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Lipidic Peptides. Ill: Lipidic Amino Acid and Oligomer Conjugates of Morphine
Ama December 1991 Volume 80, Number 12
JOURNAL OF PHARMACEUTICAL SCIENCES A publication of the American Pharmaceutical Association
Lipidic Peptides. 111: Lipidic Amino Acid and Oligomer Conjugates of Morphine R. A. HUGHES*, I. TOTH*,P. WARD5, s. J. IRELANDs, AND W. A. GIBBONS" Received July 30, 1990, from the Department of Pharmaceutical Chemistry, School of Pharmacy, University of London, 29-39 Brunswick Accepted for Square, London WClN 7A%, U.K., and SGIaxo Group Research Lfd., Greenford Road, Greenford, Middlesex UB6 OH€,U.K. publication February 13, 1991. Abstract 0 A series of lipidic morphine esters lb-lf with enhanced membrane-likecharacter were synthesized by coupling the lipidic amino acids 2 e 2 e to the phenolic hydroxyl group of the opioid analgesic morphine (la). The antinocioceptive activity of the esters l b l f was
determined in vivo following both iv and oral dosing. After iv administration, four of the conjugates, l b , l c , i d , and i f , exhibited antinocioceptive activity in the mouse abdominal constriction test, with a potency similar to that of the parent compound l a . Conjugate 1b showed activity following oral administration.
The a-amino acids with long hydrocarbon side chains, the so-called lipidic amino acids and their homo-oligomers the lipidic peptides, represent a class of compounds which combine structural features of lipids with those of amino acids.1 Several uses of lipidic amino acids and peptides have been proposed.2 Of particular interest is their potential use as a drug delivery system.3 The lipidic amino acids and peptides could be covalently conjugated to, or incorporated into, poorly absorbed peptides and drugs to enhance the passage of the pharmacologically active compounds across biological membranes and promote metabolic stability. Because of their bifunctional nature, the lipidic amino acids and peptides have the capacity to be chemically conjugated to drugs with a wide variety of functional groups. The linkage between drug and lipidic unit may either be biologically stable (i.e., a new drug is formed) or possess biological or chemical instability (i.e., the conjugate is a prodrug). In either case, the resulting conjugates could possess a high degree of membrane-like character, sufficient to facilitate their passage across membranes. The long hydrocarbon side chains may also have the additional effect of protecting a labile parent drug from enzymatic attack and, hence, increase metabolic stability. In this paper, we investigate the ability of lipidic amino acids and peptides to improve the oral absorption of the opioid analgesic morphine (la). Despite the development of analgesics considerably more potent than la,4 it is still used extensively in the clinic for the control of severe pain. In this capacity, when l a is administered orally, only 30% of the oral dose reaches the systemic circulation.5 Thus, there is consid0022-3549/91/7200-7 103$02.50/0 0 1997, American Pharmaceutical Association
erable scope for improving the oral absorption properties of l a by conjugation to lipidic amino acids and peptides.
Results and Discussion Synthesis of Morphine Conjugates-Morphine (la) possesses two hydroxyl (one phenolic and one allylic) functions. These hydroxyl groups provide suitable sites for esterification with lipidic amino acids and peptides. A series of five lipidic peptide conjugates l b - l f were prepared by reacting the appropriate N-tert-butoxycarbonyl (BOC) lipidic amino acids and peptides 2a-2e' with l a . The crude reaction mixtures were purified by TLC on silica gel, furnishing conjugates l b - l f in adequate yields. The tartaric acid salt of conjugate I d was prepared by treatment of conjugate I d with one equivalent of tartaric acid. Because of the greater reactivity of the phenolic hydroxyl group relative to the allylic one, it was anticipated that esterification of la to the lipidic amino acids and peptides 2a-2e would result primarily in phenolic esters. This expec-
i
R --NH- H-C -0 3, 1EH,hq
'
1
n
la lb lc Id le It lg lh 11
7 7 11 11 17
-
-
m
R'
R2
H BOC BOC EOC BOC BOC
H
CH3CHzC0
H H H H H H
H
CH&H&O
CH&H,CO
CH3CHzC0
Journal of Pharmaceutical Sciences I 1103
Table CAntlnocloceptlve Actlvlty of Morphine and Conjugate8 following Intravenous and Oral Adminirtratlon
ED, (95% limits)
la l a tartrate lb lc Id I d tartrate lo 11
