Synthesis, structure and structure-activity relationship analysis of caffeic acid amides as potential antimicrobials

Article abstract of DOI:10.1016/j.ejmech.2010.01.066

A series of caffeic acid amides 1-23 were synthesized and nine of which (13-17, 19-21 and 23) were reported for the first time. The chemical structures of these compounds were confirmed by means of ¹H NMR, ESI MS and elemental analyses. Compound 15 was determined by single-crystal X-ray diffraction analysis. All of the compounds were assayed for antibacterial (Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens and Staphylococcus aureus) and antifungal (Aspergillus niger, Candida albicans and Trichophyton rubrum) activities by MTT method. Compounds 10-12, 15, 18 and 21 showed considerable antibacterial activities against B. subtilis with MICs of 7.95, 6.25, 3.89, 1.18, 3.12 and 15.5?μg/mL, respectively. Structure-activity relationship analysis disclosed that caffeic acid anilides with electron-donating groups at p-position of benzene ring have better inhibitory activities.

Full text of DOI:10.1016/j.ejmech.2010.01.066

European Journal of Medicinal Chemistry 45 (2010) 2638–2643
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, structure and structure–activity relationship analysis of caffeic acid
amides as potential antimicrobials
*
Jie Fu, Kui Cheng, Zhi-ming Zhang, Rui-qin Fang, Hai-liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of caffeic acid amides 1–23 were synthesized and nine of which (13–17, 19–21 and 23) were
reported for the first time. The chemical structures of these compounds were confirmed by means of 1H
NMR, ESI MS and elemental analyses. Compound 15 was determined by single-crystal X-ray diffraction
analysis. All of the compounds were assayed for antibacterial (Bacillus subtilis, Escherichia coli, Pseudo-
monas fluorescens and Staphylococcus aureus) and antifungal (Aspergillus niger, Candida albicans and
Trichophyton rubrum) activities by MTT method. Compounds 10–12, 15, 18 and 21 showed considerable
Received 29 May 2009
Received in revised form
25 January 2010
Accepted 28 January 2010
Available online 6 February 2010
antibacterial activities against B. subtilis with MICs of 7.95, 6.25, 3.89, 1.18, 3.12 and 15.5
mg/mL,
Keywords:
Caffeic acid amides
Synthesis
Antimicrobial activities
Structure–activity relationship
respectively. Structure–activity relationship analysis disclosed that caffeic acid anilides with electron-
donating groups at p-position of benzene ring have better inhibitory activities.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
botulinum and reduce thermal processing requirements of heat
sensitive foods [6]. Lysozyme–caffeic acid conjugate shows bacte-
The rapid incidences of drug resistance of microorganisms to
antibiotics were reported [1]. For example, by the late 1950s
Staphylococcus aureus (S. aureus) became resistant to penicillin and
infections with these strains caused many deaths [2]. Until recently,
antibiotic discovery has kept ahead of microbial resistance, but
recent outbreaks of multidrug-resistant Mycobacterium tuberculosis,
penicillin resistant Streptococcus pneumoniae, methicillin-resistant
S. aureus, vancomycin-resistant enterococci and, more recently,
vancomycin-resistant S. aureus have prompted an urgent discovery
of novel active agents against these pathogens [3,4]. Therefore,
a new demand for alternative antibiotics, the structure of which
differs from conventional antibiotics is created.
Caffeic acid is one of the most widely distributed hydroxy-
cinnamate and phenylpropanoid metabolites in plant tissues. It is
usually found as various simple derivatives including amides,
esters, sugar esters, and glycosides [5]. The antimicrobial activity of
these derivatives has attracted much attention and has been
studied by many research groups in recent years [6–10]. Caffeic acid
has potential as a food additive to inhibit growth of Clostridium
ricidal effect for Escherichia coli (E. coli) and S. aureus [7]. The
dicaffeoylquinic acids and dicaffeoyltartaric acids are potent and
selective inhibitors of human immunodeficiency virus type 1
integrase [8]. The caffeic acid phenylethyl esters are demonstrated
to have higher antimicrobial activities against S. aureus, B. subtilis
(B. subtilis), and Pseudomonas aeruginosa than 5-chorogenic acid
and caffeic acid [9]. Almajano et al. has studied the effect of pH on
the antimicrobial activity and oxidative stability of oil-in-water
emulsions containing caffeic acid [10].
