HK1200753B - Lysin-glutamic acid dipeptide derivatives - Google Patents

Description

Lysine-glutamic acid dipeptide derivatives

The invention relates to compounds of formula I

Wherein the content of the first and second substances,

R¹and R²Identical or different and represent hydrogen or an ester protecting group,

R³is hydrogen or an amino protecting group, and

R⁴is C_12-20-an alkyl group,

and enantiomers, diastereomers and salts thereof.

In further embodiments, the invention relates to a process for the preparation of compounds of formula I and to the use of compounds of formula I in solid phase peptide synthesis.

The compounds of the present invention have been found to be multifunctional peptide intermediates for Solid Phase Peptide Synthesis (SPPS) of peptide drugs comprising a Glu-fatty alkyl side chain building block (building block) linked to a Lys-moiety of the peptide chain. For example, Liraglutide (Liraglutide), which is a GLP-1 analog diabetic-2 peptide drug, can be mentioned. Liraglutide Lys²⁶The position carries the Glu-hexadecanoyl side chain building unit (wikipedia, free encyclopedia of 4, 30 days 2012).

It is an object of the present invention to provide novel peptide intermediates carrying a Glu-fatty alkyl side chain and which can be easily inserted into SPPS.

The compounds of formula I of the present invention were found

Wherein the content of the first and second substances,

R¹and R²Identical or different and represent hydrogen or an ester protecting group,

R³is hydrogen or an amino protecting group, and

R⁴is C_12-20-an alkyl group,

and enantiomers, diastereomers and salts thereof have the potential to meet this goal well.

For the substituent R⁴The term "C" of_12-20The alkyl group "means a branched or straight chain monovalent saturated aliphatic hydrocarbon group of twelve to twenty carbon atoms, and particularly means a straight chain monovalent saturated aliphatic hydrocarbon group. The term may be exemplified by the groups dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and eicosyl.

In a particular embodiment, R⁴Finger C_14-16Alkyl, more particularly C₁₅An alkyl group.

More particularly, R⁴Tetradecyl, pentadecyl or hexadecyl, but pentadecyl is especially preferred.

For the substituent R³The term "amino protecting group" refers to the common substituents conventionally used to hinder the reactivity of amino groups. Suitable amino protecting groups are described in "Fmoc Solid Phase Peptide Synthesis-A practical approach" W.C&White, Oxford University Press, 2000, 2004 revamping, digital publishing.

In particular R³Is Fmoc (9H-fluoren-9-ylmethoxycarbonyl).

For the substituent R¹And R²The term "ester protecting group" as used herein refers to any substituent conventionally used to hinder the reactivity of a hydroxyl group. Suitable hydroxyl protecting groups are described in Green T., "Protective groups in organic Systhesis", Chapter 1, John Wiley and Sons, Inc., 1991, 10-142 and can be selected from C_1-4Alkyl, optionally substituted by phenyl, C_2-4-alkenyl, piperidinyl or dimethylaminoboroalkyl. For R¹And R²A specific ester protecting group of (A) is C_1-4-alkyl or C_2-4-alkenyl.

More particularly, R¹Is tert-butyl and R²Is an allyl group.

The term "salt" in connection with the compounds of the present invention includes the usual salts that will be used by those skilled in the art, such as hydrochloride, acetate, trifluoroacetate or formate.

In a particular embodiment of the invention, R¹Is hydrogen or C_1-4-alkyl and R²Is hydrogen or C_2-4-alkenyl.

In a more particular embodiment of the invention, R¹Is tert-butyl and R²Is hydrogen or allyl.

In a particular embodiment of the invention, the compound of formula I has the formula

Wherein the content of the first and second substances,

R¹，R²，R³and R⁴As described above, and enantiomers, diastereomers and salts thereof.

Compounds of formula Ia or Ib having the following substitution patterns are still more particular embodiments of the present invention:

■R¹tert-butyl radical, R²Hydrogen, R³Fmoc，R⁴C₁₅Alkyl, especially pentadecyl.

■R¹Tert-butyl radical, R²Allyl radical, R³Fmoc，R⁴C₁₅-an alkyl group,in particular pentadecyl.

