Design, synthesis and evaluation of benzoisothiazolones as selective inhibitors of PHOSPHO1

Article abstract of DOI:10.1016/j.bmcl.2014.07.013

We report the discovery and characterization of a series of benzoisothiazolone inhibitors of PHOSPHO1, a newly identified soluble phosphatase implicated in skeletal mineralization and soft tissue ossification abnormalities. High-throughput screening (HTS)

Full text of DOI:10.1016/j.bmcl.2014.07.013

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4308–4311
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of benzoisothiazolones as selective
inhibitors of PHOSPHO1
Yalda Bravo a,b, , Peter Teriete a,b, , Raveendra-Panickar Dhanya a,b, Russell Dahl a,b, Pooi San Lee a,b
,
Tina Kiffer-Moreira c, Santhi Reddy Ganji a,b, Eduard Sergienko b, Layton H. Smith b, Colin Farquharson d,
José Luis Millán c, Nicholas D. P. Cosford a,b,
⇑
a Cell Death and Survival Networks Research Program, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla,
CA 92037, USA
b Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
c Sanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
d The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, Scotland, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the discovery and characterization of a series of benzoisothiazolone inhibitors of PHOSPHO1, a
newly identified soluble phosphatase implicated in skeletal mineralization and soft tissue ossification
abnormalities. High-throughput screening (HTS) of a small molecule library led to the identification of
benzoisothiazolones as potent and selective inhibitors of PHOSPHO1. Critical structural requirements
for activity were determined, and the compounds were subsequently derivatized and measured for
in vitro activity and ADME parameters including metabolic stability and permeability. On the basis of
its overall profile the benzoisothiazolone analogue 2q was selected as MLPCN probe ML086.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 14 May 2014
Accepted 4 July 2014
Available online 15 July 2014
Keywords:
Phosphatase
Vascular calcification
Probe compound
ML086
PHOSPHO1
PHOSPHO1 is a recently identified orphan phosphatase that
belongs to the family of halo-acid dehalogenases. It is a soluble
phosphatase with specificity for phosphoethanolamine (P-Etn)
and phosphocholine (P-Cho) present in matrix vesicles (MVs).1
PHOSPHO1 is responsible for increasing the local concentration
of inorganic phosphate (Pi) inside MVs to change the phosphate/
pyrophosphate (Pi/PPi) ratio to favor precipitation of hydroxyapa-
tite (HA) seed crystals.2 Aberrations of the Pi/PPi ratio have been
associated with numerous pathologies. Low extracellular PPi (ePPi)
production has been identified as a cause in the development of
severe medial vascular calcification (MVC) known as generalized
arterial calcification of infancy (GACI; OMIM # 20,8000)3 as well
as ossification of the posterior longitudinal ligaments of the spine
(OPLL; OMIM # 60,2475) and osteoarthritis (OA).4 In addition,
low ePPi transport manifests as ankylosing vertebral hyperostosis
(DISH; OMIM # 10,6400), chondrocalcinosis (OMIM # 605145)
and ankylosing spondylitis (AS; OMIM # 10,6300).5 On the other
hand, accumulation of ePPi results in rickets or osteomalacia,
known as hypophosphatasia (HPP; OMIM # 17,1760).6 Because of
the accumulation of ePPi, HPP patients may also display chondro-
calcinosis or calcium pyrophosphate dihydrate deposition (CPPD)
disease (OMIM # 11,8600). However, many of these conditions
have been linked to deficiencies in other transporters and phos-
phatases, most notably tissue-nonspecific alkaline phosphatase
(TNAP) that is also found in the same biological compartment.7
There is therefore a significant need to identify small molecule
compounds that can probe the function of these enzymes and pro-
vide a starting point for the development of therapeutic agents.
We previously reported the synthesis and optimization of selec-
tive small molecule inhibitors of TNAP that have been employed to
investigate the role of TNAP in vascular calcification.8 We also
disclosed the synthesis and characterization of a series of com-
pounds that inhibit phosphomannose isomerase (PMI), an enzyme
implicated in therapeutically important protein glycosylation
processes.8a,9 These small molecule probes, discovered using
high-throughput screening (HTS) and chemical optimization
through the Molecular Libraries Probe Production Centers Network
(MLPCN; http://mli.nih.gov/mli/mlpcn/), have found utility for
investigating the role of intracellular and extracellular phospha-
tases. The structure of the PMI inhibitor probe ML089 (1) is shown
in Figure 1. We hypothesized that, in a similar manner, it would be
possible to discover small molecule inhibitors of PHOSPHO1 that
⇑
Corresponding author.
