Structural requirements for TLR7-selective signaling by 9-(4-piperidinylalkyl)-8-oxoadenine derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.01.037

We report the synthesis and biological evaluation of a new series of 8-oxoadenines substituted at the 9-position with a 4-piperidinylalkyl moiety. In vitro evaluation of the piperidinyl-substituted oxoadenines 3a-g in human TLR7- or TLR8-transfected HEK293 cells and in human PBMCs indicated that TLR7/8 selectivity/potency and cytokine induction can be modulated by varying the length of the alkyl linker. Oxoadenine 3f containing a 5-carbon linker was found to be the most potent TLR7 agonist and IFNα inducer in the series whereas 3b possessing a 1-carbon linker was the most potent TLR8 agonist.

Full text of DOI:10.1016/j.bmcl.2015.01.037

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1318–1323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural requirements for TLR7-selective signaling by
9-(4-piperidinylalkyl)-8-oxoadenine derivatives
à
⇑
Hélène G. Bazin , Yufeng Li , Juhienah K. Khalaf , Sandra Mwakwari, Mark T. Livesay, Jay T. Evans,
David A. Johnson
GlaxoSmithKline Vaccines, 553 Old Corvallis Road, Hamilton, MT 59840, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and biological evaluation of a new series of 8-oxoadenines substituted at the 9-
position with a 4-piperidinylalkyl moiety. In vitro evaluation of the piperidinyl-substituted oxoadenines
3a–g in human TLR7- or TLR8-transfected HEK293 cells and in human PBMCs indicated that TLR7/8
selectivity/potency and cytokine induction can be modulated by varying the length of the alkyl linker.
Received 24 November 2014
Revised 14 January 2015
Accepted 19 January 2015
Available online 31 January 2015
Oxoadenine 3f containing a 5-carbon linker was found to be the most potent TLR7 agonist and IFN
inducer in the series whereas 3b possessing a 1-carbon linker was the most potent TLR8 agonist.
a
Keywords:
TLR7/8 agonist
Oxoadenine
Ó 2015 Elsevier Ltd. All rights reserved.
Immunostimulants
Toll-like receptors (TLR) are a family of structurally related
receptors that detect highly conserved microbial components com-
mon to large classes of pathogens. These receptors are expressed
on immune cells and upon activation mobilize defense mecha-
nisms aimed at eliminating the invading pathogens. Of the more
than 10 known TLRs that have been identified in humans, five
are associated with the recognition of bacterial components (TLRs
1, 2, 4, 5, 6) and four others (TLRs 3, 7, 8, 9) appear to be restricted
to cytoplasmic compartments and are involved in the detection of
viral RNA (TLRs 3, 7, 8) and unmethylated DNA (TLR9).1,2 Activa-
tion of TLRs regulates intracellular signaling pathways leading to
the expression of inflammatory cytokines/chemokines and type I
hydroxyadenine or ‘oxoadenine’ series revealed that a hydroxy
group at the 8-position is essential for IFN-inducing activity and
that an alkylated heteroatom at the 2-position dramatically
increases IFN activity (e.g., compound 2, Fig. 1).
While an extensive evaluation of SAR in the 9-arylmethyl and
heteroarylmethyl oxoadenine series has been carried out over the
past several years,8–16 to our knowledge no systematic studies have
been performed on the corresponding saturated derivatives (i.e.,
cycloalkyl or heterocycloalkyl-substituted oxoadenines). The few
reported oxoadenines with cycloalkyl groups attached to the
nitrogen at the 9-position (directly or via a methylene unit) have
demonstrated weak or diminished IFN induction.7,11 Since alkyl lin-
ker length on the corresponding N1 of 1-phenylalkylimidazoquino-
lines is known to profoundly effect IFN-inducing activity,6 we were
particularly interested in the synthesis and biological evaluation of
a series of 4-piperidinylalkyl derivatives of 2 in which the length of
the N9 alkyl linker was systematically varied (compounds 3a–g,
Fig. 1). In addition to the ability to form water-soluble salts, the 4-
piperidinyl moiety of oxoadenines 3a–g also provides a suitable
handle for potential N-derivatization and conjugation as
substitution of the aromatic amino group in both the oxoadenine
and imidazoquinoline series abolishes IFN-inducing activity.6,17
Conjugation of TLR7/8 agonists to lipids, proteins, and other mole-
cules is known to enhance immune responses and decrease toxic
effects.6,18,19
interferons (IFNa/b), which can lead to the preferential enhance-
ment of innate anti-microbial responses and antigen-specific
humoral and cell-mediated immune responses.
