An Alternative Method for Synthesizing N,2,3-Trimethyl-2H-indazol-6-amine as a Key Component in the Preparation of Pazopanib

Abstract

Due to its application as an anti-cancer drug, pazopanib (1) has attracted the interest of many researchers, and several studies on pazopanib synthesis have been reported over the years. This paper provides a novel route for synthesizing N,2,3-trimethyl-2H-indazol-6-amine (5), which is a crucial building block in the synthesis of pazopanib from 3-methyl-6-nitro-1H-indazole (6). By alternating between the reduction and two methylation steps, compound 5 was obtained in a yield comparable (55%) to what has been reported (54%). It is noteworthy that the last step of N²-methylation also yielded N,N,2,3-tetramethyl-2H-indazol-6-amine (5′) as a novel compound. Furthermore, the data presented in this paper can serve as a valuable resource for future research aimed at further refining the process of synthesizing pazopanib and its derivatives.

1. Introduction

Pazopanib hydrochloride, marketed under the brand name Votrient^TM, is an oral anti-cancer drug that inhibits the activity of various kinase enzymes. It was approved by the United States Food and Drug Administration (FDA) for the treatment of soft tissue sarcoma progression and renal cell carcinoma in 2009 [1] and metastatic pretreated uterine leiomyosarcoma in 2012 [2]. It has also been trialled in combination with durvalumab, an anti-PD-L1 inhibitor, in the treatment of metastatic and/or recurrent soft tissue sarcoma [3]. Notably, in renal cell carcinoma, pazopanib demonstrated the ability to increase the response rates and progression-free survival of patients who had or had not received cytokine pretreatment [4,5]. The drug works by blocking vascular endothelial growth factor receptors (VEGFR-1, -2, and-3) and also platelet endothelial growth factor receptors (PDGFR-α and -β)m which leads to the inhibition of angiogenesis and subsequent tumour growth [6].

The pazopanib molecule can be divided into three parts, featuring a sulfonamide, a pyrimidine, and an indazole component (Figure 1). These fragments can be separately synthesized and modified before being combined to offer the target compound. In particular, the indazole component bears structural similarities to the adenine component of ATP, which enables the formation of hydrogen bonds within the tyrosine–kinase receptor’s binding pockets. As a result, this prevents ATP from binding to the receptor and results in the inactivation of ATP-related intracellular reactions [4,7]. Given its crucial role in expressing the biological activity of not only pazopanib but also more potent derivatives [8], the indazole fragment has received considerable attention, with its synthesis having been extensively studied [9,10,11,12,13,14].

The synthesis of pazopanib can start from either indazole derivative 4 or 5 (Scheme 1) [9,10,11,12]. While compound 4 has the inherent instability of a free aniline and usually requires conversion to a stable HCl salt form, compound 5 is a better candidate as it is not only more stable but also does not entail an extra N-methylation in the aniline group like compound 4 does (Scheme 1).

Theoretically, there are two possible methods for synthesizing compound 5 from compound 6. The first one involves a selective N² methylation on the indazole ring, followed by the reduction of the nitro group; this is completed by an Eschweiler–Clarke methylation at the amine group (Scheme 2). This has been reported by Mei Y. C. et al., who cleverly combined the last two steps into one synthetic sequence to afford compound 5 with an overall yield of around 54% [10]. In this synthetic route, while the combined nitro reduction and methylation yielded 86%, the N² methylation’s yield was limited at 63%, which was comparable to previously reported data [9]. This was due to the intrinsic tautomerism of the indazole ring, in which the methylating reagent can generate either an N¹ or N² methylation product [10,12].

The second route is our proposal, which has not been reported elsewhere. We propose that the nitro reduction should be completed before the methylations at the –NH₂ and N² positions, respectively (Scheme 2). The products at each step were purified and characterized by IR, NMR, MS, TLC and melting points, which can serve as a handy database for research on synthesising pazopanib derivatives.

