Triazole inhibitors of Cryptosporidium parvum inosine 5′- monophosphate dehydrogenase

Article abstract of DOI:10.1021/jm900410u

Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleoti

Full text of DOI:10.1021/jm900410u

J. Med. Chem. 2009, 52, 4623–4630 4623
DOI: 10.1021/jm900410u
Triazole Inhibitors of Cryptosporidium parvum Inosine 50-Monophosphate Dehydrogenase
Sushil K. Maurya,† Deviprasad R. Gollapalli,† Shivapriya Kirubakaran,† Minjia Zhang,† Corey R. Johnson,‡
Nicole N. Benjamin,† Lizbeth Hedstrom,†,‡ and Gregory D. Cuny*,§
†Department of Biology and ‡Department of Chemistry, Brandeis University, MS009, 415 South Street, Waltham, Massachusetts 02454, and
§Laboratory for Drug Discovery in Neurodegeneration and Partners Center for Drug Discovery, Brigham & Women’s Hospital and Harvard
Medical School, 65 Landsdowne Street, Cambridge, Massachusetts 02139
Received March 31, 2009
Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This
protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 50-monophos-
phate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Because
C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially
be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series
of 1,2,3-triazole containing ether CpIMPDH inhibitors are described. A structure-activity relation-
ship study revealed that a small alkyl group on the R-position of the ether was required, with the (R)-
enantiomer significantly more active than the (S)-enantiomer. Electron-withdrawing groups in the
3- and/or 4-positions of the pendent phenyl ring were best, and conversion of the quinoline containing
inhibitors to quinoline-N-oxides retained inhibitory activity both in the presence and absence of bovine
serum albumin. The 1,2,3-triazole CpIMPDH inhibitors provide new tools for elucidating the role
of IMPDH in C. parvum and may serve as potential therapeutics for treating cryptosporidiosis.
Scheme 1. IMPDH Catalyzed Conversion of 1 to 3, Which Is
Further Processed to 4
Introduction
Inosine 50-monophosphate dehydrogenase (IMPDHa) cat-
alyzes the nicotinamide-adenine dinucleotide(NADþ)-depen-
dent oxidation of inosine 50-monophosphate (IMP, 1) to
xanthosine 50-monophosphate (XMP, 3), the first committed
and rate limiting step in the biosynthesis of guanosine
monophosphate (GMP, 4, Scheme 1).1 IMPDH controls the
guanine nucleotide pool and therefore plays an integral role in
cellular proliferation.2 Human IMPDH exists in two iso-
forms, IMPDH1 and IMPDH2, which have high (85%)
sequence identity. Human IMPDH1 is most prevalent in
leukocytes and lymphocytes, while IMPDH2 is found in
greatest abundance in rapidly proliferating cells, including
neoplastic cells.3 IMPDH has emerged as an attractive ther-
apeutic target for the treatment of various conditions4 inclu-
ding cancer5 and viral infections.6 IMPDH inhibitors have
also been used clinically as immunosuppressants, promp-
ting further interest in utilizing this class of therapeutics for
treating other autoimmune diseases.7
selective inhibitors.1,8 Prokaryotic IMPDHs are resistant to
the known human IMPDH inhibitor mycophenolic acid,
demonstrating that selective inhibition is possible. IMPDH
is a promising target for the treatment of cryptosporidiosis, a
major cause of diarrhea and malnutrition initiated by the
protozoan parasites Cryptosporidium parvum and a related
pathogenic species Cryptosporidium hominis.9 These organ-
isms can cause severe gastroenteritis and diarrhea, which can
be life threatening in immunocompromised individuals. In
addition, C. parvum is a potential bioterrorism agent. Inter-
estingly, both C. parvum and C. hominis rely exclusively on the
IMPDH-mediated pathway for guanine nucleotide synthesis
by salvaging and converting adenosine to 1 and then subse-
quently to 4.10-12 C. parvum IMPDH (CpIMPDH) appears to
IMPDH may also be a target for antimicrobial chemo-
therapy, although its utility can be compromised if the
microorganism can salvage guanine and/or xanthine. Prok-
aryotic and eukaryotic IMPDHs have divergent amino
acid sequences and display significantly different kinetic
properties, suggesting that it should be possible to develop
*Corresponding author. Phone: 1-617-768-8640. Fax: 1-617-768-
8606. E-mail: gcuny@rics.bwh.harvard.edu.
a Abbreviations: Cp, Cryptosporidium parvum; BSA, bovine serum
albumin; DCM, dichloromethane; DIBAL, diisobutylaluminum hy-
dride; EDCI, 1-ethyl-3-(30-dimethylaminopropyl)carbodiimide; GMP,
guanosine monophosphate; HTS, high throughput screening; IMP,
inosine 50-monophosphate; IMPDH, inosine 50-monophosphate dehy-
drogenase; m-CPBA, m-chloroperoxybenzoic acid; NADþ, nicotin-
amide-adenine dinucleotide; XMP, xanthosine 50-monophosphate.
Figure 1. CpIMPDH inhibitor identified by HTS.
r
2009 American Chemical Society
Published on Web 07/22/2009
pubs.acs.org/jmc

4624 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Maurya et al.
have been obtained from anε-proteobacterium by lateral gene
transfer.13 These observations suggest that selective inhibition
of CpIMPDH may provide an attractive means for parasite-
specific inhibition, while minimizing potential side effects on
the human (or other mammalian) host.
Ethyl glyoxylate was allowed to react with c-PrMgBr to give
a corresponding alcohol that was subsequently converted to
bromide 7 (R=c-Pr) with carbon tetrabromide and triphe-
nylphosphine. Various other bromide derivatives of 7 were
commercially available. Treatment of 7 with 1-naphthol in
the presence of base (K2CO3) gave ester 8 (X=CH). The ester
was saponified with 3N sodium hydroxide in THF to give
acid 9 (X=CH), which was subsequently converted to amide
Our laboratories have been seeking to identify and optimize
low molecular weight molecules capable of selectively inhibit-
ing CpIMPDH in order to provide lead compounds for
therapeutic development to treat cryptosporidiosis.14 During
a high throughput screening(HTS) campaign, 5 was identified
(Figure 1) as a moderately potent and very selective inhi-
bitor of CpIMPDH (EC50=3.3 ( 0.2 μM) with no detectable
activity against the human enzyme (EC50 . 50 μM).15 The
compound demonstrated uncompetitive inhibition with re-
spect to 1 and noncompetitive (mixed) inhibition with respect
to NADþ. It was also shown to bind the nicotinamide subsite
and to directly or indirectly impose on the ADP site.15 Herein,
we report a structure-activity relationship (SAR) study for
this class of inhibitors, including a key structural change to the
amide functional group.
10 (X = CH) with the aid of EDCI HCl in anhydrous
3
dichloromethane (DCM). In the case of a quinoline analogue
of 10 (X = N), the acetyl chloride derivative 11 was first
converted to amide 12, which was treated with 4-hydroxy-
quinoline to give 10 (X=N).
Analogues of 5 that replaced the amide functional group
with a 1,2,3-triazole were prepared according to Scheme 3.
