Design and synthesis of isoform-selective phospholipase D (PLD) inhibitors. Part II. Identification of the 1,3,8-triazaspiro[4,5]decan-4-one privileged structure that engenders PLD2 selectivity

Article abstract of DOI:10.1016/j.bmcl.2009.02.125

This Letter describes the synthesis and structure-activity relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of a 1,3,8-triazaspiro[4,5]decan-4-one privileged structure, PLD inhibitors with nanomolar potency and an unp

Full text of DOI:10.1016/j.bmcl.2009.02.125

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2240–2243
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of isoform-selective phospholipase D (PLD) inhibitors.
Part II. Identification of the 1,3,8-triazaspiro[4,5]decan-4-one privileged
structure that engenders PLD2 selectivity
Robert Lavieri a, Sarah A. Scott a, Jana A. Lewis a, Paige E. Selvy a, Michelle D. Armstrong a,
H. Alex Brown a,b,d, Craig W. Lindsley a,b,c,d,
*
a Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
c Vanderbilt Program in Drug Discovery, Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA
d Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the synthesis and structure–activity relationships (SAR) of isoform-selective PLD
inhibitors. By virtue of the installation of a 1,3,8-triazaspiro[4,5]decan-4-one privileged structure, PLD
inhibitors with nanomolar potency and an unprecedented 40-fold selectivity for PLD2 over PLD1 were
developed. Interestingly, SAR for this diverged from our earlier efforts, and dual PLD1/2 inhibitors were
also discovered within this series.
Received 30 January 2009
Revised 23 February 2009
Accepted 24 February 2009
Available online 6 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PLD
Phospholipase
Cancer
Privileged structure
There are two mammalian isoforms of Phospholipase D (PLD),
referred to as PLD1 and PLD2. Despite conserved regulatory and
catalytic domains between PLD1 and PLD2, studies indicate dis-
tinct modes of activation and distinct functional roles for PLD1
and PLD2.1 From a therapeutic perspective, PLD signaling has been
implicated in a variety of human cancers (breast, renal, gastric and
colorectal), and PLD signaling is thought to regulate actin cytoskel-
eton reorganization and cell motility.2–8 Thus, small molecules that
selectively inhibit PLD1 or PLD2 are potentially a novel approach
for the treatment of cancer. However, the PLD field has been hin-
dered for decades by a lack of isoform selective direct-acting small
molecule inhibitors.
sity-oriented synthesis campaign identified the 1,3,8-triazaspi-
ro[4,5]decan-4-one scaffold as a PLD2-preferring moiety.9 Indeed,
compound 3 represents the only known PLD2 preferring (ꢀnine-
fold versus PLD1) inhibitor. Monovich and co-workers reported
on a series of PLD2 inhibitors in 2007, but the compounds proved
H
N
H
N
O
O
N
N
O
O
N
HN
Br
N
HN
In recent reports,9,10 we have disclosed the discovery and devel-
opment of dual PLD1/2 inhibitors, such as 1, and a PLD1 inhibitor 2
with unprecedented selectivity (1700-fold) over PLD2 in a cell
based assay (Fig. 1). Moreover, these compounds represent a new
class of antimetastatic agents, and data suggest they may be inhib-
iting PLD by an allosteric mechanism.9–13 After synthesizing ꢀ500
compounds within the (1-(piperidin-4-yl)-1H-benzo[d]imidazol-
2(3H)-one privileged structure scaffold represented by 1 and 2,
PLD2 selective inhibitors remained elusive until a limited diver-
Dual PLD1/2 inhibitor, 1
PLD1 selective inhibitor, 2
H
N
O
N
O
HN
N
PLD2 selective inhibitor, 3
Figure 1. Our recently reported isoform-selective PLD inhibitors: dual PLD1/2
inhibitor 1, PLD1-selective (>1700-fold) inhibitor 2, and PLD2 selective (ꢀninefold)
inhibitor 3.
* Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.125

R. Lavieri et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2240–2243
2241
Table 1
Homologate
and/or constrain
Structures and activities of analogs 9
H
N
H
N
Diverse
R2
R1
O
N
O
Amides
N
R1
O
O
HN
N
HN
N
H, Me and
other alkly
9
R1
PLD1 IC50
(nM)
PLD2 IC50
(nM)
Fold PLD2-
selective
a
b
Compd
Figure 2. Library strategy to refine PLD inhibitors to improve potency and PLD2
isoform selectivity.
