Enhanced Hydrolytic Stability and Water Solubility of an Aromatic

Chlorambucil, an aromatic nitrogen mustard, has been conjugated to putrescine- ...... Fan, J. Y., Gravatt, G. L., Grigor, B. A., and McLennan, D. J. (...
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Bioconjugate Chem. 2003, 14, 667−671

667

Enhanced Hydrolytic Stability and Water Solubility of an Aromatic Nitrogen Mustard by Conjugation with Molecular Umbrellas Saketh Vijayaraghavan,‡ Bingwen Jing,‡ Tracy Vrablik,‡ Ting-Chao Chou,† and Steven L. Regen*,‡ Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, and Laboratory of Preclinical Pharmacology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York, 10021. Received January 16, 2003

Chlorambucil, an aromatic nitrogen mustard, has been conjugated to putrescine- and spermidinebased scaffolds bearing one, two, and four persulfated cholic acid units. Those conjugates bearing two or four sterols show improved hydrolytic stability and water solubility relative to chlorambucil. A similar conjugate that contained only one sterol unit shows negligible improvement in hydrolytic stability but a significant increase in water solubility. Qualitatively, the hydrolytic stability within this series of conjugates parallels the shielding effects that have previously been found for related conjugates bearing a pendant, hydrophobic fluorescent probe. In vitro studies indicate that these conjugates possess modest to moderate activity against certain human lymphoblastic leukemia and human colon carcinoma cells.

INTRODUCTION

Scheme 1

Molecular umbrellas are a unique class of amphiphiles that are capable of shielding an attached agent from an incompatible environment. Such molecules are composed of a central scaffold that bears two or more facially amphiphilic units (1). When a hydrophobic agent is bound to a molecular umbrella, immersion in water favors a shielded conformation whereby intramolecular hydrophobic interactions are maximized (2). A stylized illustration of shielded and exposed conformers of a molecular umbrella that contains two facially amphiphilic units and a hydrophobic agent is shown in Scheme 1. Here, the shaded rectangle represents a hydrophobic face. In previous studies, we have shown that certain molecular umbrellas are effective in shielding an attached hydrophobic fluorescent probe, 5-(dimethylamino)1-naphthalenesulfonyl (Dansyl), from direct contact with an aqueous phase (2). On the basis of such findings, we hypothesized that molecular umbrellas should be capable of significantly altering the physical and chemical properties of attached hydrophobic agents. Specifically, we hypothesized that strong alkylating agents bound to molecular umbrellas should exhibit greater hydrolytic stability and water solubility. The primary aim of the work that is described herein was to test this hypothesis. For this purpose, chlorambucil (CMB) was chosen as a prototype due to its hydrolytic instability, its poor watersolubility, and its clinical importance as an anticancer agent (3). Kinetic studies of nucleophilic substitution reactions of Chlorambucil support a mechanism in which intramolecular displacement, leading to an aziridinium ion, is rate-limiting (4-7). Subsequent reaction with water or other nucleophiles (e.g., DNA) then yields hydrolyzed and alkylated products (8, 9). Because charge is developed in the transition state leading to the aziridinium ion, one * To whom correspondence should be addressed. E-mail: [email protected]. † Memorial Sloan-Kettering Cancer Center. ‡ Lehigh University.

would expect that its rate of formation should be faster in a polar microenvironment. Conversely, a slower rate would be expected in a more hydrophobic microenvironment. Thus, a shielded conformation of a molecular umbrella-chlorambucil conjugate would be expected to result in a reduced rate of hydrolysis. At the same time, by exposing the hydrophilic face of the amphiphilic units to the aqueous phase, one would expect to observe a significant increase in water solubility. The present work explores these possibilities by use of a family of suitably designed conjugates.