E-mail address: ncosford@sanfordburnham.org (N.D.P. Cosford).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.07.013
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Y. Bravo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4308–4311
4309
O
O
would help to delineate the role of this enzyme in skeletal miner-
alization and soft tissue ossification abnormalities at a fundamen-
tal level. Furthermore, selective PHOSPHO1 inhibitors would be
useful tools to elucidate the mechanism of action for the aforemen-
tioned diseases. With this in mind, a high-throughput screening
(HTS) campaign was performed through the MLPCN.
The screen employed a colorimetric assay based on the ability
of PHOSPHO1 to liberate phosphate from P-Etn and its subse-
quent? reaction with the Biomol Green reagent (Biomol Interna-
tional, Plymouth Meeting, PA, USA). The construct was designed
to express PHOSPHO1 protein fused to a V5 epitope and 6 His-
tag at the C-terminus. A diverse library of 288,481 compounds
from the MLSMR collection was tested at a single concentration
F
N
N
R2
R1
S
S
2 R1 = H, F
1 ML089
R2 = aryl
Figure 1. Phosphomannose isomerase (PMI) probe ML089 (1) and the benzothiazo-
lone scaffold of the PHOSPHO1 screening hits.
Table 1
Summary of first round of SAR for compounds synthesized and tested for PHOSPHO1
inhibition as well as selectivity against related phosphatases
of 13.3 lM (PubChem AID 1565). This provided 3,164 compounds
Compd
Ar
R1
PHOSPHO1a
IC50
PMIb
M]
PMM2b
with greater than 60% activity in the single point assay, a hit rate
of 1.1%. Hit confirmation was performed using the colorimetric
assay to verify inhibitory activity against PHOSPHO1 in dose-
response mode performed in duplicate using a 10-point 2-fold
serial dilution of the hit compounds in DMSO. Inhibitors that were
active in dose–response mode against PHOSPHO1 and soluble in
the concentration range relevant to their potency were classified
as confirmed hits. This led to the identification of several sub-
micromolar inhibitors of PHOSPHO1 (see PubChem link to AID
1565 and 1666 for details).
[l
2a
2b
2c
H
F
0.94
0.79
1.3
6.4
1.3
3.9
>20
>50
>30
H
2d
H
2.7
6.0
>20
It was noted that some of the confirmed hits fell into the ben-
zoisothiazolone class of small molecules, such as 1, that were pre-
viously identified as PMI inhibitors. For example, the unsubstituted
benzoisothiazolone 2a (Table 1) inhibited PHOSPHO1 with an IC50
2e
2f
F
6.7
4.9
>10
11
1.0
3.4
3.6
8.4
>10
>20
>50
>30
H
F
value of 0.94 lM while also inhibiting PMI (IC50 = 6.4 lM). We
2g
2h
hypothesized that within this series it might be possible to opti-
mize the potency of compounds against PHOSPHO1 while reducing
or eliminating activity at PMI and PMM2. The benzothiazolone ser-
ies was therefore prioritized for chemical optimization using both
analogue by catalogue (ABC) and synthetic chemistry.
