Fig. 27.3. a) Distribution of molecular weight, library selected by a combination of the "rule log P, number of donors and the number of of five" and diversity. Compounds with acceptors for the whole virtual library, (b) For properties above the "rule of five" limits are the library selected by the "rule of five"; (c) for represented by dark grey bars, the library selected by diversity; and (d) the ing role in supporting this process. With relatively little computational effort virtual library compounds can be optimized with respect to maximum diversity, high similarity with an already known screening hit or lead structure, or improved drug-likeness and ADME properties. Even the efficiency of virtual screening can be increased by applying appropriate filters for functional groups, molecular weight, or other physicochemical properties that reduce the number of compounds for the actual docking process. Depending on the intended purpose for which the library is designed and synthesized, a combination of different computational methods is, in most cases, appropriate.

A screening hit from a library that has been optimized using computational methods can be optimized more efficiently based on the design principles or rules used for the original library. The new library can be focused on compounds similar in structure to the screening hit while areas of the property space that have not led to bioactive compounds can more easily be avoided.

Starting from calculated physicochemical properties and standard 2D fingerprint keys, as available with chemical structure databases for structure searching, new descriptors have been developed that can address more complex properties (e.g. 3D pharmacophore keys) or encode properties more efficiently (e.g. BCUT) for library evaluation. In general, fingerprint keys or bitstrings provide an efficient way to encode properties and can be used for the diversity assessment of combinatorial libraries. They can be tailored according to the properties suitable for a particular purpose.

Given the wealth of available descriptors and methods it is already becoming more and more challenging to find the most suitable descriptors for a particular library design task and the best optimization method. This is reminiscent of the situation in classical QSAR. However, the situation is much more promising with virtual library design than with QSAR. With combinatorial chemistry a whole set of new test compounds can be synthesized and tested, whereas in classical QSAR projects typically only single compounds (usually only the estimated most active ones) become available. The educated ''shot gun'' approach with a combinatorial library has a much higher probability for success during screening. The overall risk of failure due to insufficient information on the true nature of the interaction of bioactive compounds with their biological target or system decreases when whole libraries of potentially active compounds (rather than single compounds) are synthesized and screened.

While combinatorial chemistry can efficiently be supported by computational methods, support of computer-assisted drug design can be evaluated by library synthesis and screening. As a result of the combined efforts of computer-assisted library design and combinatorial chemistry, a new quality in the overall drug discovery process has been achieved. While its usefulness has been proven for several cases, library design is an active research field that is expected to gain more importance and applicability in the future.


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Erythropoietin Sensitizer - A Case Study

Berthold Hinzen, Gabriele Bräunlich, Christoph Gerdes, Thomas Krämer, Klemens Lustig, Ulrich Nielsch, Michael Sperzel, Josef Pernerstorfer



In healthy individuals kidneys perform the vital functions of excretion of waste products from the body, maintaining fluid levels, and controlling blood pressure. Furthermore, kidneys regulate the production of red blood cells by the synthesis of erythropoietin (EPO) [1]. When kidneys fail, the production of erythropoietin and the production of red blood cells are reduced. Thus, severe anemia is often caused by insufficient production of EPO, which circulates through the blood stream to the bone marrow, where it stimulates the production of red blood cells from stem cells [2]. Insufficient amounts of EPO result in inadequate numbers of red blood cells, and thus oxygen levels are reached which are too low to maintain normal physiological conditions. Anemia leaves patients permanently fatigued and exhausted [3].

Traditional treatments for anemic patients with renal failure have been repeated blood transfusions and androgen therapy. Both can have severe side-effects: transfusion may cause iron to accumulate in organs and can furthermore result in the development of antibodies that can preclude patients from successfully receiving a kidney transplant later [4].

Currently, the first-line therapy for anemic patients is the administration of recombinant human erythropoietin (rhEPO) [5]. Since its approval, rhEPO has virtually eliminated the need for repeated blood transfusions for dialysis patients. Clinical studies have demonstrated that an increased hematocrit (percentage of red blood cells in the blood volume) resulting from rhEPO administration has a significant impact on the patients' lives. The target hematocrit range for dialysis patients is 30-36%. Healthy individuals have a hematocrit in the range of 42-48% - a range which possibly can be dangerous for anemic people due to the formation of clots, etc.

