Synthesis and activity of a metabolite of (S)-6-amino-5-(6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxamido)-3-methyl-1-phenyl-2,4-(1H,3H)-pyrimidinedione (CX-659S)

Article abstract of DOI:10.1248/cpb.50.1418

CX-659S (1) [(S)-6-amino-5-(6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxamido)-3-methyl-1-phenyl-2,4-(1H,3H)-pyrimidinedione], has been developed as a new type anti-inflammatory agent for the treatment of dermatitis. The structure of a major metabolite

Full text of DOI:10.1248/cpb.50.1418

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Notes
Chem. Pharm. Bull. 50(10) 1418—1420 (2002)
Vol. 50, No. 10
Synthesis and Activity of a Metabolite of (S)-6-Amino-5-(6-hydroxy-
2
2
,5,7,8-tetramethylchroman-2-carboxamido)-3-methyl-1-phenyl-
,4-(1H,3H)-pyrimidinedione (CX-659S)
Yoshiaki ISOBE, Masanori TOBE, Osamu TAKAHASHI, Yuso GOTO, Yoshifumi INOUE, Fumihiro OBARA,
Masami TSUCHIYA, and Hideya HAYASHI*
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation; Toda, Saitama 335–8502, Japan.
Received July 9, 2002; accepted August 6, 2002
CX-659S (1) [(S)-6-amino-5-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamido)-3-methyl-1-phenyl-2,4-
1H,3H)-pyrimidinedione], has been developed as a new type anti-inflammatory agent for the treatment of der-
(
matitis. The structure of a major metabolite of CX-659S was determined as (S)-6-amino-5-[2-hydroxy-2-methyl-
-(2,4,5-trimethyl-3,6-dioxo-1,4-cyclohexadienyl)butanamide]-3-methyl-1-phenyl-2,4-(1H,3H)-pyrimidinedione
2) by direct comparison with the synthesized authentic compound. The anti-inflammatory activity of 2 was
4
(
equipotent with that of 1 on the contact hypersensitivity reaction (CHR) induced by picryl chloride (PC) in mice,
suggesting that compound 2 contributes, at least in part, to the anti-inflammatory activity of CX-659S.
Key words CX-659S; metabolite; CHR; ROS; antioxidative activity
We have recently reported a novel pyrimidine derivative was identified to be 2 by LC/MS/MS comparison with the
(S)-6-amino-5-(6-hydroxy-2,5,7,8-tetramethylchroman-2- authentic compound. Figure 2 shows a typical reverse phase
(
carboxamido)-3-methyl-1-phenyl-2,4-(1H,3H)-pyrimidine- (RP)-HPLC elution profile of plasma samples after adminis-
dione ((1), CX-659S, Fig. 1), which showed anti-inflamma- tration of CX-659S to dogs. The main peak corresponding to
tory activities against both acute inflammation induced by ir- the metabolite of CX-659S was eluted with a retention time
ritants and delayed-type hypersensitivity in various animal characteristic of authentic 2 at 8.8 min.
1
—3)
models.
The chemical structure of 1 is quite different
It has been reported that reactive oxygen species (ROS)
from those of typical non-steroidal anti-inflammatory agents, participate in the process of inflammation in various tissues
6
)
steroid and immunosuppressant, and thus is of interest. In the including skin, and compound 1 is considered to have anti-
course of our pharmacokinetic study on 1, a metabolite was inflammatory effects on the contact hypersensitivity reaction
commonly observed in the plasma when 1 was administered (CHR) in mice by topical administration due to its potent
to animals. According to the analysis of the metabolite of 1, radical scavenging activities against the hydroxyl radical and
the m/z on liquid chromatography-mass spectrometry (LC- peroxynitrite and to its inhibitory effects on lipid peroxida-
MS) of it was larger than that of 1 by 16. On the other hand,
a number of a-tocopherol metabolites have been described.
Among them, a-tocopheronic acid, which results from ring
opening of the chroman moiety was characterized in the
4
)
urine of animals and man. On the basis of the above evi-
dence, we considered that compound 1 (exact massϭ464.2),
having a tocopherol-related chroman moiety, would be con-
verted into the corresponding quinone 2 (exact massϭ480.2)
under oxidative conditions. But this possibility needed to be Fig. 1. Structures of 1 (CX-659S) and 2
confirmed by direct comparison of this metabolite with the
synthesized authentic sample. In this present study, we pre-
pared 2 to confirm the structure of this metabolite, and addi-
tionally, evaluated its anti-inflammatory activity.
Compound 2 was prepared by 2 synthetic methods, out-
lined in Chart 1. Cerium (IV) ammonium nitrate (CAN) is a
reagent that oxidizes hydroquinone derivatives into their cor-
responding quinones, however, direct oxidation of 1 by CAN
gave 2 in very poor yield (15%) along with many by-prod-
ucts. Therefore, chroman carboxylic acid, S-(Ϫ)-3, was first
5
)
oxidized by CAN into the corresponding quinone, S-(ϩ)-4,
with retention of configuration. The resulted S-(ϩ)-4 was
then reacted with 5 using 1-[3-(dimethylamino)propyl]-3-eth-
ylcarbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) to
give S-(ϩ)-2 in moderate yield. Other reagents for oxidation
of 1 were also evaluated, and S-(ϩ)-2 was obtained in excel-
lent yield when iodine was used.
The above mentioned metabolite of 1 in guinea pig plasma Chart 1. Synthesis of the Quinone Derivative, 2
*
To whom correspondence should be addressed. e-mail: hhayashi@j-energy.co.jp
© 2002 Pharmaceutical Society of Japan

