Development of dual PLD1/2 and PLD2 selective inhibitors from a common 1,3,8-triazaspiro[4.5]decane core: Discovery of ML298 and ML299 that decrease invasive migration in U87-MG glioblastoma cells

Article abstract of DOI:10.1021/jm301782e

An iterative parallel synthesis effort identified a PLD2 selective inhibitor, ML298 (PLD1 IC₅₀ > 20 000 nM, PLD2 IC₅₀ = 355 nM) and a dual PLD1/2 inhibitor, ML299 (PLD1 IC₅₀ = 6 nM, PLD2 IC₅₀ = 20 nM). SAR studies revealed that a small structural change (incorporation of a methyl group) increased PLD1 activity within this classically PLD2-preferring core and that the effect was enantiospecific. Both probes decreased invasive migration in U87-MG glioblastoma cells.

Full text of DOI:10.1021/jm301782e

Brief Article
pubs.acs.org/jmc
Development of Dual PLD1/2 and PLD2 Selective Inhibitors from a
Common 1,3,8-Triazaspiro[4.5]decane Core: Discovery of ML298 and
ML299 That Decrease Invasive Migration in U87-MG Glioblastoma
Cells
Matthew C. O’Reilly,§ Sarah A. Scott,†,∥ Kyle A. Brown,†,‡,§ Thomas H. Oguin, III,⊥ Paul G. Thomas,⊥
J. Scott Daniels,†,‡,∥ Ryan Morrison,†,‡,∥ H. Alex Brown,†,§ and Craig W. Lindsley*,†,‡,§,∥
‡
†Department of Pharmacology and Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center,
Nashville, Tennessee 37232, United States
∥
§Department of Chemistry and Vanderbilt Specialized Chemistry for Accelerated Probe Development (MLPCN), Vanderbilt
University, Nashville, Tennessee 37232, United States
⊥Department of Immunology, St. Jude Children’s Hospital, Memphis, Tennessee 38105, United States
S
* Supporting Information
ABSTRACT: An iterative parallel synthesis effort identified a PLD2 selective inhibitor, ML298 (PLD1 IC50 > 20 000 nM, PLD2
IC50 = 355 nM) and a dual PLD1/2 inhibitor, ML299 (PLD1 IC50 = 6 nM, PLD2 IC50 = 20 nM). SAR studies revealed that a
small structural change (incorporation of a methyl group) increased PLD1 activity within this classically PLD2-preferring core
and that the effect was enantiospecific. Both probes decreased invasive migration in U87-MG glioblastoma cells.
isoform selective PLD inhibitors 2−4, with unprecedented
INTRODUCTION
■
isoform selectivity for PLD1 or PLD2 (Figure 1).2,12−14 Our
Phospholipase D (PLD) is a lipid signaling enzyme that
catalyzes the hydrolysis of phosphatidylcholine into choline and
phosphatidic acid (PA), an important lipid second messenger
that is central to a number of critical metabolic and signaling
pathways.1−3 The PLD protein is characterized by the presence
of a conserved histidine, lysine, aspartate (HKD) amino acid
domain that forms the catalytic site, and conserved phox
homology (PX) and pleckstrin homology (PH) regulatory
domains at the N-terminus.1−3 There are two mammalian
isoforms of PLD, designated PLD1 and PLD2, with ∼53%
sequence identity and subject to different regulatory mecha-
nisms and distinct physiological roles.1−3 Genetic and
biochemical studies implicate dysregulated PLD function
and/or expression as having a role in cancer (e.g., breast,
renal, colorectal, and glioblastoma)4−7 and CNS disorders (i.e.,
Alzheimer’s disease8 and stroke9). The tools available to inhibit
PLD activity have been limited to genetic/biochemical
approaches, unselective small molecules, and n-butanol
(which competes with water in a transphosphatidylation
reaction); however, none of these represent viable therapeutic
options or allow the role of individual PLD isoforms to be
dissected.1
In 2007, halopemide 1, a classical atypical antipsychotic agent
(D2 IC50 = 7 nM) that was successfully evaluated in five human
clinical trials, was reported to be a PLD inhibitor. Importantly,
the exposure of 1 in those trials was sufficient to inhibit PLD1
and PLD2, suggesting that PLD inhibition was not overtly toxic
in humans.10,11 Subsequent work in our lab demonstrated that
1 was a direct, potent dual PLD1/PLD2 inhibitor (PLD1 IC50
= 21 nM, PLD2 IC50 = 300 nM).2 A diversity-oriented
synthesis (DOS) effort around 1 led to the discovery of the first
Figure 1. Structure of halopemide (1) and optimized inhibitors after a
DOS campaign: PLD1 selective inhibitor (2), dual PLD1/PLD2
inhibitor (3), and PLD2 selective inhibitor (4).
