Special ergolines are highly selective, potent antagonists of the chemokine receptor CXCR3: Discovery, characterization and preliminary SAR of a promising lead

Article abstract of DOI:10.1016/j.bmcl.2009.09.002

The special ergoline 1 is a highly potent, selective antagonist of the chemokine receptor CXCR3. The surprising selectivity of this LSD-related compound can be explained by different electronic and steric properties of the ergoline core structure caused b

Full text of DOI:10.1016/j.bmcl.2009.09.002

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6185–6188
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Special ergolines are highly selective, potent antagonists
of the chemokine receptor CXCR3: Discovery, characterization
and preliminary SAR of a promising lead
*
Gebhard Thoma , Rolf Baenteli, Ian Lewis, Trixie Wagner, Lukas Oberer, Wolfgang Blum, Fraser Glickman,
Markus B. Streiff, Hans-Guenter Zerwes
Novartis Institutes for BioMedical Research, Forum 1, Novartis Campus, 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 special ergoline 1 is a highly potent, selective antagonist of the chemokine receptor CXCR3. The sur-
prising selectivity of this LSD-related compound can be explained by different electronic and steric prop-
erties of the ergoline core structure caused by the urea portion of the molecule. Discovery,
biopharmaceutical properties and first derivatives of this promising lead compound are discussed.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 5 August 2009
Revised 1 September 2009
Accepted 1 September 2009
Available online 6 September 2009
Keywords:
CXCR3
Ergoline
LSD
CXCL9
CXCL10
CXCL11
The chemokine receptor CXCR3 is a G-protein coupled heptah-
elical cell surface receptor binding the inflammatory chemokines
IP-10 (CXCL10), Mig (CXCL9) and I-TAC (CXCL11).1 The interaction
of CXCR3 and its ligands is involved in directing Th1 T cells to sites
of inflammation/immune injury.1 Both, CXCR3 and ligands are
upregulated and highly expressed in diseased tissues such as in
psoriasis,2 rheumatoid arthritis,3 multiple sclerosis,4 diabetes,5
and acute transplant rejection6 suggesting a causal role in patho-
logical processes. Furthermore, the involvement of CXCR3 and its
ligands in allograft rejection is claimed by literature reports on
transplantation experiments using both KO mice7 and neutralizing
principles such as anti CXCR3 monoclonal antibodies6a and anti-
sense peptide nucleic acid.8 However, these findings could not be
confirmed by us and others.9 Several reports on low molecular
weight (LMW) CXCR3 inhibitors have been recently published.10
High throughput screening of the Novartis compound collection
against CXCR3 led to the discovery of compound 1 (Fig. 1). The ori-
ginal hit 111 was resynthesized starting from the literature known
lysergic acid derivative 212 by treatment with phenyl isocyanate
(Scheme 1). The promising potency of the hit was confirmed in
binding,13 Ca2+-mobilization,14 and ligand-induced cell migration
assays15 (Table 1). The compound inhibited murine CXCR3 with
similar potencies. Its biopharmaceutical in vitro properties were
determined in a variety of profiling assays (Table 1). Compound 1
neither inhibited the cytochrome P450 enzymes nor the hERG
channel. Intrinsic clearance and log P are acceptable. The expected
oral bioavailability (Fm) based on permeability is high, water solu-
bility is low and protein binding is high. Consequently, the overall
profile is very promising for a screening hit.
As compound 1 is related to lysergic acid diethylamide (LSD) its
receptor selectivity was tested against a broad panel of >50 GPCRs.
It did not inhibit other chemokine receptors such as CCR5, CXCR4
O
N
N
O
N
N
O
N
N
H
N
H
O
N
H
N
H
N
H
1
LSD
2
* Corresponding author. Tel.: +41 61 3243342; fax: +41 61 3246735.
E-mail address: gebhard.thoma@novartis.com (G. Thoma).
