Henry J. Lee

Synthesis and Pharmacology of Anti-inflammatory Steroidal Antedrugs

The purpose of this review is to give a comprehensive understanding about the anti-inflammatory steroidal antedrugs. The concept of antedrug first came into light in 1982 and since then a few academic institutions and some pharmaceutical industries worldwide are performing active research in this avenue in search of safer therapeutic agents. Although the concept primarily focused to the discovery of safer topical anti-inflammatory steroids, the scopes has broadened involving other therapeutic drug classes as well. A recent review has outlined briefly on all different currently available approaches of antedrug discovery. Because of the increasing interests on antedrug approach and vastness of steroid chemistry, the focuses of the present review had been concentrated on anti-inflammatory steroidal antedrugs. The research efforts since 1980s in the chemical synthesis and pharmacological actions of the steroidal antedrugs have dictated the breadth of this article. For the sake of the completeness of information and consistency, historical materials and references to earlier relevant works has also been cited. After a brief introduction about the glucocorticoid therapy, especially their clinical applications and deleterious shortcomings and earlier medicinal chemical efforts to overcome those shortcomings, the antedrug approach is introduced outlining the rationale, scopes and successes. Borderline between the other related concepts such as prodrug has also been explained. The synthetic approaches of all chemical classes of anti-inflammatory steroidal antedrugs and their pharmacological activities have been the main emphasis and a section on pro-antedrugs has also been devoted. Finally, to reflect the scopes and future of the concept of antedrug, drugs in clinical use or in pipeline that are developed based on this concept has been placed before the concluding remarks. 1.1. An Introduction to Glucocorticoid Therapy The beneficial effects of glucocorticoids in the treatment of chronic inflammatory diseases such as asthma, rheumatoid arthritis, inflammatory bowel disease and autoimmune disorders have been appreciated for over 50 years but not without serious complications, which have imposed limitations on the clinical use of this class of drugs.1,2 Suppression on the hypothalamic-pituitary-adrenal (HPA) axis, the immune system, aggravation of diabetes, hypertension, osteoporosis, and retardation of growth in children are few of the most deleterious side effect.3–7 A considerable research effort has been devoted to the structural modifications of glucocorticoids, with a hope of increasing their potencies while minimizing their propensity to elicit systemic adverse effects, and some success had been evident in producing potent glucocorticoids with minimum salt-retaining activity.8–11 The introduction of fluorine at the 9α-position of the natural hydrocortisone (1) increased the binding affinity to glucocorticoid (GC) receptors and retarded the oxidation of the proximal 11-OH group, e.g., fludrocortisone (2). The introduction of 1-double bond, as in prednisolone (3) led to increased potency with reduced salt retaining activity. The incorporation of C6α-methyl, to prevent hydroxylation at this position as in 6α-methylprednisolone (4), increased both potency and duration of action. The incorporation of 16-methyl, in addition to the 9α-flouro and Δ1-double bond, resulted increase in anti-inflammatory potency by about 25 folds, e.g., dexamethasone (6) and betamethasone (7). The main challenge or shortcomings with GC therapy, however, has been in the separation of anti-inflammatory effects on target tissues or organs from systemic glucocorticoid effects, which are largely inherent in the nature of steroids themselves. Not only do they possess multiple biological activities, but structural requirements for various activities seem to be overlapping and inseparable.1–5 Furthermore, GC receptors are present in virtually all tissues, and these receptors appear to be similar or identical on the basis of the relative affinities of GC ligands for these receptors. The introduction of C16,C17-acetonides12 and esterification at C17- and/or C21-OH groups that increased lipophilicity of glucocorticoids proved to be useful for topical application.13,14 Other changes more significant from the chemistry viewpoint rather than pharmacotherapeutic viewpoint included the replacement of C21- and C11-OH with chlorine, incorporation of fused phenylpyrazole ring at C2 and C3 or fused oxazole ring at C16 and C17.15,16 The relatively newer potent anti-inflammatory agents like flurandrenolone (8), fluorometholone (9), and flucinolone (10) are used only topically for the treatment of psoriasis due to their systemic toxicities.