Rajan et al. synthesized a series of amides of caffeic acid and
evaluated their antioxidant properties as lipid peroxidation
inhibitors [11]. Some hydroxycinnamic acid amides and analogues
were synthesized and studied antibacterial activity against
S. aureus [12]. Some caffeoylglycolic and caffeoylamino acid
derivatives were synthesized for the purpose of simplifying the
structure of
L-chicoric acid as new HIV-1 integrase inhibitors [13].
All of these encouraged us to find new caffeic acid amide anti-
microbials. Therefore, in this paper, a series of caffeic acid amides
1–23 were synthesized and their antimicrobial activities were
evaluated. The results were compared with those obtained with
the corresponding p-coumaric acid amides 24 and 25, caffeic acid,
p-coumaric acid, and standard antimicrobials such as kanamycin
and penicillin. Based on theses results, structure–activity rela-
tionship (SAR) analysis was conducted. The results of this study
* Corresponding author. Tel./fax: þ86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-l. Zhu).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.066

J. Fu et al. / European Journal of Medicinal Chemistry 45 (2010) 2638–2643
2639
may be useful to researchers attempting to find new potential
antimicrobials.
Cambridge Crystallographic Data Centre, and the CCDC number is
742,862.
2.3. Antimicrobial activity
2. Results and discussion
In this study, attention is focused on the evaluation of the SAR. In
screening assay studies, caffeic acid amides 1–23 were evaluated
for their antimicrobial activities against two Gram-positive bacteria
(B. subtilis ATCC 6633 and S. aureus ATCC 6538), two Gram-negative
bacteria (E. coli ATCC 35218 and Pseudomonas fluorescens ATCC
13525), and three fungi (Aspergillus niger ATCC 16404, Candida
albicans ATCC 10231, and Trichophyton rubrum ATCC 10218) by the
3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) method [14], taking p-coumaric acid amides 24 and 25,
caffeic acid, p-coumaric acid, kanamycin and penicillin as
comparisons. The results were shown in Table 3 and the minimum
inhibitory concentrations (MICs) of the compounds differed greatly,
2.1. Chemistry
In order to investigate the antimicrobial activities of caffeic acid
amides, a series of caffeic acid amides 1–23 were synthesized from
the free acid and the amines using benzotriazole-1-yl-oxy-tris-
(dimethylamino)-phosphonium hexafluorophosphate (BOP) as
coupling reagent (Scheme 1) [11]. Among these compounds,
compounds 13–17, 19–21 and 23 are reported for the first time.
These compounds gave satisfactory elementary analyses (ꢀ0.4%).
1H NMR and ESI MS spectra were consistent with the assigned
structures. Compounds 1 and 2 were caffeic acid fatty amides.
Compounds 3–21 were caffeic acid anilides with a single different
substituent (F, Cl, Br, CH3, OCH3 or NO2) in o-, m-, or p-position of
benzene ring. Compounds 22 and 23 were caffeic acid secondary
amides with phenyl groups. In addition, to demonstrate the
importance of caffeoyl group for the activity, two p-coumaric acid
amides 24 and 25 were synthesized using the same method as for
synthesizing caffeic acid amides.
ranging from 1.18 to over 50
significant inhibition activities against E. coli, A. niger, and T. rubrum,
supported by the fact that their MICs were all over 50 g/mL (data
mg/mL. These compounds had not
m
not shown). Of these compounds, six (10–12, 15, 18 and 21)
exhibited potent antibacterial activity against B. subtilis, with MICs
comparable to kanamycin and penicillin.
Some studies [15,16] reported that Gram-positive bacteria pre-
sented higher sensitivity than Gram-negative bacteria to various
polyphenols. This higher resistance can be explained on the basis of
the different composition of the cell-wall membrane. Our results
showed that of the four bacteria, Gram-positive bacterium B. sub-
tilis was more sensitive to our compounds than P. fluorescens,
S. aureus, and E. coli. However, our compounds had showed weak
antibacterial activity against some Gram-positive bacterium such
as S. aureus.