Since its introduction for the treatment of patients with insufficient erythrocyte levels due to renal failure, the therapeutic use of rhEPO has broadened signif-

Handbook of Combinatorial Chemistry. Drugs, Catalysts, Materials. Vol. 2. Edited by K. C. Nicolaou, R. Hanko, and W. Hartwig Copyright © 2002 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30509-2

icantly. Today, patients receiving chemotherapy or those having received bone marrow transplantations are treated with rhEPO as well as human immunodeficiency virus (HIV) patients and others. In clinical trials the most frequently observed adverse side-effects with rhEPO were hypertension, headache, tachycardia, nausea/vomiting, and clotted vascular access. However, it is not clear whether these effects are directly related to treatment with rhEPO. rhEPO has to be administered three times a week. Because of its chemical nature as a peptide hormone of 165 amino acids, it has to be injected parenterally. Despite these disadvantages, rhEPO is currently one of the best-selling drugs on the market ($5 billion annual sales in 2000).

Synthetic compounds which stimulate or sensitize endogenous EPO and can overcome the disadvantages of rhEPO are highly attractive research targets for the pharmaceutical industry. They would meet the needs of a large patient population and a large market with expected annual growth rates above 10%. It is expected that an orally active EPO substitute would attract a major share of this market. Thus, the project goal was to find an orally active low-molecular-weight EPO sensitizer that would be able to increase hematocrit levels within 12 weeks by about 10% points in total to a final hematocrit of approximately 30-35% at a maximal dose of 3 mg kg-1 day-1.

28.2 Results

High-throughput Screening and Biological Evaluations

The erythropoietin program began by building up a high-throughput screening (HTS) assay using a murine cell line which was transfected with the human EPO receptor and the luciferase reporter gene under the control of a globin promoter. In this cell line, EPO causes the induction of luciferase activity. Since the HTS was designed to identify sensitizers (compounds that would enhance the effect of endogenous erythropoietin), testing of compounds was performed in the presence of low concentrations of rhEPO, and the potentiation of this response was quantified.

Approximately 650,000 compounds were tested. After hit verification cluster analysis, and structure similarity searches, 70 of the most promising compounds were further tested in vivo.

Dihydropyridazinones showed in vitro and in vivo activity as well as preliminary structure-activity relationships (SARs). The most active compound within this class, 1 (Fig. 28.1), displayed an EC50 = 0.23 mM in the luciferase assay. This activity corresponds to a 6.5- to tenfold stimulation of erythropoiesis during treatment with 10 milliunits (mU) Epo mL-1 without sensitizer. In vivo, a 4.5% point increase of hematocrit was measured after p.o. application (10 mg kg-1 d-1 for 5 days) to mice within a week. Such an increase is already in the desirable range for a sensitizer. Higher increases in hematocrits can be dangerous for anemic people because the density of their blood increases, which favors the formation of clots.

treatment with 10 mU mL_1 Epo without sensitizer; 4.5% point increase in the hematocrit after p.o. application (10 mg kg-1); IC50 (PDE3) = 0.2 mM].

Further experiments demonstrated that 1 stimulates erythropoiesis not only by means of an EPO-sensitizing effect but also by triggering an EPO release from the kidney, which was due to the inhibition of phosphodiesterase (PDE)3 (IC50 = 0.2 mM). These PDE3-associated hemodynamic side-effects are not acceptable in the treatment of anemia. Thus the lead 1 had to be optimized with respect to potency and selectivity. Therefore, the lead was divided into three parts: a central core (C), a heterocyclic moiety (D), and a rather lipophilic substituent (A) attached to the core via an amide linkage (B). The corresponding compound with the inverse amide displayed good biological activity as well. All three parts were planned to be modified in a medicinal chemistry program (Fig. 28.2).

