Synthesis and biological evaluation of papain-family cathepsin l-like cysteine protease inhibitors containing a 1,4-benzodiazepine scaffold as antiprotozoal agents

Article abstract of DOI:10.1002/cmdc.201402079

Novel papain-family cathepsin L-like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure-activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3-bromoisoxazoline moiety. Several rhodesain and falcipain-2 inhibitors displaying single-digit micromolar or sub-micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds.

Full text of DOI:10.1002/cmdc.201402079

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DOI: 10.1002/cmdc.201402079
Synthesis and Biological Evaluation of Papain-Family
Cathepsin L-Like Cysteine Protease Inhibitors Containing
a 1,4-Benzodiazepine Scaffold as Antiprotozoal Agents
Roberta Ettari,*[a] Andrea Pinto,[a] Lucia Tamborini,[a] Ilenia C. Angelo,[b] Silvana Grasso,[c]
Maria Zappalꢀ,[b] Natale Capodicasa,[d] Laura Yzeiraj,[d] Esther Gruber,[e] Makoah N. Aminake,[e]
Gabriele Pradel,[e] Tanja Schirmeister,[f] Carlo De Micheli,[a] and Paola Conti[a]
Novel papain-family cathepsin L-like cysteine protease inhibi-
tors endowed with antitrypanosomal and antimalarial activity
were developed, through an optimization study of previously
developed inhibitors. In the present work, we studied the
structure–activity relationships of these derivatives, with the
aim to develop new analogues with a simplified and more syn-
thetically accessible structure and with improved antiparasitic
activity. The structure of the model compounds was signifi-
cantly simplified by modifying or even eliminating the side
chain appended at the C3 atom of the benzodiazepine scaf-
fold. In addition, a simple methylene spacer of appropriate
length was inserted between the benzodiazepine ring and the
3-bromoisoxazoline moiety. Several rhodesain and falcipain-2
inhibitors displaying single-digit micromolar or sub-micromolar
antiparasitic activity against one or both parasites were identi-
fied, with activities that were one order of magnitude more
potent than the model compounds.
Introduction
Neglected tropical diseases (NTDs) are a group of chronic disa-
bling infections that are especially endemic in low-income
populations in developing regions of Africa, Asia, and the
Americas. Over one billion people suffer from one or more
NTDs. Two of the most important diseases are malaria and
sleeping sickness.[1] Malaria is currently the most widespread
and severe tropical disease; in humans, it is caused by several
species of the Plasmodium genus, with P. falciparum being the
most dangerous species and responsible, together with
P. vivax, for more than 95% of cases of malaria.[2] Human Afri-
can trypanosomiasis (HAT), also known as “sleeping sickness”,
is caused by parasites of the Trypanosoma genus, transmitted
by the bite of a fly of the Glossina genus. The subspecies of
Trypanosoma responsible for the transmission of the disease to
humans are T. brucei gambiense and T. b. rhodesiense, which are
widespread in West and East Africa, respectively.[3] Whereas the
first kind of parasite causes a chronic form of the disease, the
second one is responsible for the acute form, which is charac-
terized by an immediate onset, progression of the disease in
a few weeks, and mortality within a short time. Currently avail-
able therapeutic regimens suffer from many drawbacks, includ-
ing cost, poor efficacy, toxicity, route of administration, and in-
creasing threat of resistance.[4–6]
Taking into consideration all of these reasons, there is an
urgent need to develop new chemotherapeutic agents to treat
malaria and/or HAT and, in particular, to identify new targets
for drug design. To address this need, we focused our atten-
tion on the cathepsin L-like cysteine proteases, notably falci-
pain-2 (FP-2) and rhodesain, which have been recognized as
novel promising targets for the treatment of protozoal infec-
tions[7,8] because these enzymes are essential for parasite sur-
vival.[9] Falcipain-2, isolated from P. falciparum, is involved in
hemoglobin degradation to provide amino acids that are es-
sential to the parasite for protein synthesis; moreover, it is re-
sponsible for the cleavage of both ankyrin and band-4.1 pro-
tein, cytoskeletal elements that are vital to the stability of the
red-cell membrane.[10] Rhodesain is a key enzyme of T. b. rhode-
siense, and it is involved in parasite invasivity by allowing the
parasite to cross the blood–brain barrier (BBB) of the human
host and cause the late and lethal stage of the disease.[11]
Moreover, it plays an important role in immunoevasion, being
involved in the turnover of variant surface glycoproteins of the
[a] Dr. R. Ettari, Dr. A. Pinto, Dr. L. Tamborini, Prof. C. De Micheli, Prof. P. Conti
Dipartimento di Scienze Farmaceutiche
Universitꢀ degli Studi di Milano
Via Mangiagalli, 25, 20133 Milano (Italy)
E-mail: roberta.ettari@unimi.it
[b] Dr. I. C. Angelo, Prof. M. Zappalꢀ
Dipartimento di Scienze del Farmaco e Prodotti per la Salute
University of Messina, Viale Annunziata, 98168 Messina (Italy)
[c] Prof. S. Grasso
Dipartimento di Scienze Chimiche, Universitꢀ degli Studi di Messina
Via F. Stagno D’Alcontres 31, S. Agata, 98166 Messina (Italy)
[d] Dr. N. Capodicasa, Dr. L. Yzeiraj
Universiteti “Zoja e Kꢁshillit tꢁ Mirꢁ”
Kompleksi Spitalor Universitar, Rruga Dritan Hoxha, Tirana (Albania)
[e] Dr. E. Gruber, Dr. M. N. Aminake, Dr. G. Pradel
Institute of Molecular Biotechnology
RWTH Aachen Unversity, Worringerweg 1, 52074 Aachen (Germany)
[f] Prof. T. Schirmeister
Institute of Pharmacy and Biochemistry
University of Mainz, Staudinger Weg 5, 55099 Mainz (Germany)
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T. brucei coat, which prevents
a specific response by the host
cells, and in the degradation of
the immunoglobulins.[12] Both
proteases are characterized by
a left (L) and a right (R) domain;
the catalytic triad (Cys/His/Asn)
is located in a cleft between the
two domains. The general struc-
ture of FP-2 and rhodesain inhib-
itors reported in the literature is
characterized by the presence of
two moieties: 1) a moiety that is
able to fit the enzyme active site
and 2) a second one, termed the
“warhead”, that is provided with
electrophilic properties suitable
to react with a cysteine residue
present in the catalytic site.
In this context, our research
group has been actively in-
volved, in the recent past, in the
Figure 1. Model and target compounds.
development of nonpeptidic[13] or peptidomimetic[14,15] cysteine
protease inhibitors. In particular, we focused our attention on
the development of peptidomimetics, by taking into consider-
ation the fact that the use of an appropriate scaffold, incorpo-
atoms). Finally, we combined the two modifications to gener-
ate derivatives (ꢀ)-8, (ꢀ)-9, and (ꢀ)-10.
rated into a characteristic peptide sequence, could be particu- Results and Discussion
larly advantageous in terms of potency and selectivity, as de-
Chemistry
scribed also in our recent studies on threonine protease inhibi-
tors.[16]
The synthesis of compound (ꢀ)-3 (Scheme 1) was accom-
Recently, we developed the novel peptidomimetics 1 and 2
plished by using 1,4-benzodiazepine 11 as the starting materi-
al, which, in turn, was prepared according to a literature proce-
dure.[18] Benzodiazepine 11 was treated with ethyl bromoace-
tate, in the presence of NaH, to yield intermediate 12, which
was then hydrolyzed to form the corresponding acid 13. With
a standard procedure, intermediate 13 was condensed with al-
lylamine to produce unsaturated amide 14.
(Figure 1),[17a] composed of a reactive warhead and a benzodia-
zepine nucleus. A spacer connects the two moieties, and a hy-
droxymethyl side chain, linked at the C3 position of the benzo-
diazepine, is functionalized as a carbamate. For the warhead,
we selected the 3-bromoisoxazoline nucleus, which, as well as
the 3-chloroisoxazoline, was previously exploited for the cova-
lent inhibition of enzymes containing a catalytic cysteine resi-
due, for example, glutamine amidotransferases.[17b–d] Deriva-
tives 1 and 2 displayed a time-independent inhibition of the
target enzyme (inhibition constant (Ki) values in the range of
0.96–4.64 mm).[17a]
Intermediate 14 was used as the dipolarophile in a 1,3 dipo-
lar cycloaddition with bromonitrile oxide, generated in situ by
dehydrohalogenation of the stable precursor dibromoformal-
doxime,[19] to afford only the 3,5-disubstituted isoxazoline re-
gioisomer (ꢀ)-3.[20] Alternatively, acid 13 was condensed with
the bicyclic amine (ꢀ)-15[17] to yield amide derivative (ꢀ)-4.
