Synthesis of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazines that have antibacterial activity and also inhibit inorganic pyrophosphatase

Article abstract of DOI:10.1016/j.bmc.2013.11.011

Inorganic pyrophosphatases are potential targets for the development of novel antibacterial agents. A pyrophosphatase-coupled high-throughput screening assay intended to detect o-succinyl benzoic acid coenzyme A (OSB CoA) synthetase inhibitors led to the unexpected discovery of a new series of novel inorganic pyrophosphatase inhibitors. Lead optimization studies resulted in a series of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazine derivatives that were prepared by an efficient synthetic pathway. One of the tetracyclic triazine analogues 22h displayed promising antibiotic activity against a wide variety of drug-resistant Staphylococcus aureus strains, as well as activity versus Mycobacterium tuberculosis and Bacillus anthracis, at a concentration that was not cytotoxic to mammalian cells.

Full text of DOI:10.1016/j.bmc.2013.11.011

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazines
that have antibacterial activity and also inhibit inorganic
pyrophosphatase
Wei Lv a, Biplab Banerjee a, Katrina L. Molland b, Mohamed N. Seleem c, Adil Ghafoor c, Maha I. Hamed c,
Baojie Wan d, Scott G. Franzblau d, Andrew D. Mesecar b, Mark Cushman a,
⇑
a Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and The Purdue University Center for Cancer Research, Purdue University, West Lafayette,
IN 47907, United States
b Department of Biological Sciences and The Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, United States
c Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, United States
d Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Inorganic pyrophosphatases are potential targets for the development of novel antibacterial agents. A
pyrophosphatase-coupled high-throughput screening assay intended to detect o-succinyl benzoic acid
coenzyme A (OSB CoA) synthetase inhibitors led to the unexpected discovery of a new series of novel
inorganic pyrophosphatase inhibitors. Lead optimization studies resulted in a series of 3-(3-aryl-pyrroli-
din-1-yl)-5-aryl-1,2,4-triazine derivatives that were prepared by an efficient synthetic pathway. One of
the tetracyclic triazine analogues 22h displayed promising antibiotic activity against a wide variety of
drug-resistant Staphylococcus aureus strains, as well as activity versus Mycobacterium tuberculosis and
Bacillus anthracis, at a concentration that was not cytotoxic to mammalian cells.
Received 10 September 2013
Revised 23 October 2013
Accepted 5 November 2013
Available online xxxx
Keywords:
Inorganic pyrophosphatase
Inhibitors
Antibacterial agents
3-(3-Aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-
triazine
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
two phosphorus atoms and also making the attached phosphate
into a better leaving group through coordination with the pyro-
Inorganic pyrophosphatases (PPases) are essential metal-
dependent enzymes that catalyzes the hydrolysis of inorganic
pyrophosphate (PPi) to phosphate.1 They have been categorized
into three structurally and mechanistically distinct families of sol-
uble PPases in addition to membrane-integral PPases that couple
pyrophosphate hydrolysis to H+ and/or Na+ transport.2 The energy
released from pyrophosphate hydrolysis can be used to drive ener-
getically unfavorable reactions to completion.3 PPases play
essential roles in many biological processes, including lipid metab-
olism, calcium absorption, bone formation, and DNA synthesis.4–7
PPases are widely expressed in most microorganisms and they
have therefore been considered to be possible targets for the dis-
covery of antimicrobial agents.8 In general, catalysis involves coor-
dination of the pyrophosphate by three or four metal ions and does
not proceed through a covalent enzyme–phosphate intermedi-
ate.9–14 The enzyme and associated metal ions increase the nucle-
ophilicity of the substrate water molecule by facilitating
deprotonation, while increasing the electrophilicity of one of the
phosphate oxygens. The crystal structures of a wide variety of inor-
ganic PPases are available in the Protein Data Bank, including the
recently determined structures of two sodium-pumping mem-
brane-integral pyrophosphatases.13,15
A number of small inorganic substances have been reported to
inhibit inorganic pyrophosphatase, including fluoride,16–23 cal-
cium,24–27 arsenate,28 phosphonate,29 phosphate,30 and imidodi-
phosphates.31 Substituted methane derivatives can be added to
the list, including diphosphonates21,32–39 and tetranitromethane.40
Covalent inhibitors include N,N0-dicyclohexylcarbodiimide,41
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,42
fluorescein
50-isothiocyanate,43 and N-ethylmaleimide.42,44 Finally, various
amines inhibit inorganic PPase, including adenine nucleotides,45
nucleotidyl sulfates,46 amino acid esters,47 acylspermidine deriva-
tives,48 and phosphoethanolamine.49 In general, antimicrobial
activity has not been reported in the publications that list PPase
inhibitory activity for these substances. One exception is the re-
ported inhibition of streptococcal growth by several diphospho-
nates at 2–5 millimolar concentration levels, but in that case
inhibition of bacterial growth was ascribed to magnesium chela-
tion instead of PPase inhibition.37
⇑
Corresponding author. Tel.: +1 765 494 1465; fax: +1 765 494 6790.
E-mail address: cushman@purdue.edu (M. Cushman).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.011
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2
W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Our efforts to identify o-succinyl benzoic acid coenzyme A
with the triazine sulfone 5 in dry DMF, the tetracyclic compounds
12a–i were obtained in good yield. Hydrogenation of compounds
12b–e afforded the reduction products 12j–m.
(OSB-CoA) synthetase inhibitors by high-throughput screening
using a PPase-coupled assay50 led unexpectedly to the discovery
that compound 12a (Scheme 2) is a weak inhibitor of M. tuberculo-
sis PPase (45% inhibition at 100
lM, IC50 148
l
M). This result is
2.1.3. Synthesis of the adenine analogue 17
noteworthy because in contrast to many of the reported inorganic
pyrophosphatase inhibitors, 12a is not a substrate analogue and it
is also not ionic at physiological pH. Compound 12a was subse-
quently found to inhibit the growth of M. tuberculosis with a MIC
Intermediate 15 was prepared by the procedure reported by
Kocalka et al. (Scheme 3).64 The N-Boc group of 15 was removed
smoothly in 20% TFA in dichloromethane. Treatment of the
crude product 16 with the triazine sulfone 5 in the presence of
triethylamine provided the desired product 17 in high yield
(Scheme 3).
of approximately 60 lM. These results suggested that it might be
possible to design and synthesize a series of 12a analogues to opti-
mize the PPase inhibitory activity and the antimicrobial activity. A
number of 1,2,4-triazine-6-ones,51 1,3,5-triazines,52–56 and annu-
lated 1,2,4- and 1,2,3-triazines57 have previously been reported
to possess antimicrobial activity.
Although compound 12a was discovered to be a PPase inhibitor
due to its presence in a high-throughput screening library, it has
not been reported in the chemical literature. In order to provide
enough material for biological testing and confirm the structure,
a synthesis of compound 12a was devised from simple starting
materials. The route is quite versatile and has been used to make
3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazine analogues. The
PPase inhibitory activities and antibacterial activities of the result-
ing compounds against M. tuberculosis, S. aureus, and Bacillus spe-
cies were determined. These efforts were eventually rewarded
through the synthesis of compound 22h, which was active against
a wide range of drug-resistant S. aureus strains.
2.1.4. Synthesis of analogues 22a–h and 29a–d
Oxidation of the acetophenones 18a–f with selenium dioxide
proceeded smoothly to provide a mixture of glyoxals 19a–f and
their covalent monohydrate forms (Scheme 4). Without purifica-
tion, the crude products 19a–f were condensed with S-methylthio-
semicarbazide hydrogen iodide (3) to regioselectively afford the
triazinyl methyl sulfides 20a–f in good yields. Then, m-CPBA was
added to convert 20a–f to the corresponding sulfones 21a–f. The
oxidization proceeded smoothly, but the product triazine sulfones
21a–f were poorly soluble in most organic solvents (including
chloroform, dichloromethane, ethyl acetate and acetone) and
therefore could not be purified by column chromatography. The
crude products were therefore used for the next step. The coupling
reactions of 21a–f with pyrrolidine 11a were carried out in dry
DMF to afford 22a–f in good yields (Scheme 4). The nitro com-
pounds 22e–f were converted to the amines 22g–h by catalytic
hydrogenation.
Compounds 29a–c were synthesized starting from the boronic
acids 23a–c. The Suzuki coupling with the bromide 24 furnished
the bicyclic products 25a–c in high yield. The same procedures
used to make 22a–f were also employed to synthesize 29a–c
(Scheme 5).
Compound 29d was synthesized through a similar route start-
ing from 4-bromoacetophenone (30) and 3-nitrophenylboronic
acid (23a) as described in Scheme 6.
2. Results and discussion
2.1. Synthesis
2.1.1. Synthesis of the triazinyl sulfone 5
The general procedure reported by Hajbi et al.58 was used to
prepare the triazinyl sulfone 5 (Scheme 1). The oxidation of 2-acet-
yl-5-methylfuran (1) by selenium dioxide proceeded selectively on
the ketone methyl group to afford a mixture of furyl glyoxal 2 and
its covalent monohydrate. Without purification, the furyl glyoxal 2
was condensed with S-methylthiosemicarbazide hydrogen iodide
(3)59 to regioselectively provide the triazinyl methyl sulfide
4.60,61 The oxidation of 4 with meta-chloroperoxybenzoic acid
(m-CPBA) in dichloromethane proceeded selectively on the sulfur
atom to afford the triazinyl sulfone 5.
2.2. MtPPase inhibitory activity and antimicrobial activity
The inhibitory activities of the triazine analogues 12a–c,
12f–m, 17, 22a–h, and 29a–d were determined against inorganic
pyrophosphatase from M. tuberculosis (MtPPase). The percentage
inhibition of MtPPase catalytic activity in the presence of a
100 lM concentration of the triazine analogues was initially mea-
2.1.2. Synthesis of the analogues 12a–m
sured. Only those compounds with more than 35% inhibition
were subjected to IC50 determination (Table 1). The results indi-
cate that replacing the 3-chloro substituent on the phenyl ring
of 12a with a hydroxy group (12k–m) abolished the MtPPase
inhibitory activity, while other substituents including 3-OBn
(12c), 3-F (12g), 3-CF3 (12h), 3-NH2 (12j) were tolerated better
in this position. Replacing the furan ring with a substituted phe-
nyl ring usually afforded more potent inhibitors (22a–h) with IC50
The succinic acids 9a–e were synthesized based on the proce-
dure reported by Breukelman et al.62 (Scheme 2). Starting from
the corresponding benzaldehydes 6a–e,63 the Knoevenagel con-
densation with diethyl malonate was performed to afford the dies-
ters 7a–e. Michael addition with KCN in hot ethanol, followed by
hydrolysis and decarboxylation gave the nitrile esters 8a–e. Hydro-
lysis of 8a–e under acidic or basic conditions produced the succinic
acids 9a–e. The succinimides 10a–e were synthesized by treating
9a–e with urea in refluxing xylene. Further reduction of 10a–i with
borane in THF provided the pyrrolidines 11a–i (10f–i were com-
mercially available). When the pyrrolidines 11a–i were reacted
values ranging from 33
22c had the best inhibitory activity, with an IC50 of 33
l
M to 118
l
M. The 4-bromo compound
M. Two
l
of the compounds with an additional aromatic ring attached to
H2N
NH
N
HI
SCH3
N
O
O
N
N
HN
c
O
O
O
O
a
3
O
N
SO2CH3
N
SCH3
H
b
1
2
4
5
Scheme 1. Reagents and conditions: (a) SeO2, dioxanes, H2O; (b) NaHCO3, EtOH, H2O; (c) m-CPBA, CH2Cl2.
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W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
R
R
R
R
O
b
a
d
R
c
O
CO2Et
COOH
HOOC
NH
NC
OEt
CHO
CO2Et
O
9a-e
10a-e
8a-e
6a-e
7a-e
a. R = 3-Cl
a. R = 3-Cl
a. R = 3-Cl
a. R = 3-Cl
a. R = 3-Cl
b. R = 3-NO2
b. R = 3-NO2
b. R = 3-NO2
c. R = 3-OBn
d. R = 3,4-OBn
b. R = 3-NO2
c. R = 3-OBn
b. R = 3-NO2
c. R = 3-OBn
d. R = 3,4-OBn
c. R = 3-OBn
c. R = 3-OBn
d. R = 3,4-OBn
e. R = 3-OBn, 4-OMe
d. R = 3,4-OBn
e. R = 3-OBn, 4-OMe
d. R = 3,4-OBn
e. R = 3-OBn, 4-OMe
e. R = 3-OBn, 4-OMe
e. R = 3-OBn, 4-OMe
R
R
N
N
N
O
N
O
O
N
5
R
SO2Me
e
N
N
NH
NH
O
f
11a-i
10a-i
12a-i
a. R = 3-Cl
a. R = 3-Cl
b. R = 3-NO2
c. R = 3-OBn
d. R = 3,4-OBn
e. R = 3-OBn, 4-OMe
f. R = H
a. R = 3-Cl
g
g
g
g
b. R = 3-NO2
c. R = 3-OBn
d. R = 3,4-OBn
e. R = 3-OBn, 4-OMe
f. R = H
j. R = 3-NH2
b. R = 3-NO2
c. R = 3-OBn
d. R = 3,4-OBn
e. R = 3-OBn, 4-OMe
f. R = H
k. R = 3-OH
l. R = 3,4-OH
m. R = 3-OH, 4-OMe
g. R = 3-F
g. R = 3-F
g. R = 3-F
h. R = 3-CF3
i. R = 3-CN
h. R = 3-CF3
i. R = 3-CN
h. R = 3-CF3
i. R = 3-CN
Scheme 2. Reagents and conditions: (a) diethyl malonate, piperidine, toluene; (b) KCN, EtOH, H2O; (c) HCl, H2O or KOH, EtOH; (d) urea, xylene; (e) BH3, THF; (f) dry DMF; (g)
H2, Pd/C, MeOH, AcOEt.
