Stereoselective synthesis and Cytoselective toxicity of Monoterpene-fused 2-Imino-1,3-Thiazines

Article abstract of DOI:10.3390/molecules191015918

Starting from pinane-, apopinane- and carane-based 1,3-amino alcohols obtained from monoterpene-based ?-amino acids, a library of monoterpene-fused 2-imino-1,3-Thiazines as main products and 2-Thioxo-1,3-oxazines as side-products were prepared via two- or

Full text of DOI:10.3390/molecules191015918

Molecules 2014, 19, 15918-15937; doi:10.3390/molecules191015918
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Stereoselective Synthesis and Cytoselective Toxicity of
Monoterpene-Fused 2-Imino-1,3-thiazines
Zsolt Szakonyi 1,*, István Zupkó 2, Reijo Sillanpää 3 and Ferenc Fülöp 1,4,
*
1
Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös utca 6,
H-6720 Szeged, Hungary
2
3
4
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6,
H-6720 Szeged, Hungary; E-Mail: zupko@pharm.u-szeged.hu
Department of Chemistry, University of Jyväskylä, P.O. Box 35, 40351 Jyväskylä, Finland;
E-Mail: e.reijo.j.sillanpaa@jyu.fi
Stereochemistry Research Group of the Hungarian Academy of Sciences, Eötvös u. 6, H-6720
Szeged, Hungary
* Authors to whom correspondence should be addressed; E-Mails: szakonyi@pharm.u-szeged.hu (Z.S.);
fulop@pharm.u-szeged.hu (F.F.); Tel.: +36-62-545-564 (F.F.); Fax: +36-62-545-705 (F.F.).
External Editor: Jean Jacques Vanden Eynde
Received: 27 August 2014; in revised form: 22 September 2014 / Accepted: 24 September 2014 /
Published: 2 October 2014
Abstract: Starting from pinane-, apopinane- and carane-based 1,3-amino alcohols obtained
from monoterpene-based β-amino acids, a library of monoterpene-fused 2-imino-1,3-
thiazines as main products and 2-thioxo-1,3-oxazines as side-products were prepared via
two- or three-step syntheses. When thiourea adducts prepared from 1,3-amino alcohols and
aryl isothiocyanates were reacted with CDI under mild conditions, O-imidazolylcarbonyl
intermediates were isolated which could be transformed to the desired 1,3-thiazines under
microwave conditions. 1,3-Thiazines and 2-thioxo-1,3-oxazine side-products could also be
prepared in one-step reactions through the application of CDI and microwave irradiation.
The ring-closure process was extended to cycloalkane-based γ-hydroxythioureas. The
carane- and apopinane-based derivatives exhibited marked antiproliferative activity against
a panel of human adherent cancer cell lines (HeLa, A2780, MCF7 and A431).
Keywords: monoterpene; 1,3-amino alcohol; stereoselective; CDI; 1,3-thiazine; antiproliferative

Molecules 2014, 19
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1. Introduction
In the past decade, alicyclic 1,3-amino alcohols have proved to be versatile building blocks. They have
been applied as useful starting materials in the stereoselective syntheses of compounds of pharmacological
interest and they have served as chiral ligands and auxiliaries in enantioselective transformations [1–3].
Several natural chiral terpenes, including (+)-pulegone [4–6] α- and β-pinene [7–9] and fenchone-
camphor [10–12], have proved to be excellent sources for the production of various amino alcohols, which
have been successfully applied in enantioselective syntheses. The transformation of enantiomerically pure
α-pinene to β-amino acid derivatives such as 1,3-amino alcohols was recently reported [9,13]. These
synthons served as chiral auxiliaries in the enantioselective synthesis of secondary alcohols or pharmacons,
e.g., esomeprasol [14–18].
Besides their value in enantioselective catalysis, 1,3-amino alcohols are good starting points for the
synthesis of various heterocyclic ring systems, such as 1,3-oxazines, 1,3-thiazines or 1,4-oxazepams [2,19].
The 2-imino-1,3-thiazine and 2-iminothiazolidine ring systems can be found as moieties in biologically
relevant compounds, including antifungicidal and antimicrobial agents [20], BACE1 inhibitors [21], or
cannabinoid receptor agonists [22–24]. New spiro derivatives of 2-imino-1,3-thiazines have been
synthetized and shown to be potential neuroprotectors [25].
In recent years, we have devised novel pathways to synthetize new monoterpene-based chiral β-lactams
and β-amino acid derivatives derived from (–)- and (+)-α-pinene, (–)-3-carene, (–)- and (+)-apopinene and
myrtenic acid [9,13,26–30]. These amino acid derivatives have proved to be excellent building blocks for
the syntheses of compounds with multidrug resistance (MDR) antagonist activity [29].
We also found that monoterpene-based 1,3-amino alcohols prepared from the abovementioned β-amino
acid derivatives are excellent building blocks for the synthesis of 2-imino-1,3-oxazines which possess
marked anti-cancer activity [31]. The analogous pinane- or apopinane-based 2-imino-1,3-thiazines could
not be prepared by the methods applied earlier for the synthesis of cycloalkane- or norbornane-fused
analogue 1,3-thiazines [32,33].
The aim of the present work was to synthetize new chiral pinane-, apopinane- and carane-fused
2-imino-1,3-thiazines, analogues of 1,3-oxazines with noteworthy cytoselective toxicity on multiple
cancer cell lines.
2. Results and Discussion
2.1. Syntheses of Alicyclic and Monoterpene-Based 1,3-Amino Alcohols
The synthetic routes applied for the preparation of 1,3-amino alcohols 9–12 and 16–18 followed
literature methods (Figure 1) [9,13,27–29,31,33]. The corresponding β-lactams were prepared by the
stereoselective cycloaddition of chlorosulfonyl isocyanate to cyclopentene, cycloxene, α-pinene, 3-carene
and apopinene, followed by ring opening, which resulted in cis-fused β-amino esters 5–8 and 14. Under
alkaline conditions, the cis-amino ester 14 underwent fast and complete isomerization at the carboxylic
function, resulting in the trans-amino ester 15 in excellent yield [28]. Reduction of 5–8, 14 and 15 with
LAH led to the primary amino alcohols 9–12, 16 and 17. From 17, N-benzyl derivative 18 was prepared by
reductive alkylation with benzaldehyde and NaBH4 in EtOH [31].

Molecules 2014, 19
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Figure 1. Synthesis of 1,3-amino alcohol starting materials.
R1
R1
R1
NH2
refs. 1-4
NH2
OH
COOR2
9-12
1-4
5-8
(S)
NH2
NH2
refs. 1-4
O
(S)
COOEt
COOEt
15
13
14
NHR3
OH
NH2
OH
16
17,18
R1
3,7,11
4,8,12
2,6,10
1,5,9
1,2,5,6,9,10: R1 = H; 3,4,7,8,11,12: R2 = Me; 5,6: R2 = Et; 7,8: R2 = Me;
17: R3 = H; 18: R3 = CH2Ph
2.2. Syntheses of 2-Imino-1,3-thiazine Derivatives
The intermediate thiourea adducts 19–23 were prepared in good to excellent yields by the reaction of the
appropriate aryl isothiocyanates and 1,3-amino alcohols 10, 11 and 16–18 (Scheme 1, Table 1) [13,27,31].
Scheme 1. Synthesis and attempted ring closure of thioureas 19–23.
Reagents and conditions: (i) 1.05 equiv. R3NCS, toluene, rt, yield: 85%–95%; (ii) 10% HCl/EtOH,
rt to reflux; (iii) DEAD, TPP, THF, rt.

