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Design of Hybrid Molecules
for Drug Development
Design of Hybrid Molecules
for Drug Development

Edited by

Michael Decker
Julius Maximilian University of Würzburg, Würzburg, Germany
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

Copyright © 2017 Elsevier Ltd. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and retrieval system, without
permission in writing from the publisher. Details on how to seek permission, further information about the
Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance
Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher
(other than as may be noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our
understanding, changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any
information, methods, compounds, or experiments described herein. In using such information or methods they
should be mindful of their own safety and the safety of others, including parties for whom they have a professional
responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability
for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or
from any use or operation of any methods, products, instructions, or ideas contained in the material herein.
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
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A catalog record for this book is available from the Library of Congress
ISBN: 978-0-08-101011-2

For Information on all Elsevier publications visit our

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Dedication

This book is dedicated to Professor John L. Neumeyer for his mentorship and his inspirational
approach to Medicinal Chemistry and to T. W. W. for his company during the many years we
spent together at university.
List of Contributors

Luca Agnetta Julius Maximilian University of Würzburg, Würzburg, Germany

Maria Laura Bolognesi University of Bologna, Bologna, Italy

Salvatore Bongarzone King’s College London, London, United Kingdom

Giovanni Bottegoni Istituto Italiano di Tecnologia, Genova, Italy

María do Carmo Carreiras Research Institute for Medicines and

Pharmaceutical Sciences (iMed.ULisboa), Lisbon, Portugal

Andrea Cavalli Istituto Italiano di Tecnologia, Genova, Italy; Università di

Bologna, Bologna, Italy

Kelly Chibale University of Cape Town, Cape Town, South Africa

Michael Decker Julius Maximilian University of Würzburg, Würzburg,

Germany

Dominik Dolles Julius Maximilian University of Würzburg, Würzburg,

Germany

John F. Gilmer School of Pharmacy and Pharmaceutical Sciences, Trinity

College Dublin, Ireland

Liu He Virginia Commonwealth University, Richmond, VA, United States

Guozheng Huang College of Life and Environmental Sciences, Shanghai

Normal University, Shanghai, P.R. China; Xinjiang Technical Institute of Physics
and Chemistry, Chinese Academy of Sciences, Urumqi, P.R. China

Lhassane Ismaili UFR of Medical and Pharmaceutical Sciences (SMP),

Besançon, France

José Marco-Contelles Institute of General Organic Chemistry (CSIC); Juan de

la Cierva, Madrid, Spain

xi
xii List of Contributors

Diego Muñoz-Torrero University of Barcelona, Barcelona, Spain

Peter Mbugua Njogu University of Nairobi, Nairobi, Kenya

John Okombo University of Cape Town, Cape Town, South Africa

Federica Prati University of Dundee, Dundee, United Kingdom

Alejandro Romero Complutense University of Madrid, Madrid, Spain

John M. Saathoff Virginia Commonwealth University, Richmond, VA, United

States

Elisa Uliassi University of Bologna, Bologna, Italy

Shijun Zhang Virginia Commonwealth University, Richmond, VA, United

States

Qingjie Zhao Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai, P.R. China
Preface

Hybrid molecules (or multitarget-directed drugs) are stable chemical combinations of two or
more drug molecules, and they represent a comparatively new area in drug research. Their
design aims to tackle complex multifactorial diseases by polypharmacology, since these dis-
ease states cannot be adequately addressed by the classical “one target, one molecule”
strategies.
Numerous projects have aimed to design and obtain such compounds and the results are
very promising. This makes hybrid molecule design one of the hot topics in modern medici-
nal chemistry. For this book it was possible to gain some of the world-leading medicinal che-
mists and experts in the field of hybrid molecules as chapter authors, which allows this book
to cover many areas of drug development ranging from neurodegenerative disorders to can-
cer to parasitic diseases, from NO-releasing compounds to photoswitchable ones, from drug
synthesis to computational methods to in vivo studies. While not all aspects of this highly
proliferative topic could be covered in a single volume, I am confident this text will neverthe-
less illustrate and present all significant aspects enabling the reader to get both a sound over-
view as well as sufficient details on the opportunities as well as challenges for hybrid
molecule design. Numerous successful examples will be presented.
The book is intended for anyone who has an interest in hybrid compounds for medicinal
purposes, either as a drug researcher in academia or a company, or as a graduate student or
group leader who wants to apply the respective methods for successful hybrid molecule
development to their specific research problem.
I most gratefully acknowledge the time, effort, and expertise the chapter authors have
invested in their chapters. I would also like to thank Elsevier for the offer and chance to write
and edit a book on this exciting topic, and all authors express their gratitude to the universi-
ties we work at and the funding agencies that have enabled us to conduct our research on
hybrid molecules.
Michael Decker
Würzburg, Germany
November 2016

xiii
 1
Introduction
Michael Decker
JULIUS MAXIMILIAN UNIVERSITY OF WÜRZBURG, W ÜRZBURG, GERM ANY

The design and discovery of novel drug candidates represents the initial and therefore
probably most crucial step in the drug development process. The identification of a hit and
subsequently a lead structure for further development is a very risky and expensive process.
There has been enormous progress in screening technologies and assay development,
and novel high-throughput methods are based on many biochemical and pharmacological
discoveries. Nevertheless, hit identification is in most cases based on random screening of
compound libraries, sometimes—and with increasing success rates—supported by virtual
screening methods. Classical combinatorial chemistry approaches have not yet brought
a breakthrough in the success rate of hit discovery.
But this is not the only bottleneck in the identification of a hit or lead structure. It is
well recognized that numerous diseases are not caused by a defect in one specific biologi-
cal target such as an enzyme or a receptor, but based on a plethora of biochemical
and physiological processes that often even occur concomitantly. Exogenous and/or
endogenous factors can contribute to the disease state. Examples for such multifactorial
diseases are idiopathic hypertension and neurodegenerative disorders such as Parkinson’s
or Alzheimer’s disease. It is obvious that even the best assay and most promising hit
will only address one biological target, which is probably not sufficient to efficiently fight
multifactorial diseases.
Based on these challenges the concept of “hybrid molecules” was developed and is
the topic of this book. Hybrid molecules combine two distinct biologically active molecules
that act at different targets, into one new molecule/chemical entity to combine the effects
of each molecule. Hybrid molecules are usually defined as a structure in which a linker,
often a simple hydrocarbon chain, connects the two drug molecules that are not much
altered with regard to their initial chemical structure. Other terms used in current literature
for such experimental therapeutics are “multitarget-directed compounds,” “multifunctional
ligands,” “dual-acting compounds,” “chimeras,” and others. In the pharmacological context,
the term is “polypharmacology.”
A search of the scientific database SciFinder shows increasing interest in the development
of compounds able to address multiple targets. In this analysis (Fig. 1-1) only two major
keywords (“designed multiple ligands” and “multitarget ligands”) are represented, however,
as noted above, there are numerous other terms used to describe the ability of a drug to
address more than one target.

Design of Hybrid Molecules for Drug Development. DOI: http://dx.doi.org/10.1016/B978-0-08-101011-2.00001-5

© 2017 Elsevier Ltd. All rights reserved.
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2 DESIGN OF HYBRID MOLECULES FOR DRUG DEVELOPMENT

50

40
Number of publications

30

20

10

0
00

01

02

03

04

05

06

07

08

09

10

11

12

13

14

15
20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20
Year

FIGURE 1-1 Research from 2000 to 2015 showing increased interest in multifunctional compounds. The lines refer
to the common keywords “designed multiple ligands” (blue) and “multitarget ligand” (red).

