Chemical conversion of nicotinamide into type I positive allosteric modulator of α7 nAChRs

Article abstract of DOI:10.1016/j.bmcl.2019.05.046

Structural modifications of nicotinamide, a form of vitamin B3, gave rise to a series of compounds (8aa–8ce) that exhibit activities as type I positive allosteric modulators (PAMs) of human α7 nAChR expressed in Xenopus oocytes in two-electrode voltage clamp assay. The compound 8ai was a potent and efficacious PAM with an EC₅₀ = 3.34 ± 1.13 μM and the maximum activation effect of α7 current over 1474 ± 246% in the presence of acetylcholine (100 μM). It is highly specific to α7 nAChR over other subtypes of nAChR and 5-HT_3A receptors. The structure–activity relationship analysis identified a key skeleton of nicotinamide nucleus critical for biological activity. Taken together, the 8ai as a type I PAM of α7 nAChR may be beneficial for improvement of cognitive deficit.

Full text of DOI:10.1016/j.bmcl.2019.05.046

Accepted Manuscript
Chemical Conversion of Nicotinamide into Type I Positive Allosteric Modulator
of α7 nAChRs
Xin Li, Wenjun Xie, Xintong Wang, Zongze Huang, Xiling Bian, KeWei Wang,
Qi Sun
PII:
S0960-894X(19)30343-9
https://doi.org/10.1016/j.bmcl.2019.05.046
BMCL 26466
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
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8 May 2019
22 May 2019
Please cite this article as: Li, X., Xie, W., Wang, X., Huang, Z., Bian, X., Wang, K., Sun, Q., Chemical Conversion
of Nicotinamide into Type I Positive Allosteric Modulator of α7 nAChRs, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.046
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Chemical Conversion of Nicotinamide into Type I Positive Allosteric
Modulator of α7 nAChRs
Xin Lia, †, Wenjun Xiea, b, †, Xintong Wangb, Zongze Huanga, Xiling Bianb, KeWei Wangb, c* and Qi
Suna*
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China;
b
Department of Molecular and cellular Pharmacology, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China;
c
Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao 266021,
China.
Abstract: Structural modifications of nicotinamide, a form of vitamin B3, gave rise to
a series of compounds (8aa−8ce) that exhibit activities as type I positive allosteric
modulators (PAMs) of human α7 nAChR expressed in Xenopus ooctyes in
two-electrode voltage clamp assay. The compound 8ai was a potent and efficacious
PAM with an EC50 = 3.34 ± 1.13 μM and the maximum activation effect of α7 current
over 1474 ± 246% in the presence of acetylcholine (100 μM). It is highly specific to
α7 nAChR over other subtypes of nAChR and 5-HT3A receptors. The
structure−activity relationship analysis identified a key skeleton of nicotinamide
nucleus critical for biological activity. Taken together, the 8ai as a type I PAM of α7
nAChR may be beneficial for improvement of cognitive deficit.
Keywords: Nicotinamide; α7 nAChR; Positive allosteric modulators; Cognitive
impairment; Alzheimer’s disease
The α7-subtype of nicotinic acetylcholine receptors (α7 nAChR) has been well
recognized as a potential target in the central nervous system (CNS) for treatment of
neuropsychiatric disorders, such as Alzheimer’s disease (AD).1 Several studies have
shown that coincident compromises in cholinergic activity and cognition in early AD
may be mediated by the α7 nAChR, suggesting that the activation of α7 nAChR may
represent an effective treatment strategy for the cognitive impairments associated with
early AD.2 The α7 homomeric receptor demonstrates a wide-spread localization in the
brain and is characterized by a high calcium ion permeability and a fast
1

