Malaria, the most devastating of the parasitic diseases, afflicts an estimated 300-500 million people worldwide and results in more than 800,000 deaths annually. Several parasites in the genus Plasmodium infect humans, but Plasmodium falciparum has the most significant lethality. Drug resistance has compromised the efficacy of existing drugs. New antimalarial agents, especially agents that work against new targets in P. falciparum, are needed. As part of efforts to identify novel inhibitors of P. falciparum, we initiated a quantitative high-throughput screening (qHTS) campaign against the 3D7 line of P. falciparum. The robust whole parasite viability assay was screened against the Molecular Libraries Small Molecule Repository (MLSMR) as well as the Diversity-Oriented Synthesis (DOS) informer I library (approximately 8,000 compounds), which is a subset of the Broad Institute DOS library. From these efforts, we have identified a unique and novel structural class of antimalarial compounds from the Broad Institute DOS Informer I library. The probe (ML238) displayed picomolar activity in the SBYR live/dead assay against 3D7 and Dd2 P. falciparum. This activity was recapitulated in the luciferase assay, albeit at single-digit nanomolar activity, while showing no apparent mammalian cell cytotoxicity. The probe is in a novel structural class in the antimalarial field with unknown mechanism of action; therefore, this probe will be highly useful to the malaria research community in identifying new targets or developing new drug classes.

Probe Structure and Characteristics

ML238

Recommendations for scientific use of the probe

Malaria, the most devastating of the parasitic diseases, afflicts an estimated 300–500 million people worldwide and results in more than 800,000 deaths annually. More than 85% of all malaria-related mortality is among children in sub-Saharan Africa. Drug resistance has compromised the efficacy of existing drugs, and new antimalarial agents, especially those that work against new targets in Plasmodium falciparum, are needed. This agent represents a novel structural class for small molecules with antimalarial properties and may possess promise as a novel antimalarial. Further, this chemotype may provide insight into new molecular target(s) for development of additional antimalarial agents.

1. Introduction

Malaria is a serious disease caused by parasites from the Plasmodium genus, which infects particular mosquitoes that feed on humans. This disease is often fatal and is common in poor areas of tropical and subtropical regions, such as Africa, Asia, and parts of the Americas. The disease results from the multiplication of malaria parasites within red blood cells (RBCs), causing symptoms that typically include fever and headache. In severe cases, symptoms may progress to coma and death. Malaria, particularly from P. falciparum, affects hundreds of millions of people worldwide and is responsible for almost a million deaths every year (1). Chloroquine and pyrimethamine-sulfadoxine (Figure 1A) have been used as the standard treatment for malaria throughout the 20th century because of their limited host toxicity, ease of use, low cost, and effective synthesis. But, their use progressively diminished due to the appearance and spread of resistance (2). Currently, the most effective strategy recommended by the World Health Organization (WHO) is artemisinin-based combination therapies (ACT; Artemether and Lumefantrine, Figure 1B) or simplified Fixed-Dose Combination (FDC; Artesunate and Amodiaquine, Figure 1C) to avoid the development of drug resistance against artemisinin-based therapies (3).

Figure 1

Antimalarial Drugs Recommended by the WHO. Antimalarial drugs recommended by WHO: Chloroquine and the combination Pyrimethamine-Sulfadoxine (A) are the drugs of choice but can be substituted by artemisinin-based treatments such as the combination Artemether-Lumefantrine (more...)

Recent studies suggest that resistance to the endoperoxide artemisinin family is emerging, particularly in southeast Asia; the main concern is not if but when will it spread (4, 5). Thus, the development of new drugs, which could be used against artemisinin-resistant malaria is critical. In this context, high-throughput screening (HTS) campaigns aimed at discovering new antimalarial probes is of great importance. Recently, Gamo et al. screened nearly 2 million compounds from GlaxoSmithKline’s chemical library for inhibitors of P. falciparum and found more than 13,000 compounds showing inhibition of parasite growth by at least 80% at 2 μM (6). More than 8,000 of these compounds showed potent activity against the multidrug resistant Dd2 line. Another recent study performed by Guiguemde et al. described the screening of more than 300,000 compounds using a phenotypic forward chemical genetic approach (7). This screening led to the identification of potent compounds against drug-resistant P. falciparum line. They further described a detailed profiling of 172 compounds and identified 19 new inhibitors of four validated drug targets and 15 novel binders among 61 malarial proteins.

There is a wide variety of structures that are and have been used as antimalarial drugs (8). Table 1 presents a summary of currently used antimalarial drugs (9).

Table 1

Currently Used Antimalarial Drugs.

Chloroquine (CID2719, Figure 1A) is a 4-aminoquinoline, fast-acting antimalarial drug and was introduced into clinical practice in 1947. It is used both for treatment and prevention of malaria. Amodiaquine is another 4-aminoquinoline, which is used against chloroquine-resistant P. falciparum (CRPF). Quinine (Figure 2A) was the first effective treatment for malaria caused by P. falciparum, appearing in therapeutics in the 17th century, when it was extracted from the bark of the cinchona tree. Several other arylamino alcohol antimalarial agents (such as Lumefantrine; Figure 1B) have been introduced to the clinic and are now commonly used in combination therapies with artemisinines. Antibiotics have also been shown to be potent antimalarial drugs (10). Doxycycline (Figure 2D) and Clindamycin (Figure 2E) are known as delayed death antimalarial drugs. They act in a delayed fashion and are only effective on the second generation of the parasite (typically after 4 days); thus, these drugs need to be used in combination with a fast- acting antimalarial drug.

Figure 2

Structures of Other Antimalarial Drugs Currently in Use. Structures of other antimalarial drugs currently in use: Doxycycline (D) and Clindamycin (E) are particularly important as they target the second generation of parasite in a so-called delayed death (more...)

A large proportion of the antimalarial drugs currently undergoing clinical evaluation are combinations of known substances or new structures in the 4-aminoquinoline, 8-aminoquinoline, and artemisinine classes; thus, these combination therapies are very likely to lead to similar toxicity and resistance issues (9). Only three small molecules undergoing clinical evaluation are known to act against yet unexploited targets in malaria chemotherapy. Fosmidomycin (Figure 3A) is active against P. falciparum via inhibition of a new target enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, involved in isoprenoid biosynthesis (11,12). T3 (SAR97267, Figure 3B) is a bisthiazolium salt that targets membrane biogenesis during intra-erythrocytic P. falciparum (13). One of the main drawbacks of these charged species is their low oral absorption and bioavailability. Thus, several prodrugs of T3 have been synthesized recently in order to overcome these barriers (14). Recently, spiroindolones have been described as potent antimalarial drugs with a new mode of action, and NITD609 (Figure 3C) has recently entered a phase I clinical trial (15). NITD609 works by inhibiting protein synthesis in P. falciparum and was optimized to address the metabolic liabilities of the lead compound, resulting in improved stability and exposure levels in animals. As a result, NITD609 is one of only a handful of molecules capable of completely curing mice infected with P. berghei, a model of blood-stage malaria.

Figure 3

Structure of Potential New Antimalarial Drugs. Fosmidomycin (A), T3 (B), and NITD609 (C) are known to act against yet unexploited targets in malaria chemotherapy and are currently undergoing clinical evaluation.

The discovery of new structural classes of antimalarial molecules with potential novel mode of action is greatly needed by the malaria research community to better understand the biology of the parasite and, thus, to better combat its deadly mechanisms.

2. Materials and Methods

2.2. Probe Chemical Characterization

The probe (ML238) was synthesized as shown in Scheme 1 using an aldol-based build/couple/pair strategy and full account of the synthesis of the macrocycle has already been reported by the DOS group at the Broad Institute (17). Full experimental details and characterization are provided in Appendix C.

Scheme 1

Synthesis of the Probe (ML238).

The solubility of the probe (ML238) was measured at room temperature and was found to be 124.8 μM in water but <1 μM in PBS.

The stability of the probe (ML238) in PBS (0.1% DMSO) was measured over 48 hours. We noticed that the concentration of the probe steadily increased over 48 hours to about 800% (data not shown). Since the solubility of the probe in PBS is low, we believe that the gradual increase in concentration is the direct result of more compound dissolving in PBS over time. In other words, when we performed the PBS stability assay under the recommended conditions, we measured the kinetic solubility of the probe in PBS and not its stability. Thus, we decided to increase the amount of DMSO so that the compound dissolves completely at the start of the stability assay and stays in solution during the course of the experiment. We tried 5%, 10% and 15% DMSO in PBS and followed stability of the probe over time. When 15% DMSO was used, the probe rapidly dissolved and remained completely soluble over 48 hours and the results from this experiment are shown in Figure 4. From these results, the probe seems to be stable in PBS since 100% is still present after 48 hours of incubation.

Figure 4

Stability Data for the Probe (ML238) in PBS (15% DMSO) Over 48 Hours.

After preparation as described in Section 2.3, full characterization of the probe (ML238) was analyzed by UPLC, ¹H and ¹³C NMR spectroscopy, and high-resolution LC mass spectrometry. The associated spectroscopic data is provided in Appendix D.

3. Results

3.1. Summary of Screening Results

3.1.1. NCGC Screen of the MLSMR Library

The delayed death qHTS screen was run at two time points corresponding to the 48-hour and 96-hour incubation periods. The median Z′ across all the plates was 0.70 and 0.57 for the 48-hour and 96-hour time points, respectively. The signal-to-background values were 22 and 12 for the 48-hour and the 96-hour time points, respectively. Each plate had two controls: azithromycin (an antimalarial exhibiting the delayed death phenotype) and chloroquine (a classical “fast-acting” antimalarial). These controls were added as 16-point, 2-fold titrations (in duplicate) starting at 18.6 μM for azithromycin and 28.7 μM for chloroquine. The controls exhibited consistent potencies throughout the screen. In addition to the control titrations, wells containing fixed concentrations of azithromycin and chloroquine were used to normalize the 96-hour and 48-hour data, respectively. In total, 223,059 compounds were tested at both time points, at six concentrations ranging from 28.7 μM to 1.84 nM.

Following the primary qHTS screen, the concentration-response curves were classified according to a heuristic scheme (18). This scheme allows us to distinguish active, inconclusive, and inactive compounds, based on efficacy and curve-fit parameters. Class 1 curves display a complete response with well-defined upper and lower asymptotes and an R2 > 0.9. Class 2 curves are incomplete, having only one asymptote and an R2 > 0.9. Class 3 curves show poor curve fits or have a single point of activity, and are considered as low confidence. Class 4 curves either have a curve fit below threshold activity or lack one altogether, and these are considered inactive. Each class is further categorized into subclasses based on the efficacy of the response (18).

Using curve class and efficacy, we defined compounds as differentially active if they satisfied either of the following two criteria:

1. Inactive (Class 4) at the 48-hour time point but active (Class 1.1, 1.2, 2.1) with an efficacy greater than 60% at the 96-hour time point.
2. Active (Class 1.1, 1.2, 2.1) with efficacy greater than 60% at both time points, but with 5-fold lower IC₅₀ at the 96-hour time point compared with the 48-hour time point.

