Design, synthesis, and biological evaluation of halogenated N-(2-(4-Oxo-1-phenyl-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)benzamides: Discovery of an isoform-selective small molecule phospholipase D2 inhibitor

Article abstract of DOI:10.1021/jm100814g

Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to the lipid second messenger phosphatidic acid. Two mammalian isoforms of PLD have been identified, PLD1 and PLD2, which share 53% sequence identity and are subject to different regulatory mechanisms. Inhibition of PLD enzymatic activity leads to increased cancer cell apoptosis, decreased cancer cell invasion, and decreased metastasis of cancer cells; therefore, the development of isoform-specific, PLD inhibitors is a novel approach for the treatment of cancer. Previously, we developed potent dual PLD1/PLD2, PLD1-specific (>1700-fold selective), and moderately PLD2-preferring (>10-fold preferring) inhibitors. Here, we describe a matrix library strategy that afforded the most potent (PLD2 IC₅₀ = 20 nM) and selective (75-fold selective versus PLD1) PLD2 inhibitor to date, N-(2-(1-(3-fluorophenyl)-4-oxo-1, 3,8-triazaspiro[4.5]decan-8-yl)ethyl)-2-naphthamide (22a), with an acceptable DMPK profile. Thus, these new isoform-selective PLD inhibitors will enable researchers to dissect the signaling roles and therapeutic potential of individual PLD isoforms to an unprecedented degree.

Full text of DOI:10.1021/jm100814g

6706 J. Med. Chem. 2010, 53, 6706–6719
DOI: 10.1021/jm100814g
Design, Synthesis, and Biological Evaluation of Halogenated N-(2-(4-Oxo-1-phenyl-
1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)benzamides: Discovery of an Isoform-Selective Small
Molecule Phospholipase D2 Inhibitor
Robert R. Lavieri,‡,† Sarah A. Scott,‡,† Paige E. Selvy,‡ Kwangho Kim, Satyawan Jadhav,‡ Ryan D. Morrison,^
J. Scott Daniels,‡,^ H. Alex Brown,‡,§, and Craig W. Lindsley*,‡,§, ,^
‡Department of Pharmacology, §Department of Chemistry, Vanderbilt Institute of Chemical Biology, and ^Vanderbilt Program in Drug
Discovery, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600. †These authors contributed equally to this work.
Received July 1, 2010
Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to the lipid second messenger
phosphatidic acid. Two mammalian isoforms of PLD have been identified, PLD1 and PLD2, which
share 53% sequence identity and are subject to different regulatory mechanisms. Inhibition of PLD
enzymatic activity leads to increased cancer cell apoptosis, decreased cancer cell invasion, and decreased
metastasis of cancer cells; therefore, the development of isoform-specific, PLD inhibitors is a novel
approach for the treatment of cancer. Previously, we developed potent dual PLD1/PLD2, PLD1-
specific (>1700-fold selective), and moderately PLD2-preferring (>10-fold preferring) inhibitors. Here,
we describe a matrix library strategy that afforded the most potent (PLD2 IC50 = 20 nM) and selec-
tive (75-fold selective versus PLD1) PLD2 inhibitor to date, N-(2-(1-(3-fluorophenyl)-4-oxo-1,3,8-
triazaspiro[4.5]decan-8-yl)ethyl)-2-naphthamide (22a), with an acceptable DMPK profile. Thus, these
new isoform-selective PLD inhibitors will enable researchers to dissect the signaling roles and
therapeutic potential of individual PLD isoforms to an unprecedented degree.
Introduction
messenger that is strategically located at the intersection of
severalessentialsignaling and metabolicpathways.3 Increased
PLD expression and aberrant PLD enzymatic activity have
been observed in a variety of human cancers including breast
cancer,4 renal cancer,5 colorectal cancer,6 and glioblastoma.7
Additionally, PLD activity has been shown to be required for
mutant Ras driven tumorigenesis in mice.8 Experiments uti-
lizing inactivating mutations of PLD suggest that inhibiting
PLD enzymatic activity decreases cancer cell invasion9 and
increases apoptosis.10 On a molecular level PLD has been
implicated in oncogenic signaling events involving the epider-
mal growth factor receptor (EGFR),11 matrix metalloprotein-
ase (MMP) secretion,7,12 p53,13,14 the mammalian target of
rapamycin (mTOR),15,16 and Ras.17 Taken together, evidence
from genetic and biochemical experiments indicates that PLD
is an attractive target for cancer therapy. Until recently, the
tools available to inhibit PLD activity were limited to genetic
and biochemical approaches including the use of 1-butanol,
none of which are viable therapeutic options. Furthermore,
1-butanol is not a PLD inhibitor; rather 1-butanol blocks
PLD-catalyzed phosphatidic acid production by competing
with water as a nucleophile, thereby causing the formation of
phosphatidylbutanol 4 in a unique transphosphatidylation
reaction.2 Several pan-PLD inhibitors 5-11 have been
reported,18-24 but many of these compounds do not act
directly on the enzyme, lack target potency, or are not drug-
like molecules (Figure 2A). Developing isoform-selective
PLD inhibitors is a formidable task due to several factors: (1)
PLD1 and PLD2 are challenging to purify in large quanti-
ties, (2) the enzyme activity assays are labor intensive and
Cancer is the second leading cause of mortality in the
United States.1 Both academic and industrial groups have
expended significant effort in attempts to develop novel
chemotherapeutics and, more recently, to identify previously
underappreciated targets essential to disease progression.
Advancing our basic understanding of cancer as a molecular
phenomenon through the use of small molecules is an im-
portant mechanism by which to both identify new targets and
develop innovative cancer therapeutics. Historically, targeted
cancer drug discovery efforts have focused on the develop-
ment of small molecule kinase inhibitors (typically at the ATP
binding site, though allosteric inhibitors are emerging) due to
the central role many kinases play in regulating cell growth
and division. The development of kinase inhibitors into drugs
has been partially hindered by poor selectivity versus the more
than 500 members of the human kinome. The identification of
other protein targets that regulate cell survival, invasion, and
proliferation will provide alternative options for cancer drug
development.
PLDa catalyzes the hydrolysis of phosphatidylcholine (PC, 1)
into the lipid second messenger phosphatidic acid (PA, 2)
and choline 3 (Figure 1).2 PA is an essential lipid second
*To whom correspondence should be addressed. E-mail: craig.
lindsley@vanderbilt.edu. Phone: 615-322-8700, Fax: 615-343-6532.
a Abbreviations: PA, phosphatidic acid; PC, phosphatidylcholine;
PLD, phospholipase D; EGFR, epidermal growth factor receptor;
mTOR, mammalian target of rapamycin; gfp, green fluorescence pro-
tein; PAM, positive allosteric modulator; NAM, negative allosteric
modulator; FBS, fetal bovine serum.
r
pubs.acs.org/jmc
Published on Web 08/24/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6707
Figure 1. PLD catalyzed the hydrolysis of phosphatidylcholine 1 (PC) into phosphatidic acid 2 (PA) and choline 3. In the presence of a primary
alcohol, typically 1-butanol, PLD catalyzes a unique transphosphatidylation reaction to produce phosphatidylbutanol 4.
Figure 2. (A) Structures of reported PLD inhibitors. (B) Sequence of PLD1 and PLD2 highlighting the PX and PH domains, the two HKD
motifs, the two catalytic sites, and the loop in PLD1, which is absent in PLD2. Overall, homology between the two PLD isoforms is only 53%.
time-consuming, and (3) the two mammalian isoforms of
PLD share 53% sequence identity (Figure 2B). Importantly,
due to the multitude of cellular events which require PA abla-
ting all PLD enzymatic activity may not be a viable thera-
peutic approach, in which case it would be necessary to
possess isoform-selective PLD inhibitors.
A turning point for the field occurred in 2007, when a group
at Novartis disclosed halopemide 12 as a PLD2 inhibitor
discovered in a high-throughput screen;25 however, rigorous
characterization by our laboratories demonstrated that 12
and the reported analogues were dual PLD1/2 inhibitors or
even moderately PLD1 preferring (Figure 3).26 Nonetheless,
this was the first time a potent, direct-acting, drug-like, small-
molecule PLD inhibitor had been reported; therefore, we
began a campaign to optimize halopemide for isoform-
specific PLD inhibition. An initial diversity-oriented synthesis
approach yielded a library of 263 compounds containing
direct-acting, potent (IC50 values 1-50 nM) PLD1 inhibitors,
such as 13 with 160-fold selectivity versus PLD2 in cellular
enzyme activity assays.26 A subsequent iterative analogue
synthesis approach delivered 14 with improved PLD1 potency
(IC50 = 3.7 nM) and selectivity (>1700-fold selective versus
PLD2 in cells).27 The identification of a single chiral (S)-
methyl group provided a significant gain in PLD1 selectivity.

6708 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Lavieri et al.
Figure 3. Structures and activities of halopemide 12 and isoform-selective PLD inhibitors 13 and 14 (PLD1 selective) and 15a,b (PLD2
preferring).
Scheme 1a
Developing a PLD2-selective inhibitor has been significantly
more challenging. After synthesizing over 500 compounds, we
identified a 1,3,8-triazaspiro[4,5]decan-4-one scaffold that
engendered PLD2-preferring inhibition, with the best com-
pound, 15b, only possessing ∼20 fold selectivity for PLD2 in
cells (Figure 3).28 Studieswith various PLD constructs suggest
that these inhibitors may bind at an allosteric site in the
N-terminus, accounting for the high isoform selectivity and
unique, shallow SAR.26 Moreover, these inhibitors blocked
the in vitro invasive migration of a triple negative breast
cancer cell line (MDA-MB-231), and siRNA studies indicated
that PLD2 played a dominant role.26
Due to a lack of small molecule tools and a perception of
phospholipases as nondruggable targets coupled with labor
intensive and complex assay systems, little effort has been
focused on their therapeutic potential. Herein we discuss our
ongoing medicinal chemistry efforts to develop isoform-
selective phospholipase D (PLD) inhibitors, the development
of the first PLD2-selective inhibitor, and the potential for
PLD inhibitors as a new class of cancer therapeutics. Here, we
report the results of our a matrix library approach to increase
PLD2 potency and selectivity within the 1,3,8-triazaspiro-
[4,5]decan-4-one series.
Chemistry
a Reagents and conditions: (a) KCN, AcOH, ArNH2, 12 h, room
temperature; (b) H2SO4, 12 h, 68%-74% for two steps; (c) (i) trimethyl
orthoformate, AcOH, microwave, 150 ꢀC, 15 min; (ii) NaBH4, MeOH,
3 h, 12%-20%; (d) H2, Pd/C, MeOH, AcOH, 20 h, 89%-96%.
Previous work showed that SAR was shallow with respect
to the Eastern amide moiety in 15a,b;28 thus current efforts
focused on functionalization of the 1,3,8-triazaspiro[4,5]decan-
4-one scaffold by the incorporation of various halogens, as
this proved successful in the benzimidazolone-based PLD1
inhibitors 13 and 14.27 Only the unsubstituted 1-phenyl-1,3,8-
triazaspiro[4,5]decan-4-one was commercially available, so
while known in the literature, the halogenated congeners
had to be synthesized. As shown in Scheme 1, N-benzylpiper-
idinone 16 underwent a Strecker reaction with 3-fluoroaniline
to provide 17a, and acidic hydrolysis delivered the carbox-
amide 18a in 68% yield for the two steps. Closing of the
spirocyclic five-membered ring required forcing microwave-
assisted conditions (150 ꢀC for 15 min in AcOH), followed by
reduction to provide 19a in 12% yield. A final hydrogenation
with Pd/C removed the benzyl protecting group, affording
the key scaffold 20a in 96% yield. In a similar manner, key
scaffolds 20b-f were prepared in overall yields from 16
averaging 8%.
