Optimization Strategy of Novel Peptide-Based Michael Acceptors for the Treatment of Human African Trypanosomiasis

Article abstract of DOI:10.1021/acs.jmedchem.9b00908

This paper describes an optimization strategy of the highly active vinyl ketone 3 which was recognized as a strong inhibitor of rhodesain of Trypanosoma brucei rhodesiense, endowed with a k_second value of 67 × 10⁶ M^-1 min<

Full text of DOI:10.1021/acs.jmedchem.9b00908

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Article
Optimization strategy of novel peptide-based Michael
acceptors for the treatment of Human African Trypanosomiasis
Roberta Ettari, Santo Previti, Santina Maiorana, Giorgio Amendola, Annika Wagner,
Sandro Cosconati, Tanja Schirmeister, Ute A. Hellmich, and Maria Zappalà
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00908 • Publication Date (Web): 12 Nov 2019
Downloaded from pubs.acs.org on November 14, 2019
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Optimization strategy of novel peptide-based Michael
acceptors for the treatment of Human African
Trypanosomiasis
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‡
Roberta Ettari,*,† Santo Previti,† Santina Maiorana,† Giorgio Amendola, Annika Wagner,∇ ϴ
Sandro Cosconati,‡ Tanja Schirmeister,⊥ Ute A. Hellmich∇ ϴ and Maria Zappalà†
†Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of
Messina, Italy, Viale Annunziata, 98168 Messina, Italy
‡DiSTABiF, University of Campania Luigi Vanvitelli, Via Vivaldi 43, 81100 Caserta, Italy
∇ Centre for Biomolecular Magnetic Resonance (BMRZ), Max von Laue Str. 9, Frankfurt, DE
60438 Germany
ϴ Institute of Pharmacy and Biochemistry, University of Mainz, Johann-Joachim-Becherweg 30,
Mainz, DE 55128 Germany
⊥Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5 Mainz, DE 55128
Germany
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ABSTRACT
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This paper describes an optimization strategy of the highly active vinyl ketone 3 which was
recognized as a strong inhibitor of rhodesain of T. b. rhodesiense, endowed with a k2nd value of
67000·x 103 M-1 min-1 coupled with a high binding affinity (Ki = 38 pM). We now report a new
structure-activity relationship study based on structural variations on the P3, P2 and P1’ sites which
led us to identify two potent lead compounds, i.e vinyl ketones 4h and 4k. Vinyl ketone 4h showed
an impressive potency towards rhodesain (k2nd=881100 x 103) coupled to a good antiparasitic activity
(EC50 =3.6 µM), while vinyl ketone 4k proved to possess the highest binding affinity towards the
trypanosomal protease (Ki=0.6 pM) and a submicromolar antiparasitic activity (EC50= 0.67 µM), thus
representing new lead compounds in the drug discovery process for the treatment of Human African
Trypanosomiasis.
INTRODUCTION
Human African Trypanosomiasis (HAT), well-known as sleeping sickness, is a protozoan disease
endemic in more than 30 countries in sub-Saharan Africa, where it continues to be an significant
cause of illness and death, mainly in the rural areas, despite the substantial decrease of new cases
reported in the last years.1
The etiological agents of HAT are two Trypanosoma subspecies: T. brucei gambiense is
predominant in central and western Africa, where it causes a chronic form of HAT, whereas T. brucei
rhodesiense is widespread in southern and eastern Africa, where it is responsible of an acute form of
Trypanosomiasis, characterized by a fast onset and a high mortality.2
In this disease, which is transmitted by the bite of tsetse flies of the Glossina genus, two different
stages could be recognized. The first one is known as hemolymphatic stage: the protozoan is initially
present in the bloodstream (BS) and subsequently invades the hemolymphatic system, and thus causes
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fever, myalgia and weakness. The hemolymphatic stage normally persists for weeks or months and,
if untreated, can evolve into the second stage, during which the protozoan penetrates the blood-brain
barrier (BBB), inducing neurological disturbances, sleep/wake cycle alterations, coma and finally
death.3
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To date, an effective vaccine has not yet been developed; this can be partially ascribed to the fast
turnover of the variant surface glycoproteins (VSGs) present on the coat of the parasite that induces
a high level of antigenic variations.4 Consequently, the sole possible approach to treat this pathology
is represented by the chemotherapy. At present, five drugs are available for HAT treatment: suramin
and pentamidine, active in the hemolymphatic stage since they are unable to pass through the blood-
brain barrier due to unfavorable physicochemical properties; as well as melarsoprol, eflornithine and
nifurtimox, which are active in the neurological stage. Nevertheless, melarsoprol induces a reactive
encephalopathy in 5-10% cases treated whereas eflornithine is active only against T. b. gambiense.5
Currently, a combination of eflornithine and nifurtimox, represents the first-choice therapy of the
neurological-stage of the disease caused by T. b. gambiense. Moreover, on 15 November 2018, a
positive opinion has been adopted by the Committee for Medicinal Products for Human Use on the
approval of fexinidazole to treat the gambiense trypanosomiasis.6-8
In this scenario, there is a pressing demand of identifying new targets and new effective drugs for
HAT treatment.
In this context, rhodesain, a Clan CA, family C1 (papain-family) cathepsin L-like cysteine protease
of T. brucei rhodesiense, represents one of the most promising targets for the development of new
anti-HAT drugs.9-11 The importance of rhodesain is rooted in its diverse functions: it is required to
cross the BBB, thus inducing the neurological stage of HAT,12 it is involved in the turnover of the
trypanosome VSGs,13 which form a densely packed coat surrounding the parasite and whose periodic
antigenic variation allows the parasite to evade the host immune system. In addition, one of rhodesain
extracellular activities is the degradation of host anti-VSG immunoglobulins, which further
contributes to reduce the host immune response.14-15 Lastly, rhodesain also takes part in the lysosomal
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proteolysis of both protozoan proteins and host proteins transported inside the parasite.16 All these
functions clearly highlight the relevance of rhodesain both for Trypanosoma survival and at the same
time for the progression of the disease.
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The structure of rhodesain resembles that of other proteases of the papain family, with the catalytic
triad, C25/H162/N182, situated in a cleft between two domains, named left (L) and right (R). Two
crystal structures of rhodesain in complex with K11777 (1) and K11002 (2) (Chart 1), two potent
irreversible rhodesain inhibitors with a peptidyl vinyl sulfone structure, have been deposited in the
Protein Data Bank (PDB) (codes 2P7U17 and 2P8618). These structural studies were fundamental to
understand the key requisites for the binding of the inhibitors to the trypanosomal cysteine protease.
O
S
O
O
S
O
H
H
N
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O
N
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O
N
N
H
H
N
O
O
O
1
2
Chart 1. Structure of vinyl sulfones 1 and 2.
In the last decade, our group has been involved in research projects aimed at identifying new
inhibitors of rhodesain.19-26 In particular, starting from the structure of vinyl sulfones 1-2, we recently
synthesized a number of dipeptidyl derivatives with different electrophilic portions as Michael
acceptors, and identified as lead compound the vinyl ketone 3 (see chart 2),26 a potent rhodesain
covalent inhibitor which showed a k2nd value of 67000·x 103 M-1 min-1 coupled with a high binding
affinity (Ki = 38 pM). On the basis of the structure of the lead compound 3, a new series of Michael
acceptors has been designed (Chart 2). Here, we retained the vinyl ketone warhead and the
homophenylalanine (hPhe) at the P1 site, whereas at the P3 site we introduced an N-methylpiperazine
or a morpholine moiety in agreement to the structures of 1 and 2 (i.e. compounds 4a-b). We then
decided to replace the Cbz group with some benzo-fused rings (i.e. 4c-e), one of which is the 2,3-
dihydrobenzo[b][1,4]dioxine moiety (i.e. 4c) present in potent rhodesain inhibitors reported in
literature.27 A panel of aromatic moieties, variously decorated with halogen atoms (i.e. 4f-m), were
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also introduced at the P3 site, in agreement to the structure of peptidic inhibitors bearing at the P3
site 3,5-difluoro- or 4-CF3-phenyl moieties28 or taking into consideration non peptide rhodesain
inhibitors based on a triazine nucleus variously decorated with halogen–substituted aromatic nuclei.29
This approach could also allow us to investigate the size of S3 pocket and to concurrently allow for
the formation of halogen bonds and/or hydrophobic interactions. We also evaluated the relevance of
the Phe residue at the P2 site, by synthesizing the hPhe analog (i.e. 4n). A final study was also
performed on the P1’ site, thus synthesizing the corresponding ethyl analog of the lead compound 3
(i.e. 4o), so as to explore the size of S1’ pocket.
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In this paper, we describe the approach to synthesize the inhibitors 4a-o, together with the results
of the biological investigation against rhodesain and T. b. brucei as well as molecular modeling
studies.
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O
O
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N
4a
4b
4d
4c
H
F
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P2
P1'
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-CH2CH3
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CH3
H
4o
O
F
F3C
Cl
4h
4j
4i
3
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P3
P1
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4l
4m
4k
Chart 2. SAR study carried out starting on the lead compound 3.
RESULTS AND DISCUSSION
Chemistry. The synthesis of compounds 4a-m (Scheme 1) was carried out starting from methyl
esters 5a-m. Hydrolysis with LiOH provided the carboxylic acids 6a-m. Amine 7, obtained as
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previously reported,30 was reacted with carboxylic acids 6a-m using HOBt and EDCI as coupling
reagents, to provide the terminal olefins 8a-m. Cross-metathesis (CM) reaction between 8a-m and
methyl vinyl ketone 9, in the presence of 2nd generation Hoveyda-Grubbs catalyst and under
microwave irradiation,31 yielded the desired Michael acceptors 4a-m.
