Identification and physicochemical characteristics of temozolomide process-related impurities

Article abstract of DOI:10.3390/molecules181215344

In this article the crystal structures of the starting material TZ-5 and the key intermediate TZ-6 of temozolomide (TZ-7), an anticancer therapeutic agent, are presented, together with their spectroscopic and thermal characteristics. Both compounds crystallize in the triclinic P-1 space group. X-ray crystallography studies proved that the compound TZ-6 exists as a monohydrate. A complete structural assignment was obtained for the signals in the 1H-, 13C- and 15N-nuclear magnetic resonance spectra and the structures were confirmed by Fourier-Transform infrared and Raman spectroscopy. The article describes the importance of the high purity of TZ-6 during the small-scale plant production of TZ-7 in a desired polymorphic form III with the purity higher than 99.50%, according to an HPLC method.

Full text of DOI:10.3390/molecules181215344

Molecules 2013, 18, 15344-15356; doi:10.3390/molecules181215344
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Identification and Physicochemical Characteristics of
Temozolomide Process-Related Impurities
1
,
1
1
1
Marta Łaszcz *, Marek Kubiszewski , Łukasz Jedynak , Monika Kaczmarska ,
1
1
1
1
Łukasz Kaczmarek , Wojciech Łuniewski , Krzysztof Gabarski , Anna Witkowska ,
1
2
Krzysztof Kuziak and Maura Malińska
1
Pharmaceutical Research Institute (PRI), Warszawa 01-793, Poland;
E-Mails: m.kubiszewski@ifarm.eu (M.K.); l.jedynak@ifarm.eu (Ł.J.);
m.kaczmarska@ifarm.eu (M.K.); l.kaczmarek@ifarm.eu (Ł.K.); w.luniewski@ifarm.eu (W.Ł.);
k.gabarski@ifarm.eu (K.G.); a.witkowska@ifarm.eu (A.W.); k.kuziak@ifarm.eu (K.K.)
Faculty of Chemistry, University of Warsaw, Warszawa 02-093, Poland;
E-Mail: mauramalinka@gmail.com
2
*
Author to whom correspondence should be addressed; E-Mail: m.laszcz@ifarm.eu;
Tel.: +48-22-4563-903; Fax: +48-22-4563-838.
Received: 24 October 2013; in revised form: 27 November 2013 / Accepted: 3 December 2013 /
Published: 11 December 2013
Abstract: In this article the crystal structures of the starting material TZ-5 and the key
intermediate TZ-6 of temozolomide (TZ-7), an anticancer therapeutic agent, are presented,
together with their spectroscopic and thermal characteristics. Both compounds crystallize in
the triclinic P-1 space group. X-ray crystallography studies proved that the compound TZ-6
exists as a monohydrate. A complete structural assignment was obtained for the signals in
1
13
15
the H-, C- and N-nuclear magnetic resonance spectra and the structures were
confirmed by Fourier-Transform infrared and Raman spectroscopy. The article describes
the importance of the high purity of TZ-6 during the small-scale plant production of TZ-7
in a desired polymorphic form III with the purity higher than 99.50%, according to an
HPLC method.
Keywords: temozolomide intermediate; X-ray crystallography; nuclear magnetic resonance;
high-performance liquid chromatography

