Design of Gallinamide A Analogs as Potent Inhibitors of the Cysteine Proteases Human Cathepsin L and Trypanosoma cruzi Cruzain

Article abstract of DOI:10.1021/acs.jmedchem.9b00294

Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, K_i = 0.0937 ± 0.01 nM and k_inact/K_i = 8 730 000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is highly homologous. Our investigations revealed that gallinamide and its analogs potently inhibit cruzain and are exquisitely toxic toward T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC₅₀ = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for the treatment of Chagas disease.

Full text of DOI:10.1021/acs.jmedchem.9b00294

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Article
Design of gallinamide A analogs as potent inhibitors of the cysteine
proteases human cathepsin L and Trypanosoma cruzi cruzain
Paul D. Boudreau, Bailey W Miller, Laura-Isobel McCall, Jehad Almaliti, Raphael Reher,
Ken Hirata, Thu Le, Jair L Siqueira-Neto, Vivian Y. H. Hook, and William H Gerwick
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00294 • Publication Date (Web): 20 Sep 2019
Downloaded from pubs.acs.org on September 21, 2019
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Design of gallinamide A analogs as potent inhibitors of the cysteine
proteases human cathepsin L and Trypanosoma cruzi cruzain
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,†
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Paul D. Boudreau, Bailey W. Miller, Laura-Isobel McCall, Jehad Almaliti,
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Raphael Reher, Ken Hirata, Thu Le, Jair L. Siqueira-Neto , Vivian Hook , William H.
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Gerwick
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Center for Marine Biotechnology and Biomedicine, Scripps Institution of
Oceanography, University of California, San Diego, La Jolla, California 92093, United
States
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Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San
Diego, La Jolla, California 92093, United States
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Center for Discovery and Innovation in Parasitic Diseases, University of California, San
Diego, La Jolla, California 92093, United States
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Department of Chemistry and Biochemistry, University of Oklahoma, Norman,
Oklahoma 73019, United States
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Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma
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3019, United States
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Department of Pharmaceutical Sciences, College of Pharmacy, the University of Jordan,
Amman, 11942, Jordan
†
These authors contributed equally to the work.
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Keywords: Molecular-modeling, organic synthesis, structure activity relationship,
gallinamide A, cathepsin L, Trypanosoma cruzi
Abstract
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Gallinamide A, originally isolated with modest antimalarial activity, was
subsequently re-isolated and characterized as a potent, selective, and irreversible
inhibitor of the human cysteine protease cathepsin L. Molecular docking identified
potential modifications to improve binding, which were synthesized as a suite of
analogs., Resultingly, this current study produced the most potent gallinamide analog yet
tested against cathepsin L (10, Ki = 0.0937 ± 0.01 nM and kinact /Ki = 8,730,000). From a
protein structure and substrate preference perspective, cruzain, an essential
Trypanosoma cruzi cysteine protease, is a highly homologous. Our investigations
revealed that gallimamide and its analogs potently inhibit cruzain and are exquisitely
toxic towards T. cruzi in the intracellular amastigote stage. The most active compound,
5
, had an IC50 = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect
stage) and the host cell, and thus represents a new candidate for treatment of Chagas
disease.
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Introduction
Natural products (NPs) have long served an important role in drug discovery
through identifying new chemical scaffolds that interact with novel targets within
cellular systems. Indeed, NPs of different molecular weight classes have led to
approximately 70% of all clinically useful agents, either directly, by derivation, or
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inspiration. Marine life has only recently been prospected in earnest as a resource for
new drug leads, but already thirteen agents have been derived or inspired from marine
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NPs for use in the clinic, and another 31 are in various stages of clinical development.
Nevertheless, some therapeutic areas have lagged behind others in terms of
development of new pharmacotherapies; neurological disorders and parasitic disease
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, 5
represent two such conditions.
Interestingly, these two disease areas are
mechanistically connected in that cysteine proteases have been identified as excellent
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potential drug targets in both.
Gallinamide A (1) was first isolated from a Panamanian collection of marine
cyanobacteria in 2009 for activity against the malaria-causing parasite Plasmodium
falciparum. In this work the full planar structure was solved but the stereochemistry of
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the N,N-dimethylisoleucine residue was not resolved. It was subsequently and
independently isolated by Taori and coworkers from a Floridian cyanobacterial
8
collection and given the name ‘symplostatin 4’. At that time of the second isolation it
was posited on the basis of NMR data that the stereochemistry of the N,N-
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dimethylisoleucine was different between gallinamide A and symplostatin 4. Conroy
and coworkers confirmed the absolute stereochemistry of symplostatin 4 by total
synthesis, and also suggested the stereochemistry of the N,N-dimethylisoleucine tail
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was different in gallinamide A. These authors subsequently corrected this surmise and
showed by careful synthesis of all four diastereomers at this terminal amino acid that
gallinamide A also contained a 2S,3S-N,N-dimethyl isoleucine residue, and thus the two
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compounds were identical; in this publication we shall refer to this compound
exclusively as ‘gallinamide A’.
