Proc. Natl. Acad. Sci. USAVol. 93, pp. 2464-2469, March 1996Medical Sciences

High yield conversion of doxorubicin to 2-pyrrolinodoxorubicin,an analog 500-1000 times more potent: Structure-activityrelationship of daunosamine-modified derivatives of doxorubicin

(cytotoxic agents/antineoplastic drugs/design and synthesis/steric factors/alkylating agents)

ATrILA NAGY*t, PATRICIA ARMATIS*, AND ANDREW V. SCHALLY*t*Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, and tDepartment of Medicine, Tulane University School of Medicine, NewOrleans, LA 70146

Contributed by Andrew V. Schally, December 4, 1995

ABSTRACT A convenient, high yield conversion of doxo-rubicin to 3'-deamino-3'-(2"-pyrroline-1"-yl)doxorubicin isdescribed. This daunosamine-modified analog of doxorubicinis 500-1000 times more active in vitro than doxorubicin. Theconversion is effected by using a 30-fold excess of 4-iodobu-tyraldehyde in anhydrous dimethylformamide. The yield ishigher than 85%. A homolog of this compound, 3'-deamino-3'-(1l,3"-tetrahydropyridine-1"-yl)doxorubicin, was also syn-thesized by using 5-iodovaleraldehyde. In this homolog, thedaunosamine nitrogen is incorporated into a six- instead of afive-membered ring. This analog was 30-50 times less activethan its counterpart with a five-membered ring. A similarstructure-activity relationship was found when 3'-deamino-3'-(3"-pyrrolidone-1"-yl)doxorubicin (containing a five-membered ring) and 3'-deamino-3'-(3"-piperidone-1"-yl)doxorubicin (with a six-membered ring) were tested in vitro,the former being 5 times more potent than the latter. Tofurther elucidate structure-activity relationships, 3'-deamino-3'-(pyrrolidine-1"-yl)doxorubicin, 3'-deamino-3'-(isoindoline-2"-yl)doxorubicin, 3'-deamino-3'-(2"-methyl-2"-pyrroline-l"-yl)doxorubicin, and 3'-deamino-3'-(3Y-pyrroline-1"-yl)doxorubicin were also synthesized and tested. All theanalogs were prepared by using reactive halogen compoundsfor incorporating the daunosamine nitrogen of doxorubicininto a five- or six-membered ring. These highly active anti-neoplastic agents can be used for incorporation into targetedcytotoxic analogs of luteinizing hormone-releasing hormoneintended for cancer therapy.

Daunorubicin and doxorubicin (DOX) (Fig. 1) are members ofthe family of the anthracycline antibiotics (1, 2) that wereintroduced into cancer therapy about 3 decades ago. As a resultof a small structural difference, DOX is a much more potentanticancer agent than daunorubicin. Since its introduction intohuman cancer therapy, DOX is still the most widely usedchemotherapeutic drug, with the broadest spectrum of anti-tumor effect (3).The antiproliferative activity of DOX is due mainly to its

ability to intercalate into DNA and break the strands of doublehelix by inhibiting topoisomerase 11 (4). Despite its wideacceptance in the chemotherapy of various cancers, prolongeduse of DOX is severely limited by cardiotoxicity (5). Anotherlimitation of DOX is multidrug resistance (6).Thousands of anthracycline derivatives were synthesized

to overcome these limitations and in search for even moreactive analogs (3). One of the most significant milestones inthe semisynthetic development of more potent, non-cross-resistant analogs of DOX with lower cardiotoxicity was thefinding of 3-cyanomorpholinodoxorubicin (MRA-CN) (7,

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

8). This derivative was isolated as a highly potent by-productthat formed during the reductive alkylation of thedaunosamine nitrogen of DOX. MRA-CN was 100-500times more active than DOX in vitro. In fact, MRA-CNproved to be 1500 times more potent on cell lines resistantto DOX (9-11). It was revealed that the extremely highactivity of this compound, even against DOX-resistant tumorcell lines, is a result of its ability to form an aminal adductwith an amino group of a guanine base in close vicinity to itsbinding site (12, 13). Based on this knowledge, a class ofintensely potent analogs was developed, best represented byN-(5,5-diacetoxypent-1-yl)doxorubicin (14, 15). This analogis a water soluble, latent aldehyde derivative, activated intissues by carboxylate esterase enzymes to become 150 timesmore active than DOX. Other semisynthetic derivatives, likemorpholinodoxorubicin (7) and 2-methoxymorpholinodoxo-rubicin, become 50-80 times more active in vivo afteractivation by liver enzymes (16, 17).These highly potent analogs of DOX either have a latent