In the case of TLR7 and TLR8 activation, a few different classes
of small molecule mimetics of the natural uridine- and/or guano-
sine-rich viral ssRNA ligands have been identified,3–5 including
1H-imidazo[4,5-c]quinolines6 and 8-hydroxyadenines.7 Through
screening of a library of purine derivatives, Hirota7 discovered that
9-benzyl-8-hydroxyadenine 1 (Fig. 1; shown as favored keto/oxo
tautomer) possessed IFN-inducing activity in vitro. Further evalua-
tion of structure-activity relationships (SAR) in the 9-benzyl
⇑
Corresponding author.
The piperidinylalkyl oxoadenines 3a–g were synthesized from
2-n-butoxy-8-methoxyadenine (6)20 and N-t-butoxycarbonyl
(Boc)-4-piperidinyl derivatives 10 or 11 in a convergent manner
as shown in Scheme 1. Adenine 6 was prepared in 6 steps and in
Present address: GlaxoSmithKline Oncology R&D, 1250 S. Collegeville Road,
Collegeville, PA 19426, USA.
à
Present address: Omeros Corporation, 201 Elliot Avenue W., Seattle, WA 98199,
USA.
http://dx.doi.org/10.1016/j.bmcl.2015.01.037
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

H. G. Bazin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1318–1323
1319
NH2
NH2
N
NH2
H
H
N
H
N
N
N
N
N
O
O
O
N
N
N
nBuO
N
nBuO
N
( )
n
NH
2 Cl
1
2
3a-g
n= 0 to 6
Figure 1. Structures of prototypical oxoadenines 1 and 2 and piperidinyl-substituted oxoadenines 3a–g.
56% overall yield from commercially available 2,6-dichloropurine
(4).21 N-protection of 4 as the tetrahydropyranyl (THP) aminal, fol-
lowed by sequential displacement of the 6- and 2-chloro groups
with ammonia and n-butoxide gave the THP-protected adenine
520 in 73% overall yield from 4. Adenine 5 was then converted to
6 in 3 steps and in 77% overall yield by 8-bromination, bromide
displacement with methoxide, and THP-deprotection with trifluo-
roacetic acid (TFA). The requisite Boc-protected 4-piperidinylalkyl
bromides were either purchased commercially (11a,e) or prepared
from the corresponding alcohols using Appel conditions (Ph3P/
CBr4). The two piperidinyl alcohols not commercially available
(10f,g) were prepared in 3 steps from 4-bromopyridine (7) by
Sonogashira coupling of 7 with acetylenic alcohols 8f,g, followed
by hydrogenation of the alkynyl pyridines 9f,g and N-Boc protec-
tion of the resulting piperidinyl alkanols.22 Subsequent N-alkyl-
measures NFrB mediated SEAP production following TLR7- or
TLR8-specific activation. It should be noted that the HEK reporter
assay only measures the NFjB pathway so additional assay sys-
tems are necessary to evaluate IRF7 pathway activation. The hTLR7
and hTLR8 specificity and potency (EC50) of oxoadenines 3a–g are
shown in Figure 2. Oxoadenine 3a was not active on hTLR7 or
hTLR8 but the other oxoadenines 3b–g were all active. While
increasing the linker length beyond one carbon dramatically
increased hTLR7 potency, no linear correlation between linker
length and hTLR7 potency was observed in this assay. The 5-carbon
linker oxoadenine 3f was the most potent hTLR7 agonist of the ser-
ies while the 1-carbon linker oxoadenine 3b was the most potent
hTLR8 agonist of the series, with hTLR8 potency significantly
decreasing with longer linkers.
The loss of hTLR8 activity observed after stimulation with
higher doses of oxoadenines 3e–g suggested possible toxicity or
activation induced cell death in HEK293-hTLR8 cells. Live/deadÒ
fixable Aqua staining was used to evaluate potential cell death fol-
lowing HEK293-hTLR8 stimulation with oxoadenines 3e–g. Aqua
staining (cell death) at 24 h following stimulation correlated with
both oxoadenine linker length and dose (Fig. 3). Cell toxicity was
not observed after 24 h stimulation with the shorter linker oxoade-
nines 3b–d (data not shown). The oxoadenine dose triggering
HEK293-hTLR8 cell toxicity was the lowest with the 6-carbon
linker (3g) and increased with decreased linker length (3e,f). How-
ever, the longer linker length oxoadenines (3e–g) induced modest
ation of adenine
6 with the Boc-protected piperidinylalkyl
bromides 11b–g23 in the presence of potassium carbonate in
dimethylformamide (DMF) followed by cleavage of the Boc and
methoxy groups with 4 N HCl in dioxane afforded the desired
oxoadenine hydrochloride salts 3b–g24 in 41–84% overall yield
from 6. Since N-alkylation of 6 with 1-Boc-4-bromopiperidine
(11a) failed to give any product under these conditions (K2CO3,
DMF), oxoadenine 3a25 was conveniently prepared according to a
modification of a literature method26 by Mitsunobu reaction of 6
with 1-Boc-4-hydroxypiperidine (10a) in presence of diisopropyl
azodicarboxylate (DIAD) and PPh3 followed by acidic deprotection.
