New acrylamide-sulfisoxazole conjugates as dihydropteroate synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115444

New functionalized acrylamide derivatives bearing sulfisoxazole moiety were designed to target bacterial dihydropteroate synthase (DHPS). The in vitro antimicrobial activities of these compounds were assessed. The E-configuration of compound 5b was proved

Full text of DOI:10.1016/j.bmc.2020.115444

Journal Pre-proofs
New acrylamide-sulfisoxazole conjugates as dihydropteroate synthase inhibi-
tors
Tamer Nasr, Samir Bondock, Tamer M. Ibrahim, Walid Fayad, Ahmed B.
Ibrahim, Neveen A. AbdelAziz, Tamer M. Sakr
PII:
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https://doi.org/10.1016/j.bmc.2020.115444
BMC 115444
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Reference:
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Bioorganic & Medicinal Chemistry
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27 January 2020
12 March 2020
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Please cite this article as: T. Nasr, S. Bondock, T.M. Ibrahim, W. Fayad, A.B. Ibrahim, N.A. AbdelAziz, T.M.
Sakr, New acrylamide-sulfisoxazole conjugates as dihydropteroate synthase inhibitors, Bioorganic & Medicinal
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New acrylamide-sulfisoxazole conjugates as
dihydropteroate synthase inhibitors
*
1
2,3
4
5
6
Tamer Nasr , Samir Bondock , Tamer M. Ibrahim Walid Fayad , Ahmed B. Ibrahim ,
,
7
*8
Neveen A. AbdelAziz , Tamer M. Sakr
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, 11795
Helwan, Cairo, Egypt
2
Department of Chemistry, Faculty of Science, King Khalid University, 9004 Abha, Saudi
Arabia
3Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura,
Egypt
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafr El-Sheikh 33516, Egypt.
5
Drug Bioassay-Cell Culture Laboratory, Pharmacognosy Department, National Research
Centre, Dokki, Giza, 12622, Egypt.
6
Labeled Compounds Department, Hot Labs Center, Atomic Energy Authority, P.O. Box 13759,
Cairo, Egypt.
7Department of Microbiology and Immunology, Faculty of Pharmacy, Ahram Canadian
University, 12451, Sixth of October City, Giza, Egypt.
8
Radioactive Isotopes and Generators Department, Hot Laboratories Centre, Atomic Energy
Authority, P.O. Code 13759 Cairo, Egypt.
*
Corresponding authors: tamerhefni@yahoo.com (T. Nasr) (002-02-23150566),
Tamer_sakr78@yahoo.com (T. M. Sakr) (002-01006884013)
1

Abstract
New functionalized acrylamide derivatives bearing sulfisoxazole moiety were designed to
target bacterial dihydropteroate synthase (DHPS). The in vitro antimicrobial activities of these
compounds were assessed. The E-configuration of compound 5b was proved by single crystal
X-ray analysis. Compounds 5g and 5h displayed double the activity of ampicillin against B.
subtilis. Also, 5h was two times more active than gentamycin against E. coli. Interestingly,
compounds 5f-g, 7c, 8a, 8c exhibited two folds the potency of amphotericin B against S.
racemosum while 5h displayed three folds the activity of amphotericin B against S. racemosum.
Most of the synthesized compounds showed superior activities to the parent sulfisoxazole and
were non-toxic to normal cells. DHPS is confirmed to be a putative target for our compounds
via antagonizing their antibacterial activity by the folate precursor (p-aminobenzoic acid) and
product (methionine) on E. coli ATCC 25922. Docking experiments against DHPS rationalized
the observed antibacterial activity. Additionally, compound 5g was evaluated as a selective
targeting vector for 99mTc that showed a remarkable uptake and targeting ability towards the
infection site that was induced in mice.
Keywords: Antimicrobial agents, Molecular docking, Radiolabeling, Sulfisoxazole,
Acrylamide, Thioglycoside
2

1
. Introduction
Microbial drug resistance has become a threatening challenge in the last few decades [1,
]. To face this threat, scientists are searching for new potent antimicrobial agents [3]. Among
2
the known strategies to develop novel potent antimicrobial agents is to modify the chemical
structure of existing drugs [4]. In this strategy, a synergistic effect was accessed by merging
more than one antimicrobial pharmacophore [5]. Sulfonamides are antimicrobial agents that
have been used for more than 70 years and displayed a broad spectrum activity against bacterial
as well as protozoal infections [6-8]. The dihydropteroate synthase (DHPS) enzyme was
validated to be the main target for sulfonamides via competing the natural substrate p-amino
benzoic acid (pABA) [6, 9]. DHPS catalyzes the formation of 7,8-dihydropteroate, folic acid
precursor, from pABA and 6-hydroxymethyl-7,8-dihydropterin pyrophosphate [10]. After
decades of their discovery, sulfonamides suffer from resistance [9, 11]. Currently, they are still
co-administered with trimethoprim to potentiate their effect and minimize the possibility of
resistance development [12, 13]. Recently, it was established that sulfonamides could also act
by forming dead-end pterin-sulfonamide conjugates [14]. Thus; they are able to sequester
pterin pools. The crystal structure of DHPS enzyme bound to sulfathiazole-6-hydroxymethyl-
7
,8-dihydropterin-pyrophosphate (STZ-DHPP) adduct shows that the sulfathiazole moiety
occupied the pABA binding pocket while the dihydropterin moiety occupied the pterin binding
site [10]. A few years ago we designed and synthesized some sulfisoxazole derivatives
conjugated to different heterocyclic compounds and some of these derivatives showed
excellent activities and were much more potent than sulfisoxazole [14, 15]. These derivatives
were more lipophilic and consequently had better intracellular concentrations than
sulfisoxazole. Moreover, they were able to bind to both pABA and pterin binding sites of the
DHPS enzyme.
Additionally, a literature survey revealed that some acrylamide derivatives displayed
interesting antimicrobial activities. The 3-amino-2-cyano-acrylamide derivative I [16], 2-
cyano-3-phenyl-acrylamide derivative II [14], 3-(3,4-dihydroxyphenyl)acrylamide III [17]
showed antimicrobial activities in the range 12.5-50 μg/mL, 0.49-7.81 μg/mL and 3.12-50
μg/mL; respectively (Figure 1).
<
Insert Figure 1 >
3

As a continuation of our studies on the inhibition of DHPS enzyme as a potential
antimicrobial target we designed and synthesized three series of substituted acrylamide-
sulfisoxazole conjugates. Each molecule of these conjugates will be able to occupy both the
pterin and pABA binding sites of DHPS at the same time in order to improve the therapeutic
potential of sulfisoxazole (Figure 2). To validate the inhibitory effect of our compounds
against DHPS as a putative target, MIC shifts in the presence of folate precursor (pABA) and
product (methionine) were determined. Both pABA and methionine antagonized the inhibitory
effect of the compounds on the test organism E. coli ATCC 25922. Also, docking experiments
rationalized the observed in vitro inhibitory activity and demonstrated that the synthesized
conjugates were able to inhibit the DHPS enzyme by binding to its pterin and pABA binding
sites at once. In addition, the most effective compound, 5g, was studied to act as a targeting
probe for infection imaging after being radiolabelled with technetium-99m.
<
Insert Figure 2 >
2
. Results and discussion
2
.1. Chemistry
The key intermediate used in this study, compound 3, was efficiently prepared as
previously reported by us [14]. The nucleophilic addition of the active methylene group of 3
assisted by KOH to different isothiocyanate derivatives in DMF at room temperature produced
the non-isolable potassium salts of 2-cyanoketene-N,S-acetals 4a-e. Alkylation of the later
intermediates with alkyl iodides afforded, a sole product, in each case, identified as (E)-2-
cyano-3-(alkylthio)-3-(substitutedamino)acrylamide derivatives 5a-j (Scheme 1). The
1
structures of acrylamides 5a-j were inferred from microanalyses, spectral data (IR, MS, H-,
13C-NMR), and chemical transformation. As a typical example of the series prepared, the IR
spectrum of compound 5b revealed absorption bands at 3369, 3231, 3125, 2194, 1650 cm-1
attributed to three NH, conjugated CN, and amidic C=O functions, respectively. Its mass
+
spectrum exhibited a peak at m/z 435 (M ) which corresponding to its molecular weight and
1
molecular formula (C H N O S ). The H-NMR spectrum of 5b showed triplet and quartet
1
8
21
5
4 2
signals at  1.19, 3.58 ppm due to ethyl group, six singlet signals at  1.64, 2.09, 2.66, 9.67,
0.51, 10.91 ppm due to two methyl groups, thiomethyl protons, and three secondary NH
protons, respectively, besides two doublets with coupling constant value (J = 9.0 Hz) at  7.67
1
1
3
and 7.69 ppm due to four aromatic protons. In addition, C-NMR spectrum of 5b displayed
4

distinctive five aliphatic carbon peaks at  5.8, 10.4, 15.2, 17.6, 40.9 ppm and amidic carbonyl
carbon peak at  170.6 ppm among a total 16 carbon-peaks.
<
Insert Scheme 1 >
The relative E-configuration of the obtained 2-cyano-3-(alkylthio)-3-
substitutedamino)-acrylamides 5a-e was definitely assigned from the single crystal X-ray
(
analysis of compound 5b (Figure 3) [18]. It is worthwhile to report that the preference
formation of stereoisomer (E)-acrylamides is also in line with our recently published results of
analogous compounds [19, 20].
<
Insert Figure 3 >
The structure 5j, as an example, was further chemically ascertained by its conversion
to 3-aminopyrazole [21]. Thus, treatment of 5j with two equimolar amounts of hydrazine
hydrate in ethanol, under reflux, yielded the respective 3-aminopyrazole-4-carboxamide
derivative 6. The IR spectrum of 6 was free of any absorption band at approximately 2200 cm-1
due to a nitrile function and exhibited characteristic absorption bands at 3388-3317, 3266,
-1
3
210, 3150, 1656 cm owing to NH , three NH, and amidic C=O functions, respectively. The
2
1H-NMR spectrum revealed beside the expected signals of sulfisoxazole ring residue and
phenyl protons, five singlets at  6.12, 8.52, 9.12, 10.80, 11.35 ppm, exchangeable with D O,
2
1
3
assignable to NH and four NH protons, respectively. Moreover, the C-NMR spectrum of 6
2
has 17 carbon signals which are compatible with its molecular structure and mass spectrum
which exhibited a molecular ion peak at m/z 467 (M+).
Acidification of the potassium salts of 2-cyanoketene N,S-acetals 4a-e with diluted HCl
gave the respective 2-cyano-3-mercapto-3-(substituted-amino)acrylamides 7a-e in good yields.
Treatment of the later products with 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl bromide in
boiling acetone and in the presence of catalytic amounts of anhydrous potassium carbonate
afforded the respective acrylamide thioglucosides 8a-d in good yields.
In a similar manner, acrylamide thiogalactosides 9a-d were obtained by the reaction of
compounds 7a-c, 7e, respectively, with 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl bromide
in refluxing ethanol containing potassium carbonate (Scheme 2). The structures of compounds
8
a-d and 9a-d were established based on microanalyses and spectral data. The IR spectrum of
compound 8a, as an example, showed characteristic absorption bands at 3268, 3181, 3133,
-1
2
201, 1754, 1648 cm due to three NH groups, CN, ester C=O, and amidic C=O functions,
5

