Scaffold hybridization strategy towards potent hydroxamate-based inhibitors of: Flaviviridae viruses and Trypanosoma species

Page 1 of 17

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DOI: 10.1039/C9MD00200F

ARTICLE

Scaffold Hybridization Strategy towards potent hydroxamate-

based inhibitors of Flaviviridae viruses and Trypanosoma species

a

b

Erofili Giannakopoulou, Vasiliki Pardali, Efseveia Frakolaki, Vasileiοs Siozos, Vassilios

Received 00th January 20xx,

Accepted 00th January 20xx

a

c

c,†

b,‡

Myrianthopoulos, Emmanuel Mikros, Martin C. Taylor, John M. Kelly, Niki Vassilaki, Grigoris

a,§,

Zoidis

*

DOI: 10.1039/x0xx00000x

Infections with Flaviviridae viruses, such as hepatitis C virus (HCV) and dengue virus (DENV) pose global health threats.

Infected individuals are at risk of developing chronic liver failure or haemorrhagic fever respectively, often with a fatal

outcome if left untreated. Diseases caused by tropical parasites of the Trypanosoma species, T. brucei and T. cruzi, constitute

significant socioeconomic burden in sub-Saharan Africa and continental Latin America, yet drug development is under-

funded. Anti-HCV chemotherapy is associated with severe side effects and high cost, while Dengue has no clinically approved

therapy and antiparasitic drugs are outdated and difficult to administer. Moreover, drug resistance is an emerging concern.

Consequently, the need for new revolutionary chemotherapies is urgent. By utilizing a molecular framework combination

approach, we combined two distinct chemical entities with proven antiviral and trypanocidal activity into a novel hybrid

2

scaffold attached by an acetohydroxamic acid group (CH CONHOH), aiming at derivatives with dual activity. The novel spiro-

carbocyclic substituted hydantoin analogues were rationally designed, synthesized and evaluated for their potency against

three HCV genotypes (1b, 3a, 4a), DENV and two Trypanosoma species (T. brucei, T. cruzi). They exhibited significant EC50

values and remarkable selectivity indices. Several modifications were undertaken to further explore the structure activity

relationships (SARs) and confirm the pivotal role of the acetohydroxamic acid metal binding group.

Until recently, the standard of care for HCV was ribavirin and

pegylated-interferon alpha (PEG-IFN), which had severe side effects

and low sustained virologic response (SVR) rates. Since 2011, direct-

Introduction

Hepatitis C virus (HCV) is a global health burden with ~71 million

people estimated to be infected. It is a main cause of chronic liver

disease and can lead to liver cirrhosis and hepatocellular carcinoma.

HCV is a member of the Flaviviridae family and belongs to the

Hepacivirus genus. The viral genome is a 9 600 nucleotide-long

7

acting antivirals (DAAs) targeting the viral NS3A/4A protease, the

NS5B RNA-dependent RNA polymerase (RdRp) and the NS5A

1

8

-11

phosphoprotein were introduced (Figure 1)

and allowed the

2

single-stranded RNA of positive polarity and encodes a single

polyprotein precursor, which is processed into structural proteins

(core, E1 and E2), p7 required for assembly and release of virus

particles and six non-structural (NS) proteins (NS2, NS3, NS4A, NS4B,

3

, 4

NS5A and NS5B).

The NS proteins form a membrane-associated

replicase complex (RC) in association with host cell factors. HCV is

classified into 8 major genotypes and at least 86 sub/genotypes

5

(

GTs), with genotype

1

being the most predominant type

6

worldwide.

^a^.School of Health Sciences, Faculty of Pharmacy, Department of Pharmaceutical

Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis-

Zografou, GR-15771 Athens, Greece

b.

Molecular Virology Laboratory, Hellenic Pasteur Institute, Vas. Sofias Avenue, GR-

1

1521, Athens, Greece

c.

Department of Pathogen Molecular Biology, London School of Hygiene and

Tropical Medicine, Keppel Street, London WC1E 7HT, UK

Corresponding author:

*

Dr. Grigoris Zoidis (zoidis@pharm.uoa.gr )

§

‡

†

Principal investigator of the design and synthesis of the compounds

Principal investigator of the study on Flaviviridae viruses

Principal investigator of the study on Trypanosoma species

Figure 1 Representative chemical structures of clinically approved DAAs and

their respective EC50s against HCV genotype 1b.

Electronic Supplementary Information (ESI) available: [details of any supplementary

information available should be included here]. See DOI: 10.1039/x0xx00000x

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ARTICLE

Journal Name

1

2, 13

SO3Na

implementation of interferon-free treatment schedules,

which

NaO3S

SO

3

Vie w N a Article Online

1

4

DOI: 10.1039/C9MD00200F

are able to attain SVR in more than 90% of patients. DAAs efficacy

differs between genotypes, with genotype 3 elimination rate being

SO3Na HN

O

NH SO3Na

Suramin

1

5, 16

lower than the others.

O

H

N

However, there is a risk for the development of antiviral drug

resistance, depending on the regimen and the genotype.¹⁷

Moreover, available DAAs are costly, limiting their widespread

clinical use, and, as a result, therapy remains low on a global scale.

Thus, there is still a need for novel effective drugs of lower cost.

The mosquito-borne dengue virus (DENV) is another member of

the Flaviviridae family, belonging to the Flavivirus genus. It causes

widely distributed and endemic, visceral and central nervous system

diseases. Symptoms of infection range from mild (dengue fever) to

the more severe dengue hemorrhagic fever (DHF) and dengue shock

N

H

CH3

O

CH3

NH2

N

NH2

NH

N

Melarsoprol

H2N

HN

Pentamidine

1

8

S

HN

As

S

O

OH

O

H3C

F

S

O

F

NH2

OH

N

H2N

O

O2N

NECT

Eflornithine

1

9

Nifurtimox

F

N

O

OH

O

2

0

H

syndrome (DSS). The recently approved dengue vaccine has only

limited overall efficacy and there is no approved antiviral therapy.

N

O

B

N

H

O

2

1

22

O

N

H3C

CH3

CF3 O

S

CH3

H3C

O

DENV genome consists of a positive single-stranded RNA of ~11 kb in

length, encoding for the viral polyprotein, which is processed into

structural proteins (C, prM, E) and non-structural proteins (NS1,

Fexinidazole

Acoziborole

Benznidazole

Figure 2 Currently available marketed drugs for treatment of HAT and Chagas

disease. Highlighted regions are reported to play a major role in the

2

3, 24

NS2A, NS2B, NS3, NS4A, NS4B, NS5).

Bivalent metal ions are important for the stability and activity of mechanism of action. Chemical structures of potential drug candidates

Fexinidazole and Acoribozole.

various HCV and DENV proteins, including NS3 helicase/protease,

RdRp polymerase and HCV NS5A protein.²⁵^-³⁰

absence of vaccines, development of novel effective therapeutics is

an imperative for their treatment and control.

Human African Trypanosomiasis (HAT), also known as sleeping

3

sickness, is a vector-borne parasitic disease with 65 million people

being vulnerable to infection.³¹HAT occurs in 36 countries of sub-

Saharan Africa and it is caused by infection with protozoan parasites

belonging to the Trypanosoma brucei sp. T. b. gambiense is the

causative agent of a chronic form of the disease (gambiense HAT,

gHAT). It is prevalent in central and western Africa, and can take

years or decades to produce a fatal outcome. T. b. rhodesiense is

responsible for an acute form disease, causing death within weeks or

months of infection. Rhodesiense HAT (rHAT) is found in eastern and

Several groups of trypanosomatid metalloenzymes have been

identified as playing an important role in the metabolism and viability

of parasites. Amongst the most widely explored are histone

deacetylases (HDACs), trypanosome alternative oxidase (TAO) and

4

1-48

carbonic anhydrase (CA).

Metalloenzymes’ inhibition appears attractive as

a

4

7-53

chemotherapeutic approach against viruses and parasites.

It has

been proved that the vast majority of metalloenzyme inhibitors are

chelating agents employing metal binding groups to coordinate the

3

2, 33

southern Africa.

Clinical progression of both forms of HAT is

5

4, 55

active site metal ion.

chelating agents,

As a continuation of our research on metal

we were interested in developing novel

divided in two stages. The early hemolymphatic stage and the late

meningoencephalitic stage when the parasites enter the central

nervous system causing neurological damage and sleep

5

6, 57

combined scaffolds demonstrating both antiviral and antiparasitic

properties.

In this study, we report the rational design and synthesis of novel

spiro-hydantoin analogues, attached by the acetohydroxamic acid

3

4

disturbances.

Treatment approaches of HAT consist of five drugs: pentamidine,

suramin, melarsoprol, eflornithine, and nifurtimox-eflornithine

combination therapy (NECT) (Figure 2). Nevertheless, their

increasing levels of resistance, their limited efficacy, toxicity and

2

moiety (CH CONHOH) as the potential metal binding functional group

(Figure 3). Several bicyclic fused rings were used as spiro-

substituents on the basic 2,4-diketoimidazolidine scaffold, while

methyl- and benzyl- groups were incorporated at the amide nitrogen

atom of hydantoin, to enhance binding affinity and improve drug-

likeness of the compounds. The novel analogues were assessed as

antiviral and trypanocidal agents and exhibited remarkable

inhibitory activity with respect to their parent compounds I and II.

More specifically, they were evaluated for their effect on RNA

replication and cell viability on different HCV genotypes (1b, 3a, 4a)

and DENV. Their antiparasitic activity was assessed based on their

ability to inhibit proliferation of cultured bloodstream form T. brucei

and T. cruzi epimastigotes in vitro.

3

5

adverse side effects, make HAT treatment a daunting challenge.

Fexinidazole and acoziborole (Figure 2) are two potential drug

3

6, 37

candidates, currently being in advanced clinical trials.

American trypanosomiasis, or Chagas disease, is a potentially

life-threatening infection caused by the protozoan parasite

Trypanosoma cruzi. It is endemic in continental Latin America, with a

3

8

devastating effect upon 6-7 million people, and it is characterized

by two distinct clinical phases: an acute mostly asymptomatic phase,

3

9

and a chronic phase characterized mainly by cardiac disorders. The

two drugs available, nifurtimox and benznidazole (Figure 2), are only

4

0

partially effective and toxic side effects limit their use.

Unfortunately, there is no efficient disease management for both

parasitic infections and the World Health Organization has listed

them among the Neglected Tropical Diseases (NTDs). Therefore, in

2

| J. Name., 2012, 00, 1-3

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Moreover, with the aim of evaluating the contribution of the critical

View Article Online

DOI: 10.1039/C9MD00200F

parameter of lipophilicity on biological activity, extensions of the

O

H

O

indane system to tetralin or benzocycloheptane were considered

(compounds 17 and 18), while the addition of alkyl substituents such

as methyl- or benzyl- groups on N3 of hydantoin was also explored

N

O

N

OH

N

OH

N

H

O

I

II

(

compounds 16a, 17a, 18a and 16b, 17b, 18b respectively). To

Antiviral avtivity

Hepatitis C)

Trypanocidal activity

(T. brucei)

confirm the importance of the exocyclic acetohydroxamic acid

moiety on antiviral and trypanocidal activity, the derivative 4, lacking

metal chelating ability, was evaluated as a negative control (Tables 1

and 2). The derivative 16 carrying the smallest steric bulk, i.e. an

indane system with no substitution on N3, was selected for probing

the validity of our approach.

