New 16,17-pyrazolino- and 16,17-isopyrazolino-1,4-pregnadiene-derivatives have been prepared, useful as antiinflammatory agents on topical and/or systemic administration. An analogy process for the preparation of 16,17-pyrazolino-1,4-pregnadiene derivatives and the quite surprising conversion of the latter ones to 16,17-isopyrazolino-1,4-pregnadiene derivatives are also disclosed. The results of pharmacological comparative trails of some new products of the present invention v. fluocinolone acetonide are reported.

BACKGROUND OF THE INVENTION 
The 16.alpha.,17.alpha.-pyrazolino-pregnane-derivatives characterized by 
the grouping 
##STR1## 
in the 16,17-position of the steroid skeleton are widely described in the 
literature. However said derivatives are limited to steroids of the 
.DELTA..sup.5 -pregnene- or to the .DELTA..sup.4 -pregnene series, such as 
derivatives of pregnelone, progesterone, hydrocortisone and the like. See, 
for instance, British Pat. No. 901.092 (application date 22.7.1957), U.S. 
Pat. No. 3,359,287 (application date 16.11.1959), U.S. Pat. No. 3,350,394 
(application date 26.4.1961), U.S. Pat. No. 3,086,029 (application date 
12.1.1962), Brit. Pat. Nos. 923,623 and 923,624 (application date 
1.10.1958). To our best knowledge there is only one example of a 
.DELTA..sup.1,4 -pregnadiene-derivative bearing a 
16.alpha.,17.alpha.-pyrazolino-group, namely 
9.alpha.-fluoro-16.alpha.,17.alpha.-methyleneazo-.DELTA..sup.1,4 
-pregnadiene-11.beta. 21-diol-3,20-dione 21-acetate, reported in the 
literature (J.Org.Chem., 29, 3486-95, 1964). However in all the above 
reported patents and publication, the 
16.alpha.,17.alpha.-pyrazolino-derivatives have been described or reported 
as mere intermediates for the preparation of 16.beta.-methyl-, 
.DELTA..sup.16 -16 -methyl-, 16.alpha.,17.alpha.-methylene- and 
16-methylene-17.alpha.-hydroxy-derivatives. Neither direct nor indirect 
indications have been made in the literature that the 
16.alpha.,17.alpha.-pyrazolino-derivatives show pharmacological and 
therapeutic activities. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a new class of anti-inflammatory steroids 
and to the preparation thereof. It is an object of this invention to 
provide new steroid derivatives of the pregnane series which possess 
valuable pharmacological properties, particularly anti-inflammatory and 
anti-rheumatoid arthritic activity with practical absence of side-effects 
such as sodium retention, adrenal inhibition, thymolytic activity and the 
like present in certain known physiologically active steroids. The new 
compounds of the present invention may be administered by topical, by 
interarticular, and by systemic route. 
The new compounds of the present invention are 
16.alpha.,17.alpha.-pyrazolino-.DELTA..sup.1,4 -pregnadiene derivatives of 
the following 2 general formulae I and II 
##STR2## 
in which A may be a hydrogen or a bromine or chlorine atom; 
X may be a hydrogen, or a fluorine or chlorine atom; 
Y may be a .beta.-hydroxyl, or a keto-group or a chlorine atom; 
Z may be a hydrogen, or an .alpha.- or .beta.-fluorine atom or an 
.alpha.-methyl-group; 
R may be hydrogen or an acyl residue selected from the group consisting of 
a mono- or dicarboxyl organic acid having from 2 to 8 carbon atoms, of 
methasulfobenzoic acid and of phosphoric acid. 
It is a further object of the present invention to provide pharmaceutical 
compositions for use in the topical, intraarticular and systemic treatment 
of a wide range of inflammatory disorders, rheumatoid arthritis, allergic 
diseases and the like, which comprises incorporating an effective amount 
of compounds of formulae I and/or II together with a suitable carrier for 
said steroid. 