NH2
N
NH2
N
N
N
OMs
OH
N
c
a
b
N
TFA
Boc
N
Boc
N
N
N
Boc
N
HN
13
14
15
16
N
O
N
N
N
5
O
N
SO2Me
N
N
N
N
NH2
N
d
N
17
Scheme 3. Reagents and conditions: (a) MsCl, pyridine, DCM; (b) adenine, Cs2CO3, DMSO; (c) TFA, DCM; (d) NEt3, DMF.
the furan ring (29a and 29c) had poor solubility in DMSO and
therefore could not be tested. On the other hand, the trifluoro-
methyl compound 29b had good inhibitory activity against MtP-
50
lM). Five compounds (12k, 22a, 22b, 22g and 22h) showed
moderate antimicrobial activities against Bacillus anthracis and
Bacillus subtilis, with MIC values ranging from 16 to 128 lM
Pase with an IC50 of 38
l
M, while the nitrated biphenyl
(Table 2).
compound 29d was inactive.
The compound 22h with the most potent antibacterial activity
was then tested against a wide variety of antibiotic-resistant clin-
ical isolates (MRSA, VISA, and VRSA) and the results are listed in
All of the prepared triazine analogues were evaluated for
their antimicrobial activities against the M. tuberculosis strain
H
37Rv (replicating phenotype, tested by the MABA method)65,66
Table 3. Activity was observed at the MIC 16 lM potency level
and M. tuberculosis strain H37Rv-CV-lux AB (nonreplicating per-
sistent phenotype, tested by the LORA method).67,68 Most of the
compounds with PPase inhibitory activities also exhibit good to
moderate antibacterial activities against M. tuberculosis (MIC
against all of the drug-resistant strains except for the ciprofloxa-
cin-resistant, oxacillin-resistant, and linezolid-resistant NRS37
strain, against which activity was detected at MIC 8 lM.
Several of the inorganic pyrophosphatase inhibitors were as-
sayed against the HeLa human tumor cell line and the murine mac-
rophage J774.A1 cell line in order to determine the potential toxic
effect in mammalian cells. MTS cell viability assays conducted on
ranging from 5.7 to 78
lM). Compound 22h (MIC 5.7 lM) was
the most potent compound when tested in the MABA assay,
while 12j (MIC 47 lM) had the highest potency in the LORA
assay.
compounds 22g, 22a, 22h, 12k, and 22b at 64 lM concentration
The antibacterial activities against methicillin-resistant
S. aureus (MRSA) and Bacillus species were also tested. The 3-amino
compounds 22g and 22h inhibited the growth of MRSA with MIC
showed no significant decrease in percentage mean absorption rel-
ative to the DMSO control (Fig. 1), indicating that the compounds
are not toxic in vitro at this concentration, which is four times that
of the MIC displayed by 22h in most of the drug-resistant S. aureus
strains.
values of 30
lM and 16 lM, respectively (Table 2), while the
remaining compounds were all inactive (no obvious inhibition at
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4
W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
N
N
O
O
N
c
b
CHO
a
SCH3
R
R
R
18a-f
20a-f
19a-f
a, R = H
a, R = H
a, R = H
b, R = 3,4-OMe
c, R = 4-Br
b, R = 3,4-OMe
c, R = 4-Br
b, R = 3,4-OMe
c, R = 4-Br
d, R = 3,4-methylenedioxy
e, R = 3-NO2
d, R = 3,4-methylenedioxy
e, R = 3-NO2
d, R = 3,4-methylenedioxy
e, R = 3-NO2
f, R = 4-NO2
f, R = 4-NO2
f, R = 4-NO2
N
N
Cl
N
N
N
HN
N
SO2Me
Cl
11a
R
N
R
d
21a-f
22a-h
a, R = H
a, R = H
b, R = 3,4-OMe
c, R = 4-Br
b, R = 3,4-OMe
c, R = 4-Br
d, R = 3,4-methylenedioxy
e, R = 3-NO2
d, R = 3,4-methylenedioxy
e
e, R = 3-NO2
e
g R = 3-NH2
h R = 4-NH2
f, R = 4-NO2
f, R = 4-NO2
Scheme 4. Reagents and conditions: (a) SeO2, dioxanes, H2O; (b) 3, NaHCO3, EtOH, H2O; (c) m-CPBA, CH2Cl2; (d) dry DMF, (e) H2, Pd/C, MeOH, AcOEt.
R
R
O
B(OH)2
Br
a (23a-b)
b (23c)
O
O
d
O
c
O
O
R
O
H
23a-c
24
25a-c
26a-c
a, R = 3-NO2
b, R = 2-CF3
a, R = 3-NO2
b, R = 2-CF3
c, R = 3-CO2Me
a, R = 3-NO2
b, R = 2-CF3
c, R = 3-CO2Me
c, R = 3-CO2Me
HN
Cl
R
R
N
N
Cl
e
N
N
R
N
N
O
O
N
O
11a
N
N
N
f
SMe
SO2Me
27a-c
a, R = 3-NO2
b, R = 2-CF3
28a-c
29a-c
a, R = 3-NO2
b, R = 2-CF3
c, R = 3-CO2Me
a, R = 3-NO2
b, R = 2-CF3
c, R = 3-CO2Me
c, R = 3-CO2Me
Scheme 5. Reagents and conditions: (a) Pd(OAc)2, K2CO3, (tBu)4N+ Brꢀ, H2O; (b) Pd(Ph3P)4, K3PO4, toluene; (c) SeO2, dioxanes, H2O; (d) 3, NaHCO3, EtOH, H2O; (e) m-CPBA,
CH2Cl2; (f) dry DMF.
inorganic pyrophosphatase is in fact responsible for the antibacte-
rial activity. On the other hand, the results of this study are signif-
icant because many of the compounds are structurally novel
inorganic pyrophosphatase inhibitors, and also because the series
presents a new antimicrobial scaffold for further development.
3. Conclusion
High-throughput screening in a pyrophosphatase-coupled en-
zyme assay intended to detect o-succinyl benzoic acid coenzyme
A (OSB CoA) synthetase inhibitors led to the unexpected discovery
of the novel inorganic PPase inhibitor 12a. The structure of 12a was
confirmed by an efficient synthesis that also allowed access to an
array of structural analogues. The inorganic PPase inhibitory
potencies of the series were determined and the antimicrobial
activities were optimized versus M. tuberculosis, S. aureus, and
Bacillus species. The most active compound 22h displayed promis-
ing antibiotic activity against a wide variety of drug-resistant S.
aureus strains at concentrations that were not cytotoxic in two dif-
ferent mammalian cell lines. In general, there is a lack of correla-
tion between the enzyme inhibitory activities and the
antibacterial activities versus M. tuberculosis (Table 1). For exam-
ple, at the extremes, based on its inactivity as an enzyme inhibitor,
the phenol 12k should not have antibiotic activity yet it does, and
the enzyme inhibitor 29b should have antibacterial activity and it
does not. These results bring into question whether inhibition of
4. Experimental section
4.1. Inorganic pyrophosphatase inhibition assay
All chemicals used were of the highest available purity and
were ordered from Sigma-Aldrich. The enzyme MtPPase was ex-
pressed and purified by a published protocol.69 Enzyme concentra-
tion was determined using absorbance at 280 nm and an
extinction co-efficient of 30,940 L Mꢀ1 cmꢀ1 70
MtPPase was stored
.
at ꢀ80 °C in buffer containing 20 mM Tris pH 8, 150 mM NaCl, and
10% v/v glycerol. For testing the inhibition of MtPPase by the syn-
thesized compounds, a malachite green assay was used. Malachite
green stock solution was prepared according to Baykov’s proce-
dure.71 The final assay composition consisted of 50 mM Tris–HCl
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W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
O
O
O
H
c
b
a
O2N
O2N
O
(HO)2B
NO2
Br
25d
26d
30
23a
N
N
N
N
N
N
HN
SO2Me
SMe
Cl
d
11a
O2N
O2N
e
28d
27d
Cl
N
N
N
N
O2N
29d
Scheme 6. Reagents and conditions: (a) Pd(OAc)2, K2CO3, (tBu)4N+ Brꢀ, H2O; (b) SeO2, dioxanes, H2O; (c) 3, NaHCO3, EtOH, H2O; (d) m-CPBA, CH2Cl2; (e) dry DMF.
Table 1
Table 2
The inhibitory activity against MtPPase and the minimum inhibitory concentration
The minimum inhibitory concentration (MIC) and minimum bacteriocidal concen-
trations (MBC) against methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus
species
(MIC) against Mycobacterium tuberculosis
Compound
%I (100
lM) IC50
(
lM)
MIC (lM)
MABAc
LORAd
Compound
MRSA MICa
M)
Bacillus anthracis MIC/
MBC ( M)
Bacillus subtilis MIC/
MBC ( M)
(
l
l
l
12a
12b
12c
12f
12g
12h
12i
12j
45.6 3.9
N.T.a
147.5 69
63.2 6.2
61.0 0.5
>16
58.8 1.8
>16
12k
22a
22b
22g
22h
>50
>50
>50
30
64/128
16/128
16/>128
32/128
16/128
<1/<1
128/128
16/>128
16/>128
32/>128
73.2 1.2
19.7 1.4
21.3 1.6
37.8 0.7
10.3 1.0
33.4 0.7
N.A.b
N.T.e
N.T.e
40.5 10.6 54.7 0.9
38.9 11.1 47.9 5.5
52.4 6.7
64.4 1.3
16
354.5 79
57.6 0.0
50.3 2.0
Vancomycin 0.5
<1/<1
70.1 11.2 47.1 3.1
a
The highest value of triplicate MIC determinations is reported.
12k
12l
60.3 1.3
>128
58.9 1.8
>128
N.A.b
12m
17
5.0 0.0
>128
>128
>128
>128
N.A.b
reaction volume of 100
made for each compound and a volume appropriate for the in-
tended final concentration was added to 60 L substrate solution
and mixed for 2 min by shaking. To initiate the reaction, an aliquot
of 20 L enzyme solution was added and the mixture was allowed
to react for 6 min. The reaction was stopped by the addition of
20 L malachite green development solution followed by 10 min
of shaking, and then absorbance of each well at 612 nm was mea-
sured on a Synergy 5 plate reader (Biotek). In the case of negative
control wells, no MtPPase was included in the enzyme solution. In
the case of positive control wells, an equivalent volume of DMSO
lacking inhibitor was added in the first step.
Kinetic data at each inhibitor concentration were collected in
triplicate, and the percent inhibition was calculated according to
the following formula:
%I = (1 ꢀ (Abssample ꢀ Abs(ꢀ)control)/(Abs(+)control ꢀ Abs(ꢀ)control))
⁄ 100 with error represented by the standard deviation of three
data points.73 IC50 values were calculated according to the formula:
%I = %max[I]/(IC50 + [I]) in the Enzyme Kinetics module of
SigmaPlot.
lL. A 10 mM stock solution in DMSO was
22a
22b
22c
22d
22e
22f
22g
22h
29a
29b
29c
29d
50.0 2.2
58.8 0.7
53.0 1.0
N.T.a
39.4 0.4
30.3 0.9
53.4 0.2
46.6 0.0
N.T.a
101.5 20
53.2 7.4
33.3 3.6
32.0 1.2
15.4 0.0
30.7 1.0
>16
53.6 2.1
127.4
77.7 11.5
>16
>128
>128
56.3 0.1
>128
>16
>128
>16
l
l
118.2 16.4 >128
31.2 1.2
57.4 7.1
29.7 0.6
5.7 2.2
>16
>128
>16
l
88.1 11.1
67.7 2.3
38.3 9.7
N.T.a
N.A.b
>128
>128
Rifampin
Isoniazid
Nitroimidazopyran
0.08 0.02 0.24 0.01
0.45 0.03 >256
0.44 0.02 6.83 0.79
a
The inhibitory activity could not be tested because of poor compound solubility
in DMSO (<100 M).
l
b
Not active. No detectable inhibition.
MABA = microplate Alamar Blue assay.
LORA = low oxygen recovery assay (luciferase readout).
Not submitted for testing.
c
d
e
pH 7.5, 1 mM DTT, 100 mM NaCl, 1 mM MgCl2, 0.2 mg/ml BSA,
0.13% Triton X-100, 5
50 nM MtPPase.
