Design and Synthesis of Novel Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors with the Ability to Rescue Auditory Gating Deficit in Mice

Article abstract of DOI:10.1021/acs.jmedchem.7b01492

A series of novel thiazolo[4,5-d]pyrimidin-7(6H)-ones (3aa-3eq) were designed, synthesized, and evaluated as the type I positive allosteric modulators of human α7 nAChR expressed in Xenopus ooctyes by a two-electrode voltage clamp. The structure-activity relationship analysis identified the compound 3ea as a potent and efficacious PAM with the maximum activation effect of the α7 current of over 1633% in the presence of acetylcholine (100 μM) and an EC₅₀ = 1.26 μM. It is highly specific to α7 nAChR over other subtypes of nAChR, 5-HT_3A, NMDA, and GABA_A receptors. Compound 3ea showed an elimination half-life of 10.8 ± 1.5 h for 3 mg/kg, i.v., and 7.4 ± 1.1 h for 60 mg/kg, i.g. in rat. It also exhibited sufficient blood-brain barrier penetration with no significant effect on hERG channel. Most importantly, compound 3ea dose-dependently (0.1-1 mg/kg, i.p.) reversed the prepulse inhibition deficit induced by MK-801 in the mouse schizophrenia model.

Full text of DOI:10.1021/acs.jmedchem.7b01492

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The design and synthesis of novel positive allosteric
modulators of #7 nicotinic acetylcholine receptors
with the ability to rescue auditory gating deficit in mice
Yuanheng Li, Lilan Sun, Taoyi Yang, Wenxuan Jiao, Jingshu Tang, Xiaomin Huang, Zongze
Huang, Ying Meng, Laichun Luo, Xintong Wang, Xiling Bian, Fang Zhang, Kewei Wang, and Qi Sun
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01492 • Publication Date (Web): 28 Mar 2018
Downloaded from http://pubs.acs.org on March 28, 2018
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The design and synthesis of novel positive allosteric modulators of α7
nicotinic acetylcholine receptors with the ability to rescue auditory
gating deficit in mice
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†
2†
3†
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3
Yuanheng Li , Lilan Sun , Taoyi Yang , Wenxuan Jiao , Jingshu Tang , Xiaomin Huang ,
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Zongze Huang , Ying Meng , Laichun Luo , Xintong Wang , Xiling Bian , Fang Zhang , KeWei
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, 2, 3
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Wang
* and Qi Sun *
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State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China;
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Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao 266021,
China;
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Department of Molecular and cellular Pharmacology, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China.
ABSTRACT
A series of novel thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀones (3aa−3ep) were designed, synthesized
and evaluated as the type I positive allosteric modulators (PAMs) of human α7 nAChR expressed
in Xenopus ooctyes by twoꢀelectrode voltage clamp. The structureꢀactivity relationship (SAR)
analysis identified the compound 3ea as a potent and efficacious PAM with the maximum
activation effect of the α7 current of over 1633% in the presence of acetylcholine (100 ꢀM) and an
EC50 = 1.26 ꢀM. It is highly specific to α7 nAChR over other subtypes of nAChR, 5ꢀHT3A
,
NMDA and GABA receptors. 3ea showed an elimination halfꢀlife (T1/2) of 10.8 ± 1.5 h for 3
A
mg/kg, i.v. and 7.4 ± 1.1 h for 60 mg/kg, i.g. in rat. It also exhibited sufficient bloodꢀbrain barrier
penetration with no significant effect on hERG channel. Most importantly, compound 3ea
doseꢀdependently (0.1–1 mg/kg, i.p.) reversed the prepulse inhibition (PPI) deficit induced by
MKꢀ801 in the mouse schizophrenia model.
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Keywords: α7 nAChR; Positive allosteric modulators; Thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀones;
Structure−activity relationship; Prepulse inhibition; Schizophrenia disease.
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INTRODUCTION
The α7ꢀsubtype of nicotinic acetylcholine receptors (α7 nAChR) has been well recognized as a
potential pharmacological target in central nervous system (CNS) for the treatment of
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neuropsychiatric disorders, such as schizophrenia and Alzheimer’s disease. It is a homopentamer
that can form ligandꢀgated calcium permeable ion channels upon the activation with acetylcholine,
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exhibiting extremely fast desensitization as exposed to agonists. Clinical studies have shown that
targeting α7 nAChR with selective agonists or partial agonists can effectively improve the
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cognitive deficits in schizophrenia. Positive allosteric modulators (PAMs) have been
demonstrated with numerous advantages over agonists because it can maintain the normal
temporal and spatial patterns of neurotransmission due to the lack of activity in the absence of an
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endogenous agonist. Based on their channel kinetics and desensitization characteristics, the
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PAMs of nicotinic receptors are classified into type I and II. Figure 1 shows the chemical
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structures of representative type I PAMs including macrolide 1a (ivermectin) , anthridine alkaloid
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b (galantamine) , indole derivative 1c (5ꢀhydroxyindole, 5ꢀHI) , isoflavones 1d (genistein) ,
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acrylamide 1e (AVL3288, CCMI) , ureas 1f (NSꢀ1738) and 1g (SBꢀ206553) , and
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(2ꢀaminoꢀ5ꢀketo)thiazole compound 1h (LYꢀ2087101) . The type II PAMs including urea 2a
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(
PNUꢀ120596) , thiazole compound 2b (JNJꢀ1930942) , and sulfonamides 2c (TQS) and 2d
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(Aꢀ867744) are shown in Figure 2.
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Both types of PAMs have been found with in vivo efficacy in animal models of cognition,
whereas type II PAMs are less prone to induce tolerances after the chronic administration of
nicotinic agonists. Type I PAMs can maintain the rapid channel kinetics, and thus may be more
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+
19
beneficial than type II PAMs to minimizing potential Ca ꢀinduced cytotoxicity. α7 nAChR and
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other subtypes including α4β2 and α3β4 of nAChR are also expressed in the cortex, hippocampus
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and cerebellum of brain, whose functions can be activated with acetylcholine and enhanced with
nAChR PAMs. Therefore, the design and synthesis of novel selective α7 nAChR PAMs with high
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potency and efficacy are highly desired to achieve ideal therapeutic indexes and reduce
sideꢀeffects.
Figure 1
Figure 2
To identify novel PAMs, we initially synthesized a series of fused pyrimidinꢀones 3 using
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ꢀethylsulfanylꢀ5ꢀaminoꢀthiazines as the key intermediate. A small library of these compounds and
intermediates was construed and screened for the PAM activity on the human α7 nAChR
expressed in Xenopus oocytes by the twoꢀelectrode voltage clamp assay. Compound 3aa
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(LD486) , one of the intermediates, exhibited the type I PAM activity on α7 nAChR with the
EC50 of 3.20 ± 0.30 ꢀM (n=6) and the maximum effect of 320 ± 20 % in the presence of 100 ꢀM
ACh. Based on the novel structure and good efficacy of compound 3aa, we envisaged that more
efficient compounds could be synthesized by alternative modifications. Herein, we designed and
synthesized a series of thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀones 3 and evaluated their biological
activities as novel PAMs of α7 nAChR.
Figure 3
Aiming to improve the efficacy and selectivity of the hit compound 3aa, we first studied the
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SAR of thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀones. The substitutes on N ꢀ and/or N ꢀ of
thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone motif were first examined (Figure 3). The
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ꢀClꢀ2ꢀMeꢀpyrimidinꢀ4ꢀyl group of 3aa was modified with heterocyclic aromatic (3ab−3ae),
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aliphic (3af−3ag), and aromatic (3ba−3bs) groups, respectively, to explore the tolerance of N ꢀR
group in the PAM activity. Compounds of amide 3ca, sulfamide 3cb, sulfide 3da, and other type
linkers 3cc, 3cd, 3db and 3ea−3ek were designed to examine the effects of space between
aromatic group and thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone on the activity. Compounds 3el−3eq were
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synthesized to evaluate the tolerance of N ꢀ of thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone. Compounds
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6 and 19 with different skeletons were synthesized to explore the key effects of
thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone.