The idea is simple—at first glance. If a drug researcher wants to inhibit an enzyme for
treatment of a disease and at the same time block an equally important receptor, she or he
can covalently connect two known drugs (no screening of compound libraries is necessary)
into one molecule to get a hybrid or dual-acting compound. What’s the advantage of such a
hybrid molecule over administering the combination of enzyme inhibitor and receptor
antagonist? A new chemical entity with one specific pharmacokinetic profile is obtained and
thus drug drug interactions cannot occur. While these are clear advantages, there are also
some disadvantages to consider. For example, the chemical structure of both compounds
have in most cases been optimized beforehand, altering the compounds’ structure to obtain
the hybrid molecule can strongly affect their affinity and selectivity profiles, as well as their
metabolism. Furthermore, the individual compounds may have acted in very different con-
centration ranges, such as a micromolar enzyme inhibitor and a nanomolar ligand, whereas
a hybrid molecule usually requires activities in a similar concentration range. But even if the
researcher is successful in maintaining binding profiles, they may not be the appropriate
ones for the intended “dual” pharmacotherapy.
The connection of two molecules by a linker yields a fairly large molecule (in terms of
molar mass and H-bond accepting and donating moieties), which means that the hybrid
may not be sufficiently bioavailable anymore or lose its ability to pass the blood-brain
barrier. These problems are significant drawbacks as will be discussed in this volume.
As covered in the individual chapters of this book hybrids offer many more advantages
than easy design and simplified pharmacokinetics compared to drug cocktails. Hybrid mole-
cules can be much more than the sum of their components. If hybrids are successfully
 Chapter 1 • Introduction 3

designed the correct combination of molecules can improve affinity and selectivity profiles,
and even new pharmacological properties can be created. There are many more advantages
that will also be discussed. Furthermore, with the arsenal of modern medicinal chemistry
disadvantages such as high molar mass can be effectively addressed (e.g., by merging two
structures into one).
As can already be seen from this short introduction, realization of a successful hybrid
molecule must take into account the complexity of the disease to be tackled and the
medicinal chemical knowledge available.
In addition to the above topics, this book will also cover state-of-the-art examples of
hybrid molecule design, describe their underlying principles, and show the versatility of the
hybrid approach to fight neurodegenerative diseases, probably the most extensively covered
therapeutic area, and to develop anticancer, and antiparasitic drugs. A chapter on in vivo
data of hybrid molecules will present the first such properties obtained for hybrids.
Other areas not categorized as “hybrids” such as NO- and CO-donating molecules as well
as photoresponsive compounds, which are hybrids of drug molecules with a photoswitchable
unit, will be covered to illustrate the versatility of these approaches. Throughout the text, the
reader will be referred to the respective original articles as well as the most recent and
important data for further research.
The chapter authors as well as the editor hope that this book will be of interest to
medicinal and organic chemists, pharmacologists and molecular biologists, and drug
researchers who are interested in this fascinating and challenging topic. We hope that the
content and the considerations given in this book will inspire the design of novel and
successful hybrids in new therapeutic areas.
 2
Multitarget-Directed Antioxidants
as Therapeutic Agents: Putting the
Focus on the Oxidative Stress
Lhassane Ismaili1, Alejandro Romero2,
María do Carmo Carreiras3, José Marco-Contelles4
1
UFR OF MEDICAL AND PHARMACEUTICAL SCIENCES (SMP ), BESANÇON, F RANCE
2
CO MPLUTENSE UNIVERSITY OF MADRID, M ADRID, SPAIN 3 RESEARCH INSTITUT E FOR
MEDICINES AND PHARMACE UTICAL SCI ENC ES (IMED.ULISBOA), LISBON, PORTUGAL
4
INSTITUT E OF GENERAL ORGANIC CHEMISTRY (CSIC); JUAN DE LA CIERVA, M ADRID, SPAIN

Abbreviations
AβOs soluble Aβ oligomers
AChE acetylcholinesterase
AChEI acetylcholinesterase inhibitor
AChE-PAS peripheral anionic site of acetylcholinesterase
AD Alzheimer’s disease
APP amyloid precursor protein
BBB blood-brain barrier
CD concentration to double activity
CR congo red
Cys cysteine
ERK1/2 extracellular signal-regulated kinases 1/2
FA ferulic acid
GSH glutathione
H2DCFDA 2
,7
-dichlorodihydrofluorescein diacetate
HO-1 heme oxygenase
Keap1 Kelch-like Ech-associated protein 1
NAC N-acetylcysteine
nAChRs nicotinic acetylcholine receptors
Nrf2 nuclear factor (erythroid-derived 2)-like 2
OH-1 heme oxygenase-1
OGD oxygen and glucose deprivation
OGD/reox oxygen and glucose deprivation plus reoxygenation
OHCs organotypic hippocampal cultures
ORAC-FL oxygen-radical absorbance capacity by fluorescein
PD Parkinson’s disease
PI3K/Akt phosphatidylinositol-3 kinase/protein kinase B (5Akt)

Design of Hybrid Molecules for Drug Development. DOI: http://dx.doi.org/10.1016/B978-0-08-101011-2.00002-7

© 2017 Elsevier Ltd. All rights reserved.
5
6 DESIGN OF HYBRID MOLECULES FOR DRUG DEVELOPMENT

PKC protein kinase C

PyBOP benzotriazol-1-yl-oxy-trypirrolidinophosphonium hexafluorophosphate
ROS reactive oxygen species
Rot/olig rotenone and oligomycin A combination
SB styryl benzene
SnPP tin-protoporphyrin IX
TBDMS tert-butyldimethylsilyl
TBH tert-butyl hydroperoxide
-TC under tetracycline removal conditions
Thioflavin T ThT
Trolox 6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid
TE Trolox equiv/μM

2.1 Introduction
Melatonin (N-acetyl-5-methoxytryptamine) is an endogenous and pleiotropic molecule, phy-
logenetically well preserved, originally discovered as a hormone synthesized mainly in the
pineal gland.1 In recent years, a number of scientific reports have shown the therapeutic
value of melatonin in mental disorders and neurodegenerative diseases, cardiovascular dis-
eases, cancer, gastrointestinal pathologies or infectious diseases, among others.2 Evidence
has accumulated that melatonin is also produced in various extra-pineal organs including
immune system cells, brain, airway epithelium, bone marrow, gut, ovary, testes, skin, and
likely other tissues.3
Melatonin acts through nonreceptor-mediated mechanisms, serving as a powerful free
radical scavenger against reactive oxygen species (ROS) and nitrogen species at millimolar
concentrations,4 or by increasing the activity and expression of antioxidant enzymes, a
response observed at nanomolar concentrations.5 However, many melatonin actions are also
mediated through interaction with two high-affinity G protein-coupled receptors, named
MT1 and MT2, whose physiological functions and pharmacological properties are well docu-
mented.6 The distribution of receptors indicates a remarkable pleiotropy of melatonin, which
may potentially affect the majority of cells, and in addition to other receptor-independent
mechanisms, may help explain the different and tissue-specific functions of melatonin.6
The natural decline of melatonin levels with age in serum and cerebrospinal fluid or
deficiencies in melatonin receptor expression contribute to dysfunctions and pathophysio-
logical changes, such as neurodegenerative disorders.7 In age-related degenerative disorders
and pathologies of the brain, including Alzheimer’s disease (AD), Parkinson’s disease (PD),
Huntington’s disease (HD), and amyotrophic lateral sclerosis there are frequently interre-
lated processes (glutamate excitotoxicity, free radical-mediated damage, inflammation and
mitochondrial dysfunction) identified as common pathophysiological mechanisms contri-
buting to the vulnerability of neuronal systems. Its chronobiological effects against these pro-
cesses, added to its modulatory effects on circadian disturbances, would point to melatonin
as a therapeutic substance in the symptomatic treatment of neurodegenerative diseases.
 Chapter 2 • Multitarget-Directed Antioxidants as Therapeutic Agents 7

The most important property of melatonin administration in humans, and the one that
may eventually allow it to proceed to treatment, is its lack of toxicity, even at high doses.8
Almost daily, research articles report the efficiency of melatonin to counteract the toxic
reactions of drugs and other processes that generate ROS and associated reactants. Thus
considering melatonin’s low toxicity and high efficacy, its potential usage spectrum seems to
be wide for improving human health, either as a single molecule and/or in combination
with other drugs, in the form of hybrid ligands. However, the molecular and cellular
mechanisms underlying melatonin hybrids need to be further explored.
Ferulic acid (FA) [(E)-3-(4-hydroxy-3-methoxy-phenyl) prop-2-enoic acid)], a caffeic acid
derivative, was first isolated in 1866 from Ferula foetida. To date, more than 2000 research
articles have evidenced the role of FA as a potent free radical scavenger and cell stress
response activator, both in in vitro and in vivo experimental models. However, this phenolic
compound, besides its antioxidant capacity, displays multiple cytoprotective actions, amelio-
rating neuroinflammation in neurodegenerative diseases,9 serving as an antiproliferative
agent in the pathogenesis of cancer,10 as an antihypertensive and antihyperlipidemic com-
pound in cardiovascular diseases,11 or acting in the pathophysiology of cell dysfunction and
diabetes complications.12 In addition, the use of FA, by its multiplicity of functions, has been
tested in age-related pathologies such as AD, in particular. FA acts as a disaggregating agent
of amyloid structures to prevent the development and progression of AD,13 improve memory
loss in mouse after intracerebroventricular (icv) injection of Aβ,9 reduce inflammatory bio-
markers and cause downregulation of proteins involved in cell death processes, caspase-9,
-3, and -7, thus contributing to prevent neurotoxicity.14
For the second most common form of neurodegenerative disorder, PD, the neuroprotec-
tive effect through FA antioxidant and antiinflammatory properties has also been shown.15
Despite several reports showing the low toxicity FA, a few adverse effects have been seen
after oral administration.16 Another limitation is its low bioavailability, which requires new
alterations such as including FA by means of a convenient linker, but which results in a
multitarget-directed hybrid with enhanced therapeutic properties.
In this chapter, some multitarget-directed antioxidants are summarized in the context of
hybrid ligands with melatonin and/or FA. This field exposes new therapeutic strategies
against acute and chronic pathologies, where oxidative stress plays a pivotal role.