Molecules 2014, 19
Table 1. Thioureas 19–23 and O-imidazolylcarbonyl intermediates 29–33.
15921
General Structure R1
R2
H
H
H
H
H
R3
Ph
Ph
Compound No.
Me
H
H
H
H
19a
21a
21b
21c
21d
4-MeC6H4
4-FC6H4
3-MeOC6H4
H
H
H
Ph
Ph
22a
23a
CH2Ph
Me
H
Ph
20a
Me
H
H
H
H
H
H
Ph
Ph
4-MeC6H4
4-FC6H4
29a
31a
31b
31c
H
H
H
H
3-MeOC6H4
31d
H
Ph
32a
H
H
CH2Ph
Ph
H
33a
30a
H
Although a number of methods are known for the conversion of 3-hydroxypropylthioureas to the
corresponding 2-imino-1,3-thiazines, e.g., acid-promoted dehydrative cyclization [32,33] or Mitsubobu
conditions [34], in the cases of the highly constrained acid-sensitive bicyclic pinane and carane skeletons
the expected 2-imino-1,3-thiazine systems were not obtained.
Bernacki et al. recently reported that they also identified 2-aminothiazoline cyclization products besides
the expected main product thioureas in the reactions of 1,2-amino alcohols and 3-aminobenzonitrile in the
presence of thio-CDI [35]. Finally, they devised an excellent protocol for the cyclization of 1,2-amino
alcohol-based thiourea derivatives to yield 2-phenylaminothiazolines under mild conditions (rt, THF, CDI
or thio-CDI).
When we applied the above-mentioned mild conditions, we observed the formation of
O-imidazolylcarbonyl intermediates 29–33 (similar intermediates were presumed, but could not be
isolated in Bernacki’s work) as single products even when the reaction mixture was subjected to
conventional heating from room temperature to reflux (Table 1). The structures of intermediates 31c and
31d were determined by X-ray crystallography (Figure 2) and NMR measurements.

Molecules 2014, 19
15922
Figure 2. ORTEP plots of the configurations of the major diastereoisomers 31c and 31d
(the C atom of the methoxy group (C14) is disordered in a 1:1 ratio).
31c
31d
However, when microwave irradiation was applied to the isolated intermediates 29–33 in THF, we
obtained the desired 2-imino-1,3-thiazines 25–28 as main products in acceptable yields in a short time
(60 min) (Scheme 3, Table 2).
Table 2. 2-Phenylimono-1,3-thiazines 24–28.
General Structure
R1
Me
H
H
H
R2
H
H
H
H
H
R3
Ph
Ph
Compound No.
24a
26a
26b
26c
26d
4-MeC6H4
4-FC6H4
3-MeOC6H4
H
H
H
H
Ph
Ph
27a
28a
CH2Ph
Me
H
Ph
25a
The ring-closure process could be carried out in one step by applying CDI in THF under microwave
conditions. Moreover, we observed that 2-thioxo-1,3-oxazines 34–37 were also formed as minor products
in 10%–24% yields. The transformation of 20a was an exception, when thiazine 25a was isolated as a
single product. Besides NMR assignments, the structures of 35 and 36 were proved by independent
synthesis starting from the corresponding amino alcohols and thiophosgene.
The above procedure was subsequently extended to 1-(2-hydroxymethylcycloalkyl)thioureas. Starting
from cycloalkane-based 1,3-amino alcohols 9 and 10, thioureas 38 and 39 were prepared by a literature
method [33]. When 38 and 39 were treated with CDI in THF, rapid conversion to intermediates 40 and 41
was observed and in this case the ring-closure proceeded under conventional heating to yield 1,3-thiazines
42 and 43 (Scheme 2). We observed that the developed method could be applied more easily in the case of
thioureas with sterically less hindered structures.

Molecules 2014, 19
15923
Scheme 2. Extension of the ring-closure process.
H
NHPh
NH2
N
i
S
OH
OH
9,10
38,39
ii
H
NHPh
H
N
N
NPh
iii
S
S
O
O
42,43
40,41
N
N
9,38,40,42
10,39,41,43
Reagents and conditions: (i) 1.05 equiv. PhNCS, toluene, rt, 4 h; (ii) 1.5 equiv. CDI, THF, rt, 1 h;
(iii) THF, reflux, 3 h, yield: 73%–86%
2.3. Antiproliferative Activities
The novel 2-imino-1,3-thiazines 24–28 (Scheme 3) and some of their analogues were subjected to in
vitro pharmacological studies in order to investigate their antiproliferative properties on a panel of human
adherent cancer cell lines. The results of MTT assays are presented in Table 3. Carane-based compound
25a proved to be the most potent of the tested compounds, exhibiting a cell growth-inhibiting capacity
comparable to that of the reference agent cisplatin. Compounds with a pinane ring were generally less
potent, while the introduction of a methyl group at position 2 (Scheme 3) favoured the action (24a).
Substitution of the N-phenyl ring had a limited and inconsequential impact on the efficacy (26a–d), while
the introduction of a N-benzyl function onto the 1,3-thiazine skeleton was disadvantageous (28a). Since no
substantial difference was observed between the effects of 26a and 27a, the configuration of C-3 (cis or
trans ring fusion, Scheme 3) also seems irrelevant. On the other hand, the 2-thioxo-1,3-oxazine analogue
(35) was completely ineffective, indicating that the arylimino substituent is an essential part of the
molecule. Replacement of the pinane or carane ring system with cyclopentane (42) or cyclohexane (43)
also led to ineffective congeners, demonstrating the crucial role of the bicyclic monoterpene as a building
block for the design and synthesis of novel antiproliferative agents.

Molecules 2014, 19
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Scheme 3. CDI mediated ring closure of thioureas 19–23.
Reagents and conditions: (i) 1.5 equiv. CDI, THF, rt, 6 h, yield: 52%–78%; (ii) MW, 200 W,
125 °C, 1 h, THF, yield: 49%–67% for 24–28 and 8–22% for 34–37; (iii) 1.5 equiv. CDI, MW,
200 W, 125 °C, 1 h, yield: 47%–64% for 24–28 and 10%–24% for 34–37.
Table 3. Antiproliferative effects of 2-imino-1,3-thiazines 24–28, 42, 43 and 2-thioxo-1,3-
oxazine 35 on human cancer cell lines.
Growth Inhibition, % ± SEM a
Compnd.
24a
Conc.
HeLa
-
A2780
MCF7
A431
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
67.45 ± 0.83
86.02 ± 0.27
65.24 ± 1.65
96.42 ± 0.15
-
39.40 ± 2.41
62.08 ± 1.78
65.91 ± 0.96
86.09 ± 1.20
26.52 ± 2.79
50.45 + 1.79
-
73.01 ± 1.52
83.15 ± 1.25
60.54 ± 1.79
94.17 ± 0.51
67.70 ± 1.49
85.68 ± 0.74
65.20 ± 1.47
74.49 ± 1.02
32.93 ± 1.20
34.20 ± 1.02
34.92 ± 2.66
82.46 ± 1.11
77.32 ± 0.94
80.05 ± 1.15
25.59 ± 1.93
-
25a
96.40 ± 0.28
-
26a
22.03 ± 1.57
-
68.93 ± 0.97
45.45 ± 2.01
58.67 ± 1.29
40.02 ± 0.88
56.86 ± 1.17
20.87 ± 1.87
92.17 ± 0.51
37.26 ± 2.35
42.80 ± 2.78
26b
37.02 ± 2.20
44.90 ± 0.88
53.60 ± 1.07
27.40 ± 0.52
33.67 ± 2.61
-
43.66 ± 2.32
21.55 ± 0.99
30.93 ± 1.29
-
26c
26d
63.84 ± 2.36
43.83 ± 0.99
47.15 ± 2.93
27a
21.57 ± 0.92

