Dihydropyrrolo[2,3- d ]pyrimidines: Selective Toll-Like Receptor 9 Antagonists from Scaffold Morphing Efforts

Article abstract of DOI:10.1021/ml5003184

Toll-like receptors (TLRs) play important roles in the innate immune system. In fact, recognition of endogenous immune complexes containing self-nucleic acids as pathogen- or damage-associated molecular patterns contributes to certain autoimmune diseases, and inhibition of these recognition signals is expected to have therapeutic value. We identified dihydropyrrolo[2,3-d]pyrimidines as novel selective TLR9 antagonists with high aqueous solubility. A structure-activity relationship study of a known TLR9 antagonist led to the promising compound 18, which showed potent TLR9 antagonistic activity, sufficient aqueous solubility for parenteral formulation, and druggable properties. Compound 18 suppressed the production of the proinflammatory cytokine IL-6 in CpG-induced mouse model. It is therefore believed that compound 18 has great potential in the treatment of TLR9-mediated systemic uncontrollable inflammatory response like sepsis.

Full text of DOI:10.1021/ml5003184

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Letter
Dihydropyrrolo[2,3-d]pyrimidines: Selective Toll-like
Receptor 9 Antagonists from Scaffold Morphing Efforts
Manabu Watanabe, Mai Kasai, Hideyuki Tomizawa, Masakitsu
Aoki, Kazuo Eiho, Yoshiaki Isobe, and Shigehiro Asano
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml5003184 • Publication Date (Web): 02 Oct 2014
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Dihydropyrrolo[2,3-d]pyrimidines: Selective Toll-like Recep-
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†
, §
Manabu Watanabe, Mai Kasai, Hideyuki Tomizawa, Masamitsu Aoki, Kazuo Eiho, Yoshiaki Isobe, Shigehiro Asano*
†
Sumitomo Dainippon Pharma Co., Ltd., 3ꢀ1ꢀ98 Kasugade Naka, Konohanaꢀku, Osaka 554ꢀ0022, Japan
§
Sumitomo Dainippon Pharma Co., Ltd., 33ꢀ94 Enokiꢀcho, Suita, Osaka 564ꢀ0053, Japan
KEYWORDS: Toll-like receptor 9; sepsis; autoimmune disease; dihydropyrrolo[2,3-d]pyrimidine; LLE
Supporting Information Placeholder
ABSTRACT: Tollꢀlike receptors (TLRs) play important roles in the innate immune system. In fact, recognition of endogenous
immune complexes containing selfꢀnucleic acids as pathogenꢀ or damageꢀassociated molecular patterns contributes to certain autoꢀ
immune diseases, and inhibition of these recognition signals is expected to have therapeutic value. We identified dihydroꢀ
pyrrolo[2,3ꢀd]pyrimidines as novel selective TLR9 antagonists with high aqueous solubility. A structure activity relationship study
of a known TLR9 antagonist led to the promising compound 18, which showed potent TLR9 antagonistic activity, sufficient water
solubility for parenteral formulation, and druggable properties. Compound 18 suppressed the production of the proinflammatory
cytokine ILꢀ6 in CpGꢀinduced mouse model. It is therefore believed that compound 18 has great potential in the treatment of
TLR9ꢀmediated systemic uncontrollable inflammatory response like sepsis.
Tollꢀlike receptors (TLRs) are a family of type I transmembrane
receptors and the central components of the innate immune sysꢀ
Plitas and coꢀworkers reported that TLR9 knockout mice with
cecal ligation and puncture (CLP), as peritonitis model, showed
increased bacterial clearance, decreased serum cytokine producꢀ
tion, and increased granulocytes influx in the peritoneum as comꢀ
pared to wild type animals. The researchers also showed that adꢀ
ministration of an inhibitory CpG sequence to block TLR9 signals
just before CLP treatment improves mortality in the wild type
animals. These findings have provided a rationale for the pursuit
of small molecule TLR9 antagonists as potential candidates for
treatment of systemic uncontrollable inflammatory responses,
including sepsis. As part of our efforts to find new agents for the
treatment of sepsis, we focused on TLR9 selective antagonists. To
1, 2
tem. Thirteen TLRs have so far been reported as fundamental in
recognition of pathogenꢀassociated molecular patterns (PAMPs),
which are expressed by microbial pathogens, or damageꢀ
associated molecular patterns (DAMPs), which are transmitted by
3, 4
necrotic or dying cells. Among the reported TLRs, TLR1−10
have been identified in human. Human TLR3, TLR7, TLR8, and
TLR9 are expressed on endosomal membranes in the cells and5
recognize pathogenꢀderived nucleic acid molecular patterns.
