High Yield Conversion of Doxorubicin to 2-Pyrrolinodoxorubicin

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

High yield conversion of doxorubicin to 2-pyrrolinodoxorubicin, an analog 500-1000 times more potent: Structure-activity relationship 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, New Orleans, LA 70146 Contributed by Andrew V. Schally, December 4, 1995

ABSTRACT A convenient, high yield conversion of doxo- 8). This derivative was isolated as a highly potent by-product rubicin to 3'-deamino-3'-(2"-pyrroline-1"-yl)doxorubicin is that formed during the reductive alkylation of the described. This daunosamine-modified analog of doxorubicin daunosamine nitrogen of DOX. MRA-CN was 100-500 is 500-1000 times more active in vitro than doxorubicin. The times more active than DOX in vitro. In fact, MRA-CN conversion is effected by using a 30-fold excess of 4-iodobu- proved to be 1500 times more potent on cell lines resistant tyraldehyde in anhydrous dimethylformamide. The yield is to DOX (9-11). It was revealed that the extremely high higher than 85%. A homolog of this compound, 3'-deamino- activity of this compound, even against DOX-resistant tumor 3'-(1l,3"-tetrahydropyridine-1"-yl)doxorubicin, was also syn- cell lines, is a result of its ability to form an aminal adduct thesized by using 5-iodovaleraldehyde. In this homolog, the with an amino group of a guanine base in close vicinity to its daunosamine nitrogen is incorporated into a six- instead of a binding site (12, 13). Based on this knowledge, a class of five-membered ring. This analog was 30-50 times less active intensely potent analogs was developed, best represented by than its counterpart with a five-membered ring. A similar N-(5,5-diacetoxypent-1-yl)doxorubicin (14, 15). This analog structure-activity relationship was found when 3'-deamino- is a water soluble, latent aldehyde derivative, activated in 3'-(3"-pyrrolidone-1"-yl)doxorubicin (containing a five- tissues by carboxylate esterase enzymes to become 150 times membered ring) and 3'-deamino-3'-(3"-piperidone-1"- more active than DOX. Other semisynthetic derivatives, like yl)doxorubicin (with a six-membered ring) were tested in vitro, and the former being 5 times more potent than the latter. To morpholinodoxorubicin (7) 2-methoxymorpholinodoxo- further elucidate structure-activity relationships, 3'- rubicin, become 50-80 times more active in vivo after deamino-3'-(pyrrolidine-1"-yl)doxorubicin, 3'-deamino-3'- activation by liver enzymes (16, 17). (isoindoline-2"-yl)doxorubicin, 3'-deamino-3'-(2"-methyl-2"- These highly potent analogs of DOX either have a latent pyrroline-l"-yl)doxorubicin, and 3'-deamino-3'-(3Y-pyrroline- aldehyde derivative attached to the nitrogen atom in the 1"-yl)doxorubicin were also synthesized and tested. All the daunosamine moiety through an open chain polymethylene analogs were prepared by using reactive halogen compounds bridge or the nitrogen atom becomes a part of a morpholine for incorporating the daunosamine nitrogen of doxorubicin ring. Gao et al. (18) presented a high resolution x-ray diffrac- into a five- or six-membered ring. These highly active anti- tion picture of a covalently linked adduct between a synthetic neoplastic agents can be used for incorporation into targeted DNA segment and a daunorubicin derivative. This adduct cytotoxic analogs of luteinizing hormone-releasing hormone forms readily when traces of formaldehyde are present in the intended for cancer therapy. crystallization solvent. The daunosamine nitrogen atom of the anthracycline derivative was shown to be linked covalently Daunorubicin and doxorubicin (DOX) (Fig. 1) are members of through a methylene bridge to the amino group of an amino- the family of the anthracycline antibiotics (1, 2) that were adenine moiety of the DNA segment in close vicinity. introduced into cancer therapy about 3 decades ago. As a result Based on these findings, we decided to investigate further of a small structural difference, DOX is a much more potent the structural requirements for the design of intensely potent anticancer agent than daunorubicin. Since its introduction into doxorubicin analogs. Our efforts led to the high yield (85%) human cancer therapy, DOX is still the most widely used conversion of DOX to 2-pyrrolinodoxorubicin by the use of a chemotherapeutic drug, with the broadest spectrum of anti- 30-fold excess of 4-iodobutyraldehyde. 