Molecules 2013, 18
. Introduction
A complete physicochemical characterization has recently become required by both the U.S. Food
15345
1
and Drug Administration (FDA) [1] and by the European Medicine Agency (EMA) [2], not only for
active pharmaceutical ingredients (API), but also for all key synthetic intermediates. A well
documented impurity profile, the identification and physicochemical characteristic of a starting
material and intermediate products are necessary for an API registration.
Temozolomide (TZ-7, 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide) is a
cytotoxic antitumor prodrug against malignant brain tumours. At physiological pH temozolomide undergoes
decomposition to the active compound MTIC (3-methyl-(triazen-1-yl)imidazole-4-carboxamide) [3–5].
®
The drug (brand names: Temodar, Temodal and Temcad ), originally developed by Schering-Plough
Corp., first bacame available in the U.S. in August 1999 [6]. X-ray crystallography studies of
temozolomide have been described by Lowe et al. [7]. The crystal structures of temozolomide
polymorphs, as well as its cocrystals with 4,4'-bipyridine-N,N'-dioxide have been reported by
Jagadeesh Babu et al. [8].
In this article the complete physicochemical characteristics of temozolomide intermediate and
1
13
15
starting material have been determined by the following methods: H-, C- and N-nuclear magnetic
resonance (NMR), infrared (IR) and Raman spectroscopy, as well as by thermal analyses. Single
crystals of both compounds were obtained and their X-ray structures were solved. Furthermore,
high-performance liquid chromatography (HPLC) methods for the purity determination of TZ-5 and
TZ-6 were evaluated.
2
2
2
. Results and Discussion
.1. Structure Elucidation by NMR, IR and Raman Spectroscopy
.1.1. NMR Spectroscopy
1
13
15
All signals in the H-, C- and N-NMR spectra of temozolomide (TZ-7) and key intermediates
TZ-6 and TZ-5 were assigned to the respective atoms using one- and two-dimensional techniques
1
13
1
15
(
g-HSQC and g-HMBC ( H- C and H- N-NMR, Table 1).
The comparison of TZ-5, TZ-6 and TZ-7 spectra shows that these compounds have a very similar
skeletal systems, especially the TZ-7 and TZ-6 pair. Both molecules have the same imidazotetrazine
ring in the structure. Their spectra differ only in the region of the amide group. In the imidazole ring
the differences between the corresponding values of chemical shifts are usually not more than 0.5 ppm.
Only for the nitrogen at position 8 is the difference bigger, i.e., 1.88 ppm. The tert-butyl group
substitution in the TZ-7 molecule also increases the shielding of the carbonyl carbon in position 10
(
the chemical shift changes from 161.51 ppm to 158.93 ppm). Unfortunately, in our study we did not
determine any chemical shift values for the nitrogen atoms in position 2 of the molecules TZ-7 and
TZ-6. For the compound TZ-7 this value was measured using an isotopically labeled compound in the
work of Wheelhouse et al. [9]. In that article the chemical shift of the nitrogen atom 2 is −33.99 ppm
for TZ-7 and a similar value was expected for TZ-6.

Molecules 2013, 18
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1
13
15
Table 1. Signal assignment for TZ-7, TZ-6 and TZ-5 in H- C- and N-NMR spectra,
(
in DMSO-d
6
solution).
TZ-7
TZ-6
TZ-5
1
3
15
13
15
13 15
C/ N
ppm)
C/ N
C/ N
1
1
1
Position
H (ppm)
Position
H (ppm)
Position
H (ppm)
(
(ppm)
134.21
not
(ppm)
111.67
−327.03
1
2
−
−
134.58
not
1
2
−
−
1
2
−
s, 2H, 6.33
obtained
19.59
obtained
19.42
3
4
5
6
7
8
9
−
3
4
−
−
−
−
−
4
−
−
−
−180.37
139.18
−199.86
128.36
−106.03
130.50
161.51
−274.71
36.13
−180.82
139.15
−200.03
128.22
−107.91
130.87
158.93
−246.00
36.14
q, 1H, 8.44
−293.78
150.68
−202.82
125.90
−125.92
142.74
163.84
−255.99
26.56
−
5
5
−
−
6
6
−
s, 1H, 8.81
7
s, 1H, 8.80
7
s, 1H, 7.61
−
8
−
8
−
−
9
−
9
−
10
11
12
−
10
11
12
13
14
−
10
11
12
13
14
−
s, 1H, 7.67; s, 1H, 7.79
s, 1H, 7.55
s, 3H, 3.87
−
s, 1H, 6.59
s, 3H, 2.79
−
s, 3H, 3.87
−
−
−
−
50.77
49.80
−
−
s, 9H, 1.42
28.49
s, 9H, 1.36
28.94
2
.1.2. IR and Raman Spectroscopy
The structures of all the compounds were further confirmed by IR and Raman spectroscopy. Three
−1
characteristic bands at: 1678, 730 and 710 cm [10] proved the presence of the form III in the TZ-7
−1
sample (Figure 1). A doublet at 3421 and 3388 cm is presumed to have come from the primary amide
−1
N-H stretching vibrations (γ). At 1758 and 1732 cm the first amide band (C=O (γ)) from the primary
−1
and tertiary amide is manifested and the band at 1678 cm can come from the second amide band
N-H bending vibrations (δ)) as well as from C=N (γ) and C=C (γ) vibrations. It is supposed that the
(
−1
most intensive Raman band at 1577 cm can come from the N=N (γ) vibration.
−1
The band at 3568 cm is visible only in the IR spectrum of TZ-6 (Figure 2). This band is not
visible in the Raman spectrum. It is supposed that this band can indicate the presence of a water
−1
molecule incorporated into the crystal structure of TZ-6. The band at 3424 cm can indicate the
−1
presence of the secondary amide N-H (γ) vibration. At 1758 and 1710 cm the first amide band from
−1
the secondary and tertiary amide is manifested and the band at 1648 cm can come from the second
−1
amide band as well as from C=N (γ) and C=C (γ) vibrations. It is supposed that the band at 1569 cm
represents the N=N (γ) vibration.

