Jacob Bell
New Member
Inventors
Elsohly, Mahmoud A.
Turner, Carlton E.
Assignee
The University of Mississippi
Application
No. 06/044350 filed on 05/31/1979
US Classes:
549/408
Examiners
Primary: Chan, Nicky
Attorney, Agent or Firm
Stokes; William D.
International Classes
C07D 493/00 (20060101)
C07D 493/08 (20060101)
C07D 311/00 (20060101)
C07D 311/58 (20060101)
Description
BACKGROUND OF THE INVENTION
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There has been for many years an ongoing search for the discovery and development of more effective antiinflammatory and hypothermia inducing agents which can be administered to mammals in therapeutically effective dosages with minimal sideeffects. There is also an economic need for such agents which are relatively simple to produce from readily available non-costly reagents. A wide variety of compounds have demonstrated antiinflammatory properties, as for example pyrazolidinediones,arylalkanoic acids, carboxylic acid amides, and salicylates. Anthranilic acid and certain of its derivatives, such as mefenamic acid, flufenamic acid, and N-benzoyl-anthranilic acid, have also exhibited antiinflammatory activity as described, forexample in the article by M. W. Whitehouse, "Biochemical Properties of Anti-Inflammatory Drugs," Biochem. Pharmacol., 16, pp. 753-760 (1967). Aspirin, of course, is probably the most commonly used antiinflammatory and antipyretic agent. Most of theknown antipyretics have the disadvantage of often dangerous side effects such as causing circulatory collapse.
The compound 2-methyl-2(4-methyl-pent-3-enyl)-5-hydroxy-7-pentylchromene or cannabichromene (CBC) is well-known in the prior art, occurring naturally as a cannabinoid constituent of Cannabis sativa L. The reported usefulness of cannabichromene isprimarily that of intermediate in the synthesis of related compounds, such as cannabicyclol.
The known synthesis of cannabichromene have not been entirely satisfactory, owing to historical poor yields and problems associated with purification of the product.
Exemplary of such prior art synthesis routes are the cyclodehydrogenation of cannabigerol with chloranil in benzene or with 2,3-dichloro-5,6-dicyanobenzene (DDQ). Perhaps a more important route for the synthesis of cannabichromene and relatedcompounds has been the condensation of citral with a substituted resorcinol. By this method, cannabichromene is formed by heating olivetol and citral for several hours under reflux in the presence of pyridine, in molar proportions of 1
1(olivetol:citrol
yridine), with isolation of the product by direct chromatography on silica gel. The yields obtainable by this method, however, have only about 15 to 17% of theory. Methods have been proposed to increase the yield of varying theproportion of pyridine employed; however, molar proportions of 13 have only slightly increased cannabichromene yield to 20%, and molar proportions of 16 have significantly decreased the yield to about 5%. The acid-catalyzed condensation ofolivetol and citral is also known, but the products of this reaction are not known to include cannabichromene.
The pyridine-catalyzed olivetol/citral condensation reaction also provides significant amounts of at least five by-products, in addition to unreacted material from which the cannabichromene must be separated. Both unreacted citral and theby-product isobichromene present difficulties during cannabichromene recovery, owing to the nearly identical Rf values of cannabichromene and citral in different solvent systems and the very close Rf values on silica gel of cannabichromene andisocannabichromene.
Accordingly, it is an object of this invention to provide a new method of synthesizing cannabichromene.
It is another object of this invention to provide an improved method for the recovery of cannabichromene.
It is an additional object of this invention to provide a composition useful for inducing hypothermia and useful as an antiinflammatory agent.
It is a further object of this invention to provide a new compound, 2-methyl-2(4-methyl-pent-3-enyl)-5-hydroxy-7-methylchromene, or CBC-C1 as the compound will hereinafter be referred to.
It is an additional object of this invention to provide a method for reducing inflammation in mammals.
It is yet another object of this invention to provide a method for inducing hypothermia in mammals.
SUMMARY OF THE INVENTION
The invention comprises a method for preparing cannabichromene and homologues thereof in greatly improved yields by condensation of a substituted resorcinol and citral in the presence of a primary amine, and an improved method for separating theproduct from unreacted citral and isomeric by-products by first, reduction of citral to the corresponding alcohol and second, column chromatography on silica gel impregnated with 1% sodium hydroxide.
The invention further comprises a method for reducing inflammation and for inducing hypothermia in mammals comprising administering cannabichromene or its homologues to mammals either orally or by injection, in a therapeutically effective dose. Preferably, the compound is administered as a novel composition comprising a pharmaceutically-acceptable diluent carrier and cannabichromene or its homologues.
DETAILED DESCRIPTION OF THE INVENTION
According to the method for preparing cannabichromene or its homologues of the present invention (III), a substituted resorcinol (I) is condensed with citral (II) in the presence of a primary amine such as t-butylamine or n-propyl-amine accordingto the following reaction scheme: ##STR1## wherein R is hydrogen, C1 -C10 -alkyl, or C2 -C10 -alkenyl.
Of particular interest is the compound IV, CBC-C1 formed from the condensation of orcinol (R is methyl) with citral: ##STR2## In addition to the desired chromene products III, varying amounts of other by-products are formed in both the priorart and present condensation reactions, including an isomer of the chromene product (V), and a cannabicitran product (VI): ##STR3## The presence of these by-products substantially interferes with the purification of the chromene product III whenconventional isolation procedures are employed. While the process of the present invention substantially decreases the formation of the by-products V and VI in contrast to prior art procedures, at least traces are usually present in the reactionproduct, and isolation of the chromene product III from the by-products V and VI is greatly facilitated by the improved separation method of the present invention.
Preferably, equimolar amounts of substituted resorcinol, citral, and primary amine are heated under reflux for about seven to nine hours, and the chromene product recovered from the crude reaction mixture by solvent extraction followed bychromatography of the extracted and partially purified product.
Preferably, toluene is employed as solvent in refluxing the reaction mixture, although other organic solvents such as methylene chloride are suitable, but may result in decreased yields. After reflux, the solvent is evaporated and the resultantcrude reaction mixture is redissolved in an organic solvent such as benzene or cyclohexane, and this solution is then extracted with 1% aqueous sodium hydroxide and dried.
According to the process of the present invention, separation of CBC or its homologues (III) from unreacted citral (II) in this dried residue is accomplished by converting the citral to the corresponding alcohol which is much more polar than thechromene product. The citral is preferably reduced by sodium borohydride in alcohol such as ethanol, as this procedure does not significantly adversely affect the yield of the chromene III.
After reduction, the solvent is evaporated and the resultant crude dried residue partitioned between water and organic solvent and then chromatographed employing a solvent system of, for example, benzene-chloroform (1:1) or cyclohexane-chloroform(1:1). While conventional chromatographic procedures may be employed, chromatographic separation according to the present invention is preferred. By this method, the chromene product is purified by column chromatography on silica gel 60PF impregnatedwith 1% sodium hydroxide, which cleanly separates the chromene product (III) from the chromene isomer V. In the event that difficulties in separation of III from V are not contemplated, as, for example, when the isomer is present in only trace amounts,the crude product may be chromatographed by conventional methods such as on a column of processed silica gel, a column dry-packed with silica gel 60, or by high-pressure liquid chromatography. The yields of CBC by the process of the present inventionare typically about 60% of theory.