1.33(0.9&2.13) 0.71(0.45-1.17) 0.77(0.28-?.90) 1.15(0.!59-2.65) 1.93(1.49-2.60) 6.13(4.25-9.53) 5.60(3.6$10.66) 2.46(1. W . 3 5 )
4.39(2.97-7.03) 1.97(1.25-3.25) 1.39(0.W.42) 1.59(0.81-3.66) 3.16(2.434.25) 8.93(6.19-13.89) 6.70(4.34-12.75) 3.54(2.16-6.25)
4.94(2.71-8.79) 4.14(2.21-7.27) 6.07(3.41-11.07) >10 >lo >10 >lo
>10
16.30(8.94-29.00) 11.50(6.14-20.19) 10.93(6.14-19.94) 713.81' 716.37' 714.58' >11.96' 214.39'
Equivalent to 10 mg/kg. tation was confirmed by the 'H NMR spectrum of the major product of the reaction of BOC amino acid 2a to la. The proton at position 6' (the site of the allylic hydroxyl group) was found to resonate a t 4.18 ppm, consistent with its being aGacent to a free hydroxyl group. Acylation of the neighboring hydroxyl function would have shifted the resonance of C6.-Hdownfield, to around 5 ppm. In addition, there was evidence of geminal coupling of C,.-H to the allylic hydroxyl proton in the two-dimensional (COSY) NMR spectra. Similar NMR spectral evidence was obtained for the other conjugates lc-lf. In Vivo Assessment of Lipidic Amino Acid and Peptide Conjugates of M o r p h i n e T h e in vivo activity of the six morphine conjugates l b l f and I d tartrate was compared with that of the parent compounds la and morphine tartrate ( l a tartrate). The conjugates and parent compounds were tested for antinocioceptive activity in the mouse abdominal constriction test, following iv and oral administration. At an appropriate time interval after administration, the effect of each compound on the number of abdominal constrictions after an ip injection of acetylcholine was recorded. From these observations, an EDb0 value was calculated for each compound (i.e., the dose required to reduce the control response by 50%). The results are summarized in Table I. Following iv administration, conjugates l b , l c , Id, and If showed activity comparable with that of the parent compounds l a and la tartrate. Of these, the most active was conjugate lb, which had one short alkyl side chain (8 carbon atoms). Increasing the size of the lipidit moiety, either by increasing the alkyl .side chain length or by increasing the number of lipidic amino acid residues, reduced the activity. Thus, there appeared to be a greater preference for shorter chain, monomeric lipidic amino acid substitution. The remaining conjugates tested, Id tartrate and le, were less active than the parent compounds. Indeed, I d tartrate was mmolkg). active only a t the highest dose tested (8.93 x Only one Conjugate, lb, exhibited significant antinocioceptive activity after oral dosing [ED,,, of 10.93 (6.14-19.94) x mmolkgl. However, conjugate l b was no more active than l a tartrate [ED,, of 11.5 (6.14-20.19) x mmolkg]. Conjugates I d and If caused only a 37% reduction in acetylcholine-induced abdominal constrictions (results not shown). The remaining compounds I d tartrate, lc, and If were less active than the la tartrate. Coqjugate l b , the only compound to show higher activity than the parent l a following oral dosing, was also the only compound to be completely soluble in the water:DMSO solvent mixture used for oral and iv dosing. The remaining compounds were administered as fine suspensions. Thus, the lower activity of the compounds in the test following oral administration may be due to reduced aqueous solubility of the conjugates, a legacy of the long alkyl side chains of the lipidic amino acid substituents. 1104 I Journal of Pharmaceutical Sciences
Recently, studies of the antinociceptive activity of the propionyl esters of la, lg-li, were reported6 and it was proposed that the allylic ester l h was the pharmacologically active principle following sc administration. The phenolic ester I g and the dipropionyl derivative l i were quickly metabolized in the liver to l a and the allylic ester 3h, respectively. These results mirrored those obtained for the acetyl esters of la.7
Conclusions The lipidic morphine esters lb-lf were prepared with enhanced membrane-like character compared with the parent analgesic l a . However, the desired increase in lipophilicity was accompanied by reduced aqueous solubility, which may have served to limit the oral absorption of lb-lf. Future efforts to improve the absorption of otherwise poorly adsorbed drug conjugates should concentrate on maintaining adequate solubility in biological fluids.
