J. Med. Chem. 1993,36,3278-3285
3278
Non-Steroidal Glucocorticoid-like Substances: Receptor Binding and in Vivo Activity Virendra Kumar,' Malcolm R. Bell,*ll Joseph R. Wetzel, John L. Herrmann,f Ruthann McGarry, H. Phillip Schane,? Richard C. Winneker, Ben W. Snyder,$ and Anthony J. Anzalonef Sterling Winthrop Pharmaceuticals Research Division, Collegeville, Pennsylvania 19426-0900 Received April 22,19930
Compounds of general structure I, prepared by a Diels-Alder reaction with diene 3, are relatives of the known potent glucocorticoid I1 but possess a markedly modified C- and D-ring environment. Despite these structural changes, 4,5,9,10,12a, 13, and 14 bound to the glucocorticoid receptor with an affinity which approximated that of the reference standard, 6-a-methylprednisolone. Four of these compounds not only exhibited antiinflammatory activity in the a-tocopherol pouch test but also exhibited marked adrenal suppression and other typical glucocorticoid properties at doses in the same range as the effective antiinflammatory doses. The antiinflammatory steroids are effective and often life-saving drugs but still suffer from a constellation of serious side effects, the diminution of any one of which would result in an important therapeutic advance. Most of the early work in corticosteroid research was restricted to modifications of each position of the steroidal skeleton. These efforts resulted in the discovery of a number of activity-enhancing and activity-modifying substituents,l as illustrated by the attachment of [3,2-~]-2'-arylpyrazole derivatives to the A-ring of the corticosteroid. This resulted in compounds which were -2000 times more potent than hydrocrotisone in the rat.2p3 The 4-fluorophenylpyrazole I1 structural modification has emerged
Fi
4
I
+ Present address: Box 940, Stockbridge, MA 01262. t Present addreaa: Transgenic Sciences,Inc., 57 Union Street, Worceater, MA 01608. t Deceased. 1
Present addreas: RD 1, Box 156 A, East Greenbush, NY 12061.
* Abstract published in Advance ACS Abstracts, October 1, 1993.
as the most powerful activity-enhancing group with respect to antiinflammatory activitp from a large number of chemical modifications of the glucocorticoids. In a program aimed at finding novel compounds while retaining a major portion of the steroid nucleus for glucocorticoid-like activity, we have synthesized compounds represented by the general structure I. The advantage of these hybrid structures I is the possible dissociation of antiinflammatory activity and the associated side effects when compared to other compounds containing the intact steroidal skeleton. Identification of such an antiinflammatory steroid or non-steroidal compound which did not depress adrenal function would represent a significant structure-activity relationship breakthrough. We were interested in developinga Diels-Alder approach from a readily available steroid synthon l4which would yield the compounds represented by the general structure I. This would also allow us to synthesize steroid relatives in which the C- and D-ring environment could be varied widely. To increase the likelihood of achieving activity similar to that of traditional steroidal glucocorticoids, we have also incorporated the activity-enhancing group, 4-fluorophenyl pyrazole. It should be noted that earlier efforts to prepare nonsteroidal glucocorticoid-like compounds resulted in either inactive compoundsH or substances with weak activity.10-14 The topical activity (antiinflammatory and glucocorticoid) of the present compounds has been reported earlier.ls Chemistry. The target compounds were synthesized by the route shown in Schemes 1-111. The key step was the Diels-Alder condensation with both hetero- and carbodienophiles. The N-substituted pyrimidines 12-18 were prepared by alkylation of 11. Assignment of regiochemistry of the indantrione (ninhydrin) 4 and 5 and pyrimidinetetrone (alloxan) 6,7, and 11 adducts is based on their 'HNMR spectra which exhibited one allylic proton a to oxygen. The corresponding regioisomer would possess two such protons. It was not possible to determine the regiochemistry of the adducts 8-10 by this method. The assigned regiochemistry of adducts 8-10 is in accord with theoretical considerations and precedent,' that the C-8 hydrogen atom and the (2-10 methyl group16 were trans. It was possible to isolate the cis isomer 12b as a minor product from the Diels-Alder reaction which has been catalyzed by anhydrous SnC4 (12awas the major product). The structural assignments of 12a and 12b were made on
0022-262319311836-3278$04.00/0 0 1993 American Chemical Society
Non-Steroidal Glucocorticoid-like Substances
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 22 3279
Scheme 1.
