Synthesis, crystal structure and anti-fatigue effects of some benzamide derivatives

Article abstract of DOI:10.3390/molecules19011034

A series of benzamide derivatives such as 1-(1,3-benzodioxol-5-ylcarbonyl) piperidine (1-BCP) were synthesized by the reaction of substituted benzoic acids with piperidine, morpholine or pyrrolidine using a novel method. The crystals of these benzamide de

Full text of DOI:10.3390/molecules19011034

Molecules 2014, 19, 1034-1046; doi:10.3390/molecules19011034
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Crystal Structure and Anti-Fatigue Effects of Some
Benzamide Derivatives
Xianglong Wu 1,†,*, Wutu Fan 1,†, Yalei Pan 1, Yuankun Zhai 1, Yinbo Niu 1, Chenrui Li 1 and
Qibing Mei 1,2,
*
1
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern
Polytechnical University, Xi’an 710072, China
2
Department of Pharmacology, School of Pharmacy, Fourth Military Medical University,
Xi’an 710032, China
†
These authors contributed equally in this work.
* Authors to whom correspondence should be addressed; E-Mails: wuxianglong@nwpu.edu.cn (X.W.);
gahpyudx@163.com (Q.M.); Tel./Fax: +86-29-8477-4552 (Q.M.).
Received: 4 December 2013; in revised form: 10 January 2014 / Accepted: 10 January 2014 /
Published: 16 January 2014
Abstract: A series of benzamide derivatives such as 1-(1,3-benzodioxol-5-ylcarbonyl)
piperidine (1-BCP) were synthesized by the reaction of substituted benzoic acids with
piperidine, morpholine or pyrrolidine using a novel method. The crystals of these
benzamide derivatives were obtained by recrystallization. Structures of target and
1
intermediate compounds were determined via FT-IR, H-NMR and elemental analysis and
X-ray crystallography of select examples. The crystal structures of these compounds have
potential applications to identify the binding site for allosteric modulators of the
α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor. The anti-fatigue
effects of the benzamide derivatives in weight-loaded forced swimming mice were
investigated in a swimming endurance capacity test used as an indicator of fatigue. The
swimming times to exhaustion were longer in the b3, d3, and e3 groups than in the caffeine
group (p < 0.05). In conclusion, b3, d3 and e3 enhanced the forced swimming capacity of
mice. The mechanism of the anti-fatigue effects will be studied in the future.
Keywords: benzamide derivatives; 1-BCP; X-ray diffraction; synthesis; anti-fatigue effects

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1. Introduction
Ionotropic glutamate receptors are the major excitatory amino acid neurotransmitter receptors in the
vertebrate central nervous system (CNS). There are three functionally distinct receptor subclasses:
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate
(NMDA) [1]. AMPA receptors mediate most of the fast excitatory amino acid transmission in the
CNS [2,3]. Attention has focused on drugs that modulate AMPA receptors because of their potential to
enhance memory and treat certain pathologies [4]. Positive allosteric modulators of AMPA may have
an advantage over direct-acting agonists or compounds that promote glutamate release, in that such
drugs could increase glutamatergic tone, without the obvious liability for direct receptor-mediated
excitotoxicity [5].
Ampakines are small benzamide compounds that are positive allosteric modulators of AMPA
receptors in the mammalian brain. Ampakines were initially derived from the nootropic drug
aniracetam which was discovered by Ito et al. [6]. Several ampakine compounds (see Figure 1),
including 1-BCP, CX516, CX554, CX546 and CX614 can facilitate synaptic plasticity and improve
learning and memory in both animals and humans [7].
Figure 1. Chemical structures of ampakines.
O
O
O
O
O
O
O
N
N
N
O
O
O
O
HO
Aniracetam
CX554
1-BCP
O
O
O
O
O
N
N
N
N
N
O
CX516
CX546
CX614
These compounds contain as common element a benzamide core, usually in the form of a
benzoylpiperidine or a benzoylpyrrolidine. They play a potential therapeutic role in the treatment of
neurodegenerative disorders, cognitive impairment, depression, Alzheimer’s disease and schizophrenia [8].
