Discovery of selective 2,4-diaminoquinazoline toll-like receptor 7 (TLR 7) agonists

Article abstract of DOI:10.1016/j.bmcl.2018.01.014

The discovery of a novel series of highly potent quinazoline TLR 7/8 agonists is described. The synthesis and structure–activity relationship is presented. Structural requirements and optimization of this series toward TLR 7 selectivity afforded the potent agonist 48. Pharmacokinetic and pharmacodynamic studies highlighted 48 as an orally available endogenous interferon (IFN-α) inducer in mice.

Full text of DOI:10.1016/j.bmcl.2018.01.014

Accepted Manuscript
Discovery of Selective 2,4-Diaminoquinazoline Toll-like Receptor 7 (TLR 7)
Agonists
Serge Pieters, David McGowan, Florence Herschke, Frederik Pauwels, Bart
Stoops, Stefaan Last, Werner Embrechts, Annick Scholliers, Wendy Mostmans,
Kris Van Dijck, Bertrand Van Schoubroeck, Tine Thoné, Dorien De Pooter,
Gregory Fanning, Mari Luz Rosauro, Mourad Daoubi Khamlichi, Yannis
Houpis, Eric Arnoult, Tim H.M. Jonckers, Pierre Raboisson
PII:
S0960-894X(18)30022-2
https://doi.org/10.1016/j.bmcl.2018.01.014
BMCL 25540
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 November 2017
8 January 2018
11 January 2018
Please cite this article as: Pieters, S., McGowan, D., Herschke, F., Pauwels, F., Stoops, B., Last, S., Embrechts, W.,
Scholliers, A., Mostmans, W., Van Dijck, K., Van Schoubroeck, B., Thoné, T., De Pooter, D., Fanning, G., Rosauro,
M.L., Khamlichi, M.D., Houpis, Y., Arnoult, E., Jonckers, T.H.M., Raboisson, P., Discovery of Selective 2,4-
Diaminoquinazoline Toll-like Receptor 7 (TLR 7) Agonists, Bioorganic & Medicinal Chemistry Letters (2018),
doi: https://doi.org/10.1016/j.bmcl.2018.01.014
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Discovery of Selective 2,4-Diaminoquinazoline
Toll-like Receptor 7 (TLR 7) Agonists
Serge Pieters,a* David McGowan,aFlorence Herschke,aFrederik Pauwels,aBart Stoops,aStefaan Last,aWerner
Embrechts,aAnnick Scholliers,aWendy Mostmans,aKris Van Dijck,aBertrand Van Schoubroeck,aTine
Thoné,aDorien De Pooter,aGregory Fanning,aMari Luz Rosauro,c Mourad Daoubi Khamlichi,c Yannis
Houpis,aEric Arnoult,bTim H. M. Jonckers,aPierre Raboissona
aJanssen Infectious Diseases Diagnostics BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium
bJanssen Research & Development L.L.C., 1400 McKean Rd, Spring House, PA 19454
cVillapharma Research S.L., Parque Tecnológico de Fuente Álamo. Ctra. El Estrecho-Lobosillo, Km. 2.5- Av. Azul 30320 Fuente
Álamo de Murcia, Murcia, Spain
Key words. HBV, TLR7, Quinazoline
Abstract. The discovery of a novel series of highly potent quinazoline TLR 7/8 agonists is described. The
synthesis and structure–activity relationship is presented. Structural requirements and optimization of this series
toward TLR 7 selectivity afforded the potent agonist 48. Pharmacokinetic and pharmacodynamic studies
highlighted 48 as an orally available endogenous interferon (IFN-) inducer in mice.
Toll-like receptors (TLR) are expressed on multiple cell types and detect characteristic components common to
many pathogens, allowing the host to recognize the first signs of attack by an exogenous organism. Activation
of these plasma membrane or endosomal receptors leads to NF-B/IRF/AP-1 signaling and mobilizes defense
mechanisms aimed at eliminating the invading pathogens.1

There are 10 TLR paralogues identified to date in humans.2 Activation of TLRs regulate the expression of
inflammatory cytokines/chemokines with or without type I interferons (IFN-/), which can lead to the
preferential enhancement of innate anti-microbial responses and antigen-specific humoral and cell-mediated
immune responses. TLR 7 and 8, expressed in the membranes of endosomes in the cells, recognize single
stranded viral RNA and their agonists induce a TH1-type immune response.2-4 In humans, TLR 7 is mainly
expressed in plasmacytoid dendritic cells (pDC), inducing their activation and production of IFN-, and to a
lower extent on B cells, inducing their proliferation and secretion of antibodies. Conversely, TLR 8 is mainly
expressed in monocytes and myeloid DCs (mDCs), inducing a strong TH1 profile.5-7 Therefore, TLR 7/8 agonists
have been pursued for the treatment of viral infections, tumors, asthma, and as vaccine adjuvants.8-9 This report
focuses on the effort towards a small-molecule, orally available TLR7 selective agonist with a high first pass
effect, which limit the cytokine levels in the plasma, and result in an immune response that could be beneficial
in the treatment of the above described ailments.
Several chemical series of TLR 7 agonists that induce endogenous IFN- have been described in the literature
(Figure 1). Among them are the imidazoquinoline R837 (1),10 an 8-oxopurine series (e.g. 2),11-12 and the
pteridinone class of agonists represented by GS-9620 (3).13
Figure 1. Described TLR 7/8 agonists.
In this manuscript, we report on our efforts leading to a new series of TLR 7 selective agonists which are
derived from compound (4) which originates from our previous work on dual TLR 7/8 agonists,14a and is
structurally different from our lead TLR 7 selective series.14b While agonists of the TLR 8 receptor may provide
advantageous TH1 response, there may be disadvantageous consequences of proinflammatory cytokine
induction.6 Conceptually, the core structure of 4 was altered with the aim of establishing a new scaffold that
would allow us to progress toward TLR 7 selectivity by empirical structure optimization. A fused benzene ring
across the 5,6-bond of the pyrimidine of 4 led to a novel series of 2,4-diaminoquinazolines (Figure 2). Literature

