Cyclic adenosine 5′-diphosphate ribose analogs without a southern ribose inhibit ADP-ribosyl cyclase-hydrolase CD38

Article abstract of DOI:10.1021/jm501037u

Cyclic adenosine 5′-diphosphate ribose (cADPR) analogs based on the cyclic inosine 5′-diphosphate ribose (cIDPR) template were synthesized by recently developed stereo- and regioselective N1-ribosylation. Replacing the base N9-ribose with a butyl chain generates inhibitors of cADPR hydrolysis by the human ADP-ribosyl cyclase CD38 catalytic domain (shCD38), illustrating the nonessential nature of the southern ribose for binding. Butyl substitution generally improves potency relative to the parent cIDPRs, and 8-amino-N9-butyl-cIDPR is comparable to the best noncovalent CD38 inhibitors to date (IC₅₀ = 3.3 μM). Crystallographic analysis of the shCD38:8-amino-N9-butyl-cIDPR complex to a 2.05 ? resolution unexpectedly reveals an N1-hydrolyzed ligand in the active site, suggesting that it is the N6-imino form of cADPR that is hydrolyzed by CD38. While HPLC studies confirm ligand cleavage at very high protein concentrations, they indicate that hydrolysis does not occur under physiological concentrations. Taken together, these analogs confirm that the northern ribose is critical for CD38 activity and inhibition, provide new insight into the mechanism of cADPR hydrolysis by CD38, and may aid future inhibitor design.

Full text of DOI:10.1021/jm501037u

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Cyclic Adenosine 5′-Diphosphate Ribose Analogs without a
Southern” Ribose Inhibit ADP-ribosyl Cyclase−Hydrolase CD38
“
†
‡
‡
†
‡
Joanna M. Swarbrick, Richard Graeff, Hongmin Zhang, Mark P. Thomas, Quan Hao,
and Barry V. L. Potter*
,†
†Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down,
Bath, BA2 7AY, United Kingdom
‡
Department of Physiology, University of Hong Kong, Hong Kong, China
*
S Supporting Information
ABSTRACT: Cyclic adenosine 5′-diphosphate ribose
(
cADPR) analogs based on the cyclic inosine 5′-diphosphate
ribose (cIDPR) template were synthesized by recently
developed stereo- and regioselective N1-ribosylation. Replac-
ing the base N9-ribose with a butyl chain generates inhibitors
of cADPR hydrolysis by the human ADP-ribosyl cyclase CD38
catalytic domain (shCD38), illustrating the nonessential nature
of the “southern” ribose for binding. Butyl substitution
generally improves potency relative to the parent cIDPRs, and 8-amino-N9-butyl-cIDPR is comparable to the best noncovalent
CD38 inhibitors to date (IC = 3.3 μM). Crystallographic analysis of the shCD38:8-amino-N9-butyl-cIDPR complex to a 2.05 Å
5
0
resolution unexpectedly reveals an N1-hydrolyzed ligand in the active site, suggesting that it is the N6-imino form of cADPR that
is hydrolyzed by CD38. While HPLC studies confirm ligand cleavage at very high protein concentrations, they indicate that
hydrolysis does not occur under physiological concentrations. Taken together, these analogs confirm that the “northern” ribose is
critical for CD38 activity and inhibition, provide new insight into the mechanism of cADPR hydrolysis by CD38, and may aid
future inhibitor design.
INTRODUCTION
adenine dinucleotide phosphate (NAADP) from nicotinamide
11
■
1
,2
adenine dinucleotide phosphate (NADP).
Cyclic adenosine 5′-diphosphate ribose (cADPR, 1, Figure 1)
CD38 knockout studies have revealed the importance of this
pathway in a range of diseases. CD38 is a marker in AIDS
is synthesized in biological systems from nicotinamide adenine
12
progression and a negative prognostic marker of chronic
13
lymphocytic leukemia. It acts to regulate intracellular levels of
+
NAD , being implicated in energy homeostasis, signal trans-
1
4−16
duction, and aging,
and recently has been shown to be
17
critical for social behavior in mice. The emerging role of
CD38 in disease states is stimulating the search for modulators
of activity for chemical biological studies and to provide
structural clues for drug design and potential therapeutic
18
candidates. To date, CD38 inhibitors fall broadly into two
categories: mechanism-based covalent inhibitors that bind to
the catalytic residue, and reversible, competitive, noncovalent
inhibitors. Most are derived from NAD+ and designed as
inhibitors of the predominant NADase activity of CD38.
Nicotinamide-based derivatives have demonstrated nanomolar
inhibition of NADase activity by covalent modification of
Figure 1. Structure of cADPR and N9-butyl analogs.
+
dinucleotide (NAD ) by ADP-ribosyl cyclases (ADPRCs). It
1
9
3
−6
CD38
and low millimolar activity when designed as
acts as a second messenger, mobilizing intracellular calcium.
In humans, formation of cADPR is catalyzed by the
20
membrane permeable analogs. Competitive inhibitors are
more diverse in structure, with a nonhydrolyzable NAD analog
reported (IC50 ≈ 150 μM), flavonoids showing low
micromolar inhibition and the development of a hit from
+
7
multifunctional transmembrane glycoprotein ADPRC CD38.
2
1
+
CD38 also acts as an NAD glycohydrolase (NADase) and as a
22
cADPR hydrolase to generate adenosine 5′-diphosphate ribose
2
+
8−10
(
ADPR), another Ca -releasing second messenger.
Under
2
+
acidic conditions, CD38 generates the most potent Ca -
releasing second messenger known to date, nicotinic acid
Received: July 10, 2014
Published: September 16, 2014
©
2014 American Chemical Society
8517
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Figure 2. N9-Butyl-cIDPR docked into 2PGJ crystal structure. Comparison between 2PGJ crystal structure of N1-cIDPR (carbons in cyan) bound to
shCD38 and (A) docked N9-butyl cIDPR 4 (carbons in yellow) showing overlap of their structures in the binding pocket; (B) interaction of 8-NH2-
N9-butyl cIDPR 7 (carbons in pink) with critical residues in the binding pocket and additional predicted H-bond to Asp155.
commercial libraries generating the most active reported
RESULTS AND DISCUSSION
■
23
noncovalent inhibitor to date (IC50 = 4.7 μM).
As both cADPR and ADPR are derived from a common
Crystallography of shCD38 has revealed the mechanism by
+
which NAD is either cyclized to cADPR or hydrolyzed to
2
4
intermediate, we chose to design product-like inhibitors based
on the cADPR structure to exploit inhibition of CD38 cADPR
hydrolase activity. cADPR itself is unattractive for inhibitor
design, since it is hydrolyzed at the N1 link in both neutral
39−41
ADPR.
Crystal structures obtained with shCD38 and
4
2
unnatural ligands 2 (PDB code 2PGJ), cyclic adenosine 5′-
diphosphate carbocyclic ribose (cADPcR, 3UHI), and 8-
4
3
NH -cIDPR (3U4H) suggest a critical role for the base and
2
25−27
aqueous solution and under physiological conditions.
“northern” ribose in the binding of cADPR analogs to CD38, as
might be predicted for the locus of both cADPR formation and
degradation. Glu146 hydrogen-bonds to N6 and N7 and is
More stable analogs have been accessed by one of two routes:
either a chemoenzymatic route modeled on the biosynthesis of
4
4
+
critical in regulating the ADPRC multifunctionality. Glu226 is
cADPR from NAD or by total chemical synthesis. We have
the catalytic residue and interacts with the 2″- and 3″-OH of
previously reported a chemoenzymatic route to N1-cyclic
inosine 5′-diphosphate ribose (cIDPR, 2) via its 8-bromo
45
the “northern” ribose. It has recently been highlighted as
+
46
2
8
crucial in orientating NAD for cleavage of nicotinamide. The
southern” ribose appears to be accommodated in a more
derivative (8-Br-cIDPR, 3, Figure 1). Chemically and
biologically stable, 2 and 3 both inhibit cADPR hydrolysis by
the catalytic domain of CD38 (shCD38; IC50 of 276 and 158
“
flexible fashion across the open face of the pocket. Thus, we
designed analogs in which the “southern” ribose is replaced
with an N9-butyl linker (N9-butyl-cIDPRs 4−7, Figure 1) to
explore whether it is required for binding to CD38. Such
analogs would be predicted to have the further advantage that
2+
μM, respectively). Furthermore, 2 acts as an agonist for Ca -
release with almost equivalent potency to cADPR in
permeabilized T-cells and 3 is a membrane permeant
2
9,30
4
7
agonist.
the acid sensitivity of the N9-ribosyl link is eliminated.
Until recently, total synthetic approaches have required
Molecular Modeling. To predict the binding mode of
analogs 4−7, which might be more flexible because of the linear
N9-alkyl chain, they were first docked into the 2PGJ crystal
structure of shCD38 in complex with cIDPR. The docked and
minimized pose was almost identical to that of cIDPR (Figure
2A), suggesting that these analogs would mimic the critical
interactions with the binding site. Minimization of the protein
resulted in very little movement of the amino acid residues
forming the binding site.
modification of the “northern” ribose to generate a more stable
31−33
N1-link by introduction of a carbocyclic “northern” ribose,
replacement of the “northern” ribose, or both ribose sugars, by
3
4,35
an alkyl or ether bridge
or by attaching the “northern”
36
ribose to the base at C-2″. We recently reported the use of
modified Vorbruggen conditions to effect stereo- and
regiospecific introduction of an acetylated ribose at the N1-
̈
position of a protected inosine and demonstrated the utility of
37
The docked ligands display face-to-face stacking between the
hypoxanthine base and Trp189. There is one predicted
hydrogen bond from the ribose 3′-OH hydroxyl group to
this method in the first total synthesis of 3. This paves the
way for further exploration of the structure−activity relation-
ship (SAR) of cADPR, using the cIDPR template, with the
opportunity to retain an intact “northern” ribose and without
a
the catalytic acid (Glu226), although the 2′-OH does not
appear to be as closely located, and from the side chains of
Ser126 and Arg127 to the phosphates. The 8-amino group of 7
is predicted to form an additional hydrogen bond to the side
chain of Asp155. The N9-butyl chain of the docked ligand lies
across the entrance of the binding pocket in an almost identical
position to the “southern” ribose of cIDPR (Figure 2B).