The common intermediate in the synthesis of these deriva-
tives was propargyl ether 16. This intermediate was prepared
using several different routes. In the first route (R1=Me or
H), 13 was alkylated utilizing either a Mitsonobu reaction
with a propargyl alcohol or by treatment with propargyl
bromide in the presence of potassium carbonate. The route
employing the Mitsonobu reaction also afforded enantio-
merically enriched ethers starting with (S)- or (R)-but-3-yn-
2-ol. When R1=i-Pr and R2=CO2H, the acid 14 (prepared
via Scheme 2) was reduced to the corresponding alcohol with
Results and Discussion
Chemistry. Various analogues of 5 that contain the amide
functional group were prepared according to Scheme 2.
Scheme 2. General Procedure for the Synthesis of Amide Derivativesa
a Reagents and conditions: (a) (i) c-PrMgBr, THF, -20 °C, 2 h, (ii) Ph3P, CBr4, DCM, 0 °C, 2 h; (b) 1-naphthol, K2CO3, DMF, rt, 2 h; (c) 3 M NaOH,
THF:H2O (2:1), 80 °C, 6 h; (d) 4-chloroaniline, 0 °C, EDCI HCl, DCM, rt, 12 h; (e) 4-chloroaniline, cat. DMAP, DCM, rt, 2 h; (f) 4-hydroxyquinoline,
3
K2CO3, DMF, 0 °C, rt, 12 h.
Scheme 3. General Procedure for the Synthesis of 1,2,3-Triazole Derivativesa
a Reagents and conditions: X and Y = N, CH or CCl. (a) [R1 = Me] MeCH(OH)CtCH, 0 °C, Ph3P, 10 min, DEAD, rt, 12 h; (b) [R1 = H]
BrCH2CtCH, K2CO3, DMF, rt, 12 h; (c) [R1 = i-Pr, R2 = CO2H] (i) LiAlH4, THF, 0 °C, 4 h, (ii) (COCl)2, DMSO, DCM, Et3N, -78 °C, 3 h; (d) [R1 =
Et, R2 = CO2Et] DIBAL, THF, -78 °C, 6 h; (e) (i) CBr4, Ph3P, DCM, 0 °C, 2 h, (ii) n-BuLi, THF, -78 °C, 2 h; (f) R3PhN3, CH3CN, DIPEA, CuI, rt,
30 min; (g) m-CPBA, DCM, 0 °C, 12 h.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4625
LiAlH4 and then oxidized to aldehyde 15 under Swern
conditions.16 When R1 = Et, R2 = CO2Et, the ester 14
(prepared via Scheme 2) was reduced directly to aldehyde
15 with DIBAL in THF at -78 °C. Aldehyde 15 was
converted to the corresponding alkyne utilizing the two-step
procedure described by Corey and Fuchs.17 Ether 16 was
converted to 1,2,3-triazole 17 in the presence of an aryl azide
and CuI.18 In the case of N-oxide derivatives, 16 (X or Y=N)
was first treated with m-CPBA. The N-oxide of 16 was then
converted to 17 (X or Y=Nþ-O-).
Biology. IC50 Determinations for CpIMPDH Inhibition.
Evaluation of CpIMPDH inhibitory activity for the various
prepared compounds was conducted utilizing an assay
for the conversion of 1 to 3 by monitoring the production
of NADH by fluorescence in the presence of varying inhi-
bitor concentrations.15 For all the compounds reported
herein, the IC50 values were determined in three independent
experiments. The average IC50 values and standard devia-
tions are reported in Tables 1 and 2. It is commonly observed
that antimicrobial or antiparasitic activity of compounds can
be adversely affected by nonspecific binding to serum pro-
teins, which sequesters the compounds preventing interac-
tion with the target.19 This in vitro effect can translate into
poor efficacy in vivo due to low free fraction concentration
of compound that can enter the pathogenic organism.20 To
characterize the nonspecific binding of inhibitors, IC50 val-
ues were also determined in the presence of 0.05% fatty acid
free bovine serum albumin (BSA). In addition, none of the
compounds displayed inhibitory activity against human
IMPDH2 (<10% at 5 μM).
Fusing an additional benzo-ring onto the phenyl ether of
5 resulted in a compound (18), demonstrating a 3-fold
increase in CpIMPDH inhibitory activity (Table 1). How-
ever, when the methyl group on the R-carbon of the amide
was replaced with a phenyl (19), inhibitory activity was lost.
An isopropyl group (20) was well tolerated, but surprisingly
a cyclopropyl (21) was not. The naphthyl ether could also be
replaced with a 4-quinolinyl ether, resulting in a compound
(22) exhibiting an IC50 of 0.66 μM. The amide derivatives
that demonstrated CpIMPDH inhibitory activity were then
evaluated for enzyme inhibitory activity in the presence BSA.
The CpIMPDH inhibitory activities of 18, 20, and 22 were
only slightly decreased in the presence of BSA.
Table 1. IC50 Determinations for Inhibition of CpIMPDH by Amide
Derivatives
IC50 (μM)a,b
compd
R
X
(-) BSAc
(þ) BSA
Early in the SAR study of 5, attempts were made to find a
bioisostere for the amide functional group.21 Given the many
reported successes with the use of 1,2,3-triazole in ligand
design20-24 including as a replacement for amide bonds,
this heterocycle was incorporated into an analogue of 18,
resulting in 23.25-30 Gratifyingly, 23 demonstrated increa-
sed CpIMPDH inhibitory activity with an IC50 of 130 nM
(Table 2). However, the IC50 value increased significantly to
18
19
20
21
22
Me
Ph
CH
CH
CH
CH
N
1.06 ( 0.1
>5
1.64 ( 0.2
NDd
i-Pr
c-Pr
Me
0.71 ( 0.1
>5
0.83 ( 0.2
ND
0.66 ( 0.2
0.86 ( 0.2
a Positive control: 5; IC50 = 3.3 ( 0.2 μM. b IC50 ( SD were
determined from three independent experiments. c 0.05% Fatty acid
free bovine serum albumin. d Not determined.
Table 2. IC50 Determinations for Inhibition of CpIMPDH by 1,2,3-Triazole Derivatives
IC50 (μM)a,b
compd
R1
Me
R2
X
Y
(-) BSAc
(þ) BSA
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
4-Cl
4-Cl
2-Cl
4-Cl
4-Cl
4-Cl
CH
CH
0.13 ( 0.03
>5
>5
0.78 ( 0.2
i-Pr
Me
CH
CH
CCl
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
NDd
ND
Me
Me
0.087 ( 0.03
0.024 ( 0.008
0.44 ( 0.2
3.9 ( 0.08
0.23 ( 0.06
0.48 ( 0.2
0.70 ( 0.2
0.34 ( 0.1
1.4 ( 0.4
0.65 ( 0.1
0.88 ( 0.03
1.1 ( 0.2
2.3 ( 0.04
0.030 ( 0.001
0.050 ( 0.002
0.042 ( 0.003
0.059 ( 0.02
0.050 ( 0.02
0.052 ( 0.01
H
Me
N
3,4-di-Cl
4-CN
N
0.020 ( 0.01
0.14 ( 0.03
0.040 ( 0.002
0.009 ( 0.006
0.13 ( 0.03
0.031 ( 0.009
0.60 ( 0.05
0.009 ( 0.001
0.029 ( 0.01
0.018 ( 0.003
0.044 ( 0.002
0.013 ( 0.005
0.024 ( 0.005
Me
Me
N
3-Cl, 4-CN
3,4-di-Cl
3,4-di-Cl
3-Cl, 4-CN
3-Cl, 4-CN
4-Cl
N
(R)-Me
(S)-Me
(R)-Me
(S)-Me
(R)-Me
Me
N
N
N
N
CH
Nþ-O-
Nþ-O-
CH
Nþ-O-
CH
4-Cl
CH
CH
Nþ-O-
CH
Nþ-O-
Me
Me
3,4-di-Cl
4-Cl
4-Cl
(R)-Me
(R)-Me
4-Cl
a Positive control: 5; IC50 = 3.3 ( 0.2 μM. b IC50 ( SD were determined from three independent experiments. c 0.05% Fatty acid free bovine serum
albumin. d Not determined.