The compounds of formula I can be prepared from a 1,4,16-pregnatriene 
derivative of the general structural formula III: 
##STR3## 
wherein Ac indicates an acetyl-group, and A, X, Y, and Z have the same 
meaning as heretofore reported for formulae I and II. 1,4,16-Pregnatriene 
derivative is thus reacted with diazomethane according to general methods 
known from the literature involving a .DELTA..sup.16 -pregnene-substrate, 
for instance see British Pat. No. 901,092 (application date 22.7.1957). 
The compounds of formula II can be prepared from the compounds of formula 
I by reaction of the latter ones with a strong mineral acid such as 
hydrochloric acid, hydrobromic acid, hydrofluoric acid, perchloric acid 
and the like. The concentration of said acids, the temperature, and 
duration of the reaction are not critical, but it is preferred to operate 
with aqueous concentrate acids, in the cold and for a short period of 
time. 
This conversion is a quite surprising result, as it is well known from the 
literature (U.S. Pat. No. 3,086,029, column 2 and 8, lines 39-48) that by 
reaction of 16(17)-diazomethane adduct with a strong acid a 
16.alpha.,17.alpha.-methylene-pregnane derivative is obtained. 
The compounds of structure II do indeed represent one of the most 
surprising aspect of the present invention. 
In fact their structure, the simple and smooth process for preparing them, 
as well as their unexpected useful pharmacological activity are--so to 
say--the three points confirming the great interest of the present 
invention. As far as their structure is concerned, there exists only one 
example in the literature (U.S. Pat. No. 3,359,287 column 1) in which an 
`isomer` structure 
##STR4## 
is attributed to the so-called "diazomethane adduct II", instead of the 
more likely one 
##STR5## 
However in the same U.S. Pat. No. 3,359,287, column 3, lines 4-10 there is 
quoted the following sentence, which "weakens the firm statement of the 
structure reported for compound II in column 1": " . . . For convenience 
in structural representation, the double bond in the adduct portion of the 
molecule is represented as being between a carbon and a nitrogen atom. 
However the double bond may be elsewhere in the ring and it is to be 
understood that II is intended to represent the reaction product of a 
pregnadienolone (I) and diazomethane . . . ". This sentence makes the 
actual structure indeterminate and vague. 
We succeeded to ascertain unequivocally the structures of both compounds of 
formula I and II by means of an accurate investigation with mass 
spectrography, infra-red spectrophotometry and nuclear magnetic resonance. 
Compounds of formula II are moreover characterized by physico-chemical 
parameters quite different from those of compounds of formula I, 
especially as far as specific rotatory power and the E.sub.1 cm.sup.1% at 
UV. spectrophotometry are concerned. 
In fact the rotatory power values are 6 times higher, and the E.sub.1 
cm.sup.1% -values are 10% higher than the corresponding values of 
compounds of formula I. 
In particular the structure of compounds II was confirmed by means of: 
mass spectrography, which gave a molecular weight identical to that of 
compounds of structure I; 
infra-red spectrophotometry, which showed the disappearance of the peak at 
1,540 cm.sup.-1, characteristic of the double bond --N.dbd.N--; 
N.M.R., which showed a singulet at 7,90.delta. due to double bond 
--CH.dbd.N--. 
Moreover we have found that the conversion Compound I.fwdarw.Compound II is 
an irreversible process, the thermodynamic equilibrium being completely 
shifted towards Compound II, which represents the form with the lowest 
level of free energy. 
In order to distinguish compound I from compound II by chemical name, we 
have tentatively called the former one as: 
`16.alpha.,17.alpha.-pyrazolino-1,4-pregnadiene-derivative` and the latter 
one as: `16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-derivative`. 