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Subsequent to our initial discovery and structural description of 1, our interest in
this area was re-kindled by a screening program that detected it as an extremely potent
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and highly selective inhibitor of human cathepsin L. Preincubation-dilution and
activity-based probe experiments revealed an irreversible mode of cathepsin L
inhibition, and molecular docking plus molecular dynamics simulations identified a
likely pose for 1 binding to the active site. This, as well as another modeling study by
Omotunyi and coworkers, found that the cyclic methylmethoxypyrrolinone (MMP)
residue was important for binding by positioning the pharmacophore enone proximate
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to the thiolate nucleophile.
Human cathepsin L is a clan CA family C1 protease that participates in producing
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peptide neurotransmitters (neuropeptides) for synaptic function which is compromised
1
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in neurological diseases,
and functions in lysosomes which participate in
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neurodegenerative diseases. Interestingly, it shows high sequence identity and
structural similarity to many proteases found in parasites responsible for neglected
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tropical diseases. When compared by sequence identity, cathepsin L is most similar to
human cathepsin V (78% by amino acid sequence), and clades closely with human
cathepsins K and S. However, when compared by substrate selectivity, a more
appropriate metric for assessing functional similarity, cathepsins L and V separate from
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the other human enzymes and clade with cruzain (from T. cruzi) and cathepsin L (from
1
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Leishmania mexicana). Thus, it can be hypothesized that inhibitors tailored to
human cathepsin L will have significant crossover with these parasitic enzymes, and
provide useful scaffolds for the development of anti-Chagas disease and perhaps anti-
leishmaniasis agents.
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Thus, for a variety of reasons, including its antiparasitic activity, inhibition of
cathepsin L, and unique linear lipopeptide structure, there has been considerable interest
in 1 and its analogs. For example, 1 was synthesized by Stolze and coworkers, who
showed that it inhibited one or more of the Plasmodium food vacuole falcipain cysteine
proteases, predominately FP2, FP2’ and FP3 enzymes involved in heme degradation
18
and essential for the survival of the parasite. They also demonstrated a likely
irreversible mechanism of action based on an enzyme kinetics approach, and that the
principal target in mammalian cells was cathepsin L. Stolze also investigated via
chemical synthesis some of the structural features in gallinamide A necessary for
antimalarial activity, concluding that the cyclic nature of the MMP head group was
crucial for bioactivity. Further, they produced rhodamine fluorophore labeled
derivatives, and showed that these labeled food vacuoles and the key FP2, FP2’ and
FP3 protease enzymes in a manner consistent with their being the relevant biological
target. Through selective hydrogenation of the double bonds, the enamide olefin was
shown to be necessary for high inhibition potency, supporting our previous hypothesis of
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a Michael-addition like reaction with the active site cysteine thiol, as well as
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experiments described below by Conway.
The Conway group subsequently reported the synthesis of a number of analogs of 1
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and their relative potency as inhibitors of the FP2 and FP3 proteases, as well as activity
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in the drug sensitive 3D7 and drug resistant Dd2 strains of Plasmodium falciparum.
The pharmacophore for this protease inhibition was again identified as the enone of the
unusual 4(S)-amino-2(E)-pentenoic acid unit through production of saturated analogs in
this residue and separately in the pyrrolinone ring and demonstration of reduced
potency to FP2 and FP3, as well as to P. falciparum. This latter work again revealed the
critical nature of the MMP group for production of high potency agents in this class,
and by addition of a phenyl or indole ring to the methyl-pyrrolinone ring, they were
able to produce derivatives with low nM potencies to both of the P. falciparum strains.
Our previous in-silico docking and molecular dynamics simulations with 1
produced a stable and reaction-ready pose for the natural product in the active site of the
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cathepsin L protease. This pose, as well as insights from the literature pertaining to
selective cathepsin inhibitors and their substrate specificity, was used to design a new suite
of analogs for improved binding in cathepsin L as well as the falcipains. Drawing from
the literature, 1 was synthesized using a combination of steps from both Conroy et al.
9
,10,18,19
and Stolze et al.,
modifying steps as necessary due to constraints on reagent
availability or to improve convenience or yield. Informed by our modeling, new analogs
of 1 were designed and synthesized by replacement of the amino acid building blocks. As
the analogs covered in this work were mostly similar to the natural product (e.g.
phenylalanine versus alanine), minimal modification of the reaction or purification
conditions was necessary.
This effort afforded a perspicacious suite of compounds (1-16) that were available
to test against cathepsin L. The modeled protein structure of cathepsin L suggested a
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structure and substrate scope similar to cruzain, another cysteine protease necessary for
the survival of Trypanosoma cruzi, the causative agent of Chagas disease. Current
treatments for Chagas disease, benzidazole and nifurtimox, are associated with
significant adverse effects, require long treatments up to 60 days, and have controversial
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efficacy in the chronic phase of the disease. New treatments that might target the
amastigote stage of the chronic infection are much needed, so this panel of compounds
was also tested against cruzain and in a cell based in vitro T. cruzi assay. The
gallinamide A scaffold thus represents a new model for potential drug development to
this Neglected Tropical Disease (NTD).
