Synthesis and immunostimulatory activity of 8-substituted amino 9-benzyladenines as potent Toll-like receptor 7 agonists

Article abstract of DOI:10.1016/j.bmcl.2006.06.017

Several 9-benzyl adenine derivatives bearing various substituted amines at the 8-position have been prepared and evaluated for interferon induction in peripheral blood mononuclear cells (PBMC) from healthy human donors. The 8-bromoadenine derivative 5 was

Full text of DOI:10.1016/j.bmcl.2006.06.017

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4559–4563
Synthesis and immunostimulatory activity of 8-substituted amino
9-benzyladenines as potent Toll-like receptor 7 agonists
Guangyi Jin, Christina C. N. Wu, Rommel I. Tawatao, Michael Chan,
Dennis A. Carson and Howard B. Cottam*
Moores Cancer Center, University of California, San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093-0820, USA
Received 10 May 2006; revised 5 June 2006; accepted 6 June 2006
Available online 19 June 2006
Abstract—Several 9-benzyl adenine derivatives bearing various substituted amines at the 8-position have been prepared and eval-
uated for interferon induction in peripheral blood mononuclear cells (PBMC) from healthy human donors. The 8-bromoadenine
derivative 5 was used as a versatile intermediate for all substitutions. The most active 8-substituted amino compound was found
to be the 8-morpholinoethylamino derivative 19 which had an EC50 in the submicromolar range.
Ó 2006 Elsevier Ltd. All rights reserved.
In the mid 1980s, our laboratory and others began
studying a group of synthetic nucleosides that had the
unique ability to activate the innate 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]py-
rimidine,1 pyrazolo[3,4-d]pyrimidine,2 purine,3 7-dea-
zapurine,4 and 9-deazapurine5 ring systems were all
active. Examples in each group were found to be potent
antiviral agents in mouse models due to their ability to
rapidly induce the production of interferon.4,6,7 Among
these guanosine analogs, 7-thia-8-oxoguanosine (1,
TOG, Fig. 1) was studied in greatest detail and shown
to exhibit broad spectrum antiviral activity and to acti-
vate natural killer cells, macrophages, and B-lympho-
cytes.8 Other guanosine analogs extensively studied
were 7-deazaguanosine9 and 7-allyl-8-oxoguanosine
(loxoribine).10 The exact mechanism of innate immune
potentiation by these guanosine-like compounds had
not been elucidated and we were prompted to investi-
gate the possibility that these small molecules could be
acting via signaling through one or more of the known
Toll-like receptors (TLRs), of which there are more than
ten now identified, which recognize molecular patterns
commonly associated with many microbial agents. As
a result of those studies, we recently showed that TOG
and certain other guanosine analogs activate immune
cells via TLR7.11 Interestingly, our earlier studies also
showed that a sugar moiety was not required for im-
mune system potentiation, as certain alkylated purines
were also effective.3 Since that time, alkylated adenine
derivatives have been discovered12–14 which are even
more potent interferon inducers than the guanines and
guanosines. Compounds of both classes, guanines and
adenines, 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, has recently been reported to be
effective at reducing the plasma virus concentration in
patients with chronic hepatitis C virus (HCV) infec-
tions15 with minimal side effects. Our own recent studies
provide evidence that TLR7-mediated immunity against
HCV involves at least two mechanisms: one depends on
type 1 interferon production by leukocytes, and the
other is mediated by TLR7 expressed by virally infected
hepatocytes.16 Currently, one TLR7/8 agonist (Imiqui-
mod, 2) is approved for use in human subjects for cer-
tain viral infections and skin cancers, and other
agonists are in advanced stages of clinical develop-
ment.17 TLR agonists might be exploited for use against
bioterror attacks on the basis of their mechanism of ac-
tion. However, excessive activation of the innate im-
mune system can result in severe side effects,
autoimmune disease, and septic shock.18 Indeed, the
related molecule R848 (Resiquimod, 3), a more potent
analog of 2, was reported to lack antiviral effects in
HCV-infected patients, perhaps due to the poor tolera-
bility that limited the dose level and frequency.15 There-
Keywords: Innate immune response; 8-Substituted aminoadenines;
Interferon inducers; Toll-like receptor agonists.
