Optimization of Hydantoins as Potent Antimycobacterial Decaprenylphosphoryl-β-d-Ribose Oxidase (DprE1) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c00107

In search of novel drugs against tuberculosis, we previously discovered and profiled a novel hydantoin-based family that demonstrated highly promising in vitro potency against Mycobacterium. tuberculosis. The compounds were found to be noncovalent inhibitors of DprE1, a subunit of decaprenylphosphoryl-β-d-ribose-2′-epimerase. This protein, localized in the periplasmic space of the mycobacterial cell wall, was shown to be an essential and vulnerable antimycobacterial drug target. Here, we report the further SAR exploration of this chemical family through more than 80 new analogues. Among these, the most active representatives combined submicromolar cellular potency and nanomolar target affinity with balanced physicochemical properties and low human cytotoxicity. Moreover, we demonstrate in vivo activity in an acute Mtb infection model and provide further proof of DprE1 being the target of the hydantoins. Overall, the hydantoin family of DprE1 inhibitors represents a promising noncovalent lead series for the discovery of novel antituberculosis agents.

Full text of DOI:10.1021/acs.jmedchem.0c00107

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Article
Optimization of hydantoins as potent antimycobacterial
decaprenylphosphoryl-#-D-ribose oxidase (DprE1) inhibitors
Olga B Balabon, Eleni Pitta, Maciej K. Rogacki, Eugenia Meiler, Ruth Casanueva, Laura Guijarro,
Sophie Huss, Eva Maria Lopez-Roman, Angel Santos-Villarejo, Koen Augustyns, Lluis Ballell,
David Barros Aguirre, Robert Bates, Fraser Cunningham, Monica Cacho, and Pieter Van der Veken
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00107 • Publication Date (Web): 28 Apr 2020
Downloaded from pubs.acs.org on April 28, 2020
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Optimization of hydantoins as potent
antimycobacterial decaprenylphosphoryl-β-D-ribose
oxidase (DprE1) inhibitors
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Olga Balabon,† Eleni Pitta,†‡ꢁꢀ Maciej K. Rogacki,†‡ꢀ Eugenia Meiler, Ruth Casanueva, Laura
‡ꢀ
‡
‡
‡
‡
‡
‡
†
Guijarro, Sophie Huss, Eva Maria Lopez-Roman, Ángel Santos-Villarejo, Koen Augustyns,
‡
‡
‡
‡
‡*
Lluis Ballell, David Barros Aguirre, Robert H. Bates, Fraser Cunningham, Monica Cacho,
Pieter Van der Veken† *
†Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of
Antwerp, Universitieitsplein 1, 2610 Wilrijk, Belgium
‡
Global Health R&D, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, 28760 Madrid, Spain
Keywords: anti-mycobacterial, DprE1, drug discovery, hydantoin, tuberculosis, mycobacterium.
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ABSTRACT
In search of novel drugs against tuberculosis, we previously discovered and profiled a novel
hydantoin-based family that demonstrated highly promising in vitro potency against M.
tuberculosis. The compounds were found to be non-covalent inhibitors of DprE1, a subunit of
decaprenylphosphoryl-β-D-ribose-2′-epimerase. This protein, localized in the periplasmic space
of the mycobacterial cell wall, was shown to be an essential and vulnerable antimycobacterial drug
target. Here, we report the further SAR exploration of this chemical family through more than 80
new analogues. Among these, the most active representatives combined sub-micromolar cellular
potency and nanomolar target affinity with balanced physicochemical properties and low human
cytotoxicity. Moreover, we demonstrate in vivo activity in an acute Mtb infection model and
provide further proof of DprE1 being the target of the hydantoins. Overall, the hydantoin family
of DprE1 inhibitors represents a promising non-covalent lead series for the discovery of novel
antituberculosis agents.
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INTRODUCTION
Tuberculosis (TB), a disease primarily caused by the pathogen Mycobacterium tuberculosis, is
among the top 10 causes of death worldwide, while remaining the leading cause of death from a
1
single infectious agent, as reported by the World Health Organization (WHO). Around 10.0
million people developed TB globally in 2018, with an estimated 1.3 million TB deaths among
HIV-negative people and an additional 300 000 deaths among HIV-positive individuals. The drugs
in the current first-choice treatment regimen were identified over 60 years ago, and patients are
required to take medicines for at least six months, even in the case of drug-sensitive infections.
Pronounced side effects, coupled with extended treatment regimens, lead to low patient
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compliance and have increased the emergence of drug-resistant mycobacteria strains. In fact, in
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018 alone, around half a million people developed TB that was resistant to rifampicin (RR-TB),
the most effective first-line anti-TB drug. Moreover, 78% of these cases being multidrug-resistant
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tuberculosis (MDR-TB) with resistance to at least rifampicin and isoniazid. Therefore, the
development of new antimycobacterial therapeutics, preferably with novel modes of action,
remains an urgent need.
DprE1, a subunit of decaprenylphosphoryl-β-D-ribose-2′-epimerase, is a periplasmic protein
involved in the mycobacterial cell wall biosynthesis that was shown to be a new highly-promising
drug target for antimycobacterial research.2,3 The initial recognition was brought by the
benzothiazinone series (BTZ), a DprE1 covalent inhibitor class.4–7 Later on, several research
groups provided insight into the DprE1 inhibitor binding mode as well as reported numerous
structurally diverse compound series with either an irreversible (covalent) or reversible non-
covalent binding, validating DprE1 as an attractive antimycobacterial target.8–17 All relevant
DprE1-inhibitor literature to date is summarized in a recently-published comprehensive review.18
Benzothiazinones BTZ043, azaindole AZ7371, and PBTZ-169/macozinone are the most advanced
DprE1 inhibitors that have recently entered the clinical development phase (Figure 1).19,20
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Figure 1. Selected reported DprE1 inhibitors.18 aAZ DprE1 inhibitor benchmark, compound 9 in
reference 11.
