Discovery of a highly potent series of TLR7 agonists

Article abstract of DOI:10.1016/j.bmcl.2011.07.076

The discovery of a series of highly potent and novel TLR7 agonist interferon inducers is described. Structure-activity relationships are presented, along with pharmacokinetic studies of a lead molecule from this series of N9-pyridylmethyl-8-oxo-3-deazapurine analogues. A rationale for the very high potency observed is offered. An investigation of the clearance mechanism of this class of compounds in rat was carried out, resulting in aldehyde oxidase mediated oxidation being identified as a key component of the high clearance observed. A possible solution to this problem is discussed.

Full text of DOI:10.1016/j.bmcl.2011.07.076

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5939–5943
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a highly potent series of TLR7 agonists
Peter Jones a, , David C. Pryde a, Thien-Duc Tran a, Fiona M. Adam a, Gerwyn Bish a, Frederick Calo a,
⇑
Guiseppe Ciaramella b, Rachel Dixon d, Jonathan Duckworth c, David N.A. Fox a, Duncan A. Hay a,
James Hitchin d, Nigel Horscroft b, Martin Howard c, Carl Laxton b, Tanya Parkinson b, Gemma Parsons a,
Katie Proctor a, Mya C. Smith a, Nicholas Smith a, Amy Thomas b
a Worldwide Medicinal Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
b Discovery Biology, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
c Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
d SRG, London Office, 16 St. Helen’s Place, London EC3A 6DF, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of a series of highly potent and novel TLR7 agonist interferon inducers is described. Struc-
ture–activity relationships are presented, along with pharmacokinetic studies of a lead molecule from
this series of N9-pyridylmethyl-8-oxo-3-deazapurine analogues. A rationale for the very high potency
observed is offered. An investigation of the clearance mechanism of this class of compounds in rat was
carried out, resulting in aldehyde oxidase mediated oxidation being identified as a key component of
the high clearance observed. A possible solution to this problem is discussed.
Received 7 June 2011
Revised 21 July 2011
Accepted 21 July 2011
Available online 4 August 2011
Keywords:
TLR7 agonists
HCV
Ó 2011 Elsevier Ltd. All rights reserved.
Deazapurine
Aldehyde oxidase
Pyridine oxidation
The World Health Organisation estimates that about 200 mil-
lion people, 3% of the world’s population, are infected with hepati-
tis C virus (HCV) and three to four million persons are newly
infected each year with a global 170 million chronic carriers at risk
of developing liver cirrhosis and/or liver cancer.1 A combination of
weekly subcutaneous injections of long acting pegylated interferon
(peg-IFN) and oral ribavirin represents the current standard of care
for treatment of HCV.2 Therapy with IFN is associated with signif-
icant side effects including fatigue, headache, myalgia, fever and
nausea.3 Ribavirin also causes haemolytic anaemia in 10–20% of
patients. Furthermore, the administration of IFN by intramuscular
or subcutaneous injection causes pain and irritation at the site of
injection. Therefore, the development of an oral IFN inducer, which
enhances the release of endogenous IFN by oral administration,
could be an effective alternative treatment regimen for HCV
patients.
have been identified that induce IFN, including the guanosine ana-
logue isatoribine,6 the mixed TLR7/8 agonists Imiquimod7 and Res-
iquimod,8 and the TLR7 specific 8-oxo adenine SM-3603209 (Fig. 1).
In a preceding communication,10 we detailed our research ef-
forts to identify new therapeutic agents for the treatment of
HCV. In particular we reported the discovery of compound 1, the
prototype deazapurine lead candidate compound from our TLR7
agonist programme.11
NH2
C6
H
N
N7
C8
O
N
1
EC50 269nM
N
N9
F3C C2
Sol. 1µg/mL
Toll-like receptors (TLRs) are a family of pathogen recognition
receptors that mediate the innate immune response.4 To date,
there have been 11 different TLR genes identified in humans and
their stimulation shown to cause the release of multiple cytokines,
including types 1 and 2 interferons.5 Among these, TLR7 is of par-
ticular interest because small molecule ligands for this receptor
While compound 1 possesses good potency, has been shown to be
highly selective for TLR7 over other TLRs, and possesses many other
desirable properties for oral delivery, it suffers from poor solubility,
leading to difficulty in finding an appropriate formulation for oral
dosing. This manuscript describes our attempts to identify alterna-
tive compounds in this series, with improved potency and solubility
compared to compound 1.
