Rolf W. Hartmann

Pyridyl substituted benzocycloalkenes: new inhibitors of 17 alpha-hydroxylase/17,20-lyase (P450 17 alpha).

Compounds capable of inhibiting 17 alpha-hydroxylase/17,20-lyase (P450 17 alpha) are of great interest for the therapy of prostatic cancer since they block androgen biosynthesis. In order to evaluate the inhibitory activity of a series of benzocycloalkenes developed in our group, an in vitro assay was established using rat testicular microsomes as source of the enzyme, non labelled progesterone as substrate and a HPLC procedure for separation of the steroids. The inhibitory activities of 33 test compounds were compared to ketoconazole (IC50 67 microM), a known inhibitor of P450 17 alpha, which recently has been successfully used in prostate cancer patients. Several compounds of the present study were stronger inhibitors of P450 17 alpha than ketoconazole. The most active compounds were compound 12(5-methoxy-2-(4-pyridylmethyl)-1-tetralone: IC50 13 microM) and compound 13(5-methoxy-2-(4-pyridyl)-1-tetralone: IC50 13 microM).

3,4-Bis(3′-hydroxyphenyl)hexane — A new mammary tumor-inhibiting compound

SummaryThe syntheses of the hexestrol derivatives 3,4-bis-(3′-hydroxyphenyl)hexane (4a), 3,4-bis(4′-fluoro-3′-hydroxyphenyl)hexane (4b), 3,4-bis(3′, 4′dihydroxyphenyl)hexane (4c), and 3,4-bis(3′,4′-diacetoxyphenyl)hexane (4d) are described. All compounds showed a marked, competitive inhibition of the estradiol receptor interaction (Ka4c>Ka4a>Ka4d>Ka4b). Evaluated in the mouse uterine weight test compounds 4c and 4d almost reached the estrone effect, whereas 4a and 4b did not produce full uterotrophic response. Compounds 4a-d antagonized the estrone stimulated uterine growth of the immature mouse. Compound 4a (NSC-297170) exhibited a specific, dose-related growth inhibition of the estrogen responsive MCF-7 human breast tumor cell line. Tested on the 9,10-dimethyl-1,2-benzanthracene-induced hormonedependent mammary adenocarcinoma of the Sprague-Dawley rat all compounds showed marked inhibition of tumor growth. As in all experiments compounds 4a and 4b, which is resistant to hydroxylation in 4′position exhibited an identical pattern of action, which is different from that shown by compound 4c, the effect of compound 4a cannot be explained by its possible catechol metabolite 4c.

Stable Expression of the Human 5α-Reductase Isoenzymes Type I and Type II in HEK293 Cells to Identify Dual and Selective Inhibitors

A eucaryotic cell assay was established to identify novel, dual and selective inhibitors of human 5α-reductase. For this purpose the cDNAs encoding 5α-reductase type I and type II were inserted into a pRcCMV vector and expressed in human embryonic kidney (HEK293) cells. Single cell clones with substantially high enzymatic activity were selected and established as permanent cell lines. KM values were determined for both isozymes. The inhibitory potency of several steroidal and non-steroidal compounds synthesized in our group, as well as finasteride and 4MA as controls, were tested by measuring the conversion of [3H]androstenedione. Reaction products were quantified by a HPLC reversed phase technique. Using the new cell assays, selective as well as novel dual 5α-reductase inhibitors with IC50 values between 1.0 and 2.5 μM were identified.

5α-Reductase in Intact DU145 Cells: Evidence for Isozyme I and Evaluation of Novel Inhibitors

The epithelial‐like human prostatic carcinoma cell line DU145, which expresses 5α‐reductase type I isozyme, was used to test a series of potential 5α‐reductase inhibitors. The exclusive expression of the type I isozyme was confirmed by PCR and subsequent DNA sequence analysis. Culture conditions were optimized for high conversion rates. Using this whole cell assay finasteride, 4MA, and 65 steroidal and non‐steroidal compounds synthesized in our group were tested for their inhibitory activity. Inhibitors with IC50 values in the nanomolar range could be identified.

Structural modifications of salicylates: inhibitors of human CD81-receptor HCV-E2 interaction.