Compound 16 was synthesized and subsequently tested against

HCV (replicon 1b) and bloodstream form T. brucei in vitro, exhibiting

significant inhibitory activity against both targets. More specifically,

the EC50 value for HCV was in the low micromolar range (EC50 16 =

(

EC50=126.30 ± 14.20 μM

EC50=1.87 ± 0.08 μM

Metal Chelating Agents

O

4

N

R

5

O

N

R

3

2

N

OH

O

1

N

O

N

O

H

R

O

N

HN OH

O

1

6. R: H 16a. R: CH₃

HN OH

18. R: H 18a. R: CH₃

1

6b. R: CH Ph

2

1

8b. R: CH Ph

2

1

⁷^.^R^:^H¹⁷^a^.^R^:^C^H3

2.92 μM, Table 1) and for T. brucei was in the nanomolar range (EC

6 = 0.44 μM, Table 2), identifying this particular acetohydroxamic

50

1

7b. R: CH Ph

2

1

Figure 3 Indole-flutimide analogue (I) with antiviral activity, cycloheptane acid derivative as a promising and original lead compound. All the

spiro-substituted 2,6-DKP analogue (II) with trypanocidal activity and novel

synthesized analogues reported in this study. Metal binding groups are

indicated in plum elliptical shape.

aforementioned designed analogues were also synthesized and

tested against two different genotypes of HCV (1b and 4a) and

against two Trypanosoma species. Analogues 16, 17 and 18 were also

tested against HCV genotype 3a and DENV. The resulting EC50 and

cytotoxicity values are presented in Table 1 and Table 2.

Chemistry

Results and discussion

As depicted in Scheme 1, the commercially available ketones 1-3

Rational Design – Theoretical calculations

(Scheme 1A) were subjected to Bucherer-Bergs reaction with

2

Previous studies by our group have resulted in compounds ammonium carbonate and potassium/sodium cyanide in EtOH/H O.

carrying metal chelating moieties that demonstrated a highly Treatment of the resulting hydantoins 4-6 with potassium

promising inhibition capacity against either pathogenic viruses, bis(trimethylsilyl)amide in dry THF afforded the corresponding

5

6

including influenza and HCV, or parasites of the Trypanosoma potassium imidate salts. The latter intermediates were converted to

5

7, 58

genus.

N

The two above-mentioned series of analogues, although the respective benzyl esters 7-9 upon S 2 reaction with benzyl

structurally diverse, were based on rational incorporation of metal bromoacetate in dry DMF, as described in our previously published

5

9, 60

chelating moieties on scaffolds with pronounced drug-likeness, such protocols.

Catalytic hydrogenolysis (Pd/C 10%) of the

as the combination of flutimide with the indole system (I) or aforementioned benzyl esters led to the carboxylic acids 10-12,

incorporation of the acetohydroxamic acid moiety on a 2,6- which were further converted to the corresponding O-benzyl

diketopiperazine (2,6-DKP) scaffold (II) (Figure 3). In the present hydroxamates 13-15 upon coupling with O-benzylhydroxylamine·HCl

study, a conceptual approach to further exploit the bioactivity of in the presence of CDI or EDCI·HCl, HOBt. Finally, removal of the

molecules with potential metal chelating efficacy toward designing protective group was accomplished with hydrogenolysis, yielding

effectors with inhibitory activity against both disease-related viruses almost quantitatively the target acetohydroxamic acids 16-18.

and Trypanosoma parasites is reported. The design was based on

For the synthesis of the N-alkylated analogues, the precursors

combination of the exocyclic acetohydroxamic acid pharmacophore, benzyl esters 7-9 were reacted with NaH and the corresponding alkyl

bearing well proven trypanocidal activity, with a lipophilic tail based halide in dry DMF. Surprisingly, in the case of N-methylation

on a bicyclic system-substituted hydantoin that could effectively procedure, transesterification products (methyl esters) were also

mimic the antiviral flutimide scaffold yet with increased steric bulk, isolated (Scheme 1C), along with the expected N-methylated benzyl

5

9

thus affording a novel synthetically tractable heterocyclic scaffold as esters. Employment of the previously described methodology,

presented in detail in Figure 3.

catalytic hydrogenolysis of the benzyl esters 7a-9a and 7b-9b,

To counterbalance possible issues of water solubility arising from followed by amidation and deprotection of the amides 13a-15a and

the tricyclic system of the antiviral lead I, we selected to enhance the 13b-15b, afforded the desired N-alkylated acetohydroxamic acids

3

sp character of the novel scaffold by introducing a spiro connection 16a-18a and 16b-18b.

between the acetohydroxamic group-carrying hydantoin and the

indane system. In this case, resembling the spiro trypanocidal lead II Biology

would contribute toward avoiding possible Lipinski's rule-of-five

violations. Hence, the indole nitrogen atom was converted to a

carbon, giving rise to a hydrophobic indane (compound 16).

Antiviral assays

Screening of compounds using an HCV genotype 1b replicon

system. The effects of analogues 16-18, 16a-18a, 16b-18b and 4 on

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 3

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Starting ketones

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DOI: 10.1039/C9MD00200F

A

(CH2)n

O

C

n=1

n=2

n=3

1-Indanone

1-Tetralone

1-Benzosuberone

Transesterification products

7

8

a_i. n=1

a_i. n=2

(CH2)n

B

Synthetic Pathway

O

9a . n=3

i

HN

O

N

(

CH2)n

(CH2)n

a

b

(CH2)n

O

CH3

O

c

HN

O

HN

O

N

O

NH

O

7a. n=1, R:CH₃

(CH2)n

1. n=1 2. n=2 3. n=3

4. n=1 5. n=2 6. n=3

7. n=1 8. n=2 9. n=3

7b. n=1, R:CH Ph

8a.

ⁿ⁼²^,^R^:^C^H3

2

O

N

+

R

O

8b. n=2, R:CH Ph

N

2

d

O

9a. n=3, R:CH3

O

9b. n=3, R:CH Ph

2

(CH2)n

f

e

O

N

O

N

R

O

N

R

O

Transesterification products

N

O

HN OH

HN O

7

^aii^.ⁿ⁼¹

OH

(CH2)n

8a_i_i. n=2

O

9a . n=3

ii

1

6. n=1, R:H

6a. n=1, R:CH₃

6b. n=1, R:CH Ph

7. n=2, R:H

7a. n=2, R:CH₃

7b. n=2, R:CH Ph

8. n=3, R:H

8a. n=3, R:CH₃

13. n=1, R:H

13a. n=1, R:CH₃

13b. n=1, R:CH Ph

14. n=2, R:H

14a. n=2, R:CH₃

14b. n=2, R:CH Ph

15. n=3, R:H

15a. n=3, R:CH₃

10. n=1, R:H

10a. n=1, R:CH₃

10b. n=1, R:CH Ph

11. n=2, R:H

11a. n=2, R:CH₃

11b. n=2, R:CH Ph

12. n=3, R:H

12a. n=3, R:CH₃

N

H C

3

O

N

2

O

CH3

2

8b. n=3, R:CH Ph

15b. n=3, R:CH Ph

12b. n=3, R:CH Ph

2

Scheme 1 A) Starting ketones B) Synthesis of the target acetohydroxamic acid analogues 16-18, 16a-18a and 16b-18b. Reagents and Conditions: (a) Method

A: NaCN (2.27 eq), (NH CO (6 eq), EtOH/H O (1.1:1), reflux, 8 h - 11 days; | Method B: KCN (1.3 eq), (NH CO (5 eq), EtOH/H O (1:3), MW (100 W), 120

C, 25 min - 1 h; (b) (i) [(CH Si] NK (1.02 eq), dry THF (0-5 °C) and then r.t., 20 min - 1 h, Argon; (ii) BrCH COOCH Ph (1.05 eq), dry DMF, 35-38 °C, 48 h,

Argon; (c) (i) NaH (1.2 eq), dry DMF, (0-5 °C) and then r.t., 15 min, Argon; (ii) PhCH Br (1.2 eq), 60 °C, 7 days, Argon; (d) H , Pd/C 10%, EtOH/AcOEt 3:1, 50-

5 psi, 40-45 °C, 3h; (e) Method A: EDCI·HCl (1.2 eq), HOBt (1.2 eq), PhCH ONH ·HCl (1.2 eq), TEA (5.8 eq), dry CH Cl /dry DMF, 30-35 °C, 40 h, Argon; |

Method B: (i) CDI (1.2 eq), dry THF, 28-30 °C, 1 h, Argon; (ii) PhCH ONH ·HCl (1.2 eq), TEA (1.82 eq), 28-30 °C, 24 h, Argon; (f) H , Pd/C 10%, EtOH/AcOEt

:1, 50-55 psi, 40-45 °C, 3h. C) Methyl esters obtained because of unexpected transesterification reaction (pink frames).

4

)

2

3

2

4

)

2

3

2

°

3

)

3

2

5

2

3

HCV RNA replication and cell viability were determined in the stable replacement of indane ring of 16 with the bulkier tetralin (17) and

cell line Huh5-2 containing an HCV genotype 1b (strain Con1) benzocycloheptane (18) rings progressively increased the activity of

replicon encoding the firefly luciferase gene.⁶¹In this cell system, the the compounds. It is noteworthy that the activity of 18 (EC50 = 0.25

level of expressed luciferase directly correlates with the level of HCV μM) was 12-fold better than lead compound 16 (EC50 = 2.92 μM). A

marked improvement was also observed on the EC50 value of 16 (43-

RNA replication. Using serial dilutions of the compounds, the half

maximal effective concentration (EC50) and the median cytotoxic

concentration (CC50) were obtained, by measuring HCV replication-

driven luciferase activity and intracellular ATP levels, respectively

fold lower), compared to the structurally related fused indole-

5

6

flutimide analogue I (EC50 = 126.3 μM), validating the idea of

incorporating a spiro connection between hydantoin and the indane

ring. Introduction of a methyl substituent on the amide nitrogen

atom of the 2,4-diketoimidazolidine scaffold of 16 led to a 2-fold

increase in the activity (EC50 16a = 1.41 μΜ). However, this

methylation had almost no effect in the potency of 17 (EC50 17 = 1.41

μM; EC50 17a = 1.45 μM) and resulted in lower inhibitory potency for

the N-methylated analogue 18a (EC50 18a = 1.18 μM). The addition

of the bulky lipophilic benzyl substituent to the same nitrogen atom

of the hydantoin scaffold led to lower EC50 values for the resulting

analogues 16b (EC50 = 0.22 μM) and 17b (EC50 = 0.32 μM), as

(Table 1, Figure 4A). Dose-response curve analysis is presented for

the most potent analogue 18 (Figure 4B). Selectivity indices

(SI)=CC50/EC50 were calculated for all analogues synthesized.

As shown in Table 1, acetohydroxamic acid analogues were

potent against HCV-1b replication, with EC50 values ranging from

.22 to 2.92 μM. Compounds 16b, 17b and 18 exhibited the highest

inhibitory activity, with EC50s in the submicromolar range (0.22 μM,

0

.32 μM and 0.25 μM, respectively). We observed that the

4

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Table 1 In vitro inhibitory activity of compounds 16-18, 16a-18a, 16b-18b and hydantoin analogue 4 tested against HCV (genotypes 1b, 3a, 4a) and DENV

DOI: 10.1039/C9MD00200F

(serotype 2). Cytotoxicity against Huh5-2, HCV replicon cells and selectivity indices.