The data in Table 3 showed that many caffeic acid amides had
stronger antimicrobial activities than caffeic acid, suggesting
amides groups could enhance the activities. p-Coumaric acid
amides 24 and 25, and p-coumaric acid also had poorly activities,
2.2. The crystal structure of compound 15
Among these compounds, a crystal structure of compound 15
was determined by X-ray diffraction analysis. The crystal data are
presented in Table 1 and Fig.1 gave perspective views of 15 with the
ꢀ
atomic labeling system. It crystallizes in the space group Pı with the
following unit cell parameters: a ¼ 9.4460 (19) Å, b ¼ 10.701 (2) Å,
c ¼ 13.812 (3) Å,
a
¼ 78.63 (3)ꢁ,
b
¼ 76.87 (3)ꢁ,
g
¼ 89.42 (3)ꢁ,
V ¼ 13,332.1 (5) Å3, Z ¼ 4. The selected bond lengths/angles and
torsion angles are given in Table 2. The bond lengths and angles are
all in normal values. The X-ray data have been deposited at the
Scheme 1. Synthesis of caffeic acid amides and p-coumaric acid amides.

2640
J. Fu et al. / European Journal of Medicinal Chemistry 45 (2010) 2638–2643
Table 1
Table 2
Crystallographic data for compound 15.
Selected bond lengths, bond angles and torsion angles of compound 15.
Crystal data
15
Bond lengths (Å)
O1–C8
O2–C15
N1–C8
N1–C5
C1–C2
C2–C3
C8–C9
C9–C10
1.225 (2)
1.384 (2)
1.354 (2)
1.423 (2)
1.516 (3)
1.379 (3)
1.480 (2)
1.327 (3)
1.469 (2)
Formula
C16H15NO3
0.30 ꢂ 0.20 ꢂ 0.10
269.29
Triclinic
ꢀ
Pı
Crystal size (mm)
MW (g molꢃ1
)
Crystal system
Space group
Cell parameters
a (Å)
9.4460 (19)
10.701 (2)
13.812 (3)
1332.1 (5)
4
b (Å)
C10–C11
c (Å)
Bond angles (ꢁ)
H2–O2–C15
H1A–N1–C8
C5–N1–C8
V (Å3)
109.4 (7)
116.7 (7)
126.5 (2)
109.4 (7)
117.7 (2)
120.9 (2)
123.6 (2)
113.2 (2)
125.4 (2)
Z
m
r
(mmꢃ1
calcd (g cmꢃ3
)
0.093
1.343
)
H1B–C1–C2
C7–C2–C3
C1–C2–C3
N1–C8–O1
C9–C8–N1
C11–C10–C9
Selected torsion angles (ꢁ)
C3–C4–C5–N1
C8–N1–C5–C4
C8–N1–C5–C6
N1–C5–C6–C7
C5–N1–C8–O1
C5–N4–C8–C9
O1–C8–C9–C10
N1–C8–C9–C10
O3–C14–C15–O2
Data collection
limits (ꢁ)
hkl limits
No. collected refl.
q
1.55 < q < 25.27
0 ꢄ h ꢄ 11; ꢃ12 ꢄ k ꢄ 12; ꢃ16 ꢄ l ꢄ 16
5155
No. ind refl. (Rint
Refinement
R [I > 2sigma(I)]
Rw (all data)
)
4835 (0.015)
0.044
0.058
0.056(3)
1.021
ꢃ0.27
0.30
178.5 (2)
ꢃ45.2 (3)
136.2 (2)
ꢃ178.8 (2)
1.2 (3)
ꢃ177.8 (2)
ꢃ20.8 (3)
158.2 (2)
ꢃ1.7 (2)
Extinction coefficient
GOF
Drmin (e Åꢃ3
)
)
Drmax (e Åꢃ3
indicating that the presence of adjacent dihydroxy in the caffeoyl
group was very important for the antimicrobial activities.
As shown in Table 3, caffeic acid anilides 10–12, 15, 18 and 21
exhibit excellent activities against B. subtilis, whereas caffeic acid
fatty amides 1 and 2 obviously showed much lower inhibition
under the same conditions. This indicated that anilides had better
antibacterial activities than fatty amides, and supportive evidence
may be found by a comparison of other data listed in Table 3. Caffeic
acid secondary amides 2, 22 and 23 had low activities, proving that
H–N is important for the activities.