Starting point of the present paper was the result of a virtual screening using the open conformation of the large extracellular loop (LEL) of the CD81‐receptor (crystal structure: PDB‐ID: 1G8Q). After benzyl salicylate had been experimentally validated to be a moderate inhibitor of the CD81‐LEL–HCV‐E2 interaction, further optimization was performed and heterocyclic‐substituted benzyl salicylate derivatives were synthesized. The compounds were tested for their ability to inhibit the interaction of a fluorescence‐labeled antibody to CD81‐LEL using HUH7.5 cells. No compound showed an increase concerning the inhibition of the protein‐protein interaction compared to benzyl salicylate.

5α-reductase in human embryonic kidney cell line HEK293: Evidence for type II enzyme expression and activity

Human 5α-reductase catalyses the last step in androgen biosynthesis, namely the reduction of testosterone (T) to the more potent androgen dihydrotestosterone (DHT). The enzyme is therefore considered to be an important drug target for androgen related diseases such as benign prostatic hyperplasia and prostate cancer. The present study displays evidence that the human embryonic kidney cell line HEK293 which is frequently used in recombinant target protein expression contains an endogenous 5α-reductase type II activity. After an incubation of 24 h 1 × 106 HEK293 cells converted 23% of the substrate 4-androstene-3,17-dione (7.5 nM) to the product 5α-androstane-3,17-dione. Reverse transcription polymerase chain reaction was carried out to identify the mRNA of the isoform responsible for the 5α-reductase activity. Only with type II specific primers a fragment with the predicted size was amplified, while with type I specific primers no band could be observed. An antiserum against human 5α-reductase type II was raised by immunizing a rabbit with a hemocyanin-conjugated peptide corresponding to amino acid 29 to 44 of the type II enzyme. Western blot analysis of different fractions of a HEK293 homogenate performed with this antiserum detected a band at 45 kDa in the nuclear and microsomal fraction corresponding to 5α-reductase type II protein. (Mol Cell Biochem 270: 201–208, 2005)

Development of a Screening Assay for the In Vitro Evaluation of Thromboxane A2 Synthase Inhibitors

Inhibitors of thromboxane A2 (TxA2) synthase are regarded as potentially useful agents in the treatment of cardiovascular diseases and in the prevention of tumour cell metastases. We report here a novel in vitro assay for the evaluation of TxA2 synthase inhibitors. For the determination of inhibitory activity, malondialdehyde (MDA) formation by TxA2 synthase in whole blood was utilized. After reaction with thiobarbituric acid MDA was quantified spectrofluorimetrically. The blank value was obtained by incubation with a selective TxA2 synthase inhibitor. For the screening of compounds the simple MDA assay represents an alternative to the rather expensive and time consuming radioimmunoassay, HPLC and TLC methods. Only for compounds which have been shown to be good inhibitors in the MDA assay should a radioimmunoassay for selective inhibition of TxA2 synthase be performed.

Effect of 2-(4-aminophenylmethyl)-6-hydroxy-3, 4-dihydronaphthalen-1(2H)-one on all-trans and 13-cis-retinoic acid levels in plasma quantified by high perfomance liquid chromatography coupled to tandem mass spectrometry.

The effect of the titled tetralone as a retinoic acid metabolism blocking agent (RAMBA) in vivo in comparison with ketoconazole, a well known cytochrome P450 inhibitor, was studied. Development of a HPLC/MS/MS method for the quantification of retinoic acid levels extracted from rat plasma was used to demonstrate that ketoconazole and the tetralone (100 mg/kg) enhanced the endogenous plasma concentration of retinoic acid. Levels of retinoid were raised from a control value of 0.11 to 0.15 and 0.17 ng/mL after treatment with tetralone and ketoconazole respectively showing that the tetralone and ketoconazole lead to comparable effects, indicating an inhibitory activity of the tetralone on retinoic acid metabolism.