MATERIALS AND METHODS

General Methods. Unless stated otherwise, all reagents were obtained from commercial sources and used without further purification. House-deionized water was purified using a Millipore Milli-Q-filtering system containing one carbon and two ion-exchange stages. All 1H NMR spectra were recorded on a Bruker AMX 360 MHz or a Bruker Avance 500 MHz instrument; chemical shifts are reported in ppm relative to residual solvent. All UV spectra were recorded using a Carey 300 Bio UV-Visible spectrophotometer operating at ambient temperature. N-(4-Aminobutyl)choleamide (4). A mixture of 817 mg (2.0 mmol) of cholic acid, 253 mg (2.2 mmol) of

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668 Bioconjugate Chem., Vol. 14, No. 3, 2003

N-hydroxysuccinimide, and 454 mg (2.2 mmol) of N,N′dicyclohexylcarbodiimide (DCC) in 10 mL of THF was stirred at room temperature for 6 h. The insoluble urea that was formed was removed by filtration. To the filtrate was added a solution made from 377 mg (2.0 mmol) of N-Boc-1,4-diaminobutane (Fluka Chemical Company) and 2 mL of THF, followed by addition of 697 µL of N,Ndiisopropyl-N-ethylamine (DIPEA). After stirring overnight at room temperature, the mixture was concentrated under reduced pressure, and the residue was dissolved in chloroform. Subsequent washing with dilute aqueous hydrochloride and brine, concentrating under reduced pressure, and purifying the residue via column chromatography (silica gel, CHCl3/CH3OH, 10/1, v/v) afforded 851 mg (73%) of 4-N-Boc-butylcholeamide having Rf 0.41, and 1H NMR (CDCl3, 50 °C, 500 MHz) δ: 0.70 (s, 3 H), 0.90-2.25 (m, 43 H), 3.04 (m, 2 H), 3.16 (m, 2 H), 3.36 (m, 1 H), 3.78 (d, 1 H), 3.93 (d, 1 H). To an ice-cold solution of 626 mg (1.081 mmol) of 4-NBoc-butylcholeamide in 10 mL of methanol was added 4 mL of acetyl chloride over a 30 min period. The mixture was then allowed to stir at room temperature for 3 h. Subsequent purification by column chromatography (silica gel, CHCl3/CH3OH/NH4OH, 40/10/1, v/v/v) afforded 440 mg (85%) of 4 having Rf 0.22 and 1H NMR (CD3OD, 25 °C, 500 MHz) δ: 0.70 (s, 3 H), 0.90-2.25 (m, 34 H), 2.94 (t, 2 H), 3.20 (m, 2 H), 3.37 (m, 1 H), 3.79 (d, 1 H), 3.94 (s, 1 H). Conjugate 5. A mixture of 190 mg (0.625 mmol) of chlorambucil, 79 mg (0.686 mmol) of N-hydroxysuccinimide, and 132 mg (0.688 mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC‚HCl) in 4 mL of chloroform was stirred for 4 h at room temperature. To this solution was added 272 mg (0.568 mmol) of 4 followed by 198 µL of DIPEA. After stirring for an additional 12 h, the mixture was washed with aqueous hydrochloride and brine. The organic layer was then concentrated under reduced pressure, and the residue was purified by column chromatography [silica, using, initially, CHCl3/CH3CO2CH2CH3 (5/1, v/v) followed by CHCl3/CH3OH (9/1, v/v)] to give 283 mg (65%) of 5 having Rf 0.33 and 1H NMR (CD3OD, 25 °C, 500 MHz) δ: 0.65 (s, 3 H), 0.85-2.25 (m, 38 H), 2.49 (t, 2 H), 3.13 (m, 4 H), 3.34 (m, 1 H), 3.57 (t, 4 H), 3.67 (t, 4 H), 3.77 (d, 1 H), 3.90 (s, 1 H), 6.60 (dt, 2 H), 7.03 (dd, 2 H). HRMS for C42H68Cl2N3O5 (MH+): Calcd: 764.4536. Found: 764.4533. Conjugate 1. To a solution of 196 mg (0.256 mmol) of 5 in 5 mL of DMF at 0 °C was added 367 mg (2.31 mmol) of Pyr‚SO3. After stirring for 6 h at room temperature, the reaction mixture was cooled to 0 °C. The pH was then adjusted to ca. 9 via the addition of aqueous sodium bicarbonate, while maintaining the temperature of ca. 0 °C. Removal of solvent under reduced pressure (23 °C), followed by sequential column chromatographic purification of the residue (silica, CHCl3/CH3OH/H2O, 60/40/10, v/v/v) and preparative thin layer chromatograph (silica, CHCl3/CH3OH/H2O, 60/40/10, v/v/v), afforded 257 mg (93%) of 1 having Rf 0.44 and 1H NMR (CD3OD, 25 °C, 360 MHz) δ: 0.74 (s, 3 H), 0.80-2.50 (m, 40 H), 3.16 (s, 4 H), 3.61-3.72 (m, 8 H), 4.12 (bs, 1 H), 4.43 (s, 1 H), 4.64 (s, 1 H), 6.66 (d, 2 H), 7.04 (d, 2 H). Chlorambucil Benzotriazinone Activated Ester. To a solution of 393 mg (1.29 mmol) of chlorambucil and 232 mg (1.42 mmol) of 3-hydroxy-1,2,3-benzotriazin4(3H)-one in 5 mL of chloroform was added 227 mg (1.42 mmol) of DCC. After being stirred for 9 h at room temperature, the mixture was filtered and concentrated under reduced pressure. Recrystallization of the residue with ethanol afforded 448 mg (77%) of the activated ester

