J. Med. Chem. 1985,28, 3-9
3
Art ides
1H-2-Benzopyran-1-one Derivatives, Microbial Products with Pharmacological Activity. Conversion into Orally Active Derivatives with Antiinflammatory and Antiulcer Activities Yukiji Shimojima,* Takashi Shirai, Tsuneo Baba, and Hiroshi Hayashi Technical Research Laboratory, Asahi Chemical Industry Co., Ltd., 2-1, Samejima, Fuji-shi, Shizuoka 416, Japan. Received September 14, 1983 A novel gastroprotective substance, 6-[[ 1(S)-[3(S),4-dihydro-8-hydroxy-l-oxo-1H-2-benzopyran-3-yl]-3-methylbutyl]amino]-4(S),5(S)-dihydroxy-6-ox0-3(S)-ammoniohexanoate (AI-77-B, l),isolated from a culture broth of Bacillus pumilus AI-77, was chemically modified to prodrugs that are active by oral dosing. Compound 1 was lactonized and then monoalkylated at the primary amine position. Six N-alkylated y-lactone derivatives of 1 (with alkyl chains being methyl 5a, ethyl 5b, n-propyl5c, n-buty15d9n-pentyl5e, or n-hexyl50 were synthesized and eight compounds including 1 and y-lactone derivative 2 were compared for their gastroprotective activities and blood levels after oral administration in rats. Further, chloroform-water partition coefficients of 5a-f were also compared as a measure of lipid solubility. The protective effects of these compounds on stress ulcers were mutually related to blood levels of dealkylated compounds (1 and 2). Parent compound 1 was detected in blood a t 1 h after each of 5a-d was administered. When 5b or 5c was administered, high activity and high blood levels of 1 were observed in comparison with those levels obtained with 5a or 5d. Neither 5e nor 5f were detected in any amount in blood by oral administration without special formulation due to extremely low solubilities and agglutinative properties in intestinal fluid. Interestingly, 5b and 5c were found to have antiinflammatory activities in addition to potent antiulcerogenicity action.
A novel gastroprotective substance (AI-77-B,1) has been isolated from a culture broth of Bacillus pumilus AI-77 as the major component of seven related structures.l The structure of 1 has been established as 6-[ [l(S)-[3(S),4dihydro-8-hydroxy-l-oxo-lH-2-benzopyran-3-y1] -3methylbutyl] amino]-4(S),5(S)-dihydroxy-6-0~0-3(S)-ammoniohexanoate.2 Compound 1 appears to be part of a OH
0
iI 1
N
unique drug classs exhibiting noncentral suppressive, nonanticholinergic and nonantihistaminergic properties despite its potent antiulcerogenicity acting against stress ulcers induced in rats by restraint and water immersion. However, 1 shows no significant activity by oral administration apparently due to little gastrointestinal absorption. We have reported on the structural requirements for gastroprotective activity of 1 and derivatives, and it has become apparent that the presence of both the amino acid moiety of the side chain and the 1H-2-benzopyran-1-one (1) Shimojima, Y.;Hayashi, H.; Ooka, T.; Shibukawa, M. Agric. Bid. Chim. 1982,46, 1823. (2) (a) Shimojima, Y.; Hayashi, H.; Ooka, T.; Shibukawa, M.; Iitaka, Y.Tetrahedron Lett. 1982,23,5435. (b) Shimojima, Y.; Hayashi, H.; Ooka, T.; Shibukawa, M.; Iitaka, Y.Tetrahedron 1984,40, 2519.