2-Pyrrolinodoxorubicin tumor effect (3). is a stable, water soluble analog, which is 500-1000 times more The antiproliferative activity of DOX is due mainly to its active than its parent compound in vitro. ability to intercalate into DNA and break the strands of double helix by inhibiting topoisomerase 11 (4). Despite its wide acceptance in the chemotherapy of various cancers, prolonged MATERIALS AND METHODS use of DOX is severely limited by cardiotoxicity (5). Another Materials. DOX HCl salt, 1,4-diiodobutane, a,a'-dichloro- limitation of DOX is multidrug resistance (6). ortho-xylene, cis-1,4-dichloro-2-butene, 3-bromopropionyl Thousands of anthracycline derivatives were synthesized chloride, and 4-bromobutyryl chloride were purchased from to overcome these limitations and in search for even more Aldrich. Silica gel (Merck grade 9385; 230-400 mesh; pore active analogs (3). One of the most significant milestones in size, 60 A) was also from Aldrich. TLC aluminum sheets the semisynthetic development of more potent, non-cross- precoated with silicagel 60 F254 by Merck Art no. 5554 were resistant analogs of DOX with lower cardiotoxicity was the obtained from Curtin Matheson Scientific (Houston). 2-(3- finding of 3-cyanomorpholinodoxorubicin (MRA-CN) (7, Chloropropyl)-1,3-dioxolane, 2-(3-chloropropyl)-2-methyl-

The publication costs of this article were defrayed in part by page charge Abbreviations: DIPEA, N,N-diisopropylethylamine; DMF, N,N- payment. This article must therefore be hereby marked "advertisement" in dimethylformamide; DOX, doxorubicin; MRA-CN, 3-cyanomorpho- accordance with 18 U.S.C. §1734 solely to indicate this fact. linodoxorubicin; TFA, trifluoroacetic acid. 2464 Downloaded by guest on October 1, 2021 Medical Sciences: Nagy et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2465

O OH oil was dissolved in 3 ml of 0.1% TFA in H20 and extracted with ether to remove excess 1,4-diiodobutane. Purification 14 by HPLC resulted in 41.6 mg of 98% pure DOX derivative 12 CH2 -R (yield, 68%). Preparation of 3'-deamino-3'-(isoindoline-2"-yl)doxorubicin TFA salt (AN-184). DOX HCl salt (50 mg; 86 ,umol) was CH3/ dissolved in 1 ml of DMF and 226 mg (1.3 mmol; 15-fold excess) of a,a'-dichloro-ortho-xylene was added followed by 45 H3C ,tl (260 ,tmol; 3-fold excess) of DIPEA and a catalytic amount of Nal. After 16 hr, the reaction was complete and the desired end product was isolated as given above (yield, 55%). NH2 OH Preparation of 3'-deamino-3'- (3"-pyrroline-1"-yl)doxorubicin TFA salt (AN-185). DOX HCl salt (50 mg; 86 ,umol) was FIG. 1. Structures of daunorubicin (R = H) and DOX (R = OH). dissolved in 1 ml of DMF and 136.8 gl (1.3 mmol; 15-fold The daunosamine amino sugar moiety is indicated by an arrow. excess) of cis-1,4-dichloro-2-butene was added, followed by 45 ,ul (260 3-fold excess) of DIPEA. After 16 hr, the 1,3-dioxolane, and 2-(4-chlorobutyl)-1,3-dioxolane were ,tmol; reaction mixture was worked up as described above, yielding bought from Fluka. 22.6 mg of 98% pure end product (yield, 37%). Synthesis. Preparation of 1-chloro-4-bromo-2-butanone Preparation of 3'-deamino-3'-(3"-pyrrolidone-1"-yl)doxoru- and 1-chloro-5-bromo-2-butanone. 