Inspired by the encouraging antimycobacterial properties of described compounds, GSK
performed a target-based high-throughput screening (HTS) campaign in search of novel
DprE1inhibitors (paper under preparation). This led to the identification of the hydantoin-derived
compound 1 and its several analogues (2-4) as promising hits. Recently, we reported the biological
2
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profiling and initial optimization efforts on hit 1. Several potent representatives with ring A
modifications obtained during this study showed promising in vitro enzyme inhibition (pIC50 7-
7
.4) together with low micromolar whole-cell MIC values and no cytotoxicity at 100 µM in a
HepG2 assay (representative compounds 1-4, Figure 2, Table 1).
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Figure 2. Most potent representatives (1-4) published previously by our team. The previous
findings indicate that both the hydantoin core and the acetyl linker are crucial for the potency of
the series.
Table 1. In vitro activity, cytotoxicity, and physicochemical properties of selected representatives
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1
-4 from our previous report.
DprE1
pIC50
Mtb MIC HepG2 IC50 Solubility Chrom
[
a]
[b]
[c]
[d]
[e]
№
R
(µM)
(µM)
(µM)
logD
1
2
7.0
8.3
> 100
> 100
202
4.54
7.0
2.5
≥ 487
3.57
3
4
7.3
7.4
3.1
10
> 100
> 100
379
85
3.78
5.63
a
b
Inhibition of DprE1 enzyme; MIC against Mycobacterium tuberculosis (H37Rv), reference:
c
Isoniazid, MIC = 1.8 µM; Cytotoxicity against HepG2 human caucasian hepatocyte carcinoma;
d
e
Kinetic aqueous solubility (CLND); Lipophilicity - chromlogD at pH = 7.4.
Earlier, we reported that the hydantoin core functions not only as a scaffold that ensures proper
spatial orientation of the peripheral moieties but also appears to take part in protein interactions,
21
crucial for the series potency. Likewise, several modifications of the acetyl linker in these
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molecules led to significant potency loss, indicating its importance. In addition, several analogues
of 1 and 4 with varied substitutions around ring B were evaluated. Several modifications were
observed to be permitted in that part of the molecule, although none of the analogues showed
significant potency improvement.
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Overall, the obtained results suggested that the SAR around rings A and B should be further
explored to optimize the potency and properties of the series. Finally, we also demonstrated in the
previous publication that only the R-enantiomer of the hit hydantoin 1 contributes to both
enzymatic and whole-cell activity. However, the assays in this manuscript were generally run with
racemates for procedural simplicity.
RESULTS AND DISCUSSION
Chemistry and SAR. We reported earlier that replacing the ring A cyano moiety in compound 1
with different polar substituents seemed to be favorable for retaining antimycobacterial potency in
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the phenotypic MIC-assay. Therefore, we first focused on the introduction of additional polar
groups on ring A, such as a carboxylic acid 17, an ester 10 and amide function 16, a urea moiety
1
1 and several polar heterocycles, including a fused bicyclic analogue of the benzene ring 14
Scheme 1-2). Similar to the approach reported earlier, most analogues with ring A substitution
modifications (10-14) were synthesized starting from ketones according to a modified Bucherer-
(
2
2
Berg hydantoin cyclization , followed by an alkylation as shown in Scheme 1. The ketones
were either available commercially or prepared based on standard literature procedures (see
Supporting Information). The selective alkylation on the N -nitrogen was previously confirmed by
3
full NMR assignment and crystal structure analysis.21
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Scheme 1. General synthetic approach toward analogues with a modified para-substitution pattern
on ring Aa
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a
◦
Reagents and conditions: (a) KCN, (NH
4
)
2
CO
3
, EtOH-H
2
O, microwave irradiation 70 C or
◦
*
heating 55 C, 7-17 hrs; (b) K
2
CO
3
, DMF or acetone, r.t., 24-48 hrs. The main isolated reaction
product was the hydantoin sodium salt 8a, used for the alkylation.
Intermediate 15, bearing an amide group on ring A, was prepared by reaction of 5 with ammonia,
and subsequent alkylation of the hydantoin ring resulted in final product 16 (Scheme 2). In the
case of analogues 17-18, the substituents on the ring A seemed to be reactive in the last alkylation
step. Therefore, the non-substituted acid derivative 17 was prepared by base-promoted hydrolysis
of the corresponding ester 10. Lastly, the tetrazole ring in 18 was formed by zinc-promoted [3+2]
cycloaddition of the nitrile in 1 with sodium azide.
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Scheme 2. Synthetic approach toward analogues 16-18 with a modified para-substitution pattern
on ring Aa
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aReagents and conditions: (a) NH
(c) LiOH, THF, H O, rt, 1h; (d) NaN
OH, 90°C, overnight; (b) K
CO , DMF or acetone, r.t., 24 hrs;
4
2
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2
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, ZnCl , n-PrOH, 95°C, 24h
2
Inspired by methylsulfonamide 2‘s overall activity profile (Table 1), we decided to prepare a small
sub-series of compounds, covering analogues that are linked to ring A via either the N- or S- atom
of the sulfonamide functionality (compounds 20-22 and 30-36, respectively, Schemes 3 and 4).
The synthetic approach shown in Scheme 1 was anticipated to be unfit for the preparation of the
N-linked analogues. In essence, undesired alkylation of the acidic sulfonamide nitrogen was
expected to occur during the final synthetic step (N-alkylation of the hydantoin moiety). Therefore,
sulfonamides 20-22 were prepared in moderate yields from bromo-substituted precursor 19,
following a literature procedure for palladium-catalyzed amidation of aryl rings (Scheme 3).23
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Scheme 3. Synthetic approach to analogues with N-linked sulfonamide substituents on ring Aa
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aReagents and conditions: (a) [PdCl(allyl)]2, t-BuXPhos, K
◦
2
CO
3
, 2-MeTHF, 80 C, 2 hrs.
