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Poly(ethylene glycol) Prodrugs of the CDK Inhibitor, Alsterpaullone (NSC 705701): Synthesis and Pharmacokinetic Studies Richard B. Greenwald,* Hong Zhao, Jing Xia, Dechun Wu, Stephen Nervi, Sherman F. Stinson,*,† Eva Majerova,† Chris Bramhall,† and Daniel W. Zaharevitz† Enzon Pharmaceuticals Inc., Research & Development Division, 20 Kingsbridge Road, Piscataway, New Jersey 08854 and Developmental Therapeutics Program, NCI, Frederick, Maryland 21702. Received April 14, 2004; Revised Manuscript Received June 22, 2004
Two methods were devised to conjugate PEG to alsterpaullone (NSC 705701) via the N of the indole ring portion of the molecule. In the first approach, activation of the indole was accomplished by reaction with p-nitrophenyl chloroformate to produce a reactive carbamate that was then condensed with a mono blocked diamine to form a urea bond followed by deblocking and conjugation to PEG. The second route used the anion of the indole and produced a carbamate bond. Both compounds were highly water soluble, were stable in buffer, and released alsterpaullone in vitro and in vivo. Studies were conducted in mice to investigate the influence of PEGylation on the plasma pharmacokinetics of alsterpaullone. The total plasma clearance rate was decreased up to 32-fold, and the biological halflife lengthened up to 8-fold when alsterpaullone was injected i.v. as a PEG-conjugate and compared to injection of the unconjugated compound. The most pronounced effect on the pharmacokinetics of alsterpaullone was produced by a 40-kDa PEG urea-linked conjugate. When the 40- and 20-kDa urealinked conjugates were administered by i.p. injection, high relative bioavailability (46% and 99%, respectively) of alsterpaullone was observed.
INTRODUCTION
The cyclin-dependent kinases (CDKs) are a group of enzymes that are involved in cell cycle progression regulation (1). The CDKs activate host proteins through phosphorylation on serine or threonine using adenosine triphosphate as a phosphate donor (2). CDKs have attracted much attention as potential therapeutic targets, especially in treating cancer, because they are key players in the control of cell proliferation. Recently, a novel class of small molecule CDK/cyclin B inhibitors, the paullones, containing a four ring fused system were synthesized, and antitumor activity was demonstrated in vitro (3). Structure activity studies yielded 9-nitropaullone (alsterpaullone, 1) as one of the lead compounds in the series; however, due to its poor aqueous solubility (2 µg/mL), 1 could not be easily formulated, and this precluded in vivo testing. Since PEG prodrugs of molecular weight (mw) > 20 000 have been shown to solubilize and release small molecules in a predictable fashion (4), we sought to find means by which PEG could be used in a similar manner with 1, which has only indole and amide N-H functionalities available for attachment to PEG. A successful conjugation would thus solubilize alsterpaullone and permit intravenous (i.v.) or interperatoneal (i.p.) administration. To this end, we have developed two methods based on indole modification that yielded PEG prodrugs and produced usable levels of 1 in vivo as evidenced by pharmacokinetic (PK) studies. Chemistry. Synthetic approaches to indole functionalization of alsterpaullone were initiated by attempting * To whom correspondence should be addressed. Phone: (732) 980-4924. Fax: (732) 885-2950. E-mail: richard.greenwald@ enzon.com. Phone: (301)-846-1118. Fax: (301) 846-6910. E-mail:
[email protected]. † NCI.
