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. 2022 Aug 25;65(16):11111-11125.
doi: 10.1021/acs.jmedchem.2c00562. Epub 2022 Aug 5.

Discovery of Orally Bioavailable and Brain-Penetrable Prodrugs of the Potent nSMase2 Inhibitor DPTIP

Arindom Pal  1   2 , Sadakatali Gori  1   2 , Seung-Wan Yoo  1 , Ajit G Thomas  2 , Ying Wu  2 , Jacob Friedman  2 , Lukáš Tenora  1   2 , Harshit Bhasin  1   2 , Jesse Alt  2 , Norman Haughey  1   3 , Barbara S Slusher  1   2   3   4   5   6   7 , Rana Rais  1   2   4
Affiliations

Discovery of Orally Bioavailable and Brain-Penetrable Prodrugs of the Potent nSMase2 Inhibitor DPTIP

Arindom Pal et al. J Med Chem. .

Abstract

Extracellular vesicles (EVs) can carry pathological cargo and play an active role in disease progression. Neutral sphingomyelinase-2 (nSMase2) is a critical regulator of EV biogenesis, and its inhibition has shown protective effects in multiple disease states. 2,6-Dimethoxy-4-(5-phenyl-4-thiophen-2-yl-1H-imidazol-2-yl)phenol (DPTIP) is one of the most potent (IC50 = 30 nM) inhibitors of nSMase2 discovered to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), limiting its clinical development. To overcome DPTIP's PK limitations, we synthesized a series of prodrugs by masking its phenolic hydroxyl group. When administered orally, the best prodrug (P18) with a 2',6'-diethyl-1,4'-bipiperidinyl promoiety exhibited >fourfold higher plasma (AUC0-t = 1047 pmol·h/mL) and brain exposures (AUC0-t = 247 pmol·h/g) versus DPTIP and a significant enhancement of DPTIP half-life (2 h vs ∼0.5 h). In a mouse model of acute brain injury, DPTIP released from P18 significantly inhibited IL-1β-induced EV release into plasma and attenuated nSMase2 activity. These studies report the discovery of a DPTIP prodrug with potential for clinical translation.

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Conflict of interest statement

The authors declare the following financial interests which may be considered as potential competing interests: A.P., N.H., B.S.S., and R.R. are listed as inventors in patent applications filed by Johns Hopkins Technology Ventures covering novel compositions of nSMase2 inhibitors and their utility.

Figures

Figure 1:
Figure 1:. Single time-point pharmacokinetic screening of all prodrugs (P1 - P18) and DPTIP in CES1−/− mice dosed orally at 10 mg/kg DPTIP equivalent.
(A) Plasma levels of released DPTIP from the prodrugs. (B) Brain levels of released DPTIP from the prodrugs. (C) Brain to plasma ratio of released DPTIP at 2 h. Data expressed as mean ± SEM, n = 3.
Figure 2:
Figure 2:. Time-dependent in vivo PK analysis of DPTIP (A) P1 (B) P18 (C) and their PK parameters (D) in CES1−/− mice.
DPTIP, P1, and P18 were dosed orally at 10 mg/kg DPTIP equivalent dose. Data expressed as mean ± SEM, n = 3.
Figure 3:
Figure 3:. Effects of orally administered P18 in a mouse model of acute brain injury.
Quantification of GFP labeled EVs in plasma when treated with (A) DPTIP or (Bi) P18. ###p < 0.001 (compared to saline + vehicle group); ***p <0.001 (compared to IL1-β + vehicle group). (Bii) nSMase2 activity in mouse striata under different treatments following IL-1β injection. n = 4/group, bars represent mean ± SEM. ####p < 0.0001 (compared to saline + vehicle group); ****p < 0.0001, ***p <0.001 and *p < 0.05 (compared to IL-1β + vehicle group). B(iii) Plasma and brain levels of released DPTIP from P18 in the same samples. n = 4/group, bars represent mean ± SEM
Scheme-1:
Scheme-1:. Synthesis of Prodrugs P1-P13.
(a) DIPEA, triphosgene, DCE, 0 °C – rt, 4 – 20 h; (b) DIPEA, DCE, 0 °C – rt, 4 – 20 h; (c) DIPEA, DCE, rt, 8 h.
Scheme-2:
Scheme-2:. Synthesis of Prodrugs P14-P16.
(a) (i) POCl3, DIPEA, 0 °C, 2 h, (ii) K2CO3, H2O, rt, 18 h; (b) Cs2CO3, NaI, DMF, rt, 24 h; (c) TFA, DCM, rt, 12 h; (d) silica, methanol, conc. HCl (cat.), rt, 24 h.
Scheme-3:
Scheme-3:. Synthesis of Prodrug P17.
(a) EDC, DMAP, DMF, rt, 12 h; (b) CH3I, acetone, rt, 24 h; (c) Na2S2O4, NaHCO3, H2O, 0 °C, 3 h.
Scheme-4:
Scheme-4:. Synthesis of Prodrug P18.
(a) propionaldehyde, benzylamine, H2O, 0 °C – rt, 48 h; (b) piperidine, NaCNBH3, methanol, acetic acid, rt, 16 h; (c) 10% Pd/C, H2 (5 atm), 50 °C, methanol, acetic acid, 16 h; (d) triphosgene, DIPEA, DCE, 0 °C – rt, 16 h.
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