Guanosine analog in the pyrido[2,3-d]pyrimidine ring system as a potential toll-like receptor agonist

Article abstract of DOI:10.1080/15257770600918912

The synthesis of a guanosine analog in the pyrido[2,3-d]pyrimidine ring system has been accomplished by glycosylation of the preformed aromatic heterocyclic base, which was prepared in 2 steps by condensation of methyl acrylate with guanidine carbonate an

Full text of DOI:10.1080/15257770600918912

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Guanosine analog in the pyrido[2,3-
d]pyrimidine ring system As a potential
Toll-like receptor agonist
Guangyi Jin a , Christina C. N. Wu a , Dennis A. Carson a & Howard B.
Cottam a
a Moores Cancer Center , University of California , San Diego, La
Jolla, California, USA
Published online: 22 Dec 2010.
To cite this article: Guangyi Jin , Christina C. N. Wu , Dennis A. Carson & Howard B. Cottam (2006)
Guanosine analog in the pyrido[2,3-d]pyrimidine ring system As a potential Toll-like receptor agonist,
Nucleosides, Nucleotides and Nucleic Acids, 25:12, 1391-1397, DOI: 10.1080/15257770600918912
To link to this article: http://dx.doi.org/10.1080/15257770600918912
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Nucleosides, Nucleotides, and Nucleic Acids, 25:1391–1397, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600918912
GUANOSINE ANALOG IN THE PYRIDO[2,3-d]PYRIMIDINE RING
SYSTEM AS A POTENTIAL TOLL-LIKE RECEPTOR AGONIST
Guangyi Jin, Christina C. N. Wu, Dennis A. Carson, and Howard B. Cottam
Moores Cancer Center, University of California, San Diego, La Jolla, California, USA
2
The synthesis of a guanosine analog in the pyrido[2,3-d]pyrimidine ring system has been
2
accomplished by glycosylation of the preformed aromatic heterocyclic base, which was prepared in
2 steps by condensation of methyl acrylate with guanidine carbonate and methyl cyanoacetate in
the presence of sodium methoxide, followed by dehydrogenation. The analog was evaluated in vitro
for its ability to modulate the innate immune response by acting as an agonist or as an antagonist
of Toll-like receptor (TLR) signaling by measuring cytokine induction or inhibition of induction,
respectively, in mouse bone marrow-derived macrophages. Despite its structural similarity to 7-thia-8-
oxoguanosine, a known TLR7 agonist, the analog was found to antagonize TLR7-induced cytokine
induction in this cell-based assay.
Keywords Innate immunity; Guanosine analog; Pyrido[2,3-d]pyrimidine; Toll-like
receptor agonist
INTRODUCTION
In the mid-1980s, our laboratory and others began studying a group
of synthetic nucleosides that had the unique ability to activate the in-
nate immune response. These compounds were mainly derivatives and
analogs of guanosine. Structure-activity studies within this class showed
that certain compounds in the thiazolo[4,5-d]pyrimidine,[1] pyrazolo[3,4-
d]pyrimidine,[2] purine,[3] 7-deazapurine,[4] and 9-deazapurine[5] ring
systems were all active. Examples in each group were found to be po-
tent antiviral agents in mouse models due to their ability to rapidly in-
duce the production of interferon.[4,6,7] Among these guanosine analogs,
7-thia-8-oxoguanosine (TOG, Figure 1) was studied in greatest detail and
shown to exhibit broad spectrum antiviral activity and to activate natural
killer cells, macrophages, and B-lymphocytes.[8] Other guanosine analogs
Received 19 April 2006; accepted 12 June 2006.
We thank Nancy Noon for secretarial support. This work was supported in part by Grants AI57436
and AI56453, both from the National Institutes of Health.
Address correspondence to Howard Cottam, Moores Cancer Center, University of California,
San Diego, 3855 Health Science Driver, La Jolla, CA 92093-0820. E-mail: hcottam@ucsd.edu
1391

