1 Resurrection of Organophosphorus-Aged Acetylcholinesterase Via Mannich Bases Derived from Proline Thesis Presented in Partial

Home , Novichok agent

Resurrection of Organophosphorus-Aged Acetylcholinesterase via Mannich Bases

Derived from Proline

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Nathan A. Ward

Graduate Program in Chemistry

The Ohio State University

2019

Thesis Committee

Dr. Christopher M. Hadad, Advisor

Dr. Jon R. Parquette, Member

Copyrighted by

Nathan A. Ward

2019

Abstract

Organophosphorus (OP) compounds are responsible for an estimated 220,000 deaths annually. OP compounds inhibit the enzyme acetylcholinesterase (AChE) via phosphylation of a serine residue within the active site. Upon inhibition of AChE, acetylcholine accumulates in neuromuscular junctions, resulting in a cholinergic crisis and, if not treated immediately, can lead to death. Along with management of symptoms, current medical countermeasures for OP poisoning utilize nucleophilic oximes, which can displace the phosphorus moiety from the serine residue, reactivating AChE to its native state. However, following inhibition, a spontaneous O-dealkylation of the phosphylated serine residue can occur, resulting in an anionic phosphylated serine residue; this process is known as aging. There is currently no approved therapeutic that is capable of restoring OP-aged AChE to its native state (a process referred to as resurrection). Previous studies by our group have shown that a class of compounds called quinone methide precursors (QMPs) both resurrect OP-aged AChE and reactivate OP- inhibited AChE back to its native state.

A previous lead QMP from the Hadad group featured a pyrrolidine leaving group, while the current lead QMP features an (R)-2-methylpyrrolidine leaving group. Thus, the addition of a methyl group at the 2-position of the pyrrolidine ring with the (R)- configuration increased the resurrection efficacy of OP-aged AChE. This work describes the synthesis of novel QMPs that have been derived from proline. Proline was derivatized to vary the functional group at the 2-position of the pyrrolidine ring in an effort to further increase the resurrection efficacy of OP-aged AChE.

iii

More specifically, proline was derivatized to feature ester, amide, alcohol, ether, and amine functional groups. Then, the QMPs were synthesized via a Mannich reaction between 3-hydroxypyridine and the proline derivatives. The QMPs were screened against

OP-aged AChE and the resurrection efficacy was measured via Ellman’s assay.

Acknowledgments

To all my friends and family who have supported me on this arduous journey: thank you.

To my advisor, Dr. Christopher Hadad – thank you for giving me the opportunity to do research in your laboratory, and for the guidance you have provided over the past two years.

To Dr. Christopher Callam – thank you for giving me the opportunity to teach organic chemistry recitation for you; it was an invaluable experience. Thank you for all the organic synthesis advice you have given me, as well.

To everyone in the Hadad, Callam, and Dogan Ekici groups – thank you for being so supportive and for making the laboratory a fun place to work every day.

To Curt Wong – thanks for being a good friend, and for all of the synthesis advice. Also, thanks for convincing me that I was being stupid every time I even thought about quitting graduate school.

Finally, I’d especially like to thank my best friend and partner, Nicole González

Salguero – I would never have gotten this far without your endless love and support.

You’ve inspired and motivated me since the very first day of this journey. I love you.

Vita

2016…………………………………………B.S. Chemistry (ACS Certified), Northern

Kentucky University

2016 – 2017…………………………………HPLC Chemist, Advanced Testing

Laboratory

2017 – 2019…………………………………Graduate Teaching Assistant, Department

of Chemistry and Biochemistry, The Ohio

State University

Fields of Study

Major Field: Chemistry

Table of Contents

Abstract ...... iii Acknowledgments ...... v Vita ...... vi List of Tables ...... viii List of Figures ...... ix Chapter 1: Introduction ...... 1 Chapter 2: Mannich Base QMPs Derived from Proline Esters and Amides ...... 13 Chapter 3: Mannich Base QMPs Derived from Prolinol Ethers and Amines ...... 43 Chapter 4: Future Work ...... 58 Bibliography ...... 61 Appendix: NMR Spectra ...... 65

vii

List of Tables

Table 1.1 Aging half-times of various OP compounds.19 ...... 8 Table 2.1 Data for the resurrection (24 h) of DFP-aged recombinant human AChE with Mannich base QMPs derived from proline esters and amides. Each QMP was used at a 1 mM concentration at pH 7.5 and 37 °C. The standard deviation is derived from four replicate samples. Screening data for QMP-2.2 is shown for reference. N.D. = Not Determined...... 21 Table 3.1 Data for the resurrection of DFP-aged recombinant human AChE with Mannich base QMPs (1 mM, pH 7.5, 37 °C, 24 h) derived from prolinol. Screening data for QMP-2.2 is shown for reference. The negative control consisted of DFP-aged hAChE with no QMP. The positive control consisted of native hAChE. The standard deviation was determined from 4 replicate samples...... 44

viii

List of Figures

Figure 1.1 Chemical structures of DFP, PM, and PE...... 1 Figure 1.2 Chemical structures of some common G-series and V-series nerve agents. .... 3 Figure 1.3 Mechanism of AChE-catalyzed hydrolysis of ACh...... 5 Figure 1.4 Common oximes used for the treatment of OP-inhibited AChE...... 6 Figure 1.5 Mechanism of inhibition and aging of AChE by an OP compound...... 7 Figure 1.6 Resurrection of OP-aged AChE by QMPs...... 9 Figure 2.1 The chemical structures of two lead Mannich base QMPs that are capable of resurrecting OP-aged AChE...... 13 Figure 2.2 General synthesis route to make Mannich base QMPs based on various proline derivatives...... 15 Figure 2.3 General reaction scheme for the synthesis of Mannich base QMPs derived from various proline ester derivatives and the chemical structures of all synthesized ester variants...... 16 Figure 2.4 General reaction scheme for the synthesis of some Mannich base QMPs derived from various proline amide derivatives and the chemical structures of the QMPs synthesized from this route...... 18 Figure 2.5 Reaction scheme for the synthesis of Mannich base QMPs derived from L- prolinamide and D-prolinamide...... 19 Figure 2.6 The reactions of Ellman’s assay...... 20 Figure 2.7 Resurrection of DFP-aged AChE with Mannich base QMPs derived from proline esters and amides. Screening data for QMP-2.5, QMP-2.7, and QMP-2.14 is still pending...... 22 Figure 3.1 General reaction scheme for the synthesis of Mannich base QMPs derived from prolinol and the chemical structures for each variant synthesized...... 43 Figure 3.2 Resurrection of DFP-aged AChE with Mannich base QMPs derived from prolinol...... 45 Figure 3.3 General reaction scheme for the synthesis of Mannich base QMPs derived from various prolinol ether derivatives and the chemical structures of the QMPs synthesized from this route...... 46 Figure 3.4 General reaction scheme for the synthesis of Mannich base QMPs derived from various prolinol amine derivatives and the chemical structures of the QMPs synthesized from this route...... 48 Figure 4.1 Proposed scheme for synthesizing various Mannich base QMPs derived from alkylated pyrrolidines...... 60

Chapter 1: Introduction

Organophosphorus (OP) compounds are members of an extremely toxic class of compounds that have been deployed as pesticides and nerve agents. OP compounds target the central nervous system and exposure can result in permanent damage to the nervous system and death. The most common mode of OP exposure is through pesticides such as diisopropyl fluorophosphate (DFP), methyl paraoxon, and ethyl paraoxon (Figure 1.1).

Globally, there is an estimated three million cases of pesticide poisonings per year with approximately 220,000 deaths.1,2

O N O N O 2 O 2 O P P P O O O O O O F O O

Diisopropyl fluorophosphate (DFP) Paraoxon-methyl (PM) Paraoxon-ethyl (PE)

Figure 1.1 Chemical structures of DFP, PM, and PE.

While OP nerve agents do not account for as many deaths as pesticides, they are still a persistent threat to civilian and military populations. The first reported OP nerve agent, tabun (GA, Figure 1.2), was synthesized by Gerald Schrader in 1936 when he was attempting to develop new phosphorus- and cyanide-based pesticides. Although a poor pesticide, tabun exhibited lethal toxicity in mammals and could cause death within 20 minutes. This deadly compound caught the attention of the German military, prompting weaponization of the OP, along with research and development into structural variants. In

1939, Schrader reported a new OP, sarin (GB, Figure 1.2), which was both more volatile

1 and twice as toxic as tabun. In 1943, German chemist and Nobel Laureate Richard Kuhn synthesized soman (GD), an OP that was reported to be even more toxic than sarin. In the

1950s, the United States and Great Britain synthesized an analog of sarin, cyclosarin

(GF).3

The aforementioned OP agents are part of what is known as the G-series, where

“G” represents “German.” After World War II, a new class of OPs, referred to as the V- series where “V” represents “venomous,” was developed. Current members of the V- series include VX, Russian-VX (also called VR), and Chinese-VX (CVX) (Figure 1.2).

The V-series were reported to be even more toxic than the G-series. VX in particular has been shown to be three times as toxic as sarin via inhalation and one thousand times more toxic via adsorption through the skin. It is estimated that less than 10 milligrams of VX is needed to kill an adult within 15 minutes. Furthermore, due to the V-series low volatility, they can persist for a longer period of time; they have no odor, so it is more difficult to detect them; and they can be deployed as an aerosol. VX was stockpiled by the United

States during the Cold War, while the Soviet Union stockpiled its isomer, VR.3,4 In addition to G-series and V-series nerve agents are the Novichok agents; these OP compounds originate from Russia, but not much is known about them. It has been reported that the toxicity of the Novichok agents exceeds that of VX by five to eight times.5

O O O P O N O P P P O O F F O N F

Tabun (GA) Sarin (GB) Soman (GD) Cyclosarin (GF)

O O O N P N P S N P S O S O O

VX VR CVX

Figure 1.2 Chemical structures of some common G-series and V-series nerve agents.

