WO1998027095A1

WO1998027095A1 - Novel pyrrolizine derivatives, their salts or solvates having anti-tumor activity, and processes for preparing them

Info

Publication number: WO1998027095A1
Application number: PCT/KR1997/000266
Authority: WO
Inventors: Jin-Suk Woo; Chong-Ock Lee; Dea-Sil Lee; Bun-Seang Park
Original assignee: Genechem Inc.
Priority date: 1996-12-19
Filing date: 1997-12-13
Publication date: 1998-06-25
Also published as: AU5413598A; KR19980049575A; KR100225457B1

Abstract

The present invention relates to novel pyrrolizine derivatives of formula (1), their salts or their solvates, and relates to the use of the compounds as anti-tumor medicines or as site-specific DNA alkylating agents in the gene relating technologies, the compounds and their salts which are so soluble into water-soluble solvents and stable in bodies as to be capable of being used as anti-tumor medicines and in addition to this, can be so bound with the materials site-specifically bound with nucleic acid such as oligonucleotides, peptide analogs and cholesterols, as to be used in the gene relating technologies. In formula (1), R1, X, X', Y and n are the same as defined in the specification.

Description

SPECIFICATION

Title of The Invention

NOVEL PYRROLIZINE DERIVATIVES, THEIR SALTS OR SOLVATES HAVING ANTI-TUMOR ACTIVITY, AND PROCESSES FOR PREPARING THEM.

Background of The Invention

The present invention relates to novel pyrrolizine derivatives represented the following formula I, their salts or solvates, processes for preparing them and the use of the compounds as anti-tumor medicines or as site-specific DNA alkylating agents in the gene relating technologies.

Wherein, Ri is Ci-Cio alkyl, C₃-C₇ cycloalkyl, C₃-Cβ alkenyl or phenyl group,

X and X' are respectively hydroxy, alkoxy, alkylthio or silyloxy group, Y is hydroxy, mercapto, amino, halogen, carbonyl group, and n is 1 to 10, the length of the linkers between the invention's compounds and other biologically important materials.

In details, the novel pyrrolizine derivatives and their salts are so soluble into water-soluble solvents and stable in bodies, so as to have excellant anti-tumor activity and able to be bound with oligonucleotides, peptide analogies, cholesterol and other materials site-specifically bound with nucleic acid. Thus the invention's compounds are useful in the field of gene relating technologies.

Generally, many anti-tumor medicines used in the treatment of tumor have functional groups which can be reacted with nucleic acid existing in cells and thus have anti -tumor activities.

In details, cisplatin, mitomycin, cyclophosphamide and the like used widespreadly as anti-tumor medicines include functional groups reacted with nucleic acid and other structural features to show their anti-tumor activities.

Particularly, as for the natural products, mitomycin C and pyrrolizidine alkaloids being compared with each other, the unique features of the functional groups can be viewed.

Although the two kinds of compounds, mitomycin C and pyrrolizidine alkaloids have similar pyrrole substructures of each other when each is activated in cells(Carter, S. K. and Crooke, S. T., Mytomycin C, Current Status and New Development, Academic Press, New York, 1978; Woo, J. S. et al., J. Am. Chem. Soc, 1993, 115, 3407), mitomycin C shows excellent anti-tumor activity but pyrrolizidine alkaloids existing in weeds have toxicity, which is the principal cause of animal's poisoning in the world.

The different functions of the two kinds of compounds showed in cells are caused from the reactivity of them.

In details, although pyrrole metabolites derived from pyrrolizidine alkaloids have functional groups reactive to nucleic acid, they are not apt to use as anti- tumor medicines because of their high reactivity and poison (See sheme 1).

Meanwhile, mitocycin C, which has similar functional group reactive to nucleic acid, has appropriate reactivity which is able to reach its pyrrole metabolite to nuclei in cells and make it reacted directly with nucleic acid(DNA) (See scheme 2).

Scheme 1

Scheme 2 However, there are some problems to solve for mitomycin C being used as good anti-tumor medicine.

In details, it is difficult to supply mitomycin C, which exists small portion in nature and is extracted small amount like common natural products.

Furthermore, reduction enzymes existing in cells are essential to react mitomycin C with nucleic acid, but generally the reduction enzymes do not exist much in cells, thus mitomycin C shows reduced anti-tumor activity.

In 1976, based on the models of the natural products, mitomycin C and pyrrolizidine alkaloids, Anderson and Corey working at New York University of USA had easily and successfully synthesized novel pyrrolizine compounds (See formula 2) of which reactivity to nucleic acid had been similar to mitomycin C and which had showed anti-tumor activity (Anderson, W. K. and Corey, P. F., Journal of Medicinal Chemistry, 1976, 20, 812).

(2)

To control the reactivity of pyrrole metabolites of pyrrolizidine alkaloids lower, they had substituted the substituent of the pyrrole ring system with substituent R₂ which could have controlled structurally the electron density of the pyrrole ring system and with ester group (R COO^") which had been substituent substituted by reaction with nucleic acid.

Wherein, the substituent R₂ includes methyl, trifluoromethyl, phenyl, 4- chlorophenyl, 4-fluorophenyl, 4-methoxyphenyl, 3,4-dichlorophenyl group, and the substituent R₃ includes methyl, NHCH₃, NHC₂H₅, NH-«-C4H₉, NH-isopropyl, NH-cyclohexyl group.

When the variety of the compounds had been synthesized with combination of the above mentioned substituents R and R₃, some of the compounds having electron withdrawing group as the substituent R₂ had showed more excellent anti- tumor activity than the other having electron donation group as the substituent R₂, and some having carbamate as ester leaving group, R₃COO^" had been more stable and showed excellent anti-tumor activity than another having acetate as ester leaving group, R₃COO^".

Speaking repeatedly, the reactivity of the compounds decreases by the electron withdrawing group, the substituent R₂ which make the density of the pyrrole ring system lower and inhibit the unpair electron of the pyrrole ring(electron in nitrogen atom not engaged in the reaction ) engaging in the reaction, and by the stable carbamates used as ester leaving groups make the compounds have stabler reactivity than the pyrrole metabolites of the pyrrolizidine alkaloids. Through the above mentioned process, Anderson and Corey had synthesized

2, 3 -dihydro-5 -(3 ' ,4 ' -dichlorophenyl)-6, 7-bis(hydroxymethyl)- 1 H-pyrrolizine bis(isopropylcarbamate) as the representitive of the compounds, which has 3,4- dichlorophenyl group as substituent R₂ and NH-isopropyl group as substituent R₃ and its derivatives, and they had asserted that it and its derivatives showed excellent antileukemic activity for the treatment of leukemia.

However, it is difficult for these compounds to be used as anti-tumor medicines, because they have some problems that they are little soluble into water- soluble solvents and lose the anti-tumor activity at the time of substituting ester leaving group by materials in cell, water, glutathione and the other nucleophiles,

Accordingly, though these compounds showed good anti-tumor activity, by this time they have not been used as anti-tumor medicines widespreadly.

