WO1992012122A1

WO1992012122A1 - N-protected aminoalkylaldehydes

Info

Publication number: WO1992012122A1
Application number: PCT/US1991/009791
Authority: WO
Inventors: Maciej Adamczyk; Yon-Yi Chen
Original assignee: Abbott Laboratories
Priority date: 1990-12-27
Filing date: 1991-12-27
Publication date: 1992-07-23
Also published as: AU9171791A

Abstract

A method for synthesizing novel N-protected aminoalkylaldehydes having aldehyde and amino termini and which are useful as crosslinking reagents for preparing conjugates with biologically active compounds is described. N-benzyloxy-carbonylaminoalkylalcohols and N-butyloxy-carbonylaminoalkylalcohols are oxidized with a Dess-Martin periodinane reagent to provide N-protected aminoaldehydes for use as heterobifunctional crosslinking reagents.

Description

"N-PROTECTED AMINOAIJ YLALDEHYDES"

Field of the Invention

The present invention relates to the preparation and use of heterobifunctional crosslinking reagents. In particular, the present invention is related to N-protected aminoalkylaldehydes such as N-t-butylcarbonylaminoalkyl- aldehydes and N-benzyloxycarbonylaminoaldehydes, which are particularly useful as blocked crosslinking reagents.

The use of bifunctional reagents (i.e., reagents which have two reactive groups capable of reacting with, and forming bridges between, e.g., the side chains of amino acids and proteins) is a versatile method for crosslinking. Homobifunctional reagents (i.e., reagents carrying two identical reactive groups) are commonly used but their use is limited due to several inherent problems including random collisional crosslinking, crossreaction time, difficulty in controlling the reaction, and nonselective crosslinking [Kanaoka, et al., Chem. Phar . Bull., 32 (10) 3926 (1984)]. Therefore, the use of heterobifunctional reagents, where the two reactive groups are sufficiently different to permit well-controlled seguential reactions of each group, are more desirable. Even more desirable heterobifunctional reagents are those in which one of the reactive functional groups can be initially masked and subsequently can be readily unmasked and then used for coupling in a well-defined sequence of reaction steps, wherein uncontrolled crosslinking, often leading to undesired polymeric products, can be avoided [Weltman, et al., BioTechniσues, 148-152 (Sept/Oct 1983); Jou, et al., Methods in Enzymology, Academic Press, Inc., vol. 92, part E, 257-267 (1983); Gregoriadis, et al., Targeting of Drugs with Synthetic Systems, Plenum Press, pg. 176 (1986); Ishikawa, et al., J. Immunoassav, (4)3:209-327 (1983); and Ohnishi, et al., Chem. Pharm. Bull., 33(7):2871 (1985)].

A number of different types of heterobifunctional crosslinking reagents have been described. For example, Kanaoka, et al., supra, describe multifunctional crosslinking reagents which are photoactivatable, thiol—directed fluorescent reagents. Such heterobifunctional crosslinking reagents include m- maleimidobenzoyl-N-hydroxysuccinimide (MBS) for preparing antibody-beta-galactosidase conjugates, Fab'-beta- galactosidase conjugates, and the like; succinimidyl 4-(N- maleimidoethyl)cyclohexane-l-carboxylate (SMCC) for conjugating rabbit Fab' to horseradish peroxidase, conjugation of alkaline phosphatase and human IgG, and the like; sulfo-MBS for a comparison of maleimide containing heterobifunctional crosslinking reagents in the conjugation of Fab' fragments to horseradish peroxidase, and the like; and N-succinimidyl 3-2(2-pyridyldithio)propionate (SPDP) for introducing thiol groups into proteins and methods for

SUBSTITUTESHEET forming protein conjugates, preparation of antibody-toxin conjugates, and the like.