2. Results and Discussion

The target compound, 5, was prepared in three separate steps from 3-methyl-6-nitro-1H-indazole (6), with an overall yield of 55%. The reaction conditions were modified from the related references [9,11] (Scheme 3).

2.1. Process for 3-Methyl-1H-indazol-6-amine (8)

The first step was the reduction of the C6-nitro group by tin chloride in concentrated HCl. Due to its low cost and simple handling, ethyl acetate (EA) was used as the sole solvent for both the reaction and extraction instead of a combination of 2-methoxyethyl ether and diethyl ether, as previously reported [9]. In the acidic condition, the freshly formed aniline group was readily protonated to result in a stable and water soluble HCl salt. Adjusting the pH to 9 by a saturated Na₂CO₃ solution neutralized the reaction acidity and returned the free aniline that was extracted in the EA layer, leaving other by-products (SnO, Sn(OH)₂, and NaCl) in the aqueous layer. To obtain the pure product without column chromatography, the extracted product in EA was further reacted with HCl solution to get the product back into the aqueous HCl solution, which was then separated from EA. Adjusting the pH of the resulting aqueous solution to a basic range facilitated the precipitation of a pure product in aniline form, which was filtered off and dried as a yellow crystalline solid (87%). In this nitro reduction step by SnCl₂/HCl (conc), the molar ratio between SnCl₂ and compound 6 was determined to be beneficial at 4:1 due to the low chemical consumption and comparable efficiency when compared with the other ratios examined (

Table 1. The comparable yields of compound 8 generated by various molar ratios between SnCl₂ and compound 6.

Entry	Ratio (SnCl2:Compound 6)	Time (h)	Product Mass (g)	Yield (%)
1	3:1	8	1.35	81
2	4:1	3	1.45	87
3	5:1	3	1.37	83
4	6:1	3	1.39	84

). Similar nitro reduction has been reported with a yield up to 92% [9]; however, the product was isolated as a HCl salt, not a free aniline product as observed in our experiment.

2.2. Process for N,3-Dimethyl-1H-indazol-6-amine (9) via Reductive Amination

The second step was a reductive amination between the aniline functional group (compound 8) and formaldehyde, which resulted in compound 9 yielding at 87%. This reaction was performed in a basic condition due to the use of a weak organic or inorganic base as the catalyst (

Table 2. Organic and inorganic bases used as catalysts in the synthesis of compound 9.

Entry	Base Catalyst	Time (h)	Product Mass (g)	Yield (%)
1	CH3ONa	6	1.90	87
2	t-BuOK	6	1.74	79
3	K2CO3	10	1.43	65
4	Na2CO3	10	1.46	67

). We again adopted the purification method used in the synthesis of compound 8. We first protonated the product into a HCl salt, which was selectively extracted into aqueous solution to leave other organic matter in the EA layer. This aqueous product solution was then separated, and the pH was adjusted to 9 (by Na₂CO₃) to neutralize the HCl salt facilitating the precipitation of the pure secondary amine product. This purification technique required no column chromatography, as previously reported, and significantly enhanced the yield of compound 9 synthesized, from 39% [11] to 87%.

In this synthesis of compound 9, we trialed several base catalysts and the results are presented in Table 2. Replacing inorganic bases such as K₂CO₃ and Na₂CO₃ with an organic base such as potassium tert-butoxide (t-BuOK) and CH₃ONa resulted in better efficiency and a shorter reaction time (Table 2). While both organic bases (t-BuOK and CH₃ONa) enabled comparable yields (79% vs. 87%, Table 2), CH₃ONa stood out in terms of cost when scaling up as it can freshly be made from Na and methanol in our lab. Additionally, we explored the optimal molar ratio between compound 8 and NaBH₄. The shortest reaction time and highest efficiency were achieved with a molar ratio of 1:4 between compound 8 and the reducing agent NaBH₄ (

Table 3. The impact of the molar ratio between compound 8 and NaBH₄ on product yield.

Entry	Molar Ratio of Compound 8:NaBH4	Time (h)	Yield (%)
1	1:2	14	76
2	1:3	9	79
3	1:4	6	87
4	1:5	6	87

).

