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Diversification of Scaffolds and Through

Carbenoid Functionalization

A Thesis Presented to the

Honors Tutorial College,

Ohio University

In Partial Fulfillment

Of the Requirements for Graduation

From the Honors Tutorial College

With the degree of Bachelor of Science in Chemistry

By:

Andrea J. Oliver

May 2019

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This thesis is titled

Diversification of Antibiotic Scaffolds Spiramycin and Roxithromycin Through

Carbenoid Functionalization

By: Andrea J. Oliver

Has been approved by

The Honors Tutorial College

And the Department of Chemistry and Biochemistry

Dr. Mark C. McMills

Associate Professor, Thesis Advisor

______

Dr. Lauren McMills

Director of Studies, Chemistry

______

Cary Roberts Frith

Interim Dean, Honors Tutorial College

______

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ABSTRACT

OLIVER, ANDREA J., May 2019, Chemistry

Diversification of Antibiotic Scaffolds Spiramycin and Roxithromycin Through

Carbenoid Functionalization

Thesis Advisor: Dr. Mark C. McMills

Despite the constant development of new , difficulties are encountered almost immediately through the development of antibiotic resistance. Researchers must constantly work to develop new antibiotics, while diversifying old antibiotic structures in order to avoid a global crisis caused by the generation of multidrug resistant organisms.

This work described in this thesis attempts to diversify inexpensive, antibiotic scaffolds such as roxithromycin and spiramycin through the removal of sugar moieties, the functionalization of the existing ring and other structural changes.

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ACKNOWLEDGEMENTS

I would like to thank Dr. Mark McMills and Dr. Lauren McMills for their endless patience and guidance these last four years. I could not have made it this far without the support of two such caring individuals. Thank you for pushing me to try even when I was scared and getting me to the next step.

I would like to thank the students in my 1510, 1500, and 1220 labs. It was such a privilege to get to be a part of your learning experience. Thank you for your enthusiasm and giving me opportunities to laugh every week.

Thank you to Joe Tysko, you keep the oil changed in the vacuum pump, the N2 tanks filled, and the bin filled with dry ice. Thank you for only making fun of me a little when I ask you stupid questions.

To my parents, and everyone else who has loved me along the way, thank you for keeping me sane and giving me the support I needed to keep plugging along.

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TABLE OF CONTENTS

Abstract……………………………………………………………………………………3

Acknowledgements………………………………………………………………………..4

List of Figures & Tables…………………………………………………………………..6

List of Schemes……………………………………………………………………………8

List of Abbreviations……………………………………………………………………...9

Chapter 1: Introduction…………………………………………………………………..10

Chapter 2: Introduction to …………………………………………………...13

2.1 Spiramycin…………………………………………………………………...17

2.2 Roxithromycin……………………………………………………………….26

Chapter 3: Carbenoid Functionalization…………………………………………………30

3.1 Synthetic Strategy for the Preparation of Spiramycin Derivatives…………..36

3.2 Synthetic Strategy for the Preparation of Roxithromycin Derivatives………40

3.3 Experimental Determination…………………………………………………42

Chapter 4: General Experimental………………………………………………………...44

4.1 Spiramycin Experimental…………………………………………………….45

4.2 Roxithromycin Experimental………………………………………………...59

Chapter 5: Results………………………………………………………………………..66

Chapter 6: Discussion……………………………………………………………………74

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List of Figures & Tables

Figure 1: The Structure of Penicillin…………………………………………………….10

Figure 2: , A 16-Membered Macrolide………………………………………….13

Figure 3: , A 15-Membered Macrolide…………………………………...13

Figure 4: Structure of Roxithromycin’s Aminosugar, which contributes to it’s basicity..14

Figure 5: Tacrolimus: A 23-Membered Macrolide……………………………………....15

Figure 6: : A 16-Membered Macrolide……………………………………..15

Figure 7: Concanamycin: An 18-Membered Macrolide…………………………………15

Figure 8: : A 14-Membered Macrolide……………………………………16

Figure 9: 2D Structure of Spiramycin……………………………………………………17

Figure 10: Crystal Structure of Spiramycin (1KD1, Protein Data Base, RCSB.org)...... 20

Figure 11: C5 Disaccharide Chain of Spiramycin……………………………………….21

Figure 12: C4-C7 Portion of Spiramycin’s Macrocyclic Lactone Ring (Sans Sugars).....22

Figure 13: Modification of Spiramycin through C5 Triazole Arm with Various R-groups.

Derived from (Klich, et. al, 2016)……………………………………………………..…23

Figure 14: Roxithromycin (right) an Oxime Derivative of Erythromycin (left)………...26

Figure 15: Proximity of Roxithromycin groups to Peptidyl Proteins of the Peptidyl

Transferase Center. Derived from (Schlunzen, et al., 2001)………………………….…27

Figure 16: Crystal Structure of Spiramycin (1KD1, Protein Data Base, RCSB.org)…....28

Figure 17: (left) and Metal Stabilized Carbene (right)…………………………31

Figure 18: The Structure of Cyclopropane……………………………………………....33

Figure 19: p-ABSA, a Diazo-Transfer Reagent. (Davies et. al, 1992)……………..…....34

Figure 20: Proposed Product Structure with C10 -C12 Intact…………………..…48 7

Figure 21: Methyl Malonyl Chloride (ChemDraw, 2019)……………………………….50

Figure 22: Structure of Roxithromycin…………………………………………………..66

Figure 23: Figure 23: H1 NMR of Roxithromycin…...…………………………………..67

Figure 24: H1 NMR of Spiramycin ………………………..…………………………….69

Figure 25: Structure of Roxithromycin w/o Cladinose…………………………………..70

Figure 26: H1 NMR of AO13-BF (Cladinose-Free Roxithromycin).……………………71

Figure 27: IR of AO13-BF (Cladinose-Free Roxithromycin)………………..………….72

Figure 28: Acetal Formation of Spiramycin’s C6 Aldehyde…………………………….73

Table 1: Establishing Solvent System for Spiramycin…………………………………..46

Table 2: Solvent System Determined for AO-06A……………………………………....51

Table 3: Establishing Solvent System for AO-06B……………………………...………52

Table 4: Establishing Solvent System for AO-07B Post Ethyl Acetate/Methanol (9:1)

Column…………………………………………………………………………………...54

Table 5: Establishing Solvent System for AO-08 Crude………………………………...59

Table 6: Establishing Solvent System for AO-08C……………………………………...60

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List of Schemes

Scheme 1: Enzymatic Peptide Bond Formation, (forming peptide bond shown in red).

Derived from (Berg et. al, 2015)…..…………………………………………………..…19

Scheme 2: Intramolecular Ketal Formation of Anhydroerythromycin in an Acidic

Environment. (Al-Qattan, 2019)…………………………………………………………26

Scheme 3: Intermolecular Insertion into a Carbon-Hydrogen Bond. (Doyle, 1998)…….32

Scheme 4: Intermolecular Carbon Insertion into an Oxygen-Hydrogen Bond. (Doyle,

1998)……………………………………………………………………………………..32

Scheme 5: of an Alkene using Diazomethane and Palladium (II)

Catalyst. (Doyle, 1998)…………………………………………………………………..34

Scheme 6: Mechanism of Diazo-Transfer Using p-ABSA. (Davies et. al, 1992)……….35

Scheme 7: A 1,3 dipolar cycloaddition. (McMills Group, 2019)………………………..36

Scheme 8: The Acylation of Unmodified Spiramycin using Ethyl Malonyl Chloride…..36

Scheme 9: Diazotization of β -Diester Derivative of Spiramycin……….………………37

Scheme 10: The Removal of Sugar, Mycarose from Spiramycin……………………….38

Scheme 11: The Acylation of Mycarose-Free Spiramycin using Methyl Malonyl

Chloride…………………………………………………………………………………..39

Scheme 12: Diazotization of Acetylated Mycarose-Free Spiramycin Derivative……….39

Scheme 13: Tandem Ylide Formation/Cycloaddition Strategy………………………….40

Scheme 14: Removal of Cladinose from Roxithromycin………………………………..40

Scheme 15: Acylation of Cladinose-Free Roxithromycin Using Methyl Malonyl

Chloride…………………………………………………………………………………..41

Scheme 16: Diazotization of Acetylated Cladinose-Free Roxithromycin Derivative…...41 9

Scheme 17: Projected OH Insertion Products from Diazotized Roxithromycin………...42

Scheme 18: Acetal Formation of C6 Aldehyde Before Attempted Sugar Removal……..58

List of Abbreviations

MIC: Minimum Inhibitory Concentration

MPC: Mutant Prevention Concentration

MSW: Mutant Selection Window

PAMS: Periodic Antibiotic Monitoring and Supervision

A Site: Amino Site

P Site: Peptidyl Site

E Site: Exit Site p-ABSA: para-aminobenzenesulfonyl azide

HCl: Hydrochloric Acid

NaOH: Sodium Hydroxide

NaCl: Sodium Chloride

TLC: Thin Layer Chromatography dd: Doublet of Doublets ddq: Doublet of Doublets of Quartets m: Multiplet s: Singlet

CDCl3: Chloroform-D

Et3N: Triethylamine

NMR: Nuclear Magnetic Resonance 10

Chapter 1: Introduction

H H R N S CH3

O N CH3 O

OH O

Figure 1: The Structure of Penicillin.

Since the discovery of penicillin in 1928 by Alexander Fleming, antibiotics have become an indispensable tool of our healthcare system. Whether it is treating routine bacterial infections in the general population, helping immune-compromised cancer patients avoid infection, or keeping recently transplanted organs viable in the bodies of transplant patients who are often susceptible to bacterial infections, antibiotics are an essential part of today’s healthcare system.1

Fleming’s insight into the trajectory of antibiotic use is far beyond his serendipitous discovery. In his Nobel Peace Prize acceptance speech, he warned those who would listen, that antibiotics under constant use are quickly plagued by antibiotic resistance. Resistance is inevitable as thrive in the most adverse conditions due to their ability to evolve so quickly. Bacteria utilize methods such as horizontal gene transfer within and between species, as well as selective pressure for beneficial random mutation, to thrive at a rate that the immune system cannot always match. In order to preserve antibiotics as viable solutions to bacterial infection, all possible methods for limiting resistance must be explored and if found successful, applied to new and continuing structures.2 11

According to the Centers for Disease Control and Prevention, an estimated 50% percent of antibiotics are not optimally prescribed. The ideal conditions for resistance are created where an antibiotic is prescribed at a low dosage over a long period of time.

There are two values that help a clinician determine the optimal dosage and duration for administration of an antibiotic. These include the minimum inhibitory concentration,

MIC, and the mutant prevention concentration, MPC. MIC is the lowest dosage of the antibiotic that still inhibits growth. MPC is the lowest dosage of the antibiotic that inhibits growth in a single-mutation bacterial species. In between the MIC and the MPC is the mutant selection window, the MSW, which clinicians seek to avoid reaching because it will specifically select for antibiotic resistant bacteria.3

Beyond better prescribing practices, studies have been conducted on antibiotic heterogeneity or more simply put, antibiotic cycling. One of the most successful examples of antibiotic cycling was described in a project called Periodic Antibiotic

Monitoring and Supervision, or PAMS. As the name implies this required real-time supervision by a multidisciplinary team, who kept cycling different classes of antibiotics with similar targets and activities, but with varying mechanisms of antibiotic resistance.

