Nicotinic acid adenine dinucleotide phosphate analogues containing substituted nicotinic acid: Effect of modification on Ca2+ release

Article abstract of DOI:10.1021/jm1007209

Analogues of nicotinic acid adenine dinucleotide phosphate (NAADP) with substitution at either the 4- or the 5-position position of the nicotinic acid moiety have been synthesized from NADP enzymatically using Aplysia californica ADP-ribosyl cyclase or mammalian NAD glycohydrolase. Substitution at the 4-position of the nicotinic acid resulted in the loss of agonist potency for release of Ca²⁺-ions from sea urchin egg homogenates and in potency for competition ligand binding assays using [³²P]NAADP. In contrast, several 5-substituted NAADP derivatives showed high potency for binding and full agonist activity for Ca²⁺ release. 5-Azido-NAADP was shown to release calcium from sea urchin egg homogenates at low concentration and to compete with [³²P]NAADP in a competition ligand binding assay with an IC₅₀ of 18 nM, indicating that this compound might be a potential photoprobe useful for specific labeling and identification of the NAADP receptor.

Full text of DOI:10.1021/jm1007209

J. Med. Chem. 2010, 53, 7599–7612 7599
DOI: 10.1021/jm1007209
Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Containing Substituted Nicotinic Acid:
Effect of Modification on Ca2þ Release†
Pooja Jain,‡,† James T. Slama,*,‡ LeRoy A. Perez-Haddock,§ and Timothy F. Walseth§
‡Department of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Health Science Campus, MS no. 1015, 3000 Arlington
Avenue, Toledo, Ohio 43614, and Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
§
Received June 15, 2010
Analogues of nicotinic acid adenine dinucleotide phosphate (NAADP) with substitution at either the
4- or the 5-position position of the nicotinic acid moiety have been synthesized from NADP
enzymatically using Aplysia californica ADP-ribosyl cyclase or mammalian NAD glycohydrolase.
Substitution at the 4-position of the nicotinic acid resulted in the loss of agonist potency for release of
Ca2þ-ions from sea urchin egg homogenates and in potency for competition ligand binding assays using
[32P]NAADP. In contrast, several 5-substituted NAADP derivatives showed high potency for binding
and full agonist activity for Ca2þ release. 5-Azido-NAADP was shown to release calcium from sea
urchin egg homogenates at low concentration and to compete with [32P]NAADP in a competition ligand
binding assay with an IC50 of 18 nM, indicating that this compound might be a potential photoprobe
useful for specific labeling and identification of the NAADP receptor.
Introduction
release are not completely understood. Lee et al. defined the
pyridine-3-carboxylate, the adenosyl 20-phosphate, and por-
tions of the purine as important structural determinants.14
Billington further defined the requirements of 3-substituted
pyridine modification on agonist activity.15 Simple pyridi-
nium-3-carboxylate salts16,17 and pyridoxalphosphate-6-azo-
phenyl-2,4-disulfonate (PPADS)18 are reported to be low
potency antagonists. A potent and selective NAADP antag-
onist, Ned-19, was developed recently using virtual screening19
and has been applied to studies of the NAADP receptor.20
Continued progressinthe development of chemicalprobesfor
NAADP-mediated signaling and characterization of the
NAADP receptor require a better understanding of the
structure-activity relation (SAR) of NAADP-mediated cal-
cium ion release. To this end, we have synthesized a series of
nicotinic acid substituted NAADP derivatives using a che-
moenzymatic synthesis21,22 and characterized their ability
both to mobilize calcium-ion via the NAADP receptor in
sea urchin egg homogenates and to compete with NAADP in
competition ligand binding assay.
Nicotinic acid adenine dinucleotide phosphate (NAADP, 1)a
(Chart 1) was identified as a metabolite of NADP, which at low
concentration was shown to cause Ca2þ release from internal
calcium ion stores in sea urchin eggs.1 NAADP-mediated Ca2þ
release was subsequently shown to be pharmacologically distin-
guishable from calcium release mediated by either inositol-1,4,5-
triphosphate (InsP3) or by cyclic ADP-ribose (cADPR). Addi-
tionally, NAADP mobilized Ca2þ from a different subcellular
location than does cADPR or InsP3.2 These observations imply
that NAADP releases calcium ion after binding to a unique recep-
tor.3-6 NAADP is active in many organisms including mollusks,
plants, and mammals.4 In vertebrate tissue, NAADP-mediated
calcium release has been observed in pancreatic acinar cells,7
cultured human T lymphocytes,8 brain,9 heart,10 kidney,11 and
liver cells.12 In some of these systems, NAADP has met the
criteria for establishing it as an intracellular second messenger.3,13
Despite its physiological importance, the structural features
of NAADP which are required for high potency calcium
†Taken in part from the dissertation submitted by Pooja Jain to the
University of Toledo for the Ph.D. degree in Medicinal Chemistry,
December 2008.
Chemistry
Because the enzymes Aplysia californica ADP-ribosyl cyclase
and mammalian NAD glycohydrolase are known to catalyze
the exchange of the nicotinamide group of NADP with nicotinic
acid (Scheme 1),21,22 we obtained nicotinic acid derivatives with
substituents at either position 4 or 5 and used them as substrates
(Table 1) for the enzyme-catalyzed pyridine base exchange reac-
tion to produce the corresponding NAADP analogues. The
general reaction for the base exchange is shown in Scheme 1.
4-Substituted Nicotinic Acids. We sought to obtain 4-sub-
stituted nicotinic acid derivatives with alkyl, amino, or aryl
groups at the 4-position. The simple 4-methylnicotinic acid
(2a) as well as 4-aminonicotinic acid (2b) were both available
*To whom correspondence should be addressed. Phone: 419.383.1925.
Fax: 419.383.1909; E-mail: james.slama@utoledo.edu.
a Abbreviations: cADPR, cyclic adenosine diphosphate ribose; DIPEA,
diisopropylethylamine; DMF, N,N-dimethylformamide; ESI, electrospray
ionization mass spectrometry; EtOAc, ethyl acetate; EtOH, ethyl alcohol;
Fluo-3, refers to the fluorescent indicator Ca2þ dye developed by Dr. Roget
Y. Tsien; HPLC, high pressure liquid chromatography; HRMS, high mass
resolution mass spectrometry; InsP3, inositol-1,4,5-triphosphate; LC-mass
spec, liquid chromatography mass spectrometry; NAADP, nicotinic acid
adenine dinucleotide phosphate; NADase, nicotinamide adenine dinucleo-
tide glycohydrolase; PPADS, pyridoxalphosphate-6-azophenyl-2,4-disulfo-
nate; SAR, structure-activity relation; TCA, trichloroacetic acid; TEA,
triethylamine; TES, triethylsilyl group; THF, tetrahydrofuran; TLC, thin
layer chromatography; Tris, tris-(hydroxymethyl)aminomethane;
UV, ultraviolet light;
r
2010 American Chemical Society
Published on Web 10/13/2010
pubs.acs.org/jmc

7600 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Jain et al.
Scheme 1. Synthesis of NAADP Analogues Using the Enzyme Catalyzed Pyridine Base Exchange Reaction
Chart 1. Nicotinic Acid Adenine Dinucleotide Phosphate
(NAADP), 1
Table 1. 4-Substituted Pyridine-3-carboxylic Acids (2a-d) and 5-Sub-
stituted Pyridine-3-carboxylic Acids (3a-j) Obtained and Evaluated As
Potential Substrates in the Chemoenzymatic Synthesis Designed to
Produce Novel Pyridinium Substituted NAADP Derivatives
commercially. 4-n-Butylnicotinic acid and 4-phenylnicotinic
acid were synthesized by adding either n-butyllithium or
phenyllithium respectively to pyridyl-3-oxazolines derived
from nicotinic acid (Scheme 2).23 Addition of the organo-
lithium reagent was shown to favor the formation of the 1,4-
dihydropyridine-3-oxazoline, which was easily oxidized by
oxygen in air to obtain the 4-substituted pyridine-3-oxazo-
line. Deprotection results in the isolation of the desired
4-substituted nicotinic acids.24
5-Substituted Nicotinic Acids. The simple derivatives
5-methylnicotinic acid (3a) and 5-aminonicotinic acid (3b),
5-bromonicotinic acid (3c), and pyridine-3,5-dicarboxylic
acid (3d) were purchased. For the synthesis of 5-substituted
nicotinic acid derivatives, we started with commercially
available and inexpensive 5-bromonicotinic acid (3c) and
protected the carboxylic acid by its conversion into the
oxazoline group (4). Transmetalation of 4 was expected to
be difficult because of competing 4-addition reaction.23
Therefore we utilized intermediate 4 in palladium catalyzed
C-C coupling reactions with appropriate coupling partners
to produce 5-substituted pyridine-3-oxazolines.
The Sonogashira coupling has previously been used suc-
cessfully to produce ethynylpyridines,25,26 and we expected
that cross-coupling of terminal acetylenes with 4 (Scheme 3)
would provided an approach to 5-ethynylnicotinic acid (3e).
Coupling of 4 with commercially available triethylsilylace-
tylene (TES-acetylene), under conditions using a mixed
aqueous-organic solvent and sodium carbonate at 90 °C,
led to formation of disubstituted acetylene 5 instead of the
desired product as shown in Scheme 3. Formation of 5
probably occurred because in the presence of aqueous base
and high temperature the triethylsilyl group was removed
from initially formed 6, and the free acetylene liberated in
situ reacted with another molecule of 4.
When the reaction was conducted at room temperature and
by using a phase transfer catalyst with a minimum amount of
water in the reaction,27 we were able to obtain 6 in high yield.
The silyl group in 6 was removed by treatment with excess
aqueous potassium carbonate,28 followed by acid hydrolysis of
the oxazoline group29 to give free acid derivative 3e (Scheme 3).
Compound 3f was obtained by partial reduction of 3e in
the presence of poisoned Lindlar’s catalyst.30 The acetylene

