N -(6-Chloro-3-nitropyridin-2-yl)-5-(1-methyl-1 H -pyrazol-4-yl)isoquinolin-3-amine

: Here we describe the synthesis of N -(6-chloro-3-nitropyridin-2-yl)5-(1-methyl-1 H -pyrazol-4-yl)isoquinolin-3-amine via a three-step procedure including a Buchwald–Hartwig arylamination with benzophenone imine and a highly regioselective nucleophilic aromatic substitution. The title compound was analyzed by nuclear magnetic resonance spectroscopy ( 1 H, 13 C, HSQC, HMBC, COSY, DEPT90 and NOESY), high resolution mass spectrometry (ESI-TOF-HRMS) and infrared spectroscopy (ATR-IR) and its structure was conﬁrmed by single crystal X-ray diffraction. The inhibitory potency of the title compound was evaluated for selected kinases harboring a rare cysteine in the hinge region (MPS1, MAPKAPK2 and p70S6K β /S6K2).


Introduction
The monopolar spindle 1 (MPS1) kinase, also known as threonine and tyrosine kinase (TTK) [1], is a potential therapeutic target for the treatment of various malignancies such as triple negative breast cancer [2]. The ongoing research on small molecules blocking MPS1 activity has led to the identification of potent inhibitors and even clinical candidates [3].
Interestingly, the adjacent hinge residue in MPS1 is a poorly conserved cysteine (Cys604), which might be exploited in the development of selective targeted covalent inhibitors (TCIs) [5,6]. To address this rare cysteine, we sought to employ nucleophilic aromatic substitution (S N Ar) chemistry as a non-generic design approach [7]. Therefore, we combined the 5-(1-methyl-1H-pyrazol-4-yl)isoquinoline scaffold with an electrophilic 6-chloro-3-nitropyridine warhead (see compound 1). This warhead type has been shown previously to engage a cysteine with an equivalent placement in the receptor tyrosine kinase FGFR4 [8]. As deduced from the latter study and our previous work on JAK3 inhibitors [9], the nitro group is not only required for activating the chloropyridine moiety for nucleophilic Molbank 2021, 2021, M1181 2 of 6 aromatic displacement, but also to form a weak intramolecular hydrogen bond with the NH of the isoquinoline 3-amino group to promote the (re)active conformation.
Molbank 2021, 2021, x FOR PEER REVIEW 2 of 6 nase FGFR4 [8]. As deduced from the latter study and our previous work on JAK3 inhibitors [9], the nitro group is not only required for activating the chloropyridine moiety for nucleophilic aromatic displacement, but also to form a weak intramolecular hydrogen bond with the NH of the isoquinoline 3-amino group to promote the (re)active conformation.

Chemistry
The title compound 1 was prepared in three steps starting from previously reported 3-chloro-5-(1-methyl-1H-pyrazol-4-yl)isoquinoline (3) (Scheme 2) [4]. The amino group in the 3-position of the isoquinoline core was introduced following a modified protocol by Wolfe et al. [10]. To this end, 3 was subjected to a microwave-assisted Buchwald-Hartwig cross-coupling reaction with benzophenone imine generating intermediate 4, which was subsequently hydrolyzed under acidic conditions. The resulting primary arylamine 5 was then reacted with commercially available 2,6-dichloro-3-nitropyridine (6) in the presence of N,N-diisopropylethylamine (DIEA) to furnish the title compound by regioselective nucleophilic aromatic substitution.

Chemistry
The title compound 1 was prepared in three steps starting from previously reported 3-chloro-5-(1-methyl-1H-pyrazol-4-yl)isoquinoline (3) (Scheme 2) [4]. The amino group in the 3-position of the isoquinoline core was introduced following a modified protocol by Wolfe et al. [10]. To this end, 3 was subjected to a microwave-assisted Buchwald-Hartwig cross-coupling reaction with benzophenone imine generating intermediate 4, which was subsequently hydrolyzed under acidic conditions. The resulting primary arylamine 5 was then reacted with commercially available 2,6-dichloro-3-nitropyridine (6) in the presence of N,N-diisopropylethylamine (DIEA) to furnish the title compound by regioselective nucleophilic aromatic substitution.
Molbank 2021, 2021, x FOR PEER REVIEW 2 of 6 nase FGFR4 [8]. As deduced from the latter study and our previous work on JAK3 inhibitors [9], the nitro group is not only required for activating the chloropyridine moiety for nucleophilic aromatic displacement, but also to form a weak intramolecular hydrogen bond with the NH of the isoquinoline 3-amino group to promote the (re)active conformation.

