Novel Covalent Probes For Mapping α7 Nicotinic ...rx917k924/... · time drug discovery research...

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1 Novel Covalent Probes For Mapping α7 Nicotinic Acetylcholine Receptor Allosteric Site/s Master’s Thesis Research Defense by Vasantha Duggirala Advisor: Ganesh A. Thakur, Ph.D Department of Pharmaceutical Sciences Northeastern University November 2013

Transcript of Novel Covalent Probes For Mapping α7 Nicotinic ...rx917k924/... · time drug discovery research...

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Novel Covalent Probes For Mapping α7

Nicotinic Acetylcholine Receptor

Allosteric Site/s

Master’s Thesis Research Defense

by

Vasantha Duggirala

Advisor: Ganesh A. Thakur, Ph.D

Department of Pharmaceutical Sciences

Northeastern University

November 2013

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ACKNOWLEDGEMENTS

I would like to express my deep appreciation to Dr. Ganesh Thakur for giving me a wonderful

opportunity in his Drug Discovery lab and pursue Thesis under his guidance. I am grateful for

the quality time he gave me to develop me as a person with character as well as teach science. I

am obliged to sit through his medicinal chemistry classes for the strong concepts and realize real

time drug discovery research process which I would not have had if I chose to graduate by just

doing coursework. I thank him for having confidence in me and boosting my morale when the

project was under stake. He is one teacher who not only wishes his students should develop into

learned professionals but also into amazing individuals with self-esteem and personality. I would

like to thank Dr. Purnima Mungalachetty for her willingness to be my Thesis committee member

and spare her precious time out of a very busy schedule to review and guide me through Thesis. I

am inspired by her dedication and perseverance to excel in this field and be a leader ultimately. I

am very pleased to acknowledge Dr. John S. Gatley, my academic advisor and well-wisher from

the bottom of my heart for his confidence in me and to his valuable criticism which helped me

give my Thesis a good shape. I am thankful for his willingness to be my Thesis committee

member and guide me in the whole process. I am thankful for his good will and time to allow me

work with him in developing one of the probes in spite of his tight schedule. I am pleased to

thank our post doctoral fellow, Dr. Pushkar Kulkarni who assisted me in various difficult

situations like right from handling chemicals to troubleshooting reactions with his strong

chemistry knowledge, command over literature and judgment. I would thank him for all the

times he guided me during my thesis and provided his help in every aspect he can. I would like

to extend my heartfelt gratitude to a senior, friend, mini-teacher, well wisher and project-peer

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Abhijit Kulkarni for teaching me laboratory techniques, patiently answering my questions,

kindly help me overcome my mistakes, helping me manage time while juggling between

different activities at school and guiding through my coursework too. I am so happy to work with

such an amazing person and thank him for the quality time he spent for me. I would like to

thank other lab member and friend Ameya Ranade who shared the burden of course work and

thesis all along and help me in whichever way he could. I extend my gratefulness to Prisca

Mungalachetty, lab member, best friend, project-peer who stood by me and encouraged to never

give up at a point when I thought my project would not go ahead. I would also thank her for

assisting me in working with reactions.

I am pleased to thank Dr. Roger Kautz for kindly giving me access to use NMR and guide me

through basics and troubleshooting. I would like to thank Dr. Jim Glick and Dr. Michael Pollasri

who gave me an opportunity to access MS and LC-MS instruments whenever required for my

samples. My special thanks to Sarom Lay, administrative assistant who helped me a lot in all the

formalities related to the proposal and defense.

Finally, I would like to thank my loving parents and siblings for having faith in me to send me

overseas to live my dreams and giving me the mental support to keep up high spirit and

encourage me to keep going and finally accomplish what I for all throughout. Last but never

least I would like to extend my heartfelt gratitude to all my friends who supported me in various

ways and making me proud for what I am today.

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CONTENTS

1. Abbreviation -------------------------------------------------------------------------------------------5

2. List of figures-------------------------------------------------------------------------------------------7

3. List of schemes-----------------------------------------------------------------------------------------7

4. List of tables--------------------------------------------------------------------------------------------8

5. Abstract--------------------------------------------------------------------------------------------------9

6. Goals----------------------------------------------------------------------------------------------------10

7. Introduction -------------------------------------------------------------------------------------------10

7.1 α7 nicotinic acetylcholine receptors--------------------------------------------------------------10

7.2 Localization and neuronal expression of α7 nAChRs in human brain-----------------------12

7.3 α7 nAChRs- targets for treating cognition impairment-----------------------------------------13

7.4 Agonists and Antagonists--------------------------------------------------------------------------14

7.5 Properties of α7 nAChRs---------------------------------------------------------------------------16

7.6 First generation allosteric modulators of a7 nAChRs------------------------------------------17

7.7 Second generation allosteric modulators of a7 nAChRs---------------------------------------18

7.8 Chimeric subunit studies and site-directed mutagenesis---------------------------------------22

7.9 Molecular docking simulations--------------------------------------------------------------------23

7.10 Importance of recently discovered allosteric agonist and PAM, 4BP-TQS---------------24

8. Significance--------------------------------------------------------------------------------------------26

8.1 Ligand binding studies using covalent probes---------------------------------------------------26

8.2 Covalent probe approach---------------------------------------------------------------------------27

8.3 Study of radio-labelled ligand---------------------------------------------------------------------31

9. Schemes and chemistry -----------------------------------------------------------------------------32

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10. Results and discussion-------------------------------------------------------------------------------38

11. Conclusion--------------------------------------------------------------------------------------------42

12. Experimental------------------------------------------------------------------------------------------42

13. References---------------------------------------------------------------------------------------------56

1. ABBREVIATIONS:

ACh– Acetylcholine

nAChRs – Nicotinic acetylcholine receptors

AD - Alzheimer’s disease

ADHD - Attention-deficit-hyperactivity disorders

MS – Mass spectrometry

NAM – Negative Allosteric Modulator

PAM – Positive Allosteric Modulator

Ago-PAM – allosteric agonist positive allosteric modulator

TQS – 4-Naphthalene-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide

4BP-TQS – 4-(4-bromophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide

4IP-TQS – 4-(4-iodophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide

α-BTX – α-Bungarotoxin

MLA – Methylcaconitine

ECD – Extracellular domain

TM – Transmembrane

TMD – Transmembrane domain

CD – Cytoplasmic domain

ERK – Extra cellular signal regulated kinase

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CREB - cAMP response element binding

AChBP - Acetylcholine binding protein

5-HI - 5-Hydroxy indole

EC50 – Measure of the potency of a [compound]

uPAR - Urokinase plasminogen activator receptor

BSA - Bovine serum albumin

h- Hour

RT – Room temperature

5-HT – 5- Hydroxytryptamine

GABA – γ Aminobutyric acid

UV – Ultraviolet

THF – Tetrahydro furan

PCC – Pyridinium Chlorochromate

EtOAc – Ethyl acetate

DPT – Di-2-pyidyl thionocarbonate

DCM – Dichloro methane

EtOH - Ethanol

CPM – Counts per minute

mg – Milli gram

mmol – Milli moles

µCi – Micro curie

nCi – Nano curie

equiv – equivalents

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WR – working reagent

BCA – bicinchonic acid

2. LIST OF FIGURES:

Figure 1: Schematic representation of homomeric and heteromeric nicotinic acetylcholine

receptors

Figure 2: Ternary model of each unit of nicotinic acetylcholine receptors

Figure 3: Alpha-7 nicotinic acetylcholine receptor agonists

Figure 4: Alpha-7 nicotinic acetylcholine receptor antagonists

Figure 5: First generation PAMs of α7 nAChRs

Figure 6: Second generation Type I PAMs of α7 nAChRs

Figure 7: Second generation Type II PAMs of α7 nAChRs

Figure 8: Intermediate PAMs of α7 nAChRs

Figure 9: Comparison of peak responses of TQS and 4BP-TQS when co-applied with ACh

Figure 10: Photolysis of phenyl azide used as covalent probe

Figure 11: Photolysis of phenyl diazirine used as covalent probe

Figure 12: Isothiocyanate used as an electrophilic affinity covalent probe

Figure 13: Brain image of mice after autoradiography

3. LIST OF SCHEMES:

Scheme 1: General scheme for the synthesis of 4BP-TQS analogs by microwave-assisted

Povarov reaction

Scheme 2: Synthesis of 4-azidobenzaldehyde

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Scheme 3: Synthesis of 4-(4-isothiocyantephenyl)-3a,4,5,9b tetrahydro-3H-

cyclopenta[c]quinoline-8-sulfonamide from 4-(4-amino butyloxy carbonyl phenyl)-3a,4,5,9b

tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide

Scheme 4: Synthesis of 4-(4-isothiocyantephenyl)-3a,4,5,9b tetrahydro-3H-

cyclopenta[c]quinoline-8-sulfonamide from 4-(4-nitro phenyl)-3a,4,5,9b tetrahydro-3H-

cyclopenta[c]quinoline-8-sulfonamide

Scheme 5: Synthesis of 4-(4-isothiocyantephenyl)-3a,4,5,9b tetrahydro-3H-

cyclopenta[c]quinoline-8-sulfonamide from 4-(4-azido phenyl)-3a,4,5,9b tetrahydro-3H-

cyclopenta[c]quinoline-8-sulfonamide

Scheme 6: Synthesis of 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzaldehyde

Scheme 7: Synthesis of 4-(4-azido-2,3,5,6-pentafluorophenyl)-3a,4,5,9b-tetrahydro-3H-

cyclopenta[c]quinoline-8-sulfonamide

Scheme 8: Synthesis of [125

I]4-(4-iodophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-

8-sulfonamide

4. LIST OF TABLES:

Table 1: Summary of microwave-assisted Povarov reaction conditions and yields

Table 2: Result of electrophysiological study of covalent probes

Table 3: List of CPM/mg tissue of each brain section

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5. ABSTRACT:

Alpha7-nicotinic acetylcholine receptor (nAChRs), members of ligand-gated ion channels are

becoming attractive targets considering their therapeutic potential for the treatment of cognitive

deficits associated with schizophrenia, Alzheimer’s Disease (AD), Parkinson’s disease, and

attention-deficit/hyperactivity disorders (ADHD)1-3

, as well as inflammation4 and neuropathic

pain.5 Alpha7 nAChRs can be activated either by ligands binding at orthosteric site or allosteric

site/s along with enhancing the agonist evoked response. But, activation via orthosteric site

caused rapid desensitization of the receptors upon exposure to agonist for prolonged time, also

under diseased condition like AD the endogenous cholinergic tone is reduced. As an alternative

approach, allosteric potentiators are being emphasized. These allosteric ligands regulate nicotinic

cholinergic neurotransmission either in a positive or a negative way by binding to a site distinct

from orthosteric site. Some of them only potentiate agonist-evoked responses (Type I) while

others potentiate responses as well as decrease desensitization kinetics of the receptors (Type II).

Among the Type II positive allosteric modulators (PAMs) of α7 nAChRs, 4BP-TQS (4-(4-

bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) was found to

have unique allosteric agonist activity in addition to being a potent PAM. It increased the scope

of allosteric approach to a higher level. But with the lack of knowledge about structural features

of ligand-receptor binding site, it is difficult to design potent and efficacious ligands. Mechanism

of action, location of binding site and amino acids interacting at the site are all under question.

As the alpha-7 crystal structure is not available, alternative methods are being considered to

discover the structural features. One of the most reliable alternatives being the use of covalent

probes, this approach was taken ahead using 4BP-TQS scaffold. Covalent probes can be derived

by incorporating reactive groups at the judiciously selected positions of the allosteric ligands to

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help determine the amino acids involved in the binding of the molecule and their role in the

activity can be deduced. Photoreactive groups such as a azido or trifluoromethyl diazirine as well

as electrophilic groups such as isothiocyanate were incorporated at the para-position to the

phenyl ring of 4BP-TQS which was demonstrated to be a critical position for ago-PAM activity.

Identifying key amino acids would aid in mapping the allosteric binding site of α7 nAChRs.

Radio-labeled ligands have always been effective tools in understanding ligand-receptor

interactions like binding, affinity, etc. Radio-labelled ligands at orthosteric site have been

developed and successfully used in research based on acetylcholine receptor binding protein’s

crystal structure. In this work, I have also synthesized a radioiodinated analog (125

I) of the active

enantiomer (GAT164) of 4IPTQS, a potent ago-PAM of a7 nAChR to study the drug-receptor

binding interactions at this distinct site.

