Synthesis and anti-bacterial activity of a library of 1,2-benzisothiazol-3(2H)-one (BIT) derivatives amenable of crosslinking to polysaccharides

Article abstract of DOI:10.1016/j.tet.2017.02.025

1,2-Benzisothiazol-3(2H)-one (BIT) is one of the most common chemical biocides in industrial products, with a heterocyclic structure and a wide range of antimicrobial activity. A library of BIT derivatives was synthesized and characterized, from which 18 compounds were selected, tested for anti-bacterial activity relative to the parent molecule and amenable of coupling to plant polysaccharides in general and to galactomannans (GM) in particular, widely used as rheology modifiers, but with limited “biostability”. Four sites on the BIT core were targeted: the nitrogen and the oxygen atoms on the heterocyclic ring, the C5 and the C6 positions on the aromatic ring, where functional groups were introduced. The ultimate aim of this work is to establish whether by covalently linking a biocide to GM polymers, their “biostability” can be improved.

Full text of DOI:10.1016/j.tet.2017.02.025

Accepted Manuscript
Synthesis and anti-bacterial activity of a library of 1,2-benzisothiazol-3(2H)-one (BIT)
derivatives amenable of crosslinking to polysaccharides
Fiorenza Viani, Bianca Rossi, Walter Panzeri, Luca Merlini, Alessandra M. Martorana,
Alessandra Polissi, Yves M. Galante
PII:
S0040-4020(17)30157-6
10.1016/j.tet.2017.02.025
TET 28466
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 22 November 2016
Revised Date: 3 February 2017
Accepted Date: 13 February 2017
Please cite this article as: Viani F, Rossi B, Panzeri W, Merlini L, Martorana AM, Polissi A, Galante
YM, Synthesis and anti-bacterial activity of a library of 1,2-benzisothiazol-3(2H)-one (BIT) derivatives
amenable of crosslinking to polysaccharides, Tetrahedron (2017), doi: 10.1016/j.tet.2017.02.025.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
-
CH COOH
2
-
CH(CH )COOH
3
-
CH CH=CH
2
2
-
CH C
CH
2
O
-
CH CH OH
-CH CH SH
2 2
2
2
-
CH CH COOH
2 2
-
CH COOH
-CH(CH )COOH
2
3
NH
-
CH CH CH NH
2 2 2
2
S
-CH CH NH
-CH CH CH OH
2
2
2
2
2
2
5
-OCH3
5
-OH
-
CH CH=CH2 -CH C CH
2
2
6-OCH3
6-OH
N-, O-, and C- functionalizations of BIT for crosslinking to polysaccarides

ACCEPTED MANUSCRIPT
1
2
3
4
“Synthesis and anti-bacterial activity
of a library of 1,2-benzisothiazol-3(2H)-one (BIT) derivatives amenable
of crosslinking to polysaccharides”
5
6
Fiorenza Viani*, Bianca Rossi*, Walter Panzeri^, Luca Merlini*, Alessandra M. Martorana°,
Alessandra Polissi° and Yves M. Galante*
7
8
9
0
1
2
3
4
5
6
7
8
* Istituto di Chimica del Riconoscimento Molecolare, C.N.R.,
Via Mario Bianco 9, 20131 Milano, Italy
1
1
1
1
1
1
1
1
1
^ Istituto di Chimica del Riconoscimento Molecolare, C.N.R.,
Via Mancinelli 7, 20131 Milano, Italy
° Department of Biotechnology and Biosciences
University of Milano-Bicocca (UNIMIB)
Piazza della Scienza 2, 20126, Milan, Italy
19
20
21
22
23
24
25
26
27
28
FV: fiorenza.viani@polimi.it
BR: bianca.rossi@polimi.it
WP walter.panzeri@polimi.it
LM: luca.merlini@icrm.cnr.it
AM: alessandra.martorana@unimib.it
AP: alessandra.polissi@unimib.it
Correspondence should be addressed to:
2
3
9
0
Y.M. Galante
email: yves.galante@icrm.cnr.it; tel. +39028500032, +393358477648; fax +390228901239
31
32
33
34
Running title: Synthesis of BIT biocide derivatives.
1

ACCEPTED MANUSCRIPT
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
Abstract
1,2-Benzisothiazol-3(2H)-one (BIT) is one of the most common chemical biocides in
industrial products, with a heterocyclic structure and a wide range of antimicrobial activity.
A library of BIT derivatives was synthesized and characterized, from which 18 compounds
were selected, tested for anti-bacterial activity relative to the parent molecule and amenable
of coupling to plant polysaccharides in general and to galactomannans (GM) in particular,
widely used as rheology modifiers, but with limited “biostability”. Four sites on the BIT
core were targeted: the nitrogen and the oxygen atoms on the heterocyclic ring, the C5 and
the C6 positions on the aromatic ring, where functional groups were introduced. The
ultimate aim of this work is to establish whether by covalently linking a biocide to GM
polymers, their “biostability” can be improved.
Keywords: biocides; BIT derivatives; galactomannans; chemical coupling; biostability.
Abbreviations: BIT, 1,2-benzisothiazol-3(2H)-one; GG, guar gum; GM, galactomannans;
MIC, minimal inhibitory concentration; BZM, benzamide.
2

ACCEPTED MANUSCRIPT
54
1.
Introduction
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
Cellulose, starch, chitosan, galactomannans, alginates, pectins, but also exudative gums and
1
their modified forms are polysaccharides with several applications as emulsion stabilizers;
thickeners; rheology modifiers; agents for coating, binding, suspending, conditioning,
leveling; but are also used in packaging, drilling fluids; for water-retention, in a variety of
food, feed, cosmetics, pharmaceuticals and industrial fields. In particular, galactomannans
(GM) are high molecular weight polysaccharides found in the seed endosperms of some
Leguminosae (belonging to the family Fabaceae) where they serve as a reserve source for
2
carbon and energy upon germination. They have a branched polymeric structure composed
of a backbone of mannose units linked by β-1,4 glycosidic bonds with side units of galactose
3
bound to mannose by α-1,6 glycosidic bonds The species-dependent ratio of Gal : Man
makes them, to various extents, soluble in water at different temperatures,
chemically/biochemically quite reactive and flexible in application, thus commercially
4
extremely valuable. The most widely used GM is obtained from the seed endosperms of a
shrub (Cyamopsis tetragonolobus) and is commonly referred to as guar gum (GG).
However, due to microbial contaminations originating from raw materials, pipes, mixers,
industrial waters and other sources, all formulations containing these compounds are
extremely susceptible to spoilage and degradation, with complete deterioration of products,
5
which, on a large scale, poses serious issues of economic losses and of final disposal. Thus,
polysaccharides and their derivatives in general, and mostly GM-derived compounds, have a
6
limited “biostability”. Therefore, it is common practice to supplement them with various
amounts of chemical biocides. These are small molecular weight molecules, with different
7
degrees of toxicity and sensitization to humans. In their free form, biocides can be
hazardous to workers, users and the environment, and not always effective once massive
microbial growth is at an advanced stage or if they are added at the wrong step in a
production process. Therefore, it is of paramount importance to find alternatives and
sustainable solutions to protect natural polymers and/or their modified forms, as well as the
products to which they are added, from depolymerization and biodegradation throughout
their industrial and commercial lifespan.
There is a large body of literature in the medical field that supports the concept of increased
8
efficacy of antimicrobial agents linked to a polymeric support. Only recently, the binding of
3

ACCEPTED MANUSCRIPT
86
87
88
89
90
91
92
93
94
95
96
97
98
99
phenolics or anti microbial peptides to biomaterials has been described, with the aim of
stabilizing the biocides and creating new “functional” biomaterials for packaging and
9
coating, which is actually a promising, challenging and fast growing field.
The aim of our work was to improve the biostability of GM and to eliminate, or at least
drastically reduce, the amount of free biocides in products and the environment, by
establishing a stable chemical bond between a biocide and the polymers, with minimal
alterations of the solubility and rheological properties of the latter. As an initial model
system, we have focused on 1,2-benzisothiazol-3(2H)-one (BIT), one of the most commonly
used chemical biocide added to industrial products, that has a heterocyclic structure (i.e., it is
1
0
11
a cyclic sulfenamide) and a wide range of antimicrobial activity, while its 1,1-dioxide
1
2
derivative (i.e., saccharin) is a sweetener with a safe toxicological profile. BIT has been
reported to react with thiol-containing proteins on target microorganisms and is particularly
7
,13
potent against actively metabolizing cells. It is largely used in food packaging, as well as
in industrial and consumer products, such as: adhesives, laundry and dish detergents,
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
00 cleaning and disinfectants, air freshener, personal care products and sunscreens, paintings
14
01 and industrial lubricants.
Current regulations impose strict limitations on its
1
5
16
02 concentration due to its (relative) human toxicity.
03 However, the BIT molecule needs structural modifications in order to be bound to polymers.
04 Therefore, we have synthesized and characterized a library of BIT derivatives, from which
05 we selected 18 compounds amenable to coupling, that were tested for anti-bacterial activity,
06 relative to the parent molecule. Four sites on the molecule were targeted: the nitrogen and
07 the oxygen atoms on the heterocyclic ring, the C5 and the C6 positions on the aromatic ring.
1
7
08 A few examples of N-functionalized benzisothiazolones showing biological activity have
09 been described, although it can be anticipated that a chemical perturbation on the cyclic
10 sulfenamide moiety might affect the anti-microbial activity of the molecule. On the other
11 hand, reactive groups introduced on the aromatic ring should be less disruptive, but their
1
8
12 biocidal activity has nonetheless to be checked. Functional groups were introduced on the
13 nitrogen atom of the heterocyclic ring (e.g., hydroxyl, amino, carboxy, thio) able to form a
14 bond with a spacer linked to the GM structure, or allyl and propargyl functions for
15 polymeric radical and click reactions, respectively. In a second set of experiments, a
16 hydroxyl or methoxy group on the C5 and C6 carbon atoms of the aromatic ring were
17 introduced. The minimal inhibitory concentrations (MIC) of all these BIT derivatives was
4