Figure 1. Structures of selected ergolines.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.002

6186
G. Thoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6185–6188
tors, compound 1 inhibits only the 5HT2A receptor with an IC50
O
N
N
value below 1
hibit CXCR3.
lM (Table 2). Interestingly, compound 2 did not in-
O
O
N
N
H
N
This remarkable selectivity of compound 1 can be explained by
different electronic and steric properties compared to LSD and
compound 2. Under physiological conditions (pH 7.4) compound
1 is neutral whereas LSD (pKa = 7.8) is highly protonated which is
believed to be required for its hallucinogenic activity.16 Compari-
son of the X-ray crystal structures of compound 117 and LSD18 re-
vealed distinct conformations of the tetracyclic core structure
R
O
N
O
N
H
O
O
a
N
H
10: R=2-Ph-Me
11: R=3-Ph-Me
12: R=4-Ph-Me
13: R=2-pyridyl
14: R=3-pyridyl
15: R=4-pyridyl
3
4
O
N
N
b
c
N
N
(Fig. 2). The
D ring adopts a chair-like conformation in LSD and re-
H
lated ergolines18 but a boat-like conformation in compound 1. This
causes different positioning of the groups attached to N-6 and C-8.
NMR analyses in DMSO pointed towards similar conformations of
compound 1 in solution and solid state. A strong NOE observed be-
tween H-4ax and H-7ax (but no NOE between H-5ax and H-7ax
which would be required in case of a chair-like conformation of
16: R=3,4,5-PH(OMe)3
17: R=cycC6H12
N
H
2
N
H
a
the D-ring) indicated a boat-like conformation of the D-ring in solu-
O
N
N
O
N
N
tion. Furthermore, the coupling constant (J8–9 = 5.5 Hz) indicated a
torsion angle between H-8 and H-9 of 40–45° which is in good
agreement with the solid state (50°).19 In contrast, for amines such
as LSD and compound 2 a strong NOE between H-5ax and H-7ax was
H
N
H
N
d
O
O
observed confirming a chair-like conformation of the
D-ring as
N
H
N
H
found in the solid state.18 Compound 2 showed a very small cou-
pling constant (J8–9 <2 Hz) indicating a torsion angle close to 90°
which is in line with the solid state structure of LSD. In addition,
the bulky urea group at the 6 position might disturb high affinity
binding of compound 1 to most Serotonin, Dopamine and Adrener-
gic receptors.12a
5
1
g
e
f
O
N
N
N
O
N
N
The metabolic stability of compound 1 after incubation with
human, rat, mouse and dog liver microsomes was assessed by cap-
illary HPLC/MS–MS. The compound proved to be very stable with a
similar metabolite pattern in all species. The major metabolic path-
ways were N-de-alkylation of the amide, hydroxylation of the urea
phenyl and hydroxylation of the ergoline core. No evidence was
found for the cleavage of the urea leading to compound 2-like
structures which could cause undesirable side effects in the ner-
vous system.
A limited number of derivatives were prepared (Scheme 1 for
structures and Table 3 for potencies). The methylated urea 3 and
the carbamate 4 were found to be inactive or significantly less po-
tent than 1 pointing towards a key interaction of the urea hydro-
gen with the receptor. The C-8 epimer 5 and the amine 6 were
inactive indicating close contacts between amide group and pro-
tein. Hydrogenation of the double bond between C-9 and C-10
led to the considerably less potent compound 7. Thus, even rela-
tively moderate changes of the core modification strongly affect
H
N
H
N
H
N
N
O
O
O
N
H
N
N
8: R= Me
9: R= Bn
H
R
6
7
Scheme 1. Reagents and conditions: (a) Ar–NCO or Alkyl–NCO, NEt3, acetone, 3 h,
25 °C, 25–70%; (b) PhN(Me)COCl, pyridine, THF, 16 h, 25 °C, 60%; (c) PhOCOCl,
pyridine, THF, 16 h, 25°C, 65%; d) NaOH, MeOH, equilibration to ꢀ1:1 mixture of
epimers, separation by chromatography; (e) LiAlH4, THF, 0.3 h, 25 °C, 29%; (f) H2,
Pd(10%)/C, MeOH, 2 h, 25°, 28%; (g) MeI or BnBr; NaOH, Et3BnNCl, CH2Cl2, 3 h, 25 °C,
8: 56%. 9: 32%.
