EGFR INHIBITORS

Abstract

The application provides a compound having the general formula (I) or a pharmaceutically acceptable salt thereof, compositions including the compound and methods of using the compound. The compound of formula (I) can be used as a medicament.

Claims

1. A compound of formula (I) ##STR(I)## or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, wherein the compound is the compound of formula (I).

3. A process for the preparation of a compound according to any one of claims 1 to 2, comprising the coupling of a compound of formula (B1) ##STR(B1)## with a compound of formula (B2) ##STR00019## in the presence of a base and a catalyst.

4. A compound according to any one of claims 1 to 2, when manufactured according to a process of claim 3.

5. A compound according to claims 1 or 2 for use as therapeutically active substance.

6. A pharmaceutical composition comprising a compound according to claims 1 or 2 and a therapeutically inert carrier.

7. A compound according to claims 1 or 2 for use in the treatment or prophylaxis of cancer.

8. A compound according to claims 1 or 2 for use in the treatment or prophylaxis of non-small cell lung cancer.

9. The use of a compound according to claims 1 or 2 for the treatment or prophylaxis of cancer.

10. The use of a compound according to claims 1 or 2 for the treatment or prophylaxis of non-small cell lung cancer.

11. The use of a compound according to claims 1 or 2 for the preparation of a medicament for the treatment or prophylaxis of cancer.

12. The use of a compound according to claims 1 or 2 for the preparation of a medicament for the treatment or prophylaxis of non-small cell lung cancer.

13. A method for the treatment or prophylaxis of cancer, which method comprises administering an effective amount of a compound as defined in claims 1 or 2 to a patient in need thereof.

14. A method for the treatment or prophylaxis of non-small cell lung cancer, which method comprises administering an effective amount of a compound as defined in claims 1 or 2 to a patient in need thereof.

15. The invention as hereinbefore described.

Description

ASSAY PROCEDURES

[0057] The compound of formula (I) and pharmaceutically acceptable salts thereof possess valuable pharmacological properties. The compound was investigated in accordance with the test given hereinafter.

HTRF Phospho EGFR TMLRCS Assay (Cellular)

Cell Line and Media

[0058] BaF3-TMLRCS cell line were obtained from Crownbio (San Diego, CA, USA). Cells were maintained at 37° C., 5% CO.sub.2 in RPMI ATCC (Gibco 31870) + 2 mM Glutamine + 0.5 .Math.g/ml Puromycin supplemented with 10% fetal bovine serum (FBS) (Gibco).

Protocol

[0059] Cells are transferred as above to Greiner Bio-One, Nr. 784-08 micro-titerplate at 20000 cells/well in 12.5 .Math.l of growth medium/well after the plates had been pre-filled with 12.5 nl of DMSO solutions of the to be tested compound (in dose response) or DMSO only. After spinning the plates at 300 × g for 30 seconds the cells were incubated for 4 hours at 37C, 5% CO.sub.2, 95% humidity. The cells were lysed by adding to the compound mix 4 .Math.l/well of the supplemented lysis buffer (Cis-bio, Phospho-EGFR HTRF kit, 64EG1PEH), followed by incubation for 30 min at room temperature with shaking (400 rpm). The plates were then frozen and stored overnight at -80C. On the next day and after thawing the plates, 4 .Math.l of a mixture of anti-Phospho-EGFR Cryptate and of anti-Phospho-EGFR-d2 antibody solutions prepared in the supplied detection buffer was added to each well. The lidded plates were then incubated for 4 h at room temperature before reading the fluorescence emission at 616 and 665 nm using an Envision reader (Perkin Elmer). Data was analyzed in similar fashion as above using the normalized ratio of the 665 to 616 signals multiplied by 10000. The results are shown in Table 1.