0
*
a
H
o
1
d
*
b
2
YR2 R3
I
C
d R = 4-F-CsH4
3b R = G H 5 a
Reagents: (a) HCOZMe, NaOMe, THF; (b) HOAc, RNHNH2; (c) A, xylenes.
Scheme 11.
& 0
+
a
H
o
d
k
I7
21
22
Me
/
&
N( I
0
I
C
H
-
Reagents: (a) HCOZMe, NaOMe, Eh0;(b) HOAc, KOAc, 4-pyridylNHNH2; (c) NJV-dimethylalloxan, A, xylenes.
the basis of lH NMR and NOE experiments. Irradiation of (2-10methyl group resulted in 10-15% enhancement of C-11proton16 signal intensity for both the compounds. However, no enhancement of C-8 proton signal was observed for 12a. In contrast 12b showed an 11% enhancement of C-8proton thus providing the stereochemical assignment of the above two and related compounds. The 4,5-dihydro-5-a derivative16 7 was prepared as shown in Scheme I1 from the diene 21. The enones 24 and 25, the dienone 29, and for comparison the AeJ1 steroidal pyrazole 32 were prepared as outlined in Schemes I11 and IV. The physical properties of the compounds I are summarized in Table I. Biological Results and Discussion. The results of the biological evaluation of the compoundsI are presented in Table 11. Binding of a compound to the glucocorticoid receptor constitutes the primary event in the action of a glucocorticoid agonist. Depression of thymus weight,
depression of adrenal weight, depression of body weight gain without a change in food consumption, and antiinflammatory activity constitute significant aspects of the in vivo profile of a glucocorticoid in the rat. Depression of adrenal weight is an indirect measure of depression of pituitary function, and depression of body weight gain is an indirect measure of catabolic activity. Thymus involution is a direct consequence of glucocorticoid action on the thymus gland. Most of the compounds presented in Table I exhibited significant affinity to the glucocorticoid receptor. Compounds 4,5,9,10, and 12a bound with approximately the same affinity as the synthetic radioligand, dexamethasone. The pyridine derivative 8 bound more strongly than the ligand. The derivative 6 in which the fluorophenyl ring is replaced by a pyridine had much reduced affinity. The dihydro derivative 7 had no detectable affinity for the receptor. Any departures from the N-Me group of 12a among those variations examined resulted in a precipitous
Kumar et al.
3280 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 22
Scheme 111.
a
1
* 24
2 6 RR-=S s 27 O
J
d
28
25b 25a R=Me R=H
R
_7 e
29
Reagents: (a) ninhydrin, toluene; (b)LDA, 4,4'-dipyridyl disulfide, THF; (c) alloxan,A, xylenes; (d) mCPBA, CHCb; (e) NaH, DMF, MeI;
(0A, toluene. Scheme IV.
a
F Reagente: (a) HC02Me, NaOMe, MeOH; (b) 4-F-C&NHNHrHCl, NaOAc, HOAc, H2O.
decrease in binding to the receptor as did the change to the cis isomer 12b. Had the series been limited to an
examination of the pyrimidinetetrone (alloxan) and indantrione (ninhydrin)adducts a coherent receptor binding
Non-Steroidal Glucocorticoid-like Substances
Journal of-Medicinal Chemistry, 1993, Vol. 36, No.22 3281
Table I. Physical Properties of Compounds (I)
R
wmpd
R1
R2
R3 0
yield (% ) 54
~
4
4-F-CeH4
Qc 0
mp, O C 210-211
solvent EtOH
formula C2eHd"s
0
S
cas
0
48
6
4-pyridyl
0
43
213-215
EhO/EtOAc
C2aHd.504 1/3HzO
7
4-pyridyl(4,5-dihydro; 5a)
0
13
195-198
EtOAc/CH2C12
Cd-lnNaO4
8
4-F-Ca4
CHQ
30
154-156
EhO
CnHzeFNs
9 10 11
4-F-Ca4 4-F-CBF4 4-F-Ca4
CHAc C(COzEt)z 0
16 27 55
157-159 133-135 235-236
CHzCldisooctane MeOH EtOH
CdnFN202 CzeHsiFNiOi CuHaiFN104
12a
4-F-Ca4
0
42
188-189
Eta0
12b
4-F-Ca4
0
1
166-168
i-PrOH
13
4-F-Ca4
0
48
171-172
CHzClrheptane
14
4-F-Ca4
0
32
81-82
hexane-EtOAc
1s