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O
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CH3
O
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H2N
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a
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OH
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CH3
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h
i
j
k
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m
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F3C
Cl
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F
F
Scheme 1. Reagents and conditions: a) LiOH, MeOH/H2O/dioxane (1:1:1), 0° C, 10 min, then rt,
12h; b) HOBt, EDCI, dry DMF/CH2Cl2 (1:1), 0°C, 10 min, then DIPEA, 7, rt, 12h; e) 2nd generation
Hoveyda-Grubbs catalyst, dry CH2Cl2, 100 °C, MW, 2-4h.
Compound 4n was synthesized (Scheme 2) following the same procedure described above starting
from carboxylic acid 10. Similarly, compound 4o (Scheme 3) was obtained from the terminal olefin
12,26 using the commercially available ethyl vinyl ketone 13, as CM partner.
O
O
O
O
O
H
H
H2N
N
N
9
OH
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N
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H
H
c
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O
a
10
4n
11
Scheme 2. Reagents and conditions: a) HOBt, EDCI, dry DMF/CH2Cl2 (1:1), 0°C, N2, then 10 min
DIPEA, 7, rt, 12h; b) 2nd generation Hoveyda-Grubbs catalyst, dry CH2Cl2, 100 °C, MW, 2 h.
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H
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H
a
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O
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Scheme 3. Reagents and conditions: a) 2nd generation Hoveyda-Grubbs catalyst, dry CH2Cl2, 100
°C, MW, 2h.
Biological activity. The activity of all the synthesized compounds 4a-o against recombinant
rhodesain has been assessed by means of a fluorimetric assay, employing Cbz-Phe-Arg-AMC as
fluorogenic substrate.32 DMSO has been used as negative control whereas E-64 (14),33 a well-known
irreversible inhibitor of cysteine proteases of the papain family, has been employed as positive
control. The preliminary screening, realized at a fixed inhibitor concentration (i.e. 50 μM), showed
that all the tested compounds inhibited the target enzyme by 85%-100%.
We then performed continuous assays, by testing the compounds at seven diverse concentrations
ranging from the one that minimally inhibits to that which totally inhibits the enzyme; the hyperbolic
progress curves obtained (see e.g. 4h, Figure 1) are indicative of an irreversible inhibition, in
agreement with literature data of related compounds containing the same warheads.32 Therefore, the
following kinetic parameters have been measured: i) the dissociation constant (Ki) of the enzyme-
inhibitor non-covalent complex [E·I] as a measure of the binding affinity; ii) the first-order rate
constant of inhibition (kinac min-1) or inactivation rate constant as a measure of the rate of formation
of the covalent adduct E-I; iii) the second-order rate constant of inhibition (k2nd M-1min-1), obtained
from the ratio kinac/Ki, which is generally considered to be the best measure of relative inhibitor
potency or effectiveness,34 together with the Ki value which is the measure of the affinity towards the
enzyme.
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F
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time (seconds)
Figure 1. Progress curves of substrate hydrolysis in the presence of the inhibitor 4h. Inhibitor
concentrations (from top to bottom): 0, 0.005, 0.010, 0.025, 0.050, 0.075, 0.1, 0.5 nM.
A survey of the data reported in Table 1 shows that the introduction of the N-methyl piperazine
(i.e. 4a) or morpholine (i.e. 4b) nuclei resulted in a strong reduction of potency (between 2-3 order
of magnitude), with respect to the lead compound 3. A slight reduction in activity was observed also
in the case of compounds 4c and 4d, which bear a 2,3-dihydrobenzo[b][1,4]dioxine and a
benzo[d][1,3]dioxole group at the P3 site, respectively. It is worth noting that when the benzo-fused
nucleus at the P3 site is directly linked to the amide portion, the inhibitory properties are enhanced.
In contrast, compound 4e, with a methylene spacer between the amide moiety and the
benzo[d][1,3]dioxole nucleus, showed a k2nd value between one and two orders of magnitude lower
with respect to compound 4d (i.e. k2nd15300 x 103M-1 min-1and 435 x 103M-1 min-1for 4d and 4e,
respectively).
A survey of the results of rhodesain inhibition of compounds 4f-m put in evidence that the
substitution pattern on the phenyl at the P3 position has a remarkable impact on the inhibition of the
target protease. If we consider the Ki values, which express the affinity of the inhibitor for the enzyme,
the 2,4-difluoro substituted derivative 4k, with its subpicomolar binding affinity (Ki = 0.6 pM) gave
the best results.
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If we consider the k2nd value, which is the measure of the potency of the inhibitor, the 4-
fluoroderivative 4h turned out to be the most potent derivative of the series with an impressive k2nd
value of 881100 x 103 M-1 min-1, with respect to those of the other monosubstituted derivatives 4f-g
and 4i-j. All in all, we can conclude that the optimal substitution pattern on the phenyl is represented
by the presence of electron withdrawing fluorine atom or CF3 group located at 2 or 4 position.
Interestingly, the structural modifications realized on the lead compound 3 allowed to improve its
potency, as shown by the vinyl ketones 4f-4h and 4j-4m. It is worth noting that the most active
compounds 4h and 4k showed improved k2nd values towards rhodesain with respect to that of the
lead compound 3, coupled with a consistent improvement also of the binding affinity (i.e. Ki values
1.1 pM (4h) and 0.6 pM (4k) vs 38 pM of compound 3).
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Concerning the P2 site, the replacement of the Phe residue with a hPhe (i.e. 4n) seemed to be
unfruitful for the enhancement of the inhibitory properties. The same trend was observed when the
methyl group of the vinyl ketone warhead, present at the P1’ site, was replaced with an ethyl group
(i.e. 4o).
To check the selectivity towards the target enzyme, all the synthesized compounds were tested
against cathepsin L, the closest human homolog to rhodesain. A survey of the results of this assay
(Table 2) evidenced that all the tested compounds are weaker inhibitors of cathepsin L than they are
of rhodesain. The best results have been obtained with derivatives 4g, 4k and 4l that showed k2nd
values against cathepsin L three or four orders of magnitude inferior than those reported for the
inhibition of the trypanosomal protease. It is worth noting that compound 4k which showed the best
affinity to rhodesain (i.e. Ki=0.0006 nM) showed also the best selectivity profile.
For compounds with the highest k2nd values, an in vitro assay against T. b. brucei cultures has been
performed (Table 3). Compounds 4d, 4h, 4i and 4m were used in concentrations from 16.7 µM to
32.5 nM in consecutive dilutions in two independent triplicates at two timepoints (24 and 48 h). EC50
values of all four compounds are in a low micromolar range from 3.2 ± 0.5 µM to 5.1 ± 1.0 µM.
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At the same time we tested also compound 4k, which showed the highest binding affinity (Ki=0.6
pM) towards rhodesain, obtaining a submicromolar EC50 value (0.67 µM) against the parasite.
Interestingly, when we tested also compound 4n, which showed a lower binding affinity (Ki=5.5 nM),
we also obtained a submicromolar activity against the parasite. This good antiparasitic activity could
be justified by a increased cell permeability of the inhibitor or with the possibility to target another
crucial cysteine protease of T. b. rhodesiense, i.e. TbCatB.
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However, the lack of a proper match between the enzyme inhibition properties and the
antitrypanosomal activity is a problem that often occurs in medicinal chemistry field, reported in
literature also in the case of the most promising rhodesain inhibitors.10 In the case of peptidyl
inhibitors, one problem could be the susceptibility to the enzymatic hydrolysis: in this context, a
higher stability can be achieved, for example, by modifying the stereochemistry of the aminoacids
(i.e. from L to D-aminoacids) or by inserting non-natural amino acids (e.g. -aminoacids, N-methyl-
aminoacids) into the peptide scaffold. Another issue could be a low cellular uptake: in this case, both
the incorporation of an inhibitor into nanoparticles or prodrug approaches, e.g. to gain affinity toward
membrane transporters, could enhance the intracellular uptake. All these opportunities will be
considered in the near future with the ultimate goal of turning a potent rhodesain inhibitor into a
potent antitrypanosomal drug.
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Table 1. Activity of compounds 4a-4o towards rhodesain
4
Ph
5
6
7
8
9
Ph
Ph
O
O
O
O
O
H
H
H
N
N
Cbz
N
Cbz
R
N
N
N
H
H
H
O
O
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Ph
Ph
Ph
4o
3, 4a-m
4n
Rhodesain
Comp
4a
R
kinac (min-1)
Ki(nM)
k2nd (x 103 M-1 min-1)
N
N
0.00042±0.00012
0.00049±0.00011
0.0043±0.0032
0.0005±0.0001
0.00085±0.00025
0.0006±0.0001
0.0032±0.0005
0.0009±0.00002
0.0008±0.0001
0.00075±0.00005
1.8±0.9
3.1±0.6
266±70
N
4b
157±7
O
O
O
4c
0.74±0.62
7200±1800
O
4d
0.034±0.011
2.10±0.98
15300±2200
435±83
O
O
4e
O
F
4f
4g
4h
4i
0.003±0.001
0.023±0.008
0.0011±0.0002
0.029±0.002
0.0044±0.0005
230300±86200
149200±27900
881100±102450
29300±6300
174700±31400
F
F
Cl
4j
F3C
F
4k
4l
0.00007±0.00003
0.0012±0.0001
0.0006±0.0003
0.0076±0.001
107100±2200
150800±6500
F
F
F
F
4m
4n
0.0016±0.0006
0.015±0.008
5.5±1.9
112600±18600
137±16
F
-
0.00072±0.00017
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4o
-
0.00082±0.000025
0.50±0.15
1800±481
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O
326
0.00255 ± 0.00075
0.038 ± 0.011
67000 ± 432
9
1426
110
0.009 ± 0.0004
-
35 ± 5
-
261 ± 27
9000
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-
Table 2. Activity of compounds 4a-o towards Cathepsin L.
