Peptidomimetic Vinyl Heterocyclic Inhibitors of Cruzain Effect Antitrypanosomal Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b02078

Cruzain, an essential cysteine protease of the parasitic protozoan, Trypanosoma cruzi, is an important drug target for Chagas disease. We describe here a new series of reversible but time-dependent inhibitors of cruzain, composed of a dipeptide scaffold appended to vinyl heterocycles meant to provide replacements for the irreversible reactive "warheads" of vinyl sulfone inactivators of cruzain. Peptidomimetic vinyl heterocyclic inhibitors (PVHIs) containing Cbz-Phe-Phe/homoPhe scaffolds with vinyl-2-pyrimidine, vinyl-2-pyridine, and vinyl-2-(N-methyl)-pyridine groups conferred reversible, time-dependent inhibition of cruzain (K_i? = 0.1-0.4 μM). These cruzain inhibitors exhibited moderate to excellent selectivity versus human cathepsins B, L, and S and showed no apparent toxicity to human cells but were effective in cell cultures of Trypanosoma brucei brucei (EC₅₀ = 1-15 μM) and eliminated T. cruzi in infected murine cardiomyoblasts (EC₅₀ = 5-8 μM). PVHIs represent a new class of cruzain inhibitors that could progress to viable candidate compounds to treat Chagas disease and human sleeping sickness.

Full text of DOI:10.1021/acs.jmedchem.9b02078

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Article
Peptidomimetic Vinyl Heterocycle Inhibitors
of Cruzain Effect Anti-Trypanosomal Activity
BALA CHANDRA CHENNA, Linfeng Li, Drake Mellott, Xiang Zhai, Jair L Siqueira-
Neto, Claudia Calvet Alvarez, Jean Bernatchez, Emily Desormeaux, Elizabeth Alvarez
Hernandez, Jana Gomez, James McKerrow, Jorge Cruz-Reyes, and Thomas D. Meek
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02078 • Publication Date (Web): 03 Mar 2020
Downloaded from pubs.acs.org on March 4, 2020
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Peptidomimetic Vinyl-Heterocyclic Inhibitors of Cruzain Effect Anti-Trypanosomal Activity
Bala C. Chenna,†,# Linfeng Li,†,# Drake M. Mellott, Xiang Zhai, Jair L. Siqueira-Neto, Claudia Clavet,
†
†,‡
§
§
§
†,┴
†
†
Jean Bernatchez, Emily Desormeaux,
Elizabeth Alvarez Hernandez, Jana Gomez, James H.
§
†
†,*
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McKerrow, Jorge Cruz-Reyes, and Thomas D. Meek
#
*
These authors contributed equally
Corresponding author
†Department of Biochemistry & Biophysics, Texas A&M University, 301 Old Main Drive, College Station,
TX 77843, United States
§Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California-San Diego, 9500
Gilman Drive, La Jolla, CA 92093, United States
ABSTRACT
Cruzain, an essential cysteine protease of the parasitic protozoan, Trypanosoma cruzi, is an important drug
target for Chagas disease. We describe here a new series of reversible, but time-dependent, inhibitors of
cruzain, composed of a dipeptide scaffold appended to vinyl-heterocycles meant to provide replacements
for the irreversible reactive “warheads” of vinyl sulfone inactivators of cruzain. Peptidomimetic vinyl-
heterocyclic inhibitors (PVHIs) containing Cbz-Phe-Phe/homoPhe- scaffolds with vinyl-2-pyrimidine,
vinyl-2-pyridine, and vinyl-2-(N-methyl)-pyridine groups conferred reversible, time-dependent inhibition
of cruzain (K * = 0.1-0.4 M). These cruzain inhibitors exhibited moderate to excellent selectivity vs.
i
human cathepsins B, L, and S, and showed no apparent toxicity to human cells, but were effective in cell
cultures of Trypanosoma brucei brucei (EC = 1-15 μM), and eliminated Trypanosoma cruzi in infected
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murine cardiomyoblasts (EC = 5-8 μM). PVHIs represent a new class of cruzain inhibitors that could
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progress to viable candidate compounds to treat Chagas disease and human sleeping sickness.
INTRODUCTION
Chagas disease (CD), caused by the American parasitic protozoan Trypanosoma cruzi (T. cruzi), affects
nearly 8 million people in Mexico, Central and South America, resulting in 50,000 annual deaths.1-3 It is
estimated that more than 300,000 US residents are infected with T. cruzi, and cases have been reported in
California, Texas and other parts of the Southwestern US in which the infections were acquired locally.4-6
Approximately 20-30% of people infected with T. cruzi will develop debilitating and potentially fatal heart
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dilation, arrhythmias, apical aneurysms, and/or a dilated colon and esophagus. The related African
trypanosomes Trypanosoma brucei rhodesiense (T. b. rhodesiense) and Trypanosoma brucei gambiense
7
(
T. b. gambiense), cause human sleeping sickness. Sleeping sickness is endemic in 36 African countries,
threatening an estimated 60 million people, with more than 60,000 reported cases.8-10 With the absence of
vaccines, the standard treatment for Chagas disease is limited to nifurtimox and benznidazole which are so
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replete with side effects that abandonment of treatment is common.11-12
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14-15
Cysteine proteases of the papain family are of similar structure and substrate specificities,
and are
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implicated in numerous human diseases including cancer (cathepsin L), chronic obstructive pulmonary
disorder (cathepsins C17 and S ) and osteoporosis (cathepsin K ). Likewise, the highly homologous,
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cathepsin L-like cysteine proteases, cruzain (E.C. 3.4.22.51; Uniprot ID: P25779),20-22 rhodesain, and
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brucipain (TbCatL), found in the respective parasitic species T. cruzi, T. b. rhodesiense, and T. b. brucei,
are essential to the establishment and maintenance of host infection by trypanosomes.20-24 The roles of
protozoal cysteine proteases in disease include the scavenging of iron from metalloproteins,24 and the
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evasion of immune surveillance by proteolysis of NF-B. Gene deletion of cruzain demonstrated its role
in undermining the prophylactic role of macrophages during infection.26 The work of McKerrow and
colleagues demonstrated the biochemical essentiality of cruzain in the pathology of T. cruzi.26-30 They first
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expressed and characterized cruzain, including its crystal structure, and identified the drug candidate
K11777, a dipeptide vinyl sulfone (Figure 1) which undergoes irreversible thia-Michael addition to active-
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site Cys .
The progression of K11777 to human trials includes demonstration of a cure of T. cruzi-
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infected mammalian cells in culture,29 and also elimination of parasites from acutely-infected mice.28
However, K11777 has stalled in pre-clinical evaluation due to liver toxicity, possibly arising from its
irreversible mechanism.13, 34-35
Inactivators that form reversible covalent adducts with cysteine groups on enzymes have received recent
attention.36-38 Such reversible covalent inactivators demonstrate time-dependent inactivation, like
irreversible inactivators, but may exert greater selectivity for the intended target rather than homologous
“
off-target” enzymes. This is because while the initial-collision complexes of irreversible inactivators with
a panel of related enzymes may have variable affinities, over time the establishment of permanent covalent
bonds may render this initial selectivity inconsequential. However, in the case of a reversible covalent
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inhibitor, their residence times on these enzymes are likely to be variable, leading ultimately to “relief”
from covalent inactivation for off-targets.
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Figure 1. Structures of K11777, GSK2793660, and Cbz-Phe-Phe-vinyl-2Pyrmd 7 with thia-Michael addition of Cys25 to the vinyl
groups in these compounds, including the putative adduct 7a, which reverts to the fully conjugated 7 upon the reverse of adduct
formation. The common bioisosteric atoms of K11777, GSK2793660, and 7 are highlighted in red. X is an electron-donating or
electron-withdrawing substituent.
The design of dipeptide vinyl-heterocyclic inhibitors is predicated on existing irreversible covalent
inactivators of cysteine proteases such as the vinyl sulfone of K11777 and the acrylamide of
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GSK27993660, an inactivator of human cathepsin C that reached Phase I clinical trials. Both compounds
form irreversible covalent adducts with the active-site cysteines of the respective enzymes via a thia-
Michael reaction (Figure 1), and both have encountered either toxicity issues or adverse events in,
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respectively, either animals or humans. Accordingly, we sought replacements of the vinyl sulfone and
acrylamide “warheads” with less electrophilic moieties that would undergo reversible thia-Michael
addition, in order to develop inactivators of high potency for cruzain, but also with suitable selectivity for
trypanosomal over human cysteine proteases. One approach is the replacement of the vinyl sulfone and
carboxamide group of the acrylamide with a bioisosteric heterocyclic group which is conjugated to the
reactive vinyl group. A 2-vinyl pyrimidine is one such bioisosteric replacement (7, Figure 1), in which one
of the ring nitrogens mimics the amide carbonyl and sulfone oxygen while the other substitutes for the
amide nitrogen (red atoms in Figure 1). As the pyrimidine is conjugated to the vinyl group, the expected
addition of the thiol group of the cysteine to the -carbon of the vinyl group and attending protonation of
the -carbon would eliminate this conjugation. Subsequent re-establishment of conjugation via reversal of
the thia-Michael reaction would provide reversible covalent inactivation of the enzyme. Another feature
afforded by this vinyl-heterocycle is the ability to modify the reactivity of the vinyl group by the substitution
of the heterocycle with electron-donating or electron-withdrawing groups. In this study, we designed,
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synthesized, and evaluated a panel of dipeptide compounds containing a vinyl group replacing the scissile
amide group of the substrate, which is conjugated to a phenyl group or a collection of heterocycles. For
some, we investigated the ability of glutathione to form covalent adducts with their vinyl groups, in order
to explore the electronic nature of the heterocycle required for facile addition of thiols. Many of these
compounds displayed potent, time-dependent inhibition of cruzain, as well as anti-trypanosomal activity in
cell culture.
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RESULTS AND DISCUSSION
Evaluation of Dipeptide Substrates. We previously conducted a detailed investigation of the catalytic
mechanism of cruzain utilizing three dipeptide substrates, and by using solvent kinetic isotope effects in
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both pre-steady-state and steady-state kinetic modes. As with other cysteine proteases, our kinetic studies
supported a double-displacement chemical mechanism, in which a thioester intermediate of Cys25 and
substrate is first formed (enzyme acylation) concurrent with release of the amine product. This half-reaction
is followed by enzyme-catalyzed hydrolysis of the thioester (enzyme de-acylation) to afford the carboxylate
product. We determined that for Cbz-Phe-Arg-AMC (S1, Table 1), the most optimal substrate in terms of
values of k and k /K , and Cbz-Arg-Arg-AMC (S6), enzyme-catalyzed de-acylation comprised the slow
cat
cat
m
steps of the reaction (kcat = k ) while the rate of the acylation of active-site Cys (described by k ), was
dac
25
ac
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1
0-fold and 4-fold, respectively, more rapid than k . The parameter k /K contains the individual rate
cat cat m
constants found in the expression for k , and therefore reports on the rates of substrate binding and catalysis
ac
up to and including the acylation of active-site Cys . Accordingly, this kinetic parameter comprises a useful
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guide for the selection of optimal dipeptide scaffolds to be incorporated into inhibitors that are meant to
form reversible covalent complexes with Cys upon binding, a reaction that chemically mimics acylation.
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If our dipeptidic vinyl-heterocyclic inhibitors do indeed inhibit cruzain by addition of the thiolate of Cys25
to their olefinic replacements for the scissile amide bond, then the values of k /K (the specificity
cat
m
constants) for the fluorogenic substrates should provide an approximate linear correlation with values of
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kinact/K (which is the “specificity constant” of inactivation).