The optimization of the lead structure was mostly based on in vitro results. These were obtained from BaF3 cells expressing the human EPO receptor and the luciferase reporter gene under the control of the b-globin promoter. The cells were incubated with the test substances (0-1000 nM) in the presence of 10 mU mL-1 EPO for 48 h. Cells were then lysed and the luciferase activity was measured. Results will be expressed as percentage activity compared with luciferase activity at 10 mU mL-1 EPO in the presence of 0.1% DMSO. The second in vitro erythropoiesis

Fig. 28.1. Dihydopyridazinone 1 was the lead structure of the Epo-sensitizer project [EC50 = 0.23 mM (luciferase assay); 6.5-to tenfold stimulation oferythropoiesis over the

Fig. 28.1. Dihydopyridazinone 1 was the lead structure of the Epo-sensitizer project [EC50 = 0.23 mM (luciferase assay); 6.5-to tenfold stimulation oferythropoiesis over the model used human primary CD34-positive stem cells. The stimulation of the proliferation of these erythroid cells was determined by counting benzidine-positive cells after 14 days culture in methylcellulose medium. In vivo results were obtained from mice, for which the increase in the hematocrit was measured after 5 days of treatment.

Concept for Chemical Optimization

The dihydropyridazinone moiety constitutes part of known PDE3 inhibitors and was thought to be responsible for the undesired PDE3 activity of 1 [6]. Therefore, in the first phase of the project, chemistry efforts were focused on the replacement of this moiety by other heterocyclic groups and on the modification of the dihydropyridazinone scaffold to reduce PDE3 activity. The para-substituted benzene moiety was planned to remain constant during the initial phase of the program.

When the chemistry group working on this program was brought together, the question of how combinatorial chemistry in solution phase or on solid support could participate in the project was an important topic. The modification of the dihydropyridazinone moiety was planned to be performed by conventional solution-phase chemistry. In addition, the left part (A) of the dihydropyridazinone lead structure 1 was to be targeted by parallel solution-phase chemistry by synthesizing a series of amides, inverted amides, sulfonamides, and ureas.

The replacement of the dihydropyridazinone by other heterocycles was to be performed by combinatorial chemistry. This requirement was due to the fact that the conventional synthesis of a small number of compounds (1-5) with new heterocyclic head groups would not fully explore the structural space. Furthermore, the optimal substitution pattern of a new head group would not be known at the beginning of the synthesis. Therefore, a proper design and selection of the first compounds would not be possible and the optimal substitution pattern of a molecule with a new headgroup might be missed. On the other hand, we envisaged that 200-500 compounds had to be prepared within a structural class to find initial hits that would serve as starting points for further optimization. The necessity to perform multistep syntheses of libraries and the need for further optimization prompted us to use solid-phase chemistry methods. This approach would allow not only a fast but also a very thorough and broad verification of the initial hits with follow-up libraries.

4-Fluoro-3-nitroaniline as Central Core

To test a broad variety of heterocyclic head groups, the initial strategy was to use a specific single intermediate as a central core and to modify this core with a variety of heterocyclic nucleophiles as an approximate guide to which of these hetero-cycles would be worth following up. 4-Fluoro-3-nitroaniline (2), because of its functional group pattern, is a well-known building block in combinatorial chemistry

(Scheme 28.1). Using formyl-functionalized resin (3), this core was immobilized by reductive alkylation. Owing to the low nucleophilicity of the aniline moiety, the standard conditions for the imine formation [trimethylorthoformate (TMOF), room temperature, 2 h] had to be enforced slightly: the mixture of starting materials was heated to 40 °C overnight using TMOF as the dehydrating agent and solvent [7]. Reduction was subsequently performed with tetrabutylammonium boro-hydride in a mixture of acetic acid and dimethylformamide (DMF) (5:95) [8]. Acylation of the core was then performed under standard conditions to give the left-hand part of the desired analog (4) of lead structure 1 [9].

Scheme 28.1. Fluoro-nitroaniline as central core gave access to a variety of analogs of the lead structure 1.

In the next step, one of the heterocycles (5-10) was attached to the core by a nucleophilic aromatic substitution reaction to give intermediates 11a. The reaction worked well with reactive nucleophiles. Piperazine, for example, was coupled quantitatively after stirring the resin with the amine (15 equiv.) in dimethyl sulfoxide (DMSO) at room temperature overnight. However, with poor nucleophiles such as imidazole the alkylation reaction did not proceed in good yields at room temperature and gave unsatisfactory results at elevated temperatures. However, repeating the coupling reaction twice at room temperature gave products of sufficient purity [> 85% by high-performance liquid chromatography/ultraviolet (HPLC/UV) detection] for biological testing.