However, recognition of endogenous immune complexes containꢀ
ing selfꢀnucleic acids as PAMPs or DAMPs contributes to certain
autoimmune diseases, such as systemic lupus erythematosus
1
2, 13, 14, 15
date, a number of TLR9 antagonists have been reported
16
6
7
8
(
SLE) , psoriasis , arthritis and multiple sclerosis . Therefore,
with promising candidates reaching clinical development . It was
recently reported that the hydroxychloroquine 1, a TLR9 antagoꢀ
nist classified as an antiꢀmalarial drug and also used for SLE and
rheumatoid arthritis therapy, directly blocks interaction between
inhibition of these recognition signals is expected to have theraꢀ
peutic value.
O
OH
17
N
TLR9 and CpGꢀDNA. In addition, CPGꢀ52364 (2), a small molꢀ
NH
OMe
ecule TLR7/8/9 antagonist (the reported ratio of TLR7/TLR9
N
N
NH
N
18, 22
antagonism was 0.8)
, has recently completed phase 1 clinical
N
OMe
trial for SLE therapy (NCT00547014).
N
Cl
N
Me
Although there has been tremendous progress in the developꢀ
ment of selective TLR9 antagonists as a small molecule, most
known TLR9 antagonists are reported to also inhibit TLR7. Actiꢀ
vation or inhibition of TLR7 and TLR9 is very complex as these
1; Hydroxychloroquine
2: CPG-52364
Figure 1. Structures of small molecule TLR9 antagonists
1
9, 20, 21
receptors have opposing inflammatory and regulatory roles.
TLR9 has been identified as a key receptor for innate immune
9
Therefore, compounds that inhibit the signal cascades of both
receptors may induce unwanted immunosuppression. Herein we
describe the discovery of a promising TLR9 antagonist obtained
by optimization of the known TLR9 antagonist 2. While 2 exhibꢀ
response to unmethylated CpGꢀDNA. Accordingly, a number of
TLR9 antagonists have been suggested to be potentially useful in
the treatment of diseases characterized by undesired innate imꢀ
10
mune response, such as systemic autoimmune diseases, sepsis ,
22
1
1
12
its strong TLR9 antagonistic activity , it is lipophilic and has a
Graftꢀversusꢀhost disease , and malaria infection . Recently,
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large molecular weight to be used as lead compound. Also 2 has
boronates provided compounds 10a–e, 12a–c and 14. The desired
insufficient solubility (0.12 mg/mL, pH 7.4) for parenteral formuꢀ
lation. To identify a potential lead compound, we focused on
compounds lipophilic efficiency (LLE: pIC50 － clogP) as an
index, because compounds with low clogP generally exhibit high
compounds 11a–j and 13a–c were obtained by conversion of the
4ꢀchloro group into a methyl, methoxy, dimethylamino or hydroꢀ
gen group. The substituted piperazine derivatives 17 and 18 were
synthesized from the dihydropyrrolopyrimidine 9b.
2
3, 24, 25
solubility and good physiochemical properties.
As a first step towards exploration of a lead compound, we atꢀ
tempted to identify the minimal core structure of the quinazoline
framework for inhibition of TLR9 signal. TLR9 antagonistic acꢀ
tivity was evaluated in NFꢀκB reporter assay as shown in Table 1.
