Benzoquinolinone Derivatives

15 May

Among the benzoquinolinones LY 191704 and LY 266111 (Eli Lilly) (Fig. 21A and B) possess an  ideal  4-N-methyl, 8-chloro,  trans,  angular hydrogen arrangement (6) leading to a high  inhibition potency against type  1 isoenzyme (29). Analysis on human  5a-reductases expressed from  transfected cDNAs   in  simian   COS  cells indicate that  LY 191704 is a specific noncompetitive inhibitor of the human type  1 isoenzyme with  an IC50   of 8 nM (37). LY 266111 showed a corresponding IC50   of

9 nM on recombinant CHO cells (35). These benzoquinolinone derivatives demon-

strated that  potent inhibition of the  type  1 5a-reductase could  be  realized by  a compact rigid  tricyclic nucleus that  does not require the full steroid ring system (6).

LY 320236 (Eli Lilly) (Fig. 21C) is a potent dual  inhibitor with differing modes of activity  against the  two  human 5a-reductase isoenzymes. LY 320236 inhibits

5a-reductase activity  in human scalp  skin  competitively (Ki  type 1  ¼ 28.7 nM) and prostatic homogenates noncompetitively (Ki  type 2  ¼  10.6 nM)  (38). LY 320236 is currently under phase 1 clinical trials  against prostate cancer.

6-Substituted 1H-Quinolin-2-ones

Another interesting class  of nonsteroidal compounds are  the  6-substituted 1H- quinolin-2-ones (Fig. 21D) (39).

1H-Benzoquinolizine-3-one Derivatives

The 1H-benzoquinolizine-3-one derivatives are selective  inhibitors of human type 1

5a-reductase. IC50  of AS-97004 (Ares Serono)  (Fig. 22) determined on recombinant

CHO  cells accounts for approximately 300 nM.

Serono’s  antiacne program focused on  the  preclinical lead  compound AS-

601811. Phase  1 trials  began  in the  second quarter of 2001. The agent  was  orally bioavailable in rodents and  dogs  and  generally well  tolerated in animal models. At doses  up  to 15 mgkg21d21  no toxicity  was observed at 15 days  after dosing.

Phenylethyl Cyclohex-1-ene Carboxylic Acids

A novel approach to the synthesis of conformationally flexible nonsteroidal mimics of Epristeride is cyclohex-1-ene carboxylic acids (Fig. 23A). The compounds turned out   to   be   good   inhibitors  of  type   2  isoenzyme  (e.g.,   R ¼   CON(i-propyl)2, IC50  ¼ 760 nM).  Type  1 isoenzyme was  only  slightly   inhibited. The  compounds were  tested for  inhibition of  human  5a-reductase  isoenzymes, using   prostatic tissue  for type  2 and  DU 145 cells for type  1 isoenzyme. This  cell line,  deriving from  brain  metastases of an epithelial human prostate adenocarcinoma, has  been demonstrated to  express only  type  1 isoenzyme and  has  been  recently used  to test inhibitors toward this 5a-reductase isoform  (40).

Biphenyl-4-Carboxylic Acids

It was  recently demonstrated  that  in  the  class  of A-C  ring  steroidmimetics the biphenyl-4-carboxylic acids  were  appropriate 5a-reductase inhibitors. As a conse- quence  of  structure-activity  studies,  3,4-dihydro-naphtalene-2-carboxylic acids and   naphthalene-2-carboxylic  acids   (Figs  23B  and   C),  which   are  structurally based  on  the  steroidal inhibitor Epristeride, were  synthesized and  evaluated for

5a-reductase inhibitory activity.  Human prostate homogenates and  the  DU  145 cell line served as enzyme sources.

Apparently, the 3,4-dihydronaphtalene-2-carboxylic and  naphthalene-2- carboxylic  acid  moiety  mimicking the  steroidal A-B ring  is appropriate for gen- erating potent transition state  analogs of the steroidal substrate testosterone and androsterone. The  most   potent  inhibitors of  the  human  enzymes show   IC50 values   in  the   nM   range.   These   naphthalene  derivatives  exceeded  similarly substituted  1H-quinolin-2-ones in  their   inhibitory  activities.  This  was   to  be expected, since  in  the  class  of biphenyl type  inhibitors, a benzoic  acid  moiety is superior to a 2-pyridone structure as a steroidal A ring  mimetic.

The strong influence of the  type  and  the  position of the  substituents at the

6-phenyl group as well as the absence  or presence of a double bond  in 1 position on the activity  of the compounds are remarkable. The big difference in their inhibi- tory activities toward type 1 and type 2 isoenzyme in the case of single compounds demonstrates that they are appropriate to elaborate the structural requirements for a selective  or a dual  inhibition of type  1 or type  2 isoenzyme inhibition (39).

Indolizine- and  Indol-Butanoic  Acids

FR146687 (Fujisawa Pharmaceutical) (Fig. 24A) shows  a noncompetitive inhibition of both  isoenzymes in vitro  and  has  no inhibitory effects  on other  steroid oxido- reductases (41).