desensitization rate.3 Clinical studies have shown that targeting α7 nAChR with
selective agonists or partial agonists can effectively improve the cognitive deficits in
Alzheimer’s disease.4 Positive allosteric modulators (PAMs) have been demonstrated
numerous advantages over agonists for their maintenance of the normal temporal and
spatial patterns of neurotransmission and high selectivity. 5 They can also improve the
safety compared with orthosteric drugs.6 For instance, MDL-800 was reported to be a
well-characterized allosteric activator with good selectivity and high activity of
SIRT6.7 Based on their channel kinetics and desensitization characteristics, the PAMs
of nicotinic receptors are classified into types I and II.8 Both types of PAMs have
been found with in vivo efficacy in animal models of cognition deficit,9 whereas type I
PAMs can maintain the rapid channel kinetics, which may be more beneficial than
type II PAMs at minimizing potential Ca2+-induced cytotoxicity.10 α7 nAChR and
other subtypes including α4β2 and α3β4 of nAChR are also expressed in the cortex,
hippocampus, and cerebellum,11 whose functions can be activated by acetylcholine or
enhanced by α7 nAChR PAMs. Therefore, the design and synthesis of novel selective
α7 nAChR PAMs with high potency and efficacy are expected to achieve desired
therapeutic indexes with little side effects.
The chemical structures of representative type I PAMs includes acrylamide 1
(AVL-3288, CCMI, Figure 1),12 ureas
2
(NS-1738, Figure 1)13 and
(2-amino-5-keto)thiazole compound 3 (LY-2087101, Figure 1).14 Among these type I
PAMs, only AVL-3288 is currently in the clinical phase I development stage.
Therefore, it is necessary to discover and develop more potent type I PAMs with high
selectivity.
S
Cl
O
F3C
S
Cl
N
HN
O
NH
HN
H
N
Cl
NH
OH
2, NS-1738
O
O
N
Cl
F
1, AVL-3288 (CCMI)
3, LY-2087101
Figure 1. Representative type I α7 nAChR PAMs.
Nicotinamide is amide form of vitamin B3, which has recently been shown to be
2

effective in restoring cognition and memory. Nicotinamide can prevent necrosis and
apoptosis in the brains of MPTP-treated mice and promote learning and memory.15
Nicotinamide improves sevoflurane-induced cognitive impairment and has an
anti-inflammatory and anti-apoptotic effect against sevoflrane-induced damages.16 It
has also been shown that nicotinamide can forestall the pathology and cognitive
decline in mice.17 Therefore, rational use and modification of nicotinamide structure
may lead to discovering novel α7 nAChR PAMs compounds that can better improve
cognitive impairment.
We have previously synthesized a series of thiazolo[4,5-d]pyrimidin-7(6H)-ones
(Figure 2) that as type I PAMs exhibit good activity on α7 nAChR expressed in
Xenopus oocytes in two-electrode voltage clamp assay.18 Among them, compound 4
shows as a potent PAM with the maximum activation effect of α7 current over 1622 ±
106% in the presence of acetylcholine (100 μM) and an EC50 = 8.45 ± 0.078 μM.
Compound 5 exhibits the type I PAM activity on α7 nAChR with an EC50 of 1.26 ±
0.18 μM (n = 6) and enhancement rate of 1633 ± 87% (10 μM) in the presence of 100
μM ACh. A series of 3H-quinazolin-4-one derivatives 6 were designed and
synthesized on the basis of compound 4 and 5 (Figure 2). However, they show a weak
negative activity (6a: enhancement rate of 76% at 10 μM; 6b: 65%) rather than
activating effect. 19
R
O
O
N
R
S
N
N
N
X
Cl
Cl
N
4: R = 4-Me, X = NH
6a: R = 4-F
enhance rate at 10 μM = 1622 ± 106 %,
EC50 = 8.45 ± 0.078 μM
5: R = 4-H, X= -CH2S-
enhance rate at 10 μM = 1633 ± 87%,
EC50 = 1.26 ± 0.18 μM
enhance rate at 10 μM = 76 ± 9 %
6b: R = 4-OMe
enhance rate at 10 μM = 65 ± 1%
Figure 2. The representative structures reported by our research work.
In this work, we envisaged that more efficient compounds could be produced by
introducing the structure of nicotinamide to molecular 6.20 To test this hypothesis, we
started unclosing the pyrimidinone ring of 6 to obtain the compound 7 with a weak
3

