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Discovery of selective CYP11B2 (aldosterone synthase) inhibitors for the therapy of congestive heart failure and myocardial fibrosis
European Journal of Medicinal Chemistry 38 (2003) 363 /366 www.elsevier.com/locate/ejmech
Discovery of selective CYP11B2 (aldosterone synthase) inhibitors for the therapy of congestive heart failure and myocardial fibrosis Rolf W. Hartmann *, Ursula Mu¨ller, Peter B. Ehmer Pharmaceutical and Medicinal Chemistry, Saarland University, P.O. Box 151150, D-66041 Saarbrucken, Germany Received 30 September 2002; accepted 14 December 2002
Abstract An increased aldosterone concentration due to congestive heart failure leads to a further progression of the disease as well as to myocardial fibrosis. To interfere with these fatal processes selective inhibition of aldosterone synthase (CYP11B2) is required. CYP11B1, a key enzyme in glucocorticoid biosynthesis showing a high homology to the target enzyme ( /93%), must not be inhibited. Screening of our P450 inhibitor library for inhibition of bovine aldosterone synthase resulted in a high number of compounds showing reasonable inhibition. In the next step substances were tested for oral absorption using two artificial membrane assays. The inhibition of human CYP11B2 was evaluated using assays in fission yeast and V79MZ cells stably expressing the active human target enzyme. For selectivity, inhibition of CYP11B1, CYP11A1, CYP17, CYP19 and CYP5 was determined. Rather potent and selective compounds obtained in this way were structurally further optimised, finally leading to inhibitors showing IC50 values within the low nanomolar range. ´ ditions scientifiques et me´dicales Elsevier SAS. All rights reserved. # 2003 E Keywords: Congestive heart failure; Myocardial fibrosis; Aldosterone synthase (CYP11B2); 11b-hydroxylase (CYP11B1); P450 (CYP) enzymes; Selective inhibitors
1. Introduction Aldosterone synthase (CYP11B2) is a P450 enzyme which catalyses the key step in mineralocorticoid biosynthesis, the conversion of 11-deoxycorticosterone to the most potent mineralocorticoid aldosterone (Fig. 1, [1]). Aldosterone synthesis is regulated by several physiological parameters like the renin /angiotensin / aldosterone-system (RAAS) and the plasma potassium concentration. Elevations in plasma aldosterone levels play an important role in the pathophysiology of certain forms of hypertension and congestive heart failure [2]. The RAAS is pathologically activated due to an insufficient renal flow which leads to an excessive release of aldosterone [3]. Chronic elevations in plasma aldosterone increase the blood volume and may stimulate cardiac fibroblasts, resulting in cardiac hypertrophy, myocardial fibrosis and ventricular arrhythmia [4,5].
* Corresponding author. E-mail address: [email protected] (R.W. Hartmann).
The RALES trial (randomised aldactone† evaluation study, 1999) showed that the aldosterone antagonist spironolactone reduces mortality in heart failure patients by 30%. However, its use was accompanied by progestional and antiandrogenic side effects [6 /8]. In our opinion, a better target for the treatment of congestive heart failure, myocardial fibrosis and hyperaldosteronism is aldosterone synthase. Non steroidal inhibitors should be preferred for they are expected to have less side effects on the endocrine system. The new inhibitors must not affect steroid 11b-hydroxylase (CYP11B1) */the key enzyme of glucocorticoid biosynthesis */which shows more than 93% homology compared to CYP11B2 [1,9]. In recent years our group was engaged in the synthesis and evaluation of potent and selective inhibitors of other P450 enzymes, CYP19 [10,11], CYP17 [12,13] and CYP5 [14,15], all of which are important targets for major diseases like breast and prostate cancer. In this paper we are presenting the discovery of a high number of hits which were obtained by screening our P450 inhibitor library for inhibition of aldosterone synthase.
