Peptidyl aminosteroids as potential new antiarrhythmic agents Michael Mokotoff,* Ming Zhao,V Richard J. Marshall,* Eileen Winslow,~ Lan K. Wang,* and Qing-Jiang Liaot *Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA; Whina Pharmaceutical University, Nanjing, Peoples Republic of China; and SOrganon Laboratories, Ltd., Newhouse, Lanarkshire, Scotland
The synthesis of peptidyl derivatives of the aminosteroid, amafalone (Am), is described. Six analogs were synthesized: the hydrochloride salts of G&-Am (2) Ala-Gly-Am (3), D-Ala-Gly-Am (4), Pro-Am (a), Pro-Pro-Am (7), and D-Ala-Pro-Am (8). The peptide bonds were formed by the polymeric reagent method using polymeric hydroxybenzotriazole as the activating polymer. Peptidyl aminosteroids 2,6, 7, and8, when administered to rats intravenously, had protective antiarrhythmic effects similar to those of amafalone. By the oral route, less marked protection, in comparison to amafalone, was observed with 6, while 7 and 8 were disappointingly inactive. (Steroids 55:39!9-404, 1990)
Keywordsz steroids; peptidyl aminosteroids; an&rhythmic peptides; aminosteroids
IIltrochrCtiOIl
Ventricular fibrillation, a major complication of acute myocardial infarction, is responsible for many of the sudden deaths occurring in the early prehospital phase.’ In spite of the several an&u-rhythmic drugs available to the clinician, there is still a need for an effective and safe long-term drug. For many cardiologists, lidocaine is still the drug of choice during the occurrence of acute life-threatening ventricular dysrhythm&. However, it has a short duration of action and must be administered intravenously, two factors that limit its use. Studies reported in 1979on a series of aminosteroids showed that these substances possessed interesting antiarrhythmic activity in animal models.2 From these series, one compound, Org 6001 (amafalone [Am], 3aamino-2~-hydroxy-5candrostan-17-one), was developed to the stage of clinical testing, but it did not become commercialjy available because it was deemed not to have sufficient oral bioavailability.’ In China, Fang and co-workers,3 in an attempt to develop aminosteroid derivatives with more desirable ant&rhythmic properties, prepared a number of amide Address reprint requests to Dr. Michael Mokotoff, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, PA 15261, USA. Received December 28, 1989; accepted March 31, 1990.
U9 1990 Butterworth-Heinemann
agents; polymeric hydroxybenzotriazole; amafalone;
and N-alkyl derivatives of amafalone. By the intravenous route, these compounds generally exhibited lower toxicity while still retaining ant&rhythmic activity. Because of our continuing interest in peptides as potential therapeutic agents, we began a study aimed at preparing peptidyl derivatives of amafalone as potential prodrugs .4 Other groups have reported the use of peptide prodrugs for the delivery of several anticancer agents’ and for the delivery of the antimalarial, primaquine.6 In the former case, the peptide prodrugs were designed to be cleaved by the protease plasmin, which is known to be present in higher levels in many tumors. In the primaquine study, it was hypothesized that the malarial parasite might also contain increased levels of plasmin, and the prodrugs were thus designed to take advantage of that possibility. It was our intention to modify the 3-amino function of amafalone by coupling it to amino acids and peptides, thereby protecting the primary amine and perhaps altering its catabolism. Not knowing exactly how peptidyl aminosteroids would be enzymatically processed, we chose amino acids (Ala, Gly) that would be expected to be readily hydrolyzed by aminopeptidases (e.g., EC 3.4.11.14, an aminopeptidase from human liver that preferentially cleaves Ala - ) as well as those that might be more resistant (Pro, D-Ala). It is known that aminopeptidases do not readily cleave D-amino acids6 or Pro (e.g., aminopeptidase EC 3.4.11.11) when situated at the N terminus. Steroids,
1990, vol. 55, September
399
Papers Experimental The symbols and abbreviations we use generally follow the IUPAC-IUB recommendations (1nt J Pept Protein Res 1984; 24:9-37). Melting points were determined on a Fisher-Johns apparatus and are uncorrected. Optical rotations were determined on a Perkin-Elmer Model 241 automatic polarimeter using 95% EtOH as a solvent, unless stated otherwise. Elemental analyses were performed by Galbraith Laboratories, Inc. (Knoxville, TN, USA). Thin-layer chromatography (TLC) was carried out using silica gel GF on aluminum (EM Science, Cherry Hill, NJ, USA). The dimethylformamide (DMF) used was purified by drying over KOH overnight and then distilling, in vacua, from ninhydrin. Diisopropylethylamine (DIEA) was obtained from Aldrich Co. and distilled from ninhydrin. The trifluoroacetic acid (TFA) used was redistilled. Methylene chloride (CH,Cl,) was distilled from anhydrous Na,CO, . Polymeric hydroxybenzotriazole (PHBT) was a gift from Dr. Abraham Patchomik (The Weizmann Institute of Science, Rehovot, Israel). Also, some PHBT was prepared in our laboratory from polystyrene (Amberlite XE-305) obtained from Rohm & Haas Co., according to published procedures.7 The esterification of PHBT by each protected amino acid was carried out as previously described.