CA2183005A1 - Novel estradiol derivative-chlorambucil conjugate, process for preparing the same, and pharmaceutical composition - Google Patents

Abstract

An estradiol derivative-chlorambucil conjugate of the formula (II)

Description

1-- 218~

NOVEL Es~RAnIQL DEr~TVATI~ZE-rTTr~RAMRTTCIL CONJUGATE
PROCESS FOR pREPAT~NG T~TT~ ' AME
AND PHARMA~EUTI~'~T COMPQSITION
BACKGROUND OF THE INVENTION
This application i5 a divisional application of co-pending r~n~l;An Application Serial No. 2,097,137, filed May 27, 1993.
1. Field of the Invention The present invention relates to a novel estradiol derivative-chlorambucil c~njugate. More particularly, the present invention relates to a conjugate comprising a reaction product of an estradiol derivative and chlorambucil, a process for preparing the same, and a pharmaceutical composition comprising the con~ugate.

2. Description of the Related Art There are many conventional antitumor agents which inherently have strong antitumor effects, but in fact do not sufficiently exhibit their inherent effects. The main reason is that the amount of administration is limited due to their side effects. One of the attempts to solve the above problem is to bind the antitumor agent with a carrier having specific affinity to the tumor sites, and thereby form a con~ugate of an antitumor agent and a carrier. The attempt intended to accumulate the antitumor agent specificially to the tumor sites and effectively exhibit the antitumor effect while reducing the side effects.
On the basis of the above conception, an estradio~-chlorambucil conjugate and an antitumor agent containing mainly 30 t3~e conjugate were already proposed in USP 4,261,910 and USP

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4,332,797, The antitumor agent can accumulate specifically to the tumor sites and exhibit a strong antitumor effect thereat.
Further, its influence on llormal cells i6 extremely small.
Recently, H. Kosano, et al. reported that the above estradiol-chlorambucil conjugate inhibits the estrogen effect to promote the growth of MCF-7 ~human breast carcinoma cell, its growth is promoted by estrogen), irreversibly or over an extremely long period (~iroshi Kosano, et al., Cancer Resea~ch 52, 1187-1191, 1992). The reason thereof is suggested that the decrease in estrogen receptors causes a loss of the estrogen sensitivity of the cell, followed by inhibition of transforming growth factor (TGF)-c~ secretion and succeeding inhibition o~

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the cell growth. It is also suggested that the structure of the conjugate i$ necessary for the conjugate to exhibit the above effects on the estrogen receptors and inhibit the secretion of TGF-os That is, it is not suggested that the above effects are caused by chlorambucil liberated in the process cf the conjugate degradation .
Further, USP 4,921,849 discloses an injection prepared by dissolving the above estradiol-chlorambucil conjugate in an ester of iodinated poppy oil fatty acid. The injection enables the conjugate to reside for a long period at the tumor sites and to exhibit its full pharmacological effects. Further, USP
4,885,290 discloses an immunoregulator containing as an active ingredient the above conjugate which selectively suppresses immunoreactions caused specifically by isoantigen. Thus, the estradiol-chlorambucil conjugate exhibits selective physiological actiYities, such as a selective antit~lmor effect, a selective~immunosuppressive effect and the like.
When an antitumor agent is administered for a long period, however, even weak side effects, which do not pose-a problem with short term administration, in fact accumulate and become significant problems. In particular, a cancer patient lacks vital force, and therefore, such an accumulation oE weak side effects is lntensified. One of the problems with the conjugate is an adverse effect by a slight amount of estrogen released in the body from the estradiol-chlorambucil corl]ugate. For example, with long term administration of the conjugate, the slight amount of the released estrogen accumulates and may cause side effects such as gynecomastia, mastosis, nipple pain, genital bleeding, and the like. These estrogenic activities may be a problem even in the injections, immunoregulator or the like containing the above conjugate.
SUMMARY OF THE INVENTIO~
The present inventors discovered that an extremely useful, improved novel estradiol derivative chlorambucil conjugate with no or reduced estrogenic activity can be obtained by introducing one or more particular substituents into one or more estradiol rings, while maintaining the selective physiological activities ~ 2ls3nos of the conventional estradiol-chlorambucil conjugate, that is the selective antitumor effect and selective immunosuppressive ef f ect .
In accordance with one embodiment of the present invcntion there is provided a compound of the formula (II):

O ~ C ~ ~C~{2) m X
~ Rs ( R ) ( II ~
wherein R1 is alkyl or alko~yl of 1 to 4 carbon atoms; R2 is acyl, dansyl, or alkyl; R3, R4, and Rs independently are H, oxo, OH, or acyloxy; m is an integer of 1 to 3; n is an integer of 0 to 3; and X is halogen, OH, or a salt thereof; provided that when n is 0, all of R3, R4, and Rs are not H at the same time, and at least one of R3, R4, and Rs is a group other than H and OH; and further, when n is 2 or 3, the groups R1 are the same or di f f erent .
In accordance with another embodiment of the present invention there is provided a compound of the formula (III):
R OH

( R I ) ~ ! ( I I I ) ~
wherein R1 is alkyl or alkoxyl of 1 to 4 carbon atoms; R2 is acyl, dansyl, or alkyl; R3, R4, and Rs independently are H, oxo, ~ 2183~

OH, or acyloxy; and n is arl integer of 0 to 3; provided that when n i5 0, all of R3, R4, and Rs are not H at the same time, and at least one of R3, R4, and Rs i8 a group other than H and OH, and further, when n is 2 or 3, the groups R1 are the same or different.
DETArLED DESCRIPTION OF THE ~;~;K~;L) EMBODIMENTS
The estradiol derivative-chlorambucil conjugate (hereinafter referred to as the present conjugate contains an estradiol derivative moiety characterized by the chemical structures as follows:
That is, in addition to oR2 at the 3-position on the ring A of the estradiol, the estradiol derivative moiety has one or more substituents at the l-, 2-, and/or 4-position of the ring A, or at one or more positions on the rings B, C, and/or D.
The estrogenic action can bc eliminated or reduced by at least one substituent at R1 (one or more) on the ring A, R3 (other than H) on the ring B, R4 (other than H) ol~ the ring C or Rs ~other than H) on the ring D, but preferably one or more groups R1 at the l-, 2-, and/or 4-position on the ring A. R1 at the l-, 2-, and/or 4-position on the ring A may be alkyl of l to 4 carbon atoms or alkoxyl of l to 4 carbon atoms. R1 is preferably present at the l-- and/or 4-position. Further, the estradiol derivative moiety may carry one of oxo, OH, and acyloxy on one of the ring~ B, C, and D, or each of same or different two or three substituents on two or three different rings of the ring8, B, C, and/or D. In this case, oxo or acyloxy is preferable. The acyloxy is preferably acyloxy of 2 to 7 carbon atoms, for example, benzoyloxy, acetoxy, propanoyloxy, butanoyloxy, or pentanoyloxy group.
The substituent at the 3-position of the estradiol derivative moiety in the present conjugate has a structure 2i~3nQa wherein H of OH at the 3-position of the estradiol is substituted by acyl, dansyl (5-dimethylaminonaphthalene-sulfonyl), or alkyl. R2 as acyl is preferably acyl of 2 to 18 carbon atoms, for example, benzoyl, acetyl, palmitoyl, stearoyl, or linolenoyl. Further, R2 as alkyl is preferably alkyl of 1 to 4 carbon atoms, more preferably methyl, ethyl, or propyl. Further, the configuration of DH at the 17-position of the estradiol derivative moiety may be B-configuration, ~-configuration, or a mixture thereof, but B-configuration is preferable. The configuration of each of the substituents R3, R4, and Rs may also be B-configuration, ~Y-configuration, or a mixture thereof.
The present con~ugate may be prepared, for example, by the following method:
When an estradiol derivative having OH at the 3-position on the ring A of the estradiol and the 17-position on the ring D of the estradiol (this is, an estradiol derivative having R
at '~ 2ls3n~s the 1-, 2-, or 4-position on t~le ring A and/or having R3, R4, and/or R5 other than H on the ring B, C, ana/or D: hereinafter referred to- simply as a "3,17-OH estradiol derivative") is used and H of O~I at the 3-hydroxyl group is replaced by an acyl or dansyl group R2, the 3 ,17 -OH estradiol derivative is first dissolved in an organic solvel~t, and then reacted with an alkaline metal or an alkaline metal hydroxide to form a salt.
Then, the salt is reacted with an acid chloride or acid anhydride corresponding to the desired ~acyl or dansyl group.
Alternatively, an alkaline metal hydroxide is first dissolved in a mixture of acetone and water, and t~len the 3,17-OH estradiol derivative is added thereto Thereafter, the resulting salt is reacted with an acid chloride or acid anhydride corresponding to the desired acyl or dansyl group l~hen R2 is dansyl, dansyl chloride ~5-dimethylamino-1-naphthalenesulfonyl chloride) is used as the acid chloride When H of the 3 -hydroxyl group in the 3 ,17 -OH estradiol derivative is substituted wit~l the alkyl group R2, it is possible to apply a usual alkyletherification method. For example, a dialkyl sul~ate such as dimethyl sulfate and a dilute alkaline solution are reacted with the 3 ,17 -OH estradiol deriva tive Accordingly, an estradiol derivative carrying the oR2 group at the 3-position on the ring A, i.e., a compound (hereinafter referred to as a "3-oR2 estradiol derivative"~ of the formula (III):
R OH
~ ~ ~ ` s wherein R1, R2, R3, R4, RS, and n have the same meanings as above, is obtained 31D0~

A binding agent may be used to couple the 3-oR2 estradiol derivative and chlorambucil. The binding a~oent may be a hydroxylated or halogenated carboxylic acid derivative of the formula (IV):
X (cH2) mCY (IV) wherein X is halogen (for example, chlorine or bromine) or OH; Y
is ~lalogen which is the same as or different from X (for example, chlorine or bromine), OH, or a salt thereof; and m is an integer of 1 to 3. As examples of such carboxylic acid derivatives, there may be mentioned monochloroacetic acid, monobromoacetic acid, ~-monochloropropionic acid, ~-monobromopropionic acid, monochloroacetyl chloride, andmonobromoacetyl bromide.
It is possible ~o react the 3-oR2 estradiol derivative and the binding agent to esterify the 17-hydroxyl group and obtain the compound of the formula (II):
o Il R4 ` C- (Cl~2) m--X
(R1) ~ ~ (II) wherein R1, R2, R3, R4, R~, m, n, and X have the same meanings as the above~ The resulting compound will be hereinafter referred to as the "compound (II) ". The reaction may be performed in an organic sol~rent, for example, dimethyl sulfoxide, dimet~lylformamide, pyridine, acetone, or tetrahydrofuran at -10 to ~30~^. -Then, the compound (II) is reacted with chlorambucil. Thecarboxylic acid moiety of chlorambucil may be a salt with a 2l~3n~

metal ~for example, sodium, potassium, silver, or calcium), an acid halide or an acid anhydride and further the chlorambucil derivatives may be a hydrochloride thereof. The reaction may be performed in an organic solvent, for example, dimethyl sulfoxide, dimethylformamide, pyridine, benzene, acetone, toluene, carbon tetrachloride, chloroform, or tetrahydroEuran.
An aqueous alkaline solution ma~ be added, if necessary. The reaction may be performed at -30 to +150C, preferably 0 to 100C, for 0.1 to 90 hours, preferably 0.5 to 75 hours. The reaction product may be purified by a suitable method to obtain the present conjugate. As the purification method, there may be used extraction, chromatography, crystallization, reprecipitation, etc.
The order of the above reaction steps may be changed, if appropriate. Examples of the changes are as f=ollows:
(1) The chlorambucil and the binding agent are first reacted, and then the 3-oR2 estradiol derivative is coupled thereto.
~2) After the coupling of the chlorambucil and 3,17-OH
estradiol derivative via the binding agent, H of the 3-hydroxyl group of t~le estradiol derivative moiety may be substituted.