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7601
Scheme 2. Addition of Organolithium Reagents to Pyridine-3-oxazolinea
a Conditions and reagents: (a) C6H5Li or n-C4H9Li, -78°; (b) air, 0° 3 h; (c) 3 N HCl/acetic acid 95°/36 h.
Scheme 3a
a Conditions and reagents: (a) SOCl2, reflux, 18 h; (b) 2-amino-2-methylpropanol, 48 h; (c) SOCl2, 24 h; (d) diisopropylethylamine (DIPEA), 60 °C,
24 h; (e) Pd(Ph3)4, Na2CO3, CuI, TES-acetylene, dimethylformamide (DMF)/ethyl alcohol (EtOH)/H2O at 90° for 1 h; (f) Pd(OAc)2, PPh3,
benzyltributylammonium bromide, TES-acetylene, DIPEA and water, room temperature, 1 h; (g) K2CO3, tetrahydrofuran (THF)/ CH3OH, room
temperature, 1 h; (h) 3 N HCl, AcOH, 95° 1 h.
Scheme 4a
Scheme 5a
a Conditions and reagents: (a) EtOH/HCl; (b) NaNO2/HCl;
(c) NaOAc/NaN3; (d) CH3OH/NaOH/H2O.
5-Azidopyridine-3-carboxylate (3i) was an important pre-
cursor both to subsequently establish the SAR of NAADP
derivatives and for developing high potency photoaffinity
probes for receptor identification.32 Literature provided the
precedent of diazotization reaction, followed by nucleophilic
displacement by sodium azide.33,34 This approach was used
to synthesize 5-azidonicotinic acid 3i starting from 5-amino-
nicotinic acid 3a (Scheme 5). We started with protection of
the acid group in 5-aminonicotinic acid (3a) with a hydro-
phobic ethyl ester (10), followed by conversion of the amine
into an azide using diazotization chemistry to give 11.35 The
hydrophobic ester group used for acid protection aided in
extracting the compound into the organic phase, while the
salts from the reaction partitioned into the aqueous phase.
5-Azidonicotinic acid ethyl ester 11 was then subjected to
alkaline hydrolysis to give 5-azidonicotinic acid 3i.
a Conditions and reagents: (a) toluene/THF, (i-C3H7O)3B, then
n-BuLi, -70°; (b) (CH3)2COHCOH(CH3)2, Ph-CH3, reflux; (c) Pd(Ph3)4,
Na2CO3,, C6H5-Br, DMF and EtOH 90° for 4 h; (d) 3N HCl, acetic acid,
95° 36 h.
of compound 3e was completely reduced to an ethyl group
using ammonium formate in presence of Pd on carbon to
obtain 5-ethylnicotinic acid (3g).
For the synthesis of the 5-arylnicotinic acids, Pd-catalyzed
Suzuki reaction between aryl halides and boronic acids has
been used (Scheme 4).26 We synthesized 9 by means of Suzuki
coupling between the boronate of oxazoline substituted
nicotinic acid 7 and phenyl bromide. The boronate of the
protected nicotinic acid could not be completely character-
ized by itself because it was a mixture of several boronate
forms (boronic acids and boronic esters) that have been
reported to exist.31 Characterization of the boronate was
accomplished by converting the boronate into its pinacol
ester (8).31
Nicotinic acid derivative 3j was synthesized by click
chemistry involving [1,3] dipolar cycloaddition reaction36
between the 5-azidonicotinate ethyl ester (11) and tert-butyl
prop-2-ynylcarbamate 12 (Scheme 6). tert-Butyl prop-2-
ynylcarbamate (12) was prepared according to procedure
described by Holmes et al. (2003).25
The borate 7 was coupled to phenyl bromide in the
presence of Pd catalyst and aqueous base at 90 °C to give
the C-C coupled product 9. The deprotection of oxazoline
under acidic conditions gave 5-phenylnicotinic acid 3h
(Scheme 4).
The copper catalyzed cycloaddition resulted in formation of
1,4-disubstituted triazole ring at position 5 of nicotinic acid (13).
Use of copper catalyst brings regioselectivity to the reaction and
catalyzed the formation of an adduct between the azide and the
acetylene moiety producing only the 1,4-disubstituted isomer.

7602 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Jain et al.
Scheme 6a
a Conditions and reagents: (a) DIPEA, CuI, 12 h; (b) trifluoroacetic acid; (c) 2 N NaOH in H2O/CH3OH.
The competing reaction pathway producing the 1,5-disubstitu-
tion cycloaddition product is thereby eliminated.37 Deprotection
in strong anhydrous acid produced ester 14, which was saponi-
fied to the nicotinic acid derivative 3j.
these conditions, we obtained exchange product 15. Use of
this higher pH increased the concentration of the unproto-
nated and un-ionized 4-aminonicotinic acid methyl ester,
and hence it became more available to the active site of NAD
glycohydrolase. The 4-amino-NAADP methyl ester was
then subjected to treatment with triethylamine and water
to hydrolyze the ester and give the target compound 4-ami-
no-NAADP (16).
Enzyme Catalyzed Base Exchange. NAADP derivatives
were synthesized from pyridine-3-carboxylic acid derivatives
and NADP either by using the enzymatic activity of Aplysia
ADP-ribosyl cyclase or a mammalian NAD glycohydrolase
(Scheme 1). We relied primarily on the Aplysia ADP-ribosyl
cyclase as the catalyst because it was available as a stable,
soluble, and well-characterized enzyme. In cases where the
Aplysia ADP-ribosyl cyclase failed to produce the expected
exchange product, mammalian enzymes were also evaluated.
A high concentration of a nucleophilic pyridine base is
required for high yields of the exchange product. In a typical
reaction, NADP was treated with about a 30-fold excess of
the pyridine derivative and the enzyme catalyst at pH 4.0.
Under these conditions, base exchange competes success-
fully with both hydrolysis and cyclization reactions, side
reactions which destroy the substrate NADP by forming
either ADP-ribose or cADPR byproducts. The control of pH
is important because it is the un-ionized pyridine derivative
that is the substrate for the base exchange reaction. An
optimal pH must be maintained to provide high concentra-
tions of a un-ionized pyridine base. The exchange reactions
were monitored by following the disappearance of the limit-
ing substrate, NADP. In this assay, NADP present in
the mixture was reduced using glucose-6-phosphate in
the presence of glucose-6-phosphate dehydrogenase to
give NADPH. The NADPH that was produced showed
absorbance at 340 nm, without interference from any other
reactant or biproduct.3 When the exchange reaction was
complete, the catalyst was removed by ultrafiltration
(membrane cut off 10 kDa). The pH of the catalyst free
filtrate was adjusted to 7, and dinucleotides present in the
mixture were purified by anion-exchange chromatography.
Methylated nicotinic acids such as 2b or 3b could easily be
exchanged into NADP to result in the formation of NAADP
derivatives 17 and 21. 5-Aminonicotinic acid (3a) was simi-
larly an excellent exchange substrate, and the exchange
product 5-amino-NAADP (20) could be isolated in greater
than 40% yield after purification.
Base exchange reaction with 4-n-butylnicotinic acid (2c) or
4-phenyl nicotinic acid (2d) worked well to produce NAADP
derivatives 18 and 19. Enzyme catalyzed base exchange was
surprisingly more selective when substituents were placed in
the 5 position. Neither 5-phenylnicotinic acid (3h) nor bro-
monicotinic acid (3c) formed detectable base-exchanged
product. Similarly, 3e, 3f, and 3j failed to react to form a
substituted NAADP. It is well appreciated that electron
withdrawing substituents deactivate the pyridine ring to-
ward base exchange. Steric demands on disubstituted pyr-
idines are less well understood, although it is apparent from
our work that large substituents are better tolerated at the
4-position than at the 5-position.
Interestingly, 5-substitutions like azide and carboxylate
were tolerated as cosubstrates for exchange reactions, pro-
ducing reasonable yields of the corresponding NAADP
analogues. In cases where the exchange reaction catalyzed
by the Aplysia ADP-ribosyl cyclase was found to fail, we also
tried NAD glycohydrolase enzymes from porcine brain and
bovine spleen, but even when using the mammalian enzyme
as a catalyst, the result was the same.
We tried to exchange commercially available 4-aminoni-
cotinic acid (2a) into NADP at pH 4.0 to enzymatically
synthesize the 4-amino-NAADP (16)38 but were unsuccess-
ful in detecting any pyridine dinucleotide derivatives as
products. The likely explanation is that 2a, a vinylogous
amidine, is significantly more basic than is 3a and is largely
protonated and cationic under the usual conditions of the
reaction at pH 4. To circumvent this difficulty, we synthe-
sized the methyl ester of 4-aminonicotinic acid using Fischer
esterification and used the 4-aminonicotinic acid methyl
ester as the cosubstrate for the exchange at pH 7.5. Under
The results of enzyme catalyzed base exchange reactions
between NADP and our set of synthetic nicotinic acids are
summarized in Table 2. We also observed that all three
enzymes behaved very similarly, and the outcome of ex-
change reactions using different substrates was the same for
each of the catalysts. This suggests that the active site of the

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7603
Table 2. Result of the Pyridine Base Exchange Reaction
Figure 1. Fluorimetric calcium release traces from sea urchin egg homogenates induced by the addition of increasing concentrations of
NAADP and three selected NAADP analogues. Calcium-ion concentration was measured fluorimetrically using the calcium sensitive dye
Fluo-3. The effect of the addition of increasing concentrations of NAADP (1) (A), 5-NH2-NAADP (20) (B), 5-CH3-NAADP (21) (C), and
4-CH3-NAADP (17) (D) on fluorescence are shown.
three enzymes, as judged by the specificity of base exchange
reactions, is very similar.
these compounds induced a rapid release of calcium-ions
which reached a concentration dependent plateau within a
few seconds. Compounds 20 and 21 induce calcium-ion
release at concentrations as low as 2 nM. 4-CH3-NAADP
(17) was shown to be significantly less potent than was the
5-isomer 21. The maximal calcium concentration attained
after treatment was found to increase in a concentration
dependent manner. Saturating concentrations of com-
pounds 20 and 21 elicited the same amount of calcium release
as did the full agonist NAADP. Figure 2 shows concentra-
tion-response curves for compounds 16-24 compared to
Biological Activity
Calcium Release Properties of Compounds 16-24. When
tested for calcium ion-releasing activity in vitro, NAADP (1)
and its analogues 16-24 were shown to elicit calcium release
from sea urchin egg homogenates. Figure 1 shows the effect
of treatment of calcium-ion loaded microsomes with varying
concentrations of NAADP (1), 5-NH2-NAADP (20),
5-CH3-NAADP (21), and 4-CH3-NAADP (17). Each of

