Substituted 7-amino-5-thio-thiazolo[4,5- d ]pyrimidines as potent and selective antagonists of the fractalkine receptor (CX3CR1)

Article abstract of DOI:10.1021/jm3012273

We have developed two parallel series, A and B, of CX₃CR1 antagonists for the treatment of multiple sclerosis. By modifying the substituents on the 7-amino-5-thio-thiazolo[4,5-d]pyrimidine core structure, we were able to achieve compounds with high selectivity for CX₃CR1 over the closely related CXCR2 receptor. The structure-activity relationships showed that a leucinol moiety attached to the core-structure in the 7-position together with α-methyl branched benzyl derivatives in the 5-position displayed promising affinity, and selectivity as well as physicochemical properties, as exemplified by compounds 18a and 24h. We show the preparation of the first potent and selective orally available CX₃CR1 antagonists.

Full text of DOI:10.1021/jm3012273

Article
pubs.acs.org/jmc
Substituted 7‑Amino-5-thio-thiazolo[4,5‑d]pyrimidines as Potent and
Selective Antagonists of the Fractalkine Receptor (CX3CR1)
Sofia Karlstrom,*,† Gunnar Nordvall,† Daniel Sohn,† Andreas Hettman,† Dominika Turek,†
̈
Kristofer Åhlin,† Annika Kers,† Martina Claesson,† Can Slivo,† Yvonne Lo-Alfredsson,† Carl Petersson,†
Galina Bessidskaia,† Per H. Svensson,‡ Tobias Rein,‡ Eva Jerning,† Åsa Malmberg,† Charlotte Ahlgen,†
Colin Ray,† Lauri Vares,† Vladimir Ivanov,† and Rolf Johansson*,†
†CNSP iMed Science Sodertalje, AstraZeneca Research and Development, Innovative Medicines, SE-15185 Sodertalje, Sweden
̈
̈
̈
̈
‡Pharmaceutical Development, AstraZeneca Research and Development, SE-15185 Sodertalje, Sweden
̈
̈
S
* Supporting Information
ABSTRACT: We have developed two parallel series, A and B, of
CX3CR1 antagonists for the treatment of multiple sclerosis. By
modifying the substituents on the 7-amino-5-thio-thiazolo[4,5-d]-
pyrimidine core structure, we were able to achieve compounds with
high selectivity for CX3CR1 over the closely related CXCR2 receptor.
The structure−activity relationships showed that a leucinol moiety
attached to the core-structure in the 7-position together with α-methyl
branched benzyl derivatives in the 5-position displayed promising
affinity, and selectivity as well as physicochemical properties, as exemplified by compounds 18a and 24h. We show the
preparation of the first potent and selective orally available CX3CR1 antagonists.
contribute to the chronicity of tissue damage and organ
dysfunction through phagocytosis and production of cytotoxic
species. Interestingly, corticosteroids selectively suppress the
CD14+CD16+ subpopulation of monocytes in the blood
during the treatment of multiple sclerosis (MS)10 and
inflammatory bowel disease (IBD).11
The exact mechanism of the diapedesis is complex and far
from completely understood. Key elements are the chemotactic
activity of the chemokine12 as well as the ability to strongly
adhere to the luminal wall under high shear force.13 Still, a
number of other events are required in order for cells to make
their way into the surrounding tissue.14
The CX3CR1 receptor is of interest for the role in migration
and adhesion15 and hence the inhibition of extravasation of
proinflammatory lymphocytes and leukocytes, and antagonists
of FKN induced events is potentially an attractive approach to
the treatment of inflammatory diseases.16
Chemokine receptors are G-protein coupled receptors, which
are generally regarded as druggable targets with a good
feasibility for identification of antagonists.17 A number of
antagonists of the CCR (CC chemokine receptor)18−21 and
CXC22,23 family of receptors have previously been identified
through HTS (high throughput screening) campaigns, and we
were surprised that our initial high throughput screen of 150 K
compounds did not produce any productive hit series against
the CX3CR1 receptor. We therefore turned our attention to a
more focused screening effort. By testing a small collection of
INTRODUCTION
■
Fractalkine (FKN, CX3CL1), the sole member of the CX3C
family (δ-family) of chemokines, is a 355 amino acid peptide
that activates the heptahelical CX3CR1.1 FKN is unique among
the chemokines in the respect that it is formed as a membrane
bound peptide that is subsequently cleaved by proteases such as
ADAM17 (ADAM metallopeptidase domain 17, also known as
tumor necrosis factor-α-converting enzyme (TACE)) and
ADAM102 and cathepsin S,3 leaving a pharmacologically
soluble and active peptide/chemokine. The chemokine
distribution is widespread and is also regulated by environ-
mental factors such as inflammation.4
CX3CR1 is expressed in the brain, spleen, and in
subpopulations of leukocytes such as monocytes, macrophages,
and microglia,5 cells of monocytic lineage such as osteoclasts
and dendritic cells, and neutrophils but also in lymphocytes
such as the T-cell and the NK-cell. In the lymphocyte, high
expression is found in cytotoxic cells such as the NK-cell
(natural killer cell), γδ-T cells, and CD8+ (cluster of
differentiation 8+) cells.6 In monocytes, CX3CR1 is highly
expressed in cells also having high expression of the scavenger
receptor CD16 (FcγRIII). This subset of monocytes,
CD14+CD16+ cells, which corresponds to 5−15% of the
total peripheral blood monocyte population,7 have attracted
attention because they are expanded in diverse inflammatory
diseases such as atherosclerosis, rheumatoid arthritis,8 and
infective disorders6,9 as well as in other pathologies like cancer.6
Monocytes/macrophages and lymphocytes are key players in
the pathology of inflammatory diseases. By a sustained influx
from the blood into the surrounding tissue, these cells
Received: August 29, 2012
Published: March 21, 2013
© 2013 American Chemical Society
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about 1000 compounds from other in-house projects targeting
chemokine receptors, we identified compounds 1−4 as suitable
starting points for the development of potent and selective
CX3CR1 antagonists.24
run in the presence of a reducing agent such as sodium
borohydride. In this way any formed disulfide was reduced back
to free thiol 12 that could react with the alkylating agent.
Reaction of the branched alkyl halide 17 with 12 gave the
branched thioether 18 (Scheme 4). Racemic 17 gave a mixture
of diastereomers of compound 18 that could be separated on
HPLC using a nonchiral reversed phase column or a straight
phase chiral column. Alternatively, the ketones (15) were
enantioselectively reduced by (−)-B-chlorodiisopinocampheyl-
borane (DIPCl)26 or using a method employing (R)-(+)-α,α-
diphenyl(pyrrolidin-2-yl)methanol borate complex and borane
methyl sulfide as reducing agent27 to give the (S)-alcohols 16.
Employing the opposite reagent of the chiral reagent gave R-16.
Alcohols 16 were then converted to the chlorides 17 with
mainly inversion of configuration by methods employing for
example NCS (N-chlorosuccinimide) and triphenylphosphine,
or cyanuric chloride and DMF (Scheme 4). Reaction of
enantiomerically pure or enriched 17 with the thiol 12
proceeded with inversion of configuration resulting in a net
retention of configuration from 16 to 18. High diastereomeric
purity of compounds 18 could be obtained via this sequence
combined with chromatographic purification.
The thiazolonopyrimidines were derived from the corre-
sponding compounds 11, 14, or 18 by the method shown in
Scheme 5. A Sandmeyer-type reaction converted the 2-amino
group into the 2-chlorothiazole 19 or 22. The chloride was
subsequently transformed into a methoxy-group in 20 or 23
that finally was hydrolyzed to yield compounds 21 and 24.
To allow for a later stage introduction of the 5-position
substituent, the method outlined in Scheme 6 was developed.
The thiol 12 was oxidized to the disulfide 25 under the
Sandmayer conditions employed in the conversion of the 2-
amino group into a 2-chloro substituent. The dimeric
compound was then taken through the steps previously
described for the synthesis of 24 (Scheme 5) to yield the
dimer 27. Dimer 27 was reduced back to thiol 28 by addition of
a stoichiometric amount of NaBH4. The formed thiol was then
reacted with the halide 17 in situ to give compound 24.