(3) In cases where one or more acyloxy groups are introduced onto the ring B, C, and/or D, one or more hydroxyl groups are introduced to the desired position (s) on the ring B, C, and/or D, and then esterif ication there3f is performed.
The structures of the present conjugate thus obtained may be confirmed by IR (infrared) spectrum, W (ultraviolet) spectrum, NMR (nuclear magnetic resonance), elementary analysis, mass spectrum, etc.
The toxicity and pharmacological activities of the present conjugate are as follows:
( 1 ) Toxici ty Each of 18 types of the present conjugates was orally administered to Wistar rats in an amount of 5000 mg/kg. No death was observed over a period of one week. Therefore, the present conjugate is extremely safe. See Example 3.
(21 Estrogen effects (measurement of uterus weight) The estrogen efEects of 18 types of the present conjugates were investigated. No or only slight estrogen ëffect was , . . , . _ _ _, .

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observed. See Example 4.
(3) Selective antitumor eEfect In the following experiments (a) to (f ), the present conjugate exhibited a selective antitumor effect comparable or superior to that of the comparative substance. (estradiol-chlorambucil. conjugate) .
(a) Selective growth inhibitory efEect for transformed mouse cells The present conjugate considerably lowered the survival rate of transformed mouse cells (3T3SV~40), whereas it did not affect the survival rate of normal mouse cells (3T3). See Example 5 (1) .
(b) Selective growth inhibitor~y eEfect for human cancer cells The present conjugate considerably lowered the survival rate of various types of human cancer cells [human renal cancer (RC) cells, human prostatic carcinoma (PC-3) cells, human cervical cancer (HeLa) cells], ~hereas it did not affect the survival rate of normal human cells (FLOW400t)). See Example 5(2) .
(c) Effect for secretion of transforming growth factor (TGF)-rom human breast carcinoma cells (MCF-7) The present conjugate inhibited the secretion o TGF-induced by the estradiol. This shows that the present conjugate has the effect of killing cancer cells while inhibiting the secretion of the cancer transforming growth factor. See Example 6.
(d) Antitumor effect (intraperitoneal and oral administration~
The present conjugate exhibited a life-prolonging effect (macrobiotic effect) on cancer-bearing BDFl female mice into which P388 cells had been transplanted intraperitoneally. See Example 7.
(e) Antitumor effect (intra-arterial administration of injection) The present conjugate exhibited a macrobiotic effect on rat tumor Walker 256 See Example 8.
(f) Selective accumulation into cancer tissue transplanted in the ~iver and estrogen eEfect by liver intra-arterial administration . . . .

21~3!~0~
The concentration of the present conjugate in cancer tissue transFlanted in the liver was higher than the concentration in normal liver tissue. The present conjugate did not enlarge the uterus. See Example 9.

(4) Selective irlmunosuppressive effect In the :following experiments ~a) to (d), the present conjugate exhibited a selective immunosuppressive effect comparable to that of the comparative substance (estradiol-chlorar[bucil conjugate).
(a) Heteroantigen stimulating reaction The present conjugate did not considerably affect the blastogenesis reaction of the lj~mphocytes on phytohemagglutinin (PhA) . See Example 10 ~1) .
(b) Isoantigen stimulating reaction The present conjugate inhibited the mixed lymphocyte culture ~MLC) reaction. See Example lO (2) .
(c) Activity to inhibit GVH~ (graft versus host reaction) in a mouse bone marrow transplant modei The present conjugate increased the survival days of the mice after the transplant. See Example ll.
(d) Relation to TGF-,~ in MLC reaction The present conjugate exhibited a combined effect with the immunosuppressive substance (TGF ,~) . The inhibitory effect of the present conjugate was neutralized by the anti-TGF-~antibodies . Therefore, it is considered that ^h~n; srn of inhibitory effect by the present conjugate relates to TGF-~ in the MLC reaction. See Example 12.
As above, the present conjugate has selective physiological activities, that is, a selective antitumor or immunosuppress~ve effect, and so is useful as a pharmaceutical composition, in particular an antitumor agent or an In"sllrrreSsive agent. As the antitumor agent, it is effective against carcinomas-in, for example, the breast, ovary, uterus, stomach, rectum, colon, kidney, hematopoietic system, liver or u~inary organs, or other solid tumors When the present conjugate is used as an antitumor agent, various pharmaceutical compositi~ns suitable for administering ~ ~ rou~es may e ~ormulated by any conventional 21~3~

methods. As examples of the formulations, there may be mentioned oral agents such as capsules, syrups, pills or tablets, injections, external agents, and suppositories. As examples of the external agents, there may be mentioned a solid agent containing a usual base such as white vaseline and a penetration enhancer such as N,N-diethanol lauramide.
The present conjuga~e as an antitun~or agent has a characteristic feature that a cross tolerance ~ith the existing medicaments is slight.
The pharmaceutical composition may contain the present conjugate in an amount of preferably 0.01 to 75% by weight, more preferably 0.05 to 25% by weight. The present conjugate may be administered: orally, transdermally, intramuscularly, intravenously, intra-arterially, intrarectaLly, etc. The dosage varies with the administration method and the degree of treatment, but generally is as follows: for an adult, the dosage of oral administration is 0.1 to 50 mg/kg per"day, while the dosage of parenteral administration is 0 . 01 to 20 mg/kg per day.
When the present conjugate is contained in an injection for intra-ar~erial administration (i.e., an intra-arterial injection), it is preferable to dissolve the present conjugate in an ester of iodinated poppy oil fatty acid The concentration of the present conjugate in the intra-arterial injection is preferably 0.1 to 10% by weight, more preferably 1 to 5% by weight. As the ester of iodinated poppy oil fatty acid, a lower alkyl ester wherein an iodination degree is 30 to 40% by weight is preferable. Lipiodol (trade name of Lipiodol ~ltra-Fluide) (iodination degree of 38.8% by weight; ethyl ester) is more preferable. The dosage of the intra-arterial injection is preferably 0.01 to 20 mg/kg, more preferably 0.1 to 10 mg/kg. The intra-arterial injection is effective against a carcinoma in the liver, breast, gynecologic organs, gastrointestinal organs, and urogenital organs The intra-arterial injection accumulates specifically in carcinoma tissue --and stays there for a long period. Further, the use of an ester of iodinated poppy oil fatty acid makes it possible to perform diagnosis or medical treatment while observing ~he tumor cells by X- rays , CT (computer tomography), ultrasonic waves , etc .
.. . ... . ... . . .

1-- 2183~

The present conjugate is effective as an immunosuppressive agent in the organ transplantation, for example, for the prevention and treatment of rejection reactions in the transplantation of the kidney, liver, heart, skin, bone marrow, or the like, and further for the treatment of autoimmune diseases such as various types of renal diseases, thyroid diseases, chronic articular rheuma~ism, aplastic anemia, systemic lupus erythematosus, myasthenia gravis, liver diseases, polyarteritis, and dermatomyositis. Therefore, the present conjugate may be used as a preventing or treating agent for rejection in the organ transplantation, and a treating agent for autoimmune diseases.
When tlle present conjugate is used as an irnmunosuppressive agent, various pharmàceutical composi~ions suitable for administering via various routes may be formulated by conventional methods. As examples of the formulation, there may be mentioned oral agents such as tablets, granules, dispersions or capsules, suppositories, injections or external agents. The administration routes and dosages are the sama as in the antitumor agents mentioned above.
As above, the estradiol derivative moiety in the present conjugate carries one or more substituents in addition to the 3-substituent present also in the conventionally known estradiol - -derivative moiety. By virtue of such a structure, it is possible to eliminate or tremendously weaken the estrogen effect of t~le conventionally known conjugate, while maintaining the selectivQ physiological activities of the conventional estradiol-chlorambucil conjuga~e, namely, the selective --antitumor effect and the selective immunosuppressive effect.
Therefore, the usefulness as an antitumor or i~munosuppressive agent is remarkably increased.
EXAMPLE~S
The present invention now will be further illustrated by, but is by no means limited to, the following examples.
In the following examples, the physical properties of the compound were measured by the following methods:
(1) Thin layer chromatography (silica gel):
... ., ,, , _ _ ~

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Silica gel thin layer plate LK~DF (layer t~lickness = -250 mm; ~hatmRn Co. ) (2) Elementary analysis:
Yanaco CHN-CORDER MT- 3 (Yanagimoto Co . ) (3) Mass spectrum:
Mass spectrometer JMS-DX303 (JOEL) ( 4 ) NMR ( CDCl 3 ):
JNM-GSX- 500 (JOEL) (5) Fluorometric Rnalysis:
Shimadzu Spectrophotofluorometer RF- 540 (Shimadzu Co . ) Exatt Pl e 1 -~11 Prçl~R~ation of l-methvl-estra-1,3,5(10)-t~riene-3,1713-diol, 3 -b~n~Qate, 17 -monobr~mn~ tate Acetone (200 ml) Rnd l-methyl-estra-1,3,5(10)-triene-3,17~-diol (7.2 9) were placed in a four-neck flask (500 ml), agitated to dissolve them, and then lN-NaOH (0 . 028 mole as NaOH) was added thereto. A solution of benzoyl chloride (4.2 9) in acetone (20 ml) was added dropwise to ~the solution while cooling with ice. A viscous substance was precipitate~. Thus, the viscous substRnce was extracted with chloroform (200 ml + 100 ml) and the organic layers were washed with water, then treated with magnesium sulfate, and dried under reduced pressure to obtain a light yellow viscous substance (9.9 g). The Vc=o absorption was conirmed in IR spectrum of the substance. The product was developed by thin layer chromatography (silica gel;
developing solvent: cyclohexRne/ethyl acetate (5/2; v/v) ), whereupon an Rf of the product ~/as 0.27 and the disappearance of the raw material spot (Rf=0.18) was confirmed.
Then, the resulting l-methyl-estra-1,3,5(10)-triene-3,17~-diol,3-benzoate (9.9 g), dry tetrahydrofuran (250 ml) and pyridine (2.4 g) were placed in a four-neck flas]c (500 ml) ec~uipped with a cRlcium chloride tube, then a solution of bromoacetyl bromide (10 9) in dry tetrahydrofurRn (80 ml) was added dropwise at -5C to -3C. The mixture was agitated at 0C
for 1 hour. After the disappearance o the raw material had been confirmed by thin layer chromatography, the precipitated pyridine-HBr salt was filtered out and the filtrate was dried . _ _ _ . , . , ., _ _ . . , . , . _ _ . , .