Enzyme kinetic assays were performed in clear, flat-bottomed
96-well plates (Fisherbrand Cat. No. 12566601) with a total
4.2. Chemistry
lM PPi, 13.3 l lM CoA, and
M OSB,72170.5
Melting points were determined using capillary tubes with a
Mel-Temp apparatus and are uncorrected. The nuclear magnetic
resonance (1H and 13C NMR) spectra were recorded using Bruker
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011

6
W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 3
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Compound 22h Against S. aureus MRSA, VISA and VRSA Clinical Isolates
Strain name
NARSAa
strain ID
Origin Phenotypic Properties
MICb/MBC
M)
(l
Methicillin-resistant Staphylococcus aureus
(MRSA) ATCC 4330
Vancomycin-intermediate Staphylococcus aureus
(VISA) ATCC 700699
Vancomycin-intermediate Staphylococcus aureus
(VISA) HIP07256
Vancomycin-intermediate Staphylococcus aureus
(VISA) LIM 3
Linezolid-resistant Staphylococcus aureus SA
LinR12
Methicillin-resistant Staphylococcus aureus
(MRSA) USA 100
Methicillin-resistant Staphylococcus aureus
(MRSA) USA 200
Methicillin-resistant Staphylococcus aureus
(MRSA) USA 300
—
USA
Resistant to methicillin and oxacillin
16/>256
16/>256
16/>256
16/>256
8/>256
NRS1
Japan
USA
Resistant to aminoglycosides and tetracycline (minocycline) glycopeptide-
intermediate
Resistant to oxacillin, tetracycline and ciprofloxacin glycopeptide-
intermediate
NRS19
NRS37
NRS119
NRS382
NRS383
NRS384
NRS385
VRS10
France Resistant to aminoglycosides, tetracycline, and ciprofloxacin glycopeptide-
intermediate
USA
USA
USA
USA
USA
USA
Resistant to ciprofloxacin, oxacillin and linezolid
Resistant to ciprofloxacin, clindamycin, erythromycin, and oxacillin
16/>256
16/>256
16/>256
Resistant to ciprofloxacin, clindamycin, erythromycin, gentamicin, and
oxacillin
Resistant to erythromycin, oxacillin, and tetracycline
Methicillin-resistant Staphylococcus aureus
(MRSA) USA 500
Vancomycin-resistant Staphylococcus aureus
(VRSA)
Resistant to ciprofloxacin, clindamycin, erythromycin, gentamicin, oxacillin, 16/>256
tetracycline, and trimethoprim
Resistant to oxacillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, 16/>256
and vancomycin
a
NARSA = Network on Antimicrobial Resistance in Staphylococcus aureus.
The highest value of triplicate MIC determinations is reported.
b
Figure 1. MTS assays showing the percent mean absorbance at 490 nm after incubating J774A.1 cells (A) and HeLa cells (B) with compounds 22g, 22a, 22h, 12k, 22b, and
DMSO at 64 M for 2 h. DMSO was used as a control.
l
ARX300 spectrometer (300 MHz) with a QNP probe or Bruker DX-2
spectrometer (500 MHz) with a BBO probe. IR spectra were ob-
tained using a Perkin–Elmer 1600 series FTIR spectrometer. High-
resolution mass spectra were recorded on a double-focusing sector
mass spectrometer with magnetic and electrostatic mass analyz-
ers. HPLC analyses were performed on a Waters 1525 binary HPLC
pump/Waters 2487 dual k absorbance detector system using a
residues were dissolved in ethanol (10 mL), and then aqueous solu-
tions of S-methylthiosemicarbazide hydrogen iodide (3, 582 mg,
2.50 mmol) and sodium bicarbonate (553 mg, 6.58 mmol) in water
(2 mL) were added. The solutions were heated at reflux for 2 h. After
cooling down, the solvents were evaporated and the residues were
dissolved in water (20 mL) and extracted with dichloromethane
(3 ꢁ 20 mL). The organic layers were combined and dried over
Na2SO4. Evaporation of the solvents afforded the desired products,
which were pure enough for the next reaction.
5 lM C18 reverse phase column. Purities of biologically important
compounds were P95%. For purities estimated by HPLC, the major
peak accounted for P95% of the combined total peak area when
monitored by a UV detector at 254 nm. Elemental analyses were
performed at Midwest Microlab LLC (Indianapolis, IN) and the
determined values are within 0.4% of the calculated values.
4.2.2. 5-(5-Methylfuran-2-yl)-3-(methylthio)-1,2,4-triazine (4)
Yellow solid (54%): mp 91–93 °C. IR (film) 1593, 1539,
1483 cmꢀ1 1H NMR (500 MHz, CDCl3) d 9.09 (s, 1H), 7.33 (d,
;
J = 3.5 Hz, 1H), 6.22 (d, J = 3.5 Hz, 1H), 2.62 (s, 3H), 2.41 (s, 3H);
13C NMR (125 MHz, CDCl3) d 173.0, 158.4, 147.0, 145.8, 140.0,
118.2, 110.0, 14.0, 13.7; CIMS m/z 208 (MH+). Anal. Calcd for C9H9-
N3OS: C, 52.16; H, 4.38; N, 20.27. Found: C, 52.09; H, 4.34; N, 20.11.
4.2.1. General procedure for the synthesis of triazinyl methyl
sulfides 4, 20a–f and 27a–d
Solutions of selenium dioxide (808 mg, 7.28 mmol) in dioxane
(8 mL) and water (0.8 mL) were stirred at 50–55 °C until the solids
completely dissolved, and then they were combined with solutions
of the ketone (1, 18a–f, 25a–d, 2.44 mmol) in dioxane (2 mL). The
mixtures were heated at reflux for 5 h. After cooling down, the black
solids were filtered off, and the solvents were evaporated. The resi-
dues were partially dissolved in ethyl acetate (10 mL), and the white
solids were filtered off. The filtrates were evaporated and the
4.2.3. 3-(Methylthio)-5-phenyl-1,2,4-triazine (20a)61
Yellow solid (82.6%): mp 92–94 °C (lit.61 97–99 °C). 1H NMR
(500 MHz, CDCl3) d 9.30 (s, 1H), 8.07 (dd, J = 1.5, 8.5 Hz, 2H),
7.52–7.46 (m, 3H), 2.65 (s, 3H); 13C NMR (125 MHz, CDCl3) d
173.5, 154.3, 141.8, 132.9, 132.5, 129.2, 127.5, 13.7.
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011

W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
4.2.4. 5-(3,4-Dimethoxyphenyl)-3-(methylthio)-1,2,4-triazine
(20b)74
145.7, 140.0, 130.9, 130.0, 129.4, 129.1, 128.6, 125.6, 118.7,
109.3, 52.3, 13.8; EIMS m/z 327 (M+); HREIMS m/z Calcd for C16H13-
N3O3S (M+) 327.0678, Found 327.0681.
Yellow solid (46.7%): mp 147–149 °C (lit.74 150–152 °C). 1H
NMR (300 MHz, CDCl3) d 9.29 (s, 1H), 7.73–7.68 (m, 2H), 6.97 (d,
J = 8.3 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 2.70 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 173.2, 153.9, 153.1, 149.7, 141.6, 125.4, 121.4,
110.1, 109.7, 56.1, 56.0, 13.9.
4.2.12. 3-(Methylthio)-5-(30-nitrobiphenyl-4-yl)-1,2,4-triazine
(27d)
Yellow solid (87.3%): mp 197–199 °C. IR (film) 1535, 1508,
1481, 1345, 1240, 745 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.44
;
4.2.5. 5-(4-Bromophenyl)-3-(methylthio)-1,2,4-triazine (20c)74
Yellow solid (67.2%): mp 149–151 °C (lit.74 153–154 °C). 1H
NMR (300 MHz, CDCl3) d 9.32 (s, 1H), 7.98 (m, 2H), 7.64 (m, 2H),
2.69 (s, 3H).
(s, 1H), 8.53 (t, J = 1.9 Hz, 1H), 8.32–8.26 (m, 3H), 7.99 (d,
J = 7.8 Hz, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 7.8 Hz, 1H),
2.76 (s, 3H); 13C NMR (125 MHz, CDCl3) d 173.8, 153.6, 148.7,
142.6, 141.7, 141.3, 133.1, 133.0, 130.0, 128.4, 128.0, 122.9,
122.0, 13.9; EIMS m/z 324 (M+); HREIMS m/z Calcd for C16H12N4O2S
(M+) 324.0681, Found 324.0679.
4.2.6. 5-(3,4-Methylenedioxylphenyl)-3-(methylthio)-1,2,4-
triazine (20d)
Brown solid (64.9%): mp 154–157 °C. IR (film) 2903, 1537, 1488,
4.2.13. 5-(5-Methylfuran-2-yl)-3-(methylsulfonyl)-1,2,4-triazine
(5)
1441, 1277, 1246, 1147, 1034 cmꢀ1 1H NMR (500 MHz, CDCl3) d
;
9.23 (s, 1H), 7.67 (dd, J = 1.7, 8.2 Hz, 1H), 7.64 (d, J = 1.7 Hz, 1H),
6.92 (d, J = 8.2 Hz, 1H), 6.07 (s, 2H), 2.68 (s, 3H); 13C NMR
(125 MHz, CDCl3) d 173.3, 153.7, 151.6, 148.8, 141.4, 127.0,
123.0, 108.8, 107.2, 102.0, 13.8; EIMS m/z 247 (M+); HREIMS m/z
Calcd for C11H9N3O2S (M+) 247.0416, Found 247.0418.
The triazinyl methyl sulfide 4 (208 mg, 1 mmol) was dissolved
by anhydrous dichloromethane (5 mL) and the solution was
cooled to 0 °C. Then 77% m-CPBA (450 mg, 2 mmol) was added
and the mixture was stirred under argon at room temperature
for 3.5 h. The solution was filtered, and solvent was evaporated.
The residue was purified by column chromatography using 40%
ethyl acetate–hexane to afford the product as yellow solid
(160 mg, 66%): mp 125–127 °C. IR (film) 1596, 1542, 1535,
4.2.7. 3-(Methylthio)-5-(3-nitrophenyl)-1,2,4-triazine (20e)
Yellow solid (60.0%): mp 133–135 °C. IR (film) 3090, 1526,
1495, 1348, 1242 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.46 (s, 1H),
;
1319, 1140 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.51 (s, 1H), 7.63
;
8.97 (s, 1H), 8.48 (d, J = 7.8 Hz, 1H), 8.41 (m, 1H), 7.77 (t,
J = 8.0 Hz, 1H), 2.72 (s, 3H); 13C NMR (75 MHz, CDCl3) d 174.2,
152.2, 148.9, 141.5, 135.0, 133.2, 130.6, 126.9, 122.5, 13.9; EIMS
m/z 248 (M+); HREIMS m/z Calcd for C10H8N4O2S (M+) 248.0368,
Found 248.0368.
(d, J = 3.6 Hz, 1H), 6.35 (dd, J = 3.6, 0.8 Hz, 1H), 3.45 (s, 3H), 2.47
(s, 3H); 13C NMR (75 MHz, CDCl3) d 166.3, 161.0, 147.9, 146.1,
145.7, 122.3, 111.5, 39.5, 14.3; EIMS m/z 239 (M+); HREIMS m/z
Calcd for C9H9N3O3S (M+) 239.0365, Found 239.0361. Anal. Calcd
for C9H9N3O3S: C, 45.18; H, 3.79; N, 17.56. Found: C, 45.33; H,
3.71; N, 17.40.
4.2.8. 3-(Methylthio)-5-(4-nitrophenyl)-1,2,4-triazine (20f)75
Yellow solid (67.0%): mp 179–181 °C (lit.75 222–223 °C). 1H
NMR (300 MHz, CDCl3) d 9.45 (s, 1H), 8.41 (d, J = 8.8 Hz, 2H), 8.34
(d, J = 8.8 Hz, 2H), 2.76 (s, 3H).
4.2.14. General procedure for the synthesis of methylsulfonyl
triazines 21a–f and 28a–d
The triazinyl methyl sulfides (20a–f, 27a–d, 0.247 mmol) were
dissolved in dichloromethane (2 mL) and the solutions were
cooled to 0 °C. m-CPBA (77%, 125 mg, 0.558 mmol) was added in
portions and the mixtures were stirred under argon at room tem-
perature for 3 h. The solvents were evaporated and the residues
were purified by silica gel column chromatography using 50%
ethyl acetate in hexane and then 15% ethyl acetate in dichloro-
methane. Evaporation of the solvents provided the crude prod-
ucts, which were used in the next reaction without further
purification.
4.2.9. 3-(Methylthio)-5-(5-(3-nitrophenyl)furan-2-yl)-1,2,4-
triazine (27a)
Yellow solid (74.3%): mp 190–192 °C. IR (film) 1593, 1521,
1491, 1349, 1269, 1248 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.32
;
(s, 1H), 8.58 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.11 (d, J = 7.5 Hz,
1H), 7.66 (t, J = 8.0 Hz, 1H), 7.58 (d, J = 3.7 Hz, 1H), 7.06 (d,
J = 3.7 Hz, 1H), 2.70 (s, 3H); 13C NMR (75 MHz, CDCl3) d 173.5,
155.5, 149.1, 148.7, 145.5, 140.0, 130.7, 130.2, 130.0, 123.5,
119.4, 118.6, 110.4, 13.9; ESIMS m/z 315 (MH+); HRESIMS m/z
Calcd for C14H11N4O3S (MH+) 315.0552, Found 315.0556.
4.2.15. General procedure for the synthesis of 7a–e
Mixtures of diethyl malonate (4.65 g, 29.0 mmol), the appropri-
ate benzaldehyde (6a–e, 29.6 mmol), and piperidine (300 lL) in
4.2.10. 3-(Methylthio)-5-(5-(2-(trifluoromethyl)phenyl)furan-2-
yl)-1,2,4-triazine (27b)
toluene (25 mL) were stirred under reflux for 5 h using a Dean–
Stark trap. After cooling down, the reaction mixtures were washed
sequentially with aqueous 1 M HCl, saturated aqueous sodium
bicarbonate, water (each for 25 mL), and dried over sodium sulfate.
Removal of the solvents gave thick yellow oils, which were used in
the next step without further purification.
Yellow solid (91.5%): mp 114–116 °C. IR (film) 1577, 1539,
1312, 1261, 1127, 1035 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.27
;
(s, 1H), 7.80 (t, J = 7.0 Hz, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.57–7.50
(m, 2H), 6.90 (d, J = 3.6 Hz, 1H), 2.69 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 173.4, 155.3, 149.0, 145.9, 140.1, 132.0, 130.3, 129.3,
127.9, 127.4, 127.1, 127.0, 125.5, 121.9, 118.1, 113.0, 13.8; EIMS
m/z 337 (M+); HREIMS m/z Calcd for C15H10F3N3OS (M+)
337.0497, Found 337.0496.