2.3. Process for N,2,3-Trimethyl-2H-indazol-6-amine (5)

The synthesis of target compound 5 from 9 has never been reported before. It involved a selective methylation at the N² position on compound 9. A closely related reaction was reported for compound 6 using trimethyl orthoformate (TMOF) as the methylating reagent [10]. This reaction generated a mixture of N¹- and N²-methylated products due to the tautomerism of the NH group in the indazole ring, which was typical for the alkylation of an indazole derivative [13,14]. It was found that in the presence of concentrated sulfuric acid, this reaction favoured the N²-methylated product [10]. However, in our experiment, we found that beside the target N²-methylated product, 5, a new by-product, 5′, was formed depending on the equivalence of the TMOF used, whilst no N¹-methylated product (5″) was isolated (Scheme 4). Compound 5′, having not been reported in the literature, was found to contain a tertiary amine functional group attached on C6 of the indazole ring. This indicated that the methylating reagent also attacked the secondary amine of compound 9. This could be explained by the potent alkylating ability of TMOF [15], which readily attacked both the NH in the indazole and the secondary amine (Scheme 5). Although the possibility of generating multiple methylated products limited the yield of the target product 5 to 73%, this was more efficient than the similar N-methylation of a simpler compound previously reported (compound 7, 63%, [10]). Both compounds 5 and 5′ displayed very close Rf values (R_f~0.30 in n-hexane/ethyl acetate, 3:7, and 0.70 in dichloromethane/methanol, 9:1), which prompted us to use our in-house preparative chromatography to purify them before characterization (yield: 28% for 5, and 21% for 5′). In this N-methylation reaction, using concentrated H₂SO₄ as the sole catalyst was important, as changing it to either polyphosphoric acid or a mixture of p-toluenesulfonic acid (PTSA) and concentrated H₂SO₄ resulted in unknown products whose Rf values were different from those of the target compound 5. Moreover, choosing the right ratio between TMOF and the starting material 9 was crucial. Using the ratio (~6.2:1) reported for a similar methylation reaction [10] led to more new spots, including the aforementioned compound 5′, while reducing it to less than (4:1) returned unreactive starting material. As such, we utilized the ratio of (4:1), which enabled both the completion of the reaction and a cleaner TLC, which prioritised the formation of target compound 5 at 73% yield. From compound 5, we have successfully synthesized pazopanib hydrochloride with a purity of 99.0–101.0% (Figure S24, Supplementary Materials) by using a recently patented novel approach (data unpublished).

We also trialled CH₃I as an alternative methylating reagent accompanied by various base catalysts (CH₃ONa, t-BuOK or K₂CO₃). However, their reaction mixtures displayed more unknown new spots on TLC when compared with that of TMOF, while the starting material (9) was still detectable. As a result, TMOF was chosen as the methylating reagent for our synthesis.

A probable mechanism for the N-methylation reaction using TMOF is suggested in Scheme 5. In acid media, a TMOF molecule was protonated into a methoxonium intermediate that was converted into a carbocation (carbenium ion) through the cleavage of a methanol molecule. The carbocation and its resonance structure, the oxonium ion, both possess an electrophilic H₃C^δ+ center (Scheme 5). This center then received a nucleophilic attack from the lone pair of electrons located at the N² position of the indazole ring to afford the target compound 5, at the same time releasing a methanol and a methyl formate molecule as good leaving groups. When using a large amount of TMOF vs. starting material 9 (6.2:1), this methylation process was continued on the newly formed compound 5 whose nucleophilic center was the lone pair of electrons located on the secondary amine NH- connected to C6-indazole to afford a novel compound, 5′ [12,16].