ACCEPTED MANUSCRIPT
1
9
1
1
1
1
18 established against a panel of Gram-positive and -negative bacteria, and a challenge test
19 with a bacterial consortium was performed on guar gum solutions supplemented with BIT or
20 hydroxyethyl-BIT. Finally, a tentative structure/activity relationship (SAR) is proposed.
21
5

ACCEPTED MANUSCRIPT
1
1
1
1
1
22 2.
Results and Discussion
23 As depicted in Figure 1 and Figure 2, a library of twenty-eight compounds was created and
24 the MIC values of eighteen BIT derivatives were established against a panel of Gram-
25 positive and -negative bacteria.
26
R' = H, R" = H
R = CH CH OH
(6a)
(6b)
2
2
O
CH CH COOCH
3
2
2
CH CH SX
(6c) X = SCH CH N
2 2
2
2
CH COOCH
(6d)
(6e)
S
2
3
CH COOC(CH )
2
3 3
CH(CH )COOCH (6f)
3
3
O
CH CH=CH
(6g)
(6h)
(6i)
2
2
R'
CH C CH
2
N R CH CH NHBoc
2
2
S
CH CH CH NHBoc (6j)
2 2 2
R"
CH CH CH OH
(6k)
(8)
2
2
2
H
CH CH COOH
(15b)
(15c)
(15d)
(15f)
(15i)
(15j)
2
2
CH CH SH
2
2
CH COOH
2
CH(CH )COOH
3
CH CH NH
2
2
2
CH CH CH NH
2
2
2
2
R = H, R' = OCH R" = H (18)
3
,
R = H, R' = H, R" = OCH (27)
3
R = H, R' = OH, R" = H
R = H, R' = H, R" = OH
(19)
(28)
1
27
28
1
1
1
29 Figure 1. General formula of compounds 6a-k, 8, 15b-j, 18, 19, 27, and 28.
30
1
1,20c,e,d
1
1
1
1
1
1
31 Of the 18 compounds tested, ten are N-derivatives: hydroxyethyl (6a),
propanoic acid
1
7h
17a,21a,b
16a,20a
32 (15b), mercaptoethyl (15c), acetic acid (15d),
(methyl)acetic acid (15f),
allyl
11,17b,20c,20e,22
23
33 (6g),
propargyl (6h), aminoethyl (15i), aminopropyl (15j) and hydroxypropyl
11
24
34 (6k) (Figure 1). Four are O-derivatives: acetic acid (16d), (methyl)acetic acid (16f), allyl
2
3
35 (10g), and propargyl (10h) (see Figure 2).
36
6

ACCEPTED MANUSCRIPT
1
37
1
1
38
39 Figure 2. General formula of compounds 10d-h and 16d, f.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
40
41 Four are C-derivatives: 5-methoxy (18), 6-methoxy (27), 5-hydroxy (19) and 6-hydroxy (28)
42 (see Figure 1).
43 The synthesis of the benzisothiazolone core is well described in the literature and several
2
5
44 different methods have been proposed. As summarized in Table 1, we have focused on the
2
0e
45 reaction of dimethyl-2,2'-dithiobenzoate (4) and bromine with nucleophilic amines,
46 (Scheme 2, method A, entries 1, 3 and 5), on the N-alkylation of the commercially available
2
2
47 1,2-benzisothiazol-3-one (BIT) (8) (Scheme 3, method B, entries 7 – 16), and on the
2
0c
48 copper mediated sulfur-nitrogen coupling reaction (Scheme 4, method C, entries 2, 4, 6,
49 17 – 19).
50
1
51 Table 1. Methods, procedures, chemical yields and molar ratios (as determined by H NMR
52 spectra) for the synthesis of compounds 6, 19 and 28.
53
6
/7
6/10
Molar
Ratio
6/12
Molar
Ratio
Method Yield
Entry Compd
1
R
R’
R”
Molar
Ratio
(
Procd)
(%)
a
A
31
2.6
-
-
6
a
CH
2
CH
2
OH
H
H
b
2
3
C
45
-
-
-
12a n.i.*
14a n.i.
-
c
4
3
A
24
1.5
6
b
CH
2
CH
2
COOCH
3
H
H
H
H
4
5
C
A
49
32
-
-
-
12b n.i.
7c n.i.
-
6
c
CH CH S)
2 2 2
6
7
C
10
27
-
-
12c n.i.
d
e
B (1)
5.4
-
7

ACCEPTED MANUSCRIPT
f
8
9
6d
6e
CH
2
COOCH
3
H
H
H
H
H
H
B (2)
45
45
63
-
-
-
2.5
10d n.i.
2.2
-
-
-
g
B(3)
t
1
0
CH
2
COO Bu
B (2)
11
B (1)
4
-
0.1
-
6
f
CH(CH )COOCH
3 3
1
2
3
B (2)
B (2)
27
57
-
-
0.4
1.4
-
-
1
6
g
CH
2
CH=CH
2
H
H
H
H
h
1
1
4
5
B (4)
64
53
-
-
2.1
1.3
-
-
B (2)
6
h
CH CΞCH
2
1
1
6
7
B (4)
A
60
98
-
1.8
-
-
-
6i n.i.
(
7i)
6
i
CH
2
CH
2
NHBoc
H
H
H
H
1
1
8
9
C
A
45
74
-
-
-
2.3
-
6j n.i.
(
7j)
6
j
CH
2
CH
2
CH NHBoc
2
2
0
C
48
-
-
2.6
c
3
5
14j n.i.
c
2
2
2
1
2
3
6k
19
28
CH
2
CH
2
CH
2
OH
H
H
H
OMe
H
C
-
45
-
-
-
-
-
-
2.4
H
H
40
20
-
OMe
-
-
1
1
54
a
d
e
55 Method A: Br , CCl , Et N, reflux. Method B: NaH, 80 °C. Procedure 1: CF CH OH and
2
4
3
3
2
f
g
h
1
1
1
56 DMF. Procedure 2: DMF. Procedure 3: CH CN, CF CH OH. Procedure 4: K CO , DMF;
3 3 2 2 3
b
c
57
Method C: CuI, 1,10-phenanthroline, S , K CO , DMF, 110 °C. Obtained from 2-iodo
8 2 3
58 derivative. *not isolated.
59
1
1
60 The N-hydroxyethyl derivatives 6a, 6b and 6c were prepared following method A (Schemes
1
1
61 1 and 2).
62
8

ACCEPTED MANUSCRIPT
1
63
64
1
Scheme 1. Synthesis of compound 4 following Routes 1 and 2.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
65 The starting compound, dimethyl 2,2'-disulfanediyldibenzoate (4) was synthesized in good
66 yields following two alternative and convergent synthetic routes, Route 1 and Route 2, as
67 depicted in Scheme 1.
68 As shown in Scheme 2, the reaction with bromine gave the intermediate 2-
69 methoxycarbonylbenzenesulfenyl bromide (not isolated, compound A), that was added to the
70 solutions of the amines 5 to give compounds 6. From ethanolamine 5a and 3-
71 aminopropanoate 5b, the 2-N-substituted benzisoxazolones 6a and 6b were obtained in
72 rather low yield (31% and 24%, respectively), along with the sulfenamides 7a and 7b (12%
73 and 16% yields, respectively) that could be converted to 6a and 6b. The ethanolamine
74 derivative 7a underwent slow ring closure, while for the aminopropanoate derivative 7b,
75 KOH or Triton-B had to be used giving 6b in 33% yield. From 2,2'-
76 disulfanediyldiethanamine (5c), only the dimer 6c was detected and isolated in rather low
77 yield (32%). From aminoethyl- and aminopropylcarbamates 5i and 5j, only the
78 corresponding sulfenamides 7i and 7j were formed in 98% and 84% yield, respectively and
79 were quantitatively converted into 6i and 6j by using KOH. The chemical yields and the 6/7
80 ratios obtained following method A are reported in Table 1.
81
9

ACCEPTED MANUSCRIPT
1
82
1
1
1
1
83 Scheme 2. Synthesis of compounds 6 (second step) following method A (5a, R =
84 CH CH OH; 5b, R = CH CH COOCH ; 5c, R = CH CH SSCH CH NH ; 5i, R =
2
2
2
2
3
2
2
2
2
2
85 CH CH NHBoc; 5j, R = CH CH CH NHBoc).
2
2
2
2
2
86
2
1b
1
1
1
1
1
1
1
1
87 The N-derivatives 6d, 6e, 6f, 6g and 6h were prepared following method B (Table 1,
88 Scheme 3). The sodium salt of the commercially available BIT (8) was employed as the
89 nucleophilic species acting on electrophiles 9 displacing bromide ion as the leaving group.
90 As shown in Scheme 3, the starting compound 8 is an ambident nucleophile (N- nucleophile
1
2a,26
91 A and/or O-nucleophile B)
and, in order to find the best conditions to enhance the N-
92 alkylated products (6) vs the O-alkylated ones (10), three different procedures were
93 explored.
94
CF CH OH/DMF 4:1
3
2
NaH
1
5 min, 0 °C + 1 h, 80 °C
O
O
O
R
Procedure 1 (d, f)
RBr (9)
N
H
N
R +
N
DMF
NaH
S
S
S
(
8)
(6)
(10)
1
5 min, 0 °C + 1 h, 80 °C
Procedure 2 (d, e, f, g, h)
CH CN
NaH
5 min, 0 °C
3
CF CH OH
3
2
1
h, 80 °C
1
O
O
M
Procedure 3 (d)
Procedure 3
RBr (9)
(
6) + (10)
N M
S
(
8)
DMF
N
DMF
K CO
5 min, 0 °C
S
2
3
4
h, 80 °C
A
M = Na or K
B
1
Procedure 4 (g, h)
Procedure 4
1
95
96
1
1
1
1
2
97
98 Scheme 3. Synthesis of compounds 6 and 10 following method B in four different
99 procedures. (d, R = CH COOCH ; e, R = CH COOC(CH ) ; f, R = CH(CH )COOCH ; g, R
00 = CH CH=CH ; h, R = CH C≡CH).
2
3
2
3 3
3
3
2
2
2
1
0

