Development and application of novel scaffolds in drug discovery
Boltjes, André
DOI:
10.33612/diss.98161351
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Boltjes, A. (2019). Development and application of novel scaffolds in drug discovery: the MCR approach. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.98161351
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IN DRUG DISCOVERY
The MCR approach
Department of Drug Design (Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands) and was financially supported by the University of Groningen.
Printing of this thesis was financially supported by the University Library and the Graduate School of Science, Faculty of Science and Engineering, University of Groningen, The Netherlands
ISBN: 978-94-034-2005-9 (printed version) ISBN: 978-94-034-2004-2 (electronic version) Printing: Ipskamp printing, Enschede Cover design: André Boltjes
Cover Graphics: By pikisuperstar Source: Freepik
Copyright © 2019 André Boltjes. All rights are reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without the prior permission in writing of the author.
Development and application
of novel scaffolds in Drug
Discovery
The MCR approach
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. C. Wijmenga en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op vrijdag 29 november 2019 om 14.30 uur
door
André Boltjes
Prof. dr. A.S.S. Dömling Prof. dr. F.J. Dekker
Beoordelingscommissie
Prof. dr. R.V.A Orru Prof. dr. G.J. Poelarends Prof. dr. ir. A.J. Minnaard
CHAPTER 1 GENERAL INTRODUCTION 1
CHAPTER 2 FRAGMENT BASED LIBRARY GENERATION FOR THE DISCOVERY OF A PEPTIDOMIMETIC P53-MDM4 INHIBITOR
11
CHAPTER 3 UGI MULTICOMPONENT APPROACH TO SYNTHESIZE SCHISTOSOMIASIS DRUG PRAZIQUANTEL
35
CHAPTER 4 UGI 4-CR SYNTHESIS OF γ- AND δ-LACTAMS PROVIDING NEW ACCESS TO DIVERSE ENZYME INTERACTIONS, A PDB ANALYSIS
51
CHAPTER 5 Gd-TEMDO: DESIGN, SYNTHESIS AND MRI
APPLICATION 75
CHAPTER 6 THE GROEBKE-BLACKBURN-BIENAYMÉ
REACTION 97
CHAPTER 7 DIVERSE ONE-POT SYNTHESIS OF ADENINE
MIMETICS FOR BIOMEDICAL APPLICATIONS 189
CHAPTER 8 SUMMARY AND DISCUSSION 213
SAMENVATTING NEDERLANDSE SAMENVATTING 223
APPENDIX ACKNOWLEDGEMENTS 231
LIST OF PUBLICATIONS ABOUT THE AUTHOR
Chapter 1
Introduction
Multicomponent reactions
Multicomponent reactions (MCRs) are a special class of chemical transforma-tions, which produce complex scaffolds in a single step by employing three or more starting materials, in which most of the atoms are present in the final prod-uct.1 This type of chemistry can be classified as a domino reaction. Similar to
polymer chemistry, MCRs proceed through a cascade of intermediate reactions, however, yielding a complex but small molecule. The first example of a MCR reaction is the Strecker amino acid synthesis, developed almost 170 years ago in 1850. The Strecker reaction is a 3-component reaction (3-CR) between an alde-hyde or ketone, ammonia and potassium cyanide and yields an α-aminonitrile, subsequent hydrolysis gives access to synthetic α-amino acids (Scheme 1).2 The
high atom economy, efficiency, convergent nature and very high bond forming index of MCR reactions make this class extremely useful for drug exploration.3
R H O NH3 KCN H2N COOH R + +
Scheme 1. The Strecker amino acid synthesis.
MCRs are often considered to be complex, because the formation of the final products proceed via a number of intermediate steps. In order to understand the complex nature of MCRs, a breakdown of the reaction into intermediate steps could aid in comprehension of the underlying mechanism. Difference in reactiv-ity of each of the individual components is the driving force behind selective re-actions, resulting in a predefined order in which each component will react. The most common studied and therefore well-developed early type of MCRs evolve around the reactivity of imine groups, usually obtained through reacting carbon-yl compounds with amines. The resulting imines or Schiff bases are electrophiles and can react via electrophilic addition to for example α-methylene carbonyls in the Mannich 3-CR, to β-ketoesters in the Biginelli 3-CR, to boronates (from boronic acids) in the Petasis 3-CR and many other reactions, described in scheme 2.4-8 The formation of the Schiff base is performed in the presence of the third
component, the electrophile, in a one-pot fashion, which in practice comes down to adding all the reactants together in a suitable solvent and stir the multicom-ponent reaction, often under ambient conditions. The selective order generally allows for a clean reaction with little byproducts and high conversion towards a single product.
1
Strecker R1 R2 O R3 NH2 HCN N H R1 CN R3 R2 + + Mannich R1 H N R2 R3 R4 O R5 R 6 O N R1 R2 R3R4 R5 R6 O Hantsch R1 H O R2 NH2 N R1 O O R2 Staudinger R1 H O R2 NH2 R3 Cl O N O R2 R3 R1 R1 H O R2 H N R3 R4 B(OH)2 Petasis R2 N R1 R3 R4 Biginelli Ar H O H2N NH2 O EtO O O N H NH Ar EtO O O H3C + + + + + + + + + + O O 2Scheme 2. Overview of the most common MCR 3-CR of amines, carbonyl compounds and nucleophiles.
In 1920, Passerini discovered the first isocyanide based MCR reaction (IMCR) and involves an isocyanide, an oxo component and a nucleophile.9 The carbene
like properties of the isocyanide group allow for some very interesting transfor-mations, as this functional group can behave as nucleophile and then as electro-phile at the same atom, resulting to the so-called α-adduct.10 The IMCR subclass
has complemented the field of MCR due to its versatile reactivity, variability and scaffolds and is currently the most widely used/applied type of MCR. Neverthe-less it took roughly 40 years until more developments were made in this field,
considered unpleasant, even in such a way that the US government investigated the use of isocyanides as non-lethal chemical weapons.11 The considered
‘con-straint’ is limited to the liquid isocyanides, as higher molecular weight isocya-nides are often solid and have little to no smell. Regardless of this fact, the result is poor commercial availability, which is not necessarily problematic as isocya-nides can readily be prepared from primary amines or aldehydes in one or two steps.12-14 R1 R2 O R3 OH O R4 NC O R1R2 R3 O H N O R4 Passerini-3CR 1920 R1 R2 O R3 NH2 R 4 OH O R5 NC R4 N O R1R2 H N O R5 R3 Ugi-4CR 1959 R1 H O R2 NH2 R 3 Ts NC N N R1 R3 R2 Van Leusen-3CR 1977 Ugi Tetrazole-4CR 1959 R1 R2 O R3 H N HN3 R5 NC N R1R2 N NN NR5 R4 R4 R3 Groebke Blackburn Bienaymè-3CR 1998 R1 O H N NH2 5-6- R2 NC N N HN R2 R1 5- 6-R1 R2 O R3 H N R R5 NC 4 R5 H N O N R4 R3 R1R2 Ugi-3CR 1959 + + + + + + + + + + + + + +
Scheme 3. Some of the well-known IMCRs
A considerable amount of pioneering work with IMCRs was performed by Ugi in the late 1950s. The Ugi-4CR is defined as the reaction of an amine with an aldehyde or ketone, an isocyanide and a carboxylic acid (Scheme 3).15 The limits
of IMCRs were explored by Ivar Ugi and since the introduction of the Ugi 4-CR, many new IMCR reactions have found their way to the nowadays well
accept-1
ed field of MCR chemistry. The work described in this thesis is based on drug design, utilizing the Ugi reaction and several variations of the Ugi reaction to synthesize drug like compounds and medical probes.
Drug discovery and MCR.
Identification of biochemical pathways related to a disease and the intervention of potential targets is the first step in the discovery of new medicines. Luckily more and more molecular targets are being identified, allowing for target vali-dation and development of small molecules. High throughput screening (HTS) is a common method in the pharmaceutical industry to discover new leads for molecular targets. Such screenings, however, are expensive, up to $10 million per screening, show low efficiency and have limited success rate. Therefore, ac-ademia aim at development of smarter design and selection criteria to enable screening of smaller focused compound collections to provide higher success rates and lower costs. Effective selection criteria can be obtained by analysis of the binding site in a co-crystal structure to identify a pharmacophore. This pharmacophore is used subsequently to design potential binders for screening. This approach relies heavily on the medicinal chemists knowledge of molecu-lar interactions, more specifically the attractive interactions between two partner molecules in biological systems. Parallel synthesis is then applied to synthesize libraries of compounds for hit to lead optimization.
In drug discovery, synthesis of compound libraries is necessary to optimize a lead. Determination of the biological activity on a specified target can identi-fy which moieties are important and which need optimizing for increasing its activity, selectivity often via a structure activity analysis. Compiling a library of 20 compounds, where stepwise the individual parts of the target molecule have to be chemically connected, the number of reaction and purification steps quickly exceeds 100. This is first of all time consuming and second very resource demanding. Looking at the MCR approach, the philosophy is to obtain products in just a single step, where the product contains all the desired properties. With this in mind, the MCR methodology deems to be a competitive alternative to ordinary parallel multistep synthesis.