The human (h) TLR7/8 activity of new oxoadenines 3a–g was
assessed by a reporter gene assay using HEK293 cells stably trans-
fected with either hTLR7 or hTLR8 and the NFrB SEAP (secreted
embryonic alkaline phosphatase) reporter (Fig. 2).27 This assay
NFjB activation in the HEK293-hTLR8 cells suggesting that the
toxicity observed in HEK293-hTLR8 cells is dissociated from NF
jB
activity and possibly associated with activation of another intracel-
lular signaling pathway via TLR8.
NH2
NH2
Cl
N
N
N
N
a-c
N
d-f
N
OMe
N
n-BuO
N
5
N
N
N
H
n-BuO
Cl
N
H
NH2
H
N
O
N
6
4
O
N
n-BuO
N
( )
n
HO
k,m or l,m
( )n-2
NH2 Cl
OH
3a-g
( )n-2
8f,g
X
Br
( )n
h,i
n=5,6
g
Series
n
a
b
c
d
e
f
0
1
2
3
4
5
6
n=5,6
N
N
N
Boc
7
9f,g
10b-d,f,g X = OH
11b-d,f,g X = Br
j
g
Scheme 1. Reagents and conditions: (a) 3,4-dihydropyran, p-TsOH, AcOEt, 50 °C; (b) 2 M NH3 in i-PrOH, 60 °C, 86% (2 steps); (c) t-BuONa, n-BuOH, 100 °C, 85%; (d) N-
bromosucccinimide, CHCl3, rt, 88%; (e) NaOMe, MeOH, reflux; (f) TFA, MeOH, rt, 87% (2 steps); (g) (PPh3)PdCl2, CuI, Et3N, , 82% (9f), 21% (9g); (h) 10% Pd(OH)2/C, H2, AcOH,
D
90 °C; (i) Boc2O, Et3N, CH2Cl2, rt, 80% (2 steps); (j) CBr4, PPh3, CH2Cl2, rt, 92–99%; (k) Et3N, PPh3, DIAD, DMF, 70 °C, 63%; (l) K2CO3, DMF, 50 °C; (m) 4 N HCl/dioxane, MeOH, rt,
59% (3a), 75% (3b), 83% (3c), 41% (3d), 63% (3e), 64% (3f), 84% (3g).

1320
H. G. Bazin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1318–1323
hTLR7/pNifty2-SEAP Transfected HEK293
120
110
100
90
80
70
60
50
40
30
20
10
0
A
CRX-668 (1C)
3a
(0)
CRX-672 (2C)
3b
(1)
CRX-717 (3C)
3c
(2)
CRX-689 (4C)
3d
(3)
CRX-715 (5C)
3e
(4)
CRX-716 (6C)
CRX-732 (0C)
3f
(5)
(6)
3g
4
2
3
1
5
6
0
1x10-8
1x10-7
1x10-6
Concentration (M)
1x10-5
hTLR8/pNifty2-SEAP Transfected HEK293
B
100
90
80
70
60
50
40
30
20
10
0
3a
(0)
CRX-668 (1C)
3b
(1)
CRX-672 (2C)
CRX-717 (3C)
3c
(2)
2
3
CRX-689 (4C)
3d
(3)
CRX-715 (5C)
3e
(4)
CRX-716 (6C)
3f
(5)
CRX-732 (0C)
3g
(6)
4
1
6
5
0
1x10-7
1x10-6
1x10-5
Concentration (M)
0.0001
0.001
C
Figure 2. NFrB response of (A) HEK293-hTLR7 and (B) HEK293-hTLR8 cells treated for 24 h with oxoadenines 3a–g; (C) hTLR7 and hTLR8 EC50 values for oxoadenines 3a–g
are shown with 95% confidence interval. Each data point was done in triplicate and averaged. EC50 were calculated after generating dose-response curves in XLfit (IBDS). Data
is representative of four independent experiments. The maximum activity of 3b (used as a reference standard) was set to 100% NFrB and all other compound values are
relative to 3b.
oxoadenines 3e–g was evaluated using cytokine ELISA and intra-
100
cellular cytokine staining (ICS).29 Induction of TNF
a
is shown in
secretion with increasing linker
length was observed, with maximal TNF secretion observed for
3e
3f
3g
90
80
70
60
50
40
30
20
10
0
Figure 4A. A clear increase in TNF
a
a
the 5-carbon linker (Fig. 4A). ICS was also used to examine the acti-
vation status and cytokine contributions of distinct cell subsets. A
similar pattern was observed in myeloid dendritic cells (mDCs)
(HLA-DR+ CD11c+ CD123À) when comparing IL-6 (Fig. 4B), TNF
a
and IFNc induction (not shown). Taken together, these data dem-
onstrate an increase in proinflammatory cytokines as the length
of the alkyl linker is increased to 5 carbons. The 6-carbon linker
0
100 200 300 400 500 600 700 800
oxoadenines (μM)
oxoadenine 3g induced less TNFa than 3f possessing 5 carbons
but more than the 4-carbon linker oxoadenine 3e.