Synthesis of N9-Butyl-cIDPR Analogs 4−7. 6-Chlor-
opurine (11, Scheme 1) was alkylated with 4-chlorobutylacetate
38
the structural limitations of using enzymatic cyclization. We
report here the synthesis of the first analogs in which the
“southern” ribose is selectively replaced, the activity of these
analogs as inhibitors of cADPR hydrolysis by shCD38,
crystallography of the most potent inhibitor with shCD38,
and HPLC studies to examine the ability of shCD38 to
hydrolyze the cIDPR scaffold.
8
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Scheme 1. Synthesis of the N1-Ribosyl-N9-butyl-8-
bromohypoxanthine Building Block
yield. Deprotection of the three acetyl esters using methanolic
ammonia generated triol 18.
a
The 2′,3′-diol was reprotected as an isopropylidene ketal to
afford 19, which allowed selective introduction of the first
phosphate ester to the 5′-OH by treatment of 19 with N,N-
diisopropyldibenzylphosphoramidite and 5-phenyl-1-H-tetra-
zole as activator, followed by oxidation of the intermediate
phosphite with hydrogen peroxide and triethylamine (Scheme
2
). The silyl ether of 20 was then removed under neutral
a
Scheme 2. Sequential Introduction of the Phosphate Esters
a
Reagents: (i) DBU, 4-Cl-butyl-OAc, 77%; (ii) NH , MeOH, 96%;
3
(
(
iii) imidazole, TBDPS-Cl, 92%; (iv) (a) diisopropylamine, n-BuLi,
b) Br , 66%; (v) NaNO , AcOH (aq), 84%; (vi) (a) DBU, (b)
2
2
tetraacetyl-D-ribose, TMSOTf, 86%; (vii) NH , MeOH, 98%.
3
48
in the presence of 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU).
The desired major product, the N9-isomer 12, was obtained in
high yield after separation from the minor N7-isomer.
Treatment of 12 with methanolic ammonia at 80 °C efficiently
effected both amination at the 6-position and deprotection of
the N9-butyl acetyl ester to afford N9-hydroxybutyladenine
4
9
1
3. The alkyl group was then reprotected as the TBDPS ether
a
Reagents: (i) pTsOH, H CC(OMe) CH , acetone, 100%; (ii) (a)
3
2
3
using tert-butyldiphenylsilyl chloride (TBDPS-Cl) and imiza-
dole to give 14. Introduction of the 8-bromo substituent to 14
is crucial at this stage to promote the subsequent N1-
(tBuO) PN( Pr) , 5-Ph-1H-tetrazole, DCM; (b) H O , Et N, 80%;
(
i
2
2
2
2
3
iii) TBAF·3H O, AcOH, 100%; (iv) PSS, TPS-Cl, 5-Ph-1H-tetrazole,
2
pyridine, 100%; (v) 50% TFA (aq), 100%; (vi) 0.1 M NaOH−dioxane.
3
7
ribosylation. Attempted bromination of 14 using a solution
of bromine in sodium hydrogen phosphate buffer and dioxane
was not successful, and no reaction was observed. Similarly,
treatment of 14 with N-bromosuccinimide or N-bromoaceta-
mide afforded only starting material. Applying the conditions
conditions to reveal the hydroxyl group. The second protected
phosphate ester was introduced by treatment of 21 with
cyclohexylammonium S,S-diphenylphosphorodithioate (PSS),
2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl), and 5-
phenyl-1-H-tetrazole in pyridine under strictly dry conditions
to generate the fully protected precursor for cyclization, 22.
Sequential deprotection of the phosphates was carried out as
50
developed by Laufer et al. for the 8-bromination of N9-
methyl chloropurines, we first deprotonated 14 using lithium
diisopropylamide (LDA), followed by addition of 1,2-
51
dibromotetrachloroethane. On a small scale (100 mg) the
desired 8-brominated product 15 was isolated in 67% yield.
However, when this reaction was scaled up (2.5 g), an
inseparable mixture of two products was obtained after column
chromatography. Mass spectrometry (m/z = 480.1970 and
3
7
previously described using first 50% aqueous TFA to
simultaneously remove the tert-butyl esters and the isopropy-
lidene ketal, then 0.1 M sodium hydroxide in dioxane, to afford
24. The N9-butyl chain shows increased stability toward acidic
conditions, compared to the natural “southern” ribose of
cIDPR, which had initially suffered partial hydrolysis at the N9-
glycosidic bond during deprotection of the isopropylidene
3
5/37
79/81
524.1476) containing characteristic
Cl and
Br isotope
patterns confirmed this was both the 8-bromo and 8-chloro
substituted products. It was only possible to recover the starting
material by treating the mixture of products with Pd/C under
3
7,47
acetals and tert-butyl phosphate esters.
No degradation of
an atmosphere of H . We therefore sought an alternative source
of bromine that would avoid this complication and found that
the N9-butyl analogs was observed even over prolonged
periods in 50% TFA at room temperature, since the ribose
oxygen has been removed.
2
direct addition of Br to deprotonated 14 generated 15 in 66%
2
yield, with the remainder as starting material, which could be
separated by column chromatography and reused. Treatment of
Cyclization of 24 was promoted using iodine and activated 3
33
Å molecular sieves in pyridine, under very dilute conditions,
to afford 5 (21% yield over two steps, Scheme 3). Because of
the stability of our cIDPR-based analogs, modifications at the 8-
position were possible after cyclization, which allowed us to
generate three further analogs; 8-H, 8-N , and 8-NH .
1
5 with excess sodium nitrite converted 8-bromoadenine 15 to
8-bromohypoxanthine 16, which was a suitable substrate for
N1-ribosylation. Deprotonation of 16 with DBU followed by
addition of tetraacetyl-D-ribose and trimethylsilyl triflate (TMS-
3
2
3
7
OTf) afforded stereo- and regioselective N1-coupling to
generate only the desired N1-ribosylated product 17 in high
Subjecting 5 to an atmosphere of hydrogen with Pd/C catalysis
generated the 8-H analog, N9-butyl-cIDPR (4). Treatment of 5
8
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a
Scheme 3. Synthesis of N9-Butyl-cIDPR Analogs 4−7
Figure 3. Inhibition of shCD38-mediated cADPR hydrolysis by
analogs 4−7.
a
Reagents: (i) I , 3 Å molecular sieves, pyridine, 21%; (ii) H , Pd/C,
Table 1. Half Maximal Values for Inhibition of shCD38-
Mediated cADPR Hydrolysis by Novel Analogs 4−7 and
Equivalent 8-Substituted cIDPRs
2
2
NaHCO , EtOH−H O, 76%; (iii) TMSN , 57%; (iv) dithiothreitol,
0
3
2
3
.05 M TEAB, 68%.
43
cIDPR
IC50 (μM)
N9-butyl-cIDPR
IC50 (μM)
with NaN in an attempt to generate the 8-N analog 6 was not
8-H
2
3
276
158
4
5
6
7
33
3
3
successful, despite conversion to the triethylamine salt or the
free acid to improve the solubility of 5 in DMF. In each case,
the DMF solution became cloudy over time as the sodium salt
of the starting material was formed and became insoluble in the
reaction media. However, upon treatment of the free acid form
8-Br
27
8-N3
8-NH2
6.4
3.3
56
increased flexibility afforded by an alkyl chain linker, compared
to a ribose sugar. This might allow the ligand to align other key
residues more favorably with their binding targets, resulting in a
tighter affinity for CD38. Another factor may be the
hydrophobic nature of the butyl chain, which may prefer to
sit further into the binding pocket, in a less polar environment
away from bulk solvent.
of 5 with TMSN in DMF, no precipitation was observed and
3
the clear solution was stirred at 70 °C for 16 h, becoming
yellow in color as the reaction proceeded. The reaction
progression was followed by RP-HPLC which clearly showed
the shift in wavelength as the 8-bromo starting material (λmax
=
2
55 nm) was converted into the 8-azido product [8-N -N9-
3
butyl-cIDPR (6), λmax = 277 nm]. Treatment of 6 with
dithiothreitol reduced the 8-azido substituent to an 8-amino
Structure of 8-NH -N9-butyl-cIDPR (7) Complexed
with Wild-Type CD38. Preformed crystals of wild-type
2
group which gave 8-NH -N9-butyl-cIDPR (7).
shCD38 were soaked with a cryoprotectant solution containing
2
Inhibition of CD38-Mediated cADPR Hydrolysis. The
ability of the novel compounds 4−7 to inhibit shCD38-
mediated hydrolysis of cADPR was assessed in a dose−
5 mM 8-NH -N9-butyl-cIDPR (7). X-ray diffraction data were
2
collected to a resolution of 2.05 Å. The structure was solved by
the molecular replacement method revealing two molecules of
CD38 in the asymmetric unit with a ligand present in each
52
response manner using a fluorimetric cycling assay. cIDPR
42
43
(2) inhibits hydrolysis of cADPR with an IC50 of 276 μM.
active site (Figure 4A). Previous crystallographic work has
The novel N9-butyl analogs 4−7 inhibit cADPR hydrolysis in a
concentration-dependent manner, with half maximal inhibition
in the low micromolar range (Figure 3 and Table 1). They are
all more potent than 2, showing an almost 100-fold increase in
inhibition. The introduction of an 8-bromo substituent (5)
gave a marginally improved inhibition (IC50 = 27 μM)
compared to the parent 4 (33 μM), which was then further
improved by substitution to the 8-azido (6) or 8-amino (7)
group (IC50 of 6.4 and 3.3 μM, respectively). The trend of
generally shown occupancy of only one site by noncovalent
inhibitors.
