Structure-Activity Relationship Studies of Retro-1 Analogues against Shiga Toxin

Article abstract of DOI:10.1021/acs.jmedchem.0c00298

High-throughput screening has shown that Retro-1 inhibits ricin and Shiga toxins by diminishing their intracellular trafficking via the retrograde route, from early endosomes to the Golgi apparatus. To improve the activity of Retro-1, a structure-activity relationship (SAR) study was undertaken and yielded an analogue with a roughly 70-fold better half-maximal effective concentration (EC50) against Shiga toxin cytotoxicity measured in a cell protein synthesis assay.

Full text of DOI:10.1021/acs.jmedchem.0c00298

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Article
Structure-activity relationship studies of Retro-1 analogs against Shiga toxin
Hajer Abdelkafi, Aurélien Michau, Valérie Pons, Flora Ngadjeua, Alexandra Clerget, Lilia Ait Ouarab, David-
Alexandre Buisson, David Montoir, Lucie Caramelle, Daniel Gillet, Julien Barbier, and Jean-Christophe Cintrat
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00298 • Publication Date (Web): 10 Jul 2020
Downloaded from pubs.acs.org on July 11, 2020
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Structure-activity relationship studies of Retro-1 analogs
against Shiga toxin
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‡
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Hajer Abdelkafi , Aurélien Michau , Valérie Pons , Flora Ngadjeua , Alexandra Clerget , Lilia
†
†
†
‡
‡,*
Ait Ouarab , David-Alexandre Buisson , David Montoir , Lucie Caramelle , Daniel Gillet ,
‡
†,*
Julien Barbier , and Jean-Christophe Cintrat
†Université Paris-Saclay, CEA, INRAE, Médicaments et Technologies pour la Santé (MTS),
SCBM, 91191 Gif-sur-Yvette, France
‡Université Paris-Saclay, CEA, INRAE, Médicaments et Technologies pour la Santé (MTS),
SIMoS, 91191 Gif-sur-Yvette, France
ABSTRACT
High-throughput screening has shown that Retro-1 inhibits ricin and Shiga toxins by diminishing
their intracellular trafficking via the retrograde route, from early endosomes to the Golgi apparatus.
In order to improve the activity of Retro-1, a SAR study was undertaken and yielded an analog
with a roughly 70-fold better EC50 against Shiga toxin cytotoxicity measured in a cell protein
synthesis assay.
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INTRODUCTION
Shiga toxins (Stx) are a family of structurally and functionally similar protein toxins produced by
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Shigella dysenteriae and some serogroups of Escherichia coli. Gram-negative bacteria producing
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Stx are pathogenic and responsible for a number of human foodborne diseases such as bloody
diarrhea and hemolytic uremic syndrome (HUS), the most feared complication of the infection
defined by acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia.2
Stx belong to the group of AB toxins and consist of a catalytically active A-subunit (StxA) and a
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binding pentameric B-subunit (StxB). After binding of StxB to the globotriaosylceramide (Gb3)
receptor at the cell surface, Stx is endocytosed and traffics through endosomes to the endoplasmic
3
reticulum (ER) by the retrograde pathway. Once in the ER, StxA translocates to the cytosol where
it arrests protein biosynthesis of host cells by enzymatically inactivating ribosomes.
Hitherto, no specific therapies are available to treat Stx intoxication. At the hospital, management
of HUS is based on general supportive care, and early dialysis for acute renal failure. Plasma
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,5
exchange has been explored without indication of its efficacy. Yet, the massive outbreak of Stx-
producing E. coli in Germany in 2011 allowed assessment of new drugs, such as the humanized
monoclonal antibody eculizumab directed against the complement protein C5 and approved for
the treatment of the genetic related disorder atypical HUS (aHUS). However, a retrospective study
concluded that there was no benefit for eculizumab-treated HUS patients.5
Therapeutics that hamper Stx binding, uptake, trafficking, translocation or enzymatic activity are
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highly relevant for the treatment of STEC infections (for a review see ). Thus, blocking of the
intracellular retrograde trafficking of Stx can be one viable strategy to arrest the intracellular action
of the toxin, as demonstrated either by the activity of chemical compounds (Figure 1), or by
manganese ions.7–16
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Figure 1. Chemical structures of some known cellular inhibitors of Shigatoxins. EC50 values or
active concentrations are indicated. NBT: Nitrobenzylthioinosine.
Identified by high-throughput screening, Retro-1 selectively blocked Stx retrograde trafficking at
the early endosome/trans-Golgi network (TGN) interface, thus protecting exposed cells from the
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cytotoxic action of Stx. Unlike various small molecules that inhibit intracellular Stx transport,
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Retro-1 did not perturb cellular morphology nor did it affect other trafficking pathways. Here, we
report on the development of a related compound Retro-1.1 with a similar mode of action and
conferring improved protective efficacy against Stx on human cells.