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under reduced pressure. The residue was purified by silica gel column chromatography [developing solvent: chloroform/n-hexane (2/1; v/v) ] to obtain the title compound (9.3 9) . The physicochemical data was as follows:
R~: 0.70 [cyclohexane/ethyl acetate (5/2; v/v) ]
Elçmentary analysis ~or C2gH31O4Br:
Calculated (%): C: 65.77, H:6.06, Br:15.64 Found (%): C:66.0, H:6.1, Br:15.9 EI-MS: m/z 510 (parent ion peak) The intermediate products shown in Table 1 were prepared by a s imi lar method . - -Tabl ç
No.Intermediates R~ (1) m/z (2) 4-methyl-estra-1,3,5(10)- 0.70 510 triene- 3 ,17,~-diol, 3 -benzoate ,17 - monobromoacetate 22-methyl-estra-1,3,5(10)- 0.71 510 triene 3 ,17~ - diol, 3 -benzoate, 17 -monobromoacetate 32-ethyl-estra-1,3,5(10)- 0.81 524 triene- 3 ,17,~ -diol, 3 -benzoa te, 17 - monobromoace ta te 4 2-isopropyl-estra-1,3,5(10)- 0 88 538 triene - 3 ,17~-diol, 3 -benzoate, 17 -monobromoacetate 51,2-dimethyl-estra-1,3,5(10)- 0.80 524 tr~ene-3, 17,~-diol, 3-benzoate, 17 -monobromoacetate 61, 4 -dimethyl -estra- 1, 3, 5 (10) - 0 .79 524 triene - 3 ,17~3 - diol, 3 -benzoate, 17 -monobromoacetate 7 2-methoxy-i-methyl-estra- 0 79 549 1, 3, 5 ( 10 ) - triene - 3 ,17~ -diol,3-benzoate, 17 -monobromoace ta te 23~3n~

82 -methoxy- estra -1, 3, 5 (10) - 0 . 69 535 triene - 3 ,17B -diol, 3 -benzoa te, 17 - monobromoacetate 9 1-methyl-estra-1,3,5(10)- , 0 80 524 triene - 3 ,17~ -diol, 3 -benzoa te, 17 - ( ~ -monobromopropionate) 1-methyl-estra-1,3,5(10)- 0.85 538 triene - 3, 17,B - diol, 3 -benzoate, 17 - (y-monobromobutyrate) ,~ :
(1) Mobility in silica gel thin layer chromatography Silica gel LK6DF (Whatman Co. ) Developing solvent CyclohexaneJethyl acetate ( 5/2; v/v) (2) Parent ion peak in mass spectrum (EI) ~21 PreParat;on Qf l-m~othvl~estra-ll3~(lQ)-tricne-3~l7B-di 3 - (~-dimethv] ~ninona~hthalenes~ll fonat~) ,17 -monobromoac~tate Acetone (100 ml) and 1-methyl-estra-1,3,5(10)-triene-3,17,B-diol (9 9) were placed in a four-neck flask (2000 ml) and agitated to dissolve NaOH (lN: 32.5 ml) was added, and then a solution of 5-dimethylaminonaphthalenesulfonyl chloride (dansyl chloride: DNS-C1) (9 2 9) in dry acetone (200 ml) was added dropwise thereto at 10 to 15~_ The mixture was agitated for 3 hours. After the end of the reaction had been confirmed by thin layer chromatography (silica gel; developing solvent:
cyclohexane/ethyl acetate (5/2; v/v) ), the acetone was removed under reduced pressure, and then the residue was extracted with chloroform (200 ml x 3). The chloroform layers were collected, dried over magnesium sulfate, ar.d then further dried under reduced pressure to obtain a yellow viscous substance (31.3 9).
Column chromatography using silica gel [cyclohexane/ethyl acetate (5/2; v/v) ~ was carried out to obtain l methyl estra 1,3,5(10) ~triene-3,17B-diol,3- (5-dimethylaminonaphthalene-sulfonate) (12 9) Rf: 0.15 [cyclohexane/ethyl acetate (5/2, v/v)]
_ .

~83n~
.

Elementary analysis for C31H37~O4S:
Calculated (%): C: 71.64, H:7.18, N:2.70 Found (%): C:71.5, H:7.2, N:2.6 EI-MS: m/z 519 (parent ion peak) Fluorometric analysis:
Excitation: 356 nm Emission: 520 nm Then, the resulting 1-methyl-estra-1,3,5(10)-triene-3,17~-diol, 3- (5-dimethylaminonaphthalenesulfonate) (12 9), dry tetrahydrofuran (200 mI), and pyridine (5 9) were placed in a four-neck flask (500 ml) eo~uipped with a calcium chloride tube and agitated at -5C to -3C. A solution of bromoacetyl bromide (7.6 9) in dry tetrahydrofuran (40 ml) was added dropwise to the solution. The mixture was agitated at 0C for 1 hour, and further agitated at room temperature overnight After the disappearance of the raw material had been confirmed by thin layer chromatography, the precipitated pyridine-HBr salt was `
filtered out, and then the filtrate was treated with magnesium sulfate. The solvent was removed under reduced pressure to obtain a yellow-~hite solid. The solid was purified by column chromatography using silica gel [cyclohexane/ethyl acetate (5/2;
v/v) ] to obtain the title compound.
~f: 0.62 [cyclohexane/ethyl acetate (5/2; v/v)~
Elementary analysis for C33H3gNOsSBr:
Calculated (%): C:61.87, El:5.98, Br:12.47, N:2.19 Found (%): C:61.7, H:5.8, Br:13.0, N:2.2 H - NMR ( CDC13 ):
2.90 ppm (S, CH3, DNS-N (CE~3)2) 2.14 ppm (S, CH3, Ar-CH3) 0.70 ppm (S, CH3, E2-18CH3) EI-MS: m/z 639 (parent ion peak) Fluorometric analysis:
Excitation: 355 nm Emission: 525 nm Flame reaction: Halogen coloration In the same manner as mentioned above, the intermediates shown in the following Table 2 were prepared ~ ~183(1~

Tablq 2 No . Intermediates ~ Rf m/z (3 ) 1-methyl-estra-1,3,5(10)- 0.85 (1) 490 triene - 3 ,17~ -diol, 3 -acetate, 17 -monobromoacetate 2 1 -methyl -estra- 1, 3, 5 (10) - 0 .70 (2) 644 triene - 3 ,17~ - diol, 3 -palmitate, 17 -monobromoacetate 3 1-methyl-estra-1,3,5(10)- 0.80 (2) 672 triene- 3 ,17~3 -diol, 3 -stearate, 17 -monobromoacetate 4 1-methyl-estra-1,3,5(10)- 0.92 (2) 666 triene-3, 17~-diol, 3 -linolenate, 17 -monobromoacetate (1) Mobility o silica gel thin layer chromatography Silica gel LK6DF (Whatman Co. ) Developing solvent: Cyclohexane/ethyl acetate ( 5 /2; v/v) (2) Mobility of silica gel thin layer chromatography Silica gel LK6DF
Developing solvent: Chloroform/ethyl acetate (50/l;
v/v) (3) Parent ion- peak in mass spectrum (EI) ~3l Pre~aration Qf es~ra-1,3,5(10)-trie~e-3.16~,1713-triol,3- _ benzoate, 17 -mtmob~omoac~tate ~ - ~
An eggplant type flask (300 ml) was charged with estriol (5 9), acetone (45 ml), distilled water (150 ml), and lN-NaOH
(20.6 ml). The mixture was agitated to dissolve. The solution was cooled to 0C, then a solution of benzoyl chloride (2 . 68 9) in acetone (35 ml) was added dropwise over 1 hour. The mixture was agitated at the same temperature for 1 hour and at room temperature for 1.5 hours, and then allowed tQ stand overnight.
T~le precipitated crystals were filtered out. The crystals were washed with ethanol (200 ml), and then dissolved in chloroform (700 ml). The conce-ntration and crystallization were repeated ... . . . . .. _ _ _ _ .