To our surprise, the electron density within each active site
did not fit the cyclic compound 7. However, hydrolysis at N1 to
generate a linear compound, 7a, gave a structure with a good fit
(Figure 4B). After refinement, the complex clearly showed that
the catalytic residue Glu226 was still hydrogen-bonded to the
ribose. For molecule A, Glu226 H-bonds to the 1′- and 3′-OH
of the ribose (both 2.7 Å), which has previously been observed
53
improved inhibition upon 8-H → 8-Br → 8-NH substitution
in hydrolyzed intermediates or products. In molecule B,
Glu226 forms H-bonds with the 3′-OH (2.6 Å) and longer
distance interactions with the 1′-OH and 2′-OH (3.3 and 3.5
Å). In both molecules, the 2′-OH interacts with an active site
water molecule (Figure 4C). Addition of water to C-1′ has
occurred to generate the hydrolyzed product, and the -OH
group is attached to the α-face of the ribose, as has been
2
43
mimics that which was observed for the cIDPR series, the 8-
NH derivative benefiting from an additional binding site
2
interaction with Asp155. This suggests that the new analogs are
binding similarly in the active site (vide infra). They are of
comparable potency to the recently reported non-nucleoside
22,23
and flavonoid NADase inhibitors.
The improved inhibition
+
of cADPR hydrolysis suggests that the “southern” ribose is not
essential for activity.
observed previously for NAD hydrolysis products captured by
5
3,54
crystallization.
Such observations are at odds with both the
The significant increase in potency by substitution of the
southern” ribose with a butyl chain may be a result of the
proposed ionic or covalent hydrolysis mechanisms, which
predict a predominantly or entirely β-product, respectively.
55
“
8
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Figure 4. Crystal structure of PDB code 4TMF. (A) Ligand (shown as sticks) occupies each active site. (B) The electron density fits that of a
hydrolyzed ligand (carbons in cyan, key residues that interact with the ligand in the binding site shown as green sticks). (C) H-bonds (shown as
black dotted lines) are formed between the rotated hypoxanthine base and both Glu146 and the backbone nitrogen of Asp156. Interacting residues
are shown as pink sticks, and other residues are shown as green lines. The interacting binding site water is shown as a red sphere, and the other
waters as red crosses.
Notably, the hydrolyzed ligand still occupies the catalytic site in
the same manner as the cADPR, except that the hypoxanthine
base has rotated in the binding pocket after hydrolysis. While
maintaining the stacking interaction with Trp189, it has also
formed new hydrogen bonds with Glu146 and Asp156 via its 8-
Proposed Mechanism of Hydrolysis by CD38. Finding a
hydrolyzed ligand in the active site of CD38 was unexpected, as
the cIDPR template has previously been considered “non-
hydrolyzable”. The absence of a partial positive charge at N1,
compared to that in the N6-amino form of cADPR, was
thought to contribute to this stability (Figure 5).
However, a mechanism in which the anomeric oxygen assists
in breaking the N1-link could still be envisaged (Scheme 4).
This would be without prejudice as to whether the role of
Glu226 is to capture the resulting oxonium ion formally or
interact with it as an ion pair before insertion of water to
generate the final linear product. Examination of the crystal
structures 2PGJ and 3U4H suggests that both binding sites
42
NH and 6-O substituents, respectively (Figure 4C). It is
2
unusual to capture this snapshot of the ligand directly after
hydrolysis, before diffusion out of the binding site. Previously
reported crystal structures of the hydrolysis products of N7-
cGDPR and cADPR, GDPR and ADPR, respectively, have also
captured products that were not bound in a conformation
53
directly resulting from hydrolysis.
8
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Figure 5. cADPR has a partial positive charge at N1.
contain two water molecules close to the N1-link of the cyclic
ligand. One of these waters forms H-bonds to Leu123, Leu145,
and the ribose 2′-OH that is consistent with other reported
54
CD38 crystal structures. Notably, in the 3U4H crystal with 8-
NH -cIDPR, the remaining water molecule lies within 3 Å of
2
the hypoxanthine carbonyl group. This water is not observed in
the complex of the hydrolyzed ligand 7a (PDB code 4TMF);
thus, we postulate that donation of a proton from this water
molecule could be enzyme-assisted in the active site. Further
examination reveals a proximal histidine residue (His133) that
may potentially contribute to overall catalysis (Figure 6).
For the corresponding cADPR ligand to be hydrolyzed via
such a mechanism would require the ligand to be present in the
N6-imino form. Indeed, an earlier report indicated that cADPR
exists in two forms, the N6-imino and N6-amino, the latter with
Figure 6. Docking of 8-NH
removal of ligand (ligand carbons in pink sticks, protein carbons in
green).
2
-N9-butyl-cIDPR (7) into 3U4H after
43
in the cADPcR-shCD38 complex, the only cocrystal structure
of a cyclic ligand with a starting C6-substituent in the amino
form yet determined, measures 1.35 Å, also suggesting
substantial double bond character. The authors measured the
26
pK of the N6-amino dissociation in this case as 8.9. However,
a
cADPcR is a completely nonhydrolyzable ligand due to the
absence of the ribose oxygen. Since crystallization of cADPR
itself with shCD38 is not possible because of its very rapid
hydrolysis, it has been impossible to capture directly the
intermediates in this process. Previously, the surrogate substrate
5
6
a pK of 8.3. Although at physiological pH the amino form
a
2
+
will be predominant, the active form for Ca release remains
unresolved and crystallization of cADPR originally suggested
that the C6−N6 bond displayed double bond characteristics
+
(
length 1.33 Å, cf. 1.47 Å expected for an amino group) with
NGD revealed the role of Glu226 in activating and stabilizing
2
53
only one hydrogen atom bound to N6. This is somewhat
surprising given that the molecule was crystallized as its free
acid, presumably well below pH 8.3. The C6−N6 bond length
the intermediate precursor for hydrolysis or cyclization. We
originally showed that cIDPR, as expected, has no ionization
28
between 6.8 and 10.9 and it is not likely that 7 will be any
Scheme 4. Proposed Mechanism for Hydrolysis of 7 by a High Concentration of shCD38
8
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different. Thus, since it is unlikely that cIDPR or any related
analog will exist in the 6-phenolic form at the pH used with
shCD38 (vide infra) and the enzyme would not adopt a
different mechanism to hydrolyze an analog vs cADPR itself, we
can conclude that the complex of 7a with shCD38 provides the
first evidence that CD38 can bind cADPR in the N6-imino
form. This could have future implications for inhibitor design.
We clearly cannot comment upon the ability of CD38 to bind
the N6-amino form of cADPR.
Study of Ligand Hydrolysis by CD38. Following the
discovery of hydrolyzed ligand 7a in the crystal complex, it was
important to determine whether it is the cyclic or the
hydrolyzed linear compound that inhibits hydrolysis of
cADPR by shCD38. Incubation of 7 (1 mM final
concentration) with 4 mg/mL shCD38 was monitored using
RP-HPLC. The peak corresponding to 7 (tR = 9.1 min)
reduced in intensity over time, alongside the appearance of a
the enzyme assay (Supporting Information, Figure S3). Under
these conditions the ligand is not hydrolyzed, confirming that
the inhibition observed in the CD38 assay is a consequence of
the cyclic compounds, not their hydrolyzed counterparts.
CONCLUSION
■
Two of the analogs developed in this study are potent (≤10
μM) inhibitors of cADPR hydrolysis by CD38. Analogs 4−7
illustrate that the “southern” ribose can be modified to
considerably improve inhibitory activity of CD38 mediated
cADPR hydrolysis. This agrees with modeling predictions
based on the shCD38-ligand crystal structure complex
interactions and confirms that the “northern” ribose and base
are key features for the interaction of cADPR analogs with the
CD38 binding pocket. The investigation of SAR in this region
in isolation was not possible prior to development of a method
for N1-ribosylation. This method thus opens up new avenues
and the potential to introduce desirable characteristics, such as
membrane permeability, into cADPR analogs by modifications
in this region, without loss of activity at CD38. Crystallization
new peak (t = 10.6 min), which was characteristic of an ADPR
R
analog (see Supporting Information, Figure S1). The rate of
conversion depended on the concentration of CD38 present in
solution (Figure S2), and no change in the original peak was
observed in a parallel control experiment containing no
shCD38 (data not shown). Novel analogs 4, 5 and cIDPR
of 8-NH -N9-butyl-cIDPR with shCD38 revealed that the
2
cIDPR scaffold can unexpectedly be hydrolyzed at high
concentrations of shCD38, resulting in a cocrystal structure
(2) were also analyzed in this way (Figure 7). Surprisingly, we
with linear 8-NH -N9-butyl-IDPR (7a) in the active site.
2
Hydrolysis of the hypoxanthine-linked scaffold offers new
insight into the mechanism by which CD38 hydrolyzes cADPR
to ADPR and suggests for the first time that the imino form is
active in this process. However, HPLC studies suggest that
hydrolysis would not occur under assay or physiologically
relevant concentrations. Thus, this observation does not detract
from the established use of cIDPR or its derivatives as either
pharmacological tools or as nonhydrolyzable templates for
30
future CD38 inhibitor design or indeed of 8-Br-cIDPR (3) as
a stable membrane permeant agonist of Ca2+ release.
EXPERIMENTAL SECTION
■
General. All reagents and solvents were of commercial quality and
were used without further purification, unless described otherwise.