RESULTS AND DISCUSSIONS
Scheme 1
The synthesis route of thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀones 3 is outlined in Scheme 1. The
reaction of aromatic amines with chloroacetyl chloride gave 2ꢀchloroꢀNꢀphenyl acetamide 4 that
was treated with dipotassium cyanodithioimidocarbonate, followed by the addition of iodoethane,
to produce the key intermediate [(4ꢀaminoꢀ2ꢀethylthio)ꢀthiazolo]ꢀ5ꢀcarboxmide 5 via the
22a, 22b
ThorpeꢀZiegler cyclization.
Intermediate 5 was refluxed in HCOOH for 1 h to afford a crude
2
2c
2 4 2
product that, without further purification, was stirred with Na WO ·H O in 30% hydrogen
o
peroxide for 1 h at room temperature, and heated at 50 C for another 1 h to afford the key
intermediate 6. The nucleophilic substitution of the ethylsulfonyl in 6 with different aromatic and
2
1b
aliphatic amines yielded the corresponding products 3aa–3ah and 3ba–3bs, respectively.
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Scheme 2
Scheme 3
Scheme 4
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The nucleophilic substitution of ethylsulfonyl 6a with the substituted amide 7, sulfamide 8, N,
ꢀdimethylanine, 4ꢀmethylbenzylamine and 4ꢀmethylꢀthiophenol 9 (Scheme 2) gave the
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corresponding products 3ca–3cd, respectively. Compound 3db was synthesized according to the
procedure described in Scheme 3. Intermediate 5 was refluxed in HCOOH for 1 h to afford key
intermediate 6. Then, desulfurative palladiumꢀcatalyzed Liebeskind–Srögl coupling reaction of 6
2
2d
and 4ꢀmethylphenyl boric acid provided the product 3db in presence of CuTC. Compounds
3ea–3eq were prepared via an alternative method as shown in Scheme 4. Different substituted
benzylsulfonyl, 2ꢀphenylethylsulfonyl, and 3ꢀphenylpropyl groups were introduced to afford
2ꢀchloroꢀNꢀ2ꢀcholoroꢀ6ꢀmethylphenyl acetamides 4a–4g that were subjected to the thorpeꢀZiegler
cyclization with dipotassium cyanodithioimidocarbonate, followed by the addition of chloride or
bromide 11, to produce thiazoles 10a–10q. The thiazoles were treated with formic acid to yield
target products 3ea–3eq.
Scheme 5
2ꢀ(Benzylthio)ꢀNꢀ(2ꢀchloroꢀ6ꢀmethylphenyl)thiazolo[4,5ꢀd]pyrimidinꢀ7ꢀamine
16
was
22e
synthesized according to the reference procedure shown in Scheme 5.
The reaction of
dipotassium cyandithiomidocarbonate 12 with benzyl bromide in acetoneꢀwater afforded
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compound 13 without further purification. Certain amounts of chloroacetonitrile and Et N were
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then added to the solution of 13 and heated at 70 C for 4 h to produce the key intermediate,
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ꢀaminoꢀ2ꢀ(benzylthio)thiazoleꢀ 5ꢀcarbonitrile, 14 that was concentrated with DMF·DMA at 100
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C to yield intermediate 15 as a grey solid. The reaction of 15 with 2ꢀchloroꢀ6ꢀmethylaniline in
acetic acid at a higher temperature gave the desired product 16 via a oneꢀpot reaction.
Scheme 6
The synthesis route of compound 19 with another new skeleton is described in Scheme 6.
6
ꢀiodoꢀbenzo[d]pyrimidinꢀ4(3H)ꢀone 18 was obtained via the oneꢀpot condensation of
o
5ꢀiodoanthranilic acid 17, 2ꢀchloroꢀ6ꢀmethylaniline and trimethylorthoformate at 100 C in acetic
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acid. The copperꢀcatalyzed Ullmann reaction of intermediate 18 with 4ꢀmethylaniline produced
the target product 19.
The synthesized compounds were tested for the in vitro activity in Xenopus laevis oocytes
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expressing human α7 nAChR at room temperature as described in our previous work. The
compounds were inactive in eliciting α7 current in the absence of direct agonists. Adding
acetylcholine (ACh, 100 ꢀM) to 10 ꢀM test compounds resulted in the activation of α7 currents,
suggesting that the compounds were positive allosteric modulators of α7 nAChR. The
concentrationꢀresponse curves of the compounds are listed in Tables 1, 2, 3, and 4. The maximum
modulation (at 10 ꢀM) and EC50 where half maximum modulation was achieved for the
compounds with the maximum effect > 400% were also determined.
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Table 1
The substitution of 6ꢀchloroꢀ2ꢀmethylꢀpyrimidinꢀ4ꢀyl with 6ꢀchloroꢀpyrimidinꢀ4ꢀyl (3ab, Table
), 6ꢀchloroꢀpyridazinꢀ3ꢀyl (3ac, Table 1), pyridinꢀ2ꢀyl (3ad, Table 1) or pyrimidinꢀ4ꢀyl (3ae,
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Table 1) caused either lost or reduced activity. Substituting 6ꢀchloroꢀ2ꢀmethylꢀpyrimidinꢀ4ꢀyl (3aa,
Table 1) with aliphic groups, such as propyl (3af, Table 1) and benzyl (3ag, Table 1), resulted in
completely lost activity. These results indicate that suitable heterocyclic aromatic and aliphic
groups are detrimental for the allosteric modulation activity and compounds 3af−3ag are inactive
for allosteric modulation.
Based on the in vitro activities of 3aa−3ag shown in Table 1, we examined the effects of the
1
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substitutes (3ba−3bs) on the phenyl group in R . The substitutions of R in 3aa with
3ꢀchloroꢀ5ꢀmethylphenyl (3ba) and 3ꢀchloroꢀphenyl (3bb), and 3ꢀmethylꢀphenyl (3bc) (Table 1)
resulted in similar modulation effects. Compound 3bc (max effect of 1540%, EC50 = 8.39 ꢀM)
with 3ꢀmethyl exhibited a 5ꢀfold higher modulation effect than 3aa. Although the EC50 of 3bc was
not as high as that of 3aa (3.20 ꢀM), it encouraged us to further modify the phenyl group to
achieve better activities. Changing the 3ꢀmethyl of 3bc to the 2ꢀposition and 4ꢀposition gave
compounds 3bd and 3be, respectively. 3bd showed the maximum effect of 300% at 10 ꢀM as the
modulator of α7 nAChR, and 3be exhibited the highest maximum modulation effect of 1622%
with the EC50 of 8.45 ꢀM. The substitutions of 4ꢀMe in 3be with H, 4ꢀCl, 4ꢀBr, and 4ꢀF (3bf−3bi)
dramatically reduced the activity. The isomeric 2ꢀ (3bj) and 3ꢀ fluorophenyl (3bk) compounds
showed significant activities. Compound 3bk exhibited a potency with EC50 = 7.65 ꢀM and the
maximum modulation effect of 450%. Moving the fluorine atom to the 2ꢀposition (3bj)
dramatically reduced the activity to the maximum modulation effect of 200%. The substitutions of
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the 4ꢀmethyl in 3be (max effect of 1622%) with 4ꢀethyl (3bl, max effect of 250%) and 4ꢀmethoxy
(3bm, max effect of 200%) resulted in almost 10ꢀfold loss in the maximum effect. Similar results
3
were obtained when 4ꢀmethyl was substituted with 4ꢀOCF (3bn, with the max effect of 200%).
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Based on the maximum effects acquired from 3ꢀfluoro, 3ꢀmethyl and 4ꢀmethyl substitutes, we
envisaged that the introduction of methyl and /or fluoro to the metaꢀ and paraꢀpositions of phenyl
group could improve the activity. Therefore, compounds 3bo−3bs were designed and synthesized,
which, to our delightfulness, resulted in the maximum effects ranging from 300% to 600%.
