Process-Related Impurities of Pazopanib

Article abstract of DOI:10.1021/acs.oprd.9b00117

The main target of this study was to synthesize reference compounds for HPLC analysis of impurities, produced in Pazopanib synthesis. It is highly desirable that impurity standards are made available to help in developing an HPLC control method that could be suitable for use in the industrial process. To the best of our knowledge, most of these impurities are not commercially available and no synthetic methods have been reported. Potential impurities were synthesized, and their structures were established by various spectroscopy techniques.

Full text of DOI:10.1021/acs.oprd.9b00117

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Communication
Process-related Impurities of Pazopanib
Svetlana Terentjeva, Dzintra Muceniece, and Viesturs Lusis
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00117 • Publication Date (Web): 18 Jul 2019
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Svetlana Terentjeva, Dzintra Muceniece and Viesturs Lūsis*
Latvian Institute of Organic Synthesis, Riga, LV 1006, Latvia
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ABSTRACT
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The main target of this study was to synthesize reference compounds for HPLC analysis of
impurities, produced in Pazopanib synthesis. It is highly desirable that impurity standards are made
available to help in developing HPLC control method that could be suitable for the use in industrial
process.
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To the best of our knowledge, most of these impurities are not commercially available and no
synthetic methods have been reported. Potential impurities were synthesized and their structure
established by various spectroscopy techniques.
KEYWORDS Pazopanib, regioisomers, alkylation, 6-nitro-1H-indazole, indazol-6-ylamines,
amination of 2,4-dichloropyrimidine, process impurities.
INTRODUCTION
Pazopanib (developed by GlaxoSmithKline and marketed by Novartis as Votrient) is a multi–
tyrosine kinase inhibitor that is used for treatment of metastatic renal cell carcinoma. Active
pharmaceutical ingredient (API) is 5-[[4-[2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-
pyrimidinyl]amino]-2-methylbenzene sulfonamide monohydrochloride. There are many
synthetic protocols reported in the prior art literature for the preparation of API drug substance1-7
However, many of the synthetic routes are found to have primarily academic significance rather
than commercial value as could be evaluated after literature studies.
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Development of synthetic chemical process can lead to generation of unwanted organic
impurities. These usually include unreacted starting materials (SMs), impurities originating from
the SMs, unreacted intermediates (IMs), reaction side and degradation products. Thus, the
detection, identification, quantification, and control of such impurities originating in the
manufacturing process have become an important element of drug development in order to
ensure product quality and, ultimately, patient safety.8-13 Impurities should not only be measured
in the final API, but proactively controlled throughout the manufacturing process as well,
because the process understanding provides evidence that impurities originating in SMs or
previous synthesis steps can be carried to the next stage or transformed by the process into new
impurities.
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RESULTS AND DISCUSSIONS
Our investigation has been aimed to identify the structure of process-related impurities in the
API route starting the synthesis with 3-methyl-6-nitro-1H-indazole. A schematic representation
of the synthetic route is given in Scheme 1:
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Scheme 1. Preparation of pazopanib
Literature reports, predominantly disclosing the synthesis of drug substance pazopanib in patents,
do not report synthetic details, type and characteristics of impurities. Mass spectrometry is most
6,8
commonly used for characterizing impurities,
but without additional methods it alone is
incapable of distinguishing between regioisomers and identifying structures; therefore reference
compounds are needed.
Potential impurities as reference compounds are not easily accessible. There are two main
reasons. Firstly, content of the impurities is relatively low; secondly, their chemical properties
often are very similar to those of target compounds and therefore their separation is very
difficult. In addition, it has to be pointed out that side products closely related to process IM are
involved in next stages along the main products, thus generating secondary impurities.
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Based on the process pictured in Scheme 1 2-5, we carried out impurity tracking from 3-methyl-6-
nitro-1H-indazole, through IMs and finally to API of pazopanib step-by-step.
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Step 1: Methylation of 3-methyl-6-nitro-1H-indazole
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The alkylation of N-H indazoles generally yielded a mixture of N-alkyl 1H-indazole and N-alkyl
2H-indazole. The regioselectivity of indazoles is highly dependent on the nature of the methylating
agents; mixtures of methylated products are often formed due to a lack of regioselectivity.
High yields of 2-alkyl substituted 2H-indazoles were reportedly achieved using trimethyloxonium
tetrafluoroborate or triethyloxonium hexafluorophosphate as alkylating agents.3,14 However,
alkylation of 3-methyl-6-nitro-2H-indazole (1) with trimethyloxonium tetrafluoroborate in acetone
6
gave 2,3-dimethyl indazole 2 in 32% yield after purification. Besides, the reagent is rapidly
destroyed by atmospheric moisture and is unsuitable for use on large scale.
Highly regioselective methylation of indazole 1 with either Meerwein’s salt, trimethyl
2,15
orthoformate or dimethyl sulfate (DMS) is reported.
However, moderate yield (63%) of 2,3-
dimethyl indazole 2 attained with trimethyl orthoformate in the presence of sulfuric acid, is in
contradiction with the assertion that methylation is regioselective.
N
N
N
N
+
+
N
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N
O2N
O2N
O2N
O2N
H
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3
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Scheme 2. Methylation of 3-methyl-6-nitro-1H-indazole
Regioselective methylation at the 2-position of 3-methyl-6-nitro-1H-indazole by refluxing with
dimethyl carbonate (DMC) in the presence of triethylenediamine (DABCO) affording 2,3-
dimethyl indazole 2 in 81% yield is described.16 In our hands the repeated experiment resulted in
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mixture of 1,3- and 2,3-dimethyl indazoles 2 and 3 (Scheme 2), the same result has been reported
in another case17 when 1,3-dimethyl indazole 3 was isolated in 37% yield.
