METHOD FOR CLEAVING AMIDE BONDS

Abstract

A method for cleaving amide bonds, comprising: a) providing a molecule comprising an amide group; b) reacting the molecule comprising an amide group with hydroxylamine (NH.sub.2OH) or a salt thereof to cleave the amide bond of the amide group.

Claims

1-30. (canceled)

31. A method for cleaving amide bonds, comprising: a) providing a molecule comprising an amide group, wherein the amide group is a primary, secondary or tertiary amide group; b) reacting the molecule comprising an amide group with hydroxylamine (NH.sub.2OH) or a salt thereof to cleave the amide bond of the amide group.

32. The method according to claim 31, wherein the method further comprises: c) recovering a product formed by the reaction of step b).

33. The method according to claim 31, wherein the amide group is an N-acyl amide group.

34. The method according to claim 31, wherein the molecule comprising an amide group further comprises a pH sensitive chiral center.

35. The method according to claim 31, wherein the molecule comprising an amide group further comprises a pH sensitive protecting group.

36. The method according to claim 31, wherein step b) comprises reacting the molecule comprising an amide group with the hydroxylamine or salt thereof at a temperature of 100? C. or less.

37. The method according to claim 31, wherein step b) comprises reacting the molecule comprising an amide group with the hydroxylamine or salt thereof for 2-200 hours.

38. The method according to claim 31, wherein step b) comprises reacting the molecule comprising an amide group with hydroxylamine in water.

39. The method according to claim 31, wherein the molar concentration of hydroxylamine in step b) is in the range of 5-20 M.

40. The method according to claim 31, wherein step b) comprises reacting the molecule comprising an amide group with a hydroxylamine salt.

41. The method according to claim 40, wherein the hydroxylamine salt is a salt of hydroxylamine and hydroiodic acid or trifluoroacetic acid.

42. The method according to claim 31, wherein the concentration of the hydroxylamine salt in step b) is in the range of 0.1-5 M.

43. The method according to claim 31, wherein the reaction in step b) is performed in a solvent capable at least of at least partially dissolving the hydroxylamine salt.

44. The method according to claim 31, wherein the reaction in step b) is performed at a pH value in the range of 4-12.

45. The method according to claim 31, wherein the reaction in step b) is performed at a pH value in the range of 9-11.

46. The method according to claim 44, wherein the pH of the reaction is lowered to a value in the range of 4-9 by addition of a pH reducing agent selected from the group consisting of mineral acids, organic acids and pH reducing salts, and combinations thereof.

47. The method according to claim 46, wherein the pH reducing agent is a pH reducing salt.

48. Use of hydroxylamine or a salt thereof for cleaving an amide bond in a molecule comprising an amide group.

Description

EXAMPLES

[0053] Without desiring to be limited thereto, the present invention will in the following be illustrated by way of examples.

[0054] Analysis Methods

[0055] .sup.1H NMR spectra were recorded on a BRUKER Biospin AVANCE 400 spectrometer. Chemical shifts are reported as ? values downfield from internal TMS in appropriate organic solutions. The purity and the structures of the products were confirmed by LCMS (254 nm) on a Waters 2690 photodiode array detector system using the following conditions: Column, Symmetry C-18; Solvent A, water 0.1% formic acid; Solvent B, CH.sub.3CN; flow rate, 2.5 ml/min; run time, 4.5 min; gradient, from 0 to 100% solvent B; mass detector, micro mass ZMD. Purifications were carried out directly by mass-triggered preparative LCMS Waters X-Terra reverse-phase column (C-18, 5 microns silica, 19 mm diameter, 100 mm length, flow rate of 40 ml/minute) and decreasingly polar mixtures of water (containing 0.1% formic acid) and acetonitrile as eluent. The fractions containing the desired compound were evaporated to dryness to afford the final compounds usually as solids.

