Bioconjugate Chem. 1990, 1, 77-82
77
Hydrazide Pharmaceuticals as Conjugates to Polyaldehyde Dextran: Syntheses, Characterization, and Stability Ned D. Heindel,' Huiru Zhao, Jeffrey Leiby, Jacobus M. VanDongen, C. Jeffrey Lacey, Daniel A. Lima, Barakat Shabsoug, and John H. Buzby Institute for Health Sciences and Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015. Received October 16. 1989
The coupling of ellipticine and CI-921, two antineoplastic pharmaceuticals, to polyaldehyde dextran can be carried out under mild reaction conditions with novel acid hydrazides of the parent drugs. The resulting acyl hydrazones resist hydrolytic and enzymatic cleavage and offer a method for drug loading via dextran conjugates to monoclonal antibodies.
Numerous examples of the conjugation of antineoplastic pharmaceuticals to antibodies or to their fragments can be found in the burgeoning field of immunochemotherapy (1-3). Often these drug-to-antibody linkages involve the amino or carboxy functions on the glycoprotein bridging to counterpart carboxyl (or formyl) and amino moieties on the small molecule pharmaceutical (3-5). Recently, however, the chemical advantages of utilizing hydrazides in the conjugation process have been recognized. We have reported the synthesis of a no-carrier-added radioiodinated hydrazide which couples efficiently to carbonyl moieties (6). Others have noted that the very low pK of hydrazides (ca. 2.6) compared to that of primary amines (ca. 9-10) means that under mildly acidic conditions hydrazides can be linked to aldehydes without intervention of potentially competitive amino functions (7, 8). This chemistry has been exploited to permit conjugation of hydrazide anticancer agents to CHO moieties-often generated by oxidation of carbohydrate units on the antibody-with minimal intramolecular crosslinking by lysine residues on the glycoprotein (9-11). Furthermore, the acyl hydrazones thus generated are considerably more resistant to hydrolysis and do not require reduction for stabilization. In this laboratory we have been developing the use of hydrazide-based pharmaceuticals and radiotracers as well as polyaldehyde dextran (PAD) as a macromolecular carrier for these agents (12,13). Not only do drug-dextran conjugates offer a facile polymeric spacer for antibody attachment but they also offer a more soluble delivery form of the parent drug, which may now undergo tumoraffinic uptake by an endocytosis mechanism. Drug-toPAD conjugates appear to offer therapeutic advantages in several cases (14, 15). In this study we have linked the antitumor agents CI-921 (Chart I) and two ellipticines (9-methoxy and 9-H, Chart 11) as hydrazides onto PAD and have studied the stability of the conjugates. EXPERIMENTAL PROCEDURES
Materials. Ellipticine and 9-methoxyellipticine were supplied by the Drug Development Branch, National Cancer Institute, NIH, Bethesda, MD. CI-921, also known as 9-[ [2-methoxy-4-[ (methylsulfonyl)amino]phenyl]amino]-N,5-dimethylacridine-4-carboxamide, was supplied by Parke-Davis Pharmaceuticals of Ann Arbor, MI. Clinical-grade dextran (40 kDa) was supplied by Pharmachem Corp., Bethlehem, PA. All other chemicals and solvents were of the highest purity commercial grade. Combustion analyses were supplied by Robertson Microana1043- 1802f 901290 1-0077$02.50/0
Scheme I. Synthesis of (31-921 Carbazate (111)
R
?
CH,
CONHNHCOC ( W)S
I11
I 0
lytical Laboratory, Madison, NJ. 'H NMR spectra were obtained in the indicated solvents on a JEOL FX9OQ spectrophotometer. Melting points were obtained on either a Thomas-Hoover capillary or a Fisher-Johns melting point apparatus and are reported uncorrected. Syntheses. tert-Butyl N-[[9-[[2-Methoxy-4[ (methylsulfonyl)amino]phenyl]amino]-&methylacridin-4-yl]carbonyl]carbazate Hydrochloride (111). Intermediates in this pathway proved highly unstable and decomposed to red oils from initially isolated solids. The synthesis was continued in sequence until stable I11 was isolated (Scheme I). 5-Methyl-9-oxoacridan-4-carboxylic acid (1.51 g, 5.95 mmol) was dissolved in 20 mL of thionyl chloride containing one drop of dimethyl formamide and the solution was refluxed for 30 min (17). The volume was reduced to 5 mL and 15 mL of dry benzene was added. The mixture was again evaporated to about 5 mL, fresh benzene was added, and the flask contents were evaporated to dryness. The intermediate, yellow 5-methyl-9-chloroacridine-4-carbonyl chloride hydrochloride (I) (mp 143-146 "C) was used directly by immediate dissolution in 70 mL of dry methylene chloride. tert-Butyl carbazate (0.78 g, 5.9 mmol) was added as a single portion to the solution of I chilled in an ice-water bath. After 1min of mixing, the methylene chloride was vacuum evaporated a t room 0 1990 American Chemical Society
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Bioconjugate Chem., Vol. 1, No. 1, 1990
Chart I. Structures of CI-921 and Analogues OCH3
Heindel et al.
