Analogs of Natural Product FR900098 as Small Molecule Inhibitors for the Methylerythritol Phosphate (MEP) Pathway in Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf)

by Ruiqin Wang

B.Sc. in Chemistry, May 2014, University of British Columbia

A Thesis submitted to

The Faculty of The Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Master of Science

May 20th, 2018

Thesis directed by

Cynthia S. Dowd Associate Professor of Chemistry © Copyright 2018 by Ruiqin Wang All rights reserved Dedication

I would like to dedicate my thesis to my mother and father, Caiwen Ma and Ping

Wang. Thank you for being there for me every step of the way, especially during those years when I was studying in Canada and USA by myself. You have made my life a lot easier and I cannot over emphasize how grateful I feel every single day of my life.

Acknowledgments

First of all, I would like to thank Professor Dowd for letting me be a part of her group. I have learned so much in the past two years and this cannot possibly happen without this amazing opportunity you kindly offered me. Secondly, I want to express my deepest gratitude to my friend, Doctor Chofor, for his constant encouragement. I would also like to thank Professor King for offering me the teaching assistant position. The interaction I had with my students during those three semesters completed my educational experience here at GWU. I would like to thank our collaborators at NIH,

Washington University, and George Mason University for their contributions on the

Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf) whole cell assays and Mtb and Pf Dxr enzyme activities. This project cannot be done without the contribution from all of you.

ii Abstract of Thesis

Analogs of Natural Product FR900098 as Small Molecule Inhibitors for the Methyl Erythritol Phosphate (MEP) Pathway in Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf)

Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf) are very prevalent and deadly species affecting millions of people’s lives while causing lots of infection-related deaths every year. Effective drugs discovered in the past are now facing great challenges such as multi-drug resistance. Due to the urgent need to discover efficacious molecules that can kill Mtb and Pf through novel modes of actions, we have designed, synthesized, and tested a series of analogs of a natural product, FR900098.

These compounds were synthesized aiming to block 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr), the second step of the methyl erythritol 4-phosphate (MEP) pathway. Out of the eighteen molecules in this series, SRW-61 has shown the best Pf whole cell inhibition.

iii Table of Contents

Dedication ··························································································· iii

Acknowledgment ···················································································· iv

Abstract ······························································································· v

List of Figures ······················································································· vii

List of Tables and Schemes ······································································ viii

Chapter 1 ····························································································· 1

Chapter 2 ···························································································· 21

Chapter 3 ···························································································· 35

Chapter 4 ···························································································· 50

Chapter 5 ···························································································· 97

Appendix ·························································································· 106

iv List of Figures

Figure 1.1 ····························································································· 4

Figure 1.2 ····························································································· 6

Figure 1.3 ···························································································· 10

Figure 1.4 ··························································································· 11

Figure 1.5 ··························································································· 12

Figure 1.6 ··························································································· 14

Figure 1.7 ··························································································· 16

Figure 2.1 ··························································································· 22

Figure 2.2 ··························································································· 25

Figure 2.3 ··························································································· 28

Figure 2.4 ··························································································· 30

Figure 3.1 ··························································································· 37

Figure 3.2 ··························································································· 40

Figure 4.1 ··························································································· 54

Figure 4.2 ··························································································· 64

v List of Tables and Schemes

Table 3.1 ····························································································· 39

Table 3.2 ····························································································· 43

Table 3.3 ····························································································· 46

Table 4.1 ····························································································· 58

Table 4.2 ····························································································· 60

Table 4.3 ····························································································· 62

Table 4.4a ···························································································· 66

Table 4.4b ··························································································· 67

Table 4.5 ····························································································· 69

Table 5.1 ··························································································· 100

Table 5.2 ··························································································· 101

Scheme 4.1 ·························································································· 55

vi

Chapter 1: Introduction of Tuberculosis and Malaria and Related Drug Discovery

Epidemic Prevalence and Infection Mechanism

Tuberculosis (TB) and malaria have long been problematic in our society.1,2 Even with the great progress researchers have made over the years, both diseases are still showing high prevalence in regions such as Southeast Asia (e.g., on the Thai/Cambodian border) and Africa.1,2 Out of the many existing strains of mycobacteria and malaria parasites, Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf) are the most concerning due to their ability to cause high mortality rate in patients.1,2

In 2015, there were a total of 1.8 million TB and malaria related deaths reported worldwide, among which 70% of the malaria related deaths were children under the age of 5.1,2 In addition to deaths caused by TB and malaria, co-infection of Mtb, P. falciparum and other microorganisms make other diseases more severe than they would be otherwise. For example, co-infection of TB and HIV will further compromise the patient’s immune system and cause a higher death rate.1

Many non-drug approaches are available and have been put into use to stop the spread of these diseases. For tuberculosis, the Bacille Calmette-Guerin (BCG) vaccine has been shown to be effective as a preventative measure. Additional vaccine candidates are in the process of development.3,4 Scientists have discovered that mosquitoes are responsible for the transmission of malaria parasites. Therefore, lowering or even eliminating the population of mosquitoes available in malaria-prevalent areas can help decrease malaria infection rates. For malaria, insecticide-treated mosquito tents, sprays, and intermittent preventative therapy are also achieving some success.2

1 While resources are being distributed and insecticide-treated mosquito nets are being implemented, new treatments for TB and malaria are also being discovered and developed by researchers. The first-line drugs known to display great potency against TB are shown in Figure 1.1. Artemisinin Combination Therapy (ACT) is used as the most effective treatment for malaria (Figure 1.2).2 It is worth noting that as great and effective as these medications are, they show limitations such as drug resistance and severe side effects. These drawbacks became part of our motivation to discover new molecules capable of treating TB and malaria.

First line Drugs for Tuberculosis:

There are five first-line drugs for tuberculosis currently (Figure 1.1). They are isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin. Each drug utilizes a different mechanism of action with great potency. These drugs are responsible for disrupting various steps of the Mtb cell life cycle.

Isoniazid:

Meyer and Malley were the first to synthesize isoniazid in 1912,5 and its antituberculosis activity was then discovered in 1951.6 At that time, isoniazid was classified as a Monoamine Oxidase (MAO) inhibitor. Monoamine oxidase is responsible for the metabolism of neurotransmitters. Since that time, it was discovered that the mechanism of action of isoniazid requires activation by an enzyme called KatG. The final active compound is an adduct formed with NADH, called isonicotinic acyl-NADH. This

2 adduct inhibits the activity of an enoyl reductase named InhA, decreasing the synthesis of mycolic acid, a precursor for the Mtb cell wall. Thus, isoniazid is a prodrug.5

Rifampicin:

The parent structure (lead compound) Rifampicin B was discovered as a natural metabolite of Nocardia mediterranei through the studies of pine forest soil samples.7

After modifications, Rifampicin SV was obtained, and it has great antibiotic activity against Mtb. Its mechanism of action involves disruption of RNA polymerase activity.

Rifampicin does not bind directly to the active site of RNA polymerase, however, it does have allosteric effects on the enzyme that halts the synthesis of RNA.8

Pyrazinamide:

Pyrazinamide was discovered in 1936, but it was used as antitubercular treatment beginning in 1952.9 Interestingly, pyrazinamide does not show antitubercular activity in vitro but is active in vivo. Pyrazinamide is converted to pyrazinoic acid which serves as the active form of this Mtb inhibitor. Thus, pyrazinamide, like isoniazid is also a prodrug.9 The mechanism of action of pyrazinamide is considerably more complex than that for other TB drugs and has taken some time to discern. As was recently discovered, pyrazinamide inhibits trans translation.10

3 Figure 1.1: First-Line Drugs for Tuberculosis

H O N NH2 OH H N N H N HO Isoniazid Ethambutol

HO O OH O O O OH OH NH N O NH2 N O N O OH N O N

Pyrazinamide Rifampicin

OH NH2 HO O OH HO HO N NH2 O OH O N NH NHO 2 O NH2 OH Streptomycin

4 Ethambutol:

Ethambutol was discovered in 1961 in an attempt to reduce the side effects of antibiotic treatments.11 Its mechanism of action involves inhibition of arabinosyltransferases (EmbA, EmbB and EmbC) in the Mtb cell, thus stopping the synthesis of arabinogalactan and lipoarabinomannan, which are major cell wall components. This will eventually impair Mtb by causing cell wall growth defects.12

Streptomycin:

Streptomycin disrupts the synthesis of protein by binding to the Mtb ribosome.

This will affect the binding of tRNA and cause errors in protein synthesis, leading to the eventual death of Mtb.13

5 Figure 1.2: Artemisinin and Derivatives of Artemisinin

CH CH H 3 H 3 O O H3C H3C O O O O H H H H O O CH3 CH3 O O Artemisinin Artemether

CH CH H 3 H 3 O O H3C H3C O O O O H H H H O O CH3 CH3 O O OH Dihydroartemisinin

HO O Artesunate

6 Artemisinin and Artemisinin Based Combination Therapy for Malaria:

Artemisinin:

One of the most important drugs for malaria treatments is artemisinin (Figure

1.2). It was discovered by a group of scientists led by Youyou Tu in 1971.14 The discovery of this compound was very complicated due to the source of plants and the number of traditional Chinese herbal medicine treatments the researchers had to analyze.15 The process was also slowed down by the limitation of instruments. However, they were eventually able to extract artemisinin and characterize it using X-ray crystallography in 1975. Youyou Tu was awarded a Nobel Prize in physiology or medicine in 2015 for the discovery of artemisinin.

One difficulty researchers faced was the extraction of artemisinin. In the original ancient texts for malaria treatment, water was used to extract artemisinin at 60 oC. As we now know, the oxygen-oxygen bridge is very vulnerable at higher temperatures. Once the bond is broken, the compound loses its antimalarial activity. Youyou Tu utilized ethyl ether as a solvent instead of water and extracted the plant at lower temperatures. In this way, she successfully extracted artemisinin from the plant.15

Artemisinin Combination Therapy:

Artemisinin has been modified and its derivatives were made to be co- administered with other drugs. These derivatives are dihydroartemisinin, artesunate and artemether.

7 Current Drug Resistance:

Multidrug resistant Mtb and Pf have been observed in many parts of the world such as Southeast Asia and the Thailand-Cambodia Border.2 This phenomenon results from a number of factors; two of which are misuse of medications and the innovation gap.16

As shown in Figure 1.3, there was a time gap of almost 40 years between 1960s and early 2000s with no discovery of new classes of antibiotics. This allowed Mtb and Pf, as well as other pathogens, to adapt to the existing treatments and develop drug resistance. This problem persisted for many decades. We observe extensive drug resistance for TB (Figure 1.4)2. As effective as artemisinin-based combination therapy

(ACT) is, malaria also suffers from drug resistance in some parts of the world. In regions like Cambodia and Thailand, resistance has been observed for one or more ACTs (Figure

1.5).2

Drug resistance, along with the high prevalence of Mtb and Pf, is an extremely alarming issue that calls for the discovery of new antibiotics. With that in mind, there are several goals we consider in the design of an ideal drug.17

Goals:

1. Prophylaxis: For soldiers and people who travel to Mtb and Pf prevalent areas, access to medication may be limited. Therefore, protecting these people from infection with single or multi-dose treatment before their trip is crucial.

8 2. Novel mechanism of action (new biochemical interactions caused by an inhibitor which result in the cure of a disease or infection): This can help lower the chance of developing drug resistance.

3. High potency: A very active compound can lower the amount (weight) of treatment taken by the patients. This may also lower the cost of manufacture, thus making the treatment more accessible for people with lower incomes.

4. High selectivity index: this can be partly interpreted as toxicity. The inhibitors can target bacteria and parasites more than normal human cells, so they should not exhibit high toxicity for humans near their IC50 values.

5. Metabolic stability: The inhibitor needs to be stable enough so that it would last long enough in the human body to interact with Mtb and Pf. This also affects bioavailability, since most antibiotics are administered orally. If an inhibitor is prone to degradation, it may have poor bioavailability.

9 Figure 1.3: Antibiotics Innovation Gap16

10 1 Figure:: FIG. 4.131.4: Extensively-Drug Resistant (XDR) TB Cases in 2015 Number of patients with laboratory-confirmed XDR–TB started on treatment in 2015

Number of patients 0 1–19 20–199 ≥200 No data Not applicable

Drug susceptibility testing for second-line drugs and coverage of appropriate diagnosis and treatment is a fun- detection of XDR-TB damental requirement for achieving the milestones and Among MDR/RR-TB patients notified in 2015, 36% were targets of the End TB Strategy. TB treatment coverage is reported to have had DST for both fluoroquinolones and defined as the number of new and relapse cases detected second-line injectable agents. Coverage was lowest in the and treated in a given year, divided by the estimated num- WHO Western Pacific and South-East Asia regions. In ber of incident TB cases in the same year, expressed as a 2015, 7579 XDR-TB cases were reported to have been de- percentage (Table 2.1). In this section, the number of noti- tected by 74 countries. fied new and relapse cases in 2015 is used as a proxy for Treatment of XDR-TB patients was reported by 58 coun- the number of cases detected and treated. As discussed tries and territories (Fig. 4.13). Globally, 7234 patients with further below, however, there are also people with TB who XDR-TB were enrolled on treatment (more than twice the are treated but not notified to national authorities (and in level in 2014). Most of the cases in 2015 were notified by turn are not notified to WHO), and people who are notified India (2130), Ukraine (1206), the Russian Federation (1205) but who may not be started on treatment. and South Africa (719). ART is recommended for all HIV-positive TB patients, and a second-line MDR-TB treatment regimen is recom- 4.2 Treatment coverage mended for people with MDR/RR-TB. This section includes estimates of treatment coverage for these two interven- The Sustainable Development Goals (SDGs) include a tions as well. target to “Achieve universal health coverage, including fi- nancial risk protection, access to quality essential health- 4.2.1 TB treatment coverage care services and access to safe, efective, quality and afordable essential medicines and vaccines for all” (Chap- Trends in notifications of new and relapse cases and ter 2, Box 2.2). Indicators for Target 3.8 of SDG3 include estimated incidence are shown for the 30 high TB burden prevention and treatment coverage of tracer interventions,1 countries in Fig. 4.14. Estimates of TB treatment cover- one of which is TB treatment. age in 2015 (calculated as notifications of new and relapse TB treatment coverage is also one of the 10 priority in- cases divided by estimated TB incidence) are shown glob- dicators for monitoring progress in implementation of the ally, for WHO regions and the 30 high TB burden countries End TB Strategy (Chapter 2, Table 2.1). This is because, in Fig. 4.15. Globally, TB treatment coverage was 59% 2 as highlighted in the introduction to this chapter, universal (range, 50–70%) in 2015, up from 54% (range, 46–65%) in 2010 and 36% (range, 30–43%) in 2000. Three WHO 1 There are many diferent prevention and treatment interventions. In this context, a few interventions are selected to act as tracers for 2 Here and elsewhere in the report, “range” refers to the 95% progress towards UHC for all interventions. uncertainty interval.

68 :: GLOBAL TUBERCULOSIS REPORT 2016

11 In 2015–2016, pfhrp2/3 gene deletions were reported in studies from the China– Myanmar border, Ghana, in the Democratic Republic of the Congo, Eritrea, India, Uganda, and Rwanda. These reports confirm that populations of P. falciparum lacking one or both of the pfhrp2/3 genes are now present outside South America in both high and low transmission areas, and with varying prevalence across narrow geographical ranges. Only RDTs that specifically target non-HRP2 antigens including pan- or Pf-specific lactate dehydrogenase or aldolase will detect P. falciparum with pfhrp2 gene deletions. Currently, very few RDTs of this type meet WHO recommended procurement criteria or are WHO prequalified and therefore, new and/or improved RDTs that can detect these mutated parasites are needed. Treatment Plasmodium falciparum resistance to artemisinin has been detected in five countries in the Greater Mekong subregion. Artemisinin resistance is defined as delayed clearance of the parasites; it represents a partial resistance. Most patients who have delayed parasite clearance after treatment with an ACT are still able to clear their infections, except where the parasites are also resistant to the ACT partner drug. Resistance to ACT partner drugs can pose a challenge to the treatment of malaria in some areas. In Cambodia, high failure rates after treatment with an ACT have been detected for four diferent ACTs (Figure 4.8). Resistance to dihydroartemisinin- piperaquine, first detected in Cambodia in 2008, has spread eastwards and was Figure 1.5: Artemisinin Combination Therapy (ACT) Resistance for Malaria in detected in Viet Nam in 2015. Selection Southeast Asia2 of an appropriate antimalarial medicine Figure 4.8 Distribution of malarial multidrug resistance, is based on the efcacy of the medicine

2016. Source: WHO database against the malaria parasite. Monitoring the therapeutic efcacy of antimalarial medicine is therefore a fundamental component of treatment strategies. Yunnan Province, China WHO recommends that all malaria endemic countries conduct therapeutic efcacy studies at least every 2 years to inform national treatment policy (22). Studies of molecular markers of Myanmar Lao People’s Democratic Republic drug resistance can provide important additional information for detecting and tracking antimalarial drug resistance. Thailand WHO collects information on therapeutic Viet Nam efficacy and molecular markers in a Cambodia global database.

1 ACT 2 ACTs 4 ACTs

ACT, artemisinin-based combination therapy

WORLD MALARIA REPORT 2016 WORLD MALARIA REPORT 2016 33

12 Mycobacterium tuberculosis Transmission:

Tuberculosis is transmitted through droplet nuclei in the air. When a patient with

TB coughs in a poorly ventilated area, these particles which originate from the patient and stay in the environment for hours. This lag time will give these tiny particles plenty of opportunity to be inhaled by another person. The particles make their way to the alveoli of that person’s lung and cause TB (Figure 1.6).18

13 Figure 2.2 Transmission of TB TB is spread from person to person through the air. The dots in the air represent droplet nuclei containing tubercle bacilli. Figure 1.6: Tuberculosis Transmission18

Factors that Determine the Probability of M. tuberculosis Transmission Tere are four factors that determine the probability of transmission of M. tuberculosis (Table 2.1). Table 2.1 Factors that Determine the Probability of Transmission of M. tuberculosis Factor Description Susceptibility Susceptibility (immune status) of the exposed individual

Infectiousness Infectiousness of the person with TB disease is directly related to the number of tubercle bacilli that he or she expels into the air. Persons who expel many tubercle bacilli are more infectious than patients who expel few or no bacilli (Table 2.2) (see Chapter 7, TB Infection Control)

Environment Environmental factors that afect the concentration of M. tuberculosis organisms (Table 2.3)

Exposure Proximity, frequency, and duration of exposure (Table 2.4)

Chapter 2: Transmission and Pathogenesis of Tuberculosis 22

14 Plasmodium falciparum Infection Cycle:

Malaria requires the assistance of female anopheles mosquitoes to pass the parasites from one person to another. Pf parasites reside in the midgut of mosquitoes as ookinetes. As they pass through the midgut, they become oocysts and are ready to be injected into humans when the mosquito takes a bloodmeal from a human. They are transmitted into the human body as sporozoites and stay dormant in the human liver for a short period of time before bursting out as merozoites. The merozoites mature and are passed to another mosquito in the form of gametocytes. These gametocytes develop into ookinetes later and thus, complete the infection cycle (mosquito to human) of malaria

(Figure 1.7).19

15 Figure 1.7: Parasite Cycles for Malaria19

16 Conclusions:

TB and malaria are affecting modern society with their high infection rates and drug resistance. New antibiotics, alongside insecticide-treated mosquito nets for malaria and other measures, are needed to combat TB and malaria. A number of antibiotics (even first-line drugs) have been extracted from natural sources and then have been chemically modified to improve their antibiotic activities through the process of drug discovery.

Thus, once an active lead compound (parent structure) is determined through in vitro analysis, more analogs can be made to increase its enzyme binding affinity, lipophilicity and oral availability.

The mechanism of action of any new compound is of high importance as the newly synthesized inhibitor will be co-administered with other drugs to minimize drug- resistance. Therefore, a novel mode of action is extremely beneficial. The search for new

Mtb and Pf inhibitors can start with natural products. In this project, a natural product named FR900098 was modified, and these new compounds have undergone biological evaluation for their Mtb and Pf inhibitory activities.

17 References:

1. World Health Organization 2016. Global Tuberculosis Report 2016. http://apps.who.int/medicinedocs/en/d/Js23098en/.

2. World Health Organization. World Malaria Report 2016. http://www.who.int/malaria/publications/world-malaria-report-2016/report/en/.

3. Zhang, L.; Goren, M. B.; Holzer, T. J.; Andersen, B.R. Infect. Immun. 1988, 56, 2876.

4. Young, D.; Dye, C. The development and impact of tuberculosis vaccines. Cell. 2006,

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5. Suarez. J.; Ranguelova, K.; Jarzecki, A.A.; Manzerova, J.; Krymov, V.; Zhao, X.; Yu,

S.; Metlitsky, L.; Gerfen, G.J.; Magliozzo, R.S. An oxyferrous heme/protein-based radical intermediate is catalytically competent in the catalase reaction of Mycobacterium tuberculosis catalase-peroxidase (KatG). J. Biol. Chem. 2009, 284(11):7017–29.

6. Murray, J.F. A century of tuberculosis. Am. J. Respir. Crit. Care. Med. 2004,

169:1181–1186.

7. Sensi, P. History of the development of rifampin. Rev. Infect. Dis. 1983, 5 Suppl 3:

S402–6.

8. Calvori, C.; Frontali, L.; Leoni, L.; Tecce, G. Effect of rifamycin on protein synthesis.

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9. Zhang, Y.; Mitchison, D.; Shi, W.; Zhang, W. Mechanisms of Pyrazinamide Action and Resistance. Microbiol. Spectr. 2014, 2 (4).

10. Shi, W.; Zhang, X.; Jiang, X.; Yuan, H.; Lee, J. S.; Barry, C. E.; Wang, H.; Zhang,

W.; Zhang, Y. Pyrazinamide Inhibits Trans-Translation in Mycobacterium tuberculosis.

Science. 2011, 333:1630-2.

18 11. Landau, R; Achilladelis, B; Scriabine, A. Pharmaceutical Innovation: Revolutionizing

Human Health. Chemical Heritage Foundation. 1999, p. 171.

12. Goude, R.; Amin, A.G.; Chatterjee, D.; Parish, T. The arabinosyltransferase EmbC is inhibited by ethambutol in Mycobacterium tuberculosis. Antimicob. Agents Chemother.

2009, 53: 4138–4146.

13. Sharma, D.; Cukras, A.R.; Rogers, E.J.; Southworth, D.R.; Green, R. Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome".

J. Mol. Bio. 2007, 374 (4): 1065–76.

14. Miller, L.H.; Su, X. Artemisinin: discovery from the Chinese herbal garden. Cell.

2011, 146 (6): 855–858.

15. Cindy Hao, “Lasker Award Rekindles Debate Over Artemisinin's Discovery,”

Science Magazine, Sep 29, 2011. http://www.sciencemag.org/news/2011/09/lasker- award-rekindles-debate-over-artemisinins-discovery.

16. Fischbach, M.A., Walsh, C.T. Antibiotics for emerging pathogens. Science. 2009,

325(5944): 1089-1093.