Compounds 5a,b–7a,b were obtained from intermediate
(R)-16, synthesized according to our previously reported proce-
dure,[14a] which was treated with alkenyl halides 17–19, in the
presence of sodium hydride, to yield intermediates (R)-20–22,
respectively (Scheme 2). The subsequent cycloaddition reaction
with bromonitrile oxide, carried out as described above, afford-
ed, as expected,[20] only the 3-bromo-5-substituted isoxazolines.
Due to the presence of two stereogenic centers, derivatives
23a,b–25a,b were obtained as 1:1 diastereomeric mixtures
with the fixed configuration R at the C3 position of the benzo-
diazepine nucleus. The pairs of diastereomers could not be
separated at this stage.
In the present work, we studied the structure–activity rela-
tionships of these derivatives, with the aim to develop new an-
alogues with a simplified and more synthetically accessible
structure and with improved physicochemical properties. In
particular, we focused our attention on the side chain at the
C3 position and on the nature and length of the spacer be-
tween the benzodiazepine nucleus and the bromoisoxazoline.
We first decided to eliminate the carbamoyl side chain ap-
pended at the C3 position of the benzodiazepine nucleus
(compounds (ꢀ)-3 and (ꢀ)-4 in Figure 1). In parallel, we pre-
pared derivatives (ꢀ)-5a,b, (ꢀ)-6a,b, and (ꢀ)-7a,b, in which
we kept the 2-CF3-4-Cl-phenylcarbamoyl moiety but varied the
nature of the spacer; the amide group of compound 1 was re-
placed by aliphatic chains of different lengths (2–4 carbon
Removal of the silyl group and subsequent treatment with
4-chloro-2-(trifluoromethyl)phenyl isocyanate gave diastereo-
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formation on the biological activities. Lastly, by treating benzo-
diazepine 11 with bromoalkenes 17–19, we obtained inter-
mediates 26–28, which, in turn, were submitted to the 1,3 di-
polar cycloaddition with bromonitrile oxide to give final deriva-
tives (ꢀ)-8–(ꢀ)-10 (Scheme 3).
Biological activity
All of the new derivatives were tested for their inhibitory activi-
ty against rhodesain (Table 1), by using Cbz-Phe-Arg-AMC (Cbz:
benzyloxycarbonyl; AMC: 7-amino-4-methylcoumarin) as a fluo-
rogenic substrate. We also tested intermediates 23a,b–25a,b,
to gain additional information on the structure requirements
of the substituent at the C3 position of the molecule in the in-
teraction with the enzyme active site. Moreover, an increase in
lipophilicity, conferred by the TBS group, could improve the
parasite penetration with a positive effect on the antiparasitic
activity.
First, we performed a preliminary screening with an inhibitor
concentration of 20 mm. An equivalent volume of DMSO was
used as the negative control. Compounds capable of inhibiting
the enzymatic activity by more than 40% were progressed to
the continuous assays. The compounds did not show time-de-
pendent kinetics but only linear progress curves, so no rate
constants but only Ki values could be determined.
Scheme 1. Synthesis of target compounds (ꢀ)-3 and (ꢀ)-4. Reagents and
conditions: a) NaH, DMF, 08C, N2, 1 h, then ethyl bromoacetate, RT, 12 h,
64%; b) LiOH, H2O/MeOH, 08C!RT, 6 h, 99%; c) HATU, 08C, CH2Cl2, 10 min,
then allylamine, DIPEA, RT, 12 h, 59%; d) DBF, NaHCO3, EtOAc, 12 h, 45%;
e) EDCI, HOBt, 08C, CH2Cl2, 10 min, then (ꢀ)-15, DIPEA, RT, 12 h, 48%. HATU:
N-[(dimethylamino)-1H-1,2,3-triazole[4,5-b]-pyridin-1-ylmethylene]-N-methyl-
methanaminium hexafluorophosphate; DIPEA: diisopropylethylamine; DBF:
dibromoformaldoxime; EDCI: 3-(3-dimethylaminopropyl)-1-ethylcarbodii-
mide; HOBt: 1-hydroxy-1H-benzotriazole.
The most active compounds against rhodesain were proven
to be those bearing a silyl group directly linked to the hydrox-
ymethyl chain at the C3 position of the benzodiazepine nu-
cleus, particularly those with a spacer of two or four carbon
atoms (that is, 23a,b and 25a,b, with Ki values of 1.70 and
1.52 mm, respectively). This clearly suggests that the presence
of a lipophilic substituent at the P3 site strongly enhances the
interaction with the S3 site of rhodesain.
meric mixtures of carbamates 5a,b–7a,b, which also at this
stage could not be separated by chromatographic methods
and were therefore tested as obtained to gain preliminary in-
All of the compounds were also tested against FP-2 from
P. falciparum (Table 1). The data reported in Table 1 show that
the elimination of the chain linked at the C3 position
of the benzodiazepine ring of model compounds
1 and 2 (as in compounds (ꢀ)-3 and (ꢀ)-4) leads to
a drop in the inhibitory activity. On the other hand,
the amide bond of the spacer does not have any
impact on the activity, because it can be replaced by
a simple alkyl chain (as in compounds 5a,b–7a,b).
Notably, whereas compounds 5a,b and 6a,b inhibit
both rhodesain and FP-2, in analogy to model com-
pounds 1 and 2, an increase in the length of the
spacer from two to four carbon atoms results in
a potent and selective FP-2 inhibitor (that is, 7a,b,
with Ki =0.76 mm). It is noteworthy that, whereas the
deletion of the C3 side chain from reference com-
pounds 1 and 2 leads to inactive derivatives (ꢀ)-3
and (ꢀ)-4, the same modification on derivatives
5a,b–7a,b, which leads to compounds (ꢀ)-8–(ꢀ)-10,
was well tolerated, because micromolar inhibitory ac-
Scheme 2. Synthesis of target compounds 5a,b–7a,b. Reagents and conditions: a) NaH,
DMF, 08C, N2, 1 h, then 17–19, RT, 12 h, (R)-20: 85%, (R)-21: 88%, (R)-22: 82%; b) DBF,
NaHCO3, EtOAc, 12 h, 23a,b: 78%, 24a,b: 75%, 25a,b: 70%; c) TBAF, THF, RT; 6 h; d) 4-
Cl,2-CF3C6H3NCO, CH2Cl2, RT, 12 h, 5a,b: 69%, 6a,b: 67%, 7a,b: 62% (two steps). TBAF:
tetrabutylammonium fluoride; TBS: tert-butyldimethylsilyl.
tivity against both enzymes was observed. Further-
more, the results obtained with silyl derivatives
23a,b–25a,b highlight the possibility of inserting
a side chain at the C3 position with physicochemical
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potent antiparasitic activities against P. falciparum
(IC50 values in the range 0.71–1.59 mm) and are ~20-
fold more effective than model compounds 1 and 2,
with IC50 values in the low or sub-micromolar range.
Surprisingly, there is little correlation between the
observed antiplasmodial activity of compounds (ꢀ)-
8–(ꢀ)-10 and their modest potency as falcipain-2 in-
hibitors; this suggests possible off-target effects,
which are worthy of further investigation. Finally, all
of the inhibitors were also evaluated for their cyto-
toxicity against mammalian cells by using mouse
macrophages (J774.1 cell line) and, as reported in
Table 2, they all showed preference for parasitic, es-
pecially P. falciparum, versus mammalian cells.
Scheme 3. Synthesis of target compounds (ꢀ)-8–(ꢀ)-10. Reagents and conditions: a) NaH,
DMF, 08C, N2, 1 h, then 17–19, RT, 12 h, 26: 89%, 27: 81%, 28: 78%; b) DBF, NaHCO3,
EtOAc, 12 h, (ꢀ)-8: 56%, (ꢀ)-9: 53%, (ꢀ)-10: 51%.
