Pharmacologic evaluation of antidepressant activity and synthesis of 2-morpholino-5-phenyl-6H-1,3,4-thiadiazine hydrobromide

Article abstract of DOI:10.3390/ph9020027

Substituted thiadiazines exert a reliable therapeutic effect in treating stress, and a schematic description of their ability to influence all aspects of a stress response has been depicted. This study was conducted to pharmacologically evaluate compound L-17, a substituted thiadiazine, (2-morpholino-5-phenyl-6H-1,3,4-thiadiazine, hydrobromide) for possible anti-psychotic/antidepressant activity. Compound L-17 was synthesized by cyclocondensation of α-bromoacetophenone with the original morpholine-4-carbothionic acid hydrazide. Pharmacologic evaluations were conducted using methods described by E.F. Lavretskaya (1985), and in accordance with published guidelines for studying drugs for neuroleptic activity. Compound L-17 was evaluated for various possible mechanisms of action, including its effects on cholinergic system agonists/antagonists, dopaminergic neurotransmission, the adrenergic system, and 5-HT3 serotonin receptors. One or more of these mechanisms may be responsible for the beneficial effects shown by thiadiazine compounds in experiments conducted to evaluate their activity in models of acute stress and acute myocardial infarction.

Full text of DOI:10.3390/ph9020027

pharmaceuticals
Article
Pharmacologic Evaluation of Antidepressant Activity
and Synthesis of 2-Morpholino-5-phenyl-6H-1,3,4-
thiadiazine Hydrobromide
Alexey P. Sarapultsev 1,2,*, Oleg N. Chupakhin 1,3, Petr A. Sarapultsev 1,2, Larisa P. Sidorova 1
and Tatiana A. Tseitler 1
1
Ural Federal University named after the first President of Russia B. N. Yeltsin, Ekaterinburg 620002, Russia;
chupakhin@ios.uran.ru (O.N.C.); p.sarapultsev@gmail.com (P.A.S.); vlapp@isnet.ru (L.P.S.);
tseitler85@mail.ru (T.A.T.)
Institute of Immunology and Physiology of the Ural Branch of the RAS, Ekaterinburg 620219, Russia
2
3
The IJ Postovsky Institute of Organic Synthesis of the Ural Branch of the RAS, Ekaterinburg 620990, Russia
*
Correspondence: a.sarapultsev@gmail.com; Tel.: +7-343-374-0070
Academic Editor: Jean Jacques Vanden Eynde
Received: 7 March 2016; Accepted: 13 May 2016; Published: 19 May 2016
Abstract: Substituted thiadiazines exert a reliable therapeutic effect in treating stress, and
a schematic description of their ability to influence all aspects of a stress response has
been depicted. This study was conducted to pharmacologically evaluate compound L-17
a substituted thiadiazine, (2-morpholino-5-phenyl-6H-1,3,4-thiadiazine, hydrobromide) for possible
anti-psychotic/antidepressant activity. Compound L-17 was synthesized by cyclocondensation of
,
α
-bromoacetophenone with the original morpholine-4-carbothionic acid hydrazide. Pharmacologic
evaluations were conducted using methods described by E.F. Lavretskaya (1985), and in accordance
with published guidelines for studying drugs for neuroleptic activity. Compound L-17 was
evaluated for various possible mechanisms of action, including its effects on cholinergic system
agonists/antagonists, dopaminergic neurotransmission, the adrenergic system, and 5-HT3 serotonin
receptors. One or more of these mechanisms may be responsible for the beneficial effects shown by
thiadiazine compounds in experiments conducted to evaluate their activity in models of acute stress
and acute myocardial infarction.
Keywords: adrenoblockers; α-adrenoblockers; heterocycles; thiadiazines; 1,3,4-thiadiazine-2-amines;
serotonin; stress; pharmacological evaluation
1
. Introduction
A variety of biological activities have been reported for a large number of thiadiazine derivatives,
such as antimicrobial [
activity [ ]. Moreover, in vivo studies have shown their significant therapeutic effects in various
models of disease, including models of systemic inflammation [ ], myocardial infarction [10 11],
1–4] antiagregant [5] anti-inflammatory and analgesic [6] and tuberculostatic
7,8
9
,
and acute pancreatitis [12]. Besides, some compounds from the group of substituted thiadiazines were
suggested to influence of all components of a stress-induced response [13]. Results of the previously
mentioned experiments led us to hypothesize the existence of a central mechanism of action for
substituted thiadiazines, and suggest this class of compounds as a promising source of new drugs
to treat stress and diseases in which inflammation and stress play a major role, such as myocardial
infarction and stress-induced cardiomyopathy.
This study was conducted to evaluate the substituted thiadiazine compound L-17 (Figure 1) for
possible anti-psychotic/anti-depressant activity.
Pharmaceuticals 2016, 9, 27; doi:10.3390/ph9020027
www.mdpi.com/journal/pharmaceuticals

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Figure 1. Compound L-17, a 5-phenyl substituted-6H-1,3,4-thiadiazine-2-amine.
2
. Results
The results of our studies evaluating the overall effects of test drugs on exploratory/locomotor
activity, fear response, and affective aggression in animals are shown in Tables 1 and 2.
Table 1. Effects of 2-morpholino-5-phenyl-6H-1,3,4-thiadiazine (compound L-17) and Eglonyl on
overall exploratory/locomotor activity, fear response, and affective aggression.