2.2 Melatonin Hybrids

2.2.1 Tacrine
Melatonin Hybrids
By combining the free radical scavenging activity (FRSA) of melatonin and the cholinesterase
inhibition effect of tacrine (Fig. 2-1) (the first FDA-approved drug17 for the treatment of AD,
but withdrawn shortly after its approval18 due to its dose-dependent hepatotoxicity), new
tacrine
melatonin hybrids I (Fig. 2-1) have been designed and synthesized, and their antiox-
idant profile and pharmacological properties have been assessed.19,20 These hybrids were
accomplished after modification of the substituent either in tacrine’s aromatic ring (R 5 H,
8 DESIGN OF HYBRID MOLECULES FOR DRUG DEVELOPMENT

N N

R NH
NH2 HN NH
n O N
Tacrine H
O N
H OMe
Melatonin
I R′
1 n = 5, R = H, R′ = H
2 n = 6, R = H, R′ = H
3 n = 5, R = 6-Cl, R′ = H
4 n = 6, R = 6-Cl, R′ = H
5 n = 5, R = 8-Cl, R′ = H
6 n = 6, R = 6,8-di-Cl, R′ = H
7 n = 5, R = H, R′ = OMe
8 n = 6, R = H, R′ = OMe
FIGURE 2-1 Tacrine
melatonin hybrids I described by Rodríguez-Franco.19,20

6-Cl, 6,8-di-Cl, 7-F) or in indole (Rʹ 5 H, OH, OMe) rings, or even by changing the length of
the spacer (n 5 4
7) that connects the two pharmacophoric motifs.
In Fig. 2-1 the structures of the selected hybrids 1
8 are represented. According to the
oxygen-radical absorbance capacity measured by the fluorescence method (ORAC-FL),
these compounds are potent antioxidants, showing peroxyl radical absorbance capacities
in the range of 1.5- to 4-fold, the value measured for melatonin [2.3 6 0.1 trolox equiv/μM
(TE)]. Compound 5, without any substituent on the indole ring, was the most potent
(4.0 6 0.1 TE), whereas the least potent was hybrid 7, derived from 5-methoxyindole
(1.5 6 0.1 TE). Among the unsubstituted indole derivatives, the contribution of the tacrine
fragment to the antioxidant properties could be resumed as follows: the best radical scav-
enger 5 bears a chlorine atom attached to position 8; the unsubstituted tacrines 1 and 2
displayed ORAC values of 3.6 and 3.3 TE, respectively, whereas the activity of the 6,8-
dichlorotacrine 6 and 6-chloro- derivatives 3 and 4 clearly dropped to 2.2 and 2.1 TE,
respectively. It is worth noting that the most potent antioxidant serotonin derivative
(Rʹ 5 OMe) was 8 (2.7 TE).
Furthermore, tacrine-melatonin hybrids 2, 3, and 8 displayed neuroprotective properties
in a human neuroblastoma cell line against cell death induced by various toxic insults such
as Aβ25
35, H2O2, and rotenone/olygomycin (rot/olig). In particular, hybrid 2 exerted moder-
ate (19%) neuroprotection at 0.3 μM against H2O2, and hybrid 3 gave the same neuroprotec-
tion (30%) as catalase against rot/olig at 0.3 μM. Further pharmacological analysis showed
that hybrids 1
8 have very strong inhibitory activity, ranging from 0.008 to 0.87 nM and
from 1.5 to 175 nM against human acetycholinesterase (hAChE) and human butyrylcholines-
terase (hBuChE), respectively. Moreover, hybrids 2, 3, and 8 efficiently inhibited Aβ1240 self-
aggregation in a range varying from 47% to 63%, in good agreement with the molecular
modeling studies showing that these hybrids bind both to the catalytic active site (CAS) and
 Chapter 2 • Multitarget-Directed Antioxidants as Therapeutic Agents 9

the peripheral anionic site (PAS) of hAChE. In a comprehensive study,21 direct intracerebral
administration of hybrid 2 decreased the amyloid β peptide-induced cell death and the amy-
loid burden in the brain parenchyma of APP/Ps1 mice. This reduction was accompanied by
recovery in cognitive function. Finally, the new tacrine
melatonin hybrids were assesed in
the parallel artificial membrane permeability assay/blood-brain barrier (PAMPA-BBB) test,
which showed that they are permeabale and can reach their biological targets located at the
central nervous system (CNS).
Related tacrine
melatonin hybrids22 have been designed by incorporating a carbamate at
O(C5) of the melatonin moiety linked to tacrine by spacers of different length (n 5 2
11),
leading to the new target 3-[2-(acetylamino)ethyl]-1H-indol-5-yl[4-(1,2,3,4-tetrahydroacridin-
9-ylamino)yl] carbamates. Unfortunately, no investigation has been conducted to evaluate
the antioxidant activities of these heterodimers. However, these compounds showed potent
cholinesterase inhibitory activity, with IC50 values lower than 1.18 nM for hAChE and
0.24 nM for hBuChE.

2.2.2 Berberine
Melatonin Hybrids

Berberine (Fig. 2-2) is a naturally occurring alkaloid isolated from Coptis chinensis Franch, a
Chinese herb whose roots have been widely used as traditional medicine for treating gastro-
intestinal disorders.23,24 Regarding its chemical structure, berberine has three aromatic rings
and a quaternary nitrogen that might bind to the CAS or PAS of AChE by π
π stacking and
electronic interactions, and consequently could be used as a potential inhibitor of AChE.25
Thus, not surprisingly, berberine has been associated by a convenient linker to several
known natural antioxidants such as melatonin, resulting in multitarget-directed berberine-
melatonin hybrids26 9
11 (Fig. 2-2). These compounds are excellent antioxidants, based on
their activity with ORAC-FL values of 3.1
3.78 TE, showing higher capacity to scavenge per-
oxyl radicals than berberine (0.4 TE) and melatonin (2.3 TE).
Concerning other assessed pharmacological tests, berberine
melatonin hybrids 9
11 are
submicromolar inhibitors of Electrophorus electricus AChE (EeAChE) and serum horse
BuChE (eqBuChE), as well as strong inhibitors of Aβ1
42 aggregation at 20 μM with values
ranging from 75.8% to 82.8%, which compare very favorably with those with curcumin
(52.1%) and berberine (36.3%).

O − O −
Cl Br R O
O N N
+ O + H MeO N
OMe O N H

O N
OMe OMe NH H
Berberine Melatonin
9 R=H
10 R = Me
11 R = OMe
FIGURE 2-2 Berberin
melatonin hybrids I described by Li.26
10 DESIGN OF HYBRID MOLECULES FOR DRUG DEVELOPMENT

Me Me
N OMe OMe
O R2 N H
n N H
O O N
O
N N
H H
Melatonin
II (R = H, OH; n = 1-5)

R1
R
Tamoxifen (R = H, R2 = Me)
1

4-Hydroxytamoxifen (R1 = OH, R2 = Me)

Endoxifen (R1, R2 = H)
FIGURE 2-3 Tamoxifen
melatonin hybrids II.29

2.2.3 Tamoxifen
Melatonin hybrids

Tamoxifen (Fig. 2-3) was the first selective estrogen receptor modulator to be used in the first-
line treatment for estrogen-dependent breast cancer.27 Its two clinically active metabolites,
4-hydroxytamoxifen and endoxifen (Fig. 2-3), have greater affinity toward ER-α and much
higher antiestrogenic potency in breast cancer cells than the parent drug.28 Tamoxifen and its
metabolite 4-hydroxytamoxifen have been connected to melatonin by C2
C6 alkyl linear
chains between the tamoxifen amine and the carbonyl of melatonin, resulting in hybrids II29
(Fig. 2-3), which showed improved activity in the prevention and treatment of cancer, particu-
larly breast cancer. Unfortunately, their antioxidant activities were not evaluated.