aldehyde derivative attached to the nitrogen atom in thedaunosamine moiety through an open chain polymethylenebridge or the nitrogen atom becomes a part of a morpholinering. Gao et al. (18) presented a high resolution x-ray diffrac-tion picture of a covalently linked adduct between a syntheticDNA segment and a daunorubicin derivative. This adductforms readily when traces of formaldehyde are present in thecrystallization solvent. The daunosamine nitrogen atom of theanthracycline derivative was shown to be linked covalentlythrough a methylene bridge to the amino group of an amino-adenine moiety of the DNA segment in close vicinity.Based on these findings, we decided to investigate further

the structural requirements for the design of intensely potentdoxorubicin analogs. Our efforts led to the high yield (85%)conversion of DOX to 2-pyrrolinodoxorubicin by the use of a30-fold excess of 4-iodobutyraldehyde. 2-Pyrrolinodoxorubicinis a stable, water soluble analog, which is 500-1000 times moreactive than its parent compound in vitro.

MATERIALS AND METHODSMaterials. DOX HCl salt, 1,4-diiodobutane, a,a'-dichloro-

ortho-xylene, cis-1,4-dichloro-2-butene, 3-bromopropionylchloride, and 4-bromobutyryl chloride were purchased fromAldrich. Silica gel (Merck grade 9385; 230-400 mesh; poresize, 60 A) was also from Aldrich. TLC aluminum sheetsprecoated with silicagel 60 F254 by Merck Art no. 5554 wereobtained from Curtin Matheson Scientific (Houston). 2-(3-Chloropropyl)-1,3-dioxolane, 2-(3-chloropropyl)-2-methyl-

Abbreviations: DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DOX, doxorubicin; MRA-CN, 3-cyanomorpho-linodoxorubicin; TFA, trifluoroacetic acid.

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O OH

14

12 CH2 -R

CH3/H3C

NH2

OH

FIG. 1. Structures of daunorubicin (R = H) and DOX (R = OH).The daunosamine amino sugar moiety is indicated by an arrow.

1,3-dioxolane, and 2-(4-chlorobutyl)-1,3-dioxolane were

bought from Fluka.Synthesis. Preparation of 1-chloro-4-bromo-2-butanone

and 1-chloro-5-bromo-2-butanone. 3-Bromopropionyl chlo-ride (100.8 1lI; 1 mmol) was reacted with excess diazometh-ane in ether for 1 hr. The ethereal solution was developed inCHCl3/MeOH (95:5, vol/vol) on TLC. After chromatogra-phy, 2,4-dinitrophenylhydrazine reagent (19) was sprayed on

the TLC sheet. The diazomethylketone derivative thusformed showed a yellow spot with Rf = 0.3. The etherealsolution was then reacted with anhydrous HCl in ether,converting the diazomethylketone to the desired end prod-uct, 1-chloro-4-bromo-2-butanone. This compound showed a

yellow spot, characteristic of oxo compounds, with Rf = 0.8in the same solvent system and with the spot test reagentdescribed above. After evaporation of the solvent, the crudeproduct was applied on a column (2.5 x 15 cm) packed with15 g of silica gel. The liquid, mobile phase was CHCl3.Fractions containing the desired end product (characterizedby the spot test) were combined and evaporated to drynessto yield 1.5 g of clear oil. (yield, 80%.) Preparation of1-chloro-5-bromo-2-pentanone was performed similarlystarting from 4-bromobutyryl chloride.

Preparation of 4-iodobutyraldehyde, 5-iodovaleraldehyde,and 5-iodo-2-petanone. 2-(3-Chloropropyl)-1,3-dioxolane,(1.3 ml; 10 mmol) was dissolved in 200 ml of acetonecontaining 30 g (200 mmol; 20-fold excess) of Nal. Thesolution was refluxed for 24 hr followed by evaporation todryness. Diethyl ether (100 ml) was used to extract theorganic material from the inorganic solid residue. Theethereal solution was then washed with 50 ml of H20/50 mlof 5% (wt/vol) aqueous Na2S203 solution and then threetimes again with 50 ml of H20. The ether was removed invacuo and the remaining oil was dissolved in 3 ml of 50%(vol/vol) aqueous AcOH. After 1 hr, 100 ml of ether was