Table 1. Inhibition of rhodesain, falcipain-2, and human cathepsins B and
L.[a]
Conclusions
In summary, the present paper describes an optimization study
around two previously developed papain-family cathepsin L-
like cysteine protease inhibitors, 1 and 2. Our aim was to in-
crease their antiparasitic activity and possibly generate more
easily accessible derivatives. We have shown that it is possible
to significantly simplify the structure of our model compounds
by modifying or even eliminating the side chain appended at
the C3 position of the benzodiazepine scaffold and by insert-
ing a simple methylene spacer of appropriate length between
the benzodiazepine ring and the 3-bromoisoxazoline warhead.
It is noteworthy that we have identified several rhodesain and
falcipain-2 inhibitors displaying single-digit micromolar or sub-
micromolar antiparasitic activity against one or both parasites,
activities that are one order of magnitude more potent than
those of the model compounds 1 and 2.
Compd
Ki [mm] or inhibition [%] at 20 mm
Rhodesain
Falcipain-2
Cath B
Cath L
1[13]
2[13]
(ꢀ)-3
(ꢀ)-4
5a,b
3.52ꢀ0.17
0.96ꢀ0.09
7.5%
5.48ꢀ2.80
6.00ꢀ0.20
11.08ꢀ0.36
17.7%
n.i
12.03%
n.i.
n.i
n.i
n.i.
n.i.
4.6%
8.5%
5.7%
n.i.
21.0%
4.7%
16.1%
9.9%
4.1%
n.i.
n.i.
4.38ꢀ0.09
2.48ꢀ0.71
26.2%
2.85ꢀ0.32
3.33ꢀ0.05
2.44ꢀ0.06
1.70ꢀ0.16
2.84ꢀ0.50
1.52ꢀ0.04
1.63ꢀ0.05
3.40ꢀ0.72
0.76ꢀ0.10
4.86ꢀ0.60
4.49ꢀ0.54
10.6ꢀ0.22
1.17ꢀ0.24
1.36ꢀ0.12
0.23ꢀ0.01
3.5%
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
6a,b
7a,b
(ꢀ)-8
(ꢀ)-9
(ꢀ)-10
23a,b
24a,b
25a,b
[a] Values are the meanꢀSD of n=2 experiments performed in duplicate;
n.i.: no inhibition.
Table 2. Activity against T. b. brucei and P. falciparum and cytotoxicity on
macrophages.
parameters that are different from those of aromatic carba-
mates. Of particular interest is the inhibitory activity against
FP-2 shown by derivative 25a,b (Ki =0.23 mm).
Compd
IC50 [mm][a]
TC50 [mm][b]
Selectivity assays were also performed, by testing the inhibi-
tors against two papain-family human cysteine proteases, cath-
epsins B and L. None of the tested compounds passed the ini-
tial screening at 20 mm against cathepsins B and L, which dem-
onstrated the excellent selectivity of the synthesized inhibitors
toward the parasitic proteases.
T. b. brucei
P. falciparum
J774.1
1[13]
16.91ꢀ0.46
14.04ꢀ1.02
27.75
>100
9.36
n.d.
n.d.
n.d.
n.d.
2[13]
(ꢀ)-3
(ꢀ)-4
5
>40
n.d.
n.d.
10.83ꢀ0.57
3.78ꢀ0.60
3.27ꢀ0.03
18.05
3.47
5.18
1.17
1.59
0.71
4.80
1.49
3.21
5.3
>100
>100
>100
22.3ꢀ0.87
22.5ꢀ1.10
12.2ꢀ0.65
>100
6
7
With the exception of derivative (ꢀ)-4, which has no inhibi-
tory activity against either of the target enzymes, all of the
novel compounds were tested for antiparasitic activity against
T. b. brucei and P. falciparum (Table 2). Several compounds dis-
played single-digit micromolar antiparasitic activity against one
or both parasites and were more potent than model com-
pounds 1 and 2.[17] In detail, if we consider the IC50 values at
72 h, the most active compounds against T. b. brucei were
6a,b, 7a,b, 23a,b, and 24a,b (IC50 values in the range 3.27–
5.29 mm), with IC50 values similar to those evaluated on P. falci-
parum (IC50 values in the range 1.49–5.18 mm). These data cor-
relate quite well with the results obtained in the enzymatic
tests, with the exception of compound 7a,b. It is noteworthy
that compounds (ꢀ)-8–(ꢀ)-10 have highly selective and
(ꢀ)-8
(ꢀ)-9
(ꢀ)-10
23a,b
24a,b
25a,b
E-64[21]
Eflornithine[21]
>40
>40
12.22ꢀ1.53
5.29ꢀ0.18
3.54ꢀ0.11
24.58ꢀ1.35
–
43.4ꢀ1.50
9.4ꢀ0.90
–
30.7
–
>1000
[a] Half-maximal inhibitory concentration (IC50) values were determined
after treatment for 72 h; for T. b. brucei, data are the meanꢀSD of n=2
experiments performed in duplicate; for P. falciparum, the assay was car-
ried out once in triplicate; the data represent the median IC50 value calcu-
lated automatically by GraphPad Prism. [b] Half-maximal toxic concentra-
tion (TC50) values were determined against J774.1 macrophages; data rep-
resent the mean ꢀSD of n=2 experiments.
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eluent to give compound 14 as an oil (33 mg, 0.1 mmol, 59%). Rf =
0.93 (CHCl3/MeOH, 9:1); H NMR (300 MHz, CDCl3): d=3.00 (d, J=
Experimental Section
1
Chemistry
15.2 Hz, 1H), 3.51 (d, J=15.2 Hz, 1H), 3.63 (d, J=10.7 Hz, 1H),
3.78–3.88 (m, 2H), 4.15 (d, J=10.7 Hz, 1H), 5.06–5.20 (m, 2H),
5.69–5.85 (m, 1H), 6.08 (brs, 1H), 7.21–7.40 (m, 3H), 7.48–7.61 (m,
5H), 7.93–8.01 ppm (m, 1H); 13C NMR (75 MHz, CDCl3): d=43.6,
55.9, 56.2, 115.8, 117.4, 126.6, 127.2, 128.9, 129.2, 131.0, 131.2,
131.9, 134.3, 139.1, 140.5, 140.7, 169.5, 171.0 ppm.
General: All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Elemental
analyses were carried out on a C. Erba Model 1106 (Elemental Ana-
lyzer 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 column chromatography
was performed on Merck silica gel (200–400 mesh). 1H and
13C NMR spectra were recorded on a Varian Gemini 300 MHz spec-
trometer by using the residual signal of the deuterated solvent as
an internal standard. Splitting patterns are described as singlet (s),
doublet (d), doublet of doublet (dd), triplet (t), quartet (q), multip-
N-((3-Bromo-4,5-dihydroisoxazol-5-yl)methyl)-2-(2,3-dihydro-2-
oxo-5-phenylbenzo[e][1,4]diazepin-1-yl)acetamide ((ꢀ)-3): Dibro-
moformaldoxime (41 mg, 0.20 mmol) and solid NaHCO3 (34 mg,
0.40 mmol) were added to a solution of 14 (33 mg, 0.10 mmol) in
EtOAc at room temperature. The mixture was vigorously stirred for
24 h, and the progress of the reaction was monitored by TLC. H2O
was added to the reaction mixture, and the organic layer was sepa-
rated. The aqueous phase was further extracted with EtOAc, and
the combined organic layers were dried over anhydrous
Na2SO4.The reaction mixture was purified by flash column chroma-
tography by using CHCl3/MeOH (9:1) as the eluent to afford prod-
uct (ꢀ)-3 as a solid (20 mg, 0.045 mmol, 45%). Rf =0.84 (CHCl3/
let (m), or broad singlet (brs). H and 13C NMR chemical shifts (d)
1
are expressed in ppm, and coupling constants (J) are given in Hz.
MS analyses were performed on a Varian 320-MS triple-quadrupole
mass spectrometer equipped with an electron-spray ionization
(ESI) source.
