Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

Summary

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

Abstract

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. Isonicotinic acid, or its derivatives, are coupled to variable length diaminoalkane chains under an inert atmosphere in anhydrous DMF or DMSO with the use of a weak base, triethylamine, and a coupling agent, 1-propylphosphonic anhydride. The products precipitate from solution upon formation or can be precipitated by the addition of water. If desired, the ligands can be further purified by recrystallization from hot water. Dinuclear platinum complex synthesis using the bispyridine ligands is done in hot water using transplatin. The most informative of the chemical characterization techniques to determine the structure and gross purity of both the bispyridine ligands and the final platinum complexes is ¹H NMR with particular analysis of the aromatic region of the spectra (7-9 ppm). The platinum complexes have potential application as anticancer agents and the synthesis method can be modified to produce trinuclear and other multinuclear complexes with different hydrogen bonding functionality in the bridging ligand.

Introduction

Platinum anticancer drugs remain one of the most widely used family of agents in the treatment of human cancer¹. Despite their success, they are limited in their application by severe dose-limiting side effects^2-4. The limited doses that can be administered to patients also means that tumors can develop resistance⁵. As such, new drugs continue to be developed to improve the side effect profile and overcome acquired resistance, like phenanthriplatin⁶ and phosphaplatin⁷.

In the late 1990s, a trinuclear platinum drug was developed, BBR3464 (Scheme 1)⁸, that is up to 1,000x more cytotoxic in vitro than the leading platinum drug, cisplatin. BBR3464 is also able to overcome acquired resistance in a panel of human cancer cell lines⁹. Unfortunately, the increased activity of BBR3464 is matched by 50- to 100- fold higher toxicity, which limits its use^10-12. It is also easily degraded in the body, meaning little of the drug reaches cancer nuclei intact⁹.

Picoplatin is a mononuclear platinum-based drug that contains a 2-methyl-pyridine ligand (Scheme 1)¹³. The methyl group of this drug protects it from attack by biological nucleophiles; in particular cysteine and methionine containing peptides/proteins^14-16. As such, the drug is quite stable and has a much higher concentration that reaches cancer nuclei compared with both BBR3464 and cisplatin¹⁷. Its reduced reactivity also means picoplatin has a higher maximum tolerated dose compared with BBR3464 and cisplatin^10,18,19.

This project therefore sought to combine the properties of BBR3464 and picoplatin to produce new drugs that are able to overcome acquired resistance that display improved biological stability and less severe side-effects (e.g., Figure 1). In doing so, a range of dinuclear platinum complexes were prepared with bispyridine bridging ligands²⁰. The ligands are made using amide coupling reactions with isonicotinic acid, or its derivatives like 2-methyl-isonicotinic acid, variable length diaminoalkanes. Reaction of one mole equivalent of the ligands with two mole equivalents of transplatin yields the desired platinum complexes (Scheme 1).

Protocol

1. Synthesis of the N,N’-(alkane-1,n-diyl)diisonicotinamide

1. Dry a single neck or three-neck round bottom flask in an oven (100 ºC, 1 hr) to ensure all moisture is removed.
2. Add solid isonicotinic acid, or its derivative, to the flask along with a magnetic stirring bar. If the diaminoalkane ligand(s) are solids at room temperature, then 0.5 mole (to the number moles of isonicotinic acid) is added to the flask at this stage.
3. Cap the neck(s) of the flask with rubber septa and replace the air in the flask with nitrogen either through a continuous nitrogen stream or through the use of nitrogen filled balloons.
4. Use a hypodermic needle and a syringe to add anhydrous dimethylformamide or dimethylsulfoxide (4 ml per 500 mg of isonicotinic acid or 2-methyl-isonicotinic acid) to dissolve the solid. If the solids do not dissolve easily, then heat the solution gently.
5. Add 7 mole equivalents (to the amount of isonicotinic acid used) of triethylamine (weak base) and 0.5 mole equivalents of the diaminoalkane. If the solution is a liquid at room temperature, then add 1.5 mole equivalents.
6. Add one mole equivalent of 1-propylphosphonic anhydride (coupling agent) with continuous stirring and allow the reaction to complete over 5-12 hr.