Fear Response
and affect Aggr., Conventional Units, M ˘ m
(
Dose
Explor/Locomotor
Activ. for 10 m,
M ˘ m
M ˘ m
Test Sub.
mg/kg)/(N
of Animals)
Squeak in
Points
Decrease
in Activity
Threshold, V
Catalepsy
Analgesia
1
3
0/10
0/8
1.5 ˘ 0.07
2.0 ˘ 0.05
0
0
0
0
2.0 ˘ 0.06
2.5 ˘ 0.05
3.0 ˘ 0.05
3.0 ˘ 0.02
L-17
60/10
1
2
104 ˘ 11 *
15 ˘ 2 *
43 ˘ 1.5 *
48 ˘ 1.5 *
0.5
0
˘
˘
0.05 * 2.5 ˘ 0.05
3.0 ˘ 0.05
20/10
40/10
1.5 ˘ 0.05
4.0 ˘ 0.05
2.5 ˘ 0.05
Saline
/
20
309 ˘ 26 *
40 ˘ 0.8 *
0.5
0.07 *
0
.9%
3
0 ˘ 0.8
2 ˘ 0.05
1
2
4
1
0/8
0/8
0/8
00/8
0
0
0
0
0
0
250 ˘ 26
35 ˘ 0.5 *
38 ˘ 0.6 *
0.9
0.8
˘
˘
0.04 * 1.0 ˘ 0.05
0.05 * 2.0 ˘ 0.05
2.5 ˘ 0.06
Eglonil
*
189 ˘ 12 *
0.8 ˘ 0.05
1.5 ˘ 0.07
1.0 ˘ 0.05
Differences vs. control were statistically significant at a p-value < 0.05. The severity of “squeak in points”
(
the equivalent of an "emotional" fear response) for stimulation with 20 V of electricity; “Threshold, V”—the
threshold voltage at which the reaction approximates reactions of animals in the control group.
Our results (Table 1) showed that administration of compound L-17 resulted in decreased animal
motor activity (3-fold and > 20-fold decreases at doses of 60 mg/kg and 120 mg/kg, respectively),
and an increase in the threshold voltage (to 43
˘
1.5 V and 48
˘
1.5 V when administered at doses
of 60 mg/kg and 120 mg/kg, respectively). Squeak intensity (the equivalent of an "emotional" fear
response) changed significantly following administration of compound L-17 at a dose of 120 mg/kg.
Administration of Eglonyl led to a significant decrease in overall exploratory/locomotor activity
(
1.6-fold at a dose of 40 mg/kg), an increase in the threshold voltage (to 35
˘
0.5 V and 38
˘
0.6 V
when administered at doses of 20 mg/kg and 40 mg/kg, respectively), as well as changes in squeak
intensity (at a dose of 20 mg/kg).
Thus, administration of compound L-17 led to the appearance of CNS-associated signs of
depression similar to those induced by Eglonyl, which causes changes in spontaneous behavior
(
activity reduction), altered reactions to painful stimuli, and an increased threshold for a pain reaction.
Compound L-17 produced an analgesic effect when given at a dose of 10 mg/kg, while a 120 mg/kg
dose induced a cataleptic state.
Changes in apomorphine-induced stereotypy were evaluated in mice (Table 2).

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Table 2. Changes in apomorphine-induced stereotypy following administration of compound L-17.
Apomorphine (2 mg/kg)
Dose,
mg/kg
Latent Period
Duration, sec.,
M ˘ m
Stereotyped
Behavior Duration, ED50, mg/kg
min, M ˘ m
Test Sub.
LD50/ED50
3
0
55.0 ˘ 9.8 *
22.5 ˘ 1.4
19.2 ˘ 1.0 *
5.8 ˘ 0.9 *
-
80
-
7.6
6.9–8.4
-
L-17
60
67.5 ˘ 10.3 *
402.5 ˘ 50.0 *
1
20
Saline
Eglonil
Saline
28.3 ˘ 3.6
23.2 ˘ 0.4
-
-
2
4
0
0
85 ˘ 4.7 *
15.6 ˘ 1.3 *
19
10.4
8.9–12.1
150 ˘ 9.3 *
6.6 ˘ 0.9 *
42 ˘ 7.8
31.7 ˘ 1.8
*
Differences were statistically significant at a p-value < 0.05.
Animals that were administered L-17 displayed a significant increase in their latent period
starting at a 30 mg/kg dose, and also a reduced duration of stereotypy starting at a 60 mg/kg dose.
The estimated effective dose (ED50) of L-17 was 80 mg/kg. Animals which received the antipsychotic
drug Eglonyl displayed significant increases in both their latent period and duration of stereotypy.
The estimated effective dose (ED50) of Eglonyl was 19 mg/kg.
This test is specific to neuroleptic agents derived from phenothiazine and butyrophenone.
An ability to suppress stereotypy indicates that the test substance inhibits dopaminergic
neurotransmission in the nigrostriatal system of the brain. This trait was displayed by compound L-17
.
Next, apomorphine-induced hypoactivity was evaluated in rats (Table 3).
Table 3. Evaluation of apomorphine-induced hypoactivity following administration of compound L-17.
Test Sub.
Dose, mg/kg
Motor Activity (Number of Movemets for 5 min), M ˘ m
Saline
Apomorphine
-
217.75 ˘ 17.10
75.88 ˘ 19.33 *
26.4 ˘ 3.8 **
4
6
0
0
L-17 + Apomorphine (0.15 mg/kg)
16.50 ˘ 7.56 **
152.38 ˘ 12.42 **
Eglonil + Apomorphine (0.15 mg/kg) 40
*
Differences were statistically significant at a p-value < 0.05., define; ** differences were statistically significant
with apomorphine at dose 0.15 mg/kg.