2.2.4 Curcumin
Melatonin Hybridsa

According to the multitarget-directed ligands (MTDL) design30 Zhang and coworkers con-
ceived the development of hybrids of curcumin and melatonin (Fig. 2-4)31 as a way to create
more effective neuroprotective compounds as potential AD-modifying agents. The new
chemical scaffolds retain either the functional nature of the parent compounds, or produce
new agents with distinct mechanisms of action, thus representing an interesting procedure
to identify promising hits for further improvement.
Curcumin is a yellow spice isolated from the rhizome of Curcuma longa, which has
been reported to prevent Aβ-induced toxicity, lower the level of Aβ in the brain, and
reduce the extent of inflammatory cytokines and oxidative stress.31 The expected hybrids
were designed in order to keep the structural features of curcumin and melatonin that are
essential to their pharmacological properties. Thus the phenol and the β-diketone moieties
of curcumin are important for its antioxidant, antiinflammatory, and metal-chelating
a
For a more detailed discussion on this topic, please see Chapter 8, Molecular Hybridization: An Emerging Tool
for the Design of Novel Therapeutics for Alzheimer’s Disease (“Design Neuroprotective Hybrids”, by Shijun Zhang)
in this book.
 Chapter 2 • Multitarget-Directed Antioxidants as Therapeutic Agents 11

MeO
O OH
MeO OMe
H
N
HO OH HN
O
Curcumin Melatonin

O O NH
O O NH
R1
N
H N
H
R2
OMe HO
OMe
12 R1 = OMe, R2 = OH 16
13 R1 = OMe, R2 = H
14 R1 = H, R2 = OH
15 R1, R2 = H
FIGURE 2-4 Curcumin
melatonin hybrids described by Zhang et al.31

properties, whereas the acetamide group of melatonin is important for its antioxidant and
free radical scavenging properties.
Five hybrid derivatives 12
16 (Fig. 2-4) were designed and synthesized as potential neu-
roprotectants for AD. Compounds 12
15 incorporate the β-diketone of curcumin and a
β-ketone component instead of the acetamide group of melatonin. The indole ring of mela-
tonin was also used to replace one of the phenyl rings of curcumin. Compounds 13
15, ana-
logues of 12, were designed to evaluate the importance of the 4-OH and 3-OMe substitutions
on the curcumin part of 12, since structural modifications on the phenyl ring of curcumin
may significantly affect its biological activity.
Thus the neuroprotective activities of compounds 12
15 were assessed in MC65 cells,
which represents a well-established AD model for Aβ and oxidative stress-induced cellular
toxicities under tetracycline removal (-TC) conditions.32 Compound 12 was shown to protect
MC65 cells from -TC induced cell death (B61% increase in cell viability). Removal of 4-OH
from 12 as demonstrated by compound 13 led to a complete loss of neuroprotection in
MC65 cells, while removal of 3-OMe did not affect its biological activity as compound 14
showed significant neuroprotection in MC65 cells. These results clearly indicate the 4-OH
group is essential to the neuroprotective activity of 12. This assumption is further supported
by the results of the unsubstituted analogue 15, which exhibited reduced protections of
MC65 cells. Further, compound 16 was synthesized in order to evaluate the role of the dou-
ble bond between the phenyl ring and the β-ketone. In fact, 16 showed significant and com-
parable neuroprotection of MC65 cells with 14, suggesting that the double bond and the
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12 DESIGN OF HYBRID MOLECULES FOR DRUG DEVELOPMENT

conjugation system with the phenyl ring is not necessary to produce neuroprotection for
these analogues. Hence, dose-response studies of 12, 14, and 16 determined an EC50 of
134.2 6 4.5, 23.05 6 5.23, and 27.60 6 9.4 nM), respectively, for their neuroprotection of
MC65 cells. Multiple in vitro assay results established that hybrid 16 displays moderate
inhibitory effects on the production of amyloid-β oligomers (AβOs) in MC65 cells, but not on
the aggregation of Aβ species. Compounds 14 and 16 have been shown to suppress dose-
dependent intracellular oxidative stress, with an IC50 of B63 andB68 nM, respectively. In
addition, N-acetylcysteine (NAC) and Trolox were assessed in the same conditions as com-
pound 16 for protection of M65 cells. The study demonstrated that like 16, Trolox signifi-
cantly protected cells from -TC induced cytotoxicity at concentrations as low as 10 μM. NAC
only partially rescued cell viability at 8 and 16 mM concentrations.

2.2.5 N,N-Dibenzyl(N-methyl)Amine
Melatonin Hybrids

The rationale for the present research was the design of multifunctional molecules that
might improve the potential neurogenic profile of melatonin-based hybrids, which are
created with additional anticholinergic properties. Thus, in this work, the authors
designed new melatonin-N,N-dibenzyl(N-methyl)amine hybrids 17
3033 (Fig. 2-5) by
binding two fragments with interesting and complementary properties. The melatonin
framework, besides its neurogenic profile, can display antioxidant and neuroprotective
features and might also interact with the AChE-PAS because of its aromatic character, as
was observed in the tacrine
melatonin series.20 The second selected fragment was the

MeO
N
H MeO CH3
N
HN MeO O O
O
Melatonin AP2238

R2

H N
N
CH3 R1
HN
O
17 R1, =H
R2 24 R1 = 3-Cl, R2 = H
18 R1 = H, R2 = 5-OH 25 R1 = 3-Cl, R2 = 5-OMe
19 R1 = H, R2 = 5-OMe 26 R1 = 3-Cl, R2 = 6-OMe
20 R1 = H, R2 = 6-OMe 27 R1 = 2-OMe, R2 = H
21 R1 = H, R2 = 6-F 28 R1 = 2-OMe, R2 = 5-OMe
22 R1 = 2-Cl, R2 = H 29 R1 = 3-OMe, R2 = H
23 R1 = 2-Cl, R2 = 5-OMe 30 R1 = 3-OMe, R2 = 5-OMe
FIGURE 2-5 N,N-dibenzyl(N-methyl)amine
melatonin hybrids described by Rodríguez-Franco et al.33
 Chapter 2 • Multitarget-Directed Antioxidants as Therapeutic Agents 13