reactions with 40 000 mw PEG-isocyanate (5) and with SC-PEG (PEG succinimidyl carbonate) (5). In no case was any reaction detected. We therefore turned to using a model compound, 2,3-dimethylindole (2), hoping to define reaction conditions that would lead to the desired modifications. Reaction was achieved in two different instances. First, condensation of formaldehyde (3) with 2 gave the hydroxymethyl derivative 4 in situ, in essentially quantitative yield (monitored by HPLC), which was then coupled to the Boc-protected succinimidyl carbonate, 5, producing the modified indole 6 (Scheme 1). Unfortunately, deprotection of 6 with TFA caused decomposition to the unmodified indole. Unexpectedly, no reaction with 4 was observed using the base labile Bsmoc (1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl) (6) protected amine 7, or activated PEG linkers such as N-hydroxyphthalimido PEG (BSC-PEG, 15). However, when using 1 as the substrate, it was found that the formaldehyde reaction would proceed to form 8 in only 50% yield (HPLC). Generally, 8 was not isolated but reacted directly with the bifunctional activated carbonate 5, to give the indole carbamate, 9, in about 15% yield (Scheme 2). Again, attempts to remove the Boc protecting group with TFA led to decomposition. Finally, using model compound 2 the activation of the NH bond by carbonyldiimidazole (CDI) and conversion to the activated derivative 11 could be accomplished in almost quantitative yield (Scheme 3). This compound was found to be stable in solution, but was unstable toward silica gel; therefore, it was used in the next step without purification. Reaction of crude CDI derivative 11 with the Boc-protected amine 12 produced urea 13. TFA deprotection followed by condensation with 15 yielded the PEG derivative of 2,3-dimethylindole, 16. A similar reaction using 1 gave the desired CDI derivative in only 20% yield. Clearly, the model results were not reflective
10.1021/bc049906a CCC: $27.50 © 2004 American Chemical Society Published on Web 08/14/2004
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Scheme 1
of those obtained for the more highly hindered alsterpaullone, and further use of 2 was abandoned in favor of alternative approaches to modification of 1. Using the more reactive reagent, p-nitrophenyl chloride (PNP-Cl) condensation with 1 in the presence of DMAP in THF solution yielded the activated carbamate 17 (Scheme 4). This was condensed directly with 12 to give the desired Boc indole urea intermediate 18. In this case deprotection was successfully accomplished with 33% TFA in DCM to give 19. PEGylation of 19 proceeded smoothly using 15 to give 20. Alternatively, 19 was conjugated with PEG acid (21) in the presence of EDC to yield 22. A second strategy that was successfully developed is shown in Scheme 5. Reaction of 1 with KOH in THF/DMF produced the potassium salt (not isolated), which was directly condensed with 23, yielding the Bocprotected carbamate, 24. Deprotection under acid conditions proceeded smoothly, to the amine salt (25) that was conjugated with 14 and 21 to give the indole carbamate PEG conjugated paullones 26 and 27 respectively. EXPERIMENTAL SECTION
Materials and Methods. Pharmacokinetics Studies. Male, young adult (average body weight approximately 27 g) CD2F1 mice (Harlan Sprague-Dawley, Frederick, MD) were used for all studies. The animals were maintained on hardwood chip bedding in temperaturecontrolled rooms (20 °C) with a 12 h light-dark cycle. Standard diet (Rat and Mouse 18% Protein Diet, PMI Nutrition International, Inc., Brentwood, MO) and water were provided ad libitum. Housing, animal care, and all experimental procedures and manipulations were carried out in strict compliance with the National Institutes of Health guidelines for the care and use of laboratory animals. Scheme 2