positive activity (136%, Figure 3). Then, further modification of 7 was performed
with nicotinamide instead of benzamide to provide compound 8aa that showed a
5-fold improvement of activity (604%, Figure 3). Herein, we report the synthesis of a
new series of nicotinamides 8ac‒8ce and evaluation of their biological activities as
novel PAMs of α7 nAChRs.
Figure 3. The optimization of α7 nAChR modulators
The synthesis route of benzamide derivative 7 is outlined in Scheme 1. The key
intermediate 10 was obtained via one pot reaction of 2-amino-5-iodo-benzoic acid 9a,
2-chloro-6-methyl-phenylamine and trimethoxymethane in toluene in the presence of
catalytic acetic acid at 110 ºC for 24h.21 Then palladium-catalyzed Suzuki coupling
reaction of 10 with 4-fluro-phenylboronic acid provided 11 in the presence of 0.05
equiv Pd(PPh3)4 and 2 equiv K2CO3 with good yield.22 Finally, 11 was dissolved in
absolute ethyl alcohol in the presence of 5N NaOH at 73 ºC for 2 h to produce
benzamide derivative product 7.
Scheme 1.
4

Reagents and conditions: a) AcOH(cat.), Toluene, 110 oC (yield: 65%); b) Pd(PPh3)4, K2CO3,THF,
90 oC (yield: 75%); c) 5N NaOH, C2H5OH, 73 oC (yield: 66%).
The nicotinamide derivatives 8aa‒8ce were synthesized from the commercially
available nicotinic acid derivatives 9b and 9c as outlined in Scheme 2. 9b and 9c
reacted with SOCl2 in toluene at 110 ºC for 2 h gave two nicotinoyl chloride
intermediates. The nicotinoyl chlorides were stirred with corresponding anilines in
THF at 0 ºC for 0.5 h, then reflux at 90 ºC for another 4 h produced 12a‒12d, which
were treated with corresponding boronic acids in the presence of 0.05 equiv Pd(PPh3)4
and 2 equiv K2CO3 in THF at 90 ºC to afford target nicotinamide derivatives 8aa‒8ce
with 18‒68 % yields.
O
O
R2
O
R2
R3
Br
Br
N
H
OH
a)
b)
N
H
R1
N
R1
N
R1
N
9b: R1 = -NH2
9c: R1 = -H
12a: R1 = -NH2; R2 = 2-Cl-6-Me
12b: R1 = -NH2; R2 = 4-H
12c: R1 = -NH2; R2 = 2-Me
12d: R1 = -H; R2 = 2-Cl-6-Me
8aa-8aw: R1 = -NH2; R2 = 2-Cl-6-Me;R3 = R-Ph
8ba-8bc: R1 = -NH2; R2 = 2-Cl-6-Me; R3 = Het-Ar
8ca: R1 = -NH2; R2 = 4-H; R3 = 4-F-3-Me-Ph
8cb: R1 = -NH2; R2 = 2-Me; R3 = 4-F-3-Me-Ph
8cc-8ce: R1 = -H; R2 = 2-Cl-6-Me; R3 = R-Ph
Scheme 2. Reagents and conditions: a) SOCl2, Toluene, 110 oC; Corresponding anilines R2NH2,
THF, 0 oC‒90 oC(38 %‒43 %); b) R3B(OH)2, K2CO3, Pd(PPh3)4, THF, 90 oC (18 %‒68 %).
The target compounds were tested for the in vitro activities in Xenopus laevis
oocytes expressing human α7 nAChR with method described in our previous work.23
Those compounds were inactive in eliciting α7 current in the absence of direct
agonists, then adding acetylcholine (ACh, 100 μM) to 10 μM test compounds resulted
in the activation of α7 currents, suggesting that the compounds were positive
allosteric modulators of α7 nAChR. The maximum modulation (at 10 μM) and EC50
(max effect > 400%) were determined and listed in Tables 1–3.
Table 1. In vitro activities of compounds 7 and 8aa−8aw for enhancement of human
α7 nAChRa
O
R
N
H
Cl
X
NH2
5

