2-Amino-6-chloro-3,4-dihydroquinazoline: A novel 5-HT3 receptor antagonist with antidepressant character

Article abstract of DOI:10.1016/j.bmcl.2013.08.072

2-Amino-6-chloro-3,4-dihydroquinazoline HCl (A6CDQ, 4) binds at 5-HT ₃ serotonin receptors and displays antidepressant-like action in the mouse tail suspension test (TST). Empirically, 4 was demonstrated to be a 5-HT₃ receptor antagonist (two-electrode voltage clamp recordings using frog oocytes; IC₅₀ = 0.26 μM), and one that should readily penetrate the blood-brain barrier (log P = 1.86). 5-HT₃ receptor antagonists represent a potential approach to the development of new antidepressants, and 4 is an example of a structurally novel 5-HT₃ receptor antagonist that is active in a preclinical antidepressant model (i.e., the mouse TST).

Full text of DOI:10.1016/j.bmcl.2013.08.072

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5945–5948
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Amino-6-chloro-3,4-dihydroquinazoline: A novel 5-HT3 receptor
antagonist with antidepressant character q
a
a
b
b
Małgorzata Dukat a, , Katie Alix , Jessica Worsham , Shailesh Khatri , Marvin K. Schulte
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298-540, USA
b Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of Sciences, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Amino-6-chloro-3,4-dihydroquinazoline HCl (A6CDQ, 4) binds at 5-HT3 serotonin receptors and dis-
plays antidepressant-like action in the mouse tail suspension test (TST). Empirically, 4 was demonstrated
Received 17 June 2013
Revised 12 August 2013
Accepted 15 August 2013
Available online 24 August 2013
to be
IC50 = 0.26
a
5-HT3 receptor antagonist (two-electrode voltage clamp recordings using frog oocytes;
M), and one that should readily penetrate the blood–brain barrier (logP = 1.86). 5-HT3 recep-
l
tor antagonists represent a potential approach to the development of new antidepressants, and 4 is an
example of a structurally novel 5-HT3 receptor antagonist that is active in a preclinical antidepressant
model (i.e., the mouse TST).
Keywords:
5-HT3 receptors
TST (tail suspension test)
Antagonists
Ó 2013 Elsevier Ltd. All rights reserved.
Antidepressants
Depression is a serious and relatively common disorder affect-
ing up to 20% of the global population,1 and the World Health
Organization predicts that unipolar depression will be the second
most prevalent cause of illness-induced disability by 2020.2 Only
about half of depressed patients respond to current antidepressant
treatment such as TCAs (tricyclic antidepressants) and SSRIs (selec-
tive serotonin reuptake inhibitors), leaving many patients refrac-
tory to current treatment.1 Furthermore, it is commonly
acknowledged that depressed patients are particularly prone to
the many side effects of established antidepressants.3
Behavioral, neurochemical and genetic studies have implicated
5-HT3 serotonin receptors in the treatment of depression.4 How-
ever, relatively few studies have examined 5-HT3 receptor agents
and the results are somewhat conflicting. Two of the most widely
used preclinical assays for antidepressant-like action are the Por-
solt forced swim test (FST),5,6 and the mouse tail suspension test
(TST).7 The 5-HT3 receptor antagonist ondansetron (Fig. 1) has
been shown both to possess8 and lack9 antidepressant activity in
the FST, whereas in the TST ondansetron significantly decreased
immobility time (a measure of antidepressant activity).8 Bemese-
tron (Fig. 1), another 5-HT3 receptor antagonist examined in the
TST, displayed pronounced decreased immobility time, indicating
potential antidepressant-like activity.10 In addition, 5-HT3 receptor