4.2.16. General procedure for the synthesis of 8a–e
The crude oils 7a–e (26.6 mmol) were dissolved in absolute eth-
anol (35 mL) and
a solution of potassium cyanide (1.75 g,
4.2.11. Methyl 3-(5-(3-(methylthio)-1,2,4-triazin-5-yl)furan-2-
yl)benzoate (27c)
26.8 mmol) in water (5 mL) was added. The mixtures were stirred
at 70 °C overnight, filtered and acidified by 1 M HCl to pH <1. Sol-
vents were evaporated and the residues were dissolved in ethyl
acetate (40 mL), washed with brine (25 mL) and dried over sodium
sulfate. Concentration of the organic layer afforded the products,
which were used in the next step without purification.
Yellow solid (95%): mp 168–170 °C. IR (film) 3120, 1725, 1537,
1281, 1247, 753 cmꢀ1 1H NMR (500 MHz, CDCl3) d 9.29 (s, 1H),
;
8.38 (s, 1H), 8.01 (d, J = 7.7 Hz, 1H), 7.94 (dd, J = 7.7, 1.0 Hz, 1H),
7.54–7.51 (m, 2H), 6.94 (d, J = 3.7 Hz, 1H), 3.95 (s, 3H), 2.67 (s,
3H); 13C NMR (125 MHz, CDCl3) d 173.3, 166.3, 157.2, 148.3,
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011

8
W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
4.2.17. General procedure for the synthesis of 9a–b
4.2.25. 3-(3-Chlorophenyl)pyrrolidine-2,5-dione (10a)79
Light yellow solid (55%): mp 120–122 °C (lit.79 119–121 °C). 1H
NMR (500 MHz, CDCl3) d 8.98 (s, 1 H, –NH), 7.33–7.29 (m, 2H),
7.26–7.25 (m, 1H), 7.15–7.13 (m, 1H), 4.06 (dd, J = 5.5, 10.0 Hz,
1H), 3.24 (dd, J = 10.0, 18.5 Hz, 1H), 2.85 (dd, J = 5.5, 18.5 Hz, 1H);
13C NMR (125 MHz, CDCl3) d 177.5, 176.0, 138.3, 135.0, 130.4,
128.3, 127.6, 125.6, 46.7, 37.9.
The crude esters 8a–b (6.4 mmol) were added to concentrated
hydrochloric acid (10 mL), and the mixtures were heated to reflux
overnight. After the mixtures were cooled down, the solid cakes
were collected by filteration and broken up. The products were
washed with cold water and dried at 60 °C overnight.
4.2.18. 2-(3-Chlorophenyl)succinic acid (9a)76
White solid (79.2%): mp 163–165 °C (lit.76 161–162 °C). 1H NMR
(300 MHz, CD3OD) d 7.33 (s, 1H), 7.31–7.23 (m, 3H), 4.02 (dd,
J = 9.6, 5.4 Hz, 1H), 3.08 (dd, J = 17.1, 9.9 Hz, 1H), 2.64 (dd,
J = 17.1, 5.4 Hz, 1H).
4.2.26. 3-(3-Nitrophenyl)pyrrolidine-2,5-dione (10b)78
Light yellow oil (78%). IR (film) 3310, 1780, 1710, 1520, 1340,
890 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 9.20 (s, 1 H, –NH),
,
8.17–8.14 (m, 2H), 7.63–7.53 (m, 2H), 4.24 (dd, J = 9.6, 5.4 Hz,
1H), 3.30 (dd, J = 18.6, 9.9 Hz, 1H), 2.90 (dd, J = 18.6, 5.7 Hz, 1H);
13C NMR (CDCl3, 75 MHz) d 180.4, 179.1, 149.8, 140.9, 135.3,
131.2, 124.0, 123.7, 48.1, 38.8.
4.2.19. 2-(3-Nitrophenyl)succinic acid (9b)77,78
Brown solid (73.2%): mp 212–215 °C (lit.78 216–218 °C). 1H
NMR (500 MHz, methanol-d4) d 8.21–8.20 (m, 1H), 8.15–8.13 (m,
1H), 7.75–7.73 (m, 1H), 7.58 (t, J = 8.0 Hz, 1H), 4.18 (dd, J = 9.3,
5.9 Hz, 1H), 3.13 (dd, J = 17.0, 9.3 Hz, 1H), 2.72 (dd, J = 17.0,
5.9 Hz, 1H).
4.2.27. 3-(3-(Benzyloxy)phenyl)pyrrolidine-2,5-dione (10c)
Orange solid (66%): mp 113–115 °C. IR (film) 3220, 1775, 1708,
1584, 1349, 1180 cmꢀ1 1H NMR (500 MHz, CDCl3) d 8.91 (br s,
;
1H), 7.44–7.37 (m, 4H), 7.35–7.31 (m, 1H), 7.29 (t, J = 8.0 Hz,
1H), 6.94–6.91 (m, 1H), 6.88–6.87 (m, 1H), 6.84 (d, J = 7.9 Hz,
1H), 5.05 (s, 2H), 4.03 (dd, J = 9.6, 5.0 Hz, 1H), 3.20 (dd, J = 18.6,
9.6 Hz, 1H), 2.85 (dd, J = 18.6, 5.0 Hz, 1H); 13C NMR (125 MHz,
CDCl3) d 178.1, 176.4, 159.2, 138.1, 136.5, 130.2, 128.6, 128.0,
127.5, 119.8, 114.4, 114.0, 70.0, 47.2, 38.1, 30.9; CIMS m/z 282
(MH+); HREIMS m/z Calcd for C17H15NO3 (MH+) 281.1052, Found
281.1055.
4.2.20. General procedure for the synthesis of 9c–e
The crude nitrile esters 8c–e (25.3 mmol) were dissolved in
absolute ethanol (55 mL), potassium hydroxide (7.81 g, 139 mmol)
was added, and the mixtures were heated to reflux for 4 h. After
cooling down, the solvents were evaporated, the residues were dis-
solved in water (50 mL), and 3 M HCl was added to make the pH
<1. The solid precipitates were collected by filtration and dried at
90 °C overnight.
4.2.28. 3-(3,4-Bis(benzyloxy)phenyl)pyrrolidine-2,5-dione (10d)
Yellow solid (57%): mp 125–127 °C. IR (film) 3246, 1777,
4.2.21. 2-[3-(Benzyloxy)phenyl]succinic acid (9c)
1712, 1513, 1259, 1012, 696 cmꢀ1 1H NMR (300 MHz, CDCl3)
;
Yellow solid (91.6%): mp 137–139 °C. 1H NMR (300 MHz,
DMSO-d6) d 7.46–7.30 (m, 5H), 7.21 (t, J = 8.0 Hz, 1H), 6.97 (s,
1H), 6.89–6.85 (m, 2H), 5.05 (s, 2H), 3.82 (dd, J = 9.5, 5.2 Hz, 1H),
2.94 (dd, J = 16.6, 9.5 Hz, 1H), 2.45 (dd, J = 16.6, 5.2 Hz, 1H); 13C
NMR (75 MHz, DMSO-d6) d 174.5, 173.7, 159.2, 142.6, 138.0,
130.3, 129.4, 128.8, 128.7, 120.9, 115.4, 113.6, 70.1, 47.7, 38.0; neg-
ative ion ESIMS m/z 299 (MꢀH+)ꢀ; negative ion HRESIMS m/z Calcd
for C17H15O5 (MꢀH+)ꢀ 299.0920, Found 299.0926.
d 8.20 (s, 1H), 7.43–7.26 (m, 10H), 6.91 (d, J = 8.2 Hz, 1H),
6.82 (s, 1H), 6.75 (d, J = 8.2 Hz, 1H), 5.15 (s, 4H), 3.97 (dd,
J = 9.5, 5.0 Hz, 1H), 3.18 (dd, J = 18.6, 9.6 Hz, 1H), 2.77 (dd,
J = 18.6, 5.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 177.7, 175.8,
149.3, 148.8, 137.0, 129.5, 128.5, 127.9, 127.4, 127.2, 120.3,
115.2, 114.4, 71.5, 71.2, 46.7, 38.1; ESIMS m/z 410 (MNa+);
HRESIMS m/z Calcd for C24H21NO4Na (MNa+) 410.1368, Found
410.1371.
4.2.22. 2-[3,4-Bis(benzyloxy)phenyl]succinic acid (9d)
Yellowsolid(90%):mp176–179 °C.1HNMR(300 MHz,DMSO-d6)
d 7.47–7.29 (m, 10H), 7.03–6.95 (m, 2H), 6.80–6.77 (m, 1H), 5.08 (s,
4H), 3.81 (dd, J = 10.5, 4.6 Hz, 1H), 2.93 (dd, J = 16.8, 10.5 Hz, 1H),
2.45 (dd, J = 16.8, 4.6 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) d 175.0,
173.7, 149.1, 148.4, 138.2, 138.1, 132.5, 129.3, 128.7, 128.4, 121.2,
115.4, 115.2, 71.2, 71.0, 47.2, 38.5; negative ion ESIMS m/z 405
(MꢀH+)ꢀ; negative ion HRESIMS m/z Calcd for C24H21O6 (MꢀH+)ꢀ
405.1338, Found 405.1341.
4.2.29. 3-(3-(Benzyloxy)-4-methoxyphenyl)pyrrolidine-2,5-
dione (10e)
Yellow solid (50%): mp 169–171 °C. IR (film) 3247, 1778, 1713,
1516, 1258, 1140, 1020, 698 cmꢀ1 1H NMR (500 MHz, DMSO-d6)
;
d 11.23 (s, 1 H, –NH), 7.41 (d, J = 7.3 Hz, 2H), 7.36 (t, J = 7.3 Hz,
2H), 7.30 (t, J = 7.2 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 6.89 (d,
J = 8.3 Hz, 1H), 6.77 (dd, J = 8.2, 1.5 Hz, 1H), 5.04–4.97 (m, 2H),
3.99 (dd, J = 9.5, 5.6 Hz, 1H), 3.70 (s, 3H), 3.03 (dd, J = 18.0,
9.5 Hz, 1H), 2.67 (dd, J = 18.0, 5.6 Hz, 1H); EIMS m/z 311 (M+);
HRESI-TOFMS m/z Calcd for C18H17NO4Na (MNa+) 334.1055,
found 334.1063.
4.2.23. 2-(3-(Benzyloxy)-4-methoxyphenyl)succinic acid (9e)
Yellow solid (92%): mp 159–162 °C. 1H NMR (300 MHz, DMSO-
d6) d 7.46–7.32 (m, 4H), 7.06–7.05 (m, 1H), 6.88–6.81 (m, 3H), 5.01
(s, 2H), 3.76 (dd, J = 9.9, 5.3, 1H), 3.72 (s, 3H), 2.92 (dd, J = 16.6,
9.9 Hz, 1H), 2.41 (dd, J = 16.6, 5.3 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6) d 174.9, 173.8, 149.0, 148.5, 138.0, 133.6, 129.3, 128.9,
128.8, 120.9, 114.3, 113.0, 71.0, 56.5, 47.2, 38.1; negative ion ESIMS
m/z 329 (MꢀH+)ꢀ; negative ion HRESIMS m/z Calcd for C18H17O6
(MꢀH+)ꢀ 329.1025, Found 329.1031.
4.2.30. General procedure for the synthesis of 11a–i
Solutions of 10a–i (0.124 mmol) in dry tetrahydrofuran (5 mL)
were stirred under argon at 0 °C, and 1 M BH3–tetrahydrofuran
(6 mL) was added dropwise. The solutions were stirred at 0 °C for
30 min and then heated to reflux for 3 h. After cooling down, 3 M
HCl was added cautiously until pH <1, and white solids precipi-
tated. The solvents were evaporated and the white solids were
washed twice with ether (3 mL) and then dried in vacuo. The white
solids were combined with 5 M NaOH (3 mL) and the mixtures
were extracted with ethyl acetate (10 mL). The organic layers were
dried over Na2SO4, filtered and dried to give the products as pale
yellow oils that were used in the next step without further
purification.
4.2.24. General procedure for the synthesis of 10a–e
Mixtures of the succinic acid (9a–e, 10.4 mmol) and urea
(2.54 g, 42.3 mmol) in xylene (35 mL) were heated at reflux over-
night, and then concentrated to dryness in vacuo. The crude prod-
ucts were purified by silica gel column chromatography, eluting
with hexane–ethyl acetate 1:1.
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011

W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
4.2.31. General procedure for the synthesis of 12a–i, 22a–f, and
29a–d
7.43–7.41 (m, 2H), 7.36–7.32 (m, 2H), 7.29–7.27 (m, 1H), 7.23 (s,
1H), 6.86–6.84 (m, 3H), 6.20 (d, J = 2.8 Hz, 1H), 5.14 (s, 2H), 4.3–
3.4 (br s, 5H), 3.87 (s, 3H), 2.42 (s, 3H), 2.37 (br s, 1H), 2.05 (br s,
1H); 13C NMR (125 MHz, CDCl3) d 159.0, 156.9, 148.5, 148.3,
148.1, 147.0, 137.0, 134.4, 134.1, 128.4, 127.7, 127.2, 119.6,
116.0, 113.4, 111.8, 109.2, 71.1, 56.0, 52.6, 46.3, 43.2, 33.0, 14.0;
ESIMS m/z 443 (MH+); HRESIMS m/z Calcd for C26H27N4O3 (MH+)
443.2083, Found 443.2093.
