Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)

Article abstract of DOI:10.1021/jm900383s

We report the characterization and optimization of drug-like smallmolecule inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme critical for the regulation of extracellular matrix calcification during bone formation and growth.High-thro

Full text of DOI:10.1021/jm900383s

J. Med. Chem. 2009, 52, 6919–6925 6919
DOI: 10.1021/jm900383s
Discovery and Validation of a Series of Aryl Sulfonamides as Selective Inhibitors of Tissue-Nonspecific
Alkaline Phosphatase (TNAP)
Russell Dahl,† Eduard A. Sergienko,† Ying Su,† Yalda S. Mostofi,† Li Yang,† Ana Maria Simao,‡ Sonoko Narisawa,‡
†
†
†
†
§
‡
ꢀ
ꢀ
Brock Brown, Arianna Mangravita-Novo, Michael Vicchiarelli, Layton H. Smith, W. Charles O’Neill, Jose Luis Millan,
and Nicholas D. P. Cosford*,†
†Conrad Prebys Center for Chemical Genomics and ‡Sanford Children’s Health Research Center, Burnham Institute for Medical Research, 10901
North Torrey Pines Road, La Jolla, California 92037, and §Renal Division, Department of Medicine, Emory University School of Medicine,
Atlanta, Georgia 30322
Received March 27, 2009
We report the characterization and optimization of drug-like small molecule inhibitors of tissue-nonspecific
alkaline phosphatase (TNAP), an enzyme critical for the regulation of extracellular matrix calcification
during bone formation and growth. High-throughput screening (HTS) of a small molecule library led to the
identification of arylsulfonamides as potent and selective inhibitors of TNAP. Critical structural require-
ments for activity were determined, and the compounds were subsequently profiled for in vitro activity and
bioavailability parameters including metabolic stability and permeability. The plasma levels following
subcutaneous administration of a member of the lead series in rat was determined, demonstrating the
potential of these TNAP inhibitors as systemically active therapeutic agents to target various diseases
involving soft tissue calcification. A representative member of the series was also characterized in
mechanistic and kinetic studies.
Introduction
upregulation of TNAP in Enpp1-/- and ank/ank vascular
smooth muscle cells (VSMC) and also in uremic aortas,
suggesting that it is an important cause of ePPi deficiency
and medial calcification and a potential therapeutic target.8,9
Thus, an effort to find selective and potent small molecule
inhibitors of TNAP as potential therapeutics is warranted.
Herein we describe the discovery of potent small molecule
TNAP inhibitors that, on systemic administration, are likely
to cause a reduction in TNAP activity, resulting in an increase
in the local amount of ePPi to prevent or ameliorate vascular
calcification.