3-Bromopropionyl chlo- bicin TFA salt (AN-191) and 3'-deamino-3'-(3"-piperidone- ride (100.8 1lI; 1 mmol) was reacted with excess diazometh- 1"-yl)doxorubicin TFA salt (AN-195). DOX HCl salt (50 mg; ane in ether for 1 hr. The ethereal solution was developed in 86 ,umol) was dissolved in 1 ml of DMF and 241 mg (1.3 CHCl3/MeOH (95:5, vol/vol) on TLC. After chromatogra- mmol; 15-fold excess) of 1-chloro-4-bromo-2-butanone was phy, 2,4-dinitrophenylhydrazine reagent (19) was sprayed on added followed by 45 ,ul (260 ,umol; 3-fold excess) of DIPEA. the TLC sheet. The diazomethylketone derivative thus After 16 hr, the reaction mixture was purified as cited above, formed showed a yellow spot with Rf = 0.3. The ethereal yielding 20.6 mg of 98% pure end product (yield, 33%). solution was then reacted with anhydrous HCl in ether, 3'-Deamino-3'-(3"-piperidone-1"-yl)doxorubicin was pre- converting the diazomethylketone to the desired end prod- pared similarly by using 1-chloro-5-bromo-2-pentanone uct, 1-chloro-4-bromo-2-butanone. This compound showed a (yield, 28%). yellow spot, characteristic of oxo compounds, with Rf = 0.8 Preparation of 3'-deamino-3'-(2"-pyrroline-1"-yl)doxorubicin in the same solvent system and with the spot test reagent TFA salt (AN-201), 3'-deamino-3'-(1",3"-tetrahydropyridine-1"- described above. After evaporation of the solvent, the crude yl)doxorubicin TFA salt (AN-205), and 3'-deamino-3'-(2"- product was applied on a column (2.5 x 15 cm) packed with methyl-2"-pyrroline-1"-yl)doxorubicin (AN-204). DOX HCl salt 15 g of silica gel. The liquid, mobile phase was CHCl3. (50 mg; 86 ,tmol) was dissolved in 1 ml of DMF and 515 mg Fractions containing the desired end product (characterized (2.6 mmol) of 30-fold excess 4-iodobutyraldehyde was added by the spot test) were combined and evaporated to dryness followed by 45 gl (260 jLmol; 3-fold excess) of DIPEA. After to yield 1.5 g of clear oil. (yield, 80%.) Preparation of 30 min, 100 ,lI of glacial acetic acid was added to the reaction 1-chloro-5-bromo-2-pentanone was performed similarly mixture, which was then added dropwise to 5 ml of 0.1% TFA starting from 4-bromobutyryl chloride. in 70% aqueous acetonitrile. This solution was diluted with 2 Preparation of 4-iodobutyraldehyde, 5-iodovaleraldehyde, ml of 0.1% aqueous TFA solution, followed by removal of the and 5-iodo-2-petanone. 2-(3-Chloropropyl)-1,3-dioxolane, acetonitrile in vacuo. The resulting aqueous solution was (1.3 ml; 10 mmol) was dissolved in 200 ml of acetone extracted with hexane to remove the excess of the halogen containing 30 g (200 mmol; 20-fold excess) of Nal. The compound. After purification by HPLC, 52 mg of 98% pure solution was refluxed for 24 hr followed by evaporation to end product was obtained (yield, 85%). 3'-Deamino-3'-(1",3"- dryness. Diethyl ether (100 ml) was used to extract the tetrahydropyridine-1"-yl)doxorubicin TFA salt (AN-205) and organic material from the inorganic solid residue. The 3'-deamino-3'-(2"-methyl-2"-pyrroline-1"-yl)doxorubicin TFA ethereal solution was then washed with 50 ml of H20/50 ml salt (AN-204) were prepared similarly from 5-iodovaleralde- of 5% (wt/vol) aqueous Na2S203 solution and then three hyde (yield, 75%) and 5-iodo-2-pentanone (yield, 54%), re- times again with 50 ml of H20. The ether was removed in spectively. vacuo and the remaining oil was dissolved in 3 ml of 50% Purification. The final purification of all the crude products (vol/vol) aqueous AcOH. After 1 hr, 100 ml of ether was was carried out on a Beckman model 342 semipreparative added to this solution and the acetic acid as well as the HPLC system, using an Aquapore Octyl (250 x 10 mm; pore ethylene glycol were removed by washing three times with 50 size, 300 A; particle size, 15 ,um) column. The solvent system ml of H20. The main product eluted with Rf = 0.8 on TLC consisted of two components-0.1% TFA in water, and 0.1% in CHCl3. The spot test used for characterizing the aldehyde TFA in 70% aqueous acetonitrile-and was used in linear function was the same as cited above (19). The ether was then gradient mode. removed and the black oily residue was applied on a column Analytical HPLC. A Beckman analytical HPLC system packed with silica gel as described. Purification resulted in equipped with a model 168 diode array detector and System 1.6 g of yellow oil (yield, 80%.) 