL of 25mM phosphate buffer.
(4a) at pH 5 and 6. A considerable degree of hydrolysis of the in vitro, indicating that AcCP2Et (4b, d) is cleaved enzymati-
ethyl ester functionality in the AcCP2Et (4a) was observed at cally followed by non-enzymatic ring opening to liberate the
pH 4. Therefore, the AcCP2Et (4b) is quite stable to prolonged parent Cys. AcCP2Et (4b, d) was highly stable in 25mM phos-
storage at pH 5 to 7. It is noteworthy that the degradation phate buffer at pH 4 to 7 at 70°C for 5d. In addition, AcCP2Et
products are cis-(2R,4R)- and trans-(2S,4R)-isomers of AcCP (4b, d) exhibited far less odor than the original CP2Et (2a, b).
(3a, b), which is also expected to function as a prodrug of These results indicate that AcCP2Et (4b, d) may have the po-
Cys. Stability to enzymatic hydrolysis is currently under in- tential as a stable derivative of Cys for skin-whitening.
vestigation in our group, and the results will be reported in
due course.
AcCP2Et (4) comprises four stereoisomers because of its
two asymmetric centers at C2 and C4, making it necessary
Odor Test Aqueous solutions (1%) at pH 4 and 7 of trans- to characterize each stereoisomer obtained by acetylation.
enantiomers of AcCP2Et (4b, d) (Table 1, entry 2) and CP2Et N-Acetylation of cis-(2R,4R)-isomer of CP2Et (2a) proceeded
(2a, b) in sealed vials were kept in a thermostatic bath at 70°C with epimerization at C4 via an intermolecular mixed acid
for 6d. The odor of each sample was evaluated by six panel- anhydride such as 7a and d (Chart 2B), producing stable
ists, and the evaluation scores of the six panelists were totaled trans-(2R,4S)-AcCP2Et (4d) (Table 1, entry 2), when large
(see Experimental). CP2Et (2a, b) is in equilibrium with Cys excess acetyl chloride and an organic amine was used, while it
and ethyl pyruvate in aqueous solution via non-enzymatic ring proceeded with retention of the (R) configuration at C4 when
opening and hydrolysis; therefore odor originating for Cys an equivalent of acetyl chloride and an inorganic base was
may be detected. Indeed, a strong sulfur odor was detected in used, to produce cis-(2R,4R)-isomers of AcCP2Et (4a) (Table
both samples of CP2Et (2a, b), while a slight sulfur odor was 1, entry 4). HPLC analysis of AcCP2Et (4) stereoisomers, and
detected in AcCP2Et (4b, d) at pH 4 and almost no odor was single crystal X-ray structure analysis of trans-enantiomers

Vol. 64, No. 12 (2016)
Chem. Pharm. Bull.
1687
of AcCP (3b, d) derived from trans-enantiomers of AcCP2Et
1H-NMR (DMSO-d6) δ: diastereomer 1; 1.23 (3H, t,
(4b, d), confirmed that N-acetylation by both methods leaves J=7.1Hz), 1.62 (3H, s), 2.81 (1H, dd, J=8.6, 10.0Hz), 3.42 (1H,
the original configuration at C2 unchanged. These results dd, J=6.1, 10.4Hz), 4.03 (1H, dd, J=6.2, 10.0Hz), 4.20 (2H,
suggest that the formation of an intermolecular mixed acid dd, J=7.1, 13.9Hz). Diastereomer 2; 1.19 (3H, t, J=7.1Hz),
anhydride is responsible for the isomerization at the C4 asym- 1.75 (3H, s), 2.97 (1H, dd, J=8.6, 10.0Hz), 3.29 (1H, m), 4.09
metric center.
(2H, m), 4.16 (1H, m). ESI-MS m/z: 220.0 (M+H)+.