They have also been shown to be effective in animal models to treat various neuronal disorders [9] and
to enhance viability of neurons [10], probably by promoting expression of growth factors [11].
In this paper, we synthesized several benzamide derivatives which contain a benzamide core and
studied their unexpected anti-fatigue activity in the laboratory. To this end we first investigated their
anti-fatigue activity by weight-loaded forced swimming tests using a mouse model.
Numerous methods have been developed toward the synthesis of benzamide derivatives. Formation
of the amide bond has been one of the most widely studied reactions in organic chemistry [12]. It has
been taken for granted that amide bond formation is easily realized by reacting an activated carboxylic
acid with an amine.
Generally, most amides are prepared by the condensation of carboxylic acids and amines in the
presence of dehydrating reagents, such as B(OMe)3, TsCl/Py, TiCl4, Ph2POCl, Ph3P/PySSPy, N,N'-
dicyclohexylcarbodiimide (DCC), cyanuric chloride, 2-halo-N-methylpyridinium, and (benzotriazol-1-
yl) oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), or the carbonylation of aryl

Molecules 2014, 19
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halides with amines in the presence of Pd(OAc)2, and CO, etc. [13]. These methods all have some
advantages and disadvantages.
As a result, we report in this paper a convenient approach for the synthesis of amides. Substituted
benzoic acids were first treated with N-hydroxysuccinimidyl trifluoroacetate (NHS-TFA). Then, the
resulting intermediates were treated with piperidine, morpholine or pyrrolidine to give the target
compounds. Unexpectedly, crystals of the intermediate or target compounds were also obtained. All
compounds were analyzed via FT-IR, 1H-NMR and elemental analysis. The anti-fatigue activity of the
benzamide derivatives in weight-loaded forced swimming mice was then investigated. Detailed results
and discussion are elaborated in the following sections.
2. Results and Discussion
2.1. Synthesis and Structural Confirmation
Benzamide derivatives are typically prepared by chlorination of benzoic acids followed by
amidation of the resulting acid chlorides with an amine compound. Although the purified product can
be obtained by flash column chrmatography, difficulties can’t be avoided if using this method [14].
1-BCP was also prepared by the reaction of piperonaldehyde with an equimolar amount of piperidine at
100 °C in the presence of RuH2(PPh3)4 catalyst [15]. A specific catalyst was used in this method. Beller and
co-workers used CataCxium A at 80–130 °C and 2 bar CO pressure as the ligand for primary amide
synthesis from various substituted aryl bromides and heteroaryl bromides [16]. Recently, palladium-based
homogeneous catalysts were also reported for substrates such as benzyl chlorides and aryl chlorides [17].
In this paper, piperonylic acid was treated with N-hydroxysuccinimidyl trifluoroacetate (NHS-TFA)
at room temperature (Scheme 1). Then, the intermediate was reacted with piperidine to give the target
compound 1-BCP, which could be easily purified by recrystallization from ethanol. The approach
provided an efficient and environmentally friendly pathway to synthetic benzamide compounds [18] in
good to excellent yields. A total of ten benzamide derivatives were synthesized. The compounds were
characterized by elemental analysis, FT-IR, 1H-NMR and X-ray crystallography. The information provided
should be useful in the area of medical or pharmaceutical applications.
Scheme 1. Synthesis of benzamide derivatives.
