Design, synthesis and X-ray crystallographic study of NAmPRTase inhibitors as anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.01.034

NAmPRTase (PBEF/Visfatin) plays a pivotal role in the salvage pathway of NAD⁺ biosynthesis. NAmPRTase has been an attractive target for anti-cancer agents that induce apoptosis of tumor cells via a declining plasma NAD⁺ level. In this report, a series of structural analogs of FK866 (1), a known NAmPRTase inhibitor, was synthesized and tested for inhibitory activities against the proliferation of cancer cells and human NAmPRTase. Among them, compound 7 showed similar anti-cancer and enzyme inhibitory activities to compound 1. Further investigation of compound 7 with X-ray analysis revealed a co-crystal structure in complex with human NAmPRTase, suggesting that Asp219 in the active site of the enzyme could contribute to an additional interaction with the pyrrole nitrogen of compound 7.

Full text of DOI:10.1016/j.ejmech.2011.01.034

European Journal of Medicinal Chemistry 46 (2011) 1153e1164
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and X-ray crystallographic study of NAmPRTase inhibitors
as anti-cancer agents
,
,
,
,
Hyun You b 1, Hyung-Seop Youn a 1, Isak Im a 1, Man-Ho Bae a, Sang-Kook Lee c, Hyojin Ko b
**
**
,
Soo Hyun Eom a , Yong-Chul Kim
,
a,b,
*
a School of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
b Graduate-program of Medical System Engineering (GMSE), Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
c College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
NAmPRTase (PBEF/Visfatin) plays a pivotal role in the salvage pathway of NADþ biosynthesis. NAmPRTase
has been an attractive target for anti-cancer agents that induce apoptosis of tumor cells via a declining
plasma NADþ level. In this report, a series of structural analogs of FK866 (1), a known NAmPRTase
inhibitor, was synthesized and tested for inhibitory activities against the proliferation of cancer cells and
human NAmPRTase. Among them, compound 7 showed similar anti-cancer and enzyme inhibitory
activities to compound 1. Further investigation of compound 7 with X-ray analysis revealed a co-crystal
structure in complex with human NAmPRTase, suggesting that Asp219 in the active site of the enzyme
could contribute to an additional interaction with the pyrrole nitrogen of compound 7.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Article history:
Received 1 September 2010
Received in revised form
4 January 2011
Accepted 12 January 2011
Available online 31 January 2011
Keywords:
Nicotinamide phosphoribosyltransferase
(NAmPRTase)
Pre-B-cell colony-enhancing factor (PBEF)
FK866
Nicotinamide adenine dinucleotide (NADþ
X-ray crystal structure
)
1. Introduction
phosphoribosyltransferase (NAmPRTase) is also known as pre-B-
cell colony-enhancing factor 1 (PBEF) or visfatin, and it is the rate-
Nicotinamide adenine dinucleotide (NADþ) is generally
considered a key component involved in redox reactions, and it
plays various remarkable roles in a range of NADedependent
biological processes including signal transduction, DNA repair, and
post-translational protein modification [1]. It has been revealed
that NADþ is a substrate for poly (ADP-ribose) polymerase (PARP)
and a specific subclass of histone deacetylases that are known as
sirtuins [2], which are involved in genomic stability, apoptosis,
aging, stress resistance, and metabolism [3e6]. Since most cancer
cells have continuous PARP activation through DNA damage and
genomic instability [7e10] and they have higher energy
consumption demands relative to non-transformed cells [11],
cancer cells are expected to be more vulnerable to the inhibition of
NAD synthesis than non-transformed cells. Nicotinamide
limiting enzyme for nicotinamide adenine dinucleotide (NADþ)
biosynthesis from nicotinamide. Indeed, NAmPRTase over-
expression was shown to maintain adequate levels of NADþ in
cancer cells [12,13]. Eukaryotic cells possess several mechanisms to
replenish NAD including a de novo synthesis pathway from the
amino acid tryptophan, and there are at least two salvage/recycling
pathways [14,15]. One of these pathways relies on the enzyme
NAmPRTase that converts nicotinamide into nicotinamide mono-
nucleotide (NMN), which is subsequently converted to NADþ by
NMN adenylyltransferase (NMNAT).
A novel antitumor agent FK866 (1, Fig. 1) was reported to induce
apoptosis of tumor cells by reducing the steady state intracellular
NADþ levels, and this process was mediated through specific
inhibition of NAmPRTase [16]. Indeed, inhibition of this enzyme
alone appears to have an important impact on NADþ levels in
a number of cell types. Thus, the enzymes involved in NADþ
metabolism have become attractive targets for cancer therapy [17].
FK866 has successfully completed a Phase I study, and it is currently
being investigated in Phase I/II and II trials for the treatment of
various cancers [18]. Recent reports have shown some obstacles to
utilize FK866 as an anti-cancer agent: the rapid intravenous
* Corresponding author. Graduate-program of Medical System Engineering
(GMSE), Gwangju Institute of Science and Technology, Gwangju 500-712, Republic
of Korea. Tel.: þ82 62 970 2502; fax: þ82 62 970 2484.
** Corresponding authors.
E-mail address: yongchul@gist.ac.kr (Y.-C. Kim).
These authors equally contributed to the work.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.034

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H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
Due to the long, narrow binding pocket of FK866, modification of
FK866 appears to be limited to each end portion. The metabolite
FK866-N-oxide is the primary problem of FK866; thus, further
modification of the pyridine residue of FK866 has been suggested.
We previously investigated an FK866 analog, IS001 (Fig. 1), which
has an additional ribose ring at the nitrogen of the pyridine ring,
using co-crystal structure with NAmPRTase [21]. Although IS001
had an almost identical binding mode to that of FK866, the binding
affinity of IS001 is dramatically decreased [21]. The X-ray co-crystal
structure of IS001 with NAmPRTase showed that the additional
ribose ring did not have any strong interaction with the hydrophilic
residues of NAmPRTase. The additional ribose ring possibly inter-
feres with the most stable conformation of the FK866 moiety
because of the relatively bulky constrained structure that is con-
nected to the pyridine nitrogen. Herein, we have investigated the
design and synthesis of FK866 analogs by combining FK866 and
a ribose-mimicking moiety to reduce the constraints of the ribose
ring, expecting a tighter interaction by the additional hydrogen
bonds. We also replaced the pyridine ring of FK866 with various
heteroaromatic rings for combination with
a simple ribose-
mimicking moiety containing a diol group. The mode of binding
and biological activity was assessed by X-ray crystal structure and
cell- and enzyme-based assay.
Fig. 1. Structure of FK866 and IS001.
2. Results and discussion
2.1. Chemistry
clearance of FK866 through the metabolite FK866-N-oxide in vivo
and the dose limit toxicity due to thrombocytopenia. The crystal
structure of NAmPRTase with FK866 showed that FK866 is bound in
a tunnel at the interface of the NAmPRTase dimer through primarily
hydrophobic interactions [19,20]. This long (15 Å) and narrow
diameter (6 Å) tunnel ends at the active site where the natural
The general synthetic method for the FK866 analogs is described
in Scheme 1. The commercially available 4-piperidine butyric acid
hydrochloride (2) was esterified by treatment with thionyl chloride
in methanol; then, it was benzoylated using benzoyl chloride to
produce compound 3. The N-benzoated ester (3) was reduced to an
alcohol by LAH and subsequently transformed into an amine (4) by
two step reactions with potassium phthalimide and hydrazine
hydrate. Compound 4 was subjected to coupling reactions with
several heteroaromatic acrylic acids, such as pyrimidine, amino-
pyrimidine, pyrrole, pyrazole groups, urocanic acid, trans-3-indole
acrylic acids, and nitrocinnamic acids to produce FK866 analogs
substrate nicotinamide (NM) binds by
pep-stacking interactions
with Phe193 and Tyr180. In contrast, nicotinamide mononucleotide
(NMN), which is the product of the enzyme reaction, is involved in
a network of many hydrogen bonds via the ribose ring, and the
phosphate group is surrounded by a highly hydrophilic active site.
Scheme 1. Synthesis of FK866 analogs I. (a) SOCl2, MeOH, RT, 2 h, 89%; (b) benzyl chloride, pyridine, DCM, RT, 3 h, 68%; (c) LAH, THF, RT, 2 h, 65%; (d) methanesulfonic chloride, TEA,
DCM, RT, 2 h, 68%; (e) potassium phthalimide, DMF, 50 ꢀC, 8 h, 87%; (f) hydrazine hydrate, EtOH, RT, 1 day, 52%; (g) EDC, HOBt, TEA, DCM or DMF, RT, 3e4 h, 70%.

H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
Table 1 (continued).
1155
Table 1
Effects of FK866 (1) and its analogs 5e12, 14e20 on MCF7 cell viability.