Cannabichromene and its disclosed homologues has been found to be effective as antiinflammatory agents in mammals, and can be used to reduce inflammation and to relieve pain in diseases such as arthritis, as well as to reduce and control edema. Cannabichromene has also been found to be effective in inducing hypothermia which is useful, for example, when a decrease in metabolic activity is desired.
In both the rat-paw edema test and the eryrocytes membrane hemolytic test, CBC and CBC-C1 were fond to be more effective than the standard phenylbutazone in controlling inflammation, as measured by reduced edema in rats and inhibition ofheat-induced hemolysis of red blood cells.
Treatment for inflammation or to induce hypothermia is preferably by oral administration or intra-peritoneal injection, in combination with a pharmaceutically-acceptable carrier which may be solid or liquid. Examples of acceptable solid carriersinclude, but are not limited to, starch, dextrose, sucrose, lactose, gelatin, agar, stearic acid, magnesium stearate, acacia, and similar carriers. Examples of liquids include, but are not limited to, water, edible oils, such as corn or peanut oils, andthe like.
When administered in solid form, the compound and diluent carrier may be in the form of tablets, capsules, powders or lozenges prepared by standard techniques. When given as a liquid diluent carrier may be in the form of a liquid suspensionadministered as such or encapsulated.
When employed to treat an inflammatory condition in a mammal, animal, or human, the active compound is preferably administered orally in admixture with a pharmaceutically-acceptable diluent carrier as described above. When employed to inducehypothermia in a mammal, animal or human, the active compound is preferably administered by intraperitoneal injection, also an admixture with a pharmaceutically acceptable diluent carrier as described above. The compound is administered in a non-toxicdosage concentration sufficient to reduce the inflammation or edema where present, or to induce the desired degree of hypothermia. The actual dosage administered will be determined by such generally-recognized factors as the body-weight of the subject,the severity of the condition being treated, the idiosyncrasies of the particular subject, and the activity of the compound employed. With these considerations in mind, the dosage for a particular subject can be readily determined by the medicalpractitioner in accordance with conventional techniques in the medicinal art.
The following examples are illustrative of the invention.
EXAMPLES
EXAMPLE I
(Preparation of Cannabichromene)
To a three-necked round bottomed flask (100 ml capacity), fitted with a dropping funnel and a condenser was added 5 g. olivetol (27.8 mmole) and 2.03 g. (2.96 ml., 27.8 mmole) t-butyl amine in 55 ml toluene and the mixture was heated to50°-60° C., 4.23 g. (4.76 ml., 27.8 mmole) of citral was then added dropwise. The mixture was refluxed for 9 hours, after which time it was cooled to room temperature and the solvent evaporated to give 9.3 g. of crude reaction mixture. Gas chromatographic analysis of the reaction mixture showed 59.46% CBC (molar conversion), 5.04% cannabicitran and trace amount of iso-CBC.
EXAMPLE II
(Purification of Cannabichromene)
5 g. of the crude reaction mixture from Example I was dissolved in 100 ml. benzene and the solution extracted twice with 50 ml. of 1% aqueous sodium hyroxide solution followed by 50 ml. of water. The benzene solution was then dried overanhydrous sodium sulfate and the solvent evaporated. The residue was then dissolved in 50 ml. ethanol, and 250 mg. of sodium borohydride were added portion-wise while stirring.
Stirring at room temperature was continued for 30 minutes after which time the solvent was evaporated and the residue partitioned between water (50 ml.) and benzene (100 ml.). The crude reaction mixture was chromatographed on a column ofprocessed silica gel (200 g.). Processed silica gel was prepared by making a paste of silica gel -PF254 with water (equal amount) which was then dried in an oven at 110° and the resulting cake passed through 60 mesh sieve. The solvent systemused was a mixture of benzene and chloroform (1:1). Fractions were collected and combined according to their tlc similarities in the same solvent system. Fractions containing pure CBC were combined, the solvent evaporated and the residue (2.1 g.) wasanalyzed by GC method and found to be 97% pure CBC. The synthetic CBC was found to be identical in all respects (tlc, GC, ir, uv, 1 H nmr and 13 C-nmr) with authentic CBC [2-methyl-2-(4-methyl-pent-3-enyl)-5-hydroxy-7-pentyl chromene].
EXAMPLE III
The procedure of Example II was followed, except the crude reaction mixture was dissolved in cyclohexane instead of benzene. The same results were obtained.
EXAMPLE IV
(Preparation of Cannabichromene)
To a three-necked, 2 liter round bottomed flask fitted with a condenser, a dropping funnel and a mechanical stirrer, was added 90 g. (0.5 mole) of powdered olivetol and 36.5 g. (53.2 ml., 0.5 mole) t-butylamine and the mixture dissolved in 1liter toluene. The reaction mixture was then stirred for awhile when a brownish white gelatinous material appeared. The mixture was then heated to 50° C. and stirred constantly. To the resulting clear brown solution was added 76 g. (85.6 ml.,0.5 mole) citral dropwise. After complete addition of citral, the reaction mixture was refluxed for 9 hours, cooled to room temperature and the solvent evaporated to give 166.8 g. of crude reaction mixture.
Dry weight analysis of the crude mixture using gas liquid chromatography showed 62.05% CBC, 5.09% Cannabicitran and trace amount of iso-CBC. Cannabichromene was purified and chromatographed in the same manner as previously described underExample II.
EXAMPLE V
(Preparation of Cannabichromene)
The reaction of 5 g. olivetol (27.8 mmole), 1.652 g. n-propylamine (2.3 ml., 27.8 mmole) and 4.6 ml. citral (27.8 mmole) in 55 ml. toluene was carried out in the same manner as described under Example 1. The refluxing time was 7 hours. Gaschromatographic analysis of the reaction mixture showed 61.62% CBC, 4.01% Cannabicitran and trace amount of iso-CBC.
Purification and chromatography of CBC were carried out as described under Example II.
EXAMPLE VI
(Preparation of the Methyl Homologues of Cannabichromene)
Th procedure described in Example I was followed, except 3.45 g of orcinol was reacted in place of the 5 g. of olivetol. Gas chromatographic analysis of the reaction mixture showed 48.27% yield of CBC-C1.
EXAMPLE VII
(Purification of CBC-C1)
The product of Example VI was purified according to the process of Example II. The product was found to be pure CBC-C1 as a pale yellowish oil, the identity of which was determined as2-methyl-2(4-methyl-pent-3-enyl)-5-hydroxy-7-methylchromene by comparison of the spectral data with those of CBC.
