P. Oliva et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128137
R-dependent calcium mobilization in 1321 N1 human astrocytoma cells stably expressing the human P2Y R. Potencies are presented
a
Agonist potencies reflect P2Y
6
6
in the form of EC50 values, which represent the concentration of agonist at which 50% of the maximal effect on calcium mobilization is achieved. These values were
determined using a four-parameter logistic equation and the GraphPad software package (GraphPad, San Diego, CA). The results are presented as mean ± standard
error and are the average of three to six different experiments, unless noted, with each molecule.
b
c
4
EC50 values from Maruoka et al. (PLC assay).
All syntheses for indicated compounds were reported in Junker et al., as well as the P2Y
2+
R Ca assay results for compound 30.
33
6
d
2+
3
Reported in Toti et al. (Ca assay).
e
Structures of 39: 40:
n = 1 (tested once).
f
Representative concentration–response curves are shown in Fig. 1.
All of the compounds synthesized here contained a
50% binding inhibition at 10 µM) off-target activities. At 10 µM, 26
inhibited binding at the D dopamine receptor (85%), and 27 inhibited
binding at the receptor (59%). Nevertheless these nucleotides are
α
,β-methylene
3
bridge, while 14–30 and 37, 38 additionally contained an
alkyloxyimino-pyrimidine nucleobase. Several of the compounds were
σ
1
non-promiscuous in binding.
4
previously reported by us as intermediates and as P2Y
6
R agonists in the
N -Benzyloxy-CDP analogues containing a stabilizing
α,β -methylene
synthesis of CD73 inhibitors.33 Selected analogues were also measured
in a fluorescent P2Y14R binding assay by flow cytometry of whole CHO
cells expressing the hP2Y14R to establish selectivity (Table 2).
bridge were prepared and evaluated as P2Y
between cytidine and alkoxy amines afforded single condensation
products, which we represent with a Z-exo C N double bond, consistent
with previous reports, including X-ray crystallographic structures ob-
6
R agonists. The reactions
–
Reference compounds 1, 2, 9, 10, and 15 are included in Table 1.3
The P2Y
–5
tained for model compounds.3
8,39
Considerable freedom of substitution
6
R potency of UDP 1 (EC50 0.0416 µM) was reduced 12- to 19-
fold with 4-imino-oxyalkyl modifications in 11 and 13. Nevertheless, we
sought to explore the interaction of bound to a nucleotide agonist with
this distal region of the receptor, especially in combination with a sta-
of the benzyl ring is present, while substitution of the pyrimidine C5
3
position (halo, methyl) but not the N position (alkyl) is compatible with
6
P2Y R activation. SAR studies of several other P2YRs have revealed that
6
bilizing methylene phosphonate. Previously, considerable P2Y R
agonist potency was observed upon 4-imino-oxyalkylaryl substitution of
4
UDP.
Table 2
4
Although the N -methoxy
α,β-methylene analogue 9 (0.42 µM) was
4
Potency of
α
,β-methylene analogues of UDP and CDP at the human P2Y
hP2Y
6
R and
more potent than the corresponding N -benzyloxy analogue 10 (2.36
µM), we examined functional group substitution of the phenyl ring of
0. 3-Substituted benzyloxy groups (19–22) displayed EC50 values in
a
P2Y14R.
6
R
hP2Y14R
1
Compound
EC50 ± SEM (µM) %
IC50 ± SEM (µM)
the range of 0.9–2.7 µM, while 4-substituted benzyloxo groups (23–27)
displayed EC50 values in the range of 0.57–2.3 µM. Notably, the IC50
values for hP2Y14R binding of compounds 20, 23 and 25 were > 100
µM, indicating a high degree of selectivity. A 4-trifluoromethyl-benzyl
substituent in 25 provided the highest potency (0.57 µM), and a 5-fluoro
b
b
14a
14b
16
0.339
0.011
c
b
b
1.99
0.00092
0.203 ± 0.030
0.097 ± 0.011
1.45 ± 0.22
0.362 ± 0.090
16.6 ± 10.3
>100
17
2
2
2
2
0
3
5
8
1.32 ± 0.08
>100
substitution of the cytosine ring in 28 similarly enhanced potency, with
0.571 ± 0.196
0.549 ± 0.070
1.39 ± 0.223
>100
4
>
175- and 39-fold selectivity over P2Y14R, respectively. N -Benzoyl
21.5 ± 11.1
6.66 ± 4.74
derivative 30 was a potent P2Y
-pentafluorosulfanyl group (SF
tency. 5-Methyl substitution of the cytosine ring in 29 led to an EC50 of
6
R agonist (1.39 µM), but nonselective. A
30
4
5
) group36 in 27 maintained P2Y
6
R po-
%
activation at 3 µM, unless noted. Fluorescent antagonist was used as a tracer
32
for flow cytometry of hP2Y14R-CHO cells, unless noted.
a
1
.09 µM, i.e. slightly more potent than 10. 3-Alkyl or aryl alkyl (31–33,
7, 38), β-D-arabinofuranose 39, 6-aza 40 substitution, as previously
2+
6
FLIPR (Ca ) assay in hP2Y R-1231 N1 astrocytoma cells, expressed as EC50
3
or.
3
3
synthesized, was shown here to completely prevent P2Y
6
R activation.
,β-methylene series
R, in contrast to closely related, potent uracil de-
b
The 5-substituted cytosine derivatives 34–36 in the
were inactive at P2Y
α
6
Potency in a phospholipase C assay of hP2Y R activation (Maruoka et al.,
4
9
2010) or in a cAMP assay of hP2Y14R activation (Das et al., 2010).
6
3
4
rivatives (15, 16). Curiously, alkylation of N -position of N -benzyloxy
′
analogues (37, 38) led to a complete loss of P2Y
,β-methylene-UDP 16 was nonselective between P2Y
affinity 0.2–0.3 µM).
P2Y agonist 26 and 27 and P2Y14 agonist 14b were chosen as
representative UDP analogues containing a stabilizing
,β-methylene
6
R activity. 5-Fluoro-5 -
α
6
R and P2Y14R
(
6
c
Structure of 14b:
α
bridge for the determination of off-target activities at 46 receptors,
channels and transporters (Supporting information) by the Psychoactive
Drug Screening Program (PDSP).37 14b showed no significant (i.e. >
4