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For further studies 1,3-dimethyl-6-nitro-1H-indazole (3) as impurity reference compound was
isolated from mixture of 1,3- and 2,3-dimethyl indazoles formed by alkylation with DMS in
DMF in the presence of NaH at 120 °C (indazoles 2 and 3 were formed in 33 and 61% yield,
respectively).
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It should be noted that, except for indazole 3, which was isolated from reaction mixture, all other
impurity standards are synthesized by non-optimized alternative methods.
The structure of 1,3-dimethyl indazole 3 is proven by NOESY spectrum: it shows the spin
interaction between protons of 1-CH3 (4.10 ppm) and 7-H (8.30 ppm) of the indazole ring; as
expected, such interaction is not observed in the spectrum of isomer 2.
Alkylation of indazole 1 in acidic medium carried out according to patent 4 protocol gave 1,2,3-
trimethyl indazolium salt 4 along with the major reaction product dimethyl indazole 2 and the
minor one 3. The indazolium salt was prepared and isolated in form of perchlorate 4·ClO4 as
reference compound for analysis of alkylation reaction mixtures.
Fortunately, the indazolium derivative produced upon alkylation of 3-methyl indazole remains
entirely in the aqueous phase after reaction workup, thus eliminating this impurity completely.
In our hands, the highest yield of pazopanib synthesis intermediate 2,3-dimethyl indazole 2
(76%) and low byproduct level (0.25-0.50 area-% in HPLC; here and everywhere else absolute
area percentages are used) was attained in methylation with DMS in toluene at 70 ºC. Although
SM 1 is usually completely consumed, control of its remainder is important as any remaining
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amount of 1 may serve as a source of secondary impurities in API. Occurrence of such impurities
which would originate from indazoles 1 and 3, is discussed separately.
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Step 2: Hydrogenation of 6-nitroindazoles
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Following the synthesis scheme, 6-nitroindazole 2 (and indazole 3 for preparation of the
corresponding downstream impurities) was reduced to corresponding indazolamine 5 by
hydrogenation (Pd/C, methanol, ambient temperature) 16 and could be isolated as HCl salt (yield
89%, assay 97+%) upon treatment with HCl/AcOEt, or used in the next step as a base after
partial removal of the solvent (preferable amine concentration 1g/4-5 mL).
Structure of 1,3-dimethyl-1H-indazol-6-ylamine (base, prepared by catalytic hydrogenation) is
confirmed by single-crystal X-ray study 17 without reporting other characteristics. The substance
was characterized only by MS-ESI when it was prepared by reduction with hydrazine hydrate.18
We have isolated 1,3-dimethyl-1H-indazol-6-ylamine (20) as hydrochloride in 81% yield after
hydrogenation of 6-nitroindazole 3 (H2, Pd/C, methanol).
Step 3: Amination of 2,4-dichloropyrimidine
The next step in the manufacturing process of pazopanib includes reacting the key starting
material 2,3-dimethyl-6-amino-2H-indazole (5) with 2,4-dichloropyrimidine (6) in the presence
of base to get the target intermediate 7.
During the preparation of IM 7, the two chlorine atoms of 2,4-dichloropyrimidine displayed
different reactivity towards 6-aminoindazole 5 in the presence of base. The 4 position is more
prone to be attacked by nucleophile reagents under basic conditions. However, the 2 position can
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also be susceptible to attack by nucleophile reagents under the same conditions, producing minor
product 8 (we detected compound 8 at 3.0- 4.6% level in the reaction mixture by HPLC). This
impurity was expected to form due to the competitive amination at 2-Cl position versus 4-Cl
position of dichloropyrimidine 6.
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Both pyrimidine halogens reacting with indazole produce the dimer 9 (detected in average
0.15%). Formation of compound 10 is caused by reaction of 7 with methanol, which is used as
solvent. UPLC/MS analysis revealed 0.11 or 1.48% of compound 10 when performing reaction
at ambient temperature or upon heating to 40 °C, respectively.
Condensation of 2,3-dimethyl-6-aminoindazole (5) with 6 in the presence of sodium hydrogen
carbonate in methanol at room temperature resulted in formation of the intermediate 7, its
regioisomer 8 as well as minor side products 9 and 10 (Scheme 3). Starting materials 5 and 6
were completely consumed, their residual peaks were under 0.1% and 0.05% respectively
(HPLC, method B).
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Scheme 3. Reaction of 2,3-dimethyl-6-amino-2H-indazole (5) with 2,4-dichloropyrimidine
Often the emergence of mentioned impurities is not reported at all 19 or their characteristics are
not given. 6,7,20 Well-known impurity 8, the isomer of target intermediate 7, usually is isolated
from enriched mother liquor in quantity depending on solvent used for 7 synthesis: yield can be
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as high as 6.8%, when reaction is performed in butanol under reflux;6 it was 1.6% in the case of
ethanol.12 We have obtained compound 8 via independent route (Scheme 4) starting with 2-
methylsulfanyl-3H-pyrimidin-4-one without demonstrating whether the reaction intermediate is
pyrimidone or pyrimidinol (two tautomeric forms, i.e. keto-N1H and keto-N3H in exact ratio 1:1
are specific for isocytosine 21).
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POCl3
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Scheme 4. Synthesis of impurity 8
Occurrence of dimer 9 in the manufacturing process of pazopanib is often mentioned 6,7,20 and
the implemented analytical control methods are elaborated.11 Synthesis of dimer 9 by reaction of
amine 5 with dichloropyrimidine 6 is reported but the substance is characterized only by a mass
spectrum of MS-ESI.6 We obtained dimer 9 by interaction of 7 and amine 5 in the presence of
K2CO3 in propanol under reflux.