This reporter assay was carried out with HEK293ꢀhuman TLR9
cells transfected with pNFꢀκBꢀLuc reporter vectors. To obtain
other bicyclic pyrimidines, we replaced the dimethoxyphenyl
moiety of 2 with a tetrahydropyran or cyclohexane ring. The obꢀ
tained fusedꢀring compounds were less lipophilic compared to the
quinazoline core structure (2: clogP 2.96 versus 5a: 0.43, 5b:
2.15). Although compound 5a showed better LLE value than 2,
we had to optimize its selectivity against TLR7 (5a, TLR7:
a
Scheme 1. Synthesis of the Pyrimidine Derivatives
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: (a) 4ꢀ(2ꢀaminoethyl)morpholine,
N,Nꢀdiisopropylethylamine or K CO , iꢀPrOH or DMF, 60 °C; (b)
2
3
Pd(PPh ) , 4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenylboronic acid pinacol
IC =50 nM). Fortunately, compound 5b showed acceptable
3
4
50
ester, 3 M NaOH, 1,4ꢀdioxane, 120−150 °C (microwave).
TLR9 antagonistic activity with good selectivity against TLR7
(
5b, TLR9/TLR7=71 fold). These findings suggested that lipoꢀ
The pyrimidine derivatives 5a–e were synthesized as shown in
Scheme 1. Displacement of the 4ꢀchloro group in 3a–e with 4ꢀ(2ꢀ
aminoethyl)morpholine afforded 4a–e. SuzukiꢀMiyaura cross
coupling reaction with the corresponding pinacol boronates under
microwave heat yielded the desired compounds 5a–e. Dihydroꢀ
pyrrolo[2,3ꢀd]pyrimidine derivatives were prepared as show in
Scheme 2. The diethyl allylmalonate 6 was treated with urea folꢀ
philic substituents at the 5ꢀ and/or 6ꢀposition of the pyrimidine
are optimal for generation of lead compounds with high TLR9
selectivity. To reduce molecular weight, the ring of tetrahydroꢀ
quinazoline 5b was simplified by removal of the cyclohexane ring
to give the dimethyl pyrimidine 5e. Compound 5e showed imꢀ
provement in LLE value with weaker selectivity for TLR7 than 5b
(
TLR7: IC =928 nM). Based on these findings, it was concluded
lowed by chlorination of 5ꢀallylbarbituric acid using POCl to
50
3
that the pyrimidine derivatives could provide the minimum core
structure for TLR9 antagonism. Next, we turned our attention to
compounds 5c and 5d, which were obtained by removal of the
methyl group at the 5ꢀ and/or 6ꢀposition of the pyrimidine ring.
Both compounds exhibited reduced TLR9 antagonistic activity,
likely due to a decrease in hydrophobic interaction and lower
a
give trichloro compound 7 in high yield. Oxidative cleavage of
the olefin using sodium periodate in presence of potassium osꢀ
mate gave the key intermediate 8. Tandem reaction via reductive
amination with various alkyl amines followed by cyclization proꢀ
duced the desired dihydropyrrolopyrimidines 9a–e. Suzukiꢀ
Miyaura cross coupling reaction with the corresponding pinacol
Scheme 2. Synthesis of Dihydropyrrolo[2,3-d]pyrimidine Derivatives
R1
N
R1
N
N
N
e
R2
N
Cl
N
N
N
d
R1
N
N
N
Me
O
Me
N
Cl
N
Cl
N
1
0a
e
11a j
EtO2C
a
b
c
N
N
CHO
N
CO2Et
Cl