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid (cis-
Isomer of AcCP, 3a, c) (Table 1, Entry 1) Compounds
Experimental
1H-NMR spectra were recorded using a Brucker Avance 1a and b (20.0g, 105mmol) and acetic anhydride (40mL,
400 (400MHz) and electron spray ionization (ESI)-MS spectra 418mmol) were heated at 100°C for 2h. The reaction mix-
were measured using a Thermo Quest TSQ 700. The melt- ture was concentrated under reduced pressure, aqueous citric
ing point (mp) of the obtained crystal was measured using an acid (5%, 200mL) was added thereto, and the mixture stirred
IA9100 digital melting point measuring apparatus manufac- overnight at r.t. To the mixture was added concentrated hydro-
tured by Electrothermal (U.K.).
chloric acid (2.0mL) and the mixture was stirred at 70°C for
RP-HPLC Conditions Column: Inertsil ODS-3 (high- 1h. The reaction mixture was filtered to remove tar material,
pressure type, GL Sciences, Tokyo, Japan, particle size 3µm, and the filtrate was concentrated under reduced pressure to a
inner diameter 4.6mm, length 250mm). Detector: UV absorp- volume of 100mL and extracted with ethyl acetate (250mL,
tion spectrophotometer (measurement wavelength: 210nm). twice). The extract was washed with saturated brine (100mL),
Eluent: A: 0.05M KH2PO4 (adjusted to pH 2 with 85% H3PO4), dried over anhydrous MgSO4, and concentrated to give crude
B: MeOH. Gradient conditions: 0–5min: A 100%, 5–25min: 3a and c (16.3g) as an amorphous substance. The amorphous
A:B=50:50, 25–35min: A:B=50:50, 35–40min: A 100%, substance was recrystallized from water, and the obtained
40–45min: A 100%. Flow rate: 0.8mL/min. Column tem- crystals were dried under reduced pressure to give 3a and c
perature: 30°C. Sample concentration: 25–50mg/dL. Injec- (cis-isomers, 5.3g, 22.7mmol, 21.7%).
tion volume: 20µL. Retention times of the stereoisomers of
N-AcCP2Et (4): 4a, c (cis-isomers): 30.5min; 4b, d (trans- dd, J=1.0, 12.0Hz), 3.61 (1H, dd, J=6.3, 12.0Hz), 5.22 (1H,
1H-NMR (DMSO-d6) δ: 1.80 (3H, s), 2.02 (3H, s), 3.49 (1H,
1
isomers): 32.4min.
dd, J=1.0, 6.2Hz). H-NMR (D2O) δ: 1.80 (3H, s), 2.03 (3H,
Chiral HPLC Conditions Column: CHIRALPACK QN-AX s), 3.41 (1H, d, J=12.4Hz), 3.72 (1H, dd, J=6.4, 12.4Hz),
(0.46 cm×15cm). Buffer: MeOH–AcOH–AcONH4=98:2:0.5 5.05 (1H, d, J=6.4Hz). 13C-NMR (D2O) δ: 22.8, 24.2, 34.1,
(v/v/w). Flow rate: 1.0mL/min. Temperature: 40°C. Detection: 67.1, 73.7, 172.4, 174.7, 176.7. ESI-MS m/z: 234.0 (M+H)+,
UV 250nm. Sample concentration: 1% in MeOH. Injection: 232.0 (M−H)−. FAB-MS m/z: 234.0451 (M+H) (Calcd for
10 µL. Retention time of stereoisomers of N-AcCP2Et (4): 4a C8H12NO5S: 234.0436).
(cis-(2R,4R)): 3.62min; 4c (cis-(2S,4S)): 3.75min; 4b (trans-
(2S,4R)): 4.08min; 4d (trans-(2R,4S)): 4.42min.
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid An-
hydride (5a, c) Compounds 1a and b (2.0g, 10.5mmol) and
2-Methylthiazolidine-2,4-dicarboxylic Acid (CP, 1a, b) acetic anhydride (4.0mL, 42.0mmol) were heated at 100°C
Under an argon atmosphere, L-Cys (15.0g, 124mmol) was for 2h. The reaction mixture was then concentrated under
dissolved in dry ethanol (35mL), pyruvic acid (18.6mL) was reduced pressure. To the residue was added water (10mL) and
added at r.t., and the mixture was stirred for 3h. The resulting the residue was extracted with ethyl acetate (15mL, twice).
precipitate was collected by filtration, washed with ice-cooled The extract was washed with saturated sodium chloride
ethanol, and dried under reduced pressure to give 1a and b (10mL, twice), dried over anhydrous MgSO4, and concen-
(ca. 1:1 diastereomer mixture, 23.0g, 120mmol, 97% yield).
trated to give crude 5 (2.12g) as an amorphous substance. The
1H-NMR (dimethyl sulfoxide (DMSO)-d6) δ: 1.59 (1.5H, s), amorphous substance was purified by silica gel chromatogra-
1.72 (1.5H, s), 2.76 (0.5H, dd, J=10.1, 10.2Hz), 2.97 (0.5H, dd, phy (eluted with n-hexane–ethyl acetate) to give 5a and c as a
J=8.4, 9.9Hz), 3.26 (0.5H, dd, J=6.7, 10.0Hz), 3.40 (0.5H, dd, white solid (1.2g, 5.6mmol, 53.1%).