TABLE-US-00001 BaF3 cellular HTRF Phospho EGFR TMLRCS assay data Exam. Structure IC.sub.50 (BaF3 TMLRCS) 1 [00006]2-[4-chloro-6-[2-[4-[[4-(hydroxymethyl)-1-piperidyl]methyl]phenyl]ethynyl]-1-oxo-isoindolin-2-yl]-2-(6,7 -dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-N-thiazol-2-yl-acetamide 3nM

[0060] The compound of formula (I) and their pharmaceutically acceptable salts can be used as medicaments (e.g. in the form of pharmaceutical preparations). The pharmaceutical preparations can be administered internally, such as orally (e.g. in the form of tablets, coated tablets, dragées, hard and soft gelatin capsules, solutions, emulsions or suspensions), nasally (e.g. in the form of nasal sprays), rectally (e.g. in the form of suppositories) or topical ocularly (e.g. in the form of solutions, ointments, gels or water soluble polymeric inserts). However, the administration can also be effected parenterally, such as intramuscularly, intravenously, or intraocularly (e.g. in the form of sterile injection solutions).

[0061] The compound of formula (I) and their pharmaceutically acceptable salts can be processed with pharmaceutically inert, inorganic or organic adjuvants for the production of tablets, coated tablets, dragées, hard gelatin capsules, injection solutions or topical formulations Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts etc. can be used, for example, as such adjuvants for tablets, dragées and hard gelatin capsules.

[0062] Suitable adjuvants for soft gelatin capsules, are, for example, vegetable oils, waxes, fats, semi-solid substances and liquid polyols, etc.

[0063] Suitable adjuvants for the production of solutions and syrups are, for example, water, polyols, saccharose, invert sugar, glucose, etc.

[0064] Suitable adjuvants for injection solutions are, for example, water, alcohols, polyols, glycerol, vegetable oils, etc.

[0065] Suitable adjuvants for suppositories are, for example, natural or hardened oils, waxes, fats, semi-solid or liquid polyols, etc.

[0066] Suitable adjuvants for topical ocular formulations are, for example, cyclodextrins, mannitol or many other carriers and excipients known in the art.

[0067] Moreover, the pharmaceutical preparations can contain preservatives, solubilizers, viscosity-increasing substances, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.

[0068] The dosage can vary in wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, in the case of oral administration a daily dosage of about 0.1 mg to 20 mg per kg body weight, preferably about 0.5 mg to 4 mg per kg body weight (e.g. about 300 mg per person), divided into preferably 1-3 individual doses, which can consist, for example, of the same amounts, should it be appropriate. In the case of topical administration, the formulation can contain 0.001% to 15% by weight of medicament and the required dose, which can be between 0.1 and 25 mg in can be administered either by single dose per day or per week, or by multiple doses (2 to 4) per day, or by multiple doses per week It will, however, be clear that the upper or lower limit given herein can be exceeded when this is shown to be indicated.

Pharmaceutical Compositions

[0069] The compound of formula (I) or a pharmaceutically acceptable salt thereof can be used as therapeutically active substances, e.g. in the form of a pharmaceutical preparation. The pharmaceutical preparation can be administered orally, e.g. in the form of tablets, coated tablets, dragées, hard and soft gelatin capsules, solutions, emulsions or suspensions. The administration can, however, also be effected rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of injection solutions.

[0070] The compound of formula (I) or a pharmaceutically acceptable salt thereof can be processed with pharmaceutically inert, inorganic or organic carriers for the production of a pharmaceutical preparation. Lactose, corn starch or derivatives thereof, talc, stearic acids or its salts and the like can be used, for example, as such carriers for tablets, coated tablets, dragées and hard gelatin capsules. Suitable carriers for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like. Depending on the nature of the active substance no carriers are however usually required in the case of soft gelatin capsules. Suitable carriers for the production of solutions and syrups are, for example, water, polyols, glycerol, vegetable oil and the like. Suitable carriers for suppositories are, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like.

[0071] The pharmaceutical preparation can, moreover, contain pharmaceutically acceptable auxiliary substances such as preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.