3
1000
110
9.1
—
O
NH
NHBoc
O
NHBoc
a
O
+
or
n
HN
R1
9a
9b
>20,000
1900
>20,000
90
N
R1
4
5
6
21.1
H
N
N
R2
NHBoc
O
O
b, c
N
n
N
n
R1
O
R1
HN
HN
9c
2100
1600
1.3
N
N
S
7
8
9d
9e
9f
150
30
5.0
7.8
10.7
—
Scheme 1. Reagents and conditions: (a) MP-B(OAc)3, DCE, rt, 16 h (72–95%); (b) 4 N
HCl/dioxane, MeOH (98%); (c) R2COCl, DCM, DIEA, rt (60–94%) or (i) R2COH, PS-DCC,
HOBt, DCM, DIEA; (ii) MP-CO3 (55–90%).
4250
5900
>20,000
990
2À
OMe
NH2
to be dual PLD1/2 or modestly PLD1 preferring inhibitors.9,10,14 In
this Letter, we describe an iterative analog library synthesis ap-
proach based on 3,15 coupled with biochemical assays and mass
spectrometric lipid profiling of cellular responses,9,10 for the dis-
covery of PLD2 inhibitors with improved potency and PLD2 iso-
form selectivity.
550
N
N
9g
>20,000
a
Cellular PLD1 assay with Calcu-1 cells.
Cellular assay with HEK293-gfp PLD2 cells. Each IC50 was determined in trip-
b
In order to optimize 3 and evaluate if the 1,3,8-triazaspi-
ro[4,5]decan-4-one scaffold would maintain PLD2 selectivity, we
employed our iterative parallel synthesis approach,15 and synthe-
sized libraries to address the potential SAR depicted in Figure 2.
The requisite building blocks were all commercially available
and the libraries were prepared according to the general route de-
picted in Scheme 1. Synthesis begins with a reductive amination
with either N-Boc glycinal, a functionalized alinal 5 or a homolo-
gated/cyclic constrained N-Boc amino aldehyde 6 and 1,3,8-triaza-
spiro[4,5]decan-4-one 4 to provide 7 in yields ranging from 72% to
95%. Subsequent removal of the Boc group with 4 N HCl and stan-
dard acylation chemistry provides analogs 8. All compounds were
then purified to >98% purity by mass-directed preparative HPLC.16
All library members were evaluated for their ability to inhibit
PLD1 and PLD2 in a cellular assay (Calu-1 and HEK293-gfpPLD2,
respectively) as well as a biochemical assay with purified PLD1
and PLD2 enzymes.9 The cellular assays were the ‘workhorse’ as-
says which drove the SAR, with routine confirmation in the
in vitro biochemical assay. As observed in the previous series of
PLD1 selective inhibitors, represented by 2, the ethyl diamino lin-
ker was essential—homologation to the corresponding 3- and 4-
carbon tethers were inactive, as were cyclic constraints. Table 1
highlights unsubstituted ethyl diamine linker congeners 9 without
the (S)-methyl group and examines only alternative amides. Com-
pound 3 was the lead PLD2-preferring compound previously iden-
tified in a diversity-oriented synthesis campaign, with a PLD2 IC50
of 110 nM and ꢀninefold selectivity versus PLD1 (IC50 = 1000 nM).
In general, analogs 9 with PLD inhibitory activity were PLD2-pre-
ferring (1.3- to 21.1-fold). Incorporation of the PLD1 preferring
trans-phenyl cyclopropane amide moiety of 2, provides 9a, and a
licate. For details see Ref. 9.
complete loss of PLD inhibition. A 4-amino, 5-methoxybenzamide
analog 9f (PLD2 IC50 = 550 nM) displayed over 10-fold selectivity
for PLD2 over PLD1. The most potent analog in the series, 9d
(PLD2 IC50 = 30 nM), incorporated a 2-benzothiophene amide, but
displayed only fivefold PLD2 selectivity. A 2-quinoline amide con-
gener, 9b, displayed comparable PLD2 potency (PLD2 IC50 = 90 nM)
to the lead 3, but selectivity versus PLD1 (PLD1 IC50 = 1900 nM)
was improved (>20-fold). Incorporation of a second nitrogen atom
to provide the corresponding quinoxaline derivative, 9g, results in
a complete loss of PLD inhibitory activity.