Mixtures of the corresponding methylsulfonyltriazine (5, 21a–f,
or 28a–d, 0.654 mmol) and the crude pyrrolidines (11a–i,
0.917 mmol) were dissolved in dry DMF (5 mL). The solutions were
stirred under argon at room temperature overnight. Solvents were
evaporated, and the residues were further purified by silica gel col-
umn chromatography, eluting with hexane–ethyl acetate 7:3.
4.2.32. 3-[3-(3-Chlorophenyl)pyrrolidin-1-yl]-5-(5-
methylfuran-2-yl)-1,2,4-triazine (12a)
4.2.37. 5-(5-Methylfuran-2-yl)-3-(3-phenylpyrrolidin-1-yl)-
1,2,4-triazine (12f)
Dark brown oil (65%). IR (film) 2943, 2873, 1551, 1527, 1478,
Dark brown liquid (63%). IR (film) 2941, 2871, 1605, 1527, 1297,
1039 cmꢀ1, 1H NMR (CDCl3, 300 MHz) d 8.81 (s, 1H), 7.35–7.21 (m,
6H), 6.18 (d, J = 2.7 Hz, 1H), 4.35–4.12 (m, 1H), 4.10–3.85 (m, 1H),
3.80–3.60 (m, 2H), 3.58–3.46 (m, 1H), 2.46–2.36 (m, 1H), 2.40 (s,
3H), 2.25–2.05 (m, 1H); 13C NMR (CDCl3, 75 MHz) d 159.1, 156.9,
148.3, 147.0, 141.5, 134.5, 128.6 (2C), 127.0 (2C), 126.7, 116.0,
109.3, 52.6, 46.3, 43.7, 32.9, 14.0; ESIMS m/z 307 (MH+); HRESIMS
m/z Calcd for C18H19N4O (MH+) 307.1559, Found 307.1561; HPLC
purity: 96% (C-18 reverse phase, MeOH–H2O, 85:15).
1081, 784 cmꢀ1 1H NMR (500 MHz, acetone-d6) d 8.81 (s, 1H),
;
7.42 (s, 1H), 7.36–7.34 (m, 3H), 7.28–7.26 (m, 1H), 6.32 (s, 1H),
4.30–3.40 (m, 5H), 2.50–2.40 (m, 1H), 2.39 (s, 3H), 2.25–2.15 (m,
1H); 13C NMR (125 MHz, acetone-d6) d 159.1, 156.9, 148.3, 146.5,
144.4, 134.1, 133.8, 130.1, 127.2, 126.6, 125.6, 116.0, 109.2, 52.3,
46.0, 43.2, 32.5, 12.9; ESIMS m/z 341 (MH+); HRESIMS m/z Calcd
for C18H18ClN4O (MH+) 341.1169, Found 341.1172; HPLC purity:
95.8% (C-18 reverse phase, MeOH–H2O, 90:10), 96.0% (C-18 reverse
phase, MeOH–H2O, 85:15).
4.2.38. 3-(3-(3-Fluorophenyl)pyrrolidin-1-yl)-5-(5-
methylfuran-2-yl)-1,2,4-triazine (12g)
4.2.33. 5-(5-Methylfuran-2-yl)-3-(3-(3-nitrophenyl)pyrrolidin-
1-yl)-1,2,4-triazine (12b)
Dark brown liquid (88%). IR (film) 2943, 2873, 1606, 1589, 1527,
Light brown oil (51%). IR (film) 2943, 2873, 1551, 1510, 1462,
1478, 785 cmꢀ1 1H NMR (CDCl3, 300 MHz)
; d 8.82 (s, 1H),
1345, 978 cmꢀ1
,
1H NMR (CDCl3, 300 MHz) d 8.85 (s, 1H), 8.19–
7.56–7.40 (m, 4H), 7.25 (d, J = 3.1 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H),
4.32–4.21 (m, 1H), 4.13–3.84 (m, 1H), 3.80–3.65 (m, 1H),
3.63–3.56 (m, 2H), 2.52–2.44 (m, 1H), 2.40 (s, 3H), 2.23–2.01 (m,
1H); 13C NMR (CDCl3, 75 MHz) d 164.5, 161.2, 159.0, 156.9,
144.2, 134.6, 130.0, 122.7, 116.1, 114.1, 113.8, 113.4, 109.3, 52.3,
46.2, 43.4, 32.7, 14.0; ESIMS m/z 325 (MH+); HRESIMS m/z Calcd
for C18H18FN4O (MH+) 325.1465, Found 325.1463; HPLC purity:
97% (C-18 reverse phase, MeOH–H2O, 90:10).
8.11 (m, 2H), 7.64 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 8.1 Hz, 1H), 7.25
(d, J = 3.6 Hz, 1H), 6.21 (d, J = 3.6 Hz, 1H), 4.30–4.20 (m, 1H),
4.16–3.92 (m, 1H), 3.85–3.55 (m, 3H), 2.60–2.50 (m, 1H), 2.43 (s,
3H), 2.30–2.10 (m, 1H); 13C NMR (CDCl3, 75 MHz) d 159.1, 157.1,
148.4, 148.2, 147.1, 143.7, 134.9, 133.3, 129.6, 122.1, 121.9,
116.3, 109.4, 52.3, 46.2, 43.4, 32.7, 29.6, 14.0; ESIMS m/z 352
(MH+); HRESIMS m/z Calcd for C18H17N5O3 (MH+) 352.1409, Found
352.1405. HPLC purity: 96% (MeOH–H2O, 90:10).
4.2.39. 5-(5-Methylfuran-2-yl)-3-(3-(3-
4.2.34. 3-(3-(3-(Benzyloxy)phenyl)pyrrolidin-1-yl)-5-(5-
methylfuran-2-yl)-1,2,4-triazine (12c)
(trifluoromethyl)phenyl)pyrrolidin-1-yl)-1,2,4-triazine (12h)
Dark brown liquid (85%). IR (film) 2944, 2874, 1606, 1529, 1328,
1121 cmꢀ1; 1H NMR (CDCl3, 300 MHz) d 8.83 (s, 1H), 7.58–7.42 (m,
4H), 7.24 (d, J = 3.3 Hz, 1H), 6.19 (d, J = 2.7 Hz, 1H), 4.37–4.20 (m,
1H), 4.15–3.88 (m, 1H), 3.83–3.68 (m, 1H), 3.67–3.56 (m, 2H),
2.53–2.44 (m, 1H), 2.41 (s, 3H), 2.26–2.02 (m, 1H); 13C NMR (CDCl3,
75 MHz) d 159.0, 157.0, 148.2, 147.0, 142.5, 134.6, 131.0, 130.4,
129.0, 123.9, 123.8, 123.6, 116.2, 109.3, 52.4, 46.2, 43.5, 32.8,
13.9; ESIMS m/z 375 (MH+); HRESIMS m/z Calcd for C19H18F3N4O
(MH+) 375.1433, Found 375.1439; HPLC purity: 98% (C-18 reverse
phase, MeOH–H2O, 85:15).
Brown oil (67%). IR (film) 2872, 1553, 1526, 1478, 1020,
696 cmꢀ1 1H NMR (500 MHz, CDCl3) d 8.82 (s, 1H), 7.43 (d,
;
J = 7.1 Hz, 2H), 7.39–7.35 (m, 2H), 7.33–7.30 (m, 1H), 7.27–7.23
(m, 2H), 6.95–6.94 (m, 1H), 6.91 (d, J = 7.6 Hz, 1H), 6.86 (dd,
J = 8.1, 2.4 Hz, 1H), 6.19 (d, J = 3.0 Hz, 1H), 5.05 (s, 2H), 4.3–3.4
(br s, 5H), 2.42 (br s, 1H), 2.41 (s, 3H), 2.13 (br s, 1H); 13C NMR
(125 MHz, CDCl3) d 159.1, 159.0, 156.8, 148.3, 147.0, 143.3,
136.9, 134.5, 129.6, 128.5, 127.9, 127.4, 119.7, 116.0, 114.0,
112.7, 109.3, 69.9, 52.5, 46.3, 43.7, 32.7, 14.0; ESIMS m/z 413
(MH+); HRESIMS m/z Calcd for C25H25N4O2 (MH+) 413.1978, Found
413.1971.
4.2.40. 3-(1-(5-(5-Methylfuran-2-yl)-1,2,4-triazin-3-
yl)pyrrolidin-3-yl)benzonitrile (12i)
4.2.35. 3-(3-(3,4-Bis(benzyloxy)phenyl)pyrrolidin-1-yl)-5-(5-
methylfuran-2-yl)-1,2,4-triazine (12d)
Dark brown liquid (93%). IR (film) 2962, 2890, 2243, 1600, 1552,
1438, 852 cmꢀ1 1H NMR (CDCl3, 300 MHz)
, d 8.75 (s, 1H),
Brown oil (61%). IR (film) 2871, 1553, 1526, 1478, 1257,
7.53–7.47 (m, 3H), 7.40–7.36 (m, 1H), 7.18 (d, J = 3.3 Hz, 1H),
6.15 (d, J = 3.0 Hz, 1H), 4.30–4.10 (m, 1H), 4.08–3.82 (m, 1H),
3.75–3.60 (m, 1H), 3.57–3.43 (m, 2H), 2.44–2.35 (m, 1H), 2.36 (s,
3H), 2.13–2.04 (m, 1H); 13C NMR (CDCl3, 75 MHz) d 159.0, 157.0,
148.1, 147.0, 143.2, 134.7, 131.6, 130.7, 130.4, 129.4, 118.7,
116.3, 112.5, 109.4, 52.2, 46.1, 43.2, 32.6, 14.0; ESIMS m/z 332
(MH+); HRESIMS m/z Calcd for C19H18N5O (MH+) 332.1511, Found
332.1507; HPLC purity: 96% (C-18 reverse phase, MeOH–H2O,
85:15).
1019 cmꢀ1 1H NMR (500 MHz, CDCl3) d 8.84 (s, 1H), 7.46–7.44
;
(m, 4H), 7.38–7.33 (m, 4H), 7.32–7.28 (m, 2H), 7.25 (s, 1H), 6.92–
6.90 (m, 2H), 6.82 (dd, J = 8.2, 1.7 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H),
5.16 (s, 2H), 5.15 (s, 2H), 4.30–3.35 (br s, 5H), 2.43 (s, 3H), 2.39
(br s, 1H), 2.06 (br s, 1H); 13C NMR (125 MHz, CDCl3) d 159.1,
156.9, 148.9, 148.4, 147.8, 147.0, 137.3, 137.2, 135.0, 134.5,
128.4, 127.7, 127.3, 127.2, 119.9, 116.0, 115.2, 114.4, 109.3, 71.4,
71.3, 52.6, 46.3, 43.3, 33.1, 30.8, 14.0; ESIMS m/z 519 (MH+); HRE-
SIMS m/z Calcd for C32H31N4O3 (MH+) 519.2396, Found 519.2393.
4.2.41. 3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-5-phenyl-1,2,4-
triazine (22a)
4.2.36. 3-(3-(3-(Benzyloxy)-4-methoxyphenyl)pyrrolidin-1-yl)-
5-(5-methylfuran-2-yl)-1,2,4-triazine (12e)
Yellow oil (30.1%). IR (film) 1535, 1478, 1309, 1018 cmꢀ1 1H
;
Dark brown oil (64.9%). IR (film) 2936, 2872, 1553, 1515, 1478,
NMR (300 MHz, CDCl3) d 9.03 (s, 1H), 8.12 (d, J = 7.2 Hz, 2H),
7.55–7.49 (m, 3H), 7.31–7.18 (m, 4H), 4.30 (br s, 1H), 4.10 (br s,
1255, 1020, 697 cmꢀ1
;
1H NMR (500 MHz, CDCl3) d 8.82 (s, 1H),
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011

10
W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
1H), 3.90–3.45 (br s, 3H), 2.48 (br s, 1H), 2.17 (br s, 1H); 13C NMR
(125 MHz, CDCl3) d 159.4, 155.2, 143.5, 136.3, 134.5, 134.4, 131.7,
129.8, 128.9, 127.3, 126.9, 125.2, 52.4, 46.4, 43.4, 32.9; ESIMS m/z
336.9 (MH+); HRESIMS m/z Calcd for C19H18N4Cl (MH+) 337.1220,
Found 337.1224; HPLC purity: 98.7% (C-18 reverse phase, MeOH–
H2O, 95:5), 99.4% (C-18 reverse phase, MeOH–H2O, 90:10).
382 (MH+); HRESIMS m/z Calcd for C19H17N5O2Cl (MH+)
382.1071, Found 382.1075; HPLC purity: 95.06% (C-18 reverse
phase, MeOH–H2O, 90:10).