Since no literature procedures were available for the late-stage introduction of S-linked
sulfonamide groups, we decided to return to the strategy shown in Scheme 1 and evaluate its
potential for the synthesis of analogues 30-36 (Scheme 4). The sulfonamide moiety was first
installed by the reaction of 4-acetylbenzenesulfonyl chloride with different amines, followed by
applying the modified Bucherer-Berg protocol to obtain intermediates 23-29. Alkylation of the
hydantoin ring provided the desired products 30-36 in moderate to high yields. Remarkably, no
alkylation of the sulfonamide moiety was observed under these conditions.
Scheme 4. Synthetic approach to analogues with S-linked sulfonamide substituents on ring Aa
a
.
Reagents and conditions: (a) amine HCl, Et
3
N or amine with no base, DCM, r.t., 1 hr; (b) KCN,
◦
(NH4)
2
CO
3
, EtOH-H
2
O, 70 C (MW or heating), 7-17 hrs; (c) K
2
CO , acetone, r.t., 24-48 hrs.
3
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Table 2 summarizes the biological and biophysical evaluation results for the compounds that carry
a polar A-ring substituent. Overall, good solubility and no detectable cytotoxicity in the HepG2
assay (IC50 > 100 µM) were observed.
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Most polar substituents in the first subset of compounds (10-14, 16-18) nonetheless led to the loss
of both DprE1 inhibitory potency and whole-cell activity compared to hit 1. Notable exceptions
are methyl ester 10 and fused bicyclic analogue 14, both retaining the overall activity profile of 1.
Surprisingly, tetrazole 18 that is closely related to a previously reported tetrazole 3 did not retain
activity.
Table 2. In vitro activity, cytotoxicity, and physicochemical properties of the compounds with
varying substituents at the 4-position of ring A.
DprE1
pIC50
Mtb MIC HepG2 IC50 Solubility Chrom
[a]
[b]
[c]
[d]
[e]
№
R
(µM)
(µM)
(µM)
logD
10
7.1
11.2
> 100
140
4.84
1
1
5.3
6.2
5.2
> 80
> 40
80
> 100
> 100
> 100
≥ 296
≥ 440
≥ 486
2.71
3.54
3.36
12
1
1
3
4
6.8
5.2
10
> 100
> 100
≥ 369
3.38
2.68
16
> 80
≥ 454[f]
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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
21
6.4
80
100
100
44
4.90
5
7
.4
.2
> 80
154
2
3
3
2
0
1
4.96
3.19
0.7
20
80
80
> 100
> 100
> 100
> 100
≥ 486
≥ 478
224
6.1
3.88
4.60
4.48
5
5
.7
.7
32
334
3
3
4
4
.5
.7
> 80
> 80
63.1
79.4
57
55
4.78
5.03
34
3
5
6
3
4.9
>40
>100
217
4.39
a
b
Inhibition of DprE1 enzyme; MIC against Mycobacterium tuberculosis (H37Rv), reference:
Isoniazid, MIC = 1.8 µM; Cytotoxicity against HepG2 human caucasian hepatocyte carcinoma;
Kinetic aqueous solubility (CLND); Lipophilicity - chromlogD at pH = 7.4; Solubility
determination in 5% DMSO pH7.4 phosphate buffer saline and quantification of DMSO stock
concentration by Charged Aerosol Detector (CAD).
c
d
e
f
The N-linked sulfonamides 20-22 also demonstrated a significant reduction in DprE1 affinity and
antimycobacterial potency compared to the previously reported analogue 2, indicating that further
structural diversification in this compound subset was not promising. Conversely, the presence of
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
an inverted, S-linked sulfonamide group in 30 resulted in potent DprE1 inhibition (pIC50 = 7.3)
and significant improvement of the whole-cell activity (MIC = 0.6 µM). The introduction of
additional N-substituents on 30 (as in compounds 31-36) was again found to be detrimental for
cellular and DprE1 inhibitory potency. Taken together, it is likely that steric constraints in DprE1’s
active site are responsible for the observed trends in affinity and activity in the N- and S-linked
sulfonamide series. Since the S-linked sulfonamide derivative 30 was the most potent analogue
identified at this point and the first sub-micromolar DprE1 inhibitor encountered in the hydantoin
family, the aminosulfonyl group of 30 was selected as a recurrent structural feature in the
compound series that was subsequently prepared. Additionally, we decided to include some
analogues bearing hit 1’s 4-cyano substituted ring A for activity comparison. The substitution
pattern on aryl ring B was thoroughly investigated by means of analogues 38-71. For the
preparation of these molecules, the same synthetic strategy was applied again (Scheme 5).
Alkylation of hydantoin precursors 23 or 37 with an appropriately substituted haloacetophenone
derivative provided the desired products 38-71 in high to moderate yields.
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
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Scheme 5. Synthetic scheme of the synthesis of analogues with variable substitution on ring Ba
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
aReagents and conditions: (a) K
by hydrolysis of 49 in LiOH solution.
CO
, acetone or DMF, r.t., 24-48 hrs. Compound 50 was formed
*
2
3
This series was then supplemented with compounds in which ring B was replaced by saturated or
heterocyclic moieties. Compounds 72-88 were prepared following the general alkylation-based
approach (Scheme 6), in this case relying on the appropriate alkyl halides. The aromatic ring B
was changed to a simple methyl substituent in 72 or to one of several saturated ring systems in 73-
75. Moreover, the aryl moiety was replaced by a pyridine ring (76-82), 5-membered heterocycles
(
83-86), or bicyclic systems (87-88) to provide more diverse modifications in this part of the
structure and to explore the physicochemical properties of novel analogues.
All the alkyl halides utilized in Schemes 5-6 were commercially available or prepared according
to literature procedures (see Supporting Information).
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 6. Synthetic approach to analogues with saturated or heterocycle moieties replacing ring
B
a
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
aReagents and conditions: (a) K
CO , acetone or DMF, r.t., 24-48 hrs.
