of the uracil ring. Unexpectedly, the 2,3-dimethyl 13a could not
be evaluated for its anti-inflammatory activity due to poor
solubility in acetone. In addition, compound 13a seemed to have
axial chirality at the N(1)-position of the uracil ring (data not
shown). The 3,4-dimethyl 13b showed potent anti-inflammatory
activity, while the 3,5-dimethyl 13c had lower activity than 13b.
The potent activity of 13b urged us to investigate the effects of
3- or 4-methylsubstitution on the phenyl group.
activity of 13dagainst MEK1/2 in an enzymatic assay. We found
that compound 13dweakly inhibits MEK1/2 (data not shown).20
In conclusion, we have designed and efficiently synthesized
novel 1-phenyl-6-aminouracils by replacing the chroman moiety
in CX-659S with DDB to cancel CX-659S asymmetric center.
Medicinal chemistry effort on the phenyl group at the N(1)-
position of the uracil ring led to the discovery of 13dbearing a 3-
methyl group as a suitable candidate for improved anti-
inflammatory activity. Compound 13dwith good in vitro ADME
profile and moderate oral bioavailability in mice showed potent
anti-inflammatory activity in mice PC-induced CHR. The
beneficial effects of 13d were equipotent to that of tacrolimus or
prednisolone. In addition, compound 13d, having potent
hydroxyl radical-scavenging activity, showed more potent
suppressive effect on mice SP-induced pruritus than oxatomide.
At present, we are conducting further chemical, pharmacological,
and toxicological studies on the 1-phenyl-6-aminouracils and will
report the findings in future publications.
The anti-inflammatory activity of the 3-methyl 13d was
comparable to that of tacrolimus, a widely used dermatologic
drug. On the other hand, the 4-methyl 13e had weaker activity
than 13d. With 13d potent inhibitory activity in mind, we next
focused on 3-substitution on the phenyl group. Both the ethyl
(13f) and ethoxy (13g) groups led to decreased activity compared
to 13d. From these results, we assumed that groups bulkier than
the methyl group were unfavorable for better activity. However,
a small fluoro group (13h) also resulted in loss of activity
compared to 13d. Moreover, a carboxyl group (13i), which is
bulkier and more polar than 13d, decreased the anti-
inflammatory activity. These results suggested that a methyl
group was the most suitable substituent at the 1-phenyl group.
On the basis of the above-mentioned results, we selected
compound 13d and evaluated its in vivo oral efficacy. First, we
examined compound 13d water solubility, metabolic stability,
and membrane permeability in vitro. The profile of 13d from
these studies is shown in Table 2. Compound 13d exhibited no
serious issues related to ADME parameters. To investigate 13d
oral exposure, we conducted a mice pharmacokinetic (PK) study
with this compound (Table 3). Intravenous injection of 13d
resulted in low clearance (CL) and low volume of distribution at
steady state (Vdss). Given orally, the maximum concentration
(Cmax) of 13d in plasma reached 511 ng/mL with an area under
the concentration curve (AUC) of 1096 ng∙h/mL. Compound 13d
bioavailability was moderate at 12%.
Based on 13d PK profile, we next investigated this compound
oral anti-inflammatory activity in mice PC-induced CHR. As
shown in Figure 2, compound 13d dose-dependently suppressed
inflammation with significant effect at 10 mg/kg. The potency of
13d at this dose was comparable to that of prednisolone. These
good results prompted us to evaluate the suppressive effect of
13d on pruritus using substance P (SP)-induced pruritus in
mice.12−15 The results of this evaluation are summarized in Figure
3. In our experiment, oxatomide, a well-known anti-allergic
agent, showed a suppressive effect as described in the literatue.15
Compound 13dshowed significant suppressive effect on pruritus
in mice at 10 mg/kg. This effect was more potent than that of
oxatomide. These findings indicate that compound 13d has good
potential as an orally active dermatologic drug with both anti-
inflammatory and anti-pruritic effects.
Acknowledgments
We are thankful to Dr. Takayuki Fukaya, Dr. Tomoya Shiro,
Mr. Takahiro Nagasaki and Mr. Masashi Tanaka for their great
support throughout this research.
References and notes
1. Akdis, C. A.; Akdis, M.; Bieber, T.; Bindslev-Jensen, C.; Boguniewicz,
M.; Eigenmann, P.; Hamid, Q.; Kapp, A.; Leung, D. Y. M.; Lipozencic,
J.; Luger, T. A.; Muraro, A.; Novak, N.; Platts-Mills, T. A. E.;
Rosenwasser, L.; Scheynius, A.; Simons, F. E. R.; Spergel, J.;
Turjanmaa, K.; Wahn, U.; Weidinger, S.; Werfel, T.; Zuberbier, T. J.
Allergy Clin. Immunol. 2006, 118, 152.