TNAP, as with all mammalian APs, has been shown to be
inhibited by a limited number of small molecule compounds
including L-homoarginine, levamisole, and theophylline
(Figure 1).1,10 However, these known inhibitors of TNAP
are very weak binders and do not show specificity for the
TNAP isozyme. In addition, they are not particularly effective
at inhibiting the pyrophosphatase activity of TNAP. We
previously reported the results of an initial high-throughput
screening (HTS) campaign that led to the identification of
several low micromolar inhibitors of TNAP.8 We also re-
ported the results of a second HTS campaign, performed
within the Molecular Library Screening Center Network
(MLSCN), which led to the discovery of several small mole-
cule TNAP inhibitors with different mechanisms of action
(MOA).11 Subsequent work on the optimization of one of the
series discovered in this recent HTS campaign culminated in
the development of selective competitive pyrazole TNAP
inhibitors with low nanomolar potency (Figure 1).12 We now
report the structure-activity relationship (SAR) studies and
validation of a novel class of sulfonamides that are un-
competitive TNAP inhibitors showing excellent phosphatase
Among the human alkaline phosphatases, tissue-nonspeci-
fic alkaline phosphatase (TNAPa) is essential for bone matrix
mineralization.1 The biological function of TNAP is to hydro-
lyze extracellular inorganic pyrophosphate (ePPi), an inhibi-
tor of calcification, to maintain the correct ratio of Pi/ePPi in
skeletal tissues to enable normal skeletal calcification.2-4
Elsewhere in the body, high ePPi levels prevent ectopic
calcification.5 In turn, low levels of ePPi have been associated
with the development of soft-tissue calcification.6 This defi-
ciency of ePPi can be attributed to deficits in either the
production or transport of pyrophosphate, as seen in Enpp1
and Ank deficiencies.2,3 This physiological state can result in
rather severe clinical indications including idiopathic infantile
arterial calcification (IIAC), ectopic ossification in spinal
ligaments, ankylosis, and osteoarthritis.2-6 Arterial calcifica-
tion, particularly medial calcification aka Monckeberg’s
sclerosis, is a serious complication of chronic kidney disease,
obesity, diabetes, and aging.7 We have recently observed an
*To whom correspondence should be addressed. Phone: (858) 646-
3100. Fax: (858)- 646-3199. E-mail: ncosford@burnham.org.
a Abbreviations: AP, alkaline phosphatase; IAP, intestinal alkaline
phosphatase; PLAP, placental alkaline phosphatase; TNAP, tissue-
nonspecific alkaline phosphatase; IIAC, idiopathic infantile arterial
calcification; VSMC, vascular smooth muscle cells; HTS, high-through-
put screening; ADME, absorption, distribution, metabolism, excretion;
CYP2C19, cytochrome P450 2C19; MLSCN, Molecular Libraries
Screening Center Network; MLSMR, Molecular Libraries Small Mo-
lecule Repository; SAR, structure-activity relationship; MOA, me-
chanism of action; CDP-star, disodium 2-chloro-5-(50-chloro-
4-methoxyspiro[1,2-dioxetane-3,20-tricyclo[3.3.1.13,7]decan]-4-yl)-phe-
nol-1-(dihydrogen phosphate); PAMPA, parallel artificial membrane
permeability assay.
r
2009 American Chemical Society
Published on Web 10/12/2009
pubs.acs.org/jmc

6920 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Dahl et al.
Table 1. SAR of TNAP Inhibitors from HTS and Purchased Analogues
Figure 1. Structures of reported TNAP inhibitors.
selectivity and acceptable plasma levels in rat following sub-
cutaneous administration. These compounds have the poten-
tial to be developed into therapeutic agents to treat vascular
calcification.
Results and Discussion
High-throughput screening (HTS) of 66000 compounds
from the NIH Molecular Libraries Small Molecule Repository
(MLSMR) compound collection (http://www.mli.nih.gov/
mlsmr) using a luminescence-based HTS assay was performed
as a part of the MLSCN initiative. These screening efforts led to
the identification of several classes of submicromolar inhibitors
of TNAP11 (for assay details see Experimental Section and
PubChem link to AID 1056 http://pubchem.ncbi.nlm.nih.gov/
assay/assay.cgi?aid = 1056). Initial HTS was performed at a
concentration of 20 μM and was followed with dose-response
assays performed in duplicate using a 10-point 2-fold serial
dilution of the hit compounds in DMSO. Hit confirmation was
performed using luminescent and colorimetric assays to verify
inhibitory activity against TNAP in dose-response mode.
Selectivity was assessed against the isozymes placental and
intestinal alkaline phosphatase (PLAP and IAP) in lumines-
cence-based assays. Compounds that were active in dose-
response mode against TNAP, soluble in the range relevant
to their potency, and inactive against PLAP and IAP were
prioritized for synthetic chemistry follow-up.
HTS hits and purchased commercialanaloguesprovidedan
initial set of arylsulfonamides having TNAP IC50 values in the
nanomolar to low micromolar range (Table 1). Interestingly,
several compounds sharing similar structural features were
also found that had greatly reduced activities compared to the
initial lead structures, affording relevant information about
the keyrequiredstructural elements (Figure2). Analysis ofthe
nascent structure-activity relationship (SAR) present in
those analogues showing confirmed activity revealed key
features attributable to their inhibitory activity. Most notable
was the presence of either 3-pyridyl or 3-quinoline moieties on
the amine portion of the molecules and ortho-alkoxy substitu-
tion of the arylsulfonyl portion, which seemed to be required
for activity. Additional substitution of methyl or halogen on
this portion was equally tolerated in the active inhibitors.
Finally, quinoline derivatives appeared to be more active than
the corresponding pyridine analogues, as can be seen in the
comparison of compounds 1 and 2 (Table 1).
a TNAP IC50 data are presented as the geometric mean followed in
parentheses by the lower and upper limits of the mean and the number of
replicates.
movement of the 5-methoxy substituent of 1 also gave a similar
reduction of activity. Next, removal of the ortho-methoxy (15)
also resulted in a dramatic loss of activity. Finally, reversing the
sulfonamide moiety (16) or replacement with an amide or
methylene (17) also abolished the inhibitory activity. These
analogues confirmed the key attributes of our lead series,
specifically a 3-substituted quinoline or pyridine ring and an
ortho alkoxy substituent on the arylsulfonyl ring.