+
respectively. Its mass spectrum revealed a molecular ion peak at m/z 737 (M ) which agrees
with its molecular formula (C H N O S ). The ion peaks appeared at m/z 331, 266, and 111
3
0
35
5
13 2
+
were attributed to fragment ions glucose unit [C H O ] , sulfisoxazole fragment
1
4
19
9
+
+
1
[
C H N O S] , and aminoisoxazole fragment [C H N O] ; respectively. In addition, the H-
11 12 3 3 5 7 2
NMR spectrum of 8a revealed the appearance of the anomeric proton as a doublet signal at 
.90 with coupling constant value (J 1',2' = 9.2 Hz) indicating the -configuration of acrylamide
thioglucoside 8a [22].
5
The reaction mechanism of the formation of acrylamide-thioglucosides 8a-d and
2
thiogalactosides 9a-d seemed to start with S displacement reaction of the thiolate ion in 7a-
N
c and 7e with -bromosugars to afford the -thioglucoside products 8a-d and -thiogalactoside
products 9a-d [23].
Finally, our attempts to remove the protecting acetate groups in acrylamide
thioglycosides 8 and 9 by treating with hot solution of methanolic ammonia failed to obtain the
free acrylamide thioglucosides.
<
Insert Scheme 2 >
2
.2. Antimicrobial evaluation
Twenty four of the target compounds were preliminary screened for their in vitro
antimicrobial activities against a panel of human pathogenic Gram-positive and Gram-negative
bacteria as well as pathogenic fungal strains [14-15]. Ampicillin, gentamycin, amphotericin B
and sulfisoxazole were used as reference drugs and the minimum inhibitory concentration
(
MIC) for the target compounds with significant inhibition zones (>13 mm) were determined
by broth micro-dilution method [24]. Details about the screened microbial species are provided
supplementary material).
(
2
.2.1. Antibacterial activity
Results for the initial antibacterial activity screening of the target compounds were
summarized in table 1.
The 3-(ethylthio)acrylamide derivatives 5f-h (scheme 1) displayed significant
antibacterial activities against all screened Gram-positive and Gram-negative bacteria. Of
these, compounds 5g and 5h were the most potent displaying similar activity to ampicillin
toward S. pneumoniae (MIC, 0.12 µg/mL) and two folds the activity of ampicillin toward B.
subtilis (MIC, 0.12 µg/mL vs 0.24 µg/mL). Compared to gentamycin, 3-(ethylthio)acrylamide
6

derivatives 5f-h were generally less potent against Gram-negative bacteria. Exceptionally, 5h
possessed two folds the activity of gentamycin toward E. coli (MIC, 0.98 µg/mL vs 1.95
µg/mL) and 5g was equipotent to gentamycin toward E. coli (MIC, 1.95 µg/mL). However,
compounds 5f, 5g and 5h exhibited increased activity against S. epidermidis when compared
to sulfisoxazole (MIC, 0.25-1.95 µg/mL vs 7.81 µg/mL). Likewise, they were significantly
more potent than sulfisoxazole against all tested Gram-negative bacteria, specially P. vulgaris
which was totally resistant to sulfisoxazole. Nonetheless, the 3-(methylthio)acrylamide
derivatives 5a, 5c and 5e and the 3-(ethylthio)acrylamide derivative 5i were totally inactive
against all tested bacteria. Finally, the 3-(ethylthio) acrylamide derivative 5j was moderately
active against only B. subtilis and S. epidermidis. Unfortunately, transforming the 3-(ethylthio)
acrylamide derivative 5j to the pyrazole compound 6 didn’t improve its antibacterial activity.
The 3-mercapto-acrylamide derivatives 7a-d inhibited the growth of all screened Gram-
positive and Gram-negative bacteria contrary to 7e which was inactive against all microbial
strains tested. Compounds 7a and 7c displayed the best results with improved activity
compared to sulfisoxazole against most of the screened bacterial strains. For instance, 7a, 7c
were four times and two times more potent than sulfisoxazole against S. epidermidis (MIC,
1
.95 µg/mL, 3.9 µg/mL vs 7.81 µg/mL; respectively). Also, 3-mercapto-acrylamide
derivatives 7a and 7c showed moderate activity against P. vulgaris (MIC, 7.81 µg/mL and 3.9
µg/mL; respectively) in contrast to sulfisoxazole which showed no activity. Interestingly, 7a,
7
c showed three folds and four folds the activity of sulfisoxazole against K. pneumonia (MIC,
.98 µg/mL, 0.49 µg/mL vs 7.81 µg/mL; respectively). Besides, the 3-mercapto-acrylamide
0
derivative 7c possessed two folds the potency of sulfisoxazole and half the activity of
ampicillin toward B. subtilis (MIC, 0.49 µg/mL vs 0.98, 0.24 µg/mL). Compound 7a showed
equipotent activity to sulfisoxazole against B. subtilis (MIC, 0.98 µg/mL). Moreover, the
mercaptoacrylamide derivatives 7a and 7c displayed equal activity to gentamycin toward E.
coli (MIC, 1.95 µg/mL).
Glucothioglycoside 8a-d showed broad spectrum activity against all screened Gram-
positive and most of the tested Gram-negative bacteria. Both compounds 8a and 8c showed
half potency of gentamycin and four folds the potency of sulfisoxazole toward E. coli (MIC,
3
.9 µg/mL vs 1.95 µg/mL, 62.5 µg/mL; respectively). Compound 8a was twice more potent
than sulfisoxazole against S. pneumoniae and S. epidermidis (MIC, 0.98 µg/mL & 3.9 µg/mL
vs 1.95 µg/mL & 7.81 µg/mL; respectively). Also, it was eight times efficient than
sulfisoxazole toward K. pneumonia (MIC, 0.98 µg/mL vs 7.81 µg/mL). Similarly, compound
7

8
b exhibited eight times the potency of sulfisoxazole toward K. pneumonia (MIC, 0.98 µg/mL
vs 7.81 µg/mL) while 8c showed four folds the potency of sulfisoxazole toward K. pneumonia
MIC, 0.49 µg/mL vs 7.81 µg/mL). Compound 8c demonstrated moderately increased activity
(
against S. epidermidis compared to sulfisoxazole (MIC, 3.9 µg/mL vs 7.81 µg/mL).
Unexpectedly, compounds 8a, 8b and 8d had some effect against P. vulgaris whereas
sulfisoxazole didn’t show any activity.
The galactothioglycosides 9a-d showed substantial activity against all screened Gram-positive
and Gram-negative bacteria. They also had the advantage of being active against P. vulgaris
while sulfisoxazole was completely inactive. Interestingly, compound 9b demonstrated three
folds the activity of sulfisoxazole against E. coli (MIC, 7.81 µg/mL vs 62.5 µg/mL) and was
equipotent to sulfisoxazole against K. pneumonia (MIC, 7.81 µg/mL). Similarly, compound 9c
exhibited three folds the activity of sulfisoxazole against E. coli (MIC, 7.81 µg/mL vs 62.5
µg/mL) and three folds the potency of sulfisoxazole against K. pneumonia (MIC, 0.98 µg/mL
vs 7.81 µg/mL) and was equipotent to sulfisoxazole toward B. subtilis (MIC, 0.98 µg/mL).
Finally, compound 9d showed weaker activity than sulfisoxazole against all screened bacterial
strains.
To prove our hypothesis on targeting folate pathway via inhibition of DHPS enzyme,
MIC shifts in the presence of folate precursors (pABA) and product (methionine) were
explored for the E. coli ATCC 25922. Both pABA and methionine antagonized the inhibitory
effect of the test compounds. An increase by at least one fold in the MIC values of the
compounds under investigation was observed. This is consistent with previous reports [25],
supporting our assumption that the mechanism of the antibacterial activities is mediated by
DHPS enzyme.
8

Table 1. Antibacterial inhibition zone in mm ± standard deviation and minimal inhibitory
concentrations (MIC, µg/ml, between brackets) for the target compounds
Compounds
Gram-positive bacteria
Gram-negative bacteria
S. pneumoniae
B. subtilis
NA
S. epidermidis
NA
E. coli
NA
NA
P. vulgaris
NA
K. pneumonia
a
5
a
NA
NA
NA
NA
NA
NA
5
5
c
e
NA
NA
NA
NA
NA
NA
NA
5
f
20.2 ± 0.63
21.2 ± 0.58
(0.98)
26.3 ± 0.63
(0.12)
26.8 ± 0.25
(0.12)
20.9 ± 0.58
(1.95)
22.8 ± 0.25
(0.49)
24.2 ± 0.46
(0.25)
18.2 ± 0.72
(7.81)
20.6 ± 0.63
(1.95)
21.8 ± 0.44
(0.98)
NA
16.4 ± 0.63
(31.25)
19.6 ± 0.46
(3.9)
20.7 ± 0.43
(3.9)
21.3 ± 0.58
(1.95)
23.2 ± 0.53
(0.49)
24.7 ± 0.25
(0.25)
(
3.9)
24.3 ± 0.44
0.12)
24.9 ± 0.32
5
g
(
5
h
(0.12)
5
i
NA
NA
NA
NA
NA
5
j
NA
11.2 ± 0.25
10.1 ± 0.63
(NT)
NA
NA
NA
b
(NT )
6
NA
12.6 ± 0.33
NT)
13.4 ± 0.63
(15.62)
NA
NA
NA
(
7
a
20.3 ± 0.44
3.9)
13.7 ± 0.43
62.5)
21.7 ± 0.44
0.98)
16.9 ± 0.44
22.4 ± 0.58
(0.98)
15.4 ± 0.58
(62.5)
23.8 ± 0.63
(0.49)
17.4 ± 0.25
(31.25)
20.8 ± 0.32
(1.95)
17.3 ± 0.53
(31.25)
20.1 ± 0.63
(3.9)
16.2 ± 0.53
(62.5)
20.6 ± 0.63
(1.95)
14.3 ± 0.58
(62.5)
21.2 ± 0.25
(1.95)
13.3 ± 0.44
(62.5)
18.3 ± 0.58
(7.81)
12.6 ± 0.63
(NT)
20.1 ± 0.63
(3.9)
11.7 ± 0.63
(NT)
22.6 ± 0.46
(0.98)
16.4 ± 0.44
(62.5)
24.2 ± 0.58
(0.49)
16.2 ± 0.58
(62.5)
(
7
b
(
7
c
(
7
d
(31.25)
7
e
NA
NA
NA
NA
NA
NA
8
a
22.2 ± 0.43
0.98)
25.3 ± 0.44
3.9)
26.7 ± 0.34
1.95)
10.6 ± 0.72
NT)
17.3 ± 0.58
15.63)
18.9 ± 0.41
7.8)
19.3 ± 0.44
3.9)
15.1 ± 0.58
62.5)
19.6 ± 0.44
1.95)
23.8± 0.2
23.4 ± 0.53
(0.98)
27.5 ± 0.25
(1.95)
28.6 ± 0.25
(0.98)
12.4 ± 0.63
(NT)
16.8 ± 0.48
(15.63)
17.6 ± 0.63
(7.8)
20.8 ± 0.58
(0.98)
19.8 ± 0.63
(1.95)
20.4 ± 0.58
(3.9)
24.7 ± 0.63
(3.9)
25.6 ± 0.25
(3.9)
18.9 ± 0.53
(15.62)
15.4 ± 0.44
(62.5)
16.3 ± 0.25
(31.25)
17.1 ± 0.58
(15.63)
14.2 ± 0.72
(125)
20.2 ± 0.25
(3.9)
21.3 ± 0.33
(7.81)
23.4 ± 0.58
(3.9)
10.6 ± 0.58
(NT)
16.3 ± 0.48
(15.63)
17.1 ± 0.58
(7.81)
18.3 ± 0.72
(7.81)
13.2 ± 0.63
(125)
17.8 ± 0.53
(7.81)
21.4 ± 0.63
(7.81)
22.1 ± 0.44
(0.98)
18.8 ± 0.63
(0.98)
23.2 ± 0.63
(0.49)
14.2 ± 0.25
(62.5)
17.1 ± 0.41
(31.25)
19.8 ± 0.63
(7.81)
21.2 ± 0.63
(0.98)
15.8 ± 0.58
(62.5)
(
8
b
(
8
c
NA
(
8
d
8.3 ± 0.63
(NT)
11.8 ± 0.44
(NT)
13.4 ± 0.41
(125)
15.2 ± 0.63
(62.5)
(
9
a
(
9
b
(
9
c
(
9
d
11.2 ± 0.48
(NT)
(
Sulfisoxazole
Ampicillin
20.8± 0.63
(0.98)
32.4± 0.3
(0.24)
17.5± 0.72
(7.81)
25.4± 0.18
(0.06)
15.2 ± 0.25
(62.5)
NA
18.2± 0.58
(7.81)
(
NT
NT
NT
(0.12)
Gentamycin
NT
NT
NT
19.9± 0.3
23.4± 0.3
(0.24)
26.3± 0.15
(0.03)
(1.95)
a
NA, no activity.
b
NT, not tested.
2
.2.2. Antifungal activity
The antifungal activity for most of the target compounds was summarized in table 2.
The 3-(methylthio)acrylamide derivative 5c was active against all screened fungal strains. It
showed double the activity of sulfisoxazole toward S. racemosum (MIC, 62.5 µg/mL vs 125
µg/mL). Also, it was equipotent to sulfisoxazole against G. candidum (MIC, 15.63 µg/mL). On
the other hand the 3-(methylthio)acrylamide derivatives 5a and 5e didn’t show any antifungal
activity. Surprisingly, some of the 3-(ethylthio)acrylamide derivatives 5f-j were more potent
9

