Design, synthesis and biological characterization of novel inhibitors of CD38

Article abstract of DOI:10.1039/c0ob00768d

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca²⁺ messenger molecule, cyclic ADP-ribose, from NAD⁺. It is well established that this novel Ca²⁺ signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD⁺ complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound1-14). A number of these compounds exhibited moderate inhibition of the NAD⁺ utilizing activity of CD38, with Compound4 showing the highest potency. The crystal structure of CD38/Compound4 complex and computer simulation of Compound7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.

Full text of DOI:10.1039/c0ob00768d

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Organic &
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PAPER
Design, synthesis and biological characterization of novel inhibitors of CD38†
Min Dong,a Yuan-Qi Si,a Shuang-Yong Sun,a Xiao-Ping Pu,a Zhen-Jun Yang,a Liang-Ren Zhang,a
Li-He Zhang,*a Fung Ping Leung,b Connie Mo Ching. Lam,b Anna Ka Yee Kwong,b Jianbo Yue,b Yeyun Zhou,c
Irina A. Kriksunov,c Quan Haob and Hon Cheung Lee*b
Received 23rd September 2010, Accepted 17th February 2011
DOI: 10.1039/c0ob00768d
Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte
activation, but also as an enzyme catalyzing the synthesis of a Ca2+ messenger molecule, cyclic
ADP-ribose, from NAD+. It is well established that this novel Ca2+ signaling enzyme is responsible for
regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD+
complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1–14).
A number of these compounds exhibited moderate inhibition of the NAD+ utilizing activity of CD38,
with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex
and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide
moiety and the distal aromatic group of the compounds for substrate recognition by the active site of
CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced
contraction of muscle preparations from rats and guinea pigs. This study is a rational design of
inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future
drug development.
aorta,8 hormonal signaling in pancreatic acinar cells,9 migration of
dendritic cell precursors,10 bone resorption,11 insulin secretion,5,12
Introduction
and social behavior changes.13 Clinically, CD38 expression is a
negative prognostic marker for chronic lymphocytic leukemia.14,15
Moreover, CD38 is responsible for synthesizing yet another
ubiquitous Ca2+ messenger, nicotinic acid adenine dinucleotide
phosphate (NAADP), from NADP and nicotinic acid via a base-
exchange reaction.16,17 It should now be a generally accepted fact
that CD38 is expressed both in intracellular organelles, such as the
nucleus, ER, etc., as well as on the surface of some cells, particular
the blood cells. It is likely that the internal CD38 may be more
relevant for cell signaling.
That CD38 plays key roles in physiology provides an important
impetus for this study to design and synthesize inhibitors of
CD38. Inhibitors of the enzymatic activities of CD38 have
been described, but none of them have been shown to have
physiological effects (Fig. 1). Slama et al. synthesized a non-
hydrolyzable analog of NAD+, dinucleotide carbanicotinamide
adenine dinucleotide (carba-NAD+), as a competitive inhibitor
of the NAD+ glycohydrolase activity of CD38 with an IC50 of
about 100 mM.18,19 Inhibitors that form covalent intermediates
with the catalytic residues of CD38 have also been described.
These inhibitors are mainly NAD+ derivatives with modifications
at the nicotinamide ribonucleoside. For example, a series of
fluoro-substituted NAD+ derivatives has been produced.20,21 The
fluoro substitution at the 2¢-position of the nicotinamide sugar
moiety promotes the formation of a stable covalent bond between
CD38 is a trans-membrane enzyme, originally identified as a
lymphocyte differentiation antigen.1 It is now known to be ubiq-
uitously expressed in virtually all mammalian tissues examined.2
As a multi-functional protein and a member of the ADP-ribosyl
cyclase family, CD38 catalyzes the synthesis of cyclic ADP-ribose
(cADPR) from NAD+, a cyclic nucleotide messenger mediating
Ca2+ release from intracellular stores in a wide range of biological
systems from plant to human.3 Remarkably, CD38 can also
hydrolyze the product, cADPR, and the substrate, NAD+, to
produce ADP-ribose.4 That CD38 is a naturally occurring enzyme
responsible for the synthesis of cADPR has been shown by
ablation of the CD38 gene in mice, which results in large reduction
in endogenous cADPR in many tissues.5,6 The CD38 knockout
mice exhibit a variety of defects, establishing the importance of
CD38 as a regulator of diverse physiological functions,5,6 which
include immune cell differentiation,7 a-adrenoceptor signaling in
aState Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing 100191, China. E-mail:
zdszlh@bjmu.edu.cn; Fax: +86-10-82802724; Tel: +86-10-82801700
bDepartment of Physiology, University of Hong Kong, Hong Kong, China.
E-mail: leehc@hku.hk; Fax: +852-2817-1334; Tel: +852-2819-9163
cMacCHESS, Cornell High Energy Synchrotron Source, Cornell University,
Ithaca, NY 14853, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob00768d
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the ribose and Glu226, the catalytic residue of CD38, during
catalysis. The covalent intermediate has been captured by X-
ray crystallography.22 Based on the structure of ara-F NAD,
Schramm and coworkers reported that ara-F NMN and several b-
nicotinamide 2¢-deoxyribosides are also potent inhibitors of CD38
with Ki values in the nanomolar range.21,23
or bis-nicotinamide moiety (Compounds 8–11) may have sufficient
affinity for the catalytic cavity of CD38 to serve as inhibitors.
In addition, a series of derivatives was designed with aromatic
ether strands replacing the adenosine-pyrophosphate moiety of
the natural substrate, NAD+, so as to improve the membrane
permeability of the compounds. Finally, in Compounds 13 and 14,
4-amino-nicotinamide and 6-quinoline carboxylic amide replaced
and mimicked the nicotinamide ring, respectively.
Compounds 1 and 2 were synthesized as depicted in Scheme
1.25 Starting from 1,3-dioxolane and acetyl chloride, the
chloromethoxyl ethyl acetate was obtained in 62% yield, which was
stirred with nicotinamide for 10 h in DMF at room temperature26
to form Compound 1 with high yield (80%). Compound 2 was
produced by the condensation of [2-(benzyloxy)ethoxy]methyl
chloride27 and nicotinamide in DMF in 60% yield.