The Compound II of the present invention, in which R is an acyl residue 
different from acetyl, and it is selected from the group consisting of a 
mono- or dicarboxyl organic acid having from 3 to 8 carbon atoms, of 
methasulfobenzoic acid and of phosphoric acid, may be prepared by starting 
from Compound II in which R is the acetyl residue according to an analogy 
process as hereinbelow explained. The analogy process consists essentially 
in a suitable alkaline hydrolysis of Compound II in which R is the acetyl 
residue under nitrogen atmosphere to give the corresponding 21-alcohol, 
and then in a suitable acylation of said 21-alcohol to give the desired 
21-ester. The details are given in Examples from 7 to 16.

The following EXAMPLES illustrate methods of carrying out the present 
invention but it is to be understood that these examples are given for the 
purpose of illustration and not of limitation. 
EXAMPLE 1 
16.alpha.,17.alpha.-Pyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta.,21- 
diol-3,20-dione-21-acetate (compound I in which X=F, Y=OH, Z=H, A=H, 
R=--CO--CH.sub.3)--Laboratory code Pyr--F--AC) 
To a two phase solution of 53 ml of aqueous 40% potassium hydroxide and 240 
ml of ethyl ether, 17,7 g of nitroso-methylurea were slowly added with 
stirring and cooling in an ice bath. 
To the resulting ether solution of diazomethane 3 g of 
9.alpha.-fluoro-1,4,16-pregnatriene-11.beta.,21-diol-3,20-dione 21-acetate 
(compound III, in which X=F, Y=OH, Z=H, A=H) dissolved in 50 ml of 
methylene chloride were added with stirring and the temperature was kept 
at 10.degree.-15.degree. C. 
The reaction mixture was maintained at this temperature for one hour and 
the excess of diazomethane was decomposed with acetic acid at 0.degree. C. 
The solution was washed with water, dried over anhydrous sodium sulfate, 
filtered and then concentrated "in vacuo" to dryness. 
The residue was triturated with 20 ml of isopropyl ether, filtered and 
dried to give 2.8 g of the desired compound I, 
M.p. 195.degree. C. 
UV. Spectrum: .lambda.max.=230 m.mu.; E.sub.1 cm.sup.1% =367.7. 
[.alpha.]=+63.degree. (c=1%, dioxane). 
IR. Spectrum (nujol): 3315-1760-1730-1660-1615-1605-1540 cm.sup.1. 
EXAMPLE 2 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta., 
21-diol-3,20-dione-21-acetate (Compound II in which X=F, Y=OH, A=Z=H, 
R=--CO--CH.sub.3 ; laboratory code 16-17 MF-AC) 
To 15 ml of 36% aqueous hydrochloric acid cooled to 0.degree. C., 2.8 g of 
the compound I obtained as described in Example 1, were added with 
stirring. The reaction mixture was kept at 0.degree. under stirring for a 
further 15 min., then it was poured in 150 ml of water and crushed ice. 
The precipitate thus formed, was filtered, washed with water, and dried. 
Yield: 2.5 g of crude Compound II. 
Upon crystallization from chloroform-methanol 2 g of pure compound II were 
obtained showing the following characteristics: 
m.p. 225.degree. C.; [.alpha.].sub.D =+311 (c=1%, dioxane); 
UV. Spectrum: .lambda.max.=238 m.mu.; E.sub.1 cm.sup.1% =414. 
IR. Spectrum (nujol): 3530-3300-1735-1705-1665-1620-1605 cm.sup.-1. 
EXAMPLE 3 
16.alpha.,17.alpha.-Pyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluoro-1 
1.beta.,21-diol-3,20-dione 21-acetate (Compound I in which X=F, Y=OH, 
Z=.alpha.F, R=COCH.sub.3, A.dbd.H; laboratory code Pyr.Dif-AC) 
By starting from 
6.alpha.,9.alpha.-difluoro-1,4,16-pregnatriene-11.beta.,21-diol-3,20-dione 
-21-acetate (compound III, X=F, Y=OH, Z=.alpha.F, A=H) and by operating 
according to the method illustrated in Example 1, the above compound I was 
obtained, showing the following characteristics: 
m.p. 198.degree. C.; [.alpha.].sub.D =+56.6.degree. (c=1%, dioxane); 
UV. Spectrum: .lambda.max=238 m.mu.; E.sub.1 cm.sup.1% =376.16. 