*
Corresponding author. Tel.: +1 858 534 5424; fax: +1 858 534
5399; e-mail: hcottam@ucsd.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.017

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G. Jin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4559–4563
O
N
NH2
N
NH2
NH2
H
N
S
N
HN
H2N
N
N
N
O
N
N
O
N
N
O
N
O
O
OH
HO
O
OH
HO
TOG
Imiquimod
R848
SM360320
4
3
2
1
Figure 1.
fore, the ideal candidates as TLR agonists should have
the right balance of activity and tolerability.
of a few prodrugs that would eventually provide the 8-
oxo function.13 Thus, the reported modifications includ-
ed those at the 2 and 9 positions only.12 We elected to
prepare and investigate the structure–activity function
of a series of 8-substituted amino adenines while main-
taining all other structural features constant.
As part of our ongoing studies in the design and prepa-
ration of agonists of TLR signaling, we prepared a series
of 8-substituted amino adenine compounds and evaluat-
ed them in mouse and human cell-based assays. The
starting point for the selection of the compounds was
based on recent reports describing the potent activity
of 9-benzyl-8-oxo-2-alkoxyadenines as interferon induc-
ers.13,14 We first prepared 9-benzyl-2-methoxyethoxy-8-
oxoadenine (4) according to the published procedure14
and then confirmed that it was acting exclusively
through TLR7 signaling.16 A review of the literature re-
vealed that apart from the 8-oxo group, no other mod-
ifications at the 8-position had been reported for this
purine class of interferon inducers, with the exception
The versatile 8-bromoadenine derivative (5)14 was used
as an intermediate for all substitutions. The general pro-
cedures for the amine substitutions are depicted in
Scheme 1 and the final products are listed in Table 1,
each with the corresponding method of preparation
indicated.
The synthesis of compounds 7–10, 12, 14–16, 18–24, 26,
and 29 by method A was carried out in an autoclave
using water as solvent. The hydrazino compound 29 pre-
NH2
NH2
N
N
N
N
MethodA,B or C
N
NR1R2
N
Br
O
N
O
N
O
O
7-26 and 29
5
Method D
NH2
N
N
N
NH2
N
O
O
6
Method E
NH2
NH2
O
O
N
N
N
Method F
NH2
N
N
OEt
N
H
N
H
O
N
N
O
N
O
O
28
27
Scheme 1. Reagents and conditions: Method (A) NH(R1R2), H2O, 110–125 °C, 12 h. Method (B) NH(R1R2), 150–160 °C, 6 h. Method (C)
Pd2(dba)3, BINAP, secondary amine, K2CO3, t-butanol, 130 °C, 12 h. Method (D) 1—NaN3, DMF, 100 °C, 7 h; 2—H2/Raney-Ni, rt, 12 h. Method
(E) ClCOOEt, pyridine, rt, 12 h. Method (F) NH3/MeOH, 70 °C, 10 h.

G. Jin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4559–4563
4561
Table 1.
NH2
N
N
NR1R2
N
O
N
O
Compound
R1
R2
Method
IFN-aa
SEM
4
418.00
35.25
53.94
23.19
6
H
H
D
A
A
A
A
C
A
B
b
7
H
Me
Et
8
H
80.30
8.80
0.73
34.22
4.44
0.73
9
H
n-Pr
n-Bu
Et
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
DMSO
H
Et
b
H
2-Hydroxylethyl
2-Hydroxylethyl
2-Hydroxylethyl
99.21
0.82
113.18
36.11
0.00
39.73
H
H
H
H
H
H
H
H
H
H
H
3-Hydroxy-n-propyl
4-Hydroxy-n-butyl
Tetrahydrofurfuryl
Benzyl
A
A
A
B
b
b
b
Phenylethyl
A
A
A
A
A
A
A
C
A
E
1.33
110.48
0.00
30.16
2-(Morpholino)ethyl
2-(Piperidin-1-yl)ethyl
2-Methoxyethyl
b
b
b
b
b
Diethanolaminoethyl
Diethanolaminopropyl
Cyclohexylmethyl
Morpholino
0.41
0.00
0.00
b
H
H
2-(1H-Indol-3-yl)ethyl
Ethoxycarbonyl
1.03
b
Carbamoyl (8-ureido)
NH2 (8-Hydrazino)
F
H
A
0.21
0.26
0.21
0.26
a Interferon concentration in pg/mL. All compounds compared at 1 lM; see Ref. 24.
