Chapter 21. Pteridine derivatives (folic acid, methotrexate)

Pteridine derivatives are based on the following structure; pteridine is a bicyclic system.

The structure of folic acid and related compounds

1. Folic Acid

Acidum folicum (Latin: folium, “leaf”)

pteroylglutamic acid (folic acid)

Vitamins of the folic acid group differ in the amount of glutamic acid residues (up to 7 are possible).

Coenzyme forms of folic acid

1. 5,6,7,8-tetrahydrofolic acid.
2. Folinic acid is 5-formyl-5,6,7,8-tetrahydrofolic acid. Synthesized as a medicinal product.

Description

Folic acid is a yellow powder (due to pteridine), odorless, tasteless, hygroscopic, that decomposes in the light and is insoluble in water. It is an amphoteric compound with a predominance of acidic properties. Easily soluble in NaOH, NH₄OH, alkali metal carbonates. Destroyed by acids, reducing agents, oxidizing agents, light.

Identification

1. Physicochemical parameters: melting point, IR, UV spectra in 0.1 M alkali.
2. Acid-base properties ― mainly an acid:

a) dissolves in alkalis and ammonia;

b) reactions of salt formation and complexation with salts of heavy metals: substance + alkali equivalent ⇒ disodium salt (easily soluble in water) + CuSO₄ (or cobalt, iron salts) ⇒ reactions are nonspecific.

3. Hydrolysis and oxidation ― formation of pterin-6-carboxylic acid. Substance + NaOH + 0.1 M HCl + KMnO₄ ⇒ heated in a water bath ⇒
   + H₂O₂ ⇒ filtered ⇒ the filtrate has blue fluorescence in UV:

Reaction of azo dye formation ― see assay. Folic acid + HCl (or + NaOH) + zinc dust ⇒ when heated, para-aminobenzoic acid (PABA) is formed.

Assay

1. Photoelectric colorimetry ― by the reaction of azo dye formation. Sample + water + NH₄OH + KMnO₄ ⇒ then sodium nitrite in HCl is added; its excess is removed with sulfamic acid (N₂ is formed) + 1 ml N-1-naphthylethylenediamine ⇒ azo dye; optical density is determined.

Stage 1. KMnO₄ oxidation:

Stage 2:

Stage 3. Removing excess NaNO₂ + HCl:

Stage 4:

2. Fluorimetry ― formation of pterin-6-carboxylic acid.

3. HPLC.

Antivitamins

1. Aminopterin: OH is replaced by an amino group. It’s a cytostatic agent used for treatment of leukemia.
2. Ametoterin: 4-amino-10-methyl-folic acid. The basis of the drug methotrexate.

2. Methotrexate (Methotrexatum) is an antimetabolite of folic acid, a cytostatic agent for treatment of leukemia in children. It is a mixture of amethopterin and simple pterin compounds.

Assay: HPLC.

Isoalloxazine derivatives

Riboflavin (vitamin B2);

Riboflavin mononucleotide (coenzyme).

1. Riboflavin (Riboflavinum) ― B₂

Contains residual ribitol alcohol. Capable of complexation. The isoalloxazine group is involved in oxidation.

6,7-dimethyl-9-D-1’-ribitylisoalloxazine

Description: a yellow-orange powder (conjugated system of double bonds). Three crystalline forms Þ different solubility in water 1:3000-1:20,000; soluble in alcohol.

2. Riboflavin mononucleotide (Riboflavini mononucleotidum)

It’s a coenzyme of riboflavin, the sodium salt of riboflavin 5’-phosphate ester.

Identification

Riboflavin

1. UV spectrum (λ_max 267 nm, 372 nm and 444 nm).

2. Optically active.

3. Acid-base properties:

Riboflavin is an ampholyte.

A. Acidic properties are due to the imide group. Forms characteristic precipitates with AgNO₃ ― an orange-colored precipitate (as in barbiturates with nitrogen). Uncharacteristic precipitates with cobalt and copper salts.

B. Basic properties are very weak because of N⁹ and N¹⁰. The drug is soluble in CH₃COOH. Precipitation with general alkaloid reagents.

4. Redox properties:

a) substance + water ⇒ yellow-green coloration. When irradiated with UV ⇒ intense yellow-green fluorescence;

b) reduction:

Riboflavin + [H] (NaHSO₃ or Na₂S₂O₅, Na₂SO₃ + SO₂, Zn + HCl) ⇒ dihydroriboflavin (leukoriboflavin)

c) oxidation:

1) concentrated H₂SO₄ ― red coloration.

5. Fluorescence

Riboflavin exhibits green fluorescence that disappears under the influence of UV, in an acidic or alkaline medium. In an acidic environment, lumichrome (alloxazine derivative) is formed:

6. In an alkaline medium, the formation of (colored) lumiflavin, a derivative of isoalloxazine, is a specific impurity that is soluble in chloroform.

6. Riboflavin mononucleotide is different in that it’s easily soluble in water, has a reaction with Na⁺ and three reactions with phosphate ion after acid hydrolysis (see cocarboxylase).

Assay

1. Spectrophotometry in UV at l_max = 267 nm, SPM in the visible region of the spectrum at l_max = 444 nm.

2. Fluorimetry.

3. HPLC.