To unambiguously verify the configuration of the stereo-
centers in the purified diastereomers, compound 18a (Figure 1,
left) and compound 24h (Figure 1, right) were subjected to
crystallization and X-ray structure investigation. Crystals of 18a-
HCl were obtained by crystallization from MeOH/EtOAc and
the derived X-ray structure demonstrates the 5S/7R config-
CHEMISTRY
■
To find potent and CX3CR1 selective compounds, we designed
a synthetic strategy that allowed the systematic variation of the
5- and 7-substituents on the scaffolds represented by
compounds 1−4. The synthesis of the 2-aminosubstituted
analogues is outlined in Scheme 1. Commercially available
compound 5 was treated with sodium hydride and benzyl
bromide to give compound 6. Electrophilic addition of
thiocyanate followed by condensation at elevated temperatures
afforded the thiazole 8. The 7-hydroxy group was then
converted in a Vilsmeier reaction to the corresponding chloride
9 to introduce a good leaving group. The chloride was
subsequently displaced in a nucleophilic aromatic substitution
reaction to allow the introduction of a variety of aminoalcohols
10 to give compounds 11 (Table 1). By choosing an
enantiomerically pure aminoalcohol, a single enantiomer of
compound 11 was obtained without any racemization.
To obtain compound 11f, the required aminoalcohol was
prepared and coupled with compound 9 as described in
Scheme 2.
The 5-position was accessed by the removal of the S-benzyl
group in a Birch reduction25 of a compound 11 to liberate the
free thiol 12a−b, followed by a coupling of the thiol with the
appropriate alkylating agent. The easily accessible organic
chloride 13 was used to give 14 in a SN2-type reaction (Scheme
3). To prevent oxidation of the thiol 12 to the corresponding
disulfide, a process that occurs relatively fast in DMSO in air, it
was important to run the coupling reaction under an inert
atmosphere. Further improvement was seen if the reaction was
Scheme 1. Synthesis of 2-Aminosubstituted Analogues 11
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Scheme 2. Synthesis of Compound 11f
Scheme 3. Synthesis of Compounds 14
Scheme 4. Synthesis of Compounds 18
Scheme 5. Synthesis of [1,3]Thiazolo[4,5-d]pyrimidin-
2(3H)-ones 21 and 24
RESULTS AND DISCUSSION
■
Exploring the 7-Substituent. The initial hit compounds 2
and 4 containing a 2-ethyl-2-aminoalcohol were more potent
than the corresponding alaninol derivatives 1 and 3, indicating
that the lipophilicity of the amino alcohol side chain was of
importance for affinity. Thus, we decided to introduce a range
of lipophilic groups in this position while retaining the alcohol
moiety. The derived compounds were tested in a CX3CR1
receptor binding assays using [125I]-fractalkine as the radio-
ligand. The hits originated from an in-house CXCR2 antagonist
program and displayed high affinity for this receptor. Because
our aim was to develop a selective CX3CR1 antagonist, we also
included a CXCR2 receptor binding assay, using [125I]-IL8 as
the radioligand, as a selectivity assay in the screening cascade.24c
The assay results together with the selectivity for the
compounds for the CX3CR1 receptor over the CXCR2
receptor (the ratio: Ki(CX3CR1/Ki(CXCR2)) are summarized
in Table 1. By replacing the R1 methyl group in compound 1
with an ethyl (compound 2) or an isopropyl group (compound
11d), potency increased marginally, whereas larger groups such
as a n-propyl as in 11e and the isobutyl derivative 11a resulted
in a significant increase in potency. Generally, it appears that
the more lipophilic the moiety in this region is, the more potent
the compound is at the CX3CR1 receptor. Compounds
containing both enantiomers of the leucinol chain were
prepared and tested (R-11a and S-11b), which showed that
the R-enantiomer is highly preferred over the S-enantiomer for
binding to the CX3CR1 receptor. This is the same preferred
enantiomer as what has previously been reported for the
uration. Compound 24h was crystallized from MeOH as a
hemihydrate. The obtained structure also verifies the
configuration of the stereocenters.
We also made the corresponding sulfone and sulfoxide 29
and 30 from compound 21a via oxidation with oxone, either >2
equiv or 1 equiv, respectively. Compound 31 containing an
ether instead of thioether was prepared by employing the
sulfone in compound 29 as leaving group in a reaction with
benzyl alcohol as shown in Scheme 7.
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Scheme 6. Alternative Method for Synthesis of Compound 24
Figure 1. Crystal structures of compounds 18a-HCl and 24h-H2O.
Scheme 7. Synthesis of Compounds 29, 30, and 31
CXCR2-antagonists.24a,b The phenyl derivative 11c show
similar potency compared to the t-Bu derivative 11g, indicating
that also the shape and length of the R1-subtituent is of
importance for affinity. A larger R1-group not only increases
CX3CR1 affinity but also decrease the affinity for the CXCR2
receptor as can be concluded from the data in Table 1. This
observation is in accordance with what has been previously
published regarding the SAR (structure−activity relationship)
of thiazolopyrimidines targeting the CXCR2 receptor24a−c and
gives an opportunity to obtain CX3CR1 selectivity approx-
imately 10-fold over CXCR2, but we reasoned that further
optimization was needed to increase selectivity. The
thiazolonopyrimidines (series B) analogue 21a of the most
potent aminothiazolopyrimidines 11a displayed similar
CX3CR1 affinity but significantly higher CXCR2 affinity.
Exploring the 5-Substituent. After the identification of
(R)-leucinol as a potent and preferable group in the 7-position
of the 2-amino-thiazolo[4,5-d]pyrimidine core, our attention
focused on modification of the S-benzyl group in the 5-position.
We introduced a range of hydrophilic and lipophilic
substituents in the 2, 3, and 4 position of the phenyl ring
and also replaced it with the different regioisomers of pyridine
(compounds 14, Table 2). In general, lipophilic substituents in
all three positions of the phenyl ring were tolerated whereas the
more hydrophilic cyano group resulted in a 2−3-fold reduction
in potency in the 2- (compound 14e) and 3-positions
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Table 1. In Vitro Pharmacological Results of 7-Substituted Aminoalcohol Derivatives
a
b
c
compd
series
R1
absolute configuration (*)
CX3CR1
CXCR2
ratio
d
1
A
A
B
B
A
A
A
A
A
A
B
Me
Et
R
R
R
R
R
S
1100 220
770 100
29000
13
160
0.01
0.2
2
4
d
3
Me
Et
4.5
21
0.0002
0.04
11
4
560 50
6
11a
11b
11c
11d
11e
11g
21a
i-Bu
i-Bu
Ph
48
6
530
600 70
220 20
640 60
not tested
not tested
150 30
510 120
370 140
R
R
R
R
R
i-Pr
Pr
0.2
5
100
3
t-Bu
i-Bu
190 20
30 10
2
79
9
3
a
Displacement of specific [125I]-CX3CL1 ([125I]-fractalkine) binding to membrane preparations from HEK293S cells stably expressing human
b
CX3CR1, expressed as Ki SEM (nM). Displacement of specific [125I]-IL8 binding to membrane preparations from HEK293 cells stably expressing
human CXCR2, expressed as Ki SEM (nM). Ratio of Ki (CXCR2)/Ki (CX3CR1) IC50 (nM) taken from ref 24c.
c
d
increase in affinity compared to the unsubstituted compound
Table 2. In Vitro Pharmacological Results of Benzyl
Substituted Derivatives
11a. In addition, the affinity for the CXCR2 receptor was
significantly reduced for several 2-substituted compounds, thus
increasing selectivity.
Thus, it appears that a favorable lipophilic pocket exists in
the CX3CR1 receptor but not in the CXCR2 receptor and that
this pocket can be accessed from the ortho position of the
aromatic ring. However, this increase in potency and selectivity
was achieved by an increase in lipophilicity and, consequently,
low solubility remained an issue (Table 6). As could be
expected, solubility was enhanced by the introduction of a
pyridine moiety as R2 as in 14m, but CX3CR1 potency was
greatly reduced for compounds 14m−o. We hypothesized that
the lipophilic pocket in the CX3CR1 receptor could also be
reached by a substituent from the benzylic position of the linker
in the 5-position and that this alternative way of reaching this
area also could be advantageous for solubility by introducing
bulk in an aliphatic part of the molecule. We prepared a
number of compounds with methyl substitution on the benzylic
carbon with different substitution patterns on the aromatic ring
in both series of compounds, 18 and 24 (Table 3). The
branched benzyl moiety gave a further increase of CX3CR1
activity and selectivity toward CXCR2 than what could be
achieved by varying the substituent pattern on the aromatic ring
in the S-benzyl in the 5-position of the molecule (Table 2).
Interestingly, an additional increase in potency for CX3CR1 or
selectivity versus CXCR2 was not achieved for compound 18c
compared to 14a. Thus, a lipophilic 2-aryl substituent and a
branched methyl group on the linker do not show any additive
effects, indicating that they may be interchangeable and occupy
the same small lipophilic area. In series A, the preferred
stereoisomer has the (S)-configuration, which has about 4−8
fold higher potency for the CX3CR1 receptor, whereas the
CXCR2 potency is similar between the stereoisomers.
