15 May

During the past  three  decades, research has focused on understanding the biologi- cal functions and effects of 5a-reductases and their 5a-reduced metabolites in order to obtain  compounds able to block androgenic action  through the inhibition of 5a- reductases (13). A high  degree of selectivity is important to ensure that  no other enzyme is affected  and  that no interactions exist with  steroid receptors (10). Devel- opment of  specific  5a-reductase  inhibitors began  in  1983, soon  after  DHT  was reported to  be  the  major  androgen acting  in  the  periphery. Great  progress has since  been  made and   continuing  interests grow   in  designing  and   developing more potent and specific 5a-reductase inhibitors. DHT, being 2 to 10 times stronger than  testosterone in androgen activity,  was supposed to play a more important role than  testosterone in many  androgen-dependent diseases (14). Approximately 70% to 80% of serum DHT in men is produced by the type 2 isoenzyme, and 20% to 30% by the type  1 isoenzyme (21).

Since  sebum production  is  regulated  by  DHT,  inhibitors  of  type   1  5a- reductase may,  therefore, form a new  therapeutic class of agents designed to treat acne.  It is expected that  5a-reductase inhibitors will  produce fewer  side  effects than  the  presently available hormonal therapies. Inhibition of type  1 isoenzyme may  represent a means of blocking  the local production of DHT within the sebac- eous  glands. This inhibition, in turn,  may  reduce sebum production and  improve acne (21).

5a-reductase inhibitors can be classified  according to their

1.    chemical  structures (steroidal/nonsteroidal),

2.    type  of inhibitor activity  (competitive/noncompetitive/uncompetitive), and

3.    specificity  (type  1/type 2/dual inhibitors).

Notice  that  the  mentioned inhibition constant values (Ki  values)  and  IC50 values vary,  depending on the species  (e.g., human versus rat), the cells (primary cultures  versus  cell  lines),   and   the  tissue   origin   (e.g.,  testis   versus  prostate) examined.

In the following, a number of 5a-reductase inhibitors are highlighted. Due to the vast quantity of corresponding papers we are far from a position to claim a com- plete  listing  of all  published inhibitors. However, we  focus  mainly on  essential structural requirements for inhibitors, on characterized inhibitors for human type

1 isoenzyme, and  on  inhibitors that  stand in  clinical  testing   phase   or  that  are already commercially available.

Structural Requirements for Inhibitors

The  design of most  synthetic 5a-reductase inhibitors is based  on  the  transition state  inhibitor paradigm, a  concept that  states  that  the  affinity  for  the  enzyme should be greater for molecules that  mimic  the  transition state  of the  enzymatic process  (Fig. 4B) (16). Molecular modeling studies suggest that  there  is a require- ment  for

1.    Groups to mimic  the  steroid A ring,  in particular, the  C-3 carbonyl group (Fig.   13)   (26).   It   is   concluded   that    any    steroid-derived   structure, possessing  a  C-3  polar   group,  should  exhibit   5a-reductase  inhibitory activity,   whereas  other   structural  factors   determine  the   extent   of  the inhibition (27).

2.    The space below the steroidal plane  area about  C-3, C-4, C-5, and C-6. This area should be sterically hindered, since the NADPH moiety  requires access to the D4 site (26).

3.    The  area  of  the  active  site  of  the  enzyme about   the  C-17  position of  the steroid substrate, which   appears to  lack  hydrogen bonding groups and   is unrestricted (26).

In most synthetic and  natural steroids, loss of the C-19 methyl group reduces the  potency of the  steroid to be recognized by 5a-reductase. Appropriate substi- tution at C-17 is, therefore, of particular importance in inhibitor design (5). That the  C-17 hydroxyl group is not  essential for inhibition is supported by inhibitors where the  D-ring  has  been  altered to a six-membered lactam  resulting in potent activity.  The volume of space occupied by the C-17 side chain of inhibitors is exten- sive. Therefore, it is postulated that the entry/exit to the active site of 5a-reductase results from this position (26) by lodging the C-17 side chains  in specific cavities  of the  enzyme (28). A further postulation is that  the  increased inhibitory activity  is associated with  the lengthening of side chains.  It is also proposed that the two iso- enzymes possibly vary  in the positioning of the reducing NADPH moiety  (26).

Steroidal Inhibitors


The  first  generation of  synthetic inhibitors were  steroids with  a  nitrogen atom located  at different positions of the steroidal structure. Because of this characteristic, they are designated as azasteroids (Figs. 14A – C) (10). The presence of the nitrogen atom  in the steroid structure increases the nucleophilicity of the carbonyl group at

C-3 and,  thus,  favors  the interaction with  an electrophilic residue in the active site. In general, azasteroids interact with the enzyme NADPH complex and are competi- tive with  respect to testosterone (16).

4-Azasteroids  are  mimics   of  the  “product-like” transition state,   whereas

6-azasteroids and  10-azasteroids, which  have  an  enone  structure in  the  A ring, are  mimics  of the  “substrate-like” transition state  (Fig. 4B). 4- and  6-azasteroids are generally more rigid structures than 10-azasteroids, and thus  may be more suit- able for developing models of the 5a-reductase active  site (16).