From the 223,059 compounds screened, we used these constraints to identify 1,185 (0.53%) differential actives. Correspondingly, there were 121,502 (54.47%) inactive compounds (Class 4) at both time points. Figure 5 represents an overlay of the concentration-response curves (at the 96-hour time point) for the 1,185 differentially active compounds.

Figure 5

Overlay of the Concentration-response Curves for the 1,185 Active Compounds at 96 Hours.

3.1.2. Broad Screen of the DOS Informer Set I

Figure 6 displays the critical path for probe development.

Figure 6

Critical Path for HTS of the DOS Informer Set and Probe Development.

A screen of an informer set of 8000 DOS compounds was performed in duplicate in the black Greiner GNF clear-bottom, 384-well plates (Greiner Bio-One Ltd.) using a SYBR dye detection method. Screening compounds were distributed into plates by a Labcyte Echo liquid handling system (Sunnyvale, CA). Parasites were synchronized at ring stage using the sorbitol method, before performing the assay. Next, 40 μl of 1% parasitemia/1% hematocrit culture in RPMI containing 4.16 mg/ml Albumax was then robotically dispensed into wells, and the plates were briefly mixed and incubated for 72 hours in gas-controlled incubators. After incubation, 10 μl of 10X SYBR Safe DNA Stain in Lysis buffer (Saponin 0.16%, Tris-HCl [pH 7.5] 20 mM, EDTA [disodium salt] 5 mM, Triton X-100 6% v/v) was added robotically to the wells. The plates were covered with aluminum seals, briefly centrifuged at 1000 rpm for 1 minute, mixed, and then incubated overnight at room temperature. The plates were then centrifuged and read from below in a Wallac EnVision plate reader using the routine parameters for SYBR detection (EX 480 nm, EM 530 nM). The SYBR assay in 384-well format performed favorably in the NIH Genomics validation criteria for 384-well plate assays are detailed in NCGC Assay Guidance Manual (19). Z′ values were in the range of 0.65–0.75, with a signal-to-noise ratio of 1.8:2.5, and a standard deviation (SD) of control wells < 5%.

Primary hits were defined as compounds yielding a reduction in parasite growth of at least 90% in both duplicate assays. A total of 560 primary hits representing five scaffolds were identified and advanced through a four-point titration (1000, 303, 98, and 28 nM). One scaffold differentiated itself from the others, and this lead was confirmed via independent synthesis followed by iterative 12-point titrations. At the same time, untested derivatives of the lead and all of its stereoisomers were subject to full titration.

3.2. Dose Response Curves for Probe (ML238)

The dose-response curves for the probe (ML238) are presented in Figure 7.

Figure 7

Dose-Response Curves for the Probe (ML238). Dose-response curves for the probe (ML238) obtained using Luciferase assays against P. falciparum 3D7 (A) and Dd2 (B), and using a SYBR-green based assay in 3D7 (C) and Dd2 (D), low nanomolar to subnanomolar (more...)

The probe (ML238) displayed low nanomolar activities against both 3D7 and Dd2 lines of P. falciparum, when using a luciferase-based assay (Figures 7A and 7B, respectively). The probe displayed picomolar activities against both 3D7 and Dd2 lines of P. falciparum, when using a SYBR-green based assay (Figures 7C and 7D, respectively). The probe was found to have very low toxicity in a human cell line (HEPG2), with an EC₅₀ value of 34 μM (Figure 7E).

3.4. SAR Tables

The Broad Institute has synthesized close to 100,000 complex small molecules through DOS for use in HTS. This DOS collection consists of a variety of different chemical skeletons, and they are generally rich in sp³-hybrized carbon atoms compared to members in the MLPCN library. A subset of the DOS library called the ‘informer set’ (approximately 8,000 compounds) was screened at the Broad Institute in the live/dead assay against both Dd2 and 3D7 malarial strains. This effort led to the identification of the hit compound (CID44487502, Figure 8).

Figure 8

Stereochemical SAR Heat Map of the HTS Hit Compound (CID44487502) in Dd2.

A number of related analogs (based both on stereochemistry and building blocks) were then screened at the Broad Institute to identify SAR trends and to optimize activity. A vast majority of these analogs were also screened at NCGC, and the data from both institutions were remarkably close. We report data from NCGC in Figure 8 and in Tables 3–7. In Table 8, we compare data from the Broad Institute, and NCGC for the probe (ML238) and a limited set of analogs to illustrate data reproducibility across institutions and assay platforms.

Table 3

Summary of Stereochemical SAR (SSAR) on the Hit Compound.

Table 4

Summary of SAR on the Urea Portion.

Table 5

Summary of SAR on the Sulfonamide Portion.

Table 6

Summary of SAR on the Exocyclic Substituents of the Lactam Nitrogen Atom.

Table 7

SAR Study on Multiple Diversity Sites.

Table 8

Summary of Data Obtained in the SYBR Green-based Fluorescence Assay Versus the Luciferase Assay Against P. falciparum.

The informer set that was screened included all the stereoisomers of the hit compound and additional related analogs. The initial hit has four stereocenters; hence, there is a total of 16 possible stereoisomers. Results from all 16 stereoisomers are summarized in Table 3. The stereo-structure-activity relationship (SSAR) can be readily visualized from the SSAR heat map shown in Figure 8. Of the 16 stereoisomers, two stereoisomers demonstrated < 1 μM activity against the resistant Dd2 cell line. These two compounds have the same configuration (S,R,R) on all three stereocenters (C2, C8, and C9, respectively) inside the macrocycle with different configurations at the exocylic stereocenter (C11). SRRR-1 was the most active isomer with 0.49 μM activity (Table 3, entry 5); however, this isomer showed some toxicity in the HepG2 assay (4.26 μM). SRRS-1 (Table 3, entry 11) had very similar activity (0.66 μM) against Dd2 and was totally inactive in the HepG2 assay; hence, this stereoisomer was chosen for further studies.

Having identified the optimal stereoisomer, we focused on the SAR of the hit compound (CID44487502) around a) the urea, b) the sulfonamide, and c) the exocyclic primary alcohol. Table 4 presents the SAR at the phenyl urea position. Replacing the phenyl urea with an aniline (Table 4, entry 2) led to a slight increase in potency in 3D7 P. falciparum but to a slight decrease in potency in Dd2 P. falciparum. Replacement of the phenyl urea by an isopropyl urea (Table 4, entry 3) led to a 3-fold decrease in activity. Using a 4-isoxazolyl urea (Table 4, entry 4) in place of the phenyl urea improved potency slightly. A more rigid mimic of the phenyl urea portion, a benzoxazol analog (Table 4, entry 5) was also synthesized, but led to a decrease in activity and an increase in toxicity.

SAR study at the phenyl urea portion did not lead to significant improvements in activity, thus, we turned our attention to the sulfonamide portion of the hit compound (Table 5). Moving the fluoro substituent on the phenyl sulfonamide portion from the para- (Table 5, entry 1) to the meta- position (Table 5, entry 2) or the ortho- position (Table 5, entry 3) had a great influence on the activity. The meta-fluoro phenyl sulfonamide analog was found to be approximately 10-fold more potent than the hit compound but led to low micromolar toxicity in HEPG2. Introduction of a 3-pyridyl sulfonamide ring (Table 5, entry 4) in order to improve solubility led to a 15-fold decrease in activity. Replacing the para-fluoro substituent by a para-trifluoromethyl substituent (Table 5, entry 5) also led to a decrease in activity in both 3D7 and Dd2 P. falciparum lines. Next, we replaced the sulfonamide portion by carbamates (Table 5, entries 6–9) or amine (Table 5, entry 10), but these modifications did not improve potency.

We also studied SAR on the exocyclic substituents attached to the lactam nitrogen atom (Table 6). Removing the methyl at C-11 (Table 6, entry 2) led to a decrease in activity. Replacing the hydroxymethylene portion by a methyl group (Table 6, entry 3) led to a 10-fold increase in activity but also increased toxicity. Using the hydroxymethylene portion for further derivatization as esters (Table 6, entries 4–5) or ethers (Table 6, entries 6–10) led to more potent compounds, showing that this portion of the molecule could be exploited to improve activity through hydrophobic interactions. Particularly, the tert-butyl ether analog (Table 6, entry 6) was found to be the most potent compound, showing subnanomolar activity in the 3D7 strain after 96 hours. The tert-butyl dimethyl silyl analog (Table 6, entry 11) was also found to be very active with IC₅₀ values of 0.8 nM and 6 nM against 3D7 P. falciparum and Dd2 P. falciparum, respectively. In the tert-butyl dimethyl silyl series, the presence of the (S)-methyl group was found to be critical as removal (Table 6, entry 12) or inversion of its configuration (Table 6, entry 13) led to a high decrease in potency. In order to improve solubility of the molecule, we also introduced a dimethylamine group (Table 6, entry 14). Although this analog was not as potent as the t-butyl and TBDMS analogs, it still had low nanomolar potencies (2.4 nM in 3D7 and 13 nM in Dd2 after 96 hours) and was found to have good aqueous solubility (124.8 μM in water and <1 μM in PBS) and good plasma stability (98% in human and 83.1% in mouse). This compound was later designated as the probe (ML238). The azido analog (Table 6, entry 15), which was used as an intermediate in the synthesis of the dimethylamine analog, was also tested and found to be less active and less soluble than the dimethylamine analog.

Table 7 presents several analogs synthesized by modifying multiple diversity sites. Maintaining the phenyl urea on the western portion of the molecule (R₁) and the benzoyl ester on the lactam nitrogen side chain (R₂), and replacing the 4-fluoro sulfonamide by different aliphatic carbamates (Table 7, entries 1–2) led to an approximate 10-fold decrease in activity. Using a phenyl urea as R₁, a PMB-protected ether on the lactam nitrogen side chain and a p-methoxybenzylamine as R₃ (Table 7, entry 3) did not improve activity. Using a 4-isoxazolyl urea as R₁, an isopropyl as R₂ and a p-fluorophenylsulfonamide as R₃ (Table 7, entry 4) led to a reasonably active analog along with low micromolar toxicity.

Table E1 (shown in Appendix E) presents other analogs synthesized during the course of the SAR study. The macrocyclization step involves a ring closing metathesis that generally afforded a mixture of E and Z olefins. In most cases, the two diastereoisomers were not separable on silica gel and were subsequently hydrogenated. In the case of the SRRS stereoisomer, the two diastereoisomers were separable on silica gel, although it was impossible to assign the stereochemistry at the olefin position due to very complex NMR signals. Both stereoisomers (Table E1, entries 1–2) were tested but showed similar potencies.

Table 8 presents a comparison of the data obtained at the NCGC (Luciferase Assay) with the original results obtained at the Broad Institute (SYBR Assay) for the probe (ML238) and nine analogs. Overall, a good correlation is observed between the two assays performed at different institutions. In the SYBR green-based, fluorescence assay (live/dead assay), the probe (ML238; Table 8, entry 1) showed picomolar activity against both 3D7 (0.24 nM) and Dd2 P. falciparum (0.26 nM), while showing low nanomolar activity in the luciferase-based assay. Another analog (Table 8, entry 5), possessing a PMB-protected ether in place of the dimethylamine substituent, also showed picomolar activity.