search indicated that 2,4-diaminoquinazolines were described as immunomodulators in antiallergy therapy,
however their TLR activity was not disclosed.15 2-Aminoquinolines and pyrido[2,3-d]pyrimidines have also
been described as TLR 8 selective agonists.16,17
Figure 2. New series of quinazoline TLR 7/8 agonists.
We considered the quinazoline class as interesting given the advantage that each position of the fused benzene
ring can be explored by well described chemistry. Diaminoquinazolines have been described as drug-like
molecules such as HSP-90 inhibitors,18 inhibitors of -catenin/Tcf-4,19 and as anti-HIV agents.20
The strategy toward TLR 7 selectivity began by examining amine substitution on the unsubstituted scaffold
(Table 1). 2-Amino-3H-quinazolin-4-one (5, Scheme 1) was coupled with a small set of amines via BOP 21 in
anhydrous DMF in the presence of DBU to afford compounds 5a-h.
Scheme 1. Synthesis of compounds 5a-h: (i) DBU, BOP, RNH2, DMF, rt, 16h.
Table 1
SAR of the amines on the unsubstituted scaffold.
Human TLR 7 Human TLR 8 hPBMC
Entry
R1
(LEC M)
(LEC M)
(LEC M)
n-butyl
methyl
ethyl
n-propyl
n-butyl
4.7
18.7
3.07
1.07
1.1
6.87
0.77
0.93
1.43
8.88
2.27
0.67
4
5a
5b
5c
5d
0.17
0.07
0.03
0.06
0.10
0.04
5e
n-pentyl
5f
>25
2.11
1.50
0.71
0.46
0.17
0.01
0.10
0.04
5g
5h
All compounds CC50 > 25M. See supplementary information for assay conditions.

Assessment of TLR agonism was determined in a HEK-293 cell based assay transiently transfected with either
TLR 7 or TLR 8. A second assay was employed where compound was incubated with conditioned media from
human peripheral blood mononuclear cells (hPBMCs), then the supernatant transferred to assess anti-HCV
activity in the replicon system, a confirmation of IFN- induction (see supporting information). It was found
that n-butylamine analog 5d was over 15 times more potent than its congener 4 of the pyrimidine series. Among
the selected amines, n-butylamine (5d) and n-pentylamine (5e) analogs were most potent on TLR 7 and TLR 8,
while smaller alkyl chains showed considerably less agonist potential (5a, 5b, 5c). Branching the terminus of
the carbon chain with a methyl group as in 5f, or at the α-carbon (5g), resulted in increased TLR 8 selectivity. A
similar observation was made when the chain contained an ether as in 5h.
It has been previously described that any change to the 2-amino group leads to loss of activity,13,14 therefore this
group was held constant. Next, the substitution pattern of the fused benzene ring wasassessed. While the n-
pentylamine analog (5e) gave the highest potency (Table 1), it was decided to keep the n-butylamine constant
for facile comparison with our previous series,14 as well as other published work,13, 22 where the n-butylamine,
and n-butoxy analogues retained high activities. The synthesis began with commercially available anthranilic
esters or acids which were heated in acidic conditions in the presence of excess cyanamide, using an alcoholic
solvent or diglyme, analogous to described methods to afford 2-amino-4-hydroxyquinazolines 7a-l (Scheme 2,
Table 2).23
Table 2.
2-amino-4-hydroxyquinazolines
Entry
R2
R3
R4
R5
F
H
OCH3
CO2CH3
H
H
H
H
H
H
H
H
H
Cl
CH3
OCH3
Br
H
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
F
F
OCH3
Br
H
H
H
H
OCH3
F
Scheme 2. (i) HCl, NH2CN, EtOH, 100°C, 16h
7j
7k
7l
H

Scheme 3. Synthesis of compounds 8-27: (i) DBU, BOP, n-butylamine, DMF, rt, 16h; (ii) LAH, THF, -78°C to 0°C, 2h; (iii) 1.
LiOH(aq), CH3OH, THF, rt; 2. PO(OEt)2CN, amine, NEt3, DMF, rt, 2h; (iv) BH3-S(CH3)2, THF, 80°C, 3h; (v) Ac2O, reflux, 1.5h (vi)
Pd(OAc)2, dppp, KOAc, THF, CH3OH, 30 bar CO, THF, 120°C, 16h; (vii) LAH, THF, -78°C to 0°C, 2h; (viii) 1. Zn(CN)2, Pd(PPh3)4,
DMF, microwave, 160ºC, 10min; 2. NaOCH3, CH3OH, 60°C, 1h; (ix) 1. tributyl(1-ethoxyvinyl)tin, PdCl2(PPh3)2, DMF, 80ºC, 16 h; 2.
HCl (1N), rt, 2h; 3. NaOCH3, CH3OH, 60°C, 1h; (x) NaBH4, CH3OH, rt, 2h; (xi) 1. (Het)arylacetylene, Pd(PPh3)2Cl2, P(Ph)3, CuI,
Et2NH, DMF, 80°C, 16 h; 2. NaOCH3, CH3OH, 60°C, 1h; 3. Pd/C 10%, H2, THF, rt.
Table 3.
SAR of the scaffold substitution.
Human TLR7 Human TLR8
hPBMC
(LEC M)
0.03
Entry
R2
R3
R4
(LEC M)
0.17
(LEC M)
0.07
5d
8
9
H
F
H
H
H
H
H
H
H
H
0.2
0.3
0.07
0.15
0.05
OCH3
CH2OH
0.9
0.3
11
0.1
0.1