b Below lower limit of detection.
cipitated from a reaction using 20% aqueous hydrazine,
while compound 6 was the major product if a concentra-
tion of hydrazine lower than 10% was used. Products 13
and 17 were afforded by reaction of 5 with a large excess
of diethanolamine or benzylamine, respectively, as re-
agent and solvent at elevated temperatures. Compounds
11 and 25 were obtained in good yield by the palladium-
catalyzed reaction of the corresponding secondary
amine with 5 under anhydrous conditions. To our
knowledge, this is the first example of palladium-cata-
lyzed amination of an adenine system by a hindered
amine. There are existing methods for 8-aminoadenine
preparation.19–23 Reaction of 8-bromo compound 5 with
NaN3 followed by Raney-Ni-catalyzed hydrogenation
furnished 6 in good yield. Acylation of 6 with ethylchlo-
roformate led to the ethylcarbamate 27 which was con-
verted to the ureido compound 28 by reaction with
methanolic ammonia.
lytical data for these compounds are provided in the
references section below.25–28 In addition, the EC50
(the concentration of compound at which 50% of the
maximal IFN concentration was achieved) was deter-
mined for these four most active compounds and these
are shown relative to compound 4 in Table 229 along
with the average maximal IFN produced by each com-
pound. These values are a composite of data from sever-
al healthy donors.
The simple replacement of the 8-oxo function in com-
pound 4 with an amino group (compound 6) com-
pletely removed all interferon inducing activity.
Table 2.
a
Compound
EC50 (lM)
Maximal IFNb
4
6
0.14
>10
220
132
141
168
206
176
8
4.42
5.33
3.20
0.79
All compounds were tested for their ability to induce the
production of interferon a in human peripheral blood
mononuclear cells (PBMC) compared to compound 4
as a positive control.24 As indicated in Table 1, each
compound was tested at 1 lM and the most active com-
pounds were found to be 8, 12, 14, and 19. Detailed ana-
12
14
19
a See Ref. 29.
b Average value in pg/mL of interferon from PBMC of three healthy
donors. All compounds compared at 10 lM.

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G. Jin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4559–4563
8. Smee, D. F.; Alaghamandan, H. A.; Cottam, H. B.;
Sharma, B. S.; Jolley, W. B.; Robins, R. K. Antimicrob.
Agents Chemother. 1989, 33, 1487.
9. Smee, D. F.; Alaghamandan, H. A.; Ramasamy, K.;
Revankar, G. R. Antiviral Res. 1995, 26, 203.
10. Reitz, A. B.; Goodman, M. G.; Pope, B. L.;
Argentieri, D. C.; Bell, S. C.; Burr, L. E.; Chour-
mouzis, E.; Come, J.; Goodman, J. H., et al. J. Med.
Chem. 1994, 37, 3561.
11. Lee, J.; Chuang, T.-H.; Redecke, V.; She, L.; Pitha, P. M.;
Carson, D. A.; Raz, E.; Cottam, H. B. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 6646.
12. Isobe, Y.; Kurimoto, A.; Tobe, M.; Hashimoto, K.;
Nakamura, T.; Norimura, K.; Ogita, H.; Takaku, H. J.
Med. Chem. 2006, 49, 2088.
13. Kurimoto, A.; Tobe, M.; Ogita, H.; Ogino, T.; Takaku,
H.; Ichii, S.; Kawakami, H.; Isobe, Y. Chem. Pharm. Bull.
2004, 52, 466.
14. Kurimoto, A.; Ogino, T.; Ichii, S.; Isobe, Y.; Tobe, M.;
Ogita, H.; Takaku, H.; Sajiki, H.; Hirota, K.; Kawakami,
H. Bioorg. Med. Chem. 2004, 12, 1091.
15. Horsmans, Y.; Berg, T.; Desager, J.-P.; Mueller, T.;
Schott, E.; Fletcher, S. P.; Steffy, K. R.; Bauman, L. A.;
Kerr, B. M.; Averett, D. R. Hepatology (Hoboken, NJ,
United States) 2005, 42, 724.
16. Lee, J.; Wu, C. C. N.; Lee, K. J.; Chuang, T.-H.; Katakura,
K.; Liu, Y.-T.; Chan, M.; Tawatao, R.; Chung, M.; Shen,
C.; Cottam, H. B.; Lai, M. M. C.; Raz, E.; Carson, D. A.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1828.