17. Baragaña, B.; Norcross, N.R.; Wilson, C.; Porzelle, A.; Hallyburton, I.; Grimaldi, R.;

Cabello, M.O.; Norval, S.; Riley, J.; Stojanovski, L.; Simeons, F.R.C.; Wyatt, P.G.;

Delves, M.J.; Meister, S.; Duffy, S.; Avery, V.M.; Winzeler, E.A.; Sinden, R.E.; Wittlin,

S.; Frearson, J.A.; Gray, D.W.; Fairlamb, A.H.; Waterson, D.; Campbell, S.F.; Willis, P.;

Read, K.D.; Gilbert, J.H. Discovery of a Quinoline-4-carboxamide Derivative with a

Novel Mechanism of Action, Multistage Antimalarial Activity, and Potent in Vivo

Efficacy. J. Med. Chem. 2016, 59 (21), 9672-9685.

19 18. Centers for Disease Control and Prevention. Transmission and Pathogenesis of

Tuberculosis. https://www.cdc.gov/tb/education/corecurr/pdf/chapter2.pdf.

19. Cowman, A.F.; Berry, D.; Baum, J. The cellular and molecular basis for malaria parasite invasion of the human red blood cell. J. Cell. Biol. 2012, 198:961–971.

20 Chapter 2: 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)

Rationale of Target Selection:

Cell building blocks and major metabolites are important for the survival of bacteria and parasites. By terminating the synthesis of these molecules, we can successfully halt the growth of cells, leading to a cure of the infection. In addition, avoiding the selection of currently used targets can help to minimize drug resistance in

Mtb and Pf. Our work aims at developing potent inhibitors against a target not currently used by any drug in clinical use.

The MEP Pathway and 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase:

The 2C-methyl-D-erythritol-4-phosphate (MEP) pathway is responsible for the synthesis of isoprenoids (Figure 2.1),1,2 which will later be converted to terpenes through a variety of downstream, intracellular reactions. Terpenes are cell building blocks responsible for important cell functions (i.e., cell signaling). Therefore, termination of terpene synthesis would potentially kill Mtb bacteria and Pf parasites.3 1-Deoxy-D- xylulose 5-phosphate reductoisomerase catalyzes the second step of the MEP pathway, converting 1-deoxy-D-xylulose-5-phosphate (DXP) to MEP while consuming a molecule of NADPH (Figure 2.1).4

21 Figure 2.1: MEP Pathway1, 2

O CO2 NADPH/H+ NADP+ CTP PPi - OH CO2 HO HO O O OPi OPi OCDP DXS OH DXR OH OH IspD OH OH H OPi OH DXP MEP CDP-ME

ATP IspE ADP

OPPi O - 2H+ + 2e- H+ + 2e- O P O CMP O O PiO IPP P O- OPPi O OCDP IspH OH IspG OH OH IspF OH OH

HMBPP MEcPP CDP-MEP OPPi

DMAPP

22 Targeting Mtb Dxr and Pf Dxr:

As we are attempting to target Dxr in both Mtb and Pf, the structural differences between Dxr homologs become a factor when designing inhibitors. Ultimately, we would like to synthesize molecules that show significant Dxr selectivity for different enzyme homologs. These compounds would lower the probability of resistance.

Although Mtb and Pf homologs share structural similarities, the differences in their DXP binding sites may enable a compound to inhibit one species over the other. To understand the differences between Dxr homologs, we need to study how earlier compounds bind to and inhibit Dxr in order to decide where additional modifications could be done on these molecules. Inhibitor bound co-crystal structures and inhibitor bound docking structures can give us information on how the inhibitors interact with certain amino acid residues in the enzyme, how they fit in the catalytic pocket, and how they can possibly block the binding pocket for the NADPH co-factor as well.

Mtb Dxr and Its Inhibitor Bound Structure:

Mtb Dxr and Pf Dxr are both homodimers with DXP and NADPH binding pockets in the active sites.5 In 2011, a report by Andaloussi et.al. described the binding of a series of fosmidomycin analogs bound to Mtb Dxr.6 These co-crystal structures can be used to understand how the molecules interact with the enzyme, and assist in the design of novel analogs. Because the work described in this project focuses on alpha-substituted analogs, analysis of the co-crystal structure of compound 9a (Figure 2.2a) is especially relevant.

23 Our project is partly focusing on the effect of the addition of a 3,4-dichlorophenyl group to the α-position of FR900098 (Figure 2.2a). Therefore, it is necessary to gather information on how compound 9a interacts with Mtb Dxr. Compound 9a was first synthesized by the Van Calenbergh group in 2006.7 This compound inhibits E.coli Dxr activity with an IC50 value of 59nM and Pf whole cell inhibitory activity value of 90nM.

As a homolog of E.coli and Pf Dxr, Mtb Dxr can also be inhibited by compound 9a, with

7 an Mtb Dxr IC50 value of 150nM.

As a homodimer, Mtb Dxr has three domains for each monomer: an N-terminal

NADPH domain, a C-terminal α helical domain, and a DXP catalytic site.5,8 While binding to the DXP site of Mtb Dxr, the phosphonate moiety of compound 9a interacts with the nitrogen and the side chain of Ser177. The phosphonate also interacts with the side chains of Ser 213, Asn218 and Lys219. The retrohydroxamate moiety of compound

9a interacts with a bridging manganese cation to interact with the side chains of Asp151 and Ser152. The alpha position of compound 9a is a chiral center, and the R enantiomer of compound 9a was used for the modeling (Figure 2.2b). However, whether the binding of this compound is stereoselective is still undetermined. Interestingly, the alpha substituent of compound 9a can clash with Trp203 on the flexible flap of Mtb Dxr, causing loss of multiple interactions.6 This could be a key characteristic we can utilize in our design of Dxr inhibitors to make them species-specific.

24 Figure 2.2a: Structures of Fosmidomycin, FR900098 and Compound 9a used for Crystallography6 O HO OH P OH O OH O OH N O P N O P N HO Cl O H HO OH OH Cl

Fosmidomycin FR900098 Compound 9a

Journal ofFigure Medicinal 2.2b: Crystallographi Chemistryc Structure of Compound 9a Bound Mtb Dxr6 ARTICLE

The phosphonate group of each makes interactions with the backbone nitrogen of Ser177, as well as the side chains of Ser177, Ser213 (in one of its two possible conformations), Asn218, Lys219, and several waters. The hydroxamate group is bound in a very similar manner in each of the analogue complexes, coordinating the manganese ion and overlapping with two of the three metal-bound waters in apo-MtDXRb; interactions between the terminal oxygen and the side chains of Asp151 and Ser152 are also present. A sixth metal-coordinating group, the third water in apo-MtDXRb,isabsentinthecom- plexes because of lack of space. The dichlorophenyl ring shows some variability, including a 180° flip; electron density is weakest for the ring, which may arise from heterogeneity concerning the ring flip, from some small population of bound R-enantiomer, or both. Finally, the fosmidomycin propyl back- Figure 3.CarbonInteractions atoms on Dxr in are the labeled active gold and site carbon of atomsMtDXR on compound-9a-NADPH. 9a are labeled bone atoms assume a very similar conformation in the three b analogue structures. Protein carbonmaroon. atoms Water molecules are shown are represented in gold, by while red spheres those while of Mn the2+ ion inhibitor is represented 9a are maroon. Water molecules are shown as small red spheres, and the Unexpectedly, the backbone conformation seen for the R-aryl Mn2+ ionby is magenta shown sphere. in magenta. Dark grey Darkspheres grayrepres bubblesent interactions show between the observedcompound 9a and fosmidomycin analogues differs from that observed for fosmido- interactions.Mtb Dxr. mycin itself, with two of the three torsion angles, adopting different rotamers (Figure 2D). Interestingly, our modeling of

25 the R-enantiomers of 9a and 9c suggests that they would adopt highest resolution that we have yet achieved for this enzyme, and backbone conformations more similar to fosmidomycin when the latter allows us to evaluate structural changes due to the new binding to the active site, because of steric constraints. However, unit cell as well as to the removal of sulfate ions from the until the optically pure enantiomers of 9a and 9c have been crystallization conditions. In the apo-MtDXRb A subunit, a prepared and evaluated, it is not possible to say if both enantio- sulfate is located in the DXP/MEP-binding site, making five mers are active or if the binding is stereoselective. Large changes interactions with protein hydrogen bond donors (Ser177 OG, have been described previously in a particular loop near the active Ser177 N, Ser213 OG, Asn218 ND2, Lys219 NZ) and four water site of both MtDXR31 and EcDXR.33 In the present case, the most molecules. In apo-MtDXRc, the sulfate ion is replaced by a pair of dramatic change is the fact that this flap containing Trp203 is water molecules forming three hydrogen bond interactions closed (and ordered) in the fosmidomycin complex of MtDXR (Asn218 ND2 for one, Ser177 N and Lys219 NZ for the other, but open in the case of the analogues described here (residues 199 while the Ser213 side chain rotates to form an interaction with to 204 are disordered in the analogue complex structures). 209 O). This∼ difference arises because the dichlorophenyl ring of the Crystals of three complexes could be produced that were analogues would clash with the indole ring of Trp203 if the active suitable for structural work: MtDXRb-9a-NADPH, MtDXRc-9a, site flap were to adopt the closed conformation (Figure 2D). and MtDXRb-9c. The A subunits of MtDXRb structures can be Thus, a hydrogen bond between His200 and the phosphonate 31 aligned with the MtDXRb-fosmidomycin-NADPH structure group, as well as the interaction of the indole ring of Trp203 with (2JCZ) with rmsd of 0.3 Å over 375 CR atoms, excluding the the fosmidomycin backbone, is lost in the analogues. Further, the ∼ fl∼ large structural variations in the ap. The MtDXRc A subunits interaction of the hydroxamate group with the backbone nitro- superimpose with rmsd of 0.7 Å for 359 CRs, while A subunits of gen of residue 152 in fosmidomycin is lost, and only the the different crystal forms superimpose with pairwise rmsd in the interaction of the side chain hydroxyl group of Ser152 is range of 0.6 1.0 Å over 360 CR atoms. The larger deviations maintained in the analogue complexes. This is a consequence ∼ are the resultÀ of rigid body shifts in the C-terminal R-helical of fosmidomycin lying 0.5 Å deeper in the MtDXR active site domain. Unless otherwise indicated, the electron density for the than the analogues. The∼ shift also avoids clashes that would occur main chain of all five structures is of good quality, with the between the dichlorophenyl ring and Pro265. The p-chlorine fi exception of the N-terminal His6-tag, the rst 10 residues, the atom and parts of the phenyl ring are partially accessible to active site flap of the A subunit (residues A198 A208), and solvent, wedged into a depression between three ordered and residues A69 A78 in the MtDXR structures. TheÀ density for one disordered loop (containing residues 179, 245, 265 and 203, À c the cofactor in the MtDXRb-9a-NADPH structure is weaker than respectively). In the first complex structure that we solved, we observed in the equivalent fosmidomycin ternary complex MtDXRc-9a, we were concerned because this edge of the (PDB code 2JCZ).31 In particular, the NADPH in the B subunit dichlorophenyl ring was in contact with the active site flap from is best defined for the phosphate groups and is otherwise poorly a symmetry-related copy of a B subunit (Thr202 CG2 CL atom defined. contacts of 3.6 Å). To ensure that this had not producedÀ a Binding of Fosmidomycin Analogues. The two fosmido- crystallographic artifact, we obtained complexes that were crys- mycin analogues bind in a very similar manner in the DXP/MEP tallized under other conditions, as described above. In the site of the A chain, both in the presence and absence of NADPH MtDXRb-9a-NADPH and MtDXRb-9c structures, there are no (Figure 2). The detailed interactions to one of them (MtDXRb- stabilizing interactions with symmetry related molecules. How- fl 9a-NADPH) are shown in Figure 3. As for the inhibition studies, ever, the 180° ring ip that we observe in MtDXRc-9a may a racemic mixture was used in the crystallographic work. Our indeed represent an artifact of crystal packing that allows the crystallographic results indicate that 9a and 9c bind primarily, if m-chlorine to pack against the symmetry molecule, instead of being not exclusively, as the S-enantiomers (see Supporting Information). buried as in the other structures (Figure 2). The extra methyl

4968 dx.doi.org/10.1021/jm2000085 |J. Med. Chem. 2011, 54, 4964–4976 Pf Dxr and Its Inhibitor Bound Structure:

There are currently 3 types of crystal structures reported for Pf Dxr: 1) an open form of Pf Dxr with flexible loop open (no inhibitor), 2) an open form of Pf Dxr with flexible loop closed (inhibitor bound), 3) a closed form of Pf Dxr with flexible loop closed (inhibitor bound).9

As a homodimer, each monomer of Pf Dxr incorporates one molecule of NADPH and one Mg2+ ion for every inhibitor bound in the enzyme. One molecule of NADPH is needed for the catalysis of DXP to MEP.2,4 Aiming to block this NADPH co-factor binding pocket has been the focus of prior studies from our group and is an important part of this project. This project probes the NADPH binding site with different alkyl groups, discussed further in Chapter 4.

FR900098 and fosmidomycin,10,11 as inhibitors of Pf Dxr, bind to Pf Dxr through two types of interactions (Figure 2.3). The retrohydroxamate and phosphonate groups are very polar and require bridging water molecules and the Mg2+ cation to bind to Pf Dxr.

The phosphonate interacts with Ser270, Asn311 and His293 with the help of two water molecules. The retrohydroxamate interacts with Asp231, Glu233, Glu315 and the Mg2+ cation.9

The carbon backbones of FR900098 and fosmidomycin, on the other hand, are hydrophobic and interact with the indole ring of Trp296. It is worth pointing out that

Trp296 also interacts with the methyl group on the acetyl moiety of FR900098. This is an attractive interaction as there is roughly 10-fold improvement in Pf Dxr inhibition for

FR900098 compared to fosmidomycin.9

26 Inhibitor Bound Enzyme Structures and Design of New Inhibitors:

Analysis of the available co-crystal structures leads to important considerations for making analogs of FR900098 or fosmidomycin as Pf Dxr inhibitors. First, the retrohydroxamate and phosphonate moieties are necessary for the water/metal cation assisted interactions with Pf Dxr. Next, because the methyl group on the acetyl moiety appears to make a significant difference, it may be more efficacious to design FR900098 analogs (with the acetyl group) instead of fosmidomycin analogs (with the formyl group).

Moreover, more substituents can be added to the hydrophobic methylene backbone of

FR900098 analogs to increase hydrophobicity.12,13 However, Trp296 does have a big side chain and in order to avoid clashing between Trp296 and a substituent, the size of the substituent cannot be too large. A selection of substituents at the alpha position has been studied. These compounds are discussed in Chapter 3.

27 www.nature.com/scientificreports

Figure 2.3: Crystallographic Structure of Fosmidomycin-Bound Pf Dxr9

Carbon, nitrogen, oxygen and phosphorus atoms on fosmidomycin are represented by yellow, purple, red and orange spheres. Bridging water molecules and Mg2+ ion are represented by cyan and green spheres.

28

Figure 3 | The inhibitor-binding sites of the quaternary complexes of PfDXR. a, Fosmidomycin complex. The carbon atoms of fosmidomycin, the four buried water molecules, and the bound Mg21 ion are shown in yellow, cyan, and green, respectively. b, Stereo diagrams showing the | Fo | – | Fc | omit maps of bound inhibitors in fosmidomycin (top) and FR900098 (bottom) complexes at 1.8- and 2.15-A˚ resolutions, respectively. To exclude a model bias, the structures were refined in the absence of the inhibitor molecules before the map calculation. The amplitude | Fc | and the phase angle calculated from the partial structure were then used to calculate the | Fo | – | Fc | omit map. The contour levels are 2.5 s (cyan) and 10.0 s(red). c, Schematic drawing of the metal coordination system observed in the quaternary complexes of PfDXR. Bond lengths are shown in A˚ . d, FR900098 complex. The carbon atoms of the FR900098 molecule are shown in magenta. To compare the induced-fit movement of the active site residues upon inhibitor binding, LSQ fitting was performed with respect to the 11 atoms common to the inhibitors in the fosmidomycin and FR900098 complexes. The RMSD for the 11 pairs was 0.50 A˚ . The methyl group of the bound FR900098 molecule is indicated by an arrow head. The fosmidomycin complex is shown in yellow stick models. e, Multiple sequence alignment of the flexible loop region of DXRs. The abbreviations (GenBank accession numbers) are as follows: Pf (AAD03739), Plasmodium falciparum DXR; Ec (BAA77848), Escherichia coli DXR; Mt (CAA98375), Mycobacterium tuberculosis DXR; Zm (AAD29659), Zymomonas mobilis DXR; Tm (AAD35970), Thermotoga maritima DXR. The residue numbers are shown on the right.

(compound 1a in Fig. 4): the phosphonate group, the carbon back- by the present crystal structure analysis is essential for inhibitor bone, and the hydroxamate group. Most reported efforts at synthesis- binding. The failure of the carboxylate analogue to inhibit EcDXR ing fosmidomycin analogues involve modifications of the phosphate can be explained by a structural difference: a phosphonate has a and hydroxamate moieties. pyramidal structure, and a carboxylate has a planar structure. The Replacement of a phosphonate group by other isosteric groups, failure of the sulphonate analogue to inhibit EcDXR is not as clear as such as a carboxylate (2a and 2b) or a sulphonate (3a and 3b), was in the case of a carboxylate analogue. The difference between the evaluated for reverse hydroxamate analogues of fosmidomycin26. phosphonate and sulphonate groups is that the C-P and P-O bond The results showed drastically decreased inhibitory activity against lengths are shorter than those of the C-S and S-O bonds. This EcDXR and suggest that the tight hydrogen bond network around difference might prevent the sulphonate group from ideal binding. the phosphonate group of fosmidomycin or FR900098 revealed On the other hand, the phosphate analogue of FR900098 (4b) (the

SCIENTIFIC REPORTS | 1 : 9 | DOI: 10.1038/srep00009 4 Dxr Docking Structures: Other groups have published the docking structures of their fosmidomycin and

FR900098 analogs with Mtb Dxr and Pf Dxr.8,14 Because of the structural similarities of the SRW series of compounds and the published inhibitors, discussion and analysis of the structures published by Professor Dowd’s group and their docking results are most relevant.15

Fosmidomycin Bound Mtb Dxr:

Fosmidomycin docks into the DXP pocket of Mtb Dxr in the presence of NADPH

(Figure 2.4A). The distance between the formyl carbon atom of fosmidomycin and nicotinamide ring of NADPH is roughly 3.5 Å. By adding larger alkyl groups to the retrohydroxamate moiety, new compounds are designed that could extend into the

NADPH pocket, displaying increased binding affinity with the enzyme.15

FR900098 Analog Bound Mtb Dxr:

Two types of FR900098 analogs were synthesized and analyzed by Drs. Emily

Jackson and Geraldine San Jose.15 Using molecular docking, the alkyl substituents of these analogs are predicted to extend into the NADPH pocket of Mtb Dxr (Figures 2.4B and C). However, even though these inhibitors show activity against Mtb Dxr, the

Lineweaver-Burk plot later showed that one of these inhibitors (compound 8) does not bind to the NADPH pocket of Mtb Dxr. Due to the fact that Mtb Dxr and Pf Dxr are different, the bisubstrate binding mechanism for inhibitors may still work for these molecules as Pf Dxr inhibitors.15