The toxicity of OP compounds has unfortunately been exploited by various governments and terrorist organizations. During the Iran-Iraq War (1980-1988), the Iraqi government deployed over 140 tons of tabun and 600 tons of sarin against Iranian forces and civilians, resulting in nearly 5,000 deaths and 100,000 injuries. At the end of the war in 1988, Iraq deployed nerve agents against its own citizens in the Kurdish city of

Halabja, killing between 3,200 and 5,000 people and injuring between 7,000 and 10,000 others.6,7 In 1995, Tokyo, Japan suffered a sarin attack perpetrated by the Aum Shinrikyo cult when the OP agent was released in five subway commuter cars, killing 12 people and injuring nearly 3,800 others.8 In 2013, the Syrian government used sarin in an attack against Ghouta during the Syrian Civil War, killing over 1,000 people and injuring approximately 3,600.9 In 2017, the Syrian government once again used sarin in an attack against Khan Shaykhun, killing at least 83 people and injuring nearly 300 others.10 In the same year, North Korean dictator Kim Jong-un’s half-brother Kim Jong-nam was 3 assassinated when two women smeared VX on his face at the Kuala Lumpur airport.11

Most recently, in 2018, an assassination was attempted on Sergei Skripal, a former

Russian spy who became an agent for MI6, using a Novichok class nerve agent. Sergei, who was residing in Salisbury, UK at the time, and his daughter, Yulia, who was visiting from Moscow, were both exposed to the OP compound and fell into critical condition; however, they fortunately survived the attack.12 However, on June 30, 2018, in

Amesbury, UK, Dawn Sturgess and her partner Charlie Rowley were hospitalized after

Dawn found a perfume bottle and sprayed the contents onto her wrists. Unbeknownst to her, the perfume was contaminated with a Novichok agent. Sturgess died in the hospital in July while Rowley survived. It is believed that the contaminated perfume bottle that killed Sturgess is linked to the assassination attempted on Sergei Skripal.13

The toxicity of OP compounds is attributed to their ability to inhibit the enzyme acetylcholinesterase (AChE). The native function of AChE is to catalyze the hydrolysis of the neurotransmitter acetylcholine (ACh), which binds to receptors at neuromuscular junctions and triggers a neuro or muscular response. At neurosynaptic junctions, AChE hydrolyzes ACh into acetic acid and choline, effectively terminating the transmission of the synaptic signal and preventing overstimulation.14

The active site of AChE can be found 20 Å down a narrow gorge, which contains what is known as the catalytic triad of three amino acids: serine (S203), histidine

(H447), and glutamate (E334). The catalytic triad is responsible for the mechanism of action by which AChE hydrolyzes ACh (Figure 1.3). S203 first performs nucleophilic attack at the carbonyl carbon of ACh, forming a tetrahedral intermediate. Upon collapse

4 to reform the carbonyl group, the leaving group (choline) is released with the aid of a proton transfer from H447. H447 then activates water, which attacks the carbonyl, forming another tetrahedral intermediate. Then, acetic acid is released and S203 is free to bind to another molecule of ACh. The role of E334 has been hypothesized to stabilize the transition states and intermediates formed during the hydrolysis, namely the histidinium species.14,15

O N N N O HO HO H O O O N H N N NH NH NH O H O O

AChE AChE AChE

OH O HO N H N OH NH O NH

AChE AChE

Figure 1.3 Mechanism of AChE-catalyzed hydrolysis of ACh.

OP compounds inhibit AChE via phosphylation of S203 within the active site of the enzyme. Upon phosphylation, AChE is inhibited and can no longer catalyze the hydrolysis of ACh, thereby causing an accumulation of the neurotransmitter – thus, leading to a cholinergic crisis and possibly death. Current therapeutics to counteract OP poisoning involve a combination of atropine (an antimuscarinic drug), diazepam (an

5 anticonvulsant drug), and a nucleophilic oxime. Oximes (or oximates, their deprotonated form) attack the phosphorus center of the OP-serine adduct, causing the OP to leave as an

OP-oxime adduct, returning the S203 residue to its native state – a process referred to as reactivation. There are several oximes that are administered for treatment of OP exposure, namely pyridinium oximes (Figure 1.4). Pralidoxime (2-PAM) chloride is the only oxime approved by the US FDA.16,17 One significant disadvantage of these pyridinium oximes is the fact that they are permanently charged, are therefore very hydrophilic, and are not effective at crossing the lipophilic blood-brain barrier in order to reactivate OP-inhibited AChE in the central nervous system.18

OH N N O N X N N N HO OH N O N HO N O X X NH2 X X

Pralidoxime (2-PAM) Obidoxime (LuH-6) Asoxime (HI-6)

Figure 1.4 Common oximes used for the treatment of OP-inhibited AChE.

After a period of time that is dependent upon the OP, a side reaction can occur following the inhibition of AChE. A spontaneous O-dealkylation of the phosphylated

S203 residue can result in an anionic phosphylated S203 residue – a process referred to as aging (Figure 1.5). In the aged state, the phosphorus center is resistant to nucleophilic attack by oximes, preventing recovery of the native enzyme. The aging time varies between OP compounds (Table 1.1). For example, the aging half-life of VR is 139 hours

(5.8 days) while for soman, only a mere 4 minutes.19 After about 70 years of active research, there are still no approved therapeutics that are capable of restoring OP-aged

AChE to its native state.

X O X R O P O 2 R2 O R O R2 O 1 P P O P O H N HN HN aging N NH R1 NH NH NH O H O R1 O R1 O dealkylation

AChE AChE AChE AChE

OP-inhibited AChE OP-aged AChE

N O R O R P N O N O R1 O O R2 O R2 R2 P O P O N H N HN R OH NH 1 O NH R1 O NH reactivation

AChE AChE AChE

Figure 1.5 Mechanism of inhibition and aging of AChE by an OP compound.

Table 1.1 Aging half-times of various OP compounds.19

OP Aging half-time (h)

Methyl paraoxon 3.7

Ethyl paraoxon 31.5

Tabun (GA) 19.2

Sarin (GB) 3.0

Soman (GD) 0.07

Cyclosarin (GF) 7.0

VX 36.5

VR 138.6

CVX 32.2

Studies from the Hadad group have shown that a class of compounds called quinone methide precursors (QMPs) are capable of restoring OP-aged AChE back to its native state20 – a process referred to as resurrection.21,22 QMPs can form quinone methides (QMs), which are alkylating agents that we hypothesized to realkylate the anionic phosphylated S203 residue, thereby resurrecting aged AChE back to its inhibited form, which is then reactivated back to the native form. In other words, QMPs are not only capable of resurrecting OP-aged AChE, but also reactivating OP-inhibited AChE back to its native state (Figure 1.6).20 To date, such QMPs are the only compounds capable of resurrecting OP-aged AChE. Furthermore, an additional advantage of these

QMPs is that they do not feature a permanent positive charge, potentially enabling them to cross the blood-brain barrier more easily and resurrect OP-aged AChE in the central nervous system.

O S203 P O O O S203 N N N OP-aged AChE N P reactivation S203 O O realkylation OH O HO OH QMP QM OP-inhibited AChE Native AChE

Figure 1.6 Resurrection of OP-aged AChE by QMPs.

The initial QMP in Figure 1.6 has been shown to resurrect OP-aged AChE.

Results from the Hadad group have shown that QMPs featuring a pyrrolidine moiety exhibited a higher resurrection efficacy than QMPs featuring a piperidine moiety or an acyclic amine. Additionally, a methyl group at the 2-position of the pyrrolidine ring enhanced the resurrection efficacy even further. Thus, we wished to explore QMPs that featured pyrrolidine moieties with various substituents at the 2-position of the ring.

Conveniently, proline is an amino acid that is a pyrrolidine ring that features a carboxylic acid at the 2-position. Proline, then, can be derivatized to feature a variety of substituents at the 2-position, and the derivatives can then be used as synthetic precursors to create a library of new QMPs.

Proline was first derivatized in such a way so that it still featured a carbonyl- containing functional group at the 2-position of the ring, such as esters and amides (more details in Chapter 2). Then, reduced versions of proline and its derivatives were synthesized, such as alcohols, ethers, and amines (more details in Chapter 3).

By derivatizing proline and altering what substituents are at the 2-position, we hoped to learn why the 2-position is important for resurrecting OP-aged AChE, and what functional groups result in the highest resurrection efficacy. Once the most effective substituent is determined, then the aromatic framework will be optimized (more details in

Chapter 4).

References for Chapter 1

(1) Jeyaratnam, J. Acute Pesticide Poisoning: A Major Global Health Problem. Wld.

hlth. quart. 1990, 43, 139–144.

(2) Eddleston, M.; Buckley, N. A.; Eyer, P.; Dawson, A. H. Management of Acute

Organophosphorus Pesticide Poisoning. Lancet 2008, 371 (9612), 597–607.

(3) Tucker, J. B. War of Nerves; Anchor, 2007.

(4) Franjesevic, A. J.; Sillart, S. B.; Beck, J. M.; Vyas, S.; Callam, C. S.; Hadad, C. M.