1
MeOH, 9:1); H NMR (300 MHz, CDCl3): d=2.89–3.03 (m, 1H), 3.02
Ethyl 2-(2,3-dihydro-2-oxo-5-phenylbenzo[e][1,4]diazepin-1-yl)
acetate (12): A 0.1m solution of 11[18] (123 mg, 0.52 mmol) in DMF
was added to a suspension of NaH (18 mg, 0.78 mmol) in dry DMF
at 08C under nitrogen, and the mixture was then allowed to warm
to room temperature over 1 h. After this time, ethyl bromoacetate
(113 mg, 75 mL, 0.78 mmol) was added through a syringe, and the
solution was further stirred for 12 h. The reaction mixture was
quenched with NH4Cl and extracted with ethyl acetate. The organic
layer was washed with water, dried over anhydrous Na2SO4, and
concentrated, and the resulting residue was purified by flash
column chromatography by using light petroleum/EtOAc (1:1) as
the eluent to afford compound 12 as an oil (107 mg, 0.33 mmol,
64%). Rf =0.37 (CHCl3/MeOH, 9:1); 1H NMR (300 MHz, CDCl3): d=
1.17 (t, J=6.9 Hz, 3H), 3.01 (d, J=15.1 Hz, 1H), 3.53 (d, J=15.1 Hz,
1H), 3.62 (d, J=10.6 Hz, 1H), 4.10 (q, J=6.9 Hz, 1H), 4.17 (d, J=
10.6 Hz, 1H), 7.20–7.39 (m, 3H), 7.49–7.60 (m, 5H), 7.92–8.00 ppm
(m, 1H); 13C NMR (75 MHz, CDCl3): d=14.1, 54.0, 56.2, 61.1, 115.8,
126.5, 127.2, 128.8, 129.2, 131.1, 131.2, 131.9, 139.1, 140.5, 140.7,
167.7, 169.5 ppm.
(d, J=16.9 Hz, 1H), 3.18–3.33 (m, 1H), 3.42–3.58 (m, 2H), 3.64 (d,
J=11.1 Hz, 1H), 4.11 (d, J=16.9 Hz, 1H), 4.15 (d, J=11.1 Hz, 1H),
4.70–4.84 (m, 1H), 6.31 (brt J=5.7 Hz, 1H), 7.30–7.41 (m, 4H),
7.41–7.60 (m, 4H), 7.93–8.03 ppm (m, 1H); 13C NMR (75 MHz,
CDCl3): d=27.0, 47.4, 53.6, 55.9, 67.0, 121.8, 124.5, 127.3, 128.9,
129.3, 129.4, 131.1, 131.3, 137.1, 139.0, 140.8, 140.7, 169.6,
170.8 ppm; MS: m/z 455.1 [M+H]+; elemental analysis: calcd for
C21H19BrN4O3: C 55.40, H 4.21, N 12.31; found: C 55.58, H 3.98, N
12.62.
1-(2-(3-Bromo-3a,4,6,6a-tetrahydropyrrolo[3,4-d]isoxazol-5-yl)-2-
oxoethyl)-5-phenyl-1H-benzo[e][1,4]diazepin-2-(3H)-one ((ꢀ)-4):
EDCI (36 mg, 0.19 mmol) and HOBt (26 mg, 0.19 mmol) were
added to a solution of 13 (47 mg, 0.16 mmol) in CH2Cl2 (10 mL) at
08C. After 10 min, the ice bath was removed, and a solution of
amine (ꢀ)-15[17] (46 mg, 0.24 mmol) in CH2Cl2 (5 mL) was added.
The resulting mixture was stirred at room temperature for 12 h
and washed with water. The organic layer was separated, dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash chromatography by
using CHCl3/MeOH (9:1) as the eluent to give title compound (ꢀ)-4
as a solid (36 mg, 0.08 mmol, 48%). Rf =0.73 (CHCl3/MeOH, 9:1);
1H NMR (300 MHz, CDCl3): d=3.50–3.81 (m, 2H), 3.82–4.05 (m, 3H),
3.90 (d, J=10.6 Hz, 1H), 4.12 (d, J=16.2 Hz, 1H), 4.75 (d, J=
16.2 Hz, 1H), 4.80 (d, J=10.6 Hz, 1H), 5.24–5.43 (m, 1H), 7.16–
7.67 ppm (m, 9H); 13C NMR (75 MHz, CDCl3): d=27.9, 41.3, 51.4,
54.3 55.8, 60.5, 121.7, 124.5, 127.3, 128.9, 129.2, 129.4, 131.1, 131.3,
137.2, 139.0, 140.5, 140.7, 167.9, 169.5 ppm; MS: m/z 469.1 [M+
H]+; elemental analysis: calcd for C22H19BrN4O3: C 56.54, H 4.10, N
11.99; found: C 56.18, H 4.29, N 11.75.
2-(2,3-Dihydro-2-oxo-5-phenylbenzo[e][1,4]diazepin-1-yl) acetic
acid (13): LiOH (20 mg, 0.82 mmol) was added to a solution of the
ester 12 (107 mg, 0.33 mmol) in a mixture of methanol/water (1:1;
20 mL) at 08C, and the mixture was stirred at room temperature
until the disappearance of the starting material (TLC monitoring;
CHCl3/MeOH, 9:1). The solvent was concentrated under reduced
pressure, and the mixture was treated with 10% citric acid, extract-
ed with ethyl acetate (100 mL), dried over anhydrous Na2SO4, and
concentrated to give acid 13 as an oil (94 mg, 0.32 mmol, 99%).
1
Rf =0.33 (CHCl3/MeOH, 9:1); H NMR (300 MHz, CDCl3): d=3.00 (d,
J=15.1 Hz, 1H), 3.52 (d, J=15.1 Hz, 1H), 3.61 (d, J=10.6 Hz, 1H),
4.18 (d, J=10.6 Hz, 1H), 7.19–7.38 (m, 3H), 7.48–7.61 (m, 5H),
7.91–8.01 ppm (m,1H).
1-But-3-enyl-3-(R)-(tert-butyldimethylsilyloxymethyl)-5-phenyl-
1,3-dihydrobenzo[e][1,4]diazepin-2-one ((R)-20): Compound (R)-
20 was obtained from (R)-16[14a] (200 mg, 0.52 mmol), NaH (18 mg,
0.78 mmol), and 17 (105 mg, 0.78 mmol) by employing the proce-
dure described for compound 12. The reaction mixture was puri-
fied by flash column chromatography by using light petroleum/
EtOAc (7:3) as the eluent to afford compound (R)-20 as an oil
(252 mg, 0.66 mmol, 85%). Rf =0.58 (light petroleum/EtOAc, 7:3);
1H NMR (300 MHz, CDCl3): d=0.14 (s, 3H), 0.16 (s, 3H), 0.91 (s, 9H),
2.10–2.36 (m, 2H), 3.66–3.75 (m, 1H), 3.71 (t, J=6.4 Hz, 1H), 4.30
(dd, J=6.7, 9.7 Hz, 1H), 4.40–4.52 (m, 1H), 4.58 (dd, J=6.5, 9.7,
1H), 4.79–4.92 (m, 2H), 5.51–5.65 (m, 1H), 7.11–7.66 ppm (m, 9H);
N-Allyl-2-(2,3-dihydro-2-oxo-5-phenylbenzo[e][1,4]diazepin-1-yl)-
acetamide (14): HATU (72 mg, 0.19 mmol) was added to a solution
of 13 (47 mg, 0.16 mmol) in CH2Cl2 (10 mL) at 08C. After 10 min,
the ice bath was removed and allylamine (14 mg, 18 mL,
0.24 mmol) and DIPEA (31 mg, 42 mL, 0.24 mmol) were added. The
mixture was stirred at room temperature for 12 h. After this time,
the mixture was diluted with EtOAc and washed with water. The
organic layer was dried over anhydrous Na2SO4, filtered, and
evaporated under reduced pressure, and the crude residue was pu-
rified by flash chromatography with CHCl3/MeOH (9:1) as the
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13C NMR (75 MHz, CDCl3): d=ꢁ2.3, 25.9, 30.7, 31.2, 52.2, 67.3, 68.1,
115.9, 116.4, 126.5, 127.2, 128.8, 129.3, 131.0, 131.3, 132.0, 135.8,
139.1, 140.5, 140.7, 170.9 ppm.
using light petroleum/EtOAc (7:3) as the eluent to afford 24a,b as
a solid (291 mg, 0.51 mmol, 75%). Rf =0.78 (light petroleum/EtOAc,
6:4); H NMR (300 MHz, CDCl3): d=0.13 (s, 3H), 0.15 (s, 3H), 0.91 (s,
1
9H), 1.00–1.16 (m, 2H), 1.34–1.46 (m, 2H), 2.41 (dd, J=9.0, 16.9 Hz,
0.5H), 2.60 (dd, J=8.2, 16.9 Hz, 0.5H), 2.99 (dd, J=6.9, 16.6 Hz,
0.5H), 3.10 (dd, J=8.7, 16.6 Hz, 0.5H), 3.45–3.63 (m, 1H), 3.74 (t,
J=6.5 Hz, 1H), 4.29 (dd, J=6.2, 10.3 Hz, 1H) 4.35–4.52 (m, 2H),
4.57 (dd, J=6.7, 10.3 Hz, 1H), 7.17–7.63 ppm (m, 9H); MS: m/z
570.2 [M+H]+; elemental analysis: calcd for C28H36BrN3O3Si: C
58.94, H 6.36, N 7.36; found: C 59.16, H 6.25, N 7.64.