29 MedChemCommMedChemComm ConciseConcise Article Article

nicotinamidenicotinamide ring ring of NADPH of NADPH binds binds approximately approximately 3.5 3.5A˚ fromA˚ from thethe formyl formyl carbon carbon atom atom of fosmidomycin of fosmidomycin (Fig. (Fig. 3A). 3A). Hence, Hence, our our approachapproach toward toward Dxr Dxr inhibitor inhibitor development development was was based based on onthe the designdesign of fosmidomycin/FR900098 of fosmidomycin/FR900098 analogs analogs that that target target the the two two majormajor binding binding sites sites in Dxr: in Dxr: the the fosmidomycin/DXP fosmidomycin/DXP site site and and the the Fig.Fig. 2 Structures 2 Structures of fosmidomycin of fosmidomycin (1), FR900098 (1), FR900098 (2)andamide-or (2)andamide-orO-linkedO-linkedNADPHNADPH site. site. Our Our goal goal was was to bridge to bridge these these adjacent adjacent binding binding sites sites analogsanalogs (3–9 ().3–9). to yieldto yield a high a high affinity, affinity, bisubstrate bisubstrate ligand, ligand, while while considering considering the the needneed for forincreased increased lipophilicity lipophilicity compared compared with with fosmidomycin/ fosmidomycin/ FR900098.FR900098. We We report report here here the the design, design, synthesis, synthesis, and and evaluation evaluation mechanismmechanism of action, of action, and and Dxr Dxr inhibitors inhibitors would would be expected be expected to toof twoof two series series of compounds of compounds with with either either amide- amide- or O or-linkedO-linked MedChemComm be ebeffective effective against against drug-resistant drug-resistantConcise strains strains Article of Mtb. of Mtb. substituentssubstituents appended appended to tothe the retrohydroxamate retrohydroxamate moiety moiety of of FosmidomycinFosmidomycin (1) and (1) and its acetyl its acetyl derivative derivative FR900098 FR900098 (2) are(2) areFR900098FR900098 (Fig. (Fig. 2) and 2) and evidence evidence of aof novel, a novel, non-bisubstrate non-bisubstrate nicotinamide ringnaturalnatural of NADPH products products binds isolated approximately isolated from from 3.5StreptomycesA˚Streptomyces from lavendulae lavendulaemodemode of binding. of binding. the formyl carbon(Fig.(Fig. atom 2).10 2). ofThese10 fosmidomycinThese secondary secondary metabolites (Fig. metabolites 3A). Hence, are are both our both known known inhibi- inhibi- 11 approach towardtors Dxrtors of inhibitor Dxr, of Dxr,and11 developmentand fosmidomycin fosmidomycin was based is currently is oncurrently the under under clinical clinicalResultsResults design of fosmidomycin/FR900098investigationinvestigation due due to itsanalogs to activity its activity that against target against a the variety atwo variety of Gram-nega- of Gram-nega- major bindingtive sitestive and in and Dxr: Gram-positive Gram-positive the fosmidomycin/DXP bacteria, bacteria, as well as site well as and malaria as themalaria parasites. parasites.12–1412–14ModelingModeling of amide- of amide- and andO-linkedO-linked ligands ligands Fig. 2 Structures of fosmidomycin (1), FR900098 (2)andamide-orO-linked NADPH site. OurIn goal theseIn these was pathogens, to pathogens, bridge these fosmidomycin fosmidomycin adjacent binding is actively is actively sites transported transported into intoSeveralSeveral studies studies describe describe the thein silicoin silicoevaluationevaluation of inhibitors of inhibitors analogs (3–9). to yield a high acellsffinity,cellsvia bisubstrateviaa glycerol-3-phosphatea glycerol-3-phosphate ligand, while considering transporter, transporter, the GlpT. GlpT.15 Mtb,15 Mtb,againstagainst Dxr. Dxr.20,30–20,3035 These–35 These reports reports highlight highlight the the challenges challenges of of need for increasedhowever,however, lipophilicity does does not compared not have have GlpT. with GlpT. This, fosmidomycin/ This, combined combined with with both both the themodelingmodeling a protein a protein that that undergoes undergoes signi signicantcant conformational conformational FR900098. We reporthydrophilichydrophilic here the nature design, nature of synthesis, fosmidomycin of fosmidomycin and evaluation and and the the highly highly hydro- hydro-changechange upon upon cofactor cofactor binding. binding. We We sought sought to use to use docking docking to to mechanism of action, and Dxr inhibitors would be expected to of two series ofphobic compoundsphobic Mtb Mtb cell with cell wall, wall, either renders renders amide- fosmidomycin fosmidomycin or O-linked inactive inactive against againstdiscerndiscern whether whether bisubstrate bisubstrate inhibition inhibition of Mtb of Mtb Dxr Dxr with with amide amide be effective against drug-resistant strains of Mtb. substituents appendedMtb.Mtb.9,16 9,16However, toHowever, the weretrohydroxamate wehave have shown shown that moiety that lipophilic lipophilic of prodrugs prodrugs of ofor Oor-linkedO-linked compounds compounds was was feasible. feasible. Fosmidomycin (1) and its acetyl derivative FR900098 (2) are FR900098 (Fig.FR900098 2)FR900098 and evidence demonstrate demonstrate of a eff novel,ective effective antitubercular non-bisubstrate antitubercular activity activity17 and17 and act act 200200 Compounds Compounds with with the the general general structures structures shown shown in Fig. in Fig. 2 2 natural products isolated from Streptomyces lavendulae mode of binding.in ain GlpT-independent a GlpT-independent manner. manner.18 Additionally,18 Additionally, a range a range of ofwerewere docked docked into into the the Mtb Mtb Dxr Dxr structure. structure.28 NADPH28 NADPH and and fosmi- fosmi- (Fig. 2).10 These secondary metabolites are both known inhibi- syntheticsynthetic fosmidomycin fosmidomycin and and FR900098 FR900098 analogs analogs have have been beendomycindomycin were were removed removed from from the the active active site, site, while while Mn Mn2+ was2+ was kept kept tors of Dxr,11 and fosmidomycin is currently under clinical Results described,described, designed designed to compete to compete with with DXP DXP at the at the substrate- substrate-in place.in place. The The analogs analogs retained retained the the phosphonate phosphonate and and backbone backbone investigation due to its activity against a variety of Gram-nega- bindingbinding site. site.19–2719While–27 While demonstrating demonstrating potent potent inhibition inhibition of the of theof theof the parent parent compounds, compounds, designed designed to ensure to ensure affinity affinity to the to the DXP DXP 12–14 Modeling of amide- and O-linked ligands tive and Gram-positive bacteria, as well as malaria parasites. puripuriFigureed enzyme,ed 2.4: enzyme, Compou none none ofnd these of8 and these analogs,Docking analogs, toStructures our to our knowledge, knowledge,of (A) Fosmidomycin is isbindingbindingsite Bound site in the inMtb the enzyme. Dxr, enzyme. The The analogs analogs were were appended appended with with In these pathogens, fosmidomycin is actively transported into Several studiesactive describeactive against against the intactin silicointact mycobacteria.evaluation mycobacteria. of Our inhibitors Our goal goal was was to expand to expand on on(un)substituted(un)substituted aromatic, aromatic, alkylaryl, alkylaryl, or (cyclo)alkyl or (cyclo)alkyl substituents substituents cells via a glycerol-3-phosphate transporter, GlpT.15 Mtb, against Dxr.20,30this–35 this(B)These work O work- designingLinked reports designing FR900098 highlight Dxr Dxr inhibitors inhibitors Analog the challenges with Bound with a novel aMtb novel of binding Dxr binding and mech- (C) mech- N-Linkedto theto the NFR900098-hydroxyN-hydroxy oxygen Analog oxygen atom atom or the or the amide amide carbonyl carbonyl group group of of however, does not have GlpT. This, combined with both the modeling a proteinanismanism that and undergoes and suffi sucientfficient signi lipophilicity lipophilicitycant conformational to gainto gain whole whole cell cell anti- anti-thethe retrohydroxamate. retrohydroxamate. These These structural structural features features were were designed designed hydrophilic nature of fosmidomycin and the highly hydro- change upon cofactormycobacterialmycobacterialBound binding. Mtbactivity. Dxr. We activity.15 sought to use docking to to increaseto increase affinity affinity to the to the NADPH NADPH binding binding site. site. Speci Specically,cally, we we phobic Mtb cell wall, renders fosmidomycin inactive against discern whether bisubstrateSeveralSeveral crystal inhibition crystal structures structures of Mtb of Dxr ofDxr Dxr have with have been amide been reported, reported, facili- facili-werewere interested interested in testing in testing aromatic aromatic substituents substituents as mimics as mimics of of 28,29 Mtb.9,16 However, we have shown that lipophilic prodrugs of or O-linked compoundstatingtating the was the rational rationalfeasible. design design of novel of novel inhibitors. inhibitors.28,29TheThe structure structurethethe nicotinamide nicotinamide ring ring of NADPH, of NADPH, aiming aiming to increase to increase affinity affinity to to FR900098 demonstrate effective antitubercular activity17 and act 200 Compoundsof Mtbof with Mtb Dxr the Dxr in general complex in complex structures with with the shown the competitive competitive in Fig. inhibitor 2 inhibitor fosmi- fosmi-thatthat pocket. pocket. Cycloalkyl Cycloalkyl groups groups were were used used to to examine examine the the in a GlpT-independent manner.18 Additionally, a range of domycindomycin hasO has led led to the to theOH identi28 identicationcation of binding of binding sites sites in the in theimportanceimportance of an of aryl an aryl substituent. substituent. were docked into theHO Mtb Dxr structure. NADPH and fosmi- synthetic fosmidomycin and FR900098 analogs have been domycin were removedenzymeenzyme fromandP and is the an is active excellentan excellent site,N while template template Mn2+ forwas for protein-ligand kept protein-ligand dock- dock- TheThe docking docking results results predicted predicted that that our our ligands ligands would would adopt adopt a a 28 28 described, designed to compete with DXP at the substrate- in place. The analogsing.ing.NaOIn retained particular,In particular, the the phosphonate the crystal crystal structure and structure backbone has has revealed revealed chelation chelationbisubstratebisubstrate binding binding mode mode and and bridge bridge the the two two adjacent adjacent binding binding binding site.19–27 While demonstrating potent inhibition of the of the parent compounds,betweenbetween the designed the retrohydroxamate retrohydroxamate to ensureO affi moietynity moiety to of the fosmidomycin of DXP fosmidomycin and and a asites.sites. Representative Representative docking docking images images are are shown shown in Fig. in Fig. 3B 3Band and puried enzyme, none of these analogs, to our knowledge, is binding site inmetal themetal enzyme. cation, cation, The the the analogs phosphonate phosphonate were appended binding binding site, with site, and and the the close closeC. InC. general, In general, the the amide-linked amide-linked compounds compounds were were expected expected to to ffi active against intact mycobacteria. Our goal was to expand on (un)substitutedbinding aromatic,binding proximity alkylaryl, proximity or of (cyclo)alkyl of fosmidomycin fosmidomycin substituents and and NADPH. NADPH. The Thebindbind with with greater greater a nity affinity compared compared with with the theO-linkedO-linked Compound 8 this work designing Dxr inhibitors with a novel binding mech- to the N-hydroxy oxygen atom or the amide carbonyl group of anism and sufficient lipophilicity to gain whole cell anti- the retrohydroxamate. These structural features were designed mycobacterial activity. to increase affinity to the NADPH binding site. Specically, we Several crystal structures of Dxr have been reported, facili- were interested in testing aromatic substituents as mimics of tating the rational design of novel inhibitors.28,29 The structure the nicotinamide ring of NADPH, aiming to increase affinity to of Mtb Dxr in complex with the competitive inhibitor fosmi- that pocket. Cycloalkyl groups were used to examine the domycin has led to the identication of binding sites in the importance of an aryl substituent. enzyme and is an excellent template for protein-ligand dock- The docking results predicted that our ligands would adopt a ing.28 In particular, the crystal structure has revealed chelation bisubstrate binding mode and bridge the two adjacent binding between the retrohydroxamate moiety of fosmidomycin and a sites. Representative docking images are shown in Fig. 3B and metal cation, the phosphonate binding site, and the close C. In general, the amide-linked compounds were expected to binding proximity of fosmidomycin and NADPH. The bind with greater affinity compared with the O-linked Fig. 3Fig.Mtb 3 Mtb Dxr active Dxr active site and site anddocking docking results. results. (A) Active (A) Active site of site Mtb of Mtb Dxr with Dxr with fosmidomycin fosmidomycin (left (left ligand) ligand) and andNADPH NADPH (right (right ligand) ligand) bound bound (pdb (pdb 2JCZ). 2JCZ).18 Ligands18 Ligands are are separatedseparated by by3.5 A.˚3.5 (B)A.˚ Docked (B) DockedO-linkedO-linked ligand ligand4. (C)4 Docked. (C) Docked amide amide ligand ligand8.ProteinchainAisshownascartoon(blue),Mn8.ProteinchainAisshownascartoon(blue),Mn2+ as2+ aas sphere a sphere (pink), (pink), ligands ligands are shown are shown as as   stickssticks colored colored by atom by atom type, type, protein protein residues residues as lines as lines colored colored by atom by atom type. type. Hydrogen Hydrogen atoms atoms have have been been hidden hidden for clarity. for clarity.

11001100| Med.| Med. Chem. Chem. Commun. Commun.,2013,,2013,4,10994,1099–1104–1104 ThisThis journal journal is ª isTheª The Royal Royal Society Society of Chemistry of Chemistry 2013 2013

18 Fig. 3 Mtb Dxr active site and docking results. (A) Active site of Mtb Dxr with fosmidomycin (left ligand) and NADPH (right ligand) bound (pdb 2JCZ). Ligands are 2+ separated by 3.5 A.˚ (B) Docked O-linked ligand 4. (C) Docked amide ligand 8.ProteinchainAisshownascartoon(blue),MnCarbon atoms2+ ason a Mtb sphere Dxr (pink), are ligands labeled are shownblue and as Mn ion is labeled pink.  sticks colored by atom type, protein residues as lines colored by atom type. Hydrogen atoms have been hidden for clarity.

1100 | Med. Chem. Commun.,2013,4,1099–1104 This journal is ª The Royal Society of Chemistry 2013

30 Conclusions:

By analyzing inhibitor bound Mtb and Pf Dxr structures, we try to understand how the inhibitors interact with the surrounding side chains of amino acid residues. Thus, we can determine the kind of changes needed to increase the binding affinities of novel analogs. Previously, the synthesis of FR900098 analogs was attempted in order to block both the DXP catalytic pocket and the NADPH pocket in Mtb Dxr. The Lineweaver-Burk plot indicated that the alkyl side chain binds to a different pocket instead. This means that the bisubstrate mechanism could be very species-specific and may only work for Pf Dxr.

A series of Dxr inhibitors will be designed based on the structure of FR900098, because the methyl group on its acetyl moiety can enhance the interaction between inhibitor and Dxr. Moreover, the alpha position of these inhibitors will also be explored to increase interaction between the alpha substituent and Trp296 of Pf Dxr. The design, synthesis and biological evaluation of these molecules will be further discussed in

Chapters 3 and 4.

31 References:

1. Zeidler, J. G.; Schwender, J.; Müller. C., Wiesner, J.; Weidemeyer, C.; Beck, E.,

Jomaa, H.; Lichtenthaler, H. K. Inhibition of the non-mevalonate 1-deoxy-D-xylulose-5- phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Z.

Naturforsch. 1998, 53:980–986.

2. Kuzuyama, T., Shimizu, T., Takahashi, S.; Seto, H. Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis. Tetrahedron Lett. 1998, 39, 7913–7916.

3. Kuntz, L.; Tritsch, D.; Grosdemange-Billiard C.; Hemmerlin, A.; Willem, A.; Bach,

T.J.; Rohmer M. Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto- isomerase. Biochem. J. 2005, 386:127–135.

4. Takahashi, S.; Kuzuyama, T.; Watanabe, H.; Seto, H. A 1-deoxy-d-xylulose 5- phosphate reductoisomerase catalyzing the formation of 2-C-methyl-d-erythritol 4- phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis. Proc.

Natl Acad Sci. U.S.A. 1998, 95 (17) 9879-9884.

5. Henriksson, L. M., Unge, T., Carlsson, J., Aqvist, J., Mowbray, S. L.; Jones, T. A.

Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase provide new insight into catalysis. J. Biol. Chem. 2007, 282, 19905–

19916.

6. Andaloussi, M.; Henriksson, L.M.; Wieckowska, A.; Lindh, M.; Bjorkelid, C.;

Larsson, A.M.; Suresh, S.; Iyer, H.; Srinivasa, B.R.; Bergfors, T.; Unge, T.; Mowbray,

S.L.; Larhed, M.; Jones, T.A.; Karlen, A. Design, synthesis, and X-ray crystallographic

32 studies of alpha-aryl substituted fosmidomycin analogues as inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase. J. Med. Chem. 2011, 54,

4964-4976.

7. Haemers, T.; Wiesner, J.; Van Poecke, S.; Goeman, J.; Henschker, D.; Beck, E.;

Jomaa, H.; Van Calenbergh, S. Synthesis of α-substituted fosmidomycin analogues as highly potent Plasmodium falciparum growth inhibitors. Bioorg. Med. Chem. Lett. 2006,

16, 1888− 1891.

8. Deng, L.; Diao, J.; Chen, P.; Pujari, V.; Yao, Y.; Cheng, G.; Crick, D.C.; Prasad, B.V.;

Song, Y. Inhibition of 1-deoxy-D-xylulose-5- phosphate reductoisomerase by lipophilic phosphonates: SAR, QSAR, and crystallographic studies. J. Med. Chem. 2011, 54, 4721-

4734.

9. Umeda, T.; Tanaka, N.; Kusakabe, Y.; Nakanishi, M.; Kitade, Y.; Nakamura, K.T.

Molecular basis of fosmidomycin's action on the human malaria parasite Plasmodium falciparum. Sci. Rep. 2011, 1, 1-8.

10. Okuhara, M.; Kuroda, Y.; Goto, T.; Okamoto, M.; Terano, H.; Kohsaka, M.; Aoki,

H.; Imanaka, H. Studies on new phosphonic acid antibiotics. III. Isolation and characterization of FR-31564, FR-32863 and FR-33289. J. Antibiot. 1980, 33, 24-28.

11. Hemmi, K.; Akeno, H.T.; Hashimoto, M.; Kamiya, T. Studies on Phosphonic Acid

Antibiotics. IV. Synthesis and antibacterial activity of analogs of 3-(N-Acetyl-N- hydroxyamino)- propylphosphonic acid (FR-900098). Chem. Pharm. Bull. 1982, 30, 111-

118.

12. Brown, A.C.; Parish, T. Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake. BMC Microbiol. 2008, 8, 78.

33 13. Dhiman, R. K.; Schaeffer, M. L.; Bailey, A. M.; Testa, C. A.; Scherman, H.; Crick,

D.C. 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (IspC) from Mycobacterium tuberculosis: towards Understanding Mycobacterial Resistance to Fosmidomycin. J.

Bacteriol. 2005, 187, 8395.

14. Ortmann, R.; Wiesner, J.; Silber, K.; Klebe, G.; Jomaa, H.; Schlitzer, M. Novel deoxyxylulosephosphate-reductoisomerase inhibitors: fosmidomycin derivatives with spacious acyl residues. Arch. Pharm. 2007, 340, 483-490.

15. San Jose, G.; Jackson, E.R.; Uh, E.; Johny, C.; Haymond, A.; Lundberg, L.; Pinkham,

C.; Kehn-Hall, K.; Boshoff, H.I.; Couch, R.D.; Dowd, C.S. Design of Potential

Bisubstrate Inhibitors against Mycobacterium tuberculosis (Mtb) 1-Deoxy-D-Xylulose 5-

Phosphate Reductoisomerase (Dxr)-Evidence of a Novel Binding Mode. Med. Chem.

Comm. 2013, 4:1099–1104.

34 Chapter 3: Fosmidomycin, FR900098 and Their Analogs

Introduction of Fosmidomycin and FR900098

Fosmidomycin and FR900098 (Figure 3.1) are natural products produced by a strain of Streptomyces which exhibit antimicrobial activities against Gram-negative bacteria.1,2 FR900098 was tested for safety in mice by the Okuhara group and was found to have very low toxicity (4~5g/kg).1 According to Okuhara et al., five mice that were treated with a single intravenous dose of FR900098 (4 to 5g/kg) survived after 14 days without observing any adverse effects from FR900098.1 Due to its micromolar antimicrobial activity1 and low toxicity in mice, FR900098 was selected as the lead compound for this project.

Despite its promising antimicrobial activity and low toxicity, one important disadvantage of FR900098 may be its limited metabolic stability. Studies have shown that its parent compound, fosmidomycin, has a human plasma half-life of 1.6 hours.3

Because FR900098 and fosmidomycin vary only by one methyl group on the retrohydroxamate, they may exhibit similar metabolic stability in human plasma. This will likely cause FR900098 to have low oral availability (F%) in humans. Therefore, more stable analogs of this lead compound need to be made in order for them to stay intact in the human body for a longer period of time.

Previously, significant research by our group and others has been done on α,β- unsaturated analogs4,6, reverse analogs7 and β-substituted reverse analogs of FR9000987.

While these analogs exhibited significant in vitro activity against both Mtb and Pf, the current project will build on earlier work from our group, focused on O-linked

35 substituents on the retrohydroxamate. The general structure of compounds made in this project is shown in Figure 3.2.

36 Figure 3.1: Fosmidomycin and Its Analog FR900098

O OH O OH P N O P N O HO HO OH OH

Fosmidomycin FR900098

37 Prior Work on Synthesis of Saturated Analogs of Fosmidomycin and FR900098

Methylene Linker Length:

The number of methylene groups linking the retrohydroxamate and phosphonate moieties has an effect on the activity of FR900098 analogs3. In our 2011 paper, analogs with 2, 4 and 5 methylene groups in the linker region were tested against both Mtb Dxr and Mtb whole cells. The Mtb Dxr inhibition was rather poor for all three analogs, proving that 3 methylene groups separating the phosphonate and retrohydroxamate moieties was necessary to retain optimal activity for FR900098 analogs.

α-Position Substituent:

Once the main chain structure of the FR900098 analog had been determined

(retrohydroxamate and phosphonate ester separated by 3 methylene groups), we looked into the substituents on each of the methylene groups. In 2006, the Van Calenbergh group published their work on the α-aryl substituents.4 A variety of changes at the α-position

(including addition of an α-phenyl ring) were tested against E.coli Dxr (Table 3.1). All of the FR900098 analogs had micromolar activities against Pf whole cells with the α-3,4- dichlorophenyl substituted analog being the most active inhibitor of the series. This analog showed 125 nM inhibition of Pf 3D7 malarial parasites. Therefore, we chose to maintain the α-3,4-dichlorophenyl moiety in our analogs.

38 Table 3.1: Whole Cell Activities of Van Calenbergh FR900098 Inhibitors Against Plasmodium falciparum 3D74 O HO OH P OH N

R1 O R3 R2

Compound R1 R2 R3 IC50 (µM) 3D7

2a H H CH3 0.55

2b Me H CH3 0.95

1c OMe H H 0.36

2c OMe H CH3 0.85

2d Cl H CH3 0.35

1e Cl Cl H 0.090

2e Cl Cl CH3 0.25

39 Figure 3.2: General Structure of SRW Compounds

O O R3 R R1, R2 = Et, Na/H or POM 2 O P N R O R3 = Alkyl Groups 1 O O POM = Cl O Cl

40 N-Acyl and O-linked substituents:

Some time ago, our lab developed a bisubstrate approach toward Dxr inhibitor design.4,6,10 Extension of substituents from the retrohydroxamate (via ether an N-acyl or

O-linked substituent) could enable the inhibitor to bind to both the DXP and NADPH pockets in Dxr at the same time. By the addition of hydrophobic groups on the retrohydroxamate’s carbonyl group and ether’s N-hydroxyl group, the inhibitor may be able to bind to the nicotinamide pocket of the NADPH binding site, increasing the potency of the inhibitor.4,6,10

Out of these two strategies, theoretically, the N-acyl analogs would be expected to yield more potent activities against Mtb and Pf Dxr because of the chelating ability of the

N-hydroxyl oxygen (as the anion) would be retained. Substituents on the N-hydroxyl group (O-linked analogs) would prevent deprotonation of the hydroxyl group due to the absence of hydroxyl proton and the strong chelating ability of the oxygen anion would be lost. As shown in Table 3.2, while both of these strategies led to analogs with poorer inhibition of the enzyme target compared with fosmidomycin and FR900098, it is intriguing to note that both series of compounds inhibited the enzyme to some extent.

Di-POM Ester Prodrugs:

While most FR900098 analogs can be taken up by Pf cells fairly easily because of its thin cell membrane, it is rather difficult for polar inhibitors to enter Mtb cells due to the thick mycobacterial cell walls. The N-acyl/O-linked approach already made the

FR900098 analogs more lipophilic than FR900098. Adding prodrug moieties such as the pivaloyloxymethyl groups (POM) to the phosphonate moiety could further improve cell

41 wall penetration. It is hypothesized that once the POM protected inhibitor enters the cell, the POM groups are cleaved by an esterase.10 However, the specific esterase involved in this process has not yet been determined.

42 Table 3.2: FR900098 Analogs and Their Mtb Dxr Activities10 R O O 1 P N R NaO 2 OH O

Compound R1 R2 Mtb Dxr IC50

1 H H (µM)0.31

2 H CH3 2.39

3 (CH2)2Ph CH3 >50% residual

4 (CH2)3Ph CH3 >50%activity residual

5 4-ipr-benzyl CH3 activity48.4

6 CH2cyclohexyl CH3 >50% residual

7 H (CH2)2Ph activity26.9

8 H (CH2)3Ph 17.8

9 H cyclohexyl >50% residual

activity

43 The Design of SRW Compounds

The compounds designed and prepared are shown in Table 3.3. By design, these compounds share several structural features in common with Dxr inhibitors synthesized by our group in prior studies. As previously mentioned, the phosphonate is designed to bind to the DXP pocket in Dxr. Each of the analogs has the saturated propyl linker chain and acetyl group of FR900098. Each of our compounds bears an aromatic (3,4- dichlorophenyl) substituent at the alpha position (relative to the phosphorus atom). The

O-linked substituent placed on the retrohydroxamate hydroxyl group is designed to extend into the NADPH pocket to interact with the nicotinamide pocket.4 Because of this, five out of the six O-linked substituents have an aromatic moiety stemming from the N- hydroxyl group. These compounds are designed to favor potential π-π stacking interaction between the inhibitor and residues of nicotinamide binding pocket. Taken together, the goal of our compound design is to understand if the alpha aryl substituent and the O-linked substituent can both be accommodated within the Dxr active site.

Metabolic Stability

As mentioned above, one of the most significant liabilities of fosmidomycin is low metabolic stability within humans.3 With a human plasma half-life of 3.5 hr, fosmidomycin has a short half-life.11 This indicates it is easy for these compounds to be broken down and depleted from the circulatory system, thus lowering the chance of the drug actually getting to Mtb cells and Pf parasites.

With the 3,4-dichlorophenyl group at the α position and O-linked substituents on the retrohydroxamate moiety, the SRW compounds may be more stable than the parent

44 compounds and display improved half-lives. One possibility is that the 3,4- dichlorophenyl moiety creates steric hindrance, slowing down esterase cleavage of the prodrug, thus improving the metabolic stability of the compounds.