⇑
Corresponding author. Tel.: +44 1304 643577.
E-mail address: peter.jones2@pfizer.com (P. Jones).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.076

5940
P. Jones et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5939–5943
Table 1
O
NH2
NH2
NH2
TLR7 potency (EC50, nM)a for N9-pyridinylmethyl analogues
H
N
S
N
HN
H2N
HO
N
N
N
N
N
N
N
O
O
N
NH2
N
O
O
O
N
N
N
N
O
F
F
HO
HO
OH
O
R
F
Isatoribine
Imiquimod
Resiquimod
SM-360320
Compound
R1
R
Figure 1. Structure of known TLR7 agonists.
8
9
10
Following a general strategy to improve solubility of analogues
based on lead compound 1, we prepared a group of substituted iso-
meric N9-pyridinylmethyl compounds 8A–F, 9A–F and 10A–F (Ta-
ble 1) to investigate structure–activity relationships. Test
compounds were prepared by the general methodology described
in Scheme 1.
R1
N
N
R1
N
R1
O
N
A
B
121
125
81.9
6.9
The preparation of the requisite starting material for this route,
benzylaminopyridine 2, was described in our previous publica-
tion.12 2 was debenzylated in quantitative yield using concentrated
sulphuric acid to provide the amino pyridine 3, which was then
converted to the corresponding carbamate 4 by reaction with ethyl
chloroformate. Alkylation of 4 with one of three isomeric chloropy-
ridinylmethyl bromides under basic conditions provided interme-
diates 5 in good yield. Selective displacement of the chloride at
C6, which is activated by the adjacent nitro group, was carried
out in excellent yield using concentrated ammonia, providing com-
pound 6. Selective reduction of the nitro group, leaving the chloro-
pyridine intact, gave the corresponding amine. This underwent
subsequent intramolecular attack on the carbamate group to give
bicyclic product 7. Target molecules were prepared through dis-
placement of the remaining chloro group by heating with alcohols
in the presence of potassium tert-butoxide, or with neat amines,
providing ethers and amines, 8A–F, 9A–F and 10A–F (Table 1).
From this initial set of analogues, a clear potency trend
emerged. While reasonable potency could be obtained with both
O- and N-linked substituents of the pyridine ring, compounds in
rows A and B, the greatest potency in both cases resided in the
2,5 isomer, compound 10. This trend continued and became more
marked when using cyclic amino substituents, compounds in rows
C–F. In general, potencies for the 2,3 isomers, compounds in col-
umn 8, were significantly weaker (>586 nM EC50) than the other
H
N
105
28.6
2.7
O
O
N
C
586
47.0
N
O
N
D
1780
2610
45.8
55.4
4.1
9.0
N
N
E
F
N
N
N
920
2.3
1.7
N
a
Values are means of at least two experiments; the TLR7 agonist potency assay is
described in reference 13.
isomers. Good potency could be obtained in general from the 2,4
isomers, compounds in column 9, (ꢀ50 nM EC50), while excellent
levels of potency were obtained in general from the 2,5 isomers,
compounds in column 10, (<10 nM EC50).
These initial observations prompted us to explore the 2,5 iso-
meric series more widely. Using the same synthetic sequence
Cl
Cl
Cl
Cl
NO2
NO2
NO2
N
N
NO2
NH2
N
N
(b)
F
N
(c)
F
(a)
F
F
F
F
F
NH
F
N
Cl
F
4
F
2
F
3
O
O
5
O
F
N
O
(d)
NH2
H
N
N
O
O
F
F
NH2
N
NH2
H
N
NO2
F
(f)
N
N
OR
O
F
F
F
(e)
NH2
F
N
N
N
O
H
N
Cl
N
F
F
N
O
F
(g)
Cl
F
7
N
N
6
R2
N
F
N
R1
8,9,10 A-F
Scheme 1. Reagents and conditions: (a) CH2SO4, rt, 100%; (b) EtO2CCl, NEt3, 2-MeTHF, rt, 80%; (c) 3 or 4 or 5-(bromomethyl)-2-chloropyridine, K2CO3, NaI, acetone, rt, 75–
81%; (d) cNH3, 2-MeTHF, rt, 90–95%; (e) (i) Na2S2O4, EtOH, H2O, 70 °C; (ii) cHCl, 70 °C, 80–83%; (f) ROH, KOtBu, 130 °C, 50–80%; (g) HNR1R2, 130 °C.