4626 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Maurya et al.
780 nM in the presence of BSA. Also, unlike the amide series,
replacing the methyl substituent with an isopropyl (24) in the
1,2,3-triazole series was not tolerated. In addition, moving
the para-chloro group on the pendent phenyl to the ortho-
position (25) was detrimental. However, adding a chlorine
atom on the naphthyl ether (26) or replacing the naphthyl
with a 4-quinolinyl (27) resulted in further increases in
inhibitory activity, with IC50 values of 87 and 24 nM,
respectively. Unfortunately, the inhibitory activity for both
compounds was eroded in the presence of BSA. Given the
potency of 27, various other changes were made to this
compound. For example, removing the methyl group (28)
resulted in a significant loss of activity. Introduction of an
additional chloride atom in the 3-position of the pendent
phenyl (29) retained potent inhibitory activity. Introduction
of 4-cyano (30) or 3-chloro-4-cyano (31) substituents on the
pendent phenyl resulted in IC50 values of 140 and 40 nM,
respectively. Next, the enantiomers of 29 were evaluated.
The (R)-enantiomer (32), which demonstrated an IC50 of
9 nM, was significantly more potent (∼14-fold) than the (S)-
enantiomer (33). A similar finding was observed with the
enantiomers 34 and 35. The connectivity of the ether to the
quinoline ring was changed to the 5-position (36) with
retention of potent inhibitory activity. Interestingly, the
inhibitory activity of this quinoline derivative was not as
adversely affected as the other analogues by the presence of
BSA. Finally, in an attempt to increase the polarity of the
1,2,3-triazole series with the goal of mitigating the effects of
BSA on inhibitory activity, the N-oxide of 27 was evaluated.
The IC50 value of 37 was 29 nM and in the presence of BSA
only slightly increased to 50 nM. This 2-fold increase was less
than seen with many of the non-N-oxide derivatives and was
reminiscent of the results observed with the amide series.
Several other N-oxide derivatives in the 1,2,3-triazole series,
such as 38-41 also demonstrated potent inhibitory activity
both in the presence and absence of BSA.
Experimental Section
Chemistry Material and Methods. Unless otherwise noted, all
reagents and solvents were purchased from commercial sources
and used without further purification. All reactions were per-
formed under nitrogen atmosphere unless otherwise noted. The
NMR spectra were obtained using a 400 or 500 MHz spectro-
meter. All 1H NMR spectra are reported in δ units ppm and are
referenced to tetramethylsilane (TMS) if conducted in CDCl3
or to the central line of the quintet at 2.49 ppm for samples in
DMSO-d6. All chemical shift values are also reported with
multiplicity, coupling constants, and proton count. All 13C
NMR spectra are reported in δ units ppm and are referenced
to the central line of the triplet at 77.23 ppm if conducted in
CDCl3 or to the central line of the septet at 39.5 ppm for samples
in DMSO-d6. Coupling constants (J values) are reported in
hertz. Column chromatography was carried out on SILI-
CYCLE SiliaFlash silica gel F60 (40-63 μm, mesh 230-400).
High-resolution mass spectra were obtained using a SX-102A
mass spectrometer (JEOL USA, Inc., Peabody, MA), a LCT
mass spectrometer (Micromass Inc., Beverly, MA), or a Q-tof
Ultima API mass spectrometer. All melting points were taken in
glass capillary tubes on a Mel-Temp apparatus and are uncor-
rected. All test compounds had a purity g95% as determined by
either elemental analysis or high performance liquid chroma-
tography (HPLC) analysis, unless otherwise noted. The elemen-
tal composition of compounds agreed to within (0.4% of
the calculated values. Chemical and enantiomeric purities
were determined using high performance liquid chromato-
graphy (HPLC) analysis on a Hewlett-Packard 1100 series
instrument equipped with a quaternary pump and a Daicel
Chiralpak AD column (250 ꢀ 4.6 mm). UV absorption was
monitored at λ=254 nm. The injection volume was 1 μL. HPLC
gradient was 50% n-hexane and 50% i-propanol with a flow
rate of 1.0 mL/min. In some cases, chemical purity was deter-
mined using a Agilent 1100 HPLC instrument equipped with
a quaternary pump and a Zorbax SB-C8 column (30 mm ꢀ
4.6 mm, 3.5 μm). UV absorption was monitored at λ=254 nm.
The injection volume was 5 μL. HPLC gradient went from
5% acetonitrile and 95% water to 95% acetonitrile and
5% water (both solvents contain 0.1% trifluoroacetic acid)
over 1.9 min with a total run time of 2.5 min and a flow rate
of 3.0 mL/min.
Conclusions
An SAR study of the R-phenoxide anilide derivative 5 that
was previously shown to be a moderately potent and very
selective inhibitor of C. parvum IMPDH,15 an essential en-
zyme to this important human pathogenic protozoan parasite
and potential bioterrorism agent, was undertaken. Initially,
the amide was retained and addition of a fused ring onto the
phenyl ether, resulting in 1-naphthyl or 4-quinolinyl ethers,
was shown to increase CpIMPDH inhibitory activity.
The active amide analogues retained inhibitory activity in
the presence of BSA. Replacement of the amide with the
bioisostere 1,2,3-triazole resulted in increased CpIMPDH
inhibitory activity, but activity was eroded in the presence
of BSA. Further increases in CpIMPDH inhibitory activity
were accomplished with various electron-withdrawing groups
in the 3- and/or 4-positions, but not the 2-position, of the
pendent phenyl ring. Also, a small alkyl group (i.e., methyl)
was required on the R-position of the ether with the (R)-
enantiomers demonstrating significantly more activity than
the (S)-enantiomers. Finally, conversion of the quinolines
to quinoline-N-oxides retained potent inhibitory activity
both in the presence and absence of BSA. The 1,2,3-triazole
CpIMPDH inhibitors described herein can serve as new tools
for elucidating the role of IMPDH in C. parvum and related
organisms. These inhibitors could also serve as potential lead
compounds for therapeutic development for the treatment of
cryptosporidiosis.
Synthesis of Ethyl r-Bromocyclopropaneacetate (7, R=c-Pr).
A flame-dried two-neck round-bottom flask fitted with a reflux
condenser and nitrogen inlet was charged with anhydrous THF,
freshly activated Mg (120 mg, 4.95 mmol), and a catalytic
amount of iodine. A small portion of cyclopropyl bromide
dissolved in THF was added. After initiation of reflux, the
reaction mixture was cooled to -20 °C and the remaining
cyclopropyl bromide (500 mg, 4.13 mmol) was gradually added.