Our results showed that compound L-17 could potentiate the effects of presynaptic doses of
apomorphine, and significantly reduce the motor activity of animals. A 40 mg/kg dose of L-17
administered in conjunction with apomorphine (0.15 mg/kg) reduced animal motor activity by
2
6.4 ˘ 3.8 movements when compared with a control group (217.75 ˘ 17.10 movements) and also
when compared with a group of animals administered apomorphine alone at a dose of 0.15 mg/kg
75.88 19.33 movements). In contrast, a 40 m/kg dose of Eglonyl induced an increase in locomotor
activity (152.38 12.42 movements) when compared locomotor activity displayed by animals treated
(
˘
˘
with apomorphine alone (75.88 ˘ 19.33 movements).
The amphetamine-induced locomotion test was conducted with mice (Table 4).
A 100 mg/kg dose of compound L-17 produced a significant increase in the duration of an animal’s
latent period, and a 200 mg/kg dose produced a significant decrease in stereotypy. The estimated
effective dose (ED50) of compound L-17 was 170 mg/kg. A 40 mg/kg dose of Eglonyl induced a
significant decrease in the duration of stereotypy (40.3
˘
1.8 min) when compared with the duration in
control animals (118.0 ˘ 1.2 min).
Liver enzyme activity was evaluated by using the hexenal test to examine the hypnotic effect of a
barbiturate (hexenal) in mice (Table 5). Our results showed that a 60 mg/kg dose of compound L-17
significantly increased the duration of both latency (up to 254.0
˘ 75.0 s) and sleep (65.7 ˘ 8.1 min),

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when compared with those parameters in a control group (126.3
˘
6.4 sec and 39.3
˘
6.4 min,
respectively). The estimated effective dose (ED50) of L-17 was 80 mg/kg. A 25 mg/kg dose of
Eglonyl produced an increase in sleep duration (up to 39.8 1.7 min) when compared with that in
˘
control animals (25.0 ˘ 0.8 min), and the estimated ED50 of Eglonil was 42 mg/kg.
Table 4. Results of the amphetamine-induced locomotion test after administration of compound L-17
.
Phenaminum (6 mg/kg)
Dose,
Latent Period
Duration, sec.,
M ˘ m
Stereotyped
Test Sub.
mg/kg
Behavior Duration, ED50, mg/kg
min, M ˘ m
LD50/ED50
5
0
247.5 ˘ 59.5
308.3 ˘ 20.8 *
277.5 ˘ 25.9 *
76.5 ˘ 5.5
L-17
100
76.5 ˘ 5.5
170
3.5 (3.2–4.0)
2
00
60.3 ˘ 10.0
Saline
Eglonil
Saline
185.0 ˘ 26.4
35.5 ˘ 5.3
2
4
0
0
4.2 ˘ 2.2
70.7 ˘ 2.8
25
7.9 (6.7–9.2)
5.2 ˘ 0.8
40.3 ˘ 1.8 *
6.0 ˘ 1.65
118.0 ˘ 1.2
*
Differences were statistically significant at a p-value < 0.05.
As antipsychotic drugs typically potentiate the narcotic effect of barbiturates [14], we also tested
substances for their influence on the hypnogenetic effects of barbiturates (Table 5).
Table 5. Results of the hexenal tests.
Hexenal (60 mg/kg)
Latent Period
Test Sub.
Dose, mg/kg
Sleep Duration,
Duration, sec.,
ED50, mg/kg LD50/ED50
min, M ˘ m
M ˘ m
6
0
254.0 ˘ 75.0
65.7 ˘ 8.1 *
L-17
Saline
Eglonil
Saline
1
20
67.5 ˘ 4.2 *
96.0 ˘ 10.0
80
42
7.6 (6.9–8.4)
4.7 (4.0–5.5)
126.3 ˘ 6.4
39.3 ˘ 6.4
2
4
0
0
168.0 ˘ 24.5
39.8 ˘ 1.7 *
120.3 ˘ 15.8
40.0 ˘ 0.9 *
150.2 ˘ 10.4
25.0 ˘ 0.8
Hexenal (30 mg/kg)
Latent Period
Duration, sec.,
M ˘ m
110.3 ˘ 70.8
393.0 ˘ 51.4
310.0 ˘ 95.0
354.0 ˘ 30.2
210.8 ˘ 57.5 *
The Number of Animals
Fallen Asleep/Number of
Animals in a Group
Test Sub.
Dose, mg/kg
Sleep Duration,
min, M ˘ m
1
2
0
0
12.8 ˘ 5.7
28.0 ˘ 6.5
24.2 ˘ 6.7
50.2 ˘ 7.0
68.5 ˘ 8.6
3/6
5/6
5/6
5/6
6/6
L-17
30
4
0
1
20
Saline
1–6
*
Differences were statistically significant at a p-value < 0.05.
The results shown in Table 5 indicate that administration of a sub-threshold dose of hexenal
caused 16.7% of the laboratory animals to fall asleep, while a 10 mg/kg dose of L-17 administered in
addition to hexenol caused a significantly greater percentage of the animals to fall asleep. This result
suggests that compound L-17 can decrease the ability of liver microsomes to metabolize barbiturates,
which is a characteristic feature of many neuroleptics [14].

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The arecoline-evoked tremor was evaluated in mice. The results shown in Table 6 indicate that a
high dose (60 mg/kg) of compound L-17 caused a significant reduction in the mean duration of tremor
(
14
˘
0.7 s), when compared with the mean duration in a control group; providing evidence for its
anti-cholinergic effect [15,16].
Table 6. The effect of compound L-17 on arecoline-evoked tremor.
Test Sub.
