Facile syntheses of functionalized toll-like receptor 7 agonists

Article abstract of DOI:10.1016/j.tetlet.2014.11.126

Protein conjugates of toll-like receptor 7 agonists have been shown to elicit powerful immune responses. In order to facilitate our studies in this area our group has developed efficient syntheses for a number of functionalized derivatives that retain imm

Full text of DOI:10.1016/j.tetlet.2014.11.126

Accepted Manuscript
Facile syntheses of functionalized toll-like receptor 7 agonists
Babatope Akinbobuyi, Matthew R. Byrd, Charles A. Chang, Mysa Nguyen,
Zacharie J. Seifert, Anne-Laure Flamar, Gerard Zurawski, Katherine C.
Upchurch, SangKon Oh, Stephen H. Dempsey, Thomas J. Enke, JohnLe, Hunter
J. Winstead, José R. Boquín, Robert R. Kane
PII:
S0040-4039(14)02040-1
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.11.126
TETL 45510
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 November 2014
26 November 2014
28 November 2014
Please cite this article as: Akinbobuyi, B., Byrd, M.R., Chang, C.A., Nguyen, M., Seifert, Z.J., Flamar, A-L.,
Zurawski, G., Upchurch, K.C., Oh, S., Dempsey, S.H., Enke, T.J., Le, J., Winstead, H.J., Boquín, J.R., Kane, R.R.,
Facile syntheses of functionalized toll-like receptor 7 agonists, Tetrahedron Letters (2014), doi: http://dx.doi.org/
10.1016/j.tetlet.2014.11.126
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Facile syntheses of functionalized toll-like
receptor 7 agonists
Leave this area blank for abstract info.
Babatope Akinbobuyi, Matthew R. Byrd, Charles A. Chang, Mysa Nguyen, Zacharie J. Seifert,
Anne-Laure Flamar, Gerard Zurawski, Katherine C. Upchurch, SangKon Oh, Stephen H. Dempsey,
Thomas J. Enke, John Le, Hunter J. Winstead, José R. Boquín, and Robert R. Kane *

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Facile syntheses of functionalized toll-like receptor 7 agonists
Babatope Akinbobuyia, Matthew R. Byrda, Charles A. Changb, Mysa Nguyena, Zacharie J. Seiferta, Anne-
Laure Flamarc, Gerard Zurawskibc, Katherine C. Upchurchb, SangKon Ohbc, Stephen H. Dempseyd, Thomas
J. Enked, John Led, Hunter J. Winsteadd, José R. Boquínd, and Robert R. Kane abc
a Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, TX 76798, USA
b Institute of Biomedical Studies, Baylor University, One Bear Place #97224, Waco, TX 76798, USA
c Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak Street, Dallas, TX 75204, USA
d Department of Chemistry, Augustana College, 639 38th Street, Rock Island, IL 61201, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Protein conjugates of toll-like receptor 7 agonists have been shown to elicit powerful immune
responses. In order to facilitate our studies in this area our group has developed efficient
syntheses for a number of functionalized derivatives that retain immune stimulatory activity.
2009 Elsevier Ltd. All rights reserved.
Available online
Keywords:
Toll-like receptor
Adenine derivatives
Vaccines
Immune activation
Pathogens are initially detected by the innate immune system
upon the binding of their associated ‘signature’ molecules to
germline-encoded pathogen-associated molecular pattern
(PAMP) receptors.1 Toll-like receptor 7 (TLR7) is a endosomal
member of the Toll-like receptor family of PAMP recognition
proteins.2 Virus-associated single stranded RNA is the molecular
pattern that activates TLR7, thereby initiating an antiviral
immune response involving the maturation of dendritic cells
(DC), the secretion of cytokines, and the up-regulation of major
Previous work has demonstrated that TLR7 ligands can be
conjugated to a variety of molecules (lipids, peptides, and
proteins) while retaining their potent agonist activity in vitro and
in vivo.20-23 Specifically, para substituents on compounds such as
2 are well tolerated, as demonstrated by the conjugation of
aldehyde 3 to a to proteins and peptides with retention of TLR7
stimulation.22 Our group is interested in synthesizing anti-DC
receptor antibody/TLR7 agonist conjugates in order to facilitate
the targeted delivery of these immunostimulatory compounds,
thereby potentially avoiding the problems associated with
histocompatibility complex.3,4
The development of
immunotherapeutic compounds that act via the activation of
PAMP receptors, including TLR7, is an active area of research.
systemic administration.
This paper reports the efficient
synthesis of functionalized TLR7 agonists 4 that can potentially
serve as the starting point for the synthesis of antibody
conjugates.
Potent small molecule TLR7 agonists have been discovered,
including imidazoquinolines such as compound 15 and substituted
adenine derivatives such as compound 26-8 (Figure 1).
Unfortunately, the clinical utilization of these compounds is
significantly limited by side-effects resulting from the
overwhelming generation of cytokines subsequent to systemic
administration.
compound 1 has been limited to localized administration such as
formulation for topical use for the treatment of skin diseases.9
Accordingly, the clinical application of
A
wide variety of derivatives of these compounds have been
synthesized in hopes of identifying analogues that elicit
especially potent or selective immune responses10-15 or to provide
compounds with improved solubility,16 bioavailability,17 or
Figure 1. Synthetic TLR-7 agonists
pharmacokinetic properties.18,19
———
 Corresponding author. Tel.: +1-254-710-4556; fax: +1-254-710-4272; e-mail: Bob_Kane@baylor.edu