I
We have kinetically characterized 12 dipeptide fluorogenic substrates of cruzain which are of the form:
Cbz/NMePip-P -P -AMC, for which Cbz is benzyloxycarbonyl; NMePip is N-methyl-piperazinyl; P =
2
1
2
Phe, Leu, Arg or (4-Pyr)Ala (4-pyridyl-alanine); P = Phe, hPhe (homoPhe), Arg, Ala and (4-Pyr)Ala; and
1
AMC is 7-amino-4-methyl coumarin (Table 1). The largest specificity constant (k /K ) measured was that
cat
m
-1 -1
of Cbz-Phe-Arg-AMC (11 M s , relative k /K = 1.0). Substrates S2-S4 have values of k /K which
cat
m
cat
m
are 73%-35% of that of Cbz-Phe-Arg-AMC, indicating that Cbz-Phe-hPhe-AMC, Cbz-Leu-hPhe-AMC,
and Cbz-Leu-Arg-AMC all comprise highly competent substrates likely effecting rapid acylation of
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cruzain. The poorest substrate, Cbz-Phe-Ala-AMC (S12), demonstrated that a small sidechain in the P1
position is less preferable than more bulky hydrophobic moieties. Conversely, the dipeptide Cbz-Phe-hPhe-
-1 -1
AMC (S2) exhibited a value of k /K = 8 M s , indicating that a peptide substrate with the same P and
cat
m
2
P1 amino acids as K11777 is an excellent substrate of cruzain, and likely involves rapid acylation.
Interestingly, when the Cbz group of substrate S2 is replaced with NMePip (S7), a substrate mimic of
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K11777, k /K is 8-fold lower (1.3 M s ). However, its value of k exceeded that of Cbz-Phe-hPhe-
cat
m
cat
AMC (S2), suggesting that the NMePip N-terminus of S7 acts to retard cruzain acylation in comparison to
the Cbz group of S2. While Cbz-Phe-Phe-AMC (S9) had a value of k /K that was only 7% of Cbz-Phe-
cat
m
Arg-AMC, its K value of 0.34 M indicated potent binding, albeit, with slow turnover. This may indicate
m
that a P Phe substitution leads to favorable binding, but this sidechain impedes either the acylation or de-
1
acylation step in catalysis. This was observed for dipeptide-AMC substrates for the papain-like protease,
human cathepsin C.43
a
Table 1. Kinetic Parameters of Peptide Substrates for Cruzain
k
cat/K
m
-1
-1
Substrate
Structure
k
cat (s )
K
m
(M)
k
cat/K m rel k
cat rel
-1
(
M s )
S1
S2
Cbz-Phe-Arg-AMC
9.6 ± 0.2 0.89 ± 0.09
2.0 ± 0.2 0.26 ± 0.06
11 ± 1
1.00
0.73
0.36
0.35
0.18
0.17
0.12
0.10
0.07
0.06
0.02
0.00
1.00
0.21
0.21
0.88
0.76
0.75
0.42
0.55
0.03
0.51
0.29
0.09
Cbz-Phe-hPhe-AMC
Cbz-Leu-hPhe-AMC
Cbz-Leu-Arg-AMC
8 ± 2
4 ± 1
S3
3.0 ± 0.2
8.4 ± 0.4
7.5 ± 0.5
7.2 ± 0.1
4.0 ± 0.1
0.8 ± 0.2
2.2 ± 0.3
3.7 ± 0.5
3.7 ± 0.4
3.1 ± 0.3
4.6 ± 0.5
S4
3.8 ± 0.7
2.0 ± 0.4
S5
Cbz-Phe-(4-Pyr)Ala-AMC
b
S6
1.9 ± 0.2
Cbz-Arg-Arg-AMC
S7
NMePip-Phe-hPhe-AMC
1.3 ± 0.1
S8
Cbz-(4-Pyr)Ala-hPhe-AMC 5.3 ± 0.2
1.1 ± 0.1
S9
Cbz-Phe-Phe-AMC
Cbz-Arg-hPhe-AMC
NMePip-Phe-Phe-AMC
0.26 ± 0.01 0.34 ± 0.06
0.8 ± 0.2
S10
S11
S12
4.9 ± 0.1
6.8 ± 0.4
14 ± 4
0.7 ± 0.04
0.2 ± 0.04
0.023 ± 0.003
2.8 ± 0.06
b
0.89 ± 0.06
38 ± 2
Cbz-Phe-Ala-AMC
a
b
Data obtained at pH 7.5, 25°C, 10% DMSO (v/v); Data obtained at 2% DMSO (v/v) from Ref. 40.
We explored the viability of the (4-Pyr)Ala residue as a potential mimic of both Phe and Arg, for the latter
residue when the pyridine is protonated. Cbz-Phe-(4-Pyr)Ala-AMC (S5) was found to be a good substrate
(
relative k /K = 0.10), but with a higher value of K (3.7 M) compared to substrates containing Phe and
cat
m
m
hPhe in the P residue. To leverage the ability of cruzain to tolerate both charged basic and hydrophobic
1
residues in the P position, we prepared Cbz-(4-Pyr)Ala-hPhe-AMC and Cbz-Arg-hPhe-AMC (S8 and
2
-1
S10). These substrates displayed efficient turnover numbers (kcat = 5.3 and 4.9 s , respectively), but poor
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values of K (4.6 and 6.8 μM) in comparison to Cbz-Phe-Phe/hPhe-AMC (0.26 M). This demonstrates
m
that the enzyme has a strong preference for substrates that contain a hydrophobic P residue. Together, these
2
results indicated that dipeptide scaffolds in which P = Cbz-Phe or NMePip-Phe and P = Arg, hPhe, or (4-
2
1
Pyr)Ala may be among the best to incorporate into the vinyl-heterocyclic framework. Accordingly, for our
new peptidomimetic vinyl-heterocyclic inhibitors we have primarily utilized the Cbz-Phe-Phe and
NMePip/Cbz-Phe-hPhe dipeptide scaffolds.
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Computer-Assisted Inhibitor Design. To aid in the rational design of our vinyl-heterocyclic inhibitors we
employed molecular docking of these compounds to a model constructed from the crystal structure of
K11777-cruzain (PDB accession code: 2OZ2) which contains a covalent bond between the inactivator and
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Cys . Owing to our hypothesis that the vinyl-heterocyclic inhibitors have the ability to undergo a
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reversible thia-Michael addition with the active-site Cys of cruzain, it is necessary to consider scenarios
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of both non-covalent and covalent binding. To this end, we first predicted the binding patterns for NMePip-
Phe-hPhe-vinyl-2Pyrmd (9) which has the same scaffold as K11777 using Glide44-46 and CovDock47
modules embedded in the Schrodinger software package. In the covalent model, the binding of 9 with
cruzain was highly conserved when compared to that of K11777 (Figure 2A). Pyrimidine N1 of 9 was
within hydrogen bonding distance of Gln and Trp , allowing the stabilization of the vinyl-heterocycle in
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a nearly analogous fashion to the sulfone moiety in K11777. In addition, the α-carbon of the inhibitor is
positioned within 2.4 Å of His , an interatomic distance that would easily allow facile proton transfer
1
62
between this carbon and the imidazole nitrogen, supporting our hypothesis that a reversible adduct could
be formed with cruzain. The non-covalent model (Figure 2B) shared similar shape complementarity with
the covalent binding pose, except that it was slightly shifted away from the binding site as militated by the
docking algorithm to avoid clashing with Cys . This suggested that covalent bond formation would only
2
5
slightly perturb the non-covalent binding conformation. Overall, these data suggested that the binding of
our newly designed compounds containing a vinyl-heterocyclic warhead have the ability to interact with
cruzain in a very similar fashion to the characterized, irreversible inactivators of the enzyme. Similarly, we
carried out docking for five other PVH compounds (7, 11, 12, 13, and 15 in Figure S1, and their covalent-
docking affinity values (Cdock affinity) are summarized in Table S1. The corresponding inhibition
constants (predicted K ) converted from these affinity values ranged from 0.79 to 6.1 M, and with the
i
exception of compound 7, were similar with the experimental values found in Table 2. In agreement with
our dipeptide substrate kinetic data, we observed an increased Cdock affinity for 13 and 15 for which each
contained a hPhe in the P position instead of Phe. In addition, the substitution of a pyridine ring at the P ’
1
1
positions of 11, 12, 13, and 15 may subtly improve the binding compared to the pyrimidine substituent of
7
. Further, the N-methylation of pyridine resulted in a fairly large shift in Cdock affinity, possibly resulting
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9
from an additional ion-ion/dipole interaction. On the basis of these docking analyses, our inhibitor design
focused on using a dipeptidic scaffold containing Phe in the P position and either Phe or hPhe in the P
2
1
position. We varied the identity of the heterocycle in order to modify the electrophilicity of the olefin bond,
but generally maintained functional groups that possibly could afford hydrogen bonding with Gln19 to
stabilize the binding of the compounds near Cys of cruzain.
2
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Molecular models of compound 9 bound to cruzain. (A) K11777 (gray) is superimposed with a binding pose (green) in
which a covalent bond is formed between the β-carbon of the vinyl group of 9; (B) Binding pose (cyan) of 9 in which no covalent
bond is formed with Cys25. Yellow and white dashed lines represent hydrogen bonds with surrounding residues for 9 and K11777,
respectively. Red dashed lines are measurements between the catalytic dyad and vinyl moiety of 9.
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1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesis of PVHIs. The general synthetic routes employing either Wittig48 or Horner-Wadsworth-
Emmons49 reactions shown in Scheme 1 were used to synthesize peptidomimetic vinyl-heterocyclic
compounds from aldehydes and halo-methyl heterocycles.
Commercially-available Boc-protected L-amino acids phenylalanine, homophenylalanine and alanine (a)
were converted to Weinreb amides50 by T3P-catalyzed coupling to N,O-dimethylhydroxylamine
hydrochloride to afford b (GP1, General Procedure 1 in Experimental Section). Reduction of the Weinreb
amide using LAH at -10 °C in anhydrous THF provided the Boc-amino acid aldehyde (c, GP2), generally
in overall yields of ~80% (a-c).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. General synthetic route to PVHIsa
aConditions and reagents (General Procedures are detailed in Experimental Section): GP1. N,O-dimethylhydroxylamine
o
o
o
4 2 3
hydrochloride, T3P, DIPEA, DCM, 0 C; GP2. LAH, THF, -10 C; GP3. 1) NaBH , EtOH, 0 C, 2) SOCl , DCM; GP4. PPh ,
o
o
o
o
o
benzene, reflux; GP5. P(OEt)
0 oC, 2) R
3
, 150 C; GP6. LHMDS, THF, -70 C - 0 C; GP7. LHMDS, THF, -70 C - 0 C; GP8. 1) TFA, DCM,
o
1
-Xaa-OH, T3P, DIPEA, DCM, 0 C; GP9: MeI, MeCN, reflux; GP10: 1) LiOH, H
2
O, 2)ClCOOEt, NH
4
Cl.
Phosphonium salts of methyl-heterocycles were in general prepared by derivatization of either the 2-
methylcarboxy- or 2-hydroxymethyl-heterocycle (d to f, Scheme 1). Methyl 2-carboxy-pyrimidine (or
pyridine, oxazole, and thiazole) was reduced using sodium borohydride to the primary alcohol, followed
by conversion of the alcohol to the 2-chloromethyl-pyrimidine (e) using SOCl or POCl in DCM or CHCl
3
2
3
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(
GP3). Reaction of e with triphenylphosphine provided the Wittig reagent phosphonium salt (f) at overall
yields of 28 - 80% (GP4). Wittig coupling of f with a peptide aldehyde (c) using LHMDS in anhydrous
THF or sodium methoxide in benzene as base provided the peptide vinyl-heterocyclic product h (GP6),
with general overall yields of 13 - 54%. Typically, the ratio of E:Z was 4:1, and separation of these
regioisomers was readily achieved using silica gel column chromatography.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Alternatively, 2-chloromethyl-heterocycle e was converted to its phosphonate g by use of the Arbuzov
reaction with triethyl-phosphite (~80% yields, GP5). The resulting phosphonate was de-protonated with
LHDMS in THF, and then coupled with aldehyde c to provide the peptide vinyl-heterocycle h at 20% -
8
0% yield (GP7). The Boc group was removed quantitatively by treatment with TFA in DCM, then the free
amine was coupled with the P -P fragment (R -Xaa-OH) using T3P to give the inhibitor i (GP8).
3
2
1
In addition, some of the PVHIs underwent N-methylation of the heterocycle (j, GP9). Further, we also
prepared several acrylamides (k) through hydrolysis of corresponding acrylate ester and subsequent
treatment with ethyl chloroformate and NH Cl (GP10). Final products were confirmed structurally by NMR
4
and LCMS as described in Experimental Section and Supplementary Information. It is important to note
that proton NMR analysis of the products (i, j, and k) indicated negligible epimerization at the -carbon in
these products, as evidenced by the absence of diastereomers.