EXAMPLE 1

Preparation of N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(trityloxy)hexan-2-yl)acetamide

[0056] ##STR00002##

[0057] A solution of N-((2R,3S,5S)-2,4,5-trihydroxy-6-trityloxymethyl-tetrahydro-pyran-3-yl)-acetamide (556 mg, 1.20 mol, 1.00 eq.) in a mixture of THF-H.sub.2O (20 ml, 4:1) at r.t., was treated with solid sodium borohydride (49.92 mg, 1.32 mol, 1.10 eq.) [gas evolution]. The reaction mixture was stirred at r.t. for 2 h, concentrated to dryness to afford N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(trityloxy)hexan-2-yl)acetamide (500 mg, 89.54%) as a white solid that was used without further purification.

[0058] LCMS: t.sub.R=1.01 min., purity=100%; ES+, 464.26 (M?H).sup.?.

EXAMPLE 2

Deacetylation of N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(trityloxy)hexan-2-yl)acetamide

[0059] A suspension of N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(trityloxy)hexan-2-yl)acetamide (1 eq) in hydroxylamine (10 volumes) was either treated with acid additives to lower the pH to 7 or not as set out in Table 1, Examples 1-10. The mixture was heated at 80? C. until full conversion of the deacetylation was reached. Deacetylation of N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(trityloxy)hexan-2-yl)acetamide with hydrazine (pH 13) under the same conditions as in Example 2-1 is also included as Example 2-10.

[0060] The results are displayed in Table 1. The results show that the deacetylation procedure proceeds considerably faster with hydroxylamine than with hydrazine, and is significantly by the addition of a pH reducing agent.

TABLE-US-00001 TABLE 1 Solvent Time to reach Example (50 vols)* Additive pH 100% conversion 2-1 50% NH.sub.2OH (aq) None 10.2 72 h 2-2 50% NH.sub.2OH (aq) HCl 7 12 h 2-3 50% NH.sub.2OH (aq) HBr 7 9 h 2-4 50% NH.sub.2OH (aq) HI 7 5 h 2-5 50% NH.sub.2OH (aq) H.sub.2SO.sub.4 7 29 h 2-6 50% NH.sub.2OH (aq) CH.sub.3COOH 7 6 h 2-7 50% NH.sub.2OH (aq) TFA 7 4 h 2-8 50% NH.sub.2OH (aq) (CH.sub.3).sub.3COOH 7 5 h 2-9 50% NH.sub.2OH (aq) CH.sub.3CH.sub.2COOH 7 8 h 2-10 NH.sub.2NH.sub.2H.sub.2O None 13 120 h

[0061] The reaction mixtures were purified directly by Preparative LCMS to afford (2R,3R,4S)-2-amino-6-(trityloxy)hexane-1,3,4,5-tetraol as a white solid.

[0062] LCMS: t.sub.R=0.88 min., purity=99%; ES+, 422.11 (M?H).sup.?.

[0063] .sup.1H NMR (DMSO-d.sub.6) ?: 7.47-7.37 (m, 6H), 7.30 (dd, J=8.3, 6.7 Hz, 6H), 7.26-7.15 (m, 3H), 3.92 (m, 1H), 3.83-3.74 (m, 1H), 3.62-3.53 (m, 1H), 3.52-3.41 (m, 1H), 3.34-3.27 (m, 1H), 3.22-3.16 (m, 1H), 3.13-3.04 (m, 1H), 3.01-2.91 (m, 1H)

EXAMPLE 3

Preparation of N-(4-aminophenethyl)acetamide

[0064] ##STR00003##

[0065] A 4-(2-aminoethyl)aniline (1.50 g; 11.01 mmol; 1.00 eq.) was added neat p-cresyl acetate (1.65 g, 11.0 mmol, 1.00 eq.) and the reaction mixture was stirred at room temperature for 30 h. The resulting orange solution was absorbed directly on silica gel and purified by flash chromatography (silica gel, DCM/MeOH 0-5%) to afford N-(4-aminophenethyl)acetamide (1.76 g, 89.7% yield)

[0066] LCMS: t.sub.R=0.58 min., purity=99.5%; ES+, 179.5 (M+H).sup.+.

[0067] .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ? 1.78 (s, 3H), 2.50 (m, 2H hidden by DMSO signal) 3.14 (m, 2H), 4.83 (s, 2H), 6.49 (d, J=7.5 Hz, 2H), 6.84 (d, J=7.5 Hz, 2H), 7.82 (s, 1H).

EXAMPLE 4

Preparation of tert-butyl (4-(2-acetamidoethyl)phenyl)carbamate

[0068] ##STR00004##

[0069] To a stirred solution of N-[2-(4-Amino-phenyl)-ethyl]-acetamide (500 mg, 2.81 mmol, 1.00 eq.) in DCM (20 ml) at r.t., was added triethylamine (0.51 ml, 3.65 mmol, 1.30 eq.) followed by di-tert-butyl dicarbonate (673.48 mg, 3.09 mmol, 1.10 eq.). The reaction mixture is stirred at r.t. for 1 h, washed with water (5 ml), a saturated solution of NaHSO.sub.4 (aq) (5 ml) and water (3?5 ml), dried over MgSO.sub.4 and concentrated to dryness to afford tert-butyl (4-(2-acetamidoethyl)phenyl)carbamate (496 mg, 63% yield) as a pale orange solid.

[0070] LCMS: t.sub.R=1.11 min., purity=100%; ES+, 279.5 (M+H).

[0071] .sup.1H-NMR (DMSO-d.sub.6) ? 1H NMR (400 MHz, DMSO-d6) ? 1.57 (s, 9H), 1.87 (s, 3H), 2.75-2.64 (m, 2H), 3.36-3.20 (m, 2H), 7.27-7.07 (m, 2H), 7.45 (d, J=8.3 Hz, 2H), 7.94 (t, J=5.6 Hz, 1H), 9.31 (s, 1H).

EXAMPLE 5

Preparation of NH.SUB.2.OH.HI

[0072] To a stirred solution of 50% NH.sub.2OH (aq) (9.28 ml, 0.15 mol, 1.00 eq) at 0? C. was added carefully dropwise 57% HI (aq) over a period of 5 minutes until a pH of 7 was achieved. A dense white crystalline solid formed that was collected by filtration, washed carefully with ice cold water to afford hydroxylamine hydrogen iodide (6.80 g, 28%).

EXAMPLE 6

Preparation of NH.SUB.2.OH.TFA

[0073] To a stirred solution of 50% NH.sub.2OH (aq) (9.28 ml, 0.15 mol, 1.00 eq) at 0? C. was added carefully dropwise TFA over a period of 5 minutes until a pH of 7 was achieved. The reaction mixture was concentrated under nitrogen sparging to afford hydroxylamine.trifluoroacetate (11.0 g, 98%) as clear colourless oil.

EXAMPLE 7

Comparative Studies of NH.SUB.2.OH and Salts thereof Versus Commonly Used Transamidation Agents such as NH.SUB.2.NH.SUB.2..H.SUB.2.O and NaOH

[0074] ##STR00005##

[0075] To a stirred solution/suspension of tent-butyl (4-(2-acetamidoethyl)phenyl)carbamate (50 mg, 0.18 mmol) in the chosen solvent (5 volumes) was added the salt (5 eq) and the resulting mixture was heated at 80? C. for the time necessary to complete the reaction. The results are displayed in Table 2. The results show that the deacetylation procedure proceeds quickly with for example hydroxylamine hydrogen iodide (Example 7-3) or hydroxylamine trifluoroacetate (Example 7-9), even when the relative concentration of hydroxylamine in the salts is much lower than the concentration of hydroxylamine alone in Example 7-1.