Cathepsin L
Comp
kinac (min-1)
Ki (nM)
158.3±42.5
174.8±29.8
78.2±20.9
10.2±0.2
90.3±6.8
5.3±3.2
k2nd (x 103 M-1 min-1)
4a
4b
4c
4d
4e
4f
0.0047±0.0005
0.0039±0.0001
0.0076±0.0010
0.0016±0.0001
0.0053±0.0002
0.0011±0.0000
0.0019±0.0000
0.0018±0.0001
0.0018±0.0001
0.0015±0.0001
0.0015±0.0001
0.002±0.0002
0.0022±0.0001
0.0056 ±0.0002
0.0071±0.0007
31±5
23±3
102±14
157±3
59±2
313±183
41±6
4g
4h
4i
47±7
1.5±0.2
1228±136
3030 ±303
380±37
52±19
0.60±0.06
4.1±0.3
4j
4k
4l
35±14
230±65
8.9±1.6
156±34
12.0±0.8
67±6
4m
4n
4o
14.8±3.3
453.0±43.9
108.2±21.5
Table 3. Antitrypanosomal activity of selected rhodesain inhibitors
T. brucei brucei EC50 (µM)
Comp
24 h
48 h
4d
4h
4i
5.0 ± 0.9
5.1 ± 1.0
4.8 ± 0.5
0.74 ± 0.21
4.0 ± 0.8
0.55 ± 0.12
3.2 ± 2.2
3.7 ± 0.4
3.6 ± 0.2
3.2 ± 0.5
0.67 ± 0.26
3.4 ± 0.9
0.44 ± 0.15
3.0 ± 1.2
4k
4m
4n
326
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Molecular Modeling.
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To elucidate the structural basis of inhibition of the novel compounds, docking studies were
performed. For these in silico experiments, the same AutoDock4.2 (AD4) covalent docking protocol
successfully employed in our previous work was used (See Materials and Methods).26,35-37 Thus, the
compounds 4a-o were all covalently docked into the crystal structure of rhodesain (PDB code 2P86)18
through residue C25. The binding pose of the inhibitor featuring best k2nd 4h in the active site of
rhodesain is shown in Figure 2.
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Figure 2. a) Superimposition of compounds 326 (cyan sticks) and 4h (green sticks) docked
conformations in rhodesain (orange ribbons). b) Rhodesain/4h theoretical complex. The enzyme is
depicted as orange ribbons and sticks, while the ligand as green sticks. Important residues are labelled.
H-bonds are shown in blue dashed lines. The π-stacking interaction is shown in light green dashed
lines. Information on the distances of all calculated non-bonded ligand/enzyme interactions are
reported in SI (Table S1).
The achieved docked conformations for all the studied compounds are in accordance with our
previous study.26 In particular, the ligands establish a tight network of H-bonds within the active site.
The backbone atoms of G66 engage in a double bridged H-bond interaction with the ligand P2 CO
and NH atoms. The backbone NH of P1 is involved in a H-bond with the backbone CO of D161.
Furthermore, the ketone warhead accepts a H-bond from Q19 sidechain, while W184 sidechain might
form a second H-bond with the ketone warhead. Of note, the longer alkyl chain featured by compound
4o is partially solvent exposed and would point towards the hydrophilic side chain of D161, possibly
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accounting for its lower activity. Instead, the Phe P2 moiety is favorably lodged in the hydrophobic
S2 gorge lined by M68, A138, L160, and A208 residues. Analogously, AD4 calculates for 4n a
binding pose that generally resembles the one obtained for 4h, with the exception of the long hPhe
P2 residue, which is more solvent exposed and less prone to fill the S2 cavity of the enzyme, thereby
explaining the lower inhibitory activity of 4n.
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As for the P3 moieties, they generally fit in the S3 cleft lined by residues D60, F61, L67, and N70.
According to our docking study, the sole exception to this trend is represented by compound 4e,
whose bulky P3 portion was found to be lodged in the S2 region of the active site, with the Phe P2
placed in the S3 cleft. Possibly, this switch between S2 and S3 of the P2 and P3 residues could be
energetically disfavored, causing a decrease in affinity. Interestingly, in the case of the P3 aromatic
substituents of 4c-d and 4f-m, a parallel-displaced π-stacking interaction can be observed with F61.
Such an interaction should be enhanced in compounds 4f-m, featuring substituents with electron-
withdrawing groups. It is also worth noting that in compounds 4c-d and 4f-m the aromatic ring is
directly connected to an amide portion. This latter portion engages in a H-bond with G66, as we
mentioned before. Therefore, it could be postulated that, given the overall electronic conjugation,
electron-withdrawing groups at position 4 of the aromatic substituent (4h, 4j, and 4k), would enhance
the strength of the H-bond to G66.38 Additionally, the 4-fluorine atoms in 4h, 4j, and 4k are also
located in a possible H-bonding position with N70. Nevertheless, while compound 4j features an
electron-withdrawing 4-CF3, it can also be argued that its high electron density would cause in this
position an unfavorable contact with the nearby acidic D60, this should explain why 4j is a less potent
enzyme inhibitor. Moreover, in compound 4g, 3-F atom should be less efficacious in enhancing the
H-bond to G66. Thus, the 4-fluorine atom in P3 (4h and 4k) seems to be the most productive
substitution to yield high inhibitory potency at rhodesain.
The drug-likeness of compounds for which the antitrypanosomal was assessed (compounds 3, 4d,
4h, 4i, 4k, 4m, and 4n) was in silico evaluated through the QikProp tool, part of the Schrödinger suite
(Schrödinger Release 2019-1: QikProp, Schrödinger, LLC, New York, NY, 2019). The latter is useful
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to predict several physicochemical and pharmacokinetic parameters of a given compound, and the
results are also compared to the values obtained for the 95% of marketed drugs. The summary of the
calculated values are reported in Table 3.
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Table 3. Calculated Physicochemical and Pharmacokinetic Properties of Compounds 3, 4d, 4h-
k, 4m-n.
Range of
Parameter
#rotorb
3
4d
4h
4i
4k
4m
4n
recommended
valuesa
12
1
11
2
11
1
11
1
11
1
11
1
13
1
0 – 15
0 – 2
#rtvFGc
Lipinski Rule of
5 violationsd
1
1
1
1
1
1
1
N.A.
mol_MWe
484.6 498.6 472.6 489.0 490.6 490.5 498.6 130.0 – 725.0
6.7 3.6 2.4 2.3 5.2 3.1 10.7 1.0 – 12.5
893.1 806.9 833.8 849.0 835.8 837.2 917.6 300.0–1000.0
dipolef
SASAg
donorHBh
accptHBi
QPlogPo/wj
QPlogSk
1.3
6.3
6.1
-7.3
1.3
7.8
4.9
-5.5
1.3
6.3
5.8
-6.7
1.3
6.3
6.0
-7.1
1.3
6.3
5.8
-6.9
1.3
6.3
5.9
-6.9
1.3
6.3
6.6
-8.3
0.0 – 6.0
2.0 – 20.0
-2.0 – 6.5
–6.5 – 0.5
<25 poor,
>500 great
QPPCacol
522.7 795.0 739.0 738.8 568.2 578.1 1080.0
-1.5 -1.1 -1.1 -1.0 -1.1 -1.1 -1.4
413.3 585.5 1053.2 1438.4 1182.5 1482.4 598.7
QPlogBBm
QPPMDCKn
-3.0 – 1.2
<25 poor,
>500 great
Jorgensen Rule
of 3 violationso
#metabp
1
5
1
4
1
4
1
4
1
4
1
4
1
5
N.A.
1 – 8
Human Oral
Absorptionq
<25% poor,
>80% high
100
100
100
100
100
100
100
b
aFor 95% of known drugs. Number of non-trivial (not CX3), non-hindered (not alkene, amide, small ring)
rotatable bonds. cNumber of reactive functional groups. dPredicted numbers of violations of Lipinski’s rule of
five. eMolecular weight of the molecule. fComputed dipole moment of the molecule. gTotal solvent accessible
surface area (SASA) in square angstroms using a probe with a 1.4 Å radius. hEstimated number of hydrogen
i
bonds that would be donated by the solute to water molecules in an aqueous solution. Estimated number of
hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution. jPredicted
octanol/water partition coefficient. kPredicted aqueous solubility, log S. S in mol dm–3 is the concentration of
l
the solute in a saturated solution that is in equilibrium with the crystalline solid. Predicted apparent Caco-2
cell permeability in nm/sec. Caco-2 cells are a model for the gut-blood barrier. mPredicted brain/blood partition
n
coefficient for orally delivered drugs. Predicted apparent MDCK cell permeability in nm/sec. MDCK cells
are considered to be a good mimic for the blood-brain barrier. oPredicted numbers of violations of Jorgensen
rule of three. pNumber of likely metabolic reactions. qPredicted qualitative human oral absorption percentage.