7604 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Jain et al.
Figure 2. Concentration-response curves depicting maximum
Ca2þ concentration attained after treatment of calcium loaded sea
urchin egg homogenates by the addition of varying concentrations
of NAADP (1) (9), 5-NH3-NAADP (20) (0), 5-CH3-NAADP (21)
(ꢀ), 5-CH2CH3-NAADP (23) (b), 5-N3-NAADP (24) (O), 4-NH3-
NAADP (16) (light ]), 4-CH3-NAADP (17) (dark ]), 4-n-butyl-
NAADP (18) (light 3), 4-phenyl-NAADP (19) (dark 4), and
5-CO2H-NAADP (22) (dark 3). Each plotted point represents the
mean of a minimum of three determinations.
Table 3. EC50 Values for Ca2þ Release Induced by NAADP (1) and
NAADP Analogues (16-24) Measured Fluorometrically from Ca2þ
Loaded Sea Urchin Egg Homogenates
Fold increase
in EC50
compd
NAADP (1)
EC50 ( SD, nM (n)
18.7 ( 6.9 (19)
27.4 ( 13.9 (9)
43.0 ( 25.3 (11)
352 ( 219 (3)
1689 ( 1349 (9)
4413 ( 2794 (8)
42231 ( 28870 (6)
>1000000 (5)
>1000000 (6)
>1000000 (3)
1
1.5
5-amino-NAADP (20)
5-methyl-NAADP (21)
5-ethyl-NAADP (23)
5-azido-NAADP (24)
4-amino-NAADP (16)
4-methyl-NAADP (17)
4-n-butyl-NAADP (18)
4-phenyl-NAADP (19)
5-carboxy-NAADP (22)
Figure 3. Desensitization of sea urchin egg homogenate Ca2þ
release by pretreatment with subthreshold concentrations of
NAADP or NAADP analogues. (A) Effect of a 9 min pretreatment
with buffer (solid line), 1 nM NAADP (1) (dotted line), or 1 nM
5-CH3-NAADP (21) (gray line) on calcium release in response to
treatment with 1 μM NAADP. The data has been adjusted along the
Y-axis to prevent overlap. (B) Comparison of the abilities of a 9 min
preincubation with NAADP (1) or NAADP analogues (16-24) at
the stated concentrations to reduce Ca2þ release in response to the
addition of 1 μM NAADP. Values shown are mean ( SEM (n = 3).
2.3
18
90
235
2200
>50000
>50000
>50000
that of NAADP. Compounds 20 and 21 were shown to be
approximately equipotent with NAADP, compounds 23 and
24 possessing larger 5-substituents were ca. 10 to 100-fold
less potent, while the 4-substituted NAADP derivatives 16
and 17 were from 200- to 2000-fold less potent (Table 3).
EC50 values for 18, 19, and 22 were higher still and could not
be accurately determined in this experiment. Compounds 16,
20, and 21 elicit maximal calcium release at high concentra-
tion and therefore are considered to possess full-agonist
activity. The maximal Ca2þ release elicited at high concen-
trations of compounds 23, 24, and probably also 17 was
shown to plateau at concentrations significantly less than
that elicited by NAADP, indicating that these compounds
are behaving as partial agonists. Complete concentra-
tion-response curves could not be determined for the low
potency compounds 18, 19, and 22.
does NAADP itself (Figure 3A) and hence that 21 must be
releasing Ca2þ through the same receptor as NAADP itself.
Other tested compounds (16-24) were shown to be similarly
effective in desensitization when used for pretreatment at a
concentration of 1 nM or higher (Figure 3B). The concentra-
tions of analogues used in Figure 3B were chosen based on
previous findings3,39 that pretreatment with NAADP at sub-
threshold concentrations (for calcium release) were able to
completely desensitize calcium release by a maximal concentra-
tion of NAADP. In the experiment shown in Figure 3B, we
attempted to use analogue concentrations that were just below
the threshold concentration necessary to induce measurable
calcium release, approximately 20-100-fold lower than the
observed EC50.
Receptor Desensitization. NAADP and other related ago-
nists characteristically induce desensitization of the Ca2þ
release mechanism when the receptor is subjected to pre-
treatment for a few minutes using subthreshold agonist
concentrations.3 Subsequent treatment with a much higher
concentration of NAADP is then unable to release calcium.
Such desensitization is indicative of involvement of a receptor
mediated response and is also a useful tool to demonstrate that
an agonist is releasing calcium through the NAADP receptor. It
was found that pretreatment with 1 nM 5-CH3-NAADP (21)
produced the same characteristic receptor desensitization as
Competition Ligand Binding Studies. The ability of NAADP
(1) and its analogues 16-24 to compete with [32P]NAADP for
specific binding to sea urchin microsomes was determined using
a competition ligand binding assay according to the procedure
of Aarhus et al. (1996).3 The concentration response curves for
the 10 tested compounds are shown in Figure 4, and the IC50
values derived from the fitted data derived from determina-
tions made in two or more additional experiments in addition to
that depicted in Figure 4 are presented in Table 4. The bind-
ing studies show that NAADP (1), 5-NH2-NAADP (20) and
5-CH3-NAADP (21) inhibit the binding [32P]NAADP by 50%

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7605
unexpected limitation of the pyridine base exchange reaction in
this study with respect to the synthesis of the 5-substituted
NAADP derivatives. Nicotinic acids containing large groups at
the 5-position (3h or 3j) or those containing groups which are
both electron withdrawing and hydrophobic (3c, 3e (σm for
acetylene is 0.21 indicating electron withdrawal), or 3f) fail to
exchange. The interesting NAADP derivatives that would be
derived from these compounds must therefore be produced by an
alternate method. It is interesting that 5-azidonicotinic acid was
shown to exchange with the nicotinic acid base of NAADP in
good yield to produce 24 (5-azido-NAADP), a potential photo-
affinity label for the NAADP binding protein.
When tested as competitive ligands for NAADP binding or
for calcium-ion mobilizing activity, we find that NAADP
derivatives containing a nicotinic acid moiety with 4-substitu-
ents are associated with loss of binding potency. Even small
4-substituents (as in 16 and 17) are associated with a 100- to 200-
fold loss of potency. The 4-substituted compounds are appar-
ently low potency agonists, and we have not yet encountered
any 4-substituted NAADP derivatives that behave as antago-
nists. Subthreshold concentrations of the 4-substituted deriva-
tives 16-19 desensitize the NAADP receptor, indicating that
these compounds act similarly to the other agonists.
Figure 4. Competition radioligand binding curves for NAADP (1) (9)
and the nine analogues (16-24) in sea urchin egg homogenates: 5-NH3-
NAADP (20) (0), 5-CH3-NAADP (21) (ꢀ), 5-CH2CH3-NAADP (23)
(b), 5-N3-NAADP (24) (O), 4-NH3-NAADP (16) (light ]), 4-CH3-
NAADP (17) (dark ]), 4-n-butyl-NAADP (18) (light 3), 4-phenyl-
NAADP (19) (dark 4), and 5-CO2H-NAADP (22) (dark 3). Each
plotted point represents the mean of a minimum of three determinations.
Table 4. IC50 Values Determined for Competition Ligand Binding
between [32P]NAADP and NAADP Analogues (16-24)
Fold increase
in IC50
compd (structure no.)
IC50 ( SD, nM (n)
We find that the 5-position of the nicotinic acid ring is
tolerant of substitution. Introduction of an amino group (20)
or a methyl group (21) at the 5-position results in the produc-
tion of derivatives which are only slightly less potent than is
NAADP itself. An ethyl group is tolerated with only a 20-fold
loss of potency, and the azide (24) (a dipolar group containing
three linearly disposed heavy atoms) is associated with only a
11-fold increase in IC50 as measured in the competition ligand
binding assay. The concentration response for Ca2þ release
induced by 5-ethyl-NAADP (23) (Figure 2) apparently pla-
teaus at a significantly lower percent calcium ion release than
does the full agonist NAADP, indicating that 23 exhibits
partial antagonist activity. Similarly the 5-azide 24 may also
be a partial agonist. Most reported modifications of NAADP
have been observed to result in the loss of receptor binding
potency, and our observation suggests that further modifica-
tion at the nicotinic acid 5-position might produce potent
agonists that will be informative in future studies of receptor
function, receptor localization, and receptor isolation. The
EC50 for 5-azido-NAADP (24) is low enough to suggest that
when 24 is appropriately labeled, it should prove useful as a
photoaffinity label for the identification of NAADP binding
proteins (Tables 3 and 4).
NAADP (1)
1.6 ( 1.5 (9)
1.7 ( 1.3 (6)
1.9 ( 1.5 (5)
37 ( 30 (4)
1
5-amino-NAADP (20)
5-methyl-NAADP (21)
5-ethyl-NAADP (23)
5-azido-NAADP (24)
4-amino-NAADP (16)
4-methyl-NAADP (17)
4-n-butyl-NAADP (18)
4-phenyl-NAADP (19)
5-carboxy-NAADP (22)
1.1
1.2
23
18 ( 14 (5)
11
88
141 ( 116 (3)
149 ( 99 (6)
465 ( 221 (5)
859 ( 896 (6)
>100000 (4)
93
303
540
>60000
at a concentration range of 1-2 nM, whereas other analogues
are able to compete with [32P]NAADP only at much higher
concentrations. Of the compounds tested, 5-carboxy-NAADP
(22) was observed to be the least potent with an EC50 of
>100000 nM,>60000-fold higher than the EC50 of NAADP.
Discussion
Although approaches to the chemical synthesis of NAADP
and its derivatives have been developed,40 the process of
producing NAADP derivatives synthetically is still lengthy
and relatively difficult. Chemoenzymatic syntheses of NAD,
NADP, and NAADP derivatives have been effectively used to
produce novel pyridine dinucleotides since the discovery of
the NAD glycohydrolase catalyzed base exchange reaction in
the 1950s. Successful base exchange was found to require a
uncharged species, a sufficiently nucleophilic pyridine base
(pKa>3),41 and usually the absence of sterically interfering
substituents ortho- to the pyridine nitrogen. A variety of
monosubstituted pyridines with large groups in the 3- or 4-
positions were found to successfully exchange into the pyr-
idine dinucleotides using the enzyme catalyzed reaction.
Disubstituted nicotinamides were studied less often, but
5-methyl-NAD and 5-amino-NAD had been synthesized
using the porcine brain NAD-glycohydrolase as a catalyst.42
We find that 4-substituted nicotinic acids, even those with large
4-substituents, are excellent exchange substrates. One exception
is a cationic compound such as 2a, which must be converted into
an ester before it can be successfully exchanged into the pyridine
dinucleotide at a high pH under conditions where the uncharged
base is again present at high concentration. We encountered an
The EC50 values derived from Ca2þ release (Table 3) and
the IC50 values derived from competition ligand binding
(Table 4) are well correlated (see Supporting Information) ,
but the IC50 values are consistently lower. This may be due to
the fact that the measurements are made under different
experimental conditions. Alternately, it has been suggested
recently that the NAADP receptor possesses two binding sites
for NAADP, a highaffinityinhibitorysiteand the stimulatory
site with a relatively lower affinity.20 The IC50 values mea-
sured using the competition ligand binding assay might in
such a case reflect the binding potency of the high affinity
inhibitory site.
Experimental Section
General Procedures. The following procedures were used in
all reactions unless otherwise noted. Oxygen- and moisture-
sensitive reactions were carried out in oven-dried (T>100 °C)

