FI63420C - FOERFARANDE FOER FRAMSTAELLNING AV TERAPEUTISKT VERKSAMMA TETRAHYDROPYRANYLDERIVAT AV ANTRACYKLINGLYKOSIDER - Google Patents

Description

»Γ · 1 ^ ANNOUNCEMENT r 7 A n ^

Wa W (11) UTLÄGGN INGSSKMFT 6 3 4 2 0 C (45) Pi tantti:.: Y "r .: · r 'y 10 96 1933 ^ T ^ (51) K» .lk.3 / lntci.3 C 07 H 15/26, 17/04 FINLAND — FINLAND (21) Ntuttwwkwwi »-» »« iwwaiaiiiit 782388 (22) Htkwnlipllv »—An» eknlnj * dtf 02.08.78 * * (23) AlloipUvi — GUUgMtadtg 02.08.78 (41) ) Tullut JulklMfc ·) - Wlvk off «KNg 06 02 79 tattia J» rrtlrtrttallltu · (44) N »uw,. *»! *, «« »» ''

FatMlt · ocn r * gist «ratyr * lMn 'AntOfcsn utUgd och utl.» Krtfc «n pubUcarad 2Ö.02.83 (32) (33) (31) Pyydetty ttuoHt * u * -» «ftr4 priority 05.08.77

Japan-Japan (JP) Sho. 52-9 ^ 3 ^ 8 (71) Zaidanhojin Biseibutsu Kagaku Kenkyukai, lU-23, Kamiosaki 3-chome, Shinagawa-ku, Tokyo, Japan-Japan (JP) (72) Hiroshi Naganava, Tokyo, Kuniaki Tatsuta, Tokyo, Hamao Umezawa,

Tokyo, Tornio Takeuchi, Tokyo, Japan-Japan (JP) (7M Oy Borenius & Co. Ab (5 * 0 Process for the preparation of tetrahydropyranyl ether derivatives of therapeutically active anthracycline glycosides)

The invention relates to a process for the preparation of tetrahydropyranyl ethers of anthracycline glycosides for use as antimicrobial and antitumor agents, of the general formula (I) wherein R 1 represents a hydrogen atom or a hydroxyl or tetrahydropyranyl oxy group, and R 2 represents hydroxydryl or an oxy group, in which case, in the case where R1 represents a hydrogen atom or a hydroxyl group, R2 represents a tetrahydropyranyloxy group, and to prepare non-toxic acid addition salts of these compounds.

2 63420

A number of anthracycline glycosides have been described in the literature. Of these, daunomycin (U.S. Patent 3,612,212, GB Patent 1,003,383) and adriamycin (U.S. Patents 3,590,028 and 3,803,124), which are prepared by culturing certain Streptomyces microorganisms, have extensive anti-cancer activity in a variety of experimentally developed tumors. , and are used as clinically effective chemotherapeutic drugs. Although adriamycin and Dauno-mycine have several advantages as clinical and anti-cancer drugs, they are also known to have serious side effects, including hair loss, white blood cell loss, and cardiac toxicity.

The applicant has extensively studied the chemical modification of daunomycin and adriamycin with the intention of preparing new derivatives of these glycosides with improved anti-cancer activity and reduced toxicity (i.e., lower side effects). According to the invention, it has been possible to develop new tetrahydropyranyl ether derivatives of adriamycin and daunomycin which have the desired strong anti-cancer effect and low toxicity.

As used in this specification and claims, the term "non-toxic acid addition salt" refers to all those organic and inorganic acid addition salts of the compounds of formula (I) which are commonly used as predominantly non-toxic salts of amine-functional drugs. By way of example, these salts may be formed using sulfuric, phosphoric, hydrochloric, hydrobromic, nitric, phosphoric, acetic, propionic, maleic, petroleum, palmitic, lemon, amber, , fumaric, glutamine, pantothenic, laurylsulfonic, methanesulfonic, naphthalenesulfonic acid, etc.

Compounds of formula (I) in which R 2 is a tetrahydropyranyloxy group as substituents exist as separate diastereomers (arbitrarily hereinafter referred to as "isomer a" and "isomer b") which differ in the C-2 position configuration of the tetrahydropyranyloxy group, but these compounds may also be mixtures of such Isomers. The invention includes both the individual diastereomers and mixtures thereof.

5,63420

The invention is illustrated by the curves of the accompanying drawings.

Figure 1 shows the infrared absorption spectrum (KBr) of 4'-O-tetrahydropyranyl-daunomycin (isomer a).

Figure 2 shows the infrared absorption spectrum (KBr) of 4, -O-tetrahydropyranyl-daunomycin (isomer b).

Figure 3 shows the infrared absorption spectrum (KBr) of 4 ', 14-bis- (O-tetrahydropyranyl) -adriamycin (isomer a).

Figure 4 shows the infrared absorption spectrum (KBr) of 4 ', 14-bis- (O-tetrahydropyranyl) -adriamycin (isomer b).

Figure 5 shows the infrared absorption spectrum (KBr) of 14-O-tetrahydropyranyl-adriamycin.

Figure 6 shows the infrared absorption spectrum (KBr) of 4'-O-tetrahydropyranyl-adriamycin (isomer a).