Compound 3be (R = 4ꢀMeꢀPh) exhibited the highest activity with 5ꢀfold higher maximum
effect than that of the lead compound 3aa. In order to further explore the SAR of the linker
between the aromatic group and key pharmacophore motif, changing –NH– (3be, Max mod
1622%) with different type of amide (3ca, 450%), sulfamide (3cb, 97%), –NMe– (3cc, 107%),
2
–CH NH– (3cd, 111%), –S– (3da, 226%), or bond (3db, 185%) resulted in a much decrease of
activity, indicating the high influence of the space on the activity (Table 2).
Table 2
Attempts to extend the linker of ꢀSꢀ atom to ꢀCH Sꢀ group gave compounds 3ea–3ei with the
2
maximum effects and EC50 listed in Table 2. The SAR study revealed that 3ea with no substitution
on its phenyl group had a higher activity than the monoꢀsubstituted derivatives 3eb–3eh. In
2
addition, the activity of 3ea is much higher than that of 3ag (Table 1) with ꢀCH NHꢀ as the linker.
Monoꢀsubstituted fluorine derivatives 3eb–3ed exhibited similar effects compared to the diꢀfluoro
derivative 3ei (max mod of 1046% and EC50 = 3.54 ꢀM), acceptable EC50 values in the range of
2 2 2 3
2.05–7.65 ꢀM were achieved. Further extending the linker of ꢀSꢀ atom to ꢀ(CH ) Sꢀ or ꢀ(CH ) Sꢀ
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groups produced 3ej–3ek with completely lost activities. Based on these results, it can be deduced
that further modifying the 2ꢀposition of thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀones may give more
efficient α7 nAChR PAMs.
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The modifications of the 2ꢀchloroꢀ6ꢀmethyl group (3ea) in R with 2ꢀfluoro (3el), 3ꢀfluoro
(3em), 4ꢀfluoro (3en), 4ꢀchloro (3eo), 2ꢀchloro (3ep) and 2ꢀmethyl (3eq) led to dramatically
reduced activity or even complete loss of effect (Table 3), indicating that
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N ꢀ(2ꢀchloroꢀ6ꢀmethyl)ꢀphenyl group was the key pharmacophore for this novel series of
compounds. In addition, the low activities of 16 and 19 suggest that
thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone is the key biological skeleton (16: max mod of 33%, Table 4
vs. 3ea: 1633%, Table 4; 19: max mod of 63% Table 6 vs. 3be: 1622%, Table 1).
Table 3
Table 4
Based on the results presented above, it can be concluded that, among the synthesized
analogs, 3ea is the optimal PAM of α7 nAChR. A 2ꢀminute preincubation of oocytes expressing
α7 nAChR with 3ea, followed by the application of 100 ꢀM ACh, remarkably increased the peak
current and slightly prolonged the current duration (Figure 4A). In addition, such enhanced
AChꢀevoked current by 10 ꢀM 3ea was completely inhibited with 10 nM MLA, a selective α7
nAChR antagonist. To evaluate the specificity of 3ea for α7 nAChR, its effects on other nAChRs
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and 5ꢀHT3A receptor expressed in oocytes were investigated. As shown in Figure 4B, 3ea was only
able to cause potentiation to α7 nAChR. The inspection of Figure 4C and D suggests that 3ea
slightly affected the desensitization of α7 nAChR, causing a minor increase in τdesensitization (ACh:
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τdesensitization = 5.56 ± 1.27; ACh + 3ea: τdesensitization = 6.54 ± 1.31; P ＞ 0.05). We also tested the
A
effect of 3ea on NMDA and GABA channels. The results show that 3ea had little effect on
NR1/NR2B and several subtypes of GABA channels (Figure S1). Based on these results, it can
A
be concluded that 3ea is a selective type I α7 nAChR PAM.
Figure 4
The activation of α7 nAChR with 100 ꢀM ACh alone was firstly conducted as the control. 3ea
was then coꢀapplied with 100 ꢀM ACh after 2ꢀmin preꢀincubation at the initial concentration of
0
.1
ꢀM to activate α7 current. The 3ea concentration was increased at the 10ꢀmin intervals to 0.3,
1, 3, 10, and 30
ꢀM. Figure 5A shows α7 currents evoked with different concentrations of 3ea in
the presence of 100 ꢀM ACh. The 3ea concentrationꢀdependent activation of α7 current was
plotted (Figure 5B) and fitted to the Hill equation, which gave EC50 = 1.26 ± 0.18 ꢀM, Emax = 1633
±
87% and Hill coefficient (n )= 1.85 ± 0.24. To further confirm the activity of 3ea, the ACh
H
concentrationꢀresponse curves were generated in the absence and presence of 10 ꢀM 3ea. A 2ꢀmin
preꢀincubation with 3ea increased the Emax 15ꢀfold from 109 ± 2% to 1545 ± 59%, reduced the
H
EC50 of ACh from 256.4 ± 20.1 ꢀM to 186.8 ± 14.5 ꢀM, and slightly increased of n from 1.14 ±
0.10 to 1.57 ± 0.16 (Figure 5C).
Figure 5
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The voltage protocol was run for at least 1 min and perfused continuously (0.5 mL/min) with
an extracellular bath solution containing compound 3ea (1, 3, 10, or 30 ꢀM) and 100 nM cisapride,
a hERG classical inhibitor, followed by washing with the bath solution. The peak tail currents at
ꢀ40mV in the presence and absence of drugs were normalized to their respective control values
and plotted as the relative current amplitude vs. 3ea concentration. As shown in Figure 6, the
hERG current remained stable with the increase of 3ea concentration, suggesting that 3ea had no
cardiac toxicity in vitro.
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Figure 6
The assay that we previously developed for rat plasma was used to determine the
23
pharmacokinetic profile of 3ea. Mean halfꢀlife for the iv dose and oral administration (60 mg/kg)
was 10.8 and 7.4 h respectively. Considering the experimental error in vivo as well as the
clearance results from two administration pathways, these t1/2 values are likely in the acceptable
range. Low clearance (0.005 L/h/kg) for compound 3ea and the metabolic stability from the i.v.
dose in rat are translated well to gavaged dose (60 mg/kg). Since the bioavailability of the
compound 3ea is around 8%, the CL/F estimates for gavaged doses (0.06 L/h/kg) are reasonable.
The mean residence time (MRT) for gavage (9.5 h) is longer than that for intravenous injection
(7.4 h), indicating the mean absorption time is nearly 2 h. The low bioavailability of 3ea could be
explained by precipitation in the rat gut. These results suggest that suitable drug formulation of
oral administration needs further exploration during its preꢀclinical studies. The pharmacokinetic
parameters were evaluated in each individual rat by a nonꢀcompartmental approach using the
software DAS2. The administration doses of 60 mg/kg (i.g.) and 3 mg/kg (i.v.) resulted in the
maximum concentrations (Cmax) of 200 ± 80 ng/mL and 700 ± 200 ng/mL and the areas under the
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curve (AUC ) of 1100 ± 200 ng·h/mL and 680 ± 40 ng·h/mL, respectively (Table 5).
Inf
Table 5
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A frequently confronted challenge in developing CNS targeted drugs is the insufficient
bloodꢀbrain barrier (BBB) penetration. Table 6 lists the results of the CNS exposure of 3ea in
mice. The compound was administered intraperitoneally (ip) to C57BL/6J mice at 1mg/kg and its
brain and plasma concentrations were assayed at 0.5, 2 and 8 h after the administration. The
brain/plasma concentration ratios were measured to be 2.5, 2.7 and 2.3 at 0.5, 2.0 and 8.0 h
postꢀadministration, respectively, suggesting the excellent brain penetration of 3ea. Therefore, 3ea
can robustly activate central receptors, while minimizing possible side effects caused by the
interactions with the peripheral systems.
