Synthesis of isothiocyanates using DMT/NMM/TsO? as a new desulfurization reagent

Article abstract of DOI:10.3390/molecules26092740

Thirty-three alkyl and aryl isothiocyanates, as well as isothiocyanate derivatives from esters of coded amino acids and from esters of unnatural amino acids (6-aminocaproic, 4-(aminomethyl)benzoic, and tranexamic acids), were synthesized with satisfactory or very good yields (25–97%). Synthesis was performed in a “one-pot”, two-step procedure, in the presence of organic base (Et₃ N, DBU or NMM), and carbon disulfide via dithiocarbamates, with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TsO^? ) as a desulfurization reagent. For the synthesis of aliphatic and aromatic isothiocyanates, reactions were carried out in a microwave reactor, and selected alkyl isothiocyanates were also synthesized in aqueous medium with high yields (72–96%). Isothiocyanate derivatives of L-and D-amino acid methyl esters were synthesized, under conditions without microwave radiation assistance, with low racemization (er 99 > 1), and their absolute configuration was confirmed by circular dichroism. Isothiocyanate derivatives of natural and unnatural amino acids were evaluated for antibacterial activity on E. coli and S. aureus bacterial strains, where the most active was ITC 9e.

Full text of DOI:10.3390/molecules26092740

molecules
Article
−
Synthesis of Isothiocyanates Using DMT/NMM/TsO as a New
Desulfurization Reagent
1
,
2
and Beata Kolesi n´ ska 1
Łukasz Janczewski * , Dorota Kr e˛ giel
1
Faculty of Chemistry, Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116,
0-924 Lodz, Poland; beata.kolesinska@p.lodz.pl
Department of Environmental Biotechnology, Faculty of Biotechnology and Food Sciences, Lodz University
of Technology, Wolczanska 171/173, 90-924 Lodz, Poland; dorota.kregiel@p.lodz.pl
Correspondence: lukasz.janczewski@p.lodz.pl
9
2
*
Abstract: Thirty-three alkyl and aryl isothiocyanates, as well as isothiocyanate derivatives from esters of
coded amino acids and from esters of unnatural amino acids (6-aminocaproic, 4-(aminomethyl)benzoic,
and tranexamic acids), were synthesized with satisfactory or very good yields (25–97%). Synthesis
was performed in a “one-pot”, two-step procedure, in the presence of organic base (Et N, DBU
3
or NMM), and carbon disulfide via dithiocarbamates, with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
−
methylmorpholinium toluene-4-sulfonate (DMT/NMM/TsO ) as a desulfurization reagent. For
the synthesis of aliphatic and aromatic isothiocyanates, reactions were carried out in a microwave
reactor, and selected alkyl isothiocyanates were also synthesized in aqueous medium with high yields
(72–96%). Isothiocyanate derivatives of L- and D-amino acid methyl esters were synthesized, under
conditions without microwave radiation assistance, with low racemization (er 99 > 1), and their absolute
configuration was confirmed by circular dichroism. Isothiocyanate derivatives of natural and unnatural
amino acids were evaluated for antibacterial activity on E. coli and S. aureus bacterial strains, where the
most active was ITC 9e.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Janczewski, Ł.; Kr e˛ giel, D.;
Kolesi n´ ska, B. Synthesis of
Isothiocyanates Using
Keywords: isothiocyanates; desulfurization agent; 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-
−
-
DMT/NMM/TsO as a New
morpholinium toluene-4-sulfonate; DMT/NMM/TsO ; microwave-assisted synthesis of biologically
Desulfurization Reagent. Molecules
active compounds; amino acids; circular dichroism; microwave synthesis; microwave technology;
antibacterial activity
2
021, 26, 2740. https://doi.org/
1
0.3390/molecules26092740
Academic Editor: Farid Chemat
1
. Introduction
Isothiocyanates (ITCs) with the general formula R-N=C=S can be considered as com-
pounds derived from biologically inactive glucosinolates [ ]. They are produced by
cruciferous vegetables such as broccoli, radishes, wasabi, and cauliflower as part of their
defense mechanisms [ ]. In response to damage to the plant, glucosinolates are con-
verted into intermediate aglucon derivatives, which are converted into the final ITCs by
a biochemical process according to the Lossen rearrangement [ ]. Myrosinase, which is
present in separate organelles and even in the cells of undamaged cruciferous plant tissue,
plays a role in this transformation [ ]. Isothiocyanateshave many desirable characteristics,
including primarily anti-proliferative properties [ 15]. Like naturally occurring isothio-
cyanates (e.g., sulforaphane [16 20] (SFN), phenethyl isothiocyanate [21] (PEITC), or benzyl
isothiocyanate [22] (BITC)), their synthetically modified analogs with the structure of a
phosphorus atom [23 24] or fluorine atom [25 26] also exhibit anti-proliferative activity. Nat-
ural as well as synthetic ITCs may additionally have anti-bacterial [27 30], anti-fungal [31],
and anti-glioblastoma effects [32]. They are used in proteomics as probes [33 34], and in
Received: 9 March 2021
Accepted: 3 May 2021
Published: 6 May 2021
1–4
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Licensee MDPI, Basel, Switzerland.
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
,
,
–
,
organic synthesis for the preparation of thioureas [35], thioamides [36], and heterocyclic
compounds [37,38].
In recent years, several new methods have been developed for the synthesis of ITCs, us-
+
−
ing fluorine-containing reagents such as Langlois reagent (F CSO Na) [39], Ph P CF CO
3 2 3 3 2
4
.0/).
Molecules 2021, 26, 2740. https://doi.org/10.3390/molecules26092740
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2740
2 of 24
(
PDFA)/S8 [40], (Me N)SCF [41], CF SiMe /S or AgSCF [42], and BrCF CO Na/S [43].
4 3 3 3 8 3 2 2 8
However, three methods still predominate. The most common method is the Staudinger/aza-
Wittig tandem reaction, in which the starting azides react with triphenylphosphine and
are converted into iminophosphorane intermediates. Then, in the presence of carbon
disulfide, the iminophosphate intermediates are transformed into the final ITCs (Figure 1,
route a) [44–47]. The second method involves the use of a primary amine and thiophosgene
as a reagent for the transfer of the thiocarbonyl moiety in the presence of a base (Figure 1,
route b) [48–51]. Due to the toxicity of thiophosgene and the sensitivity of certain functional
groups, this reagent is increasingly replaced by surrogates such as di-(2-pyridyl) thionocar-
0
0
bamate [52], 1,1 -thiocarbonyldiimidazole [53], or 1,1 -thiocarbonyldi-2-(1H)-pyridone [54].
The third method is a two-step one-pot procedure. Primary amines are transformed in
the presence of a base and carbon disulfide into intermediate dithiocarbamates, which
after treatment with the desulfurating agent are transformed into the target ITCs (Figure 1,
route c).
Figure 1. Methods of synthesizing isothiocyanates.
Many desulfurating agents are currently available. Their choice depends on the re-
action conditions and the presence of functional groups in the reagents. The most often
used desulfurating agents include tosyl chloride [55], sodium persulfate [56], iodine [57],
mesyl chloride [58], ethyl chloroformate [59], di-tert-butyl dicarbonate [60], cyanuric chlo-
ride [61], and others [62]. Microwave radiation has been found to support the conversion
of dithiocarbamates into ITCs [63]. Coupling reagents used in peptide chemistry, such
®
as HBTU and PyBOP [64], DCC [65], and T3P [66], can also be used as desulfurating
reagents. However, despite the availability of numerous reagents, new compounds are
continually sought to provide for the efficient synthesis of structurally diverse ITCs from
dithiocarbamates, supported by microwave radiation.
Here, we describe the microwave-assisted synthesis of structurally diverse aliphatic
and aromatic isothiocyanates, as well as normal synthesis of isothiocyanate derivatives of
natural and unnatural amino acids, using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-
−
morpholinium toluene-4-sulfonate (DMT/NMM/TsO ,
1
) as a new, efficient desulfurating
agent for the indirect formation of dithiocarbamates from primary amines or hydrochlo-
rides (Figure 2).
−
Figure 2. Synthesis of ITCs using DMT/NMM/TsO (1).

Molecules 2021, 26, 2740
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−
DMT/NMM/TsO
[67
,68] (1) is an effective, and environmentally friendly coupling
reagent used for the synthesis of amides, esters, and peptides in solution or solid phase,
as well as under microwave-assisted conditions [69]. It enables the synthesis of peptides
using Z-, Boc-, and Fmoc- protected substrates as well as unnatural amino acids. Us-
−
ing DMT/NMM/TsO , synthesis of peptides occurs rapidly, with high yields, without
racemization, and often with sufficiently high purity to avoid the necessity of additional
−
chromatographic purification. Use of DMT/NMM/TsO in the synthesis of structurally
diverse isothiocyanates characterized by a wide range of biological activity could allow
for the efficient synthesis of peptide conjugates with isothiocyanates, requiring only a
single coupling reagent. Reducing the numbers of compounds utilized in the synthesis
of biologically active compounds should reduce the number and variety of by-products,
which is particularly important for the impurity profile.
2. Results and Discussion
2
.1. Optimizing the Synthesis of Aliphatic Isothiocyanates
−
In the first stage of the research, it was checked whether DMT/NMM/TsO (1) acts
as a desulfurating agent, and experiments to optimize the reaction conditions were carried
out. Phenethylamine (2a) was used as a model amine in the optimization tests. Use of
carbon disulfide (CS ) (3 equiv.) in the presence of triethylamine (Et N) (4 equiv.) in DCM
2
3
as the solvent enabled formation of intermediate dithiocarbamate 3a in just 5 min at room
temperature (rt) [63]. The obtained dithiocarbamate 3a was subjected to a microwave-
−
assisted reaction with 1 equiv. of DMT/NMM/TsO (
1
) as a desulfurating agent, producing
◦
the target isothiocyanate 4a with 90% yield in 3 min at 90 C (initial 200 W power) (Table 1,
entry 1).
Table 1. Optimization of conditions of synthesis of isothiocyanate 4a, derived from model amine 2a a
.
MW Conditions
−
Base
(Equiv.)
DMT/NMM/TsO
(Equiv.)
Yield
(%)
Entry
Solvent
b
◦
Time (min)
Temp ( C)
1 c
2
3
3
3
3
3
3
3
3
3
3
90
90
90
90
90
90
90
90
90
90
rt
Et N (4)
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
1.0
1.0
1.0
1.0
1.0
1.3
0.7
0
90
78
69
51
92
92
86
55
73
89
82
3
NMM (4)
DBU (4)
-
4
5
Et N (3)
3
d
6
Et3N (3)
7
8
Et N (3)
3
Et N (3)
3
9
Et N (3)
H O
1.0
1.3
1.0
3
2
e
1
1
0
3
30
Et N (3)
H O
3
2
f,g,h
1
Et3N (3)
DCM
a—reagents and conditions: 10 mL pressure vial, 2a (0.25 mL, 2 mmol, 1 equiv.), base, CS
equiv.), solvent (3 mL). First step: 5 min, rt., then DMT/NMM/TsO
MW) irradiation are presented in Table 1. Standard mode (initial power 200 W); —yield of 4a after flash
chromatography (hexane 100%); —elongation time to 5 and 10 min resulted the same yield (90%); —increasing
(0.36 mL, 6 mmol,
2
−
3
(
(
1
). Time and temperature of microwave
b
c
d
e
the amount of
1
to 1.6 equiv. resulted in the same yield (92%); —increasing the amount of
1
to 1.6 equiv. resulted
f
g
in the same yield (89%); —normal reaction at rt.; —prolonging the time to 60 min resulted in the same yield
82%), shortening the time to 10 min resulted in a lower yield (79%); —reaction in sealed tube (30 min, 90 C)
h
◦
(
resulted in the same yield (82%).
Extending the reaction time to 5 min and 10 min did not further increase the yield
(Table 1, entry 1, footnote c). Next, we investigated the influence of the base on the yield.

