Docstoc

STS - PowerPoint

Document Sample
STS - PowerPoint Powered By Docstoc
					Steroid Sulfatase Inhibitors:
Potential New Drugs for the Treatment of Acne
and Cancer




                                    Peter Nussbaumer
                                    Novartis Institutes for BioMedical Research Vienna
1   P. Nussbaumer, Univ Prag 2008
    Agenda
      Introduction
        • Steroid sulfatase pathway

        • Potential indications for inhibitors

        • Enzyme characteristics

      Approaches to STS Inhibition (IC50, rIC50, KI)
        • Irreversible inhibitors and associated issues

        • Reversible inhibitors: discovery and optimisation

      Clinical Proof of Concept


2   P. Nussbaumer, Univ Prag 2008
                   Production of Androgens and Estrogens in
                              Peripheral Tissues
                                  O
                                                     post                                                                                   O

                                                                                                             menopause
             O                                                                                                           O
    O                                                                                                           O
         S                                                                                                           S
             O                                                                                                           O
    O                                                                                                            O
                     DHEAS                                                                                                    Estrone sulfate
                                                                       tumor growth
    Steroid                                                                                                              Steroid
    Sulfatase                                                                                                            Sulfatase
                             O                                    OH                                    OH                                  O



                                      17 -HSD                                                               17 -HSD
                                                                            ER
    HO                                        HO                                      HO                                 HO
                     DHEA                           Androstenediol                          Estradiol                             Estrone


                            -HSD
                            -SR                                                                                        Aromatase
                            17 -HSD                                                                                                        O

                                 OH                         OH



                                        +                                             sebum production
                                                                          AR                                             O

             O                          O
                                                                                      tumor growth                            Androstenedione

                   Testosterone             Dihydrotestosterone


3                P. Nussbaumer, Univ Prag 2008
    Potential Indications for STS Inhibitors


     Androgen-dependent diseases:
      acne, androgenetic alopecia, hirsutism,
      cancer (prostate)

     Estrogen-dependent diseases:
      cancer (breast, endometrium)




4    P. Nussbaumer, Univ Prag 2008
    Steroid Sulfatase (STS, aryl sulfatase C, E.C. 3.1.6.2):
     65 kDa membrane-bound (ER) protein

     catalyzes hydrolysis of steroid sulfates (e.g., estrone, DHEA)

     ca. 30 % homology to aryl sulfatase A and B (structures solved
      by X-ray), but different substrates

     3D structure only available since 2004

     over-expressed in breast tumours,                  SH   X   XH
                                                                  2
                                                                                 S

                                                  N                        N
      acne lesions, dermal papilla                H H    O                 H H   O

                                                   Cys                           H2O
     features unusual AA oxoalanine
                                                                                 H2S
      (posttranslational modification                                            O
                                                 HO      OH
      of cysteine) in the active site
                                                 N                         N
                                                 H H     O                 H H   O
                                                                      FGly = oxoalanine


5      P. Nussbaumer, Univ Prag 2008
    Approaches to STS Inhibition
     transition state analogues:
      not feasible because of trigonal-bipyramidal transition state

     substrate analogues
                                                                               O

     active-site directed inhibition                                     HO   S   O

                                                                               O


        irreversible inhibitors
                                                         OH                                          OH
                                                              H                              H
     novel types of inhibitors                           O
                                                                  O
                                                                  S   O                     H O
      by rational design                                          O
                                                                                                      O
                                                          O                                      O    S   O
      by HTScreening                                 H            H
                                                                                                      O       R
                                                                                   HO   R
     structure-based design
                                                   oxo-Ala essential for catalysis
      (X-ray, homology modeling)                      X-ray: sulfate is bound


6     P. Nussbaumer, Univ Prag 2008
3D Structure of Steroid Sulfatase




                                        active site
                                                      membrane
                                                      anchor
    Hernandez-Guzman, Higashiyama, Pangborn,
    Osawa, Ghosh: J. Biol. Chem. 2003, 278, 22989