6. GOALS:

1) To develop covalent probe of 4BP-TQS to aid in mapping the α7 nAChR allosteric

binding site(s)

2) To develop a radio-labeled allosteric ligand that can bind and aid in understanding the

drug-receptor interaction

7. INTRODUCTION:

7.1 Alpha 7 nicotinic acetylcholine receptors:

Nicotinic receptors belong to a super family of ligand-gated ion channels present in the central

nervous system (CNS) and the peripheral nervous system (PNS) that mediate the effects of

acetylcholine in the parasympathetic nervous system. Activation of nicotinic receptors in the

brain releases neurotransmitters like dopamine, serotonin, glutamate and GABA6. These

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receptors are one of the most significant integral membrane proteins with pentameric structures

consisting of five types of subunits α2-α10, β2-β4, δ, γ, and ε

arranged either in a heterologous or homologous manner around a

central pore7, 8

. α7 and α8 subtypes are capable of forming

functional homomeric nAChRs and α7 are the important

homomeric subtype in mammalian brains. α4β2 and α7 are the

two important nAChRs in the brain which can be targeted for

cognitive enhancement9. The former has high affinity to nicotine

and ACh than the latter10

and therefore, targeting α4β2 would

enhance the addiction properties and the heteromeric nature

would increase the difficulty for target-specific drug design. So,

targeting α7 would be ideal. The CHRNA7 gene that encodes for the α7 subunit is thought to be

the ancestral gene11

and it is likely that the nAChR family must have emerged from the same

gene after genetic duplication and variations along with additional genes encoding.12

As a

common feature, nAChR subunits have an amino-terminal extracellular domain (ECD), a trans-

membrane domain (TMD) and a cytoplasmic domain (CD). The TMD is comprised of TM1-

TM4 folded as α-helices. They have ligand binding sites at the interfaces of α-subunit and non-α-

subunit13

and the allosteric binding domains are present distantly from primary orthosteric

binding sites12

. As the number of equivalent orthosteric sites depend on the subunits present (2-

5), heterologous receptors like α4β2 have unequal number of agonist binding sites with different

affinities for ligands.14

Hence when they co-exist within a particular oligomer, it’s a challenge

for targeted drug design.15

Whereas in case of α7 nAChR, all the binding sites are at the interface

of α-subunits and have similar affinities for the orthosteric ligands. Receptor subtype

Figure(1) Schematic representation of homomeric and heteromeric nAChRs

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combinations also differ in their pharmacological and kinetic properties depending on the subunit

combinations and localization. Alpha 7 nAChRs are homomeric receptors with five α subunits

and equal number of binding sites on them extra-cellularly, they can also be modulated by

allosteric ligands. As neuronal nicotinic receptors have been implicated in a number of

neurological and psychiatric disorders, there is growing interest in exploiting nicotinic receptors

to treat Alzheimer’s disease, schizophrenia, depression, attention deficit hyperactivity disorder

(ADHD)1-3

, as well as inflammation4 and pain.

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7.2 Localization and neuronal expression of α7 nAChRs in human brain:

Though there are many drugs for treating nervous-system

diseases, there is an unmet medical need for treating disorders

related to cognition impairment. Cognition impairment is a

growing concern as it is prevalent with most of the

neurological and psychiatric diseases as well as aging.

Cholinergic system is closely related to cognitive function; and

α7 nAChRs are localized in the hippocampus and cortex which

are the main cognition domains. Radiolabeled binding studies

using α-bungarotoxin (BTX) - selective α7 nAChR antagonist

confirmed that α7 nAChR are localized in rat hippocampus and

cortex.16, 17

α7 nAChRs are found in reticular nuclei of the thalamus. Thalamus upon receiving

excitatory signals from cortex, transmits inhibitory signals to dorsal thalamus nuclei.18

Reticular

nuclei mainly mediate attention and sensory gating. Enhancing the activation of α7 nAChRs

would modulate these two important cognitive processes.

Figure(2) Ternary molecular model of each subunit of nAChRs

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Alpha 7 nAChRs are present both post- as well as pre-synaptically. Pre-synaptically they

regulate neurotransmitter release and post-synaptically they regulate the receptors localized on

the GABAergic interneurons in the cortex and hippocampus where they, stimulate signaling

cascades intracellularly and affect downstream processes which control learning and memory.9

Alpha 7 nAchRs are highly permeable to Ca2+

in main brain regions which are significantly

involved in cognitive impairment. Ca2+

is involved in mediating signaling cascades, enhancing

synaptic plasticity and controlling Ca2+

levels prevents cell death due to excitotoxicity. The post

synaptic Ca2+

release takes care of the neuronal circuitry and maintains synaptic plasticity there

by influence learning and memory.19

7.3 Targeting α7 nAChRs for treating cognition impairment:

Conventionally for AD, acetylcholinesterase inhibitors like galantamine, donezepil,

rivastigmine and tacrine are used to enhance acetylcholine’s effects by inhibiting enzyme

cholinesterase thereby increasing the concentration of neurotransmitter acetylcholine in the

synapse. But their use has shown only moderate to low benefits. Donezepil was the only drug

which was recommended for advanced AD dementia in older patients for recovering cognitive

deficit to some extent.20

But, cognition is a complex mental process which includes attention,

memory, understanding, processing, problem solving and decision making which necessitates

more potent and selective drugs to take control of the symptoms. In this regard, drugs that

activate α7 nAChRs can improve performance of various cognition domains like episodic

memory, working memory, attention, and sensory gating.21, 22

Episodic memory is that which

records situational information like time, date, emotion, place etc of any particular instance and

involves hippocampus of the medial temporal lobe of the brain.9 Working memory is short term

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memory which helps in completing a task and it may decline due to the disease especially in

schizophrenia, aging or stress. It involves prefrontal cortex region where cholinergic system

mediates working memory function.9 Similarly, attention is also mediated by the nicotinic

systems in the prefrontal cortex and attention deficit is an underlying cause for ADHD and

schizophrenia. Sensory gating deficits are found significantly in schizophrenia which inhibit the

filtering process of redundant stimuli from the environment and involves the cortex region.9

Activating α7 nAChRs can restore the auditory gating by increasing GABAergic

neurotransmission in schizophrenic patients.23, 24

α7 nAChR agonist, A-582941 enhances

memory by increasing neurotransmission and triggering intracellular signaling cascades like

extracellular signal regulated kinase (ERK) ½ phosphorylation in PC12 cell line and

phosphorylation of transcription factors like cAMP response element binding (CREB) in mice.25

7.4 Agonists and Antagonists:

An archetype nAChR agonist is nicotine which has proven therapeutic performance in improving

cognition, reducing anxiety, normalizing sensory gating and neuroprotection but due its non

selectivity, side effects like seizures, gastrointestinal effects and hypertension were quite

evident.26-30

Many naturally occurring compounds like lobeline, epibatidine were identified to

have agonist activity at α7 nAChRs. Based on the scaffolds of nicotine and epibatidine, novel

compounds were researched, out of which TC-1698 emerged as a successful full agonist which

exhibited neuroprotection through nicotine-induced JAK-2/PI-3K cascade.31

Research was then

focused on finding selective potent α7 nAChR agonists. GTS-21 and its hydroxyl derivatives

were few of the first selective partial agonists which showed enhanced working memory but had

their activity through the α4β2 receptor.32-34

Further, more efficacious agonist like the

quinuclidine derivative, AR-R17779 was found to be a full agonist at α7 nAChRs with nootropic

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potential.35, 36

But it still had activity at 5HT3 receptor due to homologous nature. Extended

research resulted in finding other quinuclidine analogues like PNU-28298737

, SSR 180711A38

and PHA-543613 which had similar activity.39

A-582941 was another selective agonist which

improved cognitive performance in various domains.25

Similarly TC-5619, a partial agonist

developed and tested by Targacept for treating Schizophrenia and ADHD is currently in Phase II

clinical trials. Recently, RG3487 a partial agonist by Roche group was shown to improve

attention and was used in ADHD.40

Although many successful attempts were made to develop

selective potent agonists, the intrinsically low probability of opening of α7 nAChRs, fast

desensitization upon on high occupancy of agonist binding sites and disturbed endogenous

cholinergic tone prompted the need to go for the alternative approaches one of them being

allosteric modulation.9

Alpha 7 nAChRs have two key selective antagonists which are mainly obtained from natural

sources. One is α-bungarotoxin (α-BTX) obtained from venom of Taiwanese snake which

inhibits ion flow into post synaptic receptors,41

and the other is a diterpenoid alkaloid,

methyllycacotinine (MLA) obtained from many species of Delphinium. It was found to have a

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neuroprotective activity in AD by inhibition/desensitization.42

Other antagonists are α-conotoxin,

memantine which showed moderate-mild effect on AD.

7.5 Properties of α7 nAChRs:

The functional features of α7 nAChRs are that they have low sensitivity to agonists ACh and

nicotine, they can be rapidly activated and quickly desensitized upon prolonged exposure to

agonist.43

A typical neuronal nAChR protein was defined by Monod, Wyman, Changeux (MWC)

allostery model. 44

It exists in three basic receptor states- closed resting state in the absence of

agonist, open active state upon exposure to agonist and closed desensitized state as the receptor

is stabilized due to long exposure to agonist and the attain resting state even without re-

activation.45, 46

Later, many theories were proposed to explain the number of energy states and

nature of desensitized states upon agonist/PAM activation. Co-crystal structures of the

acetylcholine binding protein (AChBP) with orthosteric ligands (agonists like nicotine, choline,

ACh; antagonists like snake α-neurotoxins) showed that they bind in the ECD at the interface of

MLA Memantine

Figure 4. Alpha-7 nAChR antagonists

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two subunits.47-49

Orthosteric ligand binding stabilizes the receptor in its active state. Allosteric

modulators bind to a distinct site from that of the orthosteric site and modulate the effects of the

orthosteric ligand. The protein is suggested to exist in multiple conformational states and

allosteric ligand binding would change the conformation and stabilize it in a preferred

conformation.44

For nAChRs, agonists stabilized the receptor in its open active state but

competitive antagonists stabilize it in closed state. When allosteric modulators bind to the

receptor, they modulate energy barrier between different conformational states displacing the

equilibrium which can be measured using isomerization coefficients. Those effectors which

reduce the energy barrier between closed resting state and open active state and increase agonist-

evoked response are called positive allosteric modulators (PAMs). Those effectors which

increase the energy barrier between the states and reduce the agonist response are called negative

allosteric modulators (NAMs).12

Allosteric modulation is an alternative approach to direct agonism especially in patient

population where smoking is prevalent which might interfere with direct agonist approach. 60-

70% schizophrenic patients smoke as it enhances their brain activity eventually get addicted to

nicotine. Nicotine competes with orthosteric agonists in the extra cellular domain where as

PAMs avoid competitive interactions at the orthosteric site with nicotine. Alpha 7 nAChR PAMs

activate the receptors in the presence of endogenous agonist, ACh there by managing phasic

stimulation. PAMs have an advantage over orthosteric agonist that they might not cause

desensitization following stimulation and avoid loss of function.9

7.6 First generation allosteric modulators of a7 nAChRs:

Ivermectin was the first PAM identified to activate α7 nAChRs.50

It was found to increase the

peak agonist-evoked current and Hill coefficient, reduce EC50 of ACh and desensitization time

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course. Another non-selective PAM identified was 5-hydroxy indole (5-HI) which increased

agonist-evoked peak current but is weak and non-selective, hence it needs high concentrations to

activate α7 nAChRs.51

A tyrosine kinase inhibitor, genistein was identified to increase agonist-

evoked peak current similar to 5-HI.52

Nicotinic receptors are also targets for many endogenous

divalent cations like Ca2+

which potentiate the receptor by binding near the N-terminal domain in

the extra cellular space as shown by studies of subunit chimeras between nAChRs and 5-HT3

receptors and by site directed

mutagenesis.53

Endogeneous proteins and

peptides are also found to act as allosteric

modulators at the α7 nAChRs. SLURP-1,

mammalian Ly-6/urokinase plasminogen

activator receptor (uPAR) related protein secreted by human keratinocytes act as α7 nAChR

PAM.54

Other secreted proteins like lynx-1 and 2 found in circulation modulate nAChRs. These

proteins interestingly have homology with the snake toxin α-bungarotoxin, which itself is a

selective antagonist at α 7 nAChRs.

7.7 Second generation a7 nAChR selective PAMs

Allosteric modulators of α7 nAChRs are divided into two types depending on their

pharmacological profile. Type I PAMs increase the agonist-evoked response and affect peak

current.12

In other words they reduce the energy barrier for transition into active states without

changing the actual difference in energy between the states and Popen is increased transiently upon

agonist application.55

Type II PAMs not only increase the agonist evoked response but also

change the desensitization profile of the receptor response.12

In other words, they increase the

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response amplitude and prolong the response by altering the equilibrium energy levels between

states along with restoring the previously desensitized receptors.55

TYPE I PAMs:

Taking the advantage of homology between nAChRs and GABAA receptors , modulators that

activated GABA were tested for α7 nAChR PAM activity and compound 6, N-4-chlorophenyl)-

a-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazole acetamide was found. It enhances

the current evoked by ACh without any change to the desensitization profile and has efficacy for

treating various cognitive disorders.56

NS-1738, an analog from the biarylurea series showed

PAM activity at α7 nAChR

when tested to improve

recognition memory

performance in rats (Morris

water maze test - a hippocampal

learning and memory model).

NS-1738 reversed muscarinic

antagonist scopolamine-induced

learning impairment.57

Galantamine, an acetylcholine

esterase inhibitor was discovered

to act as type 1 PAM which moderately potentiate release of acetylcholine in hippocampal

neurons and thus could be used in treating AD.58

It was shown to bind at a position at N-terminal

ECD close to agonist binding site. LY-2087101 is a recently identified PAM of α7 nAChRs,

which is less selective for α7 nAChRs. LY-2087101 has little effect on the time course of

agonist-evoked responses, a feature that is typical of type I PAMs59

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TYPE II PAMs:

PNU-120596 is the well-known and well characterized type II PAM. It was found to restore

agonist responses in desensitized receptors formed due to prolonged agonist exposure. In

addition, its application during

prolonged exposure to the

agonist is able to restore a peak

current even larger than the

peak current evoked by the

agonist alone.60

Though PNU-

120956 and NS-1738 are urea

derivatives, NS-1738 has a

type I PAM profile whereas

PNU-120596 showed type-II PAM profile, which suggests that they may have distinct

mechanisms or sites of action. PNU-120596 is a selective potentiator of α7 nAChRs, with little

or no activity on other nAChR subtypes. A tetrahydroquinoline analog, 4-naphthalene-1-yl-

3a,4,5,9btetrahydro-3-H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS) was shown to be

effective treating cognition impairment. TQS increased peak current and also showed down

desensitization of peak current responses.61

Unlike TQS, its analog 4-(4-bromophenyl)-

3a,4,5,9btetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (4-BPTQS) is an allosteric

agonist which shows agonist-induced peak currents in the absence of agonist binding at

orthosteric site.62

It is called an allosteric agonist-positive allosteric modulator or ago-PAM and

is the most potent α7 nAChR ligand.

