S. De Jonghe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 145–149
149
6-aryl substituent of hit 1, without modifications of the rest of the
molecule (Table 1). Both methoxy groups of compound 1 seem to
be essential for activity, as the 4-methoxyphenyl (compound 9g),
as well as the 3-methoxyphenyl (compound 9c) derivative, are
much less active. Other types of substituent on the phenyl ring,
such as halogens (compounds 9b and 9f), electron withdrawing
groups such as trifluoromethyl and cyano (compounds 9a and
9d) or electron-donating substituents (compound 9e) do not show
biological activity in any of the two assays. In order to prove that
the presence of a dimethoxyphenyl substituent is mandatory for
immunosuppressive and anti-inflammatory activity, the closely
related 4-isopropoxy-pteridine series (compounds 9h–n) was syn-
thesized. The same trend was observed here as the dimethoxy-
phenyl containing analogue (compound 9m) resulted in superior
IC50 values in the MLR and TNF assay (IC50 values of 0.4 and
and the phenyl moiety. A two carbon linker yielded compound 20j,
displaying a TNF IC50 value of 10 nM and a MLR IC50 of 100 nM. A
phenoxyacetyl side chain on the piperazine group (compound
20k) led to an extremely potent immunosuppressive agent with
an IC50 value of 4 nM. The corresponding 4-chloro analogue (com-
pound 20l) displayed even more pronounced immunosuppressive
activity (MLR IC50 = 0.5 nM). In order to further probe into the SAR,
we expanded the synthesis towards urea derivatised analogues.
The phenyl substituted analogues (compounds 20m–q) all show
impressive activity in the MLR assay, with IC50 values in the low
nanomolar range. Especially, the 4-methyl analogue (compound
20o) was extremely potent (MLR IC50 = 0.8 nM), whereas the 4-cya-
no derivative (compound 20p) was somewhat less active (MLR
IC50 = 74 nM). The benzyl substituted urea derivatives (compounds
20r-s) are approximately 10-fold less potent in the MLR assay than
their phenyl counterparts (compounds 20m–n). These N-acyl and
N-carbamoyl-piperazine-pteridine derivatives represent the most
powerful immunosuppressive drugs known up to now. For compar-
ison, marketed immunosuppressive drugs were also evaluated in
this MLR assay. The pteridine analogues display more pronounced
immunosuppressive activity than cyclosporine (IC50 = 65 nM),
while their potency is similar to that of tacrolimus (IC50 = 1 nM)
and rapamycin (IC50 = 1 nM).
2.3 lM, respectively), when compared to other types of substitu-
tions on the phenyl ring. In addition, this isopropoxy derivative
9m is threefold more potent than the corresponding ethoxy ana-
logue 1 in both assays.
In a second round of optimization, a series of 6-(3,4-dimethoxy-
phenyl)-pteridine derivatives (compounds 19a–h) were made in
which a number of nitrogen nucleophiles is introduced at position
4 of the scaffold. As can be derived from Table 2, it seems that aro-
matic rings (e.g. compounds 19a–b and 19f) do not exhibit potent
immunosuppressive or anti-inflammatory activity. On the other
hand, a number of aliphatic and cycloaliphatic substituents are tol-
erated for activity (compounds 19c–e and 19g–h) and give rise to
With respect to the anti-inflammatory activity of the amides
and urea, it is clear that the most potent anti-inflammatory
compounds (analogues 20j–m) within this series have TNF IC50
values which are similar to the N-aryl-piperazine congeners. It
demonstrates that a carbonyl function is an indispensable struc-
tural element to obtain potent immunosuppressive drugs, whereas
this is not the case for the anti-inflammatory activity.
dual acting compounds with IC50 around 0.5
well as in the TNF assay.
lM in the MLR, as
Before starting in vivo work with this type of compounds, we
felt it necessary to further improve the potency of the compounds.
Given the low molecular weight of the lead compounds, adding
additional groups in order to exploit additional interactions was
still justified. Position 4 of the pteridine scaffold was the preferred
site to do so, as it tolerated quite some structural variety. As het-
erocyclic aliphatic substituents such as morpholino, piperidine,
thiomorpholino were found to be active in both assays, we focused
on piperazine as the 4-substituent as it represents an easy handle
for further derivatization. A number of commercially available
N-aryl (compounds 20a–d) and N-benzyl (compounds 20e–h)
piperazine derivatives were incorporated onto the pteridine core
structure. The N-phenyl-piperazine derivative 20a was the first
compound in this optimization campaign with very potent activity
in the TNF assay (IC50 = 50 nM). On the other hand, the MLR IC50 of
compound 20a did not improve when compared to smaller substit-
uents such as morpholine. The nature of the substitution pattern
on the phenyl ring determines the biological activity: the unsubsti-
tuted phenyl ring (compound 20a) and its 4-methyl (compound
20b) and 4-fluoro (compound 20d) congeners show comparable
In conclusion, a novel series of 2-amino-4-N-piperazinyl-6-(3,4-
dimethoxyphenyl)pteridine analogues have been prepared and
evaluated for their immunosuppressive (using the MLR assay) and
anti-inflammatory (using the TNF assay) properties. The herein de-
scribed SAR study indicated that the 6-(3,4-dimethoxyphenyl) moi-
ety is essential in obtaining immunosuppressive and anti-inflamma
tory activity. Further SAR studies revealed that derivatization of the
piperazine moiety as amides or urea led to extremely potent immu-
nosuppressives (sub nM IC50 values), while retaining good activity in
the TNF assay. In vivo activity in preclinical animal models of Crohn’s
disease of some representative examples (compounds 20b and 20k)