On the basis of the superior activity of compound 1, a directed
set of compounds was prepared to specifically address the key
SAR requirements of this scaffold (Figure 3). It is noteworthy
that when the two previously described SAR requirements were
altered, the activity of the resulting derivatives was greatly
diminished. Removal (12) or movement (13) of the quinoline
nitrogen resulted in loss of activity. Replacement (14) or

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6921
Table 2. SAR and ADME Profiles of Follow-up Compounds of Lead
TNAP Inhibitor 1
Figure 2. Examples of inactive arylsulfonamides which gave in-
sight into required structural elements. TNAP IC50 > 10 μM for all
examples.
a TNAP IC50 data are presented as the geometric mean followed in
parentheses by the lower and upper limits of the mean and the number of
replicates. b Percent remaining after incubation for 30 min at 37 °C.
c PAMPA flux values assessed according to previously published meth-
ods.15 d Solubility measurements performed in an aqueous buffer solution at
pH 7.4, in duplicate.
Figure 3. Elucidation of key requirements of lead TNAP inhibitor
1. TNAP IC50 > 10 μM for compounds 12-17.
After establishing the key features required for activity,
additional analogues were prepared to further optimize the
activity of this series and allow for in vitro profiling of
absorption, distribution, metabolism, and excretion
(ADME) properties. A variety of representative compounds
showed activity in the high nanomolar to single-digit micro-
molar range. Notably, one analogue (18) possessed compar-
able activity to lead compound 1 (Table 2).
Table 3. Selectivity Profile of TNAP Inhibitor 1
In addition, the compounds in Table 2 were profiled in in
vitro ADME assays to assess their drug-likeness and potential
for systemic activity in animal models of vascular calcifica-
tion. Many of the sulfonamide compounds were shown to
have suitable properties for oral administration including
acceptable metabolic and plasma stability, good permeability
across artificial lipid membranes, and good solubility. The
results of the specific ADME profiling assays are shown in
Table 2. Although the profiles of all of the synthesized
analogues had acceptable and drug-like ADME profiles, it
was clear that in addition to showing acceptable aqueous
solubility at physiological pH, the overall properties of 1 were
superior. The selectivity of 1 against other phosphatases and
across other biological targets was remarkable (Table 3).
TNAP inhibitor 1 showed no inhibition in over 250 assays
reported in the PubChem database (http://pubchem.ncbi.
nlm.nih.gov/) as well as assays performed in-house for two
other alkaline phosphatase isozymes, intestinal alkaline phos-
phatase (IAP), and placental alkaline phosphatase (PLAP).
Compound 1 showed activity in only one other assay, cyto-
chrome P450 2C19 (CYP2C19). Compound 1 was also tested
for its ability to inhibit the function of NPP1, a phosphodies-
terase that also has phosphatase activity and regulates ePPi
production in skeletal tissue,2,3 and PHOSPHO1, a phospha-
tase with specificity for phosphethanolamine and phospho-
choline, also involved in skeletal mineralization.13 At their
optimal alkaline pHs, inhibition was less than 9.5% for NPP1
and 7.4% for PHOSPHO1. Thus, TNAP inhibitor 1 was
established as a drug-like and selective probe molecule,
prompting further evaluation of its therapeutic potential as
a systemically active TNAP inhibitor.
The in vitro profile of 1 suggested that this compound had
the potential for further development because it had proper-
ties indicative of systemic availability. To evaluate the poten-
tial for systemic activity, the plasma levels of compound 1
were assessed following subcutaneous administration. Rats
were dosed subcutaneously with compound 1 at 4.13 mg/kg.
At the 1 h time point, compound 1 showed plasma levels
consistent with achieving therapeutic concentrations (Cmax
=
1.5 μg/mL), indicating the potential for in vivo efficacy. In
summary, these data indicate that compound 1 may find
utility as an in vivo tool for elucidating the role of TNAP in
vascular calcification in animal models.
Mechanistic Studies
The mechanism of a general alkaline phosphatase reaction
is outlined in Figure 4. Briefly, the initial alkaline phosphatase
(E) catalyzed reaction consists of a substrate (DO-Pi) bind-
ing step, phosphate-moiety transfer to Ser-93 (in the TNAP
sequence of its active site), and product alcohol (DOH)
release. In the second step of the reaction, phosphate
is released through hydrolysis of the covalent intermediate
(E-Pi) and the noncovalent complex (E Pi) of the inorganic
3

6922 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Dahl et al.
phosphate in the active site. Phosphate release is the rate-
limiting step of the overall reaction, resulting in accumulation
of the phosphorylated enzyme. In the presence of nitrogen-
containing alcohol molecules (AOH), such as the buffer
diethanolamine (DEA), phosphate is also released via a faster
transphosphorylation reaction without affecting the rate-
limiting step. The physiological relevance and identity of the
phosphate-acceptor molecules in vivo are not yet known.