7      P. Nussbaumer, Univ Prag 2008
     Active Site of STS with Docked Substrate




8   P. Nussbaumer, Univ Prag 2008
    Profiling of STS Inhibitors

    Primary Screen:                    IC50 on purified human STS

    All inhibitors:                    IC50 in CHO cells over-expressing STS

    Potential candidates:              STS inhibition in: fibroblasts, keratinocytes,
                                       monocytes; human skin homogenate
                                       Specificity testing: arylsulfatase A and B;
                                       species selectivity; HaCaT cell proliferation
                                       Stability & solubility
                                       Penetration studies: pig and human skin

    Development candidate: In vivo testing in pig: STS inhibition
                                       Pharmacokinetic studies after topical
                                       application (skin and systemic levels)


9      P. Nussbaumer, Univ Prag 2008
     Substrate-Based Inhibitors
      non-cleavable substrate analogues
      active site-directed inactivation
                                       O                        O




         R
             O                                   R

             R = SO3K                         R = SO3K        R = NH2
             R = PO32-, PO3-Na                R = SO2CH3      R = NHSO2NH2
                        IC50 = 0.17 - 52 µM   R = SO2NH2      R = CH2SO3H
                                              R = SO2Cl
             R = P(=S)CH3OH                   R = SH          R = COOH
                       IC50 = 75 µM           R = SSO2NH2
                                              R = SSO2N(CH3)2
             R = SO2NH2                       R = SCON(CH3)2


         Nussbaumer, Billich: Med. Res. Rev. 2004, 24 (4), 529

10     P. Nussbaumer, Univ Prag 2008
     Active Site-Directed Inhibition
      aryl sulfamates as irreversible inhibitors
                                                O                                     R

                                        H                                     H
                O                                         O
            O                       H       H        O                   H        H
                S                                         S
         H2N        O                               H2N       O
          EMATE, IC50 = 53 nM                                     IC50 > 100 µM
                  KI = 670 nM

       Howarth, Purohit, Reed, Potter:
                                                                                      O
       J. Med. Chem. 1994, 37, 219
                                                                              H
      other functional groups do not work,               O
                                                                          H       H
       except: aryl formates                          H       O
        Schreiner, Billich: BMCL 2004, 14, 4999                   IC50 = 420 nM

11      P. Nussbaumer, Univ Prag 2008
     EMATE as Lead Molecule
                                               O

                                       H

         O       O                 H       H                     O       O
             S                                                       S
      H2N         O                                          H2N         O             O    O
                  EMATE rIC50 = 1                                COUMATE rIC50 = 30


                                                                                   H
                                                                                   N
                                                                                                10
              O       O                              O       O
                  S                                      S                             O
          H2N          O               O       O   H2N       O
              667COUMATE rIC50 = 0.9                                     DU-14 rIC50 = 20


        Woo, Purohit, Reed, Potter: J. Med. Chem. 1996, 39, 1349
        Woo, Purohit, Malini, Reed, Potter: Chem. Biol. 2000, 7, 773
        Li, Milano, Kluth, Rhodes: J. Steroid Biochem. Mol. Biol. 1996, 59, 41
12     P. Nussbaumer, Univ Prag 2008
 (Thio)Chromenone-Based                                              O   R
                                                             O
 Inhibitors                                               O
                                                             S
                                                        H2 N   O
                                     EMATE, rIC50 = 1                O

                    R                        rIC50               R           rIC50

                *                             29            *                0.4


                *
                                              29            *                2.4


                *
                                               3.4           *
                                                                             1.1

                *                             12.9                           0.8
                                                             *

                *                              7.2          *                0.2


                *                              1.4          *                0.1

13   P. Nussbaumer, Univ Prag 2008
     (Thio)Chromenone-Based Inhibitors

                                  O                          H2N   O         O    R
          O                                                      S
        O                                                      O
          S                                                      O
      H2N   O
                                  O                                          O
                        rIC50 = 2.5                        R = c-hexyl, rIC50 = 113
                                                           R = n-propyl, rIC50 = 4090


                                       S   R
           O                                   R=
         O
           S                                         *            *          *
       H2N   O
                                       O            rIC50 = 1.1 rIC50 = 0.1 rIC50 = 0.006