With the requisite synthetically derived halogenated con-
geners 20a-f in hand, we initiated the synthesis of a 4 ꢀ 6
matrix library of 24 analogues based on the PLD2-preferring

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6709
Scheme 2a
a Reagents and conditions: 22(a-f) - 27(a-f) (a) (i) tert-butyl 2-oxoethylcarbamate, MP-B(OAc)3H, DCM, MeOH, 18 h, (ii) 4.0 M HCl/dioxane,
DCM, MeOH, 4 h, 58%-78%; (b) RCOCl, DIEA, DMF, room temperature, 4 h, 75%-85%.
Table 1. Structures and Cellular Assay Activities of Analogues 22a-d-27a-d
a Cellular PLD1 assay with Calu-1 cells. b Cellular assay with HEK293-gfpPLD2 cells. Cell-based assays were used to develop CRCs (from 200 pM to
20 μM) and determine IC50s for all compounds in Calu-1 or HEK293-gfpPLD2 cell lines. The geometric mean of the standard errors of the log(IC50
)
values from the curve fits of all compounds were computed and compared to the IC50s themselves. There were levels of ∼30% error for Calu-1 and ∼70%
for HEK293-gfpPLD2 IC50s. Despite the variance in the absolute values over a large number of assays, the reproducibility of the effects and relative
potency of the inhibitors were found to be robust.
inhibitors 15a,b (Scheme 2). In the event, 1,3,8-triazaspiro-
[4,5]decan-4-ones 20a-f underwent a reductive amination
reaction with tert-butyl 2-oxoethylcarbamate to provide,
after deprotection, amines 21a-f in 58-78% yields. Then,
the six amines 21a-f were acylated with four acid chlorides
(2-naphthyl, 3-quinolyl, 4-fluorobenzoyl, and 5-fluoro-2-
indolyl) to deliver the 24-member library of analogues
22a-d-27a-d in 75%-85% yields. All final compounds
were purified by mass-directed preparative HPLC to ana-
lytical purity.

6710 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Lavieri et al.
Results and Discussion
All library members 22a-d-27a-d were evaluated for
their ability to inhibit PLD1 and PLD2 in a cellular assay
(Calu-1 and HEK293-gfpPLD2 cell lines, respectively) as well
as a biochemical assay with recombinant PLD1 and PLD2
enzymes. Thecellularassayswerethe “workhorse” assays that
drove the SAR, with routine confirmation in the in vitro bio-
chemical assay to ensure compounds were direct acting
inhibitors. As shown in Table 1, SAR for the 24-member lib-
rary marked a clear departure from the SAR of the earlier
PLD1-selective benzimidazolone-based inhibitors, and all but
two of the analogues 22a-d-27a-d displayed a preference
for PLD2 inhibition, with the two exceptions, 26c and 27c,
being dual PLD1/2 inhibitors with comparable PLD1 and
PLD2 inhibition. Both PLD2 potency and selectivity were
dependent on the halogen employed, the substitution pattern
on the phenyl ring of the 1,3,8-triazaspiro[4,5]decan-4-one
scaffold, and the nature of the eastern amide moiety. As with
many allosteric ligands, SAR was shallow and unpredictable.
However, this matrix library approach identified several
PLD2 inhibitors that represented a significant improvement
over the original PLD2 inhibitor 15a and highlights the power
and utility of a matrix library approach, as the SAR would not
have informed a singleton approach toward optimal PLD2
inhibitors. For example, 23c and 25b displayed ∼50-fold
selectivity for PLD2, with PLD2 IC50s of 70 and 40 nM,
respectively; interestingly, 23c contains the 3-Cl moiety and a
4-fluorphenyl amide whereas 25b is based on a 4-F scaffold
and a 3-quinolinyl amide. Any other combination within these
scaffolds results in a decrease ineitherPLD2 potencyorPLD2
selectivity. From this effort, we discovered the most potent
and selectivePLD2inhibitor todate, 22a (VU0364739), with a
PLD2 IC50 of 20 nM and possessing 75-fold selectivity versus
PLD1 in the cellular assay (Figure 4A). In our in vitro
biochemical assay using purified PLD1 and PLD2, 22a
possessed a PLD1 IC50 of 7500 nM and a PLD2 IC50 of
100 nM, replicating the unprecedented 75-fold selectivity for
PLD2 (Figure 4B). While we could not replicate the fortuitous
1700-fold PLD1 selectivity of 14 in a PLD2-preferring inhi-
bitor, the 75-fold PLD2 selectivity of 22a afforded a small
molecule probe to effectively evaluate PLD2 pharmacology.
With potent and isoform-selective PLD1 (14) and PLD2 (22a)
inhibitors in hand, we were poised to dissect the individual
roles of PLD1 and PLD2 in a number of in vitro cancer cell
models.
Figure 4. Concentration-response curves (CRCs) for (A) cellular
PLD1 (9) (Calu-1) assay and PLD2 (2) (HEK293-gfpPLD2) assay
and (B) biochemical inhibition assay CRCs with purified (9) PLD1
and (2) PLD2 highlighting the unprecedented 75-fold PLD2 versus
PLD1 selectivity for 22a in both PLD assays. Error bars show
standard error of the mean for triplicate measurements.
In our earlier work with PLD1 inhibitor 13 and the
moderately selective PLD2 inhibitor 15a, we found that both
inhibitors blocked the in vitro invasive migration of a triple
negative breast cancer cell line (MDA-MB-231); however,
siRNA studies indicated that PLD2 played a dominant role.26
With significantly improved isoform-selective PLD1 (14) and
PLD2 (22a) inhibitors, we extended our study to dissect the
roles of PLD1 and PLD2 for proliferation and apoptosis in
MDA-MB-231 breast cancer cells. PLD2 inhibitor 22a pro-
vided a striking effect in a 48 h cell proliferation assay, wherein
inhibition of PLD2 affords a pronounced decrease in cell
proliferation of MDA-MB-231 cells, as compared to an
equivalent 10 μM concentration of the PLD1 inhibitor 14
(Figure 5A). When cultured under serum-free conditions, the
same assay in MDA-MB-231 cells resulted in almost a
complete blockade of proliferation with 22a, and under these
conditions, PLD1 inhibition has a significant effect as well
(Figure 5B). These data do show a preferential sensitization of
Figure 5. Inhibition of PLD2 with 22a leads to decreased prolif-
eration of MDA-MB-231 cells. MDA-MB-231 cells were cultured in
the presence of PLD inhibitor for 48 h after which cell viability was
assayed using WST-1 cell proliferation reagent. (A) MDA-MB-231
cells cultured in the presence of 10% FBS were fairly resistant to
PLD inhibitor treatment with only 10 μM 22a treatment leading to a
significant decrease in cell proliferation. (B) MDA-MB-231 cells
cultured under serum-free conditions had a more pronounced
response to PLD inhibition with both PLD1- (14) and PLD2-
(22a) selective compounds significantly decreasing cell prolifera-
tion. n = 3. Error bars show standard error of the mean for triplicate
measurements.
MDA-MB-231 cells to PLD2 inhibition. There was a notice-
able difference in the effect of PLD inhibitor treatment on
MDA-MB-231 cell proliferation depending on culture condi-
tions. When cells were cultured in the absence of serum with
either inhibitor 14 or 22a, there was a more pronounced
decrease in cell growth compared to the vehicle control

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6711
Figure 6. Inhibition of PLD2 leads to a time-dependent decrease in proliferation of MDA-MB-231 cells. MDA-MB-231 cells were cultured in
the presence of PLD inhibitor, and cell viability was assayed using WST-1 cell proliferation reagent over 96 h. (A) MDA-MB-231 cells cultured
in the presence of 10% FBS showed a dose-dependent attenuation of cell proliferation over time. Cultures with 10 and 5 μM 22a treatment led
to a significant decrease in cell proliferation while 1 μM inhibitor had no effect. (B) MDA-MB-231 cells cultured in the absence of serum had a
more pronounced response to PLD inhibition with all concentrations of the PLD2-selective compound significantly decreasing cell
proliferation in a dose- and time-dependent manner. Data are representative of three independent experiments. Error bars show standard
error of the mean for triplicate measurements.
Figure 7. Inhibition of PLD2 leads to increased apoptosis in MDA-MB-231 cells compared with minimal effect of PLD1 inhibition. MDA-
MB-231 cells were cultured in the presence of PLD inhibitor for 48 h after which time Caspase 3 and 7 activity was measured. (A) MDA-MB-
231 cells cultured in the presence of 10% FBS were fairly resistant to PLD inhibitor treatment (14 or 22a) with only 10 μM 22a treatment leading
to a significant increase in Caspase 3 and 7 activity compared to vehicle control. (B) MDA-MB-231 cells cultured under serum-free conditions
had increased Caspase 3 and 7 activity upon 10 μM PLD inhibitor treatment as compared to the vehicle control. *, p < 0.05; **, p < 0.01.
Data are representative of three independent experiments. Error bars show standard error of the mean for triplicate measurements.
samples or those cells cultured in the presence of 10% FBS.
There are several possible explanations for this observation.
These data may suggest that when these cells undergo the
stress of serum depravation, survival pathways in which PLD
is a key component become essential for cell proliferation, and
the inhibition of the PLD enzymatic activity causes a decrease
in cell proliferation. Alternatively, the pharmacokinetic
properties, specifically plasma protein binding, of these small
molecules have not been optimized, and it is likely that a
significant percentage of the compound in experiments con-
taining 10% FBS will be serum protein bound. We then
evaluated the effect of 22a on cell proliferation in MDA-
MB-231 cells over a 96 h time course and with a dose-
response paradigm (Figure 6). In the presence of 10% FBS,
22a displayed a dose-dependent decrease in cell proliferation
over the time course, with significant effects at both a 5 μM
and 10 μM dose (Figure 6A). Under serum-free conditions
(Figure 6B), a more pronounced effect was observed in a dose-
(1, 5, and 10 μM) and time-dependent manner, again suggest-
ing that PLD may be playing a role in the stress response of
these cells. Importantly, 22a was significantly less cytotoxic in
standard cell viability assays in nontransformed cells (datanot
shown).
Next, we evaluated the role of PLD1 and PLD2 inhibition
on apoptosis in MDA-MB-231 with and without serum,
employing Caspase 3 and 7 activity as a surrogate marker
for apoptosis (Figure 7). Once again, our isoform-selective
inhibitors were able to distinguish differential roles for PLD1
and PLD2. In the standard 48 h apoptosis assay, a 10 μM dose
of PLD2 inhibitor 22a provided a significant (3-fold increase)
increase in Caspase 3 and 7 activity, whereas inhibition of
PLD1 with 14 led to a marginal increase in Caspase 3 and
7 activity (Figure 7A). Under serum-free conditions, both 14
and 22a had similar effects on Caspase 3 and 7 activity
(Figure 7B). These data again suggest that PLD2 signaling
plays a critical role in the invasive migration, proliferation,
and survival of MDA-MB-231 breast cancer cells. Moreover,
these data were only obtainable once isoform-selective small
molecule PLD1 and PLD2 inhibitors were developed.