Compounds 3–7 were synthesized by a similar strategy. Sub-
stituted phenols or 8-hydroxyl-quinoline were condensed with
chloroethanol in a 10% NaOH solution.28 The corresponding
alcohols were chloromethylated by paraformaldehyde and dry
HCl in DCE. The chloromethylation products were used directly
to react with nicotinamide to produce Compounds 3–7 in good
yields (Scheme 2).
These inhibitors, being derivatives of NAD+, are charged
molecules with limited permeability to cells and tissues and none
has been reported to exhibit biological effects. It is the purpose
of this study to develop membrane-permeant CD38 inhibitors as
tools for physiological investigations and as potential drug candi-
dates as well. Based on the crystal structure of the CD38/NAD+
complex that we reported previously,24 we synthesized a series of
simplified N-substituted nicotinamide derivatives (Fig. 2, 1–14)
and showed that some of them, including Compounds 4 and 7, are
moderate inhibitors of the enzymatic activities of CD38. X-Ray
crystallography shows that Compound 4 binds inside the catalytic
cavity of CD38 and reveals important details of the interacting
residues at the active site. Moreover, when applied to applied to
muscle preparations from rat and guinea pig, both Compounds
4 and 7 exhibit potent relaxing effects on the agonist-induced
tension.
8-OH-quinoline was coupled with 1,4-butenediol by Mitsunobu
reaction in 70% yield. The chloromethylation product was ob-
tained as described before and then condensed with nicotinamide
to give Compound 12 in 55.8% for two steps. (Scheme 3)
The syntheses of the bis-nicotinamide Compounds 8–11 were
completed by the condensation of the bis-chloromethylation
products with two equivalents of nicotinamide in high yields. 1,2-
Bis- chloromethoxyethane, 1,4-bis-chloromethoxybutane, 1,4-bis-
chloromethoxycrotonylene and 1,6-bis-chloromethoxyhexane29
were prepared from the corresponding dihydroxyl com-
pounds by chloromethylation reaction, as described before.
(Scheme 4)
4-Phenoxyphenol was coupled with chloroethanol and then
chloromethylated. The product was condensed with quinoline-6-
carboxylic amide or 4-amino nicotinamide,30 respectively, in DMF
for 12 h at room temperature to produce Compounds 13 (63%) and
14 (43%) in two steps (Scheme 5).
Biological activities
Inhibition of the activity of NADase. The newly synthesized
Compounds 1–14 were tested for their inhibitory properties against
the NAD-glycohydrolase activity of the recombinant CD38,
which was measured using a fluorimetric and highly sensitive
coupled enzyme assay as previously described.31 As shown in
Table 1, compounds 3,4,5,7,12,13 and 14 exhibit weak inhibitory
activities. Structurally, compounds with an aromatic group at the
distal end from the nicotinamide are more effective in inhibiting
Fig. 1 Inhibitors of CD38.
Results and discussion
Chemistry
The crystal structure of the CD38/NAD+ complex shows that the
nicotinamide group of the bound NAD+ enters the catalytic cavity
first and interacts with residues Glu146 and Asp155 at the active
site through two hydrogen bonds to its amide. The interaction
is further enhanced by the parallel p interactions between its
pyridine ring and the Trp189 indole ring.24b It is reasoned that
derivatives consisting of either one nicotinamide (Compounds 1–7)
Table 1 NADase inhibitory activity of 1–14
Inhibitor
IC50/mM
Inhibitor
IC50/mM
1
2
3
4
5
6
7
NMa
8
NM
9
11.41
10
3.42
11
5.65
12
NM
13
10.00
14
NM
NM
NM
NM
3.45
2.12
4.80
a NM = not measurable.
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Fig. 2 NAD+ and N-aromatic ether substituted nicotinamides.
CD38. This is likely to be due to the enhanced hydrophobic
Physiological effects on muscle preparations from rat and guinea
pig
interactions with the active site. Compound 6 contains a strong
electron withdrawing m-CF3-phenol group that should interfere
with hydrophobic interaction, and it indeed showed no affinity
for CD38. The positively charged bis-nicotinamide moiety in
Compounds 8–11 likewise prohibit hydrophobic interaction and
the compounds were ineffective in inhibiting CD38. Interestingly,
the longer aliphatic chain in Compound 12, as compared to
Compound 7, improves this compound’s interaction with CD38.
Comparing Compounds 13 and 4, replacing the nicotinamide
with a quinoline ring, improved the affinity slightly. On the
other hand, the additional amino group on the nicotinamide
ring in Compound 14 offered no improvement as compared to
Compound 4.
It has previously been shown that CD38 gene ablation attenuates
the contraction induced by a-adrenoceptor stimulation in
mouse aorta, indicating that the contraction is mediated by
the CD38/cADPR-pathway.8 We therefore tested the effect of
compound 4 on the phenylephrine-induced contraction in rat
aortic ring preparations with intact endothelium. As shown in Fig.
3A, compound 4 produced concentration-dependent relaxation
with a half-maximal effect (pD2) at about 36 mM. The effect is
specific because the structurally similar but inactive compound
9 produced no such vascular relaxing effect. Nicotinamide,
a commonly used inhibitor of CD38, can effectively attenuate
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Scheme 1 Synthesis of analogues 1 and 2.
Scheme 2 Synthesis of analogues 3–7.
Scheme 3 Synthesis of analogues 12.
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Scheme 4 Synthesis of 8–11.
Scheme 5 Synthesis of analogues 13 and 14.
agonist-induced contraction.32 Fig. 3B shows that it can
induce vascular relaxation in a manner similar to compound 4.
The half-maximal effect was at about 1 mM.
illustrate the usefulness of these new inhibitors for investigating
the physiological roles of CD38 in cells and tissues.