Molecules 2021, 26, 2740
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Replacing triethylamine with N-methylmorpholine (NMM) or 1,8-diazabicyclo[5.4.0]undec-
7
2
5
-ene (DBU) resulted in lowering of the yield to 78% and 69%, respectively (Table 1, entry
and 3), whereas the reaction without base resulted in final isothiocyanate 4a with only
1% yield (Table 1, entry 4). Decreasing the amount of Et N to 3 equiv. resulted in a
3
slight increase the yield, to 92% (Table 1, entry 5). Increasing the amount of coupling
reagent to 1.3 equiv. (Table 1, entry 6) and to the 1.6 equiv. (Table 1, entry 6, footnote d)
1
did not affect the yield, which remained at 92% in both cases. However, the reduction of
−
DMT/NMM/TsO
(1
) to 0.7 equiv. reduced the yield to 86% (Table 1, entry 7). The reaction
produced 4a with only 55% yield (Table 1, entry 8). In the
without desulfurating agent
1
next step, we investigated the possibility of performing the reaction in aqueous media,
◦
using optimized conditions: 3 min, 90 C, CS (3 equiv.), Et N (3 equiv.) as a base, with
2
3
−
DMT/NMM/TsO (1 equiv.). In these conditions, ITC 4a was obtained in a satisfactory
yield 73% (Table 1, entry 9). It has been found that increasing the amount of agent to
.3 equiv. allowed us to obtain isothiocyanate 4a with a very high yield of 89% (Table 1,
1
1
Entry 10). Further increasing the amount of 1 to 1.6 equiv. did not affect the yield of the
reaction (Table 1, entry 10, footnote e). We also show that this reaction occurs in normal
conditions at rt, although in lower yield. Performing the reaction in 30 min at rt resulted in
obtaining the product 4a with 82% yield (Table 1, Entry 11). Prolonging the time to 60 min
did not affect the results, but shortening the time to 10 min resulted in a decrease in the
yield to 79% (Table 1, entry 11, footnote g). Additionally, performing the reaction in the
◦
sealed tube for 30 min at 90 C (Table 1, entry 11, footnote h) did not affect the yield of
reaction, which was still 82%. The results of experiments in normal conditions at room and
at high temperature undoubtedly indicate that the use of microwave radiation assistance
leads to higher yields of the ITC 4a, and also that it is possible to significantly shorten the
reaction time.
A library of isothiocyanates 4a–j, 7a–j, 9a–j, and 11a–c using structurally various
aliphatic amines 2a (Figure 3), aromatic amines 5a– j (Figure 4), hydrochlorides of methyl
–
j
and the ethyl esters of L- and D-amino acids 8a–j (Figure 5), as well as hydrochlorides of
the ethyl esters of unnatural amino acids 10a–c (Figure 6), has been synthesized. All the
compounds were isolated with good or very good yields (97–25%) after purification on a
short pad of silica gel.
2.2. Synthesis of Aliphatic Isothiocyanates 4a–j
Aliphatic isothiocyanates were synthesized in a microwave reactor using two methods
with different solvents. In Method A, DCM was used as solvent, whereas in Method B, the
synthesis of selected ITCs was performed in H O (Figure 3).
2
In Method A, isothiocyantes with an aromatic ring in the alkyl chain 4a–d were
obtained with up to 94% yield. Moreover, the synthesis of (R)-1-isothiocyanatoethylbenzene
4c) and (S)-1-isothiocyanatoethylbenzene (4d) from optically active amine 2c and 2d
occurred without racemization (>99: 1 er). Yields of alkyl ITCs 4e and volatile 4f were
slightly lower, at 88% and 72%, respectively. The yields of ITCs obtained from amines 2g
and 1-adamantylamine (2i), which are less volatile and more sterically hindered, rose to
7% for 4g, 82% for 4h, and 83% for 4i. In addition to compounds with one –NCS group,
(
–h
9
an ITC with two –NCS groups (1,6-diisothiocyanatohexane, 4j) was synthesized with high
yield (70%). In order to investigate the scope and limitations of the possible synthesis of
ITCs in water (Table 1, entry 14), the reaction was additionally performed for selected
amines 2a
g, and 4i were a few percent lower than in DCM, but still acceptably high, and they were
in the range of 96–72%.
, 2c, 2e, 2g, and 2i in an aqueous media (Method B). The yields of ITCs 4a, 4c, 4e,
4

Molecules 2021, 26, 2740
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Figure 3. Scope of aliphatic isothiocyanates 4a–ja,b. -Method A: conditions: amine 2a
a
–
j
(2 mmol),
Et N (3 equiv., 6 mmol), CS (3 equiv., 6 mmol), DCM (3 mL), first step 5 min, rt, then
3
2
−
◦
DMT/NMM/TsO
(
1
) (2 mmol, 1 equiv.); MW irradiation in standard mode 3 min, 90 C. Yield of
products isolated after flash chromatography (hexane). Method B: conditions: amine 2a
,
2c 2e 2g,
,
,
and 2i (2 mmol), Et N (3 equiv., 6 mmol), CS (3equiv., 6 mmol), H O (3 mL); first step 5 min, rt,
3
2
2
−
◦
then DMT/NMM/TsO
(
1
) (2.6 mmol, 1.3 equiv.); MW irradiation in standard mode 3 min, 90 C;
b–
c
yield in parenthesis applies to the reaction performed in H O; –conditions: amine 2j (2 mmol),
Et N (6 equiv., 12 mmol), CS (6 equiv., 12 mmol), DCM (5 mL), DMT/NMM/TsO
2
−
(1) (4 mmol,
3
2
2
equiv.).
Figure 4. Scope of aromatic isothiocyanates7a–ja. a–conditions: amine 5a
–j
(2 mmol), DBU (3 equiv.,
−
6
mmol), CS (3 equiv., 6 mmol), DCM (3 mL), first step 5 min, rt, then DMT/NMM/TsO
(1)
2
◦
(2 mmol, 1 equiv.); MW irradiation in standard mode 3 min, 90 C. Yield of products isolated after
b
flash chromatography (hexane); –conditions: amine 5j (2 mmol), DBU (6 equiv. 12 mmol), CS2
−
(6 equiv. 12 mmol), DCM (5 mL), DMT/NMM/TsO (1) (4 mmol, 2 equiv.).

Molecules 2021, 26, 2740
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Figure 5. Scope of isothiocyanate derivatives of amino acids 9a– –j
ja. a-conditions: hydrochloride 8a
(
1
2 mmol), NMM (0.66 mL, 3 equiv., 6 mmol), CS (0.36 mL, 3 equiv., 6 mmol), DCM (5 mL); first step
2
−
0 min, rt, then DMT/NMM/TsO
(1
) (0.828 g, 1 equiv., 2 mmol,), normal conditions, rt., 30 min.
b
Products isolated after flash chromatography (hexane:EtOAc 20:1); –conditions: hydrochloride 8i
(
2 mmol), NMM (1.32 mL, 6 equiv., 12 mmol), CS (0.72 mL, 6 equiv., 12 mmol), DCM (5 mL); first
2
−
step 10 min, rt, then DMT/NMM/TsO
(1) (1.656 g, 2 equiv., 4 mmol,), normal conditions, rt., 30 min.
Products isolated after flash chromatography (hexane:EtOAc 20:1).
Figure 6. Synthetized isothiocyanates 11a
hydrochloride 10a
5 mL); first step 5 min, rt, then DMT/NMM/TsO
–c
derived from unnatural amino acids a. a-conditions:
–
c
(2 mmol), Et N (0.83 mL, 3 equiv., 6 mmol), CS (0.36 mL, 3 equiv., 6 mmol), DCM
3
2
−
(
(1
) (0.828 g, 1 equiv., 2 mmol,), MW irradiation in
◦
standard mode 3 min, 90 C. Yield of products after flash chromatography (hexane:EtOAc 10:1).
Comparing the yields of aliphatic ITCs obtained in optimal conditions in Method A
with previous results using only microwave radiation to synthesis of aliphatic ITCs [63], it
−
can be concluded that application of DMT/NMM/TsO
(1) as a desulfurating agent allows
us to receive the final isothiocyanates 4a–i with higher yield. The biggest yield difference
was found for ITC 4f (72% vs. 50% [63]), and the smallest for ITC 4i (83% vs. 82% [63]). For

Molecules 2021, 26, 2740
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other ITCs, except 4j, which was not previous obtained, the yields were higher, from 5 to
14%, than in previous publication [66].
2.3. Synthesis of Aromatic Isothiocyanates 7a–j
In the next stage of the research, attempts were made to check the usefulness of
−
DMT/NMM/TsO (
1) as a desulfurating agent in reactions with aromatic amines. Per-
forming the reaction under optimized conditions (Table 1, entry 5 and Figure 3 Method A)
with aniline (5a) resulted in a low yield of final phenyl isothiocyanate (7a) at 30% (Table 2,
entry 1). For this reason, the synthesis of aromatic ITCs required optimization (Table 2).
Table 2. Optimization of reaction conditions of synthesis of isothiocyanate 7a, derived from model
amine 5a a.
MW Conditions
Time (min)
−
DMT/NMM/TsO
Entry
Base (Equiv.)
Solvent
Yield (%) b
(Equiv.)
1
2
3
4
5
6
3
3
3
3
10
3
Et N (3)
DCM
DCM
DCM
DCM
DCM
1.0
1.0
1.0
1.3
1.0
1.3
30
71
67
70
67
21
3
DBU (3)
DBU (4)
DBU (4)
DBU (3)
DBU (3)
H O
2
a–reagents and conditions: 10 mL pressure vial, 5a (0.19 mL, 2 mmol, 1 equiv.), base, CS
(0.36 mL, 6 mmol,
2
−
3
equiv.), solvent (3 mL). First step: 5 min, rt., then DMT/NMM/TsO
(1), time of microwave irradiation
see Table 2, temp. = 90 C, standard mode (initial power 200 W); b–yield of 7a after flash chromatography
◦
(hexane 100%).
Replacing Et N with DBU increased the yield to 71% (Table 2, entry 2). Increasing the
3
amount of DBU to 4 equiv. or the desulfurating agent 1 to 1.3 equiv. did not improve to
the yield (Table 2, entries 3 and 4, respectively). Prolonging the time to 10 min decreased
the yield to 67% (Table 2, entry 5). A library of aromatic isothiocyanates 7a–j substituted at
◦
ortho-, meta-, or para-positions was obtained under optimized conditions: 3 min, 90 C, CS
(
2
−
3 equiv.), DBU (3 equiv.), DMT/NMM/TsO (1 equiv.). The yields of the final products
were in the range of 40–92% (Figure 4).
Introducing the methyl group to the aromatic ring in the para position increased the
yield of ITC 7b to 87%, whereas introducing two methyl groups in positions 2 and 6 resulted
in a slight increase of the yield of 7c to 92%. Replacing the methyl with a methoxy group in
the para position gave ITC 7d with 87%. However, the methoxy group in the metha position
as well as the presence of two dimethoxy groups in positions 3 and 5 in the aromatic ring
diminished the yield of ITCs 7e and 7f to 66% and 67%, respectively. Application of this
protocol to halogenoanilins 5g–i enabled ITCs 7g–i to be obtained with 61–53% yields.
Aromatic isothiocyanate with two –NCS group – 1,3-diisothiocyanatobenzene (7j) were also
obtained, although with a moderate yield of 40%. The synthesis of aromatic isothiocyanates
in aqueous medium has failed. Model phenyl isothiocyanate (7a) was obtained with a low
yield of 21% (Table 2, entry 6), and the synthesis of other aromatic isothiocyanates in H O
2
was not carried out. Comparing the yields of obtained aromatic isothiocyanates 7a
and 7g in microwave-assisted synthesis with desulfurating agent and without it [63],
we can conclude that in almost all cases, the lack of desulfurating agent resulted in better
, 7c–e,
–j
1
yields of final isothiocyanates. The yields of ITCs 7a
desulfurating agent were from 6 to 42% higher than yields with DMT/NMM/TsO . The
,
7c
,
7e, and 7g
–
j
in reaction without
−