Figure 7 shows the infrared absorption spectrum (KBr) of 4'-tetrahydropyranyl-adriamycin (isomer b).

Figures 8 to 14 show the proton nuclear magnetic resonance spectra (100 MHz, CDCl 3) of the above compounds in the same order as their infrared absorption spectra.

The starting materials used to prepare the compounds of the invention, i.e. adriamycin and daunomycin, can be represented by the following formulas: 63420 1 9 ^ in 0 η h λ M2 J41 Jz 13 "1 2 C-CH3 3 * T 5T oi [7

OCH-j 0 OH

O daunomycin 5 'yf— * · Α> Νΐχ, HO ^ - / I 2' 1¾ 1 9 10 O lii J * J & 2 Ali i® 13 »14

2 C "CH 2 OH

I I 9> 4T 5 rr 6T (7

OCH-j 0 OH

0 adriamycin 5r-0v * 'M A, HO ^ 1— / I 2' NHg

Daunomycin has two reactive hydroxyl groups (excluding both phenolic hydroxyl groups) at positions C-9 and C-4 ', and adriamycin S has three reactive hydroxyl groups (again excluding both phenolic groups) at positions C-9, C-14 and C-4' . Applicants have found that under suitable conditions, different reactivities of the various reactive hydroxyl groups of these compounds exist, and these differences can be used to prepare the desired novel derivatives. More specifically, it has been found that when suspending or dissolving adriamycin or daunomycin free base or an acid addition salt thereof (e.g. hydrochloride) in an inert organic solvent, and reacting these compounds with 3,4-dihydro-2H-pyran in the presence of an acid catalyst, various novel tetrahydropyranyl ether derivatives of the glycosides used as starting material. The reaction products formed in each case, the ratios between the various products and the reaction products vary according to the reaction conditions used, for example, solvent, acid catalyst, ratios of reaction components, temperature, length of reaction time, etc.

According to the invention there is provided a process for the preparation of an anthracycline glycoside derivative of the general formula (I) or a non-toxic acid addition salt thereof, which process is characterized by attaching one or two tetrahydropyranyl groups at the C-4 'or C-14 position of the compound of formula (II), or both. hydroxyl groups 0 OH »· OCHj 0 OH 0 • Ö HO \ -f NH2 in which R represents a hydrogen atom or a hydroxyl group by reacting 3,4-dihydro-2H-pyran with a compound of formula II or an acid addition salt thereof in an inert organic solvent, wherein an acid catalyst is present, and optionally removing the tetrahydropranyl group at position 14 by partially hydrolyzing or alcoholizing the di-tetrahydropyranyl compound, and optionally converting the resulting compound of formula (1) to a non-toxic acid addition salt or converting the acid addition salt to the free base.

The general reactions for the preparation of the compounds of formula (I) are as follows: 6 6 3 4 2 0

Formula I

O OH ft / —— \ acid catalysis catalyst «, 0 0H i * \ _) - *„ 0 daunonysin> 0 - JT — 0 I VO-PDal / CH_j \ J ♦ • -0-PDbj yjy ™ * I 0 OH o OCH ^ 0 OH IX)

O \ - / 9-O-PD

φ HHg o <L O 63420

Formula II ^ ^ She ""

OCH ^ O 0K

0 / - \ acid catalyst · ° adriazine NHg C ~ s ° * «αλ OCH, O OH [

9 O 1A-O-PA

0 ** / = ^

VV

j ψ. ! f s / ~ \ o ^ o

OCH? O OH T

O

fi? \ fv f 4 'jlA-di-O-PAaT)

<Y *. -di-O-PAbJ

L

hydrolysis or alcoholysis V

° <? H ίϊ OCH ^ O OH f 0 qA :::: ¾ 8 63420

The reactive hydroxyl group of adriacinine or daunomycin is converted by etherification to a tetrahydropyranyloxy group.

The glycoside used as a starting material may exist as a free base or as an acid addition salt. Since the acid addition salts of the tetrahydropyranyl ether products obtained by the reaction can be converted into the corresponding free bases or non-toxic acid addition salts by known methods, the starting material need not be non-toxic.

Any non-reactive organic solvent such as benzene, toluene, xylene, dimethylformamide, acetonitrile and tetrahydrofuran can be used in the tetrahydropyranylation reaction. The reaction solvent may be a single solvent or a mixture of solvents. Anhydrous dimethylformamide is quite preferably used as the solvent.

The acid catalyst can be an organic acid (e.g. formic trifluoroacetic acid) or an inorganic acid (e.g. hydrochloric acid, phosphoric acid). Organic sulfonic acids represent a preferred class of acid catalysts, and in particular aromatic sulfonic acids are used, e.g. p-toluenesulfonic acid or benzenesulfonic acid, quite preferably p-toluenesulfonic acid.

The reaction temperature is not critical. Good results of the etherification reaction have been obtained at room temperature, although higher or lower temperatures can also be used.

The reaction time varies depending on the reaction conditions selected in each case, such as temperature, catalyst, solvent, etc. The optimal reaction time for preparing a particular product or product mixture can be determined experimentally by chromatography on a thin layer experiment to be described later. Advantageous results are generally obtained by using reaction times of about 20 to about 50 hours.