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Table 3. Cont.
Growth Inhibition, % ± SEM a
Compnd.
Conc.
HeLa
A2780
MCF7
A431
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
46.83 ± 1.42
42.24 ± 2.48
23.10 ± 1.00
50.23 ± 0.52
-
-
-
-
-
-
-
-
-
28a
35
23.84 ± 1.08
-
-
-
-
-
-
-
-
-
-
-
42
41.23 ± 1.30
-
-
-
-
43
24.07 ± 2.96
42.61 ± 2.33
99.93 ± 0.26
32.59 ± 1.49
53.03 ± 2.29
86.90 ± 1.24
83.57 ± 1.21
95.02 ± 0.28
88.54 ± 0.50
90.18 ± 1.78
Cisplatin
a
Substances eliciting less than 20% inhibition of cell proliferation were regarded as ineffective and the
results are not presented.
3. Experimental Section
3.1. General Information
1H- and 13C-NMR spectra were recorded on a Bruker Avance DRX 400 spectrometer (400 MHz, δ = 0
(TMS)), in an appropriate solvent. Chemical shifts are expressed in ppm (δ) relative to TMS as internal
reference. J values are given in Hz. FT-IR spectra were recorded on a Perkin-Elmer Spectrum 100
instrument. Microanalyses were performed on a Perkin-Elmer 2400 elemental analyser. Microwave
reactions were carried out by heating at 125 °C and 200 W for 1 h in a CEM Discover LabMate microwave
reactor. Optical rotations were obtained with a Perkin-Elmer 341 polarimeter. Melting points were
determined on a Kofler apparatus and are uncorrected. Chromatographic separations were carried out on
Merck Kieselgel 60 (230–400 mesh ASTM). Reactions were monitored with Merck Kieselgel 60 F254-
precoated TLC plates (0.25 mm thickness). All the chemicals and solvents were used as supplied.
Compounds 5–18 and thioureas 19a, 20a, 38 and 39 were prepared by literature methods; all their
spectroscopic data and physical properties were similar to those reported therein [9,13,27–29,31,33].
3.2. Synthesis
3.2.1. General Procedure for the Synthesis of Thioureas 21–23
Amino alcohol 16, 17 or 18 (1.08 mmol) and the appropriate isothiocyanate (1.14 mmol) were dissolved
in toluene (80 mL) and the mixture was stirred at room temperature for 6 h, except that in the case of
N-benzylamino alcohol 18, heating at 50 °C for 6 h was indicated. The resulting mixtures were then
evaporated to dryness, filtered and washed with n-hexane. The purities of the products obtained were
determined via NMR to be >97%.
(1R,2R,3S,5R)-1-(3-Hydroxymethyl-6,6-dimethylbicyclo[3.1.1]hept-2-yl)-3-phenylthiourea (21a): 0.31 g
(95%); mp 152–155 °C, 2D0 = +52.0 (c 0.25, MeOH), 1H-NMR (CDCl3) δ (ppm) 0.94 (1H, d, J = 9.9 Hz),
1.01 (3H, s), 1.23 (3H, s), 1.63–1.70 (1H, m), 1.72–1.79 (1H, m), 1.88–1.94 (1H, m), 1.98–2.18 (3H, m),

Molecules 2014, 19
15926
2.54–2.65 (1H, m), 3.48-3.63 (2H, m), 5.15 (1H, t, J = 8.2 Hz), 7.16 (1H, d, J = 8.5 Hz), 7.19-7.44
13
(5H, m), 7.68 (1H, s). C-NMR (CDCl3) δ (ppm) 21.1, 26.5, 26.6, 26.8, 30.0, 32.6, 39.3, 40.7, 46.4,
57.0, 65.1, 125.7, 127.6, 130.3, 136.5, 180.2. Anal. Calcd for C17H24N2OS (304.45): C, 67.07; H, 7.95;
N, 9.20; S, 10.53%; Found: C, 67.39; H, 8.13; N, 9.01; S, 10.42%.
(1R,2R,3S,5R)-1-(4-Tolyl)-3-(3-hydroxymethyl-6,6-dimethylbicyclo[3.1.1]hept-2-yl)thiourea
(21b):
0.32 g (92%); mp 98–101 °C, 2D0 = +63.0 (c 0.25, MeOH), H-NMR (CDCl3) δ (ppm) 0.92 (1H, d,
J = 10.1 Hz), 1.01 (3H, s), 1.22 (3H, s), 1.62–1.68 (1H, m), 1.73–1.86 (1H, m), 1.87–1.93 (1H, m),
1.98–2.16 (3H, m), 2.35 (3H, s), 2.54–2.64 (1H, m), 3.47–3.61 (2H, m), 5.15 (1H, br s), 7.05 (1H, d,
J = 8.5 Hz), 7.09 (2H, d, J = 8.2 Hz), 7.20 (2H, d, J = 8.2 Hz). 13C-NMR (CDCl3) δ (ppm) 21.2, 21.5,
26.8, 26.9, 30.0, 32.8, 39.4, 40.8, 46.5, 57.0, 65.3, 126.0, 131.0, 132.8, 137.7, 151.9, 180.1. Anal.
Calcd for C18H26N2OS (318.48): C, 67.88; H, 8.23; N, 8.80; S, 10.07%; Found: C, 68.13; H, 8.35; N,
8.69; S, 9.94%.
1
(1R,2R,3S,5R)-1-(4-Fluorophenyl)-3-(3-hydroxymethyl-6,6-dimethylbicyclo[3.1.1]hept-2-yl)thiourea
(21c): 0.32 g (93%); mp 162–163 °C, D20 = +55.0 (c 0.25, MeOH), H-NMR (CDCl3) δ (ppm) 0.97
1
(1H, d, J = 9.8 Hz), 1.00 (3H, s), 1.22 (3H, s), 1.62–1.72 (1H, m), 1.87–1.94 (1H, m), 1.98–2.16 (4H,
m), 2.51–2.63 (1H, m), 3.51 (1H, dd, J = 4.6, 10.5 Hz), 3.61 (1H, dd, J = 3.2, 10.7 Hz), 5.11 (1H, br s),
7.01–7.26 (4H, m). 13C-NMR (CDCl3) δ (ppm) 21.1, 26.6, 26.7, 29.9, 32.4, 39.3, 40.7, 46.3, 57.0,
65.0, 117.1 (d, J = 23.7 Hz), 128.1 (d, J = 8.5 Hz), 162.6, 180.1. Anal. Calcd for C17H23FN2OS
(322.44): C, 63.32; H, 7.19; N, 8.69; S, 9.94%; Found: C, 63.49; H, 7.29; N, 8.48; S, 9.77%.
(1R,2R,3S,5R)-1-(3-Methoxyphenyl)-3-(3-hydroxymethyl-6,6-dimethylbicyclo[3.1.1]hept-2-yl)thiourea
(21d): 0.33 g (90%); mp 118–120 °C, D20 = +57.0 (c 0.20, MeOH), H-NMR (CDCl3) δ (ppm) 0.99
1
(1H, d, J = 8.8 Hz), 1.01 (3H, s), 1.23 (3H, s), 1.65–1.74 (1H, m), 1.78–1.92 (2H, m), 1.99–2.19 (3H,
m), 2.55–2.66 (1H, m), 3.53 (1H, dd, J = 4.5, 10.7 Hz), 3.61 (1H, br d, J = 10.5 Hz), 3.79 (3H, s), 5.17
(1H, br s), 6.72–6.83 (3H, m), ), 7.23–7.34 (2H, m). 13C-NMR (CDCl3) δ (ppm) 21.1, 26.7, 26.8, 29.9,
32.6, 39.3, 40.8, 46.4, 55.8, 57.1, 65.1, 110.9, 113.1, 117.3, 128.8, 131.1, 137.6, 161.1, 179.9. Anal.
Calcd for C18H26N2O2S (334.48): C, 64.64; H, 7.84; N, 8.39; S, 9.59%; Found: C, 64.73; H, 7.99; N,
8.11; S, 9.38%.
(1R,2R,3R,5R)-1-(3-Hydroxymethyl-6,6-dimethylbicyclo[3.1.1]hept-2-yl)-3-phenylthiourea (22a): 0.31 g
(94%); mp 151–155 °C, 2D0 = −19.0 (c 0.25, MeOH), H-NMR (CDCl3) δ (ppm) 0.94 (3H, s), 1.23
1
(3H, s), 1.30 (1H, d, J = 10.2 Hz), 1.57–1.64 (1H, m), 1.71–1.88 (2H, m), 1.92–2.00 (1H, m),
2.06–2.13 (1H, m), 3.42–3.50 (1H, m), 3.66–3.83 (2H, m), 4.91 (1H, t, J = 7.7 Hz), 6.16 (1H, d,
J = 9.4 Hz), 7.18 (2H, d, J = 7.7 Hz), 7.29 (1H, t, J = 7.0 Hz), 7.43 (2H, t, J = 7.5 Hz), 7.82 (1H, s).
13C-NMR (CDCl3) δ (ppm) 19.8, 23.8, 27.1, 27.3, 39.3, 40.4, 41.1, 46.6, 56.6, 64.4, 125.3, 127.7,
130.7, 135.8, 159.6, 179.5. Anal. Calcd for C17H24N2OS (304.45): C, 67.07; H, 7.95; N, 9.20; S,
10.53%; Found: C, 67.31; H, 7.80; N, 9.30; S, 10.41%.
(1R,2R,3R,5R)-1-Benzyl-1-(3-hydroxymethyl-6,6-dimethylbicyclo[3.1.1]hept-2-yl)-3-phenylthiourea
(23a): 0.36 g (85%); mp 177–178 °C, D20 = +7.0 (c 0.25, MeOH), 1H-NMR (CDCl3) δ (ppm) 1.07 (3H,
s), 1.26 (3H, s), 1.65 (1H, d, J = 10.2 Hz), 1.75–1.86 (1H, m), 1.89–2.01 (3H, m), 2.09–2.15 (1H, m),

