Vinyl sulfone-based peptidomimetics as anti-trypanosomal agents: Design, synthesis, biological and computational evaluation

Article abstract of DOI:10.1021/jm400294w

A series of vinyl sulfone-containing peptidomimetics were rationally designed, synthesized, and evaluated against Trypanosoma brucei brucei. These electrophilic compounds are likely to exert their antitrypanosomal activity via inhibition of trypanosomal cysteine proteases, TbCatB and rhodesain, through alkylation of a key cysteine residue within the protease active site. The series was designed to present complementary groups to naturally recognized peptide substrates while probing tolerance to a range of substitutions at the P1, P1′, and P2 positions. The most potent compound, 29 (EC₅₀ = 70 nM, T. b. brucei whole cell assay), displayed minimal toxicity (>785 times selectivity) when assayed for cytotoxicity against the human promyelocytic leukemia (HL-60) cell line. Cells treated with compound 29, as with K777 (2), exhibited an increase in both the number of multinucleated cells and cells with swollen flagellar pockets. Computational analysis revealed a strong correlation between the hypothetical binding mode in TbCatB/rhodesain and trypanocidal activity in vitro.

Full text of DOI:10.1021/jm400294w

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Article
Vinyl Sulfone-based Peptidomimetics as Anti-Trypanosomal
Agents: Design, Synthesis, Biological and Computational Evaluation
Elizabeth Dunny, William Doherty, Paul Evans, Jonathan Paul.G. Malthouse, Derek Nolan, and Andrew Knox
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400294w • Publication Date (Web): 16 Aug 2013
Downloaded from http://pubs.acs.org on August 17, 2013
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Vinyl Sulfone-based Peptidomimetics as Anti-Trypanosomal Agents: Design,
Synthesis, Biological and Computational Evaluation.
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Elizabeth Dunny,a William Doherty,a Paul Evans,a* J. Paul G. Malthouse,b Derek Nolanc and Andrew J. S. Knoxc*
aCentre for Synthesis and Chemical Biology, School of Chemistry and Chemical Biology,
University College Dublin, Dublin 4, Ireland. E-mail: paul.evans@ucd.ie; Tel: +353-1-7162291
bConway Institute, School of Biomolecular and Biomedical Science,
University College Dublin, Dublin 4, Ireland
cSchool of Biochemistry and Immunology, Trinity Biomedical Sciences Institute,
Trinity College Dublin, Pearse St, Dublin 2, Ireland. E-mail: aknox@tcd.ie; Tel: +353-1-8963959
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Abstract A series of vinyl sulfoneꢀcontaining peptidomimetics were rationally designed, synthesized and evaluated
against Trypanosoma brucei brucei. These electrophilic compounds are likely to exert their antitrypanosomal activity
via inhibition of trypanosomal cysteine proteases, TbCatB and rhodesain, through alkylation of a key cysteine residue
within the protease active site. The series was designed to present complementary groups to naturally recognized
peptide substrates, whilst probing tolerance to a range of substitutions at the P1, P1’ and P2 positions. The most potent
compound, 29 (EC50 = 70 nM, T.b.brucei whole cell assay) displayed minimal toxicity (>785 times selectivity) when
assayed for cytotoxicity against the human promyelocytic leukemia (HLꢀ60) cell line. Cells treated with compound 29,
as with K777 (2), exhibited an increase in both the number of multinucleated cells and cells with swollen flagellar
pockets. Computational analysis revealed a strong correlation between the hypothetical binding mode in
TbCatB/rhodesain and trypanocidal activity in vitro.
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1. Introduction*
African sleeping sickness, or African trypanosomiasis, is a parasitic disease endemic in over 36
countries in subꢀSaharan Africa.1,2 It is caused by the flagellate kinetoplastid protozoan parasite,
Trypanosoma brucei, which resides extracellularly in the blood and tissue fluids of the mammalian
host and is transmitted by the tsetse fly. Trypanosoma brucei gambiense is widespread in central
and western Africa whereas Trypanosoma brucei rhodesiense is restricted to east and east central
Africa and is less common.1,2 The colloquial term for African trypanosomiasis ꢀ African sleeping
sickness ꢀ refers to the most serious phase of the disease which occurs when the parasite penetrates
the central nervous system causing a sense of confusion, changes in behaviour, poor coordination
and a disturbed sleep cycle. It is currently estimated that between 30,000 and 50,000 deaths per
*Abbreviations used: Cbz: benzyloxycarbonyl; Bn: benzyl; iꢀBu: isobutyl; Boc: tertꢀbutyloxycarbonyl;
EDCI: Nꢀ(3,dimethylaminopropyl)ꢀN'ꢀethylcarbodiimide; DAPI: 4',6ꢀdiamidinoꢀ2ꢀphenylindole; DIPEA:
diisopropylethylamine; DCM: dichloromethane; HLꢀ60: human promyelocytic leukemia cells; THF:
tetrahydrofuran; PCC: pyridinium chlorochromate; EWG: electron withdrawing group; Ph: phenyl; TFA:
trifluoroacetic acid.
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year result from this disease.1ꢀ3 In addition, several subspecies (Trypanosoma brucei brucei,
Trypanosoma congolense and Trypanosoma vivax) cause Nagana (a Zulu word meaning “to be
depressed”), the bovine equivalent of African trypanosomiasis. In Africa, it has been estimated that
if the tsetse fly could be eradicated, or an effective, cheap treatment were available, a doubling in
cattle numbers could be achieved that would yield an extra annual income of US $5 billion.4
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In the absence of medical intervention sleeping sickness is ultimately fatal. Current treatment for
human sleeping sickness still relies heavily on drugs developed many years ago, all of which are
toxic, show decreasing efficacy and suffer from administration/patient compliance issues.5 Based
on these points, new strategies for the control and treatment of African sleeping sickness and
Nagana could potentially benefit many people.1ꢀ5
Cysteine proteases present in T. brucei play an essential role to the parasite’s life cycle and as such
represent a potential target for pharmaceutical intervention.6ꢀ9 These proteins belong to a wide class
of proteolytic enzymes, that rely on a nucleophilic cysteine residue within the active site, to assist in
peptide bond hydrolysis (Scheme 1).6 They have been classified into three structurally distinct
groups: papainꢀlike (Clan CA), interleukinꢀ1βꢀconvertingꢀlike (Clan CD) and picornainꢀlike (Clan
PA). Clan CA, papainꢀlike, cysteine proteases represent the largest group and are widely expressed
in both plants and animals and are essential for the life cycle of several parasites, including
trypanosoma.6ꢀ9
Of the several types of compound developed and reported as parasitic cysteine protease inhibitors
most rely on covalent derivatization of the key cysteine residue. For example, peptideꢀbased
aldehydes, αꢀhalomethyl ketones, nitriles, epoxides, aziridines and Michaelꢀtype acceptors have all
been reported as effective inhibitors.6ꢀ12 Typically, these inhibitors have a peptide component (or
mimic) that binds optimally to the S1 to S3 subsite of the protease. Additionally, electrophilic
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functionality with the ability to react with and derivatize the nucleophilic sulfur atom, either
reversibly, or irreversibly, is included (see, for example, Scheme 1).
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O
H
N
O
H
N
cysteinyl
protease
His
His
cysteinyl
protease
HN
HN
S2
S
NH
NH
S
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O
P2
O
O
P2
H
O
H
H2O
S3
N
P1'
N
P1'
P3
N
N
P3
N
OH
H2N
+
H
H
hydrolysis
of peptide
H
O
O
P1
O
P1
S1
H
cysteinyl
protease
N
O
H
N
His
His
NH
cysteinyl
protease
N
S2
H
N
S
NH
Ph
Ph
S3
S
O
O
H
N
H
N
β
SO2Ph
SO2Ph
β
N
N
H
N
N
H
S-Conjugate
addition
X
O
X
O
Ph
Ph
S1
X= O; 1
X = NMe; K777 (2)
X= O; 3
X = NMe; 4
Scheme 1. Cysteine proteaseꢀbased peptide hydrolysis and the mechanismꢀbased cysteine protease inhibition by vinyl
sulfones 1 and 2.
One such class of inhibitor; peptidyl vinyl sulfones, was first introduced by Hanzlik and coꢀworkers
as a group of compounds which inhibit the cysteine protease, papain.12,13 Linked to the likely
mechanism of action, the Sꢀconjugate reaction of vinyl sulfones (as shown in Scheme 1) with
sulfurꢀbased nucleophiles (particularly under basic conditions) is a reliable chemical process.