Unless otherwise stated, all reactions were carried out under an inert
1
13
31
atmosphere of argon. H, C, and P NMR spectra were collected on
a Varian Mercury 400 MHz or Bruker Avance III 500 MHz
1
13
spectrometer. All H and C NMR assignments are based on
gCOSY, gHMBC, gHSQC, and DEPT-135 experiments. Abbrevia-
tions for splitting patterns are as follows: b, broad; s, singlet; d,
doublet; t, triplet; m, multiplet. Coupling constants are given in hertz
(Hz). High resolution time-of-flight mass spectra were obtained on a
Bruker Daltonics micrOTOF mass spectrometer using electrospray
ionization (ESI). The purity of new tested compounds was determined
to be ≥95% by analytical HPLC. Analytical HPLC analyses were
carried out on a Waters 2695 Alliance module equipped with a Waters
Figure 7. Hydrolysis of analogs 4, 5, 7, and cIDPR (2) over time with
shCD38 (4 mg/mL).
found that all the compounds, including cIDPR, are hydrolyzed
by the enzyme at 4 mg/mL but at a slower rate than 7. The
increased rate of hydrolysis of 7 could conceivably be promoted
by the additional hydrogen bond generated between the 8-NH2
substituent and Asp155 promoting elongation of the N1-bond
toward the transition state. This faster hydrolysis of 7 under
these conditions (50% hydrolysis for 7 at T = 105 min and for
cIDPR at T = 420 min), also reinforced by the extra interaction
afforded by the 8-amino group after cleavage, perhaps explains
2
996 photodiode array detector (210−350 nm). The chromatographic
system consisted of a Hichrom Guard column for HPLC and a
Phenomenex Synergi 4 μm MAX-RP 80A column (150 mm × 4.60
mm), with elution at 1 mL/min with the following ion-pair buffer:
0.17% (m/v) cetrimide and 45% (v/v) phosphate buffer (pH 6.4) in
MeOH. Synthetic phosphates were assayed and quantified by the
why the hydrolyzed ligand 7a is captured in the 8-NH -N9-
2
5
7
Ames phosphate test.
butyl-cIDPR:CD38 complex, but a cyclic ligand is observed in
4
8
42
6-Chloro-9-(4-acetoxybutyl)purine (12). To 6-chloropurine
1 (2.50 g, 16.2 mmol) and DBU (2.91 mL, 19.4 mmol) in DMF (22
the previously reported cIDPR:CD38 complex 2PGJ.
1
These conditions are representative of crystallization
concentrations of shCD38 (10 mg/mL) and are 10 000-fold
more concentrated than those used in the enzyme assay (1 μg/
mL). Therefore, further HPLC experiments were carried out
using shCD38 concentrations of 1 μg/mL and either cIDPR or
mL) was added 4-chlorobutylacetate (4.54 mL, 32.3 mmol). After the
mixture was stirred at 60 °C for 14 h, the DMF was removed under
reduced pressure, and the residue was purified by column
chromatography on silica gel, eluting with PE/EtOAc (1:0 → 0:1 v/
v) to afford the title compound (3.07 g, 71%) as a colorless oil. R =
f
1
7
to observe the effect of CD38 under conditions that reflect
0.61 (DCM/MeOH 9:1 v/v); H NMR (400 MHz, MeOD) δ 8.78 (s,
8
523
dx.doi.org/10.1021/jm501037u | J. Med. Chem. 2014, 57, 8517−8529

Journal of Medicinal Chemistry
Article
1
H), 8.16 (s, 1H), 4.37 (t, 2H, J = 7.2, CH ), 4.15 (t, 2H, J = 6.4,
and DBU (341 μL, 2.283 mmol) was added. After 30 min, 1,2,3,5-
tetra-O-acetyl-β-D-ribofuranose (266 mg, 0.837 mmol) was added and
the solution cooled to −78 °C. Trimethylsilyl trifluoromethanesulfo-
nate (551 μL, 3.044 mmol) was added dropwise and the solution
stirred for a further 45 min before warming to rt. After 1 h, NaHCO3
(sat. aq.) was added and the crude material extracted into DCM (×3).
The combined organic fractions were dried (Na SO ), and solvent was
2
CH ), 2.08−2.04 (m, 5H, CH and OAc), 1.74−1.67 (m, 2H, CH )
ppm.
2
2
2
4
9
9
-(4-Hydroxybutyl)adenine (13). A solution of 12 (3.00 g,
1
1.2 mmol) in MeOH (7 mL) was cooled to 0 °C and saturated with
NH (g), then stirred for 14 h at 80 °C. On cooling, a white solid
precipitated, which was collected by filtration and air-dried to afford
3
2
4
the title compound (2.22 g, 96%). R = 0.17 (DCM/MeOH 9:1 v/v);
H NMR (400 MHz, MeOD) δ 8.23 (s, 1H), 8.16 (s, 1H), 4.30 (t, 2H,
evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel, eluting with PE/EtOAc (1:0 →
f
1
J = 7.2, CH ), 3.61 (t, 2H, J = 6.4, CH ), 2.03−1.96 (m, 2H, CH ),
1:0 v/v) to afford the title compound (203 mg, 90%) as a colorless
2
2
2
+
1
1
2
.61−1.54 (m, 2H, CH ) ppm; HRMS (ESI ) calcd for C H N O
glass. R
f
= 0.69 (PE/EtOAc 1:3 v/v); H NMR (400 MHz, CDCl
3
) δ
2
9
14
5
1
+
08.1193 [(M + H) ], found 208.1195.
-(4-tert-Butyldiphenylsilylbutyl)adenine (14). To 13 (2.00 g,
.65 mmol) in DMF (20 mL) were added imidazole (1.71 g, 25.09
8.14 (s, 1H, 2H), 7.63 (dd, 4H, J = 7.8, 1.5), 7.40−7.34 (m, 6H) (10 ×
Ar-H), 6.39 (d, 1H, J = 4.6, H-1′), 5.47 (dd, 1H, J = 5.8, 4.6, H-2′),
5.44 (dd, 1H, J = 5.8, 4.5, H-3′), 4.42−4.38 (m, 3H, H-4′ and both H-
5′), 4.17 (t, 2H, J = 7.3, CH ), 3.70 (t, 2H, J = 6.0, CH ), 2.13 (s, 3H),
9
9
mmol) and TBDPS-Cl (3.25 mL, 12.55 mmol). After 14 h at rt, all
solvents were evaporated and the residue was purified by column
chromatography on silica gel, eluting with DCM/acetone (1:0 → 0:1
2
2
2.11 (s, 3H), 2.07 (s, 3H) (3 × OAc), 1.96−1.92 (m, 2H, CH ), 1.58−
2
13
1.54 (m, 2H, CH ), 1.03 (s, 9H) ppm; C NMR (100 MHz, CDCl )
2
3
v/v) to afford the title compound (3.96 g, 92%) as a white solid. R =
δ 170.2, 169.58, 169.57, 154.8, 148.7, 144.1, 135.5 (4C), 133.7 (2C),
f
1
0
1
2
.42 (DCM/acetone 1:3 v/v); H NMR (400 MHz, CDCl ) δ 8.21 (s,
129.7 (2C), 127.7 (4C), 126.1, 124.1, 87.3, 80.3, 74.2, 70.3, 63.0, 62.9,
3
+
H), 8.09 (s, 1H), 7.62−7.60 (m, 4H), 7.43−7.35 (m, 6H), 4.26 (t,
44.7, 29.4, 26.9 (3C), 26.3, 20.8, 20.5, 20.4 19.2 ppm; HRMS (ESI )
7
9
+
H, J = 7.0, CH ), 3.71 (t, 2H, J = 6.2, CH ), 2.05−1.97 (m, 2H,
calcd for C H N O Si Br 783.2055 [(M + H) ], found 783.2046,
2
2
36 44
4
9
13
81
+
CH ), 1.58−1.52 (m, 2H, CH ), 1.01 (s, 9H) ppm; C NMR (100
MHz, CDCl ) δ 155.3, 153.0, 150.2, 140.5, 135.5 (4C), 133.7 (2C),
calcd for C H N O Si Br 785.2035 [(M + H) ], found 785.2042.
2
2
36 44 4 9
N1-(β-D-Ribofuranosyl)-N9-[4-(tert-butyldiphenylsilyl)-
oxybutyl)-8-bromohypoxanthine (18). Intermediate 17 (600 mg,
0.766 mmol) was taken up in MeOH (6 mL) in a pressure tube. The
3
1
29.7 (2C), 127.7 (4C), 119.8, 63.0, 43.8, 29.5, 26.9 (3C), 26.7, 19.2
+
+
ppm; HRMS (ESI ) calcd for C H N OSi 446.2371 [(M + H) ],
25
32
5
found 446.2377.
-(4-tert-Butyldiphenylsilylbutyl)-8-bromoadenine (15). To
solution was saturated with NH (g) at 0 °C, then stirred at rt for 12 h.