An effort was made to administer all agents at the highest dose possible. Alsterpaullone was given at its maximum tolerated dose (10 mg/kg). Viscosity, rather than toxicity, was the factor limiting the maximum dose of the 40-kDa conjugates; 1000 mg/kg was the greatest concentration resulting in a dosing solution that was workable. The dose of the 20-kDa conjugates (500 mg/ kg) was selected so that the alsterpaullone dose would be comparable to that for the 40 kDa studies. The plasma pharmacokinetics of alsterpaullone was investigated following a 0.5 min i.v. infusion at a dose of 10 mg/kg, dissolved in a vehicle of DMSO and administered in a dose volume of 1 mL/kg. The conjugates were studied following a short (0.5 min) i.v. infusion, or bolus i.p. injection (except NSC 723780, compound 26, and NSC 726143, compound 27, for which only an i.v. study was conducted). The 40-kDa conjugates were given at a dose of 1000 mg/kg dissolved in sterile water (100 mg/mL), in a dose volume of 10 mL/kg. The 20-kDa conjugates were injected at a dose of 500 mg/kg, using the same vehicle and concentration, but in a dose volume of 5 mL/kg. The equivalent dose of alsterpaullone in each case was approximately 14 mg/kg. At selected intervals through 24 h (6 h for the i.v. study with alsterpaullone) groups of three mice were lightly anesthetized with methoxyfluorane and were exsanguinated via the retro-orbital sinus. Blood was collected into heparinized microfuge tubes that were then chilled in an ice bath for 1 min, and centrifuged under refrigeration (4 °C) at 13 000 × g for 3 min. Plasma was separated and the concentrations of alsterpaullone were immediately assayed by HPLC. Aliquots (50 µL) of mouse plasma samples were prepared for analysis by precipitation of plasma proteins with 150 µL of chilled (-20 °C) methanol. Samples were vortexed vigorously for 30 s, maintained at -20 °C for
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Scheme 3
50 min, and centrifuged under refrigeration at 13 000 × g for 12 min. Next, 180 µL of the supernatant was combined with an equal volume of 0.05 M formate buffer (pH 3.0), and 330 µL of the mixture was injected on column. The analytical system consisted of a HewlettPackard (Palo Alto, CA) model 1050 pump, a refrigerated autosampler (4 °C), and a variable wavelength ultraviolet absorbance detector with associated software for system control and chromatogram and spectrum analysis. The system was equipped with a stainless steel 4.6 × 150 mm column containing 4 µm J’Sphere ODS H-80 packing (YMC, Inc., Wilmington, NC). Chromatography was performed with an isocratic flow rate of 1.0 mL/min Scheme 4
using a mobile phase of acetonitrile/0.05 M, pH 3.0 ammonium formate buffer (38:62, v/v). The column eluent was monitored at 300 nm. Under these conditions, conversion of the conjugates to alsterpaullone during sample preparation and assay was negligible (24 h >48 h >48 h
23 min 1.0 h 19.9 h 50 h
a
% active
solubility of conjugates (mg/mL)
solubility of alsterpaullone equivalent (mg/ mL)
1.44 2.80 1.44 2.88
153 189 172 186
2.20 5.30 2.48 5.36
Average mw was used for PEG. b All experiments were done in duplicate. Standard deviation of measurements ) ( 10%.
solved in DCM (300 mL), filtered, and finally washed with 0.25 N HCl (3 × 300 mL). The organic layer was dried (MgSO4) and filtered, and the solvent was removed under reduced pressure. The residue was further purified by column chromatography to yield 18 (1.18 g, 2.08 mmol, 40.6%). 13C NMR (75.5 MHz, CDCl3) δ 28.4, 29.7, 31.6, 40.1, 41.0, 68.9, 70.0, 70.3, 79.5, 113.9, 115.1, 116.9, 119.9, 121.7, 123.0, 124.4, 126.4, 129.1, 129.6, 133.9, 135.6, 140.9, 143.4, 150.8, 155.9, 172.9. HRMS: m/z calcd for C28H33N5O8 (M+ + Na) 590.2227, found 590.2213. Compound 19. To a mixture of trifluoroacetic acid (TFA, 2 mL) and anhydrous DCM (18 mL) was added compound 18 (1.0 g, 1.76 mmol) and the reaction mixture was stirred for 45 min at room temperature. The solvents were removed under reduced pressure and the crude product was washed with ethyl ether to give 19 (1.02 g, 1.76 mmol, 99%). 