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Intermediate PAM:

2-[[4-fluoro-3-(trifluoromethyl) phenyl]amino]-4-(4-pyridinyl)-5-thiazolemethanol (JNJ-

1930942) is a novel compound added to a new PAM class of

compounds which shows properties intermediate to that of type

I and type II PAM classes. It is highly selective, efficacious

allosteric modulator at the α7 nAChRs and increases the

agonist evoked responses but doesn’t activate the receptor in

the absence of orthosteric agonist. The enhanced agonist action

is accompanied by decrease in desensitization kinetics like the type II PAMs. Thus, it is called an

intermediate PAM. It is shown to increase the synaptic transmission and aid long term

potentiation effect in rat hippocampal slices and also normalize sensory gating deficits in-vivo.63

α7 nAChR NAMs:

Negative allosteric modulators are those which increase the energy barriers between different

conformational states of a receptor by binding at allosteric sites and inhibit receptor potentiation.

In this class of compounds for α7 nAChRs- m-Chlorophenylguanidine (mCPG; MD-354) was

identified as the first small–molecule NAM of α7 nAChRs (IC50 = 7.98 µM) although it is non-

selective.64

MD-354 was shown to block the antinociceptive action of (-)nicotine by acting on

nAChRs. But binding studies proved that it lacked affinity for most abundant α4β2 to antagonize

nicotine, whereas it had affinity for α7 nAChRs. Moreover, these studies proved that its action is

through allosteric site(s).65

Later, 1,2,3,3a,4,8b-hexahydro-2-benzyl-6-N,N-dimethylamino-1-

methylindeno[1,2,-b]pyrrole (HDMP) was found to act as a potent subtype selective NAM (IC50

= 0.07 µM).66

Further investigation is being done currently to produce a class of selective α7

nAChR NAMs.

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7.8 Alpha7/5HT3 functional chimeric subunit studies and site-directed mutagenesis:

Though the α7 nAChR PAMs were explored for their therapeutic potential, limited information

is available regarding their mechanism of action. Studies were conducted using previously

constructed and expressed series of functional subunit chimeras between α7 nAChR and 5-HT3

receptors based on their sequence homology67, 68

and site directed mutagenesis studies were

conducted. Studies using two PAMs PNU-120596 and LY-2087101 concluded that the TM

regions of α7 are critical for the activation of nAChRs. In that, two amino acids present in TM1,

one in TM2 and two in TM4 were identified which when mutated reduced the potentiation of α7

nAChRs. The presence of an allosteric site within the intra subunit cavity within α-helical TM

chains seemed much similar to the site where neurosteroids and volatile anesthetics interact with

GABAA and glycine receptors. Two of the amino acids identified in this study, M253 (in TM2)

(methionine) and C459 (in TM4) (cysteine), lie in positions exactly analogous to amino acids

that have been identified as influencing the binding of allosteric modulators to GABA or glycine

receptors.69

PNU-120596 is a subtype selective type-II PAM and LY-2087101 is less selective

type-I PAM. Electrophysiological studies using Xenopus oocytes showed that TM1-TM3 are

important for potentiating the α7 nAChRs. Several specific amino acids present in these TMs

were mutated to find altered potentiation effect by PNU-120596 out of which S222M, A225D,

M253L, F455A, and C459Y mutations caused low responses. Especially A225 in TM1 or M253

in TM2 showed a drastic change in the activity of PNU-120596. Also, F455 (phenylalanine) and

C459 (cysteine) amino acids present in TM4 played a significant role as shown in mutagenesis

studies. As A225D and M253L showed a substantial decrease in PNU’s activity, scientists

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experimented the same with LY-2087101 and observed similar results of significantly reduced

potentiation.69

7.9 Molecular docking simulations:

Three α7 subunit homology models70, 71

obtained from the 4Å resolution structure of the Torpedo

nAChR72

revealed an intrasubunit cavity as a binding site. Further, considering the ability of

PAMs to bind in the intra subunit cavity, computer docking simulations were performed using a

“blind docking” approach. The above mentioned amino acids were found to be located centrally

and the side chains pointing towards the centre of intra subunit cavity between the α- helical

TMs.69

Both models suggested, PNU-120596 and LY-2087101 were found to bind within 6Å proximity

to the five key amino acids found by site-directed mutagenesis studies in their lowest binding

free energy state with high affinity.69

Further, a third open channel structure homology model

developed based on Taly et al. showed better affinity model than closed structure model

described before.73

In that, higher affinity was shown by both PAMs when the amino acid side

chains had flexibility.69

It is thought that PAMs might reduce the energy barriers between

different conformation states of the receptor or reduce the desensitization kinetics. Interestingly,

in the most favorable energy state, PNU-120596 and LY-2087101 oriented in a same manner by

positioning isoxazole heterocyclic group of PNU-120596 and thiophene heterocyclic group of

LY-2087101 in close proximity to F455 present in TM4 and their larger halogenated aromatic

group positioned higher in the intrasubunit cavity.69

Overall, with the site-directed mutagenesis

the five mutations (S222M, A225D, M253L, F455A, and C459Y) caused the most significant

decrease in the potentiation by PNU-120596. Hence these amino acids are crucial for ligand

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binding in between the α-helical TMs and are supported by homology modeling and computer

docking simulations.

7.10 Importance of recently discovered allosteric agonist, 4BP-TQS:

The most recent discovery in the class of allosteric modulators of α7 nAChR was 4BP-TQS

which has close structural similarity to TQS.62

Though TQS was a conventional type II PAM,

4BP-TQS was found to be a potent allosteric modulator along with agonist activity called ago-

PAM. While conventional agonists are competitively antagonized by metyllycaconitine (MLA),

it is a non-competitive antagonist of 4BP-TQS. 4BP-TQS, like other PAMs binds at the

transmembrane allosteric site to potentiate nAChRs in the absence of agonist at the orthosteric

site.62, 69

TQS has the ability to reactivate desensitized α7 nAChRs only when co-applied with

acetylcholine whereas, 4BP-TQS nearly showed no evidence of desensitization which can be

observed graphically by slow onset and slow response to reach a plateau (fig 9).

Dose-response curve with 4BP-TQS was shown to be substantially steeper than that with

acetylcholine.62

Though the responses obtained using either 4BP-TQS or TQS co-applied with

acetylcholine were different in their kinetics and extent of receptor desensitization, they throw

Figure 9. a) co-application of TQS with ACh shows prolonged potentiation, increased peak response and

reactivation of desensitized receptors. b) even in the absence of agonist activation, 4BP-TQS shows no

desensitization. It shows slow onset and prolonged time to reach a plateau (Ref 62)

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light on difference in the mechanism of action to activate α7 nAChRs. The point mutations

mentioned earlier that affected PAM activity of PNU-12059669

were hypothesized and proved to

affect 4BP-TQS as well. Mutation studies were performed to investigate the binding site of 4BP-

TQS in reference to agonist acetylcholine. A point mutation, M253L in the TM2 region

dramatically affected the agonist response of 4BP-TQS (complete loss) whereas no significant

effect was observed in the agonist response of ACh. A point mutation W148F in very close

proximity to the orthosteric site, brought a significant rightward shift in the agonist acetylcholine

dose-response curve when compared to wild-type, whereas rightward shift in that of 4BP-TQS

was very less.62

MLA which is a competitive antagonist of agonist ACh on α7 nAChRs acts as a

non-competitive antagonist of 4BP-TQS. This suggests that irreversible antagonism of 4BP-TQS

by MLA is because MLA binds at the orthosteric site and might change the receptor

conformation making it unfavorable for 4BP-TQS binding or MLA might stabilize the receptor

in its closed state. In contrast, when 4BP-TQS was antagonized by α-bungarotoxin the responses

were not readily reversible. 4BP-TQS when tested to potentiate acetylcholine responses, its

response was 41 ± 5- fold larger than that of a maximal concentration of acetylcholine applied in

the absence of 4BP-TQS. In addition, there was no evidence of desensitization too like type II

PAMs.62

Overall, it is expected that 4BP-TQS binds in the allosteric site similar to that observed in case of

PNU-120596, LY-2087101 and TQS as shown by site-directed mutagenesis. Furthermore,

computer docking simulations done using α7 nAChR homology model suggest that 4BP-TQS

also binds in the intra subunit cavity within the TMD but closer to the inner face of M2 helix in

very close proximity to M253.62

It was thought that in the absence of agonists, the receptor

underwent spontaneous opening and receptor stabilization might occur with an open

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conformation when ligands bind at distinct sites. Interestingly, a point mutation L247T brought

about spontaneous openings of the receptor which resulted in TQS being able to act as an agonist

like 4BP-TQS on wild-type α7 nAChRs. Though 4BP-TQS has therapeutic advantages

therapeutic over other PAMs, excessive receptor activation might lead to excitotoxicity due to

excessive Ca2+

influx.62

8. SIGNIFICANCE:

8.1 Ligand binding studies using covalent probes:

Efforts made till date in searching the allosteric binding site/s made it evident that TM1-TM3 α-

helices of the TMD are involved in the binding of PAMs. As mentioned above, different PAMs

showed different pharmacological responses. In addition, it is evident in case of PNU-120596

and LY-2087101 that variation in the binding site or even amino acids that interact while

binding, change its mechanism of action and thus the pharmacological profile of the compound.

Hence, knowledge at molecular level is extremely important to assess a compound’s PAM/ago-

PAM activity at α7 nAChRs.

Conventional methods to elucidate structure include X-ray crystallography, mass spectroscopy

and nuclear magnetic resonance spectroscopy. They require isolated purified form of the protein

specifically which is a tough task. Site directed mutagenesis facilitates in identifying the amino

acids involved and their role in the interaction which was performed previously, which resulted

in identifying five afore-mentioned amino acids. Although, it is a powerful tool, there are a

number of risks involved when amino acids or portions of protein are replaced by others. A

mutation might bring conformational changes at a distant site which might not be apparent;

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mutation might affect protein properties and/or bring new properties unrelated to original

protein. In that case designing of site-directed mutants/chimeras becomes extremely important.

In order to avoid erroneous results and/or wrong interpretation, mutagenesis studies should be

combined with other methods like molecular modeling. As an alternative method to acquire the

structural details of a ligand-receptor interactive site, is the development of covalent probes,

photo/radio affinity labels capable of binding to the ligand binding site/s. Previously,

photolabelling studies have been done for nAChRs using tritiated benzophenone and

azietomidate to identify binding sites within nAChRs, which resulted in revealing sites at the

interface of subunits in the ECD, within TMs and near CD. 74, 75

But they weren’t specific to α7

nAChR allosteric site/s. With the recent discovery of 4BP-TQS and its advantages as discussed

above, it is necessary to elucidate the structure of its binding site for the consequential

therapeutic significance. This thesis includes design, synthesis, characterization and

electrophysiological evaluation of the developed covalent probes.

8.2 Covalent probe approach:

In this approach, reactive moieties are incorporated at the different sites of the ligand which are

capable of reacting with amino acid residues at the receptor binding site(s). Information obtained

from this approach is confirmed using computer docking models and site directed mutagenesis

which gives comprehensive knowledge of ligand binding sites of different classes of ligands-

agonists, antagonists, and allosteric modulators. This method is used to identify key amino acids

that are actually involved in the drug-receptor interaction at the allosteric binding site. From the

data obtained, three dimensional structural features of the allosteric modulator and the receptor

site can be elucidated using computational models. The ligands used in this method have retained

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affinity which is sufficient to attach to the residues within the allosteric site. By attaching such

reactive groups at various positions of the ligand, a number of amino acids involved in ligand-

receptor binding can be identified. All the amino acids identified can help in mapping the

allosteric binding site.76

This method is advantageous to identify binding motifs of different

ligands like agonist, antagonists, partial/inverse agonist, and allosteric modulators. After research

on different structural changes around the phenyl ring, it was concluded that halogen at the para

position favors ago-PAM activity, thus, in our design, the reactive groups were incorporated only

at the fourth position of phenyl ring. Covalent probes recognize and interact with particular

targets with high affinity as they are designed to mimic a receptor’s ligand similar in biochemical

and pharmacological properties in order to selectively bind to a particular target.

Salient features of a successful covalent probe are:

it must be recognized well by the target (i.e., possess moderate to high affinity);

it must decompose or engage in nonselective reactions before covalently reacting with

the binding site (i.e, stability)

it should be selective and controllable in binding with the protein target

Covalent probes are classified into two groups- Photoactivatable ligands and electrophilic

ligands. Photoactivatable ligands are those which have an inert group which when irradiated

gets converted to reactive species like carbene and nitrene mostly. These reactive species bind to

the amino acids at the binding site covalently. For example, ligands with aromatic azido group,

phenyl azides, hydroxyphenyl azides, nitrophenyl azides, tetrafluorophenyl azide, aryl diazirines,

benzophenones etc., have been used as photo labeling reagents. On other hand, electrophilic

affinity ligands are those which have the prototypical pharmacophore capable of reacting with

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an amino acid residue carrying a nucleophilic group at or near the binding site to form a covalent

bond. In most cases, the isothiocyanate (NCS) group is used as an electrophile in the ligands.