Table F1 (shown in Appendix F) presents the data obtained by the Assay Provider for the probe (ML238) and five analogs against 3D7 and Dd2 lines using flow cytometry. In general, this data matches well with the data obtained by using other platforms (SYBR and luciferase).

Figure 9 presents a visual summary of the SAR performed on the 14-membered ring scaffold. Stereochemistry, as well as three points of diversity were investigated and led to the identification of a subnanomolar antimalarial probe (ML238). This probe is developed from the DOS compound collection at the Broad Institute; hence, it is structurally unique compared to all the other compounds pursued by both academic institutions and pharmaceutical companies for malaria. This DOS-based probe possesses good water solubility and exhibits picomolar activity against 3D7 and the chloroquine-resistant Dd2 P. falciparum. Research efforts are currently underway to advance this probe (ML238) to subsequent stages of drug discovery.

Figure 9

Summary of SAR Performed on the Probe (ML238; 54 Synthetic Analogs). Summary of SAR studies showing that the SRRS stereoisomer series as well as aromatic groups at the urea and sulfonamide positions are preferred for activity, while amines are preferred (more...)

The probe (ML238/MLS003448149) as well as five analogs (MLS003448150, MLS003448151, MLS003448152, MLS003448153, MLS003448154) have been submitted to the MLSMR collection.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Investigation into relevant prior art entailed searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO) PatFT and AppFT, and World Intellectual Property Organization (WIPO) databases. The search terms applied and hit statistics are provided below in Table 9. Abstracts were obtained for all references returned and were analyzed for relevance to the current project. The searches were performed on, and are current as of, March 2, 2011.

Table 9

Search Strings and Databases Employed in the Prior Art Search.

In the field of antimalarial drugs, all classes of compounds, from the 4-aminoquinoline and arylamino alcohol to the more recently developed artemisinins, have or are in the process of suffering development of resistance in various malaria lines. The discovery of new structural classes of antimalarial drugs, acting on different targets or different stages of the malaria parasite development, is of great interest. The probe (ML238) is derived from the Broad Institute DOS chemistry effort; thus, its structure is in a completely new class. The probe shows subnanomolar IC₅₀ values in 3D7 and the chloroquine-resistant Dd2 strain of P. falciparum constituting an improvement over existing antimalarial drugs, which typically show low nanomolar IC₅₀ values (20). In our hands, Atovaquone was found to have an IC₅₀ of 0.089 ± 0.030 nM in 3D7 and 0.097 ± 0.046 nM in Dd2. Chloroquine was found to have an IC₅₀ of 13.0 ± 12.0 nM in 3D7 and 252 ± 119 nM in Dd2. Thus, ML238 is more potent than chloroquine and compares with atovaquone.

4.2. Mechanism of Action Studies

4.2.1. Assessing Apicoplast Disruption Using a Whole Cell Imaging Assay

Delayed death inhibitors specifically target the parasite apicoplast; an organelle of cyanobacterial origin that employs prokaryotic-derived transcription and translation machinery (21). During asexual blood-stage development, this organelle adopts an elongated and branched structure as the parasite matures into its trophozoite form, then segregates into individual organelles that parse with the individual daughter merozoites prior to their release from the infected erythrocyte. To verify if the compounds do interfere with the apicoplast, we used a whole-cell imaging screen with a recombinant parasite line that has GFP-labeled apicoplasts. This line, ACP_L-GFP, has already been developed and benefits from chromosomal integration of the GFP fusion, producing excellent signal homogeneity. Antibiotics (such as zithromycin and doxycycline) that are known to interfere with the apicoplast have been observed to disrupt the morphology of the apicoplast in this GFP-expressing line (see Figure 10) (22).

Figure 10

Effect of Compound Treatment on Apicoplast Mmorphology. Parasites expressing GFP targeted to the apicoplast, 3D7 ACP_(L)-GFP, were treated with the indicated compounds in 200 μL of culture media at a 1% hematocrit. At 96 hours, 30 μl of (more...)

For this imaging screen, ACP_L-GFP parasites were cultured with compounds tested at approximately 2X their EC₅₀ value at 96 hours to screen for inhibition of apicoplast elongation and segregation. Azithromycin was used as a positive control, while parasites treated with drug vehicle (medium containing 0.05 – 0.1% DMSO) served as a negative control. Assays began with synchronized ring stages, and the parasites were harvested on Day 4. The parasites were incubated with 1 mg/ml Hoechst 33342 nuclear stain, and 20 nM Mitotracker-Red, washed, and deposited on poly-lysine coated slides. Images were taken on a Nikon Ti inverted microscope.

The untreated parasites exhibit bright staining for all three markers. Chloroquine is a fast-acting drug that inhibits heme detoxification in the parasite digestive vacuole. Chloroquine-treated parasites exhibit small, but intact apicoplast structures. The mitochondria are metabolically inactive and do not stain. Azithromycin is a delayed death-inducing drug that inhibits the apicoplast encoded 50s ribosomal subunit. The apicoplast has been disrupted in azithromycin treated-parasites, although many still display intact mitochondria. None of the experimental compounds including the probe (ML238) appeared to disrupt the apicoplast. The apicoplast morphology resembles the phenotype seen with chloroquine treatment; it is much smaller than in the untreated parasites but still present. Mitochondrial staining was generally diminished, though not entirely absent, in parasites treated with the probe (ML238) and some of its analogs.

As of now, the mode of action of the probe (ML238) is still unknown. But its structure, very different from known antimalarial drugs and probes, suggests that its mode of action could involve a new target. In this context, target identification will help understanding the mechanism of action of the probe and will be a priority in the near future.

5. References

1.

Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature. 2005 Mar 10;434(7030):214–7. [PMC free article: PMC3128492] [PubMed: 15759000]

2.

Fidock DA. Drug discovery: priming the antimalarial pipeline. Nature. 2010 May 20;465(7296):297–8. [PubMed: 20485420]

3.

Wikipedia contributors. Malaria [Internet] Wikipedia, The Free Encyclopedia. [Accessed 05 April 2011]. Available at: http://en​.wikipedia.org/wiki/Malaria.

4.

White NJ. Qinghaosu (artemisinin): the price of success. Science. 2008 Apr 18;320(5874):330–4. [PubMed: 18420924]

5.

Dondorp AM, Yeung S, White L, Nguon C, Day NP, Socheat D, von Seidlein L. Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol. 2010 Apr;8(4):272–80. Epub 2010 Mar 8. [PubMed: 20208550]

6.

Gamo FJ, Sanz LM, Vidal J, de Cozar C, Alvarez E, Lavandera JL, Vanderwall DE, Green DV, Kumar V, Hasan S, et al. Thousands of chemical starting points for antimalarial lead identification. Nature. 2010 May 20;465(7296):305–10. [PubMed: 20485427]

7.

Guiguemde WA, Shelat AA, Bouck D, Duffy S, Crowther GJ, Davis PH, Smithson DC, Connelly M, Clark J, Zhu F, Jiménez-Díaz MB, et al. Chemical genetics of Plasmodium falciparum. Nature. 2010 May 20;465(7296):311–5. [PMC free article: PMC2874979] [PubMed: 20485428]

8.

Robert A, Benoit-Vical F, Dechy-Cabaret O, Meunier B. From classical antimalarial drugs to new compounds based on the mechanism of action of artemisinin. Pure Appl. Chem. 2001;73(7):1173–88.

9.

Schlitzer M, Ortmann R. Feeding the antimalarial pipeline. ChemMedChem. 2010 Nov 8;5(11):1837–40. [PubMed: 20922746]

10.

Pradel G, Schlitzer M. Antibiotics in malaria therapy and their effect on the parasite apicoplast. Curr Mol Med. 2010 Apr;10(3):335–49. [PubMed: 20331433]

11.

Lell B, Ruangweerayut R, Wiesner J, Missinou MA, Schindler A, Baranek T, Hintz M, Hutchinson D, Jomaa H, Kremsner PG. Fosmidomycin, a novel chemotherapeutic agent for malaria. Antimicrob Agents Chemother. 2003 Feb;47(2):735–8. [PMC free article: PMC151759] [PubMed: 12543685]

12.

Steinbacher S, Kaiser J, Eisenreich W, Huber R, Bacher A, Rohdich F. Structural basis of fosmidomycin action revealed by the complex with 2-C-methyl-D-erythritol 4-phosphate synthase (IspC). Implications for the catalytic mechanism and anti-malaria drug development. J Biol Chem. 2003 May 16;278(20):18401–7. Epub 2003 Mar 5. [PubMed: 12621040]

13.

Nicolas O, Margout D, Taudon N, Wein S, Calas M, Vial HJ, Bressolle FM. Pharmacological properties of a new antimalarial bisthiazolium salt, T3, and a corresponding Prodrug, TE3. Antimicrob Agents Chemother. 2005 Sep;49(9):3631–9. [PMC free article: PMC1195427] [PubMed: 16127032]

14.

Caldarelli SA, Duckert JF, Wein S, Calas M, Périgaud C, Vial H, Peyrottes S. Synthesis and evaluation of bis-thiazolium salts as potential antimalarial drugs. ChemMedChem. 2010 Jul 5;5(7):1102–9. [PubMed: 20540062]

15.

Rottmann M, McNamara C, Yeung BK, Lee MC, Zou B, Russell B, Seitz P, Plouffe DM, Dharia NV, Tan J, et al. Spiroindolones, a potent compound class for the treatment of malaria. Science. 2010 Sep 3;329(5996):1175–80. [PMC free article: PMC3050001] [PubMed: 20813948]

16.

Plouffe D, Brinker A, McNamara C, Henson K, Kato N, Kuhen K, Nagle A, Adrián F, Matzen JT, Anderson P, et al. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9059–64. Epub 2008 Jun 25. [PMC free article: PMC2440361] [PubMed: 18579783]

17.

Marcaurelle LA, Comer E, Dandapani S, Duvall JR, Gerard B, Kesavan S, Lee MD 4th, Liu H, Lowe JT, Marie JC, et al. An aldol-based build/couple/pair strategy for the synthesis of medium- and large-sized rings: discovery of macrocyclic histone deacetylase inhibitors. J Am Chem Soc. 2010 Dec 1;132(47):16962–76. Epub 2010 Nov 10. [PMC free article: PMC3004530] [PubMed: 21067169]

18.

Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11473–8. Epub 2006 Jul 24. [PMC free article: PMC1518803] [PubMed: 16864780]

19.

Eli Lilly and Company and the National Institutes of Health Chemical Genomics Center. Assay Guidance Manual. NCGC High Throughput Screening Assay Guidance Criteria. 2008. [Access Date: 31 March 2011]. Available at: http://www​.ncgc.nih.gov/guidance/index​.html.

20.

Mu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, et al. Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet. 2010 Mar;42(3):268–71. Epub 2010 Jan 31. [PMC free article: PMC2828519] [PubMed: 20101240]

21.

Lim L, McFadden GI. The evolution, metabolism and functions of the apicoplast. Philos Trans R Soc Lond B Biol Sci. 2010 Mar 12;365(1541):749–63. [PMC free article: PMC2817234] [PubMed: 20124342]

22.