13C NMR (75.5 MHz, CDCl3/DMSO-d6) δ 30.4, 38.8, 40.0, 66.0, 68.3, 69.3, 69.5, 112.1, 114.4, 115.2, 118.4, 121.4, 122.3, 123.6, 125.8, 127.7, 128.6, 134.4, 135.0, 139.9, 142.5, 151.2, 172.4. HRMS: m/z calcd for C23H25N5O6 (free base M+ + Na) 490.1703, found 490.1708. Compound 20. To a solution of 19 (0.102 g, 0.176 mmol) in anhydrous dimethylformamide (DMF, 3 mL) and DCM (37 mL) was added 40 kDa PEG linker 15 (2.36 g, 0.059 mmol) and DMAP (0.022 g, 0.176 mmol). The reaction was stirred at room temperature for 12 h, the solution was concentrated under reduced pressure, and the PEG derivative was precipitated with ethyl ether (150 mL). The crude product was crystallized from IPA, (200 mL) to give 20 (2.0 g, 0.05 mmol, 87%). 13C NMR (75.5 MHz, CDCl3) δ 31.2, 40.2, 40.5, 63.5, 68.6, 69.0, 70.174.0 (PEG), 113.3, 114.6, 116.3, 119.2, 121.3, 122.9, 123.7, 126.0, 128.4, 128.9, 135.4, 140.3, 142.8, 150.5, 155.9, 171.6. Compound 22. To a solution of 19 (0.765 g, 1.64 mmol) in anhydrous DMF (20 mL) and DCM (90 mL) was added 20 kDa PEG diacid 21 (7.5 g, 0.37 mmol), 1-[3(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (EDC, 0.315 g 1.64 mmol), and DMAP (0.364 g, 2.98 mmol), and the mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure and the residue was crystallized from IPA (200 mL) to give 22 (7.2 g, 0.343 mmol, 93%). 13C NMR (75.5 MHz, CDCl3) δ 31.2, 38.0, 40.5, 68.5-74.0 (PEG), 113.3, 114.6, 116.2, 119.1, 121.2, 122.9, 123.6, 126.0, 128.4, 128.9, 133.8, 135.5, 140.3, 142.8, 150.4, 169.8, 171.6. Compound 23. To a solution of tert-butyl N-(3hydroxypropyl)-carbamate (5.0 g, 28.57 mmol), N,N′disuccinimidyl carbonate (DSC, 9.6 g, 37.5 mmol) in chloroform (125 mL) was added pyridine (3.0 mL, 37.0 mmol) and the reaction mixture was stirred at room temperature for 12 h. The mixture was washed with 0.5 N HCl (60 mL), dried (MgSO4) and filtered, and solvent was removed under reduced pressure to give compound 23 (8.2 g, 25.92 mmol, 90.7%). 13C NMR (75.5 MHz, CDCl3) δ 25.5, 28.4, 28.9, 36.8, 68.9, 79.4, 151.4, 155.8, 168.5. HRMS: m/z calcd for C13H30N2O7 (M+ + Na) 339.1168, found 339.1163.
Compound 24. Compound 23 (0.81 g, 2.6 mmol) was added to a reaction mixture of 1 (0.25 g, 0.85 mmol) and finely ground potassium hydroxide (KOH, 0.114 g, 2.03 mmol) in DMF/THF (20 mL/100 mL) previously stirred for 1 h at 0 °C in an ice-salt bath, and the resulting reaction mixture was gradually warmed to room temperature and stirred for 24 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL), filtered, and washed with 0.25 N HCl (2 × 200 mL). The organic layer was dried (MgSO4) and filtered, and the solvent was removed under reduced pressure. The residue was further purified by column chromatography to yield 24 (0.340 g, 0.706 mmol, 83%). 13 C NMR (75.5 MHz, CDCl3) δ 25.7, 28.4, 29.0, 31.4, 37.0, 65.5, 68.0, 76.6, 79.6, 114.8, 115.8, 119.4, 120.7, 122.8, 123.9, 128.0, 128.2, 129.9, 134.7, 141.1, 144.3, 150.8, 155.6, 172.7. HRMS: m/z calcd for C25H26N4O7 (M+ + Na) 517.1699, found 517.1708. Compound 25. To a solution of 24 (0.15 g, 0.30 mmol) in DCM (3 mL) cooled to 0 °C using an ice-salt bath was added TFA (3 mL) dropwise over 1 h with stirring. The solvents were removed under reduced pressure to give 25 (0.146 g, 0.295 mmol, ∼100%). HRMS: m/z calcd for C20H18N4O5 (free base M+ + H) 395.1355, found 395.1365. Compound 26. To a solution of 25 (0.15 g, 0.30 mmol) in anhydrous DMF/DCM (6 mL/9 mL) was added 40 kDa PEG linker 15 (2.98 g, 0.074 mmol) and DMAP (0.072 g, 0.60 mmol). The reaction mixture was stirred at room temperature for 12 h, diluted with DCM, and washed with 0.1 N HCl (2 × 20 mL) and brine (20 mL). Removal of the solvent under reduced pressure gave the crude product, which was crystallized from DMF/ethanol (45 mL/45 mL) to give 26 (2.5 g, 0.061 mmol, 83%). 13C NMR (75.5 MHz, CDCl3) δ 28.1, 31.4, 38.6, 63.7, 67.1, 69.4, 69.9, 70.1, 70.3-73.5 (PEG), 114.6, 115.4, 119.3, 120.4, 123.1, 123.2, 127.7, 129.0, 129.3, 135.3, 140.9, 144.0, 150.4, 155.6, 171.8. Compound 27. A solution of 25 (0.46 g, 0.92 mmol) and 20kDa PEG diacid 21 (4.6 g, 0.23 mmol) in anhydrous DMF (30 mL) and DCM (46 mL) was cooled to 0 °C. Next, EDC (0.177 g, 0.922 mmol) and DMAP (0.562 g, 4.6 mmol) were added all at once and the reaction mixture was stirred at room temperature for 12 h. The solution was washed by 0.1 N HCl (2 × 30 mL) and brine (30 mL), dried (MgSO4), and filtered. The solvent was removed under reduced pressure, and the residue was crystallized from IPA (100 mL) to give 27 (4.26 g, 0.205 mmol, 89%). 13C NMR (75.5 MHz, CDCl3) δ 28.1, 31.1, 34.9, 65.2, 68.5-73.5 (PEG), 114.4, 115.4, 118.9, 120.1, 122.7, 123.1, 127.5, 128.7, 129.4, 134.8, 135.3, 140.6, 143.8, 150.3, 169.6, 171.8. RESULTS