lab is highly interested in selective inhibition of PLD2, and
while 4 is 75-fold PLD2 selective, the selectivity is driven by
potency at PLD2 (IC50 = 20 nM), but it still inhibits PLD1
(IC50 = 1500 nM) at modest concentrations.15 Thus, in this
manuscript, we describe the further optimization of 4 to afford
an improved PLD2 inhibitor, a dual PLD1/PLD2 inhibitor
Received: December 3, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm301782e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
(based on an enantiospecific “molecular switch”)15 with
interesting activity as a metastatic agent in cellular assays.
more potent at PLD2, also inhibits PLD1 at 1.5 μM. Thus, at
standard in vitro concentrations and in vivo plasma exposures
(above 5 μM), ML298 only inhibits PLD2.
RESULTS AND DISCUSSION
Within the piperidine benzimidazolone based PLD inhibitors
such as 2,2,12 the introduction of a chiral methyl group α to the
amide dramatically increased PLD1 inhibitory activity, but
interestingly, the (R)- and (S)-enantiomers were equipotent/
selective.12 Preliminary data with a lone example indicated that
introduction of a methyl group into the ethyl linker α to the
amide of the 1,3,8-triazaspiro[4.5]decane-based PLD inhibitors
also enhanced PLD1 inhibitory activity.14 As ancillary
pharmacology was improved in the 1,3,8-triazaspiro[4.5]decane
series relative to the piperidine benzimidazolone series, we
opted to explore the impact of (R)- and (S)-chiral methyl
group introduction into analogues of 7, as SAR between the
series is highly divergent.12−14 The synthesis of analogues 9
followed Scheme 2 and employed (R)- or (S)-tert-butyl 1-
■
Chemistry. After the large DOS effort that identified the
1,3,8-triazaspiro[4.5]decane core as a PLD2-preferring motif,2 a
subsequent 4 × 6 matrix library led to the discovery of 4,
possessing a key 3-fluorophenyl moiety; however, this exercise
only produced four amide analogues of 4.16 Thus, we held the
3-fluorophenyl moiety constant and explored a diverse array of
28 amides in an effort to identify a PLD2 inhibitor with little or
no inhibition of PLD1 (Scheme 1).
a
Scheme 1. Synthesis of Amide Analogues 7
a
Scheme 2. Synthesis of Amide Analogues 9
a
Reagents and conditions: (a) (1) tert-butyl 2-oxoethylcarbamate, MP-
B(OAc)3H, DCM, rt; (2) 4.0 M HCl/dioxane, DCM, MeOH, rt; (b)
RCOCl, DIEA, DMF, rt.