EXAMPLE VIII
[Inhibition of Inflammation of CBC, as Measured by the Rat-Paw Edema Test (Intraperitoneal Injection)]
A. Procedure
The test rats were divided into test groups of 6 to 8 animals, weighed and marked so that the individual rats could be identified; all rats were given a 700 mg/kg intraperitoneal injection of ethyl urethane in distilled water to render themtractable during testing. A circle was drawn, with a felt-tipped mixer, around the hind leg of each rat just above the ankle, and each rat was dosed with a test or control compound by intraperitoneal injection. Test compounds were given at doses of 60,120, 240, or 480 mg/kg. The negative control was the vehicle which was used to give the test compounds and the two positive controls were phenylbutazone given at 120 mg/kg and 60 mg/kg. Phenylbutazone was prepared for injection by suspending it innormal saline using Tween 60. The rats were then held in group cages for 30 minutes. The volume of the left hind paw was measured using a mercury displacement pleysmograph. The paw was dipped into the mercury until the mercury touche the line abovethe ankle. Mercury was then withdrawn until the mercury returned to its original level. The amount of mercury removed was measured in milliliters (ml). The mercury can be measured accurately to 0.01 ml. The left hind paw was then injected with 0.1 mlof a 5% w/v solution of viscous carageenen in normal saline. The injection was given between the metatarsal bones using a 27 gauge needle, and the rats then held in group cages for 3 hours. The volume of the left hind paws was measured again in thepleysmograph. The results were computed in the following manner:
a. A mean is taken for both the preinjection and post-injection paw volume of each test group.
b. The mean difference in volume (MDV) for each test group is computed by subtracting the preinjection mean from the postinjection mean.
c. The percent of control is computed for each test group as: ##EQU1## The percent of control is used to compare the efficacy of the various drug treatments.
d. The percent increase in paw volume is calculated for each group as: ##EQU2## The percent increase is used to compare the amount of edma observed in one experiment to the amount of edma observed in other experiments.
B. Results
The results of the rat-paw edema test are summarized in Table 1. As can be seen, doses of 120, 240, and 480 mg/kg all produced strong antiinflammatory effects. The effects were shown to be dose related, that is, higher doses of CBC producedstronger antiinflammatory effects. All the animals receiving 480 mg/kg died within 2 days of injection, but this cannot be judged to be simply a result of CBC toxicity since the rats also received an IP injection of 750 mg/kg of ethyl urethane as partof the test procedure. Seven of the eight animals receiving 240 mg/kg of PBZ died before the test could be completed. The eighth rat died within 24 hours of injection.
The data from the rat paw test were further analyzed using a one-way analysis of varience (ANOVA) and Duncan's New Multiple Range Test. The results of these tests are given in Tables 2 and 3. The pre-injection score of each animal wassubtracted from his post-injection score and an analysis of the different scores was conducted. The analysis showed that all test groups differed significantly from the vehicle control group. The 120 mg/kg dose of CBC differed significantly from the240 and 480 mg/kg doses of CBC, and the 480 mg/kg dose of CBC differed significantly from the 120 mg/kg dosage of PBZ.
No significant effects were seen on the CNS screen at doses of CBC as large as 800 mg/kg in unanethetized mice.
TABLE 1 ______________________________________ RAT - PAW EDEMA DATA PERCENT COM- OF PERCENT POUND DOSE MDV CONTROL INCREASE ______________________________________ Vehicle 0.5 ml 0.463 100.000 42.383 Control CBC 120 MG/KG 0.139 30.00012.729 CBC 240 MG/KG 0.004 0.927 0.365 CBC 480 MG/KG -0.043 -9.189 -3.708 PBZ 120 MG/KG 0.106 22.973 10.316 ______________________________________ Percent of Control and Percent Increase Computed before Rounding MDVS or Group Means.
TABLE 2 ______________________________________ Analysis of varience for difference scores from rat-paw edema test. Source ΣX2 df MS F ______________________________________ Among 1.2291 4 0.3073 24.434** Within 0.4311 340.0128 Total 1.6602 38 ______________________________________ **p. < 0.01
TABLE 3 __________________________________________________________________________ Duncan' s Test for Difference Scores Rat-Paw Edema Test. Group -x I0 I1 I2 I3 SSR.sup. __________________________________________________________________________ Vehicle 0.170 control 0.463 -- .0324** 0.356** 0.458** 0.505** 0.128 CBC 0.167 120mg/kg 0.139 -- -- 0.033 0.134* 0.181** 0.125 PBZ 0.162 120mg/kg 0.016 -- -- --0.102 0.149* 0.121 CBC 0.156 240mg/kg 0.004 -- -- -- -- 0.047 0.115 CBC 480mg/kg 0.043 -- -- -- -- -- Vehicle CBC PBZ CBC CBC control 120mg/kg 120mg/kg 240mg/kg 480mg/kg __________________________________________________________________________ *p. <0.05 **p. <0.01 Shortest significant range for the 0.05 and 0.01 levels of significance.
EXAMPLE IX
[Inhibition of Inflammation of CBC as Measured by the Rat-Paw Edema Test (oral administration]
A. Procedure: The procedure described in Example VIII was followed, except the rats were dosed by oral gauge instead of i.p. injection. The test was conducted twice, once with nonfasted rats and once with rats that had been fasted during the 24hour period prior to dosing. The 60 mg/kg Penylbutazone control group was not used in the test with fasted animals. Tween 60 in normal saline was the vehicle control for the non-fasted rats and normal saline was the vehicle control for the fasted rats.
B. Results: The results are given in Tables 4 and 5. As can be seen from the tables, cannabichromene was active at all the doses tested. The degree of inhibition of edema increased in both tests as the amount of CBC given was increased. Thedegree of inhibition was greater in the fasted rats than it was in the nonfasted rats. This would be expected since the fasted rats should absorb the test compound more readily than the non-fasted rats.
When CBC is compared to PBZ in Tables 1 and 2 it is seen that PBZ was slightly more effective than CBC at 120 mg/kg in the nonfasted rats and that CBC and PBZ were about equally effective at 120 mg/kg in the fasted rats. The higher doses of CBCwere generally more effective in inhibiting edema than was PBZ. PBZ was not given at higher doses because of the rapid deaths produced by 240 mg/kg of PBZ given intraperitoneally in the test described in Example VIII.
TABLE 4 ______________________________________ RAT - PAW EDEMA DATA Oral CBC in Non-Fasted Rats. Run 11-6-1978 COM- PERCENT OF PERCENT POUND DOSE MDV CONTROL INCREASE ______________________________________ Vehicle 0.5 ml 0.625 100.00058.140 Control CBC 120 MG/KG 0.496 79.400 41.311 CBC 240 MG/KG 0.412 66.000 38.372 CBC 480 MG/KG 0.315 50.400 29.200 PBZ 60 MG/KG 0.489 78.200 50.257 PBZ 120 MG/KG 0.392 62.667 36.098 ______________________________________ Percent of Control andPercent Increase Computed Before Rounding MDVS or Group Means.
TABLE 5 ______________________________________ RAT - PAW EDEMA DATA Oral CBC in Fasted Rats. Run 11-14-1978 PERCENT PERCENT OF IN- COMPOUND DOSE MDV CONTROL CREASE ______________________________________ Saline Vehicle Control 0.5 ml 0.625100.000 46.339 CBC 120 MG/KG 0.409 65.400 31.382 CBC 240 MG/KG 0.209 33.400 15.981 CBC (given fol- 480 MG/KG 0.281 45.000 22.321 lowing saline infusion) PBZ 120 MG/KG 0.403 64.400 31.051 ______________________________________ Percent of Controland Percent Increase Computed Before MDVS or Group Means. *Doses are Approximate Due to an Error in Procedure.
EXAMPLE X
[Inhibition of Inflammation by CBC as Measured by Inhibition of Erythema (Red-Blood Cell Hemolysis)]
A. Procedure: The red blood cells (RBC's) were sensitized by washing twice with a volume of saline equal to the initial blood volume. CBC was suspended in a 2% ethanol in saline solution because of its insolubility in water. The procedure wasduplicated successfully 3 times using either 40% or 20% RBC suspensions.