The reaction products obtained and the reaction yields obtained depend on several factors, such as the concentration of the starting materials, the ratio of the reaction components, etc. When daunomycin is used as starting material, 4'-O-tetrahydropyranyl-daunomycin -O-tetrahydropyranyl-daunomycin (3 is hereinafter abbreviated as 9-O-PD). These products can be expressed in the reaction mixture by thin-layer chromatography on silica gel (Merck Co 60-25) using a mixture of chloroform, methanol and acetic acid in a volume ratio of 80: 20: 4. The products have an Rf value of 0.74 (4 * -O-PD) and an Rf value of 0.15 (9-0-PD).

The product 4'-O-PD was separated into two components with Rf values of 0.46 and 0.65 using thin layer chromatography on silica gel to develop a 10: 1 v / v mixture of chloroform and methanol. These components were found to be 4'-O-PD diastereomers of the product. The components Rf = 0.46 and Rf = 0.65 were arbitrarily labeled 4'-O-PDa (isomer a) and 4'-O-PDb (isomer b).

When adriamycin was used as the starting glycoside, 14-0-tetrahydropyranyl-adriamycin (hereinafter abbreviated as 14-O-PA) with »0.12 and the two components 4 ', 14- diastomers of di- (O-tetrahydropyranyl) -adriamycin, i.e. 4 ', 14-di- (O-tetrahydropyranyl) -adriamycin (isomer a), abbreviated as 4', 14-di-O-PAa, R , 55 and 4 ', 14-di- (O-tetrahydropyranyl) -adriamycin (isomer b), abbreviation 4, -14-di-O-PAb), Rf = 0.75. The compound 4 ', 14-di- (O-tetrahydropyranyl) -adriamycin can also be called 4', 14-bis- (O-tetrahydropyranyl) -adriamycin.

A diastereomeric mixture of the 4 ', 14-di-O-PAa components can also be prepared in high yield by settling 14-O-tetrahydropyranyl-adriamycin or its acid addition salt with 3,4-dihydro-2H-pyran in an inert organic solvent in the presence of an acid catalyst.

Taking advantage of the difference in reactivities between the C-14 primary hydroxyl group and the 0-4'-secondary hydroxyl group, the C-14 position of the components 41,14-di-O-PAa and 4 ', 14-di-O-PAb (or their acid addition salt) can be the tetrahydropyranyl group is selectively removed by hydrolysis or alcoholization to give the corresponding diastereomers of components 4'-O-PAa and 4 * -O-PAb in high yields. The tetrahydropyranyl oxy group can be converted to a hydroxy group, e.g., by hydrolysis with an aqueous solution of an acid, e.g., an inorganic or organic acid, or by alcoholization with an alcohol or phenol, e.g., C 1 -C 6 alkanol. According to a preferred method, the treatment is carried out with a dilute acetic acid solution or a methanolic solution of p-toluenesulfonic acid at room temperature for about 30 minutes to 5 hours. Side reactions can be reduced by performing hydrolysis or alcoholization in the dark.

The products of formula (i) can be isolated from the reaction mixture by conventional methods. Thus, the products of the tetrahydropyranylation reaction can be recovered by neutralizing the reaction mixture with a basic substance, e.g., alkali metal carbonate or bicarbonate, extracting the neutralized reaction mixture with a water-immiscible organic solvent, e.g. an acidic aqueous layer with a base, extracting the neutralized aqueous layer with a water-immiscible organic solvent and evaporating the organic extract to dryness. The dark red dried flour thus obtained can be purified by chromatography on a column containing silica gel, or in question from a small sample, by applying preparative thin layer chromatography. The products resulting from the hydrolysis or alcoholization reaction can be recovered from the reaction mixture by neutralizing with a basic substance, extracting the neutralized reaction mixture as a water-immiscible organic solvent, and evaporating the organic extract to dryness.

The products obtained as a mixture of diastereomers (isomers a and b) can be separated as described above by thin layer chromatography on silica gel to separate isomers a and b in substantially pure form.

The products obtained from the reactions described above can be recovered as the free base, an acid addition salt or a non-toxic acid addition salt. The free bases can be readily converted into non-toxic acid addition salts which are substantially as effective as the corresponding free bases in terms of their medicinal efficacy. Salts are formed, isolated, purified and formulated using methods commonly used to form salts for antibiotics, e.g., anthracycline glycoside antibiotics. Thus, the free base can be reacted with a non-toxic acid in a suitable solvent and the salt recovered by lyophilization or precipitation with a solubilizing agent, e.g., as a solvent in which the desired salt is only poorly soluble. The products in the form of an acid addition salt can be converted into the corresponding free bases by neutralization with a basic substance. Toxic acid addition salts can finally be converted to non-toxic acid addition salts by neutralization and then treatment with a non-toxic acid as described above.

Physico-chemical properties

The compounds of the invention exist in solid form as an amorphous or crystalline red powder. As free bases, these compounds are soluble in ethyl acetate, chloroform and ethanol, and sparingly soluble in water, n-hexane, petroleum ether, etc. The ethanol and acidic solutions of the compounds are red in color, give a positive ninhydrin reaction, and do not merely react. Figures 1 to H and the following Table 1 show the elemental analysis, melting point (decomposition point), specific rotation (C = 0.2 in GHG1), ultraviolet and visible absorption spectra (in methanol), infrared absorption spectra (KBr tablet) of these products. and nuclear magnetic resonance spectra (100 MHz, CDCl 3).