P. Jones et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5939–5943
5941
Table 2
Selected data for N9-2,5-pyridinylmethyl analogues
NH2
N
N
O
F
N
F
F
N
R
TLR7 EC50 (nM)a
MW
396
log D (c log P)
HLM RLM (
l
l/min/mg)
PAMPA (Â10À6 cm/s)
11
12
26.9
6.6
1.2 (2.8)
22 (24)
1.3
0.1
N
N
O
395
422
0.7 (2.5)
1.2 (3.4)
9 (16)
10 (9)
N
H
13
14
6.9
1.7
4.7
0.1
N
N
O
421
0.8 (3.2)
8.5 (<8.5)
N
H
N
15
16
17
6.6
2.7
4.1
409
407
451
1.2 (2.8)
1.8 (2.4)
2.3 (2.6)
16 (11)
0.9
12.0
9.9
N
N
30 (46)
17 (9)
N
N
N
N
O
N
18
1.5
464
0.9 (2.8)
<8 (<8.5)
0.1
N
19
20
21
22
N
N
N
N
1.6
1.0
0.4
0.2
449
437
464
435
1.8 (4.1)
1.3 (3.85)
1.5 (1.9)
1.3 (3.7)
25 (13)
24 (11)
25 (15)
13 (16)
5.2
2.5
0.7
0.3
N
N
N
N
N
H
HLM, human liver microsomes; RLM, rat liver microsomes; PAMPA, parallel artificial membrane permeability assay (passive transcellular diffusion rate).
a
Values are means of at least two experiments; the TLR7 agonist potency assay is described in reference 5.
outlined in Scheme 1, further analogues were prepared in this ser-
ies. In an effort to prepare compounds with improved solubility rel-
ative to compound 1, substituents with polar atoms and/or basic
centres were appended to the pyridine ring of these analogues. Fur-
ther properties were also explored with the view to achieving an
appropriately balanced compound for oral delivery. Potency data
along with molecular weight, lipophilicity assessment (measured
log D, c log P), metabolic stability assessment (HLM, RLM) and an
assessment of membrane permeability (PAMPA) are presented in
Table 2.
Initial analogues were made to examine the effect of the linker
atom. Comparing, for example, compound 11 with 12 and com-
pound 13 with 14, it became evident that N-linked substituents
were in general more potent than the corresponding O-linked ana-
logues. Exploring further analogues within the N-linked series, it
was discovered that both secondary and tertiary amines main-
tained excellent potency, compounds 14 and 15, as well as tertiary
cyclic amines, compound 16. The addition of polar groups or basic
centres was also tolerated, leading to analogues with excellent po-
tency. This provided a means to easily influence the overall proper-
ties of the molecules with regard to lipophilicity, metabolic
stability and membrane permeation, for example, compounds 17
and 18. Further analogues were prepared with substituents con-
taining basic centres (compounds 19–22), leading to the discovery
of compounds with exquisite potency, the most potent of which
was compound 22, with an EC50 of 0.2 nM.
It has previously been shown that TLR7 receptors are localised
inside endosomal compartments within immune cells, and that
correct endosomal maturation/acidification must occur before
activation of TLR7 can occur.14 It is therefore interesting to note

5942
P. Jones et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5939–5943
Table 3
Rat pharmacokinetics (I.V., 0.2 mg/kg)
Compound
16
Cl (ml/min/kg)
PPB (%)
162.0
80
Clu (ml/min/kg)
AUC (ng h/ml)
Vdss (l/Kg)
810
21
6.4
T1/2 (h)
1.1
Cl, clearance; PPB, rat plasma protein binding; Clu,
unbound clearance; AUC, area under curve; Vdss, vol-
ume of distribution at steady state; T1/2, half life.
basic centre, relative to very similar compounds without a base,
for example compounds 10A and 10B, 23 and 12, and 25 and 16.
A correlation of TLR7 potency against calculated basic pKa15
(cpKa) for all compounds presented in this Letter is shown in Figure
2. It is clear that simply incorporating a basic group is not sufficient
to generate highly potent compounds, since the basic compounds
from the 2,3-pyridyl series are weak (all EC50 >100 nM). However,
within the more potent 2,5-pyridyl series of analogues, there is a
clear correlation. The analysis suggests that incorporation of an
amino group with a cpKa >8 within this particular isomeric series
will lead to a much greater likelihood of finding excellent potency
(EC50 <10 nM). This strategy could represent a general design prin-
ciple when considering TLR7 as a target. Whether this observation
holds for other potent series of TLR7 agonists remains to be seen.