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RESULTS AND DISCUSSION
Synthesis of Retro-1. The synthesis of compound 6, Retro-1, was achieved starting from
the commercially available 2-amino, 5-bromo benzophenone 1 (Scheme 1) in 39% yield over 4
steps. First, regioselective bromination was performed with NBS with complete conversion.
Acetylation with bromoacetyl bromide was immediately followed by cyclization with ammonia to
yield the benzodiazepine 4 in 66% yield over two steps. Then, reduction of the imino moiety with
NaBH
3
CN yielded the two enantiomers of benzodiazepine 5 which were treated with propionyl
chloride to obtain Retro-1 as a mixture of two enantiomers. In addition, racemic Retro-1 was
1
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obtained as a 1:1 mixture of conformers as detected by H and C NMR. High-temperature NMR
allowed us to obtain coalescence of the two conformer signals (see supplementary materials).
Scheme 1. Synthetic route to Retro-1
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Evaluation of benzodiazepine drugs and derivatives. We first started to evaluate the
biological activities of a few commercially available (some are marketed drugs) related analogs of
Retro-1 along with other structural analogs that we synthesized (Figure 2). The latter were obtained
via the synthetic route depicted in Scheme 1 starting either from commercially available
aminobenzophenones or were synthesized in house (by a Friedel-Craft reaction between the
desired aniline and the corresponding benzonitrile). All tested molecules are depicted in Figure 2.
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Figure 2. Initial screening of commercially available benzodiazepinones and miscellaneous
derivatives
All of these compounds were inactive which strongly suggests that a benzodiazepine scaffold and
not a benzodiazepinone one is mandatory. All benzodiazepines were synthesized according to the
scheme 2:
Scheme 2. General procedure for the synthesis of 1,3,4,5-tetrahydro-2H-
benzo[e][1,4]diazepin-2-one.
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Modification of N4. We then checked the impact of the N4 amide substitution by
introducing small variations around the propionyl group.
Table 1. Preliminary evaluation of the substitution at N4
Yield
Protection
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EC50
Compound
R
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(µM)
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Unfortunately, the four amide analogs we synthesized proved inactive (Table 1, compounds 22,
4, 25) or less active than the parent Retro-1 (Table 1, compound 23) in Stx protection assays.
It should be reiterated that Retro-1 (see below), but also the analogs presented in Table 1
compounds 22-25), give rise to conformers, presumably due to the presence of the tertiary amide.
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(
We therefore decided to synthesize a reduced analog of Retro-1, i.e. compound 26. Compound 26
was indeed the best analog and showed slightly better protection compared to Retro-1 (6.2 µM).
This reduced analog not only yielded an active compound, but also got rid of conformers, thereby
simplifying the analysis. This propylated compound was therefore a good starting point for more
extensive SAR studies. Based on this N-propyl analog, we then investigated the impact of the N1
amide substitution (Table 2).
Substitution at N1. The synthesis of N1-substituted benzophenones were achieved via
either classical alkylation reaction from diazepinones (see experimental part for conditions) or
Ullmann arylation according to scheme 3:
Scheme 3. Synthesis of N1-substituted diazepin-2-one from 7-bromo-5-phenyl-4-propyl,
1
,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one.
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None of the analogs we synthesized in this series proved active against Shiga toxin, with
aliphatic (Table 2, compound 27), allylic or benzylic (compounds 32 and 33) or
aromatic/heteroaromatic (compounds 28-31) substituents. These results suggest that the NH bond
is crucial for bioactivity and may be involved in H bonding with the target(s) or may point to a
small hydrophilic cavity.
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Table 2. Modification on the N1 amide
Yield
Protection
(%)
EC50
(µM)
n.d.
Compound
R
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Modification of the aromatic rings. Faced with these rather disappointing results, we
decided to evaluate the impact of the BZD phenyl ring substitution. The latter was functionalized
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either by palladium-catalyzed cross-coupling, taking advantage of the bromine at C7, or by the use
of initially functionalized 2-aminobenzophenones according to scheme 4.
Scheme 4. Late stage functionalization of 7-bromo-5-phenyl-4-propyl, 1,3,4,5-tetrahydro-
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Table 3. Evaluation of the substitution at the phenyl benzodiazepine ring
Yield Protection
EC50
Compound
Structure
(%)
(%)
(µM)
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Based on palladium coupling reactions, various aromatic (compounds 34, 35, 38) and
heteroaromatic (compounds 36, 37) compounds were synthesized, but were completely inactive.
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-Bromo or 8-methoxy derivatives of compound 29 were also obtained (compounds 39, 40), but
did not protect cells against Stx. Substitution of the bromide at C7 by a chloride, iodide or azide
maintained the protection of the cells against Stx (compounds 41, 42, 43).