7606 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Jain et al.
glassware sealed under a positive pressure of dry nitrogen
supplied from a manifold. Moisture or air sensitive liquids and
solutions were transferred under a nitrogen atmosphere by
syringe through rubber septa. Reactions were stirred with a
Teflon-covered magnetic stirring bar. Reagents for reactions
were obtained from commercial sources and were used without
further purification, except as indicated. Either sure-seal bottles
of anhydrous solvents were purchased from Aldrich or they
were purified according to Perrin’s Purification of Laboratory
Chemicals. Proton (1H) NMR spectra and carbon-13 (13C)
NMR spectra were recorded at 600 MHz or at 400 MHz.
Chemical shifts are reported in ppm (δ) and are referenced to
the residual proton signal of the deuterated solvent. 31P NMR
spectra were recorded on Gemini 200 MHz spectrometer with
85% H3PO4 as external reference. The pH of the solutions was
adjusted to pH 7 prior to the determination of the 31P NMR
spectra. The chemical shifts for 13C NMR peaks are reported to
the first decimal place, while the peaks which are very close are
reported to the second decimal place. The following abbrevia-
tions are used to describe spin multiplicity: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, b = broad, dd
= doublet of doublets, dt = doublet of triplets, dq = doublet of
quartets, and ddd = doublet of doublet of doublets; other
combinations derive from those listed. Coupling constants (J)
are reported in hertz (Hz). Melting points were determined on an
Electrothermal digital melting point apparatus and are uncor-
rected. Mass spectral analyses were done at University of Toledo
Instrumentation Center. Electrospray ionization mass spectra
(ESI) were acquired on FT/ICR spectrometer. High resolution
mass spectroscopies with accurate mass analyses (HRMS) were
performed at Ohio State University Mass Spectrometry and
Proteomics facility. Analytical thin layer chromatography
(TLC) was performed using 0.25 mm Silica Gel 60 glass plates
with a 254 nm fluorescent indicator from Analtech. Plates were
developed in a covered chamber and visualized by examination
under short wavelength ultraviolet (UV) light, iodine stain,
sulfuric acid charring, or ninhydrin stain. Flash chromatogra-
phy was done using Fisher Silica Gel 60, 200-425 mesh (40-60
mm) as stationary phase. For flash column purification, the
amount of stationary phase used was about 40 times the
quantity of compound being purified. The silica gel columns
were packed in nonpolar solvent, followed by sample applica-
tion. The column was always washed with 2-3 column volumes
of nonpolar solvent to remove highly nonpolar impurities.
Solvent evaporation was done under reduced pressure using a
Buchi rotavapor either at water aspirator vacuum or at vacuum
achieved with a regular vacuum oil pump. Elemental analyses
were performed by Atlantic Microlab, Inc. (Norcross, GA), and
are regarded as being acceptable when within (0.4% of the
theoretical values.
5-substituted NAADP derivatives were calculated with respect
to NADP.
Materials. 4-Aminonicotinic acid was purchased from AKSci,
5-aminonicotinic acid was bought from Combi-Blocks, Inc., 4-me-
thylnicotinic acid was bought from Maybridge, and 3,5-pyridine-
dicarboxylate was purchased from Acros Organics. NADP used in
pyridine base exchange reactions was bought as the disodium salt in
98% purity from Roche Diagnostics (Indianapolis, IN). (4,5-
Dihydro-4,4-dimethyl-2-oxazolyl)pyridine was kindly provided by
our colleague Chris Trabbic (University of Toledo).
Synthesis of 4-Substituted Nicotinic Acid Analogues. 4-n-Bu-
tylnicotinic Acid (2c). The immediate precursor of 2c, 4-n-butyl-
3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine, was synthe-
sized by the addition of n-butyllithium to the pyridyl-3-oxazo-
line as follows: 3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine
(0.72 g, 4.10 mmol) was taken in a flask and N2 was flushed
through the flask. Anhydrous THF (8.0 mL) was added and the
mixture was stirred at room temperature until a solution was
formed. The flask was cooled in a -20 °C bath and stirred using
a magnetic stirrer. The reaction temperature was regulated using a
thermostatically controlled mechanical cryostat. n-Butyllithium
(1.6 M solution in hexanes, 2.88 mL) was added dropwise over a
period of 10 min. The reaction was allowed to stir at -20 °C for 2 h.
O2 gas was bubbled into the reaction for 3 h at 0 °C (bath tem-
perature). The reaction was quenched by adding water (25 mL),
and product was extracted into diethylether (3 ꢀ 20 mL). The
organic layer was dried over sodium sulfate and filtered. The filtrate
was concentrated under reduced pressure to an oily residue. This
was purified by column chromatography (20-50% ethyl acetate
(EtOAc) in hexanes) to obtain product as clear oil (0.67 g, 71%).
TLC Rf 0.35 in EtOAc:hexanes (2:3). 1H NMR (400 MHz, CDCl3)
δ 8.85 (s, 1H), 8.47 (d, 1H, J = 5.2), 7.11 (d, 1H, J = 5.2), 4.05 (s,
2H), 2.94 (t, 2H, J = 8.0), 1.58-1.48 (m, 2H), 1.4-1.3 (m, 2H),
1.36 (s, 6H), 0.89 (t, 3H, J = 7.2).
4-n-Butyl-3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine
(0.30 g, 1.29 mmol) was dissolved in 3 N HCl (100 mL) and
acetic acid (50 mL) and was refluxed at 95 °C for 36 h. The
solution was concentrated under reduced pressure and then
dissolved in water (20 mL) and neutralized using 2 N NaOH.
The aqueous layer was lyophilized ,and the solid was purified by
flash chromatography (silica gel, CH2Cl2:CH3OH:acetic acid,
90:9:1) to give 2c as white solid (0.16 g, 69%): mp 121-123 °C.
TLC Rf 0.62 in CH2Cl2:CH3OH:acetic acid (90:9:1). 1H NMR
(400 MHz, CD3OD) δ 8.94 (s, 1H), 8.52 (d, 1H, J = 3.6), 7.40 (d,
1H, J = 3.6), 3.05 (t, 2H, J = 5.2), 1.64-1.58 (m, 2H), 1.46-
1.38 (m, 2H), 0.96 (t, 3H, J = 5.2).
4-Phenylnicotinic Acid (2d). The immediate precursor of 2d,
4-phenyl-3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine,
was synthesized by the addition of phenyllithium to pyridyl-3-
oxazoline as follows: 3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-
pyridine (0.57 g, 3.26 mmol) was taken in a flask and N2 was
flushed through the flask. Anhydrous THF (9.5 mL) was added,
and the mixture was stirred at room temperature until it formed
a solution. The flask was cooled in an acetone/dry ice bath
(-78 °C) with stirring. Phenyllithium (2 M solution in ether, 1.6
mL) was added dropwise over a period of 10 min. The reaction
was allowed to stir at -78 °C for 1 h and then was stirred at 0 °C
for another hour. O2 gas was bubbled into the reaction for 3 h at
0 °C (bath temperature). The reaction was quenched by adding
water (15 mL), and product was extracted in EtOAc (3 ꢀ
15 mL). The organic layer was dried over sodium sulfate and
filtered. The filtrate was concentrated under reduced pressure to
oily residue. This was purified by column chromatography
(50% EtOAc in hexanes) to obtain product as colorless oil
(612.0 mg, 75%). TLC Rf 0.32 in EtOAc:hexanes (8:7). 1H
NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.67 (d, 1H, J 5.2),
7.39 (s, 5H), 7.30 (d, 1H, J = 5.2), 3.83 (s, 2H), 1.30 (s, 6H).
4-Phenyl-3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine
(0.35 g, 1.38 mmol) was dissolved in 3 N HCl (100 mL) and
acetic acid (50 mL) and was refluxed at 95 °C for 36 h. The
Anion exchange chromatography was performed using
diethylaminoethyl substituted cellulose (DE-52 cellulose from
Whatman Inc.). The dimensions of the column used for anion
exchange was 1.5 cm ꢀ 40 cm. Dowex AG1-X2 resin was ob-
tained in the Cl- form from Bio-Rad Laboratories (Hercules,
CA) was converted from the Cl- form into acetate form by
passing 1 M sodium acetate through the column until the
column effluent tested negative for chloride ions. The cation
exchange resin, Dowex 50W-X8, was purchased in Hþ form and
was converted to tetrabutylammonium form by treating it with
an excess of 40% tetrabutylammonium hydroxide.
For base exchange enzymes, 1 unit represents the amount
enzyme required to hydrolyze 1 μmol of NAD/min. For the base
exchange reaction, the pH of the solution was buffered by the
nicotinic acid analogue and was not readjusted after the addi-
tion of NADP disodium salt. The catalyst present in base
exchange reactions was removed by filtration of the mixture
using an Amicon Ultra centrifugal filter device with a molecular
weight cut of 10 kDa. The centrifugal filter device was bought
from Millipore Corporation (Bedford, MA). The yields of 4- or