Concerning the cytotoxicity evaluation (Table 1), all the non-

HCV-1b

EC50 (μΜ)^a

HCV-3a

EC50 (μΜ)^a

HCV-4a

EC50 (μΜ)^a

DENV-2

EC50 (μΜ)^a

CC50

(μΜ)

Structure

Cmpd

R

H

SI^b

c

1

6

2.92 ± 0.01

>68

2.97 ± 0.21

>67

0.58 ± 0.04

>345

96.34 ± 1.00

>2

>200

6.91 ±

0.67

2

O

1

6a

6b

CH

3

1.41 ± 0.01

19

-

0.21 ± 0.02

128

-

O

N

R

N

OH

N

7

.53 ±

O

H

1

CH

2

Ph

0.22 ± 0.01

1.41 ± 0.35

1.45 ± 0.07

34

>142

99

-

>84

-

0.14 ± 0.01

0.38 ± 0.04

0.21 ± 0.01

54

-

>3

-

1

.29

1

7

H

2.37 ± 0.17

-

>526

682

61.49 ± 3.86

-

>200

43.2 ±

1

O

17a

17b

CH

3

N

0

.37

8.90 ±

.77

98.4 ±

.24

11.6 ±

R

O

N

O

HN OH

CH

2

Ph

0.32 ± 0.01

0.25 ± 0.02

1.18 ± 0.07

28

794

95

-

2480

-

0.07 ± 0.02

0.10 ± 0.00

0.11 ± 0.01

127

1984

1015

-

0

1

8

H

0.08 ± 0.01

-

16.29 ± 2.17

-

12

-

2

N

O

18a

CH

3

R

1

2.43

N

O

5

.45 ±

1

8b

HN OH

CH

2

Ph

1.00 ± 0.05

>10

5

-

0.12 ± 0.01

-

45

-

0

.27

O

HN

4

H

-

>10

NH

O

1

26.30 ±

4.20

N

OH

I

>1.6

-

>200

1

O

^aEC50 (median effective concentration) values represent the mean ± SD of three independent experiments, each carried out in triplicate. Calculated against HCV genotype 1b

b

c

(

Huh5-2, HCV 1b replicon cells), genotype 3a (Huh7.5-3a, HCV 3a replicon cells), genotype 4a (Huh7.5-4a, HCV 4a replicon cells) Selectivity index: CC50/EC50. CC50 (median

cytotoxic concentration against Huh5-2, HCV replicon cells) values represent the mean ± SD of three independent experiments, each carried out in triplicate.

Figure 4 Α) Screening of compounds against HCV genotype 1b replication. Huh5-2 cells, harboring HCV genotype 1b (Con1) subgenomic replicon, were seeded

at 30% confluency and treated for 72 h with the compounds at 3 μΜ. Β) Activity of compound 18, against HCV genotype 1b, 3a and 4a RNA replication, using

dose–response curve analysis. Huh5-2, Huh7.5-3a and Huh7.5-4a cells, harboring subgenomic replicons of HCV genotype 1b (Con1), 3a (S52) or 4a (ED43),

respectively, were seeded at 30% confluency and treated for 72 h with serial dilutions of 18. In both A and B, as a marker of viral RNA replication, firefly

luciferase activity was determined and calculated as relative light units (RLU) per μg of total protein. Values obtained with compound-treated cells were

expressed as percentage of those obtained with cells treated with the solvent DMSO (control). Bars represent mean values obtained from three separate

experiments in triplicate. Error bars represent standard deviation (SD).

compared to the addition of the methyl substituent. On the other substituted analogues 16, 17 and 18 exhibited high CC50 values (≥ 200

hand, the benzyl group decreased the activity of compound 18b 4- μM), which resulted in remarkable selectivity indices. The most

fold with respect to the corresponding NH-analogue 18. In the case promising analogue appears to be compound 18, with SI value of

of the hydantoin 4, the lack of activity underlines the importance of 794. It is of great interest that the incorporation of an alkyl

the acetohydroxamic acid metal binding group for the antiviral substituent onto the amide nitrogen atom progressively increased

potency of the compounds.

the cytotoxicity of the compounds, in agreement with the higher

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lipophilicity determined by calculated logD values (Table 2); the

bulkier the lipophilic substituent, the lower the CC50 value.

View Article Online

DOI: 10.1039/C9MD00200F

Activity of the compounds against other HCV genotypes. As the

compounds exhibited significant potency against HCV genotype 1b,

they were selected for further testing against other HCV genotypes.

Specifically, we determined viral replication-derived luciferase

activity in Huh7.5-3a stable cell line for compounds 16, 17 and 18 and

in Huh7.5-4a stable cell line for compounds 16-18, 16a-18a and 16b-

1

8b, containing subgenomic replicons of HCV genotype 3a (isolate

S52) and 4a (isolate ED43), respectively.

Similar to genotype 1b, the activity of 16, 17 and 18 against HCV

genotype 3a ranged from low micromolar to nanomolar, and the

increase in ring size conferred higher potency. Specifically, EC50

values were below 2.97 μΜ, with the most promising analogue being

compound 18 (EC50 18 = 0.08 μM; SI 18 = 2480), whose activity was

3

-fold higher compared to the one exhibited in HCV-1b.

Biological evaluation of the hydroxamic acid derivatives on HCV-

a indicated enhanced activity as compared to genotypes 1b and 3a.

4

All the novel synthesized analogues inhibited HCV-4a replication at

submicromolar concentrations, ranging from 0.07 μM for 17b to 0.58

μM for the indane substituted analogue 16. Tetralin substitution

increased the activity by 1.5 times (EC50 17 = 0.38 μΜ) and

benzocycloheptane by 6 times (EC50 18 = 0.10 μΜ).

Moreover, N-methyl substitution on the hydantoin ring had a

favorable effect on the antiviral activity, but only in the context of

the indane- and tetralin- substituted analogues (EC50 16a = 0.21 μM;

EC50 17a = 0.21 μM). The N-methylated analogue 18a exhibited

potency similar (in the order of 0.1 μM) to the parent compound 18.

The same trend was observed for the N-benzyl counterparts 16b and

Figure 5 Inhibitory effect of compounds on HCV RNA levels and protein

expression in a subgenomic HCV Con1 replicon assay. A) Quantification of

(+) strand HCV RNA by RT-qPCR in Huh7.5-4a replicon cells treated with

serial dilutions of compounds 17a, 18 or the solvent DMSO. Values from

compound-treated cells are expressed as percentage of those obtained with

cells that received the solvent (control). mRNA levels of the housekeeping

gene YWHAZ were used for normalization. B) Indirect immunofluorescence

for NS5A (left panels) in Huh7.5-4a replicon cells, treated with serial

dilutions of compound 18 or the solvent DMSO. Nuclei were stained with

propidium iodide (PI; middle panels). Merged images are shown at the right.

Bar, 50 μm.

1

7b, which were found to be 4 and 5.5 times more potent than their

parent compounds 16 and 17, respectively, whereas compound 18b

was equipotent (EC50 18b = 0.12 μM) to the NH and N-methyl

congeners (EC50 18 = 0.10 μM and EC50 18a = 0.11 μM, respectively).

The increased activity of the compounds in HCV genotype 4a, as

compared to the other genotypes, resulted in higher SI values, which

ranged from >345 to 1984 for the NH analogues 16, 17 and 18. As for

the N-methylated analogues, the most selective were 17a (SI 682)

and 18a (SI 1015). Finally, out of all compounds, the most promising

appeared to be 18, whose SI values were significantly high: 794, 2480

and 1984 for genotypes 1b, 3a and 4a, respectively.

cells treated with compound 18, which were best visible at high

compound concentration (Figure 5B). Nuclei were stained with

propidium iodide (PI) as a cell viability control.

Validation of compounds 17a and 18 activity with additional assays.

The inhibition profile of the most potent and safe compounds from

the tetralin and benzocycloheptane groups, as measured by

luciferase assay, was confirmed by determining viral RNA and NS5A

protein levels. As the compounds showed higher activity against HCV

genotype 4, Huh7.5-4a replicon cells were used for validating

activity. Specifically, cells were treated with serial dilutions of

compounds 17a or 18, or the solvent DMSO (control) and viral RNA

was quantified by reverse transcription-quantitative polymerase

chain reaction (RT-qPCR) whereas NS5A was evaluated by indirect

immunofluorescence. We observed that compounds 17a and 18

Activity of the compounds against DENV. Taking into account the

broad activity of the compounds against different HCV genotypes,

they were also tested against the closely related DENV. Both HCV and

DENV belong to the same family (Flaviviridae) and express

metalloenzymes, which are potential targets of our compounds. To

examine the anti-DENV activity of the non-substituted analogues (16,

1

7 and 18), we determined viral replication-derived luciferase

activity in Huh7-D2 stable cell line containing the subgenomic

replicon of DENV serotype 2 (strain 16681). Interestingly, the order

of activity obtained with 16, 17 and 18 was the same when they were

tested against DENV. Compound 16 exhibited the lowest inhibitory

activity (EC50 = 96.34 μM), which was progressively increased along

caused a reduction in HCV RNA replication (Figure 5A), with EC50

s

similar to those calculated on the basis of virus-derived luciferase

activity (Table 1). Consistent results were obtained by indirect

immunofluorescence analysis of HCV NS5A in Huh7.5-4a replicon

6

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with the bulkiness of the spiro-substituent, resulting in an EC50 value substituents at position 4 of the hydantoin core were a favorable

View Article Online

DOI: 10.1039/C9MD00200F

of 16.30 μM for analogue 18.

structural modification.

Methyl substitution on the amide nitrogen atom of the

respective 2,4-diketoimidazolidine residue of the parent compound

Trypanocidal assays

Activity of the compounds against T. brucei. Acetohydroxamic 18 led to a 2-fold increase in potency for the benzocycloheptane

acid derivatives exhibited considerable trypanocidal activity against substituted analogue 18a. On the other hand, incorporation of a

T. brucei, with EC50s ranging from 0.013 to 1.91 μM. Compounds 18b methyl substituent in 16 and 17, lowered the activity observed in the

and 17b were the most potent against African trypanosomes with N-methylated analogues 16a and 17a, with EC50 values 1.22 μM and

low nanomolar EC50 values (13 nM and 50 nM respectively, Table 1), 1.91 μM respectively. They were almost 3 times less potent than the

while hydroxamates 16b, 17b and 18b were also significantly active corresponding NH-analogues (Table 2).

against T. cruzi epimastigotes, with EC50 values in the low micromolar

range.

Addition of the bulky hydrophobic benzyl substituent to the

same nitrogen atom of the hydantoin scaffold yielded analogues that

Changing the indane ring in structure 16 with the tetralin ring were extremely potent against T. brucei, as exemplified by

decreased the activity, with the EC50 value of 17 being 1.5-fold higher compounds 16b, 17b and 18b (Table 2). Their activities were

(EC50 = 0.71 μM). Somewhat surprisingly, the replacement of indane respectively 4.5, 14 and 31 times higher than the parent compounds

by benzocycloheptane led to compound 18, which was almost 16, 17 and 18. All the N-benzylated analogues retained high potency,

equipotent to lead compound 16 (EC50 16 = 0.44 μM; EC50 18 = 0.40 with EC50 values ranging between 13 and 101 nM (Table 2). It is

μM). These molecules appeared to confer a beneficial effect on the noteworthy that the incorporation of a benzyl group in the

trypanocidal potency, with respect to the structurally related corresponding non-substituted precursors 16, 17 and 18

cycloheptane spiro-substituted 2,6-DKP analogue II (EC50 = 1.87 progressively increased activity against T. brucei in vitro. This

5

7

μM), indicating that the fused bicyclic systems that were used as enhanced potency of analogues 16b, 17b and 18b reflects the

The cytotoxicity of all target compounds against mammalian

cells was determined using the rat skeletal myoblast L6 cell line.