3
9
The solvent was evaporated and the residue purified by column
chromatography on silica gel, eluting with PE/EtOAc (1:0 → 1:0 v/v)
to afford the title compound (492 mg, 98%) as a white amorphous
diisopropylamine (4.67 mL, 33.32 mmol) in THF (20 mL) at −78 °C
was added n-butyllithium (21.2 mL, 1.6 M solution, 33.97 mmol),
dropwise. After 1 h, a solution of 14 (2.97 g, 6.66 mmol) in THF (25
mL) was added dropwise and stirring continued for a further 1 h. Br2
solid. R
= 0.35 (DCM/acetone 1:1 v/v); 1H NMR (500 MHz,
f
MeOD) δ 8.79 (s, 1H, H-2), 7.62−7.60 (m, 4H), 7.42−7.35 (m, 6H)
(10 × Ar-H), 6.22 (d, 1H, J = 3.1, H-1′), 4.32−4.28 (m, 2H, H-2′, H-
(
2.04 mL, 39.96 mmol) was added dropwise and the solution allowed
to warm to rt over 4 h. The reaction was quenched by addition of NH4
aq, 2 mL), and all solvents were evaporated. The residue was taken up
3′), 4.23 (t, 2H, J = 7.1, CH ), 4.13 (ddd, 1H, J = 5.5, 2.9, 2.5, H-4′),
2
(
3.98 (dd, 1H, J = 12.3, 2.5, H-5a′), 3.83 (dd, 1H, J = 12.3, 2.9, H-5b′),
3.70 (t, 2H, J = 6.0, CH ), 1.98−1.92 (m, 2H, CH ), 1.55−1.49 (m,
2H, CH ), 1.02 (s, 9H) ppm; C NMR (125 MHz, MeOD) δ 156.9,
in DCM/H O (1:1 v/v, 100 mL) and the organic layer separated,
2
2
2
13
washed with brine, dried (Na SO ), and evaporated to dryness. The
2
2
4
crude material was purified by column chromatography on silica gel,
eluting with DCM/acetone (1:0 → 0:1 v/v) to afford the title
150.5, 147.1, 136.6 (4C), 134.8 (2C), 130.9 (2C), 128.8 (4C), 127.7,
124.6, 91.5, 86.2, 76.9, 70.6, 64.1, 61.7, 45.7, 30.4, 27.4 (3C), 27.1, 19.9
+
79
compound (2.18 g, 62%) as an amorphous cream solid. R = 0.61
ppm; HRMS (ESI ) calcd for C H N O Si Br 657.1739 [(M +
f
30 38 4 6
1
+
81
(
DCM/acetone 1:1 v/v); H NMR (400 MHz, MeOD) δ 8.31 (s, 1H,
H) ], found 657.1747, calcd for C H N O Si Br 659.1718 [(M +
30 38 4 6
+
H-2), 7.64−7.62 (m, 4H), 7.40−7.33 (m, 6H), 5.85 (bs, 2H, NH ),
H) ], found 659.1729.
2
4
2
.22 (t, 2H, J = 7.4, CH ), 3.70 (t, 2H, J = 6.1, CH ), 2.00−1.93 (m,
N1-(2′,3′-O-Isopropylidine-β-D-ribofuranosyl)-N9-[4-(tert-
butyldiphenylsilyl)oxybutyl)-8-bromohypoxanthine (19). To
18 (430 mg, 0.634 mmol) in acetone/2,2-dimethoxypropane (5 mL,
4:1 v/v) was added p-TsOH (124 mg, 0.634 mmol). After 30 min,
2
2
13
H, CH ), 1.62−1.55 (m, 2H, CH ), 1.03 (s, 9H) ppm; C NMR
2
2
(
100 MHz, CDCl ) δ 154.2, 153.0, 151.3, 135.5 (4C), 133.7 (2C),
3
1
1
29.6 (2C), 127.8 (4C), 127.3, 119.9, 63.0, 44.4, 29.4, 26.8 (3C), 26.1,
+
79
9.1 ppm; HRMS (ESI ) calcd for C H N OSi Br 524.1476 [(M +
DCM and NaHCO
3
(sat. aq) were added, the organic layer was dried
2
5
31
5
+
81
H) ], found 524.1473, calcd for C H N OSi Br 526.1455 [(M +
(Na SO ), and all solvents were evaporated. The residue was taken up
2
4
2
5
31
5
+
+
H) ], found 526.1462.
-(4-tert-Butyldiphenylsilylbutyl)-8-bromohypoxanthine
16). To 15 (4.13 g, 7.87 mmol) in AcOH/H O (20:3 v/v, 138 mL)
in MeOH (2 mL), and Dowex 50WX8 H resin (50 mg) was added.
9
After 30 min the resin was removed by filtration under gravity and the
(
solvent evaporated to obtain the title compound (455 mg, 100%) as a
2
1
was added NaNO (6.52 g, 94.48 mmol) in one portion. After 48 h, all
colorless glass. R = 0.63 (PE/EtOAc 1:3 v/v); H NMR (400 MHz,
2
f
solvents were evaporated and EtOH (100 mL) was added and
CDCl ) δ 7.87 (s, 1H, H-2), 7.55 (dd, 4H, J = 5.9, 1.5), 7.35−7.26 (m,
3
evaporated to dryness. The residue was taken up in CHCl and washed
6H) (10 × Ar-H), 5.62 (d, 1H, J = 2.8, H-1′), 5.23 (dd, 1H, J = 6.5,
3
with H O, then NaHCO (aq sat.) to pH 7. The organic layer was
2.8, H-2′), 5.08 (dd, 1H, J = 6.5, 3.6, H-3′), 4.28 (dd, 1H, J = 3.6, 2.4,
2
3
washed with brine, dried (Na SO ), and evaporated to dryness. The
H-4′), 4.09 (t, 2H, J = 7.3, CH ), 3.85 (dd, 1H, J = 12.2, 2.4, H-5a′),
2
4
2
crude material was purified by column chromatography on silica gel,
3.75 (bd, 1H, J = 12.2, H-5b′), 3.61 (t, 2H, J = 6.0, CH ), 1.89−1.82
2
eluting with PE/EtOAc (1:0 → 0:1 v/v) to afford the title compound
(m, 2H, CH ), 1.52 (s, 3H), 1.50−1.45 (m, 2H, CH ), 1.28 (s, 3H),
2
2
1
13
(
3.49 g, 84%) as a cream foam. R = 0.37 (PE/EtOAc 1:3 v/v); H
1.02 (s, 9H) ppm; C NMR (100 MHz, CDCl ) δ 155.3, 149.1, 146.5,
f
3
NMR (400 MHz, CDCl ) δ 13.07 (bs, 1H, NH), 8.16 (s, 1H, 2H),
135.5 (4C), 133.6 (2C), 129.6 (2C), 127.6 (4C), 126.5, 124.8, 114.2,
97.1, 88.1, 83.5, 80.6, 62.8, 62.8 (2C), 44.7, 29.2, 27.3, 26.8 (3C), 26.1,
3
7
3
.65−7.63 (m, 4H), 7.41−7.34 (m, 6H), 4.21 (t, 2H, J = 7.3, CH ),
2
+
79
.71 (t, 2H, J = 6.0, CH ), 1.96 (tt, 2H, J = 7.4, 7.3, CH ), 1.58 (tt, 2H,
25.2, 19.1 ppm; HRMS (ESI ) calcd for C H N O Si Br 697.2052
2
2
33 42 4 6
13
+
81
J = 6.5, 6.0, CH ), 1.04 (s, 9H) ppm; C NMR (100 MHz, CDCl ) δ
[(M + H) ], found 697.2072, calcd for C H N O Si Br 699.2031
33 42 4 6
2
3
+
1
1
58.0, 150.6, 145.5, 135.6 (4C), 133.8 (2C), 129.7 (2C), 127.7 (4C),
26.3, 124.8, 63.0, 44.8, 29.4, 26.9 (3C), 26.2, 19.2 ppm; HRMS
[(M + H) ], found 699.2032.
N1-[2′,3′-O-Isopropylidine-5′-O-(di-tert-butyl)phosphoryl-β-
D-ribofuranosyl]-N9-[4-(tert-butyldiphenylsilyl)oxybutyl)-8-
bromohypoxanthine (20). To a solution of 19 (350 mg, 0.502
mmol) in DCM (2.8 mL) was added 5-phenyl-1-H-tetrazole (147 mg,
1.003 mmol) and N,N-diisopropyldibutylphosphoramidite (238 μL,
+
79
+
(
ESI ) calcd for C H N O Si Br 525.1316 [(M + H) ], found
25 30 4 2
81
+
5
5
25.1319, calcd for C H N O Si Br 527.1295 [(M + H) ], found
25 30 4 2
27.1301.
N1-(2′,3′,5′-Tri-O-acetyl-β-D-ribofuranosyl)-N9-[4-(tert-
butyldiphenylsilyl)oxybutyl)-8-bromohypoxanthine (17). Inter-
mediate 16 (400 mg, 0.761 mmol) was taken up in DCM (4.0 mL),
0.753 mmol). After 2 h, the solution was cooled to 0 °C and Et N
3
(419 μL, 3.012 mmol) and H O (138 μL, 1.255 mmol) were added.
2
2
8
524
dx.doi.org/10.1021/jm501037u | J. Med. Chem. 2014, 57, 8517−8529

Journal of Medicinal Chemistry
Article
The solution was allowed to warm to rt and stirred for a further 2 h,
(d, J = 7.9), 85.0, 82.8 (d, 2C, J = 7.3), 81.4, 67.0 (d, J = 8.7), 66.5 (d, J
= 6.2), 43.9, 29.77 (d, 3C, J = 4.2), 29.74 (d, 3C, J = 4.2), 27.1, 27.0 (d,
after which DCM (20 mL) and H O were added. The organic layer
2
J = 6.7), 25.6, 25.2 ppm; 31P NMR (162 MHz, H decoupled, CDCl )
1
was washed with NaHCO (sat. aq), then brine, dried (Na SO ), and
3
2
4
3
+
79
evaporated to dryness. The residue was purified by column
chromatography on silica gel, eluting with PE/EtOAc (1:0 → 1:0 v/
v), where both solvents contained 0.5% v/v pyridine, to afford the title
δ 49.0, −10.3 ppm; HRMS (ESI ) calcd for C H N O P S BrNa
37
49
4
10 2 2
+
937.1441 [(M
+ H) ], found 937.1447, calcd for
81
+
C H N O P S BrNa 939.1420 [(M + H) ], found 939.1440.
