CX-5461

CX-5461 is an organic heterotetracyclic compound that is 5-oxo-5H-[1,3]benzothiazolo[3,2-a][1,8]naphthyridine-6-carboxylic acid substituted by a 4-methyl-1,4-diazepan-1-yl group at position 2 and in which the carboxy group at position 6 is substituted by a [(5-methylpyrazin-2-yl)methyl]nitrilo group. An inhibitor of ribosomal RNA (rRNA) synthesis which specifically inhibits RNA polymerase I-driven transcription of rRNA in several cancer cell lines. It is currently in phase I/II clinical trials for advanced hematologic malignancies and triple-negative breast cancer with BRCA1/2 mutation. It has a role as an antineoplastic agent, an apoptosis inducer and an EC 2.7.7.6 (RNA polymerase) inhibitor. It is a diazepine, an organic heterotetracyclic compound, a member of pyrazines, a secondary carboxamide and a naphthyridine derivative.|Pidnarulex is an orally bioavailable inhibitor of RNA polymerase I (Pol I), with potential antineoplastic activity. Upon oral administration, pidnarulex selectively binds to and inhibits Pol I, prevents Pol I-mediated ribosomal RNA (rRNA) synthesis, induces apoptosis, and inhibits tumor cell growth. Pol I, the multiprotein complex that synthesizes rRNA, is upregulated in cancer cells and plays a key role in cell proliferation and survival. Hyperactivated rRNA transcription is associated with uncontrolled cancer cell proliferation.

To a solution of Compound I (45 g; see WO 2009/046383 for synthesis) in DCM (1500 ml, ~33 parts (v/w)) was added 35% H2O2 (300 ml, ~35 equiv.) at 20-25 C and the resulting mixture was stirred for -18 hr. After phase separation, the organic layer was added 300 ml of purified process water (PPW) for extraction. After phase separation, the combined aqueous layers was added 300 ml of DCM for extraction. After phase separation, the aqueous layer was evaporated to remove DCM. The aqueous layer was lyophilized to get 48.6 g of Compound 9 (2-(4-methyl-4-oxidodiazepan-4-ium-l-yl)-/V-((5-methylpyrazin-2-yl)methyl)-5-oxo-5iT- benzo[4, 5]thiazolo[3, 2-a][l, 8]naphthyridine-6-carboxamide) in approximately 100% yield with 98.2% purity. MS: m/z 530.187 [M+H]+. NMR and 13C NMR in CDCh as shown belowTo a 1 L reactor was added Compound I (11.1 g) in acetic acid (220 ml) as a clear solution. To the mixture was added CuBr (4.61 g) to form slurry. To the mixture was added 70% /-BuOOH(aq) for ~l hr of addition time while maintaining the temperature at no more than 30 C. The color of the solution turned from yellow slurry to green slurry. The mixture was stirred for 3-5 days. The mixture was added into 1500 ml of H2O and 1600 ml of DCM. The mixture was added 28-33% NH3(aq) to adjust pH to 11 while maintaining the temperature at no more than 30 C. The mixture was settled for phase separation and some solid was observed between organic and aqueous layer. The aqueous layer was extracted with 500 ml of DCM. The combine organic layer was extracted with 600 ml of 0.15% NFh(aq). The organic layer was collected and the emulsion layer was discarded. The organic layer was evaporated to dryness. The residue was added 60 ml of DMSO and sonicated for 10 min to precipitate the product. The mixture was filtrated and the wet cake