Molecules 2014, 19
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2.23–2.31 (1H, m), 3.55–3.63 (1H, m), 3.92–3.99 (1H, m), 4.73–4.90 (2H, m), 6.01 (1H, br s), 7.03–7.45
13
(10H, m). C-NMR (CDCl3) δ (ppm) 19.8, 25.6, 27.3, 27.5, 35.6, 40.0, 42.8, 45.8, 49.0, 59.6, 62.1,
126.4, 126.5, 126.6, 128.5, 128.9, 129.7, 139.3, 139.8, 183.1. Anal. Calcd for C24H30N2OS (394.57):
C, 73.06; H, 7.66; N, 7.10; S, 8.13%; Found: C, 73.40; H, 7.86; N, 7.05; S, 8.38%.
3.2.2. General Procedure for the Reactions of Thioureas 19–23 with CDI to Yield Intermediates 29–33
To the respective thiourea (1.25 mmol) 19–23, 38 or 39 in THF solution (12 mL), CDI (0.306 g,
1.88 mmol) was added at room temperature. The reaction was stirred for 2–6 h at room temperature (TLC
monitoring), followed by careful evaporation of the solvent at 35 °C. The crude products obtained were
purified by flash column chromatography on silica gel.
(1R,2R,3S,5R)-[2,6,6-Trimethyl-2-(3-phenylthioureido)bicyclo[3.1.1]hept-3-yl]methyl
imidazole-1-
carboxylate (29a): 0.27 g (52%); oil, D20 = +32.0 (c 0.25, MeOH), H-NMR (CDCl3) δ (ppm) 0.92
(3H, s), 1.07 (1H, d, J = 10.1 Hz), 1.22 (3H, s), 1.42 (3H, s), 1.60–1.76 (1H, m), 1.92–2.20 (3H, m),
2.90–3.00 (1H, m), 4.35 (2H, d, J = 5.2 Hz), 5.26 (1H, t, J = 9.4 Hz), 6.25 (1H, d, J = 9.0 Hz), 7.05–7.39
1
13
(7H, m), 7.80 (1H, br s), 7.92 (1H, s). C-NMR (CDCl3) δ (ppm) 21.3, 22.5, 26.7, 26.6, 29.6, 30.1,
39.5, 40.8, 46.5, 55.6, 71.5, 117.5, 125.3, 128.5, 130.0, 130.4, 131.2, 135.5, 148.7, 181.5. Anal. Calcd
for C22H28N4O2S (412.55): C, 64.05; H, 6.84; N, 13.58; S, 7.77%; Found: C, 63.89; H, 6.91; N, 13.73;
S, 7.65%.
(1R,2R,3S,5R)-[2,6,6-Trimethyl-2-(3-phenylthioureido)bicyclo[3.1.1]hept-3-yl]methyl
imidazole-1-
carboxylate (30a): 0.32 g (62%); oil, D20 = –140.0 (c 0.25, MeOH), H-NMR (CDCl3) δ (ppm) 0.58
(1H, t, J = 8.5 Hz), 0.76–0.85 (2H, m), 0.93 (3H, s), 1.05 (3H, s), 1.28–1.55 (3H, m), 1.67 (3H, s),
1.72–1.82 (1H, m), 3.36 (1H, dd, J = 2.7, 11.1 Hz), 3.75 (1H,dd, J = 7.1, 14.0 Hz), 3.84 (1H, dd,
J = 11.3, 16.7 Hz), 3.95 (1H, dd, J = 2.7, 11.2 Hz), 7.19 (1H, br s), 7.22–7.46 (7H, m). 13C-NMR
(CDCl3) δ (ppm) 15.6, 19.1, 19.3, 24.0, 29.0, 30.1, 31.1, 45.4, 63.0, 69.1, 122.5, 126.6, 126.9, 127.6,
128.8, 130.1, 137.0, 143.0, 180.0. Anal. Calcd for C22H28N4O2S (412.55): C, 64.05; H, 6.84; N, 13.58;
S, 7.77%; Found: C, 64.21; H, 6.97; N, 13.23; S, 7.71%.
1
(1R,2R,3S,5R)-[6,6-Dimethyl-2-(3-phenylthioureido)bicyclo[3.1.1]hept-3-yl]methyl
imidazole-1-
carboxylate (31a): 0.39 g (78%); oil, D20 = +39.0 (c 0.32, MeOH), H-NMR (CDCl3) δ (ppm) 0.86
(1H, d, J = 10.3 Hz), 1.03 (3H, s), 1.26 (3H, s), 1.65–1.72 (1H, m), 1.95–2.26 (4H, m), 2.90–3.00 (1H,
m), 4.39 (2H, d, J = 5.4 Hz), 5.29 (1H, t, J = 9.2 Hz), 6.26 (1H, d, J = 9.0 Hz), 7.00–7.30 (7H, m), 7.85
(1H, br s), 7.92 (1H, s). 13C-NMR (CDCl3) δ (ppm) 21.1, 26.5, 26.6, 29.6, 30.1, 39.5, 40.7, 46.5, 55.8,
71.5, 117.3, 125.5, 128.1, 130.1, 130.5, 131.0, 135.9, 148.9, 181.0. Anal. Calcd for C21H26N4O2S
(398.52): C, 63.29; H, 6.58; N, 14.06; S, 8.05%; Found: C, 63.54; H, 6.60; N, 13.84; S, 7.91%.
1
(1R,2R,3S,5R)-{6,6-Dimethyl-2-[3-(4-tolyl)thioureido]bicyclo[3.1.1]hept-3-yl}methyl
imidazole-1-
carboxylate (31b): 0.36 g (69%); mp 148–150 °C, 2D0 = +81.0 (c 0.265, MeOH), 1H-NMR (CDCl3) δ
(ppm) 0.97 (3H, s), 1.19 (1H, d, J = 10.0 Hz), 1.24 (3H, s), 1.66–1.75 (1H, m), 1.91–1.99 (2H, m),
2.06–2.16 (1H, m), 2.19–2.30 (1H, m), 2.24 (3H, s), 2.79–2.91 (1H, m), 4.34–4.44 (2H, m), 5.14 (1H, t,
J = 8.8 Hz), 7.01–7.09 (3H, m), 7.25 (1H, d, J = 8.2 Hz), 7.57 (1H, s), 7.72 (1H, d, J = 8.9 Hz), 8.23