Step 4: Methylation of N-(2-chloropyrimidin-4-yl)-(2,3-dimethyl-2H-indazol-6-yl)-amine
Intermediate 7 was further methylated under mild conditions providing N-(2-chloropyrimidin-4-
yl)-N,2,3-trimethyl-2H-indazol-6-amine (11) (Scheme 5). The methylation could be realized
using methyl iodide or dimethyl carbonate.5,12,19 Our methylation experiments accomplished with
dimethyl carbonate in the presence of K2CO3 either in DMF, DMAA or DMSO afforded methyl
derivative 11 in 80-86% yield.
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Scheme 5. Methylation of intermediate 7 and impurities 8- 10
Under these conditions alkylation of other compounds (8-10) present in the reaction mixture
from previous step also take place to give corresponding methyl derivatives 12-16 (Scheme 5).
Moreover, if the conversion of secondary amine 7 into 11 is not complete, it may contaminate
the IM prepared in this step and yield process impurity 18 in the next step (Scheme 6, see below)
The impurities were revealed in crude IM 11 batches by HPLC analysis in following amounts
(method B): 7 <0.30%, 8 about 0.5%, 12 0.42–0.65%, 13 0.05–0.16%, 15 0.47–0.67 (as well as
23 0.06–0.12%, Scheme 7).
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N-(4-Chloropyrimidin-2-yl)-2,3-dimethyl-2H-indazol-6-amine (8), as pyrimidine 7 regioisomer,
is the major impurity of the previous step. Methylation of contaminant 8 affords N-(4-chloro-
pyrimidin-2-yl)-N-methyl indazolamine 12. The level of impurity 12 reached 0.42–0.65% in our
experiments. It is essential to remove this impurity before using the intermediate 11 in the next
step, otherwise, it generates secondary impurity 17 (Scheme 6), which is difficult to remove.
HPLC analysis of crude API obtained without elimination of contaminant 12 revealed impurity
17 present in amounts averaging 0.25%. Crystallization of the final product only slightly reduced
the level of contaminant (0.19%).
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Crystallization of crude methylation product should be recommended, considering that
purification of intermediates is more beneficial than that of the final product.
Crystallization of crude N-(2-chloropyrimidin-4-yl)-N-methylindazolamine 11 from ethanol
reduces the amount of contaminant 12 to <0.1% at recovery ~80%. The same assay can be
ensured by crystallization from methanol or aqueous methanol (80% v/v), however the recovery
is only 56–68% in this case. This crystallization also reduced amount of contaminant 15 by half
(<0.25% in crystallised samples versus 0.65% - the highest amount of impurity 15 ever found in
crude 11) and eliminated other impurities almost completely.
With respect to alkylation of compound 10, two supposed isomeric impurities of same molecular
mass peak m/z 284 were synthesized: methylation with iodomethane afforded N-methyl-
pyrimidone 16, while dimethyl carbonate gave 15 only with traces of 16. HPLC analysis of crude
and purified samples for API base showed content of impurity 15 as 0.11 and <0.01 area percent
correspondingly. Pyrimidone 16 as an impurity was never found.
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Methylation of bis-indazolylpyrimidinamine 9 with dimethyl carbonate in the presence of K2CO3
afforded N4-methyl derivative 13 selectively, whereas treatment of 9 with NaH followed by
interaction with methyl iodide gave N2,N4-dimethyl derivative 14. An alternative path to
compound 14 is reaction of indazolamine 5 with 2-chloropyrimidine 11 followed by methylation
of formed 13. 12 The presence of this side product was never detected in analyzed samples.
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Location of the N-methyl group in 13 was proven by ROESY NMR showing spin interaction
between NCH3 (3.61 ppm) and pyrimidine 5-H (5.82 ppm) as well as protons of N4-indazole
moiety 5-H (6.89 ppm), 7-H (7.48 ppm). It is interesting that N4-CH3 interacts also with 5-H of
the indazolyl group at N2 atom. This along with spin ineraction between N2-H and N2-inazolyl 7-
H protons indicates to s-cis disposition of the pyrimidine 3-N atom and N2-indazolyl group as
drawn in the Scheme 5.
Step 5: Amination of intermediates with 5-amino-2-methylbenzenesulfonamide
N-(2-Chloropyrimidin-4-yl)-N,2,3-trimethyl-2H-indazol-6-ylamine (11) and 5-amino-2-methyl-
benzenesulfonamide (AMBS) refluxed in methanol in the presence of HCl afford the target API
(Scheme 1). Related pyrimidines 12 and 7 can also undergo similar nucleophilic substitution
with AMBS, producing process impurities 17 and 18 (Scheme 6), respectively.
As it was already mentioned, the crucial requirement ensuring final API quality is purification of
intermediate 11 by complete removal of contaminant 12. If this material is present, the difficult
to separate impurity 17 – regioisomer of pazopanib – is formed in this step.
Intermediate 7, considered as impurity caused by incomplete conversion of the starting material in
the previous step, is capable of forming compound 18. If the reaction is started with unpurified 11,
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the obtained crude IM contains impurity 18 in 0.24% amount; the crystallisation of final API
reduces this amount to 0.04%.
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Usage of crystallized IM 11 avoids the occurrence of impurities 17 and 18 in the final API. This
is also essential with regard to the side products 13 and 15 which are transferred to the final
product in only slightly reduced amounts. However this is less important as API crystallization
removes them efficiently. Only traces (<0.03%) of residual AMBS was detected in crude API.
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AMBS
N
N
N
N
N
N
N
N
Cl
Cl
N
N
N
H
H2NO2S
N
N
N
H
H
7
18
N
AMBS
N
N
N
N
H2NO2S
N
N
N
H
12
17
N
N
N
AMBS
SO2
N
N
N
N
N
N
Cl
N
N
H
11
NH2
19
Scheme 6. Amination of intermediates with AMBS
In formation of API base and impurities 17 and 18, NH2 group of AMBS aniline moiety is
engaged in nucleophilic substitution with chlorine. However, aforementioned substitution with
participation of SO2NH2 group can not be excluded. Such interaction would lead to compound
19 (Scheme 6).