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Indeed, all the compounds showed good drug-like properties with predicted 100% oral absorption,
one violation of the Lipinski rule, one violation of the Jorgensen rule, and good lipophilic features. A
general improvement with respect to the lead compound 3 was recorded for the predicted apparent
MDCK and permeability in Caco-2 cell, and for the predicted coefficient of brain/blood partition,
especially for compounds 4d, 4h, 4i, 4k, and 4m. These data represent key features for an effective
antitrypanosomal agent. Moreover, a prospective increase in the solubility is also reported for all the
new compounds with the exception of 4n. Indeed, the lipophilicity calculated for this latter compound
might be conducive of a higher membrane penetration thereby explaining why, although being a less
potent rhodesain inhibitor, this ligand has a submicromolar antitrypanosomal activity in cell-based
experiments. Thus, compound 4h and 4k should meet all the necessary requirements to be good lead
compounds for design and discovery of new antitrypanosomal agents.
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CONCLUSIONS
In conclusion, we developed a new series of peptidyl vinyl ketones that were demonstrated to be
inactivators of rhodesain at nanomolar/picomolar level, with an EC50 against cultured T. b. brucei in
a low micromolar range. The vinyl ketone 4h, with an impressive potency towards rhodesain
(k2nd=881100 x 103) coupled to a good antiparasitic activity (EC50 =3.6 µM), and the vinyl ketone 4k,
which showed the highest binding affinity (Ki=0.6 pM) and a submicromolar antiparasitic activity
(EC50= 0.67 µM), could certainly represent new lead compounds for the discovery of new drugs to
treat Human African Trypanosomiasis.
EXPERIMENTAL SECTION
Chemistry. All reagents and solvents were obtained from commercial suppliers and were
used without further purification. Reactions under microwave irradiation were performed on
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a CEM Discover apparatus. Elemental analyses were carried out on a C. Erba Model 1106
(Elemental Analyzer for C, H and N) instrument, and the obtained results are within ± 0.4%
of the theoretical values. Merck silica gel 60 F254 plates were used for analytical TLC; flash
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column chromatography was performed on Merck silica gel (200–400 mesh). H-NMR
13
spectra were recorded on a Varian 300 MHz NMR spectrometer. C-NMR were obtained
on a Varian 300 and 500 MHz NMR spectrometer, operating at frequencies of 75 or 125
MHz . The residual signal of the deuterated solvent was used as internal standard. Splitting
patterns are described as singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartet
(q), multiplet (m), or broad singlet (bs). 1H and 13C NMR chemical shifts () are expressed
in ppm and coupling constants (J) are given in Hz. Compounds 5a-m,32,39-43 6a-d and 6f-j,32,41,43-
and 1047 were synthesized according to standard procedures and spectroscopic data are in
46
agreement to those reported in literature.
4-Methyl-N-((S)-1-oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-2-
yl)piperazine-1-carboxamide (4a). A solution of terminal olefin 8a (1 equiv.) in dry CH2Cl2 (3 mL),
in a 10 mL microwave tube equipped with a magnetic stirrer, was treated with methyl vinyl ketone 9
(10 equiv.) and 2nd generation Hoveyda–Grubbs catalyst [(1,3-bis-(2,4,6-trimethylphenyl)-2-
imidazolidinylidene)-dichloro-(o-isopropoxyphenylmethylene) ruthenium] (0.1 equiv.) under
microwave irradiation at 100°C for 4h. Subsequently, the solvent was removed under reduced
pressure and the residue was purified by flash column chromatography using CH2Cl2/MeOH (9:1) as
eluent mixture, to obtain Michael acceptor 4a. Yield: 81%; Rf = 0.34 (CH2Cl2/MeOH, 9:1).
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Consistency: white solid. 1H NMR (300 MHz, CDCl3) = : 1.82-1.99 (m, 2H), 2.23 (s, 3H), 2.29 (s,
3H), 2.32-2.43 (m, 4H), 2.54 (t, J = 7.7 Hz, 2H), 3.03-3.14 (m, 2H), 3.32-3.40 (m, 4H), 4.37-4.49 (m,
1H), 4.78-4.92 (m, 1H), 5.29 (d, J = 6.3 Hz, 1H), 5.86 (d, J = 7.6 Hz, 1H), 6.28 (d, J = 15.2 Hz, 1H),
6.91 (dd, J = 15.2 Hz, 5.2, 1H), 7.16-7.42 (m, 10H). 13C NMR (125 MHz, CDCl3) = : 28.89, 31.92,
36.37, 38.64, 43.52, 45.85, 51.29, 54.33, 56.09, 125.93, 126.93, 128.35, 128.40, 128.70, 129.40,
130.14, 137.18, 137.53, 148.19, 165.17, 172.32, 197.64. Elemental analysis calcd for C28H36N4O3: C
70.56; H 7.61; N 11.76. Found: C 70.41, H 7.33, N 12.02.
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N-((S)-1-Oxo-1-((S,E)-6-oxo-1-phenylhept-4-en-3-ylamino)-3-phenylpropan-2-
yl)morpholine-4-carboxamide (4b). Synthesis of Michael acceptor 4b was carried out following the
procedure described for compound 4a, starting from terminal olefin 8b. Yield: 84%; Rf = 0.27
(EtOAc/light petroleum, 3:2). Consistency: white solid. 1H NMR (300 MHz, CDCl3) = : 1.62-1.75
(m, 2H), 2.28 (s, 3H), 2.51 (t, J = 7.6 Hz, 2H), 2.98-3.11 (m, 2H), 3.21-3.34 (m, 4H), 3.49-3.64 (m,
4H), 4.34-4.47 (m, 1H), 4.72-4.83 (m, 1H), 5.37 (d, J = 6.6 Hz, 1H), 5.88 (d, J = 7.5 Hz, 1H), 6.31
13
(d, J = 15.2 Hz, 1H), 6.88 (dd, J = 15.2 Hz, 5.4, 1H), 7.11-7.38 (m, 10H). C NMR (125 MHz,
CDCl3) = : 27.62, 32.11, 36.64, 39.14, 44.20, 51.55, 56.17, 66.50, 126.14, 127.05, 128.46, 128.51,
128.72, 129.64, 129.97, 137.82, 141.48, 145.38, 157.38, 171.59, 197.77. Elemental analysis calcd for
C27H33N3O4: C 69.95; H 7.18; N 9.06. Found: C 70.24, H 7.42, N 8.87.
N-((S)-1-Oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-2-yl)-2,3-
dihydrobenzo[b][1,4]dioxine-6-carboxamide (4c). Synthesis of Michael acceptor 4c was carried
out following the procedure described for compound 4a, starting from terminal olefin 8c, under
microwave irradiation for 2 h. Yield: 87%; Rf = 0.17 (light petroleum/EtOAc, 1:1). Consistency:
1
white solid. H NMR (300 MHz, CDCl3) = : 1.69-1.98 (m, 2H), 2.27 (s, 3H), 2.50 (t, J = 7.6 Hz,
2H), 2.96-3.17 (m, 2H), 4.22-4.46 (m, 5H), 4.59-4.71 (m, 1H), 5.39 (d, J = 6.4 Hz, 1H), 5.88 (d, J =
7.3 Hz, 1H),6.04 (d, J = 15.2 Hz, 1H), 6.67 (dd, J = 15.2, 6.0 Hz, 1H),7.06 (d, J = 1.6 Hz, 1H), 7.11-
7.39 (m, 11H), 7.63 (d, J = 1.6 Hz, 1H). 13C (75 MHz, CDCl3) = δ: 26.83, 31.93, 36.41, 38.67, 51.94,
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56.17, 64.20, 64.56, 116.52, 116.84, 120.89, 126.34, 126.74, 127.76, 128.07, 128.27, 128.69, 128.93,
130.77, 136.81, 141.35, 146.62, 147.86, 149.16, 166.90, 170.43, 197.65. Elemental analysis calcd for
C31H32N2O5: C 72.64; H 6.29; N 5.47. Found: C 72.47, H 5.96, N 5.71.
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N-((S)-1-Oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-2-
yl)benzo[d][1,3]dioxole-5-carboxamide (4d). Synthesis of Michael acceptor 4d was carried out
following the procedure described for compound 4c, starting from terminal olefin 8d. Yield: 86%; Rf
1
=0.70(light petroleum/EtOAc, 2:3). Consistency: white powder. H NMR (300 MHz, CDCl3) = δ:
1.62-1.89 (m, 2H), 2.20 (s, 3H), 2.56 (t, J = 7.6 Hz, 2H), 3.07-3.32 (m, 2H), 4.49-4.65 (m, 1H), 4.74-
4.89 (m, 1H), 5.93 (d, J = 15.8 Hz, 1H), 6.02 (s, 2H), 6.23 (d, J = 7.6 Hz, 1H), 6.46 (dd, J = 15.8, 5.6
Hz, 1H), 6.69 (d, J = 6.4 Hz, 1H), 7.05 (d, J = 7.5 Hz, 2H), 7.13-7.38 (m, 11H). 13C (125MHz, CDCl3)
= 27.70, 31.81, 35.74, 38.04, 50.03, 55.19, 101.81, 107.59, 108.10, 121.86, 127.27, 127.60, 128.30,
128.52, 128.87, 129.25, 129.28, 129.30, 129.94, 136.46, 140.51, 145.27, 148.09, 166.77, 170.45,
197.87. Elemental analysis calcd for C30H30N2O5: C 72.27; H 6.06; N 5.62. Found: C 72.38; H 6.29;
N 5.40.