Molecules 2014, 19
15928
(1H, s), 9.28 (1H, s). 13C-NMR (DMSO–d6) δ (ppm) 21.3, 21.5, 27.1, 27.2, 29.9, 30.4, 39.3, 47.0, 53.7,
59.0, 71.8, 118.4, 124.1, 129.7, 131.0, 134.2, 137.2, 137.7, 149.3, 181.6. Anal. Calcd for C22H28N4O2S
(412.55): C, 64.05; H, 6.84; N, 13.58; S, 7.77%; Found: C, 64.39; H, 6.93; N, 13.27; S, 7.56%.
(1R,2R,3S,5R)-{6,6-Dimethyl-2-[3-(4-fluorophenyl)thioureido]bicyclo[3.1.1]hept-3-yl}methyl imidazole-
1-carboxylate (31c): 0.39 g (75%); mp 201–203 °C, 2D0 = +44.0 (c 0.35, MeOH), 1H-NMR (CDCl3)
δ (ppm) 0.87 (1H, d, J = 10.5 Hz), 1.03 (3H, s), 1.24 (3H, s), 1.65–1.72 (1H, m), 1.96–2.26 (4H, m),
2.91–3.01 (1H, m), 4.37–4.46 (2H, m), 5.26 (1H, t, J = 9.9 Hz), 6.20 (1H, br s), 6.94–7.15 (5H, m),
13
7.30 (1H, s), 7.78 (1H, br s), 8.14 (1H, br s). C-NMR (DMSO–d6) δ (ppm) 21.0, 26.5, 26.6, 29.7,
30.1, 39.4, 40.6, 46.6, 55.4, 71.6, 117.2, 117.3, 117.4, 127.7, 127.8, 148.8, 160.4, 162.9, 181.3. Anal.
Calcd for C21H25FN4O2S (416.51): C, 60.56; H, 6.05; N, 13.45; S, 7.70%; Found: C, 60.79; H, 6.41; N,
13.08; S, 7.53%.
(1R,2R,3S,5R)-{6,6-Dimethyl-2-[3-(3-methoxyphenyl)thioureido]bicyclo[3.1.1]hept-3-yl}methyl imidazole-
1-carboxylate (31d): 0.39 g (73%); mp 128–130 °C, 2D0 = +9.0 (c 0.26, MeOH), H-NMR (CDCl3)
1
δ (ppm) 0.91 (1H, d, J = 10.6 Hz), 1.03 (3H, s), 1.26 (3H, s), 1.66–1.72 (1H, m), 1.97–2.02 (1H, m),
2.06–2.28 (3H, m), 2.90–3.00 (1H, m), 3.73 (3H, s), 4.34–4.44 (2H, m), 5.30 (1H, t, J = 9.8 Hz), 6.41
(1H, d, J = 9.7 Hz), 6.60–6.69 (3H, m), 7.02 (1H, s), 7.17 (1H, t, J = 8.1 Hz), 7.22 (1H, s), 7.79 (1H,
13
s), 7.92 (1H, s). C-NMR (CDCl3) δ (ppm) 21.2, 26.6, 26.7, 29.6, 30.2, 39.6, 40.7, 46.6, 55.8, 55.9,
71.5, 110.6, 111.2, 113.4, 117.1, 117.3, 131.0, 131.3, 137.0, 148.9, 161.4, 180.8. Anal. Calcd for
C22H28N4O3S (428.55): C, 61.66; H, 6.59; N, 13.07; S, 7.48%; Found: C, 61.83; H, 6.84; N, 12.89;
S, 7.53%.
(1R,2R,3R,5R)-[6,6-Dimethyl-2-(3-phenylthioureido)bicyclo[3.1.1]hept-3-yl]methyl
imidazole-1-
carboxylate (32a): 0.33 g (67%); oil, D20 = −15.0 (c 0.30, MeOH), H-NMR (CDCl3) δ (ppm) 0.95
(3H, s), 1.26 (3H, s), 1.31 (1H, d, J = 9.5 Hz), 1.61–1.72 (1H, m), 1.94–2.19 (5H, m), 4.54 (1H, dd,
J = 6.6, 10.8 Hz), 4.65 (1H, dd, J = 6.6, 10.7 Hz), 5.04 (1H, t, J = 8.7 Hz), 6.16 (1H, d, J = 9.2 Hz),
7.06 (1H, s), 7.13 (2H, d, J = 7.8 Hz), 7.24–7.29 (1H, m), 7.40 (2H, t, J = 7.9 Hz), 7.46 (1H, br s), 8.13
1
13
(1H, br s), 8.16 (1H, s). C-NMR (CDCl3) δ (ppm) 19.9, 23.6, 26.7, 27.5, 35.6, 40.2, 40.646.2, 55.7,
70.4, 117.6, 125.2, 127.5, 130.6, 131.0, 136.5, 149.2, 180.3. Anal. Calcd for C21H26N4O2S (398.52): C,
63.29; H, 6.58; N, 14.06; S, 8.05%; Found: C, 63.43; H, 6.75; N, 13.85; S, 8.16%.
(1R,2R,3R,5R)-[6,6-Dimethyl-2-(1-benzyl-3-phenylthioureido)bicyclo[3.1.1]hept-3-yl]methyl imidazole-
1-carboxylate (33a): 0.37 g (60%); oil, 2D0 = −15.0 (c 0.30, MeOH), 1H-NMR (CDCl3) δ (ppm) 1.09
(3H, s), 1.32 (3H, s), 1.68 (1H, d, J = 10.1 Hz), 1.80–1.89 (1H, m), 1.95–2.11 (2H, m), 2.20 (1H, t,
J = 5.4 Hz), 2.33–2.50 (2H, m), 4.65 (1H, dd, J = 7.3, 11.6 Hz), 4.80–4.89 (3H, m), 6.50 (1H, br d,
13
J = 10.6 Hz), 7.03–7.55 (12H, m), 8.20 (1H, s). C-NMR (CDCl3) δ (ppm) 20.1, 25.7, 27.1, 28.1,
33.3, 39.9, 42.5, 45.8, 49.1, 58.9, 70.3, 117.7, 126.3, 126.5, 128.7, 128.9, 129.2, 130.9, 131.8, 136.1,
137.1, 140.0, 149.2, 184.2. Anal. Calcd for C28H32N4O2S (488.22): C, 68.82; H, 6.60; N, 11.47; S,
6.56%; Found: C, 68.63; H, 6.49; N, 11.72; S, 6.83%.
2-[(3-Phenylthioureido)cyclopent-3-yl]methyl imidazole-1-carboxylate (40): 0.28 g (65%); oil, 1H-NMR
(CDCl3) δ (ppm) 1.36–1.49 (2H, m), 1.54–1.75 (2H, m), 1.92–2.02 (1H, m), 2.08–2.18 (1H, m),