Considering Lipinski’s rules,16 from the set of compounds exem-
plified in Table 2, all had a molecular weight below 500 as well as
reasonable log D values, while numerous examples were highly
potent (EC50 <5 nM). Of these, a number were metabolically stable
Figure 2. TLR7 potency versus calculated basic pKa.
(e.g., compound 14, HLM/RLM <8.5
moderate metabolic turnover (e.g., compound 16, HLM/RLM = 30/
46 l/min/mg). In order to assess the impact of the N9 substituent
on aqueous solubility, two compounds were prepared and re-crys-
tallised to a highly crystalline solid form (assessed through powder
X-ray diffraction). The measured thermodynamic solubility of
ll/min/mg), while others had
that the exquisite potency observed for TLR7 activation occurs
with a set of compounds containing basic centres. Localisation of
such compounds into the acidic environment of the endosome,
where they would exist predominantly in their protonated state,
would potentially lead to a higher concentration of such com-
pounds within this cellular compartment. This could explain the
improved potencies observed for those compounds containing a
l
these solid samples at physiological pH, compound 12 (483
lg/
ml) and compound 16 (317 g/ml), was greatly improved relative
l
to compound 1 (1 lg/ml).
Table 4
Aldehyde oxidase data for N9-2,5-pyridinylmethyl analogues
NH2
N
N
N
O
F
F
F
N
R
TLR7 EC50 (nM)
121
MW
368
log D (c log P)
HLM RLM (
l
l/min/mg)
Rat cytosol T1/2 (min)
OH
N
H
23
24
1.6 (1.6)
1.8 (3.8)
<8 (<8.5)
196
N
H
31
414
395
394
407
<8 (16)
9 (16)
250
370
588
625
N
12
25
16
6.6
32.1
2.7
0.7 (2.5)
2.5 (1.9)
1.8 (2.4)
N
H
N
34 (24)
30 (46)
O
N
N

P. Jones et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5939–5943
5943
Given the higher PAMPA value of compound 16, (12.0), relative
to that for compound 12 (0.1), we reasoned that the former had a
better chance of good oral absorption. We chose to investigate the
in vivo pharmacokinetics of this compound first in rat, the results
of which are shown in Table 3.
Following intravenous administration to the rat, compound 16
demonstrated a mean elimination half-life of 1.1 h due to a blood
clearance of 162 ml/min/kg and a volume of distribution of 6.4 l/
kg. The very high in vivo clearance rate in rat (>liver blood flow)
was surprising given the moderate level of in vitro clearance in
and reasonable all round properties for good oral exposure. Based
on our work, a design principle to identify exquisite potency within
a series of TLR7 agonists is suggested. Unfortunately these com-
pounds were substrates for aldehyde oxidase which led to very
high systemic clearance in rat. A strategy to modulate the AO lia-
bility of these compounds is suggested and our efforts and even-
tual solution towards finding a stable compound with good oral
exposure are detailed in another recent communication.18
References and notes
RLM (46 ll/min/mg). This indicated that an additional nonRLM
mediated clearance mechanism was contributing to the overall
clearance of this compound.
1. http://www.who.int/vaccine_research/diseases/hepatitis_c/en/.
2. Ghany, M. G.; Strader, D. B.; Thomas, D. L.; Seeff, L. B. Hepatology 2009, 49, 1335.
3. Wang, Q. M.; Heinz, B. A. Prog. Drug Res. 2000, 55, 1.
We suspected that an additional complementary clearance
pathway could be through metabolism of the pyridine ring by alde-
hyde oxidase (AO).17 AO oxidises the sp2 carbon atom adjacent to
the aromatic nitrogen atom of aza-heterocyclic systems. Thus, this
set of N9-pyridyl analogues are all potential substrates for this
metabolic pathway. This was confirmed by testing the stability of
compound 16 in isolated rat cytosol. We determined that com-
pound 16 was turned over in vitro in a rat cytosol preparation with
a half life of approximately 625 min. In the same assay in the pres-
ence of Raloxifene, a specific AO inhibitor, the compound was com-
pletely stable. This was an indication of instability with respect to
AO for this compound, although the significance of the magnitude
of this effect in vitro is unclear.