than the reference drug amphotericin B. Compound 5h was three folds more potent than
amphotericin B toward S. racemosum (MIC, 0.98 µg/mL vs 7.81 µg/mL) while 5f-g exhibited
two fold the activity of amphotericin B toward S. racemosum (MIC, 3.9 µg/mL, 3.9 µg/mL vs
7
.81 µg/mL; respectively). Compounds 5f, 5g and 5h showed weaker activity than
amphotericin B but with improved antifungal activity compared to sulfisoxazole toward A.
fumigates (MIC, 0.49-15.63 µg/mL vs 62.5 µg/mL) and G. candidum (MIC, 1.95-7.81 µg/mL
vs 15.63 µg/mL). Unfortunately, both 5i and 5j were totally inactive against all screened fungal
strains in contrast to sulfisoxazole. Transforming compound 5j to the pyrazole compound 6
didn’t improve the antifungal activity.
It was observed that 3-mercapto-acrylamide derivatives 7a-e, in most of the cases, had
better antifungal activity than sulfisoxazole against the screened fungal strains. Fortunately,
compound 7c showed two folds the activity of amphotericin B toward S. racemosum (MIC, 3.9
µg/mL vs 7.81 µg/mL) whereas, compound 7b was equipotent to amphotericin B toward S.
racemosum (MIC, 7.81 µg/mL). The antifungal activity of compound 7a against S. racemosum
was much better than sulfisoxazole (MIC, 15.63 µg/mL vs 125 µg/mL). On the other hand, 3-
mercapto-acrylamide derivatives 7d-e almost had the same potency of sulfisoxazole.
Regarding the antifungal activity of 3-mercapto-acrylamide derivatives 7a-e against A.
fumigates, it was observed that compounds 7c, 7a, 7e were seven-folds, four folds and two
folds more potent than sulfisoxazole (MIC, 0.49 µg/mL, 3.9 µg/mL, 15.62 µg/mL vs 62.5
µg/mL; respectively). Also, it was noticed that compounds 7c and 7a were six-folds and two
folds more active than sulfisoxazole against G. candidum (MIC, 0.24 µg/mL, 3.9 µg/mL vs
1
5.63 µg/mL; respectively).
The results summarized in table 2 revealed that the glucothioglycoside derivatives 8a-
c in most of the cases displayed better antifungal activities than sulfisoxazole whereas
compound 8d was completely inactive. Auspiciously, compounds 8a and 8c were one fold and
two folds more potent than the antifungal drug amphotericin B toward S. racemosum (MIC,
3
.9 µg/mL and 1.95 µg/mL vs 7.81 µg/mL; respectively). Compounds 8a-c were four to six
times more potent than sulfisoxazole toward A. fumigatus (MIC, 0.98-3.9 µg/mL vs 62.5
µg/mL; correspondingly). Also, compounds 8a-c showed three to five folds better activity than
sulfisoxazole against G. candidum (MIC, 0.49-1.95 µg/mL vs 15.63 µg/mL; respectively).
Similarly, the galactothioglycosides 9a-c displayed broad spectrum antifungal activity
against A. fumigates, S. racemosum and G. candidum. They were one to three folds more potent
than sulfisoxazole against A. fumigates and S. racemosum (MIC, 7.81-31.25 µg/mL, 15.63-
1
0

6
2.5 µg/mL vs 62.5, 125 µg/mL; respectively). Also, compound 9c exhibited two folds the
potency of sulfisoxazole against G. candidum (MIC, 3.9 µg/mL vs 15.63 µg/mL). On the other
hand, the galactothioglycosides 9d was totally inactive.
Table 2. Antifungal inhibition zone in mm ± standard deviation and minimal inhibitory
concentrations (MIC, µg/ml, between brackets) of some new synthesized compounds
Compounds A. fumigatus
S. racemosum
G. candidum
Compounds
7d
A. fumigatus
14.9± 0.25
S. racemosum
G. candidum
14.7± 0.58
(125)
a
5
a
NA
NA
NA
13.6± 0.25
b
(62.5)
(NT )
5
5
c
e
12.6± 0.58
15.7± 0.63
(62.5)
18.2± 0.58
(15.63)
NA
7e
16.8± 0.36
(15.62)
19.3± 0.58
14.3± 0.44
(125)
19.9± 0.58
(3.9)
17.1± 0.58
(31.25)
21.2± 0.44
(1.95)
(NT)
NA
NA
8a
(3.9)
5
f
17.1± 0.19
19.9± 0.58
(3.9)
20.3± 0.58
(3.9)
22.4± 0.25
(0.98)
18.3± 0.28
(7.81)
20.4± 0.72
(3.9)
22.1± 0.16
(1.95)
8b
24.2± 0.25
(3.9)
26.1± 0.37
(0.98)
21.3± 0.58
(15.63)
24.4± 0.25
(1.95)
22.1± 0.63
(0.98)
23.2± 0.58
(0.49)
(
15.63)
21.6± 0.63
0.98)
22.9± 0.39
5
g
8c
(
5
h
8d
NA
NA
NA
(0.49)
5
i
NA
NA
NA
9a
16.3± 0.63
15.1± 0.36
(62.5)
15.9± 0.19
(31.25)
17.2± 0.58
(15.63)
16.9± 0.25
(62.5)
18.3± 0.36
(31.25)
20.2± 0.19
(3.9)
(
31.25)
17.2± 0.58
15.62)
18.3± 0.44
5
j
NA
NA
NA
NA
NA
NA
9b
(
6
9c
9d
(7.81)
7
a
18.6± 0.63
3.9)
12.6± 0.1
NT)
22.3± 0.39
0.49)
17.2± 0.58
(15.63)
13.2± 0.2
(7.81)
20.4± 0.16
(3.9)
18.9± 0.58
(3.9)
17.1± 0.1
(15.63)
25.2± 0.58
(0.24)
NA
NA
NA
(
7
b
Sulfisoxazole
15.1± 0.39
(62.5)
23.7± 0.1
(0.12)
13.2 ± 0.58
(125)
19.7± 0.2
(7.81)
16.8 ± 0.58
(15.63)
28.7± 0.2
(0.007)
(
7
c
Amphotericin
B
(
a
NA, no activity.
NT, not tested.
b
2
.3. Cytotoxicity evaluation
The percent cytotoxicity of the most active target compounds is represented in figure
. All compounds were generally safe to normal cells and none of them exceeded 30%
4
cytotoxicity. Cisplatin, a clinically used anticancer drug, showed more than 80% cytotoxicity.
We conclude that the compounds are primarily safe, however further toxicology studies are
recommended to confirm their safety.
<
Insert Figure 4 >
2
2
.4. In silico study
.4.1. Molecular Docking:
The aim of the modelling part is to rationalize the observed in vitro antimicrobial
activity on a molecular basis. Since our design strategy is based on inhibition of the putative
bacterial target DHPS, we carried out molecular docking experiments of representative
1
1

compounds to our scaffolds namely, 5g, 7c, 8a and 9c against DHPS X-ray crystal structure
PDB code: 5jq9).
(
2
.4.1.1. Selection of a model for docking:
In this study we propose new compounds based on the rationale of introducing
acrylamide thiols, acrylamide thioglycosides and pyrazole hybrids as new substructures to
occupy the pterin pocket of the DHPS binding site. This would expose new chemotype [26]
classes for DHPS inhibitors as antimicrobial candidates. Our proposed substructures possess
relatively larger topological features than the standard planar pterin moiety which were used to
occupy the flat cleft of the pterin pocket. Therefore, one would expect some conformational
changes in the pterin pocket residues to accommodate binding events of our relatively larger
moieties. Thus, selecting a suitable model of the DHPS from the protein data bank (PDB) for
docking experiments should be considered. Our selection of a particular model was based on
meta-analysis of multiple DHPS structures from the PDB. For this, we collected all bacterial
DHPS from the PDB comprising 62 structures (Table S1 in the supplementary material). Then
we selected an X-ray structure (PDB code: 5jq9) that possesses the heaviest co-crystal ligand
in its binding site with high similarity to our designed compounds with the sulfisoxazole moiety
which blocks the access towards the pABA pocket of the enzyme. Generally, such approach
would, to a certain extent, help in speculating binding site conformational changes, especially
in the pterin pocket of the enzyme, due to binding adaptation caused by relatively larger ligands.
Accordingly, we applied a flexible docking approach to rationalize the postulated binding
modes of our representative compounds 5g, 7c, 8a and 9c in the binding site of DHPS (more
details of the docking setup are discussed in the experimental section).
2
.4.1.2. Description of the docking poses
The docking pose of 5g displays favorable interactions in the binding site of DHPS.
The sulfisoxazole moiety demonstrated a tolerable overlay with the same part of the co-crystal
ligand, as observed in Figure 5A. This comprised H-bond interactions with Ser215 via the
sulfonyl group as an acceptor. Additionally, the dimethylisoxazolyl group showed a favorable
hydrophobic contact with Pro57. The central phenyl group appeared to be packed between the
side chains of Lys214 and Phe183 demonstrating hydrophobic interactions. Based on the above
data, this represents key interactions in the pABA pocket of the DHPS which guarantees a
proper blockage of this part of the enzyme. The acrylamide ethyl thiol moiety appeared to fill
the region formed by Ile110, Asp49, Asp89, Gly210, Asp178, Gly180 which blocks the access
1
2

towards the pterin pocket of DHPS. Overall, these types of interactions showed high in silico
affinity with a docking score of 61.78.
Like 5g, the docking pose of the congeneric 7c reproduced the key interactions of the
sulfisoxazole moiety in the pABA pocket of the DHPS, as seen in Figure 5B. Only major
differences in the postulated binding modes between 7c and 5g can be observed for the thiol
and the N-allyl groups of 7c. The N-allyl group showed hydrophobic interactions with the side
chain of Phe183 and the backbone of Gly210, while H-bonding interaction with the side chain
of Lys214 was observed. Overall, these types of interactions contributed to the high in silico
affinity with a docking score of 73.45.
<
Insert Figure 5 >
To investigate the effect of glycosylating some of our compounds, we analyzed the
postulated binding mode of 8a and 9c as representative examples of the glycosylated
derivatives. Since estrases are widely distributed and produced by microorganisms [27, 28],
we depicted the sugar moieties of 8a and 9c as deacetylated structures in the in silico discussion
assuming that the tested bacteria / fungi had hydrolyzed the vulnerable sugar acetyl ester groups.
As expected the sulfisoxazole moiety of both docking poses reproduced the key interaction
pattern in the pABA pocket of the DHPS in comparable fashion to 5g and 7c, as demonstrated
(Figure 6). Interestingly, the galactopyranose moiety of 9c appeared to be directed towards the
hydrophilic region near the pterin pocket of the DHPS (Figure 6B). Therefore, a network of
H-bonding interactions with the polar side chains of Asp49, Asp89, Glu53 and Thr55 with
multiple donor and acceptor hydroxy groups of 9c can be observed. Subsequently, this led to a
gain of in silico affinity especially for 9c (docking score of 92.97) when compared to its non-
glycosidic congener 7c (docking score of 73.45). Concerning 8a, its glucopyranose moiety
occupies the hydrophilic region near the pterin pocket of the binding site in a similar pattern to
9
c pose, as observed in Figure 6A. Generally, such postulated binding modes of the sugar
moieties would rationalize the profound in vitro antimicrobial activities of the glycosylated
derivatives.
Comparing 9c and 8a poses, the N-allyl group of 9c appeared to access better hydrophobic
interactions in the binding site, namely with the side chain of Phe183 and the backbone of
Gly210, compared to the smaller N-methyl group of 8a. This contributed to the superior
docking score of 9c compared to 8a (69.69).
1
3

