3-(Benzodioxan-2-ylmethoxy)-2,6-difluorobenzamides bearing hydrophobic substituents at the 7-position of the benzodioxane nucleus potently inhibit methicillin-resistant Sa and Mtb cell division

European Journal of Medicinal Chemistry 120 (2016) 227e243

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European Journal of Medicinal Chemistry

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Research paper

3-(Benzodioxan-2-ylmethoxy)-2,6-diﬂuorobenzamides bearing

hydrophobic substituents at the 7-position of the benzodioxane

nucleus potently inhibit methicillin-resistant Sa and Mtb cell division

Valentina Straniero ^a, Marco Pallavicini ^a, Giuseppe Chiodini ^a, Carlo Zanotto ^b,

c

,

ꢀ ^b

Luca Volonte , Antonia Radaelli ^d, Cristiano Bolchi ^a, Laura Fumagalli ^a,

Maurizio Sanguinetti ^e, Giulia Menchinelli ^e, Giovanni Delogu ^e, Basem Battah ^e,

Carlo De Giuli Morghen ^{b d}, Ermanno Valoti ^a

,

, *

a

ꢀ

Dipartimento di Scienze Farmaceutiche, Universita di Milano, via Mangiagalli 25, I-20133 Milano, Italy

Department of Medical Biothechnologies and Translational Medicine, Universita di Milano, via Vanvitelli 32, I-20129 Milano, Italy

Department of Pharmacological and Biomolecular Sciences, Universita di Milano, via Balzaretti 9, I-2013 Milano, Italy

CNR Institute of Neurosciences, Cellular and Molecular Pharmacology Section, Universita di Milano, via Vanvitelli 32, I-20129 Milano, Italy

b

c

ꢀ

d

e

ꢀ

Institute of Microbiology, Universita Cattolica del Sacro Cuore, largo Gemelli 8, I-00168 Rome, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

Lipophilic substituents at benzodioxane C (7) of 3-(benzodioxan-2-ylmethoxy)-2,6-diﬂuorobenzamide

improve the antibacterial activity against methicillin-resistant Staphylococcus aureus strains to MIC

Received 18 December 2015

Received in revised form

9 March 2016

Accepted 25 March 2016

Available online 28 March 2016

values in the range of 0.2e2.5

mg/mL, whereas hydrophilic substituents at the same position and

modiﬁcations at the benzodioxane substructure, excepting for replacement with 2-cromanyl, are dele-

terious. Some of the lead compounds also exhibit good activity against Mtb. Parallel SARs to those of 3-(2-

benzothiazol-2-ylmethoxy)-2,6-diﬂuorobenzamide, well known FtsZ inhibitor, and cells alterations

typical of FtsZ inhibition indicate such a protein as the target of these potent antibacterial benzodioxane-

benzamides.

Keywords:

FtsZ

Antibacterial activity

Benzodioxane

2,6-Diﬂuorobenzamide

Staphylococcus aureus

Mycobacterium tuberculosis

Methicillin resistance

1. Introduction

PC190723, developed from 2,6-diﬂuoro-3-nonyloxybenzamide

(DFNB) [2], displays a potent antistaphylococcal activity (1 mg/mL

Filamentous temperature-sensitive protein Z (FtsZ) is a highly

conserved and ubiquitous bacterial protein. When bacteria divide,

FtsZ polymerizes in a GTP-dependent manner to form the Z-ring at

the mid-point of the cell under the membrane and, after recruiting

other cell division proteins, such a cytokinetic structure contracts

resulting in septum closure and formation of two daughter cells [1].

Because of its important role in bacterial cell division, FtsZ is a

promising target for agents able of altering its proper assembly and

thus of exerting antibiotic activity by inhibition of cell division.

Among the FtsZ-targeting compounds, the benzamide derivative

MIC) and it is one of the most attractive and studied antibacterial

agent [3,4]. In the successive optimization of PC190723 [5,6], the

best option proved to be replacement of thiazolopyridine with a

phenyl substituted oxazole and hydroxymethyl substitution at the

pseudo-benzylic position of the linker, this latter modiﬁcation

allowing both advantageous chiral switch to eutomer (R)-1

(0.12 mg/mL MIC against Staphylococcus aureus) and esteriﬁcation to

succinate prodrug (R)-2 [6]. Successively to PC190723 discovery

and contemporarily with its recent optimization to oxazole-

diﬂuorobenzamide (R)-1, we have reported that the chloro-

substituted benzodioxane-diﬂuorobenzamide (S)-4 is a potent

bacterial cell division inhibitor 1 (0.25 mg/mL MIC against S. aureus)

causing cell volume increase, as potent as rac-1, with which it is

* Corresponding author.

thought to share the same FtsZ-targeting mechanism proper of

E-mail address: ermanno.valoti@unimi.it (E. Valoti).

http://dx.doi.org/10.1016/j.ejmech.2016.03.068

228

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

heteroarylmethyl ethers of 2,6-diﬂuoro-5-hydroxybenzamide [7].

We have found that 4, as a racemate, is ten-fold more potent than

the unsubstituted benzodioxane-diﬂuorobenzamide 3 and its S

enantiomer, the eutomer, is twenty-fold more potent than 3 (Chart

1).

In a continuation of our effort to further develop benzodioxane-

diﬂuorobenzamides as antibacterial agents and to consolidate their

SAR analysis, we have designed a number of analogues of 3 through

a series of isosteric, positional or substituent modiﬁcations and we

have extended the biological evaluation to strains of gram-positive

bacterial clinical isolates, with different drug-resistance, and to

Mycobacterium tuberculosis. Here we report the synthesis of

diﬂuorobenzamides 5e23, the antibacterial activity and the SAR

intramolecular cyclization (37), (4) mesylation (38), (5) O-alkyl-

ation of 2,6-diﬂuoro-3-oxybenzamide with the mesylate of 2-

hydroxymethl-7-bromo-1,4-benzodioxane 38. By the same steps

(39e42), the 7-triﬂuoromethyl analogue 18 was prepared from 2-

hydroxy-5-triﬂuoromethylbenzaldeyde. The 7-ﬂuoro derivative 13

was obtained from 2-hydroxymethyl-7-ﬂuoro-1,4-benzodioxane,

the synthesis of which has been previously reported [12], by

mesylation (43) and O- alkylation of 2,6-diﬂuoro-3-oxybenzamide

with the resultant mesylate. The 2-hydroxymethl-7-bromo-1,4-

benzodioxane intermediate 37 was used as an acetate (44) to

prepare both the 7-vinyl and the 7-ethynyl derivative, 15 and 14.

Palladium catalysed cross-coupling reaction with potassium

vinyltriﬂuoroborate provided the 7-vinyl-1,4-benzodioxane inter-

mediate 45, whose acetate function was hydrolyzed. The resultant

2-hydroxymethyl-7-vinyl-1,4-benzodioxane 46 was mesylated (47)

and used to O-alkylate 2,6-diﬂuoro-3-oxybenzamide to yield 15. On

the other hand, Sonogashira reaction with trimethylsilylacetylene

gave the 7-trimethylsilylethynyl-1,4-benzodioxane intermediate

48, which was converted into 7-ethynyl-2-hydroxymethyl-1,4-

benzodioxane 49 and then into 14 by the same two last steps as

those used to obtain 15. Reaction of 4-nitrocatechol with epi-

bromohydrin yielded 7-nitro-2-hydroxymethyl-1,4-benzodioxane

(51), which was tosylated (52) and reacted with 2,6-diﬂuoro-3-

oxybenzamide to give the 7-nitro derivative 16. Reduction with

SnCl₂converted 16 into the 7-amino derivative 17, which was

transformed into the corresponding methanesulfonamide 19 by

treatment with mesyl chloride. 7-Nitro-2-hydroxymethyl-1,4-

benzodioxane (51) was used to synthesize the 7-iododerivative

12 through acetylation (53), reduction of nitro to amino (54),

diazotation and treatment with NaI (55), acetate hydrolysis (56),

mesylation (57) and reaction with 2,6-diﬂuoro-3-oxybenzamide.

The syntheses of compounds 20e23 are shown in Scheme 3. The

synthesis of 7-methoxycarbonyl substituted 21 started from methyl

3,4-dihydroxybenzoate which was reacted with epibromohydrin to

give 7-methoxycarbonyl substituted 2-hydroxymethyl-1,4-

benzodioxane containing 15% of the 6-methoxycarbonyl

regioisomer. After conversion into the corresponding benzyl

ethers mixture and methyl ester hydrolysis, double crystallization

from toluene allowed the undesired regioisomer to be removed.

The 7-carboxy intermediate 58 was reconverted into methyl ester

(59), debenzylated (60) and condensed with 2,6-diﬂuoro-3-

oxybenzamide to 21 by Mitsunobu reaction or, alternatively, it

was converted into primary amide 61 or N-butylamide 64, deben-

zylated (62 or 65), tosylated (63) or mesylated (66) and reacted

study of 3e22, some of which showed <1

mg/mL antistaphylococcal

and ~10 g/mL antitubercular activity, associated to bacterial

m

swelling and, as revealed by electron microscopy, to inhibition of

septum formation (Chart 2).

1.1. Chemistry

The syntheses of compounds 5e10 are shown in Scheme 1. The

chromane derivatives 5 and 6 and the tetrahydronaphthalene

analogue

7

were

prepared

by

mesylation

of

2-

hydroxymethylchromane [8], 3-hydroxymethylchromane [8] and

2-hydroxymethyl-1,2,3,4-tetrahydronaphthalene [8], respectively,

and etheriﬁcation of 2,6-diﬂuoro-3-oxybenzamide [9] with the

resulting mesylates 24e26. Compound 8, in which the diﬂuor-

obenzamide portion is linked through the oxymethylene bridge to

benzodioxane benzene, was prepared by O-alkylating 2,6-diﬂuoro-

3-oxybenzamide with 1,2-ethylenedioxy-4-bromomethylbenzene

[10]. The glycerol 1,3-diphenyl ether 9 was synthesized from 1-

benzyloxy-3-phenoxy-2-propanol [11], which was mesylated (27),

reacted with 2,6-diﬂuoro-3-oxybenzamide (28) and debenzylated.

To prepare the 6,7-dichlorobenzodioxane 10, 3,4-dichlorophenol

was converted into acetate 29, submitted to Fries transposition

(30), condensed with epibromohydrin (31), BaeyereVilliger-

oxidized to phenyl acetate 32, cyclized to 6,7-dichloro-2-

hydroxymethyl-1,4-benzodioxane 33, mesylated (34) and then

used to O-alkylate 2,6-diﬂuoro-3-oxybenzamide.

The syntheses of 7-substituted benzodioxane derivatives 11e19

are shown in Scheme 2. The 7-bromo substituted benzodioxane 11

was synthesized from 2-acetyl-4-bromophenol in ﬁve steps: (1)

etheriﬁcation with epichlorohydrin (35), (2) BaeyereVilliger

oxidation to phenyl acetate 36, (3) ester hydrolysis followed by

Chart 1. Chemical structures of DFNB, PC190723, (R)-1, (R)-2, 3 and (S)-4.

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

229

Chart 2. Chemical structures of compounds 5e23.

Scheme 1. Syntheses of compounds 5e10 (a) Mesyl chloride, TEA, DCM. (b) 2,6-Diﬂuoro-3-oxybenzamide, K₂CO₃, DMF. (c) H2, 5% Pd/C, methanol. (d) AcCl, TEA, DCM. (e) AlCl₃,

DCM. (f) Epibromohydrin, K₂CO₃, DMF. (g) m-Chloroperbenzoic acid, DCM. (h) NaOH aq, methanol.

with 2,6-diﬂuoro-3-oxybenzamide to 22 or 23. The cyano deriva-

tive 20 was obtained by dehydration of 7-aminocarbonyl-2-

benzyloxymethyl-1,4-benzodioxane (67), followed by mesylation

(68) and condensation with 2,6-diﬂuoro-3-oxybenzamide.

were determined. The compounds with <1

evaluated for cytotoxicity on human MRC-5 cells and the doses (

mL) reducing the viability of these cells by 90% (TD₉₀) are reported.

The therapeutic index (TI) was also determined and deﬁned as the

ratio between TD₉₀and MBC values.

m

g/mL MIC were also

g/

m

When tested on an extended-spectrum beta-lactamase-positive

(ESBL) Escherichia coli, no compound was able to inhibit cell growth

2. Results and discussion

at the highest dose of 100 mg/mL (data not shown). Conversely, as

To test whether compounds 5e23 could inhibit bacterial

growth, both Gram-positive and Gram-negative bacteria were used.

In particular, the minimal inhibiting concentration (MIC), i.e. the

listed in Table 1, different levels of inhibition were found when they

were tested against a methicillin-resistant S. aureus strain (MRSA,

ATCC 29213). Six compounds (5, 10, 13, 16, 18 and 20) showed a MIC

lowest compound dose (

the minimal bactericidal concentration (MBC), i.e. the minimal dose

g/mL) at which cell growth is inhibited after compound removal,

mg/mL) at which growth is inhibited, and

ranging from 5 to 1.25

very active with MICs lower than 1

m

g/mL and ﬁve (11, 12, 14, 15 and 21) were

g/mL. The other eight

m

(m

230

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

Scheme 2. Syntheses of compounds 11e19. (a) Epichlorohydrin, K₂CO₃, DMF. (b) m-Chloroperbenzoic acid, DCM. (c) NaOH aq, methanol. (d) Mesyl chloride, TEA, DCM. (e) 2,6-

Diﬂuoro-3-oxybenzamide, K₂CO₃, DMF. (f) AcCl, TEA, DCM. (g) Potassium vinyltriﬂuoroborate, PdCl₂(dppe),CH₂Cl₂, t-butylamine, i-PrOH, water. (h) Ethynyltrimethylsilane,

PdCl₂, CuI, PPh₃, TEA. (i) Epibromohydrin, NaHCO₃, DMF. (j) TsCl, Py. (k) SnCl₂, EtOAc. (l) Mesyl chloride, TEA, EtOAc. (m) AcCl, TEA, EtOAc. (n) NaNO₂, H₂SO₄, NaI.

Scheme 3. Syntheses of compounds 20e23. (a) Epibromohydrin, K₂CO₃, DMF. (b) BnBr, NaH, THF. (c) NaOH aq, methanol. (d) Methanol, H₂SO₄, trimethyl orthoformate. (e) H₂, 10%

Pd/C, ethanol. (f) 2,6-Diﬂuoro-3-oxybenzamide, DIAD, PPh₃, THF. (g) Thionyl chloride and then aq NH₃(30%), DCM. (h) H₂, 5% Pd/C, acetone. (i) (CF₃CO)₂O, Py, dioxane. (j) Mesyl

chloride, TEA, DCM. (k) 2,6-Diﬂuoro-3-oxybenzamide, K₂CO₃, DMF. (l) TsCl, Py. (m) Thionyl chloride and then n-BuNH₂, DCM. (n) H₂, 10% Pd/C, methanol.

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

231

Table 1

Inhibitory activity against a methicillin-resistant S. aureus strain (MRSA, ATCC 29213) and cytotoxicity of DFNB^aand compounds 5e23.

Compd

S. aureus

MRC-5 toxicity

Compd

S. aureus

MIC ( g/mL)

MRC-5 toxicity

MIC (

m

g/mL)

MBC (

m

g/mL)

TI

TD₉₀

(m

g/ml)

m

MBC (

m

g/mL)

TI

TD₉₀(mg/ml)

DFNB

5

6

7

8

1

5

1

5

100

>200

14

15

16

17

18

19

20

21

22

23

0.313

0.625

1.25

100

2.5

>100

1.25

0.625

>100

0.625

1.25

2.5

100

2.5

640

>640

400

>800

100

>100

100

>100

2.5

0.625

0.313

2.5

100

9

10

11

12

13

5

2.5

0.625

2.5

>1280

>800

>1280

a

S. aureus MIC of DFNB is 2 mg/mL in Ref. [2]. MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration; TD₉₀, compound concentration reducing

MRC-5 viability of 90%; TI, Therapeutic Index (ratio between TD₉₀and MBC values).

compounds did not inhibit S. aureus growth up to 100

(compounds 7, 9,19, 22 and 23) or showed a modest 100 g/mL MIC

(compounds 6, 8 and 17). Identical MICs and MBCs (0.625 g/mL)

were determined for 11 and 21, whereas MBCs were found higher

than MICs for the other three very active compounds, namely 12

m

g/mL

by evaluating their activity against Mtb H37Rv. The antitubercular

activities are indicated by the MIC values reported in Table 2, where

m

the

staphylococcal activity, appears as the most active compound also

against Mtb, showing a good 8 g/mL MIC.