Although caffeic acid anilides exhibit better antibacterial activity
against B. subtilis, their MICs differ significantly. One rule could be
found: anilides with a single substitute at the p-positions of the
benzene ring exhibit the most powerful inhibitory activities, whereas
anilides with a substituent at o- or m-positions show less potent
activities. From this fact, it is reasoned that the p-postion is an
essential point for the antibacterial activities of anilides, and
substituents at the o- or m-positions decrease their inhibition. For
example, when a methyl is introduced at p-position to form 15, the
activity is the most significant. However, when a methyl was intro-
duced at o-position (or m-position) to form 13 (or 14), the activity
decreases significantly. According to further SAR analysis,
replacement of methyl group of (15) at p-position by a bromide (12)
resulted in the decreases of activity. Similarly, substitution of a chlo-
ride (11) at p-position for a methoxy group (18) also led to a decrease
of inhibitory activity. This suggested that compounds with electron-
donating groups at p-position showed better inhibitory activities
than those with electron-withdrawing groups. For more extensive
evidences, the substitution of methoxy group at p-position (18) by
a fluoride or nitro (10 or 21) produced some decrease in activity.
Table 3
Antimicrobial activities of caffeic acid amides 1–25 (MIC, mg/mL).
Compounds
B. subtilis
P. fluorescens
S. aureus
C. albicans
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
>50
>50
28.4
50
45.8
49.5
46.7
50
>50
>50
>50
>50
41.2
42.2
>50
>50
>50
>50
50
>50
>50
41.5
>50
>50
>50
>50
49.2
>50
>50
46.8
>50
>50
45.6
>50
47.4
>50
50
>50
>50
>50
>50
>50
45.4
>50
>50
>50
1.28
1.39
>50
>50
50
33.3
42.8
27.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
44.7
42.8
>50
>50
49.4
>50
>50
50
50
7.95
6.25
3.89
35.0
40.2
1.18
39.8
50
3.12
>50
>50
15.5
>50
>50
>50
>50
>50
>50
0.39
0.78
50
42.5
38.8
42.2
13.2
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
5.08
–
25
>50
>50
>50
–
Caffeic acid
p-Coumaric acid
Kanamycin
Penicillin
Fig. 1. ORTEP view showing the atom-labeling scheme with thermal ellipsoids drawn
at 30% probability for compound 15.
–

J. Fu et al. / European Journal of Medicinal Chemistry 45 (2010) 2638–2643
2641
3. Conclusions
1H), 9.16 (s, 1H), 7.57 (dd, 2H, J1 ¼ 8.2 Hz, J2 ¼ 2.0 Hz), 7.50 (d, 1H,
J ¼ 15.6 Hz), 7.12 (m, 2H), 7.08 (dd, 1H, J1 ¼ 8.1 Hz, J2 ¼ 2.0 Hz), 7.05
(d, 1H, J ¼ 2.0 Hz), 6.82 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 1.9 Hz), 6.78 (d, 1H,
J ¼ 8.3 Hz), 6.55 (d, 1H, J ¼ 15.6 Hz). Anal. Calc for C15H13NO3: C,
70.58; H, 5.13; N, 5.49%. Found: C, 70.36; H, 5.32; N, 5.45%.
A series of caffeic acid amides 1–23 were synthesized. To study
the potential antimicrobial activities of the synthesized amides,
screening experiments were performed for four bacteria and three
fungi strains. Compounds 10–12, 15, 18 and 21 (MICs ¼ 7.95, 6.25,
3.89, 1.18, 3.12 and 15.5
mg/mL) showed considerable antibacterial
4.1.6. (E)-3-(3,4-Dihydroxyphenyl)-N-(2-fluorophenyl)
acrylamide (4)
activities against B. subtilis. According to SAR analysis, it is found that
caffeic acid anilides with electron-donating groups at p-position of
benzene ring have better inhibitory activities. This study shows that
caffeic acid anilides are potential antimicrobials. We hope this
information might be helpful for the discovery of new antibiotics.
Pale yellow crystal, yield 62%, mp: 177–178 ꢁC, ESI MS: 274.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.06 (s, 1H), 9.45
(s, 1H), 9.26 (s, 1H), 7.68 (dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 2.0 Hz), 7.38 (d, 1H,
J ¼ 15.6 Hz), 7.12–7.14 (m, 2H), 7.08 (m, 1H), 7.00 (d, 1H, J ¼ 2.0 Hz),
6.90 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 1.9 Hz), 6.78 (d, 1H, J ¼ 8.3 Hz), 6.53 (d,
1H, J ¼ 15.5 Hz). Anal. Calc for C15H12FNO3: C, 65.93; H, 4.43; N,
5.13%. Found: C, 65.61; H, 4.36; N, 5.68%.
