Special ergolines efficiently inhibit the chemokine receptor CXCR3 in blood

Article abstract of DOI:10.1016/j.bmcl.2011.06.070

The structure-activity relationship of highly potent special ergolines which selectively block the chemokine receptor CXCR3 is reported. The most potent compounds showed IC₅₀ values below 10 nM in both ligand binding and Ca²⁺-mobilization assays. However, these compounds were poorly active in an assay that measures receptor occupancy in blood. Introduction of polar substituents led to derivatives with IC₅₀ values below 10 nM in this assay. Among them was compound 11a which showed both a favorable PK profile and cross reactivity with rodent CXCR3 making it a promising tool compound to further explore the role of CXCR3 in animal models.

Full text of DOI:10.1016/j.bmcl.2011.06.070

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4745–4749
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Special ergolines efficiently inhibit the chemokine receptor CXCR3 in blood
⇑
Gebhard Thoma , Rolf Baenteli, Ian Lewis, Darryl Jones, Jiri Kovarik, Markus B. Streiff, Hans-Guenter Zerwes
Novartis Institutes for BioMedical Research, Forum 1, Novartis Campus, Lichtstrasse 35, CH-4056 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure–activity relationship of highly potent special ergolines which selectively block the chemo-
kine receptor CXCR3 is reported. The most potent compounds showed IC50 values below 10 nM in both
ligand binding and Ca2+-mobilization assays. However, these compounds were poorly active in an assay
that measures receptor occupancy in blood. Introduction of polar substituents led to derivatives with IC50
values below 10 nM in this assay. Among them was compound 11a which showed both a favorable PK
profile and cross reactivity with rodent CXCR3 making it a promising tool compound to further explore
the role of CXCR3 in animal models.
Received 30 May 2011
Revised 14 June 2011
Accepted 15 June 2011
Available online 29 June 2011
Keywords:
CXCR3
Ergoline
CXCL9
Ó 2011 Elsevier Ltd. All rights reserved.
CXCL10
CXCL11
Chemokines are a group of chemotactic peptides that bind to G-
protein coupled receptors and regulate leukocyte trafficking to
lymphoid organs and tissues under resting conditions as well as
during inflammation.1 The inflammatory chemokine receptor
CXCR3 is predominantly expressed on activated inflammatory T
helper 1 (Th1) T cells, but not on resting cells or on Th2 cells.
CXCR3 binds with high affinities three different ligands, that is,
Several reports on low molecular weight CXCR3 inhibitors have
been published.10 We have reported on the discovery of the lyser-
gic acid-derived inhibitor 1a (Table 1) which is a selective and po-
tent antagonist of CXCR3.11 Contrary to basic ergoline derivatives
the neutral urea 1a did not significantly inhibit Serotonin, Andren-
ergic and Dopamine receptors. Therefore, we call this compound
‘special ergoline’. Here we describe the SAR of this promising lead
series and disclose derivatives with IC50 values below 10 nM in li-
gand binding and Ca2+-mobilization assays as well as in an assay
that measures receptor occupancy in blood.
A variety of amides were prepared from 6-nor-lysergic acid
derivative 2 (Scheme 1).12 Reaction with phenyl isocyanate gave
urea 3. Saponification led to acid 4 which was transformed into
the corresponding amides 1a–1w (Table 1). The epimers were sep-
arated on silica gel. The desired 8R-epimers generally eluted first.13
Diethylamide 1a showed promising inhibition in binding and
Ca2+-mobilization assays.14 Primary (1b) and secondary amides 1b
and 1c were inactive and significantly less potent, respectively,
whereas dimethylamide 1d showed slightly improved inhibition.
Introduction of polar groups led to decreased potency (1e and 1f).
Amides of cyclic amines were highly potent (1g–1j). Pyrrolidine-
amide 1h and piperidine-amide 1i were optimal for CXCR3 inhibi-
tion with IC50 values below 10 nM in both binding and mobilization
assays. Their unsaturated analogs 1k and 1l were similarly potent.