J=6.0, 10.2Hz), 3.98 (0.5H, dd, J=6.0, 10.1Hz), 4.19 (0.5H,
1H-NMR (DMSO-d6) δ: 1.78 (3H, s), 1.89 (3H, s), 3.63 (1H,
dd, J=0.8, 12.6Hz), 3.86 (1H, dd, J=5.8, 12.6Hz), 4.91 (1H,
dd, J=6.7, 8.3Hz). ESI-MS m/z: 190.0 (M−H)−.
2-Methylthiazolidine-2,4-dicarboxylic Acid 2-Ethyl Ester dd, J=0.9, 5.7Hz). ESI-MS m/z: 215.8 (M+H)+ (weak), 231.8
(CP2Et, 2a, b) Under an argon atmosphere, L-Cys hydro- (M+H2O−H)−. Rf 0.80 (ethyl acetate–AcOEt (1:1), Kieselgel
chloride (60.0g, 381mmol) was added to ion-exchanged water 60).
(500mL), and the pH of the solution was adjusted to 5.05
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid 2-
by adding aqueous sodium hydroxide (4N, ca. 88mL). Ethyl Ethyl Ester (trans-Isomers of AcCP2Et, 4d and b, Table
pyruvate (63.0mL, 567mmol) was added to the solution and 1, Entry 2) Compounds 2a and b (10.0g, 45.6mmol) was
the mixture was stirred for 4h at r.t. The mixture was concen- dissolved in ethyl acetate (100mL), and the solution was kept
trated under reduced pressure to ca. 150mL. Water (100mL) at 0°C. To the solution was added Et3N (12.7mL, 91.2mmol),
was added, and the mixture was extracted with ethyl acetate and acetyl chloride (4.86mL, 68.4mmol) was added dropwise
(500, 200mL). The organic layer was washed with satu- over 10min. The reaction temperature was allowed to rise
rated sodium chloride solution (50mL), dried over anhydrous gradually, and the mixture was stirred overnight at r.t. The
MgSO4, and concentrated to ca. 200mL. To the residue was reaction mixture was then concentrated under reduced pres-
added n-heptane (220mL) at 45°C and the mixture was stored sure. To the residue was added ethyl acetate (200mL), and
overnight at r.t. The precipitate was collected and dried under the organic layer was washed with water (50mL), aqueous
reduced pressure to give 2a and b (ca. 1:1 diastereomer mix- citric acid (5%, 50mL), aqueous sodium hydrogen carbonate
ture, 65.6g, 299mmol, 79% yield).
(50mL), and saturated sodium chloride (50mL). The organic

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Chem. Pharm. Bull.
Vol. 64, No. 12 (2016)
layer was dried over anhydrous MgSO4, and concentrated. The (3H, s), 2.16 (3H, s), 3.47 (1H, dd, J=1.7, 12.2Hz), 3.72 (1H,
residue (trans–cis=95:5) was crystalized from ethyl acetate dd, J=6.8, 12.2Hz), 4.33–4.48 (2H, m), 4.98 (1H, dd, J=1.7,
and n-heptane to give 4b and d (trans-isomer: 99%) as pale 6.8Hz). 13C-NMR (DMSO-d6, 100MHz) δ: cis-isomer: 14.2,
yellow crystals (7.0g, 26.8mmol, 58.8%).
24.1, 25.3, 33.0, 61.4, 65.1, 72.9, 168.8, 170.4, 170.8. FAB-MS
1H-NMR (CDCl3) δ: 1.27 (3H, t, J=7.1Hz), 1.94 (3H, s), m/z: 262.0753 (M+H) (Calcd for C10H16NO5S: 262.0749). mp:
2.18 (3H, s), 3.40 (1H, d, J=11.6Hz), 3.56 (1H, dd, J=5.5, 101–105°C (cis-isomer).