Chemistry
The title compound 1 was prepared in three steps starting from previously reported 3-chloro-5-(1-methyl-1H-pyrazol-4-yl)isoquinoline (3) (Scheme 2) [4]. The amino group in the 3-position of the isoquinoline core was introduced following a modified protocol by Wolfe et al. [10]. To this end, 3 was subjected to a microwave-assisted Buchwald-Hartwig cross-coupling reaction with benzophenone imine generating intermediate 4, which was subsequently hydrolyzed under acidic conditions. The resulting primary arylamine 5 was then reacted with commercially available 2,6-dichloro-3-nitropyridine (6) in the presence of N,N-diisopropylethylamine (DIEA) to furnish the title compound by regioselective nucleophilic aromatic substitution.

X-ray Crystallography
To demonstrate that the S N Ar reaction carried out in the final step of the synthetic route delivered the desired regioisomer, we determined the X-ray crystal structure of compound 1 (Figure 1). The data confirmed that nucleophilic substitution occurred in the 2-position of the 2,6-dichloro-3-nitropyridine precursor. The product shows the mentioned intramolecular hydrogen bond between the diarylamine NH and the nitro group. In addition, we observed two rotamers distinguished by the conformation of the methylsubstituted pyrazole ring.
To demonstrate that the SNAr reaction carried out in the final step of the synthetic route delivered the desired regioisomer, we determined the X-ray crystal structure of compound 1 (Figure 1). The data confirmed that nucleophilic substitution occurred in the 2-position of the 2,6-dichloro-3-nitropyridine precursor. The product shows the mentioned intramolecular hydrogen bond between the diarylamine NH and the nitro group. In addition, we observed two rotamers distinguished by the conformation of the methylsubstituted pyrazole ring. Figure 1. Structure of 1 determined by X-ray crystallography confirming the desired regiochemistry. The structure further demonstrates the formation of an intramolecular hydrogen bond between the nitro group and the diarylamine NH. Two rotamers were observed.

Biological Evaluation
The biological activity of compound 1 was evaluated in a radiometric HotSpot ® kinase assay (Reaction Biology Corp. (Malvern, PA, USA)) [11] on selected kinases harboring the aforementioned cysteine in the middle hinge region (Table 1). While the compound did not show substantial activity on MAPKAPK2 and the intended target MPS1, it displayed an IC50 value of 444 nM for the ribosomal s6 kinase p70S6Kβ (S6K2). As there are no selective p70S6Kβ inhibitors known so far, this compound may serve as a starting point for the design of such molecules.

General Experimental Section
The utilized chemicals and reagents were of commercial quality and used without further purification, if not stated otherwise. Dry solvents were purchased from Fisher Scientific (Schwerte, Germany) and stored in septum-sealed bottles under N2 atmosphere and over molecular sieves.
Purification via flash chromatography was performed on an Interchim PuriFlash 430 (Interchim, Montluçon, France) using Geduran Si 60-200 µm silica gel (Merck, Darmstadt, Figure 1. Structure of 1 determined by X-ray crystallography confirming the desired regiochemistry. The structure further demonstrates the formation of an intramolecular hydrogen bond between the nitro group and the diarylamine NH. Two rotamers were observed.

Biological Evaluation
The biological activity of compound 1 was evaluated in a radiometric HotSpot ® kinase assay (Reaction Biology Corp. (Malvern, PA, USA)) [11] on selected kinases harboring the aforementioned cysteine in the middle hinge region (Table 1). While the compound did not show substantial activity on MAPKAPK2 and the intended target MPS1, it displayed an IC 50 value of 444 nM for the ribosomal s6 kinase p70S6Kβ (S6K2). As there are no selective p70S6Kβ inhibitors known so far, this compound may serve as a starting point for the design of such molecules.