37
49
4
10 2 2
compound (356 mg, 80%) as a colorless glass. R = 0.43 (PE/EtOAc
N1-[5′-O-Phosphoryl-β-D -ribofuranosyl]-N9-(4-
f
1
1
:3 v/v); H NMR (400 MHz, CDCl ) δ 8.02 (s, 1H, H-2), 7.63−7.60
[(diphenylthio)phosphoryl]hydroxybutyl)-8-bromo-
hypoxanthine (23). Intermediate 22 (40 mg, 0.044 mmol) was
stirred in 50% TFA (2 mL) at 0 °C for 4 h. All solvents were
evaporated, and the residue was coevaporated with MeOH (×4). The
residue was purified by column chromatography on silica gel, eluting
3
(
5
(
m, 4H), 7.40−7.32 (m, 6H) (10 × Ar-H), 5.97 (d, 1H, J = 1.8, H-1′),
.08 (dd, 1H, J = 6.4, 1.8, H-2′), 4.99 (dd, 1H, J = 6.4, 3.9, H-3′), 4.41
dd, 1H, J = 9.6, 4.3, H-4′), 4.27−4.13 (m, 4H, both H-5′ and CH ),
2
3
1
1
1
9
6
2
.67 (t, 2H, J = 6.0, CH ), 1.93−1.89 (m, 2H, CH ), 1.57 (s, 3H),
2
2
with EtOAc/MeOH/H O (1:0:0 → 4:2:0 → 7:2:1 v/v/v) to afford
.55−1.44 (m, 2H, CH ), 1.448 (s, 9H), 1.447 (s, 9H), 1.33 (s, 3H),
2
2
.02 (s, 9H) ppm; 1 C NMR (100 MHz, CDCl ) δ 153.0, 147.2, 144.1,
3
the title compound (33 mg, 100%) as a colorless glass. R = 0.20
f
3
1
(
8
×
3
EtOAc/MeOH/H O 7:2:1 v/v/v); H NMR (500 MHz, MeOD) δ
33.7 (4C), 131.9 (2C), 127.8 (2C), 125.8 (4C), 124.2, 122.7, 112.5,
2.5, 85.1 (d, J = 7.9), 83.2, 80.9 (d, 2C, J = 7.2), 79.7, 64.7 (d, J =
.2), 61.1, 42.8, 28.0 (d, 3C, J = 2.8), 27.9 (d, 3C, J = 2.9), 27.5, 25.3,
5.0 (3C), 24.4, 23.5, 17.3 ppm; 31P NMR (162 MHz, H decoupled,
2
.59 (s, 1H, H-2), 7.43 (dd, 4H, J = 7.1, 0.7), 7.33−7.28 (m, 6H) (10
ArH), 6.21 (d, 1H, J = 3.8, H-1′), 4.24 (d, 2H, J = 2.6, H-2′ and H-
′), 4.16−4.07 (m, 7H, H-4′, both H-5′ and 2 × CH ), 1.70 (app
1
2
+
79
quintet, 2H, J = 7.1, CH ), 1.55 (app quintet, 2H, J = 5.7, CH ) ppm;
CDCl ) δ −10.2 ppm; HRMS (ESI ) calcd for C H N O PSi BrNa
9
C H N O PSi BrNa 913.2766 [(M + H) ], found 913.2763.
2
2
3
41 58
4
9
13
+
C NMR (125 MHz, MeOD) δ 157.2, 150.6, 147.1, 136.6 (d, 4C, J =
11.2786 [(M + H) ], found 911.2762, calcd for
81
+
5.3), 131.1 (d, 2C, J = 3.4), 130.7 (d, 4C, J = 2.5), 127.6, 127.0 (d, 2C,
J = 6.8), 124.5, 89.8, 85.6 (d, J = 8.6), 77.1, 71.4, 69.2 (d, J = 8.9), 65.3
(d, J = 4.8), 45.2, 28.3 (d, J = 7.0), 26.8 ppm; 31P NMR (202 MHz, H
41
58
4
9
N1-[2′,3′-O-Isopropylidine-5′-O-(di-tert-butyl)phosphoryl-β-
D-ribofuranosyl]-N9-(4-hydroxybutyl)-8-bromohypoxanthine
21). Acetic acid (12 μL, 0.213 mmol) and TBAF·3H O (64 mg, 0.203
1
−
(
decoupled, MeOD) δ 50.8, 0.8 ppm; HRMS (ESI ) calcd for
2
7
9
−
mmol) were stirred in DMF (0.5 mL) for 30 min, after which the
solution was cooled to 0 °C and 20 (60 mg, 0.068 mmol) in DMF (0.5
mL) added. The resulting solution was allowed to warm to rt and
stirred for a further 4 h. The solution was diluted with ether, and
NaHCO (sat. aq) and NH Cl (sat. aq) were added. The organic layer
C H N O P S Br 760.9911 [(M − H) ], found 760.9878, calcd
2
6
28
4
10 2 2
81 −
for C H N O P S Br 762.9890 [(M − H) ], found 762.9915.
26
28
4
10 2 2
N1-(5′-O-Phosphoryl-β-D -ribofuranosyl)-N9-(4-
[(diphenylthio)phosphoryl]hydroxybutyl)-8-bromo-
hypoxanthine (24). Intermediate 23 (20 mg, 0.026 mmol) was taken
3
3
was separated and the aqueous layer extracted with ether (×3). The
up in dioxane/H
and the solution stirred for 30 min at rt before addition of HCl (100
μL, 1M). The solution was diluted with H O and washed with hexane
(×3) before evaporation of all solvents to give a colorless glass which
2
O (1 mL, 1:1 v/v). NaOH (100 μL, 1 M) was added
combined organic layers were dried (Na SO ), evaporated to dryness
2
4
and the residue was purified by column chromatography on silica gel,
eluting with DCM/acetone (1:0 → 1:0 v/v) to afford the title
2
31
compound (44 mg, 100%) as a colorless glass. R = 0.47 (DCM/
was converted to the TEA salt as described below. P NMR (202
f
1
1
−
acetone 1:1 v/v); H NMR (400 MHz, CDCl ) δ 8.06 (s, 1H, H-2),
MHz, H decoupled, D
2
O) δ 15.6, 2.6 ppm; HRMS (ESI ) calcd for
3
79
−
5
1
4
1
2
3
1
.98 (d, 1H, J = 1.8, H-1′), 5.05 (dd, 1H, J = 6.4, 1.8, H-2′), 4.96 (dd,
H, J = 6.4, 3.9, H-3′), 4.39 (app. dd, 1H, J = 9.0, 4.5, H-4′), 4.25−
.11 (m, 4H, both H-5′ and CH ), 3.65 (t, 2H, J = 6.3, CH ), 2.34 (bs,
C
C
20
H
24
N
4
O
11
P
2
S Br 668.9826 [(M − H) ], found 668.9853, calcd for
S Br 670.9806 [(M − H) ], found 670.9863.
8
1
−
H
N
O
P
20
24
4
11
2
+
Conversion to TEA salt: The Na salt was passed through prewashed
Dowex H resin. Acidic fractions were neutralized with TEAB (2 mL,
2
2
+
H, OH), 1.89 (app. quintet, 2H, J = 7.2, CH ), 1.56 (app. quintet,
2
H, J = 6.3, CH ), 1.55 (s, 3H), 1.44 (s, 9H), 1.43 (s, 9H), 1.31 (s,
1 M). All solvents were evaporated, and the residue was coevaporated
2
H) ppm; 13C NMR (100 MHz, CDCl ) δ 154.7, 149.0, 145.9, 125.9,
with H O to remove excess buffer. The colorless glass obtained was
3
2
24.4, 114.2, 94.1, 86.8 (d, J = 7.9), 85.1, 82.71 (d, J = 7.3), 82.69 (d, J
used directly for cyclization.
=
(
7.2), 81.3, 66.4 (d, J = 6.3), 61.7, 44.5, 29.74 (d, 3C, J = 4.2), 29.72
N1-Cyclic-8-bromohypoxanthine-N9-hydroxybutyl Diphos-
phate Ribose (8-Br-N9-butyl-cIDPR, 5). Intermediate 24 (0.026
mmol) was evaporated from pyridine (2 × 2 mL). The residue was
taken up in pyridine (10 mL) and placed in a syringe. This solution
was added over 15 h to a solution of iodine (70 mg, 0.30 mmol) and 3
Å molecular sieves (0.5 g) in pyridine (20 mL), in the dark. The
d, 3C, J = 4.3), 29.1, 27.1, 26.2, 25.2 ppm; 31P NMR (162 MHz, H
1
+
decoupled, CDCl ) δ −10.4 ppm; HRMS (ESI ) calcd for
C H N O P BrNa 673.1608 [(M + H) ], found 673.1584, calcd
for C H N O P BrNa 675.1588 [(M + H) ], found 675.1570.
