Analytical Profiles of Drug Substances, 7

6-MERCAPTOPURINE

Steven A . Benezra and Penelope R . B. Foss

343 Copyright 0 1978 by Academic Press, Inc.

All rights of reproduction in any form reserved.
ISBN 012-260807-0
344 STEVEN A. BENEZRA AND PENELOPE R. B. FOSS

Table of Contents

1. Description

1.1 Name, Formula, Molecular Weight

1 . 2 Appearance, Color, Odor

2. Physical Properties

2.1 Infrared Spectrum

2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Spectrum
2.4 Mass Spectrum
2.5 Melting Point
2.6 Solubility
2.7 Dissociation Constant

3. Synthesis

4. Stability

5. Methods of Analysis

5.1 Elemental Analysis

5.2 Nonaqueous Titrhetric Analysis
5.3 Spectrophotometric Analysis
5.4 Polarography
5.5 Mass Spectrometry
5.6 Chromatography
5.61 High Performance Liquid Chromatography
5.62 Column Chromatography
5.63 Gas Chromatography
5.64 Thin Layer Chromatography

6. Pharmacokinetics and Metabolism

6.1 Excretion Rate

6.2 Tissue Distribution
6.3 Metabolism
6-MERCAPTOPURINE 345

1. Description

1.1 Name, Formula, Molecular Weight

Mercaptopurine is purine-6-thiol monohydrate

SH

C5H4N4S * H 2 0 170.19

1.2 Appearance, Color, Odor

Mercaptopurine is a yellow, practically odorless,
crystalline powder'.

2. Physical Properties

2.1 Infrared Spectrum

The infrared2spectrum of mercaptopurine (anhydrous)
is shown in Figure 1 .
It was taken as a 0.2% dispersion of
mercaptopurine in KBr with a Perkin Elmer model 457 infrared
spectrophotometer. Table I gives the infrared gssignments
consistent with the structure of mercaptopurine .
Table I
Infrared Spectral Assignments for Mercaptopurine

Band (cm-l) Assignment

3420, 3490 Aromatic NH stretch
3120, 3040, 2780 Aromatic CH stretch
1200 C=S stretch
930 CH bend

2.2 Nuclear Magnetic Resonance (NMR) Spectrum

The NMR spectrum of mercaptopurine is shown in Figure
24 It was obtained with a Varian CFT-20 80 MHz NMR spectro-
.
meter. Deuterated DMSO was used as the solvent with tetra-
methylsilane as an internal standard. Based on the NMR
spectrum, the following proton assignments for mercaptopurine
can be made.
Figure 1- Infrared Spectrum of 6-Mercaptopurine
2
*rl
k
7
a
0
U
a
cd
0
k
a,
7
\D
w
0
5
k
U
u
al
a
rn
b)
348 STEVEN A. BENEZRA AND PENELOPE R. B. FOSS

Proton Chemical Shift (ppm)

S-H 2.75-3.75 broad singlet
-
C-H (aromatic) 8.15 singlet
C-H (aromatic) 8.35 singlet
N-H 11.0-12.0 broad singlet
2.3 Ultraviolet (W)Spectrum
The W spectra of mercaptopurine in 0.1N NaOH, 0.1N
HC1, and methanol were taken with a B e c p n ACTA CIII W spec-
trophotometer and are shown in Figure 3 .
Table I1 summar-
izes the W data.
Table I1
UV Spectral Data €or Mercaptopurine

Solvent )i max (nm)

0.1N NaOH 230 (3)
14000(5)
312 19600
0.1N HC1 222 924012;
327 21300(3)
Methanol
216
329 8940(3)
19300
2.4 Mass Spectrum
The low r solution mass spectrum of mercaptopurine is
shown in Figure 4% . It was obtained with a Varian-MAT model
731 mass spectrometer. Direct probe at 145OC into the elec-
tron impact source was used. The electron energy was 70eV.
The assignment of fragment ions is given below.

m /e I25 ( 18%)
H

y>>
; H2NXLJ7+
H2N N
m/e 119 ( 16%) m /e 98 ( 10%)
+
CzNSH' (CN?
m/e71 ( 1 1 % ) m/e 26 (20%)
6-MERCAPTOPURINE 349

I .4
I .2
I .o
0.8
0.6
0.4
0.2
0
I.4
W I .2
0
7 I .o
a
m 0.8
02
0 0.6
rn
m 0.4
a 0.2
0