Although, these inhibitors show only moderate activity in vitro,
they are much more effective in vivo. This is likely because of
the hydrophobicity of the compounds, allowing them to easily
penetrate the cell membrane and accumulate inside cells and
tissues. We have calculated the AlogP using Pipeline Pilot V7.5
and the results indicate clearly that compounds 4 and 7 are more
lipophilic than compound 9.†
As is shown below, Compound 4 binds inside the catalytic pocket
of CD38. It is possible that the structure as well as the residues
constituting the active sites of rat and guinea pig CD38 may be
somewhat different from those of the human CD38, which was
used in all the in vitro assays. Currently, the structures of neither
rat nor guinea pig CD38 have been solved.
We have previously shown that Compound 7, the less potent
of the series with an IC50 of 10 mM for the inhibition of the
NADase, significantly inhibited the acetylcholine (Ach)-induced
Ca2+ increase in PC12 cells. That the effect of Ach is mediated
by CD38 is shown by RNAi knockdown of the enzyme, which
completely abrogates the ACh-induced production of cADPR.33
Airway smooth muscle contraction requires elevation of intracel-
lular Ca2+. Studies indicate that the CD38/cADPR/RyR-Ca2+
signaling pathway plays a vital role in the regulation of Ca2+
homeostasis in the airway smooth muscle cells.34 Here we use
the newly synthesized inhibitors to illustrate the role of CD38 in
mediating the Ach-induced contraction in airway smooth muscle.
The isolated tracheal strips of guinea pigs and rats were first
contracted by treatment with Ach. The ability of the CD38
inhibitors to relax the contraction was then used as a model for
the antispasmodic effects of the compounds. As shown in Fig.
3, both Compounds 4 and 7 were effective in relaxing the Ach-
induced contraction in a dose-dependent manner. Similar results
were also observed in the isolated tracheal strips of rat (data not
shown). These results are consistent with the known fact that
CD38 is important in mediating the Ach-induced contraction
in airway smooth muscle.34 But more importantly, the results
Structural study of the binding of Compounds 4 and 7 to CD38
To understand the interactions between CD38 and these in-
hibitors, we prepared the complex of Compound 4 with CD38
and analyzed it using X-ray crystallography. Pre-formed crystals
of the catalytic domain of CD38 were soaked in the cryoprotectant
buffer containing the compound to obtain the complex. We were
able to obtain only the complex with Compound 4 (the ESI shows
the statistics of data collection and structure refinement of the
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is the bound NAD previously determined by us.24 As can be
seen, the nicotinamide groups of both Compound 4 and NAD
bind at the same position. They also interact identically with the
same residues, forming hydrogen bonds with Glu146 and Asp155,
as well as hydrophobically stacking with Trp189 (Fig. 4B). The
structural results indicate that the inhibitory effect of Compound
4 is likely to be due to its specific binding to the active site. The
N-substituted biphenyl ether group in Compound 4 distal to the
nicotinamide ring, on the other hand, binds quite differently than
the ribose and phosphate groups of NAD, interacting instead
mainly with Trp176 through hydrophobic stacking (Fig. 4A).
Molecular dynamics simulation allows us to model the in-
teraction of Compound 7 with CD38, even though we were
unable to obtain the crystal complex. The stimulation was carried
out starting with the crystallographic data obtained from the
CD38/Compound 4 complex.
The result is shown in Fig. 5a and the superimposition of
Compounds 4 and 7 is shown in Fig. 5b. The docking study
indicates that the positioning of Compound 7 at the active site is
approximately the same as Compound 4 as observed in the crystal
structure. Besides the same hydrogen bonding of the nicotinamide
moieties of Compound 7 and 4 with the active site, interactions
between the nitrogen atom of the quinoline of Compound 7 with
residue Ser186 and p-stacking interaction between the quinoline
ring and Trp176 of CD38 were also observed. The similarity of the
interactions is consistent with both compounds having inhibitory
effects on CD38.
Conclusion
Fig. 3 Biological effects of compounds 4, 7 and 9 on agonist-induced
muscle contraction. A. Concentration-response curves for solvent (MQ
water) control, compound 4 (active) and compound 9 (inactive), on the
phenylephrine-induced vascular contraction in isolated rat aortic ring
preparations. B. Nicotinamide, a commonly used inhibitor of CD38,
induced similar vascular relaxation. Results are means SEM. using
tissues from three to five rats in each group. C. The relaxing effects of
Compounds 4 and 7 on the acetylcholine-induced contraction in isolated
tracheal strips of guinea pigs. Data represent the mean SEM (n = 7). *P
< 0.05, statistically significant as compared with Krebs–Henseleit solution
group.
There is a growing tendency to develop non-nucleotide com-
pounds to mimic such signaling molecules.35 This study represents
a rational design of a series of inhibitors of CD38, one of the
key enzymes in cellular Ca2+ signaling. The design was based on
the crystal structure of NAD binding to the active site of CD38
and takes into account the need for membrane permeability. We
targeted the nicotinamide portion of the NAD because it interacts
strongly with the active site via not only hydrogen bonding but
also hydrophobic stacking. The reasoning was substantiated by
the crystal structure, showing that the nicotinamide portion of
Compound 4 binds identically as NAD. The replacement of the
rest of the highly charged moieties of the NAD with aromatic
groups ensures membrane permeability and contributes to the
complex†). Fig. 4A shows that Compound 4 binds inside the
catalytic pocket of human CD38. Superimposed in the figure
Fig. 4 Structural alignment between CD38–Compound 4 and CD38–NAD complexes. (A) Surface presentation of the active pocket of CD38 (pale green).
NAD (sticks presentation in magenta) penetrated to the bottom of the active pocket of CD38, while compound 4 (sticks presentation in grey) floated over
the active site. (B) The nicotinamide group of both compound 4 (grey) and NAD (magentas) is similarly positioned and stabilized by the interactions
with residues Glu146, Asp155 and Trp189 of CD38.