* Couplings using PHBT were done by shaking the polymers using a Tekmar VXR shaker. All filtrations and washings of the PHBT polymers were carried out in an apparatus similar to that described by Stern et a1.9 The apparatus for conducting the liquid HF cleavages was constructed as previously described.” The HF was dried prior to use by distillation into the first vessel, which contained CoF,, and then distillation from there into the reaction vessel. All evaporations were performed in vacua on a rotary evaporator. Organic solutions that had been previously extracted with aqueous solutions were dried with anhydrous Na$O, prior to evaporation. The Boc groups were routinely removed by stirring the peptide with neat TFA at room temperature for 10 minutes, after which the excess TFA was evaporated, the product solidified with anhydrous ether, then dried in vacua over P,O, and KOH pellets. The progress of all peptide coupling reactions was followed by TLC. Information regarding the Wistar rats used in the biologic assays, e.g., their size and diet, has been previously described.” 3wAmino-2fi-hydroxy&r-androstan-17-one (amafalone)2. Optically active epiandrosterone was converted to an&alone in five steps and an overall yield of approximately 35% (Liao and Zhao, personal communication): mp 194” to 196” C (from CH,OH-H,O) (Lit.,2 mp 190” to 192” C); [(r]25D + 103” (c 0.74, CHCl,) (lit.,2 [czI~~D+ 110” [c 0.81, CHCl,]). The hydrochloride salt was crystallized from EtOH: [(rJ20D + 102“ (c 0.92) (lit.,2 [(w]~~D+ 103” [c 0.88, CHC&]). Boc-Gly-an&alone (1) (general coupling procedure). Boc-Gly-PHBT (9.7 g, 10.2 mmol) was suspended in 400
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1990, vol. 55, September
DMF (40 ml) and allowed to swell while shaking for 15 minutes. A mixture of Am (1.10 g, 3.6 mmol) and Am HCl (1.83 g, 5.4 mmol) was mixed with DMF (30 ml) and DIEA (0.48 g, 3.7 mmol) and immediately added to the polymer. The flask was shaken at room temperature for 15 hours. The polymer was removed by filtration9 and washed with DMF (30 ml) by shaking for 15 minutes, followed by four washes with CH,Cl, (30 ml, 15 minutes). The combined filtrates were evaporated, the residue dissolved in CHCl, (70 ml), washed successively with cold 10% citric acid, cold 5% Na,CO, , and H,O, and dried. Evaporation of the solvent and crystallization of the residue from acetone-ether gave in three crops, 3.09 g (74%) of purified 1. Recrystallization gave the analytic sample: mp 192.5” to 193.5” C; MS 463 (MH+). Analysis calculated for C,,H,,N,O,: C, 67.50; H, 9.15; N, 6.06. Found: C, 67.61; H, 9.04; N, 6.08. Gly-amafalone hydrochloride (2). Boc-Gly-Am (1) (0.80 g, 1.73 mmol) was stirred with 3.6N HCl in dioxane (10 ml) at room temperature for 30 minutes. Evaporation of the solvent and crystallization of the residue from CH,OH/acetone gave hydrochloride 2 (0.66 g). Recrystallization afforded 0.52 g (75%) of pure 2: [(r]22D +91.5” (c 0.95); MS 363 (MH+). Analysis calculated for C,,H,,N,O,Cl: C, 63.22; H, 8.84; N, 7.02. Found: C, 62.83, H, 8.98; N, 7.17. D-Ala-Gly-amafalone hydrochloride (4). Boc-Gly-Am (1.00 g, 2.16 mmol) was converted to its TFA salt. Boc-D-Ala-PHBT (4.1 g, 2.8 mmol) was suspended in CH,CL, (15 ml) and shaken for 15 minutes. The TFA salt was dissolved in a solution of CH,Cl, (10 ml) and DIEA (0.50 ml, 2.9 mmol) and immediately added to the polymer. The mixture was shaken at room temperature for 6 hours, filtered, and worked up as above, except all extractions of the polymer were done with CH,Cl, . Evaporation of the dried solvent gave Boc-DAla-Gly-Am as an oil; MS 534 (MH+). The oil was stirred with 3.6 N HCl in dioxane (10 ml) at room temperature for 2.5 hours. Evaporation of the excess reagent and crystallization of the residue from CH,OH/ ether gave 0.82 g (81%) of hydrochloride 4. Recrystallization gave the analytic sample: [a]25D + 68.9” (c 0.92). Analysis calculated for C,H,N,O,Cl . H,O: C, 56.96; H, 8.76, N, 8.30. Found: C, 56.73; H, 8.31; N, 8.07. Ala-Gly-amafalone hydrochloride (3). Boc-Ala-PHBT (2.8 g, 2.9 mmol) was allowed to swell with CH,Cl,, as described above. The TFA salt, obtained from BocGly-AM (1 .OOg, 2.16 mmol), was dissolved in CH,Cl, and DIEA and allowed to proceed exactly as above. Evaporation of the dried solvent gave solid Boc-AlaGly-Am: MS 534 (MH+). This solid was deblocked with HCI in the same manner as above and, after crystallization from CH,OH/acetone, gave 0.83 g (82%) of hydrochloride 3. Recrystallization gave the analytic sample: [u!]~~D+ 82.3” (c 0.80). Analysis calculated for C24H40N304C1* H,O: C, 59.06; H, 8.67; N, 8.61. Found: C, 59.47; H, 8.94; N, 8.64.