Thus, as a result that we, the inventors of the present invention have tried to overcome the above mentioned problems for development of novel anti-tumor medicines, we have synthesized novel pyrrolizine derivatives and their salts which are so soluble into water-soluble solvents, stable against the materials in cells and bound with nucleic acid(DNA) in nuclei as to show excellent anti-tumor activity. The compounds of the present invention can be bound with biologically important materials, oligonucleotide, peptide derivatives, cholesterol and so on, and be easily used in the studies on genetic technologies including gene therapy.

Summary of The Invention

The purpose of the present invention is to provide novel pyrrolizine derivatives of formula I, their salts and their solvates pharmaceutically allowed.

Ri vi_n

(1) Wherein, Ri is C₁-C₁0 alkyl, C₃-C₇ cycloalkyl, Cs-Cβ alkenyl or phenyl group,

X and X' are respectively hydroxy, alkoxy, alkylthio or silyloxy group, Y is hydroxy, mercapto, amino, halogen, carbonyl group, and n is 1 to 10, the length of the linkers between the present invention's compounds and other biologically important materials.

Particularly, the purpose of the invention is to provide novel pyrrolizine derivatives of formula I, wherein Ri is Cι-C₃ straight alkyl group, X and X' are respectively hydroxy, methoxy, ethoxy, methylthio, ethylthio, trimethylsilyloxy or t-butylmethylsilyloxy, Y is hydroxy, mercapto or amino, and n is 1 to 6, their salts and their solvates pharmaceutically allowed.

More particularly, the purpose of the invention is to provide 2,3-dihydro-5- [4 ' ( β -hydroxyethylmethylamino)phenyl] -6, 7-bis(hydroxymethyl)- 1 H-pyrrolizine, its salts and its solvates pharmaceutically allowed.

More particularly, the purpose of the invention is also to provide 2,3-dihydro- 5-[4'( β -hydroxyethylmethylamino)phenyl]-6,7-bis(methoxymethyl)- 1H- pyrrolizine, its salts and its solvates pharmaceutically allowed.

More particularly, the purpose of the invention is also to provide 2,3-dihydro- 5-[4'( β -acetoxyethylmethylamino)phenyl]-6,7-bis(ethoxymethyl)-lH-pyrrolizine, its salts and its solvates pharmaceutically allowed.

More particularly, the purpose of the invention is also to provide 2,3-dihydro- 5-[4'( S -hydroxyethylmethylamino)phenyl]-6,7-bis(ethoxymethyl)-lH-pyrrolizine, its salts and its solvates pharmaceutically allowed.

Particularly, the purpose of the invention is also to provide novel pyrrolizine derivatives of formula I, wherein R[ is C C₃ straight alkyl group, X and X' are respectively O-benzyl, O-tπtyl or O-dimethoxytryl group, and Y is hydroxy, t- butylmethylsilyloxy, chloro, bromo or lodo group, their salts and their solvates pharmaceutically allowed

More particularly, the purpose of the invention is to provide 2,3-dιhydro-5- [4'( β -t-butylmethylsιlyloxyethylmethylamιno)phenyl]-6,7-bιs(hydroxymethyl)-lH- pyrrolizine, its salts and its solvates pharmaceutically allowed

More particularly, the purpose of the invention is also to provide 2,3-dιhydro- 5-[4'-( /3 -hydroxyethylmethylamιno)phenyl]-6,7-bιs(benzyloxymethyl)-lH- pyrro zme, its salts and its solvates pharmaceutically allowed More particularly, the purpose of the invention is also to provide 2,3-dιhydro-

5-[4'( /S -hydroxyethylmethylamιno)phenyl]-6,7-bιs(dιmethoxytπtyl-O-methyl)-lH- pyrro zine, its salts and its solvates pharmaceutically allowed

The purpose of the invention is also to provide processes for preparing the novel pyrrolizine derivatives or their salts

The purpose of the invention is also to provide 2,3-dιhydro-6,7- bιs(methoxymethyl)-lH-pyrrolιzιne-5-[4'-(methyloxymethylamιno)phenyl]-O- [(N,N-dnsopropylamιno)- β -cyanoethoxyphosphine] or its salts and a process for preparing it The purpose of the invention is also to provide 2,3-dιhydro-6,7- bιs(methoxymethyl)-lH-pyrrolιzιne-5-[4'-(ethyloxymethylamιno)phenyl]-O-[(N,N- dnsopropylamino)- β -cyanoethoxyphosphine] or its salts and a process for preparing it

The purpose of the invention is also to provide 2,3-dιhydro-6,7- bιs(dιmethoxytπtyl-O-methyl)-lH-pyrrolιzιne-5-[4'-

(ethyloxymethylamιno)phenyl]-O-[(N,N-dnsopropylamιno)- β - cyanoethoxyphosphine] or its salts and a process for prepraπng it The pyrrolizine derivatives capable of being bound with oligonucleotide can be directly synthesized by nucleic acid synthesizer using the above mentioned compounds.

The purpose of the invention is also to provide the use of the pharmaceutical compositions as anti-tumor medicines, including the novel pyrrolizine derivatives or their salts as active ingredient.

The pyrrolizine derivatives of the invention can be so bound with biologically important materials, oHgonuleotides, peptide analogies, cholesterol and so on that can be availably used the studies on gene technologies including gene therapy.

In details, the compounds can be used as site-specific DNA alkylating agents in condition of being bound with the above mentioned materials site-specifically bound with nucleic acid.

Detailed Description of The Invention

The present invention is to provide novel pyrrolizine derivatives of formula I, which show anti-tumor activity by reacting with nucleic acid in cells.

(1) Wherein, Ri is Ci-Cio alkyl, C₃-C₇ cycloalkyl, C₃-C₆ alkenyl or phenyl group,

X and X' are respectively hydroxy, alkoxy, alkylthio or silyloxy group, Y is hydroxy, mercapto, amino, halogen, carbonyl group, and n is 1 to 10, the length of the linker between the present invention's compounds and other biologically important materials

In the formula I, alkyl group included in Ri, X and X' is straghted or branched alkyl group which includes ethyl, propyl, prop-2-yl, butyl, but-2-yl, 2-methylprop- 2-yl, pentyl, pent-3-yl and hexyl group Also, alkenyl group includes prophenyl group and cycloalkyl group includes cyclopropyl, cyclopentyl and cyclohexyl group

In the formula I, R4 of alkoxy group(ORι) included in X and X' is same to Ri or phenyl-Ci ^ alkyl, 4,4'-dιmethoxytπtyl or the like, R5 of alkylthio group(SR₅) is same to Ri, and Re, R₇ and Rg of silyloxy group(OSιR6R Rs) are same to Ri, and same or different to each other

Wherein,

alkyl group includes benzyl, phenethyl or 3-phenylpropyl group

Also, in the formula I carbonyl of Y includes carbonylchloπde or methylester group Preferably, Rj is Cι-C₃ straighted alkyl group, X and X' are respectively hydroxy, methoxy, ethoxy, methylthio, ethylthio, trimethylsilyloxy or t- butyldimethylsilyloxy group, Y is hydroxy, mercapto or ammo group and n is 1 to 6 These compounds show the most preferable activities

Also, preferably, Ri is Ci-C, straighted alkyl group, X and X' are respectively trityl or 4,4'-dιmethoxytπtyl group and Y is hydroxy, chloro, bromo, or lodo group

Particularly, the preferable compounds of the invention is the compounds of formula 3 their salts and their solvates

(3)

Wherein, R₉ is methyl, ethyl or propyl group, Rio is hydrogen, methyl, ethyl or propyl and n is 1 to 6.