Resurrection and Reactivation of Acetylcholinesterase and Butyrylcholinesterase.

Chem. - A Eur. J. 2018, 1–36.

(5) Vásárhelyi, G.; Földi, L. History of Russia’s Chemical Weapons. Aarms 2007, 6

(1), 135–146.

(6) Central Intelligence Agency. Iraq’s Chemical Warfare Program

https://www.cia.gov/library/reports/general-reports-1/iraq_wmd_2004/chap5.html

(accessed Feb 7, 2019).

(7) Stone, R. Chemical Martyrs. Science 2018, 359 (6371), 20–25.

(8) Olson, K. B. Aum Shinrikyo: Once and Future Threat? Emerg. Infect. Dis. 1999, 5

(4), 413–416.

(9) The White House. Government Assessment of the Syrian Government’s Use of

Chemical Weapons on August 21, 2013 https://www.whitehouse.gov/the-press-

office/2013/08/30/government-assessment-syrian-government-s-use-chemical-

weapons-august-21 (accessed Feb 7, 2019).

(10) Brooks, J.; Erickson, T. B.; Kayden, S.; Ruiz, R.; Wilkinson, S.; Burkle, F. M.

Responding to Chemical Weapons Violations in Syria: Legal, Health, and

Humanitarian Recommendations. Confl. Health 2018, 12 (1), 1–7.

(11) Nauert, H. Imposition of Chemical and Biological Weapons Control and Warfare

Elimination Act Sanctions on North Korea

https://www.state.gov/r/pa/prs/ps/2018/03/279079.htm (accessed Feb 7, 2019).

(12) Asthana, A.; Roth, A.; Harding, L.; MacAskill, E. Russian Spy Poisoning: Theresa

May Issues Ultimatum to Moscow. The Guardian. March 13, 2018.

(13) BBC News. Russian Spy Poisoning: What We Know so Far. October 8, 2018.

(14) Yanzi, Z.; Shenglong, W.; Yingkai, Z. Catalytic Reaction Mechanism of

Acetylcholinesterase Determined by Born-Oppenheimer Ab Initio QM/MM

Molecular Dynamics Simulations. J. Phys. Chem. B 2015, 27 (3), 320–331,

(15) Beck, J. M.; Hadad, C. M. Reaction Profiles of the Interaction between Sarin and

Acetylcholinesterase and the S203C Mutant: Model Nucleophiles and QM/MM

Potential Energy Surfaces. Chem. Biol. Interact. 2010, 187 (1–3), 220–224.

(16) Mercey, G.; Verdelet, T.; Renou, J.; Kliachyna, M.; Baati, R.; Nachon, F.; Jean,

L.; Renard, P. Y. Reactivators of Acetylcholinesterase Inhibited by

Organophosphorus Nerve Agents. Acc. Chem. Res. 2012, 45 (5), 756–766.

(17) Agency for Toxic Substances and Disease Registry. Cholinesterase Inhibitors:

Including Pesticides and Chemical Warfare Nerve Agents; 2007.

(18) Kalász, H.; Nurulain, S. M.; Veress, G.; Antus, S.; Darvas, F.; Adeghate, E.;

Adem, A.; Hashemi, F.; Tekes, K. Mini Review on Blood-Brain Barrier

Penetration of Pyridinium Aldoximes. J. Appl. Toxicol. 2015, 35 (2), 116–123.

(19) Worek, F.; Thiermann, H. The Value of Novel Oximes for Treatment of Poisoning

by Organophosphorus Compounds. Pharmacol Ther. 2013, 139, 249–259.

(20) Zhuang, Q.; Franjesevic, A. J.; Corrigan, T. S.; Coldren, W. H.; Dicken, R.; Sillart,

S.; Deyong, A.; Yoshino, N.; Smith, J.; Fabry, S.; Fitzpatrick, K.; Blanton, T.G.;

Joseph, J.; Yoder, R. J.; McElroy, C. A.; Ekici, Ö. D.; Callam, C. S.; Hadad, C. M.

Demonstration of in Vitro Resurrection of Aged Acetylcholinesterase after

Exposure to Organophosphorus Chemical Nerve Agents. J. Med. Chem. 2018, 61

(16), 7034–7042.

(21) Topczewski, J. J.; Quinn, D. M. Kinetic Assessment of N-Methyl-2-

Methoxypyridinium Species as Phosphonate Anion Methylating Agents. Org. Lett.

2013, 15 (5), 1084–1087.

(22) Topczewski, J. J.; Lodge, A. M.; Yasapala, S. N.; Payne, M. K.; Keshavarzi, P.

M.; Quinn, D. M. Reversible Inhibition of Human Acetylcholinesterase by

Methoxypyridinium Species. Bioorganic Med. Chem. Lett. 2013, 23 (21), 5786–

5789.

Chapter 2: Mannich Base QMPs Derived from Proline Esters and Amides

During the last several years, the Hadad research group has synthesized hundreds of QMPs via a Mannich reaction between an aromatic framework and an amine moiety.

The first such QMP that was capable of significantly resurrecting OP-aged AChE was a

Mannich base that featured a 3-hydroxypyridine framework and a pyrrolidine moiety

(QMP-2.1), depicted in Figure 2.1. This QMP was capable of resurrecting approximately 12% methylphosphonate-aged electric eel AChE (eeAChE) in vitro, and approximately 5% isopropyl phosphate-aged eeAChE.1 The resurrection efficacy of

QMP-2.1 was then evaluated in vitro against human AChE (hAChE) that was aged with various OP pesticides, such as DFP, PM, and PE (Figure 1.1), as well as the OP nerve agent analogue cyclohexyl methylphosphonate ester (CMP), a simulant of the G-agent cyclosarin. QMP-2.1 was observed to resurrect approximately 4%, 1%, 0.7%, and 2% relative activity of PM-aged, PE-aged, DFP-aged, and CMP-aged hAChE, respectively.

N N N N

OH OH

QMP-2.1 QMP-2.2

Figure 2.1 The chemical structures of two lead Mannich base QMPs that are capable of resurrecting OP-aged AChE.

Modification of the scaffold to feature a (R)-2-methylpyrrolidine ring rather than a pyrrolidine ring resulted in QMP-2.2 (Figure 2.1), which was observed to resurrect

13 approximately 14%, 9%, 10%, and 3% relative activity of PM-aged, PE-aged, DFP-aged, and CMP-aged human AChE, respectively. These results were encouraging as Anglister et al.2 suggested that 10% of AChE recovery is all that is needed to retain normal neuronal communication at the neuromuscular junction; therefore, a therapeutically relevant recovery was becoming within reach. Thus, the addition of a (R)-methyl group at the 2-position of the pyrrolidine ring significantly enhanced the resurrection efficacy of the QMP, prompting an investigation into what functional groups on the pyrrolidine ring could enhance the efficacy of resurrection, as well as to determine the importance of the stereochemistry at the position.

The goal of this project was to synthesize a library of QMPs that share the same structural framework as QMP-2.1 but with varying substituents at the 2-position of the pyrrolidine ring. A synthetic route was established for these compounds by utilizing the amino acid proline as a structural foundation.

Proline is a pyrrolidine ring that features a carboxylic acid at the 2-position

(Figure 2.2). The carboxylic acid functional group can be converted to a variety of other functional groups, leading to various proline derivatives that can be reacted further in a

Mannich reaction with 3-hydroxypyridine and paraformaldehyde to produce the desired

QMPs (Figure 2.2). Furthermore, the 2-position of proline is a chiral center, meaning that both enantiomers, L-proline and D-proline, can be used to synthesize two enantiomers of each desired QMP. Even more important is that both L-proline and D- proline are relatively cheap, commercially available compounds that can be purchased

14 from most major chemical suppliers. All of these factors contribute to proline being an ideal starting material for creating this QMP library.

OH O R derivatize paraformaldehyde N R N N OH N H H Mannich reaction OH

Proline Proline Derivative QMP

Figure 2.2 General synthesis route to make Mannich-base QMPs using various proline derivatives as the source of the chiral amine.

This chapter is focused on the synthesis and screening of proline ester and amide derivatives. The synthesis of the ester derivatives was done by first dissolving proline in the desired alcohol followed by slow addition of thionyl chloride and refluxing for three hours. The proline ester derivative was then either reacted in a Mannich reaction with 3- hydroxypyridine and paraformaldehyde in benzene, and refluxing for 12 hours, or in an

SN2 reaction with 2-(bromomethyl)pyridine-3-ol hydrobromide, potassium carbonate, and acetonitrile, and refluxing for 12 hours. Purification via flash column chromatography yielded the pure QMP. (R)- and (S)-enantiomers of methyl, ethyl, and isopropyl ester variants were synthesized (Figure 2.3).

1. a) O OR O 2. b) or c) N N N OH H OH R = CH3 CH2CH3 CH(CH3)2 Conditions and reagents: a) SOCl2, ROH, reflux, 3 h b) 3-hydroxypyridine, paraformaldehyde, benzene, reflux, 12 h c) 2-(bromomethyl)pyridin-3-ol hydrobromide, K2CO3, MeCN, reflux, 12 h

O O O O O O O O

N (S) N (R) N (S) N (R) N N N N

OH OH OH OH

QMP-2.3 QMP-2.4 QMP-2.5 QMP-2.6

O O O O

N (S) N (R) N N

OH OH

QMP-2.7 QMP-2.8

Figure 2.3 General reaction scheme for the synthesis of Mannich-base QMPs derived from various proline ester derivatives and the chemical structures of all synthesized ester variants.