2
3
Table 3. In vitro activity, cytotoxicity, and physicochemical properties of the analogues with ring
B substitution modifications.
DprE1 Mtb MIC
HepG2 IC50 Solubility Chrom
R
[a]
[b]
[c]
[d]
[e]
№
R
pIC50
6.6
5.9
6.7
6.1
6.8
6.1
6.8
7.2
7.2
(μM)
(μM)
(μM)
logD
2.81
4.19
2.94
4.30
3.08
4.39
3.03
3.34
3.82
38
39
40
-SO
2
NH
NH
NH
2
2
2
H
10
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
≥ 415
≥ 428[f]
340
-CN
H
> 80
2.5
40
-SO
2
2-F
41
42
43
44
45
46
-CN
2-F
≥ 312[f]
≥ 408
≥ 372[f]
≥ 387
≥ 314
362
-SO
2
3-F
5.0
80
-CN
3-F
-SO
-SO
-SO
2
NH
NH
NH
2
2
2
4-F
2.2
1.2
1.3
2
2
3,4-diF
3-Cl, 4-F
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4
4
4
7
8
9
-SO
2
2
NH
2
2
2,4-diCl
7.1
7.0
7.5
1.9
30
> 100
> 100
6.45
372
4.11
3.77
5.84
≥ 347[f]
-SO
NH
3-Br
t
N.D. [h]
-CN
2-OCO Bu, 5.3
4-F
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
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
-CN
2-OH, 4-F
2-OH, 4-F
2-Me
6.2
7.2
7.0
6.1
6.7
6.0
30
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
65.3
12
4.56
3.12
3.30
4.73
3.36
4.71
3.33
4.73
2.98
3.00
2.86
3.56
3.96
4.15
2.40
2.54
2.77
4.14
5.50
4.24
5.38
4.90
-SO
2
NH
2
2
2.5
2.5
40
≥ 496
-SO
2
NH
≥ 311
110[f]
-CN
2-Me
≥ 320[f]
50[f]
-SO
2
NH
2
2
3-Me
5.0
80[g]
-CN
3-Me
-SO
2
NH
4-Me
6.8
5.8
5.6
6.9
6.5
7.0
7.1
6.0
5.9
5.3
6.2
7.1
6.2
10
351
246[f]
-CN
4-Me
> 80
> 80
10
-SO
-SO
-SO
-SO
-SO
-SO
-SO
-SO
-SO
-SO
2
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
2
2
2
2
2
2
2
2
2
2
2-OMe
3-OMe
4-OMe
≥ 473
≥ 417
≥ 392
≥ 381
279
2
2
2
2
2
2
2
2
2
20
2-CF
3-CF
4-CF
3
3
3
2.5
0.9
20
370
3-CN
4-CN
3-NO
40
> 100
> 100
> 100
> 100
69.4
≥ 482
≥ 411
132
60
2
20
3-OCF
3
3
0.6
40
≥ 438
127[f]
≥ 287
121
-CN
3-OCF
-SO
2
NH
2
3-CF
3-CF
3
3
, 4-F 7.2
, 4-F 6.4
0.6
20
> 100
76.7
-CN
71
-SO
2
NH
2
3,5-diCF
3
5.1
> 80
74.6
95
a
Inhibition of DprE1 enzyme (DprE1 assay data was generated using a modified version of the
assay described, paper under preparation) ; MIC against Mycobacterium tuberculosis (H37Rv),
1
8
b
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1
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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
c
reference: Isoniazid, MIC= 1.8 µM; Cytotoxicity against HepG2 human caucasian hepatocyte
d
e
carcinoma; Kinetic aqueous solubility (CLND); Lipophilicity - chromlogD at pH = 7.4;
Solubility determination in 5% DMSO pH7.4 phosphate buffer saline and quantification of DMSO
stock concentration by Charged Aerosol Detector (CAD); Only partial inhibition was reached;
f
g
h
N.D. - not determined.
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
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6
7
8
9
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0
1
2
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4
5
6
7
8
9
0
As Table 3 demonstrates, several ring B substitution modifications were tolerated. In general, all
the compounds bearing a 4-aminosulfonyl moiety on ring A showed superior enzyme affinity in
comparison with the 4-cyano analogues.
Stripping off the substituents on ring B leads to a significant activity drop in 38 and 39 compared
to both hit 1 and its sulfonamide analogue 30 (see Tables 1-3). Nonetheless, it was desirable to
include these analogues in the series for relative activity comparison with the corresponding
compounds lacking fluorine atoms and containing other substituents or heterocycles. Compounds
with a single fluorine substituent (40-44) also showed a small but consistent drop in enzymatic
affinity. The presence of a 2,4-dichloro substitution pattern in 47 led to a significant drop in the
whole-cell activity (MIC = 5 µM) compared to reference 30 (MIC = 0.6 µM), suggesting particular
importance of fluorine substituents for the series’ whole-cell activity. Although we do not have a
clear rationale for this observation, it is most likely connected to small steric size and high
electronegativity of fluorine substituent. In fact, analogues with a 3,4-difluoro or 3-Cl,4-F
substitution pattern (45 and 46, respectively) were among the most active compounds obtained
(
pIC50 = 7.2, MIC = 1.2-1.3 μΜ). Interestingly, 3-Br substituted compound 48 retained relatively
high enzymatic potency (pIC50 = 7.0) with a MIC of 1.9 µM.
Compounds 52-60 with an electron-donating group (-Me or -OMe) in different ring positions (2-,
3- or 4-) demonstrated lower enzymatic and whole-cell potency in comparison to the
corresponding references 30 or 1.