In Vitro Measurements. The solubility of all PEG derivatives was determined by the previously described (8) procedure. Rates of hydrolysis in saline and rat plasma of 16, 23, 26, and 27 were carried out as described earlier (5). These values are presented in Table 1.
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of the terminal phase (82% and 18% respectively) suggests that tissue distribution rather than elimination is a controlling factor in the in vivo disposition of alsterpaullone. Administration of PEGylated alsterpaullone by the i.v. route resulted in a 3 to 32-fold reduction in the CL of unconjugated alsterpaullone (Table 3, Figure 2). The urea linkage was associated with the largest reduction in the CL. The 40- and 20-kDa urea-linked conjugates produced CL values for alsterpaullone (0.3 and 0.7 L/hr/kg, respectively), which were three to four times lower than those found with corresponding carbamate-linked conjugates of equal molecular weight. The biological halflife of alsterpaullone was also three to four times longer with the urea-linked conjugates. The CL of alsterpaullone was one-third to one-half slower, and the biological halflife two to three times longer with the 40-kDa conjugates when compared to corresponding 20-kDa conjugates. The relative bioavailability of alsterpaullone when the urealinked conjugates were given by the i.p. route was 46% for the 40-kDa compounds and 99% for the 20-kDa compounds. Peak plasma concentrations of 4 and 7 µM were achieved 6 h after administration. Levels then declined with halflives of 9 and 3 h for the 40-kDa and 20-kDa conjugates, respectively. Asterpaullone was not detected in the plasma following i.p. injection of the 20-kDa carbamate-linked conjugate. An i.p. study was not conducted with the corresponding 40-kDa conjugate.
Figure 1. Geometric mean plasma concentrations and lines of best fit observed following i.v. administration of 1 to CD2F1 mice at a dose of 10 mg/kg. Table 2. Selected Pharmacokinetic Parameters Derived from Nonlinear Least Squares Regression Analysis of Alsterpaullone (1) Plasma Concentration vs. Time Data Obtained Following i.v. Injection of 10 Mg/kg Body Weight in CD2F1 Mice parameter
value
t1/2,1 t1/2,z MRT CL V1 Vz AUC0-∞ AUC1 AUCz
11.2 min 1.6 hr 0.6 hr 9.7 l/hr/kg 3.1 l/kg 6.2 l/kg 3.5 µM × hr 82% 18%
DISCUSSION
PEG has been extensively used due to its ability to increase the solubility, circulating life, safety, and permeable tissue accumulation level for small molecule prodrug platforms (9). The use of prodrug design has been applied to numerous therapeutic areas with the goal to improve the pharmacologic properties of drugs. A prodrug is a biologically inactive derivative of a parent drug molecule that usually requires an enzymatic or chemical transformation within the body in order to release the active drug, and has improved delivery properties over the parent molecule (10). Essential to drug delivery with a prodrug is the rate of release of the active drug. A rapid breakdown of the prodrug can result in spiking of the parent drug and possible toxicity, while too slow a release rate will compromise the drug’s effectiveness. The linkages between the active drug and the carrier molecule can theoretically be chosen so that either pH or enzymatic degradation can mediate prolonged drug release. Therefore, the effectiveness of prodrug delivery is dependent on the stability of the drug conjugate linkage and its potential for controlled degradation. PEG conjugated to
t1/2,1 and t1/2,z ) half-lives for the initial and terminal disposition phases, MRT ) mean residence time, CL ) total plasma clearance rate, V1 ) apparent volume of the central compartment, Vz ) apparent volume of distribution, AUC0-∞) area under the plasma concentration-time curve extrapolated to infinity, AUC1 and AUCz ) percentage of AUC0-∞ represented by the initial and terminal disposition phases.