(S)-2-(2-(Benzo[d][1,3]dioxol-5-yl)acetamido)-N-((S,E)-6-oxo-1-phenylhept-4-en-3-yl)-3-
phenylpropanamide (4e). Synthesis of Michael acceptor 4e was carried out following the procedure
described for compound 4c, starting from terminal olefin 8e. Yield: 81%; Rf = 0.18 (light
1
petroleum/EtOAc, 1:1). Consistency: white solid. H NMR (300 MHz, CDCl3) = : 1.66-1.92 (m,
2H), 2.24 (s, 3H), 2.54 (t, J = 7.8 Hz, 2H), 2.96-3.11 (m, 2H), 3.39 (s, 2H), 4.17-4.28 (m, 1H), 4.59-
4.67 (m, 1H), 5.55 (d, J = 6.6 Hz, 1H), 5.89 (d, J = 7.3 Hz, 1H), 5.96 (s, 2H), 6.14 (d, J = 15.1 Hz,
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1H), 6.69 (dd, J = 15.1, 6.1 Hz, 1H), 6.58-6.74 (m, 3H), 7.07-7.32 (m, 10H). C NMR (75 MHz,
CDCl3) = δ: 27.51, 32.04, 38.21, 43.16, 51.54, 54.43, 101.17, 108.42, 109.49, 122.74, 125.80, 126.19,
128.29, 128.43, 128.62, 128.84, 129.66, 130.81, 136.42, 141.53, 146.95, 148.17, 149.24, 170.29,
171.33, 197.57. Elemental analysis calcd for C31H32N2O5: C 72.64; H 6.29; N 5.47. Found: C 72.40,
H 6.55, 5.19.
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yl)benzamide (4f). Synthesis of Michael acceptor 4f was carried out following the procedure
described for compound 4c, starting from terminal olefin 8f. Yield: 60 %; Consistency: white powder;
Rf = 0.15 (cyclohexane /EtOAc, 7:3); 1H NMR (500 MHz, CDCl3) = δ: 1.73-1.92 (m, 2H), 2.21 (s,
3H), 2.51-2.60 (m, 2H), 3.16 (dd, J = 13.7, 8.0 Hz, 1H), 3.23 (dd, J = 13.8, 6.5 Hz, 1H), 4.55-4.62
(m, 1H), 4.80-4.86 (m, 1H), 5.96 (dd, J = 16.0, 1.4 Hz, 1H), 6.07 (d, J = 8.3 Hz, 1H), 6.49 (dd, J =
16.0, 5.6 Hz, 1H), 7.05 (d, J = 7.1 Hz, 2H), 7.09-7.18 (m, 2H), 7.17-7.36 (m, 9H), 7.44-7.54 (m, 1H),
8.03 (td, J = 7.8, 1.7 Hz, 1H) ppm. 13C NMR (125.73 MHz, CDCl3) = δ: 27.75. 31.85, 35.75, 37.80,
50.00, 55.47, 116.23 (d, J = 24.5 Hz), 120.23, 124.89 (d, J = 3.3 Hz), 126.19, 127.26, 128.28, 128.50,
128.86, 129.32, 129.90, 131.85 (d, J = 1.8 Hz), 133.89 (d, J = 9.4 Hz), 136.35, 140.61, 145.31, 160.72
(d, J = 248.7 Hz), 163.53, 170.10, 197.88. Elemental analysis: calcd for C29H29FN2O3: C 73.71; H
6.19; N 5.93; found: C 71.60, H 6.22, N 5.92.
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3-Fluoro-N-((S)-1-oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-2-
yl)benzamide (4g) Synthesis of Michael acceptor 4g was carried out following the procedure
described for compound 4c, starting from terminal olefin 8g. Yield: 64 %; Consistency: white
powder; Rf = 0.21 (cyclohexane /EtOAc, 7:3); 1H NMR (500 MHz, CDCl3) = δ: 1.72-1.82 (m, 2H),
2.20 (s, 3H), 2.48 (d, J = 7.8 Hz, 2H), 3.11 (dd, J = 13.5, 8.5 Hz, 1H), 3.21 (dd, J = 13.6, 6.2 Hz, 1H),
4.49-4.60 (m, 1H), 4.82-4.93 (m, 1H), 5.91 (d, J = 16.0 Hz, 1H), 6.35 (d, J = 8.3 Hz, 1H), 6.45 (dd,
J = 16.0, 5.8 Hz, 1H), 7.05 (dd, J = 15.6, 7.3 Hz, 2H), 7.10-7.31 (m, 10H), 7.33-7.41 (m, 1H), 7.42-
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7.52 (m, 2H) ppm. C NMR (125.73 MHz, CDCl3) = δ: 27.65, 31.84, 35.71, 38.36, 50.08, 55.25,
114.53 (d, J = 22.5 Hz), 119.03 (d, J = 21.2 Hz), 122.52 (d, J = 2.7 Hz), 126.22, 127.31, 128.25,
128.52, 128.84, 129.29, 130.01, 130.36 (d, J = 7.7 Hz), 135.72 (d, J = 6.9 Hz), 136.23, 140.50,
145.16, 163.73, 166.08, 170.32, 197.77 ppm. Elemental analysis: calcd for C29H29FN2O3: C 73.71; H
6.19; N 5.93; found: C 71.59, H 6.29, N 5.95.
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4-Fluoro-N-((S)-1-oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-2-
yl)benzamide (4h). Synthesis of Michael acceptor 4h was carried out following the procedure
described for compound 4c, starting from terminal olefin 8h. Yield: 84%; Rf =0.77(light
petroleum/EtOAc, 2:3). Consistency: white powder. 1H NMR (300 MHz, CDCl3) = δ: 1.62-1.91 (m,
2H), 2.19 (s, 3H), 2.52 (t, J = 7.6 Hz, 2H), 3.06-3.27 (m, 2H), 4.49-4.63 (m, 1H), 4.85-4.99 (m, 1H),
6.10 (d, J = 15.8 Hz, 1H), 6.46 (dd, J = 15.8 Hz, 5.9, 1H), 6.51-6.71 (m, 1H), 6.99-7.37 (m, 13H),
7.67-7.81 (m, 2H). 13C (125 MHz, CDCl3) = δ: 27.67, 31.84, 35.47, 38.34, 50.05, 55.21, 115.76 (d, J
= 21.9) 126.23, 127.29, 128.25, 128.52, 128.83, 129.29, 129.45 (d, J = 9.1), 129.67, 129.99, 136.30,
140.52, 145.20, 165.00 (d, J = 252.4), 166.42, 170.40, 197.57. Elemental analysis calcd for
C29H29FN2O3: C 73.71; H 6.19; N 5.93. Found: C 73.65; H 6.32; N 5.61.
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4-Chloro-N-((S)-1-oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-2-
yl)benzamide (4i). Synthesis of Michael acceptor 4i was carried out following the procedure
described for compound 4c, starting from terminal olefin 8i. Yield: 82%; Rf =0.79 (light
petroleum/EtOAc, 2:3). Consistency: white powder. 1H NMR (300 MHz, CDCl3) = δ: 1.62-1.92 (m,
2H), 2.20 (s, 3H), 2.52 (t, J = 7.6 Hz, 2H), 3.06-3.26 (m, 2H), 4.49-4.61 (m, 1H), 4.84-4.98 (m, 1H),
6.10 (d, J = 15.8 Hz, 1H), 6.45 (dd, J = 15.8 Hz, 5.4, 1H), 6.53-6.66 (m, 1H), 6.99-7.12 (m, 2H), 7.13-
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7.42 (m, 11H), 7.60-7.75 (m, 2H). C (125 MHz, CDCl3) = δ: 27.68, 31.84, 35.72, 38.35, 50.07,
55.23, 126.24, 127.31, 128.25, 128.50, 128.53, 128.84, 128.94, 129.29, 130.01, 131.85, 136.23,
138.35, 140.49, 145.15, 166.37, 170.34, 197.77. Elemental analysis calcd for C29H29ClN2O3: C 71.23;
H 5.98; N 5.73. Found: C 71.65; H 5.82; N 5.60.
N-((S)-1-Oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-2-yl)-4-
trifluoromethylbenzamide (4j). Synthesis of Michael acceptor 4j was carried out following the
procedure described for compound 4c, starting from terminal olefin 8j. Yield: 78 %; Consistency:
white powder; Rf = 0.22 (cyclohexane/EtOAc, 7:3); 1H NMR (500 MHz, CDCl3) = δ: 1.76-1.93 (m,
2H), 2.21 (s, 3H), 2.50 (d, J = 7.8 Hz, 2H), 3.11 (dd, J = 13.4, 8.4 Hz, 1H), 3.23 (dd, J = 13.5, 6.3 Hz,
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1H), 4.51-4.62 (m, 1H), 4.79-4.90 (m, 1H), 5.92 (d, J = 16.0 Hz, 1H), 6.24 (d, J = 8.1 Hz, 1H), 6.45
(dd, J = 16.0, 5.8 Hz, 1H), 7.01-7.10 (m, 2H), 7.11-7.33 (m, 9H), 7.67 (d, J = 7.9 Hz, 2H), 7.83 (d, J
= 7.7 Hz, 2H) ppm. 13C NMR (125.73 MHz, CDCl3) = δ: 27.70, 31.83, 35.70, 38.38, 50.14, 55.31,
123.57 (q, J = 272.7 Hz), 125.66 (q, J = 3.7 Hz), 126.27, 127.39, 127.55, 128.54, 128.89, 129.07,
128.89, 129.27, 130.06, 133.46 (q, J = 32.6 Hz), 136.13, 140.43, 145.24, 166.05, 170.14, 197.78.