Particularly, the preferable compounds of the invention are showed as following.

2,3-dihydro-5-[4'-( δ -hydroxybutylethylamino)phenyl]-6,7- bis(hydroxymethyl)-lH-pyrrolizine,

2,3-dihydro-5-[4'-( δ -hydroxybutylethylamino)phenyl]-6,7- bis(ethoxymethyl)- 1 H-pyrrolizine,

2,3-dihydro-5-[4'-( β -hydroxybutylethylamino)phenyl]-6,7- bis(trimethylsilyloylmethyl)- 1 H-pyrrolizine, 2,3-dihydro-5-[4'-( /3 -hydroxybutylethylamino)phenyl]-6,7- bis(ethoxymethyl)- 1 H-pyrrolizine,

2,3-dihydro-5-[4'-( /3 -hydroxybutylethylamino)phenyl]-6,7-bis(DMTr-O- methyl)-! H-pyrrolizine , their salts or their solvates. Also, the particularly preferable compound of the present invention is 2,3- dihydro- 5 - [4 ' -( β -hydroxyethylmethylamino)phenyl] -6, 7-bis(hydroxymethyl)- 1 H- pyrrolizine of the following formula 4, its salts or its solvates.

(4)

The compound of the formula 4 according to the invention is so soluble into water-soluble solvents and has not the problem that the ester group leave by water as to be able to be reacted effectively with nucleic acid.

Though hydroxy group is much lower reactive leaving group than ester group, the whole reactivity of the compound is similar to 2,3-dihydro-5-(3',4'- dichlorophenyl)-6,7-bis(hydroxymethyl)-l H-pyrrolizine bis(isopropylcarbamate) and its derivatives of the prior art because the amine group, electron donating to pyrrole ring system increases the reactivity.

Accordingly, the compound of the formula 4 according to the invention exerts excellent anti-tumor activity. Another preferable compound of the invention are 2,3-dihydro-5-[4'-( /3 - hydroxyethylmethylamino)phenyl]-6,7-bis(methoxymethyl)-lH-pyrrolizine of the formula 5, its salt or its solvate

(5)

The whole reactivity of the compound of the formula 5 decreases in compared with the compound of the formula 4, because the compound of the formula 5 has methoxy group instead of hydroxy group of the compound of the formula 4

It is reported that the methoxy group, less reactive leaving group than hydroxy group, is scarcely reactive in the basic condition, but is gradually reactive in the weak acidic condition

Accordingly, considering that phosphate group constructing the skeleton of nucleic acid is weak acidic, the compound is expected to have reactivity when it reaches to nucleic acid without reaction with other materials in cells

Particularly, the compound of the formula 5 has hydroxy group, which is functional group capable of being bound with other biologically important materials Also, another preferable compound is 2,3-dihydro-5-[4'-( _/6^l hydroxyethylmethylamino) phenyl]-6,7-bis(ethoxymethyl)-l H-pyrrolizine of the formula 6, its salt or its solvate.

(6)

Also, another preferable compound of the invention is 2,3-dihydro-5-[4'-( β - hydroxyethylmethylamino)phenyl]-6,7-bis(methyl-O-methylidine)-lH-pyrrolizine of the formula 7, its salt or its solvate.

(V)

Also, another preferable compound of the invention is 2,3-dihydro-5-[4'-( 5 - hydroxyethylmethylamino)phenyl]-6,7-bis(benzyloxymethyl)-l H-pyrrolizine of the formula 8, its salt or its solvate.

(8)

Also, another preferable compound of the invention is 2,3-dihydro-5-[4'-( 3 hydroxyethylmethylamino)phenyl]-6,7-bis(dimethoxytrityl-O-methyl)-lH- pyrrolizine of the formula 9, its salt or its solvate.

(9)

The present invention is to provide processes for preparing novel pyrrolizine derivatives of the formula 1 and their salts.

In details, a process of the present invention consists of

1) doing coupling-reaction of N-(4-fluorobenzoyl)proline, which can be synthesized by Anderson and Corey's method, with the secondary amine compounds having hydroxy group to obtain the compounds having amino group introduced at the 4-position,

2) reacting the compounds resulted through the step 1 with acetic anhydride and dimethylacetylenedicarboxylate(DMAD) to obtain the pyrrolizine compounds properly substituted at 6,7-positions,

3) reducing the pyrrolizine compounds of the step 2 to obtain the pyrrolizine derivatives of the invention or their salts.

Meanwhile, the pyrrolizine compounds can be converted to another compounds having the same skeletonal structure by substituting substituents. Accordingly, as the previous process further is gone by addition of acid anhydride, other pyrrolizine derivatives or their salts can be synthesized.

Also, another pyrrolizine derivatives or their salts can be synthesized by the leaving group of the previous compounds being substituted with nucleophile.

In more details, the invention is to synthesize the novel pyrrolizine derivatives of the formula 1 as following.

1) The compounds of the formula 13 having amino group introduced at 4- position are synthesized by coupling reaction of N-(4-fluorobenzoyl)proline of the formula 1 1 with the secondary amines of the formula 12 having hydroxy group(See scheme 3).

The compounds of the formula 12 include n-methylethanolamine and N- methylbutanolamine, wherein R_n and n are the same as defined previously.

The solvent used in the reaction is most preferably dimethylsulfoxide(DMSO), wherein K₂CO₃ should be added in the reaction mixture.

The reaction temperature is preferralely 80 to 100 "C, the reaction time is proper to 7 to 10 days, and particularly the reaction is done under argon atmosphere.

Various linkers and functional groups are introduced to the compounds of the invention by using the previous reaction.

Scheme 3

2) Next, the compounds of the formula 14 are synthesized by the reaction of the compounds of the formula 13 with acetic acid anhydride and dimethylacetylenedicarboxylate(DMAD)(See scheme 4).

13 14

Scheme 4

The compounds of the formula 14 are synthesized by the 1,3 -dipolar addition method of the successive procedure of first, dissolving the compounds of the formula 13 by using acetic anhydride at about 60 ^°C, cooling the solution to the room temperature and slowly reacting the solution with dimethylacetylenedicarboxylate (DMAD)

3) Next, the compounds of the formula 15 are synthesized by reducing the compounds of the formula 14(See scheme 5)

Wherein, the reducting agents are preferably catalysts having proper reactivity such as lithium aluminiumhydride(LiAlH )

14 15

Scheme 5

4) The compounds of the formula 16 are synthesized by the reaction of the compounds of the formula 15 with acid anhydride(See scheme 6)

Preferably, when Rn is methyl and n is 2 in the formula 15, the compound of the formula 16 in which R and Rι₂ are each methyl and n is 2 can be synthesized by reaction of the compound of the formula 15 with acetic acid anhydride

The compound of the formula 16 is immediately used in next reaction after the synthesis of it, because the compound is very reactive and not preserved easily

15 16

Scheme 6

5) The compounds of the formula 17 and their derivatives are synthesized by reaction of ester leaving group existing in the compounds of the formula 16 with nucleophile(See scheme 7).