To further investigate the influence of electron-withdrawing substituents, we first prepared a
number of analogues containing -CF
3
, -CN, -NO
2
, or -OCF groups in ring B (61-68). Most of the
3
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prepared compounds showed a significant drop in enzymatic and whole-cell potency. The 2-CF -
3
substituted compound 61 showed a MIC value of 2.5 µM. Notably, the most potent representatives
were the 3-substituted analogues 62 (3-CF ) and 67 (3-OCF ), which retained sub-micromolar
whole-cell potencies (MIC = 0.6-0.9 μΜ). Keeping in mind the high activity of 3,4-dihalo
substituted analogues 45 and 46, we decided to combine the two substitution patterns (3-CF , 4-F)
in products 69-70, while two CF -groups were simultaneously introduced in 71. Sulfonamide 69
, 4-F) retained sub-micromolar whole-cell potency (MIC = 0.6 μΜ) and enzymatic activity
pIC50 = 7.2 vs. 7.1) compared to its closest analogue 62 (3-CF ), while compounds 70 and 71
3
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
3
3
(
(
3-CF
3
3
resulted in a substantial activity loss.
Overall, the majority of analogues with a modified substitution pattern on ring B (apart from 49,
3, 68, 70-71) demonstrated no detectable cytotoxicity in the HepG2 assay (IC50 > 100 µM).
6
Table 4. In vitro activity, cytotoxicity, and physicochemical properties of the analogues with
saturated or heterocyclic moieties instead of ring B.
DprE1 Mtb MIC HepG2 IC50 Solubility Chrom
[
a]
[b]
[c]
[d]
[e]
№
72
73
74
R
pIC50
(μM)
> 80
> 80
(μM)
(μM)
logD
0.54
1.51
-SO
-SO
-SO
2
2
2
NH
NH
NH
2
2
2
4.0
> 100
> 100
≥ 504
4.5
58
6
5
.7
.9
7.5
> 100
> 100
≥ 317
≥ 404
3.62
4.87
7
5
-SO
2
2
NH
2
2
> 80
76
77
-SO
NH
6
5
.3
.5
10
> 100
> 100
≥ 405
2.03
3.48
-CN
≥ 292[f]
> 80
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
7
8
8
9
0
-SO
2
NH
2
5
.5
> 80
> 100
≥ 298
1.16
-CN
≥ 334[f]
4
5
4
.6
.6
.6
>80
80
> 100
> 100
> 100
2.46
1.16
2.52
-SO
2
NH
2
2
≥ 439
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
81
-CN
≥ 310[f]
>80
8
2
-SO
2
NH
7
.1
0.6
> 100
≥ 441
2.39
8
8
3
4
-SO
2
2
NH
2
2
6
7
.7
.0
10
> 100
> 100
≥ 311
2.35
3.52
-SO
NH
2.5
≥ 437[f]
8
5
-CN
6.3
6.2
80
> 100
184[f]
4.85
8
8
6
7
-SO
-SO
-SO
2
NH
NH
NH
2
2
2
60
> 100
> 97
≥ 453
1.90
3.78
2
2
40[g]
≥ 339[f]
6
6
.7
.3
88
10
> 100
167
3.93
a
b
Inhibition of DprE1 enzyme; MIC against Mycobacterium tuberculosis (H37Rv), reference:
Isoniazid, MIC = 1.8 µM; Cytotoxicity against HepG2 human caucasian hepatocyte carcinoma;
Kinetic aqueous solubility (CLND); Lipophilicity - chromlogD at pH = 7.4; Solubility
determination in 5% DMSO pH7.4 phosphate buffer saline and quantification of DMSO stock
c
d
e
f
g
concentration by Charged Aerosol Detector (CAD); Only partial inhibition was reached.
Table 4 summarizes the biological evaluation results for compounds 72-88. Methyl- or
cyclopropyl-substituted compounds 72 and 73 showed a pronounced loss in both DprE1 and
cellular potency, while adamantyl-containing analogue 75 retained moderate DprE1 affinity (pIC50
=
5.9), but did not display significant antimycobacterial activity. Introduction of a cyclohexyl ring
in place of the B ring in 74, however, preserved the potency over its phenyl analogue 38 with pIC50
values 6.7 and 6.6, and MIC values 7.5 and 10 µM, respectively. This could indicate that
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appropriately substituted cyclohexyl-analogues may act as a potential replacement of the phenyl-
type ring B in compound 30. The introduction of a pyridine ring at this position (76-81) led to an
activity drop in most compounds, and only the 2-pyridinyl-based analogues 76 and 77 retained
comparable activity with the phenyl-analogues 33 and 4. Therefore, the 2-pyridinyl moiety was
combined with the difluoro substitution pattern of reference 30, providing its closest heterocyclic
analogue 82. The latter retained comparable, sub-micromolar whole-cell activity (MIC = 0.6 µM).
The 2-thiophenyl-substituted product 83 retained enzymatic and whole-cell potency compared to
its direct phenyl analogue 38, while the chlorine substituent addition to the 5-position of the
thiophene ring in 84 led to further potency improvement (pIC50 = 7.0, MIC = 2.5 µM). The latter
was, however, still inferior to the profile of reference 30. In contrast, the introduction of a
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
(
benzo)thiazole moiety in 86-87 was characterized by a significant loss of potency. Lastly, 2-
naphthalene-containing compound 88 preserved the same whole-cell activity (MIC = 10 µM) as
its phenyl analogue 38, suggesting that the enzymatic pocket could potentially accommodate
additional substituent expansion in this part of the molecule. It should be emphasized that, in the
majority of analogues, even those with increased lipophilicity, no considerable cytotoxicity was
detected among the reported modifications (HepG2 IC50 > 100 µM), which may indicate a
promising safety profile of this chemical series.
hERG inhibition. Potential cardiotoxicity of the series was one of our primary concerns since the
previously reported hit 1 and its most potent analogues 2-4 had all demonstrated considerable
21
hERG potassium channel inhibition (pIC50 = 4.4-5.3). To our satisfaction, all tested sulfonamide
derivatives showed no significant hERG inhibition (pIC50 < 4.3), as shown in Table 5. Overall,
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these data support our previous findings that potential cardiotoxicity is not intrinsic to the series
but rather determined by the substitution pattern on rings A and B.