´ ditions scientifiques et me´dicales Elsevier SAS. All rights reserved. 0223-5234/03/$ - see front matter # 2003 E doi:10.1016/S0223-5234(03)00049-7
364
R.W. Hartmann et al. / European Journal of Medicinal Chemistry 38 (2003) 363 /366
optimisation resulted in very potent and selective inhibitors of human CYP11B2.
2. Results
Fig. 1. Biosynthesis of mineralo- and glucocorticoids.
Taking into consideration oral absorption and selectivity towards other CYP enzymes, further structural
Fig. 2. Screening system for the evaluation of CYP11B2 inhibitors.
As we have recently described, a strong inhibitor of a P450 enzyme must be able to mimic the steroidal substrate of the corresponding enzyme and should consist of a group capable of complexing the heme iron [16]. The fact that there is a high homology in the amino acid sequences of the substrate binding sites made us decide to use our library of compounds, which have been synthesised as potential inhibitors of P450 enzymes like CYP19, CYP17 and CYP5 (see Refs. [10 /15] and literature cited therein). Several hundreds of compounds were tested using our screening system (Fig. 2). In a kind of preselection test, the compounds are evaluated first for inhibition of bovine CYP11B using mitochondria from adrenal cortex [10]. Compounds showing inhibitory activity are further tested for oral absorption [17], as a lack of oral absorption often is the reason for compounds failing at an advanced development stage. In case of high flux rates, compounds are subsequently evaluated for inhibition of the human target enzyme CYP11B2. First an assay is employed using a very robust yeast system stably expressing human CYP11B2 [18]. Compounds showing strong inhibition are further tested for activity and selectivity using the more difficult to handle V79MZ cells, stably expressing CYP11B2 and CYP11B1, respectively [18]. Tests for selectivity towards other steroidogenic CYP enzymes (CYP11A1, CYP19, CYP17) have to be performed using microsomal or mitochondrial fractions of appropriate tissues and cells recombinantly expressing the target enzyme [19 /21], before an in vivo test is taken into consideration. Approximately 100 compounds showed inhibition of bovine CYP11B. They have been further evaluated using our screening system and the most promising were structurally modified for optimising. In the following two interesting examples are presented. In the class of aryl-substituted cyclopropatetrahydronaphthalines, which originally have been developed as CYP19 inhibitors [10,22], 3- and 4-pyridyl substituted compounds showed inhibition of bovine CYP11B. Exchange of the heterocycle by a 4-imidazolyl substituent increased inhibition strongly (compound 1, Fig. 3).
Fig. 3. Structural optimisation in the class of cyclopropatetrahydronaphthalines.
R.W. Hartmann et al. / European Journal of Medicinal Chemistry 38 (2003) 363 /366
365
Table 1 Inhibition of steroidogenic CYP enzymes by cyclopropatetrahydronaphthalines No
X
CYP11B
a
CYP11B yeast b
CYP19 c
% inhibition 1 2 3 4 5
H 4-OCH3 5-OCH3 6-OCH3 / a b c d
CYP17 d
IC50 values [nM]
909/3 839/4 819/4 819/5 809/3
639/5 729/2 599/5 469/6 789/4
600 1700 1600 120 1040
1800 2200 43 2400 2800
Corticosterone 200 mM, inhibitor 1 mM. Deoxycorticosterone 100 nM, inhibitor 2.5 mM. Testosterone 2.5 mM. Progesterone 25 mM.
Fig. 4. Structure-activity-relationships in the class of arylmethylsubstituted indanes.