As would be understood by one skilled in the art, development of such crosslinking reagents is very important in the rapidly growing field of biotechnology and immunology. For example, immunochemistry crosslinking applications wherein conjugates used in drug carrier systems, antibody production and enzyme immunoassays employing SPDP have been described. Intramolecular crosslinking has been used to introduce additional tertiary structure into proteins, e.g., enzymes, in order to attempt to increase their conformational stability and to measure interresidue distances in proteins. In addition, intermolecular crosslinking can be used to bind proteins of the same or different kinds to each other (protein-protein conjugation) and to modify cell membranes or other macromolecular assemblies. However, the control of intra- versus inter- molecular crosslinking is difficult to achieve with homobifunctional reagents (when two reactive groups are identical). Conversely, with a heterobifunctional reagent, where the two reactive groups are directed toward different functional groups, the coupling and the crosslinking steps can be conducted in separate sequential steps. For example, the synthesis and properties of N-succinimidyl 3-(2-pyridyldithio) propionate, a hetero-directed heterobifunctional reagent, have been described for use as a protein-protein conjugation reagent [Carlsson, et al., Biochem. J. , 173:723 (1978)].

Although protected aminoalkylaldehydes are particularly useful as heterobifunctional crosslinking reagents, unprotected aminoalkylaldehydes known in the art will self-condense. Accordingly, without protection of one of the functional groups, such aminoalkylaldehydes are generally undesirable as heterobifunctional crosslinking reagents.

For protecting amino groups, the most widely used protecting group has been t-butoxycarbonyl (BOC) . Tarbell, et al., Proc. Nat'l. Acad. Science USA, 69:730 (1972), describe the formation of N-t-butoxycarbonyl (t-BOC) derivatives and their thiol analogs and the use of t-butyl carbamate. In addition, protection of an amino group employing t-butyl carbamate has been described [Green, T.W., "Protective Groups in Organic synthesis", John Wiley & Sons, New York, page 232 [1981]. For certain applications, however, the lability and lipophilicity of the BOC group renders it unsuitable. A more suitable moiety for some applications is the benzyloxycarbonyl (Cbz) group which is stable to basic in most aqueous acetic conditions, but which is readily removed photolytically or by hydrogenolysis. The use of carbobenzoxy chloride (C₆H₅CH₂OCOCl) has been described [Fieser & Fieser, "Reagents for Organic Synthesis", Volume 1, John Wiley & Sons, New York, page 109 [1067] for the N-protection of amino groups in peptide synthesis. Once the synthetic sequence has been completed, the N-protective Cbz group can be removed either by hydrogenolysis or by treatment with hydrogen bromide in acetic acid.

Since most biologically active compounds which are capable of being conjugated contain free amino groups and aldehyde groups, it is desirable to have the equivalent of a heterobifunctional crosslinking reagent having both aldehyde and amino termini. However, such desirable properties are not found in the heterobifunctional crosslinking reagents previously described, nor has there been any description for the preparation of N-protected aminoalkylaldehydes.

Summary of the Invention The present invention provides a method for preparing crosslinking reagents comprising novel N-protected aminoalkylaldehydes having aldehyde and amino termini which are useful for preparing conjugates with biologically active compounds having free amino and aldehyde groups. In particular, the present invention relates to a simple and efficient method for preparing N-t-butyloxycarbonyl- aminoalkylaldehydes and N-benzyloxycarbonylaminoalkyl- aldehydes which are capable of being conjugated to such biologically active compounds by reductive amination with the free amino groups thereof.

According to the method of the present invention, N- benzyloxycarbonylaminoalkylalcoholsandN-butyloxycarbonyl- aminoalkylalcohols are oxidized with a Dess-Martin periodinane reagent to provide such novel N-protected aminoaldehydes for use as heterobifunctional crosslinking reagents. The resulting crosslinking reagents are particularly useful for preparing labeled reagents useful in immunoassays, radioimmunoassays, and the like, which require the conjugation of a biologically active compound with a detectable moiety for use as a labeled reagent therein.

Brief Description of the Drawings Fig. 1 illustrates the schematic pathway for preparing

N-t-butyloxycarbonylaminoalkylaldehyde according to the present invention.