Given the nonlinear relationship observed between the linker
length and the hTLR7 EC50 values obtained with oxoadenines 3a–
Figure 3. After 24 h stimulation with oxoadenines 3e–g, HEK293-hTLR8 cells were
incubated with Aqua dye for 30 min. After extensive wash, the stained cells were
analyzed for viability using BD LSRII.
g in the HEK293 system (which is only indicative of the NF
of TLR7 signaling), IFN induction from hPBMCs was expected to
be more representative of the hTLR7 activity of these compounds.
jB side
a
The induction of cytokines in human peripheral blood
mononuclear cells (hPBMCs)28 after 24 h stimulation with

H. G. Bazin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1318–1323
1321
3000
A
3a
3b
3c (2)
3d (3)
3e
3f
3g
(0)
(1)
2500
2000
1500
1000
500
5
6
(4)
(5)
(6)
4
2
3
1
0
0
0.0001
0.001
0.01
0.1
1
10
TLR7/8 Agonist (μM)
Alkyl linker length
B
1
4
5
6
2
3
IL-6
Figure 4. (A) TNFa induction in hPBMCs, and (B) IL-6 expression in mDCs after stimulation with oxoadenines 3a–g. Data shown is representative of hPBMCs from three
different healthy donors evaluated in triplicate. Circled numbers indicate the linker length. Error bars indicate SEM. TNF
a
was assayed using FluoroKine multiplex kits and IL-
6 levels were measured by ICS in mDCs (HLA-DR+ CD11c+ CD123À).
3b
3f
7000
6000
5000
4000
3000
2000
1000
0
3a
(0)
3b(1)
3c (2)
3d(3)
3e
3f
Tinted:
Purple: 0.01 nM
Blue: 1 nM
Green: 100 nM
Red: 10 µM
0
4
2
6
(4)
(5)
(6)
1
mDC
pDC
3
3g
5
0
0.0001
0.001
0.01
0.1
1
10
TLR7/8 agonist (μM)
Figure 5. IFN
a
induction in hPBMCs after stimulation with oxoadenines 3a–g. IFN
a
levels were measured using human IFN
a
VeriKine ELISA kit. Data shown is
representative of hPBMCs from three different healthy donors evaluated in
triplicate. Circled numbers indicate the linker length. Error bars indicate SEM.
Annexin-V
Figure 6. Annexin-V staining of hPBMCs after stimulation with 3b or 3f and cell
A bimodal pattern for IFN
of these compounds (Fig. 5). Each compound exhibited a bell-
shaped dose–response curve from peak to base within a 100
dose range, except oxoadenine 3a which was inactive. The dose
required for peak IFN induction decreased with increasing linker
length, but higher concentrations led to dose-responsive
decrease in IFN . Concurrently, TNF levels in the same cell culture
supernatants increased in a dose-dependent fashion (Fig. 4A). The
dichotomy observed between TNF and IFN induction in the
a expression was observed for all but one
phenotyping (mDC: HLA-DR+ CD11c+ CD123À and pDC: HLA-DR+ CD11cÀ CD123+).
lM
dose-dependent increase in Annexin-V staining in pDC starting at
1 nM but not in mDC (Fig. 6). In contrast, only the highest dose
a
a
of 3b (10
lM) was associated with Annexin-V positive cells in both
a
a
pDC and mDC subsets. This observed cell type specific apoptosis
correlated with the dose-dependent IFN
curves and the intracellular cytokine staining of IFN
a
and TNF
a
induction
and TNF
a
a
a
a
same hPBMC supernatants could be due to the differential expres-
sion of TLR8 on human mDCs and TLR7 on human pDCs. The pDC
specific suppression of the TLR7-IRF7 signaling pathway via a reg-
ulatory feedback loop or cell-type specific activation induced cell
death (AICD) could be responsible for the unique cytokine pattern
observed in this study. To evaluate these hypotheses, hPBMCs were
stimulated with various doses of 3b (1-carbon linker) or 3f (5-car-
bon linker) and the cells evaluated for activation induced apoptosis
by Annexin-V staining. Oxoadenine 3f was associated with a
in pDCs and mDCs, respectively (data not shown). Overall, these
results demonstrated that increasing the carbon linker from 1 to
5 carbons increases the potency for IFN
a induction from pDC but
also reduces the dose threshold for apoptosis while leaving TNF
a
induction from mDC largely unaltered.