L-17
Dose, mg/kg
Duration of Arecoline-Evoked Tremor, sec., M ˘ m
3
0
-
60
20
14 ˘ 0.7 *
1
14 ˘ 0.7 *
Saline
Eglonil
Saline
24 ˘ 1.5
2
4
0
0
15,5 ˘ 0.5
17 ˘ 0.8
16 ˘ 0.9
*
Differences were statistically significant at a p-value < 0.05.
The influence of compound L-17 on the contractile activity of isolated smooth muscles (guinea
pig ileum and rat seminal vesicles) was studied using acetylcholine and epinephrine in the standard
method described by R. Blattner (Table 7) [17].
Table 7. The influence of compound L-17 on the contractile activity of isolated smooth muscles.
Eglonil
L-17 Compound
Concentr.,
mol/L
Object
Agonist
Amplitude,
mm, M ˘ m
Diff. to Contr.
Values, %
Amplitude,
mm, M ˘ m
Diff. to Contr.
Values, %
Control
57 ˘ 1
44 ˘ 1.2 *
36 ˘ 2.3
22 ˘ 1.1 *
24 ˘ 1
100
77
100
61
100
29
65 ˘ 3.4
58 ˘ 1.7
60 ˘ 1.6
45 ˘ 2.4 *
67 ˘ 3.1
23 ˘ 4.1 *
153 ˘ 6.3
150 ˘ 4.2
150 ˘ 2.3
139 ˘ 3.1
143 ˘ 2.4
130 ˘ 1.3
100
89
-
75
-
´
6
1
ˆ 10
Rat seminal
vesicles
Epinephrine
Control
´
5
´5
Control
1 ˆ 10
M
1 ˆ 10
´
4
1
ˆ 10
7 ˘ 1.3 *
34
Control
46 ˘ 2.3
42 ˘ 1.5
40 ˘ 1.7
38 ˘ 0.8
37 ˘ 0.8
36 ˘ 0.9
105 ˘ 3.5
95 ˘ 4.0
132 ˘ 2.4
73 ˘ 4.1 *
61 ˘ 1.1
12 ˘ 0.8
100
91
100
95
100
97
100
98
100
93
100
91
´
6
1
ˆ 10
Ileum
guinea pig
Acetylcholine
Control
´
5
´5
Control
1 ˆ 10
M
1 ˆ 10
´
4
1
ˆ 10
Control
100
90
100
55
100
20
42 ˘ 4.5
38 ˘ 3.2
46 ˘ 3.1
30 ˘ 2.4 *
38 ˘ 2.1
20 ˘ 1.2 *
100
90
100
65
100
53
´
6
1
ˆ 10
Ileum
guinea pig
Serotonin
Control
´
6
´5
1 ˆ 10
M
1 ˆ 10
Control
´4
1
ˆ 10
*
Differences were statistically significant at a p-value < 0.05.
´
5
When injected at a concentration of 1
ˆ
10 mol/L, compound L-17 demonstrated a moderate
ability to block adrenoreceptors, which indicated its ability to interact with adrenergic receptors, and
in particular, alpha-1-adrenoceptors. Eglonyl blocked adrenoceptors when injected at a concentration
of 1 ˆ 10 mol/L. The ability of compound L-17 to interact with 5-HT3 receptors was demonstrated
in the model of serotonin-induced spasm of isolated guinea pig ileum muscle [18]. Significant
reductions in contraction amplitude were observed after injecting either compound L-17 or Eglonyl at
a concentration of 1 ˆ 10´ mol/L.
´
6
5

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Thus, our data suggest that compound L-17, which was selected from a group of 5-phenyl
substituted-6H-1,3,4-thiadiazine-2-amines, might exert its effects via one or more mechanisms of action.
These include affecting cholinergic system agonists/antagonists (arecoline 15 mg/kg), dopaminergic
neurotransmission (apomorphine 0.1 mg/kg and amphetamine 5 mg/kg), the adrenergic system
(evaluation of smooth muscle contractile activity), and (or) 5-HT3 serotonin receptors (model of
serotonin-induced muscle spasm).
3
. Discussion
The results shown in Tables 1–7 and by assays used to differentiate groups of drugs in
initial screening tests [11] indicated that compound L-17, selected from a group of 5-phenyl
substituted-6H-1,3,4-thiadiazine-2-amines, possesses a combination of adrenergic properties, as well
as certain characteristics of choline and serotonin blockers. Compound 17 demonstrated its effects when
used in a concentration range similar to that employed when using the atypical antipsychotic agent Eglonyl
(sulpiride), as well as antidepressants such as amitriptyline, and alpha-blockers such as pyrroxanum.
Practically all effective anti-psychotic medications are known to block a1-adrenoreceptors [19].
While these receptors were initially thought to merely promote sedation, more recent data indicate that
the ability to blockade a1-adrenoceptors contributes to the therapeutic effects anti-psychotic agents [19].
A1-adrenoceptors are believed to play a regulatory role in the brain by enhancing certain excitatory
afferent inputs to brain cells. The first evidence for tonic regulation of serotonin neurons (5-HT)
by postsynaptic alpha-1-adrenergic receptors belonging to the noradrenergic system was reported
by TH Svensson in 1975 [20]. Those data were subsequently supported by several studies showing
that alpha-1-blockers suppressed the activity of serotonergic neurons [21,22], and reduced serotonin
levels in the hippocampus [23]. These findings explain why medications that act on serotonin and
noradrenaline systems demonstrate their most pronounced activity as antidepressants [19]. Moreover,
their synergistic effect may be due to the fact that alpha-1 and serotonin (5HT2A) receptors are involved
in the phosphatidyl inositol/protein kinase C intracellular pathway via Gq proteins [24].