1-(2,6-dichloro-4-hydroxyphenyl)-2-phenylethanes--new biological response modifiers for the therapy of breast cancer. Synthesis and evaluation of estrogenic/antiestrogenic properties

[meso‐1,2‐Bis(2,6‐dichloro‐4‐hydroxyphenyl)ethylenediamine]platinum(II) complexes (meso‐1‐PtLĹ; L,Ĺ= Cl or L = H2O and Ĺ = OSO3) are highly effective towards hormone‐sensitive, rodent breast cancers due to their significant estrogenic potencies. Their antitumor activities are caused by modification of the immune response. The pharmacophor of these compounds, the 1,2‐bis(2,6‐dichloro‐4‐hydroxyphenyl) ethane (23H), was used as the lead structure in a structure‐activity study with the goal of finding new biological response modifiers for the therapy of breast cancer. As intermediates for the synthesis of the 23H derivatives, the CH3O‐substituted stilbenes 12E/12Z— 16E/16Z were prepared by reaction of the related benzyltriphenylphosphonium halides with 2,6‐dichloro‐4‐methoxybenzaldehyde by the method of Wittig/Campbell and Donald, respectively. Separation of the E/Z‐mixtures was performed by fractional crystallization and/or column chromatography. The E‐1,2‐bis(2,6‐dichloro‐4‐methoxyphenyl)ethene (17E) was obtained by reductive coupling of 2,6‐dichloro‐4‐methoxybenzaldehyde with TiCl4/Zn according to the method of Mukaiyama. Illumination of the solution of 17E in benzene with UV light resulted in an E/Z‐isomerization. Compound 17Z could be isolated from this mixture. The CH3O‐substituted stilbenes were transformed into their 1,2‐diphenylethanes (12H—17H) by catalytic hydrogenation of the C1=C2 double bond. Ether cleavage of the compounds was performed with BBr3. In the estrogen receptor binding assay all OH‐substituted 1,2‐diphenylethanes showed affinity to the estrogen receptor, which was about two orders of magnitude lower than that of 17 β‐estradiol. In the uterus weight test on the immature mouse 1,2‐diphenylethanes with 4‐substituted OH groups proved to be “true” estrogens (19H: 2‐F/4‐OH; 20H: 2‐Cl/4‐OH; 23H: 2,6‐Cl2/4‐OH), while those with a 3‐substituted OH group in the 2‐phenyl ring showed the properties of a “partial” estrogen (18H: 3‐OH) or of an “impeded” estrogen (21H: 2‐Cl/3‐OH; 22H: 2‐Cl/5‐OH). The latter also showed significant additional antiestrogenic activity. The related E‐stilbenes mostly exhibit similar hormonal activities. As a rule, the replacement of the OH groups by the CH3O groups and the change from the E‐ to the Z‐configuration led to a reduction of the estrogenic potencies. Several of the 1‐(2,6‐dichloro‐4‐methoxyphenyl)‐2‐phenylethenes (12E: 3‐OCH3; 12Z: 3‐OCH3; 15E: 2‐Cl/3‐OCH3; 15Z: 2‐Cl/3‐OCH3; 16E: 2‐Cl/5‐OCH3) produced antiestrogenic effects in the uterus weight test. It is supposed that those new 1‐(2,6‐dichloro‐4‐hydroxyphenyl)‐2‐phenylethanes endowed with marked estrogenic properties are also active as biological response modifiers in animals bearing hormone‐sensitive breast cancer. The antiestrogenic derivatives presumably inhibit the breast cancer development by competing with tumor growth stimulating endogenous estrogens for the binding to the receptor. This is to be confirmed in a further study.

Screening procedure for the development of mammary tumour-inhibiting anti-oestrogens

Many human mammary tumours are hormone dependent. Consequently endocrine manipulation for disseminated breast cancer has become first line treatment in recent years. Anti-oestrogens, principally tamoxifen-have become the therapeutic drugs of choice, especially in post-menopausal women. But since the remission rate after tamoxifen treatment is only about 40% there is a need for more effective compounds. For more than 10 years our group has been working on the syntheses and pharmacological evaluation of potential mammary tumour-inhibiting anti-oestrogens. During this time hundreds of compounds have been synthesized in our laboratory. In view of such a great number of compounds it is important to have an efficient screening system available in order to select only the most promising compounds for further testing. In this paper we describe our screening system and give a short account of the action of two very promising mammary tumour-inhibiting anti-oestrogens on our test models.