Synthesis and immunoregulatory activities of conjugates of a Toll-like receptor 7 inert ligand

Article abstract of DOI:10.1016/j.bmcl.2014.10.034

In the synthesis and modification of the analogs of an adenine type of Toll-like receptor (TLR) 7 agonists, we found a special compound, 9-propionyloxy-8-hydroxy-2-(2-methoxyethoxy)-adenine (6). It is a synthesized TLR7 inert ligand, which does not respond to TLR7 itself. However, it can be coupled with protein or peptide antigens via propionyloxy functional group to promote their immunogenicity significantly. The compound was covalently coupled to protein and peptide to get the conjugates. The inductivity of cytokine production by the conjugates was 872.4-fold compared with the unconjugated antigens in vitro by mouse splenocyte. These data show that the immunostimulatory activity of inert TLR7 ligand can be endowed, and the activity of antigens can be amplified by conjugation with various proteins and peptides, thus broadening the potential therapeutic application and reducing the risk of TLR7 agonists' side effects.

Full text of DOI:10.1016/j.bmcl.2014.10.034

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5792–5795
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and immunoregulatory activities of conjugates
of a Toll-like receptor 7 inert ligand
Dong Gao a,b, , Yu Liu a, , Wang Li a, Fangshu Zhong a, Xiaoli Zhang a, Yuwen Diao a, Ningning Gao a,
Xiaodong Wang a, Wenqi Jiang a,b, , Guangyi Jin a,
⇑
⇑
a Shenzhen University Cancer Research Center, Institute of Otorhinolaryngology, Shenzhen 518060, China
b Sun Yat-sen University Cancer Center, Guangzhou 510060, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the synthesis and modification of the analogs of an adenine type of Toll-like receptor (TLR) 7 agonists,
we found a special compound, 9-propionyloxy-8-hydroxy-2-(2-methoxyethoxy)-adenine (6). It is a syn-
thesized TLR7 inert ligand, which does not respond to TLR7 itself. However, it can be coupled with protein
or peptide antigens via propionyloxy functional group to promote their immunogenicity significantly.
The compound was covalently coupled to protein and peptide to get the conjugates. The inductivity of
cytokine production by the conjugates was 872.4-fold compared with the unconjugated antigens
in vitro by mouse splenocyte. These data show that the immunostimulatory activity of inert TLR7 ligand
can be endowed, and the activity of antigens can be amplified by conjugation with various proteins and
peptides, thus broadening the potential therapeutic application and reducing the risk of TLR7 agonists’
side effects.
Received 20 May 2014
Revised 26 August 2014
Accepted 9 October 2014
Available online 17 October 2014
Keywords:
Innate immune response
Adenine derivatives
Inert ligand
Toll-like receptor
Ó 2014 Elsevier Ltd. All rights reserved.
The Toll-like receptors (TLRs) are a type of pattern recognition
receptor (PRR) that recognize highly conserved components of
diverse pathogens and induce host innate and adaptive immune
responses.1–3 These specific molecular patterns are broadly shared
by pathogen molecules but are structurally distinct from host
molecules,4,5 such as cell wall structures (recognized by TLR1,
-2, -4, -5, and -6 that respond to extracellular stimuli) and nucleic
acids (recognized by TLR3, -7, -8, and -9 that respond to intracellu-
lar stimuli).6,7 Many cancer researchers show their interest in the
profound antitumoral activity of the agonists8 because the
small-molecule agonists could activate certain TLR pathways. The
natural ligand for TLR7 was identified as G and U-rich ssRNA.9 In
addition, several synthesized low molecular weight agonists of
TLR7 were found, including imidazoquinolines,9 and purine-like
molecules.10–12
Figure 1. The molecular structures of purine class of potent TLR7 agonists.
maturation program.15 Because the systemic TLR activation could
induce a rapid and potentially toxic cytokine syndrome, the immu-
notherapeutic activities of these agonists in vitro were difficult to
translate to in vivo settings.8,16,17 Accordingly, the major applica-
tions of TLR7 ligands in vivo were agents or immune adjuvants
injected in small quantities.8,9,18 A potential effective strategy is
to conjugate the ligand to a macromolecule,8,14,19,20 or linking itself
to multimeric,21,22 offering the possibility of drastically reducing
systemic exposure of the adjuvant, yet maintaining relatively high
local concentrations at the site of vaccination.20,23,24
Synthetic small molecules, such as 9-benzyl-8-hydroxy-2-
(2-methoxyethoxy) adenine (SM360320)13 and 4-[6-amino-8-
hydroxy-2-(2-methoxyethoxy)purin-9-ylm-ethyl]benzaldehyde
(UC-1V150) have been shown to be potent and specific TLR7 ago-
nists (Fig. 1).8,14 The certain derivatives of adenine could activate
immune cells via TLR7 by triggering antigen-presenting cell
⇑
Corresponding authors. Tel.: +86 755 86671906.
E-mail addresses: wqjiang@szu.edu.cn (W. Jiang), gyjin@szu.edu.cn (G. Jin).
As the majority of purine-like potent agonists were mostly
dependent on 9-benzyl derivatives,8,10–14 the effect of 9-benzyl
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.10.034
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

D. Gao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5792–5795
5793
Scheme 1. General synthetic route for compound 6. Reagents and conditions: (i) NH3–MeOH, 80 °C; (ii) methyl 3-bromopropionate, K2CO3/DMF/KI, 25 °C; (iii)
CH3OCH2CH2CH2ONa/CH3OCH2CH2CH2OH, 80 °C, reflux; (iv) Br2/CH2Cl2, 25 °C; (v) CH3ONa/CH3OH/80 °C, reflux; HCl, 25 °C.
Scheme 2. Conjugation of compound 6 to protein or peptide. Reagents and conditions: (i) 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDCI), NHS, DMSO, 25 °C; (ii)
DMSO or PBS, 4 °C.
Figure 2. In vitro cytokine release enhancement in response to protein conjugates. Mouse splenocyte were treated with GD or GD-OVA, -BSA, -MSA conjugates at various
concentrations as indicated. Culture supernatants were harvested 24 h later, and cytokine levels were measured by ELISA. LW is UC-1V1508 as a control. Data are
means SEM and a representative experiment of three independent experiments in triplicate per treatment is shown.