5-lodovaleraldehyde and Gold chromatography software (Beckman) was used to check 5-iodo-2-pentanone were obtained exactly the same way the purity and to monitor the chemical reactions. The column starting from 2-(4-chlorobutyl)-1,3-dioxolane or 2-(3- was Dynamax C18 (250 x 4.6 mm; pore size, 300 A; particle chloropropyl)-2-methyl-1,3-dioxolane, respectively. size, 12 ,um). Preparation of 3'-deamino-3'-(pyrrolidine-1"-yl)doxorubi- Cytotoxicity Assay. The MCF-7 human breast cancer cell cin trifluoroacetic acid (TFA) salt (AN-181). DOX HCl salt line, used for the determination of the antiproliferative activity (50 mg; 86 ,umol) was dissolved in 1 ml of dimethylform- of the DOX derivatives, was obtained from the American Type amide (DMF) and 171 ,lI (1.3 mmol; 15-fold excess) of Culture Collection. MXT hormone-independent mouse mam- 1,4-diiodobutane was added, followed by 45 ul (260 ,umol; mary carcinoma cell line was a gift from Gunther Bernhardt 3-fold excess) of N,N-diisopropylethylamine (DIPEA). After (University of Regensburg, Regensburg, Germany). For eval- 16 hr, the reaction is complete as assessed by analytical uation of the activity of the analogs, a colorimetric cytotoxicity HPLC. The solvent was evaporated in vacuo and the residual assay in microtitration plates was used based on quantification Downloaded by guest on October 1, 2021 2466 Medical Sciences: Nagy et al. Proc. Natl. Acad. Sci. USA 93 (1996)

20-fold excess of Nal. After hydrolysis of the acetal, flash chromatography 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 conversion of doxorubicin to 1,3-tetrahydropyridino-DOX, a homolog of 2-pyrrolino-DOX, incorporating the daunosamine nitrogen in a six-membered ring. Interestingly, 2-pyrrolino-DOX showed antiproliferative activity in vitro 30-50 times higher than its homolog with a six-membered ring, as shown in Tables 1-3. A similar phenomenon was observed when the in vitro cytotoxic activities of 3-pyrrolidono-DOX (containing a five-membered ring) and 3-piperidono-DOX (with a six-membered ring) were compared, the former 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- 0 100 200 300 400 500 800 700 done derivatives of primary amines was studied very exten- FIG. 2. MS/MS (daughter mass) spectra of the mass M + H+ sively, the literature on the preparation of 3-pyrrolidone and 596 of the mass spectra of 2-pyrrolino-DOX. 3-piperidone derivatives is rather limited. The syntheses in- of biomass by staining cells with crystal violet. The results of previously described for preparation of such analogs volved extreme conditions (21) not applicable for derivat- this assay correlate very well with the determination of cell number (20). ization of the complex, chemically sensitive structure of Analysis. Bruker ARX300 NMR spectrometer (300 MHz DOX. The conversion of DOX to these derivatives was with reactive com- 1H frequency; 75 MHz 13C frequency) and electrospray mass achieved again by alkylation halogen spectrometer Finnigan-MAT TSQ 7000 were used for struc- pounds. A 15-fold excess of 1-chloro-4-bromo-2-butanone tural identification of the DOX derivatives (Fig. 2). was reacted with DOX in anhydrous DMF in the presence of a 3-fold excess of a tertiary base, leading to the formation of 3-pyrrolidono-DOX with a moderate yield of 33%. Its RESULTS homolog counterpart, bearing a six-membered 3-piperidone A convenient synthetic procedure was developed for con- ring at the 3' position of the daunosamine moiety, was version of DOX to 2-pyrrolino-DOX. This derivative, which prepared under similar conditions by using 1-chloro-5- has the daunosamine nitrogen of DOX incorporated in a bromo-2-pentanone. The yield was 28%. For preparation of five-membered ring, is 500-1000 times more potent than 1-chloro-4-bromo-2-butanone, 3-bromopropionyl chloride DOX (Tables 1-3). A conversion of >90% of DOX (Fig. 3) was reacted with excess diazomethane in ether and the was achieved by using a 30-fold excess of 4-iodobutyralde- resulting diazomethyl ketone was converted to the corre- hyde in anhydrous DMF in the presence of a tertiary base. sponding chloromethyl ketone by anhydrous HCl in ether. The reaction is complete within 15 min and, after purifica- Flash chromatography of the halogen derivative thus ob- tion by HPLC, the yield is >85%. 4-lodobutyraldehyde was tained lead to a clear oil in a yield of 80%. 