FK143 (Fujisawa Pharmaceutical) (Fig. 24B) was  shown to inhibit  both  types of human 5a-reductase equally. The mode  of inhibition against both the isoenzymes is noncompetitive. The inhibition constants for the human type 1 and  type 2 isoen- zymes  were 27 and  20 nM, respectively. Species selectivity between human and  rat on the inhibitory activity of FK143 was not found for both the isoenzymes. No other inhibitory effects  were  shown to  other  oxidoreductases (42). After  a single  oral administration of FK143 (100 – 500 mg) on healthy volunteers, DHT plasma concen- trations decreased to about  65% of a predose value  (43).


Zinc has long been used  in dermatology and is reported to reduce sebum secretion. Metal  ions have  numerous roles in protein function, especially at the active  site of enzymes. They  could   be  complexed to  a  substrate or  modify the  structure  of the  protein. The two  types  of human 5a-reductases were  transfected into  SW-13 cells,  a  human adrenal carcinoma cell  line,  containing  negligible endogeneous

Note: Transfected SW13  human  adrenal carcinoma cells  were  homogenized and  incubated with 200 nM [14C]androstenedione in the  presence of 50 mM sodium  phosphate buffer, pH 7.5, and  400 mM NADPH. Ki values were  deter- mined by the Cornish-Bowden method and are expressed as mean + SD. “n.i.” indicates that no significant inhibition was observed.

Source: Adapted from Ref. 44.

5a-reductase activity.  The expressed 5a-reductases were  analyzed for their  sensi- tivity  to heavy  metal  ions using  0.2 mM (14C)androstenedione as a substrate.

The addition of CuCl2,  CdCl2, and  ZnCl2 led to a potent inhibition of type  1 isoenzyme, with  Ki values of 3.47, 0.61, and  1.51 mM, respectively (Table 4). NiCl2 and  FeCl3   exerted weaker inhibition, with  Ki  values of 176 and  250 mM. MnCl2, MgCl2,  CaCl2,  and  LiCl,  however, did  not  significantly inhibit  either  type  1 or type  2 5a-reductase activities. It is somewhat surprising that  type  2 5a-reductase was not inhibited by ZnCl2 or CdCl2,  as it contains a triple  stretch  of histidine resi- dues  at amino  acid positions 229 to 231, and  thus  is more likely to bind  zinc and  its analogs than  is type  1. Of the  cations  studied, only  CuCl2 significantly inhibited type  2 5a-reductase activity  with  a Ki value  of 11 mM.

Anions only showed a moderate influence on the type 1 isoenzyme inhibition.

ZnSO4  has  an  effect  similar to ZnCl2,  whereas Zn(CH3COO)2 showed a slightly decreased inhibition (44).

Phytochemical Inhibitors

Phytotherapeutics  are   generally  well   accepted  by  consumers  and,   therefore, received considerable attention  from  the  pharmaceutical industry.  However, a limiting factor for the use and effectiveness of phytotherapy is the lack of standardi- zation  of most  of these  products.

The berries  of saw  palmetto (Serenoa repens), a small  palm  native  to the south east United States, possess a dual  5a-reductase inhibition activity,  due  to their  high content  of  phytosterols  (b-sitosterol  (Fig.  25A),  stigmasterol  (Fig.  25B),  lupeol (Fig. 25C), lupenone (Fig. 25D), and  cycloartenol (Fig. 25E). Saw palmetto was used by native  Americans as herbal  treatment for enuresis, nocturia, atrophy of testes, impotence, inflammation of the prostate, and  low libido  in men. Standardized lipo- philic  saw  palmetto extracts   are  commercially available: e.g.,  Reguw-Seb (Penta- pharm) for topical  treatment of seborrheic skin  conditions and  Permixonw  (Pierre Fabre Medicament) for systemic application to treat  benign prostatic hyperplasia. A

90-day  topical  treatment of a Reguw-Seb, containing emulsion lead  to a statistically significant decrease  of  46%  (p , 0.01)  in  severity  of  seborrheic skin  conditions

(Fig. 26). Permixonw was launched in Europe in 1984 but has no FDA approval. The lipido-sterol extract markedly inhibits both the human isoenzymes. Type 1 isoenzyme is  noncompetitively  (Ki ¼ 7.2 mg/mL)   and   type   2  isoenzyme  uncompetitively (Ki ¼ 4.9 mg/mL) inhibited (45).