Notably, the non-substituted analogues 16, 17 and 18 displayed very

low cytotoxicity against mammalian cells, having CC50s in the 125-

4

19 μM range, thus resulting in remarkable selectivity indices, which

varied from 313 (18) to 609 (16). In general, as in the case of HCV, it

was observed that the incorporation of an alkyl substituent onto the

amide nitrogen atom progressively increased the cytotoxicity of the

compounds, with the exception of compound 16a (CC50 = 550 μM, SI

strongly favorable stereoelectronic and hydrophobic effects exerted

by the benzyl substituent in the binding site.

T. brucei

T. cruzi

EC90 (μM)^a

logD

L6 cells

^C^C50 (μM)

pKa^e

Structure

Cmpd

R

(pH

c

EC50 (μM)^a

EC90 (μM)^a

SI^b

EC50 (μM)^a

SI^b

d

7

.4)

1

6

H

CH

0.44 + 0.08

1.22 + 0.39

0.80 + 0.02

1.93 + 0.45

609

451

>30

-

268 + 9

-0.24

9.1

9.0

O

550 + 24

-0.01

1

6a

3

O

N

R

N

OH

N

O

H

16b

CH

2

Ph 0.101 + 0.003

0.192 + 0.003

62

1.70 + 0.06

3.01 + 0.36

4

6.27 + 0.60

1.71

9.5

1

7

H

CH

0.71 + 0.12

1.91 + 0.23

1.27 + 0.02

4.69 + 0.10

590

15

>30

-

419 + 55

0.21

0.43

9.3

9.0

O

17a

3

27.6 + 5.3

42.1 + 2.4

1

28.2 + 1.0

N

O

N

O

17b

CH Ph 0.050 + 0.003

0.077 + 0.021

63

0.96 + 0.03

1.71 + 0.07

3

3.17 + 0.48

2.16

9.2

2

HN OH

1

8

H

0.40 + 0.04

0.23 + 0.01

0.52 + 0.05

0.30 + 0.01

313

71

>30

-

125 + 12

0.65

0.88

9.3

9.2

1

8a

8b

N

O

CH

3

10.6 + 0.6

15.3 + 0.2

2

16.4 + 1.1

R

N

O

1

CH

2

Ph 0.013 + 0.002

0.018 + 0.001

604

0.495 + 0.058

0.94 + 0.033

16

7.85 + 0.33

>500

2.60

9.4

HN OH

O

H

-

>50

-

>500

-

0.89

n.a.

HN

4

II

NH

O

H

O

N

O

1.87 + 0.08

2.53 + 0.29

-

N

OH

N

H

O

a

b

Concentrations required to inhibit growth of T. brucei and T. cruzi by 50% and 90%, respectively. EC50 and EC90 data are the mean of triplicate experiments ± SEM. Selectivity indices were

c

calculated as the ratio of the CC50 for L6 cells to EC50 for T. brucei or T. cruzi respectively. Cytotoxicity was determined by establishing the concentration required to inhibit growth of

cultured L6 cells by 50% (CC50). Data are the mean of triplicate experiments ± SEM. logD at pH 7.4 determined using Marvin. pKa values for the oxime proton determined from MM

preoptimized geometries at the DFT(b3lyp) level with the cc-pVTZ(+) basis set using the Jaguar pKa module of Maestro.

d

62 e

6

3-66

Table 2 Activity of acetohydroxamic acid analogues 16-18, 16a-18a, 16b-18b and hydantoin analogue 4 tested against cultured bloodstream form T. brucei (pH

This journal is © The Royal Society of Chemistry 20xx

.4) and T. cruzi epimastigotes. Cytotoxicity against cultured rat skeletal myoblast L6 Cells, selectivity indices, logD and pKa values.

J. Name., 2013, 00, 1-3 | 7

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4

51). Finally, the acetohydroxamic acid derivative 18b, which had

suggest that the increased size of the spiro-substituent generally

favors inhibitory activity towards the targets of interest. Moreover,

View Article Online

DOI: 10.1039/C9MD00200F

the highest activity against T. brucei (EC50 = 0.013 μM) displayed a

very good selectivity (SI 604), despite the benzyl substitution.

activity is enhanced upon increasing the lipophilicity of the

compounds, particularly via introduction of a methyl- or benzyl-

substituent at the amide nitrogen atom of the hydantoin ring. In fact,

the results suggest that increasing the bulkiness of the substituent

improves rather than retards activity. Along with the lipophilicity,

the acetohydroxamic acid moiety, capable of chelating active site

metal ion(s), is an indispensable component of this class of

inhibitors, since removal of this functional group from the imidic

nitrogen atom caused a dramatic loss of activity. Finally, the

remarkable selectivity indices observed for most of the tested

analogues indicate that the novel scaffold of an appropriately

substituted hydantoin ring and a metal binding moiety may be the

key to unlock multiple pathogen inhibition, as a successful strategy

for producing multifunctional drugs.

Activity of the compounds against T. cruzi.

Compounds were also tested against cultured T. cruzi

epimastigotes, with IC50 values at submicromolar to micromolar

levels (0.49-27.6 μΜ) (Table 2). Acetohydroxamic acid derivatives

with a substituent on the amide nitrogen atom were able to inhibit

parasite growth, an effect that was not observed with the non-

substituted derivates. More specifically, compounds 16, 17 and 18

exhibited no activity at the highest concentration tested (30 μM),

whereas the N-methylated analogues showed medium inhibitory

activity (EC50 17a = 27.6 μM; EC50 18a = 10.6 μM), with the only

exception being analogue 16a. It is of great importance that the

incorporation of a benzyl group resulted in unexpectedly potent

analogues. The EC50s obtained ranged from 0.49 μM for the bulkier

analogue 18b to 1.70 μM for the indane analogue 16, indicating the

positive effect of increasing the bulkiness and lipophilicity of the

compounds, a pattern also observed in T. brucei sp. Interestingly,

activity of the N-benzylated analogue 18b was 21.5-fold higher than

the N-methylated counterpart 18a. For the respective tetralin

analogue 17b, this increase in potency was 29 times higher. Finally,

the absence of the acetohydroxamic acid moiety was detrimental for

the activity against bloodstream form T. brucei and T. cruzi

epimastigotes (Table 2), suggesting that the hydroxamic acid unit is

essential also for the trypanocidal activity.

Concerning the physicochemical parameters of the prepared

analogues, enhanced lipophilicity quantified by calculated logD

values seems to be important to their bioactivity, and in most cases

beneficial in terms of their capacity as drug leads. Conversely, pKa

values of the various analogues do not demonstrate significant

difference and thus they do not appear to contribute to the observed

structure-activity relationships.

Biological evaluation indicates that metalloproteins may

constitute the targets of the acetohydroxamic acid-based analogues

described herein. However, binding and pulldown assays will be

helpful to fully rationalize the observed bioactivity and determine

the exact molecular targets of the novel analogues, thus facilitating

structure-based design of metalloenzyme inhibitors.

Experimental

Chemistry

Experimental methods. During the conduct of the experimental part

of the present study were used the following materials, apparatuses

and techniques. Melting points were determined using a Büchi

capillary apparatus and are uncorrected. NMR experiments were

performed to elucidate the structure and determine the purity of the

1

newly synthesized compounds. H NMR and 2D NMR spectra (COSY,

HSQC, HSQC-DEPT and HMBC) were recorded on a Bruker DRX400

1

spectrometer (400.13 MHz, H NMR) and a Bruker Ultrashield™ Plus

1

13

Avance III 600 spectrometer (600.11 MHz, H NMR). C NMR and

DEPT NMR spectra were recorded on a Bruker Avance 200

1

3

spectrometer (50.32 MHz, C NMR) and a Bruker Ultrashield™ Plus

1

3

Avance III 600 spectrometer (150.9 MHz, C NMR). Chemical shifts

δ (delta) are reported in parts per million (ppm) downfield from the

NMR solvent, with the tetramethylsilane or solvent (DMSO-d ) as

6

internal standard. Data processing including Fourier transformation,

baseline correction, phasing, peak peaking and integrations were

performed using MestReNova software v.12.0.0. Splitting patterns

are designated as follows: s, singlet; br s, broad singlet; d, doublet;

br d, broad doublet; t, triplet; q, quartet; dd, doublet of doublets;

ddd, doublet of doublets of doublets; dddd, doublet of doublets of

doublets of doublets; ddddd, doublet of doublets of doublets of

doublets of doublets; dt, doublet of triplets; dq, doublet of quartets;

ddt, doublet of doublets of triplets; ddq, doublet of doublets of

quartets; dtt, doublet of triplets of triplets; dqt, doublet of quartets

of triplets; ddtd, doublet of doublets of triplets of doublets; td,

triplet of doublets; tdd, triplet of doublets of doublets; qt, quartet of

triplets; m, multiplet; complex m, complex multiplet. Coupling

constants (J) are expressed in units of Hertz (Hz). The spectra were

recorded at 293 K (20 °C) unless otherwise specified. The solvents

Conclusions

In summary, despite the small overlapping degree between the

starting frameworks I and II, we have successfully designed and

synthesized compounds with dual activity, by merging two distinct

scaffolds into a single chemical entity. The framework combination

strategy involved the integration of structural elements from indole-

flutimide analogues, exhibiting antiviral potency, with spiro

carbocyclic 2,6-DKP acetohydroxamic acid-based trypanocidal

agents, in order to achieve both activities into a single lead molecule.

The novel bicyclic substituted hydantoin analogues not only

exhibited individually improved antiviral and antiparasitic activity

compared to analogues I and II, but also demonstrated exceptional

inhibitory activity against different HCV genotypes (1b, 3a, 4a) and

DENV, as well the two Trypanosoma species (T. brucei, T. cruzi).

Structure-activity relationships deduced from the biological results

3

used to obtain the spectra were: deuterated chloroform, CDCl (s,

1

13

7

.26 ppm, H NMR; t, 77.16 ppm, C NMR), deuterated dimethyl

1

13

sulfoxide, DMSO-d

NMR) and deuterated methanol, MeOD-d

septet, 49.00 ppm, 13_C_N_M_R₎_._A_n_a_l_y_t_i_c_a_l_t_h_i_n_-_l_a_y_e_r_c_h_r_o_m_a_t_o_g_r_a_p_h_y

TLC) was used to monitor the progress of the reactions, as well as

6

(quin, 2.50 ppm, H NMR; septet, 39.52 ppm,

C

1

4

(quin, 3.31 ppm, H NMR;

(

to authenticate the compounds. TLCs were conducted on, precoated

8

| J. Name., 2012, 00, 1-3

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with normal-phase silica gel, aluminium sheets (Silica gel 60 F254

Merck) (layer thickness 0.2 mm). Developed plates were examined

under a UV light source, at wavelengths of 254 nm and 365 nm, or

,

Method B: The appropriate carboxylic acid (11, 10a-1 _V 2 _i _e a _w ,_A 1 _r _t 0 _i _c b _l _e ,_O 1 _n 1 _l _i b _n _e )

(2.0 mmol, 1.0 eq) was treated with CDI (2.4 mmol, 1.2 eq) in dry THF

(26 mL) and the mixture was stirred for 1 h, at 28-30 °C under Argon.