3-(R)-(tert-Butyldimethylsilyloxymethyl)-1-pent-4-enyl-5-phenyl-
1,3-dihydrobenzo[e][1,4]diazepin-2-one ((R)-21): Compound (R)-
21 was obtained from (R)-16[14a] (200 mg, 0.52 mmol), NaH (18 mg,
0.78 mmol), and 18 (116 mg, 0.78 mmol) by employing the proce-
dure described for compound 12. The reaction mixture was puri-
fied by flash column chromatography by using light petroleum/
EtOAc (7:3) as the eluent to afford product (R)-21 as an oil
(305 mg, 0.68 mmol, 88%). Rf =0.67 (light petroleum/EtOAc, 7:3);
1H NMR (300 MHz, CDCl3): d=0.14 (s, 3H), 0.16 (s, 3H), 0.90 (s, 9H),
1.43–1.68 (m, 2H), 1.78–1.96 (m, 2H), 3.58–3.69 (m, 1H), 3.73 (t, J=
6.5 Hz, 1H), 4.31 (dd, J=7.4, 10.1 Hz, 1H), 4.35–4.44 (m, 1H), 4.58
(dd, J=6.4, 10.1, 1H), 4.75–4.88 (m, 2H), 5.55–5.70 (m, 1H), 7.15–
7.62 ppm (m, 9H); 13C NMR (75 MHz, CDCl3): d=ꢁ2.3, 25.9, 27.8,
30.6, 31.4, 42.9, 67.4, 68.1, 115.8, 115.9, 126.6, 127.2, 128.8, 129.3,
131.0, 131.3, 132.0, 136.5, 139.0, 140.5, 140.7, 170.9 ppm.
1-[4-((R)-3-Bromo-4,5-dihydroisoxazol-5-yl)butyl]-3-(R)-(tert-bu-
tyldimethylsilyloxymethyl)-5-phenyl-1,3-dihydrobenzo[e][1,4]di-
azepin-2-one (25a) and 1-[4-((S)-3-bromo-4,5-dihydroisoxazol-5-
yl)butyl]-3-(R)-(tert-butyl-dimethylsilyloxymethyl)-5-phenyl-1,3-
dihydrobenzo[e][1,4]diazepin-2-one (25b): The diastereomeric
mixture of 25a,b was obtained from (R)-22 (296 mg, 0.64 mmol),
DBF (260 mg, 1.28 mmol), and NaHCO3 (215 mg, 2.56 mmol) by
employing the procedure described for compound (R)-3. The reac-
tion mixture was purified by flash column chromatography by
using light petroleum/EtOAc (7:3) as the eluent to afford 25a,b as
a solid (263 mg, 0.45 mmol, 70%). Rf =0.83 (light petroleum/EtOAc,
3-(R)-(tert-Butyldimethylsilyloxymethyl)-1-hex-5-enyl-5-phenyl-
1,3-dihydrobenzo[e][1,4]diazepin-2-one ((R)-22): Compound (R)-
22 was obtained from (R)-16[14a] (200 mg, 0.52 mmol), NaH (18 mg,
0.78 mmol), and 19 (127 mg, 0.78 mmol) by employing the proce-
dure described for compound 12. The reaction mixture was puri-
fied by flash column chromatography by using light petroleum/
EtOAc (7:3) as the eluent to afford product (R)-22 as an oil
(296 mg, 0.64 mmol, 82%). Rf =0.81 (light petroleum/EtOAc, 7:3);
1H NMR (300 MHz, CDCl3): d=0.14 (s, 3H), 0.16 (s, 3H), 0.91 (s, 9H),
1.11–1.32 (m, 2H), 1.32–1.56 (m, 2H), 1.82–1.93 (m, 2H), 3.56–3.68
(m, 1H), 3.72 (t, J=6.8 Hz, 1H), 4.31 (dd, J=6.4, 10.6 Hz, 1H), 4.34–
4.44 (m, 1H), 4.57 (dd, J=7.9, 10.6 Hz, 1H), 4.78–4.87 (m, 2H),
5.50–5.65 (m, 1H), 7.14–7.63 ppm (m, 9H); 13C NMR (75 MHz,
CDCl3): d=ꢁ2.3, 25.9, 27.0, 27.5, 30.6, 33.6, 42.8, 67.3, 68.2, 115.7,
115.8, 126.5, 127.2, 128.8, 129.3, 131.0, 131.2, 131.9, 139.1, 139.1,
140.6, 140.7, 170.9 ppm.
1
6:4); H NMR (300 MHz, CDCl3): d=0.14 (s, 3H), 0.15 (s, 3H), 0.91 (s,
9H), 1.02–1.22 (m, 2H),1.34–1.59 (m, 4H), 2.39 (dd, J=9.1, 16.8 Hz,
0.5H), 2.61 (dd, J=8.3, 16.8 Hz, 0.5H), 2.96 (dd, J=10.6, 17.9 Hz,
0.5H), 3.17 (dd, J=11.0, 17.9 Hz, 0.5H), 3.56–3.69 (m, 2H), 3.73 (t,
J=6.3 Hz, 1H), 4.23–4.34 (m, 2H), 4.38–4.50 (m, 1H), 4.52–4.61 (m,
1H), 7.16–7.64 ppm (m, 9H); MS: m/z 584.2 [M+H]+; elemental
analysis: calcd for C29H38BrN3O3Si: C 59.58, H 6.55, N 7.19; found: C
59.93, H 6.28, N 7.36.
((R)-1-(2-((R)-3-Bromo-4,5-dihydroisoxazol-5-yl)ethyl)-2,3-dihy-
dro-2-oxo-5-phenyl-1H-benzo[e][1,4]diazepin-3-yl)methyl
(4-
chloro-2-(trichloromethyl)phenyl)carbamate (5a) and ((R)-1-(2-
((S)-3-bromo-4,5-dihydroisoxazol-5-yl)ethyl)-2,3-dihydro-2-oxo-5-
phenyl-1H-benzo[e][1,4]diazepin-3-yl)methyl
(4-chloro-2-(tri-
chloromethyl)phenyl)carbamate (5b): Step 1: TBAF (1m solution
in THF, 1.27 mL) was added dropwise to a solution of 23a,b
(284 mg, 0.51 mmol) in dry THF. The mixture was stirred at room
temperature until disappearance of the starting material (TLC mon-
itoring), and then it was diluted with EtOAc and washed with
water. The organic layer was separated, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure to give
the deprotected intermediate.