Compounds
Cell Viability
R
IC50 (mM)
(10
m
M)
MCF7a (% of control)
O
N
O
17
104.9 ꢁ 1.1
103.6 ꢁ 0.4
N
H
R
Compounds
(10 M)
R
Cell Viability
IC50
(
m
M)
18
19
m
MCF7a (% of control)
1, FK866
5.6 ꢁ 0.5
0.68 ꢁ 0.1
106.3 ꢁ 1.5
103.9 ꢁ 0.5
5
86.8 ꢁ 2.6
20
a
Values are given by the means of three experiments with standard deviations.
6
103.4 ꢁ 0.7
7
8
4.5 ꢁ 0.8
1.29 ꢁ 0.05
(5e12) (Scheme 1 and Table 1). Compounds 7e10 were further N-
alkylated with 13 followed by deprotections of the acetonide group
to afford compounds 14e17 containing ribose-mimicking 2-ethyl-
propane-1,3-diol moieties. Compound 18 was prepared from
a methylation reaction of 7, and compound 19 and 20 were
obtained by reductions of the nitro groups of 11 and 12, respectively
(Scheme 2 and Table 1).
101.7 ꢁ 2.4
Scheme 3 shows the preparation of the heteroaromatic acrylic
acids as building blocks. 5-bromo-pyrimidine (21a), 5-bromopyr-
imidin-2-amine (21b), Boc-pyrrole (26) and tosylated 4-bromo
pyrazole (30) were reacted with methyl acrylate (22) under
microwave-assisted Heck reaction conditions, and followed by
deprotection and hydrolysis reactions to yield each acrylic acid (23,
24, 28, and 31). Other heteroaromatic acrylic acids used in this
study were purchased from commercial resources.
9
102.9 ꢁ 0.7
103.3 ꢁ 0.5
10
The 2-ethylpropane-1,3-diol analog (13) was prepared from
decarboxylation of triethyl-1-1,2-ethane tricarboxylate (32) fol-
lowed by a protection reaction of the diol group of 33 with 2,3-
dimethoxy propane (Scheme 4).
11
102.7 ꢁ 0.2
2.2. Cell viability assay
A cell viability assay was conducted to test the synthesized
FK866 analogs [15] on MCF7 breast cancer cells. Briefly, the cells
were incubated for six days with a fixed concentration of the
compounds, and the viability was measured by an SRB assay. The
induction of delayed cell death of the tumor cells appeared prom-
inent after six days of FK866 treatment on the SRB assay. The
structure and activity of the FK866 analogs are shown in Table 1.
Substitution of the pyridine ring of FK866 with various hetero-
aromatic rings (5e10) revealed a great loss of anti-cancer activity
except for compound 7 with the pyrrole group. Even a one-point
substitution, such as carbon for nitrogen at the pyridine ring (5),
dramatically dropped the activity. However, compound 7 main-
12
14
103.1 ꢁ 0.4
105.0 ꢁ 0.7
15
16
106.6 ꢁ 1.2
104.3 ꢁ 0.9
tained an activity similar to that of FK866 at 10 mM. Although the
addition of a ribose-mimicking diol moiety in the structure (7 to 14)
would theoretically improve the activity, compound 14 turned out
to be inactive. Also, the compounds with a ribose-mimicking diol
moiety on the other heteroaromatic rings (15e17) failed to inhibit

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H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
Scheme 2. Synthesis of FK866 analogs II. (a) NaH, DMF, RT, 10 h; (b) 1 N HCl, THF, RT, 2 h; (c) NaH, CH3I, DMF, 55 ꢀC, 9 h; (d) SnCl2, EtOH, 70 ꢀC, 1.2 h.
Scheme 3. Synthesis of acrylic acid building blocks. (a) methyl acrylate, TEA, Pd(OAc)2, acetonitrile, 170 ꢀC, 20 min, microwave, 27%; (b) 5% NaOH, MeOH : Water ¼ (4:1), RT, 2 h,
70%; (c) Boc2O, TEA, DCM, RT, 2 h, 94%; (d) methyl acrylate, Pd(OAc)2, acetic acid-dioxane-DMF (3:9:1), RT, 96 h, 23%; (e) TFA-CH2Cl2 (3:10), 1 h, 75%; (f) 5% NaOH, MeOHeWater
(4:1), 4 h, 91%; (g) p-toluene sulfonyl chloride, TEA, DCM, RT, 3 h, 73%; (h) methyl acrylate, TEA, Pd(OAc)2, LiCl, p(o-tol)3, DMF, 140 ꢀC, overnight, 38%; (i) 5% NaOH, MeOHeWater
(4:1), 4 h, 78%.

H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
1157
FK866 (IC50 > 20
value of 1.4 M.
mM) in human leukemia cells (K562) with an IC50
m
2.3. Enzyme assay using HPLC
We performed an in vitro enzyme assay using an HPLC system to
evaluate the inhibitory activity of FK866 and compound 7. In the
presence of FK866, the formation of NMN as a NAmPRTase reaction
product from two reactants (NM and PRPP) was significantly
decreased in HPLC analysis by monitoring the UV absorbance at
261 nm (Fig. 3). In our data, two compounds showed similar in vitro
enzyme inhibitory activities, which was consistent to the cell-based
anti-proliferative activities. The IC50 values obtained from the
concentration-dependent inhibition curves of FK866 and
compound 7 showed comparable values, 14.8 and 20.3
mM,
respectively (Fig. 4).
Scheme 4. Preparation of ribose mimic moiety. (a) NaBH4, MeOH, tert-butyl alcohol.
reflux, 1 h, 88%; (b) 2,2-dimethoxy propane, p-toluene sulfonic acid, THF, RT, 4 h, 59%;
(c) p-toluene sulfonyl chloride, pyridine, DCM, RT, 5 h, 58%.
2.4. Crystal structure
The crystal structure of human NAmPRTase in complex with
compound 7 showed a dimer in crystal packing and 1:1 stoichi-
ometry (Fig. 5), and this is consistent with the previously reported
data of the human NAmPRTase-FK866 complex assessed using gel
filtration and dynamic light scattering [19]. Moreover, the binding
site of 7 was nearly identical to that of FK866 and NMN, which are
the active sites of NAmPRTase formed around the dimeric interface
(Fig. 6). The binding sites of both inhibitors, except the pyridine or
pyrrole ring, into NAmPRTase consisted of primarily hydrophobic
interactions at Val242(A), Ala244(A), Ile309(A), Arg349(A), Val350
(A), Ile351(A), Ala379(A), and Asp16(B). In addition, the pyrrole ring
cell viability. Therefore, even a relatively small ribose-mimicking
diol moiety in 14e17 seems to interfere with the interaction with
the enzyme (i.e. the ribose moiety in IS001).
Moreover, compound 18 with N-methylation at the pyrrole
group of 7 showed significant loss of the activity, suggesting the
hydrogen of the pyrrole group is important. Compounds 11, 12, 19,
and 20, which had nitro or aminophenyl moieties instead of
heterocycles, did not improve the cytotoxic activity, indicating that
the interaction of the pyridine moiety of FK866 with the corre-
sponding binding region of the enzyme must be tightly managed
for attempted modifications.
made
p-p stacking interactions with Phe193(A) and Tyr18(B)
identical to that on the pyridine ring of FK866. The hydrophobic
bond length between Asp16(B) of NAmPRTase and the pyridine ring
of FK866 (4.09 Å) was slightly closer than that of the pyrrole ring of
7 (4.53 Å). Interestingly, Asp219(A) interacted with the NAmPR-
Tase-7 complex through additional ionic interaction with the
pyrrole ring; however, this residue showed no interaction with the
pyridine ring of FK866, as shown in Fig. 7. This interaction might
contribute to the structural stabilization between NAmPRTase and
7, or we propose that the pyridine ring of FK866 may rotate
allowing it to make the ionic interaction with Asp219(A) of human
NAmPRTase in the complex of FK866 (PDB code 2GVJ). In addition,
although we did not determine the structures of NAmPRTase in
complex with the other candidates, the results of the cell viability
assays suggest that the addition of a bulky or hydrophilic moiety
cannot improve the interaction with NAmPRTase due to the steric
hindrance and the destabilized hydrophobic interactions. It might
be further explained by the binding mode of FK866, which has
a hydrogen bond with Asp219 through a water molecule in the
‘tunnel’. This entropically disadvantageous interaction can be
altered easily by small molecular changes derived from the modi-
fication of FK866. If the optimal binding interactions of the pyridine
We then compared FK866 and compound 7 via the concen-
tration-response curves of both compounds. The IC50 values of
FK866 and compound 7 demonstrating their cytotoxicity against
MCF7 cells were determined as 0.68 mM and 1.29 mM, respectively,
indicating that compound 7 might be another possible candidate
as an anti-cancer agent (Fig. 2). Therefore, the anti-cancer activ-
ities of compound 7 and FK866 were investigated for five other
cancer cell lines (Table 2). The effect of compound 7 in the other
cancer cells was consistent with that on the MCF7 cells in the low
micromolar range of IC50 values, whereas FK866 generally
showed higher activity with submicromolar range of IC50 values.