As can be seen from Table 1, introduction of a methoxy group into the benzene nucleus slightly decreased inhibition of the bovine enzyme, whereas in case
of human CYP11B2, the 4-OCH3 group increased inhibitory activity (compound 2). Tested for selectivity towards sex hormone synthesising enzymes CYP19 and CYP17, the compounds only showed inhibitory activity in rather high concentrations, compound 2 being the most selective one. Tested for activity and selectivity using V79MZ cells, 2 turned out to be highly active (CYP11B2, IC50 /41 nM) and rather selective (CYP11B1, IC50 /90 nM). Exchange of the benzene nucleus by a thienyl group (compound 5) further increased activity and selectivity. Interestingly in the class of aryl- and arylmethylsubstituted tetrahydronaphthalenes [23] structural modification resulted in highly potent and rather selective CYP11B1 inhibitors. In the class of arylmethylene substituted indanones, which had been synthesised as CYP19 inhibitors [24], CYP11B inhibition strongly depends on the heterocyclic
Table 2 Activity, selectivity and oral absorption profile of HB60
Assay CYP11B CYP11B2 CYP11B2 CYP11B1
mitochondria yeast a V79 a V79 a
Oral absorption, Papp values g:
PAMPA d PAMPORE e Caco-2 f
a b c d e f g
Human enzyme. Bovine. Calculated for identical substrate conditions. Lipophilic artificial membrane. Hydrophilic artificial membrane. Human cell line. [cm/s/10 6].
b
IC50 values [nM] c
Assay
100 260 59 273
CYP19 CYP17 CYP11A1 CYP5
99/1 119/2 89/2
IC50 values [nM] c microsomes a microsomes a mitochondria b whole blood a
76 000 720 000 /100 000 35 000
366
R.W. Hartmann et al. / European Journal of Medicinal Chemistry 38 (2003) 363 /366
ring and the substituent X at the benzene moeity (Fig. 4). Maximum activity of less than 40% could be observed. Hydrogenation of the double bond increases activity by a factor of about 2. Reduction of the ketone group resulted in highly active compounds, the most interesting of which was HB60. It becomes apparent from Table 2 that HB60 is a highly potent inhibitor of human CYP11B2. The compound is suitable for oral application, is rather selective towards CYP11B1 and highly selective towards other steroidogenic CYP enzymes.
3. Conclusion Inhibition of aldosterone formation (CYP11B2) is a new strategy for the treatment of congestive heart failure and myocardial fibrosis. Compounds 5 and HB60 are highly active and selective inhibitors of this target enzyme and therefore they can be seen as promising lead compounds which presently are further optimised.
Acknowledgements Thanks are due to the Saarland Ministry of Education and the Fonds der Chemischen Industrie for financial support and to Prof. R.B., Saarland University, for supplying us with the V79MZ cells.
References [1] T. Kawamoto, Y. Mitsuuchi, K. Toda, Y. Yokoyama, K. Miyakara, S. Miura, T. Onishi, Y. Ichihawa, K. Nakao, H. Imura, S. Ulick, Y. Shizuta, Proc. Natl. Acad. Sci. USA 89 (1992) 1458 /1462. [2] C.G. Brilla, Herz 25 (2000) 299 /306.
[3] M. Young, J.W. Funder, Trends Endocrinol. Metab. 11 (2000) 224 /226. [4] C.G. Brilla, Cardiovasc. Res. 47 (2000) 1 /3. [5] P. Lijnen, V. Petrov, J. Mol. Cell Cardiol. 32 (2000) 865 /879. [6] B. Pitt, F. Zannad, W.J. Remme, R. Cody, A. Castaigne, A. Perez, J. Palensky, J. Wittes, New Eng. J. Med. 341 (1999) 709 / 717. [7] R.J. MacFadden, C.S. Barr, A.D. Struthers, Cardiovasc. Res. 35 (1997) 30 /34. [8] J.E. Soberman, K.T. Weber, Curr. Hypertens. Rep. 2 (2000) 451 / 456. [9] S.E. Taymans, S. Pack, E. Pak, D.J. Torpy, Z. Zhuang, C.A. Stratakis, J. Clin. Endocrinol. Metab. 