12
H
H
0.4
2.0
0.15
13
14
H
H
H
H
0.1
0.2
0.5
0.06
0.04
0.04
15
16
20
21
22
23
H
H
H
H
H
H
H
H
H
H
H
H
F
0.3
5.6
0.9
>25
6.1
12
0.1
1.8
0.2
7.1
1.9
5.8
0.11
2.44
0.14
13.2
2.39
8.49
OCH3
-CH2OH
-CN
-(CO)CH3
-C(OH)CH3
24
H
H
H
0.2
0.5
0.15
H
0.1
0.3
0.04
25
H
H
F
F
1.8
10
0.1
1.1
0.26
2.05
26
27
OCH3
OCH3
b
All compounds CC50 > 18 M. See supplementary information for assay conditions. LEC values were averaged when determined in two or
more independent experiments.
The 2-amino-4-hydroxyquinazolines (7a-l) (Scheme 2, Table 2) were coupled with n-butylamine via BOP 21 in
anhydrous DMF in the presence of DBU to afford the initial agonists. Table 3 displays the substitution on three
positions of the scaffold and further derivatives were made using the aryl bromide or hydroxyl products as
intermediates toward new agonist products (vide infra). N4-butyl-8-fluoroquinazoline-2,4-diamine (8) was found
to have dual activity on TLR 7 and TLR 8, and in the same range as 5d. At the 7-position of the quinazoline
(Table 3, R3), methoxy (9), or hydroxymethyl (11) substituted analogs, which were made by reduction of the
corresponding ester (10) via LAH, provided no TLR 7 selectivity. Hydrolysis of ester 10, followed by reaction
with ammonia or dimethylamine, under standard coupling reaction conditions afforded amides 12 and 13
respectively, which showed a slight increase in TLR 7 selectivity. Selectivity was found to be reversed for the
basic amine 14, made by reduction of 13 with borane dimethyl sulfide, where five-fold selectivity for TLR 8
was observed. The fluorine substituted analog 15 (Table 3, R4), showed potency comparable to the unsubstituted
5d and 8. To improve the yield for Pd catalyzed reactions, toward compounds 19-25, intermediate 17 was first
acylated by refluxing acetic anhydride for 1.5 hours. The congener with the methoxy group (16), and alcohol
(20), made by reduction of ester 19 via LAH in THF, showed more potency and selectivity for TLR 8. When a
cyano group (21) was introduced by Pd catalyzed cyanation with Zn(CN)2 in DMF via the aryl bromide 18,
activity was greatly reduced. The ketone derivative 22, derived from 18, via Stille reaction with tributyl(1-

ethoxyvinyl)tin in DMF at 80°C, resulted in an overall reduction in activity. Reduction of the ketone agonist 22
with NaBH4 in methanol led to the corresponding alcohol derivative 23, which further decreased potency on
both TLR 7 and TLR 8. Derivatives 24 and 25 were built via 18, and the corresponding alkynes, formed in the
initial step under standard Sonogashira reaction conditions, followed by catalytic hydrogenation to afford the
saturated analogs 24 and 25. These analogs displayed high potency but unfortunately no TLR 7 selectivity.
Finally, disubstituted analogs 26 and 27 (Table 3) showed selectivity for TLR 8 where the difluoro substitution
(26) was observed to have greater agonist potential compared to the dimethoxy analog (27).

Scheme 4. Synthesis of compounds 28-41: (i) DBU, BOP, n-butylamine, DMF, rt, 16h; (ii) Pd(dppf)Cl2, cyclopentylmethyl-
trifluorborate, Cs2CO3, water, THF, microwave, 180°C, 1h; (iii) phenylacetylene, Pd(PPh3)2Cl2, P(Ph)3, CuI, Et2NH, DMF, 80°C, 16h;
(iv) Pd/C 10%, H2, THF, rt; (v) pyridine HCl, pyridine, 120°C, 16h; (vi) alkylbromide, Cs2CO3, DMF, rt, 16h.
Table 4.
SAR of the substituent variations on R5
hTLR7
(LEC M)
hTLR8
(LEC M)
hPBMC
(LEC M)
CC50 
M)
Metabolic
R5
Entry
stabilitya(m,h)
28
29
30
Cl
CH3
OCH3
1
0.5
0.4
0.1
0.3
0.1
0.17
0.32
0.10
>25
>25
>25
88
100 96
100 98
99
32
0.12
0.13
>25
0.09
0.27
>25
>25
nd
97
nd
24
33
0.47
0.1
0.2
0.12
>25
98
59
35
R6
R7
R8
X Y
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
OCH3
C
C
C
N
C
N
C
C
C
C
N
C
0.16
0.27
0.03
0.49
0.03
0.03
1.66
>25
>25
18.5
0.53
>25
0.04
0.33
0.018
0.043
0.011
0.034
10
>25
16
>25
>25
16
97
98
98
99
99
98
11
48
43
60
44
37
36
37
38
39
40
41
CH2OH
H
H
H
OCH3
See supplementary information for assay conditions. nd;not determined. aPercent metabolized after 15 min. at 1M in liver microsomes.
Investigation into the structure−activity relationship of the substituent variation (R5) positioned ipsilateral to the
n-butylamine explored the effect on TLR 7 selectivity and the synthesis is described in Scheme 4. Smaller
groups such as chlorine (28), methyl (29), or methoxy (30) showed selectivity for TLR 8, whereas the larger
cyclopentylmethyl analog 32, made by reacting the aryl bromide intermediate 31 with cyclopentylmethyl
trifluoroborate under modified Suzuki conditions,24 was potent and selective for TLR 7 (SI > 200 over TLR 8)
(Table 4). Phenethyl derivative 33, obtained by reaction between the bromoquinazoline 7j and phenylacetylene
under standard Sonogashira conditions, followed by reduction of the alkyne via catalytic hydrogenation,
showed reduced selectivity. The 2-methoxyethoxy derivative 35 was generated by demethylation of 30 via
pyridine hydrochloride, followed by alkylation of the resulting phenolic oxygen of 34 with 1-bromo-2-