Dahl EL, Rosenthal PJ. Multiple antibiotics exert delayed effects against the Plasmodium falciparum apicoplast. Antimicrob Agents Chemother. 2007 Oct;51(10):3485–90. Epub 2007 Aug 13. [PMC free article: PMC2043295] [PubMed: 17698630]

23.

Kato S, Harada H, Morie TM. J Chem Soc Perkin Trans. 1997:3219–25.

Appendix A. Assay Summary Table

Appendix B. Detailed Assay Protocols

Appendix C. Experimental Details of the Synthesis of the Probe

Preparation of tert-Butyl methyl(2-oxoethyl)carbamate (1): A flame-dried, three-neck, 3-liter, round-bottom flask containing a mechanical stirrer (central port), internal temperature probe, and addition funnel was charged with oxalyl chloride (176 ml, 2.01 mol, 1.5 equiv) and dichloromethane (634 ml). The flask was cooled to −70 ºC and dimethyl sulfoxide (286 ml, 4.02 mol, 3.0 equiv) was introduced at a rate that maintained an internal temperature below −65 ºC. An initial exotherm was observed, and the temperature did rise. After addition of approximately 50 ml, the addition was slowed to allow the reaction to cool. This type of intermittent addition strategy was followed to prevent overheating. The addition proceeded over 2.5 hours. Then, the mixture was stirred for an additional 15 minutes, after which t-butyl 2-hydroxyethyl (methyl)carbamate (235 g, 1.34 mol, 1.0 equiv) in methylene chloride (CH₂Cl_2; 700 ml) was added over 1 hour, followed by slow addition of triethylamine (841 ml, 6.04 mol, 4.5 equiv) over 30 minutes. Both of these reactants were added at a rate that does not cause any increase in internal temperature. After addition of the alcohol solution the reaction was cloudy. Upon initial addition of triethylamine, the reaction became colorless/clear; however, at the end, the reaction was milky white. The reaction was monitored by thin-layer chromatography (TLC; Hex:EtOAc 1:1) until the reaction was deemed complete (2.5 hours additional, −70 ºC). Saturated sodium bicarbonate solution (500 ml) was introduced to quench the reaction at −78 °C. The reaction was warmed to room temperature, and the phases were separated. The combined organics were washed with water (2 × 100 ml), dried over magnesium sulfate, and concentrated. Distillation (87–89 ºC, 3 mm Hg) provided aldehyde 1 (22.2 g, 128 mmol) as a colorless oil, which matched all data reported by Kato et al. (23).

Preparation of (2S,3R)-(1S,2R)-1-phenyl-2-(N,2,4,6-tetramethylphenylsulfonamido)propyl 4-((tert-butoxycarbonyl)(methyl)amino)-3-hydroxy-2-methylbutanoate (3): An oven-dried, 3-liter, three-neck, round-bottom flask was equipped with a mechanical stirrer and temperature probe. Under a positive flow of nitrogen, the vessel was charged with (1S,2R)-1-phenyl-2-(N,2,4,6-tetramethylphenylsulfonamido)propyl propionate (109 g, 270 mmol) and CH₂Cl₂ (932 ml). The reaction was cooled to −78 ºC with constant agitation at which point triethylamine (Et₃N; 112 ml, 810 mmol) was added dropwise, maintaining an internal reaction temperature no greater than −65 ºC (approximately 10 minutes). After 15 minutes of additional stirring, a solution of dicyclohexylboron triflate (1.0 M in CH₂Cl₂, 176 g, 540 mmol) was added dropwise, maintaining an internal reaction temperature below −67 ºC (approximately 30 minutes). The resulting yellow enolate reaction solution was stirred at −78 ºC for an additional 2 hours. At this point t-butyl methyl(2-oxoethyl)carbamate 1 (68.1 ml, 405 mmol) was added dropwise at a rate so as to maintain an internal temperature below −67 ºC (approximately 15 minutes). The reaction mixture was stirred at −78 °C for 2 hours and then was allowed to warm to 0 °C for 1 hour. The reaction was quenched by addition of methyl alcohol (MeOH; 1.09 L) and pH 7 buffer (130 ml). Then, aqueous hydrogen peroxide (35% by wt, 130 ml, 1.5 mol) was slowly added such that the internal reaction temperature did not exceed 20 ºC. After the addition was complete, the mixture was allowed to warm to ambient temperature where it was stirred for 1 hour. The resultant slurry was then concentrated in vacuo, and the residue was partitioned between water (550 ml) and CH₂Cl₂ (500 ml). The aqueous layer was extracted with CH₂Cl₂ (4 × 300 ml), and the combined organic layers were washed with water (100 ml) followed by brine (100 ml). The organic layer was dried over anhydrous MgSO₄ and the solvent was removed in vacuo to yield 1-phenyl-2-(N,2,4,6-tetramethylphenylsulfonamido)propyl 4-(tert-butoxycarbonyl(methyl)amino)-3-hydroxy-2-methylbutanoate 3 (156 g, 99%) as a yellow oil, which was deemed sufficiently pure and carried forward without any further purification.

(R,S,S,R)-(+)-3: TLC: R_f = 0.34 (30% EtOAc in hexanes). [α]_D²⁰ +6.42 (c 1.0, CHCl₃). IR (cm⁻¹) 3446 (br), 2975, 2930, 1778, 1682, 1481, 1455, 1390, 1366, 1294, 1210, 1154, 1109, 1050, 1015, 990, 969, 925, 879, 763, 749, 703. ¹H NMR (500 MHz, CDCl₃, 60 °C) δ 7.20 (d, J = 7.4, 2H), 7.05 (d, J = 7.5, 1H), 6.87 (s, 1H), 5.79 (d, J = 5.3, 1H), 4.07 (s, 1H), 3.90 (s, 1H), 3.30 (s, 2H), 2.90 (s, 3H), 2.78 (s, 3H), 2.68-2.56 (m, 1H), 2.45 (s, 6H), 2.29 (s, 3H), 1.45 (s, 9H), 1.31 (d, J = 6.8, 2H), 1.19 (d, J = 7.2, 1H). ¹³C NMR (125 MHz, CDCl₃, 60 ºC) δ 173.8, 142.5, 140.6, 138.3, 132.9, 132.1, 128.6, 128.1, 126.5, 126.3, 80.2, 78.4, 73.1, 58.8, 55.9, 53.3, 47.3, 44.3, 36.3, 35.7, 28.6, 25.7, 24.2, 22.7, 20.9, 13.6, 12.3, 8.9. HRMS (ESI) calcd for C₂₁H₃₀N₂O₆Na [M + Na]⁺: 429.1996. Found: 429.2000.

Preparation of (2S,3R)-4-((tert-butoxycarbonyl)(methyl)amino)-3-hydroxy-2-methylbutanoic acid (4): A 3-liter, three-neck, round-bottom flask was equipped with an overhead stirrer, an internal temperature probe, and an addition funnel. The vessel was charged with the crude 1-phenyl-2-(N,2,4,6-tetramethylphenylsulfonamido)propyl-4-(tert-butoxycarbonyl-(methyl)amino)-3-hydroxy-2-methylbutanoate (156.0 g, 270 mmol, 1.0 equiv) and 1:1 t-BuOH/MeOH (1700 ml) was added and cooled 0 ºC. A solution of hydrogen peroxide (H₂O_2; 30% in water, 166 ml, 1623 mmol, 6.0 equiv) was added via addition funnel, followed by aqueous sodium hydroxide (NaOH; 1.0 M, 811 ml, 811 mmol, 3.0 equiv), which was added at such a rate that the internal temperature did not rise above 10 ºC. The mixture was warmed to room temperature slowly and allowed to stir overnight. In the morning, organic solvents were removed in vacuo. The resulting aqueous layer was diluted with water (300 ml) and extracted with ethyl acetate (EtOAc; 4 × 200 ml) to remove the auxiliary. The resulting aqueous layer was then acidified with 6 M HCl to pH 4 and extracted with EtOAc (5 × 250 ml). The combined EtOAc extracts were dried (MgSO₄), filtered and concentrated, to provide the desired product 4 as a yellow oil (66.0 g, 99%). While the crude product was carried on to the next step without further purification, an analytical sample was obtained by chromatography on silica gel. (S,R)-(+)-4: TLC: R_f = 0.34 (33% EtOAc in hexanes). [α]_D²⁰ +2.1 (c 1.0, CHCl₃). IR (cm⁻¹) 3446 (br), 2977, 2933, 1678, 1483, 1456, 1393, 1367, 1249, 1154, 1056, 874, 766. ¹H NMR (500 MHz, CDCl₃, 60 °C) δ 3.99 (s, 1H), 3.91 (dd, J = 5.7, 11.4, 1H), 3.39 (m, 1H), 2.95 (s, 1.5H), 2.93 (s, 1.5H), 2.59 (dd, J = 6.2, 12.0, 1H), 1.48 (s, 4.5H), 1.47 (s, 4.5H), 1.28 (dd, J = 7.2, 3H). ¹³C NMR (125 MHz, CDCl₃, 60 °C) δ 181.1, 158.2, 81.0, 72.6, 53.4, 43.8, 36.2, 28.7, 15.0. HRMS (ESI) calcd for C₁₁H₂₁NO₅Na [M + Na]⁺: 270.1312. Found: 270.1312.

Preparation of (2S,3R)-4-((tert-butoxycarbonyl)(methyl)amino)-3-((tert-butyldimethylsilyl)oxy)-2-methylbutanoic acid (5): An oven dried, 5-liter, three-neck, round-bottom flask equipped with a magnetic stirrer and an internal temperature probe was charged with 4-(tert-butoxycarbonyl(methyl)amino)-3-hydroxy-2-methylbutanoic acid 4 (58.7 g, 237 mmol, 1.0 equiv), CH₂Cl₂ (concentration of 4 = 0.1 M) and 2,6-lutidine (91 ml, 783 mmol, 3.3 equiv) under a positive flow of nitrogen. The reaction mixture was cooled to −78 ºC and tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) (120 ml, 522 mmol, 2.2 equiv) was added dropwise over 20 minutes. The reaction mixture was stirred at −78 ºC for 1 hour and quenched with aqueous saturated sodium bicarbonate (NaHCO₃;500 ml). The layers were separated, and the aqueous layer was extracted with diethyl ether (2 × 200 ml). The combined organic extracts were washed with saturated ammonium chloride (NH₄Cl; 2 × 500 ml) and brine (2 × 500 ml), dried (MgSO₄), filtered, and the solvent evaporated. The residue was dissolved in MeOH:THF (1:1) and cooled to 0 ºC. An aqueous solution of potassium carbonate (K₂CO₃; 1.5–2.2 equiv) was added, and the mixture was stirred at 0 ºC for 1 hour. The reaction mixture was concentrated to remove volatiles, and the remaining aqueous layer was extracted with EtOAc. The organic layer was washed with 1 N HCl, then dried over MgSO₄, filtered, and concentrated in vacuo to yield the crude product as a clear oil, which was co-evaporated with toluene to remove excess TBSOH and dried to provide the TBS ether 5 as a clear oil (74.1 g, 83%). (S,R)-(+)-5: TLC: R_f = 0.67 (33% EtOAc in hexanes). [α]_D²⁰ +3.7 (c 1.0, CHCl₃). IR (cm⁻¹) 2978, 1649, 1459, 1395, 1285, 1157, 880. ¹H NMR (500 MHz, CDCl₃, 60 ºC) δ 4.15 (br s, 1H), 3.35 (dd, J = 14.5, 6.0 Hz, 1H), 3.23 (br s, 1H), 2.88 (s, 3H), 2.65 (br s, 1H), 1.44 (s, 9H), 1.21 (d, J = 7.0, 1H), 0.89 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 60 ºC) δ 176.8, 156.4, 80.3, 72.4, 52.9, 43.9, 36.7, 28.7, 26.0, 18.2, 12.8, −4.2, −4.7. HRMS (ESI) calcd for C₁₇H₂₅NO₅SiNa [M + Na]⁺: 384.2177. Found: 384.2167.