Scheme 11. Synthesis of (31-921 Hydrazide (IV)
w I
CY
C-R I/ 0 R = NHCH3
O
H
ocH3 \
II
0
(CI-921)
= NHNH2.ACL
(111)
(IV)
I
/
I
c%
(V)
Chart 11. Structures of Ellipticine and Methoxyellipticine
R
e
CONHNHCOC ( CH313
I11
= NHNH-COO-2-bu
= @CH3
T
/W
CONHNl+-HCl
IV Scheme 111. Synthesis of CI-921 Methyl Ester (V)
*tcw
d
yq coocH3
COC 1
C H3
I R
=
H
R = OCH3
Ellipticine
4 /"HSWH3
9-Methoxyellipticine
temperature and the labile tert-butyl N'-[(5-methyl-9chloroacridin-4-yl)carbonyl]carbazate hydrochloride (11) was dissolved in 50 mL of anhydrous methanol and employed directly in the condensation step with N-(4amino-3-methoxypheny1)methanesulfonamide(16) (1.29 g, 5.95 mmol) and 100 pL of concentrated hydrochloric acid. This slurry was refluxed for 20 min, the condenser was removed, and the solvent was evaporated to halfvolume. Ethyl acetate (50 mL) was added and the resultant slurry was allowed to stand for 5 h prior to filtration. The isolated product (1.99 g) was shown by 'H NMR to be a mixture of I11 and IV. Pure title product (tert-butyl carbazate 111) was obtained as a red-orange solid by preparative thick (lo00 pm) layer chromatography employing 9:4:1 chloroform-isopropyl alcohol-methanol as the moving phase. The product eluted just under the solvent front (R, -0.8) and all other impurities including the hydrazide (R, -0.4) were considerably less migratory: yield 0.122 g (3.4%) of a red-orange solid; mp 116120 OC; 'H NMR (DMSO-dJ 6 1.45 (s, 9H, t-Bu), 2.61 (9, 3 H, C-5 CH,), 3.10 (s, 3 H, SO,CH,), 3.59 ( s , 3 H, OCH,), 7.01 (9, 2 H, ArH), 7.48 (m, 4 H, ArH), 7.89 (d, 1 H, ArH), 8.11 (d, 1 H, ArH), 8.55 (d, 1 H, ArH), 8.70 (d, 1 H, ArH), 9.28 (s, 1 H, NH), 10.15 (s, 1 H, NH), 11.21 (s,1H, NH). Anal. Calcd for C2,H,,N,O6S~HC1: C, 55.85; H, 5.36; N, 11.63. Found: C, 55.73; H, 5.53; N, 11.43. 9-[ [%-Methoxy-4-[ (methylsulfonyl)amino]phenyl]amino]-bmethylacridine-4-carboxylicAcid Hydrazide Dihydrochloride (IV). This product could be obtained as a byproduct in the synthesis of I11 described above ( R , 0.40-0.45) but was best obtained by acid-catalyzed hydrolysis of purified carbazate I11 (Scheme 11). A sample of 0.122 g (0.203 mmol) of I11 was dissolved in 1.0 mL of methanol and 15 mL of 2.0 M aqueous hydrochloric acid. This acidic, methanolic solution was allowed to stand a t ambient temperature for 1 h and was evaporated in vacuo, and the resulting red solid was purified by preparative thick-layer chromatography on 1000-bm silica gel plates. The mobile phase was 102:l chloroform-
2-propanol-methanol (v:v:v) and the product was eluted as a sharp band at R, 0.74. It was eluted from the silica gel in methanol, filtered, evaporated to dryness, and dried under vacuum for 2 h to yield 0.062 g (56%) of red-orange IV: mp 109.5-111.5 "C; 'NMR (CD,OD) 6 2.81 (s, 3 H, C-5 CH,), 3.09 (s, 3 H, S02CH,), 3.58 (s, 3 H,OCH,), 7.05 (m, 3 H, ArH), 7.50 (m, 3 H, ArH), 7.87 (d, 1 H, ArH), 8.10 (d, 1 H, ArH), and 8.55 (m, 2 H, ArH) (NH signals not visible in this solvent system); MS (FAB) m / e = 465.9 (calcd mle = 465.6 for molecular ion of free base). Anal. Calcd for C2,H2,N,O,S~2HC1: C1, 13.16. Found: C1, 13.05. Methyl 9-[ [2-Methoxy-4-[(methylsulfonyl)amino]phenyllamino1-5-methyl-4-acridinecarboxylate Hydrochloride ( V ) . 