O
H3CO
H3CO
O
COOH
O
O
O
O
OH
O
O
O
H3CO
H3CO
H3CO
H3CO
R
H
O
N
O
O
R
O
O
R
O
N
R
H
Pyridine, rt, stir
Pyridine, rt, stir
O
O
a
b
b1, b3, b4
a1, a2
O
O
F
F
N
O
O
F
O
H3CO
H3CO
H3CO
Pyridine, rt, stir
O
O
H3CO
COOH
COOH
O
O
O
O
N
R
H3CO
H3CO
O
R
H
H3CO
R
H
R
O
O
N
O
OCH3
Pyridine, rt, stir
O
O
OCH3
d1, d2, d3
e
e2, e3
d
b4:
b3, d3, e3: R=
R=
a1, b1, d1:
a2, d2, e2: R=
O
N
N
N
CH3
R=
N

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2.2. Crystal Structures
Single crystals of compounds a, a2 and e2 were obtained by recrystallization and their structures
were further confirmed by X-ray diffraction determination. The data of compounds a, a2 and e2 are
deposited as CCDC Nos. 815292, 960811 and 960813, respectively. The perspectives and packing
views are shown in Figures 2 and 3, respectively. The crystal data, details concerning data collection
and structure refinement are listed in Table 1.
Figure 2. ORTEP drawings of molecules a, a2 and e2 with 30% probability displacement ellipsoids.
Figure 3. View of the molecular packing in a, a2 and e2.
Compound a
Compound a2
Compound e2

Molecules 2014, 19
1038
Table 1. Crystal data and structure refinement for a, a2 and e2.
a
815292
a2
960811
e2
960813
CCDC Deposit No.
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
C12H9NO6
263.20
C12H13NO4
235.23
C14H19NO5
281.30
296(2)
296(2)
296
0.71073
Monoclinic
P2(1)/n
0.71073
0.71073
Orthorhombic
P bca
Triclinic
P-1
8.7694(11)
6.2631(8)
22.168(3)
90
14.595(3)
7.7849(14)
20.112(4)
90
8.6577(9)
9.5460(10)
9.8578(10)
64.1760(10)
77.037(2)
71.6540(10)
692.44(12)
2
b (Å)
c (Å)
α (°)
β (°)
94.814(2)
90
90
γ (°)
Volume (Å3)
90
1213.3(3)
4
2285.1(7)
8
Z
D
calc (g/cm3)
1.441
1.368
1.349
Abs.coefficient (mm−1)
0.118
0.104
0.096
F(000)
544
992
300
Crystal size (mm3)
0.14 × 0.11 × 0.10
1.84 to 25.10
−10/10, −7/6, −26/26
0.20 × 0.10 × 0.10
2.46 to 25.00
−15/17, −9/9, −23/17
Full-matrix least-squares on F2
10446/2001/0.033
99.6%
0.39 × 0.27 × 0.20
2.31 to 25.00
−9/10, −9/11, −11/11
θ limit (°)
Ranges/indices h. k. l.
Refinement method
Reflections collected/ unique/Rint
Completeness to θ = 25.10°
Data/restraints/parameters
Goodness of fit on F2
R1, wR2
5777 / 2165/ 0.043
99.6%
3490/ 2432/0.0205
99.3%
2165/0/173
1.088
2001/0/155
1.023
2432/0/185
1.085
0.0456, 0.1199
0.020(4)
0.0589, 0.1553
0.0028(11)
0.489 and −0.219
0.0404, 0.1261
0.120(12)
Extinction coefficient
Largest diff. peak and hole(e. Å−3)
0.149 and −0.161
0.155 and −0.186
The crystals of 3-benzodioxole-5-carboxylic acid 2,5-dioxo-1-pyrrolidinyl ester (a) are monoclinic,
space group P2(1)/n with four molecules per unit cell. The O (6) atom lies within the mean plane of the
benzene, as indicated by O(6)-C(10)-C(11)-C(12) torsion angle of −179.16°. The O(5) atom is also in
the mean plane of the benzene, as indicated by the O(6)-C(10)-C(11)-O(5) torsion angle of 0.6(2)°. All
carbon and oxygen atoms of piperonyl are in a plane. The succinimide ring also forms a plane. The
dihedral angle between the mean plane of the piperonyl and succinimide rings is 74.69°. In the crystal
packing, as shown in Figure 3, weak intermolecular hydrogen bonds are observed (showed as
dashed lines).
