Synthesis and evaluation of arylpiperazines derivatives of 3,5-dioxo-(2H,4H)-1,2,4-triazine as 5-HT1AR ligands

Article abstract of DOI:10.1016/j.bmcl.2014.07.048

5-HT_1AR agonist or partial agonists are established drug candidates for psychiatric and neurological disorders. We have reported the synthesis and evaluation of a series of high affinity 5-HT_1AR partial agonist PET imaging agents with greater selectivity over α-1AR. The characteristic of these molecules are 3,5-dioxo-(2H,4H)-1,2,4-triazine skeleton tethered to an arylpiperazine unit through an alkyl side chain. The most potent 5-HT_1AR agonistic properties were found to be associated with the molecules bearing C-4 alkyl group as the linker. Therefore development of 3,5-dioxo-(2H,4H)-1,2,4-triazine bearing arylpiperazine derivatives may provide high affinity selective 5-HT_1AR ligands. Herein we describe the synthesis and evaluation of the binding properties of a series of arylpiperazine analogues of 3,5-dioxo-(2H,4H)-1,2,4-triazine.

Full text of DOI:10.1016/j.bmcl.2014.07.048

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4759–4762
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of arylpiperazines derivatives
of 3,5-dioxo-(2H,4H)-1,2,4-triazine as 5-HT1AR ligands
a
c
a,c
J. S. Dileep Kumar a,b, , Vattoly J. Majo , Jaya Prabhakaran , J. John Mann
⇑
a Division of Molecular Imaging and Neuropathology, New York State Psychiatric Institute, New York, USA
b Department of Psychiatry and Behavior Science, Stony Brook University, New York, USA
c Columbia University College of Physicians and Surgeons, New York, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
5-HT1AR agonist or partial agonists are established drug candidates for psychiatric and neurological dis-
orders. We have reported the synthesis and evaluation of a series of high affinity 5-HT1AR partial agonist
Received 15 June 2014
Revised 16 July 2014
Accepted 17 July 2014
Available online 14 August 2014
PET imaging agents with greater selectivity over
a-1AR. The characteristic of these molecules are 3,5-
dioxo-(2H,4H)-1,2,4-triazine skeleton tethered to an arylpiperazine unit through an alkyl side chain.
The most potent 5-HT1AR agonistic properties were found to be associated with the molecules bearing
C-4 alkyl group as the linker. Therefore development of 3,5-dioxo-(2H,4H)-1,2,4-triazine bearing arylpip-
erazine derivatives may provide high affinity selective 5-HT1AR ligands. Herein we describe the synthesis
and evaluation of the binding properties of a series of arylpiperazine analogues of 3,5-dioxo-(2H,4H)-
1,2,4-triazine.
Keywords:
5-HT
5-HT1AR
Agonist
Ó 2014 Elsevier Ltd. All rights reserved.
a-1AR
Arylpiperazine
The serotonin receptor, 5-HT1AR, is a G protein-coupled recep-
5-spiroimidazolidine-2,4-dione 5, indolylalkyl-amines, ergolines,
dihydrofuoroaporphines, 1,3-oxathiolane, 1,3-dioxolanes and pyr-
roloimidazoles have also been identified recently as agonist ligands
at 5-HT1AR (Fig. 1).16–23 Although several ligands with high affinity
for 5-HT1AR have been developed, a major drawback of these
ligands is their significant affinity for alpha1-adregenic receptor
tor (GPCR).1 It is widespread and heterogeneously distributed in
CNS as somatodendritic autoreceptors in raphe nuclei and postsyn-
aptically in cortical and subcortical brain regions.2 In addition to
5-HT, 5-HT1AR affects release and signaling by a variety of neuro-
transmitters, hormones and growth factors in brain.3–5 Alteration
of 5-HT1AR binding has been implicated in the pathophysiology
of a variety of neuropsychiatric diseases and neurodegenerative
disorders.6–9 5-HT1AR agonists (e.g., buspirone and tandospirone)
are currently approved drugs for anxiety and depression.10,11
5-HT1AR agonists and partial agonists are being evaluated as
antipsychotic drugs with fewer side effects for the treatment of
(a-1AR).