Introduction of heteroatoms in the ring system (1m–1r) led to de-
creased potencies compared to 1h and 1i. The morpholine amide
1q was the best compound in this sub-series. In a small series of
hydroxylated cyclic amides (1s–1w) hydroxy-piperidine amide 1s
was found to be the most potent derivative. The high potencies of se-
lected compounds (1a, 1h, 1i, 1q) were confirmed in an ITAC-in-
duced cell migration assays (74, 19, 13, 78 nM, respectively).14 All
Mig (CXCL9, monokine induced by IFN-c), IP-10 (CXCL10, IFN-c-in-
duced protein-10), and ITAC (CXCL11, IFN inducible T cell alpha
chemoattractant).2 These ligands are produced by activated leuko-
cytes (monocytes and macrophages), vascular endothelial cells and
activated tissue cells. The main inducing factor is the cytokine, IFN-
c
.3 Because IFN-
c itself and activated monocytes and macrophages
are present in most Th1-mediated inflammatory sites, high levels
of CXCR3 ligand expression as well as high numbers of CXCR3
expressing cells are observed in inflamed joints of rheumatoid
arthritis (RA) patients,4 in MS lesions in the brain,5 during pancre-
atitis in type 1 diabetes6 and in human allograft recipients during
episodes of acute rejection.7 Further evidence for an involvement
of CXCR3 and its ligands in allograft rejection stems from literature
reports on transplantation experiments utilizing both KO mice and
neutralizing principles such as anti CXCR3 monoclonal antibodies
and antisense peptide nucleic acid.8 However, these findings could
not be confirmed by us and others.9 Nevertheless, CXCR3 could be
an attractive target for pharmaceutical intervention because pre-
vention of ligand–receptor interactions may alleviate various
inflammatory conditions.
⇑
Corresponding author. Tel.: +41 61 3243342; fax: +41 61 3246735.
E-mail address: gebhard.thoma@novartis.com (G. Thoma).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.070

4746
G. Thoma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4745–4749
Table 1
Amide modifications
O
O
N
O
O
R1
N
O
H
N
a)
b)
R2
O
NH
70%
85%
O
H
N
N
N
H
N
H
2
3
N
H
R1
N
–NR1R2
Binding14
IC50 (nM)
Ca2+ mobil.14
O
OH
N
IC50 (nM)* hu (mouse)
*
O
R1
N
R2
O
c)
8R
H
R2
H
N
N
N
1a
1b
51
18 (30)
n.t.
H
N
6R
N
separation
of epimers
H
H
O
>10,000
10 - 40%
N
H
N
H
4
1a-w
for R1 and R2-groups see table 1
N
1c
379
28
71 (90)
10 (10)
H
Scheme 1. Reagents and conditions: (a) Ph-NCO, CH2Cl2, 20 °C, 16 h; (b) LiOH,
CH3OH/THF/H2O (1:2:1), 20 °C, 2 h; (benzotriazol-1-yloxy)-tripyrrolidinophospho-
nium-hexafluorophosphate (PyBOP), NEt3, CH2Cl2, 20 °C, 4 h or propanphosphonic
acid anhydride, NEt3, CH2Cl2, 0–5 °C, 1 h.
N
N
1d
O
1e
1f
305
66
187 (437)
12 (9)
O
compounds were found similarly active on murine CXCR3 in an as-
say monitoring I-TAC induced Ca2+ mobilization.
N
OH
Based on the highly active compound 1h a series of ureas were
prepared starting from lysergic acid (5). Transformation into pyr-
rolidine amide 6 was followed by N-demethylation which was
achieved applying the non-classical Polonovski protocol,15 a supe-
rior method when compared to the von Braun de-methylation
involving highly toxic and hazardous cyanogen bromide.16 Reac-
tion of 6 with perbenzoic acid led to the formation of the corre-
sponding N-oxide intermediate which was treated with Fe2+ to
give compound 7. Compounds 8b–8w were obtained from 7 (see
Scheme 2 and Table 2). Amides 8v and 8w were obtained from 7
by reaction with the corresponding acid chlorides (see Scheme 2).