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Substitution at C5. Because of the difficulty of late-stage modifications, we decided to
obtain some benzodiazepines modified at C5 from different 2-amino, 5-bromobenzophenone
analogs. The latter were either commercially available (Table 4, compounds 45, 46, 48) or obtained
by Friedel-Craft reaction between 5-bromoaniline and the corresponding benzonitrile (compound
4
4).
Table 4. Evaluation of substitution at C5
Yield Protection
EC50
Compound
R
(%)
(%)
(µM)
4
4
66
14.4
n.d.
n.d.
45
56
inactive
4
4
6
7
62
71
>100
59.6
3.87±0.01
n.d.
4
8
21
>100
12.82±0.21
Compound 47 was obtained via a directed palladium iodination reaction previously developed in
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our group. Except for a 2-monofluorinated analog (compound 46) and a 2,4-difluorinated analog
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(
compound 48), all compounds tested proved less potent than compound 29. Despite the fact that
the fluoride atom is slightly larger than the hydrogen atom, little change to the steric bulk of the
molecule is usually seen. The biggest changes in bioactivity are usually due to a large
electronegativity difference between these two atoms (besides the higher lipophilicity of fluoride),
but here there seems to be no influence of this parameter on bioactivity against Stx, though better
cell membrane permeation cannot be ruled out.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Modification at the N4 secondary amine. In a final round of the SAR study, based on
previous experience with the reduction of the propionyl substituent (see Table 2), we examined
more extensively the effect of substitution on the secondary amine. The substituents were
introduced by either reductive amination or nucleophilic substitution from compound 5 according
to scheme 5:
Scheme 5. Introduction of substituents at N4.
Table 5. Structure-activity relationship of compounds modified at N4
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1
1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Yield Protection
EC50
Compound
49
R
(%)
(%)
(µM)
50
20.6
n.d.
n.d.
n.d.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
5
0
1
79
91
5.3
38.8
52
85
74
inactive
29.9
n.d.
n.d.
5
5
3
4
86
88
92
88
58.2
12.2
n.d.
n.d.
n.d.
n.d.
55
5
5
6
7
2.9
Inactive
5
5
8
9
95
78
Inactive
Inactive
n.d.
n.d.
5
H
74
90
23
85
Inactive
Inactive
85.8
n.d.
n.d.
6
6
6
0
1
2
8.81±2.70
n.d.
29.5
6
3
62
24
Inactive
>100
n.d.
64
4.70±0.01
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64
>100
>100
5.16±0.64
0.60±0.04
66
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
46
Inactive
n.d.
6
6
7
8
9
0
57
63
72
>100
>100
1.29±0.26
3.91±0.63
n.d.
Inactive
7
7
7
1
2
3
47
100
55
Inactive
Inactive
Inactive
n.d.
n.d.
n.d.
74
38
>100
6.50±0.14
Numerous analogs were synthesized and tested. First, compounds bearing an indole ring were
obtained, but were less active than Retro-1 (compound 49). Then, pyridine substituents were
introduced (4-substituted, compound 50 or 3-substituted pyridine, compound 51), but neither
compound showed improved efficiency. The naphthyl derivative 52 was completely inactive. A
closely related structure, the quinoline moiety 53, showed lower activity than Retro-1. A few small
heterocyclic thiophenyl (compounds 54, 55) and furyl (compounds 56, 57) substituents were
synthesized and screened, but without any improvement. A phenyl substituent was introduced with
either a one-methylene (compound 58) or three-methylene (compound 59) linker, but with
complete loss of activity. Many aliphatic substituents (compounds 60-70) were introduced and we
obtained more potent inhibitors, especially with acyclic or cyclic derivatives (compounds 66, 68,
69). It should be noted that more sterically demanding ramified aliphatic chains (compounds 63,
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1
1
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2
2
2
2
2
2
3
3
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3
3
3
3
3
4
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4
4
4
4
4
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5
5
5
5
5
5
5
6
6
7) and substituted aliphatic rings (compound 70) gave lower bioactivity. The most promising
candidates in this series were cyclopentyl derivatives (compounds 66, 68, 69). Finally,
functionalized aliphatic chains were inactive (compounds 71-73), or less active (compound 74).
0
1
2
3
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8
9
0
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9
0
Halide effect. Having the best analog in hand (compound 66), we focused our attention on
the effect of bromide, since we already experienced a change in bioactivity with different halogens
at C7 (see Table 4, compounds 41, 42). Therefore, we synthesized analogs containing respectively
no halide (75), fluoride (76), chloride (77) and iodide (78). The iodinated analog (compound 78)
afforded an improved EC50 value of around 300 nM, suggesting that a putative X-bond with
electron-rich groups present in the target(s) might account for this ranking of halogenated
derivatives (Figure 3).18,19
Figure 3. Effect of halogen at C7 on the bioactivity against Shiga toxin
Since compound 78 was the best analog of Retro-1 synthesized during this SAR study, we decided
to name it Retro-1.1.