General Experimental Section
The utilized chemicals and reagents were of commercial quality and used without further purification, if not stated otherwise. Dry solvents were purchased from Fisher Scientific (Schwerte, Germany) and stored in septum-sealed bottles under N 2 atmosphere and over molecular sieves.
High-performance liquid chromatography (HPLC) was performed on an Agilent Technologies 1100 Series chromatographic system (Agilent Technologies, Santa Clara, CA, USA) equipped with an UV/Vis diode array detector (DAD) and a Phenomenex Luna ® 5 µ (150 mm × 4.6 mm, 5 µm) reversed phase C8 separation column from Phenomenex (Phenomenex, Torrance, CA, USA). The mobile phase consisted of phase A (MeOH) and phase B (0.01 M KH 2 PO 4 -Buffer, pH = 2.3) and elution was performed at a flowrate of 1.5 mL/min using the gradient described in Table 2. The injection volume was 10 µL. The purity was determined at 254 nm and 230 nm. Mass spectrometry was performed on an Advion expression ® compact mass spectrometer (Advion, Ithaca, NY, USA) with an electrospray ionization (ESI) ion-source equipped with an Advion plate express TLC plate reader (Advion, Ithaca, NY, USA). High resolution mass spectrometry (HRMS) was performed on a Bruker maXis 4G (Bruker Daltonik, Bremen, Germany) ESI-TOF high resolution mass spectrometer.
Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker Avance III HD 400 MHz NMR spectrometer (Bruker, Billerica, MA, USA). The 1 H and 13 C NMR spectra were calibrated against the residual proton or 13 C signals of the deuterated solvents. Signals are reported in parts per million (ppm) relative to tetramethylsilane (δ = 0 ppm).

5-(1-Methyl-1H-pyrazol-4-yl)isoquinolin-3-amine (5)
700 mg of 3-chloro-5-(1-methyl-1H-pyrazol-4-yl)isoquinoline [4] (2.87 mmol, 1 eq.), 64 mg of Pd(OAc) 2 (0.29 mmol, 0.1 eq.), 632 mg of BINAP (0.86 mmol, 0.3 eq.) and 830 mg of t-BuONa (8.62 mmol, 3 eq.) were suspended in 10 mL of dry toluene in a microwave tube [10]. The reaction mixture was degassed and purged with argon and 1.04 g of benzophenone imine (5.74 mmol, 2 eq.) was added. The mixture was stirred at rt and under argon atmosphere for 10 min and subsequently at 130 • C under microwave irradiation for 35 min (including 5 min ramp time). After cooling down to rt, the mixture was concentrated under vacuum, 25 mL of a 2 N HCl aq. solution was added and stirring continued at 75 • C for 1 h. After the completion of imine hydrolysis, the suspension was extracted twice with 100 mL of DCM. The aqueous layer was then basified to a pH of 8 with 30% (w/w) NaOH aq. and extracted three times with 100 mL of ethyl acetate (EtOAc). The combined EtOAc phases were dried over Na 2 SO 4 and concentrated under vacuum. The resulting crude product was then purified via flash chromatography (SiO 2 , EtOAc: MeOH, gradient elution from 0 to 8% MeOH). The solid obtained was triturated with HPLC grade pentane for further purification. The suspension was filtered, and the residue was dried under high vacuum to yield 493 mg (77%) of the desired product as a red-brown powder.  (1) 40 mg of 5-(1-methyl-1H-pyrazol-4-yl)isoquinolin-3-amine (0.18 mmol, 1 eq.) and 69 mg of 2,6-dichloro-3-nitropyridine (0.36 mmol, 2 eq.) were dissolved in 3 mL of dry 1,4-dioxane. Subsequently, 93 µL of N,N-diisopropylethylamine (DIEA) (1.25 mmol, 7 eq.) were added to the stirring solution. The reaction mixture was then heated to reflux for 26 h. After cooling down to rt, the solvent was removed under vacuum. The resulting crude product was purified via flash column chromatography (SiO2, hexane: EtOAc, gradient elution from 40 to 100% EtOAc). The obtained solid was triturated with HPLC grade pentane for further purification. The suspension was filtered, and the residue was dried under high vacuum to yield 47 mg (69%) of the desired product as a carmine-

Conclusions
In this study, we established a synthesis for N-(6-chloro-3-nitropyridin-2-yl)-5-(1methyl-1H-pyrazol-4-yl)isoquinolin-3-amine (1). The synthesis started from known 3chloroisoquinoline derivative 3, which was converted into unprecedented isoquinoline-3-amine derivative 5 via Buchwald-Hartwig arylamination with benzophenone imine followed by acid-promoted hydrolysis of the imine intermediate. From compound 5, the desired compound could be obtained via regioselective nucleophilic aromatic substitution with 2,6-dichloro-3-nitropyridine. Both 5 and 1 were fully characterized and the regiochemistry of 1 was confirmed by X-ray crystallography. Although inhibitor 1 did not show the expected inhibitory potency on the intended target kinase, MPS1, we found significant activity on the kinase p70S6Kβ, which features an equivalent cysteine. The compound may thus serve as a starting point for the development of p70S6Kβ inhibitors.

Conflicts of Interest:
The authors declare no conflict of interest.