Molecules 2021, 26, 2740
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biggest difference was for ITC 7i (53% vs. 95% [63]). Only ITC 7d was obtained with higher
yield in reaction with agent
1 than without it (87% vs. 75% [63]). It can conclude that
in contrast to aliphatic isothiocyanates, aromatic iothiocyanates do not need addition of
desulfurating agent, however its presence does not cause a significant drop of yield.
2.4. Synthesis of Isothiocyanate Derivatives of Natural 9a–j and Unnatural 11a–c Amino Acids
In next stage of the research, we attempted to synthesize ITCs using hydrochlorides of
methyl and ethyl esters of L- and D-amino acids 8a–j as substrates. As Et N causes partial
3
racemization of products [66], it was replaced with NMM. Performing the reaction on
◦
model L-alanine methyl ester hydrochloride (8a) under optimal conditions (3 min, 90 C) in
a microwave reactor produced 9a with a yield of 59% and concomitant high racemization
(
er 65:35). To prevent the loss of enantiomeric homogeneity, the reaction was performed
under normal conditions (30 min, rt). The product (S)-methyl 2-isothiocyanatopropanoate
9a) was obtained with 50% yield and low racemization (er 99 > 1) (Figure 5). Using these
(
optimal conditions, a second enantiomer (R)-methyl 2-isothiocyanatopropanoate (9b) was
produced with 51% yield and low racemization (er 99 > 1), using D-alanine methyl ester
hydrochloride (8b) as a substrate. In addition to hydrochlorides of methyl esters of alanine,
L-alanine benzyl ester hydrochloride (8c), and tert-butyl ester of L-alanine hydrochloride
(
8d), hydrochlorides of methyl esters of L-valine (8e), L-leucine (8f), L-isoleucine (8g),
L-phenylalanine (8h), and L-lysine (8i), as well as the achiral hydrochloride of the ethyl
ester of glycine (8j), were used as starting materials. Figure 5 presents the results of the
synthesis of isothiocyanate derivatives of amino acids 9a–j.
Isothiocyanates derived from the benzyl ester 9c and tert-butyl ester 9d of the L-alanine
group were synthesized with lower yields of 38% and 35%, respectively. This may have
been due to the presence of a larger ester group and thus greater steric hindrance. Isothio-
cyanate analogs of L-valine 9e, L-leucine 9f, L-isoleucine 9g, and L-phenylalanine 9h with
isopropyl, isobutyl, sec-butyl, and the benzyl moiety, respectively, were synthesized with
satisfactory yields (30–63%). During the synthesis of these compounds, it was observed
that the yield depended on the effect of steric hindrance (steric hindrance 9h
by the substituent on the amino acid side chain (yield of 9e 9f 9g 9h). In the case of
the achiral ethyl ester of the isothiocyanates of glycine 9j, the yield of the reaction was
8%. As in the case of the aliphatic and aromatic isothiocyanates with two –NCS groups
also an isothiocyanate derivative from L-lysine 9i with two –NCS groups was obtained,
>9g>9f>9e)
>
>
>
4
although with a low yield (25%). ITCs 9a
desulfurating agent T3P also in normal conditions [66]. ITCs 9a and 9b were obtained
with 10% better yield than with DMT/NMM/TsO , however, ITC 9h was synthesized
with definitely higher yield – 72% [66]. Others ITCs were not synthesized using T3P .
,
9b, and 9h were previous synthesized using as a
®
−
®
In addition to methyl and ethyl esters of natural amino acids, hydrochlorides of ethyl
esters of unnatural amino acids with aliphatic, aromatic, and cyclohexyl linkers were used
as substrates for the synthesis ITCs. Hydrochlorides of ethyl esters of 6-aminocaproic acid
(10a), 4-(aminomethyl)benzoic acid (10b) and trans-4-(aminomethyl)cyclohexanecarboxylic
acid (Tranexamic acid) (10c) were converted into target ITCs 11a
synthesis under optimal conditions (3 min, 90 C), with DMT/NMM/TsO
–
c
in microwave-assisted
◦
−
(1) as the
desulfurating agent. All ITCs 11a–c were isolated after flash chromatography with a very
good yields and high purity (Figure 6). In the case of compound 11c which is trans-isomer
1
the synthesis occurred without racemization, due to the lack of signals of cis-isomer on H
NMR spectrum.
Parental amino acids of substrates 10a
–c are characterized by hemostatic proper-
ties [70–74]. The biological activities of ITCs 11a–c were also tested and described below.
2.5. Determination of the Absolute Configuration Using Circular Dichroism
Inspired by Michalski and Cie z˙ [75], we attempted to determine the absolute con-
figuration of chiral ITCs 4c–d and 9a–i using circular dichroism (CD). All samples were
dissolved in methanol. The concentrations of the samples were between 0.52 mg/mL and

Molecules 2021, 26, 2740
9 of 24
0
.30 mg/mL. For all S enantiomers of the ITCs analogs of L-amino acids 9a and 9c
Cotton effect (CE) was positive. For the R enantiomer of the ITC analog of D-alanine 9b
the CE was negative. These results support the literature data [75 76]. The ITC 9a with the
–i, the
,
S configuration at C-2 exhibited a strong positive CE at 205 nm, and the second enantiomer
ITC 9b with an R configuration at C-2 exhibited a strong negative CE, also at 205 nm. The
CE for ITCs 9a and 9b were similar but reversed, confirming the presence of two different
enantiomers (Figure 7).
Figure 7. CD spectra of ITCs 9a and 9b.
For the other two ITC analogs of L-alanine with a benzyl 9c or tert-butyl 9d ester moiety,
strong positive CE were observed at 205 nm and 210 nm, respectively (see Supporting
Material Figures S77 and S78). A strong positive CE for ITCs 9e
–g was also observed at
210 nm (see Supporting Material Figures S79–S81). However, for ITC 9h, a strong positive
CE was observed at 225 nm (see Supporting Material Figure S82), which could be due to
the presence of an aromatic ring. For ITC 9i with two –NCS groups, a strong positive CE
was observed at 210 nm (see Supporting Material Figure S83).
As in the case of the chiral ITCs analogs of L- and D-amino acids 9a–i, the absolute
configuration of the chiral ITCs with a benzyl moiety 4c and 4d was also determined using
CD (Figure 8).
Figure 8. CD spectra of ITCs 4c and 4d.

Molecules 2021, 26, 2740
10 of 24
For both enantiomers 4c and 4d, a strong CE was observed at 215 nm. For ITC 4d,
the CE was positive; consequently, the absolute configuration was S. In the case of ITC 4c
,
the CE was negative; consequently, the absolute configuration was R. The presence of two
different enantiomers was confirmed.
The absolute configurations of all chiral ITCs 4c–d and 9a–i, as well as their optical
rotations, are presented in Table 3.
Table 3. Absolute configurations and optical rotations for ITCs 4c–d and 9a–i.
Entry
Compound
Absolute Configuration
[α]D (Concentration)
1
2
3
4
5
6
7
8
9
4c
4d
9a
9b
9c
9d
9e
9f
R
S
S
R
S
S
S
S
S
S
S
−18.1 (1.0 CHCl )
3
+17.5 (1.0 CHCl )
3
+24.1 (0.32 CHCl3)
−23.3 (0.32 CHCl )
3
+32.1 (0.31 CHCl )
3
+21.4 (0.34 CHCl3)
+16.3 (1.0 EtOH)
−21.7 (0.32 CHCl )
3
9g
9h
9i
+23.5 (1.0 EtOH)
10
1
−60.0 (1.0 toluene)
1
−18.2 (0.5 CHCl )
3
−
2
.6. Proposed Mechanism of Synthesis of ITC Using DMT/NMM/TsO
We postulate the following mechanism for the synthesis of ITCs using DMT/NMM/TsO
Figure 9). Dithiocarbamate 3b formed in a reaction amine 2b with Et N and CS reacts
−
(
3
2
−
1), affording active ester 12. Next, under the influence of Et N,
3
with DMT/NMM/TsO (
leaving group 13 is eliminated with the simultaneous formation of ITC 4b. Forming the
intermediate product which is a derivative of dithiocarbamate with desulfurating agent 12
and then formation the final isothiocyanate in the presence a base is characteristic stage
for synthesis of isothiocyanates using various desulfurating agents and it was described
,
in many publication [57,60,64,66]. Next, the anion 13 can undergo two reactions. The first
possible reaction is protonation of anion 13 by protonated triethylamine and formation if
thiol 15 which is in equilibrium with its thiocarbonyl form 16. However, due to the rapid
tautomerism identification was impossible.
Figure 9. Possible mechanism for synthesis of isothiocyanates.