In a more particular embodiment, the compound of formula I has formula Ia.

The compounds of the invention can be prepared in a manner known in principle to those skilled in the art of peptide synthesis.

For R in the formula²Preparation of a compound of formula I which is hydrogen, said process comprising

a) Glutamic acid derivatives of formula II

(wherein R is¹And R⁴As described above), or a salt thereof with a lysine derivative of formula III

(wherein R is^2’Is an ester protecting group and R³As described above), or salts thereof, to form compounds of formula Ic

(wherein R is¹，R^2’，R³And R⁴As described above), and

b) removal of the ester protecting group R^2’。

Step a)

Step a) entails coupling a glutamic acid derivative of formula II with a lysine derivative of formula III.

The glutamic acid derivatives of formula II can be prepared according to scheme 1 below starting from commercially available starting materials.

Scheme 1:

a suitable commercially available glutamic acid derivative of formula II is (S) -2-amino-glutaric acid 5-benzyl ester 1-tert-butyl ester hydrochloride.

Lysine derivatives of formula III are commercially available. Suitably, allyl (S) -6-amino-2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6-aminocaproate is used.

The coupling of the glutamic acid derivative of formula II with the lysine derivative of formula III can then be carried out using classical techniques of peptide synthesis.

Thus, initially, the glutamic acid derivative of formula II is activated with an activating agent commonly used in the art, such as with Carbonyldiimidazole (CDI), a carbodiimide selected for example from Dicyclohexylcarbodiimide (DCC) or Diisopropylcarbodiimide (DIC), or a triazole selected for example from 1-hydroxy-benzotriazole (HOBt) or 1-hydroxy-7-aza-benzotriazole (HOAt).

Good results have been obtained with CDI (1, 1' -carbonyldiimidazole) applied in suitable organic solvents, such as, for example, dichloromethane.

The coupling with the lysine derivative of formula III can then take place in the presence of an organic base such as triethylamine, usually at room temperature.

The dipeptide compound of formula Ib produced can be obtained from the organic phase by evaporation of the solvent and subsequent crystallization of the residue in a suitable organic solvent, such as diethyl ether.

Compounds of formula Ib, which are subgenera of formula Ia, as outlined above, are particular embodiments of the invention.

A particular representative of compounds of formula Ib is allyl (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitamidopentanoylamino) hexanoate, wherein R¹Tert-butyl radical, R^2’═ allyl, R³Fmoc and R⁴Pentadecyl.

Step b)

Step b) requires removal of the ester protecting group R^2’To form a compound of formula Ia.

Such reactions are well known to those skilled in the art.

Suitable systems for removing allyl groups are, for example, Pd-sources, such as solutions of tetrakis (triphenylphosphine) palladium (0) and phenylsilane in organic solvents, such as dichloromethane, tetrahydrofuran or methyltetrahydrofuran.

The reaction may take place at room temperature.

The dipeptide of formula Ia produced can be obtained from the organic phase by evaporation of the solvent and subsequent digestion of the crude product with a suitable organic solvent, such as with heptane and/or a mixture of heptane/dichloromethane.

As outlined above, the compounds of formula I may be used as multifunctional intermediates in solid phase peptide synthesis, in particular in the synthesis of peptides comprising a Glu-fatty alkyl side chain building block linked to a Lys-moiety of the peptide chain.

Even more particularly, the compounds of formula I may be used for FMOC solid phase peptide synthesis of such peptides.

Examples

Abbreviations:

r.t rt, DCM dichloromethane, THF, TBME, t-butyl methyl ether, EtOAc, thin layer chromatography