Vijayaraghavan et al.

of chlorambucil having 1H NMR (CDCl3, 25 °C, 360 MHz) δ: 2.10 (pent, 2 H), 2.72 (pent, 4 H), 3.61 (pent, 4 H), 3.69 (pent, 4 H), 6.65 (t, 2 H), 7.13 (t, 2 H), 7.83 (t, 1 H), 7.98 (q, 1 H), 8.20 (d, 1 H), 8.34 (q, 1 H). Conjugate 6. To a solution, made from 305 mg (0.33 mmol) of N1,N3-spermidine-bis[cholic acid amide], 92 µL of triethylamine, and 5 mL of DMF was added 178 mg (0.395 mmol) of chlorambucil benzotriazinone activated ester, and the mixture was allowed to stir overnight at room temperature. Subsequent removal of DMF under reduced pressure and purification of the residue by column chromatography (silica, CHCl3/CH3OH/H2O, 40/ 10/1, v/v/v) afforded 284 mg (77%) of 6 having Rf 0.65 and 1H NMR (CDCl3/CD3OD, 3/1, 45 °C, 500 MHz) δ: 0.60 (s, 6 H), 0.81-2.24 (m, 70 H), 2.49 (m, 2 H), 3.063.35 (m, 10 H), 3.54 (m, 4 H), 3.63 (m, 4 H), 3.73 (s, 2 H), 3.85 (s, 2 H), 6.58 (d, 2 H), 6.99 (d, 2H). HRMS for C69H112Cl2N4O9 (MNa+): Calcd: 1233.7699. Found: 1233.7738. Conjugate 2. To an ice-cold solution made from 253 mg (0.209 mmol) of 6 and 5 mL of DMF was added 598 mg (3.75 mmol) of Pyr‚SO3. The resulting mixture was stirred for 3 h at 0 °C and then for 15 h at room temperature. The mixture was subsequently cooled to 0 °C and 5 mL of cold water added to it. The pH was adjusted to ca. 9 by addition of a sodium bicarbonate solution, while maintaining a temperature of 0 °C. After removal of the solvents under reduced pressure, the residue was dissolved in ca. 10 mL of methanol, and the solids were removed by filtration. Concentration of the filtrate under reduced pressure afforded a residue that was purified by column chromatography (silica, CHCl3/ CH3OH/H2O, 60/40/10, v/v/v, Rf 0.26). Additional purification was made by reverse phase preparative thin-layer chromatography (C-18 silica, CH3OH/H2O, 1/1, v/v, Rf 0.37), followed by another regular phase preparative thinlayer chromatography (silica, CHCl3/CH3OH/H2O, 60/40/ 10, v/v/v, Rf 0.26) to give 256 mg (67%) of 2 having, and 1 H NMR (CD3OD, 25 °C, 500 MHz) δ: 0.74 (s, 6 H), 0.922.55 (m, 72 H), 3.10-3.34 (m, 8 H), 3.63-3.73 (m, 8 H), 4.12 (m, 2 H), 4.43 (s, 2 H), 4.64 (s, 2 H), 6.73 (d, 2 H), 7.05 (d, 2 H); 13C NMR (CD3OD, 25 °C) δ: 41.63 [N(CH2CH2Cl)2], 54.56 [N(CH2CH2Cl)2].3 Conjugate 8. To a solution made from 40 mg (0.13 mmol) of chlorambucil, 25 mg (0.15 mmol) of 3-hydroxy1,2,3-benzotriazin-4(3H)-one, and 2 mL of chloroform was added 30 mg (0.15 mmol) of N-(3-dimethylaminopropyl)N′-ethylcarbodiimide hydrochloride (EDC‚HCl). After the mixture was stirred for 4 h, 194 mg (0.10 mmol) of the tetrawalled molecular umbrella, 7, was added along with 46 µL (0.26 mmol) of DIPEA. Subsequently, ca. 1 mL of methanol was added, followed by stirring at room temperature for 12 h. Removal of solvent under reduced pressure, followed by column chromatography (silica, CHCl3/CH3OH/H2O, 35/10/1, v/v/v), afforded 182 mg (81%) of 8 having Rf 0.39 and 1H NMR (CD3OD, 25 °C, 500 MHz) δ: 0.69 (d, 12 H), 0.90-2.25 (m, 136 H), 2.52 (t, 2 H), 3.10-3.29 (m, 10 H), 3.31-3.39 (m, 10 H), 3.66 (q, 4 H), 3.71 (q, 4 H), 3.78 (s, 4 H), 3.93 (s, 4 H), 4.23 (brs, 2 H), 4.34 (m, 2 H), 6.68 (d, 2 H), 7.06 (d, 2 H). HRMS for C128H210Cl2N8O19 (MNa+): Calcd: 2256.4982. Found: 2256.5044. Conjugate 3. To an ice-cold solution made from 161 mg (0.072 mmol) of 8 and 4 mL of DMF was added 825 mg (5.18 mmol) of Pyr‚SO3. After the mixture was stirred overnight at room temperature, 2 mL of cold water was added to quench the reaction (maintaining the temperature of the mixture at ca, 0 °C), followed by addition of aqueous sodium bicarbonate to adjust the pH to ca. 9. The mixture was then concentrated under reduced pres-