knock-out mice exhibited increased immobility in the FST further
indicating potential involvement of 5-HT3 receptors in depres-
sion.11 Few compounds with 5-HT3 receptor agonist character have
been examined. In one study, the agonist SR57227A (Fig. 1) was
found effective both in the mouse and rat FST,12 whereas in an-
other study the 5-HT3 receptor partial agonist m-CPBG (Fig. 1),
due perhaps to its difficulty in penetrating the blood-brain barrier,
was inactive in the rat FST.13 To best of our knowledge SR57227A is
the only example of a 5-HT3 receptor agonist reported in the liter-
ature to display antidepressant-like activity in the mouse and rat
FST. There are several possible explanations for the antidepres-
sant-like action of SR57227A: (i) different tests were employed
(FST vs TST); (ii) the observed effect might be due to a totally dif-
ferent mechanism than 5-HT3 receptor agonist action—the exact
binding profile of SR57227A is not reported; (iii) 5-HT3 receptors
are known to undergo rapid desensitization during sustained
application of agonist, thus the observed antidepressant-like effect
of SR57227A might be due to desensitization of the 5-HT3
receptors.12
Interestingly, Rupprecht and co-workers,14 using a concentra-
tion-clamp technique and measurement of intracellular Ca2+ con-
centration, demonstrated that different classes of clinically used
antidepressants behave as functional antagonists at h5-HT3A
receptors expressed in HEK-239 cells, endogenous 5-HT3 receptors
of rat hippocampal neurons and N1E-115 neuroblastoma cells.
q
Presented in part at the ASCEPT 43rd Annual Scientific Meeting, December 2009,
Sydney, Australia
⇑
Corresponding author. Address: Department of Medicinal Chemistry, School of
Pharmacy, Virginia Commonwealth University, PO Box 980540, Richmond, VA
23298-540, USA. Tel.: +1 8048285256; fax: +1 8048287625.
E-mail address: mdukat@vcu.edu (M. Dukat).
That
is,
all
tested
compounds
effectively
reduced
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.072

5946
M. Dukat et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5945–5948
CH3
N
1.00
N
N
O
CH3
O
Cl
N
NH
CH3
IC50= 0.26 0.013 µM
0.75
N
O
N
CH3
N
CH3
Cl
ondansetron
O
bemesetron
granisetron
0.50
CH3
N
NH2 NH2
0.25
O
N
N
NH2
O
Cl
N
N
0.00
N
NH2
Cl
H
-8
-7
-6
-5
m-CPBG
tropisetron
SR57227A
log [4] (M)
NH2
NH2
NH
Figure 2. Effect ( SEM) of 4 on the peak amplitude of 5-HT (EC50 = 2.5
induced currents in m5-HT3 receptors.
lM)-
N
NH2
N
Cl
Cl
MD-354
1
90
60
Figure 1. Chemical structures of 5-HT3 receptor antagonists ondansetron,
bemesetron, granisetron, and tropisetron, and the agonists m-CPBG, SR57227A,
and MD-354. Compound 1 is a conformationally-constrained analog of MD-354.
O
NH2
NH
NH2
NH
HN
O
N
N
a
b
#
*
30
0
O
O
(4)
R
7
7
7
R
R
*
6(5)
6
6
R1
R1
R1
1
4
R = Cl, R1= H
R = H, R1= Cl
2a
2b
3a
3b
**
saline
0.1
0.3
1.0
3.0
fluoxetine
20 mg/kg
Scheme 1. Synthesis of compounds
CH3SC(NH)NH2ÁH2SO4, Na2CO3, MeCN,
1
and 4. Reagents and conditions: (a)
, 6 h; (b) BH3–THF, , 2.5 h, 6 N NaOH, CHCl3.
D
Compound 4 Dose (mg/kg)
Figure 3. Effect ( SEM) of acute fluoxetine (ip) at a standard literature dose and 4
(ip) on duration of immobility in the mouse TST (n = 10–11 mice/treatment).
serotonin—induced Na+ and/or Ca2+ currents in a noncompetitive
(with the exception of mirtazapine) dose-dependent manner. The
inhibitory effect was voltage-independent suggesting that the
examined compounds do not act as ion channel blockers.14
Furthermore, it is generally thought that the antidepressant
mirtazapine owes, at least in part, some of its action to its affinity
Asterisk denotes
a significant difference compared to control group (saline);
#,⁄P < 0.05, ⁄⁄P < 0.01.