However, compound 7 showed superior anti-cancer activity to
Table 2
IC50 (mM) cytotoxicity on various cancer cell lines.
K562a
SNU638b
HT1080c
A549d
HCT116e
FK866
7
Ellipticine
>20
1.40
0.38
<0.16
1.70
1.64
<0.16
3.20
0.21
<0.16
4.86
0.47
<0.16
0.30
0.41
a
K562 : human leukemia cell.
b
c
SNU638 : human stomach cancer cell.
HT1080 : human sarcoma cell.
A549 : human lung cancer cell.
HCT116 : human colon cancer cell.
d
e
Fig. 2. MCF7 cell viability after 6 day in the presence of increasing concentrations of
compounds FK866 and 7.

1158
H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
Fig. 3. The products of human NAmPRTase reaction were analyzed by HPLC. Elution times for each chemical were confirmed by running standards in the same HPLC conditions. The
products of reaction without FK866 were shown in pink, and the products with 100
legend, the reader is referred to the web version of this article.)
mM FK866 were shown in cyan. (For interpretation of the references to color in this figure
ring moiety through
pep
-stacking interactions (Phe193, Tyr180)
are not conserved, then the hydrogen bonds with Asp219 via
a water molecule might be disturbed by slight delocalization of the
pyridine ring moiety. In which case, an FK866 analog is not strong
enough to compete with a natural substrate since the other part of
the analog is far from the active site and it has no significant
interactions to hold the whole inhibitor inside the binding tunnel.
Fig. 4. 5, 10, 20, 50, and 100 mM FK866 and 7 were added to reaction buffer, respec-
tively. All experiments were triplicated, error bars represent standard deviation.
Fig. 5. Schematic representation of the NAmPRTase-7 complex. In ribbon diagram of
NAmPRTase dimer, two NAmPRTase monomers were shown in green (MolA) and pink
(MolB), respectively. 7 were displayed as sphere models. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article.)
Fig. 6. Modes of binding of FK866 and 7 with NAmPRTase. Both inhibitors were shown
in pink (FK866) and blue (7). Red text indicates the residue in Mol B. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version
of this article.)

H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
1159
supplied without further purification. Proton nuclear magnetic
resonance spectroscopy was performed on a JEOL JNM-LA 300 WB
and 400 WB spectrometer, and spectra were taken in CDCl3 or
DMSO-d6. Unless otherwise noted, chemical shifts are expressed as
ppm downfield from tetramethylsilane as the internal standard,
and J values are given in Hz. Data is reported as follows: chemical
shift, multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; b,
broad; app., apparent), coupling constants, and integration. Mass
spectroscopy was carried out on MALDI-TOF or FAB (fast atom
bombardment) instruments. [FAB source: JEOL FAB source and ion
gun (Cs ion beam, 30 kV acceleration)]. High-resolution mass
spectra (m/z) were recorded on a FAB (JEOL: mass range 2600 amu,
10 kV acceleration) at Korea Basic Science Institute (Daegu).
Fig. 7. Ionic interaction between His191(A), Asp210(A), and both inhibitors. Colors of
inhibitors were same as Fig. 5. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
Table 3
Data collection and refinement statistics.
Data collection statistics
Dataset
4.1.1. Methyl 4-(1-benzoylpiperidin-4-yl)butanote (3)
NAmPRTase-7 Complex
Thionyl chloride (6.7 L, 91.6 mmol) was added to 4-piperidine
butyric acid hydrochloride (2) (9.5 g, 45.8 mmol) in methanol
(30 mL) at room temperature and the reaction mixture was stirred
for 2 h. After removal of the reaction solvent, the residue was
partitioned between chloroform (3 ꢂ 400 mL) and sat NaHCO3 (aq)
(400 mL). The combined organic layers were dried over MgSO4,
concentrated and purified by silica gel column chromatography
(chloroform:methanol ¼ 8:1) to give ester (7.5 g, 89%). To a solution
of esterified product (7.5 g, 40.5 mmol) in DCM was added pyridine
(6.5 L, 81.0 mmol) and benzoyl chloride (9.4 L, 81.0 mmol). After the
mixture was stirred for 3 h at room temperature, it was partitioned
between DCM (2 ꢂ 350 mL) and NH4Cl (aq) (400 mL). The
combined organic layers were dried over MgSO4 and concentrated.
The crude residue was purified by silica gel column chromatog-
raphy (hexane:ethylacetate ¼ 3:1) to give 3 (7.9 g). Yield: 68%; 1H
X-ray source
Wavelength (Å)
Space group
PAL-4A
1.0000
P21
a ¼ 60.3, b ¼ 106.5, c ¼ 83.1,
50e3.0 (3.05e3.0)
83,924
20,888
99.9(99.9)
Unit-cell dimensions (Å, ꢀ
Resolution range (Å)
Observed reflections
Unique reflections
Completeness (%)
)
b
¼ 96.5
a
Rmerge (%)
17.2 (39.5)
6.1 (3.3)
I/
s
(I)
Refinement statistics
Resolution range
50e3.0
22.8
25.9
b
Rwork (%)
Rfree (%)
r.m.s.d. from ideal geometry
r.m.s.d. bond length (Å)
r.m.s.d. bond angle (ꢀ)
Ramachandran statistics
Most favored (%)
0.012
1.7
NMR (300 MHz, CDCl3)
d (ppm) 7.35 (s, 5H), 4.71 (m, 1H), 3.68 (s,
3H), 3.63 (m, 1H), 2.98 (m, 2H), 2.34 (t, J ¼ 7.5 Hz, 2H), 1.78e1.15 (m,
97.2
2.8
0
9H); ESI [M þ H] ¼ 290.5.
Additional (%)
Generously allowed (%)
4.1.2. (4-(4-aminobutyl)piperidin-1-yl)(phenyl)methanone (4)
3 (7.8 g, 27.0 mmol) was added to LAH (1.0 g, 27.0 mmol) in THF
(30 mL) and the resulting solution was stirred for 2 h at room
temperature. The reaction mixture was partitioned between chlo-
roform (2 ꢂ 400 mL) and NH4Cl (aq) (500 mL). The combined
organic layers were dried over MgSO4, concentrated and purified by
silica gel column chromatography (hexane:ethylacetate ¼ 1:1) to
PAL-4A, Pohang Light Source 4A HFMX beamline; r.m.s.d., root-mean-square devi-
ation. Values in parentheses refer to the highest resolution shells.
P
P
P P
a
Rmerge
¼
i｜I(h)i ꢃ <I(h)>｜/
iI(h)i, where I(h) is the intensity of
h
h
reflection h, Sh is the sum over all reflections, and Si is the sum over i measurements
of reflection h.
P
P
b
Rwork
Rfree calculation.
¼
hkljjFoj ꢃ jFcjj/ hkljFoj; 10% of the reflections were excluded for the
give alcohol (4.6 g). Yield: 65%; 1H NMR (300 MHz, CDCl3)
d (ppm)
3. Conclusion
7.35 (s, 5H), 4.07 (t, J ¼ 6.9 Hz, 2H), 3.50 (s, 2H), 2.90 (d, J ¼ 10.5 Hz,
2H),1.65e1.23 (m,11H); ESI [M þ H] ¼ 262.3. To a solution of alcohol
(4.6 g,17.6 mmol) in DCM (100 mL) methanesulfonyl chloride (3.0 g,
26.4 mmol) and TEA (4.9 L, 35.2 mmol) were added and the resulting
solution was stirred for 2 h at room temperature. The reaction
mixture was partitioned between chloroform (2 ꢂ 400 mL) and
NH4Cl (aq) (500 mL). The combined organic layers were dried over
MgSO4 and were concentrated. Resulted residue was purified by
silica gel column chromatography (hexane:ethylacetate ¼ 1:1) to
give methanesulfonyl protected compound (4.1 g, 68%). Next,
potassium phthalimide (2.6 g, 14.2 mmol) was added to the
obtained compound (4.0 g, 11.8 mmol) in DMF (80 mL). After the
reaction mixture was stirred for 8 h at 50 ꢀC, it was partitioned
between ethyl acetate (2 ꢂ 400 mL) and water (400 mL). The
combined organic layers were dried over MgSO4 and were
concentrated. Reaction compound was purified by silica gel column
chromatography (hexane:ethylacetate ¼ 3:2) to give intermediate
product (4.0 g, 87%). Hydrazine hydrate (1.0 g, 20.4 mmol) was
added to the previous product (4.0 g,10.2 mmol) in ethanol (100 mL)
and the resulting solution was stirred for 1 day at room temperature
then it was filtered. The filtrate was evaporated and purified by silica
gel column chromatography (chloroform:methanol ¼ 20:1) to give
We have described the design and synthesis of FK866 analogs
developed by modifications of the pyridine ring with various het-
eroaromatic rings. Among the newly synthesized compounds,
compound 7 showed the most potent anti-cancer activity. The
pyrrole group of 7 might be a substitution group instead of the
pyridine ring to potentially achieve better metabolic stability in
vivo, to solve the rapid metabolism of FK866 to FK866-N-oxide.