Elemental analysis: calcd for C30H29F3N2O3: C 68.95; H 5.59; N 5.36; found: C 68.99, H 5.46, N
5.52.
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2,4-Difluoro-N-((S)-1-oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-
2-yl)benzamide (4k). Synthesis of Michael acceptor 4k was carried out following the procedure
described for compound 4c, starting from terminal olefin 8k. Yield:65 %; Consistency: white powder;
Rf = 0.40 (cyclohexane /EtOAc, 6:4); 1H NMR (300 MHz, CDCl3) = δ: 1.71-1.92 (m, 2H), 2.21 (s,
3H), 2.50 (d, J = 7.8 Hz, 2H), 3.13 (dd, J = 13.5, 8.2 Hz, 1H), 3.22 (dd, J = 13.6, 6.2 Hz, 1H), 4.52-
4.64 (m, 1H), 4.78-4.91 (m, 1H), 5.95 (d, J = 15.9 Hz, 1H), 6.09-6.17 (m, 1H), 6.42-6.52 (m, 1H),
6.82-6.93 (m, 1H), 6.94-7.01 (m, 1H), 7.02-7.11 (m, 2H), 7.12-7.35 (m, 8H), 8.03 (dd, J = 8.4, 6.5
Hz, 1H) ppm. 13C NMR (125.73 MHz, CDCl3) = δ: 27.71, 31.84, 35.74, 38.10, 50.03, 55.50, 112.38,
104.50 (dd, J = 28.2, 25.7 Hz), 112.48 (dd, J = 20.9, 3.1 Hz), 126.21, 127.29, 128.25, 128.51, 128.84,
129.30, 129.96, 136.23, 137.8, 140.57, 145.25, 162.53, 166.16, 166.93, 170.06, 197.80 ppm.
Elemental analysis: calcd for C29H28F2N2O3: C 71.01; H 5.75; N 5.71; found: C 71.21, H 5.54, N
5.50.
2,6-Difluoro-N-((S)-1-oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-
2-yl)benzamide (4l). Synthesis of Michael acceptor 4l was carried out following the procedure
described for compound 4c, starting from terminal olefin 8l. Yield: 66%; Consistency: white powder;
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Rf = 0.39 (cyclohexane /EtOAc, 6:4); H NMR (500 MHz, CDCl3) = δ:1.73-1.90 (m, 2H), 2.18 (s,
3H), 2.52-2.64 (m, 2H), 3.15 (dd, J = 13.7, 8.0 Hz, 1H), 3.23 (dd, J = 13.7, 6.4 Hz, 1H), 4.53-4.63
(m, 1H), 4.93 (dd, J = 14.4, 7.8 Hz, 1H), 5.91 (dd, J = 16.0, 1.4 Hz, 1H), 6.27 (d, J = 8.4 Hz, 1H),
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6.46 (dd, J = 16.0, 5.6 Hz, 1H), 6.68 (d, J = 7.8 Hz, 1H), 6.88-6.97 (m, 2H), 7.07 (d, J = 8.0 Hz, 2H),
7.13 (dd, J = 10.6, 4.1 Hz, 1H), 7.18-7.32 (m, 7H), 7.32-7.42 (m, 1H) ppm. 13C NMR (125.73 MHz,
CDCl3) = δ: 27.73, 31.82, 35.61, 38.03, 49.99, 55.14, 112.09 (dd, J = 21.1, 4.4 Hz), 113.36 (d, J =
19.5 Hz), 126.11, 127.24, 128.33, 128.48, 128.77, 129.35, 129.79, 132.18 (t, J = 10.4 Hz), 136.06,
140.72, 145.24, 159.93 (dd, J = 253.1, 6.5 Hz), 160.42, 169.67, 197.84 ppm. Elemental analysis:
calcd for C29H28F2N2O3: C 71.01; H 5.75; N 5.71; found: C 71.25, H 5.56, N 5.52.
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3,5-Difluoro-N-((S)-1-oxo-1-(((S,E)-6-oxo-1-phenylhept-4-en-3-yl)amino)-3-phenylpropan-
2-yl)benzamide (4m). Synthesis of Michael acceptor 4m was carried out following the procedure
described for compound 4c, starting from terminal olefin 8m. Yield: 83%; Rf = 0.27 (light
petroleum/EtOAc, 4:1). Consistency:white solid. 1H NMR (300 MHz, CDCl3) = : 1.86-1.97 (m, 2H),
2.23 (s, 3H), 2.55 (t, J = 7.8 Hz, 2H), 3.01-3.22 (m, 2H), 4.16-4.29 (m, 1H), 4.51-4.60 (m, 1H), 5.36
(bs, 1H), 5.94 (d, J = 8.3 Hz, 1H), 6.01 (d, J = 15.2 Hz, 1H), 6.54 (dd, J = 15.2, 6.1 Hz, 1H), 7.02-
7.11, (m, 1H), 7.16-7.32 (m, 8H), 7.44-7.57 (m, 4H). 13C (125 MHz, CDCl3) = δ: 26.54, 32.14, 36.86,
38.41, 51.18, 56.67, 107.22 (t, J = 25.9 Hz), 110.59 (dd, J = 28.0, 3.7 Hz), 126.24, 126.97, 128.54,
128.79, 129.04, 129.51, 130.83, 136.78, 137.12, 141.29, 149.39, 163.51 (dd, J = 251.2, 9.8 Hz),
164.94, 170.22, 197.58. Elemental analysis calcd for C29H28F2N2O3: C 71.01; H 5.75; N 5.71. Found:
C 70.86, H 5.97, N 5.93.
Benzyl ((S)-1-oxo-1-((S,E)-6-oxo-1-phenylhept-4-en-3-ylamino)-4-phenylbutan-2-yl)carbamate
(4n). Synthesis of Michael acceptor 4n was carried out following the procedure described for
compound 4c, starting from terminal olefin 11. Yield:86%; Rf = 0.26 (light petroleum/EtOAc, 7:3).
Consistency:white powder. 1H NMR (300 MHz, CDCl3) = : 1.71-2.01 (m, 4H), 2.21 (s, 3H), 2.53-
2.72 (m, 4H), 4.02-4.20 (m, 1H), 4.53-4.70 (m, 1H), 5.10 (s, 2H), 5.26 (d, J = 7.4 Hz, 1H), 6.12 (d, J
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= 15.6 Hz, 1H), 6.30 (d, J = 6.9 Hz, 1H), 6.63 (dd, J = 15.6, 4.6 Hz, 1H), 7.02-7.42 (m, 15H). C
NMR (75 MHz, CDCl3) = : 27.56, 31.71, 31.97, 32.48, 35.67, 49.91, 54.45, 67.23, 126.23, 126.25,
128.04, 128.31, 128.34, 128.44, 128.48, 128.56, 128.59, 129.88, 135.91, 140.40, 140.65, 145.74,
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156.39, 171.02, 197.92; Elemental analysis calcd for C31H34N2O4: C 74.67; H 6.87; N 5.62. Found:
C 74.91, H 7. 04, N 5.47.
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((S)-1-oxo-1-(((S,E)-6-oxo-1-phenyloct-4-en-3-yl)amino)-3-phenylpropan-2-
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yl)carbamate (4o). Synthesis of Michael acceptor 4o was carried out following the procedure
described for compound 4c starting from terminal olefin 12,22 which was treated with CM partner 13,
commercially available. Yield:86%; Rf = 0.28 (light petroleum/EtOAc, 7:3). Consistency: white solid.
1H NMR (300 MHz, CDCl3) = : 1.10 (t, J = 7.2 Hz, 3H), 1.67-1.96 (m, 2H), 2.33-2.64 (m, 4H),
2.99-3.15 (m, 2H), 4.32-4.48 (m, 1H), 4.50-4.67 (m, 1H), 5.10 (s, 2H), 5.38 (d, J = 5.4 Hz, 1H), 5.88-
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NMR (75 MHz, CDCl3) = : 27.94, 32.04, 34.85, 35.87, 38.11, 51.04, 56.63, 67.21, 126.33, 127.22,
128.04, 128.38, 128.41, 128.63, 128.67, 128.86, 129.32, 130.04, 135.95, 136.21, 141.01, 146.13,
156.14, 170.47, 199.73. Elemental analysis calcd for C31H34N2O4: C 74.67, H 6.87, N 5.62. Found C
74.89, H 6.63, N 5.86.