Molecules 2013, 18
15347
Figure 1. IR and Raman spectra of TZ-7.
Figure 2. IR and Raman spectra of TZ-6.
−1
Presumably, in TZ-5 (Figure 3) the bands from 3453 to 3330 cm manifest the presence of the
N-H (γ) vibration form the amide and amine groups as well. The most intensive band in the IR spectra,
−
1
−1
at 1721 cm indicates the C=O (γ) vibration. The bands at 1651, 1630 and 1601 cm can come from
the N-H () and C=N (γ) vibrations.

Molecules 2013, 18
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Figure 3. IR and Raman spectra of TZ-5.
2.1.3. X-Ray Cyrstallography Studies
The crystal structure of temozolomide has been previously reported [7,8]. TZ-5 crystallises in the
triclinic P-1 space group with two molecules in the asymmetric unit, whereas TZ-6 crystallises as a
hydrate with one molecule of water. The molecular structures of TZ-5 and TZ-6 are depicted with the
numbering scheme in Figure 4.
The differences in the corresponding structural parameters of the two molecules in the asymmetric
unit of the TZ-5 crystal structure are within the level of errors (less than 3σ). The only significant
difference between these molecules is the disorder of the side chain present in one of them. Therefore,
the numerical values of interatomic distances will be reported only for one molecule. The TZ-5
molecules are planar and form an intramolecular hydrogen bond between the amine group and the
carboxylic oxygen atom. The distances between the acceptor and donor of the hydrogen bond are equal
to: 2.91(1)Å and 2.90(1)Å for O(4)…N2 and O(3)…N2, respectively. The planarity of the ring and
amide bond is reserved in the TZ-6 molecules where the intramolecular hydrogen bonds are
impossible. However, TZ-6 crystallises with one molecule of water per molecule of TZ-6 and the
water forms four hydrogen bonds with the precursor (N11…O1S 2.903(1)Å, O1S…N8 2.887(1)Å,
O1S…N2x,y+1,z 3.067(1)Å, O1S…O1x,y+1,z 2.719(1)Å).
Both structures reveal a similar layered packing scheme with easily distinguished hydrophilic and
hydrophobic parts with the supremacy of hydrogen bonds and dispersive interaction, correspondingly
(
see Figure 5).
The packing of TZ-5 molecules is based on four hydrogen bonds, i.e., two for one molecule from
the asymmetric unit (N4…N8A-x,-y+1,-z 2.89(1)Å, N2…O5-x+1,-y+1,-z 2.91(1)Å). The hydrogen bonds
between the TZ-6 molecule and water are responsible for the formation of the layered structure with
alternating TZ-6 moieties and water molecules (see Figure 5). In the second direction, the molecules
are hold together by π…π stacking between the heteroaromatic rings. For both reported structures, the

Molecules 2013, 18
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interactions between layers are dispersive. These are mostly the H…H interactions between side chains
of molecules. The TZ-7 molecule [7,8] does not have a bulky tert-butyl substituent, and its crystal
structure exhibits a more effective packing. In this case, the hydrogen bonds govern the packing in the
crystal lattice in all directions.
Figure 4. Molecular structure and atomic labelling of TZ-5 (a) and TZ-6 (b). Thermal
displacement ellipsoids are drawn at the 50% probability level.
(
a)
(
b)

Molecules 2013, 18
15350
Figure 5. The view of crystal packing of TZ-5 along the y axis (a) and crystal packing of
TZ-6 along the x axis (b). The hydrogen bond is marked by a green, dashed line.
(
a)
.1.4. Thermoanalitycal Studies
Figure 6 shows the DSC curves of TZ-5, TZ-6 and TZ-7. The DSC curve of TZ-5 is characterized
(b)
2
by one exothermal signal from the substance decomposition, followed by an endothermic melting peak
at 148.96 °C.
Figure 6. DSC curves of TZ-5, TZ-6 and TZ-7.