Then, O-benzylhydroxylamine hydrochloride (2.4 mmol, 1.2 eq) and

TEA (3.64 mmol, 1.82 eq) were added successively, and the stirring

was continued for 24 h, at 28-30 °C under Ar. After removal of the

solvent in vacuo, the reaction mixture was quenched with ice-water

DOI: 10.1039/C9MD00200F

f

after being stained by iodine vapors. The Retention factor (R ) of the

newly synthesized compounds, that equals to the distance migrated

over the total distance covered by the solvent, was also measured

on the chromatoplates. Column chromatography technique was

used to isolate the desired compounds from by-products of

reactions, and it was carried out using elution solvents of increasing

(

40 mL) and extracted with AcOEt (4 × 40 mL). The combined organic

phases were washed with H O (3 × 70 mL) and brine (2 × 70 mL),

dried with anh. Na SO and evaporated under reduced pressure. The

crude residue was purified by column chromatography on silica gel

using a mixture of CH Cl /AcOEt and AcOEt as eluents to afford the

2

polarity in a stationary phase of SiO (Silica gel 60, 40-63 μm, 230-

2

4

00 mesh ASTM, Silica flash). Column packing was done using the

slurry method, while the mixtures to be analyzed were loaded using

either the technique of dry deposition, or as a solution in the first

eluent where this was possible. Elemental analyses (C, H, N) were

performed by the Service Central de Microanalyse at CNRS (France),

and were within ±0.4% of the theoretical values. Elemental analysis

results for the tested compounds correspond to ˃95% purity. The

commercial reagents were purchased from Alfa Aesar, Sigma-

Aldrich, and Merck, and were used without further purification,

except for the benzyl bromoacetate. This reagent was purified by

fractional distillation in vacuo prior to use. Organic solvents used

were in the highest purity, and when necessary, were dried by the

standard methods. Solvent and reagent abbreviations: THF,

2

desired amides (14, 13a-15a, 13b, 14b respectively).

General experimental procedure for the preparation of the

hydroxamic acids 16-18, 16a-18a and 16b-18b. 10 wt. % Pd on

charcoal was added to a solution of the appropriate O-benzyl

hydroxamate (13-15, 13a-15a, 13b-15b) (1.0 mmol) in a mixture of

EtOH/AcOEt 3:1 (40 mL). After 3 h of shaking under an atmosphere

of 50-55 psi hydrogen, at 40-45 °C, the catalyst was removed by

filtration and washed with hot MeOH (3 × 15 mL). The combined

filtrates were concentrated to dryness under reduced pressure to

afford the title acetohydroxamic acid analogues (16-18, 16a-18a and

tetrahydrofuran; DMF, N,N-dimethylformamide; Et

AcOEt, ethyl acetate; EtOH, ethanol; MeOH, methanol; EDCI·HCl, N-

3-dimethylaminopropyl)-Nʹ-ethylcarbodiimide hydrochloride;

HOBt, 1-hydroxybenzotriazole hydrate; CDI, 1,1ʹ-

2

O, diethyl ether;

1

6b-18b respectively).

(

General N-benzylation procedure for the preparation of analogues

b-9b. The appropriate benzyl ester (7-9) (2.6 mmol, 1.0 eq) was

carbonyldiimidazole; TEA, triethylamine.

7

dissolved in 12 mL dry DMF and was treated with small portions of

NaH (3.12 mmol, 1.2 eq, 60% dispersion in mineral oil) under ice-

cooling. After 15 min of stirring at r.t. under Argon, benzyl bromide

was added dropwise (3.12 mmol, 1.2 eq). After 7 days of stirring at

General experimental procedures

General experimental procedure for the preparation of carboxylic

acids 10-12, 10a-12a and 10b-12b. A solution of the appropriate

benzyl ester 7-9, 7a-9a and 7b-9b (2.0 mmol) in a mixture of

EtOH/AcOEt 3:1 (40 mL) was hydrogenated for 3 h at 40-45 °C and

6

0 °C, under Argon, the mixture was poured into 120 mL ice-water

and extracted with AcOEt (3 × 90 mL). The combined organic layers

were washed with H O (3 × 150 mL) and brine (2 × 150 mL), dried

over anh. Na SO and the solvent was evaporated in vacuo. The

5

0-55 psi pressure, in the presence of Pd/C (10 wt.%) as a catalyst.

The catalyst was filtered off, washed with portions of hot MeOH (3 ×

5 mL) and the combined filtrates were evaporated under reduced

2

4

resulting oily residue was chromatographed on silica gel column to

1

afford the title N-benzylated products 7b-9b.

pressure to yield the corresponding carboxylic acids 10-12, 10a-12a

and 10b-12b.

2

-(2,5-dioxo-2',3'-dihydrospiro[imidazolidine-4,1'-inden]-1-

yl)acetic acid (10). Prepared from benzyl ester 7 (420 mg, 1.2 mmol)

by catalytic hydrogenolysis in a mixture of EtOH/AcOEt 3:1 (24 mL),

following the previously described general procedure for the

preparation of carboxylic acids. Evaporation of the solvents under

reduced pressure afforded the title compound 10 as a white

General experimental procedure for the preparation of N-

(

phelylmethoxy)acetamides 13-15, 13a-15a and 13b-15b. Method

A: To a solution of the appropriate carboxylic acid (10, 12, 12b) (2.0

mmol, 1.0 eq) in 20 mL dry CH Cl /dry DMF (4:1), EDCI·HCl (2.4

2

mmol, 1.2 eq), HOBt (2.4 mmol, 1.2 eq, 80% monohydrate), O-

crystalline solid (310 mg, almost quantitative yield); mp 237-239 °C

benzylhydroxylamine hydrochloride (2.4 mmol, 1.2 eq) and TEA

1

(

from AcOEt/n-pentane), R

DMSO-d ) δ (ppm) 2.22 (dt, 1H, J

H, J =13.2 Hz, J =6.5 Hz, H2'), 3.05 (t, 2H, J=7.2 Hz, H3'), 4.07-4.15 (q,

AB, 2H, JAB=17.5 Hz, NCH COOH), 7.19 (d, 1H, J=7.5 Hz, H7'), 7.26 (tdd,

H, J =7.0 Hz, J =1.9 Hz, J =1.0 Hz, H6'), 7.33 (td, 1H, J =7.5 Hz, J =1.2

Hz, H5'), 7.34-7.36 (m, 1H, H4'), 8.84 (s, 1H, H ), 13.12 (br s, 1H,

NCH ) δ (ppm) 29.7 (C3'), 36.0

COOH); ¹³C NMR (150.9 MHz, DMSO-d

2'), 39.1 (NCH COOH), 70.8 (C1'/C ), 123.1 (C7'), 125.0 (C4'), 127.0

6'), 129.0 (C5'), 141.0 (C7a'), 143.9 (C3a'), 155.2 (C =O), 168.7

f

= 0.06 (AcOEt). H NMR (600.11 MHz,

(11.6 mmol, 5.8 eq) were added sequentially. The reaction mixture

1 2

=13.3 Hz, J =7.8 Hz, H2'), 2.55 (dt,

was stirred for 40 h, at 30-35 °C under Argon. After removal of CH Cl

2

6

1

2

under reduced pressure, ice-water was added (40 mL) and the

mixture was extracted with AcOEt (4 × 40 mL). The combined organic

2

1

2

3

1

2

layers were washed with H

over anh. Na SO and evaporated in vacuo. The crude residue was

purified by column chromatography on silica gel using a mixture of

CH Cl /AcOEt and AcOEt as eluents to afford the desired amides (13,

5, 15b respectively).

2

O (3 × 70 mL) and brine (2 × 70 mL), dried

3

2

4

2

6

(

C

2

4

2

1

2

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J. Name., 2013, 00, 1-3 | 9

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Please Md eo d nC oh te am d Cj uo smt mm argins

Page 10 of 17

ARTICLE

Journal Name

(

NCH

2

COOH), 175.2 (C

: C, 60.00; H, 4.65; N, 10.76%

5

=O). Found: C, 60.08; H, 4.70; N, 10.79. Calc.

NCH

¹³C NMR (150.9 MHz, DMSO-d

8.0 (NCH CO, E-isomer), 38.5 (NCH

123.4 (C7'), 124.9 (C4'), 127.0 (C6'), 129.0 (C5'), 141.3 (C7a'), 143.9 (C3a'),

155.4 (C =O, E-isomer), 155.6 (C =O, Z-isomer), 163.3 (NCH CO, E-

isomer), 168.7 (NCH CO, Z-isomer), 175.5 (C =O, E-isomer), 175.7

(C =O, Z-isomer). Found: C, 56.78; H, 4.81; N, 15.30. Calc. for

: C, 56.72; H, 4.75; N, 15.27%

2

CONHOH, Z-isomer), 10.72 (s, 0.6H, CH CONHOH, E -isomer);

2

View Article Online

for C13

12

H N

2

O

4

6

) δ (ppm) 29.8 (C3'), 36.0, 36.1 (C2'),

CO, Z-isomer), 70.8 (C1'/C ),

DOI: 10.1039/C9MD00200F

3

2

4

N-(phenylmethoxy)-2-(2,5-dioxo-2',3'-dihydrospiro[imidazolidine-

,1'-inden]-1-yl)acetamide (13). Carboxylic acid 10 (260 mg, 1.0

mmol) was treated with EDCI·HCl (230 mg, 1.2 mmol), HOBt (203 mg,

.2 mmol, 80% monohydrate), O-benzylhydroxylamine

hydrochloride (192 mg, 1.2 mmol) and TEA (587 mg, 5.8 mmol) in 10

mL dry CH Cl /dry DMF (4:1) following the general procedure for the

preparation of O-benzyl hydroxamates (Method A). After removal of

CH Cl under reduced pressure, the reaction mixture was poured

into 20 mL ice-water and extracted with AcOEt (4 × 20 mL). The

combined organic layers were washed with H O (3 × 35 mL) and

brine (2 × 35 mL), dried over Na SO and concentrated to dryness in

vacuo. The oily pale-yellow residue was purified by column

chromatography on silica gel eluting first with CH Cl /AcOEt 8:1, 5:1

4

2

5

1

5

13 13 3 4

C H N O

2

2-(3-methyl-2,5-dioxo-2',3'-dihydrospiro[imidazolidine-4,1'-

inden]-1-yl)acetic acid (10a). It was prepared by hydrogenolysis of

the corresponding benzyl ester 7a (320 mg, 0.88 mmol) in a mixture

of 18 mL EtOH/AcOEt (3:1), following the general procedure for the

preparation of carboxylic acids. After removal of the solvent under

reduced pressure, the title compound 10a was obtained as a white

crystalline solid (240 mg, almost quantitative yield); mp 203-205 °C

2

4

2

and then AcOEt to give the corresponding O-benzyl hydroxamate 13

as a white foamy solid, which strongly binds the eluting solvents.