The library was first evaluated in a cell-based assay (single
point screen at 200 nM) against PLD1 (Calu-1) and PLD2
(293-PLD2).2 As with other allosteric ligands, SAR was “flat”,
and the library afforded few PLD inhibitors (Figure 2).2,12−16
a
Reagents and conditions: (a) (1) (R)- or (S)-tert-butyl 1-propan-2-
ylcarbamate, MP-B(OAc)3H, DCM, rt; (2) 4.0 M HCl/dioxane,
DCM, MeOH, rt; (b) RCOCl, DIEA, DMF, rt.
propan-2-ylcarbamate. As shown in Table 1, this exercise
provided the first examples of enantioselective PLD inhibition
and PLD isoform selectivity induced by a simple “molecular
switch”.15 Within the 1,3,8-triazaspiro[4.5]decane series, the
(S)-enantiomer was uniformly more potent than the (R),
enhancing PLD inhibitory activity >230-fold in some cases
while also enhancing PLD2 activity 2- to 10-fold, leading to a
novel series of dual PLD1/PLD2 inhibitors 9. This effort
identified 9b, a potent dual PLD1 (IC50 = 6 nM) and PLD2
(IC50 = 20 nM), with equivalent data in the biochemical assay
with purified PLD proteins (PLD1 IC50 = 48 nM and PLD2
IC50 = 84 nM), indicating that ML299 is a direct inhibitor. On
the basis of this profile, 9b was declared an MLPCN probe and
designated ML299.17
DMPK Profiling. The two probes were then profiled in a
battery of in vitro and in vivo DMPK assays to assess their
utility as in vivo tools (Table 2). Both compounds were stable
in PBS buffer up to 48 h, afforded no GHS conjugates, were
soluble in PBS buffer (>20 μM or >10 μg/mL), and in a
Ricerca radioligand binding panel of 68 GPCRs, ion channels,
and transporter,18,19 displayed significant activity (>50%
inhibition at 10 μM) at only 3 targets (opiate and hERG)
compared to 1 with significant activity at over 30 targets.18
Importantly, in follow-up functional assays, neither compound
functionally inhibited hERG (IC50 > 20 μM), and there was no
agonist activity at the opiate receptors. Both probes were highly
cleared in rat and human microsomes but possessed good free
fraction in rat and human and favorable CYP profiles. Thus, for
in vivo PK, mice (due to future oncology PD models) were
dosed ip to diminish first pass effects. This route of
administration provided excellent plasma levels for both probes,
but while ML299 was CNS penetrant (brainAUC/plasmaAUC of
0.44), ML298 was peripherally restricted (brainAUC/plasmaAUC
Figure 2. (A) Single point (200 nM) cell-based screen of amide
analogues 7 for their ability to inhibit PLD1 and PLD2. (B) Structure
and PLD inhibitory activity of 7g (ML298), a >53-fold PLD2 selective
inhibitor. (C) Cell-based PLD1 and PLD2 CRCs for 7g.
In general, aliphatic, cylcoalkyl, and heteroaryl amides were
inactive. However, the library did identify CID53393915 (7g),
a potent >53-fold selective PLD2 inhibitor (IC50 = 355 nM)
with no measurable inhibition of PLD1 (IC50 > 20 000 nM).
Comparable data were generated in the biochemical assay with
purified PLD proteins, indicating that 7g acted directly on
PLD2. On the basis of the PLD inhibitory profile, 7g was
declared an MLPCN probe and assigned as ML298.17 ML298
does not inhibit PLD1 up to 20 μM, which makes it a better
tool for in vitro and in vivo work compared to 4, which, while
B
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Journal of Medicinal Chemistry
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Table 1. Chiral Methyl Groups in Analogues 9 as “Molecular
Switches” Governing PLD Isoform Potency and Selectivity
Table 2. DMPK Characterization of ML298 and ML29918
parameter
ML298
ML299
MW
TPSA
cLogP
432.44
489.3
64.9
4.11
64.8
3.20
In Vitro Pharmacology
IC50(μM): CYP (1A2, 2C9, 3A4,
>30, >30, 17.6, 7.9 >30, 13.3, 6.8, 9.4
2D6)
In Vitro PK
)
rat CLHEP (mL min−1 kg−1
)
60.9
17.8
5.0
61.8
19.1
3.0
human CLHEP (mL min−1 kg−1
rPPB (% fu)
hPPB (% fu)
3.0
1.0
In Vivo PK, Mouse ip, Plasma, Lung, Brain (10 mg/kg, 0−6 h)
plasma (μM·h)
lung (μM·h)
brain (μM·h)
B/P
1.55
4.61
0.08
0.05
1.08
48.5
0.48
0.44
a
b
Cellular PLD1 assay with Calu-1 cells. Cellular PLD2 assay with
Figure 3. Inhibition of PLD2 leads to decreased invasive migration in
U87-MG cell lines. (A) Effect of dual PLD1/PLD2 inhibitor ML299
and (B) effect of PLD2 selective inhibitor ML298 on invasive
migration.19 Cells were plated in medium various concentrations of
ML298 or ML299 of the upper chamber of 8 μm pore Matrigel coated
Transwell filters (scale bar, 50 mm). The lower chamber contained
medium and inhibitor containing 10% fetal bovine serum. Cells
migrating to the underside were stained/counted 48 h later. Each bar
represents mean of five fields of three wells sd.