3. Materials and Methods

3.1. General Information

3-Methyl-6-nitro-1H-indazole (compound 6) was synthesized in the lab. Trimethyl orthoformate (98.0%) was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Potassium carbonate (K₂CO₃) (99.0%) and paraformaldehyde (≥95.0%) were purchased from Guangdong Guanghua Sci-Tech Co., Ltd. (Shantou, China). Ethyl acetate (99.5%) was purchased from Samchun Chemical Co., Ltd. (Pyeongtaek-si, Gyeonggi-do, Republic of Korea). Sodium borohydride (NaBH₄) (98.0%), n-hexane (≥98.0%), methanol (MeOH) (99.5%), isopropanol (IPA) (99.7%), anhydrous sodium sulfate, toluene (99.5%), tin(II) chloride (SnCl₂·2H₂O) 98.0% and N,N-dimethylformamide (DMF) (99.5%), all of which met analytical reagent (AR) purity standards, were obtained from Xilong Scientific Co., Ltd. (Shantou, China). All chemicals were used without further purification.

The melting point (M.p) was measured by using the capillary tube method with an SRS EZ-Melt apparatus (Stanford Research Systems, Sunnyvale, CA, USA). The mass spectra of compounds were recorded on an LC-MSD Trap SL machine, ionized by the ESI (electrospray ionization) method. The FT-IR spectrum was recorded by a Shimadzu spectrometer (Kyoto, Japan). Nuclear magnetic resonance (¹H, ¹³C, COSY, HSQC, HMBC, NOESY) experiments were measured on a Bruker Ascend spectrometer (Billerica, MA, USA) at 500 MHz for protons and 125 MHz for carbon-13 using CDCl₃ as the solvent and tetramethylsilane (TMS) as an internal standard. The reaction mixtures were monitored, and the purity of the compounds was checked by thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany).

3.2. Synthetic Procedure

3.2.1. 3-Methyl-1H-indazol-6-amine (8)

In a 100 mL round-bottom flask, 3-methyl-6-nitro-1H-indazole (6) (2.00 g, 11.3 mmol, 1 eq) was dispersed into ethyl acetate (10 mL) at 0 °C; then, tin(II) chloride dihydrate (10.19 g, 45.2 mmol, 4 eq) was slowly added. This was followed by the dropwise addition of concentrated HCl (1 mL) over 1 min, keeping the reaction temperature below 10 °C with an ice bath. The ice bath was taken out when the HCl addition was finished, and the mixture was stirred for three more hours. Once the reaction was complete (as shown by TLC), 50 milliliters of ethyl acetate was added and then the reaction mixture was cooled to 5 °C. The reaction mixture was then neutralized by saturated Na₂CO₃ solution to a pH of 9. The organic phase was separated, and two more fractions of ethyl acetate (2 × 50 mL) were sequentially added to carry out the extraction two more times. Following that, the organic phase was combined and rinsed three times with distilled water (3 × 20 mL). The organic phase was then transferred to a conical flask, in which 10 mL of a 20% hydrochloric acid solution was gradually added, and the mixture was shaken to facilitate the formation of HCl salt, which was soluble in the aqueous phase. The latter was separated and cooled to 5 °C and the saturated Na₂CO₃ solution was added in small portions under stirring to pH = 9, which enabled the formation of a precipitate. The precipitate was collected by vacuum filtration, washed three times with distilled water (3 × 5 mL), and dried to obtain 3-methyl-1H-indazol-6-amine (8) as a yellow–brown solid (1.45 g, 87% yield). M.p: 203.8–204.5 °C. TLC: R_f = 0.45 (n-hexane/ethyl acetate, 3:7). MS (ESI_, MeOH), m/z: Calculated for C₈H₉N₃ [M + H]⁺: 148.08, found: 147.9. FT-IR (KBr), v_max (cm⁻¹): 3382, 3180 (N-H); 1640 (C=N); 1520 (C=C); ¹H-NMR (500 MHz, CDCl₃), δ (ppm): 9.44 (s, 1H, N-NH); 7.43 (d, J = 8.5 Hz, 1H, H-4); 6.57–6.55 (m, 2H, H-5, H-7); 3.84 (s, 2H, NH₂); 2.50 (s, 3H, H-1′). ¹³C-NMR (125 MHz, CDCl₃), δ (ppm): 146.1 (C-6); 143.5; (C-7a); 142.9 (C-3); 121.0 (C-4); 116.8 (C-3a); 111.9 (C-5); 92.4 (C-7); 11.9 (C-1′) (Figures S1–S4, Supplementary Materials).