Separation of enantiomers and attribution of configuration and biological activities.
A chiral phase separation of the enantiomers of Retro-1.1 was carried out on a ChiralPak IA HPLC,
which allowed us to obtain two enantiomers, Retro-1.1.a and Retro-1.1.b (Figure S1).
In order to determine the absolute configuration of each enantiomer, we decided to take advantage
of the enantiomers of Retro-1 (scheme 6), an assignment that was previously achieved by X-ray
crystallography.17
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Scheme 6. Strategy to determine the absolute configurations of compounds 5 and 78 (Retro-
1.1)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Step 1: We synthesized the two enantiomers of Retro-1 from the separated enantiomers of 5 which
allowed us retrospectively to identify (S)-5 and (R)-5.
The configurations of
The configurations
compound 5
enantiomers were
determined thanks to
this chemical
of Retro-1
Known configurations
Unknown configurations
enantiomers were
previously known
thanks to X-ray
crystallography
transformation
Step 2: With the assignment of compound 5 enantiomers in hand, we decided to perform a two-
step chemical modification, knowing that no inversion of configuration could occur during these
transformations, which allowed us to obtain an HPLC profile of (S)-Retro-1.1 and (R)-Retro-1.1.
The configurations
The
configurations
are known
thanks to the
previous step
of Retro-1.1
enantiomers were
determined thanks to
these chemical
Known configurations
Unknown configurations
transformations
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5
5
5
5
5
5
6
HeLa cells were then challenged against Stx in the presence of each enantiomer of Retro-1.1
(Figure 4). (S)-enantiomer of Retro-1.1 proved to be the eutomer, as already experienced with
Retro-1.17
0
1
2
3
4
5
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9
0
1
2
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Evaluation of protective activity towards Stx cytotoxicity of each enantiomer of Retro-
1
.1. HeLa cells were incubated for 4 hours with racemic Retro-1.1 (grey), (R)-Retro-1.1 (white),
or (S)-Retro-1.1 (black) before the addition of Stx for 20 hours. Medium was removed and replaced
1
4
by DMEM containing [ C]-leucine at 0.5 µCi/mL for 7 hours before counting. Each data point
represents the mean of duplicate ± SD of two independent experiments.
To prove that the mode of action of Retro-1.1 obtained herein prevents the deleterious effect of
Stx by blocking its intracellular trafficking through the retrograde pathway, as reported for Retro-
1, we examined the subcellular distribution of fluorescently labeled Stx. These experiments
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showed that Shiga toxin is not able to reach the Golgi apparatus in the presence of (S)-Retro-1.1,
whereas (R)-Retro-1.1 appeared unable to block Stx trafficking inside cells (Figure 5, Figure S2).
1
1
1
1
1
1
1
1
1
1
2
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5
5
5
5
5
5
5
6
0
1
2
3
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0
1
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0
1
2
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. (±)-Retro-1.1 and (S)-Retro-1.1 block the retrograde transport of Shiga toxin. Cells were
pretreated for 1 hour with (±)-Retro-1.1 (upper panel), (S)-Retro-1.1 (middle panel) or (R)-Retro-
1
.1 (lower panel) at 1 µM before addition of Alexa488-labeled StxB (0.1 µg/mL, green). Cells
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
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4
4
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4
5
5
5
5
5
5
5
5
5
5
6
were fixed with 4% PFA, and labeled with phalloidin-Atto-550 (red) and DAPI (blue) for actin
and nuclei staining, respectively. Scale bar, 20 µm.
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
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0
CONCLUSION
Based on our SAR study, we were able to obtain benzodiazepinones that afforded cells greater
protection against Shiga toxin than the parent molecule Retro-1. This SAR study shows that a
halogen atom at C7 is mandatory. The most active compound, (S)-Retro-1.1, provides an EC50 of
9
0 nM corresponding to a 70-fold improvement compared to the parent Retro-1. We also show
that this compound blocks the retrograde trafficking of Shiga toxin. Experiments are in progress
to decipher the mode of action of this compound and to identify its cellular target(s). As Retro-1
blocks retrograde trafficking similarly to Retro-2, it would be worth testing optimized Retro-1
compounds against various pathogens, in particular viruses such as poxvirus, cytomegalovirus and
enterovirus 71, for which Retro-2 derivatives proved efficient.20–22
EXPERIMENTAL SECTION
Synthesis
All chemicals and solvents used in the syntheses were reagent grade and were used without
additional purification. THF and CH
2
Cl were distilled respectively from sodium/benzophenone
2
ketyl and calcium hydride before use. Glassware was flame-dried under vacuum and cooled under
nitrogen to room temperature. All reactions were performed under dry nitrogen gas and monitored
by thin-layer chromatography (TLC). TLC was performed with precoated TLC silica gel 60 F254,
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and organic compounds were visualized by UV light (254 nm), iodine vapor, or phosphomolybdic
acid [10% (w/v) in ethanol] staining with heating.