4-Azasteroid Derivatives

4-Azasteroids  have   been   studied  extensively  (6).  Hundreds  of  substituted

4-azasteroids have  been  published and  patented since the early  1980s, especially from  Merck,  Glaxo,  and  Pharmacia (29).  Among these  inhibitors, Finasteride (MK906)  (Fig.  15A)  was  the  first  and,  for  the  last  decade, the  only  available active  ingredient in  systemic drugs,  (Proscar   and  Propecia, Merck)  approved for clinical  use.  Finasteride is a specific  competitive type  2 5a-reductase inhibi- tor  (IC50   type  2  ¼ 9.4 nM,  IC50   type  1  ¼ 313 nm)  (10) and  has  been  shown to  be effective  for the  treatment of benign prostatic hyperplasia (Proscar)  and  andro- genetic  alopecia (Propecia), respectively (14). In  humans Finasteride decreases prostatic DHT  levels  by 70% to 90% and  reduces prostate size,  while  testoster- one  tissue  levels  remain constant (5). In males  with  benign  prostatic hyperpla- sia  treated  with   Finasteride,  there   was   no  reduction  of  sebum  production, which   may   be  related  to  the  possibility  that   5a-reductase  in  the  sebaceous gland  remains  unaffected  by  the   drug  (30).  The  corresponding  amino   acid sequences proposed  to  be  involved  in  the   Finasteride/human  5a-reductase

interaction are  Ala26-Val27-Phe28-Ala29   for  type  1  isoenzyme and   Gly21-Ala22- Leu23-Ala24  for  type  2 isoenzyme (Fig. 3) (19).

On the other  hand, Dutasteride (GI198745, GG745) (Fig. 15B), one of the most potent dual  inhibitors to  date,  is the  first  active  ingredient in  a drug (Avodart, GlaxoSmithKline) that  inhibits both  the  isoenzymes (16). Avodart received Food and  Drug  Administration (FDA) approval in 2002 and  was  launched in 2003 so that  after  a monopoly for  a decade as  the  only  available 5a-reductase inhibitor, Finasteride  will  be  joined   by  a  second  (20).  This  dual   5a-reductase  inhibitor shows  a greater inhibition of type  1 isoenzyme compared with  Finasteride and  is also  potent against type  2 isoenzyme (IC50   type  1  ¼ 2.4 nM,  IC50   type  2  ¼ 0.5 nm) (29). Biochemically, Dutasteride achieves greater and  more rapid DHT suppression compared with  Finasteride. It  is  reported to  reduce circulating DHT  levels  by

90%.  Clinically,   it   appears  to   be   at   least   as   good   in   terms   of  improving symptoms of benign prostatic hyperplasia. However, until  these two drugs are for- mally  compared, the  true  benefits  of additional type  1 isoenzyme inhibition are unknown (20).

PNU  157706 (Pharmacia & Upjohn) (Fig. 15C) is highly  potent in inhibit- ing  human recombinant type  1 and  2 5a-reductase, showing IC50   values of 3.9 and  1.8 nM,  respectively. PNU  157706 was  shown to  have  no  binding affinity for  the  rat  prostate androgen receptor and  to  be  devoid of  any  antiandrogen activity  (31).


In contrast to 4-azasteroid derivatives, compounds with 4-methyl-4-aza functional- ity are generally more potent to type 1 5a-reductase (6). Experimental data  showed that  azasteroid derivatives containing groups larger  than  methyl result  in poorer inhibition due  to the restricted access of NADPH (32).

After  the  launch of Proscar  in the  early  nineties, Merck  has  continued the research efforts  and  conducted clinical  trials  for at least  three  further compounds MK963, MK434,  and  MK386  for  the  treatment of acne,  alopecia, and  hirsutism (16), but  will probably not  bring  any  of these  to the  market. MK386 (Fig. 16A) is a selective  inhibitor of human type  1 5a-reductase with  an IC50  of 20 nM. A four- teen-day oral  application with  MK386 resulted in a dose-dependent suppression of serum and  sebum DHT,  without affecting  semen  DHT concentrations. Serum DHT concentrations were  reduced 22% by a daily  dose  of 50 mg MK386, with  no significant change in serum testosterone. Sebum  DHT was  reduced by  55% and

35% by doses  of 50 and  20 mg,  respectively (33). However, due  a certain  degree of hepatic toxicity  in men  the development of MK386 was  stopped (10). A further development is L-751788 (Fig. 16B), which  differs  from Merck’s other  5a-reductase inhibitors by having a substituent on C-16 than  on C-17. This makes  it more  selec- tive for type  1 isoenzyme.