The synthesis of the amide proline derivatives was done by several different methods. One method involved dissolving carboxybenzyl-protected proline (Cbz-proline) in benzene, followed by slow addition of thionyl chloride, and then stirring the mixture at reflux for 2 hours. After workup, the acyl chloride was dissolved in dichloromethane, the

16 desired amine and triethylamine were added, and then the mixture was stirred at room temperature (23 °C) for 12 hours.

Another method involved dissolving Cbz-proline in ethyl acetate, cooling to 0 °C, and adding ethyl chloroformate, N-methylmorpholine, and the desired amine.

Alternatively, Cbz-proline was also dissolved in tetrahydrofuran, cooled to -78 °C, and isobutyl chloroformate, N-methylmorpholine, and the desired amine were added. The mixtures were then stirred at 23 °C for 12 hours. After the amide was formed, hydrogenation removed the Cbz protecting group, producing the proline amide derivative. Finally, as with the proline ester derivatives, a Mannich reaction or SN2 was performed to furnish the QMP. Purification was performed via flash column chromatography. The (R)- and (S)-enantiomers of the dimethyl and diethyl carboxamide variants were synthesized (Figure 2.4).

O O

1. d) O N OH N NR NR a), b), or c) 2 2. e) or f) 2 O O N O O N

R = CH3 CH2CH3 Conditions and reagents: a) SOCl2, benzene, reflux, 2 h; R2NH, Et3N, CH2Cl2, 23 °C, 12 h b) Ethyl chloroformate, N-methylmorpholine, R2NH, EtOAc, 0 °C to 23 °C, 12 h c) Isobutyl chloroformate, N-methylmorpholine, R2NH, THF, -78 °C to 23 °C, 12 h (d) Pd/C, H2 (1 atm), MeOH, 23 °C, 24 h e) 3-hydroxypyridine, paraformaldehyde, benzene, reflux, 12 h f) 2-(bromomethyl)pyridin-3-ol hydrobromide, K2CO3, MeCN, reflux, 12 h

O O N N O N O N

N (S) N (R) N (S) N (R) N N N N

OH OH OH OH

QMP-2.9 QMP-2.10 QMP-2.11 QMP-2.12

Figure 2.4 General reaction scheme for the synthesis of some Mannich base QMPs derived from various proline amide derivatives and the chemical structures of the QMPs synthesized from this route.

A proline amide derivative that was not synthesized by the above route, but instead purchased directly from a chemical supplier, was L-prolinamide and D- prolinamide. These compounds were then used in a Mannich reaction with 3- hydroxypyridine and paraformaldehyde in benzene at reflux for 24 hours (Figure 2.5).

O NH2 O N paraformaldehyde N + N N NH H 2 OH benzene reflux 24 h OH

O O NH2 NH2 N (S) N (R) N N

OH OH

QMP-2.13 QMP-2.14

Figure 2.5 Reaction scheme for the synthesis of Mannich base QMPs derived from L- prolinamide and D-prolinamide.

The synthesized proline derivatives, except QMP-2.5, QMP-2.7, and QMP-2.14

(screening pending), were screened for their ability to resurrect DFP-aged AChE in vitro via Ellman’s assay (Figure 2.6). A negative control consisted of DFP-aged hAChE with no QMP. A positive control consisted of native hAChE which was not exposed to any

OP. All screening assays were performed by Dr. Qinggeng Zhuang and Dr. Andrew

Franjesevic. Prior to Ellman’s assay, hAChE is incubated with an OP compound for sufficient time such that all AChE has been aged. The OP-aged AChE is then incubated with a QMP for a finite period of time. Aliquots are withdrawn from the solution at different time points and assayed with Ellman’s reagent (5,5’-dithiobis-(2-nitrobenzoic acid), or DTNB) and acetylthiocholine. If the AChE has been resurrected back to its native state, it will catalyze the hydrolysis of acetylthiocholine into acetic acid and 19 thiocholine, which undergoes disulfide exchange with DTNB to prepare 2-nitro-5- thiobenzoic acid (TNB). TNB ionizes in neutral or basic aqueous solutions to 2-nitro-5- thiobenzoate (TNB2-) that can be quantified by absorbance at 412 nm. Thus, each mole of acetylthiocholine hydrolyzed produces one mole of TNB2-, allowing for the quantitative determination of AChE activity.3

O AChE O N + N S OH HS Acetylthiocholine Thiocholine

O OH O O N O OH O O N S N O HS HO S S + N HO S O O N O N O HS O Ellman’s Reagent 2-nitro-5-thiobenzoic acid (TNB) (DTNB)

O OH O O O H O O N 2 O N neutral or alkaline pH O HS S TNB 2-nitro-5-benzoate (TNB2-)

Figure 2.6 The reactions of Ellman’s assay.

Unfortunately, none of the ester or amide proline derivatives were able to resurrect DFP-aged AChE (Figure 2.7), even after 24 h. The presence of a carbonyl- containing functional group at the 2-position of the pyrrolidine ring appears to destroy the resurrection capabilities of the QMP. And these results are independent of the

20 stereochemistry at the 2-position, i.e., both (R) and (S) enantiomers yielded little to no resurrection of the aged form of hAChE.

Table 2.1 Data for the resurrection (24 h) of DFP-aged recombinant human AChE with

Mannich base QMPs derived from proline esters and amides. Each QMP was used at a 1 mM concentration at pH 7.5 and 37 °C. The standard deviation is derived from four replicate samples. Screening data for QMP-2.2 is shown for reference. N.D. = Not

Determined.

QMP Resurrected Activity (%) Standard Deviation

QMP-2.2 13.1 0.6

QMP-2.3 0.1 0.0

QMP-2.4 0.2 0.0

QMP-2.5 N.D. N.D.

QMP-2.6 0.2 0.0

QMP-2.7 N.D. N.D.

QMP-2.8 0.3 0.0

QMP-2.9 0.3 0.0

QMP-2.10 0.2 0.0

QMP-2.11 0.3 0.0

QMP-2.12 0.2 0.0

QMP-2.13 0.2 0.0

QMP-2.14 N.D. N.D.

Negative Control 0.2 0.0

Positive Control 100 1.0

Resurrection of DFP-Aged AChE

100 y

t 90 i

v 15

a l

e 5 R

0 2 3 4 6 8 9 0 1 2 3 l l ...... 1 1 1 1 o o -2 -2 -2 -2 -2 -2 . . . . tr tr P P P P P P -2 -2 -2 -2 n n P P P P o o M M M M M M c c Q Q Q Q Q Q M M M M e e Q Q Q Q v v ti ti a i g s e o n p

Figure 2.7 Resurrection (24 h) of DFP-aged recombinant human AChE with Mannich base QMPs (1 mM, pH 7.5, 37 °C) as derived from proline esters and amides. Screening data for QMP-2.5, QMP-2.7, and QMP-2.14 are still pending.

We therefore posited that there is some significant selectively at the 2-position of the pyrrolidine ring and that electron-withdrawing substituents (such as an ester or an amide) were ineffective. Thus, we considered reduced versions of these QMPs (i.e. ether and amine derivatives) as potential options for resurrection. Therefore, such derivatives were then explored and are discussed in the next chapter.

References for Chapter 2

(1) Zhuang, Q.; Franjesevic, A. J.; Corrigan, T. S.; Coldren, W. H.; Dicken, R.; Sillart,

S.; Deyong, A.; Yoshino, N.; Smith, J.; Fabry, S.; Fitzpatrick, K.; Blanton, T.G.;

Joseph, J.; Yoder, R. J.; McElroy, C. A.; Ekici, Ö. D.; Callam, C. S.; Hadad, C. M.

Demonstration of in Vitro Resurrection of Aged Acetylcholinesterase after

Exposure to Organophosphorus Chemical Nerve Agents. J. Med. Chem. 2018, 61

(16), 7034–7042.

(2) Anglister, L.; Stiles, J. R.; Salpeter, M. M. Acetylcholinesterase density and

turnover number at frog neuromuscular junctions, with modeling of their role in

neurosynaptic function. Neuron 1994, 12, 783-794.

(3) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70–77.

General Methods

Chemicals and solvents used in these experiments were purchased from Sigma-Aldrich,

Fisher Scientific, Acros Organics, Alfa Aesar, Matrix Scientific Synthonix, TCI, and

Combi-Blocks, and were used without further purification. Dried solvents and liquid reagents were transferred by oven-dried syringes or hypodermic syringes. Solvents were removed in vacuo under ~30 mmHg and heated with a water bath at 30-70 °C using a rotary evaporator. Unless otherwise stated, all reactions were performed under atmospheric pressure and monitored by TLC on silica gel 60Å F254 (0.25 mm). TLC plates were visualized under UV light at 254 nm or with potassium permanganate stain.

Chromatography was performed on silica gel 60 (40-60 µM) using Teledyne ISCO

1 13 CombiFlash RF+UV automated system. H and C NMR spectra were recorded on a 400

MHz Bruker spectrometer. Chemical shifts for 1H NMR are reported in parts per million

(ppm), using the residual solvent signal as a reference (CDCl3: 7.26 ppm; CD3OD: 3.31 ppm). 13C chemical shifts are reported in ppm, using the residual solvent signal as a reference (CDCl3: 77.16 ppm; CD3OD: 49.00 ppm). Multiplicities are given as: s

(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets), dt

(doublet of triplets), and br (broad). Coupling constants (J) were recorded in hertz (Hz).