Following this route, the first library of compounds with the general structure 11a was prepared using the indicated heterocycles 5-10 and aromatic carboxylic acid chlorides as building blocks. However, no biologically active compounds were obtained. Therefore, the structural space around the core of 11a was investigated more thoroughly via reduction of the nitro group to the corresponding aniline derivatives (11b) (Scheme 28.1) using SnCl2 in DMF [10]. These conditions gave much higher yields than reductions with Zn [11] or Fe reagents [12]. Derivatiza-tion of the anilines using carboxylic acid chlorides or isocyanates gave, after cleavage, final products 12 and 13. This methodology was exploited for the production of a library of 460 compounds. Despite this rather large number, neither the urea derivatives nor the amides showed biological activity. A possible explanation might be a unfavorable set of building blocks. However, the size and constitution of the library was thought to be large enough to cover such a broad range of substitution patterns that this explanation was discarded. Thus, the cause of the lack of biological activity of the whole library was thought to be either the heterocyclic moieties or the presence of the meta-nitrogen substituent in the central core. If the first explanation had been the only reason for the lack of activity, it would have been straightforward to establish the SAR of 1. However, the second reason made the interpretation difficult. The situation was clarified by the synthesis of the nitro-substituted analog of 1, which was also inactive. Obviously, a meta substituent in the central core was not tolerated. Since this substitution is a prerequisite for the described chemical route the methodology was therefore abandoned.

Libraries Around Single Heterocycles

The approach of preparing a library of compounds with different heterocyclic headgroups derived from one common intermediate had been shown to be potentially misleading. Continuing the project, new routes to heterocyclic analogs of lead structure 1 had to be developed. But rather than using one library to verify the potential of a variety of heterocycles, future work concentrated on the preparation of libraries with only one specific heterocyclic headgroup. Using this concept, misleading chemical functionalities can be avoided more easily and individual hetero-cycles can be verified more carefully. Thus, libraries of hydantoins and pyrazoles were prepared. Again solid-phase chemistry was used to investigate the potential of these structural classes. Hydantoins

The targeted library of compounds 14 (Fig. 28.3) can be prepared retrosynthetically from three types of building blocks: the hydantoin moiety can be built up from an amino acid 15 and isocyanate 16, which are coupled and then cyclized under basic conditions. The amide bond can be formed from the corresponding carbox-ylic acid derivative 17 and an amine functionality can be prepared from the nitro group in 16. Based on literature precedent [13], the ease of cyclo cleavage from the support, and the fact that immobilized amino acids are commercially available, the latter was chosen as the attachment point to the support, as indicated in Fig. 28.3.

Fig. 28.3. Building blocks for the synthesis of hydantoins and the attachment point to the solid support.

point of attachment to the solid support

Fig. 28.3. Building blocks for the synthesis of hydantoins and the attachment point to the solid support.

In the first series of experiments, the preparation of 18 and 19 started from immobilized N-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids 20, 4-nitro-phenylisocyanate 16, and carboxylic acid chlorides 21 (Scheme 28.2). The amino groups of the amino acids were liberated using piperidine in DMF and then reacted with 16 to give urea 22. Subsequently, the nitro group was reduced to 23 using SnCl2-2H2O in DMF at room temperature.

NHFmoc 20

1) piperidine, DMF, 30 min, RT

2) 4-Nitrophenylisocyanate (16), EtOAC, 18 h, RT

18 h

1) piperidine, DMF, 30 min, RT

2) 4-Nitrophenylisocyanate (16), EtOAC, 18 h, RT

18 h

2) PhCOCI (21), EtjN, DCM, 18h, RT or PhCOOH (24), TBTU, DIPEA, 18 h, RT

Scheme 28.2. Synthesis of a library of compounds having a hydantoin moiety in common and otherwise displaying the same structural pattern as lead 1.

2) PhCOCI (21), EtjN, DCM, 18h, RT or PhCOOH (24), TBTU, DIPEA, 18 h, RT

The acylation of the resulting aniline 23 was first investigated using carboxylic acid chlorides (e.g. benzoyl chloride 21), although ultimately the synthesis of the target amide 18 was envisaged to start from carboxylic acids since important building blocks were not readily available as acid chlorides (e.g. nicotinoyl chlorides). Benzoyl chloride (21) in dichloromethane and triethylamine (TEA) as base gave the amide functionality in good yield and purity. The replacement of the highly reactive acid chlorides by carboxylic acids, e.g. benzoic acid 24, proved to be possible using TBTU as the activating agent [14]. The corresponding urea func-

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