~ 2~33~5 to obtain white crystals (estradiol-3-benzoateJ (6.2 9).
An eggplant type flask (300 ml) was charged with the resulting estradiol-3-benzQate (5.6 9), pyridine (1.3 9), and dry tetrahydrofuran (160 ml), and the mixture was agitated under cooling at 0C. A solution of bromoacetyl bromide (3.5 9) in dry tetrahydrofuran (35 ml) was added dropwise to the mixture - --over 1 hour. After the end of the addition, the mixture was agitated for further 1. 5 hours, and then allowed to stand overnight in a refrigerator (about 5C). The precipitated pyridine-H}3r salt was filtered out and the filtrate was concentrated to obtain an oily substance (8.4 9). Three components were confirmed by thin layer chromatography [silica gel: cyclohexane/ethyl acetate (5/l; v/v) ~ . Using silica gel chromatography (developing solvent = the same as in the above thin layer chromatography), compounds having an Rf of 0.71, an Rf of 0.75, and an Rf of 0.87 were obtained in an amount of 1 9, 2.3 9, and 1 9, respectively. The title compound was a compound having an Rf o f 0 . 71.
Rf: 0.71 [cyclohexane~ethyl acetate (5/l; v/v) ]
EI-MS: m/z 512 (parent ion peak) ~41 Pre~aration Qf estra-1.3,5(10)-triene-3,17B-diQl-6-sne,3-benzoate, 17 -mQnobrQmoacetate - - - =
Estradiol (50 9), pyridine (90 9), and dry tetrahydrofuran (400 ml) were placed in a four-neck flask (l liter). Then, acetic anhydride (94 9) was added thereto and the mixture was refluxed for 6 hours under stirring. The pyridine in the solutiQn was concentrated to about l/3 volume. Then, ethyl acetate (400 ml) and distilled ~ater (300 ml) were added, and the mixture was shaken for extraction. The organic layer was washed two times with e~ual volumes of distilIed water. The organic layer was dried sver magnesium sulfate, and then dried under reduced pressure to obtain white crystals (estradiol-3, 1713 - diacetate; 64 . 1 9 ) .
The resulting estradiol-3,17B-diacetate (64 9), acetic acid (740 ml), and distilled water (160 ml) were placed in an eggplant type flask (1 liter), and ~hen chromium trioxide (53 8 g) was added portionwise thereto on a bath at 30 to 40C After 3 hours, chromium trioxlde (6 9) was further added, and then the _ _ , _ ... ... _ . . . .. . , _ _ _ _ _ , 21~3n~3 mixture was agitated at room temperature overnight. The reaction mixture was dispersed in distilled water (3 liters) and extracted with ethyl acetate (1 liter x 3). The organic layers were washed with a saturated aqueous solution (1 liter x 6) of sodium hydrogen carbonate and distilled water (l liter x 2), dried over magnesium sulfate, and then dried under reduced pressure to obtain a yellow oily substance. The substance was purified by silica gel column chromatography [developing solvent: cyclohexane/ethyl acetate (5,~2; v/v) ] to obtain white crystals (13.1 9). The compound was confirmed by NMR and IR
spectrum to be estradiol-3,17~ diol-6-one,3,17~-diacetate The resulting estradiol-3,17,~-diol-6-one,3,17~-diacetate (12 9) and methanol (350 ml) were placed in an eggplant type flask (1 liter) ~ solution of potassium hydroxide ~53 9) in methanol (530 mI) was added to the solution while cooling and the mixture was agitated at room temperature for 2 hours. The mixture was concentrated under reduced pressure to about half of its volume, adjusted to about a pH 4 with 2N-HCl, and then extracted with ethyl acetate ~00 ml x 3). The organic layers were washed with distilled water (500 ml), dried over magnesium sulfate, and dried under reduced pressure to obtain light yellow crystals (8.74 9). The compound was confirmed by NMR and IR
spectrum to be estradiol-3,17~-diol-6-one.
The resulting estradiol-3,17~-diol-6-one (2 9) was dissolved in acetone (50 ml) and placed in an eggplant type flask (200 ~ ). Then, an aqueous solution of NaOH (309 mg) in water (100 ml) and further, a solution of benzoyl chloride (1.04 g) in ethyl ether (10 ml) were added thereto and the mixture was vigorously agitated, whereupon a white substance was quickly precipitated. The white substance was filtered out with a G-4 filter, washed thoroughly with water and dried under reduced pressure to.obtain white crystals (2.6 9) T~le crystals (2 9) and triethylamine (2 9) were dissolved in dry tetrahydrofuran (20 ml) . The solution was placed in an Erlenmeyer flask (100 ml), and then agitated under cooling with ice water.
Thereafter, a solution of bromoacetyl bromide (4 9) in tetrahydrofuran (20 ml) was added dropwise to the solution, and the mixture was allowed to react under cooling and stirring for . .. _ .. ~ . . , ., . , . , . , _ _ _ . , ~ 218'3~Q5 2 hours, and then allowed to stand overnight in a refrigerator (about 5C). The resulting precipitate was filtered out with a G-4 filter and the filtrate was dried under reduced pressure to obtain a viscous compourld (3.4 9). The viscous compound was purified by silica gel chromatography [developing solvent:
cyclohexane/ethyl acetate (5~3; v/v) ~ . Fractions exhibiting a single spot ~Rf=0.63) on thin iayer chromatography [silica gel:
cyclohexane/ethylene acetate (5/3; v/v) ] were collected. The fractions were dried under reduced pressure. The residue was crystallized using a slight amount of ethyl acetate and ethyl ether at -20C to obtaLn white crystals of the title c:ompound (1.7 9) Rf: 0.63 [cyclohexane~ethyl acetate (5/3; v/v)~
EI-MS: m/z 510 ~parent ion peak) Exam~le 2: Pre~aration of coniu~ate - -rll Pre~ara~;on of l-me~hYl-estra-1,3,5(10)-~riene-3,17~-diol, 3 -ben~Q~e,17 - ~ ~4 - ~4 - ~bis (~-chloroethvl) aminol Phenvll -l -oxobutoxvl acetatel ~ res~nt co~l~iuRate (I) 1 ~
Chlorambucil (7.2 9), dimethylformamide (100 ml), and lN-NaOH (26.0 ml) were placed in a~l eggplant type flaslc (500 ml).
The solvent was removed under reduced pressure at 50C with a rotary evaporator and the residue was dried under reduced pressure. Then, the residue was dissolved in dry dimethylformamide (200 ml) again. A solution of the intermediate product obtained in Example l[1] ~9.3 9) in dry dimethylformamide (100 ml) was added thereto. The mixture was agitated at room temperature for 24 hours. After the disappearance of the raw material spot had been confirmed by thin layer chromatography (silica gel), the dLmethylformamide was removed under reduced pressure. The residue was dissolved in chloroform (200 ml) and washed once with 200 ml of dilute saline solution. The organic layer was treated with magnesium sulfate and dried under reduced pressure to obtain a light brown oily substance. Further, the residue was purified by silica gel column chromatography [developing solvent: cyclohexane/ethyl acetate (5/1; v/v) ] to obtain a colorless oily substance (8.8 9) exhibiting a single spot on thin layer chromatography.
A solutLon of the colorless oily substance (8.8 9) in _ _ _ . , ... _ . . .. _ _ _ ~ ~183~5 ethyl acetate (20 ml) was added portionwise to isopropyl alcohol (1.3 liters), which was agitated on a water bath o:E 60C. The mixture was gradually cooled to 0C while agitating, and then allowed to stand overnight in a refrigerator at -18C. The resulting white crystal was filtered out at a low temperature and washed with cold n-hexane (-20"C). The crystal was dried under reduced pressure at 0C for 1 hour and at room temperature for further 4 hours to obtain ~hite crystals (5.2 9) o the present conjugate (I).
Rf: 0 . 66 [cyclohexane/ethyl acetate ( 5/2; v/v) ]
Elementary analysis ~or C42H49cl2~o6:
Calculated (%): C:68.65, H:6.72, Cl:9.65, N:1.91 Found (%~: C:68.8, H:6.7, Cl:9.7, N:1.9 EI-MS: m/z 733 (parent ion pe~k) H - NMR ( CDCI 3 ):
4 . 56 ppm (S, CH2, COCH2O) 3.71-3.60 ppm (m, CH2~ N(cH2cH2cl~2 2.23 ppm (S, C~3, Ar-CH3) 0.80 ppm. (S, CH3, 18-CH3) Flame reaction: Halogen coloration In the same manner as mentioned above, the present conjugates from (II) to (XI) shown in the ~ollowing Table 3 were prepared .
Tablç 3 Conjugate Name of conjugate Rf (1) m/z (2) No .
II4-methyl-estra-1,3,5(10)- 0.65 733 triene - 3 ,17,~ - diol, 3 -benzoate, 17 - [ ~4 - [4 - [bis (2 -chloroethyl ) amino] phenyl ] -1-oxobutoxy] acetate]
III2-riethyl-estra-1,3,5~10)- 0.66 733 triene- 3 ,17~ -diol, 3 -benzoate, 17- [ [4- [4 - [bis (2-chloroethyl) amino] phenyl] -1-oxobutoxy] acetate]

- 23 - 21g30~)~
IV 2-ethyl-estra-1,3,5(10)- 0.75 747 triene-3 ,17~-diol, 3 -benzoate, 17 - [ [4 - [4- [bis (2 -chloroethyl~ amino] phenyl] -1-oxobutoxy] acetate]
2-isopropyl-éstra-1,3,5(10)- 0.89 761 triene- 3, 17~-diol, 3 -benzoate, 17 - [ [4 - [4 - [bis (2 -chloroethyl ) amino] phenyl~
oxobutoxy~ acetate~ -VI 1,2-dimethyl-estra-1,3,5(10)- ~0.74 747 triene - 3, 17fl - diol, 3 -benzoate, 17 - [ r4 - [4 - [bis (2-chloroethyl) amino~ phenyl~ -1-ox~butoxy~ acetate~
VII 1,4-dimethyl-estra-1,3,5(10)- 0.73 747 triene-3 ,17,~-diol, 3-benzoate, 17 - [ [4 - [4 - [bls (2 -chloroethyl) amino~ phenyl] - l -oxobutoxy] acetate]
VIII 2-methoxy-1-methyl-estra- 0 75 763 1,3,5(10) -triene-3,17~-diol, 3 -benzoate,-17 - [ [4 - [4 -[bis (2 -chloroethyl) amino] -phenyl ] -1- oxobu toxyj ac~tate]
2 -methoxy- estra -1, 3, 5 ( io ) - o . 65 749 triene - 3, 17,~-diol, 3 -be~ zoate, 17 - [ [4 - [4 - [bis ~2-chloro-ethyl) amino] phenyl] -1-oxobutoxy] acetate~
X 1-methyl-estra-1,3,5~10)- 0.76 747 triene-3, 17,~-diol, 3 -benzoate, 17 - [ [4 - [4 - [bis ~2 -chloroethyl) amino] phenyl~ -1-oxobutoxy~ propionatel ~lg30~

XI 1-methyl-estra-1,3,5(10)- 0.80 761 triene-3,17~-diol,3-benzoate, 17 - [ [g - [4- [bis (2-chloroethyl) amino] phenyl] -1-oxobutoxy~ - y - butylate] ~, (1) Mobility of silica gel thin layer chromatography Silica gel LK6DF (Whatman Co. ) Developing solvent: Cyclohexane~ethyl acetate ( 5/2; v/v) (2) Parent ion peak in mass spectrum (EI) ~2l Pre~aration Qf ~-methvl-est~a-1,3,s(1Q~-tri~ne-3,17B-diol, 3 - (5 -~ thyl~rn;non~nhth~l enesll~ fQnate) ,17 - r ~g - ~4- ~bis (2-chlo~ge~hvl) ;l~;nr~l Phenvl~ oxobutoxvl acetate~ resent coniuoate (XII ) T
Chlorambucil (3 . 9 9), dimethylformamide (lOO ml), and lN-NaOH (14.1 ml) were placed in an eggplant type flask (5DO ml).
The mixture was concentrated to about 1/3 volume under reduced pressure at 50C wi t~l a rotary evaporator . ~ solution of the intermediate product prepared in Example 1[2] (6. 5 9) in dry dimethylformamide (150 ml) was added thereto. The mixture was allowed to react at room temperature for 24 hours and the disappearance of the raw material was confirmed by thin layer chromatography. Then, the dimethylformamide was removed under reduced pressure. Et~iyl acetate (150 ml) was added to the residue. The mixture was washed with cQol water (100 x 2 ml).
The ethyl acetate layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure to obtain a yellow-brown oily substance. The substance was purified by silica gel chromatography rdeveloping solvent: cyclohexane/ethyl acetate (5Jli v/v) ] and a compound exhibiting a single spot by thin layer chromatography (6 2 9~ was obtained The purified product was crystallized from isopropyl alcohoi~n-hexane to obtain the present conjugate (X~I) (5.1 9).
Rf: 0.52 [cyciohexane/ethyl acetate (5/2; v/v)~
Elementary analysis for C47Hs6Cl2N207S:
Calculated (%): C:63.96, H:6.26, Cl:i3.23, N:3.2g ... . . .. ..