Table 5. hERG inhibition of selected potent analogues.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
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5
6
7
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9
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№
Structure
hERG pIC50
3
0
< 4.3
51
< 4.3
< 4.3
< 4.3
< 4.3
< 4.3
< 4.3
6
2
67
7
4
82
8
4
Enantiomeric separation. The enantiomers of the new reference 30 were separated by chiral
HPLC, the absolute configuration was determined by VCD analysis. The obtained results
confirmed that only the R-isomer is responsible for both the enzymatic and whole-cell potency
Table 6), in agreement with our previous findings.21
(
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Table 6. In vitro activity, cytotoxicity, and physicochemical properties of 30 and its enantiomers.
DprE1 Mtb MIC
HepG2 IC50 Solubility
Chrom
logD
№
Structure
[a]
[b]
[c]
[d]
[e]
pIC50
(μM)
(μM)
(μM)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
4
4
4
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4
4
4
4
4
4
5
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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
3
3
3
0
7.2
0.7
> 100
> 100
> 100
≥ 486
≥ 344
≥ 379
3.19
3.17
3.17
0R
0S
7.5
5.1
0.78
> 80
a
b
c
Inhibition of DprE1 enzyme; MIC against Mycobacterium tuberculosis (H37Rv); cytotoxicity
d
against HepG2 human caucasian hepatocyte carcinoma; kinetic aqueous solubility (CLND);
e
lipophilicity - chromlogD at pH = 7.4.
Metabolic stability. The in vitro metabolic stability assessment of compound 30 indicates the
general stability of the compound with an intrinsic clearance value lower than 3 ml/min/g in mouse
microsomes and under 0.4 ml/min/g in human microsomes, as summarized in Table 7
Table 7. Microsomal stability of compound 30.
Mouse
Human
№
Structure
Clint (ml/min/g)
Clint (ml/min/g)
30
2.67
< 0.40
Evaluation against M. tuberculosis DprE1 mutants. To provide a genetic link to the mechanism
of action, the primary hit compound 1 was tested against three M. tuberculosis DprE1 mutants
(
E221Q, G248S, and Y314H) that were generated in-house via oligonucleotide-mediated
recombineering as previously described.11,24,25 Additionally, three spontaneous DprE1 mutants
L368P, G17C, and C387S) were provided by Stewart T. Cole (Institut Pasteur, Paris, France).2,14
(
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As shown in Table 8, a clear MIC-modulation of reference hydantoin 1 is mainly visible in the
E221Q, G248S, and Y314H mutant strains. Together with the MIC-modulation that we observed
earlier in a DprE1-overexpressing strain, these data bring on additional support to our hypothesis
0
1
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9
0
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0
21
that DprE1 is the principal target of the hydantoins in M. tuberculosis. Moreover, the resistance
profile of DprE1 mutants to compound 1 is also shared by the non-covalent reference AZ DprE1
11
inhibitor and, to a lesser extent, with the covalent ligand TCA1 (Structures shown earlier in
Figure 1). While these data suggest that broader cross-resistance could be present between
representatives of these three compound families, they also indicate that some overlapping
interactions could be present for these families within DprE1’s ligand-binding site. The latter is
nonetheless not straightforwardly rationalized by looking at DprE1’s crystal structure: only Tyr314
lines the ligand pocket, while the other two amino acids (Gln 221 and Gly248) are at 6-9 Å distance
and are closer to the protein’s outer surface.17
Table 8. MIC of the primary hit 1 against a panel of M. tuberculosis DprE1 resistant mutants.
MICmutant/MICH37Rv Ratio[a]
Compd
E221Q
16
G248S
> 16
4
Y314H
16
L368P
G17C
C387S
1
2
8
1
2
<1
8
TCA1[b]
24
32
AZ
16
>63
>63
4
2
1
DprE1[
b],[c]
BTZ043[b]
4
1
1
1
1
64
aA mutant strain is considered resistant if the MICmutant/MICH37Rv Ratio ratios is 8 or higher. The
b
c
structures of reference DprE1 inhibitors AZ DprE1, TCA1 and BTZ043 are shown in Figure 1.
AZ DprE1 inhibitor benchmark is compound 9 in reference 11.
Together, the observed DprE1 inhibitory potencies, the MIC modulation observed in the DprE1-
21
overexpression strain (reported previously) , and the MIC modulation against resistant mutants
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to other classes of DprE1 inhibitors strongly support the assignment of DprE1 as the primary driver
of antimycobacterial activity in the hydantoin series.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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In vivo therapeutic efficacy. The two most potent compounds that were available at the time (3
and 30) were subsequently admitted to preclinical in vivo studies, for which approval was obtained
from the responsible local ethical committee. The efficacy of both compounds was determined in
2
6
a C57BL/6J mouse model of acute intratracheal infection with Mtb. H37Rv. Compounds were
administered once daily per os for four consecutive days, starting five days after the infection.
Moxifloxacine (100 mg/kg) was used as a positive inter-assay control for efficacy in these
experiments. Blood samples were obtained at specific time points from treated mice to measure
the levels of the assayed molecules. Lungs were harvested on day 9, 24 hours after the last
compound administration. The blood levels measured for both compounds and the lung
microorganism burden differences (log10CFUs/lung) from the treated mice compared to untreated
controls (day 9 after infection) are shown in Table 9 and Figure 3. No adverse clinical signs were
observed in any animal.
Table 9. Blood exposure levels and log10CFU reduction for compounds 30 and 3.
Blood Levels
Reduction of
log10CFUs/lung,
Dose
Cmax
AUC
(
relative to
untreated
controls)
Compound (mg/kg)a
(ng/mL)
(h*ng/mL)
Moxifloxacin 100
4.1 ± 0.3
0.5 ± 0.1
0.2 ± 0.1
30
3
200
170
6380
1870
31400b
6378c
a
b
Compounds were dosed per os, once daily; Compound 30’s blood levels were found to remain
c
above its MIC-value for 24 hrs after single oral administration; Compound 3’s blood levels were
found to be below its MIC-value within 6 hrs after single oral administration.