Preparation of tert-butyl ((2R,3S)-2-((tert-butyldimethylsilyl)oxy)-4-(((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)amino)-3-methyl-4-oxobutyl)(methyl)carbamate (6): An oven-dried, 3-liter, 3-neck, round-bottom flask was equipped with an overhead stirrer, addition funnel, and a temperature probe. Under a positive flow of nitrogen, the vessel was charged with 4-(tert-butoxycarbonyl(methyl)amino)-3-(tert-butyldimethylsilyloxy)-2-methylbutanoic acid 5 (38.1 g, 105 mmol, 1.0 equiv) dissolved in CH₂Cl₂ (80% of total solvent, final concentration of 5 = 0.2 M), followed by benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP; 60.4 g, 116 mmol, 1.1 equiv), and diisopropyl ethylamine (DIPEA;55.3 ml, 316 mmol, 3.0 equiv). The resulting mixture was cooled in an ice bath before 1-(4-methoxybenzyloxy)propan-2-amine (24.7 g, 127 mmol, 1.2 equiv) was added as a solution in CH₂Cl₂ (remaining 20% of total solvent) by addition funnel. The rate of addition was controlled so as to maintain an internal temperature between 3–5 ºC. When the addition was complete, the mixture was warmed to ambient temperature and allowed to stir for 15 hours. The reaction was quenched with water, and extracted with CH₂Cl₂. The combined organic extracts were dried over MgSO₄, filtered, and concentrated. The yellow oil was taken up in Et₂O (500 ml), and the phosphoramide byproducts were removed via filtration. The solvent was removed in vacuo, and the crude product was isolated. Flash chromatography on silica gel (4:1 Hexanes/EtOAc to 7:3 Hexanes/EtOAc) gave the product 6 as a colorless oil (51.9 g, 91%). (S,S,R)-6: [α]_D²⁰ −16.5 (c 1.0, CHCl₃). IR (cm⁻¹) 3365, 2930, 1696, 1670, 1513, 1460, 1390, 1365, 1248, 1156. ¹H NMR (500 MHz, CDCl₃, 55 ºC) δ 7.22 (d, J = 8.5, 2H), 6.86 (d, J = 8.5, 2H), 6.75 (br s, 1H, NH), 4.46 (d, J = 11.7, 1H), 4.41 (d, J = 11.7, 1H), 4.22-4.15 (m, 1H), 4.05-3.98 (m, 1H), 3.80 (s, 3H), 3.39 (d, J = 4.6, 2H), 3.34 (dd, J = 6.4, 14.0, 1H), 3.18 (dd, J = 6.3, 14.0, 1H), 2.88 (s, 3H), 2.41 (dq, J = 2.9, 7.3, 1H), 1.46 (s, 9H), 1.22 (d, J = 7.2, 3H), 1.18 (d, J = 6.7, 3H), 0.91 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 55 ºC) δ 173.4, 159.4, 156.0 (br), 130.6, 129.1 (2C), 113.9 (2C), 79.7 (br), 72.9, 72.8, 72.6 (br), 55.3, 53.5, 44.8, 44.5, 36.6 (br), 28.5 (3C), 25.9 (3C), 18.0, 17.9, 15.6, −4.5, −4.9. HRMS (ESI) calcd for C₂₈H₅₀N₂NaO₆Si [M + Na]⁺: 561.3330. Found: 561.3498.

Preparation of tert-butyl ((2R,3R)-2-((tert-butyldimethylsilyl)oxy)-4-(((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)amino)-3-methylbutyl)(methyl)carbamate (7): An oven-dried, 2-liter, one-necked, round-bottom flask was equipped with a magnetic stirrer. Under a positive flow of nitrogen, the flask was charged with tert-butyl 2-(tert-butyldimethylsilyloxy)-4-(1-(4-methoxybenzyloxy)propan-2-ylamino)-3-methyl-4-oxobutyl(methyl)carbamate 6 (36.0 g, 66.8 mmol, 1.0 equiv) and anhydrous tetrahydrofuran (THF; final concentration 0.1 M). Borane dimethylsulfide complex (BH₃·DMS) (31.7 ml, 334 mmol, 5.0 equiv) was added dropwise via syringe. Afterwards, the reaction mixture was heated at 65 ºC for 5 hours. After cooling to ambient temperature, excess hydride was quenched by the careful addition of MeOH. The mixture was concentrated under reduced pressure to afford a colorless oil, which was then co-evaporated with MeOH three times to remove excess B(OMe)₃. The oil was then re-dissolved in MeOH and 10% aqueous potassium sodium tartrate (2:3 ratio, final concentration 0.067 M). The resulting slurry was heated at reflux for 12 hours. The volatiles were removed under reduced pressure, and the aqueous layer was extracted with EtOAc (3 × 200 ml). The combined organic layers were washed with brine (1 × 200 ml), dried over magnesium sulfate, filtered and concentrated to provide the desired amine 7 as a colorless oil (31.5 g, 90%). (S,R,R)-(+)-7: [α]_D²⁰ −10.65 (c 2.46, CHCl₃). IR (cm⁻¹) 3010, 2929, 1693 1513 1461, 1389, 1247, 1152. ¹H NMR (500 MHz, CDCl₃, 60 °C) δ 7.19 (d, J = 8.5, 2H), 6.82 (d, J = 8.5, 2H), 4.40 (s, 2H), 3.89 (m, 1H), 3.74 (s, 3H), 3.29 (m, 4H), 2.98 (dd, J = 13.5, 7.5, 1H), 2.85 (m, 4H), 2.63 (m, 1H), 2.41 (dd, J = 11, 9, 1H), 1.72 (m, 1H), 1.41 (s, 9H), 0.98 (d, J = 6.5, 1H), 0.92 (d, J = 6.5, 1H), 0.86 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H). ¹³C NMR (125 MHz, CDCl₃, 60 °C) δ 159.3, 155.7, 130.8, 129.1, 113.9, 79.1, 74.6, 73.3, 72.8, 55.2, 52.5, 52.0, 49.4, 38.7, 36.4, 28.6, 26.0, 18.0, 17.6, 13.7, −4.5. HRMS (ESI) calcd for C₂₈H₅₃N₂O₅Si [M + H]⁺: 525.3718. Found: 525.3698.

Preparation of tert-butyl ((2R,3R)-2-((tert-butyldimethylsilyl)oxy)-4-(N-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-5-nitro-2-((S)-pent-4-en-2-yloxy)benzamido)-3-methylbutyl)(methyl)carbamate (8): A solution of starting material 7 (15.0 g, 28.6 mmol, 1.0 equiv) and diisopropyl ethylamine (DIEA;19.9 ml, 114 mmol, 4.0 equiv) in CH₂Cl₂ (80% of total solvent volume, final concentration of 7 = 0.12 molar) was cooled to 0 ºC in an ice/water bath. Then, the acid chloride (10.0 g, 37.2 mmol, 1.3 equiv) was added to the reaction mixture as a solution in CH₂Cl₂ (remaining 20% of solvent volume). Upon completion, the reaction was stirred for 2.5 hours while warming to room temperature. Analysis of the reaction by LC-MS indicated excellent conversion, and the formation of a new product. The reaction mixture was quenched with aqueous saturated NH₄Cl solution (300 ml), and the aqueous layer was extracted with CH₂Cl₂ (2 × 300 ml). The combined organic layers were dried over MgSO₄, filtered, and concentrated under reduced pressure to provide the crude product, which was typically taken on without further purification. An analytical sample was obtained through purification by chromatography on silica gel, which provided a pure product (19.57 g, 90%), as a clear oil. (S,R,R,S)-8: TLC: R_f = 0.45 (30% EtOAc in hexanes). IR (cm⁻¹) 2930 (w), 2856 (w), 1690 (m), 1636 (m), 1610 (w), 1514 (m), 1481 (m), 1460 (m), 1338 (s), 1248 (s), 1154 (s), 1078 (m). ¹H NMR (500 MHz, DMSO-d₆, 140 ºC) δ 8.18 (dd, J = 9.3, 2.4 Hz, 1H), 7.93 (d, J = 2.0, 1H), 7.26 (d, J = 9.3, 1H), 7.20 (br s, 2H), 6.89 (d, J = 8.3, 2H), 5.86 (ddt, J = 16.6, 9.8, 3.4, 1H), 5.14 (d, J = 17.1, 1H), 5.10 (d, J = 9.8, 1H), 4.76 (q, J = 5.9, 1H), 4.41 (s, 2H), 4.07-3.97 (m, 1H), 3.96-3.83 (m, 1H), 3.78 (s, 3H), 3.69-3.38 (br, 2H), 3.37-3.28 (m, 2H), 3.20-2.93 (br s, 2H), 2.82 (s, 3H), 2.47 (obscured m, 1H), 2.41 (dd, J = 14.2, 6.8, 1H), 2.24-1.93 (br s, 1H), 1.43 (s, 9H), 1.28 (d, J = 5.9, 3H), 1.25 (br s, 3H), 0.91 (d, J = 7.8, 3H), 0.83 (br s, 9H), 0.02 (br s, 6H). ¹³C NMR (125 MHz, DMSO-d₆, 130 ºC) δ 166.3, 158.4, 157.7, 154.4, 140.1, 132.7, 129.7, 128.5, 128.0 (2), 124.7, 122.9, 116.8, 113.3 (2), 113.1, 78.0, 74.2, 72.5, 71.4, 70.9, 54.5, 53.4, 51.0, 39.0, 34.8 (2), 27.4 (3), 24.9 (3), 18.1, 16.8, 15.1, 12.7, −5.3, −5.6. HRMS (ESI) calcd for C₄₀H₆₄N₃O₉Si [M + H]⁺: 758.4406. Found: 758.4372.