Photoactivatable/ photolabile probes that have high affinity for binding site will more effectively

label the site upon irradiation. These probes first interact noncovalently within the binding site

and after irradiating with appropriate UV light wavelength, they form carbene or nitrene reactive

species capable enough to covalently insert into C-H or N-H or multiple bonds of amino acid

residues (Figure 10).77

One of the best photoactivatable group that could be incorporated into ligands is the

aryl(trifluoro methyl) diazirine moiety. This group is gaining importance for photoaffinity

crosslinking to study the interactions of drug-receptor, nucleic acid-protein or any other

biological molecules. Upon irradiation, aryldiazirines isomerize into diazo derivatives which

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readily form highly reactive species like carbine or radicals that in turn form covalent bonds

between interacting molecules (Figure 11). Aryl (trifluoromethyl) diazirines are advantageous

over other photoactivatable probes – it creates a highly reactive carbene with a nanosecond time

scale which is favorable even for kinetic studies; trifluoromethyl group stabilizes diazo

derivative which also acts as a carbene precursor; photolysis of diazirine takes place under mild

conditions which ensures biological molecules are protected; this photolabile group is stable at

most chemical conditions.78

Azide probes form nitrene groups as the reactive species upon

irradiation, which can cause addition reactions at double bonds, insert into C-H and N-H sites.

And, unlike diazirines, aryl azides can form less reactive electrophilic dehydroazepine

intermediates by ring expansion which selectively destroyed Cys and Lys amino acid residues

(primary amines) which are nucleophilic in the interacting zone. Not all azides can be photo

activated at long UV, simple phenyl azides need short UV (254nm-275nm) to effectively activate

the probe but this wavelength range is harmful for other biological molecules. Also, azides are

not stable over wide range of chemical conditions. For example, thiol containing reducing agents

like DDT can convert azide to amine group, buffers used while photo activation should be

compatible to the reaction chemistry.

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On the other hand, electrophilic affinity ligands rely on incorporating a functional group that is

capable of reacting with protein target and form covalent bonds with the amino acids within the

binding site which carry a nucleophilic group. The most common electrophile used in affinity

labels is the sterically similar isothiocyanate (NCS) group. These probes rely on the chemical

reactivities between ligand and protein target leading to the formation of a covalent bond. NCS-

containing ligands react with amino acid residues possessing sulfhydryl or unprotonated amino

side chains when they are properly aligned capable to engage in a nucleophilic addition reaction

(Figure 12). Electrophilic probes more selective than photoactivatable congeners as they only

react in the presence of a suitably positioned nucleophilic group within the binding pocket.

8.3 Study of radio-labeled ligand:

Radiotracers are commonly used to characterize the binding site of target-selective ligands.

Radio-label ligand binding increases the knowledge about the binding and interaction between

ligand and receptor. To date, no radio-labeled ligand was developed targeting the allosteric site

of α7 nAChRs. Previously, radio-tracer compounds like 125

I-α-BTx, 3H-methyllycaconitine (

3H-

MLA) targeting the orthosteric site of α7 nAChR antagonists were developed and showed

incomplete binding selectivity for α7nAChRs; 11

C-AR-R255082 , 3-([2,4-dimethyl-5-123

I

iodo]benzylidene)anabaseine were developed and showed to fail to selectively bind in-vivo, but

125I-MLA showed encouraging in vivo biodistribution in rats, despite rapid clearance and poor

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brain penetration. Further, α7 nAChR agonists based on quinuclidine nucleus were developed to

show their selective binding to α7 nAChRs and improve an understanding of the receptor

topography.79

Similarly, allosteric site(s) can be explored and mapped using selective radio tracer

ligand to map the PAM/ago-PAM binding site(s) using 4IP-TQS. [125

I] 4IP-TQS was synthesized

by stannylation and radioiododestannylation of chiral pure enaniomers of 4IP-TQS. Bio-

distribution in mice brains was studied. The ex-vivo autoradiography sheds light on tissue

localization of the radio-labeled ligand and receptor binding. Binding affinities (Ki) for various

parts of mice brains can be obtained by in-vitro binding studies which give more insight into

understanding the correlation between affinity and efficacy of 4BP-TQS analogs.

9. SCHEMES AND CHEMISTRY:

All the analogs of 4BP-TQS were synthesized using the three-component Povarov cyclization

(aza-Diels-Alder reaction) reaction (Scheme II) assisted by microwave irradiation and the

respective aldehydes were synthesized as shown under following schemes.80

The reaction

conditions and yields for the analogs are summarized in Table (1). 4-Azidobenzaldehyde(4) was

synthesized following Scheme II.81

4-Nitro benzaldehyde 1 was reduced to 4-amino benzyl

alcohol 2 using sodium borohydride with a 75% yield. (4-Azidophenyl)methan-1-ol 3 was

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synthesized by converting the amine to azide by diazotization of 2 followed by displacement

with sodium azide which yielded 90% of the desired compound.

cAldehyde (R)

(compound #)

aReaction conditions Product

(compound#); bYield

(4)

Microwave;

50°C; 20min;

(7);

88%

(8)

RT; 18 hours;

(9);

35%

(12)

Microwave;

100°C; 20 min;

(13);

99%

(15)

Microwave;

70°C; 20min;

(16);

52%

(17)

Microwave;

100°C; 20min;

(18);

61%

(21)

Microwave;

100°C; 15min;

(23);

81%

(22)

Microwave;

50°C; 20min;

(24);

79%

Table (1)

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The azide alcohol 3 was oxidized using PCC to give aldehyde 4 in 94% yield. To obtain 4-

azidophenyl substituted TQS, compound 4 when treated with 4-aminosulphanamide 5 and

cyclopentadiene 6 in the presence of anhydrous indium chloride for 20 min, at 50°C yielded 88%

4-(4-azidophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulphonamide (4-

azidophenyl TQS) 7.

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In an attempt to synthesize 4-aminophenyl TQS 10, we tried to reduce 4-nitrophenyl TQS 13

using reducing agent (NaBH4), but it resulted in reduction of the double bond between C1-C2 i.e

pentacyclo moiety attached to quinoline. 4-BOCaminophenyl TQS 9 was synthesized from

commercially available 4-BOC amino benzyl alcohol 8 in 35% yield using Povarov cyclization

(Scheme I) at room temperature. Compound 10 was successfully synthesized by deprotecting the

terminal amino group in compound 9 using 3M HCl at 40°C in 3 hours with 75% yield.

As a electrophilic affinity ligand, phenyl-4-isothiocyanate-TQS 11 was synthesized via two

routes as shown in Schemes III, IV and V from corresponding phenyl-4-amino-TQS (III, IV) and

phenyl-4-azido-TQS (V) precursors. Scheme III utilizes afore mentioned reaction of 9 to give 10.

Compound 11 was obtained by incorporating isothiocyanate group by using di-2-pyridyl

thionocarbonate (DPT) at room temperature as shown in Scheme III. Compound 10 was also

synthesized by a two-step process including formation of 4-nitrophenyl-TQS 13 from 4-nitro

benzaldehyde 1 (using scheme II yielded 99%) and reduction of nitro group to amino group

using Zn granules which gave 84% yield (Scheme IV).

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Compound 11 was synthesized from compound 7 as a precursor using triphenyl phospine and

carbon disulphide as reagents under anhydrous conditions (Scheme V). As

aryl(trifluoromethyl)diazirine reagents are also used

as photolabelling probes which can instantly form

reactive species like carbenes or nitrenes upon

irradiation, compound 16 was synthesized. 4-

(trifluoromethyl)diazirine-benzylbromide 14 was

oxidized to 4- (trifluoromethyl)diazirine-

benzaldehyde 15 using sodium bicarbonate as

oxidant at 100°C (Scheme VI). Compound 15 was subjected to Scheme I to form compound 16

in 20min at 70ᵒC. This was a unique covalent probe developed. As the synthesis and in-vitro data

of compound 7 was promising, it was thought that fluorine substituted phenyl-TQS would

expand the horizons of understanding.

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As a result, 2,3,4,5,6-pentafluoro phenyl-TQS 23 and 4-azido-2,3,5,6-tetrafluorophenyl TQS 24

were produced which did not show results in-vitro as expected. Compound 23 was produced

from commercially available pentafluorobenzaldehyde 21 as mentioned under Scheme I yielding

81% product. After a failed attempt to directly displace 4-fluoro (in compound 23) to 4-azido

group (in compound 24) as mentioned under Scheme VII, Scheme VIII was followed. Precursor,

4-azido-2,3,5,6-tetrafluoro benzaldehyde 22 was formed by refluxing 21 in the presence of

sodium azide for 8 hours which gave 74% yield. And, compound 24 was 79% when treated with

5 and 6 in the presence of anhydrous indium chloride at 50°C for 20min.

4-[I125

] iodophenyl-TQS 20 is a radioligand, designed to bind at the allosteric site of α7 nAChRs

just like 4BP-TQS allosteric agonist and increase our knowledge on the drug-receptor

interaction. 4IP-TQS 18 was synthesized as per Scheme I yielding 61% product at 100ᵒC for

20min. The chirally pure enantiomers of 18 obtained by chiral HPLC, were used to synthesize

radioligands. Iodostannylation and destannylation using sodium - [I125

] iodide gave the product

(scheme VIII). Iodostannylation was done using hexabutylditin in the presence of palladium

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catalyst under anhydrous conditions at 120°C after 17 hours yielding 93% conversion

(compound 19). Radioiododestannylation was a quick reaction using Na125

I and chloramines-T

in aqueous HCl and ethanol with a >98% radiochemical purity (compound 20).

10. RESULTS AND DISCUSSION:

The original Povarov cyclization (aza-Diels-Alder reaction) used to synthesize 4BP-TQS gave

the desired product only in 37% yield over 24 hour reaction. Our modified microwave assisted

approach transformed the synthesis into a reproducible, high-yielding cyclopentadiene ring-fused

tetrahydroquinolines with cis-diastereomer being exclusively formed or as a major product.80

This was applied to synthesize most of the analogs of 4BP-TQS. The reaction conditions and

yields as summarized in table 1 make the application successful for covalent probes. These

analogs have three chiral centers and the cis-diastereomer is the one having cyclopentene and 4-

bromophenyl moieties oriented to each other with a coupling constant, J between H-3a and H-4

as 3.5Hz (2D NMR studies). From the previous functional characterization of enantiomers of

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4BP-TQS in our lab, it was found that cis-enantiomer is the active form for the α7 nAChRs. So,

enantiomeric resolution of racemic mixture was carried out using chiral HPLC for the potent

compounds and radio-iodinated ligand. A hexane/EtOH (60:40) mixture is used as the mobile

phase with Chiralpak 1A for baseline separation of the two enantiomers. The retention times and

enantiomeric excess were determined and extended to preparative HPLC to separate racemates.

In-vitro biological evaluation was carried using Xenopus oocytes which were genetically

modified to abundantly express human α7 nAChRs. The results of two-electrode voltage clamp

experiments are summarized in table 2.

Compound GAT # R1 Reaction conditions

and yields

Electrophysiological data

Time

(min)

Temp.

(°C)

Yield

(%)

Charge

Alone

Charge

+C

Activity

7 GAT143 Phenyl N3 20 50 88 1.38 294.56 ago-

PAM

7a GAT165 (-)enantiomer

of GAT143

0.004 1.05 NA

7b GAT166 (+)enantiomer

of GAT143

9.5 101 ago-

PAM

24 GAT161 tetrafluoroazi

de

20 50 79 1.28 7.11

inactive

16 GAT162 Phenyl

trifluorometh

yl diazirine

20 70 50 10.43

71.29

ago-

PAM

11 GAT144 NCS Synthesized

via Staudinger

aza-Wittig

1.85

30.02

PAM

9 GAT141 -NHBOC 18hr RT 35 3.96 251 ago-

PAM

18 GAT142 iodo 100 20 61 11 41 ago-

PAM

18a GAT163 (-)enantiomer

of GAT142

0.002 0.97 NA

18b GAT164 (+)enantiomer

of GAT142

36 146 ago-

PAM

Table (2)

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Among the photoaffinity probes, 7 emerged as the most efficacious molecule when coapplied

with acetylcholine, followed by the diazirine 16 although the allosteric agonist activity of 16 was

almost ten folds more than that of 7. The bioisosteric approach of substituting the hydrogen on

the phenyl ring with fluorine rendered the molecule with less activity suggesting that such

substitution on the ortho and the meta positions along with a para substitution is not tolerated.

Lastly, the electrophilic probe 11 proved to be a moderate PAM with agonist activity comparable

to ACh. Compound 7 being the best molecule among the probes, we separated the enantiomers

(enantiomer pair 7a and 7b) and found that the (+) enantiomer 7b retained all the activity of the

racemate. The intermediate compound 9 when tested, was found to be active having 4-fold

increase in ago-PAM activity as compared to 4BP-TQS which sheds light on the possibility of

having bulky groups at para-postion to the phenyl ring. Although, as an ago-PAM, compound 18

showed slightly low potentiation of net charge when compared to 4BP-TQS, it is efficacious

enough to radio-iodinate it for binding study and obtain a standard dose-response curve based on

which relative affinities of all active analogues can be measured. This can form the basis to

discover the best efficacious, potent and high-affinity compound among all the developed

analogues. The enantiomers 18a, 18b were separated on chiral HPLC and 18b retained all the

activity which is a promising ago-PAM.

Radio-iodinated compound 20 was evaluated by ex-vivo autoradiography on slide-mounted brain

sections. The images show the distribution profile of isomer-2 of 4IP-TQS in mice brains. The

chirally purified enantiomers (18a), (18b) when stannylated and radio-iodo destannylated gave

enantiomer pair 20a (-) enantiomer and 20b (+) enantiomer. Figure13 shows the distribution

of 20b. The brain image of [125

I] enantiomers (Figure13) is consistent with the binding to α7

nAChRs in hippocampus, thalamus and cortex regions shown by dark regions in the image (parts

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labeled). Although, 125

I is widely used for studying localization and bio-distribution of

compounds in animal bodies, the results don’t stand out in this application as these receptors are

present in most brain areas with major concentration in hippocampus, cortex and thalamus.