An attempt was made to re-synthesize quinazoline 1 via a two-
step reaction (Scheme 1) as previously reported,16 using the gen-
eral method of Grosso et al.17 Condensation of 4-chloroisatoic
anhydride (2a) with S-methylisothiourea provided the given qui-
nazolinone intermediate that was reduced with diborane to the
corresponding quinazoline. Curiously, the physicochemical and
spectral properties of the product were not identical to what had
been reported earlier.16 Comparison of 1H NMR spectra suggested
that the synthesis described above led to compound 1, whereas
the originally reported compound might be a positional isomer
(i.e., 4).
To confirm this hypothesis compound 4 was prepared from 5-
chloroisatoic anhydride (2b) as shown in Scheme 1. The product
was found to be identical to that reported earlier. 1H NMR
(DMSO-d6; 400 MHz) spectral analysis of the free bases of 1 and
4 indicated that the difference between the 6-chloroquinazoline
4 (6.91 (s, 1H), 6.98 (d, 1H)) and the 7-chloroquinazoline 1 (6.73
(d, 1H), 6.89 (d, 1H)) was an up-field shift of two aromatic hydro-
gen signals as well as a change in signal multiplicity. These data
were compared to the 1H NMR (DMSO-d6; 300 MHz) data of the
free base of the previously reported compound (6.95 (s, 1H), 6.98
for 5-HT3 receptors (Ki = 2.9 l
M).14
We previously identified MD-354 (i.e., m-chlorophenylguani-
dine; Fig. 1) as a 5-HT3 receptor agonist,15 and prepared several
conformationally-constrained analogs.16 For example 2-amino-7-
chloro-3,4-dihydroquinazoline hydrochloride (1) was shown to
bind at 5-HT3 receptors with an affinity comparable to MD-354
(Ki ꢀ30 nM).16
The present study was designed to address several questions.
First, will conformationally-constrained analogs of arylguanidines
such as MD-354 function as 5-HT3 receptor agonists or antago-
nists; second, will a conformationally-constrained arylguanidine
analog possess antidepressant action in an accepted preclinical
(i.e. animal) model, and third, will the conformationally-con-
strained analog possess the ability (i.e., logP) to penetrate the
blood–brain barrier to act in a central manner?
A specific goal of the present investigation was to resynthesize a
constrained analog of the agonist MD-354, such as 1, and evaluate
its actions in the mouse TST. The studies reported herein led to the
discovery of a structurally novel 5-HT3 receptor antagonist (i.e.,
A6CDQ, 4).

M. Dukat et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5945–5948
5947
90
60
30
0
Ondansetron, for example, is not particularly lipophilic and suffers
from low central bioavailability.18 In contrast, the experimentally
determined logP value for 4 (1.86) is in agreement with Hansch’s19
postulate that drugs with logP values in the range of 1.5–2.5 will,
in general, ‘find ready entry into the brain . . .’. In addition, quinaz-
oline 4 complies with Lipinski’s20 rules for CNS penetration sug-
gesting it should readily penetrate the blood–brain barrier.
Examined in the mouse TST (Fig. 3),4 (ED50 = 0.23 mg/kg) was at
least as efficacious as commonly employed doses of TCAs (imipra-
mine and desipramine)8,21 and the SSRI fluoxetine8 (Fig. 4).
Since drug-induced psychomotor stimulation or sedation may
increase or decrease immobility time relative to saline, to elimi-
nate the possibility of false positive readings the effective TST
doses (i.e., 1.0 and 3.0 mg/kg) of 4 were tested in a mouse locomo-
tor activity (LA) assay using saline as control. Statistical analysis
between vehicle and drug-treatment groups using both a one-
way ANOVA and t-test for 45 min duration for total-movement
time and total-movement distance showed no significant differ-
ence (Supplementary Fig. S1).