As probes for obtaining preliminary data, eighteen different molecules (Table 3.3) were synthesized to study the structure-activity relationship between SRW compounds and their respective Mtb/Pf whole cell and enzyme activities. More compounds will be synthesized once biological data on these molecules is obtained and analyzed. It is worth pointing out that the α-position of SRW compounds is a chiral center, and future synthesis of a particular stereoisomer may give more potent Dxr inhibition (S- versus R- stereoisomers).

45 Table 3.3: Structures of SRW Compounds

O O R R 3 2 O P N R O 1 O

Cl Cl

R3 R1/R2 =Et R1/R2=Na/H R1/R2=POM

SRW-58 SRW-61 SRW-68

SRW-59 SRW-67 SRW-69

SRW-60 SRW-65 SRW-77

SRW-80 SRW-84 SRW-91

SRW-81 SRW-85 SRW-89

SRW-83 SRW-86 SRW-90

46 Conclusions:

Based on earlier studies, the antimicrobial activities, and low toxicity in mice,

FR900098 was chosen as the lead compound for this project. The phosphonate and retrohydroxamate moieties need to be retained for the analogs due to their binding ability to the DXP pocket in Dxr. Three methylene groups are used to obtain more optimized general inhibitor activities. The 3,4-dichlorophenyl moiety on the α position of the molecule was discovered by the Van Calenbergh group in 2006. Compounds with this group displayed potent E.coli Dxr and Pf whole cell inhibition. Aromatic substituents on the N-hydroxyl group have the ability to extend into the NADPH pocket for the nicotinamide, and this group may provide a bisubstrate binding mechanism to further facilitate the inhibition of Dxr. With all these factors considered, the objectives of this project are to synthesize a series of di-substituted analogs of FR900098 and evaluate their ability to inhibit Mycobacterium tuberculosis and Plasmodium falciparum.

47 References:

1. Okuhara, M.; Kuroda, Y.; Goto, T.; Okamoto, M.; Terano, H.; Kohsaka, M.; Aoki, H.;

Imanaka, H. Studies on new phosphonic acid antibiotics. III. Isolation and characterization of FR-31564, FR-32863 and FR-33289. J. Antibiot. 1980, 33, 24-28.

2. Lell, B.; Ruangweerayut, R.; Wiesner, J.; Missinou, M. A.; Schindler, A.; Baranek, T.;

Hintz, M.; Hutchinson, D.; Jomaa, H.; Kremsner, P. G. Fosmidomycin, a novel chemotherapeutic agent for malaria. Antimicrob. Agents Chemother. 2003, 47, 735−8.

3. Kuemmerle, H.P.; Murakawa, T.; Sakamoto, H.; Sato, N.; Konishi, T.; De Santis, F.

Fosmidomycin, a new phosphonic acid antibiotic. Part II: 1. Human pharmacokinetics. 2.

Preliminary early phase IIa clinical studies. Int. J. Clin. Pharmacol. Ther. Toxicol. 1985,

23:521–528.

4. Jackson, E. R.; San Jose, G.; Brothers, R. C.; Edelstein, E. K.; Sheldon, Z.; Haymond,

A.; Johny, C.; Boshoff, H.I.; Couch, R.D.; Dowd, C. S. The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs. Bioorg. Med. Chem. Lett. 2014, 24(2), 649-653.

5. Haemers, T.; Wiesner, J.; Van Poecke, S.; Goeman, J.; Henschker, D.; Beck, E.;

Jomaa, H.; Van Calenbergh, S. Synthesis of α-substituted fosmidomycin analogues as highly potent Plasmodium falciparum growth inhibitors. Bioorg. Med. Chem. Lett. 2006,

16, 1888− 1891.

6. San Jose, G.; Jackson, E. R.; Haymond, A.; Johny, C.; Edwards, R. L.; Wang, X.;

Brothers, R.C.; Edelstein, E.K.; Odom, A.R.; Boshoff, H.I.; Couch, R.D.; Dowd, C. S.

Structure-Activity Relationships of the MEPicides: N-Acyl and O-Linked Analogs of

48 FR900098 as Inhibitors of Dxr from Mycobacterium tuberculosis and Yersinia pestis.

ACS Infect Dis. 2016, 2(12), 923-935.

7. Brücher, K.; Gräwert, T., Konzuch, S.; Held, J.; Lienau, C.; Behrendt, C.; B.

Illarionov.; Maes, L.; Bacher, A.; Wittlin, S.; Mordmüller, B.; Fischer, M.; Kurz, T. J.

Med. Chem. 2015, 58, 2025–2035

8. Schultz C. Bioorg. Med. Chem. 2003;11:885–898.

9. Kuemmerle, H. P.; Murakawa, T.; De Santis, F. Pharmacokinetic evaluation of fosmidomycin, a new phosphonic acid antibiotic. Chemioterapia. 1987, 6:113-119.

10. San Jose, G.; Jackson, E.R.; Uh, E.; Johny, C.; Haymond, A.; Lundberg, L.; Pinkham,

C.; Kehn-Hall, K.; Boshoff, H.I.; Couch, R.D.; Dowd, C.S. Design of Potential

Bisubstrate Inhibitors against Mycobacterium tuberculosis (Mtb) 1-Deoxy-D-Xylulose 5-

Phosphate Reductoisomerase (Dxr)-Evidence of a Novel Binding Mode. Med. Chem.

Comm. 2013, 4:1099–1104.

11. Armstrong, C.M.; Meyers, D.J.; Imlay, L.S.; Meyers, C.F.; Odom, A.R. Resistance to the antimicrobial agent fosmidomycin and an FR900098 prodrug through mutations in the deoxyxylulose phosphate reductoisomerase gene (dxr). Antimicrob Agents

Chemother. 2015, 59:5511–5519.

49 Chapter 4: Synthesis, Analysis, and Biological Evaluation of FR900098 Analogs

Background

As described in Chapter 3, the desired FR900098 analogs (SRW compounds) consist of both an α-3,4-dichlorophenyl substituent and an O-linked aromatic substituent on the retrohydroxamate moiety (Figure 4.1). According to our hypothesis, we expect these two moieties to interact with Trp296 and the Dxr NADPH pocket, respectively, in both Mtb and Pf Dxr to afford better enzyme inhibition. Similar FR900098 analogs have been made without either the α-3,4-dichlorophenyl substituent1,2 or the O-linked moieties.3 Some of these FR900098 analogs were able to inhibit either Pf whole cells or

Mtb Dxr. The newly synthesized FR900098 analogs from Scheme 4.1 were tested for their inhibition of Mtb and Pf Dxr and whole cell growth, as well as other properties.

Synthesis

Our general strategy for synthesizing molecules 7a-g, 8a-g and 9a-g utilizes divergent synthesis (Scheme 4.1). The phosphonate and retrohydroxamate moieties are assembled onto the carbon backbone first, followed by cleavage of the benzyl group of the retrohydroxamate to obtain a central intermediate. This intermediate was used to make three different versions of each target compound: a diethyl ester, monosodium salt, and POM prodrug.

The synthetic scheme starts with introduction of the α-3,4-dichlorophenyl ring in the first step through a Wittig reaction. Reaction between 3,4-dichlorophenyl benzaldehyde and triphenylphosphoranylidene acetaldehyde affords α,β-unsaturated

50 aldehyde 1 (Scheme 4.1). The phosphonate moiety is added through a Michael addition reaction to yield intermediate 2. The protecting phenyl groups are removed upon reaction with hydrochloric acid to afford aldehyde 3.

Hydrochloric acid was used for the reaction to make aldehyde 3, and acetone was used as the solvent. However, with acetone in the reaction mixture, the temperature of the reaction mixture should not go above 70 oC as indicated by literature experimental procedures.4 Therefore, based on my experience, this reaction cannot reach completion within 4 hours, and it would normally take approximately 3 days for it to complete.

Reductive amination was employed to elongate the backbone of the molecule while keeping the protecting benzyl group. Thus, compound 3 was treated with O- benzylhydroxylamine in anhydrous ethanol at room temperature to yield an imine. The imine was not isolated and was reduced in situ with sodium cyanoborohydride in anhydrous methanol to yield amine 4.

Acetylation using acetyl chloride and triethylamine completed the retrohydroxamate moiety, yielding compound 5. The ultimate goal of this work is to make ethers in the next step; therefore, ether cleavage of the benzyl group was done with boron trichloride in dichloromethane at -78oC. After purification by column chromatography, the central intermediate, 6, was obtained.

Williamson ether synthesis between intermediate 6 and various alkyl bromides provided the diethyl phosphonate esters of the target compounds. The O-linked products of this reaction (compounds 7) were obtained in moderate to good yields (59-88%).

Conversion of compound 7e to compound 7f was carried out using a Suzuki coupling reaction in high yield (91%). Diethyl esters 7 served as the precursors for synthesis of the

51 monosodium salts and di-POM prodrugs. Ester cleavage using trimethylsilylbromide

(TMSBr), followed by deprotonation with NaOH yielded the desired monosodium salts

(compounds 8) in quantitative yield.

The final step of the scheme is an SN2 reaction between the phosphonate anion of the monosodium salt and pivaloyloxymethylchloride (POMCl) in N,N- dimethylformamide (Scheme 4.1). This reaction typically gives very low yield (13 to

33%). Addition of six equivalents of POMCl was used to ensure complete conversion of starting material to product, with no mono-POM ester remaining.

Because DMF is used as the solvent for the reaction, the product is hard to purify.

We can, instead, utilize the property of the intended product during purification. Because the lipophilicities of the prodrug compounds in SRW series are very high (clogP of 8.8 to

10.9), extraction can be performed in order to remove almost all of the DMF in the crude product. After the reaction reached completion, the mixture was extracted with water and ethyl ether to remove most of the DMF. This is because the prodrugs are very soluble in ethyl ether. After the initial extraction, the ethyl ether is removed under reduced pressure, then the residue is extracted with hexanes. This gives the crude product with no DMF left.

All of the steps prior to the Williamson ether synthesis reactions have been previously reported.1,3 In order to eliminate the use of pyridine during the reductive amination reaction, the free amine of O-benzylhydroxylamine hydrochloride was made.

All of the products except the monosodium salts were purified using normal phase silica gel column chromatography before isolation and characterization. Using this scheme,

52 eighteen SRW compounds were successfully synthesized. These compounds were characterized using LCMS (or GCMS), HRMS, and 1H and 13C NMR.

53 Figure 4.1: General Structure of FR900098 Analogs

O O R R 3 2 O P N R O 1 O

Cl Cl

R1/R2=Et, Na/H and POM, R3=alkyl substituents

54 Scheme 4.1: Synthetic Scheme of SRW Series of FR900098 Analogs

O O EtO OEt EtO OEt O P P i ii OPh iii H O OPh O

Cl Cl Cl Cl Cl Cl Cl Cl

1 2 3

O O O OBn O OH EtO OEt O O P P EtO P N O vi EtO P N O iv v EtO EtO OBn O N H Cl Cl Cl Cl Cl Cl Cl Cl 6 3 4 5

R R R O O O O O O vii NaO P N O POMO P N O EtO P N O viii HO ix POMO EtO

Cl Cl Cl Cl Cl Cl

7a: 8a-d,f,g 9a-d,f,g

7b:

7c:

7d:

7e: Br x 7f: 7g:

Reagents and Conditions: (i) triphenylphosphoranylidene acetaldehyde, toluene, 50-80 oC; (ii) TEP, phenol, 100 oC; (iii) 2N HCl, acetone, 100 oC; (iv) (a) OBHA, EtOH, rt, (b)

o NaBH3CN, acetic acid, MeOH, rt; (v) AcCl, Et3N, DCM 0 C to rt; (vi) BCl3, DCM, -78 oC; (vii) alkyl bromide, NaH, THF, 0 oC to rt; (viii) TMSBr, DCM, 0 oC to rt; (ix)

o POMCl, Et3N, DMF, 60 C; (x) 4-isopropylphenyl boronic acid, Pd(PPh3)4, diethyl ether,

o Na2CO3, EtOH, 70 C.

Abbreviations: TEP = triethylphosphite, OBHA = O-benzylhydroxylamine, AcCl = acetyl chloride, RBr = alkyl bromide, TMSBr = trimethylsilylbromide, POMCl = pivaloyloxymethylchloride, DCM = dichloromethane, THF = tetrahydrofuran, DMF =

N,N-dimethylformamide

55 Biological Evaluation of SRW Compounds

After the compounds were synthesized, they were sent to our collaborators to evaluate inhibition of the Mtb and Pf Dxr enzymes and whole cell activities, ability to terminate MEP pathway, as well as cytotoxicity and metabolic stability. Only the monosodium salts were evaluated for enzyme activity, as these salts display the active inhibitor which should bind to Dxr. The ethyl and POM groups are not expected to bind to Dxr. We are grateful to our collaborators at Washington University, George Mason

University, and the National Institutes of Health for providing us with the preliminary data for this project.

Interpretation of the Enzyme and Whole Cell Activity Data of SRW Compounds

Mycobacterium tuberculosis Dxr Enzyme Data

In order to determine whether the SRW compounds have on-target activity, the percentage of inhibition of their respective Dxr homolog needs to be tested (Table 4.1).

As the data indicate, none of the 6 monosodium salts inhibited Mtb Dxr to an appreciable extent. Therefore, if any of the SRW compounds are active against intact Mtb cells, they must have off-target effects that are capable of killing the bacteria.

56 Plasmodium falciparum Dxr Enzyme Data

Most of the SRW monosodium salts (five out of six) are active against Pf Dxr

(Table 4.1). SRW-61 and SRW-86 are the most active compounds in the series with IC50 values of 0.34 and 1.00 µM, respectively. SRW-65, 67 and 85 are active, but less so, with low µM values. Phenylbutyl analog SRW-84 does not inhibit Pf Dxr.

57 Table 4.1: Mtb and Pf Dxr Enzyme Activities of SRW Compounds

R O O HO P N NaO O

Cl Cl

Dxr IC (µM) Compound R 50 Mtb Pf Fosmidomycin NA 0.44 0.0032 FR900098 NA 2.91 0.0018 SRW-61 >50 0.34

SRW-65 >50 1.430

SRW-67 >50 3.75

SRW-84 >50 >100

SRW-85 >50 2.49

SRW-86 >50 1.00

58 Mycobacterium tuberculosis Whole Cell MIC Data

All of the eighteen SRW compounds were tested for activity against intact Mtb cells (Table 4.2). Given that the SRW compounds did not show activity against Mtb Dxr, we were surprised to see that some of these compounds display moderate activity against the intact cells. The most active compounds were SRW-65 and 67 with MIC values of 9.4 ug/mL in the one week GAST-Fe assay. As the compounds are not active against Mtb

Dxr, there must be an additional off-target mechanism at work to yield this antimycobacterial effect.

59 Table 4.2: Mtb Whole Cell Minimum Inhibitory Concentrations (MIC) for SRW Compounds

1-week MIC 2-week MIC 1-week MIC in 2-week MIC in Compound in GAST/Fe in GAST/Fe 7H9/ADC/Tw 7H9/ADC/Tw (in µg/mL) (in µg/mL) (in µg/mL) (in µg/mL) SRW-58 74 100 >100 >100 SRW-59 74 100 100 >=100 SRW-60 100 100 148 148 SRW-61 >=200 >=200 >200 >200 SRW-65 9.4 12.4 >100 >100 SRW-67 9.4 12.5 100 >=100 SRW-68 >=200 200 >200 >200 SRW-69 >200 >200 >200 >200 SRW-77 >200 >200 >200 >200 SRW-80 100 100 148 148 SRW-81 76 148 148 148 SRW-83 100 100 148 148 SRW-84 18.8 37.6 >200 >200 SRW-85 37.6 50 >200 >200 SRW-86 18.8 18.8 148 148 SRW-89 >200 >200 >200 >200 SRW-90 >200 >200 >200 >200 SRW-91 50 50 >200 >200 Rifampicin 0.0024 0.0024 0.0024 0.0024 Isoniazid (µM) 0.23 0.47 1.9 NA

60 Plasmodium falciparum Whole Cell IC50 Data

Due to the thinness of the Pf cell membrane, all three sets of compounds are able to penetrate into the cell. According to our hypothesis, we believe the phosphonate ester moieties will pass through the cell membrane and then get cleaved by phosphonate esterases in Pf, revealing the active moiety. However, if cleavage of the esters is not complete, there will be less active moieties to bind to Dxr. Based on our data, it seems the prodrug moieties are being sufficiently cleaved by esterases. This can be confirmed by the fact that the prodrugs are generally more active than their respective monosodium salts, except for SRW-68.

SRW-61 (924nM) and SRW-86 (1.6µM) have the most potent inhibition of whole cell P. falciparum (Table 4.3). As these two compounds were the most active against Pf

Dxr, this result is very gratifying. Data for the remainder of the compounds is mixed with some esters retaining activity when the parent salt is less active. These compounds may have a more complex mechanism of action, including off-target effects. Further biological evaluation needs to be conducted to study whether they inhibit the MEP pathway. As SRW-61 displays impressive activity against both the Pf enzyme and intact cells, it was selected for IPP rescue studies.

61 Table 4.3: Pf Whole Cell Activities of SRW Compounds

O O R R 3 2 O P N R O 1 O

Cl Cl

Compound R1/R2 R3 Pf IC50 (µM)

SRW-58 Et 10.7

SRW-61 Na/H 0.9

SRW-68 POM 5.2

SRW-59 Et 3.9

SRW-67 Na/H 30.2

SRW-69 POM 13.9

SRW-60 Et 5.0

SRW-65 Na/H 35.5

SRW-77 POM 7.9

SRW-80 Et 4.5

SRW-84 Na/H >100

SRW-91 POM 14.1

SRW-81 Et 6.9

SRW-85 Na/H 12.8

SRW-89 POM 15.1*

SRW-83 Et 2.6

SRW-86 Na/H 1.6

SRW-90 POM 20.2*

*More replicates are needed to improve the precision of the analysis

62 Determining the On-Target Intracellular Effect of the SRW-61:

The isopentenyl pyrophosphate (IPP) rescue method is one useful way of determining whether the inhibitors are acting on the designated target and whether they have any off-target effect3. If the inhibitor successfully blocked the DXP binding site on

Dxr, the production of IPP will be stopped, thus blocking the entire MEP pathway. When

IPP is re-introduced into the medium, the parasite activity can be restored even without the production of IPP. We can confirm whether the inhibitor is working on Dxr based on whether cell activity can be restored with IPP in the medium. During this project, one IPP rescue study was carried out on SRW-61 (Figure 4.1). With no IPP in the medium, SRW-

61 was able to inhibit Pf cell at 0.924µM. With the addition of IPP, normal cell function was maintained until the concentration SRW-61 reached almost 10-times its IC50 value.

This differential indicates that the intracellular mechanism of action is very likely due to inhibition of the MEP pathway.

63 Figure 4.2: IPP Rescues P. falciparum strain MR4 from the effects of SRW-61

SRW-61p 150

MR4 MR4 + IPP

100

50 PicoGreen Fluorescence

0 0.01 0.1 1 10 100 Drug Concentration (µg/mL)

MR4 MR4 + IPP 0.4655 5.264

64 Comparison between SRW Compounds and Previously Synthesized Compounds:

Out of the eighteen molecules in the SRW series, nine were previously synthesized in our lab without the α-3,4-dichlorophenyl moiety.2 These nine analogs were evaluated for their antimalarial activities against 3D7 cells. Therefore, it is beneficial to examine how the addition of α-3,4-dichlorophenyl moiety affected the analogs’ Pf whole cell activities by making comparisons between the earlier structures and the more recent

SRW compounds.

As shown in Tables 4.4a and 4.4b, seven of the nine SRW compounds (SRW-58,

60, 61, 67, 77, 81, 85) are more active than the earlier des-aryl structures. This finding matched our hypothesis, which is that the α-3,4-dichlorophenyl moiety may be beneficial to Dxr binding, perhaps by involving a π-π stacking interaction between the α-phenyl ring and the indole ring of a nearby tryptophan residue in Dxr.

65 Table 4.4a: Comparison between Analogs with and without α-3,4-dichlorophenyl Group

R O O 2 R O 3 P N O R3O R 1

R1 R3 R2 Pf IC50 (µM)

SRW-67 3,4-dichlorophenyl Na/H 3.745

GSJ-76 H Na/H >200

SRW-69 3,4-dichlorophenyl POM 13.9

GSJ-96 H POM 8.07

SRW-85 3,4-dichlorophenyl Na/H 12.8

JXW-79 H Na/H >200

SRW-81 3,4-dichlorophenyl Et 6.92

JXW-74 H Et 26.44

66 Table 4.4b: Comparison between Analogs with and without α-3,4-Dichlorophenyl Group

R1 R3 R2 Pf IC50 (µM)

SRW-65 3,4-dichlorophenyl Na/H 35.471

GSJ-74 H Na/H 8.09

SRW-60 3,4-dichlorophenyl Et 4.997

JXW-77 H Et 16.92

SRW-77 3,4-dichlorophenyl POM 7.9

GSJ-97 H POM 14.37

SRW-61 3,4-dichlorophenyl Na/H 0.924

GSJ-63 H Na/H 12.06

SRW-58 3,4-dichlorophenyl Et 10.669

GSJ-28 H Et >200

67 Cytotoxicity of SRW Compounds:

All eighteen of the SRW compounds have been analyzed for their cytotoxic properties (Table 4.5). Unfortunately, all but seven compounds were toxic toward hepatocytes (HepG2) with IC50 values of 18.75 µM or lower. Only seven compounds were shown to have no toxicity (>50 µM). Interestingly, these non-toxic compounds were nearly all monosodium salts, indicating that the more lipophilic esters (ethyl and POM) display some degree of toxicity. Given this data, it was surprising to see that SRW-90, the most lipophilic molecule in the series, showed no toxicity. Additional compounds and studies are needed to fully understand the SAR between our compounds and HepG2 cytotoxicity.

Metabolic Stability of SRW-61 and SRW-68

Given the limited half-life of fosmidomycin, the stability and half-life of our compounds is an important feature to measure. In order to study the stability of the most active compound (SRW-61) and its prodrug analog (SRW-68), we have sent these two compounds to our collaborators at St. Louis University. Evaluation of their stability in human and mouse plasma, as well as toward human liver microsomes, is currently underway.

68 Table 4.5: Cytotoxicity of FR900098 Analogs

Plate A Plate B Galactose Glucose Galactose Glucose

Compounds IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM) SRW-58 9.375 18.75 9.375 18.75 SRW-59 9.375 9.375 4.6875 9.375 SRW-60 4.6875 9.375 9.375 9.375 SRW-61 >50 >50 >50 >50 SRW-65 >50 >50 >50 >50 SRW-67 >50 >50 >50 >50 SRW-68 18.75 18.75 18.75 18.75 SRW-69 18.75 18.75 18.75 37.5 SRW-77 18.75 37.5 37.5 37.5 SRW-80 9.375 9.375 9.375 9.375 SRW-81 9.375 9.375 9.375 9.375 SRW-83 9.375 9.375 9.375 9.375 SRW-84 >50 >50 >50 >50 SRW-85 >50 >50 >50 >50 SRW-86 >50 >50 >50 >50 SRW89 18.75 18.75 18.75 37.5 SRW90 >50 >50 >50 >50 SRW91 18.75 37.5 18.75 37.5

69 Conclusions

All eighteen SRW compounds were synthesized following a parallel strategy and obtained in appreciable yield and amount. The compounds have been tested against Mtb and Pf whole cells as well as the corresponding Dxr homologs for their inhibitory activities. Based on the Mtb enzyme inhibition, we can conclude that none of the SRW compounds have on-target effects on Mtb Dxr. However, five of the SRW monosodium salts are active against Pf Dxr, which means five types of O-linked ether compounds have on target effect on Pf Dxr. Out of these Pf Dxr inhibitors, SRW-61 shows the best enzyme and whole cell activities. IPP rescue studies were conducted for SRW-61 and confirmed that SRW-61 inhibits the MEP pathway. Based on the data collected from cytotoxicity studies, we concluded that SRW-61 has a selectivity index above 50 (HepG2

IC50/Pf IC50). Therefore, SRW-61 exhibits low cytotoxicity against Pf cells and high potency against Pf Dxr. More information on the metabolic stability of this compound needs to be collected and such studies are currently in progress. Taken together, SRW-61 could represent an exciting lead compound against malaria, and one that can start a new generation of molecules with a novel mode of action against the pathogen.