Molecules 2021, 26, 2740
11 of 24
The second is possible methylation, leading to preparation of 2,4-dimethoxy-6-methylthio-
1
,3,5-triazine (14), which was isolated after flash chromatography with low yield and con-
1
13
firmed by H and C NMR. The yield of the isolated compound 14 was low, indicating a
slight share this reaction.
−
2
.7. Comparison DMT/NMM/TsO vs. Selected Desulfurating Agents
−
In order to show the superiority of DMT/NMM/TsO relative to other desulfurating
agents the effectiveness of reagents was compared in the synthesis of model benzyl isoth-
iocyanate (4b). All reactions were carried out in the same optimal microwave conditions
◦
(
3 min, 90 C), using different, known desulfurating agents: cyanuric chloride (TCT) [61],
iodine [57], di-tert-butyl dicarbonate (Boc O) with catalityc amount of DMAP [62], propane
2
®
phosphonic acid anhydride (T3P ) [66], tosyl chloride (TsCl) [55], ethyl chloroformate [59],
and 30% hydrogen peroxide (H O ) [77], and using as substrate benzyl amine (2b). The
2
2
final product was isolated after flash chromatography. All experiments are presented in
Table 4.
−
Table 4. Comparison of the efficiency of DMT/NMM/TsO with selected desulfurating agents in
synthesis benzyl isothiocyanate (4b) a.
Entry
DesulfuratingAgents
Yields (%)
1 b
2
3
4
5
6
7
8
−
DMT/NMM/TsO
92
87
86
84
83
81
78
75
TCT
I2
Boc O, DMAPcat.
2
®
T3P
TsCl
Ethyl chloroformate
H O 30%
2 2
a-conditions: hydrochloride 2b (2 mmol), Et
3
2
N (0.83 mL, 3 equiv., 6 mmol), CS (0.36 mL, 3 equiv., 6 mmol), DCM
(
9
3 mL); first step 5 min, rt, then desulfurating agent (1 equiv., 2 mmol), MW irradiation in standard mode 3 min,
0 C. Yield of products after flash chromatography (hexane 100%). -this work.
◦
b
The above results show that effectiveness of all desulfurating agents in microwave-
−
conditions is very good (yield of 4b is between 92 and 75%); however, DMT/NMM/TsO
turned out to be the best desulfurating agent (yields of 4b 92%) (Table 4, Entry 1). The TCT
as well as I were also very efficient reagents; however, the yields of ITC 4b were slightly
2
lower, respectively, 87% and 86% (Table 4, Entries 2 and 3). Application of Boc O with
2
®
DMAP, T3P , and TsCl also caused the yields of 4b to be satisfactory (84–81%) (Table 4,
Entries 4–6). The lower yields below 80% obtained using ethyl chloroformate and 30%
H O (Table 4, Entries 7 and 8) show that these compounds were definitely less effective
2
2
−
−
than DMT/NMM/TsO . Although, some of the reagents (TsCl and iodine) are cheaper
than DMT/NMM/TsO , the DMT/NMM/TsO allows us to obtain the product with
higher yield, which is undoubtedly its greatest advantage.
−
2.8. Antibacterial Activity
Isothiocyanates derivatives of natural 9a–j and unnatural amino acids 11a–c were
evaluated for antibacterial activity on Gram positive bacteria strain S. aureus and Gram
negative bacteria strain E. coli, using chloramphenicol as positive control. The antibacterial
activity of ITCs 9a
TSA) is presented in Table 5, and on the pictures of Petri dishes in the (Supporting Material
Figure S87). ITCs 9a and 11a at micromolar concentration (16–27 M) in DMSO or
–j and 11a–c based on the well diffusion method on trypticase soy agar
(
–
j
–
c
µ

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12 of 24
mixture DMSO and water (4 mg/mL) [78
were reported in millimeter (mm).
,79] were used in the test. The inhibition zones
Table 5. Antibacterial activity of isothiocyanates 9a–j and 11a–c.
Compound
Concentration)
Growth Inhibition of
E. coli (Mean ± DS)
Growth Inhibition of
S. aureus (Mean ± DS)
Entry
Structure
(
9
a
1
(4 mg/mL,
7 µM)
15.3 ± 0.3
16.1 ± 0.4
22.7 ± 0.3
2
9b
2
3
(4 mg/mL,
7 µM)
22.0 ± 0.4
22.1 ± 0.1
2
9
c
(4 mg/mL,
8 µM)
18.4 ± 0.6
25.1 ± 0.3
1
9d
4
(4 mg/mL,
1 µM)
36.0 ± 0.7
2
9
e
5
6
(4 mg/mL,
3 µM)
36.2 ± 0.2
18.3 ± 0.3
32.3 ± 0.1
25.2 ± 0.3
2
9
f
(4 mg/mL,
1 µM)
2
9
g
7
8
9
(4 mg/mL,
1 µM)
20.2 ± 0.2
20.1 ± 0.3
29.3 ± 0.1
30.0 ± 0.4
20.1 ± 0.3
17.5 ± 0.3
24.5 ± 0.6
28.3 ± 0.2
32.1 ± 0.1
32.3 ± 0.3
2
9h
(4 mg/mL,
8 µM)
1
9
i
(4 mg/mL,
6 µM)
1
9
j
1
1
0
1
(4 mg/mL,
7 µM)
2
1
1a
(4 mg/mL,
0 µM)
2
1
1b
(4 mg/mL,
8 µM)
1
1
2
3
24.4 ± 0.6
16.3 ± 0.4
1
1
1c
(4 mg/mL,
7 µM)
20.8 ± 0.2
15.5 ± 0.2
1
1
1
5
4
Negative control
Positive control
(20 µg/mL,
sterile water
NO
NO
Chloramphenicol
36.2 ± 0.2
38.8 ± 0.3
0.062 µM)
Based on results in Table 5 more of the tested ITCs exhibited antibacterial activity.
For E. coli the most active ITCs were 9d and 9e > 36 mm). Additionally, good activity
characterized ITCs 9i and 9j about 30 mm). Others ITCs 9a–c 9f–h 11a 11c also had
(φ
(
φ
,
,
–

Molecules 2021, 26, 2740
13 of 24
antibacterial activity, although slightly lower (
1a exhibited very high antibacterial activity (
and 9i had weaker, but also good activity (30 >
showed moderate antibacterial activity (φ < 25 mm).
φ
< 25 mm). Additionally ITCs 9e
> 32 mm) against S. aureus. ITCs 9d 9f
> 25 mm). The other ITCs 9a–c 9g, 11b–c
,
,
9j and
1
φ
, 9h,
φ
,
For all tested compounds, the most active for both bacterial strains was ITC 9e derived
from methyl ester of L-valine. Probably the short and branched alkyl chain of isopropyl
moiety was responsible for the highest activity. Despite the satisfactory antibacterial activity
of tested ITCs 9a
control was definitely higher (0.062
action and toxicity will be continued.
–
j
and 11a
–
c
(16–27
µ
M), the activity of chloramphenicol used as a positive
M). The study on the mechanism of antimicrobial
µ
3. Materials and Methods
3.1. General Information
NMR spectra were measured on a Bruker Avance II Plus (Bruker Corporation, Billerica,
1
13
MA, USA) spectrometer (700 MHz for H NMR and 176 MHz for C NMR) in CDCl3
1
13
solution. H and C NMR spectra were referenced according to the residual peak of the
solvent, based on literature data. Chemical shifts ( ) were reported in ppm and coupling
δ
1
3
constants (J) in Hz. C NMR spectra were proton-decoupled. A monomode microwave
reactor (CEM Discover) (CEM, Matthews, NC, USA) equipped with an Intelli Vent pressure
control system was used. The standard method was applied and the maximum pressure
was set to 250 psi. The temperatures of the reaction mixtures were measured with an
external infrared sensor. Flash chromatography was performed using a glass column
packed with Baker silica gel (30–60
µm). For TLC, silica gel with a 254 nm indicator on
Al foils (Sigma Aldrich, St. Louis, MO, USA) was used. All reagents and solvents were
−
purchased from Sigma-Aldrich (Poland) and used as obtained. Optical rotations were
◦
measured at 25 C on a PolaAAr 3001 Polarimeter (A. KRÜSS Optronic GmbH, Hamburg,
D
Germany) at
λ
= 589 nm. Optical rotations were reported as follows: [
α
]
(c = g/100 mL
25
solvent). The enantiomeric ratio (er) of 4c and 4d was determined by chiral stationary phase
HPLC, using a Daicel Chiralpak ID column (hexane) and a Daicel Chiralpak IC column for
◦
compounds 9a
–b
(hexane/i-PrOH, 98:2), with a column temperature of 30 C and a flow
rate of 1.0 mL/min. Melting points were obtained using a Büchi SMP-20 apparatus. CD
studies were performed using a Jasco J–1500 spectrometer Far-UV (ABLE JASCO Polska,
Krakow, Poland) and a rectangular quartz cuvette (1 mm path length, Hellma, Müllheim,
Germany). The samples were prepared in methanol at a concentration of 0.52–0.30 mg/mL.
All studies were carried out at rt. CD spectra were measured in the range of 190–300 nm.
The experimental parameters were as follows: data pitch, 5 nm; scanning mode, continuous;
scanning speed, 100nm/min; bandwidth, 4 nm; integration time, 1 s. Mass spectrometry
analysis was performed on a Bruker microOTOF-QIII (Bruker Corporation, Billerica, MA,
USA) equipped with electrospray ionization mode and a time of flight detector (TOF). IR
−
1
spectra were measured on an FT-IR Alpha Bruker (ATR) instrument in cm
.
3.2. General Procedure and Characterization of Compounds 4a–ag
3.2.1. General Procedure for Compounds 4a–j—Method A
Amine 2a–j (2 mmol, 1 equiv.), Et N (0.84 mL, 6 mmol, 3 equiv. or 1.68 mL, 12 mmol,
3
6
equiv. for 2j), and CS (0.36 mL, 6 mmol, 3 equiv. or 0.72 mL, 12 mmol, 6 equiv. for
2
2
j
) were dissolved in dry DCM (3 mL or 5 mL for 2j) in a 10 mL pressure vial, equipped
−
with a magnetic bar, and stirred 5 min at rt. Next, DMT/NMM/TsO
(1) (0.828 g, 2 mmol,
1
equiv. or 1.656 g, 4 mmol, 2 equiv. for 2j) was added. The reaction was carried under MW
◦
conditions (standard mode, 3 min, 90 C). The reaction mixture was diluted with DCM
50 mL) and washed with H O (5 mL), 1 N HCl (2 5 mL), H O (5 mL), then dried under
(
×
2
2
anhydrous MgSO . The crude products were purified by flash chromatography on silica
4
gel (7–8 g) using hexane as an eluent. Pure isothiocyanates 4a–j were isolated after careful
evaporation of the solvent and removal of volatile residues under reduced pressure. All
the synthesized isothiocyanates have been described in the literature.