I .2 r
I .o -
0.8 -
0.6
0.4
0.2
0
:P
i-1 1 I 1 1 1 1 1 1 1 1 1 1

nm
Figure 3- Ultraviolet Spectra of 6-Mercaptopurine
Top-O.1N HC1, Middle-O.1N NaOH, Bottom-Methanol
 100

80
>
-
I-
cn
60
I-
z
-
w
>
-I- 40
a
A
w
20

0
SO I00
m /e
Figure 4- Mass Spectrum of 6-Mercaptopurine
6-MERCAPTOPURINE 351

2.5 Melting Point

Mercaptopurine decomposes above 308°C1
.
2.6 Solubility
Mercaptopurine is insoluble in water, acetone, and
ether. It is soluble in hot ethanol and dilute alkalilsolu-
tions. It is slightly soluble in dilute sulfuric acid .
2.7 Dissociation Constant
The pK and pKa of mercaptopurine qetermined poten-
1 7.77 ani 11.17 respectively
tiometrically fs .
3. Synthesis
Mercap@yyrine has been prepared by a variety of synthetic
procedures . These procedures are outlined in Figure 5.
Process 1 involves treatment of 4-amino-5-formamido-6-hydroxy-
pyrimidine with phosphorous pentasulfide, decomposition of ex-
cess phosphorous pentasulfide with base and acidification to
pH 4-5. Mercaptopurine precipitates from the acidic solution.
Process 2 treats hypoxanthine with phosphorous pentasulfide.
The solid formed is treated with NH40H to pH 5. Mercaptopur-
ine precipitates from the solution. Process 3 utilizes 4-
amino-6-chloro-5-nitropyrimidine as the starting material.
Treatment of this compound with potassium hydrosulfide gives
4,5-diamino-6-mercaptopyrimidine. The 4,5-diamino-6-mercapto-
pyrimidine is heated in concentrated formic acid to yield 7-
amino-thiazolo(5,4-d)pyrFmidine. Mercaptopurine is precipi-
tated after treatment of 7-amino-thiazolo(5,4-d)pyrimidine
with NaOH and adjusted to pH 5 with acetic acid. Process 4
is a varient of process 3. The 4,5-diamino-6-mercaptopyrimi-
dine is treated with 50% formic acid to give 4-amino-5-forma-
mido-6-mercaptopyrFmidine, which in turn can be treated as the
7-amino-thiazolo(5,4-d)pyrimidine in process 3 to give mer-
captopurine. Process 5 treats 4-aminoimidazole-5-carboxamide
with phosphorous pentasulfide. The product, 4-aminoimida-
zole-5-thiocarboxamide, is heated with formamide to form
the six-membered ring. Removal of the formamide and recrys-
tallization of the residue gives mercaptopurine.

4. Stability
The decomposition of mercaptopurine in 0.1N NaOH, 0.1y2
HC1, distilled water, and photolytically has been studied
When mercaptopurine is refluxed for 6 days in 0.1N NaOH, 4-
.
aminoimidazole-5-thiocarboxamide and 4-amino-5-cyanoimidazole
are the major products formed. When refluxed for 4 days in
0.1N HC1 and 7 days as an aqueous suspension in distilled wa-
ter, mercaptopurine decomposes primarily to 4-aminoimidazole-
5-thiocarboxamide. Mercaptopurine in 0.1N NaOH and as an
 I*&
0 ?=J
z
I
c
N d-
6-MERCAPTOPURINE 353

aqueous suspension in distilled water forms hypoxanthine when

irradiated for 72 hours with a medium pressure mercury lamp.

5. Methods of Analysis

5.1 Elemental Analysis (As the Hydrate)

Element C
- H
- N
- -S
X calculated 35.28 3.55 32.92 18.83

5.2 Nonaqueous Titrimetric Analysis

Nonaqueous titration is t e official method of analy-
P
sis in the USP for mercaptopurine . An accurately weighed
sample of mercaptopurine is dissolved in dimethylformamide.
The solution is titrated with standardized 0.1N sodium methcrx-
ide using thymol blue as an indicator. Precautions must be
taken against absorption of atmospheric carbon dioxide.