Preparation of tert-butyl ((2R,3R)-2-(allyloxy)-4-(N-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-5-nitro-2-((S)-pent-4-en-2-yloxy)benzamido)-3-methylbutyl)(methyl)carbamate (9): A solution of t-butyl ((2R,3R)-2-((tert-butyldimethylsilyl)oxy)-4-(N-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-5-nitro-2-((S)-pent-4-en-2-yloxy)benzamido)-3-methylbutyl)(methyl)carbamate (19.6 g, 25.8 mmol, 1.0 equiv) in THF (0.1 molar) was cooled to 0 ºC in an ice bath. Tetrabutylammonium fluoride (TBAF) solution (1.0 M in THF, 65 ml, 65 mmol, 2.5 equiv) was added dropwise over 3 minutes, and the reaction was stirred for 14 hours, slowly warming to room temperature. Analysis of the reaction by LC-MS indicated complete disappearance of the starting material. The reaction was quenched with saturated aqueous NH₄Cl (300 ml), and extracted with EtOAc (3 × 300 ml). The combined organic layers were dried (MgSO₄), filtered, and concentrated to provide the crude product, which was purified by flash chromatography on silica gel (13.5 g, 81%). Then, a solution of the obtained alcohol (12 g, 18.6 mmol, 1.0 equiv) in DMF (0.1 molar) was cooled to 0 ºC in an ice bath. Allyl bromide (16.1 ml, 186 mmol, 10.0 equiv) was then added, followed by sodium hydride as a solid in one portion (60% dispersion in mineral oil, 1.79 g, 48 mmol, 2.4 equiv), and the reaction was stirred for 14 hours, slowly warming to room temperature. Analysis of the reaction by LC-MS indicated complete disappearance of the starting material, and the formation of a new product. The reaction mixture was quenched with aqueous saturated NH₄Cl solution (200 ml) and extracted with EtOAc (3 × 300 ml). The combined organic extracts were dried over MgSO₄, filtered, and concentrated under reduced pressure to provide the crude product, which was purified by chromatography on silica gel to provide pure product 9 as a clear oil (12.3 g, 97%). (S,R,R,S)-9: TLC: R_f = 0.26 (30% EtOAc in hexanes). IR (cm⁻¹) 2976 (w), 2932 (w), 1688 (m), 1634 (m), 1610 (m), 1513 (m), 1481 (m), 1453 (m), 1337 (s), 1266 (s), 1247 (s), 1153 (s), 1074 (s). ¹H NMR (500 MHz, DMSO-d₆, 140 ºC) δ 8.18 (dd, J = 9.3, 2.9, 1H), 7.94 (d, J = 2.9, 1H), 7.27 (d, J = 9.3, 1H), 7.21 (br s, 2H), 6.89 (d, J = 8.3, 2H), 5.85 (ddt, J = 17.1, 10.2, 6.8, 2H), 5.14 (d, J = 18.1, 2H), 5.09 (d, J = 9.8, 2H), 4.77 (q, J = 5.9, 1H), 4.41 (br s, 2H), 4.07-3.88 (br s, 2H), 3.87-3.81 (br s, 1H), 3.77 (s, 3H), 3.70-3.01 (br, 7H), 2.83 (s, 3H), 2.53-2.45 (obscured m, 1H), 2.38 (dd, J = 14.6, 7.3, 1H), 2.28-1.97 (br s, 1H), 1.42 (s, 9H), 1.28 (d, J = 5.9, 3H), 1.25 (br s, 3H), 0.95 (br s, 3H). ¹³C NMR (125 MHz, DMSO-d₆, 130 ºC) δ 166.4, 158.4, 157.7, 154.3, 140.1, 134.7, 132.7, 129.7, 128.5, 128.0 (2), 124.7, 123.0, 116.8, 114.8, 113.3 (2), 113.1, 79.9, 77.9, 74.2, 71.4, 70.8, 70.1, 54.5, 53.4, 49.3, 39.1, 35.0, 34.5, 27.4 (3), 18.1, 15.1, 12.8. HRMS (ESI) calcd for C₃₇H₅₄N₃O₉ [M + H]⁺: 684.3854. Found: 684.3846.

Preparation of tert-butyl (((2S,8R,9R)-14-amino-11-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-2,9-dimethyl-12-oxo-2,3,4,5,6,8,9,10,11,12-decahydrobenzo[b][1,9,5]dioxaazacyclotetradecin-8-yl)methyl)(methyl)carbamate (10): A solution of the diolefin 9 (13.5 g, 19.8 mmol, 1.0 equiv) and Hoveyda Grubbs Catalyst II (1.24 g, 1.98 mmol, 0.10 equiv) in toluene (0.01 molar) was degassed with dry nitrogen for 15 minutes. The reaction was stirred for 14 hours at room temperature (23 ºC) after which analysis of the reaction by LC-MS indicated complete disappearance of the starting material, and the formation of the target material as well as some dimerized product. The solvent was evaporated under reduced pressure, and the mixture was purified by flash chromatography on silica gel using a gradient of EtOAc in hexanes. The resulting products (a mixture of geometric isomers) were redissolved in EtOAc (0.02 molar) and stirred overnight with activated carbon. The mixture was then filtered over Celite and concentrated to provide the crude products as a light brown, foamy solid (10.34 g, 80%). Then, 20 mol% Pd(OH)₂ on activated carbon (10.7 mg, 15 μmol, 0.10 equiv) was added to a solution of the obtained RCM mixture (100 mg, 0.152 mmol, 1.0 equiv) in methanol (0.02 molar). Hydrogen gas was bubbled into the mixture for 30 minutes, after which the reaction was placed under a static H₂ environment (balloon pressure) for 12–15 hours. The reaction mixture was filtered through Celite, washing the filter cake with EtOAc (50 ml), dichloromethane (50 ml), and finally hot EtOAc (50 ml). The filtrate was concentrated, and the crude residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM to provide the pure aniline 10 (48 mg, 51%). (S,R,R,R)-10: IR (cm⁻¹) 2930 (m), 1685 (s), 1612 (s), 1512 (s), 1491 (s), 1456 (s), 1247 (s), 1225 (s), 1154 (s), 1080 (m). ¹H NMR (500 MHz, DMSO-d₆) δ 7.22 (br s, 2H), 6.90 (d, J = 8.3, 2H), 6.70 (d, J = 8.8, 1H), 6.58 (dd, J = 8.8, 2.4, 1H), 6.42 (br s, 1H), 4.64-4.18 (br m, 5H), 4.06-3.82 (br m, 2H), 3.77 (s, 3H), 3.74-3.47 (br, 3H), 3.46-3.29 (br s, 1H), 3.28-3.15 (br s, 1H), 3.14-2.99 (br s 1H), 2.95-2.58 (br m, 5H), 2.15-1.85 (br, 1H), 1.84-1.45 (br 4H), 1.41 (s, 9H), 1.34-1.25 (m, 3H) 1.24-0.99 (br, 6H), 0.96-0.68 (br, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 170.2 (169.7, 169.1*), 158.8 (158.6*), 155.5 (155.2, 154.9*), 143.5, 142.1 (141.4*), 130.2, 129.5 (129.0*), 127.4 (127.3*), 114.9, 113.8 (113.6*), 112.88 (112.80*), 111.7, 83.7, 82.8, 78.4, 77.9 (77.7*), 75.3, 74.9, 72.4, 72.0, 71.9, 71.7, 71.6, 70.4, 69.5, 69.3, 68.4, 65.8, 55.0, 53.6, 51.4, 50.7, 47.6, 47.0, 42.3, 38.5, 35.7, 34.5, 34.3, 33.7, 33.1, 29.9, 29.7, 29.1, 28.5, 28.1 (28.0*), 18.7, 18.4, 18.0, 17.7, 17.3, 15.7, 14.8, 14.6, 13.0, 12.0, 11.8. HRMS (ESI) calcd for C₃₅H₅₃N₃O₇ [M + H]⁺: 628.3956. Found: 628.3987.

Preparation of Allyl-(((2S,8R,9R)-14-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-11-((S)-1-hydroxypropan-2-yl)-2,9-dimethyl-12-oxo-2,3,4,5,6,8,9,10,11,12-decahydrobenzo[b][1,9,5]dioxaazacyclotetradecin-8-yl)methyl)(methyl)carbamate (13): To a solution of the crude product 10 (9.04 g, 14.4 mmol) dissolved in dioxane (386 ml) at 0 ºC, was added 10% NaHCO₃ solution (40 ml) and fluorenylmethyloxycarbonyl chloride (Fmoc-Cl; 5.40 g, 20.9 mmol, 1.5 equiv). The reaction was stirred, slowly warming to room temperature for 15 hours, after which LC/MS analysis showed that the reaction was complete. The reaction was quenched with NH₄Cl solution (200 ml), and extracted EtOAc (3 × 100 ml). The combined organic extracts were dried over MgSO₄ or Na₂SO₄, filtered, and concentrated under reduced pressure. Purification of the crude residue by flash chromatography provided 11.16 g (91% over two steps) of pure Fmoc-protected product 11 as a foamy white solid.

To a solution of Fmoc-protected macrocycle 11 (34.0 g, 40.0 mmol, 1.0 equiv) in CH₂Cl₂ (800 ml) at 0 ºC was added 2,6-lutidine (18.63 ml, 160 mmol, 4.0 equiv) followed by tert-butyldimethylsilyl triflate (23.0 ml, 100 mmol, 2.5 equiv). The reaction was stirred for 2 hours while warming to room temperature. The reaction was then re-cooled to 0 ºC, and more 2,6-lutidine (18.63 ml, 160 mmol, 4.0 equiv) was added (to quench the triflic acid byproduct), followed by the addition of saturated aqueous NH₄Cl solution slowly. The reaction was extracted with CH₂Cl₂ (3 × 300 ml), and the combined organic layers were dried over MgSO₄, filtered, and concentrated under reduced pressure. The crude residue was then dissolved in THF (800 ml) at room temperature in a plastic bottle, and pyridine hydrofluoride (70%, 4.97 ml, 40.0 mmol, 1.0 equiv) was added. The reaction was stirred at room temperature until LC analysis showed complete conversion to the desired product. The reaction was quenched with saturated aqueous NH₄Cl solution, the layers were separated, and the aqueous layer was extracted EtOAc (3 × 200 ml). The combined organic layers were dried over MgSO₄, filtered, and concentrated under reduced pressure to provide the crude amine, which was used in the next step without further purification. The crude intermediate was dissolved in THF (200 ml), and cooled to 0 ºC. Pyridine (12.9 ml, 160 mmol, 4.0 equiv) and allyl chloroformate (12.8 ml, 120 mmol, 3.00 equiv) were added sequentially, and the mixture was stirred for 10 minutes before warming to room temperature. The reaction was stirred for 16 hours during which it turned bright orange in color, and the starting material was no longer visible by LC/MS. The reaction was quenched with NH₄Cl solution, the layers were separated, and the aqueous layer was extracted with EtOAc (3 × 100 ml). The combined organics were dried, filtered, and concentrated under reduced pressure, and the crude material was purified by flash chromatography to provide 28.0 g (84% over 2 steps) of the pure Alloc-protected macrocycle 12 as a white solid.