Example 1

(S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanamido) hexanoic acid

a) (S) -2-palmitoylamidoglutamic acid 5-benzyl ester 1-tert-butyl ester

In a 200-mL, 3-necked flask, a mixture of (S) -2-amino-glutaric acid 5-benzyl ester 1-tert-butyl ester hydrochloride (5.00g, 14.9mmol), triethylamine (3.12g, 30.7mmol) and tetrahydrofuran (100mL) was stirred at 0-5 ℃ for 15 min. Palmitoyl chloride (4.35g, 15.5mmol) was added to the suspension over 10min by syringe. The reaction mixture was stirred at 0-5 ℃ for an additional 30 min. Concerning TLC (EE/heptane 1: 1, R)_FRaw material ═ 0.1, R_FProduct ═ 0.6, detected at 254nm with the aid of Komarowsky reagent (see p.stevens, j.chromatog.1964, 14, 269)), conversion was complete. Water (60mL) and tert-butyl methyl ether (70mL) were added to the reaction mixture and the mixture was stirred at r.t for 5 min. The organic layer was separated, washed with brine (120mL), dried over sodium sulfate and evaporated to dryness to give (S) -2-palmitoylamidoglutaric acid 5-benzyl ester 1-tert-butyl ester (8.21g, > 99%) as a white solid with a chemical purity of 98.9% (LC method see below)

M.p.47℃；EI-MS：m/z＝531.39(M+H)⁺。

The LC method comprises the following steps: X-Bridge phenyl column No.823, 50x4.6mm, ID 2.5 μm; mobile phase, a: water/NCMe (95: 5), B: NCMe, C: water/glycine (pH 9); flow rate: 3 ml/min; the gradient was from 50/4/55(A/B/C) to 7/88/5(A/B/C) within 2min, and the isoconcentration was 7/88/5(A/B/C) for 0.8 min. Retention time: 0.54min ((S) -and (R) -2-amino-glutaric acid 5-benzyl ester 1-tert-butyl ester), 2.17min ((S) -and (R) -2-palmitoylamidoglutamic acid 5-benzyl ester 1-tert-butyl ester).

b) (S) -5-tert-butoxy-5-oxo-4-palmitoyl pentanoic acid

In a 250-mL 3-neck flask, a mixture of crude (S) -5-benzyl palmitoyl glutarate 1-tert-butyl ester (13.2g, 24.8mmol), 10% palladium on charcoal (1.31g, 1.20mmol), and THF (150mL) was stirred at room temperature under a hydrogen atmosphere. Concerning TLC (EE/heptane 1: 1, R)_FRaw material ═ 0.5, R_FProduct ═ 0.2, detected with the help of Komarowsky reagent (see p.stevens, j.chromanog.1964, 14, 269)), after 23h the transition was complete. The black suspension was passed through a glass fiber filter and the resulting colorless filtrate was evaporated to dryness to provide a crude product (11.3g), which was subsequently purified by crystallization from heptane to obtain (S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanoic acid (8.78g, 76% yield) as a white solid with 97.7% chemical purity (LC method see below)

M.p.63℃；EI-MS：m/z＝440.33(M-H)^-.

The LC method comprises the following steps: X-Bridge phenyl column No.823, 50x4.6mm, ID 2.5 μm; mobile phase, a: water/NCMe (95: 5), B: NCMe, C: water/glycine (pH 9); flow rate: 3 ml/min; the gradient was from 50/4/55(A/B/C) to 7/88/5(A/B/C) within 2min, and the isoconcentration was 7/88/5(A/B/C) for 0.8 min. Retention time: 0.77min ((S) -and (R) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanoic acid), 2.17min ((S) -and (R) -2-palmitoylamide glutaric acid 5-benzyl ester 1-tert-butyl ester).

c) (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid allyl ester

In a 500-mL 3-necked flask, a mixture of (S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanoic acid (8.77g, 19.4mmol), 1, 1' -carbonyldiimidazole (3.30g, 20.4mmol) and DCM (125mL) was stirred at room temperature for 90 min. A solution of (S) -6-amino-2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6-aminocaproic acid allyl ester (8.68g, 19.5mmol) and triethylamine (1.96g, 19.4mmol) in DCM (50mL)The resulting white suspension was added over 15 min. The reaction mixture was stirred at r.t for an additional 90min to complete the transformation (by TLC (EE/heptane 1: 1, R)_FRaw material 0, R_FProduct ═ 0.5, detected with the aid of Komarowsky reagent (see p.stevens, j.chromanog.1964, 14, 269). Next, DCM (50mL) and water (40mL) were added to the mixture and the layers were separated. The aqueous layer was extracted with DCM (20mL) and the combined organic layers were dried over sodium sulfate. After evaporation of the solvent, the remaining crude product (16.0g) was purified by crystallization from diethyl ether to afford allyl (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylamideglutamido) hexanoate (15.2g, 91%) as a white solid with a chemical purity of 96.5% (LC process see below) and an enantiomeric-and diastereomeric purity of > 99.9% (chiral LC process see below).