16 17

Scheme 7 Wherein, R_n is methyl, ethyl or propyl group and Rι₂ is general alkyl group. The nucleophile RS- is alkylsulfide ion such as CH₃CH₂S- and HOCH₂CH₂S- and RO- is alkoxide ion such as methoxide and ethoxide.

In details, the compound of the formula 5 is synthesized by reaction of the compound of the formula 16 in which Rn and Rι₂ are each methyl and n is 2, with nucleophile such as methoxide.

Therein, the two ester leaving groups bound to the pyrrolizine ring are substituted by methoxides and the ester group bound to the linker is hydrolyzed and converted to hydroxy group.

Also, the present invention is to provide 2,3-dihydro-6,7-bis(methoxymethyl)- lH-pyrrolizine-5-[4'-(ethyloxymethylamino)phenyl]-O-[(N,N-diisopropylamino)- β -cyanoethoxyphosphine] of the formula 10, which is capable of direct binding to oligonucleotides in nucleic acid synthesizer and a process for preparing it.

(10) The process according to the invention consists of

2) reacting the compounds resulted through the step 1 with acetic anhydride and dimethylacetylenedicarboxylate(DMAD) to obtain the pyrrolizine compounds properly substituted at 6,7-positions, 3) reducing the pyrrolizine compounds of the step 2,

4) after reaction of the reduced compounds of the step 3 with acid anhydride, reacting the ester leaving group of the compounds with nucleophile,

5) reacting the compounds of the step 4 with 2-cyanoethyl-N,N-diisopropyl chlorophosphoamidite and the like to obtain 2,3-dihydro-6,7-bis(methoxymethyl)- 1 H-pyrrolizine-5 - [4 ' -(ethyl oxymethylamino)phenyl] -O- [(N,N-diisopropylamino)- β -cyanoethoxyphosphine] of the formula 10.

The invention is to synthesize the compound of the formula 10 by reaction of the compound of the formula 5 with 2-cyanoethyl-N,N- diisopropyl chlorophosphoamidite and the like at the last step. Also, the compounds, which consist of N,N-diisopropylamino- β - cyanoethoxyphosphine group bound to the ethoxy group of the formula 7, 8 and 9, are synthesized by the same process of the above mentioned.

The compounds are capable of being bound to the 5^*-end of oligonucleotides and the like in nucleic acid synthesizer.

In addition, the pyrrolizine derivatives and their salts of the invention can be bound to cholesterol or protein analogies. Also, the anti-tumor activities of the compounds of the invention have been examined with various kinds of tumor cells.

Particularly, the anti-tumor activities of the compound of the invention, 2,3- dihydro-5-[4'-( β -hydroxyethylmethylamino)phenyl]-6,7-bis(hydroxymethyl)- 1H- pyrrolizine, its salts and its solvates have been verified by measuring the growth rate of tumor cells compared with that of standard cells(See table 1).

In addition to the above mentioned, the compounds of the invention can be used to the studies on gene relating technology by binding them to ligands site- specifically binding to nucleic acid for example, oligonucleotides, protein analogies or cholesterol.

In details, the compounds can be used in gene therapy, antisensor, antigene, site-specific gene blocking and so on.

The present invention is in details described by the following examples, but as the following examples are embodiments of the invention, the scope and boundary of the invention are not restricted by them.

<Example 1> Synthesis of 2,3-Dihydro-5-[4'-( /? -hydroxyethylmethyl amino)phenyl]-6,7-bis(hydroxymethyI)-l /-pyrrolizine

(Step 1) Synthesis of N-(4-Fluorobenzoyl)proline

A solution of L-proline (50g, 0.43mol), thymolphthalein (7mg), and Na2CO3 (170g) in water (800mL) was treated at room temperature with concentrated aqueous NaOH solution until the solution became blue. The stirred solution was placed in ice-water bath and treated portionwise with a solution of 4- fluorobenzoylchloride (81g, 0.51mmol) in ether (180mL). During the addition of 4-fluorobenzoylchloride solution, concentrated aqueous NaOH solution was added periodically as necessary to maintain the blue color of the reaction mixture The reaction mixture was stirred for an additional 30min at room temperature after the addition was completed The resulting mixture was extracted with ether (2 X 250mL) The aqueous layer was acidified to ~pH 1 with concentrated HC1 and extracted with ethyl acetate (4 X 400mL) The combined ethyl acetate solution was washed with brine (300mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo The solid residue was crystallized from hot ethyl acetate-hexane(2 1, 450mL) to afford 80g of the desired product as white powders The concentrated mother liquor, after similar treatment, gave an additional 15g of pure compound for a total yield of 93%

melting point 168 - 170°C

(Step 2) Synthesis of N-[4-( β -Hydroxyethylmethylamino)benzoyl]proline, potassium salt

14g (59mmol) of N-(4-fluorobenzoyl)proline obtained from step 1 was dissolved in anhydrous DMSO (24mL) To this were added anhydrous K₂CO₃ (8 3g) and N-methylethanolamine (12mL, 150mmol) The reaction mixture was stirred at 95 °C for 14 days under argon atmosphere RP-HPLC analysis of the reaction mixture indicated that most starting compound was converted into the desired product The undissolved salts in resulting mixture were removed by filtration The filtrate was concentrated in vacuo and resuspended in hot CH₂C1₂ (lOOmL) The undissolved impurities were removed by filtration, and the filtered solution was concentrated in vacuo The resulting residue was suspended in ether (50mL), and ether was removed in vacuo to give 17g of N-[4-( 5 - Hydroxyethylmethylamino)benzoyl]proline, potassium salt as slightly yellowish foam. RP-HPLC showed this sample was contaminated with small amount of the starting compound, N-(4-fluorobenzoyl)proline This sample was used for the next reaction without further purification

*The UV spectrum of N-[4-( β -Hydroxyethylmethylamino)benzoyl] proline, potassium salt ( λ _ma = 290nm) and N-(4-fluorobenzoyl)proline ( λ _max - 240nm) were recorded in 30% 0 1M triethylammonium acetate buffer (pH 7.5)/ CH₃CN The λ _ma of the product was bathochromically shifted by some 50nm relative to the starting compound, indicating the structural conversion of a 4-fluorobenzoyl compound to a 4-( β -Hydroxyethylmethylamino)benzoyl compound

(Step 3) Synthesis of Dimethyl-2,3-dihydro-5-[^"4'-( /g -acetoxyethylmethyl amino phenyl]-lH-pyrrolizine-6.7-dicarboxylate

10 15g (~30mmol) of N-[4-( β -Hydroxy ethylmethylamino)benzoyl] proline, potassium salt obtained from the above step was dissolved in warm acetic anhydride (35mL) in a flask equipped with a reflux condenser and a gas bubbler, and then the resulting solution was cooled to room temperature To this was added dimethyl acetylenedicarboxylate (17mL), stirred, and heated to 120 °C over 20-min period with monitoring CO₂ evolution, CO₂ evolution occurred at an increasingly rapid rate as the reaction temperature reached to 120 °C The reaction temperature was maintained at 120 °C for 2h and then concentrated in vacuo The residue was dissolved in CH₂C1₂ (20mL) and purified using silica gel column chromatography