Electrophilicity of Vinyl-Heterocycles. To evaluate the chemical reactivity of the vinyl group in our
PVHIs and K11777 we treated selected compounds with glutathione (GSH) at pH 8.0 to determine their
reactivity in a thia-Michael addition of the sulfhydryl group of GSH with the vinyl group of the inhibitors.
Normally, the addition of glutathione to an enzyme inhibitor is to be avoided, but here this serves as a
means to evaluate the electrophilicity of these inhibitors. K11777 and compounds 7, 11, 12, 15, 17, 25 and
2
6 (Table 2), which respectively contain a vinyl sulfone (R = I), a vinyl-2-pyrimidine (R = IV), a vinyl-
3 3
2
-pyridine (R = V), a vinyl-2-N-methylpyridine (R = V, R = Me), a vinyl-2-(4-trifluoromethyl)-pyridine
3 3 4
(R = V, R = CF ), a vinyl-2-thiazole (R = IX), and a vinyl-2-N-methylthiazole (R = IX, R = Me). As
3 5 3 3 3 4
51
-1
previously reported, the formation of a glutathione adduct with K11777 was very slow (k = 0.00028 s ,
Table S2, Figure 3), and we were unable to ascertain an equilibrium constant for the K11777-GSH adduct.
For the PVHIs, the reaction between GSH and the vinyl-2-pyrimidine (7), the vinyl-pyridine (11), the vinyl-
2-(4-trifluoromethyl)-pyridine (17), and the vinyl-thiazole (25), was negligible as no adduct was observed
after 90 minutes incubation with either a 2:1 or 10:1 molar ratio of GSH:inhibitor. The electron-
withdrawing 4-trifluoromethyl group on the pyridine of 17 had no effect on the electrophilicity of 11. In
contrast, addition of GSH to the vinyl group of vinyl-2-N-methylpyridine (12, 15) and vinyl-2-N-
methylthiazole (26) in a 2:1 molar ratio resulted in the rapid formation of GSH adducts at respective rates
-1 -1
-1 -1
-1 -1
of 0.037 mM s , 0.054 mM s , and 0.015 mM s , and apparent equilibrium was achieved for these
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
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5
5
5
5
5
5
5
5
6
-1
-1
-1
compounds in 90 min (K = 7400 M , 2400 M , and 930 M , respectively). This demonstrated that the N-
eq
methylation of the PVHs afforded a significant increase in the electrophilicity of the vinylic position,
enabling rapid addition to thiols, owing to the strong electron-withdrawing effect of the methylpyridinium
moiety. For example, compounds 11 and 12 are identical except for the N-methylpyridine group of
compound 12; compound 12 readily forms an adduct with GSH (97% conversion of 12 to its GSH adduct
in 20 min at a 10:1 molar ratio of GSH to compound), whereas compound 11 is unreactive towards GSH.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Interestingly, the rate of thiolation of compound 12 is 50% that of 15, while the values of K indicated that
eq
the 12-GSH adduct is three times more abundant than that of 15-GSH. This suggested that the phenylalanyl
sidechain of 12 may retard the addition of GSH to its vinyl group, and also slowed the presumed base-
catalyzed elimination of GSH from its adduct with 12. Overall, these results demonstrated that the reactivity
of vinyl-heterocycles with GSH and presumably Cys vary with the nature of the heterocycles. Hence, it
2
5
is possible to tune the electronic properties of the vinyl bond in the PVHIs, thereby allowing for
development of modifiable electrophilic inhibitors of other enzymes that have an active-site cysteine or
other nucleophile.
Figure 3. Time courses of depletion of 0.5 mM of K11777 (red circles), compound 12 (blue circles), and compound 15 (black
circles) upon formation of adducts (red, blue, and black squares, respectively) with 1 mM glutathione. Lines drawn through the
curves for substrate depletion and adduct formation, were respectively, [Substrate] = (0.5 mM -A)(1-exp(-k*t)) + C and [Adduct]
=
A*(1-exp(-k*t)) + C, with resulting kinetic parameters found in Table S3.
Kinetic Analysis of Cruzain Inhibitors and Inactivators. Scheme 2 is a kinetic depiction of inhibition
5
2
and inactivation of cruzain and the relevant kinetic parameters. The initial, and usually rapid, formation
of EI is characterized by the inhibition constant K . For time-dependent inhibitors EI progresses to a second,
i
tighter complex EI*, generally over the course of minutes, characterized by K *, for which K * < K when
i
i
i
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k < k . For irreversible covalent inactivators, k and K * ~ 0, and the kinetic parameter k / K is generally
4
3
4
i
inact
I
reported. For reversible time-dependent inhibitors, initiation of reaction by adding enzyme to substrate and
inhibitor leads to concave-downward, curvilinear time courses of product formation in which reaction rates
demonstrably decrease as the EI* complex forms. Typical data, as exemplified for compound 15, are shown
in Figure 4A. Alternatively, extended pre-incubation of enzyme and inhibitor, followed by dilution of the
inhibitor and initiation of reaction with high concentrations of substrate, leads to concave-upward
curvilinear plots of product formation as E reforms from EI* (Figure 4B). Results of this analysis for
cruzain inhibitors and inactivators are collected in Table 2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Kinetic depiction of inhibition and inactivation.
k1
k2
k3
E + I
EI
EI*
k4
K * = K k /(k + k )
K = k /k
1
i
i 4
3
4
i
2
K = (k + k )/k
1
kinact = k3
I
2
3
K11777 comprises a useful benchmark compound despite the fact that it is an irreversible inactivator of
-1 -1 32,53
cruzain (reported kinetic data: apparent IC of 2 nM, k /K = 234,000 M s ).
We replaced the P1
50
inact
I
hPhe group of K11777 with a Phe sidechain to provide vinyl sulfone 1, which had apparently equivalent
potency (K * = 3.6 nM) to that of K11777, but which, interestingly, exhibited kinetically reversible
i
inhibition of cruzain. However, a crystal structure we obtained for 1 bound to cruzain indicated the
formation of a C-S bound between Cys and 1 (Figure S4). This may indicate that a phenylalanyl group at
2
5
the P position partly impedes the ability of an adjacent vinyl electrophile to access Cys , as was observed
1
25
with the solution phase GSH addition studies to our PVH compounds.
We next evaluated three C-terminal acrylamides (R = II) within the Cbz-Phe-Phe, Cbz-Phe-hPhe, and
3
NMePip-Phe-hPhe scaffolds (2-4). The acrylamides within the Cbz-Phe-hPhe and NMePip-Phe-hPhe
-1 -1
scaffolds afforded apparently irreversible covalent inactivation (kinact/K = 1700-1900 M s ), while Cbz-
I
-1 -1
Phe-Phe-acrylamide (2) was less effective (kinact/K = 22 M s ). Comparing the values of k /K for
I
inact
I
K11777 and 4 indicated that the vinyl sulfone is overwhelmingly more effective as a covalent inactivator
than its acrylamide counterpart, possibly owing to hydrogen bond contacts of the sulfone oxygen with Gln ,
1
9
which position the vinyl group proximal to Cys of cruzain. As with 1, a Phe rather than a hPhe group at
2
5
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the P position, may retard covalent formation over the time course of kinetic analysis when one compares
1
the rates of apparent inactivation of 2 vs. 3 and 4, as was also seen with peptide substrates.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
1
2
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4
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a
Table 2. Kinetic Data of Peptidomimetic Vinyl-Heterocyclic Inhibitors of Cruzain
Ph/i-Pr
O
H
N
R3
R1
N
H
O
R2
R4
O
O
O
R5
N
S
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
N
N
IV
R4
I
II
III
V
R3
R4
N
N
N
N
N
O
R4
S
VI
VII
VIII
IX
Cruzain Inhibition or Inactivation
Compound
Structure
R
1
R
2
R
3
k
inact/K inact
-1
K
i
(M)
K
i
* (M)
-
1
(M s )
b
b,c
K11777 NMePip-Phe-hPhe-VSPh
NMePip CH
2
Bn
I
I
NA
0.002
234,000
ND
1
2
3
4
5
6
7
8
9
Cbz-Phe-Phe-VSPh
BnO
Bn
Bn
ND
0.0036 ± 0.0001
NA
Cbz-Phe-Phe-vinyl-CONH
2
BnO
II
II
37 ± 2
3 ± 1
21.7 ± 0.8
1700 ± 500
1900 ± 200
NA
Cbz-Phe-hPhe-vinyl-CONH
2
BnO
CH
2
Bn
Bn
NA
NMePip-Phe-hPhe-vinyl-CONH
Cbz-Phe-Phe-vinyl-Ph
2
NMePip CH
2
II
3.4 ± 0.4
1.8 ± 0.1
ND
NA
BnO
BnO
Bn
Bn
Bn
III
0.87 ± 0.05
0.37 ± 0.02
0.364 ± 0.004
NA
Cbz-Phe-Phe-vinyl-(4-NO
2
)Ph
III, R
4
= NO
2
NA
Cbz-Phe-Phe-vinyl-2Pyrmd
Cbz-Phe-hPhe-vinyl-2Pyrmd
BnO
IV
IV
IV
IV
V
28 ± 1
>35
NA
BnO
CH
2
Bn
Bn
NA
NMePip-Phe-hPhe-vinyl-2Pyrmd
Cbz-Phe-Ala-vinyl-2Pyrmd
Cbz-Phe-Phe-vinyl-2Pyr
NMePip CH
2
>10
2.2 ± 0.1
25 ± 1
NA
1
0
1
2
BnO
BnO
Me
Bn
Bn
58 ± 6
5.5 ± 0.4
3.8 ± 0.4
NA
1
1
0.31 ± 0.01
0.28 ± 0.08
NA
Cbz-Phe-Phe-vinyl-2PyrNMe
Cbz-Phe-hPhe-vinyl-2Pyr
BnO
V, R
4
= Me
NA
13
BnO
CH
2
2
2
2
2
2
Bn
Bn
V
V
1.06 ± 0.07 0.171 ± 0.004
ND 3.4 ± 0.1
NA
1
1
1
4
5
6
NMePip-Phe-hPhe-vinyl-2Pyr
Cbz-Phe-hPhe-vinyl-2PyrNMe
NMePip CH
NA
BnO
BnO
BnO
BnO
BnO
CH
CH
CH
CH
Bn
Bn
Bn
Bn
V, R
V, R
V, R
4
= Me 0.76 ± 0.04 0.126 ± 0.004
NA
Cbz-Phe-hPhe-vinyl-2-(4-OMe)-Pyr
5
= OMe
>5
NA
NA
NA
17
Cbz-Phe-hPhe-vinyl-2-(4-CF
Cbz-Leu-hPhe-vinyl-2Pyr
Cbz-Phe-Ala-vinyl-2Pyr
Cbz-Phe-Lys-vinyl-2Pyr
Cbz-Phe-Phe-vinyl-4Pyr
3
)-Pyr
5
= CF
3
0.57 ± 0.05
1.42 ± 0.09
4.8 ± 0.2
NA
18
19
20
21
V
V
7.8 ± 0.6
ND
NA
Me
NH
NA
BnO (CH
BnO
2
)
4
2
V
17.3 ± 0.3
ND
0.87 ± 0.02
5.5 ± 0.2
NA
Bn
Bn
Bn
Bn
Bn
Bn
VI
NA
22
Cbz-Phe-Phe-vinyl-4PyrNMe
Cbz-Phe-Phe-vinyl-4Pyrmd
Cbz-Phe-Phe-vinyl-2Oxz
Cbz-Phe-Phe-vinyl-2Thz
BnO
VI, R
4
= Me
92 ± 5
10.8 ± 1.4
10 ± 1
ND
4.0 ± 0.1
NA
23
24
25
BnO
VII
VIII
IX
1.14 ± 0.07
0.71 ± 0.01
1.71 ± 0.09
0.94 ± 0.06
NA
BnO
NA
BnO
NA
26
Cbz-Phe-Phe-vinyl-2ThzNMe
BnO
IX, R
4
= Me
ND
NA
a
b
c
-1 -1
Data obtained at 25°C, pH 7.5; Reported as apparent IC50 in Ref. 32; Reported as 32,500 M s (pH 8.0) in Ref. 53; NA, not applicable; ND, not determined; app K
i
i
and K * are respectively, the apparent initial and tight-binding inhibition constants.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
We therefore sought to explore the effects of replacement of both the vinyl-phenylsulfone and acrylamide
groups with a phenyl and heterocyclic groups conjugated to the vinyl group. The Cbz-Phe-Phe-vinyl-
benzene compound 5 is a time-dependent inhibitor of cruzain (K * = 0.87 M), but substitution of the para
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
position of the phenyl ring with an electron-withdrawing nitro group (compound 6) led to a nearly 3-fold
improvement in potency (K *= 0.34 M), suggesting that the vinyl group of 6 is more capable of thiolation
i
by the cruzain. As seen with 1, these compounds also demonstrated reversible inhibition of cruzain, possibly
due to the P phenylalanine. Due to poor aqueous solubility (solubility of 5 and 6 ≤ 2 M in 10% DMSO),
1
the inhibitors containing vinyl-benzene were not explored further.