11.0Hz), 4.20 (2H, qd, J=7.1, 7.2Hz), 5.00 (1H, d, J=5.9Hz),
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid (cis-,
1
9.10 (1H, brs). H-NMR (DMSO-d6) δ: 1.15 (3H, t, J=7.1Hz), trans-Isomers of AcCP, 3a, b, Table 1, Entry 4) Com-
1.77 (3H, s), 2.03 (3H, s), 3.36 (1H, dd, J=5.7, 11.6Hz), 3.40 pounds 4a and b (5.0g, 19.1mmol) obtained from compounds
(1H, dd, J=10.6, 11.7Hz), 4.04 (2H, qd, J=7.1, 9.0Hz), 5.34 2a and b by an operation similar to that described above was
(1H, d, J=5.2Hz). 13C-NMR (DMSO-d6, 100MHz) δ: 14.3, dissolved in ethanol (10mL), and sodium hydroxide solution
23.2, 23.9, 33.6, 61.4, 65.4, 70.7, 168.9, 171.4, 171.9. FAB-MS (4 N, 12.0mL) was added to the solution. The mixture was
m/z: 262.0753 (M+H) (Calcd for C10H16NO5S: 262.0749). mp: stirred with heating at 60°C under an argon atmosphere over-
138–141°C.
night. The mixture was allowed to cool to r.t., and the pH of
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid (trans- the solution was adjusted to ca. 7 with an ion-exchange resin
Isomers of AcCP, 3b and d, Table 1, Entry 2) Compounds (Amberlite IR 120B H AG(H+)), filtered and concentrated
4b and d (20.0g, 91.3mmol) obtained from compounds 2a and under reduced pressure to give 3a, b and small amount of 3d
b by an operation similar to that described above was dis- as a colorless amorphous (4.4g, 18.9mmol, 99.5%).
solved in ethanol (40mL), and sodium hydroxide solution (4N,
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid 2,4-Di-
57.0mL) was added to the solution. The mixture was stirred ethyl Ester (10a, Chart 3) Compounds 4a and b (cis–
with heating at 60°C under an argon atmosphere overnight. trans=48:52, 1.0g, 3.84mmol) was dissolved in dichloro-
The mixture was allowed to cool to r.t., and the pH of the so- methane (15mL), and the reaction mixture was kept at 0°C.
lution was adjusted to 1 with hydrochloric acid solution (6N). To the solution were added ethanol (0.27mL, 4.60mmol),
The solution was kept at r.t. overnight, and the resulting white 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
crystals were collected by filtration and washed with a small (EDCI·HCl, 0.89g, 4.64mmol), and 4-dimethylaminopyridine
amount of cold water. The crystals were dried under reduced (DMAP; 95mg, 0.77mmol). The reaction temperature was
pressure to give 3b and d (trans-isomers, 14.2g, 61.0mmol, allowed to gradually rise to r.t., and the mixture was stirred
66.8%).
overnight. The reaction mixture was concentrated under re-
1H-NMR (DMSO-d6) δ: 1.73 (3H, s), 2.01 (3H, s), 3.36 duced pressure, and water (10mL) was added. The mixture
1
(2H, d, J=3.6Hz), 5.28 (1H, t, J=3.6Hz). H-NMR (D2O) δ: was then extracted with ethyl acetate (10mL, three times).
1.77 (3H, s), 2.10 (3H, s), 3.43 (1H, d, J=12.0Hz), 3.50 (1H, The combined organic layer was washed with aqueous citric
dd, J=6.0, 12.0Hz), 5.24 (1H, d, J=5.6Hz). 13C-NMR (D2O) acid (5%, 10mL, twice), aqueous sodium hydrogen carbonate
δ: 22.1, 23.0, 32.9, 66.1, 70.8, 172.6, 173.6, 175.4. ESI-MS (5%, 10mL, twice) and saturated sodium chloride (10mL),
m/z: 234.0 (M+H)+, 232.0 (M−H)−. FAB-MS m/z: 234.0451 dried over anhydrous MgSO4, and concentrated to give 10a
(M+H) (Calcd for C8H12NO5S: 234.0436).
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid 2-Ethyl
as a colorless oil (cis–trans=45:55, 1.0g, 3.48mmol, 91.0%).