Removal of the entrapped solvents upon drying under high vacuum

and treatment with n-pentane at 0 °C gave 13 as a white crystalline

(melted gradually from 198 °C) (AcOEt/n-pentane), R

¹H NMR (400.13 MHz, MeOD-d

) δ (ppm) 2.40 (ddd, 1H, J

=8.4 Hz, J =6.8 Hz, H2'), 2.56 (ddd, 1H, J =14.0 Hz, J =8.3 Hz, J

Hz, H2'), 2.69 (s, 3H, NCH ), 3.07-3.22 (m, 2H, H3'), 4.21-4.34 (q, AB,

2H, JAB=17.6 Hz, NCH COOH), 5.13 (br s, 1H, NCH COOH), 7.20 (d, 1H,

J=7.3 Hz, H7'), 7.22-7.29 (m, 1H, H6'), 7.31-7.38 (m, 2H, H4', H5'); ¹³

NMR (50.32 MHz, MeOD-d ) δ (ppm) 25.4 (NCH ), 31.4 (C3'), 33.5

(C2'), 40.4 (NCH COOH), 76.5 (C1'/C ), 124.5 (C7'), 126.4 (C4'), 128.4

(C6'), 130.9 (C5'), 139.0 (C7a'), 146.4 (C3a'), 156.6 (C =O), 170.3

(NCH COOH), 176.6 (C =O). Found: C, 61.29; H, 5.21; N, 10.28. Calc.

: C, 61.31; H, 5.15; N, 10.21%

f

= 0.24 (AcOEt).

=13.9 Hz,

=5.7

4

1

J

2

3

1

2

3

solid (260 mg, 71%); mp 154-156 °C (AcOEt/n-pentane), R

f

= 0.06

) δ (ppm)

=13.1 Hz, J =6.6

Hz, H2'), 3.05 (t, 2H, J=7.1 Hz, H3'), 3.92-4.01 (q, AB, 1.55H, JAB=16.3

Hz, NCH CO), 4.25 (br s, 0.3H, NCH CO), 4.81, 4.86 (s + s, 2H,

OCH Ph), 7.25-7.30 (m, 2H, H6', H7'), 7.31-7.35 (m, 2H, H4', H5'), 7.35-

.47 (m, 5H, H2'', H3'', H4'', H5'', H6''), 8.81 (s, 1H, H ), 11.02 (s, 0.1H,

CONHOCH Ph), 11.39 (s, 0.7H, CONHOCH

Ph); ¹³C NMR (150.9 MHz,

DMSO-d ) δ (ppm) 29.7 (C3'), 36.0 (C2'), 38.0 (NCH CO), 70.8 (C1'/C ),

7.0 (OCH Ph), 123.3 (C7'), 124.9 (C4'), 127.0 (C6'), 128.3 (C3'', C4'', C5''),

28.8 (C2'', C6''), 129.0 (C5'), 135.8 (C1''), 141.1 (C7a'), 143.9 (C3a'), 155.3

=O), 163.6 (CONHOCH Ph), 175.4 (C =O). Found: C, 65.69; H, 5.30;

N, 11.53. Calc. for C20 : C, 65.74; H, 5.24; N, 11.50%

2

1

(from CH

2

Cl

2

/AcOEt 8:1). H NMR (600.11 MHz, DMSO-d

6

C

2

.22 (dt, 1H, J

1

=13.5 Hz, J

2

=7.7 Hz, H2'), 2.57 (dt, 1H, J

1

2

4

3

2

4

2

5

7

3

14 2 4

for C14H N O

2

6

2

4

N-(phenylmethoxy)-2-(3-methyl-2,5-dioxo-2',3'-

7

1

2

dihydrospiro[imidazolidine-4,1'-inden]-1-yl)acetamide (13a). Using

the general procedure for the preparation of N-

(phelylmethoxy)acetamides (Method B), the carboxylic acid

precursor 10a (200 mg, 0.73 mmol) was treated with CDI (143 mg,

(C

2

5

19 3 4

H N O

0

.88 mmol), O-benzylhydroxylamine hydrochloride (140 mg, 0.88

N-hydroxy-2-(2,5-dioxo-2',3'-dihydrospiro[imidazolidine-4,1'-

inden]-1-yl)acetamide (16). A solution of the appropriate O-benzyl

hydroxamate 13 (200 mg, 0.55 mmol) in a mixture of EtOH/AcOEt

mmol) and TEA (135 mg, 1.33 mmol) in dry THF (9 mL). After removal

of the solvent in vacuo, the reaction mixture was quenched with 15

mL ice-water and extracted with AcOEt (4 x 15 mL). The combined

(

3:1, 22 mL) was hydrogenated following the general procedure

organic phases were washed with H

mL), dried over anh. Na SO and evaporated under reduced

pressure. The pale-yellow viscous oily residue was purified by

column chromatography on silica gel using CH Cl , CH Cl /AcOEt

20:1, 10:1 and AcOEt as eluents to afford the corresponding O-

benzyl hydroxamate 13a as a colorless oil, which was further

crystallized to a white crystalline solid by treatment with n-pentane

under ice-cooling (210 mg, 76%); mp 108-110 °C (from AcOEt/n-

2

O (3 × 25 mL) and brine (2 × 25

described above for the preparation of acetohydroxamic acids.

Evaporation of the solvents in vacuo yielded the title compound 16

as a white foamy solid, which strongly binds the aforementioned

solvents. Removal of the entrapped solvents upon drying under high

vacuum gave 16 as a glass solid, which was further crystallized upon

treatment with n-pentane-dry Et O 5:1 under ice-cooling (150 mg,

2

almost quantitative yield); mp 179-182 °C (from AcOEt/n-pentane-

dry Et

1

2

4

2

1

/AcOEt 8:1). ¹H NMR (600.11

=14.4 Hz, J =8.5 Hz, J =6.7

=6.0 Hz, H2'), 2.62 (s,

2

O), R

f

= 0.29 (AcOEt). This compound appeared in the H and

pentane-dry Et

MHz, DMSO-d ) δ (ppm) 2.41 (ddd, 1H, J

Hz, H2'), 2.48 (ddd, 1H, J =14.3 Hz, J =8.3 Hz, J

3H, NCH ), 3.05-3.16 (m, 2H, H3'), 3.96-4.06 (q, AB, 1.6H, JAB=16.2Hz,

NCH CO), 4.27 (br s, 0.3H, NCH CO), 4.81, 4.86 (s + s, 2H, OCH Ph),

7.22 (d, 1H, J=7.6 Hz, H7'), 7.29 (t, 1H, J=7.2 Hz, H6'), 7.33-7.47 (m, 7H,

4', H5', H2'', H3'', H4'', H5'', H6''), 11.06 (s, 0.1H, CONHOCH Ph), 11.40

) δ (ppm)

CO), 74.3 (C1'/C ), 77.0

2 f 2 2

O), R = 0.21 (CH Cl

3

1

C NMR spectra as a mixture of E/Z conformers. H NMR (600.11

6

1

2

3

MHz, DMSO-d

ddd, 1H, J =13.4 Hz, J

Hz, J =6.5 Hz, J =2.4 Hz, H3'), 3.91-4.00 (q, AB, 1.5H, JAB=16.5 Hz,

NCH CO, E-isomer), 4.20-4.29 (q, AB, 0.45H, JAB=17.5 Hz, NCH CO, Z-

isomer), 7.23-7.35 (complex m, 4H, H4', 7'), 8.73 (s, 0.1H, H ),

CONHOH, E-isomer),

CONHOH, Z-isomer), 10.29 (s, 0.2H,

6 1

) δ (ppm) 2.22 (dt, 1H, J =13.4 Hz, J

2

=7.5 Hz, H2'), 2.56

1

2

3

(

1

2

=7.5 Hz, J =6.0 Hz, H2'), 3.04 (ddd, 2H, J

3

1

=8.2

3

2

3

2

H

5',

H

6',

H

3

H

2

8

9

.76, 8.77 (s + s, 0.9H, H

.33 (s, 0.2H, NCH

3

), 8.95 (s, 0.6H, CH

2

(s, 0.7H, CONHOCH

24.7 (NCH ), 30.0 (C3'), 32.1 (C2'), 38.4 (NCH

2

Ph); ¹³C NMR (150.9 MHz, DMSO-d

6

2

3

2

4

1

0 | J. Name., 2012, 00, 1-3

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ARTICLE

(

1

OCH

28.8 (C2'', C6''), 129.5 (C5'), 135.7 (C1''), 138.0 (C7a'), 144.7 (C3a'), 155.3

=O), 163.6 (CONHOCH Ph), 175.4 (C =O). Found: C, 66.54; H, 5.61;

N, 11.00. Calc. for C21 : C, 66.48; H, 5.58; N, 11.08%

2

Ph), 123.4 (C7'), 125.2 (C4'), 127.2 (C6'), 128.3 (C3'', C4'', C5''),

1

J=7.7 Hz, H7'), 7.10-7.13 (m, 2H, H2Bz, H6Bz), 7.14 (td, 1H, J =7.4 Hz,

View Article Online

J

2

=1.3 Hz, H6'), 7.21-7.27 (complex m, 3H, HD 3OB Iz :,1H0 4. 1B 0z ,3H9 /5 CB z9 )M, 7D.020820(d0tF,

1H, J =7.5 Hz, J =1.1 Hz, H4'), 7.31 (td, 1H, J =7.4 Hz, J =1.1 Hz, H5'),

7.34-7.41 (m, 5H, H2'', H3'', H4'', H5'', H6''); ¹³C NMR (150.9 MHz, CDCl

δ (ppm) 30.7 (C3'), 33.9 (C2'), 40.1 (NCH COO), 44.1 (NCH Ph), 67.9

(COOCH Ph), 76.0 (C1'/C ), 124.0 (C7'), 125.4 (C4'), 127.7 (C2Bz, C6Bz),

(C

2

5

1

2

1

2

21

H N

3

O

4

3

)

2

N-hydroxy-2-(3-methyl-2,5-dioxo-2',3'-

2

4

dihydrospiro[imidazolidine-4,1'-inden]-1-yl)acetamide (16a).

A

127.8 (C6'), 128.6 (C3Bz, C5Bz), 128.7 (C2'', C6'', C4Bz), 128.76 (C4''), 128.84

(C3'', C5''), 130.0 (C5'), 135.0 (C1''), 137.5 (C1Bz), 137.9 (C7a'), 145.1 (C3a'),

mixture of the O-benzyl hydroxamate 13a (160 mg, 0.42 mmol) and

Pd on charcoal (19 mg) in EtOH/AcOEt 3:1 (17 mL) was subjected to

catalytic hydrogenolysis in exactly the same procedure described

above for the preparation of the final compounds. Removal of the

solvents under reduced pressure provided the acetohydroxamic acid

155.7 (C

2

=O), 167.3 (COOCH

2

Ph), 175.0 (C

5

=O). Found: C, 73.66; H,

5.55; N, 6.30. Calc. for C27

H N O : C, 73.62; H, 5.49; N, 6.36%

24 2 4

2-(3-benzyl-2,5-dioxo-2',3'-dihydrospiro[imidazolidine-4,1'-inden]-

1-yl)acetic acid (10b). Prepared from benzyl ester 7b (400 mg, 0.91

mmol) by catalytic hydrogenolysis in a mixture of 18 mL EtOH/AcOEt

(3:1) following the procedure described for the preparation of the

above-mentioned carboxylic acids. The desired compound 10b was

afforded as a glass solid, which was further crystallized upon

1

6a as a white foamy solid. Crystallization of this product upon

treatment with n-pentane-dry Et O under ice-cooling gave a white

crystalline solid (120 mg, almost quantitative yield); mp melted

gradually from 158°C to 162 °C (AcOEt/n-pentane-dry Et O), R = 0.22

from AcOEt). This compound exhibited distinct peaks attributed to

2

f

(

each of the two E/Z conformers in the H and ¹³C NMR spectra. ¹H

NMR (600.11 MHz, DMSO-d ) δ (ppm) 2.40 (ddd, 1H, J =14.1 Hz,

=8.5 Hz, J =4.2 Hz, H2'), 2.47 (ddd, 1H, J =14.1 Hz, J =8.2 Hz, J =4.0

Hz, H2'), 2.61 (s, 3H, NCH ), 3.04-3.16 (m, 2H, H3'), 3.95-4.04 (q, AB,

.5H, JAB=16.1 Hz, NCH CO, E-isomer), 4.24-4.33 (q, AB, 0.5H,

AB=17.3 Hz, NCH CO, Z-isomer), 7.23 (d, 1H, J=7.8 Hz, H7'), 7.27 (td,

=2.0 Hz, H6'), 7.34-7.40 (m, 2H, H4', 5'), 8.98 (s, 0.7H,

CONHOH, E-isomer), 9.36 (s, 0.23H, NCH CONHOH, Z-isomer),

0.34 (s, 0.23H, NCH CONHOH, Z-isomer), 10.75 (s, 0.7H,

CH ) δ (ppm)