High-resolution mass spectra (HRMS) were recorded on a Bruker MicroTOF II spectrometer using sodium formate as an internal standard under electrospray ionization

(ESI) conditions. HRMS was reported in units of mass of charge ratio (m/z). HRMS samples were dissolved in methanol.

O O N N

QMP-2.3

Methyl ((3-hydroxypyridin-2-yl)methyl)-L-prolinate (QMP-2.3) To a solution of methanol (30 mL) was added L-proline (504.2 mg, 4.38 mmol). The reaction mixture was cooled to 0 °C and thionyl chloride (0.35 mL, 4.80 mmol) was added dropwise. The reaction mixture was refluxed for 12 h and the solvent was removed in vacuo. The residue was re-dissolved in acetonitrile (30 mL), and to the mixture was added 2-

(bromomethyl)pyridine-3-ol hydrobromide (1.30 g, 4.84 mmol), and potassium carbonate

(1.82 g, 13.19 mmol). The reaction mixture was refluxed for 24 h and the solvent was removed in vacuo. The residue was re-dissolved in CH2Cl2 (30 mL) and the insoluble solids were removed by filtering through celite via vacuum filtration. The filtrate was concentrated in vacuo and the crude product was purified via flash chromatography

(100:0-95:5, CH2Cl2/MeOH) to yield QMP-2.3 (453.3 mg, 1.92 mmol, 44%) as a

1 brown oil. H NMR (400 MHz, CDCl3): δ 10.52 (br, 1H), 7.99 (dd, J = 2.6, 1.8 Hz, 1H),

7.14-7.07 (m, 2H), 4.33 (d, J = 14.0 Hz, 1H), 3.77 (s, 3H), 3.74 (d, J = 14.0 Hz, 1H), 3.41

(dd, J = 6.5, 2.8 Hz, 1H), 3.07-3.01 (m, 1H), 2.49-2.40 (m, 1H), 2.33-2.22 (m, 1H), 2.05-

13 1.94 (m, 1H), 1.93-1.84 (m, 2H). C NMR (400 MHz, CDCl3): δ 174.3, 154.1, 143.4,

139.9, 123.9, 123.3, 65.6, 60.1, 53.5, 52.5, 29.7, 23.8. HRMS ESI: m/z Calcd for

+ C12H17N2O3 [M+H] 237.1234, found 237.1224.

O O N N

QMP-2.4

Methyl ((3-hydroxypyridin-2-yl)methyl)-D-prolinate (QMP-2.4) To a solution of methanol (40 mL) was added D-proline (502.1 mg, 4.36 mmol). The reaction mixture was cooled to 0 °C and thionyl chloride (0.38 mL, 5.23 mmol) was added dropwise. The reaction mixture was refluxed for 12 h and the solvent was removed in vacuo. The residue was re-dissolved in acetonitrile (40 mL), and to the mixture was added 2-

(bromomethyl)pyridine-3-ol hydrobromide (250.8 mg, 0.93 mmol), and potassium carbonate (2.64 g, 19.12 mmol). The reaction mixture was refluxed for 24 h and the solvent was removed in vacuo. The residue was re-dissolved in CH2Cl2 (30 mL) and the insoluble solids were removed by filtering through celite via vacuum filtration. The filtrate was concentrated in vacuo and the crude product was purified via flash chromatography (100:0-95:5, CH2Cl2/MeOH) to yield QMP-2.4 (120.2 mg, 0.51 mmol,

1 55%). Yellow oil. H NMR (400 MHz, CDCl3): δ 10.56 (br, 1H), 7.99 (dd, J = 2.6, 1.8

Hz, 1H), 7.15-7.08 (m, 2H), 4.34 (d, J = 14.0 Hz, 1H), 3.78 (s, 3H), 3.74 (d, J = 14.0 Hz,

1H), 3.41 (dd, J = 6.5, 2.8 Hz, 1H), 3.08-3.01 (m, 1H), 2.49-2.42 (m, 1H), 2.33-2.22 (m,

13 1H), 2.06-1.95 (m, 1H), 1.94-1.84 (m, 2H). C NMR (400 MHz, CDCl3): δ 174.3, 154.1,

143.4, 139.9, 123.9, 123.3, 65.7, 60.2, 53.5, 52.5, 29.7, 23.8. HRMS ESI: m/z Calcd for

+ C12H17N2O3 [M+H ] 237.1234, found 237.1225.

O O N N

QMP-2.5

Ethyl ((3-hydroxypyridin-2-yl)methyl)-L-prolinate (QMP-2.5) To a solution of ethanol (30 mL) was added L-proline (502.0 mg, 4.36 mmol). The reaction mixture was cooled to 0 °C and thionyl chloride (0.54 mL, 7.44 mmol) was added dropwise. The reaction mixture was refluxed for 12 h and the solvent was removed in vacuo. The residue was re-dissolved in acetonitrile (30 mL), and to this mixture was added was

27 added 2-(bromomethyl)pyridine-3-ol hydrobromide (1.29 g, 4.80 mmol), and potassium carbonate (1.39 g, 10.03 mmol). The reaction mixture was refluxed for 24 h and the solvent was removed in vacuo. The residue was re-dissolved in CH2Cl2 and the insoluble solids were removed by filtering through celite via vacuum filtration. The filtrate was concentrated in vacuo and the crude product was purified via flash chromatography

(100:0-95:5, CH2Cl2/MeOH) to yield QMP-2.5 (216.3 mg, 0.86 mmol, 20%). Yellow oil.

1 H NMR (400 MHz, CDCl3): δ 10.56 (br, 1H), 7.99 (dd, J = 2.5, 1.8 Hz, 1H), 7.15-7.07

(m, 2H), 4.34 (d, J = 14.0 Hz, 1H), 4.27-4.18 (m, 2H), 3.73 (d, J = 14.0 Hz, 1H), 3.42-

3.33 (m, 1H), 3.07-2.98 (m, 1H), 2.49-2.37 (m,1H), 2.33-2.20 (m, 1H), 2.04-1.93 (m,

13 1H), 1.92-1.82 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H). C NMR (400 MHz, CDCl3): δ 173.8,

154.1, 143.4, 139.8, 123.9, 123.2, 65.8, 61.5, 60.1, 53.5, 29.7, 23.8, 14.3. HRMS ESI:

+ m/z Calcd for C13H19N2O3 [M+H] 251.1390, found 251.1379.

O O N N

QMP-2.6

Ethyl ((3-hydroxypyridin-2-yl)methyl)-D-prolinate (QMP-2.6) To a solution of ethanol (40 mL) was added D-proline (304.2 mg, 2.64 mmol). The reaction mixture was cooled to 0 °C and thionyl chloride (0.29 mL, 3.96 mmol) was added dropwise. The reaction mixture was refluxed for 12 h and the solvent was removed in vacuo. The residue was re-dissolved in acetonitrile (40 mL). To the mixture was added 2-

(bromomethyl)pyridine-3-ol hydrobromide (272.0 mg, 1.01 mmol), and potassium carbonate (1.45 g, 10.50 mmol). The reaction mixture was refluxed for 24 h and the solvent was removed in vacuo. The residue was re-dissolved in CH2Cl2 (30 mL) and the insoluble solids were removed by filtering through celite via vacuum filtration. The filtrate was concentrated in vacuo and the crude product was purified via flash chromatography (100:0-95:5, CH2Cl2/MeOH) to yield QMP-2.6 (53.5 mg, 0.21 mmol,

1 21%). Yellow oil. H NMR (400 MHz, CDCl3): δ 10.55 (br, 1H), 7.99 (dd, J = 2.5, 1.8

Hz, 1H), 7.15-7.07 (m, 2H), 4.38 (d, J = 14.0 Hz, 1H), 4.28-4.20 (m, 2H), 3.74 (d, J =

14.0 Hz, 1H), 3.42-3.35 (m, 1H), 3.08-3.00 (m, 1H), 2.49-2.40 (m,1H), 2.33-2.22 (m,

1H), 2.05-1.95 (m, 1H), 1.93-1.84 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H). 13C NMR (400 MHz,

CDCl3): δ 173.8, 154.1, 143.5, 139.8, 123.9, 123.3, 65.8, 61.5, 60.1, 53.5, 29.8, 23.8,

+ 14.3. HRMS ESI: m/z Calcd for C13H19N2O3 [M+H] 251.1390, found 251.1370.

O O N N

QMP-2.7

Isopropyl ((3-hydroxypyridin-2-yl)methyl)-L-prolinate (QMP-2.7) To a solution of isopropanol (30 mL) was added L-proline (599.5 mg, 6.30 mmol). The reaction mixture was cooled to 0 °C and thionyl chloride (0.35 mL, 4.80 mmol) was added dropwise. The reaction mixture was refluxed for 12 h and the solvent was removed in vacuo. The residue was re-dissolved in benzene (30 mL), and to the mixture was added 3- hydroxypyridine (599.5 mg, 6.30 mmol), and paraformaldehyde (189.3 mg, 6.30 mmol).