1H-NMR (CDCl3) δ: cis-isomer; 1.28 (3H, t, J=7.1Hz),
Ester (cis- and trans-Isomers of AcCP2Et, 4a and b, Table 1.34 (3H, t, J=7.1Hz), 1.96 (3H, s), 2.17 (3H, s), 3.50 (1H, dd,
1, Entry 4) Compounds 2a and b (20.0g, 91.3mmol) was J=6.1, 11.8Hz), 3.61 (1H, dd, J=1.8, 11.9Hz), 4.26–4.21 (2H,
dissolved in ethyl acetate (190mL), and the solution was kept m), 4.34–4.27 (2H, m), 4.85 (1H, dd, J=1.8, 6.0Hz). trans-
at 0°C. To the solution was added potassium carbonate (15.2g, Isomer; 1.27 (3H, t, J=7.1Hz), 1.33 (3H, t, J=7.2Hz), 1.93
110mmol), and acetyl chloride (7.8mL, 110mmol) was added (3H, s), 2.12 (3H, s), 3.36 (1H, d, J=11.7Hz), 3.54 (1H, dd,
dropwise over 40min. The reaction temperature was allowed J=6.2, 11.6Hz), 4.12–4.25 (2H, m), 4.25–4.33 (2H, m), 4.95
to gradually rise, and the mixture was stirred overnight at r.t. (1H, d, J=5.84Hz). FAB-MS m/z: 290.1063 (M+H) (Calcd for
To the solution was added hydrochloric acid aqueous solution C12H20NO5S: 290.1062).
(2N) until the pH of the aqueous layer was 2. The organic
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid 2-Ethyl
layer was separated and the water layer was extracted with 4-Isopropyl Diester (10b, Chart 3) An operation similar to
ethyl acetate (200mL, twice). The combined organic layers the one described for 10a was performed using isopropanol,
were washed with water (100mL, twice) and saturated sodium and 10b was obtained in an overall yield of 86% as a colorless
chloride (100mL, twice), dried over anhydrous MgSO4, and oil (cis–trans=27:73).
concentrated. The residue (cis–trans=50:50) was crystal-
ized from ethyl acetate and n-hexane to give 4a and b (cis– (6H, d, J=6.2Hz), 1.96 (3H, s), 2.16 (3H, s), 3.49 (1H, dd,
trans=50:50) as colorless crystals (22.3g, 85.5mmol, 93.6%). J=6.1, 11.9Hz), 3.59 (1H, dd, J=2.0, 1.9Hz), 4.81 (1H, dd,
1H-NMR (CDCl3) δ: cis-isomer; 1.28 (3H, t, J=7.1Hz), 1.33
Pure trans-isomer 4b was obtained by recrystallization of J=2.0, 6.1Hz), 5.19–5.12 (1H, m). trans-Isomer; 1.28 (3H, t,
the above mentioned diastereomeric mixture from ethyl ace- J=7.1Hz), 1.31 (6H, d, J=6.2Hz), 1.93 (3H, s), 2.12 (3H, s),
tate (150mL) as colorless crystals (7.3g). The cis-isomer was 3.36 (1H, dd, J=0.7, 11.6Hz), 3.55 (1H, dd, J=6.2, 11.6Hz),
obtained by an operation similar to that outlined above, except 4.17–4.28 (2H, m), 4.91 (1H, d, J=5.6Hz), 5.12–5.19 (1H,
that dichloromethane was used as the reaction and recrystalli- m). FAB-MS m/z: 304.1208 (M+H) (Calcd for C13H22NO5S:
1
zation solvent (36% yield, cis-isomer: 96%). The H-NMR and 304.1219).
13C-NMR spectra of trans-isomer 4b are virtually identical to
those of the racemic mixture of 4b and d.
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid 2-
Ethyl Ester 4-Gly Methyl Ester Amide (13a, Chart 3)
1H-NMR (CDCl3) δ: cis-isomer; 1.39 (3H, t, J=7.1Hz), 1.99 Compounds 4a and b (cis–trans=48:52, 2.0g, 7.68mmol)

Vol. 64, No. 12 (2016)
Chem. Pharm. Bull.