Fig. 2 illustrates the schematic pathway for preparing

N-benzyloxycarbonylaminoalkylaldehyde according to the present invention.

SUBSTITUTESHEET Detailed Description of the Invention Referring to Figure 1, of the drawings, commercially available aminoalkylalcohols (I) were readily transformed to N-t-butyloxycarbonylaminoalkyl-alcohols (2.) by reacting an aminoalkylalcohol (1) with di-t-butyldicarbonate [Green T.W., "Protective Groups in Organic Synthesis", John Wiley & Sons, New York, page 232, (1981)] in a mixture of dimethylformamide and triethylamine. The N-t-butyloxy- carbonylaminoalkylalcohols (2_.) can be used for the next transformation without further purification, are stable to column chromatography on silica gel, and can be stored for weeks at room temperature without noticeable decomposition. Referring to Figure 2 of the drawings, the benzyloxycarbonyl (Cbz) group, which was used as an alternative amino blocker, is stable under basic as well as most aqueous conditions, but is readily removed by photolysis or by hydrogenolysis [Fieser L.F., Fieser M., "Reagents for Organic Synthesis", vol. 1, John Wiley & Sons, New York, page 109, (1967)]. Aminoalcohols (JL) were reacted with benzylchloroformate in 1,4-dioxane in the presence of triethylamine [Green T.W., "Protective Groups in Organic Synthesis", John Wiley & Sons, New York, page 232, (1981)] to give excellent yields of N- benzyloxycarbonylamino- alkylalcohols (5.) .

N-Boc-aminoalkylalcohols (2.), as well as N-Cbz-aminoalkylalcohols (5_.), can be readily oxidized with Dess-Martin periodinane reagents [Dess, et al., J. Org. Chem. , 48:4156-4158 (1983)] in methylene chloride to give desired N-protected aminoalkylaldehydes (3_. and 6.) .

The use of hypervalent iodine in organic synthesis is described in Moriarty, et al., Ace. Chem. Res. , 19:244 (1986). The Dess-Martin reagent, originally described by Dess, et al., supra, page 4155, is a mild oxidative procedure which does not involve acid and does not generate any undesired by-products. After mild alkaline treatment, crude products exhibited only 1 spot by thin layer chromatography (silica gel, ethyl acetate/hexanes [1:1] as an eluent) and provided satisfactory 1H NMR spectra. The products can be used as crosslinking reagents without further purification. Analytical samples were obtained by column chromatography on silica gel. In the course of oxidation of N-protected aminobutanols and aminopentanols, cyclicization products were formed which are unsuitable as crosslinking reagents.

The crosslinking reagents prepared according to the present invention are particularly useful for preparing labeled antibody conjugates for use in homogeneous and heterogeneous immunoassay systems known in the art, such as competitive immunoassays, sandwich immunoassays, immunometric assays, and the like, to determine the amount

SUBSTITUTESHEET of analyte present in a test sample. Generally, such immunoassay systems depend upon the ability of an immunoglobulin, i.e., a whole antibody or fragment thereof, to bind to a specific analyte wherein a conjugate comprising an antibody to such analyte conjugated with a label or detectable moiety known in the art is employed to determine the extent of such binding. Such detectable moieties or labels include, but are not intended to be limited to, enzymes, chromogens, luminescent compounds, phosphorescent compounds, chemiluminescent compounds, fluorescent compounds, and the like. Typically, the extent of binding is determined by the amount of the detectable moiety present in the conjugate which either has or has not participated in a binding reaction with the analyte, wherein the amount of the detectable moiety detected and measured can be correlated to the amount of analyte present in the test sample.

The heterobifunctional crosslinking reagent prepared according to Example 3 below, having the general formula of OHC(CH ) -1NH-R, where R=-CO -C(CH,)„ or -COCHPh, can be used in a variety of situations requiring both an aldehyde and an amino group terminus. For example, in the first step of a two step process, the free aldehyde of the N-protected aminoalkylaldehyde can be reacted by reductive amination with any free amino group. In the second step, the protecting group can be removed from the heterobifunctional reagent by mild reduction for the removal of Cbz and by mild acid for the removal of BOC. Once the protecting group has been removed, a free amino group is available to which can be linked any molecule having a free aldehyde group.