After 30 min, a freshly distilled solution of glyoxalate 9 (549 mg,
5.37 mmol) in THF was added over a 10 min period and the
resulting solution was stirred at -20 °C for 2 h before being
quenched with a small amount of water. After 10 min, the
reaction mixture was further diluted with water (50 mL) and
extracted with ethyl acetate (3 ꢀ 50 mL). The organic extracts
were combined, dried over anhydrous MgSO4, filtered, concen-
trated in vacuo, and purified by column chromatography elut-
ing with ethyl acetate/n-hexane (a gradient of 10-20%) to
furnish ethyl R-hydroxycyclopropaneacetate (422 mg, 71%)
as a viscous oil. The oil (350 mg, 2.43 mmol) was dissolved
in anhydrous DCM and cooled to 0 °C. Then Ph3P (2.04 g,
7.78 mmol) was added followed by CBr4 (1.20 g, 3.64 mmol).
The reaction mixture was stirred at 0 °C for 2 h and then
concentrated in vacuo. The Ph3PO was precipitated by addition
of n-hexane and removed by filtration. The crude reaction
mixture was purified by flash column chromatography eluting
with ethyl acetate/n-hexane (1:9) to furnish ethyl R-bromo-
cyclopropaneacetate (7, R=c-Pr): (311 mg, 62% yield).

Article
General Procedure for the Synthesis of 2-(1-Naphthalenyloxy)-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4627
Synthesis of 2-Bromo-N-(4-chlorophenyl)propanamide (12).
To a solution of 4-chloroaniline (400 mg, 3.13 mmol) in anhy-
drous DCM at room temperature under a nitrogen atmosphere
was added slowly 2-bromopropanoyl chloride (474 μL, 4.7
mmol). The reaction mixture was stirred at room temperature
for 2 h and then diluted with water (50 mL) and extracted with
ethyl acetate (3 ꢀ 50 mL). The organic extracts were combined,
washed with brine, dried over anhydrous MgSO4, and concen-
trated in vacuo to give 2-bromo-N-(4-chlorophenyl)propan-
amide, which was used without further purifications.
acetic Acids (9). Exemplified for 2-Cyclopropyl-2-(1-naphthale-
nyloxy)acetic Acid (9, R=c-Pr). To a solution of 1-naphthol
(170 mg, 1.18 mmol) in anhydrous DMF (10 mL) was added
K2CO3 (510 mg, 3.53 mmol) and ethyl R-bromocyclopropane-
acetate (295 mg, 1.41 mmol). The mixture was stirred at room
temperature for 2 h and then diluted with water (50 mL) and
then extracted with ethyl acetate (3 ꢀ 50 mL). The organic
extracts were combined, washed with brine, dried over anhy-
drous MgSO4, filtered, concentrated in vacuo, and purified by
flash column chromatography using a mixture of ethyl acetate/
n-hexane (1:9) to furnish ethyl 2-cyclopropyl-2-(1-naphthalenyl-
oxy)acetate (8, R=c-Pr, 296 mg, 93%) as a white solid. The ester
(250 mg, 0.92 mmol) was dissolved in 20 mL THF:H2O (2:1),
and then 3 N NaOH (111 mg, 2.77 mmol) was added. The
reaction was heated at 80 °C for 6 h. After cooling, the reaction
mixture was quenched with 1N HCl to a pH ∼7 and then
extracted with chloroform. The organic extract was dried over
anhydrous MgSO4, filtered, concentrated in vacuo, and purified
by flash column chromatography eluting with a mixture of ethyl
acetate/n-hexane (2:1) to furnish 2-cyclopropyl-2-(1-naphthale-
nyloxy)acetic acid (9, R=c-Pr) (136 mg, 61%) as a white solid.
General Procedure for theSynthesis of N-(4-Chlorophenyl)-
2-(1-naphthalenyloxy)acetamides (10, X=CH). Exemplified for
N-(4-Chlorophenyl)-2-cyclopropyl-2-(1-naphthalenyloxy)acet-
amide (21). To a solution of 2-cyclopropyl-2-(1-naphthaleny-
loxy)acetic acid (120 mg, 0.49 mmol) and 4-chloroaniline (44.0
μL, 0.49 mmol) in anhydrous DCM (10 mL) under N2 cooled at
Synthesis N-(4-Chlorophenyl)-2-[[4-quinolinyl]oxy]propanamide
(22). To a solution of 4-hydroxyquinoline (100 mg, 0.69 mmol)
in anhydrous DMF under a nitrogen atmosphere was added
K2CO3 (286 mg, 2.10 mmol) and a solution of 2-bromo-N-(4-
chlorophenyl)propanamide (218 mg, 0.83 mmol) in DMF. The
reaction mixture was stirred for 12 h at room temperature before
being diluted with water (50 mL) and extracted with chloroform
(3 ꢀ 50 mL). The combined organic extracts were dried over
anhydrous MgSO4, filtered, concentrated in vacuo, and purified
by flash chromatography using ethyl acetate/n-hexane (1:9) to
furnish 22 (207 mg, 92% yield) as a white solid; mp 170-172 °C.
1H NMR (CDCl3, 400 MHz) δ 1.83 (d, J=6.4 Hz, 3H), 5.06 (q,
J=6.8, 13.6 Hz, 1H), 6.76 (d, J=5.6 Hz, 1H), 7.28 (d, J=8.4 Hz,
2H), 7.46 (d, J=8.4 Hz, 2H), 7.61 (t, J=8.0 Hz, 1H), 7.77 (t, J=
7.6 Hz, 1H), 8.09 (d, J=7.2 Hz, 1H), 8.26 (d, J=8.4 Hz, 1H), 8.76
(d, J=5.6 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ 18.72, 75.83,
102.05, 122.27, 121.32, 121.58, 126.61, 129.34, 129.60, 130.34,
130.51, 135.40, 149.74, 151.48, 159.47, 169.20. ESI-HRMS for
C18H16N2O2Cl (M þ H)þ calcd 327.0900; found 327.0901.
General Procedure for the Preparation of Propargyl Ether 16
via the Mitsonobu Reaction. Exemplified for 1-[(1-Methyl-2-
propyn-1-yl)oxy]naphthalene (16, R1=Me, X=Y=CH). To a
solution of 1-naphthol (200 mg, 1.38 mmol) and but-3-yn-2-ol
(146 mg, 2.07 mmol) in anhydrous DCM (10 mL) under a
nitrogen atmosphere and at 0 °C was added Ph3P (435 mg,
1.66 mmol) portion wise. The reaction mixture was stirred for 10
min. and then DEAD (360 mg, 2.07 mmol) (70% solution in
toluene) was slowly added. The resulting reaction solution was
stirred at the room temperature for 24 h and then diluted with
water (50 mL) and extracted with chloroform (3 ꢀ 50 mL). The
combined organic extracts were washed with brine, dried over
anhydrous MgSO4, filtered, concentrated in vacuo, and the
residue was purified by flash column chromatography using
ethyl acetate/n-hexane (a gradient of 5-10%) to furnish 1-[(1-
methyl-2-propyn-1-yl)oxy]naphthalene (176 mg, 65%) as a
viscous oil.