Peptidyl aminosteroids: Mokotoff et al.
Bee-Pro-amafa&e (5). Using the procedure described for Boc-Gly-Am (l), Am HCl(2.70 g, 7.9 mmol), BocPro-PHBT (10.0 g, 8.5 mmol), and DIEA (1.16 g, 9.0 mmol) in DMF afforded an oil that was crystallized from CHCl,/ether to give, in three crops, 3.95 g (99%) of Boc-Pro-Am (5). Recrystallization gave 2.90 g (73%) of puritied 5: mp 215” to 216” C; MS 503 (MH+). Pro-amafaione hydroeIdoride (6). Using the same procedure described for hydrochloride 2, Boc-Pro-Am (5) (0.25 g, 0.50 mmol) was converted to its HCl salt. Crystallization of the residue from CH,OH/acetone gave 0.13 g of hydrochloride 6 (5% yield): [a]22D +61.3” (c 1.06); MS 403 (MH+). Analysis calculated for CuHrs N20,Cl: C, 65.66; H, 8.95; N, 6.38. Found: C, 65.26; H, 9.02; N, 6.21. Pro&me hydrochloride (7). Boc-Pro-Am (5) (0.82 g, 1.63 mmol) was converted to the corresponding TFA salt and reacted with Boc-Pro-PHBT (2.1 g, 1.8 mmol) as described above for the preparation of dipeptide 4. Evaporation of the dried solvent gave 1.56 g of Boc-Pro-Pro-Am as an oil: MS 600 (MH+). The oil was treated with 3.6 N HCl/dioxane, as previously described. The resulting product was crystallized from CHJOH/ether to give 0.76 g (87%) of hydrochloride 7: [a] D + 15.2” (c 1.07). Analysis calculated for C,&I, N,O&l: C, 64%; H, 8.65; N, 7.84. Found: C, 64.70; H, 8.69; N, 7.87. D-Ala-Pro-am&lone hydrochloride (8). Boc-Pro-Am (5) (1.18 g, 2.35 mmol) was converted to its TFA salt
and reacted with Boc-D-Ala-PHBT (5.2 g, 3.6 mmol) as described above for dipeptide 4. Work-up and evaporation of the dried solvent yielded 1.52 g of Boc-DAla-Pro-Am as an oil: MS 574 (MH+). Cleavage of the Boc group with 3.6 N HCl/dioxane, as described above, and crystallization of the residue from ethanoldioxane-acetone gave 1.04 g (87%) of hydrochloride 8: [a]23D + 63.7” (c 0.82). Analysis calculated for C2,Ha N,OJl: C, 63.57; H, 8.69; N, 8.24. Found: C, 62.97; H, 8.86; N, 7.91.
pentobarbital sodium anesthetized, arti8cially ventilated male Wistar rats, a fine silk suture then being placed under the main let? coronary artery. After a stabilization period (15 minutes), test drugs or vehicle (saline) were injected into a femoral vein, and changes in blood pressure and heart rate were continuously monitored. The coronary ligature was tightened 15 minutes after drug administration, and the electrocardiogram (ECG) was continuously monitored over the next 30-minute period. For the oral studies, drugs or vehicle were administered by gavage (volume, 0.1 ml/100 g body weight) before anesthesia or surgical preparation. Coronary ligation was instituted exactly 60 minutes after drug administration. Cardiovascular parameters before and after intravenous drug administration were compared using a paired t test. The incidences of ventricular fibrillation and mortalities were compared using a chi-square test. All other comparisons were made using Student’s unpaired t test. Results and Discussion
For several years, we have been preparing peptides by the use of novel polymeric reagents such as PHBT.EJ2J3 This method, which involves the activation of a protected amino acid via esterification to a specialized polymer such as PHBT, is considered to be a hybrid of both the solution and solid phase methods of peptide synthesis and combines some of the advantages inherent in each. In the present case, the amafalone amino group acts as the nucleophile, then becomes amidated with a protected amino acid on reaction with the polymeric reagent. Amafalone was prepared at the China Pharmaceutical University by Liao and Zhao (unpublished) using an improved procedure from that reported in the literature.2 Scheme 1 shows the route used in the synthesis of the Gly series of compounds. This route begins with
Mass spectrometry The DC1 mass spectra were obtained on a Finnigan-
Mat 4600 mass spectrometer with a Superincos data system. The CI reagent gas was isobutane. During the mass spectrometric analyses, the ion source was set at 110”C and the pressure was adjusted to about 0.3 torr. Electron energy was at 100 eV and filament emission current was at 0.3 mA. These source conditions generally yielded, for isobutane, an intensity ratio of m/z 57 to 43 of close to 10. Sample introduction was via a direct exposure probe. The power supply of the probe was set to deliver current to the probe tip at 50 mA/sec to effect rapid heating of the sample. The scan cycle time was 1 second. Biologic studies Methods used were similar to those previously described.” Briefly, a left thoracotomy was performed in
Schema 1