4. (a) Janeway, C. A.; Medzhitoov, R. Annu. Rev. Immun. 2002, 20, 197; (b) Beutler,
B. Nature 2004, 430, 257.
5. O’Neill, L. A. Science 2004, 303, 1481.
6. (a) Lee, J.; Chuang, T.; 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; (b) 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 2005, 42, 724.
7. Tran, H.; Moreno, G.; Shumack, S. Exp. Opin. Pharmacother. 2004, 5, 427.
8. Jurk, M.; Heil, F.; Vollmer, J.; Schetter, C.; Krieg, A. M.; Wagner, H.; Lipford, G.;
Bauer, S. Nat. Immunol. 2002, 3, 499.
9. Kurimoto, A.; Tobe, T.; Ichii, S.; Isobe, Y.; Tobe, M.; Ogita, H.; Takaku, H.; Sajiki,
H.; Hirota, K.; Kawakami, H. Bioorg. Med. Chem. 2004, 12, 1091.
10. Pryde, D. C.; Tran, T.-D.; Jones, P.; Parsons, G. C.; Bish, G.; Adam, F. M.; Smith, M.
C.; Middleton, D. S.; Smith, N. N.; Calo, F.; Hay, D.; Paradowski, M.; Proctor, K. J.
W.; Parkinson, T.; Laxton, C.; Fox, D. N. A.; Horscroft, N. J.; Giuseppe, C.; Jones,
H. M.; Duckworth, J.; Benson, N.; Harrison, A.; Webster, R. Med. Chem. Commun.
2011, 2, 185.
11. The potency discrepancy for compound 1 between this article and reference 10
is due to the use of different assay formats. In this article, a co-culture assay in
a 384 well plate format was used as described in reference 13, compared to a
96 well plates format used in reference 10.
12. Adam, F. A.; Bish, G.; Calo, F.; Carr, C. L.; Castro, N.; Hay, D.; Hodgson, P. B.;
Jones, P.; Knight, C. J.; Paradowski, M.; Parsons, G. C.; Proctor, K. J. W.; Pryde, D.
C.; Rota, F.; Smith, M. C.; Smith, N.; Tran, T.-D. Org. Process Res. Dev. 2011, 15,
788.
13. Thomas, A.; Laxton, C.; Rodman, J.; Myangar, N.; Horscroft, N.; Parkinson, T.
Antimicrob. Agents Chemother. 2007, 51, 2969.
14. Heil, F.; Ahmad-nejad, P.; Hemmi, H.; Hochrein, H.; Ampenberger, F.; Gellert,
T.; Dietrich, H.; Lipford, G.; Takeda, K.; Akira, S.; Wagner, H.; Bauer, S. Eur. J.
Immunol. 2003, 33, 2987.
15. Calculated pKa performed using ACD pKa predictor from ACD Labs (http://
www.acdlabs.com/).
16. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
1997, 23, 3.
17. Pryde, D. C.; Dalvie, D.; Hu, Q.; Jones, P.; Obach, S.; Tran, T.-D. J. Med. Chem.
2010, 53, 8441.
In order to determine whether we could find analogues that
were stable to AO within this set of compounds, a subset was
screened in the rat cytosol assay, the results of which are illus-
trated in Table 4. All analogues that were screened showed an in-
creased liability for AO metabolism relative to compound 16. This
was indicated by a shorter half life in rat cytosol for these com-
pounds, and was irrespective of the measured log D or RLM values.
Although within this set of compounds we were unable to find an
example that was stable to AO, it was interesting to note that the
relative stability to AO could be modulated with varying substitu-
tion around the pyridine ring. Even though in each case the funda-
mental structural requirement for AO oxidation was present, the
stability could be easily modified with a remote substituent. This
indicates that remote changes to a molecule could be pursued as
a general tactic to find AO stable compounds within a susceptible
series.
18. Tran, T.-D.; Pryde, D. C.; Jones, P.; Adam, F. M.; Benson, N.; Bish, G.; Calo, F.;
Ciaramella, G.; Dixon, R.; Duckworth, J.; Fox, D. N. A.; Hay, D.; Hitchin, J.;
Horscroft, N.; Howard, M.; Gardner, I.; Jones, H. M.; Laxton, C.; Parkinson, T.;
Parsons, G.; Proctor, K.; Smith, M. C.; Smith, N. N.; Thomas, A. Bioorg. Med.
Chem. Lett. 2011, 21, 2389.
In summary, a number of highly potent TLR7 agonists for the
treatment of HCV were discovered. These possessed greatly im-
proved solubility relative to our lead compound in the program