<
Insert Figure 6 >
2
.4.2. Physicochemical properties and pharmacokinetics profile predictions
This part offers a theoretical clue about the drug-likeness and the pharmacokinetic profile of
the representative compounds 5g, 7c, 8a and 9c. Normally, compounds that do not achieve the
drug-likeness criteria often do not prosper to be an effective clinical candidate due to poor
bioavailability, adverse effects or other concerns. The automated SwissADME [29] server was
used for these predictions.
Pharmacokinetics properties (Table 3) use certain models to evaluate individual ADME
behaviors of the compounds under investigation. The calculations for passive human
gastrointestinal absorption (GIA) and blood-brain barrier (BBB) permeation both were
concluded from the readout of the BOILED-Egg model [29, 30]. All compounds showed low
gastrointestinal absorption. Fortunately, these compounds fail to be permeable through BBB,
and therefore, they likely exhibit low occurrence for central nervous system (CNS) adverse
effects. P-gp substrate parameter indicates whether a compound being a substrate or non-
substrate of the permeability glycoprotein (P-gp)[29]. P-gp is crucial to assess active efflux
through biological membranes, for example, to preserve the CNS from xenobiotics, and
involved in the resistance mechanism of drugs [29, 31]. Compounds 5g and 7c (Table 3)
displayed no substrate activity towards P-gp while 8a and 9c appeared to be substrates for P-
gp.
Table 3. In silico predictions of the pharmacokinetics properties for the most active compounds.
GI
BBB
P-gp
CYP1A2
inhibitor
CYP2C19
inhibitor
CYP2C9
inhibitor
CYP2D6
inhibitor
CYP3A4
inhibitor
Compounds
absorption
permeant
substrate
5
g
Low
Low
Low
Low
No
No
No
No
No
No
No
No
No
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
7
c
8
a
Yes
Yes
9
c
No
No
No
GIA: human gastrointestinal absorption; BBB: blood-brain barrier permeation; P-gp: permeability glycoprotein; CYP1A2, CYP2C19,
CYP2C9, CYP2D6 and CYP3A4 are the five major isoforms of cytochromes P450 (CYP).
Providing information about compound interaction with the cytochromes P450 (CYP)
superfamily is of significance. It was appraised that 50-90% of therapeutic compounds are
substrate of at least one of the five major isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6,
CYP3A4) [29, 32, 33]. CYP inhibition is certainly one main cause of pharmacokinetics-based
drug-drug interactions [34, 35] resulting in toxic or adverse effects. As seen from Table 3, 8a
1
4

and 9c demonstrated inhibition of only one of CYP five major isoforms (CYP3A4) signifying
low occurrence of drug-drug interactions, while, 5g and 7c showed inhibition of some isoforms.
None of the compounds displayed any alert to be a probable PAINS (pan-assay interference
compounds) [36], as shown in Table 4. This pinpoints that their chemical nature lacks an
interfering scaffold that may produce an artefact response with protein assays.
The drug-likeness properties (Table 4) highlighted that the glycosylated compounds (8a and
9
c) demonstrated more violations for the Lipinski and Veber rules [37], compared to non-
glycosylated analogues (5g and 7c). Nevertheless, this drives us to recommend parenteral route
of administration for these candidates.
Table 4. In silico predictions of the drug-likeness properties for the most active compounds.
Compounds
LogP
ESOL class
Lipinski #violations
Veber #violations
PAINS #alerts
5
g
3.58
3.13
2.76
3.40
Moderately soluble
Moderately soluble
Moderately soluble
Moderately soluble
0
0
2
2
1
1
2
2
0
0
0
0
7
c
8
a
9
c
LogP is calculated as XLOGP3 descriptor [38]. ESOL class: is a solubility class based on LogS scale [39]. Lipinski #violations counts
the number of violations of Lipinski rule summarized as: lipophilicity (logP) ≤ 5, molecular weight ≤ 500, number of hydrogen bond
donors ≤ 5 and number of hydrogen bond acceptors ≤ 10. Veber #violations counts the number of violations of Veber rule summarized
2
as: NRB ≤ 10 and TPSA ≤ 140 Å . PAINS #alerts counts the number of pan-assay interference compounds/substrucutres. All calculations
were performed using SwissADME [29].
2
.5. Structure Activity Relationship (SAR)



Sulfisoxazole has moderate antimicrobial activity. This could be attributed to its low
lipophilic characteristic (LogP = 1.19). Moreover, it targets only the pABA binding
pocket of the DHPS enzyme.
Because the pterin binding pocket is polar [40]. Therefore, the polar acrylamide
moieties of the synthesized target compounds could be easily directed to the pterin
binding pocket and interact with it by its hydrogen bond donor/acceptor groups.
Substituting sulfisoxazole’s amino group by acrylamide derivatives, in most of the
cases, improved the antimicrobial activity because the target compounds binds to both
the pABA and pterin binding pockets at the same time (Figure 7). Moreover, some of
these acrylamide derivatives were more lipophilic than sulfisoxazole and accordingly
they have improved intracellular concentration than sufisoxazole.
1
5


Some of 3-(amino substituted)-2-cyano-3-(ethylthio) acrylamide derivatives 5f-j have
excellent antimicrobial activities compared to the parent sulfisoxazole. Excellent
activities were obtained when the substitution on the amino group is small alkyl group.
The antimicrobial activity has the following descending order: 5h (allyl amino
derivative) > 5g (ethyl amino derivative) > 5f (methyl amino derivative). Derivatives
5
i and 5j with bulky substitutions, adamantyl or phenyl, on the amino group showed
weak or even no antimicrobial activities. Molecular modeling showed that the
acrylamide ethyl thiol moiety of compound 5g fills the region formed by Ile110, Asp49,
Asp89, Gly210, Asp178, Gly180 which blocks the access towards the pterin pocket of
DHPS. Unexpectedly replacing the ethylthio moiety by the less bulky methylthio
group, compounds 5a-e, abolished the antimicrobial activity. Only compound 5c
possessed antifungal activity.

Interestingly, the 3-(mercapto)acrylamide derivatives 7a-e displayed quite less potent
antibacterial activities than the 3-(ethylthio)acrylamide derivatives 5f-j and higher
antifungal activities than 5f-h. The antibacterial activities of 7a-e were in the following
descending order 7c (allyl amino) > 7a (methyl amino) > 7d (ethyl amino) while the
phenyl amino derivative has no antibacterial activity. On the other hand, the antifungal
activity of 7a-e was in the following descending order 7c > 7a > 7b > 7e > 7d. The high
activity of compound 7c could be attributed to the hydrophobic interactions of the allyl
moiety with the side chain of Phe183 and the backbone of Gly210.

Regarding the thioglycoside derivatives 8a-d and 9a-d, the glucothioglycosides 8a-d
had better antimicrobial activities than the galactothioglycosides 9a-d. The allyl amino
glycosides 8c and 9c were the most potent derivatives. This could be in part due to the
hydrophobic interactions of the allyl moieties with the binding site of the DHPS,
namely with the side chain of Phe183 and the backbone of Gly210. Moreover, the sugar
moieties were directed towards the hydrophilic region near the pterin pocket of the
DHPS as explained by the docking experiments (Figure 6) and this could explain the
profound in vitro antimicrobial activities of the glycosylated derivatives. As expected
the bulky phenyl amino derivatives 8d and 9d showed no antifungal activities.
<
Insert Figure 7 >
2
.6 Radiolabeling and in vivo infection targeting ability of 99mTc-5g:
2
.6.1 99mTc-5g complex preparation:
1
6

The radiochemical yield 99mTc-5g was determined by evaluation the percent of 99mTc-5g, free
9
9m
TcO and reduced hydrolyzed-99mTc colloid species, (Figure 8: A-D), as follows:
-
4
2
.6.1.1 Effect of sodium dithionate content:
Sodium dithionate is employed to reduce 99mTcO to lower oxidation state of 99mTc to be ready
-
4
to complex with the ligand. As shown in Figure 8A, the radiochemical yield of 99mTc-5g was highly
suffering from the sodium dithionate content (3-20 mg). At low sodium dithionate (3 mg), the
radiochemical yield was low. By increasing sodium dithionite content to 10 mg, the maximum
radiochemical yield of 99mTc-5g (92 ± 1.34%) was obtained. More than 10 mg, the radiochemical yield
of 99mTc-5g was decreased and the free 99mTcO was increased.
-
4
2
.6.1.2 Effect of 5g compound content
The content of 5g was studied within the range of 100-900µl from a solution with concentration
mg/ml 0.1-1µg (Figure 8B). At 100 µL 5g, the radiochemical yield was low where the quantity of
1
the 5g wasn’t sufficient to complex all the reduced technetium-99m to get a high radiochemical yield
41]. By increasing the 5g amount to 600 µL, the maximum radiochemical yield of 99mTc-5g (92 ± 1.34
) was obtained. Then the radiochemical yield was slightly decrease by increasing the 5g amount over
00 µL.
[
%
6
2
.6.1.3 Effect of pH
Figure 8C represents the pH effect (ranging from 3 to 11) on the radiochemical yield of 99mTc-
5
g. At pH 3, the radiochemical yield was extremely low and increased upon increasing the pH. The
optimum pH was 6 where the maximum radiochemical yield of 99mTc-5g (92 ± 1.34 %) was obtained.
Beyond the optimum pH, the radiochemical yield was decreased upon increasing the pH.
2
.6.1.4 Effect of time
Figure 8D shows that the maximum radiochemical yield of 99mTc-5g (92 ± 1.34 %)
was obtained after 15 min incubation time of the reaction.
In conclusion, the best yield of 99mTc-5g (92 ± 1.34%) was obtained upon using 600 μL
(1mg/mL) of compound 5g and 10 mg sodium dithionite at pH 6 and allowing the reaction to proceed
for 15 min, additionally, 99mTc-5g showed in-vitro stability up to six hours.
<
Insert Figure 8 >
2
.6.2 In vivo infection targeting ability study of 99mTc-5g in infection bearing mice model:
Figure 9 shows the in vivo biodistribution of 99mTc-5g in infection bearing mice at different
time intervals. The infected muscle (T. muscle) uptake was greater than the normal muscle at
1
7

all time intervals. Liver, intestine and kidneys high radioactivity levels indicate that the
excretion route of 99mTc-5g is through hepatobiliary and urinary pathways.
The ratio of target to non-target (T/NT) indicates the selectivity and targeting ability of the
molecule toward specific organ or diseased tissue [42].
The molecule is taken into account selective when the T/NT is more than 1.5, and also the
higher the T/NT value the higher is the selectivity. In this research, the target was the infected muscle
in the left thigh of the mouse and also the non-target was the normal muscle in the other thigh of the
mouse [43].
As shown in Figure 10, the target to non-target ratio (Infected muscle/normal muscle; T/NT)
was greater than 1 at all time intervals and reached its maximum value at 1 hr post injection
(T/NT= 5.2).
<
Insert Figure 9 >
Insert Figure 10 >
<
Conclusion
Microbial resistance to the available drugs is a problem and the search for new potent
antimicrobial agents is continuous to solve this problem. Twenty three new functionalized
acrylamide derivatives and one pyrazole carboxamide derivative tagged with sulfisoxazole
moiety were synthesized. The in vitro antimicrobial activities of the target compounds were
assessed toward a panel of human pathogenic bacterial and fungal strains. The E-configuration
of compound 5b was verified by single crystal X-ray analysis. The target compounds were
designed to bind to both the pABA and pterin binding sites of DHPS at the same time for the
purpose of increasing the therapeutic potential of sulfisoxazole. Compounds 5g and 5h were
one fold more active than ampicillin toward B. subtilis (MIC, 0.12 µg/mL vs 0.24 µg/mL;
respectively) and displayed equal activity to ampicillin toward S. pneumonia (MIC, 0.12
µg/mL). Also, compound 5h was one fold more active than gentamycin toward E. coli (MIC,
0
.98 µg/mL vs 1.95 µg/mL; respectively) while compounds 5g, 7a, 7c were equipotent to
gentamycin against E. coli. Interestingly, compounds 5f, 5g, 7c, 8a and 8c exhibited two folds
the activity of amphotericin B toward S. racemosum (MIC: 3.9 µg/mL vs 7.81 µg/mL;
respectively) and compound 7b was equipotent to amphotericin B against S. racemosum.
Compounds 5h displayed three folds the activity of amphotericin B against S. racemosum.
Compounds 5f-h, 7a, 7c, 8a-c, 9c displayed better antimicrobial activities than sulfisoxazole.
1
8