1689
was dissolved in dichloromethane (20mL), and the reaction
N-Hexadecanoyl-2-methylthiazolidine-2,4-dicarboxylic
mixture was kept at 0°C. To the solution were added Gly Acid (trans-Isomer, 12, Chart 3) To the solution of com-
methyl ester hydrochloride (0.963g, 7.67mmol), EDCI·HCl pound 11 (trans-isomer, 5.12g, 11.2mmol) in EtOH (10mL)
(1.62g, 8.47mmol) and 1-hydroxy-1H-benzotriazole hydrate was added aqueous NaOH solution (4N, 6.97mL, 27.88mmol),
(HOBt·H2O, 1.14g, 8.45mmol). The reaction temperature was and the solution was stirred overnight at 60°C. The pH of the
allowed to gradually rise to r.t., and the mixture was stirred solution was adjusted to 1 by addition of aqueous HCl (6N).
overnight. The reaction mixture was concentrated under re- The solution was then extracted with ethyl acetate (30mL,
duced pressure, and water (20mL) was added. The mixture twice) and the combined organic layer was washed with water
was then extracted with ethyl acetate (80mL, three times). (30mL, twice) and saturated sodium chloride (30mL, twice),
The combined organic layer was washed with aqueous citric dried over anhydrous MgSO4, and concentrated. The residue
acid (5%, 20mL, twice), aqueous sodium hydrogen carbonate was recrystallized from mixed solvent of ethyl acetate and
(5%, 20mL, twice) and saturated sodium chloride (20mL), n-hexane to give 12 as a white solid (trans-isomer, 4.16g,
dried over anhydrous MgSO4, and concentrated to give 13a as 9.68mmol, 86.4%).
a pale-yellow oil (cis–trans=48:52, 2.5g, 7.20mmol, 93.8%).
1H-NMR(400MHz, CDCl3) δ: 0.88 (3H, t, J=6.9Hz),
1H-NMR (CDCl3) δ: cis-isomer; 1.30 (3H, t, J=7.1Hz), 2.07 1.23–1.29 (24H, m), 1.62–1.67 (2H, m), 1.94 (3H, s), 2.25–2.36
(3H, s), 2.21 (3H, s), 3.36 (1H, d, J=11.9Hz), 3.62 (1H, dd, (2H, m), 3.42 (1H, d, J=11.2Hz), 3.59 (1H, dd, J=6.1,
J=7.0, 11.9Hz), 4.35–4.14 (4H, m), 3.81(3H, s), 4.90 (1H, d, 11.9Hz), 5.04 (1H, d, J=6.7Hz). ESI-MS m/z: 430.3 (M+H)+,
J=6.8Hz), 6.89 (1H, brs). trans-Isomer; 1.36 (3H, t, J=7.1Hz), 428.3 (M−H)−. FAB-MS m/z: 430.2620 (M+H) (Calcd for
1.99 (3H, s), 2.23 (3H, s), 3.44 (1H, dd, J=1.1, 12.3Hz), 3.70 C22H40NO5S: 430.2627).
(1H, dd, J=7.0, 12.3Hz), 3.76 (3H, s), 4.35–4.14 (4H, m), 4.84
(1H, dd, J=1.0, 7.0Hz), 9.09 (1H, brs). FAB-MS m/z: 333.1118 (CP2Mt, 8, Chart 3) An operation similar to the one de-
(M+H) (Calcd for C13H21N2O6S: 333.1120). scribed for compounds 2a and b was performed using methyl
2-Methylthiazolidine-2,4-dicarboxylic Acid 2-Methyl Ester
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid 2-Ethyl pyruvate, and 8 was obtained in an overall yield of 71.5% as a
Ester 4-L-Ala Methyl Ester Amide (13b, Chart 3) An op- white solid (ca. 50:50 diastereomer mixture).
eration similar to the one described for 13a was performed
1H-NMR(DMSO-d6) δ: diastereomer 1; 1.74 (3H, s), 2.96
using L-Ala methyl ester hydrochloride, and 13b was obtained (1H, dd, J=8.7, 10.0Hz), 3.27 (1H, dd, J=6.7, 10.0Hz), 3.62
in quantitative yield of as a pale-yellow oil (cis–trans=48:52). (3H, s), 4.13 (1H, d, J=6.8, 8.5Hz). Diastereomer 2; 1.60 (3H,
1H-NMR (CDCl3) δ: cis-isomer; 1.29 (3H, t, J=7.1Hz), s), 1.60 (3H, s), 3.41 (1H, dd, J=6.1, 10.3Hz), 3.72 (3H, s), 4.01
1.48 (3H, d, J=7.4), 2.02 (3H, s), 2.21 (3H, s), 3.35 (1H, (1H, dd, J=6.1, 9.9Hz).