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5-Amino-N-[4-[(2,3-dimethylindazol-6-yl)-methylamino]pyrimidin-2-yl]-2-methylbenzene
sulfonamide (19) as reference compound was prepared by heating 2-chloropyrimidine derivative
11 and AMBS in the presence of K2CO3 in DMF at 130°C.
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Both API base and compound 19 show the same m/z [M+1]+ value, but their PMR spectra differ
significantly and were used for structure assignments. Presence of the NH2 group specific to
aniline is demonstrated by ROESY spectrum of compound 19: interaction between 5-NH2 (5.09
ppm, 2H) and aromatic 4-H (6.56 ppm) and 6-H (7.36 ppm) protons was observed; such
interaction is impossible if the molecule contains SO2NH2 group as is the case with API.
Compound 19 was not detected as an impurity in the final product.
Impurities originating from 1,3-dimethylindazole 3
The impurities resulting from 1,3-dimethyl indazole 3, which is formed in the first synthesis step,
may also be expected.
Starting with isomer 3 and following API synthesis route outlined in Scheme 1, 2-chloro-
pyrimidin-4-yl)-(1,3-dimethyl-1H-indazol-6-yl) amine (21) was obtained. Then, the
corresponding reference compounds 22 and 25 were prepared considering the necessity to
identify potential impurities of final product (m/z values in MS are identical for derivatives
originating from 1,3- and 2,3-dimethyl indazoles 2 and 3) (Scheme 7).
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3
4
5
6
7
8
9
N
6
AMBS
N
N
N
N
N
N
N
Cl
N
N
H2N
H2NO2S
N
N
N
H
H
H
20
21
22
Me2CO3
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ref.22
N
N
N
AMBS
N
N
N
N
N
N
N
Cl
N
N
H
Cl
N
H2NO2S
N
N
N
H
H
24
23
25
Scheme 7. Impurities originating from 1,3-dimethylindazole
Alternative route to intermediate 23 by alkylation of N-(2-chloro-4-pyrimidinyl)-N-(3-methyl-
1H-indazol-6-yl)amine (24) is described.22 Occurrence of mono methyl derivative 24 also could
be supposed if the conversion of starting material 1 by methylation with DMS is incomplete
(Scheme 2).
HPLC analysis of crude 11 revealed the presence of impurities 21 and 23 (0.02 and 0.06-
0.12%). Using intermediate of such quality in the API synthesis final step, the side products 22
and 25 were detected at 0.01% and 0.02- 0.05% level correspondingly. Purification of the
intermediate 11 removes the precursors and excludes the occurrence of impurities.
Several analytical HPLC methods have been developed for detection of impurities. The most
universal method suitable for simultaneous detection of multiple impurities is Method D,
employing column Allure Biphenyl. Corresponding chromatogram of mixture of impurities is
shown in Figure 1. The only impurities that cannot be distinguished individually due to peak
overlapping are 8 and 22. This drawback is an insignificant one, as the presence of impurity 8
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can be controlled using Apollo C18 column (method B and C, Fig.2 and Fig.3). These methods
are essential for assessing the quality of intermediate 11. Besides the impurities shown in Figure
2, this method is also suitable for detection of 5 and 6 (their rrt relative to 11 0.39 and 0.82). A
shortcoming of this method is poor separation of peaks corresponding to 6 and 14. However,
occurrence of 14 in synthesis of the API is considered highly unlikely (we did not detect this
contaminant in any of our experiments), and its absence can be verified by method C (Fig.3) or
D (Fig.1).
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HPLC methods used to analyze intermediates and retention times of impurities reference
standards are collected in Table 1.
Figure 1. Sample HPLC-plot of Pazopanib impurities reference standards at c0.01mg/mL.
HPLC method D, injection volume 10 L.
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Figure 2. Sample HPLC-plot of IM11 impurities reference standards (plot a, 7- 15, 23) at
c0.01mg/mL, HPLC method B, injection volume 10 L. Insertion b) shows separation of
amines 5 and 20 under these conditions.
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Figure 3. Sample HPLC-plot of IM 11 impurities reference standards 5- 15, 23 at c0.01mg/mL.
HPLC method C, injection volume 10 L.
Table 1. HPLC data of reference standards, methods and retention times
r.t. (min)
r.t. (min)
Compound
Compound
a
Method B Method C Method D
Method B Method C Method D
4.1
8.7b
8.3
4.4
10.6
10.3
11.9
8.6
6.5
5
6
16
17
20.1
11.9
17.7c
12.3
7.0
13.8
7
18
9.1
8.7
8
19
7.0
4.7
9
20
6.1
7.1
28.0
10
11
12
13
21
10.5
11.5
7.8
15.5
18.3
14.0
26.5
32.6
23.0
11.9
19.5
41.4
30.1
2.9
23
25
AMBS
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8.6b
6.7
12.4
8.0
38.8
13.0
24.4
14
15
API
6
7
8
9
a compounds 1- 3: method A, retention times 1 4.5 min, 2 4.9 min, 3 7.3 min
b insufficient resolution of peaks of 6 and 14 c overlapped by 22
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CONCLUSION
Impurities present in Pazopanib drug substance occurring from route synthesis and regioisomers
are identified and characterized. To make the impurities in question available as reference
compounds and precursors for the subsequent transformations, a facile route for their synthesis is
proposed. For the process development of active pharmaceutical ingredient, it is also of
importance to know structurally related substances. In this regard, several possible impurities are
proposed, synthesized and characterized.
EXPERIMENTAL SECTION
General information: Solvents and reagents were purchased from commercial suppliers and
used as supplied. Compounds 5,7 and 11 were synthesized according to published procedures.