Positron Emission Tomography (PET) is a noninvasive method
to measure 5-HT1AR binding in living human brain.24,25 Estimation
of in vivo occupancy of agonist drugs at 5-HT1AR is a valuable tool
for drug development and monitoring the therapeutic interven-
tion. Additionally, in vivo PET estimation of the sensitivity of bind-
ing of 5-HT1AR ligands to competition by intrasynaptic serotonin
would make it possible to compare serotonergic transmission in
healthy and diseased populations.24 A number of 5-HT1AR antago-
nist radioligands such as [carbonyl-11C] WAY-100635, [18F]MPPF
and [18F]FCWAY have been routinely used for research PET studies
in man.24,25 However, an agonist ligand would bind preferentially
to the G-protein coupled high affinity (HA) state of the receptor
and thereby offers advantages to an antagonist ligand.24
schizophrenia.12–14 Furthermore, 5-HT1A
R desensitization and
increased 5-HT1AR postsynaptic activation has also been
postulated as part of the antidepressant action of SSRIs and
perhaps other antidepressants.2,5
Many 5-HT1AR agonists and partial agonists belong to amino-
tetraline and arylpiperazine structural skeletons (Fig. 1).15 Recent
additions of compounds to these prominent structural skeletons
include CUMI-101, S14506 (1), benzo[d]oxazole 2, alkylthiobenz-
imidazole, benzothiazole, and benzofuro[7,6-g]quinoline 3.16–18
Compounds belonging to several novel structural classes such as
derivatives of 4-(aminomethyl)-4-fluoropiperidin-1-ones 4,
Several studies attempted to develop a 5-HT1AR agonist PET
imaging agent with limited success in vivo.24,25 We have recently
identified several high affinity and selective 5-HT1AR ligands as
candidates for PET ligands.26–29 Of these [11C]CUMI-101, is the only
successful partial agonist PET tracer available so far for the in vivo
measurement of high affinity 5-HT1AR agonist binding in
nonhuman primates and human subjects with PET.26,30–33 The
⇑
Corresponding author at: Division of Molecular Imaging and Neuropathology,
New York State Psychiatric Institute, New York, USA. Fax: +1 212 543 1054.
E-mail address: kumardi@nyspi.columbia.edu (J.S.D. Kumar).
http://dx.doi.org/10.1016/j.bmcl.2014.07.048
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

4760
J. S. D. Kumar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4759–4762
O
O
H
N
N
O
O
O
N
N
N
O
N
S
N
N
N
N
N
O
CUMI-101
1 (S14506)
Benzo[d]oxazole 2
F
O
N
Cl
H
O
N
N
N
H
N
H
F
N
O
N
F
O
O
4 (F15599)
Spiroimadazoline 5
benzofuro[7,6-g]quinoline 3
Figure 1. Representative examples of recent 5-HT1A R agonist ligands.
Table 1
O
O
Substituted phenyl and heterocyclic derivatives of 3,5-dioxo-(2H,4H)-1,2,4-triazine
tethered piperazines
N
N
ACN, K2CO3
NaI
+
N
N
HN
N
Ar
O
N
O
N
5
Ar
N
R1
4
Y
N
n
Cl
O
R
n
3
6
n = 1-4
7
8-43 n= 1-4
N
X
N
N
N
N
Scheme 1. Synthesis of azaurazil derivatives of aryl and heteroaryl piperazines.
n
O
n= 1-4
8-36
characteristic feature of the ligands we identified is the presence of
a 3,5-dioxo-(2H,4H)-1,2,4-triazine skeleton tethered to an arylpip-
erazine unit through a C-4 alkyl side chain. Prior to our studies 3,5-
dioxo-(2H,4H)-1,2,4-triazines were reported as 5-HT1AR ligands in
Compd
n
X
Y
R1
R
5-HT1AR Ki
(nM)
a1-AR Ki
(nM)
8 (CUMI-
101)26
927
3
3
C
C
C
C
OCH3
H
H
5,
0.15
1.1
6.75
a
patent application with limited information.34 Herein we
413
describe the synthesis and evaluation of a series of 3,5-dioxo-
(2H,4H)-1,2,4-triazine derivatives as 5-HT1AR ligands.
