Abstract:

This study utilizes a two-step synthesis of EB-Mal, a maleimide-functionalized derivative of the albumin-binding dye Evans Blue (EB). The successful synthesis was confirmed via ¹H NMR and high-resolution mass spectrometry (HRMS). EB-Mal serves as a versatile intermediate for prodrug development, facilitating conjugation with the cytotoxic payload MMAE and the targeting ligand cRGD to create an integrated drug delivery system for enhanced cancer therapy.

Introduction:

Cancer remains one of the leading causes of death in the United States; the development of more effective drug delivery strategies for cancer therapy is still urgently needed^[1]. A long-standing goal of our project is to develop a novel delivery system for the highly cytotoxic agent Monomethyl auristatin E (MMAE) by conjugating it to an Evans Blue (EB) moiety through a stimuli-responsive linker and a targeting ligand such as cRGD. Due to its high affinity for serum albumin, conjugating an EB moiety will help increase the drug’s blood circulation time and thus improve the therapeutic efficacy^[2]. To achieve this, we synthesized EB-Mal as a key intermediate for subsequent conjugation and future biomedical applications.

Experimental Details:

Scheme 1. Synthesis of the Evans Blue-maleimide (EB-Mal) intermediate

1. Synthesis of 1

o-Tolidine (400 mg, 1.88 mmol) was dissolved in 5 mL DMF in a 25 mL flask. After stirring for 5 min, the 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanoic acid (200 mg, 1.18 mmol), HATU (900 mg, 2.37 mmol) and N, N-diisopropylethyl-amine (DIPEA, 800 mg, 6.19 mmol) was dissolved in 5 mL DMF, and then dropwise to the mixture. The reaction mixture was stirred for 6h at room temperature. The yellow mixture was washed with water (1 × 100 mL), saturated NaHCO3 aqueous solution (2 × 100 mL), and saturated brine (1 × 100 mL). The organic layer was separated and dried over anhydrous Na2SO4. The solution was concentrated by rotatory evaporator, and the residue was purified by chromatography. Gradient ethyl acetate and hexane mixture (1/1) were used as eluent. Yield: 275 mg (80% yield).

Fig. 1. ¹H NMR of Compound 1

2. Synthesis of EB-Mal

A solution of 0.5 mL cold 2.0 M HCl was added dropwise to a 3 mL solution of Compound 1 (100 mg, 0.27 mmol) in DMF in a 25 mL flask in an ice bath. After stirring for 15 min, 1 mL of saturated sodium nitrite was slowly added to the reaction mixture, and was stirred for another 30 min to obtain a yellow solution. The obtained solution was then added dropwise to the mixture solution of NaHCO3 (100 mg, 1.19 mmol) and 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt hydrate (187 mg, 0.55 mmol, dissolved in 2 mL water) in an ice bath. After stirring for another 60 mins, purple reaction mixture was obtained. Purified by preparative HPLC, and lyophilized to 87 mg purple powder (yield 45.7%).

Fig. 2. High-resolution mass spectra of EB-Mal

Results and Discussion:

We successfully synthesized EB-Mal in two steps from commercially available compounds. Compound 1 was obtained from o-tolidine and 3-maleimidopropionic acid, yielding 275 mg after purification by column chromatography. EB-Mal was then synthesized from Compound 1 and 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt hydrate, affording 87 mg of product after separation by HPLC. The structures of all products were confirmed by ¹H NMR and high-resolution mass spectrometry.

Conclusion:

An albumin-binding EB moiety was synthesized in two steps with high yield. The resulting EB-Mal will be further utilized for conjugation with the cytotoxic drug MMAE and the targeting ligand cRGD to construct a stimuli-responsive, albumin-hitchhiking prodrug for efficient cancer drug delivery.

Reference

[1] R.L. Siegel, T.B. Kratzer, A.N. Giaquinto, H. Sung, A. Jemal, Cancer statistics, 2025, CA: A Cancer Journal for Clinicians 75(1) (2025) 10-45.

[2] S. Fu, A. Zheng, L. Wang, J. Chen, B. Zhao, X. Zhang, V.A.A. McKenzie, Z. Yang, R.M. Leblanc, R. Prabhakar, F. Zhang, Tuneable redox-responsive albumin-hitchhiking drug delivery to tumours for cancer treatment, J Mater Chem B 12(27) (2024) 6563-6569.