added to this solution and the acetic acid as well as theethylene glycol were removed by washing three times with 50ml of H20. The main product eluted with Rf = 0.8 on TLCin CHCl3. The spot test used for characterizing the aldehydefunction was the same as cited above (19). The ether was thenremoved and the black oily residue was applied on a columnpacked with silica gel as described. Purification resulted in1.6 g of yellow oil (yield, 80%.) 5-lodovaleraldehyde and5-iodo-2-pentanone were obtained exactly the same waystarting from 2-(4-chlorobutyl)-1,3-dioxolane or 2-(3-chloropropyl)-2-methyl-1,3-dioxolane, respectively.

Preparation of 3'-deamino-3'-(pyrrolidine-1"-yl)doxorubi-cin trifluoroacetic acid (TFA) salt (AN-181). DOX HCl salt(50 mg; 86 ,umol) was dissolved in 1 ml of dimethylform-amide (DMF) and 171 ,lI (1.3 mmol; 15-fold excess) of1,4-diiodobutane was added, followed by 45 ul (260 ,umol;3-fold excess) of N,N-diisopropylethylamine (DIPEA). After16 hr, the reaction is complete as assessed by analyticalHPLC. The solvent was evaporated in vacuo and the residual

oil was dissolved in 3 ml of 0.1% TFA in H20 and extractedwith ether to remove excess 1,4-diiodobutane. Purificationby HPLC resulted in 41.6 mg of 98% pure DOX derivative(yield, 68%).

Preparation of 3'-deamino-3'-(isoindoline-2"-yl)doxorubicinTFA salt (AN-184). DOX HCl salt (50 mg; 86 ,umol) wasdissolved in 1 ml of DMF and 226 mg (1.3 mmol; 15-foldexcess) of a,a'-dichloro-ortho-xylene was added followed by 45,tl (260 ,tmol; 3-fold excess) of DIPEA and a catalytic amountof Nal. After 16 hr, the reaction was complete and the desiredend product was isolated as given above (yield, 55%).

Preparation of 3'-deamino-3'- (3"-pyrroline-1"-yl)doxorubicinTFA salt (AN-185). DOX HCl salt (50 mg; 86 ,umol) wasdissolved in 1 ml of DMF and 136.8 gl (1.3 mmol; 15-foldexcess) of cis-1,4-dichloro-2-butene was added, followed by 45,ul (260 ,tmol; 3-fold excess) of DIPEA. After 16 hr, thereaction mixture was worked up as described above, yielding22.6 mg of 98% pure end product (yield, 37%).

Preparation of 3'-deamino-3'-(3"-pyrrolidone-1"-yl)doxoru-bicin TFA salt (AN-191) and 3'-deamino-3'-(3"-piperidone-1"-yl)doxorubicin TFA salt (AN-195). DOX HCl salt (50 mg;86 ,umol) was dissolved in 1 ml of DMF and 241 mg (1.3mmol; 15-fold excess) of 1-chloro-4-bromo-2-butanone wasadded followed by 45 ,ul (260 ,umol; 3-fold excess) of DIPEA.After 16 hr, the reaction mixture was purified as cited above,yielding 20.6 mg of 98% pure end product (yield, 33%).3'-Deamino-3'-(3"-piperidone-1"-yl)doxorubicin was pre-pared similarly by using 1-chloro-5-bromo-2-pentanone(yield, 28%).Preparation of 3'-deamino-3'-(2"-pyrroline-1"-yl)doxorubicin

TFA salt (AN-201), 3'-deamino-3'-(1",3"-tetrahydropyridine-1"-yl)doxorubicin TFA salt (AN-205), and 3'-deamino-3'-(2"-methyl-2"-pyrroline-1"-yl)doxorubicin (AN-204). DOX HCl salt(50 mg; 86 ,tmol) was dissolved in 1 ml of DMF and 515 mg(2.6 mmol) of 30-fold excess 4-iodobutyraldehyde was addedfollowed by 45 gl (260 jLmol; 3-fold excess) of DIPEA. After30 min, 100 ,lI of glacial acetic acid was added to the reactionmixture, which was then added dropwise to 5 ml of 0.1% TFAin 70% aqueous acetonitrile. This solution was diluted with 2ml of 0.1% aqueous TFA solution, followed by removal of theacetonitrile in vacuo. The resulting aqueous solution wasextracted with hexane to remove the excess of the halogencompound. After purification by HPLC, 52 mg of 98% pureend product was obtained (yield, 85%). 3'-Deamino-3'-(1",3"-tetrahydropyridine-1"-yl)doxorubicin TFA salt (AN-205) and3'-deamino-3'-(2"-methyl-2"-pyrroline-1"-yl)doxorubicin TFAsalt (AN-204) were prepared similarly from 5-iodovaleralde-hyde (yield, 75%) and 5-iodo-2-pentanone (yield, 54%), re-spectively.