Prodrug and antedrug: Two diametrical approaches in designing safer drugs

The prodrug and antedrug concepts, which were developed to overcome the physical and pharmacological shortcomings of various therapeutic classes of agents, employ diametrically different metabolic transformations. The prodrug undergoes a predictable metabolic activation prior to exhibiting its pharmacological effects in a target tissue while the antedrug undergoes metabolic deactivation in the systemic circulation upon leaving a target tissue. An increased therapeutic index is the aspiration for both approaches in designing as well as evaluation criteria. The recent research endeavors of prodrugs include the gene-directed and antibodydirected enzymatic activation of a molecule in a targeted tissue, organ specific delivery, improved bioavailabilities of nucleosides and cellular penetration of nucleotides. As for antedrugs, emphasis in research has been based upon the design and synthesis of systemically inactive molecule by incorporating a metabolically labile functional group into an active molecule.

Suppression of the Mattox rearrangement of 16α-cyanoprednisolones in acid: Synthesis of methyl 16α-prednisolonecarboxylates

Abstract Prednisolone derivatives with a 16α-methyl carboxylate group were synthesized by a novel procedure of 1,3-dipole addition of fulminic acid to 21-acetyloxy-11β-hydroxy-3,20-dioxo-1,4,16-pregnatriene, followed by base-catalyzed ring opening of the resulting isoxazoline to yield a 16α-cyanoprednisolone derivative and treatment of the nitrile with methanolic HCl. Conversion of the cyanosteroids in the acid to the corresponding methyl carboxylates was achieved with or without the Mattox rearrangement by controlling reaction temperature and protective group for the 21-OH.

Antedrugs: an approach to safer drugs.

The antedrug concept was introduced by Lee and Soliman in 1982 in designing potent, yet safer locally active anti-inflammatory steroids. Antedrug is defined as an active synthetic derivative that is designed to undergo biotransformation to the readily excretable inactive form upon entry in the systemic circulation. Thus, are minimizing systemic side effects and are increasing the therapeutic indices. Steroid-21-oate esters have been developed as the first of this kind, which quickly hydrolyzed to the corresponding inactive steroid acids thereby exerting minimal systemic side effects. Later, other steroidal and nonsteroidal antedrugs have also been developed. The cost, complexity and length in development of a new therapeutic drug, in addition to the adverse drug reactions, the potential risk factors causing the withdrawal of the drugs from the market, have made the approach of antedrug design unique and attractive to the scientists involved in drug design. Considering these factors, the concept which strictly designed to eliminate the deleterious adverse systemic effects, is becoming an affordable approach in drug development arena. This review is not intended to be comprehensive; instead the focus will be on the recent advances in steroidal as well as nonsteroidal antedrugs, which appear to offer new leverage to the development of safer and more efficacious therapeutic agents.

New steroidal anti-inflammatory antedrugs bind to macrophage glucocorticoid receptors and inhibit nitric oxide generation.

In continuing efforts to synthesize potent, anti-inflammatory steroids devoid of systemic side effects, methyl 9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16 alpha-carboxylate (FP16CM) and its 21-acetate derivative (FP16CMAc) were recently synthesized and screened in animal models of inflammation. The compounds have now been assessed for high-affinity glucocorticoid receptor binding and glucocorticoid-mediated inhibition of nitric oxide (NO) generation in an in vitro RAW 264.7 macrophage cell culture system. Relative potencies for glucocorticoid receptor binding were 1, 1.7, and 2.4 for prednisone (P) (IC50 = 287 nM), FP16CM, and FP16CMAc, respectively. Concomitant relative potencies for inhibition of NO generation by macrophages stimulated with lipopolysaccharide were 1, 0.92 and 1.9 for P (IC50 = 126 nM), FP16CM, and FP16CMAc, respectively. Collectively, results suggest that the novel antedrugs are active anti-inflammatory agents. The 9 alpha-fluoro and 21-acetate substituent may contribute to enhanced topical potency, increased receptor binding affinity and inhibitory effects on NO generation. Inhibition of vasoactive NO may be one anti-inflammatory action of the steroidal antedrugs in vivo. Collectively, results suggest that these agents may be useful for topical application in allergic/inflammatory diseases.