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7607
solution was concentrated under reduced pressure and then dis-
solved in water (20 mL) and neutralized using 2 N NaOH. The
aqueous layer was lyophilized, and the solid was purified by flash
chromatography(CH2Cl2:CH3OH:acetic acid, 90:9:1) to give 2d as
sticky solid (0.20 g, 73%). TLC Rf 0.58 in CH2Cl2:CH3OH:acetic
acid (90:9:1). 1H NMR (600 MHz, CD3OD) δ 8.90 (s, 1H), 8.67 (d,
1H, J = 4.8), 7.49 (d, 1H, J = 4.8), 7.46-7.42 (m, 5H).
5-Phenylnicotinic Acid (3h). 5-Phenyl-3-(4,5-dihydro-4,4-di-
methyl-2-oxazolyl)-pyridine (9) (0.35 g, 1.38 mmol) was dis-
solved in 3 N HCl (100 mL) and acetic acid (50 mL) and was
refluxed at 95 °C for 36 h. The solution was concentrated under
reduced pressure and then dissolved in water (20 mL) and
neutralized using 2 N NaOH. The aqueous layer was lyophi-
lized, and the solid was purified by flash chromatography
(CH2Cl2:CH3OH:acetic acid, 90:9:1) to give product as white
solid (215 mg, 78%); mp 256-257 °C (reported mp 267-
269 °C44). TLC Rf 0.58 in CH2Cl2:CH3OH:acetic acid (90:9:1).
1H NMR (600 MHz, CD3OD) δ 9.10 (d, 1H, J = 1.8), 9.00 (d,
1H, J = 2.4), 8.60 (t, 1H, J = 1.8), 7.73 (d, 2H, J = 7.2),
7.55-7.45 (m, 3H). 13C NMR (100 MHz, CD3OD) δ 150.6,
148.6, 137.3, 136.5, 135.8, 129.2, 128.7, 128.0, 127.7, 127.0.
5-Azidonicotinic Acid (3i). 5-Azidonicotinic acid ethyl ester
(11) (2.5 g, 13.02 mmol) was dissolved in CH3OH (15 mL). The
solution was stirred at room temperature for 10 min, and 2 N
NaOH (10 mL) was added to it. This solution was then stirred at
room temperature, and the reaction was monitored by TLC for
disappearance of starting material (24 h). The solution was con-
centrated under reduced pressure and was diluted with water (15
mL). The basic layer was then carefully neutralized with 2 N HCl,
resulting in precipitation of white solid. The solid was filtered and
dried under vacuum to give product (1.69 g, 79%); mp 178-180 °C.
TLC Rf 0.53 in CH2Cl2:CH3OH:acetic acid (90:9:1). 1H NMR (600
MHz, CD3OD) δ 8.88 (d, 1H, J = 1.8), 8.48 (d, 1H, J = 2.4), 8.05
(t, 1H, J = 1.8).; 13C NMR (150 MHz, CD3OD) δ 167.1, 147.4 (d,
J = 6.9), 145.4 (d, J = 6.15) 139.6, 129.4, 128.5. Analysis Calcd for
C6H4N4O2: C 43.91, H 2.46, N 34.14. Found: C 44.00, H 2.4, N
33.88. LC-MS (ESI) m/z 165.2 (M þ 1).
Synthesis of 5-Substituted Nicotinic Acid Analogues. 5-Ethy-
nylnicotinic Acid (3e). The immediate precursor of 3e, 5-ethynyl-
3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine, was produced
from 6 by removal of the TES-protecting group. Compound 6
(0.56 g, 2.06 mmol) and anhydrous K2CO3 (1.46 g, 10.5 mmol)
were added to a flask under N2. Anhydrous CH3OH (2 mL) and
anhydrous THF (2 mL) were added to the flask. The mixture
was stirred for 1 h and monitored by TLC. After the reaction
was complete, water (15 mL) was added to the flask and product
was extracted into EtOAc (2 ꢀ 30 mL). The organic layer was
dried over sodium sulfate and filtered. The filtrate was concen-
trated under reduced pressure, and the oily residue was purified
by flash chromatography (20-50% EtOAc in hexanes) to
obtain 5-ethynyl-3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyri-
dine as white solid (0.26 mg, 62%); mp 97-98 °C. TLC Rf 0.30 in
EtOAc:hexanes (1:1). 1H NMR (600 MHz, CD3OD) δ 8.99 (d,
1H, J = 1.8), 8.77 (d, 1H, J = 2.4), 8.31 (t, 1H, J = 1.8) 4.26 (s,
2H), 3.92 (s, 1H), 1.39 (s, 6H). 13C NMR (100 MHz, CD3OD) δ
161.6, 155.5, 149.1, 139.8, 125.2, 121.3, 84.2, 80.8, 80.0, 69.1,
28.5. LC-MS (ESI) m/z 201.2 (M þ 1).
5-Ethynyl-3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine
(0.8 g, 4 mmol) was dissolved in 3 N HCl (150 mL) and acetic
acid (75 mL), and the mixture was refluxed at 95 °C for 36 h. The
solution was concentrated under reduced pressure and then
dissolved in water (25 mL) and neutralized using 2 N NaOH.
The aqueous layer was lyophilized, and the solid was purified by
flash chromatography to give 3e as white solid (0.43 g, 74%); mp
210-211 °C with decomposition. TLC Rf 0.60 in CH2Cl2:
5-(4-(Aminomethyl)-1H-1,2,3,-triazol-1-yl)nicotinic Acid (3j).
A solution of ethyl 5-(4-(aminomethyl)-1H-1,2,3-triazol-1-
yl)nicotinate (14) (100 mg, 0.4 mmol) in CH3OH (2 mL) and
NaOH (2 M, 0.5 mL, 1 mmol) was stirred for 24 h at room
temperature. When no starting material was detected on TLC
(CH2Cl2:CH3OH:TEA, 8:1:1), the solution was neutralized with
2 N HCl and concentrated in vacuum. The residue was dissolved
in water (10 mL) and lyophilized. The solid obtained was then
suspended in CH3OH and filtered. The filtrate was then con-
centrated in vacuum to obtain 3j as white solid with some
1
CH3OH:acetic acid (90:9:1). H NMR (400 MHz, CD3OD) δ
9.00 (d, 1H, J = 1.6), 8.62 (d, 1H, J = 2.0), 8.34 (t, 1H, J = 2.0),
3.79 (s, 1H). 13C NMR (100 MHz, CD3OD) δ 167.0, 156.4,
150.7, 141.6, 128.3, 121.4, 84.0, 80.0. Analysis Calcd for
C8H5NO2 0.1 H2O: C 64.52, H 3.52, N 9.40. Found: C 64.29,
H 3.33, N 9.31. LC-MS (ESI) m/z 148.3 (M þ 1).
1
inorganic salts (85 mg). H NMR (400 MHz, CD3OD) δ 9.27
5-Ethenylnicotinic Acid (3f). To a cooled solution of 5-ethynyl
nicotinic acid (3e) (0.1 g, 0.68 mmol) in CH3OH (10 mL) were
added Lindlar’s catalyst (5 mg, 5%) and 2,20-(ethylenedithio)di-
ethanol (50 μg, 10 parts per thousand of Pd catalyst). The mix-
ture was shaken under H2 atmosphere (30 psi) at room tem-
perature. The reaction was monitored by TLC and was filtered
through a Celite pad after 24 h. The filtrate was concentrated
under reduced pressure at room temperature, and the crude
product was purified by column chromatography in CH2Cl2:
CH3OH:acetic acid (90:9:1) to give 3f (27.4 mg, 27%); mp
154-156 °C. TLC Rf 0.55 in CH2Cl2:CH3OH:acetic acid
(90:9:1). 1H NMR (600 MHz, CD3OD) δ 8.98 (s, 1H), 8.76 (s,
1H), 8.45 (s, 1H), 6.87-6.82 (dd, 1H, 2J = 18, 3J = 11.4), 6.02
(d, 1H, J = 17.4), 5.50 (d, 1H, J = 10.8). 13C NMR (150 MHz,
CD3OD) δ 166.6, 150.5, 148.9, 134.3, 133.9, 132.4, 127.5, 117.3.
LC-MS (ESI) m/z 150.1 (M þ 1).
(s, 1H), 9.23 (s, 1H), 8.83 (s, 1H), 8.81 (t, 1H), 4.39 (s, 1H). 13
C
NMR (150 MHz, CD3OD) δ 163.1, 151.7, 141.4, 130.4, 124.4,
119.8, 116.9, 35.5.
3-Bromo-5-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine (4).
Compound 4 was prepared from 3c in three steps according to
the procedure of Robert et al., (2006)43 and isolated as a white
solid (4.92 g, 78% over three steps); mp 91-93 °C (lit.43 90-
91 °C). TLC Rf 0.65 in EtOAc. The NMR data was in agreement
with the published data.
1,2-Bis(5-(4,4-dimethyl-4,5-dihydrooxazol-2y)pyridine-3-yl)-
ethyne (5). To a two-neck flask, triethyl(ethynyl)silane (TES-
acetylene, 182 mg, 1.3 mmol) was added and it was protected from
air under N2 atmosphere. To this flask, a solution of tetrakis-
(triphenylphosphine)-palladium(0) (45 mg, 0.04 mmol) in ethanol/
dimethoxyethane (1:1, 2 mL), aqueous sodium carbonate (2 M, 4
mL), and copper iodide (46 mg, 0.24 mmol) was added. The
resultant solution was stirred at room temperature for 15 min,
and then to this solution 3-bromo-5-(4,5-dihydro-4,4-dimethyl-2-
oxazolyl)-pyridine (4) (0.6 g, 2.5 mmol) was added under a stream
of nitrogen. The flask was then stirred at 90 °C (oil bath tempera-
ture) for 1 h, and the reaction was brought to room temperature.
The mixture was poured into a flask containing anhydrous sodium
sulfate, filtered, and evaporated in vacuum. The residue was then
purified by column chromatography (10-50% EtOAc in hexanes)
to give compound 5 (155 mg, 58%). TLC Rf 0.30 in EtOAc:hexanes
(1:1). 1H NMR (600 MHz, CDCl3) δ 9.05 (s, 1H), 8.79 (s, 1H), 8.32
(t, 1H, J = 1.8), 4.12 (s, 2H), 1.36 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 159.6, 154.0, 148.8, 138.7, 138.0, 124.1, 119.6, 89.2, 68.2,
28.6. LC-MS (ESI) m/z 375.5 (M þ 1).
5-Ethylnicotinic Acid (3g). In a flask under N2, 5-ethynylni-
cotinic acid (3e) (0.04 g, 0.3 mmol) and ammonium formate
(0.063 g, 1 mmol) were added. To this flask, a mixture of 10%
Pd-C (50 mg) and cold CH3OH (5 mL) was added and the
resulting mixture was stirred for 12 h. The Pd-C was filtered off
through a bed of Celite, and the clear filtrate was evaporated.
The residue was washed with methylene chloride several times,
and the product went into the methylene chloride. The solvent
was evaporated to give product 3g as sticky residue (25 mg,
78%). 1H NMR (600 MHz, CD3OD) δ 8.93 (s, 1H), 8.57 (s, 1H),
8.25 (s, 1H), 2.76 (q, 2H, 2J = 15.0, 3J = 7.2), 1.29 (t, 3H, J =
7.2). 13C NMR (150 MHz, CD3OD) δ 167.6, 151.6, 147.4, 140.3,
137.2, 128.4, 25.5, 14.4. LC-MS (ESI) m/z 152.1 (M þ 1).