Compounds 14 and 22a were then subjected to a battery of
DMPK assays to elucidate their respective disposition charac-
teristics, ultimately in an effort to frame these isoform-selective
PLD inhibitors as suitable candidates as in vivo probes of PLD
function. PLD1 inhibitor 14 was lipophilic (c log P = 4.5) and
yet was ∼2% free in rat and human plasma protein binding
experiments (equilibrium dialysis) with a corresponding ease of

6712 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Table 2. Pharmacokinetic Profile of 14 and 22a in Rat
Lavieri et al.
iv (pharmacokinetics)a
CL
(mL min-1 kg-1
po (plasma and brain levels)b
plasma protein
binding (% bound)
dose
(mg/kg)
Vdss
(L/kg)
dose
(mg/kg)
plasma
(ng/mL)
brain
(ng/mL)
compd
)
t1/2 (h)
brain/plasma
14
22a
98.1
97.9
1
1
60.7
61.5
0.78
1.52
4.7
8.1
10
10
29
39.9
BLQ
29
BLQ
0.73
a 20% DMSO/80% saline. b 10% Tween 80/0.5% methylcellulose.
Scheme 3a
a Reagents and conditions: (a) (S)-tert-butyl 1-oxopropan-2-ylcarbamate, MP-B(OAc)3H, DCM, MeOH, 18 h, 34%; (b) 4.0 M HCl/dioxane, DCM,
MeOH, 4 h, 98%; (c) 2-naphthoyl chloride, DIEA, DMF, room temperature, 4 h, 9%.
formulation into dose vehicles amenable to iv and po adminis-
tration. Parenteral administration to rats (n = 2) revealed 14 to
be a highly cleared compound (CL = 60 mL min-1 kg-1),
approaching that of hepatic blood flow (Qh) in the rat (Table 2).
A corresponding volume of distribution at steady state (Vdss)
for 14 of 4.7 L/kg produced a mean residence time (MRT) of 1.1
h and an effective half-life (t1/2) of 0.78 h in rat. A similar profile
was obtained for PLD2 inhibitor 22a. While less lipophilic (c log
P=3.2), 22a also displayed ∼2% free fraction in rat and human
plasma protein binding experiments (equilibrium dialysis)
and was easily formulated into acceptable vehicles. Simi-
Figure 8. Structures and activities of PLD2-preferring inhibitor
larly, parenteral administration to rats revealed 22a to be a
15a and impact of a chiral (S)-methyl group providing 28 and a
40-fold increase in PLD inhibitory activity.
highly cleared compound (CL=61 mL min-1 kg-1), with a
high volume of distribution (Vdss =8.1 L/kg), a 2.2 h MRT,
and a corresponding effective t1/2 of 1.5 h. Employing rat
hepaticmicrosomes, the intrinsic clearancevalues(CLint, eqs1
and 2) for 14 and 22a were determined to be 660 and 203 mL
min-1 kg-1, respectively (data not shown), and converted
to predicted hepatic clearance (CLhep), utilizing the well-
stirred model of hepatic clearance (eq 3), produced CLhep
values of 63 and 52 mL min-1 kg-1. The general agreement
between in vitro and in vivo clearance (CL and CLhep) values
indicate hepatic metabolism to be a likely mechanism con-
tributing to the disposition of 14 and 22a.
Recent genetic and knockout studies have suggested thera-
peutic roles for PLD inhibition in Alzheimer’s disease29,30 and
stroke;31 therefore, PLD inhibitors with exceptional CNS
bioavailability would be of great value for preclinical target
validation. To address CNS penetration, fasted Sprague-
Dawley rats (n = 2) received a single, oral gavage of 14 and
22a ata doseof 10 mg/kg in a typical 90 min singlepoint brain:
plasma (PBL) study design. While levels of 14 were below
quantitation in the brain, the napthylene analogue 22a dis-
played a brain:plasma value of 0.73, thereby representing the
first centrally penetrant PLD inhibitor. While 14 and 22a
remain important in vitro tools to probe and describe the
differential roles and pharmacology of PLD1 and PLD2,
additional optimization will be required to develop robust in
vivo proof-of-concept compounds. These data also suggest
that the differences observed in the cellular experiments
involving the presence and absence of serum could be due to
the lipophillic character of these compounds and the result
that ∼2% is displayed as free fraction in rat and human
plasma protein binding experiments.
Finally, the impact of incorporating an (S)-methyl group
was found to be important for increasing PLD inhibitory
activity in the PLD1-selective benzimidazolone series repre-
sented by 13 and 14. Installation of the (S)-methyl group into
the modestly PLD2-preferring 15a (PLD1 IC50 = 1000 nM,
PLD2 IC50 = 110 nM), within the triazaspirone series (Figure 8),
resulted in 28 with enhanced (40-fold) PLD1 inhibition and
essentially no effect on PLD2 activity (PLD1 IC50 = 25 nM,
PLD2 IC50 = 140 nM).28 This type of “molecular switch”
has been noted before for allosteric modulators of GPCRs
engendering either subtype selectivity or reversing the mode of
pharmacology (NAM to PAM or PAM to NAM). Thus, we
wanted to evaluate if the addition of the PLD1-preferring (S)-
methyl “molecular switch” would increase PLD1 inhibitory
activity in a highly PLD2-preferring compound such as 22a.
In the event, 22a underwent a reductive amination with (S)-
tert-butyl 1-oxopropan-2-ylcarbamate to provide 29, which
was then deprotected to provide 30. Acylation with 2-
naphthoyl chloride afforded the (S)-methyl analogues 31 of
22a (Scheme 3). Evaluation of 31 in our PLD1 and PLD2

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6713
cellular assays (Calu-1 and HEK293-gfpPLD2, respectively)
further highlighted the impact of the (S)-methyl group as a
“molecular switch” for these allosteric ligands, providing a
High-resolution mass spectra were recorded on a Waters QToF-
API-US plus Acquity system with electrospray ionization. Ana-
lytical thin-layer chromatography was performed on 250 μm
silica gel 60 F254 plates. Automated flash column chromato-
graphy was performed on a Teledyne ISCO combiflash Rf system.
Analytical HPLC was performed on an Agilent 1200 analytical
LC-MS equipped with a YMC Jsphere H-80 S-4 3.0 ꢀ 50 mm
column running a gradient of 5%-95% (at a flow rate of 1.25
mL/min over 4 min) acetonitrile in 0.1% trifluoroacetic acid in
water and UV detection at 214 and 254 nm along with ELSD
detection. Analytical HPLC for compounds 30 and 31 was
performed on an Agilent 1200 LC-MS with electrospray ioniza-
tion equipped with a Phenomenex Kinetex 2.1 ꢀ 50 mm C18
column running a gradient of 10%-95% (over 45 s) acetonitrile
in 0.1% trifluoroacetic acid in water and UV detection at 214 and
254 nm. Preparative purification of library compounds was
performed on a custom Agilent 1200 preparative LC-MS with
collection triggered by mass detection, or alternatively com-
pounds were purified on a Gilson 215 preparative LC system
equipped with a Phenomenx Luna 5u C18 50ꢀ30 mm column by
running a gradient of 20%-60% acetonitrile in 0.1% trifluoro-
acetic acid in water at a flow rate of 50 mL/min over approxi-
mately 5 min. All compounds described within this report
are >95% pure by HPLC (254 nm, 214 nm, and ELSD) as well as
1H NMR. All yields refer to analytically pure and fully characterized
materials (1H NMR, 13C NMR, analytical LC-MS, and HRMS).
1-Benzyl-4-((3-fluorophenyl)amino)piperidine-4-carboxamide
(18a). To a solution of 1-benzylpiperidin-4-one (13.25 g, 70 mmol)
in glacial acetic acid (70 mL) and water (12 mL) cooled to 0 ꢀC
were added 3-fluoroaniline (8.55 g, 77 mmol) and potassium
cyanide (4.55 g, 70 mmol). The reaction was allowed to warm to
room temperature and agitated for approximately 12 h. The
reaction was then cooled to 0 ꢀC, and ammonium hydroxide
(18 M) was added dropwise until the solution pH was 11 or
greater. The mixture was then extracted into dichloromethane
and dried under reduced pressure to yield the crude product as a
tan oil (20.5 g). The crude product was then immediately cooled to
0 ꢀC, and concentrated sulfuric acid (18 M, 120 mL) was added
dropwise. The reaction was allowed to warm to room temperature
and agitated for approximately 12 h. The reaction was then cooled
to 0 ꢀC, and ammonium hydroxide (18 M) was added dropwise
until the solution pH was 11 or greater. The mixture was then
extracted into dichloromethane and dried under reduced pressure
to afford a tan solid (15.78 g, 48.25 mmol, 68%). 1H NMR (400.1
MHz, CDCl3) δ (ppm): 7.51-7.37 (m, 7H), 6.67-6.47 (m, 4H),
4.27 (s, 1H), 3.64 (s, 2H), 2.95-2.87 (m, 2H), 2.53-2.44 (m, 2H),
2.29-2.21 (m, 2H), 2.07 (d, J = 13 Hz, 2H). 13C NMR (100.6
MHz, CDCl3) δ (ppm): 178.0, 162.6, 145.7, 138.3, 130.5, 129.1
(2C), 128.4 (2C), 127.2, 111.8, 106.1, 103.1, 63.1, 58.5, 48.7 (2C),
34.8, 31.5. HRMS (TOF, ESI) C19H23N3OF [M þ H]þ calculated
328.1825, found 328.1827. LC-MS: rt (min) = 1.855. LRMS
(ESI) m/z = 328.2.
150-fold increase in PLD1 inhibitory activity (PLD2 IC50
=
10 nM) while maintaining PLD2 activity (PLD2 IC50 =60nM).
Thus, a 75-fold PLD2-preferring inhibitor 22a is converted
into a potent dual PLD1/2 inhibitor 31 by the addition of a
single methyl group.
Conclusion
In summary, we have developed the most potent (PLD2
IC50 = 20 nM) and selective (75-fold versus PLD1) PLD2
inhibitor 22a described. Due to the shallow and unpredictable
SAR for these allosteric PLD inhibitors, a matrix library app-
roach enabled the rapid discovery of 22a, whereas a classical
singleton approach probably would have failed to discover
22a. As with other allosteric ligand optimization programs,
SAR proved to be shallow and somewhat unpredictable for
the triazaspirone series, with PLD potency and PLD2 selec-
tivity dependent on both the halogen substituent on the tri-
azaspirone scaffold and the nature of the amide moiety. With
potent and selective PLD1 and PLD2 inhibitors in hand, we
were able to dissect the relative contributions of PLD1 and
PLD2 signaling on proliferation and survival of the triple
negative breast cancer cell line MDA-MB-231. In all in-
stances, selective PLD2 inhibition with 22a displayed signifi-
cant effects and suggests that for this cancer cell line a PLD2
inhibitor, not a PLD1 or a dual PLD1/2 inhibitor, would be
the optimal therapeutic agent. Introduction of a “molecular
switch”, in the form of an (S)-methyl group, to 22a increased
PLD1 activity 150-fold, providing a potent PLD1/2 inhibitor
31. Current efforts are focused on employing 14 and 22a to
dissect the contributions of PLD1 and PLD2 signaling in
other cancer cell lines as well as developing additional iso-
form-selective PLD inhibitors with improved DMPK proper-
ties which will enable the discovery of new indications, such as
schizophrenia, stroke, and Alzheimer’s disease, where aber-
rant PLD activity is implicated.