12 6 3 4 2 0 m «» o M- in tn · r- o ^ v '“M ι-n' - * r-% σ \ o cm ^ o a w <! oo m (Ti tn in o cm

Ph ... o cm tn oo tn I O vo> - in • h— 'and in O vo CM' “'tn« «i ww -« T- o in o

P ^ -N Ift On O

V £>! ”+ Tn« O mo

I CM CO CO W / -N r- T-. * · VO

··· CM ^ BO ww cvj

* “H-VOH-CO — HCMn O VO O n-VO

• vo T- λ k m nm- ον cnc- «

”M · CB J C ~ CMw CM

*. ·. #. CO · O I o · ^ O ä a · · t ~ i nj o> w H- oo «-. J · »“ r- jC O w ri ir <- * in T- - O in ..

Ph O 'O cv m t- t- o o t 00 in Ov · r ~ w * ^ o ··. -M- w 00 - 'iovot- o tn o σι o- n • h vo in cvi σν m- c ~

"Ö * - * * cm« cm «m X

I VO - ”n - ^« O

N in vo CO ^ + O n in n tn v ~ h ™ n- co H-nj cm o vt o x

i · · · tn · ri Ί · tn vo r- U

- v vo i- co · o cm o wtnwr- ..

m · vo «» -n in b ^ m ^ tn Η-Ort -j in O tn o cm h, * i 4i £ ^ ® · £ · ® · w in cp mo part c- »- wo cm cm -3 · Mspo ^ • S ^ • g ..in 2 ^ a) cm 2 m. -h a 5 co m ho • ri _ _ _ rivo

Co 'n' * '-' .— O Lj C Ht cm o in - * - ^ n ho o ri O CM CM in · O «“ O oo e ... «hj-cti '-' cm +» o Ä n vo cm cm tn cm a) ^ ai is P vo vo cm «en vo Bo

p P, '- - r- «' -S -M- r ~ OfH

® i tn ^ O - m ϋ m tn o cp c- t * - σι '-v + o uv n «fis ·

• h i m- ιλ σι i— etj m tn in n o O

f · ~ · · · O · ri cm tn ^ tn in o h «- T- vo e-« o cm m T- co u ^

ri VO · * n K, in tö. > w w> o (D

• ri »-OcflJvOCMCMOCM g cj E Η-σνχιο · cm mmm ϋΛ oo _ s vo» - ^ —1 · \ ΐ o cm cv] 'ϊ m +.> 3 · * 2 om A <a μ · ~ · 0 3

Il ^ 9 -h? H in N ^ H 5 ri * - t- O m fr) d -hj- ^ cm ιη'-Η ^ -'-> φη o CM CM '- cy> · .— Ln ho -h * H ... O rt * CO ^ h— o + -> en s t- vo n in tn cm mo - 'o, >> ·? vo cm cm · σι vo S · VH Ph www t- «-> ri- f- ri i vo» - o »in s ► SO CM e— -M- σ> + in 1Λ -n · H · - φ i oo cm cm '“ri tn tn o ή> + i -. ·. O * ri CM tn v- '· Μ ~ O 6. <- »m * t- o vo cm c ^« o cm fp wm h- co • H \ 0 · * * n K, CQ ·' «—1 * 'w> im

ri h- tn ri i hk- cm cm o cm B

e ...... h- σ A ιβ · cm in co tn e o x a vo f w sj o tvi cm? · m ri ri ^ fi

• d 1 -H

o o +> -rH +> 'Ί ^ + H ri -τ' o a nj> i -p -h o in o: »» o 03 w o -3 g ί? >> on>,

ri <o>, ® + », α- ^ S

fH -H H + »fn ri s o>, -P rt tM O tn ai tn e e

Ph C 'Λ q -W P H i xt) w o

+ »Rt -h a ^ o | > S

® «ri rt n <a * o>» · * “h ~» e -pa ή ή S> ^ ri • p-ptJ p -ha rt h 3 pa + »o cu p ri ® m ri ccMrtfiPit'aio + > a riPri p <> »rH ή. i i ® h- »- m% a ^ |

H · Η H ^ H φ I S

^ Ό cd -> h h — n —s O Ja ίΰ, ί C ^ C'JO ίΛ LfN \ o Ή> η ρς »« - / -w ^ Hl 63420 13 ra § γΗ • Η -Ρ