Molecules 2014, 19
15929
2.73–2.84 (1H, m), 4.31 (1H, dd, J = 5.3, 11.0 HZ), 4.39 (1H, dd, J = 6.1, 11.1 Hz), 4.96–5.04 (1H, m),
5.96 (1H, d, J = 7.9 Hz), 7.03 (1H, s), 7.10–7.15 (2H, m), 7.19–7.25 (1H, m), 7.30–7.37 (3H, m), 7.99
13
(1H, br s), 8.03 (1H, s). C-NMR (CDCl3) δ (ppm) 22.5, 27.7, 33.0, 39.9, 57.9, 69.0, 117.5, 125.6,
128.1, 130.7, 131.1, 136.2, 137.5, 148.9, 181.3. Anal. Calcd for C17H20N4O2S (344.43): C, 59.28; H,
5.85; N, 16.27; S, 9.39%; Found: C, 59.28; H, 5.85; N, 16.27; S, 9.39%.
1
2-[(3-Phenylthioureido)cyclohex-3-yl]methyl imidazole-1-carboxylate (41): 0.26 g (57%); oil, H-NMR
(CDCl3) δ (ppm) 0.86–1.00 (1H, m), 1.06–1.20 (1H, m), 1.32–1.45 (1H, m), 1.59–1.79 (4H, m), 1.86–1.97
(1H, m), 2.24–2.36 (1H, m), 4.23–4.35 (2H, m), 4.96–5.04 (1H, m), 6.23 (1H, d, J = 9.0 Hz),
7.06–7.09 (1H, m), 7.23–7.28 (2H, m), 7.37 (1H, t, J = 7.5 Hz), 7.47–7.53 (3H, m), 8.20 (1H, s).
13C-NMR (CDCl3) δ (ppm) 21.0, 24.7, 26.4, 32.2, 32.6, 34.3, 52.5, 122.8, 123.5, 125.6, 125.7, 129.3,
135.0, 146.1, 156.4, 171.2. Anal. Calcd for C18H22N4O2S (358.46): C, 60.31; H, 6.19; N, 15.63; S,
8.95%; Found: C, 60.47; H, 6.23; N, 15.12; S, 8.69%.
3.2.3. General Procedure for the Synthesis of 2-Imino-1,3-thiazines 24–28 and 2-Thioxo-1,3-oxazines
34–37
Method A: A CEM Discover 10 mL vial containing the respective thiourea (1.25 mmol) 19–23 in THF
solution (12 mL) and CDI (0.306 g, 1.88 mmol) and sealed with a Teflon cap was irradiated at 125 °C
(200 W) for 60 min with a ramp time of 5 min. Next, the solvent was evaporated off under reduced
pressure and the residue was purified by flash column chromatography on silica gel; elution with
n-hexane/EtOAc (4:1) gave 2-imino-1,3-thiazines and 2-thioxo-1,3-oxazines.
Method B: A CEM Discover 10 mL vial containing the respective intermediates (1.25 mmol) 29–33 in
THF solution (12 mL) and sealed with a Teflon cap was irradiated at 125 °C (200 W) for 60 min with a
ramp time of 5 min. Next, the solvent was evaporated off under reduced pressure and the residue was
purified by flash column chromatography on silica gel; elution with n-hexane/EtOAc (4:1) gave 2-imino-
1,3-thiazines and 2-thioxo-1,3-oxazines.
(1R,2R,7S,9R)-(10,10-Dimethyl-5-thia-3-azatricyclo[7.1.1.02,7]undec-4-ylidene)phenylamine
(26a):
Method A: 0.197 g (55%), method B: 0.204 g (57%); mp 124–128 °C, 2D0 = +142.0 (c 0.25, MeOH),
1H-NMR (CDCl3) δ (ppm) 0.95 (3H, s), 1.24 (3H, s), 1.30 (1H, d, J = 10.5 Hz), 1.50–1.58 (1H, m),
1.92–1.99 (2H, m), 2.13–2.30 (2H, m), 2.61–2.73 (2H, m), 2.87 (1H, t, J = 13.4 Hz), 3.92 (1H, t,
13
J = 8.9 Hz), 6.88–7.30 (5H, m). C-NMR (CDCl3) δ (ppm) 20.9, 26.9, 27.0, 33.1, 34.1, 35.7, 39.0,
41.1, 47.7, 54.9, 122.9, 123.7, 129.3, 137.7, 160.4. Anal. Calcd for C17H22N2S (286.43): C, 71.28; H,
7.74; N, 9.78; S, 11.19%; Found: C, 71.39; H, 8.00; N, 9.21; S, 11.11%.
(1R,2R,7S,9R)-10,10-Dimethyl-5-oxa-3-azatricyclo[7.1.1.02,7]undecane-4-thione (35): Method A: 0.053
g (20%), method B: 0.058 g (22%); this compound was also isolated from transformations of thioureas
26c–d, in 18%–24% yield; mp 160–161 °C, 2D0 = +112.0 (c 0.25, MeOH), H-NMR (CDCl3)
1
δ (ppm) 0.91 (3H, s), 1.26 (3H, s), 1.28 (1H, d, J = 11.8 Hz), 1.45–1.52 (1H, m), 1.97–2.24 (4H, m),
2.64–2.75 (1H, m), 3.85–3.88 (1H, m), 3.91 (1H, dd, J = 10.2, 21.1 Hz), 4.24 (1H, dd, J = 6.1, 10.8 Hz),
13
7.53 (1H, br s). C-NMR (CDCl3) δ (ppm) 20.4, 25.3, 26.6, 26.7, 28.0, 39.6, 40.5, 46.1, 54.2, 72.6,

Molecules 2014, 19
15930
192.5. Anal. Calcd for C11H17NOS (211.32): C, 62.52; H, 8.11; N, 6.63; S, 15.17%; Found: C, 62.77;
H, 8.36; N, 6.41; S, 15.00%.
(1R,2R,7S,9R)-(10,10-Dimethyl-5-thia-3-azatricyclo[7.1.1.02,7]undec-4-ylidene)-p-tolylamine (26b):
Method A: 0.199 g (53%), method B: 0.203 g (54%); mp 199–203 °C, 2D0 = +170.0 (c 0.25, MeOH),
1H-NMR (CDCl3) δ (ppm) 0.94 (3H, s), 1.23 (3H, s), 1.30 (1H, d, J = 10.6 Hz), 1.49–1.57 (1H, m),
1.88–1.99 (2H, m), 2.11–2.29 (2H, m), 2.31 (3H, s), 2.59–2.71 (2H, m), 2.87 (1H, t, J = 13.4 Hz), 3.91
13
(1H, d, J = 8.3 Hz), 5.01 (1H, br s), 6.80 (1H, d, J = 8.1 Hz), 7.08 (1H, d, J = 8.1 Hz). C-NMR
(CDCl3) δ (ppm) 20.9, 21.3, 26.9, 27.0, 34.2, 35.8, 39.1, 41.2, 47.8, 54.8, 122.5, 129.9, 133.0, 146.2,
159.7. Anal. Calcd for C18H24N2S (300.46): C, 71.95; H, 8.05; N, 9.32; S, 10.67%; Found: C, 71.99; H,
8.21; N, 9.10; S, 10.70%.
(1R,2R,7S,9R)-(10,10-Dimethyl-5-thia-3-azatricyclo[7.1.1.02,7]undec-4-ylidene)-3-Methoxyphenylamine
(26c): Method A: 0.225 g (57%), method B: 0.233 g (59%); mp 154–155 °C, 2D0 = +234.0 (c 0.25,
MeOH), 1H-NMR (CDCl3) δ (ppm) 0.95 (3H, s), 1.25 (3H, s), 1.30 (1H, d, J = 10.6 Hz), 1.49–1.59 (1H,
m), 1.90–2.02 (2H, m), 2.13–2.32 (2H, m), 2.61–2.75 (2H, m), 2.88 (1H, t, J = 12.5 Hz), 3.91 (1H, d,
13
J = 9.1 Hz), 6.81–7.00 (4H, m). C-NMR (CDCl3) δ (ppm) 20.9, 26.9, 27.0, 34.2, 35.8, 39.1, 41.2,
47.8, 54.9, 55.6, 108.3, 109.6, 115.2, 129.8, 129.9, 150.6, 160.6. Anal. Calcd for C18H24N2OS
(316.46): C, 68.32; H, 7.64; N, 8.85; S,10.13%; Found: C, 68.45; H, 7.93; N, 8.50; S,9.89%.
(1R,2R,7S,9R)-(10,10-Dimethyl-5-thia-3-azatricyclo[7.1.1.02,7]undec-4-ylidene)-4-fluorophenylamine
(26d): Method A: 0.221 g (58%), method B: 0.232 g (61%); mp 201–205 °C, 2D0 = +272.0 (c 0.25,
MeOH), 1H-NMR (CDCl3) δ (ppm) 0.95 (3H, s), 1.25 (3H, s), 1.30 (1H, d, J = 10.6 Hz), 1.49–1.59 (1H,
m), 1.90–2.02 (2H, m), 2.13–2.32 (2H, m), 2.61–2.75 (2H, m), 2.88 (1H, t, J = 12.5 Hz), 3.91 (1H, d,
13
J = 9.1 Hz), 6.81–7.00 (4H, m). C-NMR (CDCl3) δ (ppm) 20.9, 26.9, 27.0, 34.2, 35.8, 39.1, 41.2,
47.8, 54.9, 55.6, 108.3, 109.6, 115.2, 129.9, 150.6, 160.6. Anal. Calcd for C17H21FN2S (304.43): C,
67.07; H, 6.95; N, 9.20; S, 10.53%; Found: C, 67.41; H, 7.13; N, 9.01; S 10.27%.
(1R,2R,7R,9R)-(10,10-Dimethyl-5-thia-3-azatricyclo[7.1.1.02,7]undec-4-ylidene)phenylamine
(27a):
Method A: 0.186 g (52%), method B: 0.197 g (55%); mp 192–195 °C, 2D0 = +165.0 (c 0.25, MeOH),
1H-NMR (CDCl3) δ (ppm) 0.82 (3H, s), 1.25 (3H, s), 1.43–1.53 (1H, m), 1.67 (1H, d, J = 10.6 Hz),
1.91–2.16 (5H, m), 2.91–3.03 (2H, m), 3.44 (1H, d, J = 8.1 Hz), 6.95–7.28 (5H, m). 13C-NMR (CDCl3)
δ (ppm) 20.1, 23.8, 27.3, 29.9, 33.8, 41.1, 41.5, 46.6, 58.1, 121.5, 123.0, 129.2, 145.2, 160.8. Anal.
Calcd for C17H22N2S (286.43): C, 71.28; H, 7.74; N, 9.78; S, 11.19%; Found: C, 71.35; H, 7.83; N, 9.65;
S, 11.17%.
(1R,2R,7R,9R)-10,10-Dimethyl-5-oxa-3-azatricyclo[7.1.1.02,7]undecane-4-thione (36): Method A:
0.053 g (20%), method B: 0.055 g (21%); mp 176–179 °C, 2D0 = +331.0 (c 0.25, MeOH), H-NMR
1
(CDCl3) δ (ppm) 0.83 (3H, s), 1.33 (3H, s), 1.54 (1H, t, J = 12.5 Hz), 1.78 (1H, d, J = 10.7 Hz), 1.87–1.95
(1H, m), 2.03–2.12 (1H, m), 2.23–2.31 (1H, m), 2.40–2.53 (1H, m), 3.62 (1H, d, J = 10.2 Hz), 4.31
13
(1H, dd, J = 10.1, 12.5 Hz), 4.52 (1H, dd, J = 4.7, 10.0 Hz), 8.29 (1H, br s). C-NMR (DMSO–d6) δ
(ppm) 19.6, 23.2, 23.5, 27.5,32.6, 41.6, 42.4, 43.1, 54.9, 75.6, 188.2. Anal. Calcd for C11H17NOS
(211.32): C, 62.52; H, 8.11; N, 6.63; S, 15.17%; Found: C, 62.73; H, 8.46; N, 6.52; S, 15.01%.