N1-[2′,3′-O-Isopropylidine-5′-O-(di-tert-butyl)phosphoryl-β-
D-ribofuranosyl]-N9-(4-[(diphenylthio)phosphoryl]-
hydroxybutyl)-8-bromohypoxanthine (22). Intermediate 21 (20
mg, 0.031 mmol) was coevaporated from pyridine (3 × 1 mL) and
taken up in pyridine (0.5 mL). This solution was added to PSS (35
mg, 0.092 mmol) which had also been coevaporated from pyridine (3
3
79
+
25
40
4
9
81
+
25
40
4
9
solution was filtered through Celite, washed with H O. After addition
2
of TEAB (2 mL) all solvents were evaporated, and the residue was
partitioned between H O and CHCl . The aqueous layer was washed
2
3
with CHCl and evaporated to dryness. The residue was purified by
3
semipreparative HPLC (1.1 cm × 25 cm C18 column), eluting with
acetonitrile/0.1 M TEAB (1:19 → 13:7 v/v) over 25 min. Fractions
were analyzed by analytical HPLC and appropriate fractions collected
and evaporated under vacuum to give the title compound (3.0 mg,
21% over two steps). UV (H O, pH 7), λ = 256 nm (ε = 19 900);
×
1 mL). 5-Phenyl-1-H-tetrazole (13 mg, 0.092 mmol) and TPS-Cl
19 mg, 0.061 mmol) were added, and the solution was stirred at rt for
h. DCM and H O were added. The organic layer was separated and
(
5
2
the aqueous layer washed with DCM (×2). The combined organic
layer was washed with brine, dried (Na SO ), and evaporated to
2
max
1
H NMR (500 MHz, D O) δ 8.81 (s, 1H, H-2), 6.08 (d, 1H, J = 1.8,
2
4
2
dryness. The residue was purified by column chromatography on silica
gel, eluting with PE/EtOAc (1:0 → 1:0 v/v) to afford the title
H-1′), 4.43 (dd, 1H, J = 4.7, 1.8, H-2′), 4.38 (dd, 1H, J = 6.9, 4.7, H-
3′), 4.35−4.19 (m, 4H), 4.17 (app septet, 1H, J = 5.1, CH H), 4.11
a
compound (28 mg, 100%) as a white foam. R = 0.73 (DCM/acetone
(dd, 1H, J = 11.0, 3.9, H-5 ′), 1.92−1.83 (m, 2H, CH ), 1.41−1.38 (m,
f
b
2
1
13
1
:1 v/v); H NMR (400 MHz, CDCl ) δ 8.04 (s, 1H, H-2), 7.52−7.49
1H, CH H), 1.13−1.07 (m, 1H, CH H) ppm; C NMR (125 MHz,
D O) δ 156.7, 150.1, 145.0, 127.7, 122.6, 91.1, 83.5 (d, J = 8.9), 75.2,
3
a
b
(
5
(
m, 4H), 7.36−7.30 (m, 6H) (10 × ArH), 5.97 (d, 1H, J = 1.8, H-1′),
.07 (dd, 1H, J = 6.4, 1.8, H-2′), 4.98 (dd, 1H, J = 6.4, 3.9, H-3′), 4.40
app. dd, 1H, J = 9.4, 5.2, H-4′), 4.25−4.17 (m, 4H, both H-5′ and
CH ), 4.11 (t, 2H, J = 7.0, CH ), 1.82 (app. quintet, 2H, J = 7.2, CH ),
2
68.1, 65.2 (d, J = 6.0), 62.8 (d, J = 4.3), 42.9, 24.8 (d, J = 9.8), 23.7
ppm; 31P NMR (202 MHz, D O, H-decoupled) δ −10.2 (d, J = 14.9
1
2
−
2
2
2
Hz), −11.4 (d, J = 14.9 Hz) ppm; HRMS (ESI ) calcd for
79
−
1
1
1
.66 (app. quintet, 2H, J = 6.3, CH ), 1.56 (s, 3H), 1.454 (s, 9H),
C H N O P Br 558.9636 [(M − H) ], found 558.9627; calcd
81 −
2
14 18
4
11 2
.449 (s, 9H), 1.32 (s, 3H) ppm; 13C NMR (100 MHz, CDCl ) δ
3
for C H N O P Br 560.9616 [(M − H) ], found 560.9612.
14 18
4
11 2
54.7, 149.0, 146.0, 135.2 (d, 4C, J = 5.2), 129.4 (d, 2C, J = 3.2), 129.3
N1-Cyclic Hypoxanthine-N9-hydroxybutyl Diphosphate Ri-
bose (N9-Butyl-cIDPR, 4). Analog 5 (7.0 mg, 0.0125 mmol) was
(
d, 4C, J = 2.5), 126.3 (d, 2C, J = 6.7), 125.8, 124.5, 114.3, 94.3, 86.8
8
525
dx.doi.org/10.1021/jm501037u | J. Med. Chem. 2014, 57, 8517−8529

Journal of Medicinal Chemistry
Article
taken up in Milli-Q (3 mL), and NaHCO (225 mg, 2.77 mmol) was
added. When all material had dissolved, EtOH (1.5 mL) and then Pd/
C (6 mg) were added and the solution was placed under an
H-5′b), 3.95 (app septet, 1H, J = 5.3, CHH), 2.00−1.85 (m, 2H,
3
1
3
CH ), 1.53−1.50 (m, 1H, CHH), 1.31−1.28 (m, 1H, CHH) ppm; C
2
NMR (125 MHz, D O) 155.3, 152.2, 148.3, 143.2, 116.7, 91.1, 83.5
2
atmosphere of H . After 5 h stirring, the solution was filtered and all
(d, J = 8.9), 75.2, 68.1, 65.1 (d, J = 5.6), 62.9 (d, J = 3.1), 39.7, 24.8 (d,
2
3
1
solvents evaporated. The residue was purified by semipreparative
HPLC (1.1 cm × 25 cm C18 column), eluting with MeCN/0.1 M
TEAB (1:19 → 13:7 v/v) over 25 min. Fractions were analyzed by
analytical HPLC and appropriate fractions collected and evaporated
J = 9.6), 22.5 ppm; P NMR (202 MHz, D O) −10.39 (br), −11.44
2
−
−
(br); HRMS (ESI ) found m/z [M − H] 496.0637; C H N O P
1
4
20
5
11 2
requires 496.0640.
Enzyme Assay for cADPR Hydrolysis. The inhibition of cADPR
hydrolysis by various concentrations of analog (0−1 mM) was
determined by incubating 1 μM cADPR with 1 μg/mL CD38 catalytic
domain for 10 min at 20−24 °C in 25 mM sodium acetate, pH 4.5.
The reaction was stopped by the addition of 150 mM HCl. The
precipitated protein was filtered, and the pH was neutralized with Tris
base. After the mixture was diluted 20-fold, the concentration of the
unhydrolyzed cADPR present in the diluted reaction mixture was
under vacuum to give the title compound (4.6 mg, 76%). UV (H O,
2
1
pH 7), λmax = 252 nm (ε = 11 400); H NMR (500 MHz, D O) δ 8.79
2
(
3
(
3
1
s, 1H, H-2), 8.03 (s, 1H, H-8), 6.10 (s, 1H, H-1′), 4.49 (d, 1H, J =
.0, H-2′), 4.40 (app. t, 1H, J = 3.0, H-3′), 4.33−4.24 (m, 4H), 4.11
dd, 1H, J = 11.8, 4.4, H-5 ′), 4.05 (app septet, 1H, J = 5.2, CH H),
b
a
.81 (app septet, 1H, J = 5.2, CH H), 1.96−1.84 (m, 2H, CH ), 1.41−
b
2
13
.35 (m, 1H, CH H), 1.11−1.05 (m, 1H, CH H) ppm; C NMR
a
b
(
125 MHz, D O) δ 158.0, 148.9, 144.7, 142.3, 122.2, 91.1, 83.6 (d, J =
2
52
assayed by the fluorimetric cycling assay as described previously.
8
=
=
.9), 75.2, 68.3, 65.1 (d, J = 5.9), 63.0 (d, J = 4.5), 42.3, 25.4, 25.0 (d, J
9.5) ppm; 31P NMR (202 MHz, D O, H-decoupled) δ −10.3 (d, J
1
Modeling of the Butyl Compounds. The 2PGJ crystal structure
of human CD38 with cIDPR was passed through the Protein
Preparation Wizard in the Schrodinger software (http://www.
̈
schrodinger.com/). Four N9-butyl compounds, where the cIDPR
“southern” ribose is replaced by a four carbon linker, were built using
2
−
14.1), −11.2 (d, J = 14.2) ppm; HRMS (ESI ) calcd for
−
C H N O P 481.0531 [(M − H) ], found 481.0522.
1
4
19
4
11 2
N1-Cyclic-8-azidohypoxanthine-N9-hydroxybutyl Diphos-
phate Ribose (8-N -N9-butyl-cIDPR, 6). Analog 5 (4.5 mg, 8.0
3
μmol) was converted to the free acid by addition of Milli-Q (5 mL)
and stirring with Dowex 50WX8 (H form) for 30 min. The resin was
̈
the Schrodinger software. The four compounds differed in the
+
substituent at the 8-position: hydrogen, bromine, azido, or an amino
group. The compounds were docked into the 2PGJ structure using
removed by filtration, washed with Milli-Q and the combined filtrate
evaporated. The residue was evaporated from dry DMF (4 × 2 mL),
5
8
GOLD. The binding site was defined as a sphere of 5 Å radius
centered on the centroid of the cIDPR ligand: the centroid of the
docked ligand has to lie within this sphere. Each ligand was docked 25
times. The best ranked pose of each of the ligands was merged with
the protein structure, and the resulting complex was passed through a
taken up in DMF (1 mL), and stirred under argon. TMSN (50 μL,
3
0.43 mmol) was added and the resulting solution stirred at 70 °C in
the dark for 16 h, after which ∼65% conversion of the starting material
to product was observed by HPLC (λ = 255 nm → λ = 277 nm). All
solvent was evaporated and the resulting residue coevaporated with
Milli-Q (2 × 5 mL). The residue was then taken up in Milli-Q (5 mL),
filtered through cotton wool, and purified by semipreparative HPLC
̈
minimization procedure using the Schrodinger software.
Crystallization, Diffraction Data Collection, and Structure
Refinement. The human CD38 catalytic domain was expressed and
(
→
1.1 cm × 25 cm C18 column), eluting with acetonitrile/Milli-Q (1:19
13:7 v/v) over 25 min. Fractions were analyzed by analytical HPLC
and appropriate fractions were collected and evaporated under vacuum
to give the title compound (2.4 mg, 57%). UV (H O, pH 7), λ
45,54
purified as described previously.