The antibiotics were sorted into three distinct classes labeled as recommended, restricted, or off-supervision and rotated during a 3-month period. PAMS proved to be a successful method for minimizing resistance. Unlike other medications, such as anticancer drugs, which can only be prescribed by oncologists, almost any clinician including dentists and ophthalmologists can prescribe antibiotics. Thus, any effort to implement a system like

PAMS on a world-wide level would require a level of coordination and cooperation that has yet to be achieved for any global issue, including the problem of climate change.3 12

In fact, antibiotic usage across the globe has increased by 65% from 2000 to 2015, with the United States having one of the highest overall rates of antibiotic use. Without any changes in common prescribing practices, at the current annual rate of growth, global antibiotic use is projected to increase by another 15% by 2030. But if consumption continues to increase at the compounded annual growth rates, an increase of 202% can be expected by 2030. As use increases, so will resistance. 4

Even if clinicians are able to achieve a significant level of responsible antibiotic use, the prevalence of antibiotic usage in the food-animal industry is considered much more problematic. In order to promote animal growth, the food-animal industry has pushed “unnecessary” antibiotics over long periods of time, creating pathogenic strains of multiple drug resistant bacteria and leading to the evolution of a number of difficult pathogens. Limiting antibiotic use in farms to sick animals (and not for weight gain), prescribing probiotics and providing living situations that make animals less susceptible to infections is key to the continued successful use of antibiotics.5

Responsible clinical use of antibiotics, antibiotic cycling, and a reworking of the food-animal industry’s approach to the use of antibiotics can help lengthen the useful lifetime of current antibiotics. These unfortunately are not permanent solutions. The only way to ensure that humanity will have effective methods to eliminate infections from evolving pathogenic bacteria is to continue to discover new entities, synthesize new antibiotics, while effectively functionalizing old ones. Unlike medicines for heart or arthritis conditions, new antibiotics do not guarantee the long-term profitability that makes such work appealing for a large pharmaceutical company to pursue. Therefore, the 13 efforts to diversify the structure of existing antibiotics and potentially increase their bioactivity should not be ignored.

Chapter 2: Introduction to Macrolides

Figure 2: Tylosin: A 16-Membered Macrolide.

Figure 3: Azithromycin: A 15-Membered Macrolide.

The macrolides are a specific class of antibiotic, characterized by a macrocyclic lactone ring of 12 or more atoms, with, at least one sugar moiety attached via a glycosidic bond. The sugars typically contribute to the macrolides ability to bind the bacterial and thus play a role in the macrolide bacteriostatic properties. The sugar moiety of macrolide often represents the major hydrophilic aspect of the molecule, otherwise the majority of the macrolide structure is highly lipophilic. This means that they dissolve in many polar organic solvents as such as dichloromethane and chloroform, and in alcohols 14 as such as methanol. They generally have some solubility in water, and, as was discovered over the course of this project, little in ethyl acetate. Macrolides tend to be basic due to the presence of amino sugars as such as the dimethylamine group of , part of azithromycin and tylosin. The nitrogen atom of the dimethylamine group contains a lone pair of electrons making it a Lewis base. Although macrolides have low solubility in water alone, they are stable in aqueous solutions that include water and other miscible organic solvents at and below room temperature, but unstable in acidic or basic conditions or at high temperatures.

N

HO HO O OR

Figure 4: Structure of Roxithromycin’s Aminosugar, which contributes to its basicity.

Macrolides are generally effective against gram-positive bacteria and to a much lesser extent, gram-negative bacteria. The specific species that each macrolide targets changes with respect to the particular macrolide used. Macrolides, compared to penicillin antibiotics, tend to have a wider spectrum of antibacterial activity, making them an acceptable replacement for patients with penicillin sensitivity and/or allergy. Macrolides have been shown to work both as antibiotics but also as immunosuppressors and immunomodulators. An example of this is the 23-membered macrocycle tacrolimus, which impairs the pathway that activates T-cells of the immune system and thus helps prevent the body from rejecting a transplanted organ.6 15

Figure 5: Tacrolimus: A 23-Membered Macrolide.

Bafilomycin and concanamycin, 16- and 18-membered macrocyclic lactones respectively, inhibit H+ATPase, inhibiting a cells ability to harness energy while indirectly causing cell death.6

Figure 6: Bafilomycin: A 16-Membered Macrolide.

Figure 7: Concanamycin: An 18-Membered Macrolide. 16

Erythromycin, the macrolide from which roxithromycin is derived, inhibits interleukin-8 expression indirectly, which reduces the recruitment of neutrophils, thus reducing the excessive activation of neutrophils in the lungs that characterize . Panbronchiolitis is an inflammation of the lungs by an unknown agent that causes breathing difficulties. Macrolides have a range of bioactive properties and these are just a few of the effects that macrolides display beyond their antibacterial properties.6

Figure 8: Erythromycin: A 14-Membered Macrolide.

Although global consumption of antibiotics is increasing, macrolide use in high- income countries as such as the United States has actually decreased by 25% from 2000 to 2015.4 This trend is reversed in low-income countries with an increase in macrolide use of 119% over the same time period.4 Reasons for this difference include better education against unnecessary antibiotic use in high-income countries as well as reduced need due to better water sanitation in place. In low-income countries, the need to fight infectious disease is often more important than a clinician’s concern about creating multidrug resistant organisms.4 Despite the evidence that macrolide use has been somewhat conservative, a 2015 report shows resistance macrolides in the erythromycin 17 family to be around 50% in the United States. Thus new, not yet susceptible to resistance forms of macrolides, will be needed in the very near future.7

Chapter 2.1: Spiramycin

Figure 9: 2D Structure of Spiramycin.

Spiramycin was discovered in 1952 and is commonly prescribed for toxoplasmosis as well as other infections of soft tissues. Spiramycin mode of action works by inhibiting protein synthesis. Spiramycin is a relativity inexpensive, readily available macrolide antibiotic, costing approximately $80 U.S. dollars per gram from chemical company Sigma Aldrich. As a macrolide, it is characterized by a 16-membered macrocyclic lactone ring at its core as well as two amino sugars, mycaminose (CA) and furosamine (CB), and one neutral sugar, mycarose (CC), attached through glyosidic bonds.

Spiramycin is sold as a complex of three familial compounds, spiramycin I, spiramycin

II, and spiramycin III, with a C3-OH, C3-O-Acetyl, and C3-O-Propionyl groups respectively. The spiramycin purchased for this project was 80% spiramycin I, while, spiramycin I is the only isomer detectable in plasma and thus the one focused on in the context of this project.8

Proteins play a crucial role in all living organisms, they serve not only as structural components as such as muscle, but also act as catalysts for various biochemical 18 processes and serve roles in the immune system as antibodies. Proteins are comprised of folded chains of amino acids. They are assembled by during a process called translation. A bacterial ribosome such as the one found in E. Coli has a mass of 2500 kDa and a diameter of roughly 250Å with a sedimentation coefficient of 70s. A sedimentation coefficient is the ratio of particles sedimentation velocity to the centrifugal acceleration it is exposed to. Although this value is dependent on the particles mass and volume it also accounts for surface area. As macrolides interact with the ribosome in the 3-D, measurements as such as sedimentation coefficient, which takes into consideration the ribosomes orientation in space in terms of surface area, can help researchers quantify an otherwise abstract concept. A bacterial ribosome is comprised of a 50s subunit which can be further broken down into 34 different proteins (L1-L34), and two RNA molecules 23s and 5s, as well as a 30s subunit that is comprised of 21 different proteins (S1-S21) and a

16s RNA molecule. The proteins, L1-L34, play an important role in the binding of macrolides, spiramycin and roxithromycin. The mechanism of this binding will be covered in more detail in each respective macrolide chapter. Together, these components work to catalyze protein synthesis.9

During transcription, DNA is used to make a complementary messenger RNA, mRNA. This mRNA serves as the template from which proteins are synthesized, it is read in the 5’ to 3’ direction, one codon (a sequence of three nucleotides) at a time. The corresponding protein is synthesized in the amino to the carboxyl functional group direction, with the new amino acid added to the carboxyl end. Transfer RNA (tRNA), works as an “adaptor molecule” which links to a specific codon and carries the corresponding amino acid through its anticodon and acceptor stem components, 19 respectively 9. The formation of a peptide bond between free amino acids is not a thermodynamically favorable process. Therefore, two ATP molecules are consumed in process of attaching and subsequently activating amino acids with tRNA. Once an amino acid is activated, there are three ribosomal binding sites at which translation will commence, the aminoacyl site (A site), the peptidyl site (P site), and the exit site (E site).

The mRNA to be translated is bound to the 30s subunit while the three aminoacyl-tRNA pairs are in contact with both subunits, 50s and 30s, and bound to the mRNA through codon-anticodon base pairs. Elongation factor Tu, EF-Tu, a 43kDa member of the G- protein family, carries the aminoacyl-tRNA to the A site. Once delivered, correct codon- anticodon pairing with the mRNA induces a structural change in the 30s subunit that releases EF-Tu from the ribosome and rotates the aminoacyl-tRNA in the A site, where it is now in prime position to bind with the aminoacyl-tRNA in the P site, in a process called “accommodation”. Thus in the ribosome, amino acids are positioned to exploit the intrinsic reactivity of the amine in the A site and the ester in the P site. By capitalizing on ideal positioning of the activated aminoacyl-tRNA in the process of accommodation, the ribosome is able to increase the rate of peptide bond formation by 107. 9

Scheme 1: Enzymatic Peptide Bond Formation, (forming peptide bond shown in red).

Derived from (Berg et. al, 2015).

With the A and P site occupied and oriented as such, peptide bond formation occurs at the center, the 23s rRNA of the 50s subunit near the peptide exit tunnel which, as the name implies, allows the growing polypeptide to exit the 20 ribosome. The growing peptide is still attached to the tRNA and in the A site on the 30s subunit after the peptide bond is formed. The 30s and 50s subunits will then rotate and push the peptidyl-tRNA in the A site into the P site, while a new aminoacyl-tRNA is delivered to the A site and another peptide bond is synthesized. The now deacylated tRNA is moved from the P site to the E site where it is exits the ribosome. Both mRNA and tRNA must be moved through the ribosome during peptide bond formation and this is performed by another elongation factor, EF-G, also known as translocase, which moves mRNA the distance of one codon, using the energy of one GTP molecule.9

Figure 10: Crystal Structure of Spiramycin (1KD1, Protein Data Base, RCSB.org)

Spiramycin binds in the peptide exit tunnel, between the peptidyl transferase center and constriction in the tunnel due to proteins L4 and L22 of the 50s subunit of the ribosome. The C5 disaccharide chain stretches out in the direction of the peptidyl- transferase center so the longer the chain, the more potential interaction.10 21

Figure 11: C5 Disaccharide Chain of Spiramycin.

The C6-ethyl aldehyde forms a covalent bond with N6 of A2103 nucleotide of the

23s RNA sequence of the ribosome forming a carbinolamine. Even though the intuitive product of an amine and aldehyde is a Schiff base (an imine), in the case of an “exocyclic primary amine of a nucleotide”, a cabinolamine is produced instead.10 The crystal structure shows continuous electron density joining the ethyl aldehyde and nucleotide, this best supports a carbinolamine product model. This covalent bond is believed to contribute the majority of the interaction between macrolide and ribosome. Beyond this, the strength of the interaction between macrolide and ribosome is determined by surface area upon binding. At least 34% of the total surface area of the macrolide is hidden due to the orientation of the macrolide within the ribosome, but up to 67% of surface area available for interaction can be contributed to the sugars attached to the lactone ring.