B. Results: CBC was screened for inhibition of red cell hemolysis. The results of the tests are shown in Table 6. Phenylbutazone (PBZ), aspirin and tolmetin were used as positive controls and all inhibited heat induced hemolysis at theconcentration tested. Inhibition of hemolysis was dose-related in the positive controls and cannabichromene groups (Test 3). CBC produced 35% inhibition of heat-induced red cell hemolysis at 10-4 M test concentration and 26% at 2×10-5 MCBC. PBZ produced 16% and 10% inhibition of red cell hemolysis at the 10-4 M and 2×10-5 M test levels, respectively. Aspirin produced a 40% inhibition at the 5×10-4 M Test concentration.
It was apparent that not all the CBC actually went into suspension. In order to determine if some of the CBC adhered to the wall of the glassware a 10 ml aliquot of the 2×10-4 (solution a) of CBC saturated with Nacl was extracted withCHcl3 and analyzed by G.C. The solution was found to be 2×10-5 M CBC or to contain 0.3 mg of the original CBC. The flask was rinsed with ethanol and the washings analyzed by GC and found to contain 1.355 mg of CBC. The remaining 1.475mg (50%) of CBC that could not be accounted for by G.C. analysis may have been lost during the extraction procedure or while adjusting the pH of the test solution. If the 1.475 mg CBC which could not be accounted for was actually in suspension then themaximum final concentration of CBC used in the highest CBC test level would be 5×10-5 M or one half the amount listed in Table 6. In summary, CBC, at a minimum, produces 2 to 21/2 times more inhibition of heat induced red cell hemolysis thandoes PBZ at equimolar concentrations. Inhibition of heat-induced hemolysis was seen over a range of 10-4 M to 2×10-6 M CBC. The actual activity of CBC may have been 3 to 30 times more protective of red cell membranes than an equivalentamount of PBZ.
TABLE 6 ______________________________________ (Inhibition of Heat-Induced Erythrocyte Hemolysis) Final Concentration of Percent Inhibition the TEST SOLUTION TEST 1* TEST 2 TEST 3 ______________________________________ Phenylbutazone 2.5× 10-4 M 54 70 49 1.0 × 10-4 M 16 2.5 × 10-5 M 31 2.0 × 10-5 M 10 Acetylsalicylic Acid 5.0 × 10-4 M 40 2.5 × 10-4 M 21 Tolmetin 2.0 × 10-4 M 27 Cannabichromene 1.0× 10-4 M 40 37 1.0 × 10-4 M 33 2.0 × 10-5 M 26 26 2.0 × 10-6 M 7 ______________________________________ *A 20% RBC suspension was used for Tests 1 and 3. A 40% RBC solution was used for Test 2.
EXAMPLE XI
(Inducement of Hypothermia by CBC in Mice)
A. Procedure: CBC (100 mg/kg was prepared in emulsion form with 3% Tween 60 and 3% Arlacel in normal saline (0.9%) in such a way as to permit a volume of 10 ml/kg of body weight to be injected intraperitoneally into male mice weighing between 32and 38 grams.
The animals were divided into groups of 10, and body temperatures were recorded with a multichannel Yellow Springs Telethermometer and Thermistor probes. During the experiment, the mice were confined in plastic restraint tubes. They wereallowed 45-60 min. for adaption to the restraint tubes and to rectal thermistor before a pre-drug baseline temperature reading was taken. Body temperature readings were obtained at 0.5, 1.0, 2.0, 3.0 and 4.0 hrs. post-CBC administration.
The mean (. -.S.E.) decrease in temperature from the preinjection baseline reading for saline-, vehicle-, and CBC-treated mice were calculated and statistical analyses were conducted using the two-tailed Student's t test.
B. Results: As summarized in Table 7, both the CBC- and vehicle control-treated groups showed a consistent drop in body temperature over the duration of the experiment, with the CBC-induced hypothermia being more pronounced and significantlydifferent from saline controls at all readings, whereas the vehicle induced decrease in body temperature was only significant from saline controls at the 2.0, 3.0 and 4.0 hr. readings.
TABLE 7 __________________________________________________________________________ Mean (. -.S.E.) Decrease in Rectal Temp. (°0) Time (hrs.) Treatment 0.5 1.0 2.0 3.0 4.0 __________________________________________________________________________ Normal Saline 0.43 . -. 0.12 0.44 . -. 0.07 0.47 . -. 0.07 0.61 . -. 0.14 0.60 . -. 0 Vehicle Control 0.58 . -. 0.16 0.80 . -. 0.16 1.62 . -. 0.16* 1.35 . -. 0.14* 1.04. -. 0* CBC (100 mg/kg) 1.86 . -. 0.18Δ * 2.52 . -. 0.26Δ * 1.93 . -. 0.21* 1.53 . -. 0.21* 1.61 . -. 0*Δ __________________________________________________________________________ *Significantly different fromsaline controls, P ≤ 0.05. ΔSignificantly different from vehicle control, P ≤ 0.05.
In spite of the vehicle's activity, the hypothermia induced by CBC was still significantly different from vehicle controls at 0.5, 1.0 and 4.0 hrs.
The peak hypothermic effect (at a dose of 100 mg/kg, i.p.) for CBC was attained within the first two hours and then declined thereafter.
EXAMPLE X
[Inhibition of Inflammation by CBC-C1 as Measured by the Rat-Paw Edema Test (intraperitoneal administration)]
A. Procedure: The procedure described in Example VIII was followed. CBC-C1 was tested at doses of 60 mg/kg and 120 mg/kg prepared in an emulsion with Tween 60, Arlacel and distilled water. The vehicle control used was Tween 60, Arlacel anddistilled water prepared without CBC-C1.
B. Results: The results are given in Table 8. CBC-C1 was active at both 60 mg/kg and 120 mg/kg. The inhibition of edema was dose related. When the effects of CBC-C1 were compared with those of PBZ it can be seen that CBC-C1 didreduce the rat paw edema with slightly less activity than PBZ at the 120 mg/kg dose and was as active as PBZ at 60 mg/kg. No toxic or adverse effects were observed in any of the rats tested.
TABLE 8 ______________________________________ RAT - PAW EDEMA DATA CBC-C1 given i.p. in fasted rats. Run 5-10-79 COM- PERCENT OF PERCENT POUND DOSE MDV CONTROL INCREASE ______________________________________ Vehicle 0.00 MG/KG 0.191100.000 13.947 Control CBC-C1 120 MG/KG 0.070 36.601 4.956 CBC-C1 60 MG/KG 0.090 47.059 6.742 PBZ 120 MG/KG 0.046 24.183 3.254 PBZ 60 MG/KG 0.090 47.059 6.742 ______________________________________ Percent of Control and PercentIncrease Computed Before Rounding MDVS or Group Means.
Although this invention has been described with reference to illustrative embodiments thereof, it will be apparent to those skilled in the art that the principles of this invention can be embodied in other forms within the scope of the followingclaims.
Other References
Kane et al., JACS, 90, 6551 (1968)
Crombie et al., J. Chem. Soc. (C), 796 (1971)
Crombie et al., J. Chem. Res. S (Synopses), 1977, (5), 114-115, (C.A. 87:102470s)
Source: Process for preparing cannabichromene - US Patent 4315862 Description
Elsohly, Mahmoud A.