OCH3
4,OCH3 414
4,OCH3 190
H
H
H
H
H
H
H
4, I
H
2, F
4, OMe 215
2, Br
H
5, OH
H
10
11
12
1329
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
2
2
2
4
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
C
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
C
C
C
C
C
C
C
C
H
OCH3
OCH2CH3
OCH2CH2F
>10,000
>10,000
1.8
21.4
280
350
450
1.8
64
103
7.3
>10,000
380
450
>10,000
>10,000
9.73
The candidate 5-HT1AR ligands characterized by an 3,5-dioxo-
(2H,4H)-1,2,4-triazine tethered to arylpiperazines through an alkyl
side chain were prepared by refluxing a solution of 2-(4-chlorobu-
tyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)-dione (6) preactivated
with sodium iodide in acetonitrile with substituted arylpiperazine
(7) in the presence of anhydrous K2CO3.28 Thirty six analogues of
3,5-dioxo-(2H,4H)-1,2,4-triazine (compounds 8–41) were synthe-
sized from the corresponding substituted arylpiperazines
(Scheme 1).
Most of the arylpiperazines were commercially available and
used as such without further purification. 2,4-Dimethoxyphenyl-
piperazine was prepared by the treatment of 2,4-dimethoxyphenyl
amine with bis(2-chloroethyl)amine hydrochloride in the presence
of potassium carbonate in 2-butoxyethanol at 150 °C.35 The naph-
thyl piperazines, for the synthesis of 3,5-dioxo-(2H,4H)-1,2,4-tri-
azine derivatives (compounds 39–43) were prepared by the
condensation of the corresponding napthylamines with bis(2-chlo-
roethylamine) hydrochloride in polyethylene glycol under micro-
wave irradiation.28 Binding affinity values (Ki) of the compounds
at 5-HT1AR were determined by competition binding studies with
[3H]8-OH-DPAT using racemic 8-OHDPAT as the reference com-
0.66
0.1
0.8
180
220
0.46
2.5
58.8
0.835
4.8
OCH2CH2CH2F
N(CH3)2
NHCH3
SCH3
H
H
CN
CN
CN
CN
OH
H
F
H
H
H
28
1.7
2.8
1.89
1.1
68
1.2
175
8
0.2
1.4
240
180
350
250
150
H
262
4, F
2, F
H
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
H
SH
H
H
3, F
3, Br
H
H
H
H
OCH3
OCH3
H
pound.26–28 Ki of the compounds at
a-IAR were determined by
competition binding studies with [3H]-7-methoxyprazosin using
phentolamine as the reference compound.26–28 The agonist proper-
ties of selected ligands were examined on 5-HT1AR using
F
H
H
OCH3
[
35S]GTP
cS binding in membranes of Chinese hamster ovary cells
stably expressing the human 5-HT1AR (CHO-h5-HT1A cells).26–28
ortho-position and exhibited nanomolar affinity toward 5-HT1AR.
A few of these ligands have meaningful selectivity over -1AR such
The unsubstituted phenyl analogue 18 has a 2.5 nM Ki for
a
5-HT1AR and 25 times selectivity over
a-1AR (Ki = 64 nM). In the
as 45, 210, 42, >10,000, and >10,000 times for compounds 8, 13, 14,
24 and 31, respectively. The methoxy (9) and hydroxyl (25) substi-
tution at meta-position of the phenyl group also resulted in high
affinity ligands with excellent selectivity for 5-HT1AR compared to
case of substituted phenyl derivatives (Table 1), a variety of
substituents such as methoxy (8), ethoxy (12), thiomethyl (17),
cyano (20), fluoro (25), fluoroethyl (13), fluoropropyl (14),
hydroxyl (24), thiohydroxy (31) groups were well tolerated at
a-1AR. The substitution of dimethylamino (15) and methylamino

J. S. D. Kumar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4759–4762
4761
Table 2
References and notes
Substituted naphthyl derivatives of 3,5-dioxo-(2H,4H)-1,2,4-triazine tethered
piperazines
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Neuropsychopharmacol. 1997, 7, 109.