Each sugar moiety of spiramycin contributes at least one hydrogen bond to the ribosome, and so these sugar moieties are where the majority of the free binding energy is thought to come from. The base of A2103 of the 23s rRNA usually acts as part of the peptide exit tunnel, where nascent peptides emerge from the ribosome. When bound to spiramycin 22 though, base A2102 reorients by as much as 90° and therefore hinders the progress of proteins as they attempt to exit the ribosome.10

Figure 12: 2-D C4-C7 Portion of Spiramycin’s Macrocyclic Lactone Ring (Sans Sugars).

Between the C2098 and A2100 bases of the ribosome is a hydrophobic cleft in which the C4-C7 portion of the macrolide binds (Figure 11). These interactions between spiramycin and the 50s subunit of the ribosome, simply put, plug up the exit tunnel so no peptide larger than a dipeptide can exit the ribosome. On a microbial level, this interaction prevents the growth of bacteria and is considered bacteriostatic. In high concentrations of spiramycin, it can even be bactericidal. Bacteriostatic means that the growth of the bacteria is inhibited, while bactericidal implies bacterial death. Some resistance to spiramycin is thought to be due to methylation of the N6 adenine residue,

A2058 (of Escherichia coli) or G2099 (of Haloarcula marismortui) on the 23s rRNA, a component of the 50s subunit where the drug interaction occurs.10 This methylation is the result is believed to be the result of a random mutation that was selected for within the bacteria population because it hinders the interaction between macrolide and ribosome thus lessening its antibacterial properties.9 Another mutation that confers resistance is the 23 replacement of A2103 (of Haloarcula marismortui) or A2062 (of Escherichia coli) both adenine (A) bases with guanine (G) bases. The change in base will not allow for the formation of the carbinolamine, the single most stabilizing interaction between ribosome and macrolide.10

Any modifications to the structure of spiramycin that increase the length of the C5 disaccharide chain or allow for stronger interactions between the compound, through the

C6 aldehyde or C3 and its target have the potential to make it a more effective drug in terms of binding to bacteria. Recent work was completed using Click methodology, to convert spiramycin’s C5-C6 ethyl aldehyde to a tetrahydrofurfuryl acetal that includes triazole conjugates instead of the pendant sugars mycaminose and mycarose.11

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Figure 13: Modification of Spiramycin through a C5 Triazole Arm with Various R- groups. Derived from (Klich, et. al, 2016).

Derivatives 11-15, with bicyclic-triazole bridged aglycones, showed the most favorable lipophilicity but no activity against bacterial panels. This lack of antibacterial activity was believed to be due to the loss of the C6 aldehyde, eliminating the possibility of carbinolamine formation and unfavorable lipophilic/hydrophilic interactions as the ribosomal exit tunnel is polar and filled with water to transport macrolide to site of action. The same issues plagued derivative 16 with a triazole conjugate containing an azidothymidine moiety, similar to those nucleoside reverse transcriptase inhibitors prescribed for patients infected with acquired immunodeficiency syndrome (AIDS).

Derivatives 6-10 containing terminal saccharides had varied antibacterial responses, but 25 researchers could not find any structural pattern to explain this using the currently accepted binding mode of spiramycin. Derivative 2, not shown, maintained antibacterial activity, although to a less extent than spiramycin. The lesser activity of derivative 2 is believed to be due to the preservation of the aldehyde at C6, which is key to the macrolide-ribosome interaction but attenuated by the loss of the saccharides that hydrogen bond with G2099 and G2540 (of Haloarcula marismortui) and contribute to macrolide-ribosome binding to a lesser extent than the C6 aldehyde. Even though a theory for new binding mode was not offered by the authors for these triazole-saccharide conjugates, the antibacterial properties of derivatives 6-10 suggest that even derivatives without the C6 aldehyde can be active against bacteria if other additional saccharides are present for binding.11

This is not the only method to make spiramycin a more effective antibiotic.

Structural modifications have affected the oral bioavailability of the drug, which is currently about 30-40%.12 Oral bioavailability refers to the fraction of an orally administered drug that actually reaches the bloodstream and can be distributed though the body until it achieves its point of pharmacological action. Increasing oral bioavailability is important because other methods of drug administration can be increasingly inconvenient and therefore increase the likelihood of non-compliance for patients.

Clinicians often must push for drugs to be in an orally-consumed form, which, for drugs with a low oral bioavailability, means that the drug must be administered in higher doses or formed as a different salt. Higher dosage increases adverse side effects of the drug as well as the potential of antibiotic resistance to develop. If spiramycin can be modified to have a greater bioavailability even by something as simple as increasing its solubility in 26 water, it could require smaller doses over a shorter period of time thus potentially prolonging the effectiveness of the antibiotic before widespread resistance can occur.13

Chapter 2.2: Roxithromycin

Figure 14: Roxithromycin; (right) an Oxime Derivative of Erythromycin (left).

Roxithromycin is a semi-synthetic macrolide antibiotic derived from

Erythromycin in the late 1980’s. Roxithromycin differs from erythromycin through the formation of a polyether oxime derived from the C9 ketone functional group (Figure 14).

Unfunctionalized, erythromycin is acid sensitive due to the Lewis basicity of the ketone at C9. This ketone, under acidic conditions, will protonate, then form an intramolecular cyclic acetal, converting erythromycin into anhydroerythromycin due to the ideal positioning of alcohols at C6 and C11 (Scheme 1).14

Scheme 2: Intramolecular Ketal Formation of Anhydroerythromycin C6-C11 in an Acidic

Environment. (Al-Qattan, 2019). 27

Oxime modification of the C9 ketone shown in Figure 14 has been synthesized as the analog, roxithromycin. Formation of the oxime prevents inactivation of the antibiotic by gastric acid during digestion, providing a clinically relevant improvement. Beyond this modification, macrolide antibiotics still tend to be less active at low pH’s. The minimum inhibitory concentration (MIC) is increased by eight-fold at an acidic pH compared to a neutral pH of 7 and drops in half at a pH of 8. This implies that structural modifications to macrolide antibiotics such as roxithromycin increases its stability in acidic conditions, such as those of the human digestive tract, would also increase the effectiveness of said antibiotic and allow for prescriptions at a lower dosage.15

Figure 15: Proximity of Roxithromycin groups to Peptidyl Proteins of the Peptidyl

Transferase Center. Derived from (Schlunzen, et al., 2001).

Protein binding studies have shown that roxithromycin is 8Åfrom peptidyl protein L4 and 9Å from peptidyl protein L22 when bound to the ribosome. At this distance, unlike spiramycin, these proteins have an indirect effect on roxithromycin’s 28 bound structure with E. coli. Instead, the C2’OH group of the pendent desosamine sugar is thought to hydrogen bond at N1 of A2058 and N6 of A2059, both nucleotides of the peptidyl transferase ring. The three hydroxyl groups on the lactone ring, C6-OH, C11-

OH, and C12-OH are able to hydrogen bond with N6 A2062, U2609, and U2609 again, respectively. C11-OH and C12-OH are also thought to bind with 50s subunit proteins

L22 and L4 respectively. The cladinose sugar of roxithromycin has not been found to hydrogen bond with the 23s rRNA of the bacterial 50s ribosome subunit. Furthermore, the desosamine sugar of roxithromycin interacts with nucleotides G2057, A2058, and

A2059 through its hydroxyl moiety as well as G2505 through its dimethylamine group.

Finally, roxithromycin’s polyether oxime chain plays a role in blocking the exit of nascent peptide chains as it points towards the exit tunnel in the bound macrolide- ribosome structure. These specific nucleotide-to-antibiotic interactions are the main contribution to the inhibitory action of the macrolide, but the bound confirmation is also in position to stall the movement of tRNAs at the peptidyl transferase center through sheer bulk of the molecule.16

Figure 16: Crystal Structure of Spiramycin (1KD1, Protein Data Base, RCSB.org) 29

Roxithromycin is currently prescribed for soft tissue, urinary tract, and respiratory tract infections. It has been found to be effective against gram-positive cocci, mainly the staphylococcus species as well as gram-negative cocci, including Neisseria meningitidis.

Other bacteria species it targets include the gram-positive bacilli as such as

Corynebacterium, Listeria monocytogenes, and Lactobacillus species, and gram-negative bacilli as such as Bordetella pertussis, Pasterurella species and Hamophillus species.

Roxithromycin has not been shown to be effective against glucose-nonfermenting gram- negative bacilli. This limited spectrum of activity against the anaerobic gram-negative bacteria that populate and even protect the human intestine makes roxithromycin especially useful in a clinical setting.15, 7

To be gram-positive, a bacterial species must have a thick peptidoglycan layer which retains crystal violet dye in the process of gram-staining by which a molecular biologist might use to differentiate between the two bacterial species. Although intuitively one might believe gram-positive bacteria would be more resistant to antibiotics due to this thicker peptidoglycan layer, this is not the case. Gram-positive bacteria actually lack the outer that makes gram-negative bacteria harder to penetrate with antibiotics.15 But, this does not mean gram-positive bacteria do not develop resistance to antibiotics by other mechanisms. One such gram-positive bacteria is

Streptococcus agalactiae, also known as group B streptococcus, or GBS. GBS is a relatively harmless bacterium for healthy human adults; it is estimated about one-third of this population carries the species in their gastrointestinal tract, or in women in their genitourinary tracts without symptoms. GBS infections becomes a serious concern when a woman is pregnant. GBS is usually the species responsible for neonatal infections17. 30

Deoxyadenosine methylase is an in bacteria that can serve as transcription regulator by preventing DNA from being cleaved through methylation of the DNA at specific points18. In GBS, rRNA methylases also act as an agent “for site-directed mutagenesis” through methylation17. These methylations occur at nucleotides of the ribosome at which macrolides of the erythromycin family bind, thus hampering the binding and subsequently making it so the MIC of the drug must be increased significantly to achieve similar antibacterial outcomes.17

Unfortunately, as of 2015, bacterial resistance to macrolides in the erythromycin family such as roxithromycin is at 48% in the United States, and 70% in Asia5.

Resistance to one macrolide in this family often translates to resistance to other macrolides in this family due to similarities of their ribosomal binding mechanism17.

Increasing the number of functional groups on the roxithromycin’s core lactone ring has the potential to make it a more effective antibiotic with more Lewis base sites to interact and thus bind with the bacterial ribosome. These modifications also diversify the macrolide, thwarting the bacteria who have been evolutionarily selected with mutations that confer resistance.

Chapter 3: Carbenoid Functionalization

A carbenoid is a molecule that still has a valence number of four but has some of the characteristics of a carbene, a carbon atom with two unshared valence electrons, along with two additional ligands. are typically very unstable so metal stabilized carbenoids serve as more stable substitutes for chemical reactions. A particular useful variation of a carbenoid is a metal carbene, where a double bond from a carbene carbon to a transition metal forms a more stable high energy intermediate for various 31 carbenoid reactions. Carbenoid or metal carbene reactions include C-H or N-H insertions, ylide formation/cycloadditions and sigmatropic rearrangements.19

Figure 17: Carbene (left) and Metal Stabilized Carbene (right).