Figure 13: brain images after 1 week exposure to film of (+)

enantiomer of GAT-142 injected mice brain

Table 3 lists out counts per minute per μg of brain tissue protein obtained by Packard COBRA

gamma counter for each tissue section. 20b is very well bound hippocampus and cortex which

are the main areas where α7 nAChRs are localized. Further, radio-ligand binding assay will give

us the affinity.

(+) GAT-142 isomer-2

(mean CPM/μg)

Hippocampus (HP) 21.26

Thalamus (TH) 21.97

Cortex (CX) 19.63

Frontal cortex (FCX) 24.97

Striatum (ST) 23.98

Rest of the brain 21.12

Table(3)

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11. CONCLUSION:

Modified microwave assisted approach was successfully applied for covalent probe synthesis.

The results were reproducible, high-yielding compounds with cis-form being exclusively formed

or as a major product. Enantiomeric resolution of racemic mixture was carried out using chiral

HPLC for the potent compounds and iodinated ligand for radio-labeling. Furthermore, in-vitro

biological evaluation by two-electrode voltage clamp experiments using Xenopus oocytes which

were genetically modified to abundantly express human α7 nAChRs were carried out. Among

all, GAT-143 emerged as the most efficacious molecule when co-applied with acetylcholine.

GAT-164 is the first radio-labelled ligand developed for allosteric site of α7 nAChR and an agent

for radio-ligand binding assay. Autoradiography showed brain penetration of the ligand.

12. EXPERIMENTAL SECTION:

Material and Methods:

Chemistry

All commercial chemicals and solvents were purchased from Sigma-Aldrich Inc. and Alfa Aesar,

and unless otherwise specified they were used without further purification. Biotage Initiator

microwave system was used for the synthesis. The progress of the reaction was monitored by

thin layer chromatography (TLC) using commercially prepared silica gel 60 F254 glass-backed

plates. All compounds were visualized under ultraviolet (UV) light. NMR spectra and other 2D

spectra were recorded in DMSO-d6, unless otherwise stated, on a Varian 500 MHz. Chemical

shifts are recorded in parts per million (δ) relative to internal tetramethylsilane (TMS).

Multiplicities reported in hertz (Hz). LCMS analysis was performed using a Waters Alliance

reverse-phase HPLC (electrospray ionization). The CD spectra of enantiomers (dissolved in

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CHCl3) were measured on a Jasco J-815 CD spectropolarimeter. Specific rotations of

enantiomers were measured at 589 nm with a Jasco polarimeter model P-2000 equipped with a

Na lamp. The volume of the cell was 100 mm and specific rotations were recorded in methanol

(c = 1.0).

Method for separation of racemic mixtures by chiral HPLC

Analytical Setting. Analytical HPLC was performed using a Chiralpak IA (Daicel) column (4.6

x 250 mm, 5µm) with ethanol/hexane (40/60) as a mobile phase at a 1.0 mL/min flow rate (diode

array detector; wavelength 273 nm both enantiomers; retention times were obtained ). These

conditions were used for enantiomeric excess determination after separation and it was >99% in

both cases.

Preparative Setting. Preparative HPLC was performed using a Chiralpak IA (Daicel) column (30

x 250 mm, 5µm) with ethanol/hexane (40/60) as a mobile phase at a 25.0 mL/min flow rate (30

mg/injection with runtime of 25 min/injection with stacking).

cDNA Clones and RNA.

The human α7 nAChR receptor clone was obtained from Dr. Jon Lindstrom (University

of Pennsylvania, Philadelphia, PA), and the RIC-3 clone from Dr. Millet Treinin (Hebrew

University, Jerusalem, Israel) for the purpose of co-injection with α7 to improve the level and

speed of receptor expression (Halevi et al., 2003). After linearization and purification of cloned

cDNA's, RNA transcripts were prepared using the appropriate mMessage mMachine kit from

Ambion (Austin, TX).

Expression in X. laevis oocytes.

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Oocytes were obtained from mature (>9cm) female Xenopus laevis African frogs (Nasco,

Ft. Atkinson, WI). Frogs were anesthetized in 0.7 g/L solution of ethyl 3-aminobenzoate

methanesulfonate buffered with sodium bicarbonate, and oocytes were surgically removed

through an abdominal incision. Harvested oocytes were treated with 1.25 mg/ml collagenase

(Worthington Biochemicals, Freehold, NJ) in calcium-free Barth's solution (88 mM NaCl, 1 mM

KCl, 2.38 mM NaHCO3, 0.82 mM MgSO4, 15 mM HEPES, 12 mg/L tetracycline, pH 7.6) for 3-

4 hours to remove the follicular layer. Stage-5 oocytes were isolated and injected with 50 nl (5-

20 ng) of human α7 and RIC-3 cRNA. Suitable levels of receptor expression were typically

achieved 2-6 days after injection of cRNA. For experiments involving the PAM 4BP-TQS/GAT-

107, where standard levels of expression result in ion currents too large to be recorded in voltage

clamp, experiments were typically conducted 1-3 days after RNA injection.

Electrophysiology.

Two-electrode voltage clamp experiments were conducted using OpusXpress6000A

(Molecular Devices, Sunnyvale, CA), an integrated system that provides automated impalement

and voltage clamp of up to eight oocytes in parallel. Both the current and voltage electrodes were

filled with 3 M KCl, and oocytes were clamped at a holding potential of -60 mV. Data were

collected at 50 Hz and filtered at 20 Hz. The oocytes were bath-perfused with Ringer's solution

(115 mM NaCl, 10 mM HEPES, 2.5 mM KCl, 1.8 mM CaCl2, pH 7.3), and agonist solutions

were delivered from a 96-well drug plate using disposable tips. Flow rates were set at 2 ml/min,

with each drug or control application delivered in 12 s durations followed by 181 s washouts

with Ringer's unless noted otherwise.

Experimental Protocols and Data Analysis.

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Responses of human α7 receptors to agonists were measured as both peak current and net

charge measured over a 120 s period beginning with the compound application (Papke and

Papke, 2002). Note that for data obtained in the absence of PAMs, net charge data are a more

reliable measurement of response since the fast desensitization, characteristic of α7, results in the

peak current being reached before agonist solution exchange is complete, giving values which do

not correspond to activation produced by the ligand at the final concentration applied. In

experiments involving positive allosteric modulators (PAM), the fast desensitization of α7 is

eliminated, allowing peak currents is to be used as a valid measurement of the receptor-mediated

responses (Wang et al., 2012; Williams et al., 2011).

Oocytes received two control ACh applications prior to receiving any drug in order to

establish a steady reference response, and they received one or more control ACh applications at

the end of all experiments.

For experiments involving drug incubation through bath applications, all responses were

normalized to the average of two ACh control responses taken immediately prior to the switch of

bath solution, for each cell individually. These normalization procedures had the effect of

compensating for differing levels of receptor expression among the multiple oocytes used in each

experiment. Each experiment was conducted on at least four oocytes, with mean values and

standard errors (S.E.M.) calculated from their normalized responses.

Animals and drugs.

Autoradiographic studies, tissue dissection and counting studies were performed using male

Swiss-Webster mice weighing 25-30g. [125

I] 4IP-TQS was synthesized in our lab with >98%

radiochemical purity as tested by analytical HPLC. Two mice for each isomer were sacrificed.

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[125

I]-4IP-TQS Brain autoradiography in mice.

Mice were administered 5µCi of [125

I] 4IP-TQS in ethanol: emulphor: saline = 1:1:18 via tail

vein and sacrificed by cervical dislocation after 15min. Brains were dissected and washed in ice

cold saline. The frozen brains were sectioned using (WPI) World Precision Instruments,

VibroSlicer into 400μ thickness. The sections were carefully mounted on slides and air dried

overnight and exposed to Kodak X-ray film for a week. The films were removed and developed

in a PhosphorImager at 600dbi resolution, to image radioactivity. To quantify the radioactivity

levels in various brain sections, different parts of the brain like cortex, hippocampus, thalamus,

striatum, frontal cortex, the dried slices were scratched out from the slide using a blade. The

tissue sections were taken in vials and counting using Packard COBRA gamma counter, energy

windows set at 15-75 keV. Counts per minute (CPM) were obtained for radioactivity from each

brain region of drug-treated mice.

BCA assay protocol.

Protein concentrations are determined and reported with reference to standards of bovine serum

albumin (BSA) using the standard microplate BCA assay protocol (working range= 125-

2000μg/mL). A series of dilutions of known concentration are prepared from the protein and

assayed alongside the proteins from brain tissue sections. The concentration of each unknown

was determined based on a standard curve. 2mg/mL albumin standard was diluted to 25, 125,

250, 500 and 1000μg/mL concentrations using PBS buffer. Working reagent was prepared using

1:50= BCA reagent A: BCA reagent B sufficient for all wells. The tissue samples were dissolved

in Solvable and diluted 10-fold making sure the radioactivity in each is <1nCi. 10μL of standard

dilutions were added in duplicate and alongside 10μL of tissue samples in triplicate in a 96-well

cell cuture plate. 200μL of working reagent was added (1:20 = sample:WR) and incubated at

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37°C for 30min. The absorbances were measured at 562nm on a Biotek plate reader using Gen5

software. Standard curve was plotted using mean values of blank-corrected 562nm measurement

for each BSA standard vs concentration in μg/mL and unknown protein concentrations were

determined.

General Procedure for Povarov reaction.

To a microwave vial were added cyclopentadiene (3.0 equiv.), corresponding aldehyde (1.0

equiv.), 4-aminosulfonamide (1.0 equiv.), anhydrous indium trichloride (0.3 equiv.) and

acetonitrile (5 mL). The reaction vial was placed in a microwave synthesizer and heated to the

desired temperature for a specified amount of time. The reaction was quenched by pouring it to

the aqueous Na2CO3 solution (20%; 10 mL) and extracted with DCM (3 × 20 mL). The

combined organic layer was washed with water (10 mL), dried (Na2SO4) and concentrated under

reduced pressure. The crude was purified by flash chromatography (ethylacetate/hexane).

4-Aminobenzylalcohol (2): Nickel chloride hexahydrate (3.15 g, 13.23 mmol) was added to a

solution of 4-nitro benzaldehyde (1 g, 6.62 mmol) in dry THF (40mL) and dry methanol (8-

10mL) and stirred for 1 hr under argon. 50mg fractions of sodium borohydride (0.98 g, 26.47

mmol) were added to the reaction one by one at 0°C letting the effervescences fade out. The

reaction was quenched with saturated ammonium chloride and filtered. Organic layer was

separated after diluting with water and DCM . Organic layers were combined and solvent

evaporated under reduced pressure (610 mg, 75% yield). Rƒ= 0 (EtOAc/Hexane = 20/80). 1H

NMR (500 MHz, CDCl3) (d, J = 8 Hz, 2H), 6.68 (d, J = 8.5 Hz, 2H), 4.55 (s, 2H), 3.69 (s,

2H), 1.52 (s, 1H).

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4-Azidobenzylalcohol (3): To a suspension of 4-amino benzyl alcohol (88 mg, 0.71 mmol) in

freshly prepared 4N H2SO4 (about 5mL) at 0-5°C, sodium nitrite (73.95 mg, 1.07mmol) was

added slowly while stirring. After obtaining a clear solution, sodium azide ( 69.7 mg, 1.07 mmol)

was added drop by drop with gas evolution and yellow solid precipitating out while maintaining

temperature. Reaction mixture was stirred at room temperature for an hour. Reaction was diluted

with water and extracted diethyl ether layer. Organic layers were combined, dried over sodium

sulphate and solvent evaporated under reduced pressure (53 mg, 49.7% yield). Rƒ= 0.5

(EtOAc/Hexane = 40/60). 1H NMR (500 MHz, CDCl3) (d, J = 8.5 Hz, 2H), 7.02 (d, J = 9

Hz, 2H), 4.67 (s, 2H), 1.83 (s, 1H).

4-Azidobenzaldehyde (4): Pyridium chlorochromate (98.3 mg, 0.46 mmol) was added to a

solution of 4-azido benzyl alcohol ( 40 mg, 0.27 mmol) in DCM (5-6 mL) while stirring at room

temperature and left stirring for about an hour. Reaction was diluted with DCM and passed

through silica gel packed phase separator after the starting material was consumed (as shown on

TLC). Solvent was evaporated under reduced pressure and product was stored under argon at -

20°C (37 mg, 93.8% yield). Rƒ= 0.6 (EtOAc/Hexane = 20/80). 1H NMR (500 MHz, CDCl3)

(s, 1H), 7.89 (d, J = 8 Hz, 2H), 7.17 (d, J = 8 Hz, 2H).

4-(4-azidophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (7): To a

reaction mixture of 4-azido benzaldehyde (50 mg, 0.34 mmol), sulphanilamide (58.5 mg,0.34

mmol) and anhydrous indium chloride ( 22.6 mg, 0.10 mmol) in acetonitrile (4-5 mL) was added

cyclopentadiene (67.4 mg, 1.02 mmol) and taken in a microwave reaction vial. The reaction was

microwaved for 22 min at 55°C. Reaction was quenched with 20% sodium bicarbonate solution

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and diluted with DCM. Organic layer was separated and aqueous layer extracted with DCM.

Combined organic layers were dried (Na2SO4) and solvent evaporated under reduced pressure.