The crystals of compound a2 are orthorhombic, space group Pbca with eight molecules per unit
cell. The torsion angles of C(12)-N(1)-C(9)-C(10) and C(11)-O(4)-C(10)-C(9) [53.7(3)° and 58.5(3)°,
respectively] show that the morpholine ring is in a typical chair conformation. The dihedral angle
between the mean plane of benzene and a plane formed by N(1), C(9) and C(12) is 89.8°. In the crystal

Molecules 2014, 19
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packing, as shown in Figure 3, adjacent molecules are crossed stacked through strong offset π···π
aromatic stacking interactions. Intermolecular hydrogen bonds are also observed.
The crystals of compound e2 are triclinic, space group P-1 with two molecules per unit cell. The
morpholine ring is also is a typical chair conformation. In the crystal packing, as shown Figure 3
intermolecular hydrogen bonds are also observed (showed as dashed lines).
2.3. Effect on Forced Swimming Capacity
The forced swimming capacities are shown in Figure 4. There are significant differences in the
swimming time to exhaustion between the control group and each treatment group. The swimming
times to exhaustion of the caffeine (positive control group), b3, d3, and e3 groups were 1052 ± 148 s,
1310 ± 85 s, 1348 ± 139 s, and 1378 ± 105 s, respectively. Thus, the swimming times to exhaustion of
the b3, d3, and e3 groups were significantly longer than those of the caffeine group
(p < 0.05). It is worthwhile to note that b3, d3, and e3 have a common pyrrolidine ring. But what is the
real reason? Further observation and study are needed. On the other hand, the swimming times to
exhaustion of the a2, b1, b4, d1, d2, and e2 groups were longer than that of the normal saline group.
However, there was no significant difference in between the caffeine group and each of the other
treatment groups.
Figure 4. Effects of compounds on the forced swimming time. ** p < 0.01 compared with
the saline group, * p < 0.05 compared with the saline group, # p < 0.05 compared with the
caffeine group.
3. Experimental
3.1. General Information
1
Melting points were taken on a X-4 micro melting point apparatus and are uncorrected. H-NMR
spectra were recorded out in CDCl3 on a Varian Inova-400 spectrometer and chemical shifts were
reported relative to internal Me4Si. Infrared spectra were obtained as KBr pellets on an Equiox-55
FTIR spectrometer. Elemental analyses were performed with a Vario EL-III instrument. The X-ray
diffraction data were collected on a Bruker SMART AREX II CCD diffractometer equipped with a

Molecules 2014, 19
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graphite monochromated Mo Kα radiation (λ = 0.71073 Å) source by using the ω-2θ scan technique at
room temperature. The structure was solved by direct methods with SHELXS-97 [19], and refined
using SHELXL-97 [20]. Hydrogen atoms were generated geometrically. Molecular illustrations were
prepared using the XP package [21]. CCDC 815292 (for compound a), 960811 (for compound a2) and
960813 (for compound e2) contain the supplementary crystallographic data for this paper. These data
can be obtained free of charge from http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223
336033).
3.2. Synthesis
3.2.1. Preparation of N-Hydroxysuccinimidyl Trifluoroacetate (NHS-TFA)
The synthesis of NHS-TFA is shown in Scheme 2. To trifluoroacetic anhydride (15 ml, 71.2 mmol),
N-hydroxysuccinimide (5 g, 42.6 mmol) was slowly added with vigorously stirring. On a larger scale,
a condenser equipped with a drying tube may be needed. The solution was stirred at room temperature
about 4 h until the reaction was completed. The reaction solution was evaporated in vacuo to remove
trifluoroacetic anhydride and trifluoroacetic acid to give a thick oil. Then, it was further dried below 70 °C
until a free-flowing colorless solid was obtained. The product is of good quality if it gives bubbles
when dissolved in methanol.
Scheme 2. Preparation of N-hydroxysuccinimidyl trifluoroacetate.