S enantiomer of 4, endowed with the highest anti-

m

and 14 (0.625 vs 0.313

m

g/mL) and 15 (1.25 vs 0.625

m

g/mL). All of

The study of the compounds 5e8 was aimed at understanding

the role of the benzodioxane substructure. Previous researches on

2,6-diﬂuorobenzamides 3-substituted with different benzoheter-

oaryl methoxy groups had shown very different antistaphylococcal

activities depending on the benzo-condensed heterocycle. Consis-

tently, the present results indicate that the benzodioxane sub-

structure of our compounds is not a mere scaffold correctly

positioning pharmacophoric groups, but also a target-interacting

part of the molecule, in particular, through its O(1). In fact, the

chromane 5 maintains unaltered the good activity of the previously

the ﬁve most active compounds showed a low cytotoxicity on

mammalian cells and a favourable TI. In particular, compounds 11,

12 and 21 had a TI higher than 1280, while 14 and 15 higher than

640. To obtain cytotoxicity in 90% of the cells (TD₉₀), 11, 12, 15 and

21 had to be used at a concentration higher than 800

higher than 400 g/mL.

mg/mL and 14

m

The investigation on the antimicrobial activity was successively

extended to clinical isolates of Gram-positive bacteria by using a

panel of 30 strains including 10 sensitive S. aureus (MSSA) and 10

methicillin-resistant S. aureus (MRSA), 5 vancomycin-sensitive

Enterococcus faecalis (VSE) and 5 vancomycin-resistant E. faecalis

(VRE). DFNB, 3, (S)-4, 10 and 12 were selected for testing: MICs and

MCBs are reported in Table 2. All the compounds exhibited from

moderate to high antistaphylococcal activity against both MSSA

and MRSA and the MIC₉₀and MBC₉₀values are consistent with the

MICs and MBCs reported in Table 1 (10 and 12) and with those we

have previously published (3 and (S)-4). In particular, the reported

potent submicromolar activity of the lead compound (S)-4 against

reported unsubstituted benzodioxane 3 (5

mg/mL MIC against

S. aureus), whereas the chromane 6, lacking of O(1), the tetrahy-

dronaphthalene 7, lacking of both oxygens, and the ‘reversed’

benzodioxane 8 are modestly active or inactive. Neither can the

phenoxyethyl residue efﬁcaciously replace the 2-chromanylmethyl

and 2-benzodioxanylmethyl substructures, although the oxygen

position is not changed: compound 9, unlike 3 and 5, is inactive.

This can be ascribed to the ﬂexibility of the phenoxyethyl residue or

to the interference with correct interaction by intramolecular

hydrogen bonding between its oxygen and the hydroxymethyl

substituent, beta positioned on the ethylene bridge to resemble 1

where the hydroxymethyl group has the same position relative to

oxazole heteroatoms.

MSSA (0.25

and now found to be almost equally exerted also against clinical

isolates of MRSA (0.39 g/mL; 1 M). Within this group of com-

mg/mL; 0.7 mM) was conﬁrmed (0.19 mg/mL; 0.5 mM)

m

pounds, high anti-MSSA and anti-MRSA activities, almost identical

to those of (S)-4, were shown by 12. Inhibition of VSE and VRE

growth was observed at higher concentrations (6e25 mg/mL) and it

is noteworthy that 12, potent antistaphylococcal agent, exhibits

also a moderate anti-VSE and anti-VRE activity.

After investigating the role of the benzodioxane oxygen atoms,

we considered a series of meta-substituents relative to the benzo-

dioxane O(1), since introducing chlorine at the 7-position of ben-

zodioxane had been a breakthrough in our previous search for new

DFNB, 3, (S)-4 and 10 were tested also as antitubercular agents

antibacterial 2,6-diﬂuorobenzamides. Compound 4 (0.5 mg/mL

Table 2

In vitro susceptibilities of S. aureus methicillin-susceptible (MSSA), -resistant (MRSA), and E. faecalis vancomycin-susceptible (VSE) or eresistant (VRE) isolates and of Mtb

H37Rv to DFNB and compounds 3, (S)-4, 10 and 12.^a

Compd

S. aureus MSSA

S. aureus MRSA

E. faecalis VSE

E. faecalis VRE

Mtb H37Rv

MIC₉₀

(m

g/ml)

MBC₉₀

(m

g/ml)

MIC₉₀

(m

g/ml)

MBC₉₀

(m

g/ml)

MIC₉₀

(m

g/ml)

MBC₉₀

(m

g/ml)

MIC₉₀

(

m

g/ml)

MBC₉₀

(

m

g/ml)

MIC (mg/mL)

DFNB

3

(S)-4

10

0.19

3.12

0.19

3.12

0.78

0.19

12.5

0.39

6.25

3.12

0.39

3.12

0.39

3.12

0.39

0.78

6.25

0.78

6.25

1.56

6.25

25

6.25

12.5

50

12.5

25

12.5

6.25

25

50

25

16

8

16

12

a

As determined by the CLSI M27-A3 broth microdilution method [13]. MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration.

232

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

MIC) is ten-fold more potent than its des-chloro analogue 3 as a

racemate and twenty-fold as S enantiomer (0.25 g/mL MIC). Based

the inhibition of FtsZ assembly and cell division. By light micro-

scopy, an increase in cell volume of S. aureus cultured in the pres-

ence of (S)-4, 14 and 21 was observed (Fig. 1A, B, C), which was

comparable to the effect of DFNB (Fig. 1E). Bacterial swelling was

also accompanied by cytocidal activity as indicated by cell deseg-

regation (Fig. 1B, C, E). As expected, no morphological change could

be instead observed in untreated S. aureus (Fig. 1D). These results

were conﬁrmed by transmission electron microscopy, which visu-

alized alterations in the septum formation in most bacteria exposed

to (S)-4 and DFNB (Fig. 2A, D) and complete bacterial desegregation

(ghosts) with total loss of the cytoplasmatic content upon treat-

ment with 21 (Fig. 2B). On the other hand, normal cell morphology

and septum formation were observed in the untreated negative

control (Fig. 2C). Signiﬁcantly altered bacterial morphology was

also visible upon exposure of Mycobacterium smegmatis at subMIC

concentrations of DFNB and (S)-4 (Fig. 3B and C), as shown by the

presence of elongated cells with an enlarged bacterial pole. These

changes are compatible with the perturbation of the FtsZ poly-

m

on these results, we initially selected alternative substituents hav-

ing positive s_mlike chlorine and from negligible to high lip-

ophilicity such as CO₂Me, CN, NO₂, F, CF₃, Br, I, ethinyl and vinyl. The

bromo- and iodo-analogues, 11 and 12, the methoxycarbonyl

analogue 21 and the alkinyl and alkenyl substituted compounds 14

and 15 showed high potency, similar to that of 4, with <1 mg/mL,

antistaphylococcal MICs, closely followed behind by the ﬂuoro,

nitro, triﬂuoromethyl and cyano derivatives, 13, 16, 18 and 20, with

antistaphylococcal MICs ranging between 1.25 and 2.5 mg/mL. The

higher activity of the 7-susbtituted analogues relative to unsusb-

stituted 3 was conﬁrmed also against clinical isolates of MSSA and

MRSA, which were found sensible to (S)-4 and 12 at submicromolar

concentrations, 4e16-fold lower than those of 3. The signiﬁcant

inﬂuence of the 7-substitution and the preference for hydrophobic

substituents at this position were conﬁrmed by the dramatic loss of

activity resulting from their replacement with hydrophilic and

hydrogen bond donor substituents, either electron-releasing or

attracting, such as NH₂, NHSO₂Me and CONH₂(compounds 17, 19

and 22): a detrimental effect which could not be overcome neither

by N-alkylation as indicated by the inactivity of the N-butyl de-

rivative 23. An almost identical trend in antistaphylococcal MICs

can be observed for the substitutions at the ﬁve position of ben-

zothiazole in the benzothiazole analogue of PC190723: chlorine,

bromine, CF₃and COOMe increase the antistaphylococcal activity

merization.

A similar, though less evident pattern was also

observed when M. tuberculosis was subjected to subMIC concen-

trations of (S)-4 (Fig. 3F) and DFNB (Fig. 3E).

3. Conclusions

We have tested

a

series of 3-(benzodioxan-2-yl)-2,6-

diﬂuorobenzamides substituted at benzodioxane C(7) or modiﬁed

at the benzodioxane substructure for the activity against a strain of

methicillin resistant S. aureus and, after selecting the most active

compounds, against clinical isolates of either antibiotic sensitive or

antibiotic resistant gram-positive bacteria (S. aureus, E. faecalis) and

against Mtb H37Rv. Among the compounds modiﬁed at the bicyclic

scaffold, only the 2-cromanyl analogue 5 maintains the good ac-

from 2 mg/mL to 0.1e1 mg/mL, whereas NH₂and CONH₂annul it.

The PC190723 carba analogue and 3 have identical structures

except for the benzoheterocycle substructure, benzothiazole and

benzodioxane respectively, and the benzothiazole C(5) of the latter

is superimposable to the benzodioxane C(7). Therefore, the

observed parallel shifts in antibacterial activity resulting from

parallel substitutions at these two carbons strongly suggests that 3

and the PC190723 carba analogue exert their antibacterial activity

interacting in a similar fashion with the same biological target, that

has been proven to be, in the case of PC190723, the bacterial cell

division protein FtsZ. Such a statement, based on the shared 3-

benzoheteroarylmethoxy-2,6-diﬂuorobenzamide molecular struc-

ture and on very similar SARs, is consistent with the results of the

morphometric analysis, which reveals that bacteria treated with

some of our most potent compounds show alterations indicative of

tivity

of

the

parent

unsubstituted

benzodioxane-

diﬂuorobenzamide 3 thus indicating the importance of benzo-

dioxane O(1) for target-activity. Within the series of the 7-

substituted analogues, high antibacterial activity, much higher

than that of 3, against MRSA and Mtb is showed by the derivatives

bearing a lipophilic substituent at that position, whereas hydro-

philic substituents have a highly detrimental effect. These results

are consistent with the previously observed enhancement in anti-

bacterial activity resulting from chlorination of benzodioxane C(7)

Fig. 1. Phase-contrast light microscopy of S. aureus after over night incubation in the presence of compounds (S)-4 (0.5

compound (NT) or in the presence of DFNB (0.25

g/mL) were used as negative and positive control respectively. All picture were taken at 630 ꢀ optical enlargement. De-

segregations: black arrows; bacterial swelling: white arrows.

mg/mL), 14 (0.5 mg/mL), 21 (1 mg/mL). Bacteria with no

m

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

233

Fig. 2. Ultrastructural analysis of S. aureus cultured in the presence of (S)-4 (0.5

m

g/mL) and 21 (1

m

g/mL). Bacteria with no compound (NT) or in the presence of DFNB (0.25

mg/mL)

were used as negative and positive control respectively. Cells were ﬁxed and processed after overnight growth, and alterations were examined at the ultrastructural level by

transmission electron microscopy. Arrows indicate septum formation. Scale bar: 0.5

m.

Fig. 3. Pictures of M. smegmatis (A, B, C) and M. tuberculosis (D, E, F) expressing GFP when untreated (A and D) or exposed at subMIC concentrations of (S)-4 (C and F: 4

DFNB (B: 0.5 g/mL; E: 8 g/mL).

mg/mL) and

m

234

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

of 3 and suggest that this is a crucial position for hydrophobic

substituents as C(5) in the benzothiazole analogues of PC190723, a

well-known FtsZ inhibiting diﬂuorobenzamide. The common

mechanism of action of these two classes of compounds,

NaHCO₃(20 mL), and brine (20 mL), dried over Na₂SO₄, and

concentrated. The resulting residue was puriﬁed by ﬂash chroma-

tography on silica gel. Elution with 70/30 cyclohexane/ethyl acetate

gave 1.03 g (88.8%) of 25 as a colourless oil: ¹H NMR (CDCl₃)

d 7.15

benzodioxane-diﬂuorobenzamides

and

benzothiazole-

(m, 2 H), 6.85 (m, 2 H), 4.24 (m, 3 H), 4.08 (dd, 1 H, J ¼ 11.0, 6.3 Hz),

diﬂuorobenzamides, is then supported by morphometric analyses

of Sa and Mtb treated with (S)-4, 14 and 21 by optical and electron

microscopy, which show cell alterations typical of FtsZ inhibition.

2.99 (m, 4 H), 2.65 (dd, 1 H, J ¼ 16.5, 6.3 Hz), 2.54 (m, 1 H).

3-(Chroman-3-yl)methoxy-2,6-diﬂuorobenzamide (6). Po-

tassium carbonate (700 mg, 5.05 mmol) was added to a solution of

2,6-diﬂuoro-3-hydroxybenzamide (800 mg, 4.63 mmol) in DMF

(7 mL). After stirring at room temperature for 15 min, a solution of

25 (1.02 g, 4.63 mmol) in DMF (7 mL) was added. The reaction

mixture was stirred at 60 ^ꢁC for 6 h, concentrated, and diluted with

water. The aqueous phase was extracted with ethyl acetate

(3 ꢀ 20 mL). The combined organic phases were washed with 1 M

NaOH (40 mL), and brine (40 mL), dried over Na₂SO₄, and

concentrated. The resulting solid residue was crystallized from

DCM to give 650 mg (48.5%) of 5 as a white solid: mp 124.5 ^ꢁC; ¹H

4. Experimental

4.1. Chemistry

Melting points were determined by DSC analysis and corre-

spond to the peak maximum. ¹H NMR spectra were recorded

operating at 300 MHz and ¹³C NMR at 75 MHz. Chemical shifts are

reported in ppm relative to residual solvent (CHCl₃or DMSO) as

internal standard. Signal multiplicity is designed according to the

following abbreviations: s ¼ singlet, d ¼ doublet, t ¼ triplet,

q ¼ quartet, m ¼ multiplet, dd ¼ doublet of doublets, dq ¼ doublet

of quartets, qd ¼ quartet of doublets, dt ¼ doublet of triplets,

td ¼ triplet of doublets, ddd 0 doublet of doublet of doublets,

ddt ¼ doublet of doublet of triplets, br s ¼ broad singlet. Coupling

constants are reported in Hertz (Hz). Optical rotations were

determined by a PerkineElmer 241 Polarimeter at 25 ^ꢁC. Elemental

analyses (C, H, N, Br, Cl, I, S) of the new substances are within 0.40%

of theoretical values. Puriﬁcations were performed by ﬂash chro-

NMR (CDCl₃) d 7.11 (m, 3 H), 6.85 (m, 3 H), 6.18 (br s, 1 H), 6.02 (br s,

1 H), 4.33 (dd, 1 H, J ¼ 10.7, 2.7 Hz), 4.14 (dd, 1 H, J ¼ 11.0, 6.6 Hz),

4.03 (d, 1 H, J ¼ 6.6 Hz), 3.00 (dd, 1 H, J ¼ 16.5, 5.0 Hz), 2.72 (dd, 1 H,

J ¼ 16.5, 7.2 Hz), 2.61 (m, 2 H). ¹³C NMR (DMSO-d)

d 162.5), 154.6,

153.8 (dd, J ¼ 245.4, 5.2 Hz, 150.4 (dd, J ¼ 253.1, 6.8 Hz), 144.0 (dd,

J ¼ 11.1, 3.4 Hz), 130.3, 127.7, 120.9, 120.6, 117.6 (d, J ¼ 9.7 Hz), 116.9,

114.5 (d, J ¼ 16.5 Hz), 111.4 (d, J ¼ 23.6 Hz), 70.9, 67.4, 32.7, 27.5.

Anal. Calcd for C₁₇H₁₅F₂NO₃(319.30).

2-Mesyloxymethyl-1,2,3,4-tetrahydronaphthalene

Mesyl chloride (0.47 mL, 6.05 mmol) was added dropwise to a

solution of 2-hydroxymethyl-1,2,3,4-tetrahydronaphthalene

(26).

matography using silica gel (particle size 40e63

2-Mesyloxymethylchromane (24). Mesyl chloride (0.44 mL,

5.77 mmol) was added dropwise to solution of 2-

mm, Merck).