Molecules 2013, 18
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The endothermic melting effect of TZ-6 is at 137.02 °C, in the range of decreasing baseline from
1
00 to 130 °C. A thermogravimetric analysis proved a mass loss of 6.70% in this range. This value
agrees well with water content in the sample and theoretical water content for a monohydrate (6.71%).
Endothermic effects visible in the temperature range from 160 to 180 °C come from the decomposition
of the substance. The DSC curve of temozolomide only exhibits the exothermic decomposition effect
at 206.35 °C. The mass loss value calculated from the thermogravimetric curve in the range of this
effect is above 50%.
3
3
3
. Experimental
.1. Synthesis
.1.1. Synthesis of 5-amino-N-(tert-butyl)-3-(methylcarbamoyl)imidazole-4-carboxamide (TZ-5) [11]
(
Scheme 1).
Scheme 1. The snthesis of TZ-5.
The mixture of the compounds TZ-3 [12] (96.0 g, 0.5 mol) and TZ-4 [13]: (95.0 g, 0.6 mol) was
diluted with dichloromethane (1.0 L), then acetic acid (100%, 5 mL) was added. The whole mixture
was stirred at ambient temperature for 1 h, refluxed for an 1 h and then cooled to ambient temperature.
The mixture was neutralized with aqueous potassium carbonate (pH 4–5), the organic layer was
separated and the aqueous one extracted with dichloromethane (2.0 L). The combined extracts were
evaporated in vacuo to approx. 20% of their volume, the precipitate collected and washed with
dichloromethane (75 mL). The filtrate was evaporated to approx. 50% of volume and refrigerated at
−
10 °C for 16 h. The precipitate was collected and washed with dichloromethane (25 mL). The
combined precipitates were dried in vacuo at 30 °C to afford 87 g (73%) of the compound TZ-5 in the
form of yellow crystals. Elementary analysis: 50.07% (C), 7.18% (H), 29.06% (N). ESI-MS: calcd. for
+
(
M+H) : 239.14, found 239.14.
3
.1.2. Synthesis of N-(tert-butyl)-3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d]-1,2,3,5-tetrazine-8-
carboxamide (TZ-6) [11] (Scheme 2).
The compound TZ-5 (50.0 g, 0.21 mol) was added at 10 °C to the stirred solution of lithium
chloride hydrate (378 g) and acetic acid (24 mL) in water (1 L). The mixture was stirred for 30 min
and cooled to 4 °C, then sodium nitrite (22 g, 0.32 mol) was added in one portion. The whole mixture
was stirred for 1 h at 4–5 °C and for next 18 h at ambient temperature, then diluted with

Molecules 2013, 18
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dichloromethane (1.0 L) and stirred vigorously for 10 min. The organic layer was separated and the
aqueous one extracted twice with 500 mL and 250 mL portions of dichloromethane.
Scheme 2. The synthesis of TZ-6.
The combined extracts were then washed with 10% aqueous sodium dithionite (1.0 L) and
evaporated to dryness in vacuo at temperature not exceeding 25 °C. The crude product was suspended
in the mixture of 2-propanol–water–36% hydrochloric acid (1,000 mL–225 mL–25 mL) and refluxed
for 20 min. After filtration the solution was left at ambient temperature for 24 hrs, the precipitate
collected, washed with 80% aqueous 2-propanol (125 mL) and dried in vacuo at ambient temperature
to afford the compound TZ-6 of HPLC 99.9% + purity in the yield 28.5 g (54%). Elementary analysis:
+
4
4.77% (C), 6.02% (H), 31.23% (N). ESI-MS: calcd. for (M+H) : 250.12, found 250.12.
3
.1.3. Synthesis of 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide (TZ-7) [11]
(
Scheme 3).
Scheme 3. The synthesis of TZ-7.
The compound TZ-6 (25,0 g, 0,10 mol) was added portionwise to stirred sulfuric acid (d 1.8, 70 mL)
at 15–25 °C. The whole mixture was stirred at ambient temperature for 3 h and then poured into
6% ethanol (800 mL) at 0 °C. The suspension was stirred for 30 min. at 0 °C, the precipitate
9
collected, washed with cold ethanol (50 mL) and dried on air for 16 h. The obtained crude TZ-7 was
dissolved in the refluxed mixture of acetone (450 mL), water (150 mL) and acetic acid (38 mL), the
solution was filtered, the filtrate refluxed again, cooled to ambient temperature and stirred for 4 h.
The precipitate was then collected, washed with acetone/water 3:1 (80 mL) and dried in vacuo for 16 h
at 25 °C to afford 10.1 g (52%) of pharmaceutically pure TZ-7. Elementary analysis: 36.99% (C),
+
3
.00% (H), 43.02% (N). ESI-MS: calcd. for (M+H) : 194.06, found 194.06.