To date there are many marketed drugs that can be prepared through a MCR (Figure 1). The local anesthetic lidocaine (Xylocaine®) is for example prepared by a Ugi-3CR in a single step by reacting formaldehyde, diethyl amine and 2,6-di-methyl-phenylisocyanide.16 This example is an early adaption of IMCR in
com-mercial drug production. Other examples of MCR directed/assisted drug synthe-sis are shown in figure 1.
N N N O N H t-Bu OH Ph O H N HO * Crixivan® U-4CR H N O N Lidocaine U-3CR N N N NH O H2N F F KAF156 GBB-3CR O O H N NH O N O H N O N H O N N Telaprevir P-3CR and U-3CR HN N S N N Olanzapine Gewald-3CR N H O O O O NO2 Nifedipine Hantsch-3CR O H N O Cl O O Mandipropamid Passerini-3CR
Figure 1. Various drug examples produced through the MCR methodology. The blue and purple color assigns the part of the molecule constructed with MCR chemistry.
Aim of the research described in this thesis
Drug design aims at the development of druglike compounds and probes which act as agonists or antagonists in biological processes. A pharmacophore mod-el and complementing MCR methodologies can prove to be an invaluable asset in drug design. With the current state of the art in MCR chemistry, design and synthesis of vast libraries of compounds, targeting various identified biological targets is made possible. In turn, the MCR toolbox used for drug discovery is continuously expanded by the development of new scaffolds, simplification of existing chemistry with broader scope, better toleration towards sensitive func-tional groups and enables targeting of otherwise difficult to target binding sites such as flat surfaces. Noteworthy is the recent discovery of the catalytic enanti-oselective U-4CR, which proves that the rational design of MCR’s has never been so important and widely applicable in drug discovery.17-18 Development of new
1
probes and scaffolds using MCR chemistry and its application for the discovery of new drug like compounds is the main theme in this thesis.
Thesis Outline
In Chapter 2 selective inhibitors of Mdm4 are presented. The Mdm4 protein is a closely related protein to Mdm2 and it also binds to the same epitope of p53. Both Mdm2 and Mdm4 (MdmX) were characterized as druggable by analysis of the Mdm2-p53 co-crystal structure and are considered as an important oncology target.19 All current known binders are highly specific for Mdm2. In a
combinato-rial synthetic approach, the U-4CR was applied to generate a library of peptido-mimetic small molecules as potential Mdm2/4 binders. A selective Mdm4 binder was identified and subjected to subsequent hit-to-lead optimization.
Chapter 3 describes the improvements made in the preexisting U-4CR assisted synthesis of the anti-helminthic drug praziquantel. There are multiple methods to prepare praziquantel on industrial scale. The U-4CR method was developed in 2009, but was not adopted for industrial production up to date, likely due to the requirement of isocyanide chemistry. With the introduction of the in situ isocyanide methodology by Neochoritis et al., this constraint could be overcome, which was demonstrated in a preparation procedure, published in Organic Syn-theses.20-21
Chapter 4 presents the post-MCR modification of ester containing UT-4CR prod-ucts to obtain N-unsubstituted γ-and δ-lactams. The well-known UT-4CR allows for the introduction of the tetrazole moiety, which serves as a bio-isostere for carboxylic acids. In the experimental design tritylamine was used as a convert-ible amine component and an ester containing aldehyde as bi-functional building block, containing either 2 or 3 methylene groups. Removal of the tritylgroup re-sults in compounds with both an amine and ester, which readily undergo ami-nolysis upon basic treatment, , resulting in intramolecular cyclization, thus for-mation of lactams. The peptidomimetic nature of the resulting tetrazolo-lactam motif can be applied as potent drugs attributed to the peptidomimetic proline cis-amide functionality.
In chapter 5 a new generation of MRI contrast agents was introduced on the basis of the known metal chelator tetraxatan. In the current application of gadoteric acid, MRI contrast enhancement is achieved by the reduction of T1 relaxation times, more specifically by fast water exchange on the 9th coordination site of the
gadolinium complex. Although the gadoteric acid complex is very stable with a stability constant (log Keq) of 25.8, swift clearance from the body is important to prevent severe nephrotoxic adverse effects due to release of gadolinium ions by metabolic degradation of the gadolinium complex. Patients with renal failure are therefore susceptible to these side effects. Replacement of the appendant car-boxylate arms with tetrazole bio-isosteres would tackle this unfavored leaching and its subsequent toxicity. The tetrazole is introduced in a two-step synthetic
complex was determined and the contract enhancing abilities were tested by in
vivo experiments.
Chapter 6 highlights the developments of the GBB-3CR since its discovery in 1998. During the course of two decades the GBB-3CR reaction has emerged as a very important MCR, resulting in over a hundred patents and a great number of publications in various fields of interest. To celebrate this event, we would like to present an overview of the developments in the GBB-3CR, including an analysis of each of the three starting material classes, solvents and catalysts described. Additionally, a list of patents and their applications and a more in-depth sum-mary of the biological targets that were addressed, including structural biology analysis, is given.
Chapter 7 focusses on the application of bis-amidines in the GBB-3CR. With multiple possible regioisomers, this amidine component could yield various un-precedented heterocyclic scaffolds. Pyrazine-2,3-diamine, however, is the only amidine affording products that did not show quick degradation at room tem-perature when exposed to air. The scaffold from this amidine, 2,3-di-substituted imidazo[1,2-a]pyrazin-8-amines represent a similar shape to adenine and thus could be applied for development of novel kinase inhibitors, mimicking the ade-nosine part in the ATP binding site. To strengthen our hypothesis we performed comprehensive docking studies with multiple potential targets where our imid-azo[1,2-a]pyrazin-8-amine scaffold can bind.
1
References
1 I. Ugi, A. Dömling, W. Hörl, Endeavour 1994, 18, 115-122. 2 A. Strecker, Justus Liebigs Annalen der Chemie 1850, 75, 27-45. 3 L. F. Tietze, Chemical Reviews 1996, 96, 115-136.
4 N. A. Petasis, I. Akritopoulou, Tetrahedron Letters 1993, 34, 583-586. 5 A. Hantzsch, Berichte der deutschen chemischen Gesellschaft 1881, 14,
1637-1638.
6 P. Biginelli, Berichte der deutschen chemischen Gesellschaft 1891, 24, 2962-2967.
7 C. Mannich, W. Krösche, Archiv der Pharmazie 1912, 250, 647-667. 8 H. Staudinger, J. Meyer, Helvetica Chimica Acta 1919, 2, 635-646. 9 M. Passerini, L. Simone, Gazz. Chim. Ital. 1921, 51, 126-129. 10 A. Dömling, Chemical Reviews 2006, 106, 17-89.
11 M. C. Pirrung, S. Ghorai, Journal of the American Chemical Society 2006,
128, 11772-11773.
12 A. W. Hofmann, Ann. Chem. 1868, 146, 107.
13 I. Ugi, U. Fetzer, U. Eholzer, H. Knupfer, K. Offermann, Angewandte
Che-mie International Edition in English 1965, 4, 472-484.
14 C. G. Neochoritis, T. Zarganes-Tzitzikas, S. Stotani, A. Domling, E. Herdt-weck, K. Khoury, A. Domling, Acs Comb Sci 2015, 17, 493-499.
15 I. Ugi, R. Meyr, U. Fetzer, C. Steinbrückner, Angew Chem Int Edit 1959, 71, 386-386.
16 A. Dömling, W. Wang, K. Wang, Chemical Reviews 2012, 112, 3083-3135. 17 S. Shaabani, A. Dömling, Angewandte Chemie International Edition 2018,
57, 16266-16268.
18 J. Zhang, P. Yu, S.-Y. Li, H. Sun, S.-H. Xiang, J. Wang, K. N. Houk, B. Tan,
Science 2018, 361, eaas8707.
19 C. F. Cheok, C. S. Verma, J. Baselga, D. P. Lane, Nature Reviews Clinical
Oncology 2010, 8, 25.
20 A. Boltjes, H. X. Liu, H. P. Liu, A. Dömling, Org Synth 2017, 94, 54-65. 21 C. G. Neochoritis, S. Stotani, B. Mishra, A. Dömling, Org Lett 2015, 17,
Chapter 2
Fragment based Library
Generation for the Discovery of
a Peptidomimetic p53-Mdm4
Inhibitor
André Boltjes, Yijun Huang, Rob van de Velde, Laurie Rijkee, Siglinde Wolf, James Gaugler,Katarzyna Lesniak, Katarzyna Guzik, Tad A. Holak,d Alexander
Dömling
Abstract
Based on our recently resolved first co-crystal structure of Mdm4 in complex with a small molecule inhibitor (PDB ID: 3LBJ), we devised an approach for the generation of potential Mdm4 selective ligands. We performed the Ugi four-com-ponent reaction (Ugi-4CR) in 96-well plates with an indole fragment, which is specially designed to mimic Trp23, a key amino acid for the interaction between p53 and Mdm4. Generally the reaction yielded mostly precipitates collected by 96-well filter plates. The best hit compound was found to be active and selective for Mdm4 (Ki = 5 µM, 10 fold stronger than Mdm2). This initial hit may serve as the starting point for designing selective p53-Mdm4 inhibitors with higher affinity.