Cl
Cl
Cl
Cl
N
Cl
N
O
6
7
8
9a e
N
N
f
g
N
N
R3
2a
N
Cl
R3
13a
N
1
c
c
N
N
N
O
O
O
N
N
N
N
O
N
h
N
i
N
j
N
N
N
Cl
N
N
1
7 (R4 = Me)
Cl
N
Cl
N
N
N
16 (R4 = TBS)
HN
HN
4
N
18 (R4 = H)
k
R O
9b
14
15
a
Reagents and Conditions: (a)(1) urea, NaOEt, EtOH, reflux; (2) POCl , N,Nꢀdimethylaniline, 120 °C; (b) K OsO , NaIO , acetone, H O,
3
2
4
4
2
3
rt; (c) R NH , NaBH CN, AcOH, MeOH, rt; (d) Pd(PPh ) , 4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenylboronic acid pinacol ester, Na CO , H O,
2
3
3 4
2
3
2
1
,4ꢀdioxane, 110−120 °C (microwave); (e) for 11a–d, 2 M MeZnCl or MeZnBr/THF, Pd(tꢀBu P) , THF; for 11e, NaOMe, MeOH, 120 °C
3 2
(
microwave); for 11f, 2 M Me NH/THF, Nꢀmethylpyrrolidone, 180 °C (microwave); for 11g, 11i, 11j, 10% Pd/C, H , trifluoroacetic acid,
2
2
MeOH, rt; for 11h, 10% Pd/C, HCO NH , trifluoroacetic acid, MeOH, 50 °C; (f) Pd(PPh ) , substituted phenylboronic acid pinacol ester,
2
4
3 4
3
M Na CO , 1,4ꢀdioxane, 110−120 °C (microwave); (g) for 13a, lithium aluminum hydride, THF, reflux; for 13b, (1) 10% Pd/C,
2 3
HCO NH , MeOH, 50 °C; (2) lithium aluminum hydride, THF, reflux; for 13c, (1) 4 M HCl/1,4ꢀdioxane, MeOH, rt; (2) 10% Pd/C,
2
4
HCO NH , MeOH, 50 °C; (3) 30% formaldehyde solution, NaBH CN, AcOH, MeOH, rt; (h)(1) Pd(PPh ) , tꢀbutyl 4ꢀ(4ꢀ(4,4,5,5ꢀ
2
4
3
3 4
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)piperazineꢀ1ꢀcarboxylate, 3 M Na CO , 1,4ꢀdioxane, 120 °C (microwave); (2) 4 M HCl/1,4ꢀ
2
3
4
dioxane, MeOH; (i) 10% Pd/C, HCO NH , MeOH, 50 °C; (j) R OCH CHO, NaBH CN, AcOH, MeOH, rt; (k) 1 M TBAF/THF, THF, rt.
2
4
2
3
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basicity of the pyrimidine nitrogen than 5e (Table S2). Therefore
order of activity was consistent with the basicity of the terminal
amino group. This finding indicated that basicity of the amino
group could be an important factor for high TLR9 antagonistic
activity (Table S3). Despite its strong antagonism of TLR9
(IC =3.5 nM), the pyrrolidine derivative 11d displayed slightly
we focused on substituted or fused pyrimidine derivatives. In
general, conformationally locked compounds with appropriate
2
6
interaction are expected to improve the activity. In order to reꢀ
duce the number of rotatable bonds in the 4ꢀamino group, we
cyclized the 5ꢀmethyl group of compound 5e with the nitrogen at
the 4ꢀposition to form the dihydropyrrolopyrimidine 11a. As exꢀ
pected, 11a showed potent TLR9 antagonistic activity with a lowꢀ
er clogP relative to compound 5e. We speculate that a fixed 4ꢀ
substituted side chain led to strong interaction with the receptor.
Furthermore, the metabolic stability of compound 11a was imꢀ
proved compared to that of 5e (human MS: 11a <0.010 vs 5e
50
lower LLE and metabolic stability than the corresponding morꢀ
pholine derivative 11b, presumably due to its higher lipophilicity
(human MS: 11d 0.035 versus 11b < 0.01 mL/min/mg protein).