1-[2-((R)-3-Bromo-4,5-dihydroisoxazol-5-yl)ethyl]-3-(R)-(tert-butyl-
dimethylsilyloxymethyl)-5-phenyl-1,3-dihydrobenzo[e][1,4]
diazepin-2-one (23a) and 1-[2-((S)-3-bromo-4,5-dihydroisoxazol-
5-yl)ethyl]-3-(R)-(tert-butyldimethylsilyloxymethyl)-5-phenyl-1,3-
dihydrobenzo[e][1,4]diazepin-2-one (23b): The diastereomeric
mixture of 23a,b was obtained from (R)-20 (252 mg, 0.66 mmol),
DBF (268 mg, 1.32 mmol), and NaHCO3 (222 mg, 2.64 mmol) by
employing the procedure described for compound (ꢀ)-3.The crude
material was purified by flash column chromatography by using
light petroleum/EtOAc (7:3) as the eluent to afford 23a,b as a solid
(284 mg, 0.51 mmol, 78% yield). Rf =0.82 (light petroleum/EtOAc,
Step 2: The latter product was used without any further purifica-
tion in
a reaction with 4-Cl,2-CF3C6H3NCO (221 mg, 151 mL,
1.0 mmol) in dry CH2Cl2 and under a nitrogen atmosphere,. The re-
sulting mixture was stirred for 12 h at room temperature. After this
time, it was washed with water and dried over anhydrous Na2SO4.
The crude material, obtained after evaporation of the solvent, was
purified by flash chromatography by using light petroleum/EtOAc
(8:2) as the eluent to give a diastereomeric mixture of 5a,b as
a solid (249 mg, 0.35 mmol, 69% yield). Rf =0.58 (light petroleum/
1
6:4); H NMR (300 MHz, CDCl3): d=0.13 (s, 3H), 0.15 (s, 3H), 0.91 (s,
9H), 1.81–1.95 (m, 1H), 2.00–2.13 (m, 1H), 2.81 (dd, J=8.3, 17.1 Hz,
1H), 3.09 (dd, J=10.4, 17.1 Hz,1H), 3.73 (t, J=6.5 Hz, 1H), 3.80–
3.92 (m, 1H), 4.23–4.44 (m, 3H), 4.52–4.60 (m, 1H), 7.18–7.64 ppm
(m, 9H); MS: m/z 556.2 [M+H]+; elemental analysis: calcd for
C27H34BrN3O3Si: C 58.27, H 6.16, N 7.55; found: C 58.09, H 6.40, N
7.24.
1
EtOAc, 6:4); H NMR (300 MHz, CDCl3): d=1.80–1.95 (m, 1H), 1.98–
2.16 (m, 1H), 2.68–2.88 (m, 1H), 3.06–3.25 (m, 1H), 3.78–3.91 (m,
1H), 3.94 (t, J=6.3 Hz, 1H), 4.32–4.46 (m, 1H), 4.61–4.74 (m, 1H),
4.91 (dd, J=6.5, 10.8 Hz, 1H), 5.05 (dd, J=7.2, 10.8 Hz, 1H), 6.94
(brs, 1H), 7.27–7.64 (m, 11H), 8.11 ppm (d, J=8.9 Hz, 1H); MS: m/z
663.1 [M+H]+; elemental analysis: calcd for C29H23BrClF3N4O4: C
52.47, H 3.49, N 8.44; found: C 52.17, H 3.64, N 8.19.
1-[3-((R)-3-Bromo-4,5-dihydroisoxazol-5-yl)propyl]-3-(R)-(tert-bu-
tyldimethylsilyloxymethyl)-5-phenyl-1,3-dihydrobenzo[e][1,4]di-
azepin-2-one (24a) and 1-[3-((S)-3-bromo-4,5-dihydroisoxazol-5-
yl)propyl]-3-(R)-(tert-butyldimethylsilyloxymethyl)-5-phenyl-1,3-
dihydrobenzo[e][1,4]diazepin-2-one (24b): The diastereomeric
mixture of 24a,b was obtained from (R)-21 (305 mg, 0.68 mmol),
DBF (276 mg, 1.36 mmol), and NaHCO3 (228 mg, 2.72 mmol) by
employing the procedure described for compound (ꢀ)-3. The
crude material was purified by flash column chromatography by
((R)-1-(3-((R)-3-Bromo-4,5-dihydroisoxazol-5-yl)propyl)-2,3-dihy-
dro-2-oxo-5-phenyl-1H-benzo[e][1,4]diazepin-3-yl)methyl
(4-
chloro-2-(trichloromethyl)phenyl)carbamate (6a) and ((R)-1-(3-
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((S)-3-bromo-4,5-dihydroisoxazol-5-yl)propyl)-2,3-dihydro-2-oxo-
5-phenyl-1H-benzo[e][1,4]diazepin-3-yl)methyl (4-chloro-2-(tri-
chloromethyl)phenyl)carbamate (6b): The diastereomeric mixture
of the intermediate was obtained from 24a,b (291 mg, 0.51 mmol)
and TBAF (1m solution in THF, 1.27 mL) by employing the proce-
dure described for 5a,b. The deprotected mixture was then treated
with 4-Cl,2-CF3C6H3NCO (226 mg, 154 mL, 1.02 mmol). The crude
residue was purified by flash column chromatography by using
light petroleum/EtOAc (8:2) as the eluent to afford 6a,b as a solid
(247 mg, 0.34 mmol, 67%). Rf =0.52 (light petroleum/EtOAc, 6:4);
1H NMR (300 MHz, CDCl3): d=1.35–1.49 (m, 2H), 1.52–1.65 (m, 2H),
2.43 (dd, J=8.5, 17.4 Hz, 0.5H), 2.62 (dd, J=8.3, 17.4 Hz, 0.5H),
3.00 (dd, J=10.8, 17.1 Hz, 0.5H), 3.03 (dd, J=10.6, 17.1 Hz, 0.5H),
3.63–3.77 (m, 1H), 3.94 (t, J=6.7 Hz, 1H), 4.34–4.61 (m, 2H), 4.92
(dd, J=6.9, 11.3 Hz, 1H), 5.06 (dd, J=7.1, 11.3 Hz, 1H), 6.94 (brs,
1H), 7.27–7.65 (m, 11H), 8.12 ppm (d, J=9.1 Hz, 1H); MS: m/z
677.1 [M+H]+; elemental analysis: calcd for C30H25BrClF3N4O4: C
53.15, H 3.72, N 8.26; found: C 53.01, H 3.94, N 8.03.
NaH (18 mg, 0.78 mmol), and 18 (116 mg, 0.78 mmol) by employ-
ing the procedure described for compound 12. The crude residue
was purified by flash column chromatography by using light petro-
leum/EtOAc (4:6) as the eluent to afford product 27 as an oil
(128 mg, 0.42 mmol, 81%). Rf =0.80 (light petroleum/EtOAc, 4:6);
1H NMR (300 MHz, CDCl3): d=1.41–1.75 (m, 2H), 1.78–2.00 (m, 2H),
3.57–3.69 (m, 1H), 3.76 (d, J=10.3 Hz, 1H), 4.32–4.44 (m, 1H), 4.78
(d, J=10.3 Hz, 1H), 4.79–4.89 (m, 2H), 5.56–5.72 (m, 1H), 7.15–
7.63 ppm (m, 9H); 13C NMR (75 MHz, CDCl3): d=27.1, 30.6, 45.9,
57.2, 114.4, 115.3, 122.1, 124.2, 128.3, 129.2, 129.4, 130.3, 130.4,
131.3, 137.3, 138.8, 140.5, 170.1 ppm.
1-(Hex-5-enyl)-5-phenyl-1H-benzo[e][1,4]diazepin-2-(3H)-one
(28): Compound 28 was obtained from 11[18] (123 mg, 0.52 mmol),
NaH (18 mg, 0.78 mmol), and 19 (127 mg, 0.78 mmol) by employ-
ing the procedure described for compound 12. The crude residue
was purified by flash column chromatography by using light petro-
leum/EtOAc (4:6) as the eluent to afford product 28 as an oil
(127 mg, 0.40 mmol, 78%). Rf =0.86 (light petroleum/EtOAc, 4:6);
1H NMR (300 MHz, CDCl3): d=1.35–1.69 (m, 4H), 1.84–1.95 (m, 2H),
3.56–3.69 (m, 1H), 3.76 (d, J=10.3 Hz, 1H), 4.32–4.44 (m, 1H), 4.77
(d, J=10.3 Hz, 1H), 4.80–4.88 (m, 2H), 5.51–5.66 (m, 1H), 7.16–
7.62 ppm (m, 9H); 13C NMR (75 MHz, CDCl3): d=25.8, 27.4, 33.2,
46.3, 57.2,112.8, 114.7, 122.1, 124.2, 128.3, 129.4, 129.8, 130.4, 130.5,
131.9, 138.2, 138.7, 141.7, 170.1 ppm.