At the same time, it should be noted that the affinity of norepinephrine for alpha-1 adrenoceptors
is significantly less than its affinity for alpha-2-adrenoceptors. As a result, a substantial increase in
norepinephrine levels (e.g., as induced by a stress reaction) is required for norepinephrine to interact
with alpha-1-receptors [19]. This explains why administration of an alpha-1-adrenergic receptor’s
antagonist produces almost no effect under normal (non-stress) conditions, but has a protective effect
during stressful conditions [25].
The mechanisms described above may be responsible for the beneficial effects of thiadiazine
compounds, as previously demonstrated in experimental models of acute stress [13] and acute
myocardial infarction [10,11].
4
. Materials and Methods
4
.1. Chemical Synthesis
Work on the synthesis of 1,3,4-thiadiazine derivatives has been ongoing for several decades at
Ural Federal State University in Russia (Figure 2) [26–29].
Figure 2. Chemical synthesis of thiadiazine derivatives.

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The molecule 2-morpholino-5-phenyl-6H-1,3,4-thiadiazine hydrobromide (compound L-17
)
(
III) was synthesized by cyclocondensation of -bromoacetophenone ( ) with the original
α
I
morpholine-4-carbothionic acid hydrazide molecule (II). Progress of the cyclization reaction was
monitored via thin layer chromatography (TLC) performed using a standard solvent system
(butanol:acetic acid:water; 4:1:5) and Sifufol UV-254 TLC plates (Kavalier, Savaza, Czech Republic).
4
.1.1. Method for Synthesis of α-Bromacetophenone (I)
Acetophenone (0.3 mol) in glacial HOAc (200–250 mL) was stirred at 25 C, and then treated
˝
˝
dropwise with an equimolar amount of Br . This mixture was first stirred for 1 h at 25 C, and then
again for 2 h at 50 C–55 C; after which, it was cooled, diluted with a 4- to 6-fold volume of ice water,
and stored for 3–5 h. Following storage, the mixture was filtered to isolate a light gray residue, which
2
˝
˝
was allowed to dry. Next,
α-bromacetophenone was crystallized from 50 mL of absolute ethanol.
The yield of the final product was 40%.
4
.1.2. Method for Synthesis Morpholine-4-Carbothionic Acid Hydrazide (II)
A solution of 55 g (1 mol) of potassium hydroxide in 50 mL of water and 1 mole of
morpholine in 150 mL of ethanol was mixed with 1 mol of carbon disulfide with sufficient
˝
cooling to maintain its temperature at < 20 C; this solution was then stored for 8 h. Next, dried
potassium-morpholine-4-carbodithioate was added to a solution of 95 g (1 mol) of chloroacetic acid
in 300 mL of water, neutralized with potassium carbonate. This mixture was stored for ~ 8 h. Then,
a 50 mL volume of hydrazine hydrate was added, and the solution was evaporated to ~ 50% of its
original volume. After cooling, morpholine-4-carbothionic acid hydrazide (II) crystals were formed,
and the white solid precipitate was removed by filtration. The yield of morpholine-4-carbothionic acid
hydrazide (II) was 79%. The isolated compound II was a white solid with the following characteristics:
˝
1
m.p.176–177 C, H-NMR (DMSO-d , 400 MHz, δ, ppm): 3.56–3.59 (m, 4H, 2CH ), 3.68–3.70 (m, 4H,
2
6
2
CH ), 4.82 (s, 2H, NH ), 9.14 (s, br, 1H, NH). Anal. Calcd. for C H N OS (161.23): C, 37.25; H, 6.88;
2 2 5 11 3
N, 26.06. Found: C, 37.22; H, 6.85; N, 26.02.
4
.1.3. Method for Synthesis 2-Morpholino-5-phenyl-6H-1,3,4-thiadiazine Hydrobromide (compound
L-17) (III)
α
-Bromoacetophenone (20 mmols) in 20 mL of absolute ethanol was added to a solution
consisting of 20 mmols of morpholine-4-carbothionic acid hydrazide (II) in 30 mL of absolute
ethanol, and the mixture was refluxed for 20 minutes. After hot filtration and cooling, the
white powdery precipitate was filtered and crystallized from absolute ethanol. The yield of
2
-morpholino-5-phenyl-6H-1,3,4-thiadiazine hydrobromide was 75%. Compound III showed the
˝
1
following characteristics: m.p.191–192 C, H-NMR (DMSO-d , 400 MHz,
δ
, ppm): 3.81–3.97 (m, 8H,
6
morpholino); 4.45 (s, 2H, CH S); 7.5–8.0 (m, 5H, C H ); Anal. Calcd. for C H BrN OS (342.2):
2
6
5
13 16
3
C, 45.60; H, 4.70; N, 12.30. Found: C, 45.70; H, 4.65; N, 12.25.
4
.2. Biological Experiments
4
.2.1. Animal Preparation
Animal studies were conducted using healthy sexually mature, nonlinear albino male rats, mice,
and guinea pigs obtained from the vivarium of the Institute of Immunology and Physiology of the
Ural Division of RAS.
After arrival at the research facility, the animals were quarantined in the vivarium, and carefully
observed to ensure they showed symptoms of any disease. All animals were housed under the same
conditions, and fed according to a customary schedule. The protocols for all animal studies were
approved by the Institute of Animal Care and Use Committee of the Institute of Immunology and
Physiology of the Ural Division of RAS (F-1-2014-25). All studies were performed in accordance with

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guidelines developed at the European Convention for the Protection of Vertebrate Animals used for
Experimental and Other Scientific Purposes (Strasbourg, France, 18.03.1986), APS s Guiding Principles
'
in the Care and Use of Vertebrate Animals in Research and Training, and the Laboratory Practice
Regulations of RF (Ministry of Public Health Order no. 267 from 19.06.2003) [30].