Experimental Section
All reactions were carried out employing standard chemical
techniques. Unless otherwise noted, reactions were run in an-
hydrous solvents. Solvents for extraction, washing, and chromato-
graphy were HPLC grade. All reagents were purchased from
Sigma-Aldrich and Biotage at the highest commercial quality and
were used without purification. Microwave-assisted reactions
were conducted using a Biotage Initiator-60 single mode micro-
wave synthesizer. All NMR spectra were recorded on a 400 MHz
Bruker AMX NMR. 1H chemical shiftsarereportedas δvalues in
ppm relative to the solvent residual peak (MeOD =3.31, DMSO-
d6 = 2.50, CDCl3 = 7.26). Data are reported as follows: chemical
shift, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet), coupling constant (Hz), and inte-
gration. 13C chemical shifts are reported as δ values in ppm re-
lative to the solvent residual peak (MeOD = 49.0, DMSO-d6 =
39.5, CDCl3 = 77.16). Low-resolution mass spectra were ob-
tained on an Agilent 1200 LC-MS with electrospray ionization
equipped with a YMC Jsphere H-80 S-4 3.0 ꢀ 50 mm column
running a gradient of 5%-95% (over 4 min) acetonitrile in 0.1%
trifluoroacetic acid in water. Low-resolution mass spectra for
compounds 30 and 31 were obtained on an Agilent 1200 LC-MS
with electrospray ionization equipped with a Phenomenex
Kinetex 2.1 ꢀ 50 mm C18 column running a gradient of 10%-
95% (over 45 s) acetonitrile in 0.1% trifluoroacetic acid in water.
1-(3-Fluorophenyl)-1,3,8-triazaspiro[4.5]decan-4-one (20a). 1-
Benzyl-4-((3-fluorophenyl)amino)piperidine-4-carboxamide 18a
(15.78 g, 48.25 mmol), trimethyl orthoformate (80 mL), and
glacial acetic acid (40 mL) were combined and subjected to
microwave irradiation at 150 ꢀC for 15 min. The mixture was
adjusted to pH 12 with ammonium hydroxide (18 M) and
extracted into dichloromethane and dried under reduced pres-
sure. This material was then added to a suspension of sodium
borohydride (4.56 g, 120.6 mmol) in methanol (150 mL) and
stirred for about 3 h. The reaction was quenched with water,
extracted into dichloromethane, and dried under reduced pres-
sure. The material was then chromatographed on a 330 g flash
column (Teledyne) as follows: (1) a gradient from 0% to 80%
ethyl acetated in hexanes over 10 min was run, and on the same
column (2) a gradient from 0% to 10% methanol in dichloro-
methane was run. The purity of the isolated intermediate com-
pound was established via LC-MS: rt (min) 1.723; LRMS (ESI)
m/z = 340.1. This intermediate 19a (1.94 g) was immediately

6714 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Lavieri et al.
dissolved in methanol (40 mL) and glacial acetic acid (10 mL)
and treated with palladium on carbon (cat., 80 mg) under an
atmosphere of hydrogen. After about 36 h the reaction mix-
ture was filtered through Celite, concentrated under reduced
pressure, diluted with water, made alkaline with saturated
sodium bicarbonate, and extracted 8 times into dichloro-
methane to afford a white solid (1.37 g, 5.49 mmol, 11%).
1H NMR (400.1 MHz, DMSO-d6) δ (ppm): 8.67 (s, 1H), 7.20
(q, J = 8 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.62 (d, J = 13 Hz,
1H), 6.52-6.46 (m, 1H), 4.57 (s, 2H), 3.20-3.09 (m, 3H),
2.91-2.82 (m, 2H), 2.46-2.36 (m, 2H), 1.48 (d, J = 14 Hz,
2H). 13C NMR (100.6 MHz, DMSO-d6) δ (ppm): 176.0, 164.3,
145.0, 130.1, 109.3, 103.1, 100.2, 58.8, 58.6, 42.1 (2C), 28.9 (2C).
HRMS (TOF, ESI) C13H17N3OF [M þ H]þ calculated
250.1356, found 250.1351. LC-MS: rt (min) = 1.394. LRMS
(ESI) m/z = 250.1.
8.04-7.92 (m, 4H), 7.64-7.56 (m, 2H), 7.11 (q, J = 8 Hz, 1H),
6.68-6.63 (m, 1H), 6.58-6.52 (m, 1H), 6.49-6.43 (m, 1H), 4.58
(s, 2H), 3.48 (q, J=6 Hz, 2H), 2.91-2.83 (m, 2H), 2.80-2.72 (m,
2H), 2.64-2.53 (m, 4H), 1.58 (d, J = 14 Hz, 2H). 13C NMR
(100.6 MHz, DMSO-d6) δ (ppm): 175.9, 166.2, 164.3, 161.9,
145.0, 134.1, 132.2, 130.3, 128.8, 127.8, 127.6, 127.5, 127.3,
126.7, 124.1, 109.3, 103.3, 100.3, 58.7, 58.1, 56.9, 49.4 (2C),
37.3, 28.2 (2C). HRMS (TOF, ESI) C26H28N4O2F [M þ H]þ
calculated 447.2196, found 447.2195. LC-MS: rt (min) = 2.287.
LRMS (ESI) m/z = 447.2. Analogues 22b-d were made follow-
ing the same protocol starting from 21a and were purified via
reversed-phase chromatography to greater than 95% purity
(as trifluoroacetate salts) as analyzed by ELSD and UV at both
214 and 254 nM.
N-(2-(1-(3-Fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-
8-yl)ethyl)-2-naphthamide Hydrochloride (22a HCl). N-(2-(1-(3-
3
8-(2-Aminoethyl)-1-(3-fluorophenyl)-1,3,8-triazaspiro[4.5]de-
can-4-one Dihydrochloride (21a). 1-(3-Fluorophenyl)-1,3,8-tri-
azaspiro[4.5]decan-4-one 20a (1370 mg, 5.49 mmol) and tert-
butyl (2-oxoethyl)carbamate (961 mg, 6.03 mmol) were com-
bined and dissolved in dichloromethane (25 mL) and methanol
(10 mL) and stirred for about 30 min at room temperature. After
about 30 min macroporous triacetoxyborohydride (3 g, 7.26
mmol) was added to the reaction, and after 14 h an additional
amount of tert-butyl (2-oxoethyl)carbamate (200 mg, 1.25 mmol)
was added to drive the reaction to completion. After about 24 h
the reaction mixture was filtered through Celite and concen-
trated under reduced pressure. The crude compound was chro-
matographed on an 80 g flash column eluting in a gradient of
0%-10% methanol in dichloromethane to afford a white solid
(1.64 g, 4.18 mmol, 76%). 1H NMR (400.1 MHz, DMSO-d6) δ
(ppm): 8.69 (s, 1H), 7.22 (q, J = 8 Hz, 1H), 6.72-6.63 (m, 2H),
6.60-6.49 (m, 2H), 4.58 (s, 2H), 2.83-2.75 (m, 2H), 2.74-2.65
(m, 2H), 2.61-2.48 (m, 2H), 2.42-2.35 (m, 2H), 1.91 (s, 2H),
1.55 (d, J = 13 Hz, 2H), 1.39 (s, 9H). 13C NMR (100.6 MHz,
DMSO-d6) δ (ppm): 175.8, 161.9, 155.6, 145.0, 130.4, 109.4,
103.2, 100.3, 77.5, 58.7, 58.1, 57.4, 49.3 (2C), 37.6, 28.3 (3C),
28.1 (2C). HRMS (TOF, ESI) C20H30N4O3F [M þ H]þ calcu-
lated 393.2302, found 393.2301. LC-MS: rt (min) = 1.966.
LRMS (ESI) m/z = 393.2. tert-Butyl (2-(1-(3-fluorophenyl)-4-
oxo-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)carbamate (1.64 g,
4.18 mmol) was dissolved in dichloromethane (40 mL) and a
minimal amount of methanol added dropwise. Hydrochloric
acid was added (4 M in dioxane, 20 mL), and the reaction was
stirred for approximately 36 h at room temperature. The reac-
tion was concentrated under reduced pressure to afford a white
solid (1.34 g, 3.66 mmol, 88%). 1H NMR (400.1 MHz, DMSO-
d6) δ (ppm): 9.12 (s, 1H), 8.47 (s, 2H), 7.18 (q, J = 8 Hz, 1H),
7.07-7.02 (m, 1H), 6.79-6.72 (m, 1H), 6.57-6.50 (m, 1H), 4.63
(s, 2H), 3.72-3.56 (m, 4H), 3.45-3.38 (m, 4H), 3.10-3.00 (m,
2H), 1.90 (d, J = 15 Hz, 2H), 13C NMR (100.6 MHz, DMSO-d6)
δ (ppm): 174.4, 162.3, 144.4, 130.3, 109.8, 103.8, 100.2, 69.0,
56.5, 53.3, 49.1 (2C), 33.8, 25.6 (2C). HRMS (TOF, ESI)
C15H22N4OF [M þ H]þ calculated 293.1778, found 293.1776.
LC-MS: rt (min) = 1.405. LRMS (ESI) m/z = 293.1.
Fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)-2-
naphthamide 22a (1.25 mg, 2.80 mmol) was stirred in methanol
(30 mL) at room temperature and treated with hydrochloric acid
(4 M in dioxane, 4 mL). After about 25 min the compound was
dried under reduced pressure to afford a white solid (1.31 g, 2.72
mmol, 97%). 1H NMR (400.1 MHz, DMSO-d6) δ (ppm): 10.99
(s, 1H), 9.14 (t, J = 5 Hz, 1H), 9.11 (s, 1H), 8.60 (s, 1H),
8.06-7.97 (m, 4H), 7.65-7.57 (m, 2H), 7.21 (q, J = 8 Hz, 1H),
7.05-7.01 (m, 1H), 6.83-6.77 (m, 1H), 6.58-6.52 (m, 1H), 4.64
(s, 2H), 3.85-3.75 (m, 2H), 3.74-3.64 (m, 4H), 3.41-3.36 (m,
2H), 3.11-2.99 (m, 2H), 1.92 (d, J = 14 Hz, 2H). 13C NMR
(100.6 MHz, DMSO-d6) δ (ppm): 174.4, 166.6, 164.6, 162.2,
144.6, 134.3, 132.1, 131.2, 130.5, 128.9, 127.9, 127.8, 127.6,
126.8, 124.2, 109.9, 104.0, 100.3, 59.0, 56.6, 55.7, 48.7 (2C),
34.4, 25.7 (2C). HRMS (TOF, ESI) C26H28N4O2F [M þ H]þ
calculated 447.2196, found 447.2186. LC-MS: rt (min) = 2.264.
LRMS (ESI) m/z = 447.2.
8-(2-Aminoethyl)-1-(3-chlorophenyl)-1,3,8-triazaspiro[4.5]-
decan-4-one Dihydrochloride (21b). 1-(3-Chlorophenyl)-1,3,8-
triazaspiro[4.5]decan-4-one 20b (127 mg, 0.47 mmol) and tert-
butyl (2-oxoethyl)carbamate (83.8 mg, 0.51 mmol) were com-
bined and dissolved in dichloromethane (1.5 mL) and methanol
(0.05 mL) and stirred for about 30 min at room temperature.