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
01 In procedure 1 (Table 1, entries 7 and 11), a 4 : 1 CF CH OH and DMF mixture was used as
3
2
02 solvent; in procedure 2 (Table 1, entries 8, 10, and 12), only DMF was employed and in
03 procedure 3 (Table 1, entry 9), CH CN was the solvent of choice for the sodium salt
3
04 formation, while the nucleophilic substitution was performed in CF CH OH alone. For the
3
2
05 synthesis of 6g and 6h, two different procedures were followed: procedure 2 (Table 1,
06 entries 13 and 15), and procedure 4 (K CO in DMF, Table 1, entries 14 and 16). The
2
3
07 chemical yields and the 6/10 ratios obtained following the different procedures are reported
08 in Table 1.
09 As shown in Scheme 4, the compounds 6a, 6b and 6c were prepared also following method
10 C (Table 1, entries 2, 4, and 6), as well as 6i (Table 1, entry 17), 6j (Table 1, entry 18) and
11 6k (Table 1, entry 19). The first step involved the synthesis of a library of 2-bromo (12) or 2-
12 iodo (14) benzamides.
13
2
14
2
2
2
2
2
15
16 Scheme 4. Synthesis of compounds 6 following method C (a, R = CH CH OH; b, R =
17 CH CH COOCH ; c, R = CH CH S CH CH NH ; i, R = CH CH NHBoc; j, R =
18 CH CH CH NHBoc; k, R = CH CH CH OH).
19
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
20 The commercially available 2-bromo (11) and/or 2-iodo benzoyl chlorides (13) were reacted
21 with the amines 5 and the intermediates 12 and/or 14 were isolated. The chemical yields are
22 reported in Table 2. In the second step, the benzamides (BZM) 12 and/or 14 were treated in a
23 3 to 1 molar ratio with preformed Cu/1,10-phenanthroline complex, sulfur and K CO to
2
3
24 promote the copper-mediated S-N linkage giving compounds 6a-c and 6i-k.
25
11

ACCEPTED MANUSCRIPT
2
26
27
2
Table 2. Chemical yields in the synthesis of the BZM 12 and 14.
Entry
BZM
X
R
Yield (%)
a
1
2
3
4
5
6
7
8
9
12a
12b
12c
12i
Br
Br
Br
Br
Br
I
CH CH OH
94
2
2
a
CH CH COOCH
3
98
2
2
a
CH CH S)
2
58
2
2
a
CH CH NHBoc
94
2
2
a
12j
CH CH CH NHBoc
98
2
2
2
b
14a
14b
14c
CH CH OH
95
2
2
b
I
CH CH COOCH
3
96
2
2
b
I
CH CH S)
2
60
2
2
b
14j
I
I
CH CH CH NHBoc
85
2
2
2
b
10
14k
CH CH CH OH
86
2
2
2
a
b
2
28
29
From 11; from 13
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
30 The compounds 6a-c were obtained in rather low yields (from 10 to 49%, see Table 1,
31 entries 2, 4 and 6) and, in case of compounds 6i-k (Table 1, entries 18, 20 and 21), high
32 amounts of the starting compounds 12i-k were recovered unreacted (see Experimental). The
33 mixtures of the starting compounds 12i-k and the corresponding products 6i-k could hardly
34 be separated by flash chromatography. In case of 6i and 6k, fractional crystallization could
35 resolve the 6/12 mixture, but in case of 6j, only analytical samples were obtained. Because
36 of this drawback and even if in two steps, the A procedure to obtain the aminopropyl
37 derivative 6j is preferable. Starting from 14j, the compound 6j was isolated in pure form but
38 in lower yield (35%, see Table 1, entry 20). BZM 12j was also treated with the Cu/1,10-
39 phenanthroline complex in a 20 : 1 molar ratio, obtaining 6j in 45% yield but in a low molar
40 ratio over the starting compound (see Experimental). The chemical yields of compounds 6
41 are reported in Table 1.
42 The final step was the removal of the protecting groups. As shown in Scheme 5, the
43 compounds 6b, 6d and 6f were submitted to hydrolysis of the methyl esters by LiOH in 1,4-
27
44 dioxane to give, respectively, 15b, 15d and 15f. Compound 6e was submitted to a 1 : 4
1
2

ACCEPTED MANUSCRIPT
2
8
2
2
2
2
2
45 mixture of trifluoroacetic acid and CH Cl at 0 °C affording 15d in 93% yield. The same
2 2
2
9
46 procedure was performed on compounds 6i and 6j to remove the N-Boc group, obtaining,
47 respectively, 15i and 15j as the corresponding trifluoroacetic salts. Compound 15c was
3
0
48 obtained from 6c by reduction of the disulfide moiety with tributylphosphine.
49
LiOH
1,4-dioxane
rt
b, d, f
O
O
(
Bu) P
3
N R
N R'
DMK/CH OH 1:1
3
S
S
(
6)
rt
c
(15)
TFAA/CH Cl 1:4
2
2
0
°C
e, i, j
2
50
51 Scheme 5. Removal of protecting groups from compounds 6 (b, R = CH CH COOCH ; c, R
2
2
2
2
2
2
2
2
3
52 = CH CH S CH CH N(CO)PhS; d, R = CH COOCH ; e, R = CH COOC(CH ) ; f, R =
2
2
2
2
2
2
3
2
3 3
53 CH(CH )COOCH ; i, R = CH CH NHBoc; j, R = CH CH CH NHBoc) to give compounds
54 15 (b, R’ = CH CH COOH; c, R’ = CH CH SH; d, R’ = CH COOH; f, R’ =
55 CH(CH )COOH; i, R’ = CH CH NH .CF COOH; j, R’ = CH CH CH NH .CF COOH).
3
3
2
2
2
2
2
2
2
2
2
2
3
2
2
2
3
2
2
2
2
3
56
2
2
2
2
57 The O-substituted compounds 10d and 10f (Scheme 6) underwent the same reactions (LiOH
58 in 1,4-dioxane) described above, giving 16d and 16f, while the O-substituted derivative 10e
59 was treated with TFAA in CH Cl yielding 16d.
2
2
60
2
2
61
62 Scheme 6. Removal of protecting groups from compounds 10 (d, R = H; f, R = CH ) to give
3
2
2
2
2
63 compounds 16.
64
65 The chemical yields and the methods followed are shown in Table 3.
66
1
3

ACCEPTED MANUSCRIPT
2
67
Table 3. Synthetic methods and chemical yields in protecting group removals.
Yield
Entry
1
Compd.
15b
R
R’
R”
Method
(
%)
LiOH
CH
CH
COOH
H
H
75
2
2
1
,4-dioxane
2
3
15c
15d
CH
2
CH
2
SH
H
H
H
H
(Bu)
3
P
95
67
LiOH
,4-dioxane
LiOH
CH
COOH
2
1
1
4
5
6
7
8
9
15f
15i
CH(CH
3
)COOH
H
H
H
H
H
H
H
H
H
H
H
H
56
86
60
80
,4-dioxane
TFAA
CH CH NH
2 2
2
CH Cl2
2
TFAA
15j
16d
16f
16d
CH
2
CH
2
CH
2
NH
2
CH Cl2
2
LiOH
,4-dioxane
LiOH
CH COOH
2
1
1
CH(CH
)COOH
55
3
,4-dioxane
TFAA
a
CH
COOH
90
2
CH Cl2
2
b
1
0
19
28
H
H
OH
H
H
_
_
76
20
c
11
OH
a
b
c
2
68
From 10e; from 18; from 20.
2
2
69
70
2
2
2
2
2
2
2
71 The syntheses of the derivatives with a functionalized aromatic ring, 19 and 28, followed
72 two different synthetic routes. The 5-O-hydroxy compound 19 was prepared in a two-step
73 sequence (Scheme 7): the 5-O-methoxy precursor 18 was synthesized in one step from the
74 commercially available α-bromo amide 17, using CuI as the precatalyst, 1,10-
75 phenanthroline as the ligand, DABCO as the base, Bu NBr as the additive, and H O as the
4
2
31
76 solvent at 140 °C.
77
1
4

ACCEPTED MANUSCRIPT
2
78
2
2
79 Scheme 7. Two-step synthesis of compound 19.
80
2
2
2
2
2
2
2
81 The 6-hydroxy derivative 28 required an eight-step synthetic sequence (Scheme 8). Starting
82 from commercially available 4-methoxy salicylic acid 20 and according to known
3
2
33
83 procedures, the intermediates 21, 22, 23 and 24, were isolated and used for the next steps.
32a
84 The last four steps, described in the literature for the synthesis of similar compounds, gave
85 27 in 30% overall yield from 20. The conversions of 18 into 19 and of 27 into 28 (Schemes
3
4
86 7 and 8) were carried out using boron tribromide in dichloromethane.
87
SOCl2
MeOH
(CH ) NCSCl
H CO
OH
H3CO
OH
3 2
H3CO
O
N
3
DBU
3
h, rt
3 d, rt
S
COOH
COOCH3
COOCH3
(
22)
(
20)
(21)
2
10 °C
O
I2
i
NaOH 4N
MeOH
H CO
S
2
3
H CO
SH
H CO
N
3
3
EtOH/ PrOH
3
h, rt
2 h, rt
S
COOH
COOH
COOCH3
(
25)
(24)
(23)
SOCl /toluene/DMF
2
1
2 h, 75 °C
O
O
NH
Br2
BBr3
CH Cl
2
H CO
S
2
3
NH OH
4
2
NH
CH Cl
12 h, rt
2
2
S
S
H CO
HO
COCl
1 h, rt
3
(
26)
(27)
(28)
2
88
89
2
2
2
90 Scheme 8. Eight-step synthesis of compound 28.
91
292 The library of N- (6a, 6g, 6h, 6k, 15b-d, 15f, 15i, 15j), O- (10g, 10h, 16d, 16f), and C- (17,
293 18, 27, 28) derivatives along with 8 (BIT), were submitted to antibacterial activity. 0.1 mL
294 of BIT and each derivative (dissolved at a concentration of 1000 ppm in the mixture
295 indicated in Table 4) was mixed with 0.1 mL of Mueller-Hinton culture broth, and tested
296 against a panel of Gram-positive and Gram-negative bacteria, namely: Escherichia coli,
1
5