IR. Spectrum (nujol): 3500-1740-1725-1665-1630-1605-1550 cm.sup.-1. 
EXAMPLE 4 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluor 
o-11.beta.,21-diol-3,20-dione 21-acetate (compound II: X=F, Y=OH, 
Z=.alpha.F, A=H, R= --COCH.sub.3 ; laboratory code 16-17 M Dif-AC) 
By starting from 
16.alpha.,17.alpha.-pyrazolino,1,4-pregnadiene-6.alpha.,9.alpha.-difluoro- 
11.beta.,21-diol-3,20-dione 21-acetate (prepared according to Example 3) 
and by operating according to the method illustrated in Example 2, the 
title compound II was prepared, showing the following characteristics: 
m.p. 224.5.degree. C.; [.alpha.].sub.D =309.5.degree. C. (c=1%, dioxane); 
UV. Spectrum: .lambda.max.=238 m.mu.; E.sub.1 cm.sup.1% =416. 
IR. Spectrum (nujol): 3580-3280-1735-1725-1670-1620-1605 cm.sup.-1. 
EXAMPLE 5 
16.alpha.,17.alpha.-Pyrazolino-1,4-pregnadiene-11.beta.,21-diol-3,20-dione 
21-acetate (compound I: A=X=Z=H, R.dbd.COCH.sub.3 ; laboratory code 
Pyr-T.sub.1) 
By starting from 1,4,16-pregnatriene-11.beta.,21-diol-3,20-dione 21-acetate 
(compound III: A=X=Z=H, R=COCH.sub.3) and by operating according to the 
method illustrated in Example 1., the title compound I was prepared, 
showing the following characteristics: 
IR. Spectrum (nujol): 3400-1740-1725-1655-1620-1600-1550 cm.sup.-1. 
EXAMPLE 6 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-11.beta.,21-diol-3,20-dio 
ne 21-acetate (compound II: A=X=Z=H, R=COCH.sub.3 ; laboratory code 16-17 
MT.sub.1) 
By starting from the Compound I prepared in the previous Example and by 
operating according to the method illustrated in Example 2, the title 
compound II was obtained. 
EXAMPLE 7 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta., 
21-diol-3,20-dione (compound II in which X=F, Y=OH, A=Z=H, R=--H; 
laboratory code 16-17 MF) 
To a suspension of 10 g of 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta. 
,21-diol-3,20-dione-21-acetate (prepared according to Example 2) in 200 ml 
of methanol kept at 0.degree..+-.2.degree. C. under continuous bubbling of 
nitrogen and with stirring a solution of 2 g of sodium bicarbonate in 20 
ml of water was added. Stirring was continued for a further 45 minutes. 
The reaction mixture was neutralized with acetic acid and then the 
methanol was distilled off "in vacuo". The resulting suspension was cooled 
to 0.degree. C. and filtered, the crude product was washed with water and 
dried "in vacuo". 
Yield 7,1 of 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta. 
,21-diol-3,20-dione. 
EXAMPLE 8 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluor 
o-11.beta.,21-diol-3,20-dione (Compound II: X=F, Y=OH, Z=F, A=H, E=--H; 
laboratory code 16-17 M Dif) 
By starting from 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluo 
ro-11.beta.,21-diol-3,20-dione 21-acetate (prepared according to Example 4) 
and by operating according to the method illustrated in Example 7, the 
title compound II was prepared. 
EXAMPLE 9 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta., 
21-diol-3,20-dione-21-propionate (compound II in which X=F, Y=OH, A=Z=H, 
R=--CO--CH.sub.25 ; laboratory code 16-17 MF-Pr) 
To a solution of 2 g of 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta. 