was washed with 10 ml MeOH. The wet cake was added 10 ml of DCM for slurry and then evaporated to dryness to remove the residual MeOH. Compound 10 (1.8 g; 2-(4-methyl-l, 4-diazepan-l-yl)-/V-((5-methylpyrazin-2- yl)carbonyl)-5-oxo-5i7-benzo[4, 5]thiazolo[3, 2-a][l, 8]naphthyridine-6-carboxamide) was obtained in 15% yield with 93.6% purity. MS: m/z 528.183 [M+H]+. NMR and 13C NMR in CDCb as shown below.Compound I was dissolved in a solvent mixture at an elevated temperature, then HCl was added. The suspension was stirred overnight then filtered and washed to obtain an HCl salt of Compound I as a white powder (62percent yield). The resulting salt was characterized by 1H NMR (FIG. 23), XRPD (FIG. 21), and TGA (FIG. 25). The TGA showed HCl salt of Compound I demonstrated up to 0.9 wt percent loss at 140° C.Preparation of (E)-2-(4-methyl-1, 4-diazepan-1-yl)-N-((5-methylpyrazin-2- yl)methyl)-5-(phenylimino)-5H-benzo[4, 5]thiazolo[3, 2-a][1, 8]naphthyridine-6-carboxamide (Compound 10; See, Table 1 and Figure 3B):2-(4-methyl- 1 , 4-diazepan- 1 -yl)-N-((5 -methylpyrazin-2-yl)methyl)-5 -oxo-5H-benzo[4, 5 ]thiazolo[3 , 2-a] [1, 8]naphthyridine-6-carboxamide (100 mg) was suspended in3 mL of phosphoryl chloride, and the resulting mixture was refluxed for 3 hours. Thephosphoryl chloride was thereafter removed by evaporation and the resulting crude product was dissolved in MeOH. Aniline was then added (2 mL) and the resulting solution was stirred for 5 minutes. The compound was purified by preparative HPLC to yield 56 mg (50 percent yield).MS [ESI]: calcd. 589.7 found [M+H] 589.4Preparation of Compound 9 (see, Table 1):2-(4-methyl- 1 , 4-diazepan- 1 -yl)-N-((5 -methylpyrazin-2-yl)methyl)-5 -oxo-5H-benzo[4, 5]thiazolo[3 , 2-a] [1 , 8]naphthyridine-6-carboxamide (Compound 1) (100 mg) was suspended in 3 mL of phosphoryl chloride, and the obtained mixture was refluxed for 3 hours. The phosphoryl chloride was thereafter removed by evaporation and the obtained crude product was dissolved in MeOH. 100 mg of urea were added and the resulting solution was left to stir for 4 hours. The title compound was purified by preparative HPLC to yield 78 mg (75percent yield)MS [ESI]: calcd. 537.2 found [M+H] 537.6Preparation of Compound 8 (see, Table 1):2-(4-methyl- 1 , 4-diazepan- 1 -yl)-N-((5 -methylpyrazin-2-yl)methyl)-5 -oxo-5H-benzo[4, 5]thiazolo[3 , 2-a] [1 , 8]naphthyridine-6-carboxamide (Compound 1) (100 mg) was suspended in 3 mL of phosphoryl chloride, and the obtained mixture was refluxed for 3 hours. The phosphoryl chloride was thereafter removed by evaporation and the obtained crude product was dissolved in MeOH. 3 mL of Triethylamine and 100 mg of [Methoxylamine hydrogen chloride] were then added and the resulting solution wasstirred for 5 minutes. The obtained compound 5 was purified by preparative HPLC to yield 34 mg (32 percent yield).MS [ESI]: calcd. 