Analogues of the benzoisothiazolone hits were synthesized by a
method originally reported by Correa et al.10 that utilized a key
cyclization step using phenyliodine bis(trifluoroacetate) (PIFA) to
H
2i
2j
H
H
5.2
4.0
4.8
6.6
>30
>20
generate
a N-acylnitrenium ion followed by intramolecular
trapping by sulfur (Scheme 1a). The synthesis of the analogues
was carried out in a semi-convergent manner. The required aniline
precursors were either purchased or, in the case of the amides, pre-
pared via EDCI mediated coupling of amines with the appropriate
benzoic acid derivatives (Scheme 1b).11 This product was then
coupled with methyl 2-mercaptobenzoate and subjected to the
cyclization conditions (Scheme 1a). This synthetic methodology
allowed for preparation of the benzoisothiazolone derivatives
shown in Tables 1 and 2.12
2k
F
3.3
1.1
7.3
2l
F
4.7
1.8
9.9
>20
2m
H
>50
>100
The potency and selectivity of the benzoisothiazolone ana-
logues were assessed by in vitro enzymatic assays using purified
human PHOSPHO1, PMI, or PMM213 to establish a preliminary
SAR. As noted previously, the hit compound 2a inhibited
PHOSPHO1 with good potency but also significantly inhibits PMI
(Table 1). Fluoro substitution (R1 = F) of the benzoisothiazolone
2n
H
1.1
3.3
52
2o
2p
2q
H
H
H
0.82
1.2
4.9
23
12
>50
>50
O
O
O
A
6, AlMe3
PIFAa
CH2Cl2
TFA
Ar
OCH3
N
H
N
Ar
CH2Cl2
0-60oC
R1
SH
R1
SH
R1
S
0.14
>50
3
4
2
O
O
B
HNR3R4
EDCI, HOBT,
Hunig's base
NR3R4
OH
2r
H
2.3
2.3
18
H2N
H2N
5
6
a
See http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1666 for details of
assay protocol.
Scheme 1. General synthetic sequences for the synthesis of small molecule
b
See Dahl et al., 20118a for assay protocol.
PHOSPHO1 inhibitors. aPIFA phenyliodine bis(trifluoroacetate).

4310
Y. Bravo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4308–4311
Table 2
lessened potency at PHOSPHO1 while retaining the unwanted
activity at PMI. Substitution at the 4-position of the phenyl ring
with methoxy (2e), fluoro (2f, 2g), or NMe2 (2h) effectively wors-
ened potency against PHOSPHO1 while in general increasing
potency at PMI. Substitution at the 3-position with chloro, as in
2i, provided a compound with similar potency at both PHOSPHO1
and PMI. Interestingly, none of the initial set of compounds 2a-2i
had significant activity at PMM2. The 2,5- or 2,3-dimethyl substi-
tution patterns (2j and 2l, respectively) gave compounds that were
similarly potent at both PHOSPHO1 and PMI. Introduction of
R1 = fluoro, as in 2k, unfortunately enhanced potency at both PMI
and PMM2. In contrast to 2i, the 3-chloro-4-fluorophenyl deriva-
tive 2m was potent at PHOSPHO1 but inactive at both PMI and
PMM2. Carboxylic acid substitution at the 3-position (2n) provided
a potent PHOSPHO1 inhibitor with micromolar activity at PMI and
no activity at PMM2. The methyl ester derivative 2o exhibited sub-
micromolar potency at PHOSPHO1 but also micromolar activity at
both PMI and PMM2, whereas the ethyl ester derivative 2p was
essentially devoid of activity at PMI and PMM2. The breakthrough
came, however, with the dimethyl amide derivative 2q, exhibiting
an IC50 value of 140 nM at PHOSPHO1 and no activity at PMI or
PMM2. Interestingly, the corresponding benzylamide derivative
2r, while potent at PHOSPHO1 also showed activity at PMI and
PMM2.
Based on the promising data for the first set of compounds, and
in particular 2q, we next tested a series of analogues containing a
sulfonamide moiety at the 3-position of the phenyl ring. The
results of these efforts are shown in Table 2. Several analogues in
this series exhibited good potency as PHOSPHO1 inhibitors, with
the dimethyl (2s) and diethyl (2t) analogues being especially
potent. Interestingly, while compound 2s was active at PMI and
PMM2 at micromolar levels, the diethyl sulfonamide 2t was devoid
of activity at these phosphatases. The anthranilic acid sulfonamide
2u exhibited submicromolar potency at both PHOSPHO1 and PMI.
Sulfonamide derivatives 2v–2y were less potent at PHOSPHO1 and
all had some level of activity at PMI and PMM2.
Data for analogues with a sulfonamide substituent at the 3-position of the phenyl ring
Compd
Ar
PHOSPHO1a
PMIb
M]
PMM2b
IC50
[l
2s
2t
0.50
0.56
0.81
2.8
13
>50
0.71
>100
5.2
2u
2v
2w
2x
1.2
1.8
1.1
2.6
3.1
8.6
9.9
11
63
2y
7.5
7.5
30
a
See http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1666 for details of
assay protocol.
b
See Dahl et al., 20118a for assay protocol.
moiety, as in 2b, provides a marginal improvement in potency at
PHOSPHO1 but also increased potency at PMI (IC50 = 1.3 M).