The reaction mixture was refluxed for 24 h and the solvent was removed in vacuo. The crude product was purified via flash chromatography (100:0-95:5, CH2Cl2/MeOH) to

1 yield QMP-2.7 (171.0 mg, 0.65 mmol, 15%). Yellow oil. H NMR (400 MHz, CDCl3): δ

10.58 (br, 1H), 7.99 (dd, J = 2.5 Hz, 1.8 Hz, 1H), 7.15-7.06 (m, 2H), 5.15-5.05 (m, 1H),

4.34 (d, J = 14.0 Hz, 1H), 3.73 (d, J = 14.0 Hz, 1H), 3.38-3.31 (m, 1H), 3.07-2.99 (m,

1H), 2.48-2.39 (m, 1H), 2.33-2.20 (m, 1H), 2.06-1.78 (m, 3H), 1.28 (dd, J = 4.2 Hz, 2.0

13 Hz, 6H). C NMR (400 MHz, CD3OD): δ 174.9, 155.5, 144.8, 140.0, 125.3, 124.8, 70.1,

66.7, 59.5, 54.3, 30.6, 24.5, 21.99, 21.95. HRMS (ESI): m/z Calcd

+ for C14H21N2O3 [M+H] 265.1547, found 265.1536.

O O N N

QMP-2.8

Isopropyl ((3-hydroxypyridin-2-yl)methyl)-D-prolinate (QMP-2.8) To a solution of isopropanol (40 mL) was added D-proline (301.7 mg, 2.62 mmol). The reaction mixture was cooled to 0 °C and thionyl chloride (0.29 mL, 3.93 mmol) was added dropwise. The reaction mixture was refluxed for 12 h and the solvent was removed in vacuo. The residue was re-dissolved in acetonitrile (40 mL). To the mixture was added 2-

(bromomethyl)pyridine-3-ol hydrobromide (279.4 mg, 1.04 mmol), and potassium carbonate (1.43 g, 10.34 mmol). The reaction mixture was refluxed for 24 h and the solvent was removed in vacuo. The residue was re-dissolved in CH2Cl2 and the insoluble solids were removed by filtering through celite via vacuum filtration. The filtrate was concentrated in vacuo and the crude product was purified via flash chromatography

(100:0-95:5, CH2Cl2:MeOH) to yield QMP-2.8 (69.9 mg, 0.26 mmol, 25%). Yellow oil.

1 H NMR (400 MHz, CDCl3): δ 10.57 (br, 1H), 7.99 (dd, J = 2.5 Hz, 1.8 Hz, 1H), 7.15-

7.07 (m, 2H), 5.16-5.05 (m, 1H), 4.34 (d, J = 14.0 Hz, 1H), 3.73 (d, J = 14.0 Hz, 1H),

3.39-3.31 (m, 1H), 3.09-2.98 (m, 1H), 2.49-2.38 (m, 1H), 2.33-2.20 (m, 1H), 2.02-1.93

(m, 1H), 1.92-1.83 (m, 2H), 1.28 (dd, J = 4.2 Hz, 2.0 Hz, 6H). 13C NMR (400 MHz,

CDCl3): δ 173.3, 154.2, 143.5, 139.8, 123.9, 123.3 69.1, 66.0, 60.2, 53.5, 29.8, 23.7, 21.9,

+ 21.9. HRMS ESI: m/z Calcd for C14H21N2O3 [M+H] ] 265.1547, found 265.1534.

N OH O O

INT-2.1

((Benzyloxy)carbonyl)-L-proline (INT-2.1) To an aqueous solution of sodium hydroxide (2 M, 40 mL) was added L-proline (5.01 g, 43.52 mmol) at 0 °C. To this solution was simultaneously added benzyl chloroformate (7.45 mL, 52.22 mmol) and more aqueous sodium hydroxide (2 M, 40 mL) over 15 minutes. The mixture was stirred for 1 h at 23 °C. The solution was washed with diethyl ether (2 × 50 mL), and then acidified to approximately pH 4 with 6 M HCl. The solution was extracted with ethyl acetate (2 × 50 mL) and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to yield INT-2.1 (7.68 g, 30.82 mmol, 71%).

1 Clear oil. H NMR (400 MHz, CDCl3): δ 9.80 (br, 1H), 7.36-7.30 (m, 5H), 5.21-5.13 (m,

2H), 5.44-4.36 (m, 1H), 3.66-3.42 (m, 2H), 2.33-2.06 (m, 2H), 2.03-1.84 (m, 2H). 13C

NMR (400 MHz, CDCl3): δ 178.0, 176.6, 155.7, 154.6, 136.5, 136.4, 128.6, 128.5, 128.2,

128.0, 127.7, 67.5, 67.2, 59.3, 58.7, 47.0, 46.7, 30.9, 29.6, 24.3, 23.5. HRMS ESI: m/z

+ Calcd for C13H16NO4 [M+H] 250.1074, found 250.1065.

N O O O

INT-2.2

Benzyl (S)-2-(dimethylcarbamoyl)pyrrolidine-1-carboxylate (INT-2.2) To a solution of benzene (30 mL) under an atmosphere of N2 was added INT-2.1 (2.01 g, 8.06 mmol).

Thionyl chloride (0.70 mL, 9.67 mmol) was added dropwise and the solution was refluxed for 2 h. The solvent was removed in vacuo. The resulting residue was dissolved in anhydrous CH2Cl2 (20 mL) under an atmosphere of N2 and the solution was cooled to

0 °C. To this solution was added dropwise dimethylamine (2.0 M in THF, 4.84 mL, 9.67 mmol) and triethylamine (2.25 mL, 16.12 mmol). The mixture was stirred for 12 hours at

23 °C and was then washed with aqueous ammonium chloride (2 × 20 mL), dried with anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified via flash chromatography (9:1, CH2Cl2/MeOH) to yield INT-2.2 (1.76 g, 6.37 mmol, 79%).

1 Orange oil. H NMR (400 MHz, CDCl3): δ 9.80 (br, 1H), 7.36-7.30 (m, 5H), 5.21-5.13

(m, 2H), 5.44-4.36 (m, 1H), 3.66-3.42 (m, 2H), 2.33-2.06 (m, 2H), 2.03-1.84 (m, 2H). 13C

NMR (400 MHz, CDCl3): δ 178.0, 176.6, 155.7, 154.6, 136.5, 136.4, 128.6, 128.5, 128.2,

128.0, 127.7, 67.5, 67.2, 59.3, 58.7, 47.0, 46.7, 30.9, 29.6, 24.3, 23.5. HRMS ESI: m/z

+ Calcd for C13H16NO4 [M+H] 250.1074, found 250.1065.

O N N N

QMP-2.9

(R)-1-((3-hydroxypyridin-2-yl)methyl)-N,N-dimethylpyrrolidine-2-carboxamide

(QMP-2.9) Into a Parr hydrogenator flask was added INT-2.2 (1.76 g, 6.37 mmol), palladium on activated carbon (10 wt. %) (82.0 mg, 0.77 mmol), and methanol (50 mL).

The air in the flask was vacuumed and backfilled with hydrogen three times before placing the reaction under 50 psi of hydrogen for 12 h. The reaction mixture was vacuum filtered through celite and washed with methanol. The solvent was removed in vacuo and the resulting residue was dissolved in acetonitrile (30 mL). To this mixture was added 2-

(bromomethyl)pyridine-3-ol hydrobromide (254.0 mg, 0.94 mmol) and potassium carbonate (3.00 g, 21.67 mmol). The mixture was refluxed for 24 hours and the solvent was removed in vacuo. The residue was re-dissolved in CH2Cl2 and the insoluble solids were removed by filtering through celite via vacuum filtration. The filtrate was concentrated in vacuo and the crude product was purified via flash chromatography

(100:0-95:5, CH2Cl2/MeOH) to yield QMP-2.9 (184.6 mg, 0.74 mmol, 79%). Yellow oil.

1 H NMR (400 MHz, CDCl3): δ 7.93 (dd, J = 3.0, 1.5 Hz, 1H), 7.14-7.04 (m, 2H),

4.22 (d, J = 13.4 Hz, 1H), 3.51 (d, J = 13.4 Hz, 1H), 3.48-3.43 (m, 1H), 3.02 (s, 3H),

3.00 (s, 3H), 2.99-2.95 (m, 1H), 2.41-2.33 (m, 1H), 2.28-2.18 (m, 1H), 1.87-1.74 (m,

13 3H). C NMR (400 MHz, CDCl3): δ 173.4, 154.2, 144.3, 139.2, 123.8, 123.4, 64.0, 59.2,

+ 52.9, 36.6, 36.1, 29.3, 23.7. HRMS ESI: m/z Calcd for C13H20N3O2 [M+H] 250.1550, found 250.1563.

O N O O

INT-2.3

Benzyl (R)-2-(dimethylcarbamoyl)pyrrolidine-1-carboxylate (INT-2.3) To a solution of THF (50 mL) was added N-Cbz-D-proline (1.00 g, 4.01 mmol). The mixture was cooled to 0 °C and N-methylmorpholine (0.53 mL, 4.81 mmol) and ethyl chloroformate

(0.33 mL, 4.81 mmol) were added dropwise followed by dimethylamine (2.0 M in THF,

0.83 mL, 8.04 mmol). The mixture was stirred for 12 h at 23 °C. The solvent was removed in vacuo and re-dissolved in water (30 mL). The mixture was extracted with ethyl acetate (3 × 30 mL) and washed with brine (30 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified via flash column chromatography (9:1, CH2Cl2/MeOH) to yield INT-2.3

1 (617.4 mg, 2.24 mmol, 56%). Yellow oil. H NMR (400 MHz, CDCl3): δ 7.41-7.22 (m,

5H), 5.23-4.99 (m, 2H), 3.72-3.41 (m, 4H), 3.10 (s, 1H), 2.97 (s, 1H), 2.85 (d, J = 2.7 Hz,

13 1H), 2.32-1.78 (m, 4H). C NMR (400 MHz, CDCl3): δ 176.4, 175.0, 172.6, 172.3,

156.0, 155.1, 154.5, 154.3, 136.9, 136.8, 136.7, 136.4, 128.6, 128.5, 128.5, 128.4, 128.2,

128.0, 128.0, 128.0, 128.0, 127.9, 127.7, 67.6, 67.2, 67.1, 59.4, 58.7, 57.0, 56.3, 47.3,

47.0, 46.7, 37.1, 36.9, 36.2, 36.0, 31.0, 30.5, 29.6, 29.3, 24.4, 24.4, 23.7, 23.5. HRMS

+ ESI: m/z Calcd for C15H21N2O3 [M+H] 277.1547, found 277.1534; m/z Calcd for

+ C15H20N2O3Na [M+Na] 299.1366, found 299.1355.