(0.88 g, 5.42 mmol) and TEA (0.99 mL, 7.05 mmol) in DCM

(15 mL) at 4 ^ꢁC. The mixture was stirred at 4 ^ꢁC for 15 min and then

at room temperature for 30 min, cooled at 4 ^ꢁC, and diluted with

water (20 mL) and DCM (10 mL). The organic layer was separated,

washed with 10% HCl (15 mL), saturated aqueous NaHCO₃(15 mL),

and brine (15 mL), dried over Na₂SO₄, and concentrated to give

a

hydroxymethylchromane (0.63 g, 3.84 mmol) and TEA (0.80 mL,

5.77 mmol) in DCM (10 mL) at 0 ^ꢁC. The mixture was stirred at room

temperature for 30 min, cooled at 4 ^ꢁC, and diluted with water

(20 mL) and DCM (10 mL). The organic layer was separated, washed

with 10% HCl (10 mL), saturated aqueous NaHCO₃(10 mL), and

brine (10 mL), dried over Na₂SO₄, and concentrated to give 866 mg

1.31 g of 26 (99.2%) as a yellow oil: ¹H NMR (CDCl₃)

d 7.11 (m, 4 H),

4.21 (m, 2 H), 3.04 (s, 3 H), 2.88 (m, 3 H), 2.58 (dd, 1 H, J ¼ 16.5,

of 24 (93.1%) as a light brown oil: ¹H NMR (CDCl₃)

d 7.09 (m, 2 H),

10.5 Hz), 2.24 (m, 1 H), 2.03 (m, 1 H).

6.84 (m, 2 H), 4.42 (m, 2 H), 4.30 (m, 1 H), 3.10 (s, 3 H), 2.90 (m, 1 H),

2.78 (m, 1 H), 2.05 (m, 1 H), 1.91 (m, 1 H).

2,6-Diﬂuoro-3-(1,2,3,4-tetrahydronaphthalen-2-yl)methox-

ybenzamide (7). Potassium carbonate (460 mg, 2.50 mmol) was

added to a solution of 2,6-diﬂuoro-3-hydroxybenzamide (530 mg,

3.06 mmol) in DMF (4 mL). After stirring at room temperature for

15 min, a solution of 26 (670 mg, 2.78 mmol) in DMF (5 mL) was

added. The reaction mixture was stirred at 70 ^ꢁC for 7 h, concen-

trated, and diluted with water. The aqueous phase was extracted

with ethyl acetate (3 ꢀ 20 mL). The combined organic phases were

washed with 1 M NaOH (30 mL), and brine (30 mL), dried over

Na₂SO₄, and concentrated. The resulting solid residue was crystal-

lized from 70/30 cyclohexane/ethyl acetate to give 330 mg (37.5%)

3-(Chroman-2-yl)methoxy-2,6-diﬂuorobenzamide (5). Po-

tassium carbonate (304 mg, 2.20 mmol) was added to a solution of

2,6-diﬂuoro-3-hydroxybenzamide (380 mg, 2.20 mmol) in DMF

(6 mL). After stirring at room temperature for 15 min, a solution of

23 (485 mg, 2 mmol) in DMF (3 mL) was added. The reaction

mixture was stirred at 80 ^ꢁC for 2 h, concentrated, and diluted with

water and ethyl acetate. The organic phase was separated, washed

with brine, dried over Na₂SO₄, and concentrated. The resulting

residue was puriﬁed by ﬂash chromatography on silica gel. Elution

with 55/45 cyclohexane/ethyl acetate gave 386 mg (60.5%) of 5 as a

of 7 as a white solid: mp 165.1 ^ꢁC; ¹H NMR (CDCl₃)

d 7.06 (m, 5 H),

white solid: mp 159.1 ^ꢁC; ¹H NMR (CDCl₃)

d

7.13 (dd, 1 H, J ¼ 9.3,

6.88 (dt, 1 H, J ¼ 9.1, 1.9 Hz), 5.98 (br s, 2 H), 3.98 (d, 2 H, J ¼ 6.5 Hz),

5.3 Hz), 7.08 (m, 2 H), 6.91 (dd, 1 H, J ¼ 9.0, 2.1 Hz), 6.84 (m, 2 H),

5.96 (br s, 2 H), 4.40 (ddd, 1 H, J ¼ 5.4, 5.1, 2.4 Hz), 4.27 (dd, 1 H,

J ¼ 10.0, 5.4 Hz), 4.18 (dd,1 H, J ¼ 10.0, 5.1 Hz), 2.88 (m, 2 H), 2.16 (m,

3.00 (dd, 1 H, J ¼ 17.9, 5.7 Hz), 2.87 (dd, 1 H, J ¼ 8.1, 4.7 Hz), 2.65 (dd,

1 H, J ¼ 16.3, 10.4 Hz), 2.34 (m, 1 H), 2.11 (m, 1 H), 1.60 (m, 2 H). ¹³

C

NMR (DMSO-d)

d

162.0, 152.4 (dd, J ¼ 239.7, 6.9 Hz), 148.6 (dd,

1 H), 1.99e1.82 (m, 1H). ¹³C NMR (CDCl₃)

d

162.2, 154.4, 154.1 (dd,

J ¼ 246.4, 8.6 Hz), 144.0 (dd, J ¼ 10.6, 2.9 Hz), 136.9, 136.0, 129.7,

129.4, 126.3, 117.3 (dd, J ¼ 24.7, 20.5 Hz), 116.2 (d, J ¼ 9.4 Hz), 111.6

(d, J ¼ 9.1 Hz), 74.4, 34.4, 32.8, 32.4, 28.6, 26.1. Anal. Calcd for

J ¼ 245.9, 5.4 Hz), 150.6 (dd, J ¼ 253.2, 7.0), 144.0 (dd, J ¼ 11.3,

3.6 Hz), 129.8, 127.6, 121.9, 120.8, 118.5 (d, J ¼ 8.3 Hz), 117.0, 111.6

(m), 111.3 (m), 74.2, 73.0, 24.41, 24.38. Anal. Calcd for C₁₇H₁₅F₂NO₃

(319.30).

C₁₈H₁₇F₂NO₂(317.33).

3-(1,4-Benzodioxan-6-ylmethoxy)-2,6-diﬂuorobenzamide

3-Mesyloxymethylchromane (25). Mesyl chloride (0.44 mL,

(8). Potassium carbonate (362 mg, 2.62 mmol) was added to a so-

lution of 2,6-diﬂuoro-3-hydroxybenzamide (453 mg, 2.62 mmol) in

DMF (3 mL). After stirring at room temperature for 15 min, a so-

lution of 1,2-ethylenedioxy-4-bromomethylbenzene (500 mg,

2.18 mmol) in DMF (3 mL) was added. The reaction mixture was

stirred at 100 ^ꢁC for 1 h, cooled to room temperature and then

poured into iced water (80 mL). A precipitate was formed which

5.77 mmol) was added dropwise to

a

solution of 3-

hydroxymethylchromane (0.79 g, 4.81 mmol) and TEA (0.90 mL,

6.25 mmol) in DCM (15 mL) at 4 ^ꢁC. The mixture was stirred at 4 ^ꢁC

for 15 min and then at room temperature for 30 min, cooled at 4 ^ꢁC,

and diluted with water (20 mL) and DCM (10 mL). The organic layer

was separated, washed with 10% HCl (20 mL), saturated aqueous

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

235

was collected by ﬁltration, washed with water, dried under vacuum

and crystallized from CHCl₃to give 426 mg (60.8%) of 8 as a pale

at 0 ^ꢁC. After stirring at 0 ^ꢁC for 30 min, the organic phase was

separated and the aqueous phase was extracted with DCM (30 mL).

The organic extracts were combined, washed with brine, dried over

Na₂SO₄, and concentrated to give 1.87 g (93.4%) of 30 as a light pink

pink solid: mp 130.0 ^ꢁC; ¹H NMR (DMSO-d)

d

8.11 (br s,1 H), 7.83 (br

s, 1 H), 7.25 (dd, 1 H, J ¼ 9.2, 5.4 Hz), 7.03 (dd, 1 H, J ¼ 9.2, 1.7 Hz),

6.88 (m, 3 H), 5.03 (s, 2 H), 4.22 (s, 4 H). ¹³C NMR (DMSO-d)

162.0,

d

solid: ¹H NMR (CDCl₃)

(s, 3 H).

d

12.17 (s, 1 H), 7.79 (s, 1 H), 7.12 (s, 1 H), 2.62

152.5 (dd, J ¼ 239.7, 6.8 Hz),148.7 (dd, J ¼ 247.0, 8.4 Hz),144.1,143.9,

143.4 (dd, J ¼ 10.8, 3.1 Hz), 129.9, 121.8, 117.7 (d, J ¼ 4.3 Hz), 117.3,

117.0, 116.4 (dd, J ¼ 9.4, 2.3 Hz), 111.5 (dd, J ¼ 23.0, 3.8 Hz), 71.1,

64.73, 64.71. Anal. Calcd for C₁₆H₁₃F₂NO₄(321.28).

3-(2-Acetyl-4,5-dichlorophenoxy)-1,2-epoxypropane

(31).

Potassium carbonate (1.45 g, 10.53 mmol) was added to a solution

of 30 (1.89 g, 8.78 mmol) in DMF (20 mL). After stirring at room

temperature for 30 min, epibromohydrin (1.44 g, 10.53 mmol) was

added. The mixture was heated at 40 ^ꢁC for 18 h, cooled to room

temperature, and concentrated under reduced pressure. Ethyl ac-

etate and brine were added to the resulting residue. The organic

phase was separated, washed with brine twice, dried over Na₂SO₄,

and concentrated. The resultant solid residue was puriﬁed by ﬂash

chromatography on silica gel. Elution with 70/30 cyclohexane/ethyl

acetate gave 1.52 g (66.2%) of 31 as a light yellow solid: ¹H NMR

1-Benzyloxy-2-mesyloxy-3-phenoxypropane (27). Mesyl

chloride (0.21 mL, 2.71 mmol) was added dropwise to a solution of

1-benzyloxy-2-hydroxy-3-phenoxypropane (580 mg, 2.25 mmol)

and TEA (0.4 mL, 2.87 mmol) in DCM (6 mL) at 4 ^ꢁC. The mixture

was stirred at 4 ^ꢁC for 15 min and then at room temperature for

30 min, cooled at 4 ^ꢁC, and diluted with water (15 mL) and DCM

(10 mL). The organic layer was separated, washed with 10% HCl

(10 mL), saturated aqueous NaHCO₃(10 mL), and brine (10 mL),

dried over Na₂SO₄, and concentrated to give 670 mg (88.5%) of 27 as

(CDCl₃)

d

7.84 (s, 1 H), 7.07 (s, 1 H), 4.40 (dd, 1 H, J ¼ 11.0, 2.5 Hz),

a colourless oil: ¹H NMR (CDCl₃)

d

7.41e7.28 (m, 7 H), 6.98 (t, 1 H,

3.95 (dd, 1 H, J ¼ 11.0, 6.3 Hz), 3.42e3.38 (m, 1 H), 2.96 (t, 1 H,

J ¼ 4.5 Hz), 2.77 (dd, 1 H, J ¼ 4.7, 2.8 Hz), 2.63 (s, 3 H).

J ¼ 7.2 Hz), 6.89 (d, 2 H, J ¼ 7.2 Hz), 5.08 (m, 1 H), 4.62 (d, 1 H,

J ¼ 9.1 Hz), 4.58 (d, 1 H, J ¼ 9.1 Hz), 4.24 (d, 2 H, J ¼ 6.5 Hz), 3.82 (d,

2 H, J ¼ 6.5 Hz), 3.09 (s, 3 H).

3-(2-Acetyloxy-4,5-dichlorophenoxy)-1,2-epoxypropane

(32). m-Chloroperbenzoic acid (4.8 g, 21.45 mmol) was added

portion wise to a solution of 31 (1.40 g, 5.36 mmol) in DCM (14 mL)

at room temperature. The mixture was reﬂuxed for 18 h, cooled to

room temperature and then to 0 ^ꢁC. After 2 h, the precipitate of 3-

chlorobenzoic acid was removed by ﬁltration and washed on the

ﬁlter with ethyl acetate. The whole ﬁltrate was washed with 10%

aqueous NaHCO₃, dried over Na₂SO₄and concentrated to give 1.48 g

3-(1-Benzyloxy-3-phenoxy-2-propyloxy)-2,6-

diﬂuorobenzamide (28). Potassium carbonate (275 mg,

2.00 mmol) was added to

a solution of 2,6-diﬂuoro-3-

hydroxybenzamide (345 mg, 2.00 mmol) in DMF (5 mL). After

stirring at room temperature for 15 min, a solution of 27 (345 mg,

2.00 mmol) in DMF (5 mL) was added. The reaction mixture was

stirred at 100 ^ꢁC for 7 h, concentrated, and diluted with water

(15 mL). The aqueous phase was extracted with ethyl acetate

(3 ꢀ 20 mL). The combined organic phases were washed with 1 M

NaOH (30 mL), and brine (30 mL), dried over Na₂SO₄, and

concentrated. The resulting residue was puriﬁed by ﬂash chroma-

tography on silica gel. Elution with 80/20 toluene/ethyl acetate gave

(~100%) of 32 as a yellow oil: ¹H NMR (CDCl₃)

d 7.15 (s, 1 H), 7.08 (s,

1 H), 4.26 (dd, 1 H, J ¼ 11.3, 2.8 Hz), 3.91 (dd, 1 H, J ¼ 11.0, 5.8 Hz),

3.31e3.28 (m, 1 H), 2.88 (t, 1 H, J ¼ 4.5 Hz), 2.71 (dd, 1 H, J ¼ 5.0,

2.8 Hz), 2.30 (s, 3 H).

6,7-Dichloro-2-hydroxymethyl-1,4-benzodioxane (33). Water

(3 mL) and 2.5 M NaOH (4 mL) were added to a solution of 32 (1.4 g,

5.05 mmol) in methanol (15 mL). The mixture was stirred at room

temperature for 3 h and concentrated. Ethyl acetate and 2.5 M

NaOH were added to the residue. The organic phase was separated,

washed with brine, dried over Na₂SO₄and concentrated. The res-

idue was crystallized from DCM to give 776 mg (65.4%) of 33 as a

420 mg (50.8%) of 28 as a colourless oil: ¹H NMR (CDCl₃)

d 7.39e7.18

(m, 8 H), 6.95 (t, 1 H, J ¼ 7.2 Hz), 6.85 (m, 3 H), 5.90 (br s, 2 H), 4.64

(m, 1 H), 4.60 (s, 2 H), 4.23 (d, 2 H, J ¼ 6.8 Hz), 3.83 (d, 2 H,

J ¼ 6.8 Hz).

2,6-Diﬂuoro-3-(1-hydroxy-3-phenoxy-2-propyloxy)benza-

mide (9). A solution of 28 (260 mg, 0.63 mmol) in methanol (7 mL)

was added with 5% Pd/C (50 mg) and vigorously shaken under

hydrogen at room temperature for 3 h. The catalyst was removed by

ﬁltration and the ﬁltrate concentrated to give 194 mg (95.2%) of 9 as

white solid:: mp 97.0 ^ꢁC; ¹H NMR (CDCl₃)

d 6.99 (s, 1 H), 6.98 (s,

1 H), 4.30 (dd, 1 H, J ¼ 11.3, 2.2 Hz), 4.27e4.20 (m, 1 H), 4.15e4.06

(m, 2 H), 3.93e3.80 (m, 2 H).

6,7-Dichloro-2-mesyloxymethyl-1,4-benzodioxane

(34).

a yellow oil: ¹H NMR (DMSO-d)

d

8.08 (br s, 1H), 7.79 (br s, 1H),

Mesyl chloride (0.35 mL, 4.47 mmol) was added dropwise to a so-

lution of 33 (700 mg, 2.98 mmol) and TEA (0.62 mL, 4.47 mmol) in

DCM (7 mL) at 0 ^ꢁC. The mixture was stirred at room temperature

for 2 h, cooled at 4 ^ꢁC, and diluted with 10% HCl (10 mL) and DCM

(10 mL). The organic layer was separated, washed with saturated

aqueous NaHCO₃(10 mL), and brine (10 mL), dried over Na₂SO₄,

and concentrated to give 887 mg (95.0%) of 34 as a yellow oil: ¹H

7.36e7.32 (m, 3H), 7.03 (dt, 1H, J ¼ 9.1, 1.8 Hz), 6.92 (m, 3H), 5.03 (t,

1 H, J ¼ 5.5 Hz), 4.6 (m, 1H), 4.23 (dd, 1 H, J ¼ 10.5, 3.6 Hz), 4.14 (dd,

1 H, J ¼ 10.5, 5.2 Hz), 3.69 (m, 2 H). ¹³C NMR (DMSO-d)

d 161.8,

158.8,152.6 (dd, J ¼ 239.9, 6.3 Hz),149.3 (dd, J ¼ 247.4, 9.1 Hz),143.0

(dd, J ¼ 11.4, 3.4 Hz), 130.0, 121.3, 118.8 (d, J ¼ 9.1 Hz), 117.1 (dd,

J ¼ 25.2, 20.6 Hz), 114.9, 111.4 (dd, J ¼ 22.9, 3.4 Hz), 80.4, 67.6, 60.3.