83 (1998) 1033 /1036. [10] R.W. Hartmann, H. Bayer, G. Gru¨n, T. Sergejew, U. Bartz, M. Mitrenga, J. Med. Chem. 38 (1995) 2103 /2111. [11] H. Bayer, C. Batzl, R.W. Hartmann, A. Mannschreck, J. Med. Chem. 34 (1991) 2685 /2691. [12] R.W. Hartmann, M. Hector, B.G. Wachall, A. Palusczak, M. Palzer, V. Huch, M. Veith, J. Med. Chem. 43 (2000) 4437 /4445. [13] B.G. Wachall, M. Hector, Y. Zhuang, R.W. Hartmann, Bioorg. Med. Chem. 7 (1999) 1913 /1924. [14] R.W. Hartmann, M. Frotscher, Arch. Pharm. Pharm. Med. Chem. 332 (1999) 358 /362. [15] C. Jacobs, M. Frotscher, G. Dannhardt, R.W. Hartmann, J. Med. Chem. 43 (2000) 1841 /1851. [16] R.W. Hartmann, P.B. Ehmer, S. Haidar, M. Hector, J. Jose, C.D.P. Klein, S.B. Seidel, T.F. Sergejew, B.G. Wachall, G.A. Wa¨chter, Y. Zhuang, Arch. Pharm. Pharm. Med. Chem. 335 (2002) 119 /128. [17] For information see: http://www.pharmacelsus.com. [18] P.B. Ehmer, M. Bureik, R. Bernhardt, U. Mu¨ller, R.W. Hartmann, J. Steroid Biochem. Mol. Biol. 81 (2002) 173 /179. [19] R.W. Hartmann, C. Batzl, J. Med. Chem. 29 (1986) 1362 /1369. [20] T. Sergejew, R.W. Hartmann, J. Enz. Inhib. 81 (1994) 113 / 122. [21] P.B. Ehmer, J. Jose, R.W. Hartmann, J. Steroid Biochem. Mol. Biol. 75 (2000) 57 /63. [22] G.A. Wa¨chter, R.W. Hartmann, T. Sergejew, G.L. Gru¨n, D. Ledergerber, J. Med. Chem. 39 (1996) 834 /841. [23] R.W. Hartmann, M. Frotscher, D. Ledergerber, G.A. Wa¨chter, G.L. Gru¨n, T.F. Sergejew, Arch. Pharm. Pharm. Med. Chem. 339 (1996) 251 /261. [24] R.W. Hartmann, H. Bayer, G. Gru¨n, J. Med. Chem. 37 (1994) 1275 /1281.
Discovery of selective CYP11B2 (aldosterone synthase) inhibitors for the therapy of congestive heart failure and myocardial fibrosis Rolf W. Hartmann *, Ursula Mu¨ller, Peter B. Ehmer Pharmaceutical and Medicinal Chemistry, Saarland University, P.O. Box 151150, D-66041 Saarbrucken, Germany Received 30 September 2002; accepted 14 December 2002
Abstract An increased aldosterone concentration due to congestive heart failure leads to a further progression of the disease as well as to myocardial fibrosis. To interfere with these fatal processes selective inhibition of aldosterone synthase (CYP11B2) is required. CYP11B1, a key enzyme in glucocorticoid biosynthesis showing a high homology to the target enzyme ( /93%), must not be inhibited. Screening of our P450 inhibitor library for inhibition of bovine aldosterone synthase resulted in a high number of compounds showing reasonable inhibition. In the next step substances were tested for oral absorption using two artificial membrane assays. The inhibition of human CYP11B2 was evaluated using assays in fission yeast and V79MZ cells stably expressing the active human target enzyme. For selectivity, inhibition of CYP11B1, CYP11A1, CYP17, CYP19 and CYP5 was determined. Rather potent and selective compounds obtained in this way were structurally further optimised, finally leading to inhibitors showing IC50 values within the low nanomolar range. ´ ditions scientifiques et me´dicales Elsevier SAS. All rights reserved. # 2003 E Keywords: Congestive heart failure; Myocardial fibrosis; Aldosterone synthase (CYP11B2); 11b-hydroxylase (CYP11B1); P450 (CYP) enzymes; Selective inhibitors
1. Introduction Aldosterone synthase (CYP11B2) is a P450 enzyme which catalyses the key step in mineralocorticoid biosynthesis, the conversion of 11-deoxycorticosterone to the most potent mineralocorticoid aldosterone (Fig. 1, [1]). Aldosterone synthesis is regulated by several physiological parameters like the renin /angiotensin / aldosterone-system (RAAS) and the plasma potassium concentration. Elevations in plasma aldosterone levels play an important role in the pathophysiology of certain forms of hypertension and congestive heart failure [2]. The RAAS is pathologically activated due to an insufficient renal flow which leads to an excessive release of aldosterone [3]. Chronic elevations in plasma aldosterone increase the blood volume and may stimulate cardiac fibroblasts, resulting in cardiac hypertrophy, myocardial fibrosis and ventricular arrhythmia [4,5].