Purification. The final purification of all the crude productswas carried out on a Beckman model 342 semipreparativeHPLC system, using an Aquapore Octyl (250 x 10 mm; poresize, 300 A; particle size, 15 ,um) column. The solvent systemconsisted of two components-0.1% TFA in water, and 0.1%TFA in 70% aqueous acetonitrile-and was used in lineargradient mode.

Analytical HPLC. A Beckman analytical HPLC systemequipped with a model 168 diode array detector and SystemGold chromatography software (Beckman) was used to checkthe purity and to monitor the chemical reactions. The columnwas Dynamax C18 (250 x 4.6 mm; pore size, 300 A; particlesize, 12 ,um).

Cytotoxicity Assay. The MCF-7 human breast cancer cellline, used for the determination of the antiproliferative activityof the DOX derivatives, was obtained from the American TypeCulture Collection. MXT hormone-independent mouse mam-mary carcinoma cell line was a gift from Gunther Bernhardt(University of Regensburg, Regensburg, Germany). For eval-uation of the activity of the analogs, a colorimetric cytotoxicityassay in microtitration plates was used based on quantification

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0 100 200 300 400 500 800 700

FIG. 2. MS/MS (daughter mass) spectra of the mass M + H+596 of the mass spectra of 2-pyrrolino-DOX.of biomass by staining cells with crystal violet. The results ofthis assay correlate very well with the determination of cellnumber (20).

Analysis. Bruker ARX300 NMR spectrometer (300 MHz1H frequency; 75 MHz 13C frequency) and electrospray massspectrometer Finnigan-MAT TSQ 7000 were used for struc-tural identification of the DOX derivatives (Fig. 2).

RESULTS

A convenient synthetic procedure was developed for con-version of DOX to 2-pyrrolino-DOX. This derivative, whichhas the daunosamine nitrogen of DOX incorporated in a

five-membered ring, is 500-1000 times more potent thanDOX (Tables 1-3). A conversion of >90% of DOX (Fig. 3)was achieved by using a 30-fold excess of 4-iodobutyralde-hyde in anhydrous DMF in the presence of a tertiary base.The reaction is complete within 15 min and, after purifica-tion by HPLC, the yield is >85%. 4-lodobutyraldehyde wasobtained from the commercially available 2-(3-chloropro-pyl)-1,3-dioxolane by refluxing it in acetone containing a

20-fold excess of Nal. After hydrolysis of the acetal, flashchromatography was used to isolate an oil with 80% yield.5-Iodovaleraldehyde was similarly prepared from 2-(4-chlorobutyl)-1,3-dioxolane. This reagent was used for con-version of doxorubicin to 1,3-tetrahydropyridino-DOX, a

homolog of 2-pyrrolino-DOX, incorporating thedaunosamine nitrogen in a six-membered ring. Interestingly,2-pyrrolino-DOX showed antiproliferative activity in vitro30-50 times higher than its homolog with a six-memberedring, as shown in Tables 1-3. A similar phenomenon was

observed when the in vitro cytotoxic activities of 3-pyrroli-dono-DOX (containing a five-membered ring) and 3-piperi-dono-DOX (with a six-membered ring) were compared, theformer being -5 times more active than the latter. 3-Pyr-rolidono-DOX was also 10 times more active than DOX(Table 3). While the chemistry of 2-pyrrolidone or 2-piperi-done derivatives of primary amines was studied very exten-sively, the literature on the preparation of 3-pyrrolidone and3-piperidone derivatives is rather limited. The synthesespreviously described for preparation of such analogs in-volved extreme conditions (21) not applicable for derivat-ization of the complex, chemically sensitive structure ofDOX. The conversion of DOX to these derivatives was