ACCEPTED MANUSCRIPT
2
2
2
3
3
3
3
97 Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Streptococcus faecium,
98 as described in § 4.4, and results are reported in Table 4.
99
00 Table 4. Biocide activity of compounds 6, 8, 10, 15, 16, 17, 18, 27, 28 and solvent mixtures
01 used in the preparation of the samples.
02
03
a
MIC (ppm)
Solvent
Gram-negative
Gram-positive
Entry Compound MeOH/H
µL/µL))
O
2
Escherichia Pseudomonas
Bacillus
subtilis
Staphylococcus
aureus
Streptococcus
faecium
(
coli
aeruginosa
1
2
3
4
5
6
7
8
9
BIT (8)
6a
100/1900
20/1980
50/1950
250/1750
40/1960
7.8
62.5
7.8
31.3
31.3
15.6
31.3
250
500
500
500
500
500
125
500
500
500
31.3
250
15.6
125
31.3
125
125
500
500
500
nt
7.8
62.5
125
125
125
500
500
500
500
500
500
250
500
500
500
31.3
125
31.3
31.3
62.5
62.5
250
500
500
500
500
500
250
500
500
500
62.5
250
250
500
500
500
500
500
500
500
500
500
250
500
500
500
250
500
6g
6h
6k
b
10g
10h
15b
15c
15d
15f
15i
15j
16d
16f
18
200/1800
200/1800
b
1000/1000
35/1965
1
0
450/1550
225/1775
25/1975
125
125
500
500
125
500
125
250
11
1
2
3
4
5
6
7
1
1
1
1
1
25/1975
100/1900
120/1880
300/1700
500/1500
19
1
6

ACCEPTED MANUSCRIPT
1
1
8
9
27
28
400/1600
400/1600
62.5
500
250
15.6
62.4
62.5
125
62.5
125
62.5
a
b
3
3
04
05
MIC, Minimal Inhibitory Concentration. 30 µL of DMSO were added.
3
3
3
3
3
3
3
06
07 The most active and promising BIT derivatives were found to be the hydroxyethyl-6a, allyl-
08 6g, propargyl-6h, hydroxypropyl-6k, 5-methoxy-18 and 6-methoxy-27, as summarized in
09 Table 5. Consequently, a tentative structure-activity relationship (SAR) analysis was made.
10
11 Table 5.
MIC and polarity ranking (in parenthesis) of bioactive compounds
12
6h, 6g, 18, 27, 6k, 8 and 6a.
MIC (ppm) of Bioactive Compounds
(Lipophilicity Ranking)
Strain
6h
6g
18
27
6k
8
6a
(
1°)
(2°)
31.3
500
31.3
31.3
125
(3°)
62.5
250
31.3
125
31.3
(3°)
62.5
500
15.6
62.5
62.5
(7°)
62.5
500
31.3
125
125
(8°)
7.8
(9°)
31.3
250
31.3
125
62.5
E. coli
62.5
P. aeruginosa 500
62.5
7.8
B. subtilis
S. aureus
S. faecium
15.6
125
125
15.6
7.8
3
3
3
3
3
3
3
3
3
3
3
3
13
14 Lipophilicity is one of the parameters evaluated in Quantitative Structure–Activity
3
5
15 Relationship (QSAR) studies. The relative position of the 18 compounds tested was
16 determined chromatographically by its R (retardation factor), as follows: a) lipophilicity
F
17 with protic and polar eluent (MeOH/H O 1:1) on RP-18 stationary phase, and b) polarity
2
18 with aprotic and polar eluents (n-hexane/AcOEt 6:4 or pure AcOEt) on silica gel (Figure 1S
19 in Supplementary Material whose R values are also reported in italic in Experimental).
F
20 Those compounds, plus the parent BIT, were ordered in an experimentally established
21 ranking order of: a) increasing lipophilicity, and b) decreasing polarity (Figure 2S in
22 Supplementary Material).
23 Considering only the microbiologically active BIT derivatives amenable to coupling
24 reported in Table 5, there appears to be a correlation between lipophilicity and
1
7

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
25 microbiological activity, for which the most lipohilic 6h shows the relatively highest activity
26 against both Gram-negative and -positive bacteria (i.e., E. coli, B. subtilis and S. faecium).
27 However, even if the parent BIT molecule is the most active biocide, but not the most
28 lipophilic neither the least polar (R values of 0.30 in MeOH/H O 1:1 and 0.26 in n-
F
2
29 hexane/AcOEt 6:4, respectively), as such, it cannot be coupled to GM.
30 The hydroxyethyl-BIT (HE-BIT) derivative 6a was submitted to a challenge test in order to
31 confirm its biocide activity. Challenge tests are a customery way to evaluate the protection
32 provided to an industrial product by a given biocide at specified concentrations or by a
33 combination of biocides to cover a wider range of possible microbial contaminations. In
34 these tests, a microbial consortium of bacteria, yeasts or fungi is repeatedly inoculated in the
35 sample at regular intervals, followed by incubation. Microbial growth is regularly checked
36 by streaking an aliquot of the sample on agar plate. The more inocula are needed to obtain
37 cell growth, the better protected is the sample. In our case, a 0.66% (w/v) of unprotected GG
38 solution was compared to the same supplemented with either 500 ppm of 8 (BIT) or 500
39 ppm of 6a (HE-BIT), one of the library compounds with the lowest MIC (see Table 4). All
40 were challenged with three rounds of a bacterial consortium, as detailed in § 4.1.1.2. The
41 results showed that while bacterial growth in unprotected GG occurred after the first
42 inoculum, the samples supplemented with BIT or HE-BIT remained cell-free even after the
43 third inoculum (not shown), thus confirming the results of the MIC evaluation (Table 4).
44 Preliminary coupling experiments are in progress using the following active compounds
3
6
45 from Table 5: 6h with click chemistry and 6g with radical polymerization.
46
47 3.
Conclusions
48 A library of compounds, N-, O- and C-derivatives of the biocide BIT, amenable to coupling
49 to polysaccharides were synthesized and evaluated for their anti microbial efficacy against
50 Gram-positive and -negative bacteria. Six of them showed promising activity with a selected
51 bacteria panel, with the exception of P. aeruginosa, which is well known for its intrinsic
52 resistance to BIT. Therefore, these six compounds were selected for further development,
53 namely coupling to guar gum (GG) and other GM, in order to obtain new “functional”
54 polysaccharides, from renewable sources, with antimicrobial properties and improved
55 biostability. The coupling of the allyl and propargyl BIT derivatives to GM’s is under study,
1
8

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
56 as well as the biostability of the coupled polysaccharides, and will be reported in a
57 forthcoming publication.
58
59 4.
Experimental
60 4.1. General procedures
1
13
61
H and C NMR experiments were performed using a Bruker AV 400 (400 MHz)
62 instrument equipped with a BBFO probe with automatic autotuning and autoshimming,
63 equipped with autosampler [T = 305 K, chemical shift reference = internal TMS
64 (tetramethylsilane)] and a Bruker AVANCE 500 (500 MHz) instrument equipped with a 5
1
19
31
13
65 mm four-nuclei probe H, F, P, C) [T = 305 K, chemical shift reference = internal TMS
66 (tetramethylsilane)]. ESI-MS Experiments were performed using a Bruker Esquire 3000
n
67 PLUS (ESI Ion Trap LC/MS System), equipped with an ESI source and a quadrupole ion
-1
68 trap detector (QIT). Acquisition parameters: needle: 4.5 kV, N flow rate: 10 L x h , cone
2
-1
69 voltage: 40 V, scan resolution 13,000 (m/z) x s over a mass range m/z 35–500, by direct
-1
70 infusion of methanol solution of compounds at a rate of 4 µL x min . The assignment of
71 some of the detected species was confirmed by collision-activated decomposition (CAD)
72 mass spectrometry experiments (MS^2). IR spectra were performed on solid samples using a
73 Fourier Transform Infrared Spectrometer (Jasco FT/IR-610) equipped with accessories for
74 ATR-FT/IR and a Fourier transform Agilent Resolution Pro Spectrum. Non-purified gum
75 powder from guar (GG) with indicative Brookfield viscosity of 1% (w/v) aqueous solutions
76 at 20 rpm and 25 °C of 5000 mPa x s was from commercial sources and kindly provided by
77 Lamberti S.p.A. Actual GM content of unpurified guar gum varied between 76 and 80%
78 (w/w). It was purified by dispersion in ethanol/H O (7:3), followed by vacuum filtration,
2
79 further dispersion in acetone and final recovery by vacuum filtration. Before use, it was
80 oven-dried at 60 °C overnight. The yield of this procedure was 85–90% (w/w). Purified GG
81 was dissolved in MilliQ water at room temperature at 1200 rpm with an IKA overhead
82 stirrer. The solution was kept standing 12 hours at room temperature without stirring before
83 any further experiment. All solvents used were of reagent grade purity and/or purified or
TM
84 dried according to standard procedures. BBL Mueller Hinton Broth, used for determining
85 MIC, was from DIFCO.
86 MIC determination. Minimal inhibitory concentrations (MIC) (defined as the lowest
87 concentration of an antimicrobial that will inhibit the visible growth of a microorganism after
1
9