2
Introduction
The protein-protein interaction between the transcription factor p53 and the neg-ative regulator Mdm2 is an important recent oncology target.1 The interaction is
crystallographically characterized and druggable and several compounds are in late preclinical and early clinical evaluation.2 The Mdm4 protein is a closely
re-lated protein to Mdm2 and it also binds to the same epitope of p53. The sequence homology, the shape, dimension and size is similar. Nevertheless all current com-pound scaffolds characterized by co-crystal structure analysis are highly specific for Mdm2 and show no or very little Mdm4 binding which, is surprising and not well understood regarding the great similarity between the two proteins (Figure 1).3-4 Nutlin-3a 1, for example, binds to Mdm4 too but with a roughly 1000-fold
lower affinity of about 25 µM.5 Several Novartis compounds 2-4 show weak low
µM Mdm4 affinity while being very potent binders to Mdm2 (again ~1000-fold difference, Figure 2).6-7 Other described Mdm4 selective compounds are either
covalent binders or not validated (5, 6).8-9 Surprisingly, pyrazolone compound6
5 loses activity to Mdm4 in the presence of a reducing reagent, dithiothreitol (DTT). Incubation of these compounds with Mdm4 under non-reducing condi-tions lead to a time dependent change of Mdm4 structure determined by NMR; concomitantly, the MS analysis showed the presence of covalent adducts of the compound with Mdm4. Additionally, we have found out, by 1H NMR, that the
pyrazolone reacts with β-mercaptoethanol in chloroform.
Figure 1. Alignment of Mdm4 (green cartoon, PDB-3DAB) and Mdm2 (magenta car-toon PDB-1YCR) with the Mdm2 peptide (yellow carcar-toon and sticks) highlighting the similarities and differences of the two binding sites (the Mdm4 binding p53 peptide is omitted for clarity). The main differences between the two receptor p53 binding sites are
the M>V exchange. The key amino acids of Mdm2, Mdm4 and p53 are shown as sticks.
Selective Mdm4 antagonists are highly sought after since Mdm4 and Mdm2 pro-teins are differentially over-expressed in several cancers and both play a promi-nent but presumably different role in apoptosis induction.10 Herein, we describe
the discovery of B1, a selective p53-Mdm4 inhibitor (with ~5 µM affinity to Mdm4 but 54 µM affinity to Mdm2) with reversed selectivity compared with most p53-Mdm2 inhibitors, which we believe is a good starting point to elaborate Mdm4 selective compounds.
2
Results and Discussion
Based on our previously generated insight into the binding of small molecules into Mdm2 and Mdm4 and our recently developed Mdm2 and Mdm4 fluo-rescence polarization assay, we planned to synthesized libraries of potential Mdm2/4 binding compounds.5, 11-21 Thus, we generated a 96-member library of
peptidomimetic small molecules via Ugi four-component reaction (Ugi-4CR) (Scheme 1). Each compound contains the indole or p-halobenzyl fragment to mimic the Trp23 “anchor”, which is the key anchor residue in the p53 Mdm2 and Mdm4 protein-protein interaction interface, respectively. Figure 3 illustrates the structure of amine and isocyanide inputs, as well as the experimental setting in a 96-well microliter plate. Since the reaction products regularly precipitated, the compounds were collected by a 96-well filter plate, and washed with ether to remove unreacted starting materials. The yields of the isolated products were between low (6%) and good (56%) with an average of 28% over all 96 wells. In addition, the purities of the isolated materials were considered sufficient for an initial screening. The collected samples were dissolved as a 10 mM stock solution in DMSO for the screening.
HCHO R2NH 2 R1NC N O R2 O N H R1 NH OH O N H
Scheme 1. Ugi-4CR for high throughput synthesis
Compound B1 was identified as a p53/Mdm4 inhibitor (Ki = 19 µM) via our
re-cently reported fluorescence polarization assay. The hit compound was
re-syn-thesized and purified by flash chromatography, which was further confirmed by the binding with Mdm4 (Ki = 5.5 µM), as shown in Figure 4.5 Although the p53-binding sites within the Mdm4 and Mdm2 proteins are closely related, known Mdm2 small-molecule inhibitors have been shown experimentally not or very poorly to bind to its homolog Mdm4. This hit compound may provide a new avenue for the design of potential selective inhibitors of the p53-Mdm4 interaction. Further studies are ongoing in our lab to uncover the puzzle of the Mdm2 and Mdm4 selectivity.
A NH2 Cl NH2 Cl NH2 F NH2 NH2 NH2 NH2 1 2 3 4 5 6 7 8 9 10 11 12 NH2 NH2 NH2 Cl Cl NH2 Br NH2 O B NC NC NC NC NC A B C D E F G H NC CO2Me Ph Ph NC NC O N
Figure 3. Parallel synthesis of “anchor” biased compound library via Ugi-4CR. Structures of the amine A and isocyanide B starting materials used.
For further optimizing purposes a second library was synthesized, that follows the structure of hit compound B1, yielding a total of 38 new compounds. Mi-nor changes were made in the indole moiety (from the carboxylic acid com-ponent) and different halogen substituted benzylamines were employed, keeping the cyclohexyl fragment intact, as shown in figure 5. This time a se-quential approach was used, which made it possible to run 1 mmol scale reac-tions as opposed to 0.2 mmol scale in the 96-well plate. Increased yields up to 79% were observed, in average 46%, which confirms that larger scale Ugi re-actions in general give better yields. Unfortunately, all the other compounds synthesized in Figure 5 showed worse (>50 µM) or no activity in the FP assay.
A B C N O OHN Cl N H
Figure 4. Hit compound as p53/Mdm4 inhibitor. A: Structure of B1; B: Ki = 54 µM (Mdm2) C: Ki = 5.5 µM (Mdm4) as determined by FP.
2
A H N O O S COOH HO COOH MeO COOHCOOH COOH COOH
H N COOH N COOH H N COOH N H N COOH Cl HO I J K L M N O P Q R N N N COOH COOH S T S COOH COOH Cl H N COOH U V W B H2N CF3 H2N F H2N Cl Cl 13 14 15
Figure 4. Starting materials for the second library of compounds with the structures of A the carboxylic acids and B the amines.
Conclusions
In summary, this work demonstrates that the Ugi four-component reaction (Ugi-4CR) can be used to address the requirements for efficient high-throughput syn-thesis of diverse compounds in a cost- and time-effective manner. Integrated with biochemical screening assays, a peptidomimetic p53-Mdm4 inhibitor B1 was identified from a 96-membered library generated via Ugi-4CR of an indole fragment. This approach provides an efficient strategy for the discovery of small molecule probes selectively targeting protein-protein interactions. Further opti-mization studies on B1 are ongoing and will be reported in due course.
Experimental procedures and Spectral Data
All reagents were purchased from commercial sources and used without fur-ther purification. Proton and carbon NMR spectra were determined on Bruker Avance™ 600 MHz NMR spectrometer. Chemical shifts are reported as δ values in parts per million (ppm) as referenced to residual solvent. 1H NMR spectra are
tabulated as follows: chemical shift, number of protons, multiplicity (s = singlet, br.s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet), and cou-pling constant. High Resolution Mass spectra were obtained at the University of Pittsburgh Mass Spectrometry facility. LC-MS analysis was performed on an SHIMADZU instrument (reverse-phase HPLC coupled to electrospray ioniza-tion-mass spectrometry), using an analytical C18 column (Dionex Acclaim 120 Å, 2.1 × 50 mm, 3.0 µm) coupled to an Applied Biosystems API2000 mass spectrom-eter (ESI-MS). Acetonitrile/water mixtures were used as the mobile phase for reverse-phase HPLC, the flow rate was maintained at 0.2 mL/min. The column temperature was kept at 40 °C. The UV detection was at 254 nm.
High throughput synthesis of a 96-member library.
Add indole-2-carboxylic acid (200 µL, 1M methanol stock solution; 0.2 mmol), amine (200 µL, 1M methanol stock solution; 0.2 mmol), isocyanide (200 µL, 1M methanol stock solution; 0.2 mmol), formaldehyde (120 µL, 2M methanol stock solution; 0.24 mmol) into each well of 96 deep well plate with printed labeling (VWR D108839, 1.2 mL).The plated was sealed by aluminum foil sealing film and was sonicated for 1h, then stand overnight under RT. After the partial evapora-tion of the solvent, the product was collected by filtraevapora-tion and washed by ether (AcroPrepTM 96 filter plate from PALL (0.2 um GHP, 1 mL well)) was used, 96
deep well plate was used as the receiver). For the second library equal conditions were used, but performed in 4 mL vials.