Turner, Carlton E.
Assignee
The University of Mississippi
Application
No. 06/044350 filed on 05/31/1979
US Classes:
549/408
Examiners
Primary: Chan, Nicky
Attorney, Agent or Firm
Stokes; William D.
International Classes
C07D 493/00 (20060101)
C07D 493/08 (20060101)
C07D 311/00 (20060101)
C07D 311/58 (20060101)
Description
BACKGROUND OF THE INVENTION
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There has been for many years an ongoing search for the discovery and development of more effective antiinflammatory and hypothermia inducing agents which can be administered to mammals in therapeutically effective dosages with minimal sideeffects. There is also an economic need for such agents which are relatively simple to produce from readily available non-costly reagents. A wide variety of compounds have demonstrated antiinflammatory properties, as for example pyrazolidinediones,arylalkanoic acids, carboxylic acid amides, and salicylates. Anthranilic acid and certain of its derivatives, such as mefenamic acid, flufenamic acid, and N-benzoyl-anthranilic acid, have also exhibited antiinflammatory activity as described, forexample in the article by M. W. Whitehouse, "Biochemical Properties of Anti-Inflammatory Drugs," Biochem. Pharmacol., 16, pp. 753-760 (1967). Aspirin, of course, is probably the most commonly used antiinflammatory and antipyretic agent. Most of theknown antipyretics have the disadvantage of often dangerous side effects such as causing circulatory collapse.
The compound 2-methyl-2(4-methyl-pent-3-enyl)-5-hydroxy-7-pentylchromene or cannabichromene (CBC) is well-known in the prior art, occurring naturally as a cannabinoid constituent of Cannabis sativa L. The reported usefulness of cannabichromene isprimarily that of intermediate in the synthesis of related compounds, such as cannabicyclol.
The known synthesis of cannabichromene have not been entirely satisfactory, owing to historical poor yields and problems associated with purification of the product.
Exemplary of such prior art synthesis routes are the cyclodehydrogenation of cannabigerol with chloranil in benzene or with 2,3-dichloro-5,6-dicyanobenzene (DDQ). Perhaps a more important route for the synthesis of cannabichromene and relatedcompounds has been the condensation of citral with a substituted resorcinol. By this method, cannabichromene is formed by heating olivetol and citral for several hours under reflux in the presence of pyridine, in molar proportions of 1
1(olivetol:citrolyridine), with isolation of the product by direct chromatography on silica gel. The yields obtainable by this method, however, have only about 15 to 17% of theory. Methods have been proposed to increase the yield of varying theproportion of pyridine employed; however, molar proportions of 13 have only slightly increased cannabichromene yield to 20%, and molar proportions of 16 have significantly decreased the yield to about 5%. The acid-catalyzed condensation ofolivetol and citral is also known, but the products of this reaction are not known to include cannabichromene.The pyridine-catalyzed olivetol/citral condensation reaction also provides significant amounts of at least five by-products, in addition to unreacted material from which the cannabichromene must be separated. Both unreacted citral and theby-product isobichromene present difficulties during cannabichromene recovery, owing to the nearly identical Rf values of cannabichromene and citral in different solvent systems and the very close Rf values on silica gel of cannabichromene andisocannabichromene.
Accordingly, it is an object of this invention to provide a new method of synthesizing cannabichromene.
It is another object of this invention to provide an improved method for the recovery of cannabichromene.
It is an additional object of this invention to provide a composition useful for inducing hypothermia and useful as an antiinflammatory agent.
It is a further object of this invention to provide a new compound, 2-methyl-2(4-methyl-pent-3-enyl)-5-hydroxy-7-methylchromene, or CBC-C1 as the compound will hereinafter be referred to.
It is an additional object of this invention to provide a method for reducing inflammation in mammals.
It is yet another object of this invention to provide a method for inducing hypothermia in mammals.
SUMMARY OF THE INVENTION
The invention comprises a method for preparing cannabichromene and homologues thereof in greatly improved yields by condensation of a substituted resorcinol and citral in the presence of a primary amine, and an improved method for separating theproduct from unreacted citral and isomeric by-products by first, reduction of citral to the corresponding alcohol and second, column chromatography on silica gel impregnated with 1% sodium hydroxide.
The invention further comprises a method for reducing inflammation and for inducing hypothermia in mammals comprising administering cannabichromene or its homologues to mammals either orally or by injection, in a therapeutically effective dose. Preferably, the compound is administered as a novel composition comprising a pharmaceutically-acceptable diluent carrier and cannabichromene or its homologues.
DETAILED DESCRIPTION OF THE INVENTION
According to the method for preparing cannabichromene or its homologues of the present invention (III), a substituted resorcinol (I) is condensed with citral (II) in the presence of a primary amine such as t-butylamine or n-propyl-amine accordingto the following reaction scheme: ##STR1## wherein R is hydrogen, C1 -C10 -alkyl, or C2 -C10 -alkenyl.
Of particular interest is the compound IV, CBC-C1 formed from the condensation of orcinol (R is methyl) with citral: ##STR2## In addition to the desired chromene products III, varying amounts of other by-products are formed in both the priorart and present condensation reactions, including an isomer of the chromene product (V), and a cannabicitran product (VI): ##STR3## The presence of these by-products substantially interferes with the purification of the chromene product III whenconventional isolation procedures are employed. While the process of the present invention substantially decreases the formation of the by-products V and VI in contrast to prior art procedures, at least traces are usually present in the reactionproduct, and isolation of the chromene product III from the by-products V and VI is greatly facilitated by the improved separation method of the present invention.
Preferably, equimolar amounts of substituted resorcinol, citral, and primary amine are heated under reflux for about seven to nine hours, and the chromene product recovered from the crude reaction mixture by solvent extraction followed bychromatography of the extracted and partially purified product.
Preferably, toluene is employed as solvent in refluxing the reaction mixture, although other organic solvents such as methylene chloride are suitable, but may result in decreased yields. After reflux, the solvent is evaporated and the resultantcrude reaction mixture is redissolved in an organic solvent such as benzene or cyclohexane, and this solution is then extracted with 1% aqueous sodium hydroxide and dried.
According to the process of the present invention, separation of CBC or its homologues (III) from unreacted citral (II) in this dried residue is accomplished by converting the citral to the corresponding alcohol which is much more polar than thechromene product. The citral is preferably reduced by sodium borohydride in alcohol such as ethanol, as this procedure does not significantly adversely affect the yield of the chromene III.
After reduction, the solvent is evaporated and the resultant crude dried residue partitioned between water and organic solvent and then chromatographed employing a solvent system of, for example, benzene-chloroform (1:1) or cyclohexane-chloroform(1:1). While conventional chromatographic procedures may be employed, chromatographic separation according to the present invention is preferred. By this method, the chromene product is purified by column chromatography on silica gel 60PF impregnatedwith 1% sodium hydroxide, which cleanly separates the chromene product (III) from the chromene isomer V. In the event that difficulties in separation of III from V are not contemplated, as, for example, when the isomer is present in only trace amounts,the crude product may be chromatographed by conventional methods such as on a column of processed silica gel, a column dry-packed with silica gel 60, or by high-pressure liquid chromatography. The yields of CBC by the process of the present inventionare typically about 60% of theory.