1-Chloro-5- obtained from the commercially available 2-(3-chloropro- bromo-2-pentanone was prepared similarly from 4-bro- pyl)-1,3-dioxolane by refluxing it in acetone containing a 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 10-7 Compound time, h M M M M M M DOX 70 98 82 54 120 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 bovine serum in 24-well plates. Relative cell number in treated and control plates was determined by crystal violet staining and was expressed as T/C values where T/C = T - Co/C - Co x 100. [T = absorbance of treated cultures, C = absorbance of control cultures, Co = absorbance of cultures at the start of incubation (t = 0). Measured absorbance is proportionate to cell number.] Negative T/C values indicate a cell number smaller than the number originally seeded at t = 0-i.e., a cytocidal effect. Downloaded by guest on October 1, 2021 Medical Sciences: Nagy et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2467 Table 2. Effects of DOX and its daunosamine-modified derivatives on proliferation of MXT estrogen-independent mouse mammary carcinoma 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 10-7 Compound time, hr M M M M M M M M DOX 24 84 77 65 69 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 number in 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 design DISCUSSION of intensely potent daunosamine-modified DOX analogs, 2-methyl-2-pyrrolino-DOX and 3-pyrrolino-DOX, a struc- A thorough investigation of the mechanism of action of tural isomer of 2-pyrrolino-DOX, were also synthesized. intensely potent DOX analog MRA-CN revealed that this 2-Methyl-2-pyrrolino-DOX has a methyl substituent on the compound can "alkylate" an amino function of a guanine base reactive carbon atom 2 of the ring and, surprisingly, it was in close vicinity of the binding site of the daunosamine even less active than DOX. The synthesis was done similarly nitrogen (13). It was assumed, that MRA-CN may act as such to that of 2-pyrrolino-DOX, but using 5-iodo-2-pentanone or, after hydrolysis, as a latent aldehyde in a closed six- for alkylation. In the structure of 3-pyrrolino-DOX, the membered ring, like 3-hydroxymorpholine. It was considered double bond is between carbon atoms 3 and 4 of the conceivable that MRA-CN and cyanopiperidino-DOX, a five-membered ring. This analog was -5 times more active closely related analog, as well as N-(5,5-diacetoxypent-1- than DOX in inhibiting growth of the MCF-7 human breast yl)doxorubicin, could react with nucleophils more distant from cancer cell line in vitro. Synthesis was accomplished by reacting a 15-fold excess of cis-1,4-dichloro-2-butene (22) Peak Retenion Peak Peak Area Height with DOX in the presence of an excess of tertiary base. The No. Time roe Height Pwecnt Percent 1 3.121 0.15115 0.00122 0.065 0.275 yield was 37% after purification by HPLC. 2 11.235 4.77011 0.01135 2.067 2.561 Pyrrolidino-DOX was synthesized by using the alkylating 3 12.879 214.10022 0.41002 92.760 92.467 agent 1,4-diiodobutane. The yield was 70%. The five- 4 21.352 11.7979 0.02083 5.08 4.697 membered ring, attached to the 3' position of the daunosamine Totals 230.81237 0.44343 100.000 100.000 moiety of DOX in this analog, had no reactive double bond or nAn . oxo in The of 0) function its structure. activity pyrrolidino-DOX ON was only slightly better than that of DOX in vitro. A more lipophilic DOX analog, isoindolino-DOX, was also 0.30, prepared in a satisfactory yield (55%) by using an excess of a,a'-dichloro-ortho-xylene. This analog was more potent than DOX at higher concentrations but less active at lower con- 020- centrations. .0 0 .0n Table 3. Growth inhibition by DOX and its derivatives < 0.10- incorporating the daunosamine nitrogen in five- and six-membered rings of MXT estrogen-independent mouse mammary carcinoma 7 0b and MCF-7 human breast cancer cell lines in vitro 0.00- f . | 1C*0, 10-10 M -V Compound MXT MCF-7 [l