N-(4-chlorobenzyl)-N-(2-(cyclohexylamino)-2-oxoethyl)-1H-indole-2-carbox-amide (B1) NH O N O H N
Cl White solid, 20.6 mg; yield: 24%. HPLC/MS: tR = 11.61
min; m/z = 424.2 [M+H]+. HRMS: C 24H26N3O2ClNa, 446.1611 (calcd.), 446.1634 (found). 1H NMR (600 MHz, d6-DMSO): 1.18-1.27 (m, 5H), 1.53-1.74 (m, 4H), 3.58 (m, 1H), 3.94 (m, 1H), 4.18 (m, 1H), 4.66 (m, 1H), 5.00 (m, 1H), 6.53 (m, 1H), 6.75 (m, 1H), 7.03 (m, 1H), 7.18 (m, 1H), 7.37-7.56 (m, 4H), 7.84 (m, 1H), 8.05 (m, 1H), 11.70 (m, 1H). 13C NMR (150 MHz, d6-DMSO): 24.4, 25.1, 32.2, 47.5, 49.7, 51.2, 54.9, 103.9, 111.99, 112.04, 119.8, 121.4, 123.5, 126.8, 128.3, 129.8, 131.8, 135.9, 136.3, 163.8, 166.9. N-(tert-butyl)-N-(2-(cyclohexylamino)-2-oxoethyl)-1H-indole-2-carboxamide (B7):
2
H N O N O H N 1H NMR (600 MHz, DMSO): 1.13-1.21 (m, 3H), 1.27-1.33 (m, 2H), 1.46 (s, 9H), 1.55-1.57 (m, 1H), 1.68-1.70 (m, 2H), 1.77-1.79 (m, 2H), 3.63-3.66 (m, 1H), 4.21 (s, 2H), 6.67 (s, 1H), 7.00-7.02 (t, 1H, J = 7.2Hz), 7.14-7.16 (t, 1H, J = 7.5 Hz), 7.37-7.38 (d, 2H, J = 8.4Hz), 7.53-7.54 (d, 1H, J = 8.4Hz), 7.93-7.94 (d, 1H, J = 7.8Hz), 11.52 (s, 1H). 13C NMR (150 MHz, DMSO): 24.9, 25.7, 28.0, 32.6, 48.0, 50.6, 58.0, 100.0, 103.3, 112.4, 120.0, 121.7, 123.4, 127.2, 133.2, 136.2, 166.0, 169.9. HRMS: C21H29N3O2Na, 378.2157 (calcd.), 378.2140 (found). N-(2-(benzylamino)-2-oxoethyl)-N-(tert-butyl)-1H-indole-2-carboxamide (C7): N H O N O N H 1H NMR (600 MHz, DMSO): 1.47 (s, 9H), 4.32 (s, 2H), 4.37-4.38 (d, 2H), 6.60 (s, 1H), 7.02 (m, 1H), 7.15 (m, 1H), 7.26-7.39 (m, 5H), 7.37-7.39 (d, 1H, J = 8.4Hz), 7.47-7.48 (d, 1H, J = 8.4Hz), 8.58-8.60 (t, 1H, J = 6.0Hz), 11.51 (s, 1H). 13C NMR (150 MHz, DMSO): 28.0, 42.8, 50.7, 58.0, 103.1, 112.4, 120.1, 121.7, 123.4, 127.2, 127.4, 127.9, 128.8, 133.0, 136.2, 139.8, 165.9, 170.9. HRMS: C22H25N3O2Na, 386.1844 (calcd.), 386.1812 (found).N-(tert-butyl)-N-(2-(mesitylamino)-2-oxoethyl)-1H-indole-2-carboxamide (E7): H N O N O H N 1H NMR (600 MHz, DMSO): 1.29 (s, 9H), 1.90 (s, 6H), 2.00 (s, 3H), 4.27 (s, 2H), 6.52 (s, 1H), 6.67 (s, 2H), 6.78-6.81 (t, 1H, J = 7.2Hz), 6.92-6.95 (t, 1H, J = 7.2Hz), 7.16-7.17 (d, 1H, J = 8.4Hz), 7.26-7.28 (d, 1H, J = 8.4Hz), 9.16 (s, 1H), 11.29 (s, 1H). 13C NMR (150 MHz, DMSO): 17.9, 20.5, 27.5, 57.5, 62.8, 102.7, 111.9, 119.7, 121.0, 123.0, 126.7, 128.6, 131.9, 132.6, 134.8, 135.6, 135.8, 165.5, 169.3. HRMS: C24H30N3O2, 392.2338 (calcd.), 392.2341 (found). N-(2-(cyclohexylamino)-2-oxoethyl)-N-phenethyl-1H-indole-2-carboxamide (B12): NH O N O N H 1H NMR (600 MHz, DMSO): 1.13-1.29 (m, 6H), 1.55-1.57 (d, 1H, J = 12.6Hz), 1.68-1.70 (m, 2H), 1.75-1.77 (d, 2H, J = 12.0Hz), 2.92 (s, 2H), 3.03 (s, 1H), 3.64 (s, 1H), 3.89 (m, 1H), 4.09 (s, 1H) 4.24 (s, 1H), 6.68 (s, 1H) 7.03-7.05 (t, 1H, J = 7.32Hz), 7.18-7.26 (m, 2H), 7.26-7.36 (m, 4H), 7.42-7.44 (d, 1H, J = 8.4Hz), 7.57 (m, 1H), 8.11 (m, 1H), 11.64 (s, 1H). 13C NMR (150 MHz, DMSO): 24.5, 25.2, 32.3, 41.3, 47.6, 49.6, 52.0, 103.5, 112.0, 119.7, 121.3, 123.3, 126.2, 126.9, 128.5, 128.7, 135.8, 163.3, 167.4. HRMS: C25H30N3O2, 404.2338 (calcd.), 404.2328 (found).
Table 1. The obtained U-4CR products.
Compound Structure Mass (mg) Yield (%) LC/MS
A1 N O OHN Cl N H 22.3 28 tR = 11.61 min; m/z = 398.3 [M+H]+ B1 N O O NH Cl H N 20.6 24 tR = 11.61 min; m/z = 424.2 [M+H]+ C1 N O OHN Cl N H 32.4 38 tR = 11.48 min; m/z = 432.3 [M+H]+ D1 N O OHN Cl N H 15.6 18 tR = 11.65 min; m/z = 446.2 [M+H]+ E1 N O OHN Cl N H 28.6 31 tR = 12.01 min; m/z = 460.2 [M+H]+ F1 N O OHN Cl N H CO2Me 34.7 42 tR = 10.75 min; m/z = 414.3 [M+H]+ G1 N O OHN Cl N H N O 30.8 34 tR = 10.63 min; m/z = 453.2 [M+H]+
2
H1 N O OHN Cl N H Ph Ph 19.7 19 tR = 12.18 min; m/z = 508.2 [M+H]+ A2 N O OHN N H 25.4 35 tR = 11.18 min; m/z = 364.4 [M+H]+ B2 N O OHN N H 30.1 39 tR = 11.44 min; m/z = 390.4 [M+H]+ C2 N O OHN N H 10.5 13 tR = 11.10 min; m/z = 398.4 [M+H]+ D2 N O OHN N H 12.3 15 tR = 11.32 min; m/z = 412.3 [M+H]+ E2 NOHN O N H 12.8 15 tR = 11.66 min; m/z = 426.4 [M+H]+ F2 N O OHN N H CO2Me 27.6 36 tR = 10.31 min; m/z = 380.2 [M+H]+ G2 N O OHN N H N O 34.2 41 tR = 10.44 min; m/z = 419.4 [M+H]+ H2 N O OHN N H Ph Ph 19.1 20 tR = 11.87 min; m/z = 474.4 [M+H]+A3 N O OHN N H Cl 41.6 51 tR = 11.78 min; m/z = 412.2 [M+H]+ B3 N O OHN N H Cl 23 26 tR = 11.98 min; m/z = 438.2 [M+H]+ C3 N O OHN N H Cl 5.5 6 tR = 11.65 min; m/z = 446.3 [M+H]+ D3 N O OHN N H Cl 7.4 8 tR = 11.82 min; m/z = 460.3 [M+H]+ E3 N O OHN N H Cl 17.2 18 tR = 12.15 min; m/z = 474.3 [M+H]+ F3 N O OHN N H CO2 Me Cl 30.2 35 tR = 10.90 min; m/z = 428.3 [M+H]+ G3 N O OHN N H N O Cl 37.7 40 tR = 10.40 min; m/z = 467.4 [M+H]+ H3 N O OHN N H Ph Ph Cl 10.4 10 tR = 12.33 min; m/z = 522.3 [M+H]+ A4 N O OHN N H F 23.9 31 tR = 11.24 min; m/z = 382.2 [M+H]+
2
B4 N O OHN N H F 12.4 15 tR = 11.48 min; m/z = 408.4 [M+H]+ C4 N O OHN N H F 21.2 26 tR = 11.16 min; m/z = 416.3 [M+H]+ D4 N O OHN N H F 10.7 12 tR = 11.38 min; m/z = 430.1 [M+H]+ E4 N O OHN N H F 49.2 56 tR = 11.69 min; m/z = 444.4 [M+H]+ F4 N O OHN N H F CO2Me 30.5 38 tR = 10.39 min; m/z = 398.3 [M+H]+ G4 N O OHN N H F N O 19 22 tR = 10.31 min; m/z = 437.4 [M+H]+ H4 N O OHN N H F Ph Ph 19.5 20 tR = 11.88 min; m/z = 492.3 [M+H]+ A5 N O OHN N H 21.1 29 tR = 11.79 min; m/z = 370.5 [M+H]+B5 N O OHN N H 10.6 13 tR = 11.97 min; m/z = 396.5 [M+H]+ C5 N O OHN N H 18.3 23 tR = 11.62 min; m/z = 404.3 [M+H]+ D5 N O OHN N H 27.9 33 tR = 11.84 min; m/z = 418.2 [M+H]+ E5 NOHN O N H 27.4 32 tR = 12.18 min; m/z = 432.4 [M+H]+ F5 N O OHN N H CO2Me 17.6 23 tR = 10.82 min; m/z = 386.3 [M+H]+ G5 N O OHN N H N O 14.5 17 tR = 10.68 min; m/z = 425.3 [M+H]+ H5 N O OHN N H Ph Ph 26.3 27 tR = 12.34 min; m/z = 480.4 [M+H]+ A6 N O O NH N H 26.9 38 tR = 11.42 min; m/z = 356.4 [M+H]+ B6 N O OHN N H 14.7 19 tR = 11.63 min; m/z = 382.2 [M+H]+ C6 N O OHN N H 28.8 37 tR = 11.28 min; m/z = 390.3 [M+H]+