methoxyethane in DMF and Cs2CO3 as a base. Compounds 32 and 33 implied that TLR 7 selectivity could be
achieved with a distal, sterically hindered moiety, placed one or two atoms away from the quinazoline scaffold
and adjacent to the butylamine. In view of those results, a small subset of benzyl ethers was prepared
(compounds 36-41). Benzylether analog 36, synthesized in the same manner as 35, showed TLR 7 selectivity
(SI = 10 over TLR 8) but unfortunately toxicity in the HEK cell line (HEK293 CC50 = 10 M). Potency and
selectivity could be further enhanced with the 2-methoxybenzyl group (37), hinting at a possible influence of
the rotation around this bond, (SI = 90 over TLR 8). To further build on this trend toward TLR 7 selectivity, the
(2-methoxy-4-(hydroxymethyl) benzyl congener 38 led to a further increase in potency and concomitant
increase in SI > 200 over TLR 8. The pyridine analogs (39, 40) demonstrated that TLR 7 selectivity varied with
the position of the nitrogen on the aromatic ring, and were overall less selective. Addition of methoxy groups to
the pyridine ring, as in 41, resulted in an enormous boost in TLR 7 selectivity compared to 39. The activity and
selectivity profile of the benzyl ethers 36-41 was attractive, however inhibition of certain CYP450 isozymes
(e.g. compound 36: CYP450 1A2 IC50 = 0.3 M, CYP450 2D6 IC50 = 5.7 M) was observed. This was less of a
concern since our target product profile consisted of a TLR 7 selective agonist with a high first pass effect.
Moreover, literature data suggests that dosing regimens less than once daily may be sufficient.25 Despite this,
further optimization was desired. Exploration in moieties at the carbon adjacent to the n-butylamine persisted
with the design of carboxylic amides, where variable amines could be installed, allowing the one to two bond
distance from the quinazoline scaffold to target optimal activity and TLR 7 selectivity.

Scheme 5. Synthesis of compounds 42-50: (i) amine, Pd(OAc)2, dppp, KOAc, DIPEA, 50 bar CO, THF, 100°C, 16h; (ii) DBU, BOP,
n-butylamine, DMF, rt, 16h.
Table 5
SAR of the amide variations
hTLR8 hPBMC
hTLR7
Metabolic
(LEC
(LEC
Entry
Amines
M)
M)
stabilitya(m,h)
(LEC M)
0.35
0.39
0.15
90
23
42

43
44
45
46
2.05
5.62
0.06
0.53
15.55
>25
1.6
0.56
1.92
0.03
0.27
45
nd
92
99
14
nd
28
98
>25
47
48
2.29
0.09
>25
>25
>25
75
68
32
31
0.14
0.37
0.28
>25
>25
0.5
96
99
97
99
49
50
0.14
All compounds CC50 > 25 M. nd;not determined. aPercent metabolized after 15 min. at 1M in liver microsomes.
The carboxylic amide variations were constructed via palladium catalyzed aminocarbonylation in an autoclave
by reacting bromoquinazoline 31 with excess amine, and 50 bar CO(g) in THF, as depicted in Scheme 5.
Strategically, many amines are commercially available with a range of size, morphology and basicity to rapidly
scan for TLR 7 selectivity in parallel. The carboxylic amide variations demonstrated that the methyl amide (42),
showed potent dual agonist potential, but the more sterically hindered isopropylamide analog 43 decreased
activity. The N-methyl, N-isopropyl analog 44, essentially blocking the amide hydrogen bond donor,
demonstrated a further loss in potency. Benzylamide 45 showed intriguing potency but meager TLR 7
selectivity. The N-methyl, N-benzylamide analog 46 as compared with congener 45, again implied that the
hydrogen bond donor is important for TLR 7 potency. Relocating the methyl group to the alpha-position
revealed enantiospecific activity (47 vs. 48) where the (S)--methylbenzyl isomer 48 was clearly superior in