Next, we evaluated the effects of a substitution at the 4ꢀposition of
11d. The chloro (10d), methoxy (11e), and dimethylamino (11f)
compounds showed significantly reduced TLR9 antagonistic acꢀ
tivity. Although the unsubstituted compound 11g exhibited strong
activity, its metabolic stability was not sufficient (human MS:
0.071 mL/min/mg protein). These results suggested that morphoꢀ
line derivatives were more favorable as drug candidates than pyrꢀ
rolidine derivatives. As 4ꢀhydogen substitution gave good result
for LLE, we replaced the 4ꢀmethyl group with a hydrogen in the
morpholine compounds 11a and 11b, which had high LLE value
and low clogP. Remarkably, compound 11i showed strong TLR9
antagonistic activity with the best LLE value among the comꢀ
pounds in Table 2. In addition, the solubility of 11i (1.1 mg/mL at
pH 7.4) was much improved compared to that of compound 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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.069 mL/min/mg protein), probably due to interrupted oxidation
of the 6ꢀmethyl group (metabolic site) by cyclization steric hinꢀ
drance. As for receptor selectivity, compound 11a showed good
selectivity against TLR7 (TLR9/TLR7=93 fold). Overall, the
dihydropyrrolo[2,3ꢀd]pyrimidine was found to be superior to
other core structures in terms of TLR9 selectivity, low lipophiliciꢀ
ty, and ligand efficiency.
Table 1. IC50 Values, LLE, and clogP of the Prepared Py-
rimidine Analogues
(0.12 mg/mL at pH 7.4), and its TLR9 selectivity remained over
5
0ꢀfold that for TLR7 (11i, IC =480 nM). Therefore, we selected
50
1
1i as lead compound for further investigation.
1
2
Table 2. Optimization of the Substituents in the R and R
Positions of the Dihydropyrrolo[2,3-d]pyrimidine
hTLR9:
IC50 (nM)
c
Comp
2
A
a
LLE
5.38
clogP
4.6
2.96
hTLR9:
1
2
c
Comp
R
R
LLE clogP
a
IC50 (nM)
b
5
5
a
190
6.30
5.42
5.16
5.49
5.86
0.43
2.15
1.56
1.43
1.60
11a
11b
Me
Me
Me
Me
Cl
20
6.26
6.16
5.47
5.77
4.14
3.77
3.65
6.01
6.00
6.72
6.18
1.43
1.64
2.48
2.69
2.93
3.13
3.48
2.40
1.14
1.35
1.77
c
16
b
b
27
1
1c
11
NH
5
c
N
190
120
35
1
1
1d
0d
3.5
N
b
84
5d
5e
b
1
1e
1f
OMe
NMe2
H
120
b
1
73
1
1g
1h
3.9
73
1
1a
20
6.26
1.43
1
H
a
b
c
Average (N=2), MEAN (N=3), Calculated by ACD/LogP.
As we had a good lead compound (11a) in our hand, we looked
c
11i
11j
H
8.6
at the SAR of the side chains. The compounds listed in Table 2
were designed and synthesized to extensively explore suitable
c
1
2
H
11
substituents at the 7ꢀposition (R ) and 4ꢀposition (R ) of the dihyꢀ
dropyrrolo[2,3ꢀd]pyrimidine core structure. Among 11a–d, the
a
b
c
d
Average (N=2). N=1. N=3. Calculated by ACD/LogP.
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2
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2
2
2
2
2
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3
3
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4
4
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5
5
5
5
5
5
5
5
5
6
3
Table 3. Optimization of the Substituents in the R Position
of the Dihydropyrrolo[2,3-d]pyrimidine
with excellent water solubility and showed remarkable efficacy in
CpGꢀinduced mouse model. Although further efforts are required
to assess the in vivo safety profile of this compound, it is believed
that compound 18 has great potential in the treatment of TLR9ꢀ
mediated systemic uncontrollable inflammatory response like
sepsis. Besides, compound 18 would be a useful reagent for studꢀ
ying the physiological roles of TLR9.
Table 4. In vitro Profile of Compound 18
hTLR9:
3
c
Comp
R
LLE
5.55
clogP
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
IC50 (nM)
IC50
nM)
Evaluation
a
(
b
1
3a
3b
56
1.71
human TLR9
human TLR7
13
b
970
b
1
56
5.99
-
1.27
1.19
1.15
0.65
b
human TLR8
>10000
74
CpG-induced IL-6 inhibition in mouse spleen
CpG-induced IL-6 inhibition in human PBMC
1
3c
>1000
28
b
244
a
b
Average (N=2). N=1.