2
D6 NOHN O N H 32.8 41 tR = 11.51 min; m/z = 404.2 [M+H]+ E6 NO O NH H N 33 40 tR = 11.88 min; m/z = 418.5 [M+H]+ F6 N O OHN N H CO2Me 23.7 32 tR = 10.43 min; m/z = 372.3 [M+H]+ G6 OHNN O N H N O 21 26 tR = 10.34 min; m/z = 411.4 [M+H]+ H6 N O OHN N H Ph Ph 25.1 27 tR = 12.07 min; m/z = 466.5 [M+H]+ A7 N O OHN N H 7.2 11 tR = 11.23 min; m/z = 330.5 [M+H]+ B7 N O OHN N H 11.8 17 tR = 11.44 min; m/z = 356.2 [M+H]+ C7 NOHN O N H 15.7 22 tR = 10.98 min; m/z = 364.3 [M+H]+ D7 N O OHN N H 14.8 20 tR = 11.22 min; m/z = 378.4 [M+H]+ E7 N O OHN N H 28.8 37 tR = 11.58 min; m/z = 392.3 [M+H]+ F7 N O OHN N H CO2Me 35 51 tR = 9.95 min; m/z = 346.3 [M+H]+G7 N O OHN N H N O 22.1 29 tR = 9.90 min; m/z = 385.3 [M+H]+ H7 OHNN O N H Ph Ph 16.6 19 tR = 11.81 min; m/z = 440.4 [M+H]+ A8 N O OHN N H MeO 8.3 13 tR = 10.30 min; m/z = 332.2 [M+H]+ B8 N O OHN N H MeO 13.5 19 tR = 10.60 min; m/z = 358.5 [M+H]+ C8 N O OHN N H MeO 9.6 13 tR = 10.32 min; m/z = 366.2 [M+H]+ D8 NOHN O N H MeO 14.9 20 tR = 10.56 min; m/z = 380.3 [M+H]+ E8 NOHN O N H MeO 36.5 46 tR = 10.92 min; m/z = 394.3 [M+H]+ F8 N O OHN N H MeO CO2Me 30.1 43 tR = 9.35 min; m/z = 448.3 [M+H]+ G8 N O OHN N H MeO N O 32.6 42 tR = 9.28 min; m/z = 387.2 [M+H]+ H8 N O OHN N H MeO Ph Ph 19.1 22 tR = 11.48 min; m/z = 442.3 [M+H]+
2
A9 N O OHN N H 21.7 33 tR = 10.80 min; m/z = 328.4 [M+H]+ B9 N O OHN N H 17.8 25 tR = 11.03 min; m/z = 354.3 [M+H]+ C9 N O OHN N H 13.8 19 tR = 10.73 min; m/z = 362.3 [M+H]+ D9 N O OHN N H 9.4 13 tR = 10.93 min; m/z = 376.4 [M+H]+ E9 N O OHN N H 29.4 38 tR = 11.32 min; m/z = 390.4 [M+H]+ F9 N O OHN N H CO2Me 35.2 51 tR = 9.84 min; m/z = 344.5 [M+H]+ G9 OHNN O N H N O 25.8 34 tR = 9.75 min; m/z = 383.2 [M+H]+ H9 N O OHN N H Ph Ph 15.1 17 tR = 11.58 min; m/z = 438.4 [M+H]+ A10 N O OHN N H Br 30.2 34 tR = 11.68 min; m/z = 442.1 [M+H]+ B10 N O OHN N H 29.1 31 tR = 11.91 min; m/z = 468.3 [M+H]+C10 N O OHN N H Br 44.9 47 tR = 11.58 min; m/z = 476.2 [M+H]+ D10 N O OHN N H Br 24.3 25 tR = 11.76 min; m/z = 490.2 [M+H]+ E10 N O OHN N H Br 28.7 29 tR = 12.11 min; m/z = 504.3 [M+H]+ F10 N O OHN N H Br CO2Me 28.3 31 tR = 10.88 min; m/z = 458.2 [M+H]+ G10 N O OHN N H Br N O 25.8 26 tR = 10.73 min; m/z = 497.1 [M+H]+ H10 N O OHN N H Br Ph Ph 17.9 16 tR = 12.27 min; m/z = 552.3 [M+H]+ A11 N O OHN N H Cl Cl 12.1 14 tR = 11.93 min; m/z = 432.0 [M+H]+ B11 N O OHN N H Cl Cl 51.4 56 tR = 12.13 min; m/z = 458.4 [M+H]+ C11 N O OHN N H Cl Cl 17.8 19 tR = 11.80 min; m/z = 466.3 [M+H]+
2
D11 N O OHN N H Cl Cl 22 23 tR = 11.98 min; m/z = 480.3 [M+H]+ E11 N O OHN N H Cl Cl 33.7 34 tR = 12.28 min; m/z = 494.2 [M+H]+ F11 N O OHN N H Cl Cl CO2Me 38 42 tR = 11.06 min; m/z = 448.1 [M+H]+ G11 N O OHN N H Cl Cl N O 30.2 31 tR = 10.91 min; m/z = 487.2 [M+H]+ H11 N O OHN N H Cl Cl Ph Ph 22.4 21 tR = 12.47 min; m/z = 542.3 [M+H]+ A12 N O OHN N H 28.9 38 tR = 11.32 min; m/z = 378.3 [M+H]+ B12 N O OHN N H 28.5 35 tR = 11.55 min; m/z = 404.3 [M+H]+ C12 N O OHN N H 14.2 17 tR = 11.23 min; m/z = 412.2 [M+H]+ D12 N O OHN N H 41.7 49 tR = 11.43 min; m/z = 426.3 [M+H]+E12 N O OHN N H 48.2 55 tR = 11.76 min; m/z = 440.3 [M+H]+ F12 N O OHN N H CO2Me 19.9 25 tR = 10.48 min; m/z = 394.2 [M+H]+ G12 N O OHN N H N O 21 24 tR = 10.34 min; m/z = 433.3 [M+H]+ H12 N O OHN N H Ph Ph 31.3 32 tR = 11.96 min; m/z = 488.3 [M+H]+
2
References
1 C. F. Cheok, C. S. Verma, J. Baselga, D. P. Lane, Nat Rev Clin Oncol 2011,
8, 568-568.
2 K. Khoury, G. M. Popowicz, T. A. Holak, A. Domling, MedChemComm 2011, 2, 246-260.
3 G. M. Popowicz, A. Dömling, T. A. Holak, Angewandte Chemie
Internatio-nal Edition 2011, 50, 2680-2688.
4 K. Khoury, G. M. Popowicz, T. A. Holak, A. Dömling, Medchemcomm 2011, 2, 246-260.
5 G. M. Popowicz, A. Czarna, U. Rothweiler, A. Szwagierczak, M. Krajew-ski, L. Weber, T. A. Holak, Cell Cycle 2007, 6, 2386-2392.
6 J. Berghausen, N. Buschmann, P. Furet, F. Gessier, L. J. Hergovich, P. Holzer, E. Jacoby, J. Kallen, K. Masuya, S. C. Pissot, H. Ren, S. Stutz, p. 448pp.
7 G. Bold, P. Furet, F. Gessier, J. Kallen, L. J. Hergovich, K. Masuya, A. Vaupel, p. 274pp.
8 D. Reed, Y. Shen, A. A. Shelat, L. A. Arnold, A. M. Ferreira, F. Zhu, N. Mills, D. C. Smithson, C. A. Regni, D. Bashford, S. A. Cicero, B. A. Schul-man, A. G. Jochemsen, R. K. Guy, M. A. Dyer, Journal of Biological
Chemis-try 2010, 285, 10786-10796.
9 J. H. Lee, Q. Zhang, S. Jo, S. C. Chai, M. Oh, W. Im, H. Lu, H.-S. Lim,
Journal of the American Chemical Society 2010, 133, 676-679.
10 J.-C. W. Marine, M. A. Dyer, A. G. Jochemsen, Journal of Cell Science 2007,
120, 371-378.
11 S. Srivastava, B. Beck, W. Wang, A. Czarna, T. A. Holak, A. Dömling,
Journal of Combinatorial Chemistry 2009, 11, 631-639.
12 G. M. Popowicz, A. Czarna, S. Wolf, K. Wang, W. Wang, A. Dömling, T. A. Holak, Cell Cycle 2010, 9, 1104-1111.
13 Y. Huang, S. Wolf, M. Bista, L. Meireles, C. Camacho, T. A. Holak, A. Dömling, Chemical Biology & Drug Design 2010, 76, 116-129.