The majority of the target compounds showed activity against P. vulgaris while sulfisoxazole
was completely inactive. The highly potent target compounds were safe to normal cells. Most
of the target compounds had improved antimicrobial activities than the parent sulfisoxazole.
Inhibition of DHPS enzyme as a putative target for antimicrobial activity was confirmed by
exploring MIC shifts in the presence of folate precursors (pABA) and product (methionine) for
the E. coli ATCC 25922. Docking experiments rationalized the observed antimicrobial activity
and provided insights on binding capabilities to both pABA and pterin binding pockets of
DHPS at the same time. Finally, the in vivo infection targeting ability of 99mTc-5g was studied.
Biodistribution results showed a remarkable high accumulation of radioactivity in infected
muscle compared to normal muscle (high T/NT). Thus 99mTc-5g could be used as infection
diagnostic agent.
3
. Experimental
3
.1. Chemistry
1
The instruments used for measuring the melting points, spectral data (IR, Mass, H-NMR and
13C-NMR, X-ray) and elemental analysis are provided (Supplementary material). Sulfisoxazole
1
, acetobromo-α-D-glucose and acetobromo-α-D-galactose were obtained from Sigma-
Aldrich. 1-Cyanoacetyl-3,5-dimethylpyrazole [44] and 2-Cyano-N-(4-{[(3,4-
2
dimethylisoxazole-5-yl)amino]sulfonyl}phenyl)acetamide 3 [14] were synthesized according
to the reported procedures. The procedures for the synthesis of (E)-3-(substituted amino)-3-
(alkylthio)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)acrylamide
derivatives (5a-j), 3-amino-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-5-
(
phenylamino)-1H-pyrazole-4-carboxamide (6), (E)-3-(alkylamino/phenylamino)-2-cyano-N-
4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-mercaptoacrylamides (7a-e), (E)-
(
3
-(substituted amino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-
(2΄,3΄,4΄,6΄-tetra-O-acetyl-β-D-glucopyranosylthio)acrylamide (8a-d), (E)-3-(substituted
amino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(2΄,3΄,4΄,6΄-
tetra-O-acetyl-β-D-galactopyranosylthio)acrylamide (9a-d) are provided (Supplementary
material).
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(methylamino)-3-
(methylthio)-acrylamide (5a)
1
9

-1
Yellow powder, yield (85%), mp 183-184˚C; IR (KBr) νmax/cm : 3362 (NH), 3234 (NH), 3129
3
1
(
NH), 3065 (CH-Ar), 2978 (CH- sp ), 2198 (CN), 1652 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.66 (s, 3H, CH ), 2.10 (s, 3H, CH ), 2.66 (s, 3H, SCH ), 3.61 (s, 3H, NCH ), 7.65
3
3
3
3
(d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 7.78 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 9.58
2 6 3 5
+
(
s, 1H, NH), 10.55 (s, 1H, NHSO ), 11.00 (s, 1H, CONH); MS m/z (%): 422 ([M+H] , 0.28),
2
+
4
1
4
21 (M , 0.77), 374 (1.2), 325 (0.79), 310 (5.0), 294 (1.9), 246 (3.2), 155 (79.6), 127 (17.5),
11 (19.6), 96 (9.9), 89 (5.2), 81 (100); Anal. Calcd. for C H N O S (421.49): C, 48.44; H,
1
7
19
5
4 2
.54; N, 16.62 %, Found: C, 48.49; H, 4.58; N, 16.59%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(ethylamino)-3-
(methylthio)-acrylamide (5b)
-1
Yellow powder, yield (79%), mp 182-183˚C; IR (KBr) νmax/cm : 3369 (NH), 3231 (NH), 3125
3
1
(
NH), 3067 (CH-Ar), 2972 (CH- sp ), 2194 (CN), 1650 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.19 (t, J= 7.15 Hz, 3H, CH ), 1.64 (s, 3H, CH ), 2.09 (s, 3H, CH ), 2.66 (s, 3H,
3
3
3
SCH ), 3.58 (q, J= 7.15 Hz, 2H, NCH ), 7.67 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 7.79
3
2
2
6
(
d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 9.67 (s, 1H, NH), 10.51 (s, 1H, NHSO ), 10.91 (s,
3 5 2
1
3
1
H, CONH); C-NMR (125 MHz, DMSO-d ): δ = 5.8 (CH ), 10.4 (CH ), 15.2 (CH ), 17.6
6 ppm 3 3 3
(
SCH ), 40.9 (NCH ), 74.5 (Acrylamide-C ), 115.0 (CN), 119.1, 120.2, 127.4, 133.5, 143.2,
3 2 2
1
55.5, 161.3, 165.9, 170.6 (11C, C H , Isoxazole-C , Isoxazole-C , Isoxazole-C , Acrylamide-
6 4 4 3 5
+
C , CO); MS m/z (%): 435 (M , 1.8), 339 (31.7), 324 (7.9), 251 (13.2), 184 (5.1), 175 (3.2),
3
1
1
11 (25.3), 96 (9.9), 95 (100); Anal. Calcd. for C H N O S (435.52): C, 49.64; H, 4.86; N,
18 21 5 4 2
6.08 %, Found: C, 49.69; H, 4.91; N, 16.14%.
(E)-3-(Allylamino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-
(methylthio)-acrylamide (5c)
-1
Yellow powder, yield (68%), mp 178-179˚C; IR (KBr) νmax/cm : 3372 (NH), 3228 (NH), 3121
3
1
(
NH), 3069 (CH-Ar), 2981 (CH- sp ), 2199 (CN), 1653 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.64 (s, 3H, CH ), 2.09 (s, 3H, CH ), 2.65 (s, 3H, SCH ), 4.23 (m, 2H, CH N), 5.19-
3
3
3
2
5
7
.22 (m, 2H, CH =), 5.90-5.97 (m, 1H, CH=), 7.67 (d, J= 8.5 Hz, 2H, Phenyl-H , Phenyl-H ),
2 2 6
.80 (d, J= 8.5 Hz, 2H, Phenyl-H , Phenyl-H ), 9.71 (s, 1H, NH), 10.64 (s, 1H, NHSO ), 10.91
3
5
2
1
3
(
s, 1H, CONH); C-NMR (125 MHz, DMSO-d ): δ = 5.8 (CH ), 10.2 (CH ), 17.8 (SCH ),
6 ppm 3 3 3
4
1
7.8 (NCH ), 75.5 (Acrylamide-C ), 105.0 (CN), 116.6, 118.9, 120.3, 127.4, 133.7, 134.0,
2 2
43.1, 155.5, 161.3, 165.9, 171.3 (13C, CH =, CH=, C H , Isoxazole-C , Isoxazole-C ,
2
6
4
4
3
+
Isoxazole-C , Acrylamide-C , CO); MS m/z (%): 447 (M , 3.2), 406 (12.3), 351 (2.3), 294
5
3
2
0

(
0.9), 272 (21.9), 266 (11.1), 175 (100), 96 (55.0); Anal. Calcd. for C H N O S (447.53): C,
19 21 5 4 2
5
0.99; H, 4.73; N, 15.65 %, Found: C, 50.96; H, 4.77; N, 15.71%.
(E)-3-(Adamantylamino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-
3
-(methylthio)-acrylamide (5d)
-1
Yellow powder, yield (71%), mp 143-144˚C; IR (KBr) νmax/cm : 3363 (NH), 3225 (NH), 3135
3
1
(
NH), 3044 (CH-Ar), 2911 (CH-sp ), 2190 (CN), 1651 (CO); H-NMR (500 MHz, DMSO-d ):
6
δppm = 1.64-1.67 (m, 12H, CH , 9 x Adamantyl-H), 2.01 (m, 3H, 3 x Adamantyl-H), 2.08-2.09
3
(
m, 6H, CH , 3 x Adamantyl-H), 2.67 (s, 3H, SCH ), 7.69 (d, J= 9.0 Hz, 2H, Phenyl-H ,
3 3 2
Phenyl-H ), 7.81 (d, J= 8.5 Hz, 2H, Phenyl-H , Phenyl-H ), 9.70 (s, 1H, NH), 10.59 (s, 1H,
6
3
5
+
+
NHSO ), 10.95 (s, 1H, CONH); MS m/z (%): 543 ([M+2] , 6.7), 541 (M , 10.4), 406 (6.7), 366
2
(8.5), 290 (7.7), 275 (6.4), 175 (0.5), 141 (100), 96 (3.3); Anal. Calcd. for C H N O S
26 31 5 4 2
(541.69): C, 57.65; H, 5.77; N, 12.93 %, Found: C, 57.69; H, 5.81; N, 12.99%.
(
E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(methylthio)-3-
phenylamino)acrylamide (5e)
(
-1
Yellow powder, yield (83%), mp 110-111˚C; IR (KBr) νmax/cm : 3386 (NH), 3246 (NH), 3133
3
1
(
NH), 3076 (CH-Ar), 2987 (CH-sp ), 2186 (CN), 1654 (CO); H-NMR (500 MHz, DMSO-d ):
6
δppm = 1.66 (s, 3H, CH ), 2.08 (s, 3H, CH ), 2.64 (s, 3H, SCH ), 7.34-7.56 (m, 5H, C H ), 7.71
3
3
3
6
5
(
(
(
(
d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 7.92 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 9.98
2 6 3 5
+
s, 1H, NH), 10.55 (s, 1H, NHSO ), 10.98 (s, 1H, CONH); MS m/z (%): 483 (M , 5.7), 457
2
11.6), 387 (2.9), 372 (17.0), 308 (16.8), 217 (100), 151 (1.9); Anal. Calcd. for C H N O S
4 2
2
2
21
5
483.56): C, 54.64; H, 4.38; N, 14.48 %, Found: C, 54.61; H, 4.43; N, 14.52%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(ethylthio)-3-
(methylamino)-acrylamide (5f)
-1
Yellow powder, yield (88%), mp 180-181˚C; IR (KBr) νmax/cm : 3360 (NH), 3256 (NH), 3168
3
1
(
NH), 3087 (CH-Ar), 2928 (CH- sp ), 2191 (CN), 1648 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.30 (t, J= 7.5 Hz, 3H, CH ), 1.63 (s, 3H, CH ), 2.09 (s, 3H, CH ), 3.15 (m, 2H,
3
3
3
SCH ), 3.36 (s, 3H, NCH ), 7.67 (d, J= 8.5 Hz, 2H, Phenyl-H , Phenyl-H ), 7.79 (d, J= 9.0
2
3
2
6
Hz, 2H, Phenyl-H , Phenyl-H ), 9.64 (s, 1H, NH), 10.51 (s, 1H, NHSO ), 10.91 (s, 1H, CONH);
3
5
2
1
3
C-NMR (125 MHz, DMSO-d ): δ = 5.8 (CH ), 10.2 (CH ), 14.7 (CH ), 28.8 (SCH ), 32.9
6
ppm
3
3
3
2
(
NCH ), 76.0 (Acrylamide-C ), 105.0 (CN), 119.2, 120.2, 127.4, 133.5, 143.2, 155.5, 161.3,
3 2
1
65.7, 168.9 (11C, C H , Isoxazole-C , Isoxazole-C , Isoxazole-C , Acrylamide-C , CO); MS
6 4 4 3 5 3
2
1