d, J=11.9Hz), 3.60 (1H, dd, J=6.9, 11.9Hz), 3.78 (3H, s),
N-Acetyl-2-methylthiazolidine-2,4-dicarboxylic Acid 2-
4.30–4.36 (2H, m), 4.85 (1H, d, J=6.6Hz), 6.83 (1H, brs), Methyl Ester (AcCP2Me, 9, Chart 3) An operation similar
9.00 (1H, brs). trans-Isomer; 1.35 (3H, t, J=7.1Hz), 1.48 (3H, to the one described for compounds 4a and b was performed
d, J=7.2Hz), 1.99 (3H, s), 2.24 (3H, s), 3.41 (1H, dd, J=0.9, using compounds 8 and 9 was obtained in an overall yield
12.2Hz), 3.67 (1H, dd, J=7.0, 12.2Hz), 3.74 (3H, s), 4.19–4.24 of 85.2% (cis–trans=50:50) as colorless crystals (10.3g,
(2H, m), 4.62–4.68 (1H, m), 4.52–4.60 (1H, m), 4.80 (1H, dd, 41.5mmol). The pure cis-isomer and trans-isomer were ob-
J=0.8, 6.9Hz), 9.00 (1H, brs). FAB-MS m/z: 347.1285 (M+H) tained by repeated recrystallization of the diastereomeric mix-
(Calcd for C14H23N2O6S: 347.1277).
N-Hexadecanoyl-2-methylthiazolidine-2,4-dicarboxylic
ture from ethyl acetate.
1H-NMR (CDCl3) δ: cis-isomer; 2.01 (3H, s), 2.17 (3H, s),
Acid 2-Ethyl Ester (trans-Isomer, 11, Chart 3) Compounds 3.49 (1H, dd, J=1.7, 12.3Hz), 3.72 (1H, dd, J=6.7, 12.2Hz),
2a and b (trans–cis=50:50, 10.0g, 45.6mmol) was dissolved 3.95 (3H, s), 5.00 (1H, dd, J=1.7, 6.6Hz). trans-Isomer; 1.95
in ethyl acetate (90mL), and the solution was kept at 0°C. To (3H, s), 2.19 (3H, s), 3.49 (1H, d, J=11.6Hz), 3.58 (1H, dd,
the solution was added Et3N (12.7mL, 91.2mmol), and n-hexa- J=6.7, 12.2Hz), 3.76 (3H, s), 5.01 (1H, d, J=5.8Hz). FAB-MS
decanoyl chloride (20.7mL, 68.4mmol) was added dropwise m/z: 248.0607 (M+H) (Calcd for C9H14NO5S: 248.0593).
over 30min. The reaction temperature was allowed to gradu-
Single Crystal X-Ray Study Single crystals of the mix-
ally rise to r.t., and the mixture was stirred overnight. Citric ture of cis-(2R,4R)- and cis-(2S,4S)-isomers of AcCP (3a,
acid solution (5%, 30mL) was then added to the reaction mix- c) (Table 1, entry 1) and the mixture of trans-(2S,4R)- and
ture, and the organic layer was separated. The aqueous layer trans-(2R,4S)-isomers of AcCP (3b, d) (Table 1, entry 2) suit-
was extracted with ethyl acetate (60mL, twice). The combined able for X-ray analysis were obtained from MeOH via slow
organic layer was washed with water (50mL, twice) and satu- evaporation.
rated sodium chloride (50mL, twice), dried over anhydrous
X-Ray diffraction (XRD) data were collected using a
MgSO4, and concentrated. The residue was purified by silica Rigaku RAXIS RAPID imaging plate area detector with
gel chromatography (eluted with n-hexane–ethyl acetate=9:1 filtered CuKα radiation (λ=1.54187Å) at a temperature of
to 1:1, 1% AcOH) to give a pale yellow solid. The obtained 20 1°C. The crystal-to-detector distance was 127.40mm.
solid was washed with n-hexane to give 11 as a pale yellow Readout was performed in the 0.100mm pixel mode. The ex-
wax (trans-isomer, 13.2g, 28.8mmol, 63.2%).