Molecules 2013, 18
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3
.2. Techniques
3.2.1. X-Ray Crystallography
The deposition numbers CCDC 806024 (TZ-5) and CCDC 805454 (TZ-6) contain the
supplementary crystallographic data. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.htmL (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk).
The data of TZ-5 and TZ-6 were collected using the Bruker KAPPA APEXII ULTRA
diffractometer controlled by the APEXII software [14], equipped with the MoKα rotating anode X-ray
source (λ = 0.71073 Å, 50.0 kV, 22.0 mA) monochromatized by multi-layer optics and APEX-II CCD
detector. The data collection was carried out at 100 K using the Oxford Cryostream cooling device.
Indexing, integration and initial scaling were performed with the SAINT [15] and SADABS [16]
software (Bruker AXS, Madison, WI, USA, 2008). The structures were solved by direct methods using
the SHELXS-97 [17] program and refined with the SHELXL-97 [15]. A multi-scan absorption
correction has been applied in the scaling procedure. Crystal structure parameters are reported in
Table 2 for the corresponding structures.
Table 2. Crystal structure parameters for TZ-5 and TZ-6.
Crystal Data
TZ-5
TZ-6
Chemical formula
C
10
H
17
N
5
O
2
C
10
H
14
N
6
O
2
2
·H O
M
r
239.29
268.29
Crystal system, space group
Temperature (K)
a, b, c (Å)
Triclinic, P-1
Triclinic, P-1
100
100
10.4130 (11), 11.425 (2), 12.2354 (12) 6.5042 (3), 7.5629 (4), 13.1645 (7)
α, β, γ, (°)
99.703 (7), 114.649 (5), 102.470 (7)
78.729 (3), 85.280 (3), 87.848 (3)
3
V (Å )
1,235.7 (3)
4
632.77 (6)
2
Z
3
.2.2. Nuclear Magnetic Resonance (NMR)
1
13
The H- and C-NMR Heteronuclear Single Quantum Coherence (g-HSQC) and Heteronuclear
Multiple Bond Correlation (g-HMBC) (1H-13C i 1H-15N) spectra were acquired on the Varian vnmrs
1
6
00 spectrometer at 599.94 MHz transmitter frequency for H at the Institute of Organic Chemistry
Polish Academy of Science in Warsaw. The spectra were measured in the DMSO-d
6
solution at room
1
temperature. The chemical shift standards were as follows: liquid TMS (0.00 ppm for H-NMR),
1
3
15
residual signal of DMSO (39.50 ppm for C-NMR) and liquid nitromethane (0.00 ppm for N-NMR).
.2.3. Infrared Spectroscopy (IR)
The infrared spectra were obtained in the range from 4,000 to 400 cm with the spectral resolution
3
−1
−1
of 4 cm using a Nicolet iS10 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA) and
KBr pellets.