17. Tran, H.; Moreno, G.; Shumack, S. Expert Opin. Phar-
macother. 2004, 5, 427.
18. Amlie-Lefond, C.; Paz, D. A.; Connelly, M. P.; Huffnagle,
G. B.; Dunn, K. S.; Whelan, N. T.; Whelan, H. T. J.
Allergy Clin. Immunol. 2005, 116, 1334.
19. Holmes, R. E.; Robins, R. K. J. Am. Chem. Soc. 1965, 87,
1772.
However, further substitution of this amino function
yielded several active compounds with structure–activ-
ity trends that are apparent even within this relatively
small group. First, the homologous series of N-methyl,
N-ethyl, N-n-propyl, and N-n-butyl derivatives showed
that, by comparison, the 2-carbon chain (compound
8) was superior to the others. However, when one
adds a second ethyl group (compound 11), the activity
is lost. When a terminal hydrophilic group, such as
OH, is added to the ethyl group (compound 12), the
activity is enhanced somewhat. In this series, the
hydroxypropyl is about equally active but the hydrox-
ybutyl is devoid of activity. Addition of a second
hydroxyalkyl group (compound 13) again abrogates
activity. The ring-containing secondary amine, mor-
pholino compound 25, was also not active. Interest-
ingly, addition of a 2-carbon alkyl chain between the
8-amino and the morpholino ring (compound 19)
resulted in the highest activity of the entire group of
8-substituted amino compounds studied to date, with
an EC50 of 0.79 lM. Other compounds with ring-
chain combinations besides morpholinoethyl, such as
phenylethyl (18), piperidin-1-ylethyl (20), and 2-(1H-
indol-3-yl)ethyl (26), were not active. All active com-
pounds are thought to be working exclusively through
TLR7 signaling, as was shown earlier for compound
4,16 since experiments in which bone marrow derived
macrophages from TLR7 knockout mice were exposed
to the active compounds showed no enhancement of
cytokine (TNFa, IL-6, IL-12, etc.) production (data
not shown). Studies to optimize the lead compound
19 are presently underway.
20. Young, R. C.; Jones, M.; Milliner, K. J.; Rana, K. K.;
Ward, J. G. J. Med. Chem. 1990, 33, 2073.
21. Janeba, Z.; Holy, A.; Masojidkova, M. Collect. Czech.
Chem. Commun. 2001, 66, 517.
Acknowledgments
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.
22. de Ligt, R. A. F.; van der Klein, P. A. M.; von Frijtag
Drabbe Kunzel, J. K.; Lorenzen, A.; Ait El Maate, F.;
Fujikawa, S.; van Westhoven, R.; van den Hoven, T.;
Brussee, J.; Ijzerman, A. P. Bioorg. Med. Chem. 2004, 12,
139.
23. Kelley, J. L.; McLean, E. W.; Linn, J. A.; Krochmal, M.
P.; Ferris, R. M.; Howard, J. L. J. Med. Chem. 1990, 33,
196.
References and notes
1. Nagahara, K.; Anderson, J. D.; Kini, G. D.; Dalley, N.
K.; Larson, S. B.; Smee, D. F.; Jin, A.; Sharma, B. S.;
Jolley, W. B.; Robins, R. K.; Cottam, H. B. J. Med. Chem.
1990, 33, 407.
2. Bontems, R. J.; Anderson, J. D.; Smee, D. F.; Jin, A.;
Alaghamandan, H. A.; Sharma, B. S.; Jolley, W. B.;
Robins, R. K.; Cottam, H. B. J. Med. Chem. 1990, 33,
2174.
3. Michael, M. A.; Cottam, H. B.; Smee, D. F.; Robins, R.
K.; Kini, G. D. J. Med. Chem. 1993, 36, 3431.
4. Smee, D. F.; Alaghamandan, H. A.; Gilbert, J.; Burger, R.
A.; Jin, A.; Sharma, B. S.; Ramasamy, K.; Revankar, G.
R.; Cottam, H. B., et al. Antimicrob. Agents Chemother.
1991, 35, 152.
5. Girgis, N. S.; Michael, M. A.; Smee, D. F.; Alaghaman-
dan, H. A.; Robins, R. K.; Cottam, H. B. J. Med. Chem.