Monomethyl substitution of the phenyl ring, as in 2c and 2d,
l
Select PHOSPHO1 inhibitors (2n, 2o, 2q, 2s) were comprehen-
sively profiled in in vitro absorption, distribution, metabolism
Table 3
Characterization of a selection of synthesized analogues for drug-like properties in a range of in vitro ADME assays
Cmpd. Structure
Aqueous Solubility
in pION’s buffer
PAMPA Permeability
[Â10À6 cm/s] Acceptor
pH 7.4
Plasma
Protein Binding
[% Bound]
Plasma Stability
[% Remaining at
3 h]
Hepatic
Microsome
Stability [%
Remaining at
1 h]
Toxicity towards
Fa2 N-4
Immortalized
Human
[
lg/ml]
Plasma: 1Â PBS,
pH 7.4,
Hepatocytes
1:1 1ÂPBS, pH
7.4
(NADPH minus) LC50 [lM]
pH5.0 pH6.2 pH7.4 pH5.0 pH6.2 pH7.4 Human
M/
Mouse
1 lM/
Human Mouse Human Mouse
1
l
10
lM
10 lM
n.d./
59.4
18.8/
30.1
50.2
55.5
100
71.2
2n
2o
2q
2s
>27
12.6
>30
23.4
>27
18.9
>30
24.6
>27
20.8
>30
23.8
157
1294
94
18
<4.4
1300
97
100
100
>50
>50
>50
>50
41.2/
57.3
48.0/
37.6
15.4
49.3
59.6
55.2
72.4
31.5
45.9
44.6
1231
92
41.3/
44.2
48.0/
46.7
18.9
60.0
76.2
56.9
45.7
72.4
56.6
100
n.d./
56.5
35.6/
34.1
11.4
32.3
65.3
40.0
781
817
777
For details of assay protocols see Khan et al., 201114
.

Y. Bravo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4308–4311
4311
Table 4
Stewart, A. J.; Farquharson, C. Bone 2004, 34, 629; (c) Roberts, S. J.; Stewart, A. J.;
Sadler, P. J.; Farquharson, C. Biochem. J. 2004, 382, 59; (d) Stewart, A. J.; Roberts,
S. J.; Seawright, E.; Davey, M. G.; Fleming, R. H.; Farquharson, C. Bone 2006, 39,
1000.
Activity of 2q (MLPCN Probe ML-086) on the target (PHOSPHO1) compared with
counter-target enzymes and a pathway screen
PHOSPHO1
TNAP
NPP-1
PMI
PMM2
AID1028a
2. Roberts, S.; Narisawa, S.; Harmey, D.; Millan, J. L.; Farquharson, C. J. Bone Miner.
Res. 2007, 22, 617.
3. McKusick, V. A. Am. J. Hum. Genet. 2007, 80, 588.
IC50
[
lM]
0.14
—
>100
>719
>30
>215
62
442
76
549
24
176
Selectivity
4. (a) Terkeltaub, R. A. Am. J. Physiol. Cell Physiol. 2001, 281, C1; (b) Rutsch, F.; Ruf,
N.; Vaingankar, S.; Toliat, M. R.; Suk, A.; Hohne, W.; Schauer, G.; Lehmann, M.;
Roscioli, T.; Schnabel, D.; Epplen, J. T.; Knisely, A.; Superti-Furga, A.; McGill, J.;
Filippone, M.; Sinaiko, A. R.; Vallance, H.; Hinrichs, B.; Smith, W.; Ferre, M.;
Terkeltaub, R.; Nurnberg, P. Nat. Genet. 2003, 34, 379; (c) Rutsch, F.; Vaingankar,
S.; Johnson, K.; Goldfine, I.; Maddux, B.; Schauerte, P.; Kalhoff, H.; Sano, K.;
Boisvert, W. A.; Superti-Furga, A.; Terkeltaub, R. Am. J. Pathol. 2001, 158, 543.
5. Tsui, H. W.; Inman, R. D.; Paterson, A. D.; Reveille, J. D.; Tsui, F. W. Arthritis Res.
Ther. 2005, 7, R513.