skelton are important for a ~ t i v i t y . ~For the purpose of obtaining drugs that are more effective on oral dosing, we have prepared prodrugs of 1 by introducing various kinds of lipophilic groups onto the primary amine moiety. As a result of these studies a series of N-monoalkylated ylactone derivatives of 1 was found to be suitable. Interestingly, some of those compounds exhibited antiinflammatory activity in addition to gastroprotective activity. In this paper, the preparation of N-monoalkyl-substituted y-lactone derivatives of 1 (with alkyl chains = methyl 5a, ethyl 5b,n-propyl5c, n-butyl5d, n-pentyl5e) or n-hexyl 5f) is described. Their gastroprotective activities, concentration in blood following oral administration, and chloroform-water partition coefficients as a measured of lipid solubility are compared. Furthermore, antiinflammatory activity of 5b and 5c is evaluated. These are selected for their high absorption from the gastrointestinal tract. Chemistry. The N-monoalkylated derivatives 5a-f with alkyl chains of methyl, ethyl, n-propyl, n-butyl, npentyl, or n-hexyl were prepared by two different methods as shown in methods 1and 2. Treatment of an amide with 1equiv of triethyloxonium tetrafluoroborate (Meerwein's reagent)4in methylene ,chlorideis known to give the imino ether tetrafluoroborate in excellent yield.5 In the case of imino ethers from secondary and tertiary amides, replacement of the methylene chloride by absolute ethanol, followed by treatment with excess sodium borohydride at 0 or 25 "C, gives the corresponding amine in essentially (3) Shimojima, Y.;Hayashi, H. J. Med. Chem. 1983, 26, 1370. (4) Meerwein, H. Org. Synth. 1966,46, 113. (5) (a) Karplus, M. J. Chem. Phys. 1959, 30, 11. (b) Hanessian, S. Tetrahedron Lett. 1967, 1549. (c) Meerwein, H.; Borner, P.; Fuchs, 0.; Sasse, H. J.; Schrodt. H.; Spille, J. Ber. Dtsch. Chem. Ges. 1956,89,2060.
0022-2623/85/1828-0003$01.50/00 1984 American Chemical Society
4 Journal of Medicinal Chemistry, 1985, VoE. 28, No. 1
Method 1
Shimojima et al. Table I. N-Alkylated y-Lactone Derivatives of 1
R
OH
I
+ cI O H HN+
BF4 OH
'oEt
0
(HCI) NHCHZR
5a- f
'v
% yield
b,
R =CH3
C.
R zC2H5
d,
R
n-C3H7
e,
R
In - c 4 ~ 9
f,
R
n-C5~11
Method 2 HO
R method 1 method 2 mp, "C dec formulaa CH3 b 10 149-151 CzlHzsNz07 CzH6 69 58 162-164 C22H30N207 T I - C ~ H ~ 58 75 137-139 CZaH3zNz07 5d T I - C ~ H ~ 42 62 157-158 C24H34N207 5e n-C6H11 45 54 159-161 CzbH36N207 5f n-C6HlS 42 54 166-168 C2eH38N207 "Elemental analyses obtained for C, H, and N were in agreement (0.4%) with the theoretical values. *An attempted alkylation by method 1 was unsuccessful. no. 5a 5b 5~
(HCI) NHR
ug/mL
I
C
5a-f
m,
rv
30.
+
h
C
E-
20.
0
10. 0
HO
+*
COO-
h
6a-f
N
quantitative yield.6 For preparing 5b, method 1was superior to method 2 in yield (Table I). It is very fortunate that triethyloxonium tetrafluoroborate reacts with the amide on (2-10' with high selectivity at room temperature. Even in the treatment of N-acetyl derivative 3b with 3 equiv of triethyloxonium tetrafluoroborate, neither the amide at the center of the molecule nor the phenol on C-8 were attacked. This difference in reactivity between the two amide moieties may be due to steric hindrance around the center amide. If alkylation of the phenol on C-8 had occurred, the resulting derivative would lack the characteristic fluorescence of 3,4-dihydro-8-hydroxy-lH-2benzopyran-1-0ne;~however, no products without fluorescence could be detected from the above reaction. Regarding the possibility of alkylation of the secondary alcohol on C-8' by the reagent, no clear evidence was obtained. The imino ethers 4 easily suffered hydrolysis to the corresponding amine; therefore, they had to be reduced directly without isolation. Triethylamine is superior to pyridine as a base for acylation of 1, because residual pyridine consumes Meerwein's reagent in the next reaction. Attempted alkylation via method 1of the formamide resulting from treatment of 1 with dicyclohexylcarbodiimide and formic acid, resulted in recovering only the unchanged formamide. A possible reason for this unsuccessful result ~~
(6) (a) Borch, R. F. Tetrahedron Lett. 1968,61. (b) Monteiro, H. J. Synthesis, 1974, 137.