The concentration–response-curves (CRCs) for inhibition in
PLD1 and PLD2 cellular assay (Calu-1 and 293-PLD2, respectively)
for 9f (10.7-fold PLD2 selective) and 9b (21.1-fold PLD2 selective)
are shown in Figure 3. In an in vitro biochemical assay using puri-
fied PLD1 and PLD2 enzymes, 9b possessed a PLD1 IC50 of >20 lM
and a PLD2 IC50 of 500 nM, or >40-fold PLD2 selective. While not at
the level of isoform selectivity as the PLD1 inhibitor 2 (1700-fold
selective),10 compound 9b represents the most potent and selec-
tive PLD2 inhibitor ever described.17
While this data was very exciting, the results from a directed li-
brary exploring the impact of incorporation of the (S)-methyl
group (PLD1-preferring)9,10 on the diamino ethyl linker with the
1,3,8-triazaspiro[4,5]decan-4-one scaffold (PLD2-preferring) were
completely unexpected. Incorporation of the (S)-methyl group into
analogs 10 had significant impact. While affording a series with
shallow SAR and few PLD inhibitors, PLD1 inhibitory activity was

2242
R. Lavieri et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2240–2243
Table 2
H
N
H
N
H
N
Structures and activities of analogs 10
H
N
R1
O
O
N
N
O
O
NSO2Me
HN
N
O
11
12
13
10
Compd
R1
PLD1 IC50 (nM)
PLD2 IC50 (nM)
a
b
N
NH
NH
NH
N
N
N
N
H
N
10a
25
140
14
15
16
Figure 4. Alternative privileged structures evaluated 11–16.
10b
10c
2600
150
30,000
F
now possessed measureable PLD1 inhibitory activity (PLD1
IC50 = 2.6 lM) with no effect, relative to 9a, on PLD2 inhibition. A
3,4-difluorobenzamide analog, 10c, also displayed dual PLD1/2
200
F
Me
inhibition (PLD1 IC50 = 150 nM, PLD2 IC50 = 200 nM).
10d
3400
27,000
The ability of different GPCR privileged structures18 to engender
either PLD1- or PLD2-preferring inhibition, and the ability of a sin-
gle chiral (S)-methyl group to override PLD2-preferring scaffolds to
serve as a ‘molecular switch’ to dial-in PLD1 inhibition, led us to
evaluate additional GPCR privileged structures (Fig. 4). This effort
was disappointing, as 10 new scaffolds were surveyed, but with lit-
tle success. With the exception of two heteroaryl piperazines 15
a
Cellular PLD1 assay with Calu-1 cells.
Cellular assay with HEK293-gfpPLD2 cells. Each IC50 is avg of three determi-
nations. For details see Ref. 9.
b
dramatically increased with the previously PLD2-preferring 1,3,8-
triazaspiro[4,5]decan-4-one privileged structure (Table 2). For
example, compound 10a, the (S)-methyl analog of the ꢀninefold
PLD2 selective 3, displayed comparable PLD2 inhibitory activity
(PLD2 IC50 = 140 nM), but PLD1 inhibitory activity increased 40-
fold (PLD1 IC50 = 25 nM), relative to 3. Thus, 10a is considered a
PLD1/2 dual inhibitor, and the in vitro biochemical assay verified
this result (PLD1 IC50 = 299 nM, PLD2 IC50 = 235 nM). Another clas-
sical PLD1-preferring moiety, the trans-phenyl cyclopropane moi-
ety of 2, was inactive on both PLD isoforms in analogs such as
9a, but in combination with the (S)-methyl group, congener 10b
and 16 (PLD1 IC50s in the 3.6- to 18 lM range), none of these ana-
logs displayed PLD inhibition.