M.p.118℃；EI-MS：m/z＝832.55(M+H)⁺。

The LC method comprises the following steps: X-Bridge phenyl column, 50x4.6mm, ID 2.5 μm; mobile phase, a: water/NCMe (95: 5), B: NCMe, C: 0.1% aqueous formic acid; flow rate: 2 ml/min; the gradient is from 65/25/10(A/B/C) to 10/80/10(A/B/C) within 10min, and the concentration is 10/80/10(A/B/C) for 2 min. Retention time: 9.59min ((S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoate)).

Chiral LC method: chiracel OD-RH columns No.745 and No.702, 150x4.6mm, ID 5 μm; mobile phase, a: NCMe, B: Water/HClO₄(pH 2); flow rate: 1ml/min, isoconcentration 68: 32(A/B)32min, gradient from 68/32(A/B) to 75/25(A/B) within 0.5min, isoconcentration 75/25(A/B)29.5 min. Retention time: 45.39min ((R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((R) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid allyl ester), 47.75min of allyl ((R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoate), 51.98min ((S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((R) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanamide).Allyl) hexanoate), 55.66min ((S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoate).

Example 2

(S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanamido) hexanoic acid

In a 500-mL 3-necked flask, a mixture of (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) allyl hexanoate (10.0g, 11.4mmol), phenylsilane (7.02g, 62.9mmol), tetrakis (triphenylphosphine) palladium (0) (1.00g, 0.85mmol) and DCM (250mL) was stirred at room temperature. For TLC (EE/heptane 3: 1, R)_FRaw material ═ 0.2, R_FProduct ═ 0, detected by UV at 254 nm), conversion completed after 11 min. The reaction mixture was diluted with DCM (50mL) and washed successively with water (50mL), aqueous sodium diethyldithiocarbamate (0.5%, 30mL) and brine (30mL), dried over sodium sulfate and rotary evaporated to dryness. The residual crude product was digested first with heptane (25mL) and then with heptane/DCM (9: 1) at r.t. to afford, after filtration and drying, crude (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanamido) hexanoic acid (8.92g) having a chemical purity of 77.2% (LC method see below). The crude product contained 11% triphenylphosphine oxide as the major impurity. Preparative supercritical fluid chromatography (SFC, procedure see below) of a 1g sample of the crude product afforded pure (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid (0.75g, 72%) as having a chemical purity of 96.7% (LC procedure see below), 98.0% enantiomer and 99.8% diastereomeric purity (chiral LC procedure see below)) White solid of (2).

M.p.119℃；EI-MS：m/z＝792.52(M+H)⁺。

The LC method comprises the following steps: X-Bridge phenyl column No.823, 50x4.6mm, ID 2.5 μm; mobile phase, a: water/NCMe (95: 5), B: NCMe, C: 0.1% aqueous formic acid; flow rate: 2 ml/min; the gradient was from 65/25/10(A/B/C) to 10/80/10(A/B/C) within 10min, and the concentration was 10/80/10(A/B/C) for 2 min. Retention time: 8.65min ((S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid), 9.59min ((S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid allyl ester)).

Chiral LC method: chiracel OD-RH columns No.745 and No.702, 150x4.6mm, ID 5 μm; mobile phase, a: NCMe, B: Water/HClO₄(pH 2); flow rate: 1ml/min, isoconcentration 68: 32(A/B)32min, gradient from 68/32(A/B) to 75/25(A/B) at 0.5min, isoconcentration 75/25(A/B)29.5 min. Retention time: 21.56min ((R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((R) -5-tert-butoxy-5-oxo-4-palmitamido pentanamido) hexanoic acid), 23.52min ((R) -2- ((((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitamido pentanamido) hexanoic acid), 25.68min ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((R) -5-tert-butoxy-5-oxo-4-palmitamido pentanamido) hexanoic acid), 28.32min ((S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid).