achieved again by alkylation with reactive halogen com-

pounds. A 15-fold excess of 1-chloro-4-bromo-2-butanonewas reacted with DOX in anhydrous DMF in the presence ofa 3-fold excess of a tertiary base, leading to the formation of3-pyrrolidono-DOX with a moderate yield of 33%. Itshomolog counterpart, bearing a six-membered 3-piperidonering at the 3' position of the daunosamine moiety, was

prepared under similar conditions by using 1-chloro-5-bromo-2-pentanone. The yield was 28%. For preparation of1-chloro-4-bromo-2-butanone, 3-bromopropionyl chloridewas reacted with excess diazomethane in ether and theresulting diazomethyl ketone was converted to the corre-

sponding chloromethyl ketone by anhydrous HCl in ether.Flash chromatography of the halogen derivative thus ob-tained lead to a clear oil in a yield of 80%. 1-Chloro-5-bromo-2-pentanone was prepared similarly from 4-bro-mobutyryl chloride.

Table 1. Effects of DOX and its daunosamine-modified derivatives on the growth of MCF-7 human breast cancer cell line in vitro

T/C value

Incubation At 3 x 10-10 At 10-9 At 3 x 10-9 At 10-8 At 3 x 10-8 At 10-7Compound time, h M M M M M M

DOX 70 98 82 54120 95 66 33

Pyrrolidino-DOX 70 97 25 -26(AN-181) 120 94 17 -19

Isoindolino-DOX 70 118 86 -11(AN-184) 120 108 77 -29

3-Pyrrolino-DOX 70 106 72 -3(AN-185) 120 97 65 -5

3-Pyrrolidono-DOX 70 87 30 -28(AN-191) 120 67 25 -10

3-Piperidono-DOX 70 96 80 59(AN-195) 120 97 70 43

2-Pyrrolino-DOX 70 50 -3 -18(AN-201) 120 26 2 -9

2-Methyl-2-pyrrolino-DOX 70 96 96 91(AN-204) 120 119 114 89

1,3-Tetrahydropyridino-DOX 70 96 88 69(AN-205) 120 99 93 62

Cells were incubated in Eagle's improved minimal essential medium containing 5% heat-inactivated dextran-coated charcoal-treated fetal bovineserum in 24-well plates. Relative cell number in treated and control plates was determined by crystal violet staining and was expressed as T/C valueswhere T/C = T - Co/C - Co x 100. [T = absorbance of treated cultures, C = absorbance of control cultures, Co = absorbance of cultures atthe start of incubation (t = 0). Measured absorbance is proportionate to cell number.] Negative T/C values indicate a cell number smaller thanthe number originally seeded at t = 0-i.e., a cytocidal effect.

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Table 2. Effects of DOX and its daunosamine-modified derivatives on proliferation of MXT estrogen-independent mouse mammarycarcinoma cell line in vitro

T/C value

Incubation At 3 x 10-11 At 10-10 At 3 x 10-10 At 10-9 At 3 x 10-9 At 10-8 At 3 x 10-8 At 10-7Compound time, hr M M M M M M M M

DOX 24 84 77 6569 73 47 33

Pyrrolidino-DOX 24 51 43 14(AN-181) 69 20 10 -13

3-Pyrrolidono-DOX 24 63 28 25(An-191) 69 -2 -6 -10

3-Piperidono-DOX 24 85 56 27(AN-195) 69 38 -2 1

2-Pyrrolino-DOX 28 90 78 56(AN-201) 69 52 6 -13

1,3-Tetrahydropyridino-DOX 24 97 94 85(AN-205) 69 79 53 14

Cells were incubated in RPMI 1640 medium containing 6 mM L-glutamine and 10% fetal bovine serum in 96-well plates. Relative cell numberin treated and control plates was determined by crystal violet staining and is expressed as T/C values as defined in Table 1.

To further elucidate structural requirements for the designof intensely potent daunosamine-modified DOX analogs,2-methyl-2-pyrrolino-DOX and 3-pyrrolino-DOX, a struc-tural isomer of 2-pyrrolino-DOX, were also synthesized.2-Methyl-2-pyrrolino-DOX has a methyl substituent on thereactive carbon atom 2 of the ring and, surprisingly, it waseven less active than DOX. The synthesis was done similarlyto that of 2-pyrrolino-DOX, but using 5-iodo-2-pentanonefor alkylation. In the structure of 3-pyrrolino-DOX, thedouble bond is between carbon atoms 3 and 4 of thefive-membered ring. This analog was -5 times more activethan DOX in inhibiting growth of the MCF-7 human breastcancer cell line in vitro. Synthesis was accomplished byreacting a 15-fold excess of cis-1,4-dichloro-2-butene (22)with DOX in the presence of an excess of tertiary base. Theyield was 37% after purification by HPLC.