In summary, we have described the synthesis and the struc-
ture-activity relationship of a series of oxoadenines substituted
at the 9-position with a 4-piperidinylalkyl moiety. A minimum
of 1-carbon linker was required for hTLR7 and hTLR8 activity.

1322
H. G. Bazin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1318–1323
17. Kazaoka, K.; Sajiki, H.; Hirota, K. Chem. Pharm. Bull. 2003, 51, 608.
18. Chan, M.; Hayashi, T.; Kuy, C. S.; Gray, C. S.; Wu, C. C. N.; Corr, M.; Wrasidlo, W.;
Cottam, H. B.; Carson, D. A. Bioconjugate Chem. 2009, 20, 1194.
19. Wille-Reece, U.; Wu, C. y.; Flynn, B. J.; Kedl, R. M.; Seder, R. A. J. Immunol. 2005,
174, 7676.
The 5-carbon linker oxoadenine was the most potent hTLR7
agonist while the 1-carbon linker was the most potent hTLR8
agonist of the series. Proinflammatory cytokines and IFN
a
induction in hPBMCs increased with increasing linker length,
with the 5-carbon linker oxoadenine being the most active
cytokine inducer. These results suggest that it is possible to
modulate hTLR7/8 specificity and cytokine induction in the
oxoadenine series with non-aromatic groups at N9 using minor
structural modification. Insights from these types of studies will
help to identify safer, more selective TLR7/8 agonists with tai-
lored biological properties. More extensive structure–activity
relationship studies in the 8-oxoadenine series and molecular
modeling studies using the recently described TLR8 crystal struc-
ture30 are currently underway.
Author contributions: All authors participated in the design or
implementation or analysis, and interpretation of the study results.
All authors were involved in drafting the manuscript or revising it
critically for important intellectual content. All authors had full
access to the data and approved the manuscript before it was
submitted by the corresponding author.
20. McInally, T.; Thom, S.; Wada, H. WO 2007031726 A1, 2007.
21. Synthesis of 6: p-Toluenesulfonic acid (0.01 equiv) was added to a suspension of
2,6-dichloropurine in ethyl acetate (0.5 M). The mixture was heated to 50 °C
and 3,4-dihydro-2H-pyran (1.5 equiv) was added. The reaction mixture was
stirred at 50 °C for 2 h then concentrated and dried under vacuum. The
resulting yellow solid was heated with 2 M ammonia in isopropanol
(7.0 equiv) at 50 °C for 7 h and the reaction mixture poured into water. After
standing at rt overnight, the yellow solid was filtered off, washed with
isopropanol, dried under vacuum and purified by chromatography on silica gel
(0–2% CHCl3 in CH3OH) to give 2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-amine31 in 86% yield. Sodium t-butoxide (4.0 equiv) was added
portionwise to n-butanol (20 equiv) (Note: exothermic reaction). The suspen-
sion was stirred until homogeneous (ꢀ20 min) and 2-chloro-9-(tetrahydro-
2H-pyran-2-yl)-9H-purin-6-amine (1.0 equiv) was added. The reaction mix-
ture was then stirred at 100 °C overnight and concentrated under vacuum.
After aqueous work-up (CH2Cl2/H2O) and purification by chromatography on
silica gel (0–1.2% CH3OH in CHCl3), compound 520 was isolated in 85% yield. 1
H
NMR (CDCl3): d 7.86 (1H, s), 6.10 (broad s, 2H), 5.63 (d, J = 10.0 Hz, 1H), 4.36
(m, 2H), 4.14 (d, J = 9.6 Hz, 1H), 3.75 (t, J = 11.2 Hz, 1H), 2.06 (m, 3H), 1.37–1.81
(m, 7H), 0.92 (t, J = 7.2 Hz, 3H). N-Bromosuccinimide (1.05 equiv) was slowly
added to a solution of 5 in CHCl3 (0.65 M) at 0 °C. The reaction mixture was
stirred at 0 °C for 20 min then at rt for 4 h. After aqueous work-up (CH2Cl2/
H2O) and purification by chromatography on silica gel (0–1.5% CH3OH in
CHCl3) 8-bromo-2-butoxy-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine20
was obtained in 88% yield. A solution of 8-bromo-2-butoxy-9-(tetrahydro-2H-
pyran-2-yl)-9H-purin-6-amine in CH3OH (0.35 M) was heated to reflux with a
solution of sodium methoxide in CH3OH (2.7 equiv) for 4 h. The reaction
mixture was concentrated and after aqueous work-up (ethyl acetate and
saturated NH4Cl solution) the crude was purified by chromatography on silica
gel (0–1.5% CH3OH in CHCl3) to give 2-butoxy-8-methoxy-9-(tetrahydro-2H-
pyran-2-yl)-9H-purin-6-amine.20 TFA (10% v/v TFA/CH3OH) was added to a
solution of 2-butoxy-8-methoxy-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-
amine in CH3OH (0.23 M) and stirred at rt for 3 days. The reaction mixture
was concentrated, diluted with ethyl acetate and filtered. The yellow solid was
washed with a small volume of ethyl acetate and dried under vacuum to give
620 as an off-white solid in 87% yield (2 steps). 1H NMR (CD3OD/CDCl3) d 4.50
(t, J = 6.8 Hz, 2H), 4.15 (s, 3H), 1.80 (m, 2H), 1.48 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H).
