Synthesis of 7-deaza-8-bromo cyclic adenosine 5′-diphosphate ribose: The first hydrolysis resistant antagonist at the cADPR receptor

Article abstract of DOI:10.1039/a700336f

7-Deaza-8-bromo cyclic adenosine 5′-diphosphate ribose is synthesised from 7-deazaadenosine via 7-deaza-8-bromo nicotinamide adenine dinucleotide; it is both a more potent antagonist than the 8-bromo derivative and has the advantage of chemical and enzyma

Full text of DOI:10.1039/a700336f

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Synthesis of 7-deaza-8-bromo cyclic adenosine 5A-diphosphate ribose: the first
hydrolysis resistant antagonist at the cADPR receptor
Victoria C. Bailey,a Jaswinder K. Sethi,b Antony Galioneb and Barry V. L. Potter*a
a Department of Medicinal Chemistry, School of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, UK
BA2 7AY
b Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK OX1 3QT
7-Deaza-8-bromo cyclic adenosine 5A-diphosphate ribose is
synthesised from 7-deazaadenosine via 7-deaza-8-bromo
nicotinamide adenine dinucleotide; it is both a more potent
antagonist than the 8-bromo derivative and has the ad-
vantage of chemical and enzymatic hydrolytic stability.
to the increased hydrophobic nature of the modified purine ring
(an extra CH group and the bromo substituent), 8 eluted at a
higher concentration of buffer than expected, namely 250–300
mm TEAB as opposed to the 200–250 mm observed for
adenosine 5A-monophosphate, and was not contaminated by
inorganic phosphate, often a problem with this method.
Dicyclohexylcarbodiimide coupled 8 to nicotinamide mono-
nucleotide (NMN).16 The novel pyrophosphate 9† was purified
from monophosphates and symmetrical pyrophosphates by ion
exchange chromatography. 7-Deaza-8-bromo-NAD+ eluted at
120–150 mm TEAB as its triethylammonium salt.
Cyclic adenosine 5A-diphosphate ribose (cADPR, 1, Fig. 1) is a
Ca2+-mobilising agent known to act via ryanodine receptors to
release internal stores of Ca2+ in a wide variety of cell types,1–3
independent of the second messenger myo-inositol 1,4,5-tris-
phosphate.4 The role of cADPR is not yet fully understood, but
it may act as a second messenger for a number of receptor
agonists, including nitric oxide.5 The binding protein for
cADPR is unknown, and there is doubt whether cADPR acts
directly at the ryanodine receptor, although its action involves
calmodulin.6 Hence it is important to build up a structure–
activity profile for cADPR in relation to Ca2+ release and to
design molecules to probe its biological role.
Finally, the NAD+ analogue was cyclised using the Aplysia
enzyme.11 7-Deaza-8-bromo-NAD+ cyclised at a similar rate to
cADPR. The product was purified by ion exchange chromato-
graphy, at 130–150 mm TEAB, as the triethylammonium salt.
Phosphates 8, 9 and 5‡ were quantified using total phosphate
analysis9,17 from which the extinction coefficients were calcu-
Analogues substituted at the 8-position including the
8-bromo 2 and 8-amino 3 derivatives are documented as
antagonists at the cADPR receptor.7 Importantly, 8-amino-
cADPR inhibits cardiac excitation–contraction coupling.8 Re-
cently, we reported 7-deaza-cADPR 4 as the first partial
agonist, highlighting the N-7 position as an essential motif for
effective Ca2+ release.9 Furthermore, 7-deaza-cADPR is resis-
tant to both chemical and enzymatic hydrolytic attack, a
property desirable to confer upon an antagonist, to improve its
physiological value. We have combined these two modifica-
tions and we now report 7-deaza-8-bromo-cADPR 5 as the first
hydrolysis resistant cADPR antagonist.
There is no effective chemical synthesis of cADPR, the only
published route produces the correct stereoisomeric product in
very low yield.10 We, like others, have relied upon a
chemoenzymatic route to develop structural diversity7,11 utilis-
ing Aplysia ADP-ribosyl cyclase.12 The enzyme is extremely
flexible in its substrate specificity.11–13
NH2
NH2
N
N
Br
H
O
HO
HO
i
N
N
N
O
N
OH
OH
OH
OH
6
7
ii
NH2
N
O
Br
–O
P
O
N
N
O
O–
OH
OH
8
iii
NH2
O
N
NH2
O
O
Br
7-Deazaadenosine (Tubercidin, 6) was selectively bromi-
nated at the pseudo-8-position (6-position) using
N-bromosuccinimde (Scheme 1). After chromatography, dry 7
was phosphorylated at the 5A-hydroxy group using essentially
the general method described by Yoshikawa et al.15 The novel
8‡ was purified by ion exchange chromatography using
triethylammonium hydrogen carbonate buffer (TEAB). Owing
+
N
N
O
P
O
P
O
N
O
O–
O–
O
OH
OH
9
OH
OH
iv
OH
OH
O
3′R
OH OH
2′R
+
NH2
O
N
O
P
4′R
1′R
–O
N
O
O
1
1 X = N, Y = H, cADPR
NH2
6
O
5′R
N
P
O
O
+
P
2
3
O
2 X = N, Y = Br, 8-Bromo-cADPR
3 X = N, Y = NH2, 8-Amino-cADPR
4 X = CH, Y = H, 7-Deaza-cADPR
5 X = CH, Y = Br, 7-Deaza-8-bromo-cADPR
N
O–
O
–O
5
N
Br
O
P
O–
X 7
8
4
O
5′A
O
N
9
OH
OH
O
Y
4′A
5
1′A
OH OH
3′A 2′A
Scheme 1 Reagents and conditions: i, NBS, KOAc, DMF, 10 min, 40%; ii,
PO(OEt)3 50 °C, 15 min, then POCl3 (1.5 equiv.), 0 °C, 2 h, then H2O, 44%;
iii, DCC, NMN, pyridine–H2O (4:1), 7 d, 28%; iv, 1.5 mm solution in 25
mm HEPES, ADP-ribosyl cyclase, 30 min, 31%
Fig. 1 Structures of cADPR 1, 8-bromo-cADPR 2, 8-amino-cADPR 3,
7-deaza-cADPR 4 and 8-bromo-7-deaza-cADPR 5
Chem. Commun., 1997
695