O
R
N
N
N
N
n
N
O
5. Blier, P.; Abbott, F. V. J. Psychiatry Neurosci. 2001, 26, 37.
6. Glennon, R. A.; Dukat, M.; Westkaemper, R. B. (2000-01-01) Am. Coll.
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Frecentese, F.; Perissutti, E. J. Med. Chem 2014. http://dx.doi.org/10.1021/
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C. P. L. H. Brain Res. 2003, 974, 82.
37-41
n = 2-4
Compd
n
R
5-HT1AR Ki (nM)
a1-AR Ki (nM)
826
39
0.15
0.44
1.6
2.1
240
175
6.75
24.5
32.0
48
>10,000
>10,000
3
3
3
4
2
OH
4028,34
41
OCH3
OCH2CH3
OCH3
9. Meltzer, H. Y.; Li, Z.; Kaneda, Y.; Ichikawa, J. Prog. Neuropsychopharmacol. Biol.
Psychiatry 2003, 27, 1159.
42
43
OCH3
10. Schatzberg, A. F.; Nemeroff, C. B. The American Psychiatric Publishing Textbook of
Psychopharmacology, 4th ed.; American Psychiatric Publishing Inc, 2009.
11. Savitz, J.; Lucki, I.; Drevets, W. C. Prog. Neurobiol. 2009, 88, 17.
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13. Millan, M. J. J. Pharmacol. Exp. Ther. 2000, 295, 853.
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T. Psychopharmacol. (Berl) 2009, 202, 315.
15. Caliendo, G.; Santagada, V.; Perissutti, E.; Fiorino, F. Curr. Med. Chem. 2005, 12,
1721.
16. Czopek, A.; Byrtus, H.; Kolaczkowski, M.; Pawlowski, M.; Dybala, M.; Nowak,
G.; Tatarczynska, E.; Wesolowska, A.; Chojnacka-Wojcik, E.; Eur, E. J. Med.
Chem. 2010, 45, 1295.
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Nowak, M.; Bojarski, A. J.; Mereghetti, I.; Cagnotto, A.; Mennini, T. J. Med. Chem.
2008, 51, 4529.
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Crawford, T.; Denny, L.; Evans, L. M.; Gray, D. L.; Lee, P.; Lenoir, E. A.; Xu, W.;
Bioorg, W. Med. Chem. Lett. 2009, 19, 1159.
Table 3
Agonist properties of selected 5-HT1AR ligands
Compd
GTP
c
S EC50, (nM)
GTPcS Emax (%)
8
9
0.1
0.7
0.65
0.3
0.08
0.8
0.2
80
95
77
93
85
80
95
95
13
18
27
29
32
40
0.05
19. Dawson, L. A.; Watson, J. M. CNS Neurosci. Ther. 2009, 15, 107.
20. Fornaretto, M. G.; Caccia, C.; Marchi, G.; Brambilla, E.; Mantegani, S.; Post, C.
Ann. N. Y. Acad. Sci. 1997, 812, 226.
21. Liu, Z.; Zhang, H.; Ye, N.; Zhang, J.; Wu, Q.; Sun, P.; Li, L.; Zhen, X.; Zhang, A. J.
Med. Chem. 2010, 53, 1319.
22. Franchini, S.; Prandi, A.; Sorbi, C.; Tait, A.; Baraldi, A.; Angeli, P.; Buccioni, M.;
Cilia, A.; Poggesi, E.; Fossa, P.; Brasili, L. Bioorg. Med. Chem. Lett. 2010, 20, 2017.