Cannabichromene and its disclosed homologues has been found to be effective as antiinflammatory agents in mammals, and can be used to reduce inflammation and to relieve pain in diseases such as arthritis, as well as to reduce and control edema. Cannabichromene has also been found to be effective in inducing hypothermia which is useful, for example, when a decrease in metabolic activity is desired.
In both the rat-paw edema test and the eryrocytes membrane hemolytic test, CBC and CBC-C1 were fond to be more effective than the standard phenylbutazone in controlling inflammation, as measured by reduced edema in rats and inhibition ofheat-induced hemolysis of red blood cells.
Treatment for inflammation or to induce hypothermia is preferably by oral administration or intra-peritoneal injection, in combination with a pharmaceutically-acceptable carrier which may be solid or liquid. Examples of acceptable solid carriersinclude, but are not limited to, starch, dextrose, sucrose, lactose, gelatin, agar, stearic acid, magnesium stearate, acacia, and similar carriers. Examples of liquids include, but are not limited to, water, edible oils, such as corn or peanut oils, andthe like.
When administered in solid form, the compound and diluent carrier may be in the form of tablets, capsules, powders or lozenges prepared by standard techniques. When given as a liquid diluent carrier may be in the form of a liquid suspensionadministered as such or encapsulated.
When employed to treat an inflammatory condition in a mammal, animal, or human, the active compound is preferably administered orally in admixture with a pharmaceutically-acceptable diluent carrier as described above. When employed to inducehypothermia in a mammal, animal or human, the active compound is preferably administered by intraperitoneal injection, also an admixture with a pharmaceutically acceptable diluent carrier as described above. The compound is administered in a non-toxicdosage concentration sufficient to reduce the inflammation or edema where present, or to induce the desired degree of hypothermia. The actual dosage administered will be determined by such generally-recognized factors as the body-weight of the subject,the severity of the condition being treated, the idiosyncrasies of the particular subject, and the activity of the compound employed. With these considerations in mind, the dosage for a particular subject can be readily determined by the medicalpractitioner in accordance with conventional techniques in the medicinal art.
The following examples are illustrative of the invention.
EXAMPLES
EXAMPLE I
(Preparation of Cannabichromene)
To a three-necked round bottomed flask (100 ml capacity), fitted with a dropping funnel and a condenser was added 5 g. olivetol (27.8 mmole) and 2.03 g. (2.96 ml., 27.8 mmole) t-butyl amine in 55 ml toluene and the mixture was heated to50°-60° C., 4.23 g. (4.76 ml., 27.8 mmole) of citral was then added dropwise. The mixture was refluxed for 9 hours, after which time it was cooled to room temperature and the solvent evaporated to give 9.3 g. of crude reaction mixture. Gas chromatographic analysis of the reaction mixture showed 59.46% CBC (molar conversion), 5.04% cannabicitran and trace amount of iso-CBC.
EXAMPLE II
(Purification of Cannabichromene)
5 g. of the crude reaction mixture from Example I was dissolved in 100 ml. benzene and the solution extracted twice with 50 ml. of 1% aqueous sodium hyroxide solution followed by 50 ml. of water. The benzene solution was then dried overanhydrous sodium sulfate and the solvent evaporated. The residue was then dissolved in 50 ml. ethanol, and 250 mg. of sodium borohydride were added portion-wise while stirring.
Stirring at room temperature was continued for 30 minutes after which time the solvent was evaporated and the residue partitioned between water (50 ml.) and benzene (100 ml.). The crude reaction mixture was chromatographed on a column ofprocessed silica gel (200 g.). Processed silica gel was prepared by making a paste of silica gel -PF254 with water (equal amount) which was then dried in an oven at 110° and the resulting cake passed through 60 mesh sieve. The solvent systemused was a mixture of benzene and chloroform (1:1). Fractions were collected and combined according to their tlc similarities in the same solvent system. Fractions containing pure CBC were combined, the solvent evaporated and the residue (2.1 g.) wasanalyzed by GC method and found to be 97% pure CBC. The synthetic CBC was found to be identical in all respects (tlc, GC, ir, uv, 1 H nmr and 13 C-nmr) with authentic CBC [2-methyl-2-(4-methyl-pent-3-enyl)-5-hydroxy-7-pentyl chromene].
EXAMPLE III
The procedure of Example II was followed, except the crude reaction mixture was dissolved in cyclohexane instead of benzene. The same results were obtained.
EXAMPLE IV
(Preparation of Cannabichromene)
To a three-necked, 2 liter round bottomed flask fitted with a condenser, a dropping funnel and a mechanical stirrer, was added 90 g. (0.5 mole) of powdered olivetol and 36.5 g. (53.2 ml., 0.5 mole) t-butylamine and the mixture dissolved in 1liter toluene. The reaction mixture was then stirred for awhile when a brownish white gelatinous material appeared. The mixture was then heated to 50° C. and stirred constantly. To the resulting clear brown solution was added 76 g. (85.6 ml.,0.5 mole) citral dropwise. After complete addition of citral, the reaction mixture was refluxed for 9 hours, cooled to room temperature and the solvent evaporated to give 166.8 g. of crude reaction mixture.
Dry weight analysis of the crude mixture using gas liquid chromatography showed 62.05% CBC, 5.09% Cannabicitran and trace amount of iso-CBC. Cannabichromene was purified and chromatographed in the same manner as previously described underExample II.
EXAMPLE V
(Preparation of Cannabichromene)
The reaction of 5 g. olivetol (27.8 mmole), 1.652 g. n-propylamine (2.3 ml., 27.8 mmole) and 4.6 ml. citral (27.8 mmole) in 55 ml. toluene was carried out in the same manner as described under Example 1. The refluxing time was 7 hours. Gaschromatographic analysis of the reaction mixture showed 61.62% CBC, 4.01% Cannabicitran and trace amount of iso-CBC.
Purification and chromatography of CBC were carried out as described under Example II.
EXAMPLE VI
(Preparation of the Methyl Homologues of Cannabichromene)
Th procedure described in Example I was followed, except 3.45 g of orcinol was reacted in place of the 5 g. of olivetol. Gas chromatographic analysis of the reaction mixture showed 48.27% yield of CBC-C1.
EXAMPLE VII
(Purification of CBC-C1)
The product of Example VI was purified according to the process of Example II. The product was found to be pure CBC-C1 as a pale yellowish oil, the identity of which was determined as2-methyl-2(4-methyl-pent-3-enyl)-5-hydroxy-7-methylchromene by comparison of the spectral data with those of CBC.
