Evaluation of a series of bicyclic CXCR2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.11.039

The CXCR2 SAR of a series of bicyclic antagonists such as the 2-aminothiazolo[4,5-d]pyrimidine 3b was investigated by systematic variation of the fused pyrimidine-based heterocyclic cores. Replacement of the aminothiazole ring with a 2-thiazolone alternat

Full text of DOI:10.1016/j.bmcl.2007.11.039

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 798–803
Evaluation of a series of bicyclic CXCR2 antagonists
Iain Walters,* Caroline Austin, Rupert Austin, Roger Bonnert, Peter Cage, Mark Christie,
Mark Ebden, Stuart Gardiner, Caroline Grahames, Steven Hill, Fraser Hunt,
Robert Jewell, Shirley Lewis, Iain Martin,à David Nicholls and David Robinson
Departments of Medicinal Chemistry, Molecular Biology, and Discovery BioScience, AstraZeneca R&D Charnwood,
Bakewell Road, Loughborough, Leicestershire LE11 5RH, United Kingdom
Received 3 October 2007; revised 9 November 2007; accepted 10 November 2007
Available online 17 November 2007
Abstract—The CXCR2 SAR of a series of bicyclic antagonists such as the 2-aminothiazolo[4,5-d]pyrimidine 3b was investigated by
systematic variation of the fused pyrimidine-based heterocyclic cores. Replacement of the aminothiazole ring with a 2-thiazolone
alternative led to a series of thiazolo[4,5-d]pyrimidine-2(3H)-one antagonists with markedly improved biological and pharmacoki-
netic properties, which are suitable pharmacological tools to probe the in vivo effects of CXCR2 antagonism combined with the
associated CCR2 activity.
Ó 2007 Elsevier Ltd. All rights reserved.
Chemokines are a set of small proteins typically com-
prising 70–80 amino acids which play an important role
in the recruitment and activation of inflammatory cells.
They may be classified according to the nature of con-
served cysteine motifs, and generally fall into two main
categories, the CC and CXC chemokines. Both subfam-
ilies include a number of potent chemoattractants and
activators of different leukocyte subsets. For example,
the CXC chemokines interleukin-8 (IL-8) and growth-
related oncogene-a (GRO-a) modulate the activity of
neutrophils, whereas the CC chemokine monocyte che-
moattractant protein-1 (MCP-1) influences the behav-
iour of monocytes and T lymphocytes. Upregulation
of these chemokines has been associated with several
inflammatory diseases in man.1,2 In such diseases leuko-
cytes are directed to the site of infection or injury by a
chemical gradient of chemokines, a process which is
mediated by interaction with G-protein coupled recep-
tors on the leukocyte surface. Blockade of these chemo-
kine receptors represents an attractive strategy for
therapeutic intervention in inflammatory diseases, pro-
vided the key chemokine–receptor interactions can be
targeted. The CXCR2 receptor, found on neutrophils,
binds a number of neutrophil activating chemokines
including IL-8 with high affinity.3 The CCR2 receptor,
found on monocytes, likewise interacts with several
CC ligands such as MCP-1.4 Both of these pathways
independently present tempting approaches for small
molecule antagonism; the development of an antagonist
with activity at both receptors would offer a particularly
novel opportunity for disease modification, since no
such dual antagonists have been reported to date.
We recently disclosed a series of potent 2-aminothiazol-
o[4,5-d]pyrimidine CXCR2 antagonists such as
1
(Fig. 1) derived from a hit-to-lead study on the HTS
hit 2.5 Subsequent exploration of the 5-thio- and 7-ami-
no-substituent SAR resulted in the optimised aminothi-
azole antagonist 3b,6 which possessed improved potency
but still suffered from relatively poor rat bioavailability
R
OH
HN
OH
Keywords: CXCR2; Thiazolo[4,5-d]pyrimidine-2(3H)-one.
S
S
N
N
H2N
*
Corresponding author. Tel.: +44 1509 644000; fax: +44 1509
645571; e-mail: iain.walters@astrazeneca.com
Present address: UCB Granta Park, Great Abington, Cambridge
CB21 6GS, UK.
H2N
N
N
S
N
S
N
F
2
1 R = Me
3b R = H
F
à
Present address: Organon Research Scotland, Newhouses, Lanark-
shire ML1 5SH, UK.