70 Experimental Procedures for Synthesis of the Intermediates

SRW-71 (Intermediate 1): (2E)-3-(3,4-Dichlorophenyl) prop-2-enal

O Toluene, 80 oC H O O + PPh3 Cl 73% Cl Cl Cl

1

A solution of 3,4-dichlorophenyl benzaldehyde (3g, 17.1mmol) and triphenylphosphoranylidene acetaldehyde (5.73g, 18.9mmol) in 100mL anhydrous toluene was stirred at 50 oC for 1 hour and at 80 oC for 3 days. The toluene was removed under reduced pressure. The crude residue was purified by normal phase silica chromatography with 1:1 dichloromethane: hexane to afford SRW-71 as a yellow solid

(2.51g, 73%). The GCMS and 1H-NMR data match literature values.4,5

SRW-72 (Intermediate 2): Diethyl [1-(3,4-dichlorophenyl)-3,3-diphenoxypropyl] phosphonate

O EtO OEt P OPh TEP, phenol, 100 oC O OPh

Cl 81% Cl Cl Cl

1 2

71 (2E)-3-(3,4-dichlorophenyl) prop-2-enal (1) (2.5g, 12.4mmol) was stirred with phenol

(3.04g, 32.2mmol) and triethylphosphite (2.6g, 15.5mmol) at 100 oC for 18h. The crude residue was directly purified by normal phase silica chromatography with 6:4 Hexanes:

Ethyl acetate to yield a yellow oil as the product (5.15g, 81%). The LCMS and 1H-NMR data matched literature values.4,5

SRW-73 (Intermediate 3): Diethyl [1-(3,4-dichlorophenyl)-3-oxopropyl] phosphonate

O O EtO OEt EtO OEt P P OPh 2N HCl, acetone, 60 oC OPh O

Cl 66% Cl Cl Cl

2 3

Diethyl [1-(3,4-dichlorophenyl)-3,3-diphenoxypropyl] phosphonate (2) (5.15g, 15mmol) was dissolved in 71mL acetone. 16.4mL of 2M HCl acid and 14.2mL of water were added to the solution. The mixture was heated at 60 oC for 3 days. After removing the acetone under reduced pressure, the residue was mixed with 100mL of water and then extracted with 100mL of dichloromethane 3 times. The organic phase was dried over sodium sulfate and filtered. Dichloromethane was removed under reduced pressure and the residue was purified by normal phase silica chromatography with pure ethyl acetate, afforded a colorless oil as the product (2.24g, 66%). The LCMS and 1H-NMR data matched literature values.4,5

72 SRW-74: O-benzylhydroxylamine

NH + Cl- NaOH NH O 3 O 2

Sodium hydroxide (0.5g, 12.5mmol) and O-benzylhydroxylamine hydrochloride (1.99g,

12.5mmol) were dissolved in 50mL of water, and stirred for 30 minutes at room temperature. 150mL of ethyl ether was added to the reaction mixture to extract the aqueous solution 3 times. The organic solution was dried over sodium sulfate and filtered. Diethyl ether was removed under reduced pressure to yield the desired colorless liquid as a product (1.28g, 83%). The GCMS and 1H-NMR data matched literature values.

SRW-76 (Intermediate 4): Diethyl {3-[(benzyloxy) amino]-1-(3,4-dichlorophenyl) propyl} phosphonate

O O EtO OEt O O P 1. OBHA, EtOH P 2. NaBH3CN, acetic acid, MeOH OBn O N H Cl Cl Cl Cl

3 4

Diethyl [1-(3,4-dichlorophenyl)-3,3-diphenoxypropyl] phosphonate (3) (1.59g,

4.69mmol) was dissolved in 50mL anhydrous ethanol and O-benzylhydroxylamine

(0.577g, 4.69mmol) was added and stirred at room temperature for 1 hour. The ethanol

73 was removed under reduced pressure. Sodium cyanoborohydride (1.03g, 16.4mmol) and

50ml of anhydrous methanol were added to the crude reaction mixture and stirred for 30 minutes. Acetic acid (20mL) was added to the mixture and the reaction mixture was stirred under a nitrogen atmosphere at room temperature for 3 hours. The methanol was removed under reduced pressure. The crude mixture was extracted with 20mL of 0.1M

NaOH aq and 100mL of dichloromethane. The organic layer was dried over sodium sulfate and filtered. After the dichloromethane was removed under reduced pressure, the crude product was purified by normal phase silica chromatography using 99:1 dichloromethane: methanol and gave the desired compounds as a colorless oil (1.88g,

89%). The LCMS and 1H-NMR data matched literature values.4,5

SRW-78 (Intermediate 5): Diethyl {3-[N-(benzyloxy) acetamido]-1-(3,4- dichlorophenyl) propyl} phosphonate

O O OBn O O P EtO P N O EtO OBn AcCl, Et3N, DCM N H Cl 92% Cl Cl Cl 4 5

Diethyl {3-[(benzyloxy) amino]-1-(3,4-dichlorophenyl) propyl} phosphonate (4) (1.44g,

3.23mmol) was dissolved in 50 mL of anhydrous dichloromethane. Triethylamine

(326.4mg, 3.23mmol) and acetylchloride (253.2mg, 3.23mmol) were added to the reaction mixture at 0 oC. The mixture was warmed up to room temperature and allowed

74 to stir for 12 hours. The crude product was extracted with 50mL of water and 100mL of dichloromethane 3 times. The organic layer was dried over sodium sulfate and filtered.

After dichloromethane was removed under reduced pressure, the crude product was purified by normal phase silica chromatography using 70:30 dichloromethane: ethyl acetate. The desired product was obtained as a colorless oil (1.45g, 92.4%). The LCMS and 1H-NMR data matched literature values.4,5

SRW-79 (Intermediate 6): Diethyl [1-(3,4-dichlorophenyl)-3-(N-hydroxyacetamido) propyl] phosphonate

O OBn O OH EtO P N O EtO P N O EtO EtO DCM, BCl3

89% Cl Cl Cl Cl 6 5

Diethyl {3-[N-(benzyloxy) acetamido]-1-(3,4-dichlorophenyl) propyl} phosphonate (5)

(1.45g, 2.97mmol) was dissolved in 50mL of anhydrous dichloromethane and boron trichloride (1.22g, 10.4mmol) was added at -78 oC dropwise. The reaction mixture was

o stirred at -78 C for 4 hours and then was quenched with 20mL of NaHCO3. The mixture was extracted with 100mL of dichloromethane 3 times. The organic phase was dried over sodium sulfate and filtered. The dichloromethane was removed under reduced pressure.

The crude product was purified by normal phase silica chromatography using 95:5

75 dichloromethane: methanol to afford an orange, viscous oil as the product (1.05g, 89%).

The LCMS and 1H-NMR data matched literature values.4,5

SRW-58 (7a): Diethyl [1-(3,4-dichlorophenyl)-3-[N-(1phenylethoxy) acetamido] propyl] phosphonate

O O O O P O O NaH, THF, 1-bromo-ethyl benzene P OH O N N

Cl O Cl O Cl Cl 6 7a

Diethyl [1-(3,4-dichlorophenyl)-3-(N-hydroxyacetamido) propyl] phosphonate (6)

(132.8mg, 0.334mmol) was dissolved in anhydrous tetrahydrofuran (25mL) under a nitrogen atmosphere. Sodium hydride (8 mg, 0.334mmol) and 1-bromo-ethyl benzene

(62mg, 0.334mmol) were added to the solution at 0 oC. The mixture was warmed to room temperature and was stirred overnight. After removing the solvent under reduced pressure, the residue was mixed with water (50mL) and extracted with dichloromethane

(50mL) 3 times. The organic layer was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by normal phase silica column chromatography using methanol: dichloromethane (5:95) to yield a

1 colorless oil (89.8mg, 54%). H NMR (400 MHz, CDCl3) δ (ppm): 1.11-1.16 (q, 3H , J =

5.44Hz), 1.24-1.29 (t, 3H, J = 7.6Hz), 1.52 (q, 4H), 1.97 (m, 4H), 2.29 (m, 1H), 2.85 (m,

2H), 3.49 (m, 1H), 3.93 (m, 3H), 4.69 (s, 1H), 7.09-7.34 (m, 8H). 13C NMR (100 MHz,

76 CDCl3) δ (ppm): 16.3, 20.3, 26.2, 40.7, 42.1, 62.2, 62.7, 82.9, 127.1, 127.2, 128.6, 128.6,

128.8, 130.3, 130.9, 131.1.

SRW-60 (7b): Diethyl {3-[N-({[1,1'-biphenyl]-4-yl} methoxy) acetamido]-1-(3,4- dichlorophenyl) propyl} phosphonate

O O O O P O O NaH, THF, 4-bromomethy biphenyl bromide P OH O N N

Cl O Cl O Cl Cl 6 7b

Diethyl [1-(3,4-dichlorophenyl)-3-(N-hydroxyacetamido) propyl] phosphonate (6)

(30mg, 0.076mmol) was dissolved in anhydrous tetrahydrofuran (25mL) under a nitrogen atmosphere. Sodium hydride (1.8mg, 0.076mmol) and 4-bromo-methybiphenyl (18.7mg,

0.076mmol) was added to the solution at 0 oC. The mixture was warmed to room temperature and was allowed to stir overnight. After removing the solvent under reduced pressure, the residue was mixed with water (50mL) and extracted with dichloromethane

(50mL) 3 times. The organic layer was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by normal phase silica column chromatography using methanol: dichloromethane (5:95) to yield a

1 colorless oil (36.5mg, 86%). H NMR (400 MHz, CDCl3) δ (ppm): 1.147 (t, 3H, J =

7.72Hz), 1.266 (t, 3H, J = 7.72Hz), 2.183 (m, 4H), 2.455 (m, 1H), 3.034 (m, 1H), 3.519

(m, 2H), 3.795-4.091 (4H), 4.753 (s, 2H), 7.167-7.606 (m, 12H). 13C NMR (100 MHz,

77 CDCl3) δ (ppm): 16.310, 20.439, 26.762, 62.711, 62.779, 127.124, 127.427, 127.663,

128.650, 128.862, 129.629, 130.448, 131.048, 165.608.

SRW-59 (7c): Diethyl [1-(3,4-dichlorophenyl)-3-(N-{[4-(propan-2-yl) phenyl] methoxy} acetamido) propyl] phosphonate

O O O O O O P P NaH, THF, 4-isopropyl benzyl bromide OH O N N

Cl O Cl O Cl Cl 6 7c

Diethyl [1-(3,4-dichlorophenyl)-3-(N-hydroxyacetamido) propyl] phosphonate (6)

(30mg, 0.076mmol) was dissolved in anhydrous tetrahydrofuran (25mL) under a nitrogen atmosphere. Sodium hydride (1.8 mg, 0.076mmol) and 4-isopropyl benzyl bromide (16.1 mg, 0.076mmol) was added to the solution at 0 oC. The mixture was warmed to room temperature and was stirred overnight. After removing the solvent under reduced pressure, the residue was mixed with water (50mL) and extracted with dichloromethane

(50mL) 3 times. The organic layer was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by normal phase silica column chromatography using methanol: dichloromethane (5:95) to yield a

1 colorless oil (30.9mg, 88%). H NMR (400 MHz, CDCl3) δ (ppm): 1.144 (t, 3H, J =

7.52Hz), 1.254 (m, 10H), 1.633 (s, 1H), 2.023 (s, 3H), 2.185 (m, 1H), 2.431 (m, 1H),

2.950 (m, 2H), 4.102 (m, 4H), 4.669 (t, 2H, J = 9.8Hz), 7.203-7.411 (m, 7H). 13C NMR

(100 MHz, CDCl3) δ (ppm): 16.401, 20.371, 23.878, 26.739, 33.920, 40.744, 42.125,

78 62.225, 62.756, 126.790, 128.650, 129.317, 130.433, 131.116, 131.420, 135.921,

150.009, 165.608.

SRW-81 (7d): Diethyl [1-(3,4-dichlorophenyl)-3-{N-[(naphthalen-2-yl) methoxy] acetamido} propyl] phosphonate

O O O O P O O NaH, THF, 2-bromo-methyl naphthalene P OH O N N

Cl O Cl O Cl Cl 6 7d Diethyl [1-(3,4-dichlorophenyl)-3-(N-hydroxyacetamido) propyl] phosphonate (6)

(205mg, 0.52mmol) was dissolved in anhydrous tetrahydrofuran (25mL) under a nitrogen atmosphere. Sodium hydride (12.1mg, 0.52mmol) and 2-bromomethyl naphthalene

(114.2 mg, 0.52mmol) was added to the solution at 0 oC. The mixture was warmed to 70 oC and was stirred overnight. After removing the solvent under reduced pressure, the residue was mixed with water (50mL) and extracted with dichloromethane (50mL) 3 times. The organic layer was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by normal phase silica column chromatography using methanol: dichloromethane (5:95) to yield the pure

1 colorless oil (209.5 mg, 75%). H NMR (400 MHz, CDCl3) δ (ppm): 1.13 (t, 3H, J =

8.08Hz), 1.24 (t, 3H, J = 8.08Hz), 2.193 (s, 3H), 2.461 (m, 1H), 2.992 (m, 1H), 3.461 (m,

2H), 3.777-4.064 (m, 4H), 4.871 (s, 3H), 7.096-7.866 (m, 10H). 13C NMR (100 MHz,

CDCl3) δ (ppm): 16.295, 20.485, 26.770, 40.752, 42.141, 62.233, 62.301, 62.696, 62.764,

79 126.304, 126.562, 126.714, 126.761, 128.035, 128.566, 128.596, 128.619, 128.680,

130.403, 130.426, 131.025, 131.094, 133.060, 133.363, 135.823, 135.898.

SRW-82 (Intermediate 7e): Diethyl (3-{N-[(4-bromophenyl) methoxy] acetamido}-1-

(3,4-dichlorophenyl) propyl) phosphonate

O O O O O O Br P P NaH, THF, 4-bromobenzyl bromide OH O N N

Cl O Cl O Cl Cl 6 7e

Diethyl [1-(3,4-dichlorophenyl)-3-(N-hydroxyacetamido) propyl] phosphonate (6)

(200mg, 0.5mmol) was dissolved in 50mL of anhydrous tetrahydrofuran at room temperature, and the solution was cooled to 0 oC. Sodium hydride (12.1mg, 0.5mmol) and 4-bromobenzyl bromide (126mg, 0.5mmol) were added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. The tetrahydrofuran was removed under reduced pressure, and the residue was mixed with water (50mL) and extracted with 100mL of dichloromethane 3 times. The organic layer was dried with sodium sulfate and filtered. After the dichloromethane was removed under reduced pressure, the crude product was purified by silica column chromatography using 98:2 dichloromethane: methanol to afford a yellow oil as product (204.8mg, 71.7%). 1H NMR

(400 MHz, CDCl3) δ (ppm): 1.10-1.32 (m, 6H), 2.01 (s, 3H), 2.16 (m, 1H), 2.41 (m, 1H),

2.99 (m, 1H), 3.49 (m, 2H), 3.75-4.10 (m, 4H), 4.65 (t, 2H), 7.06-7.56 (m, 7H).

80 SRW-83 (7f): Diethyl (3-{N-[(4-bromophenyl)methoxy]acetamido}-1-(3,4- dichlorophenyl) propyl) phosphonate

O O O Br O P Diethyl ether, EtOH, O O P O 4-isopropyyl phenyl bronic acid, N Na CO , Pd(PPh ) O 2 3 3 4 N Cl O Cl O Cl Cl 7e 7f Diethyl (3-{N-[(4-bromophenyl) methoxy] acetamido}-1-(3,4-dichlorophenyl) propyl) phosphonate (7e) (198.5mg, 0.35mmol) was dissolved in diethyl ether (8mL). Pd(PPh3)4

(40.2mg, 0.035mmol) was added to the solution and stirred at room temperature. 4- isopropylphenyl boronic acid (287mg, 1.8mmol) in ethanol (3mL) was added. 0.5mL 2M

o Na2CO3 was then added. The reaction mixture was stirred at 70 C while it was refluxed.

The Reaction reached completion overnight. The mixture was filtered with a membrane filter then the diethyl ether was removed under reduced pressure. The residue was mixed with water (50mL) and extracted with dichloromethane (100mL) 3 times. The organic layer was dried over sodium sulfate and filtered. Dichloromethane was removed under reduced pressure and the crude product was purified by normal phase silica column using

97:3 dichloromethane: methane and a yellow oil was obtained as the product (199.2mg,

1 91%). H NMR (400 MHz, CDCl3) δ (ppm): 1.147-1.285 (m, 12H), 1.581 (t, 2H), 2.008

(s, 3H), 2.453 (m, 1H), 2.978 (m, 2H), 3.483 (m, 2H), 3.795-4.104 (m, 3H), 4.790 (t, 2H),

13 7.093-7.738 (m, 11H), C NMR (101 MHz, CDCl3) δ (ppm): 16. 340, 23.802, 23.961,

33.806, 59.918, 113.863, 126.957, 127.033, 127.253, 128.445, 128.566, 129.606,

131.056, 132.035, 132.134, 133.796.

81 SRW-80 (7g): Diethyl [1-(3,4-dichlorophenyl)-3-[N-(4-phenylbutoxy) acetamido] propyl] phosphonate

O O O O P O O NaH, THF, phenylbutyl bromide P OH O N N

Cl O Cl O Cl Cl 6 7g Diethyl [1-(3,4-dichlorophenyl)-3-(N-hydroxyacetamido) propyl] phosphonate (6)

(200mg, 0.5mmol) was dissolved in anhydrous tetrahydrofuran (25mL) under a nitrogen atmosphere. Sodium hydride (12.1mg, 0.5mmol) and 4-phenylbutyl bromide (107.4mg,

0.5mmol) was added to the solution at 0 oC. The mixture was warmed to 70 oC and was stirred overnight. After removing the solvent under reduced pressure, the residue was mixed with water (50mL) and then extracted with dichloromethane (50mL) 3 times. The organic layer was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by normal phase silica column chromatography using methanol: dichloromethane (5:95) to yield the pure colorless oil

1 (80mg, 33.6%). H NMR (400 MHz, CDCl3) δ (ppm): 1.134-1.254 (m, 6H), 1.289-1.556

(7H), 2.042 (s, 3H), 2.177 (m, 1H), 2.432 (1H), 2.914 (m, 2H), 3.687 (m, 3H), 3.836 (m,

13 1H), 3.990 (m, 1H), 7.155-7.512 (m, 8H). C NMR (100 MHz, CDCl3) δ (ppm): 16.310,

16.356, 26.800, 27.643, 27.863, 35.628, 62.301, 62.772, 74.028, 125.970, 128.369,

128.422, 128.589, 128.657, 130.388, 131.078, 131.139.

82 SRW-61 (8a): Sodium Hydrogen-[1-(3,4-dichlorophenyl)-3-[N-(1-phenylethoxy) acetamido]propyl phosphonate

O O O O NaO OH P P O O N TMSBr, DCM, 0 oC to RT N

Cl O Cl O Cl Cl

7a 8a Diethyl [1-(3,4-dichlorophenyl)-3-[N-(1phenylethoxy) acetamido] propyl] phosphonate

(7a) (136.3mg, 0.27mmol) was dissolved in anhydrous dichloromethane (20mL) and cooled down to 0 oC. Trimethylsilyl bromide (415mg, 2.7mmol) was added to the mixture dropwise and the mixture was warmed to room temperature and stirred overnight. Trimethylsilyl bromide and dichloromethane were removed under reduced pressure after the reaction reached completion. An aqueous solution of sodium hydroxide

(11mg, 0.27mmol) was added to the reaction intermediate and stirred for 1 hour. The

1 product was obtained after lyophilization (127mg, 100%). H NMR (400 MHz, CD3OD)

δ (ppm): 1.51 (d, 3H), 2.03 (s, 3H), 2.03-2.15 (m, 2H), 2.80-3.01 (m, 2H), 3.45 (m, 1H),

13 3.58 (m, 1H), 7.150-7.507 (m, 8H), C NMR (100 MHz, CD3OD) δ (ppm): 18.937,

27.082, 127.124, 127.162, 128.172, 128.232, 128.377, 128.468, 129.819, 130.843,

131.056.

SRW-65 (8b): Sodium Hydrogen-{3-[N-({[1,1'-biphenyl]-4-yl} methoxy) acetamido]-

1-(3,4-dichlorophenyl)}propylphosphonate

83 O O O O NaO OH P P o O TMSBr, DCM, 0 C to RT O N N

Cl O Cl O Cl Cl

7b 8b Diethyl {3-[N-({[1,1'-biphenyl]-4-yl} methoxy) acetamido]-1-(3,4-dichlorophenyl) propyl} phosphonate (7b) (166.8mg, 0.3mmol) was dissolved in anhydrous dichloromethane (20mL) and cooled down to 0 oC. Trimethylsilyl bromide was added to the mixture dropwise (453mg, 3 mmol) and the mixture was warmed to room temperature and stirred overnight. Trimethylsilyl bromide and dichloromethane were removed under reduced pressure after the reaction reached completion. An aqueous solution of sodium hydroxide (12mg, 0.3mmol) was added to the reaction intermediate and stirred for 1 hour.

The product was obtained after lyophilization (133.7mg, 100%). 1H NMR (400 MHz,

CD3OD) δ (ppm): 1.99 (m, 3H), 2.04 (m, 1H), 2.51 (m, 1H), 2.875 (q, 2H), 3.439 (m,

13 3H), 7.226-7.665 (m, 12H), C NMR (100 MHz, CD3OD) δ (ppm): 18.960, 75.509,

126.608, 126.729, 127.177, 128.483, 129.045, 129.485, 129.720, 131.003, 140.377.

SRW-67 (8c): Sodium Hydrogen-[1-(3,4-Dichlorophenyl)-3-(N-{[4-(propan-2-yl) phenyl] methoxy} acetamido) propyl] propyl phosphonate

O O O O NaO OH P P O TMSBr, DCM, 0 oC to RT O N N

Cl O Cl O Cl Cl 7c 8c

84 Diethyl [1-(3,4-dichlorophenyl)-3-(N-{[4-(propan-2-yl) phenyl] methoxy} acetamido) propyl] phosphonate (7b) (156.3mg, 0.3mmol) was dissolved in anhydrous dichloromethane (20mL) and cooled down to 0 oC. Trimethylsilyl bromide was added to the mixture dropwise (453mg, 3 mmol) and the mixture was warmed to room temperature and stirred overnight. Trimethylsilyl bromide and dichloromethane were removed under reduced pressure after the reaction reached completion. An aqueous solution of sodium hydroxide (12mg, 0.3mmol) was added to the reaction intermediate and stirred for 1 hour.