3,5,16 Warning! Several synthetic descriptions require use of sodium hydride in DMF – this
combination is suitable for small-scale synthesis only, and may be hazardous if scaled up. 23
Flash chromatography was performed with the indicated solvents on SiO2 60 (35−70 m; 15−40
m for compounds 13 and 14). Yields reported are for isolated, NMR pure compounds.
Melting points were recorded (uncorrected) on a Kofler block. Elemental analysis was done on a
Carlo Erba EA 1108 analyzer. HRMS were obtained on an Agilent UPLC (6230 TOF LC/MS)
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instrument with an Acquity UPLC liquid chromato-mass spectrometer of the (Waters)-Q-TOF
(Micromass) system with an Acquity UPLC BEH C18 chromatographic column (1.7 μm, 2.1×50
mm), gradient elution MeOH–HCOOH (0.1%) in water, ESI ionization.
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NMR spectra were recorded on Bruker AMX-400 spectrometer. Chemical shifts in NMR
analyses were referenced to the residual 1H solvent signals (CDCl3, δ 7.27; DMSO–d6, δ 2.50)
and solvent 13C signals (CDCl3, δ 77.0; DMSO–d6, δ 39.5). Chemical shifts are reported in parts
per million (δ), coupling constants (J-values) are reported in hertz (Hz).
Reaction monitoring and product purity were analyzed by reverse phase HPLC on a Waters
Alliance 2695 liquid chromatograph with a Waters 2489 UV/Vis detector, an Apollo C18 column
(5 μm, 4.6×150 mm) and gradient elution with 0.1% H3PO4 water solution/ acetonitrile or an
Allure Biphenyl column (5 μm, 4.6×150 mm) and gradient elution with 0.1% TFA water
solution/ acetonitrile.
Method A.
Column: Apollo C18 (150x4.6 mm, 5 ); mobile phase A: 0.1% aqueous H3PO4,
B: acetonitrile; elution: isocratic A/B 55/45 for 4min followed by linear gradient from 55% A to
5%A over 5min, flow rate 1.0 mL/min; oven 40 ºC; detector UV 210 nm and 254 nm.
Method B.
Apollo C18 column (150x4.6 mm, 5 ); mobile phase A: 0.1% H3PO4, B:
acetonitrile, gradient program A/B 95:5 to 5:95 (0 to 15 min), composition A/B 5:95 held for 5
min; flow rate 1.0 mL/min; oven 40 ºC; detector UV 210 nm.
Method C.
Apollo C18 column (150x4.6 mm, 5 ); mobile phase A: 0.1% H3PO4, B:
acetonitrile, gradient program given below, flow rate 1.0 mL/min; oven 40 ºC; detector UV 254
nm.
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Time, min
% A
% B
Time, min
% A
% B
5
6
7
8
0
6
95
70
45
5
28
33
35
5
5
95
95
5
30
65
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26
95
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Method D.
Column: Allure BiPh (150x4.6 mm, 5 ); mobile phase A: 0.1%TFA, B:
acetonitrile, gradient program given below, flow rate 1.0 mL/min; oven 40 ºC; detector UV 254
nm.
Time, min
% A
% B
Time, min
% A
% B
0
1
85
85
78
75
70
15
15
22
25
30
42
52
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35
50
95
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2
12
32
85
85
Procedures and Characterizations
1,3-Dimethyl-6-nitro-1H-indazole (3) and 2,3-dimethyl-6-nitro-2H-indazole (2).
Sodium hydride (0.27 g, 60%, 1.2 eq.) was added portionwise to solution of 3-methyl-6-
nitroindazole 1 (1.00 g, 5.6 mmol) in DMF (5 mL) over 15 minutes. After the mixture was
stirred at room temperature for 0.5 h dimethyl carbonate (2.4 mL, 5 eq) was added and stirring
was continued at 120 °C for 5 h. Reaction mixture was poured into ice, the resulting solid was
filtered off and dried to obtain mixture of isomers 2 and 3 (1.10 g). The isomers were resolved by
chromatography (hexane/EtOAc 1:1). 1,3-Dimethyl-6-nitro-1H-indazole (3) as a yellow solid,
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(0.67 g, 61%) was obtained from the first fraction, the second fraction afforded indazole 2 (0.36
g, 33%). All 2,3-dimethyl-6-nitro-2H-indazole properties correspond to described 2,16 ones.
7
8
9
1,3-Dimethyl-6-nitro-1H-indazole (3); mp 151–152 °C (MeOH).
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1H NMR (300 MHz, CDCl3): δ 2.61 (s, 3H, 3-CH3), 4.10 (s, 3H, 1-CH3), 7.72 (d, 1H, J = 8.9 Hz,
4-H), 7.97 (dd, 1H, J = 8.9; 1.9 Hz, 5-H), 8.30 (d, 1H, J = 1.9 Hz, 7-H).
13C NMR (100 MHz, CDCl3): δ 11.9 (3-CH3), 35.8 (1-CH3), 105.7 (7-C), 114.5 (5-C), 121.3 (3a-
C), 126.5 (4-C), 139.6 (7a-C), 142.1 (3-C), 146.7 (6-C).
MS (ES+) m/z: 192 [M+H]+ (100%).
Anal. Calcd for C9H9N3O2: C, 56.54; H, 4.74; N, 21.98. Found: C, 56.35; H, 4.66; N, 21.89.
HPLC: method A, 1 tR 4.5 min, 2 tR 4.9 min, 3 tR 7.3 min.
1,2,3-Trimethyl-6-nitro-1H-indazol-2-ium perchlorate (4∙ClO4).
