2-Azidoalkoxy-7-hydro-8-oxoadenine derivatives as TLR7 agonists inducing dendritic cell maturation

Article abstract of DOI:10.1016/j.bmcl.2009.02.095

The synthesis of an array of 2-azidoalkoxy substituted 7-hydro-8-oxoadenines is described. The relation of the structure of these compounds and their ability to induce maturation of dendritic cells is evaluated.

Full text of DOI:10.1016/j.bmcl.2009.02.095

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2249–2251
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Azidoalkoxy-7-hydro-8-oxoadenine derivatives as TLR7 agonists inducing
dendritic cell maturation
Jimmy J. Weterings a, Selina Khan b, Gerbrand J. van der Heden van Noort a, Cornelis J. M. Melief b,
Herman S. Overkleeft a, Sjoerd H. van der Burg c, Ferry Ossendorp b, Gijsbert A. van der Marel a,
,
*
Dmitri V. Filippov a,
*
a Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
b Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
c Department of Clinical Oncology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of an array of 2-azidoalkoxy substituted 7-hydro-8-oxoadenines is described. The relation
of the structure of these compounds and their ability to induce maturation of dendritic cells is evaluated.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 20 January 2009
Revised 23 February 2009
Accepted 24 February 2009
Available online 27 February 2009
Keywords:
7-Hydro-8-oxoadenine
TLR-7
Dendritic cell
Toll-like receptor 7 (TLR7), one of the thirteen mammalian
TLRs1 currently known, can be activated by specific imidazoquino-
lines.2 As such, TLR7 is the first TLR for which small molecule mod-
ifiers were identified, a result that has spawned great interest
worldwide in the search after specific agents to either enhance a
specific immune response (through activating a given TLR) in bat-
tling cancer or infectious disease3, or reduce an immune response
(through inactivating a TLR) in autoimmune disease therapies.4–6
TLRs appear to have evolved to recognize (partially degraded) bio-
molecules from microbial origin and upon recognition initiate a
signaling cascade leading to a specific immune response against
these microbial invaders.7 The natural TLR7 ligands are single
strand RNA (ssRNA) oligonucleotides8 and the therapeutic poten-
tial of ssRNA molecules is thought to be limited due to their low
enzymatic and chemical stability and high cost. It is therefore for-
tuitous that TLR7 also recognizes imidazoquinolines and 7-hydro-
8-oxoadenines.9 Whether TLR7 activation by these adenine ana-
logues (see for representative examples structure 1–4, Fig. 1) oc-
curs by interaction with the ssRNA binding site or by occupation
of an altogether different site of the TLR 7 receptor remains to be
established. The archetypal imidazoquinoline-based TLR agonist
is imiquimod 110, also known as R-837. Imiquimod is a component
H2N
N
H2N
H2N
N
H
H
N
6
N
N
N 8
N
O
O
N
N
2
H
N
O
N
R1
1
2
1
3:
4:
R = CH3CH2
1
R = CH3O
H2N
H2N
H
N
N
H
N
N
N
N
O
O
O
N3
N3
O
N
O
N
m
n
5f: m = 1
5g:
5d: n = 4
5a: n = 1
m = 2
5e:
5b:
5c:
n = 5
n = 2
n = 3
Figure 1. Adenine analogues known to be TLR-7 agonists.
* Corresponding authors.Tel.: +31 71 5274280; fax: +31 71 5274307 (G.A. van der
Marel), tel.: +31 71 5274768; fax: +31 71 5274307 (D.V. Filippov).
E-mail addresses: marel_g@chem.leidenuniv.nl (G.A. van der Marel), filippov@
chem.leidenuniv.nl (D.V. Filippov).
of the therapeutic ointment Aldara11 that is currently in use as a
treatment for several skin diseases12,13 including superficial basal
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.095

2250
J. J. Weterings et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2249–2251
(1) bromination, (2) displacement of Br with OMe and (3) acidic
R-OH
7a-g
NH2
NH2
hydrolysis with aqueous HCl. Although the yields vary for the indi-
vidual steps, (see the Scheme 1 and Supplementary data for spe-
cific yields) each target compound was prepared in sufficient
quantities and purity for ensuing TLR-binding studies.