Large-scale purification was performed on a CombiFlash with a UV-visible detector with RediSep
columns. The samples were adsorbed on Celite or silica and loaded into solid load cartridges. An
ethyl acetate/cyclohexane or methanol/methylene chloride gradient was employed. Fractions were
collected based on UV detection at 254 nm.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HPLC-MS analysis and purification were performed using a Waters system (2525 binary gradient
module, in-line degasser, 2767 sample manager, 2996 Photodiode Array Detector) with a binary
gradient solvent delivery system. This system was coupled with a Waters Micromass ZQ system
with a ZQ2000 quadrupole analyzer. The ionization was performed by electrospray and the other
parameters were as follows: source temperature 120 °C, cone voltage 20 V, and continuous sample
injection at 0.3 mL/min flow rate. Mass spectra were recorded in both positive and negative ion
mode in the m/z 100-2,000 range and treated with the Mass Lynx 4.1 software.
The eluent was a gradient of (99.9% water / 0.1% HCOOH) and (99.9% MeCN / 0.1% HCOOH)
or (99.9% water / 0.1% HCOOH) and (99.9% MeOH / 0.1% HCOOH). Each compound was
applied to a 100 x 4.6 mm (5 µm) WATERS XBridge C18 column equilibrated with H
or H O/MeOH 95:5.
2
O/MeCN
2
Gradient A: Samples were eluted by increasing MeOH to 100% (25 min) then 100% (5 min).
Gradient B: Samples were eluted by increasing MeOH to 90% (24 min), then 100% (1 min) and
then staying at 100% (5 min).
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9
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1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Gradient C: Samples were eluted by increasing MeOH to 80% (24 min), then 100% (1 min) and
then staying at 100% (5 min).
Gradient D: Samples were eluted by increasing MeCN to 100% (25 min) then 100% (1 min).
Gradient E: Samples were eluted by increasing MeCN to 90% (24 min), then 100% (1 min) and
then staying at 100% (5 min).
0
1
2
3
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1
2
3
4
5
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8
9
0
Gradient F: Samples were eluted by increasing MeCN to 80% (24 min), then 100% (1 min) and
then staying at 100% (5 min).
Gradient G: Samples were eluted by increasing MeCN to 60% (24 min), then 100% (1 min) and
then staying at 100% (5 min).
HPLC (chiral) analyses were performed on a system equipped with a binary gradient solvent
delivery system (LC-20AB, Shimadzu), an SIL-20A autosampler (Shimadzu), and a photodiode
array detector (SPD-20A, Shimadzu).
The purity of the compounds was assessed by UPLC/UV (DAD 210 – 400 nm) using a Waters
Acquity system equipped with a BEH XBridge C18 column (1.7 µM, 2.1*50 at 40 °C) and purity
was ≥ 95% from the analysis detection mode (compounds 65, 67, 68, 69 were mixtures of
diastereomers). Elution conditions were as follow:
Solvent:
T0
A: H
2
O+1/1000 HCO
2
H, B: ACN+1/1000 HCO H
2
0.4 mL/min
0.4 mL/min
0.6 mL/min
0.6 mL/min
95%A 5%B
T3min
T3.1min
T4min
0%A 100%B
0%A 100%B
0%A 100%B
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1
NMR experiments were performed on a Bruker Avance 400 Ultrashield spectrometer. H-NMR
1
3
and C spectra were recorded at room temperature at 400 MHz and 100 MHz, respectively, with
samples dissolved in DMSO-D6 at a concentration of approximately 5 mM. The DMSO singlet
signal was set up at 2.50 ppm. Chemical shifts are given in ppm and the coupling constants in Hz.
Spectral data are consistent with assigned structures.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
High-resolution mass spectrometry (HRMS) was performed on an ESI/TOF LCP premier XE mass
spectrometer (Waters) using flow injection analysis mode.
Physicochemical properties were calculated using MarvinSketch 5.4.1.1 software (ChemAxon)
High-resolution mass spectrometry (HRMS) was performed using the imagif platform (CNRS,
Gif-sur-Yvette, France), and recorded on an ESI/TOF LCP premier XE mass spectrometer
(Waters) using flow injection analysis mode.