1392
G. Jin et al.
FIGURE 1 Structural features of guanosine analog.
extensively studied were 7-deazaguanosine[9] and 7-allyl-8-oxoguanosine
(loxoribine).[10] The exact mechanism of immune potentiation by these
guanosine-like compounds had not been elucidated, and we were prompted
to investigate the possibility that these small molecules could be acting via
signaling through one or more of the known TLRs. As a result of those
studies, we recently showed that TOG and certain other guanosine analogs
activate immune cells via TLR7.[11] Thus, compounds of this type might
be clinically useful in immune-based therapy or prophylaxis against viral
diseases and other infectious diseases and cancer. Indeed, TOG, also known
as isatoribine, recently has been reported to be effective at reducing the
plasma virus concentration in patients with chronic hepatitis C virus (HCV)
infections[12] with minimal side effects. Our own recent studies provide evi-
dence that TLR7-mediated immunity against HCV involves at least 2 mecha-
nisms: one depends on type 1 interferon production by leukocytes, and the
other is mediated by TLR7 expressed by virally infected hepatocytes.[13]
As part of our ongoing studies in the design and preparation of
agonists of TLR signaling, we investigated the guanosine analog in the
pyrido[2,3-d]pyrimidin-7-one ring system, which may be considered bioisos-
teric with TOG, since a ring sulfur atom may be replaced by a carbon-
carbon double bond.[14,15] A review of the literature revealed no reports
of the fully aromatic guanine base analog or any nucleosides thereof.
However, the preparation of this analog was facilitated by the recent
report[16] of the one-pot preparation of the dihydro derivative 2-amino-
5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (1) from guanidine
carbonate, methyl acrylate and methyl cyanoacetate (Scheme 1).
Dehydrogenation of compound 1 using 10% palladium on carbon
in acetic acid gave a good yield of the aromatic guanine analog (2).
Glycosylation of 2 was accomplished by a Lewis acid-catalyzed Vorbruggen-
type procedure using TMS triflate as the catalyst and 1-O-acetyl-2,3,5-tri-O-
benzoyl-D-ribfuranose as the glycon to yield the protected ribonucleoside 3.
Deprotection of 3 using sodium methoxide in methanol gave the desired
guanosine analog 4. The structure assignment of 4 as the N-8 β isomer was
confirmed by proton NMR and UV spectra. The NMR spectrum of 4 in

Pyrido[2,3-d] pyrimidine Nucleoside as TLR Agonist
1393
SCHEME 1 Reagents and conditions: a) NaOMe / MeOH, Reflux, 36 hours b) Pd/C (10%) / HOAc,
Reflux, 30 hours c) 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose, HMDS/TMStriflate/CH3CN, 78◦C, 18
hours d) NaOMe/MeOH, RT, 36 hours.
deuterated methanol displayed a doublet at 7.00 ppm of 4.0 Hz coupling
constant, characteristic of the anomeric proton for a nucleoside of this
ring system. Similar downfield chemical shifts for the anomeric proton have
been observed for the adenosine analog in this ring system.[17] The small
coupling constant of this doublet is consistent with ribonucleosides of the
β configuration.[18] In addition, the UV spectrum of 4 resembles that of
the corresponding base 2, especially considering the absorption maxima
at longer wavelengths. It has been shown previously for 7-deazapurines,[19]
purines, and 7-thia-8-oxopurines[1] that substitution at N-9 (purine number-
ing, analogous to 4) results in a slight hypsochromic or no shift relative to
the aglycon, whereas substitution at N-3 (purine numbering, analogous
to N-1 of the pyridopyrimidine system) leads to a bathochromic shift
relative to the aglycon. Thus, the site of glycosylation and configuration of
the product (4) are N-8 and β, respectively.
The guanosine analog 4 was first evaluated in vitro for its ability
to stimulate the innate immune response by acting as an agonist of
Toll-like receptor (TLR) signaling by measuring cytokine induction in
mouse bone marrow-derived macrophages. Cells were cultured in the
presence or absence of compound 4 and then assayed for the production of
certain cytokines (IL-6, TNFα, IL-12, interferon, etc.). In this assay, no signif-
icant increase was observed for any of the cytokines tested even at concentra-
tions of 4 up to 100 µM, whereas TOG was active as the positive control (data
not shown). Since compound 4 did not appear to be active as an agonist for
TLR signaling, it also was tested for potential antagonist activity. Therefore,
cells were stimulated with a known inducer of cytokine production, such
as R848 (resiquimod),[20] in the presence or absence of compound 4 and
cytokine levels were measured as before. Here, compound 4 did show