The 2-fluorophenyl derivative 8b was less potent than 1h
whereas the 3- and 4-fluorophenyl ureas 8c and 8d showed com-
parable inhibition. Methoxy substituents resulted in a significant
drop of activity (8e–8g). Amino groups were not tolerated (8h–
8j). Thiophenyl ureas 8k and 8l showed only slightly reduced
potencies compared to 1h whereas pyrrole derivative 8m was
found to be completely inactive. In a small series of aliphatic deriv-
atives (8n–8s) only the cyclohexyl and cycloheptyl ureas 8l and
8m showed promising activities. Both the benzyl derivative 8t
and furanyl methyl derivative 8u were poorly active. Introduction
of amides instead of the urea led to poorly active derivatives 8v
and 8w. Thus the phenyl urea seems to be optimal for CXCR3
inhibition.
Several of our more potent CXCR3 antagonists were tested in
assays that measure receptor occupancy in blood. The assay prin-
ciple was based on the attribute that staining of blood lymphocytes
with anti-CXCR3 antibodies is prevented when I-TAC is added to
the blood samples and that the anti-CXCR3 staining was dose-
dependently restored when the samples were pre-incubated with
inhibitors before the addition of I-TAC.17 Very disappointingly,
compound 1h which showed IC50 values of 5 and 4 nM in binding
an mobilization assays, respectively, only modestly inhibited ITAC
binding to CXCR3 in both rat blood (IC50 = 3300 nM) and human
blood (IC50 = 700 nM). Related piperidine amide 1i showed an
IC50 value of 840 nM in human blood. Interestingly, the less potent
derivative 1t (c log P = 1.2) which is more polar than 1h
(c log P = 2.7) and 1i (c log P = 3.2) showed improved inhibition in
blood (rat: 410 nM; hu: 200 nM). Thus, we aimed at compounds
N
N
1g
1h
61
5
8 (14)
4 (4)
N
N
1i
1j
9
5 (6)
81
14 (13)
N
N
N
1k
1l
17
3 (3)
12
4 (2)
NH
1m
1010
457 (1730)
O
1n
255
74
N
N
NH
N
1o
1p
1q
131
825
22
83 (112)
286
O
N
N
N
O
16 (12)
N
N
O
1r
1160
216
O
S
1s
1t
400
14
67 (534)
5 (8)
O
N
N
OH
OH
1u
50
200 (40)
OH
N
N
1v
61
47
22
OH
1w
12 (19)
OH
*
Mean values of at least two independent measurements.

G. Thoma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4745–4749
4747
Table 2 (continued)
O
OH
N
O
N
–R3
Binding14
IC50 (nM)
Ca2+ mobil.14
IC50 (nM) hu (mouse)
*
*
a)
b)
N
N
56%
38%
8j
n.t.
5400
H
N
S
N
H
8k
8l
26
37
7 (11)
8 (10)
N
H
5
6
S
H
N
H
N
NH
8m
8n
8o
>10,000
160
>10,000
73 (81)
11(31)
O
N
N
N
O
H
N
c)
40 - 80%
R3
H
N
NH
24
O
H
N
8p
28
14 (16)
N
H
N
H
7
8b-w
for R-groups see table 2
H
N
OH
8q
8r
8s
>10,000
>10,000
n.t.
9475
H
N
CH3
Scheme 2. Reagents and conditions: (a) pyrrolidine (2 equiv), propanphosphonic
acid anhydride (1.5 equiv), NEt3 (2 equiv), CH2Cl2, 0–5 °C, 30 min; (b) (1) meta-
chlor-perbenzoic acid (1.25 equiv), 0 °C, 10 min, (2) FeSO4ꢀ7 H2O (0.5 equiv) CH3OH,
20 °C, 2 h; (c) R3-NCO, THF, 20 °C, 10 min; or diphosgene (1.5 equiv), NEt3, CH2Cl2,
20 °C, 20 min; then H2N–R3 (5 equiv), 20 °C, 2 h; or R3–COCl, NEt3, CH2Cl2, 20 °C,
30 min.