Chlorambucil−Molecular Umbrella Conjugates

sure (at 23 °C) and the residue purified by preparative thin-layer chromatography (silica, CHCl3/CH3OH/H2O, 5/4/1, v/v/v, Rf 0.23), followed by preparative reverse phase thin-layer chromatography (C-18 silica, CH3OH/ H2O, 1/1, v/v Rf 0.47) and one final regular phase preparative thin-layer chromatography (silica, CHCl3/ CH3OH/H2O, 5/4/1, v/v/v, Rf 0.23) to give 135 mg (54%) of 3 having 1H NMR (CD3OD, 25 °C, 360 MHz) δ: 0.75 (d, 12 H), 0.90-2.50 (m, 138 H), 3.15 (m, 8 H), 3.35 (m, 8 H), 3.65-3.71 (m, 8 H), 4.12 (brs, 4 H), 4.22 (s, 2 H), 4.32 (s, 2 H), 4.43 (s, 4 H), 4.65 (s, 4 H), 6.65 (d, 2 H), 7.05 (d, 2 H). Water Solubility. Water solubilities were estimated at ambient temperature by adding ca. 1.0 mg of 1, 2, or 3 in a v-vial (microvial, Wheaton, NJ), and adding, incrementally, 1 µL volumes of water. The mixture was shaken by hand, and a maximum solubility was recorded by visual inspection; that is, the minimum volume of water needed to obtain a clear solution was noted. Hydrolysis of the Nitrogen Mustards. In a typical procedure, a vial was charged with 5.00 mL of a 0.1 M aqueous phosphate buffer (pH 7.4) and immersed in a temperature bath that was maintained at 37 °C. To this vial was added 50 µL of a 10 mM acetone solution of chlorambucil. Aliquots (300 µL) were then withdrawn as a function of time and added to separate vials, which contained 3 µL of a 0.5 M acetone solution of 4-(4nitrobenzyl)pyridine (NBP). These vials were vigorously shaken for 0.25 min and then incubated at 37 °C for 2 h. The contents of these vials were then transferred to centrifuge tubes, which was followed by the sequential addition of 100 µL of a 10 M aqueous NaOH solution and 600 µL of 1-octanol. The mixture was quickly shaken and centrifuged (total time of 1 min), and the UV absorbance (λmax 534 nm) of the 1-octanol layer was immediately recorded. A similar procedure was used in the case of 2, except that the conjugate was directly added to the aqueous phosphate solution (maintained at 37 °C) as a solid, such that its final concentration was 100 µM. Also, with 2, the volume of the 0.5 M NBP solution was increased to 6 µL and the time that was used for reaction with NBP increased to 3 h. It should be noted that due to the enhanced water solubility, UV absorption measurements (λmax 567 nm) were recorded using the aqueous phase, containing the violet-colored, alkylated product derived from 2. In the case of 1 and 3, 20 µL of 20 mM solutions [made using CH3OH and H2O, respectively] were added to the aqueous phosphate buffer. These stock solutions were prepared at room temperature and used immediately (within 3 min) to minimize decomposition; with 1 and 3, 9 µL of the 0.5 M acetone solution of NBP was used for a reaction time of 2 h. Similar to 2, the alkylated, violet-colored products in these cases were present in the aqueous phase. Biological Activity. Activity for inhibiting cell growth in vitro were conducted in human lymphoblastic leukemia cells (CCRF-CEM) and its subline that was 80-fold resistant to vinblastine (CCRF-CEM/VBL), and in human colon carcinoma cells HCT-116. For the solid tumor cells, HCT-116, growing in a monolayer, cytotoxicity of the drug was determined in 96-well microtiter plates by using the sulforhodamine B (SRB) method described by Skehan et al. (10). For leukemia CCRF-CEM and its subline growth in suspension, cytotoxicity was measured by XTT-microculture method (11). Dose-effect relationship data were analyzed with the median-effect plot by using a previously described computer program (12, 13).