Molecules 2013, 18
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3
.2.4. Raman Spectroscopy
FT Raman spectra were recorded on a Nicolet NXR 9650 instrument (Thermo Scientific), using
−1
1
,064 nm excitation from Nd:YVO laser, in the range from 3,700 to 150 cm with a spectral
4
−1
resolution of 8 cm .
3
.2.5. Thermal Analysis (DSC, TGA)
Thermal analyses were carried out on a DSC 822 differential scanning calorimeter equipped with
IntraCooler and on a TGA/SDTA 851cells thermogravimeter (Mettler Toledo GmbH, Schwerzenbach,
Switzerland) under nitrogen atmosphere. Accurately weighed samples (4–7 mg) were packed in
aluminum pans with pierced lids. TGA measurements were blank curve corrected.
3.2.6. Water Content
Water content determination was done by Karl Fischer volumetric titration using the Methrohm 701
KF Titrino apparatus (Methrohm AG, Herisau, Switzerland).
3
.2.7. High-Performance Liquid Chromatography (HPLC)
The purity of temozolomide produced in PRI according to an HPLC method [18] is not less than
9.50%. It has been proved experimentally, in a small-plant scale, that in order to obtain such high
9
purity of TZ-7 in a desired polymorph form III [10], the purity of the intermediate TZ-6 and the
starting material TZ-5 should not be less than 99.7% and 97.5%, respectively. Lower purity of TZ-6
leads to a higher level of impurities in TZ-7 which are difficult to remove during crystallisation. The
purification of TZ-7 leads to its degradation to 5-amino-4-imidazolecarboxamide (AIC) or to the
partial transformation, mainly into polymorph IX [10].
The determination of the chemical purity of TZ-7, TZ-6 and TZ-5 was carried out using Waters
Alliance 2695e HPLC systems (Waters, Milford, MA, USA) equipped with gradient pumps, column
heaters, autosamplers and either a UV-Vis 2487 or a PDA 2998 detector. The separations during the
determination of TZ-7 purity were performed using Aqua C18, 250 × 4.6 mm, 5 µm column
(Phenomenex, Torrance, CA, USA). Temozolomide-related substances were separated using water
with 0.5% acetic acid and acetonitrile as Eluent A and Eluent B, respectively. The following gradient
elution program was applied, t (min)/%B: 0/8; 8/8; 28/40; 31/8; 36/8. Other chromatographic
parameters were as follows: column temperature: 25 °C; autosampler temperature: 5 ± 2 °C; flow rate:
−
1
1
0
1
5
mL min ; injection volume: 20 µL; UV detection wavelength: λ = 254 nm; diluent: water with
.5% HAc:ACN (4:1, v/v); the concentration of temozolomide in the investigated sample solutions:
−
1
mg mL . The chemical purity of TZ-6 was determined with the use of a XTerra C18, 250 × 4.6 mm,
µm column (Waters). 0.1% H PO and methanol were used as Eluent A and Eluent B, respectively.
3
4
The following gradient elution program was applied, t (min)/%B: 0/5; 20/50; 30/50; 32/5; 40/5. Other
chromatographic parameters were as follows: column temperature: 25 °C; autosampler temperature:
−1
1
5 °C; flow rate: 1 mL min ; injection volume: 20 µL; UV detection wavelength: λ
1
= 254 nm,
λ
2
= 201 nm; diluent: water with 0.1% H PO -MeOH (9:1, v/v); the concentration of TZ-6 in the
3
4
−1
investigated sample solutions: 0.5 mg mL . The chemical purity of TZ-5 was investigated using Aqua