[0072] Medicaments containing a compound of formula (I) or a pharmaceutically acceptable salt thereof and a therapeutically inert carrier are also provided by the present invention, as is a process for their production, which comprises bringing one or more compound of formula (I) and/or pharmaceutically acceptable salts thereof and, if desired, one or more other therapeutically valuable substances into a galenical administration form together with one or more therapeutically inert carriers.

[0073] The dosage can vary within wide limits and will, of course, have to be adjusted to the individual requirements in each particular case. In the case of oral administration the dosage for adults can vary from about 0.01 mg to about 1000 mg per day of a compound of general formula (I) or of the corresponding amount of a pharmaceutically acceptable salt thereof. The daily dosage may be administered as single dose or in divided doses and, in addition, the upper limit can also be exceeded when this is found to be indicated.

[0074] The following examples illustrate the present invention without limiting it, but serve merely as representative thereof. The pharmaceutical preparations conveniently contain about 1-500 mg, particularly 1-100 mg, of a compound of formula (I). Examples of compositions according to the invention are:

Example A

[0075] Tablets of the following composition are manufactured in the usual manner:

TABLE-US-00002 possible tablet composition ingredient mg/tablet 5 25 100 500 Compound of formula (I) 5 25 100 500 Lactose Anhydrous DTG 125 105 30 150 Sta-Rx 1500 6 6 6 60 Microcrystalline Cellulose 30 30 30 450 Magnesium Stearate 1 1 1 1 Total 167 167 167 831

Manufacturing Procedure

[0076] 1. Mix ingredients 1, 2, 3 and 4 and granulate with purified water.

[0077] 2. Dry the granules at 50° C.

[0078] 3. Pass the granules through suitable milling equipment.

[0079] 4. Add ingredient 5 and mix for three minutes; compress on a suitable press.

Example B-1

[0080] Capsules of the following composition are manufactured:

TABLE-US-00003 possible capsule ingredient composition ingredient mg/capsule 5 25 100 500 Compound of formula (I) 5 25 100 500 Hydrous Lactose 159 123 148 - Corn Starch 25 35 40 70 Talk 10 15 10 25 Magnesium Stearate 1 2 2 5 Total 200 200 300 600

Manufacturing Procedure

[0081] 1. Mix ingredients 1, 2 and 3 in a suitable mixer for 30 minutes.

[0082] 2. Add ingredients 4 and 5 and mix for 3 minutes.

[0083] 3. Fill into a suitable capsule.

[0084] The compound of formula (I), lactose and corn starch are firstly mixed in a mixer and then in a comminuting machine. The mixture is returned to the mixer; the talc is added thereto and mixed thoroughly. The mixture is filled by machine into suitable capsules, e.g. hard gelatin capsules.

Example B-2

[0085] Soft Gelatin Capsules of the following composition are manufactured:

TABLE-US-00004 possible soft gelatin capsule ingredient composition ingredient mg/capsule Compound of formula (I) 5 Yellow wax 8 Hydrogenated Soya bean oil 8 Partially hydrogenated plant oils 34 Soya bean oil 110 Total 165

TABLE-US-00005 possible soft gelatin capsule composition ingredient mg/capsule Gelatin 75 Glycerol 85 % 32 Karion 83 8 (dry matter) Titan dioxide 0.4 Iron oxide yellow 1.1 Total 116.5

Manufacturing Procedure

[0086] The compound of formula (I) is dissolved in a warm melting of the other ingredients and the mixture is filled into soft gelatin capsules of appropriate size. The filled soft gelatin capsules are treated according to the usual procedures.

Example C

[0087] Suppositories of the following composition are manufactured:

TABLE-US-00006 possible suppository composition ingredient mg/supp. Compound of formula (I) 15 Suppository mass 1285 Total 1300

Manufacturing Procedure

[0088] The suppository mass is melted in a glass or steel vessel, mixed thoroughly and cooled to 45° C. Thereupon, the finely powdered compound of formula (I) is added thereto and stirred until it has dispersed completely. The mixture is poured into suppository moulds of suitable size, left to cool; the suppositories are then removed from the moulds and packed individually in wax paper or metal foil.