DOX 270 540 O.c6i-I--o I 2-Pyrrolino-DOXt 0.3 1 0.00 10.00 20.00 30.00 1,3-Tetrahydropyridino-DOXt 11 (46)§ 3-Pyrrolidono-DOXt (21) 49 Retention Time (minutes) 3-Piperidono-DOXf (72) 230 FIG. 3. Chromatograms of the conversion of DOX to 2-pyrrolino- *Cell growth inhibition data, determined at three different concen- DOX: reaction mixture after 15 min (trace b) and DOX control (trace trations shown in Tables 1 and 2, were used to calculate the drug a). Conditions of chromatography: column, Dynamax C18 250 x 4.6 concentration that inhibited cell growth by 50% compared to un- mm; pore size, 300 A; particle size, 12 ,um. Eluents: A, 0.1% TFA in treated control cultures. water; B, 70% acetonitrile in 0.1% aqueous TFA. The system was run tAnalog with five-membered ring. at a 1.2 ml/min flow rate with an eluent mixture changing from 30% tAnalog with six-membered ring. B to 60% B in 30 min in linear gradient mode. UV absorption was §Numbers in parentheses were obtained by extrapolation. detected at 480 nm. Downloaded by guest on October 1, 2021 2468 Medical Sciences: Nagy et al. Proc. Natl. Acad. Sci. USA 93 (1996) R ax reactive carbon atom vicinal to the nitrogen are markedly more active than those having a CH2 group between these two atoms. Thus, 2-pyrrolino-DOX is - 100 times more active than 3-pyrrolino-DOX. The high yield, clear-cut conversion of DOX to 2-pyrro- R N b lino-DOX was facilitated by use of 4-iodobutyraldehyde. An excessive amount of the aldehyde derivative has to be reacted first with a salt of the amino alcohol in an anhydrous polar H solvent like DMF. This is followed by addition of a tertiary base. When the base was added to the reaction mixture prior to addition of the aldehyde, the formation of several by- products was observed. The reaction with the aldehyde leads FIG. 4. Steric representation of N-alkyl-2-pyrroline (a) and N-al- to formation of a five-membered oxazolidine ring having a kyl-1,3-tetrahydropyridine (b) analogs. R = alkyl. Position of the iodopropyl side chain at its carbon atom 2. In this ring, the reactive carbon atom 2 of the rings is indicated by asterisk. daunosamine nitrogen is a secondary amine. Basification of the reaction mixture leads quickly to construction of a fused the daunosamine nitrogen, in the form of open-chain N- ring system by alkylation of the secondary nitrogen with the alkanal derivatives (7, 15, 23). reactive iodopropyl side chain. The quick formation of a To investigate further the structural requirements of in- thermodynamically favorable five-membered ring protects tensely potent DOX derivatives, analogs with five- and the secondary nitrogen from derivatization by excess of the six-membered rings at the 3' position of the daunosamine reactive halogen compound present. Acidification of the moiety were designed and synthesized. Interestingly, analogs reaction mixture with dilute aqueous acid leads to formation that incorporated the daunosamine nitrogen in a five- of 2-pyrrolino-DOX, a stable, water soluble derivative that membered ring, having a reactive "alkylating" function, were is 500-1000 times more potent than DOX. Analogous reac- markedly more active in vitro than their counterparts with a tions take place between 5-iodovaleraldehyde or 5-iodo-2- six-membered ring (Table 3). The reactive function was pentanone and vicinal amino alcohols. The corresponding either a double bond or an oxo group. 2-Pyrrolino-DOX has morpholino derivative may be prepared by using 2-(2- a double bond between carbon atoms 2" and 3" of its iodoethoxy)acetaldehyde. five-membered ring. Its homolog, 1,3-tetrahydropyridino- The utilization of this reaction for formation of targeted DOX, also has a double bond between carbon atoms 2" and cytotoxic luteinizing hormone-releasing hormone analogs (24) 3" of its six-membered ring. Both analogs are enamines-that with lower peripheral toxicity has been accomplished, and the is, latent aldehydes-but the former was 30-50 times more chemistry of the conjugation and the in vivo activity of the active than the latter, as shown in Table 3. An explanation resulting hybrids, intended for cancer therapy, will be the for this great difference in activities may be found in