Preparative SFC method: viridis 2-ethylpyridine OBD column, 150x30mm, ID 5 μm; column temperature 50 ℃; mobile phase, a: CO 2₂And B: MeOH; flow rate: 60ml/min, gradient from 80: 20(A/B) to 60/40(A/B) over 10 min.

Example 3

(S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanamido) hexanoic acid

In a 250-mL 3-necked flask, a mixture of (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) allyl hexanoate (12.0g, 13.7mmol), phenylsilane (2.28g, 20.4mmol), tetrakis (triphenylphosphine) palladium (0) (96.0mg, 0.08mmol) and DCM (120mL) was stirred at r.t. For TLC (DCM/MeOH 9: 1, R)_FRaw material ═ 0.9, R_FProduct ═ 0.3, detected with UV at 254 nm), conversion was complete after 3 h. The reaction mixture was then washed successively with aqueous sodium diethyldithiocarbamate (0.5%, 20mL) and brine (75mL), dried over sodium sulfate and rotary evaporated to dryness to yield crude (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitamidopentanoylamino) hexanoic acid (11.6g) having a chemical purity of 93.5% (LC method see example 2), > 99.9% enantiomer and 99.7% diastereomeric purity (chiral LC method see example 2) containing 1.2% residual triphenylphosphine oxide. The crude product was then suspended in heptane (230mL) at r.t for 1H, the mixture was filtered and the filter cake was washed with heptane (50mL) to yield (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitamidopentanoylamino) hexanoic acid (10.9g, 97% yield) as a yellow solid with 96.2% chemical purity (LC method see example 2), > 99.9% enantiomer and 99.8% diastereomeric purity (chiral LC method see example 2) containing 0.8% residual triphenylphosphine oxide.

M.p.119℃；EI-MS：m/z＝792.52(M+H)⁺。

Example 4

((R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid allyl ester

In a 25-mL 3-necked flask, a mixture of (S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanoic acid (500mg, 1.12mmol), 1-hydroxybenzotriazole (175mg, 1.14mmol), 1, 1' -carbonyldiimidazole (200mg, 1.23mmol) and DCM (10mL) was stirred at room temperature for 90 min. To the resulting white suspension, a solution of (R) -6-amino-2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6-aminocaproic acid allyl ester (507mg, 1.12mmol) and triethylamine (113mg, 1.12mmol) in DCM (5mL) was added over 5 min. The reaction mixture was stirred at room temperature for an additional 60min to complete the transition (by TLC (DCM/MeOH 95: 5, R)_FRaw material 0, R_FProduct ═ 0.2, detection by UV at 254 nm). Next, water (10mL) was added to the mixture and the layers were separated. The aqueous layer was extracted with DCM (30mL) and the combined organic layers were dried over sodium sulfate. After evaporation of the solvent, the residual crude product (983mg) was purified by crystallization from diethyl ether to afford (R) -allyl 2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoate (686mg, 70%) as a white solid with a chemical purity of 94.2% (LC method see below) and an enantiomeric-and diastereomeric purity of > 99.9% (chiral LC method see example 1 c).

M.p.114℃；EI-MS：m/z＝832.54(M+H)⁺。

The LC method comprises the following steps: X-Bridge phenyl column, 50x4.6mm, ID 2.5 μm; mobile phase, a: water/NCMe (95: 5), B: NCMe, C: 0.1% aqueous formic acid; flow rate: 2 ml/min; the gradient is from 65/25/10(A/B/C) to 10/80/10(A/B/C) within 10min, and the concentration is 10/80/10(A/B/C) for 2 min. Retention time: 9.55min ((R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoate)).