5794
D. Gao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5792–5795
Figure 3. In vitro cytokine release enhancement in response to peptide conjugates, as described of Figure 2.
Table 1
Cytokine ratio in vitro treated with conjugated versus unconjugated antigensa
Drug (lM)
GD-OVA
GD-BSA
GD-MSA
GD-MG7
IFN-
GD-thymosin
IL-12 IFN-c
IL-12
IFN-c
IL-12
IFN-
c
IL-12
IFN-c
IL-12
c
0.001
0.01
0.1
1
10
1.50
0.79
6.08
44.34
14.47
1.00
1.11
2.64
31.40
9.00
1.31
1.72
1.64
3.56
4.43
0.85
0.64
2.00
89.37
2.35
2.76
1.16
2.75
1.75
2.00
2.40
0.67
0.17
1.57
7.51
0.50
1.08
0.38
4.00
74.44
0.07
0.21
1.83
36.70
872.40
1.01
1.30
1.72
3.33
20.28
0.79
0.55
9.46
91.80
5.70
a
1 Â 106/mL cells were incubated with serially diluted TLR7 conjugates or un-conjugates for 24 h. The levels of IL-12 and IFN-
c in the culture supernatants were measured
by ELISA. Data are ratio of cytokine production in vitro mouse splenocyte treated with conjugated versus unconjugated antigens.
on adenine needs to be examined. We designed Scheme 1 to syn-
thesize 9-propionyloxy-8-hydroxy-2-(2-methoxyethoxy)-adenine
(6), an inert ligand of TLR7. Compound 6 was synthesized in five
steps from 2,6-dichloropurine (Scheme 1), amount to C replace
phenyl of substituent group at the C9 of UC-1V150. The structure
of 2–6 were determined by NMR and MS.
The 9-propionyloxy of compound 6 enabled us to couple it with
many different macromolecules, including proteins and peptides
through EDCI.25 It was covalently coupled to ovalbumin (OVA),
bovine serum albumin (BSA), mouse serum albumin (MSA), mono-
clonal gastric cancer7 antigen (MG7) and Thymosin (Scheme 2).
The coupling ratios of ligand to OVA, BSA, MSA, MG7 and Thymosin
were 5, 6, 5, 1, and 4, respectively (detailed results were reported
in Supplementary data).
All conjugates were then tested employing in vitro mouse
splenocyte. Incubation of the mouse splenocyte with conjugates
alone stimulated cytokine release. The free compound 6 did not
stimulate cytokine release, and lost TLR7-agonistic properties
(Figs. 2 and 3). After conjugated to macromolecules, such as
proteins (OVA, BSA and MSA) (Fig. 2) and peptides (MG7 and
Thymosin) (Fig. 3), comparison of the cytokine production profiles
We sequentially prolongated the C9-carboxylic acid chain
length and gained the C9-(4-carboxybutyl) and C9-(5-carboxyam-
yl) derivatives. The two derivatives have high activity self, but their
conjugates have low activity compared with compound 6 in
stimulating cytokines production (data not shown).
Overall, we synthesized inert purine-like ligands through minor
modifications of the 9H-Purine scaffold resulting in compound 6,
amounted –(CH2)3– tether to replace the phenyl group of
UC-1V150. This compound did not stimulate cytokine release
in vitro, which means it has no agonistic effect. However its prop-
erties were totally reconverted once it was conjugated to proteins
or peptides. To be more specifically, the immunostimulatory activ-
ity of such inactive TLR7 ligand can be endowed, and the activity of
low immunogenicity antigens can be amplified through such con-
jugations. This coupling method can effectively avoid hypersensi-
tivity reactions caused by agonist in vivo. We hypothesized the
mechanism of this phenomenon lies in that the dendritic cells
and macrophages in the splenetic cultures proteolyzing the conju-
gates, and leaving behind the pharmacore attached to a few
epitope peptides. One of the metabolites may have potent TLR7
activity compared to the starting material. By analogy, antibody-
drug conjugates are degraded in target cells, leaving behind a tubu-
lin inhibitor attached to a small peptide, that mediate effects. The
novelty of our results is that we found a type of prodrug that is
inert until activated within the TLR7 expressing target cells. Our
results will hopefully broaden the potential therapeutic applica-
tion of TLR7 ligands by reducing the risk of unexpected side effects
and increasing sustained release time of adjuvant.
(IL-12 and IFN-
c) between original antigens vs. conjugates at var-
ious dosages (0.001–10
lM) demonstrated that the conjugates
enhanced the potency 872.4 times most (Table 1). We also tested
the effect of different substitution level on the immunostimulatory
response. There was no significant difference statistically in pro-
duction of IL-12 among 3 different coupling ratios (detailed results
were reported in Supplementary data).

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We appreciate the advice and assistance of Benjamin M. Carson.
This work was supported by grants from Basic Research Program of
Shenzhen (Grant Nos. JCYJ20130326110139687, 20120613113228732
and JC201005250060A) Shenzhen Development and Reform
Commission (Grant Nos. 2012553 and 2012886), the China
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.10.034.
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