Boc-Gly-PHBT, which was prepared by esterifying Boc-Gly to PHBT via the condensing agent diisopropylcarbodiimide. The use of the latter reagent was a modification of the previously reported procedure.* Boc-Gly-PHBT was then allowed to react with amafalone, affording Boc-Gly-Am (1) in a 74% yield. GlyAm HCl (2) was readily prepared by cleavage of the protecting group with HCl/dioxane. The corresponding dipeptide derivatives were prepared by removal of the Boc group from peptidyl steroid 1 using TFA, followed by reaction with either Boc-Ala-PHBT or its correSteroids, 1990, vol. 55, September
401
Papers
Pro-Pro-Am
(1)
mc-Pro-Pro-Am
(Q)
D-Ala-Pro-Am mc-Ala-Pro-A”
D-Ala-Gly-Am
(3)
Bee-Ala-Gly-Am
Ala-ay-Am
(1)
tWJ
(M+Hl z&Q
A
Q
500
482
411
103
212
600
582
431
403
312
C
Q
474
156
130
403
186
574
556
431
401
286
434
116
391
363
318
116
531
516
391
363
318
246
131
116
391
363
348
146
Figure 1 lsobutane DCI mass spectral fragmentation the peptidyl amafalone derivatives.
H
x
544
526
518
500
478
160
ions indicative of the peptide skeleton are discernible in all cases. These fragment ions are more intense in the Boc derivatives. The key features of the DC1 mass spectra are shown in Figure 1. In almost all cases, the fragment ions A, B, C, D, and X involve double hydrogen transfers. Fragment ion C is absent in Pro-Pro-Am and D-AlaPro-Am because of the cyclic nature of Pro. The ions at m/z 289 and 306 verify the presence of the amafalone moiety, while the fragment ion D gives information on the peptidyl portion of these molecules. It should be noted that DC1 mass spectrometry, using isobutane as the reagent gas, is quite suitable for the characterization of peptidyl amafalones (and presumably other peptidyl aminosteroids); any metabolic modification of the aminosteroid portion can be readily detected by inspection of the fragment ions at m/z 289 and 306. Alternatively, information on the metabolic modification of the peptide portion can be obtained from fragment ions A, B, C, and D.
pattern of
sponding enantiomer, Boc-D-Ala-PHBT. The resulting dipeptides, Boc-Ala-Gly-Am and Boc-D-Ala-Gly-Am, were then converted to their HCl salts 3 and 4 in overall yields, for the three steps, of 82% and 81%, respectively . Similarly, amafalone was reacted with Boc-ProPHBT and gave Boc-Pro-Am (5) in a 73% yield
Biologic testing The effects of test compounds 2 through 4 and 6 through 8 on ventricular fibrillation, evoked by acute coronary artery ligation in pentobarbital-anesthetized rats, were determined using the methods of Kane and Winslow.” Carotid arterial pressure and a standard lead II ECG were recorded simultaneously. Heart rates were obtained from the ECG signals.
Intravenous
(Scheme 2). As above, Pro-Am HCl (6) was readily obtained from peptidyl steroid 5 via HCl cleavage of the protecting group. Removal of the Boc group from 5 with TFA and condensation with Boc-Pro-PHBT or Boc-D-Ala-PHBT followed by deblocking with HCl/ dioxane afforded Pro-Pro-Am HCl(7) and D-Ala-ProAm HCI (8), both in overall yields of 87%. To confirm the structures of the peptidyl aminosteroids described above, and because of their novelty, we subjected all of the dipeptide compounds to desorption chemical ionization (DCI) mass spectrometry. The isobutane DC1 mass spectra of these aminosteroids all exhibited M + H ions as their base peaks (Figure 1). It should be noted that the electron impact mass spectra of these compounds did not show any molecular ions. In the DC1 mass spectra, fragment
402
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administration
As has been shown in a previous study,’ amafalone (5 or 10 mg/kg) produces a dose-dependent reduction in both ventricular ectopic activity and the incidence of ventricular fibrillation, as seen in the 30-minute period after acute coronary ligation (Table 1). Similar protective effects were seen with Pro-Am (6), Pro-Pro-Am (7), and D-Ala-Pro-Am (8) (Table 1). While Gly-Am (2) also conferred some protection, both Ala-Gly-Am (3) and D-Ala-Gly-Am (4) were inactive at the doses tested. When comparing the biologic activities of these peptidyl amafalone compounds, it should be recognized that the actual amafalone content of peptides 2 through 4 and 6 through 8 is approximately 84%, 70%, 70%, 75%, 60%, and 65%, respectively. This difference in amalfalone content might account for the fact that when given intravenously, these analogs were slightly less potent than the parent an&alone, but the results shown in Table 1 suggest that several of the compounds were still active antiarrhythymic agents. Standardizing the doses of all analogs for amafalone content would not change these results. Amafalone and all peptide analogs tested produced slight and transient (