2. Simpson, E. L. Curr. Med. Res. Opin. 2010, 26, 633.
3. (a) Tobe, M.; Isobe, Y.; Goto, Y.; Obara, F.; Tsuchiya, M.; Matsui, J.;
Hirota, K.; Hayashi, H. Bioorg. Med. Chem. 2000, 8, 2037. (b) Isobe,
Y.; Tobe, M.; Inoue, Y.; Isobe, M.; Tsuchiya, M.; Hayashi, H. Bioorg.
Med. Chem. 2003, 11, 4933. (c) Inoue, Y.; Isobe, M.; Shiohara, T.; Goto,
Y.; Hayashi, H. Br. J. Dermatol. 2002, 147, 675.
4. Goto, Y.; Watanabe, N.; Kogawa, N.; Tsuchiya, M.; Takahashi, O.;
Uchi, H.; Furue, M.; Hayashi, H. Eur. J. Pharmacol. 2002, 438, 189.
5. (a) Trenam, C. W.; Blake, D. R.; Morris, C. J. J. Invest. Dermatol. 1992,
99, 675. (b) Mittal, M.; Siddiqui, M. R.; Tran, K.; Reddy, S. P.; Malik,
A. B. Antioxid. Redox Sign. 2014, 20, 1126.
6.
(a) McConnell, O.; Bach, A. II; Balibar, C.; Byrne, N.; Cai, Y.; Carter,
G.; Chlenov, M.; Di, L.; Fan, K.; Goljer, I.; He, Y.; Herold, D.; Kagan,
M.; Kerns, E.; Koehn, F.; Kraml, C.; Marathias, V.; Marquez, B.;
McDonald, L.; Nogle, L.; Petucci, C.; Schlingmann, G.; Tawa, G.;
Tischler, M.; Williamson, R. T.; Sutherland, A.; Watts, W.; Young, M.;
Zhang, M.-Y.; Zhang, Y.; Zhou, D.; Ho, D. Chirality 2007, 19, 658. (b)
FDA’s guidances for the development of new stereoisomeric drugs can
be
found
at:
http://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/gu
idances/ucm122883.htm
Next, we evaluated a hydroxyl radical-scavenging activity of
compound 13d (Table 4).16 As a result of the evaluation of the
several reference compounds, both Trolox® and CX-659S
showed more potent activity than L-ascorbic acid which is a
well-known hydroxyl radical scavenger. In addition, the order of
the activities among the three compounds was consistent with
that of the previous report.17 On the other hand, the hydroxyl
radical-scavenging activity of compound 13d was more potent
than that of Trolox® or CX-659S. These results suggest that the
hydroxyl radical-scavenging activity of 13d is one of the
contributing factors toward both its potent anti-inflammatory and
its anti-pruritic effects.4,18
7. (a) Tamura, K.; Kato, Y.; Ishikawa, A.; Kato, Y.; Himori, M.; Yoshida,
M.; Takashima, Y.; Suzuki, T.; Kawabe, Y.; Cynshi, O.; Kodama, T.;
Niki, E.; Shimizu, M. J. Med. Chem. 2003, 46, 3083. (b) Niki, E.;
Fukuhara, A.; Omata, Y.; Saito, Y.; Yoshida, Y. Bioorg. Med. Chem.
Lett. 2008, 18, 2464. (c) Ohuchida, S.; Nambu, F.; Toda, M. JP Patent
07112980, 1995. (d) Hasegawa, T.; Hachitani K.; Nambu, F. Onada, S.
JP Patent 08109179, 1996.
8. Isobe, Y.;Tobe, M.; Isobe M. WO Patent 026842, 2004.
9. Hirose, N.; Nakamura, T.; Banba, T.; Mergalet, D.; Nakamura, T.; Sano,
Y.; Miyauchi, Y.; Kijima, S. JP Patent 05178848, 1993.
10. Rieche, A.; Gross, H.; Höft, E. Chemische Berichte 1960, 93, 88.
11. For more information, see Supporting Information.
12. Toyoda, M.; Nakamura, M.; Makino, T.; Hino, T.; Kagoura, M.;
Morohashi, M. Br. J. Dermatol. 2002, 147, 71.
13. Kuraishi, Y.; Nagasawa, T.; Hayashi, K.; Satoh, M. Eur. J. Pharmacol.
1995, 275, 229.
14. Hägermark, Ö.; Hökfelt, T.; Pernow, B. J. Invest. Dermatol. 1978, 71,
233.
Finally, we set studies to investigate the target molecule of 13d.
Uchi et al. previously reported that CX-659S inhibits
phosphorylation
of
mitogen-activated
protein
kinase/extracellular-signal regulated kinase (MEK) 1/2 in
15. Inagaki, N.; Nagao, M.; Nakamura, N.; Kawasaki, H.; Igeta, K.; Musoh,
K.; Nagai, H. Eur. J. Pharmacol. 2000, 400, 73.
keratinocytes.19 Based on this report, we examined the inhibitory
4