Metal carbenes can serve as effective tools for insertion into heteroatom-hydrogen bonds, including C-H, N-H, and O-H bonds 20. As carbons and hydrogens have similar electronegativities, the bond between carbon and hydrogen is not particularly polar and therefore unreactive in most reactions, carbenoid reactions being a notable exception. The polarity of the heteroatom-hydrogen bond changes the mechanism by which the insertion can occur. Typically, O-H and N-H insertion reactions can be “better described as an ylide transformation” 20. The usefulness of being able to create carbon-carbon bonds cannot be over emphasized, but the selectivity of this insertion remains difficult to achieve. By using carbenoids generated from rhodium (II)catalysis of diazo compounds, can provide some degree of selectivity. The insertion is believed to proceed through “an electrophilic metal carbine intermediate” 20. The metal carbene p-orbital can overlap with the σ-orbital of the C-H to which the carbene will insert. As the metal dissociates from the carbene and takes its electrons with it, the metal departs, the carbene formed is very electrophilic. This increased electrophilicity of the carbene decreases its selectivity as it quickly forms carbon-hydrogen and carbon-carbon bonds to compensate for the loss of electrons due to the metal ligand dissociation. Rhodium catalysts decrease the electron withdrawal caused by the dissociation of the metal ligand and can help to stabilize the transition state, thus allowing for greater insertion selectivity. 20 32

Scheme 3: Intermolecular Insertion into a Carbon-Hydrogen Bond. (Doyle, 1998).

Unlike the nonpolar carbon-hydrogen bonds, when inserting into polar X-H bonds as such as a hydrogen halide, a catalyst is often not even required. H-O and H-N bonds are not quite as polar as hydrogen-halide bonds and therefore still require a catalyst for to occur. When inserting into an oxygen-hydrogen bond using a metal catalyst such as Rh2L4 (MC), the nucleophilic alcohol attacks the “electrophilic metal carbene to form an oxonium ylide” 21. Once the ylide forms, the intermediate will rearrange and the proton of the O-H bond will rearrange to the carbon while the catalyst is regenerated (Scheme 4). The same mechanism can be used in the case of insertion into nitrogen-hydrogen bonds. 20

Scheme 4: Intermolecular Carbon Insertion into an Oxygen-Hydrogen Bond. (Doyle,

1998).

Cyclopropane is a structurally simple molecule. All three cyclopropane carbons exist in the same plane. While sp3 hybridized carbon atoms typically have bond angles of

109.5°, cyclopropane is special in that its bond angles are closer to 60°. These angles smaller than the optimal 109.5° cause both “angle strain” of about 50°, along with 3 sets 33 of eclipsing hydrogens which make the cyclopropane bonds much weaker and therefore more reactive than those of unstrained sp3 carbons. This unusual structure also causes the sp3 orbitals to become distorted and allows the carbon-carbon bonds to have more π-bond character, while the attached carbon-hydrogen bonds are shorter.21

Figure 18: The Structure of Cyclopropane.

Cyclopropane was originally found useful as an anesthetic. It is no longer used as an anesthetic due to its high volatility and flammability which makes it a high risk in any surgery. Cyclopropane is rated a 4 (the same as hydrogen gas) as found in Material

Safety Data Sheets, MSDS. Cyclopropane is also costly to produce, especially in the volumes necessary for anesthesia. Other medicinal uses have been found for cyclopropane as a ligand for medicinal compounds. When cyclopropane rings are attached to drugs, as such as macrolides, they can “enhance potency, reduce off-target effects, increase metabolic stability, increase brain permeability, and decrease plasma clearance” 21.

One effective method for synthesizing cyclopropane from various ,

− + involves diazomethane ( CH2 N2) as the carbene source upon reaction with palladium(II) catalysts (Scheme 4). This reaction works best with alkenes that are electron-poor or contain increasing ring strain which tend to be more reactive than non-strained alkenes.20

34

Scheme 5: Cyclopropanation of an Alkene using Diazomethane and Palladium (II)

Catalyst. (Doyle, 1998).

Diazomethane is a highly toxic and explosive gas at room temperature, while the precursors to diazomethane are carcinogenic. Owing to these difficulties it has been prudent to prepare alternative reagents that can generate diazo-containing substrates. One of the most useful reagents for this purpose is para-aminobenzenesulfonyl azide (p-

ABSA), developed by Davies et.al.22

Figure 19: p-ABSA, a Diazo-Transfer Reagent. (Davies et. al, 1992).

The p-ABSA reagent has been particularly useful when using a 1,3- β

-dicarbonyl group as such as dimethyl malonate with its activated methylene(CH2). The presence of the two carbonyl groups separated by one carbon provides for highly acidic methylene hydrogens (pKa ~9-13 depending on the carbonyl functional group). As such the methylene hydrogens can be abstracted by simple bases such as triethylamine, Et3N.

The enolate reacts with the terminal nitrogen of the sulfonylazide, p-ABSA. This is followed by a proton transfer to produce an β-diazocarbonyl compound. 35

Scheme 6: Mechanism of Diazo-Transfer Using p-ABSA. (Davies et. al, 1992).

This product, an β-diazocarbonyl moiety, can then be used as a pendant carbenoid precursor and then used for further reactions. Cycloaddition can be achieved using a rhodium-based catalyst with a 1,3-dipolar species. 1,3-dipolar species or better known as an ylide, a compound with both a positive and a negative charge on adjacent carbon atoms as was created in Scheme 6. An example of a [1,3]-dipolar cycloaddition is shown below in Scheme 7. 36

Scheme 7: A 1,3-dipolar cycloaddition. (McMills Group, 2019)

The carbonyl ylide (a dipole) is created when an oxygen lone pair attacks the electrophilic carbenoid, by way of the rhodium carbenoid formed through dirhodium catalysis. The ylide then undergoes an intramolecular [1,3]-dipolar cycloaddition across the alkene π-bond. This reaction can be used produce a number of cyclic heteroatom compounds with functional groups that lend themselves well to bioactive binding.23

Chapter 3.1 Synthetic Strategy for the Preparation of Spiramycin Derivatives

Scheme 8: The Acylation of Unmodified Spiramycin using Ethyl Malonyl Chloride. 37

The original plan for modifying spiramycin is shown in Scheme 8. This plan provides a method to modify the structure of spiramycin by acylation followed by generation of a carbenoid precursor of the C3 alcohol. This particular group has been chosen to modify due to the ease of functionalization as well as its potential interaction various intramolecular functional groups of spiramycin itself and with the 50s subunit the bacterial ribosome. As covered in the introduction, it is believed that between the C2098 and A2100 bases of the ribosome is a hydrophobic cleft in which the C4-C7 portion of the lactone ring of the macrolide binds.10 The first series of reactions includes the acylation of the C3 alcohol, this provides a carbenoid precursor to further functionalize the molecule. The β-dicarbonyl of the malonyl group provided through acylation affords an acidic methylene unit to be diazotized, then utilized as a carbenoid precursor group via rhodium catalysis. Through utilization of a panel of different rhodium catalysts, we will be able to diversify alkenes of the macrolide scaffold with formation of cyclopropanes,

C-H/O-H/N-H insertion, through ylide formation/cycloaddition or by utilizing a sigmatropic rearrangement.

Scheme 9: Diazotization of β -Diester Derivative of Spiramycin. 38

It was found that following initial experimentation the sugar moieties of spiramycin add complexity due to the presence of additional Lewis bases. It was decided that removal of 1-3 sugar groups through hydrolysis could provide alternative substrates of interest for functionalization. After recognizing the difficultly of reacting with the C3 alcohol alone on spiramycin when each sugar moiety contributes a similar secondary alcohol, a synthetic strategy was devised to simplify spiramycin down to its lactone ring scaffold by the removal of one or more sugar groups.

Scheme 10: Removal of Mycarose Sugar from Spiramycin.

After the removal of mycarose from spiramycin, and subsequent removal of a sugar bound secondary alcohol, it was believed that acylation would now occur at the preferred alcohol, C3. It was hoped that this could also make a simpler substrate to alleviate chromatographic problems. 39

Scheme 11: The Acylation of Mycarose-Free Spiramycin using Methyl Malonyl

Chloride.

Considering additional NMR signals present owing to the ethyl group of the acid chloride, it was decided to change to the methyl malonyl group to simplify the NMR to help with structural identification.

Scheme 12: Diazotization of Acetylated Mycarose-Free Spiramycin Derivative.

Once the acylated product of Scheme 12 is formed, with its β-diazocarbonyl moiety, it can undergo one of the various rhodium(II) catalyzed reactions as such as a cycloaddition (Scheme 13). The ylide/cycloaddition methodology is a natural subsequent step for spiramycin given its presence of conjugated double bonds. 40

O O O O

O O O O O O

H3CO H3CO

Scheme 13: Tandem Ylide Formation/Cycloaddition Strategy

Chapter 3.2 Synthetic Strategy for the Preparation of Roxithromycin Derivatives

Having the benefit of working through reactions of spiramycin prior to work with roxithromycin, it was determined that initial studies with roxithromycin should begin preparing the des-sugar analog. The synthetic strategy developed for roxithromycin began with hydrolysis of the cladinose sugar. We presumed this was the more important modification, as the cladinose sugar of roxithromycin does not play a role in binding with the bacterial ribosome and was determined to be the target of any glycosidic cleavage methodology16.

Scheme 14: Removal of Cladinose from Roxithromycin. 41

Glycosidic cleavage was to then be followed by an acylation of the newly revealed secondary alcohol at C3 where cladinose had previously been bound to the core macrocyclic lactone ring of roxithromycin.

Scheme 15: Acylation of Cladinose-Free Roxithromycin Using Methyl Malonyl

Chloride.

Following essentially the same scheme used previously with spiramycin, the β- dicarbonyl formed through from acylation with methyl malonyl chloride afforded an acidic methylene unit, poised to be diazotized, then utilized as a carbenoid group through rhodium catalysis and loss of nitrogen gas (Scheme 15 & 16).

Scheme 16: Diazotization of Acetylated Cladinose-Free Roxithromycin Derivative.

Once the product of Scheme 16 is formed, with its β-diazocarbonyl moiety, it can undergo one of the various rhodium(II) catalyzed reactions as such as a OH insertion 42

(Scheme 17). The OH insertion is a natural next step for spiramycin with the availability of both the C6-OH and C11-OH to create a heterocyclic substrate.

O O O O O O N OCH N OCH N OCH3 3 3 C C C9 9 9 OH HO OH HO OH HO C11 C11 C11 2+ C OH C Rh C12 OH C6 C12 O C6 12 6 and/or CO2CH3 a H C O O O H C O OH H5C2 O OH O 5 2 5 2 b C C O C3 3 3 O O O OCH O O N2 O O 3 O O a Product (C5) OH Insertion b Product (C11) OH Insertion

Scheme 17: Projected OH Insertion Products from Diazotized Roxithromycin.