Further  dual   phytotherapeutic  5a-reductase  inhibitors  include,  among other  extracts  of Pygeum africanum, Artocarpus incisus (14), Thuja orientalis, Lami- naria   saccharina,  Arnica   Montana,   Cinchona  succirubra,   Eugenia   caryophyllata, Humulus  lupulus,  Hypericum  perforatum,  Mentha  piperata,  Rosmarinus  officinalis, Salvia officinalis and  Thymus officinalis (46); furthermore, diterpens (29), flavins (25),  and   isoflavonoids such  as  genistein (Fig.  27A)  and   daidzein (Fig.  27B), lignans,  resveratrol  (Fig.   28)  (14),  curcumin  (Fig.   29)  (IC50     type   1  ¼ 3 mM, IC50    type  2  ¼ 5 mM) (47), and  certain   polyunsaturated fatty  acids.  The  relative inhibitory potencies of unsaturated fatty  acids  are,  in decreasing order:  g-lino- lenic  acid  (Fig. 30A), a-linolenic acid  (Fig. 30B), linoleic  acid  (Fig. 30C), palmi- toleic acid  (Fig. 30D), oleic acid  (Fig. 30E), and  myristoleic acid  (Fig. 30F). Other unsaturated  fatty   acids   such   as  undecylenic  acid,  erucic   acid,  and   nervonic acid,  are  inactive  (48).

inhibitory effect  of  zinc  sulfate  and  azelaic  acid  on  5a-reductase activity  was shown (49). Azelaic  acid  also  possesses bacteriostatic activity   to  both  aerobic and   anaerobic  bacteria  including  Propionibacterium  acnes.  Moreover,  azelaic acid  is  an  antikeratinizing agent,  displaying  antiproliferative cytostatic effects on  keratinocytes, and  modulating the  early  and  terminal phases of epidermal differentiation. Therefore, several  topical  dermatological antiacne preparations containing  a  combination  of  a  zinc  salt  and   high   concentrations  of  azelaic acid  are  available.


The most challenging and urgent work to achieve  rigorous progress in the develop- ment  of novel  drugs, based  on the  inhibition of 5a-reductase, will  be the  purifi- cation  of these  enzymes, for which  all efforts  have  failed  so far  because  of their unstable nature during purification. Crystallization and X-ray diffraction structural analysis of  the  purified enzymes will  be  another important  topic.  Computer- supported  molecular modeling, then,  should provide directory data  for  devel- oping  more  potent and  selective  inhibitors. On  the  other  hand, the  mechanism of action  of enzymes and  inhibitors needs  to be further investigated. Steroidal inhibitors have  already been  extensively studied. However, developing of more efficient  nonsteroidal compounds with  simpler molecular structures and  higher selectivity  at  lower   costs  is  needed. This  will  also  provide new  information about   enzyme and  inhibitor interactions, and  might   lead  to  novel  drugs that act  differently from  steroidal derivatives (6).

The prevalence of symptomatic benign prostatic hyperplasia is around 14% in men in their 40s, 24% in men in their 50s, and 43% in men beyond the age of 60. The number of patients undergoing operations for  benign  prostatic hyperplasia has decreased by approximately 60% in the  United States  over  the  past  decade, due to  the  introduction of 5a-reductase  inhibitors. Moreover, prostate cancer  is  the second leading cause  of cancer  death in males  in the United States (20). Although acne is the most  common skin  disorder treated by dermatologists, it is, therefore, not  surprising that  the  majority of the  publications focus  on the  investigation  of novel  type  2 and  dual  acting  5a-reductase inhibitors. The contribution of DHT in the  serum, which  is partially derived from  type  1 5a-reductase in the  liver  and the small  amount of type  1 5a-reductase in the prostate, may  play  a role in main- taining prostatic enlargement. Thus,  it is an effort to increase efficacy of treatment for benign prostatic hyperplasia that  inhibits both  types  of 5a-reductase.

Beside the phytotherapeutic preparation Permixonw, to date  Finasteride and Dutasteride are  still  the  only  synthetic 5a-reductase inhibitors, which  are  on  the market as active ingredients in drugs. Anyhow, some inhibitors of type 1 isoenzyme have,  meanwhile, been  developed and  studied in men  with  acne.  To our  knowl- edge,  however, no successful outcomes have  been  published yet. The potential of drugs, such  as Dutasteride that  inhibits both  the isoenzymes, for use in acne treat- ment  remains to be determined.

Though systemically applied inhibitors of 5a-reductase could  prove  to  be safe in women, they  are currently not used  because of potential fetal feminization and  teratogenic risks (1). Currently published clinical results on acne treatment are rather disappointing (50,51).  Therefore, effective  topical   applications may  be  a useful    alternative.   In   humans,   clinical    responses   to   topical    5a-reductase inhibitors have  been  moderate, yet  measurable. Actually, there  exist  no  topical

pharmaceuticals  claiming exclusively 5a-reductase  inhibition activity,   although there  are  antiacne products on  the  market containing, for  example, zinc  acetate and  azelaic acid, respectively. Most of the synthetic or phytochemical 5a-reductase inhibitors are  still  under in  vitro  investigation, animal studies, or  clinical  trials. Finally,  it  nevertheless remains to  be  pointed out  that  current research in  acne therapy favors  investigations of topical  androgen receptor blockers  over  topical

5a-reductase inhibitors.

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