Notable among the analogs synthesized, a modification was made to introduce a
terminal alkyne to serve as a handle for potential click chemistry, as well as another
analog that exhibited reversible binding characteristics to the enzyme. Two analogs were
measured as sub-nanomolar inhibitors of human cathepsin L (analogs 10, 11), with one
that displays single digit nanomolar antiparasitic activity against T. cruzi in a cell-based
assay (analog 5) without any toxicity towards host cells up to 10 micromolar. Cathepsin
L inhibition is not fatal, as demonstrated by cathepsin L gene knockout mice which are
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viable and live through development to adulthood and reproduce progeny.
Studies of
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cruzain, the most abundant cysteine protease of T. cruzi and a validated drug
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target,
along with the findings presented in this manuscript, illustrate the productive
strategy of investigating related human and parasitic cysteine proteases for the design
and synthesis of potent inhibitors as candidate pharmaceutical agents.
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Results
Molecular modeling and rational design of analogs
The co-crystal structure of human cathepsin L with a bound covalent nitrile inhibitor
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(2xu3.pdb) was imported to the integrated drug discovery and computational
chemistry software package Molecular Operating Environment (Computational
Chemistry Group). The nitrile ligand was removed, and the protein was prepared for
docking using MOE’s Structure Preparation module to fix structural errors, protonated
with the Protonate 3D module set to pH 5.5 (maximal activity for cathepsin L), and then
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energy minimized using the Amber14:EHT force field equations. Compound 1 was
imported into MOE and similarly protonated, energy minimized using the same criteria,
and then docked to the prepared cathepsin L enzyme using an induced fit protocol to
allow a flexible ligand and receptor. The docked poses were filtered based on proximity
of the reactive carbon of the enamide to the cysteine thiol, and the top pose (S=-10.70
score for binding energy using GBVI/WSA dG) was compared to the pose reported
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previously. The orientation of the core enamide in both cases was identical, and the
carbonyl of this functional group was well supported by hydrogen bonding in the
oxyanion hole of the active site cleft, an important feature for stabilization of the
intermediate inhibitory species (Figure 1).
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Figure 1: Gallinamide A docked pose in cathepsin L and ligand interactions. A. Docked pose for
gallinamide A in the active site cleft of human cathepsin L. The pose was generated through the
induced- fit docking protocol, followed by energy minimization. The enzyme surface map is colored
by electrostatic potential, with red indicating a negative and blue indicating positive surface potential
B. Detailed representation of the hydrogen bond network (blue dotted lines) and proximity of the
enamide pharmacophore to the active site cysteine (yellow sphere). The red line indicates the
distance between the sulfur atom and reactive carbon atom of the enone. C. 2D ligand interactions.
Importantly, the carbonyl of the reactive enamide core is stabilized by hydrogen bonds in the enzyme
oxyanion hole.
Using this as a starting binding pose, in silico modifications were made to the
structure of 1, energy minimized and then re-scored to assess predicted changes to
binding affinity. The R' and R'' residues (Figure 2), both L-alanine in the natural product
(
1), were each changed to L-phenylalanine based on comparisons with common
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cathepsin substrates and examination of the binding sites by MOE. Inclusion of L-
phenylalanine at the R'' (3) residue showed modest improvement to the binding score (S
= -10.95), as well as at the R' position (6) (S = -11.29). Inclusion of L-phenyalanine at
both positions (8) further improved binding, resulting in an S score of -11.47. While
changing the R'' position residue to D-phenylalanine resulted in only mildly worse binding
compared to the opposite configuration (S = -10.46 versus S = -10.95 for 3), inclusion of a
bulky D-amino acid in the R' position appeared to cause a steric clash that prevented the
reactive carbon of the enamide from being accessed by the cysteine-25 thiol. This
modification reduced the binding score to -7.58 and the distance between the reactive
carbon and the sulfur increased from 3.64 Å in 6 to 5.11 Å in this analog.
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Interestingly, a lipophilic surface was identified beyond the binding site of the
methylmethoxypyrrolinone (MMP) group, which could be accessed by extensions to the
methoxy group on the pyrrolinone ring, while simultaneously including a click handle
for later structural modifications. An analog with an O-hexynyl moiety at this position
(16) was modeled and the binding energy was slightly improved to S = -11.09 (Figure
2
). Not modeled was the analog where the fourth residue alkene in the enamide was
hydrogenated to the alkane (17); mechanistically, this was expected to be a reversible
11,18,19
inhibitor.
Based on these in silico data, summarized in Figure 2, a prioritized set of
analogs incorporating various combinations of these modifications was identified for
total synthesis (compounds 2-16). These compounds were designed to evaluate whether
incorporation of phenylalanine residues would improve binding in vitro against
cathepsin L, or if the incorporation of D-amino acids would interfere with binding.
Leucine was included as a replacement for the phenylalanine residue to test whether the
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aromatic system, or only the hydrophobic residue, was important to binding. Finally,
analogs were designed wherein several of these modifications were combined to
evaluate for potential synergistic interactions.
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Figure 2: In silico structural modifications to gallinamide A and their resulting docking scores. The
residues within the gallinamide analogs are labeled according to Schechter-Berger nomenclature,
with the fourth and fifth residues residing at P1 and P1', respectively. Changes to the substrate
residues (at R, R', R'' and R''') are shown with a corresponding binding score, calculated with
GBVI/WSA dG. Binding scores with a lower number (more negative) are predicted to bind with
higher affinity.