* Corresponding author. E-mail address: [email protected] (R.W. Hartmann).
The RALES trial (randomised aldactone† evaluation study, 1999) showed that the aldosterone antagonist spironolactone reduces mortality in heart failure patients by 30%. However, its use was accompanied by progestional and antiandrogenic side effects [6 /8]. In our opinion, a better target for the treatment of congestive heart failure, myocardial fibrosis and hyperaldosteronism is aldosterone synthase. Non steroidal inhibitors should be preferred for they are expected to have less side effects on the endocrine system. The new inhibitors must not affect steroid 11b-hydroxylase (CYP11B1) */the key enzyme of glucocorticoid biosynthesis */which shows more than 93% homology compared to CYP11B2 [1,9]. In recent years our group was engaged in the synthesis and evaluation of potent and selective inhibitors of other P450 enzymes, CYP19 [10,11], CYP17 [12,13] and CYP5 [14,15], all of which are important targets for major diseases like breast and prostate cancer. In this paper we are presenting the discovery of a high number of hits which were obtained by screening our P450 inhibitor library for inhibition of aldosterone synthase.
´ ditions scientifiques et me´dicales Elsevier SAS. All rights reserved. 0223-5234/03/$ - see front matter # 2003 E doi:10.1016/S0223-5234(03)00049-7
364
R.W. Hartmann et al. / European Journal of Medicinal Chemistry 38 (2003) 363 /366
optimisation resulted in very potent and selective inhibitors of human CYP11B2.
2. Results
Fig. 1. Biosynthesis of mineralo- and glucocorticoids.
Taking into consideration oral absorption and selectivity towards other CYP enzymes, further structural
Fig. 2. Screening system for the evaluation of CYP11B2 inhibitors.
As we have recently described, a strong inhibitor of a P450 enzyme must be able to mimic the steroidal substrate of the corresponding enzyme and should consist of a group capable of complexing the heme iron [16]. The fact that there is a high homology in the amino acid sequences of the substrate binding sites made us decide to use our library of compounds, which have been synthesised as potential inhibitors of P450 enzymes like CYP19, CYP17 and CYP5 (see Refs. [10 /15] and literature cited therein). Several hundreds of compounds were tested using our screening system (Fig. 2). In a kind of preselection test, the compounds are evaluated first for inhibition of bovine CYP11B using mitochondria from adrenal cortex [10]. Compounds showing inhibitory activity are further tested for oral absorption [17], as a lack of oral absorption often is the reason for compounds failing at an advanced development stage. In case of high flux rates, compounds are subsequently evaluated for inhibition of the human target enzyme CYP11B2. First an assay is employed using a very robust yeast system stably expressing human CYP11B2 [18]. Compounds showing strong inhibition are further tested for activity and selectivity using the more difficult to handle V79MZ cells, stably expressing CYP11B2 and CYP11B1, respectively [18]. Tests for selectivity towards other steroidogenic CYP enzymes (CYP11A1, CYP19, CYP17) have to be performed using microsomal or mitochondrial fractions of appropriate tissues and cells recombinantly expressing the target enzyme [19 /21], before an in vivo test is taken into consideration. Approximately 100 compounds showed inhibition of bovine CYP11B. They have been further evaluated using our screening system and the most promising were structurally modified for optimising. In the following two interesting examples are presented. In the class of aryl-substituted cyclopropatetrahydronaphthalines, which originally have been developed as CYP19 inhibitors [10,22], 3- and 4-pyridyl substituted compounds showed inhibition of bovine CYP11B. Exchange of the heterocycle by a 4-imidazolyl substituent increased inhibition strongly (compound 1, Fig. 3).