Experimental Section Infrared spectra were recorded with a Perkin-Elmer 841 spectrophotometer.' Melting points are not given for diastereomeric mixtures. The 'H NMR spectra were obtained on Varian XL-300 and Bruker AM500 instruments operating at fields of 300 and 500 MHz, respectively; chemical shifts are reported in ppm downfield from internal TMS. Mass spectra were run on a VG Analytical ZAB-SE instrument, using fast atom bombardment (FAB)techniques. ReacP wing tion progress was monitored by TLC on Kieselgel ,F dich1oromethane:methanol(1O:l) as the mobile phase. Purification WBB achieved by TLC using Kieeelgel PFzM+sas (Merck) on 20 x 20-cm plates of thickness 1.5mm, or column or flash chromatography through Kieeelgel G (System,dichloromethanexnethanol,1O:l). Solvents were evaporated under reduced pressure with a rotary evaporator. Melting points are given uncorrected. Infrared spectra gave characteristic carbonyl and aromatic absorbances. Purity of the compounds was determined by TLC and HPLC. The HPLC separation was carried out on a Whatman Partisil 5 FtAC silica column. The HPLC-grade dichloromethane (Aldrich) and methanol (Rathburn) were filtered through a 25-pm membrane filter and deaerated with helium flow prior to use. Separation was achieved with a solvent gradient beginning with 0% methanol, increasing conetantly to 50% methanol at 15 min, and decreasing steadily to 0% methanol from 17 to 20 min a t a constant flow of 3 mL * min-'. The gradient was effected by two microprocessorsontrolled Gileon 302 single piston pumps. Compounds were detected with a Holochrome UV-VIS detector at 254 nm. Chromatographs were recorded with an LKB 2210 single channel chart recorder. The ED,, values were calculated using the Glaxo Group Research Ltd.Library VAX computer software POTPNEW. Method A-MorphinJ'-yl2-(tert-butoxycarbonylamino)icosanwte f1O-h a 50-mL round-bottomed flask equipped with magnetic stirrer, morphine monohydrate (90 mg, 0.293 mmol), l e (125 mg, 0.293 mmol), 1-hydroxybenzotriazole hydrate (39.6mg,0.293mmol). triethylamine (29.6 mg, 0.293 mrnol), and l-ethyl-3-(3-dimethylaminopropylkarbodiimide hydrochloride (112.3 g, 0.586 mmol) in
CHa I (CHn I n
I (CH3)aCOCO(-NH-CH-CO-)~OH 2
n 2a 2b 2c
2d 2e
7 7 11 11 17
m 1 2 1 2 1
dichloromethane (15 mL) were stirred a t room temperature for 5 h. The reaction mixture was washed with water, the organic layer was evaporated after drying (MgSO,), and the residue was purified by TLC: R, = 0.33; yield 122 mg (58%) gel-like solid; 'H NMR (CDC1,): 6 6.75, 6.65 (2H,s,aromatic H), 5.75 (lH,d,C,-H), 5.28 (lH,d,C,-H), 5.07 (lH,m,NH), 4.95 (lH,m,C,-H), 4.49 (lH,m,CH), 3.45 (1H,m,C9H), 3.08,2.4 (2H,m,C,,-H), 2.5 (3H,s,NCH,), 1.45 [SH,s,(CH,)&I, 1.3 (34H, m,17 CH,), and 0.85 (3H,t,CH3); MS, m/z (%): 696(35), 695(M+H)+(77), 639(35), 621(46), 595(10), 577(10), 285(86), 268(46), 226(14), 209(11), 181(17), 162(19), 83(11), 81(15), 74(11), 70(15), 41(44), and 29(19). AnaLCalc. for C,,H,,N,O, (694.9): C, 72.58; H, 9.57; N, 4.03. Found: C, 72.39; H, 9.31; N, 4.01. Morphin-3'-yl 2-(tert-butoxycarbonylamino)decanoate (1b)Compound 2a (412 mg, 1.44 mmol) was reacted with morphine monohydrate (435 mg, 1.44 mmol) as described above in method A; yield: 500 mg (63%)of a pale yellow oil; 'H NMR(CDC1,): 6 6.75,6.65 (2H,m,aromatic H), 5.75 (1H,d,C7-H), 5.28 (1H,d,C6-H), 4.91 (lH,m,C,-H), 4.80 (lH,m,OCONH), 4.18 (lH,m,C,-H), 4.05 (lH,m,aCH), 3.45 (lH,m,C,-H), 3.08, 2.40 (2H,m,C,,-H), 2.46 (3H,s,NCH3), 1.45 [9H,d,(CH,),C], 1.30 (22H,m,ll CH,), 0.85 (3H,t,CH3); MS, mlz (%I: 555 [M+H]+ (loo%), 499(15), 481(18), 285(53), 268(13), 226(12), 186(12), 142(33), 57(78), 44(22), 41(15). Anal.-Calc. for C32H46N206(554.7): C, 69.28; H, 8.36; N, 5.05. Found: C, 69.11; H, 8.23; N, 5.03. Morphin-J'-yl 2-12-(tert-butoxycarbonylamino~decanoylaminoldecanoate (IckCompound 2b (649 mg, 1.475 mmol) was reacted with morphine monohydrate (450 mg, 1.475 mmol) as described above in method A; yield: 690 mg (65%) of a pale yellow oil; 'H NMR(CDC1,): 66.75,6.65 (2H,m,aromaticH), 6.51 (lH,m,CONH), 5.75 (1H,d,C7-H), 5.28 (1H,d,C6-H), 5.05 (lH,m,OCONH), 4.91 (lH,m,C,-H), 4.73 (lH,m,-CH), 4.18 (lH,m,C,-H), 4.07 (lH,m,a-CH), 3.45 (lH,m,Cg-H), 3.08,2.40 (2H,m,C,,-H), 2.46 (3H,s,N-CH3), 1.45 [gH,t,(CHJ&I, 1.30 (28H,m,14 CH,), 0.85(3H,t,CH3); MS, m/z (%I: 742[M+Hl+(72), 555(13), 307(28), 289(19), 286(29), 285(29), 268(10), 226(10), 186(10), 142(100), 65(34), 58(11),57621, 44(18), 30(16), 29(15), 27(11). Anal.-Calc. for C4,H6,N307 (724.0): C, 69.67; H, 9.50; N, 5.80. Found: C, 69.49; H, 9.37; N, 5.78. (1d)Morphin-3'-yl 2-(tert-butozycarbonylamino~tetmdecanoate Compound 2c (492 mg, 1.44 mmol) was reacted with morphine monohydrate (435 mg, 1.44 mmol) as described above in method A; yield 500 mg (57%) of a pale yellow oil; 'H NMR(CDC1,): 6 6.75, 6.65 (2H,m,aromatic-H), 5.75 (1H,d,C7-H), 5.28 (lH,d,C,-H), 5.05 (lH,m,OCONH), 4.91 (1H,m,C,-H), 4.59 (lH,m,a-CH), 4.18 (lH,m,C,-H), 3.45 (lH,m,Cg-H), 3.08, 2.40 (2H,m,CIo-H), 2.46 (3H,s,N-CH3), 1.45 [9H,d,(CH3),CI, 1.30 (14H,m,7 CHd, 0.85 (3H,t,CH,); MS, m/z (%I: 611[M+Hl+ (28),555(10),537(10), 285(51), 268(18), 258(26), 242(13), 198(47), 74(11), 57(100), 43(15), 41(26), 29(14). Anal.-Calc. for C3,H66N206 (610.8): c, 70.78; H, 8.91; N, 4.59. Found: C, 70.93; H, 9.00, N, 4.62. Morph in -3' -y1 2-12-(tert -butoxycarbonylamino) tetmdecanoylami-