[0076] LCMS: t.sub.R=0.81 min., purity=100%; ES+, 237.51(M+H).sup.+.

[0077] .sup.1H-NMR (DMSO-d.sub.6) ? 1H NMR (400 MHz, DMSO-d.sub.6) ? 9.26 (s, 1H), 8.40 (s, 1H), 7.38 (d, J=8.0 Hz, 2H), 7.11 (d, J=8.0 Hz, 2H), 2.89 (m, 2H), 2.80-2.63 (m, 2H), 1.47 (s, 9H) (isolated as formate salt).

TABLE-US-00002 TABLE 2 Solvent 1 h 2 h (% 4 h Example (5 vols)* Additive pH (% conv.) conv.) (% conv.) 7-1 50% NH.sub.2OH None 10.2 34.8 64.7 83.0 (aq) 7-2 50% NH.sub.2OH 5 eq 9 48.6 83.5 97.0 (aq) NH.sub.2OHHI 7-3 EtOH/H.sub.2O 5 eq 7 63.8 85.8 98.9 (4:1) NH.sub.2OHHI 7-4 NH.sub.2NH.sub.2H.sub.2O None 13 13.6 34.9 35.2 7-5 NH.sub.2NH.sub.2H.sub.2O 5 eq 13 57.9 86.9 97.4 NH.sub.2OHHI 7-6 EtOH (4 4N NaOH (aq) 14 3.7 11.63 14.5 vols) (1 vol) 7-7 EtOH/H.sub.2O 5 eq 7 3.4 5.8 17.2 (4:1) NH.sub.2OHHCl 7-8 EtOH/H.sub.2O 5 eq 7 0 0.2 0.7 (4:1) NH.sub.2OHH.sub.2SO.sub.4 7-9 EtOH/H.sub.2O 5 eq 7 34.2 72.4 91.3 (4:1) NH.sub.2OHTFA 7-10 EtOH/H.sub.2O 5 eq NH.sub.4I 7 0 0 0 (4:1) *Volume = 1 g = 1 ml = 1 volume

EXAMPLE 8

Preparation and Deacetylation of Reduced HA-4 by Hydroxylamine

[0078] Diaminotetra-HA (DA-4HA) was synthesized according to the below scheme:

##STR00006##

[0079] Step 1

[0080] A solution of HA-4 (500 mg, 0.61 mmol) in water (5 ml) at room temperature was treated with sodium borohyride (23.05 mg, 0.61 mmol) and the resulting solution was stirred for 3 h, concentrated to dryness to afford the reduced product 1 (532 mg, assumed 100%) as a white foam.

[0081] LCMS (t.sub.r=0.28 min., ES+=779.4 (M?2Na+2H)

[0082] Step 2

[0083] The reduced product 1 (532 mg) was dissolved in aqueous NH.sub.2OH (5 ml, 50% v/v/) and solid NH.sub.4I (100 mg) was added. The resulting suspension was heated at 70? C. for 48 h, cooled to room temperature and concentrated to dryness to afford a residue. The residue was precipitated in neat EtOH and the resulting precipitate was collected by filtration and dried to a constant weight to afford the a 1:1 mixture of diamine 2 and mono-amine 3 in quantitative yield.

[0084] 2: LCMS (t.sub.r=0.16 min., ES+=695.36 (M?2Na+2H)

[0085] 3: LCMS (t.sub.r=0.19 min., ES+=737.47 (M?2Na+2H)

EXAMPLE 9

Deacetylation of Reduced HA-4 by NH.SUB.2.OH.HI

[0086] Reduced HA-4 (532 mg) prepared as described in Step 1 of Example 26, is dissolved in EtOH-H.sub.2O (2.5 ml, 1:1) and solid NH.sub.2OH.HI (491 mg, 3.05 mmol) is added. The resulting suspension is heated at 80? C. for 6 h, cooled to room temperature and the reaction mixture is purified by Preparative HILIC chromatography to afford deacetylated HA-4 as a white solid. Deacetylated HA-4: LCMS (t.sub.r=0.16 min., ES+=695.36 (M?2Na+2H)