5-Methyl-9-chloroacridine-4carbonyl chloride hydrochloride (11) (prepared in situ by chlorination as described above from 1.51 g, 5.95 mmol of 5-methylacridone-4-carboxylicacid) was slurried in 50 mL of dry methanol with 5 mL of triethylamine for 1 h at room temperature (Scheme 111). The yellow solid which formed, the methyl ester of the acridine, was filtered, washed with dry methanol, and suspended in 50 mL of dry methanol. N-(4-amino-3-methoxyphenyl)methanesulfonamide (1.29 g, 5.95 mmol) and one drop of concentrated hydrochloric acid were added to this suspension. The reaction mixture was distilled to 50% volume and 50 mL of ethyl acetate was added. The resulting slurry was allowed to stand at room temperature overnight. A tangerine solid (V) was filtered and washed with chloroform: yield 0.41 g (14%); mp 232-234 OC dec; 'H NMR (DMSO-d,) 6 2.61 ( s , 3 H, C-5 CH,), 3.05 (s, 3 H, SO,CH,), 3.50 (s, 3 H, OCH,), 4.02 (s, 3 H, COOCH,), 7.01
Hydrazide Pharmaceuticals as Conjugates
Scheme IV. Synthesis of Ellipticinium Hydrazides VI11 and IX
Bloconlugate Chem., Vol. 1, No. 1 , 1990
79
Scheme V. Conjugation of Hydrazides to Polyaldehyde Dextran
rtkoj+ 0
~ ~ c H /d /4 Brc o o c H .
I+"HCR
H VI VI1
0 0
CY
I
X
R = OCH3
Dextran Aldehyde
R = H
I COR
R may be f o r example C I 921 o r E l l i p t i c i n e ..
H VI11
IX
CY
R = OCH3 R ' H
(s, 2 H, ArH), 7.19 (m, 4 H, ArH), 7.65 (d, 1 H, ArH), 7.85 (d, 1 H, ArH), 8.40 (d, 2 H, ArH), and 9.78 (9, 1 H, NH) (other NH signals not seen). Anal. Calcd for C24H,4C1N305S~1/2H,0:C, 56.41; H, 4.93; N, 8.22. Found: C, 56.66; H, 4.82; N, 8.20.
2-(4-Methoxy-4-oxobutyl)-9-methoxyellipticinium Bromide (VI). A mixture of 9-methoxyellipticine (0.838 g, 3.04 mmol), methyl 5-bromopentanoate (3.41 g, 17.5 mmol), 2-propanol (48 mL), and dimethylformamide (16 mL) was refluxed for 72 h, cooled, and the precipitate was filtered and washed successively with 2propanol and acetone. Evaporation of the combined mother liquors and washings to ca. 25 mL yielded, upon cooling, additional orange-brown crystals of 2-(4-methoxy-4-oxobutyl)-9-methoxyellipticinium bromide (VI) for a total of 1.02 g (71%), mp 200-202 "C (from CHC1,). Anal. Calcd for C,,H,,BrN,O,: C, 61.15; H, 5.77; N, 5.94. Found: C, 60.99; H, 5.56; N, 5.78. 2-(4-Methoxy-4-oxobutyl)ellipticiniumBromide (VII). The above method with ellipticine (3.25 mmol) and methyl 5-bromopentanoate (18.2 mmol) gave VI1 (78%), mp 273-275 "C (from CHC1,). Anal. Calcd for C,,H,,BrN,O,: C, 62.59; H, 5.71; N, 6.35. Found C, 62.36; H, 5.51; N, 6.28. 2-(4-Hydrazino-4-oxobutyl)-9-met hoxyellipticinium Bromide (VIII). A solution of 0.753 g (1.60 mmol) of VI in 80 mL of methanol was treated to the dropwise addition over 5 min of 4.5 mL of hydrazine hydrate (Scheme IV). The mixture was then stirred at room temperature for 48 h, concentrated to 15 mL, diluted with 60 mL of anhydrous benzene, and evaporated repeatedly (3X) in vacuo to 20 mL to remove residual water. The solid was taken up in a minimum volume of anhydrous methanol, the solution was chilled in ice, and ethyl ether was added dropwise to induce crystallization of 0.635 g (84%) of salt VIII: mp 174-177 OC; 'H NMR (DMSOd,) 6 1.7-2.3 (m, 6 H, (CHJ3) 2.75 (s, 3 H, CH,), 3.20 (s, 3 H, CH,), 3.38 (br s, 2 H, NH,), 3.91 (s, 3 H, CH,O), 4.72 (br t, 2 H, CH2N+),7.2-8.6 (m, 5 H, ArH), 9.25 (br s, 1 H, NH), 10.06 (s, 1 H, C-1 H), and 11.95 (br s, 1 H, NH). Anal. Calcd for C,,H,,BrN,O,-H,O: C, 56.44; H, 5.97; N, 11.44. Found: C, 56.78; H, 5.62; N, 11.03. 2-(4-Hydrazino-4-oxobutyl)ellipticinium Bromide (IX). Following the method for the preparation of VIII, the title compound was prepared from 0.206 g (0.467 mmol) of VI1 and 4.5 mL of hydrazine hydrate, in 50 mL of methanol. The product (IX) was precipitated