,21-diol-3,20-dione (prepared according to Example 7) in 30 ml of anhydrous 
pyridine at room temperature a mixture of 1 ml of propionic anhydride and 
10 ml of anhydrous tetrahydrofuran was added dropwise over a period of 30 
minutes with stirring. 
The reaction mixture was kept for a further three hours, then it was poured 
into a separatory funnel containing 300 ml of icy distilled water and 10 
ml of concentrated sulfuric acid. The mixture was thoroughly shaken 3 
times with 50 ml of methylisobutylketone each time. 
The combined organic layer was shaken with a saturate aqueous solution of 
sodium bicarbonate and with water, then it was dried over anhydrous 
magnesium sulfate. The solution was concentrated "in vacuo" to a volume of 
about 10 ml. The residue was taken up with 50 ml of isopropyl ether and 
kept at 0.degree. C. for three hours. The precipitate thus formed was 
filtered and washed with isopropyl ether. The crude product was 
recrystallized from acetone-hexane. 
Yield 1,5 g of the title compound showing the following characteristics: 
IR. Spectrum (nujol): 3570-3350-3290-1740-1725-1670-1620-1605 cm.sup.-1. 
EXAMPLE 10 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta., 
21-diol-3,20-dione-21-butyrate (laboratory code 16-17 MF Bu) 
To a suspension of 2 g of 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta. 
,21diol-3,20-dione (prepared according to Example 7) in 20 ml of anhydrous 
pyridine at room temperature a mixture of 1,2 ml of butyrric anhydride and 
12 ml of anhydrous tetrahydrofuran was added dropwise over a period of 20 
minutes with stirring. 
The reaction mixture was kept for a further 4 hours, at room temperature 
then it was poured into a flask containing 200 ml of icy distilled water 
and 7 ml of concentrated sulfuric acid. The solid thus separated was 
filtered, washed with water and dried "in vacuo" to constant weight. The 
crude product (2,1) was dissolved in 20 ml of acetone, decoulorized with 
50 mg of charcoal DARCO G 60. 
After filtration the solution was concentrated, then 45 ml of hexane were 
added and kept at 0.degree. C. overnight. 
The crystalline product was filtered and dried. Yield 1,3 g of the title 
compound showing the following characteristics: 
IR. Spectrum (nujol): 3370-3295-1740 (s)-1725-1670-1620-1605 cm.sup.-1. 
EXAMPLE 11 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta., 
21-diol-3,20-dione-21-valerate (laboratory code 16-17 MF-Va) 
In a flask there were poured 20 ml of anhydrous pyridine, 2 g of 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta. 
,21-diol-3,20-dione (prepared according to Example 7) and 4 ml of valeric 
acid anhydride. 
The reaction mixture was kept for a further 3 hours at room temperature, 
then it was poured into a separatory funnel containing 200 ml of icy 
distilled water and 7 ml of concentrated sulfuric acid. The mixture was 
thoroughly shaken 4 times with 50 ml of methylene chloride each time. The 
combined organic layer was shaken with a saturate aqueous solution of 
sodium bicarbonate and with water, then it was dried over anhydrous sodium 
sulfate. The solution was concentrated "in vacuo" to an oily residue, 
which was chromatographed over a column containing 150 g of Florisil. The 
column was then eluted with methylene chloride/acetone 9:1. 
The eluates were concentrated "in vacuo" to dryness. 
The residue was recrystallized from isopropyl ether/methylene chloride. 
Yield 1,3 of the title compound showing the following characteristics: 
IR. Spectrum: 3600-3315 (s)-3295-1750-1730-1720-1665-1620-1605 cm.sup.-1. 
EXAMPLE 12 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta., 
21-diol-3,20-dione-21-benzoate (laboratory code 16-17 MF-Bz) 
To a solution of 2 g of 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta. 