3.2.2. N,3-Dimethyl-1H-indazol-6-amine (9)

In a 100 mL single neck round-bottom flask, 3-methyl-1H-indazol-6-amine (8) (2.00 g, 13.6 mmol, 1 equivalent) was dissolved in 20 mL of methanol. Then, CH₃ONa (3.67 g, 67.9 mmol, 5 eq) and paraformaldehyde (2.04 g, 67.9 mmol, 5 eq) were added to the reaction vessel, respectively. The mixture was then refluxed for 5 min, and stirred at room temperature for 4 h. After that, the reaction was cooled to 5 °C, and NaBH₄ (2.06 g, 54.4 mmol, 4 eq) was slowly added under stirring for 5 min. The reaction mixture was refluxed for approximately 2 h, and the solvent was removed by rotary evaporation. The residue was redistributed in water and washed three times with ethyl acetate (3 × 50 mL). The ethyl acetate extracts were combined and rinsed three times with purified water (3 × 30 mL). Then, 20% HCl solution was added dropwise to the combined ethyl acetate layer under stirring until pH = 1. Subsequently, the aqueous phase was separated using a decanter. After the aqueous solution was cooled to room temperature, saturated Na₂CO₃ solution was gradually added under stirring until pH = 9, which enabled the precipitation of the base product (9). The precipitate was filtered using a Buchner filter funnel, collected, and dried under an infrared lamp. Compound 9 was obtained as a pink–white solid (1.90 g, 87% yield). M.p: 215.6–217.3 °C. TLC: R_f = 0.60 (n-hexane/ethyl acetate, 3:7). MS (ESI, MeOH), m/z: Calculated for C₉H₁₁N₃ [M+H]⁺: 162.10, found: 161.8. FT-IR (KBr), v_max (cm⁻¹): 3338, 3221 (N-H); 2974, 2939, 2900 (C-H sp³), 1624 (C=N); 1589, 1510 (C=C); ¹H-NMR (500 MHz, CDCl₃), δ (ppm): 7.42 (d, J = 8.5 Hz, 1H, H-4); 6.52 (dd, J₁ = 8.8 Hz, J₂ = 1.8 Hz, 1H, H-5); 6.43 (d, J = 1.5 Hz, 1H, H-7); 2.90 (s, 3H, H-2′); 2.52 (s, 3H, H-1′).¹³C-NMR (125 MHz, CDCl₃), δ (ppm): 149.1 (C-7a); 143.3 (C-3); 120.7 (C- 4); 115.7 (C-3a); 111.5 (C-5); 87.9 (C-7); 30.8 (C-2′); 11.9 (C-1′) (Figures S5–S8, Supplementary Materials).