no]tetmdecanoate (1e)-Compound 2d (745mg, 1.311 mmol) was reacted with morphine monohydrate (400 mg, 1.311 mmol) as described above in method A; yield: 740 mg (68%) of a pale yellow oil; 'H NMR (CDCl,): 6 6.75, 6.65 (2H,m,aromatic H), 6.51 (1H,m, CONH), 5.75 (1H,d,C7-H), 5.28 (lH,d,CB-H),5.05 (lH,m,OCONH), 4.91 (1H,m,C6-H), 4.73 (lH,m,a-CH), 4.18 (1H,m,C6-H), 4.07 (lH,m,a-CH), 3.45 (1H,m,C9-H), 3.08, 2.40 (2H,m,CIo-H), 2.46(3H,s,NCH3), 1.45 [9H,d,(CH,),Cl, 1.30 (44H,m,22 x CH,), 0.85 (3H,t,CH,); MS, m/z (%): 836[M+Hl+ (20), 736(4), 285(18), 284(14), 268(14), 199(17), 198(100), 57(52), 56(12), 55(14), 44(11), 43(11), 41(18), 30(10). Anal.-Calc. for C,,H8,N307 (835.6): C, 71.85; H, 9.77; N, 5.03. Found: C, 71.97; H, 9.88; N, 5.04. In Vivo Assessment of Morphine Conjugates-Male mice (19-27 g, CFUH strain, Glaxo) were allocated randomly to treatment groups (six or eight mice per group) and housed in pairs in sawdust-lined plastic cages (12 x 13 x 30 cm). Each mouse received drug solution or vehicle either orally or iv. Then, 60 min after oral dosing or 30 min after iv dosing, each animal was given an ip injection of acetyl choline (3 mgkg) and a record was made of the number of abdominal constrictions occurring during the next 3.5 (oral dose group) or 4 min (iv dose group). Immediately after the end of the observation period, animals were killed using a recommended humane method. For oral administration (dose volume 20 mL/kg), drugs were dissolved in distilled water containing a minimal quantity of dimethyl sulfoxide (DMSO, up to 1% v/v). For iv administration (dose volume 10 mLikg), drugs were dissolved in sterile saline solution (0.9% w/v, Steriflex, Boots), containing, where necessary, a minimal quantity of DMSO (up to 10% v/v). Compounds lc, Id, I d tartrate, l e , and If were not fully soluble under the oral administration conditions and thus were administered as fine suspensions, supposing either that the conjugates gradually dissolved in the gastric juices or alternatively were taken up as micellar drugs. Acetylcholine iodide (Sigma) was dissolved in sterile saline. An ED,, value was calculated for each compound; that is, the dose required to reduce the control response by 50%. The effects of an opioid antagonist against antinociceptive effects of the compounds in the abdominal constriction test have not been studied, but it has been reported for morphine itself.8 Our attribution of the apparent antinociceptive activity of the analogues to an action at opioid receptors is therefore probable, but not definitive at this stage.
References and Notes 1. Gibbons, W. A.; Hughes, R. A.; Charalambous, M.; Christodoulou, M.; Szeto, A.; Aulabaugh, A.; Mascagni, P.; Toth, I. Liebigs Ann. Chem. 1990, 1175-1183. 2. Gibbons, W. A. U.K. Pat. Appl. GB 2217319 A, Int. Pat. Appl. PCT/AU89/00166. 3. Toth, I.; Hughes, R. A.; Munday, M. R.; Mascagni, P.; Gibbons, W. A. InPe tides, Chemistry, Structure and Biology; Rivier, J. E.; Marshall, R., Eds.; ESCOM: Leiden, 1990; pp 1078-1079. 4. Lenz, G. R.; Evans, S.M.; Walters, D. E.; Hopfinger, A. J . Opiates; Academic: London, 1986. 5. Stanski, D. R.; Greenblatt, D. J.; Lowenstein, E. Clin. Phurmacol. Ther. 1978,24, 52-59. 6. Whitehouse, L. W.; Paul, C. J.; Gottschling, K. H.; Lodge, B. A.; By, A. W. J. Phurm. Sci. 1990, 79,349-350. 7. Mav. E. L.: Jacobson. A. E. J. Phurm. Sci. 1977.66.285-286. 8. Birch, P. J:; Hayes, A: G.; Sheehan, M. J.; Tyers,M. B. J.Pharm. Phurmacol. 1988,40, 213-214.
6.
Acknowledgments The authors express their thanks to Dr. K. J. Welham for runnin the mass spectra, Mr. C. H. James for the NMR spectra, and Mr. Baldeo for the microanal ses. In vivo assessment of morphine co 'u ates was erformed gy Miss J. Stables, Mrs. S.M. Harrison, an! drs. R. L. 8argent. This work was sup orted by Glaxo Group Research La., U.K. (Grant No. ESAD056). Toth is on leave from Central Research Institute for Chemistry of the Hungarian Academy of Sciences.
d
f.