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
88 overnight incubation) were determined by standard protocols for testing susceptibility to
1
9
89 antibiotic agents.
90 Challenge tests. The challenge tests were performed following a standard protocol in which
91 an aqueous solutions of 0.66% (w/v) guar gum, without or with the addition of 500 ppm of
92 BIT or 500 ppm of hydroxyethyl-BIT, were inoculated on a weekly basis for three
9
93 consecutive weeks, with a 1.0 x 10 CFU/mL bacterial consortium composed of: E. coli, Ps.
94 aeruginosa, Ps. autida, B. cepacia, A. aureus, A. faecalis (involving a two orders of
7
95 magnitude dilution factor down to 1.0 x 10 CFU/ml), followed by incubation at 30 °C,
96 plating in Petri dish after 2 and 6 days on tryptic soy agar (40 g/L of Sigma, T4536) and
97 counting colony formation.
98
99 4.2. Synthesis of N-substituted benzisothiazol-3(2H)-ones.
00 4.2.1. Method A.
01 4.2.1.1. Synthesis of dimethyl 2,2'-disulfanediyldibenzoate (4).
02 Route 1. To a solution of thiosalicylic acid 1 (15.4 g, 100 mmol) in 95% ethanol (800 mL)
03 at room temperature 400 mL of a saturated slurry of I (38.1 g, 150 mmol) prepared by
2
04 suspending neat I (100 g, 390.0 mmol) in 95% ethanol (1000 mL) were added dropwise.
2
05 The solution was stirred at room temperature until a persistent yellow color formed (30
06 min). A colorless precipitate slowly formed, was filtered, collected and dried under vacuum
07 (11.3 g, 37%). The mother liquor was evaporated in vacuo and purified by flash
08 chromatography (CH Cl /AcOEt/CH COOH 98:2:0.1) to give another portion of 2,2’-
2
2
3
+
.
09 disulfanediyldibenzoic acid (2) (17.1 g, 56%). ESI-MS (m/z): 329 [M+Na]. The other
37
10 chemical and analytical data were identical to those of the already described compound 2.
11 To a solution of disulfide 2 (17.1 g, 56.0 mmol) in methanol (600 mL) neat 98% H SO (10
2
4
12 mL) was added and the reaction mixture was heated at reflux for 24 h (TLC in n-
13 hexanes/AcOEt 9:1), poured into a cold K CO /H O mixture (300 g, 600 mL) and the
2
3
2
14 organics were extracted in CHCl (3 x 500 mL), dried over anhydrous Na SO , filtered and
3
2
4
15 evaporated to dryness in vacuo to give the title compound 4 as a colorless solid (17.0 g,
16 91%). The mother liquor was acidified to pH 2 by diluted HCl (as needed) and extracted
17 with CHCl to recover the starting compound 2 (1.71 g, 10% ). δ (400 MHz, CDCl ) 7.25 –
3
H
3
+
.
+.
18 8.27 (8H, m, Ar), 4.00 (6H, s, OCH ); ESI-MS (m/z): 335 [M], 357 [M+Na], 373
3
2
0

ACCEPTED MANUSCRIPT
+
.
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
19 [M+K]. The other chemical and analytical data were identical to those of the already
3
8
20 described compound.
21 Route 2. To a solution of thiosalicilic acid 1 (15.4 g, 100 mmol) in methanol (600 mL) neat
22 98% H SO (10 mL) was added and the reaction mixture was heated at reflux for 72 h (TLC
2
4
23 in n-hexanes/AcOEt 7:3), poured into cold H O (500 mL) and the organics were extracted
2
24 from acidic (by diluted HCl) water (pH<3) in CHCl (3 x 500 mL), dried over anhydrous
3
25 Na SO , filtered and evaporated to dryness in vacuo. After flash chromatographic
2
4
26 purification of the crude (n-hexane/ethyl acetate 7:3), methyl 2-mercaptobenzoate (3) was
+
. 1
13
27 isolated as a colorless solid (15.9 g, 95% ). ESI-MS (m/z): 167 [M – H]; H and C NMR
3
9
28 data were similar to those of the already described compound. To a solution of 3 (15.9 g,
29 95.0 mmol) in CCl (100 mL) at 0 °C, neat Br (7.82 g, 50.0 mmol) in CCl (250 mL) was
4
2
4
30 added dropwise in 30 min. The reaction mixture was stirred at rt for additional 30 min while
31 a colorless precipitate formed and was filtered to give the title compound 4 (28.5 g, 90%).
32
33 4.2.1.2. Cyclization Reaction. General Procedure. To a solution of 4 (20.0 g, 60.0 mmol)
34 in CCl (100 mL), neat Br (10.3 g, 66.0 mmol) in CCl (40 mL) was added dropwise in 30
4
2
4
35 min. The brownish slurry was stirred at rt for 30 min and added dropwise to a solution of the
36 amines 5a-c, i, j (72.0 mmol) and Et N (72.0 mmol) in CCl (40 mL). The reaction mixture
3
4
37 was stirred 1 h at rt and heated at reflux for the suitable amount of time, poured into H O
2
38 (300 mL) and extracted in CHCl (3 x 100 mL). The combined organic phases were dried
3
39 over anhydrous Na SO , filtered and evaporated to dryness under reduced pressure to give a
2
4
40 residue that was purified by flash chromatography.
41
42 4.2.1.2.1. Synthesis of 2-(2-hydroxyethyl)benzisothiazol-3(2H)-one (6a). Starting from
43 ethanolamine (5a) (4.39 g), in 1 h, the N-hydroxyethyl derivative 6a was purified by flash
44 chromatography (CH Cl /AcOEt 1:9) and obtained (3.63 g, 31%) as a colorless solid, along
2
2
45 with methyl 2-{[(2-hydroxyethyl)amino]thio}benzoate 7a isolated as a pale yellowish solid
1
0
20e
46 (1.63 g, 12%). 6a: m.p. 116 – 118 °C (acetone) (lit. 112 – 112.5 °C, lit. 112 – 114 °C);
47 R 0.35 (0.48 in AcOEt; 0.34 in MeOH/H O 1:1 on RP-18); ν (ft-ATR) 3356 (s, br), 3068
F
2
max
48 (w), 2971 (m), 2928 (m), 2875 (m), 1631 (ss), 1568 (w), 1460 (w), 1446 (m), 1343 (m),
-1
49 1251 (m), 1075 (m), 1047 (s), 739 (m), 672 (w) cm (Figure 3S in Supplementary Material);
50 δ (400 MHz, DMSO-d ) 7.93 (1H, ddd, J 7.9, 0.9, 0.7 Hz, 4-H ), 7.85 (1H, ddd, J 7.9, 1.3,
H
6
Ar
2
1

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
51 0.9 Hz, 7-H ), 7.66 (1 H, ddd, J 7.9, 7.0 and 1.3 Hz, 6-H ), 7.41 (1H, ddd, J 7.9, 7.0, 0.9
Ar
Ar
52 Hz, 5-H ), 4.99 (1H, t, J 5.3 Hz, OH), 3.85 (2H, t, J 5.5 Hz, CH N), 3.64 (2H, td, J 5.5, 5.3
Ar
2
+
.
53 Hz, CH OH) (Figure 4S in Supplementary Material); ESI-MS (m/z): 196 [M+H], 218
2
+
.
+.
54 [M+Na], 234 [M+K]. The other chemical and analytical data of compound 6a were
1
0b, 20d,e
55 identical to those already published.
7a: m.p. 80 – 82 °C (acetone); [Found: C, 52.82;
56 H, 5.73; N, 6.14, O, 21.09; S, 14.10. C H NO S Requires C, 52.85; H, 5.77; N, 6.16; O,
10
13
3
57 21.12; S, 14.11%]; R 0.50; ν
(ft-ATR) 3356 (br, s), 2972 (s), 2940 (m), 2900 (m), 1704
max
F
-
1
58 (w), 1638 (s), 1447 (m), 1077 (m), 1048 (ss), 879 (w), 740 (m) cm (Figure 5S in
59 Supplementary Material); δ (400 MHz, DMSO-d ) 7.92 (1H, dd, J 7.8, 1.2 Hz, 4-H ), 7.82
H
6
Ar
60 – 7.75 (1H, m, 7-H ) 7.60 (1H, ddd, J 7.6, 7.3, 1.5 Hz, 6-H ), 7.19 (1h, ddd, J 7.8, 7.6, 1.0
Ar
Ar
61 Hz, 5-H ), 4.58 – 4.65 (1H, br signal, NH), 4.43 (1H, t, J 5.4 Hz, OH), 3.82 (3H, s, OCH ),
Ar
3
62 3.51 (1H, t, J 5.7 Hz,CH NH), 2.89 (1H, td, J 5.7, 5.4 Hz, CH OH) (Figure 6S in
2
2
63 Supplementary Material); δ (100.6 MHz, DMSO-d ) 166.4, 149.8, 133.0, 131.3, 123.9,
C
6
+.
64 123.3, 61.1, 53.7, 52.4 (Figure 7S in Supplementary Material); ESI-MS (m/z): 228 [M+H],
+
.
+.
65 250 [M+Na], 266 [M+K]. 7a (1.00 g, 4.40 mmol) Dissolved in MeOH (20 mL) gave
66 spontaneously 6a (0.85 g, 94%) in one week at rt.
67
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
68 4.2.1.2.2. Synthesis of methyl 3-(3-oxobenzisothiazol-2(3H)-yl)propanoate (6b). Starting
69 from methyl 3-aminopropanoate (5b) (7.42 g), after 5 h, the N-carboxymethylethyl
70 derivative 6b was purified by flash chromatography (n-hexane/AcOEt 7:3) and obtained as a
71 colorless solid (3.41 g, 24%). Methyl 2-{[(3-methoxy-3-oxopropyl)amino]thio}benzoate 7b
72 was isolated as a yellowish oil (2.58 g, 16%). 6b: m.p. 90 – 92 °C (acetone); [Found: C,
73 55.62; H, 4.63; N, 5.94, O, 20.29; S, 13.50. C H NO S requires C, 55.68; H, 4.67; N, 5.90;
11
11
3
74 O, 20.23; S, 13.51%]; R 0.25; ν
(ft-ATR) 3676 (br, m), 2972 (ss), 2900 (ss), 1728 (m),
max
F
75 1648 (m), 1436 (w), 1408 (w), 1251 (w), 1231 (w), 1172 (w), 1052 (ss), 878 (w), 735 (w)
-1
76 cm (Figure 8S in Supplementary Material); δ (400 MHz, CDCl ) 8.02 (1H, d, J 7.8 Hz, 4-
H
3
77 H ), 7.60 (1H, dd, J 7.6, 7.5 Hz, 6-H ), 7.53 (1H, d, J 7.5 Hz, 7-H ), 7.39 (1H, dd, J 7.8,
Ar
Ar
Ar
78 7.6 Hz, 5-H ), 4.18 (2H, t, J 6.6 Hz, CH N), 3.71 (3H, s, OCH ), 2.81 (2H, t, J 6.6 Hz, CH )
Ar
2
3
2
79 (Figure 9S in Supplementary Material); δ (100.6 MHz, CDCl ) 171.5, 165.4, 140.6, 131.9,
C
3
80 126.6, 125.5, 124.2, 120.3, 52.0, 39.9, 33.8 (Figure 10S in Supplementary Material); ESI-
+
.
+.
+.
81 MS (m/z): 238 [M+H], 260 [M+Na], 276 [M+K]; 7b: [Found: C, 53.62; H, 5.63; N,
82 5.24, O, 23.79; S, 11.80. C H NO S Requires C, 53.52; H, 5.61; N, 5.20; O, 23.76; S,
12
15
4
2
2

