TEGOBUVIR ..IN PHASE II FOR HEPATITIS C

Hebner CM, Han B, Brendza KM, Nash M, Sulfab M, Tian Y, Hung M, Fung W, Vivian RW, Trenkle J, Taylor J, Bjornson K, Bondy S, Liu X, Link J, Neyts J, Sakowicz R, Zhong W, Tang H, Schmitz U.

PLoS One. 2012;7(6):e39163. doi: 10.1371/journal.pone.0039163. Epub 2012 Jun 13.

Wong KA, Xu S, Martin R, Miller MD, Mo H.

Virology. 2012 Jul 20;429(1):57-62. doi: 10.1016/j.virol.2012.03.025. Epub 2012 Apr 28.

Zeuzem S, Buggisch P, Agarwal K, Marcellin P, Sereni D, Klinker H, Moreno C, Zarski JP, Horsmans Y, Mo H, Arterburn S, Knox S, Oldach D, McHutchison JG, Manns MP, Foster GR.

Hepatology. 2012 Mar;55(3):749-58. doi: 10.1002/hep.24744.

Shih IH, Vliegen I, Peng B, Yang H, Hebner C, Paeshuyse J, Pürstinger G, Fenaux M, Tian Y, Mabery E, Qi X, Bahador G, Paulson M, Lehman LS, Bondy S, Tse W, Reiser H, Lee WA, Schmitz U, Neyts J, Zhong W.

Antimicrob Agents Chemother. 2011 Sep;55(9):4196-203. doi: 10.1128/AAC.00307-11. Epub 2011 Jul 11.

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http://www.google.com/patents/WO2013040492A2

ompound 1 can be prepared using synthetic methods and intermediates like those described in US 7,754,720. Compound 1 can also be prepared as described in the following Example.

Compound 1 is:

Compound 1 may also be referred to as 5-((6-(2,4-bis(trifluoromethyl)phenyl)pyridazin-3-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine, 5-[[6-[2,4-bis (trifluoromethyl)phenyl]pyridazin=3-yl]methyl]-2-(2-fluorophenyl).

Example 1 : 5-({6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl}methyl)-2-(2-fluorophenyl)-5H- imidazo[4,5-c]pyridi

Compound 103 was dissolved in dimethoxyethane (DME). To this solution was added 2,4-bis(trifluromethyl)phenylboronic acid 105 and a 2N aq. Na₂C0₃ solution. To the resulting biphasic mixture was added Pd(PPh₃)₄ and the reaction was then heated at 80°C for 72 hrs. The reaction was cooled to room temperature and filtered through Celite and the Celite washed with EtOAc. The filtrate was concentrated in vacuo. The residue was purified on 6g Si0₂ using MeOH/CH₂CI₂ to elute compound. The compound thus obtained was contaminated with PPh₃(0). The product was repurified on a 1 mm Chromatotron plate with 0 to 5%

MeOH/CH₂CI₂ in 1 % steps. The pure fractions were combined and concentrated in vacuo, then dried on high vacuum for 12 hrs. 11.8 mg of the free base of compound 1 was obtained with no PPh₃ contamination. ¹H NMR (300MHz,CD₃OD) δ 6.20 (s, 2), 7.32 (m, 3), 7.52 (m, 1 ), 7.78 (d, 1), 7.89 (d, 1), 7.95 (s, 2), 8.15 (m, 3), 8.35 (d, 1), 9.12 (s, 1); LC/MS M+H = 518.

The intermediate compound 104 was prepared as follows, a. Preparation of Compound 10

101 102

To a solution of the commercially available starting material 101 in CHCI₃, trichloroisocyanuric acid (TCCA) was added at 60°C. Then the solution was stirred for 1.5 hrs, cooled, and filtered with HiFlo-Celite. The filtrate was concentrated and dried with vacuum. The yield was 5.037 g of compound 102. b. Preparation of Compound 104.

102 104

To a solution of compound 103 in DMF (dimethylformamide), NaOH was added.

Compound 102 was dissolved in DMF (20 mL) and added to the solution slowly. The reaction was stirred for 3 hrs, was diluted with water and extracted with EtOAc. The organic layer was dried with Na₂S0 . The solvent was removed and the product recrystallized with

dichloromethane. The yield was 5.7 g of compound 103.