O
O
O
O
O
F
F
F
F
O
F
N
F
F
O
OH
N
F
Room teperature
F
O
O
3.2.2. Synthesis of 3-Benzodioxole-5-carboxylic acid, 2,5-dioxo-1-pyrrolidinyl Ester (a)
Piperic acid (1.66 g, 10 mmol) was dissolved in anhydrous THF (50 mL) followed by the addition of
anhydrous pyridine (4 mL) and N-hydroxysuccinimidyl trifluoroacetate (NHS-TFA) (6.33 g, 30 mmol).
The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated
under reduced pressure. The residue was dissolved in CH2Cl2 (80 mL) and washed with 1mol/L HCl (3
× 80 mL), followed by saturated brine (3 × 80 mL). The organic layer was dried over anhydrous
Na2SO4, filtered and concentrated to give 3-benzodioxole-5-carboxylic acid, 2,5-dioxo-1-pyrrolidinyl
ester (a). Single crystals of (a) for X-ray diffraction experiments were grown from ethanol-water
mixture (1:1). White solid; Yield: 88%; m.p. 152–154 °C; FTIR (cm−1): 3103, 2995, 2913, 1771, 1730,
1
1623, 1485, 1442, 1375, 1263, 1210, 1073, 994, 830; H-NMR (CDCl3) δ: 2.91 (s, 4H), 6.09 (s, 2H),
6.90 (d, 1H), 7.51 (s, 1H), 7.76 (d,1H); Anal. calcd. For C12H9NO6: C, 54.76; H, 3.45; N, 5.32; Found
C, 54.72; H, 3.41; N, 5.27.

Molecules 2014, 19
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3.2.3. Synthesis of 3,4-Dimethoxybenzoic acid, 2,5-dioxo-1-pyrrolidinyl ester (b)
Compound b was prepared from 3,4-dimethoxybenzoic acid (1.82 g, 10 mmol) and NHS-TFA (6.33 g,
30 mmol) by the procedure utilized for compound a. Compound b was obtained as a white solid in
84% yield. FTIR (cm−1): 3094, 3023, 2940, 2841, 1734, 1599, 1513, 1458, 1448, 1417, 1373, 1273,
1206, 1143, 1071, 1017, 878, 810; 1H-NMR (CDCl3) δ: 2.92 (s, 4H), 3.94 (s, 6H), 6.94 (d, 1H, J = 8.8 Hz),
7.57 (s, 1H), 7.82 (d, 1H, J = 6.8 Hz); Anal. calcd. For C13H13NO6: C, 55.91; H, 4.69; N, 5.02; Found
C, 55.89; H, 4.67; N, 5.04.
3.2.4. Synthesis of 4-Methoxybenzoic acid, 2,5-dioxo-1-pyrrolidinyl ester (d)
This compound was prepared from 4-methoxybenzoic acid (1.52 g, 10 mmol) and NHS-TFA (6.33 g,
30 mmol) by the procedure utilized for compound a. Compound (d) was obtained as a white solid in
86% yield. m.p. 142–144 °C; FTIR (cm−1): 3433, 3111, 2940, 2848, 1766, 1735, 1602, 1514, 1457,
1
1427, 1377, 1271, 1255, 1216, 1183, 1073, 1022, 984, 847, 757; H-NMR (CDCl3) δ: 2.89 (s, 4H),
3.89 (s, 3H), 6.97 (d, 2H, J = 8.8 Hz), 8.09 (d, 2H, J = 8.8 Hz).
3.2.5. Synthesis of 3,4,5-Trimethoxybenzoic acid, 2,5-dioxo-1-pyrrolidinyl Ester (e)
This compound was prepared from 3,4,5-trimethoxybenzoic acid (2.12 g, 10 mmol) and NHS-TFA
(6.33 g, 30 mmol) by the procedure utilized for compound a. Compound (e) was obtained as white
solid in 80% yield. m.p. 125–127 °C; FTIR (cm−1): 2997, 2952, 1763, 1734, 1588, 1504, 1469, 1422,
1
1339, 1260, 1241, 1203, 1166, 1128, 1078, 989, 892, 750; H-NMR (CDCl3) δ: 2.91(s, 4H), 3.92(s,
9H), 7.38(s, 2H).