The synthesis of peptidyl derivatives of the aminosteroid, amafalone (Am), is described. Six analogs were synthesized: the hydrochloride salts of G&-Am (2) Ala-Gly-Am (3), D-Ala-Gly-Am (4), Pro-Am (a), Pro-Pro-Am (7), and D-Ala-Pro-Am (8). The peptide bonds were formed by the polymeric reagent method using polymeric hydroxybenzotriazole as the activating polymer. Peptidyl aminosteroids 2,6, 7, and8, when administered to rats intravenously, had protective antiarrhythmic effects similar to those of amafalone. By the oral route, less marked protection, in comparison to amafalone, was observed with 6, while 7 and 8 were disappointingly inactive. (Steroids 55:39!9-404, 1990)
Keywordsz steroids; peptidyl aminosteroids; an&rhythmic peptides; aminosteroids
IIltrochrCtiOIl
Ventricular fibrillation, a major complication of acute myocardial infarction, is responsible for many of the sudden deaths occurring in the early prehospital phase.’ In spite of the several an&u-rhythmic drugs available to the clinician, there is still a need for an effective and safe long-term drug. For many cardiologists, lidocaine is still the drug of choice during the occurrence of acute life-threatening ventricular dysrhythm&. However, it has a short duration of action and must be administered intravenously, two factors that limit its use. Studies reported in 1979on a series of aminosteroids showed that these substances possessed interesting antiarrhythmic activity in animal models.2 From these series, one compound, Org 6001 (amafalone [Am], 3aamino-2~-hydroxy-5candrostan-17-one), was developed to the stage of clinical testing, but it did not become commercialjy available because it was deemed not to have sufficient oral bioavailability.’ In China, Fang and co-workers,3 in an attempt to develop aminosteroid derivatives with more desirable ant&rhythmic properties, prepared a number of amide Address reprint requests to Dr. Michael Mokotoff, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, PA 15261, USA. Received December 28, 1989; accepted March 31, 1990.
U9 1990 Butterworth-Heinemann
agents; polymeric hydroxybenzotriazole; amafalone;
and N-alkyl derivatives of amafalone. By the intravenous route, these compounds generally exhibited lower toxicity while still retaining ant&rhythmic activity. Because of our continuing interest in peptides as potential therapeutic agents, we began a study aimed at preparing peptidyl derivatives of amafalone as potential prodrugs .4 Other groups have reported the use of peptide prodrugs for the delivery of several anticancer agents’ and for the delivery of the antimalarial, primaquine.6 In the former case, the peptide prodrugs were designed to be cleaved by the protease plasmin, which is known to be present in higher levels in many tumors. In the primaquine study, it was hypothesized that the malarial parasite might also contain increased levels of plasmin, and the prodrugs were thus designed to take advantage of that possibility. It was our intention to modify the 3-amino function of amafalone by coupling it to amino acids and peptides, thereby protecting the primary amine and perhaps altering its catabolism. Not knowing exactly how peptidyl aminosteroids would be enzymatically processed, we chose amino acids (Ala, Gly) that would be expected to be readily hydrolyzed by aminopeptidases (e.g., EC 3.4.11.14, an aminopeptidase from human liver that preferentially cleaves Ala - ) as well as those that might be more resistant (Pro, D-Ala). It is known that aminopeptidases do not readily cleave D-amino acids6 or Pro (e.g., aminopeptidase EC 3.4.11.11) when situated at the N terminus. Steroids,
1990, vol. 55, September
399
Papers Experimental The symbols and abbreviations we use generally follow the IUPAC-IUB recommendations (1nt J Pept Protein Res 1984; 24:9-37). Melting points were determined on a Fisher-Johns apparatus and are uncorrected. Optical rotations were determined on a Perkin-Elmer Model 241 automatic polarimeter using 95% EtOH as a solvent, unless stated otherwise. Elemental analyses were performed by Galbraith Laboratories, Inc. (Knoxville, TN, USA). Thin-layer chromatography (TLC) was carried out using silica gel GF on aluminum (EM Science, Cherry Hill, NJ, USA). The dimethylformamide (DMF) used was purified by drying over KOH overnight and then distilling, in vacua, from ninhydrin. Diisopropylethylamine (DIEA) was obtained from Aldrich Co. and distilled from ninhydrin. The trifluoroacetic acid (TFA) used was redistilled. Methylene chloride (CH,Cl,) was distilled from anhydrous Na,CO, . Polymeric hydroxybenzotriazole (PHBT) was a gift from Dr. Abraham Patchomik (The Weizmann Institute of Science, Rehovot, Israel). Also, some PHBT was prepared in our laboratory from polystyrene (Amberlite XE-305) obtained from Rohm & Haas Co., according to published procedures.7 The esterification of PHBT by each protected amino acid was carried out as previously described.