2. Purification of the Ligands

1. For bispyridine ligands made using diaminoalkane ligands with 10 or more methylene groups, wait for the products to precipitate from solution as the reaction progresses.
2. For bispyridine ligands made using diaminooctane, precipitate the product by adding ~40 ml of water.
3. For bispyridine made using diaminoalkanes of two to six methylene groups, add ~40 ml of water and allows the compounds to crystallize over 1-3 days.
4. Collect each bispyridine ligand by vacuum filtration and recrystallize from approximately 400-500 ml of boiling water per 200 mg of ligand. Note: More water is needed for the longer bispyridine ligands due to their much reduced water solubility.
5. Add NaOH and KOH (pH 9) to the solution to ensure that the compounds are free bases upon recrystallization.

3. Synthesis and Purification of the Dinuclear Platinum Complexes

1. Fully dissolve trans-diamminodichloridoplatinum(II), transplatin, in hot (70-80 ºC) water (150 ml per 200 mg of transplatin) to produce a clear strongly yellow colored solution.
2. Add a 0.5 mole equivalent of the bispyridine ligand and stir the solution at temperature until the ligand dissolves (clear solution). Wait for the solution to turn near colorless, turn off the heat, and stir at room temperature for a few additional hours.
3. Remove the solvent by rotary evaporation, which will yield a yellow colored powder.
4. Purify, the platinum complex(es) by dissolving in a minimum amount of warm water (~50 ºC). If remaining yellow or white colored solids are present, then filter these off.
5. Add acetone to the solution until a white precipitate is formed which appears to be a polymeric form of the metal complexes and represents up to 10% of the reaction product. Continue the addition of acetone (~extra 20-30 ml) until no more precipitate appears. Note: this white precipitate is an impurity.
6. Remove this precipitate by filtering the contents through nylon filter paper (0.2 µm pore size) and rotary evaporating the remaining solution to dryness, which will yield a pure product. Note: If necessary, then additional acetone precipitation steps can be performed until the complex is pure.

Results

The bispyridine ligands and their respective dinuclear platinum complexes are characterized by ¹H, ¹³C and ¹⁹⁵Pt NMR (Tables 1 and 2), and electrospray ionization mass spectroscopy. Accurate melting points can be determined using differential scanning calorimetry and purity is best determined by elemental analysis for C, H and N percentage content. Of most use is ¹H NMR as it is quick and easy to use, giving results within minutes of isolation ...

Discussion

In this work dinuclear platinum complexes have been synthesized as potential anticancer agents. In doing so bispyridine bridging ligands were synthesized via an amide coupling reaction using isonicotinic acid and variable length diaminoalkanes. Previously the synthesis of bispyridine ligands and their methyl analogues with 2 to 8 methylene groups and their respective platinum complexes have been reported. In this paper, the synthesis and purification method has been revised making it faster and cheaper and have demonstra...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
D2O	Aldrich	151882	99.9% D
DMSO-d6	Aldrich	156914	99.96% D
1,8-diaminooctane	Aldrich	D22401	98%
1,10-diaminodecane	Aldrich	D14204	98%
1,12-diaminododecane	Aldrich	D1,640-1	98%
Isonicotinic acid	Aldrich	I17508	99%
1-Propylphosphonic anhydride solution	Aldrich	431303	50 wt% in ethyl acetate
Trans-diaminodichloridoplatinum(II)	Aldrich	P1525
Dimethylsulfoxide	Sigma-Aldrich	Z76855	>99.9%, anhydrous
N,N’-dimethylformamide	Sigma-Aldrich	227056	99.8%, anhydrous
Triethylamine	Sigma-Aldrich	T0886	>99%
Nylon filter membranes	Whatman	7402-004	Pore size, 0.2 µm
Magnetic stirring hotplate
Magnetic stirring bar
Round bottom or three neck flask
Rubber septums of sufficient size for chosen round bottom or three neck flask
5 ml hypodermic syringes
Hypodermic needles
Rubber party ballons
Rubber bands
A source of N2 gas
Rotary evaporator
Drying oven
NMR tubes
NMR spectrometer
500 ml beakers
Glass or plastic pipettes