6. (a) Whyte, M. P. Ann. N. Y. Acad. Sci. 2010, 1192, 190; (b) Whyte, M. P.;
Greenberg, C. R.; Salman, N. J.; Bober, M. B.; McAlister, W. H.; Wenkert, D.; Van
Sickle, B. J.; Simmons, J. H.; Edgar, T. S.; Bauer, M. L.; Hamdan, M. A.; Bishop, N.;
Lutz, R. E.; McGinn, M.; Craig, S.; Moore, J. N.; Taylor, J. W.; Cleveland, R. H.;
Cranley, W. R.; Lim, R.; Thacher, T. D.; Mayhew, J. E.; Downs, M.; Millan, J. L.;
Skrinar, A. M.; Crine, P.; Landy, H. N. Engl. J. Med. 2012, 366, 904.
7. (a) Bobryshev, Y. V.; Orekhov, A. N.; Sobenin, I.; Chistiakov, D. A. Curr. Pharm.
Des. 2014; (b) Kiffer-Moreira, T.; Narisawa, S. Methods Mol. Biol. 2013, 1053,
125.
8. (a) Dahl, R.; Sergienko, E. A.; Su, Y.; Mostofi, Y. S.; Yang, L.; Simao, A. M.;
Narisawa, S.; Brown, B.; Mangravita-Novo, A.; Vicchiarelli, M.; Smith, L. H.;
O’Neill, W. C.; Millan, J. L.; Cosford, N. D. J. Med. Chem. 2009, 52, 6919; (b)
Sidique, S.; Ardecky, R.; Su, Y.; Narisawa, S.; Brown, B.; Millan, J. L.; Sergienko,
E.; Cosford, N. D. P. Bioorg. Med. Chem. Lett. 2009, 19, 222.
9. Sharma, V.; Ichikawa, M.; He, P.; Scott, D. A.; Bravo, Y.; Dahl, R.; Ng, B. G.;
Cosford, N. D.; Freeze, H. H. J. Biol. Chem. 2011, 286, 39431.
10. Correa, A.; Tellitu, I.; Dominguez, E.; SanMartin, R. Org. Lett. 2006, 8, 4811.
11. (a) Chemler, S. R.; Trauner, D.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2001, 40,
4544; (b) Kotha, S.; Lahiri, K. Biopolymers 2003, 69, 517.
a
http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1028.
and excretion (ADME) assays (Table 3).14 The data in Table 3 pro-
vide insight into the drug-likeness and potential for systemic activ-
ity of compounds, thus enabling advanced testing and future target
validation efforts. The selected compounds were shown to have
properties indicative of the potential for oral availability including
acceptable metabolic stability, good permeability across artificial
lipid membranes, and good solubility. No significant cell toxicity
could be detected for any of the analogues. With these data indic-
ative of drug-like behavior, good potency and selectivity, this series
may be suitable for in vivo proof-of-concept studies. Of note is the
esterification of the carboxylic group on 2o, which essentially
forms a pro-drug prone to facile metabolic cleavage and subse-
quent formation of 2n. Since 2o exhibits improved permeability
parameters compared with 2n, the data suggest that the develop-
ment of a series of pro-drug analogues may be a viable approach to
develop compounds with in vivo activity.
Compound 2q was additionally tested for selectivity against the
enzymes TNAP and ectonucleotide pyrophosphatase/phosphodies-
terase-1 (NPP-1) (Table 4). Gratifyingly, 2q was found to be devoid
of any significant inhibitory activity against these counter targets.
With respect to testing in other screens through the MLPCN pro-
gram, 2q (CID16749996) was found to have weak activity in a
screen for inhibitors of the mevalonate pathway in Streptococcus
pneumonia (AID1028). As shown in Table 4, 2q was at least 170-
fold selective for PHOSPHO1 versus counter targets.