Interestingly, for compound 24a and 24b from the series B,
no preference between stereoisomers is seen for the CX3CR1
affinity but a 6-fold difference is observed in CXCR2 affinity,
leading to the (R)-isomer being the most selective. In addition,
a
b
c
compd series
R2
CX3CR1
CXCR2
ratio
14a
14b
14c
14d
14e
14f
A
A
A
A
A
A
A
A
A
A
A
A
B
A
A
A
2-Br-phenyl
2-Cl-phenyl
2-F-phenyl
4.0 2.7
51
1750 140
1790 50
220 32
435
35
10
21
35
6
23 10
70 2.0
120 10
99 27
32 10
91 53
47 36
d
2-Me-phenyl
2-CN-phenyl
2-MeO-phenyl
3-Br-phenyl
3-CN-phenyl
4-Br-phenyl
4-CN-phenyl
2-F,4-Br-phenyl
2,3-diF-phenyl
2,3-diF-phenyl
2-pyridyl
1500
4100 570
590
14g
14h
14i
190
6
1980 100
1600 130
258
22
34
0.2
70
1
14j
1200 900
8.1 0.5
71 26
14k
14l
567
79 20
4.1 1.6
64
21b
14m
14n
14o
10 3.7
0.4
0.2
8
360 10
670 440
900 130
3-pyridyl
5400 300
4-pyridyl
9000 1500
10
a
Displacement of specific [125I]-CX3CL1 ([125I]-fractalkine) binding
to membrane preparations from HEK293S cells stably expressing
b
human CX3CR1, expressed as Ki
SEM (nM). Displacement of
specific [125I]-IL8 binding to membrane preparations from HEK293
cells stably expressing human CXCR2, expressed as Ki SEM (nM).
c
d
Ratio of Ki (CXCR2)/Ki (CX3CR1). n = 1.
(compound 14h) and a 25-fold reduction in potency in the
para-position (compound 14j). Thus, the para-position appears
to be directed toward a more lipophilic region of the receptor.
The 2-position appeared to be the most promising region for
substitution. The 2-Br derivative 14a displayed a 12-fold
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Table 3. In Vitro Pharmacological Results of Methyl Substituted Derivatives
a
b
c
compd
series
R1
R2
absolute configuration (*)
CX3CR1
CXCR2
ratio
18a
18b
24a
24b
18c
18d
18e
18f
A
A
B
B
A
A
A
A
B
A
A
B
B
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
n-Pr
n-Pr
i-Bu
i-Bu
i-Bu
n-Pr
Ph
Ph
Ph
Ph
S
R
S
R
S
R
S
S
S
S
S
S
S
3.9 0.0
18 3.3
7.8 1.9
8.0 2.4
13 2.5
2800 340
2100 180
210 60
1360 30
2600 200
6000 1200
1400 530
13000 2000
920 20
>100000
720
120
30
170
200
60
2-Br-phenyl
2-Br-phenyl
102
1
2-F-phenyl
4.7 0.6
42 21
300
310
65
3-CN-phenyl
3-CN-phenyl
3-SO2Me-phenyl
4-Cl-2-pyridyl
4-Cl-2-pyridyl
4-Cl-3-pyridyl
24f
14
28
1
6
18g
18h
24h
24i
>3500
180
80
6.2 1.3
5.8 0.4
1129
489
21
5
220 110
10
a
Displacement of specific [125I]-CX3CL1 ([125I]-fractalkine) binding to membrane preparations from HEK293S cells stably expressing human
b
CX3CR1, expressed as Ki SEM (nM). Displacement of specific [125I]-IL8 binding to membrane preparations from HEK293 cells stably expressing
human CXCR2, expressed as Ki SEM (nM). Ratio of Ki (CXCR2)/Ki (CX3CR1).
c
the introduction of a methyl group in the benzylic position had
a positive effect on solubility (Table 6).
leads to retained CX3CR1 potency and an approximately 4-fold
increased selectivity toward CXCR2.
Exploring Linkers. Increasing the 5-position linker length
by one additional carbon (compound 14p) in the substituted
aminothiazolo[4,5-d]pyrimidines series gave a moderate
enhancement of CX3CR1 activity and retained selectivity
toward CXCR2 compared to 11a (Table 4). A further increase
in distance between the core and the aromatic ring gives
reduced CX3CR1-affinity (compound 14q). The oxidation of
the sulfur in 21a to the sulfone 29 and sulfoxide 30 reduced the
affinity for CX3CR1 about 4−10-fold, indicating that a polar
group in this position is not favorable. However, exchanging the
thioether in compound 21a for an ether as in compound 31
Exploring the Alcohol Group. Coupling of compound 9
with other amino acid derivatives and functional group
transformations gave rise to compounds 11f and 32−36
(Table 5). We explored the importance of the alcohol moiety in
the 7-position by varying the position and hydrogen bond
donating/accepting properties. Extending the alcohol moiety by
an additional methylene group (32) gave only a small decrease
in CX3CR1 affinity compared to compound 11a. A significant
Table 5. In Vitro Pharmacological Results of Derivatives
Modified in the Aminoalcohol Part
Table 4. In Vitro Pharmacological Results of Derivatives
with Modified Aryl Linkers
a
b
c
compd
R2
R4
CX3CR1
CXCR2
ratio
11f
32
Ph
2-Cl-
phenyl
2-Cl-
phenyl
C(CH3)2OH
(CH2)2OH
1200 300
91 15
not tested
not tested
a
b
c
compd
series
X
CX3CR1
CXCR2
ratio
14p
14q
29
A
A
B
B
B
SCH2
S(CH2)2
SO2
18 4.0
110 20
110 24
280 28
44 7.0
349
523
20
5
33
C(O)OMe
43
5
4200 500
100
100 29
460 160
580 56
0.9
1.6
13
34
35
36
Ph
Ph
Ph
C(O)OMe
C(O)NH2
C(O)OH
8.3 0.8
220
320 15
1940 200
2400 100
5900 800
230
11
30
S(O)
O
1
31
20
a
a
Displacement of specific [125I]-CX3CL1 ([125I]-fractalkine) binding
to membrane preparations from HEK293S cells stably expressing
Displacement of specific [125I]-CX3CL1 ([125I]-fractalkine) binding
to membrane preparations from HEK293S cells stably expressing
b
b
human CX3CR1, expressed as Ki
SEM (nM). Displacement of
human CX3CR1, expressed as Ki
SEM (nM). Displacement of
specific [125I]-IL8 binding to membrane preparations from HEK293
cells stably expressing human CXCR2, expressed as Ki SEM (nM).
specific [125I]-IL8 binding to membrane preparations from HEK293
cells stably expressing human CXCR2, expressed as Ki SEM (nM).
c
c
Ratio of Ki (CXCR2)/Ki (CX3CR1).
Ratio of Ki (CXCR2)/ Ki (CX3CR1).
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Table 6. Solubility, Clint in Human Microsomes, and Caco-2 Permeability of Selected Compounds
a
b
c
compd
series
X
R1
R2
R3
R4
solubility
Clint
Caco-2 A−B Papp
11a
11e
11g
14a
14b
14e
14i
14m
18a
18h
21a
24a
24h
29
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
A
A
A
S
S
S
S
S
S
S
S
S
S
S
S
S
i-Bu
n-Pr
t-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
Ph
Ph
Ph
H
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
C(O)OMe
C(O)NH2
<1
19
ND
23
ND
6
H
H
11
17
8
2-Br-phenyl
H
3
48
ND
ND
ND
ND
1.6
2-Cl-phenyl
H
<1
3
ND
ND
ND
99
2-CN-phenyl
H
4-Br-phenyl
H
<1
>100
22
2-pyridyl
H
d
Ph
Me
Me
H
34
high
4-Cl-2-pyridyl
63
81
1
Ph
1
ND
46
ND
20
Ph
Me
Me
H
34
4-Cl-2-pyridyl
79
77
>33
1
SO2
SO
O
Ph
>100
>100
79
ND
4
30
Ph
H
1
31
Ph
H
67
23
33
S
2-Cl-Ph
Ph
H
<1
10
ND
62
ND
ND
ND
35
S
H
36
S
Ph
H
C(O)OH
>100
1
a
b
c
HT solubility (μM). In vitro metabolic stability in human microsomes (μL/min/mg). Permeability Papp (apparent permeability coefficient) A−B
d
in Caco-2 cells at pH 6.5 (10−6 cm/s). A permeability value could not be reported due to low recovery of the compound in the experiments.
decrease in CX3CR1 affinity was seen for the tertiary
dimethylalcohol 11f, thus the receptor interaction is sensitive
to increasing the steric bulk in this area. The hydrogen bond
donating ability of the alcohol does not appear to be essential
for activity because compounds 33 and 34 with an ester
functionality retained the same potency as the alcohol analogue
14b. The 4−6-fold reduction in potency seen for the
carboxamide 35 and carboxylic acid 36 could be due to higher
polarity and hence higher desolvation energy penalty. Thus,
there is an apparent need for an acceptor but not for a donor
moiety, however, the replacement of the alcohol with an ester
did not improve solubility (Table 6).
metabolic stability and solubility and high Caco-2 permeability.