Journal of Pharmaceutical Sciences I 1105
JOURNAL OF PHARMACEUTICAL SCIENCES A publication of the American Pharmaceutical Association
Lipidic Peptides. 111: Lipidic Amino Acid and Oligomer Conjugates of Morphine R. A. HUGHES*, I. TOTH*,P. WARD5, s. J. IRELANDs, AND W. A. GIBBONS" Received July 30, 1990, from the Department of Pharmaceutical Chemistry, School of Pharmacy, University of London, 29-39 Brunswick Accepted for Square, London WClN 7A%, U.K., and SGIaxo Group Research Lfd., Greenford Road, Greenford, Middlesex UB6 OH€,U.K. publication February 13, 1991. Abstract 0 A series of lipidic morphine esters lb-lf with enhanced membrane-likecharacter were synthesized by coupling the lipidic amino acids 2 e 2 e to the phenolic hydroxyl group of the opioid analgesic morphine (la). The antinocioceptive activity of the esters l b l f was
determined in vivo following both iv and oral dosing. After iv administration, four of the conjugates, l b , l c , i d , and i f , exhibited antinocioceptive activity in the mouse abdominal constriction test, with a potency similar to that of the parent compound l a . Conjugate 1b showed activity following oral administration.
The a-amino acids with long hydrocarbon side chains, the so-called lipidic amino acids and their homo-oligomers the lipidic peptides, represent a class of compounds which combine structural features of lipids with those of amino acids.1 Several uses of lipidic amino acids and peptides have been proposed.2 Of particular interest is their potential use as a drug delivery system.3 The lipidic amino acids and peptides could be covalently conjugated to, or incorporated into, poorly absorbed peptides and drugs to enhance the passage of the pharmacologically active compounds across biological membranes and promote metabolic stability. Because of their bifunctional nature, the lipidic amino acids and peptides have the capacity to be chemically conjugated to drugs with a wide variety of functional groups. The linkage between drug and lipidic unit may either be biologically stable (i.e., a new drug is formed) or possess biological or chemical instability (i.e., the conjugate is a prodrug). In either case, the resulting conjugates could possess a high degree of membrane-like character, sufficient to facilitate their passage across membranes. The long hydrocarbon side chains may also have the additional effect of protecting a labile parent drug from enzymatic attack and, hence, increase metabolic stability. In this paper, we investigate the ability of lipidic amino acids and peptides to improve the oral absorption of the opioid analgesic morphine (la). Despite the development of analgesics considerably more potent than la,4 it is still used extensively in the clinic for the control of severe pain. In this capacity, when l a is administered orally, only 30% of the oral dose reaches the systemic circulation.5 Thus, there is consid0022-3549/91/7200-7 103$02.50/0 0 1997, American Pharmaceutical Association
erable scope for improving the oral absorption properties of l a by conjugation to lipidic amino acids and peptides.
Results and Discussion Synthesis of Morphine Conjugates-Morphine (la) possesses two hydroxyl (one phenolic and one allylic) functions. These hydroxyl groups provide suitable sites for esterification with lipidic amino acids and peptides. A series of five lipidic peptide conjugates l b - l f were prepared by reacting the appropriate N-tert-butoxycarbonyl (BOC) lipidic amino acids and peptides 2a-2e' with l a . The crude reaction mixtures were purified by TLC on silica gel, furnishing conjugates l b - l f in adequate yields. The tartaric acid salt of conjugate I d was prepared by treatment of conjugate I d with one equivalent of tartaric acid. Because of the greater reactivity of the phenolic hydroxyl group relative to the allylic one, it was anticipated that esterification of la to the lipidic amino acids and peptides 2a-2e would result primarily in phenolic esters. This expec-
i
R --NH- H-C -0 3, 1EH,hq
'
1
n
la lb lc Id le It lg lh 11
7 7 11 11 17
-
-
m
R'
R2
H BOC BOC EOC BOC BOC
H
CH3CHzC0
H H H H H H
H
CH&H&O
CH&H,CO
CH3CHzC0
Journal of Pharmaceutical Sciences I 1103
Table CAntlnocloceptlve Actlvlty of Morphine and Conjugate8 following Intravenous and Oral Adminirtratlon
ED, (95% limits)
la l a tartrate lb lc Id I d tartrate lo 11
1.33(0.9&2.13) 0.71(0.45-1.17) 0.77(0.28-?.90) 1.15(0.!59-2.65) 1.93(1.49-2.60) 6.13(4.25-9.53) 5.60(3.6$10.66) 2.46(1. W . 3 5 )