Palmer et al. expanded on these studies synthesizing a library of peptidyl vinyl sulfones that contain
“functional scaffolds,” combining inhibitorꢀprotease complementarity in terms of binding with
enzymatic deactivation.14 In this important study, it was shown that vinyl sulfones specifically
inhibit cysteine proteases over serine proteases (despite the fact that the serine proteases often
possess a similar substrate profile, in terms of the type of amino acid sideꢀchain preferentially
recognized). The irreversibility of cysteine protease inactivation by vinyl sulfones was also
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explored and it was shown that cathepsin B was completely inactivated by 1, which following
purification by column chromatography demonstrated no recovery of enzymatic activity. Finally, in
terms of selectivity and possible nonꢀdiscriminate toxicity, the stability of selected vinyl sulfones
toward the ubiquitous tripeptide glutathione was explored. Preꢀincubation of vinyl sulfone 1 with
glutathione for 22 hours showed no subsequent loss of inhibitory potency, suggesting selectivity for
the protease.14,15
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Rosenthal and colleagues showed in 1993 that a peptidyl αꢀfluoromethyl ketone cured 80% of
malariaꢀinfected mice when it was administered subcutaneously over 4 days. Unfortunately
however, the high doses required to cure malarial infection resulted in significant toxic effects to the
host.16 Subsequently, the same group revealed that a peptidyl vinyl sulfone was a potent inhibitor of
falcipain, a cysteine protease found in Plasmodium falciparum.17 This compound markedly delayed
the progression of malaria and cured about 40% of mice when they were treated orally twiceꢀaꢀday
for four days without the associated toxicity with the previous study. In 1998, McKerrow et al.
reported that dipeptide vinyl sulfone 2 rescued mice from a lethal Trypanosoma cruzi infection as a
result of the inhibition of the cysteine protease, cruzain.18 Now known as K777 (also K11777), it
was subsequently shown that this vinyl sulfone 2 protected Beagle dogs from cardiac damage
during infection by T. cruzi.19 In 2009 the results of a preclinical trial were reported: K777 proved
to be nonꢀmutagenic, wellꢀtolerated, and demonstrated efficacy in models of acute and chronic
Chagas’ disease in both mice and dogs.20 Since 2007, FDA approval has been sought to allow K777
to enter Phase I trials in humans.20
Specifically for African trypanosomiasis, in 1999 it was reported that a peptideꢀbased, diazomethyl
ketone, killed bloodstream forms of T. brucei in vitro and in vivo.21 Although, after cessation of
treatment the effect proved not to be permanent, infected mice treated with the inhibitor survived
nearly twice as long as those treated with placebo.21 Importantly, the trypanocidal action of the
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compound was associated with almost complete inhibition of brucipain (also known as rhodesain,
trypanopainꢀtb), the cysteine protease in T. brucei. Coetzer also evaluated a range of αꢀ
electrophilic ketone, and vinyl sulfone derivatives for the bloodstream form of Trypanosoma brucei
brucei. Although T. b. brucei causes Nagana, it is also a suitable model for T. b. gambiense and T.
b. rhodesiense.4 The ketone derivatives were moderately potent, with the most potent compound in
the study being vinyl sulfoneꢀbased, in particular 2 (IC50 0.1 ꢁM).4 In an effort to identify the
possible intracellular targets of these inhibitors, biotinylated derivatives were incubated with live
and/or lysed T. b. brucei. The results demonstrated that the major intracellular target was
trypanopainꢀTb, a key lysosomal cysteine proteinase.4 In several publications, the type of peptide
recognized and digested by the papainꢀlike cysteine proteases presented by T.b. rhodesiense and T.
cruzi have been profiled using fluorogenic tagged peptides and microarray techniques.9,26
Summarizing this data, it can be concluded that both enzymes prefer basic, lysine and arginine
residues in the S1 position, and bulky, hydrophobic residues in S2 (see Schemes 1 and 2).
Rhodesain equally accepted leucine and phenylalanine whereas cruzain prefers phenylalanine.
Stereochemistry was also interrogated and a preference for the naturally occurring amino acid
stereogenicity was demonstrated.26a Noteworthy, the human cysteine protease; cathepsin L, shares
many similarities with these parasitic enzymes, both in terms of substrate specificity and degree of
amino acid homology.26b In 2009 the first crystal structure of rhodesain bound to 2 was published
providing additional understanding of the substrateꢀbinding site profile.20
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S2
Hydro-
phobic
R
O
O
O
S1'
H
N
H
N
O
O
R
S
S
N
H
NHR'
S
Ph
O
N
H
Ar
Modification
O
O
Hydro-
phobic
Basic
R = Ph, i-Pr etc.;
Ar = Ph etc.
Diversification
NH3
S1
S3
Scheme 2. Trypanosome cysteine protease substrate specificity and lysineꢀbased vinyl sulfones inhibitors.
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Based on the clear preference for a basic residue in the parasitic enzyme binding pocket (S1) and
the demonstration that vinyl sulfones are an efficacious, biologically active scaffold upon which to
base irreversible inhibitor design27, we felt that appropriately designed inhibitors of this type would
be of interest (Scheme 2). To the best of our knowledge only one example of a lysineꢀvinyl
sulfone4 and one example of an arginineꢀvinyl sulfone28 based cysteine protease inhibitor have been
reported. In addition, it was also envisaged that this basic group would represent a functional
handle, in order to both explore the S1 subsite and also to tune physiochemical properties. Thus, a
series of lysineꢀcontaining dipeptide vinyl sulfones were synthesised, assessed for trypanocidal
activity and toxicity, and finally rationalised via novel computational methods.
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Results and discussion
Chemistry
As outlined in Scheme 3, using commercially available protected Lꢀlysine 8 peptide bond
formation, mediated by the water soluble carbodiimide EDCI29 was efficiently achieved with Cbzꢀ
protected Lꢀphenylalanine 5, Lꢀleucine 6 and Lꢀproline 7. On treatment with lithium borohydride30
the resultant dipeptides 9 to 11 were efficiently converted to their corresponding primary alcohols
12 to 14. The next task was an alcohol to aldehyde oxidation, followed by an olefination process.
In our hands, Swern oxidation (including its variants) and DessꢀMartin periodinane led to poor
conversion and the formation of additional reaction products. Eventually it was found that
pyridinium chlorochromate (PCC) in the presence of Celite, to facilitate product retrieval from the
chromium byꢀproducts, proved optimal.31 Using these conditions the PheꢀLys aldehyde (not
shown) could be reproducibly isolated in approximately 50% yield, after purification by flash
column chromatography.
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NaBH4, LiCl,
THF-EtOH (2:1),
rt, 16 h
EDCI·HCl,
DIPEA, DCM,
rt, 16 h
R
NHBoc
O
NHBoc
CbzN
CO2H
ClH·H2N
R
CbzN
CO2Me
NHBoc
N
H
CO2Me
R'
R'
5: R = H; R' = Bn;
6: R = H; R' = i-Bu;
7: R = R' = (CH2)3
8
9: R = H; R' = Bn; 90%;
10: R = H; R' = i-Bu; 91%;
11: R = R' = (CH2)3; 90%
9
(1) PCC, Celite,
DCM, rt, 6 h
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O
NHBoc
EWG
O
R
CbzN
R
CbzN
OH
N
H
N
H
(2) 15-18, NaH,
THF, 0 °C to rt,
16 h
R'
R'
19: R = H; R' = Bn; EWG = SO2Ph; 29%;
20: R = H; R' = Bn; EWG = SO2(2-Naphthyl); 55%;
21: R = H; R = Bn; EWG = SO2(1-Naphthyl); 41%;
22: R = H; R = Bn; EWG = COPh; 13%;
23: R = H; R' = i-Bu; EWG = SO2Ph; 46%;
24: R = R' = (CH2)3; EWG = SO2Ph; 53%
12: R = H; R' = Bn; 92%;
13: R = H; R' = i-Bu; 97%;
14: R = R' = (CH2)3; 87%
O
O2
S
O2
S
O2
S
PO(OEt)2
PO(OEt)2
PO(OEt)2
PO(OEt)2
16
18
17
15
Scheme 3. Synthesis of dipeptide vinyl sulfones.
This material could then be converted into the corresponding vinyl sulfone 19 following Hornerꢀ
WadsworthꢀEmmons olefination32 using the sodium salt of phosphonate 15 (for the synthesis of
phosphonates 15ꢀ18 see electronic supporting information). As anticipated, good transꢀselectivity
was observed and 19 was isolated in 52% yield (29% over two steps). The likely explanation for
the modest yield observed over these two steps is associated with the instability of the aldehyde.33
Consequently, subsequent attempts circumvented its purification and under these conditions the
crude aldehyde could be more efficiently converted into the 2ꢀ and 1ꢀnaphthyl vinyl sulfones 20 and
21 in 55 and 41% isolated yields respectively. In order to compare activity of the vinyl sulfoneꢀ
based inhibitors with the more electrophilic α,βꢀunsaturated ketone, the synthesis of 22 was
attempted. However, even under the optimized procedure, only small amounts of 22 could be
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isolated. Linked to a desire to probe effects associated with the identity and conformation of the
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second amino acid, leucine 23 and proline 24 based vinyl sulfones were similarly accessed.