,21-diol-3,20-dione (prepared according to Example 7) in 30 ml of anhydrous 
pyridine cooled to -10.degree. C. a mixture of 1,2 ml of benzoyl chloride 
and of 12 ml of tetrahydrofuran was added dropwise over a period of 30 
minutes with stirring. The reaction mixture was kept for a further two 
hours at -5.degree. C., then it was poured into 300 ml of ice/water and 12 
ml of concentrated sulfuric acid. 
The solid thus separated was filtered, washed with water and dried "in 
vacuo" to constant weight. 
The crude product was chromatographed over a column containing 150 g of 
Florisil, then eluted with methylene chloride/ethyl ether 96/4. The 
eluates were concentrated "in vacuo" to dryness. The residue was 
recrystallized from acetone/ethylether. 
Yield 1,15 g of the title compound showing the following characteristics: 
IR. Spectrum (nujol): 3560-3330-1730-1710-1620-1615-1605-725 cm.sup.-1. 
EXAMPLE 13 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluor 
o-11.beta.,21-diol-3,20-dione-21-butyrate (laboratory code 16-17 M Dif-Bu) 
By starting from 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluo 
ro-11.beta.,21-diol-3,20-dione (prepared according to Example 8) and by 
operating according to the method illustrated in Example 10, the title 
compound was prepared, showing the following characteristics: 
IR. Spectrum (nujol): 3370-3320-1750-1720-1665-1620 (s)-1610 cm.sup.-1. 
EXAMPLE 14 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluor 
o-11.beta.,21-diol-3,20-dione-21-valerate (laboratory code 16-17 M Dif-Va) 
By starting from 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluo 
ro-11.beta.,21-diol-3,20-dione (prepared according to Example 8) and by 
operating according to the method illustrated in Example 11, the title 
compound was prepared, showing the following characteristics: 
IR. Spectrum (nujol): 3335-3300-1750-1720-1665-1620 (s)-1615 cm.sup.-1. 
EXAMPLE 15 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluor 
o-11.beta.,21-diol-3,20-dione-21-pivalate (laborabory code 16,17 M Dif-Piv) 
By starting from 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-6.alpha.,9.alpha.-difluo 
ro-11.beta.,21-diol-3,20-dione (prepared according to Example 8), by using 
pivaloyl chloride as acylating agent, and by operating as indicated in the 
previous Examples, the title compound was prepared showing the following 
characteristics: 
IR. Spectrum (nujol): 3660-3570-3350 (s)-3295-1740-1725-1670-1640-1605. 
EXAMPLE 16 
16.alpha.,17.alpha.-Isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta., 
21-diol-3,20-dione-21-succinic acid ester (laboratory code 16-17 MF-HS) 
By starting from 
16.alpha.,17.alpha.-isopyrazolino-1,4-pregnadiene-9.alpha.-fluoro-11.beta. 
,21-diol-3,20-dione (prepared according to Example 7), by using succinic 
anhydride as acylating agent, and by operating as indicated in the 
previous Examples, the title compound was prepared. 
Biological assays 
The topical anti-inflammatory activity of the new compounds of this 
invention was determined using the cotton granuloma assay. This test was 
performed according to C. A. Winter and C. C. Porter J. Am. Pharm. Ass. 
Sci., Ed. 46, 515. 1957 using adult female albino rats (Sprague-Dawley) 
average body weight 150 grams. 8 animals per group. 
The method consists in the subcutaneous implantation in the dorsal region 
of 2 sterile cotton pelletts. The pellets employed were 5 mm section cut 
from dental cotton rolls weighing about 45 mg each. The tested compound 
dissolved in ethyl alcohol, was absorbed on the pellets before 
implantation. Fluocinolone acetonide and Dexamethasone-21-acetate was used 
as comparison products and their activities were conventionally taken 
equal to 1 for each of the various parameters herein considered. 
Comparison substance and tested new compounds were absorbed on the pellets 
at the following scalar dosage per each pellet: 20-2-0.2 mcg/cotton 
pellet. For the controls the pellets were soakened in the pure solvent 
(ethyl alcohol) and then dried as usually. 