The product was obtained after lyophilization (125.9mg, 100%). 1H NMR (400 MHz,

CD3OD) δ (ppm): 1.23 (m, 6H), 1.957 (t, 3H), 2.01-2.49 (m, 2H), 2.90 (m, 2H), 3.487

13 (m, 3H), 7.176-7.467 (m, 7H), C NMR (100 MHz, CD3OD) δ (ppm): 14.003, 22.899,

33.784, 65.474, 126.236, 129.401.

SRW-85 (8d): Sodium Hydrogen-[1-(3,4-Dichlorophenyl)-3-{N-[(naphthalen-2-yl) methoxy]acetamido}propyl]propylphosphonate

O O NaO OH O O P P o O O TMSBr, DCM, 0 C to RT N N Cl O Cl O Cl Cl 8d 7d

Diethyl [1-(3,4-dichlorophenyl)-3-{N-[(naphthalen-2-yl) methoxy] acetamido} propyl] phosphonate (7d) (160mg, 0.3mmol) was dissolved in anhydrous dichloromethane

(20mL) and cooled down to 0 oC. Trimethylsilyl bromide was added to the mixture dropwise (457mg, 3mmol) and the mixture was warmed to room temperature and stirred

85 overnight. Trimethylsilyl bromide and dichloromethane were removed under reduced pressure after the reaction reached completion. An aqueous solution of sodium hydroxide

(12mg, 0.3mmol) was added to the reaction intermediate and stirred for 1 hour. The

1 product was obtained after lyophilization (150mg, 100%). H NMR (400 MHz, CD3OD)

δ (ppm): 1.96 (m, 3H), 2.02-2.49 (m, 2H), 2.80-3.20 (m, 2H), 3.510-3.582 (m, 3H),

13 7.154-7.934 (m, 10H), C NMR (100 MHz, CD3OD) δ (ppm): 29.541, 60.988, 126.069,

126.282, 126.395, 126.623, 127.329, 127.397, 127.716, 127.785, 128.027, 128.513,

129.955, 131.739, 133.189, 133.447.

SRW-86 (8f): Sodium Hydrogen-[1-(3,4-Dichlorophenyl)-3-(N-{[4'-(propan-2-yl)-

[1,1'-biphenyl]-4-yl]methoxy}acetamido)propyl]propylphosphonate

O O O O NaO OH P P o O TMSBr, DCM, 0 C to RT O N N

Cl O Cl O Cl Cl 7f 8f Diethyl (3-{N-[(4-bromophenyl) methoxy] acetamido}-1-(3,4-dichlorophenyl) propyl) phosphonate (7f) (173mg, 0.29mmol) was dissolved in anhydrous dichloromethane

(20mL) and cooled down to 0 oC. Trimethylsilyl bromide was added to the mixture dropwise (438mg, 2.9mmol, 10 equiv) and the mixture was warmed to room temperature and stirred overnight. Trimethylsilyl bromide and dichloromethane were removed under reduced pressure after the reaction reached completion. An aqueous solution of sodium hydroxide (11.4mg, 0.29mmol, 1 equiv) was added to the reaction intermediate and stirred for 1 hour. The product was obtained after lyophilization (148mg, 90%). 1H NMR

86 (400 MHz, CD3OD) δ (ppm): 1.265 (m, 6H), 1.997 (s, 3H), 2.01-2.49 (m, 2H), 2.934 (m,

13 2H), 3.315 (m, 4H), 7.273-7.655 (m, 11H), C NMR (100 MHz, CD3OD) δ (ppm):

22.975, 33.663, 126.350, 126.433, 126.517, 126.540, 128.483, 128.612, 129.720,

131.603, 131.701, 132.362.

SRW-84 (8g): Sodium Hydrogen-[1-(3,4-Dichlorophenyl)-3-[N-(4-phenylbutoxy) acetamido] propyl] propyl phosphonate

O O O O NaO OH P P o O TMSBr, DCM, 0 C to RT O N N

Cl O Cl O Cl Cl 8g 7g

Diethyl [1-(3,4-dichlorophenyl)-3-[N-(4-phenylbutoxy) acetamido] propyl] phosphonate

(7g) (80mg, 0.15mmol) was dissolved in anhydrous dichloromethane (20mL) and cooled down to 0 oC. Trimethylsilyl bromide was added to the mixture dropwise (231mg, 1.5 mmol) and the mixture was warmed to room temperature and stirred overnight.

Trimethylsilyl bromide and dichloromethane were removed under reduced pressure after the reaction reached completion. An aqueous solution of sodium hydroxide (6mg,

0.15mmol) was added to the reaction intermediate and stirred for 1 hour. The product was

1 obtained after lyophilization (71mg, 100%). H NMR (400 MHz, CD3OD) δ (ppm):

1.173-1.312 (m, 4H), 2.094 (m, 3H), 2.646 (m, 2H), 3.01-3.07 (m, 2H), 3.556 (m, 2H),

13 3.615-3.836 (m, 2H), 7.163-7.553 (m, 8H), C NMR (100 MHz, CD3OD) δ (ppm):

87 27.211, 27.659, 35.158, 62.985, 73.566, 125.454, 127.967, 128.058, 128.984, 129.819,

130.988, 131.056, 141.857.

SRW-68 (9a): ({[1-(3,4-dichlorophenyl)-3-[N-(1-phenylethoxy) acetamido] propyl]({[(2,2-dimethylpropanoyl) oxy] methoxy}) phosphoryl} oxy) methyl 2,2- dimethylpropanoate (9a)

O O NaO OH POMO OPOM P P DMF, Et N, POMCl, 60oC O 3 O N N

Cl O Cl O Cl Cl 8a 9a

Sodium hydrogen-[1-(3,4-dichlorophenyl)-3-[N-(1-phenylethoxy) acetamido] propyl phosphonate (8a) (113mg, 0.24mmol) was dissolved in anhydrous dimethylformamide

(DMF) (20mL). Triethyl amine (147mg, 1.45mmol) and pivaloyloxymethyl chloride

(POMCl) (218mg, 1.45mmol) were added to the solution at room temperature. The reaction mixture was heated to 60 oC and was stirred overnight. The reaction mixture was extracted with 50mL of water and 50mL of diethyl ether 3 times. After removing diethyl ether from the organic layer under reduced pressure, the crude compound was extracted with 50mL of hexanes and 50mL of water 3 times. The organic layer was dried over sodium sulfate and filtered. The hexanes was removed under reduced pressure and the residue was purified by normal phase silica column chromatography with 60:40 hexanes: ethyl acetate. A colorless oil was obtained as the product (28.1mg, 17.3%). 1H NMR (400

MHz, CDCl3) δ (ppm): 1.159-1.205 (m, 21H), 1.513 (m, 3H), 1.893 (t, 3H), 2.996 (m,

1H), 4.675 (1H), 5.288-5.632 (m, 5H), 7.037-7.374 (m, 8H), 13C NMR (100 MHz,

88 CDCl3) δ (ppm): 20.196, 26.747, 26.808, 26.853, 29.677, 38.641, 81.717, 127.162,

127.207, 128.581, 128.657, 128.794, 128.877, 130.509, 130.980.

SRW-77 (9b): [({3-[N-({[1,1'-biphenyl]-4-yl} methoxy) acetamido]-1-(3,4- dichlorophenyl) propyl}({[(2,2-dimethylpropanoyl)oxy] methoxy}) phosphoryl)oxy] methyl-2,2-dimethylpropanoate

O O NaO OH POMO OPOM P P o O DMF, Et3N, POMCl, 60 C O N N

Cl O Cl O Cl Cl

8b 9b

Sodium hydrogen-{3-[N-({[1,1'-biphenyl]-4-yl} methoxy) acetamido]-1-(3,4- dichlorophenyl)} propyl phosphonate (8b) (111mg, 0.21mmol) was dissolved in anhydrous dimethylformamide (DMF) (20mL). Triethyl amine (127mg, 1.25mmol) and pivaloyloxymethyl chloride (POMCl) (190mg, 1.27mmol) were added to the solution at room temperature. The reaction mixture was heated to 60 oC and was stirred overnight.

The reaction mixture was extracted with 50mL of water and 50mL of diethyl ether 3 times. After removing diethyl ether from the organic layer under reduced pressure, the crude compound was extracted with 50mL of hexanes and 50mL of water 3 times. The organic layer was dried over sodium sulfate and filtered. The hexanes was removed under reduced pressure and the residue was purified by normal phase silica column chromatography with 60:40 hexanes: ethyl acetate. A colorless oil was obtained as the

89 1 product (38.5mg, 25%). H NMR (400 MHz, CDCl3) δ (ppm): 1.158 (s, 9H), 1.192 (s,

9H), 2.034 (s, 3H), 2.327 (m, 1H), 3.167 (m, 1H), 3.443 (m, 2H), 4.739 (2H), 5.408 (m,

13 4H), 7.215-7.530 (m, 12H), C NMR (100 MHz, CDCl3) δ (ppm): 26.739, 26.739,

38.641, 38.687, 81.695, 81.763, 127.124, 127.443, 127.655, 128.695, 128.854, 129.667,

130.580, 131.094.

SRW-69 (9c): ({[1-(3,4-Dichlorophenyl)-3-(N-{[4-(propan-2-yl) phenyl] methoxy} acetamido)propyl]({[(2,2-dimethylpropanoyl)oxy]methoxy})phosphoryl}oxy) methyl

2,2-dimethylpropanoate

O O NaO OH POMO OPOM P P o O DMF, Et3N, POMCl, 60 C O N N

Cl O Cl O Cl Cl 8c 9c

Sodium hydrogen-[1-(3,4-Dichlorophenyl)-3-(N-{[4-(propan-2-yl) phenyl] methoxy} acetamido) propyl] propyl phosphonate (8c) (138mg, 0.28mmol) was dissolved in anhydrous dimethylformamide (DMF) (20mL). Triethyl amine (169mg, 1.67mmol) and pivaloyloxymethyl chloride (POMCl) (252mg, 1.67mmol) were added to the solution at room temperature. The reaction mixture was heated to 60 oC and was stirred overnight.

The reaction mixture was extracted with 50mL of water and 50mL of diethyl ether 3 times. After removing diethyl ether from the organic layer under reduced pressure, the crude compound was extracted with 50mL of hexanes and 50mL of water 3 times. The organic layer was dried over sodium sulfate and filtered. The hexanes was removed under reduced pressure and the residue was purified by normal phase silica column

90 chromatography with 60:40 hexanes: ethyl acetate. A colorless oil was obtained as the

1 product (62mg, 31.7%). H NMR (400 MHz, CDCl3) δ (ppm): 1.18 (d, 18H, J =

14.08Hz), 1.25 (d, 6H, J = 6.96Hz), 1.996 (s, 3H), 2.382 (1H), 2.863 (m, 1H), 3.091 (m,

1H), 3.445 (m, 2H), 4.682 (s, 2H), 5.497-5.656 (m, 4H), 7.116-7.372 (m, 7H), 13C NMR

(100 MHz, CDCl3) δ (ppm): 20.310, 23.916, 26.739, 33.912, 40.827, 42.216, 81.687,

81.748, 81.809, 126.797, 128.695, 129.355, 130.555, 130.585, 131.094, 131.162,

131.367, 134.411, 134.487, 150.009.

SRW-89 (9d): ({[1-(3,4-Dichlorophenyl)-3-{N-[(naphthalen-2-yl) methoxy] acetamido} propyl]({[(2,2-dimethylpropanoyl)oxy] methoxy})phosphoryl} oxy) methyl-2,2-dimethylpropanoate

O O NaO OH POMO OPOM P P DMF, Et N, POMCl, 60oC O 3 O N N

Cl O Cl O Cl Cl 8d 9d

Sodium hydrogen-[1-(3,4-Dichlorophenyl)-3-{N-[(naphthalen-2-yl)methoxy] acetamido} propyl] propyl phosphonate (8d) (155mg, 0.308mmol) was dissolved in anhydrous dimethylformamide (DMF) (20mL). Triethyl amine (187mg, 1.85mmol) and pivaloyloxymethyl chloride (POMCl) (278mg, 1.85mmol) were added to the solution at room temperature. The reaction mixture was heated to 60 oC and was stirred overnight.

The reaction mixture was extracted with 50mL of water and 50mL of diethyl ether 3

91 times. After removing diethyl ether from the organic layer under reduced pressure, the crude compound was extracted with 50mL of hexanes and 50mL of water 3 times. The organic layer was dried over sodium sulfate and filtered. The hexanes was removed under reduced pressure and the residue was purified by normal phase silica column chromatography with 60:40 hexanes: ethyl acetate. A colorless oil was obtained as the

1 product (36.7mg, 16.8%). H NMR (400 MHz, CDCl3) δ (ppm): 1.079-1.298 (m, 18H),

1.638 (s, 1H), 2.041 (t, 3H), 2.417 (m, 1H), 3. 083 (m, 1H), 3.506 (m, 2H), 4.846 (s, 2H),

13 5.447-5.628 (m, 4H), 7.071-7.849 (m, 10H), C NMR (100 MHz, CDCl3) δ (ppm):

26.246, 26.732, 26.777, 40.835, 81.687, 81.809, 126.555, 126.714, 127.761, 128.619,

128.657, 128.725.

SRW-90 (9f): ({[1-(3,4-Dichlorophenyl)-3-(N-{[4'-(propan-2-yl)-[1,1'-biphenyl]-4-yl] methoxy}acetamido)propyl]({[(2,2-dimethylpropanoyl)oxy]methoxy})phosphoryl} oxy)methyl 2,2-dimethylpropanoate

O O NaO OH POMO OPOM P P DMF, Et N, POMCl, 60oC O 3 O N N

Cl O Cl O Cl Cl 8f 9f

Sodium hydrogen-[1-(3,4-Dichlorophenyl)-3-(N-{[4'-(propan-2-yl)-[1,1'-biphenyl]-4- yl]methoxy}acetamido)propyl]propylphosphonate (8f) (103mg, 0.18mmol) was dissolved in anhydrous dimethylformamide (DMF) (20mL). Triethyl amine (109mg,

1.08mmol) and pivaloyloxymethyl chloride (POMCl) (162mg, 1.08mmol) were added to

92 the solution at room temperature. The reaction mixture was heated to 60 oC and was stirred overnight. The reaction mixture was extracted with 50mL of water and 50mL of diethyl ether 3 times. After removing diethyl ether from the organic layer under reduced pressure, the crude compound was extracted with 50mL of hexanes and 50mL of water 3 times. The organic layer was dried over sodium sulfate and filtered. The hexanes was removed under reduced pressure and the residue was purified by normal phase silica column chromatography with 60:40 hexanes: ethyl acetate. A colorless oil was obtained

1 as the product (36.5mg, 26%). H NMR (400 MHz, CDCl3) δ (ppm): 1.156-1.300 (m,

24H), 1.669 (s, 1H), 2.028 (s, 3H), 2.248 (m, 1H), 2.426 (m, 1H), 3.162 (m, 1H), 3.494

(m, 2H), 4.726 (s, 2H), 5.470-5.638 (m, 4H), 7.111-7.591 (m, 11H), 13C NMR (100 MHz,

CDCl3) δ (ppm): 23.969, 26.739, 26.793, 29.685, 33.806, 38.687, 81.755, 126.957,

127.033, 127.260, 129.644, 130.600.

SRW-91 (9g): ({[1-(3,4-Dichlorophenyl)-3-[N-(4-phenylbutoxy) acetamido] propyl]({[(2,2-dimethylpropanoyl) oxy] methoxy}) phosphoryl}oxy) methyl 2,2- dimethylpropanoate

O O NaO OH POMO OPOM P P O DMF, Et N, POMCl, 60oC O N 3 N

Cl O Cl O Cl Cl 8g 9g

Sodium hydrogen-[1-(3,4-Dichlorophenyl)-3-[N-(4-phenylbutoxy) acetamido] propyl] propyl phosphonate (8f) (152mg, 0.31mmol) was dissolved in anhydrous

93 dimethylformamide (DMF) (20mL). Triethyl amine (185mg, 1.83mmol) and pivaloyloxymethyl chloride (POMCl) (276mg, 1.83mmol) were added to the solution at room temperature. The reaction mixture was heated to 60 oC and was stirred overnight.

The reaction mixture was extracted with 50mL of water and 50mL of diethyl ether 3 times. After removing diethyl ether from the organic layer under reduced pressure, the crude compound was extracted with 50mL of hexanes and 50mL of water 3 times. The organic layer was dried over sodium sulfate and filtered. The hexanes was removed under reduced pressure and the residue was purified by normal phase silica column chromatography with 60:40 hexanes: ethyl acetate. A colorless oil was obtained as the

1 product (26.9mg, 12.5%). H NMR (400 MHz, CDCl3) δ (ppm): 1.155-1.273 (m, 18H),

1.609-1.789 (m, 4H), 2.049 (s, 3H), 2.232 (m, 1H), 2.431 (m, 1H), 2.699 (m, 2H), 3.235

(m, 1H), 3.550 (m, 2H), 3.773 (m, 2H), 5.520-5.704 (m, 4H), 7.196-7.445 (m, 8H), 13C

NMR (100 MHz, CDCl3) δ (ppm): 26.276, 26.755, 26.808, 27.620, 27.825, 38.702,

42.247, 74.119, 81.687, 81.748, 81.816, 125.970, 128.376, 128.422, 128.642, 128.710,

130.570, 131.132, 131.200, 134.396.

94 References:

1. Jackson, E. R.; San Jose, G.; Brothers, R. C.; Edelstein, E. K.; Sheldon, Z.; Haymond,

A.; Johny, C.; Boshoff, H.I.; Couch, R.D.; Dowd, C. S. The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs. Bioorg Med Chem Lett. 2014, 24(2), 649-653.

2. San Jose, G.; Jackson, E. R.; Haymond, A.; Johny, C.; Edwards, R. L.; Wang, X.;

Brothers, R.C.; Edelstein, E.K.; Odom, A.R.; Boshoff, H.I.; Couch, R.D.; Dowd, C. S.

Structure-Activity Relationships of the MEPicides: N-Acyl and O-Linked Analogs of

FR900098 as Inhibitors of Dxr from Mycobacterium tuberculosis and Yersinia pestis.

ACS Infect Dis. 2016, 2(12), 923-935.

3. Haemers, T.; Wiesner, J.; Poecke, S.V.; Goeman, J.; Henschker, D.; Beck, E.; Jomaa,

H.; Calenbergh, S.V. Synthesis of alpha-substituted fosmidomycin analogues as highly potent Plasmodium falciparum growth inhibitors. Bioorg. Med. Chem. Lett. 2006, 16,

1888–91.

4.Jansson, A. M.; Wieckowska, A.; Bjorkelid, C.; Yahiaoui, S.; Sooriyaarachchi, S.;

Lindh, M.; Mowbray, S. L. DXR inhibition by potent mono- and disubstituted fosmidomycin analogues. J. Med. Chem. 2013, 56(15), 6190-6199.

5. Andaloussi, M.; Henriksson, L. M.; Wieckowska, A.; Lindh, M.; Bjorkelid, C.;

Larsson, A. M.; Karlen, A. Design, synthesis, and X-ray crystallographic studies of alpha-aryl substituted fosmidomycin analogues as inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase. J. Med. Chem. 2011,

54(14), 4964-4976.

95 6. Yeh, E.; DeRisi, J. L. Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum. PLoS. Biol. 2011,

9(8), e1001138.

96 Chapter 5: Summary and Future Efforts

Synthesis of α–3,4–Dichlorophenyl FR900098 Analogs:

Eighteen SRW analogs were successfully synthesized and characterized. The variations were introduced using the Williamson Ether Synthesis. This route led to three sets of compounds (diethyl esters, monosodium salts and prodrugs with POM moieties) with six different O-linked substituents.

The synthesis of these α-aryl substituted analogs was successful, with only three obstacles listed as the following:

1. Deprotection of acetal SRW-72 was a low yielding reaction, which sometimes could not reach completion even after 3 days of heating at 75oC. This was resolved by decreasing the volume of acetone used for the reaction.

2. Ester cleavage using TMSBr was an issue throughout the synthesis of all six monosodium salts. Twenty equivalents of TMSBr were normally used for this reaction.

In order to obtain the pure monosodium salts, an ethyl acetate wash was required after lyophilization. This approach was taken due to the fact that the monosodium salts with one ethyl group left intact on the phosphonate are highly soluble in ethyl acetate.

3. The most difficult purification steps were the reaction with POMCl in DMF. The aryl ether prodrugs are very lipophilic; therefore, they are very soluble in hexanes, which is immiscible with DMF. We can take advantage of this important feature through extraction of the reaction mixture first with diethyl ether and water, and then followed by hexanes. Almost all of the DMF can be removed before silica column chromatography.

97 Biological Analysis of SRW Compounds:

P. falciparum:

The SRW compounds have micromolar activity against Pf whole cells ranging from 0.92 to 35.47µM (except SRW-84 which is inactive against Pf whole cells). Only

SRW-61 and SRW-86 exhibit comparable Pf activity to the parent structure. Pf Dxr activities of these two molecules confirmed their on-target effects. Further IPP rescue studies on SRW-61 confirmed that the compound actually inhibits the MEP pathway.

Therefore, more studies on its metabolic stability in human and mouse plasma are being conducted currently.

M. tuberculosis:

The SRW compounds are active against Mtb whole cells, however, none of the monosodium salts showed potent enzyme inhibition of Mtb Dxr. As shown in Table 5.1,

Mtb Dxr has over 20% residual activity after being treated with all six SRW monosodium salts. Therefore, we believe that the SRW compounds with antimycobacterial activity must be acting through non-MEP pathway targets.

98 Future Projects:

Plasmodium falciparum (Pf):

For future Plasmodium falciparum projects, if we were to continue with the synthesis of alpha-aryl substituted ether compounds, the following aspects need to be considered:

1. Improving inhibitor activity:

Even though SRW-61 exhibits potent Pf whole cell and enzyme activities, it is still helpful to improve the Dxr binding affinity. One of the main problems affecting the binding affinity is that the 3,4-dichlorophenyl ring may interfere with amino acid residue side chains in Pf Dxr. If we were to maintain the 3,4-di-halophenyl moiety, we could use fluorine atoms in the place of the chlorine atoms. The fluorine substituents would be smaller than the current chlorine substituents, which may minimize steric interference with the enzyme. However, this minor change may also affect the π-π stacking ability of the α-substituent negatively, due to the stronger electron-withdrawing ability of fluorine atoms.

99 Table 5.1: SRW Compounds and Mtb Dxr Inhibition

R O O HO P N NaO O

Cl Cl

Compound R % Residual Mtb Dxr Activity

SRW-61 23

SRW-65 90

SRW-67 90

SRW-84 95

SRW-85 75

SRW-86 70

100 Table 5.2: clogP of SRW Compounds

R O O HO P N NaO O

Cl Cl

Compound R clogP

SRW-61 4.24

SRW-65 5.51

SRW-67 5.02

SRW-84 4.87

SRW-85 4.83

SRW-86 6.70

101 2. Increasing the water solubility of the inhibitors:

The monosodium salts in the SRW series have very poor solubility in water. This feature may be due to the high lipophilicity of these compounds (clogP values in Table

5.2). While these compounds may have longer half-lives in animals (to be determined), the low solubility may limit the distribution of the inhibitors. Addition of one or two hydrophilic functional groups to either the propyl linker or the aryl ether substituent may help to resolve this issue.