In series B, compound 24h was the most potent compound and
showed 80-fold selectivity vs CXCR2 and adequate in vitro
DMPK properties. These compounds displayed promising in
vitro characteristics and were selected for further character-
ization.
Pharmacokinetics in rodents indicated moderate and low
volume of distribution for series A and B, respectively, as
exemplified for compounds 18a and 24h in Table 7. Clearance
Table 7. Pharmacokinetic Parameters for Compounds 18a
and 24h in Rats
DMPK Properties. Table 6 displays solubility, metabolic
stability in human microsomes and Caco-2 permeability data
for selected compounds previously discussed in Tables 1−5.
Metabolic stability was similar in both series A and B. A trend
was observed between molecular weight and metabolic stability,
hence the benzyl substituted analogues displayed somewhat
increased metabolism in in vitro systems. Compounds with low
lipophilicity, for example, compounds 30 and 36, also showed
low metabolic turnover. Studies of metabolic pathways
indicated significant contributions of alkylchain oxidation,
dealkylation of the benzyl group, and glucuronidation of the
free hydroxyl.
The permeability in Caco-2 cells systems was generally high
for the acidic series B, whereas the neutral series A showed
moderate to poor permeability. For both series, a trend of lower
permeability with increased molecular weight was observed,
consistent with earlier reported findings.28 Compound 18a, a
representative for the series A, displayed high potency and good
selectivity versus the CXCR2 receptor together with adequate
compd CL (mL/min × kg) t1/2 iv (h) t1/2 po (h) VSS (Lkg−1
)
F (%)
18a
24h
13
1.9
5.5
2.7
4.0
1.1
0.4
39
78
1.3
was moderate and low for series A and B, respectively. These
findings are consistent with the expected differences in plasma
protein binding between the two ion classes as acids (series B)
are known to have higher plasma protein binding than neutrals
(series A).29 The oral bioavailability (F%) was acceptable for
both series, slightly higher for series B, in line with observed
Caco-2 results. For the treatment of MS, the compounds
should also preferably access the brain. Permeability results for
several of the compounds from an in vitro BBB-model showed
low to moderate permeabilities but were inconclusive.
However, as discussed below, compound 18a has nevertheless
showed promising results in an in vivo disease model for MS. It
should also be kept in mind that the BBB is disrupted in MS-
patients.30
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Series Properties. The SAR in series A and B is broadly
parallel at both the CX3CR1 and the CXCR2 receptor,
indicating that the receptor interactions of the neutral
aminothiazole (series A) and acidic 2-thiazolone (series B)
are similar despite the fact that they belong to different ion
classes. The physicochemical differences between the series are
reflected in a generally lower Caco-2 permeability for the
neutral series A, probably due to the larger number of hydrogen
bond donors/higher PSA. As demonstrated in Table 7,
examples from both series show good oral bioavailibility. For
both series, the high clogP (>3.5) of many of the compounds
presented have negative implications on properties such as
CONCLUSIONS
■
We have from a small set of HTS hits originally targeting the
CXCR2 receptor developed a number of CX3CR1 antago-
nists.31 Two series with overlapping SAR but different
physicochemical properties have been explored. We have
extensively explored three areas of the molecules. The
aminoalcohol moiety in the 7-position requires a lipophilic
region as well as a hydrogen bond acceptor. In the benzyl
linker, we discovered that a methyl substituted linker improved
selectivity as well as solubility. The aromatic ring in the 5-
position was the most tolerable position for introducing polar
groups to balance lipophilicity, solubility, and potency. We were
able to reduce the CXCR2 affinity and improve the CX3CR1
affinity of the substance classes. The structure−activity
relationships show that a leucinol moiety attached to the core
structure in the 7-position together with α-methyl branched
benzyl derivatives in the 5-position show promising affinity and
selectivity as well as physicochemical properties. Thus, we have
prepared the first potent and selective orally available CX3CR1
antagonists.
solubility (Table 6) and hERG (the human ether-a-
̀
go-go-
related gene) inhibition (data not shown).
In vitro receptor binding with [3H]18a and mechanism
studies with [125I] CX3CL1 indicate that 18a and FKN binds to
separate binding sites on the receptor and thus this compound
is an allosteric antagonist. Determination of the mechanism of
antagonism was performed using binding experiments with
increasing concentrations of [125I]CX3CL1. The results suggest
that compounds in this class inhibit the binding of [125I]-
CX3CL1 to the hCX3CR1 receptor through a noncompetitive
mechanism as the IC50 values do not increase with increasing
concentration of [125I]CX3CL1.
EXPERIMENTAL SECTION
■
Crystal Data for 18a-HCl. C19H25N5OS2·HCl, Mr = 440.0 g/mol,
monoclinic, space group P21 (no. 4), a = 13.913(1) Å, b = 9.156(1) Å,
c = 18.750(1) Å, β = 106.85(1)°, V = 2285(3) Å3, Z = 4, Dcalc = 1.279
g cm−3, μ(Mo Kα) = 3.69 cm−1 R = 0.041, wR2 = 0.074, GOF = 1.773.
The data were collected at 293K on a KappaCCD diffractometer with
graphite monochromatized Mo Kα radiation (λ = 0.71073 Å) and
employing a 0.02 mm × 0.29 mm × 0.46 mm crystal (6200 unique,
Rint = 0.03).
Compound 18a appear to be selective versus other screened
receptors including other chemokine receptors. In vitro
receptor binding studies showed 246-fold selectivity versus
hCCR1 and 187-fold versus hCCR2 and no significant
antagonism of the CCR4, CCR5, CCR6, CXCR3, and
CXCR5 receptors. Compound 18a did not show significant
interactions with the majority of the targets in a broad screen
with 65 different receptors, enzymes, or ion channels (MDS
Pharma Services), but a significant interaction (>50% activity at
10 μM) was observed for the adenosine A1 receptor and the
selectivity was later determined to be 33-fold (Ki 132 nM).
Other interactions were observed with dopamine, norepinephr-
ine, serotonin transporters, and sodium channel site 2, but
selectivities were greater than 200-fold.
Further in vitro pharmacological characterization of 18a
included inhibition of fractalkine stimulated GTPγS binding in
the low nanomolar range, potent inhibition of human
peripheral blood monocyte adhesion to a FKN-coated surface,
and potent inhibition of soluble FKN-induced monocyte
migration. In addition, FKN-induced actin polymerization was
used as an ex vivo biomarker for the functional inhibition of
CX3CR1 by 18a in whole blood. Compound 18a, in a
concentration-dependent fashion, inhibited FKN-induced actin
polymerization in monocytes from human and rat blood. In
vivo characterization of 18a was performed in the rat
experimental autoimmune encephalomyelitis (EAE) disease
model of MS. Continuous subcutaneous (sc) administration of
18a has repeatedly shown strong and dose dependent effects in
the MOG-induced EAE model. The compound has completely
reversed clinical symptoms, and the treatment effect has been
confirmed histologically through reduced spinal cord neuro-
inflammation and demyelination. The calculated effective IC50
concentrations in the EAE rats were in the range of 1.5−4 μM
(mean 2 μM). The details of the in vitro and in vivo
characterization of 18a will be presented in separate papers
currently in preparation.
Crystal Data for 24h-H2O. C18H22ClN5O2S2·1/2H2O, Mr = 458.0
g/mol, tetragonal, space group P43212 (no. 96), a = 10.3691(3) Å, b =
10.3691(3) Å, c = 39.7170(13) Å, V = 4270.2(2) Å3, Z = 8, Dcalc
=
1.397 g cm−3, μ(Mo Kα) = 4.01 cm−1, R = 0.083, wR2 = 0.184, GOF =
1.19. The data were collected at 200K on a KappaCCD diffractometer
with graphite monochromatized Mo Kα radiation (λ = 0.71073 Å) and
employing a 0.09 mm ×0.11 mm × 0.15 mm crystal (3346 unique, Rint
= 0.051).
CCDC-889652 and CCDC-889653 contains the supplementary
crystallographic data for 18a-HCl and 24h-H2O, respectively. These
data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/
cif.