*
*
*
*
**
We have identified a novel 5-HT3 receptor antagonist with anti-
depressant-like character. Although the exact mechanism underly-
ing the antidepressant action of A6CDQ (4) is unknown, one
plausible explanation (on the basis of currently available radioli-
gand binding and functional data) involves a 5-HT3 receptor antag-
onist mechanism, with the added benefit of diminished side effects
because 5-HT3 antagonists have been demonstrated to lack overt
side effects in humans. These studies also confirm that the 5-HT3
receptor antagonist ondansetron is active in the mouse TST, and
suggest that the novel 5-HT3 receptor antagonist 4 possesses anti-
depressant potential. It would seem that a 5-HT3 receptor antago-
Drug Dose (mg/kg)
Figure 4. Effect ( SEM) of acute administration of standard doses of clinically
employed antidepressants, the 5-HT3 receptor antagonist ondansetron, and com-
pound 4 (ip), on duration of immobility in the mouse tail suspension test (n = 7–11
mice/treatment). Asterisk denotes a significant difference compared to control
group (0.9% saline; 10 mL/kg); ⁄P < 0.05, ⁄⁄P < 0.01; one way ANOVA
(F(4,41) = 3.255) followed by Newman–Keuls multiple comparison test.
(s, 1H)) which supports the conclusion that the structure had been
misassigned and that 2-amino-6-chloro-3,4-dihydroquinazoline
hydrochloride (4), not 1, had been originally synthesized. A
possible explanation for the confusion is that commercially avail-
able 4-chloroisatoic anhydride is also known as 7-chloroisatoic
anhydride. In any event, the structure of the compound described
earlier by us was misassigned as 116 where, here, we show it to be
4. A corrigendum has been published.16
Compound 4, to which structure 1 had been originally assigned,
was reported to bind at 5-HT3 (NG108-15, [3H]GR65630) receptors
with an affinity (Ki = 34 nM) comparable to that of MD-354
(Ki = 32 nM).16 The newly synthesized 4 and 1 were re-evaluated
(under different conditions: m5-HT3A; [3H]LY278584) at m5-HT3A
receptors and 4 was found to bind with roughly similar affinity
(Ki = 80 11 nM) whereas 1 binds with 20 times lower affinity
(Ki = 1,975 168 nM) than 4. As a consequence, compound 4 rather
than compound 1 was selected for further evaluation. When exam-
ined for selectivity against more than 40 proteins (G-protein cou-
pled receptors, ligand-gated ion channel receptors, transporters) 4
lacked affinity (Ki >10,000 nM) to the majority of them except: sero-
tonin 5-HT1D, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5A, 5-HT7 (Ki = 684,
nist mechanism is still
a viable approach for the potential
development of novel antidepressants.
Acknowledgments
We are grateful to Professor Richard A. Glennon for fruitful dis-
cussions and proof reading of the manuscript. This work was sup-
ported, in part, by the Jeffress Memorial Trust RG J-778 (MD), and
A. D. Williams Trust funds (MD). Ki values were generously pro-
vided by the National Institute of Mental Health’s Psychoactive
Drug Screening Program, Contract #NO1MH32004 (NIMH PDSP).
Supplementary data
Supplementary data (methods: synthetic detail, logP analysis,
functional characterization of 4 in Xenopus oocytes, ligand binding
assays, animals, tail suspension test (TST), locomotor activity, sta-
tistical analysis. Fig. S1) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
08.072.
1,322, 565, 644, 466, 1159 nM, respectively), a2A,B,C-adrenoceptors
References and notes
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expressed in Xenopus oocytg) was at least as efficacious as com-
monly es using two-electrode voltage clamp recordings. The peak
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