Purification of the crude by gravity column chromatography (15%-35% EtOAc: Hexane) gave a

reddish orange solid (110 mg, 88% yield). Rƒ= 0.31 (EtOAc/Hexane = 50/50). 1H NMR (400

MHz, DMSO) (d, J = 8.8 Hz, 2H), 7.43 (s, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8Hz,

2H), 6.96 (s, 2H), 6.37 (s, 1H), 5.89 (s, 1H), 5.62 (s, 1H), 4.64 (s, 1H), 4.06 (d, J = 8.8 Hz, 1H),

2.87-2.98 (m, 1H), 1.64 (dd, J = 8 Hz, 15.6 Hz, 1H), Mass spectrum m/z – 368.10 [M+H]+

4-(4-amino butyloxy carbonyl phenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-

sulfonamide (9): To a reaction mixture of 4-amino butyloxy carbonyl benzaldehyde (100 mg,

0.45 mmol), sulphanilamide (71 mg, 0.45 mmol) and anhydrous indium chloride (30mg, 0.14

mmol) in acetonitrile (4-5 mL) was added cyclopentadiene (89.61 mg, 1.36 mmol) and taken in a

clean and dry round-bottomed flask. The reaction was stirred for 18 hours at RT. Reaction was

quenched with 20% sodium bicarbonate solution and diluted with DCM. Organic layer was

separated and aqueous layer extracted with DCM. Combined organic layers were dried (NaSO4)

and solvent evaporated under reduced pressure. Purification of the crude by flash

chromatography (10%-45% EtOAc: Hexane) gave an off-white solid (70 mg, 35% yield).

Rƒ=0.77 (EtOAc/Hexane = 50/50 2 runs). 1H NMR (500 MHz, DMSO) (s, 1H), 7.45 (d,

J = 8.5 Hz, 2H), 7.42 (d, J = 2 Hz, 1H), 7.32 (dd, J = 8.5 Hz, 2 Hz, 1H), 7.30 (d, J = 9 Hz, 2H),

6.94 (s, 2H), 6.79 (d, J = 8.5 Hz, 1H), 6.29 (s, 1H), 5.84-5.91 (m, 1H), 5.59-5.63 (m, 1H), 4.55

(d, J = 3.5 Hz, 1H), 4.04 (d, J = 7 Hz, 1H), 2.84-2.95 (m, 1H), 2.37 (ddd, J = 16 Hz, 9.5 Hz, 2

Hz, 1H), 1.65 (dd, J = 15 Hz, 9 Hz, 1H), 1.47 (s, 9H), Mass spectrum m/z – 442.15 [M+H]+

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4-(4-aminophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (10): A

solution of compound (9) (40 mg, 0.09 mmol) in ethyl acetate was heated with freshly prepared

3M HCl at 40°C for 3 hours. After starting was consumed, the reaction was diluted with

saturated sodium bicarbonate solution while pH was 5. Ethyl acetate layer was extracted and

aqueous layer was washed with ethyl acetate twice. Combined organic layers were dried over

Na2SO4 and solvent evaporated under reduced pressure (23 mg; 74.65% yield). Rƒ= 0.24

(EtOAc/Hexane = 50/50).

(Procedure using Zn granules): A solution of compound (13) (1.5g, 4.038 mmol) in

100mL of acetone and 100mL of saturated ammonium chloride solution diluted with water (1:1)

was heated to 70°C. 1-1.5g of Zn granules were added to the reaction mixture over a period of

15min while heat was off. The reaction mixture was refluxed under argon (half-filled balloon)

for 18-20h. It was diluted with water after solvent evaporated. The product was extracted with

ethyl acetate, washed with brine twice and dried over Na2SO4 to give a white solid. Purification

using flash chromatography (ethylacetate/hexane) (0.87g; 63.1% yield) Rƒ= 0.24

(EtOAc/Hexane = 50/50). 1H NMR (400 MHz, DMSO) (s, 1H), 7.30 (d, J = 8.8 Hz, 1H),

7.07 (d, J = 8 Hz, 2H), 6.91 (s, 2H), 6.77 (d, J = 8 Hz, 1H), 6.56 (d, J = 8 Hz, 2H), 6.17 (s, 1H),

5.82-5.91 (m, 1H), 5.58-5.66 (m, 1H), 4.97 (s, 2H), 4.44 (s, 1H), 4.01 (d, J = 6.4 Hz, 1H), 2.78-

2.89 (m, 1H), 2.34-2.46 (m, 1H), 1.72 (dd, J = 16.4 Hz, 8.8 Hz, 1H), Mass spectrum m/z –

343.09 [M+H]+

4-(4-isothiocyantephenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide

(synthesis from compound (7)) (11): Argon was bubbled through a solution of dried Compound

(7) (30 mg, 0.08 mmol) in anhydrous benzene (5 mL). Triphenyl phosphine (21.4 mg, 0.82

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mmol) was added and the reaction was refluxed for 4h at 70°C. After the substrate was

consumed as shown on TLC, carbon disulphide (excess) was added and refluxed for 12 hours at

40°C. Solvent evaporated and crude purification was done using flash column chromatography

(15% - 40% EtOAc/hexane ) gave a green solid.

(synthesis from compound (10)) (11): A solution of 4-amino phenyl TQS (30 mg, 0.088

mmol) in DCM (2-3 mL) was added slowly drop-wise to a solution of di-2-pyridyl thiono

carbonate (20.46 mg, 0.088 mmol) in DCM (2-3 mL) and stirred at room temperature for 40-50

min. Solvent evaporated under reduced pressure and purification of crude using flash column

chromatography (15% - 40% EtOAc/hexane) TLC (EtOAc/Hexane = 30/70 4 runs). 1H NMR

(500 MHz, CDCl3) (d, J = 9 Hz, 2H), 7.43 (d, J = 2.0 Hz, 1H), 7.34 (dd, J = 8 Hz, 2 Hz,

1H), 6.96 (s, 2H), 6.80 (d, J = 8.5 Hz, 1H), 6.42 (s, 1H), 5.87 – 5.91 (m, 1H), 5.59 – 5.64 (m,

1H), 4.67 (d, J = 3.5 Hz, 1H), 4.06 (d, J = 8.5 Hz, 1H), 2.98 – 2.90 (m, 1H), 2.32 (ddd, J = 18,

12, 2 Hz, 1H), 1.62 (dd, J = 15.5, 9 Hz, 1H), Mass spectrum m/z – 384.09 [M+H]+

4-(4-nitrophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (13): To a

reaction mixture of 4-nitro benzaldehyde (1.6g, 10.87mmol), sulphanilamide (1.87g, 10.87mmol)

and anhydrous indium chloride (0.72g, 3.26mmol) in acetonitrile (4-5 mL) was added

cyclopentadiene (2.15g, 32.61mmol) and taken in a microwave reaction vial. The reaction was

microwaved for 20 min at 100°C. Reaction was quenched with 20% sodium bicarbonate solution

and diluted with DCM. Organic layer was separated and aqueous layer extracted with DCM.

Combined organic layers were dried (NaSO4) and solvent evaporated under reduced pressure.

Purification of the crude by gravity column chromatography (25%-35% EtOAc: Hexane) gave a

lemon yellow solid (3.96g, 99% yield). Rf =0.6 (EtOAc/Hexane = 50/50). 1H NMR (500 MHz,

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DMSO) δ 8.27 (d, J = 8.5 Hz, 2 H), 7.74 (d, J = 9.0 Hz, 2H), 7.46 (d, J = 2.0 Hz, 1H), 7.36

(dd, J = 8.5 Hz, 2.5 Hz, 1H), 6.98 (s, 2H), 6.82 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.95 – 5.86 (m,

1H), 5.65 – 5.57 (m, 1H), 4.80 (d, J = 3.0 Hz, 1H), 4.10 (d, J = 8.5 Hz, 1H), 3.07 – 2.95 (m, 1H),

2.33 (ddd, J = 16.5Hz, 10Hz, 2Hz, 1H), 1.60 (dd, J = 15.5 Hz, 9.5 Hz, 1H), Mass spectrum m/z –

372.08 [M+H]+

4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzaldehyde (15): A solution of NaHCO3 (1.6g,

19.7mmol) in DMSO (3-4mL) was heated to 100°C. A solution of compound (14) (400mg,

1.43mmol) in DMSO (1mL) was added and reaction was stirred at 100°C for 30min and cooled

to RT. Reaction mixture was added to ice cold brine and product wa extracted with Et2O twice. It

was washed with saturated NaHCO3 solution and brine and solvent evaporated. Crude was

purified by flash chromatography (5-6% EtOAc/Hexane) to give a colorless liquid. (120mg, 39%

yield). Rf = 0.53 (EtOAc/ hexane = 20/80). 1H NMR (400 MHz, CDCl3) (s, 1H), 7.92 (d,

J = 8 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H).

4-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-3a,4,5,9b-tetrahydro-3H

cyclopenta[c]quinoline-8-sulfonamide (16): To a reaction mixture of compound (15) (120mg,

0.56mmol), sulphanilamide (96.43mg, 0.56mmol) and anhydrous indium chloride (37.16mg,

0.17mmol) in acetonitrile (4-5 mL) was added cyclopentadiene (111.05mg, 1.68mmol) and taken

in a microwave reaction vial. The reaction was microwaved for 20 min at 70°C. Reaction was

quenched with 20% sodium bicarbonate solution and diluted with DCM. Organic layer was

separated and aqueous layer extracted with DCM. Combined organic layers were dried (NaSO4)

and solvent evaporated under reduced pressure. Purification of the crude by gravity column

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chromatography (25%-35% EtOAc: Hexane) gave a colorless semi-solid (125mg, 52% yield). Rf

= 0.34 (EtOAc/Hexane = 40/60). 1H NMR (500 MHz, CDCl3) 7.61 (d, J = 2Hz, 1H), 7.53 (dd,

J = 9 Hz, 2.5 Hz, 1H), 7.45 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.5 Hz, 1

H), 5.86 – 5.92 (m, 1H), 5.64 – 5.70 (m, 1H), 4.78 (s, 2H), 4.72 (d, J = 3 Hz, 1H), 4.19 (s, 1H),

4.12 (d, J = 7.5 Hz ,1H), 2.95-3.04 (m, 1H), 2.50 (qdd, J = 16.5 Hz, 10 Hz, 2.5 Hz, 1H), 1.78

(qdd, J = 16.5 Hz, 9 Hz, 1.5 Hz, 1H), Mass spectrum m/z – 435.01 [M+H]+

4-(4-iodophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (18): To a

reaction mixture of 4-iodo benzaldehyde (200 mg, 0.86 mmol), sulphanilamide (148 mg,0.86

mmol) and anhydrous Indium chloride ( 60mg, 0.26 mmol) in acetonitrile was added

cyclopentadiene (170 mg, 2.58mmol) and taken in a microwave reaction vial. The reaction was

microwaved for 15min at 100°C. Reaction was quenched with 20% sodium bicarbonate solution

and diluted with DCM. Organic layer was separated and aqueous layer extracted with DCM.

Combined organic layers were dried (NaSO4) and solvent evaporated under reduced pressure.

Purification of the crude by flash column chromatography (10%-40% EtOAc: Hexane) gave a

pale white solid (238 mg, 60.67% yield). (EtOAc/Hexane = 50/50). 1H NMR (500 MHz,

DMSO) (d, J = 8.5 Hz, 2H), 7.43 (d, J = 2.0 Hz, 1H), 7.34 (dd, J = 8.5 Hz, 2 Hz, 1H),

7.26 (d, J = 8.5 Hz, 2H), 6.96 (s, 2H), 6.79 (d, J = 8.5 Hz, 1H), 6.37 (s, 1H), 5.86 – 5.91 (m, 1H),

5.59 – 5.64 (m, 1H), 4.6 (d, J = 3 Hz, 1H), 4.06 (d, J = 9.5 Hz, 1H), 2.97 – 2.88 (m, 1H), 2.33

(ddd, J = 16, 10, 2 Hz, 1H), 1.68-1.59 (m, 1H), Mass spectrum m/z – 453.01 [M+H]+

4-(4-tributylstannyl phenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide

(19): A solution of compound (18) (100 mg, 0.22 mmol) in anhydrous toluene (about 5 mL) was

bubbled with argon for 15 min. Hexabutylditin ( 50 mol%) and tetra triphenylphosphine

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palladium was added under anhydrous conditions and reaction was refluxed for 17 hours.

Solvent evaporated under reduced pressure and crude was purified by gravity column

chromatography (15% - 30% EtOAc/Hexane ) which gave pale brown solid (126 mg, 92.8%

yield ). Rƒ= 0.42 (EtOAc/Hexane = 40/60). 1H NMR (500 MHz, DMSO) (d, J = 2.5 Hz,

1H), 7.52 (dd, J = 8 Hz, 1.5 Hz, 1H), 7.48 (d, J = 8 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 6.64 (d, J =

8.5 Hz, 1H), 5.86-5.91 (m, 1H), 5.66-5.71 (m, 1H), 4.68 (s, 2H), 4.67 (s, 1H), 4.24 (s, 1H), 4.11

(d, J = 9 Hz, 1H), 2.88-3.08 (m, 1H), 2.58 (qdd, J = 16.5 Hz, 10 Hz, 2.5 Hz, 1h), 1.87 (ddd, J =

16.5 Hz, 9 Hz, 2.5 Hz, 1.5 Hz, 1H), 1.51-1.59 (m, 6H), 1.29-1.39 (m, 6H), 1.04-1.10 (m, 6H),

0.89 (t, J = 7.5 Hz, 9H)

4-azido-2,3,5,6-tetrafluorobenzaldehyde (22): A mixture of 2,3,4,5,6-penta fluoro

benzaldehyde (200 mg, 1.02 mmol) and sodium azide (71 mg, 1.2 mmol) in acetone:water (3:1)

(6-7 mL) was refluxed for 8 hours. Reaction was diluted with ether and water. Ether was

separated and aqueous layer was extracted with ether twice. Combined organic layers were dried

over Na2SO4 and solvent evaporated at reduced pressure. Purification of crude using gravity

column chromatography (15% - 30% EtOAc/hexane ) which gave a off-white solid (120 mg,

53.7% yield). Rƒ= 0.8 (EtOAc/Hexane = 40/60). 1H NMR (500 MHz, CDCl3) (s, 1H).