4.39(2.97-7.03) 1.97(1.25-3.25) 1.39(0.W.42) 1.59(0.81-3.66) 3.16(2.434.25) 8.93(6.19-13.89) 6.70(4.34-12.75) 3.54(2.16-6.25)
4.94(2.71-8.79) 4.14(2.21-7.27) 6.07(3.41-11.07) >10 >lo >10 >lo
>10
16.30(8.94-29.00) 11.50(6.14-20.19) 10.93(6.14-19.94) 713.81' 716.37' 714.58' >11.96' 214.39'
Equivalent to 10 mg/kg. tation was confirmed by the 'H NMR spectrum of the major product of the reaction of BOC amino acid 2a to la. The proton at position 6' (the site of the allylic hydroxyl group) was found to resonate a t 4.18 ppm, consistent with its being aGacent to a free hydroxyl group. Acylation of the neighboring hydroxyl function would have shifted the resonance of C6.-Hdownfield, to around 5 ppm. In addition, there was evidence of geminal coupling of C,.-H to the allylic hydroxyl proton in the two-dimensional (COSY) NMR spectra. Similar NMR spectral evidence was obtained for the other conjugates lc-lf. In Vivo Assessment of Lipidic Amino Acid and Peptide Conjugates of M o r p h i n e T h e in vivo activity of the six morphine conjugates l b l f and I d tartrate was compared with that of the parent compounds la and morphine tartrate ( l a tartrate). The conjugates and parent compounds were tested for antinocioceptive activity in the mouse abdominal constriction test, following iv and oral administration. At an appropriate time interval after administration, the effect of each compound on the number of abdominal constrictions after an ip injection of acetylcholine was recorded. From these observations, an EDb0 value was calculated for each compound (i.e., the dose required to reduce the control response by 50%). The results are summarized in Table I. Following iv administration, conjugates l b , l c , Id, and If showed activity comparable with that of the parent compounds l a and la tartrate. Of these, the most active was conjugate lb, which had one short alkyl side chain (8 carbon atoms). Increasing the size of the lipidit moiety, either by increasing the alkyl .side chain length or by increasing the number of lipidic amino acid residues, reduced the activity. Thus, there appeared to be a greater preference for shorter chain, monomeric lipidic amino acid substitution. The remaining conjugates tested, Id tartrate and le, were less active than the parent compounds. Indeed, I d tartrate was mmolkg). active only a t the highest dose tested (8.93 x Only one Conjugate, lb, exhibited significant antinocioceptive activity after oral dosing [ED,,, of 10.93 (6.14-19.94) x mmolkgl. However, conjugate l b was no more active than l a tartrate [ED,, of 11.5 (6.14-20.19) x mmolkg]. Conjugates I d and If caused only a 37% reduction in acetylcholine-induced abdominal constrictions (results not shown). The remaining compounds I d tartrate, lc, and If were less active than the la tartrate. Coqjugate l b , the only compound to show higher activity than the parent l a following oral dosing, was also the only compound to be completely soluble in the water:DMSO solvent mixture used for oral and iv dosing. The remaining compounds were administered as fine suspensions. Thus, the lower activity of the compounds in the test following oral administration may be due to reduced aqueous solubility of the conjugates, a legacy of the long alkyl side chains of the lipidic amino acid substituents. 1104 I Journal of Pharmaceutical Sciences
Recently, studies of the antinociceptive activity of the propionyl esters of la, lg-li, were reported6 and it was proposed that the allylic ester l h was the pharmacologically active principle following sc administration. The phenolic ester I g and the dipropionyl derivative l i were quickly metabolized in the liver to l a and the allylic ester 3h, respectively. These results mirrored those obtained for the acetyl esters of la.7
Conclusions The lipidic morphine esters lb-lf were prepared with enhanced membrane-like character compared with the parent analgesic l a . However, the desired increase in lipophilicity was accompanied by reduced aqueous solubility, which may have served to limit the oral absorption of lb-lf. Future efforts to improve the absorption of otherwise poorly adsorbed drug conjugates should concentrate on maintaining adequate solubility in biological fluids.