Pharmacokinetics Studies. Nonlinear regression analysis indicated that plasma concentrations of alsterpaullone declined in a biexponential manner following i.v. administration of 10 mg/kg, from nearly 10 µM at 5 min to 20 nM by 6 h (Figure 1). The half-lives for the initial and terminal disposition phases were 11 and 94 min, respectively (Table 2). The total plasma clearance rate (CL) was 9.7 l/hr/kg. The high ratio of the proportion of the area under the plasma concentration-time curve (AUC0-∞) represented by the initial phase relative to that
Table 3. Selected Pharmacokinetic Data Derived from Alsterpaullone (1) Plasma Concentration Time Curves Obtained Following i.v. or i.p. Administration of PEG-conjugates in CD2F1 Mice molecular weight linker dose (mg/kg) alsterpaullone equivalent dose (mg/kg) i.v. studies AUC0-∞ (µM x hr) CL (l/hr/kg) t1/2,z (hr) i.p. studies AUC0-∞ (µM x hr) t1/2,z (hr) F (%)
NSC 723779 (20)
NSC 723780 (26)
NSC 726142 (22)
NSC 726143 (27)
40 998 urea 1000 14.3
40 842 carbamate 1000 14.3
21 012 urea 500 13.9
20 754 carbamate 500 14.1
149 0.3 12.9
54 0.9 4.8
73 0.7 6.6
17 2.9 1.8
69 9.2 46
nd -
72 3.1 99
bld -
AUC0-∞ ) area under the curve extrapolated to infinity, CL ) total plasma clearance rate, t1/2,z ) terminal disposition phase halflife, F ) relative bioavailable fraction, nd ) study not done, bld ) below the limit of detection.
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Figure 2. Geometric mean plasma concentrations of 1 and lines of best fit observed following i.v. (O) or i.p. (9) administration of PEG-conjugates to CD2F1 mice at doses equivalent to 14 mg/kg 1 (A, 20, B, 26; C, 22; D, 27).
amino prodrugs that function via a 1,4- or 1,6-elimination has been demonstrated to be a feasible methodology to deliver anticancer drugs (5). Another approach based on PEG technology also has now been proven to be useful for the delivery of insoluble indole-containing systems. PEG linkers were designed to target the indole N-H bond and resulted in a PEG-linked-drug bipartate system that provided alteration of plasma pharmacokinetics, while providing substantial aqueous solubility to the modified alsterpaullone. The synthetic program conducted for this initial investigation of PEG-alsterpaullone prodrugs provided two distinct types of PEG-alsterpaullone prodrugs: those formed with a carbamate linker and those with a urea connection. These PEG conjugates were highly soluble in saline, (>2.2 mg/mL), and both of these systems demonstrated prolonged stability (>24 h) in saline at 25 °C; thus, physically the conjugates have potential clinical utility as injectable agents. Alsterpaullone is a potent CDK inhibitor and produces cytotoxicity in sensitive human tumor cell lines exposed to 6 uM concentrations for brief periods (0.75 to 1.5 h) (11). An important objective of this investigation was to determine the feasibility of producing effective concentrations of alsterpaullone in vivo. When given to mice at its maximum tolerated dose, plasma concentrations of alsterpaullone were in the effective range for only 10 min. In contrast, when the 20- and 40-kDa urea conjugates were administered, alsterpaullone concentrations exceeded effective levels for 3 or 8 hours, respectively. Furthermore, administration of 1 as a PEG conjugate significantly reduced its plasma clearance rate and lengthened its biological halflife, thereby greatly increasing the in vivo residence time of 1, and enhancing its potential for producing a therapeutic effect. Thus, this investigation revealed that PEG conjugated to 1 via an indole results in prodrugs that demonstrate significant in vivo release of 1 (Tables 2 and 3).