+
m/z (%): 435 (M , 0.7), 420 (15.6), 339 (23.3), 251 (13.2), 184 (16.9), 175 (8.4), 111 (33.6),
9
6 (18.3), 81 (100); Anal. Calcd. for C H N O S (435.52): C, 49.64; H, 4.86; N, 16.08 %,
18 21 5 4 2
Found: C, 49.60; H, 4.90; N, 16.11%.
(
E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(ethylamino)-3-
ethylthio)-acrylamide (5g)
(
-1
Yellow crystals, yield (85%), mp 166-167˚C; IR (KBr) νmax/cm : 3354 (NH), 3235 (NH), 3158
3
1
(
NH), 3069 (CH-Ar), 2976 (CH- sp ), 2190 (CN), 1646 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.19 (t, J= 7.0 Hz, 3H, CH ), 1.31 (t, J= 7.5 Hz, 3H, CH ), 1.64 (s, 3H, CH ), 2.09
3
3
3
(
s, 3H, CH ), 3.19 (q, J= 7.5 Hz, 2H, SCH ), 3.59-3.62 (m, 2H, NCH ), 7.67 (d, J= 9.0 Hz,
3 2 2
2
1
H, Phenyl-H , Phenyl-H ), 7.79 (d, J= 8.5 Hz, 2H, Phenyl-H , Phenyl-H ), 9.65 (s, 1H, NH),
2 6 3 5
1
3
0.64 (s, 1H, NHSO ), 10.91 (s, 1H, CONH); C-NMR (125 MHz, DMSO-d ): δ = 5.8
ppm
2
6
(
CH ), 10.2 (CH ), 14.7 (CH ), 15.2 (CH ), 29.2 (SCH ), 40.8 (NCH ), 75.9 (Acrylamide-C ),
3 3 3 3 2 2 2
1
05.0 (CN), 119.1, 120.3, 127.4, 133.6, 143.2, 155.5, 161.3, 165.9, 168.1 (11C, C H ,
6 4
+
Isoxazole-C , Isoxazole-C , Isoxazole-C , Acrylamide-C , CO); MS m/z (%): 449 (M , 3.3),
4
3
5
3
4
20 (320), 294 (0.7), 266 (8.1), 175 (13.1), 155 (3.3), 95 (100); Anal. Calcd. for C H N O S
19 23 5 4 2
(449.55): C, 50.76; H, 5.16; N, 15.58 %, Found: C, 50.80; H, 5.19; N, 15.64%.
(E)-3-(Allylamino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-
(ethylthio)-acrylamide (5h)
-1
Yellow powder, yield (71%), mp 110-111˚C; IR (KBr) νmax/cm : 3390 (NH), 3246 (NH), 3179
3
1
(
NH), 3090 (CH-Ar), 2979 (CH- sp ), 2193 (CN), 1644 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.33 (t, J= 7.5 Hz, 3H, CH ), 1.64 (s, 3H, CH ), 2.09 (s, 3H, CH ), 3.20 (q, J= 7.5
3
3
3
Hz, 2H, SCH ), 4.30 (m, 2H, CH N), 5.20-5.23 (m, 2H, CH =), 5.88-5.95 (m, 1H, CH=), 7.67
2
2
2
(
(
(
d, J= 8.5 Hz, 2H, Phenyl-H , Phenyl-H ), 7.80 (d, J= 8.5 Hz, 2H, Phenyl-H , Phenyl-H ), 9.70
2 6 3 5
+
s, 1H, NH), 10.66 (s, 1H, NHSO ), 10.93 (s, 1H, CONH); MS m/z (%): 461 (M , 0.9), 405
2
5.4), 350 (11.5), 294 (2.6), 266 (2.6), 210 (6.6), 195 (19.4), 167 (6.3), 128 (100), 111 (11.2),
1
07 (21.3), 96 (23.1); Anal. Calcd. for C H N O S (461.56): C, 52.04; H, 5.02; N, 15.17 %,
20 23 5 4 2
Found: C, 52.08; H, 5.08; N, 15.13%.
(E)-3-(Adamantylamino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-
3
-(ethylthio)acrylamide (5i)
-1
Yellow powder, yield (69%), mp 129-130˚C; IR (KBr) νmax/cm : 3405 (NH), 3287 (NH), 3100
3
1
(
NH), 3060 (CH-Ar), 2911 (CH-sp ), 2192 (CN), 1641 (CO); H-NMR (500 MHz, DMSO-d ):
6
2
2

δppm = 1.35 (t, J= 7.0 Hz, 3H, CH ), 1.63-1.67 (m, 12H, CH , 9 x Adamantyl-H), 2.09-2.14 (m,
3
3
9
H, CH , 6 x Adamantyl-H), 3.46 (q, J= 7.0 Hz, 2H, SCH ), 7.67 (d, J= 9.0 Hz, 2H, Phenyl-
3 2
H , Phenyl-H ), 7.77 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 9.73 (s, 1H, NH), 10.92 (s,
2
6
3
5
1
3
1
H, NHSO ), 11.10 (s, 1H, CONH); C-NMR (125 MHz, DMSO-d ): δ = 6.1 (CH ), 10.9
2 6 ppm 3
(
CH ), 14.3 (CH ), 18.5, 28.8, 29.0, 30.5, 35.2, 39.0, 39.1, 40.0, 40.1, 41.8, 42.2 (11C, SCH ,
3
3
2
1
1
0 x Adamantyl-C), 76.1 (Acrylamide-C ), 104.9 (CN), 119.2, 120.4, 127.4, 133.7, 143.0,
2
55.5, 161.3, 166.5, 168.2 (11C, C H , Isoxazole-C , Isoxazole-C , Isoxazole-C , Acrylamide-
6
4
4
3
5
C , CO); Anal. Calcd. for C H N O S (555.71): C, 58.36; H, 5.99; N, 12.60 %, Found: C,
3
27 33
5
4 2
5
8.32; H, 5.96; N, 12.66%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(ethylthio)-3-
(phenylamino)-acrylamide (5j)
-1
Yellow powder, yield (74%), mp 105-106˚C; IR (KBr) νmax/cm : 3380 (NH), 3219 (NH), 3104
3
1
(
NH), 3059 (CH-Ar), 2974 (CH-sp ), 2195 (CN), 1644 (CO); H-NMR (400 MHz, DMSO-d ):
6
δppm = 1.22 (t, J= 7.2 Hz, 3H, CH ), 1.68 (s, 3H, CH ), 2.14 (s, 3H, CH ), 2.77 (q, J= 7.2 Hz,
3
3
3
2
H, SCH ), 7.24-7.27 (m, 1H, C H ), 7.39-7.44 (m, 4H, C H ), 7.71 (d, J= 8.8 Hz, 2H, Phenyl-
2 6 5 6 5
H , Phenyl-H ), 7.78 (d, J= 8.8 Hz, 2H, Phenyl-H , Phenyl-H ), 10.07 (s, 1H, NH), 10.51 (s,
2
6
3
5
+
1
H, NHSO ), 10.88 (s, 1H, CONH); MS m/z (%): 497 (M , 4.9), 420 (3.3), 405 (1.1), 266 (9.3),
2
2
51 (2.6), 175 (1.2), 128 (100), 96 (47.6); Anal. Calcd. for C H N O S (497.59): C, 55.52;
2
3
23
5
4 2
H, 4.66; N, 14.07 %, Found: C, 55.56; H, 4.70; N, 14.05%.
3
-amino-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-5-(phenylamino)-1H-
pyrazole-4-carboxamide (6)
-1
White powder, yield (65%), mp 102-103˚C; IR (KBr) νmax/cm : 3475 (NH), 3388-3317 (NH ),
2
3
1
3
266 (NH), 3210 (NH), 3150 (NH), 3053 (CH-Ar), 2995 (CH-sp ), 1656 (CO); H-NMR (400
MHz, DMSO-d ): δ = 1.64 (s, 3H, CH ), 2.08 (s, 3H, CH ), 6.12 (s, 2H, NH ), 6.80 (m, 1H,
6
ppm
3
3
2
C H ), 7.19-7.32 (m, 4H, Aromatic-H), 7.65-7.74 (m, 4H, Aromatic-H), 8.52 (s, 1H, NH), 9.12
6
4
1
3
(
s, 1H, NH), 10.80 (s, 1H, NHSO ), 11.35 (s, 1H, CONH); C-NMR (100 MHz, DMSO-d ):
2
6
δppm = 6.4 (CH ), 10.8 (CH ), 89.0 (Pyrazole-C ), 104.0, 117.0, 119.7, 119.9, 127.5, 128.0,
3
3
4
1
29.3, 133.5, 143.7, 149.6, 150.1, 156.0, 161.6, 163.6 (18C, C H , C H , Isoxazole-C ,
6 4 6 5 4
+
Isoxazole-C , Isoxazole-C , Pyrazole-C , Pyrazole-C , CO); MS m/z (%): 467 (M , 0.1), 266
3
5
3
5
(
4.6), 216 (0.1), 111 (1.7), 96 (4.7), 96 (4.2), 64 (100); Anal. Calcd. for C H N O S (467.50):
21 21 7 4
C, 53.95; H, 4.53; N, 20.97 %, Found: C, 53.98; H, 4.59; N, 20.94%.
2
3

(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-mercapto-3-
(methylamino)acrylamide (7a)
-1
Yellow powder, yield (71%), mp 110-111˚C; IR (KBr) νmax/cm : 3237 (NH), 3205 (NH), 3106
3
1
(
NH), 3058 (CH-Ar), 2933 (CH- sp ), 2186 (CN), 1652 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.66 (s, 3H, CH ), 2.11 (s, 3H, CH ), 3.63(s, 3H, NCH ), 6.68 (d, J= 9.0 Hz, 2H,
3
3
3
Phenyl-H , Phenyl-H ), 7.39 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 8.10 (broad s, 1H,
2
6
3
5
+
NH), 10.41 (s, 1H, NHSO ), 10.75 (s, 1H, NHCO), 10.98 (s, 1H, SH); MS m/z (%): 407 (M ,
2
7
4
.6), 294 (3.2), 251 (4.2), 80 (100); Anal. Calcd. for C H N O S (407.47): C, 47.16; H,
16 17 5 4 2
.21; N, 17.19 %, Found: C, 47.20; H, 4.24; N, 17.22%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(ethylamino)-3-
mercapto-acrylamide (7b)
-1
Yellow powder, yield (76%), mp 97-98˚C; IR (KBr) νmax/cm : 3244 (NH), 3204 (NH), 3106
3
1
(
NH), 3053 (CH-Ar), 2981 (CH- sp ), 2187 (CN), 1653 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.15 (t, J= 7.5 Hz, 3H, CH ), 1.64 (s, 3H, CH ), 2.10 (s, 3H, CH ), 3.56 (q, J= 7.5
3
3
3
Hz, 2H, CH N), 6.61 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 7.37 (d, J= 9.0 Hz, 2H, Phenyl-
2
2
6
H , Phenyl-H ), 7.96 (s, 1H, NH), 10.48 (s, 1H, NHSO ), 10.70 (s, 1H, NHCO), 10.90 (s, 1H,
3
5
2
1
3
SH); C-NMR (125 MHz, DMSO-d ): δ = 5.8 (CH ), 10.2 (CH ), 12.4 (CH ), 35.7 (CH N),
6
ppm
3
3
3
2
1
1
04.3 (Acrylamide-C ), 105.3 (CN), 112.8 (2C, Phenyl-C and Phenyl-C ), 122.2 (Phenyl-C ),
2 2 6 4
27.8 (Phenyl-C ), 128.6 (2C, Phenyl-C and Phenyl-C ), 141.0 (Acrylamide-C ), 142.8
1
3
5
3
+
(
Isoxazole-C ), 156.2 (Isoxazole-C ), 161.4 (Isoxazole-C ), 162.2 (CO); MS m/z (%): 421 (M ,
4 3 5
1
4
2.7), 392 (3.3), 266 (5.2), 251 (4.2), 80 (100); Anal. Calcd. for C H N O S (421.49): C,
17 19 5 4 2
8.44; H, 4.54; N, 16.62%, Found: C, 48.48; H, 4.57; N, 16.67%.
(E)-3-(Allylamino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-
mercapto-acrylamide (7c)
-1
Yellow powder, yield (73%), mp 119-120˚C; IR (KBr) νmax/cm : 3243 (NH), 3190 (NH), 3105
3
1
(
NH), 3049 (CH-Ar), 2978 (CH- sp ), 2189 (CN), 1649 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.63 (s, 3H, CH ), 2.09 (s, 3H, CH ), 4.03-4.18 (m, 2H, CH N), 5.15-5.27 (m, 2H,
3
3
2
CH =), 5.86-5.93 (m, 1H, CH=), 7.65 (d, J= 9.0 Hz, 2H, Phenyl-H and Phenyl-H ), 7.82 (d,
2
2
6
J= 9.0 Hz, 2H, Phenyl-H and Phenyl-H ), 9.52 (s, 1H, NH), 10.70 (s, 1H, NHSO ), 10.90 (s,
3
5
2
1
3
1
4
H, CONH), 11.00 (s, 1H, SH); C-NMR (125 MHz, DMSO-d ): δ = 5.8 (CH ), 10.2 (CH ),
6 ppm 3 3
1.2 (CH N), 105.0 (Acrylamide-C ), 112.9 (CN), 118.9-133.2 (8C, Ar-C and -CH=CH ),
2
2
2
2
4