Figure 1.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.039

I. Walters et al. / Bioorg. Med. Chem. Lett. 18 (2008) 798–803
799
(Table 1). In order to address this issue, a programme of
structural variation of the central bicyclic heterocycle
was undertaken, the results of which are presented in
this paper.
produce the desired target. A second general route
hinged on incorporation of a suitable amino alcohol
substituent earlier in the synthetic process, as shown
in Scheme 2, which allowed the construction of an
alternative set of heterocyclic ring systems. Thus, alkyl-
ation of the mono-thio pyrimidine 6 followed by chlo-
rination and displacement with (R)-alaninol gave the
diamino pyrimidine intermediate 7. This could be
annulated with a cyanopyridone ring directly to give
25, or converted via nitrosation and reduction8 to the
useful triamino pyrimidine intermediate 8 which was
used to construct a range of different pteridine-based
analogues.9 The synthesis of heterocycles inaccessible
by either of these two general routes is outlined in
Schemes 3 and 4. Thus, the amino-pteridinone 910
was diazotised giving the pteridine-dione 10 as a by-
product. Bis-chlorination and alaninol displacement
were followed by a Suzuki coupling with methylboron-
ic acid to afford the 7-methyl-pteridine 18. The ‘reverse’
aminothiazole 21 was derived from the dichloronitro-
pyrimidine intermediate 1111 by sequential chloro-dis-
The synthetic routes to the chemokine antagonists de-
scribed in this paper are depicted in Schemes 1–4.
Although substituent variation could be readily carried
out by parallel synthetic chemistry methodology,
changes to the heterocyclic core required a more indi-
vidual approach. However, two synthetic pathways
proved to be particularly useful for the construction
of a range of different fused heterocyclic systems, the
first of which, shown in Scheme 1, offered rapid access
to several pteridine and purine templates. Alkylation of
the commercially available bis-thio pyrimidine 4 affor-
ded the common dibenzyl intermediate 5, which could
be converted into several different bicyclic systems by
condensation with a range of electrophiles.7 In each
case, the peri-thiobenzyl substituent could be selectively
displaced with a primary amine such as (R)-alaninol to
Table 1. CXCR2 potency,14 rat oral bioavailability (F), rat in vivo clearance (Cl), human plasma protein binding (hPPB), pKa and logD for
15
compounds 3b, 15–29, 30b
Compound
CXCR2 IC50, nM
F, %
Cl, ml/min/kg
hPPB, %free
pKa
logD
3b
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30b
4
9
5
17
31
2.6
2.3
3.3
3.2
13
630
7
6
24
5.2
2.9
280
140
280
850
5
<5
44
44
21
1.1
1.2
4.7
7.5
0.9
3.1
3.4
3.3
2.5
2.5
2.6
3.1
3.2
1
350
16
2
10.5
7.9
1.7
6.6
6.5
45
2500
220
1
9.1
11.4
6.7
22
6
0.29
SH
N
OH
OH
H2N
H2N
HN
N
HN
N
N
N
N
N
SH
4
N
H
S
O
N
H
N
S
i
F
16
F
23
F
F
ii-iv
F
viii,iv
F
S
OH
OH
HN
H2N
v-vii,iv
HN
N
ix,x,iv
N
N
N
N
N
H2N
H2N
N
S
N
N
S
H2N
N
S
H
F
5
F
F
15
F
17
F
F
Scheme 1. Reagents: (i) KOH, 2,3-difluorobenzyl bromide, MeOH (47%); (ii) Ac2O; (iii) SOCl2 (44%); (iv) (R)-alaninol, Hunig’s base, NMP (40–
50%); (v) ethoxycarbonyl isothiocyanate, MeCN; (vi) diisopropylcarbodiimide (73%); (vii) LiOH, THF, water (65%); (viii) ethyl glyoxalate, Na,
MeOH (46%); (ix) BrCH2CN, Hunig’s base, DMSO (29%); (x) KOH, MeOH (26%).

800
I. Walters et al. / Bioorg. Med. Chem. Lett. 18 (2008) 798–803
OH
OH
HN
OH
HN
NC
i-iii
iv
N
N
N
H2N
N
SH
O
N
H
N
S
H2N
N
S
6
F
25
F
7
F
F
OH
OH
v,vi
HN
HN
H
N
N
N
O
O
N
N
vii
N
S
N
H
N
S
x
OH
HN
F
F
19
27
H2N
H2N
F
F
N
N
S
F
8
ix
viii
F
OH
OH
HN
HN
MeO2C
O
N
N
N
N
N
H
N
S
N
N
S
F
26
F
20
F
F
Scheme 2. Reagents: (i) KOH, 2,3-difluorobenzyl bromide, aq DMF (89%); (ii) POCl3, 2-picoline (44%); (iii) (R)-alaninol, Hunig’s base, NMP (90%);
(iv) H2CO, ethyl cyanoacetate, Et3N, EtOH (17%); (v) NaNO2, aq AcOH (92%); (vi) Na2S2O4, aq DMF (69%); (vii) diethyl oxalate (13%); (viii)
diethyl ketomalonate, Na, MeOH (27%); (ix) glyoxal, NMP (13%); (x) 1,3-dihydroxyacetone, NaOAc, MeOH (47%).