To test the reversibility of binding of 1 to TNAP and to
qualitatively estimate the rate at which equilibrium was
achieved, the compound was serially diluted and preincubated
with TNAP at a concentration 12.5-fold higher than in the
final assay. Incubation at higher concentrations of a mixture
of enzyme and inhibitor is a standard method to accelerate the
binding process compared to the assay at normal concentra-
tions. The rate of binding is proportional to the concentration
of reactants, an important consideration if the enzyme is
inactivated through a collision-based mechanism by the com-
pound itself or a product of its degradation. In addition,
higher concentrations of inhibitor shifts the equilibrium of
binding toward higher saturation within the enzyme-inhibi-
tor complex, promoting any slow conversion of the enzyme-
inhibitor complex.
The data for mechanistic experiments performed with
TNAP inhibitor 1 are summarized in Figure 5. The binding
of the compound appears to obey fast-equilibrium kinetics
and lacks any time-dependent component. Compound pote-
ncy in the assay was unchanged either right after mixing with
the enzyme (EI 0:0) or after a preincubation for 1 h in the
presence of elevated concentrations of compound 1 with (EI
1:1) or without (EI 1:0) an additional 1 h incubation post-
dilution to the final compound concentrations.
In addition, we completed detailed mechanism of action
(MOA) studies with 1 at different concentrations of phos-
phate-donor substrate, 1,2-dioxetane reagent disodium 2-
chloro-5-(50-chloro-4-methoxyspiro[1,2-dioxetane-3,20-
tricyclo[3.3.1.13,7]decan]-4-yl)-phenol-1-(dihydrogen phos-
phate) (CDP-star), or phosphate-acceptor substrate, dietha-
nolamine (DEA). Defining the binding mode and site of
action is important for building a predictive pharmacophore
model to facilitate future medicinal chemistry efforts. The
artificial substrate CDP-star is expected to provide an ade-
quate system forstudying the effectsofcompound1 on TNAP
because the rate-limiting step for the reaction is downstream
of active site phosphorylation, as noted previously. As shown
in Figure 6A, [CDP-star]-dependent curves obtained in the
presence of different concentrations of 1 and plotted using
Lineweaver-Burk coordinates yields parallel lines. This con-
firms the uncompetitive nature of the compound binding
versus the CDP-star substrate arising from the compound’s
binding to enzyme-substrate complex, most likely after the
Figure 4. Catalytic mechanism of the alkaline phosphatase reac-
tion. See text for details.
active site phosphorylation and targeting either E-Pi or E Pi.
3
This mode of inhibition was also observed for levamisole,
theophylline and L-homoarginine and some inhibitors identi-
fied in the earlier rounds of screening.8,10 Interestingly, all
other series identified in the current HTS campaign demon-
strated competitive inhibition with respect to the phosphate-
donor substrate.11,12 A similar study was performed to char-
acterize the mode of TNAP inhibition by compound 1 with
respect tothe phosphate-acceptor substrate DEA. Compound
1 demonstrated noncompetitive inhibition against DEA
(Figure 6B), and this is consistent with the activity profile of
the sulfonamide series in the in vitro luminescent assay
performed at different concentrations of DEA. This MOA
indicates that the inhibitor could bind either before or after
Figure 5. Time-dependent mechanistic studies of TNAP inhibition
with 1. The enzyme was added with compound 1 serially diluted
immediately prior to activity measurement (b, EI (0:0)) or preincu-
bated with the compound for 1 h at 12.5-fold of the concentrations
and then diluted to final concentrations with ([, EI (1:1)) or without
(1, EI (1:0)) an additional 1 h incubation after the dilution and prior
to activity measurement. The dose-response curves were analyzed
using the Hill equation; best-fit parameters (IC50 and Hill coefficient
nH) and their standard errors of fit are shown in the Table.
Figure 6. Lineweaver-Burk plots for compound 1 MOA experiments. Inhibition of TNAP was measured in the presence of the following
concentrations of 1: 0 μM (O), 0.195 μM (b), 0.39 μM (0), 0.78 μM (9), 1.56 μM (]). The concentrations of DEA and CDP-star were equal to
100 mM (A) and 50 μM (B), respectively. The Ki values for compound 1 are 0.34 μM (Figure 6A) and 0.59 μM (Figure 6B).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6923
DEA binding (E-Pi or E-Pi AOH), suggesting that compound
incubation, the reaction was terminated by adding 2,3-dimer-
capto-1-propanesulfonate (a compound that binds Zn2þ with
high affinity thus inactivating the enzyme) to a concentration
of 100 μM. TNAP activity was measured using absorbance at
405 nm.