14 A. Czarna, B. Beck, S. Srivastava, G. M. Popowicz, S. Wolf, Y. Huang, M. Bista, T. A. Holak, A. Dömling, Angewandte Chemie-International Edition 2010, 49, 5352-5356.
15 M. Bista, K. Kowalska, W. Janczyk, A. Dömling, T. A. Holak, Journal of the
16 U. Rothweiler, A. Czarna, M. Krajewski, J. Ciombor, C. Kalinski, V. Kha-zak, G. Ross, N. Skobeleva, L. Weber, T. A. Holak, ChemMedChem 2008, 3, 1118-1128.
17 G. Popowicz, A. Czarna, T. Holak, Cell Cycle 2008, 7, 2441-2443.
18 U. Rothweiler, A. Czarna, L. Weber, G. M. Popowicz, K. Brongel, K. Ko-walska, M. Orth, O. Stemmann, T. A. Holak, Journal of Medicinal
Chemis-try 2008, 51, 5035-5042.
19 A. Czarna, G. M. Popowicz, A. Pecak, S. Wolf, G. Dubin, T. A. Holak, Cell
Cycle 2009, 8, 1176-1184.
20 Y. Huang, S. Wolf, D. Koes, G. M. Popowicz, C. J. Camacho, T. A. Holak, A. Dömling, ChemMedChem 2012, 7, 49-52.
21 D. Koes, K. Khoury, Y. Huang, W. Wang, M. Bista, G. M. Popowicz, S. Wolf, T. A. Holak, A. Dömling, C. J. Camacho, PLoS ONE 2012, 7, e32839.
Chapter 3
Ugi multicomponent approach to
synthesize schistosomiasis drug
praziquantel
André Boltjes, Haixia Liu, Haiping Liu and Alexander Dömling
Abstract
Praziquantel is currently the only effective drug for the treatment of schistoso-miasis, a neglected tropical disease, affecting over 200 million people worldwide. The current production method to produce praziquantel is via the Merck pro-cess which consist of a five-step synthesis, in which the key step is a Reissert reaction requiring large excess of toxic KCN. The accompanying environmental treats of potential cyanide pollution cannot be ignored and therefore a more effi-cient three-step MCR Ugi-4CR was presented. The methodology incorporates the novel in situ formation of the phenethylisocyanide component. The procedure is described in a thorough detail oriented way to meet the requirements for publi-cation in Organic Syntheses and was checked for reproducibility in the laborato-ry of a member of the Board of Editors.
3
Introduction
Schistosomiasis is a parasitic infection which affects over 200 million people worldwide and in particular sub-Saharan Africa.1-3 The infection is classified as
a neglected tropical disease and manifests itself by flu-like symptoms due to egg deposition of schistosomes, into the tissues of the host, mostly in the intestine, liver and genitourinary system. Untreated infections will result in chronic schis-tosomiasis. The immune response of the host causes the formation of granulomas around the eggs ultimately leading to fibrosis in the affected tissues and calcifica-tion in the genitourinary tracts. It is currently one of the most common infectious diseases, which is efficiently treated by administering praziquantel. Praziquantel was initially developed as a potential tranquilizer by E. Merck in the 1970’s.4 It is,
however, not uncommon that drugs are repurposed for other conditions such as sildenafil and thalidomine.5-6 Praziquantel’s anthelmintic properties were tested
and proved by Bayer A.G. and initially marketed for the veterinary market and then for the human market.7
In terms of drug safety, studies have shown that praziquantel is currently the saf-est drug available.8 Production of praziquantel is performed via a slight adaption
of the original Merck process, developed in 1983 by Shin Poong Pharmaceutical company as depicted in scheme 1. This process is fairly cheap. Due to the safe use, amount of affected people, efficacy of treatment, praziquantel is placed on the WHO list of Essential medicines.9
N N CN Cy O NH N H N HN O Cl N N O O KCN,
CyCOCl H2/Ni 70 atm
90 oC Cl O Cl 1 2 3 4 5 O Cy O Cy Et3N
Scheme 1. The Merck industrial method is still the most widely applied. Isoquinoline (1) is converted to the Reissert compound (2) and then reduced by hydrogenation with nickel. N-alkylation with chloroacetyl chloride followed by a base assisted cyclisation yielding praziquantel (5).
Current production sites are located in Asia (China, Korea) and in the process large quantities of KCN are used. Incidents with spills of cyanide waste have resulted in environmental pollution, killing life in rivers for many kilometers downstream. Efforts in finding alternative synthesis routes resulted in a MCR ap-proach in 2010.10 The Ugi multicomponent reaction of amines, oxo components,
dependent on the nature of the acid component.11 For example carboxylic acids
yield α-amino acylamides (Scheme 1). The Ugi multicomponent reaction is gain-ing increasgain-ing attention due to the rapid and convergent assembly of functional structures based on four classes of widely available starting material classes.12
In addition, our recent work on generating the isonitrile in situ is making the Ugi reaction even more accessible, as the use of the infamous isonitrile is cir-cumvented in this methodology.13 While several approaches towards α-amino
acylamides are possible the Ugi approach is faster and impresses by a very large substrate scope.14-15 Here the schistosomiasis drug praziquantel (PQZ,
2-(cyclo-hexanecarbonyl)-2,3,6,7-tetrahydro-1H-pirazino[2,1-a]isoquinolin-4(11bH)-one) is prepared in just three steps using the key Ugi and Pictet-Spengler reactions.10
It comprises a short synthetic route from the readily available bulk starting ma-terials, aminoacetaldehyde dimethylacetal (6), formaldehyde (7), 2-phenylethyl formamide (8) and cyclohexancarboxylic acid (9). Employing the high yielding formylation of phenethylamine with ethyl formate, N-phenethylformamide can be achieved in 99% yield. N-phenethylformamide reacts with triphosgene which in turn reacts with paraformaldehyde, aminoacetaldehyde dimethylacetal, and cyclohexylcarboxylic acid in a Ugi four component reaction to the advanced pre-cursor 10 in 41% yield. In methanesulfonic acid at 70 °C for 6h, praziquantel 5 was afforded with 52% yield. These reactions were carried out under mild con-ditions and the sequence is atom economic, yielding only water and two equiva-lents of methanol as side products.
H N H2N O O H H O HO O + HN O N O O O N O N O MSA 70 °C MeOH, DCM, -10 °C to r.t. 6 7 8 9 5 10 Triphosgene, Et3N O
Scheme 2. The Ugi-4CR with the in situ generated phenethylisocyanide from the
for-mamide 8, followed by subsequent Pictet-Spengler reaction to yield praziquantel 5.
An Ugi-4CR reaction was used to construct the typical praziquantel function-al groups, however, as the dipeptidic scaffold associated with the U-4CR. The
3
protected aldehyde moiety was subsequently deprotected and under the same conditions cyclized via a Pictet-Spengler reaction yielding praziquantel in only two steps (Scheme 2).16
Results and Discussion
Here the schistosomiasis drug praziquantel (PQZ, 2-(cyclohexanecarbon-yl)-2,3,6,7-tetrahydro-1H-pirazino[2,1-a]isoquinolin-4(11bH)-one) is prepared in just three steps using the key Ugi and Pictet-Spengler reactions.10, 17-18 It
com-prises a short synthetic route from the readily available bulk starting materials, 2-phenylethyl formamide, formaldehyde, cyclohexancarboxylic acid and ami-noacetaldehyde dimethylacetal. Employing the high yielding formylation of phenethylamine with ethyl formate, N-phenethylformamide can be achieved in 99% yield. N-phenethylformamide reacts with triphosgene which in turn reacts with paraformaldehyde, aminoacetaldehyde dimethylacetal, and cyclohexylcar-boxylic acid in a Ugi four component reaction to the advanced precursor 2 in 41% yield. In methanesulfonic acid at 70 °C for 6h, praziquantel was afforded with 52% yield. These reactions were carried out under mild conditions and the sequence is atom economic, yielding only water and two equivalents of methanol as side products.
A. Preparation of N-phenethylformamide (8).
A 500-mL single-necked round-bottom flask equipped with a 3 cm egg-shaped Teflon-coated stirring bar and a reflux condenser was charged with phenethyl-amine (95 mL, 0.75 mol) and ethyl formate (181 mL, 2.25 mol 1.8 eq.) (Note 1). The mixture was heated to reflux using an oil bath at 60 °C for 20 hours until TLC indicates full consumption of the amine. The mixture was concentrated by rotary evaporation (40 °C, 10 mbar) to remove the excess of ethyl formate and ethanol byproduct (Note 2). N-phenethylformamide 111 g (99%) is afforded as pale yel-low oil (Note 3).
B. Preparation of N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(phenethylamino)ethyl) cylcohexanecarboxamide (10).
In a 500 mL 3-necked round-bottom flask equipped with a 7 cm Teflon blade overhead stirrer, a 100 mL pressure equalizing dropping funnel holding a nitro-gen inlet and a temperature sensor (see photo below), a mixture of N-pheneth-ylformamide (7.46g, 50 mmol) and triethylamine (16.8 mL, 120 mmol 2,4 eq.) in dichloromethane (50 mL) (Note 4) was cooled to -10 °C using an ethanol-ice bath (Note 5).