Chapter 3.3 Experimental Determination

For both roxithromycin and spiramycin, reaction progress was monitored using

Thin Layer Chromatography (TLC). This is a common technique in organic chemistry used to separate compounds via acid-base interactions with the silica bound plates. One deposits the sample (usually in solvent) onto a silica plate using a glass capillary tube, then the TLC plate is placed in a chamber using a predetermined solvent system, allowing the sample to interact with the predetermined solvent system and allowing the compounds to differentially move with the solvent depending on size, functional group interactions and polarity. The solvent relies on capillary action to rise up the TLC plate, where the intermolecular forces between the solid silica and solvent move the solvent against gravity and carry the sample up the plate. The plate is then stained (to visualize the compound being separated), while the “Retardation factor” (Rf) is calculated using the distance traveled by the solvent front versus the distance traveled by each fraction in the sample, thus helping to characterize and separate the compounds.

Due to the highly polar nature of spiramycin and roxithromycin, determining a solvent system that can be used to separate highly polar compounds is challenging. Once 43 a reasonable solvent system is determined, separation can occur and the reaction mixture can be separated using column chromatography. Column Chromatography utilizes similar principles to that of Thin Layer Chromatography (TLC) in a 3-dimensional system, capable of much larger scale separations of milligram to gram quantities of material. As macrolides are notoriously hard to work with using traditional silica-based TLCs and flash column chromatography, one of the goals of this project was to exhaust possible solvent systems for traditional silica. In normal phase liquid chromatography, the mobile phase (solvent system) is less polar than the stationary phase (silica). The less polar the solvent system is, the longer polar analytes, such as macrolides are retained on the stationary phase. As macrolides suffer from poor solubility in most organic solvents, except with the most polar solvents as such as chloroform, this project required an exploration of “hydrophilic interaction chromatography”. Hydrophilic interaction chromatography uses the same stationary phase as classical flash column chromatography, silica, but the mobile phase is used is typically more similar to those used in reversed-phase chromatography and involves water, acetic acid, formic acid, and or acetonitrile. This form of chromatography has been successfully used in the analysis of polar pharmaceuticals, so it was a logical next step from traditional flash column chromatography, to work with macrolides spiramycin and roxithromycin.24

Nuclear Magnetic Resonance spectroscopy, NMR, is used to determine if a desired structure has been synthesized and isolated. 1H NMR relies on the local proton magnetic fields around the 1H hydrogen or 13C, the carbon nuclei to provide information regarding the structure of the compound. COSY NMR is used to detect close hydrogen- hydrogen through bond interactions to help determine structure in terms of connectivity. 44

Using these two techniques as well as 13C NMR, one is able to ascertain if the reactions were successful and if preparation of the desired structures were achieved.

Once the novel analog structures have been synthesized and purified (and confirmed by NMR) they will be sent to Professor Nigel Priestley’s group (University of

Montana), who will perform a cadre of assays for antibiotic activity.

Chapter 4: General Experimental

All reactions were carried out under an inert atmosphere of Nitrogen (N2). All glassware was flame dried or placed in the oven overnight to ensure anhydrous conditions. Solvents used, as such as 1,2-dichloromethane and methanol, CH3OH, were cleaned via a simple distillation and were then placed in a round bottom with molecular sieves to maintain anhydrous conditions. Starting material, spiramycin was purchased from Sigma-Aldrich in 1g quantities, Lot # MKCG3562. Starting material, roxithromycin was purchased through Alfa Aesar in 5g quantities, Lot # W10E052. Thin Layer

Chromatography was used to determine reaction progress. Traditional TLC plates were ordered from Agela Technologies, Spec 200x200mm with aluminum backing with 10 µm silica bonded with different functional groups. C18-W TLC plates used for reverse phase chromatography were provided by Sorbent Technology, Spec 10cm x10cm plates with aluminum backing. Visualization of TLC plates was afforded by several methods including an ultraviolet lamp (fluorescence) for compounds with conjugated-pi systems.

Compounds without conjugation were visualized using various stains including phosphomolybdic acid (PMA, 10g of PMA/100mL of absolute ethanol). PMA is a universal stain that is sensitive to low concentrations of material, making it a good fit for identification of antibiotic analogs, given that most reactions were run at milligram 45 scales. Flash column chromatography used Merck silica 60 (230-400 mesh). 1H NMR,

13C NMR and COSY spectrum were collected using a 500MHz Bruker ASCEND-500 and a 300MHz Bruker AVANCE-300. Chemical shifts are quoted in parts per million downfield from chloroform-d (CDCl3).

Chapter 4.1: Spiramycin Experimental

Scheme 8: The Acylation of Unmodified Spiramycin using Ethyl Malonyl Chloride.

Reaction: AO-02

In a 10mL flamed dried round bottom flask, 0.01mL of ethyl malonyl chloride

(0.01mL, 0.078mmol, 3.25 equivalents) was dissolved in dichloromethane (dried over molecular sieves) at room temperature. Spiramycin (20.2mg, 0.024 mmol, 1 equivalents) and triethylamine (0.01mL, 0.072mmol, 3 equivalents) were added to the acid chloride.

The mixture was stirred at room temperature overnight to provide 32.4mg of crude material. The crude material was transferred to a column with a solvent system of acetic acid, water, and ethyl acetate (1:1:2). This provided four different compounds AO-02A,

AO-02B, AO-02C, and AO-02D with Rf values of 1, 0.43, 0.27, and 0 respectively. NMR indicated the reaction was a complex mixture of compounds, that included solvent. It was decided that a more efficient solvent system must be established for working with spiramycin, therefore a large panel of solvent systems were considered. 46

Solvent System Retention Factor (Rf )

Hexane-Isopropanol-Triethylamine (10:10:1) 0.53

Methanol-Ethyl Acetate-Acetone (5:3:2) 0.00

Chloroform-Acetone (6:4) 0.00

Ethyl-Acetate-Methanol (17:3) 0.00

Benzene-Acetone (1:1) 0.00

Benzene-Acetone (3:1) 0.00

Benzene-Acetone (6:1) 0.00

Acetone 0.00

Benzene 0.00

Hexane-Isopropanol-Triethylamine (20:20:1) 0.49

Hexane-Isopropanol-Triethylamine (30:30:1) 0.44

Hexane-Isopropanol-Triethylamine (40:40:1) 0.38

Hexane-Isopropanol-Triethylamine (67:67:1) 0.22

Hexane-Isopropanol-Triethylamine 0.18 (100:100:1)

Hexane-Isopropanol-Triethylamine 0.00 (200:200:1)

Chloroform-Methanol (1:1) 0.00

Chloroform-Methanol-Triethylamine 0.14 (200:200:1)

Chloroform-Methanol-Triethylamine 0.31 (100:100:1)

Isopropanol-Triethylamine (400:1) 0.26

Isopropanol-Triethylamine (200:1) 0.31

Methanol-Triethylamine (400:1) 0.07

Methanol-Triethylamine (200:1) 0.09 47

Methanol-Triethylamine (133:1) 0.32

Methanol-Triethylamine (100:1) 0.43

Hexane-Methanol-Triethylamine (200:200:1) 0.12

Hexane-Methanol-Triethylamine (100:100:1) 0.20

Hexane-Methanol-Triethylamine (67:67:1) 0.559

Hexane-Triethylamine (83:1) 0.28

Table 1: Establishing Solvent System for Spiramycin

After establishing Hexane-Isopropanol-Triethylamine (200:200:1) as polar enough solvent system to move spiramycin from the baseline of a silica plate, acetylation of Spiramycin was attempted again. This time using “Chemical Modification of

Spiramycin: VI. Synthesis and Antibacterial Activities of 3,3”-di-o-acyl-4”-o-sulfonyl and 3,3”-di-o-acyl-4”-o-alkyl Derivatives of Spiramycin I” as reference.25

Reaction: AO-03

To a flame dried 10 mL round bottom with a vigreux-type air condenser attached,

0.23 mL of 1,2-dichloroethane and spiramycin (60.0mg, 0.071 mmol, 1 equivalents) were added and cooled to 0°C. At this desired temperature, triethylamine (0.09mL, 0.640 mmol, 9.1 equivalents) and ethyl malonyl chloride (0.08mL, 0.625 mmol, 8.8 equivalents) were added to the reaction flask and were heated to 80°C using a sand bath.

The reaction remained at reflux overnight. After 12 hours of reflux, 1.00 mL methanol was added and left to stir for 15 minutes to quench the reaction. The reaction mixture was diluted with 5 mL of chloroform then washed twice in a separatory funnel with sodium bicarbonate and deionized water respectively. The reaction mixture was dried over

Na2SO4 then dissolved in 3.4 mL of 70% methanol/deionized water and heated to 50°C 48 for 93 (based of the time stated by Sano et. al) minutes. The reaction mixture was again diluted with 5 mL chloroform, washed with sodium bicarbonate and deionized water, then dried over Na2SO4. The resulting 18.9mg of crude material provided an orange/yellow, gummy product. Purification was attempted through two separate columns of Hexane-Isopropanol-Triethylamine (200:200:1). The presumed desired compound, AO-03 showed an Rf value of 0.48 while the starting material, on the same

TLC plate, showed an Rf value of 0.41. Both spots were found to be UV active using an ultraviolet lamp, which was to be expected if the conjugated double bond, C10 and C12 of spiramycin wasn’t compromised.

Figure 20: Proposed Product Structure with C10 -C12 Alkene Intact.

NMR was taken of the 8.0mg of “isolated” compound, NMR AO-03 Post-

Column#2 (Oct11-2018-MCM). The NMR showed more than one compound present, despite two attempted separations by column chromatography. It was assumed that one of these compounds present was the desired structure. Rather than attempt yet another column, it was decided to diazotize the -diester structure with the hope that it might be more easily separated post diazotization. 49

Scheme 9: Diazotization of β -diester derivative of spiramycin.

Reaction: AO-04

To a flame dried 10mL round bottom flask under argon, (8.09mg, 0.0096 mmol, 1 equivalent) (assuming the original molecular weight of spiramycin to err on the side of more equivalents) of product from the previous reaction (AO-03) and 0.08mL of acetonitrile (4 equivalents) were added and cooled to 0°C. Then p-

(acetamido)benzenesulfonylazide (2.4mg, 0.01 mmol, 1 equivalent) and triethylamine

(0.01mL, 0.072 mmol, 7.5 equivalents) were added and the mixture stirred for 1 hour.

The reaction was then removed from the ice bath and the reaction was allowed to come to room temperature, 22.7°C, then stirred for an additional 2 hours. The reaction flask was evacuated and weighed, providing 6.1mg. An NMR was obtained, NMR-AO-04 Crude diazotization Attempt (Oct16-2018-MCM). NMR did not show any of spiramycin’s lactone ring as identifiable peaks.

In the hopes of reducing the number of products and the number of peaks on the

H1 NMR, reaction AO-02 was reworked to include methyl malonyl chloride instead of ethyl malonyl chloride since methyl malonyl chloride presents with one less peak on H1

NMR. 50

O O

CH3 Cl O

Figure 21: Methyl Malonyl Chloride (ChemDraw, 2019).

Reaction: AO-05

To a flame dried 10 mL round bottom with a vigreux-type air condenser attached,

1,2-dichloroethane (0.41mL, 5.117mmol, 43 equivalents) and spiramycin (100.60 mg,

0.119 mmol, 1 equivalent) were added and cooled to 0°C. Once at temperature,, triethylamine (0.15mL, 1.075mmol, 9 equivalents) and methyl malonyl chloride

(0.12mL, 1.12mmol, 9.4 equivalents) were added to the reaction flask and then heated to

80°C using a sand bath. The reaction was heated overnight. After approximately 12 hours, 1.00 mL methanol was added and stirred for an additional 15 minutes to quench the reaction. The reaction mixture was diluted with 5 mL of chloroform, then washed twice with sodium bicarbonate and deionized water respectively. The reaction mixture was dried over Na2SO4 and the solvent evaporated in vacuo. After being left overnight under high vacuum, a yield of 93.5mg of a pale-yellow gel-like material was obtained.