General Procedure for the Preparation of Propargyl Ether 16
via the Corey-Fuchs Reaction. Exemplified for the Synthesis of
1-(1-Methylethyl)-2-propyn-1-yl]oxy]naphthalene (16, R1=i-Pr,
X=Y=CH). A solution of 14 (R1=i-Pr, R2=CO2H, X=Y=
CH, 880 mg, 3.27 mmol) in anhydrous THF was cooled to 0 °C,
and then LiAlH4 (311 mg, 8.18 mmol) was added. The reaction
mixture was stirred for 5 h at 0 °C and then quenched with water
(50 mL). The mixture was stirred until the organic and aqueous
layers separated. The mixture was extracted with chloroform
(3 ꢀ 100 mL). The combined organic layers were washed with
brine, dried over anhydrous MgSO4, filtered, concentrated in
vacuo, and purified by flash column chromatography using
ethyl acetate/n-hexane (2:1) to give alcohol 14 (R1=i-Pr, R2=
CH2OH, X=Y=CH, 466 mg, 62%) as a thick viscous oil.
A flame-dried two-neck round-bottom flask containing ox-
alyl chloride (370 μL, 4.34 mmol) in anhydrous DCM was
cooled at -78 °C under a nitrogen atmosphere. Next, anhydrous
DMSO (679 mg, 8.7 mmol) was added dropwise via a syringe.
The resulting solution was allowed to stir at -78 °C for 10 min,
and then alcohol 14 (R1 =i-Pr, R2 = CH2OH, X=Y =CH,
400 mg, 1.74 mmol) dissolved in anhydrous DCM was added
gradually via a syringe. The resulting reaction mixture was
allowed to stir for 1 h at -78 °C and then quenched with
triethylamine (1.95 mL, 13.9 mmol) before being allowed to
0 °C was added EDCI HCl (187.9 mg, 0.98 mmol) portion wise.
3
The resulting solution was stirred at room temperature for 12 h.
The reaction mixture was diluted with water (50 mL) and extrac-
ted with ethyl acetate (3 ꢀ 100 mL). The organic extracts were
combined, washed with brine, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography using ethyl acetate/n-hexane (a
gradient of 5-10%) to furnish 21 (148 mg, 86%) as a white solid;
mp 204-206 °C. 1H NMR (CDCl3, 400 MHz) δ 0.65-0.78 (m,
4H), 1.52 (m, 1H), 4.41 (d, J=6.4 Hz, 1H), 6.70 (d, J=8.0 Hz,
1H), 7.25 (d, J=6.0 Hz, 3H), 7.43 (d, J=8.4 Hz, 2H), 7.61-7.70
(m, 3H), 7.97 (s, 1H), 8.22 (d, J=8.4 Hz, 1H), 8.34 (d, J=8.0 Hz,
1H). 13C NMR (CDCl3, 100 MHz) δ 2.70, 3.16, 14.42, 82.72,
108.01, 115.68, 121.47, 122.03, 122.96, 127.64, 128.43, 129.28,
129.63, 130.07, 132.96, 135.61, 152.83, 169.27. ESI-HRMS for
C21H17ClNO2 (M - H)þ calcd 350.0948; found 350.0956.
N-(4-Chlorophenyl)-2-(1-naphthalenyloxy)propanamide (18).
Melting point 146-148 °C. 1H NMR (CDCl3, 400 MHz) δ
1.79 (d, J=6.8 Hz, 3H), 4.98 (q, J=6.8, 13.2 Hz, 1H), 6.87 (d, J=
7.6 Hz, 1H), 7.26 (d, J=8.4 Hz, 2H), 7.37 (t, J=8.4 Hz, 1H), 7.46
(d, J=8.8 Hz, 2H), 7.56 (dd, J=7.8, 12.5 Hz, 3H), 7.86 (m, 1H),
8.24-8.32 (m, 2H). 13C NMR (CDCl3, 100 MHz) δ 19.08, 76.26,
107.41, 121.42, 121.54, 122.47, 125.91, 126.04, 126.16, 127.03,
128.14, 129.26, 129.95, 134.93, 135.75, 152.61, 170.58. Anal.
(C19H16ClNO2) C, H, N.
N-(4-Chlorophenyl)-r-(1-naphthalenyloxy)benzeneacetamide
(19). Yield 92%; mp 176-178 °C. 1H NMR (CDCl3, 400 MHz)
δ 5.86 (s, 1H), 6.88 (d, J=8.0 Hz, 1H), 7.26-7.61 (m, 11H), 7.68
(d, J=7.2 Hz, 2H), 7.87 (d, J=7.2 Hz, 1H), 7.37 (d, J=8.0 Hz,
1H), 8.44 (s, 1H). 13C NMR (CDCl3, 100 MHz) δ 81.09, 107.87,
121.41, 121.47, 122.58, 125.66, 126.01, 126.27, 126.61, 127.02,
128.25, 129.12, 129.15, 129.27, 130.06, 134.94, 135.71, 135.98,
152.34, 168.11. Anal. (C24H18ClNO2) C, H, N.
N-(4-Chlorophenyl)-3-methyl-2-(1-naphthalenyloxy)butanami-
1
de (20). Yield 91%; mp 150-152 °C. H NMR (CDCl3, 400
MHz) δ 1.22 (dd, J=6.8, 22.8 Hz, 6H), 2.54 (m, 1H), 4.68 (d, J=
4.4 Hz, 1H), 6.83 (d, J=8.0 Hz, 1H), 7.25 (m, 2H), 7.35 (t, J=8.0
Hz, 1H), 7.40 (d, J=8.8 Hz, 2H), 7.52 (d, J=8.0 Hz, 1H), 7.54-
7.60 (m, 2H), 7.86 (m, 1H), 8.01 (s, 1H), 8.38 (m, 1H). 13C NMR
(CDCl3, 100 MHz) δ 17.47, 19.54, 32.48, 84.51, 106.81, 121.53,
122.20, 125.71, 126.12, 127.04, 128.15, 129.21, 129.97, 134.92,
135.57, 153.52, 169.68. Anal. (C21H20ClNO2) C, H, N.

4628 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Maurya et al.
warm to room temperature. The reaction mixture was extracted
with DCM (3 ꢀ 50 mL), washed with brine, dried over anhy-
drous MgSO4, filtered, and concentrated in vacuo to give
aldehyde 15 (R1=i-Pr, X=Y=CH), which was used without
further purification.
4-(1-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quino-
line (27). Yield 91%; mp 128-130 °C. H NMR (CDCl3, 400
1
MHz) δ 1.95 (d, J=6.4 Hz, 3H), 5.97 (q, J=6.4, 12.8 Hz, 1H),
6.89 (d, J=5.2 Hz, 1H), 7.47 (d, J=8.8 Hz, 2H), 7.53 (t, J=8.0
Hz, 1H), 7.64 (d, J=8.8 Hz, 2H), 7.71 (t, J=7.2 Hz, 1H), 7.91 (s,
1H), 8.03 (d, J=8.4 Hz, 1H), 8.28 (d, J=7.6 Hz, 1H), 8.70 (d, J=
4.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ 22.21, 70.05,
102.42, 119.17, 121.92, 121.99, 125.93, 129.23, 130.06, 130.14,
134.98, 135.48, 149.59, 150.01, 151.51, 160.17. ESI-HRMS for
C19H16ClN4O (M þ H)þ calcd 351.1013; found 351.1002. Purity
as determined by HPLC analysis was 94.7%.