Table 6
To further evaluate the modulation effects of 3ea, the mouse model of schizophrenia with
auditory gating deficit induced by NMDA antagonist MKꢀ801 was measured for the prepulse
inhibition the acoustic startle chambers before and after the administration of 3ea. Three 20ꢀms
prepulses of different intensities (8, 12 and 16 above 68 dB background noise) at 80 ms delay
intervals appeared before the application of the startle stimulus (pulse alone) at 120 dB for 40 ms
(Figure 7A). The intraperitoneal injection of MKꢀ801 (0.1 mg/kg) dramatically reduced the PPI
from 36% to 11%. The administration of different doses of 3ea (0.1–1.0 mg/kg) reversed the
reduced PPI to 22%, 26% and 28%, respectively, while clozapine (1 mg/kg, i.p.), the positive
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control, reversed the reduced PPI to 32% (Figure 7B). These results indicate that type I PAM 3ea
can rescue the auditory gating deficit in schizophreniaꢀlike behavior of mice.
Figure 7
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In order to verify whether the in vivo activity is directly related to the activation of alpha7
3d
nACh receptors, we redesigned the experimental group, as shown below in Figure 8.
Intraperitoneal injection of MKꢀ801 (0.1 mg/kg, i.p.) 30 minutes before the test resulted in a
significant reduction of PPI from 41% to 8.7%, administration of 3ea (1 mg/kg, i.p.) 60 minutes
before the test reversed the reduction of PPI to 30%. Pretreatment with MLA (3 mg/kg, i.p.) 30
minutes prevented the reversal by 3ea of MKꢀ801ꢀinduced auditory gating impairment to 14%.
Injection of 3ea or MLA alone did not significantly alter the percentage of prepulse inhibition
compared with control group. These results suggest that in vivo activity of compound 3ea is
directly related to the activation of alpha7 nACh receptors.
Figure 8
CONCLUSIONS
A series of novel thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀones (3aa−3eq) were designed, synthesized
and evaluated as the type I positive allosteric modulators (PAMs) of human α7 nAChR expressed
in Xenopus ooctyes in twoꢀelectrode voltage clamp assay. The structureꢀactivity relationship (SAR)
6
analysis revealed that N ꢀ(2ꢀchloroꢀ6ꢀmethyl)ꢀphenyl was the key pharmacophore for the activity
of these compounds. Compound 3ea exhibited high selectivity for α7 nAChR over other subtypes
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of α4β2, α3β4 nAChRs and 5ꢀHT , NMDA, GABA receptors with an excellent pharmacokinetic
3
A
A
profile and good brain tissue distributions. It was able to successfully reverse the PPI impairments
induced by MKꢀ801 in mouse acoustic startle model of schizophrenia. Our findings demonstrate
that the novel compound 3ea, which is suggestive as type I PAM, is a potential drug candidate for
improvement of cognitive function in neurodegenerative models of rodents.
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EXPERIMENTAL SECTION
General: Commercial reagents were purchased from Ouhe, Aladdin and J&K Scientific and used
1
13
without further purification. H and C NMR spectra were recorded with a Bruker AVIIIꢀ400
spectrometer at ambient temperature with CDCl or DMSOꢀd as the solvent. Chemical shifts
3
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3
were referenced to the residual peaks of the solvent [CDCl : δ = 7.26 ( H), 77.16 ppm ( C);
1
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6
DMSOꢀd : δ = 2.50 ( H), 39.52 ppm ( C)]. Highꢀresolution mass spectra were recorded with a
Bruker Apex IV Fourier transform ion cyclotron resonance mass spectrometer. Flash column
chromatography was performed with 200–300 mesh silica gel. All the final compounds were
tested by HPLC and the purity in every case was ≥95%. The reverse phase HPLC was conducted
on Agilent Technologies 1260 Infinity, which was equipped with C18 column (Aglilent Eclipse
plus C18, 3.5 ꢁm, 4.6 mm × 100 mm). The mobile phase A was water, and mobile phase B was
acetonitrile. The gradient of 70% B was run at a fl T rate of 1.0 mL/min over 10 min.
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.1 2ꢀ((6ꢀChloroꢀ2ꢀmethylpyrimidinꢀ4ꢀyl)amino)ꢀ6ꢀ(2ꢀchloroꢀ6ꢀmethylphenyl)thiazolo[4,5ꢀd]
pyrimidinꢀ7(6H)ꢀone (3aa).
Compound 6a (370 mg, 1.0 mmol) and 4ꢀaminoꢀ6ꢀchloroꢀ2ꢀmethylpyrimidine (287 mg, 2.0
mmol) were dissolved in anhydrous THF (3 mL). Then, NaH (48 mg, 2.0 mmol) was added to the
solution, the reaction was held at reflux for 0.5h with stirring, then cooled and quenched with
ethanol. The reaction mixture was concentrated under reduce pressure. The crude material was
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2 2
purified by silica gel chromatography (CH Cl /MeOH = 50:1) to afford the compound 3aa. Yield:
o
1
64%. Yellow solid, m.p.: > 300 C. H NMR (400 MHz, DMSOꢀd
6
): δ (ppm) 12.80 (bs, 1H), 8.44
(
(
s, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.51 (t, J = 8 Hz, 1H), 7.46 (d, J = 7.2 Hz, 1H), 6.99 (s, 1H), 2.56
1
3
6
s, 3H), 2.15 (s, 3H). C NMR (101 MHz, DMSOꢀd ): δ (ppm) 167.8, 164.0, 159.5, 158.1, 155.9,
150.9, 139.1, 134.0, 132.3, 131.4, 130.3, 128.1, 112.0, 104.6, 25.5, 18.2. HRMS: m/z calcd. for
+
C
H13Cl N
17 2 6
OS [M + H] 419.0243; found 419.0252. HPLC purity: 97.8%, retention time = 1.63
min.
1
.2
6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((3ꢀchloroꢀ5ꢀmethylphenyl)amino)thiazolo[4,5ꢀd]
pyrimidinꢀ7(6H)ꢀone (3ba).
Compound 6a (370 mg, 1.0 mmol) was dissolved in acetic acid (7 mL). Then,
3
4
ꢀchloroꢀ5ꢀmethylaniline (284 mg, 2.0 mmol) was added to the solution. The reaction was held at
o
0 C for 6 h with stirring. After completion of the reaction (TLC), the reaction mixture was
poured into ice water. The precipitated solid was collected and filtered. The product was washed
with water, dried under vacuum. The crude material was recrystallized to afford the compound
o
1
6
3ba. Yield: 89%. White solid, m.p.: 260–262 C. H NMR (400 MHz, DMSOꢀd ): δ (ppm) 11.26
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(
bs, 1H), 8.42 (s, 1H), 7.82 (s, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.43–7.52 (m, 2H), 7.34 (s, 1H), 7.01
13
(s, 1H), 2.33 (s, 3H), 2.13 (s, 3H). C NMR (101 MHz, DMSOꢀd
6
): δ (ppm) 167.0, 165.7, 154.7,
1
50.4, 140.8, 140.6, 138.6, 133.5, 133.3, 131.8, 130.9, 129.8, 127.6, 123.6, 117.4, 115.2, 108.3,
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+
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20.9, 17.7. HRMS: m/z calcd. for C19H15Cl N OS [M + H] 417.0338; found 417.0342. HPLC
purity: 97.2%, retention time = 1.36 min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((3ꢀchlorophenyl)amino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
3bb).