543.6 found [M+H] 543.2Preparation of (E/Z)-2-(4-methyl-1, 4-diazepan-1-yl)-5-(methylimino)-N-((5-methylpyrazin-2-yl)methyl)-5H-benzo[4, 5]thiazolo[3, 2-a] [1, 8]naphthyridine-6-carboxamide (referred to herein, interchangeably, as POL1-I/3; or Compound5):2-(4-methyl- 1 , 4-diazepan- 1 -yl)-N-((5 -methylpyrazin-2-yl)methyl)-5 -oxo-5H-benzo[4, 5]thiazolo[3 , 2-a] [1 , 8]naphthyridine-6-carboxamide (Compound 1) (100 mg) was suspended in 3 mL of phosphoryl chloride, and the obtained mixture was refluxed for 3 hours. The phosphoryl chloride was thereafter removed by evaporation and the obtained crude product was dissolved in MeOH. Methylamine was then added (3 mL)and the resulting solution was stirred for 5 minutes. The obtained compound 5 was purified by preparative HPLC to yield 74 mg (73 percent yield).MS [ESI]: calcd. 527.6 found [M+H] 527.3Preparation of 5-imino-2-(4-methyl-i, 4-diazepan-i-yl)-N-((5- methylpyrazin-2-yl)methyl)-5H-benzo[4, 5]thiazolo[3, 2-a][i, 8]naphthyridine-6-carboxamide (referred to herein, interchangeably, as POL1-I/4; RAM-i orCompound 4):2-(4-methyl- 1 , 4-diazepan- 1 -yl)-N-((5 -methylpyrazin-2-yl)methyl)-5 -oxo-5H-benzo[4, 5]thiazolo[3 , 2-a] [1 , 8]naphthyridine-6-carboxamide (Compound 1) (100 mg) was suspended in 3 mL of phosphoryl chloride, and the obtained mixture was refluxed for 3 hours. The phosphoryl chloride was thereafter removed by evaporation and theobtained crude product was dissolved in MeOH. Gaseous ammonia was then bubbled into the methanol for 1 minute and the resulting solution was stirred for 5 minutes. The obtained compound 4 was purified by preparative HPLC to yield 64 mg (64 percent yield).?H NIVIR (400 MHz, CDC13 ppm): 9.28 (d, J = 8.21 Hz, 1H), 8.58 (m, 1H), 8.44 (m, 1H), 8.04 (d, J = 8.72 Hz, 1H), 7.64 (dd, J = 7.62, 1.35 Hz, 1H), 7.34 (m, 2H), 6.68 (d, J = 9.23 Hz, 1H), 4.86 (s, 2H), 3.98-3.74 (m, 4H), 2.84 (m, 2H), 2.66-2.60 (m, 2H), 2.54 (s, 3H), 2.41 (s, 3H), 2.12 (m, 2H), MS [ESI]: calcd. 513.2 found [M+H] 513.3.Preparation of 2-(4-methyl-1, 4-diazepan-1-yl)-N-((5-methylpyrazin-2- yl)methyl)-5-thioxo-5H-benzo[4, 5]thiazolo[3, 2-a][1, 8]naphthyridine-6- carboxamide (referred to herein interchangeably as POL1-112; RAM-3; orCompound 3): 2-(4-methyl- 1 , 4-diazepan- 1 -yl)-N-((5 -methylpyrazin-2-yl)methyl)-5 -oxo-5H- benzo[4, 5]thiazolo[3 , 2-a] [1 , 8]naphthyridine-6-carboxamide (Compound 1) (100 mg) was suspended in 3 mL of phosphoryl chloride, and the obtained mixture was refluxed for 3 hours. The phosphoryl chloride was thereafter removed by evaporation and the obtained crude product was dissolved in MeOH. Sodium hydrosulfide was then added (100 mg) and the resulting solution was stirred for 5 minutes. The obtained compound3 was purified by preparative HPLC to yield 82.4 mg 80 percent yield).?H NMR (500 MHz, CDC13): oe = 13.02 (t, J = 5.58 Hz, 1H), 9.50 (d, J = 7.01 Hz, 1H), 9.23 (d, J = 9.38 Hz, 1H), 8.64 (d, J = 1.13 Hz, 1H), 8.43 (s, 1H), 7.75 (m, 1H), 7.45 (m, 2H), 6.82 (d, J = 9.42 Hz, 1H), 4.89 (d, J = 5.61 Hz, 2H), 4.17-3.64 (m, 4H), 2.88-2.79 (m, 2H), 2.64-2.57 (m, 2H), 2.56-2.53 (s, 3H), 2.39 (s, 3H), 2.11 (m, 2H)ppm.MS [ESI]: calcd. 530.6 found [M+H] 530.2General procedure: General Synthetic Procedure:POLl-I (

Molecular Weight:513.6
XLogP3:3.3
Hydrogen Bond Donor Count:1
Hydrogen Bond Acceptor Count:9
Rotatable Bond Count:4
Exact Mass:513.19469430
Monoisotopic Mass:513.19469430
Topological Polar Surface Area:120
Heavy Atom Count:37
Complexity:915
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes