AU2012201979A1 - Method for Preparing Fmoc-based Hydrolysable Linkers - Google Patents

Abstract

A novel process for the production of Fmoc (9H-fluoren-9-ylmethoxycarbonyl)-based compounds is provided, wherein a protecting group for the 9-hydroxymethyl group of the fluorene ring system is utilized. These compounds are useful for the modification of protein and peptide drugs.

Description

METHOD FOR PREPARING FMOC- BASED HYDROLYSABLE LINKERS CROSS REFERENCE TO RELATED APPLICATION This application is a divisional of Australian Patent Application No. 2008267361 5 which claims the benefit of priority to U.S. provisional application Serial No. 60/937,125, filed June 26, 2007, which is incorporated herein by reference in its entirety. FILED OF THE INVENTION 10 The present invention relates to the preparation of a hydrolysable linker, which is useful for the modification of protein and peptide drugs. BACKGROUND OF THE INVENTION Most peptide and protein drugs are short-lived and have often a short circulatory 15 half-life in vivo. Considering that peptide and protein drugs are not absorbed orally, prolonged maintenance of therapeutically active drugs in the circulation is a desirable feature of obvious clinical importance. An attractive strategy for improving clinical properties of protein or peptide drugs 20 is a modification of protein or peptide drugs with polymers e.g. polyalkyleneoxides (Roberts et al., Advan Drug Rev 54: 459-476 (2002)) or polysaccharides like Polysialic acid (Fernandes et Gregoriadis, Biochim Biophys Acta 1341: 26-34 (1997), dextranes or hydroxyl ethyl starch. The modification with polyethyleneglycol (PEG) has been known for a while. However, modification of proteins with PEG often leads to reduction 25 of the activity of the protein. Therefore alternative systems were developed allowing the releasable coupling of the polymer to the protein or peptide drug using hydrolysable or degradable chemical linkers (US 6,515,100, US 7,122,189, WO 04/089280, WO 06/138572). The protein-polymer conjugate can be regarded as a prodrug and the activity of the protein can be released from the conjugate via a 30 controlled release mechanism. Using this concepts improved pharmacokinetic properties of the drug can be obtained (Zhao et al., Bioconjugate Chem 17: 341-351 (2006)). Therefore, WO 04/089280 suggested the use of a hydrolysable PEG-linker. (All documents cited in the specification are incorporated by reference.) 35 Tsubery et al. (J Biol Chem 279(37): 38118-38124 (2004)) demonstrated a hy- - 2 drolysable PEG-linker for derivatization of proteins based on the Fmoc (9 fluorenylmethyl carbamate) group. A fluorene group is reacted with maleimido propionic anhydride and N-hydroxysuccinimide, which is further reacted with poly(ethylene glycol) (PEG) and proteins by their amino groups. However, the syn 5 thesis of the hydrolysable linker, named MAL-FMS-OSU (9-Hydroxymethyl-2 (amino-3-maleimido-propionate)-7-sulfo fluorene N-hydroxysuccinimidyl carbon ate), suffers from low yield and reduced reproducibility. The key problem with the synthesis according to Tsubery et al. is the introduction of the maleimide group by reaction of 9-Hydroxymethyl-2-amino fluorene with maleimido propionic acid anhy 10 dride. In this step undesired side reactions like esterification of the OH group in position 9 occurred. Thus, an improved synthesis containing an additional step for protection of the OH group was developed. SUMMARY OF THE INVENTION 15 The present invention provides a new method for the synthesis of a com pound of general formula 1: 6 5 4 -3 7 2 8 9 1 PG (formula 1) wherein PG is a protecting group and at least one of position 1, 2, 3, 4, 5, 6, 7 or 8 is bound to radical Y. Y is a radical containing a N-maleimidyl-moiety. 20 In addition to being bound to radical Y the compound of formula 1 may op tionally be bound to radical X in at least one of the available position 1, 2 ,3, 4, 5, 6, 7 or 8. X is -S0 3

-R

3

R

3 is selected from the group consisting of hydrogen, (C 1 -Cs)-alkyl and 25 (C-Cs)-alkyl-R 4 .

R

4 is a polymer. Compounds of the invention are prepared according to a multi-step protocol - 3 wherein a protecting group for the 9-hydroxymethyl group of the fluorene ring sys tem is being utilized. These derivatives can be further modified to yield an acti vated ester such as an succinimidyl ester. 5 DETAILED DESCRIPTION OF THE INVENTION The present invention allows to overcome the above problems by a synthe sis which utilizes a protecting group to yield a compound of formula 1. A compound of formula 1 is a suitable precursor for subsequent reaction steps yielding a hydro lysable linker, like MAL-FMS-OSU and other MAL-Fmoc-OSU-derivatives contain 10 ing an active maleimide (MAL) and a N-hydroxysuccinimide (OSU = NHS) group and provides the desired products in high yield and purity. These linkers can be further modified with one or more polymers and can then be used to modify a pep tide or protein drug. The compounds of formula 1 are prepared starting from an amino 15 substituted fluorene. The new synthetic protocol introduces a protecting group for the hydroxylmethyl group at the 9 position of the fluorene in step 4 of the protocol shown below. The synthetic scheme below illustrates the preparation of a com pound of formula 1 as an example: NaH Boc-Anhydde /Ethyformate NH2 Dioxane/H 2 0 NH-Boc NBH 2N HC/AcetonitrIle IMi Cl NH-Boc I NH 2 DMF OH OH - 4

NH

2 NH / Maleimidopropionic acid DCC 0 1-Hydroxybenzotriazole THF yj)

CH

3 --- CH 3 CH 3 - -CH 3 BF 3 'EO CH3CCH3 CH3 CH3 Dichloromethane

CH

3

CH

3 Further reaction steps: Triphosgene NH ' / N-Hydroxysuccnimide / NH - NH - - N Pyridine THF NH .OH O Chlorosuffonic acid Dethylether HO 3 S NH 0 ~O 5 Step 1: In a first step (see scheme above), an amine group of an amino-substituted fluorene is protected by a BOC group (tert-Butyloxycarbonyl), for example by reac tion with BOC-anhydride or the like. Any other suitable protecting group (Greene et 10 el., Protective Groups in organic synthesis, Jon Wiley & Sons, Inc., Third Edition, New York (1999)) for amines can be used. An additional example is the Z (Benzy loxycarbonyl) group.

- 5 Multi-amino-substituted fluorene derivatives can be used in a similar reac tion in order to synthesize a compound of formula 1 having more than one radical Y in position 1, 2 ,3, 4, 5, 6, 7 or 8. Step 2: 5 In a second step a hydroxymethyl group is introduced at position 9 of the fluorene core, for example, by reaction with NaH or Lithiumdiisopropylamid (LDA) and ethylformiate and subsequent reaction with NaBH 4 or other reductive agents like DIBAL (Diisobutylaluminium hydride) in MeOH. Step 3: 10 In a third step the BOC protecting group is cleaved, for example, with HCI

CF

3 COOH or p-toluolsulfonic acid. Step 4: In a fourth step the 9-hydroxymethyl group is protected, for example, by re action with a silyl halogenide such as tBDMS-CI (Corey et al., J Am Chem Soc. 94, 15 6190-6191 (1972)) or 4,4'-Trimethoxytritylchlorid. In one embodiment the silyl halogenide is tBDMS-CI (tert-butyldimethylsilyl chloride). Preferably, the reaction with tBDMS-CI is performed with imidazole in DMF (dimethylformamide). The use of a silyl protecting group makes the molecule more lipophilic, thus facilitating the preparation of compounds, which are bound to 20 more than one radical Y. Step 5: In a fifth step a N-maleimidyl-moiety is introduced, for example, by reacting the amino group with a maleimidoalkylic acid or a maleimidoalkylic acid anhydride. The maleimidyl group is reactive towards thiole groups. Therefore, modified 25 polymers such as PEG-SH can be covalently bound to the hydrolysable linker to yield a polymer-modified hydrolysable linker. Step 6: In this optional step, radical X (-S0 3

R

3 ) is introduced in the fluorene ring system. This acidic group makes the compound more hydrophilic and allows to 30 perform subsequent coupling reactions in aqueous solvents. In addition a sulfonic acid group allows the coupling of a second polymer by esterification with a OH group of a polymer. This procedure allows to introduce radical X to yield a compound of formula - 6 1, comprising in addition to radical Y in position 1, 2 ,3, 4, 5, 6, 7 or 8 also radical X in at least one of the available position 1, 2 ,3, 4, 5,6, 7 or 8. In one embodiment, at least one radical X is bound to positions 2, 4, 5 and/or 7. In another embodiment, radical X is bound to position 7. 5 In another embodiment radical X (-S0 3 R) is introduced in the fluorene ring system after step 4. If -S0 3

R

3 is -SO 3 H the -SO 3 H group can be protected by es terification. Further reaction steps: 10 After the condensation with maleimidoalkylic acid, the 9 -hydroxymethyl can be deprotected by removing the protecting group (PG) to yield compounds of for mula 2. Deprotection is preferably performed with BF 3 , e.g. BF 3 'Et 2 O (Boron tri fuorid etherate). 5 4 63 7 2 8 9 1 OH (formula 2) MAL-FMS-OSU or its derivatives may be synthesized by reacting a com 15 pound of formula 2 with N-hydroxysuccinimide or its derivatives, such as N,N' Disuccinimidyl carbonate. Reaction conditions for the formation of a succinimidyl ester are well known in the art. A succinimidyl-modified compound for formula 2 can be further modified by reaction with SH-polymers and subsequently reacted with amino groups of peptide or protein drugs to yield conjugates of peptide or pro 20 tein drugs having a hydrolysable linker containing a polymer. The protocol exemplified above yields a compound of formula 1: - 7 6 5 4 3 7 2 8 9 1 PG (formula 1) wherein PG is a protecting group and at least one of position 1, 2 ,3, 4, 5, 6, 7 or 8 is bound to radical Y. Y is a N-maleimidyl-containing moiety. Compounds of formula 1, which are substituted with at least one radical Y 5 may also be bound to radical X in at least one of the available position 1, 2 ,3, 4, 5, 6, 7 or 8. X is -S0 3

-R

3 .

R

3 is independently selected from the group consisting of hydrogen, (C-C 8

)

alkyl and (CrCe)-alkyl-R 4 . 10 "Cr-C 8 -alkyl" refers to monovalent alkyl groups having 1 to 8 carbon atoms. This term is exemplified by groups such as methyl, ethyl, propyl, butyl, hexyl and the like. Linear and branched alkyls are included.

R

4 is a polymer. Examples of polymers are poly(ethylene glycol) (PEG), polysialic acid (PSA), hydroxyalkyl starch (HAS) and the like. 15 In another embodiment the invention relates to a compound of formula 1, wherein PG is a silyl group. Examples of a silyl group are trimethylsilyl, triethylsilyl or t-butyldiphenylsilyl. In another embodiment PG is a tert-butyldimethylsilyl group. In one embodiment Y is: 0 R---R--N 20 0 R' is at each occurrence independently a (C-C 8 )-alkyl.

- 8 In one embodiment R' is at each occurrence independently selected from the group consisting of methyl, ethyl, propyl, butyl, and hexyl.

R

2 is independently selected from the group consisting of -C(O)NR-, C(O)NR-(C 1

-C

8 )-alkyl-NR-, -NRC(O)- and -NRC(O)-(C-Cs)-alkyl-NR, wherein R is 5 independently either hydrogen or 0 1

-C

8 -alkyl. In one embodiment R 2 is -C(O)NH-. In another embodiment R 2 is -NHC(O)-. In one embodiment the compound of formula 1 is bound to radical Y in at least one of position 1, 2, 3 or 4. 10 In another embodiment the compound of formula 1, which is bound to radi cal Y in at least one of position 1, 2, 3, or 4 and is further bound to radical X in at least one of position 5, 6, 7, or 8. In another embodiment the compounds of formula 1, which are substituted with at least one radical Y in at least one of position 2 or 3 may also be bound to 15 radical X in at least one of position 7 or 8. In another embodiment the compound of formula 1, which are substituted with at least one radical Y, radical X is bound to position 7. In another embodiment the compounds of formula 1 are bound to radical Y in positions 2 and 7. 20 In another embodiment the compounds of formula 1 are bound to radical Y and radical X in positions 2 and 7, respectively. In another embodiment the compound of formula 1 is: The present invention is illustrated by the following examples without being 25 limited thereto. NH NH P0 0 PG 0 1i -9 0 H R 1 0 IV PG -N 0 00 05, H0 3 S ~ - NH S NH 0 V 0 0 0 0 0=0 H S0 3 H H H0 3 S 0 0ii 1

PG

- 10 NH NH / x 0 PG o 0 H 0L H H NH 0 PG O PG EXAMPLES MAL-Fmoc-OSu molecules can be synthesised according to the following 5 protocol (Examples 1-8): Example 1: Synthesis of 2-(Boc-amino)fluorene (Albericio et al., Synth Com mun. 31, 225-32 (2001)) 10 Q ~NH-Boc A suspension of 2- Aminofluorene (14.4 g, 79.7 mmol) was prepared in a mixture of 145 ml dioxane-H 2 0 (2:1 / V:V), cooled with an ice bath and 42.5 ml 2 N NaOH under gentle stirring. Boc 2 0 (19.1 g, 87.7 mmol, 1.1 equiv) was then added and the stirring was continued at 25 0 C. The evolution of the reaction was followed 15 by TLC (Rf = 0.75 for 2-(Boc-amino) fluorene. CHCl 3 -MeOH-HOAc (95:5:3)] and the pH was maintained between 9-10 by addition of 2 N NaOH. After 24 h TLC - 11 analysis showed the presence of 2-aminofluorene [Rf = 0.60, CHCl 3 -MeOH-HOAc (95:5:3)], so another 5.2 g Boc 2 0 (23.8 mmol, 0.3 equiv) were added and the reac tion was continued for additional 3 h until the total disappearance of the starting product. The suspension was acidified with 1 M KHSO 4 to pH 3. The solid was fil 5 tered and washed with 30 ml cold H 2 0, 30 ml dioxane-H 2 0 (2:1), 30 ml hexane and dried in vacuum. The product, a pale yellow powder (30.1 g, 90 % yield) was shown to be pure by TLC [Rf = 0.75; CHCla/MeOH/HOAc 95:5:3], and character ized by NMR. 10 1H NMR (200 MHz/DMSO) S = 9,45 (1H; s; NH); 7,84 - 7,77 (3H; m; H1, H4, H5); 7,59 - 7,17 (4H; m; H2,H6-H8); 3,86 (2H; s; CH 2 ); 1,49 (9H, s, t-Bu) 13C NMR (50 MHz/DMSO) S = 152,8 (Amid-C); 143,8 (C9a); 142,6 (C8a); 141,2 (C4b); 138,7 (C2); 135,2 (C4a); 126,7 (C6); 125,8 (C7); 124,9 (C8); 120,0 (C4); 15 119,2(C5); 116,9 (C3); 114,8 (Cl); 79,02 (CH 2 ); 39,5 (Cqt-Bu); 28,2 (3xCH 3 t-Bu) Example 2: Synthesis of 9-Hydroxymethyl-2-(Boc-amino) fluorene (Albericio et al., Synth Commun. 31, 225-32 (2001)1 NH-Boc 20 OH A solution of 2-(Boc-amino) fluorene (13.49 g, 47.9 mmol) in 140 ml dry THF (freshly distilled from sodium) was carefully added to a suspension of 6.3 g 60% NaH (160 mmol, 3.3 equiv) in 20 ml dry THF under argon atmosphere. Gas evolu tion and spontaneous warming were observed. After the complete addition of 2 25 (Boc-amino)fluorene the reaction mixture was stirred for 1 h at 400C. Then the re action mixture was cooled to room temperature and 9.7 ml ethyl formiate (120 mmol, 2.5 equiv) were slowly added to avoid vigorous hydrogen bubbling. The ini tially thick, light brown suspension rapidly clarified to a dark brown solution upon addition of ethyl formiate and was stirred for 1 h. The evolution of the reaction was 30 followed by TLC [Rf = 0.52 for the intermediate product, CHCl 3 -MeOH-HOAc (95:5:3)]. The reaction was quenched with ice chips and 100 ml H 2 0 and the or ganic solvent was removed by rotary evaporation. To the aqueous phase 10 ml 2 N NaOH were added and it washed with 3 x 50 ml diethylether, cooled in an ice bath and acidified with 25 ml glacial HOAc until pH 5. The off-white precipitate that - 12 then appeared was dissolved in 300 ml EtOAc. The aqueous phase was extracted with 50 ml EtOAc and the organic phase was washed with 2 x 75 ml sat. NaHCO 3 and 1 x 75 ml brine and dried over Na 2

SO

4 . The solvent was eliminated under re duced pressure. 5 9-Formyl-2-(Boc-amino) fluorene was suspended in 100 ml methanol and 2.0 g NaBH 4 [52.9 mmol, 1.1 equiv with respect to the starting 2-(Boc-amino) fluo rene] was added portion wise. The suspension that rapidly cleared up was mag netically stirred until the starting product disappeared for 4 h at room temperature [TLC, Rf=0.57, PE:MTBE (1:2)]. The reaction mixture was diluted with 300 ml H 2 0 10 and acidified with 15 ml glacial HOAc to pH 5.0, and the precipitate was directly dissolved in 150 ml EtOAc. The organic phase was washed with 3 x 50 ml sat. NaHCO and 1 x 50 ml brine and dried over anhydrous Na 2

SO

4 . The solvent was rotary evaporated to give a solid, which could be used without further purification. The product was analyzed by NMR (13.1g, 88% yield). 15 H NMR (200 MHz/DMSO) 8 = 9,42 (1H; s; NH); 7,87 (1H; s; Hi); 7,79 - 7,66 (2H; m; H4, H5); 7,61 (1H; d; J = 7,71 Hz; H8); 7,48 - 7,15 (3H, m, H3, H6, H7;); 5,07 (1H, t, J = 4,80 Hz; OH); 4,00 - 3,88 (1H; m; H9); 3,82 - 3,63 (2H; m; CH 2 ); 1,49 (9H; s; t-Bu) 20 C NMR (50 MHz/DMSO) S = 152,88 (Amid-C); 146,05 (C9a); 144,90 (C8a); 140,81 (C4b); 138,64 (C2); 134,90 (C4a); 127,13 (C6); 125,85 (C7); 125,05 (C8); 119,93 (C4); 119,16 (C5); 117,47 (C3); 115,24 (Cl); 79,00 (Cq t-Bu); 63,85 (CH 2 ); 50,19 (C9); 28,20 (3x CH 3 t-Bu) 25 Example 3: Synthesis of 9-Hydroxymethyl-2-amino fluorene

NH

2 OH 13.0 g 9-Hydroxymethyl-2-(Boc-amino)fluorene was dissolved in 110 ml 30 acetonitrile and stirred under reflux. 42 ml 2 N HCI (2.0 equiv, 84 mmol) was added drop-wise. The reaction mixture was stirred under reflux for 45 min. The reaction mixture was cooled to room temperature and the reaction was monitored by TLC [Rf=0.1 PE-MTBE (1:2)]. The solvent was partially eliminated by rotary evaporation and the residue was dissolved in 70 ml 2 N HCI. The solution was carefully washed - 13 with 2 x 50 ml MTBE. The aqueous phase was adjusted to pH 9 by Na 2

CO

3 and extracted with 2 x 70 ml EtOAc. The organic phase was washed with 50 ml brine and dried over Na 2

SO

4 . The solvent was eliminated by rotary evaporation. The product was used without further purification. The structural identity was verified by 5 NMR (8.76 g, 99% yield). 'H NMR (200 MHz/DMSO) 5 = 7,69 - 7,40 (3H; m; 3xAr-H); 7,37 - 7,02 (2H; m; 2x Ar-H); 6,87 (1H; s; Ar-H); 6,68 (1H; d; J = 8,34 Hz; Ar-H); 5,19 (2H; s; NH 2 ); 5,03 (1H; t; J = 4,93 Hz; OH); 3,93 - 3,58 (3H; m; H9, CH 2 ) 10 13C NMR (50 MHz/DMSO) 8 = 148,36 (Ar-Cqu); 146,81 (Ar-Cqu); 143,97 (Ar-Cqu); 141,87 (Ar-Cqu); 129,03 (Ar-Cqu); 126,92 (Ar-CH); 124,85 (Ar-CH); 124,24 (Ar CH); 120,40 (Ar-CH); 117,81 (Ar-CH); 113,00 (Ar-CH); 110,76 (Ar-CH); 64,27

(CH

2 ); 49,90 (CH) 15 Example 4: Synthesis of tert-Butyldimethylsi loxy-9-methyl-2-amInofluorene

NH

2

CH

3

CH

3

CCH

3

HCH

3 CH 3 5.91 g Imidazole (86.8 mmol, 2.1 equiv) was dissolved in 24 ml dry DMF 20 and stirred 10 min in an iced bath under argon atmosphere. 7.47 g tert Butyldimethylsilyl chloride (49.6 mmol, 1.2 equiv) dissolved in dry DMF was added. After 15 min stirring on ice 8.73 g 9-Hydroxymethyl-2-amino fluorene (41.3 mmol) dissolved in 40 ml dry DMF was added drop wise under cooling and argon atmos phere. The reaction was continued 15 min on ice and then at room temperature. 25 The reaction was monitored by TLC [title product Rf=0.6, PE-MTBE (1:2)]. After 2 hours the starting product [Rf=0.1 PE-MTBE (1:2)] had disappeared and the reac tion mixture was diluted with 400 ml CH 2

C

2 and 100 ml 5% NaHCO 3 was added. The organic phase was washed with 5 x 200 ml H 2 0 and dried over Na 2

SO

4 .

CH

2 Cl 2 was eliminated by rotary evaporation and DMF was eliminated by 30 azeotropic distillation with toluene. The residual brown oil (13.4 g, 99% yield) was analyzed by NMR and was used without further purification.

S14 'H NMR (200 MHz/DMSO) S = 7,67 - 7,40 (3H; m; 3x Ar-H); 7,34 - 7,00 (2H; m; 2x Ar-H); 6,81 (1H; s; 1x Ar-H); 6,59 (1x; dd; J = 8,02 Hz & 1,83 Hz; 1x Ar-H); 5,19 (2H; s; NH 2 ); 3,97 - 3,76 (2H; m; CH 2 ); 3,75 - 3,57 (1H; m; CH); 0,88 (9H; s; 3x 5 CH 3 ); 0,03 (6H; s; 2x CH 3 ) 13C NMR (50 MHz/DMSO) S = 148,40 (Ar-Cqu); 145,81 (Ar-Cqu); 143,67 (Ar-Cqu); 141,88 (Ar-Cqu); 129,08 (Ar-Cqu); 127,10 (Ar-CH); 124,97 (Ar-CH); 124,16 (Ar CH); 120,47 (Ar-CH); 117,87 (Ar-CH); 113,22 (Ar-CH); 110,58 (Ar-CH); 66,04 10 (CH 2 -OH); 49,60 (C9); 25,88 (3xCH 3 ; t-Bu); 18,04 (Cqu; t-Bu). -5,04 (2CH 3 ; Si

CH

3 ) Example 5: Synthesis of tert-Butyldimethylsiloxy-9-methyl-2-(amino-3 maleimidopropionate) fluorene 15 -- NH CH3- --- CH3 CH3 CH3

CH

3 To a solution of 13.5 g 9-tert-Butyldimethylsiloxymethyl-2-aminofluorene (41.5 mmol) in dry THF (freshly distilled from sodium) 9.42 g N,N' dicyclohexylcarbodiimide (75.7 mmol, 1.1 equiv) and 651 mg 1 20 hydroxybenzotriazole (4.8 mmol, 0.1 equiv) were added. 13.5 g (41.5 mmol, 1.1 equiv) 3-maleimidopropionic acid was dissolved in 50 ml dry THF and added drop wise. The reaction mixture was stirred at room temperature over night under argon atmosphere and the product formation was monitored by TLC [starting material Rf=0.6, title product Rf=0.18, PE-MTBE (1:2)]. 25 As soon as the starting material could not be detected, dicyclohexylurea was filtered out and THF was eliminated by rotary evaporation. The residual solid was dissolved in 200 ml CH 2 0 2 , washed with 50 ml 5% NaHCO 3 and 50 ml brine and dried over Na 2

SO

4 . The brown crystals were digerated in 20 ml MTBE. After filtration the residue was washed with small portions of MTBE until the washing so 30 lution maintained colorless. The yellow crystals (10.5 g, 53% yield) were analyzed by NMR.

- 15 'H NMR (200 MHz/DMSO) 8 = 10,06 (1H; s; NH); 7,92 (1H; s; Hi); 7,82 - 7,70 (2H; m; H4 & H5); 7,62 (1H; d; J = 7,20 Hz; H3); 7,49 (1H; d; J = 8,08 Hz; H8); 7,41 7,18 (2H; m; H6 & H7); 7,03 (2H; s; 2x Mal-CH); 4,02 (1H; t; J = 6,63 Hz; H9); 3,95 - 3,66 (4H; m; Prop-CH 2 -N & CH 2 -OTBDMS); 2,61 (2H; t; J = 7,07 Hz; Prop-CH 2 5 C=0); 0,84 (9H; s; 3x t-Bu CH 3 ); 0,10 - -0,56 (6H; m; 2x CH 3 -Si) 13C NMR (50 MHz/DMSO) 8 = 170,81 (C=O Mal); 168,30 (C=0 Amid); 145,12 (C9a); 144,62 (C8a); 140,68 (C4b); 138,05 (C2); 136,02 (C4a); 134,62 (2xCH Mal); 127,34 (06); 126,02 (C7); 125,15 (C8); 119,99 (C4); 119,37 (C5); 118,63 10 (C3); 116,31 (Cl); 65,54 (CH 2 OTBDMS); 49,85 (C9); 35,01 (Prop-CH 2 -N); 33,91 (Prop-CH 2 -CO); 25,84 (3x CH 3 (t-Bu); 18,01 (Cqu t-Bu); -5,40 (CH 3 -Si); -5,44 (CH 3 Si) Example 6: Synthesis of 9-Hydroxymethyl-2-(amino-3-maleimidopropionate) 15 fluorene NH 0 OH 10.3 g tert-Butyldimethylsiloxy-9-methyl-2-(amino-3-maleimidopropionate) fluorene (21.6 mmol) was dissolved in 230 ml CH 2 0 2 under argon atmosphere. 35 ml boron trifluoride etherate were added drop-wise over 30 min. The reaction was 20 monitored by TLC [starting material Rf=0.6, title product Rr=0.38, CH 2 Cl 2 -Methanol (10:1)]. As soon as the starting material had disappeared the solution was hydro lyzed with sat. NaHCO 3 solution. The resulting crystals were filtered. The organic phase of the mother liquor was evaporated. This residue and the filter cake were re-dissolved in 250 ml EtOAc and 120 ml 5% NaHCO 3 .The organic phase was 25 washed with 1x 50 ml 5% NaHCO 3 , 50 ml H 2 0 and 50 ml brine and dried over Na 2

SO

4 . The solvent was eliminated by rotary evaporation. The structure of the product was verified by NMR and mass spectroscopy. 'H NMR (200 MHz/DMSO) 8 = 10,09 (1H; s; NH); 7,88 (1H; s; H1); 7,82 - 7,69 (2H; 30 m; H4 & H5); 7,66 - 7,52 (2H; m; H3 & H8); 7,41 - 7,17 (2H; m; H6 & H7); 7,03 (2H; s; 2x Mal-CH); 5,09 (1H; t; J = 4,93 Hz; OH); 5,08 (1H; t; J = 6,06 Hz; H9); 3,97 - 3,53 (4H; m; Prop-CH 2 -N& CH 2 -OH); 2,61 (2H; t; J = 6,82 Hz; Prop-CH 2 C=0) - 16 13C NMR (50 MHz/DMSO) 8 = 170,82 (C=O Mal); 168,38 (C=O Amid); 146,00 (C9a); 144,89 (C8a); 140,63 (C4b); 138,05 (C2); 135,92 (C4a); 134,62 (2xCH Mal); 127,16 (C6); 126,09 (C7); 125,03 (C8); 119,93 (C4); 119,33 (C5); 118,39 5 (C3); 116,36 (Cl); 63,86 (CH 2 OH); 50,14 (C9); 35,07 (Prop-CH 2 -N); 33,92 (Prop