Conversely, the free aldehyde of the N-protected amino alkylaldehyde can be linked to the amino group of a protein rather than, for example, a column support. Once linked, the protecting group can be removed from the heterobifunctional linker to expose a free amino group which can be reacted with any free aldehyde groups present, such as on a column support.

Other intended uses of the crosslinking reagents of the present invention include, but are not intended to be limited to, antibody mediated delivery systems by providing a therapeutic antibody conjugate that selectively localizes to tumor cell sites and requires that an adequate number of the drug molecules reach their site of action within the tumor cells where they can then exert a cytotoxic or cytostatic effect; covalent modification of antibodies and the design and synthesis of specialized crosslinking reagents in order to retain the homogeneous binding properties of the antibody conjugate; in vivo imaging applications which require discrete functional elements

SUBSTITUTESHEET and, in particular, must have chelator groups capable of strong association to any one of the family of radioactive metal cations; and the like.

The present invention will now be illustrated, but is not intended to be limited, by the following examples. Underlined numbers in parenthesis refer to the structural formulae as used in the figures and/or the specification:

EXAMPLE 1 Preparation of N-t-Butoxycarbonylaminoalkylalcohols (2_.)

To a stirred solution of 0.02 mole aminoalcohol (JL) and triethylamine (0.02 mole) in 15 L of dimethylformamide was added di-tert-butyl-dicarbonate (0.02 mole) dissolved in 5 mL of dimethylformamide. After stirring for 30 minutes at room temperature, the reaction mixture was diluted with 20 L of water and the pH carefully adjusted to pH 7.0 with 10% hydrochloric acid. Thirty milliliters (30 mL) of saturated sodium chloride solution was added to the reaction mixture and the reaction mixture was extracted three times with 40 mL of ethyl acetate. The combined ethyl acetate extracts were dried with anhydrous magnesium sulfate and after the solvent was removed, crude products were purified by column chromatography on silica gel using ethyl acetate/hexanes [1:1] as an eluent (85-95% yield) to obtain the following compounds:

(a) BOC-NH(CH₂)₂OH: oil; IH NMR (200 MHz, CDC1₃) delta:1.44(s, 9H, t-BuO), 2.62 (br.s, IH), 3.26 (m,2H), 3.67 (m,2H), 5.00 (br.s, IH); MS, m/e 162 (M+H)+;

(b) BOC-NH(CH₂)₃OH: oil; ^XH NMR (200 MHz, CDC1₃) delta:1.45 (s, 9H, t-BuO), 1.67 (m,3H), 3.27 (m,2H), 3.66 (m,2H), 4.95 (br.s, IH); MS, m/e 176 (M+H)+;

(c) BOC-NH(CH₂)₄OH: oil; IH NMR (200 MHz, CDCL₃) delta:1.44(s, 9H, t-BuO), 1.56 (m,4H), 2.95 (br.s, IH), 3.13 (m,2H), 3.64 (m,2H), 4.88(br.s, IH); MS, m/e 190(M+H)+;

(d) BOC-NH(CH₂)₅OH: oil, IH NMR(200 MHz, CDCl₃) delta:1.45 (s, 9H, t-BuO), 1.20-1.70 ( , 6H), 1.87(t,lH), 3.10(q, 2H, J=6Hz), 3.61(q, 2H, J=6Hz), 4.85(br.s, IH); MS, m/e 204(M+H)+; and

(e) BOC-NH(CH₂)₆OH; mp.56-58C; IH NMR (200 MHz, CDCl₃) delta:1.44 (s, 9H, t-BuO), 1.20-1.70 ( , 8H), 3.11 (m, 2H), 3.62 ( , 2H), 4.70 (br.s, IH); MS, m/e 218(M+H)+; and

EXAMPLE 2 Preparation of N-Benzyloxycarbonylaminoalkylalcohols (5.)