NMR A0-05 Acetylation Attempt #3 Crude (Oct25-2018-MCM). The NMR showed that the reaction was not successful and the resulting product was identical with the starting material, spiramycin.

Reaction AO-06

In order to achieve the desired product, it was determined that a stronger base was needed. The reaction was repeated using sodium hydride rather than triethylamine. To a flame dried 10 mL round bottom, 1,2-dichloroethane (0.41mL, 5.117mmol, 46 equivalents) and spiramycin (9.35mg, 0.011mmol, 1 equivalent) were added and cooled to 0°C. At the desired temperature, sodium hydride (0.6mg, 0.025mmol, 2.3 equivalents) 51 and methyl malonyl chloride (0.11mL, 0.093mmol, 8.5 equivalents) were added to the reaction. The reaction mixture was allowed to warm to room temperature. After an hour the mixture turned a dark purple color. The reaction was left to stir overnight. After 12 hours, 2.00 mL methanol was added and stirred for an additional 15 minutes to complete the quench. The reaction mixture was diluted with 6 mL of chloroform, then washed twice with sodium bicarbonate and deionized water respectively. The reaction mixture was then dried over Na2SO4 and then the solvent evaporated. After being left overnight under high vacuum, a yield of 64.1mg of brown gel-like material was obtained. NMR

AO-06 Acetylation Attempt #4 w/ NaH Crude (Oct30-2018 MCM).

Using a solvent system of Methanol-Triethylamine (154:1), four spots were isolated using a column, AO-06A Rf 0.80 0.0357g, AO-06B Rf 0.0.625 0.0374g, AO-06C Rf 0.49, and AO-06D Rf 0.216. NMR AO-06 Post Column (Nov02-2018-MCM) showed that AO-

06A, AO-06B, and AO-06C all retained the aldehyde peak at 9.6 ppm but still appeared to contain more than more compound in the NMR. Starting with AO-06A a new solvent system was needed to optimize separation of multiple compounds.

Solvent System Retention Factor (Rf )

Ethyl Acetate 0.71* and 0.00*

Methanol 0.87* and 0.00* *UV active

Table 2: Solvent System Determined for AO-06A

With the hopes of an easy separation of the 0.71Rf fraction, A0-06A was added to a column of pure ethyl acetate with a silica to compound ratio of 100:1. The first fraction departed the column quickly and did not display any desired aldehyde H-C=O peak via 52

NMR, NMR AO-06A(E) Post Column #2 (Nov05-2018-MCM). The polarity of the solvent system was increased adding triethylamine and the baseline spot being flushed out. Again, it did not show the desired aldehyde peak on NMR AO-06A Post Column #2.

Concerned that the compound was not totally soluble in ethyl acetate or that it was decomposing on the column, a new solvent system had to be established for working with

AO-06B.

Solvent System Retention Factor (Rf )

Ethyl Acetate 0.00* and 0.78*

Ethyl Acetate-Methanol (1:1) 0.00* and 0.82*

Ethyl Acetate-Chloroform (1:1) 0.00* and 0.74* *UV active

Table 3: Establishing Solvent System for AO-06B

ethyl acetate-methanol and ethyl acetate-chloroform had comparable separation of components on the thin layer chromatography, but ethyl acetate-chloroform provided less streaking so it was chosen as the solvent system for the A0-06B column. Packing a column with a 100:1 silica to compound ratio AO-06B(H) 0.74Rf was quickly flushed through the column while polarity was increased with triethylamine in order to flush AO-

06B(I) through. NMRs were collected, AO-06B(H) Post Column #2 and AO-06B(I) Post

Column #2 (Nov13-2018-MCM). AO-06B(H) did not exhibit the desired aldehyde peak and AO-06B(I) had a peak in the 9ppm region but lacked other peaks indicating the core lactone ring of spiramycin.

Rather than constantly recalibrating the solvent system to move spiramycin and our desired derivatives from the baseline, it was decided that we would remove the one or 53 more sugars from the spiramycin core to reduce its polarity and limit the number of side reactions that can occur.

Scheme 10: The Removal of Sugar, Mycarose from Spiramycin.

Reaction AO-07

In a flame dried 10mL round bottom placed under argon, spiramycin (19.70 mg,

0.023mmol, 1 equivalents) was dissolved in a 3% HCl/methanol solution (1mL). The reaction was stirred at room temperature for 24 hours. The resulting reaction mixture was diluted with chloroform, washed with Na2CO3 and then distilled to remove H2O from the solution. The solution recovered from the distillation was dried over Na2SO4 providing

16.7mg of several products. NMR of AO-07 (Sugar Removal) crude mixture provided several compounds. A column of the crude reaction product using a solvent system of methanol-triethylamine (154:1) provided three fractions, Rf’s 0.82. 0.53, and 0.26 for

AO-07A, AO-07B, and AO-07C respectively. The 1H NMR of AO-07B’s provided the most promising spectra, so it was chosen for further purification. The first column of AO-

07B was comprised of a solvent system of isopropanol-triethylamine (286:1) and did not achieve any further separation. The material was collected from flushing the column, leading to a secondary column for the remaining 10.1mg of AO-07B with a different 54 solvent system (ethyl acetate-methanol, (9:1)) yielding 7.4mg. NMR AO-07B Post

Column #2 EA/MeOH 90/10 (Nov30-2018MCM) implied AO-07B still contained multiple compounds. Further tests were run with different solvent system to achieve a further degree of purification.

Solvent System Retention Factor (Rf )

Ethyl Acetate 0.00*

Isopropanol-Triethylamine (167:1) 0.32*

Chloroform-Methanol (9:1) 0.00*

Chloroform-Methanol-Triethylamine 0.08* (1125:125:1)

Chloroform-Methanol-Triethylamine 0.16* (692:77:1) *UV active

Table 4: Establishing Solvent System for AO-07B Post Ethyl Acetate/Methanol (9:1)

Column

chloroform-methanol-triethylamine (692:77:1) and isopropanol-triethylamine (167:1) were both used as solvent system for running a 2-dimensional (2-D) TLC’s. The 2- dimensional TLC is used to help determine if the compound is decomposing on the column or using a certain solvent system. Neither of these 2-D TLC’s showed more than one spot. So despite a suboptimal NMR, without the solvent system to show or separate

AO-07B into its presumed multiple fractions, it was decided to treat the sample as a single compound and proceed to the next step of the synthetic strategy, the acetylation of

C3. 55

Scheme 11: The Acylation of Mycarose-Free Spiramycin using Methyl Malonyl

Chloride.

Reaction AO-08

To a flame dried 10 mL round bottom with a vigreux air condenser attached, AO-

07B (7.4mg, 0.0104 mmol, 1 equivalents) and 1,2-dichloroethane (0.5 mL) were added together and cooled to 0°C. Methyl malonyl chloride (0.01mL, 0.0932mmol, 9 equivalents) and triethylamine (0.01mL, 7.3mg, 0.0717mmol, 7 equivalents) were added to the reaction flask at 0°C and then were heated to 80°C using a heater with a sand bath.

The reaction was left to stir for 48 hours. Once completed, 0.5 mL methanol was added and left to stir for 15 minutes to quench the reaction. The reaction mixture was diluted with an additional 1 mL of chloroform then washed twice in a separatory funnel with sodium bicarbonate and deionized water respectively. The reaction mixture was dried over Na2SO4, then evaporated under vacuum. After being left overnight under high vacuum, 2.6mg was obtained. Unfortunately, NMR A0-08 Crude (Dec06-2018-MCM) did not display any of spiramycin’s core lactone ring’s easily identifiable alkene peaks in the 6ppm region. Concerned that the reaction had been run on too small of a scale it was 56 decided to repeat the removal of mycarose from spiramycin and then acylate on a 100mg scale reaction.

Reaction AO-12

In a flame dried 25mL round bottom placed under argon, spiramycin (100.30 mg,

0.119mmol, 1 equivalent) was dissolved in a 1% HCl/methanol solution (5mL). The reaction was stirred at room temperature for 24 hours. The resulting reaction mixture was diluted with saturated Na2CO3 (5mL) and then rotavaped to remove solvent. Extraction was then performed with chloroform (5mL) three times. The reaction mixture was then washed twice with 3M HCl (10mL each), then neutralized with 3M NaOH (20mL). The resulting solution was then saturated with NaCl and the product extracted with chloroform (10mL) three times. The reaction mixture was then dried over Na2SO4, and evaporated under vacuum. After being left overnight under high vacuum, a yield of

16.7mg was obtained. TLC of the crude reaction product in a solvent system of H2O-

Acetic Acid-Ethyl Acetate (1:1:3) provided 4 fractions, Rf’s 0.96, 0.73, 0.28, and 0.08 for

AO-12A, AO-12B, AO-12C, and AO-12D respectively. Only AO-12B and AO-12C were

UV active implying spiramycin’s core lactone alkenes were still present. After running a column on the reaction mixture using the same solvent system, H2O-Acetic Acid-Ethyl

Acetate (1:1:3), on a 1:100 product to silica ratio, the fractions were combined and rotavaped until H2O and acetic acid were the last solvents present. This was titrated with

1M NaOH until pH strips showed a pH of 7, saturated with NaCl, then extracted with chloroform. The chloroform was then dried over Na2SO4, then evaporated under vacuum and 9.7mg of AO-10C was obtained. 1H-NMR of AO-10C looked promising and so the reaction was repeated on a 500mg scale. 57

Reaction: AO-14

In a flame dried 25mL round bottom placed under argon, spiramycin (484.9 mg,

0.575mmol, 1 equivalent) was dissolved in a 1% HCl/methanol solution (25mL). The reaction was stirred at room temperature for 24 hours. The resulting reaction mixture was diluted with saturated Na2CO3 (25mL) and then rotavaped to remove solvents. Extraction was then performed with chloroform (12.5mL, 3X). The reaction mixture was then washed (2X) with 3M HCl (20mL each), then neutralized with 3M NaOH (40mL). The resulting solution was then saturated with NaCl and then the product extracted with chloroform (12.5mL) three times. The reaction mixture was then dried over Na2SO4, then evaporated under vacuum. After being left overnight under high vacuum, a yield of

72.4mg was obtained. TLC of the crude reaction mixture in a solvent system of H2O- acetic acid-ethyl acetate (1:1:3) provided 2 fractions, Rf’s 0.71 and 0.34 for AO-14A and

AO-14C respectively. Only AO-14C was UV active, implying the presence of the spiramycin’s core lactone ring and alkenes were still present. After running a column on the reaction mixture using the same solvent system, H2O-acetic acid-ethyl acetate (1:1:3), on a 1:100 product to silica ratio, the fractions were combined and rotavaped until only

H2O and acetic acid were present. This was titrated with 1M NaOH until pH strips showed a p of 7, saturated with NaCl, then extracted with chloroform. The chloroform was then dried over Na2SO4, then evaporated under vacuum and 4.7mg of AO-14C was obtained. 1H-NMR of AO-14C looked promising considering the small amount of material left but given the cost of spiramycin as a starting material, a new method for removing sugar from spiramycin needed to be found before continuing.