((R)-1-(4-((R)-3-Bromo-4,5-dihydroisoxazol-5-yl)butyl)-2,3-dihy-
dro-2-oxo-5-phenyl-1H-benzo[e][1,4]diazepin-3-yl)methyl
(4-
chloro-2-(trichloromethyl)phenyl)carbamate (7a) and ((R)-1-(4-
((S)-3-bromo-4,5-dihydroisoxazol-5-yl)butyl)-2,3-dihydro-2-oxo-5-
phenyl-1H-benzo[e][1,4]diazepin-3-yl)methyl
(4-chloro-2-(tri-
chloromethyl)phenyl)carbamate (7b): The diastereomeric mixture
of the intermediate was obtained from 25a,b (263 mg, 0.45 mmol)
and TBAF (1.12 mL of 1m solution in THF, 1.12 mmol) by employ-
ing the procedure described for 5a,b. The deprotected mixture
was then treated with 4-Cl,2-CF3C6H3NCO (199 mg, 136 mL,
0.90 mmol). The crude residue was purified by flash column chro-
matography by using light petroleum/EtOAc (8:2) as the eluent to
afford 7a,b as a solid (207 mg, 0.28 mmol, 62%). Rf =0.60 (light pe-
troleum/EtOAc, 6:4); 1H NMR (300 MHz, CDCl3): d=1.07–1.18 (m,
2H), 1.35–1.52 (m, 4H), 2.41 (dd, J=9.2, 16.7 Hz, 0.5H), 2.62 (dd,
J=8.7, 16.7 Hz, 0.5H), 2.96 (dd, J=9.2, 17.9 Hz, 0.5H), 3.17 (dd, J=
9.7, 17.9 Hz, 0.5H), 3.58–3.73 (m, 1H), 3.94 (t, J=6.5 Hz, 1H), 4.08–
4.24 (m, 1H), 4.36–4.55 (m, 1H), 4.92 (dd, J=7.1, 10.9 Hz, 1H), 5.07
(dd, J=6.9, 10.9 Hz,1H), 6.94 (brs, 1H), 7.31–7.67 (m, 11H),
8.12 ppm (d, J=8.9 Hz, 1H); MS: m/z 691.1 [M+H]+; elemental
analysis: calcd for C31H27BrClF3N4O4: C 53.81, H 3.93, N 8.10; found:
C 53.45, H 3.99, N 7.78.
1-(2-(3-Bromo-4,5-dihydroisoxazol-5-yl)ethyl)-5-phenyl-1H-
benzo[e][1,4]diazepin-2-(3H)-one ((ꢀ)-8): Compound (ꢀ)-8 was
obtained from 26 (133 mg, 0.46 mmol), DBF (187 mg, 0.92 mmol),
and NaHCO3 (154 mg, 1.84 mmol) by employing the procedure de-
scribed for compound (ꢀ)-3. The crude residue was purified by
flash column chromatography by using light petroleum/EtOAc
(6:4) as the eluent to afford compound (ꢀ)-8 as a solid (107 mg,
0.26 mmol, 56%). Rf =0.32 (light petroleum/EtOAc, 6:4); 1H NMR
(300 MHz, CDCl3): d=1.48–1.78 (m, 2H), 2.40 (dd, J=7.3, 17.2 Hz,
1H), 2.63 (dd, J=7.8, 17.2 Hz, 1H), 3.04–3.20 (m, 2H), 3.53 (d, J=
11.3 Hz, 1H), 4.14 (d, J=11.3 Hz, 1H), 4.17–4.45 (m, 1H), 7.19–7.40
(m, 5H), 7.43–7.59 (m, 3H), 7.98–8.08 ppm (m, 1H); 13C NMR
(75 MHz, CDCl3): d=29.7, 45.1, 46.6, 50.3, 79.5, 123.4, 126.5, 126.8,
127.8, 128.7, 129.5, 130.9, 131.0, 137.0, 138.2, 139.4, 139.8,
163.8 ppm; MS: m/z 412.1 [M+H]+; elemental analysis: calcd for
C20H18BrN3O2: C 58.27, H 4.40, N 10.19; found: C 57.96, H 4.56, N
10.02.
5-Phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one (11): The synthesis
of compound 11 was carried out according to a literature proce-
dure.[18] 1H NMR (300 MHz, CDCl3): d=4.10 (s, 2H), 7.08–7.28 (m,
3H), 7.34–7.60 (m, 6H), 10.54 ppm (brs, 1H); 13C NMR (75 MHz,
CDCl3): d=58.2, 121.8, 124.1, 127.5, 129.1, 130.1, 130.4, 132.4,
133.4, 139.6, 140.6, 140.7, 172.3 ppm.
1-(3-(3-Bromo-4,5-dihydroisoxazol-5-yl)propyl)-5-phenyl-1H-
benzo[e][1,4]diazepin-2-(3H)-one ((ꢀ)-9): Compound (ꢀ)-9 was
obtained from 27 (128 mg, 0.42 mmol), DBF (171 mg, 0.84 mmol),
and NaHCO3 (141 mg, 1.68 mmol) by employing the procedure de-
scribed for compound (ꢀ)-3. The crude residue was purified by
flash column chromatography by using light petroleum/EtOAc
(6:4) as the eluent to afford compound (ꢀ)-9 as a solid (94 mg,
0.22 mmol, 53%). Rf =0.29 (light petroleum/EtOAc, 6:4); 1H NMR
(300 MHz, CDCl3): d=1.34–1.49 (m, 2H), 1.46–1.74 (m, 2H), 2.50–
2.76 (m, 1H), 2.88–3.19 (m, 2H), 3.21–3.45 (m, 1H), 3.54 (d, J=
11.2 Hz, 1H), 4.13 (d, J=11.2 Hz, 1H), 4.29–4.52 (m, 1H), 7.19–7.39
(m, 5H), 7.44–7.57 (m, 3H), 7.98–8.07 ppm (m, 1H); 13C NMR
(75 MHz, CDCl3): d=24.1, 32.0, 45.8, 48.3, 51.9, 81.8, 123.1, 126.3,
126.9, 127.2, 128.7, 129.6, 130.7, 131.1, 136.6, 138.7, 139.4, 139.8,
163.6 ppm; MS: m/z 426.1 [M+H]+; elemental analysis: calcd for
C21H20BrN3O2: C 59.17, H 4.73, N 9.86; found: C 58.89, H 5.04, N
9.64.
1-(But-3-enyl)-5-phenyl-1H-benzo[e][1,4]diazepin-2-(3H)-one
(26): Compound 26 was obtained from 11[18] (123 mg, 0.52 mmol),
NaH (18 mg, 0.78 mmol), and 17 (105 mg, 0.78 mmol) by employ-
ing the procedure described for compound 12. The crude residue
was purified by flash column chromatography by using light petro-
leum/EtOAc (4:6) as the eluent to afford product 26 as an oil
(133 mg, 0.46 mmol, 89%). Rf =0.73 (light petroleum/EtOAc, 4:6);
1H NMR (300 MHz, CDCl3): d=2.08–2.42 (m, 2H), 3.62–3.71 (m, 1H),
3.75 (d, J=11.3 Hz, 1H), 4.40–4.52 (m, 1H), 4.77 (d, J=11.3 Hz, 1H),
4.79–4.93 (m, 2H), 5.50–5.66 (m, 1H), 7.16–7.73 ppm (m, 9H);
13C NMR (75 MHz, CDCl3): d=30.5, 45.9, 57.2, 114.3, 115.2, 122.1,
124.2, 128.3, 129.3, 129.4, 130.2, 130.4, 131.2, 137.3, 138.8, 140.8,
170.1 ppm.