To a stirred solution of 0.02 mole aminoalcohol (JL) and triethylamine (0.025 mol) in 40 mL of 1,4-dioxane was added dropwise benzyl chloroformate (0.02 mol). After 4

SUBSTITUTESHEET hours of stirring at room temperature, precipitated triethylamine hydrochloride was filtered off and the filtrate was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel using ethyl acetate/hexanes (1:1) as an eluent (85-93% yield) to obtain the following compounds:

(a) HO(CH₂)₂NH-Cbz: IH NMR (200 MHz, CDC1₃) delta:2.40(br.s, IH), 3.34 (q, J=6Hz, 2H), 3.71 (t, J=6Hz, 2H), 5.10 (s, 2H, CH₂Ph), 5.25 (br.s, IH), 7.34 (s, 5H); MS, m/e 195 (M+);

(b) HO(CH₂)₃NH-Cbz; IH NMR (200 MHz, CDC1₃) delta:1.69(m, 2H), 2.57 (t, J=6Hz, IH), 3.35 (q, J=7Hz, 2H), 3.66 (q, J=7Hz, 2H), 5.05 (br.s, IH), 5.09 (s, 2H, CH₂Ph), 7.34 (s,5H); MS, m/e 209 (M+);

(c) HO(CH₂)₄NH-Cbz; IH NMR(200 MHz, CDC1₃) delta:1.60(m, 5H) 3.25 (q, J=6Hz, 2H), 3.67 (q, J=6Hz, 2H), 4.90 (br.s, IH), 5.10 (s, 2H, CH.Ph), 7.35 (s, 5H); MS, m/e 223(M+);

(d) HO(CH₂)₅NH-Cbz; IH NMR(200 MHZ, CDC1₃) delta:1.20-1.70 (m, 7H), 3.20 (q, J=6Hz, 2H), 3.64 (q, J=6HZ, 2H), 4.78 (br.s, IH), 5.09 (s, 2H, CH.,Ph), 7.35 (s, 5H); MS, m/e 237(M+); and

(e) HO(CH₂)6NH-Cbz; IH NMR (200 MHz, CDC1₃) delta:1.20-1.70 (m, 9H), 3.20 (q, J=5Hz, 2H), 3.63 (q, J=6Hz, 2H), 4.75 (br.s, IH), 5.10 (ε, 2H, CH.,Ph), 7.35 (s, 5H); MS, m/e 251 (M+)

EXAMPLE 3

Preparation of N-t-Butyloxycarbonylaminoalkylaldehydes (3.) and N-Benzyloxycarbonylaminoalkylaldehydes (jj.)

To a stirred solution of 2 mmole N-protected aminoalcohol (2. or 5_.) in dichloromethane (8 mL) was added a solution of Dess-Martin periodinane (2.2 mmol) in dichloromethane (8 L) in one portion. The reaction mixture was stirred at room temperature for 2 hours, then diluted with 70 ml of diethyl ether and hydrolyzed for 20 minutes with 70 mL of saturated aqueous sodium bicarbonate solution containing 1.1 g of sodium thiosulfate. The organic layer was separated and washed with 70 mL of saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The crude product was purified by gravity column chromatography on silica gel using ethyl acetate/hexanes (1:1) as an eluent (65-75% yield) to obtain the following products:

(a) BOC-NHCH₂CHO; 1NMR (200 MHz, CDCl₃)w:1.45 (s, 9H, t-BuO), 4.57(m, 2H), 5.25 (br.s, IH), 9.65 (s,lH, CHO); MS, m/e 160 (M+H)+;

(b) BOC-NHCH₂CH₂CHO; IH NMR (200 MHz, CDCl₃)

SUBSTITUTESHEET delta:1.44(s, 9H, t-BuO), 3.70 (t, J=6Hz, 2H), 3.42 (q, J=6Hz, 2H), 4.88 (br.s, IH), 9.81 (s, IH, CHO); MS, m/e 174