The effects of new local anti‐inflammatory steroids on leucocyte migration and prostanoid liberation in rats

The local anti‐inflammatory potency of steroid‐21‐oate esters derived from prednisolone was determined by cotton pellet granuloma bioassay. The doses which inhibited granuloma formation by 50% (ID50) were: prednisolone, 0.5 mg/pellet; methyl 20α‐dihydroprednisolonate, 5.8 mg/pellet; methyl 20 β‐dihydroprednisolonate, 1.2 mg/pellet; methyl 17,20α‐acetonidodihydroprednisolonate, 6.0 mg/pellet. When administered at these equipotent local anti‐inflammatory doses, only prednisolone depressed plasma corticosterone and promoted thymus involution. Prednisolone reduced neutrophil migration into saline‐soaked polyester sponges and depressed the levels of 6‐keto PGF1α, PGE2 and elastase in the sponge‐induced inflammatory exudate. Methyl 20α‐ and 20β‐dihydroprednisolonate had no effect on cell migration, but depressed the levels of 6‐keto PGF1α and elastase. The liberation of 6‐keto PGF1α, PGE2 and elastase and neutrophil migration were inhibited by methyl 17,20α‐ and β‐acetonidodihydroprednisolonate, but the effect of the β‐epimer on cell migration was transient. These data suggest that steroid acid esters which have local and topical anti‐inflammatory properties exert their effect in a similar fashion to glucocorticoids with a ketol side‐chain (e.g. prednisolone) with respect to liberation of prostaglandins and lysosomal enzymes. However, their effect on neutrophil migration was variable, depending on their structural features. Furthermore, the systemic effects of these new derivatives were drastically reduced indicating that they may be of potential benefit in prolonged treatment.

New steroidal anti-inflammatory antedrugs: methyl 21-desoxy-21-chloro-11β,17α-dihydroxy-3,20-dioxo-1,4-pregnadiene-16α-carboxylate, methyl 21-desoxy-21-chloro-11β-hydroxy-3,20-dioxo-1,4-pregnadiene-16α-carboxylate, and their 9α-fluoro derivatives☆ ☆

Abstract To a series of 21-desoxy-21-chloro-corticosteroids, a metabolically labile methoxycarbonyl group at C-16 has been incorporated. The approach is to synthesize locally active compounds that are hydrolyzed to inactive and readily excretable acid metabolites upon entry into the systemic circulation. Novel antedrugs were evaluated for anti-inflammatory activity and their adverse effects in an acute and semichronic croton oil-induced ear edema bioassay. Binding affinity to glucocorticoid receptors and induction of l -tyrosine-2-oxoglutarate aminotransferase were studied in hepatoma tissue culture cells. After a single topical application in the croton oil-induced ear edema bioassay, treatment with all the compounds resulted in dose-dependent inhibition of edema. From these dose-response profiles, the following ID 50 values (nmol/ear resulting in a 50% reduction of edema) were calculated: 540, 618, 454, and 346 nmol for prednisolone (P), methyl 21-desoxy-21-chloro-11β,17α-dihydroxy-3,20-dioxo-1,4-pregnadien-16α-carboxylate (PClCM), methyl 21-desoxy-21-chloro-11β,17α-dihydroxy-9α-fluoro-3,20-dioxo-1,4-pregnadien-16α-carboxylate (FPClCM), and methyl 21-desoxy-21-chloro-9α-fluoro-11β-hydroxy-3,20-dioxo-1,4-pregnadien-16α-carboxylate (FDPClCM), respectively. Results of the 5-day rat croton oil ear edema bioassay indicated that, in contrast with the parent compound P, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights, or plasma corticosterone levels. The binding affinities for cytosolic hepatoma tissue culture glucocorticoid receptors were 33, 201, 471, 5304, and 3765 nM for P, PClCM, FPClCM, methyl 21-desoxy-21-chloro-11β-hydroxy-3,20-dioxo-1,4-pregnadien-16α-carboxylate (DPClCM), and FDPClCM, respectively. Collectively, results of these investigations suggest that modifications of P, which included replacement of 21-hydroxyl group with chlorine and addition of 16-methoxycarbonyl group with or without 17-hydroxyl moiety, retained the topical anti-inflammatory activity of the parent compound P without significant adverse systemic effects.