In summary, the discovery of these potent and selective PLD2
inhibitors highlights the power of an iterative analog library synthe-
sis approach for lead optimization, coupled with biochemical assays
and mass spectrometric lipid profiling of cellular responses. PLD
inhibitor 9b (cellular assay: PLD IC50 = 1900 nM, PLD2 IC50 = 90 nM,
21-fold selective; in vitro biochemical assay: PLD IC50 = >20,000 nM,
PLD2 IC50 = 500 nM, >40-fold selective) represents the most potent
and selective PLD2 small molecule inhibitor described to date.
PLD2 selective inhibitor 9b and the PLD1 selective inhibitor 2 will
serve as invaluable tools to study and dissect the role of PLD1 and
PLD2 in blocking invasiveness in metastatic beast cancer models
and other signaling pathways in which PLD is thought to play key
regulatory roles. Work in this field, and additional experiments to
confirm an allosteric mode of inhibition are in progress and will be
reported in due course.
125
Calu-1
293-PLD2
100
75
50
25
0
Acknowledgments
The authors thank the Vanderbilt Department of Pharmacology,
the Vanderbilt Institute of Chemical Biology and the A.B. Hancock
Family Foundation for Cancer Research and the Sartain-Lanier
Family Foundation (C.W.L.) for support of this research. J.A.L. and
R.L. are supported by an NIH ITTD training Grant (T90DA022873).
-10
-9
-8
-7
-6
-5
-4
Log[ 9f]
P.E.S. is supported by
5T326M007628-30).
a Pharmacology Training Grant (NIH
125
100
75
50
25
0
References and notes
1. Ponting, C. P.; Kerr, I. D. Protein Sci. 1996, 5, 914.
2. Noh, D. Y.; Ahn, S. J.; Lee, R. A.; Park, I. A.; Kim, J. H.; Suh, P. G.; Lee, K. H.; Han, J.
S. Cancer Lett. 2000, 161, 207.
3. Uchida, N.; Okamura, S.; Nagamachi, Y.; Yamshita, S. J. Cancer Res. Clin. Oncol.
1997, 123, 280.
4. Zhao, Y.; Ehara, H.; Akao, Y.; Shamoto, M.; Nakagawa, Y.; Banno, Y.; Deguchi, T.;
Ohishi, N.; Yagi, K.; Nozawa, Y. Biochem. Biophys. Res. Commun. 2000, 278, 140.
5. Uchida, N.; Okamura, S.; Kuwano, H. Anticancer Res. 1999, 19, 671.
6. Yamada, Y.; Hamajima, N.; Kato, T.; Iwata, H.; Yamamura, Y.; Shinoda, M.;
Suyama, M.; Mitsudomi, T.; Tajima, K.; Kusakabe, S.; Yoshida, H.; Banno, Y.;
Akao, Y.; Tanaka, M.; Nozawa, Y. J. Mol. Med. 2003, 81, 126.
7. Min, D. S.; Kwon, T. K.; Park, W. S.; Chang, J. S.; Park, S. K.; Ahn, B. H.; Ryoo, Z. Y.;
Lee, Y. H.; Lee, Y. S.; Rhie, D. J.; Yoon, S. H.; Hahn, S. J.; Kim, M. S.; Jo, Y. H.
Carcinogenesis 2001, 22, 1641.
-11 -10
-9
-8
-7
-6
-5
-4
Log[ 9f]
Figure 3. CRCs for cellular PLD1 j (Calu-1) assay and PLD2
assay, highlighting the unprecedented PLD2 versus PLD1 selectivity for: (A) 9f
(10.7-fold) and (B) 9b (21.1-fold).
(HEK293-gfpPLD2)

R. Lavieri et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2240–2243
2243
8. Buchanan, F. G.; McReynolds, M.; Couvillon, A.; Kam, Y.; Holla, V. R.; DuBois, R.
N.; Exton, J. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1638.
9. Scott, S. A.; Selvy, P. E.; Buck, J. R.; Cho, H. P.; Criswell, T. L.; Thomas, A. L.;
Armstrong, M. D.; Arteaga, C. L.; Lindsley, C. W.; Brown, H. A. Nat. Chem. Biol.
2009, 5, 108.