For procedure (a): see the synthesis of Compound 4
For procedure (b): see the synthesis of Compound 6
For procedure (c): see the synthesis of Compound 26
For procedure (d): see the synthesis of Compound 28
For procedure (e): see the synthesis of HA467
For procedure (f): see the synthesis of Compound 34
For procedure (g): see the synthesis of HA244
7
-Bromo-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (4)
To a solution of 2-aminobenzophenone (10.14 mmol, 2 g) in dichloromethane (100 mL) was added
N-bromosuccinimide (10.14 mmol, 1.8 g) at 0 °C. The mixture was stirred for 1 hour at this
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5
6
temperature and for 2 hours at room temperature. The organic layer was washed with water (20
mL), dried over Na
2
SO , filtered and concentrated under vacuum. The crude mixture was used in
4
the next step without purification.
0
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3
4
5
6
7
8
9
0
Procedure (a): To a solution of 5-bromo-2-aminobenzophenone 2 (10.14 mmol) in
dichloromethane (100 mL) was added bromoacetyl bromide (12.16 mmol, 1.27 mL) followed by
a 2M aqueous solution of Na
2
CO
3
at 0 °C. The mixture was stirred for 2 hours at this temperature.
SO , filtered and
The organic layer was separated and washed with water, dried over Na
2
4
concentrated under vacuum to give 5-bromo-2-bromoacetamidebenzophenone 3 as a yellowish
solid. At 0 °C, 5-bromo-2-bromoacetamidebenzophenone 3 (10.14 mmol) was dissolved in a
solution of NH (7M in MeOH, 130 mL) and the mixture was stirred for 1 hour at this temperature
3
and then allowed to warm up to room temperature overnight. The crude mixture was dried under
vacuum, diluted in ethyl acetate and washed with water. The organic layer was concentrated under
vacuum, dried over Na
2
SO , filtered and concentrated under vacuum. The crude mixture was
4
purified by flash chromatography (cyclohexane-ethyl acetate, 1-1). The desired compound 4 was
obtained as a white solid (2.11 g, 66%).
1H-NMR (400 MHz, (CD
)
2
SO) δ ppm: 10.66 (s, 1H), 7.76 (dd, J = 8.7 Hz, J = 2.3 Hz, 1H),
3
7
.61-7.30 (m, 5H), 7.33 (d, J = 2.3 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 4.16 (s, 2H)
13C-NMR (400 MHz, (CD
3
2
) SO) δ ppm: 170.6, 168.8, 139.5, 139.1, 133.08, 129.6, 128.6, 123.8,
114.8, 57.5
-1
I.R. (neat, cm ) 3430, 2951, 1680, 1605, 1476, 1381, 1355, 1319, 1285, 1259, 1233, 1193, 1082,
1
012, 946, 895
+
HRMS m/z [(M+H) ] calcd for C15
H
11BrN O 315.0133, found 315.0134
2
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-Bromo-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (5)
To a solution of 4 (1.58 mmol, 500 mg) in methanol (15 mL) was added NaBH CN (2.37 mmol,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
150 mg) followed by acetic acid (7.9 mmol, 440 µL) dropwise. The mixture was stirred at room
temperature until complete conversion of the starting material. The mixture was than evaporated
to dryness, diluted in ethyl acetate and washed with a saturated solution of NaHCO
3
, then water.
The organic layer was concentrated under vacuum, dried over Na SO , filtered and concentrated
2
4
under vacuum. The crude mixture was purified by flash chromatography (cyclohexane-ethyl
acetate, 1-1 to 1-2). The desired compound 5 was obtained as a white solid (370 mg, 74%).