Analysis of the X-ray co-crystal structures of NAmPRTase-FK866
complex and NAmPRTase-7 complex showed that Asp219 in the
active site of the enzyme could contribute to an additional ionic
interaction with the pyrrole nitrogen of compound 7. The mode of
binding interactions might be applied to the strategy related to
developing an anti-cancer agent based on the FK866 structure.
Further investigations including binding affinity, solubility, and
pharmacokinetic characteristics of FK866 and 7 are in progress.
4. Experimental protocols
4.1. Chemistry
Starting materials, reagents, and solvents were purchased from
Aldrich Chemical Co. (Milwaukee, WI) and TCI (Tokyo) and used as
4 (1.4 g). Yield: 52%; 1H NMR (300 MHz, CDCl3)
d (ppm) 7.39 (s, 5H),

1160
H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
4.71 (m, 1H), 3.75 (m, 1H), 2.96 (m, 1H), 2.73 (m, 3H), 1.81e1.13 (m,
11H), NH peak was not shown; ESI [M þ H] ¼ 261.5.
proton of imidazole was not shown.; 13C NMR (400 MHz, CDCl3)
(ppm) 193.73, 189.11, 151.64, 150.89 (3C), 142.64 (2C), 141.32 (2C),
139.12 (2C), 128.78, 40.82, 34.03, 31.88 (2C), 30.10, 25.61 (2C), 17.94,
10.54; ESI [M ꢃ H] ¼ 379.2; HRMS (FAB) (C22H29N4O2): calcd
381.2291, found 381.2288.
d
4.1.3. General procedure for the synthesis of FK866 analogs (5e12)
EDC (2.0 equiv), HOBt (1.5 equiv), and TEA (1.5 equiv) were add
to various acrylic acids (1.0 equiv) in DCM (3 mL) or DMF. Then, 4
(1.2 equiv) was add to the resulting solution and the reaction
mixture was stirred for 3e5 h at room temperature. The reaction
mixture was added to NaHCO3 (aq) (100 mL) and extracted with
ethyl acetate (2 ꢂ 100 mL). The combined organic layers were dried
over MgSO4 and were concentrated. Compounds 5e12 were
obtained by silica gel column chromatography.
4.1.3.6. (E)-N-(4-(1-benzoylpiperidin-3-yl)butyl)-4-(1H-indol-3-yl)
but-2-enamide (10). Compound 10 (417.0 mg) was obtained from
trans-3-indole acrylic acid (200.0 mg, 0.53 mmol). Yield: 90%; 1H
NMR (300 MHz, CDCl3)
d (ppm) 8.58 (s, NH of indole, 1H), 7.91
(s, 1H), 7.89 (d, J ¼ 15.6 Hz, 1H), 7.37 (s, 5H), 7.23 (m, 4H), 6.43
(d, J ¼ 15.6, 1H), 5.78 (s, NH, 1H), 4.71 (m, 1H), 3.78 (m, 1H), 3.48
(q, J ¼ 6.6 Hz, 2H), 2.98 (m, 2H), 1.45e1.11 (m, 11H); 13C
4.1.3.1. (E)-N-(4-(1-benzoylpiperidin-3-yl)butyl)-3-(pyrimidin-5-yl)
acrylamide (5). Compound 5 (60.0 mg) was obtained from 23
(30.0 mg, 0.18 mmol). Yield: 84%; 1H NMR (300 MHz, CDCl3)
NMR (400 MHz, CDCl3) d (ppm) 193.63, 189.71, 152.10, 151.25,
148.94, 142.50, 141.25 (2C), 141.00, 139.24 (2C), 137.40, 134.56,
132.11, 131.12, 125.86, 122.68, 120.49, 40.86, 31.95, 30.23 (2C),
25.81 (2C), 20.64, 18.30, 10.72; ESI [M ꢃ H] ¼ 428.0; HRMS (FAB)
(C27H32N3O2): calcd 430.2495, found 430.2491.
d
(ppm) 9.18 (s, 1H), 8.85 (s, 2H), 7.65 (d, J ¼ 15.5 Hz, 1H), 7.39 (s,
5H), 6.55 (d, J ¼ 15.6 Hz, 1H), 5.75 (m, 1H), 4.75(s, 1H), 3.78 (s, 1H),
3.41 (q, J ¼ 6.9 Hz, 2H), 2.96 (m, 2H), 1.41e1.08 (m, 11H); 13C NMR
(400 MHz, CDCl3)
d
(ppm) 193.62, 186.19, 179.18, 174.92 (2C), 151.18,
4.1.3.7. (E)-N-(4-(1-benzoylpeperidin-4-yl)butyl)-3-(2-nitrophenyl)
acrylamide (11). Compound 11 (92.3 mg) was obtained from 2-
nitrocinnamic acid (50.0 mg, 0.25 mmol). Yield: 85%; 1H NMR
147.83, 142.57, 141.76, 141.26 (2C), 139.21 (2C), 136.60, 40.81, 33.86,
31.96 (2C), 30.49, 25.83 (2C), 17.99, 10.68; ESI [M ꢃ H] ¼ 391.2;
HRMS (FAB) (C23H29N4O2): calcd 393.2291, found 393.2287.
(300 MHz, CDCl3)
d
(ppm) 8.03 (d, J ¼ 7.8 Hz, 1H), 7.97 (d,
J ¼ 15.6 Hz, 1H), 7.64 (m, 2H), 7.53 (t, J ¼ 6.6 Hz, 1H), 7.39 (s, 5H),
6.34 (d, J ¼ 15.6 Hz, 1H), 5.86 (s, NH, 1H), 4.73 (m, 1H), 3.75 (m, 1H),
3.42 (q, J ¼ 6.9 Hz, 2H), 2.98 (m, 2H), 1.60e1.22 (m, 11H); 13C NMR
4.1.3.2. (E)-3-(2-aminopyrimidin-5-yl)-N-(4-(1-benzoylpiperidine-4-
yl)butyl)acrylamide (6). Compound 6 (14.5 mg) wasobtained from 24
(29.5 mg, 0.18 mmol). Yield: 20%; 1H NMR (300 MHz, CDCl3)
d
(ppm)
(400 MHz, CDCl3) d (ppm) 193.59, 186.88, 166.08, 151.22, 150.73,
8.45 (s, 2H) 7.48 (d, J ¼ 15.9 Hz, 1H), 7.39 (s, 5H), 6.30 (d, J ¼ 15.6 Hz,
1H), 5.58 (m,1H), 5.24 (s, NH2, 2H), 4.70 (m,1H), 3.74 (m,1H), 3.41 (q,
J ¼ 6.9 Hz, 2H), 2.96 (m, 2H), 1.41e1.08 (m, 11H); 13C NMR (400 MHz,
147.43, 142.91, 142.50, 141.13, 141.24 (2C), 139.22 (2C), 138.55,
136.81, 131.78, 40.76, 33.85, 31.95 (2C), 30.41, 25.80 (2C), 17.99,
10.68; ESI [M þ H] ¼ 436.0; HRMS (FAB) (C25H30N3O4): calcd
436.2236, found 436.2232.
CDCl3)
d (ppm) 193.59, 192.71, 184.41, 177.23 (2C), 151.22, 149.17,
142.51,141.24 (2C),139.23 (2C),134.82,129.11, 40.82, 33.87, 31.94 (2C),
29.97, 25.81 (2C), 17.94, 10.53; ESI [M ꢃ H] ¼ 406.2; HRMS (FAB)
(C23H30N5O2): calcd 408.2400, found 408.2398.