23. Valhondo, M.; Marco, I.; Fontecha, M. M.; Villa, H. V.; Ramos, J. A.; Berkels, R.;
Lauterbach, T.; Benhamú, B.; Rodríguez, M. L. L. J. Med. Chem. 2013, 56, 7851.
24. Kumar, J. S. D.; Mann, J. J. Drug Develop. Today 2007, 12, 748.
25. Paterson, L. M.; Kornum, B. R.; Nutt, D. J.; Pike, V. W.; Knudsen, G. M. Med.
Chem. Rev 2013, 33, 54.
26. Kumar, J. S. D.; Prabhakaran, J.; Majo, V. J.; Milak, M. S.; Hsiung, S. C.; Tamir, H.;
Simpson, N. R.; Van Heertum, R. L.; Mann, J. J.; Parsey, R. V. Eur. J. Nucl. Med.
Mol. Imaging 2007, 34, 1050.
(16) analogues did not provide any 5-HT1AR specificity or selectiv-
ity. Substitution on the phenyl moiety resulted in a sharp decrease
in affinity for the 5-HT1AR receptor for 4-methoxy, 1,4-dimethoxy,
1-cyano-4-methoxy, 4-iodo analogs 10, 11, 23 and 19, respectively.
The introduction of a pyridyl ring results in high affinity 5-HT1AR
binding with excellent selectivity over a-1AR (27, 29, 32, 33).
However, the pyridyl derivative with meta-substituted fluoro
analog 28 had modest 5-HT1AR binding affinity. Similarly replace-
ment of pyrimidine ring instead of phenyl group results in a
remarkable decrease in 5-HT1AR binding affinity (30). Several
substituted naphthyl derivatives also exhibited nanomolar affinity
27. Prabhakaran, J.; Parsey, R. V.; Majo, V. J.; Hsiung, S. C.; Milak, M. S.; Tamir, H.;
Simpson, N. R.; Van Heertum, R. L.; Mann, J. J.; Kumar, J. S. D. Bioorg. Med. Chem.
Lett. 2006, 16, 2101.
at 5-HT1AR as well as possess reasonable selectivity over
a-1AR
(39–41, Table 2). The ideal length of alkyl linker was identified to
be 4. When the chain length was decreased to 3 (35–37, 43), there
was a dramatic drop in the binding affinity at 5-HT1AR (Tables 1
and 2). Similarly we did not see considerable improvement in
5-HT1AR binding affinity for linker with length 5 as shown for
molecules 38 and 42.
28. Kumar, J. S. D.; Majo, V. J.; Hsiung, S. C.; Millak, M. S.; Liu, K. P.; Tamir, H.;
Prabhakaran, J.; Simpson, N. R.; Van Heertum, R. L.; Mann, J. J.; Parsey, R. V. J.
Med. Chem. 2006, 49, 125.
29. Majo, V. J.; Prabhakaran, J.; Milak, M. S.; Mali, P.; Parsey, R. V.; Mann, J. J.;
Kumar, J. S. D. Bioorg. Med. Chem. 2013, 17, 5598.
30. Milak, M. S.; DeLorenzo, C.; Zanderigo, F.; Prabhakaran, J.; Kumar, J. S. D.; Majo,
V. J.; Mann, J. J.; Parsey, R. V. J. Nucl. Med. 2010, 52, 1892.
31. Milak, M. S.; Severance, A. J.; Ogden, T. R.; Prabhakaran, J.; Kumar, J. S. D.; Majo,
V. J.; Mann, J. J.; Parsey, R. V. J. Cer. Blood Flow Metab. 2011, 31, 241.
32. Kumar, J. S. D.; Majo, V. J.; Milak, M. S.; Prabhakara, J.; Mali, P.; Savenkova, L.;
Mann, J. J.; Parsey, R. V. J. Pharm. Sci. 2012, 120, 254.
