Synthesis and evaluation of new 4-peptidamido-2-fluorobenzyl phosphoramide mustard conjugates as prodrugs activated by prostate-specific antigen

Abstract

In our continued efforts to develop targeted prodrugs activated by prostate-specific antigen (PSA), we designed and synthesized novel phosphoramide mustard peptide conjugates using previously optimized PSA substrates. Initial Nu/Nu mouse PK studies indicated that prodrug I (glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) exhibits high clearance with significant extrahepatic metabolism in vivo. Substrate optimization studies were thus carried out to further improve PSA specificity and enable the design of prodrugs with reduced in vivo clearance and enhanced tumor selectivity. To assess the utility of the newly optimized sequences as promoieties, they were coupled to phosphoramide mustard using a 4-amino-2-fluorobenzyl alcohol linker akin to prodrug I. In the presence of human PSA, prodrug I was rapidly cleaved with a half-life (t1/2) of 35 min. Prodrugs II (glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) and III (GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) were hydrolyzed at slower rates with t1/2 values of 80 and 107 min, respectively. These results we observed here are different from our previously reported data but may be explained by the fact that PSA-activated release of phosphoramide mustard and reactive quinonimine methides resulted in mechanism-based inhibition of PSA, thereby preventing further hydrolysis of prodrugs I–III. Prodrug I was cytotoxic to PSA-producing LNCaP cells with an IC50 value of 7.3 μM and demonstrated 14-fold selectivity over the non-PSA-producing DU145. Despite its poor in vitro antiproliferative activity (IC50 = 30 µM), prodrug III was found to be more stable against non-PSA-mediated hydrolysis compared with prodrug I as revealed by metabolite profiling studies, which was in agreement with its improved stability in human hepatocyte cultures. These results suggested that a combination of the peptide sequence GABA ← mGly-Ala-Ser-Chg-Gln with optimal linkers and/or other cytotoxic agents can help achieve an adequate balance between PSA cleavage rate and enhanced resistance to non-PSA-mediated hydrolysis.

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Abbreviations

AMC:

7-amino-4-methylcoumarin

ACN:

acetonitrile

Boc:

tert-butyloxycarbonyl

Bn:

benzyl

CDI:

carbonyldiimidazole

Chg:

L-cyclohexylglycine

DCM:

dichloromethane

DEA:

diethylamine

DIEA:

N,N-diisopropylethylamine

DMF:

dimethylformamide

DMSO:

dimethyl sulfoxide

EDC:

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

FCC:

Flash column chromatography

Fm:

fluorenylmethyl

Fmoc:

fluorenylmethoxycarbonyl

GABA:

γ-aminobutyric acid

HBTU:

O-benzotriazole N,N,N’,N’-tetramethyluronium hexa-fluorophosphate

HOBt:

N-hydroxybenzotriazole

HOSu:

N-hydroxysuccinimide

HPLC:

high performance liquid chromatography

HRMS:

high-resolution mass spectrometry

IPCF:

isopropyl chloroformate

Hyp:

trans-4-hydroxy-L-proline

LC-MS:

liquid chromatography-mass spectrometry

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

IS:

internal standard

NMP:

N-methyl-2-pyrrolidone

NMR:

nuclear magnetic resonance

NTR:

nitroreductase

PSA:

prostate-specific antigen

RPMI:

Roswell Park Memorial Institute

TCM:

tissue culture medium

MRM:

multiple reaction monitoring

THF:

tetrathydrofuran

TFA:

trifluoroacetic acid

TLC:

thin-layer chromatography.

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Acknowledgements

We gratefully acknowledge the financial support of grant SNJ‐CCR 700–009 from the State of New Jersey Commission on Cancer Research, a pilot grant from the Gallo Prostate Cancer Center of the Cancer Institute of New Jersey, and grant RSG‐03–004–01‐CDD from the American Cancer Society.

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Correspondence to Longqin Hu.

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The authors have filed patents on some of the compounds discussed in this paper.

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Dedicated to Professor Robert P. Hanzlik on the occasion of his retirement after 49 years of dedicated service on the faculty of the Department of Medicinal Chemistry, University of Kansas School of Pharmacy.

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Aloysius, H., Hu, L. Synthesis and evaluation of new 4-peptidamido-2-fluorobenzyl phosphoramide mustard conjugates as prodrugs activated by prostate-specific antigen. Med Chem Res 29, 1264–1279 (2020). https://doi.org/10.1007/s00044-020-02572-x

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Keywords

  • Prostate-specific antigen
  • Phosphoramide mustard
  • Prodrug
  • Peptide-linked drug conjugate