Molecules 2021, 26, 2740
14 of 24
2
-Isothiocyanatoethylbenzene (4a). Colorless oil. Yield 92% (0.3 g, 1.84 mmol) after flash
1
chromatography (hexane). H NMR (700 MHz, CDCl ):
δ = 7.36–7.34 (m, 2 H, CHAr), 7.30–
3
7
.27 (m, 1 H, CHAr), 7.23–7.22 (m, 2 H, CHAr), 3.73 (t, JHH = 7.0 Hz, 2 H, CH NCS), 3.00 (t,
2
JHH = 7.0 Hz, 2 H, CH2). 13C NMR (176 MHz, CDCl3):
28.9 (s, 2 C H), 128.8 (s, 2 C H), 127.3 (s, C H), 46.5 (s, CH NCS), 36.6 (s, CH ).
The analytical data are in agreement with those reported previously in the literature [63].
δ = 137.1 (s, C ), 131.0 (s, NCS),
Ar
1
×
×
Ar
Ar
Ar
2
2
(
Isothiocyanatomethyl)benzene (4b). Colorless oil. Yield 92% (0.273 g, 1.83 mmol) after flash
chromatography (hexane). 1H NMR (700 MHz, CDCl3):
= 7.42–7.39 (m, 2 H, CH ),
.37–7.34 (m, 1 H, CH ), 7.33–7.32 (m, 2 H, CH ), 4.71 (s, 2 H, CH NCS). C NMR
δ
Ar
1
3
7
Ar
Ar
2
(
1
176 MHz, CDCl3):
δ
= 134.3 (s, C ), 132.4 (s, NCS), 129.1 (s, 2
×
C H), 128.5 (s, C H),
Ar
Ar Ar
26.9 (s, 2
×
C H), 48.8 (s, CH NCS). The analytical data are in agreement with those
Ar 2
reported previously in the literature [63].
(
R)-1-isothiocyanatoethylbenzene (4c). Colorless oil. Yield 94% (0.306 g, 1.87 mmol) after flash
chromatography (hexane). The er was determined by HPLC using a Chiralpak ID column,
D
(
[
7
hexane); t
= 9.36 min, t
= 7.95 min (>99: 1 er). [
α
]
−
18.1 (c 1.0, CHCl ) (lit.
major
minor
25
3
D
1
α
]
−
18.3 (c 1.0, CHCl )). H NMR (700 MHz, CDCl ):
δ
= 7.41–7.38 (m, 2 H, CH ),
25
3
3
Ar
.34–7.32 (m, 3 H, CH ), 4.92 (q, J
= 6.8 Hz, 1 H, CHNCS), 1.68 (d, JHH = 6.8 Hz, 3 H,
= 140.3 (s, C ), 132.5 (s, NCS), 129.0 (s, 2
28.3 (s, C H), 125.5 (s, 2 C H), 57.2 (s, CHNCS), 25.1 (s, CH ). The concentration of
the sample for CD analysis was 0.51 mg/mL. The analytical data are in agreement with
those reported previously in the literature [63].
Ar
HH
13
CH3). C NMR (176 MHz, CDCl3):
1
δ
×
C H),
Ar
Ar
×
Ar
Ar
3
(S)-1-isothiocyanatoethylbenzene (4d). Colorless oil. Yield 92% (0.3 g, 1.84 mmol) after flash
chromatography (hexane). The er was determined by HPLC using a Chiralpak ID column
D
(
[
hexane); t
= 7.95 min, t
= 9.36 min (>99: 1 er). [
α
]
+17.5 (c 1.0, CHCl ) (lit.
major
minor
1
25 3
D
α
]
+17.6 (c 1.0, CHCl )). H NMR (700 MHz, CDCl ):
δ
= 7.41–7.38 (m, 2 H, CHAr),
25
3
3
7
.34–7.32 (m, 3 H, CH ), 4.92 (q, J
= 6.8 Hz, 1 H, CHNCS), 1.68 (d, JHH = 6.8 Hz, 3 H,
Ar
HH
δ
13
CH3). C NMR (176 MHz, CDCl3):
= 140.3 (s, C ), 132.5 (s, NCS), 129.0 (s, 2
×
C H),
Ar
Ar
128.3 (s, CArH), 125.5 (s, 2 CArH), 57.1 (s, CHNCS), 25.1 (s, CH ). The concentration of the
×
3
sample for CD analysis was 0.5 mg/mL. The analytical data are in agreement with those
reported previously in the literature [63].
1
-Isothiocyanatohexane (4e). Colorless oil. Yield 88% (0.251 g, 1.76 mmol) after flash chro-
1
matography (hexane). H NMR (700 MHz, CDCl ):
δ = 3.50 (t, JHH = 6.7 Hz, 2 H, CH NCS),
3
2
1
.71–1.67 (m, 2 H, CH ), 1.43–1.39 (m, 2 H, CH ), 1.35–1.27 (m, 4 H, 2
×
CH ), 0.90 (t,
2
2
2
13
JHH = 7.1 Hz, 3 H, CH3). C NMR (176 MHz, CDCl3):
δ = 129.7 (s, NCS), 45.2 (s, CH NCS),
2
3
1.1 (s, CH ), 30.1 (s, CH ), 26.3 (s, CH ), 22.6 (s, CH ), 14.0 (s, CH ). The analytical data are
2
2
2
2
3
in agreement with those reported previously in the literature [63].
2
-Isothiocyanatobutane (4f). Colorless oil. Yield 72% (0.166 g, 1.44 mmol) after flash chro-
1
matography (hexane). H NMR (700 MHz, CDCl ):
δ = 3.33 (d, JHH = 6.2 Hz, 2 H, CH NCS),
3
2
1.99 (sept, JHH = 6.5 Hz, 1 H, CH), 1.00 (t, JHH = 6.7 Hz, 6 H, 2
×
CH3). 13C NMR (176 MHz,
= 129.8 (s, NCS), 52.5 (s, CH NCS), 29.7 (s, CH), 19.8 (s, 2 CH ). The analytical
CDCl3):
δ
×
2
3
data are in agreement with those reported previously in the literature [63].
2
-Isothiocyanatooctane (4g). Colorless oil. Yield 97% (0.330 g, 1.94 mmol) after flash chro-
1
matography (hexane). H NMR (700 MHz, CDCl3):
δ = 3.77–3.72 (m, 1 H, CHNCS),
1
.65–1.60 (m, 1 H, H from CH ), 1.57–1.52 (m, 1 H, H from CH ), 1.47–1.42 (m, 1 H, H from
2
2
CH ), 1.39–1.33 (m, 1 H, H from CH ), 1.34 (d, J
= 6.5 Hz, 3 H, CH CH), 1.32–1.25 (m,
3
2
2
HH
1
3
6
H, 3
×
CH ), 0.88 (t, J
= 7.1 Hz, 3 H, CH3). C NMR (176 MHz, CDCl3): δ = 129.9 (s,
2
HH
NCS), 54.2 (s, CHNCS), 37.7 (s, CH ), 31.7 (s, CH ), 28.8 (s, CH CH), 26.1 (s, CH ), 22.6 (s,
2
2
3
2
CH ), 21.9 (s, CH ), 14.1 (s, CH ). The analytical data are in agreement with those reported
2
2
3
previously in the literature [63].
3
-Isothiocyanatopentane (4h). Colorless oil. Yield 82% (0.211 g, 1.64 mmol) after flash
1
chromatography (hexane). H NMR (700 MHz, CDCl ):
δ = 3.53–3.49 (m, 1 H, CHNCS),
3

Molecules 2021, 26, 2740
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1
.66–1.60 (m, 4 H, 2
CDCl3): = 130.0 (s, NCS), 61.9 (s, CHNCS), 28.6 (s, 2
analytical data are in agreement with those reported previously in the literature [63].
×
CH ), 1.01 (t, J
= 7.4 Hz, 6 H, 2
×
CH ). 13C NMR (176 MHz,
2
HH
3
δ
×
CH ), 10.6 (s, 2
×
CH ). The
2
3
◦
◦
1
(
-Isothiocyanatoadamantane (4i). White solid, mp 167–169 C (lit. 167–169 C). Yield 83%
1
0.321 g, 1.66 mmol) after flash chromatography (hexane). H NMR (700 MHz, CDCl ):
3
δ
3
3
= 2.11 (s, 3 H, 3
×
CH), 1.98 (d, JHH = 2.7 Hz, 6 H, 3
CH2). 13C NMR (176 MHz, CDCl ):
= 129.7 (s, NCS), 58.6 (s, CNCS), 43.9 (s, 3
CH). The analytical data are in agreement with those reported
×
CH ), 1.64 (q, J
= 12.5 Hz, 6 H,
2
HH
×
δ
×
CH2),
3
5.7 (s, 3
×
CH ), 29.4 (s, 3
×
2
previously in the literature [63].
1
,6-Diisothiocyanatohexane (4j). Colorless oil. Yield 70% (0.280 g, 1.4 mmol) after flash
1
chromatography (hexane). H NMR (700 MHz, CDCl ):
2
δ = 3.52 (t, JHH = 6.5 Hz, 4 H,
3
1
3
×
CH NCS), 1.73–1.69 (m, 4 H, 2
×
CH ), 1.46–1.44 (m, 4 H, 2
×
CH2). C NMR
2
2
(176 MHz, CDCl ):
δ
= 130.2 (s, NCS), 45.0 (s, 2
×
CH NCS), 29.8 (s, 2 CH ), 26.0 (s,
×
3
2
2
2
×
CH ). The analytical data are in agreement with those reported previously in the
2
literature [66].
3
.2.2. General Procedure for Compounds 4a, 4c, 4e, 4g and 4i—Method B
Amine 2a 2c 2e 2g, or 2i (2 mmol, 1 equiv.), Et N (0.84 mL, 6 mmol, 3 equiv.),
and CS (0.36 mL, 6 mmol, 3 equiv.) were dissolved in H O (3 mL) in a 10 mL pressure
,
,
,
3
2
2
−
vial equipped with a magnetic bar, and stirred for 5 min at rt. Next, DMT/NMM/TsO
(
1
) (1.077 g, 2.6 mmol, 1.3 equiv.) was added. The reaction was carried under MW
◦
conditions (standard mode, 3 min, 90 C). The reaction mixture was extracted with DCM
(
3
2
×
15 mL). The combined organic layers were washed with 1 N HCl (2
×
5 mL) and
H O (5 mL), then dried under anhydrous MgSO . The crude product was purified by flash
4
chromatography on silica gel (7–8 g) using hexane as eluent. Pure isothiocyanates 4a
4g, and 4i were isolated after careful evaporation of the solvent and volatile residues
under reduced pressure.
, 4c,
4e,
2
-Isothiocyanatoethylbenzene (4a). Colorless oil. Yield 89% (0.290 g, 1.78 mmol) after flash
chromatography (hexane).
(
R)-1-Isothiocyanatoethylbenzene (4c).Colorless oil. Yield 88% (0.286 g, 1.76 mmol) after flash
chromatography (hexane).
1
-Isothiocyanatohexane (4e). Colorless oil. Yield 86% (0.222 g, 1.72 mmol) after flash chro-
matography (hexane).
2
-Isothiocyanatooctane (4g). Colorless oil. Yield 96% (0.329 g, 1.92 mmol) after flash chro-
matography (hexane).
1
-Isothiocyanatoadamantane (4i). White solid. Yield 72% (0.190 g, 1.48 mmol) after flash
chromatography (hexane).
3
.2.3. General Procedure for Compounds 7a–j
Amine 5a (2 mmol, 1 equiv.), DBU (0.9 mL, 6 mmol, 3 equiv. or 1.8 mL, 12 mmol,
equiv. for 5j), and CS (0.36 mL, 6 mmol, 3 equiv. or 0.72 mL, 12 mmol, 6 equiv. for 5j
–
j
6
)
2
were dissolved in dry DCM (3 mL or 5 mL for 5j) in a 10 mL pressure vial equipped with a
−
magnetic bar, and stirred 5 min at rt. Next, DMT/NMM/TsO
(1) (0.828 g, 2 mmol, 1 equiv.
or 1.656 g, 4 moml, 2 equiv. for 5j) was added. The reaction was carried out under MW
◦
conditions (standard mode, 3 min, 90 C). The reaction mixture was diluted with DCM
(
50 mL) and washed with H O (5 mL), 1 N HCl (2
×
5 mL), and H O (5 mL), then dried
2
2
under anhydrous MgSO . The crude products were purified by flash chromatography on
4
silica gel (7–8 g) using hexane as an eluent. Pure isothiocyanates 7a–j were isolated after
careful evaporation of the solvent and removal of volatile residues under reduced pressure.
All the synthesized isothiocyanates have been described in the literature.