EXAMPLE 10

Deacetylation of Hyaluronic Acid by Hydroxylamine

[0087] 0.2 g or 20 g of HA (Mw 2 500 kDa, DoA 100%) was solubilised in hydroxylamine (Sigma-Aldrich 50 vol % solution), or a mixture of hydroxylamine/water as set out in Table 3. The solution was incubated in darkness and under argon at 30-70? C. for 5-353 hours. After incubation, the mixture was precipitated by ethanol. The obtained precipitate was filtered, washed with ethanol and then re-dissolved in water. The solution was purified by ultrafiltration and subsequently lyophilized to obtain the deacetylated HA (de-Ac HA) as a white solid. Examples 10-1 to 10-14 were performed using approx. 0.2 g HA and examples 10-15 to 10-16 were performed using 20 g HA. The results are displayed in Table 3. Deacetylation by hydroxylaminolysis is more efficient, and conserves the Mw of the HA backbone better as compared to hydrazinolysis (Example 11) and alkaline methods (Examples 12-13).

TABLE-US-00003 TABLE 3 Start Temp Time Mw NMR Mw Example (? C.) (h) pH Conditions (kDa) DoA (%) (kDa) 10-1 30 24 10 NH.sub.2OH 2500 99 970.sup.a (50 wt % in water) 10-2 30 72 10 NH.sub.2OH 2500 98 1060.sup.a (50 wt % in water) 10-3 30 196 10 NH.sub.2OH 2500 95 1060.sup.a (50 wt % in water) 10-4 40 24 10 NH.sub.2OH 2500 98 1050.sup.a (50 wt % in water) 10-5 40 72 10 NH.sub.2OH 2500 95 980.sup.a (50 wt % in water) 10-6 40 353 10 NH.sub.2OH 2500 80 490.sup.a (50 wt % in water) 10-7 40 24 10 NH.sub.2OH 2500 99 1090.sup.a (35 wt % in water) 10-8 40 24 10 NH.sub.2OH 2500 100 1130.sup.a (20 wt % in water) 10-9 40 24 10 NH.sub.2OH 1000 98 670.sup.b (50 wt % in water) 10-10 55 5 10 NH.sub.2OH 2500 99 1010.sup.a (50 wt % in water) 10-11 55 72 10 NH.sub.2OH 2500 86 740.sup.a (50 wt % in water) 10-12 55 120 10 NH.sub.2OH 2500 78 400.sup.b (50 wt % in water) 10-13 60 24 10 NH.sub.2OH 2500 92 930.sup.b (50 wt % in water) 10-14 70 24 10 NH.sub.2OH 2500 86 720.sup.b (50 wt % in water) 10-15 40 72 10 NH.sub.2OH 2500 95 1870.sup.b (50 wt % in water) 10-16 55 72 10 NH.sub.2OH 2500 89 1050.sup.b (50 wt % in water) .sup.aSEC-UV .sup.bSEC-MALS

EXAMPLE 11

Deacetylation of Hyaluronic Acid by HydrazinolysisComparative Example

[0088] 0.2 g of HA (Mw 2 500 kDa, DoA 100%) was solubilised in 10 mL of a 1% solution of hydrazine sulphate in hydrazine monohydrate. The reaction took place in dark and under argon at 30-55? C. for 24-120 hours. The mixture was precipitated by ethanol. The precipitate obtained was filtered, washed with ethanol and then re-dissolved in water. The final deacetylated HA product was obtained after ultrafiltration, and freeze-dried. The results are displayed in Table 4. Deacetylation by hydrazinolysis gives more degradation of the HA backbone, i.e. lower Mw of the deacetylated product as compared to hydroxylaminolysis (Examples 10-1 to 10-16).