Table 1
Properties of compound 1
Assay
Result
hu CXCR3 (binding)
m CXCR3 (binding)
IC50 = 54 nM*
IC50 = 200 nM*
IC50 = 18 nM*
IC50 = 30 nM*
IC50 = 74 nM*
IC50 >10,000 nM
IC50 >10,000 nM
IC50 >10,000 nM
IC50 >10,000 nM
IC50 >10,000 nM
IC50 >30,000 nM
4.2
60
120
5 mg/L
5 mg/L
Log Pe = ꢁ4
Free fraction: 1%
Free fraction: 2%
hu CXCR3 (Ca2+
)
)
m CXCR3 (Ca2+
hu CXCR3 (I-TAC-induced migration)
CYP1A2
CYP2C9
CYP2C19
CYP2D6
Table 2
Selectivity profiles of compounds 1 and 2
Receptor
Compound 1 IC50 (nM)
Compound 2 IC50 (nM)
CXCR3
54
3600
750
2470
>10,000
9580
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
3
6
54
1290
16
143
4900
1590
660
2650
4030
1930
215
5760
CYP3A4
Serotonin 5HT1A
Serotonin 5HT2A
Serotonin 5HT2B
Serotonin 5HT2C
Serotonin 5HT6
Serotonin 5HT7
Dopamine D1
Dopamine D2
Dopamine D3
Dopamine D4.4
hERG
Log P
Intrinsic clearance (hu)
Intrinsic clearance (rat)
Solubility (pH 1.0)
Solubility (pH 6.8)
Permeability (PAMPA)
Protein binding (hu)
Protein binding (rat)
l
l
L minꢁ1 mgꢁ1
L minꢁ1 mgꢁ1
*
Adrenergic
Adrenergic
Adrenergic
a
a
a
1
2A
2C
Mean values of at least two independent measurements.
and CXCR2. Contrary to LSD and its close derivative 2 which effi-
ciently bind to various Serotonin, Dopamine and Adrenergic recep-
Adrenergic b1

G. Thoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6185–6188
6187
2
1
J8,9<2.0 Hz
J8,9=5.5 Hz
X-Ray Structures
7
6.5
6.2
6.5
6.2
H-9: 1H-NMR signal
H-9: 1H-NMR signal
6
7
O
R
7
O
6
8
HN
6
+
N
7
6
N
D
H
N
8
N
O
9
5
4
3
10
9
C
5
4
3
10
2
A
B
2
1
N
1
H
N
LSD (prot.)
1
LSD: R=Me
2: R=H
H
1
Figure 2. Molecular structures of protonated LSD and compound 1 and 1H NMR signals of H-8 of close LSD derivative 2 and compound 1.
Table 3
Potencies of derivatives of compound 1
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Compd
Binding IC50 (nM)
Ca2+-mobilization IC50 (nM)
*
*
1
2
3
54
18
n.t.
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>10,000
>10,000
803
>10,000
809
n.t.
>10,000
1930
60
510
28
45
100
62
67
6800
564
78
4
5
6
7
8
>10,000
>10,000
5300
220
9
590
10
11
12
13
14
15
16
17
146
231
313
428
285
2000
3050
179
*
Mean values of at least two independent measurements.
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potency. Methylation of the indole nitrogen gave the slightly less
potent derivative 8. The sterically more demanding benzyl group
of compound 9 led to a more pronounced drop of potency. We also
prepared a small series of compounds with modified urea groups.
Methyl-substitution of the phenyl ring was tolerated resulting in
slightly less potent derivatives 10–12. Introduction of a pyridyl in-
stead of the phenyl ring also led to reduced potency (13–15) par-
ticularly if the pyridine nitrogen is in the 4-position. The
trimethoxy-substituted compound 16 showed modest potency
whereas the cyclohexyl urea 17 was only slightly less active than
compound 1. Thus, the unsubstituted phenylurea seems to be opti-
mal for potency to CXCR3.
In conclusion, we discovered the special ergoline 1 which is a
highly selective, potent antagonist of the chemokine receptor
CXCR3. A preliminary structure activity relationship has been
established. Compound 1 was thoroughly profiled and is consid-
ered to be a very promising lead structure.
References and notes
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G. Thoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6185–6188
11. Analytical data of compound 1: 1H NMR (400 MHz, DMSO)
d
1.03 (3H, t,
Ca2+ mobilization in the presence of CXCR3 antagonists is reported as IC50
values i.e. the concentration of compound which reduced the maximal Ca2+
response to 50%.