Subsequently, six heterocyclic groups (R = IV-IX) conjugated to the presumed electrophilic vinyl group
3
were evaluated within several dipeptide scaffolds. The vinyl-2-pyrimidine (R = IV), vinyl-2-pyridine (R
3
3
=
V), vinyl-2-oxazole (R = VIII), and vinyl-2-thiazole (R = IX) groups, unlike the vinyl-4-pyridine (R
3 3
3
= VI) and vinyl-4-pyrimidine (R = VII), maintain bioisosteric similarity to the reactive acrylamides and
3
vinyl sulfones, which is reflected in their more potent inhibition of cruzain as detailed below. Most of these
compounds induced time-dependent inhibition on cruzain and were found to be kinetically reversible with
residence times (τ) of 6-20 minutes.
The vinyl-2-pyrimidine moiety (R = IV) in the Cbz-Phe-Phe scaffold afforded compound 7, which exerted
3
time-dependent inhibition of cruzain with an initial value of K = 5 M and subsequent tight-binding
i
inhibition of K * = 0.38 M. Substitution of the phenyl group of 5 by a pyrimidine group greatly improved
i
the solubility of 7 (≥100 M in 10% DMSO). Extended pre-incubation with 7, followed by dilution, and
addition of an excess of substrate, resulted in slow recovery of cruzain activity, indicating that any covalent
-1
reaction between cruzain and 7 was kinetically reversible (k = 0.0018 ± 0.0003 s ; τ = 9 min). Interestingly,
4
when the 2-pyrimidinyl moiety is appended to Cbz-Phe-hPhe (8), the resulting compound is a poor inhibitor
of cruzain (K > 35 M); however, when the 2-pyrimidinyl group is attached to afford the same scaffold as
i
K11777, we obtained an inhibitor of low micro-molar potency (9, K * = 2.2 M). Substitution of the P Phe
i
1
with Ala (10, K = 25 M) produced a poor inhibitor of cruzain, indicating the essentiality of a larger
i
sidechain in the P position, as was observed with dipeptide substrates. To probe the importance of the vinyl
1
group for the inhibition of cruzain, we prepared an analogue in which the vinyl group of 7 was reduced
(
compound 27). This inhibitor lacked time-dependent behavior (K = 22 M), and was 100-fold less potent
i
than its vinyl analogue 7, which demonstrated the importance of the vinyl group for the inhibition of
cruzain. We prepared inhibitor 23 which contains a vinyl-4-pyrimidinyl (R = VII) group that does not
3
maintain bioisosteric similarity to the acrylamides. 23 exhibited three-fold less potency than the bioisosteric
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2
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4
5
6
7
8
9
vinyl-2-pyrimidine (7). Similarly, inhibitors containing the vinyl-4-pyridyl (R = VI) (21, 22) lack
3
bioisosteric equivalence to the acrylamides and were found to be only modest inhibitors of cruzain.
Inhibitors containing a vinyl-2-pyridinyl group (R = V) were explored more widely. Cbz-Phe-Phe-vinyl-
3
2
-pyridine 11 exhibited time-dependent inhibition of cruzain with an initial value of K = 5.5 M and
i
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
subsequent tight-binding inhibition of K * = 0.31 M (k = 0.0012 ± 0.0002 s ; τ = 13 min), and solubility
i
4
of 11 was ≤ 30 M in 10% DMSO. Unlike the vinyl-pyrimidinyl group of 7, placement of the vinyl-2-
pyridinyl group in the Cbz-Phe-hPhe scaffold improved inhibition by 3-fold (13, K * = 0.17 M), while the
i
vinyl-2-pyridinyl group was much less effective in the NMePip-Phe-Phe scaffold (14, K * = 3.4 M).
i
Substitution of an electron-donating methoxy group on the pyridine ring (16) of the Cbz-Phe-hPhe scaffold
diminished the inhibitory activity of the vinyl-2-pyridinyl heterocycle compared to its unsubstituted
counterpart 13 by >50-fold, suggesting that the methoxy group is large enough to create a steric barrier to
inhibitor binding. In contrast, the substitution at C-4 of the pyridine with the electron-withdrawing
trifluoromethyl group resulted in better inhibition (17, K * = 0.57 M), but nonetheless was less potent than
i
the unsubstituted pyridine 13. Apparently, this result arises from steric crowding as OMe > CF > H,
3
implicating that substitution at the C-4 position of the pyridine heterocycles are not well tolerated.
We next investigated how the P and P sidechains of these vinyl-2-pyridinyl inhibitors effect inhibition.
1
2
The replacement of the P Phe with Leu resulted in diminished potency (18, K * = 1.42 M) compared to
2
i
the Cbz-Phe-Phe and Cbz-Phe-hPhe scaffolds, overall demonstrating that inhibitors with bulky
hydrophobic substituents in P and P enhanced binding to cruzain. To analyze how short alkyl and charged
1
2
groups effected inhibition, we prepared Cbz-Phe-Ala-vinyl-2-pyridine (19) and Cbz-Phe-Lys-vinyl-2-
pyridine (20). We found that the Cbz-Phe-Lys scaffold, which mimics our most optimal substrate, Cbz-
Phe-Arg-AMC, exhibited good inhibition (K * = 0.87 M), whereas 19 was a poor inhibitor (K * = 4.8 M),
i
i
in concert with the poor substrate activity of Cbz-Phe-Ala-AMC.
Seeking to improve the electrophilicity of the vinyl-2-pyridinyl group, we prepared N-methylated analogues
2 and 15. This modification resulted in improved aqueous solubility (≥ 50 M in 10% DMSO), and
provided potent time-dependent inhibition of cruzain (12, K * = 0.28 M; 15, K * = 0.126 M) comparable
1
i
i
to, or exceeding, the inhibition exerted by their un-methylated counterparts (11 and 13).
Inhibition data for compound 15 were fitted by all methods outlined in Experimental Section, and as shown
in Figure 4. We fitted each curve in Figure 4A to eq 3, and the resulting values of kobs were re-plotted vs.
[
15] (Figure 4A, inset), which demonstrated a hyperbolic dependence of the inhibitor (fitting to eq 4: K =
i
-1
2
.0 ± 0.9 M, k = 0.004 ± 0.001 s , and k ~ 0). Alternatively, global fitting of these curves to eq 6 provided
3
4
-1
-1
values of K = 4.3 ± 0.1 M, k = 0.0012 ± 0.0004 s , and k = 0.00019 ± 0.00005 s , from which was
i
3
4
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
calculated a value of K * = 0.6 ± 0.2 M. Fitting of the data globally for 15 and other potent PVHIs using
i
Kintek Explorer is found in Supplementary Information. Pre-incubation of cruzain and variable
concentrations of 15, followed by initiation of reaction by the addition of substrate, produced time courses
like that shown in Figure 4B. These data demonstrated a significant lag phase for recovery of cruzain
activity, indicative of the slow desorption of the inhibitor, with or without the formation of a covalent bond
with Cys . Finally, analysis of inhibition of cruzain by 15 at early and late phases of the time courses in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
Figure 4A by fitting to eq 5 provided values of K = 0.76 ± 0.04 M and K * = 0.126 ± 0.004 M (Table
i
i
2
) Of note, in pre-incubation studies, all PVHIs which contain the vinyl-pyridinyl substituent displayed
kinetic reversibility.
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2
3
4
5
6
7
8
9
Figure 4. Time-dependent inhibition of cruzain by 15. (A) Reaction initiated by addition of cruzain (100 pM) with Cbz-Phe-Arg-
AMC (10 M) and 0-20 M 15 (pH 7.5). Lines drawn through the experimental data points were from fitting of each inhibitor
concentration to eq 3, from which the replot of kobs vs. [15] is shown in the inset (fitting to eq 4: K
i
3
= 2.00 ± 0.9 M, k = 0.004 ±
-1
0
.001 s , and k ~ 0); (B) Following 1 hr pre-incubation of cruzain (100 pM) with 0-6 M 15, reaction was initiated by addition
4
of Cbz-Phe-Arg-AMC (10 M). (C) Fitting of cruzain inhibition by compound 15 for v
results of this found in Table 2.
i 0 s 0
/v (black) and v /v (red) using eq 5 with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We investigated 5-membered ring heterocycles that are bioisosteric with acrylamide inactivators. The
syntheses of the vinyl-2-oxazole (24), the vinyl-2-thiazole (25) and its N-methylated counterpart (26) into
the Cbz-Phe-Phe scaffold proved facile, and provided useful inhibitors. Vinyl-2-oxazole 24 was a sub-
micromolar inhibitor of cruzain (K * = 0.71 M). Vinyl-2-thiazole inhibitors 25 and 26 were inhibitors of
i
similar potency (K * = 1.71 and 0.94 M, respectively), for which N-methylation of the thiazole improved
i
potency by nearly two-fold.
Cruzain inhibitors 5-26 allowed the evaluation of six heterocyclic groups (R = IV-IX) appended to the
3
presumed electrophilic vinyl group within several dipeptide scaffolds. The vinyl-2-pyrimidine, vinyl-2-
pyridine, vinyl-2-N-methylpyridinium, vinyl-2-oxazole, and vinyl-2-thiazole substituents, unlike the vinyl-
4
-pyrimidine and vinyl-4-pyridine heterocycles, maintain bioisosteric similarity to the reactive acrylamides,
and provided potent, time-dependent inhibitors in accord with our hypothesis. Of these PVHIs, the 2-
pyridine, the charged 2-N-methylpyridine, and the vinyl-2-pyrimidine presented the most interesting
heterocycles for further exploration. The inhibition of cruzain displayed by these PVHIs may be due to the
reversible formation of an adduct with active-site Cys , as is supported by the loss of time dependent
2
5
inhibition when the vinyl group is saturated. Importantly, we have no evidence that such a reversible
covalent bond is formed, and ongoing studies are underway to address this point.