3
1 occupies a distinct binding site. In contrast, the pyrazole
series demonstrated competitive inhibition with respect to
DEA, potentially occupying the site of DEA binding.12
General Synthetic Procedures. All solvents and chemicals
used were purchased from Sigma-Aldrich, Acros, or Chem-
bridge and were used as received without further purification.
Purity and characterization of compounds were established by a
combination of liquid chromatography-mass spectroscopy
(LC-MS) and NMR analytical techniques and was >95% for
all tested compounds. Silica gel column chromatography was
carried out using prepacked silica cartridges from RediSep
Conclusion
High-throughput screening utilizing luminescent and colori-
metric assays led to the identification of several potent and
selective, small molecule inhibitors of the TNAP enzyme. Hit
validation and directed synthesis led to the establishment of 1
(2,5-dimethoxy-N-(quinolin-3-yl)benzenesulfonamide) as a
potent TNAP inhibitor with excellent selectivity over other
alkaline phosphatases, favorable ADME profiles, acceptable
aqueous solubility, and good plasma levelsafter subcutaneous
dosing. Inaddition, an equallypotentinhibitor(119nM) from
this series, 18 (5-bromo-2-methoxy-N-(quinolin-3-yl)ben-
zenesulfonamide), was also found through lead validation
studies. These molecules may find utility to determine the
potential of TNAP inhibitors as therapeutics for vascular
calcification in target validation studies. Future work will
describe the continued in vitro characterization and in vivo
efficacy studies of this promising series of TNAP inhibitors.
1
(ISCO Ltd.) and eluted using an Isco Companion system. H
NMR spectra were acquired on a Varian Inova 300 MHz.
Chemical shifts are reported in ppm from residual solvent peaks
(δ 7.27 for CDCl3 1H NMR). High-resolution ESI-TOF mass
spectra were acquired at the Center for Mass Spectrometry at
The Scripps Research Institute (La Jolla, CA). HPLC-MS
analyses were performed on a Shimadzu 2010EV LCMS using
the following conditions: Kromisil C18 column (reverse phase,
4.6 mm ꢀ 50 mm); a linear gradient from 10% acetonitrile and
90% water to 95% acetonitrile and 5% water over 4.5 min; flow
rate of 1 mL/min; UV photodiode array detection from 200 to
300 nm.
General Method for the Synthesis of Sulfonamide TNAP
Inhibitors. To a stirred solution of the sulfonyl chloride (0.5
mmol in DMF), the appropriate aryl amine (0.5 mmol) was
added, followed by N,N-diisopropylethylamine (0.75 mmol),
and stirred at room temperature for 18 h. Solvents were removed
by rotary evaporation, and the products were isolated by flash
chromatography and reverse phase HPLC and lyophilized to
provide the final compounds, which were determined to be
>95% pure by HPLC-UV, HPLC-MS, and 1H NMR.
2,5-Dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (1). Pre-
pared according to the general procedure to yield a yellow solid
Experimental Section
Compound Screening Library. The compound library was
supplied by the NIH Molecular Libraries Small Molecule
Repository (MLSMR, http://www.mli.nih.gov/mlsmr). The
MLSMR, funded by the NIH, is responsible for the selection
of small molecules for HTS screening, their purchase and QC
analysis, library maintenance, and distribution within the NIH
Molecular Libraries Screening Center Network (MLSCN,
http://www.mli.nih.gov/mlscn). Both MLSMR and MLSCN
are components of the Molecular Libraries Initiatives (MLI,
http://nihroadmap.nih.gov/molecularlibraries) within the NIH
Roadmap Initiative (www.nihroadmap.nih.gov). MLSMR
compounds are acquired from commercial and, in part, from
academic and government sources and are selected based on the
following criteria: samples are available for resupply in 10 mg
quantity, are at least 90% pure, have acceptable physicochem-
ical properties, and contain no functional groups or moieties
that are known to generate artifacts in HTS (http://mlsmr.glpg.
com/). Compounds are selected to represent diversified chemical
space with clusters of closely related analogues to aid in the
HTS-based SAR analysis.
Expression and Preparation of Test Enzymes. Expression
plasmids containing a secreted epitope-tagged TNAP were
transfected into COS-1 cells for transient expression.10 The
medium was replaced with Opti-MEM 24 h later, and the
serum-free media containing secreted proteins were collected
60 h after electroporation. The conditioned medium was dia-
lyzed against TBS containing 1 mM MgCl2 and 20 mM ZnCl2
(to remove phosphate) and filtered through a 0.22 μm cellulose
acetate filter.