>10,000
>10,000
H
N
N
8t
2200
2600
2200
H
N
Table 2
Urea modifications
O
8u
10,000
H
N
O
N
N
8v
>10,000
427
6120
170
R3
8w
O
*
Mean values of at least two independent measurements.
N
H
–R3
Binding14
IC50 (nM)
Ca2+ mobil.14
IC50 (nM) hu (mouse)
*
*
equipotent to 1h with significantly increased polarity. As polar
groups in both the amide portion (1m–1w) and the urea portion
(8h–8j, 8m, 8q, and 8s) generally led to less potent derivatives
we decided to explore the indole nitrogen as a potential attach-
ment point. Starting from compound 1h a small series of deriva-
tives was prepared (Scheme 3). Isopropyl acetate was introduced
applying phase transfer conditions. The resulting ester 9 was re-
duced with lithium borohydride to give alcohol 10 which was tosy-
lated and transformed into amines 11a–11e.
Introduction of a relatively large lipophilic residue (compound
9, Table 3) led to reduced potency compared to 1h but did not com-
pletely abolish inhibition of CXCR3. However, introduction of a
neutral, hydrophilic residue (compound 10, Table 3) did not signif-
icantly affect inhibition in both binding and Ca2+-mobilization as-
says but significantly improved potency in blood. The same trend
was observed for compounds 11a18–11e containing basic amino
functions (Table 3). It is worthwhile to note that the improved
activities of compounds 10 and 11a–11e in the presence of blood
compared to 1h can not be explained by reduced protein binding
as the free fractions in rat and human serum for these compounds
were similar (1h: rat 4%, hu 3%; 10: rat 7, hu 8%; 11a rat 4, hu 7%;).
The in vivo PK properties of 11a were assessed in Sprague–
Dawley rats after intravenous and oral administration of the com-
pound (Table 4). The oral bioavailability was 97% and blood levels
(AUC normalized to a dose of 1 mg/kg was 2658 nM h), clearance
and half life (T1/2 = 8.9 h) were found to be favorable. The maximal
concentration (Cmax) normalized to a dose of 1 mg/kg was 137 nM.
In contrast to many basic ergoline derivatives lacking the urea
H
N
1h
8b
5
4 (4)
F
H
N
43
20 (16)
F
H
N
8c
8d
8e
4
3 (2)
4 (4)
60
H
N
F
7
MeO
H
N
523
OMe
H
N
8f
278
393
27 (74)
89
H
N
OMe
8g
N
8h
8i
n.t.
n.t.
>10,000
6200
H
N
N
H
N

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G. Thoma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4745–4749
Table 4
PK parameters of compound 11a
N
N
N
O
O
Parameter
11a
13
CL (mL minꢁ1 kgꢁ1
)
3
a)
b)
H
N
H
N
N
VSS (L kgꢁ1
t1/2term (h)
)
8.3 1.6
8.9 0.9
65%
64%
O
O
AUC iv d.n. (nM h)
20729 877
20658 447
97 16
137 30
3.1 1.8
AUC po d.n. (nM h)
F (%)
Cmax d.n. (nM)
Tmax (h)
N
N
H
1h
9
O
O
Formulations: iv 1 mg/kg, solution in NMP: PEG200 (30:70), volume 0.5 mL kgꢁ1
po 3 mg/kg, suspension in CMC/water/Tween (0.5:99:0.5), volume 2.5 mL kgꢁ1
CL (total blood clearance), VSS (apparent volume of distribution at steady state),
t1/2term (terminal half-life for elimination, AUC (area under the curve extrapolated to
;
N
N
N
N
O
O
.
c)
40 - 80%
H
N
H
N
infinity),
F (oral bioavailability), Cmax (maximal blood concentration after po
administration), Tmax (time of peak blood concentration after po administration, d.n.
(dose-normalized, i.e., calculated to a dose of 1 mg kgꢁ1).