1394
G. Jin et al.
FIGURE 2 Inhibition of R848-induced cytokine induction by compound 4.
modest inhibition of TNFα and IL-6 production, but had no significant
effect on IL-12 production (data not shown) in this system (Figure 2). Thus,
the analog was found to antagonize cytokine production resulting from
TLR signaling.
The mechanism of this inhibition presently is unknown and may or
may not be a result of direct inhibition of TLR signaling. Further studies
to elucidate this mechanism are underway.
EXPERIMENTAL SECTION
Chemistry
Melting points were obtained on a Mel-temp II capillary melting point
apparatus and are uncorrected. Proton nuclear magnetic resonance spectra
were obtained on a Varian Mercury (Electrothermal, Dubuque, IA, USA)
at 400.06 MHz. The chemical shifts are expressed in δ values (parts per
million) relative to tetramethylsilane (TMS) as internal standard. Mass
spectrometric analyses were performed on a Finnigan MAT900XP mass
spectrometer using electro- spray ionization. UV spectra were measured
using a Kontron Uvikon 9330 spectrophotometer. Elemental analyses
were performed by NuMega Resonance Labs (San Diego, CA, USA).

Pyrido[2,3-d] pyrimidine Nucleoside as TLR Agonist
1395
Thin-layer chromatography was performed on silica gel 60 F-254 plates (EM
Reagents). E Merck silica gel (230–400 mesh) was used for flash column
chromatography.
2-Amino-5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (1). A mix-
ture of Guanidine carbonate (3.10 g, 17.3 mmol), methyl acrylate (1.31 g,
15.0 mmol) and methyl cyanoacetate (1.46 g, 15 mmol) in a fresh solution
of NaOMe, prepared by addition of Na (1.14 g, 49.5 mmol) to 150 mL of dry
MeOH, was heated at reflux for 36 hours. The reaction was cooled to RT,
filtered, the resulting solid was washed with MeOH (3×10 mL), and dried
to yield 1 (1.16 g) as a white powder. The filtrate was concentrated in vacuo
and the precipitate was treated as above to obtain a second crop of 1 (0.327
g). Yield 55% (1.48 g). mp: >350oC. UV λmax (pH 11): 220 nm, 291 nm;
€12,600; 3,100. MS (ESI) 181 (MH+). The spectral and analytical data were
in agreement with literature.[21]
2-Amino-pyrido[2,3-d]py- rimidine-4,7(3H,8H)-dione (2). A mixture of 1
(0.90 g, 5 mmol) and palladium on carbon (10%) in acetic acid (250
mL) was heated at reflux for 30 hours. The reaction mixture was cooled
to RT and filtered. The filtered solid was added to water (25 mL) and
basified with 2N NaOH to pH 10, the catalyst was filtered out and the clear
solution was acidfied to pH 5 with acetic acid, and cooled at 0◦C for 2 hours.
The precipitate was filtered and washed with water and dried to provide 2
(0.79 g) as a white solid. Yield 88%, mp > 300◦C (dec); UV λmax (pH 11):
211, 326 nm. €9,400; 11,000. 1H-NMR (DMSO-d6) δ 11.65 (s, 1H), 10.91 (s,
1H), 7.70 (d, J = 12 Hz, 1H), 6.96 (br., 2H), 5.95 (d, J = 12 Hz, 1H). MS
(ESI) m/z: 179.19 (MH+). Anal. Calcd. for C7H6N4O2: C 47.19, H 3.39, N
31.45. Found: C 46.80, H 3.72, N 31.05.
2-Amino-8-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine-4,7
(3H,8H)-di-one (3). Compound (2) (60 mg, 0.34 mmol) and HMDS
(162 mg, 1.0 mmol) were mixed in acetonitrile (6 mL). Trimethylsilyl-
trifluromethanesulfonate (TMStriflate) (222 mg, 1.0 mmol) was added and
the reaction mixture was heated at 78◦C until a clear solution was obtained.
1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (172 mg, 0.34 mmol) and
TMStriflate (74 mg, 0.34 mmol) were then added to the reaction mixture.
The reaction was heated at 78◦C for 18 hours. The reaction was cooled
to RT, solvent was removed in vacuo, the residue was dissolved in DCM
(30 mL), washed with Sat. NaHCO3, and dried with anhdra. Na2SO4.
Solvent was distilled off and the crude product was separated by column
chromatography on silica gel (30 g) (EtOAc) to get 3 (120.6 mg) as
colorless oil, yield 57%. Compound 3 was used directly in the next reaction.
1H-NMR (CDCl3) δ 8.01 (m, 3H), 7.92 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0
Hz, 1H), 7.29–7.52 (m, 12H), 6.23 (d, J = 8.0 Hz, 1H), 5.72 (dd, J = 4.0
Hz, 1H), 5.06 (dd, J = 4.0 Hz, 1H), 4.62 (m, 2H), 4.48 (d, J = 8 Hz, 2H),
4.12 (m, 1H). MS (ESI) m/z: 645.10 (MNa+).
2-Amino-8-(β-D-ribofuranosyl)-3H,8H-pyrido[2,3-d]pyrimidine-4,7-dione (4).
Compound (3) (100 mg, 0.16 mmol) was dissolved in anhydrous methanol