activity (TLR 7 LEC = 90 nM) to the (R)-enantiomer 47 and more selective (SI > 200 over TLR 8). In contrast
with the previous subseries of benzyl ethers (36-41), installation of a methoxy group in this amide subseries
afforded agonists of lower potency (49 and 50) with respect to 48. Generally, all modifications afforded
metabolically less stable compounds, suitable for the targeted high clearance profile. 48 was 31% metabolized
in human liver microsomes compared to 98% for the close analog 46. To validate these interesting findings,
pharmacokinetic studies in rat were conducted, where the dual agonist 5d was compared with the TLR 7
selective agonists 46, and the more puissant and metabolically stable 48. Compound 48 displayed no CYP
inhibition up to 10 M, while 46 possessed some degree of inhibition (e.g. CYP450 3A4 IC50 = 8.5 M,
CYP450 2D6 IC50 = 5.6 M). Subsequently, pharmacokinetic and pharmacodynamic evaluation in mice are
reported for agonists 5d and 48.
Table 6
Rat Pharmacokinetic Parameters.
Tmax,
plasma (h)
Metabolic
stabilitya
L/P*
ratio
Cmax
(ng/mL or g)
AUC0−last
(ng·h/mL or g)
Entry
%F
Plasma
Portal
Plasma
Systemic
Plasma
Portal
Plasma
Systemic
Liver
Liver
h
r
0.5
0.5
4
583
209
276
33
9
2030
369
1520
621
56
37
6450
1064
5.4
1.2
20
95
99
54
90
98
31
>100
30
5d
46
48
87
4930
902
439
15841
36
*L/P Based on AUC(0-last), aPercent metabolized after 15 min. at 1M in liver microsomes.
A pharmacokinetic study in healthy male Sprague-Dawley rats investigated the exposure of 5d, 46, and 48 in
the systemic circulation following intravenous administration of 2.5 mg/kg and in the portal vein, liver and
systemic circulation following an oral administration of 10 mg/kg. High plasma clearance of 5d and 46 resulted
in very short half-life and low exposure in the systemic circulation, where clearance of 5d (Cl = 163
mL/min/kg) exceeded rat liver blood flow (LBF), and 46 (Cl = 55 mL/min/kg) was 79% LBF, resulting in a
very short terminal half-life and low systemic concentrations. The -methylbenzyl analog 48 showed higher
Cmax and AUC values in the plasma given the same oral dose. 48 showed higher Cmax (4930 ng/g) and AUC (15,
841 ng.h/g) in the liver, based on the comparison of the oral data of 46 and 48. The increased systemic exposure
and exposure observed before in the liver correlates with improved in vitro metabolic stability in rat liver
microsomes for compound 48.

Table 7.
Mouse Pharmacokinetic and Pharmacodynamic Parameters.
Entry
Dose*
Tmax
Cmax
AUC0−last
mIFN-α
Plasma
Liver
Plasma
Liver
Plasma Fmax Liver Fmax
IFN-
Tmax (h)
Plasma (h)
(ng/mL) (ng/mg) (ng.h/mL) (ng.h/g)
(pg/ml)
(pg/mg)
1
5
0.5
1
3
142
207
3227
3
365
133
6619
315
2800
200
325
1
4
5d
48
*oral, mg/kg
A mouse in vivo model was used to demonstrate the initial proof of concept to induce endogenous IFN-.
Single oral administration of 1 mg/kg dose of 5d and 5 mg/kg dose of the TLR 7 selective and potent 48 were
given to healthy C57BL/6 mice. Both compounds were found to be rapidly absorbed given the Tmax between 0.5
and 1 h and 5d was found to be rapidly cleared, resulting in a Cmax of 3 ng/mL, and a negligible exposure (AUC
= 3 ng.h/mL) in the plasma. Levels of mIFN-αpeaked at 1h for 5d and four hours post administration for 48, in
both the plasma and in the liver. 48 showed high levels of mIFN-αsystemically, low systemic and relatively
higher liver exposure in mice (liver to plasma ratio = 23), confirming the findings in rat.
Tyr356
Val381
Gln354
Phe351
Phe349
Phe408
Leu557
Thr586
Asp555
Figure 3. 48, in stick representation, docked into monkey-TLR 7. Monkey-TLR 7 and its dimerization partner
are colored in cyan and green, respectively. Hydrogen bonds are shown as yellow, dashed lines.
48 was docked in the Resiquimod binding site identified in the monkey TLR 7 co-crystal structure (PDB
5GMH).26 This in silico experiment was accomplished using the Glide docking software.27 The docking poses
generated by the protocol were ranked using the GBVI/WSA G scoring function available in MOE.28 The best

ranked pose of compound 48 was imported, with the monkey TLR 7 protein structure, into a Pymol session to
create the Figure 3, which reveals a similar binding mode as described for Resiquimod.26,29
The binding of 48 is governed by a network of hydrogen bonds involving the 2-amino group and the nitrogen in
the 3-position of the quinazoline scaffold together with the residues Asp555 and Thr586. Our docking study
confirmed that the n-buylamine moiety fits in the hydrophobic pocket formed by residues Phe349, Phe351 and
Val381. The quinazoline scaffold has π-stacking interaction with Phe408 and Leu557, and CH-πinteractions
with the side chains of those residues. The (S)-1-phenethylamine moiety bulges outside of the binding pocket
and is conceivably stabilized by amide-πinteractions with the backbone between Gln354 and Ile355. An attempt
was made to dock 48 into the human TLR 8 structure (PDB 3W3N), yet none of the docking poses obtained
showed the expected binding mode nor the expected interactions, especially the n-buylamine moiety that did not
fit in the hydrophobic sub-pocket formed by Phe349, Phe351, and Val381. This behavior in the docking
simulation could explain the poor potency of compound 48 on hTLR 8, which cannot fit properly in the
Resiquimod binding site.
In summary, we have described the synthesis and the structure-activity relationship of a series of orally
available aminoquinazoline immunomodulators. The pathway toward TLR 7 selectivity was described to
include bulky groups ipsilateral to the butylamine group and culminated to the potent and selective (S)-2-amino-
4-(butylamino)-N-(1-phenylethyl)quinazoline-5-carboxamide 48. Agonist 48 was over 250-fold selective for
TLR 7, compared to the reported 30-fold TLR7 selectivity of 3.30 The exceptional potency of 48 on TLR7
rivaled that of 2, and was superior to 1.31,32 Low exposure was observed during in vivo pharmacokinetic studies
in rat and mice after oral administration which is in line with the target profile and expected display low
exposure in human despite the slightly higher stability in human liver microsomes. Pharmacodynamic study in
mice highlighted 48 as a strong IFN- inducer after relatively low oral dose. These findings warrant further
investigation into the properties of TLR 7 selective immunomodulator 48.
Acknowledgements
The authors would like to thank the analytical teams of Janssen and Villapharma.

Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:....
References and notes
1. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity.
Nat. Immunol. 2001;2:675-680.
2. Pasare C, Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Microbes Infect.
2004;6:1382-1387.
3. Diebold S, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-
mediated recognition of single-stranded RNA. Science 2004;303:1529-1531.
4. Lund JM, Alewxopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA. Recognition
of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. U. S. A. 2004;101:5598-5603.
5. Bekeredjian-Ding I, Roth SI, Gilles S, Giese T, Ablasser A, Hornung V, Endres S, Hartmann GT. T cell-
independent, TLR-induced IL-12p70 production in primary human monocytes. J. Immunol. 2006;176:7438-
7446.
6. Gorden KB, Gorski KS, Gibson SJ, Kedl RM, Kieper WC, Qiu X, Tomai MA, Alkan SS, Vasilakos JP.
Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J. Immunol.
2005;174:1259-1268.
7. Xu S, Koldovsky U, Xu M, Wang D, Fitzpatrick E, Son G, Koski G, Czerniecki BJ. High-avidity antitumor
T-cell generation by toll receptor 8–primed, myeloid- derived dendritic cells is mediated by IL-12
production. Surgery 2006;140:170-178.
8. Hood JD, Warshakoon HJ, Kimbrell MR, Shukla NM, Malladi SS, Wang X, David SA. Immunoprofiling
toll-like receptor ligands: Comparison of immunostimulatory and proinflammatory profiles in ex vivo
human blood models. Human vaccines 2010;6:322-335.
9. Hotz C, Bourquin C. Systemic cancer immunotherapy with Toll-like receptor 7 agonists. Oncolmmunology
2012;1:227-228.

10. Weterings JJ, Khan S, van der Heden van Noort G, Melief CJM, Overkleeft HS,van der Burg SH,
Ossendorp F, van der Marel GA, Filippov DV. 2-Azidoalkoxy-7-hydro-8-oxoadenine derivatives as TLR7
agonists inducing dendritic cell maturation. Bioorg. Med. Chem. Lett. 2009;19:2249-2251.
11. Isobe Y, Kurimoto A, Tobe M, Hashimoto K, Nakamura T, Norimura K, Ogita H, Takaku H. Synthesis and
biological evaluation of novel 9-substituted-8-hydroxyadenine derivatives as potent interferon inducers. J.
Med. Chem. 2006;49:2088-2095.
12. Kurimoto A, Ogino T, Ichii S, Isobe Y, Tobe M, Ogita H, Takaku H, Sajiki H, Hirota K, Kawakami H.
Synthesis and evaluation of 2-substituted 8-hydroxyadenines as potent interferon inducers with improved
oral bioavailabilities. Bioorg. Med. Chem. Lett, 2004;12:1091-1099.
13. Roethle PA, McFadden RM, Yang H, Hrvatin P, Hui H, Graupe M, Gallagher B, Chao J, Hesselgesser J,
Duatschek P, Zheng J, Lu B, Tumas DB, Perry J, Halcomb RL. Identification and optimization of
pteridinone Toll-like receptor 7 (TLR7) agonists for the oral treatment of viral hepatitis. J. Med. Chem.
2013;56:7324-7333.
14. (a) McGowan DC, Herschke F, Pauwels F, Stoops B, Last S, Pieters S, Scholliers A, Thoné T, Van
Schoubroeck B, De Pooter D, Mostmans W, Khamlichi MD, Embrechts W, Dhuyvetter D, Smyej I, Arnoult
E, Demin S, Borghys H, Fanning G, Vlach J, Raboisson P. Novel pyrimidine Toll-like receptor 7 and 8 dual
agonists to treat hepatitis B virus. J. Med. Chem. 2016;59:7936-7945. (b) McGowan DC, Herschke F,
Pauwels F, Stoops B, Smyej I, Last S, Pieters S, Embrechts W, Khamlichi MD, Thoné T, Van Schoubroeck
B, Mostmans W, Wuyts D, Verstappen D, Scholliers A, De Pooter D, Dhuyvetter D, Borghys H, Tuefferd
M, Arnoult E, Hong J, Fanning G, Bollekens J, Urmaliya V, Teisman A, Horton H, Jonckers T, Raboisson
P. Identification and optimization of pyrrolo[3,2‑d]pyrimidine Toll-like receptor 7 (TLR7) selective
agonists for the treatment of hepatitis B. J. Med. Chem. 2017;60:6137-6151. DOI
10.1021/acs.jmedchem.7b00365
15. Antoku F, Iwai K, Tanaka H, Fujita K, Kawakami H. JP 2000053653A, 2000
16. Kokatla HP, Sil D, Tanji H, Ohto U, Malladi SS, Fox LM, Shimizu T, David SA. Structure-based design of
novel human Toll-like receptor 8 agonists. ChemMedChem. 2014;9:719-723.

