FIELD OF THE INVENTION
This invention describes isolation and characterization of a novel urea impurity compound 1, 1-(1R,2S)-2-(3, 4-Difluorophenyl)cyclopropyl-3--(1R,2S)-2- (3,4-Dif luorophenyl) cyclopropyl urea related to (1R, 2S),-2-(3, 4-Difluorophenyl)cyclopropyl amine (CP-Amine) 2, a process for its preparation and its use as Reference Standard to control the quality of CP-Amine 2. The invention further relates to. quality of CP-Amine 2, having a low amount of compound 1, as well as the HPLC methods for identifying 1 in 2, for purity and assay determinations- of 2, either in free base or its salts forms. The present invention also describes the use of above solid urea compound 1, for the synthesis of specific intermediates suitable for the prepration of triazolo-pyrimidines, such as Ticagrelor 3.


Background of the Invention:
Ticagrelor, (IS,2S,3R,5S)-3-[7-[{ (1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino-5-(propylthio)-3H-1,2,3-triazolo [4,5-d] pyrimidine-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentariediol 3, has the above mentioned chemical structure. Ticagrelor is currently marketed by Astra Zeneca under the trade name BRILINTA and BRILIQUE. Its CAS Number -is 274693-27-5. Ticagrelor is- a reversibly binding oral P2Y12 ADP receptor antagonist. Ticagrelor is indicated for the treatment or prevention of thrombotic events eg. stroke, heart attack, acute coronary syndrome or myocardial infarction with ST elevation, other coronary artery diseases and arterial thrombosis plus other disorders related to platelet aggregation (WO 00/34283).
WO 99/05143 discloses a series of triazolo [4,5-d]pyrimidine compounds, including Ticagrelor 3. WO 01/92262 discloses crystalline and amorphous forms of compound of Ticagrelor 3.
Most of synthetic strategies reported for Ticagrelor involve (1R,.2S)-2-(3, 4-difluorophenyl) cyclopropyl amine 2 (CP-Amine) as one of the key intermediate. This key intermediate 2 and its salts has also been synthesized by various routes of synthesis described below.
Chiral auxiliary based asymmetric cyclopropanation and subsequent synthetic transformations was reported by WO. 01/92200 and WO 01/92263, involving (1R, 2S, 5R)'-2-isopropyl-5-methyl cyclohexanol 6 as described in Scheme 1.

Similarly Oppolzer's Sultam 11 has been utilized as chiral auxiliary by Bioorg. Med. Chem. Lett. 2007,17, 6013-6018 and WO 11/017108.,


Asymmetric chiral catalytic reduction by CBS-Catalyst, chiral oxazaborolidine and borane dimethyl sulfide complex, followed ' by . enantio and ' Serospecific triethylphosphonoacetate rearrangement in the presence or sodium hydride in toluene gave Ethyl (lR,2R)-2 (3,4 difluorophenyDcyclopropyl carboxylate 10, as per wo 08/018822 and WO 08/018823 (Scheme 3).


Another alternative involving asymmetric chiral catalytic reduction by CBS-catalyst reported in WO 11/132083, describes the use of nitro group to obtain amine,, by reduction ' (Scheme 4). Thus, chiral reduction of nitro ketone 12 gave 13, which on cyclization under Mitsunobu conditions gave desired chiral nitro cyclopropane 14, which on further reduction by Pd/C or Zn/HCl gave 2.


WO 12/001531 describes yet another alternative to CP-Amine (Scheme 5), involving asymmetric' cyclopropanation with ruthenium catalyst and (S,S)-2,6-bis(4-isopropyl-2-oxazolin-2-yl)pyridine 15, to get chiral ethyl ester 10. Basic hydrolysis of ester 10, followed by coupling of NH2OH under mixed anhydride anhydrous and subsequent rearrangements gave the desired CP-Amine 2 (Scheme-5).

Recently WO 2013/144295 describes hydrazide 18 route to acylazide 19 formation and subsequent heating to form acyl nitrene 20 intermediate for Curtius Rearrangement, which
produces isocyanate 21 and react with H2O under acidic
condition to form carbamate 22", which on decarboxylation

produces the HC1 salt of amine 23. On bas'if ication, isolation of desired CP-amine 2 is accomplished (Scheme 6).