* Couplings using PHBT were done by shaking the polymers using a Tekmar VXR shaker. All filtrations and washings of the PHBT polymers were carried out in an apparatus similar to that described by Stern et a1.9 The apparatus for conducting the liquid HF cleavages was constructed as previously described.” The HF was dried prior to use by distillation into the first vessel, which contained CoF,, and then distillation from there into the reaction vessel. All evaporations were performed in vacua on a rotary evaporator. Organic solutions that had been previously extracted with aqueous solutions were dried with anhydrous Na$O, prior to evaporation. The Boc groups were routinely removed by stirring the peptide with neat TFA at room temperature for 10 minutes, after which the excess TFA was evaporated, the product solidified with anhydrous ether, then dried in vacua over P,O, and KOH pellets. The progress of all peptide coupling reactions was followed by TLC. Information regarding the Wistar rats used in the biologic assays, e.g., their size and diet, has been previously described.” 3wAmino-2fi-hydroxy&r-androstan-17-one (amafalone)2. Optically active epiandrosterone was converted to an&alone in five steps and an overall yield of approximately 35% (Liao and Zhao, personal communication): mp 194” to 196” C (from CH,OH-H,O) (Lit.,2 mp 190” to 192” C); [(r]25D + 103” (c 0.74, CHCl,) (lit.,2 [czI~~D+ 110” [c 0.81, CHCl,]). The hydrochloride salt was crystallized from EtOH: [(rJ20D + 102“ (c 0.92) (lit.,2 [(w]~~D+ 103” [c 0.88, CHC&]). Boc-Gly-an&alone (1) (general coupling procedure). Boc-Gly-PHBT (9.7 g, 10.2 mmol) was suspended in 400
Steroids,
1990, vol. 55, September
DMF (40 ml) and allowed to swell while shaking for 15 minutes. A mixture of Am (1.10 g, 3.6 mmol) and Am HCl (1.83 g, 5.4 mmol) was mixed with DMF (30 ml) and DIEA (0.48 g, 3.7 mmol) and immediately added to the polymer. The flask was shaken at room temperature for 15 hours. The polymer was removed by filtration9 and washed with DMF (30 ml) by shaking for 15 minutes, followed by four washes with CH,Cl, (30 ml, 15 minutes). The combined filtrates were evaporated, the residue dissolved in CHCl, (70 ml), washed successively with cold 10% citric acid, cold 5% Na,CO, , and H,O, and dried. Evaporation of the solvent and crystallization of the residue from acetone-ether gave in three crops, 3.09 g (74%) of purified 1. Recrystallization gave the analytic sample: mp 192.5” to 193.5” C; MS 463 (MH+). Analysis calculated for C,,H,,N,O,: C, 67.50; H, 9.15; N, 6.06. Found: C, 67.61; H, 9.04; N, 6.08. Gly-amafalone hydrochloride (2). Boc-Gly-Am (1) (0.80 g, 1.73 mmol) was stirred with 3.6N HCl in dioxane (10 ml) at room temperature for 30 minutes. Evaporation of the solvent and crystallization of the residue from CH,OH/acetone gave hydrochloride 2 (0.66 g). Recrystallization afforded 0.52 g (75%) of pure 2: [(r]22D +91.5” (c 0.95); MS 363 (MH+). Analysis calculated for C,,H,,N,O,Cl: C, 63.22; H, 8.84; N, 7.02. Found: C, 62.83, H, 8.98; N, 7.17. D-Ala-Gly-amafalone hydrochloride (4). Boc-Gly-Am (1.00 g, 2.16 mmol) was converted to its TFA salt. Boc-D-Ala-PHBT (4.1 g, 2.8 mmol) was suspended in CH,CL, (15 ml) and shaken for 15 minutes. The TFA salt was dissolved in a solution of CH,Cl, (10 ml) and DIEA (0.50 ml, 2.9 mmol) and immediately added to the polymer. The mixture was shaken at room temperature for 6 hours, filtered, and worked up as above, except all extractions of the polymer were done with CH,Cl, . Evaporation of the dried solvent gave Boc-DAla-Gly-Am as an oil; MS 534 (MH+). The oil was stirred with 3.6 N HCl in dioxane (10 ml) at room temperature for 2.5 hours. Evaporation of the excess reagent and crystallization of the residue from CH,OH/ ether gave 0.82 g (81%) of hydrochloride 4. Recrystallization gave the analytic sample: [a]25D + 68.9” (c 0.92). Analysis calculated for C,H,N,O,Cl . H,O: C, 56.96; H, 8.76, N, 8.30. Found: C, 56.73; H, 8.31; N, 8.07. Ala-Gly-amafalone hydrochloride (3). Boc-Ala-PHBT (2.8 g, 2.9 mmol) was allowed to swell with CH,Cl,, as described above. The TFA salt, obtained from BocGly-AM (1 .OOg, 2.16 mmol), was dissolved in CH,Cl, and DIEA and allowed to proceed exactly as above. Evaporation of the dried solvent gave solid Boc-AlaGly-Am: MS 534 (MH+). This solid was deblocked with HCI in the same manner as above and, after crystallization from CH,OH/acetone, gave 0.83 g (82%) of hydrochloride 3. Recrystallization gave the analytic sample: [u!]~~D+ 82.3” (c 0.80). Analysis calculated for C24H40N304C1* H,O: C, 59.06; H, 8.67; N, 8.61. Found: C, 59.47; H, 8.94; N, 8.64.