Receptor Binding Assays. Material. Recombinant human
[
125I]CX3CL1 (specific activity 2200 Ci/mmol) was from PerkinElm-
er, LifeSciences Inc., Boston, MA, USA. Unlabeled recombinant
human fractalkine and human CXCL8 (IL8) was purchased from
PeproTech EC Ltd. (London, UK). Human embryonic kidney
suspension (HEK-293S) cells stably transfected with human
CX3CR1 and cotransfected with Gαqi5 (hCX3CR1/Gαqi5) were
obtained from Molecular Sciences, AstraZeneca R&D Sodertalje,
̈
̈
Sweden. HEK293-cells stably transfected with hCXCR2 were obtained
from AstraZeneca R&D Charnwood. Recombinant human [125I]-IL8
(CXCL8), (spec. activity 2200 Ci/mmol) was from PerkinElmer,
LifeSciences Inc., Boston, MA, USA. Wheatgerm Agglutinin (WGA)
SPA beads were purchased from Amersham Biosciences, Sweden.
Cell Culture and Membrane Preparation. The hCX3CR1/Gαqi5
cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
containing Glutamax and supplemented with 10% fetal bovine serum
(FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (PEST).
Selection was performed with 250 μg/mL Zeocin and 100 μg/mL
hygromycin for hCX3CR1/Gαqi5. Cells were incubated at 37 °C and
5% CO2. To avoid reduced receptor expression, the confluence was
not allowed to exceed 80%. The cells were detached with 0.05%
trypsin and 0.02% EDTA in phosphate-buffered saline (PBS). Cells
were rinsed twice with PBS, scraped, and pooled in harvesting buffer
(10 mM Tris-HCl, 5 mM EDTA, 0.1 mg/mL Bacitracin, pH 7.4),
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followed by centrifugation at 300g for 10 min (4 °C). Collected cells
were resuspended in harvesting buffer before homogenization using a
Dounce homogenizer. The homogenate was centrifuged at 48000g for
10 min (4 °C). The pellet was suspended in harvesting buffer, and
aliquots were stored at −70 °C. Protein concentration was determined
using a modified method by Lowry.32 Cell membranes were thawed,
homogenized using an Ultra-Turrax homogenizer, and diluted in the
appropriate buffer on the day of experiment.
concentration of 1% (v/v). Nonspecific binding was defined with 10
nM unlabeled peptide. The assays were incubated at room
temperature for 24 h and terminated by rapid filtration through
Whatman GF/B filters pretreated with 0.3% PEI, followed by
subsequent washing with cold buffer (10 mM Hepes, 500 mM
NaCl, pH 7.4 at 4 °C) using a TomTec harvester. Meltilex melt-on
scintillator was added and the radioactivity determined in a
scintillation counter (Wallac 1205 Betaplate). The Ki values
(inhibition constants) of the test compounds were calculated from
the observed IC50 of the test compound using the Cheng−Prusoff
equation. The Kd values used in the calculation of the Ki values were
determined in saturation binding experiments under the correspond-
ing competition assay conditions, and were 93, 13, and 19 pM for
CXCR2, CCR1, and CCR2 respectively.
Solubility Assay. The HT (high throughput) solubility screening
protocol33 used a 10 mM DMSO stock solution of the compound and
was performed in a 96-well format. A concentration of 100 μM (1%
DMSO) was incubated in 0.1 M phosphate buffer, pH 7.4, for 24 h
under gentle shaking (<500 rpm) on a plate bed shaker at room
temperature in duplicate (2 μL till 200 μL applied in a Costar PP 96-
well plate, 350 μL). The solution was filtered from undissolved
material (Multiscreen-R4, 0.4 μm Hydrophilic PTFE LCR Membrane,
Glass-Filled PP filtration plate, Millipore AB) prior to LC-UV-DAD-
MS analysis. UV quantification was performed at the most sensitive
wavelength chosen from the DAD spectrum relative to two standard
solutions, 10 μM and 100 μM in DMSO, with a detection limit of 0.01
μM. If the solubility was found to be >100 μM, it is reported as “100
μM”. Estimated variation of solubility rank value: 10−20%.
Analysis. The physical chemical properties (identity, purity,
stability, and solubility) were run simultaneously using a generic LC-
UV-DAD-MS method (Waters 2790 Alliance HPLC-996 DAD-ZQ
MS system). The chromatographic separation was performed on RP
narrow bore columns, XTerra MS C18 2.1 mm × 100 mm, 3.5 m or
equivalent, with a fast HPLC gradient in a formic acid/acetonitrile
system (5−100%) at a flow rate of 0.3 mL/min. Mass spectrometry
was performed with electrospray ionization under generic conditions
with confirmation of the molecular weight with full scan acquisition in
positive and negative ion mode. The sample preparation was
performed from 10 mM in DMSO or using solid material. The
DMSO stock solutions as well as diluted solutions were checked for
precipitation. For each sample or standard, an injection of 20 μL was
made.
Permeability Assay. Permeability in Caco-2 cells was measured in
the A−B direction at pH 6.5.34 Permeability experiments were carried
out in Hank’s Buffered Salt Solution buffered supplemented with 25
mM MES at pH 6.5 (buffer A) and Hank’s Buffered Salt Solution
buffered supplemented with 25 mM HEPES at pH 7.4 (buffer B).
Compounds were dissolved in DMSO and diluted to 10 μM in buffer
A. Three inserts with cells and one without were used for each
experiment. The experiments were carried out on filter-grown cell
monolayers that were equilibrated in transport buffer at 37 °C for 10
min in a shaking incubator. The basolateral wells contained 0.80 mL of
buffer B, and the apical wells contained 0.20 mL of buffer A. After
removal of buffer from both sides, the test compound in buffer A was
added to the donor side and fresh buffer B was added to the receiver
side. The experiments were carried out in a humidified atmosphere of
CO2/air (5%/95%) at 37 °C for 90 min. Samples were taken from the
donor side in the beginning and at the end (90 min.) of the
experiment, and samples were taken from the receiver side at 45 and
90 min. Equal volume of fresh buffer was replenished. The integrity of
the epithelial cell monolayer was monitored by measurements of
passive transmembrane diffusion of radiolabeled [14C]mannitol.
Concentrations of the compound in both donor and receiver samples
were analyzed by LC-MS. Liquid scintillation was used for analysis of
[14C]mannitol. Data is presented as means of triplicate measuments.
Mass balances calculations were based on total recovery of parent
molecule at the end of the experiment. All incubations contained less
than 1% total DMSO.
HEK293-cells stably transfected with hCXCR2 were cultured in
EMEM containing Glutamax and supplemented with 10% FBS (from
PAA, Austria), 1% nonessential amino acids (NEAA), 100 U/ml
penicillin, and 100 μg/mL streptomycin (PEST) and 500 μg/mL
Geneticin/G418. HEK293-cells stably transfected with human CCR1
and CCR2 were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) containing pyruvate and Glutamax and supplemented with
10% FBS (from PAA, Austria), 100 U/ml penicillin, and 100 μg/mL
streptomycin (PEST). Selection was performed with 330 μg/mL
Geneticin. Cells were harvested at 60% confluence in buffer containing
10 mM Tris-HCl pH 7.4, 5 mM EDTA, and 0.1 mg/mL bacitracin and
centrifuged at 100g for for 10 min (4 °C). The cell pellet was
resuspended in harvesting buffer, pooled, and homogenized using a
Dounce homogenizer. The homogenate was centrifuged in 48000g for
10 min. (4 °C) and resuspended in harvesting buffer using Ultra-
Turrax T8. Membrane aliquots were stored at −80 °C. Protein
concentration was determined using a modified method by Lowry.32
Binding Studies Using [125I]CX3CL1. Saturation binding studies
were carried out in duplicates in polypropylene tubes in a total volume
of 1 mL of binding buffer containing 50 mM HEPES, 10 mM MgCl2, 1
mM EDTA, and 0.1% gelatin (pH 7.4) and 0.05−150 pM
[
125I]CX3CL1 (8−12 concentrations). Nonspecific binding was
determined in the presence of 10 nM unlabeled CX3CL1. The
reaction was started by the addition of 1 μg/tube of hCX3CR1/Gαqi5
membranes and incubated for 24 h in room temperature. The reaction
was terminated by rapid filtration through Whatman GF/B filters
(pretreated with 0.3% polyethyleneimine, PEI) followed by sub-
sequent washing with cold washing buffer (10 mM Hepes, 500 mM
NaCl, pH 7.4 at 4 °C) using a Brandel cell harvester. Scintillation
cocktail (Packard Ultima Gold, 4 mL) was added and the radioactivity
determined in a scintillation counter (Packard 2900TR liquid
scintillation counter). Competition studies were performed with
[
125I]CX3CL1 (15−37 pM) in the presence of increasing concen-
tration of test compound diluted in DMSO (final concentration of
1%) and 1.5 pM hCX3CR1 receptors per tube in a total volume of 1
mL binding buffer. The competition binding experiments were run for
24 h in room temperature and terminated as described for the
saturation binding studies. The Ki values (inhibition constants) of the
test compounds were calculated from the observed IC50 of the test
compound using the Cheng−Prusoff equation. The Kd value for
[
125I]CX3CL1 used for calculation of Ki were determined as described
above for saturation binding studies, and were 9.55 pM (pKd 11.02
0.05, n = 3)
Competition Binding with CXCR2, CCR1, and CCR2 Receptors.