4.1.3.8. (E)-N-(4-(1-benzoylpeperidin-4-yl)butyl)-3-(3-nitrophenyl)
acrylamide (12). Compound 12 (94.5 mg) was obtained from was
obtained from 3-nitrocinnamic acid (50.0 mg, 0.25 mmol). Yield:
4.1.3.3. (E)-N-(4-(1-benzoylpeperidin-4-yl)butyl)-3-(1H-pyrrol-2-yl)
acrylamide (7). Compound 7 (23.0 mg) was obtained from 28
(22.0 mg, 0.16 mmol). Yield: 38%; 1H NMR (300 MHz, CDCl3)
87%; 1H NMR (300 MHz, CDCl3)
d (ppm) 8.37 (s, 1H), 8.21 (d,
J ¼ 8.4 Hz, 1H), 7.77 (d, J ¼ 7.8 Hz, 1H), 7.69 (d, J ¼ 15.6 Hz, 1H), 7.58
(t, J ¼ 8.1 Hz, 1H), 7.39 (s, 5H), 6.55 (d, J ¼ 15.3 Hz, 1H), 5.92 (s, NH,
1H), 4.70 (m, 1H), 3.72 (m, 1H), 3.43 (q, J ¼ 6.6 Hz, 2H), 2.97 (m, 2H),
d
(ppm) 8.83 (s, NH of pyrrole, 1H), 7.52 (d, J ¼ 15.0 Hz, 1H), 7.39 (s,
5H), 6.86 (s, 1H), 6.51 (s, 1H), 6.25 (s, 1H), 6.03 (d, J ¼ 15.6 Hz, 1H),
5.56 (s, NH, 1H), 4.71 (m, 1H), 3.76 (m, 1H), 3.39 (q, J ¼ 6.3 Hz, 2H),
2.96 (m, 2H), 1.56e1.04 (m, 11H); 13C NMR (400 MHz, CDCl3)
1.60e1.24 (m, 11H); 13C NMR (400 MHz, CDCl3)
d (ppm) 193.60,
188.82, 166.49, 153.54, 151.57, 151.18, 148.33, 143.12, 142.53, 141.25
(2C), 139.20 (2C), 135.62, 135.42, 132.61, 40.83, 33.83, 31.95 (2C),
30.42, 25.75 (2C), 17.99, 10.67; ESI [M þ H] ¼ 436.0; HRMS (FAB)
(C25H30N3O4): calcd 436.2236, found 436.2232.
d
(ppm) 193.64, 189.14, 151.15, 144.35, 142.57, 141.61 (2C), 141.27,
139.22 (2C), 132.48, 123.71, 120.87, 118.84, 40.85, 33.88, 31.91 (2C),
30.19, 25.78 (2C), 18.14, 10.62; ESI [M þ H] ¼ 380.1; HRMS (FAB)
(C23H30N3O2): calcd 380.2338, found 380.2336.
4.1.4. General method for N-alkylation of FK-866 analogs (14e17)
To a solution of compounds 7e10 (1.0 equiv) were added
sodium hydride (3.0 equiv) in DMF. Then 13 (1.5 equiv) was added
to the resulting solution. After the reaction mixture was stirred for
10e15 h at room temperature, it was partitioned between ethyl
acetate (2 ꢂ 40 mL) and NaHCO3 (aq) (40 mL). The combined
organic layers were dried over MgSO4 and were concentrated. The
residues were added 1 N HCl was added in the residues dissolved
THF. The solution was stirred for 2e3 h at room temperature and
was partitioned between ethyl acetate (2 ꢂ 30 mL) and NaHCO3
(aq) (40 mL). The combined organic layers were dried over MgSO4
and were concentrated. 14e17 were obtained by silica gel column
chromatography.
4.1.3.4. (E)-N-(4-(1-benzoylpiperidin-3-yl)butyl)-3-(1H-pyrazol-4-yl)
acrylamide (8). Compound 8 (22.5 mg) was obtained from 31
(28.0 mg, 0.20 mmol). Yield: 28%; 1H NMR (300 MHz, CDCl3)
d (ppm)
7.74 (s, 2H), 7.57 (d, J ¼ 13.5 Hz, 1H), 7.41 (s, 5H), 6.20 (d, J ¼ 15.3 Hz,
1H), 5.65 (s, NH, 1H), 4.68 (m, 1H), 3.72 (m, 1H), 3.40 (q, J ¼ 7.2 Hz,
2H), 2.96 (m, 2H), 1.55e1.20 (m, 11H), NH proton of pyrazole was not
shown.; 13C NMR (400 MHz, CDCl3)
d (ppm) 193.64, 188.42, 151.19
(2C), 147.45, 144.70, 142.54, 141.26 (2C), 139.22 (2C), 129.53, 128.38,
40.83, 34.07, 31.93 (2C), 30.19, 25.80 (2C), 18.13, 10.65; ESI
[M þ H] ¼ 381.0; HRMS (FAB) (C22H29N4O2): calcd 381.2291, found
381.2293.
4.1.3.5. (E)-N-(4-(1-benzoylpeperidin-4-yl)butyl)-3-(1H-imidazol-4-
yl)acrylamide (9). Compound 9 (85.0 mg) was obtained from uro-
canic acid (100.0 mg, 0.72 mmol). Yield: 31%; 1H NMR (300 MHz,
4.1.4.1. (E)-N-(4-(1-benzoylpeperidin-4-yl)butyl)-3-(1-(4-hydroxy-3-
(hydroxymethyl)butyl)-1H-pyrrol-2-yl)acrylamide (14). Compound
14 (3.2 mg) was obtained from 7 (10.8 mg, 0.028 mmol). Yield: 23%;
CDCl3)
d
(ppm) 7.59 (s, 1H), 7.54 (d, J ¼ 15.3 Hz, 1H), 7.39 (s, 5H), 7.15
1H NMR (300 MHz, CDCl3)
d
(ppm) 7.59 (d, J ¼ 15.3 Hz, 1H), 7.39 (s,
(s, 1H), 6.54 (d, J ¼ 14.7 Hz, 1H), 5.75 (s, NH, 1H), 4.69 (s, 1H), 3.78
5H), 6.78 (m, 1H), 6.59 (m, 1H), 6.17 (m, 1H), 6.16 (d, J ¼ 15.6 Hz, 1H),
(m,1H), 3.39 (q, J ¼ 6.6 Hz, 2H), 2.97 (m, 2H),1.62e1.03 (m,11H), NH
5.75 (m, NH, 1H), 4.70 (m, 1H), 4.11 (m, 2H), 3.85 (m, 2H), 3.75 (m,

H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
1161
2H), 3.39 (q, J ¼ 6.0 Hz, 2H), 2.97 (m, 3H), 1.84 (m, 3H), 1.57e1.15 (m,
11H); 13C NMR (400 MHz, CDCl3)
(ppm) 194.65, 185.97, 151.86,
4.1.6. (E)-3-(2-aminophenyl)-N-(4-(1-benzoylpiperidin-4-yl)butyl)
acrylamide (19)
d
145.72, 143.01, 141.25 (2C), 140.44, 139.28 (2C), 133.97, 131.98,
131.10, 123.10, 61.69 (2C), 43.54, 40.48, 38.25, 34.68, 28.63(2C),
26.75, 25.11 (2C), 17.89, 16.70, 10.88; ESI [M þ H] ¼ 482.0; HRMS
(FAB) (C28H40N3O4): calcd 482.3019, found 482.3017.
Tin chloride (155.4 mg, 0.69 mmol) was added to 11 (30.0 mg,
0.07 mmol) in ethanol (3 mL). Reaction mixture was stirred for 1.2 h
at 70 ꢀC and evaporated for removing ethanol. Then, reaction
mixture was partitioned between ethyl acetate (2 ꢂ 20 mL) and
NaHCO3 (aq) (20 mL). The combined organic layers were dried over
MgSO4, concentrated and purified by silica gel column chroma-
tography (chloroform:methanol ¼ 40:1) to give 19 (18.3 mg). Yield:
4.1.4.2. (E)-N-(4-(1-benzoylpeperidin-4-yl)butyl)-3-(1-(4-hydroxy-3-
(hydroxymethyl)butyl)-1H-pyrazol-4-yl)acrylamide (15). Compound
15 (5.0 mg) was obtained from 8 (20.0 mg, 0.05 mmol). Yield: 20%;
62%; 1H NMR (400 MHz, CDCl3)
d
(ppm) 7.77 (d, J ¼ 15.2 Hz, 1H),
1H NMR (300 MHz, CDCl3)
d
(ppm) 7.65 (s, 1H), 7.54 (s, 1H), 7.49 (d,
7.38 (s, 5H), 7.33 (d, J ¼ 8.0 Hz, 1H), 7.15 (t, J ¼ 8.0 Hz, 1H), 6.75 (t,
J ¼ 8.0 Hz, 1H), 6.69 (d, J ¼ 8.0 Hz, 1H), 6.29 (d, J ¼ 14.8 Hz, 1H), 5.66
(s, NH, 1H), 4.69 (m, 1H), 3.94 (s, NH2, 2H), 3.71 (m, 1H), 3.39 (q,
J ¼ 6.8 Hz, 2H), 2.94 (m, 2H), 1.58e1.24 (m, 11H); 13C NMR
J ¼ 15.9 Hz, 1H), 7.39 (s, 5H), 6.15 (d, J ¼ 15.6 Hz, 1H), 5.71 (s, NH,
1H), 4.70 (m, 1H), 4.24 (t, J ¼ 6.9 Hz, 2H), 3.77 (m, 2H), 3.70 (m, 2H),
3.39 (q, J ¼ 6.9 Hz, 2H), 2.97 (m, 3H), 1.99 (m, 2H), 1.79 (m, 1H),
1.57e1.12 (m, 11H); 13C NMR (400 MHz, CDCl3)
d
(ppm) 193.61,
(400 MHz, CDCl3) d (ppm) 193.60, 188.32, 162.41 (2C), 151.46,
188.41, 153.29, 151.22, 149.34, 147.78, 144.73, 142.52, 141.26 (2C),
139.24 (2C), 128.79, 61.59 (2C), 43.58, 40.81, 31.96, 30.24 (2C), 25.81
(2C), 18.13 (2C), 16.27 (2C), 10.63; ESI [M þ H] ¼ 483.2; HRMS (FAB)
(C27H39N4O4): calcd 483.2971, found 483.2968.