1
1
4
41.5 (Acrylamide-C ), 143.6 (Isoxazole-C ), 155.5 (Isoxazole-C ), 161.3 (Isoxazole-C ),
3 4 3 5
64.4 (CO); Anal. Calcd. for C H N O S (433.50): C, 49.87; H, 4.42; N, 16.16%, Found: C,
1
8
19
5
4 2
9.83; H, 4.39; N, 16.12%.
(E)-3-(Adamantylamino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}-
phenyl)-3-mercapto-acrylamide (7d)
-1
Yellow powder, yield (68%), mp 128-129˚C; IR (KBr) νmax/cm : 3221 (NH), 3186 (NH), 3120
3
1
(
NH), 3055 (CH-Ar), 2969 (CH- sp ), 2182 (CN), 1647 (CO); H-NMR (500 MHz, DMSO-
d6): δppm = 1.60-1.70 (m, 9H, 9 x Adamantyl-H), 1.90-2.15 (m, 6H, CH , 3 x Adamantyl-H),
3
2
1
.26 (s, 3H, 3 x Adamantyl-H), 2.50 (s, 3H, CH ), 7.65-7.80 (m, 4H, Ar-H), 8.01 (s, 1H, NH),
3
1
3
0.70 (s, 1H, NHSO ), 10.90 (s, 1H, CONH), 11.00 (s, 1H, SH); C-NMR (125 MHz, DMSO-
2
d6): δppm = 5.8 (CH ), 10.2 (CH ), 28.9-36.0 (6C, Adamantyl-C), 40.1-42.9 (4C, Adamantyl-
3
3
C), 103.5 (Acrylamide-C ), 108.5 (CN), 117.0 (Phenyl-C ), 119.0 (2C, Phenyl-C and Phenyl-
2
4
2
C ), 128.0 (2C, Phenyl-C and Phenyl-C ), 132.0 (Phenyl-C ), 141.5 (Acrylamide-C ), 143.0
6
3
5
1
3
(
Isoxazole-C ), 156.5 (Isoxazole-C ), 161.3 (Isoxazole-C ), 162.5 (CO); Anal. Calcd. for
4 5 5
C H N O S (527.66): C, 56.91; H, 5.54; N, 13.27%, Found: C, 56.96; H, 5.57; N, 13.30%.
2
5
29
5
4 2
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-mercapto-3-
(phenylamino)-acrylamide (7e)
-1
Yellow powder, yield (86%), mp 133-134˚C; IR (KBr) νmax/cm : 3211 (NH), 3163 (NH), 3103
3
1
(
NH), 3061 (CH-Ar), 2977 (CH- sp ), 2191 (CN), 1649 (CO); H-NMR (400 MHz, DMSO-
d6): δppm = 1.64 (s, 3H, CH ), 2.10 (s, 3H, CH ), 7.10-7.90 (m, 10H, Ar-H and NH), 10.80 (s,
3
3
1
3
1
H, NHSO ), 10.90 (s, 1H, CONH), 11.40 (s, 1H, SH); C-NMR (100 MHz, DMSO-d ): δ
2 6 ppm
=
6.3 (CH ), 10.7 (CH ), 105.5 (Acrylamide-C ), 112.5 (CN), 119.5-131.0 (12C, C H and
3 3 2 6 4
C H ), 143.0 (Isoxazole-C ), 145.0 (Acrylamide-C ), 155.6 (Isoxazole-C ), 161.5 (Isoxazole-
6
5
4
3
3
+
+
C ), 167.0 (CO); MS m/z (%): 470 ([M+H] , 6.8), 469 (M , 3.9), 436 (4.0), 391 (5.1), 358 (4.4),
5
2
94 (4.0), 266 (5.4), 251 (4.9), 203 (1.4), 175 (1.0), 80 (100); Anal. Calcd. for C H N O S
21 19 5 4 2
(469.54): C, 53.72; H, 4.08; N, 14.92 %, Found: C, 53.76; H, 4.13; N, 14.96%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(methylamino)-3-
(2΄,3΄,4΄,6΄-tetra-O-acetyl-β-D-glucopyranosylthio)acrylamide (8a)
-1
Yellow powder, yield (48%), mp 110-111˚C; IR (KBr) νmax/cm : 3268 (NH), 3181 (NH), 3133
3
1
(
NH), 3068 (CH-Ar), 2955 (CH-sp ), 2201 (CN), 1754 (CO), 1648 (CO); H-NMR (500 MHz,
DMSO-d ): δ = 1.85 (s, 3H, CH ), 1.95-2.01 (4s, 12H, 4 x CH CO), 2.09 (s, 3H, CH ), 3.48
6
ppm
3
3
3
2
5

(
s, 3H, NCH ), 3.95-4.32 (m, 2H, Glucose-H , Glucose-H ), 4.85-4.90 (m, 1H, Glucose-H ),
3 6a′ 6b′ 5′
5
.0-5.20 (m, 2H, Glucose-H , Glucose-H ), 5.36-5.48 (m, 1H, Glucose-H ), 5.90 (d, J= 9.20
4′ 3′ 2′
Hz, 1H, Glucose-H ), 7.68 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 7.78 (d, J= 9.0 Hz, 2H,
1
′
2
6
Phenyl-H , Phenyl-H ), 9.60 (broad s, 1H, NH), 10.70 (s, 1H, NHSO ), 10.90 (s, 1H, CONH);
3
5
2
+
MS m/z (%): 737 (M , 18.7), 707 (9.2), 694 (8.2), 406 (6.1), 331 (17.8), 266 (100), 111 (7.4);
Anal. Calcd. for C H N O S (737.75): C, 48.84 H, 4.78; N, 9.49 %, Found: C, 48.87; H,
3
0
35
5
13 2
4
.73; N, 9.55%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(ethylamino)-3-
(2΄,3΄,4΄,6΄-tetra-O-acetyl-β-D-glucopyranosylthio)acrylamide (8b)
-1
Yellow powder, yield (55%), mp 126-127˚C; IR (KBr) νmax/cm : 3270 (NH), 3176 (NH), 3123
3
1
(
NH), 3069 (CH-Ar), 2951 (CH-sp ), 2195 (CN), 1752 (CO), 1644 (CO); H-NMR (500 MHz,
DMSO-d ): δ = 1.31 (t, J= 7.15 Hz, 3H, CH ), 1.71 (s, 3H, CH ), 1.95-1.99 (4s, 12H, 4 x
6
ppm
3
3
CH CO), 2.09 (s, 3H, CH ), 2.57 (q, J= 7.15 Hz, 2H, NCH ), 3.90-4.22 (m, 2H, Glucose-H ,
3
3
2
6a′
Glucose-H ), 4.90-5.10 (m, 3H, Glucose-H , Glucose-H , Glucose-H ), 5.32-5.40 (m, 1H,
6
b′
5′
4′
3′
Glucose-H ), 5.85 (d, J= 9.20 Hz, 1H, Glucose-H ), 7.71 (d, J= 9.0 Hz, 2H, Phenyl-H ,
2
′
1′
2
Phenyl-H ), 7.83 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 9.61 (s, 1H, NH), 10.69 (s, 1H,
6
3
5
+
NHSO ), 11.01 (s, 1H, CONH); MS m/z (%): 751 (M , 1.7), 722 (2.9), 708 (0.7), 692 (11.1),
2
6
40 (3.6), 420 (0.8), 331 (15.8), 266 (56.4), 155 (100), 111 (44.1); Anal. Calcd. for
C H N O S (751.78): C, 49.53 H, 4.96; N, 9.32 %, Found: C, 49.57; H, 5.01; N, 9.38%.
3
1
37
5
13 2
(E)-3-(Allylamino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-
(2΄,3΄,4΄,6΄-tetra-O-acetyl-β-D-glucopyranosylthio)acrylamide (8c)
-1
Yellow powder, yield (44%), mp 130-131˚C; IR (KBr) νmax/cm : 3299 (NH), 3186 (NH), 3139
3
1
(
NH), 3084 (CH-Ar), 2949 (CH-sp ), 2197 (CN), 1753 (CO), 1644 (CO); H-NMR (500 MHz,
DMSO-d ): δ = 1.83 (s, 3H, CH ), 1.95-2.04 (4s, 12H, 4 x CH CO), 2.09 (s, 3H, CH ), 3.90-
6
ppm
3
3
3
4
.25 (m, 2H, Glucose-H , Glucose-H ), 4.30-4.40 (m, 2H, CH N), 4.75-5.0 (m, 3H, Glucose-
6a′ 6b′ 2
H , Glucose-H , Glucose-H ), 5.10-5.25 (m, 2H, CH =), 5.30-5.45 (m, 1H, Glucose-H ),
5
′
4′
3′
2
2′
5
.85-5.98 (m, 2H, CH=, Glucose-H ), 7.71 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 7.83 (d,
1′ 2 6
J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 9.60 (s, 1H, NH), 9.80 (s, 1H, NHSO ), 10.8 (s, 1H,
3
5
2
1
3
CONH); C-NMR (100 MHz, DMSO-d ): δ
= 6.2 (CH ), 10.4 (CH ), 20.1-20.4 (4C, 4 x
6
ppm
3
3
CH CO), 49.0 (NCH ), 61.7, 65.2, 66.9, 71.9, 74.4, 79.8 (6C, Galactose-C), 85.0 (Acrylamide-
3
2
C ), 113.0 (CN), 117.0, 127.4, 129.0, 130.0, 137.7, 139.0, (8C, CH =, CH= and C H ), 152.1,
2
2
6
4
1
55.2, 161.4, 165.7, 169.0 (5C, Isoxazole-C , Isoxazole-C , Isoxazole-C , Acrylamide-C ,
4 3 5 3
2
6