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US7754720

Example 1a Synthesis of 5-({6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl}methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridineIn this method, dimethoxyethane or its related solvents, all having the general formula R¹OR²O(R⁴O)_aR³ wherein each of R¹, R², R³ and R⁴ are independently selected from C1-C6 alkyl and a is 0 or 1, have been found to be particularly advantageous over the conventional solvent DMF. Typically, each of R¹, R², R³ and R⁴ are independently C₁-C₂ alkyl and usually a is 0. C₁-C₆ alkyl includes fully saturated primary, secondary or tertiary hydrocarbon groups with 1 to 6 carbon atoms and thereby includes, but is not limited to methyl, ethyl, propyl, butyl, etc.Step 1

Compound	MW	Amount	mmoles	Equivalents
SM	128.56	5	g	38.9	1
TCCA	232.41	3.62	g	15.6	0.4
CHCl3		130	ml

To a solution of the commercially available starting material (SM) in CHCl₃, trichloroisocyanuric acid (TCCA) was added at 60° C. Then the solution was stirred for 1.5 hrs., cooled down and filtered with HiFlo-Celite. The filtrate was concentrated and dried with vacuum. The yield was 5.037 g.

Step 2

Compound	MW	Amount	mmoles	Equivalents
S.M.	163	5.073	g	31.12	1
Core	213.2	6.635	g	31.12	1
NaOH (10%)	40	1.245	g	31.12	1
DMF		320	ml

To a solution of core (obtained as described in literature in DMF (dimethylformamide), NaOH was added. Then SM for this step (obtained from step 1) was dissolved in DMF (20 ml) and added to the solution slowly. The reaction was stirred for 3 hrs, was diluted with water and extracted with EtOAc. The organic layer was dried with Na₂SO₄. The solvent was removed and the product recrystallized with DCM (dichloromethane). The yield was 5.7 g.

Step 3

Compound	MW	Amount	Moles	Equivalents
A	453.79	95 mg	0.209	1
DME	500 ul
2 N aq. Na2CO3		313ul	0.626	3
2,4-bisCF3-	257.93	80.9 mg	0.313	1.5
phenylboronic
acid
Pd(PPh3)4	1155	12 mg	0.0104	0.05

Compound A was dissolved in dimethoxyethane (DME). To this solution was added 2,4-bis(trifluromethyl)phenylboronic acid and a 2N aq. Na₂CO₃ solution. To the resulting biphasic mixture was added Pd(PPh₃)₄ and the reaction was then heated at 80° C. for 72 hrs. The reaction was cooled to room temperature and filtered through Celite and the Celite washed with EtOAc. The filtrate was concentrated in vacuo. The residue was purified on 6 g SiO2 using MeOH/CH2Cl2 to elute compound. The compound thus obtained was contaminated with PPh₃(O). The product was repurified on a 1 mm Chromatotron plate with 0 to 5% MeOH/CH₂Cl₂ in 1% steps. The pure fractions were combined and concentrated in vacuo, then dried on high vacuum for 12 hrs. 11.8 mg of the free base of compound (1) was obtained with no PPh₃ contamination.

¹H NMR (300 MHz, CD₃OD)

6.20 (s, 2)

7.32 (m, 3)

7.52 (m, 1)

7.78 (d, 1)

7.89 (d, 1)

7.95 (s, 2)

8.15 (m, 3)

8.35 (d, 1)

9.12 (s, 1)

LC/MS M+H=518

Example 1b Synthesis of 5-({6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl}methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridineThis example is directed to an additional method for making compound (1), employing the following schemes.

Methanesulfonic acid was added to 2-fluorobenzoic acid in a reactor with active cooling keeping T≦50° C. 3,4-Diaminopyridine was then added portionwise to this cooled slurry, keeping T≦35° C. The contents of the reactor were then heated to 50° C. Phosphorus pentoxide was added in a single charge. The reaction was then heated at 90-110° C. for at least 3 hours. The reaction was sampled for completion by HPLC analysis. The reaction was cooled to ambient temperature and water was added portionwise slowly to quench the reaction. The reaction was then diluted with water. In solubles were removed by filtration. The pH of the filtrate was adjusted to 5.5-5.8 with ammonium hydroxide. The reaction was allowed to self-seed and granulate for ˜4 hours at ambient temperature. The pH was then adjusted to 8.0-9.3 with ammonium hydroxide. The slurry was held at ambient temperature for at least 2 hours. The solids were isolated by filtration and washed with water, followed by IPE. The wet cake was dried in vacuo at not more than 60° C. until ≦1% water remains. The dry product is core (2).