Molecules 2021, 26, 2740
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Isothiocyanatobenzene (7a). Colorless oil. Yield 71% (0.192 g, 1.42 mmol) after flash chro-
1
matography (hexane). H NMR (700 MHz, CDCl ):
(
δ
= 7.37–7.34 (m, 2 H, CHAr), 7.29–7.27
= 135.5 (s, NCS),
CArH). The analytical
data are in agreement with those reported previously in the literature [63].
3
13
m, 1 H, CHAr), 7.23–7.21 (m, 2 H, CHAr). C NMR (176 MHz, CDCl3):
31.4 (s, CArNCS), 129.6 (s, 2 CArH), 127.4 (s, CArH), 125.8 (s, 2
δ
1
×
×
1
-Isothiocyanato-4-methylbenzene (7b). Colorless oil. Yield 87% (0.260 g, 1.74 mmol) after
1
flash chromatography (hexane). H NMR (700 MHz, CDCl ):
2
δ
= 7.14 (d, J
= 8.7 Hz,
3
HH
13
H, CH ), 7.11 (d, J
= 8.4 Hz, 2 H, CH ), 2.35 (s, 3 H, CH ). C NMR (176 MHz,
Ar
HH
Ar
3
CDCl3):
s, 2
previously in the literature [80].
δ
= 137.6 (s, CArOMe), 134.7 (s, NCS), 130.2 (s, 2
×
CArH), 128.5 (s, CArNCS), 125.6
(
×
C H), 21.3 (s, CH ). The analytical data are in agreement with those reported
Ar 3
2
-Isothiocyanato-1,3-dimethylbenzene (7c). Colorless oil. Yield 92% (0.3 g, 1.84 mmol) after
1
flash chromatography (hexane). H NMR (700 MHz, CDCl3):
δ
= 7.09–7.04 (m, 3 H,
= 135.7 (s, NCS), 135.2 (s,
CArH), 127.0 (s, CArH), 18.7 (s, 2 CH ). The
analytical data are in agreement with those reported previously in the literature [63].
CH ), 2.38 (s, 6 H, 2
×
CH3). 13C NMR (176 MHz, CDCl3):
CArCH3), 129.7 (s, CArNCS), 128.1 (s, 2
δ
Ar
2
×
×
×
3
1
-Isothiocyanato-4-methoxybenzene (7d). Colorless oil. Yield 87% (0.287 g, 1.74 mmol) after
1
flash chromatography (hexane). H NMR (700 MHz, CDCl ):
2
δ
= 7.16 (d, J
3
= 9.1 Hz,
3
HH
1
3
H, CH ), 6.85 (d, J
= 9.1 Hz, 2 H, CH ), 3.80 (s, 3 H, OCH ). C NMR (176 MHz,
Ar
HH
Ar
CDCl3):
s, 2
previously in the literature [63].
δ
= 158.7 (s, CArOCH ), 134.3 (s, NCS), 127.0 (s, 2
×
CArH), 123.7 (s, CArNCS), 114.9
3
(
×
C H), 55.6 (s, CH O). The analytical date are in agreement with those reported
Ar 3
1
-Isothiocyanato-3-methoxybenzene (7e). Colorless oil. Yield 66% (0.218 g, 1.32 mmol) after
1
flash chromatography (hexane). H NMR (700 MHz, CDCl ):
CH r), 6.83–6.81 (m, 2 H, CHAr), 6.73 (t, JHH = 2.2 Hz, 1 H, CHAr), 3.79 (s, 3 H, CH O).
δ = 7.23 (t, JHH = 8.3 Hz, 1 H,
3
1
3
C
A
3
NMR (176 MHz, CDCl3):
δ = 160.4 (s, CArOMe), 135.5 (s, NCS), 132.2 (s, CArNCS), 130.3 (s,
CArH), 118.2 (s, CArH), 113.7 (s, CArH), 111.2 (s, CArH), 55.6 (s, CH O). The analytical data
3
are in agreement with those reported previously in the literature [63].
◦
◦
1
6
δ
-Isothiocyanato-3,5-dimethoxybenzene (7f). White solid, mp 44–45 C (lit. 48–49 C). Yield
1
7% (0.262 g, 1.34 mmol) after flash chromatography (hexane). H NMR (700 MHz, CDCl ):
3
13
= 6.38–6.36 (m, 3 H, CHAr), 3.77 (s, 6 H, 2
×
CH O). C NMR (176 MHz, CDCl3):
C H), 100.4 (s, C H),
CH O). The analytical data are in agreement with those reported previously in
δ
= 161.3
3
(s, 2
×
C OMe), 135.5 (s, NCS), 132.6 (s, C NCS), 104.1 (s, 2
×
Ar
Ar
Ar
Ar
5
5.6 (s, 2
×
3
the literature [81].
◦ ◦
1
(
δ
-Chloro-4-isothiocyanatobenzene (7g). White solid, mp 42–43 C (lit. 44–45 C). Yield 61%
1
0.207 g, 1.22 mmol) after flash chromatography (hexane). H NMR (700 MHz, CDCl ):
3
13
= 7.32 (d, JHH = 8.9 Hz, 2 H, CHAr), 7.15 (d, JHH = 8.9 Hz, 2 H, CHAr). C NMR (176 MHz,
= 137.0 (s, NCS), 133.1 (s, C ), 130.2 (s, C ), 129.9 (s, 2 C H), 127.1 (s,
CDCl3):
2
literature [63].
δ
×
Ar
Ar
Ar
×
C H). The analytical data are in agreement with those reported previously in the
Ar
1
-Fluoro-4-isothiocyanatobenzene (7h). Colorless oil. Yield 59% (0.180 g, 1.18 mmol) after flash
1
chromatography (hexane). H NMR (700 MHz, CDCl ):
δ
= 7.21–7.18 (m, 2 H, CHAr), 7.05–
= 161.2 (d, JCF = 248.8 Hz, CArF), 136.2
s, NCS), 127.5 (s, C NCS), 127.4 (d, J = 8.3 Hz, C H), 116.7 (d, J = 23.9 Hz, C H).
3
13
7
(
.02 (m, 2 H, CHAr). C NMR (176 MHz, CDCl3): δ
Ar
CF
Ar
CF
Ar
The analytical data are in agreement with those reported previously in the literature [63].
◦
◦
1
(
δ
-Bromo-4-isothiocyanatobenzene (7i). White solid, mp 55–56 C (lit. 59–60 C). Yield 53%
1
0.227 g, 1.06 mmol) after flash chromatography (hexane). H NMR (700 MHz, CDCl ):
3
13
= 7.47 (d, JHH = 8.9 Hz, 2 H, CHAr), 7.09 (d, JHH = 8.8 Hz, 2 H, CHAr). C NMR (176 MHz,
= 137.2 (s, NCS), 132.9 (s, 2 C H), 130.7 (s, C NCS), 127.3 (s, 2 C H),
20.9 (s, CArBr). The analytical data are in agreement with those reported previously in the
literature [63].
CDCl3):
1
δ
×
×
Ar
Ar
Ar

Molecules 2021, 26, 2740
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◦
◦
1
0
,3-Diisothiocyanatobenzene (7j). White solid, mp 49–50 C (lit. 50–51 C). Yield 40% (0.154 g,
1
.8 mmol) after flash chromatography (hexane). H NMR (700 MHz, CDCl ):
δ
= 7.33
3
(
t, JHH = 8.1 Hz, 1 H, CH ), 7.12 (dd, J
= 8.1 Hz, JHH = 2.0 Hz, 2 H, CH ), 7.06 (t,
Ar
HH Ar
JHH = 2.0 Hz, 1 H, CHAr). 1 C NMR (176 MHz, CDCl3):
CArNCS), 130.8 (s, CArH), 124.6 (s, 2
in agreement with those reported previously in the literature [63].
3
δ
= 138.1 (s, 2
×
NCS), 133.0 (s,
2
×
×
CArH), 122.8 (s, CArH). The analytical data are
3
.2.4. General Procedure for Compounds 9a–j
Hydrochloride 8a (2 mmol, 1 equiv.), NMM (0.66 mL, 6 mmol, 3 equiv. or 1.32 mL,
2 mmol, 6 equiv. for 8i) and CS (0.36 mL, 6 mmol, 3 equiv. or 0.72 mL, 12 mmol, 6 equiv.
–j
1
2
for 8i) were dissolved in dry DCM (5 mL) in 10 mL round bottom flask equipped with
-
a magnetic bar, and stirred 10 min at rt. Next, DMT/NMM/TsO (
1) (0.828 g, 2 mmol,
1
equiv. or 1.656 g, 4 mmol, 2 equiv. for 8i) was added. The reaction was mixed 30 min at
rt. After that the reaction mixture was diluted with DCM (50 mL) and washed by H O
2
(
5 mL), 1 N HCl (2
products were purified by flash chromatography on silica gel (7–8 g) using mixture hexane:
EtOAc 20: 1 as eluent. Pure isothiocyanates 9a were isolated after careful evaporation of
the solvent and removal of volatile residues under reduced pressure.
×
5 mL), H O (5 mL), and dried under anhydrous MgSO . The crude
2
4
–
j
(
S)-Methyl2-Isothiocyanatopropanoate (9a). Colorless oil. Yield 50% (0.145 g, 1.0 mmol) after
flash chromatography (hexane/EtOAc 20:1). The er was determined by HPLC using a
Chiralpak IC column (hexane/i-PrOH, 98:2); tmajor = 6.92 min, tminor = 6.81 min (>99: 1 er).
1
H NMR (700 MHz, CDCl3):
.60 (d, JHH = 7.1 Hz, 3 H, CH3). 13C NMR (176 MHz, CDCl3):
NCS), 54.9 (s, CH O), 53.3 (s, CHNCS), 19.6 (s, CH ). IR (ATR): 2041 (NCS), 1744 (CO),
δ
= 4.35 (q, J
= 7.1 Hz, 1 H, CHNCS), 3.81 (s, 3 H, CH O),
HH 3
1
δ = 169.5 (s, CO), 137.5 (s,
3
3
−
1
D
D
1
0
450, 1435, 1289, 1208, 1149, 1053 cm . [
α
]
+24.1 (c 0.32, CHCl ) (lit. [
α
]
+25.8 (c
25
3
25
+
+
.32, CHCl )). HRMS: 145.0197, ([M] , C H NO S ; calc. 145.0204). The concentration of
3
5
7
2
sample for CD analysis was 0.52 mg/mL (MeOH). The analytical data are in agreement
with those reported previously in the literature [66].
(R)-Methyl 2-Isothiocyanatopropanoate (9b). Colorless oil. Yield 51% (0.147 g, 1.02 mmol)
after flash chromatography (hexane/EtOAc 20:1). The er was determined by HPLC using a
Chiralpak IC column (hexane/i-PrOH, 98:2); tmajor = 6.81 min, tminor = 6.92 min (>99: 1 er).
1
H NMR (700 MHz, CDCl3):
δ
= 4.35 (q, J
= 7.1 Hz, 1 H, CHNCS), 3.81 (s, 3 H, CH O),
HH 3
1
.60 (d, JHH = 7.1 Hz, 3 H, CH3). 13C NMR (176 MHz, CDCl3):
δ = 169.5 (s, CO), 137.5 (s,
NCS), 54.9 (s, CH O), 53.3 (s, CHNCS), 19.6 (s, CH ). IR (ATR): 2045 (NCS), 1746 (CO),
3
3
−
+
1
D
D
1
0
450, 1435, 1289, 1209, 1150, 1054 cm . [
.32, CHCl )). HRMS: 145.0197, ([M] , C H NO S ; calc. 145.0196). The concentration of
α
]
−
23.3 (c 0.32, CHCl ) (lit. [
α
]
−
22.8 (c
25
3
25
+
3
5
7
2
sample for CD analysis was 0.51 mg/mL (MeOH). The analytical data are in agreement
with those reported previously in the literature [66].
(
S)-Benzyl 2-isothiocyanatopropanoate (9c). Colorless oil. Yield 38% (0.168 g, 0.76 mmol) after
1
flash chromatography (hexane/EtOAc 20:1). H NMR (700 MHz, CDCl ):
δ
= 7.40–7.34
3
(
1
m, 5 H, CH ), 5.23 (d, J
.60 (t, JHH = 7.1 Hz, 3 H, CH3). C NMR (176 MHz, CDCl3):
= 1.5 Hz, 2 H, CH ), 4.37 (q, J
= 7.1 Hz, 1 H, CHNCS),
Ar
HH 2 HH
13
δ
= 168.9 (s, CO), 137.8 (s,
NCS), 134.9 (s, C ), 128.8 (s, 3 C, 3 C H), 128.5 (s, 2 C, 2
×
×
C H), 68.1 (s, CH ), 55.0
Ar
Ar
Ar
2
(
s, CHNCS), 19.6 (s, CH ). IR (ATR): 2049 (NCS), 1744 (CO), 1497, 1452, 1286, 1190, 1146,
3
−
1
D
+
+
1
052, 741, 695 cm . [
α
]
+32.1 (0.31 CHCl ). HRMS: 221.0510, ([M] , C H NO S ;
25 3 11 11 2
calc. 221.0501). The concentration of sample for CD analysis was 0.3 mg/mL (MeOH). The
analytical data are in agreement with those reported previously in the literature [82].
(
S)-tert-butyl 2-isothiocyanatopropanoate (9d). Colorless oil. Yield 35% (0.131 g, 0.7 mmol)
1
after flash chromatography (hexane/EtOAc 20:1). H NMR (700 MHz, CDCl ):
JHH = 7.1 Hz, 1 H CHNCS), 1.54 (d, JHH = 7.1 Hz, 3 H, CH ), 1.50 (s, 9 H, (CH ) ). C NMR
δ = 4.18 (q,
3
1
3
3
3 3
(176 MHz, CDCl3):
δ = 168.0 (s, CO), 137.2 (s, NCS), 83.6 (s, C(CH ) ), 55.6 (s, CHNCS), 28.1
3 3
(
s, 3 C, 3 CH ), 19.5 (s, CH ). IR (ATR): 2053 (NCS), 1738 (CO), 1477, 1454, 1394, 1223,
×
3
3