Table I. Conjugation of Drug to Polyaldehyde Dextran molar ratio loading (hydrazide/ (mol of hydrazide/ CHO) drug solvent mol of glucose) 0.04/1 IX H,O (H+)" 0.02d 0.10/1 IX EtOH-H,O (l:l)/H+ " 0.06d 0.14/1 IX PBS buffer (pH 7.4)" 0.02d 0.14/1 VIII NaCl solution (H+)" 0.03d 0.14/1 IX H,O (H+)" 0.04d 0.14/1 IX EtOH-H 0 (l:l)/H+" 0.09' 0.21/1 IX H,O (H+;b 0.08" 0.21/1 IX H,O (H+)' 0.13' 0.21/1 VI11 H,O (H+)' 0.09' 0.28/1 IX H,O (H+)b 0.094' 0.42/1 IX H,O (H+)" insoluble (H,O) 0.07/1 IV H,O (H+)" 0.04d 0.10/1 IVf H,O (H+)" 0.05d 0.12/1 IVf H,O (H+)" 0.08d 0.14/1 IV H,O (H+)" 0.15'
" Separation by dialysis. Separation by Bio-gel P-60. Separation by ultrafiltration. Dissolved in both water and 0.01 M phosphate buffer (pH 7.35). e Soluble in water but not in 0.01 M phosphate buffer (pH 7.35). Hydrazide IV generated in situ by cleavage of carbazate 111. from methanol by the dropwise addition of ether: yield 0.164 g (80%) of a red-orange solid; mp 203-206 OC (from methanol). Anal. Calcd for C2,H,,BrN,0: C, 59.87; H, 5.72. Found: C, 60.19; H, 6.20. Methods. Polyaldehyde dextran with approximately 0.36 formyl functions per glucose unit was prepared as reported from 40 kDa dextran (22). Hydrazide Conjugations to Polyaldehyde Dextran. PAD was dissolved in distilled water and incubated with the requisite hydrazide IV, VIII, or I X with continuous stirring a t ambient temperature a t pH 4 (or in one case a t pH 7.4 in PBS buffer) for 48 h (Scheme V). Several solvent media were employed (see Table I). Conjugates were purified by techniques previously described for dextran-drug conjugates (14,22)involving either exhaustive dialysis against water, purification on a Bio-Gel P-60 column, or membrane ultrafiltration. Fractions eluted from the Bio-Gel P-60 column were tested by the anthrone reagent for carbohydrate and by UV absorption at the appropriate ,A for the respective drugs. The conjugate product fraction was taken as that which tested positive in both monitoring methods. The drugPAD conjugate was then lyophilized for analysis and storage. The degree of substitution of the druglrepeating glucose unit was determined by the UV absorbances a t 447 nm for IV and at 305 nm for VI11 and IX. Indirect Hydrazide Conjugation with CI-921 Carbazate on PAD. CI-921 hydrazide (IV) was unstable and difficult to obtain in a pure, high-yield form. Thus, a convenient method was developed in which its carbazate precursor (111)was deblocked and conjugated in one
80
Heindel et
Bioconjugate Chem., Vol. 1, No. 1, 1990 -.
100
.-.-.
..
-\
.
-
,.
,
- . . ~ ..
,
.., .. - - ,, -- -P
-
:oo
-
m
b -
30
n
-
20
-
io
r
L
O
.-t .-cn L
0 L 0
n
50
0
20
40
60
-
\ \
\ 4 VIII-PAD (aged sevum)
4
30 40
20
io
t_ i-i-.
-
60-
V 0
al.
-
-
L - - L L - L
80
100
120
240
Incubation Time (hours)
Figure 1. Hydrolyses of hydrazide conjugates to PAD (in PBS, pH 7.4, at 37 "C).