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Compound 30 demonstrated the best blood exposure with a Cmax value of 6380 ng/mL and an
AUC value of 31400 h*ng/mL. Moreover, the same compound showed the highest reduction of
Log10CFUs (0.5). Although this value reflects limited in vivo activity compared to reference
moxifloxacin, it demonstrates that the hydantoin series is capable of reaching the lungs of mice
after oral administration to achieve a statistically significant bacterial load reduction. The
bioavailability of 3 (reflected in Table 9 by its lower AUC value and fast clearance) is significantly
lower: within six hours, blood levels drop below the compound’s MIC value. This factor most
likely contributes to the absence of efficacy for this molecule. Overall, these data also show that
compound optimization, especially with respect to in vivo activity, is required before the future
development of the hydantoin family into a drug candidate is possible.
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9
0
Figure 3. Antitubercular efficacy in an acute infection murine model of tuberculosis. Each point
represents data from an individual mouse that received each product administered in a once a day
schedule (qd). Treatment was administered for 4 days as detailed in the figure.* p<0.05. ANOVA
analysis with Dunnett´s posttest.
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CONCLUSIONS
Herein, we have reported an expanded SAR exploration of a hydantoin-derivative series, recently
discovered by GSK. Previously, we demonstrated that these compounds are potent and selective
antimycobacterials that act via inhibition of DprE1. Our medicinal chemistry research effort
described herein resulted in 69 novel hydantoin analogues and led to the identification of potent
representatives with high in vitro enzymatic potency (pIC50 7-7.4) and whole-cell MIC values in
the low micromolar range (0.6-0.9 μΜ). The most potent representatives were compound 30 and
its close analogues 67, 69 and 82. This chemical family is characterized by no appreciable
cytotoxicity or cardiotoxicity (hERG), satisfactory metabolic stability, and a reasonable
physicochemical profile. In vivo proof of concept for compound 30 was achieved by using an acute
murine model of intratracheal infection. Although encouraging, currently available data indicate
that additional research, mainly focussing on the improvement of in vivo efficacy response, is
required before preclinical development for this class of compounds can be considered successful.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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EXPERIMENTAL SECTION
General Information. Laboratory reagent grade solvents were employed unless stated otherwise.
Reagents were purchased from Sigma-Aldrich, Fluorochem, Acros Organics, TCI, Enamine, or
Apollo Scientific and were used without further purification unless specified otherwise. Reaction
progression was monitored by TLC on silica gel and/or by UPLC-MS. Silica gel TLC analysis was
performed using Polygram® precoated silica gel TLC sheets SIL G/UV254 with detection by UV
light (254 nm).
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1
Structural determination and characterization of all compounds were performed with H NMR and
1
3
1
13
C NMR spectroscopy and mass spectrometry. H NMR (400 MHz) and C-NMR (100 MHz)
spectra were recorded on a Bruker Avance III Nanobay Ultrashield 400 or a Bruker DPX 400
0
1
2
3
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0
spectrometer. The chemical shift () values are reported in parts per million (ppm), and coupling
1
constants are expressed in Hertz (Hz). The chemical shifts  were given relative to the residual H
1
3
1
and C signals of the solvent peak as an internal standard: in H NMR (400 MHz)  2.50 ppm
quin, C HOS) for DMSO-d ,  2.05 ppm (quin, C HO) for Acetone-d ,  3.31 ppm (quin,
CD HOD) for Methanol-d ; in C NMR (101 MHz)  39.51 ppm (sept) for DMSO-d ,  29.84
ppm (sept),  206.26 ppm (s) for Acetone-d ,  49.00 ppm (sept) for Methanol-d . Legend: s =
(
2
D
5
6
3
D
5
6
13
2
4
6
6
4
singlet, d = doublet, t = triplet, q = quartet, quin = quintet, sept = septet, m = multiplet (denotes
complex pattern), br = broad signal, dd = doublet of doublets, dt = doublet of triplets, td = triplet
of doublets, etc.
UPLC-MS analysis was performed according to the methods A, B, or C. In all cases, ESI
+
-
(
electrospray ionization) was used. The quasi-molecular ions [M+H] or [M-H] were typically
detected, unless stated otherwise. Retention time R is specified for each described method. Method
t
A involved the Waters Acquity UPLC system coupled to a Waters SQ detector. A Waters Acquity
UPLC BEH C18 1.7 μm, 3 mm × 50 mm column was employed. The sample concentration was
0
.1 mg/ml, the flow was 0.8 mL/min. Solvent A consisted of aqueous ammonium acetate 25mM
and 10% acetonitrile at pH 6.6, and Solvent B was acetonitrile. The method was as follows: 0.0-
0.2 min A:B 99.9:0.1, 0.2-1.0 min 10:90, 1.0-1.8 min 10:90, 1.9-2.0 min 99.9:0.1 at temperature
4
0ºC. The UV detection was an averaged signal from the wavelength of 210 nm to 400 nm. In
methods B and C, ESI mass spectra were obtained with an Esquire 3000plus ion trap mass
spectrometer (Bruker Daltonics), using the direct infusion mode. A Waters acquity H-class UPLC
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system coupled to a waters TQD ESI mass spectrometer and a Waters TUV detector were used
with a Waters acquity UPLC BEH C18 1.7µm 2.1´50 mm column. Solvent A consisted of water
with 0.1% formic acid. Solvent B consisted of acetonitrile with 0.1% formic acid. Method B
involved the following: flow 0.7 mL/min, 0.15 min isocratic elution (A:B = 95:5), then gradient
elution during 1.85 min (A:B = from 95:5 to 0:100), followed by 0.25 min of isocratic elution (A:B
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
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3
3
3
3
4
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0
1
2
3
4
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8
9
0
=
0:100), then 0.75 min of isocratic elution (A:B = 95:5). Method C involved the following: flow
.4 mL/min, 0.15 min isocratic elution (A:B = 95:5), then gradient elution during 4.85 min (A:B
from 95:5 to 0:100), followed by 0.25min of isocratic elution (A:B = 0:100), then 0.75 min of
0
=
isocratic elution (A:B = 95:5). In methods B and C the wavelength for UV detection was 254 nm
unless stated otherwise.