CH

2 -CO) ESI-MS: found: (M+Na)*: 385.2; calculated: (M+Na)*: 385 10 Example 7: Synthesis of 9-Hydroxymethyl-2-(amino-3-maleimidopropionate) fluorene N-hydroxysuccinim idyl carbonate NH 00 From a solution of 1.8 g pyridine in 7.3 ml abs THF 1.7 ml (5.1 mmol) were 15 added drop wise to a stirred solution of 0.93 g 9-Hydroxymethyl-2-(amino-3 maleimidopropionate) fluorene (2.6 mmol) and 1.1 g triphosgene (3.6 mmol, 1.4 equiv) in 75 ml dry THF (freshly distilled from sodium). After 40 min the precipi tated pyridine hydrochloride salt was filtered out over celite, and the THF was re moved by rotary evaporation. The oil obtained was dissolved in 75 ml dry tetrahy 20 drofuran with 1.1 g N-hydroxysuccinimide (13.6 mmol, 5.3 equiv). 2.6 ml of the pyridine solution (8.2 mmol) were then added, and the solution was stirred for 40 min. Some additional precipitated pyridine hydrochloride salt was filtered out over celite, and the THF was removed by rotary evaporation. The oil obtained was dis solved in 70 ml chloroform and washed with 4 x 40 ml 0.1 N HCI, 3 x 50 ml of an 25 aqueous 5% NaHCO 3 solution, then with 1 x 40ml water, 40 ml brine and dried over Na 2

SO

4 . The chloroform was removed by rotary evaporation. Structural iden tity was verified by NMR and mass spectroscopy. 'H NMR (500 MHz/CDC 3 ) 5 = 8,35 (1H; s; NH); 8,00 - 7,95 (1H; m; H3); 7,71 30 7,65 (2H; m; H4 & H5); 7,50 - 7,46 (1 H; d; J = 7,25 Hz; H8); 7,42 - 7,36 (2H; m; Hi & H6); 7,30 - 7,25 (1 H; m; H7); 6,69 (2H; s; 2x Mal-CH); 4,66 (1 H; dd; J = 10,40 Hz - 17 & 5,99 Hz; Ha-CH 2 0); 4,14 (1H; t; J = 10,09 Hz; Hb-CH 2 0); 4,27 (1H; dd; J = 9,46 Hz & 5,99 Hz; H9); 3,95 (2H; t; J = 7,09 Hz; Prop-CH 2 -N); 2,86 (4H; s; 2x Succ

CH

2 ); 2,80 - 2,54 (2H; m; Prop-CH 2 -C=O) 5 "C NMR (125 MHz/CDCla) 6 = 170,53 (C=O Mal); 168,21 (C=O Amid); 151,41 (C=O Carbonate); 143,79 (C2); 141,13 (C9a); 140,94 (C8a); 137,36 (C4b); 137,11 (C4a); 134,20 (2xCH Mal); 128,33 (C6); 126,82 (C7); 124,62 (C8); 120,63 (C4); 120,23 (C3); 119,96 (C5); 116,50 (C1); 72,67 (CH 2 0); 46,49 (C9); 35,62 (Prop

CH

2 -N); 34,15 (Prop-CH 2 -CO); 25,47 (2x Succ-CH 2 ) 10 ESI-MS (calculated): (M+H)*: 504; (M+Na)*: 526 ESI-MS (found): (M+H)*: 504.1; (M+Na)*:526.1 Example 8: Synthesis of 9-Hydroxymethyl-2-(amino-3-maleimidopropionate) 7-sulfo fluorene N-hydroxysuccinimidyl carbonate 15 HO1 3 S -- - NH 0 0== 0 To a solution of 1.2 g 9-Hydroxymethyl-2-(amino-3-maleimidopropionate) fluorene N-hydroxysuccinimidyl carbonate (2.1 mmol) in 60 ml trifluoroacetic acid 7 ml chlorosulfonic acid was added. After 30 min the reaction mixture was cooled to 20 40C and 350 ml cold diethylether was added. The precipitated product was filtered and washed twice with diethylether and dried in vacuum. Structural identity was verified by mass spectroscopy. ESI-MS (found): (M+H)*: 583.9 ESI-MS (calculated): (M+H)*: 583 25 The synthesis of MAL2-Fmoc-OSu starting from 2,7 Diaminofluorene is illus trated in Example 9.