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(1R,2R,7R,9R)-(3-Benzyl-10,10-dimethyl-5-thia-3-azatricyclo-[7.1.1.02,7]undec-4-ylidene)phenylamine
(28a): Method A: 0.221 g (47%), method B: 0.231 g (49%); mp 108–109 °C, 2D0 = +567.0 (c 0.25,
MeOH), 1H-NMR (CDCl3) δ (ppm) 0.77 (3H, s), 1.20 (3H, s), 1.47 (1H, dt, J = 5.1, 13.6 Hz), 1.52 (1H,
d, J = 10.8 Hz), 1.91–1.97 (1H, m), 2.04–2.14 (2H, m), 2.25–2.42 (2H, m), 2.88–2.99 (2H, m), 3.81
(1H, d, J = 8.1 Hz), 4.51 (1H, d, J = 16.4 Hz), 5.31 (1H, d, J = 16.6 Hz), 6.78–6.83 (2H, m), 6.94–6.99
13
(1H, m), 7.15–7.32 (7H, m). C-NMR (CDCl3) δ (ppm) 20.1, 24.2, 27.5, 31.1, 34.0, 35.8, 40.3, 40.4,
42.5, 49.0, 60.2, 122.7, 122.9, 126.7, 12.8, 128.7, 129.1, 140.2, 150.5, 152.6. Anal. Calcd for
C24H28N2S (376.56): C, 76.55; H, 7.49; N, 7.44; S, 8.52%; Found: C, 76.86; H, 7.63; N, 7.21;
S, 8.30%.
(1R,2R,7R,9R)-3-Benzyl-10,10-dimethyl-5-oxa-3-azatricyclo[7.1.1.02,7]undecane-4-thione
(37):
Method A: 0.053 g (14%), method B: 0.060 g (16%); mp 94–97 °C, 2D0 = +132.0 (c 0.25, MeOH),
1H-NMR (CDCl3) δ (ppm) 0.71 (3H, s), 1.23 (3H, s), 1.42–1.57 (2H, m), 1.83–1.91 (1H, m), 1.95–2.06
(2H, m), 2.20 (1H, t, J = 5.14), 2.43–2.51 (1H, m), 3.64 (1H, d, J = 9.6 Hz), 4.17 (1H, dd, J = 9.6, 12.2 Hz),
4.36 (1H, dd, J = 4.9, 9.6 Hz), 4.49 (1H, d, J = 16.2 Hz), 4.68 (1H, d, J = 16.2 Hz), 7.19–7.32 (5H, m).
13C-NMR (CDCl3) δ (ppm) 19.5, 23.5, 24.0, 27.8, 34.1, 40.6, 41.5, 41.6, 48.1, 57.1, 72.2, 127.3, 127.4,
128.9, 138.7, 155.8. Anal. Calcd for C18H23NOS (301.45): C, 71.72; H, 7.69; N, 4.65; s, 10.64%;
Found: C, 71.98; H, 7.83; N, 4.39; S, 10.39%.
(1R,2R,7S,9R)-Phenyl-(2,10,10-trimethyl-5-thia-3-azatricyclo[7.1.1.02,7]undec-4-ylidene)amine (24a):
Method A: 0.240 g (64%), method B: 0.252 g (67%); mp 182–186 °C, 2D0 = –123.0 (c 0.25, MeOH),
1H-NMR (CDCl3) δ (ppm) 1.05 (3H, s), 1.28 (3H, s), 1.38 (1H, d, J = 12.5 Hz), 1.41 (3H,s), 1.69–1.98
(2H, m), 2.10–2.23 (2H, m), 2.42–2.53 (1H, m), 2.63 (1H, dd, J = 8.0, 12.8 Hz), 2.97 (1H, dd, J = 3.9,
12.9 Hz), 5.04 (1H, br s), 6.87–7.06 (3H, m), 7.22–7.30 (2H, m). 13C-NMR (CDCl3) δ (ppm) 24.1, 27.8,
28.2, 31.7, 33.4, 33.9, 40.3, 40.6, 55.3, 60.9, 122.7, 123.4, 129.2, 148.6, 158.4. Anal. Calcd for
C18H24N2S (300.46): C, 71.95; H, 8.05; N, 9.32; S, 10.67%; Found: C, 72.19; H, 8.33; N, 9.11;
S, 10.60%.
(1R,2R,7S,9R)-2,10,10-Trimethyl-5-oxa-3-azatricyclo[7.1.1.02,7]undecane-4-thione (34): Method A:
0.028 g (10%), method B: 0.023 g (8%); mp 120–123 °C, 2D0 = +36.0 (c 0.25, MeOH), H-NMR
1
(CDCl3) δ (ppm) 0.97 (1H, d, J = 11.1 Hz), 1.06 (3H, s), 1.31 (3H, s), ), 1.35 (3H, s), 1.79–1.87 (1H,
m), 1.95–2.03 (2H, m), 2.14–2.42 (3H, m), 4.08 (1H, d, J = 11.0 Hz), 4.18 (1H, d, J = 10.7 Hz), 7.43
13
(1H, br s). C-NMR (CDCl3) δ (ppm) 20.9, 26.7, 27.2, 33.1, 34.1, 35.5, 39.3, 40.2, 41.2, 47.4, 55.1,
158.2. Anal. Calcd for C12H19NOS (225.35): C, 63.96; H, 8.50; N, 6.22; S, 14.23%; Found: C, 64.25;
H, 8.79; N, 6.01; S, 13.88%.
(1aR,2aR,6aS,7aS)-Phenyl-(1,1,6a-trimethyloctahydro-4-thia-6-azacyclopropa[b]naphthalen-5-ylidene)
amine (25a): Method A: 0.225 g (60%), method B: 0.233 g (62%); mp 157–160 °C, 2D0 = −18.0 (c 0.25,
1
MeOH), H-NMR (CDCl3) δ (ppm) 0.71–0.83 (2H, m), 0.98 (3H, s), 1.07 (3H, s), 1.22 (1H, dd,
J = 4.5, 15.8 Hz), 1.28 (3H, s), 1.53–1.71 (2H, m), 1.89–2.01 (2H, m), 2.65 (1H, d, J = 3.4, 12.4 Hz),
3.29 (1H, d, J = 3.5, 11.7 Hz), 7.02–7.12 (3H, m), 7.28–7.34 (2H, m). 13C-NMR (CDCl3) δ (ppm) 15.6,
17.6, 18.2, 19.6, 21.8, 28.8, 28.9, 31.6, 35.4, 52.1, 123.2, 123.4, 123.9, 129.3, 155.0. Anal. Calcd for