posure rate was set to 2.0s/°. The XRD data of each crystal
1H-NMR (CDCl3) δ: 0.90 (3H, t, J=6.9Hz), 1.28 (3H, t, were recorded to a total of 45 oscillation images. A first sweep
J=7.1Hz), 1.27–1.36 (24H, m), 1.62–1.67 (2H, m), 1.96 (3H, of data was performed using ω scans from 80.0 to 260.0°
s), 2.27–2.33 (2H, m), 3.40 (1H, d, J=11.6Hz), 3.59 (1H, dd, in 20.0° steps at χ=0.0° and ϕ=0.0°. A second sweep was
J=6.27, 11.8Hz), 4.18–4.28 (2H, m), 5.06 (1H, d, J=6.0Hz). performed using ω scans from 80.0 to 260.0° in 20.0° steps
ESI-MS m/z: 458.4 (M+H)+, 456.3 (M−H)−. FAB-MS m/z: at χ=54.0° and ϕ=0.0°. A third sweep was performed using
458.2950 (M+H) (Calcd for C24H44NO5S: 458.2940).
ω scans from 80.0 to 260.0° in 20.0° steps at χ=54.0° and

1690
Chem. Pharm. Bull.
Vol. 64, No. 12 (2016)
ϕ=90.0°. A fourth sweep was performed using ω scans from at all, 2 points: a slight sulfur odor was detected, 1 point:
80.0 to 260.0° in 20.0° steps at χ=54.0° and ϕ=180.0°. A fifth sulfur odor was detected, and 0 point: strong sulfur odor was
sweep was performed using ω scans from 80.0 to 260.0° in detected. The results of the 6 panelists were totaled as follows;
20.0° steps at χ=54.0° and ϕ=270.0°. An empirical absorption AcCP2Et (4b, d) at pH 4: 8 points, AcCP2Et (4b, d) at pH 7:
correction was applied to the data. The data were also cor- 17 points, CP2Et (2a, b) at pH 4: 1 point, CP2Et (2a, b) at pH
rected for Lorentz and polarization effects and the secondary 7: 1 point.
extinction effect.
Crystal structures were solved using direct methods26) and
Acknowledgments We would like to thank Toshihisa
expanded using Fourier techniques.27) Furthermore, crystallo- Kato, Eiji Nakanishi, and Yasuhiro Kimura for their encour-
graphic refinement of full-matrix least-squares was performed. agement and continued support of this work. We are grateful
The non-hydrogen atoms were refined anisotropically. Some to Shinji Kuroda, Eko Suzuki, Mandai Yamada, and Yumiko
hydrogen atoms were refined isotropically, some were refined Suzuki for their helpful discussion and assistance.
using the riding model, and the rest were included in fixed
positions. In the final cycles of the refinement, a least-squares
Conflict of Interest All authors were employees of Aji-
function of ∑w(Fo2−Fc2)2 was adopted, where w is the least- nomoto Co., Inc. when this study was conducted and have no
squares weights based on the Sheldrick weighting scheme. All further conflicts of interest to declare.
calculations were performed using the CrystalStructure crys-
tallographic software package.28,29)
Supplementary Materials The online version of this ar-
Suppression of Eumelanin Production Testing B16 mel- ticle contains supplementary materials.
anoma cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM; high glucose) supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin (10000U/mL)–strepto-
mycin (10mg/mL) solution. Cells were incubated in a humidi-
fied 5% CO2 atmosphere at 37°C.
B16 melanoma cells were plated in 96-well microplates at
8.0×103 cells per well in 200µL of DMEM containing 10%
FBS without phenol red. After overnight incubation, the me-
dium was replaced with 200µL of DMEM containing each
sample at a predetermined concentration. Cells were treated
with a predetermined concentration of each sample for 3d.
The absorbance of the medium at 450nm after 3d was mea-
sured by a microplate reader.
The concentration necessary for suppressing eumelanin
production in each sample by 50% was calculated relative to
the amount of eumelanin in the control as 100%. Cytotoxic-
ity was determined by a neutral red assay as follows: After
incubation for 3d following the addition of each sample, the
cells were incubated with neutral red dye (50µg/mL) for 2h
and subsequently fixed with 1% formaldehyde and 1% CaCl2
for 1min. The dye was extracted with the solvent solution (1%
acetic acid in 50% ethyl alcohol) and was quantitated by mea-
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