4-F-Ca4
0
40
95-96
hexane-EtOAc
16
4-F-Ca
0
37
70-72
Eta0
17
4-F-Ca4
0
33
109-111
CHCb
18
4-F-Ca4
0
44
145-146
EhO
19
cas
0
26
253-254
MezCO
model could probably have been developed. However, inclusion in the group of the high affinity and structurally divergent group of adducts 8-10 apparently undermines that endeavor. A A4-&ketone or A1p4-3-ketoneare structural features present in all the potent nonheterocyclic steroidal glucocorticoids. These functional groups are present in the compounds 24,2Sa, 2Sb,and 29 which lack the arylpyrazole moiety. None of them exhibited receptor binding. That a double bond at C-9.C-11 is compatible with binding to the receptor in this 3-keto series is demonstrated by the significant affinity for the receptor of compound 32. The indantrione adduct 4 exhibited a profound effect on all four parameters at low doses. In comparison with
6-a-methylprednisolone 20, adduct 4 was about 5 times less potent as an antiinflammatory agent but considerably more potent as an adrenal suppressant. In a side by side assay, 4 was 17 (7.0-39.3)" times as potent as 20 as a suppressant of adrenal weight and 2 (0.7-4.1) times as potent as a thymolytic agent as 20. Compound 4 is apparently also significantly more catabolic than 20. Removal of the fluorine atom in 4 resulted in compound 5 which is markedly less potent than 4. The corresponding alteration of a steroidal pyrazole also results in a sharp decrease in potency.ls The activity exhibited by compounds 9-12 was not notable with respect to potency but did serve to illustrate how widely the structure of the dienophile could be varied
Kumar et al.
3282 Journal of Medicinal Chemistry, 1993, Vol. 36,No.22
Table 11. Receptor Affinity, Glucocorticoid Profile, and Antiinflammatory Activity of I
compd 4 5
RBAa (%) for the glucocorticoid receptor 72.5 f 9.3 77.1 f 8.5
mg/kg (PO) 1
5 5 25
0.72 f 0.W
6 7 8
180 f 49.1
100
9 10
118 f 14.0 82 f 9.0
5 5 100
11
18.7 f 3.1
1
12a
oc
118 f 5.3
12b 13
4 f 1.5 118 f 1.3
14
118 f 3.5
5 25 0.2
glucocorticoid profile (rat) % change from controlb thymus adrenals -61 -56 -64 -79 -51 -38 -62
-129 -224 -62 -86
NTd NT -64
-48
-88
-59
-40
0 -80 0 -72 -84
0
-80 0
-28
1
-71
5
-83 NT -34 -34
25 100 50
-50
M Y wt gain
-47 -30 -53 -25 -57 -63
-186 -65 -140 -180 -55 -127 -236
0
0
0
-45
-75
0
0
0
0
0
-37
-58
15
2.6 f 1.3
50
0
16
2.9 f 1.1
50
-34
17
1.8 f 0.1
5
0
0
0
18
3.2 f 1.5
25
0
0
0
19
9.3 f 3.5
50 100
-34
0 0
0 0 0
0
antiinflammatory activity (rat) a-tocopherol pouch EDm, mg/kg (pol 10 inactive NT NT inactive @ 100 23 inactive @ 100 4 2
NT inactive @ 25 inactive 0 25 inactive @ 25 inactive @ 48 41% inhibition @ 29 inactive @ 70 inactive a.60
1 -41 32 -35 NT 5 -60 -145 -76 (SchemeIV) 24 0.04 f O.Ole NT (Scheme 111) 0.02 f 0.001e NT 25a (Scheme 111) NT 0.03 f 0.03' 25b (Scheme111) NT 0.02 f 0.006c 29 (Scheme 111) NT 9.6 f 1.8 hydrocortisone 18 99f 14 1 -40 0 -45 2 20 5 -42 -70 (6a-methylprednisolone) -79 0 RBA is defined as relative binding affinity: concentration of dexamethasone @ I 50% binding inhibition + concentration of competitor @ 50% binding inhibition at the rat thymusglucocorticoid receptor. The RBA of dexamethasonewas set at 100. See ref 15for the experimental procedure. b See ref 15 for the experimental details. c These compounds were evaluated by incubation with glucocorticoid receptor derived from liver cytoeol from adrenalectomized rata. [aH]RU28362 was used as the ligand. Dexamethasone has an RBA = 57 f 2% in this away. A "zero" means that binding was too low to measure. d NT means not tested.