protonable N,N-dibenzyl(N-methyl)amine, which is present in the AChE inhibitor

AP2238, as its interaction with the AChE-CAS has been investigated and reported.34
36
For the synthesis of N,N-dibenzyl(N-methyl)amine
melatonin hybrids, the alkylation of
differently substituted N-methylbenzylamines was accomplished with commercially available
4-(bromomethyl)-benzonitrile obtaining five 4-{[benzyl(methyl)amino]methyl}benzonitriles,
which afforded the corresponding acid derivatives after hydrolysis. These intermediate acids
were activated with PyBOP and subsequently combined with commercial tryptamines to
afford the N,N-dibenzyl(N-methyl)amine
melatonin hybrids 17
3033 (Fig. 2-5). All new
hybrids (17
30) were modest or poor inhibitors of hAChE and hBuChE, respectively, dis-
playing IC50s in the low micromolar range and showing little modification after the introduc-
tion of different substituents in the benzene or indole rings. The most active against hAChE
was unsubstituted hybrid 17 (IC50 5 1.9 6 0.1 μM), which displayed low micromolar inhibi-
tion of hBuChE (IC50 5 3.7 6 0.1 μM), whereas the introduction of any substituent in com-
pounds 18
30 penalized the enzyme-hybrid interactions by one order of magnitude and
displaying IC50s around 10 μM or higher. The experimental affinity of all hybrids 17
30 for
AChE-PAS was evaluated at 0.3, 1.0, and 3.0 μM concentrations. All compounds displaced
the propidium cation from the AChE-PAS better than 4,4
-(3-oxopentane-1,5-diyl)bis(N,N-
dimethyl-N-prop-2-en-1-ylanilinium) dibromide, a PAS ligand.37 These results suggest that
hybrids 17
30 were able to bind to AChE-PAS and thus could prevent Aβ aggregation trig-
gered by this enzyme. A concentration
response relationship was obtained for the majority
of compounds, yielding a maximum at 3.0 μM, which is a concentration very close to the
IC50 values observed for hAChE inhibition (1.9
6.8 μM). The best result was shown by
hybrid 19 with 67% propidium displacement at 3.0 μM. Other excellent results were dis-
played by hybrids 17 (51%), 27 (38%), and 29 (44%).
According to the ORAC-FL method, all hybrids 17
30 showed potent peroxyl radical
absorbance capacities ranging from 1.5- to 4.3-fold of the Trolox value and thus can be con-
sidered as potent antioxidant agents. The ORAC of these compounds is mainly located in the
melatonin-like fragment, where the presence of a phenolic group clearly yielded the best
ORAC value (18, R
5 5-OH, ORAC 5 4.3 TE). It was found that the introduction of a halogen
or a methoxy group in the indole and/or the N,N-dibenzyl(N-methyl)amine fragment penal-
ized the ORAC ability between 0.3
1.7 TE compared to unsubstituted hybrid 17
(ORAC 5 3.2 TE). The 5-methoxyindole derivatives 19 and 25 showed better ORAC values
(2.5 and 2.6 TE, respectively) than their 6-methoxy counterparts 20 and 26 (1.5 and 1.8 TE,
respectively), suggesting the involvement of the indolic position 5 in the trapping mechanism
of these hybrids.
The neuroprotective capacity of hybrids 17
30 against mitochondrial oxidative stress was
evaluated using the human neuroblastoma cell line SH-SY5Y with rot/olig as the toxic insult.
The compounds were assessed at 1.0 μM, a concentration close to their IC50 in hAChE inhi-
bition. All hybrids 17
30 displayed good levels of protection, ranging from 12% to 36%.
When derivatives with an unsubstituted N,N-dibenzyl(N-methyl)amine fragment (17
21)
were introduced with a 5- or a 6-substituent in the indole ring the protection percentage
increased, especially in the case of the 5-methoxy group. Indeed, hybrid 19, derived from
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 “Not more than usual. I am getting old, old and broken,” answered
Mr Bayre, fretfully, with a glance at his unwelcome nephew. “There
are plenty of folk who rejoice in that fact, doubtless,” he added
grimly.
Bartlett reddened, but said nothing.
“Ah, well, we must not worry ourselves upon those points,” said
Monsieur Blaise, cheerfully. “We have come to congratulate you on
having found your niece. She has returned, has she not?”
“Um, I believe so,” replied Mr Bayre, without enthusiasm. “Was it
your doing, her going away?”
“I! What a question! No. I told her to come back, and I have come
to suggest some final arrangements regarding her marriage with
me.”
“Ah!”
As he spoke Monsieur Blaise had gradually drawn his chair nearer
and nearer, and young Bayre, watching him intently, was surprised to
see a sudden change which came over his fat face when he was
close to the old man.
As for the recluse, he kept his eyes on the floor, or on the points of
his own slippers, so that he noted nothing of this close scrutiny, of
this change of expression on his visitor’s face.
 All at once, without the slightest warning, Monsieur Blaise stood
up. Bayre, still watching him, thought that he was going to denounce
his host where he stood. But instead of that, Monsieur Blaise said
abruptly, after drawing one of those stertorous breaths which the
slightest exertion evoked from him,—
“Well, I will not trouble you now. I—I will come again when you are
better. Till then, au revoir, au revoir.”
Old Mr Bayre looked up in surprise. His visitor was already at the
door, and the younger man, agitated and curious, was by this time
on his feet.
“You will have some coffee with me,” said the host, placing his
hand upon a spring-bell on a table beside him.
“No, no, my friend, another time, another time.”
Monsieur Blaise was already out of the room, and as old Mr Bayre
at once turned away and looked at the fire with no more interest in
the visitors, his nephew, with a formal bow of which his host took no
notice, followed his companion out of the room.
They passed through the handsome outer saloon, and the smaller
one, and found Marie Vazon waiting in the hall. The girl looked from
the one face to the other with sly eyes, but Monsieur Blaise said
nothing until he and the younger man were out of the house.
Half-way down the avenue he drew a long breath, took off his hat,
and wiped his bald forehead with a large coloured silk handkerchief.
His face was pale, almost haggard, and his eyes still had the same
scared expression as before.
“Mon Dieu!” cried he, “I have had such a shock! I have made such
a discovery!”
“Well, what is it? what is it?” cried Bayre, in a fever.
But the older man drew in his lips, recovered himself, and shook
his head.
“I am no spy upon my neighbours,” said he. “I would not bring
disgrace upon them, no, no matter what they had done. But—I would
not marry into that family—for fifty thousand pounds!”
 CHAPTER XIII.
PRUDENCE V. PASSION