test compounds Figure 1. Blood levels of 1, 2, and N-alkylated y-lactone derivatives (200 mg/kg, PO in rats). Test compounds were administered as formulations composed of 5x.HCl/glycerin/HCO-60 = 1/12/0.05 (in weight). (0) The cumulative amount of two or three compounds detected in blood samples collected 1 h after administration of test compound; (m) the cumulative amount at 3 h after treatments. Compositions are shown in Table IV. Each column shows the mean drug level in blood samples taken from six to seven rats; each vertical bar indicates the standard error.
may reside in an acceleration of hydrolysis of an intermediate imino ether due to a reduction in bulk of the amide substitutent. Therefore N-methylated derivative 5a was synthesized with excess methyl iodide by method 2.
In preparing compounds Sc-f with alkyl chains longer than ethyl, method 2 gave higher yields (reaction yield 54-75%) than method 1 (42-58%). The reason for low yields of the imino ethers 4c-f is not clear. In order to identify metabolites of 5a-f by high-performance liquid chromatography (HPLC) analyses, compounds 6a-f were prepared from corresponding compounds 5a-f by hydrolysis with aqueous NaOH at pH 7-9. Biological Results and Discussion Compound 1, y-lactone derivative 2,233and six N-alkyl derivatives 5a-f were compared for relative potency of gastroprotective activity against stress ulcers and their concentration in blood following oral administration in rats
Journal of Medicinal Chemistry, 1985, Vol. 28, No. 1 5
1H-2-Benzopyran-1 -one Derivatives Table 11. Protective Effects on Stress-Induced Ulcers in Rats protection, %
at the following dosese
dose, mg/kg, ip 12.5 50 25 12.5 NT~ 94 100 78 0 0 1 17 NT 83 83 66 66 44 2c 78 17 56 33 NT 56 11 5aC 66 33 100 76 66 78 56 5bC 89 17 89 66 44 94 78 5c' 100 17 89 44 NT 66 33 5dc 89 NT 44 56 NT 33 44 50' 56 NT 11 17 NT 44 11 5fe 44 "Five rats were treated as a group for each dosage. bTreatments in which protective values were more than 60% were evaluated as significantly effective (Student's t test, P < 0.01). CThesecompounds were administered as HCl salts. dNT = not tested. eTest compounds were administered as formulation composed of drug/glycerin/HCO-60 = 1/12/0.05 (in weight). no.
100
dose, mg/kg, PO 25
50
Table 111. Solubilitv and Distribution Coefficient distribution coeff CHC13/ solubility, pg/mL phosphate n0.O DhosDhate bufferb HoO bufferb 5a 9.9 x 102 1.5 x 103 3750 5b 1.1 x 104 >LO x 105 43 5c 1.6 x 103 >LO x 105 260 5d 3.7 x 102 1.0 x 105 1700 5e 3.3 x 102 7.3 x 103 1900 5f 5.0 X 10' 1.6 x 103 3000 "Compounds were used as HC1 salts. *pH 7.5 (10 mM). 1
.