After about 30 min macroporous triacetoxyborohydride (600
mg, 1.4 mmol) was added to the reaction, and after 14 h an
additional amount of tert-butyl (2-oxoethyl)carbamate (41.9
mg, 0.25 mmol) was added to drive the reaction to completion.
After about 24 h the reaction mixture was filtered through Celite
and concentrated under reduced pressure. The crude compound
was chromatographed on a 12 g flash column eluting in a
gradient of 0%-10% methanol in dichloromethane to afford
a white solid (72 mg, 0.18 mmol, 37%). 1H NMR (400.1 MHz,
MeOD) δ (ppm): 7.21 (t, J = 9 Hz, 1H), 6.95-6.90 (m, 2H),
6.86-6.78 (m, 1H), 4.69 (s, 2H), 3.23-3.02 (m, 4H), 2.81-2.67
(m, 4H), 1.97 (s, 2H), 1.78 (d, J = 14 Hz, 2H), 1.45 (s, 9H). 13C
NMR (100.6 MHz, MeOD) δ (ppm): 178.2, 158.5, 145.8, 136.2,
141.3, 119.5, 115.4, 114.2, 80.3, 60.4, 60.1, 58.4, 50.7 (2C), 38.1,
29.2 (2C). 28.7 (3C). HRMS (TOF, ESI) C20H30N4O3Cl [M þ
H]þ calculated 409.2006, found 409.1996. LC-MS: rt (min) =
1.984. LRMS (ESI) m/z = 409.2. tert-Butyl (2-(1-(3-chloro-
phenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)carbamate
(72 mg, 0.17 mmol) was dissolved in dichloromethane (5 mL)
and a minimal amount of methanol added dropwise. Hydro-
chloric acid was added (4 M in dioxane, 1.0 mL), and the re-
action was stirred for approximately 16 h at room temperature.
The reaction was concentrated under reduced pressure to afford
a white solid (60 mg, 0.16 mmol, 93%). 1H NMR (400.1 MHz,
MeOD) δ (ppm): 7.25 (t, J = 8 Hz, 1H), 7.20-7.15 (m, 1H),
6.88-6.81 (m, 2H), 4.74 (s, 2H), 3.96-3.86 (m, 2H), 3.73-3.65
(m, 2H), 3.51 (s, 4H), 3.20-3.10 (m, 2H), 2.05 (d, J = 15 Hz,
2H). 13C NMR (100.6 MHz, MeOD) δ (ppm): 176.9, 145.2, 135.6,
131.5, 119.9, 115.1, 114.4, 60.5, 58.4, 54.9, 51.2 (2C), 35.4, 27.8
(2C). HRMS (TOF, ESI) C15H22N4OCl [M þ H]þ calculated
N-(2-(1-(3-Fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-
8-yl)ethyl)-2-naphthamide (22a). 8-(2-Aminoethyl)-1-(3-fluoro-
phenyl)-1,3,8-triazaspiro[4.5]decan-4-one dihydrochloride 21a
(1.23 g, 3.37 mmol), 2-naphthoyl chloride (641 mg, 3.37 mmol),
and N,N-diisopropylethylamine (2.05 mL, 11.7 mmol) were all
dissolved in N,N-dimethylformamide (20 mL) at 0 ꢀC. The re-
action mixture was allowed to warm to room temperature and
stirred for about 12 h. The reaction mixture was diluted with
water and extracted into dichloromethane 5 times. The dichloro-
methane layer was then washed 3 times with a solution of lithium
chloride (3 M) and dried under reduced pressure. The reaction
mixture was chromatographed on an 80 g flash column eluting
in 0%-5% methanol in dichloromethane to afford a white solid
1
(1.25 g, 2.80 mmol, 83%). H NMR (400.1 MHz, DMSO-d6)
δ (ppm): 8.69 (s, 1H), 8.60 (t, J = 5 Hz, 1H), 8.45 (s, 1H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6715
309.1482, found 308.1480. LC-MS: rt (min) = 1.413. LRMS
(ESI) m/z = 309.1.
(S)-N-(1-(1-(3,4-Difluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]-
decan-8-yl)propan-2-yl)-2-naphthamide 2,2,2-Trifluoroacetate (24a).
(S)-8-(2-Aminopropyl)-1-(3,4-difluorophenyl)-1,3,8-triazaspiro-
[4.5]decan-4-one dihydrochloride 21c (40 mg, 0.10 mmol),
2-naphthoyl chloride (19.1 mg, 0.10 mmol), and N,N-diisopro-
pylethylamine (0.073 mL, 0.42 mmol) were all dissolved in N,N-
dimethylformamide (1 mL) at 0 ꢀC. The reaction mixture was al-
lowed to warm to room temperature and stirred for about 12 h.
The reaction mixture was diluted with water and extracted into
dichloromethane 5 times. The dichloromethane layer was then
washed 3 times with a solution of lithium chloride (3 M) and
dried under reduced pressure. The reaction mixture was sub-
jected to reversed-phase chromatography to afford a white solid
(29.2 mg, 0.05 mmol, 49%). 1H NMR (400.1 MHz, DMSO-d6)
δ (ppm): 9.69 (s, 1H), 9.08 (s, 1H), 8.77 (d, J = 8 Hz, 1H), 8.50 (s,
1H), 8.05-7.95 (m, 4H), 7.66-7.58 (m, 2H), 7.25 (q, J = 10 Hz,
1H), 7.02-6.95 (m, 1H), 6.74-6.69 (m, 1H), 4.59 (s, 2H), 3.85-
3.68 (m, 4H), 3.60-3.53 (m, 1H), 3.43-3.31 (m, 2H), 2.78-2.57
(m, 2H), 2.01-1.89 (m, 2H), 1.31 (d, J = 7 Hz, 3H). 13C NMR
(100.6 MHz, DMSO-d6) δ (ppm): 174.3, 166.5, 158.2, 148.6,
140.1, 134.3, 132.1, 131.4, 128.8, 127.8, 127.7 (2C), 127.6, 126.8,
124.4, 117.6, 117.5, 110.9, 104.1, 103.9, 60.7, 59.2, 56.7, 49.8,
48.6, 41.2, 26.1, 26.0, 19.0. HRMS (TOF, ESI) C27H29N4O2F2
[Mþ H]þ calculated 479.2259, found 479.2262. LC-MS: rt (min) =
2.286. LRMS (ESI) m/z = 479.2. Analogues 24b-d were made
following the same protocol starting from 21c and were purified
via reversed-phase chromatography to greater than 95% purity
(as trifluoroacetate salts) as analyzed by ELSD and UV at both
214 and 254 nM.
N-(2-(1-(3-Chlorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8-yl)-
ethyl)-2-naphthamide 2,2,2-Trifluoroacetate (23a). 8-(2-Amino-
ethyl)-1-(3-chlorophenyl)-1,3,8-triazaspiro[4.5]decan-4-one di-
hydrochloride 21b (60 mg, 0.15 mmol), 2-naphthoyl chloride
(30.0 mg, 0.15 mmol), and N,N-diisopropylethylamine (0.115
mL, 0.66 mmol) were all dissolved in N,N-dimethylformamide
(1 mL) at 0 ꢀC. The reaction mixture was allowed to warm to
room temperature and stirred for about 12 h. The reaction mix-
ture was diluted with water and extracted into dichloromethane
5 times. The dichloromethane layer was then washed 3 times
with a solution of lithium chloride (3 M) and dried under redu-
ced pressure. The reaction mixture was subjected to reversed-
phase chromatography to afford a white solid (43.4 mg, 0.075
mmol, 50%). 1H NMR (400.1 MHz, DMSO-d6) δ (ppm): 9.32
(s, 1H), 9.13 (s, 1H), 9.01 (t, J = 5 Hz, 1H), 8.50 (s, 1H),
8.06-7.94 (m, 4H), 7.66-7.58 (m, 2H), 7.22 (t, J = 8 Hz, 1H),
6.98-6.94 (m, 1H), 6.86-6.80 (m, 2H), 4.65 (s, 2H), 3.79-3.68
(m, 6H), 3.44-3.39 (m, 2H), 2.88-2.75 (m, 2H), 1.97 (d, J = 15
Hz, 2H). 13C NMR (100.6 MHz, DMSO-d6) δ (ppm): 174.3,
167.0, 158.6, 144.2, 134.3, 134.2, 132.1, 131.2, 130.4, 128.9 (2C),
128.0, 127.8, 127.7 (2C), 126.9, 124.1, 117.7, 113.2, 112.5, 59.0,
56.6, 55.3, 48.9 (2C), 34.6, 26.0 (2C). HRMS (TOF, ESI) C26-
H28N4O2Cl [M þ H]þ calculated 463.1901, found 463.1894. LC-
MS: rt (min) = 2.266. LRMS (ESI) m/z = 463.1. Analogues
23b-d were made following the same protocol starting from 21b
and were purified via reversed-phase chromatography to greater
than 95% purity (as trifluoroacetate salts) as analyzed by ELSD
and UV at both 214 and 254 nM.
(S)-8-(2-Aminopropyl)-1-(3,4-difluorophenyl)-1,3,8-triazaspiro-
[4.5]decan-4-one Dihydrochloride (21c). 1-(3,4-Difluorophenyl)-
1,3,8-triazaspiro[4.5]decan-4-one 20c (104.8 mg, 0.39 mmol)
and (S)-tert-butyl (1-oxopropan-2-yl)carbamate (74.6 mg, 0.43
mmol) were combined and dissolved in dichloromethane (1.5 mL)
and methanol (0.05 mL) and stirred for about 30 min at room
temperature. After about 30 min macroporous triacetoxy-
borohydride (600 mg, 1.4 mmol) was added to the reaction, and
after 14 h an additional amount of (S)-tert-butyl (1-oxopropan-
2-yl)carbamate (37.3 mg, 0.22 mmol) was added to drive the
reaction to completion. After about 24 h the reaction mixture
was filtered through Celite and concentrated under reduced
pressure. The crude compound was chromatographed on a 12 g
flash column eluting in a gradient of 0%-10% methanol in
dichloromethane to afford a white solid (60 mg, 0.14 mmol,
36%). 1H NMR (400.1 MHz, MeOD) δ (ppm): 7.15 (q, J = 10
Hz, 1H), 7.00-6.92 (m, 1H), 6.76-6.70 (m, 1H), 4.67 (s, 2H),
3.98-3.80 (m, 1H), 3.18-3.09 (m, 1H), 2.83-2.52 (m, 5H), 1.96
(s, 2H), 1.83 (d, J = 14 Hz, 2H), 1.45 (s, 9H), 1.17 (d, J = 7 Hz,
3H). 13C NMR (100.6 MHz, MeOD) δ (ppm): 178.0, 158.1,
152.9, 150.6, 141.5, 118.4, 113.2, 106.3, 80.5, 63.8, 60.8, 59.9,
51.4 (2C), 44.6, 29.1 (2C), 28.7 (3C), 19.6. HRMS (TOF, ESI)
C21H31N4O3F2 [M þ H]þ calculated 425.2364, found 425.2367.