17. (a) Kazushi F, Fujio A, Norio F, Kiyotaka I, Hiroshi T, Hajime K. JP 2000063381 A1, 2000. (b)
Aktoudianakis E, Chin G, Mackman RL, Metobo SE, Mish, MR, Pyun H-J, Zablocki J. WO 2016
141092 A1
18. Thorat DA, Doddareddya MR, Seo SH, Hong TJ, Cho YS, Hahn JS, Pa AN. Synthesis and biological
evaluation of 2,4-diaminoquinazoline derivatives as novel heat shock protein 90 inhibitors. Bioorg. Med.
Chem. Lett. 2011;21:1593-1597.
19. Chen Z, Venkatesan AM, Dehnhardt CM, Santos OD, Santos ED, Ayral-Kaloustian S, Chen L, Geng Y,
Arndt KT, Lucas J, Chaudhary I, Mansour TS. 2,4-Diamino-quinazolines as inhibitors of beta-catenin/Tcf-4
pathway: potential treatment for colorectal cancer. Bioorg. Med. Chem. Lett. 2009;19:4980-4983.
20. Yan SJ, Zheng H, Huang C, Yan YY, Lin J. Synthesis of highly functionalized 2,4-diaminoquinazolines as
anticancer and anti-HIV agents. Bioorg. Med. Chem. Lett. 2010;20:4432-4435.
21. Wan Z, Wacharasindhu S, Binnun E, Mansour T. An efficient direct amination of cyclic amides and cyclic
ureas. Org. Lett. 2006;8:2425-2428.
22. Hirota K, Kazaoka K, Niimoto I, Kumihara H, Sajiki H, Isobe Y, Takaku H, Tobe M, Ogita H, Ogino T,
Ichii S, Kurimoto A, Kawakami H. Discovery of 8-hydroxyadenines as a novel type of interferon inducer. J.
Med. Chem. 2002;45:5419-5422.
23. Dempcy R, Skibo E. Regioselective synthesis of imidazo[ 4,5-g]quinazoline quinone nucleosides and
quinazoline amino nucleosides. Studies of their xanthine oxidase and purine nucleoside phosphorylase
substrate activity. J. Org. Chem. 1991;56:776-785.
24. Molander GA, Pfeiffer D. Organolanthanide-catalyzed cyclization/boration of 1,5- and 1,6-Dienes.
Org.Lett. 2001;3:361-363.
25. Lanford RE, Guerra B, Chavez D, Giavedoni L, Hodara VL, Brasky KM, Fosdick A, Frey CR, Zheng J,
Wolfgang G, Halcomb RL, Tumas DB. GS-9620, an oral agonist of Toll-like receptor-7, induces prolonged
suppression of hepatitis B virus in chronically infected chimpanzees. Gastroenterology 2013;144:1508-
1517.

26. Zhang Z, Ohto U, Shibata T, Krayukhina E, Taoka M, Yamauchi Y, Tanji H, Isobe T, Uchiyama S, Miyake
K, Shimizu T. Structural analysis reveals that Toll-like receptor 7 is a dual receptor for guanosine and
single-stranded RNA. Immunity 2016;45:737-748.
27. Small-Molecule Drug Discovery Suite 2016-3: Glide, version 6.9, Schrödinger, LLC, New York, NY, 2015.
28. (a) Corbeil CR, Williams CI, Labute PJ. Variability in docking success rates due to dataset preparation.
Comput. Aided Mol. Des. 2012;6:775-786. (b) Molecular Operating Environment (MOE) 2016.0802;
Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7,
2017
29. The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.
30. Fosdick A, Zheng J, Pflanz S, Frey CR, Hesselgesser J, Halcomb RL, Wolfgang G, Tumas DB.
Pharmacokinetic and pharmacodynamic properties of GS-9620, a novel Toll-like receptor 7 agonist,
demonstrate interferon-stimulated gene induction without detectable serum interferon at low oral doses. J.
Pharmacol. Exp. Ther. 2014;348:96-105.
31. Kurimoto A, Hashimoto K, Nakamura T, Norimura K, Ogita H, Takaku H, Bonnert R, McInally T, Wada H,
Isobe Y. Synthesis and biological evaluation of 8-oxoadenine derivatives as toll-like receptor 7 agonists
introducing the antedrug concept. J. Med. Chem. 2010;53:2964-2972.
32. Gibson SJ, Lindh JM, Riter TR, Gleason RM, Rogers LM, Fuller AE, Oesterich JL, Keith B, Gorden KB,
Qiu X, McKane SW, Noelle RJ, Miller RL, Kedl RM, Bocarsly PF, Tomai MA, Vasilakosa JP.
Plasmacytoid dendritic cells produce cytokines and maturein response to the TLR7 agonists, imiquimod and
resiquimod. Cell. Immunol. 2002;218:74–86.
33. Experimental procedure for the synthesis of 48 Step 1. A 75mL stainless steel autoclave was charged under
N2 atmosphere with 7j (1 g, 4.12 mmol), (S)-(-)-1-phenylethylamine (2.52 g, 20.8 mmol), Pd(OAc)2 (9.4 mg,
0.042 mmol), 1,3 bis(diphenylphosphino)propane (34.4 mg, 0.083 mmol), potassium acetate (818 mg, 8.33
mmol) in THF (15 mL). The autoclave was closed and pressurized to 50 bar CO and the reaction was heated
for 16 hours at 100°C. The mixture was cooled and the precipitates were removed by filtration and the
filtrate was concentrated to dryness. The residue was partitioned between CH2Cl2 and water (set to pH=5
with acetic acid). The organic layer was dried over MgSO4, the solids were removed by filtration and