(3 1 hexanes EtOAc) The fractions containing the desired product were combined and concentrated to afford 7g of the title compound as slightly yellowish solid Crystallization from hot methanol provided the analytical sample as a white needles

melting point 109 - 1 10 °C Η NMR (CDCU) δ 2 03(s, 3H), 2 49(p, 2H), 3 01(s, 3H), 3 l l(t, 2H), 3 62(t, 2H), 3 75(s, 3H), 3 80(s, 3H), 3 93(t, 2H), 4 28(t, 2H), 6 73(d, 2H), 7 29(d, 2H)

(Step 4) Synthesis of 2,3-dihydro-5-[4'-( ^ -hydroxyethylmethylamino)ρhenyl]- 6.7-bisfhydroxymethyl)-l H-pyrrolizine

A solution of Dimethyl-2,3-dihydro-5-[4'-( /3 -acetoxyethylmethyl amino )phenyl]-lH-pyrrolizιne-6,7-dicarboxylate (5g, 12mmol) in anhydrous

CH₂C1₂ (lOmL) was added dropwise to a stirred solution of LiAlH₄ (1 6g,

43mmol) in anhydrous ether (15mL) The reaction mixture was heated at reflux for

30min after the addition was completed and cooled to room temperature The excess hydride was decomposed with wet ether and then with water until the salts were white The inorganic residue was removed by filtration through sintered glass and washed with several portions of hot CH₂C1₂ The filtrate was concentrated in vacuo, redissolved in CH₂C1₂ (5mL), and then purified by using silca gel column chromatography (7 2 0 5 0 5 EtOAc hexanes Et^N MeOH) The fractions containing the desired product were combined and concentrated to afford 3g of the title compound as slightly yellowish solid

melting point 112 - 1 13 °C

Η NMR (CDC1₃) δ 2 46(t, 2H), 2 88(t, 2H), 3 01 (s, 3H), 3 51(t, 2H), 3 81 (t, 2H), 3 89(t, 2H), 4 57(s, 2H), 4 59(s, 2H), 6 81(d, 2H), 7 23(d, 2H)

LRMS m/e 316(M ), 285 <Example 2> Synthesis of 2,3-dihydro-5-[4'-( β -hydroxyethylmethyl amino)phenyl]-6,7-bis(methoxymethyl)-L/-pyrrolizine

A solution of 2,3-dihydro-5-[4'-( 3 -hydroxyethylmethylamino)phenyl]- 6,7- bis(hydroxymethyl)-lH-pyrrolιzine (460mg, 1 5mmol), obtained from the above example 1, dry Et₃N (2 8mL), and DMAP (lOmg) in CH₂C1₂ (lOmL) was treated with acetic anhydride (lmL) The acylation was carried at room temperature for lh and then treated with MeOH (20mL) The reaction mixture was concentrated in vacuo and treated with IM Na^~MeO^" in MeOH (lOmL) The resulting mixture was stirred overnight at room temperature, concentrated in vacuo, and then chromatographed using silca gel column (1 1 0 1 hexanes EtOAc Et₃N) The fractions containing the desired product were combined and concentrated to afford 200mg of the title compound as slightly yellowish solid

Η NMR (CDCH) δ 2 42(p, 2H), 2 84(t, 2H), 2 99(s, 3H), 3 30(s, 6H), 3 48(t, 2H), 3 71(t, 2H), 3 88(t, 2H), 4 24(s, 2H), 4 47(s, 2H), 6 78(d, 2H), 7 20(d, 2H)

<Example 3> Synthesis of 2,3-dihydro-5-[4'-( /3 -acetoxyethylmethyl amino)phenyl]-6,7-bis(ethoxymethyl)-l//-pyrroIizine

A solution of 2,3-dihydro-5-[4'-( 3 -hydroxyethylmethylamino)phenyl]- 6,7- bis(hydroxymethyl)- 1 H-pyrrolizine (lg, 3mmol), obtained from the above example 1, dry E N (5mL), and DMAP (15mg) in CH₂C1₂ (20mL) was treated with acetic anhydride (2mL) The acylation was carried at room temperature for lh and then treated with EtOH (40mL) The reaction mixture was concentrated in vacuo and treated with IM NaΕtO^" in EtOH (20mL) The resulting mixture was stirred overnight at room temperature, concentrated in vacuo, and then chromatographed using silca gel column (2 1 0 1 hexanes EtOAc Et N) The fractions containing the desired product were combined and concentrated to afford 600mg of the title compound as white solid In this condition, acetoxy group in amino linker was not hydrolyzed by NaΕtO^" unlike by Na⁺MeO^" in the above example 2

Melting point 52 - 53 ^°C

*H NMR (DMSO-d₆) δ 1 10(t of d, 6H), 1 94(s, 3H), 2 37(p, 2H), 2 76(t, 2H), 2 93(s, 3H), 3 40(q of d, 4H), 3 60(t, 2H), 3 88(t, 2H), 4 16(s, 2H), 4 18(t, 2H), 4 28(s, 2H), 6 76(d, 2H), 7 25(d, 2H)

LRMS m/e 414(M^÷), 369, 325, 282

<ExampIe 4> Synthesis of 2,3-dihydro-5-[4'-( /3 -hydroxyethylmethyl amino)phenyl]-6,7-bis(ethoxymethyl)-l//-pyrrolizine

LiAlH₄ (140mg) was suspended in anhydrous ether (5mL) To this was added dropwise (2,3-dihydro-5-[4'-( 3 -acetoxyethylmethylamιno)phenyl]-6,7- bis(ethoxy methyl)- 1 H-pyrrolizine (500mg, 1 2mmol), obtained from the above example 3, in CH₂C1₂ (5mL) The resulting mixture was stirred at room temperature for 30min The excess hydride was decomposed with wet ether and then with water until the salts were white The inorganic residue was removed by filtration through sintered glass and washed with several portions of hot CH₂C1₂ The filtrate was concentrated in vacuo, redissolved in CH C1₂ (3mL), and then purified by using silca gel column chromatography (1 1 0 1 EtOAc hexanes Et₃N) The fractions containing the desired product were combined and concentrated to afford 420mg of the title compound as slightly yellowish solid

melting point 63 - 65 °C

1H MNR (DMSO-d₆) δ 1 10(t of d, 6H), 2 37(p, 2H), 2 76(t, 2H), 2 94(s,

3H), 3 40(m, 6H), 3 54(q, 2H), 3 87(t, 2H), 4 16(s, 2H), 4 28(s, 2H), 4 66 (t, 1H), 6 72(d, 2H), 7 23 (d, 2H)

LRMS m/e 372(M⁺), 341, 283

<Example 5> Synthesis of 2,3-dihydro-5-[4'-( _yS -t- butyldimethylsilyloxyethylmethylamino)phenyl]-6,7-bis(hydroxymethyl)-lH- pyrrolizine