EXAMPLE VIII
[Inhibition of Inflammation of CBC, as Measured by the Rat-Paw Edema Test (Intraperitoneal Injection)]
A. Procedure
The test rats were divided into test groups of 6 to 8 animals, weighed and marked so that the individual rats could be identified; all rats were given a 700 mg/kg intraperitoneal injection of ethyl urethane in distilled water to render themtractable during testing. A circle was drawn, with a felt-tipped mixer, around the hind leg of each rat just above the ankle, and each rat was dosed with a test or control compound by intraperitoneal injection. Test compounds were given at doses of 60,120, 240, or 480 mg/kg. The negative control was the vehicle which was used to give the test compounds and the two positive controls were phenylbutazone given at 120 mg/kg and 60 mg/kg. Phenylbutazone was prepared for injection by suspending it innormal saline using Tween 60. The rats were then held in group cages for 30 minutes. The volume of the left hind paw was measured using a mercury displacement pleysmograph. The paw was dipped into the mercury until the mercury touche the line abovethe ankle. Mercury was then withdrawn until the mercury returned to its original level. The amount of mercury removed was measured in milliliters (ml). The mercury can be measured accurately to 0.01 ml. The left hind paw was then injected with 0.1 mlof a 5% w/v solution of viscous carageenen in normal saline. The injection was given between the metatarsal bones using a 27 gauge needle, and the rats then held in group cages for 3 hours. The volume of the left hind paws was measured again in thepleysmograph. The results were computed in the following manner:
a. A mean is taken for both the preinjection and post-injection paw volume of each test group.
b. The mean difference in volume (MDV) for each test group is computed by subtracting the preinjection mean from the postinjection mean.
c. The percent of control is computed for each test group as: ##EQU1## The percent of control is used to compare the efficacy of the various drug treatments.
d. The percent increase in paw volume is calculated for each group as: ##EQU2## The percent increase is used to compare the amount of edma observed in one experiment to the amount of edma observed in other experiments.
B. Results
The results of the rat-paw edema test are summarized in Table 1. As can be seen, doses of 120, 240, and 480 mg/kg all produced strong antiinflammatory effects. The effects were shown to be dose related, that is, higher doses of CBC producedstronger antiinflammatory effects. All the animals receiving 480 mg/kg died within 2 days of injection, but this cannot be judged to be simply a result of CBC toxicity since the rats also received an IP injection of 750 mg/kg of ethyl urethane as partof the test procedure. Seven of the eight animals receiving 240 mg/kg of PBZ died before the test could be completed. The eighth rat died within 24 hours of injection.
The data from the rat paw test were further analyzed using a one-way analysis of varience (ANOVA) and Duncan's New Multiple Range Test. The results of these tests are given in Tables 2 and 3. The pre-injection score of each animal wassubtracted from his post-injection score and an analysis of the different scores was conducted. The analysis showed that all test groups differed significantly from the vehicle control group. The 120 mg/kg dose of CBC differed significantly from the240 and 480 mg/kg doses of CBC, and the 480 mg/kg dose of CBC differed significantly from the 120 mg/kg dosage of PBZ.
No significant effects were seen on the CNS screen at doses of CBC as large as 800 mg/kg in unanethetized mice.
TABLE 1 ______________________________________ RAT - PAW EDEMA DATA PERCENT COM- OF PERCENT POUND DOSE MDV CONTROL INCREASE ______________________________________ Vehicle 0.5 ml 0.463 100.000 42.383 Control CBC 120 MG/KG 0.139 30.00012.729 CBC 240 MG/KG 0.004 0.927 0.365 CBC 480 MG/KG -0.043 -9.189 -3.708 PBZ 120 MG/KG 0.106 22.973 10.316 ______________________________________ Percent of Control and Percent Increase Computed before Rounding MDVS or Group Means.
TABLE 2 ______________________________________ Analysis of varience for difference scores from rat-paw edema test. Source ΣX2 df MS F ______________________________________ Among 1.2291 4 0.3073 24.434** Within 0.4311 340.0128 Total 1.6602 38 ______________________________________ **p. < 0.01
TABLE 3 __________________________________________________________________________ Duncan' s Test for Difference Scores Rat-Paw Edema Test. Group -x I0 I1 I2 I3 SSR.sup. __________________________________________________________________________ Vehicle 0.170 control 0.463 -- .0324** 0.356** 0.458** 0.505** 0.128 CBC 0.167 120mg/kg 0.139 -- -- 0.033 0.134* 0.181** 0.125 PBZ 0.162 120mg/kg 0.016 -- -- --0.102 0.149* 0.121 CBC 0.156 240mg/kg 0.004 -- -- -- -- 0.047 0.115 CBC 480mg/kg 0.043 -- -- -- -- -- Vehicle CBC PBZ CBC CBC control 120mg/kg 120mg/kg 240mg/kg 480mg/kg __________________________________________________________________________ *p. <0.05 **p. <0.01 Shortest significant range for the 0.05 and 0.01 levels of significance.
EXAMPLE IX
[Inhibition of Inflammation of CBC as Measured by the Rat-Paw Edema Test (oral administration]
A. Procedure: The procedure described in Example VIII was followed, except the rats were dosed by oral gauge instead of i.p. injection. The test was conducted twice, once with nonfasted rats and once with rats that had been fasted during the 24hour period prior to dosing. The 60 mg/kg Penylbutazone control group was not used in the test with fasted animals. Tween 60 in normal saline was the vehicle control for the non-fasted rats and normal saline was the vehicle control for the fasted rats.
B. Results: The results are given in Tables 4 and 5. As can be seen from the tables, cannabichromene was active at all the doses tested. The degree of inhibition of edema increased in both tests as the amount of CBC given was increased. Thedegree of inhibition was greater in the fasted rats than it was in the nonfasted rats. This would be expected since the fasted rats should absorb the test compound more readily than the non-fasted rats.
When CBC is compared to PBZ in Tables 1 and 2 it is seen that PBZ was slightly more effective than CBC at 120 mg/kg in the nonfasted rats and that CBC and PBZ were about equally effective at 120 mg/kg in the fasted rats. The higher doses of CBCwere generally more effective in inhibiting edema than was PBZ. PBZ was not given at higher doses because of the rapid deaths produced by 240 mg/kg of PBZ given intraperitoneally in the test described in Example VIII.
TABLE 4 ______________________________________ RAT - PAW EDEMA DATA Oral CBC in Non-Fasted Rats. Run 11-6-1978 COM- PERCENT OF PERCENT POUND DOSE MDV CONTROL INCREASE ______________________________________ Vehicle 0.5 ml 0.625 100.00058.140 Control CBC 120 MG/KG 0.496 79.400 41.311 CBC 240 MG/KG 0.412 66.000 38.372 CBC 480 MG/KG 0.315 50.400 29.200 PBZ 60 MG/KG 0.489 78.200 50.257 PBZ 120 MG/KG 0.392 62.667 36.098 ______________________________________ Percent of Control andPercent Increase Computed Before Rounding MDVS or Group Means.
TABLE 5 ______________________________________ RAT - PAW EDEMA DATA Oral CBC in Fasted Rats. Run 11-14-1978 PERCENT PERCENT OF IN- COMPOUND DOSE MDV CONTROL CREASE ______________________________________ Saline Vehicle Control 0.5 ml 0.625100.000 46.339 CBC 120 MG/KG 0.409 65.400 31.382 CBC 240 MG/KG 0.209 33.400 15.981 CBC (given fol- 480 MG/KG 0.281 45.000 22.321 lowing saline infusion) PBZ 120 MG/KG 0.403 64.400 31.051 ______________________________________ Percent of Controland Percent Increase Computed Before MDVS or Group Means. *Doses are Approximate Due to an Error in Procedure.
EXAMPLE X
[Inhibition of Inflammation by CBC as Measured by Inhibition of Erythema (Red-Blood Cell Hemolysis)]
A. Procedure: The red blood cells (RBC's) were sensitized by washing twice with a volume of saline equal to the initial blood volume. CBC was suspended in a 2% ethanol in saline solution because of its insolubility in water. The procedure wasduplicated successfully 3 times using either 40% or 20% RBC suspensions.