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7
8
9
As mentioned in the introduction, based on the substrate specificity we were interested in accessing
the compound in which a free amino group was present on the lysine sideꢀchain (Scheme 2). Thus,
the tert-butyloxycarbonyl (Boc) group in vinyl sulfone 19 was chemoselectively removed on
treatment with trifluoroacetic acid (TFA) in acetic acid (Scheme 4). This process efficiently
generated the corresponding ammonium salt as its trifluoroacetate salt. Associated with
characterization and improved solubility, using hydrochloric acid treated Amberlite resin, the
counterion was exchanged with a chloride, according to a literature procedure,34 in order to generate
25. This compound was stored and handled as the ammonium salt since upon adjustment of the pH
proton NMR indicated that the vinyl group was substantially lost. Mass spectrometry of this
basified material, however, indicated the same molecular ion. We attributed this data to an
intramolecular conjugate addition process generating azepine 26, presumably as a mixture of
diastereoisomers.
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Since the vinyl sulfone inhibitors were designed to alkylate the catalytically active cysteine residue
in the cysteine protease peptide binding pocket, it was of interest to investigate how removal of the
potential for this mode of action impacted on the overall biological activity of the dipeptide
compound.
(1) TFA, AcOH,
O
NHBoc
O
NH2·HCl
SO2Ph
rt, 1 h;
CbzHN
CbzHN
N
H
SO2Ph
N
H
(2) Amberlite,
H2O, rt, 48 h,
89%
19
25
Ph
Ph
H2, Pd/C, EtOH,
rt, 26 h, 42%
NaOH(aq)
O
O
NHBoc
SO2Ph
CbzHN
NH
N
CbzHN
H
N
H
Ph
SO2Ph
Ph
26
27
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Scheme 4. The Bocꢀremoval and hydrogenation of 19.
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5
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7
8
9
To this end, alkene hydrogenation of 19 was performed in order to convert the vinyl sulfone into its
saturated counterpart. In relation to this conversion, it is notable that under the reaction conditions
alkeneꢀhydrogenation occurs more rapidly than hydrogenolysis of the Cbz group and thus 27 may
be isolated directly in reasonable yield.
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ArSO2Cl, Et3N,
DCM, 0 °C to rt,
15 h
O
NH2·HCl
SO2Ph
O
NHSO2Ar
SO2Ph
CbzHN
CbzHN
N
H
N
H
25
Ph
Ph
28: Ar = 4-MeC6H4; 58%;
29: Ar = 1-Naphthyl; 67%;
30: Ar = 2-BrC6H2(4,5-OMe2); 55%
Scheme 5. NꢀDerivatization of ammonium salt 25.
The efficient preparation of 25 also presented the possibility of structural diversification by
elaboration of the amino group, however, if this were to prove successful the desired intermolecular
process would need to proceed at a faster rate than the intramolecular conjugate addition (forming
26). As shown in Scheme 5, it did indeed prove possible to convert the ammonium salt in 25
directly to the corresponding sulfonamides 28, 29 and 30 in moderate yield with base in the
presence of the corresponding sulfonyl chlorides.
Biochemistry
The effect of compounds 19-25 and 27-30 on the viability of in vitro cultures of Trypanosoma
brucei brucei were tested and compared with a known cysteine protease inhibitor, K777 (2),
currently being developed as an antiꢀtrypanosomal therapy for Chagas’ disease.35 Parasiteꢀmediated
reduction of AlamarꢀBlue was used as an indicator of cell viability. A doseꢀdependent effect on
trypanosome viability was observed with EC50’s ranging from 0.07ꢀ26.4 ꢀM for the series, which
compared favourably with the EC50 of 5.56 ꢀM obtained for 2 (Figure 1). Additionally, the
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compounds exhibited a >2ꢀfold selective inhibition of parasite versus mammalian cell (HLꢀ60 cell
line) proliferation in vitro (Figure 2), with the most selective compound, 29, being >785 times more
potent for T.b.brucei (Table 1). Within the series the only significant effect on HLꢀ60 cells was
evident at micromolar concentration with the α,βꢀunsaturated ketone 22. Nwaka and Hudson
recently recommended a selectivity index of >100 to pursue animal studies36 in the Special
Programme for Research and Training in Tropical Diseases at the World Health Organization
(WHO/TDR) pointing to compound 29 potentially being an excellent candidate for further studies.
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50
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27
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0
29
30
-11 -10 -9 -8 -7 -6 -5 -4 -3 -2
K777
Log [Concentration] (M)
Figure 1. Response (4ꢀParameter logistic nonꢀlinear regression) of Trypanosoma brucei brucei to compounds 19-25,
27-30. Cells (2 x 103/well) were incubated in the presence of inhibitors for 24 h and AlamarꢀBlue added approximately
18 h before determining percentage inhibition of the fluorescent signal compared to controls minus inhibitor.
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-8
-7
-6
-5
-4
Log [Concentration] (M)
Figure 2. Response of human promyelocytic leukemia cells (HLꢀ60) to compounds 19-25, 27-30. Cells (2 x 103/well)
were incubated in the presence of inhibitors for 48 h and AlamarꢀBlue added approximately 18 h before determining
percentage inhibition of the fluorescent signal compared to controls minus inhibitor.
Generally, all compounds exhibited the ability to perturb Trypanosoma brucei brucei growth versus
controls, however, in terms of relating structure to biological behaviour several aspects associated
with their efficacy are evident. Linked to the mechanismꢀbased action of these cysteine protease
inhibitors (Scheme 1); comparing the EC50 values of vinyl sulfone 19 against its saturated
counterpart 27 (0.66 ꢀM versus 26.40 ꢀM) the influence of the electronꢀpoor vinyl group is clear.
Comparison between 19 and its corresponding ammonium salt 25 (0.66 ꢀM versus 22.40 ꢀM)
appears to indicate that the more polar 25 is not taken up by the cells as efficiently since the
substrate profiling studies, discussed above, clearly demonstrate that positively charged residues in
S1 are happily accommodated by this type of parasitic protease. Structural alteration in the S1’
pocket across compounds 19, 20 and 21 seems to lead to only modest alteration of activity (see also
Figure 5, A). Contrasting Michaelꢀtype acceptors, 19 versus 22, does not dramatically alter EC50
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values (0.66 ꢀM versus 0.82 ꢀM). However, α,βꢀunsaturated ketone 22 is significantly more active
in the HLꢀ60 cell line (Figure 2). This latter effect may be attributed to its enhanced
electrophilicity, versus the corresponding sulfone members from the compound panel. Probing the
S2 pocket, EC50 values for leucine 23 and more dramatically, proline 24, are higher than
phenylalanine 19 (see also Figure 5, C). Contrasting the group appended from the lysine nitrogen
proved worthwhile: Sulfonamides 28, 30 and particularly 29 all killed T.b.b. cells with lower EC50
values than carbamate (Boc) 19 which may be explained by more pronounced affinity within the S1
binding region. The activities of our synthetic compounds were compared with the benchmark
vinyl sulfone, K777 (2).
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NHR''
EWG
Ph
O
MeN
O
R
N
H
N
7
O
N
N
H
N
H
SO2Ph
6
O
R'
O
Bn
Compounds: 19-25, 27-30
K777 (2)
Selectivity
vs HL-60
Compound
R''
R
H
R'
EWG
SO2Ph
T.b.b. EC50 (ꢀM)
Bn (Phe)
Boc
Boc
0.66
>83
19
20
21
22
23
(Phe)
Bn (Phe)
SO2(2-Naphthyl)
SO2(1-Naphthyl)
COPh
H
H
H
H
Bn
1.03
>53.4
Boc
Boc
Boc
0.68
0.82
1.18
>80.1
>67.1
>46.6
(Phe)
(Leu)
Bn
i-Bu
SO2Ph
24
-(CH2)3- (Pro)
SO2Ph
Boc
9.96
22.40
26.40
0.25
0.07
0.31
>5.7
>2.4
H
Bn (Phe)
H·HCl
25
27a
28
29
30
SO2Ph
SO2Ph
SO2Ph
SO2Ph
(Phe)
Bn
H
H
H
H
Boc
Ts
>2.1
Bn (Phe)
Bn (Phe)
Bn (Phe)
>220.0
>785.7
>177.4
SO2(1-Naphthyl)
SO2[2-BrC6H2(4,5-OMe)2] SO2Ph
-
5.56
K777 (2)
>9.9
a
6,7-Saturated (see Scheme 4)
Table 1. EC50 values and selectivity indices for inhibitors 19ꢀ25, 27ꢀ30 and 2.
Based on the trends summarized in Table 1, compound 29, when directly compared with K777 (2),
they both exhibited similar effects in terms of morphological changes to the parasite assessed using
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epifluorescence microscopy. In the untreated sample the asynchronous population comprised
parasites containing one kinetoplast and one nucleus (1K1N), two kinetoplasts and one nucleus
(2K1N) and two kinetoplasts with two nuclei respectively (2K2N). However, treatment with either
K777 (2) or 29 appeared to cause a failure in cytokinesis as indicated by the dramatic increase in the
number of multinucleate cells observed as depicted in Panels AꢀD. In addition, many cells
exhibited a morphology similar to the “bigeye” phenotype observed upon knockdown of the
clathrin heavy chain.37 This morphology follows the loss of cell polarity and most likely emerges
from a block in endocytic recycling.