4
.2.2. Methods of Pharmacological Evaluation
Pharmacological evaluations were performed using methods described by E.F. Lavretskaya (1985),
and in accordance with guidelines followed when studying the neuroleptic activity of drugs [14,31].
The injections of the compound L-17 were made intraperitoneally. Injections were made in the lower
right abdominal quadrant away from the midline to avoid inadvertent injection into the urinary
bladder or cecum [32,33].
General locomotor activity, fear response, and affective aggression were evaluated in mice in an
open field test chamber (Opto-Varimex, Columbus Instruments, Columbus, OH, USA) in accordance
with standard protocols [14,31,34].
Changes in the apomorphine-induced stereotypy were evaluated in mice. The dopamine receptor
agonist apomorphine was subcutaneously injected at a dose of 2 mg/kg, 20 min prior to testing [35–37].
Apomorphine-induced hypoactivity was evaluated on rats. The animals were placed in an open
field chamber 10 minutes after administration of apomorphine (0.15 mg/kg) [14]. Low doses of
apomorphine (0.01–0.15 mg/kg) were administered to induce chewing movements resulting from the
effect on presynaptic dopamine receptors. Antipsychotic compounds inhibit the effects of apomorphine.
This test is extremely sensitive and is used by investigators to identify new compounds that block
presynaptic dopamine receptors [14].
The amphetamine-induced locomotion test was conducted with mice. Fifteen minutes after
administration of amphetamine, the mice were placed into a cage containing a camera equipped with
a multichannel video analyzer [14].
When administered to mice, amphetamine (phenaminum) causes an increase in spontaneous
locomotor activity, which is associated with increased dopaminergic neurotransmission in the
mesolimbic system of the brain. Typical neuroleptics inhibit the effects of amphetamine in a
dose-dependent manner, whereas atypical neuroleptics have a weaker effect or fail to inhibit
amphetamine-induced hyperactivity. In contrast, substances with antidepressant activity may enhance
the effect of amphetamine [14].
Liver enzyme activity was evaluated by using the hexenal test to examine the hypnotic effect of
barbiturates in mice. Many anti-psychotic compounds are known to potentiate the narcotic effect of
barbiturates. In this assay, the test compound was injected intraperitoneally 10 min prior to hexenal
administration (60 mg/kg, i.p.), and the percentage of dormant mice, and their time spent in a lateral
position (in minutes) after administration of hexenal were recorded [38,39].
As there exists a subthreshold dose of hexenal that causes only a certain percentage of animals to
sleep, increasing the percentage of animals that fall asleep after dosing testifies to an increased effect of
the barbiturate. A test compound that fails to affect the duration of hexenal-induced sleep does not
inhibit liver microsome activity, and can be classified as a "true potentiator" based on the classification
system developed by Brodie BB (1955) [40].
Arecoline-evoked tremor was evaluated in mice to detect the central M-anti-cholinergic action of
a compound. A single dose of the test compound was administered 30 min prior to a subcutaneous
injection of arecoline (25 mg/kg); after which, the duration of tremor was recorded [14]. Anticholinergic
activity manifests as a decrease in the duration or the complete elimination of tremor [14].
The effect of a test substance on the contractile activity of isolated smooth muscle organs (guinea
pig ileum and rat seminal vesicles) was evaluated in rats by using the standard R. Blattner method
with acetylcholine and epinephrine [17,18].

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5
. Conclusions
The relatively novel therapeutic concept of using combined autoreceptor antagonists that act on
a1-adrenoceptors, serotonin, and choline receptors to treat cardiovascular diseases deserves further
exploration in both preclinical and clinical studies.
Acknowledgments: The reported study was supported by Russian Science Foundation, research project
No. 16-15-00039.
Author Contributions: O.C. designed the experiments and chemical synthesis, L.S. and T.T. conducted
chemical synthesis, P.S. and A.S. designed and conducted the biological part of experiments, AP Sarapultsev
wrote the manuscript. The authors would like to thank E.F. Lavretskaya for initiating the research and the
methodological help.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Abdou, W.M.; Ganoub, N.A.; Sabry, E. Design, Synthesis and Pharmacological Screening of
-Amino-, Thiadiazole/Thiadiazine-Phosphonate Based Triazole Motifs as Antimicrobial/Cytotoxic Agents.
Acta Pharm. 2014, 64, 267–284. [CrossRef] [PubMed]
β
Karegoudar, P.; Prasad, D.J.; Ashok, M.; Mahalinga, M.; Poojary, B.; Holla, B.S. Synthesis, antimicrobial and
anti-inflammatory activities of some 1,2,4-triazolo [3,4-b][1,3,4] thiadiazoles and 1,2,4-triazolo [3,4-b][1,3,4]
thiadiazines bearing trichlorophenyl moiety. Eur. J. Med. Chem. 2008, 43, 808–815. [CrossRef] [PubMed]
Rahimizadeh, M.; Shiri, A.; Ranaei, M.; Bakavoli, M. Synthesis and antibacterial evaluation of new
1
1
heterocyclic system:[1,2,4] triazolo [3 ,4 :6,1] pyridazino [4,3-e][1,3,4] thiadiazine. Heterocycl. Commun.