Figure 1. Reaction Set-up for step B
While stirring (500 rpm) triphosgene (5.94 g, 20 mmol, 0,4 eq.) (Note 6) in di-chloromethane (20 mL) was added dropwise via the dropping funnel over a period of 40 minutes (Note 7), resulting in a pale yellow solution. The reaction mixture was stirred at -10 °C for an additional 30 minutes (Note 8). In a separate 50 ml round bottom flask a mixture of aminoacetaldehyde dimethyl acetal (5.52 g, 52 mmol, 1.05 eq.) and paraformaldehyde (1.50 g, 50 mmol, 1 eq.) (Note 9) in 50 mL methanol was heated shortly with a heating gun until a clear solution de-veloped; then cyclohexanecarboxylic acid (6.72 g, 52 mmol, 1.05 eq) was added; the resulting solution was then added together with another 50 mL MeOH to the in situ formed isocyanide reaction mixture at -10 °C and a slightly orange clear solution is formed. The reaction mixture was left to warm to room temperature. After stirred at room temperature for 48 h (Note 10), the mixture is concentrated (40 °C, 10 mbar) to remove the methanol. Then the mixture was redissolved in 50 mL CH2Cl2 and transferred to a 250 mL separatory funnel, washed with water (3x 50mL), saturated NaHCO3 (3x 50mL), and dried over 5g MgSO4. The drying agent was removed by filtration over a P4 sintered glass filter and concentrated by rotary evaporation (40 °C, 10 mbar), yielding 9.05 gram crude product as an orange oil (Note 11), which crystalized on standing.
3
Figure 2. Appearance of the Ugi product obtained in step BFurther trituration of the crude crystals with 10 mL cold Et2O, filtering through a P4 glass filter and washing with another 10 mL cold Et2O yields after drying (0.5 mbar) 7.56 g (40%) pure N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(phenethylamino) ethyl)cylcohexanecarboxamide as pale yellow crystals (picture above) (Note 12). C. Preparation of 2-(cyclohexanecarbonyl)-2,3,6,7-tetrahydro-1H-piraz-ino[2,1-a]isoquinolin-4(11bH)-one (5).
In a 100 mL single-necked round-bottom flask under N2 atmosphere, N-(2,2-di-methoxyethyl)-N-(2-oxo-2-(phenethylamino)ethyl)cyclohexanecarboxamide 2 (7.54 g, 20 mmol, 1 eq.) and 10 g activated molecular sieves (Note 13) was added at once to a solution of methanesulfonic acid (26.0 mL, 400 mmol, 20 eq.) at room temperature (Note 14). The mixture was heated to 70 °C (measured externally) in an oil bath for 6 h, then after cooling to room temperature the reaction mixture is gravity filtered through a slotted sieve filter into an 2L petri dish containing ice-water (200 mL) and NaHCO3 (40 g, 476 mmol) (Figure 3. Left).
Figure 3. Left: Removal of the sieves and stirring. Right: Appearance of the crude Prazi-quantel obtained in step C.
200 mL Et2O is added and the mixture is stirred for 15 minutes using a 7 cm rod-shaped Teflon-coated stirring bar to dissolve most of the solids (Note 15). The solution is transferred to a 1L separation funnel and extracted with diethyl ether (3×100 mL) (Note 16). The combined organic layers are washed with brine (100 mL), dried over 10 g anhydrous magnesium sulfate and concentrated to dryness (40 °C, 600 =>10 mbar) to afford praziquantel (4.0 g, 64%) as orange oil, which crystalized on standing overnight (right picture above). The solid was triturated by adding 2x 10 mL 20% acetone in heptane, and filtered through a P4 sintered glass filter to obtain, after vacuum drying (0.5 mbar), 2.46 g pure praziquantel (picture below). The mother liquor was concentrated (1.5g orange oil) and puri-fied by flash chromatography (Note 17). The fractions containing product were combined and yielded another 1.05 g praziquantel as an orange solid. Trituration with 5 mL Et2O yields 0.80 g pure praziquantel as a white solid. The combined yield is 3.51 g, 52% (Note 18) (Note 19).
3
Notes
1. Phenethylamine (99%) and ethyl formate (>98%) were obtained from Sig-ma Aldrich and Acros Organics respectively and used as received.
2. Removal of the byproduct EtOH requires extended time (2 hours) on the rotary evaporator, 1H NMR is used to visualize traces of EtOH seen at 1.20
(trip-let) and 3.66 (quartet) ppm. 3. N-phenethyl formamide
O N
H
Rf = 0.45 (EtOAc), 1H NMR (500 MHz, Chloroform-d) δ 8.04
(s, 1H), 7.37-7.24 (m, 2H), 7.26-7.02 (m, 3H), 6.34-5.88 (m, 1H), 3.52 (m, 2H), 2.89-2.70 (m, 2H) (only the major peaks of the formamide rotamers were described); 13C NMR (125 MHz,
Chloroform-d) δ 1, 161.4, 138.6, 137.7, 128.7, 128.6, 126.6, 39.2, 35.5 ppm.
4. The used solvents in step B and C: dichloromethane, ethyl acetate, pe-troleum ether 40-60 and methanol are technical grade and were purchased from Biosolve.
5. In a 1L dewar, 275 g ice and 150 g ethanol were used to obtain a tempera-ture of at least -10 °C for the duration of the reaction.
6. The solid reagent triphosgene is a less hazardous substitute for highly toxic gaseous phosgene, however should be handled very carefully. The reaction should be performed in a well ventilated fume hood.
7. Faster addition or temperatures above 0 °C will reduce the yield dramat-ically. Color is a good indicator of proper addition speed: slightly yellow color is good, orange towards brown indicates too fast addition of triphosgene.
8. The isocyanide intermediate Rf = 0.74 (EtOAc) appears exclusively indi-cating full consumption of the formamide.
9. Not all reagents are fully consumed, but after 48 h no change in spot in-tensity, checked by TLC Merck silica gel 60 F254 plates (visualized with 254 nm UV lamp), was observed.
10. Reagents and solvents used in this preparation were commercially avail-able and used without further purification, including triphosgene (98%) and ami-noacetaldehyde dimethyl acetal (98%) from AK Scientific Inc., trimethylamine and paraformaldehyde (96%) from Fluka and cyclohexane carboxylic acid from Sigma Aldrich ( 98%).
11. The bulk of solvent should be removed. However, leaving a trace of sol-vent (~0.5 g) is actually beneficial in allowing the product to crystalize. The crys-tallization process takes some time (2 weeks) to complete.
12. N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(phenethylamino)ethyl) cylcohex-anecarboxamide HN N O O O O mp = 86-88 °C Rf = 0.42 (EtOAc) 1H NMR (500 MHz, Chloro-form-d) δ 7.36-7.25 (m, 2H), 7.25- 7.15 (m, 3H), 7.03 (t, J = 5.8 Hz, 0.5H), 6.51 (t, J = 6.0 Hz, 0.5H), 4.57 (t, J = 5.1 Hz, 0.5H), 4.39 (t, J = 5.2 Hz, 0.5H), 3.99 (d, J = 7.4 Hz, 2H), 3.55 (q, J = 6.8 Hz, 1H), 3.48 (q, J = 6.8 Hz, 1H), 3.42 (dd, J = 7.0, 5.1 Hz, 2H), 3.37 (s, 3H), 3.33 (s, 3H), 2.80 (dt, J = 21.9, 7.2 Hz, 2H), 2.58 (tt, J = 11.6, 3.4 Hz, 0.5H), 2.25 (tt, J = 11.5, 3.3 Hz, 0.5H), 1.85- 1.71 (m, 2H), 1.71-1.56 (m, 2H), 1.53-1.37 (m, 2H), 1.35- 1.15 (m, 3H); 13C NMR (125 MHz, Chloroform-d) δ 178.0, 177.8, 169.5, 169.2, 138.8, 138.6, 128.7, 128.7, 128.6, 126.6, 126.4, 103.5, 102.7, 77.2, 55.4, 55.1, 54.0, 52.1, 51.5, 50.3, 41.0, 40.7, 40.5, 40.3, 35.6, 35.6, 29.4, 29.3, 25.7, 25.7, 25.6 ppm; HRMS (ESI). [M + Li]+ calcd. for C22H32O4N2Li: 383.2517. Found: 383.2514. Elemental Anal. calcd for C19H24N2O2: C, 66.99; H, 8.57; N, 7.44. Found: C, 66.89; H, 8.66; N, 7.43. The tertiary amide rotamers are clearly visible in the NMR spectrum and show a 1:1 ratio.
13. 3Å Molecular sieves from Sigma Aldrich were activated by washing the sieves with CH2Cl2, removing the majority of solvent, decantation, and drying by rotary evaporation (40 °C, 10 mbar) and oven for 2 hours at 120 °C. Next the molecular sieves were heated in a household microwave 3 times one minute at 600W with cooling periods (5 min each) in between with the microwave door opened. The sieves were left to cool in an evacuated desiccator and were used immediately. The activation was confirmed by putting a few sieves on the hand, adding a few drops of water and pressing with a finger on the sieves. The sieves should get hot.