Summary of the Invention
The present invention provides isoiation and characterization of a novel urea impurity compound 1, related to (1R,2S)-2-(3,4-Difluorophenyl)cyclopropylamine (CP-Amine 2), processes for its preparation and its use as Reference Standard to control the quality of CP-Amine 2. In another embodiment, a method of analysis, involving High Pressure Liquid Chromatography (HPLC) is disclosed, for identification of 1 in 2. In yet another embodiment, the invention describes CP-Amine 2_, comprising from 0.01 to 0.6 percent of " Impurity 1 . In a preferred embodiment, the invention discloses a novel urea analogue 1 In a most preferred embodiment, the invention provides the use of CP-Amine 2, comprising from. 0.01 to 0.6 percent of impurity of novel urea analogue 1, for the preparation (manufacture) of Ticagrelor.
Detailed Description of the Invention
In all these synthetic strategies discussed in prior art (see previous . section of "Background ' of Invention")involving Curtius or Curtius type rearrangement of Schmidt (hydrazide route) or Hofmann rearrangement (amide route) the intermediacy of acyl nitrene 20 is involved. The precursors of nitrene 20 are acyl azide 19 in Scheme 6, acyl chloride 10 in Scheme 1, mixed anhydride with ethyl chloroformate in Scheme 2, or diphenyl phosphoryl azide in Scheme 2, or amide 11 in Scheme 3, or Boc-anhydride and hydroxylamine in Scheme 5, or hydrazide 18 in Scheme 6, which require extractive work-up, mostly in toluene and large volume 20-30 fold of solvents per unit of substrates is require. Curtius rearrangement or. similar type of rearrangements involving nitrene intermediates eg. 20, required anhydrous conditions as the moisture present in the reaction mixture may react with isocyanate under reflux, condition. For large scale manufacturing, drying of huge volume of toluene is operationally very difficult. Even 0.2 to 0.3% of water in large volume of toluene creates enough problems, which.can trap fairly good amount of nitrene to form carbamate 22, and under refluxing conditions, this can generate enough amount of CP-amine 2, internally. Now this CP-amine internally traps, the in-situ generated isocyanate 21 and forms urea type of compound 1. In fact, in our hands this type of intermediate compound 1

was forming between 30-50% yield, which was very stable under acidic conditions, in which Curtius rearrangement or Curtius type rearrangements (Schmidt, Hoffmann etc.) are usually carried out. The positive aspect of this novel compound was that it was a very stable solid and therefore could be easily isolated and stored. In these synthetic sequences , most of the intermediates of acyl azide 19, amides, isocyanates 21 and carbamate 22 are liquid or very unstable. None of the patents mentioned above describe any physicochemical properties melting point/boiling point or any spectroscopic data for the characterization o.f these key intermediates in these synthesis of CP-amine 2. This is not a desirable property for bulk manufacturing and for process chemistry, where unstable intermediates are involved and there is very little in-process control. The purification of these liquid intermediates, with chiral centers by distillation, is also difficult. There is always a possibility of thermal degradation, isomerization or 'racemization during distillation. Considering all these limitations of prior art, this invention reports the isolation and characterization of a novel solid and stable compound 1, which is urea, analogue of CP-amine 2. The formation of 1 can be depicted as shown in Scheme 7.

Alternatively it is p.os.sible to get urea analogue 1 from the reaction of CP-amine 2 and its carbamate 22 (Scheme 6) available in plenty, under the reaction'conditions, also.
The urea analogue of 1 has been fully characterized by M.P., IR, MS,- 1H NMR & 13C-NMR. The data
Some of the characterization and selective spectroscopic data for 1 are given below:
IR (in cm-1) : 3311.9, 2991.7, 1633.8, 1560, 1514 cm"1.
MS: 364 for Molecular--ion peak.
169 for cyclopropylamine fragment as base peak.
1H NMR (CDC13) : 6.84 to 7.26 δ (6H, m, aromatic)., 5.3 δ
(2H, s, N-H) , 2.62 δ (s, 2H) , 2.0 δ (m, 2H),'1.86 δ (m,
4H) . •
13C NMR (CDC13, DEPT135): 122.3, 117.1, 115.3 (all the three peaks due to- aromatic C-H) , 32.5, 24.9 (both cyclopropyl C-H) and 16.4 (Cyclopropyl CH2)
It is possible to develop a reaction condition, where this compound 1, becomes a major product during Curtius or Curtius type rearrangement by adjusting water % age in toluene or by controlling pH of medium.
Since this compound 1 was a solid, operationally it became very easy to isolate, store and purify to proceed to next step for CP-amine 2 and finally to Ticagrelor 3. One can make different salts of this novel intermediate 1 eg. HC1, H2S04, pTSA, BSA etc. easily and these salts can be further characterized. On basification, one can get quantitative recovery of 1 from its salts.
In another aspect, the invention is directed to a process for preparing compound 1, by reacting pure CP-amine 2, with pure Isocyanate 21.
Alternatively, this compound 1 can be utilized as Reference Standard to control the quality of CP-Amine 2.
The invention also provides a method for determining the purity of CP-amine 2, which ' is free from this novel