012, 18, 39–42. [CrossRef]
Sahu, J.K.; Ganguly, S.; Kaushik, A. Synthesis and antimicrobial activity of some novel fused heterocyclic
,2,4-triazolo [3, 4-b][1,3,4] thiadiazine derivatives. J. Adv. Pharm. Technol. Res. 2014, 5, 90–95. [CrossRef]
PubMed]
2
1
[
Vasilyeva, T.M.; Makarov, V.A.; Chupakhin, O.N.; Sidorova, L.P.; Perova, N.M.; Rusinov, V.L. Platelet
aggregation inhibitor properties of 1,3,4-thiadiazine thiadiazines. Gematol. Transfuziol. 2008, 53, 12–15.
(In Russian)
El Shehry, M.F.; Abu-Hashem, A.A.; El-Telbani, E.M. Synthesis of 3-((2,4-dichlorophenoxy) methyl)-1, 2,
4
2
-triazolo (thiadiazoles and thiadiazines) as anti-inflammatory and molluscicidal agents. Eur. J. Med. Chem.
010, 45, 1906–1911. [CrossRef] [PubMed]
Khudina, O.G.; Ivanova, A.E.; Burgart, Y.V.; Saloutin, V.I. Synthesis of Pyrimido [2,1-b][1,3,5] Thiadiazines
Containing Polyfluoroalkyl-and Amino Acid Fragments. Chem. Heterocyc. Compd. 2014, 50, 901–906.
[
CrossRef]
Khudina, O.G.; Ivanova, A.E.; Burgart, Y.V.; Saloutin, V.I.; Kravchenko, M.A. 6-Polyfluoroalkylated
-thiouracils in the synthesis of pyrimido [2,1-b][1,3,5] thiadiazines by the double Mannich reaction.
2
J. Fluorine Chem. 2013, 147, 31–35. [CrossRef]
Sarapultsev, P.A.; Chupakhin, O.N.; Sarapultsev, A.P.; Rantsev, M.A.; Medvedeva, S.U.; Sidorova, L.P.;
Danilova, I.G. The effects of the compound of the group of substituted 5R1,6R2–1,3,4-thiadiazine-2-amines
on the course of systemic inflammation. Cytokines Inflam. 2013, 12, 40–45. (In Russian)
1
0. Sarapultsev, P.; Chupakhin, O.; Sarapultsev, A.; Rantsev, M.; Sidorova, L.; Medvedeva, S.; Danilova, I. New
insights in to the treatment of myocardial infarction. Int. J. Exp. Pathol. 2012, 93, 18–23. [CrossRef] [PubMed]
1. Sarapultsev, A.P.; Chupakhin, O.N.; Sarapultsev, P.A.; Rantsev, M.A.; Medvedeva, S.U.; Sidorova, L.P.;
Abidov, M.T.; Danilova, I.G. Modulation of inflammatory response improves myocardial infarct healing in
rats. Curr. Pharm. Design 2014, 20, 1980–1986. [CrossRef]
1
1
2. Rantsev, M.A.; Sarapultsev, P.A.; Chupakhin, O.N.; Sarapultsev, A.P.; Khodakov, V.V.; Danilova, I.G.;
Sidorova, L.P. The treatment of focal and extensive pancreonecrosis in the experiment (Lechenie ochagovogo
I rasprostranennogo pankreonekroza v jeksperimente). Med. Almanac (Medicinskij Al'manah) 2013, 5, 92–94.
(In Russian)

Pharmaceuticals 2016, 9, 27
10 of 11
1
1
3. Sarapultsev, P.A.; Chupakhin, O.N.; Medvedeva, S.U.; Mukhlynina, E.A.; Brilliant, S.A.; Sidorova, L.P.;
Danilova, I.G.; Sarapultsev, A.P. The impact of immunomodulator compound from the group of substituted
thiadiazines on the course of stress reaction. Int. Immunopharmacol. 2015, 25, 440–449. [CrossRef] [PubMed]
4. Ostrovskaya, R.W.; Raevsky, K.S.; Voronina, T.A.; Garibova, T.L.; Kovalev, G.I.; Kudrin, V.S.; Narkevich, V.B.;
Klodt, P.M. Guidelines for the study of neuroleptic activity of drugs. In Guidelines for Pre-Clinical Studies of
Drugs; Part one; Mironov, A.N., Ed.; Grif i K: Moscow, Russia, 2012; pp. 251–264. (In Russian)
5. Anichkov, S.V.; Grebyonkina, M.A. Pharmacological characterization of cholinergic receptors of central
nervous system. Bull. Exp. Biol. Med. (USSR) 1946, 3, 28–29.
6. Bukatin, M.V. The influence of benzimidazole derivatives on the reproductive system of male rats.
Ph.D. Thesis, The Volgograd State Medical University, Volgograd, Russia, October 2013. (In Russian)
7. Blattner, R.; Classen, H.G.; Dehnert, H.; Doering, H.G. Experiments on Isolated Smooth Muscle; Preparations
Hugo Sachs Elektronik KG: Freiburg, Germany, 1978; pp. 55–59, English edition prepared by Barnden IM,
Colson R. 1980.