To a solution of the Alloc-protected compound 12 (28.0 g, 33.6 mmol, 1.0 equiv) in CH₂Cl₂ (336 ml) and pH 7 buffer (35 ml) at 0 °C was added dichlorodicyanobenzoquinone (DDQ) (9.15 g, 40.3 mmol, 1.2 equiv) slowly. The reaction was stirred for 15 hours, slowly warming to room temperature until analysis of the reaction showed complete conversion to product. The reaction was quenched with saturated aqueous NaHCO₃ solution, and the layers were quickly separated to avoid a difficult emulsion. The aqueous layer was extracted with CH₂Cl₂ (3 × 100 ml), and the combined organic layers were washed with NaHCO₃ solution, dried over MgSO₄, filtered, and concentrated. The crude residue was purified by flash chromatography, which 22.0 g (92%) of the pure product 13 as a white solid.(S,R,R,S)-13: IR (cm⁻¹) 3400 (br w), 3290 (br w), 2938 (w), 1684 (m). 1608 (m), 1541 (m), 1493 (m), 1450 (m), 1219 (s), 1058 (m). ¹H NMR (500 MHz, DMSO-d₆, 130 °C) δ 9.03 (s, 1H), 7.95-7.82 (m, 3H), 7.82-7.65 (m, 2H), 7.65-7.55 (m, 1H), 7.48-7.24 (m, 3H), 6.92 (d, J = 9.0, 1H), 6.72 (d, J = 10.0, 1H), 6.60 (d, J = 10.0, 1H), 6.21 (s, 1H), 6.03-5.84 (m, 1H), 5.38-5.09 (m, 2H), 4.65-4.45 (m, 4H), 4.30 (t, J = 6.7, 1H), 4.28-4.18 (m, 2H), 4.13-4.02 (m, 1H), 3.90-3.55 (m, 4H), 3.52-3.22 (m, 2H), 3.20-2.70 (m, 4H), 2.52-2.48 (m, 1H), 2.27-2.19 (m, 1H), 1.99 (s, 1H), 1.86-1.48 (m, 4H), 1.42-1.16 (m, 5H), 0.86-0.68 (m, 4H). ¹³C NMR (125 MHz, DMSO-d₆, 130 °C) δ 169.3, 155.0, 153.0, 144.7, 143.3, 142.3, 140.2, 138.9, 137.0, 134.2, 133.0, 132.7, 128.5, 128.0, 126.8, 126.4, 126.2, 126.2, 125.9, 124.3, 124.3, 123.3, 122.9, 120.4, 120.1, 119.2, 119.0, 118.8, 115.7, 115.3, 114.8, 113.0, 111.2, 107.8, 81.7, 74.9, 73.3, 68.2, 65.4, 65.2, 64.3, 63.4, 63.2, 55.2, 54.8, 49.8, 46.4, 42.3, 38.1, 34.7, 33.7, 33.1, 28.7, 28.0, 19.7, 18.3, 17.8, 14.3, 13.6, 13.2, 11.3. HRMS (ESI) calcd for C₄₁H₅₂N₃O₈ [M + H]⁺: 714.3749. Found: 714.3742.

Preparation of [(S)-2-(-14-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-((((allyloxy)carbonyl)(methyl)amino)methyl)-2,9-dimethyl-12-oxo-3,4,5,6,9,10-hexahydrobenzo[b][1,9,5]dioxaazacyclotetradecin-1112H)-yl)propyl benzoate (14): To a solution of alcohol 13 (2.00 g, 2.80 mmol) in dry CH₂Cl₂ (23 ml) was added pyridine (2.3 ml, 28 mmol) followed by benzoyl chloride (0.98 ml, 8.4 mmol) under N₂ atmosphere at room temperature. The resulting mixture was stirred at room temperature for 1 h. The reaction was quenched with sat. aq. NaHCO₃ (30 ml) and diluted with CH₂Cl₂ (30 ml). The phases were separated and the aq. layer was washed with CH₂Cl₂ (3 × 20 ml). The combined organic phases were washed with water (2 × 20 ml), dried (Na₂SO₄), and the solvent evaporated. The crude residue was purified by flash chromatography on silica gel using EtOAc in hexanes to provide 1.92 g (84%) of the desired product 14 as a clear resin. ¹H NMR (300 MHz, CDCl₃) δ 8.05 (d, 1H), 8.00 (d, 1H), 7.77 (d, 2H), 7.66-7.50 (m, 3H), 7.48 – 7.37 (m, 4H), 7.36-7.28 (m, 2H), 7.22-7.11 (m, 1H), 6.89-6.66 (m, 2H), 6.03-5.78 (m, 1H), 5.26 – 5.06 (m, 1H), 4.72-4.40 (m, 4H), 4.32-4.22 (m, 1H), 4.18 – 4.02 (m, 1H), 4.00-3.84 (m, 1H), 3.80-3.50 (m, 1H), 3.47 (d, 2H), 3.41 – 3.15 (m, 1H), 3.12-2.73 (m, 3H), 2.19 (m, 1H), 2.04 (m, 1H), 1.91-1.69 (m, 2H), 1.67-1.45 (m, 3H), 1.37-1.08 (m, 9H), 0.99-0.80 (m, 6H), 0.77 (m, 2H). LRMS calculated for C48H55N3O9: 818 [M+H]⁺. Found 818

Preparation of (S)-2-(-8-((((allyloxy)carbonyl)(methyl)amino)methyl)-2,9-dimethyl-12-oxo-14-(3-phenylureido)-3,4,5,6,9,10-hexahydrobenzo[b][1,9,5]dioxaazacyclotetradecin-1112H)-yl)propyl benzoate (15): A mixture of Fmoc-protected aniline 14 (1.61 g, 1.968 mmol) and piperidine (0.390 ml, 3.94 mmol) in dry DMF (19.68 ml) was stirred under N₂ atmosphere at room temperature for 40 minutes. Then, phenyl isocyanate (0.209 ml, 1.914 mmol) was introduced to the reaction mixture, which was stirred at room temperature for 3 hours. The reaction was diluted with water/EtOAc (10 ml) and extracted with EtOAc (3 × 20 ml). The organic phase was separated, washed with water, brine, dried (Na₂SO₄), and evaporated to dryness. The crude residue was purified by flash chromatography on silica gel using MeOH in CH₂Cl₂ to provide the desired product 15 (0.77 g, 60% over two steps). ¹H NMR (300 MHz, CDCl₃) δ 8.06 (m, 1H), 8.04 – 7.89 (m, 1H), 7.81 (m, 1H), 7.59 (m, 2H), 7.51 – 7.30 (m, 2H), 7.23 (m, 2H), 6.96 (m, 1H), 6.82 (m, 1H), 6.69 (m, 1H), 5.89 (m, 1H), 5.45 – 5.08 (m, 2H), 4.83 (m, 1H), 4.55 (m, 3H), 4.18 – 4.02 (m, 1H), 3.93 (m, 2H), 3.50 (m, 1H), 3.34 (m, 1H), 3.26 – 2.95 (m, 3H), 2.88 (m, 3H), 2.26 (m, 1H), 2.04 (m, 1H), 1.77 (m, 2H), 1.62 (m, 2H), 1.38 (m, 3H), 1.24 (m, 5H), 1.14 – 1.00 (m, 2H), 0.99 – 0.85 (m, 2H), 0.77 (m, 2H). LRMS calculated for C40H50N4O8: 715 [M+H]⁺. Found 715.

Preparation of (S)-2-(-8-((4-fluoro-N-methylphenylsulfonamido)methyl)-2,9-dimethyl-12-oxo-14-(3-phenylureido)-3,4,5,6,9,10-hexahydrobenzo[b][1,9,5]dioxaazacyclotetradecin-1112H)-yl)propyl benzoate (16): A mixture of Alloc-protected amine 15 (0.770 g, 1.08 mmol), 1,3-dimethylpyrimidine-2,4,65H)-trione (1.26 g, 8.08 mmol) and Pd(PPh₃)₄ (0.249 g, 0.215 mmol) in dry CH₂Cl₂ (15 ml) was stirred under a N₂ atmosphere at room temperature for 1 hour. The reaction mixture was diluted with CH₂Cl₂ (30 ml) and washed with saturated NaHCO₃ (2 × 20 ml) and water (2 × 20 ml). The organic phase was separated, dried (Na₂SO₄), and the solvent evaporated. The crude product was then re-dissolved in dry CH₂Cl₂ (10.8 ml), then 2,6-lutidine (0.878 ml, 7.54 mmol) was added, followed by 4-fluorobenzene-1-sulfonyl chloride (0.419 g, 2.154 mmol) under a N₂ atmosphere. The resulting mixture was stirred at room temperature for 15 hours. The reaction was diluted with CH₂Cl₂ (25 ml) and washed with water (2 × 20 ml). The organic layer was separated, dried (Na₂SO₄), and the solvent evaporated. This material was chromatographed on silica gel, using a gradient of EtOAc in hexanes to give 0.53 g (62%) of the title product 16 as colorless resin. ¹H NMR (300 MHz, CDCl₃) δ 8.11 – 7.84 (m, 2H), 7.79 (m, 1H), 7.62 (m, 3H), 7.37 (m, 2H), 7.34 – 7.16 (m, 2H), 7.04 (m, 4H), 6.87 – 6.63 (m, 2H), 6.52 (m, 1H), 4.89 – 4.48 (m, 1H), 4.45 – 4.18 (m, 1H), 4.06 (m, 1H), 3.77 (m, 2H), 3.50 (m, 1H), 3.35 (m, 1H), 2.94 (m, 2H), 2.64 (m, 5H), 1.88 (m, 1H), 1.56 (m, 2H), 1.43 (m, 2H), 1.31 (m, 4H), 1.04 (m, 3H), 0.88 (m, 2H), 0.75 (m, 1H), 0.60 (m, 2H), 0.37 (m, 1H). LRMS calculated for C42H49FN4O8S: 789 [M+H]⁺. Found 789.

Preparation of 3-[-11-[(2S)-1-hydroxypropan-2-yl]-2,9-dimethyl-8-{[N-methyl(4-fluorobenzene)sulfonamido]methyl}-12-oxo-2,3,4,5,6,8,9,10,11,12-decahydro-1,7,11-benzodioxazacyclotetradecin-14-yl]-1-phenylurea (19): To a solution of Bz-protected alcohol 16 (0.490 g, 0.621 mmol) in MeOH (12.4 ml) was added potassium carbonate (K₂CO₃; 0.472 g, 3.42 mmol) under a N₂ atmosphere. The suspension was stirred at room temperature for 15 hours. The reaction mixture was then quenched with saturated NH₄Cl (20 ml) and extracted with EtOAc (2 × 20 ml). The organic phase was separated, washed with water and brine, dried (Na₂SO₄), and the solvent evaporated. This material was chromatographed on silica, using MeOH in CH₂Cl₂ to afford the desired product 17 (0.40 g, 94%) as colorless resin.¹H NMR (300 MHz, CDCl₃) δ 7.95 – 7.51 (m, 3H), 7.39 (m, 1H), 7.34 – 7.07 (m, 5H), 7.07 – 6.91 (m, 1H), 6.91 – 6.60 (m, 2H), 4.65 (s, 1H), 4.33 (s, 1H), 3.93 (m, 4H), 3.56 (m, 2H), 3.10 (m, 2H), 2.96 – 2.55 (m, 5H), 2.47 – 2.27 (m, 1H), 2.04 (m, 1H), 1.64 (m, 3H), 1.47 (m, 4H), 1.38 – 1.22 (m, 2H), 1.22 – 1.09 (m, 3H), 1.09 – 0.90 (m, 2H), 0.79 (m, 2H). LRMS calculated for C₃₅H₄₅FN₄O₇S: 685 [M+H]+. Found 685.

