Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases

Abstract

A new series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. The best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat toward the same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these seven-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents.

Figure 1

Projection view of 10(α) with 30% thermal ellipsoids (disorder atoms omitted for clarity). This crystal structure has been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 873939.

Figure 2

Effect of the incubation time on the inhibition level of Cutinase (♦), Rv0183 (□) and LipY (●) by compounds 1, 7(β) and 11, respectively. Each enzyme was pre-incubated with the corresponding phosphonate compound at a fixed inhibitor molar excess x_I = 4, and the residual activity was measured over a 60-min. period at various time intervals using the pH-stat technique. Results are expressed as mean values of at least two independent assays (CV% < 5.0%).

Figure 3

In silico molecular docking of compound 7(β) into crystallographic structures of (A) DGL (PDB: 1K8Q), (B) HPL (PDB: 1LPB) and (C) GPLRP2 (PDB: 1GPL) in a van der Waals surface representation. Hydrophobic residues (alanine, leucine, isoleucine, valine, tryptophan, tyrosine, phenylalanine, proline and methionine) are highlighted in white. The side chain of catalytic serine residue is shown as sticks and colored in magenta. Compound 7(β) is represented in atom color-code sticks (carbon, yellow; phosphorus, orange, oxygen, red). Structures were drawn using the PyMOL Molecular Graphics System (Version 1.3, Schrödinger, LLC). The [Ser-Oγ / P] calculated distances are indicated in blue.

Figure 4

Peptide mass fingerprint spectra of the digested Cutinase, before (upper panels, A) and after (lower panels, B) a 30-min incubation period with compound 4 at an inhibitor molar excess x_I = 40; and (C) the corresponding confirmed reaction mechanism with the active site Ser¹²⁰. (A–B): Zoom on the region of interest. Left panels, region of the unmodified peptide C¹⁰⁹-R¹³⁸ (C¹⁰⁹PDATLIAGGYS¹²⁰QGAALAAASIEDLDSAIR¹³⁸) containing the catalytic Ser¹²⁰ and detected at 2,977.52 Da. This multiplet peak is overlaid with a first one corresponding to peptide V¹⁶⁹-R¹⁹⁶, detected at 2,974.51 Da. Right panels, region in which a new peptide was expected to occur resulting from the covalent binding of compound 4 to the catalytic serine. Mass shifts were calculated as the difference between the experimental m/z of the modified peptide and the theoretical m/z of the unmodified catalytic peptide. The expected exact mass shift is +234.07 Da.

Scheme 1

Rationale for synthesis of Cyclophostin and Cyclipostins analogs, and related structures investigated: Cyclophostin molecule (1) and phosphonate analogs (2); monocyclic phosphonate analogs to either Cyclophostin (3–4, 5–10) or Cyclipostins (11–12).

Scheme 2

Synthesis of novel monocyclic enol-phosphonate analogs of either Cyclophostin (5–10) or Cyclipostins (11–12).

Scheme 3

Relative Stereochemistry of the monocyclic phosphonates 5–10.