O N N N

QMP-2.10

(S)-1-((3-hydroxypyridin-2-yl)methyl)-N,N-dimethylpyrrolidine-2-carboxamide

(QMP-2.10) To a solution of methanol (30 mL) was added palladium on activated carbon

(10 wt. %) (225.0 mg) and INT-2.3 (584.5 mg, 2.12 mmol). The air in the flask was evacuated and backfilled with hydrogen three times. A hydrogen-filled balloon was attached to the flask and the reaction mixture was stirred for 24 h. The reaction mixture was vacuum filtered through celite and washed with methanol. The solvent was removed in vacuo and the resulting residue was dissolved in benzene (30 mL). To this mixture was added 3-hydroxypyridine (109.2 mg, 1.15 mmol) and paraformaldehyde (36.5 mg, 1.22 mmol). The mixture was refluxed for 24 h and the solvent was removed in vacuo. The crude product was purified via flash column chromatography (100:0-9:1, CH2Cl2/MeOH) to yield QMP-2.10 (104.7 mg, 0.42 mmol, 37%). Brown oil. 1H NMR (400 MHz,

CDCl3): δ 7.95 (dd, J = 3.0, 1.5 Hz, 1H), 7.16-7.12 (m, 1H), 7.10-7.06 (m, 1H),

4.24 (d, J = 13.4 Hz, 1H), 3.55-3.44 (m, 2H), 3.04 (s, 3H), 3.01 (s, 3H), 3.00-2.97 (m,

1H), 2.43-2.35 (m, 1H), 2.28-2.19 (m, 1H), 1.90-1.77 (m, 3H). 13C NMR (400 MHz,

CDCl3): δ 173.4, 154.2, 144.3, 139.2, 123.8, 123.4, 63.9, 59.2, 52.9, 36.6, 36.1, 29.3,

+ 23.7. HRMS ESI: m/z Calcd for C13H20N3O2 [M+H] 250.1550, found 250.1544.

N O O O

INT-2.4

Benzyl (S)-2-(diethylcarbamoyl)pyrrolidine-1-carboxylate (INT-2.4) To a solution of ethyl acetate (30 mL) was added INT-2.1 (1.07 g, 4.29 mmol). The mixture was cooled to 0 °C and N-methylmorpholine (0.57 mL, 5.15 mmol) and ethyl chloroformate (0.49 mL, 5.15 mmol) was added dropwise, followed by diethylamine (0.53 mL, 5.15 mmol).

The mixture was stirred at 23 °C for 12 h and washed with water (30 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified via flash column chromatography (9:1, CH2Cl2/MeOH) to

1 yield INT-2.4 (866.7 mg, 2.85 mmol, 66%). Yellow oil. H NMR (400 MHz, CDCl3): δ

7.41-7.23 (m, 5H), 5.23-4.99 (m, 2H), 3.74-3.09 (m, 5H), 2.32-1.78 (m, 4H), 1.27 (t, J =

7.1 Hz, 1H), 1.12 (t, J = 7.1 Hz, 1H), 1.01 (t, J = 7.1 Hz, 1H), 0.94 (t, J = 7.1 Hz, 1H).

13 C NMR (400 MHz, CDCl3): δ 171.9, 171.9, 155.0, 154.4, 137.0, 136.7, 128.6, 128.5,

128.5, 128.4, 128.3, 128.1, 127.9, 127.8, 127.7, 67.3, 67.0, 56.9, 56.5, 47.4, 46.9, 42.0,

41.7, 41.0, 40.9, 31.2, 30.3, 29.1, 24.5, 23.7, 14.7, 14.3, 13.1, 13.0. HRMS ESI: m/z

+ Calcd for C17H24N2O3Na [M+Na] 327.1679, found 327.1710.

O N N N

QMP-2.11

(S)-N,N-diethyl-1-((3-hydroxypyridin-2-yl)methyl)pyrrolidine-2-carboxamide

(QMP-2.11) To a solution of methanol (50 mL) was added palladium on activated carbon

(10 wt. %) (822.0 mg) and INT-2.4 (818.8 mg, 2.69 mmol). The air in the flask was evacuated and backfilled with hydrogen three times. A hydrogen-filled balloon was attached to the flask and the reaction mixture was stirred for 24 h. The reaction mixture was vacuum filtered through celite and washed with methanol. The solvent was removed in vacuo and the resulting residue was dissolved in benzene (30 mL). To this mixture was added 3-hydroxypyridine (158.2 mg, 1.66 mmol) and paraformaldehyde (53.5 mg, 1.78 mmol). The mixture was refluxed for 24 h and the solvent was removed in vacuo. The crude product was purified via flash column chromatography (100:0-9:1, CH2Cl2/MeOH) to yield QMP-2.11 (153.7 mg, 0.55 mmol, 33%). Brown oil. 1H NMR (400 MHz,

CDCl3): δ 7.94 (dd, J = 3.0, 1.6 Hz, 1H), 7.14-7.04 (m, 2H), 4.20 (d, J = 13.5 Hz, 1H),

3.61-3.48 (m, 2H), 3.43-3.20 (m, 4H), 3.05-2.97 (m, 1H), 2.43-2.33 (m, 1H), 2.25-2.14

(m, 1H), 1.94-1.75 (m, 3H), 1.19 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H). 13C NMR

(400 MHz, CDCl3): δ 172.5, 154.3, 144.2, 139.2, 123.8, 123.5, 63.9, 59.0, 52.9, 41.2,

+ 40.5, 29.8, 23.7, 14.7, 13.1. HRMS ESI: m/z Calcd for C15H24N3O2 [M+H]

278.1863.1550, found 278.1846.

O N O O

INT-2.5

Benzyl (R)-2-(diethylcarbamoyl)pyrrolidine-1-carboxylate (INT-2.5) To a solution of

THF (40 mL) N-Cbz-D-proline (1.00 g, 4.02 mmol). The mixture was cooled to -78 °C and N-methylmorpholine (0.88 mL, 8.04 mmol) and isobutyl chloroformate (1.04 mL,

8.04 mmol) was added dropwise followed by diethylamine (0.83 mL, 8.04 mmol). The mixture was stirred for 12 h at 23 °C. The solvent was removed in vacuo and re-dissolved in ethyl acetate (30 mL). The mixture was washed with water (3 × 30 mL) and brine (30 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified via flash column chromatography

1 (9:1, CH2Cl2/MeOH) to yield INT-2.5 (480.8 mg, 1.58 mmol, 39%). Yellow oil. H

NMR (400 MHz, CDCl3): δ 7.40-7.21 (m, 5H), 5.21-4.96 (m, 2H), 4.67-4.45 (m, 1H),

3.73-3.62 (m, 1H), 3.61-3.31 (m, 3H), 3.29-3.07 (m, 2H), 2.23-2.20 (m, 2H), 1.93-1.78

13 (m, 2H), 1.30-0.90 (m, 6H). C NMR (400 MHz, CDCl3): δ 171.8, 171.7, 154.9, 154.3,

137.0, 136.7, 128.5, 128.4, 128.4, 128.0, 127.9, 127.8, 56.9, 56.4, 47.4, 46.9, 41.9, 41.6,

40.8, 40.8, 31.2, 30.3, 24.4, 23.6, 14.7, 14.3, 13.1, 13.0. HRMS ESI: m/z Calcd for

+ C17H24N2O3Na [M+Na] 327.1679, found 327.1677.

O N N N

QMP-2.12

(R)-N,N-diethyl-1-((3-hydroxypyridin-2-yl)methyl)pyrrolidine-2-carboxamide

(QMP-2.12) To a solution of methanol (50 mL) was added palladium on activated carbon

(10 wt. %) (168.0 mg) and INT-2.5 (480.8 mg, 1.58 mmol). The air in the flask was evacuated and backfilled with hydrogen three times. A hydrogen-filled balloon was attached to the flask and the reaction mixture was stirred for 24 h. The reaction mixture was vacuum filtered through celite and washed with methanol. The solvent was removed in vacuo and the resulting residue was dissolved in benzene (30 mL). To this mixture was added 3-hydroxypyridine (194.2 mg, 2.04 mmol) and paraformaldehyde (63.0 mg, 2.10 mmol). The mixture was refluxed for 24 h and the solvent was removed in vacuo. The crude product was purified via flash column chromatography (100:0-9:1, CH2Cl2/MeOH) to yield QMP-2.12 (284.6 mg, 1.03 mmol, 65%). Brown oil. 1H NMR (400 MHz,

CDCl3): δ 7.93 (dd, J = 3.0, 1.5 Hz, 1H), 7.14-7.09 (m, 1H), 7.08-7.04 (m, 1H),

4.19 (d, J = 13.6 Hz, 1H), 3.60-3.48 (m, 2H), 3.42-3.20 (m, 4H), 3.04-2.97 (m, 1H), 2.41-

2.32 (m, 1H), 2.24-2.13 (m, 1H), 1.95-1.74 (m, 3H), 1.18 (t, J = 7.2 Hz, 3H), 1.11 (t, J =

13 7.0 Hz, 3H). C NMR (400 MHz, CDCl3): δ 172.4, 154.2, 144.2, 139.1, 123.7, 123.4,

63.9, 59.0, 52.9, 41.1, 40.5, 29.8, 23.6, 14.6, 13.0. HRMS ESI: m/z Calcd for

+ C15H24N3O2 [M+H] 278.1863, found 278.1857.