Fig. 3. Structural optimisation in the class of cyclopropatetrahydronaphthalines.
R.W. Hartmann et al. / European Journal of Medicinal Chemistry 38 (2003) 363 /366
365
Table 1 Inhibition of steroidogenic CYP enzymes by cyclopropatetrahydronaphthalines No
X
CYP11B
a
CYP11B yeast b
CYP19 c
% inhibition 1 2 3 4 5
H 4-OCH3 5-OCH3 6-OCH3 / a b c d
CYP17 d
IC50 values [nM]
909/3 839/4 819/4 819/5 809/3
639/5 729/2 599/5 469/6 789/4
600 1700 1600 120 1040
1800 2200 43 2400 2800
Corticosterone 200 mM, inhibitor 1 mM. Deoxycorticosterone 100 nM, inhibitor 2.5 mM. Testosterone 2.5 mM. Progesterone 25 mM.
Fig. 4. Structure-activity-relationships in the class of arylmethylsubstituted indanes.
As can be seen from Table 1, introduction of a methoxy group into the benzene nucleus slightly decreased inhibition of the bovine enzyme, whereas in case
of human CYP11B2, the 4-OCH3 group increased inhibitory activity (compound 2). Tested for selectivity towards sex hormone synthesising enzymes CYP19 and CYP17, the compounds only showed inhibitory activity in rather high concentrations, compound 2 being the most selective one. Tested for activity and selectivity using V79MZ cells, 2 turned out to be highly active (CYP11B2, IC50 /41 nM) and rather selective (CYP11B1, IC50 /90 nM). Exchange of the benzene nucleus by a thienyl group (compound 5) further increased activity and selectivity. Interestingly in the class of aryl- and arylmethylsubstituted tetrahydronaphthalenes [23] structural modification resulted in highly potent and rather selective CYP11B1 inhibitors. In the class of arylmethylene substituted indanones, which had been synthesised as CYP19 inhibitors [24], CYP11B inhibition strongly depends on the heterocyclic
Table 2 Activity, selectivity and oral absorption profile of HB60
Assay CYP11B CYP11B2 CYP11B2 CYP11B1
mitochondria yeast a V79 a V79 a
Oral absorption, Papp values g:
PAMPA d PAMPORE e Caco-2 f
a b c d e f g
Human enzyme. Bovine. Calculated for identical substrate conditions. Lipophilic artificial membrane. Hydrophilic artificial membrane. Human cell line. [cm/s/10 6].
b
IC50 values [nM] c
Assay
100 260 59 273
CYP19 CYP17 CYP11A1 CYP5
99/1 119/2 89/2
IC50 values [nM] c microsomes a microsomes a mitochondria b whole blood a
76 000 720 000 /100 000 35 000
366
R.W. Hartmann et al. / European Journal of Medicinal Chemistry 38 (2003) 363 /366
ring and the substituent X at the benzene moeity (Fig. 4). Maximum activity of less than 40% could be observed. Hydrogenation of the double bond increases activity by a factor of about 2. Reduction of the ketone group resulted in highly active compounds, the most interesting of which was HB60. It becomes apparent from Table 2 that HB60 is a highly potent inhibitor of human CYP11B2. The compound is suitable for oral application, is rather selective towards CYP11B1 and highly selective towards other steroidogenic CYP enzymes.
3. Conclusion Inhibition of aldosterone formation (CYP11B2) is a new strategy for the treatment of congestive heart failure and myocardial fibrosis. Compounds 5 and HB60 are highly active and selective inhibitors of this target enzyme and therefore they can be seen as promising lead compounds which presently are further optimised.
Acknowledgements Thanks are due to the Saarland Ministry of Education and the Fonds der Chemischen Industrie for financial support and to Prof. R.B., Saarland University, for supplying us with the V79MZ cells.