Figure 2. Enzymatic hydrolysis of hydrazide conjugates to PAD (in sera, pH 7.4, at 37 "C).
pot (see Table I). Carbazate hydrochloride I11 (20.0 mg, 0.033 mmol) was dissolved in 5 mL of 90% trifluoroacetic acid-10 ?& water, stirred at ambient temperature for 20 min, and evaporated to dryness in vacuo. The residue was dissolved in 40 mL of distilled water, adjusted to pH 4, and added to 52.0 mg of PAD. The reaction was brought about by stirring/incubation at room temperature for 24 h and the conjugate was isolated by exhaustive dialysis against water (4 days, 2 changes of water/ day) with subsequent lyophilization. The UV spectrum of the pure conjugate matched that of authentic CI-921
unfavorable biodistributions into solid tumors (20, 21). Coupling of ellipticine and CI-921 to polymeric or immunologically based carrier systems offers the prospect of improved tumor uptake. While the syntheses of the ellipticinium hydrazides could be effected by directed hydrazinolysis of their corresponding esters, the synthesis of the CI-921 hydrazide in this fashion was impossible. Despite five attempts under varying reaction conditions, no clean, direct displacement of methyl ester V was capable of producing IV. Heating V with 0.5 M hydrazine hydrate in methanol for 24 h or even 4 h produced nearly total loss of starting material with concomitant conversion to a yellow solid displaying more than five spots on TLC. The reaction of CI-921 with 0.5 M hydrazine hydrate/methanol at reflux gave a similar mixture as did the reaction of CI-921 with tertbutyl carbazate and V with 0.5 M hydrazine standing a t room temperature for 2 days. Refluxing V with 0.02 M hydrazine hydrate in methanol for 2 days or contact of V with 0.1 M hydrazine hydrate/methanol for 2 weeks at 4 "C resulted in total recovery of the starting material. Other related variations gave either complex mixtures or no evidence of reaction. The successful approach to IV involved initial preparation of 5-methyl-9-chloroacridine-4-carbonyl chloride (I) and its condensation with an equimolar quantity of tert-butyl carbazate. This protected hydrazide I1 was reacted upon the C-9 position in a subsequent step with the pendant anilino moiety to give CI-921 carbazate I11 and the latter was deprotected to the target IV. PAD was prepared by the periodate oxidation of dextran (22). We have observed, however, that it is essential to completely purify the PAD employed for hydrazide linking of iodate or periodate contaminants. No dextran hydrazones were formed when oxidizing impurities were present and model studies on the coupling of phenylacetylhydrazide to PAD contaminated by residual oxidant gave 1,2-bis(phenylacetyl)hydrazine,an observation supported by previous studies (16). Dimers were avoided if PAD was freed of iodate by exhaustive dialysis or by anion exchange. Hydrazide conjugation load or degree of substitution upon the PAD was sensitive to the reactant ratios, expressed as moles of hydrazide employed per formyl equivalent on the PAD, as well as to solvent system and the purification method utilized. Membrane ultrafiltration, the most rapid purification technique studied, appears to yield a PAD with the highest loading. Ethanol-water reaction solvent at pH 4 was superior to water at pH 4,
(16).
Hydrolytic and Enzymatic Cleavage of the PADHydrazide Conjugates. The stability of the conjugates to hydrolysis in PBS (pH 7.4) was determined by incubating a selected hydrazide-PAD conjugate in PBS a t 37 "C for the indicated times. Studies were performed on a IV-PAD conjugate with 0.07 drug/glucose unit, a VIII-PAD conjugate of 0.03 drug/glucose unit, and a IX-PAD conjugate of 0.04 drug/glucose unit at an effective concentration of 4 mg of conjugate in 10-mL fluid volume. The free-drug and dextran-bound-drug concentrations were determined by UV absorbance after separation in a centrifugal microconcentrator (molecular weight cutoff = 10 kDa) (Figure 1). The stability of drug-PAD conjugates VI11 and IX mentioned above to plasma enzymes was determined by incubating 5 mL of rat sera (freshly prepared and in one case 2-days old) and 10 mL of an aqueous solution (pH 7.4) of the requisite hydrazide-PAD conjugate containing 0.1 % sodium azide at 37 "C. Effective conjugate concentrations were 4 mg of each in 10 mL of fluid volume. Onemilliliter aliquots were withdrawn at the indicated times, charged to a centrifuge tube, treated with 0.5 mL of a 10% solution of trichloroacetic acid, and spun. The supernatant from the resulting protein precipitation was transferred to a microconcentrator tube (molecular weight cutoff 10 kDa) and centrifuged. Free-drug and dextranbound-drug concentrations were determined by UV analysis (Figure 2). DISCUSSION