Anal. Calcd for C₁₆H₁₅F₂NO₄(323.29).

NMR (CDCl₃)

d 7.01 (s, 2 H), 4.49e4.41 (m, 3 H), 4.32 (dd, 1 H,

3,4-Dichlorophenyl acetate (29). Acetyl chloride (0.89 mL,

J ¼ 11.6, 2.3 Hz), 4.11 (dd, 1 H, J ¼ 11.6, 6.3 Hz), 3.09 (s, 3 H).

12.5 mmol) was added dropwise to

a solution of 3,4-

3-(6,7-Dichloro-1,4-benzodioxan-2-yl)methoxy-2,6-

dichlorophenol (1.63 g, 10 mmol) and triethylamine (1.74 mL,

12.5 mmol) in DCM (15 mL) at 0 ^ꢁC. The mixture was stirred at room

temperature for 20 h, diluted with DCM (15 mL), washed with 10%

HCl (10 mL), saturated aqueous NaHCO₃(10 mL), and brine (10 mL),

dried over Na₂SO₄, and concentrated to give 2.05 g (~100%) of 29 as

diﬂuorobenzamide (10). Potassium carbonate (388 mg,

2.81 mmol) was added to

a solution of 2,6-diﬂuoro-3-

hydroxybenzamide (486.5 mg, 2.81 mmol) in DMF (4 mL). After

stirring at room temperature for 15 min, a solution of 34 (800 mg,

2.55 mmol) in DMF (4 mL) was added. The reaction mixture was

stirred at 70 ^ꢁC for 18 h, concentrated under vacuum, and diluted

with water and ethyl acetate. The organic phase was washed with

brine, dried over Na₂SO₄, and concentrated to give a residue which

was puriﬁed by ﬂash chromatography on silica gel. Elution with 40/

60 cyclohexane/ethyl acetate gave 10 as a white solid, which was

further puriﬁed by crystallization from chloroform yielding 299 mg

(30.0%) of the desired product: mp 176.0 ^ꢁC; ¹H NMR (DMSO-d)

a yellow oil: ¹H NMR (CDCl₃)

d

7.42 (d, 1 H, J ¼ 8.7 Hz), 7.24 (d, 1 H,

J ¼ 2.6 Hz), 6.96 (dd, 1 H, J ¼ 8.7, 2.6 Hz), 2.27 (s, 3 H).

2-Acetyl-4,5-dichlorophenol (30). Aluminium trichloride

(1.56 g,11.7 mmol) was added portion wise to 29 (2 g, 9.75 mmol) in

DCM (2 mL) at room temperature under nitrogen atmosphere. The

mixture was stirred at 120 ^ꢁC for 3 h, cooled to room temperature,

and diluted with DCM (20 mL). 10% HCl (20 mL) was slowly added

236

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

d

8.11 (br s, 1 H), 7.83 (br s, 1 H), 7.27 (dt, 1 H, J ¼ 8.9, 5.3 Hz), 7.22 (s,

stirred at 60 ^ꢁC for 18 h, concentrated under vacuum, and diluted

with water (15 mL) and ethyl acetate (15 mL). The aqueous phase

was separated and extracted with ethyl acetate (2 ꢀ 15 mL). The

combined organic phases were washed with 1 M NaOH (15 mL),

and brine (15 mL), dried over Na₂SO₄, and concentrated to give a

residue which was puriﬁed by ﬂash chromatography on silica gel.

Elution with 60/40 cyclohexane/ethyl acetate gave 540 mg (62.1%)

1 H), 7.21 (s, 1 H), 7.06 (dt, 1 H, J ¼ 8.9,1.9 Hz), 4.64 (m 1 H), 4.46 (dd,

1H, J ¼ 11.7, 2.4 Hz), 4.32 (m, 2 H), 4.17 (dd, 1 H, J ¼ 11.7, 6.9 Hz). ¹³

C

NMR (DMSO-d)

d

161.9 (d, J ¼ 0.8 Hz), 152.9 (dd, J ¼ 240.6, 6.8 Hz),

148.7 (dd, J ¼ 247.5, 8.4 Hz), 143.3 (dd, J ¼ 11.0, 3.0 Hz), 143.3, 143.1,

123.6, 123.4, 119.1, 119.0, 117.4 (dd, J ¼ 25.1, 20.2 Hz), 116.8 (m), 111.7

(d, J ¼ 21.6 Hz), 72.3, 68.8, 65.2. Anal. Calcd for C₁₆H₁₁Cl₂F₂NO₄

(390.17).

of 11 as a white solid: mp 114.5 ^ꢁC; ¹H NMR (CDCl₃)

d 7.06 (m, 2 H),

3-(2-Acetyl-4-bromophenoxy)-1,2-epoxypropane (35). Potas-

sium carbonate (1.41 g, 10,2 mmol) and epichlorohydrin (1.46 mL,

18.6 mmol) were added to a solution of 2-acetyl-4-bromophenol

(2 g, 9.3 mmol) in DMF (22 mL). After stirring at 40 ^ꢁC for 64 h,

the mixture was poured into water (60 mL) and extracted with

ethyl acetate (3 ꢀ 30 mL). The combined organic extracts were

washed with 1 M NaOH (30 mL) and brine (30 mL), dried over

Na₂SO₄, and concentrated to give a brownish oil, which was puri-

ﬁed by ﬂash chromatography. Elution with 70/30 cyclohexane/ethyl

acetate gave 1.21 g (48.0%) of 35 as a white wax: ¹H NMR (CDCl₃)

6.93 (m, 2 H), 6.77 (d, 1 H, J ¼ 8.6 Hz), 6.02 (br s, 2 H), 4.55 (m, 1 H),

4.38 (dd, 1 H, J ¼ 11.6, 2.4 Hz), 4.23 (m, 3 H). ¹³C NMR (CDCl₃)

d

162.9, 154.3 (dd, J ¼ 246.5, 5.4 Hz), 150.6 (dd, J ¼ 253.7, 7.1 Hz),

143.6, 143.1 (dd, J ¼ 11.7, 3.7 Hz), 142.6, 124.8, 120.6, 118.8; 118.5 (d,

J ¼ 8.8 Hz), 114.5 (dd, J ¼ 20.8, 16.3 Hz), 113.5, 111.6 (d, J ¼ 23.6 Hz),

71.6, 69.0, 65.0. Anal. Calcd for C₁₆H₁₂BrF₂NO₄(400.17).

3-(2-Formyl-4-triﬂuoromethylphenoxy)-1,2-epoxypropane

(39). Potassium carbonate (1.66 g, 12.0 mmol) and epibromohydrin

(1.04 mL, 12.0 mmol) were added to a solution of 2-hydroxy-5-

triﬂuoromethylbenzaldehyde (1.90 g, 10.0 mmol) in DMF (30 mL).

The reaction mixture was stirred at 40 ^ꢁC overnight, concentrated,

diluted with water (20 mL) and ethyl acetate (20 mL). The aqueous

phase was separated and extracted with ethyl acetate (2 ꢀ 20 mL).

The combined organic phases were washed with 1 M NaOH

(40 mL), and brine (40 mL), dried over Na₂SO₄, concentrated to give

a residue which was puriﬁed by ﬂash chromatography on silica gel.

Elution with 70/30 cyclohexane/ethyl acetate gave 1.80 g (73.2%) of

d

7.84 (d, 1 H, J ¼ 2.6 Hz), 7.53 (dd, 1 H, J ¼ 8.8, 2.6 Hz), 6.85 (d, 1 H,

J ¼ 8.8 Hz), 4.37 (dd, 1 H, J ¼ 10.9, 2.7 Hz), 3.97 (dd, 1 H, J ¼ 10.9,

6.0 Hz), 3.39 (m, 1 H), 2.95 (t, 1 H, J ¼ 4.5 Hz), 2.76 (dd, 1 H, J ¼ 4.7,

2.6 Hz), 2.64 (s, 3 H).

3-(2-Acetyloxy-4-bromophenoxy)-1,2-epoxypropane (36). m-

Chloroperbenzoic acid (2.58 g, 11.52 mmol) was added portion wise

to a solution of 35 (1.21 g, 5.76 mmol) in DCM (13 mL) at room

temperature. The mixture was reﬂuxed overnight, cooled to room

temperature and then to 4 ^ꢁC. Saturated aqueous Na₂S₂O₅(20 mL)

was added. The mixture was ﬁltered to remove a white precipitate.

The organic phase was diluted with DCM (20 mL), washed with

saturated aqueous NaHCO₃, dried over Na₂SO₄and concentrated to

39 as a colourless oil: ¹H NMR (CDCl₃)

d 10.52 (s, 1 H), 8.13 (d, 1 H,

J ¼ 2.5 Hz), 7.79 (d, 1 H, J ¼ 8.8, 2.5 Hz), 7.12 (d, 1 H, J ¼ 8.8 Hz), 4.50

(dd, 1 H, J ¼ 11.2, 2.7 Hz), 4.11 (dd, 1 H, J ¼ 11.2, 5.9 Hz), 3.43 (ddt,

1 H, J ¼ 5.9, 4.2, 2.6 Hz), 2.98 (dd, 1 H, J ¼ 4.7, 4.2 Hz), 2.81 (dd, 1 H,

J ¼ 4.8, 2.6 Hz).

give 1.55 g (94.0%) of 36 as a yellow oil: ¹H NMR (CDCl₃)

d

7.30 (dd,

3-(2-Formyloxy-4-triﬂuoromethylphenoxy)-1,2-

1 H, J ¼ 8.7, 2.4 Hz), 7.20 (d, 1 H, J ¼ 2.4 Hz), 6.88 (d, 1 H, J ¼ 8.7 Hz),

4.25 (dd, 1 H, J ¼ 11.2, 2.9 Hz), 3.95 (dd, 1 H, J ¼ 11.2, 5.6 Hz), 3.30

(ddd, 1 H, J ¼ 5.6, 2.8, 1.4 Hz), 2.88 (dd, 1 H, J ¼ 4.9, 4.2 Hz), 2.71 (dd,

1 H, J ¼ 4.9, 2.6 Hz), 2.32 (s, 3 H).

epoxypropane (40). m-Chloroperbenzoic acid (3.61 g, 14.65 mmol)

was added portion wise to a solution of 39 (1.80 g, 7.31 mmol) in

DCM (18 mL) at room temperature. The mixture was reﬂuxed

overnight, and cooled to 4 ^ꢁC. Saturated aqueous Na₂S₂O₅(30 mL)

was added. The mixture was ﬁltered to remove a white precipitate.

The organic phase was diluted with ethyl acetate (30 mL), washed

with saturated aqueous NaHCO₃(4 ꢀ 30 mL), dried over Na₂SO₄

and concentrated to give 1.90 g (98.9%) of 40 as a yellow oil: ¹H

7-Bromo-2-hydroxymethyl-1,4-benzodioxane (37). 2.5

M

NaOH (2.9 mL) was added dropwise to a solution of 36 (1.55 g,

5.4 mmol) in methanol (16 mL). After stirring at room temperature

for 15 min, the reaction mixture was heated at 50 ^ꢁC for 2.5 h,

concentrated under vacuum, and diluted with water (15 mL) and

ethyl acetate (15 mL). The aqueous phase was separated and

extracted with ethyl acetate (2 ꢀ 15 mL). The combined organic

phases were washed with 1 M NaOH (30 mL), brine (2 ꢀ 30 mL),

dried over NaSO₄, and concentrated under vacuum to give 1.1 g

NMR (CDCl₃)

d

7.95 (m, 1 H), 7.19 (d, 1 H, J ¼ 2.2 Hz), 7.11 (m, 1 H),

6.93 (d, 1 H, J ¼ 8.4 Hz), 4.39 (dd, 1 H, J ¼ 11.4, 2.6 Hz), 4.04 (dd, 1 H,

J ¼ 11.4, 5.9 Hz), 3.40 (m, 1 H), 2.97 (m, 1 H), 2.81 (dd, 1 H, J ¼ 4.7,

2.7 Hz).

2-Hydroxymethyl-7-triﬂuoromethyl-1,4-benzodioxane (41).

2.5 M NaOH was added dropwise to a solution of 40 (1.90 g,

7.25 mmol) in methanol (20 mL). After stirring at room temperature

for 15 min, the reaction mixture was heated at 60 ^ꢁC for 2 h,

concentrated under vacuum, and diluted with water (20 mL) and

ethyl acetate (20 mL). the aqueous phase was extracted with ethyl

acetate (2 ꢀ 20 mL). The combined organic phases were washed

with 1 M NaOH (20 mL), and brine (2 ꢀ 20 mL), dried over Na₂SO₄,

and concentrated to yield a residue, which was puriﬁed by ﬂash

chromatography on silica gel. Elution with 70/30 cyclohexane/ethyl

acetate gave 700 mg (41.2%) of 41 as a colourless liquid: ¹H NMR

(83.1%) of 37 as a yellow oil: ¹H NMR (CDCl₃)

d 7.05 (d, 1 H,

J ¼ 2.3 Hz), 6.95 (dd,1 H, J ¼ 8.6, 2.3 Hz), 6.76 (d,1 H, J ¼ 8.6 Hz), 4.26

(m, 2 H), 4.11 (m, 1 H), 3.86 (m, 2 H), 1.87 (br s, 1 H).

7-Bromo-2-mesyloxymethyl-1,4-benzodioxane (38). Mesyl

chloride (0.22 mL, 2.82 mmol) was added dropwise to a solution of

37 (550 mg, 2.24 mmol) and TEA (0.43 mL, 3.04 mmol) in DCM

(8 mL) at 4 ^ꢁC. The mixture was stirred at 4 ^ꢁC for 15 min and then at

room temperature for 30 min. After cooling to 4 ^ꢁC, water (10 mL)

and DCM (10 mL) were added. The organic layer was separated,

washed with 10% HCl (10 mL), saturated aqueous NaHCO₃(10 mL),

and brine (10 mL), dried over Na₂SO₄, and concentrated to give

(CDCl₃)

d

7.17 (d, 1 H, J ¼ 2.1 Hz), 7.11 (m, 1 H), 6.95 (d, 1 H,

700 mg (97.2%) of 38 as a colourless oil: ¹H NMR (CDCl₃)

d

7.05 (d,

J ¼ 7.9 Hz), 4.36 (dd,1 H, J ¼ 12.1, 4.6 Hz), 4.28 (m,1 H), 4.14 (m, 2 H),

3.90 (qd, 1 H, J ¼ 12.1, 4.6 Hz), 1.83 (br s, 1 H).

1 H, J ¼ 2.3 Hz), 6.98 (dd,1 H, J ¼ 8.6, 2.3 Hz), 6.78 (d, 1 H, J ¼ 8.6 Hz),

4.46 (m, 3 H), 4.31 (dd, 1 H, J ¼ 11.7, 2.3 Hz), 4.11 (dd, 1 H, J ¼ 11.7,

6.2 Hz), 3.09 (s, 3 H).

3-(7-bromo-1,4-benzodioxan-2-yl)methoxy-2,6-

diﬂuorobenzamide (11). Potassium carbonate (330 mg,

2-Mesyloxymethyl-7-triﬂuoromethyl-1,4-benzodioxane (42).

Mesyl chloride (0.28 mL, 3.59 mmol) was added dropwise to a

solution of 41 (700 mg, 2.99 mmol) and TEA (0.55 mL, 3.89 mmol)

in DCM (10 mL) at 4 ^ꢁC. The mixture was stirred at 4 ^ꢁC for 15 min

and then at room temperature for 30 min. After cooling to 4 ^ꢁC,

water (10 mL) and DCM (10 mL) were added. The organic layer was

separated, washed with 10% HCl (10 mL), saturated aqueous

NaHCO₃(10 mL), and brine (10 mL), dried over Na₂SO₄, and

2.40 mmol) was added to

a solution of 2,6-diﬂuoro-3-

hydroxybenzamide (419 mg, 2.40 mmol) in DMF (5 mL). After

stirring at room temperature for 15 min, a solution of 38 (700 mg,

2.17 mmol) in DMF (5 mL) was added. The reaction mixture was

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

237

concentrated to yield a residue which was puriﬁed by ﬂash chro-

matography on silica gel. Elution with 70/30 cyclohexane/ethyl

acetate gave 710 mg (76.3%) of 42 as a white wax: ¹H NMR (CDCl₃)

(530 mg, 2.17 mmol) and TEA (0.42 mL, 2.93 mmol) in DCM (10 mL)

at 4 ^ꢁC. The mixture was stirred at 4 ^ꢁC for 15 min and then at room

temperature for 1 h. After cooling to 4 ^ꢁC, water (15 mL) and DCM

(10 mL). The organic layer was separated, washed with 10% HCl

(15 mL), saturated aqueous NaHCO₃(15 mL), and brine (15 mL),

dried over Na₂SO₄, and concentrated to yield a residue which was

puriﬁed by ﬂash chromatography on silica gel. Elution with 80/20

cyclohexane/ethyl acetate gave 540 mg (87.1%) of 44 as a colourless

d

7.15 (m, 2 H), 6.98 (d, 1 H, J ¼ 8.3 Hz), 4.50 (m, 3 H), 4.38 (dd, 1 H,

J ¼ 11.7, 2.3 Hz), 4.17 (dd, 1 H, J ¼ 11.7, 6.5 Hz), 3.10 (s, 3 H).