For the High-Resolution Mass Spectrometry (HRMS) measurements, positive ion mass spectra
were acquired using a QSTAR Elite System (AB Sciex Instruments) mass spectrometer, equipped
with a turbospray source, over a mass range of 250–700.
Where necessary, flash purification was performed on a Biotage ® ISOLERA One or Four flash
systems equipped with an internal variable dual-wavelength diode array detector (200-400nm).
For normal phase purifications Biotage SNAP (10-340g), Sylicicle SiliaSep (4-120g) or Götec-
Labortechnik EasyVarioFlash (5-100g) cartridges were used (flow rates 10-100mL/min).
Reversed-phase purifications were performed with Biotage KP-C18 containing cartridges.
Gradients used varied for each purification. However, typical gradients used for normal phase were
gradient of 0−100% ethyl acetate in n-heptane or cyclohexane, or 0-15% methanol in ethyl acetate.
Typically, a gradient of 5% MeCN in water to 50% MeCN in water was used for reverse-phase.
The preparative HPLC purification was conducted on the Agilent 1200 or Agilent 1100 instrument,
employing either on an X-Bridge C18 column (19 mm x 150 mm, i.d 5 µm packing diameter or 30
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1
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2
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2
2
2
2
2
2
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3
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3
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3
3
4
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4
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5
5
5
5
5
5
6
mm x 150 mm, i.d. 5 µm packing diameter) or a SunFire C18 column (19 mm x 150 mm or 30 mm
x 250 mm) at 35C. The solvents employed were: A = 10 mM ammonium bicarbonate in water; B
= acetonitrile (“basic” method) or A = 0.1 M formic acid in water; B = 0.1 M formic acid in
acetonitrile (“acidic” method) respectively. The purification was run as a gradient (A:B) typically
from 40 to 100% over either 20 min or 25 min, with a flow rate of 17 mL/min (19 mm column) or
35 mL/min (30 mm column). The UV detection wavelengths were 210 nm and 254 nm.
Microwave radiation-assisted reactions were performed in a Biotage Initiator instrument. The
initial absorption was set as ‘high,’ and 2 min of pre-stirring was applied before heating
commenced.
0
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The isolated yields are reported. Purity of final compounds was 95% or higher (verified by UPLC-
MS), except for compounds 10 (>85%) , 33 (>90%), 34 (>90%), 80 (>90%), 87 (>90%). These
molecules displayed low potency in the DprE1- and MIC-assays, further purification was therefore
not carried out. All products were obtained as amorphous solids, and melting points were not
measured. The following section reports the synthetic procedures and analytical data for all final
compounds and some representative intermediates reported in this publication. Complementary
data for the rest of the intermediate compounds can be found in the Supporting Information.
Synthetic procedures that were used in the preparation of several products are summarized here as
“
General methods ”.
The literature benchmark DprE1 inhibitor (‘AZ DprE1’, Figure 1, N-(2-fluoroethyl)-1-((6-
methoxy-5-methylpyrimidin-4-yl)methyl)-1H-pyrrolo[3,2-b]pyridine-3-carboxamide was
1
1
synthesized by the procedure described previously in Shirude et al. TCA1 and BTZ043 were
purchased from corresponding commercial sources: TCA1 (Chemexpress (Shanghai Haoyuan)
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Co., Ltd., Ref. HY-12904, CAS 864941-32-2) and BTZ043 (Selleck Chemicals LLC, Ref.
S1097, 957217-65-1).
General method A: hydantoin core synthesis. A modified Bucherer-Berg protocol22 was
1
1
1
1
1
1
1
1
1
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0
employed. The suitable ketone (1.5.0-4.0 mmol, 1.0 eq.), ammonium carbonate (NH
eq.) and potassium cyanide KCN (1.3 eq.) were dissolved in a mixture of ethanol and water (1:1)
reaction molarity ~0.25-0.4 mol/l). The reaction mixture was heated at 55°C for 18-48 hr or
4
)
2
CO
3
(9.0
(
irradiated in microwave oven at 70°C for 3-9 hours. After the reaction was complete, the reaction
mixture was cooled down to room temperature and neutralized with 6M hydrochloric acid to
pH~7-8. In the case of precipitate formation, the product was collected by filtration, washed with
water and dried. Otherwise, the solvent was removed under reduced pressure and the residue was
diluted with water and extracted with ethyl acetate; the combined organic phase was washed with
brine, dried over Na
2
SO , filtered and evaporated to dryness. When necessary the product was
4
additionally dried in the vacuum oven (40°C, 0-10 mbar). Typically, no additional purification was
performed.
General method B: hydantoin core alkylation. A mixture of the appropriate hydantoin (0.2-3.7
mmol, 1 eq.) and potassium carbonate K
2
CO (1.1-2.0 eq.) was dissolved in DMF or acetone. After
3
10-15 min, the corresponding alkyl halide was added in a slight excess (1.1-1.5 eq.) (reaction
molarity ~0.08-0.2 mol/l). The reaction mixture was stirred at room temperature for 20-72 hr. Upon
reaction completion, the solvent was removed under reduced pressure and the residue was diluted
with saturated ammonium chloride solution or water and extracted with ethyl acetate. The
combined organic phase was typically washed with 1M NaOH, brine, dried over Na
2
SO , filtered
4
and evaporated under vacuum. The residue was purified by normal phase flash chromatography
on silicagel (gradient c-Hex/Hep:EtOAc = 100:0 to 10:90) or reversed-phase flash
ACS Paragon Plus Environment
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