4. Experimental
4.1. Chemistry
4.1.7. (E)-N-(2-Chlorophenyl)-3-(3,4-dihydroxyphenyl)
acrylamide (5)
4.1.1. Chemistry general
All chemicals (reagent grade) used were purchased from Aldrich
(USA). Separation of the compounds by column chromatography
was carried out with silica gel 60 (200–300 mesh ASTM, E. Merck,
Germany). The quantity of silica gel used was 50–100 times the
weight charged on the column. TLC was run on the silica gel coated
aluminum sheets (silica gel 60 GF254, E. Merck, Germany) and
visualized in UV light (254 nm). Melting points (uncorrected) were
obtained by using an XT4 MP apparatus (Taike Corp. Beijing, China).
1H NMR spectra were recorded at 300 MHz on 1H-Varian-Mercury-
300 spectrometers at 25 ꢁC, using TMS as internal standard. ESI MS
were recorded with a Mariner System 5304 mass spectrometer.
Elementary analyses were performed on a CHN–O-Rapid instru-
ment and were within ꢀ0.4% of the theoretical values.
Pale yellow powder, yield 73%, mp: 188–189 ꢁC, ESI MS: 290.0
[M þ H]þ,1H NMR (300 MHz, DMSO-d6,
d ppm): 10.12 (s,1H), 9.48 (s,
1H), 9.16 (s, 1H), 7.76 (dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 2.2 Hz), 7.56 (dd, 1H,
J1 ¼8.0 Hz, J2 ¼ 2.1 Hz), 7.39 (d,1H, J ¼ 15.5 Hz), 7.25 (m,1H), 7.16 (m,
1H), 7.00 (d,1H, J ¼ 2.2 Hz), 6.91 (dd,1H, J1 ¼8.5 Hz, J2 ¼ 2.2 Hz), 6.79
(d,1H, J ¼ 8.5 Hz), 6.55 (d,1H, J ¼ 15.5 Hz). Anal. Calc for C15H12ClNO3:
C, 62.19; H, 4.17; N, 4.83%. Found: C, 62.16; H, 4.62; N, 4.61%.
4.1.8. (E)-N-(2-Bromophenyl)-3-(3,4-dihydroxyphenyl)
acrylamide (6)
Yellow powder, yield 76%, mp: 200–201 ꢁC, ESI MS: 334.0
[M þ H]þ,1H NMR (300 MHz, DMSO-d6,
d ppm): 10.15 (s,1H), 9.50 (s,
1H), 9.20 (s, 1H), 7.80 (dd, 1H, J1 ¼ 8.1 Hz, J2 ¼ 2.1 Hz), 7.65 (dd, 1H,
J1 ¼8.0 Hz, J2 ¼ 2.1 Hz), 7.40 (d,1H, J ¼ 15.6 Hz), 7.36 (m,1H), 7.08 (m,
1H), 7.01 (d,1H, J ¼ 2.1 Hz), 6.92 (dd,1H, J1 ¼8.5 Hz, J2 ¼ 2.2 Hz), 6.81
(d,1H, J ¼ 8.5 Hz), 6.57 (d,1H, J ¼ 15.6 Hz). Anal. Calc for C15H12BrNO3:
C, 53.91; H, 3.62; N, 4.19%. Found: C, 53.63; H, 3.72; N, 4.16%.
4.1.2. General synthesis method of caffeic acid amides and
p-coumaric acid amides
The hydroxycinnamic acid (5 mmol) is dissolved in 10 mL of
DMF and 0.7 mL (5 mmol) of triethylamine. The solution is cooled in
an ice-water bath and 5 mmol of the amine are added followed by
a solution of 5 mmol of BOP in 10 mL of CH2Cl2. The mixture is
stirred at 0 ꢁC for 30 min and then at room temperature for 2–5 h
CH2Cl2 is removed under reduced pressure and the solution is
diluted with 80 mL of water. The products are extracted with ethyl
acetate. The extract is washed successively with 1 N HCl, water, 1 M
NaHCO3, and water, dried over MgSO4, filtered and evaporated. The
residue is purified on a silica gel column (eluent: ethyl acetate–
petroleum ether).