To a stirred suspension of indazole 1 (0.3 g, 1.7 mmol) in toluene (10 mL) DMS (0.8 mL, 5 eq)
was added. The reaction mass was refluxed for 48 h. Toluene was evaporated and aqueous NaClO4
solution (0.7 g in 2 mL water) was added to the oily residue. Precipitate formed was separated and
crystallized from methanol. Yield of perchlorate 4∙ClO4 was 86% (0.45 g); mp 208–209 °C dec
1H NMR (300 MHz, DMSO-d6): δ 2.92 (s, 3H, 3-CH3), 4.30 (s, 3H, 2-CH3), 4.37 (s, 3H, 1-CH3),
8.20 (dd, 1H, J = 9.1; 1.9 Hz, 5-H), 8.44 (dd, 1H, J = 9.1; 0.5 Hz, 4-H), 9.08 (dd, 1H, J = 1.9; 0.5
Hz, 7-H).
13C NMR (100 MHz, DMSO-d6): δ 10.7 (3-CH3), 34.2 (1-CH3), 35.4 (2-CH3), 108.5 (7-C), 117.8
(5-C), 121.7 (3a-C), 124.6 (4-C), 138.1 (7a-C), 143.3 (3-C), 150.3 (6-C).
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MS (ES+) m/z: 206 [M+H]+ (100%).
5
6
7
8
9
Anal. Calcd for C10H12N3O2·ClO4: C, 39.29; H, 3.96; N 13.75. Found: C, 39.41; H, 3.94; N,
13.63.
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(4-Chloropyrimidin-2-yl)-(2,3-dimethyl-2H-indazol-6-yl)amine (8).
Step A 2-Methylsulfanyl-1H-pyrimidin-6-one (3.3 g, 23.2 mmol) and indazolylamine 5
hydrochloride (5.00 g, 1.1 eq) in AcOH (10 mL) were refluxed with stirring for 17 h. The
reaction mixture was evaporated to dryness. The residue was triturated with metanol (50 mL)
upon stirring for 15 min. The white solid was filtered and dried to obtain 2-[(2,3-
dimethylindazol-6-yl)amino] pyrimidin-4-ol (1.0 g, 17%); mp >250 °C (MeOH).
1H-NMR (300MHz, DMSO- d6), δ: indazolyl 2.56 (s, 3H, 3-CH3), 3.99 (s, 3H, 2-CH3), 6.92 (d,
1H, J = 8.8 Hz, 5-H), 7.54 (d, 1H, J = 8.8 Hz, 4-H), 8.01 (s, 1H, 7-H); pyrimidone 5.81 (d, 1H, J
= 6.1 Hz, 5-H), 7.79 (d, 1H, J = 6.1 Hz, 6-H), 8.80 (s, 1H) and 10.73 (bs, 1H) NH.
Step B The pyrimidine obtained in the previous step (1.0 g. 3.9 mmol) in POCl3 (10 mL) was
stirred at 100 °C for 2 h. The reaction mixture was poured into mixture of ice and NaHCO3 (36
g). When the ice melted the mixture was extracted with hot ethyl acetate (3×100 mL). The
extract was dried and evaporated. The residue was purified by chromatography (EtOAc/i-PrOH
20:1). The second fraction afforded compound 8 (0.9 g, 84%); mp 206–208 °C.
1H-NMR (400MHz, DMSO), δ: pyrimidinyl 6.96 (d, 1H, J = 5.2 Hz, 5-H), 8.46 (d, 1H, J = 5.2
Hz, 4-H); indazole 2.55 (s, 3H, 3-CH3), 3.98 (s, 3H, 2-CH3), 7.12 (dd, 1H, J = 9.0; 1.9, 5-H),
7.54 (dd, 1H, J = 9.0; 0.6 Hz, 4-H), 8.08 (dd, 1H, J = 1.9; 0.6 Hz, 7-H), 9.99 (s, 1H, NH).
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13C-NMR (100MHz, DMSO), δ: pyrimidinyl 111.3 (5-C), 159.8, 159.9, 160.0 (6-C); indazole
9.4 (3-CH3), 37.0 (2-CH3), 103.4 (7-C), 115.8 (5-C), 117.2 (3a-C), 120.0 (4-C), 131.3 (3-C),
136.8 (6-C), 147.2 (7a-C).
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HRMS (ESI+) m/z: [M+H]+ calcd for C13H13ClN5 [M+1]+ 274.0859; found 274.0863.
HPLC: method B, tR 9.1 min (7 tR 8.3 min); method C, tR 11.9 min (7 tR 10.3 min); method D, tR
17.7 min (7 tR 11.9 min).
N2,N4-Bis-(2,3-dimethyl-2H-indazol-6-yl)-pyrimidine-2,4-diamine (9).
Indazole 5 hydrochloride (0.7 g, 3.5 mmol), compound 7 (1.0 g, 3.6 mmol) and K2CO3 (1.5 g, 3
eq) in propanol (20 mL) were refluxed overnight to obtain dimer 9 (1.3 g, 87%); mp >260°C (i-
PrOH).
1H NMR (300 MHz, DMSO-d6): δ pyrimidine 6.26 (d, 1H, J = 5.7 Hz, 5-H), 8.04 (d, 1H, J = 5.7
Hz, 6-H), 9.13 (s, 1H, 2-NH), 9.27 (s, 1H, 4-NH); N2-indazolyl 2.55 (s, 3H, 3-CH3), 3.97 (s, 3H,
2-CH3), 7.27 (dd, 1H, J = 9.0; 1.3 Hz, 5-H), 7.49 (d, 1H, J = 9.0 Hz, 4-H), 8.11 (d, 1H, J = 1.3
Hz, 7-H); N4-indazolyl 2.57 (s, 3H, 3-CH3), 4.00 (s, 3H, 2-CH3), 7.12 (dd, 1H, J = 9.0; 1.3 Hz, 5-
H), 7.57 (d, 1H, J = 9.0 Hz, 4-H), 8.08 (bs, 1H, 7-H).