12. General synthesis of PHOSPHO1 inhibitors: To a stirred solution of benzoic acid
derivative 5 (300 mg, 2.18 mmol) in CH2Cl2 was added an amine (266 mg,
3.27 mmol) followed by the coupling agents; HOBT (442 mg, 3.27 mmol) and
EDCI (630 mg, 3.27 mmol) and then Hunig’s base (1.14 mL, 6.54 mmol). The
reaction was stirred at room temperature over night, then quenched with
saturated NaHCO3 solution and CH2Cl2, then dried over Na2SO4. The solvents
were removed by rotary evaporation, no further purification was needed. To a
stirred solution of the aniline 6 (260 mg, 1.58 mmol) in CH2Cl2 at 0 °C under
nitrogen was added AlMe3 (2 mL, 1 M in THF) dropwise and the reaction was
slowly warmed to room temperature. The mixture was stirred continuously for
In conclusion, we have identified and developed a series of
PHOSPHO1 inhibitors with sub-micromolar potencies and promis-
ing drug-like properties. Medicinal chemistry efforts were applied
to the optimization of a benzoisothialozone core scaffold initially
identified through HTS. A carefully guided SAR study yielded a
series of highly active inhibitors with proven ability to inhibit
PHOSPHO1 in ex vivo models.15 On the basis of its overall profile
compound 2q was selected as MLPCN probe ML086. This work pro-
vides an example of a successful strategy using medicinal chemis-
try to develop a useful biological probe. Furthermore, the drug-like
properties of the resulting compounds provide an opportunity to
lay the foundation for the development of therapeutic agents suit-
able for the treatment of diseases caused by MVC.
an additional 30 min. Methyl thiosalicylate (130 lL, 0.790 mmol) was added
and the reaction was heated to 60 °C and then heated under reflux overnight.
The reaction was quenched with HCl (5% aq) and CH2Cl2 was added (50 mL).
The organic layer was separated and washed with saturated NaHCO3 solution,
then brine, and dried over Na2SO4. The solvents were removed by rotary
evaporation. The products were purified by flash chromatography or reverse
phase HPLC and lyophilized to provide thiols 4 which were determined to be
>95% pure by HPLC-UV, HPLC-MS, and 1H NMR. A solution of PIFA in CH2Cl2
was added to 0 °C solution of the benzamide (250 mg, 0.833 mmol) and TFA
(0.185 mL, 16 M) in CH2Cl2. The reaction mixture was stirred at room
temperature overnight. The solvents were removed in vacuo and the product
isolated by flash chromatography or reverse phase HPLC and lyophilized to
provide the final compounds (2) which were determined to be >95% pure by
HPLC-UV, HPLC-MS, and 1H NMR.
13. (a) Chung, T. D.; Sergienko, E.; Millan, J. L. Molecules 2010, 15, 3010; (b) Dahl,
R.; Bravo, Y.; Sharma, V.; Ichikawa, M.; Dhanya, R. P.; Hedrick, M.; Brown, B.;
Rascon, J.; Vicchiarelli, M.; Mangravita-Novo, A.; Yang, L.; Stonich, D.; Su, Y.;
Smith, L. H.; Sergienko, E.; Freeze, H. H.; Cosford, N. D. J. Med. Chem. 2011, 54,
3661.
Acknowledgments
14. Khan, P. M.; Correa, R. G.; Divlianska, D. B.; Peddibhotla, S.; Sessions, E. H.;
Magnuson, G.; Brown, B.; Suyama, E.; Yuan, H.; Mangravita-Novo, A.;
Vicchiarelli, M.; Su, Y.; Vasile, S.; Smith, L. H.; Diaz, P. W.; Reed, J. C.; Roth, G.
P. ACS Med. Chem. Lett. 2011, 2, 780.
15. Kiffer-Moreira, T.; Yadav, M. C.; Zhu, D.; Narisawa, S.; Sheen, C.; Stec, B.;
Cosford, N. D.; Dahl, R.; Farquharson, C.; Hoylaerts, M. F.; Macrae, V. E.; Millan,
J. L. J. Bone Miner. Res. 2013, 28, 81.
This work was supported by NIH grants HG005033 (NIGMS/
NIMH), AR53102 (NIAMS) and an American Recovery and Rein-
vestment Act (ARRA) Challenge grant RC1HL10899 from the
National Heart, Lung, and Blood Institute (NHLB).
References and notes
1. (a) Houston, B.; Seawright, E.; Jefferies, D.; Hoogland, E.; Lester, D.; Whitehead,
C.; Farquharson, C. Biochim. Biophys. Acta 1999, 1448, 500; (b) Houston, B.;