     Nussbaumer, Lehr, Billich: J. Med. Chem. 2002, 45, 4310


14     P. Nussbaumer, Univ Prag 2008
     Synthesis of 6-Hydroxychromenones
                                         R                           R
     Method A
                                     O       O                   O       O
        2.5 equ. RCOCl                           NaH, DMF

         pyridine, rt                                                            R
                                                   0 - 5 °C
                             O       O       O                   OH O        O         1) HCOOH or MeOH/HCl
                                                                                           2) 10 % aqu. KOH, rt
      OH                         R
                                                                                                             O    R

                                                                                                HO
      OH O                                                                                                   O
        12
                                                 1) RCOCl,                                1) HCOOH, 100 °C
         PhCOCl, Et3N                O       O    pyridine, rt   O       O           2) 10 % aqu. KOH, rt

             CH2Cl2, rt                          2) NaH, DMF
                                                    0 - 5 °C                     R

     Method B                        OH O
                                                                 OH O        O




15   P. Nussbaumer, Univ Prag 2008
     Synthesis of (Thio)Chromenones and
     Sulfamates
           Method C                                                       O                                    O
                                         O        AlCl3                           R HC(OEt)                        R
                                 +                                                          3
                                     R
           HO               OH               Cl             HO            OH                       HO          O



           Method D
                                                                                               S       R
                            SH               O    O       1) PPA, 90 °C
                                 +                                                HO
                                         O            R   2) BBr3, CH2Cl2
             O
                                                                                               O




                                             ClSO2NCO + HCOOH

                                                                                  O
                                     X   2
                                                                              O                    X       2
                        7                             ClSO2NH2                             7
                                                                                  S
                   HO                    R                                H2N          O                   R
                        6                3        DMF, base, rt                            6               3

                                     O                                                             O


16     P. Nussbaumer, Univ Prag 2008
     SAR for Non-Steroidal Aryl Sulfamates:
     Aryl sulfamate              Linker Side chain




 no substitution                                     optional
 allowed                                             space
                                                                                              S
                                                                                O
                                                                           O
                                                                                S
                                                                          H2N       O
                      aryl                                                                    O

                                                                                        IC50 = 0.3 nM
                                                  bulky aliphatic group

                                        linker should contain hetero atom,
                                        bicyclic ring structures preferred

         Nussbaumer, Billich: Med. Res. Rev. 2004, 24 (4), 529

17      P. Nussbaumer, Univ Prag 2008
 Inhibition of DHEAS Metabolism by STS Inhibitor




     Metabolism of DHEAS in human skin in vitro
     blue trace: no inhibitor added
     red trace: incubation in the presence of 10 nM inhibitor

18    P. Nussbaumer, Univ Prag 2008
     Inhibition of STS: Problem Solved?

      Remaining issues:

       Estrogenicity
              EMATE has strong estrogenic activity in vivo
              Aryl sulfamates are produgs of phenols
              Phenols have to be considered as well


       Clastogenic potential

       Chemical instability


19   P. Nussbaumer, Univ Prag 2008
     Estrogenicity Issue Solved!
     non-estrogenic arylsulfamates + phenols:
                                                  O                                  OH
                                                                                          R
                                      H                                      H
                  O
              O                                             O
          O                       H       H            O                 H       H
              S                                             S
       H2N        O                                   H2N       O

            2-methoxy EMATE                             C17-subst. E2 sulfamates


                                                                        O
                                                            O
              O
                  O                                    O
                                                            S
                  S                                   H2N       O
          H2N         O               O       O
                                                                        O

                      667COUMATE                                Nussbaumer, Winiski, Billich:
                                                                J. Med. Chem. 2003, 46, 5091

20    P. Nussbaumer, Univ Prag 2008
     SAR for Estrogenicity of Chromenone Sulfamates

                           O       R       R           STS activity     Estrogenicity
     O
   O
     S
 H2N   O
                           O                              + + +              +
                                       *

                                       *                    + +              -

                                                          + + +              -
                                       *

                                                          + + +             + +
                                       *

                                                          + + +              -
                                       *

                    Nussbaumer, Winiski, Billich: J. Med. Chem. 2003, 46, 5091
21     P. Nussbaumer, Univ Prag 2008
     Inhibition of STS: Problem Solved?