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Total synthesis of gallinamide A and analogs
9
,10,18,19
Guided by previous syntheses of 1,
a retrosynthetic analysis sectioned the molecule
into three components: an enamide core, the cyclized head group, and the lipophilic tail comprised
of aliphatic amino acids. With the exception of L-leucic acid, all of the starting materials
were variously protected standard amino acids. To accomplish the total synthesis of 1,
reactions were appropriated from the published routes with additional modifications
introduced that were found to improve or facilitate the reactions, or necessary due to
other constraints (Figures3, 4).
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Figure 3: Comparison of the synthetic routes to the enamide core of gallinamide A (1).
An example of this hybrid scheme towards the synthesis is provided by the first
steps involving the formation of the enamide core from Boc-L-Alanine-OH (Figure 3).
The first reaction formed the Weinreb amide (20a), which was then reduced to an
aldehyde and reacted with an anylidene to form the conjugated system of the enamide as
a methyl ester (21a). Conway’s scheme was emulated for forming 20a as the purification
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via crystallization produced better results; however, Stolze’s route of reacting the
aldehyde without purification with the ester anylidene to form 21a proved more
effective than purifying the aldehyde and subsequently reacting it with free acid
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anylidene. It was also found that refluxing the Wittig reaction, rather than running it at
room temperature, improved the yield and shortened the reaction time.
Figure 4: Total synthesis of gallinamide A. The total synthesis was completed using reactions drawn
from the Conroy (shown in magenta) and Stolze (shown in blue) synthetic routes, as well as new
reactions to improve efficiency and yield (shown in green).
The synthetic route for the head group was largely drawn from Stolze’s route, using an
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt (EDC) based
coupling reaction to append an alanine and form the enamide core (25a), which was
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subsequently deprotected, extended with Meldrum’s acid, and cyclized. The resulting
enol was trapped via a Mitsunobu reaction to complete the head group (26a). In the final
steps to form the tail section and couple it to the enamide core, the route largely followed
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Stolze’s procedure, but with modifications. For example, piperidine was used in
10
Conroy’s work to deprotect the Fmoc group whereas thionyl chloride was employed in
1
8
Stolze’s route as a coupling reagent; both are controlled substances in California, and
thus these reaction steps were modified for the convenience of working with
unregulated chemicals.
To append the tail section, Boc-L-Leu-OH was first attached via EDC coupling
to the free amine derived from trifluoracetic acid (TFA) deprotection of 26a to afford
the leucine extended species (27a). Compound 27a was then coupled to a dipeptide
ester prepared similarly to Stolze’s and Conroy’s routes, but using different protecting
groups. L-Leucic acid was protected as the benzyl ester (22) rather than a tert-butyl or
methyl ester because synthesis of the tert-butyl ester was difficult to replicate whereas
cleavage of the methyl ester with lithium hydroxide risked cleavage of the desired ester
product. The benzyl ester of L-leucic acid was connected to Fmoc-protected L-
isoleucine (or a different amino acid) via carbondiimide coupling using N,N-
diisopropylcarbodiimide (DIC) to afford the desired ester. The product was filtered to
remove DIC derived urea, but was not otherwise purified as this was not observed to
improve the yield before Fmoc deprotection. Fmoc deprotection with diisopropyl amine
gave a free amine (23a). The amine was then dimethylated with methyl iodide (24a).
Finally the benzyl group was removed via hydrogenation to provide the desired ester
intermediate. The free acid of this two-residue ester was used immediately in an EDC
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coupling with the free amine derived from the TFA deprotection of 27a, to afford the
final product 1.
By replacing the various starting amino acids, a variety of analogs could be
synthesized. The modifications focused on four components of 1: the first residue, the
amino acid bearing the demethylated amino terminus; the fourth residue, the amino acid
extended into the enamide core; the fifth residue, the amino acid in the cyclic head group;
and the alcohol used to trap the enol via a Mitsunobu reaction in the head group (R”’).
To modify the R substituent, Fmoc-L-isoleucine was replaced as the first residue
with Fmoc-L-valine or Fmoc-L-phenylalanine. The enamide core residue R’ could easily
be modified by replacing the starting Boc-L-alanine by the equivalent carbamates of D-
and L-phenylalanine; however, the Weinreb amides derived from phenylalanine (20b and
20c) could not be crystallized from N,N-dimethyl formamide (DMF), and thus were
purified by flash chromatography. In the same fashion, the head group extension to form
the initial enamide core could be modified by using an amino acid methyl ester other than
L-alanine methyl ester. Analogs were synthesized with the equivalent esters of L-leucine
and D- or L-phenylalanine, which combined with different enamide starting materials,
led to the production of nine different core-head group pairings (25b-i, see Methods and
Supporting Information). The final modification was the use of an alcohol other than
methanol to trap the enol in the Mitsunobu reaction; 5-hexyn-1-ol was used to create a
new analog (26j, see Methods and Supporting Information) at this position (R”’) as it also
provided the opportunity to attach a probe via click chemistry in future work.