Bayre stared at Monsieur Blaise as he stood shivering and wiping

his face with a trembling hand under the bare trees of the avenue.
That he was suffering from severe excitement was evident. All the
pink colour had left his face; his eyes looked dull and glassy. If he
had seen a ghost, or if he had been witness of some frightful crime,
he could not have looked less like the comfortable, placid Monsieur
Blaise of every day.
“Surely,” said the younger man, persuasively, “you can have no
scruples about confiding your discovery to me. Remember I am a
member of his family; I am his nephew. I am therefore the last
person who could or would help to bring disgrace upon the house.”
But Monsieur Blaise shook his head with decision.
“You say you are his nephew? Well, I don’t doubt it, I never have
doubted it since you told me so, but Monsieur Bayre did not receive
you as a relation; he did not even speak to you. Hein!”
This was undeniable. Bayre was silent.
“If you belong to his family,” went on Monsieur Blaise, after a short
pause, during which he had put on his hat and resumed his walk
down the avenue, “it is for you to make inquiries, to consult your
lawyers, if you choose. But it is not for me to interfere in the matter,
neither is it for me to discuss it. We will, if you please, converse upon
some other topic.”
But Bayre was not to be put off like this.
“Surely,” he said, “you have told me too much, or not enough. You
have confessed that you received a great shock while sitting with me
in my uncle’s presence; how can it, then, be indiscreet to admit what
discovery it was that affected you so much?”
“I may have been mistaken,” said Monsieur Blaise, beginning to
recover his normal colour, and turning his small eyes cunningly
towards his companion, as if to find out how much he had betrayed.
“I was excited, nervous. We had talked ourselves, you and I, into a
sort of feverish, suspicious state, in which trifles seemed to become
mountains. What I saw was nothing; well, what I fancied may have
been nothing too.”
Bayre tried to recall every smallest fact connected with their short
visit. His uncle had sat between the fireplace and a high window;
behind him and his table Bayre remembered that there had hung a
curtain, or piece of tapestry, which fell in deep folds. The corner was
a dark one; his own attention had been riveted upon his uncle and
Monsieur Blaise. It was quite possible that some person might have
peeped out from behind the hangings during that short interview, and
that Monsieur Blaise might have seen and recognised the face of the
intruder.
“Did you see, or fancy you saw, anyone in the room besides our
three selves?” he asked abruptly.
By the sudden access of agitation in his companion Bayre saw
that his guess was a good one.
“What do you mean? I saw nothing, nobody, no, of course not,” he
stammered out incoherently. “It is like that, monsieur, I do not see
nothing nor nobody. And I will not be interrogated as if by a judge of
instruction. If you have the desire of making inquiries you will do so
without my assistance. I have seen nothing and I will say nothing.”
And he made as if to button up his own mouth by pressing the
large, loose lips together until they looked like a long white seam.
They had reached the open road, and were about to turn to the
right, in the direction of the landing-place, when they heard certain
sounds behind them which made them look guiltily, anxiously, at
each other.
A girl’s voice that spoke in a sort of sigh, a girl’s light footsteps on
the hard road. That was all. It was with a guilty look that they met
Miss Eden when she called to them to stop. She had followed them
from the château, her hat held on with one hand and no sort of wrap
round her shoulders. She was out of breath, and her eyes were full
of distress and anxiety.
Monsieur Blaise raised his hat in silence and would have pushed
on without further greeting. But she stood in front of them, with
determination in her set face.
 “Monsieur Blaise, Mr Bayre!” cried she, passionately, “you shall
not pass me without speaking. I demand to know why you came and
why you went away so quickly. What have you learnt? What have
you found out?”
It maddened Bayre to hear the cold and cutting tone in which his
companion replied to the unhappy girl.
“Found out, mademoiselle! Found out! I do not understand. We
have found nothing out. Is there anything to find?”
She brushed aside these incoherent evasions with an impatient
gesture.
“What nonsense!” she cried passionately. “Do you forget that you
discussed my guardian’s eccentricity this very morning, and in my
presence?”
Monsieur Blaise looked uncomfortable.
“We may have done,” he said vaguely. “I do not remember. We are
all eccentric more or less.”
“Why did you come to see him?”
“Why? Why? I—I—we go to see him, to—to—ma foi,
mademoiselle, since you have developed eccentricities yourself,
since you have the habit of to disappear from your guardian’s house
without to inform your friends, I go to your guardian for to formally
renounce my pretensions to your hand.”
Bayre was furious at the coolly insolent manner in which Monsieur
Blaise made this false statement. But Miss Eden, without allowing
him to interfere, went on quietly, her temper quite unruffled,—
“Indeed? You might have withdrawn them this morning when you
met me. But then you were not in that mind.”
“Mademoiselle! You accuse me of—”
“Oh, no, no, I accuse you of nothing. But I appeal to you to tell me
why you left the house so quickly. I know from Marie Vazon—”
“Ah! What?”
Both Monsieur Blaise and Bayre awaited her answer eagerly.
“Only this, that your abrupt departure has thrown her into a state of
the greatest alarm. I found her in the hall, sobbing and screaming,
and rocking herself to and fro. Of course she wouldn’t tell me
anything, so I’ve come after you to beg you to relieve my anxiety. Mr
Bayre, surely you will speak out!”
 “Do you think I wouldn’t if I could?” said Bayre, speaking with as
much passion as she had shown herself. “I’ve come away because
Monsieur Blaise came away. I know nothing. I’m just as much in the
dark as yourself.”
She looked incredulously up into his face.
“I shouldn’t have thought you capable of deceiving me, certainly,”
said she, “in a matter so important to me. If my guardian were mad,
as you seemed to think, you couldn’t, in mercy, keep such a terrible
secret to yourself, for my sake. And if—if it were anything else, why,
surely you would give me some idea of what was hanging over us,
wouldn’t you? If you knew the frightful state of anxiety in which I’m
living!”
And her voice suddenly broke and she burst into tears.
Bayre lost his head.
“My darling, my darling, don’t! If you knew what I feel for you,
you’d never believe I could deceive you,” cried he, only conscious of
her, and not even aware of the fact that poor Monsieur Blaise,
brought thus inopportunely face to face with the girl he wished to jilt,
had slipped away and was puffing and panting along towards the
shore where the boat was waiting.
Miss Eden sobbed on.
“Don’t, don’t,” whispered Bayre, putting his hand upon the girl’s
shoulder with diffident tenderness. “Listen. I don’t know anything;
you know more than I do. I can only guess what it was that that
miserable old panting rhinoceros”—he had by this time discovered
the escape of his ponderous companion—“saw in your guardian’s
room.”
And he related the events of their short visit, and told how
obstinately Monsieur Blaise had refused to confess what it was that
frightened him.
Miss Eden did not look up, but she presently spoke from under the
handkerchief which she was pressing to her eyes. Bayre’s hand was
still hovering near her shoulder; he was still bending over her in a
coaxing attitude.
“Do you think he’s mad?” she asked.
“He gave no sign of insanity just now. It was old Blaise and I who
behaved like lunatics, running away within a few seconds of being
introduced into the room, and flying from the house as if we’d been
thieves with the police at our heels!”
Miss Eden dried her eyes and looked up.
“Yes,” said she. “And I behaved more like a lunatic than he, too.
For when I came back here two hours ago I went into the salle à
manger and found him there, and it was I who stammered and spoke
brokenly and in confusion. He watched me quietly, and asked me
where I had learnt such erratic habits, and whether I expected a
husband to put up with them when I married.”
“Did he say that?”
“Yes, quite quietly, and not as if he cared much whether I went
away or not. He asked me where I’d been, and I told him. But he
didn’t ask me why I went away, or why I came back again. He didn’t
seem to care.”
“Did he say anything about me?”
“Ye-es.”
And then she hesitated.
“Something not very complimentary?”
“He said that his young fool of a nephew had been ‘sniffing about’
the château, and, and—”
“Well?”
“He said after all you ought to be welcome to look at the outside of
his house as you’d never be a penny the better for what was inside.”
“Well, I’ve never expected to be,” retorted Bayre. “I suppose he’ll
find out that I’ve been talking to you, and he’ll be angry with you for
that.”
“I don’t care.”
“But I do. Look here. Let me go to him boldly and tell him I want to
take you away.”
“Oh, no!”
She tried to run away from him. But Bayre caught her by the wrist
and held her, and finding that she was shivering, took off his own
overcoat, and insisted, although she struggled and protested, on
wrapping her in it.
“Now,” said he, coolly, though the coolness did not extend to his
eyes, “you can’t run away without robbing me of my property. I’ll let
you go in five minutes, but you must be good and listen to me first.
You say you are miserable here—”
“Only lately. It’s all changed suddenly,” said she in a piteous tone.
“Well, you are miserable, and now that you know there’s
something wrong, and that you’ve thrown over old Blaise—”
“I didn’t. He’s thrown me over!”
“Well, well, you may congratulate yourself anyhow, I think. For
though I may not be a great match, I’m a little more presentable than
he is.”
“What you are has nothing to do with it.”
“Oh, but it has, though. Look here, Miss Eden. By-the-bye, haven’t
you got a Christian name?”
She hesitated again for a moment, and then said, in a low voice,—
“Olwen.”
Perhaps it was the fact that she was wearing his overcoat which
gave him a sort of proprietary feeling. At any rate it was with the
simplest straightforwardness that he proceeded, having learnt her
Christian name, to call her by it.
“Look here, Olwen, I’m not a rich man; I’ve not even begun to be a
successful one. Perhaps I never shall be anything but a struggling
man all my life. I tell you so frankly. Perhaps, I say. But I do feel
something in me which tells me that if I had the woman I want to
struggle for I should be so strong, so dogged, that I should make my
way in the long run; I should live for her, I should fight for her, ah!
and in the long run I should grow rich for her.”
“I don’t want to be rich,” remarked Miss Eden, plaintively, from
under the overcoat.
“Well, then, I could remain poor for her, which would be easier still.
Come, come, don’t you think I’d try to make you happy?”
“I—I don’t think my guardian would say so?”
Bayre laughed.
“Would you ask him then?”
“I should have to. You forget, or you don’t understand. Although he
hasn’t been very affectionate to me since I’ve been here—since his
own troubles, that is to say—Mr Bayre has been a good and kind
friend to me ever since my own father’s death. It is he who has paid
for my education, too, so that the little my father left should be
allowed to accumulate for me. Now you see why I feel an obligation
to consult his wishes as far as possible, and why I feel that I did
wrong in running away as I did.”
“Well, you had reason to be alarmed. You have reason still. It’s not
safe for you to be in the same house with a madman.”
“You don’t know that he’s mad! I don’t know it!”
“Well, there’s something wrong about him, something that makes
me very unwilling to leave you under the same roof with him. Yet it’s
a delicate business too. For, after all, he’s my own relation; and even
old Blaise felt a reluctance about speaking out in the case of a
neighbour and friend.”
The two looked gravely into each other’s face.
“It’s full of difficulties,” she said with a sigh. “I feel that I’m pulled
first in one direction and then in the other. Though I’ve never been
able to be fond of my guardian, I feel I owe a duty to him. And
believe me, I should never have run away, as you call it, if I hadn’t
been seized with a sort of terror of what was going on, and felt that I
must have time to think—to think by myself. And now, what has my
thinking brought me to? Nowhere. I’ve had to come back in a sort of
disgrace, and I feel that he looks upon me as a traitor.”
Bayre looked uneasy.
“If even one could trust the servants it wouldn’t be so bad,” said
he. “But I loathe those Vazons, and the two other women about the
place don’t look very intelligent.”
“They’re both in terror of my guardian, and abjectly servile to Marie
Vazon,” explained Miss Eden.
“I wonder if they know anything?”
“If they do, they won’t own it. You don’t understand these cunning
peasants as well as I do. As long as they get a living here they’ll be
content to know nothing, to see nothing, but what they are told to
hear and to see. Now, let me give you back your coat. I’m going back
to the château.”
She had assumed a very precise, matter-of-fact manner, which
Bayre had to accept as a sign that all sentimental subjects were to
be shelved for the present. He submitted in silence to take his coat.
Then she held out her hand.
“When are you going back?”
 “I don’t know yet. In the meantime, I’m staying at the house by the
landing-place.”
The look of joy that flashed over her face showed him how much
relieved she was to find that a friend upon whom she could rely was
to be so near.
But she would not give him time to profit by this discovery. With
one quick, shy, blushing glance at him she fled away in the direction
of the château, leaving him strangely excited, and yet comforted, too.
 CHAPTER XIV.
TAKE THE BULL BY THE HORNS