LC-MS: rt (min) = 2.028. LRMS (ESI) m/z = 425.2. (S)-tert-
Butyl (1-(1-(3,4-difluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-
8-yl)propan-2-yl)carbamate (60 mg, 0.14 mmol) was dissolved
in dichloromethane (5 mL) and a minimal amount of methanol
added dropwise. Hydrochloric acid was added (4 M in dioxane,
1.0 mL), and the reaction was stirred for approximately 16 h at
room temperature. The reaction was concentrated under redu-
ced pressure to afford a white solid (51 mg, 0.13 mmol, 93%). 1H
NMR (400.1 MHz, DMSO-d6) δ (ppm): 9.12 (s, 1H), 8.61 (br s,
2H), 7.18 (q, J= 9 Hz, 1H), 7.12-7.05(m, 1H), 7.01-6.95(m, 1H),
4.60 (s, 2H), 3.96-3.57 (m, 5H), 3.56-3.46 (m, 2H), 3.06-2.92 (m,
2H), 1.91 (d, J = 15 Hz, 2H), 1.32 (d, J = 7 Hz, 3H). 13C NMR
(100.6 MHz, DMSO-d6) δ (ppm): 174.4, 151.2, 148.8, 139.9, 117.2,
110.2, 103.2, 59.4, 59.2, 56.6, 50.6, 48.6, 42.5, 25.8, 25.6, 17.1. HRMS
(TOF, ESI) C16H23N4OF2 [M þ H]þ calculated 325.1840, found
325.1839. LC-MS: rt (min) = 1.344. LRMS (ESI) m/z = 325.1.
8-(2-Aminoethyl)-1-(4-fluorophenyl)-1,3,8-triazaspiro[4.5]-
decan-4-one Dihydrochloride (21d). 1-(4-Fluorophenyl)-1,3,8-
triazaspiro[4.5]decan-4-one 20d (54.8 mg, 0.22 mmol) and tert-
butyl (2-oxoethyl)carbamate (38.2 mg, 0.24 mmol) were com-
bined and dissolved in dichloromethane (1.5 mL) and methanol
(0.05 mL) and stirred for about 30 min at room temperature.
After about 30 min macroporous triacetoxyborohydride (600
mg, 1.4 mmol) was added to the reaction, and after 14 h an addi-
tional amount of tert-butyl (2-oxoethyl)carbamate (19.1 mg,
0.12 mmol) was added to drive the reaction to completion. After
about 24 h the reaction mixture was filtered through Celite and
concentrated under reduced pressure. The crude compound was
chromatographed on a 12 g flash column eluting in a gradient of
0%-10% methanol in dichloromethane to afford a white solid
1
(41 mg, 0.10 mmol, 47%). H NMR (400.1 MHz, MeOD) δ
(ppm): 7.10-7.02 (m, 4H), 4.67 (s, 2H), 3.28-3.24 (m, 2H),
3.20-3.12 (m, 2H) 2.86-2.80 (m, 2H), 2.44-2.37 (m, 2H), 1.95
(s, 2H), 1.88 (d, J = 14 Hz, 2H), 1.44 (s, 9H). 13C NMR (100.6
MHz, MeOD) δ (ppm): 178.4, 160.7, 158.4, 140.8, 122.3, 122.2,
116.7, 116.5, 80.4, 61.2, 60.1, 58.1, 50.6 (2C), 37.5, 29.5 (2C),
28.7 (3C). HRMS (TOF, ESI) C20H30N4O3F [M þ H]þ calcu-
lated 393.2302, found 393.2300. LC-MS: rt (min) = 1.850.
LRMS (ESI) m/z = 393.2. tert-Butyl (2-(1-(4-fluorophenyl)-4-
oxo-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)carbamate (41 mg,
0.10 mmol) was dissolved in dichloromethane (5 mL) and a
minimal amount of methanol added dropwise. Hydrochloric
acid was added (4 M in dioxane, 0.5 mL), and the reaction was
stirred for approximately 16 h at room temperature. The reac-
tion was concentrated under reduced pressure to afford a white
solid (34 mg, 0.093 mmol, 93%). 1H NMR (400.1 MHz, MeOD)
δ (ppm): 7.16-7.08 (m, 2H), 7.08-7.00 (m, 2H), 4.72 (s, 2H),
3.92-3.81 (m, 2H), 3.72-3.63 (m, 2H), 3.50 (s, 4H), 2.93-2.81
(m, 2H), 2.05 (d, J = 15 Hz, 2H). 13C NMR (100.6 MHz,
MeOD) δ (ppm): 177.3, 160.3, 140.1, 120.5, 120.4, 116.9, 116.7,
61.1, 58.6, 54.9, 51.3 (2C), 35.4, 28.5 (2C). HRMS (TOF, ESI)
C15H22N4OF [M þ H]þ calculated 293.1778, found 293.1769.
LC-MS: rt (min) = 1.260. LRMS (ESI) m/z = 293.2.
N-(2-(1-(4-Fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-
8-yl)ethyl)-2-naphthamide 2,2,2-Trifluoroacetate (25a). 8-(2-
Aminoethyl)-1-(4-fluorophenyl)-1,3,8-triazaspiro[4.5]decan-4-
one dihydrochloride (34 mg, 0.09 mmol), 2-naphthoyl chloride

6716 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Lavieri et al.
(17.8 mg, 0.09 mmol), and N,N-diisopropylethylamine (0.067
mL, 0.385 mmol) were all dissolved in N,N-dimethylformamide
(1 mL) at 0 ꢀC. The reaction mixture was allowed to warm to
room temperature and stirred for about 12 h. The reaction mix-
ture was diluted with water and extracted into dichloromethane
5 times. The dichloromethane layer was then washed 3 times
with a solution of lithium chloride (3 M) and dried under reduced
pressure. The reaction mixture was subjected to reversed-phase
chromatography to afford a white solid (25.8 mg, 0.04 mmol,
51%). 1H NMR (400.1 MHz, DMSO-d6) δ (ppm): 9.92 (s, 1H),
9.02 (s, 1H), 8.97 (t, J = 5 Hz, 1H), 8.48 (s, 1H), 8.06-7.94 (m,
4H), 7.66-7.58 (m, 2H), 7.09 (t, J = 9 Hz, 2H), 7.03-6.97 (m,
2H), 4.61 (s, 2H), 3.76-3.63 (m, 6H), 3.38-3.34 (m, 2H),
2.63-2.51 (m, 2H), 1.97 (d, J = 14 Hz, 2H). 13C NMR (100.6
MHz, DMSO-d6) δ (ppm): 174.7, 166.9, 157.6, 155.3, 139.3,
134.3, 132.1, 131.2, 128.9 (2C), 128.0, 127.8, 127.7 (2C), 126.9,
124.1, 118.2, 118.1, 115.7, 115.5, 59.2, 56.7, 55.3, 50.0 (2C), 34.6,
26.5 (2C). HRMS (TOF, ESI) C26H28N4O2F [M þ H]þ calcu-
lated 447.2196, found 447.2196. LC-MS: rt (min) = 2.140.
LRMS (ESI) m/z = 447.2. Analogues 25b-d were made follow-
ing the same protocol starting from 21d and were purified via
reversed-phase chromatography to greater than 95% purity (as
trifluoroacetate salts) as analyzed by ELSD and UV at both 214
and 254 nM.
8-(2-Aminoethyl)-1-(4-chlorophenyl)-1,3,8-triazaspiro[4.5]decan-
4-one Dihydrochloride (21e). 1-(4-Chlorophenyl)-1,3,8-triaz-
aspiro[4.5]decan-4-one 20e (152 mg, 0.57 mmol) and tert-butyl
(2-oxoethyl)carbamate (100 mg, 0.63 mmol) were combined and
dissolved in dichloromethane (1.5 mL) and methanol (0.05 mL)
and stirred for about 30 min at room temperature. After about
30 min macroporous triacetoxyborohydride (600 mg, 1.4 mmol)
was added to the reaction, and after 14 h an additional amount
of tert-butyl (2-oxoethyl)carbamate (76 mg, 0.32 mmol) was
added to drive the reaction to completion. After about 24 h the
reaction mixture was filtered through Celite and concentrated
under reduced pressure. The crude compound was chromato-
graphed on a 12 g flash column eluting in a gradient of 0%-10%
methanol in dichloromethane to afford a white solid (108 mg,
0.26 mmol, 46%). 1H NMR (400.1 MHz, MeOD) δ (ppm): 7.23
(d, J = 9 Hz, 2H), 6.96 (d, J = 9 Hz, 2H), 4.69 (s, 2H), 3.39-3.32
(m, 2H), 3.26-3.17 (m, 2H), 2.90-2.83 (m, 2H), 2.78-2.67 (m,
2H), 1.97 (s, 2H), 1.84 (d, J = 14 Hz, 2H), 1.45 (s, 9H). 13C NMR
(100.6 MHz, MeOD) δ (ppm): 178.0, 158.5, 143.1, 130.1 (2C),
125.4, 118.1 (2C), 80.5, 60.6, 59.6, 58.2, 50.6 (2C), 37.6, 28.8
(2C), 28.7 (3C). HRMS (TOF, ESI) C20H30N4O3Cl [M þ H]þ
calculated 409.2006, found 409.2006. LC-MS: rt (min) = 2.002.
LRMS (ESI) m/z = 409.2. tert-Butyl (2-(1-(4-chlorophenyl)-4-
oxo-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)carbamate (108 mg,
0.26 mmol) was dissolved in dichloromethane (5 mL) and a
minimal amount of methanol added dropwise. Hydrochloric
acid was added (4 M in dioxane, 1.5 mL), and the reaction was
stirred for approximately 16 h at room temperature. The reac-
tion was concentrated under reduced pressure to afford a white
solid (94 mg, 0.25 mmol, 95%). 1H NMR (400.1 MHz, MeOD) δ
(ppm): 7.25 (d, J = 9 Hz, 2H), 7.06 (d, J = 9 Hz, 2H), 4.73 (s,
2H), 3.95-3.85 (m, 2H), 3.72-3.64 (m, 2H), 3.51 (s, 4H), 3.19-
3.08 (m, 2H), 2.03 (d, J = 15 Hz, 2H). 13C NMR (100.6 MHz,
MeOD) δ (ppm): 177.1, 142.5, 130.3 (2C), 125.3, 117.5 (2C),
60.6, 58.4, 55.0, 51.3 (2C), 35.5, 27.8 (2C). HRMS (TOF, ESI)
C15H22N4OCl [M þ H]þ calculated 309.1482, found 309.1479.
LC-MS: rt (min) = 1.420. LRMS (ESI) m/z = 309.1.
dichloromethane 5 times. The dichloromethane layer was then
washed 3 times with a solution of lithium chloride (3 M) and
dried under reduced pressure. The reaction mixture was
subjected to reversed-phase chromatography to afford a white
solid (62.9 mg, 0.11 mmol, 45%). 1H NMR (400.1 MHz, DMSO-
d6) δ (ppm): 10.24 (s, 1H), 9.10 (s, 1H), 9.01 (t, J = 5 Hz, 1H),
8.49 (s, 1H), 8.06-7.94 (m, 4H), 7.65-7.57 (m,2 H), 7.23 (d,
J = 9 Hz, 2H), 6.94 (d, J = 9 Hz, 2H), 4.63 (s, 2H), 3.78-3.65
(m, 6H), 3.40-3.33 (m, 2H), 2.85-2.72 (m, 2H), 1.96 (d, J =
14 Hz, 2H). 13C NMR (100.6 MHz, DMSO-d6) δ (ppm): 174.5,
166.9, 158.9, 141.7, 134.3, 132.1, 131.2, 128.9 (2C), 128.7 (2C),
128.0 (2C), 127.8, 127.7 (2C), 126.8, 124.1, 122.1, 115.7, 59.0,
56.5, 55.2, 48.9 (2C), 34.6, 26.0 (2C). HRMS (TOF, ESI)
HRMS (TOF, ESI) C26H28N4O2Cl [M þ H]þ calculated
463.1901, found 463.1897. LC-MS: rt (min) = 2.249. LRMS
(ESI) m/z = 463.2. Analogues 26b-d were made following
the same protocol starting from 21e and were purified via
reversed-phase chromatography to greater than 95% purity
(as trifluoroacetate salts) as analyzed by ELSD and UV at
both 214 and 254 nM.