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An interesting aspect of the spectroscopic data for all synthetic compounds
possessing the pyrrolidone ring (e.g. all compounds in series 26 and 27 as well as 1) was
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the appearance of 1:1 twinned signals for several of the H and C NMR resonances
assigned to the acyclic enone as well as the pyrrolidone ring structure (Figure S65). This
observation of twinning was also made by Stolze who created the pyrrolidone system
1
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using this same sequence of synthetic reactions (personnel communication). We
reasoned that this was either due to racemization of C-4 of the 4-(S)-amino-2-(E)-
pentenoic acid (Apa) unit or the presence of two stable conformers about the amide
bond connecting the Apa unit to the pyrrolidone ring (Figure S67). To distinguish
between these two possibilities, we subjected synthetic gallinamide A (1) to ozonolysis,
hydrolysis and Marfey’s analysis. This revealed that the alanyl residue released from
the Apa unit was present as a 6:1 S:R mixture, and was thus inconsistent with the 1:1
13
twinning observed by C NMR (Figure S66). This was additionally probed by variable
1
o
temperature H NMR in which the -OCH
3
group appeared as a pair of signals at 22 C;
o
heating of the sample to 55 C in CDCl
3
resulted in their partial coalescence to a
broadened single peak. Calculation of the energy barrier to rotation about this tertiary
amide also supports this interpretation (between 12.2 and 13.5 kcal/mol using the
1
3
B3LYP method with the 6-31+G(d) basis set). As the C NMR of natural product
gallinamide A (1) did not display twinned resonances in this section of the molecule, it
suggests that the final pyrrolidone biosynthetic cyclization reaction occurs with this
secondary amide in the E or Z conformation, and upon formation of the tertiary amide, it
becomes locked as a single conformer at room temperature.
Kinetic analysis of cathepsin L inhibition
Irreversible inhibitors display a time-dependent shift in the observed IC50 with
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increased incubation of inhibitor and enzyme concentrations, making end-point dose-
1
2
response experiments inappropriate for assessing inhibitor potency. Thus, a kinetic
approach was used to determine the rate and potency of inhibition, in which inhibitor
and enzyme were mixed with 10 µM of the fluorogenic substrate cbz-FR-AMC, and
product formation monitored for 90 minutes. Progress curves were produced with the
concentration of inhibitor ranging from 0.0169 nM to 1000 nM and fit to a non-linear
least-squares regression based on the 2-step irreversible reaction scheme (Figure 5A and
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B). The kinetic constants kaI, kaS, and kbS, were fixed at 200, 20, and 1, respectively.
The constants kcat , kbI and kinact were explicitly estimated by the model, and used to
calculate Ki, the association constant for initial reversible inhibitor binding.
Furthermore, the kinact /Ki value was calculated to more accurately assess the complete
inhibitory potential of each compound. The regressed Ki = 4.67 ± 0.40 nM for
gallinamide A (1) was in agreement with our previously reported IC50 value of 5.0 nM,
7
,11
and the kinact/Ki = 901,000 confirms that it is an extremely potent inhibitor.
The
regression failed to fit the progress curve data to reaction schemes involving reversible
binding or 1-step reaction mechanisms.
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Figure 5: Kinetic analysis of gallinamide A inhibition of cathepsin L. A. Reaction
mechanism and differential equations used to explicitly estimate kinetic constants from
product formation curves. B. Representative data for gallinamide A (1) inhibition of
cathepsin L. Boxes are experimentally measured progress curves of cathespin L activity
with various concentrations of inhibitor, and lines were fit by non-linear regression.
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Note that I = 0 concentration overlaps with the lowest doses at 0.0169 and 0.0508 nM.
Alteration to the first residue, the amino acid bearing the dimethyl amine, had
modest but consistent effects on the cathepsin L inhibitory potency of the analogs
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(
Figure 8). Substitution of the isoleucine at this position with a valine residue resulted in
decreased potency with all other structural features held constant (e.g. compare: 1 and 2,
0 and 11), whereas replacement with a phenylalanine increased potency moderately (e.g.
1
compare: 3 and 4, 8 and 9). Interestingly, and in contrast to the docking results, inclusion
of an L-phenylalanine at the fourth residue was moderately detrimental to potency (e.g.
compare: 1 and 6). Inversion of the fifth residue stereocenter increased potency in every
case, as demonstrated by the improvement in kinact /Ki from compound 8 to 12 from
7
19,000 ± 72,000 to 1,030,000 ± 77,000. Comparing these two compounds to the analogs
14 and 15 where there is inversion of the stereocenter at the fourth residue reveals
reduced inhibitory potency as demonstrated by kinact/Ki values of 6,900 ± 587 and 23,900
±
1840, respectively. This result was consistent with the modeling and likely results
from a steric clash caused by the fourth residue’s side chain, which forces the reactive
carbon of the enamide core to be positioned further from the active site cysteine.