4.2.47. 3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-5-(5-(3-
nitrophenyl)furan-2-yl)-1,2,4-triazine (29a)
Light yellow solid (63.5%): mp 206–208 °C. IR (film) 2924, 1552,
4.2.42. 3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-5-(3,4-
dimethoxyphenyl)-1,2,4-triazine (22b)
1524, 1477, 1347 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.02 (s, 1H),
;
8.58 (d, J = 1.8 Hz, 1H), 8.21–8.18 (m, 1H), 8.12–8.09 (m, 1H),
7.65 (t, J = 8.0 Hz, 1H), 7.47 (d, J = 3.6 Hz, 1H), 7.32–7.19 (m, 4H),
7.02 (d, J = 3.6 Hz, 1H), 4.3–3.4 (br s, 5H), 2.50 (br s, 1H), 2.18 (br
s, 1H); 13C NMR (75 MHz, CDCl3) d 159.1, 154.4, 150.5, 148.7,
146.5, 143.5, 134.6, 131.1, 130.1, 129.9, 129.8, 127.3, 127.1,
125.3, 123.0, 119.2, 116.6, 110.0, 52.5, 46.5, 43.5, 32.7; ESIMS m/
Yellow oil (84.9%). IR (film) 2937, 1531, 1508, 1478, 1276 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 8.98 (s, 1H), 7.71–7.68 (m, 2H), 7.31–
7.18 (m, 4H), 6.96 (d, J = 8.6 Hz, 1H), 4.4–3.4 (br s, 5H), 3.96 (s, 3H),
3.95 (s, 3H), 2.48 (br s, 1H), 2.17 (br s, 1H); 13C NMR (75 MHz,
CDCl3) d 159.4, 154.8, 152.3, 149.4, 143.7, 136.1, 134.4, 129.9,
127.4, 127.0, 125.3, 121.0, 111.0, 109.6, 56.0, 55.9, 52.4, 46.1,
43.5, 32.6; ESIMS m/z 397 (MH+); HRESIMS m/z Calcd for C21H22N4-
O2Cl (MH+) 397.1431, Found 397.1429; HPLC purity: 99.09% (C-18
reverse phase, MeOH–H2O, 90:10), 99.11% (C-18 reverse phase,
MeOH–H2O, 85:15).
z
448 (MH+); HRESIMS m/z Calcd for C23H19ClN5O3 (MH+)
448.1176, Found 448.1182; HPLC purity: 97.5% (C-18 reverse
phase, MeOH–H2O, 90:10), 95.6% (C-18 reverse phase, MeOH).
4.2.48. 3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-5-(5-(2-
(trifluoromethyl)phenyl)furan-2-yl)-1,2,4-triazine (29b)
Yellow solid (80.8%): mp 131–133 °C. IR (film) 2873, 1530,
4.2.43. 5-(4-Bromophenyl)-3-(3-(3-chlorophenyl)pyrrolidin-1-
yl)-1,2,4-triazine (22c)
1478, 1312, 1130 cmꢀ1 1H NMR (500 MHz, CDCl3) d 8.96 (s, 1H),
;
Yellow glass (72%). IR (film) 1573, 1530, 1479, 1071, 1008 cmꢀ1
;
7.80 (d, J = 8.4 Hz, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.6 Hz,
1H), 7.43 (d, J = 3.5 Hz, 1H), 7.30–7.17 (m, 4H), 6.87 (d, J = 3.5 Hz,
1H), 4.4–3.4 (br s, 5H), 2.46 (br s, 1H), 2.13 (br s, 1H); 13C NMR
(125 MHz, CDCl3) d 159.0, 154.0, 150.3, 146.7, 143.6, 134.7,
134.4, 131.9, 130.1, 129.8, 128.8, 128.2, 127.3, 127.1, 126.9,
126.8, 126.6 (CF3), 125.2, 124.8 (CF3), 122.6 (CF3), 120.5 (CF3),
116.0, 112.6, 52.3, 46.3, 43.4, 32.9; ESIMS m/z 471 (MH+); HRESIMS
m/z Calcd for C24H19N4OClF3 (MH+) 471.1200, Found 471.1193;
HPLC purity: 97.9% (C-18 reverse phase, MeOH–H2O, 90:10),
95.5% (C-18 reverse phase, MeOH–H2O, 95:5).
1H NMR (300 MHz, CDCl3) d 8.97 (s, 1H), 7.96 (d, J = 8.5 Hz, 2H),
7.61 (d, J = 8.5 Hz, 2H), 7.34–7.16 (m, 4H), 4.4–3.4 (br s, 5H), 2.48
(br s, 1H), 2.16 (br s, 1H); 13C NMR (75 MHz, CDCl3) d 159.3,
154.2, 143.4, 135.7, 134.4, 133.3, 132.3, 129.9, 128.8, 127.3,
127.0, 126.6, 125.3, 52.5, 46.5, 43.4, 32.9; ESIMS m/z 415 (MH+);
HRESIMS m/z Calcd for C19H17N4BrCl (MH+) 415.0325, Found
415.0319; HPLC purity: 97.80% (C-18 reverse phase, MeOH–H2O,
90:10), 97.24% (C-18 reverse phase, MeOH–H2O, 95:5).
4.2.44. 5-(3,4-Methylenedioxyphenyl)-3-(3-(3-
chlorophenyl)pyrrolidin-1-yl)-1,2,4-triazine (22d)
Yellow solid (67.6%): mp 169–171 °C. IR (film) 2874, 1537,
4.2.49. Methyl 3-(5-(3-(3-(3-chlorophenyl)pyrrolidin-1-yl)-
1,2,4-triazin-5-yl)furan-2-yl)benzoate (29c)
1530, 1501, 1487, 1439 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 8.93
Light yellow solid (66%): mp 169–171 °C. IR (film) 1722, 1526,
(s, 1H), 7.66–7.63 (m, 2H), 7.30–7.17 (m, 4H), 6.92–6.89 (m, 1H),
6.05 (s, 2H), 4.4–3.4 (br s, 5H), 2.48 (br s, 1H), 2.16 (br s, 1H); 13C
NMR (75 MHz, CDCl3) d 159.3, 154.6, 150.9, 148.6, 143.6, 135.9,
134.4, 129.9, 128.6, 127.3, 127.0, 125.3, 122.4, 108.6, 107.2,
101.8, 52.4, 46.3, 43.5, 32.8; ESIMS m/z 381 (MH+); HRESIMS m/z
Calcd for C20H18N4O2Cl (MH+) 381.1118, Found 381.1121; HPLC
purity: 98.45% (C-18 reverse phase, MeOH–H2O, 90:10), 96.79%
(C-18 reverse phase, MeOH–H2O, 95:5).
1477, 1297, 1248 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.01 (s, 1H),
;
8.41 (s, 1H), 8.03–7.95 (m, 2H), 7.53 (t, J = 7.8 Hz, 1H), 7.44 (d,
J = 3.6 Hz, 1H), 7.31–7.18 (m, 4H), 6.93 (d, J = 3.6 Hz, 1H), 4.4–3.5
(br s, 5H), 3.97 (s, 3H), 2.48 (m, 1H), 2.15 (br s, 1H); 13C NMR
(125 MHz, CDCl3) d 166.4, 159.1, 156.1, 149.7, 146.7, 143.6,
134.6, 134.4, 130.9, 129.8, 129.5, 129.0, 128.4, 127.3, 127.0,
125.5, 125.2, 116.6, 108.7, 52.4, 52.3, 46.3, 43.4, 32.8; ESIMS m/z
461 (MH+); HRESIMS m/z Calcd for C25H22ClN4O3 (MH+)
461.1380, Found 461.1374; HPLC purity: 95.8% (C-18 reverse
phase, MeOH–H2O, 90:10).
4.2.45. 3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-5-(3-
nitrophenyl)-1,2,4-triazine (22e)
Yellow oil (54.1%). IR (film) 2872, 1542, 1525, 1478, 1348,
4.2.50. 3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-5-(30-
nitrobiphenyl-4-yl)-1,2,4-triazine (29d)
1082 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.06 (s, 1H), 8.92 (s, 1H),
;
8.42–8.34 (m, 2H), 7.70 (t, J = 8.0 Hz, 1H), 7.29–7.18 (m, 4H), 4.4–
3.5 (br s, 5H), 2.50 (br s, 1H), 2.19 (br s, 1H); 13C NMR (75 MHz,
CDCl3) d 159.3, 152.7, 148.8, 143.3, 136.5, 135.6, 134.5, 132.9,
130.1, 130.0, 127.3, 127.1, 126.0, 125.3, 122.2, 52.6, 46.7 (46.2),
43.4, 32.9 (32.4); ESIMS m/z 382 (MH+); HRESIMS m/z Calcd for C19-
H17N5O2Cl (MH+) 382.1071, Found 382.1078; HPLC purity 97.10%
(C-18 reverse phase, MeOH–H2O, 90:10), 98.31% (C-18 reverse
phase, MeOH–H2O, 85:15).
Yellow oil (56.2%). IR (film) 2925, 1530, 1477, 1349, 1308 cmꢀ1
;
1H NMR (500 MHz, CDCl3) d 9.06 (s, 1H), 8.47 (s, 1H), 8.24–8.23 (m,
3H), 7.95 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 7.7 Hz, 2H), 7.64 (t,
J = 8.0 Hz, 1H), 7.31–7.19 (m, 4H), 4.4–3.5 (br s, 5H), 2.50 (br s,
1H), 2.17 (br s, 1H); 13C NMR (125 MHz, CDCl3) d 159.4, 154.3,
148.7, 143.5, 141.7, 141.5, 136.1, 134.6, 134.4, 132.9, 130.0,
128.1, 127.7, 127.3, 127.0, 125.2, 122.7, 121.9, 53.4, 46.5, 46.1 (con-
former), 43.4, 32.9, 32.4 (conformer); ESIMS m/z 458 (MH+); HRE-
SIMS m/z Calcd for
C
25H21ClN5O2 (MH+) 458.1384, Found
4.2.46. 3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-5-(4-
nitrophenyl)-1,2,4-triazine (22f)
458.1389; HPLC purity: 95.6% (C-18 reverse phase, MeOH–H2O,
90:10).
Yellow oil (47.4%). IR (film) 2873, 1542, 1478, 1346, 1070,
858 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 9.07 (s, 1H), 8.35 (d,
4.2.51. General procedure for the synthesis of 12j–m, 22g–h
Palladium on carbon (10%, 42.4 mg) was added to a solution of
12b–e, 22e–f (0.243 mmol) in methanol (6 mL) and ethyl acetate
(1.5 mL). The reaction mixture was vigorously stirred at room tem-
perature under atmospheric pressure of hydrogen overnight. The
J = 8.5 Hz, 2H), 8.28 (d, J = 8.5 Hz, 2H), 7.31–7.18 (m, 4H), 4.4–3.4
(br s, 5H), 2.51 (br s, 1H), 2.22 (br s, 1H); 13C NMR (75 MHz, CDCl3)
d 159.3, 152.8, 149.7, 143.3, 140.6, 136.0, 134.5, 130.0, 128.3,
127.3, 127.1, 125.3, 124.1, 52.6, 46.2, 43.4, 32.9, 32.4; ESIMS m/z
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011

W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
11
catalyst was removed by filtration and the filtrate was concen-
trated and further purified by silica gel column chromatography,
eluting with hexane–ethyl acetate (1:1).
(MH+); HRESIMS m/z Calcd for C19H19N5Cl (MH+) 352.1329, Found
352.1325; HPLC purity: 97.3% (C-18 reverse phase, MeOH–H2O,
90:10), 98.0% (C-18 reverse phase, MeOH–H2O, 85:15).
4.2.52. 3-(1-(5-(5-Methylfuran-2-yl)-1,2,4-triazin-3-
yl)pyrrolidin-3-yl)aniline (12j)
4.2.57. 4-(3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-1,2,4-triazin-5-
yl)aniline (22h)
Yellow oil (26%). IR (film) 3490, 3310, 2920, 2870, 1540, 1505,
Yellow solid (68.6%): mp 179–181 °C. IR (film) 3332, 3211,
854 cmꢀ1
,
1H NMR (CDCl3, 300 MHz) d 8.83 (s, 1H), 7.23 (d,
1605, 1522, 1477, 1313, 1294 cmꢀ1 1H NMR (500 MHz, CDCl3) d
;
J = 3.3 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H),
6.64–6.57 (m, 2H), 6.21 (d, J = 2.7 Hz, 1H), 4.40–3.90 (m, 2H),
3.85–3.52 (m, 2H), 3.50–3.41 (m, 1H), 2.44–2.33 (m, 1H), 2.44 (s,
3H), 2.29–2.08 (m, 1H); 13C NMR (CDCl3, 75 MHz) d 159.1, 156.9,
148.3, 147.0, 146.6, 142.8, 134.4, 129.5, 117.2, 116.0, 113.7,
113.5, 109.3, 52.5, 46.3, 43.7, 32.8, 14.0; ESIMS m/z 322 (MH+);
HRESIMS m/z Calcd for
322.1664; HPLC purity: 97% (C-18 reverse phase, MeOH–H2O,
85:15).
8.91 (s, 1H), 7.96 (d, J = 8.5 Hz, 1H), 7.30–7.17 (m, 4H), 6.72 (d,
J = 8.5 Hz, 1H), 4.4–3.4 (br s, 7H), 2.45 (br s, 1H), 2.14 (br s, 1H);
13C NMR (125 MHz, CDCl3) d 159.3, 154.9, 150.2, 143.7, 135.8,
134.3, 129.8, 129.0, 127.3, 126.9, 125.2, 123.6, 114.6, 52.3, 46.2,
43.4, 32.7; ESIMS m/z 352 (MH+); HRESIMS m/z Calcd for C19H19N5-
Cl (MH+) 352.1329, Found 352.1330; HPLC purity: 97.5% (C-18 re-
verse phase, MeOH–H2O, 90:10), 98.8% (C-18 reverse phase,
MeOH–H2O, 85:15).