(S)-2-(2-(Benzo[d][1,3]dioxol-5-yl)acetamido)-3-phenylpropanoic acid (6e). To a solution of
5e (1 equiv.) in a mixture of methanol/water/dioxane (1:1:1), LiOH as powder (3 equiv.) was added
at 0°C. The reaction mixture was then stirred at rt for 12h. After this time, the solvents were
evaporated in vacuo. The residue was treated with 10% solution citric acid (x 2), and the organic
phase was extracted with EtOAc (x 3), dried over Na2SO4 and concentrated to afford the pure
carboxylic acid 6e. Yield: 94%; Rf = 0.04 (light petroleum/EtOAc, 1:1). Consistency: white solid. 1H
NMR (300 MHz, CD3OD) = : 2.86 (dd, J = 13.8, 5.9 Hz, 1H), 3.17 (dd, J = 13.8, 5.9 Hz, 1H), 3.39
(s, 2H), 4.66 (dd, J = 13.8, 5.9 Hz, 1H), 5.91 (s, 2H), 6.58-6.74 (m, 3H), 7.08-7.24 (m, 5H).
(S)-2-(2,4-Difluorobenzamido)-3-phenylpropanoic acid (6k). Synthesis of acid 6k was carried
out following the procedure described for compound 6e starting from ester 5k. Yield: 98 %;
Consistency: white solid; 1H NMR (500 MHz, CDCl3) = : 3.23 (dd, J = 14.0, 6.3 Hz, 1H), 3.34 (dd,
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J = 14.0, 5.4 Hz, 1H), 5.06 – 5.11 (m, 1H), 6.87 – 6.79 (m, 1H), 6.97 (dt, J = 16.5, 4.8 Hz, 1H), 7.10
(dd, J = 12.2, 7.2 Hz, 1H), 7.18 – 7.34 (m, 5H), 8.04 – 8.11 (m, 1H).
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out following the procedure described for compound 6e starting from ester 5l. Yield: 99 %;
Consistency: white solid; 1H NMR (500 MHz, CDCl3) =  : 3.26 (dd, J = 14.1, 5.4 Hz, 1H), 3.38 (dd,
J = 14.3, 5.7 Hz, 1H), 5.08 – 5.21 (m, 1H), 6.49 (bd, J = 6.7 Hz, 1H), 6.93 (t, J = 8.2 Hz, 2H), 7.19 –
7.42 (m, 6H).
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(S)-2-(3,5-Difluorobenzamido)-3-phenylpropanoic acid (6m). Synthesis of acid 6m was carried
out following the procedure described for compound 6e starting from ester 5m. Yield: 96%; Rf = 0.02
(light petroleum/EtOAc, 9:1). Consistency: white solid. 1H NMR (300 MHz, CD3OD) = : 3.07 (dd,
J = 13.9, 10.1 Hz, 1H), 3.35 (dd, J = 13.9, 4.9 Hz, 1H), 4.84 (dd, J = 10.1, 4.9 Hz, 1H), 7.06-7.37 (m,
8H).
4-Methyl-N-((S)-1-oxo-3-phenyl-1-((S)-5-phenylpent-1-en-3-ylamino)propan-2-
yl)piperazine-1-carboxamide (8a). To a solution of carboxylic acid 6a (1.2 equiv.) in DMF/CH2Cl2
(1:1) in nitrogen atmosphere, HOBt (1.2 equiv.) and EDCI (1.2 equiv.) were added at 0°C. After this
time, DIPEA (1.5 equiv.) and amine 730 (1 equiv.) were added and the reaction mixture was stirred
over night at rt. Then, solvents were evaporated and the resulting residue was diluted with EtOAc,
washed with brine (x 5), dried over Na2SO4, filtered and concentrated in vacuo. The crude product
was purified by flash column chromatography using CH2Cl2/MeOH (9:1) as eluent mixture, to give
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the terminal olefin 8a. Yield: 74%; Rf = 0.49 (CH2Cl2/MeOH, 9:1). Consistency: white solid. H
NMR (300 MHz, CDCl3) = : 1.58-1.80 (m, 2H), 2.23 (s, 3H), 2.24-2.30 (m, 4H), 2.49 (t, J = 7.7 Hz,
2H),3.02-3.10 (m, 2H), 3.28-3.36 (m, 4H), 4.28-4.42 (m, 1H), 4.70 (q, J = 7.3 Hz, 1H). 4.87-5.08 (m,
2H), 5.51-5.65 (m, 2H), 6.99-7.29 (m, 11H).
N-((S)-1-Oxo-3-phenyl-1-(((S)-5-phenylpent-1-en-3-yl)amino)propan-2-yl)morpholine-4-
carboxamide (8b). Synthesis of terminal olefin 8b was carried out following the procedure described
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for compound 8a. Yield: 71%; Rf = 0.20 (light petroleum/EtOAc, 1:1). Consistency: white solid. 1H
NMR (300 MHz, CDCl3) = : 1.58-1.71 (m, 2H), 2.46 (t, J = 7.8 Hz, 2H), 2.94-3.04 (m, 2H), 3.15-
3.26 (m, 4H), 3.46-3.55 (m, 4H), 4.23-4.36 (m, 1H), 4.53 (q, J = 7.1 Hz, 1H), 4.80-4.99 (m, 2H), 5.31
(d, J = 6.2 Hz, 1H), 5.43-5.58 (m, 1H), 6.31 (d, J = 7.4 Hz, 1H), 6.97-7.26 (m, 10H).
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N-((S)-1-Oxo-3-phenyl-1-((S)-5-phenylpent-1-en-3-ylamino)propan-2-yl)-2,3-
dihydrobenzo[b][1,4]dioxine-6-carboxamide (8c). Synthesis of terminal olefin 8c was carried out
following the procedure described for compound 8a, using 6c as acid. Yield: 80%; Rf = 0.31 (light
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petroleum/EtOAc, 1:1). Consistency: white solid. H NMR (300 MHz, CDCl3) = : 1.68-1.94 (m,
2H), 2.52 (t, J = 7.6 Hz, 2H), 3.00-3.19 (m, 2H), 4.18-4.42 (m, 5H), 4.67-4.81 (m, 1H), 4.88-5.09 (m,
2H), 5.27 (d, J = 6.7 Hz, 1H), 5.58-5.70 (m, 1H), 5.88 (d, J = 7.3 Hz, 1H), 7.03 (d, J = 1.5 Hz, 1H),
7.12-7.22 (m, 7H), 7.25-7.34 (m, 4H), 7.63 (d, J = 1.5 Hz, 1H).
N-((S)-1-Oxo-3-phenyl-1-(((S)-5-phenylpent-1-en-3-yl)amino)propan-2-
yl)benzo[d][1,3]dioxole-5-carboxamide (8d). Synthesis of terminal olefin 8d was carried out
following the procedure described for compound 8a, using 6d as acid. Yield: 70%; Rf= 0.49
(cyclohexane/EtOAc, 7:3). Consistency: white solid. 1H NMR (300 MHz, CDCl3) =δ: 1.55-1.83 (m,
2H), 2.48 (t, J = 8.2 Hz, 2H), 3.02-3.30 (m, 2H), 4.33-4.47 (m, 1H), 4.79-4.89 (m, 1H), 4.90-5.15 (m,
2H), 5.54-5.74 (m, 1H), 6.01 (s, 2H), 6.08-6.23 (m, 1H), 6.79 (d, J = 7.6 Hz, 1H), 6.88-7.02 (m, 1H),
7.04- 7.32 (m, 12 H).
(S)-2-(2-(Benzo[d][1,3]dioxol-5-yl)acetamido)-3-phenyl-N-((S)-5-phenylpent-1-en-3-
yl)propanamide (8e). Synthesis of terminal olefin 8e was carried out following the procedure
described for compound 8a, using 6e as acid. Yield: 83%; Rf = 0.33 (light petroleum/EtOAc, 1:1).
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Consistency: white solid. H NMR (300 MHz, CDCl3) = : 1.59-1.81 (m, 2H), 2.51 (t, J = 7.6 Hz,
2H), 3.01-3.16 (m, 2H), 3.41 (s, 2H), 4.20-4.29 (m, 1H), 4.64-4.76 (m, 1H), 4.90-5.09 (m, 2H),5.44
(d, J = 6.8 Hz, 1H), 5.55-5.71 (m, 1H), 5.80 (d, J = 7.4 Hz, 1H),5.94 (s, 2H), 6.58-6.74 (m, 3H), 7.07-
7.32 (m, 10H).
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2-Fluoro-N-((S)-1-oxo-3-phenyl-1-(((S)-5-phenylpent-1-en-3-yl)amino)propan-2-
yl)benzamide (8f). Synthesis of terminal olefin 8f was carried out following the procedure described
for compound 8a, using 6f as acid. Yield: 36%; Consistency: white crystals; Rf = 0.31 (light petroleum
/EtOAc, 8:2); 1H NMR (500 MHz, CDCl3) = δ:1.67-1.83 (m, 2H), 2.54 (t, J = 7.8 Hz, 2H), 3.13 (dd,
J = 13.7, 8.0 Hz, 1H), 3.25 (dd, J = 13.7, 6.2 Hz, 1H), 4.40 – 4.50 (m, 1H), 4.81 (ddd, J = 9.4, 8.0,
1.9 Hz, 1H), 4.93 – 5.08 (m, 2H), 5.61 (ddd, J = 17.1, 10.5, 5.8 Hz, 1H), 5.74 (d, J = 8.4 Hz, 1H),
7.06– 7.17 (m, 4H), 7.18 – 7.34 (m, 9H), 7.44-7.51 (m, 1H), 8.03 (td, J = 7.8, 1.9 Hz, 1H).