1H-NMR (400 MHz, (CD
.83 (d, J = 2.14 Hz, 1H), 5.23 (d, J = 4.9 Hz, 1H), 3.68 (s br, 1H), 3.39 (dd, J = 15.7 Hz, J = 5.4
Hz, 1H), 3.26 (dd, J = 15.7 Hz, J = 8.0 Hz, 1H)
3
2
) SO) δ ppm: 9.96 (s, 1H), 7.44-7.26 (m, 6H), 7.06 (d, J = 8.5 Hz, 1H),
6
13C-NMR (400 MHz, (CD
3
2
) SO) δ ppm: 173.6, 141.9, 136.6, 128.8, 127.8, 122.9, 115.5, 61.7,
50.8
-1
I.R. (neat, cm ) 3441, 3309, 3258, 3208, 3150, 3095, 3064, 2947, 2825, 1675, 1578, 1482, 1380,
1
284, 1248, 1227, 1173, 119, 1076, 1052, 1027, 948, 913, 880, 855
+
HRMS m/z [(M+H) ] calcd for C15
H
14BrN O 317.0290, found 317.0291
2
Procedure (b): 7-Bromo-5-phenyl-4-propionyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-
-one (6)
2
To a solution of compound 5 (0.315 mmol, 100 mg) in dichloromethane (3 mL) was added
propionyl chloride (0.410 mmol, 36 µL) followed by triethylamine (0.315 mmol, 44 µL). The
mixture was allowed to stir at room temperature overnight. A solution of saturated NaHCO
3
was
added and the organic layer was extracted with dichloromethane, dried over Na
2
SO
4
, filtered, then
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
concentrated under vacuum. The crude mixture was washed with diethyl ether. The crude mixture
was purified by flash chromatography (cyclohexane-ethyl acetate, 5-1 to 1-1) furnishing the
desired compound 6 as a white solid (374 mg, 79%).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1H-NMR (400 MHz, (CD
J = 1.9 Hz, 1H), 7.23-7.32 (m, 3H), 7.04 (dd, J = 8.5 Hz, J = 14 Hz, 3H), 6.57 (s, 1H), 4.09 (q, J
15.7 Hz, 2H), 1.05 (t, J = 7.3 Hz, 3H)
3
2
) SO) δ ppm 100 °C: 9.65 (s, 1H), 7.65 (s, 1H), 7.47 (dd, J = 8.7 Hz,
=
13C-NMR (400 MHz, (CD
133.8-133.4, 132.0-131.7, 131.7-131.6, 128.7-128-4, 127.5-127.2, 126.6-126.4, 123.5- 123.5,
15.9-115.8, 61.4-59.2, 49.2-46.1, 26.2-25.7, 9.3-9.2
3
2
) SO) δ ppm: 172.9-172.3, 168.8-168.3, 139.3-139.1, 136.7- 136.5,
1
-1
I.R. (neat, cm ) 3198, 3131, 3055, 2973, 2935, 1684, 1628, 1491, 1449, 1421, 1382, 1331, 1315,
1
244,
1
201, 1129, 1082, 1023, 964, 921, 892, 827
HRMS m/z [(M+H)+] calcd for C18
H18BrN
2
2
O 373.0552, found 373.0554
7
,9-Dibromo-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (19)
To a solution of 2-amino-5-bromobenzophenone 2 (1.014 mmol, 200 mg) in dichloromethane (10
mL) was added N-bromosuccinimide (1.014 mmol, 189 mg) at 0 °C. The mixture was stirred for
1
hour at this temperature and for 2 hours at room temperature. The organic layer was washed with
water (20 mL), dried over Na SO , filtered and concentrated under vacuum. The crude mixture
2
4
was used in the next step without purification. Then, according to procedure (a), 120 mg of the
desired compound was obtained (0.304 mg, 30% over 2 steps).
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1H-NMR (400 MHz, CDCl
) δ (ppm) = 9.98 (s, 1H), 8.19 (d, J = 2.2 Hz, 1H), 7.57-7.41 (m, 5H),
3
7
.37 (d, J = 2.2 Hz, 1H), 5.23 (s, 1H), 4.49 (m, 1H), 3.89 (m, 1H)
13C-NMR (150 MHz, CDCl
) δ (ppm) = 169.3, 167.5, 138.1, 137.0, 136.6, 130.6, 129.1, 128.4,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
117.8, 115.5, 56.7, 26.3
-1
I.R. (neat, cm ) 3367, 3204, 3073, 1688, 1607, 1579, 1461, 1446, 1379, 1317, 1231, 1175, 1151,
1
011, 858, 736
+
HRMS m/z [(M+H) ] calcd for C15
H11Br
2
2
N O 329.9238 found 329.9233
5
-(2-Iodophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (15)
To a solution of 5-phenyl-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (30 mg, 0.12
mmol) in CH CN (1.2 mL) were added Pd(OAc) (2.7 mg, 0.012 mmol) and N-iodosuccinimide
54 mg, 0.24 mmol). The mixture was stirred at 100 °C for 15 minutes. The crude mixture was
3
2
(
evaporated, diluted in ethyl acetate (10 mL), and washed with a 2M aqueous solution of NaOH (5
mL). The residue was purified by flash chromatography (cyclohexane/ethyl acetate 1:1), affording
3
0 mg (69%) of the desired compound.