J = 7.0 Hz, NCHaHb–CH3), 1.27 (3H, t, J = 7.0 Hz, NCHaHb–CH0 ), 2.93 (1H, t (br s),
J = 12.5 Hz, H-4ax), 3.18–3.27 (3H, m, H-7ax, NCHaHb–CH33, H-4eq), 3.33–3.42
(1H, m, NCHaHb–CH3), 3.48 (1H, dq, J = 14.0/7.0 Hz, NCH0 Hb–CH3), 3.59 (1H, dq,
J = 14.0/7.0 Hz, NCHaH0b–CH3), 3.71 (1H, dd, J = 5.5/4.0aHz, H-8), 4.42 (1H, d,
J = 14.0 Hz, H-7eq), 4.83 (1H, dd, 12.0/4.0 Hz, H-5), 6.40 (1H, d, J = 5.5 Hz, H-9),
6.91 (1H, t, J = 7.5 Hz, Ar–H), 7.02–7.11 (3H, m, Ar–H), 7.22 (3H, m, Ar–H), 7.39
(1H, d, J = 8.0 Hz), 8.95 (1H, s, NHurea), 10.76 (1H, s, NHindole). MS/HR HRMS: m/z
calcd for C26H28N4O2 [M+H]+: 429.2285; found: 429.2285.
15. Migration assay: the directed cell migration induced by CXCR3 ligands, for
example, I-TAC was assessed using 96-well disposable chemotaxis chambers
(Multiscreen MIC, Costar) with polycarbonate membranes containing pores of
5 lM diameter. Chemokine (I-TAC) was placed in the bottom well of the
chamber and cells (e.g., CXCR3 transfected L-1.2 cells) were placed in the top
compartment of the chemotaxis chamber. Cell migration across the porous
membrane was allowed for 4 h at 37 °C. Cells migrated from the top
compartment to the bottom compartment were quantified by flow
cytometry. When LMW inhibitors were tested, compounds were added to
both compartments at identical concentrations; Serial dilutions of compounds
were tested to assess their inhibitory effect on CXCR3 dependent cell
12. (a) Hoffman, A. J.; Nichols, D. E. J. Med. Chem. 1985, 28, 1252; (b) Nakahara, Y.;
Niwaguchi, T. Chem. Pharm. Bull. 1971, 19, 2337.
13. Ligand binding assay: cell membranes were prepared from CHO cells
transfected with human CXCR3. The binding of the 125I labeled CXCR3 ligand
I-TAC (CXCL11) to CXCR3 was assessed using the Scintillation Proximity Assay
(SPA) technology (Amersham Pharmacia Biotech). Buffer or serial dilutions of
compound were incubated for 2 h at room temperature with labeled CXCR3
ligand (e.g., I-TAC), CXCR3 expressing membranes and WGA coated PVT beads.
migration. The concentration of LMW CXCR3 inhibitors which led to
reduction of migrated cells by 50% was reported as IC50.
a
16. Baker, R. W.; Chothia, C.; Pauling, P.; Weber, H. P. Science 1972, 178, 614.
The plates were then centrifuged and counted in
a
Topcount (Packard)
17. Figure created with PLATON: Spek, A.L. Acta Crystallogr., Sect. C 1990, 34.
instrument. The data are reported as the concentration of compound required
to achieve 50% inhibition of 125I ligand binding. This assay has been adapted to
high throughput screening.
Crystallographic data for compound 1 have been deposited with the Cambridge
Crystallographic Data center as supplementary publication number CCDC
746865.
14. Ca2+-mobilization assay: CXCR3 ligand-induced Ca2+ mobilization was assessed
in CXCR3 transfected L1.2 cells (a mouse pre B cell line). For this, cells were
loaded with the Ca2+-sensitive fluorochrome Fluo-4 (Molecular Probes). After
washing, the cells were pre-incubated with low molecular weight inhibitors
for 2 h at room temperature. The transient increase in intracellular Ca2+ after
the addition of the CXCR3 ligand (I-TAC) was monitored in a fluorescence
image plate reader (FLIPR) instrument. The inhibition of CXCR3 ligand induced
18. It has been shown by X-ray structure analysis of LSD and a variety of related
ergoline derivatives that the conformation of their rigid fused ring system is
nearly identical in the solid state. Thus, it can be assumed that LSD and
compound 2 have very similar core structures in the solid state. Zhu, N.;
Johnson, L.; White, J.; Klein-Stevens, C. L.; Struct. Chem. 2002, 13, 491.
19. Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870.