HEK293-gfp-PLD2 cells. Cell-based assays were used to devleop
CRCs (from 200 pM to 20 μM) and determine IC50.
of 0.05).18 Thus, ML298 complements 4, which is highly CNS
penetrant, providing key tools to dissect selective PLD2 in the
periphery and in the CNS.
Efficacy in Cell-Based Assays. In our earlier work with
PLD1 inhibitor 2 and the dual PLD1/2 inhibitor 3, we found
that both inhibitors blocked invasive migration in a triple
negative breast cancer cell line (MDA- MB-231) and a U87-
MG glioblastoma cell line in vitro; however, siRNA studies
indicated that PLD2 played a dominant role.2 Now, with
ML299, a far more potent dual PLD1 and PLD2 inhibitor and
ML298, a selective PLD2 inhibitor, we found that both probes
had no effect (relative to DSMO) at concentrations up to 10
μM on U87-MG cell viability (in the presence of 10% FBS), so
cytotoxicity as a driver of decreasing invasive migration can be
ruled out.18 Also, ML299 robustly increased caspase 3/7
activation under serum-free conditions.2,18 However, as shown
in Figure 3A, inhibition of PLD1 and PLD2 by ML299 provides
a dose-dependent decrease (100 nM to 10 μM) in invasive
migration in U87-MG cells, with statistical significance reached
at the 1 and 10 μM doses.18 At these doses, PLD1 and PLD2
are inhibited. In Figure 3B, selective inhibition of PLD2 by
ML298 also provides a dose-dependent decrease in invasive
migration in U87-MG cells, with statistical significance reached
at a 10 μM dose. At this dose, only PLD2 is inhibited,
suggesting a key role for this PLD isoform in decreasing
invasive migration; however, the effects with ML299 are more
robust than with ML298, as the potency for PLD2 inhibition is
∼16-fold greater.18
C
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Journal of Medicinal Chemistry
Brief Article
DMPK work. S.A.S. and T.H.O. performed the molecular
pharmacology.
CONCLUSION
■
In summary, we have developed a new, direct acting >53-fold
selective PLD2 inhibitor (ML298) with no inhibition of PLD1
and an attractive DMPK profile, making it a valuable tool to
further dissect PLD2 function in multiple cellular and in vivo
environments. In the course of these efforts, we also discovered
a key enantiospecific “molecular switch” in the classically
PLD2-preferring 1,3,8-triazaspiro[4.5]decane scaffold, which
enhanced PLD1 inhibition up to 230-fold and afforded a potent
dual PLD1/PLD2 probe, ML299, with a good DMPK profile.
Both probes decreased invasive migration in U87-MG
glioblastoma cells, suggesting the centrally penetrant ML299
as a possible tool to assess therapeutic utility in brain cancer.
Further in vivo studies with these probes are in progress and
will be reported in due course.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.W.L. thanks the Warren family for support of the research in
his laboratory. Vanderbilt is a member of the MLPCN and
houses the Vanderbilt Specialized Chemistry Center for
Accelerated Probe Development, and the probes ML298 and
ML99 are freely available upon request. This work was
generously supported by Grant NIH/MLPCN U54
MH084659 (C.W.L.) and the McDonnell Foundation.