compound 1. In yet another aspect, the present invention provides the quality of CP-amine 2, having about 0.01% to about 0.6% of compound 1 % area of HPLC. In yet another aspect, the present invention provides an HPLC method for the analysis of CP-amine 2 for its purity and assay determinations .
Novel compound 1 (CP-Amine urea analogue) may be used in a method for analyzing a sample of CP-Amine 2, by performing chromatography- on the sample.to obtain data and comparing the- data with the chromatography data of compound 1,, This impurity used may or may not be with CP-Amine 2, ie. Data may be generated for both the impurity and CP-Amine 2_ simultaneously (as part of the same solution/chromatogram) or separately. A person skilled- in the' art may prepare a solution of CP-Amine 2_, containing novel impurity compound 1, subjecting the solution to a high pressure liquid chromatography (HPLC) to obtain a chromatogram and comparing the peak obtained in chromatogram to a peak resulting from compound 1, by its retention time. Further, a person skilled in the art may also prepare a solution of CP-Amine 2_, containing the novel urea analogue ]L, and subject the solution to thin layer chromatography (TLC) and compare TLC' spots to each other. The presence of novel urea analogue impurity 1. may be used to select desirable batches of high purity of CP-Amine 2_, for production and manufacturing.
The present invention also provides a gradient elution HPLC method for quantifying by area percent, the amount of impurities present in CP-Amine 2. The method for determining the purity of CP-Amine 2! includes steps of (a) preparing a sample solution of CP-Amine 2_ in acetonitrile (HPLC grade), (b) injecting the sample solution on to an HPLC column, preferably a C18 column; (c) eluting the sample wi'th a solvent, preferably a mixture of acetonitrile and water; (d) adding a base, such as triethyl amine, and adjusting the pH. to about 6; and (e) measuring the amount of each impurity, especially the novel urea analogue 1 with a detector, preferably UV detector, attached with an appropriate recording device.
Preferably, the method of determining the quantity of novel
urea analogue impurity 1 in a sample of CP-Amine 2
comprises the steps of; (a) preparing a sample solution of

CP-Amine 2 in acetonitrile; (b) injecting the sample solution (around lOul) onto an about 100.0mm x 4.6mm, 3.5u C18 HPLC column; (c) gradient eluting the sample with a mixture of acetonitrile and water; (d) adding about 0.1% triethyl amine by weight in water and adjusting the pH to about 6 (preferably 6.1) with the help of diluted ortho-phosphoric acid (mobile phase A) and combining with HPLC grade acetonitrile (mobile phase B) to a ratio of about 80:20; and (e) measuring of amount of novel urea analogue impurity of 1 at 210 nm wavelength with a UV detector (having an appropriate recording device). The flow rate in HPLC was around 1.2- ml/min. The column temp, was around 3 5°C.
The mobile phase A (buffer).may be prepared by mixing about 1L water with about 1ml of triethyl amine and adjusting pH to 6.1 with diluted ortho-phosphoric acid.
The HPLC profile' for determining the purity and assay of CP-Amine 2__is exemplified in Table 1..
Table 1

Time (min) Mobile Phase' A Mobile Phase B
o.-oi 80 20
20 10 90
25 80 20
In the method described above, CP-Amine 2 has retention
time of about 5.75 min (± 0.2 min) , where as novel urea analogue 1 has retention time of 13'. 61 min (± 0.2 min) .
Another embodiment of the invention encompasses a method* for determining the purity of CP-Amine 2, comprising using novel urea analogue compound 1, as a reference marker.
Yet another embodiment of the invention discloses a method for determining the assay of CP-Amine, comprising using novel urea analogue compound 1, as a reference standard.
The method of analysis of 1 in 2 has been developed to determine the limits of 1 in 2, up to 0.001%.
Another embodiment. of the present invention is to make