Example D

[0089] Injection solutions of the following composition are manufactured:

TABLE-US-00007 possible injection solution composition ingredient mg/injection solution. Compound of formula (I) 3 Polyethylene Glycol 400 150 acetic acid q.s. ad pH 5.0 water for injection solutions ad 1.0 ml

Manufacturing Procedure

[0090] The compound of formula (I) is dissolved in a mixture of Polyethylene Glycol 400 and water for injection (part). The pH is adjusted to 5.0 by acetic acid. The volume is adjusted to 1.0 ml by addition of the residual amount of water. The solution is filtered, filled into vials using an appropriate overage and sterilized.

Example E

[0091] Sachets of the following composition are manufactured:

TABLE-US-00008 possible sachet composition ingredient mg/sachet Compound of formula (I) 50 Lactose, fine powder 1015 Microcrystalline cellulose (AVICEL PH 102) 1400 Sodium carboxymethyl cellulose 14 Polyvinylpyrrolidon K 30 10 Magnesium stearate 10 Flavoring additives 1 Total 2500

Manufacturing Procedure

[0092] The compound of formula (I) is mixed with lactose, microcrystalline cellulose and sodium carboxymethyl cellulose and granulated with a mixture of polyvinylpyrrolidone in water. The granulate is mixed with magnesium stearate and the flavoring additives and filled into sachets.

EXAMPLES

[0093] The following examples are provided for illustration of the invention. They should not be considered as limiting the scope of the invention, but merely as being representative thereof.

Abbreviations

[0094] AcOH = acetic acid; DCM = dichloromethane; DIPEA = diisopropylethylamine; DMAP = dimethylaminopyridine; DMF = dimethylformamide; DMSO = diemethyl sulfoxide; ESI = electrospray ionization; EtOAc = ethyl acetate; EtOH = ethanol; GTP = guanosine triphosphate; HATU = hexafluorophosphate azabenzotriazole tetramethyl uronium; HPLC = high performance liquid chromatography; MeOH = methanol; MS = mass spectrometry; NMP = N-methyl-2-pyrrolidone; NMR = nuclear magnetic resonance; RT = room temperature; THF = tetrahydrofuran; TPP = triphenylphosphine; TRIS = tris(hydroxymethyl)aminomethane.

Example 1

[0095] (2RS)[4-Chloro-6-[2-[4-[[4-(hydroxymethyl)-1-piperidyl]methyl]phenyl]ethynyl]-1-oxo-isoindolinyl](6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-N-thiazolyl-acetamide

##STR00007##

Step 1: Ethyl 2-(6,7-dihydro-5H-pyrrolo[1,2-climidazol-1-yl)-2-oxo-acetate

##STR00008##

[0096] To a solution of ethyl 2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate (20.0 g, 102.97 mmol) dissolved in 200 ml of 1,4-dioxane was added selenium dioxide (22.85 g, 205.94 mmol, 2 equiv.). The reaction mixture was stirred for 5 hours at 80° C. The reaction mixture was concentrated under vacuum to give a residue. The crude product was purified by flash chromatography on a silica gel column eluting with petroleum ether:ethyl acetate 2:1 to ethyl acetate:ethanol 10:1 gradient to obtain the desired ethyl 2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-2-oxo-acetate (quant. yield) as a light brown oil, MS: m/e = 209.1 (M+H.sup.+).