      Remaining issues:

       Estrogenicity
              EMATE has strong estrogenic activity in vivo
              Aryl sulfamates are produgs of phenols
              Phenols have to be considered as well


       Clastogenic potential

       Chemical instability


22   P. Nussbaumer, Univ Prag 2008
     Micronucleus Test Screen of Sulfamates

      Clastogenic
                                                            O
                                                        O
                                                            S
                                                    H2N         O           O       O




      Weakly
                                                        H                                               O
      clastogenic                     O
                                                        N       R
                                                                                O
                                                                                    O
                                  O                                                 S
                                      S                     O               H2N         O
                                H2N       O
                                                                                                        O



      Clean                                                             R
                                                                    O

                                                                        O                                   N
                      O                             O                                           O
                  O                            O                                            O
                      S                             S                                           S                O
                H2N       O                   H2N       O                               H2N         O

                                                                                                        VAC527

23    P. Nussbaumer, Univ Prag 2008
     Synthesis & X-ray Structure of VAC527
                                                                                  OH
                          O                      NH2OH.HCl/NaAc               N
                                                      H2O

                                                        86 %
                       HO                 OH                              HO               OH


                                               1. TMSCl/Hunig base, THF
                                                                                        POCl3
                                               2. NaHMDS, -78 °C                  81%
                                                                O                       DMA/AcCN



                      N                                                       N
                                               3. TFA/toluene, reflux
                  O                                   75%                 O
                                      OH                                                     OH

              86 %    NH2SO2Cl


                      N
                                           O

                  O                        S
                                      O         NH2
                                           O

     Overall yield: 45%
     No chromatographic purification involved
                                               Schreiner, Billich: BMCL 2003, 13, 4313
24    P. Nussbaumer, Univ Prag 2008
     VAC527 – In Vitro and In Vivo Profile
 Purified STS, relative IC50                                                         4.6
             inhibition constant (Ki)                                               6 µM
              rate constant of inactivation (kinact)                             0.097 sec-1
 STS in CHO cells, relative IC50                                                 1.25 (32 nM)
 STS in HaCaT keratinocytes, IC50                                                  0.77 nM
 STS in human sebocytes cell line                                                  0.15 nM
 STS in human primary fibroblasts                                                  0.75 nM
 STS in human monocytes, IC50                                                      154 nM
 Anti-proliferative effect in HaCaT cells, IC50                                     15 µM
 Binding to estrogen receptor  and , IC50                                        >30 µM
 Conc. in human skin 48 hrs after application (solvent H2O/EtOH 1:1)             50.6 µg/cm2
 Oral bioavailability, rat                                                          20 %
 Biological data, in vivo
 Inhibition of STS 5 hr after single application of a 0.36 % solution to            99 %
 mouse skin
 Inhibition of STS 6 hr after single application of a 1 % solution to pig skin      100 %


25     P. Nussbaumer, Univ Prag 2008
     In Vivo Activity after Topical Application to Pigs
                                            100                                                                80
      STS Activity [% of vehicle control]




                                                                                Concentration in skin [µg/g]
                                             80
                                                                                                               60

                                             60
                                                                                                               40
                                             40

                                                                                                               20
                                             20


                                              0                                                                 0
                                                  3          1            0.3                                         3          1            0.3
                                                      Applied Conc. [%]                                                   Applied Conc. [%]

                        inhibition of STS activity in                                                               skin concentration
                        skin at 6 hrs post treatment                                                                    of inhibitor

                                 Billich, Meingassner, Desrayaud, Nussbaumer, Lam, Schreiner:
                                 J. Steroid Biochem. Mol. Biol. 2004, 92, 29
26                           P. Nussbaumer, Univ Prag 2008
     VAC527 Induces Atrophy of the Sebaceous Gland
     Minipig; topical treatment

     Dosage: 1% in isopropanol/propylene glycol 1:1 for 2 weeks




              Control animal                Animal treated with VAC527
              (treated with placebo)

      VAC527 was well tolerated and did not cause treatment-
      related histopathological findings in other organs.