2,6-Diﬂuoro-3-(7-triﬂuoromethyl-1,4-benzodioxan-2-yl)

methoxybenzamide (18). Potassium carbonate (345 mg,

2.50 mmol) was added to

a solution of 2,6-diﬂuoro-3-

hydroxybenzamide (433 mg, 2.50 mmol) in DMF (5 mL). After

stirring at room temperature for 15 min, a solution of 42 (710 mg,

2.27 mmol) in DMF (5 mL) was added. The reaction mixture was

stirred at 65 ^ꢁC for 7 h, concentrated under vacuum, and diluted

with water (15 mL) and ethyl acetate (15 mL). The aqueous phase

was separated and extracted with ethyl acetate (2 ꢀ 15 mL). The

combined organic phases were washed with 1 M NaOH (20 mL),

and brine (15 mL), dried over Na₂SO₄, and concentrated to give a

residue which was puriﬁed by ﬂash chromatography on silica gel.

Elution with 60/40 cyclohexane/ethyl acetate gave 500 mg (56.6%)

oil: ¹H NMR (CDCl₃)

d

7.06 (d, 1 H, J ¼ 2.2 Hz), 6.96 (dd, 1 H, J ¼ 8.6,

2.2 Hz), 6.76 (d,1 H, J ¼ 8.6 Hz), 4.39 (m,1 H), 4.28 (m, 3 H), 4.04 (dd,

1 H, J ¼ 11.5, 6.8 Hz), 2.12 (s, 3 H).

2-Acetyloxymethyl-7-vinyl-1,4-benzodioxane (45). Potassium

vinyl triﬂuoborate (400 mg, 2.98 mmol), PdCl₂(dppe),CH₂Cl₂

(170 mg, 0.3 mmol), t-butylamine (0.94 mL, 8.94 mmol) were added

to a solution of 44 (590 mg, 2.98 mmol) in 2-propanol (16 mL) and

water (10 mL) under nitrogen. The mixture was reﬂuxed overnight,

concentrated under vacuum, and diluted with water (10 mL) and

ethyl acetate (10 mL). The aqueous layer was separated, extracted

with ethyl acetate (2 ꢀ 10 mL). The combined organic phases were

washed with 10% HCl (10 mL), and brine (3 ꢀ 10 mL), dried over

Na₂SO₄, and concentrated to give 400 mg (57.3%) of 45 as a green

of 18 as a white solid: mp 125.8 ^ꢁC; ¹H NMR (DMSO-d)

d 8.12 (br s,

1 H), 7.84 (br s, 1 H), 7.29 (dt, 1 H, J ¼ 9.4, 5.4 Hz), 7.24 (s, 1 H), 7.19

(dd,1 H, J ¼ 8.7,1.5 Hz), 7.10 (s,1 H), 7.06 (dt,1 H, J ¼ 9.4, 2.1 Hz), 4.67

(m, 1 H), 4.51 (dd, 1 H, J ¼ 11.7, 2.1 Hz), 4.39e4.28 (m, 2 H), 4.22 (dd,

oil: ¹H NMR (CDCl₃)

d

6.96 (m, 3 H), 6.60 (dd, 1 H, J ¼ 17.6, 10.8 Hz),

1 H, J ¼ 11.7, 6.9 Hz). ¹³C NMR (DMSO-d)

d

16.7, 152.6 (dd, J ¼ 240.5,

5.59 (dd,1 H, J ¼ 17.5, 0.8 Hz), 5.14 (dd,1 H, J ¼ 10.8, 0.8 Hz), 4.27 (m,

6.8 Hz), 148.4 (dd, J ¼ 247.4, 9.2 Hz), 146.5, 143.3, 143.1 (dd, J ¼ 10.3,

3.4 Hz), 124.5 (q, J ¼ 270.0 Hz), 122.7 (q, J ¼ 31.4 Hz), 118.9 (q,

J ¼ 4.3 Hz), 118.3, 117.4 (dd, J ¼ 25.1, 20.4 Hz), 116.4 (d, J ¼ 9.1 Hz),

114.8 (q, J ¼ 3.4 Hz), 111.5 (dd, J ¼ 22.9, 4.6 Hz), 72.0, 68.6, 65.1. Anal.

Calcd for C₁₇H₁₂F5NO₄(389.27).

2 H), 4.10 (m, 1 H), 3.88 (m, 2 H), 2.12 (s, 3 H).

2-Hydroxymethyl-7-vinyl-1,4-benzodioxane (46). 2.5 M NaOH

(1.0 mL) and water (3.0 mL) were added to a solution of 45 (400 mg,

2.01 mmol) in methanol (5 mL). The reaction mixture was stirred at

room temperature for 30 min, concentrated, and diluted with water

(10 mL) and ethyl acetate (10 mL). The aqueous layer was separated

and extracted with ethyl acetate (2 ꢀ 10 mL). The combined organic

phases were washed with brine (2 ꢀ 10 mL), dried over Na₂SO₄, and

concentrated to yield a residue which was puriﬁed by ﬂash chro-

matography on silica gel. Elution with 80/20 cyclohexane/ethyl

acetate gave 140 mg (36.2%) of 46 as a colourless wax: ¹H NMR

7-Fluoro-2-mesyloxymethyl-1,4-benzodioxane (43). Mesyl

chloride (0.40 mL, 5.15 mmol) was added dropwise to a solution of

7-ﬂuoro-2-hydroxymethyl-1,4-benzodioxane (790 mg, 4.29 mmol)

and TEA (0.785 mL, 5.58 mmol) in DCM (10 mL) at 4 ^ꢁC. The mixture

was stirred at 4 ^ꢁC for 15 min and then at room temperature for

90 min. After cooling to 4 ^ꢁC, water (10 mL) and DCM (10 mL) were

added. The organic layer was separated, washed with 10% HCl

(10 mL), saturated aqueous NaHCO₃(10 mL), and brine (10 mL),

dried over Na₂SO₄, and concentrated to yield a residue which was

puriﬁed by ﬂash chromatography on silica gel. Elution with 70/30

cyclohexane/ethyl acetate gave 860 mg (76.4%) of 43 as a colourless

(CDCl₃)

d

6.89 (m, 3 H), 6.60 (dd, 1 H, J ¼ 17.6, 10.8 Hz), 5.59 (dd, 1 H,

J ¼ 17.5, 0.9 Hz), 5.14 (dd,1 H, J ¼ 10.8, 0.9 Hz), 4.27 (m, 2 H), 4.12 (m,

1 H), 3.88 (m, 2 H), 1.90 (br s, 1 H).

2-Mesyloxymethyl-7-vinyl-1,4-benzodioxane (47). Mesyl

chloride (0.09 mL, 1.17 mmol) was added dropwise to a solution of

46 (140 mg, 0.73 mmol) and TEA (0.17 mL, 1.17 mmol) in DCM

(5 mL) at 4 ^ꢁC. The mixture was stirred at 4 ^ꢁC for 15 min and then at

room temperature for 30 min. After cooling to 4 ^ꢁC, water (10 mL)

and DCM (10 mL) were added. The organic layer was separated,

washed with 10% HCl (10 mL), saturated aqueous NaHCO₃(10 mL),

and brine (10 mL), dried over Na₂SO₄, and concentrated to yield a

residue which was puriﬁed by ﬂash chromatography on silica gel.

Elution with 80/20 cyclohexane/ethyl acetate gave 110 mg (55.7%)

oil: ¹H NMR (CDCl₃)

d

6.83 (dd, 1 H, J ¼ 8.9, 5.4 Hz), 6.61 (m, 2 H),

4.61 (m, 3 H), 4.10 (dd, 1 H, J ¼ 11.7, 6.1 Hz), 3.09 (s, 3 H).

2,6-Diﬂuoro-3-(7-ﬂuoro-1,4-benzodioxan-2-yl)methox-

ybenzamide (13). Potassium carbonate (498 mg, 3.61 mmol) was

added to a solution of 2,6-diﬂuoro-3-hydroxybenzamide (625 mg,

3.28 mmol) in DMF (10 mL). After stirring at room temperature for

15 min, a solution of 43 (860 mg, 3.28 mmol) in DMF (5 mL) was

added. The reaction mixture was stirred at 75 ^ꢁC for 4 h, concen-

trated under vacuum, and diluted with water (20 mL) and ethyl

acetate (20 mL). The aqueous phase was separated and extracted

with ethyl acetate (2 ꢀ 20 mL). The combined organic phases were

washed with 1 M NaOH (30 mL), and brine (30 mL), dried over

Na₂SO₄, and concentrated to give a residue which was puriﬁed by

ﬂash chromatography on silica gel. Elution with 1/1 cyclohexane/

ethyl acetate gave 620 mg (55.7%) of 13 as a white solid: mp

of 47 as a colourless wax: ¹H NMR (CDCl₃)

d 6.91 (m, 3 H), 6.59 (dd,

1 H, J ¼ 17.6, 10.8 Hz), 5.60 (dd, 1 H, J ¼ 17.5, 0.9 Hz), 5.15 (dd, 1 H,

J ¼ 10.8, 0.9 Hz), 4.47 (m, 3 H), 4.31 (dd, 1 H, J ¼ 11.6, 2.2 Hz), 4.13

(dd, 1 H, J ¼ 11.6, 6.1 Hz), 3.09 (s, 3 H).

2,6-Diﬂuoro-3-(7-vinyl-1,4-benzodioxan-2-yl)methox-

ybenzamide (15). Potassium carbonate (62 mg, 0.45 mmol) was

added to a solution of 2,6-diﬂuoro-3-hydroxybenzamide (78 mg,

0.45 mmol) in DMF (2 mL). After stirring at room temperature for

15 min, a solution of 47 (110 mg, 0.41 mmol) in DMF (3 mL) was

added. The reaction mixture was stirred at 70 ^ꢁC for 6.5 h,

concentrated under vacuum, and diluted with water (10 mL) and

ethyl acetate (10 mL). The aqueous phase was separated and

extracted with ethyl acetate (2 ꢀ 10 mL). The combined organic

phases were washed with 1 M NaOH (20 mL), and brine (20 mL),

dried over Na₂SO₄, and concentrated to give a residue which was

puriﬁed by ﬂash chromatography on silica gel. Elution with 60/40

cyclohexane/ethyl acetate gave 100 mg (69.9%) of 15 as a white

120.1 ^ꢁC; ¹H NMR (CDCl₃)

d

7.07 (dt, 1 H, J ¼ 9.1, 5.1 Hz), 6.90 (dt, 1 H,

J ¼ 9.1, 2.0 Hz), 6.83 (dd, 1 H, J ¼ 8.9, 5.4 Hz), 6.60 (m, 2 H), 5.96 (br s,

2 H), 4.57 (dd, 1 H, J ¼ 11.2, 2.4 Hz), 4.36 (dd, 1 H, J ¼ 11.2, 2.4 Hz),

4.23 (m, 3 H). ¹³C NMR(CDCl₃)

d

162.1, 157.6 (d, J ¼ 238.0 Hz), 154.3

(dd, 246.5, 5.2 Hz), 150.6 (dd, J ¼ 253.6, 7.1 Hz), 143.5 (d, J ¼ 7.9 Hz),

143.1 (d, J ¼ 12.0 Hz), 139.5, 118.4 (d, J ¼ 8.0 Hz), 117.8 (d, J ¼ 9.4 Hz),

114.4 (dd, J ¼ 19.9, 16.3 Hz), 111.6 (d, J ¼ 23.6 Hz), 108.4 (d,

J ¼ 23.3 Hz), 104.8 (d, J ¼ 26.5 Hz), 71.7, 69.0, 64.9. Anal. Calcd for

C

₁₆H₁₂F3NO₄(339.27).

2-Acetyloxymethyl-7-bromo-1,4-benzodioxane (44). Acetyl

chloride (0.20 mL, 2.71 mmol) was slowly added to a solution of 37

wax: ¹H NMR (CDCl₃)

d 7.06 (m, 1 H), 6.90 (m, 3 H), 6.59 (dd, 1 H,

238

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

J ¼ 17.5, 10.9 Hz), 6.00 (br s, 2 H), 5.59 (d, 1 H, J ¼ 17.6 Hz), 5.14 (d,

1 H, J ¼ 10.9 Hz), 4.56 (m, 1 H), 4.39 (dd, 1 H, J ¼ 11.5, 2.4 Hz), 4.25

(m, 4 H). ¹³C NMR (CDCl₃) 162.1, 154.3 (dd, J ¼ 247.0, 5.7 Hz), 150.6

(dd, J ¼ 253.4, 7.0 Hz), 143.6 (dd, J ¼ 11.4, 3.6 Hz), 143.1, 142.8, 136.2,

132.2, 122.1 (d, J ¼ 13.9 Hz), 120.2, 118.6, 117.5, 115.0 (dd, J ¼ 16.8,

12.0 Hz), 112.8, 111.5 (dd, J ¼ 23.8, 2.1 Hz), 71.5, 69.3, 65.1. Anal.

Calcd for C₁₈H₁₅F₂NO₄(347.31).

12.89 mmol) in DMF (28 mL). After cooling to 4 ^ꢁC, epibromohydrin

(1.4 mL, 16.37 mmol) was added dropwise. The mixture was stirred

at 4 ^ꢁC for 15 min and then at 80 ^ꢁC for 8 h. Water (100 mL) was

added at 4 ^ꢁC and extraction was accomplished by ethyl acetate

(3 ꢀ 30 mL). The combined organic extracts were washed with 1 M

NaOH (30 mL), and with brine (2 ꢀ 50 mL), dried over Na₂SO₄, and

concentrated. The resulting residue (2.28 g) was crystallized twice

from DCM to give 0.45 g (16.5%) of 51 as a white solid: mp 136.0 ^ꢁC;

2-Acetyloxymethyl-7-trimethylsilylethynyl-1,4-

benzodioxane (48). Ethynyltrimethylsilane (0.33 mL, 2.33 mmol),

PdCl₂(20 mg, 0.1 mmol), CuI (15 mg, 0.07 mmol), and PPh₃(50 mg,

0.19 mmol) were added to a solution of 44 (540 mg, 1.88 mmol) in

TEA (2.5 mL). The mixture was heated to 55 ^ꢁC overnight. After

cooling to room temperature, ethyl acetate (20 mL) and water

(15 mL) were added. The organic layer was separated, washed with

10% HCl (15 mL) and brine (3 ꢀ 15 mL), dried over Na₂SO₄, and

concentrated to give a residue which was puriﬁed by ﬂash chro-

matography on silica gel. Elution with 90/10 cyclohexane/ethyl

acetate provided 320 mg (55.9%) of 48 as a red oil: ¹H NMR (CDCl₃)

¹H NMR (CDCl₃)

d

7.80 (m, 2 H), 6.96 (d, 1 H, J ¼ 8.6 Hz), 4.43 (dd,

1 H, J ¼ 11.1,1.9 Hz), 4.27 (m,1 H), 3.93 (qd, 2 H, J ¼ 12.2, 4.4 Hz),1.78

(br s, 1 H).

7-Nitro-2-tosyloxymethyl-1,4-benzodioxane (52). TsCl (0.30 g,

1.52 mmol) was added to a solution of 51 (0.32 g, 1.52 mmol) in

pyridine (3 mL) at 4 ^ꢁC. The mixture was stirred at room temper-

ature for 3 h, diluted with water (20 mL) and DCM (20 mL). The

organic phase was separated, washed with 10% HCl (20 mL), satu-

rated aqueous NaHCO₃(20 mL), and brine (20 mL), dried over

Na₂SO₄, and concentrated. The oily residue was puriﬁed by ﬂash

chromatography on silica gel. Elution with 80/20 cyclohexane/ethyl

acetate gave 270 mg (48.6%) of 52 as a white solid: mp 101.2 ^ꢁC; ¹H

d

7.05 (d, 1 H, J ¼ 2.1 Hz), 6.95 (dd, 1 H, J ¼ 8.5, 2.2 Hz), 6.76 (d, 1 H,

J ¼ 8.5 Hz), 4.35 (m, 4 H), 4.04 (dd, 1 H, J ¼ 11.2, 6.6 Hz), 2.12 (s, 3 H),

0.23 (s, 9 H).