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Fig. 5 (a) The molecular dynamics simulation binding mode of compound 7 in the active pocket of human CD38. The carbon atoms of 7 are indicated
in cyan. All the nitrogen atoms are blue; oxygen atoms are red. Hydrogen bonds are represented by blue dotted lines. Corresponding residues are labeled
and shown in blue lines. (b) Superimposition the binding poses of compound 4 in the enzyme–inhibitor cyrstal with the molecular dynamics simulation
result of 7. 4 is shown as the magenta stick; 7 is shown as the cyan stick.
greatly increased in vivo potency of the inhibitors. The potency
increase is observed both in cultured PC12 cells as we have
previously reported33 and muscle preparations from rat and guinea
pig described here, verifying the general applicability of the new
inhibitors. Structure–activity comparison of all the compounds in
this series provides clues for the next iteration to produce even
more effective inhibitors and set the stage toward developing drug
candidates for CD38-related diseases.
(d, 1H, J = 5.1 Hz), 9.53 (s, 1H).13C NMR (75 MHz, CD3OD) d
20.7, 63.9, 70.8, 90.7, 129.3, 135.8, 144.2, 145.8, 146.5, 165.0, 172.4.
ESI-MS: [M - Cl]+: 239.1. Anal. (C11H15ClN2O4·0.3H2O) C, H, N.
1-[(2-Benzyloxyethoxy)methyl]-3-(aminocarbonyl)-pyridinium
chloride (Compound 2). To a solution of 2-(benzyloxy)ethanol
(0.85 ml, 6 mmol) in dry 1,2-dichloroethane (8 ml), paraformalde-
hyde (0.18 g, 6 mmol) was added. Dry HCl gas, obtained in situ
from H2SO4 and NaCl, was bubbled through the reaction mixture
over 10 h at 0 ◦C. The solution was then dried with anhydrous
Na2SO4, filtered and concentrated under reduced pressure to give
a yellow oily residue. The residue was added to a solution of
nicotinamide (0.73 g, 6 mmol) in DMF (15 ml). The mixture
was stirred at room temperature for 4 h. The white precipitates
were collected by filtration and washed with dry ethyl acetate and
ethanol, then recrystallized from EtOH by freezing to give the
desired Compound 2 (1.25 g, 65% for two steps) as a white solid.
1H NMR (300 MHz, CD3OD) d 3.64 (m, 2H), 3.99 (m, 2H), 4.36
(s, 2H), 6.01 (s, 2H), 7.18–7.32 (m, 5H), 8.05 (dd, 1H, J = 8.1, 6.3
Hz), 8.82 (d, 1H, J = 8.1 Hz), 9.12 (d, 1H, J = 6.3 Hz), 9.45 (s,
1H).13C NMR (75 MHz, CD3OD) d 165.0, 146.0, 145.7, 144.1,
139.0, 135.3, 129.5, 129.1, 129.0, 128.9, 128.8, 91.4, 74.2, 73.3,
70.3. ESI-MS: [M - Cl]+ 287.1. Anal. (C16H19ClN2O3) C, H, N.
Experiment section
Chemistry
All solvents and reagents were obtained from commercial sources
and used without further purification unless otherwise stated.
HR-ESI-MS (electrospray ionization) was performed with Bruker
BIFLEX III. 1H NMR and 13C NMR were recorded with a
Bruker AVANCE III 400 or a JEOL AL300 spectrometer. CD3OD,
DMSO-d6 or D2O were used as a solvent. Chemical shifts are
reported in parts per million downfield from TMS (1H and
13C). 19F NMR spectra were recorded on a Varian VXR-500
spectrometer. Chemical shifts of 19F NMR are reported in ppm
with reference to CF3COOH as an external standard. Anhydrous
solvents were obtained as follows: DMF was dried with CaH2 at
room temperature before being distilled in vacuo; ethyl acetate was
dried with P2O5 before distillation. 1,2-Dichlorethane, ethanol and
methanol were distilled over CaH2.
1-{[2-(4-Methoxy-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-
pyridinium chloride (Compound 3). 4-Methoxyphenol (0.56 g, 4.5
mmol) was dissolved in 10% sodium hydroxide solution (16 ml,
40 mmol). 2-Chloroethanol (2.68 ml, 40 mmol) was then added
and the mixture stirred for 24 h at room temperature. The solution
was extracted with CH2Cl2. The extract was washed with water
three times then dried by evaporation. The product obtained, 2-
(4-methoxyphenoxy)ethanol (0.68 g, 4.1 mmol, 90%), was used
in the next step directly and dissolved in 1,2-dichloroethane (10
ml). Paraformaldehyde (0.13 g, 4.1 mmol) was added and dry HCl
gas was bubbled through the reaction mixture for 10 h at 0 ◦C.
The solution was then treated as in the synthesis of Compound 2
and reacted with nicotinamide (0.5 g, 4.1 mmol) in DMF (8 ml).
The mixture was stirred at room temperature for 8 h. The white
precipitates were collected by filtration and washed with dry ethyl
1-[(2-Acetoxyethoxy)methyl]-3-(aminocarbonyl)-pyridinium ch-
loride (Compound 1). Dry nicotinamide (122 mg, 1 mmol) was
dissolved in dry DMF (3 mL), chloromethoxyl ethyl acetate (0.14
mL, 1 mmol) was added under N2. The mixture was stirred at room
temperature for 1 h and a large amount of solid appeared. Dry
ethyl acetate (3 ml) was added and the mixture was stirred for 30
min. The white precipitates were collected by filtration and washed
with dry ethyl acetate, then recrystallized from EtOH/EA to give
the desired Compound 1 (221 mg, 80%) as a white solid. 1H NMR
(300 MHz, CD3OD) d 2.03 (s, 3H), 3.98 (m, 2H),4.25 (m, 2H), 6.06
(s, 2H), 8.30 (dd, 1H, J = 7.2, 5.1 Hz), 9.07 (d, 1H, J = 7.2 Hz), 9.21
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acetate and ethanol. The crude product was recrystallized from