Ο '' O LA

✓-Χ ✓ "Ν. ^ Χ. 00 Ο 'Φ -Φ ^ CM Ο Γ—' -'Τ '" ΙΛ τ- τ- —'-'-' Ο Ο

• · · Ο • d -'— 1 00 CO

jQ CT CD CM O t ^ o CJMA

<s1 la m CM OVd- · «Il

^ CM '' »^ O W

O τ— «d" LA (T \ --x + ΙΑ '-' ίηΟ O

| [- CM O T- «J LOOfA-r- LA

_. . . o · rt ia Lala · »---- W

-d- CTi vOCMC— · O CMP '—LA' - 'LA O

LA · · · η t \ CT 03'— '03 · Γ— OOrt Si d "OCMO'- ··

...... CM 00 Λ I o 1 CMLAC0LA tA

O K S M) τ- - '' —I O CM CM xj · LA (H

w o "-χο rt

LA CM '' N -H

, ^ χ τ ~ τ- O ^ "x 1H

CM O Π— LA— "VO O rt LA H · x- wO Τ- LA u •. · O 'd- CA' - ^ '- b0

rt σι vo cm O lacm F-c— O

* ai ia m CM 1- <AC— -P

p, v_ ^ s S— / t— xd "LA rt

| '' "X O · 1 · - S

0 1Λ 1Λ r C ~ ^ P

| CO to OJ -r- rt LTV ί <Λ O O f-j _. . . o · rt ld tA' — ia O rW 'd1

CT 'VO CM C— »O CMP ---------- 01 LA

LA · 1 -na CM 03 · '- ^' O CM

[- CM rt | tA o CM O CM in Ph

...... CM [-, £) IÖ · CM LP »CO 1 ° 1 fH O

O W Ϊ3 CD V- x- 'o CMCM'd-LPi 03 CD

• P ·

'-n P? O

'-'LA' · - 'P O

o <- O o S — N tc \ 1- C ~ ^ -N · Η

CM O ^ O LA '- "x ~ IA HO

and \ τ— Γ— LA x —'CD '-' CD 03 rt. . τ- · • d-CJM · --- '03 03

CJ ~ \ cD · CM CA CM (Τ 'C— M S

<3} la cm cd CM • .'d'C'— O

p. —1 ^ - '-—f τ— x «LA p | CM ^ -xO ^ -x - P '-x O τ- la tA o' "χ + οοο'-χ rt oi 1 f— CM fA CM rt xd-xd-LDLA P 03, φ ... o · rt xt IA —'T- CA Ή Λ

i- CA LO CM f- · O CMP x-AA ^ t- -H P

LA · · 1n> Ks, CM CO · CO'- 'P CO

Γ— LAOS I x “O CM O CM M

...... CM CA P lö · CM LA 00 IA M

O W 5¾ LD τ- X- 'χχ ^ Ι O CM CM xd "LA

CÖ i rt

O -H

• H -P

P> -P

• H O P rf +> co o rt rt o> »o χ- ^ o rt t3 >> rt o ro t3 03 H -H 03 -P LA P- ^ x>,

• H rt rt-P rt rd rtR P

-pew pm oi g rt o • rt -H -H -HKM trt'- 'rt w -P 03 ΗΡΛ! 01> op curtrt >> «> ω o >> · η a rt ra -h -p> i - H -h> ^ U -v.

-h rt-P rtcMrt: rt-P „a rt +> a o; 03 rt rt1 rt 0 ^^ 03 -p

T- 03 -PÄ AC H H -HO I I Of'- -P

to S -d rt P CO o II P = J ho ^ M

H -H rd H 3 pTirt ^ A ^ '-χ ^ -χο- ~ χ ^ χ rt rtPrt' - ^ CMtA xd- IA g lp)

Ed> d -rt x— X- »χ- 'χ-' X- 'w H 63420

The 4'-O-PDa, 41 2-O-PDb, 4 ', 14-di-O-PAα, 4', H-di-O-PAb, 14-O-PAb, 4'-O- The structure of PAa and 4'-O-PAb can be studied by analyzing the amount of tetrahydropyranyl groups bound to the compounds, which is found to be either one or two of the methine proton at the C-2 position of the tetrahydropyranyl group and C-3, C-4 »C-5 and C-6 based on the signal strength of the methylene proton positions. The binding position of the tetrahydropyranyl group can be analyzed due to the chemical shift of the C-4 'proton in the daunosamine moiety towards the lower field (compared to daunomycin) due to the glycosidic bond formed at the C-4' position.

The difference in the structure of 4'-O-PDa and 41'O-PDb, 4'-O-PAb and 4'-O-PAb and 4'rH-di-O-PAa and 4'-H-di-O-PAb is thought to be due to the difference in the R- and S-absolute structure of the tetrahydropyranyl group at the C-2 position because the chemical shifts and coupling constants (J-value) at the C-2 and C-3 protons differ. The absolute structure of isomers a and b is still unknown. Table 2 shows the chemical shifts of the C-2 position of the tetrahydropyranyl group and the C-4'-proton of the daunosamine moiety (based on Figures 8 to H).

Table 2

Proton 4'-THP1 14-THP1 DS4, s1 __ (parts per million) _ (parts per million) (parts per million)

Compound 4'-O-PDa 4.38 - 3.62 4 1-O-PAb 4.72 - 3.64 4 f-O-PAb 4.36 - 3.70 4 1-O-PAb 4.72 - 3.70 4 ', 14-di-O-PAa 4.38 4.70 3.60 4 ', H-di-O-PAb 4.72 4.72 3.66 14-0-PA - 4.71 3.48

Daunomycin - - 3.49 THP: chemical transfer of the C-2-methine of the substituted tetrahydropyranyl group 2 DS4 ': chemical transfer of the C-4'-methine of daunosamine

Based on the above, it was found that the structure of the compounds of the invention corresponds to the above formulas.