58

Reaction AO-16

Scheme 18: Acetal Formation of C6 Aldehyde Before Attempted Sugar Removal.

According to the work of Klich et. al, the most effective way to remove the sugars from spiramycin starts with the protection of the C6 aldehyde.26 In a flame dried 25mL round bottom under nitrogen, spiramycin (100.8mg, 0.120mmol, 1 equivalent) and pyridinium p-toluene sulfonate (PPTs) (74.5mg, 0.298mmol, 2.5 equivalents) were dissolved in methanol (2mL, 417 equivalents) and trimethylorthoformate (6mL, 461 equivalents). The reaction was stirred at 45°C, for 36 hours. The resulting reaction mixture was evaporated then dissolved in chloroform and washed twice with NaHCO3.

The resulting solution dried over Na2SO4, to produce 124.3mg of product. NMR AO-16

Crude (April04-2019-MCM) looked promising with the corrected integral of the single aldehyde peak reduced from 1.00 to .39, implying about a 60% yield of the desired acetal product with 40% still as an aldehyde. This was purified using a column with solvent system of H2O-acetic acid-ethyl acetate (1:1:3) that yielded Rf’s 0.7, 1.15, and 3.7 for

AO-16C, AO-16B, and AO-16A respectively. AO-16A (43.6mg) showed the most promising 1H-NMR with the aldehyde peak entirely eliminated.

59

Chapter 4.2: Roxithromycin Experimental

Scheme 14: Removal of Cladinose from Roxithromycin.

Reaction: AO-09

In a flame dried 10mL round bottom under argon, roxithromycin (28.9mg,

0.034mmol, 1 equivalent) of was dissolved in a 1% HCl/methanol solution (1.5mL). The reaction was stirred at room temperature, for 24 hours. The resulting HCl/methanol mixture was diluted with chloroform (5mL), washed with Na2CO3, and then with distilled

H2O. The resulting solution was dried over Na2SO4, to give 25.5mg crude reaction product. NMR AO-09 Roxithromycin Sugar Removal (1% HCl) Crude looked promising but also implied multiple compounds are present. A solvent system capable of differentiating between the compounds was sought.

Solvent System Retention Factor (Rf )

Chloroform-Methanol (1:1) 0.00, 0.83

Chloroform-Methanol (4:1) 0.00, 0.83

Ethyl Acetate 0.00, 0.64, 0.82

Ethyl Acetate-Chloroform (1:1) 0.00, 0.58, 0.86

Dichloromethane-Methanol (4:1) 0.00, 0.88

Ethyl Acetate-Methanol (4:1) 0.00, 0.79, 0.88 Table 5: Establishing Solvent System for AO-08 Crude 60

Solvent systems that include ethyl acetate afforded separation of three different spots compared to those solvent systems without ethyl acetate. Using a column of pure ethyl acetate was run on AO-08, with two fractions eliminated as the first two fractions at

Rf 0.64 and 0.82 AO-09A and AO-09B respectively. The column was flushed with triethylamine to push the baseline fraction, AO-09C through the column. NMR was collected, AO-09C Post DCM:MeOH:TEA Column (Jan19-2019-MCM), it implied further chromatography was necessary. Having collected the top fractions with the ethyl acetate column, a more polar solvent system had to be established in order to collect AO-

09C from the baseline and ideally separate any additional compounds present.

Solvent System Retention Factor (Rf )

Dichloromethane-Triethylamine (200:1) 0.00, 0.62

Dichloromethane-Triethylamine (100:1) 0.00, 0.67

Dichloromethane-Triethylamine (80:1) 0.06, 0.89

Dichloromethane-Triethylamine-Methanol 0.28 1.00 (80:1:8)

Dichloromethane-Triethylamine-Methanol 0.16, 1.00 (80:1:4) Table 6: Establishing Solvent System for AO-08C

Due to difficulties of solvent systems with a high percentage of methanol while working with spiramycin, it was decided to lower the percentage by adding additional solvents to prepare a solvent system including dichloromethane-triethylamine-methanol

(80:1:4), and this was chosen as the solvent system. With only 0.0060(mg)g of AO-09C, this would be a test column in order to see if this solvent system was capable of purifying the reaction, before committing to redo the reaction on a much larger scale. The column was run on a 500:1, silica to compound ratio and afforded a yield of 0.0028. NMR was 61 collected Jan19th, 2019. Although the small yield made the NMR difficult to interpret, it was promising enough to merit repeating the reaction at a much larger scale.

Reaction: AO-10

In a flame dried 25mL round bottom under argon, roxithromycin (150.70mg,

0.180mmol, 1 equivalent) of was dissolved in a 1% HCl/methanol solution (7.5mL). The reaction was stirred at room temperature, for 24 hours. The resulting HCl/methanol mixture was diluted with chloroform (15mL), washed with Na2CO3, and then with distilled H2O. The resulting solution dried over Na2SO4, resulting in160mg of a crude reaction product. NMR AO-10 Crude (Jan24-2019-MCM) looked promising but again implied multiple compounds. TLC of the crude reaction product in a solvent system of dichloromethane-methanol-triethylamine (80:4:1) provided three fractions, Rf’s 0.57.

0.46, and 0.36 for AO-10A, AO-10B, and AO-10C respectively. From NMR AO-10C

Post Column (DCM/MeOH/TEA) (Jan25-2019-MCM), AO-10C (0.0788mg) looked to be the most promising and contained the core macrocyclic lactone ring but was still not acceptably purified. NMR AO-10A Post Column (DCM/MeOH/TEA) (Jan25-2019-

MCM) ) contained peaks that belong to the cladinose sugar. Another column was run on

AO-10C using the same solvent system, dichloromethane-methanol-triethylamine

(80:4:1), and a 1:100 compound to silica ratio. NMR AO-10C Post Column #2 (Feb01-

2019-MCM) was slightly “cleaner” and TLC only showed one spot so product was moved to the next step, acylation. 62

Scheme 15: Acylation of Cladinose-Free Roxithromyicn Using Methyl Malonyl

Chloride.

Reaction: AO-11

To a flame dried 10 mL round bottom with a vigreux-type air condenser attached,

1,2-Dichloroethane (0.10mL, 1.33mmol, 43 equivalents) and roxithromycin (21.00 mg,

0.03mmol, 1 equivalent) were added and cooled to 0°C. At this desired temperature, triethylamine (0.04mL, 28.33mg, 0.287mmol, 9.5 equivalents) and methyl malonyl chloride (0.03mL, 0.280mmol, 9.3 equivalents) were added to the reaction flask and heated to 80°C using a sand bath. The reaction was left overnight. In the morning 1.00 mL methanol was added and left to stir for 15 minutes to quench the reaction. The reaction mixture was diluted in 5 mL of chloroform then washed twice in a separatory funnel with sodium bicarbonate and deionized water respectively. The reaction mixture was then dried over Na2SO4 and the solvent evaporated. After being left overnight under high pressure vacuum, a yield of 23.3mg of a pale-yellow gel-like material was obtained.

NMR AO-11 Crude (Feb06-2019-MCM) had promising peaks in the 2.5-4ppm region.

This was enough to merit an increase in reaction scale size to 500mg from the cladinose removal step to the acylation.

63

Reaction: AO-13

In a flame dried 25mL round bottom placed under argon, roxithromycin (499.0 mg, 0.596mmol, 1 equivalent) was dissolved in a 1% HCl/methanol solution (25mL). The reaction was stirred at room temperature for 24 hours. The resulting reaction mixture was diluted with saturated Na2CO3 (25mL) and then rotavaped to remove residual solvent.

Extraction was then performed with chloroform (12.5mL, 3X). The reaction mixture was then washed twice with 3M HCl (20mL each), then neutralized with 3M NaOH (40mL).

The resulting solution was then saturated with NaCl and then the product extracted with chloroform (12.5mL, 3X). The reaction mixture was then dried over Na2SO4, then evaporated under vacuum. After 12 hours under high vacuum, a crude yield of 319.2mg was obtained. TLC of the crude reaction product in a solvent system of dichloromethane- methanol (10:1) provided 2 fractions, Rf’s 0.76 and 0.00 for AO-13A and AO-13B respectively. A column was run using this solvent system with a compound to silica ratio of 1:100. 1H-NMR showed peaks that for AO-13A that corresponded to the cladinose sugar. 1H-NMR for AO-13B showed resonances for AO-14B that corresponded with roxithromycin’s macrocyclic lactone ring, but, was missing peaks for cladinose. With these promising results, it was hoped that AO-13B could be further purified. AO-13B was washed with 1M HCl that was then neutralized with 1M NaOH and staturated with

NaCl then extracted with chloroform and rotovaped to provide 331.4mg brown gel. TLC of the crude reaction product in a solvent system of H2O-acetic acid-ethyl acetate (1:1:3) provided 3 fractions, Rf’s 0.43, 0.33, and 0.28 for AO-13BE, AO-13BF and AO-13BG respectively. After the massive loss of material using this solvent system with spiramycin during a column, it was decided to only use 100mg of the reaction mixture for the 64 column. After running a column on the reaction mixture using the same solvent system,

H2O-acetic acid-ethyl acetate (1:1:3), on a 1:100 product to silica ratio, the fractions were combined and rotavaped until only H2O and acetic acid were present. This was titrated with 1M NaOH until pH strips showed a pH of 7, saturated with NaCl, then extracted with chloroform. The chloroform was then dried over Na2SO4, then evaporated under vacuum and 82.9mg of AO-13BF was obtained. 1H-NMR of AO-13BF looked excellent.

It was decided that 50mg of AO-14C would be used to attempt an acylation with methyl malonyl chloride (Scheme 12).

Reaction: AO-15

To a flame dried 10 mL round bottom with a vigreux-type air condenser attached,

1,2-Dichloroethane (0.25mL, 3.20mmol, 44 equivalents) and roxithromycin (50.0 mg,

0.072 mmol, 1 equivalent) were added and the mixture cooled to 0°C. At this desired temperature, triethylamine (0.09mL, 0.65mmol, 9 equivalents) and methyl malonyl chloride (0.07mL, 0.65mmol, 9 equivalents) were added to the reaction flask and were heated to 80°C using a sand bath. Reaction was left for 48 hours. After the reaction was determined to be finished by TLC, 1.00 mL of methanol was added and left to stir for 15 minutes to quench the reaction. Reaction mixture was diluted in 5 mL of chloroform then washed twice in a separatory funnel with sodium bicarbonate and deionized water respectively. The reaction mixture was then dried over Na2SO4 and the solvent evaporated. After 12 hours under high vacuum, a yield of 59.3mg of a pale yellow gel- like material was obtained. TLC of the crude reaction product in a solvent system of

H2O-acetic acid-ethyl acetate (1:1:6) provided 4 fractions, Rf’s 1.0, 0.91, 0.83, and 0.32 for AO-15A, AO-15B, AO-15C and AO-15D respectively. After running a column on the 65

reaction mixture using the same solvent system, H2O-Acetic Acid-Ethyl Acetate (1:1:6), on a 1:100 product to silica ratio, the fractions were combined and rotavaped until only

H2O and acetic acid were present. This was titrated with 1M NaOH until pH strips showed a pH of 7, saturated with NaCl, then extracted with chloroform. The chloroform was then dried over Na2SO4, then evaporated under vacuum. This produced 191.2mg of product with a strong order of acetic acid. The NMR for A0-15D looked promising but it was apparent that acetic acid was still present. The material was rinsed again with distilled H2O and extracted with chloroform to give 33.1mg of material and another NMR was run, AO-15D Post Column, Post Wash (Mar19-2019-MCM).