3.2.6. Synthesis of Compounds a1 and a2
3-Benzodioxole-5-carboxylic acid, 2,5-dioxo-1-pyrrolidinyl ester (2.63 g, 10 mmol) was dissolved
in CH2Cl2 (50 mL) followed by the addition of piperidine (2 mL). The resultant mixture was stirred at
room temperature for 2 h. The mixture was washed with 1 mol/L HCl (3 × 80 mL) and saturated
brine (3 × 80 mL), and then dried over anhydrous Na2SO4. The solvent was removed under reduced
pressure and the residue was crystallized from ethanol as needles. The compound a1 was obtained as a
white powder. Compound a2 were prepared according to the same procedure. Single crystals of
compound (a2) for X-ray diffraction experiments were grown from ethanol.
N-(3,4-methylenedioxybenzoyl) piperidine (a1). Needle-like solid; Yield: 83%; m.p. 51–52 °C
(lit. [22]: 51–52 °C); FTIR (cm−1): 3071, 3006, 2926, 2853, 1713, 1607, 1445, 1282, 1248, 1077,
1
1034, 929, 802; H-NMR (CDCl3) δ: 1.56–1.67 (m, 6H), 3.43 (t, 4H), 6.00 (s, 2H), 6.80 (d, 1H),
6.89 (s, 1H), 6.92 (d, 1H). Anal. calcd. For C13H15NO3: C, 66.97; H, 6.52; N, 5.97; Found C, 66.94; H,
6.48; N, 6.00.
N-(3,4-methylenedioxybenzoyl) morpholine (a2). Needle-like solid; Yield: 80%; m.p. 82–84 °C
(lit. [23]: 83–84 °C); FTIR (cm−1): 3433, 3040, 2974, 2899, 2859, 1620, 1494, 1444, 1368, 1342, 1283,
1
1246, 1108, 1037,940, 833; H-NMR (CDCl3) δ: 3.69 (s, 8H), 6.00 (s, 2H), 6.83 (d, 1H, J = 8.0 Hz),

Molecules 2014, 19
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6.91 (s, 1H), 6.93 (d, 1H, J = 8Hz); Anal. calcd. For C12H13NO4: C, 61.27; H, 5.57; N, 5.95; Found C,
61.59; H, 5.52; N, 5.98.
3.2.7. Synthesis of Compounds b1, b3 and b4
These compounds were prepared from 3,4-dimethoxybenzoic acid, 2,5-dioxo-1-pyrrolidinyl ester
and piperidine (pyrrolidine or 4-methylpyridine) by the procedure utilized for compound a1.
N-(3,4-Dimethoxybenzoyl) piperidine (b1). White solid; Yield: 84%; m.p. 35–36 °C (lit. [24]: 35–37 °C);
1
FTIR (cm−1): 3443, 2943, 2855, 1617, 1580, 1518, 1432, 1331, 1267, 1137, 1018, 870; H-NMR
(CDCl3) δ: 1.55–1.68 (m, 6H), 3.45–3.65 (m, 4H), 3.86 (s, 6H), 6.86 (d, 1H, J = 8.0 Hz), 6.97 (d, 1H,
J = 8.0 Hz), 6.98 (s, 1H).
N-(3,4-Dimethoxybenzoyl) pyrrolidine (b3). Colorless oil; Yield: 83%; FTIR (cm−1): 3437, 2972,
2935, 2875, 1612, 1575, 1512, 1454, 1417, 1384, 1328, 1249, 1176, 1120, 1022, 869, 821, 758;
1H-NMR (CDCl3) δ: 1.87 (t, 2H, J = 6.4 Hz), 1.96 (t, 2H, J = 6.4 Hz), 3.50 (t, 2H, J = 6.0 Hz), 3.64 (t,
2H, J = 6.4 Hz), 3.91 (s, 6H), 6.85 (d, 1H, J = 8.0 Hz), 7.12 (d, 1H, J = 8.8 Hz), 7.14 (s,1H).