Ellipticine and its N-2-alkylated ellipticinium salts with wide structural variations on the isoquinoline ring have shown substantial activity against malignant cell lines from solid tumors in culture (18). Similar success has been reported with amsacrine species like CI-921 (19). Nevertheless the in vivo activity of both drug types has been marginal at best, occasioned a t least in part by their
Hydrazide Pharmaceuticals as Conjugates
while the higher pH of PBS detracted from loading efficiency. CI-921 hydrazide (IV) conjugated more efficiently than either ellipticine hydrazide and, in fact, the variation in which IV was produced in situ from I11 in the PAD-containing medium proved even more efficient and facile. The drug-PAD conjugates were readily soluble in both water and PBS (pH 7.35) but above a loading ratio of 0.08 mol of hydrazide/mol of glucose units the conjugates lost solubility in buffer. Attempts to produce the highest drug load from a 0.42/1 molar ratio of the reactants gave a conjugate which lost both water and buffer solubility. The degree of drug substitution could not be measured. All conjugates demonstrated considerable resistance to hydrolysis in PBS and, in fact, the PAD conjugate of CI-921 hydrazide (IV) was the most resistant. 9-Methoxyellipticinium hydrazide VI11 was 50% released by 100 h of incubation while CI-921 hydrazide (IV) was only 25% released. Under hydrolytic and enzymatic cleavage conditions there was a biphasic loss of drug from the conjugates. For example, VI11 and IX (Figure 1) are first released with an initial half-life of ca. 1 day with a subsequent slower release half-life of ca. 15 days. Release of IV is slower and decidedly less biphasic. Similar biphasic scission rates are seen in the enzymatic cleavages (Figure 21, where the contrast between the more active enzymes in freshly drawn mouse sera and that allowed to stand for 48 h is evident. An explanation for these biphasic rates may rest in the original structure of PAD which, by virtue of the lack of selectivity in the periodate oxidation which generates it from dextran, can have any paired combination of hydroxyls a t carbons 2,3, or 4 converted to formyl functions. Furthermore, there is no reason to expect the condensation of the hydrazide to take place upon any one of these CHO’s specifically. It is therefore probable that hydrolytic and enzymic cleavage kinetics represent a complex function of loss from multiple sites of widely varying steric accessibility (23). It is interesting to note that this behavior is observed in the two hydrazide conjugates of the agents with extended pentamethylene side chains, Le., VI11 and IX, and virtually absent in the conjugate of IV. Clearly the hydrazide in IV at carbon 4 of an acridine, peri to a methyl at carbon 5, is a much more hindered function which may show greater selectivity in which formyl moiety on the PAD it initially attacks. This increased steric hindrance may be manifest in a slower and nearly monophasic cleavage of IV-PAD conjugates. CONCLUSIONS
Hydrazides derived from the antineoplastic pharmaceuticals ellipticine and CI-921 form stable, soluble conjugates with formyl functions on polyaldehyde dextran. One can regulate the degree of substitution of the pharmaceutical on the biopolymer by control of the reaction ratios in the conjugation medium. Similarly, the specific choice of pharmaceutical can alter the release kinetics in vitro and in vivo, with the more hindered acridinederived pharmaceutical showing the greater hydrolytic stability. Dextran conjugates of these agents and other closely related structures are under study, linked to tumorassociated monoclonal antibodies, for therapeutic drug targeting. ACKNOWLEDGMENT
The Quaker Chemical Foundation provided fellowships t o B.S. and H.Z. This work has been supported by
Bioconjugate Chem., Vol. 1, No. 1, 1990 81