EtOH/EA to obtain the desired Compound 3 (1.14 g, 82% for
two steps) as a white solid. 1H NMR (300 MHz, CD3OD) d 3.72
(s, 3H), 4.11 (m, 4H), 6.11 (s, 2H), 6.72–6.83 (m, 4H), 8.23 (dd,
1H, J = 8.4, 6.0 Hz), 8.99 (dt, 1H, J = 8.4, 1.5 Hz), 9.22 (d, 1H,
J = 6.0 Hz), 9.55 (s, 1H).13C NMR (75 MHz, CD3OD) d 165.0,
155.8, 153.5, 146.3, 145.7, 144.2, 135.5, 129.1, 91.2, 72.3, 68.5,
56.0. ESI-MS: [M - Cl]+ 303.1. Anal. (C16H19ClN2O4) C, H, N.
recrystallized from MeOH/EA, Compound 7 was obtained as a
yellow solid. (78% for two steps). 1H NMR (300 MHz, CD3OD)
d 4.36 (t, 2H, J = 4.2 Hz), 4.67 (m, 2H, J = 4.2 Hz), 6.19 (s, 2H),
7.70 (dd, 1H, J = 6.0, 2.7 Hz), 7.91 (m, 2H), 8.16 (dd, 1H, J = 8.4,
5.7 Hz), 8.25 (dd, 1H, J = 8.1, 6.0 Hz), 9.00 (d, 1H, J = 8.1 Hz),
9.18–9.27 (m, 3H), 9.58 (s, 1H).13C NMR (75 MHz, CD3OD) d
164.9, 150.0, 148.5, 146.2, 145.9, 145.5, 144.1, 135.7, 131.8, 131.7,
131.0, 129.3, 123.8, 122.0, 115.3, 90.7, 71.1, 70.0. ESI-MS: [M -
Cl]+ 324.2. Anal. (C18H18ClN3O3·3H2O) C, H, N.
1-{[2-(4-Phenoxy-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-
pyridinium chloride (Compound 4). The procedure used was
similar to that for the synthesis of Compound 3. 4-Phenoxyphenol
was condensed with 2-chloroethanol in 83% yield. The alcohol
obtained was chloromethylated and reacted with nicotinamide
successively to yield Compound 4 (71.5% for two steps). 1H NMR
(300 MHz, CD3OD) d 4.07–4.15 (m, 4H), 6.13 (s, 2H), 6.82–6.93
(m, 6H), 7.04 (t, 1H, J = 7.5 Hz) 7.29 (dd, 2H, J = 8.5, 7.5 Hz) 8.26
(dd, 1H, J = 8.1, 6.0 Hz), 9.02 (d, 1H, J = 8.1 Hz), 9.24 (d, 1H,
J = 6.0 Hz), 9.58 (s, 1H).13C NMR (75 MHz, CD3OD) d 165.0,
159.7, 155.7, 152.2, 146.3, 145.7, 144.2, 135.6, 130.8, 129.1, 123.8,
121.6, 118.8, 116.7, 91.1, 72.2, 68.5. ESI-MS: [M - Cl]+ 365.1.
Anal. (C21H21ClN2O4) C, H, N.
1,2-Dimethoxy-ethylene-bis-N,N¢-3-(aminocarbonyl)-pyridi-
nium dichloride (Compound 8). 1,2-Bis-chloromethoxy-ethane
was prepared as described in the literature.29 The bis-
chloromethylation product (0.78 ml, 5 mmol) was added to a
solution of nicotinamide (1.34 g, 11 mmol) in 20 ml DMF. The
mixture was stirred for 12 h at room temperature. Dry ethyl acetate
(10 ml) was added and the mixture was stirred a further 30 min.
The white precipitate was collected by filtration and washed with
dry ethyl acetate and ethanol to give the desired Compound 8 (1.71
g, 85%) as a white solid. 1H NMR (300 MHz, D2O) d 3.80 (s, 4H),
5.87 (s, 4H), 8.11 (dd, 2H, J = 8.1, 6.3 Hz), 8.84 (d, 2H, J = 8.1 Hz),
8.96 (d, 2H, J = 6.3 Hz), 9.26 (s, 2H). 13C NMR (75 MHz, CD3OD)
d 165.5, 146.5, 146.0, 144.4, 135.9, 129.4, 90.7, 71.4. ESI-MS: [M
- Cl]+ 367.1. Anal. (C16H20Cl2N4O4·2H2O) C, H, N.
1-{[2-(4-Nitro-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-py-
ridinium chloride (Compound 5). The procedure used was similar
to that for the synthesis of Compound 3. 4-Nitrophenol was con-
densed with 2-chloroethanol in 87% yield. The alcohol obtained
was chloromethylated and reacted with nicotinamide successively
to yield the Compound 5 (82% for two steps). 1H NMR (300 MHz,
CD3OD) d 4.19 (m, 2H), 4.32 (m,2H), 6.14 (s, 2H), 7.05 (d, JAB
= 9.3 Hz,2H,A of aryl A2B2), 8.18 (d, JAB = 9.3 Hz,2H,B of aryl
A2B2), 8.30 (dd, 1H, J = 8.1, 6.0 Hz), 9.05 (d, 1H, J = 8.1 Hz), 9.25
(d, 1H, J = 6.0 Hz), 9.58 (s, 1H).13C NMR (75 MHz, CD3OD)
d 165.0, 164.8, 146.5, 145.8, 144.2, 143.1, 135.7, 129.3, 129.2,
126.8, 115.8, 90.9, 71.4, 68.8. ESI-MS: [M - Cl]+ 318.1. Anal.
(C15H16ClN3O5) C, H, N.
1,4-Dimethoxy-butylene-bis-N,N¢-3-(aminocarbonyl)-pyridi-
nium dichloride (Compound 9). The same procedure as for
Compound 8 was followed to afford the desired Compound 9 (52.7%
for two steps). 1H NMR (300 MHz, D2O) d 1.57 (s, 4H), 3.58 (s,
4H), 5.86 (s, 4H), 8.14 (dd, 2H, J = 8.4, 6.0 Hz), 8.86 (d, 2H, J = 8.4
Hz), 8.99 (d, 2H, J = 6.0 Hz), 9.26 (s, 2H). 13C NMR (125 MHz,
CD3OD) d 165.1, 146.5, 145.8, 144.2, 135.8, 129.4, 91.0, 72.4, 26.8.