HTS Assays. Assays were developed and performed as des-
cribed in detail elsewhere.11 Briefly, the TNAP luminescent
assay was performed in 384-well white plates (784075, Greiner)
at 50 μM CDP-star substrate in assay buffer containing 100 mM
DEA-HCl, pH 9.8, 1 mM MgCl2, and 20 μM ZnCl2. The
luminescence signal was measured after a 30 min incubation at
room temperature on an EnVision plate reader (Perkin-Elmer,
Inc.). Analogous luminescent assays were optimized and utilized
for PLAP and IAP with the CDP-star concentration adjusted to
their respective Km values, 85 μM and 177 μM, respectively.
The colorimetric TNAP assay was performed as previously
described8 with slight modifications. TNAP in assay buffer
containing 1 M DEA-HCl, pH 9.8, 1 mM MgCl2, and 20 uM
ZnCl2 was added with 0.5 mM pNPP substrate, and after a 1 h
1
(67%). H NMR (300 MHz, CDCl3): δ 8.698 (s, 1H), 8.30 (d,
J = 8.1 Hz, 1H), 8.10 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.90 (t,
J = 7.5 Hz, 1H), 7.79 (s, 1H), 7.42 (d, J = 2.7 Hz, 1H), 7.00 (m,
2H), 3.95 (s, 3H), 3.77 (s, 3H). ESI-MS m/z 345 [M þ H]þ.
HRMS m/z calcd for C17H17N2O4S [M þ H]þ: 345.0903.
Found: 345.0903.
2,5-Dimethoxy-N-(pyridin-3-yl)benzenesulfonamide (2). Pre-
pared according to the general procedure to yield a white solid
(72%). 1H NMR (300 MHz, CDCl3): δ 9.01 (d, J = 2.4 Hz, 1H),
8.19 (m, 1H), 7.6 (m, 4H), 6.85 (m, 3H), 3.86 (s, 3H), 3.73 (s, 3H).
ESI-MS m/z 295 [M þ H]þ. HRMS m/z calcd for C13H15N2O4S
[M þ H]þ: 295.0747. Found: 295.1435.
2,5-Dimethoxy-N-(naphthalen-2-yl)benzenesulfonamide (12).
Prepared according to the general procedure to yield a white
powder (6%). 1H NMR (300 MHz, CDCl3): δ 7.73 (m, 3H), 7.57
(d, J = 2.1 Hz, 1H), 7.42 (m, 4H), 7.29 (m, 1H), 6.98 (m, 2H),
4.05 (s, 3H), 3.72 (s, 3H). ESI-MS m/z 344 [M þ H]þ. HRMS m/z
calcd for C18H18NO4S [M þ H]þ: 344.0951. Found: 344.0949.
2,5-Dimethoxy-N-(quinolin-6-yl)benzenesulfonamide (13).
Prepared according to the general procedure to yield a yellow
solid (35%). 1H NMR (300 MHz, CDCl3): δ 9.09 (s, 2H), 8.43
(d, J = 7.8 Hz, 1H), 8.30 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 15.6
Hz, 3H), 7.44 (s, 1H), 7.0 (m, 2H), 3.93 (s, 3H), 3.75 (s, 3H). ESI-
MS m/z 345 [M þ H]þ. HRMS m/z calcd for C17H17N2O4S [M þ
H]þ: 345.0903. Found: 345.0903.
5-Chloro-2-methoxy-N-(quinolin-3-yl)benzenesulfonamide (14).
Prepared according to the general procedure to yield a yellow
solid (17%). 1H NMR (300 MHz, DMSO-d6): δ 10.72 (s, 1H),
8.68 (s, 1H), 7.95 (m, 2H), 7.77 (m,1H), 7.65 (m, 2H), 7.21 (d,
J = 9.3 Hz, 1H), 3.85 (s, 3H). ESI-MS m/z 349 [M þ H]þ.
HRMS m/z calcd for C16H14ClN2O3S [M þ H]þ: 349.0408.
Found: 349.0403.
3-Methoxy-N-(quinolin-3-yl)benzenesulfonamide (15). Pre-
pared according to the general procedure to yield a yellow solid

6924 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Dahl et al.