3~Q~

Found (~): C:64.0, H:6.3, Cl:8.2, N:3.2 EI-MS: m/z 862 (parent ion peak) lH.NME~ (CDC13): ~
4 . 59 ppm (S, CH2, COCH20) 3.70-3.59 ppm (m, CH2, N(cH2cH2cl)2) 2.90 ppm (S, CH3, DNs-N(cE~3)2) 2.14 ppm (S, CH3, Ar-CH3) 0.70 ppm (S, CH3, E2-18CH3) Flame reaction: Halogen- coloration In the same manner~ as mentioned above, the present conjugates from (XIII) to (XVI) shown in the following Table 4 were prepared.
Table 4 ~ - `=
Con~ugate . Name of conjugate F~f m/z (3) No . ~
XIII ~ 1 methyl-estra-1,3,5~10)- 0.80(l) 671 triene-3,17,~-diol,3-acetate, 17 - [ [4 - [4 - [bis (2-chloroethyl) amino~ phenyl] -1-oxobutoxy] acetate]
XIV 1-methyl~estra-1,3,5(10)- 0.63(2) 867 triene - 3 ,17,~-diol, 3 -palmitate, 17 - [ [4 - [4 - [bis (2-chloroethyl) amino] phenyl] -1-o~obutoxy] acetate]
XV 1-methyl-estra-1,3,5(10)- o 75(2~ 895 triene-3,17~-diol,3-stearate, 17 - [ [4 - ~4 - [bis (2 -chloroethyl ) amino] phenyl ] - l -oxobutoxy~ acetate]
XVI 1-met~lyl-estra-1,3,5(10)- o.37(2) 889 triene-3,17,~-diol, 3-linolenate, 17 - [ [4 - [4 - [bis ~2-chloroethyl ) amino] phenyl ] -1-oxobutoxyl acetate] ~
~1) Mobility of silica gel thin layer chromatography . . . . . _ _ .

2ls3n~

Silica gel LK6DF (Whatman Co. ) Developing solvent: Cyclohexane/ethyl acetate ( 5/2; v/v) (2) Mobility of 6ilica gel thin layer chromatography Silica gel LK6DF
Developing solvent: Chloro~orm/ethyl acetate t50/1;
v/v) (3) Parent ion peak in mass spectrum (EI) ~31 Pr~l~aration Qf 1-me~hvl -çstra- 1, 3, 5 (10) - triene-3 ,17B-disl, - -3 -~Alm; tate, 17 - ~ ~4 - ~4 - ~bis (2 -ohlo~oethvl) Aminol l~herlvll -1-oxQbutQxvlacçtatel ~=~resent colliuaa~e (XIV~ 1 Pyridine (1.1 9) and 1-methyl-estra-1,3,5~10)-triene-3,17B-diol (2 9) were dissolved in dry tetrahydrofuran (50 ml) and agitated while cooling on an ice-salt bath, A solution of bromoacetyl bromide t2 . 8 9) in dry tetrahydrofuran (20 ml) was added dropwise thereto. After the addition, the mixture was allowed to react overnight in a ref rigerator (about 5C) . The resulting pyridine-Br salt was filterçd out and the tetrahydrofuran was removed under reduced pressure. The residue was washed with ethanol to obtain 1-methyl-estra 1,3,5(10)-triene-3,17B-diol,3,17-dibromoacetate (3.1 9). The resulting compound was dissolved in acetone (200 ml) and cooled to 0C.
Distilled water was addea portionwise thereto to make ~he condition immediately be~ore precipitation. T~len, lN-NaHCO3 (0.45 ml) was added thereto and the mixture was agitated for 30 minutes. The acetone was removed under reduced pressure, whereupon white crystals were precipitated. The thin layer chromatography showed that the raw material spot remained slightly. The crystals were filtered out, washed, and dried under reduced. pressure. The product was purified by silica gel column chromatography [developing solvent: cyclohexane/ethyl acetate (5/1, v/v) ~ to obtain white crystals (2.5 9) . By NMR
and elementary analysis, the resulting compound was confirmed to be 1 -methyl - estra -1, 3, 5 ( 10) - triene - 3 ,17B -diol ,17 -monobromoacetate .
Then, a sodium salt of chlorambucil (1 9) was dispersed in dry tetrahydrofuran (50 ml). A 601ution of the above-mentioned _ _ . . ... .. . . . _ . .

2i.8~0~

1-methyl-estra-1,3,5(10) -triene-3,17~-diol717-monobromoacetate (1.2 9~ in dry tetrahydrofuran (10 ml) was added thereto while being agitated. The mixture was heated at 40C for 30 minutes, and then allowed to react at room temperature for further 24 hours. After the disappearance of the raw material had been confirmed by thin layer chromatography, the resulting salt was filtered out and the tetrahydrofuran was removed under reduced pressure to obtain a viscous substance (2.0 9). The product was purified by silica gel column chromatography [developing solvent: cyclohexane/ethyl acetate (5/2; v/v) ] to obtain 1-methyl-estra-1,3,5(10) -triene-3,17~-diol,17- [[4- [4- [bis(2-chloroethyl)-amino]phenyl]-1-oxobutoxy]acetate] (1 5 9).
The resulting compound (1.0 9) and dry pyridine (0.25 9) were dissolved in dry tetrahydrofur~n (50 ml), and cooled to 0C
while being agitated. A solution of palmitate chloride (0.87 9) in dry tetrahydrofuran (10 ml) was added dropwise thereto.
After the addition, the mixture was agitated at 30C for 3 hours, and then allowed to react at room temperature overnioht.
After the disappearance-of the raw material had been confirmed by thin layer chromatography, tetrahydrofuran was removed under reduced pressure. The residue ~as dissolved again in ethyl acetate (50 ml), washed with water (25 ml x 2), then the ethyl acetate layer was dried over anhydrous sodium sulfate and further dried under reduced pressure. The residue was purified by silica gel column chror~atogr~phy [developing solvent:
chloroform/ethyl acetate (50/1; v/v) ] to obtain 1.1 9 of the present conjugate (XIV).
Rf: 0 . 63 [chlorofoLm/ethyl acetate ( 50/1; v/v) ]
Elementary analysis for C51H756Cl2:
Calculated (%): C:70.48, H:8.70, Cl:8.16, N:1.61 Found (%): C:70 2, H:8.8, Cl:8 2, N:1 6 lH NMR (cDcl3 ): S
4 . 57 ppm (S, C~l2~ CocH2o) 3 77 - 3 53 ppm (m, C~2, N(cH2cH2cl)2) 2 . 17 ppm ( S, CH3, Ar - CH3 ) 1. 5 - 1. 2 ppm (m, CH2, (CH2 ) 14 ) 0.83 ppm (S, CH3, 18CH3) EI-MS: m/z :867 ~parent ion peak) . . _ ..... . . . . .

~183~5 Flame reaction: Halogen coloration In the same manner as above, the present conjugates ~XIII), (XV), and (XVI) listed in Table 4 were prepared.
~4l Pre~aration o estra-1,3,5(10)-triene-3,160~.17~-~risl,3-benzoate, 16~x-ace~At~, 17 ~ r~ - r4 - ~bis ~2 -chloro~thvl) aminol -~henvll-l-Qxobutoxvlacetatel ~=r,resent cQniuaate (XVII)l The intermédiate compound (1 9) prepared in Example 1[3]
and a potassium salt of chlorambucil (0.65 9) were dissolved in dimethylformamide (80 ml), and reacted while being agitated at room temperature for 24 hours. The reaction solution was concentrated to l/3 volume, and ethyl acetate (250 ml) was added to the concentrated solution. The mixture was washed with cold water (150 ml x 3), and the organic layer was:~dried over magnesium sulfate and further dried under reduced pressure to obtain an oily substance (1.9 9). The residue was purified by silica gel column chromatography [developing solvent:
cyclohexane/ethyl acétate (5/2; v/v) ] and the product was crystallized from isopropanol to obtain a white crystalline precursor (0 . 6 9), estra- l, 3, 5 (10) - triene-3 ,16c, 17~- triol, 3 -benzoate, 17 - [ [4 - [4 [bis (2 -chloroethyl) amino] phenyl] -1-oxobutoxy] acetate] [precursor of present conjugate (XVII) ] .
Rf: 0.75 [cyclohexane/ethyl acetate (5/Z; v/v) ]
Elementary analysis for C41E~47C12NO7:
Calculated (9i): C:66 85, H:6 39, C1:9.65, N:1.90 Found (9~): C:67.0, H:6.5, Cl:9.7, N:l.9 EI-MS: m/z 735 (parent ion peak) H-NMR (CDCl3): ~
4 . 56 ppm (S, CH2, COCH2O) 3.70 - 3.60 ppm (m, CH2, N(cH2cH2cl)2) 0.81 ppm (S, CH3, 18-CH3) Flame reaction: Halogen coloration The precursor -~400 mg), dr~ tetrahydrofuran (15 ml), dry pyridine ~1.5 ml), and acetic anhydride (1.5 ml) were placed in an eggplant type flask (50 ml). Then, a condensation tube with a calcium chloride tube was attached to the flask. Thereafter, the mixture was heated and agitated at 60C for 6 hours, and then allowed to stand at room temperature overnight. The reaction mixture was dried under reduced pressure and the . _ .. ..... _ ... . . _ _ 2183()~

residue was purified by silica gel column chromatography [developing solvent: cyclohexane/ethyl acetate (5/2; v/v) ] . The resulting oily substance (Q.6 9) was crystallized from ethyl acetate and isopropanol to obtain white crystals of the present conjugate (XVII) (0.32 g).
Rf: 0.45 [cyclohexane/ethyl acetate (5/2; v/v) ]
Elementary analysis for C43H4gC12NOg:
Calculated (%): C:64.99, M:8.94, Cl:8.94, N:1.76 Found (%): C:65.1, H:9.1, Cl:8.8, N:1.8 EI-MS: m/z 793 (parent iQn peak) lH NMR (CDC13 ):
4 . 60 ppm (S, CH2, COCH20) 3.71 to 3.60 ppm (m, CH2~ N(cH2cH2cl)2) 2 . 0 ppm ( S, CH3, COCH3 ) 0.83 ppm (S, CH3, 18-CH3) Flame reaction: Halogen coloration ~51 Pre~aration Qf e~tra-1,3.5(1Q)-triene-3,1713-diol-6-one,3-benzoate, 17 - ~ ~4 - ~4 - ~bis ~2-clll oroethvl) aminQl ~henvll -1-oxobu~o~svl acetatel ~=~resent coniuqate (XVIII) 1 Estra-1,3,5(10) -triene-3,17,~-diol-6-one,3-benzoate,17-monobromoacetate (500~ mg) prepared in Example~ 1[4] and a potassium salt of cElorambucil (340 mg) were added to dry tetrahydrofuran (20 ml), and agitated at 40C for 2 hours.
Then, the mixture was allowed to react overnight at room temperature. The resulting potassium salt was filtered out with G-4 filter, and the filtrate was dried under reduced pressure.
The residue was purified by silica gel chromatography [developing solvent: cyclohexane/ethyl acetate (50/15; v/v) ] .
The fractions of a single spot showing Rf = 0 24 by a thin layer chromatography with the same developing solvent as above were collected, concentrated under reduced pressurë, and crystallized from n-hexane to obtain the present conjugate (XVIII) as white crystals (370 mg).
Rf: 0.24 [cyclohexane/ethyl ace',.ate (50/15; v/v)]
Elementary analysis for C41H4sC12NO7:
Calculated (%): C:67.03, H:6.13, Cl:9.67, N:1.91 Found (%): C:66.9, H:6.4, Cl:10 0, N:1.9 EI-MS: m/z 733 (parent ion peak) 21830~