grants from the W. W. Smith Charitable Trust and the Brady Cancer Research Fund. LITERATURE CITED (1) Ram, B. P. and Tyle, P. (1987) Immunoconjugates: applications in targeted drug delivery for cancer therapy. Pharm. Res. 4, 181-188. (2) Kanellos, J., Pietersz, G. A., Cunningham, Z., and McKenzie, I. F. (1987) Anti-tumor activity of aminopterin-monoclonal antibody conjugates. Immunol. Cell Biol. 65, 483493. (3) Borlinghaus, K. P., Fitzpatrick, D. A., Heindel, N. D., Mattis, J. A., Mease, B. A., Schray, K. J., Shealy, D. J., Walton, H. L., Jr., and Woo, D. V. (1987) Radiosensitizer conjugation to CA 19-9 monoclonal antibody. Cancer Res. 47, 40714075. (4) Bumol, T. F., Baker, A. L., Andrews, E. L., DeHerdt, S. V., Briggs, S. L., Spearman, M. E., and Apelgren, L. D. (1988) KSl/CDAVLB, A Monoclonal Antibody-Vinca Alkaloid Conjugate for Site-Directed Therapy of Epithelial Malignancies. Antibody-Mediated Delivery Systems (J. D. Rodwell, Ed.) pp 55-59, Dekker, New York. (5) Reisfeld, R. A. and Cheresh, D. A. (1985) Human tumorassociated antigens as targets for monoclonal antibody-mediated cancer therapy. Cancer Surv. 4, 271-280. (6) Heindel, N. D. and Van Dort, M. (1985) Prosthetic group radioiodination a t “no carrier added” level of carbonyl containing molecules. J . Org. Chem. 50, 1988-1990. (7) Wilchek, M. and Bayer, E. A. (1987) Labeling glycoconjugates with hydrazide reagents. Methods Enzymol. 138,429. (8) O’Shannessy, D. J. (1988) Hydrazides as specific reagents for the labeling of glycoproteins. ISBT Commun. 3, 4-6. (9) Bumol, T. F., Laguzza, B. C., DeHerdt, S. V., Andrews, E. L., Baker, A. L., Marder, P., Nichols, C. L., and Apelgren, L. D. (1987) Preclinical studies with DAVLB-hydrazide for sitedirected therapy of human melanoma. (1987) Second Znter-
national Conference on Monoclonal Antibody Immunoconjugates for Cancer (San Diego, CA) p 38, University of California-San Diego, San Diego, CA. (10) Trouet, A., Dejonghe, J. P., Collard, M. P., and Bhushana, R. K. (1987) Preparation of alkylvinblastines as antitumor agents. Eur. Pat. Appl. E P 233,101. [(1988) Chem. Abstr. 107, 237105dl. (11) Johnson, D. A. and Laguzza, B. C. (1987) Antitumor xenograft activity with a conjugate of a vinca derivative and the squamous carcinoma-reactive monoclonal antibody PF1/ D. Cancer Res. 47,3118-3122. (12) Heindel, N. D. and Van Dort, M. (1985) Indirect iodinations with oxadiazoles precursors of hydrazides. Org. Prep. Proc. Int. 17, 230-235. (13) Heindel, N. D., Zhao, H., Miller, M. R., Lautenslager, G. T., Nau, E. A., Burton, J. L., and Abel, J. H. (1988) Synthesis and conjugation of ellipticines for tumor cell targeting. Pharm. Res. 5 , S-63. (14) Takakura, Y., Takagi, A., Hashida, M., and Sezaki, H. (1987) Disposition and tumor localization of mitomycin Cdextran conjugates in mice. Pharm. Res. 4, 293-300. (15) Hurwitz, E., Kashi, R., Arnon, R., Wilchek, M., and Sela, M. (1985) The covalent linking of two nucleotide analogs to antibodies. J . Med. Chem. 28, 137-140. (16) Qureshi, S. Z. and Hasan, T. (1987) Detection and Spectrophotometric determination of anthranilohydrazide using periodic acid. Acta Pharm. Jugosl. 37, 223-225. (17) Sample was a gift from Parke-Davis Co., Ann Arbor, MI. (18) Kansal, V. K. and Potier, P. (1986) The biogenetic, synthetic and biochemical aspects of ellipticine. Tetrahedron 42,2389-2408. (19) Denny, W. A,, Atwell, G. J., and Baguley, B. C. (1984) Potential antitumor agents. 40. Orallv active 4.5-disbustituted derivatives of amsacrine. J . Med. Chem. 27, 363-367. (20) McNally, W., Whitfield, L, and Chang, T. (1987) Biodistribution of a radiolabeled amsacrine analog. Proc. Annu. Meet. Am. Assoc. Cancer Res. 28, 434. (21) Heindel, N. D., Emrich, J. G., Woo, D. V., Garnes, K., Landvatter, S. W., Wilson, A. A., and Burns, H. D. (1986)
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a2
Synthesesand melanoma avidities of 1-125derivativesof ellipticine and m-Amsa. J. Nucl. Med. 27, 1076. (22) Bernstein, A., Hurwitz, E., Maron, R., Arnon, R., Sela, M., and Wilchek, M. (1978) Higher antitumor efficacy of daunomycin linked to dextran. J. Natl. Cancer Znst. 60, 379-383. (23) A reviewer has suggested another plausible explanation for the biphasic cleavage rates. An Amadori rearrangement, occurring during the syntheses of the hydrazide conjugates, might generate ketone hydrazones or other rearranged product(s) with different hydrolysis kinetics.
Registry No. I, 124536-15-8;11,124536-16-9;111, 124536-17-
0; IV, 124536-18-1;V, 124536-19-2;VI, 124536-20-5;VII, 124536-
21-6; VIII, 124536-22-7;IX, 124536-23-8;tert-butyl carbazate, 870-46-2; N-(4-amino-3-methoxyphenyl)methanesulfonamide, 57165-06-7;5-methyl-9-oxoacridan-4-carboxylic acid, 24782-669; 9-methoxyellipticine,10371-86-5;ellipticine, 519-23-3;methyl 5-bromopentanoate,5454-83-1; hydrazine, 302-01-2;2,3-dialdehydodextran, 37317-99-0; IV dextran dialdehyde derivative, 124820-50-4;VI11 dextran dialdehyde derivative, 124820-49-1; IX dextran dialdehyde derivative, 124820-48-0.