[0097] Step 2: Ethyl 2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-2-hydroxyimino-acetate

##STR00009##

To a solution of ethyl 2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-2-oxo-acetate (Example 1, step 1) (17.5 g, 84.05 mmol) dissolved in 145 ml of ethanol was added hydroxylamine hydrochloride (6.42 g, 92.45 mmol, 1.1 equiv.) and sodium acetate (13.79 g, 168.1 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred for 3.5 hours at 80° C. The reaction mixture was concentrated and extracted with water and five times with a mixture of ethanol/THF/ethyl acetate 1: 1:8. The organic layers were concentrated to dryness. The desired ethyl 2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-2-hydroxyimino-acetate (15 g, 80 % yield) was obtained as a yellow solid, MS: m/e = 224.1 (M+H.sup.+) and used directly in the next step.

[0098] Step 3: Ethyl (2RS)-2-amino-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate

##STR00010##

To a solution of ethyl 2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-2-hydroxyimino-acetate (Example 1, step 2) (15.0 g, 67.2 mmol) dissolved in 225 ml of ethanol and 120 ml of THF was added Pd/C (30.0 g, 67.2 mmol, 1 eq, 10 %) at room temperature. The mixture was hydogenated with H.sub.2 for 24 hours at 45° C. The reaction mixture was filtered and the filtrate was concentrated under vacuum. The desired ethyl (2RS)-2-amino-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate (quant. yield) was obtained as a brown oil, MS: m/e = 210.1 (M+H.sup.+) and used directly in the next step.

[0099] Step 4: Ethyl (2RS)-2-amino-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate hydrochloride

##STR00011##

A solution of ethyl (2RS)-2-amino-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate (Example 1, step 3) (15.0 g, 82.79 mmol) in HCl/EtOH (300 ml, 1200 mmol, 14.5 equiv., 2.5 mol/L) was stirred at 25° C. for 36 hours. The reaction mixture was concentrated under vacuum below 25° C. to give a residue as brown oil. 150 ml of acetonitrile were added to the residue and the precipitated yellow solid was collected and dried under vacuum below 25° C. to give the desired ethyl (2RS)-2-amino-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate hydrochloride (quant. yield) as yellow solid, MS: m/e = 210.1 (M+H.sup.+).

[0100] Step 5: Ethyl 2-(bromomethyl)-3-chloro-5-iodo-benzoate

##STR00012##

3-Chloro-5-iodo-2-ethyl-benzoate (57.3 g, 176 mmol) was dissolved in 400 ml tetrachloroethylene and N-bromosuccinimide (46.9 g, 265 mmol, 1.5 equiv.) and AIBN (13.4 g, 88.3 mmol, 0.5 equiv.) were added at room temperature. The mixture was stirred at 80° C. for 16 hours. The reaction mixture was concentrated to dryness. The crude product was purified by flash chromatography on a silica gel column eluting with a petroleum ether:ethyl acetate 1:0 to 10:1 gradient to obtain the desired product (56 g, 76 % yield) as a pink solid.

[0101] Step 6: Ethyl (2RS)-2-(4-chloro-6-iodo-1-oxo-isoindolin-2-yl)-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate

##STR00013##

Ethyl (2RS)-2-amino-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate hydrochloride (Example 1, step 4) (12 g, 48.8 mmol, 1 equiv.) was dissolved in 120 ml of dioxane and 20 ml of DMF. Ethyl 2-(bromomethyl)-3-chloro-5-iodo-benzoate (Example 1, step 5) (19.7 g, 48.8 mmol) and diisopropylethylamine (34 ml, 195 mmol, 4 equiv.) were added at room temperature. The mixture was stirred at room temperature for 30 minutes and at 60° C. for 2 hours. The reaction mixture was extracted with water and two times with ethyl acetate. The organic layers were extracted with brine, dried over sodium sulfate and concentrated to dryness. The crude product was purified by flash chromatography on a silica gel column eluting with an ethyl acetate:methanol 100:0 to 90:10 gradient to obtain the desired product (14.5 g, 55 % yield) as a light red solid, MS: m/e = 486.2 (M+H.sup.+).