13C NMR (100 MHz, DMSO- d6): δ pyrimidine 98.8 (5-C), 155.6 (6-C), 159.8 (2-C), 160.7 (4-
C); N2-indazolyl 9.4 (3-CH3), 36.9 (2-CH3), 102.5 (7-C), 116.4 (5-C), 116.8 (3a-C), 119.4 (4-C),
130.9 (3-C), 138.3 (6-C), 147.6 (7a-C); N4-indazolyl: 9.4 (3-CH3), 37.0 (2-CH3), 104.4 (7-C),
116.4 (5-C), 117.3 (3a-C), 120.1 (4-C), 131.3 (3-C), 137.3 (6-C), 147.4 (7a-C).
HRMS (ESI+) m/z: [M+1]+ calcd for C22H23N8 399.2046; found, 399.2049.
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MS (ES+) m/z: 399 [M+1]+ (40%), 200 (100%), (fragmentation pattern is in agreement with
5
6
ref.8).
7
8
9
HPLC: method B, tR 7.0 min; method C, tR 8.6 min; method D, tR 12.3 min.
(2,3-Dimethyl-2H-indazol-6-yl)-(2-methoxypyrimidin-4-yl)-amine (10).
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To a solution of compound 7 (2.0 g, 7.3 mmol) in aqueous methanol (60 ml, water-MeOH 1:5
v/v) K2CO3 (3.0 g, 3 eq) was added. The mixture was refluxed for 42 h to obtain
methoxypyrimidine 10 as a white solid (1.6 g, 85%); mp > 250 °C (n-PrOH).
1H NMR (400 MHz, DMSO-d6): δ pyrimidinyl 3.87 (s, 3H, OCH3), 6.45 (d, 1H, J = 5.7 Hz, 5-
H), 8.06 (d, 1H, J = 5.7 Hz, 6-H); indazolyl 2.56 (s, 3H, 3-CH3), 3.99 (s, 3H, 2-CH3), 7.02 (dd,
1H, J = 8.8; 1.5 Hz, 5-H), 7.57 (d, 1H, J = 8.8 Hz, 4-H), 8.09 (bs, 1H, 7-H), 9.58 (s, 1H, NH).
13C NMR (100 MHz, DMSO-d6): δ pyrimidinyl 54.0 (OCH3), 101.4 (C-5), 156.5 (C-6), 161.5
(C-2), 164.9 (C-4); indazolyl 9.4 (3-CH3), 37.0 (2-CH3), 104.4 (C-7), 115.9 (C-3a), 117.3 (C-5),
120.3 (C-4), 131.5 (C-3), 136.9 (C-6), 147.2 (C-7a).
HRMS (ESI+) m/z: [M+1]+ calcd for C14H16N5O, 270.1355; found, 270.1357.
MS (ES+) m/z: 270 [M+1]+ (100%).
HPLC: method B, tR 6.1 min; method C, tR 7.1 min; method D, tR 7.0 min.
N-(4-Chloropyrimidin-2-yl)-N,2,3-trimethyl-2H-indazol-6-amine (12).
To a solution of compound 8 (0.9 g, 3.3 mmol) in THF (50mL) sodium hydride (0.3 g, 60%, 2.4
eq) was added portionwise. The mixture was stirred at room temperature for 1 h, then methyl
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iodide (2 mL, 10 eq) was added and stirring continued for 18 h. The reaction was quenched by
adding brine (30 mL) and extracted with DCM (3x30 mL). The extract was dried and
evaporated. Oily residue was chromatographed (EtOAc). The third fraction afforded
methylamine derivative 12 (0.3 g, yield 33%); mp 151-153 °C (i-PrOH).
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1H NMR (400 MHz, CDCl3): δ pyrimidinyl 6.58 (d, 1H, J = 5.1 Hz, 5-H), 8.14 (d, 1H, J = 5.1
Hz, 6-H); indazol 3.57 (s, 3H, N-CH3), 2.59 (s, 3H, 3-CH3), 4.08 (s, 3H, 2-CH3), 6.96 (dd, 1H, J
= 8.8; 1.6 Hz, 5-H), 7.47 (d, 1H, J = 1.6 Hz, 7-H), 7.55 (d, 1H, J = 8.8 Hz, 4-H).
13C-NMR (100MHz, CDCl3), δ, ppm: pyrimidinyl 110.2 (C-5), 158.9 (C-6), 161.1 (C-4), 162.1
(C-2); indazole 10.0 (3-CH3), 37.5 (2-CH3), 39.3 (N-CH3), 113.6 (C-7), 119.7 (C-3a), 120.4 (C-
4), 121.0 (C-5), 131.8 (C-3), 143.2 (C-6), 148.0 (C-7a).
HRMS (ESI+) m/z: [M+1]+ calcd for C14H15ClN5, 288.1016; found, 288.1016.
MS (ES+) m/z: 288 [M+H]+ (100%), 290 (30%).
HPLC: method B, tR 11.5 min (11 tR 10.5 min); method C, tR 18.3 min (11 tR 15.5 min); method
D, tR 32.6 min (11 tR 26.5 min).
N2,N4-Bis-(2,3-dimethyl-2H-indazol-6-yl)-N4-methylpyrimidine-2,4-diamine (13).
To a solution of diamine 9 (0.56 g, 1.4 mmol) in DMAA (3 mL) K2CO3 (0.58 g, 3 eq) and
dimethyl carbonate (1.2 mL, 10 eq) were added sequentially. The mixture was heated at 130°C
for 24 h, then cooled and diluted with water. Isolated crude product was purified by column
chromatography (EtOAc/MeOH, 2:1) to isolate amine 13 as a white solid (0.5 g, 83%); mp 244–
246 °C.