CONHOH, E-isomer); ¹³C NMR (150.9 MHz, DMSO-d

4.8 (NCH ), 30.1 (C3'), 32.1 (C2'), 38.5 (NCH CO, E-isomer), 39.0

NCH CO, Z-isomer), 74.3 (C1'/C ), 123.5, 123.6 (C7'), 125.2 (C4'), 127.2

6'), 129.5 (C5'), 138.2 (C7a'), 144.7 (C3a'), 154.6 (C =O, E-isomer),

54.7 (C =O, Z-isomer), 163.2 (NCH CO, E-isomer), 168.6 (NCH CO,

Z-isomer), 174.6 (C =O, E-isomer), 174.8 (C =O, Z-isomer). Found: C,

8.23; H, 5.26; N, 14.50. Calc. for C14 : C, 58.13; H, 5.23; N,

4.53%

1

2

treatment with n-pentane-dry Et O (5:1) under ice-cooling to give a

6

1

white crystalline solid (316 mg, almost quantitative yield); mp 144-

1

J

2

3

1

2

3

147 °C (from AcOEt /n-pentane), R

MHz, DMSO-d ) δ (ppm) 2.14 (ddd, 1H, J

Hz, H2'), 2.44 (ddd, 1H, J =14.5 Hz, J =8.9 Hz, J

1H, J =15.8 Hz, J =8.7 Hz, J =5.9 Hz, H3'), 3.00 (ddd, 1H, J

=8.9 Hz, J =5.6 Hz, H3'), 3.96 (d, 1H, J=16.3 Hz, NCHHPh), 4.19-4.27

(q, AB, 2H, JAB=17.4 Hz, NCH COOH), 4.58 (d, 1H, J=16.3 Hz,

f

= 0.22 (AcOEt). H NMR (600.11

3

6

1

=14.1 Hz, J

=5.9 Hz, H2'), 2.93 (ddd,

=15.0 Hz,

2 3

=8.7 Hz, J =5.6

1

2

1

2

3

J

2

1

2

3

1

H, J =7.3 Hz, J

2

H

J

2

3

CH

2

1

2

NCHHPh), 7.07-7.14 (m, 3H, H7', H2Bz, H6Bz), 7.15-7.26 (complex m,

2

6

4H, H6', H3Bz, H4Bz, H5Bz), 7.30-7.35 (m, 2H, H4', H5'), 13.33 (br s, 1H,

2

3

2

NCH

(C2'), 40.1 (NCH

125.2 (C4'), 127.1 (C6', C2Bz, C4Bz, C6Bz), 128.2 (C3Bz, C5Bz), 129.5 (C5'),

137.6 (C1Bz), 138.3 (C7a'), 144.7 (C3a'), 155.1 (C =O), 168.7

(NCH COOH), 174.3 (C =O). Found: C, 68.60; H, 5.14; N, 8.13. Calc.

18 2 4

for C20H N O : C, 68.56; H, 5.18; N, 8.00%

2

COOH); ¹³C NMR (150.9 MHz, DMSO-d

6

) δ (ppm) 30.0 (C3'), 33.0

(

2

4

2

COOH), 43.2 (NCH

2

Ph), 74.9 (C1'/C ), 123.6 (C7'),

4

C

2

1

2

5

2

5

1

15

H N

3

O

4

N-(phenylmethoxy)-2-(3-benzyl-2,5-dioxo-2',3'-

Benzyl 2-(3-benzyl-2,5-dioxo-2',3'-dihydrospiro[imidazolidine-4,1'-

dihydrospiro[imidazolidine-4,1'-inden]-1-yl)acetamide

(13b).

inden]-1-yl)acetate (7b). A stirred solution of benzyl ester 7 (600 mg,

Prepared from carboxylic acid 10b (290 mg, 0.83 mmol) upon

treatment with CDI (162 mg, 1.0 mmol), O-benzylhydroxylamine

hydrochloride (160 mg, 1.00 mmol) and TEA (153 mg, 1.51 mmol) in

dry THF (11 mL), following the general procedure for the synthesis

of O-benzyl hydroxamates (Method B). After removal of THF under

reduced pressure, the reaction mixture was quenched with 20 mL

ice-water and extracted with AcOEt (4 x 20 mL). The combined

1

.71 mmol) in dry DMF (7.6 mL) was treated with NaH (82 mg, 2.05

mmol, 60% dispersion in mineral oil) and benzyl bromide (351 mg,

.05 mmol) by employing the N-alkylation reaction described above.

2

The reaction mixture was poured into an ice-water mixture (80 mL)

and extracted with AcOEt (3 × 60 mL). The combined organic phases

were washed with H

over anh. Na SO and evaporated under reduced pressure. The

brownish-yellow crude oily residue was purified by silica gel column

chromatography. CH Cl was used as eluent to afford the target

2

O (3 × 100 mL) and brine (2 × 100 mL), dried

2

4

organic layers were washed with H

mL), dried with anh. Na SO and evaporated in vacuo. The resulting

viscous oily residue was chromatographed on silica gel using CH Cl

CH Cl /AcOEt 20:1, 10:1 and AcOEt as eluents to give the

corresponding O-benzyl hydroxamate 13b as glass solid.

Crystallization of this material upon treatment with a n-pentane-dry

Et O mixture (5:1) gave a white crystalline solid (310 mg, 82%); mp

2

O (3 × 35 mL) and brine (2 × 35

2

4

2

,

compound 7b as a white crystalline solid (510 mg, 68%); mp 129-131

2

1

°

(

C (from AcOEt/n-pentane), R

600.11 MHz, CDCl ) δ (ppm) 2.05 (ddd, 1H, J

=5.5 Hz, H2'), 2.52 (ddd, 1H, J =13.8 Hz, J =9.1 Hz, J

.84 (ddd, 1H, J =16.0 Hz, J =8.9 Hz, J =5.6 Hz, H3'), 3.07 (ddd, 1H,

=15.9 Hz, J =9.1 Hz, J =5.5 Hz, H3'), 3.81 (d, 1H, J=15.9 Hz, NCHHPh),

.41-4.49 (q, AB, 2H, JAB=17.3 Hz, NCH COO), 4.82 (d, 1H, J=15.9 Hz,

f

= 0.76 (CH

2

Cl

2

/AcOEt 8:1). H NMR

a

3

1

=14.2 Hz, J

2

=8.9 Hz,

=5.6 Hz, H2'),

J

3

1

2

3

2

1

2

3

melted gradually from 60 °C to 78 °C (from AcOEt/n-pentane-dry

1

J

1

2

3

Et

δ (ppm) 2.13 (ddd, 1H, J

(ddd, 1H, J =14.5 Hz, J =8.9 Hz, J

2

O), R

f

= 0.41 (CH

2

Cl

2

/AcOEt 8:1). H NMR (600.11 MHz, DMSO-d

6

)

4

2

1

=14.0 Hz, J

=6.0 Hz, H2'), 2.93 (ddd, 1H, J

1

2

=8.6 Hz, J

3

=5.3 Hz, H2'), 2.45

NCHHPh), 5.20-5.26 (q, AB, 2H, JAB=12.1 Hz, OCH

2

Ph), 7.06 (d, 1H,

1

2

3

=15.8

J. Name., 2013, 00, 1-3 | 11

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Journal Name

Hz, J

=5.4 Hz, H3'), 3.94 (d, 1H, J=16.3 Hz, NCHHPh), 4.04-4.13 (q, AB,

.65H, JAB=16.2 Hz, NCH CO), 4.35 (br s, 0.35H, NCH CO), 4.58 (d, 1H,

J=16.2 Hz, NCHHPh), 4.82, 4.89 (s + s, 2H, OCH Ph), 7.13 (d, 1H, J=7.2

Hz, H2Bz, H6Bz), 7.16-7.26 (m, 5H, H6', H7', H3Bz, H4Bz, H5Bz), 7.30-7.35

2

=8.6 Hz, J

3

=6.0 Hz, H3'), 2.99 (ddd, 1H, J

1

=14.9 Hz, J

2

=8.8 Hz,

translation of the HCV NS3 to NS5B coding region, fo _V l _i l _e o _w w _A e _r _t d _i _c _l _e by _O _n t _l h _i _n e _e

J

1

3

HCV 3’ UTR. The expression of the luc-ubi-neDoOfIu: 1s0io.1n0p39ro/Cte9iMnDe0n0a2b0l0eFs

the selection for cells containing the replicon using neomycin/G418

and the assessment of viral RNA replication by measuring the activity

of the reporter protein firefly luciferase (F-Luc). Stable cell lines

Huh7.5-3a and Huh7.5-4a harbor the HCV bicistronic replicons S52-

SG (Feo) (AII) and ED43-SG (Feo)(VYG) (kindly provided by C.M. Rice,

2

(

0

m, 2H, H4', H5'), 7.35-7.49 (m, 5H, H2'', H3'', H4'', H5'', H6''), 11.09 (s,

_P_h₎_;13_C_N_M_R

2 2

.15H, CONHOCH Ph), 11.46 (s, 0.75H, CONHOCH

6

7

The Rockefeller University, NY) of the analogous design and were

(150.9 MHz, DMSO-d

6

) δ (ppm) 30.0 (C3'), 33.0 (C2'), 38.6 (NCH

2

CO),

6

8

previously described. HCV sequences were derived from the

genotype 3a strain S52 and the genotype 4a strain ED43. DENV

bicistronic replicon plasmid pD2- hRUPac (kindly provided by C.M.

4

1

3.1 (NCH

2

Ph), 74.9 (C1'/C ), 77.0 (OCH Ph), 123.8 (C7'), 125.1 (C4'),

4

2

27.1 (C6', C2Bz, C4Bz, C6Bz), 128.2 (C3Bz, C5Bz), 128.3 (C2'', C4'', C6''), 128.8

3'', C5''), 129.5 (C5'), 135.7 (C1''), 137.6 (C1Bz), 138.4 (C7a'), 144.7 (C3a'),

(C

6

9

Rice) has been described previously. The structural protein-coding

region between capsid gene codon 28 and the last 26 codons at the

1

2 2

55.2 (C =O), 163.5 (CONHOCH Ph), 174.5 (C

5

=O). Found: C, 71.25;

H, 5.60; N, 9.32. Calc. for C27

25

H N

3

O

4

: C, 71.19; H, 5.53; N, 9.22%

3

’ end of the envelope gene from an infectious clone of dengue 2

virus (DEN2) 16681 have been replaced by a humanized Renilla

luciferase-ubiquitin-puromycin acetyltransferase (hRUPac) cassette.