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lated, and used to quantify reaction yields and solutions for the
biological evaluation of 5.
We thank the Medical Research Council for a Project Grant
(A. G., B. V. L. P.) and two studentships (V. C. B., J. K. S.).
A. G. is a Wellcome Trust Fellow and B. V. L. P. is a Lister
Institute Research Professor.
The pharmacology of 5 was investigated using sea urchin egg
homogenate.18 7-Deaza-8-bromo-cADPR antagonised the ac-
tion of 100 nm cADPR-induced Ca2+ release (Fig. 2),
desensitised the receptor, and was apparently more potent and
efficacious than 8-bromo-cADPR (IC50 values of 0.73 ± 0.05
and 0.97 ± 0.04 mm respectively). Furthermore, it was resistant
to chemical hydrolysis and induced antagonism even after being
incubated in buffer at 85 °C for 90 min. HPLC analysis of a
control incubation showed that 7-deaza-8-bromo-cADPR was
still present confirming that cyclic material, and not a
metabolite, caused antagonism even after heat treatment. In
contrast, after identical treatment, 8-bromo-cADPR was no
longer able to induce antagonism and HPLC showed no cyclic
material remaining. Resistance to enzymatic hydrolysis was
tested by pre-incubating the antagonist with homogenate
overnight, when an aliquot was removed and its ability to
antagonise cADPR-induced Ca2+ was tested. Whilst the
8-bromo-cADPR antagonism had reduced significantly,
7-deaza-8-bromo-cADPR was still a significant antagonist. Full
biological details will be reported elsewhere.
In 7-deaza analogues of adenosine the glycosidic bond is
more resistant to acid catalysed hydrolysis than in adenosine.19
Similarly, the increased hydrolytic resistance of 7-deaza
analogues of cADPR is thought to arise from increased stability
of the N1 ribosyl linkage. The N1 position of 7-deazaadenosine
is more basic than that of adenosine (pKa = 5.2 and 3.6
respectively20) and similarly this position is more basic in the
8-bromo substituted analogues than in adenosine (pKa = 4.02
for 8-bromoadenosine21). The combination of modifications,
therefore, makes the heterocycle substantially more basic at N1
than in adenine. Hydrolysis of the N1 ribosyl linkage probably
involves participation of a ribosyl oxygen lone pair to form a
carbocation which is then captured by water. The ability of the
N1-ribosyl linkage to be hydrolysed should therefore depend
upon the leaving group ability of the purine. The more acidic the
purine, the better leaving group it will be and, conversely, with
the more basic character of N1 in 7-deaza-8-bromo-cADPR, the
harder it will be to cleave the N1 ribosyl bond.
Footnotes
* E-mail: B.V.L.Potter@bath.ac.uk
† Spectroscopic data for 8: dH (400 MHz, D2O): 4.08–4.15 (3 H, m, H4A and
H5A), 4.53 (1 H, dd, J 5.7, 5.8 Hz, H3A), 5.07 (1 H, dd, J 5.3, 5.8 Hz, H2A),
6.04 (1 H, d, J 5.3 Hz, H1A), 6.42 (1 H, s H7), 7.92 (1 H, s, H2). dP (161.7
MHz, D2O): 2.35 broadens when proton coupled. dC (100.4 MHz, D2O):
65.2 (d, JCP 3.7 Hz, C5A), 70.3 (C3A), 72.1 (C2A), 83.6 (d, JCP 7.4 Hz, C4A),