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Phase
DAPI
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A
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Figure 3. All cells were visualized using a Zeiss Axiovert 100 fluorescence microscope. The images were captured and
processed using Axiovision software. Bar = 5 ꢂm; Panel AꢀE. The DAPI column presents the fluorescence image with
the nucleus/kinetoplast (blue). The merged column presents the corresponding merge with the phase contrast view.
Panels A and B and C and D depict the predominant two morphological changes observed in T.b.brucei postꢀtreatment
with K777 (2) and compound 29 respectively. Panel E shows an untreated control.
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Computational
The genomes of Trypanosoma spp. encode two Clan CA C1 family cysteine proteases, a cathepsin
Lꢀlike enzyme brucipain (a.k.a. rhodesain/trypanopainꢀTb) and a cathepsin Bꢀlike enzyme termed
TbCatB, the only proteases produced by the parasite during mammalianꢀhost infection. In 2009, the
first crystal structure of rhodesain bound to a vinyl sulfone inhibitor,
NꢀmethylꢀpiperazineꢀPheꢀ
hPheꢀphenyl vinyl sulfone 2, was released providing additional understanding of the substrateꢀ
binding site profile.20 The vinyl sulfone inhibitor spans the S1’ꢀS3 subsites and forms a Michaelꢀ
type adduct with the active site cysteine thiol, Cys25. The ability of each subsite in accommodating
a range of substituents has been assessed by numerous groups (see also above) and has been
recently reviewed by NicollꢀGriffith.38 In particular, T.b.brucei proteases were subsequently
crystallized by Kerr et al 39
enabling delineation of the key interactions between enzyme and
.
inhibitor and rational design of newer higher affinity inhibitors by several other groups.40ꢀ44 Yang et
al. reported a strong correlation between the potency of rhodesain inhibition and wholeꢀcell
trypanocidal activity.45 As a result, we have undertaken a computational study utilizing Xꢀray
structures of TbCatB and rhodesain available in the protein data bank (www.wwpdb.org) to
correlate wholeꢀcell activity for a series of our compounds against T.b.brucei with ‘onꢀtarget’
binding mode to both TbCatB and rhodesain and in agreement with Yang et al.45, a clear structureꢀ
activity relationship (SAR) emerges.
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Figure 4. Binding pose of compound 29 in TbCatB (left) compared with Rhodesain (right) with compound K777 (2)
also depicted (white).
As shown in Figure 4, compound 29 exhibits the same key hydrogenꢀbonding interactions with both
TbCatB and rhodesain as observed with K777 (2), locking the peptide backbone in position via
Gly281 (Asp161). Likewise, the vinyl sulfone oxygen atoms form crucial hydrogen bonds with
Trp304 (Trp184) and Gln116 (Gln19). A clear difference between compound 29 binding to
rhodesain compared with TbCatB is observed, whereby, Asn163 is capable of forming a hydrogen
bond with the naphthylsulfonylamido moiety via a waterꢀmediated interaction. In rhodesain, this is
not possible since Asn163 is mutated to a glycine residue (Gly64) which bears no sideꢀchain for
interaction. Compounds 28-30 all bound in this manner suggesting one possible explanation for
their nM trypanocidal activity compared to other members within the series.
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Figure 5. Binding poses of: A; compounds 20 (white) and 21 (cyan) in rhodesain (colored by electrostatics), B and C;
compounds 19 (white), 23 (green) and 24 (cyan) in TbCatB (gold surface) and rhodesain (colored by electrostatics).
The 2ꢀvinylsulfonylnapthalene of 20 versus 1ꢀvinylsulfonylnapthalene of 21 causes a modest
reduction in trypanocidal activity due to its less than ideal positioning against the wall formed by
Cys25 (Cys122) as highlighted in Figure 5A. Changing from a phenylalanine sidechain in 19 to
leucine, 23, or proline, 24, causes a significant loss in trypanocidal activity and can be rationalized
by the depth to which the S2 site is penetrated by the different sideꢀchains at P2. Figure 5B depicts
proline barely occupying the deep hydrophobic S2 pocket and we hypothesize that this would result
in a significant loss in affinity and subsequent loss in trypanocidal activity. The S2 pocket has a
clear preference for bulky nonꢀpolar residues at P2, also noted by Brömme et al. in Cathepsin L,15
which correlates well with the observed increase in trypanocidal potencies on replacement of P2
with Pro<Leu<Phe.
Conclusion
In summary, a series of lysineꢀcontaining dipeptide vinyl sulfones were synthesized and their
trypanocidal effects investigated. Optimum potency was observed with phenylalanine, as opposed
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to leucine or proline, as the additional amino acid fragment. Whilst the free amine 25, itself was not
particularly active, more lipophilic, nonꢀbasic Nꢀderivatives such as tert-butyloxycarbamate 19 and
sulfonamides 28 to 30 demonstrated potent killing of Trypanosoma brucei brucei in vitro. In
addition, low cytotoxicity levels were observed in HLꢀ60 cells (typically 2 orders of magnitude
higher than the EC50 values recorded). The importance of the thiophilic vinyl sulfone unit was
demonstrated, since upon hydrogenation the resultant saturated sulfone, 27, proved significantly
less active than its electrophilic precursor 19. Finally, a novel computational study was performed
which was successful in correlating binding preference for both TbcatB and rhodesain with wholeꢀ
cell trypanocidal activity.
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5. Experimental
General
Reagents were obtained from commercial suppliers and were used without further purification. Dry
THF was distilled from sodium benzophenone ketyl radical and dry DCM distilled from calcium
hydride, under nitrogen. Thinꢀlayer chromatography was performed on silica coated aluminium
sheets (60 F254) supplied by Merck. Compounds were visualised with UV light and basic aqueous
potassium permanganate, followed by heating. Flash column chromatography was performed using
1
flash silica 60 Å (230ꢀ400 mesh) 9385 supplied by Merck. H and 13C NMR spectra were recorded
using Varian Inova 300 MHz, 400 MHz and 500 MHz instruments, as indicated.
Deuterochloroform and d6ꢀdimethyl sulfoxide were used as the solvents and chemical shifts are
given in parts per million (ppm) relative to the standard reference TMS or residual protonated
solvent. Peak assignment was achieved using 2ꢀdimensional, 1Hꢀ1H and 1Hꢀ13C NMR experiments.
Infrared spectra were recorded on a Varian Instruments Excalibur series FTꢀIR 3100 spectrometer.
Samples were prepared neat between NaCl discs or deposited as films using dichloromethane.
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Melting points were recorded on a Gallenkamp electrothermal melting point apparatus. High
resolution mass spectra were carried out on a VG analytical 70ꢀE mass spectrometer. Optical
rotation data was obtained using a Perkin Elmer Model 343 polarimeter, with the sample solvent
and values are quoted in units of 10ꢀ1degcm2gꢀ1. The purity of compounds 19-25 and 27-30 was
determined by HPLC. The analysis was performed on an LCꢀ2010A Shimadzu HPLC and an
Agilent microsorbꢀMV 100ꢀ5 Cꢀ18 250 x 4.6 mm column was used for all samples. For
compounds 19-24 and 27-30, the column was eluted with an isocratic mixture of 60% acetonitrile,
30% water and 10% 0.1 M ammonium bicarbonate solution in water. For the ammonium salt 25,
the column was eluted with an isocratic mixture of 50% acetonitrile, 40% water and 10% of a 2%
TFA solution in water. Detection was at 220 and 254 nm and the average peak area was used to
determine purity: 19, 97.0%; 20, 98.0%; 21, 98.8%; 22, 91.6%; 23, 95.5%; 24, 90.8%; 25, 99.0%;
27, 93.2%; 28, 94.1%; 29, 96.3%; 30, 97.8%.
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Methyl
2S-(2S-benzyloxycarbonylamino-3-phenylpropanamido)-6-(tert-
butoxycarbonylamino)hexanoate 9.
Under nitrogen, DIPEA (1.28 mL, 7.4 mmol, 1.1 equiv.) was added to a stirred solution of 5 (2.0 g,
6.7 mmol, 1 equiv.), 8 (2.0 g, 6.7 mmol, 1 equiv.) and EDCIꢃHCl (1.28 g, 6.7 mmol, 1 equiv.) in
dichloromethane (65 mL). This mixture was left to stir at room temperature overnight under a
balloon of nitrogen. 1 M HCl (20 mL) was added along with dichloromethane (20 mL) and the
solution washed with water (20 mL). After drying the organic layer with MgSO4 and filtering, the
solvent was removed in vacuo to yield a white solid 9 (3.27 g, 90%); m.p. 98ꢀ104°C [lit. m.p.