(3,4-Dimethoxyphenyl)(4-methyl-1-piperidinyl) methanone (b4). Colorless oil; Yield: 82%; FTIR
1
(cm−1): 3444, 2957, 2863, 1631, 1517, 1437, 1324, 1271, 1141, 1072, 818; H-NMR (CDCl3) δ: 0.98
(d, 3H, J = 5.6 Hz), 1.15–1.24 (m, 2H), 1.61–1.79 (m, 4H), 3.90 (s, 6H,), 6.85 (d, 1H, J = 8.0 Hz), 6.97
(d, 1H, J = 8.4 Hz), 6.98 (s, 1H).
3.2.8. Synthesis of Compound d1–d3
These compounds were prepared from 4-methoxybenzoic acid, 2,5-dioxo-1-pyrrolidinyl ester and
piperidine (morpholine or pyrrolidine) by the procedure utilized for compound a1.
1
N-(4-Methoxybenzoyl) piperidine (d1). Colorless oil; Yield: 80%; H-NMR (CDCl3) δ: 1.58–1.67 (m,
6H), 3.30–3.71 (m, 4H), 3.83 (s, 3H), 6.90 (d, 2H, J = 8.4 Hz), 7.37 (d, 2H, J = 8.4 Hz). The properties
match those reported in the literature [25].
N-(4-Methoxybenzoyl) morpholine (d2). Colorless oil. Yield: 82%; FTIR (cm−1): 3427, 3188, 2931,
2859, 1764, 1690, 1645, 1610, 1494, 1459, 1321, 1254, 1229, 1168, 1109, 1051, 1028, 838, 757, 706;
1H-NMR (CDCl3) δ: 3.69 (s, 4H), 3.84 (s, 3H), 6.92 (d, 2H, J = 8.4 Hz), 7.39 (d, 2H, J = 8.4 Hz). The
properties match those reported in the literature [13].
N-(4-Methoxybenzoyl) pyrrolidine (d3). White solid; Yield: 85%; m.p. 76–77 °C (lit. [26]:
78.4–78.8 °C); FTIR (cm−1): 3521, 3111, 3082, 2847, 1766, 1735, 1602, 1514, 1457, 1427, 1377,
1271, 1255, 1217, 1183, 1074, 1022, 984, 847, 757; 1H-NMR (CDCl3) δ: 1.85-1.90 (m, 2H), 1.92–1.97
(m, 2H), 3.48 (t, 2H, J =6.4 Hz), 3.65 (t, 2H, J = 6.8 Hz), 3.84 (s, 3H), 6.90 (d, 2H, J = 8.8 Hz), 7.52
(d, 2H, J = 8.8 Hz).

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3.2.9. Synthesis of Compounds e2 and e3
Compounds e2 and e3 were prepared from 3,4,5-trimethoxybenzoic acid, 2,5-dioxo-1-pyrrolidinyl
ester and morpholine or pyrrolidine by the procedure utilized for compound a1. Single crystals of
compound e2 for X-ray diffraction experiments were grown from ethanol.
N-(3,4,5-Trihydroxybenzoyl) morpholine (e2). White solid; Yield: 79%; m.p. 97–99 °C (lit. [12]:
96–98 °C); FTIR (cm−1): 3430, 2974, 2934, 2871, 1765, 1637, 1586, 1449, 1416, 1327, 1233, 1132,
958, 854; 1H-NMR (CDCl3) δ: 3.69–3.71 (m, 4H), 3.87 (s, 9H), 6.63 (s, 2H).