Radioligands [125I]-humanIL8 ([125I]-hIL8), [125I]MIP-1α, and
[
125I]MCP-1 are used to study the CXCR2, CCR1, and CCR2
receptors, respectively. The CXCR2 competition binding assay was
performed in white clear bottom 96-well isoplates in a total volume of
200 μL/well. Each well contained 150 pM [125I]-hIL8, membrane
preparation equivalent to a receptor concentration of 20 pM, 1.5 mg of
WGA SPA-beads in assay buffer [50 mM HEPES-KOH pH 7.4, 10
mM MgCl2, 1 mM EDTA, 0.5% (w/v) gelatin], and increasing
concentrations of test compounds diluted in DMSO (final
concentration of 1% (v/v)). The nonspecific binding (min binding)
was defined by 500 nM unlabeled hIL8. The assay was incubated at
room temperature for 4 h, and then the assay plates were counted in
Microbeta Trilux 1450. The CCR1 and CCR2 competition binding
assays were performed in 2 mL deep well plates in a total volume of 1
mL. Each well contained 15−20 pM [125I]MIP-1α or [125I]MCP-1, 2
pM hCCR1, or 1.5 pM CCR2 receptors in assay buffer (50 mM
Hepes, 10 mM MgCl2, 1 mM EDTA, 0.1% gelatin, pH 7.4) and
increasing concentrations of test compounds diluted in DMSO (final
Metabolic Stability Assay. Metabolic stability was measured in
human microsomes. Microsome incubations for Clint (intrinsic
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Table 8. Gradient Details for LC methods A, B, and C
LC gradient method A
vol % A
LC gradient method B
LC gradient method C
vol % A
time (min)
vol % B
time (min)
vol % A
vol % B
time (min)
vol % B
0
100
65
65
0
0
35
0
100
70
65
0
0
30
0
100
65
60
0
0
35
0.1
0.1
11
0.1
11
18
35
35
40
18.8
19.5
19.8
100
100
0
12
100
100
0
12
100
100
0
0
12.8
13
0
12.8
13
0
100
100
100
clearance) determinations and metabolic profiling experiments were
conducted as previously described.35 Briefly, microsomes at the
concentration 0.5 mg protein/mL supplemented with 1 mM NADPH
were incubated at 37 °C with gentle shaking in a 5% CO2 atm. All
incubations were stopped by the addition of two volumes of ice-cold
acetonitrile and were then centrifuged prior to LC-MS/MS analysis.
The metabolic capacity of the microsomes was checked in-house using
a set of probe substrates. Clint values were obtained from
disappearance curves of the parent molecule.36 LC-MS analysis for
Clint value estimations was performed with a Micromass Quattro
Micro triple quandropole as previously described.36
In Vivo Pharmacokinetic Assay. In vivo pharmaocokinetics in rat
was assessed in male Sprague−Dawley rats:37 Male Sprague−Dawley
rats (Scanbur B&K AB, Sollentuna, Sweden) were housed with up to
five animals per cage and were allowed to acclimatize to the new
environment for at least one week upon arrival. In the conditioned
Varian) fitted with a probe of suitable configuration. 1H NMR spectra
were recorded at 400, 500, or 600 MHz. 13C NMR spectra were
recorded at 100 or 126 MHz. Chemical shifts are given in ppm down-
and upfield from TMS (0.00 ppm). The following reference signals
were used: TMS δ 0.00, or the residual solvent signal of DMSO-d6 δ
2.50 (1H), δ 39.51 (13C), CD3OD δ 3.30 (1H), δ 49.00 (13C), CDCl3 δ
7.26 (1H), δ 77.16 (13C), CD3CN δ 1.94 (1H), δ 118.26 (13C), or TFA
δ 11.50 (1H), δ 164.2 (13C) (unless otherwise indicated). Resonance
multiplicities are denoted s, d, t, q, m, br, and app for singlet, doublet,
triplet, quartet, multiplet, broad, and apparent, respectively.
The mass spectra were typically recorded utilizing thermospray
(Finnigan MAT SSQ 7000; buffer, 50 nM NH4OAc in CH3CN:H2O;
3:7), electron impact (Finnigan MAT SSQ 710), or electrospray (LC-
MS; LC, Waters 2790, column XTerra MS C8 2.5 μm 2.1 mm × 30
mm, buffer gradient H2O + 0.1%TFA:CH3CN + 0.04%TFA; MS,
micromass ZMD) ionization techniques.
animal facility, room temperature was kept at 20
2 °C, relative
Preparative HPLC was run on a Waters autopurifier HPLC with a
diode array detector. Column: Xterra MS C8, 19 mm × 300 mm, 10
μm. Narrow gradients with MeCN/water were used at a flow rate of
20 mL/min.
Thin layer chromatography (TLC) was performed on Merck TLC-
plates (Silica gel 60 F254) and UV visualized the spots.
Column chromatography was performed using Merck Silica gel 60
(0.040−0.063 mm).
GC-analyses were performed on a GC 6890 (Agilent technologies)
equipped with a flame ionization detector. The column used for
analysis of enantiomeric purity was a Cyclodex B (ID 0.25 mm × 30
m, 0.25 μm, Agilent technologies) using the specified temperature
program.
Analytical separation of diastereoisomers of compounds 18 and 24:
LC-MS analyses were performed on an LC-MS system consisting of a
Waters Alliance 2795 HPLC, a Waters PDA 2996 diode array detector,
and a ZQ 2000 single quadrupole mass spectrometer. The mass
spectrometer was equipped with an electrospray ion source (ES)
operated in positive and negative ion mode. The capillary voltage was
set to 3.0 V and the cone voltage to 30 V. The mass spectrometer was
scanned between m/z 160−600 with a scan time of 0.5 s. The diode
array detector scanned from 210 to 350 nm. The column oven
temperature was set to 40 °C Separation was performed on a Waters
XTerraMS, C8, 3.5 μm, 2.1 mm × 50 mm. Mobile phase A: formic
acid (10.6 mM) in water/acetonitrile (95:5). Mobile phase B: formic
acid (10.6 mM) in water/acetonitrile (2:98). For each structure a
special separation LC gradient was developed, details on the gradients
are given Table 8 for LC methods A, B, and C.
Representative experimental procedures for the synthesis of
compounds discussed in this publication are presented below; further
experimental details and analytical data can be found in the Supporting
Information.
4-Amino-2-(benzylsulfanyl)-6-oxo-1,6-dihydropyrimidin-5-yl thi-
ocyanate (7). 6-Amino-2-(benzylsulfanyl)pyrimidin-4-ol38 (6) (70 g,
300.1 mmol) and KSCN (120 g, 1.23 mol) were suspended in DMF
(1.5 L) and heated to 65 °C. Pyridine (40.4 g, 510 mmol) was added,
and the solution was cooled to 5 °C. Bromine (43.2 g, 270 mmol) was
added dropwise, and the solution was stirred for 2 h at 5−10 °C and
then overnight at room temperature. The mixture was poured into ice
water and stirred for 1 h. The solid product was collected by filtration,
washed with cold water, and then dried in vacuo. The procedure was
repeated once more starting from 67 g of 6-amino-2-(benzylsulfanyl)-
humidity at 60 20%, and a 12 h light−dark cycle was maintained
including a 0.5 h dusk or dawn period (lights on at 6.30 a.m.). Water
and standard rodent diet (R70 Lactamin, Stockholm, Sweden) were
freely accessible. All animal handling and experiments were performed
in full compliance with authorial and ethical guidelines (Ethical Board
of South Stockholm). Diet was removed from the animals 1 h prior to
dose administration and replaced 6 h after dose administration. Rats
(∼300 g) were weighed and divided into two groups: one group (n =
3) received 3 μmol/kg of the test compound intravenously (iv) as a
bolus injection in the tail vein, the other group received 10 μmol/kg
orally (po) via gavage in a parallel study design. All compounds were
given discretely. The dose volume was 4 mL/kg for both dose routes.