15

Antibiotic effect 634 20

The compounds of formula (I) have been found to have antimicrobial activity against a variety of pathogenic microorganisms. The following Table 3 shows the minimum inhibitory concentrations of representative compounds of the invention, determined by the broth dilution method.

Table 3 Minimum inhibitory concentration (MIC, mcg./ml.)

Tested compound

Test organism _ ^ - O-PDa 4'-0-PDb 4'-0-PAa 4'-0-PAb

Staph, aureus 6.25 6.25 6.25 6.25

PDA 209P

Staph.aureus 12.5 5.12 6.25 6.25

Smith

Bacillus subtilis 3.12 1.56 3.12 3.12 mra-

Bacillus cereus 6.25 6.25 6.25 6.25 2OTH7J702-

Bacillus megaterium 6.25 3.12 3.12 3.12

IFF

Sareina lutea 0.39 0.39 0.78 0.78

Pel 1ÖÖ1

Micrococcus flavus 0.78 1.56 3.12 3.12 PDA 16

Corynebacterium bovis 0.78 0.78 3.12 3.12 1810

Pseudomonas aeruginosa> 100> 50 to 100> 100 Å3

Escherichia coli> 100> 100> 100> 100 m _---

Mycobacterium smegmatis 6.25 6.25 100 100 atoct ϊτπ ---

Candida albicans> 50> 25> 100 ^ 50 According to this Table 3, the compounds of the invention can be used as antibiotics, especially against gram-positive bacteria.

Anti-cancer effect

The compounds of formula (i) have clear anti-cancer activity and low toxicity based on standard experiments.

16 63420 A. The compounds of the invention have been found to have a clear inhibitory effect on the growth and nucleic acid synthesis of 11210 leukemia cells in culture.

Thus, 11210 cells (5 x 10 6 cells / ml) were seeded in RPMI 1640 medium (Roswell Park Memorial Institute 1640) containing 20% calf serum at 37 ° C in the presence of 0.1 and 0, respectively. 5 μg / nl of compounds of the invention in a CO 2 incubator. The number of cells was counted periodically, and the percentage inhibition of the plant was determined as shown in Table 4.

Table 4

Growth inhibitory effect of tetrahydropyranyl derivatives on 11210 cells in culture

Concentration Blocking amount%

Compounds 0.1 0.3 μg / ml 4'-O-PDa 79.2 95.0 4 * -O-PDb 74.6 88.8

Daunomycin 68.8 72.7 4'-O-PAa 65.9 81.1 4'-O-PAb 78.1 92.9 14-0-PA 7.6 58.3 4'vH-di-O-PAa 37.5 80.8 4 ', 14-di-O-PAb 25.5 72.1

Adriamycin 70.7 84.2

The effect of the compounds of the invention on nucleic acid synthesis was studied as follows:

1 x 10 6 cells / ml 1210 cells were suspended in RPMI medium with 10% calf serum, precultured at 37 ° C for 1-2 hours in a CO 2 incubator, after which the compounds of the invention were added to the medium at various concentrations. After incubation for 15 minutes, N-uridine (0.05 YuCl / ml) or N-thymidine (0.05 YuCl / ml) was added and incubated at 37 ° C for 60 minutes. Trichloroacetic acid (TOA, 10%) was then added to the medium to quench the reaction and precipitate the acid-insoluble materials, after which the precipitate was washed three times with 5-10% formic acid solution of TCA. Cadioactivity was measured and expressed as 50% of the added substance 31 n ko n s e n t r aa t i o n a.

63420 17

Table 5

Inhibitory concentration of added β-thymidine and β-uridine 505b on cultured 11210 cells 505b inhibitory concentration

Compounds Uridine Thymidine 4'-0-PDa 0.13 0.28 4'-0-PDb 0.20 0.32

Daunorayzine 0.40 0.70 41-0-PAa 0.20 0.37 4'-0-PA'o 0.24 0.50 14-0-PA 0.17 0.42 4 ', 14-di-O-PAa 0.23 0.55 4', 14-di-O-PAb 0.24 0.97

Adriarayin 0.50 2.1 B. When the compounds of the invention were tested on various experimentally used animal tumors, these compounds were found to have clear anti-cancer activity and reduced toxicity compared to adriamycin and daunomycin. Thus, these compounds can be used in medicine to control mammalian tumors in a plant.

As an example, BDP .. mice were inoculated intraperitoneally with L1210-g 'leukemia cells at 1 x 10 cells / mouse. Twenty-four hours after inoculation, mice were administered the compounds of the invention intraperitoneally once daily for ten consecutive days, and experimental animals were inspected for 45 days. The anti-cancer effect was observed with an increase in survival days (ϊ / C, compared to the survival days of control mice treated with physiological saline. The results are shown in Table 6.