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3-Fluoro-N-((S)-1-oxo-3-phenyl-1-(((S)-5-phenylpent-1-en-3-yl)amino)propan-2-yl)benzamide
(8g). Synthesis of terminal olefin 8g was carried out following the procedure described for compound
8a, using 6g as acid. Yield: 70%; Consistency: white crystals; Rf = 0.27 (light petroleum /EtOAc,
8:2); 1H NMR (500 MHz, CDCl3) = δ: 1.65-1.84 (m, 2H), 2.49 (t, J = 7.8 Hz, 2H), 3.09 (dd, J = 13.6,
8.7, 1H), 3.22 (dd, J = 13.7, 5.8 Hz, 1H), 4.34 – 4.48 (m, 1H), 4.73-4.85 (m, 1H), 4.94 (d, J = 17.2
Hz, 1H), 5.08 (m, 1H), 5.58 (ddd, J = 17.1, 10.5, 5.8 Hz, 1H), 5.72 (d, J = 8.3 Hz, 1H), 6.94 (d, J =
7.4 Hz, 1H), 7.02– 7.17 (m, 3H), 7.18 – 7.33 (m, 8H), 7.35-7.42 (m, 1H), 7.45-7.54 (m, 2H).
4-Fluoro-N-((S)-1-oxo-3-phenyl-1-(((S)-5-phenylpent-1-en-3-yl)amino)propan-2-
yl)benzamide (8h). Synthesis of terminal olefin 8h was carried out following the procedure described
for compound 8a, using 6h as acid. Yield: 65%; Rf = 0.62 (cyclohexane/EtOAc, 3:2). Consistency:
white solid. 1H NMR (300 MHz, CDCl3) = δ: 1.57-1.89 (m, 3H), 2.49 (t, J = 7.9 Hz, 2H), 3.03-3.28
(m 2H), 4.34-4.48 (m, 1H), 4.84-5.22 (m, 3H), 5.52-5.78 (m, 1H), 6.97-7.35 (m, 13 H), 7.75 (dd, J =
8.6, 5.3 Hz, 2H).
4-Chloro-N-((S)-1-oxo-3-phenyl-1-(((S)-5-phenylpent-1-en-3-yl)amino)propan-2-
yl)benzamide (8i). Synthesis of terminal olefin 8i was carried out following the procedure described
for compound 8a, using 6i as acid. Yield:68%; Rf = 0.42 (light petroleum /EtOAc, 3:2). Consistency:
white solid. 1H NMR (300 MHz, CDCl3) = δ: 1.64-1.86 (m, 3H), 2.50 (t, J = 7.9 Hz, 2H), 3.03-3.15
(m 1H), 3.17-3.29 (m, 1H), 4.33-4.48 (m, 1H), 4.71-4.87 (m, 1H), 4.89-5.16 (m, 1H), 5.51-5.88 (m,
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2H), 6.93 (d, J = 7.0 Hz, 1H), 7.04-7.13 (m, 2H), 7.18-7.34 (m, 8H), 7.36-7.43 (m, 2H), 7.65-7.73
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N-((S)-1-Oxo-3-phenyl-1-(((S)-5-phenylpent-1-en-3-yl)amino)propan-2-yl)-4-
trifluoromethylbenzamide (8j). Synthesis of terminal olefin 8j was carried out following the
procedure described for compound 8a, using 6j as acid. Yield: 55%; Consistency: white solid; Rf =
0.55 (light petroleum /EtOAc, 8:2); 1H NMR (500 MHz, CDCl3) = δ: 1.66-1.85 (m, 2H), 2.50 (t, J =
8.1 Hz, 2H), 3.09 (dd, J = 13.8, 8.7 Hz, 1H), 3.24 (dd, J = 13.6, 5.8 Hz, 1H), 4.35–4.46 (m, 1H), 4.72-
4.89 (m, 1H), 4.89-5.15 (m, 2H), 5.58 (ddd, J = 17.1, 10.5, 5.8 Hz, 1H), 5.64-5.86 (m, 2H), 7.04-7.34
(m, 11 H), 7.67 (d, J = 8.1 Hz, 2H), 7.85 (d, J = 8.1 Hz, 2H).
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2,4-Difluoro-N-((S)-1-oxo-3-phenyl-1-((S)-5-phenylpent-1-en-3-ylamino)propan-2-
yl)benzamide (8k). Synthesis of terminal olefin 8k was carried out following the procedure described
for compound 8a, using 6k as acid. Yield: 72%; Consistency: white powder; Rf = 0.28
(cyclohexane/EtOAc, 8:2); 1H NMR (500 MHz, CDCl3) = δ: 1.65-1.83 (m, 2H), 2.49 (t, J = 8.1 Hz,
2H), 3.11 (dd, J = 13.6, 8.2, 1H), 3.24 (dd, J = 13.7, 6.0 Hz, 1H), 4.43 (ddd, J = 13.7, 7.6, 6.2 Hz,
1H), 4.74-4.89 (m, 1H), 4.93-5.13 (m, 2H), 5.59 (ddd, J = 17.1, 10.4, 5.9 Hz, 1H), 5.72-5.90 (m, 1H),
6.84-6.90 (m, 1H), 6.94-6.99 (m, 1H), 7.05-7.17 (m, 3H), 7.18-7.33 (m, 8H), 8.00-8.09 (m, 1H).
2,6-Difluoro-N-((S)-1-oxo-3-phenyl-1-((S)-5-phenylpent-1-en-3-ylamino)propan-2-
yl)benzamide (8l). Synthesis of terminal olefin 8l was carried out following the procedure described
for compound 8a, using 6l as acid. Yield: 78%; Consistency: white powder; Rf = 0.57
(cyclohexane/EtOAc, 7:3); 1H NMR (500 MHz, CDCl3) = δ: 1.68-1.88 (m, 2H), 2.49 (t, J = 7.8 Hz,
2H), 3.12 (dd, J = 13.7, 8.1, 1H), 3.18 (dd, J = 13.7, 8.1 Hz, 1H), 4.35-4.48 (m, 1H), 4.85 (dd, J =
14.0, 7.9 Hz, 1H), 4.89-5.13 (m, 2H), 5.59 (ddd, J = 17.1, 10.7, 6.0 Hz, 1H), 5.80 (d, J = 8.5 Hz, 1H),
6.66 (d, J = 7.6 Hz, 1H), 6.93 (t, J = 8.1 Hz, 2H), 7.06-7.42 (m, 11H).
3,5-Difluoro-N-((S)-1-oxo-3-phenyl-1-((S)-5-phenylpent-1-en-3-ylamino)propan-2-
yl)benzamide (8m). Synthesis of terminal olefin 8m was carried out following the procedure
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described for compound 8a, using 6m as acid. Yield: 74%; Rf = 0.43 (light petroleum/EtOAc, 4:1).
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Consistency: white solid. H NMR (300 MHz, CDCl3) = : 1.88-1.98 (m, 2H); 2.58 (t, J = 7.6 Hz,
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2H), 2.99-3.18 (m, 2H), 4.18-4.27 (m, 1H), 4.60-4.72 (m, 1H), 4.96-5.12 (m, 2H), 5.42 (d, J = 6.7
Hz, 1H), 5.57-5.70 (m, 1H), 5.86 (d, J = 8.3 Hz, 1H),6.98-7.09, (m, 1H), 7.18-7.34 (m, 8H), 7.40-
7.51 (m, 4H).
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Benzyl ((S)-1-oxo-4-phenyl-1-((S)-5-phenylpent-1-en-3-ylamino)butan-2-yl)carbamate (11).
Synthesis of terminal olefin 11 was carried out following the procedure described for compound 8a,
using 1047 as acid. Yield: 79%; Rf = 0.41 (light petroleum/EtOAc, 9:1). Consistency: white powder.
1H NMR (300 MHz, CDCl3) = : 1.70-2.00 (m, 3H), 2.08-2.24 (m, 1H), 2.54-2.72 (m, 4H), 4.12 (dd,
J = 14.3, 7.5 Hz, 1H), 4.42-4.56 (m, 1H), 5.03-5.22 (m, 2H), 5.09 (s, 2H), 5.32 (d, J = 7.6 Hz, 1H),
5.68-5.84 (m, 1H), 5.99 (d, J = 8.6 Hz, 1H), 7.09-7.37 (m, 15H).
Enzyme assays against rhodesain. Preliminary screening with rhodesain was performed with 50
µM inhibitor concentrations using an equivalent amount of DMSO as negative control. The enzyme
was recombinantly expressed as previously described.16,32,48-49 Product release from substrate
hydrolysis (Cbz-Phe-Arg-AMC, 10 µM) was determined continuously over a period of 30 min.
Compounds showing at least 85% inhibition at 50 µM were subjected to detailed assays. Second-
order rate constants of inhibition were determined with 7 different inhibitor concentrations ranging
from those that minimally inhibited to those that fully inhibited the enzyme. The assay buffer
contains: 50 mM sodium acetate, pH = 5.5, 5 mM EDTA, 200 mM NaCl and 0.005 % Brij 35 to avoid
aggregation and wrong-positive results. Enzyme buffer contains 5 mM DTT rather than Brij 35.
Product formation was monitored continuously for 30 min at room temperature. To determine the
first-order inactivation rate constants (kobs) for the irreversible inhibition, progress curves
(fluorescence (F) versus time) were analyzed by non-linear regression analysis using the equation F
= A (1-exp (-kobst)) +B.50 Fitting of the kobs values against the inhibitor concentrations to the
hyperbolic equation kobs = kinac [I]/(Kiapp+[I]) gave the individual values of Kiapp and kinac.50 The Kiapp
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