H-MMR (CDCl3):
4 . s7 ppm (S, C~2, COCH2O~
3.70 ~ 3.59 ppm (m, CH2, li(c~l2c~2cl)2) 0.80 ppm (S, CH3, 18-CH3) Flame reaction: Halogen coloration E~ le 3. To~;citv - ~
A suspension of the present conjugate (I) in a 0 . 5 %
methyl cellulose aqueous solution was once orally administered to Wistar rats (male; five weeks old; average weight = i30 9;
each group consisting of 10 rats) in an amount of 5000 mg/kg, using a metallic stomach tube. Rats were observed for 7 days after administration, but no death was observed. Similar results were observed for the present conjugates from (II) to (XVIII ) as well .
Exam~le 4: Estros~en efEe~t :(measurement o~ ute~us wei~ht) Test substances werè subcutaneously administered to Wistar/slc rats (female; 3 . 5 weeks old) for three consecutive days (namely, 50 mg/kg/day, 100 mg/kg/day, and 200 mg/kg/day, respectively). The hormone activity of the present conjugates was compared with that of a comparative substance, by measuring the weights of the uterus on the 4th day. It~ is to be noted that the above method was described as the excellent method by Koyama (Yoshihiko Koyama, Folia Endocrinologica Japonica, 37 (8):
826, 1961) From the relation between the `'uterus weight/body weight"
ratio on the 4th day and the amount of the substance administered~ the amounts of the substances to be administered were calculated that increase the "uterus weight/body weight"
ratio to twice the ratio of the control groups. When the present con~ugates were administered subcutarleously, sesame oil was used as the solvent. Further, as t~le comparative substance of conjugate, estra-1,3,5(10)-triene-3,17~-diol,3-benzoate,17-[ r4 - [4 - [bis (2 -chloroethyl) amino) -phenyl] -1 ~oxobutoxy] acetate]
(hereinafter referred to as the comparative substance I) was used. The comparative substance I corresponds to the compound of the formula (I) wherein all of R1, R3, R~, and R5 are hydrogen atoms (which is not the present conjugate). Further, estradiol- 17~ was also used for the comparative test. The _ _ _ ... .. . . _ . ... . _ _ . .

3n~
results are shown in Table 5.
Tablç S - `
Test substances Number Amount required to administered ~ ` of rats increase the tested "uterus weight/body weight" ratio to twice that of control (mg/kg) Present conjugate (I) S x 3 >200 Present conjugate (II) S x 3 >200 Present conjugate (III) S x 3 25 Pres ent con j uga te ( IV ) 5 x 3 5 0 Present conjugate (V) S x 3 100 Present conjugate (VI) S x 3 >200 Present conjugate ~VII) S x 3 >200 Present conjugate (VIII) S x 3 >Z00 Present conjugate (IX) S x 3 100 Present conjugate ~X) S x 3 >200 Present conjugate (XI) S x 3 >200 Present conjugate (XII) S x 3 >200 Present conjugate ~XIII) S x 3 >200 Present conjugate (XIV) S x 3 >200 Present con~-ugate (XV) S x 3 >200 Present conjugate ~XVI) S x 3 >200 Present conjugate (XVII) S x 3 4 . 0 Present conj,ugate (XVIIIl S x 3 Comparative substance I 5 x 3 0.20 Estradiol-17~ ~ ~10 x 3 0.02 A hormone activity was observed for the comparative substance I in the degree of abQut 1~10 that of the estradiol-17~. On the other hand, it was observed that the present conjugates carrying a substituent Rl at the 2-position on the ring A of the estradiol and not carrying a substituent on the rings }3, C and D exhibited the weakened hormone activity in the degree of 1/100 to 1/500 that of the comparative substance I.
No hormone activity was observed for the present conjugate having a substituent at the 1- or 4-position on the ring A or having plural substituents in the above experiments. Further, ~ 0~

the hormone activity of the present conjugate not carrying the substituent R1 on the ring A, ~ut carrying substituents on the ring B, C or D, that i5, the present conjugates (XVII) and (XVIII), was extremely low, i . e., about 1/20 to 1/5 that of the comparative substance I.
E:xaml~le 5: SelectiYe ~ntitumor effect - - -(1) Selective growth inhibitory effect for transformed mouse cells (normal mouse cells and transformed cells) Mouse 3T3 normal cells and transformed cells 3T3SV-40 in the logarithmic growth phase ~lere treated with 0.25 % trypsin.
The cells were dispersed in medium (MEM) at 2 x 104 cells/ml.
The cell disperslons were inoculated in test tubes ~Ikemoto Rika) in an amount of 1 ml/tube. The tubes were stuffed with cotton and allowed to stationary culture in a carbon dioxide gas incubator ~95% air and 5% carbon dioxide gas; constant temperature of 37C; humidity of 95%) at an angle of 5 from the horizontal .
The medium was removed after one day elapsed from the inoculation of the cells, and a fresh medium (p~l 7.4) was added.
Then, each test substance dissolved in dimethyl sulfoxide (10 ~g/ml or 50 ,ugtml ) was added . ~hereupon the concentration of the dimethyl sulfoxide was 1 percent with respect to the medium.
To a control test tube, only dimethyl sulfoxide was added. The samples were incubated for 5 days after the addition of the test substances. The living cells were countea by counting cells not stained by trypan blue in a Bur]~er-Turk hemocytometer. An average of 3: samples was obtained for each concentration of the test substances. The survival rate (%) was calculated on the basis of the control. The results are shown in Table 6. It was observed that the present conjugates maintained the same activity to selectively inhibit the growth of transformed cells as the comparative substance I.

._ 33 _ 2 1 8 3 ~ ~ ~
Tal~l e 6 . - ~ ~
Test Amount Survival rate Survival rate substances ad~r~inistered of 3T3 o~ 3T3SC-40 (~Ig/ml) ~, ~%) ,~, .. ( ) , =
Present con- 10 102 55 jugate (I) 50 95 14 Present cf~n- 10 100 50 jugate (II) 50 97 19 Present con- 10 100 54 jugate (III~ 50 90 15 Present con- 10 100 60 jugate (IV) 50 90 20 Present con= 10 103 61 jugate (V) 50 89 21 Present con- 10 100 49 jugate (VI) 50 97 20 Present con- lO 98 53 jugate (VII) 50 89 10 Present con- 10 95 5Z
jugate (VIII) 50 85 13 Present con- 10 95 50 jugate (IX) 50 86 15 Present con- 10 - 90 60 jugate (X) 50 85 11 Present con- 10 85 61 jugate (XI) 50 75 15 Present con- 10 100 52 jugate (XII) 50 95 18 Present con- 10 75 39 jugate (XIII~ 50 69 8 Present con- 10 98 53 jugate (XIV) 50 89 10 Present con- 10 100 50 jugate (XV) 50 92 19 Present con- 10 97 58 jugate (XVI) 50 91 24 Present con- 10 100 45 jugate (XVII) 50 93 15 21~3~

Present con- 10 105 49 jugate ~XVIII) 50 95 21 ~ _ Comparative 10 100 50 substance I 50 90 15 Chlorarnbucil 4.2 25 20 10 . 5 . lQ _c 7 Control 100 , ~ 100 (2) Selective growth inhibitory efect for human cancer cells (normal human cells and cancer cells) A growth inhibitory effect Isurvival ratel of the present conjugates were evaluated for llormal human cells (FLOW4000) and various types of human cancer cells [human renal cancer (RC) cells, human prostatic carcinoma (PC-3) cells, and human cervical cancer (He~a) cells].
Cells subcultured in a culture flask and in the logarithmic growth phase were treated with 0.25% trypsin. The cells were dispersed in medium at 2 x 104 cells/ml. The cell dispersions were inoculated in test tubes (Ikemoto Rika) in an amount of 1 ml/tube. The tubes were stuffed with cotton and allowed to stationary culture in a carbon dio~cide gas incubator (95% air and 5% carbon dioxide gas; constant te~nperature of 37C; humidity of 95%) at an angle of 5 from the horizontal.
Each test substance was dissolved in dimethyl sulfoxide and added to the culture systems. The concentration of the dimethyl sulfoxide was 1 percent with respect to the medium.
The medium was removed af ter one day elapsed f rom the inoculation of the cells, and a fresh medium containing the test substances and having a pH valua listed in Table 7 was added.
To a control test tube, only dimethyl sulfoxide was added. The samples were incubated for 5 days after the addition of the test substances. The results were e~aluated as in Example 3 (1) . The survival rate (%) of the present conjugates for the above cells are shown in Table 7. It was observed that the present conjugates maintained the same activity to selectively inhibit the growth of human cancer cells as the comparative substance I.