22. Synthesis of 10f and 10g: 4-Bromopyridine hydrochloride (2.5 g) was
partitioned between 1 N sodium hydroxide (20 mL) and ethyl acetate
(3 Â 20 mL). The organic layer was separated, dried over Na2SO4 and
concentrated under vacuum. The resulting oil was dissolved in Et3N (2.6 M)
and degassed under nitrogen. 4-Pentyn-1-ol or 5-hexyn-1-ol (1.1 equiv) was
added followed by bis(triphenylphosphine)palladium(II) chloride (0.01 equiv)
and copper(I) iodide (0.02 equiv) and the reaction mixture stirred at reflux for
20 min. Aqueous work-up (ethyl acetate/H2O) and purification by
chromatography on silica gel (gradient 0–30% ethyl acetate in heptane) led
to 9f and 9g in 82% and 21% yield, respectively. 9f: 1H NMR (400 MHz, CDCl3) d
8.52 (m, 2H), 7.25 (m, 2H), 3.82 (m, 2H), 2.58 (t, J = 7.2 Hz, 2H), 1.88 (m, 2H),
1.62 (m, 1H). 9g: 1H NMR (400 MHz, CDCl3) d 8.52 (m, 2H), 7.25 (m, 2H), 3.73
(m, 2H), 2.48 (t, J = 7.2 Hz, 2H), 1.74 (m, 4H), 1.51 (m, 1H). Compound 9f or 9g
was dissolved in acetic acid (0.05 M) and the solution hydrogenated using a H-
CubeÒcontinuous-flow hydrogenation reactor (ThalesNano) (20% Pd(OH)2/C
cartridge, 100 bars H2, 90 °C, 1 mL/min). Once the hydrogenation was
complete, the reaction mixture was concentrated and dried under vacuum.
The resulting crude was dissolved in CH2Cl2 (0.4 M) and reacted with Et3N
(1.5 equiv) and di-t-butyl dicarbonate (1.2 equiv) at rt for 30 min. After
aqueous work-up (CH2Cl2/H2O) and purification by chromatography on silica
gel (gradient 0–30% ethyl acetate in heptane), compounds 10f and 10g were
isolated in 80% yield. 10f: 1H NMR (400 MHz, CDCl3) d 4.06 (s, 2H), 3.64 (t,
J = 6.8 Hz, 2H), 2.66 (t, J = 11.4 Hz, 2H), 1.54–1.66 (m, 4H), 1.45 (s, 9H), 1.24–
1.39 (m, 8H), 1.08 (m, 2H). 10g: 1H NMR (400 MHz, CDCl3) d 4.05 (s, 2H), 3.64
(dd, J = 6.2, 10.6 Hz, 2H), 2.63 (t, J = 12.0 Hz, 2H), 1.53–1.65 (m, 4H), 1.45 (s,
9H), 1.20–1.40 (m, 10H), 1.04 (m, 2H).
Acknowledgments
This work was supported in part by the National Institute of
Allergy and Infectious Diseases (NIAID) under contract
HHSN272200900036C (to Corixa Corporation d/b/a GlaxoSmithK-
line Vaccines). Any opinions, findings, conclusions or recommenda-
tions expressed in this Letter are those of the authors and do not
necessarily reflect the views of the NIAID.
We thank Dr. Hardeep Oberoi for formulation of the compounds
described in this study and Jon Ward and Van Cybulski for techni-
cal assistance with immunology assays.
References and notes
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23. Synthesis of 11f and 11g: PPh3 (1.2 equiv) was slowly added to a cold solution
(0 °C) of 10f or 10g and CBr4 (1.6 equiv) in CH2Cl2 (0.45 M) and the resulting
solution stirred at rt for 45 min. The concentrated reaction mixture was
directly purified by chromatography on silica gel (gradient 0–30% ethyl acetate
in heptane) and 11f and 11g were isolated in 92% and 99% yield, respectively.