18 · 6 3420

Table 6

Anticancer effect of tetrahydropyranyl derivatives (T / C,%)

Dose (mg / kg / day)

Compounds 5 2.5 1.25 0.6 0.5 0.15 4 * -0-PBa> 520> 520 122 115 96 90 4 '-0-PDb> 520 256 122 115 105 90 4'-0-PAa - 175 180 187 120 127 4' -0 -PAb> 375> 360> 573 293 160 113 14-0-PA 142 130 126 113 110 103 4 ', H-di-O-PAa 154 115 109 96 103 96 4', 14-di-O-PAb 161 109 103 103 96 115

Toxic Death of Adriamycin 231 218 230 165 128 The results of the toxic death and weight loss of the mice used in this experiment show that the toxicity of the derivatives of the invention is 1/3 to 1/2 lower than that of the adriamycin and daunomycin used as starting materials of the invention.

C. The clear anti-cancer effect shown in A and B above was demonstrated by examining the stability of the compounds of the invention to the inactivation of hepatic NADPH cytochrome P450 reductase. More specifically, NADPH-cytochrome P450 reductase purified from rat liver homogenate was incubated with the compounds of the invention at 25 ° C for 25 minutes in nitrogen gas, and the resulting incubation product 7-deoxyglycone was determined by the results shown in Table 7.

Table 7 63420 19 Stability of tetrahydropyranyl derivatives against rat NADPH cytochrome P450 reductase

Product (ninol / tube)

Compounds (7-deoxyglycone) 4'-O-PDa 37.3 4'-O-PDb 46.6

Daunomycin 65.8 4'-O-PAa 10.9 4'-O-PAb 15.8 14-O-PA 18.4 4 ', 14-di-O-PAa 13.1 4', 14-di-O-PAb 15.8

Adriamycin 47.2

Composition of the reaction mixture:

NADPH 0.2 mM

tris-HCl (pH 8.0) 0.1 mM

substrate 0.1 mM

enzyme 4.6 g / ml (tris-HCl = tris- (hydroxymethyl) aminomethane)

The following examples illustrate the invention.

Example 1

Process for the preparation of 4'-O-tetrahydropyranyl-daunomycin (4'-O-PDa and 4'-O-PDb)

To a solution of 60 mg of daunomycin hydrochloride in 5 ml of anhydrous dimethylformamide were added 1 ml of 3,4-dihydro-2H-pyran and a catalytic amount of p-toluenesulfonic acid. The reaction mixture was allowed to stand overnight at room temperature in the dark, then added to 20 ml of 0.1 N aqueous sodium bicarbonate solution and extracted with chloroform (10 ml x 4). The chloroform extract was then extracted with 1% aqueous acetic acid (10 ml x 10), and the resulting acidic aqueous layer was neutralized with sodium bicarbonate and then re-extracted with chloroform (20 ml x 10). The chloroform layer was dried over anhydrous sodium sulfate and concentrated. There was thus obtained 44 mg of a residue which was subjected to preparative thin layer chromatography on silica gel (Merck Co. eOFgt 3) and developed with a 10: 1 by volume mixture of chloroform and methanol.

Silica gel bands corresponding to Rf values = 0.46 and 0.65 were scraped off from the thin layer, then eluted with a 10: 1 by volume mixture of chloroform and methanol and evaporated to dryness.

Each residue was dissolved in methylene chloride, frozen by the addition of freezing t-butanol, and dried under reduced pressure.

Thus, 10.1 mg of a red-brown solid product of 4'-O-PDa and 10.5 mg of a red solid product of 4'-O-PDb were obtained from the corresponding R 1 fractions of 0.46 and 0.65.

4 * -O-PDa and 4'-O-PDb are diastereomers of 4'-O-tetrahydropyranyl-daunomycin, the physicochemical properties of which are shown in Table 1 above.

Example 2

Method 4 ', 14-O-bis- (tetrahydropyranyl) -adriamycin (4', 14-di-O-PAa and 4 ', 14-di-O-PAb) and 14-O-tetrahydropyranyladriamycin (14-0 -PA) from adriamycin

To a solution of 150 mg of adriamycin hydrochloride in 10 ml of anhydrous dimethylformamide were added 2 ml of 5,4-dihydro-2H-pyran and a catalytic amount of p-toluenesulfonic acid. The reaction mixture was allowed to stand at room temperature for 48 hours, then added to 20 ml of 0.1 N aqueous sodium bicarbonate solution and extracted with ethyl acetate (20 ml x 5). The ethyl acetate layer was then extracted with 1% aqueous acetic acid (40 ml x 4), the acidic aqueous layer was neutralized with sodium bicarbonate and extracted with chloroform (20 ml x 10). The chloroform layer was dried over anhydrous sodium sulfate and evaporated to dryness. There was thus obtained 160 mg of a solid which was developed and purified by preparative thin layer chromatography on silica gel using a 10: 1 by volume mixture of chloroform and methanol. The thin layer was scraped with strips Rf = 0.12 and 0.55 »and purified as described in Example 1.

Thus 35 mg of red solid HO-PA, 16 mg of red solid 4 *, 14-di-O-PAa and 14 mg of red solid 4 * »14-di-O-PAb were obtained from the corresponding Rf fractions 0.12, 0.55 and 0.73.

4 ', 14-di-O-PAa and 4' »14-di-O-PAb are diastereomers of 4 '» 14-bis- (O-tetrahydropyranyl) -adriamycin, the physicochemical properties of which are shown in Table 1 above.