1990, 33, 2750.
24. Human blood samples were obtained from the San Diego
Blood Bank. PBMC were isolated by density-gradient
centrifugation over Ficoll-Hypaque (Amersham Pharma-
cia). Cells were resuspended in RPMI 1640 medium,
supplemented with 10% fetal bovine serum (FBS), L-
glutamine, and penicillin/streptomycin (RP10; Invitrogen,
Carlsbad, CA), plated at 106 cells/well in 96-well plates,
and stimulated with compounds at 1 lM final concentra-
tion for 24 h at 37 °C, 5% CO2. The IFN-a level in the
supernatants was measured by Luminex (Austin, TX)
using the Beadlyte Human MultiCytokine kit (Upstate,
Charlottesville, VA), according to the manufacturer’s
instructions. Results presented in the table are averages
of data composited from four different donors.
25. Selected data for compound 12: mp: 145–146 °C. 1H
NMR (500 MHz, CDCl3) d 7.17–7.33 (m, 5 H), 5.18 (s,
2H), 5.11 (s, 2H), 4.44 (t, J = 5 Hz, 2H), 4.36 (br, 1H), 3.75
(q, J = 5 and 5 Hz, 4H), 3.48 (t, J = 5 Hz, 2H), 3.41 (s,
3H). MS (ESI) m/z: 359.3 (MH+). Anal. Calcd for
C17H22N6O3: C, 56.97; H, 6.19; N, 23.45%. Found: C,
56.59; H, 6.29; N, 23.06%.
6. Smee, D. F.; Huffman, J. H.; Gessaman, A. C.; Huggins, J.
W.; Sidwell, R. W. Antiviral Res. 1991, 15, 229.
7. Smee, D. F.; Alaghamandan, H. A.; Jin, A.; Sharma, B.
S.; Jolley, W. B. Antiviral Res. 1990, 13, 91.

G. Jin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4559–4563
4563
26. Compound 8: mp: 156–158 °C. 1H NMR (500 MHz,
CDCl3) d 7.19–7.35 (m, 5H), 5.15 (s, 2 H), 5.11 (s, 2H),
4.45 (t, J = 5 Hz, 2H), 3.74 (t, J = 5 Hz, 2H), 3.69 (br, 1H),
3.41 (s, 3H), 3.37 (m, 2H), 1.13 (t, J = 5 Hz, 3H). MS (ESI)
m/z: 343.4 (MH+). Anal. Calcd for C17H22N6O2Æ0.5 H2O:
C, 58.05; H, 6.54; N, 23.90%. Found: C, 58.45; H, 6.44; N,
23.64%.
28. Compound 19: mp: 148–151 °C. 1H NMR (500 MHz,
CDCl3) d 7.23–7.34 (m, 5H), 5.19 (br, 2H), 5.12 (s, 2H),
4.75 (br, 1H), 4.46 (t, J = 5 Hz, 2H), 3.75 (t, J = 5 Hz, 2H),
3.47 (m, 4H), 3.41 (s, 3H), 3.38 (t, J = 5 Hz, 2H), 2.51 (t,
J = 5 Hz, 3H), 2.30 (m, 4H). MS (ESI) m/z: 450.6 (MNa+).
Anal. Calcd for C21H29N7O3Æ0.2H2O: C, 58.45; H, 6.81;
N, 22.73%. Found: C, 58.51; H, 6.85; N, 22.51%.
27. Compound 14: mp: 128–130 °C. 1H NMR (500 MHz,
CDCl3) d 7.18–7.33 (m, 5H), 5.11 (br, 4 H), 4.44 (t,
J = 5 Hz, 2H), 3.73 (t, J = 5 Hz, 2H), 3.52 (m, 4H), 3.40 (s,
3H), 1.61 (m, 2H). MS (ESI) m/z: 373.8 (MH+). Anal.
Calcd for C18H24N6O3Æ0.5H2O: C, 56.63; H, 6.55; N,
22.02%. Found: C, 57.01; H, 6.42; N, 22.22%.
29. For dose response study, cells were treated with com-
pounds at concentrations ranging from 10 lM to 10 nM
for 24 h. Results are a composite from three different
donors and non-linear regression curve fit analysis was
performed using GraphPad Prism software version 4.0b
(San Diego, CA) to determine the EC50.