1H-NMR (400 MHz, CD
CN) δ (ppm) = 7.83 (d, J = 8.0 Hz, 1H), 7.56 (dt’, J = 1.5Hz, J = 8.7
3
Hz, 1H), 7.51-7.45 (m, 2H), 7.42 (d, J = 8.2 Hz, 1H), 7.15 (ddd, J = 9.2 Hz, J = 6.4 Hz, J = 2.8
Hz, 1H), 7.09 (dt’, J = 7.9Hz, J = 0.9Hz, 1H), 6.94 (dd, J = 7.8 Hz, J = 1.4 Hz, 1H), 4.58 (d, J =
1
0.6 Hz, 1H), 3.76 (d, J = 10.6 Hz, 1H), 3.39 (s, 3H)
13C-NMR (75 MHz, CD
CN) δ (ppm) = 173.5, 170.1, 145.5, 140.3, 132.5, 131.6, 130.1, 129.6,
3
129.3, 124.9, 122.6 (C-9), 118.3 (C-9a), 96.8 (C-2’), 57.7 (C-3), 35.1 (1-CH )
3
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-1
I.R. (neat, cm ) 3057, 2988, 2850, 1676, 1611, 1573, 1489, 1449, 1361, 1324, 1280, 1201, 1167,
1
128, 1076, 1046, 1014, 984, 939, 915
+
HRMS m/z [(M+H) ] calcd for C16
H
14IN O 377.0151 found 377.0145
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5-(p-Tolyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (20)
According to procedure (a): From 2-amino-4’-methylbenzophenone (0.946 mmol, 200 mg), 124
mg of the desired product was obtained (0.870 mmol, 92% over 2 steps)
1H-NMR (400 MHz, (CD
.37 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 6.9 Hz, 4H), 7.17 (t, J = 7.4 Hz, 1H), 5.23 (s, 1H), 4.14-4.01
(m, 2H), 2.35 (s, 3H)
3
2
) SO) δ (ppm) = 10.52 (s, 1H), 7.56 (dt, J = 8.4 Hz, J = 1.4 Hz, 1H),
7
13C-NMR (150 MHz, CDCl
29.0, 127.0, 123.4, 121.4, 56.2, 21.5
) δ (ppm) = 172.1, 171.5, 141.1, 139.1, 136.2, 132.1, 131.7, 130.0,
3
1
-1
I.R. (neat, cm ) 3182, 3104, 3059, 2973, 2923, 2843, 1675, 1603, 1577, 1484, 1442, 1428, 1322,
1299, 1181, 1020, 1006, 924
+
HRMS m/z [(M+H) ] calcd for C30
H
27
N
3
2
O 251.1184 found 251.1172
7
-Chloro-5-(2-chlorophenyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (16)
According to procedure (a): From 2-amino-2’,5-dichlorobenzophenone (1.87 mmol, 500 mg),
20 mg of the desired product was obtained (0.729 mmol, 39% over 2 steps)
2
1H-NMR (400 MHz, (CD
3
2
) SO) δ (ppm) = 10.79 (s, 1H), 7.62-7.49 (m, 5H), 7.25 (d, J = 8.7 Hz,
1
H), 6.91 (d, J = 2.4 Hz, 1H), 4.21 (s, 2H)
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-1
I.R. (neat, cm ) 3210, 3123, 3072, 2928, 2851, 1688, 1616, 1591, 1569, 1482, 1434, 1387, 1325,
1
230, 1195, 1059, 951
+
HRMS m/z [(M+H) ] calcd for C15
H11Cl
2
2
N O 305.0248 found 305.0240
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7-Bromo-5-(p-tolyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (21)
To a solution of 2-amino-4’-methylbenzophenone (0.946 mmol, 200 mg) in dichloromethane (10
mL) was added N-bromosuccinimide (0.946 mmol, 168 mg) at 0 °C. The mixture was stirred for
1
hour at this temperature and 2 hours at room temperature. The organic layer was washed with
water (20 mL), dried over Na SO , filtered and concentrated under vacuum. The crude mixture
2
4
was used in the next step without purification. Then, according to procedure (a), 209 mg of the
desired product was obtained (0.633 mmol, 67%)
1H-NMR (400 MHz, (CD
(d, J = 8.1 Hz, 2H), 7.32 (d, J = 2.2 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 8.7 Hz, 1H),
.18-4.04 (s, 2H), 2.36 (s, 3H)
3
2
) SO) δ ppm: 10.63(s, 1H), 7.75 (dd, J = 8.7 Hz, J = 2.3 Hz, 1H), 7.37
4
13C-NMR (400 MHz, CDCl
28.9, 123.1, 116.2, 56.5, 21.2
) δ ppm: 172.3, 169.8, 141.1, 138.0, 136.0, 134.7, 129.7, 129.2,
3
1
-
1
IR (neat, cm ) 3209, 3117, 3049, 2922, 2853, 1682, 1604, 1567, 1478, 1381, 1346, 1320, 1230,
1
182, 1021, 1011, 945
+
HRMS m/z [(M+H) ] calcd for C16
H
14BrN O 329.0290 found 329.0282
2
7
-Chloro-5-(2-chlorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (17)
To a suspension of NaH (17 mg, 0.708 mmol) in THF (4 mL) was added at 0 °C 7-chloro-5-(2-
chlorophenyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (100 mg, 0.329 mmol). After 30
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