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
83 11.91%]; R 0.50; νmax (ft-ATR) 3338 (br, s), 2972 (s), 2885 (m), 1701 (m), 1655 (w), 1459
F
-1
84 (w), 1433 (m), 1270 (m), 1255 (m), 1088 (s), 1047 (ss), 880 (s), 738 (m) cm (Figure 11S in
85 Supplementary Material); δ (400 MHz, CDCl ) 8.00 (1H, d, J 7.9 Hz, 4-H ), 7.83 (1H, d, J
H
3
Ar
86 7.6 Hz, 7-H ), 7.52 (1H, dd, J 7.6, 7.6 Hz, 6-H ), 7.15 (1H, dd, J 7.9, 7.6 Hz, 5-H ), 3.95
Ar
Ar
Ar
87 (3H, s, OCH ), 3.73 (3H, s, OCH ), 3.20 – 3.30 (2H, m, CH NH), 2.65 (2H, t, J 6.5 Hz,
3
3
2
88 CH ) (Figure 12S in Supplementary Material); δ (100.6 MHz, CDCl ) 167.0, 164.5, 134.9,
2
C
3
+
.
89 132.6, 132.5, 131.1, 127.5, 120.3, 54.4, 52.2, 42.9, 41.0; ESI-MS (m/z): 270 [M+H], 292
+
.
90 [M+Na]. Compound 7b (2.69 g, 10 mmol) was dissolved in methanol (10 mL) and solid
91 KOH (0.61 g, 11 mmol) was added. The reaction mixture was heated at reflux for 10 h,
92 cooled and MeOH was removed under reduced pressure. The residue was suspended in
93 water (5 mL), the pH was adjusted to 5 with 0.1 N HCl and the organics were extracted in
94 AcOEt (3 x 5 mL). The residue was purified by flash chromathography (n-hexane/AcOEt
95 1:1) to give 6b (0.78 g, 33%) and a trace of 16b (eluted in CH Cl /AcOEt/CH COOH
2
2
3
96 3:7:0.1) was also isolated (0.11 g, 5%). Using Triton-B (solution 40% w/w in methanol)
97 (0.18 mL, 1 mmol) instead of KOH, after 2.5 h under reflux in methanol (20 mL), only 6b
98 was isolated (0.83 g, 35%).
4
5
5
5
5
5
5
5
5
5
5
5
5
99 4.2.1.2.3. Synthesis of 2,2'-{disulfanediylbis[ethane-2,1-diyl)]bis(benzisothiazol-3(2H)-
00 one} (6c). Starting from 2,2'-disulfanediyldiethanaminium iodide (5c) (29.4 g), after 6.5 h
01 and flash chromatographic purification (n-hexane/AcOEt 4:6), the title compound 6c was
02 obtained as a colorless solid (8.06 g, 32%), m.p. 123 – 125 °C (n-hexane/AcOEt 1:1);
03 [Found: C, 51.42; H, 3.83; N, 6.64, O, 7.69; S, 30.60. C H N O S Requires C, 51.40; H,
1
8
16
2
2 4
04 3.83; N, 6.66; O, 7.61; S, 30.50%]; R 0.25; ν (ft-ATR) 3369 (br, w), 2973 (s), 2900 (m),
F
max
05 1655 (s), 1441 (m), 1424 (m), 1338 (m), 1291 (m), 1248 (m), 1181 (m), 1086 (m), 1049 (ss),
-1
06 874 (w), 734 (s), 668 (s) cm (Figure 13S in Supplementary Material); δ (400 MHz,
H
07 CDCl ) 8.01 (2H, d, J 7.3 Hz, 4-H ), 7.57 (2H, dd, J 7.8, 6.6 Hz, 6-H ), 7.52 (2H, d, J 7.8
3
Ar
Ar
08 Hz, 7-H ), 7.37 (2H, dd, J 7.3, 6.6 Hz, 5-H ), 4.17 (4H, t, J 6.3 Hz, CH N), 3.08 (4H, t, J
Ar
Ar
2
09 6.3 Hz, CH S) (Figure 14S in Supplementary Material); δ (100.6 MHz, CDCl ) 165.5,
2
C
3
10 140.5, 131.9, 126.7, 125.6, 124.2, 120.4, 43.0, 37.1 (Figure 15S in Supplementary Material);
+
.
+.
+.
11 ESI-MS (m/z): 421 [M+H], 443 [M+Na], 459 [M+Na].
5
12 4.2.1.2.4.
Synthesis
of
methyl
5
13 2-(((2-((tert-butoxycarbonyl)amino)ethyl)amino)thio)benzoate (7i). Starting from tert-
2
3

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
14 butyl (2-aminoethyl)carbamate (5i) (11.5 g), after 4 h and flash chromatographic purification
15 (n-hexane/AcOEt 7:3), only the title compound 7i was obtained as a yellowish oil (19.3 g,
16 98%); [Found: C, 55.25; H, 6.74; N, 8.62, O, 19.55; S, 9.80. C H N O S Requires C,
1
5
22
2
4
17 55.19; H, 6.79; N, 8.58; O, 19.61; S, 9.82%]; R 0.35; ν
(ft-ATR) 3371 (m), 2976 (m),
max
F
18 2950 (m), 1697 (ss), 1511 (m), 1458 (w), 1434 (m), 1365 (w), 1307 (m), 1270 (s), 1249 (s),
-1
19 1163 (s), 1143 (s), 1099 (m), 1055 (m), 1038 (m), 961 (m), 740 (s), 691 (w) cm (Figure 16S
20 in Supplementary Material); δ (400 MHz, DMSO-d ) 7.93 (1H, dd, J 7.8, 1.2 Hz, 4-H ),
H
6
Ar
21 7.82 (1H, dd, J 8.2, 1.3 Hz, 7-H ), 7.61 (1H, ddd, J 8.2, 7.2, 1.3 Hz, 6-H ), 7.21 (1H, ddd,
Ar
Ar
22 J 7.8, 7.2, 1.1 Hz, 5-H ), 6.90 (1H, t, J 5.2 Hz, NHCO), 4.56 (1H, t, J 5.0 Hz, NHS), 3.82
Ar
23 (3H, s, OCH ), 3.06 (2H, dt, J 6.6, 5.2 Hz CH NHCO), 2.83 (2H, dt, J 6.6, 5.0 Hz,
3
2
24 CH NHS), 1.37 (9H, s, Me C) (Figure 17S in Supplementary Material); δ (100.6 MHz,
2
3
C
25 DMSO-d ) 166.4, 146.7, 134.1, 133.1, 131.7, 131.4, 125.4, 124.2, 78.1, 52.8, 50.8, 41.0,
6
+.
26 28.7 (x 3C) (Figure 18S in Supplementary Material); ESI-MS (m/z): 349 [M+Na], 365
+.
27 [M+K]. Compound 5i (3.26 g, 10 mmol) was dissolved in methanol (10 mL) and solid
28 KOH (0.61 g, 11 mmol) was added. The reaction mixture was stirred at room temperature
29 for 36 h and MeOH was removed under reduced pressure. The residue was suspended in
30 water (5 mL), the pH was adjusted to 5 with 0.1 N HCl and the organics were extracted in
31 AcOEt (3 x 5 mL). The residue was purified by flash chromathography (n-hexane/AcOEt
32 3:7) to give 6i (2.73 g, 93%) as a high density colorless oil; [Found: C, 57.32; H, 6.15; N,
33 9.50; O, 16.60; S, 10.82. C H N O S Requires C, 57.12; H, 6.16; N, 9.52; O, 16.31; S,
1
4
18
2
3
34 10.89%]; R 0.35; ν
(ft-ATR) 3327 (s), 2976 (m), 2930 (m), 1697 (s), 1646 (ss), 1599
max
F
35 (w), 1519 (m), 1447 (w), 1391 (w), 1365 (w), 1340 (w), 1278 (m), 1249 (m), 1168 (s), 740
-1
36 (m) cm (Figure 19S in Supplementary Material); δ (400 MHz, DMSO-d ) 7.97 (1H, d, J
H
6
37 8.1 Hz, 4-H ), 7.87 (1H, d, J 7.8 Hz, 7-H ), 7.68 (1H, dd, J 8.1, 7.3 Hz, 5-H ), 7.43 (1H,
Ar
Ar
Ar
38 dd, J 7.8, 7.3 Hz, 6-H ), 6.98 (1H, t, J 5.0 Hz), 3.83 (2H, t, J 5.9 Hz, CH N), 3.23 (2H, dt, J
Ar
2
39 5.9, 5.0 Hz, CH NH), 1.35 (9H, s, Me C) (Figure 20S in Supplementary Material); δ (100.6
2
3
C
40 MHz, DMSO-d ) 164.9, 156.1, 141.3, 132.1, 126.0, 125.8, 124.5, 122.2, 78.3, 43.6, 31.1,
6
+.
41 28.7 (x 3C) (Figure 21S in Supplementary Material); ESI-MS (m/z): 317 [M+23], 333
+.
42 [M+39].
43 4.2.1.2.5.
5
5
5
Synthesis
of
methyl
44 2-(((3-((tert-butoxycarbonyl)amino)propyl)amino)thio)benzoate (7j). Starting from tert-
45 butyl (2-aminopropyl)carbamate (5j) (12.5 g), after 6 h and flash chromatographic
2
4