121°C].46 Rf = 0.4 (cꢀHexꢀEtOAc; 1:1). IR (film): = 3416, 3326, 3056, 2932, 1709, 1511, 739 cmꢀ
ꢀ̅
1
1. H NMR (400 MHz, CDCl3): δ = 1.16ꢀ1.28 (m, 2H, CH2), 1.40ꢀ1.48 (m, 11H, tꢀBu, CH2), 1.56ꢀ
1.84 (m, 2H, CH2), 3.01ꢀ3.14 (m, 4H, CH2), 3.69 (s, 3H, CH3), 4.40ꢀ4.56 (m, 2H, CH), 4.66 (s (br),
1H, NH), 5.09 (s, 2H, CH2), 5.40 (s (br), 1H, NH), 6.35 (s (br), 1H, NH), 7.16ꢀ7.35 (m, 10H, ArH)
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ppm. 13C NMR (100 MHz, CDCl3): δ = 22.2 (CH2), 28.7 (CH3), 29.5 (CH2), 32.0 (CH2), 38.5
(CH2), 40.2 (CH2), 52.2 (CH), 52.6 (CH3), 56.5 (CH), 67.2 (CH2), 79.0 (C), 127.1 (CH), 128.1
(CH), 128.2 (CH), 128.5 (CH), 128.7 (CH), 129.3 (CH), 136.1 (C), 136.2 (C), 155.9 (CO), 156.1
(CO), 170.7 (CO), 172.1 (CO) ppm. HRMS (ES+): C29H40N3O7 calcd. 542.2866; found 542.2852.
[α]D = ꢀ9.8 (c = 0.1, CHCl3), [α]D = ꢀ7.2 (c = 1.0, MeOH), [lit. [α]D = ꢀ9.6 (c = 1.0, MeOH)].46
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2
S
-(2
S-Benzyloxycarbonylamino-3-phenylpropanamido)-6-(tert-
butoxycarbonylamino)hexanol 12.
Under nitrogen, 9 (1.6 g, 3.0 mmol, 1 equiv.) was stirred with LiCl (1.0 g, 23.6 mmol, 8 equiv.) in
THF (12 mL) and EtOH (6 mL). Once all solids had dissolved NaBH4 (0.9 g, 23.8 mmol, 8 equiv.)
was added and the solution left to stir overnight at room temperature. The solution was cooled to
0°C and 1 M HCl was added until the pH was ~2. The solution was diluted with dichloromethane
(20 mL), washed with water (20 mL), dried with MgSO4, filtered and the solvent removed in vacuo
to yield 12 (1.39 g, 92%) as a white solid; m.p. 110ꢀ114°C. Rf = 0.1 (cꢀHexꢀEtOAc; 1:1). IR (film):
ꢀ̅
= 3321, 3064, 2930, 2861, 1693, 1531, 1455, 748 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ = 1.15ꢀ
1.56 (m, 15H, tꢀBu, CH2), 2.06 (s (br), 1H, OH), 2.97ꢀ3.18 (m, 4H, CH2), 3.35ꢀ3.43 (m, 2H, CH2),
3.77ꢀ3.84 (m, 1H, CH), 4.35ꢀ4.43 (m, 1H, CH), 4.75 (s (br), 1H, NH), 5.06 (app. t, J = 12.5 Hz, 2H,
13
CH2), 5.65 (s (br), 1H, NH), 6.13 (d, J = 7.5 Hz, 1H, NH), 7.16ꢀ7.36 (m, 10H, ArH) ppm. C NMR
(100 MHz, CDCl3): δ = 22.8 (CH2), 28.5 (CH3), 29.6 (CH2), 30.1 (CH2), 38.7 (CH2), 39.8 (CH2),
51.4 (CH), 56.6 (CH), 64.1 (CH2), 67.0 (CH2), 79.2 (C), 127.0 (CH), 128.0 (CH), 128.1 (CH), 128.5
(CH), 128.6 (CH), 129.3 (CH), 136.1 (C), 136.6 (C), 156.1 (CO), 156.3 (CO), 171.1 (CO) ppm.
HRMS (ES+): C28H40N3O6 calcd. 514.2917; found 514.2905. [α]D = ꢀ18.1 (c = 0.35, CHCl3).
2
S-(2
S-Benzyloxycarbonylamino-3-phenylpropanamido)-6-(tert-
butoxycarbonylamino)hexanal.
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At room temperature PCC (0.26 g; 1.2 mmol, 3 equiv.) was stirred with Celite (0.26 g) in
dichloromethane (20 mL). Thereafter, 12 (0.2 g; 0.4 mmol, 1 equiv.), dissolved in dichloromethane
(5 mL), was added. This was left to stir at room temperature for 6 hours. Excess solvent was
removed in vacuo and the mixture takenꢀup in diethyl ether (10 mL) and filtered through silica
washing with ethyl acetate (50 mL). The solvent was removed in vacuo to yield the aldehyde (0.11
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g, 55%) as a creamy coloured solid; m.p. 110ꢀ124°C. Rf = 0.2 (cꢀHexꢀEtOAc; 1:1). IR (film): ꢀ̅ =
3424, 2924, 2851, 1729, 1686, 1648, 1527 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ = 1.10ꢀ1.85 (m,
15H, t-Bu, CH2), 2.90ꢀ3.10 (m, 4H, CH2), 4.28ꢀ4.44 (m, 2H, CH), 4.57 (s (br), 1H, NH), 5.03 (s
(br), 2H, CH2), 5.32 (s (br), 1H, NH), 6.34 (d, J = 6.0 Hz, 1H, NH), 7.10ꢀ7.30 (m, 10H, ArH), 9.32
(s, 1H, CHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 22.2 (CH2), 28.3 (CH2), 28.6 (CH3), 29.9
(CH2), 38.6 (CH2), 40.1 (CH2), 56.4 (CH), 58.7 (CH), 67.3 (CH2), 79.3 (C), 127.3 (CH), 128.2
(CH), 128.4 (CH), 128.7 (CH), 128.9 (CH), 129.5 (CH), 136.4 (C), 136.7 (C), 156.2 (CO), 156.3
(CO), 171.6 (CO), 198.9 (CO) ppm. HRMS (ES+): C28H38N3O6 calcd. 512.2761; found 512.2770.
[α]D = ꢀ10.3 (c = 0.1, CHCl3).
trans-5S-(2’S-Benzyloxycarbonylamino-3’phenylpropanamido)-7-(phenylsulfonyl)hept-6-en-
1-tert-butyloxycarbonylamine 19.
Under nitrogen, 60% w/w NaH in mineral oil (13 mg, 0.35 mmol, 1.2 equiv.) was stirred in THF (3
mL). At 0°C diethyl phenylsulfonylmethylphosphonate 15 (111 mg, 0.38 mmol, 1.3 equiv.),
dissolved in THF (2 mL), was added and the mixture was left to stir for 30 minutes. Then the above
aldehyde (149 mg, 0.29 mmol, 1 equiv.), dissolved in THF (6 mL), was added to the solution which
was left to stir overnight during which time room temperature was gradually reached. Water (20
mL) was added to dilute the solution and it was washed with dichloromethane (20 mL) and brine
(20 mL). After drying with MgSO4 and filtering, the solvent was removed in vacuo. Purification
by silica gel column chromatography (cꢀHexꢀEtOAc; 1:1) yielded 19 (98 mg, 52%) as a white solid;
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m.p. 130ꢀ136°C. Rf = 0.3 (cꢀHexꢀEtOAc; 1:1). IR (film): ꢀ̅ = 3375, 3068, 2928, 1663, 1529, 1366,
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1147, 1084 cmꢀ1. H NMR (300 MHz, CDCl3): δ = 1.12ꢀ1.66 (m, 15H, t-Bu, CH2), 2.94ꢀ3.13 (m,
4H, CH2), 4.33 (app. q, J = 7.0 Hz, 1H, CH), 4.55ꢀ4.70 (m, 2H, NH, CH), 5.08 (app. t, J = 14.0 Hz,
2H, CH2), 5.37 (s (br), 1H, NH), 5.88 (d, J = 8.0 Hz, 1H, NH), 6.05 (d, J = 15.0 Hz, 1H, CH), 6.75
(dd, J = 5.0, 15.0 Hz, 1H, CH), 7.10ꢀ7.40 (m, 10H, ArH), 7.50ꢀ7.68 (m, 3H, ArH), 7.85 (d, J = 7.5
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Hz, 2H, ArH) ppm. C NMR (75 MHz, CDCl3): δ = 22.5 (CH2), 28.4 (CH3), 29.3 (CH2), 33.5
(CH2), 38.2 (CH2), 40.0 (CH2), 49.2 (CH), 56.6 (CH), 67.2 (CH2), 79.0 (C), 127.3 (CH), 127.6
(CH), 128.0 (CH), 128.3 (CH), 128.5 (CH), 128.8 (CH), 129.2 (CH), 129.3 (CH), 130.4 (CH),
133.5 (CH), 136.0 (C), 136.1 (C), 140.1 (C), 145.4 (CH), 156.0 (CO), 156.1 (CO), 170.8 (CO) ppm.