17. Experimental for PLD2 selective inhibitor 9b: N-(2-(4-oxo-1-phenyl-1,3,8-
triazaspiro[4.5]decan-8-yl)ethyl)quinoline-3-carboxamide: A flask was charged
with 8-(2-aminoethyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (7, 695 mg,
2.00 mmol), quinoline-3-carboxylic acid (381 mg, 2.20 mmol), and O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
10. Lewis, J. A.; Scott, S. A.; Lavieri, R.; Buck, J. R.; Selvy, P. E.; Stoops, S. L.;
Armstrong, M. D.; Brown, H. A.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2009, 19,
1916.
11. Lewis, J. A.; Lebois, E. P.; Lindsley, C. W. Curr. Opin. Chem. Biol. 2008, 12, 269.
12. Lindsley, C. W.; Zhao, Z.; Leister, W. H.; Robinson, R. G.; Barnett, S. F.; Defeo-
Jones, D.; Jones, R. E.; Hartman, G. D.; Huff, J. R.; Huber, H. E.; Duggan, M. E.
Bioorg. Med. Chem. Lett. 2005, 15, 761.
13. Zhao, Z.; Duggan, M. E.; Barnett, S. F.; Defeo-Jones, D.; Huber, H. E.; Huff, J. R.;
Hartman, G. D.; Leister, W. H.; Kral, A.; Fu, S.; Hancock, P. J.; Jones, R. E.;
Robinson, R.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2005, 15, 761.
14. Monovich, L.; Mugrage, B.; Quadros, E.; Toscano, K.; Tommasi, R.; LaVoie, S.;
Liu, E.; Du, Z.; LaSala, D.; Boyar, W.; Steed, P. Bioorg. Med. Chem. Lett. 2007, 17,
2310.
(HATU, 1.14 g, 3.00 mmol) before adding DIPEA (775 mg, 1.045 mL,
6.00 mmol) and diluting with a 2:1 mixture of anhydrous CH2Cl2 (14.0 mL)
and anhydrous DMF (7.0 mL) to an amine concentration of 95 mM. The
mixture was stirred at room temperature for at least 4 h. The desired product
precipitated out of solution as an off-white solid and was isolated by vacuum
filtration. Yield: 28% (242 mg). 1H NMR (DMSO-d6, 400 MHz, 25 °C): d 9.26 (d,
J = 2.0 Hz, 1H), 8.96 (t, J = 5.2 Hz, 1H), 8.77 (br s, 2H), 8.52 (dd, J = 4.1, 1.0 Hz, 1H),
8.31 (dd, J = 8.2, 1.0 Hz, 1H), 8.06 (t, J = 8.4 Hz, 2H), 7.86 (td, J = 7.7, 1.2 Hz, 1H),
7.68 (t, J = 7.6 Hz, 1H), 7.32 (dd, J = 8.4, 4.4 Hz, 1H), 7.09 (t, J = 7.7 Hz, 2H), 6.86 (d,
J = 8.2 Hz, 2H), 6.67 (t, J = 7.3 Hz, 1H), 4.58 (s, 2H), 3.61 (q, J = 6.2 Hz, 2H), 3.14 (br
s, 1H), 2.93 (br t, J = 6.2 Hz, 2H), 2.70 (m, 2H), 1.70 (d, J = 13.9 Hz, 2H). 13C NMR
DMSO-d6, 100 MHz, 25 °C): d 176.1, 165.4, 149.5, 149.2, 148.8, 143.5, 139.8,
135.7, 134.9, 131.5, 129.5, 129.4, 128.4, 127.3, 120.1, 118.0, 114.5, 59.1, 57.9,
56.6, 49.6, 36.6, 27.8. LCMS, single peak, 2.135 min, ES-API: m/z 430.3 [M+H]. ES-
HRMS calcd for C25H27N5O2Na (M+Na): 452.2062. Found, 452.2055.
15. Kennedy, J. P.; Williams, L.; Bridges, T. M.; Daniels, R. N.; Weaver, D.; Lindsley,
C. W. J. Comb. Chem. 2008, 10, 345.
16. Leister, W. H.; Strauss, K. A.; Wisnoski, D. D.; Zhao, Z.; Lindsley, C. W. J. Comb.
Chem. 2003, 5, 322.
18. Patchett, A. A.; Nargund, R. P. Annu. Rep. Med. Chem. 2000, 35, 289. and
references cited therein.