7608 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Jain et al.
4,4-Dimethyl-2-(5-((triethylsilyl)ethynyl)pyridine-3-yl)-4,5-di-
hydrooxazole (6). A two-neck flask, under N2, was charged with
3-bromo-5-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine (4)
(1.6 g, 6.27 mmol) and benzyltributyl ammonium bromide (1.8
g, 5.05 mmol). To this mixture, degassed acetonitrile (9 mL) and
degassed water (0.9 mL) were added. This was followed by
addition of TES-acetylene (1.4 mL, 7.7 mmol) and DIPEA
(7.2 mL). The mixture was stirred well at ambient temperature.
In another flask, Pd(OAc)2 (222.5 mg, 1 mmol) and PPh3
(525 mg, 2 mmol) were taken and dissolved in degassed acet-
onitrile (45 mL) and water (4.5 mL). The latter was added to the
two-neck flask and was stirred at ambient temperature for 8 h.
The reaction was monitored by TLC, and when the starting
material had disappeared, water (20 mL) was added to quench
the reaction. Product was extracted into EtOAc (3 ꢀ 30 mL),
and the organic layer was dried over sodium sulfate and filtered.
The filtrate was concentrated under reduced pressure, and the
oily residue was purified by flash chromatography (20-50%
EtOAc in hexanes). Product was obtained as brown oil (1.16 g,
68%) that solidified on freezing; mp 67-69 °C. TLC Rf 0.54 in
was dissolved in degassed EtOH (6 mL) and added to the catalyst
mixture. The mixture was allowed to stir at room temperature for 15
min. The flask was sealed with a rubber septum and transferred to oil
bath at 90 °C to stir for 4 h. The reaction was monitored by TLC, and
when almost all starting material disappeared, the reaction mixture
was filtered through Celite, washing with CH2Cl2. The organic layer
was dried over sodium sulfate and filtered. The filtrate was concen-
trated in vacuum, and the residue was purified by column chroma-
tography (40-70% EtOAc in hexanes) to obtain 9 as oil (490 mg,
61%). TLC Rf 0.55 in EtOAc:hexanes (7:3). 1H NMR (400 MHz,
CDCl3) δ 9.07 (d, 1H, J = 2.0), 8.89 (d, 1H, J = 2.4), 8.39 (t, 1H,
J= 2.4), 7.6 (d, 2H, J= 7.2), 7.46-7.37 (m, 3H) 4.13 (s, 2H), 1.38 (s,
6H). 13C NMR (100 MHz, CDCl3) δ 160.4, 150.5, 148.2, 134.1,
129.3, 128.7, 127.5, 79.5, 68.2, 28.6.
5-Aminonicotinic Acid Ethyl Ester (10). 5-Aminonicotinic
acid (3a) (5.0 g, 0.036 mol) was dissolved in EtOH (200 mL)
and conc HCl (40 mL). The solution was then refluxed for 36 h.
The reaction was monitored by TLC and was concentrated
under reduced pressure upon completion. The resulting residue
was dissolved in distilled water (25 mL) and neutralized using 1
N NaOH. The aqueous layer was then extracted with EtOAc
(3 ꢀ 50 mL). The organic layer was dried over sodium sulfate
and filtered. The filtrate was concentrated under reduced pres-
sure to obtain white crystalline solid. The solid was dried under
vacuum to obtain 10 (4.9 g, 82%); mp 95-97 °C. TLC Rf 0.66 in
CH3OH:CH2Cl2 (1:9). 1H NMR (600 MHz, CD3OD) δ 8.32 (d,
1H, J = 1.8), 8.10 (d, 1H, J = 2.4), 7.59-7.60 (m, 1H), 4.36 (q,
2H, 2J = 14.4, 3J = 7.2), 1.38 (t, 3H, J = 7.2). 13C NMR (150
MHz, CD3OD) δ 167.0, 146.8, 140.6, 138.8, 128.4, 122.6 (d, J =
8.25) 62.56, 14.7. Analysis calcd for C8H10N2O2: C 57.82, H
6.07, N 16.86. Found: C 57.80, H 6.17, N 16.67.
5-Azidonicotinic Acid Ethyl Ester (11). 5-Aminonicotinic acid
ethyl ester (10) (3.0 g, 18.05 mmol) was stirred with concentrated
HCl (100 mL) in a 0 °C bath. A solution of NaNO2 (1.38 g, 19.86
mmol) in ice water (8 mL) was added dropwise to the stirred
slurry of the hydrochloride salt, resulting in a clear solution.
This solution was then added to stirred slurry of NaN3 (1.77 g,
27.09 mmol) and sodium acetate (3.9 g) in ice water (3.5 mL).
After a few minutes of stirring, a red oil separated from the
mixture. The mixture was made basic by the addition of con-
centrated ammonium hydroxide solution after 10 min, and the
product was extracted into EtOAc. The organic layer was dried
over sodium sulfate, filtered, and the filtrate concentrated under
reduced pressure. The residue was purified by column chroma-
tography (10% EtOAc in hexanes) to give 11 as golden oil34
(1.66 g, 48%). TLC Rf 0.48 in EtOAc:hexanes (1:5). 1H NMR
(400 MHz, CDCl3) δ 8.96 (d, 1H, J = 1.6), 8.47 (d, 1H, J = 2.8),
7.94 (d, 1H, J = 2.0), 4.41 (q, 2H, 2J = 14.4, 3J = 7.2), 1.40 (t,
3H, J = 7.2). 13C NMR (100 MHz, CDCl3) δ 164.7, 147.0,
144.9, 137.5, 127.2, 126.7, 62.1, 14.4. Analysis calcd for
C8H8N4O2 0.1 EtOAc: C 50.20, H 4.41, N 27.88. Found: C
49.82, H 4.21, N 27.92.
Ethyl 5-(4-((tert-Butyloxycarbonylamino)methyl)-1H-1,2,3-
triazol-1-yl)nicotinate (13). To a solution of 5-azidonicotinic
acid ethyl ester (11) (150 mg, 0.78 mmol) and tert-butyl prop-
2-ynylcarbamate (12) (124 mg, 0.8 mmol) in THF (1 mL) were
added CuI (300 mg, 1.6 mmol) and DIPEA (0.4 mL, 2.4 mmol).
The reaction was stirred at room temperature for 12 h, by which
time all the starting material was consumed. The reaction mixture
was diluted with water (10 mL) and saturated aqueous NH4Cl
(10 mL) and was extracted with CH2Cl2 (3 ꢀ 20 mL). The organic
layers were combined and washed with brine, dried over anhydrous
sodium sulfate, and filtered. The filtrate was concentrated under
reduced pressure, and the residue was purified by column chroma-
tography to obtain impure material (160 mg), which was used for
next step without further purification.
1
EtOAc:hexanes (1:1). H NMR (600 MHz, CDCl3) δ 8.96 (s,
1H), 8.70 (s, 1H), 8.25 (t, 1H, J = 1.8), 4.09 (s, 2H), 1.34 (s, 6H),
0.99 (t, 9H, J = 8.4), 0.63 (q, 6H, 2J = 16.2, 3J = 7.8). 13C NMR
(150 MHz, CDCl3) δ 159.6, 154.4, 147. 9, 138.4, 123.5, 120.2,
101.6, 96.8, 79.3, 67.9, 28.3, 7.42 (d, J = 1.95), 4.18. LC-MS
(ESI) m/z 315.3 (M þ 1).
5-(4,5-Dihydro-4,4-dimethyl-2-oxazolyl)-3-pyridinylboronic
Acid (7). 3-Bromo-5-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-
pyridine (4) (1.5 g, 5.90 mmol) was taken in a flask and flushed
with N2. Anhydrous toluene (5 mL) and anhydrous THF
(6.5 mL) were added to the flask, and the mixture was stirred
until the solid dissolved. The flask was then transferred to
a -40 °C cooling bath and stirred rapidly. Some of the solid
started to precipitate. Triisopropyl borate (1.8 mL, 7.8 mmol)
was added to the flask. When the reaction mixture had been
cooled for 15 min, n-butyllithium (1.6 M in hexanes, 4.5 mL,
7.2 mmol) was added dropwise over a period of 1 h. By the time
butyllithium addition was complete, the mixture turned into
a yellowish viscous liquid. It was stirred for another 30 min at
-40 °C, brought to -20 °C, and 2 N HCl (20 mL) added. The
acidic layer was extracted with toluene (20 mL), the toluene
extract was discarded, and then the aqueous layer was neutra-
lized with 2 N NaOH. The product was extracted into THF (4 ꢀ
15 mL). The THF layer was evaporated to dryness, and the
residue was dissolved in mixture of CH3OH/THF (1:1) and
filtered. The filtrate was evaporated under reduced pressure and
the residue crystallized from acetonitrile to obtain boronic acid 7 as
yellow solid (0.69 g, 53%); mp 270-272 °C with decomposition.
Pinacol Ester of 5-(4,5-Dihydro-4,4-dimethyl-2-oxazolyl)-3-
pyridinyl Boronic Acid (Pinacol Ester 8). 5-(4,5-Dihydro-4,4-
dimethyl-2-oxazolyl)-3-pyridinylboronic acid (7) (0.1 g, 0.4 mmol),
(CH3)2COHCOH(CH3)2, (190 mg, 1.6 mmol), and toluene (4 mL)
were taken in a two-neck flask equipped with N2 inlet and
condenser with a Dean-Stark trap. The mixture was heated in a
120 °C oil bath and refluxed for 2.5 h. The reaction was monitored
with TLC, and upon completion the solution was concentrated in
vacuum. The residue was purified using column chromatography
(10-50% EtOAc in hexanes) to obtain product as yellow oil (63.2
1
mg, 46%). TLC Rf 0.30 in EtOAc:hexanes (1:1). H NMR (400
MHz, CDCl3) δ 9.14(s, 1H,), 8.97 (s, 1H,), 8.59 (d, 1H, J = 1.6)
4.10 (d, 2H, J = 1.6), 1.36 (d, 6H, J = 1.2), 1.32 (d, 12 H, J = 1.2).
LC-MS (ESI) m/z 303.3 (M þ 1).
5-Phenyl-3-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-pyridine (9).
A two-neck flask fitted with a N2 inlet was charged with tetrakis-
(triphenylphosphine)palladium(0) (230 mg, 0.2 mmol). Degassed
dimethoxyethane (6 mL) and degassed sodium carbonate (2 M,
4 mL) were added to the flask. Bromobenzene (0.45 mL, 4.2 mmol)
was added to the mixture, and the suspension was stirred at room
temperature for 15 min. In a separate flask, 5-(4,5-dihydro-4,4-
dimethyl-2-oxazolyl)-3-pyridinyl boronic acid (7) (0.7 g, 3.18 mmol)
Ethyl 5-(4-(Aminomethyl)-1H-1,2,3-triazol-1-yl)nicotinate (14).
To a solution of 13 (160 mg) in anhydrous CH2Cl2 (2 mL) was
added triflouroacetic acid (2 mL), and it was stirred at room
temperature for 1.5 h. The reaction was concentrated in vacuum