ESI-MS: [M - Cl]+ 395.1. Anal. (C18H24Cl2N4O4·1.4H2O) C, H, N
1,4-Dimethoxy-butyne-bis-N,N¢-3-(aminocarbonyl)-pyridinium
dichloride (Compound 10). The same procedure as for Compound
8 was followed to afford the desired Compound 10 (46% for two
steps). 1H NMR (300 MHz, CD3OD) d 4.48 (s, 4H), 6.10 (s, 4H),
8.31 (dd, 2H, J = 8.1, 6.3 Hz), 9.09 (dt, 2H, J = 8.1, 1.5 Hz), 9.25
(d, 2H, J = 6.3 Hz), 9.61 (s, 2H). 13C NMR (75 MHz, CD3OD) d
165.1, 146.8, 146.6, 145.0, 135.7, 129.3, 90.0, 83.7, 59.8. ESI-MS:
[M - Cl]+ 391.1. Anal. (C18H20Cl2N4O4·0.8H2O) C, H, N.
1-{[2-(3-Trifluoromethyl-phenoxy)ethoxy]methyl}-3-(aminocar-
bonyl)-pyridiniumchloride (Compound 6). The procedure follows
the synthesis of Compound 3. 3-Trifluoromethylphenol was con-
densed with 2-chloroethanol in 95% yield. The alcohol obtained
was chloromethylated and reacted with nicotinamide successively
to yield the Compound 6 (40% for two steps). 1H NMR (300 MHz,
CD3OD) d 4.16–4.25 (m, 4H), 6.13 (s, 2H), 7.12–7.25 (m, 3H),
7.45 (m, 1H) 8.27 (dd, 1H, J = 8.1, 6.0 Hz), 9.03 (dt, 1H, J = 8.1,
1.5 Hz), 9.24 (d, 1H, J = 6.0 Hz), 9.59 (s, 1H). 13C NMR (100
MHz, CD3OD) d 164.9, 160.0, 146.4, 145.8, 144.2, 135.7, 132.9
1,4-Dimethoxy-hexamethylene-bis-N,N¢-3-(aminocarbonyl)-
pyridinium dichloride (Compound 11). The same procedure as for
Compound 8 was followed to afford the desired Compound 11 (52%
for two steps). 1H NMR (300 MHz, CD3OD) d 1.40 (m, 4H), 1.67
(m, 4H), 3.69 (t, 4H, J = 6.3 Hz), 6.01 (s, 4H), 8.29 (dd, 2H, J =
8.4, 6.3 Hz), 9.07 (dt, 2H, J = 8.4, 1.5 Hz), 9.20 (d, 2H, J = 6.3
Hz), 9.50 (s, 2H). 13C NMR (75 MHz, CD3OD) d 165.1, 146.5,
145.8, 144.2, 135.8, 129.3, 91.0, 72.6, 30.2, 26.6. ESI-MS: [M -
Cl]+ 423.2. Anal. (C20H28Cl2N4O4) C, H, N.
1
(q, JCF = 32 Hz), 131.6, 129.2, 125.4 (q, JCF = 270 Hz), 119.3,
118.8 (q, JCF = 4 Hz), 112.4 (q, JCF = 4 Hz), 91.0, 71.8, 68.4. 19F
NMR (470 MHz, CD3OD) d 21.3. ESI-MS: [M - Cl]+ 341.1. Anal.
(C16H16ClF3N2O3) C, H, N.
1-{[2-(8¢-Quinolyloxy)ethoxy]methyl}-3-(aminocarbonyl)-pyri-
dinium chloride (Compound 7). The procedure follows the
synthesis of Compound 3. 8-Hydroxyqunoline was condensed
with 2-chloroethanol in 65% yield. A suspension of 2-(8¢-
quinolinoxy)ethanol (1.28 g, 6.77 mmol), paraformaldehyde (0.2
(E)-1-{[4-(8¢-Quinolyloxy)but-2-enyloxy]methyl}-3-(aminocar-
bonyl)-pyridinium chloride (Compound 12). To a solution of
1,4-butenediol (0.88 g, 10 mmol), 8-hydroxyquinoline (0.725 g,
5 mmol) and triphenylphosphine (PPh3) (1.57 g, 6 mmol) in
THF (25 ml), diisopropyl azodicarboxylate (1.21 g, 6 mmol)
was added dropwise. The reaction was stirred for 4 h at room
temperature and refluxed for 12 h. The solution was concentrated
under reduced pressure. The crude mixture was purified by
˚
g, 6.77 mmol) and 1.5 g 3 A MS in 10 ml 1,2-dichloroethane was
◦
bubbled with HCl gas for 10 h at 0 C. The molecular sieve was
filtered and washed with DCE, the filtrate was concentrated under
reduced pressure and treated with nicotinamide as before. After
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flash column chromatography (2 : 1–3 : 1 hexanes : ethyl acetate
eluent) to furnish pure alcohol (0.72 g, 70%). The alcohol was
chloromethylated and treated with nicotinamide as in the synthesis
of Compound 7. After recrystallized from MeOH/EA, Compound
Dawley rats were also purchased from the Laboratory Animal
Center of Peking University Health Science Center (Beijing,
China). Rats were anesthetized by pentobarbitone sodium (50
mg kg-1, by intraperitoneal injection) and then sacrificed by
cervical dislocation. All experiments performed in this study were
approved by the Committee on the Use of Live Animals in
Teaching and Research of the University of Hong Kong and by the
Beijing animal committee with the confirmation number: SCXK
(Jing) 2006-0008.
Male Hartley guinea pigs were purchased from Beijing
Fangyuanyuan Laboratory Animal Company and approved by
the local animal committee with the confirmation number: SCXK
(Jing) 2009-0014. Animals were housed under standard conditions
(temperature 22 2 ◦C, relative humidity 55 5%, 12 h light/dark
cycle) with food and water available ad libitum. In the present
study, all experiments were performed under the guidelines
of the Experimental Laboratory Animal Committee of Peking
University Health Science Center and were in strict accordance
with the principles and guidelines of the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
Reagents: phenylephrine (Phe) and acetylcholine chloride (ACh)
were purchased from Sigma Chemicals (St. Louis, MO, USA).