B. Results: CBC was screened for inhibition of red cell hemolysis. The results of the tests are shown in Table 6. Phenylbutazone (PBZ), aspirin and tolmetin were used as positive controls and all inhibited heat induced hemolysis at theconcentration tested. Inhibition of hemolysis was dose-related in the positive controls and cannabichromene groups (Test 3). CBC produced 35% inhibition of heat-induced red cell hemolysis at 10-4 M test concentration and 26% at 2×10-5 MCBC. PBZ produced 16% and 10% inhibition of red cell hemolysis at the 10-4 M and 2×10-5 M test levels, respectively. Aspirin produced a 40% inhibition at the 5×10-4 M Test concentration.
It was apparent that not all the CBC actually went into suspension. In order to determine if some of the CBC adhered to the wall of the glassware a 10 ml aliquot of the 2×10-4 (solution a) of CBC saturated with Nacl was extracted withCHcl3 and analyzed by G.C. The solution was found to be 2×10-5 M CBC or to contain 0.3 mg of the original CBC. The flask was rinsed with ethanol and the washings analyzed by GC and found to contain 1.355 mg of CBC. The remaining 1.475mg (50%) of CBC that could not be accounted for by G.C. analysis may have been lost during the extraction procedure or while adjusting the pH of the test solution. If the 1.475 mg CBC which could not be accounted for was actually in suspension then themaximum final concentration of CBC used in the highest CBC test level would be 5×10-5 M or one half the amount listed in Table 6. In summary, CBC, at a minimum, produces 2 to 21/2 times more inhibition of heat induced red cell hemolysis thandoes PBZ at equimolar concentrations. Inhibition of heat-induced hemolysis was seen over a range of 10-4 M to 2×10-6 M CBC. The actual activity of CBC may have been 3 to 30 times more protective of red cell membranes than an equivalentamount of PBZ.
TABLE 6 ______________________________________ (Inhibition of Heat-Induced Erythrocyte Hemolysis) Final Concentration of Percent Inhibition the TEST SOLUTION TEST 1* TEST 2 TEST 3 ______________________________________ Phenylbutazone 2.5× 10-4 M 54 70 49 1.0 × 10-4 M 16 2.5 × 10-5 M 31 2.0 × 10-5 M 10 Acetylsalicylic Acid 5.0 × 10-4 M 40 2.5 × 10-4 M 21 Tolmetin 2.0 × 10-4 M 27 Cannabichromene 1.0× 10-4 M 40 37 1.0 × 10-4 M 33 2.0 × 10-5 M 26 26 2.0 × 10-6 M 7 ______________________________________ *A 20% RBC suspension was used for Tests 1 and 3. A 40% RBC solution was used for Test 2.
EXAMPLE XI
(Inducement of Hypothermia by CBC in Mice)
A. Procedure: CBC (100 mg/kg was prepared in emulsion form with 3% Tween 60 and 3% Arlacel in normal saline (0.9%) in such a way as to permit a volume of 10 ml/kg of body weight to be injected intraperitoneally into male mice weighing between 32and 38 grams.
The animals were divided into groups of 10, and body temperatures were recorded with a multichannel Yellow Springs Telethermometer and Thermistor probes. During the experiment, the mice were confined in plastic restraint tubes. They wereallowed 45-60 min. for adaption to the restraint tubes and to rectal thermistor before a pre-drug baseline temperature reading was taken. Body temperature readings were obtained at 0.5, 1.0, 2.0, 3.0 and 4.0 hrs. post-CBC administration.
The mean (. -.S.E.) decrease in temperature from the preinjection baseline reading for saline-, vehicle-, and CBC-treated mice were calculated and statistical analyses were conducted using the two-tailed Student's t test.
B. Results: As summarized in Table 7, both the CBC- and vehicle control-treated groups showed a consistent drop in body temperature over the duration of the experiment, with the CBC-induced hypothermia being more pronounced and significantlydifferent from saline controls at all readings, whereas the vehicle induced decrease in body temperature was only significant from saline controls at the 2.0, 3.0 and 4.0 hr. readings.
TABLE 7 __________________________________________________________________________ Mean (. -.S.E.) Decrease in Rectal Temp. (°0) Time (hrs.) Treatment 0.5 1.0 2.0 3.0 4.0 __________________________________________________________________________ Normal Saline 0.43 . -. 0.12 0.44 . -. 0.07 0.47 . -. 0.07 0.61 . -. 0.14 0.60 . -. 0 Vehicle Control 0.58 . -. 0.16 0.80 . -. 0.16 1.62 . -. 0.16* 1.35 . -. 0.14* 1.04. -. 0* CBC (100 mg/kg) 1.86 . -. 0.18Δ * 2.52 . -. 0.26Δ * 1.93 . -. 0.21* 1.53 . -. 0.21* 1.61 . -. 0*Δ __________________________________________________________________________ *Significantly different fromsaline controls, P ≤ 0.05. ΔSignificantly different from vehicle control, P ≤ 0.05.
In spite of the vehicle's activity, the hypothermia induced by CBC was still significantly different from vehicle controls at 0.5, 1.0 and 4.0 hrs.
The peak hypothermic effect (at a dose of 100 mg/kg, i.p.) for CBC was attained within the first two hours and then declined thereafter.
EXAMPLE X
[Inhibition of Inflammation by CBC-C1 as Measured by the Rat-Paw Edema Test (intraperitoneal administration)]
A. Procedure: The procedure described in Example VIII was followed. CBC-C1 was tested at doses of 60 mg/kg and 120 mg/kg prepared in an emulsion with Tween 60, Arlacel and distilled water. The vehicle control used was Tween 60, Arlacel anddistilled water prepared without CBC-C1.
B. Results: The results are given in Table 8. CBC-C1 was active at both 60 mg/kg and 120 mg/kg. The inhibition of edema was dose related. When the effects of CBC-C1 were compared with those of PBZ it can be seen that CBC-C1 didreduce the rat paw edema with slightly less activity than PBZ at the 120 mg/kg dose and was as active as PBZ at 60 mg/kg. No toxic or adverse effects were observed in any of the rats tested.
TABLE 8 ______________________________________ RAT - PAW EDEMA DATA CBC-C1 given i.p. in fasted rats. Run 5-10-79 COM- PERCENT OF PERCENT POUND DOSE MDV CONTROL INCREASE ______________________________________ Vehicle 0.00 MG/KG 0.191100.000 13.947 Control CBC-C1 120 MG/KG 0.070 36.601 4.956 CBC-C1 60 MG/KG 0.090 47.059 6.742 PBZ 120 MG/KG 0.046 24.183 3.254 PBZ 60 MG/KG 0.090 47.059 6.742 ______________________________________ Percent of Control and PercentIncrease Computed Before Rounding MDVS or Group Means.
Although this invention has been described with reference to illustrative embodiments thereof, it will be apparent to those skilled in the art that the principles of this invention can be embodied in other forms within the scope of the followingclaims.
Other References
Kane et al., JACS, 90, 6551 (1968)
Crombie et al., J. Chem. Soc. (C), 796 (1971)
Crombie et al., J. Chem. Res. S (Synopses), 1977, (5), 114-115, (C.A. 87:102470s)
Source: Process for preparing cannabichromene - US Patent 4315862 Description