TABLE-US-00004 TABLE 4 Mw (SEC Temp Time DoA MALS) Example (? C.) (h) pH Conditions (%) (kDa) 11-1 30 24 13 NH.sub.2NH.sub.2 + 100 220 NH.sub.2NH.sub.2H.sub.2SO.sub.4 11-2 30 120 13 NH.sub.2NH.sub.2 + 96 320 NH.sub.2NH.sub.2H.sub.2SO.sub.4 11-3 40 48 13 NH.sub.2NH.sub.2 + 96 260 NH.sub.2NH.sub.2H.sub.2SO.sub.4 11-4 40 120 13 NH.sub.2NH.sub.2 + 92 170 NH.sub.2NH.sub.2H.sub.2SO.sub.4 11-5 55 24 13 NH.sub.2NH.sub.2 + 93 60 NH.sub.2NH.sub.2H.sub.2SO.sub.4 11-6 55 48 13 NH.sub.2NH.sub.2 + 89 70 NH.sub.2NH.sub.2H.sub.2SO.sub.4 11-7 55 72 13 NH.sub.2NH.sub.2 + 83 40 NH.sub.2NH.sub.2 H.sub.2SO.sub.4 11-8 55 120 13 NH.sub.2NH.sub.2 + 77 50 NH.sub.2NH.sub.2H.sub.2SO.sub.4

EXAMPLE 12

Deacetylation of Hyaluronic Acid by Homogeneous Alkaline HydrolysisComparative Example

[0089] HA (1 000 kDa) was weighed to a reaction vessel, NaOH solution was added and the reaction was mixed until a homogenous solution was obtained. The mixture was incubated without stirring and subsequently diluted with water and EtOH. The mixture was neutralized by adding 1.2 M HCl, precipitated by adding EtOH. The precipitate was washed with ethanol (70 w/w %) followed by ethanol and dried in vacuum over night to obtain a solid. The results are displayed in Table 5.

[0090] Deacetylation by homogenous alkaline hydrolysis gives more degradation of the HA backbone, i.e. lower Mw of the deacetylated product as compared to hydroxylaminolysis (Examples 10-1 to 10-16).

TABLE-US-00005 TABLE 5 Mw Temp Time DoA (SEC UV) Example (? C.) (h) pH Conditions (%) (kDa) 12 65 4 13 1M NaOH 99 10 (aq.)

EXAMPLE 13

Deacetylation of Hyaluronic Acid by Heterogeneous Alkaline HydrolysisComparative Example

[0091] HA (1 000 kDa) was weighed to a reaction vessel and NaOH in EtOH (70% w/w %) was added. The heterogeneous mixture was incubated and subsequently neutralized by addition of 1.2 M HCl. The precipitate was washed with ethanol (75 w/w %) followed by ethanol and dried in vacuum over night to obtain a solid. The results are displayed in Table 6.

[0092] Deacetylation by heterogeneous alkaline hydrolysis gives more degradation of the HA backbone, i.e. lower Mw of the deacetylated product as compared to hydroxylaminolysis (Examples 10-1 to 10-16).

TABLE-US-00006 TABLE 6 Mw Temp Time DoA (SEC UV) Example (? C.) (h) Conditions (%) (kDa) 13 35 24 1.0M NaOH 99 60 (70% EtOH)

EXAMPLE 14

Preparation and deacetylation of benzyl(4-(2-acetamidoethyl)phenyl)-carbamate

[0093] ##STR00007##

[0094] To a stirred solution of N-(4-aminophenethyl)acetamide (200 mg, 1.12 mmol, 1 eq) and TEA (in DCM (10 ml) at 0? C., is added benzyl chloroformate (173 mg, 1.01 mmol) over a period of 5 minutes. The reaction mixture is stirred at room temperature overnight, diluted with DCM 10 ml), washed with a saturated aqueous solution of NaHSO.sub.4 (5 ml), water (3?5 ml), dried over MgSO.sub.4 and concentrated to dryness to afford the title compound as a white solid.