All chemicals (including compound 4, 7 and compound 9) were
dissolved in miliQ water.
1
12 was obtained as a yellow-green solid (78% for two steps). H
NMR (500 MHz, D2O) d 4.50 (d, 2H, J = 7.0 Hz), 4.94 (d, 2H,
J = 6.5 Hz), 5.99 (m, 1H), 6.09 (s, 2H), 7.50 (d, 1H, J = 6.5 Hz),
7.86 (m, 2H), 8.11 (dd, 1H, J = 8.0, 5.5 Hz), 8.25 (t, 1H, J = 7.0
Hz), 8.91 (d, 1H, J = 8.5 Hz), 9.03 (d, 1H, J = 5.0 Hz), 9.12 (d, 1H,
J = 8.5 Hz), 9.19 (d, H, J = 6.5 Hz), 9.40 (s, 1H). 13C NMR (125
MHz, D2O) d 165.8, 148.6, 148.0, 146.3, 145.6, 143.9, 143.4, 134.4,
131.2, 130.6, 130.1, 130.0, 129.3, 128.8, 123.1, 121.3, 114.9, 89.6,
67.2, 66.0. ESI-MS: [M - Cl]+ 350.2. Anal. (C20H20ClN3O3·3H2O)
C, H, N.
1-{[2-(4-Phenoxy-phenoxy)ethoxy]methyl}-6-(aminocarbonyl)-
quinolinium chloride (Compound 13). The corresponding alco-
hol was chloromethylated as in Compound 4 and reacted with
quinoline-6-carboxamide successively to yield Compound 13 (63%
for two steps).1H NMR (300 MHz, DMSO) d 4.07 (s, 4H), 5.77 (s,
2H), 6.54 (s, 2H), 6.73–7.38 (m, 9H) 7.93 (s, 1H), 8.31 (dd, 1H, J =
8.4, 5.7 Hz), 8.53 (s, 1H), 8.65 (s, 2H), 9.00 (s, 1H), 9.44 (d, 1H, J
= 8.4 Hz), 9.78 (s, 1H). 13C NMR (125 MHz, D2O) d 169.8, 160.1,
155.6, 152.3, 151.5, 150.5, 141.3, 136.8, 135.3, 131.4, 131.3, 130.8,
123.8, 123.5, 121.5, 121.0, 118.8, 116.6, 89.2, 71.7, 68.6. ESI-MS:
[M - Cl]+ 415.2. Anal. (C25H23ClN2O4·1.4H2O) C, H, N.
2. Tension measurement on isolated rat aortic ring preparations.
The thoracic aorta was excised. After the surrounding connective
tissue had been carefully cleaned off, four 3 mm wide ring segments
were prepared from each aorta. Each was dispensed between two
stainless wire hooks in a 5 mL organ bath. The upper wire was
connected to a force-displacement transducer (RM6240 system,
Chengou Instrument Factory ) and the lower one was fixed at the
bottom of the organ bath. The organ bath was filled with Krebs
solution of the following composition (in mM): 119 NaCl, 4.7
KCl, 25 NaHCO3, 2.5 CaCl2, 1 MgCl2, 1.2 KH2PO4, and 11 D-
glucose. The bathing solution was gassed with 95% O2–5% CO2
at 37 ◦C (pH ª 7.4). The rings were placed under an optimal
basal tone of 15 mN, determined from previous length–tension
experiments. Changes in isometric tension were measured with a
Grass force transducer and stored on RM6240 software for later
data analysis. Twenty minutes after mounting in organ baths, the
rings were first contracted with 0.3 mM phenylephrine (Phe) to
test the contractility and then relaxed by 1 mM ACh. They were
rinsed several times until baseline tone was restored. The rings
were thereafter allowed to equilibrate for 60 min. Baseline tone
was readjusted to 15 mN when necessary. Each set of experiments
was performed on rings prepared from different rats. The use of
laboratory animals was approved by the Animal Research Ethical
Committee of the University of Hong Kong.
1-{[2-(4-Phenoxy-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-
4-amino-pyridinium chloride (Compound 14). The corresponding
alcohol was chloromethylated as in Compound 4 and reacted with
4-aminonicotinamide successively to yield the Compound 14 (43%
for two steps). 1H NMR (300 MHz, DMSO) d 3.92 (s, 2H), 4.10 (s,
2H), 5.55 (s, 2H), 6.90–7.10 (m, 8H), 7.35 (t, 2H, J = 7.6 Hz), 7.91 (s,
1H), 8.30 (d, 2H, J = 6.9 Hz), 8.43 (bs, 1H), 9.11 (m, 3H). 13C NMR
(100 MHz, DMSO) d 166.5, 158.6, 157.9, 154.4, 149.6, 143.9,
141.6, 129.9, 122.7, 120.6, 117.3, 115.7, 112.0, 110.3, 85.7, 68.0,
66.9. ESI-MS: [M - Cl]+ 380.2. Anal. (C21H22ClN3O4·0.5H2O) C,
H, N.
Experimental details for the antagonist assay
Recombinant CD38 was prepared by a yeast expression system
as described in ref. 36. Inhibitors were dissolved in 50 mM Hepes
buffer (pH 7), except Compound 5, which was dissolved in 50%
DMSO (v/v). Recombinant CD38 (0.04 mg ml-1) and BSA (50 mg
ml-1) were mixed with different concentrations of the inhibitors
and incubated for 1 h at room temperature. NAD (2 mM) was
added to initiate the reactions and aliquots were collected at t = 0,
4, 8 and 12 min. The aliquots were immediately stopped with an
equal volume of 0.6 M HCl followed by neutralization with two
volumes of 0.5 M sodium phosphate buffer (pH 8). The amounts of
NAD in the samples were measured by a coupled enzyme assay.31
The percentages of inhibition of the NADase activity as compared
to the control without the inhibitor were plotted against various
concentrations of the inhibitor to obtain the IC50 value.
In this set of experiments, relaxation of Phe (1 mM)-contracted
endothelium-intact rings was induced by compound 4 (30–300
mM) or compound 9 (30–300 mM) or nicotinamide (0.01–6 mM).