In vivo investigations of these PEG prodrugs are currently in progress. The results will be reported at a later date. ACKNOWLEDGMENT
We would like to thank Drs. Laurent Meijer and Conrad Kunick for general discussions of the paullones, and Carol Aitken for her assistance in preparing this manuscript. LITERATURE CITED (1) Kunick, C., Schultz, C., Lemcke, T., Zaharevitz, D. W., Gussio, R., Jalluri, R. K., Sausville, E. A., Leost, M., and Meijer, L. (2000) 2-Substituted Paullones: CDK1/Cyclin B-Inhibiting Property and In Vitro Antiproliferative Activity. Bioorg. Med. Chem. Lett. 567-569. (2) Sielecki, T. M., Boylan, J. F., Benfield, P. A., and Trainor, G. L. (2000) Cyclin-Dependent Kinase Inhibitors: Useful Targets in Cell Cycle Regulation. J. Med. Chem. 42, 1-18. (3) Schultz, C., Link, A., Leost, M., Zaharevitz, D. W., Gussio, R., Sausville, E. A., Meijer, L., and Kunick, C. (1999) Paullones, a Series of Cyclin-Dependent Kinase Inhibitors: Synthesis, Evaluation of CDK1/Cyclin B Inhibition, and in vitro Antitumor Activity. J. Med. Chem. 42, 2909-2919. (4) Greenwald, R. B., Choe, Y. H., McGuire, J., and Conover, C. (2003) Effective Drug Delivery by PEGylated drug conjugates. Adv. Drug Delivery Rev. 55, 217-250. (5) Greenwald, R. B., Pendri, A., Conover, C. D., Zhao, H., Choe, Y. H., Martinez, A., Shum, K., and Guan, S. (1999) Drug Delivery Systems Employing 1, 4- or 1, 6-Elimination: Poly (ethylene glycol) Prodrugs of Amine-Containing Compounds. J. Med. Chem. 42, 3657-3667. (6) Carpino, L. A., Ismail, M., Truran, G. A., Mansour, E. M. E., Iguchi, S., Ionescu, D., El-Faham, A., Riemer, C., and Warass, R. (1999) The 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc) amino protecting group. J. Org. Chem. 64, 4324-4328. (7) Stinson, S. F., House, T., Bramhall, C., Saavedra, J. E., Keefer, L. K., and Nims, R. W. (2002) Plasma Pharmacoki-
Poly(ethylene glycol) Prodrugs of Alsterpaullone netics of a Liver-Selective Nitric Oxide-Donating Diazeniumdiolate in the Male C57BL/6 Mouse. Xenobiotica 3, 339-347. (8) Conover, C. D., Zhao, H., Longley, C., Shum, K. L., and Greenwald, R. B. (2003) The Utility of Poly (ethylene glycol) Conjugation to Create Prodrugs of Amphotericin B. Bioconjugate Chem. 14, 661-666. (9) Greenwald, R. B., Conover, C. D., and Choe, Y. H. (2001) Poly (ethylene glycol) Conjugated Drugs and Prodrugs: A Comprehensive Review. Crit. Rev. Ther. Drug 17, 101-161.
Bioconjugate Chem., Vol. 15, No. 5, 2004 1083 (10) Stella, V. J., Charman, W. N., and Naringrekar, V. H. (1985) Prodrugs, Do They Have Advantages in Clinical Practice? Drugs 29, 455-473. (11) Sausville, E. A., Johnson J., Alley M., Zaharevitz D., and Senderowicz A. M. (2000) Inhibition of CDKs as a therapeutic modality. Ann. N.Y. Acad. Sci. 910, 207-221.
BC049906A