References [1] T. Kawamoto, Y. Mitsuuchi, K. Toda, Y. Yokoyama, K. Miyakara, S. Miura, T. Onishi, Y. Ichihawa, K. Nakao, H. Imura, S. Ulick, Y. Shizuta, Proc. Natl. Acad. Sci. USA 89 (1992) 1458 /1462. [2] C.G. Brilla, Herz 25 (2000) 299 /306.
[3] M. Young, J.W. Funder, Trends Endocrinol. Metab. 11 (2000) 224 /226. [4] C.G. Brilla, Cardiovasc. Res. 47 (2000) 1 /3. [5] P. Lijnen, V. Petrov, J. Mol. Cell Cardiol. 32 (2000) 865 /879. [6] B. Pitt, F. Zannad, W.J. Remme, R. Cody, A. Castaigne, A. Perez, J. Palensky, J. Wittes, New Eng. J. Med. 341 (1999) 709 / 717. [7] R.J. MacFadden, C.S. Barr, A.D. Struthers, Cardiovasc. Res. 35 (1997) 30 /34. [8] J.E. Soberman, K.T. Weber, Curr. Hypertens. Rep. 2 (2000) 451 / 456. [9] S.E. Taymans, S. Pack, E. Pak, D.J. Torpy, Z. Zhuang, C.A. Stratakis, J. Clin. Endocrinol. Metab. 83 (1998) 1033 /1036. [10] R.W. Hartmann, H. Bayer, G. Gru¨n, T. Sergejew, U. Bartz, M. Mitrenga, J. Med. Chem. 38 (1995) 2103 /2111. [11] H. Bayer, C. Batzl, R.W. Hartmann, A. Mannschreck, J. Med. Chem. 34 (1991) 2685 /2691. [12] R.W. Hartmann, M. Hector, B.G. Wachall, A. Palusczak, M. Palzer, V. Huch, M. Veith, J. Med. Chem. 43 (2000) 4437 /4445. [13] B.G. Wachall, M. Hector, Y. Zhuang, R.W. Hartmann, Bioorg. Med. Chem. 7 (1999) 1913 /1924. [14] R.W. Hartmann, M. Frotscher, Arch. Pharm. Pharm. Med. Chem. 332 (1999) 358 /362. [15] C. Jacobs, M. Frotscher, G. Dannhardt, R.W. Hartmann, J. Med. Chem. 43 (2000) 1841 /1851. [16] R.W. Hartmann, P.B. Ehmer, S. Haidar, M. Hector, J. Jose, C.D.P. Klein, S.B. Seidel, T.F. Sergejew, B.G. Wachall, G.A. Wa¨chter, Y. Zhuang, Arch. Pharm. Pharm. Med. Chem. 335 (2002) 119 /128. [17] For information see: http://www.pharmacelsus.com. [18] P.B. Ehmer, M. Bureik, R. Bernhardt, U. Mu¨ller, R.W. Hartmann, J. Steroid Biochem. Mol. Biol. 81 (2002) 173 /179. [19] R.W. Hartmann, C. Batzl, J. Med. Chem. 29 (1986) 1362 /1369. [20] T. Sergejew, R.W. Hartmann, J. Enz. Inhib. 81 (1994) 113 / 122. [21] P.B. Ehmer, J. Jose, R.W. Hartmann, J. Steroid Biochem. Mol. Biol. 75 (2000) 57 /63. [22] G.A. Wa¨chter, R.W. Hartmann, T. Sergejew, G.L. Gru¨n, D. Ledergerber, J. Med. Chem. 39 (1996) 834 /841. [23] R.W. Hartmann, M. Frotscher, D. Ledergerber, G.A. Wa¨chter, G.L. Gru¨n, T.F. Sergejew, Arch. Pharm. Pharm. Med. Chem. 339 (1996) 251 /261. [24] R.W. Hartmann, H. Bayer, G. Gru¨n, J. Med. Chem. 37 (1994) 1275 /1281.