The relaxant effects of the vasodilators were expressed as 100
minus percentage reduction from the Phe-induced contractile
response. Non-linear regression curve fitting was performed on
individual cumulative concentration–response curves (GraphPad
software, Version 5.0). pD2 values (IC50) were calculated as
negative log molar of dilator that induced 50% of the maximal
Biological effects on muscle preparations from rat and guinea pig
1. Materials and methods. Animals: Male Sprague–Dawley
rats weighing 250–300 g were supplied from the Laboratory Ani-
mal Unit of the University of Hong Kong. Some male Sprague–
relaxation. All data were shown as means
SEM. Statistical
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significance was determined by two-tailed Student’s t-test or one-
way ANOVA followed by the Newman–Keuls test when more than
two treatments were compared. A P value of less than 0.05 was
regarded as significant.
lographic refinements were done with the program REFMAC5.39
All substrates and products were built using the Program COOT.39
Computer simulation of compound 7 for the docking to CD38
3. Tension measurement on isolated tracheal strips of guinea
pigs. Guinea pigs weighing 250–350 g were sacrificed by an
overdose of sodium pentobarbital (75 mg kg-1 intraperitoneally).
The tracheas were removed and placed in ice-cold Krebs–Henseleit
solution bubbled through with 95% O2/5% CO2. The trachea was
then isolated from surrounding connective tissue and cut spirally
into two strips 3 mm wide and 15 mm long. The composition of
Krebs–Henseleit solution was (in mM): NaCl 118.00, KCl 4.70,
CaCl2 2.50, MgSO4·7H2O 1.20, KH2PO4, 1.20, NaHCO3 25.00,
and glucose 11.00. The ends of each tracheal strip were then fixed,
via two small clips, to the bottom of the chamber and to a force
displacement transducer for recording tension with a polygraph.
The chamber (50 mL capacity) was filled with Krebs–Henseleit
solution at 37 ◦C and bubbled through with 95% O2/5% CO2.
Each strip was subjected to a load of 2 g for at least 1 h, with
frequent changes of the bath fluid until a stable baseline tension
was obtained. 10 mM acetylcholine chloride was added into the
Krebs–Henseleit solution to induce contraction of tracheal strips.
After the tension become stable, compound 4 and compound 7
of 10-8, 10-7, 10-6, 10-5 and 10-4 M were added every 10 min in
sequence, respectively. The tension of tracheal strips was recorded
by MedLab-U4C501H bio-signals collecting–processing system in
the whole experiment.37
The structure of Compound 7 first was constructed based
upon the crystal coordinates of Compound 4 followed by en-
ergy minimization using Discovery Studio 2.1. It was then
docked into the binding pocket of CD38 using the AutoDock
3.0.540 program. Considering the interactions with the key
residues in CD38, one conformation with a relatively low en-
ergy was selected as the starting conformation for the sub-
sequent molecular dynamics simulation. The molecular topol-
ogy file for Compound 7 was generated by the PRODRG241
server (http://davapc1.bioch.dundee.ac.uk/prodrg/). The partial
atomic charges of the compound were calculated by Gaussian03
program42 at the level of HF/6-31G*. The simulations were
performed with the GROMACS43 (version: 3.3.1) software and
the force field GROMOS9644 43a1 was applied for the protein.
The complex was put into a cubic periodic box with edge
˚
approximately 10 Afrom the system’s periphery in each dimension.
Then 19 974 SPC water molecules were added into the box and
2 Cl- were also added in order to ensure the charge neutrality
of the system. The final system contains 62 601 atoms. During
the entire simulation, all bond lengths were constrained by the
LINCS algorithm.45 Long-range electrostatic interactions were
calculated using the PME method.46 The Berendsen thermostat47
was applied using a coupling time of 0.1 ps to maintain the
systems at a constant temperature of 300 K and the pressure
was also maintained by coupling to a reference pressure of 1 bar
by Berendsen thermostat. The simulations began with 2000 steps
of steepest-descent algorithm to reach the tolerance. Then, the
solvent equilibration was performed in 50 ps with the protein and
the ligand fixed. Following that, a second 50 ps simulation was
carried out with the main chain and the ligand fixed. Another
20 ps simulation was used to relax the whole system except for
the Ca atoms and the ligand. The equilibration was completed
after the 10 ps relaxation for the ligand. Finally, the production
simulation of 5 ns was performed on the whole system. The
system was equilibrated after about 2 ns and the average structure
was obtained and minimized, which was considered as the stable
binding mode of Compound 7.
4. Statistical evaluation. Antispasmodic percentage = (ten-
sion before addition of compounds - tension after addition of
compounds)/(tension before given compounds - basal tension)
¥ 100%. Tension changes of tracheal strips obtained from ad-
ministration groups and the blank group were compared using
Student’s t test. All values are represented as mean SEM. Two
means were considered significantly different when P value was
<0.05 or <0.01.
Protein crystallography
The catalytic domain of human CD38 was expressed in a yeast
expression system and purified as reported previously.36 Using
the hanging vapor drop diffusion method, CD38 crystals were
obtained by mixing 1 ml 10 mg ml-1 protein with 1 ml crystallization
solution containing 100 mM MES, pH 6.0, 10% PEG4000 at room
temperature. To obtain CD38–Compound 4 complex, native CD38
crystals were soaked for several minutes at room temperature in
the crystallization mother liquid mixed with 40 mM Compound 4
and 30% glycerol.
Acknowledgements
This study was supported by grants from the National Natu-
ral Sciences Foundation of China to LH Zhang (NSFC-RGC
20831160506), and the NSFC/RGC grant N_HKU 722/08 and
General Research Fund of Hong Kong: 769107, 768408, 769309,
770610 (to H.C. Lee and Q Hao). MacCHESS is supported by an
NIH grant RR001646.
Data collection, reduction and structure refinement. All X-
ray diffraction data were collected at the Cornell High-Energy
Synchrotron Source (CHESS) A1 station under cryo-protection
˚
at 100 K with a fixed wavelength of 0.976 A. A total of 360
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apo structure served as the initial model for structure solution
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