8-(2-Aminoethyl)-1-(4-bromophenyl)-1,3,8-triazaspiro[4.5]decan-
4-one Dihydrochloride (21f). 1-(4-Bromophenyl)-1,3,8-triaz-
aspiro[4.5]decan-4-one 20f (177 mg, 0.57 mmol) and tert-butyl
(2-oxoethyl)carbamate (100 mg, 0.63 mmol) were combined and
dissolved in dichloromethane (1.5 mL) and methanol (0.05 mL)
and stirred for about 30 min at room temperature. After about
30 min macroporous triacetoxyborohydride (600 mg, 1.4 mmol)
was added to the reaction, and after 14 h an additional amount
of tert-butyl (2-oxoethyl)carbamate (76 mg, 0.32 mmol) was
added to drive the reaction to completion. After about 24 h the
reaction mixture was filtered through Celite and concentrated
under reduced pressure. The crude compound was chromato-
graphed on a 12 g flash column eluting in a gradient of 0%-10%
methanol in dichloromethane to afford a white solid (163 mg,
0.36 mmol, 63%). 1H NMR (400.1 MHz, MeOD) δ (ppm): 7.35
(d, J = 9 Hz, 2H), 6.90 (d, J = 9 Hz, 2H), 4.68 (s, 2H), 3.29-3.21
(m, 2H), 3.20-3.10 (m, 2H), 2.86-2.68 (m, 4H), 1.97 (s, 2H),
1.80 (d, J = 14 Hz, 2H), 1.45 (s, 9H). 13C NMR (100.6 MHz,
MeOD) δ (ppm): 176.2, 155.6, 143.4, 129.0 (2C), 117.6, 114.3
(2C), 77.5, 58.6, 58.2, 57.4, 49.3 (2C), 37.7, 28.3 (2C), 28.3 (3C).
HRMS (TOF, ESI) C20H30N4O3Br [M þ H]þ calculated
453.1501, found 453.1504. LC-MS: rt (min) = 2.048. LRMS
(ESI) m/z = 455.1. tert-Butyl (2-(1-(4-bromophenyl)-4-oxo-
1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)carbamate (163 mg, 0.35
mmol) was dissolved in dichloromethane (5 mL) and a minimal
amount of methanol added dropwise. Hydrochloric acid was
added (4 M in dioxane, 1.5 mL), and the reaction was stirred for
approximately 16 h at room temperature. The reaction was
concentrated under reduced pressure to afford a white solid
(140 mg, 0.33 mmol, 94%). 1H NMR (400.1 MHz, DMSO-d6) δ
(ppm): 9.09 (s, 1H), 8.48 (br s, 2H), 7.29 (d, J = 9 Hz, 2H), 7.09
(d, J = 9 Hz, 2H), 4.61 (s, 2H), 3.71-3.58 (m, 4H), 3.47-3.38
(m, 4H), 3.09-2.97 (m, 2H), 1.89 (d, J = 14 Hz, 2H). 13C NMR
(100.6 MHz, DMSO-d6) δ (ppm): 174.5, 141.8, 131.5 (2C), 115.9
(2C), 109.1, 58.9, 56.4, 53.3, 49.2 (2C), 33.7, 25.5 (2C). HRMS
(TOF, ESI) C15H22N4OBr [M þ H]þ calculated 353.0977, found
353.0977. LC-MS: rt (min) = 1.467. LRMS (ESI) m/z = 353.1.
N-(2-(1-(4-Bromophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-
8-yl)ethyl)-2-naphthamide 2,2,2-Trifluoroacetate (27a). 8-(2-
Aminoethyl)-1-(4-bromophenyl)-1,3,8-triazaspiro[4.5]decan-4-one
dihydrochloride 21f (66 mg, 0.15 mmol), 2-naphthoyl chloride
(29.5 mg, 0.15 mmol), and N,N-diisopropylethylamine (0.126
mL, 0.73 mmol) were all dissolved in N,N-dimethylformamide
(1 mL) at 0 ꢀC. The reaction mixture was allowed to warm to
room temperature and stirred for about 12 h. The reaction
mixture was diluted with water and extracted into dichloro-
methane 5 times. The dichloromethane layer was then was-
hed 3 times with a solution of lithium chloride (3 M) and dried
under reduced pressure. The reaction mixture was subjected
to reversed-phase chromatography to afford a white solid
N-(2-(1-(4-Chlorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-
8-yl)ethyl)-2-naphthamide 2,2,2-Trifluoroacetate (26a). 8-(2-
Aminoethyl)-1-(4-chlorophenyl)-1,3,8-triazaspiro[4.5]decan-
4-one dihydrochloride 21e (94 mg, 0.24 mmol), 2-naphthoyl
chloride (47.1 mg, 0.24 mmol), and N,N-diisopropylethylamine
(0.182 mL, 1.05 mmol) were all dissolved in N,N-dimethyl-
formamide (1 mL) at 0 ꢀC. The reaction mixture was allowed
to warm to room temperature and stirred for about 12 h. The
reaction mixture was diluted with water and extracted into

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6717
(66.1 mg, 0.11 mmol, 71%). 1H NMR (400.1 MHz, DMSO-d6)
δ (ppm): 10.23 (s, 1H), 9.10 (s, 1H), 9.00 (t, J = 5 Hz, 1H), 8.49
(s, 1H), 8.06-7.93 (m, 4H), 7.65-7.58 (m, 2H), 7.34 (d, J = 9 Hz,
2H), 6.89 (d, J = 9 Hz, 2H), 4.62 (s, 2H), 3.78-3.66 (m, 6H),
3.46-3.40 (m, 2H), 2.86-2.74 (m, 2H), 1.95 (d, J = 14 Hz,
2H). 13C NMR (100.6 MHz, DMSO-d6) δ (ppm): 174.5, 166.9,
158.9, 142.1, 134.3, 132.1, 131.6 (2C), 131.2, 128.9 (2C), 128.0
(2C), 127.8, 127.7 (2C), 126.8, 124.1, 116.1, 109.6, 58.9, 56.5,
6.60-6.53 (m, 1H), 4.62 (s, 2H), 3.88-3.69 (m, 3H), 3.61-3.54
(m, 1H), 3.43-3.33 (m, 3H), 2.93-2.71 (m, 2H), 2.01-1.82 (m,
2H), 1.31 (d, J = 7 Hz, 3H). 13C NMR (100.6 MHz, DMSO-d6) δ
(ppm): 174.4, 166.7, 162.1, 144.7, 134.3, 132.1, 131.4, 130.6, 130.5,
128.9, 127.9 (2C), 127.8 (2C), 127.7, 126.9, 124.4, 109.8, 104.3,
100.9, 60.8, 59.0, 56.6, 49.8, 48.7, 41.3, 26.0, 25.8, 19.0. HRMS
(TOF, ESI) C27H30N4O2F [M þ H]þ calculated 461.2353, found
461.2354. LC-MS: rt (min) = 0.445. LRMS (ESI) m/z = 461.2.
Pharmacology Methods. Cell Culture. Calu-1 and MDA-231
cells were purchased from American Type Culture Collection
(Manassas, VA). Calu-1 and MDA-231 cells were maintained in
DMEM supplemented with 10% FBS, 100 μg/mL penicillin-
streptomycin, and 0.25 μg/mL amphotericin. HEK293 cells
stably expressing GFP-tagged human PLD2A were generated
in the laboratory. To sustain selection pressure low-passage-
number HEK293-gfpPLD2 cells were maintained in DMEM sup-
plemented with 10% FBS, 100 μg/mL penicillin-streptomycin,
2 μg/mL puromycin, and 600 μg/mL G418. All HEK293-
gfpPLD2 experiments were done on tissue culture plates that
had been coated with low levels of polylysine. All cells were
maintained in a humidified 5% CO2 incubator at 37 ꢀC.
Cellular Phospholipase D Activity Assays. PLD1 and PLD2
cellular IC50 values were determined as described previously.26
Assessment of Cell Proliferation via WST-1 Assay. Cells are
plated into 96-well tissue culture plates at 15000 cells/well in
tissue culture treated 96-well black wall/clear bottom assay
plates (Corning Inc. Costar plates) in complete growth medium
and allowed to grow overnight. After 24 h of growth media were
removed, and cells were treated with PLD inhibitor or DMSO
vehicle control in 100 μL of DMEM, 1% AA, ( 10% FBS.
Media and inhibitor were replenished every 24 h, and after 48 h
cells were treated with 10 μL/well of a modified MTT reagent,
WST-1 cell proliferation reagent (Roche Diagnostics Corp.,
Indianapolis, IN). Plates were then incubated for 1 h at 37 ꢀC.
After incubation UV absorbance was measured at 450 nm with a
BioTek Synergy HT plate reader (BioTek Inc., Winooski, VT).
Background signal was subtracted from wells with no cells
present. Data are expressed as absorbance at 450 nm.
55.2, 48.9 (2C), 34.6, 25.9 (2C). HRMS (TOF, ESI) C26H28
-
N4O2Br [M þ H]þ calculated 507.1396, found 507.1393. LC-
MS: rt (min) = 2.279. LRMS (ESI) m/z = 507.1. Analogues
27b-d were made following the same protocol starting from
21f and were purified via reversed-phase chromatography to
greater than 95% purity (as trifluoroacetate salts) as analyzed
by ELSD and UV at both 214 and 254 nM.