(Tables I1 and IV and Figure 1). Furthermore, as a measure of lipid solubility, chloroform-water partition coefficients of 5a-f were determined (Table 111). (HCI)
2
N
Ring-opened compounds of the y-lactone as 1 exhibitel. no significant activity by oral administration. On the other hand y-lactone derivative 2 exhibited potent gastroprotective activity by oral administration. However, the protective value of 2 often showed significant differences between individual rata, which was probably attributed to variable absorption of 2 from the gastrointestinal tract. Introduction of an alkyl function at the primary amine of 2 resulted in more reproducible gastroprotective action and higher blood levels (Table I1 and Figure 1). The highest blood level of each compound was found in blood samples
collected 1 h after treatment (data are not shown). NEthyl derivative 5b showed the highest blood level of the six N-alkyl derivatives 5a-f. In each compound 5a-c, an approximate linear relationship existed between the concentration detected in blood and the dosage up to an oral dosage of 200 mglkg. However, at high dosages of 5d (over 100 mg/kg), saturation of absorption was observed. In the cases of 5e and 5f administered without formulation, no compound related to 1H-2-benzopyran-1-onewas detected in blood by high-performance liquid chromatography (HPLC) analysis. By means of in vivo tests in rats such as perfusion of small intestines or stomachs, the absorption site of these compounds was proved to be the small intestine. However, 5d-f showed extremely low solubilities and were found to easily agglutinate in intestinal fluid. Therefore, we studied methods to suitably disperse 5d-f in intestinal fluid. A formulation consisting of drug, glycerin, and HCO-60 in the ratio of 1:12:0.05 (weight) was selected. Figure 1 shows the levels of 5a-f in blood at 1 and 3 h after oral administration. Each value plotted is the cumulative amount of three kinds of compounds including the drug, its corresponding ring-opened compound 6, and dealkylated metabolite 1,which is the parent. When 5a, 5c, and 5d were administered, 1 was detected as shown in Table IV. In HPLC analysis, complete separation between the peak of 6b and that of l was unsuccessful under the condition in which both peaks were free from interference derived from serum components. Judging from the above results, dealkylation of 5b could also occur in vivo. On the other hand, no significant amount of 1 was detected in the blood samples collected after administration of the 5e formulation. Even trace amounts of 5f were not detected in blood in the administration of the 5f formulation. It is generally accepted that N-alkylated drugs undergo dealkylation in liver microsomes by a mechanism that is NADPH dependent and requires oxygen. The system
Table IV. Compositions of Compounds Detected in Blood compounds detected in bloody 1 h after administration 200mgi kg PO, total no. 2 5xb 6x 1 mean t SE 2 1 N D ~ ND 2 0.8 0.7 1.5 t (0.6) 0.6 5a 3.8 3.1 3 .O 9.9 t (2.5) 5b 17.5 21.0c 38.5 * (6.5) 5c 14.2 6.4 2.0 22.2 t (3.6) 5d 2.3 2.0 trace 4.3 + (2.0) 5e 1.2 0.8 ND 2.0 * (1.0) 5f ND ND ND ND t a Each value shows the mean of blood samples taken from six to seven rats. The cumulative amount of 6b and 1. ND = not detected.
pg/mL 3 h after administration total mean t SE ND 1.1 * (0.9) 1.2 1.1 2.7 2 (1.4) 8.3 7.9c 16.2 t (1.5) 3.4 3.4 4.2 11.0 t (1.5) 2.3 1.8 ND 4.1 * (1.4) 0.8 0.6 ND 1.4 t (0.7) ND ND ND ND x represents the corresponding one of a-f. 5x
6x
1 ND 0.5 0.4
6 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 1
Shimojima et al.
Table V. Evaluation of the Antiinflammatory Activity and Acute Toxicity of 5b and 5c EDm, mg/kg, no. 5bb
CPEc (rats) >200
5Cb
124 (84-182)
IDMh
1.3
BPEd (rats) 182 (107-309)s 69 (54-88) >25
PO
DPEe (rats) 47 (33-66) 43 (32-58) >25
UV erythema (guinea pigs) 13 (7-23)
EDmin: mg/kg, PO adju arthf (rats) 20
12
LDSo,mg/kg, PO (rats) 1250 (893-1750)
15
(9-7)