All compounds were formulated in 5% dimethylamine, 20%
hydroxypropyl-β-cyclodextrine in 0.1 M Meglumin for both iv and
po adminstration. For each dose occasion a fresh formulation was
prepared. To verify the concentration of the administered dose, an
aliquot of each formulation was taken, transferred to a polypropylene
tube (Cryotubes, A/S Nunc, Denmark) and diluted 10 times with
MeCN before analysis. Blood samples (400 μL) from the tail vein
were collected in microtainer tubes (BD, Plymouth, UK) containing
EDTA at 0.02, 0.08, 0.33, 0.67, 1, 3, 6, and 24 h following iv
administration, or at 0.25, 0.5, 0.75, 1, 1.5, 2.5, 6, and 24 h following po
administration. The blood samples were centrifuged for 5 min at
2000g (4 °C). The supernatant was transferred to polypropylene tubes
and stored at −20 °C until analysis. Polypropylene tubes (1.5 mL per
tube) in 96-well format used for storage of plasma samples were
obtained from Biotech Solutions (Vineland, NJ, USA). The samples
were then analyzed by LC-MS as previously described.37,38
Chemistry. General Experimental Section. Starting materials used
were either available from commercial sources or prepared according
to literature procedures. Reactions were typically run under an inert
atmosphere of nitrogen or argon in commercially available anhydrous
solvents that were used without further purification or drying. Room
temperature refers to 20−25 °C. Final compounds were purified to
>95% purity as determined by HPLC analysis.
Microwave heating was performed in a Creator or Smith
Synthesizer single mode microwave cavity producing continuous
irradiation at 2450 MHz at the indicated temperature in the
recommended microwave tubes.
1
The H and 13C NMR spectra were recorded at room temperature
unless otherwise stated, using an NMR spectrometer (Bruker or
3186
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pyrimidin-4-ol. The combined batches of the title compound (170.5 g)
was used directly in the next step without further purification. H
NMR (400 MHz, DMSO-d6) δ ppm 12.34 (br s, 1H), 8.00−7.50 (br s,
2H), 7.46 (d, J = 7.6 Hz, 2H), 7.39−7.21 (m, 3H), 4.38 (s, 2H). 13C
NMR (101 MHz, DMSO-d6) δ ppm 163.5, 162.7, 160.7, 137.2, 129.2,
128.4, 127.4, 111.9, 72.8, 33.5.
mixture was filtered, and the resulting clear solution was neutralized
(pH 7) with concd HCl. The precipitated solid was collected by
filtration, washed with water and acetonitrile, and dried at 50 °C in
vacuo for 2 h and over P2O5 overnight in vacuo to give the title
compound (8.57 g, 86% yield) as a yellow solid. 1H NMR (400 MHz,
DMSO-d6) δ ppm 12.74 (s, 1H), 8.44 (br s, 2H), 7.41 (d, J = 8.59 Hz,
1H), 4.75−4.87 (m, 1H), 4.31−4.43 (m, 1H), 3.37−3.46 (m, 2H,
overlapping with the water signal), 1.51−1.65 (m, 1H), 1.33−1.47 (m,
2H), 0.82−0.94 (m, 6H). 13C NMR (101 MHz, DMSO-d6) δ ppm
177.1, 172.6, 158.1, 155.9, 154.1, 92.1, 63.7, 50.2, 24.4, 23.4, 22.0. MS
(ES+) m/z 300 [M + H]+.
1
2-Amino-5-(benzylsulfanyl)[1,3]thiazolo[4,5-d]pyrimidin-7-ol (8).
4-Amino-2-(benzylsulfanyl)-6-oxo-1,6-dihydropyrimidin-5-yl thiocya-
nate (7) (170.5 g, 587 mmol) was suspended in water (0.33 L) and
DMF (1 L) and heated at 120 °C for 27 h. The reaction mixture was
poured onto ice, and the resulting pale-yellow precipitate was collected
by filtration and washed with water. The solid material was suspended
in water (2.5 L) and heated to 75 °C, and sodium hydroxide (10 M
solution in water, 170 mL) was added. The resulting slightly cloudy
suspension was filtered, and the product was precipitated by addition
of concd HCl until pH 4. The solids were collected by filtration and
dried in vacuo over P2O5. Excess water was coevaporated with
acetonitrile, and the resulting material was dried in vacuo overnight to
General Method 2. Synthesis of compounds 14 and 18.
1
give the title compound (170 g, 100% yield, 98% purity). H NMR
(400 MHz, DMSO-d6) δ ppm 12.53 (br s, 1H), 8.17 (s, 2H), 7.43 (d,
d, J = 7.1 Hz, 2H), 7.20−7.35 (m, 3H), 4.41 (s, 2H). 13C NMR (101
MHz, DMSO-d6) δ ppm 171.9, 168.4, 157.2, 137.4, 129.2, 129.1,
128.4, 127.3, 103.0, 33.7. MS (ES+) m/z 291.0 [M + H]+.
(2R)-2-[(2-Amino-5-sulfanyl[1,3]thiazolo[4,5-d]pyrimidin-7-yl)-
amino]-4-methylpentan-1-ol (12a) (30 mg, 100 μmol) was suspended
in DMSO (450 μL). Optionally, sodium borohydride (0.1−1 equiv)
was added. Diisopropylethylamine (19 μL, 110 μmol) was added,
followed by the quick addition of the organic halide (13 or 17) (105
μmol). The reaction was stirred for 1−20 h at room temperature or 40
°C. The mixture was poured onto ice, and the solid product was
collected by filtration after the ice had melted and then washed with
water to give the title compound after drying in vacuo. Alternatively,
the crude product was purified by reverse phase preparative HPLC or
silica gel chromatograpy to give the title compound 14 or 18.
(2R)-2-{[5-(Benzylsulfanyl)-2-chloro[1,3]thiazolo[4,5-d]pyrimidin-
7-yl]amino}-4-methylpentan-1-ol (19a). The reaction was split into
two separate 2 L round-bottomed flasks, and the two reaction mixtures
were pooled before work up. To a solution of (2R)-2-{[2-amino-5-
(benzylsulfanyl)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino}-4-methyl-
pentan-1-ol (11a) (42.0 g, 10.8 mmol) in acetonitrile (1.1 L) and 37%
hydrochloric acid (1.5 L) was added sodium nitrite (14.9 g, 21.6
mmol, dissolved in a small amount of water) dropwise. The mixture
was kept on ice during the addition to keep the temperature <5 °C and
was then stirred at 0 °C for 5 h. The reaction flask was kept in the
refrigerator overnight and was thereafter stirred at 5 °C until
completion (27 h in total). The reaction mixture was filtered. The
filtrate was poured onto ice. The mixture was filtered after the ice had
melted and the collected solid was washed with water. Acetonitrile was
added, and the solvent was evaporated. The product was dried at 50
°C in vacuo to give the title compound (34.7 g, 79% yield) as a pink
solid. The product was used as such in the next step. MS (ES+) m/z
409 [M + H]+.
5-(Benzylsulfanyl)-7-chloro[1,3]thiazolo[4,5-d]pyrimidin-2-amine
(9). 2-Amino-5-(benzylsulfanyl)[1,3]thiazolo[4,5-d]pyrimidin-7-ol (8)
(8.0 g, 27.6 mmol) was suspended in POCl3 (80 mL), and N,N-
dimethylaniline (8 mL) was added. The mixture was heated at reflux
for 3 h. After cooling to room temperature, the reaction mixture was
slowly added to a stirred mixture of ice and water. The resulting
precipitate was collected by filtration. The solid material was purified
by silica gel column chromatography using 30−50% EtOAc in CHCl3
to give the title compound as a yellow solid (5.11 g, 60% yield, 95%
1
purity). H NMR (400 MHz, DMSO-d6) δ ppm 8.96 (br s, 2H),
7.40−7.47 (m, 2H), 7.19−7.34 (m, 3H), 4.37 (s, 2H). 13C NMR (101
MHz, DMSO-d6) δ ppm 172.8, 170.9, 167.4, 148.9, 137.7, 129.0,
128.4, 127.1, 117.6, 34.5. MS (ES+) m/z 309.0 [M + H]+.
General Method 1. Synthesis of 5-(benzylsulfanyl)[1,3]thiazolo-
[4,5-d]pyrimidin-2-amine aminoalcohol derivatives 11.
To an approximately 0.05−0.6 M solution of 5-(benzylsulfanyl)-7-
chloro[1,3]thiazolo[4,5-d]pyrimidin-2-amine (9) (1.0 equiv) in
anhydrous N-methylpyrrolidone were added an aminoalcohol (10)
(1.4−4.0 equiv) and N,N-diisopropylethylamine (1.1−4.0 equiv). The
reaction mixture was stirred and heated at 100−130 °C (oil bath
temperature) for 18−96 h or heated in a microwave oven at 120−200
°C for 5 min−1 h. The mixture was poured into an ice/water slurry,
stirred until the ice was melted, and then it was filtered. The resulting
precipitate was purified for example by coevaporation with toluene/
ethanol, purification by silica gel column chromatography, recrystal-
lization in dichloromethane, or washing with water, followed by an
organic solvent and dried in vacuo over P2O5 to give the 5-
benzylsulfanyl)[1,3]thiazolo[4,5-d]pyrimidin-2-amine aminoalcohol
derivatives 11a−f.