(Step 1) Synthesis of Dimethyl-2,3-dihydro-5-|^"4'-( /3 -hydroxyethylmethyl amino phenyl]-lH-pyrrolizine-6.7-dicarboxylate

Dimethyl-2,3-dihydro-5-[4'-( 6' -acetoxyethylmethylamιno)phenyl]-lΗ- pyrrolizine-6,7-dicarboxylate (5g, 12mmol), obtained from the step 3 in example 1, in MeOH (15mL) was treated with Na⁺MeO^" (1 3g) The reaction mixture was stirred for 3h at room temperature The resulting mixture was concentrated in vacuo, redissolved in CH₂C1₂ (100ml), and extracted with water (50mL) The organic phase was washed with brine and dried over anhydrous Na₂SO , and concentrated in vacuo The residue was purified by using silca gel column chromatography (1 1 EtOAc hexanes) The fractions containing the desired product were combined and concentrated to afford 4 5g of the title compound as white solid Η NMR (CDCH) δ 2 44(p, 2H), 2 94(s, 3H), 2 97(t, 2H), 3 40(t, 2H), 3 54(q, 2H), 3 60(s, 3H), 3 66(s, 3H), 3 95(t, 2H), 4 68(t, 1H), 6 70(d, 2H), 7 21(d, 2H)

(Step 2) Synthesis of Dimethyl-2,3-dihydro-5- 4'-( _i6' -t-butyldimethylsilyloxy ethylmethyl amino phenyl]- lH-pyrrolizine-6.7-dicarboxylate

A solution of dιmethyl-2,3-dihydro-5-[4'-( _/3 -hydroxyethylmethylamino) phenyl]-lH-pyrrolizιne-6,7-dicarboxylate (7 2g, 19mmol), obtained from the above step 1, and imidazol (8 lg, 1 lOmmol) in DMF(lOmL) was treated with TBDMSiCl (8 5g, 86mmol) The reaction mixture was stirred at room temperature for 1 5h The resulting mixture was treated with 5% aqueous sodium bicarbonate (lOmL) and extracted with CΗ C1₂ (3X20mL) The organic phase was washed with brine and dried over anhydrous Na₂SO₄, and concentrated in vacuo to afford 8g of the title compound as white powder This sample was used for the next reaction without furthur purification

*H NMR (CDCL₃) δ 0 02(s, 6H), 0 82(s, 9H), 2 44(p, 2H), 2 94(s, 3H), 2 97(t, 2H), 3 46(t, 2H), 3 59(s, 3H), 3 66(s, 3H), 3 73 (t, 2H), 3 94(t, 2H), 6 70(d, 2H), 7 21(d, 2H)

(Step 3) Synthesis of 2,3-dιhydro-5-[4'-( β -t-butyldimethylsilyloxyethyl methylamino)phenyl]-6.7-bisfhydroxymethvD-lH-pyrrolizιne

A solution of Dιmethyl-2,3-dihydro-5-[4'-( /j^> -t-butyldimethylsilyloxy ethylmethylamino)phenyl]-lH-pyrrolizine-6,7-dicarboxylate (2 6g, 5 2mmol), obtained from the above step 2, in anhydrous CH₂C1 (lOmL) was added dropwise to a stirred solution of LiAlH (513mg, 13 5mmol) in anhydrous ether (20mL) The reaction mixture was stirred at room temperature for lh The excess hydride was decomposed with wet ether and then with water until the salts were white The inorganic residue was removed by filtration through sintered glass and washed with several portions of hot CH C1₂ The filtrate was concentrated in vacuo, redissolved in CH C1₂ (5mL), and then purified by using silca gel column chromatography (1 1 0 1 EtOAc hexanes Et₃N) The fractions containing the desired product were combined and concentrated to afford 7g of the title compound as white solid

melting point 94 - 95 °C

1H NMR (DMSO-d₆) 5 0 01 (s, 6H), 0 84 (s, 12H), 2 37 (p, 2H), 2 78 (t, 2H), 2 95 (s, 3H), 3 47 (t, 2H), 3 76 (t, 2H), 3 85 (t, 2H), 4 29 (d, 2H), 4 39 (d, 2H), 4 44 (t, 1H), 4 47 (t, 1H), 6 68 (d, 2H), 7 72 (d, 2H)

LRMS m/e 431 (M⁺+l), 430(M⁺), 413, 397, 383, 285

<Example 6> Synthesis of 2,3-dihydro-5-[4'-( β -hydroxyethylmethyl amino)phenyl]-6,7-bis(benzyloxynιethyl)-l /-pyrrolizine

(Step 1 ) Synthesis of 2,3-dihydro-5-[4'-( /9 -t-butyldimethylsilyloxyethyl methylamιno)phenyl]-6.7-bis(benzyloxymethyO-lH-pyrrolizine

A solution of 2,3-dihydro-5-[4'-( 5 -t-butyldimethylsilyloxyethylmethyl ammo)phenyl]-6,7-bis(hydroxymethyl)-lH-pyrrohzine (lg, 2 3mmol), obtained from the above example 4, and sodium hydride (280mg, 6 9mmol) in anhydrous CH₂C1₂ (lOmL) was treated with benzyl bromide (513mg, 3 Ommol) The reaction mixture was stirred for 3 days at room temperature The resulting mixture was quenched with 5% aqueous sodium bicarbonate (lOmL) and extracted with CH C1₂ (2X20mL) The organic phase was washed with brine and dried over anhydrous Na₂SO₄, concentrated in vacuo The residue was purified by using silica gel column chromatography (8 1 0 5 hexanes EtOAc Et₃N) to afford 150mg of the title compound as yellowish oil

(Step 2) Synthesis of 2, 3-dιhydro-5- 4'-( -hydroxyethylmethylamino) phenyl]- 6.7-bιs(benzyloxymethyl -lH-pyrrolizine

2,3-Dihydro-5-[4'-( β -t-butyldimethylsilyloxyethylmethylamino) phenyl]-6,7- bis(benzyloxymethyl)-lH-pyrrolizine (150mg, 0 24mmol), obtained from the above step 1, in anhydrous CΗ₂C1₂ (3mL) was treated with IM tetrabutyl ammonium fluoride in THF (2mL) The reaction mixture was stirred for 4h at room temperature The resulting mixture was quenched 5% aqueous sodium bicarbonate (5mL) and extracted with CH₂C1₂ (2 X lOmL) The organic phase was washed with brine and dried over anhydrous Na₂SO₄, concentrated in vacuo The liquid residue was crystallized from hot hexane/CH Cl₂ (50mL/5mL) to afford 80mg of the desired product as yellowish solid

melting point 88 - 89 °C

*H NMR (DMSO-d₆) δ 2 36 (p, 2H), 2 76 (t, 2H), 2 92 (s, 3H), 3 38 (t, 2H),

3 54 (q, 2H), 3 88 (t, 2H), 4 23 (s. 2H), 4 36 (s, 2H), 4 41 (s, 4H), 4 47 (t, 1H), 6 68 (d, 2H), 7 32 (m, 12H) <Example 7> Synthesis of 2,3-dihydro-5-[4'-( /3 -hydro xyethylmethyl amino)phenyl]-6,7-bis(dimethoxytrityl-O-methyl)-lH-pyrroIizine