EC50
Max effect
Compd.
R
X
(μM)
(%, at 10 μM)
4-F
4-F
CH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
ND
4.57 ± 1.43
ND
136 ± 36
604 ± 110
86 ± 3
7
8aa
8ab
8ac
8ad
8ae
8af
3-F
2-F
ND
119 ± 7
4-Cl
ND
278 ± 47
164 ± 7
4-CF3
ND
4-OCF3
4-NO2
4-F-2-Me
4-F-3-Me
2-Cl-4-F
3-Cl-4-F
4-Cl-3-F
3-F-4-Me
2-F-4-Me
4-OMe
3-OMe
2-OMe
4-SMe
4-OH
ND
153 ± 22
135 ± 2
ND
8ag
8ah
8ai
ND
225 ± 67
1474 ± 246
186 ± 5
3.34 ± 1.13
ND
8aj
5.49 ± 0.45
ND
495 ± 88
229 ± 82
609 ± 123
372 ± 14
531 ± 96
212 ± 8
8ak
8al
3.31 ± 1.22
ND
8am
8an
8ao
8ap
8aq
8ar
8as
8at
8.50 ± 2.59
ND
4.32 ± 0.84
4.96 ± 2.57
ND
1008 ± 85
774 ± 183
257 ± 28
539 ± 105
455 ± 101
109 ± 9
4-Me
3.19 ± 1.77
1.35 ± 0.18
ND
4-Et
8au
8av
8aw
4-NHCOCH3
4-H
ND
273 ± 39
aData were collected from 2–5 individual oocytes expressing 7 current recorded by
two-electrode voltage clamp. EC50 is the compound concentration where the half of maximum
activation effect (max effect) was achieved. ND: not determined.
The substitution of the benzamide with nicotinamide (8aa, Table 1) resulted in an
6

obvious increase in activity. This result indicates that nicotinamide may be beneficial
for allosteric modulation activity of α7 nAChR. Based on the in vitro activities of 7
and 8aa as shown in Table 1, we examined the effects of the substitutes (8ab−8aw) on
the phenyl group in R. Moving the fluorine atom of 8aa to the 3-positon (8ab) and
2-position (8ac) dramatically reduced the activity. The substitutions of R in 8aa with
4-Cl, 4-CF3, 4-OCF3, 4-NO2 (8ad‒8ag) reduced the activity. Based on the maximum
effects acquired from 4-fluoro, we envisaged that the introduction of methyl or
chlorine to the ortho- and meta-positions of phenyl group may improve the activity.
Therefore, compounds 8ah−8ak were designed and synthesized and provided the
maximum effects ranging from 186% to 1474%. Among them, 8ai (maximum effect
of 1474% and EC50 = 3.34 ± 1.13 μM) exhibited the highest activity. Not surprisingly,
when we exchanged the 4-fluoro with the 3- or 2-position substituents, the activity of
two compounds decreased (maximum effect: 1474% for 8ai vs 609% for 8am, 495%
for 8ak vs 229% for 8al) with one compound increased slightly ( maximum effect:
225% for 8ah vs 372% for 8an). Changing the 4-F of 8aa (604%) to 4-OMe (8ao,
531%), 3-OMe (8ap, 212%), 4-OH (8as, 257%), 4-Me (8at, 539%), 4-Et (8au, 455%),
4-NHCOCH3 (8av, 109%), 4-H (8aw, 273%) did not obviously improve activities.
However, Changing the 4-F of 8aa (604%) to 2-OMe (8aq, 1008%), 4-SMe (8ar,
774%) can achieve better activities.
Table 2. In vitro activities of compounds 8ba−8bc for enhancement of human α7
nAChRa
O
R
N
H
Cl
N
NH2
EC50
Max effect
Compd.
R
(μM)
(%, at 10 μM)
Thiophen-3-yl
Furan-3-yl
ND
ND
ND
153 ± 12
111 ± 6
8ba
8bb
8bc
Pyridin-3-yl
138 ± 12
aSame as that in Table 1.
7