C
18H19N5O (MH+) 322.1668, Found
4.2.58. 9-(1-(5-(5-Methylfuran-2-yl)-1,2,4-triazin-3-
yl)pyrrolidin-3-yl)-9H-purin-6-amine (17)
4.2.53. 3-(1-(5-(5-Methylfuran-2-yl)-1,2,4-triazin-3-
yl)pyrrolidin-3-yl)phenol (12k)
A solution of 1564 (80 mg, 0.26 mmol) in 20% (v/v) TFA in CH2Cl2
(5 mL) was stirred for one day. The solvent was evaporated, the
Yellow foam (64.6%). IR (film) 3127, 2966, 2946, 2874, 1602,
1555, 1533, 1478, 1453 cmꢀ1
;
1H NMR (300 MHz, methanol-d4) d
crude oil was mixed with triethylamine (200 lL, 1.43 mmol), and
8.77 (s, 1H), 7.39 (d, J = 3.3 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 6.79
(d, J = 7.8 Hz, 1H), 6.76 (s, 1H), 6.66 (d, J = 7.9 Hz, 1H), 6.32 (d,
J = 2.8 Hz, 1H), 4.13–4.07 (m, 1H), 3.91–3.87 (m, 1H), 3.69–3.49
(m, 3H), 2.43 (s, 3H), 2.41 (br s, 1H), 2.18–2.15 (m, 1H); 13C NMR
then a solution of 5 (39.7 mg, 0.166 mmol) in dry DMF (2 mL) was
added. The mixture was stirred under argon at room temperature
for 3 days. Solvents were evaporated and the residue was further
purified by silica gel preparative TLC, eluting with MeOH–CH2Cl2,
1:9, to provide the product as light yellow solid (33.2 mg, 56.4%):
mp 173–175 °C. IR (KBr) 3326, 3178, 2924, 1648, 1598, 1532,
(125 MHz, methanol-d4)
d 158.8, 157.6, 157.2, 147.8, 143.0,
133.6, 129.1, 117.8, 116.8, 113.4, 113.2, 109.2, 52.3, 46.0, 43.7,
32.3, 12.3; ESIMS m/z 323 (MH+); HRESIMS m/z Calcd for
1509, 1478, 1252 cmꢀ1 1H NMR (300 MHz, CDCl3 and methanol-
;
C
18H19N4O2 (MH+) 323.1508, Found 323.1514; HPLC purity:
d4) d 8.63 (s, 1H), 8.06 (s, 1H), 7.76 (s, 1H), 7.12 (d, J = 3.3 Hz, 1H),
6.06 (d, J = 3.0 Hz, 1H), 5.19–5.15 (m, 1H), 4.04–4.02 (m, 2H),
3.78–3.73 (m, 2H), 2.50–2.41 (m, 2H), 2.23 (s, 3H); 13C NMR
(75 MHz, CDCl3 and methanol-d4) d 158.9, 157.7, 155.4, 152.4,
149.1, 147.6, 147.4, 138.1, 134.8, 118.9, 117.3, 109.5, 53.6, 50.8,
44.4, 30.8, 13.5; ESIMS m/z 386 (MNa+); HRESIMS m/z Calcd for C17-
H17N9ONa (MNa+) 386.1454, Found 386.1452; HPLC purity: 99.1%
(C-18 reverse phase, MeOH–H2O, 90:10).
95.2% (C-18 reverse phase, MeOH–H2O, 90:10); 96.7% (C-18 re-
verse phase, MeOH, 100).
4.2.54. 4-(1-(5-(5-Methylfuran-2-yl)-1,2,4-triazin-3-
yl)pyrrolidin-3-yl)benzene-1,2-diol (12l)
Yellow foam (33%). 1H NMR (300 MHz, methanol-d4) d 8.73 (s,
1H), 7.36 (d, J = 3.4 Hz, 1H), 6.75–6.70 (m, 2H), 6.65–6.61 (m,
1H), 6.30 (d, J = 3.1 Hz, 1H), 4.05 (br s, 1H), 3.88 (br s, 1H), 3.62
(br s, 1H), 3.47–3.31 (br s, 2H), 2.41 (s, 3H), 2.35 (br s, 1H), 2.09
(br s, 1H); 13C NMR (125 MHz, methanol-d4) d 158.7, 157.6,
147.8, 147.7, 144.9, 143.6, 133.5, 132.8, 117.8, 116.8, 114.9,
113.6, 109.2, 52.6, 46.0, 42.8, 32.6, 12.3; ESIMS m/z 339 (MH+);
HRESIMS m/z Calcd for C18H19N4O3 (MH+) 339.1457, Found
339.1453; HPLC purity: 95.2% [C-18 reverse phase, MeOH (with
1% TFA)–H2O, 70:30].
4.2.59. General procedure for the synthesis of 25a–b and 25d
Mixtures of the aryl bromides 24 or 30 (2.65 mmol), the boronic
acids 23a or 23b (2.23 mmol), tetrabutylammonium bromide
(796 mg, 2.47 mmol), and K2CO3 (2.9 g, 21 mmol) were suspended
in distilled water (10 mL) under argon for 20 min. Then Pd(OAc)2
(9.2 mg, 0.04 mmol) was added, and the resulting suspensions
were heated to 70 °C and stirred under argon for 3 h. After cooling
down, the solutions were diluted with distilled water (10 mL) and
extracted with CH2Cl2 (3 ꢁ 15 mL). The combined organic layers
were dried over Na2SO4 and the solvents were evaporated. The
crude residues were further purified by silica gel column chroma-
tography, eluting with hexane–ethyl acetate 8:2, to provide the
products 25a–b or 25d.
4.2.55. 2-Methoxy-5-(1-(5-(5-methylfuran-2-yl)-1,2,4-triazin-3-
yl)pyrrolidin-3-yl) phenol (12m)
Brown oil (54%). 1H NMR (300 MHz, CDCl3) d 8.81 (s, 1H), 7.23
(d, J = 3.0 Hz, 1H), 6.90 (d, J = 1.7 Hz, 1H), 6.83–6.78 (m, 2H), 6.20
(d, J = 3.0 Hz, 1H), 4.3–3.4 (br s, 5H), 3.88 (s, 3H), 2.43 (s, 3H),
2.40 (br s, 1H), 2.10 (br s, 1H); 13C NMR (125 MHz, CDCl3) d
159.0, 156.8, 148.3, 147.0, 145.6, 145.3, 134.8, 134.4, 118.3,
116.0, 113.3, 110.6, 109.2, 55.9, 52.6, 46.4, 43.1, 32.9, 14.0; ESIMS
m/z 353 (MH+); HRESIMS m/z Calcd for C19H21N4O3 (MH+)
353.1614, Found 353.1617; HPLC purity: 95.2% (C-18 reverse
phase, MeOH–H2O, 90:10).
4.2.60. 1-(5-(3-Nitrophenyl)furan-2-yl)ethanone (25a)80
Yellow solid (32.5%): mp 112–113 °C (lit.80 116–117 °C). 1H
NMR (300 MHz, CDCl3) d 8.55 (t, J = 1.9 Hz, 1H), 8.19–8.15 (m,
1H), 8.10–8.07 (m, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.28 (d, J = 3.7 Hz,
1H), 6.93 (d, J = 3.7 Hz, 1H), 2.54 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 186.4, 154.6, 152.6, 148.6, 130.9, 130.3, 130.1, 123.4, 119.6,
119.2, 109.3, 26.1.
4.2.56. 3-(3-(3-(3-Chlorophenyl)pyrrolidin-1-yl)-1,2,4-triazin-5-
yl)aniline (22g)
Light yellow glass (37.8%). IR (film) 3337, 3217, 1535, 1477,
1327 cmꢀ1 1H NMR (300 MHz, CDCl3) d 8.97 (s, 1H), 7.46–7.43
;
4.2.61. 1-(5-(2-(Trifluoromethyl)phenyl)furan-2-yl)ethanone
(25b)80
(m, 2H), 7.30–7.17 (m, 5H), 6.86–6.83 (m, 1H), 4.4–3.4 (br s, 7H),
2.48 (br s, 1H), 2.16 (br s, 1H); 13C NMR (75 MHz, CDCl3) d 159.4,
155.4, 147.1, 143.6, 136.6, 135.5, 134.4, 129.9, 127.4, 127.0,
125.3, 118.4, 117.5. 113.3, 52.5, 46.4, 43.5, 32.8; ESIMS m/z 352
Light yellow oil (49.6%). 1H NMR (300 MHz, CDCl3) d 7.71–7.67
(m, 2H), 7.54–7.50 (m, 1H), 7.43–7.39 (m, 1H), 7.18 (dd, J = 1.1,
3.6 Hz, 1H), 6.72 (d, J = 3.6 Hz, 1H), 2.44 (s, 3H).
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011

12
W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
4.2.62. 1-(30-Nitrobiphenyl-4-yl)ethanone (25d)81
reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of
viable cells after treatment with each compound was expressed
as a percentage of the control, DMSO.
White crystals (96.5%): mp 109–110 °C. 1H NMR (300 MHz,
CDCl3) d 8.43 (t, J = 1.9 Hz, 1H), 8.22–8.18 (m, 1H), 8.05 (d,
J = 8.3 Hz, 2H), 7.95–7.91 (m, 1H), 7.69 (d, J = 8.3 Hz, 2H), 7.63 (t,
J = 8.0 Hz, 1H), 2.63 (s, 3H); 13C NMR (75 MHz, CDCl3) d 197.4,
148.7, 142.9, 141.4, 136.7, 133.1, 130.0, 129.1, 128.9, 127.3,
122.8, 122.0, 26.7.
Acknowledgments
This work was made possible by the National Institutes of
Health (NIH) through support with Research Grant UO1AI077949.
This work was supported in part by an award from the Floss
Endowment, provided to the Department of Medicinal Chemistry
and Molecular Pharmacology.
4.2.63. Methyl 3-(5-acetylfuran-2-yl)benzoate (25c)82
A
mixture of 1-(5-bromofuran-2-yl)ethanone (24, 1.57 g,
8.3 mmol), 3-(methoxycarbonyl)phenylboronic acid (23c, 1.71 g,
9.5 mmol), (2-biphenyl)dicyclohexylphosphine (0.29 g, 0.83
mmol) and K3PO4 (7.10 g, 33.5 mmol) was suspened in toluene
(36 mL) under argon for 20 min. Then Pd(OAc)2 (114.5 mg,
0.51 mmol) was added, and the resulting suspension was heated
to 90 °C and stirred under argon overnight in a sealed tube. After
cooling down, the solvent was evaporated. The crude residue was
further purified by silica gel column chromatography, eluting with
hexane–ethyl acetate 8:2, to provide the product as light yellow
solid (1.00 g, 49%): mp 117–118 °C. 1H NMR (300 MHz, CDCl3) d
8.38 (t, J = 1.5 Hz, 1H), 8.01–7.93 (m, 2H), 7.49 (t, J = 7.8 Hz, 1H),
7.25 (d, J = 3.6 Hz, 1H), 6.84 (d, J = 3.6 Hz, 1H), 3.93 (s, 3H), 2.52
(s, 3H); 13C NMR (75 MHz, CDCl3) d 186.3, 166.4, 156.4, 152.1,
130.9, 129.9, 129.6, 129.0, 128.9, 125.9, 119.3, 108.2, 52.3, 26.0.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.11.011.
References and notes
1. Harold, F. M. Bacteriol. Rev. 1966, 30, 772.
2. Kajander, T.; Kellosalo, J.; Goldman, A. FEBS Lett. 2013, 587, 1863.
3. Heinonen, J. K. Biological Role of Inorganic Pyrophosphate; Kluwer Academic
Publishers, Inc.: Boston, 2001.
4. Orimo, H.; Ohata, M.; Fujita, T. Endocrinology 1971, 89, 852.
5. Poole, K. E. S.; Reeve, J. Curr. Opin. Pharmacol. 2005, 5, 612.
6. Ko, K. M.; Lee, W.; Yu, J. R.; Ahnn, J. FEBS Lett. 2007, 581, 5445.
7. Usui, Y.; Uematsu, T.; Uchihashi, T.; Takahashi, M.; Takahashi, M.; Ishizuka, M.;
Doto, R.; Tanaka, H.; Komazaki, Y.; Osawa, M.; Yamada, K.; Yamaoka, M.;
Furusawa, K. J. Dent. Res. 2010, 89, 504.
4.3. M. tuberculosis MABA and LORA assay
Detailed experimental procedures for M. tuberculosis MABA and
8. Rodina, E. V.; Vainonen, L. P.; Vorobyeva, N. N.; Kurilova, S. A.; Sitnik, T. S.;
Nazarova, T. I. Biochemistry (Moscow) 2008, 73, 897.
LORA assay can be found in our previous published papers.83
9. Ahn, S.; Milner, A. J.; Futterer, K.; Konopka, M.; Ilias, M.; Young, T. W.; White, S.
A. J. Mol. Biol. 2001, 313, 797.
10. Merckel, M. C.; Fabrichniy, I. P.; Salminen, A.; Kalkkinen, N.; Baykov, A. A.;
Lahti, R.; Goldman, A. Structure 2001, 9, 289.
4.4. Activity against MRSA and Bacillus strains
The minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC) of PPase inhibitors were tested in
triplicate against methicillin-resistant S. aureus (MRSA), vancomy-
cin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus
(VRSA), and Bacillus strains [Bacillus anthracis Sterne strain (34F2)
and Bacillus subtilis] in the micro dilution broth method, using Mul-
ler Hinton broth (MHB) and 96 well-plates according to Clinical
and Laboratory Standards (CLSI).84 The bacterial cultures were
incubated at 37 °C for 20 h.
11. Heikinheimo, P.; Lehtonen, J.; Baykov, A.; Lahti, R.; Cooperman, B. S.; Goldman,
A. Structure 1996, 4, 1491.
12. Baykov, A. A.; Kasho, V. N.; Bakuleva, N. P.; Rea, P. A. FEBS Lett. 1994, 350, 323.
13. Lin, S. M.; Tsai, J. Y.; Hsiao, C. D.; Huang, Y. T.; Chiu, C. L.; Liu, M. H.; Tung, J. Y.;
Liu, T. H.; Pan, R. L.; Sun, Y. J. Nature 2012, 484, 399.