NMR (CDCl₃)

d

7.78 (m, 3 H), 7.64 (d, 1 H, J ¼ 2.6 Hz), 7.37 (d, 2 H,

7-Ethynyl-2-hydroxymethyl-1,4-benzodioxane (49). 2.5

M

J ¼ 8.0 Hz), 6.93 (d, 1 H, J ¼ 8.8 Hz), 4.42 (m, 2 H), 4.26 (m, 2 H), 4.14

(dd, 1 H, J ¼ 11.7, 6.8 Hz), 2.46 (s, 3 H).

NaOH (0.51 mL) was added to a solution of 48 (320 mg, 1.05 mmol)

in methanol (4 mL). After stirring at room temperature for 1 h, the

reaction mixture was concentrated and diluted with water (10 mL)

and ethyl acetate (10 mL). The aqueous layer was separated and

extracted with ethyl acetate (3 ꢀ 10 mL). The combined organic

phases were washed with brine (30 mL), dried over Na₂SO₄, and

concentrated to give 198 mg (99.1%) of 49 as a brownish oil: ¹H

2,6-Diﬂuoro-3-(7-nitro-1,4-benzodioxan-2-yl)methox-

ybenzamide (16). Potassium carbonate (110 mg, 0.81 mmol) was

added to a solution of 2,6-diﬂuoro-3-hydroxybenzamide (140 mg,

0.81 mmol) in DMF (2 mL). After stirring at room temperature for

15 min, a solution of 52 (270 mg, 0.74 mmol) in DMF (2 mL) was

added. The reaction mixture was stirred at 75 ^ꢁC for 4 h, concen-

trated under vacuum, and diluted with water (10 mL) and ethyl

acetate (10 mL). The aqueous phase was separated and extracted

with ethyl acetate (2 ꢀ 10 mL). The combined organic phases were

washed with 1 M NaOH (15 mL), and brine (15 mL), dried over

Na₂SO₄, and concentrated to give a residue which was puriﬁed by

ﬂash chromatography on silica gel. Elution with 1/1 cyclohexane/

ethyl acetate yielded 160 mg (59.0%) of 16 as a white solid: mp

NMR (CDCl₃)

d

7.02 (m, 2 H), 6.81 (d, 1 H, J ¼ 8.3 Hz), 4.28 (m, 2 H),

4.12 (dd, 1 H, J ¼ 11.6, 7.2 Hz), 3.88 (m, 2 H), 2.97 (s, 1 H), 1.98 (br s,

1 H).

7-Ethynyl-2-mesyloxymethyl-1,4-benzodioxane (50). Mesyl

chloride (0.10 mL, 1.26 mmol) was added dropwise to a solution of

49 (198 mg, 1.05 mmol) and TEA (0.20 mL, 1.36 mmol) in DCM

(6 mL) at 4 ^ꢁC. The mixture was stirred at 4 ^ꢁC for 15 min and then at

room temperature for 30 min. After cooling to 4 ^ꢁC, water (10 mL)

and DCM (10 mL) were added. The organic layer was separated,

washed with 10% HCl (10 mL), saturated aqueous NaHCO₃(10 mL),

and brine (10 mL), dried over Na₂SO₄, and concentrated to give

172.2 ^ꢁC; ¹H NMR (DMSO-d)

d 8.11 (br s,1H), 7.83 (br s,1H), 7.78 (dd,

1 H, J ¼ 9.1, 2.8 Hz), 7.70 (d, 1 H, J ¼ 2.8 Hz), 7.29 (dt, 1 H, J ¼ 9.3,

5.2 Hz), 7.10 (d, 1H, J ¼ 9.1 Hz), 7.07 (dt, 1 H, J ¼ 9.3, 1.8 Hz), 4.71 (m,

1H), 4.59 (dd, 1 H, J ¼ 11.5, 2.5 Hz), 4.41e4.26 (m, 3 H). ¹³C NMR

279 mg (98.1%) of 50 as a brownish oil: ¹H NMR (CDCl₃)

d

7.02 (m,

(DMSO-d)

d

161.4, 152.4 (dd, J ¼ 240.4, 7.0 Hz), 149.2, 148.2 (dd,

2 H), 6.83 (d, 1 H, J ¼ 8.0 Hz), 4.44 (m, 3 H), 4.33 (m, 1 H), 4.13 (dd,

J ¼ 247.3, 8.4 Hz), 142.8 (dd, J ¼ 10.8, 3.2 Hz), 142.7, 141.5, 117.77,

117.70, 116.9 (dd, J ¼ 25.4, 20.5 Hz), 116.2 (d, J ¼ 9.4 Hz), 112.9, 111.2

(dd, J ¼ 23.2, 3.8 Hz), 71.8, 68.3, 65.2. Anal. Calcd for C₁₆H₁₂F₂N₂O₆

(366.27).

1 H, J ¼ 11.5, 6.1 Hz), 3.09 (s, 3 H), 2.98 (s, 1 H).

2,6-Diﬂuoro-3-(7-ethynyl-1,4-benzodioxan-2-yl)methox-

ybenzamide (14). Potassium carbonate (160 mg, 1.16 mmol) was

added to a solution of 2,6-diﬂuoro-3-hydroxybenzamide (201 mg,

1.16 mmol) in DMF (3 mL). After stirring at room temperature for

15 min, a solution of 50 (279 mg, 1.04 mmol) in DMF (3 mL) was

added. The reaction mixture was stirred at 60 ^ꢁC for 20 h,

concentrated under vacuum, and diluted with water (10 mL) and

ethyl acetate (10 mL). The aqueous phase was separated and

extracted with ethyl acetate (2 ꢀ 10 mL). The combined organic

phases were washed with 1 M NaOH (20 mL), and brine (20 mL),

dried over Na₂SO₄, and concentrated to give a residue which was

puriﬁed by ﬂash chromatography on silica gel. Elution with 60/40

cyclohexane/ethyl acetate yielded 220 mg (60.8%) of 14 as a pale

3-(7-Amino-1,4-benzodioxan-2-yl)methoxy-2,6-

diﬂuorobenzamide (17). SnCl₂(430 mg, 2.29 mmol) was added to

a solution of 16 (210 mg, 0.57 mmol) in ethyl acetate (5 mL). The

mixture was stirred at room temperature for 15 min, reﬂuxed for

2 h, cooled to room temperature and poured into an ice-cooled

saturated aqueous NaHCO₃(20 mL). The resulting suspension was

ﬁltered and the ﬁltrate was extracted with ethyl acetate

(3 ꢀ 20 mL). The combined organic extracts were dried over Na₂SO₄

and concentrated to give a residue which was puriﬁed by ﬂash

chromatography on silica gel. Elution with 60/40 toluene/ethyl

acetate provided 190 mg (99.1%) of 17 as a white solid: mp 141.3 ^ꢁC;

yellow wax: ¹H NMR (CDCl₃)

1 H), 6.03 (br s,1 H), 4.55 (m,1 H), 4.41 (dd,1 H, J ¼ 11.5, 2.3 Hz), 4.24

(m, 3 H), 2.97 (s, 1 H). ¹³C NMR (DMSO-d)

161.9, 152.8 (dd,

d

7.05 (m, 3 H), 6.85 (m, 2 H), 6.14 (br s,

¹H NMR (DMSO-d)

d 8.09 (br s, 1H), 7.81 (br s, 1H), 7.25 (dt, 1 H,

J ¼ 9.1, 5.3 Hz), 7.04 (dt, 1 H, J ¼ 9.1, 1.8 Hz), 6.50 (d, 1 H, J ¼ 8.4 Hz),

6.10 (d, 1H, J ¼ 2.4 Hz), 6.05 (dd, 1 H, J ¼ 8.4, 2.4 Hz), 4.62 (br s, 2H),

4.47e4.59 (m, 1 H), 4.25e4.19 (m, 3 H), 3.98 (dd, 1 H, J ¼ 11.4,

d

J ¼ 240.5, 6.7 Hz), 148.6 (dd, J ¼ 247.3, 8.4 Hz), 144.4, 143.4 (dd,

J ¼ 3.3,10.9 Hz),143.1,126.0,121.0, 118.1,117.3 (dd, J ¼ 24.9, 20.2 Hz),

116.6 (d, J ¼ 9.1 Hz),115.4,111.7 (dd, J ¼ 22.8, 3.8 Hz), 83.9, 79.9, 72.1,

68.8, 65.3. Anal. Calcd for C₁₈H₁₃F₂NO₄(345.08).

2-Hydroxymethyl-7-nitro-1,4-benzodioxane (51). NaHCO₃

(1.16 g,13.5 mmol) was added to a solution of 4-nitrocatechol (2.0 g,

6.6 Hz). ¹³C NMR (DMSO-d)

d

161.7, 152.5 (dd, J ¼ 240.5, 6.9 Hz),

148.4 (dd, J ¼ 247.4, 8.0 Hz), 143.9, 143.2 (dd, J ¼ 11.5, 4.6 Hz), 143.1,

134.1, 117.4, 117.1 (d, J ¼ 4.6 Hz), 116.3 (d, J ¼ 6.9 Hz), 111.4 (dd,

J ¼ 22.9, 4.6 Hz), 108.0, 102.8, 71.9, 68.8, 64.6. Anal. Calcd for

C₁₆H₁₄F₂N₂O₄(336.29).

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

239

2,6-Diﬂuoro-3-(7-methanesulfonamido-1,4-benzodioxan-2-

2 H, J ¼ 12.1, 4.7 Hz), 1.47 (br s, 1 H).

yl)methoxybenzamide (19). Mesyl chloride (0.05 mL, 0.67 mmol)

was added dropwise to a solution of 17 (190 mg, 0.56 mmol) and

TEA (0.1 mL, 0.73 mmol) in ethyl acetate (8 mL) at 4 ^ꢁC. The mixture

was reﬂuxed for 30 min. After cooling to 4 ^ꢁC, 10% HCl was added.

The aqueous phase was separated and extracted with ethyl acetate

(3 ꢀ 15 mL). The combined organic phases were dried over Na₂SO₄,

and concentrated. The resulting residue was puriﬁed by ﬂash

chromatography on silica gel. Elution with 1/1 toluene/ethyl acetate

gave 100 mg (43.1%) of 19 as a colourless wax: ¹H NMR (DMSO-d)

7-Iodo-2-mesyloxymethyl-1,4-benzodioxane (57). Mesyl

chloride (0.06 mL, 0.74 mmol) was slowly added to a solution of 56

(0.18 g, 0.62 mmol) and TEA (0.11 mL, 0.81 mmol) in DCM (5 mL) at

4

^ꢁC. The mixture was stirred for 15 min at 4 ^ꢁC and for 30 min at

room temperature. After cooling to 4 ^ꢁC, water (10 mL) and DCM

(10 mL) were added. The organic phase was separated, washed with

10% HCl (10 mL), saturated aqueous NaHCO₃(10 mL), and brine

(10 mL), dried over Na₂SO₄, and concentrated. The resulting residue

was puriﬁed by ﬂash chromatography on silica gel. Elution with 70/

30 cyclohexane/ethyl acetate gave 0.19 g (82.8%) of 57 as a col-

d

9.40 (br s, 1H), 8.11 (br s, 1H), 7.84 (br s, 1H), 7.27 (dt, 1 H, J ¼ 9.0,

5.2 Hz), 7.06 (dt, 1 H, J ¼ 9.0, 1.5 Hz), 6.9 (d, 1 H, J ¼ 8.9 Hz), 6.8 (d,

1 H, J ¼ 2.5 Hz), 6.70 (dd, 1 H, J ¼ 8.9, 2.5 Hz), 4.57 (m, 1H), 4.39 (dd,

1 H, J ¼ 11.6, 2.5 Hz), 4.31 (m, 2H), 4.10 (dd,1 H, J ¼ 11.6, 7.2 Hz), 2.88

ourless oil: ¹H NMR (CDCl₃)

J ¼ 8.6, 2.1 Hz), 6.65 (d, 1 H, J ¼ 8.5 Hz), 4.45 (m, 3 H), 4.30 (dd, 1 H,

J ¼ 11.7, 2.3 Hz), 4.11 (dd, 1 H, J ¼ 11.7, 6.2 Hz), 3.09 (s, 3 H).

2,6-Diﬂuoro-3-(7-iodo-1,4-benzodioxan-2-yl)methox-

d

7.23 (d, 1 H, J ¼ 2.1 Hz), 7.16 (dd, 1 H,

(s, 3 H). ¹³C NMR (DMSO-d)

d

161.9, 152.8 (dd, J ¼ 240.3, 6.8 Hz),

148.6 (dd, J ¼ 247.1, 8.3 Hz), 143.4 (dd, J ¼ 10.6, 3.2 Hz), 143.3, 140.6,

132.7, 118.1, 117.5 (dd, J ¼ 24.7, 20.5 Hz), 116.5 (d, J ¼ 9.1 Hz), 115.1,

111.7 (dd, J ¼ 23.1, 4.0 Hz), 110.8, 71.5, 69.0, 65.1. The methyl signal is

obscured by the overlap with the DMSO signal. Anal. Calcd for

ybenzamide (12). Potassium carbonate (77 mg, 0.56 mmol) was

added to a solution of 2,6-diﬂuoro-3-hydroxybenzamide (97 mg,

0.56 mmol) in DMF (2 mL). After stirring at room temperature for

15 min, a solution of 57 (190 mg, 0.51 mmol) in DMF (2 mL) was

added. The reaction mixture was stirred at 75 ^ꢁC for 4 h, concen-

trated under vacuum, and diluted with water (10 mL) and ethyl

acetate (10 mL). The aqueous phase was separated and extracted

with ethyl acetate (2 ꢀ 10 mL). The combined organic phases were

washed with 1 M NaOH (15 mL), and brine (15 mL), dried over

Na₂SO₄, and concentrated to give a residue which was puriﬁed by

ﬂash chromatography on silica gel. Elution with 1/1 cyclohexane/

ethyl acetate yielded 120 mg (52.6%) of 12 as a white wax: ¹H NMR

C

₁₇H₁₆F₂N₂O₄S (414.38).

2-Acetyloxymethyl-7-nitro-1,4-benzodioxane

(53).

TEA

(0.36 mL, 2.56 mL) was added to a solution of 51 (0.45 g, 2.13 mmol)

in ethyl acetate (5 mL). After cooling to 4 ^ꢁC, acetyl chloride

(0.18 mL, 2.56 mmol) was added dropwise. The mixture was stirred

at 4 ^ꢁC for 15 min and diluted with water (15 mL) and ethyl acetate

(20 mL). The organic layer was separated, washed with 10% HCl

(20 mL), saturated aqueous NaHCO₃(20 mL), and brine (20 mL),

dried over Na₂SO₄, and concentrated to give 0.51 g (94.6%) of 53 as a

(CDCl₃)

d

7.22 (d, 1 H, J ¼ 2.2 Hz), 7.15 (dd, 1 H, J ¼ 8.5, 2.2 Hz), 7.06

colourless oil: ¹H NMR (CDCl₃)

d

7.80 (m, 2 H), 6.96 (d, 1 H,

(dt, 1 H, J ¼ 9.1, 5.1 Hz), 6.90 (dt, 1 H, J ¼ 9.1, 2.0 Hz), 6.65 (d, 1 H,

J ¼ 8.8 Hz), 4.40 (m, 4 H), 4.14 (m, 1 H), 2.12 (s, 3 H).

J ¼ 8.5 Hz), 6.00 (br s, 2 H), 4.55 (m, 1 H), 4.38 (dd, 1 H, J ¼ 11.6,

7-Amino-2-acetyloxymethyl-1,4-benzodioxane (54). SnCl₂

(1.53 g, 8.06 mmol) was added to a solution of 53 (0.51 g,

2.01 mmol) in ethyl acetate (8 mL). The mixture was reﬂuxed for

2 h. Saturated aqueous NaHCO₃(25 mL) was added and the

resulting suspension was ﬁltered. The ﬁltrate was extracted with

ethyl acetate (3 ꢀ 20 mL). The combined extracts were dried over

Na₂SO₄, and concentrated. The residue was puriﬁed by ﬂash chro-

matography on silica gel. Elution with 1/1 cyclohexane/ethyl ace-

tate gave 0.28 g (62.4%) of 54 as a pale yellow oil: ¹H NMR (CDCl₃)

2.4 Hz), 4.23 (m, 3 H). ¹³C NMR (DMSO-d)

d 161.9, 154.4 (dd,

J ¼ 246.5, 5.4 Hz), 150.6 (dd, J ¼ 253.8, 7.0 Hz), 143.8, 143.5 (dd,

J ¼ 11.4, 3.8 Hz), 143.4, 130.8, 126.4, 123.3, 119.4; 114.4 (dd, J ¼ 20.2,

16.3 Hz),114.5 (dd, J ¼ 20.8,16.3 Hz),111.5 (dd, J ¼ 19.4, 4.3 Hz), 71.5,

69.0, 65.1. Anal. Calcd for C₁₆H₁₂F₂INO₄(447.17). Anal. Calcd for

C₁₆H₁₂F₂INO₄(447.17).