Example 3

Process for the preparation of 4 ', 14-bis- (O-tetrahydropyranyl) -adriamycin from 14-O-tetrahydropyranyl-adriamycin

To a solution of 35 mg of 14-O-PA in 2 ml of anhydrous dimethylformamide were added 0.5 ml of 3,4-dihydro-2H-pyran and a catalytic amount of p-toluenesulfonic acid. The reaction mixture was allowed to stand at room temperature in the dark for 40 hours, then added to 10 ml of 0.02 N aqueous sodium bicarbonate solution and extracted with ethyl acetate (5 ml x 4). The ethyl acetate layer was extracted with 1 # aqueous acetic acid (10 ml x 3), the acidic aqueous layer was neutralized with sodium bicarbonate and extracted with chloroform (10 ml x 5). The chloroform layer was dried over anhydrous sodium sulfate and evaporated to dryness.

The obtained residue was chromatographed using a thin layer of silica gel as described in Example 2 to give 8.4 mg of red solid 4 *, 14-di-O-PA and 8.1 mg of red solid 4 ', 14-di-O-PAb from the corresponding R fractions of 0.55 and 0.75. The physicochemical properties of these products were the same as those of the compounds obtained according to Example 2.

Example 4

Process for the preparation of 4'-O-tetrahydropyranyl-adriamycin from 4 ', β-bis- (O-tetrahydropyranyl) -adriamycin a) 12.4 mg of 4', 14-di-O-PA were dissolved in 1.5 ml of acetic acid 10 # aqueous solution, and the reaction mixture was allowed to stand for 4.5 hours at room temperature in the dark. The reaction mixture was then added to 10 ml of water, neutralized with sodium bicarbonate powder and extracted with chloroform (15 mix 2).

28 63420

The chloroform layer was dried over anhydrous sodium sulfate and evaporated to dryness. There was thus obtained 11 mg of a residue which was purified by thin layer chromatography on silica gel as described above and using a 10: 1 by volume mixture of chloroform and methanol. The main band at * 0.32 was removed by scraping and eluted with a 10: 1 by volume mixture of chloroform and methanol. The eluate was evaporated to dryness. The residue was dissolved in methylene chloride, t-butanol was added under cooling to freezing point, and dried under reduced pressure. There was thus obtained 7 mg of red solid 4'-O-PA, the physicochemical properties of which are shown in Table 1.

b) 16 mg of 4 ', H-di-O-PAb was dissolved in 5 ml of a 0.005 N methanolic solution of p-toluenesulfonic acid, and the reaction mixture was allowed to stand for one hour at room temperature in the dark. The reaction mixture was then neutralized with 10 ml of 0.01 N aqueous sodium bicarbonate solution and extracted with chloroform (10 ml x 4). The chloroform layer was dried over anhydrous sodium sulfate, and the work-up was continued as described in a). There was thus obtained 7.2 mg of a red solid 4'-O-PAb having an Rf value of 0.49 in a thin layer of silica gel under the conditions described above. The physicochemical properties of this compound are shown in Table 1.

4 * -O-PAa and 4'-O-PAb are diastereomers of 4'-O-tetrahydropyranyl-adriamycin.

Example 5 Salt formation

To illustrate methods that can be used to prepare acid addition salts, 4'-O-PDa, 4'-O-PDb, 4 ', 14-di-O-PAa, 4', 14-di-O-PAb, Dissolve the free bases -O-PA, 4'-O-PAa or 4'-O-PAb in ethyl acetate and add one equivalent of hydrochloric acid. Lyophilization gives the corresponding hydrochloride salt.

Claims (2)

A process for the preparation of tetrahydropyranyl ethers of anthracycline glycosides for use as antimicrobial and antitumor agents, having the general formula (I) O * 2 * 1 OCH, 0 OH L 5 (I) 0 NH2 in which formula represents a hydrogen atom or hydroxyl or tetrahydropyranyl and R2 represents a hydroxyl or tetrahydropyranyloxy group, wherein in the case where R1 represents a hydrogen atom or a hydroxyl group, R2 represents a tetrahydropyranyloxy group, and for the preparation of non-toxic acid addition salts of these compounds, characterized in that one or two tetrahydropyranyl groups in the C-4 'or C-14 position of the compound of formula (II), or the hydroxyl groups in these two positions 0 w 0. OH 5 OCH 2 OH 0 NH 2 in which R represents a hydrogen atom or a hydroxyl group such that 3,4-dihydro-2H-pyran is reacted with a compound of formula II or an acid addition salt thereof in an inert organic solvent in the presence of an acid catalyst, and optionally removing the tetrahydropyranyl group from position 14 by partially hydrolyzing or alcoholizing the di-tetrahydropyranyl compound obtained, and if necessary converting the obtained compound of formula (I) into a non-toxic acid addition salt or converting the acid addition salt into the free base. For example, a mixture of microorganisms and tumors is used to give tetrahydropyranyl ether and anthracyclyl glycosides with all of the compounds of formula (I) as a hydrogen-bonded tetrahydrocarbon in the form of a mixture of 0CH3 or more »0 0 m2 1 R2 is a hydroxyl group or a tetrahydropyranyloxy group, which can be found in the fall of R * is a hydroxyl group or a hydroxyl group, but R2 is a tetrahydropyranyloxy group ib £ da ställningarna account en förening med formeln (II)