Experimental Section Infrared spectra were recorded with a Perkin-Elmer 841 spectrophotometer.' Melting points are not given for diastereomeric mixtures. The 'H NMR spectra were obtained on Varian XL-300 and Bruker AM500 instruments operating at fields of 300 and 500 MHz, respectively; chemical shifts are reported in ppm downfield from internal TMS. Mass spectra were run on a VG Analytical ZAB-SE instrument, using fast atom bombardment (FAB)techniques. ReacP wing tion progress was monitored by TLC on Kieselgel ,F dich1oromethane:methanol(1O:l) as the mobile phase. Purification WBB achieved by TLC using Kieeelgel PFzM+sas (Merck) on 20 x 20-cm plates of thickness 1.5mm, or column or flash chromatography through Kieeelgel G (System,dichloromethanexnethanol,1O:l). Solvents were evaporated under reduced pressure with a rotary evaporator. Melting points are given uncorrected. Infrared spectra gave characteristic carbonyl and aromatic absorbances. Purity of the compounds was determined by TLC and HPLC. The HPLC separation was carried out on a Whatman Partisil 5 FtAC silica column. The HPLC-grade dichloromethane (Aldrich) and methanol (Rathburn) were filtered through a 25-pm membrane filter and deaerated with helium flow prior to use. Separation was achieved with a solvent gradient beginning with 0% methanol, increasing conetantly to 50% methanol at 15 min, and decreasing steadily to 0% methanol from 17 to 20 min a t a constant flow of 3 mL * min-'. The gradient was effected by two microprocessorsontrolled Gileon 302 single piston pumps. Compounds were detected with a Holochrome UV-VIS detector at 254 nm. Chromatographs were recorded with an LKB 2210 single channel chart recorder. The ED,, values were calculated using the Glaxo Group Research Ltd.Library VAX computer software POTPNEW. Method A-MorphinJ'-yl2-(tert-butoxycarbonylamino)icosanwte f1O-h a 50-mL round-bottomed flask equipped with magnetic stirrer, morphine monohydrate (90 mg, 0.293 mmol), l e (125 mg, 0.293 mmol), 1-hydroxybenzotriazole hydrate (39.6mg,0.293mmol). triethylamine (29.6 mg, 0.293 mrnol), and l-ethyl-3-(3-dimethylaminopropylkarbodiimide hydrochloride (112.3 g, 0.586 mmol) in
CHa I (CHn I n
I (CH3)aCOCO(-NH-CH-CO-)~OH 2
n 2a 2b 2c
2d 2e
7 7 11 11 17
m 1 2 1 2 1
dichloromethane (15 mL) were stirred a t room temperature for 5 h. The reaction mixture was washed with water, the organic layer was evaporated after drying (MgSO,), and the residue was purified by TLC: R, = 0.33; yield 122 mg (58%) gel-like solid; 'H NMR (CDC1,): 6 6.75, 6.65 (2H,s,aromatic H), 5.75 (lH,d,C,-H), 5.28 (lH,d,C,-H), 5.07 (lH,m,NH), 4.95 (lH,m,C,-H), 4.49 (lH,m,CH), 3.45 (1H,m,C9H), 3.08,2.4 (2H,m,C,,-H), 2.5 (3H,s,NCH,), 1.45 [SH,s,(CH,)&I, 1.3 (34H, m,17 CH,), and 0.85 (3H,t,CH3); MS, m/z (%): 696(35), 695(M+H)+(77), 639(35), 621(46), 595(10), 577(10), 285(86), 268(46), 226(14), 209(11), 181(17), 162(19), 83(11), 81(15), 74(11), 70(15), 41(44), and 29(19). AnaLCalc. for C,,H,,N,O, (694.9): C, 72.58; H, 9.57; N, 4.03. Found: C, 72.39; H, 9.31; N, 4.01. Morphin-3'-yl 2-(tert-butoxycarbonylamino)decanoate (1b)Compound 2a (412 mg, 1.44 mmol) was reacted with morphine monohydrate (435 mg, 1.44 mmol) as described above in method A; yield: 500 mg (63%)of a pale yellow oil; 'H NMR(CDC1,): 6 6.75,6.65 (2H,m,aromatic H), 5.75 (1H,d,C7-H), 5.28 (1H,d,C6-H), 4.91 (lH,m,C,-H), 4.80 (lH,m,OCONH), 4.18 (lH,m,C,-H), 4.05 (lH,m,aCH), 3.45 (lH,m,C,-H), 3.08, 2.40 (2H,m,C,,-H), 2.46 (3H,s,NCH3), 1.45 [9H,d,(CH,),C], 1.30 (22H,m,ll CH,), 0.85 (3H,t,CH3); MS, mlz (%I: 555 [M+H]+ (loo%), 499(15), 481(18), 285(53), 268(13), 226(12), 186(12), 142(33), 57(78), 44(22), 41(15). Anal.-Calc. for C32H46N206(554.7): C, 69.28; H, 8.36; N, 5.05. Found: C, 69.11; H, 8.23; N, 5.03. Morphin-J'-yl 2-12-(tert-butoxycarbonylamino~decanoylaminoldecanoate (IckCompound 2b (649 mg, 1.475 mmol) was reacted with morphine monohydrate (450 mg, 1.475 mmol) as described above in method A; yield: 690 mg (65%) of a pale yellow oil; 'H NMR(CDC1,): 66.75,6.65 (2H,m,aromaticH), 6.51 (lH,m,CONH), 5.75 (1H,d,C7-H), 5.28 (1H,d,C6-H), 5.05 (lH,m,OCONH), 4.91 (lH,m,C,-H), 4.73 (lH,m,-CH), 4.18 (lH,m,C,-H), 4.07 (lH,m,a-CH), 3.45 (lH,m,Cg-H), 3.08,2.40 (2H,m,C,,-H), 2.46 (3H,s,N-CH3), 1.45 [gH,t,(CHJ&I, 1.30 (28H,m,14 CH,), 0.85(3H,t,CH3); MS, m/z (%I: 742[M+Hl+(72), 555(13), 307(28), 289(19), 286(29), 285(29), 268(10), 226(10), 186(10), 142(100), 65(34), 58(11),57621, 44(18), 30(16), 29(15), 27(11). Anal.-Calc. for C4,H6,N307 (724.0): C, 69.67; H, 9.50; N, 5.80. Found: C, 69.49; H, 9.37; N, 5.78. (1d)Morphin-3'-yl 2-(tert-butozycarbonylamino~tetmdecanoate Compound 2c (492 mg, 1.44 mmol) was reacted with morphine monohydrate (435 mg, 1.44 mmol) as described above in method A; yield 500 mg (57%) of a pale yellow oil; 'H NMR(CDC1,): 6 6.75, 6.65 (2H,m,aromatic-H), 5.75 (1H,d,C7-H), 5.28 (lH,d,C,-H), 5.05 (lH,m,OCONH), 4.91 (1H,m,C,-H), 4.59 (lH,m,a-CH), 4.18 (lH,m,C,-H), 3.45 (lH,m,Cg-H), 3.08, 2.40 (2H,m,CIo-H), 2.46 (3H,s,N-CH3), 1.45 [9H,d,(CH3),CI, 1.30 (14H,m,7 CHd, 0.85 (3H,t,CH,); MS, m/z (%I: 611[M+Hl+ (28),555(10),537(10), 285(51), 268(18), 258(26), 242(13), 198(47), 74(11), 57(100), 43(15), 41(26), 29(14). Anal.-Calc. for C3,H66N206 (610.8): c, 70.78; H, 8.91; N, 4.59. Found: C, 70.93; H, 9.00, N, 4.62. Morph in -3' -y1 2-12-(tert -butoxycarbonylamino) tetmdecanoylami-