HRMS (ES+): C35H44N3O7S calcd. 650.2900; found 650.2930. [α]D = ꢀ11.5 (c = 0.1, CHCl3). HPLC
Analysis (Cꢀ18) MeCNꢀH2Oꢀ0.1 M NH4HCO3(aq); 60:30:10 (0.4 mL/min): tr = 26.79 min.
trans-5S-(2’S-Benzyloxycarbonylamino-3’phenylpropanamido)-7-(2’’-naphthylsulfonyl)hept-
6-en-1-tert-butyloxycarbonylamine 20.
PCC (291 mg, 1.40 mmol, 3 equiv.) was stirred with Celite (291 mg) in dichloromethane (15 mL).
Alcohol 12 (229 mg; 0.50 mmol, 1 equiv.) was dissolved in dichloromethane (10 mL) and added to
the PCC solution. This was left to stir at room temperature for 6 hours. Excess solvent was
removed in vacuo and the mixture redissolved in diethyl ether (15 mL). It was filtered through
silica and washed with ethyl acetate (50 mL). The solvent was removed in vacuo to yield the crude
dipeptide aldehyde. Under nitrogen, 60% w/w NaH in mineral oil (22 mg, 0.55 mmol, 1.2 equiv.)
was stirred in THF (2 mL). At 0°C, diethyl (naphthalenꢀ2ꢀylsulfonyl)methylphosphonate 16 (200
mg, 0.60 mmol, 1.3 equiv.), dissolved in THF (3 mL), was added to the NaH mixture and left to stir
for 30 minutes. The crude dipeptide aldehyde, dissolved in THF (5 mL), was added to the solution
and it was left to stir overnight warming gradually to room temperature. Water (15 mL) was added
to dilute the solution and it was washed with dichloromethane (3 x 15 mL) and brine (15 mL).
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After drying with MgSO4 and filtering, the solvent was removed in vacuo. Purification by silica gel
column chromatography (cꢀHexꢀEtOAc; 9:1 to 1:1) yielded 20 (173 mg, 55%) as a white
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amorphous solid; m.p. 121ꢀ125°C. Rf = 0.4 (cꢀHexꢀEtOAc; 1:1). IR (film): ꢀ̅ = 3309, 3056, 2932,
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2840, 1701, 1523, 1452, 1310, 1253, 1162, 1035, 739 cmꢀ1. 1H NMR (500 MHz, CDCl3) δ = 1.15ꢀ
1.65 (m, 15H, t-Bu, CH2), 2.95ꢀ3.10 (m, 4H, CH2), 4.33 (app. q, J = 7.5 Hz, 1H, CH), 4.55ꢀ4.70 (m,
2H, CH, NH), 5.00ꢀ5.10 (m, 2H, CH2), 5.41 (s (br), 1H, NH), 6.00 (s (br), 1H, NH), 6.14 (d, J =
15.5 Hz, 1H, CH), 6.80 (dd, J = 4.5, 15.5 Hz, 1H, CH), 7.08ꢀ7.36 (m, 10H, ArH), 7.60ꢀ7.69 (m, 2H,
ArH), 7.76 (d, J = 8.5 Hz, 1H, ArH), 7.91 (d, J = 8.0 Hz, 1H, ArH), 7.98 (t, J = 8.0 Hz, 2H, ArH),
8.45 (s, 1H, ArH) ppm. 13C NMR (125 MHz, CDCl3): δ = 22.5 (CH2), 28.4 (CH3), 29.4 (CH2), 33.5
(CH2), 40.0 (CH2), 40.1 (CH2), 49.3 (CH), 56.6 (4ꢀCH), 67.2 (CH2), 79.2 (C), 122.5 (CH), 127.2
(CH), 127.7 (CH), 128.0 (CH), 128.1 (CH), 128.3 (CH), 128.6 (CH), 128.8 (CH), 129.20 (CH),
129.25 (CH), 129.3 (CH), 129.4 (CH), 129.6 (CH), 130.6 (CH), 132.3 (C), 135.2 (C), 136.00 (C),
136.05 (C), 137.0 (C), 145.4 (CH), 156.0 (CO), 156.1 (CO), 170.7 (CO) ppm. HRMS (ES+):
C39H45N3O7SNa calcd. 722.2876; found 722.2859. [α]D = ꢀ5.8 (c = 0.1, CHCl3). HPLC Analysis (Cꢀ
18), MeCNꢀH2Oꢀ0.1 M NH4HCO3(aq); 60:30:10 (0.4 mL/min): tr = 41.24 min.
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trans-5S-(2’S-Benzyloxycarbonylamino-3’phenylpropanamido)-7-(1’’-naphthylsulfonyl)hept-
6-en-1-tert-butyloxycarbonylamine 21.
PCC (259 mg, 1.2 mmol, 3 equiv.) was stirred with Celite (259 mg) in dichloromethane (5 mL).
Alcohol 12 (208 mg; 0.4 mmol, 1 equiv.) was dissolved in dichloromethane (10 mL) and added to
the PCC solution. This was left to stir at room temperature for 6 hours. Excess solvent was
removed in vacuo and the mixture redissolved in diethyl ether (15 mL). It was filtered through
silica and washed with ethyl acetate (50 mL). The solvent was removed in vacuo to yield the crude
dipeptide aldehyde. Under nitrogen, 60 % w/w NaH in mineral oil (19 mg, 0.5 mmol, 1.2 equiv.)
was stirred in THF (2 mL). At 0°C, diethyl (naphthalenꢀ1ꢀylsulfonyl)methylphosphonate 17 (180
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mg, 0.5 mmol, 1.3 equiv.), dissolved in THF (3 mL), was added to the NaH mixture and left to stir
for 30 minutes. The crude dipeptide aldehyde, dissolved in THF (5 mL), was added to the solution
and it was left to stir overnight warming gradually to room temperature. Water (15 mL) was added
to dilute the solution and it was washed with dichloromethane (3 x 15 mL) and brine (15 mL).
After drying with MgSO4 and filtering, the solvent was removed in vacuo. Purification by silica gel
column chromatography (cꢀHexꢀEtOAc; 9:1 to 1:1) yielded 21 (114 mg, 41%) as a colourless oil
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which gradually solidified; m.p. 88ꢀ90°C. Rf = 0.3 (cꢀHex: EtOAc; 1:1). IR (film): ꢀ̅ = 3309, 3056,
2932, 2840, 1701, 1523, 1452, 1310, 1253, 1162, 1035, 739 cmꢀ1. 1H NMR (500 MHz, CDCl3) δ =
1.10ꢀ1.60 (m, 15H, t-Bu, CH2), 2.90ꢀ3.08 (m, 4H, CH2), 4.29 (app. q, J = 7.5 Hz, 1H, CH), 4.56ꢀ
4.69 (m, 2H, CH, NH), 5.00ꢀ5.08 (m, 2H, CH2), 5.40 (s (br), 1H, NH), 6.00 (s (br), 1H, NH), 6.23
(d, J = 15.0 Hz, 1H, CH), 6.86 (dd, J = 5.0, 15.0 Hz, 1H, CH), 7.05ꢀ7.35 (m, 10H, ArH), 7.57ꢀ7.67
(m, 3H, ArH), 7.95 (d, J = 8.0 Hz, 1H, ArH), 8.12 (d, J = 8.0 Hz, 1H, ArH), 8.32 (d, J = 8.0 Hz, 1H,
ArH), 8.53 (d, J = 8.0 Hz, 1H, ArH) ppm. 13C NMR (125 MHz, CDCl3): δ = 22.5 (CH2), 28.4
(CH3), 29.3 (CH2), 33.6 (CH2), 38.2 (CH2), 40.1 (CH2), 49.3 (CH), 56.6 (CH), 67.2 (CH2), 79.1 (C),
124.2 (CH), 124.5 (CH), 127.0 (CH), 127.2 (CH), 128.1 (CH), 128.3 (CH), 128.5 (CH), 128.6
(CH), 128.8 (CH), 129.16 (CH), 129.19 (CH), 129.9 (CH), 130.9 (CH), 134.2 (C), 134.9 (C), 135.3
(CH), 136.00 (C), 136.05 (C), 136.1 (C), 145.4 (CH), 156.0 (CO), 156.1 (CO), 170.7 (CO) ppm.
HRMS (ES+): C39H45N3O7SNa calcd. 722.2876; found 722.2858. [α]D = ꢀ14.0 (c = 0.1, CHCl3).
HPLC Analysis (Cꢀ18), MeCNꢀH2Oꢀ0.1 M NH4HCO3(aq); 60:30:10 (0.4 mL/min): tr = 39.29 min.
trans-4
S-(2’S-Benzyloxycarbonylamino-3’phenylpropanamido)-8-(tert-
butyloxycarbonylamino)oct-2-en-1-one 22.
PCC (556 mg, 2.6 mmol, 3 equiv.) was stirred with Celite (556 mg) in dichloromethane (15 mL).