(
)
N
N
N
N
N
N
Cl
N
O
R
N
NaH,
DME
8a-g
6
The ability of the new purine derivatives to stimulate TLR7
mediated dendritic cell activation was assessed in a comparative
study in which the literature compounds 3 and 4 were included.
Upon stimulation of TLR7 by its natural agonist (ssRNA) a series
of events takes place resulting in, amongst others, the secretion
of the cytokine IL122,26 and upregulation of surface markers asso-
ciated with dendritic cell maturation.27 Briefly, murine bone mar-
row derived dendritic cells (BMDCs) were incubated for 24 h
with compounds 3, 4 and 5a–g at different concentration (see
Fig. 2 legends). The supernatants were harvested in the next step
and the amounts of IL12 in these were assessed using a standard
sandwich ELISA assay28 or phenotypic characterization of the den-
dritic cells was done after 48 h stimulation with the different TLR7
derivatives by staining for different surface markers associated
with DC maturation.28
Br2, DCM
NH2
H2N
N
H
N
1) MeONa
N
N
N
Br
O
N
O
R
N
O
R
N
2) HCl
(conc.)
5a-g
9a-g
8a (59%)
8b (55%)
8c (39%)
8d (42%)
8e (62%)
8f (38%)
5a
5b
5c
8a
)
(57% from
(73% from
(6% from
8b
)
)
8c
5d (17% from 8d)
5e (16% from 8e)
5f (31% from 8f)
5g (61% from 8g)
As shown in Figure 2A, all compounds were able to induce
upregulation of surface markers CD86, MHC class II (Fig. 2A) and
8g
(69%)
CD40 (data not shown) on dendritic cell at 1 lM. At this concentra-
tion minor distinction between the potencies of the different com-
pounds can be made, except for dendritic cells incubated with
compound 5-g which showed less pronounced upregulation of
the maturation markers. Next, the ability of the dendritic cells to
produce IL-12 upon stimulation with the individual compounds
was addressed (Fig. 2B). As is evident all compounds except for
N3
a: R=
b:
N3
N3
c:
d:
e:
f:
N3
N3
compound 5-g induced comparable level of IL-12 at 5
ever, more subtle differences become apparent at lower concentra-
tions (0.1 M), with analogues 5a, and 5b expressing activities
lM. How-
O
O
N3
O
N3
l
g:
similar to that of reference compound 3. At the lowest concentra-
tion used (10 nM) the superior activity of 3 compared to all others
comes to light.
Scheme 1. Synthesis of 7-hydro-8-oxoadenines derivatives 5a–g.
Returning to the dual aim referred to in the introduction the
small panel of compounds generated here does not contain a more
potent TLR7 agonist compared to the literature compound 3, at
least not in the assays employed here. However, introduction of
an azide moiety does not necessarily impede TLR7 activating prop-
erties, as is evidenced by the rather potent IL12 stimulating activ-
ities of derivatives 5a and 5b. This holds promise for the further
derivatization of these new lead TLR7-ligands, and two separate
strategies can be envisaged.
In the first, the azide can be reduced to the amine which is then
condensed with a range of acids to give a number of amides. In an
alternative approach the azides in 5a–g may be employed in a
Huisgen [3+2] cycloaddition mediated derivatization approach.
Here, a library of acetylenes should give access to a library of tria-
zoles. We have previously shown that 5g is a suitable substrate for
Huisgen reaction.29
cell carcinoma. Imiquimod, however is not an optimal TLR7 ligand
and some side effects that could be linked to imiquimod14,15
spurned research towards alternative small molecule TLR7 agonists.
