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Synthesis and evaluation of new peptide-linked doxorubicin conjugates as prodrugs activated by prostate-specific antigen

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Abstract

The targeted delivery of cytotoxic agents to prostate cancer cells via selective activation of peptide-linked prodrugs by prostate-specific antigen (PSA) has been previously demonstrated. In our continued efforts to design prodrugs with improved prostate tumor specificity, we developed GABA ← mGly-Ala-Ser-Chg-Gln and glutaryl-Ser-Ala-Ser-Chg-Gln as promoieties with enhanced PSA specificity starting from the known substrate sequence glutaryl-Hyp-Ala-Ser-Chg-Gln. Based on their PSA cleavage rates and resistance to non-PSA-mediated hydrolysis in plasma, GABA ← mGly-Ala-Ser-Chg-Gln and glutaryl-Ser-Ala-Ser-Chg-Gln were selected as optimal promoieties and coupled to doxorubicin (Dox) as PSA-targeted prodrugs, using Ser-Leu linkers. Following 72-h incubations with Dox prodrugs, there was insignificant cytotoxicity in non-PSA-producing DU145 cells. The Dox prodrugs, glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (I), glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox (II) and GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox (III) demonstrated comparable PSA cleavage rates (t1/2 values <23 min), and were cytotoxic against PSA-producing LNCaP cells with IC50 values of 0.18, 0.27 and 0.082 μM, respectively. To mitigate neprilysin-mediated hydrolysis of the Ser-Leu linker in prodrugs I–III and further improve PSA specificity, 3-aminooxypropionate was incorporated between the promoiety and Dox (prodrug IV). Despite its slower PSA cleavage rate (t1/2 value of 67 min), GABA ← mGly-Ala-Ser-Chg-Gln-NH-O-CH2-C(Me)2C(O)-14-O-Dox (IV) was equipotent (IC50 value of 0.19 μM) to prodrug I at killing PSA-producing LNCaP cells due to its ability to release free Dox through a cyclization activation mechanism. Further metabolism and PK/PD studies will be conducted to evaluate the tumor specificity of the novel Dox prodrugs (II–IV) reported herein.

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Abbreviations

AMC:

7-amino-4-methylcoumarin

ACN:

acetonitrile

Alloc-OSu:

(N-allylcarbonyloxy)succinimide

Boc:

tert-butyloxycarbonyl

CDI:

carbonyldiimidazole

Chg:

L-cyclohexylglycine

DBU:

1,8-diazabicycloundec-7-ene

DCM:

dichloromethane

DEA:

diethylamine

DIAD:

diisopropyl azodicarboxylate

DIEA:

N,N-diisopropylethylamine

DMF:

dimethylformamide

DMAP:

4-dimethylaminopyridine

Dox:

doxorubicin

DMSO:

dimethyl sulfoxide

EDC:

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

EDU:

1-Ethyl-3-(3-dimethylaminopropyl)urea

FCC:

flash column chromatography

Fm:

fluorenylmethyl

Fmoc:

fluorenylmethoxycarbonyl

FUDR:

floxuridine or 5-fluoro-2’-deoxyuridine

GABA:

γ-aminobutyric acid

HBTU:

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

HOSu:

N-hydroxysuccinimide

HPLC:

high performance liquid chromatography

HRMS:

high-resolution mass spectrometry

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

PSA:

prostate-specific antigen

PyBOP:

(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

RPMI:

Roswell Park Memorial Institute

TCM:

tissue culture medium

MRM:

selected 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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Authors and Affiliations

  1. Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA

    Herve Aloysius & Longqin Hu

  2. Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, 08901, USA

    Longqin Hu

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  1. Herve Aloysius
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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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Abbreviations for common amino acids are in accordance with IUPAC recommendations

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 peptide-linked doxorubicin conjugates as prodrugs activated by prostate-specific antigen. Med Chem Res 29, 1280–1299 (2020). https://doi.org/10.1007/s00044-020-02573-w

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