27      P. Nussbaumer, Univ Prag 2008
     Stability Profile of VAC527

     Half-life in aqueous solution, pH 7.5        69 hr

     Degradation after 2 weeks at 25°C in
     propylene glycol / isopropanol 1:1           9.2 %

     Degradation of solid after 4 weeks at 80°C   2.8 %




       VAC527 was abandoned due to insufficient
        stability for standard topical development!


28   P. Nussbaumer, Univ Prag 2008
     Inhibition of STS: Problem Solved?

      Remaining issues:

       Estrogenicity
              EMATE has strong estrogenic activity in vivo
              Aryl sulfamates are produgs of phenols
              Phenols have to be considered as well


       Clastogenic potential

       Chemical instability


29   P. Nussbaumer, Univ Prag 2008
     Chemical (In)Stability of Aryl sulfamates

      Aryl sulfamates are stable in the solid state
      In solution aryl sulfamates are degraded to the
       corresponding phenols:
       T1/2= 24 hrs to 8 days at pH 7.5 / 37 °C
      Limited stability in polar solvents, e.g. DMSO, PEG 400
      Susceptibility to hydrolysis is an inherent property of the
       aryl sulfamates
      No correlation between chemical reactivity (hydrolysis)
       and enzyme inhibitory activity


30     P. Nussbaumer, Univ Prag 2008
     Chemical Stability of Aryl Sulfamates
     Relative inhibitory activity of test compounds against STS vs.
     rate of hydrolysis
                                            10


                                             1


                                            0.1
                              k [hr-1]




                                           0.01


                                          0.001


                                         0.0001
                                                  0.1   1   10       100   1000
                                                             rIC50


31     P. Nussbaumer, Univ Prag 2008
     Search for Non-sulfamate Inhibitors
     “Rational approach“ → stable inhibitors

       R     O                   O                        O                           S                      O                    S
                                                                                                                                                  N
           N S       *   R       S       *       Cl       S           *       OH P              *   OH P              *                   *                   *
                 O                   O                        O                            O                     O        H2N         O                   O
       R O                   O                        O                           R                     OH


             O                       O                            O                             O
                                                                                                                 HO               H2N                         *
       H2N S   *          H2N S   *                       R       S           *                          *                *                   *
             S                  N                                         N               H2N       N                                                 N
          O                  O  H                             O           H                         H



             O                       O                        O                   O         *           O                     R
                                                                                                                  *               O                   R
       H2N S         *   OH S                *        HO P                *
                                                                                      O                                                   *                   *
          O                O                          HO                          R                          OH               O       O           O       O




                                 lead
                                                                                                                      Potent, stable, reversible STS inhibitor:
                                                                  O
                                                                                                                      purified STS: Ki = 0.5 µM
                         HO
                                                                                                                      ... but only poorly active in cells!
                                 O                                O

     Horvath et al.: J. Med. Chem. 2004, 47, 4268
32         P. Nussbaumer, Univ Prag 2008
     Reversible STS Inhibitors from (HT)Screening
                                                                                            Cl
                                        O       OH                                               NH2
                                                         H       H
                                  HO                     N       N              HO
                      O         HO                   N                                           Cl
      HO                                                     S
                                                                                    N
                                                                           N

              OH      O             O
                                                                           CN

                                                                                            OH
       Cl
                                                     O       O
                            O       O                                               H
                        S                   N                                                         R
                                N       N                                       H       H
                    O           H       H                            HO

                                                                          R = H, alkyl, Cl, Br


33      P. Nussbaumer, Univ Prag 2008
     Sulfonylureas as Reversible Inhibitors
     High-througput screening hit:
                       O

                                           H       H O
                       O              N    N       N
                                                     S          Cl   rIC50 = 8.4
                                                       O
                                               O                     Ki = 0.87 µM
                     PKF017-433

                                          Reversible, competitive inhibitor  LEAD


        first derivative:
                       O                                             rIC50 = 16.2
                                               O
                       O              N            O                 Ki = 2.4 µM
                                               N   S       Cl
                                               H                     CHO cells: rIC50 = 224
                                                   O
                                                                     monocytes: rIC50 = 129

34    P. Nussbaumer, Univ Prag 2008
     SAR of Sulfonylurea-Type Inhibitors
                                                            O
                                   O                    H
                                                        N   S        Cl
                                    O               N       O                 IC50 = 0.9 µM
                                                        O