and yet afford an adduct which possessed, at least qualitatively, some of the properties of a glucocorticoid. With respect to antiinflammatory activity the most potent compounds were the pyrimidinetetrone adduct 11 and ita NJV-dimethylderivative 12a. Other closely related N,N-disubstituted derivatives 13-19 were inactive or at best weakly active. The doses of 11 and 12 which caused significant adrenal suppression in the two-week glucocorticoid profile test were comparable to those which resulted in significant activity in the 5-day antiinflammatory test. Furthermore, when antiinflammatory activity and adrenal suppressant activity were determined in the same test following Steelman's procedure,lg significant adrenal suppression was also observed at doses which caused an antiinflammatory response. It has not been possible, therefore, to separate adrenal suppressant and antiinflammatory activity with these compounds. In conclusion, we have demonstrated that it is not
necessary to retain an intact steroid nucleus in order to retain significant affinity for the glucocorticoid receptor. Indeed, major departures from the steroid structures are permitted in both the C- and D-ring regions. In vivo, in the rat, administration of the compounds caused a significant antiinflammatory response and also resulted in adrenal suppression,thymolysis, and a catabolic effect. Experimental Section 'H NMR spectra were recorded on a Varian Model HA-100 spectrometer with Me&i as external standard. Chemical shifte are expressed in 6 units. Coupling constants (J)are expressed in Hertz (Hz).MS determinations were carried out wing a JEOLCO JMS-OISC Model instrument. Preparative liquid chromatography wae performed on a Waters Prep LC 600 instrument wing two Prep PAK columns. Analyees are indicated by symbols of the elements and are within f0.4% of the theoretical values. Mp's are uncorrected.
Non-Steroidal Glucocorticoid-like Substances 5-Ethenyl-3-(hydroxymet hylene)-4,4a,7,8-tetrahydro-4amethyl-2(3H)-naphthalenone (2). A solution of 5-ethenyl4,4a,7,8-tetrahydro-4a-methyl-2(3H)-naphthalenone (1)(50.0 g, 0.265 mol) in 350 mL of THF was cooled to -5 “C in an ice-
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 22 3283
washed with HzO, followed by saturated NaCl solution, and dried over MgSO4. Removal of solvent gave 22 as a brown oil, 21.5 g (75%). A mixture of 22 (4.0 g, 0.018 mol), 4-hydrazinopyridine MeOH bath and stirred under NZatmosphere while 57.2 g (1.06 hydrochloride (2.6 g, 0.018 mol), and KOAc (1.8 g, 0.018 mol) in 100 mL of AcOH waa stirred at room temperature for 16 h and mol) of NaOMe was added. The resulting mixture waa stirred for 30 min at -5 OC, and then a solution of methyl formate (114 then heated on a steam bath for 4 h. The solvent waa removed mL, 1.85 mol) in 100 mL of THF was added slowly. The mixture under reduced pressure, and the product was extracted with was stirred overnight at room temperature and then poured onto EtOAc, washed with HzO, and dried over MgSO4. Removal of a mixture of ice-HzO (1500 mL) and 6 N HCl(265 mL). The solvent gave a crude brown oil which was purified on a silica gel product was extracted with EBO, and the combined extracts column with CH&h to give 23 as a light yellow oil, 5.0 g (96%). were washed with H2O. The dried (MgSO4) extract was conA solution of 23 (5.0 g, 0.0172 mol) and dimethylalloxanZ1(4.85 centrated in vacuo to afford an oil. This oil was triturated with g, 0.0258 mol) in 50 mL of dry toluene was heated on a steam hexane (4 X 250 mL), and the combined triturates were dried bath under a NOatmosphere for 4 h. The solvent was removed (MgS04) and concentrated in vacuo to afford 55.37 g of a red oil under reduced pressure and the residue was dissolved in CH2Cl2, washed with water, dried over MgSO4 and evaporated to dryness (2): ‘H NMR (CDCh) 6 13.58 (broad s, lH), 7.54 (8, lH, =CHCO), 6.30 (dd, lH, J = 9.0 Hz, CH==CH2), 6.04-5.91 (m, to give a brown oil. The product was filtered through a Florisil 2H, COCH- and C