The promising compounds with higher ratios of 5-HT1AR vs.
a
-1AR were tested for 5-HT1A
Agonist increased [35]GTP
S binding over basal level in a concen-
tration dependent manner. Maximal ligand stimulated [35]GTP
R
functional measurements.36
c
33. Kumar, J. S. D.; Parsey, R. V.; Majo, V. J.; Milak, M. S.; Prabhakaran, J.; Kassir, S.
A.; Underwood, M. A.; Mann, J. J.; Arango, V. Brain Res. 2013, 11, 1507.
34. J.-F.P. Castres, C. F. Toulouse; E. D. P. Castres, F. C. Corransac, W. K. V. les-
Montagnes, 1999, US 5,977,106.
cS
binding (Emax) for the selected compounds were in the range of
80–95% of that seen with 5-HT (Table 3).
35. Typical experimental procedure: 4-Methyl-2-(4-(4-(pyridin-2-yl)piperazin-1-
yl)butyl)-1,2,4-triazine-3,5(2H,4H)-dione (11). A mixture of 2-(4-chlorobutyl)-
4-methyl-1,2,4-triazine-3,5(2H,4H)-dione, (217 mg, 1 mmol) and NaI (225 mg,
1.5 mmol) in acetonitrile (3 mL) was stirred under reflux for 30 min. Then, 2-
pyridylpiperazine (325 mg, 2 mmol) and anhydrous K2CO3 (420 mg, 3 mmol)
were added. The reaction mixture was stirred at 60 °C for 24 h. After cooling,
the reaction mixture was diluted with EtOAc, filtered to remove excess K2CO3.
The organic layer was dried over anhydrous MgSO4 and the solvent was
removed under vacuum. The crude mixtures were purified by silica gel column
chromatography using chloroform/methanol 98:2 (v/v) as the eluent. The
combined product fractions were concentrated to yield the desired product as
a colorless solid (320 mg, 93%); mp: 85 °C. 1H NMR (400 MHz, CDCl3) d:
1.61(pentet, 2H, J = 7.6 Hz); 1.84 (pentet, 2H, J = 7.6 Hz); 2.45 (t, 2H, J = 7.6 Hz);
2.57 (t, 4H, J = 5.2 Hz); 3.37 (s, 3H); 3.57 (t, 4H, J = 5.2 Hz); 4.05 (t, 2H,
J = 7.6 Hz); 6.63–6.67 (m, 2H); 7.42 (s, 1H); 7.50 (dt, 1H, J = 1.6, 7.2 Hz); 8.20–
8.22 (m, 1H). HRMS Calcd for C17H25N6O2 (MH+): 345.2039; Found: 345.2018.
In summary, a novel series of 3,5-dioxo-(2H,4H)-1,2,4-triazine
tethered arylpiperazines has been identified as candidate agonist
ligands with high affinity for 5-HT1AR. Several members of the
series such as 32 have nanomolar affinity for 5-HT1AR, higher
selectivity to 5-HT1AR over
a-1AR in comparison to CUMI-101
and robust agonist profile as measured by GTPcS binding.
Acknowledgment
This work was partially supported by National Institute of
Health grants MH062185 and MH077161.

4762
J. S. D. Kumar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4759–4762
36. Agonist-stimulated
[
35S]GTP
c
S
binding of the ligands were measured as
Elmer Life Science, Boston, MA) was added and the incubation was continued
for 60 min at room temperature. Experiments were terminated by rapid
filtration through Whatman GF/B filters followed by three washes with ice-
cold 20 mM HEPES buffer, pH 7.4, using a cell harvester (Brandel, M-24R,
Gaithersburg, MD). Bound radioactivity was determined by liquid scintillation
spectrometry (Beckman, LS9000).
described previously.28 Chinese hamster ovary (CHO) cells expressing 5-
HT1A receptors (CHO-h5-HT1A) membranes (30
specific concentrations of the test ligand for 5 min at room temperature in a
buffer containing 20 mM HEPES pH 7.4, 3 mM MgCl2, 100 mM NaCl, and 3
GDP in a final volume of 0.5 mL. [35S]GTP
S (0.1 nM; 1250 Ci/mmol Perkin
lg) were preincubated with
lM
c