Selectivity of PVHIs for Cruzain over Homologous Human Cathepsins. Cruzain has, respectively, 25%,
1
5%, and 23% amino acid identity with human cathepsins L, B, and S.31, 55 It is preferable to proceed with
cruzain inhibitors that do not readily inhibit these human lysosomal cathepsins which might engender
cellular toxicity. We evaluated selected cruzain inhibitors vs. the human cysteine proteases cathepsins L,
B, and S (Table 3). For this selectivity comparison all inhibition data were obtained at pH 5.5, for which
K * values were invariant for all inhibitors except compound 15 (K * = 88 nM). The cruzain inhibitors
i
i
demonstrated moderate selectivity vs. cathepsins L and S (generally, 3-fold or greater), while all of these
inhibitors displayed 40-fold or higher selectivity vs. cathepsin B. The vinyl-2-pyridine inhibitors 13 and 15
are particularly selective as their K values are over 10-fold lower than the corresponding values with the
i
three human cathepsins. In contrast, K11777 showed potent inactivation at nanomolar concentrations for
all three human cathepsins; this apparent lack of selectivity possibly arising from its irreversible mode of
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7
8
9
1
1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
inactivation. These results suggest that suitable selectivity for reversible cruzain inhibitors may be more
easily attained than for irreversible ones.
a
Table 3. Enzymatic Selectivity of Cruzain Inhibitors
K * (µM)
i
Compound
Structure
Human
Human
Human
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cruzain
Cathepsin L Cathepsin B Cathepsin S
b
b
b
b
K11777 NMePip-Phe-hPhe-VSPh
IC50 = 0.2 nM IC50 = 0.2 nM IC50 = 5.7 nM IC50 = 0.6 nM
7
Cbz-Phe-Phe-vinyl-2Pyrmd
Cbz-Phe-Phe-vinyl-2Pyr
0.28 ± 0.01
0.28 ± 0.02
1.1 ± 0.1
4.3 ± 0.5
32 ± 3
28 ± 4
0.37 ± 0.02
1.8 ± 0.3
11
1
1
1
2
3
5
Cbz-Phe-Phe-vinyl-2PyrNMe 0.30 ± 0.02
Cbz-Phe-hPhe-vinyl-2Pyr 0.123 ± 0.004
0.70 ± 0.04
1.9 ± 0.2
19 ± 4
0.87 ± 0.07
1.41 ± 0.06
0.32 ± 0.04
6.5 ± 0.9
37 ± 6
Cbz-Phe-hPhe-vinyl-2PyrNMe 0.088 ± 0.002 0.88 ± 0.06
a
b
Inhibition data obtained at pH 5.5, 25°C in 10% DMSO (v/v); Reported as apparent IC50 in Ref. 32.
Effects of PVHIs in axenic cultures of T. cruzi and in a cell model of T. cruzi infection. Initially, we
tested selected compounds against epimastigotes of T. cruzi (strain Y, ATCC 50832GFP) in axenic culture.
As is observed here (Table 4), and has been shown previously, K11777 weakly inhibited the growth of T.
5
4
cruzi epimastigotes (EC ~ 60 M). PVHIs 7, 12, and 15 inhibited the growth of epimastigotes of T.
5
0
cruzi (EC = 2-20 M) while 11, 13, and 24 were poorly effective. Compounds 12 and 15 were comparably
5
0
potent against cultures of T. cruzi (EC = 8.6 and 2.1 M, respectively), and were, at a minimum, 10-fold
50
more active than K11777.
a
Table 4. Effects of Cruzain Inhibitors on Trypanosome and Human Cell Growth
T. cruzi
axenic
culture
T. cruzi-infected
cardiomyoblasts
(C2C12)
T. brucei
brucei
PCFs
T. brucei
brucei
BSFs
Human
Cell
Cytotoxicity
C2C12
Cytotoxicity
Selectivity
Index
Cruzain
* (M)
Compound
K
i
CC50 (M) CC50/EC50
EC50 (M)
EC50 (M)
EC50 (M) EC50 (M) CC50 (M)
b
K11777
>20
20
0.7 ± 0.2
9.0 ± 0.5
ND
1.7 ± 0.5
7.1 ± 0.9
15 ± 2
5 ± 1
0.09 ± 0.06
10.4 ± 0.2
>20
60-100
>100
ND
>10
>10
ND
>10
>10
>10
>10
ND
140
>10
ND
>20
>10
>20
>20
ND
IC50 = 2 nM
0.364 ± 0.004
2.2 ± 0.1
7
9
20
1
1
1
2
0.31 ± 0.01
0.28 ± 0.08
0.171 ± 0.004
0.126 ± 0.004
0.71 ± 0.01
>20
4.9 ± 0.2
9.9 ± 0.5
5.9 ± 0.3
5.4 ± 0.9
ND
5 ± 4
>100
>100
>100
>100
>100
8.7 ± 0.1
>20
13 ± 3
>10
6.6 ± 0.6
4 ± 2
13
15
24
2.1 ± 0.1
5.9 ± 0.2
27 ± 5
2.8 ± 0.1
ND
>20
a
Effects of inhibitors were evaluated as EC50 for axenic T. cruzi, PCFs/BSFs of T. b. brucei and T. cruzi-infected murine cardiomyoblasts. The Selectivity Index is the
b
ratio of inhibitor cytotoxicity in human dermal fibroblasts (CC50)/ trypanosomacidal activity (EC50) in infected cardiomyoblasts. Ref. 32.
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Selected cruzain inhibitors were further evaluated in a more relevant cellular model of Chagas disease: T.
cruzi-infected murine cardiomyoblasts (C2C12 cells) (Table 4, Figure 5). Inhibitors 7, 11, 12, 13, and 15
exhibited antiparasitic efficacy at values of EC = 5-10 M, while displaying no cytotoxicity against the
5
0
host cardiomyoblasts (CC > 10 M). These EC values demonstrated that the anti-trypanosomal activities
5
0
50
of the reversible PVHIs are within an order of magnitude of potency of the irreversible inactivator, K11777
EC50 = 0.7 M), despite the large difference in activity vs. cruzain. Accordingly, the PVHIs, while
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
reversible in action, and with no apparent mammalian or human cytotoxicity, are nearly as effective as the
potent, irreversible inactivator K11777. Further, the best of the PVHIs are less than 3-fold less potent than
55
the currently-used anti-chagasic drug benznidazole (LD = 1.5 M) suggesting that a second generation
5
0
of PVHIs may provide clinical candidates.
Figure 5. Effects of cruzain inhibitors on growth of T. cruzi-infected murine cardiomyoblasts in which growth of inhibition of T.
cruzi (red) is superimposed with the viability of the cardiomyoblasts (blue).
Effects of PVHIs in axenic cultures of T. b. brucei. We additionally tested our cruzain inhibitors in axenic
cultures of the related protozoan T. b. brucei, owing to the high structural similarity and reported essentiality
of the cysteine proteases brucipain (TbCatL) in T. b. brucei.24, 34, 57 It has been demonstrated that the cruzain
inhibitor K11777 is active in cellular cultures of both T. b. brucei and T. cruzi, supporting the notion that
our PVHIs could be effective in growth inhibition of both species of parasite. For insect procyclic forms
(PCFs) of T. b. brucei (ATCC PRA-381), compounds 7, 9, 11, 12, and 15 demonstrated growth inhibition
at EC values of 5-15 M (Table 4, Figure 6). When compared to K11777 (EC = 1.7 M), these PVHIs
5
0
50
exhibited potent cell-growth inhibition. For example, compound 15 (EC = 5.9 M) was only 3-fold less
5
0
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potent vs. T. b. brucei than K11777. Values of EC for these PVHIs roughly correlated with their values
5
0
of K *, with the exception of compound 13.
i
We next evaluated these inhibitors in axenic cultures of human bloodstream forms (BSFs) of T. b. brucei
ATCC PRA-383). All PVHIs that were active vs. procyclic forms of T. b. brucei were also trypanocidal
vs. the bloodstream forms, but with equal or lower EC values compared to the procyclic forms (Table 4).
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
Compared to PVHIs that had similar potencies in both PCFs and BSFs, K11777 was nearly 20-fold more
potent in T. b. brucei BSFs than in PCFs. These results suggested that a cathepsin L-like cysteine protease
2
4
in T. b. brucei, such as brucipain (or TbCatL), is essential for growth of procyclic and bloodstream T. b.
brucei, but perhaps an additional cysteine protease, such as TbCatB, is also essential in BSFs of T. b. brucei,
as this enzyme is sensitive to K11777 but not to the PVHIs. This is similar to the findings of Yang et al58
who showed using an activity-based protein probe of K11777 that TbCatB and brucipain (TbCatL) are both
labeled in BSFs of T. b. brucei while only brucipain is labeled in PCFs. This could explain the exceptional
trypanocidal activity of K11777 in BSFs. This will be the focus of our future studies. Nonetheless, the
activity of the PVHIs vs. T. b. brucei BSFs may hold promise for progression to their evaluation in models
of African trypanosomiasis.
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4
5
6
7
8
9
Figure 6. Cell-growth inhibition of T. b. brucei. (A) Inhibition of bloodstream forms by 15; (B) Inhibition of bloodstream forms
by 7; (C) Inhibition of procyclic forms by 15; (D) A correlation plot of values of EC50 for trypanocidal activity vs. T. b. brucei
2
BSFs (closed circles, r = 0.979, slope = 0.80) and T. cruzi in murine cardiomyoblasts (open circles).
Interestingly, the values of EC obtained for PCFs of T. b. brucei and amastigotes of T. cruzi were nearly
50
identical for most PVHIs, despite their more modest inhibition of cruzain. Shown in Figure 6D is a
correlation plot of log EC for anti-trypanosomal activity for bloodstream forms of T. b. brucei and the
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amastigote forms of T. cruzi from the murine cardiomyoblast infection model. For the former, the
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correlation is excellent (r = 0.979, slope = 0.80), and the activity against parasites is nearly a 1:1 correlation
with logK with these inhibitors. This result provided support that our PVHIs are targeting a cruzain-like
i
protease in T. b. brucei. For T. cruzi, this correlation is not as strong, in part due to an absence of a sufficient
range of data. We have also compared the cytotoxicity of selected inhibitors in human dermal fibroblasts
vs. T. cruzi-infected cardiomyoblasts (Selectivity Index in Table 4), which demonstrates the PVHIs are
more than 10-fold selective for trypanosomes vs. human cells.
CONCLUSIONS
We have developed a novel class of reversible inhibitors for the essential cysteine protease of Trypanosoma
cruzi, cruzain. These compounds, the peptidomimetic vinyl heterocycles, contain bioisosteric replacements
for the acrylamide and vinyl sulfone warheads present in irreversible, covalent inactivators such as K11777.
We also demonstrated that PVHIs containing vinyl-2-N-methylpyridine or vinyl-2-N-methylthiazole
groups, unlike other inhibitors, readily form Michael adducts with glutathione. Our survey demonstrated
that the most optimal cruzain inhibitors contained vinyl-2-pyrimidine, vinyl-2-pyridine, and vinyl-2-N-
methylpyridinium groups. These PVHIs proved to be potent, time-dependent inhibitors of cruzain, albeit,
fully reversible in terms of mode of action. These PVHIs are significantly active in both axenic cultures of
T. b. brucei and in a cell infection model of T. cruzi, and further optimization may produce more potent
anti-trypanosomal agents. Importantly, the concept of reversible covalent inactivation by vinyl-heterocycles
is potentially expandable to other enzymes which contain active-site cysteines, such as EGFR, G12C K-ras
and other protein kinases for which irreversible acrylamide inactivators comprise effective drugs.56
EXPERIMENTAL SECTION
General Synthetic Chemistry Methods and Compound Characterization. All reagents and starting
materials were obtained from commercial suppliers and used without further purification unless otherwise
stated. Reactions were run under an atmosphere of nitrogen or argon and at ambient temperature unless
otherwise noted. Reaction progress was monitored using thin layer chromatography and by analysis
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employing an HPLC-MS (UltiMate 3000 equipped with a diode array coupled to a MSQ Plus Single
Quadrupole Mass Spectrometer, ThermoFisher Scientific) using electrospray positive and negative
ionization detectors. Reported liquid chromatography retention times (Rt) were established using the
following conditions: column: Phenomenex Luna 5 m C18(2) 100 Å, 4.6 mm, 50 mm, Mobile phase A:
water with 0.1% formic acid (v/v). Mobile phase B: MeCN with 0.1% formic acid (v/v). Temperature: 25
0
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°
C. Gradient: 0−100% B over 6 min, then a 2 min hold at 100% B. Flow: 1 mL/min. Detection: MS and
UV at 254, 280, 214, and 350 nm.
Semi-preparative HPLC purification of compounds was performed on a Thermo Fisher Scientific UltiMate
3
000 with a single wavelength detector coupled to a fraction collector. Purifications were conducted using
the following conditions: column: Phenomenex Luna 5 um C18(2) 100 Å, 21.2 mm, 250 mm, Mobile phase
A: water with 0.1% formic acid (v/v). Mobile phase B: MeCN with 0.1% formic acid (v/v). Temperature:
Room temperature. Gradient: 0−100% B over 30 min, then a 5-min hold at 100% B. Flow: 20 mL/min.
Detection: UV (254 nm).