Preparation of N-((-11-((S)-1-azidopropan-2-yl)-2,9-dimethyl-12-oxo-14-(3-phenylureido)-2,3,4,5,6,8,9,10,11,12-decahydrobenzo[b][1,9,5]dioxaazacyclotetradecin-8-yl)methyl)-4-fluoro-N-methylbenzenesulfonamide (18): To a solution of alcohol 17 (0.401 g, 0.586 mmol) in dry THF (2.9 ml) under N₂ was added 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (0.265 ml, 1.76 mmol) followed by diphenyl phosphorazidate (DPPA; 0.189 ml, 0.878 mmol). The reaction mixture was stirred for 16 hours, then concentrated and chromatographed on silica, using MeOH/CH₂Cl₂ to afford the desired product 18 (0.36 g, 87%) as a colorless resin. ¹HNMR (300 MHz, CDCl₃) δ 8.24 – 7.64 (m, 2H), 7.57 – 7.33 (m, 2H), 7.31 - 7.05 (m, 4H), 7.03 – 6.77 (m, 3H), 6.77 – 6.53 (m, 1H), 4.67 – 4.27 (m, 3H), 4.23 – 4.09 (m, 1H), 4.01 – 3.84 (m, 2H), 3.83 – 3.61 (m, 4H), 3.61 – 3.29 (m, 2H), 3.25 – 3.02 (m, 2H), 3.06 – 2.55 (m, 4H), 2.27 – 1.94 (m, 1H), 1.92 – 1.32 (m, 6H), 1.30 – 1.14 (m, 3H), 1.14 – 1.01 (m, 2H), 0.98 – 0.85 (m, 1H), 0.83 – 0.62 (m, 1H). LRMS (ESI) calcd for C₃₅H₄₄FN₇O₆S [M + H]⁺: 710.3. Found: 710.0.

Preparation of N-((-11-((S)-1-aminopropan-2-yl)-2,9-dimethyl-12-oxo-14-(3-phenylureido)-2,3,4,5,6,8,9,10,11,12-decahydrobenzo[b][1,9,5]dioxaazacyclotetradecin-8-yl)methyl)-4-fluoro-N-methylbenzenesulfonamide (19): To a solution of azide 18 (0.33 g, 0.47 mmol) in THF (15 ml) and H₂O (0.9 ml) was added triphenylphosphine (PPh₃) (0.305 g, 1.16 mmol). The reaction mixture was stirred 16 hours, then diluted with EtOAc (30 ml), dried (Na₂SO₄), and the solvent evaporated. This material was chromatographed on silica, using MeOH/CH₂Cl₂ to afford the 0.25 g (79%) of 19 as a colorless resin.¹H NMR (300 MHz, CDCl₃) δ 8.96 – 8.20 (m, 2H), 7.92 – 7.65 (m, 2H), 7.58 – 7.33 (m, 2H), 7.33 – 7.05 (m, 4H), 7.08 – 6.85 (m, 1H), 6.84 – 6.62 (m, 1H), 4.73 – 4.32 (m, 1H), 4.16 – 3.53 (m, 4H), 3.48 – 3.24 (m, 1H), 3.21 – 2.92 (m, 4H), 2.91 – 2.79 (m, 2H), 2.80 – 2.51 (m, 4H), 2.31 – 1.89 (m, 2H), 1.89 – 1.37 (m, 6H), 1.37 – 1.14 (m, 6H), 1.10 – 0.89 (m, 2H), 0.67 – 0.32 (m, 2H). LRMS (ESI) calcd for Chemical Formula: C₃₅H₄₆FN₅O₆S [M + H]+:684.3. Found: 684.5.

Preparation of N-((-11-((S)-1-(dimethylamino)propan-2-yl)-2,9-dimethyl-12-oxo-14-(3-phenylureido)-2,3,4,5,6,8,9,10,11,12-decahydrobenzo[b][1,9,5]dioxaazacyclotetradecin-8-yl)methyl)-4-fluoro-N-methylbenzenesulfonamide (20): To a solution of amine 19 (223 mg, 0.326 mmol) in dry CH₂Cl₂ (6.5 ml) was added formaldehyde (37% in water, 0.146 ml, 1.96 mmol) followed by MgSO₄ (471 mg, 3.91 mmol). The mixture was stirred for 1 hour, then sodium triacetoxyborohydride (691 mg, 3.26 mmol) was added and the mixture stirred for 16 hours. The mixture was diluted with water (25 ml) and CH₂Cl₂ (30 ml), the phases separated, and the aqueous phase was extracted with CH₂Cl₂ (2 × 20 ml). The combined organic phases were washed with water, dried (Na2SO4), and the solvent evaporated. This material was chromatographed on silica gel, using a gradient of MeOH in CH₂Cl₂ to afford the probe 20 in 93% purity as a colorless resin. Additional purification by preparative HPLC yielded product >99 % purity (73.1 mg, 31% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.97 (s, 1H), 7.89 – 7.82 (m, 1H), 7.80 – 7.73 (m, 1H), 7.70 – 7.60 (m, 1H), 7.47 – 7.35 (m, 2H), 7.34 – 7.11 (m, 4H), 7.03 – 6.87 (m, 1H), 6.85 – 6.65 (m, 2H), 4.67 – 4.47 (m, 1H), 4.46 – 4.20 (m, 1H), 4.19 – 3.97 (m, 1H), 3.96 – 3.54 (m, 2H), 3.52 – 3.34 (m, 1H), 3.33 – 3.18 (m, 1H), 3.17 – 2.99 (m, 1H), 2.91 (s, 1H), 2.84 – 2.69 (m, 4H), 2.69 – 2.35 (m, 1H), 2.32 (s, 3H), 2.19 – 2.01 (m, 3H), 1.96 – 1.65 (m, 2H), 1.64 – 1.48 (m, 2H), 1.48 – 1.34 (m, 3H), 1.35 – 1.18 (m, 2H), 1.18 – 1.07 (m, 2H), 1.7 – 0.92 (m, 2H), 0.91 – 0.75 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 172.7, 172.4, 166.0, 164.0, 153.3, 147.5, 139.4, 133.3, 130.0, 128.8, 128.6, 126.1, 122.1, 119.8, 119.4, 119.0, 118.8, 116.6, 116.4, 116.2, 113.4, 111.9, 83.2, 74.3, 71.6, 71.2, 69.8, 69.4, 68.4, 63.6, 63.1, 62.7, 53.6, 53.1, 51.9, 51.2, 51.0, 50.7, 50.4, 46.4, 46.0, 45.8, 45.2, 43.4, 43.1, 40.8, 38.4, 37.3, 36.7, 34.6, 34.4, 30.5, 29.3, 28.8, 18.4, 18.3, 18.2, 17.9, 17.2, 16.9, 15.8, 14.7, 13.6, 12.7. HRMS (ESI) calcd for Chemical Formula: C₃₇H₅₀FN₅O₆S [M + H]⁺:712.3539. Found: 712.3511.

Appendix D. NMR and LC Data of Probe and Analogs

¹H NMR Spectrum (CDCl₃, 500 MHz) of the Probe (ML238)

¹³C NMR Spectrum (CDCl₃, 500 MHz) of the Probe (ML238)

LCMS Chromatogram of the Probe (ML238) showing 100% purity

Appendix E. Activity of Several Other Analogs Synthesized During the Optimization Phase

Table E1Other Analogs Synthesized During the Course of SAR Study

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Entry No.	CID	SID	Broad ID	Structure	Potency (μM) Mean ± S.E.M.				Fold Selectivity HepG2/Dd2
					3D7 48 h (n=1)	3D7 96 h (n=1)	Dd2 72 h (n=1)	HepG2 48 h (n=1)
1	49849910	104179280	BRD-K15006730		0.15	0.12	0.67	IA	NA
	Solubility (PBS): <0.5 μM, (water): <0.5 μM; Purity: 98%
2	49849926	104179281	BRD-K08173135		0.36	0.30	1.32	11.79	8.9
	Solubility (PBS): <0.5 μM, (water): <0.5 μM; Purity: 97%
3	49849937	104179264	BRD-K72547289		0.28	0.18	0.61	IA	NA
	Solubility (PBS): <0.5 μM, (water): <0.5 μM; Purity: 100%
4	49849927	104179263	BRD-K95450288		1.37	1.09	3.71	IA	NA
	Solubility (PBS): <0.5 μM, (water): <0.5 μM; Purity: 98%
5	49849933	104179285	BRD-K12861152		4.33	3.47	6.60	12.01	1.8
	Solubility (PBS): <0.5 μM, (water): 129.6 μM; Purity: 92%
6	44488207	104179261	BRD-K32582686		0.42	0.050	0.52	IA	NA
	Solubility (PBS): <0.5 μM, (water): <0.5 μM; Purity: 94%

IA = Inactive; (m) = mouse; NA = Not applicable; ND = Not determined; PPB (h) = Plasma protein binding in human; PS (h) = Plasma stability in human

Appendix F. Summary of Data Obtained by Assay Provider on 3D7 and Dd2 Cell Lines

Table F1Summary of Data Obtained in the Flow Cyclometry Assay Against P. falciparum

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Entry No.	CID	SID	Broad ID	Structure	Potency (μM) Mean ± S.E.M.
					3D7		Dd2
					48 h (n=1)	96 h (n=1)	48 h (n=1)	96 h (n=1)
1	49849912	104179292	BRD-K45399554		0.00164	0.00214	0.00042	0.00057
2	49849932	104179275	BRD-K06180729		>16.7	>16.7	15.7	13.4
3	49843020	104179266	BRD-K05563014		1.15	1.53	0.70	0.70
4	44483977	104179269	BRD-K81592585		0.19	0.18	0.092	0.088
5	49849909	104179286	BRD-K95212245		0.0022	0.0027	0.0011	0.0011
6	49849931	104179288	BRD-K91442916		0.0081	0.0088	0.0044	0.0051

Entry No.	CID	SID	Broad ID	Structure	Potency (μM)
					Kidney Epithelial Cells	Dermal Fibroblasts	Lung Epithelial Cancer Cells (A549)	Erythrocyte Lysis
1	49849912	104179292	BRD-K45399554		2 4	27 63	2 >63	>40
2	49843020	104179266	BRD-K05563014		>63 >63	>63 >63	>63 >63	>40
3	49849909	104179286	BRD-K95212245		>63 >63	>63 >63	>63 >63	>40

Appendix G. Summary of Other Cytotoxicity Data

Table G1Summary of Cytotoxicity Data in Kidney Epithelial Cells, Dermal Fibroblasts, Lung Epithelial Cancer Cells (A549) and Erythrocytes

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Entry No.	CID	SID	Broad ID	Structure	Potency (μM)
					Kidney Epithelial Cells	Dermal Fibroblasts	Lung Epithelial Cancer Cells (A549)	Erythrocyte Lysis
1	49849912	104179292	BRD-K45399554		2 4	27 63	2 >63	>40
2	49843020	104179266	BRD-K05563014		>63 >63	>63 >63	>63 >63	>40
3	49849909	104179286	BRD-K95212245		>63 >63	>63 >63	>63 >63	>40

Appendix H. Compounds Provided to BioFocus