4-(2,3,4,5,6-pentafluorophenyl)-3a,4,5,9b tetrahydro-3H-cyclopenta[c]quinoline-8-

sulfonamide (23): To a reaction mixture of 2,3,4,5,6-pentafluorobenzaldehyde (200 mg, 1.02

mmol), sulphanilamide (175.64 mg,1.02 mmol) and anhydrous Indium chloride (67.7 mg, 0.31

mmol) in acetonitrile was added cyclopentadiene (202.3 mg, 3.06 mmol) and taken in a

microwave reaction vial. The reaction was microwaved for 15min at 100°C. Reaction was

quenched with 20% sodium bicarbonate solution and diluted with DCM. Organic layer was

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separated and aqueous layer extracted with DCM. Combined organic layers were dried (NaSO4)

and solvent evaporated under reduced pressure. Purification of the crude by flash column

chromatography (15%-35% EtOAc: Hexane) gave a white solid (345 mg, 81.23% yield). Rƒ=

0.6 (EtOAc/Hexane = 50/50). 1H NMR (500 MHz, DMSO) 8.01 (d, J = 2 Hz, 1H), 7.90 (dd, J

= 8 Hz, 2 Hz, 1H), 7.22 (d, J = 9 Hz, 1H), 6.68 (s, 2H), 6.36-6.41 (m, 1H), 6.36 (s, 1H), 6.12-

6.17 (m, 1H), 5.65 (d, J = 3.5 Hz, 1H), 4.62 (d, J = 9 Hz, 1H), 3.50-3.60 (m, 1H), 3.14-3.23 (m,

1H), 2.62 (ddt, J = 15 Hz, 8 Hz, 2.5 Hz, 1H), Mass spectrum m/z – 417.03 [M+H]+

4-(4-azido-2,3,5,6-pentafluorophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-

sulfonamide (24): To a reaction mixture of 4-azido-2,3,5,6-pentafluorobenzaldehyde (30 mg,

0.14 mmol), sulphanilamide (23.57 mg, 0.14 mmol) and anhydrous Indium chloride (9.07 mg,

0.04 mmol) in acetonitrile was added cyclopentadiene (27.2 mg, 0.41 mmol) and taken in a

microwave reaction vial. The reaction was microwaved for 20min at 50°C. Reaction was

quenched with 20% sodium bicarbonate solution and diluted with DCM. Organic layer was

separated and aqueous layer extracted with DCM. Combined organic layers were dried (NaSO4)

and solvent evaporated under reduced pressure. Purification of the crude by flash column

chromatography under low pressure and low flow rate (15%-35% EtOAc: Hexane) gave a

slightly yellow liquid Rƒ= 0.36 (EtOAc/Hexane = 40/60). (79% yield). 1

H NMR (500 MHz,

DMSO) 7.45 (d, J = 2.0 Hz, 1H), 7.35 (dd, J = 8.5 Hz, 2.0 Hz, 1H), 6.97 (s, 2H), 6.69 (d, J =

8.0 Hz, 1H), 6.65 (s, 1H), 2.95 (dq, J = 8.5 Hz, 2.5 Hz, 1H), 2.60 – 2.50 (m, 1H), 2.08 (dd, J =

15.5 Hz, 7.5 Hz, 1H), Mass spectrum m/z – 440.06 [M+H]+

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13. REFERENCES:

1. Arneric, S. P.; Holladay, M.; Williams, M. Neuronal nicotinic receptors: a perspective on two decades of drug discovery research. Biochem Pharmacol 2007, 74, 1092-101. 2. Levin, E. D.; Rezvani, A. H. Nicotinic interactions with antipsychotic drugs, models of schizophrenia and impacts on cognitive function. Biochem Pharmacol 2007, 74, 1182-91. 3. Romanelli, M. N.; Gratteri, P.; Guandalini, L.; Martini, E.; Bonaccini, C.; Gualtieri, F. Central nicotinic receptors: structure, function, ligands, and therapeutic potential. ChemMedChem 2007, 2, 746-67. 4. Shytle, R. D.; Mori, T.; Townsend, K.; Vendrame, M.; Sun, N.; Zeng, J.; Ehrhart, J.; Silver, A. A.; Sanberg, P. R.; Tan, J. Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors. J Neurochem 2004, 89, 337-43. 5. Reuter, G.; Fodor, D.; Katai, A.; Szucs, G. Identification of a novel variant of human hepatitis E virus in Hungary. J Clin Virol 2006, 36, 100-2. 6. Cascio, M. Structure and function of the glycine receptor and related nicotinicoid receptors. J Biol Chem 2004, 279, 19383-6. 7. Graham, A.; Court, J. A.; Martin-Ruiz, C. M.; Jaros, E.; Perry, R.; Volsen, S. G.; Bose, S.; Evans, N.; Ince, P.; Kuryatov, A.; Lindstrom, J.; Gotti, C.; Perry, E. K. Immunohistochemical localisation of nicotinic acetylcholine receptor subunits in human cerebellum. Neuroscience 2002, 113, 493-507. 8. Le Novere, N.; Changeux, J. P. Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J Mol Evol 1995, 40, 155-72. 9. Wallace, T. L.; Porter, R. H. Targeting the nicotinic alpha7 acetylcholine receptor to enhance cognition in disease. Biochem Pharmacol 2011, 82, 891-903. 10. Wonnacott, S. alpha-Bungarotoxin binds to low-affinity nicotine binding sites in rat brain. J Neurochem 1986, 47, 1706-12. 11. Chini, B.; Raimond, E.; Elgoyhen, A. B.; Moralli, D.; Balzaretti, M.; Heinemann, S. Molecular cloning and chromosomal localization of the human alpha 7-nicotinic receptor subunit gene (CHRNA7). Genomics 1994, 19, 379-81. 12. Bertrand, D.; Gopalakrishnan, M. Allosteric modulation of nicotinic acetylcholine receptors. Biochem Pharmacol 2007, 74, 1155-63. 13. Corringer, P. J.; Galzi, J. L.; Eisele, J. L.; Bertrand, S.; Changeux, J. P.; Bertrand, D. Identification of a new component of the agonist binding site of the nicotinic alpha 7 homooligomeric receptor. J Biol Chem 1995, 270, 11749-52. 14. Gotti, C.; Moretti, M.; Gaimarri, A.; Zanardi, A.; Clementi, F.; Zoli, M. Heterogeneity and complexity of native brain nicotinic receptors. Biochem Pharmacol 2007, 74, 1102-11. 15. Gotti, C.; Riganti, L.; Vailati, S.; Clementi, F. Brain neuronal nicotinic receptors as new targets for drug discovery. Curr Pharm Des 2006, 12, 407-28. 16. Cimino, M.; Marini, P.; Fornasari, D.; Cattabeni, F.; Clementi, F. Distribution of nicotinic receptors in cynomolgus monkey brain and ganglia: localization of alpha 3 subunit mRNA, alpha-bungarotoxin and nicotine binding sites. Neuroscience 1992, 51, 77-86. 17. Spurden, D. P.; Court, J. A.; Lloyd, S.; Oakley, A.; Perry, R.; Pearson, C.; Pullen, R. G.; Perry, E. K. Nicotinic receptor distribution in the human thalamus: autoradiographical localization of [3H]nicotine and [125I] alpha-bungarotoxin binding. J Chem Neuroanat 1997, 13, 105-13. 18. Ferrarelli, F.; Tononi, G. The thalamic reticular nucleus and schizophrenia. Schizophr Bull 2011, 37, 306-15.

Page 57: Novel Covalent Probes For Mapping α7 Nicotinic ...rx917k924/... · time drug discovery research process which I would not have had if I chose to graduate by just doing coursework.

57

19. Kandel, E. R. Calcium and the control of synaptic strength by learning. Nature 1981, 293, 697-700. 20. <Donepezil for dementia due to Alzheimer's disease - Cochrane Database of Systematic Reviews - Birks - Wiley Online Library.pdf>. 21. Fernandes, C.; Hoyle, E.; Dempster, E.; Schalkwyk, L. C.; Collier, D. A. Performance deficit of alpha7 nicotinic receptor knockout mice in a delayed matching-to-place task suggests a mild impairment of working/episodic-like memory. Genes Brain Behav 2006, 5, 433-40. 22. Young, J. W.; Crawford, N.; Kelly, J. S.; Kerr, L. E.; Marston, H. M.; Spratt, C.; Finlayson, K.; Sharkey, J. Impaired attention is central to the cognitive deficits observed in alpha 7 deficient mice. Eur Neuropsychopharmacol 2007, 17, 145-55. 23. Martin, L. F.; Kem, W. R.; Freedman, R. Alpha-7 nicotinic receptor agonists: potential new candidates for the treatment of schizophrenia. Psychopharmacology (Berl) 2004, 174, 54-64. 24. Hajos, M.; Hurst, R. S.; Hoffmann, W. E.; Krause, M.; Wall, T. M.; Higdon, N. R.; Groppi, V. E. The selective alpha7 nicotinic acetylcholine receptor agonist PNU-282987 [N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] enhances GABAergic synaptic activity in brain slices and restores auditory gating deficits in anesthetized rats. J Pharmacol Exp Ther 2005, 312, 1213-22. 25. Tietje, K. R.; Anderson, D. J.; Bitner, R. S.; Blomme, E. A.; Brackemeyer, P. J.; Briggs, C. A.; Browman, K. E.; Bury, D.; Curzon, P.; Drescher, K. U.; Frost, J. M.; Fryer, R. M.; Fox, G. B.; Gronlien, J. H.; Hakerud, M.; Gubbins, E. J.; Halm, S.; Harris, R.; Helfrich, R. J.; Kohlhaas, K. L.; Law, D.; Malysz, J.; Marsh, K. C.; Martin, R. L.; Meyer, M. D.; Molesky, A. L.; Nikkel, A. L.; Otte, S.; Pan, L.; Puttfarcken, P. S.; Radek, R. J.; Robb, H. M.; Spies, E.; Thorin-Hagene, K.; Waring, J. F.; Ween, H.; Xu, H.; Gopalakrishnan, M.; Bunnelle, W. H. Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties. CNS Neurosci Ther 2008, 14, 65-82. 26. Newhouse, P.; Singh, A.; Potter, A. Nicotine and nicotinic receptor involvement in neuropsychiatric disorders. Curr Top Med Chem 2004, 4, 267-82. 27. Newhouse, P. A.; Potter, A.; Singh, A. Effects of nicotinic stimulation on cognitive performance. Curr Opin Pharmacol 2004, 4, 36-46. 28. Cooper, R. G. Effect of tobacco smoking on renal function. Indian J Med Res 2006, 124, 261-8. 29. Hanna, S. T. Nicotine effect on cardiovascular system and ion channels. J Cardiovasc Pharmacol 2006, 47, 348-58. 30. Wu, W. K.; Cho, C. H. The pharmacological actions of nicotine on the gastrointestinal tract. J Pharmacol Sci 2004, 94, 348-58. 31. Marrero, M. B.; Papke, R. L.; Bhatti, B. S.; Shaw, S.; Bencherif, M. The neuroprotective effect of 2-(3-pyridyl)-1-azabicyclo[3.2.2]nonane (TC-1698), a novel alpha7 ligand, is prevented through angiotensin II activation of a tyrosine phosphatase. J Pharmacol Exp Ther 2004, 309, 16-27. 32. Briggs, C. A.; Anderson, D. J.; Brioni, J. D.; Buccafusco, J. J.; Buckley, M. J.; Campbell, J. E.; Decker, M. W.; Donnelly-Roberts, D.; Elliott, R. L.; Gopalakrishnan, M.; Holladay, M. W.; Hui, Y. H.; Jackson, W. J.; Kim, D. J.; Marsh, K. C.; O'Neill, A.; Prendergast, M. A.; Ryther, K. B.; Sullivan, J. P.; Arneric, S. P. Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo. Pharmacol Biochem Behav 1997, 57, 231-41. 33. Meyer, E. M.; Tay, E. T.; Papke, R. L.; Meyers, C.; Huang, G. L.; de Fiebre, C. M. 3-[2,4-Dimethoxybenzylidene]anabaseine (DMXB) selectively activates rat alpha7 receptors and improves memory-related behaviors in a mecamylamine-sensitive manner. Brain Res 1997, 768, 49-56. 34. Meyer, E. M.; Kuryatov, A.; Gerzanich, V.; Lindstrom, J.; Papke, R. L. Analysis of 3-(4-hydroxy, 2-Methoxybenzylidene)anabaseine selectivity and activity at human and rat alpha-7 nicotinic receptors. J Pharmacol Exp Ther 1998, 287, 918-25. 35. Mullen, G.; Napier, J.; Balestra, M.; DeCory, T.; Hale, G.; Macor, J.; Mack, R.; Loch, J., 3rd; Wu, E.; Kover, A.; Verhoest, P.; Sampognaro, A.; Phillips, E.; Zhu, Y.; Murray, R.; Griffith, R.; Blosser, J.; Gurley, D.;

Page 58: Novel Covalent Probes For Mapping α7 Nicotinic ...rx917k924/... · time drug discovery research process which I would not have had if I chose to graduate by just doing coursework.