O
O
N
OH
N
R4
10
11a-e
pH 6.8) and did not inhibit the hERG channel (IC50 >30
automated patch clamp assay).
lM in an
for R4-groups see table 3
Starting from the promising lead compound 1a we have discov-
ered highly potent, selective special ergolines such as 1h that inhi-
bit CXCR3 with IC50 values as low as 5 nM in both binding and
mobilization assays. However, 1h and related compounds showed
only modest potency in an assay that measures receptor occupancy
in blood. Introduction of polar, solubilizing residues led to deriva-
tives such as 10 and 11a with comparable potencies in binding and
mobilization assays but with highly promising activities in blood.
Favorable PK properties of 11a in rats make it a promising tool
compound to explore the role of CXCR3 in various disease models.
Scheme 3. Reagents and conditions: (a) BrCH2CO2iPr (4.0 equiv), BnNEt3Cl, CH2Cl2,
NaOH (40%), 0–5 °C, 1 h; (b) LiBH4 (4.0 equiv), THF, 0–5 °C, 5 h; (c) (1) TsCl
(1.5 equiv), DMAP (1.5 equiv), CH2Cl2, 25 °C, 2 h, (2) amine (20 equiv), CH3CN, 25–
55 °C, 1–5 h.
Table 3
Indole modifications
O
N
N
References and notes
H
N
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O
N
R4
–R4
Binding14 Ca2+ mobil.
IC50
Rat blood
IC50 (nM)
14
*
*
IC50
(nM)
(nM) hu (mouse)
1h
9
5
4 (4)
3300
n.t.
H
O
O
145
53 (53)
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10
6
2
5 (4)
2 (2)
7
5
OH
N
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11a
11b
11c
11d
11e
3
3 (2)
5 (3)
5 (3)
8 (5)
14
16
23
40
N
N
N
N
2
5
N
O
24
*
Mean values of at least three independent measurements.
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normal behavior indicating the absence of a metabolic conversion
into a basic ergoline. Compound 11a is highly soluble (>1 mM at
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14. For assay details see Ref. 9.
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17. ITAC binding assay in blood: EDTA anti-coagulated blood samples were
incubated with or without (controls) serial dilutions of low molecular weight
inhibitors for 1 h at 37 °C. ITAC was directly added to the samples followed by a
further incubation for 1 h at 37 °C. Subsequently the samples were stained
with fluorescence labeled anti-CXCR3 antibodies and the fluorescence signals
of lymphocytes measured by flow cytometry. We measured the degree of
restoration of the anti-CXCR3 stain by the inhibitors compared to the samples
that were not incubated with I-TAC. The calculated IC50 values for each
compound indicated at which concentration 50% of I-TAC binding to CXCR3
was inhibited.
18. Analytical data of compound 11a: 1H NMR (400 MHz, DMSO) d = 1.75–2.03 (4H,
m, pyrrolidine–CH2–CH2–CH2–CH2–), 2.18 (6 H, s, N(CH3)2), 2.60 (2 H, t,
J = 6.0 Hz, N–CH2–CH2–N(CH3)2), 2.93 (1H, t (br), J = 12.5 Hz, H-7ax), 3.18–3.28
(2 H, m), 3.28–3.38 (including H2O signal, m), 3.62–3.81 (3 H, m, H-8,
pyrrolidine–CH2–CH2–CH2–CH2–), 4.20 (2H, t, J = 6.0 Hz, N–CH2–CH2–N(CH3)2),
4.41 (1H, t (br), J = 12.5 Hz, H-7eq), 4.75 (1H, m, H-5), 6.52 (1H, d, J = 5.5 Hz, H-
9), 6.91 (1H, t, J = 7.5 Hz, Ar-H-9), 7.07–7.11 (3H, m, Ar-H), 7.22 (3H, m, Ar-H),
7.28 (1 H, dd, J = 6.0 Hz, J = 1.5 Hz), 7.38 (2 H, d, J = 7.0 Hz), 9.12 (1H, s, NHurea);
MS/HR HRMS: m/z calcd for C30H35N5O2 [M+H]+: 498.2864; found: 498.2864.