1396
G. Jin et al.
(25 mL) and anhydrous NaOCH3 (130 mg, 2.3 mmol) was added. The
reaction mixture was stirred at RT for 36 hours. The pH of the reaction
solution was adjusted to 4–5 using acetic acid and the solvent was removed
in vacuo, the residue was separated by column chromatography on silica gel
(20 g) (EtOAc-MeOH-Acetone-H2O: 7:1:1:1) and then recrystallized from
isopropanol, compound 4 (31 mg) was obtained as a white solid, yield 62%,
mp: 256–260oC. UV λmax (MeOH): 216 nm, 292 nm, 327 nm. €11,500;
1
5,000; 6,700. H-NMR (CD3OD) δ 7.91 (d, J = 12 Hz, 1H), 6.14 (d, J =
12 Hz, 1H), 4.79 (dd, J = 4 and 4 Hz, 1H), 4.49 (d, J = 12 Hz, 1H), 3.90
(dd, J = 4 Hz, 1H), 3.82 (m, 1H), 3.72 (d, J = 12 Hz, 2H). MS (ESI) m/z:
.
333.08 (MNa+). Anal. Calcd. for C12H14N4O6 2.5H2O: C 40.53, H 5.35, N
15.76. Found: C 40.81, H 5.79, N 15.47.
Biological Evaluation
R848 was from GL Synthesis (Worchester, MA, USA). Bone marrow was
isolated from the femur and tibia of C57BL/6 mice. Cells were plated on
nontissue culture treated petri dishes and cultured in DMEM high glucose
medium supplemented with 10% fetal bovine serum (FBS), L-glutamine,
penicillin/streptomycin (all from Invitrogen, San Diego, CA, USA) and
30% L929 cell conditioned medium. Cells were grown at 37◦C, 5% CO2
for 7 days without replacing the media. The 7-day-old bone marrow-derived
macrophages (BMDM) were harvested with trypsin/EDTA, counted and
replated at a density of 5 × 104 cells per well in 96-well plates and grown for
another 3 days before stimulation with compounds. For cytokine induction
study, compounds were prepared in medium at a final concentration
ranging from 1 to 10 µM and cells were incubated in such medium for
24 hours. For antagonist study, cells were first incubated with compounds
at 1, 3, or 10 µM in the medium for 30 minutes, R-848 was then added at
1 µM and incubation was continued for 24 hours. Cell supernatants were
collected and IL-6, IL-12p40/p70, and TNF-alpha levels determined by
ELISA. Duplicates were included in each experiment and at least 3 separate
experiments were carried out. The data presented is representative of the
experiments. Error bars indicate SEM.
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