2-Benzyloxymethyl-7-carboxy-1,4-benzodioxane (58). Potas-

sium carbonate (5.45 g, 39.4 mmol) and epibromohydrin (3.37 mL,

39.4 mmol) were added to solution of methyl 3,4-

a

d

6.68 (d, 1 H, J ¼ 8.6 Hz), 6.27 (d, 1 H, J ¼ 2.7 Hz), 6.22 (dd, 1 H,

dihydroxybenzoate (5.52 g, 32.8 mmol). The mixture was stirred

at room temperature for 15 min and at 60 ^ꢁC overnight, and then

concentrated. Water (100 mL) and ethyl acetate (100 mL) were

added. The aqueous layer was separated and extracted with ethyl

acetate (2 ꢀ 100 mL). The combined organic phases were washed

with 1 M NaOH (50 mL) and with brine (2 ꢀ 50 mL), dried over

Na₂SO₄and concentrated. The TLC analysis (silica, 60/40 cyclo-

hexane/ethyl acetate) of the residue indicated the presence of a

major spot with R_f0.25, which was isolated, as a colourless oil, in

amount of 5.19 g by ﬂash chromatography on silica gel with the

same eluent. ¹H NMR spectrum showed that it was a mixture of two

regioisomers: desired 2-hydroxymethyl-7-methoxycarbonyl-1,4-

benzodioxane (~85%) and, likely, undesired 2-hydroxymethyl-6-

methoxycarbonyl-1,4-benzodioxane (~15%). Such a mixture was

dissolved in dry THF (10 mL) and dropped into a suspension of NaH

(610 mg, 25.47 mmol) in dry THF (10 mL) under nitrogen. After

stirring at room temperature for 30 min, a solution of benzyl bro-

mide (3.03 mL, 25.47 mmol) in dry THF (10 mL) was added. After

15 min at room temperature, the mixture was reﬂuxed for 3 h, and

then cooled to room temperature. Ethyl acetate (30 mL) and 10%

HCl (30 mL) were added. The organic phase was separated, washed

with brine (2 ꢀ 30 mL), dried over Na₂SO₄, and concentrated. The

residue was puriﬁed by ﬂash chromatography on silica gel. Elution

with 90/10 cyclohexane/ethyl acetate gave 4.30 g of colourless oil,

corresponding to a TLC spot faster running than the starting alco-

hols mixture (silica, 60/40 cyclohexane/ethyl acetate, R_f0.58) and

J ¼ 8.6, 2.7 Hz), 4.31 (m, 3 H), 3.97 (dd, 1 H, J ¼ 11.5, 6.9 Hz), 3.06 (br

s, 2 H), 2.13 (s, 3 H).

2-Acetyloxymethyl-7-iodo-1,4-benzodioxane (55). A solution

of NaNO₂(95 mg, 1.38 mmol) in water (2 mL) was slowly added to a

solution of 54 (0.28 g, 1.25 mmol) in 0.29 M H₂SO₄(4.68 mL)

at ꢂ5 ^ꢁC. After 15 min, a solution of NaI (320 mg, 2.13 mmol) in

water (2 mL) was added and the mixture was stirred at room

temperature overnight. Saturated aqueous Na₂S₂O₅(10 mL) was

added and extraction was accomplished with ethyl acetate

(3 ꢀ 20 mL). The combined organic extracts were dried over Na₂SO₄

and concentrated to give a residue which was puriﬁed by ﬂash

chromatography on silica gel. Elution with 80/20 cyclohexane/ethyl

acetate gave 0.24 g (57.5%) of 55 as a colourless oil: ¹H NMR (CDCl₃)

d

7.23 (d, 1 H, J ¼ 2.1 Hz), 7.14 (dd, 1 H, J ¼ 8.5, 2.1 Hz), 6.63 (d, 1 H,

J ¼ 8.5 Hz), 4.37 (m, 1 H), 4.28 (m, 3 H), 4.03 (dd, 1 H, J ¼ 11.5,

6.9 Hz), 2.07 (s, 3 H).

2-Hydroxymethyl-7-iodo-1,4-benzodioxane

(56).

Water

(3 mL) and 2.5 M NaOH (0.37 mL) were added to a solution of 55

(0.24 g, 0.72 mmol) in methanol (3 mL). The reaction mixture was

stirred at room temperature for 1 h, concentrated, diluted with

water (10 mL), and extracted with ethyl acetate (3 ꢀ 10 mL). The

organic extracts were washed with brine (20 mL), dried over

Na₂SO₄, and concentrated to give 0.18 g (85.6%) of 56 as a brownish

oil: ¹H NMR (CDCl₃)

2.1 Hz), 6.63 (d, 1 H, J ¼ 8.5 Hz), 4.25 (m, 2 H), 4.11 (m, 1 H), 3.86 (qd,

d

7.22 (d, 1 H, J ¼ 2.1 Hz), 7.13 (dd, 1 H, J ¼ 8.5,

240

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

analogously composed of 2-benzyloxymethyl-7-methoxycarbonyl-

1,4-benzodioxane (~85%) and its undesired 6-methoxycarbonyl

regioisomer (~15%) as inferable from the ¹H NMR spectrum. Such

a benzyl ethers mixture was dissolved in methanol (40 mL); 2.5 M

NaOH (6.9 mL) and water (10 mL) were added dropwise. The

mixture was stirred at room temperature for 15 min and at 50 ^ꢁC for

2 h, and concentrated. Ethyl acetate (20 mL) and 10% HCl (15 mL)

were added to the residue. The aqueous layer was separated and

extracted with ethyl acetate (2 ꢀ 20 mL). The combined organic

phases were washed with brine (2 ꢀ 20 mL), dried over Na₂SO₄and

concentrated. The resulting solid (4.43 g) was twice crystallized

from toluene to give 1.64 g (16.6% of starting methyl 3,4-

dihydroxybenzoate) of 58 as a white solid: mp 132.3 ^ꢁC; ¹H NMR

puriﬁed by ﬂash chromatography on silica gel. Elution with 1/1

cyclohexane/ethyl acetate gave 650 mg (52.0%) of 61 as a white

wax: ¹H NMR (CDCl₃)

d

7.33 (m, 7 H), 6.91 (d, 1 H, J ¼ 8.5 Hz), 5.75

(br s, 2 H), 4.60 (s, 2 H), 4.35 (m, 2 H), 4.13 (dd, 1 H, J ¼ 11.8, 7.7 Hz),

3.71 (m, 2 H).

7-Aminocarbonyl-2-hydroxymethyl-1,4-benzodioxane (62).

A solution of 61 (650 mg, 2.17 mmol) in acetone (10 mL) was added

with 5% Pd/C (70 mg) and vigorously shaken under hydrogen at

room temperature for 8 h. The catalyst was removed by ﬁltration

and the ﬁltrate concentrated to give 430 mg (93.5%) of 62 as a white

wax: ¹H NMR (DMSO-d₆)

d 7.78 (br s, 1 H), 7.37 (m, 2 H), 7.18 (br s,

1 H), 6.87 (d, 1 H, J ¼ 8.2 Hz), 5.05 (t, 1 H, J ¼ 5.8 Hz), 4.35 (dd, 1 H,

J ¼ 11.3, 2.2 Hz), 4.16 (m, 1 H), 4.03 (dd, 1 H, J ¼ 11.3, 7.2 Hz), 3.61 (m,

2 H).

(CDCl₃)

d

7.63 (m, 2 H), 7.33 (m, 5 H), 6.92 (d, 1 H, J ¼ 8.5 Hz), 4.61 (s,

2 H), 4.39 (m, 2 H), 4.17 (dd, 1 H, J ¼ 11.8, 7.7 Hz), 3.72 (m, 2 H).

2-Benzyloxymethyl-7-methoxycarbonyl-1,4-benzodioxane

(59). Trimethyl orthoformate (0.12 mL, 1.07 mmol) and concen-

trated sulfuric acid (0.1 mL) were added to a solution of 58 (320 mg,

1.07 mmol) in methanol (6 mL). The mixture was heated at 65 ^ꢁC

overnight. After cooling to room temperature, DCM (10 mL) and

saturated aqueous NaHCO₃(10 mL) were added. The organic phase

was separated, washed with saturated aqueous NaCl (2 ꢀ 10 mL),

dried over Na₂SO₄and concentrated to give 290 mg (86.3%) of 59 as

7-Aminocarbonyl-2-tosyloxymethyl-1,4-benzodioxane (63).

Tosyl chloride (501 mg, 2.63 mmol) was added to a solution of 62

(500 mg, 2.39 mmol) in pyridine (2 mL). After stirring at 0 ^ꢁC for 2 h,

10% HCl (30 mL) and AcOEt (30 mL) were added. The aqueous layer

was separated and extracted with ethyl acetate (20 mL). The

combined organic phases were washed with 10% NaHCO₃and

brine, then dried over Na₂SO₄and concentrated. The residue was

crystallized from 1/1 2-propanol/butanone to give 230 mg (26.5%)

of 63 as a white solid: mp 180.2 ^ꢁC; ¹H NMR (CDCl₃)

d 7.78 (m, 1 H),

an oil: ¹H NMR (CDCl₃)

d

7.56 (m, 2 H), 7.32 (m, 5 H), 6.89 (d, 1 H,

7.36 (m, 5 H), 6.87 (d, 1 H, J ¼ 8.5 Hz), 5.57 (br s, 2 H), 4.46 (m, 1 H),

J ¼ 6.6 Hz), 4.60 (m, 2 H), 4.37 (m, 2 H), 4.15 (m, 1 H), 3.86 (s, 3 H),

4.33 (m, 3 H), 4.19 (m, 1 H), 2.49 (s, 3 H).

3.71 (m, 2 H).

2,6-Diﬂuoro-3-(7-aminocarbonyl-1,4-benzodioxan-2-yl)

methoxybenzamide (22). Potassium carbonate (105 mg,

2-Hydroxymethyl-7-methoxycarbonyl-1,4-benzodioxane

(60). A solution of 59 (290 mg, 0.92 mmol) in ethanol (10 mL) was

added with 10% Pd/C (50 mg) and vigorously shaken under

hydrogen at room temperature for 6 h. The catalyst was removed by

ﬁltration and the ﬁltrate concentrated to give 200 mg (97.0%) of 60

0.76 mmol) was added to

a solution of 2,6-diﬂuoro-3-

hydroxybenzamide (120 mg, 0.70 mmol) in DMF (3 mL). After

stirring at room temperature for 15 min, a solution of 63 (230 mg,

0.63 mmol) in DMF (7 mL) was added. The reaction mixture was

stirred at 75 ^ꢁC for 3 h, then concentrated under vacuum, and

diluted with water (20 mL) and ethyl acetate (20 mL). The aqueous

phase was separated and extracted with ethyl acetate (2 ꢀ 20 mL).

The combined organic phases were washed with 1 M NaOH

(20 mL), and brine (2 ꢀ 20 mL), dried over Na₂SO₄, and concen-

trated to give 22 as a white solid: mp 218.8 ^ꢁC; ¹H NMR (DMSO-d₆)

as an oil: ¹H NMR (CDCl₃)

4.39 (m, 4 H), 4.13 (m, 1 H), 3.86 (s, 3 H).

d

7.58 (m, 2 H), 6.90 (d, 1 H, J ¼ 8.2 Hz),

2,6-Diﬂuoro-3-(7-methoxycarbonyl-1,4-benzodioxan-2-yl)

methoxybenzamide (21). Triphenylphosphine (351 mg,

1.34 mmol) and 2,6-diﬂuoro-3-hydroxybenzamide (154 mg,

0.89 mmol) were added to a solution of 60 (200 mg, 0.89 mmol) in

THF (3 mL). A solution of DIAD (0.263 mL, 1.34 mmol) in THF (5 mL)

was added dropwise at 0 ^ꢁC. After stirring overnight at rt, the

mixture was concentrated and water (30 mL) and ethyl acetate

(30 mL) were added to the residue. The aqueous layer was sepa-

rated and extracted with ethyl acetate (3 ꢀ 20 mL). The combined

organic phases were dried over Na₂SO₄and concentrated. The

residue was puriﬁed by chromatography on silica gel; elution with

1/1 cyclohexane/ethyl acetate and successive crystallization from

methanol gave 90 mg (26.6%) of 21 as a white solid: mp 154.7 ^ꢁC; ¹H

d

8.09 (br s,1 H), 7.81 (br s,1 H), 7.78 (br s,1 H), 7.4 (d,1 H, J ¼ 2.1 Hz),

7.39 (dd, 1 H, J ¼ 8.3, 2.2 Hz), 7.27 (dt, 1 H, J ¼ 9.0, 5.2 Hz), 7.17 (br s,

1 H), 7.05 (dt, 1 H, J ¼ 9.0, 1.7 Hz), 6.90 (d, 1 H, J ¼ 8.4 Hz), 4.61 (m,

1 H), 4.46 (dd, 1 H, J ¼ 11.8, 2.5 Hz), 4.33 (m, 2 H), 4.17 (dd, 1 H,

J ¼ 11.5, 7.2 Hz). ¹³C NMR (DMSO-d)

d 167.4, 161.7, 152.6 (dd,

J ¼ 240.5, 6.9 Hz), 148.4 (dd, J ¼ 247.4, 8.0 Hz), 145.8, 143.2 (dd,

J ¼ 10.3, 3.4 Hz),142.4,128.2,121.7,117.1, 117.1 (dd, J ¼ 25.3, 20.4 Hz),

117.0, 116.4 (dd, J ¼ 10.3, 2.3 Hz), 111.5 (dd, J ¼ 22.9, 3.5 Hz), 71.8,

68.7, 65.1. Anal. Calcd for C₁₇H₁₄F₂N₂O₅(364.30).

NMR (DMSO-d)

d

8.09 (br s, 1H), 7.81 (br s, 1H), 7.46 (dd, 1 H, J ¼ 8.4,

7-Cyano-2-hydroxymethyl-1,4-benzodioxane (67). Triﬂuoro-

acetic anhydride (1.6 mL, 11.5 mmol) was added dropwise to a so-

lution of 62 (550 mg, 2.63 mmol) in in 4/1 dioxane/pyridine (10 mL)

at 4 ^ꢁC. The mixture was stirred at room temperature for 1 h, and

then diluted with water (20 mL) and ethyl acetate (20 mL). The

organic layer was separated, washed with 10% HCl (20 mL), satu-

rated aqueous NaHCO₃(20 mL), and brine (20 mL), dried over

Na₂SO₄and concentrated. The residue was puriﬁed by ﬂash chro-

matography on silica gel. Elution with 70/30 cyclohexane/ethyl

acetate gave 350 mg (46.4%) of 67 as a white wax: ¹H NMR (CDCl₃)

2.4 Hz), 7.40 (d, 1 H, J ¼ 2.4 Hz), 7.28 (dt, 1 H, J ¼ 9.1, 5.4 Hz), 7.05 (dt,

1H, J ¼ 9.1, 1.9 Hz), 7.00 (d, 1 H, J ¼ 8.4 Hz), 4.66e4.60 (m, 1H), 4.50

(dd, 1 H, J ¼ 11.6, 2.3 Hz), 4.38e4.27 (m, 2H), 4.21 (dd, 1 H, J ¼ 11.9,

7.0 Hz), 3.8 (s, 3H). ¹³C NMR (DMSO-d)

d 166.0, 161.6, 152.6 (dd,

J ¼ 240.5, 6.9 Hz), 148.4 (dd, J ¼ 247.3, 8.0 Hz), 147.6, 143.1 (dd,

J ¼ 10.8, 2.9 Hz), 142.8, 123.5, 118.56, 117.71, 117.69, 117.1 (dd,

J ¼ 24.0, 20.6 Hz), 116.5 (dd, J ¼ 9.1, 2.3 Hz), 111.5 (dd, J ¼ 22.4,

4.1 Hz), 71.8, 66.8, 65.2, 52.4. Anal. Calcd for C₁₈H₁₅F₂NO₆(379.31).

7-Aminocarbonyl-2-benzyloxymethyl-1,4-benzodioxane

(61). The carboxylic acid 58 (1.26 g, 4.19 mmol) was added to SOCl₂

(12 mL). The mixture was stirred at room temperature for 15 min

and then at 50 ^ꢁC for 2 h. After concentrating under vacuum, the

residue was dissolved into DCM (15 mL) and the resulting solution

was cooled to 4 ^ꢁC and added with 30% ammonia (7 mL). After

stirring at room temperature overnight, water (15 mL) and DCM

(15 mL) were added. The organic layer was separated, washed with

10% HCl (15 mL), saturated aqueous NaHCO₃(15 mL), and brine

(15 mL), dried over Na₂SO₄and concentrated. The residue was

d

7.17 (m, 2 H), 6.93 (d, 1 H, J ¼ 8.2 Hz), 4.38 (dd, 1 H, J ¼ 11.0, 1.9 Hz),

4.19 (m, 2 H), 3.90 (dq, 2 H, J ¼ 12.1, 4.1 Hz), 1.59 (br s, 1 H).