NH2
O
OH
N
HN
N
N
i
NH
ii-iv
N
N
N
O
N
H
N
S
O
N
H
N
S
N
S
F
9
F
10
18
F
F
F
F
Cl
OH
HN
O2N
Cl
v-vii
N
N
N
H2N
N
S
S
N
S
11
21
viii, vii
Cl
OH
HN
N
H2N
H2N
H
N
N
S
ix, iii
N
O
N
N
H
S
12
28
Scheme 3. Reagents: (i) isopentylnitrite, bromoform, DMSO (30%); (ii) POCl3 (68%); (iii) (R)-alaninol, Hunig’s base, NMP (40–50%); (iv)
CH3B(OH)2, Pd(PPh3)4, K2CO3, dioxane (10%); (v) (R)-alaninol, Hunig’s base, THF (64%); (vi) KSCN, DMF (95%); (vii) iron, NH4Cl, aq EtOH
(80–90%); (viii) NH3, Hunig’s base, dioxane (52%); (ix) triphosgene, Et3N, DCM (70%).
placement with alaninol and thiocyanate followed by
nitro reduction and spontaneous cyclisation. The same
intermediate 11 also gave rise to the imidazolidinone 28
via displacement with ammonia and nitro reduction to
give 12 followed by cyclisation with triphosgene and
alaninol incorporation. The pyridone 24 was made
from the diaminopyrimidine 1310 by condensation with
ethyl 3-ethoxyacrylate12 followed by diazotisation/bro-
mination and alaninol displacement (Scheme 4). The
pyrazolopyrimidine 29 was readily formed from the
commercially available precursor 14 by alkylation,
chlorination and alaninol displacement. Conversion of
the aminothiazole antagonists 3a–f into the corre-
sponding thiazolones 30a–f was simply effected by diaz-
otisation of the 2-amino group to the bromide,
displacement with methoxide and acidic hydrolysis.
Finally, synthesis of the methylsulfonate derivative 22
of 3b started with the mono-thio pyrimidine 6 which
was treated to a sequence of alkylation, thiocyanation
and ring closure steps.13 Subsequent chlorination,
sulfonation and reaction with (R)-alaninol gave the
sulfonamide 22.

I. Walters et al. / Bioorg. Med. Chem. Lett. 18 (2008) 798–803
801
across different heterocyclic systems was demonstrated
explicitly in the one case of the thiazolo[4,5-d]pyrimi-
dine-2(3H)-one antagonists described below (Table 2).
The prior survey of thiazolopyrimidine SAR5,6 had also
highlighted the importance of the primary 2-amino sub-
stituent for potency against CXCR2. This principal
activity requirement was found to be shared by other
potent neutral antagonists in this series (Table 1). Thus,
simply exchanging the thiazolo sulfur atom of 3b for a
nitrogen to generate the corresponding 8-aminopurine
15 maintains activity against CXCR2. However, replac-
ing the amino group of 15 with a methyl substituent
afforded the markedly less active compound 16. Simi-
larly, changing the amino group of the potent pteridine
antagonist 17 to a methyl group at either of the two free
positions to give 18 and 19 results in a pair of com-
pounds with greatly reduced potency, which is also the
case for the unsubstituted pteridine 20. The presence
of an amino group is no guarantee of potency, however,
as can be seen with the ‘reversed’ 2-aminothiazolo[5,4-
d]pyrimidine 21. This compound is significantly less po-
tent than its [4,5-d] isomer 3d (Table 2), suggesting a
preference for a particular spatial orientation of the
hydrogen bonding elements of the aminothiazole ring.