a steric subject of subsequent publications. distinction between the five- and the six-membered rings. It is well known that five-membered rings are almost planar We thank Prof. J. Engel, Dr. M. Bernd, Dr. E. Busker, and Dr. A. while six-membered rings take the three-dimensional shape Muller (Degussa AG and Asta Medica AG, Frankfurt am Main) for of a "chair" or a "tub." As is shown in Fig. 4, the six- mass spectra and NMR analyses and for their help in preparation of membered ring contains an extra CH2 group compared to the this manuscript. We thank Dr. H. Reile for establishing a reliable five-membered ring. It is reasonable to believe that the cytotoxicity assay in our institute. Some work described in this paper difference in the activity is due to the presence of the two was supported by the Medical Research Service of the Veterans hydrogen atoms protruding from the 4" carbon atom of the Affairs. As a result of steric the piperidino ring. hindrance, tight 1. of the six-membered into the can Di Marco, A., Gaetani, M., Orezzi, P., Scarpinato, B. M., Silves- fitting ring binding site trini, R., Soldati, M., Dasdia, T. & Valentini, L. (1964) Nature possibly drive the reactive 2" carbon atom slightly away from (London) 201, 706-707. the amino group of the guanine residue of the DNA. In the 2. Arcamone, F., Cassinelli, G., Fantini, G., Grein, A., Orezzi, P., structure of 2-pyrrolino-DOX, this CH2 group is missing. In Pol, C. & Spalla, C. (1969) Biotechnol. Bioeng. 11, 1101-1110. the structure of MRA-CN, an oxygen atom replaces the 3. Weiss, R. B. (1992) Semin. Oncol. 19, 670-686. more bulky methylene group. These structural characteris- 4. Cummings, J. & Anderson, L. (1991) Eur. J. Cancer 27, 532-535. tics possibly allow the reactive carbon atom in these rings a 5. Bristow, M. R., Thompson, P., Martin, R. P., Mason, J. W., favorable position for formation of a covalent linkage, Billingham, M. E. & Harrison, D. C. (1978) Am. J. Med. 65, reinforcing the primary binding stability of DOX. The 823-832. presence of a bulky CH3 group on the reactive carbon atom 6. Moscow, J. A. & Cowan, K. H. (1988) J. Natl. Cancer Inst. 80, 2 of makes the formation of this 14-20. 2-methyl-2-pyrrolino-DOX, 7. Acton, E. M., Tong, G. L., Mosher, C. W. & Wolgemuth, R. L. covalent linkage impossible. In fact, because of steric hin- (1984) J. Med. Chem. 27, 638-645. drance at the binding site, the activity of this analog is -5 8. Sikic, B. I., Ehsan, M. N., Harker, V. G., Friend, N. F., Brown, times lower than that of DOX. The hypothesis outlined B. W., Newman, R. A., Hacker, M. P. & Acton, E. M. (1985) above is further supported by our finding, that 3-pyrroli- Science 228, 1544-1546. dono-DOX (five-membered ring) is -5 times more active in 9. Johnston, J. B., Habernicht, B., Acton, E. M. & Glazer, R. I. vitro than 3-piperidono-DOX (six-membered ring). Both (1983) Biochem. Pharmacol. 32, 3255-3258. compounds have a reactive oxo function attached to the 3" 10. Streeter, D. G., Taylor, D. L., Acton, E. M. & Peters, J. H. (1985) carbon atom of their respective five- and six-membered ring. Cancer Chemother. Pharmacol. 14, 160-164. These results strongly indicate that the active form of these 11. Jesson, M. I., Johnston, J. B., Anhalt, C. D. & Begleiter, A. analogs is not a freely moving open chain derivative but a (1987) Cancer Res. 47, 5935-5938. closed It 12. Jesson, M. I., Johnston, J. B., Robotham, E. & Begleiter, A. ring. is also apparent that analogs with smaller, (1989) Cancer Res. 49, 7031-7036. better fitting rings attached to the 3' position of the 13. Cullinane, C. & Phillips, D. R. (1993) Nucleic Acids Res. 21, daunosamine moiety have higher activity. 1857-1862. In addition, our results demonstrate that analogs having the 14. Zwelling, L. A., Altshuler, E., Cherif, A. & Farquhar, D. (1991) daunosamine nitrogen incorporated in a ring that has a Cancer Res. 51, 6704-6707. Downloaded by guest on October 1, 2021 Medical Sciences: Nagy et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2469

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