Remarkably, inclusion of a leucine side chain at the fifth residue significantly
improved potency, with the most active compound (10) possessing a Ki = 0.094 ± 0.01
nM and kinact /Ki = 8,730,000 ± 918,000 (Figure 8). Additionally, the inclusion of an
alkynyl tail at the R”’ position as in 16, predicted to interact with the lipophilic pocket
beyond the prime-side binding pockets, resulted in a sub-nanomolar Ki and a kinact /Ki
=
3,600,000 ± 283,000. These results suggested that the most important alterations in
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terms of improving inhibitory potency were those that modified the head-group at the
amino acid side chain and R”’ positions, where the structure of the side chain, its
stereochemistry, and the inclusion of the O-hexynyl tail had the most significant
impacts. Future SAR studies could combine several of these structural modifications to
explore their collective impact on inhibition of cathepsin L activity and toxicity to T. cruzi.
Interestingly, the kinact was remarkably similar for most of the compounds
1
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1
produced in this study, rarely deviating more than 0.001 s . This indicates that the
driving force for inhibitor potency among these gallinamide analogs is the rate of
formation of the reversible E-I complex, as opposed to the rate of covalent modification.
Leveraging this information, future analogs could be designed with this characteristic
in mind. A summary of all kinetic parameters calculated for each analog can be found
in Table S63.
Saturation of the enamide olefin produces a reversible gallinamide analog
It has been proposed that gallinamide A is an irreversible inhibitor of the
1
1,18,19
cysteine cathepsins based on 1,4-nucleophilic addition to the enamide core.
Thus,
we hypothesized that hydrogenation of the enamide olefin would produce an analog
with reversible binding characteristics. Analog 17 was synthesized from gallinamide A
(1) by palladium catalyzed hydrogenation, selectively reducing the enamide double
bond. Reversibility was assessed using a preincubation and dilution experiment
adapted from Copeland et al., in which concentrated enzyme and inhibitor are
2
8
incubated before a rapid dilution with buffer containing substrate (Figure 6A).
Reversible inhibitors are released from the active site upon dilution and the resulting
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activity is proportional to the final concentration at 0.1 x IC50. By contrast, an
irreversible inhibitor is not released upon dilution, and thus no enzymatic function will
be recovered because the activity remains proportional to the initial concentration of
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0 x IC50.
Upon dilution, cathepsin L incubated with natural 1 displayed approximately
5
% catalytic activity, and displayed no signs of recovery after 1 hour of kinetic readings.
Conversely, incubation of 17 with cathepsin L followed by dilution gave
approximately 75% of initial activity, and remained linear over 1 hour of monitoring.
E-64c, a known irreversible inhibitor of cathepsin L, was used as a control, and matched
the response of natural product 1 (Figure 6C). These results clearly demonstrated the
conversion of the irreversible natural product inhibitor (1), into a reversible analog
(17), confirming that the enamide core is the pharmacophore of the natural product.
Figure 6: Saturation of the enamide olefin produces a reversible gallinamide analog (17). A. A
concentrated solution of the enzyme and inhibitor was incubated in assay buffer before a rapid
dilution with assay buffer that contained substrate at the standard assay concentration. As noted in
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section B, this dilution reduces the gallinamide concentration from approximately 90% enzyme
inhibition to a predicted 10% of enzyme inhibition. C. Following this dilution, product formation is
monitored over 2 hours. Compound 17, the hydrogenated gallinamide analog (red), reduced the
enzymatic reaction rate by 25% compared to vehicle control (black), while the intact gallinamide A
1
1
1
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(
green) reduced product formation by 95%, and this activity was not restored over a 2 hour period
post-dilution. This latter result matched that of the known irreversible inhibitor E-64c (blue).
Gallinamide A and analog activities against Chagas’ disease (Trypanosoma cruzi)
Based on the structural and functional similarities between cathepsin L and
cruzain, gallinamide A (1) and its synthetic analogs were assayed against the amastigote
stage of T. cruzi. Murine myoblast host cells were infected with T. cruzi trypomastigotes
beforeaddition of test compounds, and after a 3-day incubation the plates were fixed and
stained for analysis (Figure 7A).
Figure 7: Gallinamide A (1) potently eliminates T. cruzi from murine host cells. A. Representative
images of gallinamide A inhibition of T. cruzi infection. The nuclear stain allows visualization of the
host and parasite nuclei in the presence of DMSO vehicle or 20 nM gallinamide A; the latter
treatment eliminates the majority of parasites in infected murine cells. B. The dose response curve to
intracellular parasite with IC50 = 14.7 nM ± 2.3.
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Gallinamide A (1) showed remarkable activity in a cell-based intracellular T. cruzi
amastigote assay with an LD50 = 14.7 nM ± 2.3 (Figure 7B) and confirmed inhibitory
activity against recombinant cruzain with a sub-nanomolar IC50 (0.26 nM ± 0.02). This
0
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is significantly better compared to the positive control, benznidazole, which had an
LD50 of 1.5 µM in the same cell-based assay. Furthermore, no cytotoxicity to the host
murine cells was observed at concentrations up to 10 µM. Compound 5 was the most
potent analog with an LD50 of just 5.1 ± 1.4 nM, again without toxicity to host cells up
to 10 µM. While cytotoxicity to the host murine cells does not necessarily relate to
reduced toxicity in a whole animal system, the presence of highly homologous enzymes
in murine cells (e.g. murine cathepsin L) indicates that inhibition of these enzymes does
not necessarily lead to cell death. This lack of mammalian cell toxicity is likely due to
the redundancy of proteolytic enzymes in these latter cells which can compensate for the
3
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inhibition of specific proteases in the lysosome or elsewhere. Cruzain, however, is
indispensable to the survival of the parasite, and thus its inhibition rapidly leads to cell
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death of T. cruzi.