3. Creating a list of ether substituents from which we may obtain structure-activity relationships:

SRW-61 is the most active SRW compound so far. Additional modifications can be made on the phenyl ring of the ether substituent. We are currently making these changes under the guidance of Topliss Decision Tree.1 We plan to follow the first two steps of the decision tree by adding electron-withdrawing and electron-donating groups to the 4-position of the phenyl ring. We will conduct biological studies on these new molecules to examine their effects on Pf whole cell growth and Dxr activity.

Biological Analysis of SRW-61:

As mentioned above, SRW-61 was the most active compound from this study. A co-crystal structure of SRW-61 bound to Pf Dxr structure would help us understand how the 3,4-dichlorophenyl group fits in the DXP pocket while also accommodating the ether substituent. Also, because the α-position on SRW-61 is a chiral center, we may also learn more about which stereoisomer would be favored in the DXP pocket. In our hypothesis,

102 we predicted the O-linked aryl group would extend to the NADPH pocket. We can also investigate what interactions are responsible for the inhibitor binding in the Dxr NADPH pocket. We are pursuing crystal structures in collaboration with Dr. Sherry Mowbray’s group at Uppsala University in Sweden.

Mycobacterium tuberculosis (Mtb) Project:

Although twelve of the SRW compounds have activity against Mtb whole cells, the Mtb Dxr enzyme data shows that these inhibitors most likely have off-target effects instead of acting through Dxr. SRW-61 exhibits the most potent enzyme inhibition against Mtb Dxr. With small groups like fluoro- or chloro- on the 4-position, certain

SRW-61 analogs may retain activity against the enzyme, as these groups are not likely to disrupt binding to Mtb Dxr. This prediction comes from the fact that, in addition to SRW-

61, all of the other five SRW monosodium salts tested bear relatively large O-linked aryl substituents. Therefore, if the bisubstrate binding mechanism is occurring for SRW-61, it is reasonable to assume that smaller O-linked aryl substituents could facilitate the binding of inhibitors to Mtb Dxr.

103 Conclusions:

SRW-61 is the most promising compound in this series for further modifications.

With a Pf IC50 value of 0.92 µM, SRW-61 is less active than its lead compound

FR900098. In order to increase the Plasmodium falciparum activity of SRW-61, changes can be made on the ortho, meta, and para position of the 2-ethyl phenyl substituent.

Structure-activity relationship studies can also be done on SRW-61 analogs to further investigate the effects caused by different functional groups on the 2-ethyl phenyl substituent. The prodrug moiety can be maintained for these analogs as the POM group is likely to improve inhibitor activities in general.

Taken together, our data suggest that compounds bearing both the alpha-phenyl and O-linked substituents display remarkable P. falciparum activity. This activity can likely be improved upon with additional SAR exploration. These compounds will give us insight into the binding requirements for Pf Dxr, as well as lead compounds to make the next generation of antimalarial agents.

104 References:

1. Topliss, J. G. Utilization of Operational Schemes for analogue synthesis in drug design. J. Med. Chem. 1972, 15, 1006-1011.

105 Appendix

Experimental Information for Bioassays:

The Plasmodium falciparum growth inhibition and isopentenyl pyrophosphate rescue studies were carried out as previously described.1 Assays were carried out by Dr. Rachel

Edwards in the Odom lab at Washington University. Briefly, asynchronous P. falciparum cultures were diluted to 1% parasitemia and treated with inhibitors at concentrations ranging from 0.25ng/mL–100 µg/mL. Growth inhibition assays were conducted in opaque 96-well plates at 100µL volume per culture. Parasite growth was quantified by measuring DNA content using PicoGreen (Life Technologies) after 3 days. Fluorescence was measured on a FLUOstar Omega microplate reader (BMG Labtech) at 485nm excitation and 528nm emission. Half maximal inhibitory concentration (IC50) values were calculated by nonlinear regression analysis using GraphPad Prism sofware. For isopentenyl pyrophosphate (IPP) (Echelon) rescue experiments, 250µM IPP was added to the appropriate wells for the duration of the experiment.1

Mycobacterium tuberculosis growth inhibition assay:

Evaluation of compounds against intact M. tuberculosis was performed as described previously2 by Dr. Helena Boshoff at NIH. Briefly, Mtb H37Rv ATCC27294 was grown in Middlebrook 7H9 broth supplemented with 0.2% glycerol, 0.4% glucose, 0.5% BSA fraction V, 0.08% NaCl and 0.5% Tween or in GAST/Fe medium (PMID 10655517) to an OD650 of 0.2. Cells were then diluted to 100,000 cells/mL in their respective medium.

An equal volume (50 uL/well) of cell dilution was added to clear polystyrene round-

106 bottom 96-well plates containing 50 uL/well of the respective medium and drug as a 2- fold 12-point dilution series in duplicate. Plates were incubated at 37°C for 1 and 2 weeks after which growth was visualized using an enlarging mirror. The minimum inhibitory concentration (MIC99) is scored as the lowest concentration that completely inhibits all visible growth, taken as 99%.2

Dxr inhibition Assay:

Dxr inhibition assays were previously conducted at George Mason University at professor Robin Couch’s lab.2 Dxr activity was assayed at 37 °C by spectrophotometrically monitoring the enzyme-catalyzed oxidation of NADPH. To determine percent inhibition for each inhibitor, 120 µL of assay solutions were used, containing 100 mM Tris, pH 7.8, 25 mM MgCl2, 150 µM NADPH, 0.89 µM Dxr, 100

µM inhibitor, and the appropriate KM value of DXP (Echelon Biosciences, Salt Lake

City, Utah), which is 47 µM for Mtb Dxr. The assays were performed by preincubating the mixture of Tris, MgCl2, Dxr, and inhibitor for 10 min (37 °C) to facilitate binding of the inhibitor, followed by the addition of NADPH, a 5 min incubation (37 °C), and then the addition of DXP. The half maximal inhibition (IC50) values were determined by plotting enzyme fractional activity as a function of inhibitor concentration (1 nM to 100

µM) and using GraphPad Prism (La Jolla, CA) nonlinear curve fitting to a sigmoidal dose−response curve.2

107 Cytotoxicity assays:

HepG2 cells (ATCC HB-8065) were grown in DMEM supplemented with 4mM L- glutamine (Gibco #11966-025) with either 4.5g/L D-glucose or 1.8g/L galactose as carbon source. Cells were trypsinized, resuspended in the respective medium

(DMEM/glutamine/glucose or DMEM/glutamine/galactose) to 4×105 cells/mL and 50 mL/well transferred to flat-bottom white opaque tissue culture plates (Falcon #353296) containing 50 mL/well of the respective medium with test compound. Compound concentrations were two-fold dilutions ranging from 50 mM to 0.049 mM as well as the drug-free DMSO-only control. All concentrations were tested in duplicate for each carbon source. After 24h incubation at 5% CO2, 37◦C, 10 mL/well of Celltiter-Glo reagent (Promega #G9241) was added and luminescence recorded after 20 min incubation in the dark.

108 Annotated NMR Spectra:

Compound 7a: 3 l

PROTON_01 O c D d c SRW58P H 9

9 3 0 1 1 0 280 5 3 8 9 9 9 8 0 8 6 8 1 3 8 8 0 3 6 0 4 7 6 6 0 3 9 9 2 7 8 3 9 0 5 0 0 3 5 8 6 0 1 9 2 7 3 2 7 6 3 7 6 3 4 4 4 0 8 4 7 5 7 6 8 8 7 9 5 3 0 8 0 9 8 2 8 7 9 3 1 1 3 1 9 3 7 6 2 1 9 8 4 3 6 8 3 1 0 4 6 5 7 5 1 6 2 8 8 0 9 7 1 8 6 4 3 2 0 9 7 3 1 5 4 3 0 2 2 1 1 8 8 6 5 4 3 3 3 2 2 1 1 6 1 4 0 2 1 1 0 8 7 7 7 6 6 5 2 2 1 1 1 0 0 8 8 8 8 6 6 6 6 5 5 4 4 6 6 4 4 4 3 2 2 1 1 . 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 3 3 3 3 3 3 3 3 3 3 9 9 5 5 5 5 6 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 ...... 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 4 4 4 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 - 260

240 e O e c O O c P 220 a O g i N 200 a f h Cl O c 180 Cl b b 160

TMS 140 c 120

100

80

60 a e 40 g f i h 20

0 6 4 4 2 6 8 6 5 6 7 3 0 5 1 5 3 7 0 9 0 8 9 8 8 8 4 2 0 7 7 5 0 8 0 6 4 3 7 1 1 ......

...... -20 6 1 1 0 1 0 0 1 1 0 1 0 1 1 1 1 3 3 3 0

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

109 3 3 l l CARBON_01 3 l c c c d d 450 d c SRW58PC2 c

c 4 8 1 5 6 3 2 1 7 3 1 6 5 9 5 4 0 3

7 1 7 1 4 8 5 6 4 5 9 3 6 7 6 4 5 4 3 0 9 4 0 2 3 8 8 3 3 3 8 9 3 5 5 8 6 6 6 7 1 7 3 9 0 3 1 1 1 1 ...... 8 0 2 8 6 2 2 5 7 6 2 3 9 6 7 1 1 4 2 2 3 3 0 3 ...... 9 5 5 5 1 1 1 1 0 0 0 0 0 . 8 8 8 8 8 8 8 7 7 . 5 2 2 0 0 2 2 2 2 2 6 0 0 0 0 7 6 6 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 6 6 6 6 6 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 7 7 7 6 6 6 6 4 4 4 4 4 2 2 2 2 2 1 1 1 1 1 - 400

350

300

250

200

150

100

50

0

-50

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

110 Compound 7b:

190 3 l

PROTON_01 O c D d H c

SRW60P 4 3

180 5 1 9 6 2 9 2 1 8 3 4 3 7 1 3 5 4 9 0 4 5 6 5 3 2 1 9 5 3 8 3 7 0 1 7 2 1 7 2 3 0 7 1 9 0 7 1 0 8 6 4 6 7 1 7 3 9 2 6 9 0 5 7 4 8 4 5 7 5 5 7 1 6 9 2 1 3 9 8 6 8 7 5 1 5 0 3 0 6 1 7 6 3 8 4 2 4 2 0 0 9 7 7 2 4 3 3 5 7 8 7 2 8 0 4 6 0 6 7 1 8 6 5 3 7 2 9 8 6 5 4 4 3 3 2 1 4 2 6 9 9 5 4 5 5 5 3 3 3 2 1 1 0 0 7 7 9 9 9 8 8 7 7 8 8 8 7 6 6 5 4 4 0 0 4 5 5 8 6 5 4 7 6 6 4 6 6 4 4 4 2 2 . . 0 0 0 0 0 0 0 0 9 9 2 2 7 7 0 5 5 5 5 5 5 5 6 6 4 4 4 4 4 4 4 4 4 4 4 2 2 3 3 3 3 3 3 3 3 3 5 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 ...... 0 0 ...... 170 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 4 4 4 4 4 4 4 4 4 4 3 3 2 1 1 1 1 1 1 1 1 1 1 1 1 - - 160 O a b O O f 150 f P b g O a 140 e N a d h 130 Cl O c Cl TMS 120 110

100

90 c b 80

70

a 60

50 h 40 30 f 20 e g d 10

0 6 2 3 2 8 5 7 1 2 6 2 9 5 6 9 2 8

5 -10 5 5 5 2 2 3 3 2 0 4 3 7 1 4 4 3 7 1 ...... 3 3 2 1 1 0 0 2 4 2 1 1 1 0 2 0 3 2

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

111 3 3 3 l l CARBON_01 l 400 c c c d d d c c

SRW-60PC c

3 3 9 5 5 3 2

2 2 4 0 0 1 8 6 5 0 5 1 4 4 3 8 8 6 6 0 6 4 4 1 1 1 ...... 9 6 2 7 3 0 3 . . . . 5 . 1 1 0 0 . 9 8 8 8 7 7 7 7 . 2 7 6 6 6 6 3 3 3 3 2 2 2 2 2 2 2 2 6 0 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 7 6 1 - 350

300

250

200

150

100

50

0

-50

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

112 Compound 7c: 3 PROTON_01 l c d

SRW59P c 3

450 6 0 5 1 4 0 1 0 3 2 3 7 9 0 0 5 6 9 0 8 6 4 8 5 9 7 2 5 6 6 6 5 5 3 6 5 4 9 2 0 7 7 4 7 1 2 2 1 5 4 5 3 0 6 9 5 3 1 2 7 7 5 1 9 9 1 8 2 0 9 5 9 6 2 6 2 0 2 8 3 9 4 5 3 3 4 2 5 3 8 4 2 0 4 3 2 5 5 3 3 1 0 6 0 0 4 8 3 2 9 8 0 4 5 4 2 6 0 9 4 4 4 3 7 1 0 6 5 5 4 4 3 3 3 6 6 2 2 1 4 0 5 3 3 1 2 2 3 9 1 2 8 1 0 9 8 8 6 6 3 0 5 3 3 2 1 0 7 7 6 6 5 3 3 4 4 4 9 8 8 6 6 5 4 3 3 6 6 4 4 2 2 . 6 6 0 0 0 0 0 0 0 0 0 9 9 9 9 2 8 8 8 8 9 9 8 0 4 4 2 2 2 2 2 2 3 3 3 3 3 6 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ...... 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 - 400

b O 350 f O O f i P a b b e O h N 300 a d g Cl O Cl c 250

c 200

b 150 a TMS 100 g f 50 h i e d

0 3 0 4 2 8 1 3 4 0 8 7 7 0 8 9 9 4 8 6 2 5 0 4 8 3 1 5 8 4 3 ...... 1 3 1 0 2 2 2 2 2 1 1 2 0 3 8

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

113 3 3 l l CARBON_01 3 l c c

c 450 d d d c SRW-59PC c

c 9 2 9 3 1 9 3 0 8 0 4 0

1 1 8 9 7 1 7 7 9 2 6 5 0 9 5 4 9 4 0 4 3 8 3 6 3 4 1 7 7 2 4 0 1 ...... 9 6 2 2 7 9 3 9 8 6 3 7 1 4 2 3 3 0 3 ...... 9 5 . . . . 5 5 1 1 1 0 0 . 9 8 8 6 . 2 0 2 2 2 2 3 3 3 0 7 6 6 6 6 4 3 3 3 3 3 3 3 2 2 2 2 6 6 6 6 0 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 7 6 6 6 6 4 4 3 2 2 2 1 1 1 1 - 400

350

300

250

200

150

100

50

0

-50

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

114 Compound 7d: 3 PROTON_01 l c 150 d

SRW81P c 2

4 8 9 1 4 6 2 5 1 5 3 4 7 1 1 8 4 4 9 7 6 5 8 4 3 8 9 0 7 7 3 1 0 2 3 9 2 5 3 5 1 0 1 8 6 9 4 2 5 6 7 4 2 4 6 5 3 2 9 4 4 5 2 6 0 5 2 7 5 9 1 7 0 4 2 7 8 6 4 0 8 4 4 9 7 6 8 6 1 5 8 4 9 4 1 6 6 2 7 1 0 7 1 3 1 3 8 1 2 9 3 2 4 4 6 9 6 2 6 4 0 2 7 3 9 7 1 2 4 3 3 0 0 0 7 6 2 2 1 1 3 3 9 4 2 2 0 1 1 6 8 9 6 5 5 4 4 4 3 2 2 3 2 2 1 1 0 9 8 8 6 6 3 2 1 0 8 0 5 6 2 4 3 3 4 2 1 1 0 4 2 1 . 8 8 0 0 0 0 0 0 0 0 0 0 9 9 9 9 2 8 8 8 8 8 0 140 5 5 5 5 5 5 8 8 8 8 8 8 8 8 8 2 0 3 3 3 3 3 3 3 3 3 6 2 2 2 7 7 7 1 1 1 1 1 1 1 ...... 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 1 1 1 1 1 1 1 - 130 c b 120 b O b a O O 110 g P g a f O 100 e N a d h 90 Cl O c Cl 80

70

h 60 a TMS 50 40

g 30 20 f e d 10

0 1 6 5 5 1 9 8 7 9 0 6 9 6 9 2 8 6 6 2 1 6 4 2 5 5 4 4 8 3 1 1 1 1 5 8 8 2 2 4 3 3 1 ...... -10 2 1 1 1 1 1 0 2 0 2 2 2 0 1 1 1 2 0 1 3 2

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

115 3 3 l 3 CARBON_01 l l 210 c c c d d d c

SRW81PC1 c

5 c 1 8 1 4 9 4 5

7 0 2 8 5 9 0 8 1

5 9 9 0 1 8 3 4 5 6 9 3 4 8 8 3 5 0 3 2 9 3 5 5 6 6 0 0 200 1 7 3 . 0 0 7 ...... 6 2 3 7 4 8 6 7 1 2 2 3 3 0 3 ...... 2 ...... 5 5 3 3 1 1 0 0 8 8 8 8 8 7 6 6 6 . . 2 0 2 2 2 2 0 0 7 7 6 6 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 6 6 6 6

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 7 6 6 6 6 4 4 2 1 1 1 1 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 -30

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

116 Compound 7f: 3 PROTON_01 l c d

SRW83H c 200 8 7

2 8 4 8 6 4 4 1 6 0 4 3 5 5 1 6 5 5 8 7 2 4 8 7 8 1 4 6 5 7 8 4 7 8 7 1 8 3 2 9 4 4 1 7 0 2 1 1 9 9 2 4 3 6 7 6 7 9 1 9 8 4 6 3 4 2 4 7 7 0 9 3 1 3 8 8 3 2 7 3 1 3 2 0 6 2 0 5 1 3 7 2 8 5 9 8 6 2 2 3 5 1 6 4 2 2 5 1 2 6 6 7 5 7 2 3 3 9 4 3 6 4 6 4 3 7 9 8 4 8 6 5 0 4 8 6 1 0 8 4 3 9 7 3 2 1 1 8 8 7 6 6 4 2 2 2 2 1 0 0 9 9 0 0 8 5 3 1 0 5 0 9 8 8 6 5 3 3 1 1 0 4 0 6 9 8 6 6 4 4 2 2

. . 190 0 4 4 7 0 0 1 5 5 5 5 5 5 6 6 6 4 4 4 4 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 5 2 2 2 2 2 2 2 2 2 2 2 0 0 3 3 3 3 1 1 1 1 1 1 1 1 1 ...... 0 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 4 3 3 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 - - 180 170 c 160 g 150 d O d a c O O h 140 h P a f O 130 g N a e i 120 Cl O 110 Cl b d 100 TMS 90 b c 80 70 60 a 50 i 40 g 30 h 20 f e 10 0

5 -10 2 5 5 4 4 4 2 4 6 3 7 7 0 0 1 . . 9 5 6 4 8 4 0 1 1 4 0 4 ...... 1 0 . . . 0 1 0 1 3 2 2 0 0 0 1 0 1 1 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

117 3 3 3 l l

CARBON_01 l c c c d d d c c

SRW83C c 14

4 3 2 5 5 9 9

8 2 4 4 2 1 6 8 0 6 9 5 5 0 5 5 4 7 0 1 2 2 0 ...... 9 9 9 8 6 3 . . . . . 3 2 2 9 9 8 8 8 7 7 7 6 . 9 3 3 7 6 6 3 3 3 2 2 2 2 2 2 2 2 2

1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 5 2 2 13

12

11

10

9

8

7

6

5

4

3

2

1

0

-1

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

118 Compound 7g: 3 l

PROTON_01 O c

D 600 d H SRW80P c 1 1

6 5 5 4 4 3 2 0 7 5 3 3 4 5 4 2 1 1 2 1 1 6 6 4 3 7 3 1 0 0 6 3 2 9 9 7 7 7 5 5 5 0 9 9 7 7 5 5 8 7 7 7 6 5 5 5 9 9 7 7 5 5 3 3 . . 0 0 0 0 0 0 0 0 9 9 9 0 3 6 6 6 0 7 4 4 4 2 2 2 2 2 2 2 2 2 3 3 3 3 5 6 6 6 2 2 2 2 2 2 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ...... 0 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 4 4 4 4 4 4 4 4 3 3 3 3 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 - - 550

O 500 b g O O g b P f e O d 450 N a f d g d a 400 Cl O c Cl 350

300

TMS 250

200

150 b 100

a 50 g f e d 0 8 3 1 5 5 3 9 0 7 1 6 3 4 1 7 2 8 9 3 9 3 7 9 0 9 3 2 2 2 3 3 0 6 0 7 1 1 9 8 2 8 6 3 3 7 1 ...... -50 0 1 2 3 1 0 0 0 1 1 2 1 2 0 2 1 1 4 1 1 0 6 0

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

119 3 3 3 l l

CARBON_01 l c c c d d d c c

SRW80PC2 c

8 7 6 1 4 8 5

6

3 7000 3 8 6 9 2 7 6 1 6 3 3 5 6 9 4 5 6 4 3 0 0 1 ...... 6 2 9 6 7 0 2 3 3 0 8 6 3 ...... 5 . . . 1 1 0 . 8 8 8 8 5 . . . 2 2 5 7 7 7 6 6 4 6 3 3 3 2 2 2 2 2 6 6 6 0 1 1 1 1 1 1 1 1 1 7 7 7 7 6 6 3 2 2 1 1 1 - 6500

6000

5500

5000

4500

4000

3500

3000

2500

2000

1500

1000

500

0

-500

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

120 Compound 8a: d d d d PROTON_01 d 380 o o o o o O 3 3 3 3 3 D d d d d SRW61 d H c c c c c

360 6 5 4 8 8 5 3 1 1 1 9 8 6 8 7 5 3 3 4 1 0 0 8 5 2 0 9 9 2 2 0 1 9 8 7 4 2 2 1 1 0 0 0 9 3 1 8 2 1 0 0 6 2 7 4 5 7 5 7 6 5 5 4 3 9 8 8 5 5 5 8 8 4 4 4 4 2 2 0 9 9 9 3 3 3 3 8 8 8 8 0 0 1 5 4 4 4 2 2 2 3 3 3 3 3 3 3 3 3 3 9 5 5 5 5 8 4 4 4 2 1 1 1 1 1 1 1 1 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 340 320 300 O NaO OH P 280 e O a g 260 a f d N Cl O b 240 Cl c 220 200 180 160 140 120 100 c a 80 60 b d 40 g f e 20 0 5 5 3 1 3 3 3 9 9 4 6 9 4 2 0 5 6 2 2 2

9 2 -20 5 9 6 4 8 8 4 4 8 8 3 3 2 2 0 3 3 8 2 7 1 1 ...... 0 2 1 2 0 0 0 0 0 1 1 1 1 0 0 2 2 2 0 0 0 0

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

121 3 3 0 2 4 5 2 CARBON_01 0 3000 9 6 5 8 2 2 8 0 7 7 0 3 1 . . . . 9 . . 2 9 5 3 0 7 7 1 . . 1 0 ...... 9 9 8 7 8 6 SRW61PC2 7 7 7 7 7 7 3 3 2 2 2 2 9 1 1 1 1 1 1 4 4 4 4 4 4 4 4 1 2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0

-200

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

122 Compound 8b:

PROTON_01 d o O 3 D SRW65 d 450 H c

9 8 6 4 3 1 8 4 7 4 0 9 5 5 4 1 0 0 9 9 9 5 9 8 6 5 5 4 3 3 1 1 0 5 2 1 1 0 0 9 9 7 6 6 5 4 2 1 9 8 6 6 5 5 4 3 9 4 5 7 8 7 5 2 8 8 8 8 8 8 4 4 4 2 2 2 2 3 3 3 3 3 3 3 7 7 0 0 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 4 4 4 4 4 4 4 4 4 2 3 3 3 3 3 3 3 3 9 9 2 1 1 1 1 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 1 1 1 1 1 1 1

400

O NaO OH a P a 350 d O e N a c f Cl O 300 Cl b

250

200

150

b 100

a 50 f e d c 0 0 3 0 2 5 1 0 5 7 0 5 0 5 0 1 8 1 0 4 3 5 9 5 9 5 9 6 4 0 2 7 0 8 6 2 8 7 7 7 1 ...... 1 1 1 3 1 1 9 0 1 0 3 2 0 1 0 0 3 1 1 0

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

123 d

d 23 d CARBON_01 d o o o o 3 3 3 3 d d d SRW65C d 22 c c c c

9

6 9 7

2 1 6 4 4 3 1 7 2 0 6 9 3 8 7 5 4 7 7 9 5 1 ...... 9 9 0 . 4 2 0 5 5 9 3 7 1 ...... 0 0 . . . . . 9 8 7 6 6 21 8 8 8 6 6 7 7 7 7 4 5 4 3 2 2 2 2 2 8 1 1 1 1 1 1 1 7 4 4 4 4 4 4 4 4 4 3 1 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

124 Compound 8c: d d d PROTON_01 d d o o o o o O O 750 3 3 3 3 3 D D d d d SRW67msH d d H H c c c c c

3 8 3 9 9 5 4 7 7 4 2 0 6 5 5 4 4 4 4 4 1 1 0 0 1 9 9 6 5 5 4 4 8 8 5 3 4 2 0 7 6 1 8 3 0 9 8 8 3 3 2 0 4 8 9 9 8 8 7 7 7 5 5 2 9 8 8 5 5 5 4 4 4 4 4 4 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 7 7 7 7 7 0 4 4 4 2 2 2 9 9 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 ...... 700 7 7 7 7 7 7 7 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

650 b O NaO OH g 600 P e a b O 550 a h N d f 500 Cl O Cl c 450

400

350

300

250

200

150 b c 100 f h g 50 e d 0 0 5 5 3 8 1 0 8 6 7 6 6 2 5 5 4 4 3 3 2 5 3 7 1 7 7 ......