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
46 purification (n-hexane/AcOEt 7:3), only the title compound 7j was obtained as a yellowish
47 oil (17.1 g, 84%); [Found: C, 56.38; H, 8.27; N, 8.25, O, 18.70; S, 9.38. C H N O S
16
24
2
4
48 Requires C, 56.45; H, 7.11; N, 8.23; O, 18.80; S, 9.42%]; R 0.30; νmax (ft-ATR) 3342 (br,
F
49 s), 2973 (m), 2933 (m), 2873 (w), 1693 (ss), 1519 (m), 1458 (w), 1439 (w), 1365 (m), 1269
-1
50 (ss), 1249 (ss), 1166 (s), 1099 (w), 1042 (m), 744 (m) cm (Figure 22S in Supplementary
51 Material); δ (400 MHz, DMSO-d ) 7.96 (1H, dd, J 7.8, 1.3 Hz, 4-H ), 7.54 (1H, dd, J 8.1,
H
6
Ar
52 0.7 Hz, 7-H ), 7.64 (1H, ddd, J 8.1, 7.2, 1.3 Hz, 6-H ), 7.23 (1H, ddd, J 8.2, 8.1, 1.1 Hz, 5-
Ar
Ar
53 H ), 6.75 – 6.85 (1H, m, NH), 4.57 (1H, t, J 5.1 Hz, NH), 3.84 (3H, s, OCH ), 3.01 (2H, dt,
Ar
3
54 J 6.6, 6.5 Hz CH NH), 2.83 (2H, dt, J 6.9, 5.1 Hz, CH NH), 1.60 – 1.70 (2H, m, CH ), 1.40
2
2
2
55 (9H, s, Me C) (Figure 23S in Supplementary Material); δ (100.6 MHz, DMSO-d ) 166.3,
3
C
6
56 156.1, 149.6, 133.0, 131.4, 124.1, 124.0, 123.4, 77.9, 52.4, 48.7, 38.2, 30.9, 28.7 (x3C)
+
.
+.
57 (Figure 24S in Supplementary Material); ESI-MS (m/z): 341 [M+H], 363 [M+Na], 379
+.
58 [M+K]. Compound 5j (3.40 g, 10 mmol) was dissolved in methanol (10 mL) and solid
59 KOH (0.61 g, 11 mmol) was added. The reaction mixture was stirred at room temperature
60 for 18 h and MeOH was removed under reduced pressure. The residue was suspended in
61 water (5 mL), the pH was adjusted to 5 with 0.1 N HCl and the organics were extracted in
62 AcOEt (3 x 5 mL). The residue was purified by flash chromathography (n-hexane/AcOEt
63 3:7) to give 6j (2.68 g, 87%) as a high density colorless oil; [Found: C, 58.37; H, 6.55; N,
64 9.10; O, 15.60; S, 10.42. C H N O S Requires C, 58.42; H, 6.54; N, 9.08; O, 15.56; S,
1
5
20
2
3
65 10.40%]; R 0.35; ν (ft-ATR) 3353 (br, ss), 2972 (m), 2930 (m), 1691 (s), 1638 (ss), 1599
F
max
66 (w), 1518 (s), 1447 (m), 1365 (w), 1277 (m), 1250 (m), 1165 (ss), 878 (w), 784 (w), 739 (s),
-1
67 673 (w) cm (Figure 25S in Supplementary Material); δ (400 MHz, DMSO-d ) 7.98 (1H,
H
6
68 d, J 8.2 Hz, 4-H ), 7.87 (1H, d, J 7.8 Hz, 7-H ), 7.69 (1H, ddd, J 8.2, 7.5, 1.2 Hz, 5-H ),
Ar
Ar
Ar
69 7.45 (1H, ddd, J 7.8, 7.5, 0.8 Hz, 6-H ), 6.84 (1H, t, J 5.2 Hz), 3.83 (2H, t, J 7.1 Hz,
Ar
70 CH N), 3.03 – 2.94 (2H, m, CH NH), 1.84 – 1.74 (2H, m, CH ), 1.38 (9H, s, Me C) (Figure
2
2
2
3
71 26S in Supplementary Material); δ (100.6 MHz, CDCl ) 165.8, 156.0 (CO), 140.2, 131.9,
C
3
72 126.6, 125.6, 124.3, 120.4, 79.1, 41.1, 37.0, 29.6, 28.4 (x 3C) (Figure 27S in Supplementary
+.
+.
73 Material); ESI-MS (m/z): 331 [M+23], 347 [M+39].
5
5
5
5
74 4.2.2. Method B.
75 4.2.2.1. Nucleophilic Substitution on brominated electrophiles. General Procedures.
76 Procedure 1. A solution of benzisothiazol-3(2H)-one (8) (15.4 g, 100 mmol), bromo ester
77 9e (21.23 g, 110 mmol) or 9d (16.6 g, 110 mmol) and 2,2,2-trifluoroethanol (400 mL) in
2
5

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
78 DMF (100 mL) was added dropwise at 0 °C to a slurry of NaH (60% dispersion in mineral
79 oil, 4.8 g, 120 mmol), previously washed in n-hexanes (3 x 20 mL) at 0°C to remove the
80 mineral oil, in DMF (300 mL). The reaction mixture was stirred at the same temperature for
81 15 min and heated at 80 °C for 1 h. The mixture was poured into a cold saturated NH Cl
4
82 solution (200 mL) and extracted with AcOEt (3 x 100 mL). The combined organics were
83 dried over anhydrous Na SO , filtered and evaporated to dryness under reduced pressure to
2
4
84 give a residue that was purified by flash chromatography.
85 Procedure 2. Identical to 1, but without using 2,2,2-trifluoroethanol.
86 Procedure 3. A solution of 8 (15.4 g, 100 mmol), in dry acetonitrile (140 mL) was added
87 dropwise at 0 °C to a slurry of NaH (60% dispersion in mineral oil, 4.8 g, 120 mmol)
88 previously washed in n-hexanes (3 x 20 mL) at 0 °C to remove the mineral oil. The mixture
89 was stirred at 0 °C for 15 min and acetonitrile was removed in vacuo. The residue was
90 cooled at 0 °C and 2,2,2-trifluoroethanol (400 mL) and bromo ester 9d (16.6 g, 110 mmol)
91 were added. The reaction mixture was heated at reflux for 1 h. The work-up procedure was
92 identical to that of procedure 1.
93 Procedure 4. A solution of 9g (47.6 g, 400 mmol) or 9h (46.8 g, 400 mmol) in DMF (10
94 mL) was added dropwise at rt to a slurry of 8 (15.4 g, 100 mmol) and K CO (13.8 g, 100
2
3
95 mol) in DMF (200 mL). The reaction mixture was stirred at 80 °C for 4 h. An acetic buffer
96 solution (pH 4) was added dropwise into the reaction mixture up to pH 7, water (150 mL)
97 was added and the combined organic phases were extracted in AcOEt (3 x 100 mL), washed
98 with water (3 x 70 mL), dried over anhydrous Na SO , filtered and evaporated to dryness
2
4
99 under reduced pressure to give a residue that was purified by flash chromatography.
00
01 4.2.2.1.1. Synthesis of methyl 2-[3-oxobenzisothiazol-2(3H)-yl]acetate (6d). Following
02 procedure 2 and starting from bromomethylacetate 9d (16.6 g, 110 mmol), after flash
03 chromatographic purification (n-hexane/AcOEt 6:4), 6d (11.0 g, 45%) was isolated in
1
7a
04 colorless small needles, m.p. 83 – 84 °C (isopropylether/AcOEt 1:1) (lit. 82 – 83 °C);
05 [Found: C, 53.82; H, 4.03; N, 6.24, O, 21.59; S, 14.40. C H NO S Requires C, 53.80; H,
10
9
3
06 4.06; N, 6.27; O, 21.50; S, 14.36%]; R 0.35; ν
(ft-ATR) 3383 (br, w), 2954 (m), 2923
max
F
-1
07 (m), 1745 (s), 1649 (ss), 1597 (m), 1445 (m), 1209 (s), 739 (s) cm (Figure 28S in
17b
08 Supplementary Material); δ (400 MHz, CDCl ) 8.02 (1H, d, J 7.8 Hz, 4-H ), 8.03 (1H,
H
3
Ar
09 d, J 7.8 Hz, 7-H ), 7.61 (1H, ddd, J 8.1, 7.1, 1.1 Hz, 6-H ), 7.53 (1H, d, J 8.1 Hz, 7-H ),
Ar
Ar
Ar
2
6