Hirota and co-workers16 conducted a study in which a com-
pound library assembled from modified purines and pyrimidines
were assessed on their IFN-
a inducing properties. From these
studies compound 2 emerged as a new lead (Fig. 1). Ensuing
optimization studies17–24 led, amongst others to 7-hydro-8-
oxoadenine derivative 325 as the most potent IFN-
a inducer
for in vitro assays, and to the analogue compound 416 with im-
proved bioavailability for in vivo applications. Subsequent work
suggested the TLR7 agonizing activity as the molecular basis of
the biological action of 7-hydro-8-oxoadenine derivatives.23 To-
wards our goal to discover TLR-ligands capable to induce den-
dritic cell (DC) maturation we set out to synthesize an array
of analogues of compound 3 and evaluate their potency as DC
maturating agents. The aim of the study described here is
two-fold: a) is it possible to enhance TLR7 binding and agonis-
tic activity by directed modification of the alkyl substituents at
C-2 in 4, and b) to introduce in derivatives of 3 a ligation han-
dle for conjugation purposes, such as the TLR-ligand-peptide
constructs as leads for vaccine development alluded to in the
introduction.
Acknowledgements
This work has been funded by Netherlands Organisation for Sci-
entific Research (NWO) as a part of the ‘From Molecule to Cell’ pro-
gram (to DVF, GAvdM SHvdB and FO), the DC-THERA program
contract nr. LSHB-CT-2004-512074 from the European Commis-
sion (to FO) and the framework of project D1-101 within the Dutch
Top Institute Pharma (to SK).
Target compounds 5a–g were prepared starting from known25
9-benzyl-2-chloro-6-aminopurine (6) in a straightforward fashion
as outlined in Scheme 1. Nucleophilic aromatic substitution of
the chlorine at C2 in 6 with a range of alkoxides (7a–g) proceeded
uneventfully to give 2-azidoalkoxypurine derivatives 8a–g in 38–
69% yield. These intermediates were transformed into the target
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.095.
8-oxopurines 5a–g by means of a reported three step procedure24
:

J. J. Weterings et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2249–2251
2251
Figure 2. Activation of dendritic cells by compounds 3, 4 and 5a–g. (A) BMDCs were incubated with 1 lM of the indicated compounds for 48 h or left untreated. Cells were
stained with CD86 or MHC II antibodies and subjected to flow cytometry analysis (Supplementary data), R848 (resiquimod) a known2 derivative of 1 was used as positive
*
control; (B) BMDC were incubated with the indicated compounds for 24 h supernatant was harvested and the concentration of IL-12p40 was determined.28 Indicate that the
values were higher than the standard curve (>10 ng/ml IL-12). Results are means of triplicates SEM.
15. Witt, P. L.; Ritch, P. S.; Reding, D.; McAuliffe, T. L.; Westrick, L.; Gossberg, S. E.;
References and notes
Borden, E. C. Cancer Res. 1993, 53, 5176.
16. Hirota, K.; Kazaoka, K.; Niimoto, I.; Kumihara, H.; Sajiki, H.; Isobe, Y.; Takaku,
1. Gay, N. J.; Gangloff, M. Annu. Rev. Biochem. 2007, 76, 141.
H.; Tobe, M.; Ogita, H.; Ogino, T.; Ichii, S.; Kurimoto, A.; Kawakami, H. J. Med.
2. Hemmi, H.; Kaisho, T.; Takeuchi, O.; Sato, S.; Sanjo, H.; Hoshino, K.; Horiuchi, T.;
Chem. 2002, 45, 5419.
Tomizawa, H.; Takeda, K.; Akira, S. Nat. Immunol. 2002, 3, 196.
17. Isobe, Y.; Ogita, H.; Masanori, T.; Takaku, H.; Ogino, T.; Kurimoto, A.;
3. Chen, K. Q.; Huang, J.; Gong, W. H.; Iribarren, P.; Dunlop, N. M.; Wang, J. M. Int.
Kawakami, H. US patent, 7,157,465, 2007.
Immunopharmacol. 2007, 7, 1271.
18. Hirota, K.; Kazaoka, K.; Niimoto, I.; Sajiki, H. Org. Biomol. Chem. 2003, 1, 1354.
4. Baccala, R.; Hoebe, K.; Kono, D. H.; Beutler, B.; Theofilopoulos, A. N. Nat. Med.
19. Hirota, K.; Kazaoka, K.; Sajiki, H. Bioorg. Med. Chem. 2003, 11, 2715.
2007, 13, 543.