N-(3,4,5-Trihydroxybenzoyl) pyrrolidine (e3). White solid; Yield: 84%; m.p. 182–183 °C. (lit. [27]:
183–186 °C); 1H-NMR (CDCl3) δ: 1.89 (dd, 2H, J = 7.4 Hz, 6.0 Hz), 1.97 (dd, 2H, J = 6.8 Hz, 6.4 Hz),
3.46 (t, 2H, J = 6.8 Hz), 3.64 (t, 2H, J = 6.8 Hz), 3.86 (s, 9H), 6.75 (s, 2H).
3.3. Anti-Fatigue Activity Assessment of Benzamide Derivatives
3.3.1. Animals
Six-week-old male Kunming mice (18–22 g, specific pathogen-free grade, SPF) were obtained from
the Academy of Experimental Animal Center of The Fourth Military Medical University (Xi’an,
China). Mice were maintained under normal conditions: 21–26 °C temperature, 60%–70% relative
humidity, 12:12 h dark/light cycle, and free access to laboratory standard diet and water. All mice were
quarantined and adapted for 7 days after arriving. All animal experiments were conducted under
institutional guidelines and approved by the Ethical Committee for Animal Care and Use of the Fourth
Military Medical University.
3.3.2. Anti-Fatigue Activity
The anti-fatigue activity of benzamide derivatives was evaluated by the weight loaded swimming
model in mice. The model is a reliable measure of anti-fatigue treatment as established in both
laboratory animals and humans [28–30]. All mice were randomly divided into 12 groups, two control
groups and ten treatment groups, seven mice per group. Benzamide derivatives were dissolved in
carboxymethylcellulose sodium (CMC) 5 g/L aqueous solution, respectively. One control group
received the same volume of saline solution. Another control group received caffeine as a positive
control group. Benzamide derivatives were given to mice at concentrations of 0.1 mmol/kg body
weight. Samples were orally administered into mice using a feeding atraumatic needle, once per day at
1:00–3:00 pm for one week. After each treatment, all groups of the mice were allowed to rest 30 min
and were forced to swim for ten minutes to become accustomed to swimming. The size of swimming
pool was designed as 50 cm × 50 cm × 40 cm, filled with fresh water at 30 ± 2 °C. A tin wire (5% of
body weight) was loaded on the tail root of the mouse. The mice were assessed to be exhausted when
they failed to rise to the surface of water to breathe within a 7 s period [31]. At the end of the session,
the mice were removed from the water, dried with a paper towel, and placed back in their home cages.
Water in the container was drained after each session. The swimming time to exhaustion was used as
the index of the forced swimming capacity.

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3.3.3. Statistical Analysis
Data were analyzed using SPSS 13.0 version. The results were expressed as the mean ± S.E.M. The
data were subjected to one-way analysis of variance (ANOVA) followed by Tukey–Kramer post-hoc
analysis. The level of p < 0.05 was used as the criterion of statistical significance.
4. Conclusions
In the current study, we have established for the first time a rapid and highly efficient method for
the synthesis of benzamide derivatives. The structures of the compounds have been determined by
1
FTIR and H-NMR spectroscopy, elemental analysis and X-ray diffraction of select examples
(compounds a, a2 and e2). The crystal structures have potential applications in identifying the binding
site for allosteric modulators of the AMPA receptor, and should be useful in the area of medical or
pharmaceutical applications.
Preliminary studies suggest that compound b3, d3 and e3 increased swimming time to exhaustion in
weight-loaded forced swimming mice. They have potential application in developing new drug
candidates with anti-fatigue activity. Because a large number of complex mechanisms may be involved
in the exercise-induced fatigue, further research including molecular study is required to evaluate the
anti-fatigue mechanism.
Acknowledgments
The project was supported by the National Natural Science Foundation of China (No. 21202130,
81073037, and 81202457), the National Special Foundation of State Center for Drug Research and
Development (No. 2008ZXJ09004-024, 2009ZXG09001-020). Guo-ping Yang and Xiang-han Zhang
are thanked for their warm support.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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