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Analog
Residues
Fourth
(R’)
Headgroup
Cathepsin L
(nM)
T. cruzi LD50
(nM), SEM
Cruzain IC50
(nM), SEM
First
(R)
Fifth
(R’’)
R”’
K
i
k
inact/K
i
-
1 -1
(M s /1000)
901 ± 49
487 ± 35
1,300 ± 64
3,480 ± 267
2,510 ±133
818 ± 43
1
2
3
4
5
6
7
8
9
L-Ile
L-Val
L-Ile
L-Phe
L-Ile
L-Ile
L-Phe
L-Ile
L-Phe
L-Ile
L-Val
L-Ile
L-Phe
L-Ile
L-Ile
L-Ile
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Phe
L-Phe
L-Phe
L-Phe
L-Phe
L-Phe
L-Phe
L-Phe
D-Phe
D-Phe
L-Ala
L-Ala
L-Ala
L-Phe
L-Phe
L-Leu
L-Ala
L-Ala
L-Phe
L-Phe
L-Leu
L-Leu
D-Phe
D-Phe
L-Phe
D-Phe
L-Ala
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
4.67 ± 0.40
6.64 ± 0.44
4.15 ± 0.24
1.35 ± 0.09
1.73 ± 0.14
3.53 ± 0.22
3.54 ± 0.22
3.13 ± 0.34
1.31 ± 0.39
0.094 ± 0.01
0.632 ± 0.04
1.68 ± 0.13
1.00 ± 0.15
376 ± 25
14.7 ± 2.3
12.9 ± 1.2
19.1 ± 1.2
18.6 ± 1.3
5.1 ± 1.4
35.8 ± 1.2
60.3 ± 4.1
33.8 ± 1.2
>10000
61.8 ± 1.1
16.4 ± 2.5
33.3 ± 1.2
>10000
1083.9 ± 1.0
537.0 ± 1.2
17.0 ± 1.2
0.26 ± 0.02
2.89 ± 0.32
0.76 ± 0.04
0.73 ± 0.04
0.49 ± 0.05
0.84 ± 0.09
1.20 ± 0.15
1.15 ± 0.04
1.35 ± 0.07
1.08 ± 0.09
0.25 ± 0.0002
1.07 ± 0.37
3.69 ± 0.61
81.0 ± 11.9
37.14 ± 8.29
0.47 ± 0.07
819 ± 93
719 ± 72
1,060 ± 135
8,730 ± 918
1,780 ± 925
1,030 ± 77
1,390 ± 169
6.90 ± 0.587
23.9 ± 1.84
3,600 ± 283
1
1
1
0
1
2
13
1
1
1
4
5
6
77 ± 6.6
0.79 ± 0.05
-(CH
2
)
4
CCH
Figure 8: Structure-activity relationships of synthetic gallinamides, listing structural modifications to
the gallinamide A (1) structure and corresponding inhibitory activities to cathepsin L and T. cruzi CA-
i i
I/72. kinact/K values for cathepsin L have been divided by 1000 to allow easier viewing of the data. K
and IC50 values are given in nM. The LD50’s to the murine host cells were all over 10 µM. N/A = Not
assessed.
Discussion and Conclusions
An integrated approach of molecular modeling and total chemical synthesis
identified several structural features in 1 that could be optimized to improve binding to
human cathepsin L (Figure 8). The structure-activity relationships seen to cathepsin L
were partly mirrored in the Chagas’ disease antiparasitic activity data (Figure 8). This
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can be rationalized by recognition of the close similarity in substrate binding and
specificity between cathepsin L and cruzain, the cysteine protease target of 1 in
1
6
Chagas.
0
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While changing the first residue from L-Ile to L-Val did not improve potency
against cathepsin L, replacing this with a L-Phe residue sometimes improved and
sometimes decreased activity when in combination with other residue replacements. The
fourth residue (R’) shows a distinct preference for L-amino acids, with D-Phe at this
position leading to a substantial (up to >100 fold) reduction in activity (e.g. comparing 8
with 14). Substitution of the L-Ala derived residue at this position with a larger
aromatic group, such as L-Phe, had a relatively neutral impact on potency (e.g.
comparing 1 with 6 and 7). Our finding that the activity to cathepsin L increases when
the fifth residue is a larger aliphatic or aromatic residue (e.g. comparing 6 and 10) is
1
9
consistent with Conway’s finding that a Trp residue at this location enhances potency.
This is also consistent with our molecular modeling results of cathepsin L with bound
11
gallinamide. Finally, and again consistent with our modeling results, placement of a
lipophilic group at R”’ enhances binding affinity and potency of the resulting agent (e.g.
comparing 1 and 16). Ultimately, it appears that interaction between these various
residues can lead to a maximally potent ligand; in our case L-Ile at the first residue, L-
Phe at the fourth residue and L-Leu at the fifth provides the highest affinity binding
i
agent to cathepsin L (10, K = 0.094 ± 0.01 nM). Interestingly, this highest affinity
analog also showed an enhanced binding score by molecular modeling (-11.31).