20. Isobe, Y.; Tobe, M.; Ogita, H.; Kurimoto, A.; Ogino, T.; Kawakami, H.; Takaku, H.;
5. Abdollahi-Roodsaz, S.; Joosten, L. A. B.; Roelofs, M. F.; Radstake, T. R. D. J.;
Sajiki, H.; Hirota, K.; Hayashi, H. Bioorg. Med. Chem. 2003, 11, 3641.
Matera, G.; Popa, C.; van der Meer, J. W. A.; Netea, M. G.; van den Berg, W. B.
21. Kurimoto, A.; Ogino, T.; Ichii, S.; Isobe, Y.; Tobe, M.; Ogita, H.; Takaku, H.; Sajiki,
Arthritis Rheum. 2007, 56, 2957.
H.; Hirota, K.; Kawakami, H. Bioorg. Med. Chem. 2003, 11, 5501.
6. Barrat, F. J.; Coffman, R. L. Immunol. Rev. 2008, 223, 271.
22. Kurimoto, A.; Tobe, M.; Ogita, H.; Ogino, T.; Takaku, H.; Ichii, S.; Kawakami, H.;
7. Kawai, T.; Akira, S. Semin. Immunol. 2007, 19, 24.
Isobe, Y. Chem. Pharm. Bull. 2004, 52, 466.
8. Diebold, S. S.; Massacrier, C.; Akira, S.; Paturel, C.; Morel, Y.; Sousa, C. R. E. Eur. J.
23. Jin, G.; Wu, C. C. N.; Tawatao, R. I.; Chan, M.; Carson, D. A.; Cottam, H. B. Bioorg.
Immunol. 2006, 36, 3256.
Med. Chem. Lett. 2006, 16, 4559.
9. Lee, J.; Wu, C. C. N.; Lee, K. J.; Chuang, T.-H.; Katakura, K.; Liu, Y.-T.; Chan, M.;
24. Isobe, Y.; Kurimoto, A.; Tobe, M.; Hashimoto, K.; Nakamura, T.; Norimura, K.;
Tawatao, R.; Chung, M.; Shen, C.; Cottam, H. B.; Lai, M. M. C.; Raz, E.; Carson, D.
Ogita, H.; Takaku, H. J. Med. Chem. 2006, 49, 2088.
A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1828.
25. Kurimoto, A.; Ogino, T.; Ichii, S.; Isobe, Y.; Tobe, M.; Ogita, H.; Takaku, H.; Sajiki,
10. Kang, H. Y.; Park, T. J.; Jin, S. H. J. Invest. Dermatol. 2009, 129, 243.
H.; Hirota, K.; Kawakami, H. Bioorg. Med. Chem. Lett. 2004, 12, 1091.
11. Harrison, C. J.; Jenski, L.; Voychehovski, R.; Bernstein, D. I. Antiviral Res. 1988,
26. Gorden, K. B.; Gorski, K. S.; Gibson, S. J.; Kedl, R. M.; Kieper, W. C.; Qiu, X. H.;
10, 209.
Tomai, M. A.; Alkan, S. S.; Vasilakos, J. P. J. Immunol. 2005, 174, 1259.
12. Beutner, K. R.; Geisse, J. K.; Helman, D.; Fox, T. L.; Ginkel, A.; Owens, M. L. J. Am.
27. Banchereau, J.; Briere, F.; Caux, C.; Davoust, J.; Lebecque, S.; Liu, Y. J.; Pulendran,
Acad. Dermatol. 1999, 41, 1002.
B.; Palucka, K. Annu. Rev. Immunol. 2000, 18, 767.
28. See Supplementary data.
13. Ulrich, C.; Busch, J. O.; Meyer, T.; Nindl, I.; Schmook, T.; Sterry, W.; Stockfleth, E.
Br. J. Dermatol. 2006, 155, 451.
29. Weterings, J. J.; Khan, S.; van der Heden, G. J.; Drijfhout, J. W.; Melief, C. J. M.;
14. Strominger, N. L.; Brady, R.; Gullikson, G.; Carpenter, D. O. Brain Res. Bull. 2001,
Overkleeft, H. S.; van der Burg, S. H.; Ossendorp, F.; van der Marel, G. A.;
55, 445.
Filippov, D. V. Bioorg. Med. Chem. Lett. 2006, 16, 3258.