Bioconjugate Chem., Vol. 14, No. 3, 2003 669 Scheme 2

RESULTS AND DISCUSSION

Chlorambucil-Molecular Umbrella Conjugates. Specific molecules that were chosen as targets for this study (1, 2, and 3) were conjugates derived from cholic acid, putrescine, spermidine, and chlorambucil. By analogy to related conjugates bearing a hydrophobic fluorescent probe, we expected that 2 would provide significant shielding of the nitrogen mustard, that 3 would provide even greater shielding, and that the shielding effects associated with 1 would be relatively modest. Thus, we predicted that the relative rates of hydrolysis would be chlorambucil ∼ 1 > 2 > 3. Because of the introduction of multiple sulfate groups, we further expected that the water solubility of 1, 2, and 3 would be substantially greater than that of chlorambucil.

The synthetic route that was used to prepare 1 is summarized in Scheme 2. In brief, condensation of cholic acid with N-Boc-1,4-diaminobutane, followed by deprotection, coupling to chlorambucil, and sulfation afforded the requisite conjugate. Similarly, conjugate 2 was

670 Bioconjugate Chem., Vol. 14, No. 3, 2003

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Scheme 3

Scheme 4

prepared by acylation of N1,N3-spermidine-bis[cholic acid amide] with chlorambucil, followed by sulfation (Scheme 3). An analogous sequence of reactions (Scheme 4) was used to prepare 3, starting from a tetrasterol conjugate (7), whose synthesis has previously been reported (14). Hydrolytic Stability. To judge the hydrolytic stabilities of chlorambucil, 1, 2, and 3, we used a simplified method in which reaction with 4-(4-nitrobenzyl)pyridine (NBP), and subsequent deprotonation affords intensely colored products (Scheme 5) (3). This method assumes that the reactivity of both chloroethyl groups within each nitrogen mustard are the same and also that the molar absorptivity of the alkylated NBP moieties in mono- and dialkylated products are the same (8). Experimentally, each nitrogen mustard was incubated in an aqueous phosphate buffer (pH 7.4) at 37 °C, and aliquots were withdrawn as a function of time. These aliquots were then immediately reacted with a 25-fold excess of NBP. Since UV absorbances should then be directly proportional to the concentration of unhydrolyzed chloroethyl groups, and since the rate disappearance of these groups is controlled by the formation of aziridinium ions, a firstorder decrease in the UV absorbance is expected. As shown in Figure 1, such kinetic behavior has, in fact, been found for chlorambucil as well as for each of the chlorambucil conjugates. From the slopes of these plots, the observed first-order rate constants, kobsd, were found to be 7.03 × 10-4, 6.90 × 10-4, 2.95 × 10-4, and 2.35 × 10-4 s-1, for chlorambucil, 1, 2 and 3, respectively, which

correspond to half-lives of 16.4, 16.7, 39.2, and 49.1 min, respectively. Additional evidence for enhanced hydrolytic stability was obtained by thin-layer chromatography. Specifically, an examination of an aqueous solution of chlorambucil by TLC (silica, CHCl3/CH3OH, 9/1,v/v) revealed the complete disappearance of the starting mustard (Rf 0.41) after ca. 50 min of incubation; with 2, complete loss of the starting material (silica, CHCl3/CH3OH/H2O, 30/20/5, v/v/v, Rf 0.39) required ca. 3 h. In the case of 1, the complete loss of the starting conjugate (silica, CHCl3/CH3OH/H2O, 30/20/5, v/v/v, Rf 0.31) was apparent after ca. 70 min. Thus, as predicted, the relative rates of hydrolysis for chlorambucil, 1, 2, and 3 parallel the shielding efficiencies found for analogous conjugates bearing a hydrophobic fluorescent group.