Pyrrolidino-DOX was synthesized by using the alkylatingagent 1,4-diiodobutane. The yield was 70%. The five-membered ring, attached to the 3' position of the daunosaminemoiety of DOX in this analog, had no reactive double bond oroxo function in its structure. The activity of pyrrolidino-DOXwas only slightly better than that of DOX in vitro.A more lipophilic DOX analog, isoindolino-DOX, was also

prepared in a satisfactory yield (55%) by using an excess ofa,a'-dichloro-ortho-xylene. This analog was more potent thanDOX at higher concentrations but less active at lower con-centrations.

Table 3. Growth inhibition by DOX and its derivativesincorporating the daunosamine nitrogen in five- and six-memberedrings of MXT estrogen-independent mouse mammary carcinomaand MCF-7 human breast cancer cell lines in vitro

1C*0, 10-10 M

Compound MXT MCF-7

DOX 270 5402-Pyrrolino-DOXt 0.3 11,3-Tetrahydropyridino-DOXt 11 (46)§3-Pyrrolidono-DOXt (21) 493-Piperidono-DOXf (72) 230

*Cell growth inhibition data, determined at three different concen-trations shown in Tables 1 and 2, were used to calculate the drugconcentration that inhibited cell growth by 50% compared to un-treated control cultures.tAnalog with five-membered ring.tAnalog with six-membered ring.§Numbers in parentheses were obtained by extrapolation.

DISCUSSIONA thorough investigation of the mechanism of action ofintensely potent DOX analog MRA-CN revealed that thiscompound can "alkylate" an amino function of a guanine basein close vicinity of the binding site of the daunosaminenitrogen (13). It was assumed, that MRA-CN may act as suchor, after hydrolysis, as a latent aldehyde in a closed six-membered ring, like 3-hydroxymorpholine. It was consideredconceivable that MRA-CN and cyanopiperidino-DOX, aclosely related analog, as well as N-(5,5-diacetoxypent-1-yl)doxorubicin, could react with nucleophils more distant from

Peak Retenion PeakNo. Time roe

Peak Area HeightHeight Pwecnt Percent

1 3.121 0.15115 0.00122 0.065 0.2752 11.235 4.77011 0.01135 2.067 2.5613 12.879 214.10022 0.41002 92.760 92.4674 21.352 11.7979 0.02083 5.08 4.697

Totals 230.81237 0.44343 100.000 100.000nAn .

0.30,

020-.0

0n.0< 0.10-

0.00-

o I0.00 10.00 20.00 30.00

Retention Time (minutes)

FIG. 3. Chromatograms of the conversion of DOX to 2-pyrrolino-DOX: reaction mixture after 15 min (trace b) and DOX control (tracea). Conditions of chromatography: column, Dynamax C18 250 x 4.6mm; pore size, 300 A; particle size, 12 ,um. Eluents: A, 0.1% TFA inwater; B, 70% acetonitrile in 0.1% aqueous TFA. The system was runat a 1.2 ml/min flow rate with an eluent mixture changing from 30%B to 60% B in 30 min in linear gradient mode. UV absorption wasdetected at 480 nm.

ON

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f . |-V

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R ax

R N b

H

FIG. 4. Steric representation of N-alkyl-2-pyrroline (a) and N-al-kyl-1,3-tetrahydropyridine (b) analogs. R = alkyl. Position of thereactive carbon atom 2 of the rings is indicated by asterisk.