Bayre went straight back to the landing-place, where, as he had

expected, he found that Monsieur Blaise had pushed off without him
and was now some distance on his way back to Guernsey. He could
even see the recumbent figure of the fickle lover in the stern of the
boat; for Monsieur would not undertake the management of the tiller,
but left the business of the boat to the two men, and displayed the
better part of valour by lying with his head upon the seat and his
eyes closed, subduing qualms, both physical and mental, with what
success he could.
Bayre found lodgings, as he had proposed, at the little house of a
man who lived close to the landing-place, who made money in the
summer by bringing visitors to Creux, and filled up his time in the
winter and autumn by fishing.
The young man had only a few days’ leave from his duties in town,
and was doubtful as to whether he should be able to get an
extension, which indeed he scarcely dared to ask for. But he could
not tear himself away from the islands without some clearer
knowledge than he yet possessed on the subject of the mystery
which surrounded his uncle, on account of the effect it might have
upon Olwen Eden.
So deeply intent was he upon the solution of this uncanny mystery,
so entirely absorbed by his thoughts of the girl, that when he crossed
to St Luke’s that evening, and found a letter at Madame Nicolas’
house from Jan Repton, he felt a pang of guilt on discovering that he
had almost entirely neglected the mission on which he was
supposed to have come.
Repton’s letter ran thus:—

“Dear B.,—Southerley’s going to punch your head for you

when you get back, and I think you jolly well deserve it. Here
have we been waiting for the wire you promised to send as
soon as you found out who it was that landed us with this brat;
and I’m ready to bet you’ve forgotten all about us, and it and
everything but your own affairs!
“I hate a fellow who can’t think of anything else when he’s
got a love-affair on hand! Why, I can manage half a dozen at
a time, and never miss an appointment or forget to post a
letter!
“I don’t say Southerley isn’t as bad as you. And there’s
another thing. He’s got it into his head that this Miss Merriman
is moping because you’re away; so if you get off with a whole
bone in your body when he’s done with you, you may think
yourself lucky. And I jolly well hope you won’t, for I know very
well it was you got us into this mess, and here you’ve left us
to bear the brunt of it, and the chaff, and the rest of it! The
way that minx Susan grins and cheeks us now is intolerable.
And she has the impudence to run up when there’s a Punch
and Judy show in the street to ask if we would like her to tell
the man to give a show ‘to please the baby!’
“And there’s some idiot who’s got a room on the top floor,
who will sit with his door open singing some doggerel about
‘Molly and I and the baby!’ at the top of his voice as soon as
he hears us on the stairs.
“Southerley pretends he doesn’t care, and I don’t suppose
he would care as long as he had an excuse to go down and
talk to Miss Merriman under pretence of seeing the brat—‘the
co-operative kid’ as he calls it. But Miss Merriman seems to
be getting rather anxious at our not hearing from you, so I
suppose she’s getting tired of the bother of the animal, and no
wonder!
“What we’re going to do with it when she refuses to look
after it any longer I don’t know. But unless I get a wire from
you within forty-eight hours, I shall take it to the workhouse
myself in a brown-paper parcel and give your name with it. So
look out! Yours till the breaking of heads,

“Jan R.”
 Bayre did not quite place implicit confidence in Jan’s veracity, or
pay too much heed to his dark threats. But he thought it best to send
a telegram of a reassuring but vague character, and then he
reflected that he had really better be pushing his inquiries in the
direction Jan desired.
So on the following morning he went to the house of the Vazons,
and getting inside by a ruse, with a boy who was delivering logs for
fuel, he found himself in the presence not only of Marie Vazon of the
sly eyes, but of the baby.
And having perhaps become both more suspicious, more
observant, and more experienced of late in the matter of infants, he
had no difficulty in coming to the conclusion that the baby in the
cradle was not his cousin, but was a peasant’s child of an age more
tender than that of the hero of their adventure.
He jumped at once to the conclusion that Marie was passing this
child off as old Monsieur Bayre’s for the sake of the payment she got
from him. It was strange that a father could be thus deceived, he
thought; but old Mr Bayre was not an ordinary man, so that it was
perhaps too much to expect that he should be an ordinary father.
“What has become of Monsieur Bayre’s child?” he asked the girl
point-blank, seeing at once, by the guilty look in her eyes, that she
knew she was found out.
“This is M. Bayre’s son, monsieur,” said she, promptly.
“Oh, no, it’s not. I don’t suppose this child is more than ten or
twelve months old,” hazarded he, making a guess which was still
over the mark. “And this child’s hair is red, what there is of it, while
Mr Bayre’s son has hair almost flaxen.”
The girl frowned sulkily, and her eyes shifted uneasily from his
face to the child’s and back again.
“It’s no business of yours, at any rate,” she said defiantly, at last.
“Oh, yes, it is. You are being paid to look after one child, and you
seem unable or unwilling to tell what you’ve done with it, and you try
to pass off a much younger one for that given into your care. Such a
matter is everybody’s business, and mine especially, as I am a
member of Monsieur Bayre’s family.”
“You’d better complain to Monsieur Bayre, then,” said Marie,
sullenly.
 “That’s just what I’m going to do.”
The girl looked scared for a moment; then she shrugged her
shoulders.
“Go then, tell him, and see what thanks you get,” said she,
insolently. “Go, I say, if you dare.”
And she shot a steely glance at him out of her blue eyes.
Defiant as her manner was, Bayre detected in the girl’s face an
even greater uneasiness than he would have expected, considering
the hold the Vazons, father and daughter, appeared to have over
their nominal master. He pondered this fact as he left the cottage,
and determined to carry out his threat at once.
This he found to be impracticable, however, for on presenting
himself at the château he found himself confronted by Pierre Vazon
himself, who surlily refused him admittance, saying that Monsieur
Bayre had given strict orders that he was not to enter the house.
Thus denied, Bayre considered himself justified in further attempts
to obtain information by outside means, and after passing an uneasy
day on the island, without one glimpse either of Olwen or of his
uncle, he returned to the neighbourhood of the château after dark, in
the hope that when lighting up time came he might be able to make
more discoveries.
The great house looked desolate indeed with only a room lighted
here and there, and with whole suites in darkness. The great hall
with the long row of high windows, in which he had seen the groping
figure which he believed to have been that of his uncle tearing up the
floor-boards, had no light glimmering behind the dusky panes.
The room in which Bayre and Monsieur Blaise had been received,
and the two apartments through which they had passed on their way
thither, were equally in darkness.
But at the corner of the mansion, where the strings of dead
Virginia creeper hung over the two narrow barred windows high up in
the wall, there was a moving light behind the closed shutters.
Bayre’s attention was instantly attracted. This was the room, this
closed room at the end of the house, on the first floor, at the window
of which Olwen had seen, or fancied she saw, a woman’s hand
thrust out.
 Was it a woman who was moving about inside now? It was only
the flickering of the light above and between the cracks of the
shutters which betrayed the presence of something human within.
But slight as the indication was, it was unmistakable, and Bayre felt
that he could not rest until he should have discovered whether some
one was really imprisoned there.
He stood back on the broad path and calculated his chances of
reaching these windows as he had done those of the great hall.
But the walls here were of brick, offering no foothold, and the
creeper did not appear to be strong enough to bear his weight.
While he was considering what action to take next, the flickering
light became stationary, and remained so for some minutes.
Stepping further back to get a better look at the barred and
shuttered windows, and at the narrow slit of light above them, Bayre
presently perceived a faint glimmer appearing in like manner above
the shutters of the French windows on his right. By the flickering he
could see that while the light above the higher windows was still, that
behind the French windows was being carried about.
He crossed the path and came close to the glass, listening. For
there were certain sounds to be made out, as of the pushing about of
heavy furniture, with an occasional succession of short, sharp raps,
as of some person knocking for admittance at a closed door.
Bayre took out his pocket-knife and tried to slip the catch of the
window nearest to him. After a few attempts, during which the
sounds within became louder, he succeeded; but the slight noise he
made over this coincided with a sudden cessation of the sounds
within.
There was a rapid step across the floor, and he heard someone
breathing heavily on the other side of the still closed shutters. Then
the footsteps retreated quickly, and Bayre stood listening, shaking
the shutters gently, preparatory to making an attempt to burst them
open.
That he was on the track of the mystery at last he felt certain.
These strange nocturnal sounds, this haunting of the house by a
being who was declared by Olwen to be a woman, would be
satisfactorily explained if only he could effect an entrance now while
the disturbance was in full swing.
 So thought Bayre, and after only a few seconds’ pause he stepped
back, with the intention of dealing such a blow upon the shutters as
would probably force them open.
But before he could do this he heard a click and the fall of the iron
bar with a clanking sound against the wood, and the next moment
the shutters flew back and his uncle, with a small lantern in his right
hand, stood face to face with him.
Bayre was startled, and an exclamation broke from his lips; for he
had never seen on any human face such an expression of rage and
defiance, proud, menacing, savage, as now distorted the rugged
features, the light eyes, wrinkled cheeks, and long hatchet chin of old
Bartlett Bayre.
His voice was hoarse and broken with passion as he cried,—
“I thought so. I thought so. You rascal, you thief! It is you who play
the spy upon me, who haunt my house and listen at my doors, you,
you, you.”
And as he spoke the old man shuffled out upon the path, lantern in
hand, and shook his clenched fist in the young man’s face, panting
and husky with rage.
Bayre was taken aback. This was an unexpected turn of events,
but one which he felt he ought to have been prepared for. As it was,
the angry old man certainly appeared to have right on his side as he
stood, his face still convulsed with rage, in front of the man who had
thus been caught in an attempt at burglarious entry into his
premises.
Bayre saw at once that this loose dark dressing-gown, tied round
the waist by a frayed cord, was the very garment in which he had
seen his uncle groping on the floor of the great hall on the first day of
his investigations; the old man shivered as he stood, slippered and
hatless, with his lank and sparse grey locks ruffled by the night wind,
clutching at the sides of his collar and holding them together against
his lean throat.
“What do you want here? What do you want?” croaked out he,
after a pause of a few moments, during which his nephew reflected
upon the answer he should give to his accuser.
“I want,” cried young Bayre, boldly, suddenly resolving on the bold
course of telling the whole truth without disguise, “to know who the
woman is that you have shut up in your house.”
To his surprise, the whole demeanour of the old man changed at
once. The convulsive twitchings of his features gave place to a
sudden calmness, while he peered into the face of the younger man
with a sly intentness which prepared the other for the fact that he
had a crafty antagonist to deal with.
Coming quite close to young Bayre, and staring up into his face
with the lantern held high enough for them to see clearly into each
other’s eyes, he croaked out, in a jeering voice,—
“What’s that to you?”
Young Bayre was thunderstruck. He was prepared for denial, for
indignation, for a torrent of abuse. But this cynical speech, which he
took for an avowal, struck him dumb. The old man saw his
advantage, and went quietly on, in the same aggressive, jibing tone,
—
“What business is it of yours if I keep half a dozen women shut up
in my house, eh? Are you the master of my house, or the head of my
family, that you should interfere with me? If you’ve found a mare’s
nest, my friend, don’t come here straining your precious eyes by
looking through brick walls and wooden doors, but go to the police,
go, go, go.” And with each insulting repetition of the word the old
man thrust a skinny finger into his face. “And lay information against
me, me, me, master of Creux and benefactor to my neighbours! Say
that you, a stranger, a distant relation of the man whose property you
covet—”
“I do not covet your property. I’ve never asked you for a shilling!”
cried Bayre, hotly.
“No, because you knew very well you would never get it if you did,”
retorted the old man, grimly. “Tell the police, I say, that you, a
penniless adventurer—”
“I’m no adventurer.”
“A penniless adventurer,” repeated old Mr Bayre, his voice going
up into a squeak of rage, “have a notion that there’s a woman
concealed in my house! See what they’ll say to you, you pitiful sneak
and spy! See what honour and credit you will win for yourself by
trying to foul your own nest, to bring disgrace upon the head of your
own family!”
 Again young Bayre was for the moment dumb; the passion which
possessed the old man, the torrent of wounded pride which gushed
forth in his speech and glowed in his sunken eyes, impressed his
nephew with a sort of respect and remorse, and he began to wonder
whether he had not taken fancies, both his own and those of others,
for facts too readily.
But even as he began to hunt for suitable words in which to make
a sort of apology, there passed suddenly over the withered old face
below him an expression of cunning and malice combined which
revived all his suspicions and made him stand to his guns.
“If I’ve played the spy, sir,” he said boldly, “you’ve brought my
action upon yourself by your own outrageous behaviour. If a sane
man will behave like an insane one, if he will surround himself with
dubious people and behave in a suspicious way, he has no one to
blame but himself if a stranger to the place comes to the conclusion
that there’s something about his way of life that wants inquiring into.”
Again the old man appeared to be impressed by his words, and for
a moment he remained silent, holding his lantern hanging by his
side, and pulling the sides of his dressing-gown yet more closely
over his throat.
Then he spoke again, not angrily, not loudly, but with a keen
suspicion in his tones.
“Who told you about this woman?” he asked abruptly.
The young man hesitated. He did not wish to implicate Olwen; yet
what could he say?
He pointed suddenly to the barred windows above their heads.
“Who is in that room?” he asked sharply.
The old man turned and looked up. There was just a second’s
pause. Then he turned again, so abruptly that the lantern nearly
swung out of his hand.
“Come and see,” said he in a low voice.
And beckoning his nephew to follow him, he stepped into the
house through the open doors of the French window, and setting
down his lantern on the polished floor, barred the shutters behind
them without another word.
 CHAPTER XV.
THE HOSPITALITY OF MR BAYRE