Alcohol 12 (442 mg, 0.9 mmol, 1 equiv.) was dissolved in dichloromethane (15 mL) and added to
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the PCC solution. This was left to stir at room temperature for 6 hours. Excess solvent was
removed in vacuo and the mixture redissolved in diethyl ether (15 mL). It was filtered through
silica and washed with ethyl acetate (60 mL). The solvent was removed in vacuo to yield the crude
dipeptide aldehyde. Under nitrogen, 60% w/w NaH in mineral oil (47 mg, 1.2 mmol, 1.3 equiv.)
was stirred in THF (2 mL). At 0°C diethyl 2ꢀoxoꢀ2ꢀphenylethylphosphonate 18 (300 mg, 1.2 mmol,
1.3 equiv.), dissolved in THF (3 mL), was added to the NaH mixture and left to stir for 30 minutes.
The crude dipeptide aldehyde, dissolved in THF (5 mL), was added to the solution and it was left to
stir overnight warming gradually to room temperature. Water (10 mL) was added to dilute the
solution and it was washed with dichloromethane (20 mL) and brine (50 mL). After drying with
MgSO4 and filtering, the solvent was removed in vacuo. Purification by silica gel column
chromatography (cꢀHexꢀEtOAc; 2:1 to 1:1) yielded 22 (74 mg, 13%) as a yellow, amorphous solid;
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m.p. 85ꢀ87°C. Rf = 0.4 (cꢀHexꢀEtOAc; 1:1). IR (film): ꢀ̅ = 3440, 2929, 2861, 1690, 1625, 1527,
1454, 1042 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ = 1.20ꢀ1.85 (m, 15H, t-Bu, CH2), 2.90ꢀ3.20 (m,
4H, CH2), 4.43 (app. q, J = 7.0 Hz, 1H, CH), 4.60ꢀ4.81 (m, 2H, CH, NH), 5.00ꢀ5.10 (m, 2H, CH2),
5.35ꢀ5.55 (m, 1H, NH), 6.08 (s (br), 1H, NH), 6.68 (dd, J = 5.5, 15.5 Hz, 1H, CH), 6.80 (d, J = 15.5
Hz, 1H, CH), 7.10ꢀ7.35 (m, 10H, ArH), 7.47 (t, J = 8.0 Hz, 2H, ArH), 7.57 (t, J = 7.5 Hz, 1H, ArH),
7.91 (d, J = 7.5 Hz, 2H, ArH) ppm. 13C NMR (100 MHz, CDCl3): δ = 22.7 (CH2), 28.4 (CH3), 29.5
(CH2), 33.8 (CH2), 38.3 (CH2), 40.1 (CH2), 50.3 (CH), 56.6 (CH), 67.2 (CH2), 79.2 (C), 125.2
(CH), 127.2 (CH), 127.3 (CH), 128.0 (CH), 128.2 (CH), 128.5 (CH), 128.6 (CH), 128.7 (CH),
128.8 (CH), 133.0 (CH), 135.6 (C), 136.0 (C), 137.5 (C), 146.7 (CH), 156.05 (CO), 156.10 (CO),
170.6 (CO), 190.2 (CO) ppm. HRMS (ES+): C36H44N3O6 calcd. 636.3050; found 636.3031. [α]D = ꢀ
6.7 (c = 0.1, CHCl3). HPLC Analysis (Cꢀ18), MeCNꢀH2Oꢀ0.1 M NH4HCO3(aq); 60:30:10 (0.4
mL/min): tr = 43.28 min.
Methyl
2S-(2S-benzyloxycarbonylamino-4-methylpentanamido)-6-(tert-
butoxycarbonylamino)hexanoate 10.
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Under nitrogen, DIPEA (1.75 mL, 10.0 mmol, 1.1 equiv.) was added to a stirred solution of 6 (2.4
g, 9.1 mmol, 1 equiv.), 8 (2.7 g, 9.1 mmol, 1 equiv.) and EDCIꢃHCl (1.8 g, 9.4 mmol, 1.05 equiv.)
in dichloromethane (90 mL). This mixture was left to stir at room temperature overnight under a
balloon of nitrogen. 1 M HCl (10 mL) was added along with dichloromethane (20 mL) and the
solution washed with water (20 mL). After drying the organic layer with MgSO4 and filtering, the
solvent was removed in vacuo to yield 10 (4.2 g, 91%) as a white solid; m.p. 68ꢀ71°C [lit. m.p.
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84.5ꢀ85°C].47 Rf = 0.4 (cꢀHexꢀEtOAc; 1:1). IR (film):
ꢀ̅ = 3339, 2955, 2931, 2870, 1708, 1665,
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1528, 1519, 1438, 1454 cmꢀ1. H NMR (400 MHz, CDCl3): δ = 0.90ꢀ0.98 (m, 6H, CH3), 1.20ꢀ1.90
(m, 18H, t-Bu, CH2, CH), 3.00ꢀ3.10 (m, 2H, CH2), 3.74 (s (br), 3H, CH3), 4.15ꢀ4.25 (m, 1H, CH),
4.54ꢀ4.61 (m, 1H, CH), 4.71ꢀ4.78 (m, 1H, NH), 5.12 (s (br), 2H, CH2), 5.27ꢀ5.34 (m, 1H, NH),
6.48ꢀ6.56 (m, 1H, NH), 7.28ꢀ7.38 (m, 5H, ArH) ppm. 13C NMR (100 MHz, CDCl3): δ = 22.0 (CH3),
22.2 (CH3), 22.9 (CH2), 24.6 (CH), 28.4 (CH3), 29.1 (CH2), 31.9 (CH2), 40.1 (CH2), 41.2 (CH2),
51.8 (CH), 52.4 (CH3), 53.5 (CH), 67.1 (CH2), 79.2 (C), 128.0 (CH), 128.2 (CH), 128.5 (CH), 136.2
(C), 156.0 (CO), 156.2 (CO), 172.0 (CO), 172.5 (CO) ppm. HRMS (ES+): C26H42N3O7 calcd.
508.3023; found 508.3007. [α]D = ꢀ12.6 (c = 0.1, CHCl3) [lit. [α]D = ꢀ5.6 (c = 1.0, EtOAc)].47
2
S-(2
S-Benzyloxycarbonylamino-4-methylpentanamido)-6-(tert-
butoxycarbonylamino)hexanol 13.
Under nitrogen, 10 (3.9 g, 7.7 mmol, 1 equiv.) was stirred with LiCl (2.6 g, 61.5 mmol, 8 equiv.) in
THF (52 mL) and EtOH (26 mL). Once all solids had dissolved NaBH4 (3.3 g, 61.5 mmol, 8
equiv.) was added and the solution left to stir overnight at room temperature. Product formation
was confirmed by TLC. The solution was cooled to 0°C and 1 M HCl (40 mL) was added until the
pH was ~2. The solution was diluted with dichloromethane (30 mL), washed with water (30 mL),
dried with MgSO4, filtered and the solvent removed in vacuo to yield 13 (3.6 g, 97%) as a
colourless foam; m.p. 75ꢀ83°C. Rf = 0.1 (cꢀHexꢀEtOAc; 1:1). IR (film):
ꢀ̅
= 3331, 2956, 2934,
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2870, 1694, 1659, 1531, 1455 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ = 0.88ꢀ0.97 (m, 6H, CH3), 1.24ꢀ
1.78 (m, 18 H, t-Bu, CH2, CH), 2.85ꢀ3.20 (m, 3H, CH2, OH), 3.52ꢀ3.65 (m, 2H, CH2), 3.84ꢀ3.94 (m,
1H, CH), 4.12ꢀ4.20 (m, 1H, CH), 4.73 (s (br), 1H, NH), 5.11 (s (br), 2H, CH2), 5.37ꢀ5.44 (m, 1H,
NH), 6.35ꢀ6.41 (m, 1H, NH), 7.29ꢀ7.38 (m, 5H, ArH) ppm. 13C NMR (100 MHz, CDCl3): δ = 22.0
(CH3), 22.6 (CH2), 22.9 (CH3), 24.7 (CH), 28.4 (CH3), 29.6 (CH2), 30.0 (CH2), 39.7 (CH2), 41.3
(CH2), 51.4 (CH), 53.8 (CH), 64.5 (CH2), 67.1 (CH2), 79.3 (C), 128.1 (CH), 128.2 (CH), 128.5
(CH), 136.1 (C), 156.3 (CO), 156.4 (CO), 172.6 (CO) ppm. HRMS (ES+): C25H41N3O6Na calcd.
502.2893; found 502.2905. [α]D = ꢀ35.6 (c = 0.1, CHCl3).
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trans-5S-(2’S-Benzyloxycarbonylamino-4’-methylpentanamido)-7-(phenylsulfonyl)hept-6-en-
1-tert-butoxycarbonylamine 23.