Steroidal anti-inflammatory antedrugs: Synthesis and pharmacological evaluation of 16α-alkoxycarbonyl-17-deoxyprednisolone derivatives

In a continuing effort to minimize the systemic adverse effects of potent anti-inflammatory steroids, a series of 16 alpha-alkoxycarbonyl-17-deoxyprednisolone derivatives: methyl (8a), ethyl (8b), isopropyl (8c), and benzyl (8d) 11 beta,21-dihydroxy-3,20-dioxo-1,4-pregnadiene-16 alpha-carboxylate, was synthesized and evaluated for their topical and local anti-inflammatory activities. In the acute croton oil-induced ear edema dose-response bioassay, the topical anti-inflammatory potencies of these esters relative to prednisolone, 1, were: 8a:1.0, 8b:1.3, 8c:4.0, 8a:4.7 and 1:1.0. The putative metabolite, 11 beta,21-dihydroxy-3,20-dioxo-1,4-pregnadiene-16 alpha-carboxylic acid, 7, was inactive in this test. A seven day cotton pellet granuloma bioassay was employed to study the local and systemic anti-inflammatory activities of these steroids. The local anti-inflammatory potencies of these esters relative to prednisolone, 1, were 1.3, 1.5, 2.3, 2.5, and 1.0 for 8a, 8b, 8c, 8d, and 1, respectively. In this semi-chronic study, only prednisolone exhibited significant untoward side effects, such as reduction in thymus weights, normal body weight gain, and normal plasma corticosterone levels. The increase in the topical and local potencies of these steroid esters was consistent with the increase in their 1-octanol/buffer partition coefficient. The ratio of local to systemic anti-inflammatory activity of 8c and 8d was four times that of prednisolone. The effects of increasing the size of the alkoxy group of these new steroids on both topical and local anti-inflammatory activity and their concomitant decrease in untoward systemic effects were unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and pharmacological evaluations of new steroidal anti-inflammatory antedrugs: 9α-Fluoro-11β,17α,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16α-carboxylate (FP16CM) and its derivatives

In continuing efforts to develop potent anti-inflammatory steroids without systemic adverse effects, methyl 9alpha-fluoro-11beta,17alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16alpha-carboxylate (FP16CM) and its 16-alkoxycarbonyl derivatives (FP16CE, FP16CP and FP16CB) were synthesized based on the antedrug concept. The steroids were evaluated for their pharmacological activities and adverse systemic effects. All steroidal antedrugs showed both binding affinity to the glucocorticoid receptor in liver cytosol and inhibitory effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cell. These compounds also inhibited croton-oil-induced ear edema and showed no systemic effects such as thymus atrophy and suppression of corticosterone level after 5-day treatment. Among those compounds tested, FP16CM showed the highest activities in receptor binding, NO inhibition and ear edema, these activities were comparable to those of prednisolone. Hydrolysis study in plasma showed that FP16CB was hydrolyzed rapidly, with the half-live (T1/2) of 3.2 min and the half-lives of other compounds were between 16.9 and 29.4 min. These results support the antedrug concept, of which the decrease in systemic adverse effects is attributed to fast hydrolysis to inactive metabolite in the systemic circulation.

AN IMPROVED SYNTHESIS OF AZIDOTHYMIDINE

Abstract A convenient and high yielding procedure is described for a direct conversion of thymidine (1) into 2,3′-anhydrothymidine (2) using the Mitsunobu reaction. Isolation and characterization of two new compounds, 3 and 4, are discussed. AZT has been synthesized from 1 in two steps, in 62% overall yield, by heating 2 with NaN3 in DMF.