[0095] To a stirred solution of the benzyl (4-(2-acetamidoethyl)phenyl)carbamate (1 eq) in EtOH-H.sub.2O (4:1, 5 volumes) is added NH.sub.2OH.HI (5 eq) and the suspension is heated at 80? C. for 5 h, cooled to room temperature and purified by Mass Triggered Preparative LCMS.

EXAMPLE 15

Preparation and deacetylation of allyl(4-(2-acetamidoethyl)phenyl)-carbamate

[0096] ##STR00008##

[0097] To a stirred solution of N-(4-aminophenethyl)acetamide (200 mg, 1.12 mmol, 1 eq) and TEA (in DCM (10 ml) at 0? C., is added allyl chloroformate (122 mg, 1.01 mmol) over a period of 5 minutes. The reaction mixture is stirred at room temperature overnight, diluted with DCM 10 ml), washed with a saturated aqueous solution of NaHSO.sub.4 (5 ml), water (3?5 ml), dried over MgSO.sub.4 and concentrated to dryness to afford the title compound as a white solid. To a stirred solution of the allyl (4-(2-acetamidoethyl)phenyl)carbamate (1 eq) in EtOH-H.sub.2O (4:1, 5 volumes) is added NH.sub.2OH.HI (5 eq) and the suspension is heated at 80? C. for 5 h, cooled to room temperature and purified by Mass Triggered Preparative LCMS.

EXAMPLE 16

Preparation and deacetylation of (9H-fluoren-9-yl)methyl(4-(2-acetamidoethyl)phenyl)carbamate

[0098] ##STR00009##

[0099] To a stirred solution of N-(4-aminophenethyl)acetamide (200 mg, 1.12 mmol, 1 eq) and TEA (in DCM (10 ml) at 0? C., is added 9-fluorenylmethyl chloroformate (261 mg, 1.01 mmol) over a period of 5 minutes. The reaction mixture is stirred at room temperature overnight, diluted with DCM 10 ml), washed with a saturated aqueous solution of NaHSO.sub.4 (5 ml), water (3?5 ml), dried over MgSO.sub.4 and concentrated to dryness to afford the title compound as a white solid.

[0100] To a stirred solution of the (9H-fluoren-9-yl)methyl (4-(2-acetamidoethyl)phenyl)-carbamate (1 eq) in EtOH-H.sub.2O (4:1, 5 volumes) is added NH.sub.2OH.HI (5 eq) and the suspension is heated at 80? C. for 5 h, cooled to room temperature and purified by Mass Triggered Preparative LCMS.

EXAMPLE 17

Preparation and deacetylation of 2-(trimethylsilyl)ethyl (4-(2-acetamidoethyl)phenyl)carbamate

[0101] ##STR00010##

[0102] To a stirred solution of N-(4-aminophenethyl)acetamide (200 mg, 1.12 mmol, 1 eq) and TEA (in DCM (10 ml) at 0? C., is added 4-nitrophenyl 2-(trimethylsilyl)ethyl carbonate (286 mg, 1.01 mmol) over a period of 5 minutes. The reaction mixture is stirred at room temperature overnight, diluted with DCM (10 ml), washed with a saturated aqueous solution of NaHSO.sub.4 (5 ml), a saturated solution of NaHCO.sub.3 (aq) (5 ml), water (3?5 ml), dried over MgSO.sub.4 and concentrated to dryness to afford the title compound as a white solid.

[0103] To a stirred solution of the 2-(trimethylsilyl)ethyl (4-(2-acetamidoethyl)phenyl)-carbamate (1 eq) in EtOH-H.sub.2O (4:1, 5 volumes) is added NH.sub.2OH.HI (5 eq) and the suspension is heated at 80? C. for 5 h, cooled to room temperature and purified by Mass Triggered Preparative LCMS.