14. Methanesulfonic acid is commercial available from Sigma Aldrich ( 99.5%.). 1H-NMR indicates a big peak at 3.33 ppm, likely water. Therefore it must be carefully dried before use, with activated 3Å molecular sieves (see note 10 for activation). Use of ‘wet’ methanesulfonic acid directly from the fresh bottle lead to a dramatic reduction of yield. 100 ml Methanesulfonic acid was dried with 10 gram molecular sieves for at least 1 month. 1H NMR analysis of proper dried methanesulfonic acid will only show 1 peak in the 3 ppm region (3.15 ppm). 15. Remaining solids consisting of powdered molecular sieves and prazi-quantel were collected by decanting and extracted by vigorously stirring with 20 mL 1:1 Et2O:water in a 50 mL round bottom flask for 10 minutes. After filtration through a P4 sintered glass filter, the filtrate was added to the main solution. 16. Diethyl ether is superior as an extraction solvent than ethyl acetate be-cause the impurity is better soluble in ethyl acetate.
17. Flash chromatography was performed on a Grace Reveleris X2 using a Reveleris® Silica 12g column and elution with accomplished with a gradient of ethyl acetate/petroleum ether (bp 40-60 °C). The mixture was absorber on 2 grams silica. The gradient starts with 30% ethyl acetate and goes to 100% over 12
3
minutes in a total run time of 25 minutes. Only ELSD visible peaks are collected as fractions of ~15 mL. The desired product is collected in fraction 7-15 and con-centrated by rotary evaporation (40 °C, 10 mmHg).
18. Praziquantel 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 f1 (ppm) 3.07 2.01 4.98 0.95 1.72 1.96 0.22 0.22 0.71 0.21 0.72 2.13 0.69 4.04 N N O O 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 f1 (ppm) 25.67 25.70 25.72 28.71 29.00 29.23 29.53 38.64 39.09 40.76 45.14 46.30 49.01 49.53 54.93 55.78 77.16 CDCl3 125.44 126.95 127.42 129.26 132.79 134.72 164.37 174.71 N N O O
3
O N N O mp 136-138 °C; Rf = 0.46 (EtOAc); 1H NMR (500 MHz, Chloro-form-d) δ 7.43-7.05 (m, 4H), 5.15 (dd, J = 13.4, 2.4 Hz, 0.35H), 4.95-4.70 (m, 2H), 4.42 (dd, J = 44.4, 15.5 Hz, 1H), 3.97 (dd, J = 111.7, 18.1 Hz, 1H), 3.26 (t, J = 12.2 Hz, 0.25H), 3.03-2.85 (m, 2H), 2.85-2.75 (m, 2H), 2.65- 2.41 (m, 1H), 1.95-1.65 (m, 5H), 1.65-1.44 (m, 2H), 1.44-1.15 (m, 3H) ppm; 13C NMR (125 MHz, Chloro-form-d) δ 174.7, 164.4, 134.7, 132.8, 129.3, 127.4, 126.9, 125.4, 77.2, 55.8, 54.9, 49.5, 49.0, 46.3, 45.1, 40.8, 39.1, 38.6, 29.5, 29.2, 29.0, 28.7, 25.7, 25.7, 25.7 ppm; HRMS (ESI). [M + H]+ calcd. for C19H25O2N2: 313.19095. Found: 313.19105. Elemental Anal. calcd for C19H24N2O2: C, 73.05; H, 7.74; N, 8.97. Found: C, 72.79; H, 7.89; N, 8.95.19. Two rotamers steaming from the presence of a tertiary amide group can be seen in the NMR spectra, resulting in minor and major peaks in the proton NMR and doublets in the carbon NMR, these were not assigned due to the com-plexity of the spectrum. Alignment of the 1H NMR of an analytical sample
(Flu-ka) of praziquantel with our sample proofs identity.
Conclusions
In conclusion, an effective, shortest approach to synthesize praziquantel is de-scribed, starting with easily available materials. Based on the substrate scope of the Ugi reaction 30 praziquantel derivatives have been synthesized using the same reaction sequence in moderate to good yields (Table 1).
References
1 K. J. Wilby, S. E. Gilchrist, M. H. H. Ensom, Clinical Pharmacokinetics 2013,
52, 647-656.
2 D. G. Colley, A. L. Bustinduy, W. E. Secor, C. H. King, The Lancet 2014,
383, 2253-2264.
3 M. Njoroge, N. M. Njuguna, P. Mutai, D. S. B. Ongarora, P. W. Smith, K. Chibale, Chemical Reviews 2014, 114, 11138-11163.
4 E. Groll, Advances in pharmacology and chemotherapy 1984, 20, 219-238. 5 J. B. Bartlett, K. Dredge, A. G. Dalgleish, Nature Reviews Cancer 2004, 4,
314-322.
6 I. Goldstein, A. L. Burnett, R. C. Rosen, P. W. Park, V. J. Stecher, Sexual
Medicine Reviews 2019, 7, 115-128.
7 J. Seubert, R. Pohlke, F. Loebich, Experientia 1977, 33, 1036-1037.
8 D. Cioli, L. Pica-Mattoccia, A. Basso, A. Guidi, Molecular and Biochemical
Parasitology 2014, 195, 23-29.
9 A. Dömling, K. Khoury, Chemmedchem 2010, 5, 1420-1434.
10 H. Cao, H. Liu, A. Dömling, Chemistry – A European Journal 2010, 16, 12296-12298.
11 I. Ugi, R. Meyr, U. Fetzer, C. Steinbrückner, Angew. Chem. 1959, 71, 386. 12 A. Dömling, Chemical Reviews 2006, 106, 17-89.
13 C. G. Neochoritis, S. Stotani, B. Mishra, A. Dömling, Org Lett 2015, 17, 2002-2005.
14 I. Ugi, C. Steinbrückner, Angew. Chem. Int. Ed. 1960, 72, 267. 15 S. Marcaccini, T. Torroba, Nature Protocols 2007, 2, 632.
16 J. H. Kim, Y. S. Lee, C. S. Kim, Heterocycles 1998, 48, 2279-2285.
17 A. Pictet, A. Gams, Berichte der deutschen chemischen Gesellschaft 1911, 44, 2480-2485.
18 H. Liu, S. William, E. Herdtweck, S. Botros, A. Dömling, Chemical Biology
Chapter 4
Ugi 4-CR Synthesis of γ- and
δ-Lactams providing new access to
diverse enzyme interactions, a PDB
analysis
André Boltjes, George P. Liao, Ting Zhao, Eberhardt Herdtweck, and Alexander Dömling
Abstract
A three step synthesis of N-unsubstituted tetrazolo γ- and δ-lactams involving a key Ugi-4CR is presented. The compounds, otherwise difficult to access, are conveniently synthesized in overall good yields by our route. PDB analysis of the N-unsubstituted γ- and δ-lactam fragment reveals a strongly tri-directional hydrogen bond donor acceptor interaction with the amino acids of the binding sites.
4
Introduction
The γ-and δ-lactam moiety is an often found fragment in bioactive compounds. Examples of compounds with a lactam fragment include the anti-Parkinson drug oxotremorin1 or the anti-rhinoviral and -enteroviral rupintrivir.2 Generally, the
lactam nitrogen can be either unsubstituted or substituted, which influences its hydrogen bonding profile in the receptor binding site. For example in the crystal structure of rupintrivir with the human rhinovirus 3C protease, the γ-lactam-N forms a short hydrogen bond to the Thr142 backbone carbonyl, whereas the δ-lac-tam-O forms two short contacts to side chain His161-NH and the Thr142-OH, re-spectively (Fig. 1).3 In our ongoing efforts in structure- and computational-based
design of bioactive compounds we were interested in a short and versatile synthe-sis of N-unsubstituted γ- and δ-lactams with potential multiple hydrogen bond interactions.4 Combining the lactam functionality with a 1,5 disubstituted
tetra-zolo peptidomimetic could enhance the affinity of this tetrazole analog which is a well-known cis-amide bond mimic, which provides new synthetic probes for a series of biological targets.5-9 Multicomponent reactions (MCR) were found
to be an excellent tool to rapidly access a large and versatile drug-like chemical space to address the corresponding biological space.5 A convenient and versatile
synthesis of N-substituted γ- and δ-lactams using convergent Ugi-type MCR’s was recently introduced by Marcos et al. and refined by others.10-12 However no
MCR-based synthesis of N-unsubstituted γ- and δ-lactams has been described up to date. Clearly many synthetic approaches towards N-unsubstituted γ- nd δ-lactams are described, however these contain limitations regarding variability d length of the synthetic routes.13
Figure 1.Trifurcated hydrogen bonding interactions (red dotted lines) of an N-unsubsti-tuted γ-lactam (cyan sticks) with some protease amino acids in the example of rupintrivir (PDB ID 1CQQ).
Ammonia is one of the few amine components which does not regularly give convincing results in the Ugi MCRs.14-18 Therefore we recently introduced
trityl-amine as an ammonia surrogate in the Ugi tetrazole MCR.19 This reaction forms
the core of our design of a synthetic pathway towards N-unsubstituted γ- and δ-lactams (Scheme 1). The first step comprises the Ugi tetrazole MCR using tr-itylamine, followed by cleavage of the trityl group. The formed primary amine should easily undergo cyclisation to form the target γ- and δ-lactam compounds.