                                                                                                    IC50 [µM]

                                   HO           N       > 100             H   O               Me            16.6
                                                                          N
                   > 100
                                                                                    9
                                                                          O
           O                                                                                  F              50
                                   HO
                                                N               13
                                        N
                                                                          O                   H           > 100
          O
                   > 100
                                                                          S         6.2
          O                                                               O
                                                        > 100         O
                                            N   N                                             3,5-diCF3     0.08


     Caveat: in cellular system substantially less active
     Nussbaumer, Geyl, Horvath, Lehr, Wolff, Billich: BMCL 2003, 13, 3673
35      P. Nussbaumer, Univ Prag 2008
     From Sulfonylureas to Acylsulfonamides as
     Reversible STS Inhibitors
                                                                       O
                                                  O                H
                                                                   N   S            Cl
                                                  O            N       O
                                                                   O
                                                                           IC50 = 0.9 µM


                                          O                                                  O
                                      H                                                  H
                     O                N   S               Cl           O                 N   S   Cl
                          N       N       O                                N    N            O
                    O                 O                                O                 O

                          IC50 > 100 µM                                    IC50 = 3.6 µM

                                                                       O
                                                                   H
                                              O                    N   S            Cl
                                                      N                O
                                              O                    O

                                              IC50 = 8.6 µM, KI = 16.1 µM

36    P. Nussbaumer, Univ Prag 2008
     Parallel Synthesis to Optimize the Aryl Moiety and
     to Evaluate 3 Central Scaffolds
                                                        1000
      Cl                               BOC
                                  N                     500
                         H
                         N             Ki = 16.1 µM
                    S
                   O O O
                                                        154 -                  starting
                                                        100                    point

              Cl                                         50
                    Cl                 BOC
                                   N                  rIC50
                         H
                         N             Ki = 0.22 µM
                    S
                   O O O                                 10

                                                          5

            lipophilic meta-substituents                2.4 -

             preferred                                                         BOC        O                   O
                                                                           N                        BOC H O
            acids with 1,4 orientation                    HO                        HO         N
             preferred                                           O   1,4                  1,3
                                                                                                          1,4 homo
                                                                                                                     NHBOC
                             Lehr, Billich, Wolff, Nussbaumer: BMCL 2005, 15, 1235
37         P. Nussbaumer, Univ Prag 2008
     Further Refinement of the Central Scaffold
     Isonipecotic, 4-piperidinylacetic, 4-piperidinylenacetic acid-type

                  R1           O
                                        CH3
                                                               O                     O   R2                             Cl
                                                  O                 O
                                                                                                        ~     Cl              ~
                                                      N                  N
                               N
                                                  O                 O
                                                                                           Cl
                           O        O
                                                                                                                   Br
                                          O                    O                     O    F3 C          ~                    ~
                                                                                                             Cl
                          O                       O                 O
                                N                     N                  N                                          S
                                                                                                  CF3                        Cl
                           O                      O                 O




                                                                     O O                                      O O
                               O H
                                   N          O                     S                                        S
                                S                         R2          N                          R2            N
                                                                   O H                                      O H
                                  O
                               R1                                   R1                                       R1
                   R2
                                         N                                   N                                          N
                                    O         O                          O       O                                  O        O


38      P. Nussbaumer, Univ Prag 2008
     Highly Potent, Reversible STS Inhibitors – Profiles
     Selected compounds

                                                                O       O

                                        Comound 1                   N            Compound 3       Compound 4


                                                                        VAC624
                                                          O     NH
                                                              O S O



                                                      F                      F
                                                      F                      F
                                                          F              F

      Isolated enzyme,                     3 nM               16 nM                 40 nM           0.4 nM
      IC50
      Human skin                          0.6 µM              0.27 µM              0.66 µM         0.10 µM
      homogenate, IC50
      Skin                                24 µM               520 µM               280 µM           127 µM
      concentration
      Stability                          sufficient       sufficient             sufficient (?)    insuffient

                                                       Superior in
                                                      human Skin!

