Large-scale asymmetric synthesis of the bioprotective agent JP4-039 and analogs

Abstract

JP4-039 is a novel nitroxide conjugate capable of crossing lipid bilayer membranes and scavenging reactive oxygen species (ROS). An efficient and scalable one-pot hydrozirconation-transmetalation-imine addition methodology has been developed for its asymmetric preparation. Furthermore, this versatile methodology allows for the synthesis of cyclopropyl and fluorinated analogs of the parent lead structure.

The 4-amino-Tempo (4-AT) derivative JP4-039 ((S)-6a, Scheme 2) is a lead structure among a new generation of peptidomimetic conjugates targeted to mitochondria and effective at scavenging reactive oxygen species (ROS) such as the superoxide radical anion.^1,2 In particular, JP4-039 has been shown to protect cells from radiation damage^2b and to prolong survival of mice subjected to high levels of ionizing irradiation.^2c Moreover, the ameliorating effects of JP4-039 in irradiation-induced delay of bone wound healing were demonstrated in a murine model of combined bone wound/irradiation injury.^2d JP4-039 and its larger congener, XJB-5-131, are found to enrich in mitochondria by a factor of 30–600 times over the cytosolic concentration, at least in part due to their affinity to the mitochondrial lipid, cardiolipin.^1b,2a There, they serve to scavenge ROS and prevent hydroperoxidation of cardiolipin by cycling between nitroxide, hydroxylamine, and nitroxonium redox states.³ Their design was based on the structure of the antibiotic gramicidin S; the alkene peptide isostere replaces a polar internal amide bond and thus increases conformational regidity and membrane permeability.^1a,g In order to further explore the therapeutic potential of JP4-039, a robust synthetic route was required to prepare multigram quantities of highly pure material.

Scheme 2.

Synthesis of the (E)-alkene isosteres (S)-6a-c

JP4-039 contains a single asymmetric carbon atom as part of an alkene isostere dipeptide moiety composed of leucine and glycine residues. Numerous methods have been developed to generate these α-chiral allylic amines.⁴ For example, Overman developed the rearrangement of allylic trichloroacetimidates,^4b Berkowitz described an approach which relies on a Ni(0)-mediated allylic amination,^4c and Krische reported a C-C bond-forming hydrogenation for this purpose.^4k Overall, vinyl addition to imines remains the most commonly used strategy to prepare chiral allylic amines.^4d-k Highly effective protocols for this route are based on the reductive coupling of alkynes,^4d-f the diastereoselective addition of vinylorganometallic species,^4g-i and the acylvinyl anion addition.^4j

Our group has previously developed an efficient Zr-based method for the asymmetric synthesis of allylic amines.⁵ Hydrozirconation of alkyne 2⁶ with Cp₂ZrHCl,⁷ followed by transmetalation to trimethylaluminum and subsequent addition to chiral sulfinimine⁸ (R)-1 provided a diastereomerically pure (>20:1 dr) allylic amine according to ¹H NMR analysis of the crude reaction mixture (Scheme 1). A four-membered chelate model has been proposed to account for this excellent diastereoselectivity.^5a,c Treatment of the crude reaction mixture with HCl in diethyl ether yielded the amine hydrochloride (S)-3 on multigram scale and in 81% yield over 3 steps from 3-butyn-1-ol. The N-tert-butylsulfinimine (R)-1 could be easily obtained from isovaleraldehyde in 90% yield and proved remarkably stable to storage.

Scheme 1.

Synthesis of the common amine intermediate (S)-3

We were able to use the allylic sulfinylamine (S)-3 as a linchpin intermediate for the preparation of JP4-039 as well as a first series of analogs. Thus, acylation of (S)-3 with either a Boc, Cbz or diphenylphosphinoyl group, followed by TBAF-mediated deprotection of the silyl ether, afforded the alcohols (S)-5a-c (Scheme 2). Jones oxidation to the corresponding carboxylic acids and final coupling with 4-AT using the EDCI/HOBt/DMAP protocol provided the desired alkene isosteres (S)-6a-c in moderate to good yields.