58

Machulskis, A.; Zongrone, J.; Rosen, A.; Gordon, J. (-)-Spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin-2'-one], a conformationally restricted analogue of acetylcholine, is a highly selective full agonist at the alpha 7 nicotinic acetylcholine receptor. J Med Chem 2000, 43, 4045-50. 36. Levin, E. D.; Bettegowda, C.; Blosser, J.; Gordon, J. AR-R17779, and alpha7 nicotinic agonist, improves learning and memory in rats. Behav Pharmacol 1999, 10, 675-80. 37. Hajos, M. Targeting information-processing deficit in schizophrenia: a novel approach to psychotherapeutic drug discovery. Trends Pharmacol Sci 2006, 27, 391-8. 38. Pichat, P.; Bergis, O. E.; Terranova, J. P.; Urani, A.; Duarte, C.; Santucci, V.; Gueudet, C.; Voltz, C.; Steinberg, R.; Stemmelin, J.; Oury-Donat, F.; Avenet, P.; Griebel, G.; Scatton, B. SSR180711, a novel selective alpha7 nicotinic receptor partial agonist: (II) efficacy in experimental models predictive of activity against cognitive symptoms of schizophrenia. Neuropsychopharmacology 2007, 32, 17-34. 39. Wishka, D. G.; Walker, D. P.; Yates, K. M.; Reitz, S. C.; Jia, S.; Myers, J. K.; Olson, K. L.; Jacobsen, E. J.; Wolfe, M. L.; Groppi, V. E.; Hanchar, A. J.; Thornburgh, B. A.; Cortes-Burgos, L. A.; Wong, E. H.; Staton, B. A.; Raub, T. J.; Higdon, N. R.; Wall, T. M.; Hurst, R. S.; Walters, R. R.; Hoffmann, W. E.; Hajos, M.; Franklin, S.; Carey, G.; Gold, L. H.; Cook, K. K.; Sands, S. B.; Zhao, S. X.; Soglia, J. R.; Kalgutkar, A. S.; Arneric, S. P.; Rogers, B. N. Discovery of N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide, an agonist of the alpha7 nicotinic acetylcholine receptor, for the potential treatment of cognitive deficits in schizophrenia: synthesis and structure--activity relationship. J Med Chem 2006, 49, 4425-36. 40. Wallace, T. L.; Callahan, P. M.; Tehim, A.; Bertrand, D.; Tombaugh, G.; Wang, S.; Xie, W.; Rowe, W. B.; Ong, V.; Graham, E.; Terry, A. V., Jr.; Rodefer, J. S.; Herbert, B.; Murray, M.; Porter, R.; Santarelli, L.; Lowe, D. A. RG3487, a novel nicotinic alpha7 receptor partial agonist, improves cognition and sensorimotor gating in rodents. J Pharmacol Exp Ther 2011, 336, 242-53. 41. Young, H. S.; Herbette, L. G.; Skita, V. Alpha-bungarotoxin binding to acetylcholine receptor membranes studied by low angle X-ray diffraction. Biophys J 2003, 85, 943-53. 42. Teeling, E. C.; Springer, M. S.; Madsen, O.; Bates, P.; O'Brien, S. J.; Murphy, W. J. A molecular phylogeny for bats illuminates biogeography and the fossil record. Science 2005, 307, 580-584. 43. Couturier, S.; Bertrand, D.; Matter, J. M.; Hernandez, M. C.; Bertrand, S.; Millar, N.; Valera, S.; Barkas, T.; Ballivet, M. A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX. Neuron 1990, 5, 847-56. 44. Monod, J.; Wyman, J.; Changeux, J. P. On the Nature of Allosteric Transitions: A Plausible Model. J Mol Biol 1965, 12, 88-118. 45. Chemouilli, P.; Heidmann, T.; Changeux, J. P.; Bachy, A.; Morre, M. Allosteric effects of diprobutine on acetylcholine receptors. Eur J Pharmacol 1985, 117, 205-14. 46. Heidmann, T.; Changeux, J. P. Characterization of the transient agonist-triggered state of the acetylcholine receptor rapidly labeled by the noncompetitive blocker [3H]chlorpromazine: additional evidence for the open channel conformation. Biochemistry 1986, 25, 6109-13. 47. Hansen, S. B.; Sulzenbacher, G.; Huxford, T.; Marchot, P.; Taylor, P.; Bourne, Y. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J 2005, 24, 3635-46. 48. Bourne, Y.; Talley, T. T.; Hansen, S. B.; Taylor, P.; Marchot, P. Crystal structure of a Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J 2005, 24, 1512-22. 49. Celie, P. H.; van Rossum-Fikkert, S. E.; van Dijk, W. J.; Brejc, K.; Smit, A. B.; Sixma, T. K. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 2004, 41, 907-14.

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50. Krause, R. M.; Buisson, B.; Bertrand, S.; Corringer, P. J.; Galzi, J. L.; Changeux, J. P.; Bertrand, D. Ivermectin: a positive allosteric effector of the alpha7 neuronal nicotinic acetylcholine receptor. Mol Pharmacol 1998, 53, 283-94. 51. Zwart, R.; De Filippi, G.; Broad, L. M.; McPhie, G. I.; Pearson, K. H.; Baldwinson, T.; Sher, E. 5-Hydroxyindole potentiates human alpha 7 nicotinic receptor-mediated responses and enhances acetylcholine-induced glutamate release in cerebellar slices. Neuropharmacology 2002, 43, 374-84. 52. Charpantier, E.; Wiesner, A.; Huh, K. H.; Ogier, R.; Hoda, J. C.; Allaman, G.; Raggenbass, M.; Feuerbach, D.; Bertrand, D.; Fuhrer, C. Alpha7 neuronal nicotinic acetylcholine receptors are negatively regulated by tyrosine phosphorylation and Src-family kinases. J Neurosci 2005, 25, 9836-49. 53. Galzi, J. L.; Bertrand, S.; Corringer, P. J.; Changeux, J. P.; Bertrand, D. Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor. EMBO J 1996, 15, 5824-32. 54. Chimienti, F.; Hogg, R. C.; Plantard, L.; Lehmann, C.; Brakch, N.; Fischer, J.; Huber, M.; Bertrand, D.; Hohl, D. Identification of SLURP-1 as an epidermal neuromodulator explains the clinical phenotype of Mal de Meleda. Hum Mol Genet 2003, 12, 3017-24. 55. Williams, D. K.; Wang, J.; Papke, R. L. Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations. Biochem Pharmacol 2011, 82, 915-30. 56. Ng, H. J.; Whittemore, E. R.; Tran, M. B.; Hogenkamp, D. J.; Broide, R. S.; Johnstone, T. B.; Zheng, L.; Stevens, K. E.; Gee, K. W. Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators. Proc Natl Acad Sci U S A 2007, 104, 8059-64. 57. Timmermann, D. B.; Gronlien, J. H.; Kohlhaas, K. L.; Nielsen, E. O.; Dam, E.; Jorgensen, T. D.; Ahring, P. K.; Peters, D.; Holst, D.; Christensen, J. K.; Malysz, J.; Briggs, C. A.; Gopalakrishnan, M.; Olsen, G. M. An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo. J Pharmacol Exp Ther 2007, 323, 294-307. 58. Lopes, C.; Pereira, E. F.; Wu, H. Q.; Purushottamachar, P.; Njar, V.; Schwarcz, R.; Albuquerque, E. X. Competitive antagonism between the nicotinic allosteric potentiating ligand galantamine and kynurenic acid at alpha7* nicotinic receptors. J Pharmacol Exp Ther 2007, 322, 48-58. 59. Broad, L. M.; Zwart, R.; Pearson, K. H.; Lee, M.; Wallace, L.; McPhie, G. I.; Emkey, R.; Hollinshead, S. P.; Dell, C. P.; Baker, S. R.; Sher, E. Identification and pharmacological profile of a new class of selective nicotinic acetylcholine receptor potentiators. J Pharmacol Exp Ther 2006, 318, 1108-17. 60. Hurst, R. S.; Hajos, M.; Raggenbass, M.; Wall, T. M.; Higdon, N. R.; Lawson, J. A.; Rutherford-Root, K. L.; Berkenpas, M. B.; Hoffmann, W. E.; Piotrowski, D. W.; Groppi, V. E.; Allaman, G.; Ogier, R.; Bertrand, S.; Bertrand, D.; Arneric, S. P. A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J Neurosci 2005, 25, 4396-405. 61. Gronlien, J. H.; Hakerud, M.; Ween, H.; Thorin-Hagene, K.; Briggs, C. A.; Gopalakrishnan, M.; Malysz, J. Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes. Mol Pharmacol 2007, 72, 715-24. 62. Gill, J. K.; Savolainen, M.; Young, G. T.; Zwart, R.; Sher, E.; Millar, N. S. Agonist activation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site. Proc Natl Acad Sci U S A 2011, 108, 5867-72. 63. Dinklo, T.; Shaban, H.; Thuring, J. W.; Lavreysen, H.; Stevens, K. E.; Zheng, L.; Mackie, C.; Grantham, C.; Vandenberk, I.; Meulders, G.; Peeters, L.; Verachtert, H.; De Prins, E.; Lesage, A. S. Characterization of 2-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-4-(4-pyridinyl)-5-thiazolemethanol (JNJ-1930942), a novel positive allosteric modulator of the {alpha}7 nicotinic acetylcholine receptor. J Pharmacol Exp Ther 2011, 336, 560-74.

Page 60: Novel Covalent Probes For Mapping α7 Nicotinic ...rx917k924/... · time drug discovery research process which I would not have had if I chose to graduate by just doing coursework.

60

64. Dukat, M.; Abdel-Rahman, A. A.; Ismaiel, A. M.; Ingher, S.; Teitler, M.; Gyermek, L.; Glennon, R. A. Structure-activity relationships for the binding of arylpiperazines and arylbiguanides at 5-HT3 serotonin receptors. J Med Chem 1996, 39, 4017-26. 65. Dukat, M.; Glennon, R. A.; Young, S. MD-354: what is it good for? CNS Drug Rev 2007, 13, 1-20. 66. Abdrakhmanova, G. R.; Blough, B. E.; Nesloney, C.; Navarro, H. A.; Damaj, M. I.; Carroll, F. I. In vitro and in vivo characterization of a novel negative allosteric modulator of neuronal nAChRs. Neuropharmacology 2010, 59, 511-7. 67. Eisele, J. L.; Bertrand, S.; Galzi, J. L.; Devillers-Thiery, A.; Changeux, J. P.; Bertrand, D. Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. Nature 1993, 366, 479-83. 68. Gee, V. J.; Kracun, S.; Cooper, S. T.; Gibb, A. J.; Millar, N. S. Identification of domains influencing assembly and ion channel properties in alpha 7 nicotinic receptor and 5-HT3 receptor subunit chimaeras. Br J Pharmacol 2007, 152, 501-12. 69. Young, G. T.; Zwart, R.; Walker, A. S.; Sher, E.; Millar, N. S. Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site. Proc Natl Acad Sci U S A 2008, 105, 14686-91. 70. Taly, A.; Delarue, M.; Grutter, T.; Nilges, M.; Le Novere, N.; Corringer, P. J.; Changeux, J. P. Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism. Biophys J 2005, 88, 3954-65. 71. Cheng, X.; Lu, B.; Grant, B.; Law, R. J.; McCammon, J. A. Channel opening motion of alpha7 nicotinic acetylcholine receptor as suggested by normal mode analysis. J Mol Biol 2006, 355, 310-24. 72. Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 2005, 346, 967-89. 73. Taly, A.; Corringer, P. J.; Grutter, T.; Prado de Carvalho, L.; Karplus, M.; Changeux, J. P. Implications of the quaternary twist allosteric model for the physiology and pathology of nicotinic acetylcholine receptors. Proc Natl Acad Sci U S A 2006, 103, 16965-70. 74. Ziebell, M. R.; Nirthanan, S.; Husain, S. S.; Miller, K. W.; Cohen, J. B. Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic. J Biol Chem 2004, 279, 17640-9. 75. Garcia, G., 3rd; Chiara, D. C.; Nirthanan, S.; Hamouda, A. K.; Stewart, D. S.; Cohen, J. B. [3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure. Biochemistry 2007, 46, 10296-307. 76. <Ligand based structural studies of the CB1 can.pdf>. 77. Korshunova, G. A.; Sumbatian, N. V.; Topin, A. N.; McHedlidze, M. T. [Photoactivated reagents based on aryl(trifluoromethyl)diazirines: synthesis and use for studying nucleic acid-protein interactions]. Mol Biol (Mosk) 2000, 34, 966-83. 78. Platz, M.; Admasu, A. S.; Kwiatkowski, S.; Crocker, P. J.; Imai, N.; Watt, D. S. Photolysis of 3-aryl-3-(trifluoromethyl)diazirines: a caveat regarding their use in photoaffinity probes. Bioconjug Chem 1991, 2, 337-41. 79. Pomper, M. G.; Phillips, E.; Fan, H.; McCarthy, D. J.; Keith, R. A.; Gordon, J. C.; Scheffel, U.; Dannals, R. F.; Musachio, J. L. Synthesis and biodistribution of radiolabeled alpha 7 nicotinic acetylcholine receptor ligands. J Nucl Med 2005, 46, 326-34. 80. Thakur, G. A.; Kulkarni, A. R.; Deschamps, J. R.; Papke, R. L. Expeditious Synthesis, Enantiomeric Resolution, and Enantiomer Functional Characterization of (4-(4-Bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (4BP-TQS): An Allosteric Agonist-Positive Allosteric Modulator of alpha7 Nicotinic Acetylcholine Receptors. J Med Chem 2013. 81. Spletstoser, J. T.; Flaherty, P. T.; Himes, R. H.; Georg, G. I. Synthesis and anti-tubulin activity of a 3'-(4-azidophenyl)-3'-dephenylpaclitaxel photoaffinity probe. J Med Chem 2004, 47, 6459-65