The modifications of the 4-fluorophenyl group in 8aa to aromatic heterocycle
substituents (8ba−8bc) led to dramatic reduction of activity or even complete loss of
the effect (Table 2), indicating that the substituted phenyl group was the key
pharmacophore for this novel series of compounds.
Table 3. In vitro activities of compounds 8ca−8ce for enhancement of human α7
nAChRa
O
R1
R2
N
H
N
R3
EC50
Max effect
Compd.
R1
R2
R3
(μM)
(%, at 10 μM)
4-F-3-Me
4-F-3-Me
4-F
4-H
-NH2
-NH2
-H
ND
ND
ND
94 ± 10
156 ± 22
248 ± 31
603 ± 191
150 ± 18
8ca
8cb
8cc
8cd
8ce
2-Me
2-Cl-6-Me
2-Cl-6-Me
2-Cl-6-Me
4-Me
-H
1.97 ± 0.96
ND
4-Cl
-H
aSame as that in Table 1.
Changing the 2-chloro-6-methyl group (8ai, 1474%) in R2 to 4-H (8ca, 94%) or
2-Me (8cb, 156%) remarkably reduced the activity, indicating that the
2-chloro-6-methyl group was important for the activity. When we changed the –NH2
to –H in R3, the activity of two compounds decreased (maximum effect: 604% for 8aa
vs 248% for 8cc, 278% for 8ad vs 150% for 8ce) with one compound increased
(maximum effect: 539% for 8at vs 603% for 8cd).
A
8

B
C
D
10
8
ns
ACh alone
ACh+10 μM 8ai
16
12
8
6
4
4
2
0
0
hR
hR
ChR
AC
AC
h
+
h
receptor
3A
C
i
C
α7 nA
A
a
A
8
M
α3β4 n
α4β2 n
μ
M
M
μ
μ
0
5-HT
0
0
0
1
0
1
1
Figure 4. Selective enhancement of human α7 nAChR current by 8ai over other subtypes of nAChRs
and 5-HT3A expressed in Xenopus oocytes. (A) 7 currents were recorded by TEVC in response to 100
μM ACh alone (first trace) or in the presence of 8ai (second trace). The currents activated by 8ai were
recorded in the absence (third trace) and presence (fourth trace) of 10 nM MLA (right). (B)
Fold-increases of α7 nAChR (evoked by 100 μM ACh, n = 5), α3β4 nAChR (100 μM ACh, n = 5),
α4β2 nAChR (100 μM ACh, n = 5), and 5-HT3A receptors (10 μM 5-HT, n = 5) after incubation with 10
μM 8ai. (C) Superimposition of scaled α7 current traces evoked with 100 μM ACh in the absence
(black trace) and presence (red trace) of 10 μM 8ai. (D) Comparison between the desensitization time
constants (τdesensitization) in the absence and presence of 8ai (ACh, τdesensitization = 6.37 ± 2.56; ACh + 8ai,
τdesensitization = 3.49 ± 0.50; p = 0.1092 > 0.05, n = 5).
Based on the results presented above, it can be concluded that, among the
synthesized analogs, 8ai is the optimal PAM of α7 nAChR. A preincubation of 8ai for
2 min before application of 100 μM ACh remarkably increased the peak current of α7
nAChR (Figure 4A). The examination of the channel desensitization kinetics showed
that 8ai had a little effect the desensitization of α7 nAChR, causing a minor decrease
in τdesensitization(Figure 4C, D).
A
9