The dual  inhibitor 4-MA (Fig. 16C) (Merck) has been studied extensively and progressed to  clinical  trials  for  the  treatment of benign prostatic hyperplasia. It showed a high activity for both isoenzymes (IC50  ¼ 8.5 nM) (16) and a very low affi- nity  for the androgen receptor and  was  thus  not expected to produce undesirable antiandrogenic effects. However, 4-MA was subsequently shown to be an inhibitor of 3b-hydroxysteroid dehydrogenase, another steroid metabolizing enzyme, and to cause  hepatoxicity, like MK386 (15).

Turosteride (FCE 26073, GlaxoSmithKline) (Fig. 16D), a promising new  drug currently in  clinical  studies, is  a  selective   inhibitor of  type  2  isoenzyme (29).

Turosteride caused inhibition of the human enzyme with  IC50  values  of 53 nM and does  not show  relevant binding affinity  to prostate androgen receptor.

EM-402 strongly inhibits human type  1 5a-reductase (IC50  ¼ 7.3 nM)  and shows  low potency on the type 2 isoenzyme in transfected cells in vitro. Topically applied EM-402 (Fig. 16E) exerts a potent local antiandrogenic effect without any systemic action in the hamster. EM-402 decreased the size of flank organs of ham- sters  and  also  of the  underlying sebaceous glands. Twice  daily  applications for four  weeks  at  doses  of  30, 100, and  300 mg reduce the  size  of  the  sebaceous glands by 38%, 42%, and  59%, respectively. Comparable results were  observed on the sebaceous glands of the ears. In addition, a concentration-dependent inhi- bition of 5a-reductase activity  of 47% to 80% in the flank organs and ears, respect- ively,  were  observed. EM-402  had   no  effect  on  prostatic and   seminal vesicle weights (34).

6- and  10-Azasteroids

6-  and   10-azasteroids 5a-reductase  inhibitors  have   been  synthesized  showing different degree of 5a-reductase inhibition and  different selectivity, some  of them are under investigation for clinical application (10).

The presence of the nitrogen atom at position 10, conjugated with the carbonyl at  C-3 (Fig. 14C), is an  essential feature for  good  inhibition since,  providing an increase of the  negative partial charge  on  the  oxygen,  it probably determines a strong interaction with  an electrophilic residue in the enzyme active  site (35).

Among 6-azasteroids, the  aryl  substituted derivative (Fig. 17A) is a potent dual  inhibitor of both  isoenzymes and  17b-N-(t-butyl)carbamoyl substituted 10- azasteroid (Fig. 17B) displayed an inhibitory potency against type  2 5a-reductase with  that  of Finasteride (IC50  ¼ 10 – 122 nM,  depending on  the  isomer),  and  was more  active  against type  1 isoenzyme (IC50  ¼ 127 nM) (16).

4-Oxa- and  4-Thiasteroids

A series of irreversible inhibitors related to 4-azasteroids but with a different heteroa- tom at the C-4 position (sulphur and  oxygen)  has also been reported by Merck (29).

Pregnatriene Derivatives

Flores et al. (5) synthesized novel pregnadiene-dione and pregnatriene-dione deriva- tives (Fig. 18A and B). The trienones showed consistently higher 5a-reductase inhibi- tory activity  than  the corresponding dienones. Unfortunately, there  is no hint on the enzyme specificity.  It is believed that the trienones inactivate the enzyme by an irre- versible  Michael  type  addition of the  nucleophilic portion of the  enzyme to  the

conjugated double bond  of the steroid. The trienones having a more  coplanar struc- ture  react faster with  the enzyme and  thus  show  a higher inhibitory activity.

Steroidal Carboxylic Acids

A number of 3-androsten-3-carboxylic acids (Fig. 19A) were designed to mimic the putative enzyme bound enolate  intermediate. Because  of favorable electrostatic interactions between the carboxylate and  the positively charged oxidized cofactor, the  acrylate preferentially binds  in a ternary complex with  enzyme and  NADPþ, which  leads  to the observed uncompetitive kinetic  (6).

Epristeride  (ONO-9302,  SK&F 105657,  GlaxoSmithKline)  (Fig.  19B) is  an uncompetitive, potent selective  5a-reductase type  2 inhibitor and  is under phase III clinical testing  for the treatment of benign prostatic hyperplasia (36). Epristeride has exhibited the ability  to lower  serum DHT levels by 50% (5).

Steroidal Oxime Inhibitors

Novel   steroidal  oxime  inhibitors (Fig.  20)  show   an  excellent   inhibitor  activity toward both  types  of the human 5a-reductases (5).

Nonsteroidal Inhibitors

Benzoquinoline Derivatives

In the field of nonsteroidal inhibitors, the most interesting molecules are the group of benzoquinolinone and  benzo[c]quinolizinone derivatives. Benzoquinolinone derivatives have   been  designed starting from  the  related parent  4-azasteroids and   the  benzo[c]quinolizinone derivatives  from  10-azasteroids.  They  are  very potent  inhibitors of  human  5a-reductases with,   in  most  cases,  higher activity against the type  1 isoenzyme. Some compounds are dual  inhibitors (35).

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