7-Cyano-2-mesyloxymethyl-1,4-benzodioxane (68). Mesyl

chloride (0.17 mL, 2.20 mmol) was slowly added to a solution of 67

(350 mg, 1.83 mmol) and TEA (0.34 mL, 2.38 mmol) in DCM (6 mL)

at 4 ^ꢁC. The mixture was stirred for 15 min at 4 ^ꢁC and for 30 min at

room temperature. After cooling to 4 ^ꢁC, water (10 mL) and DCM

(10 mL) were added. The organic phase was separated, washed with

10% HCl (10 mL), saturated aqueous NaHCO₃(10 mL), and brine

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

241

(10 mL), dried over Na₂SO₄, and concentrated to give 480 mg

(97.4%) of 68 as a colourless oil: ¹H NMR (CDCl₃)

7.22 (m, 2 H), 6.96

J ¼ 6.8 Hz), 3.09 (s, 3 H), 1.51 (m, 2 H), 1.40 (m, 2 H), 0.95 (t, 3 H,

J ¼ 7.1 Hz).

d

(d, 1 H, J ¼ 8.5 Hz), 4.44 (m, 4 H), 4.17 (dd, 1 H, J ¼ 11.3, 6.6 Hz), 3.14

3-(7-N-Butylaminocarbonyl-1,4-benzodioxan-2-yl)methoxy-

2,6-diﬂuoro-benzamide (23). Potassium carbonate (221 mg,

(s, 3 H).

3-(7-Cyano-1,4-benzodioxan-2-yl)methoxy-2,6-diﬂuoro-

benzamide (20). Potassium carbonate (296 mg, 2.14 mmol) was

added to a solution of 2,6-diﬂuoro-3-hydroxybenzamide (370 mg,

2.14 mmol) in DMF (5 mL). After stirring at room temperature for

15 min, a solution of 68 (480 mg, 1.78 mmol) in DMF (5 mL) was

added. The reaction mixture was stirred at 75 ^ꢁC for 6 h, concen-

trated under vacuum, and diluted with water (20 mL) and ethyl

acetate (20 mL). The aqueous phase was separated and extracted

with ethyl acetate (2 ꢀ 20 mL). The combined organic phases were

washed with 1 M NaOH (15 mL), and brine (15 mL), dried over

Na₂SO₄and concentrated. Crystallization of the solid residue from

1/1 toluene/ethyl acetate gave 175 mg (30.8%) of 20 as a white solid:

1.60 mmol) was added to

a

solution of 2,6-diﬂuoro-3-

hydroxybenzamide (254 mg, 1.47 mmol) in DMF (3 mL). After

stirring at room temperature for 15 min, a solution of 66 (460 mg,

1.34 mmol) in DMF (7 mL) was added. The reaction mixture was

stirred at 75 ^ꢁC for 6 h, concentrated under vacuum, and diluted

with water (20 mL) and ethyl acetate (20 mL). The aqueous phase

was separated and extracted with ethyl acetate (2 ꢀ 20 mL). The

combined organic phases were concentrated and the resulting grey

solid was crystallized from ethyl acetate to give 350 mg (63.6%) of

23 as a white solid: mp 173.5 ^ꢁC; ¹H NMR (DMSO-d₆)

d 8.24 (t, 1 H,

J ¼ 5.5 Hz), 8.11 (br s, 1 H), 7.84 (br s, 1 H), 7.33 (m, 3 H), 7.07 (dt, 1 H,

J ¼ 8.8, 1.9 Hz), 6.94 (d, 1 H, J ¼ 8.5 Hz), 4.61 (m, 1 H), 4.46 (dd, 1 H,

11.8, 2.5 Hz), 4.31 (m, 2 H), 4.17 (dd, 1 H, J ¼ 11.5, 7.2 Hz), 3.20 (m,

2H), 1.51 (m, 2 H), 1.35 (m, 2 H), 0.87 (t, 3H, J ¼ 7.2 Hz). ¹³C NMR

mp 158.2 ^ꢁC; ¹H NMR (CDCl₃)

d 8.10 (br s, 1 H), 7.82 (br s, 1 H), 7.40

(d, 1 H, J ¼ 1.9 Hz), 7.32 (dd, 1 H, J ¼ 8.4, 2.1 Hz), 7.26 (dt, 1 H, J ¼ 9.3,

5.1 Hz), 7.07 (dt, 1 H, J ¼ 9.3, 2.0 Hz), 7.1 (d, 1H, J ¼ 8.4 Hz), 4.67 (m,

1 H), 4.52 (dd,1 H, J ¼ 11.6, 2.5 Hz), 4.27 (m, 3 H). ¹³C NMR (DMSO-d)

(DMSO-d)

d

165.5, 161.7, 152.6 (dd, J ¼ 240.5, 6.9 Hz), 148.4 (dd,

J ¼ 247.3, 9.2 Hz), 145.6, 143.2 (dd, J ¼ 11.4, 3.4 Hz), 142.4, 128.6,

121.3, 117.1 (dd, J ¼ 25.8, 20.0 Hz), 117.1, 116.6, 116.4 (dd, J ¼ 9.2,

2.3 Hz), 111.5 (dd, J ¼ 22.9, 3.5 Hz), 71.8, 68.7, 65.1, 39.3, 31.7, 20.1,

14.1. Anal. Calcd for C₂₁H₂₂F₂N₂O₅(420.41).

d

161.9, 152.9 (dd, J ¼ 241.1, 7.0 Hz), 148.6 (dd, J ¼ 247.9, 8.3 Hz),

147.9, 143.7, 143.3 (d, J ¼ 11.1 Hz), 126.8, 121.7, 119.4, 119.0, 117.4 (dd,

J ¼ 24.7, 20.2 Hz), 116.6 (d, J ¼ 9.1 Hz), 111.7 (d, J ¼ 22.8 Hz), 104.4,

72.2, 68.8, 65.5. Anal. Calcd for C₁₇H₁₂F₂N₂O₄(346.29).

2-Benzyloxymethyl-7-N-butylaminocarbonyl-1,4-

4.2. Cells

benzodioxane (64). The carboxylic acid 58 (1.00 g, 3.32 mmol) was

added to SOCl₂(10 mL). The mixture was stirred at room temper-

ature for 15 min and then at 50 ^ꢁC for 2 h. After concentrating under

vacuum, the residue was dissolved into DCM (15 mL) and the

resulting solution was cooled to 4 ^ꢁC and added with a solution of

n-butylamine (2.0 mL, 20.23 mmol) in DCM (10 mL). After stirring

at room temperature overnight, water (15 mL) and DCM (15 mL)

were added. The organic layer was separated, washed with 10% HCl

(15 mL), saturated aqueous NaHCO₃(15 mL), and brine (15 mL),

dried over Na₂SO₄and concentrated. The oily residue was puriﬁed

by ﬂash chromatography on silica gel. Elution with 60/40 cyclo-

hexane/ethyl acetate gave 670 mg (56.8%) of 64 as a white wax: ¹H

Normal human lung ﬁbroblasts (MRC-5) were grown in Dul-

becco's modiﬁed Eagle's medium (DMEM) supplemented with 10%

heat-inactivated calf serum, 100 U/ml penicillin and 100 mg/ml

streptomycin in an incubator at 5% CO₂atmosphere and 37 ^ꢁC. The

Gram-positive S. aureus (S. aureus) and the Gram-negative E. coli

(E. coli) bacterial cells were grown in LuriaeBertani Broth (LB)

medium at 37 ^ꢁC under constant shaking at 300 rpm.

4.3. Antibacterial activity

The antibacterial activity of compounds 5e23 and DFNB was

tested by using a methicillin-resistant S. aureus strain (MRSA, ATCC

29213), and an extended-spectrum beta-lactamase-positive (ESBL)

E. coli clinical isolate. All of the compounds were dissolved at the

ﬁnal concentration of 20 mg/ml in dimethyl sulphoxide (DMSO)

and serially diluted in LB. After incubation at 37 ^ꢁC for 16 h in

aerobic culture tubes, cell concentration was determined by optical

density measurement at 600 nm (OD₆₀₀) in a SmartSpec™ 3000

spectrophotometer (Bio-Rad, Oceanside, CA, USA). Fresh cell cul-

tures were used at 10³cells/ml in a ﬁnal volume of 3 ml. Each

bacterial sample was grown with different compound concentra-

NMR (CDCl₃)

d

7.31 (m, 7 H), 6.88 (d, 1 H, J ¼ 8.5 Hz), 5.96 (br s, 1 H),

4.60 (s, 2 H), 4.35 (m, 2 H), 4.12 (dd, 1 H, J ¼ 11.8, 7.7 Hz), 3.70 (m,

2 H), 3.42 (t, 2 H, J ¼ 6.9 Hz), 1.57 (m, 2 H), 1.41 (m, 2 H), 0.95 (t, 3 H,

J ¼ 7.2 Hz).

7-N-Butylaminocarbonyl-2-hydroxymethyl-1,4-

benzodioxane (65). A solution of 64 (670 mg, 1.88 mmol) in

methanol (10 mL) was added with 10% Pd/C (70 mg) and vigorously

shaken under hydrogen at room temperature for 24 h. The catalyst

was removed by ﬁltration and the ﬁltrate concentrated to give

460 mg (92.2%) of 65 as a white wax: ¹H NMR (CDCl₃)

d 7.34 (d, 1 H,

J ¼ 2.2 Hz), 7.24 (dd,1 H, J ¼ 8.5, 2.2 Hz), 6.89 (d,1 H, J ¼ 8.5 Hz), 6.06

(br s, 1 H), 4.34 (dd, 1 H, J ¼ 11.3, 2.2 Hz), 4.25 (m, 1 H), 4.13 (dd, 1 H,

J ¼ 11.3, 7.4 Hz), 3.70 (m, 2 H), 3.41 (q, 2 H, J ¼ 6.9 Hz),1.89 (br s,1 H),

1.56 (m, 2 H), 1.40 (m, 2 H), 0.95 (t, 3 H, J ¼ 7.2 Hz).

tions, that ranged from 100 to 0.1

with intermediate concentrations thereafter for 11,12,14,15 and 21,

that were active up to 1 g/ml but not at 0.1 g/ml. After overnight

mg/ml for the ﬁrst screening and

m

incubation at 37 ^ꢁC, an aliquot of each sample was harvested under

sterile conditions and the OD₆₀₀was measured to determine the

minimal inhibitory concentration (MIC), i.e. the lowest compound

dose at which bacterial growth is inhibited. To also determine the

minimal bactericidal concentration (MBC), i.e. the minimal dose at

which cell growth is inhibited after removing the compound, the

bacteria were then washed three times with LB, centrifuged at

900 ꢀ g for 10 min at 4 ^ꢁC, and the pellet resuspended in fresh LB.

After overnight incubation at 37 ^ꢁC, the absence of growth was

conﬁrmed by OD measurement.

7-N-Butylaminocarbonyl-2-mesyloxymethyl-1,4-

benzodioxane (66). Mesyl chloride (0.16 mL, 2.08 mmol) was

slowly added to a solution of 65 (460 mg, 1.73 mmol) and TEA

(0.32 mL, 2.25 mmol) in DCM (10 mL) at 4 ^ꢁC. The mixture was

stirred for 15 min at 4 ^ꢁC and for 30 min at room temperature. After

cooling to 4 ^ꢁC, water (10 mL) and DCM (10 mL) were added. The

organic phase was separated, washed with 10% HCl (10 mL), satu-

rated aqueous NaHCO₃(10 mL), and brine (10 mL), dried over

Na₂SO₄, and concentrated. The oily residue (680 mg) was puriﬁed

by ﬂash chromatography on silica gel. Elution with 1/1 cyclo-

hexane/ethyl acetate gave 460 mg (77.4%) of 66 as a colourless wax:

The antimicrobial activity of compounds 3, (S)-4, 10 and 12 was

determined against a panel of 30 strains of clinical isolates

including 10 methicillin-sensitive S. aureus (MSSA) and 10

methicillin-resistant S. aureus (MRSA), 10 vancomycin-sensitive E.

faecalis (VSE) and 10 vancomycin-resistant E. faecalis (VRE). The

¹H NMR (CDCl₃)

d

7.38 (d, 1 H, J ¼ 2.2 Hz), 7.29 (dd, 1 H, J ¼ 8.5,

2.2 Hz), 6.91 (d, 1 H, J ¼ 8.5 Hz), 6.0 (br s, 1 H), 4.49 (m, 3 H), 4.35

(dd, 1 H, J ¼ 11.3, 2.3 Hz), 4.14 (dd, 1 H, J ¼ 11.3, 7.4 Hz), 3.45 (q, 2 H),

242

V. Straniero et al. / European Journal of Medicinal Chemistry 120 (2016) 227e243

minimal inhibitory concentration (MIC) for liquid growth inhibition

assay was determined by standard procedures (CLSI) using serial

dilutions of the compounds in a 96 well ﬂat-bottom Microtiter®

plate. The compounds were dissolved in appropriate buffer and

4.6. Thiazolyl blue tetrazolium bromide cytotoxicity assay

Compounds 11, 12, 14, 15 and 21, showing antibacterial activity

at a concentration lower than 1

mg/ml, were serially diluted in

then diluted in LB medium to reach a ﬁnal concentration of 200

m

g/

DMEM and tested on MRC-5 cells by the thiazolyl blue tetrazolium

bromide (MTT) cytotoxicity assay (Sigma, St Louis, MO, USA). Cells

(10⁴cell/well) were tested in a 96-well plate using serially two-

mL. Logarithmic phase bacterial cultures were suspended in saline

solution to a ﬁnal concentration of 1-2 ꢀ 10⁵CFU/mL. The assay

mixture contained 100

mL diluted compounds suspension in LB

fold-diluted concentrations of the compound in 100

medium. After a 24-h incubation, the compound was removed and

the cells were overlaid with 1 mg/ml MTT in 100 l serum-free

ml DMEM

medium and 100

m

L serial diluted bacterial suspension. The ﬁnal

g/mL.

concentration of the compounds ranged from 0.097 to 100

m

After 24 h of incubation at 37 ^ꢁC, the MIC was deﬁned as the lowest

concentration of the compound that totally inhibited the growth.

To determine the minimum bactericidal concentration (MBC), an

DMEM for 3 h at 37 ^ꢁC. The MTT solution was then replaced with

DMSO for 10 min, and the absorbance was measured at 570 nm. The

percentage of cytotoxicity was calculated by the formula 100 e

(sample OD/untreated cells OD) x 100. The compound concentra-

tion reducing cell viability by 50 or 90% was deﬁned as the TD₅₀or

TD₉₀toxic dose. The therapeutic index (TI) was also determined and

deﬁned as the ratio between TD₉₀and the minimal bactericidal

concentration (MBC) values.

aliquot (100

m

L) of the wells with no visible microbial growth was

ꢁ

plated onto Trypticase Soy agar 5% sheep blood plate (bioMerieux)

and incubated at 37 ^ꢁC for 18 h. MBC was deﬁned as the lowest

concentration of the compound at which more than 99.9% of the

cells were killed compared with a non-treated control. All tests

were performed in triplicate and for each series of experiments,

both positive (no compounds) and negative (no bacteria) controls

were included.

Appendix A. Supplementary data

Activity against M. smegmatis and M. tuberculosis was carried out

in 7H9 liquid media supplemented with ADC (Difco) and Tween 80

(0.05%) and MICs were determined by the microplate Alamar Blue

assay [14]. M. smegmatis and M. tuberculosis expressing the green

ﬂuorescent protein (GFP) [15] were grown at subMIC concentration

on glass coverslips, ﬁxed with a phosphate buffered 4% para-

formaldehyde solution and then analysed at the ﬂuorescence mi-

croscope (100ꢀ magniﬁcation).

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2016.03.068.

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Article Doi

3-(Benzodioxan-2-ylmethoxy)-2,6-difluorobenzamides bearing hydrophobic substituents at the 7-position of the benzodioxane nucleus potently inhibit methicillin-resistant Sa and Mtb cell division

DOI: 10.1016/j.ejmech.2016.03.068

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