Ticagrelor 3 from highly pure CP-Amine 2^ by employing the stable, novel solid urea analogue _1.
Following examples and HPLC method of analysis discloses the key aspect of the invention. These examples and HPLC methods are non-optimized processes and are only illustrative in nature and do not limit the scope and spirit-of invention. It will be apparent for those skilled in the - art that many obvious minor modifications, both in materials and methods, may be possible and practiced without departing from the scope of the invention.
Example-1 Preparation of 1-(1R,2S)-2-<3,4-Difluorophenyl)cyclopropyl-3--(1R,2S)-2-(3, 4-Difluorophenyl)cyclopropyl urea.
A mixture of cycloproopyl carboxylic acid 9 (20gm, 0.1 mole), triethyl amine (0.11 mole) and ethyl chloroformate _(0.12 mole) was dissolved in acetone 250 ml and 20 ml of of water at 0°C to 5°C. Sodium azide (0.22eq) in water was added to the reaction mixture and stirred until starting material is consumed. The acyl azide was extracted in -400ml of toluene, after pour in the reaction -mixter in water, Toluene extract was dried over sodium sulfate and heated to 100°C till the acyl azide disappears. Concentration of toluene layer and working up with water gave the urea analogue 1 (l'5gm) . Further acidic reflux and basification gave 2gm of cyclopropyl amine 2.
Example-2 Preparation of 1-(1R,2S)-2-(3/4-Difluorophenyl)cyclopropyl-3--(lR,2S)-2-(3,4-Difluorophenyl)cyclopropyl urea.
A mixture of cyclopropyl carboxylic acid 9 (20gm, 0.1 mole), dilsopropylethylamine (0.11 mole) and ethylchloroformate (0.12 mole) was dissolved in acetone 250 ml and 20 ml of water at -5°C to 0°C, sodium azide (0.2 mole) dissolved in water, a saturated solution, was added to the reaction mixture at 0°C and stirred until starting material'is consumed. The reaction mixture was poured into large excess of ice-water and extracted with 3x200ml of toluene. Toluene extracts were combined and dried over anyThe - filtered toluene solution slowly dropped over refluxing toluene (~100ml) . TLC showed

formation of 1, as major product. Concentration of. toluene layer and acid-base purification gave I6gm of 1.
Example-3 Preparation of 1- (1R,2S)-2-(3,4-
Difluorophenyl)cyclopropyl-3—(1R,2S)-2-(3,4-
Difluorophenyl)cyclopropyl urea.
A mixture of cyclopropyl carboxylic acid 9 (20gm, 0.1'mole) and triethyl amine (0.11 mole) was dissolved in 250ml of acetone and 20ml of water. The clear solution was cooled to around 0°C and ethyl chloroformate '(0.12 mole) in 50ml acetone was slowly dropped to the reaction mixture. After 30 min of stirring, a saturated solution of sodium azide (0.2 eq) in water was added to the reaction mixture of around 0°C. After 1 hour of stirring the reaction mixture was poured into large excess of ice-water and was extracted with toluene 3x150ml subsequently/ toluene extracts were dried over anhydrous sodium sulfate. The toluene solution was slowly dropped over refluxing toluene (~100ml) and TLC shows immediate formation of 1. Concentration of toluene and precipitation gave 16gm of 1.
Example-4 Preparation of 1- (1R, 2S) -2- (3, 4-Difluorophenyl)cyclopropyl-3--(1R, 2S)-2-(3, 4-Difluorophenyl)cyclopropyl urea.
2gm of 1 was dissolved into 20ml of methanol and 30ml conc. HC1 was added to it. A solid precipitate was obtained, which was washed with minimum amount of chilled methanol. The solid HCl salt of 1 was dried under vacuum. The yield was quantitative. Basification of HCl salt of lwith aqueous NaOH and extraction with ethylacetate. Drying of organic extract, and removal of solvent gave solid gave pure 1.
Example-5 Preparation of 1.
A pure sample of cyclopropyl amine 2 (1 eq.) from example-1 and cylcopropyl isocyanate (1 eq.) were stirred at RT in Toluene. There was instantaneous formation of an off-white solid 1, which was filtered and washed with hexane.

Brief Description of Annexures:
1. Annexure-1 is a representative IR spectrum for Compound 1.
2. Annexure-2 is a representative GC/MS spectrum for Compound 1_.
3. Annexure-3 is a representative 1H NMR spectrum for Compound 1.
4. Annexure-4 is a representative 13C NMR (DEPT' 135) spectrum' for Compound 1.

We claim:
(1) The compound (1R, 2S)-2- (3,4-diflurophenyl)cyclopropyl amine (CP-Amine 2) or its salts
comprising from 0.01 to 0.6 percent of impurity of formula 1^
(2) The use of above compound, according' to above claim 1, for the preparation of Ticagrelor 3.
(3) The compound 1, an urea analogue of CP-Amine 2.
(4) The process to make compound 3, according to claim 3.
(5) The use of compound 1,.
(6) The use of compound 1, according to claim 3, as "Reference Standard".
(7) A method of analysis by HPLC, according to claim 1, for identification and for guantifying the amount of 1 in 2_, comprising steps of
(a) preparing a sample solution of CP-Amine 2_ in acetonitrile.
(b) Injecting the sample solution of about 10 ul onto
an about 100mm x 4.6mm, 3 . 5u C18 HPLC column
(c) Gradient eluting the sample with a mixture of
a acetonitrile (mobile phase B) and a buffer with pH
6.1, adjusted with about 0.1% Triethyl amine and