October 2002
1419
Fig. 2. Representative HPL Chromatograms of the Plasma Samples
(
A) standard, (B) blank plasma, (C) plasma after subcutaneous administration of 1.
Table 1. Inhibitory Effects of Compounds 1 and 2 on PC-Induced CHR in
Mice
In conclusion, we identified a metabolite structure of CX-
59S, a new type of anti-inflammatory compound, and exam-
6
a)
ined its biological activity on the CHR in mice. As a result,
the potency of 2 was found to be equipotent to that of 1 in
this model. These findings suggest that this metabolite con-
tributes, at least in part, to the anti-inflammatory activities of
CX-659S in vivo. An investigation on the action mechanisms
of CX-659S and its metabolite is now in progress.
Compound
Dose (mg/ear)
0.03
Inhibition (%)
1
21
35
0
0
.1
.3
69*
Ϫ9
42
70*
85**
2
0.03
0
0
.1
.3
Prednisolone
0.1
Experimental
General All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. Melting points were mea-
sured with a BÜCHI 535 melting point apparatus and were uncorrected.
Proton NMR spectra were recorded on a JEOL GSX270 FT NMR spectrom-
eter. Chemical shifts were given in parts per million (ppm) using tetram-
ethylsilane as the internal standard for spectra obtained in DMSO-d6 and
a) Percent inhibition was calculated from the values of percent responses of drug-
treated and control groups (nϭ6). * pϽ0.05, ** pϽ0.01 versus control (Dunnet’s test).
3
)
tion. However, compound 2 had no anti-oxidative activities,
and the IC value against lipid peroxidation was over 100 mM
CDCl . TOF MS (time-of-flight mass spectrometry) were recorded on a
3
5
0
Kompact MALDI 3 V4.0.0 spectrometer. Optical rotation was recorded on a
DIP-370 spectrometer. Elemental analyses were performed at the Toray Re-
(
data not shown). Therefore, we expected that compound 2,
an oxidized product of 1, would show a weaker or lesser ac- search Center. Wakogel C-200 (Wako; 70—150 mm) was used for column
tivity on the CHR in mice. However, contrary to our expecta- chromatography. Monitoring of reactions was carried out by using Merck 60
F254 silica gel, glass-supported TLC plates, and visualization with UV light
tion, compound 2 also suppressed the CHR in mice by topi-
cal administration, and the potency of 2 was equipotent to
that of 1 (Table 1), suggesting that compound 2 may have an
(
254 and 365 nm).
S)-(؉)-6-Amino-5-[2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxo-
,4-cyclohexadienyl)butanamide-3-methyl-1-phenyl-2,4-(1H,3H)-pyrim-
(
1
action mechanism other than the anti-oxidative activity to in- idinedione (2) A solution of 1 (13.94 g, 30 mmol) in CH Cl (200 ml) and
2
2
hibit the CHR.
NaHCO (6.30 g, 75 mmol) in water (150 ml) were mixed and agitated vigor-
3