[0102] Step 7: (2RS)-2-(4-Chloro-6-iodo-1-oxo-isoindolin-2-yl)-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-l-yl)-N-thiazol-2-yl-acetamide

##STR00014##

Ethyl (2RS)-2-(4-chloro-6-iodo-1-oxo-isoindolin-2-yl)-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)acetate (Example 1, step 6) (14.5 g, 29.9 mmol) was dissolved in 70 ml of methanol and 70 ml of THF. LiOH (1M in water) (32.8 ml, 32.8 mmol, 1.1 equiv.) was added at room temperature. The mixture was stirred for 30 minutes at room temperature. The reaction mixture was concentrated in vacuo to dryness and the residue was dissolved in 140 ml of DMF. Thiazol-2-amine (3.1 g, 31.3 mmol, 1.05 equiv.), Hunig’s base (15.6 ml, 89.6 mmol, 3 equiv.) and HATU (13.6 g, 35.8 mmol, 1.2 equiv.) were added at room temperature. The mixture was stirred at room temperature for 90 minutes. The reaction mixture was extracted with water and two times with ethyl acetate. The organic layers were extracted with water, dried over sodium sulfate and concentrated to dryness. The crude product was purified by flash chromatography on a silica gel column eluting with a dichloromethane:methanol 100:0 to 90:10 gradient to obtain the desired product (10.5 g, 59 % yield) as a light brown solid, MS: m/e = 540.1 (M+H.sup.+).

[0103] Step 8: [1-[(4-Ethynylphenyl)methyl]-4-piperidyl]methanol

##STR00015##

4-Ethynylbenzaldehyde (500 mg, 3.84 mmol) was dissolved in 3 ml of dichloromethane. Piperidin-4-ylmethanol (490 mg, 4.23 mmol, 1.1 equiv.) and sodium triacetoxyborohydride (1.3 g, 6.15 mmol, 1.6 equiv.) were added at room temperature. The mixture was stirred at room temperature for 1.5 hours. The reaction mixture was extracted with water and two times with dichloromethane. The organic layers were extracted with brine, dried over sodium sulfate and concentrated to dryness. The crude product was purified by flash chromatography on a silica gel column eluting with a dichloromethane:methanol 100:0 to 90:10 gradient to obtain the desired product (900 mg, 94 % yield) as an orange oil, MS: m/e = 230.2 (M+H.sup.+).

[0104] Step 9: (2RS)-2-[4-Chloro-6-[2-[4-[[4-(hydroxymethyl)-1-piperidyl]methyl]phenyl]ethynyl]-1-oxo-isoindolin-2-yl-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-N-thiazol-2-yl-acetamide

##STR00016##

(2RS)-2-(4-Chloro-6-iodo-1-oxo-isoindolin-2-yl)-2-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-1-yl)-N-thiazol-2-yl-acetamide (Example 1, step 7) (70 mg, 0.13 mmol) and [1-[(4-ethynylphenyl)methyl]-4-piperidyl]methanol (Example 1, step 8) (39 mg, 0.17 mmol, 1.3 equiv.) were dissolved in 1 ml of DMF. Triethylamine (0.05 ml, 0.39 mmol, 3 equiv.), bis-(triphenylphosphine)-palladium(II)dichloride (4.6 mg, 0.007 mmol, 0.05 equiv.), triphenylphosphine (3.4 mg, 0.013 mmol, 0.1 equiv.) and copper(I)iodide (1 mg, 0.007 mmol, 0.05 equiv.) were added and the mixture was stirred for 3 hours at 80° C. The reaction mixture was extracted with water and two times with ethyl acetate. The organic layers were extracted with brine, dried over sodium sulfate and concentrated to dryness. The crude product was purified by flash chromatography on a silica gel column eluting with a dichloromethane:methanol 100:0 to 90:10 gradient to obtain the desired product (26 mg, 30 % yield) as a white solid, MS: m/e = 641.4 (M+H.sup.+).