1
13
H/ C NMR magnetic resonance spectra were obtained in CDCl , CD OD, or DMSO-d at
3
3
6
4
00MHz/100MHz at 298 K on a Bruker Avance III NanoBay console with an Ascend magnet unless
otherwise noted. The following abbreviations were utilized to describe peak patterns when appropriate: br
= broad, s = singlet, d = doublet, q = quartet, t = triplet, and m = multiplet. All final compounds used for
testing in assays and biological studies had purities that were determined to be >95% as evaluated by their
proton NMR spectra and their HPLC/MS based on ultraviolet detection at 254 nm (see Supplementary
Information). Similar RP-HPLC conditions were used for the experiments of GSH addition to vinyl-
heterocycles. Masses detected were in the range 100 – 1000 Da and were detected in positive or negative
mode depending on the ionization of the molecule.
General procedures (GP1-GP10 in Scheme 1) of synthesizing PVHIs were detailed below. Each GP
described the synthesis of one representative compound. In addition, substrate synthesis and
characterization were provided in Supplementary Information.
GP1. Synthesis of Weinreb amides (a to b). A solution of Boc-L-homophenylalanine (12.02 g, 43.03 mmol)
in anh. DCM (200 mL) was cooled to 0 °C under an N atmosphere. Et N (18.1 mL, 129.09 mmol, 3 eq.)
2
3
was added slowly, followed by addition of N,O-dimethylhydroxylamine hydrochloride (6.3 g, 64.5 mmol,
.5 eq.) and dropwise addition of T3P (50% (w/v) in MeCN, 41.1 mL, 64.55 mmol, 1.5 eq.). The resulting
1
mixture was stirred at 0°C for 30 min to 1h until TLC analysis (EtOAc/hexane=1:1, v/v) showed the
disappearance of starting material. The reaction mixture was diluted with DCM and washed with H O. The
2
organic layer was dried over anh. Na SO and filtered. The filtrate was concentrated in vacuo to afford the
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crude product. Purification of the crude product by silica gel column chromatography using a gradient of
5% - 50% of EtOAc in hexane as eluent yielded the pure Weinreb amide tert-butyl (S)-(1-
(methoxy(methyl)amino)-1-oxo-4-phenylbutan-2-yl)carbamate (b, 13.3 g, 41.31 mmol, 96% yield) as a
colorless gum.
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GP2. LAH reduction of Weinreb amides (b to c). To a solution of tert-butyl (S)-(1-
(methoxy(methyl)amino)-1-oxo-4-phenylbutan-2-yl)carbamate (b, 6.7 g, 20.78 mmol) in anh. THF (120
mL) at -10°C under a N atmosphere was added dropwise LAH (2.0 M in THF, 12.5 mL, 24.93 mmol, 1.2
2
eq.). The resulting mixture was stirred at -10°C for 30 min. Upon completion of reaction as shown by TLC
analysis (EtOAc/Hexane=1:1, v/v), the reaction was quenched at the same temperature by adding dropwise
1
N HCl, followed by removal of THF by rotary evaporation. Diethyl ether (500 mL) was added to the solid
residue, and the solution was washed with aq. NaHCO (1 X 50 mL) and brine (1 X 50 mL). The organic
3
layer was dried over anh. Na SO and filtered. The filtrate was concentrated in vacuo to afford the crude
2
4
product. Purification of the crude material by silica gel column chromatography using a gradient of 10% -
0% of EtOAc in hexane as eluent yielded the pure aldehyde tert-butyl (S)-(1-oxo-4-phenylbutan-2-
6
yl)carbamate (c, 4.89 g, 18.57 mmol, 89% yield) as a white solid.
GP3. Preparation of chloromethyl-heterocycles (d to e). To a suspension of methyl pyrimidine-2-
carboxylate (d, 1.156 g, 8.37 mmol) in anh. EtOH (20 mL) at 0°C under N atmosphere, was added portion-
2
wise NaBH (0.443 g, 11.72 mmol, 5 eq.). The reaction mixture was stirred at 25°C for 2h. Upon the
4
completion of the reaction as shown by TLC analysis (EtOAc/Hexane=1:1, v/v), the reaction solvents were
removed by rotary evaporation. To the resultant colorless gummy residue was added ice cold H O (20 mL)
2
and extracted with DCM (5 X 50 mL). The organic layer was dried over anh. Na SO and filtered. The
2
4
filtrate was concentrated in vacuo to afford the crude product pyrimidin-2-yl-methanol (0.900 g, 8.17
mmol). To this pyrimidin-2-yl-methanol in CHCl (20 mL) at 0°C under N atmosphere was added dropwise
3
2
POCl (1.95 mL, 3.21 g, 2.5 eq.). The reaction mixture was stirred at 25°C for 1h, followed by refluxing
3
for an additional 3h under gentle heating until TLC analysis (EtOAc/Hexane=3:1, v/v) showed the
completion of the reaction. The reaction was quenched by a careful addition of aq. NaHCO and further
3
addition of solid NaHCO to afford a basic pH. The aqueous layer was extracted with CHCl (3 X 50 mL),
3
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and the organic layer was dried over anh. Na SO and filtered. The filtrate was concentrated in vacuo to
2
4
afford the pure product 2-(chloromethyl)pyrimidine (e, 0.948 g, 7.43 mmol, 63% yield) as a light yellow
semi-solid, which was used further without any purification.
GP4. Preparation of heterocyclic phosphonium ylides (e to f, Wittig reagents). A mixture of 2-
(chloromethyl)pyrimidine (e, 0.92 g, 7.22 mmol) and triphenyl phosphine (2.1 g, 7.94 mmol, 1.1 eq.) in
anh. benzene (25 mL) was refluxed under N atmosphere for 24h until TLC analysis (MeOH/DCM=1:19,
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v/v) showed the completion of the reaction. The reaction mixture was concentrated via rotary evaporation,
and the gummy residue was triturated with diethyl ether (3 X 10 mL). The solid obtained was purified by
silica gel column chromatography using a gradient of 1% - 10% of MeOH in DCM as eluent to afford the
pure product triphenyl(pyrimidin-2-ylmethyl)phosphonium chloride (g, 0.797 g, 2.039 mmol, 28% yield).
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GP5. Preparation of heterocyclic phosphonates (e to g, HWE reagents). 2-(chloromethyl)pyridine
hydrochloride (e, 16.5 g, 100.6 mmol) in DCM (100 mL) was treated with aq. NaHCO (20 mL), and the
3
DCM layer was dried over anh. Na SO . The filtrate was concentrated by rotary evaporation. The alkyl
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4
halide thus obtained along with triethyl phosphite (35 mL, 201.2 mmol, 2.0 eq.) were heated at 150°C under
N atmosphere for 5h until TLC analysis (MeOH/DCM=1:19, v/v) showed the completion of the reaction.
2
The reaction mixture was purified by silica gel column chromatography using a gradient of 10% - 100% of
EtOAc in hexane and later 1% - 10% of MeOH in DCM as eluent to yield the pure product 2-pyridyl methyl
phosphonate (g, 18.26 g, 79.66 mmol, 79% yield).
GP6. Wittig reaction (c + f to h). To a suspension of the Wittig reagent triphenyl(pyrimidin-2-
ylmethyl)phosphonium chloride (f, 0.719 g, 1.839 mmol) in anh. THF (40 mL) at -70°C under an N2
atmosphere, was added dropwise LHMDS (1.0 M in THF, 2.03 mL, 2.024 mmol, 1.1 eq.), which was stirred
at the same temperature for 15 min. To this mixture a solution of Boc-Phe-H (c, 0.321 g, 1.287 mmol, 0.7
eq.) in THF (10 mL) was added, and stirred over 2 h until the temperature reached -40°C. Upon completion
of reaction as revealed by TLC analysis (EtOAc/hexane=1:1, v/v), the reaction was quenched by addition
of 0.1 mL of glacial acetic acid, followed by aq. NaHCO . Most of the THF was removed carefully using a
3
rotary evaporator, and the residue was extracted with EtOAc (2X). The organic layer was dried over anh.
Na SO and filtered. The filtrate was concentrated in vacuo to afford the crude material, which was purified
2
4
by silica gel column chromatography using a gradient of 5% - 30% of EtOAc in hexane as eluent, yielding
the pure olefin tert-butyl (S,E)-(1-phenyl-4-(pyrimidin-2-yl)but-3-en-2-yl)carbamate (h, E-isomer, 0.060 g,
14%). The other Z-isomer (0.014 g) was isolated as a side product and the ratio of E to Z isomers was
typically 4:1.
GP7. Horner–Wadsworth–Emmons reaction (c + g to h). To a solution of the 2-pyridyl methyl phosphonate
ester (g, 1.30 g, 5.65 mmol) in anh. THF (25 mL) at -70°C under N atmosphere, was added dropwise
2
LHMDS (1.0 M in THF, 6.22 mL, 6.22 mmol, 1.1 eq.). The reaction was stirred at the same temperature
for 15 min, followed by dropwise addition of a solution of Boc-hPhe-H (c, 1.34 g in 10 mL THF, 5.09
mmol, 0.9 eq.). The reaction was stirred until it reached the temperature -20°C over 2 h. Upon completion
of reaction revealed by TLC analysis (EtOAc/hexane=1:1, v/v), to the reaction mixture at 0°C was added
glacial acetic acid (0.5 mL), followed by addition of 20 mL of saturated NaHCO . The aqueous layer was
3
extracted with EtOAc (3 X 100 mL). Extracts were washed with brine (1 X 50 mL), and the organic layer
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was dried over anh. Na SO and filtered. The filtrate was concentrated in vacuo to afford the crude product,
2
4
which was purified by silica gel column chromatography using a gradient of 10% - 50% of EtOAc in hexane
as eluent to yield the pure product tert-butyl (S,E)-(5-phenyl-1-(pyridin-2-yl)pent-1-en-3-yl)carbamate (h,
0.344 g, 1.016 mmol, 20% yield).
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GP8. Amide coupling with P -P fragment (h to j). To a solution of tert-butyl (S,E)-(5-phenyl-1-(pyridin-
3
2
2
-yl)pent-1-en-3-yl)carbamate (h, 0.143 g, 0.423 mmol) in anh DCM (5 mL) at 0°C, was added dropwise
TFA (1.5 mL in 1 mL DCM) with stirring at the same temperature for 1h. Upon the completion of the
reaction as revealed by TLC analysis (EtOAc/hexane=1:1, v/v), the reaction solvent was removed by a
rotary evaporator. The resulting oil was co-evaporated on a rotary evaporator with CHCl (3X) and ether
3
(3X). The solid product was dried on high vacuum to yield the TFA salt (S,E)-5-phenyl-1-(pyridin-2-
yl)pent-1-en-3-aminium trifluoroacetate (0.149 g, 0.423 mmol), which was used in subsequent synthetic
steps without further purification. To a solution of above TFA salt in anh. DCM (5 mL) at -10°C under N2
atmosphere, was added dropwise DIPEA (0.6 mL, 0.3.44 mmol, 8 eq.), followed by addition of Cbz-Phe-
OH (0.13 g, 0.43 mmol, 1 eq.) and T3P (50% in EtOAc, 0.41 mL, 1.5 eq.). The reaction was stirred at 0°C
for an additional 1 h. Upon the completion of reaction revealed by TLC analysis (EtOAc/hexane=1:1, v/v),
the reaction mixture was diluted with DCM (50 mL), and then washed with H O (3X) and brine (3X). The
2
organic layer was dried over anh. Na SO and filtered. The filtrate was concentrated in vacuo to afford the
2
4
crude product, which was purified by silica gel column chromatography using a gradient of 10% - 50% of
EtOAc in hexane as eluent to yield the pure product benzyl ((S)-1-oxo-3-phenyl-1-(((S,E)-5-phenyl-1-
(pyridin-2-yl)pent-1-en-3-yl)amino)propan-2-yl)carbamate (i, 0.113 g, 0.217 mmol, 51%).
GP9. N-methylation using methyl iodide (i to j). To a suspension of benzyl ((S)-1-oxo-3-phenyl-1-(((S,E)-
1
-phenyl-4-(pyridin-2-yl)but-3-en-2-yl)amino)propan-2-yl)carbamate (i, 0.049 g, 0.098 mmol) in anh.