Peptidyl aminosteroids: Mokotoff et al.
Bee-Pro-amafa&e (5). Using the procedure described for Boc-Gly-Am (l), Am HCl(2.70 g, 7.9 mmol), BocPro-PHBT (10.0 g, 8.5 mmol), and DIEA (1.16 g, 9.0 mmol) in DMF afforded an oil that was crystallized from CHCl,/ether to give, in three crops, 3.95 g (99%) of Boc-Pro-Am (5). Recrystallization gave 2.90 g (73%) of puritied 5: mp 215” to 216” C; MS 503 (MH+). Pro-amafaione hydroeIdoride (6). Using the same procedure described for hydrochloride 2, Boc-Pro-Am (5) (0.25 g, 0.50 mmol) was converted to its HCl salt. Crystallization of the residue from CH,OH/acetone gave 0.13 g of hydrochloride 6 (5% yield): [a]22D +61.3” (c 1.06); MS 403 (MH+). Analysis calculated for CuHrs N20,Cl: C, 65.66; H, 8.95; N, 6.38. Found: C, 65.26; H, 9.02; N, 6.21. Pro&me hydrochloride (7). Boc-Pro-Am (5) (0.82 g, 1.63 mmol) was converted to the corresponding TFA salt and reacted with Boc-Pro-PHBT (2.1 g, 1.8 mmol) as described above for the preparation of dipeptide 4. Evaporation of the dried solvent gave 1.56 g of Boc-Pro-Pro-Am as an oil: MS 600 (MH+). The oil was treated with 3.6 N HCl/dioxane, as previously described. The resulting product was crystallized from CHJOH/ether to give 0.76 g (87%) of hydrochloride 7: [a] D + 15.2” (c 1.07). Analysis calculated for C,&I, N,O&l: C, 64%; H, 8.65; N, 7.84. Found: C, 64.70; H, 8.69; N, 7.87. D-Ala-Pro-am&lone hydrochloride (8). Boc-Pro-Am (5) (1.18 g, 2.35 mmol) was converted to its TFA salt
and reacted with Boc-D-Ala-PHBT (5.2 g, 3.6 mmol) as described above for dipeptide 4. Work-up and evaporation of the dried solvent yielded 1.52 g of Boc-DAla-Pro-Am as an oil: MS 574 (MH+). Cleavage of the Boc group with 3.6 N HCl/dioxane, as described above, and crystallization of the residue from ethanoldioxane-acetone gave 1.04 g (87%) of hydrochloride 8: [a]23D + 63.7” (c 0.82). Analysis calculated for C2,Ha N,OJl: C, 63.57; H, 8.69; N, 8.24. Found: C, 62.97; H, 8.86; N, 7.91.
pentobarbital sodium anesthetized, arti8cially ventilated male Wistar rats, a fine silk suture then being placed under the main let? coronary artery. After a stabilization period (15 minutes), test drugs or vehicle (saline) were injected into a femoral vein, and changes in blood pressure and heart rate were continuously monitored. The coronary ligature was tightened 15 minutes after drug administration, and the electrocardiogram (ECG) was continuously monitored over the next 30-minute period. For the oral studies, drugs or vehicle were administered by gavage (volume, 0.1 ml/100 g body weight) before anesthesia or surgical preparation. Coronary ligation was instituted exactly 60 minutes after drug administration. Cardiovascular parameters before and after intravenous drug administration were compared using a paired t test. The incidences of ventricular fibrillation and mortalities were compared using a chi-square test. All other comparisons were made using Student’s unpaired t test. Results and Discussion
For several years, we have been preparing peptides by the use of novel polymeric reagents such as PHBT.EJ2J3 This method, which involves the activation of a protected amino acid via esterification to a specialized polymer such as PHBT, is considered to be a hybrid of both the solution and solid phase methods of peptide synthesis and combines some of the advantages inherent in each. In the present case, the amafalone amino group acts as the nucleophile, then becomes amidated with a protected amino acid on reaction with the polymeric reagent. Amafalone was prepared at the China Pharmaceutical University by Liao and Zhao (unpublished) using an improved procedure from that reported in the literature.2 Scheme 1 shows the route used in the synthesis of the Gly series of compounds. This route begins with
Mass spectrometry The DC1 mass spectra were obtained on a Finnigan-
Mat 4600 mass spectrometer with a Superincos data system. The CI reagent gas was isobutane. During the mass spectrometric analyses, the ion source was set at 110”C and the pressure was adjusted to about 0.3 torr. Electron energy was at 100 eV and filament emission current was at 0.3 mA. These source conditions generally yielded, for isobutane, an intensity ratio of m/z 57 to 43 of close to 10. Sample introduction was via a direct exposure probe. The power supply of the probe was set to deliver current to the probe tip at 50 mA/sec to effect rapid heating of the sample. The scan cycle time was 1 second. Biologic studies Methods used were similar to those previously described.” Briefly, a left thoracotomy was performed in
Schema 1
Boc-Gly-PHBT, which was prepared by esterifying Boc-Gly to PHBT via the condensing agent diisopropylcarbodiimide. The use of the latter reagent was a modification of the previously reported procedure.* Boc-Gly-PHBT was then allowed to react with amafalone, affording Boc-Gly-Am (1) in a 74% yield. GlyAm HCl (2) was readily prepared by cleavage of the protecting group with HCl/dioxane. The corresponding dipeptide derivatives were prepared by removal of the Boc group from peptidyl steroid 1 using TFA, followed by reaction with either Boc-Ala-PHBT or its correSteroids, 1990, vol. 55, September
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Papers
Pro-Pro-Am
(1)
mc-Pro-Pro-Am
(Q)
D-Ala-Pro-Am mc-Ala-Pro-A”
D-Ala-Gly-Am
(3)
Bee-Ala-Gly-Am
Ala-ay-Am
(1)
tWJ
(M+Hl z&Q
A
Q
500
482
411
103
212
600
582
431
403
312
C
Q
474
156
130
403
186
574
556
431
401
286
434
116
391
363
318
116
531
516
391
363
318
246
131
116
391
363
348
146
Figure 1 lsobutane DCI mass spectral fragmentation the peptidyl amafalone derivatives.