(M+);

(c) BOC-NH(CH₂)₅CHO; IH NMR (200 MHz, CDC1₃), (s, 9H, t-BuO), 1.20-1.80 (m, 6H), 2.45 (dubl. of t., J=6Hz, J=2Hz, 2H), 3.12 (q, J=6Hz, 2H), 4.50 (br.s, IH), 9.77 (t, J=2Hz, IH, CHO); MS, m/e 216 (M+);

(d) Cbz-NHCH₂CHO; IH NMR (200 MHz, CDC1₃) delta:4.11 (d, J=6Hz, 2H), 5.11 (s, 2H), 5.50 (br.s, IH), 7.33 (s, 5H), 9.61 (s, IH, CHO); MS, m/e; 193 (M+);

(e) Cbz-NH(CH₂)₂CHO; IH NMR (200 MHz, CDC1₃) delta:2.72 (t, J=6Hz, 2H), 3.48(q, J=6Hz, 2H), 5.09 (s, 2H) , 5.15 (br.s, IH), 7.34 (s, 5H), 9.79 (s, IH, CHO); MS, m/e 207 (M+); and

(f) Cbz-NH(CH₂)₅CHO; IH NMR (200 MHz, CDC1₃) delta:1.10-1.80(m, 6H), 2.41 (t, J=6Hz, 2H), 3.17 (q, J=6Hz, 2H), 4.74 (br.s, IH), 5.06 (s, 2H), 7.30 (s, 5H), 9.72 (s, IH, CHO); MS, m/e 249 (M+).

It will be apparent that many modifications and variations of the invention as herein set forth are possible without departing from the spirit and scope thereof, and that, accordingly, such limitations are imposed only as indicated by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An N-protected aminoalkylaldehyde.

2. An N-protected aminoalkylaldehyde of the general formula:

R-NH(CH₂)_{n l}CHO where n can be an integer from 2 through 6; and R can be -CO 2C(CH3)',3 or -CO2CH2Ph.

The aminoalkylaldehyde of claim 2 wherein R is

-C0₂C(CH₃)₃.

The aminoalkylaldehyde of claim 2 wherein R is

5. The aminoalkylaldehyde of claim 2 which is N-t- butoxycarbonylaminoalkylaldehyde.

6. The aminoalkylaldehyde of claim 2 which is N-benzyloxycarbonylaminoalkylaldehyde.

7. A method for preparing an N-protected aminoalkylaldehyde, said method comprising the steps of:

(a) transforming an aminoalkylalcohol to an N-protected aminoalkylalcohol; and

(b) oxidizing said N-protected aminoalkylalcohol to yield an N-protected aminoalkylaldehyde.

8. The method of claim 7 wherein said N-protected aminoalkylaldehyde is of the general formula:

R-NH(CH₂)_π_₁CH0 where n can be an integer from 2 through 6; and

SUBSTITUTESHEET R can be -CO 2C(CH 3) ' 3, or -CO 2CH 2Ph.

9. The method of claim 8 wherein R is

-C0₂C(CH₃)₃

10. The method of claim 8 wherein R is

11. The method of claim 8 wherein said N-protected aminoalkylaldehyde is N-t-butoxycarbonylaminoalkylaldehyde .

12. The method of claim 8 wherein said a m i n o a l k y l a l d e h y d e i s N-benzyloxycarbonylaminoalkylaldehyde. 13. The method of claim 8 wherein said N-protected aminoalkylalcohol is N-t-butyloxycarbonylaminoalkylalcohol or N-benzyloxycarbonylaminoalkylalcohol.

14. The method of claim 8 wherein said transforming agent comprises di-t-butyldicarbonate in a mixture of dimethylformamide and triethylamine.

15. The method of claim 8 wherein said transforming agent comprises benzylchloroformate in 1,4-dioxane in the presence of triethylamine.

16. The method of claim 8 wherein said oxidizing reagent is Dess-Martin periodinane.

17. The method of claim 8 wherein said oxidizing reagent is methylene chloride.