MeCN (5 mL) and under N atmosphere, was added MeI (0.03 mL, 0.490 mmol, 5 eq.), and the reaction
2
mixture was heated under reflux for 9h. Upon the completion of the reaction revealed by TLC analysis
(EtOAc/hexane=1:1, v/v), the solvents were removed by rotary evaporation. The resulting gummy residue
was dissolved in CHCl (1 mL), and precipitated with ether (5 mL). The solvents were decanted, and this
3
procedure was repeated twice. The solid obtained was dried under high vacuum to give pure product 2-
((S,E)-3-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-4-phenylbut-1-en-1-yl)-1-
methylpyridin-1-ium iodide as a yellow solid (j, 0.039 g, 61%).
GP10. Preparation of peptide acrylamide (i to k). A solution of ethyl (S,E)-4-((S)-2-
(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-5-phenylpent-2-enoate (i, 0.346 g, 0.69 mmol) in
THF (6mL) at 0°C was treated with LiOH (1N in H O, 0.83mL, 0.83mmol, 1.2 eq) and stirred overnight.
2
The reaction was concentrated by rotary evaporation and the aqueous layer was added water and acidified
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to pH 1-2, and extracted with EtOAc (3X). The combined organic layers were dried and concentrated to
yield the crude acrylic acid. To a solution of this acrylic acid (0.124 g, 0.262 mmol) in THF (6mL) at -15°C
was added Et N (0.11 mL, 0.787 mmol, 3 eq.) and dropwise addition of ClCO Et (0.035 mL, 0.367 mmol),
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2
which resulted in a white precipitate. The reaction mixture was stirred at the same temperature for an
additional 30 min, then aq. 1M NH Cl (0.4 mL) was added dropwise with continuous stirring over 3h until
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a temperature of 25°C was attained. Upon completion of reaction as revealed by TLC analysis
(EtOAc/hexane=1:1, v/v), most of the reaction solvent was removed using a rotary evaporator, and the solid
residue was extracted with EtOAc. The organic layer was washed with aq. NaHCO (2X), H O (1X) and
3
2
brine (1X), and was dried over anh. Na SO and filtered. The filtrate was concentrated in vacuo to afford
2
4
the crude product. Purification of the crude product by silica gel chromatography using a gradient of 20%
- 100% of EtOAc in hexane as eluent, yielded the pure product benzyl ((S)-1-(((S,E)-5-amino-5-oxo-1-
phenylpent-3-en-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (0.027 g, 0.057 mmol, 22% yield).
4-Methyl-N-((S)-1-oxo-3-phenyl-1-(((S,E)-1-phenyl-4-(phenylsulfonyl)but-3-en-2-yl)amino)propan-2-
1
yl)piperazine-1-carboxamide (1, Cbz-Phe-Phe-VSPh). White solid, 0.115 g, 0.202 mmol, 56% yield. H
NMR (400 MHz, CDCl ) δ 2.79 (d, 2H, J = 6.8 Hz), 2.85 - 3.12 (m, 2H), 4.26 (q, 1H, J = 7.3 Hz), 4.79 –
3
4
1
.95 (m, 1H), 5.04 (s, 2H), 5.13 (s, 1H), 5.75 (s, 1H), 5.96 (dd, 1H, J = 1.8 Hz, J = 15.1 Hz), 6.78 (dd,
1 2
H, J = 4.8 Hz, J = 15.1 Hz), 6.95 – 7.03 (m, 2H), 7.05 – 7.11 (m, 2H), 7.12 – 7.23 (m, 6H), 7.27 – 7.39
1
2
1
3
(
m, 5H), 7.47 – 7.56 (m, 2H), 7.57 – 7.67 (m, 1H), 7.72 – 7.84 (m, 2H); C NMR (100 MHz, CDCl ) δ
3
3
8.4, 40.3, 50.4, 56.7, 67.4, 127.3, 127.4, 127.8, 128.2, 128.5, 128.7 (2C), 128.8, 129.0, 129.3, 129.4,
+
131.1, 133.6, 135.5, 136.1, 136.2, 140.2, 144.6, 156.0, 170.5; LC-MS t 7.27 min, m/z 569.31 [M+H] ,
R
+
(
C H N O S Calcd 569.21).
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2
5
Benzyl ((S)-1-(((S,E)-5-amino-5-oxo-1-phenylpent-3-en-2-yl)amino)-1-oxo-3-phenylpropan-2-
1
yl)carbamate (2, Cbz-Phe-Phe-vinyl-CONH ). White solid, 0.027 g, 0.057 mmol, 22% yield. H NMR
2
(400 MHz, DMSO-d ) δ 2.64 – 2.77 (m, 1H), 2.84 (d, 2H, J = 7.2 Hz), 2.96 (dd, 1H, J = 3.9 Hz, J =
6
1
2
1
3.7 Hz), 4.19 – 4.31 (m, 1H), 4.62 (pentet, 1H, J = 6.8 Hz), 4.95 (s, 1H), 5.85 (d, 1H, J = 15.5 Hz), 6.56
13
(
dd, 1H, J = 5.9 Hz, J = 15.5 Hz), 6.93 (s, 1H), 7.12 – 7.46 (m, 17H), 8.25 (d, 1H, J = 8.2 Hz); C NMR
1 2
(
100 MHz, DMSO-d ) δ 28.7, 37.6, 50.9, 56.07, 65.1, 124.1, 126.2, 126.4, 127.4, 127.6, 127.9, 128.1,
6
1
28.2, 129.2, 129.5, 137.0, 137.8, 138.0, 142.0, 155.6, 166.2, 170.7; LC-MS t 4.71 min, m/z 472.46
R
+
+
[M+H] , (C H N O Calcd 472.22).
2
8
29
3
4
Benzyl ((S)-1-(((S,E)-6-amino-6-oxo-1-phenylhex-4-en-3-yl)amino)-1-oxo-3-phenylpropan-2-
1
yl)carbamate (3, Cbz-Phe-hPhe-vinyl-CONH ). White solid, 0.013 g, 0.027 mmol, 15% yield. H NMR
2
(
400 MHz, DMSO-d ) δ 1.64 – 1.92 (m, 2H), 2.54 – 2.72 (m, 2H), 2.81 (dd, 1H, J = 10.6 Hz, J = 13.6
6
1
2
Hz), 3.03 (dd, 1H, J = 4.0 Hz, J = 13.6 Hz), 4.25 – 4.33 (m, 1H), 4.35 – 4.43 (m, 1H), 4.85 – 5.04 (m,
1
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H), 5.90 (d, 1H, J = 15.5 Hz), 6.54 (dd, 1H, J = 5.7 Hz, J = 15.5 Hz), 6.94 (s, 1H), 7.15 – 7.35 (m,
1
2
15H), 7.41 (s, 1H), 7.49 (d, 1H, J = 8.5 Hz), 8.22 (d, 1H, J = 8.2 Hz); LC-MS t 4.89 min, m/z 486.24
R
+
+
[M+H] , (C H N O Calcd 486.24).
29 31 3 4
N-((S)-1-(((S,E)-6-amino-6-oxo-1-phenylhex-4-en-3-yl)amino)-1-oxo-3-phenylpropan-2-yl)-4-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
methylpiperazine-1-carboxamide (4, NMePip-Phe-hPhe-vinyl-CONH ). White solid, 0.022 g, 0.048
2
1
mmol, 18% yield. H NMR (400 MHz, CDCl ) δ 1.66 – 1.91 (m, 2H), 2.23 (s, 3H), 2.25 – 2.34 (m, 4H),
3
2
.50 – 2.64 (m, 2H), 2.99 – 3.15 (m, 2H), 3.25 – 3.41 (m, 4H), 4.44 – 4.57 (m, 1H), 4.62 (q, 1H, J = 7.4
Hz), 5.37 (d, 1H, J = 7.6 Hz), 5.60 (dd, 1H, J = 1.3 Hz, J = 15.3 Hz), 5.75 (s, 1H), 6.01 (s, 1H), 6.62
1
2
1
3
(dd, 1H, J = 5.5 Hz, J = 15.3 Hz), 6.93 (d, 1H, J = 8.2 Hz), 7.04 – 7.31 (m, 11H); C NMR (100 MHz,
1 2
DMSO-d ) δ 32.0, 36.1, 38.5, 43.9, 46.1, 49.9, 54.6, 56.2, 122.9, 126.2, 126.9, 128.5, 128.6, 128.8, 129.7,
6
+
+
1
4
37.3, 141.1, 144.4, 157.3, 167.5, 172.0; LC-MS t 2.54 min, m/z 478.36 [M+H] , (C H N O Calcd
R 27 35 5 3
78.28).
Benzyl ((S)-1-(((S,E)-1,4-diphenylbut-3-en-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (5, Cbz-
1
Phe-Phe-vinyl-Ph). Off-white solid, 0.054 g, 0.107 mmol, 35% yield. H NMR (400 MHz, CDCl ) δ 2.87
3
(dt, J = 2.8, 6.3 Hz, 2H), 2.96 – 3.16 (m, 2H), 4.35 (q, J = 7.6 Hz, 1H), 4.80 – 4.96 (m, 1H), 5.29 (d, J =
9
.6 Hz, 1H), 5.69 (d, J = 9.9 Hz, 1H), 5.94 (ddt, J = 3.0, 6.3, 15.9 Hz, 1H), 6.27 (d, J = 15.9 Hz, 1H), 7.03
1
3
– 7.14 (m, 2H), 7.14 – 7.28 (m, 10H), 7.28 – 7.40 (m, 9H); C NMR (100 MHz, CDCl ) δ 29.7, 38.6,
3
4
1.3, 51.8, 67.1, 126.4, 126.6, 127.1, 127.6, 128.0, 128.2, 128.3, 128.4, 128.5, 128.7, 129.3, 129.4, 130.9,
+
+
136.5, 136.8, 169.8; LC-MS t 6.20 min, m/z 505.31 [M+H] , (C H N O Calcd 505.25).
R
33 32
2
3
Benzyl ((S)-1-(((S,E)-4-(4-nitrophenyl)-1-phenylbut-3-en-2-yl)amino)-1-oxo-3-phenylpropan-2-
1
yl)carbamate (6, Cbz-Phe-Phe-vinyl-(4-NO )Ph). White fluffy solid, 0.530 g, 0.964 mmol, 55% yield. H
2
NMR (400 MHz, DMSO-d ) δ 2.76 (dd, 1H, J = 9.7 Hz, J = 13.4 Hz), 2.85 – 2.97 (m, 3H), 4.18 – 4.35
6
1
2
(m, 1H), 4.68 (pentet, 1H, J = 6.7 Hz), 4.99 (s, 2H), 6.41 (d, 1H, J = 16.1 Hz), 6.50 (dd, 1H, J = 5.4 Hz,
1
J2 = 16.1 Hz), 7.13 – 7.37 (m, 15H), 7.42 (d, 1H, J = 8.5 Hz), 7.58 (d, 2H, J = 8.5 Hz), 8.14 – 8.25 (m,
13
3
1
H); C NMR (100 MHz, DMSO-d ) δ 37.7, 39.0, 51.9, 56.2, 65.2, 123.9, 126.1, 126.2, 127.0, 127.4,
6
27.6, 127.9, 128.0, 128.1, 128.2, 129.2, 129.3, 135.6, 137.0, 137.8, 138.0, 143.3, 146.2, 155.6, 170.6;
+
+
LC-MS t 6.12 min, m/z 550.28 [M+H] , (C H N O Calcd 550.23).
R
33 31
3
5
Benzyl ((S)-1-oxo-3-phenyl-1-(((S,E)-1-phenyl-4-(pyrimidin-2-yl)but-3-en-2-yl)amino)propan-2-
1
yl)carbamate (7, Cbz-Phe-Phe-vinyl-2Pyrmd). White solid, 0.026 g, 0.0513 mmol, 33% yield. H NMR
(400 MHz, CDCl ) δ 2.73 – 2.96 (m, 2H), 3.01 (d, 2H, J = 4.8 Hz), 4.23 – 4.50 (m, 1H), 4.91 – 5.02 (m,
3
1
H), 5.05 (s, 2H), 5.30 (s, 1H), 6.05 (s, 1H), 6.48 (d, 1H, J = 15.7 Hz), 7.00 (dd, 1H, J = 5.6 Hz, J =
1 2
1
3
15.7 Hz), 7.05 – 7.37 (m, 16H), 8.63 (d, 2H, J = 4.0 Hz); C NMR (100 MHz, CDCl ) δ 38.6, 41.0, 51.6,
3
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
6.5, 67.3, 119.0, 126.9, 127.2, 128.2, 128.3, 128.6, 128.7, 128.9, 129.4, 129.5, 130.3, 136.3. 136.5,
+
+
136.7, 139.2, 156.1, 157.1, 164.2, 170.3; LC-MS t 5.14 min, m/z 507.26 [M+H] , (C H N O Calcd
R
31 30
4
3
5
07.24).