2~311~5 Tabl~ 7 =~= =
Test An~ount Normal Cancer cells substance added cell ( ~g J tube ) FLOW~I 0 0 0 RC PC - 3 HeLa ~H7 ~ Q) (pH7 . 0) r _ (pH7 . 0) (pH7 . 6) Present con- 5 101 75 78 50 jugate (I) 50 90 24 27 11 Present con- 5 103 73 82 60 jugate (II) S0 94 21 28 10 Present con- 5 100 79 80 69 jugate (III~ 50 95 28 32 19 Present con~- 5 100 80 78 75 jugate (IV) 50 98 35 36 25 Present con- 5 100 85 82 75 jugate (V) 50 97 39 38 30 Present con- 5 108 69 85 56 jugate (VI) 50 90 19 40 22 Present con- 5 100 70 80 59 jugate (VII) 50 95 25 39 29 Present con- 5 100 90 89 90 jugate 50 100 40 55 42 (VIII) Present con- 5 100 73 80 55 jugate (IX) SQ g2 29 30 16 Present con- 5 95 7a 82 60 jugate (X) 50 90 25 29 19 Present con- 5 90 70 75 70 jugate (XI) 50 90 20 35 30 Present con- 5 98 79 ~ 80 S5 jugate (XII) S0 92 25 35 20 Present con- 5 85 60 65 55 jugate : 50 75 9 11 8 (XIII) Present con- 5 102 79 85 71 jugate (XIV) 50 95 35 40 30 Present con- 5 100 80 85 75 jugate (XV) S0 95 35 38 35 Present con- 5 io2 75 80 73 jugate (XVI) 50 89 30 31 21 2183~5 - 3~ --Present con- 5 100 79 80 51 jugate 50 90 25 29 15 (XVII ) Present con- 5 102 81 84 74 jugate 50 89 32 35 32 ~XVIII ) = - =
Comparative - 5 92 79 81 52 substance I 50 91 23 31 13 Chloram- 2 1 54 30 39 35 bucil 4 . 2 , 29 29 18 20 Control lQ0 . 10D . 100 100 Ex~mr~le ~: Effect fQr sccret;on of ~r~nsfor~;nq qrowth f~c~or f ~ h~-nAn breast c~rcinoma c~e~ ~5 The efect of the present conjugate (I) on the secretion o transorming growth actor (TGF-a) from a human breast carcinoma cell MCF-7 was examined.
MCF-7 was cultured or 7 days in an MEM medium containing 5% DCC-N}3S ~dextran-coated charcoal treated newborn bovine serum) and estradiol (10-8M) and not containing phenol red. The medium was changed on the 2nd and a~th days On the 7th day ater the cultivation began, the medium was removed and an experimental medium containing 5% DCC-NBS, estradiol (10-8M) and a solution o a test substance in dimethyl sulfoxide was added On the ~th day after the test substance was added, the experimental medium was taken out and centrifuged (3500 rpm for 10 minutes1. The supernatant was dialyzed, using a SpectraPor 3 membrane ~MW 3500 cut off, Fisher Scientific) against distilled water or 3 days. A~ter the dialysis had been ~inished, the dialysate was lyophilized to obf:ain a powder. Then, ~he powder was dissolved in 1/10 volume of PBS (phosphate-bufered saline) containing 1% bovine serum albumin ~BSA).
The TGF-a was measured, using rabbit antibody and mouse monoclonal antibody (ATG - 2S) by a sandwich en7yme - linked immunoabsorbent (EIA) method in accordance Wit~l Inagaki et al.
(Inagaki, ~, et. al., J. Immunol. Method., 128: 27-37, 1990).
The results are shown in Table 8.

~ls3na~

Tablç 8 - - -Test substances Protein TGF-o~ secretion (mg/dish) (ng/mg-protein) Control 0 . 62 1. 32+0 . 08 E2 10 - 8M 0 . 63 2 . 02+0 . 09 Present cor~jugate (I) 10 - 6M
+E2 10 8M 0.59 1.20+0.09 Comparative substance I 10 - 6M
+E2 10-8M 0.65 1.40+0.10 Chlorambucil 10 - 6M
+E2 10-8M 0 56 . 1.93+0.11 .
n= 3, average + SD
It was observed that the present conjugate (I) completely inhibited, at a concentration of lO ~ 6M, the production and secretion of TGP-(x induced by 10-8M estradiol ("E2" in the table), in the same mmanner as the comparative substance I, but an equal molar concentration o~ chlorambucil did not affect the production and secretion of TG~
Exam~lç 7~ Antitumor ef~ect (intra~eri~Qneal ~3nr~ Qr~l m; n; s tra tion ) A cell suspension prepared by adjus~ing the number of P388 cells to 1 x 106 cells/D.05 ml ~anks' solution was intraperitoneally transplanted to a mouse (female; BDF1; 7.5 weeks old). A group for each test consisted of 6 mice, and a control group consisted of lO mice.
The comparative substance I was dispersed in a physiological saline solution containing 0.5~6 methyl cellulose.
50 mg/kg or lO0 mg/kg of the dispersion was administered intraperitoneally on the 1st, 4th and 7th days after the above transplant of the tumor. Purther, the present conjugates were administered in the same manner in an amount (molar ratio) corresponding to 50 mg/kg or lO0 mg/kg of the comparative substance I. When administering the present conjugates, a dispersion containing 0 . 5% methyl cellulQse in a physiological saline solution was used if such a dispersion can be prepared.
When administering the present conjugates (XIrI), (XIV~, (XV), _ _ . .... .. . .

21~300~

and (XVI) which are semisolid at room temperature, the conjugates were dissolved in sesame oil to adjust the concentration thereof so as to correspond an~ amount of administration o:E 10 mg/kg and 20 mgtkg of the comparative substance I that was dissolved or dispersed in sesame oil.
These preparations were administered orally to P388 tumor-bearing mice for 8 consecutlve days. From the number of deaths in each group, the median survival time (MST) was found and referred to as "T". In the salne manner, the MST of the control group (group to which only physiological saline solution or sesame oil was administered) was found and referred to as "C".
From "T" and "C", T/C x 100 (%) was calculated. The survival checking was ceased on the 45th day from the start of administration of the substances to be tested.
The results of the intraperitoneal administration of the test substances to P388 intraperitoneally transplanted mice are shown in Table 9 and those of the oral administration are shown in Table 10. It was observed in both administrations that the present conjugates exhibit a li~e-prolonging (macrobiotic) effect comparable or superior to that of the comparative substance I. Further, a life-prolonging effect superior to that of the comparative substance I was observed in the present conjugates (XVII) and (XVIII).
Table 9 ~ --Test substance Dose (mg/kg) Life-prolonging effect (T/C) x 1009 Present conjugate (I) 51 288 . 3 102 >405.4 Present conjugate (II) 51 270 . 0 102 >405.4 Present conjugate (III) 51 2~5.0 102 305 . 5 Present conjugate (IV) 52 280 . 0 104 300 . 0 Present conjugate (V) 53 250 . 0 106 305 . 5 ~ 2183~

Present conjugate (VI) 52 278 . 0 104 >405.4 Present con~ugate (VII) 52 260 . 0 104 399 . 0 Present conjugate (VIII) 53 270.3 106 >~05.4 Present conjugate (IX) 52 255.0 104 >405.4 Present conjugate (X) 52 200 . 0 104 260 . 0 Present conjugate (XI) 53 205.0 106 255.0 Present conjugate (XII) 60 261.3 120 291 . 0 Present conjugate (XVII) 55 220.0 110 299.0 Present conjugate (XVIII) 51 215.2 102 3~2. 1 Comparative substance. I S0 14S.1 100 = . 212.7 Table 10 Test substance Dose (mg/kg) Li~e-prolonging effect (T/C) x 100%
Present con~-ugate (XIII) 9.3 245.0 18 . 6 318 . 2 Present con~ugate :(XIV) 12.0 256.0 24 . 1 >405 . 4 Present conjugate (XV) 12.4 222.0 24.9 >405.4 Present conjugate (XVI) 12 . 3 225 . 0 24.7 .. 380 0 Comparative substance I lO 226 . 0 289 . 0 Exam~le ~3: Anti~m~r e~fect (int~a-arterial ~rn;n;~tratiQn Qf ~n ini ection) Walker 256 carinosarcoma (solid Lor ~, about 3 mm x 3 mm x ~ 2i830~S

3 mm) was prepared and subcutalleously transplanted in the lef t upper arm-partion and left thigh portion of Wistar rats (6 weeks old, female; 5 rats a group), by a transplanting needle. On the 8th or 9th day af ter the transplantation, the test substances were injected (0.1 ml/body) by a catheter from the right femoral artery. The test substances were dissolved or dispersed in Lipiodol (6.5 9) to form injection formulations.
The median survival time (MST) and the life-prolonging rate (T/C) were calculated in the same manner as in Example 7.
The results are shown in Table 11. Adriamycin was insoluble in Lipiodol and difficult to retain for a long period in the tumor tissue, and thus did not show the effect sufficiently. The present conjugate (I) was dissolved as sufficiently as the comparative substance I/ and eYhibited a sustained and suf f icient ef f ect Table 11 - ~
Experi~ Test substance ~ MST T/C
ment No. , (days) (96) Control 17 . 5 (physiological saline solution) 2 Lipiodol 18 . 5 105 .7 3 Present conjugate (I) (100 mg) (1) 45 0 257.1 4 Comparative substance 1: (100 mg) (1) 41.0 234.3 5 Adriamycin (10 mg) (2) 24.5 140.7 6 Adria~ycin (100 mg) (2) <1 <5.7 1) Dissolved in 6 . 5 9 of Lipiodol 2) Dispersed in 6 . 5 9 of Lipiodol ~xarnple 9: Select;ve accumulation into ~ncçr- tissue trisncplanted in the 1;v~r ~nfl estrQ~en effçc~ bv liver intra- -arter;~l ~flm;n;stratiQn The Walker 256 carcinosarcoma (solid tumor) same as that used in Example 8 was transplanted into the liver of Wistar rats and t~le reagents from A to E (0.05 ml/body) listed in Tables 12 and 13 were injected into the liver artery. On the 7th day after the injection, the blood sample was collected from each rat, and the amounts of GOT (glutamic oxaloacetic tr~nC~rn;n~ce) ~lg'30~

and GPT (glutamic pyruvic transaminase) were measured Further, the livers were excised from t~le rats and the concentrations of the reagents in the tumor and normal tissues in the liver tissue were measured by high performance liquid chromatography. The results are shown in Tables 12 and 13.
Table 12 Formul Concentration o test Concentration of test ation substance in tumor tissue substance in normal tissue 9/9wet tissue) (2) (~19~9 wet tissue) (3) A N.D. (5) N.D. (S) B 13.61 + 6.21 - 4.30 + 2.01 C 12.87 + 5.17 4.78 + 1.98 D 6.25 + 1.89 8.94 + 2.12 E 7.25 + 1.69 ~ 9.83 + 1.72 Averag e + SD: =
Table 13 . .- -~
Formulation GOT ~mU/ml) GPT (mU/ml) Ratio of ( 1 ) uterus weight ~. ~ (Control=1) (4) A 234+45.0 32+2.10 Q.9+0.2 B 189+59.84 49+8.91 0.9-~0.1 C 192-~76.74 52+9.07 ~ 1.4+0.2 218+69.21 ~0+7.06 : 1.0+0.2 E 227+7l.12 53+5.69 ~ 1.5+p.1 1) A: Injection prepared b~ dissolving 10 mg of adriamycin in 0 . 5 ml of Urografin, adding 1. 5 ml of Lipiodol and emulsifying by ultrasonic waves.
B: Injection prepared by dissolving 10 mg of the present conjugate (I) in 2 ml of Lipiodol.
C: Injection prepared by dissolving comparative substance I :(10 mg) in 2 ml of lipiodol.
D: Injection prepared by dissolving 10 mg of the present conjugate (I) in 2 ml of sesame oil.
E: Injection prepared by dissolving 10 mg of comparative substance I :(10 mg) in 2 ml of sesame oil.