The title compound was prepared from compound 6a and 3ꢀchloroaniline following the general
(
o
1
6
procedure of 3ba. Yield: 81%. White solid, m.p.: 248–251 C. H NMR (400 MHz, DMSOꢀd ): δ
(ppm) 11.33 (bs, 1H), 8.41 (s, 1H), 8.02 (s, 1H), 7.37–7.60 (m, 5H), 7.16 (d, J = 7.9 Hz, 1H), 2.13
13
(
s, 3H). C NMR (101 MHz, DMSOꢀd
6
): δ (ppm) 167.4, 166.2, 155.2, 150.9, 141.4, 139.1, 134.0,
134.0, 132.3, 131.4, 131.3, 130.2, 128.1, 123.4, 118.4, 117.5, 108.9, 18.2. HRMS: m/z calcd. for
+
C
H13Cl N
18 2 4
OS [M + H] 403.0181; found 403.0169. HPLC purity: 99.6%, retention time =
2
.34min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ(mꢀtolylamino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone (3bc).
The title compound was prepared from compound 6a and mꢀtoluidine following the general
o
1
6
procedure of 3ba. Yield: 78%. White solid, m.p.: 203–206 C. H NMR (400 MHz, DMSOꢀd ): δ
(ppm) 11.09 (bs, 1H), 8.37 (s, 1H), 7.42–7.61(m, 5H), 7.29 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 7.4 Hz,
13
1
H), 2.34 (s, 3H), 2.13 (s, 3H). C NMR (101 MHz, DMSOꢀd ): δ (ppm) 168.0, 166.5, 155.2,
6
150.8, 140.0, 139.1, 134.1, 132.4, 131.4, 130.3, 129.6, 128.1, 124.9, 119.7, 116.5, 108.2, 21.8,
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4
5
5
5
5
5
5
5
5
5
5
6
+
1
8.2. HRMS: m/z calcd. for C H ClN OS [M + H] 383.0727; found 383.0719. HPLC purity:
19
16
4
98.9%, retention time = 2.01 min.
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6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ(oꢀtolylamino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone (3bd).
The title compound was prepared from compound 6a and oꢀtoluidine following the general
o
1
procedure of 3ba. Yield: 75%. White solid, m.p.: 268–270 C. H NMR (400 MHz, DMSOꢀd ): δ
6
(
ppm) 10.53 (bs, 1H), 8.33 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.56 (dd, J = 7.7, 1.2 Hz, 1H),
7
.41–7.51 (m, 2H), 7.26–7.37 (m, 2H), 7.22 (dd, J = 10.7, 4.0 Hz, 1H), 2.30 (s, 3H), 2.12 (s, 3H).
13
6
C NMR (101 MHz, DMSOꢀd ): δ (ppm) 171.3, 166.7, 155.0, 150.7, 139.1, 138.0, 134.1, 132.6,
1
32.3, 131.5, 131.3, 130.2, 128.0, 127.4, 126.9, 125.0, 107.9, 18.2, 18.2. HRMS: m/z calcd. for
+
C H ClN OS [M + H] 383.0727; found 383.0717. HPLC purity: 99.7%, retention time =
1
9
16
4
1
.70min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ(pꢀtolylamino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone (3be).
The title compound was prepared from compound 6a and pꢀtoluidine following the general
o
1
procedure of 3ba. Yield: 88%. White solid, m.p.: 241–243 C. H NMR (400 MHz, DMSOꢀd ): δ
6
(
ppm) 11.10 (s, 1H), 8.39 (s, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.57 (dd, J = 7.8, 1.7 Hz, 1H), 7.50 (t,
13
J = 7.7 Hz, 1H), 7.44–7.47 (m, 1H), 7.23 (d, J = 8.4 Hz, 2H), 2.31 (s, 3H), 2.14 (s, 3H). C NMR
101 MHz, DMSOꢀd ): δ (ppm) 168.0, 166.5, 155.1, 150.7, 139.1, 137.5, 134.0, 133.2, 132.3,
(
6
4
131.4, 130.2, 130.1, 128.0, 119.4, 108.0, 20.9, 18.2. HRMS: m/z calcd. for C19H16ClN OS [M +
+
H] 383.0727; found 383.0717. HPLC purity: 96.9%, retention time = 2.16min.
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ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((3,4ꢀdimethylphenyl)amino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀ
one (3bo).
The title compound was prepared from compound 6a and 3,4ꢀdimethylaniline following the
1
1
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7
8
9
0
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2
3
4
5
6
7
8
9
0
o
1
general procedure of 3ba. Yield: 87%. White solid, m.p.: 226–228 C. H NMR (400 MHz,
DMSOꢀd ): δ (ppm) 11.03 (bs, 1H), 8.38 (s, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.43–7.55 (m, 4H),
.17 (d, J = 7.8 Hz, 1H), 2.25 (s, 3H), 2.21 (s, 3H), 2.13 (s, 3H). C NMR (101 MHz, DMSOꢀd ):
6
1
3
7
6
δ (ppm) 168.2, 166.6, 155.1, 150.7, 139.1, 137.7, 137.6, 134.1, 132.3, 131.4, 130.6, 130.2, 128.1,
+
120.6, 117.0, 107.9, 20.2, 19.3, 18.2. HRMS: m/z calcd. for C20
H18ClN
4
OS [M + H] 397.0884;
found 397.0876. HPLC purity: 99.6%, retention time = 2.27min.
6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((3,5ꢀdimethylphenyl)amino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀ
one (3bp).
The title compound was prepared from compound 6a and 3,5ꢀdimethylaniline following the
o
1
general procedure of 3ba. Yield: 95%. White solid, m.p.: 152–154 C. H NMR (400 MHz,
DMSOꢀd ): δ (ppm) 11.03 (bs, 1H), 8.37 (s, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.41–7.52 (m, 2H),
6
13
7
1
1
.32 (s, 2H), 6.78 (s, 1H), 2.30 (s, 6H), 2.13 (s, 3H). C NMR (101 MHz, DMSOꢀd ): δ (ppm)
6
68.0, 166.4, 155.1, 150.7, 139.8, 139.1, 138.9, 134.1, 132.3, 131.4, 130.2, 128.0, 125.8, 117.0,
+
08.1, 21.6, 18.2. HRMS: m/z calcd. for C20
H
18ClN
4
OS [M + H] 397.0884; found 397.0872.
HPLC purity: 97.6%, retention time = 2.43 min.
6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((3,4,5ꢀtrimethylphenyl)amino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6
H)ꢀone (3bq).
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6
The title compound was prepared from compound 6a and 3,4,5ꢀtrimethylaniline following the
o
1
general procedure of 3ba. Yield: 82%. White solid, m.p.: 260–262 C. H NMR (400 MHz,
DMSOꢀd ): δ (ppm) 10.97 (bs, 1H), 8.37 (s, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.49 (d, J = 7.4 Hz, 1H),
7.45 (d, J = 7.0 Hz, 1H), 7.31 (s, 2H), 2.26 (s, 6H), 2.13 (s, 3H), 2.10 (s, 3H). C NMR (101 MHz,
6
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
DMSOꢀd
6
): δ (ppm) 168.4, 166.6, 155.1, 150.7, 139.1, 137.4, 136.9, 134.1, 132.3, 131.4, 131.0,
+
1
30.2, 128.0, 118.8, 107.8, 21.0, 18.2, 15.2. HRMS: m/z calcd. for C H ClN OS [M + H]
21 20 4
4
11.1040; found 411.1035. HPLC purity: 99.3%, retention time = 2.71 min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((3,4ꢀdifluorophenyl)amino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀo
ne (3br).
The title compound was prepared from compound 6a and 3,4ꢀdifluoroaniline following the
o
1
general procedure of 3ba. Yield: 67%. White solid, m.p.: 255–258 C. H NMR (400 MHz,
DMSOꢀd
6
): δ (ppm) 11.35 (s, 1H), 8.43 (s, 1H), 8.00 (ddd, J = 13.0, 7.2, 2.5 Hz, 1H), 7.58 (d, J =
13
7
.6 Hz, 1H), 7.45–7.54 (m, 3H), 7.42 (d, J = 8.9 Hz, 1H), 2.15 (s, 3H). C NMR (101 MHz,
DMSOꢀd
6
): δ (ppm) 167.4, 166.1, 155.1, 150.9, 150.9, 150.8, 147.0, 144.6, 144.5, 139.1, 137.0,
1
33.9, 132.3, 131.4, 130.2, 128.0, 118.5, 118.3, 115.4, 108.8, 108.3, 108.0, 18.2. HRMS: m/z
+
calcd. for C18H12ClF N
2 4
OS [M + H] 405.0382; found 405.0376. HPLC purity: 99.7%, retention
time = 2.00min.