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1H NMR (400 MHz, CDCl3): δ pyrimidine 3.61 (s, 3H, N4-CH3), 5.82 (d, 1H, J = 6.0 Hz, 5-H),
7.80 (d, 1H, J = 6.0 Hz, 6-H); N2-indazolyl 2.55 (s, 3H, 3-CH3), 4.04 (s, 3H, 2-CH3), 7.05 (dd,
1H, J = 8.8; 1.7 Hz, 5-H), 7.24 (s, 1H, NH), 7.43 (dd, 1H, J = 8.8; 0.6 Hz, 4-H), 8.20 (dd, 1H, J
=1.7; 0.6 Hz, 7-H); N4-indazolyl 2.62 (s, 3H, 3-CH3), 4.10 (s, 3H, 2-CH3), 6.89 (dd, 1H, J = 8.8;
1.7 Hz, 5-H), 7.48 (dd, 1H, J = 1.7, 0.6 Hz, 7-H), 7.60 (dd, 1H, J = 8.8, 0.6 Hz, 4-H).
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13C NMR (100 MHz, CDCl3): δ pyrimidine 38.6 (N4-CH3), 97.3 (C-5), 155.5 (C-6), 159.9 (C-2),
163.2 (C-4); N2-indazolyl 10.0 (3-CH3), 37.2 (2-CH3), 103.3 (C-7), 116.9 (C-5), 117.6 (C-3a),
119.7 (C-4), 131.4 (C-3), 138.3 (C-6), 148.7 (C-7a); N4-indazolyl: 10.1 (3-CH3), 37.6 (2-CH3),
114.7 (C-7), 120.6 (C-5), 120.0 (C-3a), 121.4 (C-4), 132.0 (C-3), 142.8 (C-6), 148.0 (C-7a).
HRMS (ESI+) m/z: [M+H]+ calcd for C23H25N8, 413.2202; found, 413.2207.
MS (ES+) m/z: 413 [M+H]+ (8%), 227 (42%), 207 (100%).
HPLC: method B, tR 7.8 min; method C, tR 14.0 min; method D, tR 23.0 min.
N2,N4-Bis-(2,3-dimethyl-2H-indazol-6-yl)- N2,N4 -dimethylpyrimidine-2,4-diamine(14).
Suspension of diamine 9 (0.33 g, 0.83 mmol) and NaH (0.16 g, 5 eq) in THF (30 mL) was stirred
at room temperature for 1 h. Methyl iodide (0.3 mL, 6 eq) was added, and the stirring was
continued for 18 h. The reaction was quenched by adding brine (10 mL) and DCM (10 mL).
Layers were separated; the aqueous one was extracted with DCM (2x10 mL). Organic solution
was dried and evaporated; the residue was purified by chromatography (EtOAc/MeOH, 2:1) and
crystallization to obtain N2,N4-dimethylpyrimidine-2,4-diamine 14, 0.1 g, (28%); mp 220 °C
(MeOH). Synthesis of diamine 14 hydrochloride is described. 12
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1H-NMR (300MHz, CDCl3), δ: pyrimidine 3.37 (s, 3H, N4-CH3), 3.61 (s, 3H, N2-CH3), 5.73 (d,
1H, J = 6.0 Hz, 5-H), 7.77 (d, 1H, J = 6.0, 6-H); N2-indazolyl: 2.57 (s, 3H, 3-CH3), 4.05 (s, 3H,
2-CH3), 7.12 (dd, 1H, J = 8.9; 1.8, 5-H), 7.47 (dd, 1H, J = 8.9; 0.7, 4-H), 7.48 (dd, 1H, J = 1.8,
0.7, 7-H); N4-indazolyl 2.59 (s, 3H, 3-CH3), 4.08 (s, 3H, 2-CH3), 6.86 (dd, 1H, J = 8.9; 1.8, 5-H),
7.44 (dd, 1H, J = 1.8; 0.7, 7-H), 7.55 (dd, 1H, J = 8.9; 0.7, 4-H).
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13C-NMR (100MHz, CDCl3), δ: pyrimidine 37.6 (N4-CH3), 38.7 (N2-CH3), 95.98 (C-5), 155.6
(C-6), 161.8 (C-2), 162.5 (C-4); N2-indazolyl 10.0 (3-CH3), 37.3 (2-CH3), 112.3 (C-7), 119.0 (C-
4), 119.1 (C-3a), 122.3 (C-5), 131.4 (C-3), 144.8 (C-6), 148.3 (C-7a); N4-indazolyl 10.0 (3-CH3),
37.5 (2-CH3), 114.4 (C-7), 119.8 (C-3a), 121.1 (C-4), 120.7 (C-5), 131.9 (C-3), 143.0 (C-6),
148.1 (C-7a).
HRMS (ESI+) m/z: [M+H]+ calcd for C24H27N8, 427.2359; found, 427.2371.
MS (ES+) m/z: 427 [M+H]+ (100%), 235 (13%), 214 (52%), 207 (13%).
HPLC: method B, tR.8.6 min; method C, tR 12.4 min; method D, tR 38.8 min.
(2,3-Dimethyl-2H-indazol-6-yl)-(2-methoxypyrimidin-4-yl)methylamine (15).
Methoxypyrimidine 10 (0.47 g, 1.75 mmol), dimethyl carbonate (1.5 mL, 10 eq), and K2CO3
(0.90 g, 6.5 mmol) in DMAA (3 mL) were stirred at 130 C ºfor 13 h. The reaction mixture was
diluted with water (40 mL) and extracted with DCM (3x25 mL). The organic extract was washed
with brine (25 mL) and dried. The extract was evaporated; the residue was purified by
chromatography (DCM/i-PrOH 20:1). From the second fraction alkyl derivative 15 was isolated,
0.26 g (52%); mp 155–157 °C (i-PrOH).
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