NHCO), 169.1, 169.4, 169.9, 170.0 (4C, 4 x CH CO); Anal. Calcd. for C H N O S
3
32 37
5
13
2
(763.79): C, 50.32 H, 4.88; N, 9.17 %, Found: C, 50.36; H, 4.93; N, 9.21%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(2΄,3΄,4΄,6΄-tetra-
O-acetyl-β-D-glucopyranosylthio)-3-(phenylamino)acrylamide (8d)
-1
Yellow powder, yield (63%), mp 150-151˚C; IR (KBr) νmax/cm : 3254 (NH), 3186 (NH), 3123
3
1
(
NH), 3070 (CH-Ar), 2962 (CH-sp ), 2177 (CN), 1752 (CO), 1639 (CO); H-NMR (400 MHz,
DMSO-d ): δ = 1.86 (s, 3H, CH ), 1.94-2.01 (4s, 12H, 4 x CH CO), 2.15 (s, 3H, CH ), 3.98-
6
ppm
3
3
3
4
.05 (m, 2H, Glucose-H , Glucose-H ), 4.22-4.26 (m, 1H, Glucose-H ), 4.74-4.95 (m, 2H,
6a′ 6b′ 5′
Glucose-H , Glucose-H ), 5.40-5.41 (m, 1H, Glucose-H ), 5.89 (d, J= 9.20 Hz, 1H, Glucose-
4
′
3′
2′
H ), 6.95-7.30 (m, 5H, C H ), 7.67 (d, J= 8.8 Hz, 2H, Phenyl-H , Phenyl-H ), 7.80 (d, J= 8.8
1
′
6
5
2
6
Hz, 2H, Phenyl-H , Phenyl-H ), 8.63 (s, 1H, NH), 10.10 (s, 1H, NHSO ), 12.91 (s, 1H,
3
5
2
1
3
C``ONH); C-NMR (100 MHz, DMSO-d ): δ = 6.3 (CH ), 10.7 (CH ), 20.2-20.4 (4C, 4 x
6
ppm
3
3
CH CO), 61.8, 65.1, 66.8, 71.7, 74.6, 79.2 (6C, Glucose-C), 84.9 (Acrylamide-C ), 113.1 (CN),
3
2
1
1
1
19.7, 125.0, 127.9, 128.5, 129.3, 130.6, 138.6, 139.3 (12C, C H , C H ), 153.7, 155.9, 156.7,
6 4 6 5
61.6, 161.8 (5C, Isoxazole-C , Isoxazole-C , Isoxazole-C , Acrylamide-C , NHCO), 168.0-
3
4
5
3
+
70.2 (4C, 4 x CH CO); MS m/z (%): 800 ([M+1] , 7.4), 799 (3.8), 548 (4.0), 533 (3.9), 468
3
(3.5), 363 (5.1), 331 (2.7), 266 (5.9), 251 (0.1), 175 (3.4), 111 (22.4), 97 (100), 96 (65.2);Anal.
Calcd. for C H N O S (799.82): C, 52.56 H, 4.66; N, 8.76 %, Found: C, 52.59; H, 4.63; N,
3
5
37
5
13 2
8
.72%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(methylamino)-3-
(2΄,3΄,4΄,6΄-tetra-O-acetyl-β-D-galactopyranosylthio)acrylamide (9a)
-1
Yellow powder, yield (61%), mp 133-134˚C; IR (KBr) νmax/cm : 3274 (NH), 3178 (NH), 3139
3
1
(
NH), 3063 (CH-Ar), 2949 (CH-sp ), 2197 (CN), 1754 (CO), 1649 (CO); H-NMR (500 MHz,
DMSO-d ): δ = 1.83 (s, 3H, CH ), 1.93-2.04 (4s, 12H, 4 x CH CO), 2.08 (s, 3H, CH ), 2.49
6
ppm
3
3
3
(
s, 3H, NCH ), 4.03-4.27 (m, 2H, Galactose-H , Galactose-H ), 4.91-4.98 (m, 1H, Galactose-
3 6a′ 6b′
H ), 5.0-5.16 (m, 2H, Galactose-H , Galactose-H ), 5.32-5.40 (m, 1H, Galactose-H ), 5.85 (d,
5
′
4′
3′
2′
J= 9.20 Hz, 1H, Galactose-H ), 7.74 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 7.82 (d, J=
1
′
2
6
9
.0 Hz, 2H, Phenyl-H , Phenyl-H ), 9.75 (s, 1H, NH), 10.76 (s, 1H, NHSO ), 10.91 (s, 1H,
3 5 2
1
3
CONH); C-NMR (125 MHz, DMSO-d ): δ = 6.2 (CH ), 10.4 (CH ), 20.1-20.4 (4C, 4 x
6
ppm
3
3
CH CO), 29.5 (NCH ), 61.1, 67.3, 68.4, 70.8, 73.7, 80.4 (6C, Galactose-C), 86.7 (Acrylamide-
3
3
C ), 112.5 (CN), 121.0, 129.4, 130.0, 139.0 (6C, Phenyl-C), 153.0, 158.0, 162.0, 165.0, 168.0
2
(
5C, Isoxazole-C , Isoxazole-C , Isoxazole-C , Acrylamide-C , NHCO), 169.6, 169.8, 169.8,
4
3
5
3
2
7

1
69.9 (4C, 4 x CH CO); Anal. Calcd. for C H N O S (737.75): C, 48.84 H, 4.78; N, 9.49
3
30 35
5
13 2
%, Found: C, 48.89; H, 4.81; N, 9.56%.
(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(ethylamino)-3-
(2΄,3΄,4΄,6΄-tetra-O-acetyl-β-D-galactopyranosylthio)acrylamide (9b)
-1
Yellow powder, yield (56%), mp 134-135˚C; IR (KBr) νmax/cm : 3271 (NH), 3177 (NH), 3127
3
1
(
NH), 3068 (CH-Ar), 2950 (CH-sp ), 2195 (CN), 1752 (CO), 1645 (CO); H-NMR (500 MHz,
DMSO-d ): δ = 1.32 (t, J= 8.0 Hz, 3H, CH ), 1.82 (s, 3H, CH ), 2.03-2.06 (4s, 12H, 4 x
6
ppm
3
3
CH CO), 2.08 (s, 3H, CH ), 2.59 (q, J= 8.0 Hz, 2H, NCH ), 4.01-4.25 (m, 2H, Galactose-H ,
3
3
2
6a′
Galactose-H ), 4.90-4.97 (m, 1H, Galactose-H ), 5.14-5.19 (m, 2H, Galactose-H , Galactose-
6
b′
5′
4′
H ), 5.32-5.45 (m, 1H, Galactose-H ), 5.85 (d, J= 9.20 Hz, 1H, Galactose-H ), 7.75 (d, J= 9.0
3
′
2′
1′
Hz, 2H, Phenyl-H , Phenyl-H ), 7.85 (d, J= 9.0 Hz, 2H, Phenyl-H , Phenyl-H ), 9.75 (s, 1H,
2
6
3
5
1
3
NH), 10.75 (s, 1H, NHSO ), 10.92 (s, 1H, CONH); C-NMR (125 MHz, DMSO-d ): δ =
ppm
2
6
6
7
1
.2 (CH ), 10.4 (CH ), 19.4 (CH ), 22.3-23.7 (4C, 4 x CH CO), 36.6 (NCH ), 61.2, 68.1, 68.6,
3 3 3 3 2
0.7, 73.4, 80.5 (6C, Galactose-C), 86.2 (Acrylamide-C ), 113.0 (CN), 119.0, 121.0, 129.0,
2
33.0 (6C, Phenyl-C), 151.0, 153.0, 162.0, 164.0, 168.0 (5C, Isoxazole-C , Isoxazole-C ,
4
3
Isoxazole-C , Acrylamide-C , NHCO), 169.5-169.9 (4C, 4 x CH CO); Anal. Calcd. for
5
3
3
C H N O S (751.78): C, 49.53 H, 4.96; N, 9.32 %, Found: C, 49.56; H, 5.02; N, 9.38%.
3
1
37
5
13 2
(E)-3-(Allylamino)-2-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-
(2΄,3΄,4΄,6΄-tetra-O-acetyl-β-D-galactopyranosylthio)acrylamide (9c)
-1
Yellow powder, yield (47%), mp 144-145˚C; IR (KBr) νmax/cm : 3301 (NH), 3187 (NH), 3142
3
1
(
NH), 3080 (CH-Ar), 2970 (CH-sp ), 2195 (CN), 1752 (CO), 1644 (CO); H-NMR (500 MHz,
DMSO-d ): δ = 1.83 (s, 3H, CH ), 1.90-2.06 (4s, 12H, 4 x CH CO), 2.08 (s, 3H, CH ), 3.95-
6
ppm
3
3
3
4
.10 (m, 2H, Galactose-H , Galactose-H ), 4.30-4.42 (m, 2H, CH N), 4.90-5.14 (m, 3H,
6a′ 6b′ 2
Galactose-H , Galactose-H , Galactose-H ), 5.21-5.55 (m, 3H, CH =, Galactose-H ), 5.85 (d,
5
′
4′
3′
2
2′
J= 9.20 Hz, 1H, Galactose-H ), 5.90-6.0 (m, 1H, CH=), 7.74 (d, J= 8.5 Hz, 2H, Phenyl-H ,
1
′
2
Phenyl-H ), 7.84 (d, J= 8.5 Hz, 2H, Phenyl-H , Phenyl-H ), 8.45 (s, 1H, NH), 9.85 (s, 1H,
6
3
5
1
3
NHSO ), 10.80 (s, 1H, CONH); C-NMR (100 MHz, DMSO-d ): δ = 6.7 (CH ), 10.5 (CH ),
2
6
ppm
3
3
2
8
0.2-20.4 (4C, 4 x CH CO), 49.0 (NCH ), 61.1, 65.6, 67.4, 70.8, 71.5, 74.0 (6C, Galactose-C),
3 2
4.6 (Acrylamide-C ), 113.0 (CN), 117.0, 118.0, 120.3, 128.7, 131.0, 134.0 (8C, CH =, CH=
2
2
and C H ), 153.0, 159.0, 161.5, 164.0, 168.0 (5C, Isoxazole-C , Isoxazole-C , Isoxazole-C ,
6
4
4
3
5
Acrylamide-C , NHCO), 169.2, 169.3, 169.6, 169.8 (4C, 4 x CH CO); Anal. Calcd. for
3
3
C H N O S (763.79): C, 50.32 H, 4.88; N, 9.17 %, Found: C, 50.30; H, 4.85; N, 9.14%.
3
2
37
5
13 2
2
8

(E)-2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}phenyl)-3-(2΄,3΄,4΄,6΄-tetra-
O-acetyl-β-D-galactopyranosylthio)-3-(phenylamino)acrylamide (9d)
-1
Yellow powder, yield (54%), mp 161-162˚C; IR (KBr) νmax/cm : 3261 (NH), 3184 (NH), 3129
3
1
(
NH), 3071 (CH-Ar), 2967 (CH-sp ), 2198 (CN), 1752 (CO), 1644 (CO); H-NMR (400 MHz,
DMSO-d ): δ = 1.88 (s, 3H, CH ), 1.96-2.12 (4s, 12H, 4 x CH CO), 2.22 (s, 3H, CH ), 3.96-
6
ppm
3
3
3
4
2
.30 (m, 2H, Galactose-H , Galactose-H ), 4.48-4.57 (m, 1H, Galactose -H ), 5.27-5.31 (m,
6a′ 6b′ 5′
H, Galactose-H , Galactose-H ), 5.36-5.38 (m, 1H, Galactose-H ), 5.87 (d, J= 9.20 Hz, 1H,
4
′
3′
2′
Galactose-H ), 7.32-7.85 (m, 9H, C H and C H ), 8.70 (s, 1H, NH), 10.18 (s, 1H, NHSO ),
1
′
6
4
6
5
2
1
3
1
2
2.96 (s, 1H, CONH); C-NMR (100 MHz, DMSO-d ): δ = 6.7 (CH ), 10.9 (CH ), 20.7-
6 ppm 3 3
0.9 (4C, 4 x CH CO), 61.9, 66.1, 67.8, 71.2, 71.7, 74.5 (6C, Galactose-C), 85.0 (Acrylamide-
3
C ), 113.2 (CN), 120.1, 122.6, 123.7, 128.4, 129.3, 130.8, 138.4, 140.0 (12C, C H , C H ),
2
6
4
6
5
1
47.0, 156.0, 162.0, 164.0, 168.5 (5C, Isoxazole-C , Isoxazole-C , Isoxazole-C , Acrylamide-
3 4 5
C , NHCO), 169.8, 170.0, 170.1, 170.3 (4C, 4 x CH CO); Anal. Calcd. for C H N O S
13 2
3
3
35 37
5
(799.82): C, 52.56 H, 4.66; N, 8.76 %, Found: C, 52.62; H, 4.69; N, 8.81%.
3
3
.2. Antimicrobial evaluation
.2.1. Antimicrobial screening
Disk diffusion method was used for the preliminary screening of the antimicrobial activities of
the target compounds. Standard sterilized disks of Whatman filter paper were impregnated with
a solution of the investigated compound in DMF (100 µL, 5 mg/mL) and used for antibacterial
and antifungal assay according to the protocol described in our previous study [14]. S.
pneumoniae, B. Subtilis, S. epidermidis, E. coli, P. vulgaris and K. pneumonia were used as
representative examples of Gram-positive and Gram-negative bacteria. While, A. fumigatus, S.
racemosum and G. candidum were used as representative fungal strains. Sulfisoxazole,
ampicillin, gentamycin and amphotericin B were used as reference standard antimicrobial
agents. DMF was used as a control. [14]
3
.2.2. Minimal inhibitory concentration (MIC) measurement
Minimal inhibitory concentrations (MICs) were determined for compounds having growth
inhibition zones greater than 13 mm. Broth micro-dilution was used following the same
procedure we published before [14]. Details are provided (supplementary material).
Subsequently, we attempted to prove that the antimicrobial effect of the tested compounds
relies on the inhibition of the DHPS enzyme by performing MIC shift experiments in the
presence of pABA and methionine [45]. Therefore, we determined the MIC values of the
2
9