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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2014 March 28.
Published in final edited form as:
PMCID: PMC3632306
NIHMSID: NIHMS455946

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

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 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.

Keywords: Phospholipase D, PLD1, PLD2, ML299, ML298, MLPCN probe

INTRODUCTION

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.13 The PLD protein is characterized by the presence of a conserved histidine, lysine, aspartate (HKD) amino acid domain that forms the catalytic site, as well as conserved phox homology (PX) and pleckstrin homology (PH) regulatory domains at the N-terminus.13 There are two mammalian isoforms of PLD, designated PLD1 and PLD2, with ~ 53% sequence identity, and subject to different regulatory mechanisms and distinct physiological roles.13 Both genetic and biochemical studies implicate dysregulated PLD function and/or expression as having a role in cancer (e.g., breast, renal, colorectal and glioblastoma)47 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 allows 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 both PLD1 and PLD2, suggesting 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 isoform selective PLD inhibitors 24, with unprecedented isoform selectivity for either PLD1 or PLD2 (Figure 1).2,1214 Our 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 still inhibits PLD1 (IC50 = 1,500 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 (based on an enantiospecific ‘molecular switch’)15 with interesting activity as a metastatic agent in cellular assays.

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).

RESULTS AND DISCUSSION

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 analogs 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).

Scheme 1
Synthesis of amide analogs 7

The library was first evaluated in a cell-based assay (single point screen at 200 nM concentration) against both 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).2,1216 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 was generated in the biochemical assay with purified PLD proteins, indicating that 7g acted directly on PLD2. Based on 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 as compared to 4, which while more potent at PLD2, also inhibits PLD1 at 1.5 μM concentrations. Thus, at standard in vitro concentrations and in vivo plasma exposures (above 5 μM), ML298 only inhibits PLD2.

Figure 2
A) Single point (200 nM) cell-based screen of amide analogs 7 for their ability to inhibit both 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 ...

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, both 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 both (R)- and (S)-chiral methyl group introduction into analogs of 7, as SAR between the series is highly divergent.1214 The synthesis of analogs 9 followed scheme 2 and employed either (R)- or (S)-tert-butyl 1-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. Based on this profile, 9b was declared an MLPCN probe, and designated ML299.17

Scheme 2
Synthesis of amide analogs 9
Table 1
Chiral Methyl Groups in Analogs 9 as ‘Molecular Switches’ Governing PLD Isoform Potency and Selectivity.

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 hours, 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 @10 μM) at only 3 targets (opiate and hERG) as 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 both rat and human as well as favorable CYP profiles. Thus, in vivo PK in mice (due to future oncology PD models) was dosed IP to diminish first pass effects. This route of administration provided excellent plasma levels for both probes, but while ML299 was CNS penetrant (Brain-AUC/PlasmaAUC of 0.44), ML298 was peripherally restricted (BrainAUC/PlasmaAUC of 0.05).18 Thus, ML298 compliments 4, which is highly CNS penetrant, providing key tools to dissect selective PLD2 in the periphery as well as in the CNS.

Table 2
DMPK Characterization of ML298 and ML299.18

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 both 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 both 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 both the 1 μM and 10 μM doses.18 At these doses, both 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

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 ...

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 an attractive tool compound 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, that 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 compound to assess therapeutic utility in brain cancer. Further in vivo studies with these probes are in progress and will be reported in due course.

EXPERIMENTAL SECTION

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 is >95%.

3,4-Difluoro-N-(2-(1-(3-fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8yl)ethyl)benzamide, 7g (ML298)

8-(2-aminoethyl)-1-(3-fluorophenyl)-1,3,8- triazaspiro[4.5] decan-4-one dihydrochloride 6 ( 146.5 mg, .5 mmol), DMF (5 mL), triethylamine (.257 mL, 2.55 mmol, 5 eq.) followed by 3,4 difluoro benzoyl chloride 29 (136.3 mg, 0.772 mmol, 1.5 eq.) were added to this stirred mixture and the reaction was quenched in less than 30 minutes, determined by consumption of starting material seen via LC-MS. The reaction was quenched with water/brine and was extracted 3X 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 mmol, 80%). 1H 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]+ calc. mass 433.1851, found 433.1855.

Supplementary Material

1_si_001

Acknowledgments

Funding Sources

This work was generously supported by the 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).

Dr. Lindsley 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.

ABBREVIATIONS USED

PLD
phospholipase D
U87-MG
human glioblastoma cell line
CRC
concentration-response-curve

Footnotes

Author Contributions

Professors Lindsley directed and designed the chemistry, Dr. Daniels designed the pharmacokinetic studies, Dr. Brown and Dr. Thomas directed the molecular pharmacology. Mr. O’Reilly and Mr. Brown performed the synthetic chemistry and Mr. Morrison performed the bioanalytical DMPK work. Dr. Scott and Mr. Oguin performed the molecular pharmacology.

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.

References

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17. For information on the MLPCN, please see: http://mli.nih.gov/mli
18. See Supporting Information for full details.
19. For information on Ricerca, see: www.ricerca.com