14. Salminen, T.; Kapyla, J.; Heikinheimo, P.; Kankare, J.; Goldman, A.; Heinonen, J.;
Baykov, A. A.; Cooperman, B. S.; Lahti, R. Biochemistry 1995, 34, 782.
15. Kellosalo, J.; Kajander, T.; Kogan, K.; Pokharel, K.; Goldman, A. Science 2012,
337, 473.
16. Baykov, A. A.; Tamvilloslado, J. J.; Avaeva, S. M. Biochem. Biophys. Acta 1979,
569, 228.
17. Baykov, A. A.; Alexandrov, A. P.; Smirnova, I. N. Arch. Biochem. Biophys. 1992,
294, 238.
4.5. Cytotoxicities of inorganic pyrophosphatase inhibitors
in vitro
18. Baykov, A. A.; Artjukov, A. A.; Avaeva, S. M. Biochem. Biophys. Acta 1976, 429,
982.
19. Baykov, A. A.; Bakuleva, N. P.; Nazarova, T. I.; Avaeva, S. M. Biochem. Biophys.
Acta 1977, 481, 184.
20. Baykov, A. A.; Artjukov, A. A.; Avaeva, S. M. Biochem. Biophys. Acta 1977, 481,
195.
21. Baykov, A. A.; Dubnova, E. B.; Bakuleva, N. P.; Evtushenko, O. A.; Zhen, R. G.;
Rea, P. A. FEBS Lett. 1993, 327, 199.
22. Samygina, V. R.; Moiseev, V. M.; Rodina, E. V.; Vorobyeva, N. N.; Porov, A. N.;
Kurilova, S. A.; Nazarova, T. I.; Avaeva, S. M.; Bartunik, H. D. J. Mol. Biol. 2007,
366, 1305.
23. Pohjanjoki, P.; Fabrichniy, I. P.; Kasho, V. N.; Cooperman, B. S.; Goldman, A.;
Baykov, A. A.; Lahti, R. J. Biol. Chem. 2001, 276, 434.
24. Baykov, A. A.; Volk, S. E.; Unguryte, A. Arch. Biochem. Biophys. 1989, 273, 287.
25. Avaeva, S. M.; Vorobyeva, N. N.; Kurilova, S. A.; Nazarova, T. I.; Polyakov, K. M.;
Rodina, E. V.; Samygina, V. R. Biochemistry (Moscow, Russ. Fed.) 2000, 65, 373.
26. Davidson, A. M.; Halestrap, A. P. Biochem. J. 1989, 258, 817.
27. Maeshima, M. Eur. J. Biochem. 1991, 196, 11.
28. Eaton, R. H.; Moss, D. W. Biochem. J. 1967, 102, 917.
29. Martin, H.; Grant, B. R.; Stehmann, C. Pestic. Biochem. Physiol. 1998, 61, 65.
30. Grigorieva, O. V.; Mit’kevich, V. A.; Sklyankina, V. A.; Avaeva, S. M. Russ. J.
Bioorg. Chem. 2001, 27, 27.
31. Zyryanov, A. B.; Lahti, R.; Baykov, A. A. Biochemistry (Moscow) 2005, 70, 908.
32. Smirnova, I. N.; Kudryavtseva, N. A.; Komissarenko, S. V.; Tarusova, N. B.;
Baykov, A. A. Arch. Biochem. Biophys. 1988, 267, 280.
33. Gordon-Weeks, R.; Parmar, S.; Davies, T. G. E.; Leigh, R. A. Biochem. J. 1999, 337,
373.
34. Kelly, S. J.; Feldman, F.; Sperow, J. W.; Butler, L. G. Biochemistry 1973, 12, 3338.
35. Woltgens, J. H. M.; Bervoets, T. J. M.; Devries, W. J. Periodont. Res. 1977, 12, 462.
36. Kotsikorou, E.; Song, Y. C.; Chan, J. M. W.; Faelens, S.; Tovian, Z.; Broderick, E.;
Bakalara, N.; Docampo, R.; Oldfield, E. J. Med. Chem. 2005, 48, 6128.
Inorganic pyrophosphatase inhibitors were assayed at concen-
trations of 8, 16, 32, and 64 lM against human tumor cell line HeLa
and murine macrophage cell line (J774.A1) to determine the poten-
tial toxic effect in mammalian cells.85 Briefly, ꢂ2 ꢁ 104 cells sus-
pended in 200
serum (FBS),
lL of DMEM supplemented with 10% fetal bovine
L
-glutamine, NaHCO3, pyridoxine–HCl, and
45,000 mg/L glucose and preserved with 1% penicillin–streptomy-
cin solution were seeded in 96-well plates and incubated at 37 °C
in a 5% CO2 atmosphere. The cells were cultured for 24 h (60% con-
fluency) before the assays. The cells were further incubated with 8,
16, 32, and 64
DMSO as a control for 2 h. The culture media was discarded, and
the cells in each well were washed with PBS and 100 L of cell cul-
lM of compounds 22g, 22a, 22h, 12k, 22b and
l
ture media were added prior to addition of the assay reagent
MTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium) (Promega). The plates were incu-
bated for 4 h at 37 °C in a humidified 5% CO2 atmosphere. The
absorbance at 490 nm was recorded using a 96-well Elisa plate
reader (SoftMax ProInc., USA). Corrected absorbance readings (ac-
tual absorbance readings for each treatment subtracted from back-
ground absorbance) were taken using a kinetic ELISA microplate
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011

W. Lv et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
13
37. Hsu, M. T.; Sturr, M. G.; Curran, T. M.; Marquis, R. E. Oral Microbiol. Immunol.
1995, 10, 47.
63. Chang, C. Y.; Kuo, S. C.; Lin, Y. L.; Wang, J. P.; Huang, L. J. Bioorg. Med. Chem. Lett.
1971, 2001, 11.
38. Zhen, R. G.; Baykov, A. A.; Bakuleva, N. P.; Rea, P. A. Plant Physiol. 1994, 104,
153.
39. Yatsimirsky, K. B.; Manorik, P. A.; Gaivoronskaya, G. G.; Lopatina, O. I.;
Davidenko, N. K.; Komissarenko, S. V. Dopovv. Akad. Nauk Ukr. 1983, 11, 55.
40. Yang, S. J.; Jiang, S. S.; Tzeng, C. M.; Kuo, S. Y.; Hung, S. H.; Pan, R. L. Biochem.
Biophys. Acta 1996, 1294, 89.
64. Kocalka, P.; Pohl, R.; Rejman, D.; Rosenberg, I. Tetrahedron 2006, 62, 5763.
65. Collins, L. A.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
66. Falzari, K.; Zhu, Z. H.; Pan, D. H.; Liu, H. W.; Hongmanee, P.; Franzblau, S. G.
Antimicrob. Agents Chemother. 2005, 49, 1447.
67. Jayaprakash, S.; Iso, Y.; Wan, B.; Franzblau, S. G.; Kozikowski, A. P.
ChemMedChem 2006, 1, 593.
41. Zhen, R. G.; Kim, E. J.; Rea, P. A. J. Biol. Chem. 1997, 272, 22340.
42. GordonWeeks, R.; Steele, S. H.; Leigh, R. A. Plant Physiol. 1996, 111, 195.
43. Yang, S. J.; Jiang, S. S.; Van, R. C.; Hsiao, Y. Y.; Pan, R. L. Biochem. Biophys. Acta
2000, 1460, 375.
44. Kim, E. J.; Zhen, R. G.; Rea, P. A. J. Biol. Chem. 1995, 270, 2630.
45. Nordlie, R. C.; Lardy, H. A. Biochem. Biophys. Acta 1961, 53, 309.
46. Shoyab, M.; Marx, W. Arch. Biochem. Biophys. 1971, 146, 71.
47. Burobin, A. V.; Lomonova, M. V.; Sklyankina, V. A.; Avaeva, S. M. Bioorg. Khim.
1997, 23, 104.
48. Hirono, M.; Ojika, M.; Mimura, H.; Nakanishi, Y.; Maeshima, M. J. Biochem.
2003, 133, 811.
49. Kuznetsov, A. V.; Sklyankina, V. A.; Avaeva, S. M. Bioorg. Khim. 1979, 5, 1396.
50. Tian, Y.; Suk, D. H.; Cai, F.; Crich, D.; Mesecar, A. D. Biochemistry 2008, 47,
12434.
68. Bate, A. B.; Kalin, J. H.; Fooksman, E. M.; Amorose, E. L.; Price, C. M.; Williams, H.
M.; Rodig, M. J.; Mitchell, M. O.; Cho, S. H.; Wang, Y. H.; Franzblau, S. G. Bioorg.
Med. Chem. Lett. 2007, 17, 1346.
69. Tammenkoski, M.; Benini, S.; Magretova, N. N.; Baykov, A. A.; Lahti, R. J. Biol.
Chem. 2005, 280, 41819.
70. Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M. R.; Appel, R. D.;
Bairoch, A. In The Proteomics Protocols Handbook; Walker, J. M., Ed.; Humana
Press: Totowa, NJ, 2005; p 571.
71. Baykov, A. A.; Evtushenko, O. A.; Avaeva, S. M. Anal. Biochem. 1988, 171, 266.
72. Leete, E.; Bodem, G. B. J. Am. Chem. Soc. 1976, 98, 6321.
73. Pegan, S. D.; Tian, Y.; Sershon, V.; Mesecar, A. D. Comb. Chem. High Throughput
Screening 2010, 13, 27.
74. Heilman, W. P.; Heilman, R. D.; Scozzie, J. A.; Wayner, R. J.; Gullo, J. M.; Ariyan,
Z. S. J. Pharm. Sci. 1980, 69, 282.
51. Dawane, B. S.; Kadam, S. N.; Shaikh, B. M. Pharm. Lett. 2010, 2, 126.
52. Chohan, Z. H.; Farooq, M. A. J. Chem. Soc. Pak. 1995, 17, 14.
53. Chohan, Z. H.; Pervez, H.; Rauf, A.; Khan, K. M.; Maharvi, G. M.; Supuran, C. T. J.
Enzyme Inhib. Med. Chem. 2004, 19, 161.
75. Pellegatti, L.; Vedrenne, E.; Leger, J. M.; Jarry, C.; Routier, S. Tetrahedron 2010,
66, 4383.
76. Citterio, A.; Cominelli, A.; Bonavoglia, F. Synthesis 1986, 308.
77. Kamlet, M. J. J. Am. Chem Soc. 1955, 77, 4896.
54. Sawant, R. L.; Ramdin, S. S.; Rathi, S. R. Int. J. PharmTech. Res. 2012, 4, 1096.
55. Patel, R. V.; Kumari, P.; Rajani, D. P.; Pannecouque, C.; De Clercq, E.; Chikhalia,
K. H. Future Med. Chem. 2012, 4, 1053.
56. Sun, X. B.; Cao, Z. B.; Sun, Y. Y. Ind. Eng. Chem. Res. 2009, 48, 607.
57. Aly, A. A. Curr. Org. Chem. 2009, 13, 1758.
78. Lange, J.; Lapszewicz, J. Acta Pol. Pharm. 1978, 35, 289.
79. Lapszewicz, J.; Lange, J.; Mazur, W. Acta Pol. Pharm. 1981, 38, 269.
80. Obushak, N. D.; Gorak, Y. I.; Matiichuk, V. S.; Lytvyn, R. Z. Russ. J. Org. Chem.
2008, 44, 1689.
81. Hebel, A.; Haag, R. J. Org. Chem. 2002, 67, 9452.
58. Hajbi, Y.; Suzenet, F.; Khouili, M.; Lazar, S.; Guillaumet, G. Tetrahedron 2007, 63,
8286.
82. Haddach, M.; Michaux, J.; Schwaebe, M. K.; Pierre, F.; O’Brien, S. E.; Borsan, C.;
Tran, J.; Raffaele, N.; Ravula, S.; Drygin, D.; Siddiqui-Jain, A.; Darjania, L.;
Stansfield, R.; Proffitt, C.; Macalino, D.; Streiner, N.; Bliesath, J.; Omori, M.;
Whitten, J. P.; Anderes, K.; Rice, W. G.; Ryckman, D. M. ACS Med. Chem. Lett.
2012, 3, 135.
83. Zhao, Y.; Bacher, A.; Illarionov, B.; Fischer, M.; Georg, G.; Ye, Q.-Z.; Fanwick, P.
E.; Franzblau, S. G.; Wan, B.; Cushman, M. J. Org. Chem. 2009, 74, 5297.
84. NCCLS: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow Aerobically. NCCLS Approved Standard M7-A9.; 9th Ed. ed.; NCCLS,
Wayne, Pa., 2012.
59. Wu, W.; Sil, D.; Szostak, M. L.; Malladi, S. S.; Warshakoon, H. J.; Kimbrell, M. R.;
Cromer, J. R.; David, S. A. Bioorg. Med. Chem. 2009, 17, 709.
60. Gullo, J. M.; Heilman; W. P.; Wayner, R. J.; Moser, R. E. (Diamond Shamrock
Corporation), Pharmacologically Active Substituted 1,2,4-Triazines. U.S. Patent
4157392 A1, June 5, 1979.
61. Alphonse, F. A.; Suzenet, F.; Keromnes, A.; Lebret, B.; Guillaumet, G. Synthesis
2004, 2893.
62. Breukelman, S. P.; Meakins, G. D.; Roe, A. M. J. Chem. Soc., Perkin Trans. 1 1985,
1627.
85. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
Please cite this article in press as: Lv, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.011