Scheme 5

Chlorambucil−Molecular Umbrella Conjugates

Bioconjugate Chem., Vol. 14, No. 3, 2003 671 CONCLUSIONS

The covalent attachment of chlorambucil to molecules bearing one, two, or four persulfated cholic acid moieties increases its water solubility by more than 3 orders of magnitude. When two or four sterol units are included in the conjugate, a significant increase in hydrolytic stability is also observed. If only one sterol unit is present, the hydrolytic stability of the conjugate is essentially the same as that found for chlorambucil. These results closely parallel the shielding efficiencies that have previously been found for analogous cholic acid conjugates bearing a hydrophobic fluorescent probe.

Figure 1. Plot of ln(abs) as a function of hydrolysis time for (9) chlorambucil, (b) 1, (2) 2, and (1) 3. See Materials and Methods for experimental details. Table 1. Potency for Inhibiting Cancer Cell Growth in Vitroa human lymphoblastic leukemia cells

human colon carcinoma

mustard agent

CCRF-CEM

CRF-CEM/VBL

HCT-116

CMBb 1 2 3

2.15 97.9 172.7 35.6

1.36 74.0 110.1 113.2

11.4 68.3 53.7 23.4

a IC 50 values represent the micromolar concentration needed to inhibit 50% growth. b Chlorambucil.

Water Solubility. The increase in the water solubility of chlorambucil, upon conjugation with one or more of these sulfated sterols, is dramatic. In particular, whereas chlorambucil was found to have a solubility in water that was less than 0.55 mg/mL, conjugates 1, 2, and 3 showed water solubilities corresponding to 560, 1210, and 1000 mg/mL. Thus, conjugation leads to increase in watersolubility by more than 3 orders of magnitude! In Vitro Anticancer Activity. In preliminary studies, we have examined the activity of chlorambucil, 1, 2, and 3 in inhibiting the growth of human lymphoblastic leukemia cells, as well as human colon carcinoma cells. Table 1 summarizes our principal findings. When CCRFCEM cells were used as targets, the conjugate that contained four sterol units (i.e., 3), showed moderate activity and was ca. 16 times less active than chlorambucil. Decreasing the number of sterol units to two led to a decrease in activity. Further removal of one sterol (i.e., 1) resulted in a modest increase in activity. While 1 also showed greater activity than 2 toward a subline that was 80-fold resistant to vinblastine (CCRF-CEM/VBL), the activity of 3 toward these target cells was similar to that of 2. Perhaps the most striking aspect of the data shown in Table 1 is that whereas chlorambucil was more effective against leukemia cells than the human colon carcinoma cells, the conjugates showed exactly the reverse behavior. Also, in contrast to their activity against the leukemia cells, the conjugates showed a clear trend against the colon carcinoma cells such that the activity increased with increasing numbers of sterol units. It is noteworthy that the activity of 3 compared favorably with that of chlorambucil toward the colon carcinoma cells. Although we do not presently understand these differences from a mechanistic point of view, it would appear, nonetheless, that at least one of these conjugates (i.e., 3) warrants in vivo testing.

In vitro studies have shown that conjugates 1, 2, and 3 exhibit modest to moderate anticancer activity against certain tumor cells. On the basis of an increase in water solubility by a more than 3 orders of magnitude, it is likely that the biodistribution of such conjugates, in vivo, would be very different from chlorambucil. Whether such differences would lead to an increase or decrease in the therapeutic efficacy, however, remains to be determined. In a broader context, the present findings significantly extend the molecular umbrella concept by showing how a molecular umbrella can enhance the stability and water solubility of a hydrolytically sensitive agent. ACKNOWLEDGMENT

We are grateful to the National Institutes of Health (PHS Grant GM51814) for support of this research. 1

H NMR spectra for 1, 2, and 3. This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Information Available:

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