Although the amino group of the neutral antagonists
such as 3b, 15 and 17 may have a beneficial influence
on their CXCR2 antagonist potencies, it does not ap-
pear to be conducive to achieving good rat oral bioava-
ilabilities (Table 1). These three compounds all have
bioavailabilities below 10% despite possessing physico-
chemical properties (such as logD) usually concomitant
with good absorption, and in vivo clearances low en-
ough to limit the impact of first pass metabolism. As a
consequence, an effort was made to find potent CXCR2
antagonists within this series which lacked this primary
amino group, in the hope of obtaining an improved
in vivo PK profile. An attractive approach entailed sub-
stitution of the primary amino group to reduce the num-
ber of H-bond donors, in case this factor was
absorption-limiting. Although alkylation of the amino
group was known to reduce potency,6 it was found that
the sulfonamide derivative 22 retained all its activity.
While no improvement in rat bioavailability was seen
with this compound, perhaps due to its much lower
logD and/or higher clearance, its potency encouraged
an extensive survey of acidic heterocyclic ring systems
in order to explore the relationships between pKa, po-
tency and bioavailability (Tables 1 and 2).
NH2
N
OH
HN
i-iii
N
H2N
N
S
O
N
N
S
F
H
13
F
F
24
OH
F
HN
N
OH
N
iv,v,iii
N
N
N
N
N
H
S
N
SH
H
F
14
29
F
OH
HN
OH
HN
N
ii,vi,vii
S
N
N
S
H2N
N
O
N
S
Ar
N
S
Ar
H
3a-f
30a-f
OH
OH
HN
O
viii-x
O
N
S
S
N
F
v,xi,iii
N
H2N
N
SH
F
H
N
N
S
6
22
Scheme 4. Reagents: (i) ethyl 3-ethoxyacrylate, AcOH (61%); (ii)
isopentylnitrite, bromoform, DMSO (25–50%); (iii) (R)-alaninol,
Hunig’s base, NMP (50–70%); (iv) NaH, 2,3-difluorobenzyl bromide,
DMF (51%); (v) POCl3, PhNMe2 (50–80%); (vi) KOH, MeOH (79%);
(vii) aq HCl, dioxane (54%); (viii) KOH, 2,3-difluorobenzyl bromide,
DMF, water (89%); (ix) KSCN, Br2, pyridine, DMF (95%); (x) DMF,
water (88%); (xi) MeSO2Cl, Hunig’s base, THF (50%).
The set of bicyclic CXCR2 antagonists described herein
may be subdivided into two broad categories of hetero-
cycle according to the presence or absence of an acidic
hydrogen atom. The utility of this distinction is borne
out by the different physicochemical and pharmacoki-
netic properties of the two subclasses of compounds,
most notably the poor rat bioavailability of the neutral
series and the high plasma protein binding of the acidic
heterocycles, and so the SAR of the two types of antag-
onist will be outlined in turn. It should be noted that
these compounds were designed as part of an investiga-
tion specifically targeting CXCR2 antagonism. Subse-
quent selectivity profiling of particular antagonists
revealed a correspondingly high CCR2 activity. How-
ever, these compounds were not optimised against
CCR2 and so the focus of this discussion will be SAR
at the CXCR2 receptor.
The marked influence of pKa on potency can be clearly
seen in the 6,6-fused heterocyclic compounds 23–27.
Thus, the least acidic example prepared, the pyridone
24, was a relatively weak antagonist. Lowering the pKa
by adding a 6-cyano substituent to give 25 brought
about a significant improvement in potency, a change
that was all the more striking in the two more acidic pte-
ridinone analogues 23 and 26, which achieved nanomo-
lar potencies. Gratifyingly, the pteridinone 23 also
possessed good rat bioavailability, in contrast to all of
the neutral congeners tested. pKa is not the only relevant
factor in determining the activity of this series, however,
as demonstrated by the drop in potency of the acidic
pteridinedione antagonist 27. The 5,6-fused heterocyclic
Previous studies on the series of 2-aminothiazolopyrim-
idines such as 15,6 had determined the optimal combina-
tion of substituents attached to the central heterocyclic
ring system. A 5-thiobenzyl substituent was necessary
for good potency, with 2,3-difluoro substitution proving
optimal, and a 7-amino-alcohol group was required,
with (R)-alaninol the most potent. To facilitate fair com-
parison, these two substituents were incorporated in the
majority of compounds prepared for this study. The ta-
cit assumption that the substituent SAR is parallel

802
I. Walters et al. / Bioorg. Med. Chem. Lett. 18 (2008) 798–803
Table 2. CXCR2 potency,14 rat oral bioavailability (F), rat in vivo clearance (Cl), human plasma protein binding (hPPB) and CCR2 potency16 for
compounds 3a–f, 30a–f
OH
OH
HN
HN
S
S
N
N
H2N
O
N
N
S
Ar
N
H
N
S
Ar
30a-f
3a-f
Compound
Ar
CXCR2 IC50, nM
F, %
81
Cl, ml/min/kg
hPPB, %free
0.08
CCR2 pA2, nM
3a
30a
3
1
4
8
F
Cl
F
3b
30b
4
1
9
22
17
6
2.6
0.29
16
6
F
F
3c
30c
13
2
25
10
39
0.16
3d
30d
13
5
5
56
32
7
5.5
0.27
3e
30e
35
5
12
O
41
17
1.8
3f
30f
120
60
<1
17
43
6
N
S
0.87
analogues 28–30 also appear to show some dependence
on pKa. The essentially neutral pyrazole 29 has a similar
CXCR2 potency to the comparable pyridone 24.