3.2.3. N,2,3-Trimethyl-2H-indazol-6-amine (5)

Here, 30 mL of toluene, 3 mL of N,N-dimethylformamide (DMF) and 5.4 mL of trimethyl orthoformate (49.6 mmol; 4 eq) were added to a 50 mL round-bottom flask under stirring; then, 0.5 mL of 98% H₂SO₄ solution (8.7 mmol; 0.7 eq) was dropwise added to the mixture, which was stirred at 5 °C for 5 min before 2.00 g of N,3-dimethyl-1H-indazol-6-amine (9) (12.4 mmol; 1 eq) was added into the reaction vessel. The reaction mixture was then heated and stirred at 60 °C for 5 h. The solvent was removed under reduced pressure and a thick, dark-pink residue was obtained. Then, 100 mL of distilled water was added to this residue to form a clear, wine-red solution. The solution was washed with ethyl acetate (3 times × 120 mL/time) and the aqueous phase was separated. The saturated Na₂CO₃ solution was slowly added to the aqueous phase until pH = 8. The resulting aqueous solution was washed with ethyl acetate (3 × 120 mL). The ethyl acetate fractions were combined and then washed with distilled water (5 × 70 mL) to remove DMF and water-soluble impurities. Finally, the organic phase was dried with sodium sulfate and the solvent was removed using an rotary evaporator to obtain a yellow powder (1.58 g, 73% yield). M.p: 144.2–145.8 °C. TLC: R_f = 0.30 (n-hexane/ethyl acetate, 3:7); 0.65 (dichloromethane/methanol, 9:1). MS (ESI, MeOH), m/z: Calculated for C₁₀H₁₃N₃ [M+H]⁺: 176.11, found: 175.9. FT-IR (KBr), v_max (cm⁻¹): 3422 (N-H); 2982, 2904 (C-H sp³), 1645 (C=N); 1560, 1513 (C=C); ¹H-NMR (500 MHz, CDCl₃), δ (ppm): 7.28 (d, J = 9.0 Hz, 1H, H-4); 6.52 (s, 1H, H-7); 6.45 (dd, J₁ = 8.8 Hz, J₂ = 1.8 Hz, 1H, H-5); 3.97 (s, 3H, H-2′); 2.86 (s, 3H, H-3′); 2.49 (s, 3H, H-1′). ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 149.6 (C-7a); 147.8 (C-6); 131.3 (C-3); 119.9 (C-4); 115.5 (C-3a); 114.8 (C-5); 91.7 (C-7); 36.7 (C-3′); 30.8 (C-2′); 9.8 (C-1′) (Figures S9–S16, Supplementary Materials).

3.2.4. N,N,2,3-Tetratramethyl-2H-indazol-6-amine (5′)

When using a larger ratio between TMOF and the starting material 9, namely 6.2:1, beside target compound 5 (28%), the novel compound, 5′, was isolated with a 21% yield.

Data for compound 5′: TLC R_f = 0.30 (n-hexane/ethyl acetate, 3:7); 0.70 (dichloromethane/methanol, 9:1). MS (ESI_, MeOH), m/z: Calculated for C₁₁H₁₅N₃ [M + H]⁺: 190.1, found: 189.9. FT-IR (KBr), v_max (cm⁻¹): 2926 (C-H sp³); 1650 (C=N); 1559 (C=C). ¹H-NMR (500 MHz, DMSO-d₆), δ (ppm): 7.43 (d, J = 9.5 Hz, 1H, H-4); 6.77 (dd, J₁ = 9.5 Hz, J₂ = 1.5 Hz, 1H, H-5); 6.45 (d, J = 1.5 Hz,1H, H-7); 3.91 (s, 3H, H-2′); 2.88 (s, 6H, H-3′, H-4′), 2.50 (s, 3H, H-1′). ¹³C-NMR (125 MHz, DMSO-d₆) δ (ppm): 149.6 (C-7a); 148.9 (C-6); 131.2 (C-3); 120.5 (C-4); 115.4 (C-3a); 113.2 (C-5); 94.9 (C-7); 41.4 (C-3′,4′); 37.1 (C-2′); 9.8 (C-1′) (Figures S17–S23, Supplementary Materials).

4. Conclusions

In conclusion, we have successfully developed a novel alternative approach to converting 3-methyl-6-nitro-1H-indazole (6) into N,2,3-trimethyl-2H-indazol-6-amine (5), an important intermediate in the synthesis of pazopanib. This approach involved a nitro reduction (87%), a reductive amination (87%), and a N-methylation at the N² position on the indazole ring (73%), yielding an overall yield of the target compound 5 of 55%. This yield was comparable to what has been reported (54%). We successfully optimized the reaction conditions so that column chromatography could be negated, which makes the synthesis suitable for upscaling. In addition, we discovered a new structure, 5′, due to the potent methylating capability of TMOF, which facilitated multiple methylation on various nucleophilic centers on the indazole derivative. The products in each step were characterized using melting point determination, mass spectrometry, FT-IR spectroscopic analysis, and ¹H-NMR, ¹³C-NMR, COSY, HSQC, HMBC, NOESY spectroscopy, whose data can be found in the Supplementary Materials.