Against T. cruzi, the highest potency gallinamide derivative was achieved with the first
and fourth residues comprised of L-Ile and L-Ala with the fifth residue as L-Leu (5, LD50
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=
5.1 ± 1.4 nM). It will be of interest to continue to evaluate the combination of
optimized substituents at R, R’ and R” as in compound 10 with the enhancing properties
of an aliphatic group at R”’ as in compound 16.
1
1
1
1
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Chemical synthesis of an analog of 1 lacking the mid-section enone (17) allowed
biochemical experiments that provided conclusive proof that this electrophilic group is
1
1
the pharmacophore of the molecule. As predicted by our previous modeling studies,
the active site cysteine thiol serves as the nucleophile and adds via 1,4-conjugate
addition to this enamide. As discussed above, previous work by Stolze using a kinetic
1
8
19
approach, and Conway using compound potency as a metric, also identified this
same conjugated system as the electrophilic pharmacophore of 1.
Because of the close structural and functional relationship of human cathepsin L
and the T. cruzi cruzain, we also evaluated 1 and its analogs to the intracellular, disease
relevant, form of the parasite. Previous studies with 1, including the original isolation
7
workin2005, also evaluated the compound for activity against T. cruzi. However, those
7
studies reported very low or no activity to this parasite, and the focus was shifted toward
9
,18
its anti-malarial properties.
In these previous studies, however, the assay utilized the
epimastigotes (insect) stage of T. cruzi. This is likely the cause for the observed lack of
activity, as epimastigotes do not rely on cruzain for catabolism of host proteins at this life
3
1
stage, highlighting the importance of screening for antiparasitic agents using disease-
relevant stages of the parasite.
Preliminary docking studies with 1 in cruzain resulted in a similar binding score of
S = -9.568 as found for cathepsin L, with a strong hydrogen bonding network in the
oxyanion hole (Figure S69). The parasite enzyme contains a deep S2 binding pocket
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lined by a prominent glutamate residue, a feature that could be taken advantage of
through inclusion of arginine in place of the third residue, which corresponds to the P2
3
2
residue of natural substrates. Modifications could also be made to increase the
compound’s stability and solubility in vivo. For example, the ester linkage is not
indicated to be important for drug binding in the enzyme (docking scored this analog
very similar to the parent ester compound at -10.74), and could be replaced with a
0
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standard amide bond to increase plasma stability. In fact, this modification was
previously made by Conway, and resulted in an analog of increased stability, although
1
9
of two-fold reduced potency to P. falciparum.
Summarizing, the gallinamide A skeleton, comprised of amino acid and polyketide
sections, represents an inspiring scaffold with potent binding and inhibition properties to
cysteine proteases, including human cathepsin L and T. cruzi cruzain. Moreover, the
gallinamide skeleton is distinct from other anti-Chagas disease drugs and lead
33
molecules. As a result, this natural product and its analogs have significant potential in
diverse therapeutic areas, such as regulation of pain through modulation of neuropeptide
processing and antiparasitic activity through inhibition of an enzyme involved in cell
23
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invasion, parasite differentiation and metabolism. Continuing efforts are focused at
further enhancement of the selectivity of this drug class as well as improvement of
fundamental pharmaceutical properties and stability.
Experimental Section
General Experimental. Unless specifically noted otherwise, synthetic reactions were
carried out in flame or oven-dried glassware under Argon, with the exception of reactions
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run in water as co-solvent. All reactions were stirred using a magnetic stir bar.
Deuterated chloroform for NMR, with 0.03% TMS as reference, was purchased from
Cambridge Isotope Laboratories. NMR spectra were collected on a Varian 500 MHz
spectrometer or a Varian 500 MHz spectrometer equipped with an inverse cryo-probe.
HR-ESI-TOF-MS data were collected by the Molecular Mass Spectrometry Facility at
UCSD. Purity of the final products 1-17 was assessed as being above ≥95% by
1
1
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evaluation of their H NMR and C NMR spectra.
Docking protocol. Gallinamide A was positioned into the cathepsin L active site using
the docking module of MOE. No hydrogen bond constraints were applied. Initial
placements were scored with London dG, induced fit refinement was used with the
default parameters, and final poses were scored with GBVI/WSA dG during the final
step. Poses were assessed individually and filtered based on distance between the
reactive carbon of the enamide Michael acceptor system and the cysteine thiol.
General Chemical Synthesis
Final Coupling Reaction (1-16). As a general procedure, a vial was charged with the
benzyl ester protected dimer then this material was dissolved in EtOH, the palladium
(as a 1% loading on carbon) was added in one portion, and the atmosphere was
replaced with hydrogen. The reaction was then stirred overnight and the reaction
mixture was passed through a ca. 1 cm diatomaceous earth plug, prepared in a pipette
with glass wool, into a 25 mL pear flask using three 1 mL DCM rinses. This material was
then dried under N (g) flow. In a separate vial the carbamate of the leucine extended
2
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