concentrated to dryness to obtain 2-amino-4-hydroxy-N-[(1S)-1-phenylethyl]quinazoline-5-carboxamide
(789 mg, 61.4% yield). The intermediate was used without further purification in the next step.
Step 2. A 50mL vial was charged with the intermediate (598 mg, 1.94 mmol), anhydrous DMF (15 mL),
DBU (1.0 g, 2.68 mmol), and BOP (1.12 g, 2.53 mmol). The mixture stirred at room temperature for 2h, n-
butylamine (1.34 mL, 13.57 mmol) was added and the reaction stirred at room temperature for 15h. The
mixture was concentrated in vacuo. A purification was performed via Prep HPLC (Stationary phase: RP
XBridge Prep C18 OBD-10µm,30x150mm, Mobile phase: 0.25% NH4HCO3 solution in water, CH3OH), the
desired fractions were collected, evaporated to obtain 48 (93 mg, 10% yield) as a solid.
1H NMR (400 MHz, DMSO-d6) δppm 0.83 (br t, J=7.0 Hz, 3 H), 1.16 - 1.31 (m, 4 H), 1.46 (d, J=7.0 Hz, 3
H), 3.08 - 3.21 (m, 1 H), 3.25 - 3.33 (m, 1 H), 5.17 (quin, J=7.3 Hz, 1 H), 6.10 (s, 2 H), 7.01 (dd, J=7.0, 1.3
Hz, 1 H), 7.23 - 7.29 (m, 1 H), 7.31 (dd, J=8.5, 1.2 Hz, 1 H), 7.33 - 7.43 (m, 4 H), 7.49 (dd, J=8.4, 7.0 Hz, 1
H), 7.54 (t, J=4.7 Hz, 1 H), 9.41 (d, J=7.9 Hz, 1 H).
34. Assessment of TLR7 and TLR8 activity. The ability of compounds to activate human TLR7 and/or TLR8
was assessed in a cellular reporter assay using HEK293 cells transiently transfected with a TLR7 or TLR8
expression vector and NFB-luc reporter construct. In one instance, the TLR expression construct expresses
the respective wild type sequence or a mutant sequence comprising a deletion in the second leucine-rich
repeat (dlRR2) of the TLR. Such mutant TLR proteins have previously been shown to be more susceptible
to agonist activation (US 7498409). Briefly, HEK293 cells were grown in culture medium (DMEM
supplemented with 10% FCS and 2 mM glutamine). For transfection of cells in 10 cm dishes, cells were
detached with Trypsin-EDTA, transfected with a mix of CMV-TLR7 or TLR8 plasmid (750 ng), NFB-luc
plasmid (375 ng) and a transfection reagent and incubated overnight at 37°C in a humidified 5% CO2
atmosphere. Transfected cells were then detached with Trypsin-EDTA, washed in PBS and resuspended in
medium to a density of 1.67 x 105 cells/mL. Thirty microliters of cells were then dispensed into each well in
384-well plates, where 10 µL of compound in 4% DMSO was already present. Following 6 hours incubation
at 37°C, 5% CO2, the luciferase activity was determined by adding 15 µL of Steady Lite Plus substrate
(Perkin Elmer) to each well and readout performed on a ViewLux ultraHTS microplate imager (Perkin

Elmer). Dose response curves were generated from measurements performed in quadruplicates. Lowest
effective concentrations (LEC) values, defined as the concentration that induces an effect which is at least
two-fold above the standard deviation of the assay, were determined for each compound. The Z’-factor of
the assay is 0.51 and the %CV of the LEC is between 14 and 50%. Compound toxicity was determined in
parallel using a similar dilution series of compound with 30 µL per well of cells transfected with the CMV-
TLR7 construct alone (1.67 x 105 cells/mL), in 384-well plates. Cell viability was measured after 6 hours
incubation at 37°C, 5% CO2 by adding 15 µL of ATP lite (Perkin Elmer) per well and reading on a ViewLux
ultraHTS microplate imager. Data was reported as CC50.
35. Suppression of HCV replicon replication. Activation of human TLR7 results in robust production of
interferon by plasmacytoid dendritic cells present in human blood. The potential of compounds to induce
interferon was evaluated by looking at the antiviral activity in the HCV replicon system upon incubation
with conditioned media from peripheral blood mononuclear cells (PBMC). The HCV replicon assay is
based on a bicistronic expression construct, as described by Lohmann et al. (Science (1999) 285: 110-113;
Journal of Virology (2003) 77: 3007-15 3019) with modifications described by Krieger et al. (Journal of
Virology (2001) 75: 4614-4624). The assay utilized the stably transfected cell line Huh-7 luc/neo harboring
an RNA encoding a bicistronic expression construct comprising the wild type NS3-NS5B regions of HCV
type 1b translated from an Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus (EMCV),
preceded by a reporter gene (Firefly-luciferase) and a selectable marker gene (neoR, neomycine
phosphotransferase). The construct is flanked by 5’ and 3’ NTRs (non-translated regions) from HCV type
1b. Continued culture of the replicon cells in the presence of G418 (neoR) is dependent on the replication of
the HCV RNA. The stably transfected replicon cells that replicate HCV RNA autonomously and to high
levels, encoding inter alia luciferase, were used for profiling of the conditioned cell culture media.
Briefly, PBMCs were prepared from buffy coats of at least two donors using a standard Ficoll centrifugation
protocol. Isolated PBMCs were resuspended in RPMI medium supplemented with 10% human AB serum
and 2 x 105 cells/well were dispensed into 384-well plates containing compounds (70 µL total volume).
After overnight incubation, 10 µL of supernatant was transferred to 384-well plates containing 2.2 x 103
replicon cells/well in 30 µL (plated the day before). Following 24 hours of incubation, replication was

measured by assaying luciferase activity using 40 µL/well Steady Lite Plus substrate (Perkin Elmer) and
measured with ViewLux ultraHTS microplate imager (Perkin Elmer). The inhibitory activity of each
compound on the Huh7-luc/neo cells were reported as EC50 values, defined as the compound concentration
applied to the PBMCs resulting in a 50% reduction of luciferase activity which in turn indicates the degree
of replication of the replicon RNA on transfer of a defined amount of PBMC culture medium. Recombinant
interferon α-2a (Roferon-A) was used as a standard control compound. All compounds showed CC50 of >24
M in the HEK 293 TOX assay described above.
36. Mouse in vivo study. IFN-αproduction was measured with an ELISA kit from PBL Assay Science. Levels of
interferon in plasma and in liver homogenates were assessed. For each timepoint measure, 3 mice were used
to calculate the geometric mean of the compound. Studies were performed in AAALAC-accredited sites,
and ethical approval by the corresponding ethical committee was obtained.