2
Tetrahedron
The synthesis of the known6-8 non-functionalized TLR
arenes 4f, 4g, and 4h (Scheme 4). Notably, compounds 4g and
4h contain primary amines that can be used to conjugate to
proteins or other compounds.
agonist 4a was initially explored. Benzylation of commercially
available chloroadenine 5 (which can also be conveniently
synthesized on
a
large scale by amination of 2,6-
dichloropurine18) proceeded smoothly, affording the desired
product in good yield by simple precipitation (Scheme 1). This
route avoids the formation of isomers (requiring chromatographic
separation) observed upon benzylation of 2,6-dichloropurine.
Introduction of the alkoxy substituent (again, with a simple
isolation by precipitation) followed by bromination provides 6a
in good yield. In our hands the bromination proceeded very
slowly in CH2Cl2 or CHCl3 requiring a very large excess of
bromine and multiple Na2S2O3 washes to isolate the pure product.
However, in acetic acid with sodium acetate the bromination
proceeds very efficiently to afford pure product that can be
isolated by filtration from the reaction mixture. Finally, while 6a
could be hydrolyzed using a two-step procedure (methanolysis
followed by acidic cleavage of the methyl ether), we found that
simple treatment with a solution of NaOH in water/methanol
solvent gave direct access to the desired product in one step
(Scheme 2). Overall, our optimized procedure was scalable and
afforded the desired TLR7 agonist 4a in four steps with 70%
overall yield without chromatographic purification.
Scheme 3. Reagents and conditions: i. CH3ONa, CH3OH, reflux, 61%,
ii. HCl (conc), 43%, iii. HCl (conc), 15%.
Scheme 1. Reagents and conditions: i. ArCH2Br, K2CO3, DMSO, rt, ii.
n-BuONa/n-BuOH, reflux, iii. n-BuONa/n-BuOH, reflux, then add H2O,
reflux, iv. Br2, CH2Cl2, rt 12 h or Br2, AcOH, AcONa, rt, 1 h.
A similar reaction sequence also readily afforded iodide 4b
(55% for 4 steps). However when preparing 4c we observed that
yields were adversely impacted by the concomitant formation of
nitrile hydrolysis/alcoholysis side-products during the reactions
with sodium butoxide and methanolic sodium hydroxide. Nitrile
4c was therefore most reproducibly isolated via a two-step
methanolysis/hydrolysis protocol (which also provided amide 4e;
Scheme 3). In order to avoid the formation of similar mixtures
during the preparation of 4d, water was added directly to the
sodium butoxide reaction mixture after chloride displacement
was complete. Refluxing the butoxide/water mixture completed
the nitrile hydrolysis to afford, after bromination under standard
conditions, carboxylic acid 6d in good yield. Hydrolysis of 6d
then afforded the useful TLR7 agonist 4d.
Scheme 4. Reagents and conditions: i. CH3ONa, CH3OH, reflux, 86%,
ii. Pd(PPh3)2Cl2, CuI, Et3N, then propargyl alcohol, reflux, iii. NaI, TMSCl,
CH3CN, reflux, iv. Pd(PPh3)2Cl2, CuI, Et3N, then BOC-propargyl amine,
reflux, v. Pd/C, H2, MeOH, rt
Finally, carboxylic acid 4d has been linked, using COMU as a
coupling agent, to two different mono-BOC-protected diamines
which were cleanly deprotected using TFA to afford two
additional amino-substituted TLR7 agonists 4i and 4j (Scheme
5).
Scheme 5. Reagents and conditions: i. BOC-ethylenediamine, DIPEA,
COMU, DMSO, DCM, 0°-rt, 2 h, ii. TFA, DCM, rt, 1 h,
iii. BOC-NH-CH2CH2OCH2CH2OCH2CH2-NH2, DIPEA, COMU, DMSO,
DCM, 0°-rt, 2 h.
Preliminary biological evaluations (TLR7 reporter cell line
assays and cytokine release from peripheral blood mononuclear
cells) have demonstrated that each of these new compounds (4b-
j) are active TLR7 agonists (data not shown). Several of these
new compounds are well functionalized for protein conjugation,
and we are presently attaching them to targeting proteins and
characterizing their biological properties.
Scheme 2. Reagents and conditions: NaOH, CH3OH, reflux.
Iodide 7, which was prepared in good yield by the reaction of
6b with methoxide, was prepared in order to investigate
functionalization using Sonogashira conditions.24 We were
pleased to find that 7 reacted cleanly with functionalized alkynes
(followed by deprotection of BOC-protected amines) to provide

3
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This work was supported by funding from Baylor University
(URC), the Baylor Health Care System Foundation, and the NIH
(U19 AI057234).
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