Molecules 2021, 26, 2740
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144, 1054, 866 cm 1. [ ]D +21.4 (0.34 CHCl ). HRMS: 187.0667, ([M] , C H NO S ;
−
α
+
+
1
25 3 8 13 2
calc. 187.0663). The concentration of sample for CD analysis was 0.37 mg/mL (MeOH).
The analytical data are in agreement with those reported previously in the literature [83].
(
S)-Methyl 2-isothiocyanato-3-methylbutanoate (9e). Colorless oil. Yield 63% (0.218 g, 1.26
1
mmol) after flash chromatography (hexane/EtOAc 20:1). H NMR (700 MHz, CDCl ):
3
δ
= 4.16 (d, JHH = 4.2 Hz, 1 H, CHNCS), 3.79 (s, 3 H, CH O), 2.35–2.28 (m, 1 H, CH),
3
13
1
.06 (d, JHH = 6.9 Hz, 3 H, CH ), 0.96 (d, J
= 6.8 Hz, 3 H, CH3). C NMR (176 MHz,
HH
3
CDCl3):
δ = 168.6 (s, CO), 136.9 (s, NCS), 65.7 (s, CH O), 53.0 (s, CHNCS), 32.7 (s, CH),
3
1
1
9.7 (s, CH ), 17.2 (s, CH ). IR (ATR): 2052 (NCS), 1746 (CO), 1436, 1392, 1258, 1205, 1181,
3
3
−
1
D
D
146, 1062 cm .[
α
]
+16.3 (1.0 EtOH) (lit. [
α
]
+4.1 (1.0 EtOH). HRMS: 173.0510,
25
25
+
+
([M] , C H NO S ; calc. 173.0513). The concentration of sample for CD analysis was
7
11
2
0.46 mg/mL (MeOH). The analytical data are in agreement with those reported previously
in the literature [56].
(
S)-Methyl 2-isothiocyanato-4-methylpentanoate (9f). Colorless oil. Yield 57% (0.213 g, 1.14 mmol)
1
after flash chromatography (hexane/EtOAc 20:1). H NMR (700 MHz, CDCl ):
δ = 4.30
3
(
2
×
d, JHH = 9.7 Hz, JHH = 9.6 Hz, 1 H, CHNCS), 3.80 (s, 3 H, CH O), 1.88–1.81 (m, 2 H,
3
CH ), 1.73–1.69 (m, 1 H, CH(CH ) ), 0.97 (d, J
3
= 6.5 Hz, 3 H, CH ), 0.95 (d, J
= 6.5 Hz,
2
3 2
HH
3
HH
13
H, CH3). C NMR (176 MHz, CDCl3): δ = 169.4 (s, CO), 137.0 (s, NCS), 58.1 (s, CH O),
3
5
3.2 (s, CHNCS), 42.5 (s, CH ), 25.2 (s, CH), 22.8 (s, CH ), 21.2 (s, CH ). IR (ATR): 2052
2
3
1
3
−
D
(NCS), 1748 (CO), 1437, 1270, 1229, 1203, 1149, 983 cm . [
α
]
−
21.7 (0.32 CHCl ) (lit.
25
3
D
+
+
[α
]
−
18.0 (0.016 M CHCl ). HRMS: 187.0667, ([M] , C H NO S ; calc. 187.0661). The
25
3 8 13 2
concentration of sample for CD analysis was 0.41 mg/mL (MeOH). The analytical data are
in agreement with those reported previously in the literature [75].
(
0
2S)-Methyl 2-isothiocyanato-3-methylpentanoate (9g). Colorless oil. Yield 46% (0.172 g,
1
.92 mmol) after flash chromatography (hexane/EtOAc 20:1). H NMR (700 MHz, CDCl ):
3
δ
= 4.20 (d, JHH = 4.5 Hz, 1 H, CHNCS), 3.81 (s, 3 H, CH O), 2.11–2.06 (m, 1 H, CHCH ),
3
3
1
3
.48–1.43 (m, 1 H, H
α
from CH ), 1.34–1.27 (m, 1 H, H from CH ), 1.05 (d, J = 6.8 Hz,
2
β
2
HH
13
H, CH CH), 0.92 (t, J
= 7.5 Hz, 3 H, CH CH ). C NMR (176 MHz, CDCl3):
δ
= 168.6 (s,
3
HH
3
2
CO), 136.8 (s, NCS), 64.9 (s, CH O), 53.0 (s, CHNCS), 39.1 (s, CH ), 24.6 (s, CH), 16.3 (s, CH ),
3
2
3
−
1.4 (s, CH ). IR (ATR): 2054 (NCS), 1746 (CO), 1457, 1436, 1248, 1204, 1144 cm . [α]
3
1
D
1
+
1
25
56
D
+
+
23.5 (1.0 EtOH) (lit. . [
α
]
+15.3 (1.0 EtOH). HRMS: 187.0667, ([M] , C H NO S ; calc.
25 8 13 2
87.0684). The concentration of sample for CD analysis was 0.52 mg/mL (MeOH). The
analytical data are in agreement with those reported previously in the literature [56].
(
S)-Methyl 2-isothiocyanato-3-phenylpropanoate (9h). Colorless oil. Yield 30% (0.132 g, 0.6 mmol)
1
after flash chromatography (hexane/EtOAc 20:1). H NMR (700 MHz, CDCl3):
δ
= 7.36–7.34
(m, 2 H, CHAr), 7.31–7.29 (m, 1 H, CHAr), 7.23–7.22 (m, 2 H, CHAr), 4.48 (dd, JHαHβ = 8.4 Hz,
JHαHγ = 4.8 Hz, 1 H, CH
H, CH
CDCl3):
α
NCS), 3.80 (s, 3 H, CH O), 3.25 (dd, J
Ph), 3.13 (dd, JHβHγ = 13.8 Hz, JHβHα = 8.4 Hz, 1 H, CH Ph). C NMR (176 MHz,
= 13.8 Hz, J
= 4.7 Hz,
3
HγHβ
HγHα
13
1
γ
β
δ
= 168.4 (s, CO), 138.1 (s, NCS), 135.1 (s, C ), 129.4 (s, C H), 128.9 (s, C H),
Ar
Ar
Ar
1
27.7 (s, C H), 60.9 (s, CH O), 53.2 (s, CHNCS), 39.8 (s, CH ). IR (ATR): 2038 (NCS), 1745
Ar 3 2
−
1
D
D
(CO), 1436, 1271, 1208, 1175, 1117, 697 cm . [
α]
–60.0 (c 1.0, toluene) (lit. [
α
] –62.2 (c
25
25
+
+
1.0, toluene)). HRMS: 211.0510, ([M] , C H NO S ; calc. 221.0506). The concentration of
11
11
2
sample for CD analysis was 0.41 mg/mL (MeOH). The analytical data are in agreement with
those reported previously in the literature [66].
(
S)-Methyl 2,6-diisothiocyanatohexanoate (9i). Colorless oil. Yield 25% (0.122 g, 0.5 mmol)
1
after flash chromatography (hexane/EtOAc 20:1). H NMR (700 MHz, CDCl3):
δ
= 4.32
(
2
×
d, JHH = 7.8 Hz, JHH = 7.8 Hz, 1 H, CHNCS), 3.83 (s, 3 H, CH O), 3.56 (t, J
= 6.5 Hz,
3 HH
2
H, CH NCS), 1.99–1.91 (m, 2 H, CH ), 1.78–1.73 (m, 2 H, CH ), 1.61–1.57 (m, 2 H, CH ).
2
2
2
2
1
3
C NMR (176 MHz, CDCl3):
δ = 168.7 (s, CO), 138.2 (s, NCSCH), 130.9 (s, NCSCH ), 59.2
2
(
s, CH O), 53.4 (s, CHNCS), 44.8 (s, CH NCS), 32.7 (s, CH ), 29.3 (s, CH ), 22.8 (s, CH ).
3 2 2 2 2
−
1
IR (ATR): 2167 (NCS), 2053 (NCS), 2036 (NCS), 1744 (CO), 1435, 1346, 1206, 1175 cm
.