6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((3ꢀfluoroꢀ4ꢀmethylphenyl)amino)thiazolo[4,5ꢀd]pyrimidinꢀ7(
6
H)ꢀone (3bs).
The title compound was prepared from compound 6a and 3ꢀfluoroꢀ4ꢀmethylaniline following
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the general procedure of 3ba. Yield: 91%. White solid, m.p.: 222–225 C. H NMR (400 MHz,
DMSOꢀd
6
): δ (ppm) 11.27 (bs, 1H), 8.41 (s, 1H), 7.74 (d, J = 12.6 Hz, 1H), 7.57 (dd, J = 7.7, 1.0
13
Hz, 1H), 7.43–7.53 (m, 2H), 7.28–7.34 (m, 2H), 2.22 (s, 3H), 2.13 (s, 3H). C NMR (101 MHz,
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4
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5
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5
5
5
6
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DMSOꢀd
6
): δ (ppm) 167.5, 166.3, 161.0 (d, J = 241 Hz), 155.2, 150.9, 139.3 (d, J = 11 Hz), 139.1,
1
1
9
34.0, 132.4, 132.3, 131.4, 130.2, 128.1, 119.3, 119.1, 114.8, 108.5, 106.0 (d, J = 27 Hz), 18.2,
+
4.1. HRMS: m/z calcd. for C H ClFN OS [M + H] 401.0633; found 401.0622. HPLC purity:
19 15
4
8.4%, retention time = 2.25min.
1
.3
Nꢀ(6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ7ꢀoxoꢀ6,7ꢀdihydrothiazolo[4,5ꢀd]pyrimidinꢀ2ꢀyl)ꢀ4ꢀ
methylbenzamide (3ca).
Compound 6a (370 mg, 1.0 mmol) and pꢀtolylamide (270 mg, 2.0 mmol) were dissolved in
anhydrous THF (3 mL). Then, NaH (48 mg, 2.0 mmol) was added to the solution, the reaction was
held at reflux for 0.5 h with stirring, then cooled and quenched with ethanol. The reaction mixture
was concentrated under reduce pressure. The crude material was purified by silica gel
chromatography (CH
2
Cl
2
/MeOH = 50:1) to afford the compound 3ca. Yield: 47%. White solid,
o
1
m.p.: 272–274 C. H NMR (400 MHz, DMSOꢀd ): δ (ppm) 13.41 (bs, 1H), 8.49 (s, 1H), 8.08 (d,
6
13
J = 8.0 Hz, 2H), 7.43–7.61 (m, 3H), 7.37 (d, J = 8.0 Hz, 2H), 2.38 (s, 3H), 2.15 (s, 3H). C NMR
(
101 MHz, DMSOꢀd ): δ (ppm) 166.6, 165.0, 163.8, 156.1, 151.0, 144.3, 139.1, 134.0, 132.3,
6
1
4 2
31.5, 130.3, 129.8, 129.0, 128.7, 128.1, 111.7, 21.6, 18.2. HRMS: m/z calcd. for C20H16ClN O S
+
[M + H] 411.0677; found 411.0680. HPLC purity: 98.4%, retention time = 1.85min.
Nꢀ(6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ7ꢀoxoꢀ6,7ꢀdihydrothiazolo[4,5ꢀd]pyrimidinꢀ2ꢀyl)
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ꢀ
4ꢀmethylbenzenesulfonamide (3cb).
The title compound was prepared from compound 6a and 4ꢀmethylbenzenesulfonamide
o
1
following the general procedure of 3ca. Yield: 84%. White solid, m.p.: 263–264 C. H NMR (400
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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3
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5
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9
0
MHz, DMSOꢀd
6
): δ (ppm) 8.38 (s, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.53–7.57 (m, 1H), 7.48 (t, J =
13
7
.8 Hz, 1H), 7.43–7.44 (m, 1H), 7.35 (d, J = 8.0 Hz, 2H), 2.36 (s, 3H), 2.10 (s, 3H). C NMR
(101 MHz, DMSOꢀd ): δ (ppm) 167.7, 153.7, 151.6, 142.8, 138.7, 138.6, 133.1, 131.7, 131.1,
6
+
1
29.8, 129.5, 127.6, 126.1, 104.5, 21.0, 17.6. HRMS: m/z calcd. for C19
4 3 2
H15ClN O S [M + H]
4
47.0346; found 447.0350. HPLC purity: 97.4%, retention time = 0.97 min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ(methyl(pꢀtolyl)amino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
(3cc).
The title compound was prepared from compound 6a and N,4ꢀdimethylaniline following the
o
1
general procedure of 3af. Yield: 89%. Yellow solid, m.p.: >300 C. H NMR (400 MHz, CDCl
3
): δ
(ppm) 7.87 (s, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.33 (t, J = 6.0 Hz, 1H), 7.30 (m, 4H), 7.27 (d, J =
1
3
7
1
4
3
.2 Hz, 1H), 3.67 (s, 3H), 2.41 (s, 3H), 2.18 (s, 3H). C NMR (101 MHz, CDCl ): δ (ppm) 174.0,
66.5, 155.2, 149.0, 141.9, 138.8, 138.6, 133.8, 132.9, 131.0, 130.6, 129.4, 127.9, 125.8, 109.5,
+
0.9, 21.2, 18.3. HRMS: m/z calcd. for C20
H
18ClN
4
OS [M + H] 397.0884; found 397.0885.
HPLC purity: 95.7%, retention time = 2.38min.
6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((4ꢀmethylbenzyl)amino)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
3cd).
The title compound was prepared from compound 6a and pꢀtolylmethanamine following the
(
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general procedure of 3af. Yield: 83%. Yellow solid, m.p.: >300 C. H NMR (400 MHz,
DMSOꢀd ): δ (ppm) 9.38 (s, 1H), 8.27 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H),
.42 (d, J = 7.2 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 7.9 Hz, 2H), 4.57 (s, 2H), 2.29 (s,
6
7
1
1
1
1
1
1
1
1
1
1
2
2
2
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2
2
2
2
2
2
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3
3
3
3
3
4
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4
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4
4
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
3H), 2.10 (s, 3H). C NMR (101 MHz, DMSOꢀd
6
): δ (ppm) 166.4, 154.5, 150.0, 138.6, 136.5,
1
34.8, 133.7, 131.9, 130.8, 129.7, 129.1, 128.8, 127.6, 127.2, 106.7, 20.7, 17.7. HRMS: m/z calcd.
+
for C H ClN OS [M + H] 397.0884; found 397.0881. HPLC purity: 97.9%, retention time =
20 18
4
1
.81 min.
1
.4 6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ(pꢀtolylthio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone (3da).
Compound 6a (370 mg, 2.0 mmol) was dissolved in THF (10 mL). Then,
4ꢀmethylbenzenethiol (250 mg, 3.0 mmol) and Et N (2 mL) were added to the solution. The
3
o
reaction was held at 65 C for 6 h with stirring. After completion of the reaction (TLC), the
reaction mixture was concentrated under reduce pressure. The crude material was purified by
silica gel chromatography (petroleum ether/EtOAc = 10:1) to afford the compound 3da. Yield:
o
1
6
4%. White solid, m.p.: 266–268 C. H NMR (400 MHz, CDCl
3
): δ (ppm) 7.92 (s, 1H), 7.62 (d, J
=
8.1 Hz, 2H), 7.39–7.44 (m, 1H), 7.32–7.38 (m, 3H), 7.28 (d, J = 7.0 Hz, 1H), 2.45 (s, 3H), 2.16 (s,
1
3
3
3
H). C NMR (101 MHz, CDCl ): δ (ppm) 181.6, 166.9, 155.1, 149.4, 142.4, 138.5, 135.9, 133.4,
1
3 2
32.8, 131.5, 130.9, 129.7, 128.1, 124.8, 117.9, 21.6, 18.3. HRMS: m/z calcd. for C19H15ClN OS [M
+
+
H] 400.0339; found 400.0341. HPLC purity: 96.9%, retention time = 3.93 min.