Photo- Cross- Linking of Psoralen- Derivatized Oligonucleoside Methylphosphonates to Single-Stranded DNA Purshotam Bhan and Paul S. Miller’ Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, 615 N. Wolfe St., Baltimore, Maryland 21205. Received August 21, 1989
The preparation of oligodeoxyribonucleoside methylphosphonates derivatized with 3-[(2-aminoethyl)carbamoyl]psoralen [ (ae)CP] is described. These derivatized oligomers are capable of cross-linking with single-stranded DNA via formation of a photoadduct between the furan side of the psoralen ring and a thymidine of the target DNA when the oligomer-target duplex is irradiated with 365-nm light. The photoreactions of (ae)CP-derivatized methylphosphonate oligomers with single-stranded DNA targets in which the position of the psoralen-linking site is varied are characterized and compared to results obtained with oligomers derivatized with 4’-[[N-(aminoethyl)amino]methyl]-4,5’,8trimethylpsoralen [(ae)AMT]. It appears that the psoralen ring can stack on the terminal base pair formed between the oligomer and its target DNA or can intercalate between the last two base pairs of the oligomer-target duplex. Oligomers derivatized with (ae)CP cross-link efficiently to a thymidine located in the last base pair ( n position) or 3’to the last base pair ( n + 1 position) of the target, whereas the (ae)AMT-derivatized oligomers cross-link most efficiently to a thymidine located in the n + 1 position. The results show that both the extent and kinetics of cross-linking are influenced by the location of the psoralen-linking site in the oligomer-target duplex.
Psoralens, a class of naturally occurring photoreactive furocoumarins found in a variety of plants, were used as medicinal agents by the ancient Egyptians to treat the skin disorder vitiligo (1). In more recent times, these compounds have been used clinically in the treatment of a number of skin diseases including psoriasis ( I ) and cutaneous T-cell lymphoma (2). Psoralens are planar molecules which are able to intercalate into double-stranded regions of DNA and RNA. Upon irradiation with long-wavelength ultraviolet light, the 3,4 and 4‘,5‘ double bonds of the pyrone or furan ring, respectively, can undergo 2+2 cycloadditions with the 5,6 double bond of pyrimidine nucleosides to form cyclobutane type monoadducts (3). If the psoralen has intercalated into a suitable site, the furan-side monoadducts can undergo a further cycloaddition reaction to form a cross-link between the two strands of the doublestranded nucleic acid. Thus psoralens have been found to be useful in mapping the secondary structure of large RNA molecules ( 4 ) . Psoralens, such as 8-methoxypsoralen, show a preference for cross-linking with -TpA- sequences in doublestranded DNA ( 5 ) ;however, their ability to specifically recognize long, unique nucleic acid sequences is limited. When conjugated to oligonucleotides or oligonucleotide analogues, either by attachment through linker arms or
by monoadduct formation with thymine bases of the oligomer, the psoralens can be targeted to specific sites in the nucleic acid (6-10). We have used this approach to prepare oligonucleoside methylphosphonates which are capable of cross-linking to complementary sequences on single-stranded DNA and RNA in a sequence-specific manner (11-13). The oligomer is conjugated through its 5’phosphoryl group with 4’-[ [N-(aminoethy1)aminolmethyl]-4,5’,8-trimethylpsoralen[ (ae)AMT], l which, upon irradiation at 365 nm, forms a cyclobutane adduct between the pyrone ring of the (ae)AMT and a thymine or a cytosine base of the targeted nucleic acid. The oligonucleoside methylphosphonates themselves are nuclease-resistant oligonucleotide analogues which are Abbreviations used: (ae)AMT, 4‘- [ [N-(2-aminoethyl)amino]methyl]-4,5’,8-trimethylpsoralen;(ae)CP, 3-[(2-aminoeth-
yl)carbamoyl]psoralen;3-CP, 3-carboxypsoralen;CDI, l-ethyl-
3- [3-(dimethylamino)propyl]carbodiimide; DEAE, diethylami-
noethyl; EDA, ethylenediamine; EDTA, ethylenediaminetetraacetic acid; PAGE, polyacrylamide gel electrophoresis; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride; TLC, thin-layer chromatography; HPLC, highperformance liquid chromatography; TBE, 0.089 M Trisborate, 0.002 M EDTA (pH 8.0);TEAB, triethylammonium bicarbonate buffer, CPG, control pore glass.
1043-1802/90/2901 -0082$02.50/0 0 1990 American Chemical Society