~ 2~83~

2) Concentration of test substance in tumor tissue in liver tissue (,ug/g wet tissue) 3) Concentration of test substance in normal liver tissue (119/9 wet tissue) 4) Ratio of the uterus weight of the medicament-administered group to that of the control group to which only Lipiodol was intra-arterially injected, i.e., the uterus weight of the control group being 1 5) Not detected 6) Data is average + SD
When the present conjugate (I) was administered in the form of the Lipiodol injection [Formulation (B) ], the concentration thereof in the tumor tissue was higher than that in the normal liver tissue. It was the same result as the Lipiodol injection of the comparative substance I [Formulation (C) ] . When the adriamycin injection [Formulation (A) ] was used, the remaining reagent was not detected. When t~le sesame oil injections [Formulations (D) and (E) ] were used, no difference was observed between the concentrations of the test substances in the normal liver and those in the tumor tissues. Regarding the influence on uterus weights, a slight uterotrophic effect of the comparative substance I was observed, whereas no uterotrophic effect of the present conjugate was observed.
Exam~lç 1~): r In~)su~pressi~fe èffe~t (1) Heteroantigen stimulating reaction [effect of present conjugate II) on mitogen reaction]
The present Example examined the effect oE the present conjugate (I) on the blastogenesis reaction of lymphocytes responsible to heteroantigen stimulation (measured by the intake of 3H-thymidine into cells), that is, the irnmunoreaction responsible to bacteria or the like. As a mitogen, phytohemagglutinin (PHA) was used because it exhibits the strongest reaction on human lymphocytes (in particular, T
cells ) .
Peripheral venous blood was collected from a healthy male person into a vacuum blood collecting tube and centrifuged (2000 rpm, ordinary temperature, 20 minutes). The serum was further centrifuged (3500 rpm, 4C, 20 minutes) and the resulting - -3~

supernatant was added to a medium (RPMI 1640 ) so that the concentration of tl~e supernatarlt is 20 percent. The residue obtained by removing the above serum was diluted with the same amount of the same medium as above an~ the mixture was gently laid over a lymphoprep. The w~ole was centrifuged (lS00 rpm, 20~C, 30 minutes). The monocyte layer was taken out and washed twice or three times with the medium, and then the concentration of the cells was adjusted to S x 105 cells/ml.
The resulting cell floating liguid was poured into a 96 well U-bottomed microplate in an amount of 200 ,uI~well. The present conjugate (I) and comparative substance I were dissolved respectively in dimethyl sulfoxide and added to the cell floating liquid in an amount of 0 . S percent as a concentration of dimethyl sulfoxide. For a control group, only dimethyl sulfoxide was added. The test was carried out in triplicate for each group. PHA was added to each well in an amount of lO
,ug/ml . Af ter incubation for 2 days in a carbon dioxide gas incubator, 3H- thymidine was added in an amount of l ~Ci/well .
After cultivation for further 18 hours, the cells were harvested on a glass filter with a cell harvester. The radioactivity was measured with a liquid scintillation counter. The results are shown in Table 14. The present conjugate (I) and the comparative substance I did not considerably affect the blastogenesis reaction of lymphocytes responsible to heteroantigen stimulation.
(2) Isoantigen stimulating reaction [effect of present conjugate (I) on mixed lymphocyte culture (MLC) ]
The activity of the present conjugate (I) in a mixed lymphocyte culture, a so-called isoantigen stimulating reaction, was investigated as a model of immunoreaction in the transplantation of organ.
Monocytes were obtained from two healthy male persons, respectively, in the same manner as in the above PE~A reactions .
The cells obtained from one of two persons were treated by 15 Gy radiation. After the cells had been washed with the culture medium, the number of the cells was adjusted to 5 x 105 cells~ml. 100 ,ul of each of two types of the cells was poured into each of a same well of a microplate, i.e., 200 ,ul as a ~ ~183005 whole. In the same manner as in the above PHA reactions, the present con~ugate (I) and the comparative substance I were dissolved respectively in the dimethyl sulfoxide and added to each well_ After incubation for 6 days, 3H-t~lymidine (1 IlCi/ml) was added. After incubation for further 18 hours, the cells were harvested on a glass filter with a cell ~larvester. The radioactivity was measured with a liquid scintillation counter.
The results are shown in Table 14. It was observed that the present con~ugate (I) inhibited the MLC reaction in the same manner as the comparative substance I.
Tablç 14 Concentration Present conjugate (I) Comparative substance I
of test sub-stance (,ug/ml) PHA reac- MLC reac- PHA reac- MLC reac-tion (%) * tion (%) ~ tion (%) * tion (%) A
0 (control) 100 100 100 100 25 65.1 5.2 59.9 7.3 50 90.2 6.2 87.3 10.2 100 85.4 5.3 75.7 7.2 200 70.3 2.1 66.4 7 2 Percentage of intake of 3H- thymidine into lymphocytes:
relative percentage when the value of the control is 100.
Example 11: ~VHR inhibitsrv effeçt in bQrç ma~row transPlantation model i~ mr1us-e The present Example examined the GVHR (graft versus host reaction) inhibitory effect of the present conjugate in a bone marrow transplantation model, using 8-week-old male C3H/He mice and male B6C3F1 mice.
From the spleens of C3H/He mice, spleen cells were obtained by a metal mesh and added to RPMI 1640 containing 10%
FBS (fetal bovine serum). From the femur of the same mice, bone marrow cells were washed out using a syringe and added to an RPMI 1640 medium. After: each of the floating cells had been washed twice or three times, the concentration of cells was adjusted to 8 x 106 cells/ml. These were mixed in a ratio of 2183~

1:1 and then 0.5 ml of the mixture was injected to B6C3F1 mice in the tail vein. The mice had been pretreated with 9Gy of radiation. Starting from the day before the above transplantation operation, except the day of transplantation, until the 30th day after the transplantation, the present conjugate ~I) or the comparative substance I was orally administered each day in an amount of 2 mg/kg in the form of a dispersion in a physiological saline solution containing 0 . 5%
methyl cellulose. To the control group, only 0 . 5% methyl cellulose physiological saline solution was administered. The survival of the mice was observed. The results are shown in Table 15.
Table 15 Test substance ~ Amount of Number Median value oral admini- of mice ~ of survival stration (1) days after transplanta -tion (2) Control 0 6 8 Present conjugate (I) 2 6 30<
Comparative substance I 2 6 27<
(1): mg/kg/day ( 2 ): days In the control test, the median value of survival days was as low as 8 days, and early deaths believed to be caused by the occurrence of GVHD (graft versus host disease~ were observed.
On the other hand, in the present conjugate (I), a clear improvement of the survival rate was observed in the mouse bone marrow transplantation model, as in the comparative substance I.
Exam~le 12: Relation to TGP-~ on inh;hitorv effect in MLC
reactions To elucidate the mechanism of the selective inhibitory activity of the present conjugate (I) on the MLC reaction, a solution was prepared by dissolving TGF~ R&D Systems) and anti-TGF-,I~ antibodies (R&D Systems) in RPMI 1540, and added under the same experimental conditions as in Example 10 (2) . The ~ 218~

results are shown in Table 16.
Table 16 Test substance MLC reaction (%) (1) Present con- Comparative jugate (I) substance I
Control 100+13. 100 ~13 TGF~ 10 ng/ml) 44+26 4g+26 Anti-TGF-,~ antibody (lO ng/ml) 93+22 93+22 Conjugates 20 ,ug/ml (2) 46+18 34+22 Conjugates + TGF-~ (2) 16+14 7~7 Conjugates + anti-TGF-,~ antibody (2) 83+13 97+29 1) Percentage of intake of 3H-thymidine into lymphocytes:
Control = 100. n = 3, average + SD.
2) The "conjugates" indicates the present conjugate or the comparative substance. - -It was observed that the present conjugate exhibited theinhibition of the MLC reaction comparable to that by the immunosuppressive substance (TGF-~), and further a co~bined effect with the TGF-~. The anti-TGF-,B antibody did not affect the MLC rea-ction. The combination of the present conjugate (I) and the anti-TGF-~3 antibody su]~stantially neutralized the inhibitory activity of the MLC reaction obtained by the single use of the present conjugate (I). The above behavior of the prese-nt con~ugate is similar to that of the comparative substance I. Therefore, it is considered that rl-~rh~n; cnn of inhibitory effect by the present conjugate relates to TGF-,~ in the MLC reaction.
Formulation ~-~aml~le 1: Tablet ~ =
Present conjugate (I) 30 parts Mannitol~ 3 5 parts Sorbitol 25 parts Carboxymethylcellulose 5 parts Magnesium stearate 5 parts Talc . 40 parts The above components were thoroughly mixed and the mixture 2l~3nn~

was compressed to form tablets of a diameter of 10 mm.
Form-]lation Exam~le ~: Tniection The present conjugate (I~ (100 mg) was added to Lipiodol (6.5 g) at 25C and the mixture was stirred for 15 minutes to obtain a solution as an injection of the present conjugate (I).
The solution was filled in sterilized vials.
Formula~ion ~x;~mnle 3: ~a~sule The mixture prepar-ed in Formulation Example 1 was inserted and sealed in a No. O capsule to obtain a capsule.
Formulation ExamPle 4; Extern~l A~ent The present conjugate (I) (1 9), sesame oil (10 g), white vaseline (9D g), and N,N-diethanol lauramide (penetration enhancer: -10 g) were heated to melt on a water ba~h and mixed.
The mixture was cooled to room temperature to obtain an external agent .
Form~llat;on E~mr~le ~: ~u~1:7ositorv The present con~ugate (I) (100 mg), sesame oil (5 9), and Witepsol (90 9) were heated to melt and mixed on a water bath.
1.33 9 of the melted mixture was filled in a plastic suppository container and cooIed to room temperature to obtain a suppository.
Although the present invention has been :described with reference to specific examples, various changes and modifications obvious to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention.

Claims (4)

1. A compound of the formula (II):

(II) wherein R1 is alkyl or alkoxyl of 1 to 4 carbon atoms; R2 is acyl, dansyl, or alkyl; R3, R4, and R5 independently are H, oxo, OH, or acyloxy; m is an integer of 1 to 3; n is an integer of 0 to 3; and X is halogen, OH, or a salt thereof; provided that when n is 0, all of R3, R4, and R5 are not H at the same time, and at least one of R3, R4, and R5 is a group other than H and OH; and further, when n is 2 or 3, the groups R1 are the same or different.

2. A compound according to claim 1, wherein oxygen atom at the 17-position of the estradiol derivative is in .beta.-configuration.

3. A compound of the formula (III):

(III) wherein R1 is alkyl or alkoxyl of 1 to 4 carbon atoms; R2 is acyl, dansyl, or alkyl; R3, R4, and R5 independently are H, oxo, OH, or acyloxy; and n is an integer of 0 to 3; provided that when n is 0, all of R3, R4, and R5 are not H at the same time, and at least one of R3, R4, and R5 is a group other than H and OH, and further, when n is 2 or 3, the groups R1 are the same or different.

4. A compound according to claim 3, wherein oxygen atom at the 17-position of the estradiol derivative is in .beta.-configuration.