1
.5
6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ(4ꢀmethylꢀphenyl)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
(3db)
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Compound 5 (984 mg, 3 mmol) was refluxed in HCOOH (5mL) for 1 h to afford key
intermediate 6 (839 mg), which was purified via a column chromatography (petroleum ether: ethyl
o
1
3
acetate = 10: 1), in a yield of 83 % as a white solid. MP: 170–171 C. H NMR (400 MHz, CDCl ):
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
δ (ppm) 7.94 (s, 1H), 7.27–7.46 (m, 3H), 3.41 (q, J = 7.4 Hz, 2H), 2.18 (s, 3H), 1.51 (t, J = 7.4 Hz,
13
3
H). C NMR (101 MHz, CDCl
3
): δ (ppm) 176.6, 166.0, 155.1, 149.2, 138.4, 133.3, 132.7, 130.8,
129.6, 128.0, 117.2, 28.3, 18.2, 14.3.
Then, 6 (169 mg, 0.5 mmol) and 4ꢀmethylphenyl boric acid (135 mg, 1.5equiv) was stirred in a
sealed tube with THF (5mL) in presence of Pd(Ph
.5equiv) for 16h . After the reaction, the crude product was purified by column chromatography
petroleum ether: ethyl acetate = 30: 1) to provide the product 3db (126 mg) as a white solid in a
3 4
P) (29 mg, 5 mol%) and CuTC (285 mg,
1
(
o
1
yield of 69%. MP: 287–288 C. H NMR (400 MHz, CDCl ): δ (ppm) 8.04 (d, J = 8.2 Hz, 2H),
3
8
.01 (s, 1H), 7.45 (d, J = 7.9, 1.0 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.35 – 7.30 (m, 3H), 2.44 (s,
13
3H), 2.23 (s, 3H). C NMR (101 MHz, CDCl
3
): δ (ppm) 175.4, 166.9, 155.9, 149.3, 143.1, 138.5,
133.4, 132.8, 130.9, 129.9, 129.9, 129.6, 128.1, 127.4, 117.9, 21.6, 18.3. HRMS: m/z calcd. for
+
C
19 3
H15ClN OS [M + H] 368.06183; found: 368.06188. HPLC: purity: ˃99.9%, retention time =
4
.33min.
1
.6 6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ(benzylthio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone (3ea).
Compound 5a (1.10 g, 5 mmol) was dissolved in acetone (10 mL). Then, the solution of
dipotassium cyandithiomidocarbonate (970 mg, 5 mmol, 10ml) and 2 mol/L NaOH aqueous
solution (2.5 mL) were added to the solution. The reaction mixture was stirred for 2 h at room
o
temperature, then heated to 80 C for 0.5 h. Afterward, the reaction mixture was cooled to room
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temperature. To the mixture Benzyl bromide (855 mg, 5 mmol) was added drop wise with
continuous stirring. After completion of the reaction (TLC), the reaction mixture was poured into
ice water. The precipitated solid was collected and filtered. The product was washed with water.
The crude product so obtained was dried under vacuum to obtain the intermediate compound 10a.
Compound 10a (1.20 g, 3 mmol) was dissolved in HCOOH (2 mL). The reaction was held at
reflux for 1 h with stirring, then cooled and quenched with water. The reaction mixture was
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0
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extracted with ethyl ether (20 mL×5), dried with Na
2 4
SO , and concentrated under reduce pressure.
The crude material was purified by silica gel chromatography (petroleum ether/EtOAc = 10:1) to
o
1
afford the compound 3ea. Yield: 75%. Yellow solid, m.p.: 134–138 C. H NMR (400 MHz,
DMSOꢀd ): δ (ppm) 8.52 (s, 1H), 7.56–7.60 (m, 1H), 7.53–7.56 (m, 2H), 7.48–7.53 (m, 1H),
6
7.44–7.48 (m, 1H), 7.35–7.41 (m, 2H), 7.31 (ddd, J = 7.3, 3.7, 1.2 Hz, 1H), 4.70 (s, 2H), 2.13 (s,
13
3
H). C NMR (101 MHz, DMSOꢀd
6
): δ (ppm) 175.2, 165.7, 154.5, 150.8, 138.6, 135.7, 133.1,
131.7, 131.1, 129.8, 129.1, 128.6, 127.8, 127.6, 116.3, 37.0, 17.6. HRMS: m/z calcd. for
+
19 3
C H15ClN OS
2
[M + H] 400.0339; found 400.0340. HPLC purity: 99.5%, retention time = 3.15
min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((2ꢀfluorobenzyl)thio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
3eb).
The title compound was prepared from compound 5a and 2ꢀfluorobenzyl bromide (compound
(
o
1
10b) following the general procedure of 3ea. Yield: 44%. Yellow solid, m.p.: 184–186 C. H
NMR (400 MHz, DMSOꢀd ): δ (ppm) 8.53 (s, 1H), 7.64 (d, J = 6 Hz, 1H), 7.58 (d, J = 8 Hz, 1H),
6
7.50 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 8 Hz, 1H), 7.33–7.42 (m, 1H), 7.18–7.27 (m, 2H), 4.73 (s,
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3
2
H), 2.12 (s, 3H). C NMR (101 MHz, DMSOꢀd ): δ (ppm) 174.6, 165.7, 160.5 (d, J = 247 Hz),
6
154.6, 150.9, 138.6, 133.1, 131.7, 131.5 (d, J = 3 Hz), 131.2, 130.3 (d, J = 8 Hz), 129.9, 127.6,
1
24.7 (d, J = 4 Hz), 122.8 (d, J = 14 Hz), 116.5, 115.6 (d, J = 21 Hz), 30.8, 17.7. HRMS: m/z
0
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2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
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2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
calcd. for C19
H
14ClFN
3
OS
2
[M + H] 418.0245; found 418.0248. HPLC purity: 95.2%, retention
time = 3.31min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((3ꢀfluorobenzyl)thio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
3ec).
The title compound was prepared from compound 5a and 3ꢀfluorobenzyl bromide (compound
(
o
1
1
0c) following the general procedure of 3ea. Yield: 32%. Yellow solid, m.p.: 177–179 C. H
NMR (400 MHz, DMSOꢀd ): δ (ppm) 8.53 (s, 1H), 7.58 (d, J = 8 Hz, 1H), 7.48 (t, J = 8 Hz, 1H),
6
13
7
.44 (d, J = 6.8 Hz, 1H), 7.38–7.42 (m, 3H), 7.14 (m, 1H), 4.71 (s, 2H), 2.12 (s, 3H). C NMR
(101 MHz, DMSOꢀd
6
): δ (ppm) 174.9, 165.7, 160.8, 154.6, 150.9, 138. 9 (d, J = 8 Hz), 138.6,
1
33.1, 131.7, 131.1, 130.6 (d, J = 8 Hz), 129.9, 127.6, 125.3 (d, J = 3 Hz), 116.4, 115.9 (d, J = 22
+
Hz), 114.7 (d, J = 21 Hz), 36.3, 17.7. HRMS: m/z calcd. for C19
3 2
H14ClFN OS [M + H] 418.0245;
found 418.0246. HPLC purity: 97.8%, retention time = 3.12min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((4ꢀfluorobenzyl)thio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
3ed).