144.06, 142.50, 141.24 (2C) 140.33, 139.25 (2C), 131.78, 131.13,
129.19, 126.43, 40.85, 34.08, 31.96 (2C), 30.31, 25.84 (2C), 18.15,
10.71; ESI [M þ H] ¼ 406.1; HRMS (FAB) (C25H32N3O2): calcd
406.2495, found 406.2497.
4.1.4.3. (E)-N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(1-(4-hydroxy-
3-(hydroxymethyl)butyl)-1H-imidazol-4-yl)acrylamide (16). Com-
pound 16 (5.0 mg) was obtained from 9 (20.0 mg, 0.05 mmol).
4.1.7. (E)-3-(3-aminophenyl)-N-(4-(1-benzoylpiperidin-4-yl)butyl)
acrylamide (20)
Tin chloride (129.5 mg, 0.57 mmol) was added to 12 (25.0 mg,
0.06 mmol) in ethanol (3 mL). Reaction mixture was stirred for 1.2h
at 70 ꢀC and evaporated for removing ethanol. Then, reaction
mixture was partitioned between ethyl acetate (2 ꢂ 20 mL) and
NaHCO3 (aq) (20 mL). The combined organic layers were dried over
MgSO4, concentrated and purified by silica gel column chroma-
tography (chloroform:methanol ¼ 40:1) to give 20 (11.0 mg). Yield:
Yield: 31%; 1H NMR (300 MHz, CDCl3)
d (ppm) 7.43 (s, 2H), 7.39 (s,
5H), 7.04 (s, 1H), 6.56 (d, J ¼ 15.3 Hz, 1H), 6.11 (s, NH, 1H), 4.69 (m,
1H), 4.04 (m, 2H), 3.78 (m, 5H), 3.38 (q, J ¼ 6.3 Hz, 2H), 3.01 (m, 2H),
2.04 (m, 1H), 1.92 (m, 2H), 1.60e1.11 (m, 11H); 13C NMR (400 MHz,
CDCl3) d (ppm) 193.62, 185.29, 153.31, 151.25, 149.15, 145.71, 142.50,
141.25 (2C), 139.25 (2C), 131.79, 129.19, 62.10 (2C), 37.46, 32.64,
30.09, 29.82 (2C), 25.78 (2C), 25.71, 20.30, 18.01, 17.64, 10.58; ESI
[M þ H] ¼ 483.4; HRMS (FAB) (C27H39N4O4): calcd 483.2971, found
483.2970.
46%; 1H NMR (300 MHz, CDCl3)
d
(ppm) 7.55 (d, J ¼ 15.6 Hz, 1H),
7.39 (s, 5H), 7.17 (t, J ¼ 7.8 Hz, 1H), 6.92 (d, J ¼ 7.5 Hz, 1H), 6.80 (t,
J ¼ 2.1 Hz, 1H), 6.69 (d, J ¼ 7.8 Hz, 1H), 6.34 (d, J ¼ 15.6 Hz, 1H), 5.61
(s, NH, 1H), 4.72 (m, 1H), 3.73 (m, NH2 and piperidine proton, 3H),
3.41 (q, J ¼ 6.9 Hz, 2H), 2.95 (m, 2H), 1.56e1.24 (m, 11H); 13C NMR
4.1.4.4. (E)-N-(4-(1-benzoylpeperidin-4-yl)butyl)-3-(1-(4-hydroxy-3-
(hydroxymethyl)butyl)-1H-indol-3-yl)acrylamide (17). Compound 17
(28.0 mg) was obtained from 10 (96.0 mg,1.86 mmol). Yield: 12%; 1H
(400 MHz, CDCl3)
d (ppm) 193.60, 188.15, 164.12, 157.30, 151.27,
150.54, 142.87, 142.50, 141.25 (2C), 139.25 (2C), 131.25, 128.35,
126.33, 123.48, 44.39, 33.61, 31.98 (2C), 30.28, 25.83 (2C), 18.15,
10.70; ESI [M þ H] ¼ 406.3; HRMS (FAB) (C25H32N3O2): calcd
406.2495, found 406.2494.
NMR (300 MHz, CDCl3)
d
(ppm) 7.89 (d, J ¼ 7.8 Hz, 1H), 7.83 (d,
J ¼ 15.6 Hz, 1H), 7.37 (s, 5H), 7.35 (m, 2H), 7.24 (m, 2H), 6.42 (d,
J ¼ 15.3 Hz, 1H), 5.83 (s, NH, 1H), 4.63 (m, 1H), 4.20 (m, 2H), 3.77 (m,
5H), 3.41 (q, J ¼ 6.6 Hz, 2H), 2.94 (m, 2H), 1.95 (m, 2H), 1.81 (m, 1H),
1.57e1.26 (m, 11H); 13C NMR (400 MHz, CDCl3)
d
(ppm) 193.63,
4.1.8. Procedure for the synthesis of pyrimidine acrylic acid building
blocks
190.22,152.19,151.08,148.55,145.20,142.54,141.24 (2C),139.18 (2C),
138.39, 134.06, 131.81, 131.34, 124.93, 120.84, 118.38, 61.53 (2C),
40.82, 36.39, 34.03, 31.88 (3C), 30.18, 25.69 (2C), 18.13, 15.98, 10.64;
ESI [M þ H] ¼ 532.0; HRMS (FAB) (C32H42N3O4): calcd 532.3725,
found 532.3177.
Methyl acrylate (22) (22.0 mmol), TEA (11.0 mmol), and palla-
dium (II) acetate (1.1 mmol) were added to a solution of 5-bromo-
pyrimidine (21) (5.5 mmol) in acetonitrile (10 mL). After stirring
for 20 min under microwave reactor at 170 ꢀC, the reaction mixture
was filtered and worked up by adding ethyl acetate (2 ꢂ 400 mL)
and NaHCO3(aq) (400 mL). The combined organic layers were
dried over MgSO4 and were concentrated. Resulted compound
(27%) was obtained by silica gel column chromatography
(hexane:ethylacetate ¼ 1:1). Then this product was stirred in 5%
sodium hydroxide in 20% water in methanol (5 mL) for 2 h at room
temperature. The reaction mixture was quenched by careful
addition of 1 N HCl to neutralize. After remove the solvent, the
remaining residues are dissolved with chloroform:methanol ¼ 5:1
and filtered. The filtrate was evaporated and purified by silica gel
column chromatography to give 23e24.
4.1.5. (E)-N-(4-(1-benzoylpeperidin-4-yl)butyl)-3-(1-methyl-1H-
pyrrol-2-yl)acrylamide (18)
To a solution of 7 (8.0 mg, 0.02 mmol) was added sodium
hydride (1.4 mg, 0.06 mmol) and methyl iodide (2.5 ml, 0.04 mmol)
in DMF. The reaction mixture was stirred for 9 h at 55 ꢀC and it was
partitioned between ethyl acetate (2 ꢂ 20 mL) and NH4Cl (aq)
(20 mL). The combined organic layers were dried over MgSO4 and
were concentrated. The residue was obtained by silica gel column
chromatography (chloroform:methanol ¼ 80:1) to give 18 (2.3 mg).
Yield: 29%; 1H NMR (300 MHz, CDCl3)
d
(ppm) 7.59 (d, J ¼ 15.0 Hz,
1H), 7.39 (s, 5H), 6.71 (m, 1H), 6.58 (m, 1H), 6.16 (m, 1H), 6.12 (d,
J ¼ 15.0 Hz, 1H), 5.47 (s, NH, 1H), 4.71 (m, 1H), 3.70 (s, 3H), 3.62 (m,
1H), 3.40 (q, J ¼ 6.6 Hz, 2H), 2.97 (m, 2H), 1.44e1.10 (m, 11H); 13C
4.1.8.1. (E)-3-(pyrimidin-5-yl)acrylic acid (23). Compound 23
(59.0 mg) was obtained from 21a (356.5 mg, 2.24 mmol). Yield: 18%;
NMR (400 MHz, CDCl3)
d
(ppm) 193.60, 186.40, 151.27, 144.03,
1H NMR (300 MHz, CDCl3)
d (ppm) 9.17 (s, 1H), 8.89 (s, 2H), 7.65 (d,
142.49, 141.92, 141.25 (2C), 139.25 (2C), 138.15, 128.72, 127.31,
116.91, 40.84, 33.24, 30.23 (2C), 29.03, 26.50, 25.84 (2C), 18.23,
10.70; ESI [M þ H] ¼ 394.1; HRMS (FAB) (C24H32N3O2): calcd
394.2495, found 394.2491.
J ¼ 16.4 Hz, 1H), 6.62 (d, J ¼ 16.2 Hz, 1H); ESI [M ꢃ H] ¼ 148.9.