O NH2 N N

QMP-2.13

(S)-1-((3-hydroxypyridin-2-yl)methyl)pyrrolidine-2-carboxamide (QMP-2.13) To a solution of benzene (30 mL) was added L-prolinamide (313.3 mg, 2.74 mmol), 3- hydroxypyridine (320.5 mg, 3.37 mmol), and paraformaldehyde (104.6 mg, 3.48 mmol).

The mixture was refluxed for 24 h and the solvent was removed in vacuo. The crude product was purified via flash column chromatography (100:0-9:1, CH2Cl2/MeOH) to yield QMP-2.13 (278.6 mg, 1.26 mmol, 46%). Yellow oil. 1H NMR (400 MHz,

CDCl3): δ 7.98 (dd, J = 1.6, 3.0 Hz, 1H), 7.16-7.06 (m, 2H), 6.12 (br, 2H), 4.28 (d, J =

13.7 Hz, 1H), 3.69 (d, J = 13.6 Hz, 1H), 3.30-3.21 (m, 1H), 3.08-3.01 (m, 1H), 2.50-2.40

13 (m, 1H), 2.28-2.16 (m, 1H), 2.02-1.80 (m, 3H). C NMR (400 MHz, CDCl3): δ 176.3,

153.8, 144.0, 139.7, 124.0, 123.5, 66.7, 58.9, 53.6, 30.4, 23.7. HRMS ESI: m/z Calcd for

+ C11H16N3O2 [M+H] 222.1237, found 222.1228.

O NH2 N N

QMP-2.14

(S)-1-((3-hydroxypyridin-2-yl)methyl)pyrrolidine-2-carboxamide (QMP-2.14) To a solution of benzene (30 mL) was added D-prolinamide (306.4 mg, 2.68 mmol), 3- hydroxypyridine (278.0 mg, 2.92 mmol), and paraformaldehyde (93.2 mg, 3.10 mmol).

The mixture was refluxed for 24 h and the solvent was removed in vacuo. The crude product was purified via flash column chromatography (100:0-9:1, CH2Cl2/MeOH) to yield QMP-2.14 (218.9 mg, 0.99 mmol, 37%). Yellow oil. 1H NMR (400 MHz,

CDCl3): δ 8.00 (dd, J = 2.9, 1.6 Hz, 1H), 7.18-7.07 (m, 2H), 5.92 (br, 2H), 4.31 (d, J =

13.8 Hz, 1H), 3.71 (d, J = 13.8 Hz, 1H), 3.31-3.24 (m, 1H), 3.12-3.01 (m, 1H), 2.53-2.42

13 (m, 1H), 2.32-2.19 (m, 1H), 2.05-1.84 (m, 3H). C NMR (400 MHz, CDCl3): δ 175.7,

153.9, 143.7, 139.8, 124.0, 123.5, 66.8, 59.3, 53.6, 30.4, 23.7. HRMS ESI: m/z Calcd for

+ C11H16N3O2 [M+H] 222.1237, found 222.1228.

Chapter 3: Mannich Base QMPs Derived from Prolinol Ethers and Amines

Once it was discovered that the Mannich base QMPs derived from the proline esters and amides were not capable of resurrecting OP-aged AChE, the reduced versions of these QMPs were synthesized (i.e. prolinol ether and amine derivatives). This chapter is therefore focused on the synthesis and screening of these Mannich-base QMPs.

The first reduced Mannich base QMPs synthesized were the hydroxymethyl derivatives (Figure 3.1). First, L- and D-proline were reduced with LiAlH4 to produce L- and D-prolinol, respectively. Both L-prolinol and D-prolinol were then used in a Mannich reaction with 3-hydroxypyridine and paraformaldehyde to produce QMP-3.1 and QMP-

3.2.

1. LiAlH4, THF, 0 °C, 3 h

2. 3-hydroxypyridine, paraformaldehyde, benzene OH O reflux, 24 h N N N OH H OH

OH OH

N (S) N (R) N N

OH OH

QMP-3.1 QMP-3.2

Figure 3.1 General reaction scheme for the synthesis of Mannich-base QMPs derived from prolinol and the chemical structures for each variant synthesized.

QMP-3.1 and QMP-3.2 were screened for their ability to resurrect DFP-aged hAChE via Ellman’s assay. Dr. Qinggeng Zhuang and Dr. Andrew Franjesevic performed the screening assays. While the (R)-enantiomer, QMP-3.2, did not exhibit any activity, the alcohol derivative QMP-3.1, with the (S) stereochemistry, was able to resurrect 13% of DFP-aged AChE (Figure 3.2) after 24 h when used at 1 mM concentration (pH 7.5 and 37 °C). While QMP-3.1 was not quite as effective as QMP-

2.2, the results are still quite promising.

Indeed, the Mannich base QMPs in Chapter 2 that featured functional groups containing carbonyl groups were not active; however, the results of QMP-3.1 suggest that, without the carbonyl present, substituted QMPs can resurrect OP-aged hAChE.

Table 3.1 Data for the resurrection of DFP-aged recombinant human AChE with

Mannich base QMPs (1 mM, pH 7.5, 37 °C, 24 h) derived from prolinol. Screening data for QMP-2.2 is shown for reference. The negative control consisted of DFP-aged hAChE with no QMP. The positive control consisted of native hAChE. The standard deviation was determined from 4 replicate samples.

QMP Relative Activity (%) Standard Deviation

QMP-2.2 13.1 0.6

QMP-3.1 10.7 0.2

QMP-3.2 0.5 0.0

Negative Control 0.2 0.0

Positive Control 100 1.0

Resurrection of DFP-Aged AChE

100

y t

i 90

i t

c 15

e 10

a l

e 5 R

0 l l .2 .1 .2 o o -2 -3 -3 tr tr n n P P P o o M M M c c Q Q Q e e v v ti ti a i g s e o n p

Figure 3.2 Resurrection (24 h) of DFP-aged recombinant human AChE with Mannich base QMPs (1 mM, pH 7.5, 37 °C) as derived from prolinol.

Since a hydroxymethyl derivative was active, we also considered further derivatization to an ether functional group. Thus, ethers derived from prolinol were also synthesized and subsequently used in a Mannich reaction to produce additional QMPs for screening (Figure 3.3).

In the first step, a Williamson ether synthesis was performed by deprotonating N-

Boc-prolinol with sodium hydride and then performing an SN2 reaction with an alkyl

45 iodide compound. In the second step, the Boc group is removed under strongly acidic conditions. In the final step, a Mannich reaction is performed with 3-hydroxypyridine and paraformaldehyde. Currently, only the (S)-enantiomers of the methyl ether (QMP-3.3) and ethyl ether (QMP-3.4) variants have been synthesized. The isopropyl variants have proven to be more challenging to synthesize due to elimination occurring as a competing reaction. As the (S)-enantiomers showed more promise, they were prioritized for initial synthesis. The (R)-enantiomers will be considered in the future.

1. NaH, R-I, THF 23 °C, 12 h

2. TFA, CH2Cl2, 23 °C, 3 h OH 3. 3-hydroxypyridine, paraformaldehyde, benzene, OR reflux, 12 h N N O N O R = OH CH3 CH2CH3

O O N (S) N (S) N N

OH OH

QMP-3.3 QMP-3.4

Figure 3.3 General reaction scheme for the synthesis of Mannich-base QMPs derived from various prolinol ether derivatives and the chemical structures of the QMPs synthesized from this route.

The final class of synthesized prolinol derivatives were the amines (Figure 3.4).

The first step was the DMAP-catalyzed tosylation of the hydroxy group. This was followed by an SN2 reaction with an amine. As with the ether derivatives, the Boc group is removed under strongly acidic conditions and then the Mannich reaction is performed with 3-hydroxypyridine and paraformaldehyde. Currently, only the (S)-enantiomers with the dimethylamine (QMP-3.5) and diethylamine (QMP-3.6) variants at the proline segment have been synthesized. The (R)-enantiomers will be synthesized in the future.

O 1. b) or c) OH O S 2. d) NR2 N a) O 3. e) N N O N O O O OH

R = CH3 CH2CH3 Conditions and reagents: a) TsCl, DMAP, Et3N, CH2Cl2, 23 °C, 3 h b) R2NH, K2CO3, MeCN, reflux, 12 h c) R2NH, K2CO3, THF, reflux, 12 h d) TFA, CH2Cl2, 23 °C, 3 h e) 3-hydroxypyridine, paraformaldehyde, benzene, reflux, 12 h

N N N (S) N (S) N N

OH OH

QMP-3.5 QMP-3.6

Figure 3.4 General reaction scheme for the synthesis of Mannich base QMPs derived from various prolinol amine derivatives and the chemical structures of the QMPs synthesized from this route.

The ether and amine Mannich base QMPs derived from prolinol have yet to be screened for their ability to resurrect OP-aged AChE. The results of these compounds are highly anticipated due to the activity of the hydroxy derivatives, which also do not feature a carbonyl group, showing resurrection activity.