(2R)-2-{[5-(Benzylsulfanyl)-2-methoxy[1,3]thiazolo[4,5-d]-
pyrimidin-7-yl]amino}-4-methylpentan-1-ol (20a). To a solution of
(2R)-2-{[5-(benzylsulfanyl)-2-chloro[1,3]thiazolo[4,5-d]pyrimidin-7-
yl]amino}-4-methylpentan-1-ol (19a) (34.7 g, 84.8 mmol) in
methanol (870 mL) was added potassium hydroxide (9.5 g, 170
mmol), and the mixture was heated at 55 °C for 20 min. The solvent
was evaporated. Dichloromethane (350 mL) and water (350 mL) were
added, resulting in a thick slurry. The organic solvent was evaporated,
and the aqueous mixture was filtered. The solid was collected and
recrystallized in acetonitrile (350 mL) and dried at 40 °C in vacuo for
4 h to give the title compound (26.5 g, 77% yield) as a pale-orange
1
solid. H NMR (500 MHz, DMSO-d6) δ ppm 7.54 (d, J = 8.51 Hz,
(2R)-2-[(2-Amino-5-sulfanyl[1,3]thiazolo[4,5-d]pyrimidin-7-yl)-
amino]-4-methylpentan-1-ol (12a). Liquid ammonia (500 mL) was
condensed into a 1 L flask (dry ice/acetone cooling bath) and (2R)-2-
{[2-amino-5-(benzylsulfanyl)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]-
amino}-4-methylpentan-1-ol (11a) (13.0 g, 33.4 mmol) was carefully
added under a N2-atmosphere. To the clear solution was added an
excess of sodium metal in small pieces, until a blue color persisted.
When the blue color had remained for 25 s, solid ammonium chloride
was added to quench the reaction. The ammonia was removed under a
stream of N2, and the remains were dissolved in water (300 mL). The
1H), 7.42 (d, J = 7.25 Hz, 2H), 7.30 (t, J = 7.57 Hz, 2H), 7.21−7.26
(m, 1H), 4.74 (t, J = 4.73 Hz, 1H), 4.26−4.41 (m, 3H), 4.16 (s, 3H),
3.35−3.46 (m, 2H, overlapping with the water signal), 1.53−1.65 (m,
1H), 1.35−1.49 (m, 2H), 0.87 (d, J = 6.62 Hz, 3H), 0.83 (d, J = 6.62
Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ ppm 177.5, 166.8, 164.7,
156.1, 138.3, 128.6, 128.2, 126.7, 100.3, 63.3, 59.4, 50.3, 34.0, 24.2,
23.2, 21.8. MS (ES+) m/z 405 [M + H]+.
5-(Benzylsulfanyl)-7-{[(2R)-1-hydroxy-4-methylpentan-2-yl]-
amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one (21a). To a solution
of (2R)-2-{[5-(benzylsulfanyl)-2-methoxy[1,3]thiazolo[4,5-d]-
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Article
pyrimidin-7-yl]amino}-4-methylpentan-1-ol (20a) (26.5 g, 65.4
mmol) in 1,4-dioxane (790 mL) and water (16 mL) 37% hydrochloric
acid (14 mL) was added, and the mixture was heated at 45 °C for 18 h.
The solvent was evaporated, and the crude product was crystallized
from ethyl acetate/dichloromethane, 3:7 resulting in crop 1. The
mother liquid was concentrated, and the residue was crystallized from
ethyl acetate/tetrahydrofuran resulting in crop 2. The mother liquid
was once again concentrated, and the residue was added to a silica gel
column and was eluted with 30% ethyl acetate in dichloromethane,
resulting in crop 3. The crops were pooled, tetrahydrofuran was added,
and the solvent was evaporated. The product was dried at 40 °C in
vacuo for 5 h to give the title compound (13.6 g, 53% yield) as a pink
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and experimental data for intermedi-
ates and final compounds not present in the Experimental
Section. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*For S. K.: phone, +46-(0)70-3355543; E-mail, Sofia.
Karlstrom@medivir.com. For R. J.: phone, +46-(0)-73-
3542157; E-mail, daggstigen@gmail.com.
■
1
solid. H NMR (500 MHz, DMSO-d6) δ ppm 12.38 (br s, 1H), 7.42
(d, J = 7.57 Hz, 2H), 7.20−7.31 (m, 4H), 4.25−4.37 (m, 3H), 3.30−
3.44 (m, 2H), 1.53−1.63 (m, 1H), 1.32−1.47 (m, 2H), 0.86 (d, J =
6.62 Hz, 3H), 0.82 (d, J = 6.62 Hz, 3H). 13C NMR (126 MHz,
DMSO-d6) δ ppm 169.4, 166.7, 155.0, 154.4, 138.6, 129.0, 128.5,
127.1, 91.0, 63.7, 50.6, 34.2, 24.5, 23.5, 22.1. MS (ES+) m/z 391 [M +
H]+.
Present Address
The research unit where this work was conducted, AstraZeneca
Research and Development, Sodertalje, Sweden, was closed in
̈
̈
2012. Contact with the authors can be made using the
information below. For S.K.: Medivir AB, P.O. Box 1086, SE-
141 22 Huddinge, Sweden. For R.J.: Daggstigen 8B, SE-141 38
Huddinge, Sweden. For G.N.: AlzeCure Discovery, NVS
Huddinge Hospital, Novum lv 5, SE-141 57 Huddinge,
Sweden. For P.H.S.: Department of Applied Physical
Chemistry, Royal Institute of Technology, SE-10044 Stock-
holm, Sweden and SP Process Development, SE-151 21
General Method 3. Synthesis of compound 22.
Sodertalje, Sweden.
̈
̈
Concd HCl (2.5 mL/mmol of a compound 18) was added to
compound 18 (1.0 equiv) in CH3CN. The reaction mixture was
cooled in an ice bath, and sodium nitrite (2.0 equiv) dissolved in a
minimal amount of water was added dropwise. The reaction was
stirred at 0 °C until the reaction was complete (monitored by LC-MS,
HPLC, or TLC) and was then poured into ice water, neutralized with
sodium bicarbonate, and extracted with DCM or EtOAc. The
combined organic phases were dried and concentrated in vacuo to
give product compound 22.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. S.K., G.N., and R.J. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
General Method 4. Synthesis of compound 23.
We thank Margareta Bergh and the Physiochemical Character-
ization Team at the Department of Medicinal Chemistry at
AstraZeneca Sodertalje for physiochemical characterization
̈
̈
including solubility measurements, and the Analytical &
Purification Sciences Team for separations, purifications, and
NMR analyses of compounds. We also acknowledge Sanna
Laine and Eva Blomberg for in vitro characterizations of the
compounds.
Potassium hydroxide (2.0 equiv) dissolved in methanol was added
dropwise to a cooled (0 °C) solution of compound 22 (1.0 equiv) in
methanol. The resulting mixture was stirred at 0 °C until the reaction
was complete (monitored by LC-MS, HPLC, or TLC). The solvent
was evaporated and the product 23 was used in the next reaction step
without further purification.
ABBREVIATIONS USED
■
ADAM, a disintegrin and metalloproteinase domain-containing
protein; CCL, CC chemokine ligand; CCR, CC chemokine
receptor; CD, cluster of differentiation; Clint, intrinsic clearance;
CXCR, CXC chemokine receptor; CX3CL1, chemokine (C-X3-
C motif) ligand 1; CX3CR1, CX3C chemokine receptor 1,
fractalkine receptor; DIPCl, B-chlorodiisopinocampheylborane;
General Method 5. Synthesis of compound 24.
̀
FKN, fractalkine; hERG, the human ether-a-go-go-related gene;
HT, high throughput; HTS, high throughput screening; NCS,
N-chlorosuccinimide; NK-cell, natural killer cell; Papp,
apparent permeability coefficient; TACE, tumor necrosis
factor-α-converting enzyme
A solution of concentrated HCl (1.0 equiv) was added to a cooled
(0 °C) solution of compound 23 (1.0 equiv) in 1,4-dioxane. The
resulting mixture was stirred at 40 °C until the reaction was complete
(monitored by LC-MS, HPLC, or TLC). The reaction mixture was
neutralized with saturated NaHCO3 (aq), and the dioxane was
evaporated. The residue was dissolved in DCM or EtOAc, washed
with brine, dried, and concentrated in vacuo. The crude product 24
was, if necessary, purified using preparative HPLC or by flash silica gel
column chromatography.
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