M.C.O. acknowledges funding from a Predoctoral ACS
Medicinal Chemistry Fellowship (2011−2012).
EXPERIMENTAL SECTION
ABBREVIATIONS USED
■
■
Chemistry. The synthesis of ML298 is described below. The
general chemistry, experimental information, and syntheses of all other
compounds are supplied in the Supporting Information. Purity of all
final compounds was determined by HPLC analysis and is >95%.
3,4-Difluoro-N-(2-(1-(3-fluorophenyl)-4-oxo-1,3,8-
triazaspiro[4.5]decan-8yl)ethyl)benzamide, 7g (ML298). A
mixture of 8-(2-aminoethyl)-1-(3-fluorophenyl)-1,3,8-triazaspiro[4.5]-
decan-4-one dihydrochloride 6 (146.5 mg, 0.5 mmol), DMF (5 mL),
and triethylamine (0.257 mL, 2.55 mmol, 5 equiv), followed by
addition of 3,4 difluorobenzoyl chloride 29 (136.3 mg, 0.772 mmol,
1.5 equiv), was stirred, and the reaction was quenched in less than 30
min, determined by consumption of starting material seen via LC−
MS. The reaction was quenched with water/brine and was extracted
3× with ethyl acetate. The organic extract was concentrated and the
product was purified via reverse phase HPLC, eluting with MeCN/
H2O/TFA to afford the product ML298 as a white solid (177 mg, 0.41
PLD, phospholipase D; U87-MG, human glioblastoma cell line;
CRC, concentration−response curve
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1
mmol, 80%). H NMR (400.1 MHz, DMSO-d6) δ (ppm): 8.73 (s,
1H); 8.59 (t, J = 5.4 Hz, 1H); 7.90−7.83 (m, 1H); 7.76−7.70 (m,
1H); 7.56−7.48 (m, 1H); 7.10 (q, J = 8.0 Hz, 1H); 6.65 (dd, J1 = 8.0
Hz, J2 = 1.9, 1H); 6.58−6.52 (m, 1H); 6.48 (td, J1 = 8.5 Hz, J2 = 2.3
Hz, 1H); 4.57 (s, 2H); 3.44 (q, J = 5.7, 2H); 2.99−2.91 (m, 2H);
2.90−2.79 (m, 2H); 2.68−2.55 (m, 4H); 1.60 (d, J = 13.7). 13C NMR
(100.6 MHz, CDCl3) δ (ppm): 176.08, 164.45, 162.30, 151.62 (dd, J1
= 250.5 Hz, J2 = 12.9 Hz); 149.50 (dd, J1 = 246.3, J2 = 13.0); 145.3 (d,
J = 11.4); 132.43−132.28 (m); 130.63 (d, J = 10.6); 124.95 (dd, J1 =
7.3, J2 = 3.3); 118.01 (dd, J1 = 91.4, J2 = 17.5); 117.38 (dd, J1 = 93.15,
J2 = 17.9); 109.69, 103.68 (d, J = 21.22); 100.54 (d, J = 27.46); 59.14,
58.28, 56.83, 49.56, 37.21, 28.17. HRMS (TOF, ES+) C22H24N4O2F3
[M + H]+ calcd mass 433.1851, found 433.1855.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and spectroscopic data for selected
compounds, detailed pharmacology, and DMPK methods. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 615-322-8700. Fax: 615-343-3088. E-mail: craig.
lindsley@vanderbilt.edu.
Author Contributions
M.C.O. and S.A.S. contributed equally to this work.
C.W.L. directed and designed the chemistry. J.S.D. designed
the pharmacokinetic studies. H.A.B. and P.G.T. directed the
molecular pharmacology. M.C.O. and K.A.B. performed the
synthetic chemistry, and R.M. performed the bioanalytical
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Journal of Medicinal Chemistry
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(17) For information on the MLPCN, see http://mli.nih.gov/mli.
(18) See Supporting Information for full details.
(19) For information on Ricerca, see www.ricerca.com.
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