- 18 Example 9: Synthesis of 9-Hydroxymethyl-2,7-Di-(amino-3 malelmidopropionate) fluorene N-hydroxysuccinimidyl carbonate NH \i NH 0 O 0 5 9-Hydroxymethyl-2,7-Di-(amino-3-maleimidopropionate) fluorene N hydroxysuccinimidyl carbonate is prepared under the conditions as described in Examples 1-8. The amino groups in 2,7-Diaminofluorene are protected with

BOC

2 0 as described by Albericio et al., Synth Commun. 31, 225-32 (2001). Then the 10 formyl-group is introduced in position 9 by reaction of Lithiumdiisopropylamide (LDA) and ethylformiate. The aldehyde obtained is reduced with sodiumborhydride to the corresponding alcohol to form 9-Hydroxymethyl-2,7-di-(Boc-amino)fluorene. Subsequently the BOC protecting groups are cleaved with 2N HCI in CH 3 CN and 9-Hydroxymethyl-2,7-diaminofluorene Is obtained. Then the OH-group is protected 15 by reaction with tert.-Butyldimethylsilylchloride as described in Example 5 to form tert-Butyldimethylsifoxy-9-methyl-2,7-diaminofluorene. Then the reaction of the free amino groups with maleimidopropionic acid in the presence of N,N' dicyclohexylcarbodiimide and hydroxybenzotriazole is performed and tert Butyldimethylsiloxy-9-methyl-2,7-di-(amino-3-maleimidopropionate) fluorene is ob 20 tained. After deprotection of the OH group in position 9 with Boron trifluoride etherate 9-Hydroxymethyl-2,7-di-(amino-3-maleimidopropionate) fluorene is formed. Finally the reaction with Triphosgene and N-hydroxysuccinimide is carried out and 9-Hydroxymethyl-2,7-di-(amino-3-maleimidopropionate) fluorene N hydroxysuccinimidyl carbonate is prepared. 25 - 19 The term 'comprise' and variants of the term such as 'comprises' or 'comprising' are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required. 5 Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

Claims (12)

1. A method of preparing a compound according to formula 1: 6 5 4 3 7 2 8 9 1 PG (formula 1) 5 wherein PG is a protecting group and at least one of position 1, 2 ,3, 4, 5, 6, 7 or 8 is bound to radical Y; Y is a radical containing a N-maleimidyl-moiety; and at least one of an available position 1, 2 ,3, 4, 5, 6, 7 or 8 is optionally bound to 1o radical X; X is -S0 3 -R 3 ; R 3 is independently selected from the group consisting of hydrogen, (C-C) alkyl and (C-Cs)-alkyl-R 4 ; and R 4 is a polymer; 15 said method comprises the step of reacting the 9-hydroxymethyl group of a com pound of formula 2: 6 5 4 7 2 8 9 1 OH (formula 2) wherein at least one of position 1, 2, 3, 4, 5, 6, 7, or 8 is bound to an amine, with a protecting reagent introducing PG, - 21 and subsequently reacting said compound with an N-maleimidyl derivative.

2. The method according to claim 1, wherein said protecting reagent is a silyl halogenide. 5

3. The method according to claim 2, wherein said silyl halogenide is tert butyldimethylsilyl chloride.

4. The method according to claim 3, wherein said reaction with tBDMS-Cl is 10 performed with imidazole in DMF.

5. The method according to claim 4, wherein said N-maleimidyl-derivative is maleimidoalkylic acid. 15

6. The method according to claim 1, wherein PG is a silyl derivative.

7. The method according to claim 6, wherein PG is a is tert-butyldimethylsilyl group. 20

8. The method of according to claim 7, wherein at least one of an available position 1, 2 ,3, 4, 5, 6, 7 or 8 is optionally bound to radical X.

9. The method according to claim 7 or 8 wherein Y is 0 0 ,wherein 25 R' is at each occurrence independently a (0 1 -C 8 )-alkyl, and R2 is independently selected from the group consisting of -C(O)NR-, C(O)NR-(C-C 8 )-alkyl-NR-, -NRC(O)- and -NRC(O)-(Cr 1 Cs)-alkyl-NR, wherein R is independently either hydrogen or 0 1 -C 8 -alkyl. - 22

10. A compound of the formula 1, 6 5 4 3 8 9 PG (formula 1) wherein PG is a protecting group and at least one of position 1, 2 ,3, 4, 5, 6, 7 or 8 is bound to radical Y; and at least one of an available position 1, 2 ,3, 4, 5, 6, 7 or 8 is optionally bound to radical X; 5 Y is a N-maleimidyl-containing moiety; X is -SO 3 -R 3 ; and R 3 is independently selected from the group consisting of hydrogen, (C-C)-alkyl and (C-C8)-alkyl-R 4 . 1o

11. The compound according to claim 10, wherein PG is a silyl group, Y is: 0 ---R2--R1--N 0 R' is at each occurrence independently a (C-C 8 )-alkyl; and 15 R 2 is independently selected from the group consisting of -C(O)NR-, C(O)NR-(C-C 8 )-alkyl-NR-, -NRC(O)- and -NRC(O)-(Cr-C 8 )-alkyl-NR-, wherein R is independently either hydrogen or C-C 8 -alkyl.

12. The compound according to claim 11, wherein PG is a t-butyldimethylsilyl 20 group. Date: 5 April 2012