A stable cell line (Huh7-D2) was generated by transfecting Huh7 cells

with the above replicon RNA and pooling colonies after selection

with 0.5 μg/ml puromycin. Cells were cultured in high glucose (25

mM) Dulbecco’s modified minimal essential medium (Invitrogen),

supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino

acids, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% (v/v)

fetal calf serum (referred to as complete DMEM). Complete DMEM

was supplemented with 500 μg/mL G418 for Huh5-2, 750 μg/ml

G418 for Huh7.5-3a, 350 μg/mL G418 for Huh7.5-4a and 0.5 μg/ml

puromycin for Huh7-D2.

N-hydroxy-2-(3-benzyl-2,5-dioxo-2',3'-dihydrospiro[imidazolidine-

,1'-inden]-1-yl)acetamide (16b). The N-benzyloxy precursor 13b

250 mg, 0.55 mmol) was subjected to catalytic hydrogenation in a

mixture of EtOH/AcOEt 3:1 (22 mL) according to the previously

described procedure for the synthesis of hydroxamate analogues.

Concentration to dryness of the solvents under reduced pressure

yielded the title compound 16b as a white crystalline solid (200 mg,

4

(

almost quantitative yield); mp melted gradually from 174 °C to 181

1

°

C (from AcOEt/n-pentane-dry Et

2

O), R

f

= 0.49 (AcOEt). Ιn the H and

1

3

C NMR spectra of this compound, double set of characteristic

peaks are distinguished for each of the two E/Z conformers. H NMR

1

(

600.11 MHz, DMSO-d

=5.4 Hz, H2'), 2.44 (ddd, 1H, J

.92 (ddd, 1H, J =16.0 Hz, J =8.5 Hz, J

=14.4 Hz, J =8.9 Hz, J =5.4 Hz, H3'), 3.93 (d, 1H, J=16.3 Hz, NCHHPh),

.04-4.12 (q, AB, 1.5H, JAB=16.0 Hz, NCH CO, E-isomer), 4.32-4.40 (q,

CO, Z-isomer), 4.57 (dd, 1H, J =16.3 Hz,

=8.8 Hz, NCHHPh), 7.12 (d, 1H, J=6.7 Hz, H2Bz, H6Bz), 7.15-7.25

complex m, 5H, H6', H7', H3Bz, H4Bz, H5Bz), 7.29-7.34 (m, 2H, H4', H5'),

.03 (s, 0.68H, CH CONHOH, E-isomer), 9.41 (s, 0.22H,

NCH CONHOH, Z-isomer), 10.37 (s, 0.22H, NCH CONHOH, Z-isomer),

0.80 (s, 0.68H, CH

CONHOH, E-isomer); ¹³C NMR (150.9 MHz,

DMSO-d ) δ (ppm) 30.0 (C3'), 32.9, 33.0 (C2'), 38.6 (NCH CO, E-

isomer), 39.1 (NCH CO, Z-isomer), 43.1 (NCH Ph), 74.9 (C1'/C ), 123.9

7'), 125.0 (C4'), 127.1 (C6', C2Bz, C4Bz, C6Bz), 128.2 (C3Bz, C5Bz), 129.5

5'), 137.6 (C1Bz), 138.5 (C7a'), 144.7 (C3a'), 155.3 (C

=O, Z-isomer), 163.2 (NCH CO, E-isomer), 168.6 (NCH

Z-isomer), 174.6 (C =O, E-isomer), 174.8 (C =O, Z-isomer). Found: C,

5.79; H, 5.18; N, 11.55. Calc. for C20 : C, 65.74; H, 5.24; N,

1.50%

6

) δ (ppm) 2.13 (ddd, 1H, J

=13.9 Hz, J =8.9 Hz, J

=6.3 Hz, H3'), 2.98 (ddd, 1H,

1

=13.9 Hz, J

2

=8.6 Hz,

In vitro transcription. Bicistronic DENV constructs were linearized

with XbaI and used for in vitro transcription as described previously.

J

2

J

4

3

1

2

3

=6.1 Hz, H2'),

1

2

3

7

0

1

2

3

2

Transfection with in vitro transcribed RNA. Electroporation with

AB, 0.5H, JAB=17.4 Hz, NCH

J

2

1

bicistronic DENV RNA into Huh7 cells was performed as described

2

7

1

elsewhere.

(

9

2

Cell-based antiviral assays. Replicon assays were performed by

2

4

seeding 1 × 10 cells per well in a 96-well flat bottom plate, cultured

1

2

in 200 μL complete DMEM supplemented with selection antibiotic.

After 24 h incubation at 37 °C (5% CO ), medium was removed and

2

6

2

4

serial dilutions (without G418) of the test compounds in complete

DMEM were added, in a total volume of 100 μL. After 3 days of

incubation at 37 °C, cells were lysed and F-Luc or R-Luc activity was

measured. Relative luminescence units (RLU) were calculated as

percentage of the respective values from DMSO-treated control

cells. The half maximal effective concentration (EC50) was defined as

the concentration of compound that reduced the luciferase signal by

(

C

2

=O, E-isomer),

CO,

1

55.5 (C

2

5

6

1

19 3 4

H N O

5

0%. EC50 values were determined by nonlinear regression analysis

after converting the drug concentrations into log-X using the Prism

.0 software (GraphPad Software Inc.).

Biology

5

Cell culture. Huh5-2⁶¹cells (kindly provided by R. Bartenschlager,

University of Heidelberg, Germany) contain the subgenomic HCV

reporter replicon I389luc-ubi-neo/NS3-3’/Con1/5.1 (genotype 1b;

strain Con1). This replicon is composed of the HCV 5’ UTR possessing

an internal ribosome entry site (IRES) that directs the expression of

a firefly luciferase-ubiquitin-neomycin phosphotransferase fusion

protein (luc-ubi-neo). Downstream of this reporter protein is the

IRES of the encephalomyocarditis virus (EMCV) that mediates

Luciferase and Bradford assays. Firefly luciferase (F-Luc) activity in

cell lysate was measured using Luciferase Assay System (Promega),

as recommended by the manufacturer. Renilla luciferase (R-Luc)

activity in cell lysates was measured using 12 μM coelenterazine

(Promega) in assay buffer (50 mM potassium phosphate, pH 7.4, 500

mM NaCl, 1 mM EDTA). Measurements were taken in a GloMax

20/20 single-tube luminometer (Promega) for 10 s. Luciferase

1

2 | J. Name., 2012, 00, 1-3

Please do not adjust margins

Page 13 of 17

Please Md eo d nC oh te am d Cj uo smt mm argins

Journal Name

ARTICLE

5

−1

activities were normalized to the total protein amount determined

using the Bradford assay reagent (Bio-Rad).

Experiments were initiated by seeding the parasites at 2.5 × 10 ml ,

View Article Online

DOI: 10.1039/C9MD00200F

and after the addition of test compounds, cultured at 28 °C for 4

days. Resazurin was added, the plates were incubated for a further

2 days, and then assessed as above. Fluorescence intensities were

determined using a BMG FLUOstar Omega (excitation 545 nm,

emission 590 nm). Data were analyzed using Graph Pad Prism 7

software. Values are expressed as EC50 ± SD and are the average of

three independent replicates.

Cytotoxicity assay. Cytotoxicity of the compounds was determined

by measuring intracellular ATP levels. Specifically, 10 cells per well

were seeded in 96-well flat bottom plates in total volume of 100 μL

complete DMEM. After 24 h, cells were incubated with the

4

compounds for 72 h at 37 °C (5% CO

measurement. Calculation of the compound concentration causing

0% cell death (CC50) was performed using cells treated with DMSO

2

) and lysed for ATP

5

In vitro cytotoxicity assays on rat skeletal myoblast L6 cells.

as control sample. CC50 values were determined by nonlinear

regression analysis after converting the drug concentrations into log-

X using the Prism 5.0 software (GraphPad Software Inc.).

Cytotoxicity against L6 cells was assessed using microtiter plates.

4

−1

Briefly, cells were seeded in triplicate at 1 × 10 mL in growth

medium containing different compound concentrations. The plates

were incubated for 6 days at 37 °C and resazurin then added to each

Measurement of intracellular ATP levels. ATP was measured using

the ViaLight HS BioAssay kit (Lonza) according to the manufacturer’s

protocol in a GloMax 20/20 single-tube luminometer (Promega) for

well. After a further 8 h incubation, the fluorescence was

determined using a BMG FLUOstar Omega plate reader.

1

s. ATP levels were normalized to total protein amounts.

Conflicts of interest

Indirect immunofluorescence. Indirect immunofluorescence

analysis of HCV-4a NS5A was performed as described elsewhere.

There are no conflicts to declare.

7

1

DNA was stained with propidium iodide (Sigma-Aldrich). Images

were acquired with the Leica TCS-SP8 Confocal Microscope.

Acknowledgements

E.G. would like to thank the European Federation for Medicinal

Chemistry (EFMC) and the editors of the journal for their kind

invitation. This work was partially supported by the General

Secretariat for Research and Technology (grant KRIPIS II-

MIS5002486), by the International Pasteur Network grant ACIP 18-

Total RNA extraction and quantification of viral replicons. Total

RNA was extracted from Huh7.5-4a cells using TRIzol reagent

(Ambion), according to the manufacturer’s instructions. Replicon

RNA was quantified with reverse-transcription (RT) and quantitative

real-time polymerase chain reaction (qPCR). For RT, the IRES specific

primer 5'-GGATTCGTGCTCATGGTGCA-3' (reverse) and Moloney

Murine Leukemia Virus (MMLV) reverse transcriptase (Promega)

were used. For qPCR, the IRES specific primers 5'-

2

015 and by the Empirikion Foundation. We would like to thank the

State Scholarships Foundation for providing a Ph.D fellowship to E.F.

MIS-5003404) and V.P. (MIS-5000432). We are grateful to Prof. Ralf

(

Bartenschlager (Heidelberg University, Germany) for kindly

providing the Huh5-2 cell line and Prof. Charles M. Rice for kindly

providing Huh7.5 cells, replicon plasmids S52-SG (Feo) (AII), ED43-SG

GGCCTTGTGGTACTGCCTGATA-3'

(forward)

and

5'-

GGATTCGTGCTCATGGTGCA-3' (reverse) and KAPA SYBR FAST qPCR

Master Mix (Kapa Biosystems) were used. The housekeeping gene

YWHAZ was employed as an internal control (primers 5'-

(Feo) (VYG) and pD2-hRUPac, as well as the 9E10 HCV NS5A-specific

antibody.

GCTGGTGATGACAAGAAAGG-3'

GGATGTGTTGGTTGCATTTCCT -3').

and

5'-

Notes and references

Statistical analysis. In all diagrams, bars represent mean values of at

least two independent experiments in triplicate. Error bars

represent standard deviation. Only results subjected to statistical

analysis using Student’s t-test with p≤0.05 were considered as

statistically significant and presented. Statistical calculations were

carried out using Excel Microsoft Office®.

1

.

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Trypanocidal assays. Bloodstream form T. brucei (strain 221) were

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Eight-point potency curves were performed in 96-well plates (200 μL

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Scaffold hybridization strategy towards potent hydroxamate-based inhibitors of: Flaviviridae viruses and Trypanosoma species

DOI: 10.1039/c9md00200f

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