4.1.8.2. (E)-3-(2-aminopyrimidin-5-yl)acrylic acid (24). Compound
24 (29.5 mg) was obtained from 21b (180.0 mg, 1.03 mmol). Yield:

1162
H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
17%; 1H NMR (300 MHz, CDCl3)
d
(ppm) 8.52 (s, 2H), 7.51 (d,
NaHCO3 (aq) (40 mL). The combined organic layers were dried over
J ¼ 16.5 Hz, 1H), 6.38 (d, J ¼ 16.5 Hz, 1H), 5.54 (s, NH2, 2H); ESI
MgSO4, concentrated and purified by silica gel chromatography
(hexane:ethylacetate ¼ 5:1, 38.2 mg, 38%). The purified compound
(38.0 mg, 0.25 mmol) was stirred with 5% sodium hydroxide in
methanol (2 mL) for 4 h at room temperature. The reaction mixture
was neutralized by 1 N HCl. After removal of neutralized water, the
residue was dissolved with chloroform:methanol 5:1 and stirred
for a while. The reaction mixture was evaporated and purified by
silica gel column chromatography (hexane:ethylacetate ¼ 4:1) to
give 31 (28.0 mg). Yield: 78%; 1H NMR (300 MHz, DMSO-d6)
[M ꢃ H] ¼ 164.0.
4.1.9. tert-butyl 1H-pyrrole-1-carboxylate (26)
Boc2O (9.8 g, 44.7 mmol) and TEA (6.3 mL, 44.7 mmol) was
added to pyrrole (25) (3.0 g, 29.8 mmol) in DCM (15 mL) and the
resulting mixture was stirred for 2h at room temperature and then
worked up with DCM (2 ꢂ 400 mL) and water (400 mL). The
combined organic layers were dried over MgSO4 and were
concentrated to obtain 26 as white solid (7.5 g). Yield: 94%; 1H NMR
d
(ppm) 8.66 (s, NH, 1H), 8.03 (s, 2H), 7.52 (d, J ¼ 15.9 Hz, 1H), 6.24
(300 MHz, CDCl3)
J ¼ 2.4 Hz, 1H), 1.60 (d, J ¼ 11.7 Hz, 9H); ESI [M þ H] ¼ 169.0.
d
(ppm) 7.26 (s, 2H), 7.24 (t, J ¼ 2.4 Hz,1H), 6.22 (t,
(d, J ¼ 15.9 Hz, 1H); ESI [M ꢃ H] ¼ 137.9.
4.1.14. 2-(Hydroxymethyl) butane-1,4-diol (33)
4.1.10. (E)-tert-butyl 2-(3-methoxy-3-oxoprop-1-enyl)-1H-pyrrole-
1-carboxylate (27)
To a reflux solution of triethyl 1,1,2-ethane tricarboxylate (32)
(5.0 g, 20.3 mmol) and sodium borohydride (2.3 g, 60.9 mmol) in
tert-butyl alcohol (40 mL), methanol (2.5 mL) was added in the
reaction mixture in three aliquots over 0.5 h. The resulting solution
was heated under reflux condition for additional 0.5 h and allowed
to cool down to room temperature. 1 N HCl was added with care
until the solution was neutral. The solution was filtered and filtrate
was extracted with ethanol. The residue was purified by silica gel
column chromatography (chloroform:methanol ¼ 7:1) to give 33
Palladium (II) acetate (1.0 g, 4.5 mmol) was added to a solution of
methyl acrylate (22) (2.0 mL, 22.4 mmol) and 26 (7.5 g, 44.8 mmol) in
acetic acid, dioxane, and DMSO (3:9:1, 30 mL). The resulting mixture
was stirred at 35 ꢀC for 96 h in a flask left open to the atmosphere. The
reaction mixture was diluted with diethyl ether (100 mL) and water
(20 mL). The reaction mixture was filtered through a plug of celite.
The combined organic layers were dried over MgSO4, concentrated
and purified by silica gel column chromatography (hexane:ethyl-
acetate ¼ 30:1) to give 27 (2.5 g). Yield: 23%; 1H NMR (300 MHz,
(2.1 g). Yield: 88%; 1H NMR (300 MHz, CDCl3)
d (ppm) 3.80 (m, 6H),
1.93 (q, J ¼ 5.4 Hz, 1H), 1.71 (m, 2H); ESI [M þ H] ¼ 121.3.
CDCl3)
d
(ppm) 8.33 (d, J ¼ 16.2 Hz,1H), 7.40 (q, J ¼ 1.5 Hz,1H), 6.71 (t,
J ¼ 1.8 Hz,1H), 6.23 (d, J ¼ 15.9 Hz, 1H), 6.22 (t, J ¼ 3.3 Hz,1H), 3.78 (s,
4.1.15. 2-(2.2-dimethyl-1,3-dioxan-5-yl) ethanol (34)
3H), 1.63 (s, 9H); ESI [M ꢃ H] ¼ 250.0.
2,2-Dimethoxy propane (2.5 mL, 20 mmol) and p-toluene
sulfonic acid monohydrate (180.0 mg, 0.9 mmol) were added to
a solution of 33 (2.1 g, 17.9 mmol) in THF (10 mL). The resulting
solution was stirred for 4 h at room temperature. The reaction
mixture was neutralized by addition of TEA. The solvent was
removed and the residue was purified by silica gel column chro-
matography (hexane:ethylacetate ¼ 4:1) to give 34 (1.7 g). Yield:
4.1.11. (E)-3-(1H-pyrrol-2-yl) acrylic acid (28)
After 27 (100.0 mg, 0.4 mmol) was stirred with TFA in DCM (3 mL)
for1hatroomtemperature, thereactionmixturewasworked upwith
DCM (2 ꢂ 40 mL) and NaHCO3 (aq) (40 mL). The combined organic
layers were dried over MgSO4 and were concentrated. Resulted
residue was purified by silica gel chromatography (chloroform:-
methanol ¼ 50:1). Next, the obtained compound (45.3 mg, 75%) was
stirred with 5% NaOH inmethanol (2 mL) for 4 h at room temperature.
The reaction mixture was neutralized by 1 N HCl. After evaporation
neutralized water, the residue was dissolved with chloroform:-
methanol ¼ 5:1 and purified silica gel column chromatography
(chloroform:methanol ¼ 30:1) to give 28(25 mg). Yield:91%; 1H NMR
59%; 1H NMR (300 MHz, CDCl3)
d (ppm) 3.97 (m, 2H), 3.75 (m, 4H),
1.95 (m, 1H), 1.43 (s, 6H); ESI [M ꢃ H] ¼ 158.0
4.1.16. 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl 4-methylbenzene-
sulfonate (13)
p-Toluenesulfonyl chloride (2.4 g, 12.7 mmol) and pyridine
(4.3 mL, 53.0 mmol) were added to a solution of 34 (1.7 g, 10.6 mmol)
in DCM (10 mL). After the reaction mixture was stirred for 5 h at
room temperature, it was partitioned between ethyl acetate
(2 ꢂ 150 mL), water (50 mL) and 1 N HCl (25 mL). The combined
organic layers were dried over MgSO4, concentrated and purified by
silica gel column chromatography (hexane:ethylacetate ¼ 7:1) to
(300 MHz, CDCl3)
d
(ppm) 10.29 (s, NH, 1H), 6.15 (d, J ¼ 15.9 Hz, 1H),
5.76 (s, 1H), 5.26 (s, 1H), 4.97 (d, J ¼ 16.2 Hz, 1H), 4.94 (m, 1H); ESI
[M þ H] ¼ 138.0.
4.1.12. 4-Bromp-1-tosyl-1H-pyrazole (30)
TEA (380.0
m
l, 2.72 mmol) was added to 4-bromo pyrazole (29)
give 13 (1.9 g). Yield: 58%; 1H NMR (300 MHz, CDCl3)
d (ppm) 7.80 (d,
(200.0 mg, 1.36 mmol) in DCM. Then, p-toluene sulfonyl chloride
(260.0 mg, 1.36 mmol) was added to the reaction mixture. After the
reaction mixture was stirred for 3 h at room temperature, it was
partitioned between ethyl acetate (2 ꢂ 40 mL) and NaHCO3 (aq)
(40 mL). The combined organic layers were dried over MgSO4 and
were concentrated. It was purified by silica gel column chroma-
tography (hexane:ethylacetate ¼ 25:1) to give 30 (290.0 mg). Yield:
J ¼ 6.9 Hz, 2H), 7.37 (d, J ¼ 8.1 Hz, 2H), 4.02 (m, 2H), 3.83 (m, 3H), 3.52
(q, J ¼ 5.1 Hz,1H), 2.62 (m,1H), 2.46 (s, 3H), 2.04 (m, 2H),1.57 (s, 6H);
ESI [M ꢃ H] ¼ 311.0.