PCC (0.4 g, 1.8 mmol, 3 equiv.) was stirred with Celite (0.4 g) in dichloromethane (20 mL). A
solution of 13 (0.3 g; 0.6 mmol, 1 equiv.) dissolved in dichloromethane (~10 mL) was added. This
was left to stir at room temperature for 6 hours. Excess solvent was removed in vacuo and the
mixture takenꢀup in diethyl ether (10 mL). It was filtered through silica and washed with ethyl
acetate (50 mL). The solvent was removed in vacuo to yield the crude dipeptide aldehyde. Under
nitrogen, 60% w/w NaH in mineral oil (28 mg, 0.7 mmol, 1.2 equiv.) was stirred in THF (3 mL).
At 0°C diethyl phenylsulfonylmethylphosphonate 15 (239 mg, 0.8 mmol, 1.3 equiv.), dissolved in
THF (2 mL), was added to the NaH mixture and left to stir for 30 minutes. The crude dipeptide
aldehyde, dissolved in THF (6 mL), was added to the solution and it was left to stir overnight
warming gradually to room temperature. Water (40 mL) was added to dilute the solution and it was
washed with dichloromethane (30 mL) and brine (70 mL). After drying with MgSO4 and filtering,
the solvent was removed in vacuo. Purification by silica gel column chromatography (cꢀHexꢀ
EtOAc; 2:1 to 1:1) yielded 23 (168 mg, 46%) as a colourless oil which gradually solidified; m.p.
88ꢀ92
°C. Rf = 0.5 (cꢀHexꢀEtOAc; 1:1). IR (film):
ꢀ̅
= 3337, 3068, 2955, 2918, 1656, 1630, 1447,
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1366, 1148, 778 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ = 0.82ꢀ0.96 (m, 6H, CH3), 1.20ꢀ1.70 (m, 18H,
t-Bu, CH2, CH), 2.98ꢀ3.08 (m, 2H, CH2), 4.12ꢀ4.19 (m, 1H, CH), 4.61ꢀ4.69 (m, 1H, CH), 4.75ꢀ4.82
(m, 1H, NH), 5.04ꢀ5.12 (m, 2H, CH2), 5.44ꢀ5.50 (m, 1H, NH), 6.45 (d, J = 15.0 Hz, 1H, CH), 6.60ꢀ
6.70 (m, 1H, NH), 6.87 (dd, J = 5.0, 15.0 Hz, 1H, CH), 7.26ꢀ7.37 (m, 5H, ArH), 7.48ꢀ7.63 (m, 3H,
ArH), 7.82ꢀ7.88 (m, 2H, ArH) ppm. 13C NMR (100 MHz, CDCl3): δ = 21.9 (CH3), 22.6 (CH2), 22.8
(CH3), 24.6 (CH), 28.4 (CH3), 29.2 (CH2), 33.3 (CH2), 40.1 (CH2), 40.8 (CH2), 49.1 (CH), 53.6
(CH), 67.2 (CH2), 79.2 (C), 127.6 (CH), 128.0 (CH), 128.2 (CH), 128.5 (CH), 129.3 (CH), 130.6
(CH), 133.5 (CH), 136.0 (C), 140.1 (C), 145.7 (CH), 156.1 (CO), 156.5 (CO), 172.2 (CO) ppm.
HRMS (ES+): C32H46N3O7S calcd. 616.3056; found 616.3085. [α]D = ꢀ25.6 (c = 0.1, CHCl3). HPLC
Analysis (Cꢀ18), MeCNꢀH2Oꢀ0.1 M NH4HCO3(aq); 60:30:10 (0.4 mL/min): tr = 26.77 min.
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Benzyl 2
S-(6-tert-butoxycarbonylamino)-1-methoxy-1-oxohexan-2S-ylcarbamoyl)pyrrolidine-
1-carboxylate 11.
Under nitrogen, DIPEA (0.8 mL, 4.6 mmol, 1.1 equiv.) was added to a stirred solution of 7 (1.0 g,
4.0 mmol, 1 equiv.), 8 (1.2 g, 4.0 mmol, 1 equiv.) and EDCIꢃHCl (0.7 g, 4.0 mmol, 1 equiv.) in
dichloromethane (40 mL). This mixture was left to stir at room temperature overnight under a
balloon of nitrogen. 1 M HCl (10 mL) was added along with dichloromethane (15 mL) and the
solution washed with water (10 mL). After drying the organic layer with MgSO4 and filtering, the
solvent was removed in vacuo to yield 11 (1.8 g, 90%) as a white solid; m.p. 80ꢀ82°C, [lit. m.p. 92ꢀ
93°C].48 Rf = 0.5 (cꢀHexꢀEtOAc; 1:5). IR (film):
ꢀ̅ = 3477, 3393, 2917, 1739, 1659, 1542, 1423,
876 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ = 1.20ꢀ1.50 (m, 13H, t-Bu, CH2), 1.50ꢀ2.34 (m, 6H, CH2),
3.00ꢀ3.10 (m, 2H, CH2), 3.40ꢀ3.72 (m, 5H, CH3, CH2), 4.30ꢀ4.35 (m, 1H, CH), 4.49ꢀ4.57 (m, 1.4H,
CH, NH), 4.80 (s (br), 0.6H, NH), 5.17 (s (br), 2H, CH2), 6.47 (s (br), 0.35H, NH), 7.05 (s (br),
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0.65H, NH), 7.28ꢀ7.39 (m, 5H, ArH) ppm. C NMR (100 MHz, CDCl3): δ = 22.3 (CH2), 24.6
(CH2), 28.4 (CH3), 29.3 (CH2), 31.85 (CH2), 31.90 (CH2), 40.1 (CH2), 47.0 (CH2), 52.0 (CH), 52.3
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Journal of Medicinal Chemistry
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(CH3), 60.4 (CH), 67.3 (CH2), 78.9 (C), 127.8 (CH), 128.0 (CH), 128.5 (CH), 136.4 (C), 156.0 (2 x
CO), 171.5 (CO), 172.6 (CO) ppm. HRMS (ES+): C25H37N3O7 calcd. 492.2710; found 492.2686.
[α]D = ꢀ40.0 (c = 0.1, CHCl3), [lit. [α]D = ꢀ24.4 (c = 1.0, DMF)].48 Note NMR spectra complicated
due to mixtures of rotamers.
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Benzyl
2S-(6-tert-butoxycarbonylamino)-1-hydroxyhexan-2S-ylcarbamoyl)pyrrolidine-1-
carboxylate 14.
Under nitrogen, methyl ester 11 (1.5 g, 3.0 mmol, 1 equiv.) was stirred with LiCl (1.0 g, 23.6 mmol,
8 equiv.) in THF (20 mL) and EtOH (10 mL). Once all solids had dissolved NaBH4 (0.9 g, 23.7
mmol, 8 equiv.) was added and the solution left to stir overnight at room temperature. Product
formation was confirmed by TLC. The solution was cooled to 0°C and 1 M HCl (20 mL) was
added until the pH was ~2. The solution was diluted with dichloromethane (20 mL), washed with
water (20 mL), dried with MgSO4, filtered and the solvent removed in vacuo to yield 14 (1.2 g,
87%) as a white powder; m.p. 85ꢀ87°C. Rf = 0.1 (cꢀHexꢀEtOAc; 1:2). IR (film): ꢀ̅ = 3695, 3342,
3099, 2932, 2876, 1690, 1530, 1421 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ = 1.21ꢀ1.63 (m, 15H, t-
Bu, CH2), 1.85ꢀ2.33 (m, 4H, CH2), 2.82 (s (br), 0.6H, OH), 2.99ꢀ3.17 (m, 2H, CH2), 3.37ꢀ3.69 (m,
4H, CH2), 3.80ꢀ3.90 (m, 1H, CH), 4.27ꢀ4.34 (m, 1H, CH), 4.52ꢀ4.70 (m, 1H, NH), 5.03ꢀ5.26 (m,
2H, CH2), 6.18 (s (br), 0.35H, NH), 6.59 (s (br), 0.65H, NH), 7.28ꢀ7.40 (m, 5H, ArH) ppm. 13C
NMR (100 MHz, CDCl3): δ = 22.8 (CH2), 24.6 (CH2), 28.4 (CH3), 28.8 (CH2), 29.7 (CH2), 30.2
(CH2), 40.0 (CH2), 47.1 (CH2), 51.8 (CH), 61.0 (CH), 65.0 (CH2), 67.4 (CH2), 79.1 (C), 128.0
(CH), 128.2 (CH), 128.5 (CH), 136.3 (C), 156.2 (2 x CO), 172.3 (CO) ppm. HRMS (ES+):
C24H37N3O6Na calcd. 486.2580; found 486.2570. [α]D = ꢀ64.8 (c = 0.1, CHCl3). Note NMR spectra
complicated due to mixtures of rotamers.
trans-Benzyl
2S-[(1-tert-butoxycarbonylamino)-7-(phenylsulfonyl)hept-6-en-5S-
ylcarbamoyl]pyrrolidine-1-carboxylate 24.
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