H
x
544
526
518
500
478
160
ions indicative of the peptide skeleton are discernible in all cases. These fragment ions are more intense in the Boc derivatives. The key features of the DC1 mass spectra are shown in Figure 1. In almost all cases, the fragment ions A, B, C, D, and X involve double hydrogen transfers. Fragment ion C is absent in Pro-Pro-Am and D-AlaPro-Am because of the cyclic nature of Pro. The ions at m/z 289 and 306 verify the presence of the amafalone moiety, while the fragment ion D gives information on the peptidyl portion of these molecules. It should be noted that DC1 mass spectrometry, using isobutane as the reagent gas, is quite suitable for the characterization of peptidyl amafalones (and presumably other peptidyl aminosteroids); any metabolic modification of the aminosteroid portion can be readily detected by inspection of the fragment ions at m/z 289 and 306. Alternatively, information on the metabolic modification of the peptide portion can be obtained from fragment ions A, B, C, and D.
pattern of
sponding enantiomer, Boc-D-Ala-PHBT. The resulting dipeptides, Boc-Ala-Gly-Am and Boc-D-Ala-Gly-Am, were then converted to their HCl salts 3 and 4 in overall yields, for the three steps, of 82% and 81%, respectively . Similarly, amafalone was reacted with Boc-ProPHBT and gave Boc-Pro-Am (5) in a 73% yield
Biologic testing The effects of test compounds 2 through 4 and 6 through 8 on ventricular fibrillation, evoked by acute coronary artery ligation in pentobarbital-anesthetized rats, were determined using the methods of Kane and Winslow.” Carotid arterial pressure and a standard lead II ECG were recorded simultaneously. Heart rates were obtained from the ECG signals.
Intravenous
(Scheme 2). As above, Pro-Am HCl (6) was readily obtained from peptidyl steroid 5 via HCl cleavage of the protecting group. Removal of the Boc group from 5 with TFA and condensation with Boc-Pro-PHBT or Boc-D-Ala-PHBT followed by deblocking with HCl/ dioxane afforded Pro-Pro-Am HCl(7) and D-Ala-ProAm HCI (8), both in overall yields of 87%. To confirm the structures of the peptidyl aminosteroids described above, and because of their novelty, we subjected all of the dipeptide compounds to desorption chemical ionization (DCI) mass spectrometry. The isobutane DC1 mass spectra of these aminosteroids all exhibited M + H ions as their base peaks (Figure 1). It should be noted that the electron impact mass spectra of these compounds did not show any molecular ions. In the DC1 mass spectra, fragment
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1990, vol. 55, September
administration
As has been shown in a previous study,’ amafalone (5 or 10 mg/kg) produces a dose-dependent reduction in both ventricular ectopic activity and the incidence of ventricular fibrillation, as seen in the 30-minute period after acute coronary ligation (Table 1). Similar protective effects were seen with Pro-Am (6), Pro-Pro-Am (7), and D-Ala-Pro-Am (8) (Table 1). While Gly-Am (2) also conferred some protection, both Ala-Gly-Am (3) and D-Ala-Gly-Am (4) were inactive at the doses tested. When comparing the biologic activities of these peptidyl amafalone compounds, it should be recognized that the actual amafalone content of peptides 2 through 4 and 6 through 8 is approximately 84%, 70%, 70%, 75%, 60%, and 65%, respectively. This difference in amalfalone content might account for the fact that when given intravenously, these analogs were slightly less potent than the parent an&alone, but the results shown in Table 1 suggest that several of the compounds were still active antiarrhythymic agents. Standardizing the doses of all analogs for amafalone content would not change these results. Amafalone and all peptide analogs tested produced slight and transient (