1-(4-(3-Bromo-4,5-dihydroisoxazol-5-yl)butyl)-5-phenyl-1H-
benzo[e][1,4]diazepin-2-(3H)-one ((ꢀ)-10): Compound (ꢀ)-10 was
obtained from 28 (127 mg, 0.40 mmol), DBF (163 mg, 0.80 mmol),
1-(Pent-4-enyl)-5-phenyl-1H-benzo[e][1,4]diazepin-2-(3H)-one
(27): Compound 27 was obtained from 11[18] (123 mg, 0.52 mmol),
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1817 – 1825 1823

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and NaHCO3 (134 mg, 1.60 mmol) by employing the procedure de-
scribed for compound (ꢀ)-3. The crude residue was purified by
flash column chromatography by using light petroleum/EtOAc
(6:4) as the eluent to afford compound (ꢀ)-10 as a solid (88 mg,
0.20 mmol, 51%). Rf =0.34 (light petroleum/EtOAc, 6:4); 1H NMR
(300 MHz, CDCl3): d=1.02–1.17 (m, 2H), 1.30–1.57 (m, 4H), 2.74
(dd, J=8.7, 16.3 Hz, 1H), 2.88–3.03 (m, 1H), 3.18 (dd, J=10.3,
16.3 Hz, 1H), 3.24–3.38 (m, 1H), 3.52 (d, J=11.4 Hz, 1H), 4.13 (d,
J=11.4 Hz, 1H), 4.43–4.58 (m, 1H), 7.17–7.38 (m, 5H), 7.43–7.58 (m,
3H), 8.03 ppm (d, J=6.7 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=
22.5, 26.4, 34.3, 46.3, 48.3, 50.4, 81.7, 123.0, 126.2, 126.8, 127.1,
128.6, 129.5, 130.7, 131.1, 136.5, 138.7, 139.4, 139.8, 163.5 ppm;
MS: m/z 440.1 [M+H]+; elemental analysis: calcd for C22H22BrN3O2:
C 60.01, H 5.04, N 9.54; found: C 60.13, H 4.79, N 9.81.
described previously.[31] The assay was performed with synchron-
ized ring stage parasites, plated in triplicate in 96-well plates at
a parasitemia of 1% and 5% hematocrit. The compounds (dis-
solved in DMSO) were fivefold serially diluted and screened at con-
centrations ranging between 100 mm–1 nm. CQ (dissolved in
double-distilled water) was used as an internal control and 0.5%
DMSO was used as a negative control, which corresponded to the
final concentration of DMSO in all of the treated wells. The cultures
were incubated for 72 h at 378C under the above-mentioned con-
ditions. After incubation, a 20 mL aliquot of culture from each well
was transferred to a new 96-well plate, and the Malstat reagent
(100 mL) was added to each well. The assessment of parasite lac-
tate dihydrogenase (pLDH) activity was obtained by adding a mix-
ture of nitro blue tetrazolium (NBT)/diaphorase (20 mL; 1:1;
1 mgmLꢁ1 stock each) to the Malstat reaction, and the optical den-
sity (OD) values were measured at 630 nm (Infinite M200, Tecan).
The OD values were plotted against logarithmic concentrations of
the compounds, a dose–response curve was automatically generat-
ed, and the IC50 values were deducted by using GraphPad Prism.[32]
Biology
Inhibitory activity: The preliminary screening was performed with
20 mm inhibitor concentration, with an equivalent amount of
DMSO used as a negative control. The enzymes were recombinant-
ly expressed as previously described[22,23] Product release from sub-
strate hydrolysis (Cbz-Phe-Arg-AMC; 10 mm for rhodesain and
40 mm for FP-2) was determined continuously over a period of
10 min. Compounds showing at least 40% inhibition at 20 mm
were subjected to detailed assays. These were performed in
a 50 mm sodium acetate buffer (pH 5.5) containing 10 mm 1,4-di-
thiothreitol (DTT) with Cbz-Phe-Arg-AMC (10 or 40 mm) as the sub-
strate.[24] The Michaelis constant (Km) values used to calculate the Ki
values from the IC50 values were determined to be 0.9 mm (rhode-
sain)[25] and 21.5 mm (FP-2).[26]
Drug screening on T. b. brucei cultures: Trypomastigote forms of
T. b. brucei laboratory strain TC 221 were cultured in Baltz medium
according to standard conditions.[33] Trypanocidal activity was de-
termined by using the Alamar Blue assay[34,35] in a 96-well plate
format with a MR 700 Microplate ELISA Reader. Trypanosomes
were added to culture medium containing various concentrations
of test compound and 1% solvent to give a cell concentration of
104 cellsmLꢁ1 in a final volume of 200 mL. Positive controls com-
prised wells containing medium, 1% solvent, and trypanosomes;
negative controls comprised wells with test compounds but with-
out trypanosomes. After 24 h, Alamar Blue (20 mL) was added to
each well and the plates were incubated again for a further 48 h.
The absorbance was then measured at 550 nm with a reference
wavelength of 630 nm. IC50 values were calculated by linear inter-
polation as described previously.[36] Experiments were repeated
twice.
Inhibitor solutions were prepared from stocks in DMSO. Each assay
was performed twice in 96-well plates in a total volume of 200 mL.
The fluorescence of the AMC product of the substrate hydrolyses
was measured by using an Infinite 200 PRO microplate reader
(Tecan, Mꢂnnedorf, Switzerland) at room temperature with
a 380 nm excitation filter and a 460 nm emission filter. The dissoci-
ation constants (Ki) of the noncovalent enzyme–inhibitor complex
were obtained from progress curves (10 min) at various concentra-
tions of inhibitor by fitting the progress curves to the four-parame-
ter IC50 equation [Eq. (1)], in which y [dFminꢁ1] is the substrate hy-
drolysis rate, ymax is the maximum value of the dose–response
curve that is observed at very low inhibitor concentrations, ymin is
the minimum value that is obtained at high inhibitor concentra-
tions, and s is the Hill coefficient,[27] with correction to zero sub-
strate concentration from Ki =IC50/(1+[S]Kmꢁ1).
Cytotoxicity on J774.1 macrophages:[37] The macrophage cell line
J774.1 was maintained in complete Click RPMI medium. For the ex-
perimental procedures, cells were detached from the flasks with
a rubber police, washed twice with phosphate-buffered saline
(PBS), and suspended at 2ꢃ106 cells per mL in complete Click
RPMI medium. J774.1 macrophages were plated in complete RPMI
medium (200 mL) without phenol red in 96-well plates in the ab-
sence or presence of various concentrations of the compounds
and incubated for 24 h at 378C, 5% CO2, and 95% humidity. After
addition of Alamar Blue (20 mL), the plates were further incubated
under similar conditions. The plates were then read 24 and 48 h
later. Control experiments to examine the effect of cell density, in-
cubation time, and DMSO concentration were performed. Absorb-
ance in the absence of compounds was set as 100% of growth
control.
ymax ꢁ ymin
ꢀ
ꢁ
y ¼
þ ymin
s
½Iꢂ
ð1Þ
1 þ
IC50
Assays with cathepsins B and L (Calbiochem) were performed as
described previously.[28] Cbz-Phe-Arg-AMC was used as the sub-
strate (80 mm for cathepsin B; 5 mm for cathepsin L). The Km values
used to calculate the Ki values from the IC50 values were 150 mm
(cathepsin B) and 6.5 mm (cathepsin L).
Acknowledgements
Financial support from the Ministero dell’Istruzione, dell’Universi-
tꢀ e della Ricerca (MIUR), Italy (PRIN 2012) and from the Minis-
tero degli Affari Esteri, Direzione Generale per la Promozione del
Sistema Paese, Italy is gratefully acknowledged. T.S. acknowledg-
es financial support by the Deutsche Forschungsgemeinschaft
(DFG) (SFB 630). M.N.A. was funded by the Alexander von Hum-
boldt Foundation TPA4. The authors thank Antje Fuss and Svetla-
na Sologub from the Z1 project of the SFB 630, University of
Drug screening on P. falciparum cultures: Compounds were
screened for growth inhibition activity against P. falciparum NF54
by using the Malstat assay as previously described.[29,30] Parasites
were cultivated in Roswell Park Memorial Institute (RPMI) 1640
medium (Gibco) supplemented with hypoxanthine (Sigma–Aldrich),
A+ human serum, and gentamicin (Invitrogen), and cultures were
maintained at 378C in an atmosphere of 5% O2, 5% CO2, and 90%
N2. Cultures were synchronized by repeated sorbitol treatment as
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1817 – 1825 1824

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
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Wꢂrzburg, Germany, for performing the activity tests against Try-
panosoma and macrophages.
Keywords: antiprotozoal agents
· inhibitors · malaria ·
peptidomimetics · structure–activity relationships
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Received: March 21, 2014
Published online on June 11, 2014
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