(S)-tert-Butyl (1-(1-(3-Fluorophenyl)-4-oxo-1,3,8-triazaspiro-
[4.5]decan-8-yl)propan-2-yl)carbamate (29). 1-(3-Fluorophenyl)-
1,3,8-triazaspiro[4.5]decan-4-one 20a (366 mg, 1.46 mmol) and
(S)-tert-butyl (1-oxopropan-2-yl)carbamate (356 mg, 2.00 mmol)
were combined and dissolved in dichloromethane (10 mL) and
methanol (1 mL) and stirred for about 30 min at room tempera-
ture. After about 30 min macroporous triacetoxyborohydride
(2 g, 4.84 mmol) was added to the reaction. After about 24 h the
reaction mixture was filtered through Celite and concentrated
under reduced pressure. The crude compound was chromato-
graphed on an 80 g flash column eluting in a gradient of 0%-
10% methanol in dichloromethane to afford a white solid (191
mg, 0.47 mmol, 34%). 1H NMR (400.1 MHz, MeOD) δ (ppm):
7.23 (q, J = 8 Hz, 1H), 6.76-6.68 (m, 2H), 6.55-6.49 (m, 1H),
4.69 (s, 2H), 3.96-3.84 (m, 1H), 3.25-3.07 (m, 2H), 2.90-2.63
(m, 4H), 1.96(s,2H), 1.79 (d, J = 14 Hz, 2H), 1.46 (s, 9H), 1.18(d,
J = 7 Hz, 3H). 13C NMR (100.6 MHz, MeOD) δ (ppm): 178.2,
164.0, 158.3, 146.2, 131.6, 111.3, 105.7, 102.4, 80.5, 64.2, 60.5
(2C), 59.8, 51.5, 50.5, 28.9 (2C), 28.7 (3C), 19.6. HRMS (TOF,
ESI) C21H32N4O3F [M þ H]þ calculated 407.2458, found
407.2449. LC-MS: rt (min) = 1.932. LRMS (ESI) m/z = 407.2.
(S)-8-(2-Aminopropyl)-1-(3-fluorophenyl)-1,3,8-triazaspiro-
[4.5]decan-4-one Dihydrochloride (30). (S)-tert-Butyl (1-(1-(3-
fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8-yl)propan-
2-yl)carbamate 29 (166 mg, 0.40 mmol) was dissolved in dichloro-
methane (15 mL) and a minimal amount of methanol added
dropwise. Hydrochloric acid was added (4 M in dioxane, 2.0 mL),
and the reaction was stirred for approximately 16 h at room tem-
perature. The reaction was concentrated under reduced pressure
to afford a light tan solid (150 mg, 0.39 mmol, 98%). 1H NMR
(400.1 MHz, DMSO-d6) δ (ppm): 9.12 (s, 1H), 8.63 (s, 2H),
7.22-7.12 (m, 1H), 7.08-7.01 (m, 1H), 6.79-6.72 (m, 1H),
6.56-6.49 (m, 1H), 4.62 (s, 2H), 3.95-3.58 (m, 4H), 3.54-3.41
(m, 3H), 3.15-3.00 (m, 2H), 1.89 (d, J = 14 Hz, 2H), 1.32 (d,
J = 5 Hz, 3H). 13C NMR (100.6 MHz, DMSO-d6) δ (ppm):
174.3, 162.1, 144.4, 130.2, 109.8, 103.9, 100.5, 59.4, 58.9, 56.5,
50.5, 48.5, 42.4, 25.7, 25.5, 17.1. HRMS (TOF, ESI) C16H24-
N4OF [M þ H]þ calculated 307.1934, found 307.1934. LC-MS:
rt (min) = 0.255. LRMS (ESI) m/z = 307.1.
(S)-N-(1-(1-(3-Fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-
8-yl)propan-2-yl)-2-naphthamide 2,2,2-Trifluoroacetate (31). (S)-
8-(2-Aminopropyl)-1-(3-fluorophenyl)-1,3,8-triazaspiro[4.5]decan-
4-one dihydrochloride 30 (140 mg, 0.37 mmol), 2-naphthoyl
chloride (76 mg, 0.40 mmol), and N,N-diisopropylethylamine
(0.225 mL, 1.29 mmol) were all dissolved in N,N-dimethylforma-
mide (5 mL) at 0 ꢀC. The reaction mixture was allowed to warm to
room temperature and stirred for about 12 h. The reaction mixture
was diluted with water and extracted into dichloromethane
5 times. The dichloromethane layer was then washed 3 times with a
solution of lithium chloride (3 M) and dried under reduced
pressure. The reaction mixture was subjected to reversed-phase
chromatography to afford a white solid (19.3 mg, 0.03 mmol, 9%).
1H NMR (400.1 MHz, DMSO-d6) δ (ppm): 9.74 (s, 1H), 9.10 (s,
1H), 8.78 (d, J = 8 Hz, 1H), 8.50 (s, 1H), 8.06-7.95 (m, 4H),
7.66-7.58 (m, 2H), 7.20 (q, J = 8 Hz, 1H), 6.76-6.69 (m, 2H),
For time course experiments cells were seeded at 7500 cells/
well into media containing PLD inhibitor or DMSO vehicle
control. Media were removed and replaced every 24 h, and at set
time points (24, 48, 72, and 96 h) cells were treated with WST-1
reagent as described above.
Assessment of Caspase 3/7 Activity. Caspase 3/7 activity was
measured using a homogeneous bioluminescent method accord-
ing to manufacturer’s directions (Caspase-Glo 3/7 assay;
Promega, Madison, WI). In this assay, caspase 3/7 activity is
measured by the ability to cleave the proluminescent Caspase
3/7 specific DEVD-aminoluciferin substrate to liberate the free
aminoluciferin which is then consumed by luciferase generating
a luminescent signal. The luminescent signal is directly propor-
tional to the amount of Caspase 3/7 activity. Cells were plated at
15000 cells/well in tissue culture treated 96-well black wall/clear
bottom assay plates (Corning Inc. Costar plates) in 50 μL of
growth medium at 37 ꢀC. After 24 h media were removed and
replaced with DMEM, 1% AA, ( 10% FBS with either PLD
inhibitor or DMSO vehicle control. Media were replenished
every 24 h to account for metabolism of the compounds. After
48 h growth in the presence of PLD inhibitor 50 μL Caspase-Glo
3/7 reagent was added to each well, plates were incubated at
room temperature for 1 h, and luminescent signal was then
detected with a BioTek Synergy HT plate reader (BioTek Inc.,
Winooski, VT). Caspase 3/7 activity was normalized to vehicle
control and expressed as fold stimulation of Caspase activity.
Pharmacokinetic Studies. (A) In Vitro. The metabolism of
PLD inhibitors 14 and 22a was investigated in rat hepatic
microsomes (BD Biosciences, Billerica, MA) using substrate
depletion methodology (percent test article remaining). A potas-
sium phosphate-buffered reaction mixture (0.1 M, pH 7.4) of test
article (1 μM) and microsomes (0.5 mg/mL) was preincubated

6718 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Lavieri et al.
(5 min) at 37 ꢀC prior to the addition of NADPH (1 mM). The
incubations, performed in 96-well plates, were continued at
37 ꢀC under ambient oxygenation, and aliquots (80 μL) were
removed at selected time intervals (0, 3, 7, 15, 25, and 45 min).
Protein was precipitated by the addition of chilled acetonitrile
(160 μL), containing glyburide as an internal standard (50 ng/
mL), and centrifuged at 3000 rpm (4 ꢀC) for 10 min. Resulting
supernatants were transferred to new 96-well plates in prepara-
tion for LC-MS-MS analysis. The in vitro half-life (t1/2, min,
eq 1), intrinsic clearance (CLint, mL min-1 kg-1, eq 2), and sub-
50 μL aliquots from each chamber were diluted 1:1 (50 μL) with
either plasma (cis) or buffer (trans) and transferred to a new 96-
well plate, at which time ice-cold acetonitrile (2 volumes) was
added to extract the matrices. The plate was centrifuged (3000
rpm, 10 min), and supernatants were transferred to a new 96-
well plate. The sealed plate was stored at -20 ꢀC until LC-MS-
MS analysis.
Liquid Chromatography/Mass Spectrometry Analysis. (A)
In Vivo Experiments. PLD inhibitors 14 and 22a were analyzed
via electrospray ionization (ESI) on an AB Sciex API-4000
(Foster City, CA) triple-quadrupole instrument that was coup-
led with Shimadzu LC-10AD pumps (Columbia, MD) and a
Leap Technologies CTC PAL autosampler (Carrboro, NC).
Analytes were separated by gradient elution using a Fortis C18
2.1 ꢀ 50 mm, 3.5 μm column (Fortis Technologies Ltd., Che-
shire, U.K.) thermostated at 40 ꢀC. HPLC mobile phase A was
0.1% NH4OH (pH unadjusted); mobile phase B was aceto-
nitrile. The gradient started at 30% B after a 0.2 min hold and was
linearly increased to 90% B over 0.8 min, held at 90% B for 0.5
min, and returned to 30% B in 0.1 min followed by a reequili-
bration (0.9 min). The total run time was 2.5 min, and the HPLC
flow rate was 0.5 mL/min. The source temperature was set at
500 ꢀC, and mass spectral analyses were performed using multi-
ple reaction monitoring (MRM), with transitions for 14 (m/z
497.5 f 202.3) and 22a (m/z 447.4 f 198.1) utilizing a Turbo-
Ionspray source in positive ionization mode (5.0 kV spray
voltage). All data were analyzed using AB Sciex Analyst 1.4.2
software.
sequent predicted hepatic clearance (CLhep, mL min-1 kg-1
eq 3) were determined employing the equations:
,
t1=2 ¼ lnð2Þ=k
ð1Þ
where k represents the slope from linear regression analysis
(percent test article remaining)
CLint ¼ ð0:693=t1=2Þðreaction volume=mg of microsomesÞ
ꢀ ð45 mg of microsomes=g of liverÞ
ꢀ ð20 g of liver=kg body weightÞ
ð2Þ
with scale-up factors of 20 (human) and 45 (rat)
QðCLint
Þ
CLhep
¼
ð3Þ
Q þ CLint
(B) In Vivo. Male Sprague-Dawley rats (n = 2) weighing
around 300 g were purchased from Harlon Laboratories
(Indianapolis, IN) and implanted with catheters in the carotid
artery and jugular vein. The cannulated animals were accli-
mated to their surroundings for approximately 1 week before
dosing and provided food and water ad libitum. Compounds 14
and 22a were administered intravenously (iv) to rats via the
jugular vein catheter in 20% DMSO/80% saline at a dose of
1 mg/kg and a dose volume of 1 mL/kg. Blood collections via the
carotid artery were performed at predose and at 2 min, 7 min,
15 min, 30 min, and 1 h, 2 h, 4 h, 7 h, and 24 h postdose. Samples
were collected into chilled, EDTA-fortified tubes and centri-
fuged for 10 min at 3000 rpm (4 ꢀC), and resulting plasma was
aliquoted into 96-well plates for LC-MS-MS analysis. All
pharmacokinetic analysis was performed employing noncom-
partmental analysis. For oral exposure studies, measuring both
systemic plasma and CNS tissue exposure, compounds 14 and
22a were administered (oral gavage) to fasted rats (n = 2) as
suspensions in 10% Tween 80/0.5% methylcellulose at a dose of
10 mg/kg and in a dosing volume of 10 mL/kg; blood and whole
brain samples were collected at 1.5 h postdose. Blood was
collected into chilled, EDTA-fortified tubes, centrifuged for 10
min at 3000 rpm (4 ꢀC), and stored at -80 ꢀC until LC-MS-MS
analysis. The brain samples were rinsed in PBS, snap-frozen,
and stored at -80 ꢀC. Prior to LC-MS-MS analysis, brain
samples were thawed to room temperature and subjected to
mechanical homogenation employing a Mini-Beadbeater and
1.0 mm zirconia/silica beads (BioSpec Products). All animal
studies were approved by the Vanderbilt University Medical
Center Institutional Animal Care and Use Committee. The
animal care and use program is fully accredited by the Associa-
tion for Assessment and Accreditation of Laboratory Animal
Care, International.
(B) In Vitro Experiments. The PLD inhibitors were analyzed
similarly to that described above (In Vivo Experiments) with the
following exceptions: LC-MS-MS analysis was performed em-
ploying a TSQ QuantumULTRA that was coupled to a Thermo-
Surveyor LC system (Thermoelectron Corp., San Jose, CA) and
a Leap Technologies CTC PAL autosampler (Carrboro, NC).
Chromatographic separation of analytes was achieved with
an Acquity BEH C18 2.1 ꢀ 50 mm, 1.7 μm column (Waters,
Taunton, MA).
Acknowledgment. R.R.L. was supported by an NIH
integrative training in therapeutic discovery training grant
(T90DA022873). H.A.B. and C.W.L. were supported by the
John S. McDonnell Foundation. The authors thank Katrina
Brewer and Matthew Mulder for in vitro drug metabolism
stability and protein binding data.
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