The protein was diluted to 10
mg/mL for crystallization trials. Crystals of wild type CD38 were
obtained by the hanging droplet vapor diffusion method with the
reservoir buffer in 0.1 M sodium acetate, pH 4.0, 15% PEG 10K, 0.2 M
ammonium acetate, and 3% isopropanol. They were harvested and
soaked in 0.1 M sodium acetate, pH 4.0, 15% PEG 10K, 16% ethylene
glycerol, and 5 mM compound 7 for 1 h at 295 K and then flash-frozen
in liquid nitrogen. The diffraction data were collected at 100 K on
beamline BL17U at the Shanghai Synchrotron Radiation Facility and
=
max
2
1
2
6
1
3
52 nm (ε = 12 700); H NMR (500 MHz, D O) δ 8.75 (s, 1H, H-2),
2
.08 (d, 1H, J = 1.9, H-1′), 4.46 (dd, 1H, J = 4.8, 1.9, H-2′), 4.39 (dd,
H, J = 6.5, 4.8, H-3′), 4.33−4.30 (m, 2H, H-4′, CHH), 4.12−4.05 (m,
H, H-5′a, 2 × CHH), 4.00 (dt, 1H, J = 14.5, 5.2, H-5′b), 3.81 (app
septet, 1H, J = 5.0, CHH), 1.81−1.76 (m, 2H, CH ), 1.40−1.36 (m,
1
D O) 156.6, 149.0, 146.4, 144.2, 120.3, 91.2, 83.6 (d, J = 8.9), 75.2,
2
13
H, CHH), 1.14−1.09 (m, 1H, CHH) ppm; C NMR (125 MHz,
5
9
processed with HKL2000. Molecular replacement was performed
2
6
0
61
using the program Phaser from the CCP4 suite, and the wild-type
human CD38 (PDB code 1YH3) was used as the search model. The
6
8.2, 65.1 (d, J = 6.5), 62.9 (d, J = 4.4), 40.7, 24.9 (d, J = 9.1), 23.5
31
ppm; P NMR (202 MHz, D O) −10.4 (d, J = 14.1), −11.4 (d, J =
1
C H N O P requires 522.0545.
2
−
−
62
4.1) ppm; HRMS (ESI ) found m/z [M − H] 522.0549;
model was refined with Refmac and then cycled with manual
6
3
building in Coot. Hydrolyzed compound 7 was built into positive
difference electron-density maps of the CD38 model after a few
restrained refinement runs with the stereochemical restraints
14
18
7
11 2
N1-Cyclic-8-aminohypoxanthine-N9-hydroxybutyl Diphos-
phate Ribose (8-NH -N9-butyl-cIDPR, 7). Analog 5 (4.5 mg, 8.0
2
6
4
65
μmol) was converted to the free acid by addition of Milli-Q (5 mL)
generated from the program PRODRG. TLS refinement was
incorporated into the later stages of the refinement process. Solvents
were added automatically in Coot and then manually inspected and
+
and stirring with Dowex 50WX8 (H form) for 30 min. The resin was
removed by filtration, washed with Milli-Q and the combined filtrate
evaporated. The residue was evaporated from dry DMF (4 × 2 mL),
66
modified. The final model was analyzed with MolProbity, showing
taken up in DMF (1 mL), and stirred under argon. TMSN (50 μL,
3
that 98% residues were in the Ramachandran favored region with only
one residue (D202 in chain B) in the Ramachandran outlier region.
Data collection and model refinement statistics are summarized in
Supporting Information Table 1. The coordinates and structure factors
are deposited in the Protein Data Bank with the code 4TMF.
HPLC Studies. The solution containing the CD38 catalytic domain
was adjusted to the desired concentration using Tris-HCl buffer (20
mM, pH 8), and 50 μL therefrom was added to the inhibitor (0.05
μmol) in an Eppendorf tube at room temperature. At a given time
point, a sample of 5 μL was removed and diluted with 95 μL of Milli-Q
water. Then 10 μL of this sample was injected directly into the
analytical HPLC system (see section General in Experimental
Section), eluting at 1 mL/min with an isocratic ion-pair buffer:
0.17% (m/v) cetrimide and 45% (v/v) phosphate buffer (pH 6.4) in
MeOH.
0
.43 mmol) was added and the resulting solution stirred at 70 °C in
the dark for 16 h, after which ∼70% conversion of the starting material
to product was observed by HPLC (λ = 255 nm → λ = 277 nm). All
solvent was evaporated, and the resulting residue was taken up in
TEAB (5 mL, 0.05 M). Dithiothreitol (12 mg, 0.08 mmol) was added
and the solution stirred under an atmosphere of argon for 16 h. The
crude material was directly purified by semipreparative HPLC (1.1 cm
×
1
25 cm C18 column), eluting with acetonitrile/0.1 M TEAB (1:19 →
3:7 v/v) over 25 min. Fractions were analyzed by analytical HPLC
and appropriate fractions collected and evaporated under vacuum to
give the title compound (2.7 mg, 68% over two steps). UV (H O, pH
7
1
4
4
2
1
), λmax = 252 nm (ε = 14 700); H NMR (400 MHz, D O) δ 8.80 (s,
2
H, H-2), 6.19 (d, 1H, J = 2.1, H-1′), 4.56 (dd, 1H, J = 4.7, 2.1, H-2′),
.50 (dd, 1H, J = 6.4, 4.7, H-3′), 4.43−4.41 (m, 2H, H-4′, CHH),
.27−4.20 (m, 3H, H-5′a, 2 × CHH), 4.11 (ddd, 1H, J = 15.0, 5.3, 4.9,
8
526
dx.doi.org/10.1021/jm501037u | J. Med. Chem. 2014, 57, 8517−8529

Journal of Medicinal Chemistry
Article
permeable LTRPC2 channel revealed by Nudix motif homology.
ASSOCIATED CONTENT
Supporting Information
■
Nature 2001, 411, 595−599.
*
S
(9) Kim, H.; Jacobson, E. L.; Jacobson, M. K. Synthesis and
Figures S1−S3, crystallographic data collection, model refine-
degradation of cyclic ADP-ribose by NAD glycohydrolases. Science
993, 261, 1330−1333.
10) Howard, M.; Grimaldi, J.; Bazan, J.; Lund, F.; Santos-Argumedo,
1
13
ment statistics, HPLC traces, and H NMR and C NMR
spectra for all novel compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
1
(
L.; Parkhouse, R.; Walseth, T.; Lee, H. Formation and hydrolysis of
cyclic ADP-ribose catalyzed by lymphocyte antigen CD38. Science
1993, 262, 1056−1059.
Accession Codes
The structure of the shCD38:7a complex has been deposited in
the PDB with the code 4TMF.
(11) Cosker, F.; Cheviron, N.; Yamasaki, M.; Menteyne, A.; Lund, F.
E.; Moutin, M. J.; Galione, A.; Cancela, J. M. The ecto-enzyme CD38
is a nicotinic acid adenine dinucleotide phosphate (NAADP) synthase
AUTHOR INFORMATION
Corresponding Author
Phone: ++44-1225-386639. Fax: ++44-1225-386114. E-mail:
B.V.L.Potter@bath.ac.uk.
■
*
2+
that couples receptor activation to Ca mobilization from lysosomes
in pancreatic acinar cells. J. Biol. Chem. 2010, 285, 38251−38259.
(12) Savarino, A.; Bensi, T.; Chiocchetti, A.; Bottarel, F.; Mesturini,
̀
R.; Ferrero, E.; Calosso, L.; Deaglio, S.; Ortolan, E.; Butto, S.; Cafaro,
Notes
A.; Katada, T.; Ensoli, B.; Malavasi, F.; Dianzani, U. Human CD38
interferes with HIV-1 fusion through a sequence homologous to the
V3 loop of the viral envelope glycoprotein gp120. FASEB J. 2003, 17,
461−463.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(13) Malavasi, F.; Deaglio, S.; Damle, R.; Cutrona, G.; Ferrarini, M.;
We acknowledge Grants RCGAS 201105159001 (to R.G.),
HK-GRF 766911 (to Q.H.), and HK-GRF HKU 785110M (to
H.Z.). BVLP is a Wellcome Trust Senior Investigator (Grant
Chiorazzi, N. CD38 and chronic lymphocytic leukemia: a decade later.
Blood 2011, 118, 3470−3478.
(14) Aksoy, P.; White, T. A.; Thompson, M.; Chini, E. N. Regulation
1
01010).
of intracellular levels of NAD: a novel role for CD38. Biochem. Biophys.
Res. Commun. 2006, 345, 1386−1392.
ABBREVIATIONS USED
■
5
(15) Young, G. S.; Choleris, E.; Lund, F. E.; Kirkland, J. B. Decreased
+
−/−
ADPR, adenosine 5′-diphosphate ribose; ADPRC, adenosine
cADPR and increased NAD in the Cd38 mouse. Biochem. Biophys.
Res. Commun. 2006, 346, 188−192.
′-diphosphate ribosyl cyclase; cADPR, cyclic adenosine 5′-
(16) Chini, E. N. CD38 as a regulator of cellular NAD: a novel
diphosphoribose; cADPcR, cyclic adenosine 5′-diphosphate
potential pharmacological target for metabolic conditions. Curr.
Pharm. Des. 2009, 15, 57−63.
carbocyclic ribose; cIDPR, cyclic inosine 5′-diphosphoribose;
+
NAADP, nicotinic acid adenine dinucleotide phosphate; NAD ,
(17) Jin, D.; Liu, H. X.; Hirai, H.; Torashima, T.; Nagai, T.; Lopatina,
nicotinamide adenosine 5′-dinucleotide; NADP, nicotinamide
adenine dinucleotide phosphate; N9-butyl-cIDPR, cyclic-N9-
butylhypoxanthine 5′-diphosphate ribose; shCD38, CD38
catalytic domain
O.; Shnayder, N. A.; Yamada, K.; Noda, M.; Seike, T.; Fujita, K.;
Takasawa, S.; Yokoyama, S.; Koizumi, K.; Shiraishi, Y.; Tanaka, S.;
Hashii, M.; Yoshihara, T.; Higashida, K.; Islam, M. S.; Yamada, N.;
Hayashi, K.; Noguchi, N.; Kato, I.; Okamoto, H.; Matsushima, A.;
Salmina, A.; Munesue, T.; Shimizu, N.; Mochida, S.; Asano, M.;
Higashida, H. CD38 is critical for social behaviour by regulating
oxytocin secretion. Nature 2007, 446, 41−45.
ADDITIONAL NOTE
Note that for the N9-butyl analogs, the prime notation refers
to the N1-ribose, in contrast to the natural products where the
prime notation refers to the N9-ribose.
■
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