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7609
and again diluted with CH2Cl2 (5 mL). This solution was then
neutralized with triethylamine (TEA) and concentrated in va-
cuum. The residue was purified using column chromatography
(CH2Cl2:CH3OH:TEA, 8:1:1) to give 14 as colorless oil (92.6 mg,
48% over two steps with respect to 5-azidonicotinic acid ethyl
HPLC using this same system before analysis. HPLC traces are
shown for all tested compounds in the Supporting Information.
This chromatographic system was capable of separating the
pyridine dinucleotide precursor, the cyclic ADP-ribose deriva-
tive, and the linear ADP-ribose derivative in each case. Pyridine
dinucleotides were also judged pure by determining that the
aromatic and anomeric regions of the 1H NMR spectrum were
free from extraneous signals and that the 31P NMR exhibited no
unexplained signals besides the expected phosphate monoester
and the pyrophosphoryl resonance.
4-Amino-NAADP Methyl Ester (15). The requisite pyridine
base, 4-aminonicotinic acid methyl ester, was produced by
esterification of the acid 2a. A solution of 4-aminonicotinic acid
(2a) (200 mg, 1.45 mmol) in anhydrous methanol (25 mL) and
conc HCl (3 mL) was refluxed for 24 h with constant stirring.
The condenser was fitted with a Drierite trap at the top. When
the starting material had converted to a nonpolar material, as
shown by TLC, all the volatiles were removed in vacuum. The
residue was again dissolved in distilled water and was neutra-
lized with NaOH (1 M). The aqueous layer was then extracted
with EtOAc (3 ꢀ 20 mL). All the organic extracts were com-
bined, dried over anhydrous sodium sulfate, and evaporated to
give white crystalline material (92.5 mg, 42%); mp 172-174 °C.
TLC Rf 0.65 in CH3OH:CH2Cl2 (1:9). 1H NMR (600 MHz,
CD3OD) δ 8.67 (s, 1H), 8.00 (d, 1H, J = 6.0), 6.68 (d, 1H, J =
6.0), 3.88 (s, 3H). 13C NMR (150 MHz, CD3OD) δ 167.6, 156.4,
151.9, 150.2, 110.9, 107.0, 51.0.
A mixture of 4-aminonicotinic acid methyl ester (30 mg, 0.19
mmol) was made in distilled water (1.5 mL) and tris-(hydroxy-
methyl)aminomethane (Tris) HCl buffer at pH 7.5 (10 mM,
1.5 mL). The pH of the mixture was readjusted to 7.5 with Tris
base (0.5 M) and the mixture turned to solution on stirring at 37 °C.
To this solution, NADP (13 mg, 0.017 mmol) and Aplysia cyclase
(60 μL, 0.25 U) enzyme were added. The solution was stirred for
4.5 h at 37 °C. The reaction was monitored by an assay to determine
the amount of NADP left in the reaction. Upon completion, the
catalyst was removed from the reaction solution by centrifuging in
Amicon Ultra centrifugal filter device (10000 molecular weight cut)
at the speed of 3200g for 15 min. The filtrate contained the product,
and the retentate was discarded. The clear filtrate was diluted,
adjusted to pH 7.0, and purified by anion exchange chro-
matography on DEAE 52 cellulose. A gradient of 10-600 mM
NH4HCO3 (700 mL) was applied and the product eluted at around
290 mM NH4HCO3. The appropriate fractions were lyophilized to
obtain product as white solid (6 mg, 44%). 1H NMR: 8.70 (s, 1H),
8.25 (s, 1H), 8.08 (s, 1H), 8.06 (d, 1H, J = 7.2), 6.79(d, 1H, J=7.2),
6.09 (d, 1H, J= 5.4), 5.57 (d, 1H, J =6.0), 4.95 (m, 1H), 4.18-4.60
(9H), 3.89 (s, 3H). 31P NMR: 0.56, -11.20.
1
ester). TLC Rf 0.65 in CH2Cl2:CH3OH:TEA (8:1:1). H NMR
(400 MHz, CD3OD) δ 9.30 (d, 1H, J = 2.4), 9.22 (d, 1H, J = 1.6),
8.82 (s, 1H), 8.79 (t, 1H, J = 2.0), 4.47 (q, 2H, 2J = 14.4, 3J = 7.2),
4.39 (s, 1H), 1.44 (t, 3H, J=7.2). 13CNMR(100MHz, CD3OD) δ
165.3, 151.3, 146.1, 142.9, 135.1, 130.0, 129.0, 124.3, 63.4, 35.5,
14.6. LC-MS (ESI) m/z 248 (M þ 1).
Synthesis of NAADP Analogues. Enzymatic Assay of NAD
Glycohydrolase Activity.45 Recombinant Aplysia ADP-ribosyl
cyclase expressed in yeast was produced according to procedure
of Lee et al.46 NAD glycohydrolase (NADase) from pig brain
was bought from Sigma-Aldrich as an acetone powder. The dry
acetone powder was hydrated and dispersed into a fine suspen-
sion each time immediately before use. The stock mixture
consisted of 60 mg of dry acetone powder in 2 mL of distilled
water. The mixture was kept at 37 °C for 0.5 h and then subjected
to homogenization until fine milky suspension was obtained.
The NADase from beef spleen was isolated according to proce-
dure of Kaplan and Colowick.47 The NADase activity of
Aplysia cyclase, NADase (pig brain), and NADase (beef spleen)
were measured by determining the amount of NAD present at
timed intervals in the assay by the reduction of NAD to NADH
catalyzed using yeast alcohol dehydrogenase. The NAD glyco-
hydrolase test solution was made by addition of sodium dihy-
drogen phosphate pH 7.4 (0.1 M, 0.8 mL), NADase enzyme (80
μL of stock made up to 0.2 mL with buffer), and NAD (0.2 mL,
6 mg/mL of buffer, 1.808 μmol). The reaction was incubated at
37 °C for 10 min and trichloroacetic acid (TCA) (25% w/v,
0.3 mL) was added to quench the reaction. The blank solution
was treated exactly the same way, except that TCA was added
even before initiating the reaction. Both the test and the blank
solutions were centrifuged at the speed of 3200g for 5 min,
and 0.3 mL of aliquot was removed from the clear supernatant.
This was added to a cuvette with Tris-ethanol buffer pH 10
(2.7 mL, equal volumes of 0.5 M Tris and 0.5 M ethyl alcohol).
To this solution, yeast alcohol dehydrogenase was added (10 μL,
10 mg/mL) and the OD was measured at 340 nm.
Enzymatic Assay for NADP Estimation.48 NADP, glucose-6-
phosphate, and yeast glucose-6-phosphate dehydrogenase (2
units in 10 mL solution) were obtained commercially. In the
chemoenzymatic synthesis of NAADP analogues, NADP was
incubated with huge excess of nicotinic acid in the presence of
NAD glycohydrolase. The reaction was monitored by determi-
nation of the amount of NADP left in the reaction mixture. A 20
μL aliquot was removed from the reaction mixture (with con-
centration of about 0.01 mmol of NADP/mL) at intervals of
0.5 h and the amount of NADP determined until complete
disappearance of NADP was observed. For the determination,
2-(N-morpholino)ethanesulfonic acid buffer (MES) (50 mM,
1 mL) was taken in a cuvette. To this the reaction aliquot and
glucose-6-phosphate (0.1 M, 40 μL) were added. The NADP
present in the reaction mixture is converted into NADPH by
addition of 10 μL of a solution (5 mg/mL of enzyme) of yeast
glucose-6-phosphate dehydrogenase at room temperature. The
NADPH that was formed showed absorbance at 340 nm, and
the time when no absorbance was seen in the assay was
indicative of reaction completetion. This is a very sensitive assay
which is capable of detecting nM concentrations of NADP. At
the time at which the starting NADP concentration was deter-
mined to be zero, all the NADP was either exchanged into
NAADP or was converted into ADPRP as a result of a competing
hydrolyzing reaction.
4-Amino-NAADP (16). 4-Amino-NAADP methyl ester (15)
(6 mg, 0.008 mmol) was dissolved in 3 mL of a 0.2 M solution of
TEA dissolved in H2O/CH3OH. The solution was stirred at
35 °C for precisely 12 h. The reaction mixture was diluted with
distilled water, and CO2 was bubbled through it until the pH
went down to 7.0. This was followed by lyophilization to give
white solid with some triethyl ammonium bicarbonate buffer.
Repeated lyophilizations gave the product as tris(triethyl-
ammonium) salt in quantitative yields. 1H NMR: 8.37 (s, 1H),
8.34 (s, 1H), 8.08 (s, 1H), 7.88 (d, 1H, J = 7.2), 6.63 (d, 1H, J =
7.2), 6.13 (d, 1H, J = 5.4), 5.52 (d, 1H, J = 6.0), 5.05 (m, 1H), 4.6
(m, 1H), 4.15-4.40 (8H). 31P NMR: 2.42, -10.59.
4-Methyl-NAADP (17). A solution of 4-methylnicotinic acid
HCl salt (2b HCl, 70 mg, 0.51 mmol) was made in distilled water
3
(4 mL). The pH of the mixture was adjusted to 4.0 with 0.5 M
NaOH and a solution resulted. Then 2.5 mL of this solution was
added to another vial, followed by solid NADP (20 mg, 0.027
mmol) and Aplysia cyclase enzyme (60 μL, 0.25 U). The pH of
the solution was not readjusted and was buffered by the
nicotinate solution. The reaction was stirred for 2.5 h at 37 °C.
The reaction was monitored by an enzymatic assay to determine
the amount of NADP left in the reaction. Upon completion, the
Characterization of Pyridine Dinucleotide Analogues 16-24.
All compounds were examined by high pressure liquid chroma-
tography (HPLC) using AG MP-1 chromatography49 immedi-
ately prior to testing and if necessary repurified by preparative