66

Chapter 5: Results

Figure 22: 2-D Structure of Roxithromycin.

Roxithromycin: 1H NMR (500 MHz, CDCl3) 5.18 (q, 2H) 5.10 (d, 1H) 4.41 (d, 1H)

4.33 (s, 1H) 4.021 (dq, 1H) 3.10 (d, 1H) 3.82 (d, 1H) 3.79 (t, 2H) 3.74 (ddq, 1H)

3.57 (t, 2H) 3.54 (d, 1H) 3.48 (ddq, 1H) 3.42 (s, 3H) 3.38 (s, 1H) 3.32 (s, 3H)

3.22 (dd 1H) 3.14 (s, 1H) 3.01 (t, 2H) 2.91 (dq, 1H) δ2.68 (dq, 1H) 2.42 (ddd, 1H)

2.36 (d, 2H) 2.28 (s, 6H) 2.21 (d, 1H) 2.02 (ddq, 1H) δ1.92 (ddq 1H) 1.64 (ddd,

2H) 1.56 (m, 8H) 1.50 (s, 3H) 1.29 (d, 3H) 1.23 (m, 7H) 1.18 (t, 6H) 1.14 (s, 3H)

1.10 (d, 2H) 1.03 (d, 3H) 0.84 (t, 3H). 67

Figure 23: H1 NMR of Roxithromycin 68

Figure 9: 2D Structure of Spiramycin. (ChemDraw, 2018).

Spiramycin: 1H NMR (500 MHz CDCl3) 9.81 (s, 1H) 6.24 (dd 1H) 6.01 (dd, 1H)

5.68 (dd, 1H) 5.55 (ddd, 1H) 5.28 (m, 1H) 5.06 (d, 1H) 4.47 (d, 1H) 4.37 (dd, 1H)

4.06 (m, 4H) 3.80 (d, 1H) 3.52 (m, 1H) 3.49 (s, 3H) 3.41 (m, 1H) 3.27 (m, 2H)

3.06 (d, 1H) 2.93 (d, 1H) 2.78 (ddd, 1H) 2.67 (dd, 2H) 2.47 (s, 6H) 2.43 (m, 2H)

2.36 (m, 0.5H) 2.33 (m, 0.6H) 2.22 (m, 1H) 2.20 (s, 6H) 2.09 (m, 1.7H) 2.02 (d,

2H) 1.93 (m, 1H) 1.82 (m, 2.5H) 1.74 (dd, 1.4H) 1.46 (m, 3.8H) 1.29 (d, 3H) 1.28

(d, 3H) 1.22 (m, 7H) 1.20 (s, 3H) 0.98 (d, 3H). 69

Figure 24: H1 NMR of Spiramycin 70

Figure 25: Structure of Roxithromycin w/o Cladinose.

AO-13BF: 1H NMR (500 MHz CDCl3) δ5.20 (dd, 1H) δ5.16 (t, 1H) δ4.49 (d, 1H) δ3.79

(s, 1H) δ3.73 (m, 3H) δ3.54 (m, 3H) δ3.49 (s, 1H) δ3.46 (s, 4H) δ3.37 (s, 3H) δ3.33 (dd,

1H) δ2.96 (m, 1H) δ2.68 (m, 1H) δ2.63 (m, 1H) δ2.49 (s, 6H) δ2.16 (m, 1H) δ2.01 (s,

5H) δ1.92 (ddq, 1H) δ1.78 (m, 1H) δ1.62 (m, 1H) δ1.48 (m, 1H) δ1.39 (s, 3H) δ1.24 (m,

10H) δ1.18 (m, 6H) δ1.06 (d, 3H) δ1.03 (d, 3H) δ0.82 (t, 3H) 71

Figure 26: H1 NMR of AO13-BF (Cladinose-Free Roxithromycin). 72

Figure 27: IR of AO13-BF (Cladinose-Free Roxithromycin). 73

Figure 28: Acetal Formation of Spiramycin’s C6 Aldehyde.

AO-16C: 1H NMR (500 MHz CDCl3) 8.24 (s, 1H) 6.18 (dd 1H) 6.01 (dd, 1H) 5.70

(dd, 1H) 5.53 (ddd, 1H) 5.28 (m, 1H) 5.10 (s, 1H) 4.72 (d, 1H) 4.515 (m 3H) 4.42

(d, 1H) 4.18 (dd, 1H)  (d, 1H) 3.92 (m, 1H) 3.76 (d, 1H) 3.58 (t, 1H) 3.50 (s,

3.5H) 3.44 (s, 4.3H) 3.34 (m, 6H) 3.06 (d, 2H) 2.66 (t, 2H) 2.50 (s, 9H) 2.26 (m,

10H) 2.07 (m, 6.5H) 1.86 (m, 4.5H) 1.74 (m, 2.4H) 1.61 (m, 2.6H) 1.49 (m, 4.7H)

1.257 (m, 50H) 0.97 (d, 5.5H) 0.88 (m, 9H) 0.06 (s, 2.7H).

74

Chapter 6: Discussion

The first paper referenced while working on this project was “Simultaneous

Identification and Quantitative Determination of Azithromycin, ,

Roxithromycin, Spiramycin and by Thin-Layer Chromatography and

27 Densitometry” . This paper suggests that a 1:1:3 ratio of H2O, acetic acid, and ethyl acetate is sufficiently polar to move Spiramycin from the baseline on a T.L.C. plate to an

Rf value of 0.31. Unfortunately, this was not the case, an Rf of 0.0 (Baseline) was obtained with the listed solvent system. Comparable Rf values were obtained only after setting up the T.L.C. chamber with solvent, then running the T.L.C. the following morning when the majority of the ethyl acetate had evaporated. This solvent system provided some degree of separation but was a difficult TLC and chromatography, leaving residual acetic acid solvent peaks in the NMR, despite rinsing the reaction mixture with distilled water and leaving it on high vacuum for 24 hours. It was decided to attempt a different solvent system that utilized triethylamine as the most polar component to move the macrolides from the baseline, hoping for a less problematic clean up. Chloroform-

Triethylamine combinations would typically provide a spot on the baseline and another close to the solvent line. Only after adding methanol, did the spot move from the baseline. Even with a baseline to solvent line separation between spots, columns on reaction mixtures for the acylation using this solvent system were only ever able to separate the products to a degree and never able to purify them completely.

As the solvent system remained a work in progress, we sought to identify an alternative synthetic strategy to better suit the difficulties encountered with separation and clean-up of the reaction mixtures. Since the polarity of spiramycin and roxithromycin 75 antibiotics forced us to resort to complicated solvent systems, we decided to reduce the polarity of the compounds by removing sugars. Using the method provided in

“Glycosidic Cleavage Reaction on Erythromycin A. Preparation of Erythronolide A” by

LeMahieu et.al, we chose to hydrolyze the glycosidic sugar bond using protic acid HCl dissolved in methanol28. With this methodology we were able to remove cladinose sugar from roxithromycin through hydrolysis (Figure 26: H1 NMR of AO13-BF (Cladinose-

Free Roxithromycin). Unfortunately, this method proved to be less effective for spiramycin. Roxithromycin is a derivative of erythromycin, having a similar solubility, so roxithromycin was better suited to the work up of this reaction that involved washing with 1M HCl to extract the basic product then retrieving it again with chloroform. We have found that most of the literature procedures do not work for the systems we have chosen to use. The reaction work-up must be revised to avoid losing so much material, especially for spiramycin. Klich et. al’s work has been promising for the removal of sugars from Spiramycin and further work can be done in this regard.26 As for the acylation reaction, our original use of ethyl malonyl chloride as our -dicarbonyl diazo precursor was amended to the methyl ester version to help simplify identification through the NMR. Since the 1H-NMR for both spiramycin and roxithromycin are both complex, even at 500MHz, we decided to use methyl malonyl chloride in order to reduce the number of peaks in the NMR spectra.

A major road-block for this research has been the limited separation of compounds using the various solvent systems with triethylamine, it was decided to revert back to the H2O, acetic acid, and ethyl acetate solvent system. This change was motivated following our work to remove various sugars of roxithromycin. After using the water, 76 ethyl acetate, acetic acid system for chromatography, it was found that this provided a more successful outcome. This seemed to be successful and was used to provide the caldinose-free Roxithromycin, AO-13BF and when used on the acylated product of AO-

13BF, AO-15, it was also somewhat successful but not to the same degree. When using the water, ethyl acetate, acetic acid solvent system on spiramycin we were able to achieve purification to a degree with product AO-16C, spiramycin with acetal formation on C6 aldehyde.

In order to try and maximize fraction separation, a compound to silica ratio of

1:100 or greater was used and likely was partially responsible for large mass balance loss.

This most likely contributed to the low yields, as was the fact that we were generally required to conduct several columns using this ratio on each reaction mixture. The silica used was Merck silica 60 (230-400 mesh). Normal phase silica is slightly acidic and roxithromycin being slightly basic, the columns were typically more effective on roxithromycin than spiramycin due to this acid-base attraction (but this also leads to greater mass loss). Due to the high polarity of the solvent systems used, the compounds interactions with the silica were minimal in comparison to the compound to solvent system interaction. We believe this lead to poor separations as the silica’s retention time of the different compounds were not as pronounced and therefore they eluted together.

One method available to avoid some of the loading problems seen previously was to attempt a “dry load” to minimize the solvent-compound interaction. Dry-loading seemed to help, but not completely rectify the problem. Experimenting with larger silica mesh sizes and therefore smaller pores of the silica could be useful in increasing the efficacy of the flash column, by eliminating smaller side products of the reactions. One important 77 method to try is using alternative stationary phases such as C18 (reversed phase), also known as a reversed phase. In these stationary phases, the Si-OH bonds of the silica are capped as an Si-OR (where R is the 8 or 18 carbon cap for the Si-O bond). Alternatively, alumina could offer better separation as it is possible to have acidic, basic and neutral forms of alumina. Likely, the best option for purification of these highly polar/high molecular weight compounds will be high-performance liquid chromatography (HPLC).

The availability of many normal and reversed phase columns as well as preparatory columns will provide the best methods for purification would allow us to maximize yield and purity.

The McMills group has never previously worked with macrolides. This thesis documents the first foray of the group into this class of compounds. Learning how to read the complex 1H-NMRs, avoid losing product through the compounds finicky solubility, and run columns on a plethora of solvent systems commanded the majority of my time.

By removing sugars, adding the simplest possible useful functional groups (methyl malonyl chloride), dry loading the chromatography columns, and using a water, ethyl acetate, acetic acid solvent system, it is possible to change and purify these macrolides.

Although the use of something like HPLC could speed us the process, what is expedient is not always necessarily the right, or only way to go about something. Using the different work arounds, I have listed above, there is substantial evidence that anyone with basic organic chemistry lab experience should be able to modify antibiotics. Given the importance of antibiotics in healthcare, and the need for new versions as well as variations of old versions, I believe that is the real success of this project.

78

References

1 American Chemical Society International Historic Chemical Landmarks. Discovery and

Development of Penicillin. http://www.acs.org/content/acs/en/education/what is

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