1
7
8
6.41
7.24
1
13
a
b
c
Average (N=2). N=1. Calculated by ACD/LogP.
The strong TLR9 antagonistic activity and good aqueous soluꢀ
bility of compound 11i encouraged us to further investigate subꢀ
stitutions on the phenyl ring at the 2 position of the pyrimidine. It
is reported that the aqueous solubility of a compound can be imꢀ
proved by disruption of its molecular symmetry. Accordingly,
we decided to change the position of the piperazine group to the
meta- or ortho- position. Unfortunately, the resulting compounds
2
7
Figure 2. Compound 18 doseꢀdependent inhibition of CpGꢀ
induced ILꢀ6 production in mice. (N=4).
1
3a and 13b showed 6ꢀ to 7ꢀfold decline in the antagonistic activꢀ
ity. Compound 13c, a keto piperidine derivative, had no TLR9
antagonistic activity. These results suggested that the basic nitroꢀ
gen (an aniline group) at the para-position of the benzene ring is
crucial for interaction with TLR9. Finally, to further improve the
solubility of 11i for intravenous administration, the methyl group
on the piperazine was replaced by a methoxyethyl or hydroxyethyl
group that can act as a hydrophilic group. Among the prepared
compounds, the 2ꢀhydroxyethyl derivative 18 showed strong
TLR9 antagonistic activity with high LLE value. It is noteworthy
to mention here that compound 18 exhibited enough water soluꢀ
bility for parenteral formulation (>10 mg/mL, pH 7.4) with the
lowest lipophilicity (clogP=0.65). Next, we determined compound
18 selectivity for TLR9. Compound 18 IC50 values for inhibition
of the offꢀtarget receptors TLR7 and TLR8 were 970 nM and
ASSOCIATED CONTENT
Supporting Information: Synthetic procedures, analytical data,
and procedures for all biological assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Tel: +81ꢀ6ꢀ6337ꢀ5865. Fax: +81ꢀ6ꢀ6337ꢀ6538. Eꢀmail: shigehiꢀ
roꢀasano@dsꢀpharma.co.jp.
>
10000 nM, respectively. Compound 18 IC50 (TLR7)/IC50 (TLR9)
Author Contributions
ratio was 75 fold. In in vitro experiments, compound 18 inhibited
CpGꢀinduced ILꢀ6 production in mouse spleen and human peꢀ
ripheral blood mononuclear cells (PBMC) with IC50 values of 74
and 244 nM, respectively (Table 4). Based on these results, we
decided to evaluate the efficacy of compound 18 in vivo by measꢀ
uring CpGꢀinduced ILꢀ6 production in the peritoneal lavage fluid
(PLF) and plasma of mice. To our delight, compound 18 inhibited
The manuscript was written through all authors contributions. /
All authors have given approval to the final version of the manuꢀ
script.
ACKNOWLEDGMENT
We thank Dr. H. Toda for collecting data from MS spectra.
CpGꢀinduced ILꢀ6 production in a doseꢀdependent manner (ED50
(plasma)=0.8 mg/kg, Figure 2 and S1).
ABBREVIATIONS
In summary, we describe here the discovery of dihydroꢀ
pyrrolo[2,3ꢀd]pyrimidine derivatives as novel TLR9 antagonists.
The representative compound 18 possessed high TLR9 selectivity
TLR9, tollꢀlike receptor 9; PAMP, pathogenꢀassociated molecular
patterns; DAMP, damageꢀassociated molecular patterns; SLE,
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For Table of Contents Use Only:
Dihydropyrrolo[2,3-d]pyrimidines: Selective Toll-like Receptor 9 Antagonists from Scaffold
Morphing Efforts
Manabu Watanabe, Mai Kasai, Hideyuki Tomizawa, Masamitsu Aoki, Kazuo Eiho, Yoshiaki Isobe, Shigehiro Asano*
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