7610 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Jain et al.
catalyst was removed from the reaction solution by centrifuging
in an Amicon Ultra centrifugal filter device (10000 molecular
weight cut) at the speed of 3200g for 15 min. The filtrate
contained the product, and the retentate was discarded. The
clear filtrate was diluted, adjusted to pH 7.0, and purified by
anion exchange chromatography on DE-52 cellulose. A gradi-
ent of 10-600 mM NH4HCO3 (700 mL) was applied and the
product eluted at around 300 mM NH4HCO3. The appropriate
fractions were lyophilized to obtain product as white solid
anion exchange chromatography on DE-52 cellulose. A gradi-
ent of 10-600 mM NH4HCO3 (700 mL) was applied and the
product eluted at around 330 mM NH4HCO3. The appropriate
fractions were lyophilized to obtain product as white solid (7 mg,
43%). 1H NMR: 8.40 (s, 1H), 8.27 (s, 1H), 8.20 (s, 1H), 8.13 (s,
1H), 7.77 (s, 1H), 6.14 (d, 1H, J = 4.8), 5.80 (d, 1H, J = 4.2), 5.0
(m, 1H), 4.58-4.18 (9H). 31P NMR: 0.51, -10.81. HRMS:
760.231 (M þ 1).
5-Methyl-NAADP (21). The pH of a mixture of 5-methylni-
cotinic acid (3b) (70 mg, 0.51 mmol) and water (4 mL) was
adjusted to 4.0 with 0.5 M NaOH, and the mixture formed a
solution. Two mL of this solution was added to another vial,
followed by solid NADP (16 mg, 0.021 mmol) and Aplysia
cyclase enzyme (60 μL, 0.25 U). The pH of the solution was not
adjusted again and was buffered by the 5-methylnicotinate
solution. The solution was stirred for 2.5 h at 37 °C. The reaction
was monitored by an assay to determine the amount of NADP
left in the reaction. Upon completion, the catalyst was removed
from the reaction solution by centrifuging in an Amicon Ultra
centrifugal filter device (10000 molecular weight cut) at the
speed of 3200g for 15 min. The filtrate contained the product,
and the retentate was discarded. The clear filtrate was diluted,
adjusted to pH 7.0, and purified by anion exchange chromatog-
raphy on DE-52 cellulose. A gradient formed between 10-600
mM NH4HCO3 (700 mL) was applied and the product eluted
into ca. 300 mM NH4HCO3. The appropriate fractions were
lyophilized to obtain product as white solid (9.6 mg, 59%). 1H
NMR: 8.91 (s, 1H), 8.80 (s, 1H), 8.58 (s, 1H), 8.41 (s, 1H), 8.13 (s,
1H), 6.15 (d, 1H, J = 4.8), 5.93 (d, 1H, J = 4.2), 5.05 (m, 1H),
4.18-4.60 (9H), 2.48 (s, 3H). 31P NMR: 0.67, -10.60. HRMS:
759.031 (M þ 1).
1
(8.8 mg, 43%). H NMR: 8.73 (s, 1H), 8.63 (d, 1H, J = 6.6),
8.41 (s, 1H), 8.08 (s, 1H), 7.76 (s, 1H, J = 6.0), 6.14 (d, 1H, J =
6.0), 5.84 (d, 1H, J = 5.4), 5.10 (m, 1H), 4.63-4.15 (9H), 2.60 (s,
3H). 31P NMR: 0.66, -10.46. HRMS: 781.0575 (M þ Na).
4-n-Butyl-NAADP (18). A solution of 4-butylnicotinic acid
(2c, 30 mg, 0.17 mmol) was made in distilled water (2.5 mL). The
pH of the solution was adjusted to 4.0 with 0.5 M NaOH. Then
this solution was added to another vial, followed by addition of
NADP (14 mg, 0.02 mmol) and Aplysia cyclase enzyme (60 μL,
0.25 U). The solution was buffered by the nicotinate solution.
The reaction was stirred for 1 h at 37 °C. The reaction was
monitored by an assay to determine the amount of NADP left in
the reaction. Upon completion, the catalyst was removed from
the reaction solution by centrifuging in an Amicon Ultra
centrifugal filter device (10000 molecular weight cut) at the
speed of 3200g for 15 min. The filtrate contained the product
and the retentate was discarded. The clear filtrate was diluted,
adjusted to pH 7.0, and purified by anion exchange chromatog-
raphy on DE-52 cellulose. A gradient of 10-600 mM
NH4HCO3 (700 mL) was applied and the product eluted at
around 360 mM NH4HCO3. The appropriate fractions were
1
lyophilized to obtain product as white solid (9 mg, 60%). H
NMR: 8.69 (s, 1H), 8.66 (d, 1H, J = 6.4), 8.41 (s, 1H), 8.09 (s,
1H), 7.79 (d, 1H, J = 6.4), 6.14 (d, 1H, J = 5.6), 5.84 (d, 1H, J =
5.2), 5.10 (m, 1H), 4.62-4.10 (9H), 2.93 (m, 2H), 1.51 (m, 2H),
1.26 (m, 2H), 0.84 (t, 3H, J = 7.6). 31P NMR: 0.66, -10.46.
HRMS: 801.1229 (M þ Na).
5-Carboxy-NAADP (22). 3,5-Pyridine-dicarboxylic acid (3d,
70 mg, 0.42 mmol) was mixed in distilled water (6 mL). The
mixture was stirred at 37 °C for 1 h and the pH was adjusted to
4.0 with 0.5 M KOH, and stirring continued at 37 °C for 0.5 h.
The pH had decreased to around 3.5 as the solid of the mixture
was slowly dissolving. The pH was again adjusted to 4.0, and
this was repeated until the mixture had formed a clear solution.
To this solution, solid NADP (16 mg, 0.02 mmol) was added,
followed by Aplysia cyclase enzyme (100 μL, 0.42 U). The pH
was not readjusted and was buffered by nicotinate solution. The
reaction was stirred for 3.5 h at 37 °C. The reaction was
monitored by an assay to determine the amount of NADP left
in the reaction. Upon completion, the catalyst was removed
from the reaction solution by centrifuging in an Amicon Ultra
centrifugal filter device (10000 molecular weight cut) at the
speed of 3200g for 15 min. The filtrate contained the product,
and the retentate was discarded. The clear filtrate was diluted,
adjusted to pH 7.0, and purified by anion exchange chromatog-
raphy on DE-52 cellulose. A gradient of 10-600 mM NH4H-
CO3 (700 mL) was applied and the product eluted at around 500
mM NH4HCO3. The appropriate fractions were lyophilized to
obtain product as white solid (5.5 mg, 32.4%). 1H NMR: 9.41 (s,
2H), 9.24 (s, 1H), 8.54 (s, 1H), 8.38 (s, 1H), 6.22 (d, 1H, J = 5.2),
6.14 (d, 1H, J = 5.6), 5.1 (m, 1H), 4.62-4.18 (9H). 31P NMR:
0.26, -10.1. HRMS 811.0353 (M þ Na).
4-Phenyl-NAADP (19). A solution of 4-phenylnicotinic acid
(2d, 30 mg, 0.15 mmol) was made in distilled water (2.5 mL). The
pH of the solution was adjusted to 4.0 with 0.5 M KOH solution.
Then this solution was added to another vial, followed by
addition of solid NADP (14 mg, 0.02 mmol) and Aplysia cyclase
enzyme (60 μL, 0.25 U). The solution was stirred for 1 h at 37 °C.
The reaction was monitored by an assay to determine the
amount of NADP left in the reaction. Upon completion the
reaction mixture was centrifuged and filtered in centrifugal filter
tubes at 3200g for 15 min. The clear filtrate was diluted, adjusted
to pH 7.0, and then applied to DEAE 52 cellulose anion
exchange purification. A gradient of 10-600 mM NH4HCO3
(700 mL) was applied and the product eluted at around 400 mM
NH4HCO3. The appropriate fractions were lyophilized to ob-
tain product as white solid (8 mg, 51%). 1H NMR: 8.78 (d, 1H,
J = 6.4), 8.68 (s, 1H), 8.19 (s, 1H), 7.87 (s, 1H), 7.77 (d, 1H, J =
6.4), 7.34-7.26 (m, 5 H) 5.86 (t, 2H, J = 4.8), 5.9-5.8 (m, 1H),
4.43-4.00 (9H). 31P NMR: 0.51, -10.62. HRMS: 821.0932 (M
þ Na).
5-Amino-NAADP (20). A mixture of 5-aminonicotinic acid
(3a, 69 mg, 0.50 mmol) was made in distilled water (6 mL). The
mixture was stirred at 37 °C for 1 h, but some of the solid did not
dissolve. The clear supernatant (3 mL) was taken and the pH
was adjusted to 4.0 with 0.5 M KOH. To this solution, solid
NADP (16 mg, 0.021 mmol) was added, followed by Aplysia
cyclase enzyme (80 μL, 0.33 U). The reaction was buffered by the
nicotinate solution. The reaction was stirred for 3.5 h at 37 °C.
The reaction was monitored by an assay to determine the
amount of NADP left in the reaction. Upon completion, the
catalyst was removed from the reaction solution by centrifuging
in an Amicon Ultra centrifugal filter device (10000 molecular
weight cut) at the speed of 3200g for 15 min. The filtrate
contained the product, and the retentate was discarded. The
clear filtrate was diluted, adjusted to pH 7.0, and purified by
5-Ethyl-NAADP (23). The pH of a mixture of 5-ethylnicotinic
acid (3h, 20 mg, 0.13 mmol) and water (2 mL) was adjusted to 4.0
with 0.5 M NaOH, and the mixture turned into solution. This
solution was then added to another vial, followed by solid
NADP (8 mg, 0.01 mmol) and Aplysia cyclase enzyme (60 μL,
0.25 U). The pH of the solution was not adjusted again and was
buffered by the 5-methylnicotinate solution. The solution was
stirred for 2.5 h at 37 °C. The reaction was monitored by an
assay to determine the amount of NADP left in the reaction.
Upon completion, the catalyst was removed from the reaction
solution by centrifuging in an Amicon Ultra centrifugal filter
device (10000 molecular weight cut) at a centrifugal force of
3200g for 15 min. The filtrate contained the product, and the
retentate was discarded. The clear filtrate was diluted, adjusted

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7611
to pH 7.0, and purified by anion exchange chromatography on
DE-52 cellulose. A gradient of 10-600 mM NH4HCO3 (700
mL) was applied and the product eluted at around 300 mM
NH4HCO3. The appropriate fractions were lyophilized to ob-
tain product as white solid (4.0 mg, 59%). H NMR: 8.92 (s,
1H), 8.75 (s, 1H), 8.63 (s, 1H), 8.40 (s, 1H), 8.07 (s, 1H), 6.1 (d,
activity. This material is available free of charge via the Internet
at http://pubs.acs.org.
1
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