Molecules 2013, 18
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C18, 250 × 4.6 mm, 5 µm column. The separations were carried out using 0.1% H PO and methanol
3
4
as Eluent A and Eluent B, respectively. The following gradient elution program was applied,
t (min)/%B: 0/0; 4/0; 15/50; 30/50; 32/0; 40/0. Other chromatographic parameters were as follows:
−1
column temperature: 25 °C; autosampler temperature: 5 ± 2 °C; flow rate: 1 mL min ; injection
volume: 10 µL; UV detection wavelength: λ = 254 nm, λ = 201 nm; diluent: ACN; the concentration
of TZ-5 in the investigated sample solutions: 0.5 mg mL .
1
2
−1
4
. Conclusions
The structures of TZ-5 and TZ-6 were confirmed by X-ray diffraction and spectroscopic methods.
The compounds were compared to temozolomide in the structural aspect. The thermal characteristics
of the compounds were also presented. TZ-5 and TZ-6 were successfully obtained on a validated
small-plant scale. The process is well documented as sets of operational instructions, batch reports and
validation protocols. Two methods for the purity determination of both compounds were developed.
The high purity of TZ-6 is crucial for obtaining temozolomide in polymorph form III with purity
higher than 99.50%.
Acknowledgments
The support from European and Regional Funds under the project UDA-POIG.01.03.01-14-069/08-00
grant is gratefully acknowledged.
Conflicts of Interest
The authors declare no conflict of interest.
References
1
2
.
.
U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug
Evaluation and Research. ANDAs: Impurities in Drug Substances; Silver Spring, MD, USA, 2009.
The International Conference on Harmonisation of Technical Requirements for Registration of
Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline: Impurities in New Drug
Substances Q3A (R2); London, UK, 2006.
3
4
.
.
Newlands, E.S.; Stevens, M.F.G.; Wedge, S.R.; Wheelhouse, R.T.; Brock, C. Temozolomide:
A review of its discovery, chemical properties, pre-clinical development and clinical trials.
Cancer Treat. Rev. 1997, 23, 35–61.
Darkes, M.J.M.; Plosker, G.L.; Jarvis, B. Temozolomide a review of its use in the treatment of
malignant gliomas, malignant melanoma and other advanced cancers. Am. J. Cancer 2002, 1,
5
5–80.
5
.
.
Stupp, R.; Gander, M.; Leyvraz, S.; Newlands, E. Current and future developments in the use of
temozolomide for the treatment of brain tumours. Lancet Oncol. 2001, 2, 552–560.
®
6
FDA Approval for Temozolomide. Product information. TEMODAR (temozolomide)
CAPSULES. Available online: http://www.cancer.gov/cancertopics/druginfo/fda-temozolomide
(
accessed on 15 March 2005).

Molecules 2013, 18
15356
7
.
Lowe, P.R.; Sansom, C.E.; Schwalbe, C.H.; Stevens, M.F.G.; Clark, A.S. Antitumor
Imidazotetrazines. Crystal structure of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-
(3H)-one (Temozolomide) and ctructural comparisons with the related drugs mitozolomide and
4
DTIC. J. Med. Chem. 1992, 35, 3377–3382.
8
.
.
Babu, N.J.; Reddy, L.S.; Aitipamula, S.; Nangia, A. Polymorphs and polymorphic cocrystals of
temozolomide. Chem. Asian J. 2008, 3, 1122–1133.
9
Wheelhouse, R.T.; Wilman, D.E.V.; Thomson, W.; Stevens, M.F.G. Antitumour imidazotetrazines.
1
13
15
Part 31. The synthesis of isotopically labelled temozolomide and a multinuclear ( H, C, N)
magnetic resonance investigation of temozolomide and mitozolomide. J. Chem. Soc. Perkin Trans.
1
995, 1, 249–252.
0. Adin, I.; Iustain, C. Novel Crystalline Forms of Temozolomide. Patent WO 2005/063757,
0 December 2004.
1. Kuo, S.-C.; Mas, J.L.; Hou, D. Synthesis of Temozolomide and Analogs. Patent WO
002/057269 (EP 135392 A1), 16 January 2002.
1
1
1
3
2
2. Cichy, B.; Gabarski, K.; Lipner, G.; Kaczmarek, L.; Rasinska, M. An Improved Process for the
Preparation of 2-amino-N-tert-butyl-2-cyanoamide Hydrochloride. Polish Patent Application No.
P-395 425 A1, 24 June 2011.
1
3. Whitehead, C.W. The reactions of orthoesters with ureas. A new synthesis of pyrimidines. J. Am.
Chem. Soc. 1953, 75, 671–675.
1
1
1
4. APEXII-2008, v 1.0; Bruker Nonius: Madison, WI, USA, 2007.
5. SAINT V7.34A; Bruker Nonius: Madison, WI, USA, 2007.
6. SADABS-2008/1, Bruker AXS area detector scaling and absorption correction; Bruker AXS:
Madison, WI, USA, 2008.
1
7. Sheldrick, G.M. A short history of SHELX. Acta Cryst. 2008, A64, 112–122.
8. Jedynak, Ł.; Puchalska, M.; Zezula, M.; Łaszcz, M.; Łuniewski, W.; Zagrodzka, J. Stability of
sample solution as a crucial point during HPLC determination of chemical purity of temozolomide
drug substance. J. Pharmaceut. Biomed. Anal. 2013, 83, 19–27.
1
Sample Availability: Samples of the compounds TZ-5 and TZ-6 are available from the authors.
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