This methodology was further adapted for a >150 g scale preparation of JP4-039 ((S)-6a), obtained in 32% overall yield, 99% purity and >99% ee based on HPLC analysis. At this scale, the target compound was isolated from a 20:3 mixture of n-hexane/EtOAc as a crystalline solid. The X-ray structure of (S)-6a is in good agreement with a type II’ β-turn (Figure 1). The distance of 3.30 Å for the i to i+3 intramolecular hydrogen bond between the carbonyl oxygen of the Boc function and the nitrogen atom of 4-AT is indicative of a weak covalent component in this H-bond.

Figure 1.

X-ray structure of JP4-039.

The (R)-enantiomer of JP4-039 (R)-6a was obtained in 52% overall yield according to an analogous synthetic route starting from (S)-sulfinimine (S)-1 (Scheme 3). Chiral SFC on a Chiralpak-IC phase was used to determine an ee of 96.6% for alcohol (S)-5a and 98.0% for its enantiomer (R)-5a.

Scheme 3.

Synthesis of (R)-6a

An isosteric replacement of the (E)-alkene bond with a cyclopropyl moiety was also investigated as part of our medicinal chemistry program.⁹ Charette-modified Simmons-Smith cyclopropanation¹⁰ of the Cbz-protected allylic amine (S)-4b with Zn(CH₂I)₂•DME complex¹¹ provided the cyclopropyl analog 7b on gram scale in 54% yield (65% based on recovered intermediate (S)-4b) over 2 steps (Scheme 4). Only one diastereomer was isolated after chromatography on SiO₂ (>20:1 dr by ¹H NMR). Since 7b was not crystalline and spectroscopic analysis did not allow an unambiguous assignment of its configuration, we resorted to an X-ray analysis of a suitable crystalline derivative. Hydrogenolysis, coupling with p-bromobenzoyl chloride, TBAF-desilylation and treatment with 1-naphthylisocyanate afforded urea 10, which gave fine colorless needles suitable for X-ray diffraction (Scheme 5). The X-ray analysis confirmed the anti-configuration of the cyclopropane vs the C-N bond (Figure 2). This diastereoselectivity had been previously noted by our group for the dimethylzinc-mediated addition of alkenylzirconocenes to N-diphenylphosphinoyl imines, which provided diastereomerically pure C-cyclopropylalkylamines.^5b,9f The high level of anti-selectivity is also consistent with diastereoselectivities observed in the Simmons-Smith cyclopropanation of allylic ethers.¹²

Scheme 4.

Synthesis of the cyclopropyl isosteres 8a-b

Scheme 5.

Synthesis of 10

Figure 2.

X-ray structure of 10

Somewhat surprisingly, the Boc group on (S)-4a was not compatible with the Simmons-Smith conditions. The desired intermediate 7a was therefore prepared by hydrogenolysis of 7b followed by Boc protection of the resulting amine (Scheme 4). Subsequent TBAF-desilylation of 7a and 7b, Jones oxidation, and coupling with 4-AT afforded the cyclopropyl isosteres 8a and 8b.

Finally, a difluorinated analog of JP4-039 was envisioned to enhance the bioavailability of the agent. The methyl ester (S)-11, prepared from alcohol (S)-5a by Jones oxidation and esterification of the acid with TMS-diazomethane, was treated with 3 equiv. of the fluorinating agent N-fluoro-N-(phenylsulfonyl)benzene-sulfonamide (accufluor, NFSi)¹³ and 2.3 equiv. of NaHMDS in THF at −78 °C to afford the desired α,α-difluoroester (S)-12 in 86% yield (Scheme 6).¹⁴ Saponification with tetra-butylammonium hydroxide (TBAH)¹⁵ and condensation with 4-AT provided the difluorinated JP4-039 analog (S)-13 in good yield. The biological activities of (S)-13 as well as (R)-6a are currently under investigation.

Scheme 6.

Synthesis of the difluoro analog (S)-13

In summary, the hydrozirconation-transmetalation-imine addition methodology was extended to the preparation of both enantiomers of a chiral allylic amine and the corresponding dipeptide alkene and cyclopropane isosteres. This straightforward approach enabled the synthesis of the bioprotective Tempo-conjugate JP4-039 on a 160 g scale. Noteworthy is also the ready access to diverse analogs of the parent compound, including the β,γ-cyclopropylamine isosteres and difluoromethylene derivatives. SAR studies of the JP4-039 scaffold are ongoing, and the synthesis of further derivatives will be reported in due course, along with their antioxidant and biological activities.