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C18H24N2OS (300.46): C, 71.95; H, 8.05; N, 9.32; S,10.67%; Found: C, 72.20; H, 8.19; N, 9.02;
S, 10.54%.
3.2.4. Alternative Procedure for the Synthesis of 2-Thioxo-1,3-oxazines 35 and 36
The respective amino alcohol 16 or 17 (1.18 mmol) in dry toluene solution (20 mL), TEA (3.54 mmol)
and thiophosgene (0.181 mL, 2.36 mmol) was stirred at room temperature for 6 h. Next, the solvent was
evaporated off under reduced pressure and the residue was purified by flash column chromatography on
silica gel; elution with n-hexane/EtOAc (4:1) gave 2-thioxo-1,3-oxazine 35 or 36.
3.2.5. General Procedure for the Synthesis of 2-Imino-1,3-thiazines 42 and 43
Method A: The respective thiourea (1.25 mmol) 38 or 39 in THF solution (12 mL) and CDI (0.306 g,
1.88 mmol) was heated to reflux for 3 h. Next, the solvent was evaporated off under reduced pressure and
the residue was purified by flash column chromatography on silica gel; elution with n-hexane/EtOAc (4:1)
gave the pure 2-imino-1,3-thiazines 42 or 43.
Method B: A CEM Discover 10 mL vial containing the respective intermediates (0.63 mmol) 40 or 41
in THF solution (12 mL) was heated to reflux for 3 h. Next, the solvent was evaporated off under reduced
pressure and the residue was purified by flash column chromatography on silica gel; elution with n-hexane/
EtOAc (4:1) gave the pure 2-imino-1,3-thiazines 42 or 43.
(Hexahydrocyclopenta[d][1,3]thiazin-2-ylidene)phenylamine (42): 0.218 g (75%); mp 180–181 °C
1
(Lit. [33] 180–181 °C), H-NMR (CDCl3) δ (ppm): 1.47–1.71 (3H, m), 1.74–2.10 (3H, m), 2.52–2.66
(1H, m), 2.77 (1H, dd, J = 9.3, 12.5 Hz), 2.92 (1H, dd, J = 4.5, 12.5 Hz), 3.81–3.93 (1H, m), 6.95–7.10
(3H, m), 7.26–7.34 (2H, m). 13C-NMR (CDCl3) δ (ppm): 24.0, 31.4, 31.7, 35.7, 39.7, 57.3, 122.5,
123.4, 129.2, 148.5, 157.6. Anal. Calcd for C13H16N2S (232.34): C, 67.20; H, 6.94; N, 12.06; S,
13.80%; Found: C, 67.47; H, 6.81; N, 12.30; S, 13.42%.
(Octahydrobenzo[d][1,3]thiazin-2-ylidene)phenylamine (43): 0.222 g (72%); mp 183–186 °C (Lit.[33]
1
187–188 °C), H-NMR (CDCl3) δ (ppm): 1.30–1.81 (7H, m), 1.83–1.97 (1H, m), 2.05–2.15 (1H, m),
2.81 (1H, dd, J = 4.8, 12.4 Hz), 3.12 (1H, dd, J = 4.1, 12.5 Hz), 3.57–3.65 (1H, m), 6.98–7.10 (3H, m),
13
7.21–7.30 (2H, m). C-NMR (CDCl3) δ (ppm): 21.0, 24.7, 26.4, 32.2, 32.6, 34.3, 52.5, 122.8, 123.5,
129.3, 146.1, 156.4. Anal. Calcd for C14H18N2S (246.34): C, 68.25; H, 7.36; N, 11.37%; Found: C,
68.31; H, 7.51; N, 11.02%.
3.2.6. Determination of Antiproliferative Activities
Antiproliferative effects against four human cancer cell lines were determined as published
recently [36]. Briefly, HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma), A2780 (ovarian
carcinoma) and A431 (skin epidermoid carcinoma; all cell lines purchased from ECACC; Salisbury, UK)
cells were cultivated in minimal essential medium (Sigma-Aldrich, Budapest, Hungary) supplemented with
10% foetal bovine serum, 1% non-essential amino acids and an antibiotic-antimycotic mixture.
Near-confluent cells were seeded into a 96-well plate (5000 cells/well) and, after overnight standing, the
medium (200 µL) containing the tested compound (at 10 or 30 µM) was added. Following a 72-h

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incubation in a humidified atmosphere of 5% CO2 at 37 °C, the living cells were assayed by the
addition of 20 µL of 5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
solution [37]. During a 4-h contact period, the MTT was converted by intact mitochondrial reductase and
precipitated as blue crystals. The medium was then removed, the precipitated formazan crystals were
solubilized in DMSO (100 µL) during a 60-min period of shaking at 25 °C, and the absorbance was read at
545 nm with a microplate reader. Wells with untreated cells were utilized as controls. All in vitro
experiments were carried out on two microplates with at least five parallel wells. Stock solutions of the
tested substances (10 mM) were prepared with DMSO. The DMSO concentration (0.3%) of the medium
did not have any significant effect on cell proliferation. Cisplatin was used as reference compound.
3.2.7. X-ray Crystallographic Studies
Crystallographic data were collected at 123 K for 31c and 31d by using a Bruker Nonius-Kappa
CCD diffractometer with an APEXII area detector and graphite-monochromatized Mo-Kα radiation
(λ = 0.71073 Å), as reported earlier [38].
Crystal data for 31c: C21H25FN4O2S, Mr = 416.51, triclinic, space group P-1 (no. 2), a = 8.5831(5),
b = 10.7338(6), c = 11.9244(5) Å, α = 86.414(3)°, β = 69.835(3)°, γ = 85.729(3)°, V = 1027.59(9) Å3,
T = 123 K, Z = 2, μ(Mo-Kα) = 0.192 mm−1, 6132 unique reflections (Rint = 0.0211) which were used in
calculations. The final R1 (for the data with F2 > 2δ(F2) was 0.0426 and wR2(F2) (all data) was 0.1034.
Crystal data for 31d: C22H28N4O3S, Mr = 428.54, monoclinic, space group C2/c (no. 15),
a = 23.1162(6), b = 8.7978(3), c = 21.4232(5) Å, β =92.4232(5)°, V = 4352.8(2) Å3, T = 123 K, Z = 8,
μ(Mo-Kα) 0.180 mm−1, 7979 unique reflections (Rint = 0.0279) which were used in calculations. The final
R1 (for the data with F2 > 2δ(F2) was 0.0454 and wR2(F2) (all data) was 0.1063.
The structures were solved by direct methods by use of the SHELXS-97 program [39], and full-matrix,
least-squares refinements on F2 were performed by use of the SHELXL-97 program [39]. The CH
hydrogen atoms were included at fixed distances from their host atoms with the fixed displacement
parameters. The NH hydrogen atom positions were refined with the fixed displacement parameters. In 31d,
the methoxy C atom has two different orientations in a 1:1 ratio (due to packing reasons). The graphics
were drawn with ORTEP3 for Windows [40]. The depositions numbered CCDC 1000179 (for 31c) and
CCDC 1000178 (for 31d) contain the supplementary crystallographic data for this paper. These data can be
obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html (or from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif).
4. Conclusions
In conclusion, we have developed a mild and efficient method for the synthesis of 2-imino-1,3-thiazines
by the ring closure of thiourea adducts of 1,3-amino alcohols in the presence of CDI under microwave
condition. Besides the main product 1,3-thiazines, the formation of 2-thioxo-1,3-oxazines as side-products
was observed. The ring-closure process was extended to cycloalkane-based γ-hydroxythioureas and the
method developed for the synthesis of 2-imino-1,3-thiazines containing an acid-sensitive structure proved
to be comparable to those reported in the literature. The resulting 1,3-thiazines exert marked
antiproliferative action on a panel of human cancer cell lines.

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Acknowledgments
We are grateful for financial support from the Hungarian Research Foundation (OTKA NK81371,
K112442 and K109293) and from the European Union for cofunding by the European Social Fund
(TÁMOP-4.2.2/A-11/1/KONV-2012-0035).
Author Contributions
The listed authors contributed to this work as described in the following. FF designed, planed research
and interpreted the results. ZS planed research, interpreted the results and carried out of the synthetic work.
IZ performed and interpreted the biological experiments. RS performed X-ray experiments. All authors
discussed the results, prepared and commented on the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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