89.8 (C1A), 104.0 (C5), 105.2 (C7), 110.4 (C8), 149.9 (C4), 150.0 (C2),
154.5 (C6). lmax 276 nm, e = 13.3 3 103 dm3 mol21 cm21. m/z (FAB+)
425, 427 (M + H)+, (FAB2) 422, 424 (M 2 H)2, 341 (M 2 H 2 Br)2.
For 9: dH (400 MHz, D2O): 4.03–4.32 (8 H, m, HN2A, HN3A, HA4A, HN4A,
HA5A HN5A), 4.56 (1 H, dd, J 5.8, 5.3 Hz, HA3A), 5.16 (1 H, dd, J 5.9, 5.8 Hz,
HA2A), 5.99 (1 H, d, J 4.4 Hz, HN1A), 6.06 (1 H, d, J 5.9 Hz, HA1A), 6.60 (1
H, s, HA7), 8.05 (1 H, s, HA2), 8.14 (1 H, dd, J 8.3, 6.4 Hz, HN3), 8.73 (1
H, d, J 8.3 Hz, HN4), 9.09 (1 H, d, J 6.4 Hz, HN6), 9.25 (1 H, s, HN2). dP
(161.7 MHz, D2O): 210.5, 210.9 (2d, JPP 20.1 Hz). dC (100.4 MHz, D2O):
65.7 (d, J 3.7 Hz, CA5A), 66.5 (CN5A), 70.0 (CA3A), 71.4 (CN3A), 71.9 (CA2A),
78.5 (CN2A), 83.5 (d, J 9.2 Hz, CA4A), 87.8 (d, J 9.2 Hz, CN4A), 89.8 (CA1A),
100.9 (CN1A), 104.3 (CA5), 105.0 (CA7), 110.5 (CA8), 129.6 (CN4), 140.6
(CN5), 143.1 (CN6), 146.3 (CN2), 150.4 (CA2), 150.7 (CA4), 155.5 (CA6),
166.1 (CO). lmax 270 nm, e
=
13.3 3 103 dm3 mol21 cm21. m/z
(electrospray+) 741, 743 (M + H)+, (electrospray2) 739, 741 (M 2 H)2.
For 5: dH (400 MHz, D2O): 3.89–3.93 (1 H, m, HA5A), 3.98–4.03 (1 H, m,
HR5A), 4.15–4.20 (1 H, m, HA4A), 4.23–4.27 (1 H, m, HR5A), 4.30–4.40 (2 H,
m, HA5A, HR3A), 5.44 (1 H, dd, J 5.2, 6.1 Hz, HA2A), 5.97 (1 H, d, J 4.0 Hz,
HR1A), 6.03 (1 H, d, J 6.1 Hz, HA1A), 6.91 (1 H, s, HA7), 8.75 (1 H, s, HA2),
all other protons are obscured by the water peak at d 4.8. dP (161.7 MHz,
D2O): 210.8, 211.6 (2d, JPP 18 Hz). lmax = 277 nm, e = 10.85 3 103 dm3
mol21 cm21. m/z (electrospray+) 619, 621 (M + H)+, (electrospray2) 617,
619 (M 2 H)2, 79, 81 (Br)2.
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Combined modifications at the 7 and 8 positions of cADPR
thus produce slightly better antagonistic properties, with the
marked added advantage of hydrolytic resistance. One of the
most commonly employed antagonists, 8-amino-cADPR, is
unsuitable for microinjection work due to its chemical instabil-
ity.22 7-Deaza-8-bromo-cADPR, resistant to both chemical and
enzymatic hydrolysis, should be a powerful tool to investigate
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2 µM 8-Br-7-deaza-cADPR
2 µM 8-Br-cADPR
2 nmol
Ca
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100 nM cADPR
100 nM cADPR
100 nM cADPR
Fig. 2 Lytechinus pictus egg homogenates (2.5% v/v) (ref. 18) containing
Fluo-3 (3 mm) were incubated at 17 °C. Homogenate (500 ml) was
challenged with cADPR (final concentration 100 nm). Ca2+ release was
monitored by a change in fluorescence. 7-Deaza-8-bromo-cADPR was
tested for its antagonism by pre-treating the homogenate (500 ml) with the
sample (final concentration 2 mm) 3 min prior to addition of cADPR (final
concentration 100 nm). A subsequent addition of cADPR (100 nm)
demonstrated the desensitisation of the Ca2+ release mechanism. The traces
shown are representative of several such experiments.
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Received, 14th January 1997; Com. 7/00336F
696
Chem. Commun., 1997