(15%). 1H NMR (300 MHz, DMSO-d6): δ 10.83 (s, 1H), 8.64 (d,
J = 2.4 Hz, 1H), 8.03 (m, 1H), 7.94 (m, 2H), 7.65 (m, 2H), 7.45
(m, 1H), 7.35 (m, 1H), 7.17 (m, 1H), 3.75 (s, 3H). ESI-MS m/z
315 [M þ H]þ. HRMS m/z calcd for C16H15N2O3S [M þ H]þ:
315.0798. Found: 315.0792.
determinations were carried out in triplicate, and the initial
velocities were constant for at least 90 min, provided that less
than 5% of substrate was hydrolyzed. Controls without added
enzyme were included in each experiment to allow for the
nonenzymatic hydrolysis of substrate. We used the inhibitor
compound 1 at a final concentration of 10 μM to assess its effect
on PHOSPHO1 and NPP1 activities.
Assessment of Plasma and Microsomal Stability and Perme-
ability. To assess plasma stability, the test compounds were
incubated in fresh rat plasma at 37 °C. To determine metabolic
stability, the compounds in triplicate were incubated with
pooled rat liver microsomes (BD Scientific) in the presence of
NADPH (Sigma) at 37 °C. Liquid chromatography/mass spec-
trometry (LC/MS) was applied in quantitative analysis of
compounds in the biological matrix. Mass spectrometry
(Shimadzu 2010EV) was used to conduct mass analysis in
multiple-reaction-monitoring (MRM) mode. The compound
stabilities, expressed as the percentage of the remaining com-
pound to the initial concentration, were calculated using
Shimadzu LabSolutions software and Excel. The perme-
ability was determined using the PAMPA method described
previously.15
Solubility. Solubility analysis was performed using a direct
UV kinetic solubility method in a 96-well plate format. All liquid
dispense and transfer steps were performed with the Freedom
Evo automated liquid handler (Tecan US). Solubility measure-
ments were performed in an aqueous buffer solution (System
Solution, pION Inc.) at pH 7.4 in duplicate. Samples were
incubated at room temperature for a minimum of 18 h to achieve
equilibrium and then filtered (filter plate, pION Inc.) to remove
any precipitate formed. The concentration of the compounds
was measured by UV absorbance (250-498 nm) using the
Infinite M200 (Tecan US) and compared to the spectra of the
precipitation-free reference solutions. Spectroscopically pure
1-propanol (Sigma) was used as a cosolvent to suppress pre-
cipitation in the reference solutions. The solubility of each
compound was determined using μSOL Evolution Plus software
v3.2 (pION Inc.) and is expressed as the concentration (μg/mL)
of a solute in a saturated solution.
Plasma Concentration Analysis. For evaluation of in vivo
compound concentration, 400 μL of a 10 μM solution of 1 (4.13
mg/kg; 50% DMSO in normal saline) were subcutaneously
injected in each rat. Blood samples were drawn and frozen
immediately. Plasma was harvested and kept at -20 °C until
assayed. Concentrations of 1 in plasma were determined using
a validated analytical procedure based on high-performance
liquid chromatography. LC-MS/MS analyses were carried out
using a SCIEX API3000 triple quadrupole mass spectrometer
(PE Sciex Instruments, Boston, MA) operating in electrospray
ionization mode. Chromatography was carried out using gra-
dient elution (water-acetonitrile) on a Kromisil C18 reverse-
phase column at a flow rate of 1 mL/min. Plasma compound
concentrations were determined using a seven-point calibration
curve derived from peak areas obtained from serially diluted
solutions of 1.
N-(2,5-Dimethoxyphenyl)quinoline-3-sulfonamide (16). To a
stirred solution of the quinoline 3-sulfonyl chloride (0.5 mmol,
115 mg) in DMF, the 2,5-dimethoxy aniline (0.5 mmol, 76 mg)
was added, followed by N,N-diisopropylethylamine (0.75
mmol). This mixture was stirred at room temperature overnight.
1
Reverse phase HPLC afforded a white solid (7%). H NMR
(300 MHz, CDCl3): δ 9.16 (d, J = 2.4 Hz, 1H), 8.60 (d, J = 2.4
Hz, 1H), 8.15 (m, 1H), 7.89 (m, 1H), 7.65 (m, 1H), 7.26 (d, J =
2.4 Hz, 2H), 7.19 (s, 1H), 6.60 (m, 2H), 3.79 (s, 3H), 3.52 (s, 3H).
ESI-MS m/z 345 [M þ H]þ. HRMS m/z calcd for C17H17N2O4S
[M þ H]þ: 345.0903. Found: 345.0902.
5-Bromo-2-methoxy-N-(quinolin-3-yl)benzenesulfonamide (18).