Example 5

(R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanamido) hexanoic acid

In a 25-mL 3-necked flask, a mixture of (R) -allyl 2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentanamido) hexanoate (675mg, 0.76mmol), phenylsilane (351mg, 3.14mmol), tetrakis (triphenylphosphine) palladium (0) (20.0mg, 0.02mmol) and DCM (7mL) was stirred at 10 ℃. For TLC (DCM/MeOH 95: 5, R)_FRaw material ═ 0.8, R_FProduct ═ 0.2, detection with UV at 254 nm), conversion completed after 25 min. After an additional 15min, the reaction mixture was diluted with DCM (10mL) and washed successively with water (10mL), aqueous sodium diethyldithiocarbamate (0.5%, 10mL) and brine (10 mL). The organic solution was dried over sodium sulfate and rotary evaporated to dryness to yield crude (R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid (631mg) with a chemical purity of 87.4 (LC method see below)), > 99.9% enantiomer and 98.8% diastereomeric purity (chiral LC method see example 2). The crude product contained 6% triphenylphosphine oxide as the major impurity. Preparative supercritical fluid chromatography (SFC, procedure see example 2) of 603mg of crude product sample provided pure (R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid (348mg, 59%) as a white solid with 98.7% chemical purity (LC procedure see below), > 99.9% enantiomer and 99.4% diastereomeric purity (chiral LC procedure see example 2).

M.p.125℃；EI-MS：m/z＝792.52(M+H)⁺。

The LC method comprises the following steps: X-Bridge phenyl column, 50x4.6mm, ID 2.5 μm; mobile phase, a: water/NCMe (95: 5), B: NCMe, C: 0.1% aqueous formic acid; flow rate: 2 ml/min; the gradient is from 65/25/10(A/B/C) to 10/80/10(A/B/C) within 10min, and the concentration is 10/80/10(A/B/C) for 2 min. Retention time: 8.33min ((R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid), 9.35min ((R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- ((S) -5-tert-butoxy-5-oxo-4-palmitoylaminopentylamido) hexanoic acid allyl ester)).

Claims (9)

1. A compound of formula I

Wherein the content of the first and second substances,

R¹is a tertiary butyl group, and is,

R²is a hydrogen or an allyl group,

R³is hydrogen or 9H-fluoren-9-ylmethoxycarbonyl, and

R⁴is C_12-20-an alkyl group,

or an enantiomer, diastereomer or salt thereof.

2. The compound of claim 1, wherein R⁴Is C_14-16-an alkyl group.

3. A compound of formula Ia according to claim 1,

wherein the content of the first and second substances,

R¹，R²，R³and R⁴As defined in claim 1, or an enantiomer, diastereomer or salt thereof.

4. A compound of claim 1 of formula Ib,

wherein the content of the first and second substances,

R¹，R²，R³and R⁴As defined in claim 1, or an enantiomer, diastereomer or salt thereof.

5. The compound of claim 3 or 4, wherein,

R¹is tert-butyl, R²Is hydrogen, R³Is 9H-fluoren-9-ylmethoxycarbonyl and R⁴Is C₁₅-an alkyl group;

R¹is tert-butyl and R²Is allyl, R³Is 9H-fluoren-9-ylmethoxycarbonyl and R⁴Is C₁₅-an alkyl group.

6. The compound of claim 5, wherein,

R¹is tert-butylRadical and R²Is hydrogen, R³Is 9H-fluoren-9-ylmethoxycarbonyl and R⁴Is pentadecyl;

R¹is tert-butyl and R²Is allyl, R³Is 9H-fluoren-9-ylmethoxycarbonyl and R⁴Is pentadecyl.

7. Preparation of wherein R²A process for preparing a compound of formula I according to claim 1 which is hydrogen, said process comprising

a) In which R is¹And R⁴Glutamic acid derivatives of formula II as defined in claim 1

Or a salt thereof with R^2’Is an ester protecting group and R³Lysine derivatives of the formula III as defined in claim 1

Or a salt thereof to form wherein R¹，R^2’，R³And R⁴A compound of formula Ic as defined in claim 1

And

b) removing the ester protecting group R^2’。

8. Use of a compound of formula I according to claim 1 in solid phase peptide synthesis of a peptide comprising a Glu-fatty alkyl side chain building block linked to a Lys-moiety of the peptide chain.

9. The use of claim 8, wherein the solid phase peptide synthesis is FMOC solid phase peptide synthesis.