5.3 Spectrophotometric Analysis

The official USP analysis of mercaptopurine
. - in tab-
lets is a spectrophotometric analysis. A portion of powdered
tablets is weighed. Twenty ml distilled water, and 1.5 m l
NaOH TS is added to the powder in a 100 m l volumetric flask.
The flask is brought to volume with distilled water, filtered,
and a portion of the filtrate diluted with dilute hydrochlor-
ic acid. The solution is compared against a Reference Stan-
dard prepared in a similar manner at 325 nm in 1 cm cells.

5.4 Polarography
Alternating current polarography has beep3used to de-
termine decomposition kinetics of mercaptopurine .
A cata-
lytic wave with Q=0.45 was observed for mercaptopurine in 1N
H2S04.

5.5 Mass Spectrometry

Quantitative analysif40f mercaptopurine in plasma has
been accomplished with GCfMS . Mercaptopurine was extracted
from plasma, derivatized with methyl iodide, and separated by
gas chromatography using a 1.83 M, 3% OV-225 column at 2OO0C,
and detected with a mass spectrometer equipped with a peak
monitor. Limit of detection was 20 ng/ml of mercaptopurine
in plasma.

5.6 Chromatography
5.61 High Performance Liquid Chromatography
High performance liquid chromatography was used
to determine mercapSopurine and metabolites in cultured cells
and animal tissues . A 0.18 x 100 cm colunm packed with
Beckman M71 strong cation exchange resin was eluted with 0.4M
354 STEVEN A. BENEZRA AND PENELOPE R. B. FOSS

ammonium formate (pH 4.6) at 8 ml/hr. The column was kept at

50°C. The retention time of mercaptopurine under these con-
ditions was 39 minutes. Detection was accomplished with a UV
detector at 322 nm.
5.62 Column Chromatography
Column chromatography has been yEed to separate
mercaptopurine from its metabolites in urine , A cation ex-
change resin, Zeo-carb 225, eluted with 20% (v/v) ammonium
hydroxide separated mercaptopurine and other 6-thiopurines
from a concentrated urine sample.
5.63 Gas Chromatography
A gas chrornatog7aphic analysis of mercaptopurine
in serum has been described . A 1.5M x 6.3 mm 0.d. column
packed with 10% w/w SE-30 maintained at 135OC was used. Mer-
captopurine derivatized with trimethylanilinium hydroxide had
a retention time of 22 minutes.
5.64 Thin Layer Chromatography
The separation of mercavtovurine
. - from mixtures of
purines and pyrimidines has been accomplished by thin layer
chroma graphy using anion exchange ECTEOLA cellulose
platesie . The plates were developed in acetone:O.lM H SO :
ethyl acetate (45:10:45). A second development was doie In
deionized water:acetone (8:2). The R value of mercaptopurine
was 0.36. Cellulose plates developedf in 0.1N HC1, H20, and
isopropano1:methanol:H 0:NH OH (60:22:20:1) gave R valyes
for mercaptopurine of 8 . 4 3 , 40.26, and 0.55 respectfvely .
6. Pharmacokinetics and Metabolism

6.1 Excretion Rate

In the rat, by the end of 48 hours after injection,
55% of mercaptopurine was excreted in the uri28. The largest
proportion was excreted in the first 24 hours .
3Jor an in-
traperitoneal injection in the mouse of 1 mg of S -6-mercap-
topurine, 43.5% of the radioactive material was excreted in
the urin in the first four hours. At the end of 2 days over
55
60% of S was excreted. Approximately the same urinary ex-
cretion rate was found with oral dofps, but some radioactive
C02 was detected in the expired air .
6.2 Tissue Distribution
In the mouse the concentration of radioactive mercap-
topurine was highest in the gut, almost twice as high as in
the blood, and lowest in the brain. Mercaptopurine has some
difficulty passing the blood-brain barrier. The brain2fon-
centration is one-tenth the concentration in the blood .
The presence of a tumor in different sites in rats, as well as
in mice, causes lower blood levels of mercaptopurine when com-
pared to non-tumor bearing animals. The apparent volume of
6-MERCAPTOPURINE 355

distr is markedly increased in the presence of a

tumor
$9ution
.
6.3 Metabolism
The pathway of metabolism of mercaptopurine is by hy-
droxylation v i a the enzymes xanthine oxidase and aldehyde oxi-
dase. Mercaptopurine is transformed into mercaptopurine ribo-
side, 6-tltf~ypf2~acid,sulfates, and nucleotide metabolites in
the liver
356 STEVEN A. BENEZRA AND PENELOPE R.B. FOSS

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