1
420
Vol. 50, No. 10
ously. Iodine (8.38 g, 33 mmol) in CH Cl (200 ml) was added to the mixture in the serum were assayed by an HPLC method. Briefly, 1 ml of serum was
2
2
dropwisely for 3 h under ice-cooling. Na S O (6.3 g, 75 mmol) was then extracted with 5 ml of diethylether, and centrifuged at 724 g for 5 min at
2
2
3
added to the mixture to reduce the surplus iodine. The organic layer was sep- 4 °C. The aqueous layer was extracted with the same volume of diethylether
arated and washed with water and brine, dried over MgSO , and concen- again. The combined organic layers were concentrated and dissolved in
4
trated to give 2 (14.12 g, 98%) as an orange foam. mp 125—127 °C (dec.). 200 ml of H O/MeOH (1 : 1, v/v), and a portion (100 ml) was injected into
2
2
5
1
Optical rotation [a]D 10.5° (cϭ1, MeOH). H-NMR (CDCl ) d: 1.54 (3H,
the chromatograph. Chromatographic separations were achieved on a Shi-
madzu LC-10A HPLC (Shimadzu, Japan) using a Capcell Pak C18 SG120
(150ϫ4.6 mm, Shiseido, Japan). The mobile phase consisted of acetonitrile :
water (7 : 3, v/v), with the pH adjusted to 5.0 by 0.05 M ammonium acetate.
3
s), 1.68—1.72 (1H, m), 1.96 (3H, s), 1.99 (3H, s), 2.01 (3H, s), 2.46—2.48
1H, m), 2.65—2.69 (1H, m), 3.36 (3H, s), 3.89 (2H, s), 5.32 (2H, s), 7.37—
.40 (2H, m), 7.56—7.62 (3H, m), 8.55 (1H, br s). MS (TOF) m/z 481
(
7
(
ϩ
MϩH) . Anal. Calcd for C H N O ·0.5H O: C, 61.34; H, 5.87; N, 11.45. The flow rate of the mobile phase was maintained at 1.0 ml/min, and the ef-
25 28 4 6 2
Found: C, 61.37; H, 5.97; N, 11.35.
S)-(؉)-2-Hydroxy-2-methyl-4-(3,4,6-trimethyl-2,5-dioxocyclohexa-
fluent was monitored at a UV detection wavelength of 267 nm.
Picryl Chloride-Induced Contact Hypersensitivity Reaction PC-in-
(
7)
1
,3-dienyl)-butanoic Acid (4) To an ice-cooled solution of 3 (5 g, duced CHR was assessed by the method of Asherson and Ptak. Male ICR
2
0 mmol) in CH CN/H O (40 ml/20 ml) was added dropwisely a solution of mice were sensitized by applying 100 ml of 7% (w/v) PC solution in acetone
3
2
CAN (32.9 g, 60 mmol) in H O (36 ml), and the mixture was then stirred for to the shaved abdomen. Seven days later, the mice were challenged by apply-
2
3
0 min at room temperature. After partitioning with CH Cl2 (100 ml) and ing 20 ml of 1% (w/v) PC solution in acetone to the left ear. The ear thick-
2
water (100 ml), and dried over MgSO . The solvent was concentrated, and ness was measured with a digital thickness gauge before and 24 h after the
4
the residue was purified by column chromatography on silica gel challenge, and the difference in thickness was calculated. Test compounds
(
hexane : ethyl acetateϭ7 : 1) to give 4 (4.53 g, 85%) as a white solid. Opti-
were dissolved in acetone and were administered immediately after the chal-
25
1
cal rotation [a] 21.8° (cϭ1, MeOH). H-NMR (CDCl ) d: 1.52 (3H, s), lenge.
1
2
D
3
.73—1.83 (1H, m), 1.90—1.96 (1H, m), 2.00 (6H, s), 2.03 (3H, s), 2.44—
ϩ
.54 (1H, m), 2.63—2.72 (1H, m). MS (TOF) m/z 251 (MϩH) .
References and Notes
Condensation of 4 and 5 To a mixture of 4 (293 mg, 1.1 mmol) and 5
232 mg, 1 mmol) in N,N-dimethylformamide (DMF) (10 ml) were added
HOBt (149 mg, 1.1 mmol) and EDC·HCl (211 mg, 1.1 mmol), and the mix-
ture was then stirred for 8 h at room temperature. Next, the solvent was con-
1) Tobe M., Isobe Y., Goto Y., Obara F., Tsuchiya M., Matsui J., Hirota
K., Hayashi H., Bioorg. Med. Chem., 8, 2037—2047 (2000).
2) Goto Y., Inoue Y., Tsuchiya M., Isobe M., Ueno T., Uchi H., Furue M.,
Hayashi H., Int. Arch. Allergy Immunol., 123, 341—348 (2000).
3) Goto Y., Watanabe N., Kogawa N., Tsuchiya M., Takahashi O., Uchi
H., Furue M., Hayashi H., Eur. J. Pharmacol., 438, 189—196 (2002).
4) Simon E. J., Gross C. S., Milhorat A. T., J. Biol. Chem., 221, 797—
801 (1956).
(
centrated and partitioned with CH Cl2 (20 ml) and 5% NaHCO3 solution
2
(
20 ml). The organic layer was washed with brine, dried over MgSO , and
4
concentrated. Finally, the crude product was recrystallized from ether/ethyl
acetate to give 2 (300 mg, 63%).
Pharmacokinetic Study Male beagle dogs (9 months of age, 9.5—
1.2 kg) were obtained from Ridglan Research Farms Inc. (Wisconsin,
5) R-(Ϫ)-4 has been reported. Noal C., Rocco J. L., Christian N., J. Org.
Chem., 46, 2445—2450 (1981).
1
U.S.A.). Compound 1 dissolved in 0.5% propyleneglycol was administered
subcutaneously to the dogs. Blood samples (4 ml) were taken from a jugular
vein, and the serum was obtained by centrifugation at 1630 g for 10 min at
6) Trenam C. W., Blake D. R., Morris C. J., J. Invest. Dermatol., 99,
675—682 (1992).
7) Asherson G. L., Ptak W., Immunology, 15, 405—416 (1968).
4
°C, and kept at Ϫ20 °C until drug analysis. The concentrations of 1 and 2