no]tetmdecanoate (1e)-Compound 2d (745mg, 1.311 mmol) was reacted with morphine monohydrate (400 mg, 1.311 mmol) as described above in method A; yield: 740 mg (68%) of a pale yellow oil; 'H NMR (CDCl,): 6 6.75, 6.65 (2H,m,aromatic H), 6.51 (1H,m, CONH), 5.75 (1H,d,C7-H), 5.28 (lH,d,CB-H),5.05 (lH,m,OCONH), 4.91 (1H,m,C6-H), 4.73 (lH,m,a-CH), 4.18 (1H,m,C6-H), 4.07 (lH,m,a-CH), 3.45 (1H,m,C9-H), 3.08, 2.40 (2H,m,CIo-H), 2.46(3H,s,NCH3), 1.45 [9H,d,(CH,),Cl, 1.30 (44H,m,22 x CH,), 0.85 (3H,t,CH,); MS, m/z (%): 836[M+Hl+ (20), 736(4), 285(18), 284(14), 268(14), 199(17), 198(100), 57(52), 56(12), 55(14), 44(11), 43(11), 41(18), 30(10). Anal.-Calc. for C,,H8,N307 (835.6): C, 71.85; H, 9.77; N, 5.03. Found: C, 71.97; H, 9.88; N, 5.04. In Vivo Assessment of Morphine Conjugates-Male mice (19-27 g, CFUH strain, Glaxo) were allocated randomly to treatment groups (six or eight mice per group) and housed in pairs in sawdust-lined plastic cages (12 x 13 x 30 cm). Each mouse received drug solution or vehicle either orally or iv. Then, 60 min after oral dosing or 30 min after iv dosing, each animal was given an ip injection of acetyl choline (3 mgkg) and a record was made of the number of abdominal constrictions occurring during the next 3.5 (oral dose group) or 4 min (iv dose group). Immediately after the end of the observation period, animals were killed using a recommended humane method. For oral administration (dose volume 20 mL/kg), drugs were dissolved in distilled water containing a minimal quantity of dimethyl sulfoxide (DMSO, up to 1% v/v). For iv administration (dose volume 10 mLikg), drugs were dissolved in sterile saline solution (0.9% w/v, Steriflex, Boots), containing, where necessary, a minimal quantity of DMSO (up to 10% v/v). Compounds lc, Id, I d tartrate, l e , and If were not fully soluble under the oral administration conditions and thus were administered as fine suspensions, supposing either that the conjugates gradually dissolved in the gastric juices or alternatively were taken up as micellar drugs. Acetylcholine iodide (Sigma) was dissolved in sterile saline. An ED,, value was calculated for each compound; that is, the dose required to reduce the control response by 50%. The effects of an opioid antagonist against antinociceptive effects of the compounds in the abdominal constriction test have not been studied, but it has been reported for morphine itself.8 Our attribution of the apparent antinociceptive activity of the analogues to an action at opioid receptors is therefore probable, but not definitive at this stage.
References and Notes 1. Gibbons, W. A.; Hughes, R. A.; Charalambous, M.; Christodoulou, M.; Szeto, A.; Aulabaugh, A.; Mascagni, P.; Toth, I. Liebigs Ann. Chem. 1990, 1175-1183. 2. Gibbons, W. A. U.K. Pat. Appl. GB 2217319 A, Int. Pat. Appl. PCT/AU89/00166. 3. Toth, I.; Hughes, R. A.; Munday, M. R.; Mascagni, P.; Gibbons, W. A. InPe tides, Chemistry, Structure and Biology; Rivier, J. E.; Marshall, R., Eds.; ESCOM: Leiden, 1990; pp 1078-1079. 4. Lenz, G. R.; Evans, S.M.; Walters, D. E.; Hopfinger, A. J . Opiates; Academic: London, 1986. 5. Stanski, D. R.; Greenblatt, D. J.; Lowenstein, E. Clin. Phurmacol. Ther. 1978,24, 52-59. 6. Whitehouse, L. W.; Paul, C. J.; Gottschling, K. H.; Lodge, B. A.; By, A. W. J. Phurm. Sci. 1990, 79,349-350. 7. Mav. E. L.: Jacobson. A. E. J. Phurm. Sci. 1977.66.285-286. 8. Birch, P. J:; Hayes, A: G.; Sheehan, M. J.; Tyers,M. B. J.Pharm. Phurmacol. 1988,40, 213-214.
6.
Acknowledgments The authors express their thanks to Dr. K. J. Welham for runnin the mass spectra, Mr. C. H. James for the NMR spectra, and Mr. Baldeo for the microanal ses. In vivo assessment of morphine co 'u ates was erformed gy Miss J. Stables, Mrs. S.M. Harrison, an! drs. R. L. 8argent. This work was sup orted by Glaxo Group Research La., U.K. (Grant No. ESAD056). Toth is on leave from Central Research Institute for Chemistry of the Hungarian Academy of Sciences.
d
f.
Journal of Pharmaceutical Sciences I 1105