4.2. Cell viability assay
4.2.1. Cell culture
73%; 1H NMR (300 MHz, CDCl3)
d
(ppm) 8.11 (s, 1H), 7.91 (d,
MCF-7 cells were grown as suspension cultures in RPMI 1640.
The adherent cell line HepG2 cells were cultured in DMEM
medium. Both media were supplemented with 10% FBS (Invitrogen,
Co.), and antibiotic-antimycotic (Invitrogen, Co.). All cell lines were
incubated in a humidified atmosphere containing 5% CO2 at 37 ꢀC.
The adherent cells were detached from the culture flasks by
removal of the growth medium and addition of 3 mL trypsin/EDTA
solution (0.05% w/v trypsin, 0.016% w/v EDTA). After w5e10 min
incubation at 37 ꢀC, when the cells had detached from the surface,
trypsinization was stopped by the addition of 3 mL of DMEM
medium containing 10% FBS.
J ¼ 8.4 Hz, 2H), 7.65 (s, 1H), 7.37 (d, J ¼ 7.8 Hz, 2H), 2.44 (s, 3H); ESI
[M þ H] ¼ 302.8.
4.1.13. (E)-3-(1H-pyrazol-4-yl)acrylic acid (31)
To a solution of 30 (200.0 mg, 0.66 mmol) in DMF was added
methyl acrylate (22) (297.4
ml, 3.30 mmol), TEA (184.0 ml,
1.32 mmol), palladium (II) acetate (29.6 mg, 0.13 mmol), LiCl
(28.0 mg, 0.66 mmol), and tri-ortho-tolyl phosphine (200.9 mg,
0.66 mmol). After the reaction mixture was stirred for overnight at
140 ꢀC it was partitioned between ethyl acetate (2 ꢂ 40 mL) and

H. You et al. / European Journal of Medicinal Chemistry 46 (2011) 1153e1164
1163
4.2.2. SRB assay
centrifugation at 14,000 g for 1 h, 200
ml of sample was filtered
The cells were plated at a density of 50,000 cells per mL and well
in 96-well plates and incubated with inhibitors for the time indi-
cated. The SRB assay was carried out as described by Skehan et al.
[22]. The drug incubation period of the cells was stopped by the
addition of 50 ml of ice cold TCA solution into the growth medium.
After 1 h incubation in the refrigerator, the supernatant was dis-
using Millex-GV (0.22 mm, Millipore) and injected onto the column
equilibrated in 50 mM phosphate buffer (pH 7.0) and initially eluted
with 6 mL of this buffer. Further elution was performed with
a 10 mL linear gradient to 20% (v/v) methanol and then 8 mL linear
gradient to 95% methanol in the same buffer. A flow rate of 1 mL/
min was performed. The products from NAmPRTase reaction were
monitored by absorbance at 261 nm. NMN elutes 4.1 mL after
injection, PRPP at 8.9 mL, and NAm at 13.4 mL (Fig. 3). To observe
inhibitory activities, each previously synthesized inhibitor was
added to reaction buffer in addition to using the same method for
detection of the reaction products. Percent inhibition by each drug
candidate was measured by a relative decrease of the amount of
NMN, based on the peak area compared with a standard as the peak
area when no inhibitor was added to reaction buffer.
carded, the dishes were rinsed five times with deionized water,
dried at room temperature (RT). 100 ml of SRB solution was pipetted
into each well and incubated at room temperature for 30 min. Then,
the staining solution was decanted, the dishes were washed four
time with 1% (v/v) acetic acid and dried again at RT. SRB stain
unspecifically bound to protein was released by adding 1 mL of
10 mM. Tris buffer per well and gentle shaking for 20 min. Finally
the light extinction at 515 nm wavelength was read in an ELISA-
reader. The mean values of four duplicated samples were calcu-
lated. These results were expressed as a percentage, relative to
solvent treated control incubations, and IC50 values were calculated
using nonlinear regression analysis (percent survival versus
concentration).
4.5. Crystallization
Crystallization trials were performed in a 24-well plate using
the hanging-drop vapor diffusion method. NAmPRTase (10 mg/mL)
was incubated with 0.4 mM compound 7 (protein/inhibitor molar
ratio of 1:2) at 4 ꢀC for 30 min before crystallization setup. Crystals
of human NAmPRTase in complex with 7 were grown at 21 ꢀC in
4.3. Overexpression and purification of human NAmPRTase
The human NAmPRTase gene was amplified by PCR using
a GeneAmp PCR system 2400 thermocycler (Perkin Elmer). PCR was
run in ThermoPol buffer (New England Biolabs) using NQ DNA
polymerase (Anygen, Korea). The forward and reverse primers (50-
TAC CTA GGA TCC ATG AAT CCT GCG GCA GAA-30 and 50-TGA CTC
GAG CTA ATG ATG TGC TGC TTC CAG-30) were designed from the
NAmPRTase nucleotide sequence in GenBank, accession no.
NM005746. The amplified gene and the modified pET28a expres-
sion vector were digested using BamHI and XhoI, and then the
NAmPRTase gene was ligated into expression vector and used to
transform Escherichia coli strain BL21 (DE3). Transformed BL21
(DE3) cells were grown overnight at 37 ꢀC in 50 mL Luria broth
2
ml of the drop containing equal volumes of protein/inhibitor
solution and reservoir solution comprised of 100 mM TriseHCl (pH
9.0), 19% (w/v) polyethylene glycol 3350, and 175 mM MgCl2. The
crystals were cryo-protected by transferring to the reservoir solu-
tion supplemented with 15% (v/v) ethylene glycol and flash-frozen
in liquid nitrogen for data collection.
4.6. Structure determination
A complete data set up to 3.0 Å resolution was collected at 100 K
at BL-4A of the Pohang Accelerator Laboratory, Korea. Data set was
indexed and processed using the HKL-2000 package [23]. We found
crystals of NAmPRTase in complex with 7 belonged to the P21 space
group and have the unit-cell dimensions a ¼ 60.3, b ¼ 106.5,
containing 50
resuspended in 2
OD600 of 0.6. His-tagged recombinant proteins were then induced
using 0.5 mM isopropyl- -thiogalactoside (Pharmacia). After
m
g/mL kanamycin (Duchefa Biochemie). Cells were
m
l of the same media, and grown at 37 ꢀC to an
c ¼ 83.1 Å,
b
¼ 96.5ꢀ. Assuming one dimeric NAmPRTase molecule is
b-D
contained in an asymmetric unit, the Matthews coefficient was
calculated to be 2.41 Å3/Da; the estimated solvent content was thus
48.9% [24]. Initial molecular replacement calculations were carried
out with MOLREP [25] using search models based on the structure
of human NAmPRTase as free-form (PDB code 2E5B). Refinement of
this structure was performed with REFMAC and CNS [26]; final
crystallographic R value was 22.8% (Rfree ¼ 25.9%). All molecular
graphics were created using PyMol [27]. The statistics for data
collection and refinement were summarized in Table 3.
inducing for 20 h at 15 ꢀC, cells were harvested and resuspended in
lysis buffer [50 mM phosphate buffer pH 8.0, 300 mM NaCl, 5 mM
imidazole, and 10% (v/v) glycerol]. The lysate was then produced
with an ultrasonic processor (Sonics) and cleared by centrifugation
at 14,000 g for 1 h. His-tagged NAmPRTase recombinant proteins
were purified with Ni-NTA chelating agarose resin (Peptron, Korea)
by washing with lysis buffer and eluting with 50 mM phosphate
buffer (pH 8.0), 300 mM NaCl, 150 mM imidazole, 2 mM dithio-
threitol (DTT), and 10% glycerol. After which eluted NAmPRTase
proteins were further loaded onto a Superdex S200 16/60 column
(GE Healthcare) equilibrated in 20 mM Hepes-NaOH (pH 7.5),
150 mM NaCl, 2 mM DTT, and 10% glycerol. Fractions containing
human NAmPRTase was pooled and concentrated to 15 mg/mL.
Acknowledgement
We thank Drs. Heung-Soo Lee and Yeon-Gil Kim for their kind
support with X-ray data collection at BL-4A of Pohang Accelerator
Laboratory (Pohang, Korea). This study was supported by a grant of
the Korea Healthcare technology R&D Project, Ministry for Health,
4.4. HPLC assay
Welfare
& Family Affairs, Republic of Korea (A062442 and
HPLC was performed with LC-6AD pumps and an SPD-M10A
diode array detector (Shimadzu) with a Shim-pack VP-ODS column
A092006) and by a grant from the Institute of Medical System
Engineering (iMSE) in the GIST, Republic of Korea.
(250 mm ꢂ 4.6 mm i.d., and 4.6
m
m particle size, Shimadzu). The
NAmPRTase reaction was conducted at 37 ꢀC for 15 min in 200
ml of
reaction buffer [50 mM Hepes-NaOH (pH 7.5), 10 mM MgCl2, 5 mM
NAm, and 5 mM phosphoribosyl pyrophosphate (PRPP)] with
50 mM of the purified NAmPRTase protein. The reaction was
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