(Step 1) Synthesis of 2,3-dιhydro-5-^r4'-( /3 -t-butyldιmethyl silyloxyethylmethylamino phenyl]-6.7-bιs(dιmethoxytπtyl-O-methyl)-lH- pyrro zine

A solution of 2,3-dιhydro-5-[4'-( /j -t-butyldimethyl silyloxyethylmethylamino) phenyl]-6,7-bιs(hydroxymethyl)-lH-pyrrohzιne (lg, 2 3mmol), obtained from the above example 4, and tπethylamine (841 μ L) in anhydrous pyπdine (5mL) was treated with 4,4'-dιmethoxytπtyl chloride (1 73 g, 5 lmmol) in pyπdine (5mL) The reaction mixture was stirred for 3h at room temperature The resulting mixture was quenched with 5% aqueous sodium bicarbonate (lOmL) and extracted with hexanes (2 X 20mL) The organic phase was washed with brine and dried over anhydrous Na₂SO₄, concentrated in vacuo The residue was purified by using silica gel column chromatography (4 1 5 0 5 hexanes EtOAc Et^N) to afford 1 8g of the title compound as white powder

(Step 2) Synthesis of 2,3-dιhydro-5-[4'-( /3 -hydroxyethylmethylamino) phenyl]- 6.7-bιsfdιmethoxytπtyl-O-methyl)-lH-pyrrolιzιne

2,3-Dιhydro-5-[4'-( β -t-butyldimethylsilyloxyethylmethylarmno) phenyl]-6,7- bιs(dιmethoxytπtyl-O-methyl)-lH-pyrrolιzme(l 8g,2 lmmol), obtained from the above step 1, in anhydrous CH₂C1₂ (5mL) was treated with IM tetrabutyl ammonium fluoride in THF (4 2mL) The reaction mixture was stirred for 4h at room temperature The resulting mixture was quenched with 5% aqueous sodium bicarbonate (lOmL) and extracted with CH₂C1₂ (2 X 20mL) The organic phase was washed with brine and dried over anhydrous Na₂SO₄, concentrated in vacuo The residue was purified by using silica gel column chromatography (1.1 5 0 05 hexanes EtOAc. Et₃N) to afford lg of the title compound as white powder

melting point 80 -83 °C

Η NMR (DMSO-d₆) δ 2 41 (p, 2H), 2 91 (s, 3H), 2 94 (t, 2H), 3 36 (t, 2H), 3 52 (q, 2H), 3 68 (s, 6H), 3 70 (s, 6H), 3 74 (s, 2H), 3 84 (t, 1H)4 09 (s, 2H), 6 5-7 4 (m)

<Example 8> 2,3-dihydro-5-[4'-( β -hydroxyethylmethylamino)phenyl]- 6,7- bis(methyl-O-methylidine)-lH-pyrrolizine

melting point 196 - 197 °C

Η NMR (DMSO-de) δ 2 36 (p, 2H), 2 77 (t, 2H), 2 94 (s, 3H), 3 46 (t, 2H), 3 74 (q, 2H), 3 88 (t, 2H), 4 58 (s, 2H), 4 59 (s, 2H), 4 64 (t, 1H), 5 35 (s, 2H), 6 72 (d, 2H), 7 23 (d, 2H)

LRMS of 2,3-dihydro-5-[4'-( /5 -t-butyldimethylsilyloxyethyl methylamιno)phenyl]-6,7-bis(methyl-O-methylidιne)-lH-pyrrolizine m/e 442 (M⁺), 385, 355, 297

<Example 9> Synthesis of 2,3-dihydro-5-6,7-bis(methoxymethyι)-lH - pyrrolizine- 5-[4'-(ethyloxymethylamino)phenyl]-O-[(N,N-diisopropyl amino)- β -cyanoethoxyphosphine] A solution of 2,3-dihydro-5-[4'-( ^ -hydroxyethylmethyl amino)phenyl] -6,7- bis(methoxymethyl)- 1 H-pyrrolizine (lOOmg, 0 28mmol), obtained from the above example 2, and anhydrous diisopropylethylamine (0 2mL) in anhydrous CH₂C1₂ (5mL) was treated dropwise with 2-cyanoethyl NN'-diisopropylchloro phosphoramidite (0 lmL, 0 45mmol) under a positive pressure of argon The reaction mixture was stirred for lh at room temperature The reaction was stopped by addition of 5% aqueous sodium bicarbonate (2 5mL) The organic phase was washed with 5% aqueous sodium bicarbonate (2 5mL), dried over anhydrous sodium sulfate, and concentrated in vacuo The residue was dissolved in CH₂C1 (lmL) and purified by silica gel column chromatography It gave 90mg of the title compound as yellowish oily liquid

Η ΝMR (CDC1₃) δ 1 1 ~ 1 2 (m, 12H), 2 42(p, 2H), 2 57(t, 2H), 2 87(t, 2H), 3 02(s, 3H), 3.36(s, 6H), 3 5 ~3 7(m), 3 75(m, 2H), 3 89(t, 2H), 4 26(s, 2H), 4 39(s, 2H), 6 74(d, 2H), 7 27(d, 2H)

LRMS m/e 545(M"+1), 544(M^T), 326, 313, 295, 265, 250, 237

FAB m/e 597 (M⁺+l + p-nitrobenzylalcohol-Ν,Ν-diisopropylamine), 596,

560, 461

<Example 10> In vitro anti-tumor activity evaluation

The compounds according to the invention were dissolved in a small portion of DMSO and the solutions were diluted with culture medium for experiment to have the final DMSO concentration less than 5% They were filtered by 0 22um filter to dilute to each concentration

The SK-MEL-2 huma melanoma, A 549 non-small cell lung, SKOV-3 ovarian, HCT-15 colon, and XF-498 CNS tumor cell lines were maintained as stocks in RPMI 1640(Gιbco) supplemented with 10% fetal bovine serum(Gιbco) to be cultured in 5% CO₂ incubator at 37 °C Cell cultures were passaged once or twice weekly using the solution of 0 25% trypsιn-3mM CDTA in PBS(phosphate buffered solution) to detach the cells from their culture flasks

SRB (sulforhodamine B) assay, developed for measuring the cellular protein content of the cultures, is applied for the measurement of the cytotoxicity of the compounds against tumor cells The rapidly growing cells were harvested, counted, and inoculate at the appropriate concentratιons(l-2 x 10⁴ cells/well) into 96 well microtiter plates After incubation for 24h at 37 °C under 5% CO₂ atmosphere and eliminating each culture medium of the wells, the compounds (200μl per each) dissolved in culture medium were applied to the culture wells in triplicate followed by incubating for 48h at 37 °C under 5% CO₂ atmosphere When the compounds were applied, Tz (Time zero) plate was collected The cultures fixed with cold TCA were stained by 0 4% SRB and dissolved in 1% acetic acid After dissolving the bound dye with 1 OmM unbuffered tπs base by gyratory shaker, the absorbance at 520nm was measured with a microplate reader (Dynatech Model MR 700) Fifty percent inhibitory concentration (ED₅₀) was defined as the concentration which reduced absorbance by 50% of untreated wells as of the control in the SRB assay