the daunosamine nitrogen, in the form of open-chain N-alkanal derivatives (7, 15, 23).To investigate further the structural requirements of in-

tensely potent DOX derivatives, analogs with five- andsix-membered rings at the 3' position of the daunosaminemoiety were designed and synthesized. Interestingly, analogsthat incorporated the daunosamine nitrogen in a five-membered ring, having a reactive "alkylating" function, weremarkedly more active in vitro than their counterparts with asix-membered ring (Table 3). The reactive function waseither a double bond or an oxo group. 2-Pyrrolino-DOX hasa double bond between carbon atoms 2" and 3" of itsfive-membered ring. Its homolog, 1,3-tetrahydropyridino-DOX, also has a double bond between carbon atoms 2" and3" of its six-membered ring. Both analogs are enamines-thatis, latent aldehydes-but the former was 30-50 times moreactive than the latter, as shown in Table 3. An explanationfor this great difference in activities may be found in a stericdistinction between the five- and the six-membered rings. Itis well known that five-membered rings are almost planarwhile six-membered rings take the three-dimensional shapeof a "chair" or a "tub." As is shown in Fig. 4, the six-membered ring contains an extra CH2 group compared to thefive-membered ring. It is reasonable to believe that thedifference in the activity is due to the presence of the twohydrogen atoms protruding from the 4" carbon atom of thepiperidino ring. As a result of steric hindrance, the tightfitting of the six-membered ring into the binding site canpossibly drive the reactive 2" carbon atom slightly away fromthe amino group of the guanine residue of the DNA. In thestructure of 2-pyrrolino-DOX, this CH2 group is missing. Inthe structure of MRA-CN, an oxygen atom replaces themore bulky methylene group. These structural characteris-tics possibly allow the reactive carbon atom in these rings afavorable position for formation of a covalent linkage,reinforcing the primary binding stability of DOX. Thepresence of a bulky CH3 group on the reactive carbon atom2 of 2-methyl-2-pyrrolino-DOX, makes the formation of thiscovalent linkage impossible. In fact, because of steric hin-drance at the binding site, the activity of this analog is -5times lower than that of DOX. The hypothesis outlinedabove is further supported by our finding, that 3-pyrroli-dono-DOX (five-membered ring) is -5 times more active invitro than 3-piperidono-DOX (six-membered ring). Bothcompounds have a reactive oxo function attached to the 3"carbon atom of their respective five- and six-membered ring.These results strongly indicate that the active form of theseanalogs is not a freely moving open chain derivative but aclosed ring. It is also apparent that analogs with smaller,better fitting rings attached to the 3' position of thedaunosamine moiety have higher activity.

In addition, our results demonstrate that analogs having thedaunosamine nitrogen incorporated in a ring that has a

reactive carbon atom vicinal to the nitrogen are markedly moreactive than those having a CH2 group between these twoatoms. Thus, 2-pyrrolino-DOX is -100 times more active than3-pyrrolino-DOX.The high yield, clear-cut conversion of DOX to 2-pyrro-

lino-DOX was facilitated by use of 4-iodobutyraldehyde. Anexcessive amount of the aldehyde derivative has to be reactedfirst with a salt of the amino alcohol in an anhydrous polarsolvent like DMF. This is followed by addition of a tertiarybase. When the base was added to the reaction mixture priorto addition of the aldehyde, the formation of several by-products was observed. The reaction with the aldehyde leadsto formation of a five-membered oxazolidine ring having aiodopropyl side chain at its carbon atom 2. In this ring, thedaunosamine nitrogen is a secondary amine. Basification ofthe reaction mixture leads quickly to construction of a fusedring system by alkylation of the secondary nitrogen with thereactive iodopropyl side chain. The quick formation of athermodynamically favorable five-membered ring protectsthe secondary nitrogen from derivatization by excess of thereactive halogen compound present. Acidification of thereaction mixture with dilute aqueous acid leads to formationof 2-pyrrolino-DOX, a stable, water soluble derivative thatis 500-1000 times more potent than DOX. Analogous reac-tions take place between 5-iodovaleraldehyde or 5-iodo-2-pentanone and vicinal amino alcohols. The correspondingmorpholino derivative may be prepared by using 2-(2-iodoethoxy)acetaldehyde.The utilization of this reaction for formation of targeted

cytotoxic luteinizing hormone-releasing hormone analogs (24)with lower peripheral toxicity has been accomplished, and thechemistry of the conjugation and the in vivo activity of theresulting hybrids, intended for cancer therapy, will be thesubject of subsequent publications.

We thank Prof. J. Engel, Dr. M. Bernd, Dr. E. Busker, and Dr. A.Muller (Degussa AG and Asta Medica AG, Frankfurt am Main) formass spectra and NMR analyses and for their help in preparation ofthis manuscript. We thank Dr. H. Reile for establishing a reliablecytotoxicity assay in our institute. Some work described in this paperwas supported by the Medical Research Service of the VeteransAffairs.

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