It was an apartment which he had not yet seen in which young

Bartlett now found himself shut in with his elder namesake. More like
one of the galleries of a museum than a room in a private house, this
saloon with its panelled walls of white and gold, its pillars and its
painted ceiling, was bare in the centre, but lined all along the sides
with cabinets and show-cases, full of treasures of all kinds.
The most casual glance at the contents of these cases showed to
the least experienced eye that the collection was one of great value.
Exquisite specimens of rare porcelain, beautiful enamels, ancient
jewels of all countries, weapons of great price, treasures of lace and
of embroidery, all were represented here.
Bayre was attracted, in spite of his eagerness to solve the mystery
surrounding his uncle, by what he saw. He was just enough of a
connoisseur to appreciate and to wish to examine more closely the
rare and costly objects around him; and even as his uncle occupied
himself in replacing the heavy iron bar across the shutters, he drew
near to the first cabinet on the left hand and peered with interest and
curiosity at the carved ivories within.
The light was very bad, being supplied solely by the lantern which
old Mr Bayre carried with him and had now placed on the floor, but
the young man made out enough to prove that report had not
exaggerated the beauty and the value of the collection.
As he looked the light grew a little better, and he found old Mr
Bayre standing by his side, holding the lantern aloft. The young man
was for the moment under the impression that his uncle, softened a
little by the interest the treasures excited in his visitor, was
courteously enabling him to see them more distinctly.
He turned, pointing with one hand to one of those fantastic jewels
which mediæval art loved to devise out of huge mis-shapen pearls,
when, coming thus suddenly face to face with the owner of the
treasures, he was surprised to find that the light was held, not for him
to inspect the jewels, but for their owner to inspect him.
And again young Bayre asked himself whether this lined, haggard,
crafty old countenance, with its furrows and the lurking malignity in
its half-closed eyes, was that of a sane man or of one who was the
victim of mania.
Old Mr Bayre seemed to understand that he had in some measure
betrayed himself by the expression of his own countenance, for he
tried to laugh away the look of sudden consternation which he saw
on his nephew’s face. Showing the gaps between his yellow teeth in
a mirthless opening of the mouth which was meant for a genial
laugh, he said, in a more conciliatory tone than he had yet used,—
“Some nice things there, eh? Good things, uncommon things? Do
you know much about objects of that sort?”
“Not perhaps more than the average Londoner is bound to know
through the museums and collections he’s visited,” said the young
man, “but enough to know that you have something to be proud of
here.”
The old man laughed again, cunningly, as if with some secret
enjoyment.
“Worth some thousands of pounds, pictures and furniture and
what you see,” he said, waving his hand in the direction of the rest of
the cabinets.
“Yes,” said young Bayre, shortly.
He was surprised and disappointed to hear in the old man’s tone
something more like the pride of a tradesman at the market value of
the goods around him than the tender enthusiasm of the real
connoisseur.
Mr Bayre rubbed his chin thoughtfully, looking at his nephew out of
the corners of his eyes, and then beckoned to him to follow across
the room in the direction of a door at the further end, above which
there was an elaborate device in white and gold.
Young Bayre followed slowly, reflecting that he did not know by
what door he should be finally ejected from the building, and that this
might be his last chance of seeing the treasures through which he
was passing. His guide, perceiving that he lingered, stopped near
the door to say, impatiently and almost contemptuously,—