Prepared according to the general procedure to yield a white
powder (36%). 1H NMR (300 MHz, DMSO-d6): δ 10.71 (s, 1H),
8.69 (d, J = 3.0 Hz, 1H), 7.94 (m, 3H), 7.75 (m, 1H), 7.65 (m, 1H),
7.52 (m, 1H), 7.15 (d, J = 8.7 Hz, 1H), 3.85 (s, 3H). ESI-MS m/z
394.8[M þ H]þ. HRMS m/z calcd for C16H14BrN2O3S [M þ H]þ:
392.9903. Found: 392.9906.
2-Methoxy-5-methyl-N-(quinolin-3-yl)benzenesulfonamide (19).
Prepared according to the general procedure to yield a yellow solid
(3%). 1H NMR (300 MHz, CDCl3):δ8.70 (s, 1H), 8.30 (d, J= 8.1
Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.89 (m, 1H), 7.77 (m, 1H), 7.70
(s, 1H), 7.32 (m, 2H), 3.97 (s, 3H), 2.89 (s, 3H). ESI-MS m/z 329
[M þ H]þ. HRMS m/z calcd for C17H17N2O3S [M þ H]þ:
329.0954. Found: 329.0960.
2-Methoxy-4-nitro-N-(quinolin-3-yl)benzenesulfonamide (20).
Prepared according to the general procedure to yield a yellow
powder (9%).1H NMR (300 MHz, DMSO-d6): δ 8.68 (d, J =
2.4 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 2.4 Hz, 1H),
7.92 (m, 3H), 7.84 (m, 1H), 7.65 (m, 1H), 7.55 (m, 1H), 3.99 (s,
3H). ESI-MS m/z 360 [M þ H]þ. HRMS m/z calcd for
C16H14N3O5S [M þ H]þ: 360.0649. Found: 360.0644.
2-Methoxy-5-methyl-N-(pyridin-3-yl)benzenesulfonamide (21).
Prepared according to the general procedure to yield a yellow
1
powder (43%). H NMR (300 MHz, CDCl3): δ 8.35 (m, 2H),
7.70 (m, 1H), 7.62 (s, 1H), 7.30 (m, 2H), 6.93 (m, 1H), 4.02 (s,
3H), 2.29 (s, 3H). ESI-MS m/z 279 [M þ H]þ. HRMS m/z calcd
for C13H15N2O3S [M þ H]þ: 279.0798. Found: 279.0801.
2-Methoxy-4-nitro-N-(pyridin-3-yl)benzenesulfonamide (22).
Prepared according to the general procedure to yield a yellow
solid (53%). 1H NMR (300 MHz, CDCl3): δ 8.73 (d, J = 4.8 Hz,
1H), 8.66 (s, 1H), 8.23 (m, 1H), 7.99 (m, 1H), 7.89 (s, 1H), 7.83
(m, 1H), 7.54 (m, 1H), 3.85 (s, 3H). ESI-MS m/z 310 [M þ H]þ.
HRMS m/z calcd for C12H10N3O5S [M - H]-: 308.0347.
Found: 308.0347.
Enzymatic Counterscreening Assays. Phosphoethanolamine
(PEA) activity for PHOSPHO1 was assayed discontinuously, at
37 °C, by measuring the amount of inorganic phosphate re-
leased, according to the previously described procedure,14 ad-
justing the assay medium to a final volume of 0.15 mL. Standard
conditions were 20 mM Tris buffer, pH 7.2, containing 25%
glycerol (w/v), 25 μg/mL BSA, 2 mM MgCl2, and 2.5 mM PEA.
The reaction was initiated by the addition of the enzyme and
stopped with 0.075 mL of cold 30% TCA at appropriate time
intervals. The reaction mixture was centrifuged at 4000g, and
phosphate was quantified in the supernatant after pH neutrali-
zation with 0.1 M NaOH. p-Nitrophenylthymidine 50-mono-
phosphate (pNP-TMP) activity for NPP1 was assayed
discontinuously, at 37 °C by following the liberation of
p-nitrophenolate ion (ε 1 M, pH 13=17600 M-1 cm-1) at 410
nm. Standard conditions were 50 mM Tris buffer, pH 7.4, or 100
mM Tris buffer, pH 8.9, containing 2 mM MgCl2 and 5 mM
pNP-TMP, in a final volume of 0.15 mL. The reaction was
initiated by the addition of the enzyme and stopped with
0.15 mL of 1 M NaOH at appropriate time intervals. All
Acknowledgment. This work was supported in part by
NIH grants U54HG003916, DE12889, AR47908, AR53102
and HL101899.
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