A solution of 15 (R1=i-Pr, X=Y=CH, 360 mg, 1.58 mmol) in
DCM (10 mL) was cooled at 0 °C, and then Ph3P (1.24 g,
4.74 mmol) and CBr4 (785 mg, 2.37 mmol) were sequentially
added. The resulting mixture was stirred at room temp for 2 h.
The reaction mixture was concentrated in vacuo, and the Ph3PO
was precipitated by addition of n-hexane and removed by
filtration. The filtrate was concentrated and the residue purified
using a filter column (ethyl acetate/n-hexane as eluent).
The resulting material (360 mg, 1.58 mmol) was dissolved in
anhydrous THF and cooled to -78 °C. Next, n-BuLi (121 mg,
1.90 mmol) was gradually added, and the resulting solution
stirred for 2 h at -78 °C. The mixture was quenched with water
(50 mL), allowed to stir at room temperature for 30 min, and
then extracted with ethyl acetate (3 ꢀ 100 mL). The combined
organic extracts were dried over anhydrous MgSO4, filtered,
concentrated in vacuo, and purified by flash column chro-
matography eluting with ethyl acetate/n-hexane (5:95) to fur-
nish 1-(1-methylethyl)-2-propyn-1-yl]oxy]naphthalene (16, R1=
i-Pr, X=Y=CH, 282 mg, 72%) as a white solid.
4-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)quino-
1
line (28). Yield 89%; mp 230-232 °C. H NMR (CDCl3, 400
MHz) δ 5.50 (s, 2H), 6.34 (d, J=8.0 Hz, 1H), 7.39 (t, J=7.2 Hz,
1H), 7.47 (d, J=8.4 Hz, 2H), 7.57-7.65 (m, 4H), 7.74 (d, J=8.0
Hz, 1H), 7.77 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ 48.81, 111.20, 115.77, 120.21, 121.92,
124.28, 127.49, 127.58, 130.24, 132.75, 135.29, 139.87, 143.11,
143.93, 178.46. ESI-HRMS for C18H14ClN4O (M þ H)þ calcd
337.0856; found 337.0847.
4-(1-(1-(3,4-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quino-
line (29). Yield 89%; mp 62-64 °C. 1H NMR (CDCl3, 400 MHz) δ
1.95 (d, J=6.4 Hz, 3H), 5.97 (q, J=6.4, 12.8Hz, 1H),6.87(d,J=5.6
Hz, 1H), 7.26 (s, 1H), 7.53 (t, J=7.6 Hz, 1H), 7.57 (s, 2H), 7.71 (t,
J=7.2 Hz, 1H), 7.85 (s, 1H), 7.91 (s, 1H), 8.03 (d, J=8.4 Hz, 1H),
8.28 (d, J=8.4 Hz, 1H), 8.70 (d, J=4.4 Hz, 1H). 13CNMR(CDCl3,
100 MHz) δ 22.21, 69.96, 102.37, 119.13, 119.69, 121.72, 121.97,
122.50, 125.98, 129.26, 130.10, 131.68, 133.35, 134.23, 135.98,
149.61, 150.28, 151.50, 160.10. Anal. (C19H14Cl2N4O) C, H, N.
4-(4-(1-(Quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzo-
nitrile (30). Yield 87%; mp 176-178 °C. 1H NMR (CDCl3, 400
MHz) δ 1.96 (d, J=6.8 Hz, 3H), 5.98 (q, J=6.0, 12.8 Hz, 1H),
6.87 (d, J=4.8 Hz, 1H), 7.26 (s, 1H), 7.54 (t, J=7.2 Hz, 1H), 7.72
(t, J=7.2 Hz, 1H), 7.80 (d, J=8.8 Hz, 2H), 7.88 (d, J=8.0 Hz,
2H), 8.03 (m, 1H), 8.28 (d, J=8.0 Hz, 1H), 8.70 (s, 1H). 13C
NMR (CDCl3, 100 MHz) δ 22.16, 69.90, 102.35, 112.86, 117.78,
118.98, 120.82, 121.94, 126.02, 129.25, 130.15, 134.11, 139.74,
149.59, 150.55, 151.45, 160.07. ESI-HRMS for C20H16N5O (M
þ H)þ calcd 342.1355; found 342.1355.
General Procedure for the Preparation of 1H-1,2,3-Triazoles
17. Exemplified for 1-(4-Chlorophenyl)-4-(2-methyl-1-(1-naph-
thalenyloxy)propyl)-1H-1,2,3-triazole (24). A single-neck round-
bottom flask under an argon atmosphere was charged with 16
(R1 = i-Pr, X = Y = CH, 114 mg, 0.51 mmol), anhydrous
acetonitrile (5 mL), 1-azido-4-chlorobenzene (78.3 mg, 0.51
mmol), and DIPEA (254 μL, 1.53 mmol). The reaction mixture
was allowed to stir at room temperature for 10 min, and then
finely powdered CuI (194.2 mg, 1.02 mmol) was added portion
wise. After 30 min of stirring at room temperature, the reaction
mixture was quenched with saturated aqueous NH4Cl, diluted
with water (50 mL), and extracted with chloroform (3 ꢀ 50 mL).
The combined organic extracts were washed with brine, dried
over anhydrous MgSO4, filtered, concentrated in vacuo, and
purified by flash column chromatography using ethyl acetate/n-
hexane (a gradient of 10-20%) to furnish 24 (167 mg, 87%) as a
gelatinous solid. 1H NMR(CDCl3, 400 MHz) δ 1.16, 1.20 (dd, J=
6.5, 22.0 Hz, 6H), 2.47-2.53 (m, 1H), 5.55 (d, J=5.0Hz, 1H), 6.80
(d, J=8.0 Hz, 1H), 7.25 (m, 1H), 7.37-7.43(m, 3H), 7.49 (m, 2H),
7.61 (d, J=9.0 Hz, 2H), 7.79 (m, 2H), 8.38 (m, 1H). ESI-HRMS
for C22H21ClN3O (M þ H)þ calcd 378.1373; found 378.1383.
1-(4-Chlorophenyl)-4-(1-(naphthalene-1-yloxy)ethyl)-1H-1,2,3-
triazole (23). Melting point 98-100 °C. 1H NMR (CDCl3, 400
MHz) δ 1.91 (d, J=6.0 Hz, 3H) 5.90 (q, J=6.8, 12.8 Hz, 1H),
6.92 (d, J=7.2 Hz, 1H), 7.26 (s, 1H), 7.31 (t, J=8.8 Hz, 1H),
7.41-7.51 (m, 5H), 7.63 (d, J=8.0 Hz, 2H), 7.80 (m, 1H), 7.88 (s,
1H), 8.34 (m, 1H). 13C NMR (CDCl3, 100 MHz) δ 22.56, 69.90,
107.02, 119.08, 120.98, 121.87, 122.19, 125.53, 126.07, 126.14,
126.66, 127.80, 130.07, 134.73, 134.82, 135.64, 151.38, 153.21.