have been described by us before.8,10
References and notes
1. Carter, P. H.; Zhao, Q. Expert Opin. Invest. Drugs 2010, 19, 195.
2. Kumar, S.; Boehm, J.; Lee, J. C. Nat. Rev. Drug Disc. 2007, 2, 717.
3. Karin, M.; Yamamoto, Y.; Wang, Q. M. Nat. Rev. Drug Disc. 2004, 3, 17.
4. Murumkar, P. R.; DasGupta, S.; Chandani, S. R.; Giridhar, R.; Yadav, M. R. Expert
Opin.Ther. Patents 2010, 20, 31.
5. Kahan, B. D. Nat. Rev. Immunol. 2003, 3, 831.
6. O’Neill, L. A. J. Nat. Rev. Drug Disc. 2006, 5, 549.
activity in the MLR assay (IC50 values ranging from 0.1 to 0.4 lM)
and the TNF assay (IC50 values ranging from 10 to 50 nM), whereas
the 3,5-dichlorophenyl analogue 20c is approximately 10-fold less
active in the MLR as well as in the TNF assay (IC50 of 3.4 and
7. Waer, W.; Vanrenterghem, Y.; Van der Schueren, E.; Michielsen, P.; Vandeputte,
M. Transplant. Proc. 1987, 19, 1570.
8. Shen, C.; Dillissen, E.; Kasran, A.; Lin, Y.; Herman, J.; Sienaert, I.; De Jonghe, S.;
Kerremans, L.; Geboes, K.; Boon, L.; Rutgeerts, P.; Ceuppens, J. L. Clin. Immunol.
2007, 122, 53.
9. Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T. F.; Kumar, S.; Green, D.;
McNulty, D.; Blumenthal, M. J.; Keys, J. R.; Landvatter, S. W.; Strickler, J. E.;
McLaughlin, M. M.; Siemens, I. R.; Fisher, S. M.; Livi, G. P.; White, J. R.; Adams, J.
L.; Young, P. R. Nature 1994, 372, 739.
10. Shen, C.; Dillissen, E.; Kasran, A.; Lin, Y.; Clydesdale, G.; Sienaert, I.; De Jonghe,
S.; Gao, L. J.; Geboes, K.; Boon, L.; Rutgeerts, P.; Ceuppens, J. L. J. Interferon
Cytokine Res. 2006, 26, 575.
11. Pfleiderer, W.; Lohrmann, R. Chem. Ber. 1961, 94, 12.
12. Mohr, D.; Kazimierczuk, Z.; Pfleiderer, W. Helv. Chim. Acta 1992, 75, 2317.
13. Taghavi-Moghadam, S.; Pfleiderer, W. Tetrahedron Lett. 1997, 38, 6835.
14. Jang, M. Y.; De Jonghe, S.; Gao, L. J.; Rozenski, J.; Herdewijn, P. Eur. J. Org. Chem.
2006, 4257.
15. (a) Vorbrüggen, H.; Krolikiewicz, K.; Nieballa, U. Angew. Chem., Int. Ed. Engl.
1971, 10, 657; (b) Vorbrüggen, H.; Krolikiewicz, K.; Nieballa, U. Justus Liebigs
Ann. Chem. 1975, 988.
0.5
20e–h) have been prepared, from which the unsubstituted benzyl
ring (compound 20e) seems to be most potent (MLR IC50 = 0.9
and TNF IC50 = 0.09 M). The 2-fluoro analogue (compound 20g)
has a very similar profile (MLR IC50 and TNF IC50 values of 0.7
and 0.2 M, respectively). The 2,6-dichloro-benzyl (compound
lM, respectively). Similarly, N-benzyl derivatives (compounds
lM
l
l
20f) and the 4-tert-butyl derivative (compound 20h) are clearly
less potent in both assays.
A series of amides was also prepared. The benzoyl derivative
(compound 20i) is a pure dual acting compound with equipotent
activity in the MLR and TNF assay (IC50 of 0.4 lM in both assays). A
big step forward in the immunosuppressive activity was achieved
by introduction of appropriate linkers between the carbonyl group