Increasing the acidity a hundredfold in the imidazolidi-
none 28 was accompanied by a drop in activity, but this
is presumably due in part to the non-optimal 5-thioben-
zyl substituent. However, lowering the pKa further by
replacing one of the NH moieties of the imidazolidinone
with a sulfur atom gave the most potent class of CXCR2
antagonists, the thiazolo[4,5-d]pyrimidine-2(3H)-ones
30a–f (Table 2). Thus, the direct analogue of 28, the
thiazolone 30d (pKa = 7.0), is over a hundred times more
potent, and incorporation of optimal 5-substituents in
30a and 30b once more afforded low nanomolar CXCR2
antagonists with acceptable rat bioavailabilities. The
thiazolo[4,5-d]pyrimidine-2(3H)-one-based antagonists
were found to possess the best combination of potency
and pharmacokinetic properties, and several analogues
were prepared bearing different substituents. Some of
the properties of these compounds are tabulated in Ta-
ble 2 and compared with the corresponding 2-amino-
thiazolo[4,5-d]pyrimidines. The broadly parallel nature
of the substituent SAR of these two series is readily
apparent, perhaps an unexpected property given the pre-
sumably different binding nature of the neutral amino-
thiazole and acidic 2-thiazolone key pharmacophores.
Also noteworthy is the fact that representative com-
pounds from these series show comparable activity at
the CCR2 receptor (Table 2). The higher intrinsic
potency of the thiazolo[4,5-d]pyrimidine-2(3H)-ones is
partially offset by their higher plasma protein binding.
However, this is more than compensated for by their
higher rat bioavailabilities and lower metabolic clear-
ances, offering the potential for detailed in vivo profiling
of this series in animal models of inflammatory
disorders.
In conclusion, this survey of the heterocyclic core of a
series of bicyclic CXCR2 antagonists has resulted in
the discovery of a series of potent, orally bioavailable
compounds based on
a thiazolo[4,5-d]pyrimidine-
2(3H)-one core, which additionally possess high affinity
for the CCR2 receptor. The favourable pharmacokinetic
and physicochemical properties of this series render
them excellent investigative tools for the in vivo assess-
ment of combined CXCR2 and CCR2 antagonism.
The results of such studies will form the basis of forth-
coming publications.
References and notes
1. (a) Bizzarri, C.; Allegretti, M.; Di Bitondo, R.; Neve
Cervellera, M.; Colotta, F.; Bertini, R. Curr. Med. Chem.
Anti-Inflamm. Anti-Allergy Agents 2003, 2, 67; (b) Pease, J.
E.; Sabroe, I. Am. J. Resp. Med. 2002, 1, 19; (c)
Yamamoto, C.; Yoneda, T.; Yoshikawa, M.; Fu, A.;
Tokuyama, T.; Tsukaguchi, K.; Narita, N. Chest 1997,
112, 505; (d) Hay, D. W. P.; Sarau, H. M. Curr. Opin.

I. Walters et al. / Bioorg. Med. Chem. Lett. 18 (2008) 798–803
803
Pharmacol. 2001, 1, 242; (e) Takayama, F.; Miyazaki, T.;
Aoyama, I.; Tsukushi, S.; Sato, M.; Yamazaki, C.;
Shimokata, K.; Niwa, T. Kidney Int. 1998, 53, 1007.
2. Dawson, J.; Miltz, W.; Mir, A. K.; Wiessner, C. Exp.
Opin. Ther. Targets 2003, 7, 35.