ESI-HRMS for C20H17ClN3O (M þ H)þ calcd 350.1060; found
350.1074.
2-Chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)-
benzonitrile (31). Yield 85%; mp 194-196 °C. 1H NMR (CDCl3,
400 MHz) δ 1.96 (d, J=6.8 Hz, 3H), 5.98 (q, J=6.0, 12.8 Hz, 1H),
6.84 (d, J=4.8 Hz, 1H), 7.54 (t, J=7.2 Hz, 1H), 7.72 (t, J=8.0 Hz,
1H), 7.79 (q, J=8.4, 15.2 Hz, 2H), 7.97 (s, 1H), 8.00 (s, 1H), 8.04 (d,
J=8.8 Hz, 1H), 8.23 (d, J=8.0 Hz, 1H), 8.69 (d, J=4.8 Hz, 1H).
13C NMR (CDCl3, 100 MHz) δ 22.14, 69.81, 102.29, 113.53,
115.12, 118.60, 119.02, 121.51, 121.65, 121.91, 126.06, 129.27,
130.18, 135.53, 138.93, 140.24, 149.60, 150.82, 151.43, 159.98.
ESI-HRMS for C20H15ClN5O (M þ H)þ calcd 376.0965; found
376.0975.
(R)-4-(1-(1-(3,4-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)-
quinoline (32). Yield 91%; mp 62-64 °C. 1H NMR (CDCl3, 400
MHz) δ 1.95 (d, J=6.8 Hz, 3H), 5.97 (q, J=6.4, 12.8 Hz, 1H),
6.87 (d, J=5.2 Hz, 1H), 7.54 (t, J=7.2 Hz, 1H), 7.58 (s, 2H), 7.72
(t, J=7.2 Hz, 1H), 7.85 (s, 1H), 7.91 (s, 1H), 8.05 (d, J=8.8 Hz,
1H), 8.28 (d, J = 8.4 Hz, 1H), 8.70 (bs, 1H). ESI-HRMS for
C19H15N4OCl2 (M þ H)þ calcd 385.0623; found 385.0605.
(S)-4-(1-(1-(3,4-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)-
quinoline (33). Yield 89%; mp 64-66 °C. 1H NMR (CDCl3,
400 MHz) δ 1.95 (d, J=6.8 Hz, 3H), 5.98 (q, J=6.8, 12.8 Hz, 1H),
6.88 (d, J=4.8Hz, 1H), 7.54 (t, J=6.4Hz, 1H), 7.58 (d, J=1.2 Hz,
2H), 7.72 (m, 1H), 7.85 (m, 1H), 7.91 (s, 1H), 8.04 (d, J=8.0 Hz,
1H), 8.28 (d, J = 8.4 Hz, 1H), 8.70 (d, J = 5.6 Hz, 1H). ESI-
HRMS for C19H15N4OCl2 (M þ H)þ calcd 385.0623; found
385.0628.
1-(2,6-Dichlorophenyl)-4-(1-(naphthalen-1-yloxy)ethyl)-1H-
1,2,3-triazole (25). Yield 86%; gelatinous solid. 1H NMR
(CDCl3, 500 MHz) δ 1.98 (d, J=6.0 Hz, 3H), 5.92 (q, J=6.5,
13.0 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 7.26 (s, 1H), 7.31 (t, J=8.0
Hz, 1H), 7.37-7.49 (m, 6H), 7.79 (m, 1H) 8.32 (m, 1H). ESI-
HRMS for C20H16Cl2N3O (M þ H)þ calcd 384.0670; found
384.0672.
4-(1-(4-Chloronaphthalen-1-yloxy)ethyl)-1-(4-chlorophenyl)-
1H-1,2,3-triazole (26). yield 84%; gelatinous solid. H NMR
1
(CDCl3, 500 MHz) δ 1.91 (d, J=6.5 Hz, 3H), 5.86 (q, J=6.5,
13.5 Hz, 1H), 6.85 (d, J=8.0 Hz, 1H), 7.26 (s, 1H), 7.39 (d, J=
8.5 Hz, 1H), 7.46 (d, J=8.5 Hz, 1H), 7.56 (t, J=8.5 Hz, 1H),
7.62 (t, J=8.0 Hz, 3H), 7.87 (s, 1H), 8.20 (d, J=8.5 Hz, 1H),
8.36 (d, J=8.5 Hz, 1H). ESI-HRMS for C20H16Cl2N3O (M þ
H)þ calcd 384.0670; found 384.0684.
(R)-2-Chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-tri-
azol-1-yl)benzonitrile (34). Yield 92%; mp 176-178 °C. 1H
NMR (CDCl3, 400 MHz) δ 1.96 (d, J=6.0 Hz, 3H), 5.98 (q,
J=6.0, 12.8 Hz, 1H), 6.84 (d, J=5.6 Hz, 1H) 7.55 (t, J=7.2 Hz,
1H), 7.71-7.83 (m, 3H), 7.97 (m, 2H), 8.04 (d, J=8.4 Hz, 1H),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4629
8.28 (d, J=8.8 Hz, 1H), 8.70 (d, J=5.2 Hz, 1H). ESI-HRMS for
C20H15N5OCl (M þ H)þ calcd 376.0965; found 376.0964.
(S)-2-Chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-
1-yl)benzonitrile (35). Yield 91%; mp 176-178 °C. H NMR
0.61 mmol) in anhydrous DCM under a nitrogen atmosphere
was added m-chloroperbenzoic acid (163 mg, 0.73 mmol, 77%).
The reaction mixture was stirred at room temperature for 2 h,
concentrated in vacuo, and purified by flash column chro-
matography eluting with methanol/chloroform (a gradient of
5-10%) to furnish (R)-5-(but-3-yn-2-yloxy)quinoline 1-oxide
(16, R1=(R)-Me, X=Nþ-O-, Y=CH, 120 mg, 93%) as a white
solid; mp 156-158 °C. 1H NMR (CDCl3, 400 MHz) δ 1.82 (d,
J=6.8 Hz, 3H), 2.54 (s, 1H), 5.05 (q, J=6.8, 13.6 Hz, 1H), 7.20
(d, 1H, J=8.0 Hz, 1H) 7.26 (t, J=8.0 Hz, 3H), 7.68 (t, J=9.2 Hz,
1H), 8.15 (d, J=9.2 Hz, 1H), 8.35 (d, J=8.8 Hz, 1H), 8.54 (d, J=
5.2 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ 22.32, 64.68, 75.09,
81.96, 109.52, 112.49, 120.09, 121.28, 123.85, 130.57, 136.32,
142.64, 153.36. ESI-HRMS for C13H12NO2 (M þ H)þ calcd
214.0868; found 214.0875.