B
C
20
15
10
5
ACh alone
ACh+10 μM 8ai
30
25
20
15
10
5
0
0
-5
0.1
1
10
10
100
1000
ACh (μM)
8ai (μM)
Figure 5. Concentration-dependent enhancement of α7 current by 8ai. (A) Representative α7 currents
were evoked by 100 μM ACh in the absence and presence of 8ai at various concentrations. Xenopus
oocytes were preincubated with 8ai for 2 min, followed by the coapplication with 100 μM ACh (10 s).
(B) Relationship between 8ai concentration and activity of α7 nAChR. Xenopus oocytes expressing α7
nAChR were stimulated with 100 μM ACh in the absence and presence of increasing concentrations of
8ai. Peak currents were measured and normalized with the amplitude of currents elicited by 100 μM
ACh alone. The maximum efficacy of 8ai for enhancement of 7 current was 1474 ± 246% with an
EC50 of 3.34 ± 1.13 μM, and Hill coefficient (nH) of 1.67 ± 0.67 (n = 5 for all data points). (C) ACh
concentration−response curves in the absence and presence of 10 μM 8ai. Peak currents were measured
and normalized with the amplitude of currents elicited by 100 μM ACh alone. The curve parameters
were determined as follows: ACh alone: Emax = 339 ± 0%, EC50 = 231.8 ± 2 μM, nH = 2.00 ± 0.00. ACh
+ 8ai: Emax = 2167 ± 577%, EC50 = 257.6 ± 162.2 μM, nH = 0.82 ± 0.12 (n =4 for each data point).
The activation of α7 nAChR by 100 μM ACh alone was first recorded as a control
before different concentrations (0.1~30 µM) of compound 8ai were co-applied with
100 μM ACh after 2 min preincubation. As shown in Figure 5A, 8ai enhanced α7
currents in dose-dependent manner in the presence of 100 μM ACh. Fitting the
concentration-dependent activation of α7 current by 8ai gave rise to an EC50 = 3.34 ±
1.13 μM, Emax = 1474 ± 246%, and Hill coefficient (nH) =1.67 ± 0.37 (Figure 5B). To
further confirm the activity of 8ai, concentration-dependent activation of α7 by ACh
was generated in the absence or presence of 10 μM 8ai. Preincubation of 8ai for 2 min
increased α7 current about 7-fold from 339 ± 0% to 2167 ± 577%, without significant
alteration of the EC50 value of ACh from 231.8 ± 2 μM to 257.6 ± 162.2 μM (Figure
5C).
In conclusion, our chemical modifications of nicotinamide identified a novel
10

compound 8ai that functions as type I PAM of α7 nAChR. The structure−activity
relationship (SAR) analysis revealed that the substitutions of phenyl group at the
5-position and the 2-chloro-6-methylcarbamoyl at the 3-position of the pyridine ring
are important for the activity of these compounds. The compound 8ai may serve as a
lead compound for further development of potential candidate for improving
cognitive function.
Author Information
Corresponding Authors
For K. W. Wang: phone, +86 (532) 82991070; E-mail, wangkw@qdu.edu.cn;
For Q. Sun: phone, +86 (10) 82805549; E-mail, sunqi@bjmu.edu.cn.
Author Contributions
Xin Li† and Wenjun Xie† contributed equally to this work. The manuscript was written through
contributions of all authors.
Conflict of interest
The authors declare that this study was carried out only with public funding. There is
no funding or no agreement with commercial for profit firms.
Acknowledgements
The project is supported by the funds of National Natural Science Foundation of
China (NSFC 21572011, 81573410, 31370741, and 21272009) and from Ministry of
Science and Technology of China (2013CB531302).
Supplementary data
Chemical data of compounds; NMR spectra, HPLC reports and HRMS spectra of the
target compounds. The Supporting Information is available free of charge on the
website at DOI: xxxxxxxxxxxxxxx. (PDF)
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Graphic Abstract
Selective enhancement of human 7 nAChR
expressed in Xenopus oocytes by 8ai
ACh alone
ACh+10 μM 8ai
16
12
8
4
0
hR
hR
ChR
AC
AC
receptor
α7 nA
3A
α3β4 n
α4β2 n
5-HT
14