39      P. Nussbaumer, Univ Prag 2008
     Profile of VAC624  Clinical Candidate
     Purified STS, IC50                                                  16 nM
     Inhibition constant (KI)                                           8.6 nM
     STS in CHO cells, IC50                                             4.0 nM
     STS in HaCaT keratinocytes, IC50                                   24 nM
     STS in human primary fibroblasts, IC50                              5 nM
     STS in human sebocyte cell line, IC50                              10 nM
     STS in human U937 monocytes, IC50                                  20 nM
     STS in human skin homogenate, IC50                                270 nM
     STS in pig skin homogenate                                         29 µM
     Anti-proliferative effect in HaCaT cells, IC50                    >30 µM
     Binding to estrogen receptor  and , IC50                        50µM
     Concentration in human skin 48 hrs after application             290 µg/g
     STS inhibition at 6 hrs after application to human skin             92%
     Concentration in pig skin 8 hrs after top. application in vivo   37.1 µg/g

40     P. Nussbaumer, Univ Prag 2008
     Synthesis of VAC624
                                                             O
                       O                                                                                         O
                                Ph3P=CHOMe                                CeCl3 x 7H2O/NaI
     O                           THF, 0 °C       O                          acetonitrile     O
              N                                      N                                           N
                                     71%                                       95%
     O                                           O                                           O



                   1. CH3CHClOCOCl                                                                     1. NaClO2/NaH2PO4
                      (CH2Cl)2                                    F                                       tBuOH/water
                                                         F            F                          86%
                   2. MeOH                                                                             2. crystallization from
                   3. Boc2O/Na2CO3                                            O                           MeOH/water
                      THF/water                                               S
                                                                                Cl                       H      O
                      O                                   F                  O
                                             O           F
                             Mannich                              F                                              OH
                             react.
                                                                                             O
     Br       +
                                                                     aqu. NH3,
            N                                                    99%
                                                                     EtOAc
                                                                                                 N
            H                                                                                O
                                                                  F
                                                         F            F
                                                                              O
                                                                                                       PPA/DMAP/DIEA
                                                                              S                        DMA, rt
                                                                                NH2
                                                          F                  O
             Overall yield: 40%                          F
                                                                                                        69%
                                                                  F
                                                                                                 VAC624


41        P. Nussbaumer, Univ Prag 2008
 Summary
  Based on EMATE as lead, we discovered highly potent, non-estrogenic,
   irreversible STS blockers featuring a (thio)chromenone scaffold,
        but weak clastogenicity is an unacceptable safety risk

  Benzoxazole-type inhibitors (VAC527) were found to be non-clastogenic
   and sufficiently potent in vivo,
       but the intrinsic instability of aryl sulfamates in topical formulations was
   a “no go” for further development

  HTScreen delivered lead-like N-sulfonyl ureas as reversible inhibitors

  Scaffold morphing and subsequent optimisation led to the discovery of a
   clinical candidate (VAC624) for the indication acne

  While clinical PoC for the indication acne is still pending,
   PoC in breast cancer was achieved with a prototype inhibitor

42    P. Nussbaumer, Univ Prag 2008
     Clinical PoC of an STS Inhibitor in Breast Cancer
     M. Reed et al: Clin. Cancer Res. 2006, 12, 1585


      PhI: STX64 (= 667COUMATE)                               O

                                                             H2N
                                                                   S
                                                                       O

                                                                       O   O   O

         • 9 (5mg) + 5 (20 mg) patients (postmenopausal women with BC)

      Preliminary results:
         • well tolerated, only minor drug-related adverse effects
         • 98 % inhibition of STS in PBLs
           99 % inhibition of STS in breast tumor tissue
         • significant reduction serum levels of estrone, estradiol,
           androstenediol, and DHEA
         • clinical evidence of stable disease in 4 subjects who previously
           progressed on aromatase inhibitor treatment


43       P. Nussbaumer, Univ Prag 2008
Acknowledgements
Chemistry:               A. Horvath, P. Lehr, P. Nussbaumer, E. Schreiner
Biology:                 A. Billich, J. Meingassner, A. Winiski, B. Wolff-Winiski
Modeling:                A. Aszodi, A.Berces




44   P. Nussbaumer, Univ Prag 2008

				
DOCUMENT INFO