After seven days the animals were sacrified. The pellets were removed and 
the exudate weights were recorded as a measure of the granuloma formation. 
The degree of granuloma inhibition reflects the anti-inflammatory activity 
of the tested compounds. 
In the following Table 1. there are reported the achieved results with 
regard to the anti-inflammatory activity, the thymolitic activity, and the 
LD.sub.50 values: 
TABLE 1 
______________________________________ 
TOPICAL ADMINISTRATION 
Anti- 
inflammatory LD.sub.50 
activity Thymolytic 
(mg/kg body 
Product (*) (**) activity 
weight) 
______________________________________ 
Dexamethasone 21- 
1 1 1 259 
acetate 
Fluocinolone 
2 2 0.83 166 
acetonide 
Pyr-F-Ac .gtoreq.10 
.gtoreq.20 
0.14 721 
16-17 MF-AC &gt;10 &gt;20 0.02 &gt;2,000 
Pyr Dif-AC &gt;10 &gt;20 0.52 439 
16,17 M Dif-AC 
&gt;10 &gt;20 0.26 1,120 
______________________________________ 
(*)In comparison with Fluocinolone acetonide; 
(**)In comparison with Dexamethasone21-acetate. 
From the above data it results that after topical administration the new 
compounds showed significant anti-inflammatory activity and that they are 
practically free of thymolytic activity and remarkably less toxic than the 
comparative substance. 
EXPERIMENTAL RESULTS IN HUMAN VOLUNTEERS 
Some of the esters have been tested in human volunteers for their 
vasoconstrictive activity using the McKenzie and Stoughton's test (1) or 
the Falconi and Rossi's Paper-Patch Test (2) in comparison with a known 
anti-inflammatory corticosteroid, namely fluocinolone acetonide. 
In the first experiment 2 dilutions of 4 esters have been tested on 
volunteers and 10 dilutions have been applied for each subject. 
In the second trial only 16-17 M-Dif-Va which showed the best activity in 
the first trial has been further tested on 5 subjects using 5 dilutions of 
the test compound and the same dilutions of triamcinolone acetonide. 
The results of the experiments are reported in table 2 and 2a respectively. 
______________________________________ 
Results 
(Positive/ 
Compound Dilution No. of subjects 
% Positive 
______________________________________ 
Table 
Fluocinolone 
10.sup.4 5/5 100 
acetonide 10.sup.5 3/5 60 
16-17-M-F-Va 
10.sup.4 5/5 100 
10.sup.5 1/5 40 
16-17-M-Dif-Va 
10.sup.4 5/5 100 
10.sup.5 2/5 40 
16-17-Dif-Bu 
10.sup.4 3/5 60 
10.sup.5 1/5 20 
16-17-M-Dif-Piv 
10.sup.4 1/5 20 
10.sup.5 0/5 0 
Table 2a 
Triamcinolone 
10.sup.3 5/5 100 
acetonide 10.sup.4 5/5 100 
10.sup.5 3/5 60 
10.sup.6 2/5 40 
16-17-M-Dif-Va 
10.sup.3 5/5 100 
10.sup.4 4/5 80 
10.sup.5 4/5 80 
10.sup.6 3/5 60 
______________________________________ 
1. Mc Kenzie, A.W., Stoughton, R.B.: Method for comparing percutaneous 
absorption of Steroids. Arch. Derm. 86, 608, 1962. 
2. Falconi, G., Rossi, G.L.: PaperPatch Test for evaluating 
vasoconstrictive activity of Corticosteroids. Arch. Derm. 105, 856, 1972. 
From the above table it appears that all the esters tested but 
16-17-M-Dif-Piv. has a good vasoconstrictive activity as compared with the 
test compound beeing active at the higher concentrations used. One of 
them, 16-17-Dif-Va showed the same potency as fluocinolone acetonide even 
at the lower concentrations employed in the trials.