11f: 1H NMR (400 MHz, CDCl3) d 4.06 (s, 2H), 3.41 (t, J = 6.7 Hz, 2H), 2.66 (t,
J = 11.7 Hz, 2H), 1.86 (m, 2H), 1.65 (m, 2H), 1.45 (s, 9H), 1.29–1.42 (m, 5H), 1.25
(m, 2H), 1.07 (m, 2H). 11g: 1H NMR (400 MHz, CDCl3) d 4.06 (s, 2H), 3.41 (t,
J = 7.0 Hz, 2H), 2.66 (t, J = 11.7 Hz, 2H), 1.85 (m, 2H), 1.63 (m, 2H), 1.45 (s, 9H),
1.20–1.43 (m, 7H), 1.23 (m, 2H), 1.05 (m, 2H).
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24. General procedure for N-alkylation and acidic deprotection for 3b–g32: To a
solution of 6 in DMF (0.25 M) was added K2CO3 (325 mesh, 3.0 equiv). The
reaction mixture was sonicated several seconds to obtain a fine suspension and
stirred at 60 °C for 1 h. After cooling to 50 °C, bromide 11b–g (1.2 equiv) was
added and the reaction mixture stirred overnight at 50 °C. After cooling to rt
and aqueous work-up (ethyl acetate/H2O) the resulting crude was purified by
chromatography on silica gel (gradient 0–10% CH3OH in CHCl3). The purified
product was dissolved in CH3OH (0.1 M) and reacted with 4 N HCl in dioxane
16. Weterings, J. J.; Khan, S.; van der Heden van Noort, G.; Melief, C. J. M.;
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H. G. Bazin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1318–1323
1323
(6.0 equiv) at rt for 1 h. The reaction mixture was concentrated and dried under
vacuum and the residue purified by chromatography on silica gel (0–100%
CHCl3/CH3OH/H2O 85/15/0.5 in CHCl3/CH3OH/H2O 75/25/1.0). 3b: 1H NMR
(400 MHz, CD3OD) d 4.27 (t, J = 6.8 Hz, 2H), 3.77 (d, J = 7.2 Hz, 2H), 3.40 (d,
J = 12.8 Hz, 2H), 2.96 (dt, J = 2.8, 13.0 Hz, 2H), 2.21 (bs, 1H), 1.93 (d, J = 12.0 Hz,
2H), 1.75 (q, J = 6.8 Hz, 2H), 1.55 (m, 2H), 1.50 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H);
positive ES TOF-MS calcd for [M+H]+ 321.2039, found 321.2226; 3c: 1H NMR
(400 MHz, CD3OD) d 4.26 (t, J = 6.4 Hz, 2H), 3.88 (t, J = 7.0 Hz, 2H), 3.34 (d,
J = 12.8 Hz, 2H), 2.93 (dt, J = 3.2, 12.8 Hz, 2H), 2.08 (bd, J = 14.4 Hz, 2H), 1.71–
1.77 (m, 4H), 1.60 (bs, 1H), 1.48 (m, 2H), 1.41 (m, 2H), 0.98 (t, J = 7.6 Hz, 3H);
positive ES TOF-MS calcd for [M+H]+ 335.2195, found 335.2182; 3d: 1H NMR
(400 MHz, CD3OD) d 4.27 (t, J = 6.4 Hz, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.35 (d,
J = 12.4 Hz, 2H), 2.95 (dt, J = 2.8, 13.0 Hz, 2H), 1.67–1.80 (m, 6H), 1.31-1.51 (m,
7H), 0.98 (t, J = 7.2 Hz, 3H); positive ES TOF-MS calcd for [M+H]+ 349.2352,
found 349.3217; 3e: 1H NMR (400 MHz, CD3OD) d 4.26 (t, J = 6.4 Hz, 2H), 3.81
(t, J = 6.8 Hz, 2H), 3.33 (t, J = 12.4 Hz, 2H), 2.93 (dt, J = 2.8, 13.0 Hz, 2H), 1.89 (d,
J = 12.8 Hz, 2H), 1.36–1.74 (m, 13H), 0.97 (t, J = 7.2 Hz, 3H); positive ES TOF-MS
calcd for [M+H]+ 363.2508, found 363.2594; 3f: 1H NMR (400 MHz, CD3OD) d
4.28 (t, J = 6.4 Hz, 2H), 3.83 (t, J = 6.8 Hz, 2H), 3.35 (m, 2H), 2.93 (t, J = 13.2 Hz,
2H), 1.91 (d, J = 13.2 Hz, 2H), 1.75 (m, 4H), 1.31–1.60 (m. 11H), 0.98 (t,
J = 7.4 Hz, 3H); positive ES TOF-MS calcd for [M+H]+ 377.2665, found 377.3884;
3g: 1H NMR (400 MHz, CD3OD) d 4.28 (t, J = 6.4 Hz, 2H), 3.82 (t, J = 7.0 Hz, 2H),
3.38 (m, 2H), 2.94 (t, J = 12.4 Hz, 2H), 1.92 (d, J = 13.2 Hz, 2H), 1.75 (m, 4H),
1.35–1.58 (m. 13 H), 0.98 (t, J = 7.2 Hz, 3H); positive ES TOF-MS calcd for
[M+H]+ 391.2821, found 391.3733.