Benzyl ((S)-1-oxo-3-phenyl-1-(((S,E)-5-phenyl-1-(pyrimidin-2-yl)pent-1-en-3-yl)amino)propan-2-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
yl)carbamate (8, Cbz-Phe-hPhe-vinyl-2Pyrmd). Off-white solid, 0.280 g, 0.538 mmol, 29% yield. H
NMR (400 MHz, CDCl ) δ 1.76 – 2.03 (m, 2H), 2.63 (t, J = 7.9 Hz, 2H), 3.10 (t, J = 8.4 Hz, 2H), 4.47 (s,
3
1
H), 4.74 (h, J = 7.3 Hz, 1H), 5.10 (d, J = 7.9 Hz, 2H), 5.53 (s, 1H), 6.27 (s, 1H), 6.56 – 6.72 (m, 1H),
6.96 – 7.05 (m, 1H), 7.06 – 7.14 (m, 3H), 7.19 (d, J = 7.3 Hz, 3H), 7.21 – 7.34 (m, 10H), 8.66 (dd, J =
1
3
4
1
1
.9, 15.8 Hz, 2H); C NMR (100 MHz, CDCl ) δ 32.0, 36.2, 38.6, 50.5, 56.6, 67.1, 118.9, 126.0, 127.0,
3
28.0, 128.1, 128.3, 128.4, 128.5, 128.7, 128.8, 129.3, 129.4, 130.1, 136.4, 139.6, 141.2, 156.9, 164.1,
+
+
70.3; LC-MS t = 5.56 min, m/z 521.24 [M+H] , (C H N O Calcd 521.26).
R
32 32
4
3
4-Methyl-N-((S)-1-oxo-3-phenyl-1-(((S,E)-5-phenyl-1-(pyrimidin-2-yl)pent-1-en-3-yl)amino)propan-2-
yl)piperazine-1-carboxamide (9, NMePip-Phe-hPhe-vinyl-2Pyrmd). Off-white gum, 0.054 g, 0.105 mmol,
1
5
1
4% yield. H NMR (400 MHz, CDCl ) δ 1.82 – 1.99 (m, 2H) ,2.29 (s, 3H), 2.34 – 2.46 (m, 4H), 2.61 (t,
3
H, J = 7.5 Hz), 3.09 (d, 2H, J = 7.5 Hz), 3.39 (s, 4H), 4.58 – 4.74 (m, 2H), 5.47 (d, 1H, J = 6.3 Hz), 6.61
(d, 1H, J = 15.7 Hz), 6.87 (d, 1H, J = 8.2 Hz), 6.98 (dd, 1H, J = 6.3 Hz, J = 15.7 Hz), 7.06 – 7.31 (m,
1 2
1
3
11H), 8.66 (d, 2H, J = 4.8 Hz); C NMR (100 MHz, CDCl ) δ 32.1, 36.5, 38.8, 43.5, 45.6, 50.6, 54.3,
3
5
6.1, 118.9, 126.0, 126.9, 128.4, 128.5 (2C), 128.6, 129.6, 130.1, 137.2, 140.1, 141.3, 157.0, 164.3,
+
+
171.8; LC-MS t 2.96 min, m/z 513.17 [M+H] , (C H N O Calcd 513.30).
R
30 36
6
2
Benzyl ((S)-1-oxo-3-phenyl-1-(((S,E)-4-(pyrimidin-2-yl)but-3-en-2-yl)amino)propan-2-yl)carbamate (10,
1
Cbz-Phe-Ala-vinyl-2Pyrmd). White fluffy solid, 0.017 g, 0.039 mmol, 14% yield. H NMR (400 MHz,
CDCl + MeOD) δ 1.32 (d, 3H, J = 6.6 Hz), 2.96 (dd, 1H, J = 8.0 Hz, J = 13.6 Hz), 3.10 (dd, 1H, J =
3
1
2
1
6.4 Hz, J = 13.6 Hz), 4.36 – 4.44 (m, 4H), 4.71 (pentet, 1H, J = 6.4 Hz), 4.97 – 5.12 (m, 2H), 6.52 (d,
2
1
H, J = 15.7 Hz), 6.98 (dd, 1H, J = 5.8 Hz, J = 15.7 Hz), 7.13 – 7.36 (m, 11H), 8.70 (d, 2H, J = 4.9 Hz);
1 2
13
C NMR (100 MHz, CDCl + MeOD) δ 19.6, 38.7, 46.1, 56.1, 66.7, 119.0, 126.7, 127.6, 127.9, 128.1,
3
+
1
28.3 (2C), 129.2, 136.3, 141.6, 156.9 (2C), 157.0, 163.9, 171.0; LC-MS t 4.68 min, m/z 430.91 [M+H] ,
R
+
(C H N O Calcd 431.21).
2
5
26
4
3
Benzyl ((S)-1-oxo-3-phenyl-1-(((S,E)-1-phenyl-4-(pyridin-2-yl)but-3-en-2-yl)amino)propan-2-
1
yl)carbamate (11, Cbz-Phe-Phe-vinyl-2Pyr). White solid, 0.554 g, 1.096 mmol, 81% yield. H NMR (400
MHz, CDCl ) δ 2.87 (dq, 1H, J = 6.8 Hz, J = 13.7 Hz), 3.01 (d, 2H, J = 7.1 Hz), 4.35 (q, 1H, J = 7.0
3
1
2
Hz), 4.91 (pentet, 1H, J = 6.8 Hz), 5.06 (s, 2H), 5.24 (s, 1H), 5.88 (d, 1H, J = 8.4 Hz), 6.32 (d, 1H, J =
15.7 Hz), 6.58 (dd, 1H, J = 6.1 Hz, J = 15.7 Hz), 7.08 – 7.36 (m, 17H), 7.59 (dt, 1H, J = 1.6 Hz, J =
1
2
1
2
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.7 Hz), 8.53 (d, 1H, J = 4.3 Hz); C NMR (100 MHz, CDCl ) δ 38.4, 41.1, 51.7, 56.4, 67.1, 122.1,
3
122.2, 126.7, 127.0, 128.0, 128.2, 128.4, 128.5 (2C), 128.8, 129.4 (2C), 130.5, 133.0, 136.1, 136.4, 136.8,
+
+
1
49.5, 154.8, 155.9, 170.0; LC-MS t 4.39 min, m/z 506.24 [M+H] , (C H N O Calcd 506.24).
R 32 31 3 3
2
-((S,E)-3-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-4-phenylbut-1-en-1-yl)-1-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
methylpyridin-1-ium iodide (12, Cbz-Phe-Phe-vinyl-2PyrNMe). Yellow solid, 0.039 g, 0.060 mmol, 61%
1
yield. H NMR (400 MHz, CDCl ) δ 3.01 – 3.15 (m, 3H), 3.19 (dd, 1H, J = 7.7 Hz, J = 13.6 Hz), 4.19
3
1
2
(
s, 3H), 4.51 (q, 1H, J = 7.0 Hz), 4.90 - 5.02 (m, 2H), 5.06 (s, 1H), 5.81 (s, 1H), 6.69 (d, 1H, J = 15.7 Hz),
6.86 (dd, 1H, J = 4.6 Hz, J = 15.7 Hz), 7.08 – 7.28 (m, 14H), 7.65 – 7.79 (m, 2H), 7.89 – 8.01 (m, 1H),
1
2
1
3
8
.21 – 8.31 (m, 1H), 8.97 – 9.09 (m, 1H); C NMR (100 MHz, CDCl ) δ 38.1, 39.8, 47.5, 52.6, 57.1,
3
6
6.8, 119.9, 126.1, 126.3, 126.9, 127.1, 127.5, 127.6, 127.9, 128.5, 128.6, 128.8, 129.6, 129.7, 136.7,
+
+
1
36.9, 144.9, 146.0, 147.6, 152.8, 156.2, 171.5; LC-MS t 3.25 min, m/z 520.32 [M+H] , (C H N O
R
33 34
3
3
Calcd 520.26).
Benzyl ((S)-1-oxo-3-phenyl-1-(((S,E)-5-phenyl-1-(pyridin-2-yl)pent-1-en-3-yl)amino)propan-2-
1
yl)carbamate (13, Cbz-Phe-hPhe-vinyl-2Pyr). White solid, 0.113 g, 0.217 mmol, 51% yield. H NMR
(
400 MHz, CDCl ) δ 1.73 – 1.95 (m, 2H), 2.59 (t, 2H, J = 7.9 Hz), 3.04 (d, 2H, J = 7.0 Hz), 4.38 – 4.52
3
(
m, 1H), 4.65 (pentet, 1H, J = 7.1 Hz), 5.03 (s, 2H), 5.59 (d, 1H, J = 6.4 Hz), 6.34 (d, 1H, J = 5.1 Hz),
6.43 (d, 1H, J = 15.7 Hz), 6.53 (dd, 1H, J = 6.2 Hz, J = 15.7 Hz), 7.02 – 7.32 (m, 17H), 7.57 (dt, 1H, J
1
1
2
1
3
=
1.4 Hz, J = 7.7 Hz), 8.51 (d, 1H, J = 4.4 Hz); C NMR (100 MHz, CDCl ) δ 32.1, 36.4, 38.7, 50.8,
2 3
56.6, 67.1, 122.1, 122.3, 126.0, 127.0, 128.0, 128.2, 128.4, 128.5 (2C), 128.7, 129.5, 130.5, 133.8, 136.2,
+
1
36.5 (2C), 141.4, 149.5, 155.0, 156.1, 170.4; LC-MS t 4.68 min, m/z 518.74, 520.41 [M+H] ,
R
+
(C H N O Calcd 520.26).
3
3
33
3
3
4
-Methyl-N-((S)-1-oxo-3-phenyl-1-(((S,E)-5-phenyl-1-(pyridin-2-yl)pent-1-en-3-yl)amino)propan-2-
yl)piperazine-1-carboxamide (14, NMePip-Phe-hPhe-vinyl-2Pyr). Off-white solid, 0.090 g, 0.176 mmol,
1
4
2
4% yield. H NMR (400 MHz, CDCl ) δ 1.77 – 1.94 (m, 2H), 2.21 (s, 3H), 2.22 – 2.27 (m, 4H), 2.59 (t,
3
H, J = 8.0 Hz), 3.08 (d, 2H, J = 7.0 Hz), 3.28 (m, 4H), 4.61 (pentet, 1H, J = 7.3 Hz), 4.7 (q, 1H, J = 7.3
Hz), 5.39 (d, 1H, J = 7.7 Hz), 6.46 (d, 1H, J = 15.8 Hz), 6.56 (dd, 1H, J = 6.2 Hz, J = 15.8 Hz), 6.98 (d,
1
2
1
H, J = 8.4 Hz), 7.06 – 7.24 (m, 12H), 7.59 (dt, 1H, J = 1.8 Hz, J = 7.7 Hz), 8.53 (d, 1H, J = 4.3 Hz);
1 2
1
3
C NMR (100 MHz, CDCl ) δ 32.1, 36.6, 39.0, 43.8, 46.1, 50.7, 54.6, 55.9, 122.0, 122.2, 125.9, 126.8,
3
1
28.4 (2C), 128.5, 129.6, 130.3, 134.2, 136.4, 137.2, 141.4, 149.5, 155.1, 157.0, 171.6; LC-MS t 2.60
R
+
+
min, m/z 512.28 [M+H] , (C H N O Calcd 512.30).
3
1
37
5
2
2
-((S,E)-3-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-5-phenylpent-1-en-1-yl)-1-
methylpyridin-1-ium (15, Cbz-Phe-hPhe-vinyl-2PyrNMe). Yellow solid, 0.029 g, 0.044 mmol, 89% yield.
29
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