3. (a) Loetscher, P.; Seitz, M.; Clark-Lewis, I.; Baggiolini,
M.; Moser, B. FEBS Lett. 1994, 341, 187; (b) Ahuja, S. K.;
Lee, J. C.; Murphy, P. M. J. Biol. Chem. 1996, 271, 225;
(c) Wolf, M.; Belen Delgado, M.; Jones, S. A.; Dewald, B.;
Clark-Lewis, I.; Baggiolini, M. Eur. J. Immunol. 1998, 28,
164.
4. (a) Combadiere, C.; Ahuja, S. K.; Van Damme, J.; Lee
Tiffany, H.; Gao, J.-L.; Murphy, P. M. J. Biol. Chem.
1995, 270, 29671; (b) Ward, S. G.; Westwick, J. Biochem.
J. 1998, 333, 457.
5. Baxter, A.; Cooper, A.; Kinchin, E.; Moakes, K.; Unitt, J.;
Wallace, A. Bioorg. Med. Chem. Lett. 2006, 16, 960.
6. Hunt, F.; Austin, C.; Austin, R.; Bonnert, R.; Cage, P.;
Christie, J.; Christie, M.; Dixon, C.; Hill, S.; Jewell, R.;
Martin, I.; Robinson, D.; Willis, P. Bioorg. Med. Chem.
Lett. 2007, 17, 2731.
7. (a) Albert, A.; Brown, D. J. J. Chem. Soc. 1954, 2060; (b)
Albert, A.; Lister, J. H.; Pedersen, C. J. Chem. Soc 1956,
4621; (c) Ram, S.; Evans, W.; Wise, D. S., Jr.; Townsend, L.
B.; McCall, J. W. J. Heterocycl. Chem. 1989, 26, 1053; (d)
Bonnert, R.; Walters, I. Int. Patent Appl. WO 02/32507.
8. Baker, B. R.; Joseph, J. P.; Schaub, R. E. J. Org. Chem.
1954, 19, 631.
9. (a) Forrest, H. S.; Hull, R.; Rodda, H. J.; Todd, A. R.
J. Chem. Soc. 1951, 3; (b) Albert, A.; Serjeant, E. P.
J. Chem. Soc. 1964, 3357; (c) Ladd, D. L. J. Heterocycl.
Chem. 1982, 19, 917; (d) Forrest, H. S.; Walker, J.
J. Chem. Soc. 1949, 2077.
11. Bonnert, R.; Cage, P.; Hunt, F.; Walters, I.; Willis, P. Int.
Patent Appl. WO 01/58902.
12. Anderson, G. L.; Richardson, S. G. J. Heterocycl. Chem.
1985, 22, 1735.
13. Baker, J. A.; Chatfield, P. V. J. Chem. Soc. C 1970, 2478.
14. CXCR2 binding affinity was determined via a scintillation
proximity assay (SPA) using
[
125I]IL-8 and human
recombinant CXCR2 (hrCXCR2) receptor expressed in
HEK 293 membranes. For full details, see: Austin, R.;
Baxter, A.; Bonnert, R.; Hunt, F.; Kinchin, E.; Willis, P.
Int. Patent Appl. WO 00/09511. IC50 values are means of
at least two independent observations. Errors are within
20%.
15. pKa’s were determined from changes in UV spectra as a
function of pH using a GLpKa auto titrator with DPAS
attachment (Sirius Analytical Instruments Ltd, Forest
Row, UK).
16. CCR2 potency was determined as follows: THP-1 cells
were grown in RPMI medium containing 10% (v/v) FCS
and 2 mM L-glutamine in a humidified incubator at 37 °C,
5% CO2. THP-1 cells were harvested by centrifugation at
300g for 5 min at room temperature. The cells were
resuspended in Tyrode’s Buffer (10 mM Hepes, pH 7.4,
137 mM NaCl, 2.7 mM KCl, 0.4 mM sodium dihydrogen
phosphate, 1.8 mM CaCl2, 1 mM MgCl2) containing 5 lM
Fluo3-AM and incubated at room temperature for 60 min.
The Fluo-3AM solution was removed by centrifugation at
300g for 5 min at room temperature and the cell pellet
resuspended in Tyrode’s Buffer. The assay was performed
in 96-well poly-D-lysine coated FLIPR plates. Compounds
in 0.75% DMSO were incubated with 200,000 cells per well
for 30 min at room temperature. Calcium transients in
response to various concentrations of MCP-1 were then
measured using a FLIPRTM (Molecular Devices) at room
temperature, and pA2 values for antagonism determined.
10. Bonnert, R.; Gardiner, S.; Hunt, F.; Walters, I. Int.Patent
Appl. WO 01/62758.