Evaluation of CpIMPDH Inhibition. Determination of IC50
Values. Inhibition of recombinant CpIMPDH, purified from
E. coli,12 was assessed by monitoring the production of NADH
by fluorescence at varying inhibitor concentrations (25 pM to
5 μM). IMPDH was incubated with inhibitor for 5 min at room
temperature prior to addition of substrates. The following
conditions were used: 50 mM Tris-HCl, pH 8.0, 100 mM KCl,
3 mM EDTA, 1 mM dithiothreitol (assay buffer) at 25 °C, 10
nM CpIMPDH, 300 μM NAD, and 150 μM 1. To characterize
the nonspecific binding of inhibitors, assays were also carried
out in the presence of 0.05% BSA (fatty acid free). IC50 values
were calculated for each inhibitor according to eq 1 using the
SigmaPlot program (SPSS, Inc.):
1
(CDCl3, 400 MHz) δ 1.96 (d, J=6.8 Hz, 3H), 5.98 (q, J=6.0,
12.8 Hz, 1H), 6.84 (d, J=5.6 Hz, 1H), 7.54 (t, J=8.0 Hz, 1H),
7.71-7.82 (m, 3H), 7.98 (m, 2H), 8.04 (d, J=8.0 Hz, 1H), 8.28
(d, J=8.4 Hz, 1H), 8.70 (d, J=4.8 Hz, 1H). ESI-HRMS for
C20H15N5OCl (M þ H)þ calcd 376.0965; found 376.0974.
(R)-5-(1-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)-
quinoline (36). Yield 82%; mp 94-96 °C. 1H NMR (CDCl3, 400
MHz) δ 1.92 (d, J=6.4 Hz, 3H), 5.89 (q, J=6.0, 12.8 Hz, 1H),
7.00 (d, J=8.0 Hz, 1H), 7.40 (dd, J=3.6, 8.0 Hz, 1H), 7.46 (d, J=
8.4 Hz, 2H), 7.55 (t, J=8.4 Hz, 1H), 7.65 (d, J=8.0 Hz, 2H), 7.69
(d, J=8.8 Hz, 1H), 7.88 (s, 1H), 8.65 (d, J=8.0 Hz, 1H), 8.91 (s,
1H). 13C NMR (CDCl3, 100 MHz) δ 22.36, 70.17, 107.39,
119.10, 120.52, 121.40, 121.87, 122.32, 129.56, 130.10, 131.00,
134.85, 135.56, 149.36, 150.76, 150.95, 152.95. ESI-HRMS for
C19H16N4OCl (M þ H)þ calcd 351.1013; found 351.1002.
4-(1-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quino-
line 1-Oxide (37). Yield 88%; mp 158-160 °C. 1H NMR
(DMSO-d6, 400 MHz) δ 1.85 (d, J=6.8 Hz, 3H), 6.09 (q, J=
6.0, 12.8 Hz, 1H), 7.20 (d, J=6.8Hz, 1H), 7.67 (d, J=8.4 Hz, 2H),
7.74 (t, J=7.6 Hz, 1H), 7.86 (t, J=8.4 Hz, 1H), 7.95 (d, J=9.2 Hz,
2H), 8.25 (d, J=8.8 Hz, 1H), 8.50 (dd, J=6.4, 12.0 Hz, 2H), 9.06
(s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ 20.58, 69.53, 103.30,
119.27, 121.56, 121.82, 122.67, 122.80, 128.14, 129.87, 130.79,
133.06, 135.32, 135.48, 140.72, 148.38, 150.38. ESI-HRMS for
C19H16N4O2Cl (M þ H)þ calcd 367.0962; found 367.0948.
4-(1-(1-(3,4-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)-
υi ¼ υo=ð1þ½Iꢁ=IC50Þ
ð1Þ
1
quinoline 1-Oxide (38). Yield 92%; mp 216-218 °C. H NMR
where υi is initial velocity in the presence of inhibitor (I) and υo is
the initial velocity in the absence of inhibitor. Inhibition at
each inhibitor concentration was measured in quadruplicate
and averaged; this value was used as υi. The IC50 values were
determined three times; the average and standard deviations are
reported.
(DMSO-d6, 400 MHz) δ 1.85 (d, J=6.0 Hz, 3H), 6.09 (q, J=6.0,
12.8 Hz, 1H), 7.19 (d, J=6.4 Hz, 1H), 7.74 (t, J=8.0 Hz, 1H),
7.86 (m, 2H), 7.98 (d, J=9.2 Hz, 1H), 8.26 (m, 2H), 8.50 (dd, J=
7.2, 12.0 Hz, 2H), 9.12 (s, 1H). 13C NMR (DMSO-d6, 100 MHz)
δ 20.58, 69.47, 103.33, 119.28, 120.16, 121.74, 121.85, 122.67,
122.78, 128.15, 130.76, 131.09, 131.81, 132.33, 135.39, 136.03,
140.71, 148.53, 150.21. ESI-HRMS for C19H15N4O2Cl2 (M þ
H)þ calcd 401.0571; found 401.0566.
Acknowledgment. This work was supported by funding
from the National Institute of Allergy and Infectious Diseases
(U01AI075466) to LH. Also, CRJ and NNB were supported
by NSF REU CHE-0552767. GDC thanks the New England
Regional Center of Excellence for Biodefense and Emerging
Infectious Diseases (NERCE/BEID), the Harvard NeuroDis-
covery Center and the Partners Center for Drug Discovery for
financial support. The authors would also like to thank Prof.
BarrySnider (Department of Chemistry, Brandeis University)
for helpful suggestions and Prof. Li Deng (Department of
Chemistry, Brandeis University) for use of instrumentation.
5-(1-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quino-
line 1-Oxide (39). Yield 81%; mp 165-167 °C. 1H NMR
(CDCl3, 400 MHz) δ 1.93 (d, J=6.0 Hz, 3H), 5.90 (q, J=6.8
12.8 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H) 7.26 (t, J=6.0 Hz, 1H), 7.47
(d, J=8.4 Hz, 2H), 7.60 (t, J=8.4 Hz, 1H), 7.65 (d, J=8.4 Hz,
2H), 7.91 (s, 1H), 8.20 (d, J=8.4 Hz, 1H) 8.29 (d, J=9.2 Hz, 1H),
8.53 (d, J=5.2 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ 22.27,
70.58, 109.66, 112.34, 119.20, 120.13, 121.03, 121.91, 123.94,
130.26, 130.73, 135.01, 135.49, 136.33, 142.76, 150.15, 153.56.
ESI-HRMS for C19H16N4O2Cl (M þ H)þ calcd 367.0962; found
367.0977. Purity as determined by HPLC analysis was 94.3%.
(R)-4-(1-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)-
1
quinoline 1-Oxide (40). Yield 91%; mp 172-174 °C. H NMR
Supporting Information Available: Detailed information for
combustion analyses, HPLC methods and purity assessments,
including % ee determinations. This material is available free of
charge via the Internet at http://pubs.acs.org.
(CDCl3, 400 MHz) δ 1.96 (d, J = 8.8 Hz, 3H), 5.92 (q,
J=6.0, 12.8 Hz, 1H), 6.88 (d, J=6.8 Hz, 1H), 7.48 (d, J=8.8
Hz, 2H), 7.66 (d, J=8.8 Hz, 3H), 7.80 (t, J=7.2 Hz, 1H), 7.96 (s,
1H), 8.28 (d, J=8.0 Hz, 1H), 8.39 (d, J=6.8 Hz, 1H), 8.73 (d, J=
8.4 Hz, 1H). ESI-HRMS for C19H16N4O2Cl (M þ H)þ calcd
367.0962; found 367.0948.
References
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quinoline 1-Oxide (41). Yield 88%; mp 184-186 °C. H NMR
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