At the culture starting point of adding the drugs, Time zero(Tz) was defined as the value of the amount of SRB protein by measuring the collected Tz plate Control value(C) was defined as the OD value of the well untreated with the drugs

Drug-treated test value(T) was defined as the OD value of the well treated with the drugs The activities of the drugs such as growth stimulation, net growth inhibition and net killing were evaluated from Tz, C and T

If T>=Tz, the cellular response function is 100 x (T-Tz)/(C-Tz), and if T<Tz, it is l00 x (T-Tz)/Tz

table 1 Net growth as percent of control

• A549 human lung, SKOV-3 human ovarian, SKMEL-2 human melanoma

• HCT15 ^• human colon, XF498 human CNS

• Method SRB(sulforhodamιne B) assay

<Example 11> In vivo anti-tumor activity evaluation

DBA/2 (57RL/6 x DBA/6 FI) and BDFl mice were used for antitumor testing All mice were obtained from Toxicology Research Center, KRICT,

KOREA

P388 leukemia as a tumor was used and it was maintained in ascitic form in

DBA/2 mice 1 x 10^β cells of P388 leukemia in ascitic fluid were implanted intraperitoneally

(i p ) in 6 week aged BDFl mice and the test compound was injected intraperitoneally at 1, 3, 5, 7 and 9 days after leukemia inoculation.

Mice were observed daily, and antitumor drug activity was determined based upon the T/C. T/C was expressed as the ratio of the median survival time of treated animals to the median control time multiplied by 100.

Table 2. In vivo activities against mouse leukemia P388.

The MST of the compared group is 10 days.

Claims

What is claimed is

1 A novel pyrrolizine derivative of the following formula I, its salt or its solvate pharmaceutically allowed

(1)

Wherein, Ri is Ci -Cio alkyl, C-,-C₇ cycloalkyl, GrC₆ alkenyl or phenyl group, X and X are respectively hydroxy, alkoxy, alkylthio or silyloxy group, Y is hydroxy, mercapto, ammo, halogen or carbonyl group, and n is 1 to 10, the length of the linkers between the present invention^'s compounds and other biologically important materials

2 A pyrrolizine derivative, its salt or its solvate pharmaceutically allowed according to claim 1, wherein R] is Cι-C_]0 straighted alkyl group, X and X' are respectively hydroxy, methoxy, ethoxy, methylthio, ethyothio, trimethylsilyloxy or t-butyldιmethylsιlyloxy group, Y is hydroxy, mercapto or amino group and n is 1 to 6

3 2,3-dιhydro-5-[4'-( 9 -hydroxyethylmethyl amιno)phenyl]-6,7- bιs(ethoxymethyl)-lH-pyrrolιzιne, its salt or its solvate pharmaceutically allowed according to claim 1

4 2,3-dιhydro-5-[4'-( β -t-butyldimethylsilyl oxyethylmethylamιno)phenyl]- 6,7-bιs(hydroxymethyl)-lH-pyrrohzιne, its salt or its solvate pharmaceutically allowed according to claim 1

5 2,3-dιhydro-5-[4'-( 3 -hydroxyethylmethyl amιno)phenyl]-6,7- bιs(benzyloxymethyl)-lH-pyrrolιzιne, its salt or its solvate pharmaceutically allowed according to claim 1

6 2,3-dιhydro-5-[4'-( /3 -hydroxyethylmethyl amιno)phenyl]-6,7- bιs(dιmethoxytπtyl-O-methyl)-lH-pyrrohzιne its salt or its solvate pharmaceutically allowed according to claim 1

7 A pyrrolizine derivative, its salt or its solvate pharmaceutically allowed according to claim 1 , wherein Ri is

straighted alkyl group, X and X' are respectively trityl or 4,4'-dιmethoxytπtyl group, Y is hydroxy, chloro, bromo, lodo group

8 A process for preparing pyrrolizine derivative as defined in claim 1 which comprises the consequent steps of 1) doing coupling-reaction N-(4-fluorobenzoyl)proline with the secondary amine having hydroxy group to obtain the compound having amino group introduced at the 4-position,

2) reacting the compound of the step 1 with acetic anhydride and dimethylacetylenedicarboxylate(DMAD) to obtain the pyrrolizine compound substituted at the 6 and 7 position,

3) reducing the compound of the step 2

9 A process for preparing pyrrolizine derivative according to claim 8, which futher comprises reacting the compound with acid anhydride and reacting its ester leaving group with nucleophile

10 2,3-dihydro-6,7-bis(methoxymethyl)-lH-pyrrolizine-5-[4'- (ethyloxymethylamino) phenyl]-O-[N,N-diisopropylamino]- β - cyanoethoxyphosphine] which is the pyrrolizine derivative capable of binding to oligonucleotides in nucleic acid systhesizer

1 1 2,3-dihydro-6,7-(methyl-O-methyledine)-lH-pyrrolizine-5-[4'- (ethyloxymethyl amino)phenyl]-O-[N,N-diisopropylamino]- β - cyanoethoxyphosphine] which is the pyrrolizine derivative capable of binding to oligonucleotides in nucleic acid systhesizer

12 2,3-dihydro-6,7-bis(benzyloxymethyl)-lH-pyrrolizine-5-[4'- (ethyloxymethylamino) phenyl]-O-[N,N~diisopropylamino]- β - cyanoethoxyphosphine] which is the pyrrolizine derivative capable of binding to oligonucleotides in nucleic acid systhesizer

13 2,3 -dihydro-6, 7-bis(dimethoxytrityl-O-methyl)- 1 H-pyrrolizine-5 - [4 ' - (efhyloxymethyl amino)phenyl]-O-[N,N-diisopropylamino]- β - cyanoethoxyphosphine] which is the pyrrolizine derivative capable of binding to oligonucleotides in nucleic acid systhesizer

14 A process for pyrrolizine derivative as defined in claim 10, 11, 12 or 13, which comprises the consequent steps of

1) doing coupling-reaction of N-(4-fluorobenzoyl)proline with the secondary amine having hydroxy group to obtain the compound having amino group introduced at the 4-position,

2) reacting the compound of the step 1 with acetic anhydride and dimethylacetylenedicarboxylate(DMAD) to obtain the pyrrolizine compound substituted at the 6 and 7 position, 3) reducing the compound of the step 2,

4) after reaction of the compound of the step 3 with acid anhydride, reacting its ester leaving group with nucleophile, and

5) reacting the resulting compoun Xd of the step 4 with 2-cyanoethyl-N,N- diisopropyl chlorophosphoamidite or the like

15 The use of pyrrolizine derivative or its salt according to claim 1 , 10, 11, 12 or 13 as anti-tumor medicine

16 The use of pyrrolizine derivative or its salt according to claim 1 , 10, 1 1, 12 or 13 as site-specific DNA alkylating agent in gene relating technology, wherein the pyrrolizine derivative is bound with the material capable of being site- specifically bound with nucleic acid

17. The use of pyrrolizine derivative or its salt according to claim 16, wherein the material capable of being bound with nucleic acid is selected from a group consisting of oligonucleotides, peptide analogies and cholesterol.