1
1
1
1
1
2
2
2
2
2
8. Yoshida, S.; Wetanabe, T.; Seto, Y. Regulatory molecules for the 5-HT3 receptor ion channel geting system.
Bioorg Med. Chem. 2007, 15, 3515–3523. [CrossRef] [PubMed]
9. Arnsten, A.F. Adrenergic targets for the treatment of cognitive deficits in schizophrenia. Psychopharmacology
2
004, 174, 25–31. [CrossRef] [PubMed]
0. Svensson, T.H.; Bunney, B.S.; Aghajanian, G.K. Inhibition of both noradrenergic and serotonergic neurons in
brain by the α-adrenergic agonist clonidine. Brain Res. 1975, 92, 291–306. [CrossRef]
1. Baraban, J.M.; Aghajanian, G.K. Suppression of firing activity of 5-HT neurons in the dorsal raphe by
alpha-adrenoceptor antagonists. Neuropharmacology 1980, 19, 355–363. [CrossRef]
2. Baraban, J.M.; Aghajanian, G.K. Noradrenergic innervation of serotonergic neurons in the dorsal raphe:
demonstration by electron microscopic autoradiography. Brain Res. 1981, 204, 1–11. [CrossRef]
3. Claustre, Y.; Rouquier, L.; Serrano, A.; Bénavidès, J.; Scatton, B. Effect of the putative 5-HT 1A receptor
antagonist NAN-190 on rat brain serotonergic transmission. Eur. J. Pharmacol. 1991, 204, 71–77. [CrossRef]
4. Duman, S.; Nestler, E.J. Signal transduction pathways for catecholamine receptors. In Psychopharmacology:
the Fourth Generation of Progress; Bloom, F.E., Kupfer, D.J., Eds.; Raven Press: New York, NY, USA, 1995;
pp. 303–320.
2
5. Birnbaum, S.G.; Gobeske, K.T.; Auerbach, J.; Taylor, J.R.; Arnsten, A.F.T. A role for norepinephrine in stress-
induced cognitive deficits: alpha-1-adrenoceptor mediation in prefrontal cortex. Biol. Psychiatry 1999, 46,
1
266–1274. [CrossRef]
6. Novikova, A.P.; Sidorova, L.P.; Perova, N.M.; Chupakhin, O.N. Synthesis and properties of derivates of
,3,4-thiadiazolov and their condensed derivates. Khim Geterotsikl Soedin 1991, 11, 1443–1457. (In Russian)
7. Chupahin, O.N.; Sidorova, L.P.; Perova, N.M.; Charushin, V.N.; Rusinov, V.L.; Mulyar, A.G. Substituted 5-R1,
6-R2-thiadiazin-2-amines and pharmacological compositions containing them as pharmacologically active
2
2
1
-
agents with anticoagulant and antiplatelet activity. RF Patent 2259371, 27 August 2005. (In Russian)
8. Sidorova, L.P.; Tseitler, T.A.; Perova, N.M.; Emel’yanov, V.V.; Savateeva, E.A.; Maksimova, N.E.;
Mochul’skaya, N.N.; Chereshnev, V.A.; Chupakhin, O.N. Synthesis of New 1, 3, 4-Thiadiazines Capable of
Inhibiting Nonenzymatic Glycosylation of Proteins. Pharm. Chem J. USSR 2015, 49, 501–505. [CrossRef]
9. Kazakov, V.Y.; Postovskii, I.Y. Syntheses and some reactions of 4-substituted thiosemicarbazides. Dokl. Akad.
Nauk SSSR 1960, 134, 824–827.
2
2
3
3
3
0. Flecknell, P. Laboratory Animal Anaesthesia, 3rd ed.; Academic Press: London, UK, 2009.
1. Lavretskaya, E.F. Pharmacological Regulation of Mental Processes; Nauka: Moscow, Russia, 1985. (In Russian)
2. Turner, P.V.; Brabb, T.; Pekow, C.; Vasbinder, M.A. Administration of Substances to Laboratory Animals:
Routes of Administration and Factors to Consider. J. Am. Assoc. Lab. Anim. Sci. 2011, 50, 600–613. [PubMed]
3. Coria-Avila, G.A.; Gavrila, A.M.; Ménard, S.; Ismail, N.; Pfaus, J.G. Cecum location in rats and the
implications for intraperitoneal injections. Lab. Anim. (NY) 2007, 36, 25–30. [CrossRef] [PubMed]
4. Bailey, K.R.; Crawley, J.N. Methods of Behavior Analysis in Neuroscience; CRC Press: Boca Raton, FL, USA, 2009.
5. Blumstein, L.K.; Crawley, J.N.; Davis, L.G.; Baldino, F. Neuropeptide modulation of apomorphine-induced
stereotyped behavior. Brain Res. 1987, 404, 293–300. [CrossRef]
3
3
3
3
6. Georgiev, V.P.; Markovska, V.L.; Petkova, B.P. Apomorphine stereotypies and transmitter mechanisms in
the striatum. I. Changes in the apomorphine stereotypies caused by drugs acting on the GABA-ergic,
dopaminergic and cholinergic transmission. Acta Physiol. Pharmacol. Bulg. 1977, 4, 56–64.

Pharmaceuticals 2016, 9, 27
11 of 11
3
7. Kulkarni, S.K. Handbook of Experimental Pharmacology, 3rd ed.; Vallabh Prakashan: Delhi, India, 2007.
3
8. Winter, C.A. The potentiating effect of antihistaminic drugs upon the sedative action of barbiturates.
J. Pharmacol. Exp. Ther. 1948, 94, 7–11. [PubMed]
3
9. Guzevatyh, L.S.; Ostrovsky, R.W.; Gudasheva, T.A.; Zaitsev, N.I.; Voronina, T.A. Neyroleptikopodobnaya
Activity tripeptoidnogo neurotensin analog GZR-123. Exp. Clin. Pharmacol. 2002, 65, 3–6. (In Russian)
0. Brodie, B.B.; Shore, P.A.; Silver, S.L. Potentiating action of chlorpromazine and reserpine. Nature 1955, 175,
4
1
133–1134. [CrossRef] [PubMed]
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