Molecules 2021, 26, 2740
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]D
−
18.2 (0.5 toluene). HRMS: 244.0340, ([M] , C H N O S ; calc. 244.0343). The
9 12 2 2 2
+
+
[
α
25
concentration of sample for CD analysis was 0.52 mg/mL (MeOH). New compound.
Ethyl 2-isothiocyanatoacetate (9j). Colorless oil. Yield 48% (0.139 g, 0.96 mmol) after flash chro-
1
matography (hexane/EtOAc 20:1). H NMR (700 MHz, CDCl3):
δ
= 4.27 (q, J
= 7.1 Hz,
HH
2
H, CH ), 4.21 (s, 2 H, CH NCS), 1.30 (t, J
= 7.1 Hz, 3 H, CH3). 13C NMR (176 MHz,
2
2
HH
CDCl3):
δ
= 166.2 (s, CO), 138.4 (s, NCS), 62.6 (s, CH CH ), 46.5 (s, CHNCS), 14.1 (s, CH ). IR
2
3
3
−
1
+
+
(
ATR): 2052 (NCS), 1744 (CO), 1394, 1197, 1018 cm . HRMS: 145.0197, ([M] , C H NO S ;
5
7
2
calc. 145.0189). The concentration of sample for CD analysis was 0.33 mg/mL (MeOH).
The analytical data are in agreement with those reported previously in the literature [41].
3.2.5. General Procedure for Compounds 11a–c
Hydrochloride 10a (2 mmol, 1 equiv.), Et N (0.84 mL, 6 mmol, 3 equiv.) and CS
–
c
3
2
(
0.36 mL, 6 mmol, 3 equiv.) were dissolved in dry DCM (3 mL) in 10 mL pressure vial,
−
equipped with a magnetic bar, and stirred 5 min at rt. Next, DMT/NMM/TsO
(1) (0.828 g,
2
mmol, 1 equiv.) was added. The reaction was carried under MW conditions (standard
◦
mode, 3 min, 90 C). After that, the reaction mixture was diluted with DCM (50 mL) and
washed by H O (5 mL), 1 N HCl (2 5 mL), H O (5 mL), and dried under anhydrous
MgSO . The crude product were purified by flash chromatography on silica gel (7–8 g)
×
2
2
4
using hexane as eluent. Pure isothiocyanates 11a–c were isolated after evaporation of the
solvent under reduced pressure.
Ethyl 6-isothiocyanatohexanoate (11a). Colorless oil. Yield 75% (0.301 g, 1.5 mmol) after
1
flash chromatography (hexane/EtOAc 10:1). H NMR (700 MHz, CDCl3):
δ
= 4.12 (q,
JHH = 7.1 Hz, 2 H, CH O), 3.51 (t, J
= 6.6 Hz, 2 H, CH NCS), 2.31 (t, J
= 7.4 Hz,
2
HH
2 HH
CH CO), 1.73–1.79 (m, 2 H, CH ), 1.67–1.63 (m, 2 H, CH ), 1.47–1.42 ((m, 2 H, CH ), 1.25
2
2
2
2
(
t, JHH = 7.1 Hz, 3 H, CH3). 13C NMR (176 MHz, CDCl3):
δ
= 173.3 (s, CO), 130.1 (s, NCS),
6
0.4 (s, CH O), 44.9 (s, CH NCS), 34.1 (s, CH ), 29.7 (s, CH ), 26.1 (s, CH ), 24.2 (s, CH ),
2 2 2 2 2 2
−
4.3 (s, CH ). IR (ATR): 2175 (NCS), 2088 (NCS), 1728 (CO), 1452, 1180, 1155 cm . HRMS:
3
1
1
2
+
+
01.0823, ([M] , C H NO S ; calc. 201.0825). New compound.
9
15
2
◦
Ethyl 4-(isothiocyanatomethyl)benzoate (11b). White solid, mp 45–46 C. Yield 81% (0.358 g,
1
1
.62 mmol) after flash chromatography (hexane/EtOAc 10:1). H NMR (700 MHz, CDCl3):
δ
= 8.06 (d, JHH = 8.5 Hz, 2 H, CH ), 7.39 (d, J
= 8.6 Hz, 2 H, CH ), 4.78 (s, 2 H,
= 7.2 Hz, 3 H, CH3). C NMR
HH
Ar
HH Ar
1
3
CH ), 4.38 (q, J
= 7.1 Hz, 2 H, CH O), 1.40 (t, J
2
HH
2
(
2
176 MHz, CDCl3):
C, 2 C H), 126.7 (s, 2 C, 2
IR (ATR): 2197 (NCS), 2118 (NCS), 1698 (CO), 1610, 1411, 1273, 1175, 1103, 1018, 746 cm
δ = 166.0 (s, CO), 139.1 (s, C ), 133.6 (s, NCS), 130.7 (s, C ), 130.3 (s,
Ar Ar
×
×
C H), 61.2 (s, CH O), 48.5 (s, CH NCS), 14.4 (s, CH ).
Ar 2 2 3
Ar
−
1
.
+
+
HRMS: 221.0510, ([M] , C H NO S ; calc. 221.0509). New compound.
11
11
2
Trans-ethyl 4-(isothiocyanatomethyl)cyclohexanecarboxylate (11c). Colorless oil. Yield 92%
1
(
0.418 g, 1.84 mmol) after flash chromatography (hexane/EtOAc 10:1). H NMR (700 MHz,
CDCl3):
δ
= 4.11 (q, JHH = 7.1 Hz, 2 H, CH O), 3.38 (d, J
= 6.2 Hz, 2 H, CH NCS),
HH 2
2
2
2
.23 (tt, JHH = 12.3 Hz, JHH = 3.6 Hz, 1 H, CH), 2.05 (dd, JHH = 13.7 Hz, JHH = 3.5 Hz,
H, CH ), 1.87 (dd, J = 13.7 Hz, JHH = 3.4 Hz, 2 H, CH ), 1.70–1.64 (m, 1 H, CH), 1.45
2
HH
2
(
qd, JHH = 13.4 Hz, JHH = 3.5 Hz, 2 H, CH ), 1.24 (t, J
= 7.1 Hz, 3 H, CH ), 1.08 (qd,
2
HH 3
JHH = 13.2 Hz, JHH = 3.5 Hz, 2 H, CH2). 13C NMR (176 MHz, CDCl3):
δ
= 175.5 (s, CO), 130.2
(
(
1
s, NCS), 60.3 (s, CH O), 50.9 (s, CH NCS), 42.9 (s, CH), 37.9 (s, CH), 29.4 (s, 2 CH ), 28.2
×
2
2
2
s, 2
×
CH ), 14.2 (s, CH ). IR (ATR): 2180 (NCS), 2090 (NCS), 1724 (CO), 1449, 1175, 1065,
2 3
−
040, 681 cm . HRMS: 227.0980, ([M] , C H NO S ; calc. 227.0989). New compound.
11 17 2
1
+
+
3
.2.6. 2,4- dimethoxy-6-methylthio-1,3,5-triazine (14)
Yield 7% (0.027 g, 0.14 mmol) after flash chromatography (hexane/EtOAc 10:1). H
1
13
NMR (700 MHz, CDCl3):
MHz, CDCl3):
The analytical data are in agreement with those reported previously in the literature [84].
δ
= 4.00 (s, 6 H, 2
×
OCH ), 2,53 (s, 3 H, SCH ). C NMR (176
3
3
δ
= 185.5 (s, CS), 171.4 (s, 2
×
COCH ), 55.4 (s, 2
×
OCH ), 13.5 (s, SCH ).
3
3
3

Molecules 2021, 26, 2740
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3.3. Study of Antimicrobial Activity
Antibacterial activities of solutions of tested compounds were evaluated using well
diffusion method on trypticase soy agar TSA (Merck). The inhibition zones were reported
in millimeter (mm). The 24 h single colonies on agar plates were used to prepare the
8
bacterial suspensions with the turbidity of 0.5 McFarland degree (~1.5
Turbidity of the bacterial suspensions were measured using a DEN
×
10 CFU/mL).
−
1 densitometer (Merck).
Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 8739) were used as references
for the antibacterial assay. TSA agar plates were inoculated with bacterial strain under
aseptic conditions and wells (diameter = 12mm) were filled with 250
and incubated at 37 C for 24 h. After the incubation period, the diameter of the growth
inhibition zones was measured.
µ
L of the test samples
◦
The sterile distilled water was used as a negative control while chloramphenicol
(Sigma-Aldrich) at the concentration (20
µg/mL) 0.062 µM in DMSO was used as a positive
standard. All tests were performed in triplicate.
4. Conclusions
To conclude, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-
−
sulfonate (DMT/NMM/TsO ) can be recognized as a new and efficient desulfurating agent
suitable for microwave-assisted, one-pot synthesis of aliphatic and aromatic isothiocyanates
and classical synthesis (normal reaction). The synthetic procedures presented in this
study enabled the rapid and simple acquisition of a library of 20 structurally diverse
isothiocyanates with good or very good yields (40–97%), high purity, and in the case
of optically active compounds, low racemization (er 99 > 1). The synthesis of aliphatic
isothiocyanates also took place in aqueous medium in the presence of organic base, with
only slightly lower yields compared to those obtained in DCM.
−
DMT/NMM/TsO was also used to synthesis of isothiocyanate derivatives from
methyl, ethyl, benzyl, and tert-butyl esters of L- and D- natural amino acids (alanine, valine,
leucine, isoleucine, phenylalanine, and lysine) as well as achiral glycine, with satisfactory
yields (25–63%), high purity, and low racemization (er 99 > 1). Due to the observed
racemization under microwave-assisted syntheses, these reactions were performed under
normal conditions. The absolute configuration of chiral ITCs was determined by CD. The
Cotton Effect was strongly positive for ITCs with the S configuration, and for ITCs with
the R configuration it was strong negative. The optical rotation of all chiral ITCs was also
measured. As well as isothiocyanate derivatives of natural amino acids, isothiocyanate
derivatives of ethyl esters of 6-aminocaproic acid, 4-(aminomethyl)benzoic acid, and
−
tranexamic acid were synthesized with DMT/NMM/TsO in a microwave reactor, rapidly
and with very high yields (75–92%), and in the case of trans ITC 11c without racemization.
Antibacterial activity against E. coli and S. aureus bacterial strains showed that the
most of tested isothiocyanates derivatives of natural and unnatural amino acids are active;
however, their activity was worse than activity of positive control–chloramphenicol. The
most active for both strains was ITC 9e. Further research is ongoing to determine the
biological properties of ITCs derived from both natural and unnatural amino acids.
Supplementary Materials: The following are available online. Figures S1–S68: H and 13C NMR
1
spectra of compounds 4a
compounds 4c and 9a
–
b
j
,
7a
–
j
,
9a
–
j, 11a
–
c
, and 14; Figures S69–S74: HPLC chromatograms of
–
d
–
; Figures S75–S86: CD spectra of compounds 4c
–
d
and 9a ; Figure S87:
–j
Pictures of Petri dishes, tests for antibacterial activity against Staphylococcus aureus (ATCC 6538) and
Escherichia coli (ATCC 8739).
Author Contributions: Formal analysis, Ł.J.; conceptualization, Ł.J.; investigation, Ł.J. and D.K.;
methodology, Ł.J. and D.K.; visualization, Ł.J.; project administration, Ł.J.; writing—original draft,
Ł.J.; funding acquisition, B.K.; supervision, B.K.; writing—review and editing, B.K. and Ł.J. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Centre for Research and Development under
Project POIR.04.01.02-00-0004/17.

Molecules 2021, 26, 2740
21 of 24
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: Thanks to Tadeusz Gajda and Zbigniew Kaminski for their helpful discus-
sions, Łukasz Albrecht for access to chiral stationary phase HPLC, and Sebastian Frankowski for
HPLC analysis.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 4a–j, 7a–j, 9a–j, 11a–c, and 14 are available from
the authors.
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