. -50 0 1 5 3 5 3 6 0 2 0 0 2 8

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

125 1 8 1 1 2 9 9 0 5 9 2

CARBON_01 8 9 9 6 6 5 2 2 8 3 8 4 . . . . 9 2 9 9 9 7 8 4 9 9 5 5 3 7 1 .

. 34 ...... 9 9 6 6 8 6 2 2 7 7 7 7 7 7 7 3 2 2 SRW67carbon3 2 2 2 2 2 1 1 1 1 7 6 6 4 4 4 4 4 4 4 4 4 3 2 2 32

30

28

26

24

22

20

18

16

14

12

10

8

6

4

2

0

-2

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

126 Compound 8d: d d d d PROTON_01 d o o

o o 260 o O 3 3 3 3 3 D d d d d SRW85ms d H c c c c c

6 4 3 7 8 6 4 1 8 8 7 5 4 1 0 0 2 2 1 0 9 9 3 5 4 3 2 8 7 6 5 4 2 2 1 5 4 3 3 3 2 1 1 1 0 0 7 5 3 9 8 2 1 1 1 7 6 6 5 5 3 8 7 5 9 9 9 8 5 5 5 5 2 4 4 4 4 2 2 3 3 3 3 0 0 0 9 9 5 5 5 5 5 5 5 5 5 5 5 8 8 8 8 8 8 8 4 4 4 4 3 3 9 9 9 9 9 8 8 6 6 6 6 6 6 2 1 1 ...... 240 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1

220

O a 200 NaO OH P a 180 O d N a e c f 160 Cl O b Cl 140

120

100

80

60

40 a b f e 20 d c 0 6 4 9 0 2 5 3 7 7 6 6 9 6 8 8 6 0 7 8 9 6 0 9 5 5 5 9 8 8 4 2 4 3 3 0 0 7 6 2 6 3 1 ......

0 3 4 0 0 0 0 1 0 1 1 1 0 1 0 0 3 0 0 0 0 -20

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

127 1 4 6 1 0 3 8 1 2 0 7 5 1 4 9 8 2 7 3 7 0 3 6 CARBON_01 0 2 7 9 7 5 4 4 6 2 0 3 9 9 5 9 0 7 7 1 8 4 6 5 5 9 3 3 7 4 7 7 ...... 9 . . . 5 . . . . 2 7 7 1 1 1 9 5 3 7 1 ...... 1 3 3 3 1 1 0 . . . . . 9 8 8 8 8 7 7 7 7 6 6 6 6 5 3400 8 6 0 7 7 7 7 7 2 0 9 SRW85MSC3 6 6 6 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 7 6 4 4 4 4 4 4 4 3 3 2 3200

3000

2800

2600

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2200

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1600

1400

1200

1000

800

600

400

200

0

-200

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

128 Compound 8f: d d d d PROTON_01 d 240 o o o o o O 3 3 3 3 3 D d d d d SRW86 d 230 H c c c c c

3 7 9 7 7 6 0 8 4 3 1 0 0 1 9 9 6 5 5 4 4 3 3 2 2 2 1 1 0 8 8 8 6 6 5 5 4 4 4 3 3 2 2 2 1 9 8 8 7 6 2 0 7 8 7 6 6 2 2 1 4 4 9 7 8 4 8 8 8 8 2 2 3 3 3 3 3 3 0 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 2 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 ...... 220 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 4 4 3 3 3 3 3 3 3 3 2 2 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 210 200 190 c 180 170 f 160 O a c 150 NaO OH P 140 a 130 e O h N g 120 a d 110 Cl O b 100 Cl c 90 80 70 60 50 40 a b 30 g h f 20 e d 10 0 -10 9 3 6 3 6 9 2 7 1 8 2 9 3 2 1 5 0 5 6 3 5 5 5 8 2 4 2 8 3 3 3 3 0 9 9 6 3 7 1 1 ...... 1 3 2 1 1 0 0 0 0 5 0 1 0 0 1 0 4 1 0 1 -20 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

129 4 2 3 0 5 7 9 0 9 8 3 0 2 6 1 CARBON_01 7 10 5 7 6 5 5 4 5 3 4 7 9 5 6 5 ...... 9 . . 4 2 0 6 9 5 3 7 1 ...... 8 2 1 1 . . . . 9 8 8 6 6 6 8 8 8 6 7 7 7 7 3 SRW86msC 2 9 3 3 3 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 4 4 4 4 4 4 4 4 3 2

9

8

7

6

5

4

3

2

1

0

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

130 Compound 8g: d d d d PROTON_01 d o o o o o 200 O 3 3 3 3 3 D d d d d SRW84H d c c c c H c

190 7 0 2 4 6 4 2 0 1 8 5 4 3 0 5 7 7 4 4 3 3 2 7 8 6 5 1 0 8 6 4 2 0 5 2 8 0 9 7 5 3 2 1 9 7 5 1 0 3 1 8 6 6 3 1 4 5 3 2 0 8 6 9 7 8 9 9 5 5 8 8 8 8 0 0 0 9 3 3 3 3 3 3 3 6 6 6 4 2 2 0 0 1 1 7 7 5 5 4 4 4 2 2 2 2 2 3 3 6 6 6 6 6 2 2 2 2 2 2 3 7 7 7 7 1 1 1 1 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 180 170 160 O NaO OH 150 P 140 O c e N 130 a f d g c a Cl O 120 Cl b 110 100 90 80 70 b 60 50 a 40 30 g f c 20 d e 10 0 9 0 4 3 2 2 9 3 9 2 0 5 6 4 1 0 3 1 3 9 4 8 -10 4 4 5 2 2 2 2 2 6 8 4 6 0 3 6 3 7 1 1 5 2 6 2 7 7 1 ...... 0 1 1 5 0 0 0 2 0 1 0 0 1 0 1 0 0 0 2 3 0 1 1 0

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

131 8 4 4 0 7 5 5 2 6 7 0

CARBON_01 6 9 1 6 5 2 1 4 2 9 2 7 6 5 3 9 8 5 3 6 9 8 4 0 8 5 0 7 5 8 5 8 9 9 9 4 ...... 9 . 2 2 0 5 1 1 1 9 5 5 5 8 8 8 8 6 6 4 4 2 0 3 3 3 3 6 2 7 7 1 1 1 . . . . .

. . 3500 . 1 0 ...... 9 8 8 7 5 . . 8 8 8 8 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 7 7 SRW84PC2 3 4 3 2 2 2 2 2 1 1 1 1 1 1 1 7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 2 2

3000

2500

2000

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1000

500

0

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

132 Compound 9a: 3 l

PROTON_01 O 1200 c D d H SRW68P c

1

8 8 8 5 5 2 1 0 9 9 8 8 7 6 1 0 9 6 5 5 4 3 3 3 2 2 1 1 0 0 5 9 5 5 4 0 2 1 1 0 8 7 7 7 6 5 2 2 1 1 0 0 0 8 7 5 2 1 0 4 8 7 6 6 . 5 5 5 5 5 5 5 5 6 6 6 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0 3 3 3 3 3 3 3 3 3 3 3 9 5 5 5 5 8 8 8 6 2 2 2 2 2 2 2 1 1 1 1 ...... 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 - 1100

1000

900 O c c O 800 O g O O g O P a 700 O e O f N a d g 600 Cl O c b 500 Cl c 400

300

b 200

a 100 g f e d 0 2 8 5 9 8 4 6 9 9 6 5 9 4 3 4 3 3 . 8 8 5 5 8 3 7 7 9 5 9 4 4 3 1 ...... 6 ...... -100 6 1 4 0 1 0 1 0 1 1 1 1 2 3 1 0 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

133 3 7 6 4 6

8 190 0 5 5 1 8 8 1 CARBON_01 6 9 5 4 5 8 6 0 7 9 1 2 1 ...... 8 8 3 9 6 6 7 1 7 3 ...... 0 0 . 8 8 8 8 7 7 . 1 9 6 6 6 0 0 7 SRW68PC 6 6 3 3 2 2 2 2 2 2 180 1 1 1 1 1 1 1 1 8 7 7 7 2 2 2 2 2 2 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 -30 -40

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

134 Compound 9b:

3 2400 PROTON_01 l c d

SRW77P c 2300

7 9 9 5 3 4 3 2 2 5 3 7 9 4 9 1 9 8 8 2 4 0 6 5 9 5 8 3 5 4 9 8 1 9 6 6 6 6 6 7 7 0 9 5 5 3 2 0 6 5 0 0 7 1 9 8 4 4 4 2 5 2 5 3 6 5 2 9 1 5 0 2 3 2 4 7 7 6 9 8 5 3 6 9 5 9 9 4 7 3 2 8 3 9 5 0 7 2 8 7 3 0 0 5 0 2 7 0 9 5 6 0 3 2 8 5 7 0 6 9 0 1 7 5 2 6 0 8 4 9 9 6 4 2 1 0 7 9 9 7 6 4 2 1 0 2 0 3 8 3 3 5 5 4 3 3 3 0 0 7 7 9 9 9 8 8 7 7 7 8 8 8 8 7 7 6 6 5 5 4 3 3 0 5 4

4 4 3 2 2 1 2200 9 5 . 5 5 5 5 5 5 5 5 5 5 6 6 6 6 4 4 4 4 2 7 7 0 5 5 5 5 5 5 5 5 6 6 4 4 4 4 4 4 4 4 2 3 3 3 3 3 3 3 3 3 3 3 3 3 2 1 1 1 1 1 1 1 1 ...... 0 . . . 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 3 3 2 1 1 1 - 2100 2000 1900 c 1800 c O 1700 O a 1600 O O O O P g 1500 g a O O 1400 e N a h 1300 d f 1200 Cl O b 1100 Cl 1000 900 800 c 700 600 b 500 400 a 300 g f TMS 200 h e d 100 0 -100 9 1 2 4 3 6 7 7 2 2 0 1 0 4 8 2 6 . 9 6 9 4 4 6 3 7 1 5 5 4 3 3 1 ...... 7 ......

3 1 5 1 2 2 0 2 2 1 1 1 2 2 1 1 -200 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

135 0 1 0 8 4 8 5 5 8 3 4 5 1 3 8 4 8 8

CARBON_01 9 3 7 5 9 9 4 5 3 3 1 9 0 4 8 6 6 0 7 5 0 6 4 1 1 ...... 8 6 6 2 2 3 9 6 6 2 7 7 8 6 0 7 3 ...... 2 0 4 1 1 0 . . . . 9 8 8 8 7 7 7 . 2 0 1 1 1 8 8 9 6 6 6 0 7 SRW77PC2 6 6 6 4 4 3 3 3 3 2 2 2 2 2 2 2 0 750 1 1 1 1 1 1 1 1 1 1 1 1 1 8 8 8 7 7 7 7 4 4 3 3 2 2 2 2 2 -

700

650

600

550

500

450

400

350

300

250

200

150

100

50

0

-50

-100

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

136 Compound 9c: 3 PROTON_01 l 1300 c d

SRW69P c 1

4 6 5 1 5 1 6 5 4 9 0 3 2 6 3 6 9 0 8 4 0 8 5 5 6 1 7 4 6 9 7 1 1 7 1 6 6 0 6 7 1 7 6 4 1 1 2 6 7 9 0 8 2 8 0 8 4 8 0 0 1 1 2 4 4 7 8 5 1 5 9 0 2 3 2 6 5 6 1 7 2 7 1 2 3 5 7 5 6 4 2 6 3 1 6 0 9 5 4 3 3 6 8 2 8 4 1 8 1 0 9 3 2 2 8 7 1 2 8 0 4 2 8 8 6 7 6 0 5 5 9 8 6 5 3 2 0 9 8 9 6 4 9 7 6 3 2 0 0 3 7 0 9 1 8 8 7 7 6 6 6 9 0 6 5 3 3 2 1 1 5 3 7 5 4 1 4 9 9 8 7 7 4 3 3 2 1 1 9 7 5 0 . 1200 6 6 5 5 5 5 5 5 5 6 6 6 4 4 4 4 4 4 4 4 0 0 9 9 8 4 1 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 3 9 5 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ...... 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 3 3 3 3 3 3 3 3 3 3 2 2 2 2 1 1 1 1 1 1 1 1 1 -

1100

b 1000 O c b O 900 O O O i O i P g a c 800 O f O g N a e h 700 Cl O d Cl b 600

500

c 400

d 300 a 200 h i 100 g f e 0 8 5 3 5 4 0 2 3 7 5 4 9 3 0 3 7 0 0 5 . 2 6 4 4 0 0 4 6 7 5 0 2 3 3 7 1 1 ...... 2 ...... -100 1 1 3 1 4 2 2 1 1 1 0 0 2 1 0 1 7 0 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

137 9 7 9 7 4 8 4 8 5 5 5 0 8 6 5 2 3 1 7 1 1 1 8 1

CARBON_01 9 9 4 9 3 5 5 0 8 4 6 0 3 1 7 7 7 7 9 4 0 3 1 ...... 8 9 6 2 2 9 3 8 6 3 7 7 8 6 0 7 3 ...... 9 4 4 2 1 1 1 1 0 0 . . . 9 8 8 6 . . 6 2 0 1 1 1 8 3 6 6 6 3 3 0 7 7 SRW69PC 6 6 7 4 3 3 3 3 3 3 3 3 3 2 2 2 2 700 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 8 8 7 7 7 7 4 4 3 3 2 2 2 2 2 2

650

600

550

500

450

400

350

300

250

200

150

100

50

0

-50

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

138 Compound 9d: 3 l

PROTON_01 O c D d 35000 c SRW89PH2 H 1 1

6 5 9 9 8 6 6 1 1 9 9 8 8 8 6 6 1 1 4 4 9 9 5 4 3 3 2 3 3 2 2 2 2 1 1 1 8 8 6 5 5 2 0 0 0 0 0 7 5 5 5 3 3 4 9 9 8 8 7 6 6 5 4 4 . . 8 8 5 5 5 5 5 5 5 5 5 6 6 4 4 4 4 4 2 0 0 5 5 5 5 5 5 5 5 5 8 8 8 8 8 2 2 0 3 3 3 3 3 3 3 3 3 5 2 7 7 7 1 1 1 1 1 1 1 1 1 1 ...... 0 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 2 2 1 1 1 1 1 1 1 1 1 1 1 1 - -

30000 c O c O a O h O O O 25000 P h a O O f e N a d g 20000 Cl O c b Cl 15000

10000 b

a g 5000 h f e d 0 0 0 4 4 7 5 6 1 8 6 1 0 5 4 4 6 5 1 0 6 . 9 8 6 6 4 0 0 7 7 7 1 9 4 3 3 7 1 1 ...... 5 ...... 2 0 1 3 1 2 2 2 1 0 1 1 1 2 0 1 1 0 0 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

139 4 6 4 6 3 4 6 4 0 4 0 6000 3 0 1 3 6 5 4 2 3 7 3 3 8 CARBON_01 8 2 5 7 4 7 2 4 3 5 5 3 0 5 8 0 3 3 6 6 0 0 7 7 7 7 7 4 0 7 1 . . 0 ...... 8 6 6 2 2 4 9 6 8 6 7 7 8 6 0 7 3 . 1 ...... 4 4 3 3 2 1 1 0 0 . . . . 8 8 8 8 7 6 6 6 . . 6 6 2 0 1 1 1 8 8 9 6 6 6 0 7 7 SRW89PC3 6 6 6 7 7 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 8 8 7 7 7 7 7 4 4 3 3 2 2 2 2 2 - 5500

5000

4500

4000

3500

3000

2500

2000

1500

1000

500

0

-500

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

140 Compound 9f: 3 l

PROTON_01 O c D

d 850 H c 6 6

SRW90PH2

9 2 8 1 1 6 5 9 3 3 5 8 8 1 9 5 2 4 5 0 6 1 8 6 8 9 4 8 3 6 9 4 4 1 6 1 4 6 7 7 2 1 3 1 0 4 4 6 9 9 0 3 0 7 3 6 7 8 6 6 6 6 7 5 7 6 3 7 5 8 8 0 1 8 6 6 6 1 7 3 6 2 4 6 2 9 4 8 4 8 5 0 0 3 7 4 4 1 9 5 2 1 0 2 0 9 3 4 8 3 5 7 2 1 0 8 5 4 9 3 2 8 3 6 8 0 6 4 5 9 8 6 5 3 2 0 0 0 9 8 7 3 2 0 0 5 2 2 2 8 8 7 6 2 2 1 0 8 7 7 5 5 3 2 1 1 0 0 0 0 6 1 5 5 5 9 9 8 8 7 1 1 4 6 6 6 5 3 1 9 8 5 5 . . . 800 5 5 5 5 5 5 5 6 6 6 6 6 4 4 4 4 9 7 7 0 1 1 1 1 5 5 5 5 5 5 5 5 2 2 2 3 3 3 3 3 3 3 3 3 3 5 2 2 2 2 2 2 2 2 3 1 1 1 1 1 1 ...... 0 0 0 ...... 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 - - - 750

700

650

c 600 O b f 550 O a c b O j O O O P j 500 a O O g N 450 a h e i 400 Cl O d Cl b 350 c 300

250 a d 200 f 150 i j 100 g h e 50

0 4 8 5 6 4 7 7 3 8 1 3 7 1 0 7 1 8 3 -50 9 . 8 9 2 4 5 0 8 7 7 1 5 9 5 4 3 7 1 ...... 4 ...... 3 4 1 1 4 1 2 1 1 1 2 2 0 9 1 0 1 0 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

141 9 8 9 6 4 8 3 5 4 8 2 5 8 5 3 1 0 8 9 8 2 7 7 CARBON_01 5 2 9 3 4 5 5 1 9 9 4 6 6 0 7 5 9 0 2 0 7 1 . 8 ...... 8 9 2 8 2 9 3 9 6 6 3 7 7 8 6 7 3 . 1 ...... 8 1 7 4 1 1 0 0 . . . 9 8 8 7 7 6 . . 2 0 1 1 1 3 0 9 6 6 6 3 0 7 7 SRW90PC5 6 6 6 4 4 3 3 3 3 3 3 2 2 2 2 2 2 3600 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 8 8 7 7 7 7 7 4 4 3 3 2 2 2 2 2 2 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 -400

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

142 Compound 9g:

PROTON_01 600 SRW-91P 6 5 5 9 7 7 6 6 4 9 7 6 6 3 3 6 5 5 5 8 2 1 0 5 4 2 2 0 0 5 5 3 2 2 2 9 9 8 7 7 1 2 2 8 8 7 7 7 6 3 3 3 3 2 2 2 4 4 4 4 2 1 0 4 5 5 5 5 5 5 0 0 0 6 6 6 6 6 6 6 0 0 0 0 7 7 7 4 4 4 4 4 4 2 2 2 2 3 3 3 3 3 3 6 6 6 6 6 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 7 7 7 7 ......

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 5 5 5 5 5 5 5 5 5 5 3 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 550

500 b O 450 O b O O O i O i P 400 d g O f O N g 350 a e h d a Cl O c 300 Cl

250

200 c 150 b 100 a i f d h g TMS 50 e 0 0 5 0 6 3 3 3 0 4 9 1 5 1 0 7 6 6 . 8 6 6 4 3 1 1 1 1 1 5 6 6 2 3 ...... 9 . . . . .

2 1 1 3 5 2 2 1 2 1 1 3 6 1 0 0 -50 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

143 8 6 6 1 3 6 0 9 2 2 1 8 CARBON_01 6 4 0 5 3 5 8 3 9 1 6 4 3 0 7 5 1 1 1 ...... 6 2 8 9 7 6 7 1 6 8 6 7 3 ...... 4 1 1 0 . . 8 8 8 8 5 . . . 2 1 1 8 5 7 7 6 6 7 SRW-91PC3 6 6 4 3 3 3 3 2 2 2 2 2 1 1 1 1 1 1 1 1 1 8 8 7 7 7 7 4 3 3 2 2 2 2 350

300

250

200

150

100

50

0

-50

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

144 References:

1. Edwards, R. L.; Brothers, R. C.; Wang, X.; Maron, M. I.; Ziniel, P. D.; Tsang, P. S.;

Kraft, T. E.; Hruz, P. W.; Williamson, K. C.; Dowd, C. S.; and John, A. R. O.

MEPicides: potent antimalarial prodrugs targeting isoprenoid biosynthesis. Sci. Rep.

2017, 7 (1), 8400.

2. San Jose, G.; Jackson, E. R.; Haymond, A.; Johny, C.; Edwards, R. L.; Wang, X.;

Brothers, R.C.; Edelstein, E.K.; Odom, A.R.; Boshoff, H.I.; Couch, R.D.; Dowd, C. S.

Structure-Activity Relationships of the MEPicides: N-Acyl and O-Linked Analogs of

FR900098 as Inhibitors of Dxr from Mycobacterium tuberculosis and Yersinia pestis.

ACS Infect Dis. 2016, 2(12), 923-935.

145