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
10 7.38 (1H, ddd, J 7.8, 7.1, 1.0 Hz, 5-H ), 4.61 (2H, s, CH N), 3.76 (3H, s, OCH ) (Figure
Ar
2
3
11 29S in Supplementary Material); δ (100.6 MHz, CDCl ) 168.1, 165.7, 140.8, 132.3, 126.9,
C
3
12 125.6, 123.4, 120.4, 52.6, 44.5 (Figure 30S in Supplementary Material); ESI-MS (m/z): 224
+.
+.
13 [M+H], 246 [M+Na]. Another compound, methyl 2-(benzo[d]isothiazol-3-yloxy)acetate
14 (10d), was isolated in colorless cristals (4.41 g, 18%), m.p. 250 - 252 °C
15 (diisopropylether/AcOEt 1:1); [Found: C, 53.77; H, 4.08; N, 6.25, O, 21.56; S, 14.42.
16 C H NO S Requires C, 53.80; H, 4.06; N, 6.27; O, 21.50; S, 14.36%]; R 0.50; ν (ft-
max
10
9
3
F
17 ATR) 3365 (br, s), 2972 (ss), 2918 (ss), 2901 (m), 1736 (s), 1652 (m), 1504 (m), 1386 (s),
-1
18 1322 (m), 1257 (s), 1147 (w), 880 (m), 739 (m), 710 (w) cm (Figure 31S in Supplementary
19 Material); δ (400 MHz, CDCl ) 7.99 (1H, d, J 7.8 Hz, 4-H ), 7.76 (1H, d, J 7.2 Hz, 7-H ),
H
3
Ar
Ar
20 7.52 (1H, ddd, J 7.8, 7.2, 1.8 Hz, 6-H ), 7.40 (1H, ddd, J 7.8, 7.8, 0.9 Hz, 5-H ), 5.11 (2H,
Ar
Ar
21 s, CH O), 3.80 (3H, s, OCH ) (Figure 32S in Supplementary Material); δ (100.6 MHz,
2
3
C
22 CDCl ) 168.8, 161.7, 152.1, 128.8, 124.7, 124.6, 123.1, 120.1, 64.8, 52.2 (Figure 33S in
3
+
.
+.
23 Supplementary Material); ESI-MS (m/z): 224 [M+H], 246 [M+Na]. Following procedure
24 1, 6d (6.62 g, 27%) and 10d (1.23 g, 5%) were isolated. Following procedure 3, only 10d
25 (11.0 g, 45%) was isolated.
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
26 4.2.2.1.2. Synthesis of tert-butyl 2-(3-oxobenzisothiazol-2(3H)-yl)acetate (6e). Following
27 procedure 2 and starting from 9e (15.9 g, 110 mmol), after flash chromatographic
28 purification (n-hexane/AcOEt 95:5 followed by n-hexane/AcOEt 8:2), 6e (18.4 g, 63%) was
29 isolated as a colorless powder, m.p. 88 – 90 °C (EtOH); R 0.35; ν (ft-ATR) 3368 (br, w),
F
max
30 2975 (s), 2871 (m), 1739 (ss), 1660 (s), 1626 (m), 1444 (m), 1352 (m), 1227 (s), 1149 (s),
-1
31 1088 (m), 1048 (s), 739 (ss) cm (Figure 34S in Supplementary Material); δ (400 MHz,
H
2
1a
32 CDCl3) 7.98 (1H, d, J 7.9 Hz, 4-H ), 7.54 (1H, ddd, J 8.0, 7.9, 1.2 Hz, 6-H ), 7.49 (1H,
Ar
Ar
33 d, J 7.9 Hz, 7-H ), 7.32 (1H, ddd, J 8.0, 7.9, 1.1 Hz, 5-H ), 4.46 (2H s, CH N), 1.43 (9H, s,
Ar
Ar
2
34 Me C) (Figure 35S in Supplementary Material); δ (100.6 MHz, CDCl ) 166.7, 165.7,
3
C
3
35 140.9, 132.1, 126.8, 125.4, 123.5, 120.3, 82.8, 45.3, 28.0 (x3C) (Figure 36S in
+
.
+.
+.
36 Supplementary Material); ESI-MS (m/z): 210 [M+H], 232 [M+Na], 248 [M+K]; 208
+.
37 [M-H]. 10e (8.45 g, 29%) Was isolated as a colorless powder, m.p. 85 - 87 °C (EtOH);
38 [Found: C, 58.87; H, 5.68; N, 5.25, O, 18.06; S, 12.02. C H NO S Requires C, 58.85; H,
13
15
3
39 5.70; N, 5.28; O, 18.09; S, 12.09%]; R 0.50; ν
(ft-ATR) 3205 (br, ss), 2942 (s), 2856
max
F
40 (m), 2840 (m), 1647 (ss), 1637 (ss), 1532 (w), 1485 (w), 1430 (m), 1327 (m), 1257 (m),
2
7

ACCEPTED MANUSCRIPT
-
1
6
6
6
6
6
6
41 1063 (s), 1022 (m), 929 (w), 914 (w), 827 (m), 745 (s), 658 (s), 612 (s) cm (Figure 37S in
42 Supplementary Material); δ (400 MHz, CDCl ) 7.85 (1H, d, J 8.1 Hz, 4-H ), 7.60 (1H, d, J
H
3
Ar
43 8.2 Hz, 7-H ), 7.34 (1H, ddd, J 8.2, 7.9, 1.1 Hz, 6-H ), 7.23 (1H, ddd, J 8.1, 7.1, 0.6 Hz, 5-
Ar
Ar
44 H ), 4.86 (2H, s, CH O), 1.35 (3H, s, Me C) (Figure 38S in Supplementary Material); δ
C
Ar
2
3
45 (100.6 MHz, CDCl ) 167.4, 161.8, 151.9, 128.7, 124.8, 124.5, 123.1, 120.1, 82.1, 65.3, 28.0
3
+
.
46 (x3C) (Figure 39S in Supplementary Material); ESI-MS (m/z): 208 [M-H].
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
47 4.2.2.1.3. Synthesis of methyl 2-[3-oxobenzisothiazol-2(3H)-yl]propanoate (6f).
48 Following procedure 2 and starting from methyl 2-bromopropanoate (9f) (18.1 g, 110
49 mmol), after flash chromatography (n-hexane/AcOEt 7:3), 6f (6.40 g, 27%) was isolated as a
50 colorless soft solid, m.p. 106 – 108 °C (diisopropylether); [Found: C, 55.67; H, 4.68; N,
51 5.95, O, 20.26; S, 13.56. C H NO S Requires C, 55.68; H, 4.67; N, 5.90; O, 20.23; S,
11
11
3
52 13.51%]; R 0.35; νmax (ft-ATR) 3376 (br, s), 2972 (ss), 2918 (ss), 1735 (s), 1504 (s), 1375
F
-1
53 (s), 1322 (m), 1258 (m), 1231 (m), 1088 (ss), 1047 (ss), 739 (m), 575 (ss) cm (Figure 40S
1
13
54 in Supplementary Material); H and C NMR data in CDCl were identical to those already
3
1
7a, 21a
55 described:
δ (400 MHz, CDCl ) 8.0 (1H, d, J 7.6 Hz, 4-H ), 7.57 (1H, ddd, J 8.1,
H 3 Ar
56 7.9, 1.0 Hz, 6-H ), 7.53 (1H, d, J 7.9 Hz, 7-H ), 7.35 (1H, ddd, J 8.1, 7.6, 1.0 Hz, 5-H ),
Ar
Ar
Ar
57 5.42 (1H q, J 7.3 Hz, CHCH ), 3.71 (3H, s, OCH ), 1.64 (3H, d, J 7.3 Hz, CHCH ) (Figure
3
3
3
58 41S in Supplementary Material); δ (100.6 MHz, CDCl ) 171.0, 165.6, 140.9, 132.1, 126.7,
C
3
59 125.5, 120.4, 52.7, 51.2, 17.1 (Figure 42S in Supplementary Material); δ (100.6 MHz,
C
60 DMSO-d ) 171.2, 170.0, 141.5, 133.0, 132.9 (x2C), 132.6, 129.6, 62.2, 51.6, 16.9 (Figure
6
+
.
+.
+.
61 43S in Supplementary Material); ESI-MS (m/z): 238 [M+H], 260 [M+Na], 276 [M+K].
62 Methyl 2-(benzisothiazol-3-yloxy)propanoate (10f) (16.1 g, 68%) was isolated as a
63 yiellowish oil, [Found: C, 55.72; H, 4.70; N, 5.94, O, 20.20; S, 13.50. C H NO S Requires
11
11
3
64 C, 55.68; H, 4.67; N, 5.90; O, 20.23; S, 13.51%]; R 0.50; ν
(ft-ATR) 3370 (br, w), 2973
max
F
65 (ss), 2900 (s), 1736 (ss), 1502 (m), 1386 (s), 1323 (m), 1257 (s), 1147 (w), 1086 (s), 1045
-1
66 (ss), 970 (w), 880 (m), 836 (m), 770 (m), 739 (m) cm (Figure 44S in Supplementary
67 Material); δ (400 MHz, CDCl ) 7.96 (1H, d, J 7.9 Hz, 4-H ), 7.69 (1H, d, J 8.2 Hz, 7-H ),
H
3
Ar
Ar
68 7.43 (1H, dd, J 8.2, 7.5 Hz, 6-H ), 7.33 (1H, dd, J 7.9, 7.5 Hz, 5-H ), 5.53 (1H q, J 6.9 Hz,
Ar
Ar
69 CHCH ), 3.74 (3H, s, OCH ), 1.71 (3H, d, J 6.9 Hz, CHCH ) (Figure 45S in Supplementary
3
3
3
70 Material); δ (100.6 MHz, CDCl ) 171.9, 161.7, 151.9, 128.7, 124.6, 124.5, 123.1, 120.1,
C
3
2
8