The title compound was prepared from compound 5a and 4ꢀfluorobenzyl bromide (compound
(
o
1
1
0d) following the general procedure of 3ea. Yield: 25%. Yellow solid, m.p.: 193–195 C. H
NMR (400 MHz, CDCl
3
): δ (ppm) 7.96 (s, 1H), 7.46 (dd, J = 8.7, 5.4 Hz, 3H), 7.38 (t, J = 7.8 Hz,
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H), 7.31 (d, J = 7.5 Hz, 1H), 7.04 (t, J = 8.6 Hz, 2H), 4.64 (s, 2H), 2.20 (s, 3H). C NMR (101
MHz, CDCl ): δ (ppm) 175.6, 166.0, 162.5 (d, J = 247.2 Hz), 155.2, 149.5, 138.6, 133.4, 132.8,
31.4 (d, J = 3.2 Hz), 131.1, 131.0 (d, J = 8.3 Hz), 129.8, 128.2, 115.9 (d, J = 21.7 Hz), 37.3, 18.4.
3
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0
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0
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0
+
3 2
HRMS: m/z calcd. for C19H14ClFN OS [M + H] 418.0245; found 418.0261. HPLC purity:
95.4%, retention time = 3.11min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((4ꢀmethylbenzyl)thio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
(3ee).
The title compound was prepared from compound 5a and 4ꢀmethylbenzyl bromide (compound
o
1
1
0e) following the general procedure of 3ea. Yield: 63%. Yellow solid, m.p.: 238–240 C. H
NMR (400 MHz, DMSOꢀd ): δ (ppm) 8.52 (s, 1H), 7.58 (d, J = 8 Hz, 1H), 7.51 (t, J = 8 Hz, 1H),
6
7
.46 (d, J = 6.8 Hz, 1H), 7.42 (d, J = 8 Hz, 2H), 7.18 (d, J = 7.8 Hz, 2H), 4.65 (s, 2H), 2.28 (s, 3H),
1
3
2.12 (s, 3H). C NMR (101 MHz, DMSOꢀd
6
): δ (ppm) 175.4, 165.8, 154.6, 150.8, 138.6, 137.2,
1
33.1, 132.6, 131.7, 131.2, 129.9, 129.3, 129.1, 127.6, 116.3, 36.9, 20.7, 17.7. HRMS: m/z calcd.
+
3 2
for C20H17ClN OS [M + H] 414.0496; found 414.0500. HPLC purity: 99.3%, retention time =
4
.20 min.
6
ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((4ꢀmethoxybenzyl)thio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
(3ef).
The title compound was prepared from compound 5a and 4ꢀmethoxybenzyl bromide
(
compound 10f) following the general procedure of 3ea. Yield: 27%. Yellow solid, m.p.: 296–298
o
1
C. H NMR (400 MHz, DMSOꢀd
6
): δ (ppm) 8.52 (s, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.42–7.55 (m,
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4
H), 6.94 (d, J = 8.6 Hz, 2H), 4.64 (s, 2H), 3.74 (s, 3H), 2.12 (s, 3H). C NMR (101 MHz,
DMSOꢀd
6
): δ (ppm) 175.4, 165.8, 158.9, 154.6, 150.8, 138.6, 133.1, 131.7, 131.1, 130.5, 129.9,
+
127.6, 127.3, 114.1, 104.5, 55.1, 36.7, 17.7. HRMS: m/z calcd. for C20
3 2 2
H17ClN O S [M + H]
0
1
2
3
4
5
6
7
8
9
0
1
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0
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0
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0
430.0445; found 430.0451. HPLC purity: 98.5%, retention time = 3.00 min.
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ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((4ꢀchlorobenzyl)thio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
3eg).
The title compound was prepared from compound 5a and 4ꢀchlorobenzyl bromide (compound
(
o
1
1
0g) following the general procedure of 3ea. Yield: 84%. White solid, m.p.: 284–286 C. H NMR
400 MHz, CDCl ): δ (ppm) 7.96 (s, 1H), 7.39–7.47 (m, 3H), 7.34–7.39 (m, 1H), 7.28–7.34 (m,
3H), 4.62 (s, 2H), 2.20 (s, 3H). C NMR (101 MHz, CDCl ): δ (ppm) 175.5, 165.9, 155.3, 149.5,
(
3
13
3
1
38.6, 134.3, 134.0, 133.4, 132.9, 131.1, 130.7, 129.8, 129.1, 128.3, 117.8, 37.2, 18.4. HRMS:
+
m/z calcd. for C19
H
2
14Cl N
3
OS
2
[M + H] 433.9949; found 433.9952. HPLC purity: 96.1%,
retention time = 4.27 min.
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ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((4ꢀbromobenzyl)thio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
3eh).
The title compound was prepared from compound 5a and 4ꢀbromobenzyl bromide (compound
(
o
1
1
0h) following the general procedure of 3ea. Yield: 68%. White solid, m.p.: 294–296 C. H NMR
(400 MHz, CDCl
3
): δ (ppm) 7.96 (s, 1H), 7.46 (t, J = 10.4 Hz, 3H), 7.38 (t, J = 9.2 Hz, 3H), 7.31
13
(
d, J = 7.5 Hz, 1H), 4.60 (s, 2H), 2.20 (s, 3H). C NMR (101 MHz, CDCl ): δ (ppm) 175.4, 165.9,
3
155.3, 149.5, 138.6, 134.8, 133.4, 132.9, 132.1, 131.1, 131.1, 129.8, 128.3, 122.2, 117.8, 37.3,
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8.4. HRMS: m/z calcd. for C H BrClN OS [M + H] 477.9444; found 477.9459. HPLC purity:
19
14
3
2
99.3%, retention time = 4.61min.
1
1
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6ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ((2,4ꢀdifluorobenzyl)thio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone
(3ei).
The title compound was prepared from compound 5a and 2,4ꢀdifluorobenzyl bromide
(
compound 10i) following the general procedure of 3ea. Yield: 74%. Yellow solid, m.p.: 249–251
o
1
C. H NMR (400 MHz, CDCl
3
): δ (ppm) 7.97 (s, 1H), 7.58 (td, J = 8.7, 6.4 Hz, 1H), 7.42–7.46
(
m, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.29–7.33 (m, 1H), 7.27 (s, 1H), 6.86 (dddd, J = 11.1, 9.1, 4.6,
13
2
3
.8 Hz, 2H), 4.67 (s, 2H), 2.20 (s, 3H). C NMR (101 MHz, CDCl ): δ (ppm) 175.3, 165.8, 155.2,
149.5, 138.5, 133.4, 132.8, 132.4, 132.4, 132.3, 131.0, 129.8, 128.2, 119.3, 117.8, 111.8, 111.7,
11.6, 111.5, 104.5, 104.3, 104.0, 30.7, 30.6, 18.3. HRMS: m/z calcd. for C19 OS [M +
1
H13ClF
N
2 3
2
+
H] 436.0151; found 436.0161. HPLC purity: 97.7%, retention time = 3.49min.
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ꢀ(2ꢀChloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ(phenethylthio)thiazolo[4,5ꢀd]pyrimidinꢀ7(6H)ꢀone (3ej).
The title compound was prepared from compound 5a and (2ꢀbromoethyl)benzene (compound
o
1
1
0j) following the general procedure of 3ea. Yield: 91%. White solid, m.p.: 122–124 C. H NMR
(400 MHz, CDCl
3
): δ (ppm) 7.96 (s, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H),
1
3
7
.22–7.36 (m, 6H), 3.67 (t, J = 7.6 Hz, 2H), 3.17 (t, J = 7.6 Hz, 2H), 2.21 (s, 3H). C NMR (101
MHz, CDCl
3
): δ (ppm) 176.5, 166.1, 155.3, 149.4, 139.2, 138.6, 133.5, 132.9, 131.1, 129.8, 128.8,
+
1
28.8, 128.2, 126.95, 117.5, 35.4, 35.3, 18.4. HRMS: m/z calcd. for C H ClN OS [M + H]
20 17 3 2
414.0496; found 414.0505. HPLC purity: 99.1%, retention time = 3.85min.
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