(dt, J = 2.8, 10.4 Hz, 2H), 2.80 (dq, J = 3.8, 8.8 Hz, 2H), 2.01 (d, J = 10.8 Hz,
2H), 1.73 (m, 2H), 1.50 (m, 2H), 0.99 (t, J = 7.6 Hz, 3H).
26. Rückle, T.; Biamonte, M.; Grippi-Vallotton, T.; Arkinstall, S.; Cambet, Y.; Camps,
M.; Chabert, C.; Church, D. J.; Halazy, S.; Jiang, X.; Martinou, I.; Nichols, A.;
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27. HEK293 cells expressing human TLR7 or TLR8 and NFjB responsive SEAP
reporter gene were obtained from Invivogen (San Diego, CA). These cells were
maintained in culture media of DMEM (Invitrogen, Grand Island, NY), 10% FBS
(Sigma, St. Louis, Missouri) and selection antibiotics (Invitrogen, and
Invivogen). HEK293 stably transfected with hTLR7 or hTLR8 were stimulated
for 24 h with aqueous formulations of oxoadenines 3a–f and culture
supernatants were analyzed for NF
jB activation using the colorimetric SEAP
detection kit QuantBlue (Invivogen).
28. Primary human PBMCs were isolated from fresh blood from healthy donors via
Ficoll gradient separation and plated at 0.5 Â 106 cells/well in 96-well tissue
culture plates (RPMI-1640 plus 10% FBS). hPBMCs were maintained with RPMI-
1640 culture media (Invitrogen, Grand Island, NY), antibiotics (Invitrogen) and
10% FBS (Sigma). hPBMCs were stimulated for 24 h with aqueous formulations
of oxoadenines 3a–f. Culture supernatants were analyzed for cytokine/
chemokine induction using multiplex kits (FluoroKine multiplex kits from
R&D Systems, Minneapolis, MN) and human IFN
Biomedical Laboratories, Inc., Piscataway, NJ).
a VeriKine ELISA kit (Pestka
29. Human PBMCs were stimulated with aqueous formulation of oxoadenines
3a–g for 24 h and GolgiPlug (10 ng/ml, BD Bioscience) was added for the final
6 h stimulation. After the 24 h stimulation cells were washed and stained for
lineage-specific cell surface markers. BD Perm/Fix buffer (BD Bioscience) was
used for permeabilization and fluorochrome conjugated anti-cytokine/
chemokine antibodies were added. Data were acquired with a BD LSRII flow
cytometer and analyzed with FlowJo (TreeStar, Boston, MA). Dead cells were
excluded by FSC gating and Aqua exclusion.
30. Tanji, H.; Ohto, U.; Shibata, T.; Miyake, K.; Shimizu, T. Science 2013, 339, 1426.
31. Borrmann, T.; Abdelrahman, A.; Volpini, R.; Lambertucci, C.; Alksnis, E.;
Gorzalka, S.; Knospe, M.; Schiedel, A. C.; Cristalli, G.; Müller, C. E. J. Med.
Chem. 2009, 52, 5974.
25. Synthesis of 3a: DIAD (10 equiv) was added dropwise to a stirred solution of
PPh3 (10 equiv) in DMF (0.28 M) at 0 °C and the reaction mixture stirred at
this temperature for 30 min. 1-Boc-4-hydroxypiperidine (10a, 10 equiv) was
added and the reaction mixture stirred at 0 °C for 10 min then at rt for
30 min. A suspension of 6 (1.0 equiv) and Et3N (2 equiv) in DMF (0.28 M)
was added. The reaction mixture was stirred at 60 °C overnight,
concentrated and purified by chromatography on silica gel (50–80% ethyl
acetate in heptane followed by 0–2% CH3OH in CHCl3) to give the alkylated
derivative in 63% yield. Subsequent acidic deprotection following the general
procedure described above gave 3a in 59% yield. 3a: 1H NMR (CD3OD,
400 MHz) d 4.53 (m, 1H), 4.29 (t, J = 6.4 Hz, 2H), 3.53 (d, J = 8.4 Hz, 2H), 3.14
32. Bazin-Lee, H.; Coe, D. M.; Lazarides, L.; Mitchell, C. J.; Smith, S. A.; Trivedi, N.
WO 2010. 20100118134 A1.