	Summary of Materials	M.W.	Wt. Ratio	Mole ratio
	3,4-Diaminopyridine	109.13	1.0	1.0
	2-Fluorobenzoic acid	140.11	1.4	1.1
	Methanesulfonic acid	96.1	7.0	8.0
	Phosphorus pentoxide	141.94	1.3	1.0
	Water	18.02	40	—
	Isopropyl ether	102.17	5.0	—
	Ammonium hydroxide	35.09	~10	—

A solution of compound (2a) in 1,2-dichloroethane was heated to 40-45° C. Trichloroisocyanuric acid was added and the mixture was heated at 60-70° C. for at least 2 hours. The reaction was sampled for completion by HPLC analysis. The reaction was cooled to ambient temperature. Celite was added to absorb insolubles, then solids were removed by filtration. The filtrate was washed with 0.5 N sodium hydroxide solution. The organic layer was concentrated to lowest stirrable volume and displaced with DMF. Core (2) and 10% aqueous sodium hydroxide solution were added. The reaction was stirred at ambient temperature for at least 8 hours. The reaction was sampled for completion by HPLC analysis. An additional 10% charge of 10% sodium hydroxide solution was added to the reaction. The reaction was then charged into water to isolate the crude product. After granulating for at least 1 hour, the solids were isolated and washed with water and isopropyl ether. Ethyl acetate was added and refluxed (internal T=70-77° C.) for 1-5 hours to dissolve product, then cooled to 18-23° C. slowly over 4-8 hours. The reactor contents were agitated at 18-23° C. for 8-20 hours and solids collected by filtration and rinsed with ethyl acetate. Low melt (i.e., DSC about 220 degrees C.) amorphous compound (1) was discharged. Amorphous compound (1) was dissolved in ethyl acetate by heating at reflux (internal T=70-77° C.) for 1-5 hours. Water content is controlled to about 0.2% by azeotropically removing water (with ethyl acetate the upper limit on water content is about 0.6% by weight; at about 0.9% by weight water the amorphous material will reprecipitate and crystals will not be obtained). The reactor contents are cooled slowly to 18-23° C. over 4-8 hours, then agitated at 18-23° C. for 8-20 hours and solids collected by filtration. The solids were rinsed with ethyl acetate and dried in vacuo at not more than 60° C. to obtain the dry crystalline compound (1).

Summary of Materials	M.W.	Wt. Ratio	Mole ratio
3-chloro-6-methylpyridazine	128.56	1.0	1.0
2,4bis(trifluromethyl)phenylboronic	257.93	4.0	2.0
acid
X-Phos	476.72	0.18	0.05
Palladium acetate	224.49	0.04	0.025
1,2-Dimethoxyethane	90.12	16.7	—
Potassium carbonate	138.21	2.15	2.0
Water	18.02	7.8	—
Copper iodide	190.45	0.037	0.025
Celite	—	0.25	—
Heptane	100.2	22.4	—

Nuclear Magnetic Resonance (¹H-, ¹³C-, and ¹⁹F-NMR) SpectraNuclear magnetic resonance (NMR) spectra of compound (1) is consistent with the proposed structure. The ¹³C, ¹⁹F, and ¹H-NMR spectra of compound (1) in DMSO-d₆ were measured using a Varian UnityInova-400 FT-NMR spectrometer. Spectra are shown in the table below. The NMR chemical shift assignments were established using 2D correlation experiments (COSY, HSQC, HMBC and HSQCTOCSY).

¹H- and ¹³C-NMR Chemical Shift Assignments for Compound (1) Reference Standard

Atom	δC/ppm (DMSO-d6)	δF/ppm (DMSO-d6)	δH/ppm (DMSO-d6)
1A	140.16
2A	128.32	(qa, JCF = 32 Hz)
3A	123.61,	m		8.24	(m, 1 H)
4A	130.27	(q, JCF = 34 Hz)
5A	129.54	(q, JCF = 3 Hz)		8.22	(m, 1 H)
6A	133.36		7.88	(m, 1 H)
7A	123.20	(q, JCF = 273 Hz)	−56.4b
8A	123.02	(q, JCF = 275 Hz)	−62.0b
1B	158.76
2B	128.16		8.01	(d, 1 H, J = 8.4 Hz)
3B	126.20		7.95	(d, 1 H, J = 8.8 Hz)
4B	157.70
5B	60.49		6.17	(s, 2 H)
2C	131.86		8.31	(m, 1 H)
3C	112.63		7.86	(m, 1 H)
4C	155.44
6C	168.11	(d, JCF = 6 Hz)
8C	145.08
9C	133.06		9.25	(s, 1 H)
1D	123.11	(d, JCF = 10 Hz)
2D	160.46	(d, JCF = 254 Hz)	−111.7
3D	116.59	(d, JCF = 22 Hz)		7.29	(m, 1 H)
4D	130.84	(d, JCF = 8 Hz)		7.46	(m, 1 H)
5D	124.13	(d, JCF = 4 Hz)		7.31	(m, 1 H)
6D	131.72	(d, JCF = 2 Hz)		8.35	(m, 1 H)
amultiplicity, s: singlet, d: doublet, q: quartet, m: multiplet
binterchangeable signals