4.1.9. (E)-3-(3,4-Dihydroxyphenyl)-N-(3-fluorophenyl)
acrylamide (7)
Pale yellow powder, yield 72%, mp: 197–198 ꢁC, ESI MS: 274.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.06 (s, 1H), 9.45
(s, 1H), 9.26 (s, 1H), 7.58–7.60 (m, 2H), 7.38 (d, 1H, J ¼ 15.6 Hz), 7.32
(dd, H, J1 ¼ 8.0 Hz, J2 ¼ 2.0 Hz), 7.00 (d, 1H, J ¼ 2.0 Hz), 6.94 (m, 1H),
6.88 (dd, 1H, J1 ¼8.2 Hz, J2 ¼ 2.0 Hz), 6.79 (d, 1H, J ¼ 8.2 Hz), 6.51 (d,
1H, J ¼ 15.5 Hz). Anal. Calc for C15H12FNO3: C, 65.93; H, 4.43; N,
5.13%. Found: C, 65.68; H, 4.82; N, 4.91%.
4.1.3. (E)-3-(3,4-Dihydroxyphenyl)-N-ethylacrylamide (1)
4.1.10. (E)-N-(3-Chlorophenyl)-3-(3,4-dihydroxyphenyl)
acrylamide (8)
Pale green crystal, yield 84%, mp: 117–118 ꢁC, ESI MS: 208.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d
ppm): 9.36 (s, 2H), 7.47 (d,
Yellow powder, yield 75%, mp: 205–206 ꢁC, ESI MS: 290.0
1H, J ¼ 15.8 Hz), 7.06 (d, 1H, J ¼ 2.2 Hz), 7.00 (dd, 1H, J1 ¼ 8.1 Hz,
J2 ¼ 2.0 Hz), 6.76 (d, 1H, J ¼ 8.0 Hz), 6.26 (d, 1H, J ¼ 15.9 Hz), 4.16 (q,
2H, J ¼ 6.8 Hz), 1.24 (t, 3H, J ¼ 7.1 Hz). Anal. Calc for C11H13NO3: C,
63.76; H, 6.32; N, 6.76%. Found: C, 63.36; H, 5.39; N, 6.86%.
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.10 (s, 1H), 9.51
(s, 1H), 9.24 (s, 1H), 7.76 (m, 1H), 7.50 (m, 1H), 7.41 (d, 1H,
J ¼ 15.4 Hz), 7.35 (m, 1H), 7.28 (m, 1H), 7.01 (d, 1H, J ¼ 2.1 Hz), 6.90
(dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 2.1 Hz), 6.80 (d, 1H, J ¼ 8.3 Hz), 6.52 (d, 1H,
J ¼ 15.4 Hz). Anal. Calc for C15H12ClNO3: C, 62.19; H, 4.17; N, 4.83%.
Found: C, 62.70; H, 4.82; N, 4.51%.
4.1.4. (E)-3-(3,4-Dihydroxyphenyl)-N,N-diethylacrylamide (2)
White powder, yield 74%, mp: 134–135 ꢁC, ESI MS: 236.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d
ppm): 7.67 (d, 1H,
4.1.11. (E)-N-(3-Bromophenyl)-3-(3,4-dihydroxyphenyl)
acrylamide (9)
J ¼ 15.4 Hz), 7.24 (d, 1H, J ¼ 1.8 Hz), 6.99 (dd, 1H, J1 ¼ 8.2 Hz,
J2 ¼ 1.8 Hz), 6.88 (d, 1H, J ¼ 8.2 Hz), 6.65 (d, 1H, J ¼ 15.3 Hz), 3.41 (m,
4H), 1.45 (m, 6H). Anal. Calc for C13H17NO3: C, 66.36; H, 7.28; N,
5.95%. Found: C, 66.66; H, 7.35; N, 5.61%.
Yellow powder, yield 61%, mp: 208–209 ꢁC, ESI MS: 334.0
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.14 (s, 1H), 9.52
(s, 1H), 9.25 (s, 1H), 7.81 (m, 1H), 7.55 (m, 1H), 7.42 (d, 1H,
J ¼ 15.5 Hz), 7.36 (m, 1H), 7.19 (m, 1H), 7.03 (d, 1H, J ¼ 2.1 Hz), 6.91
(dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 2.1 Hz), 6.82 (d, 1H, J ¼ 8.4 Hz), 6.52 (d, 1H,
J ¼ 15.5 Hz). Anal. Calc for C15H12BrNO3: C, 53.91; H, 3.62; N, 4.19%.
Found: C, 54.29; H, 3.30; N, 4.08%.
4.1.5. (E)-3-(3,4-Dihydroxyphenyl)-N-phenylacrylamide (3)
Pale yellow powder, yield 72%, mp: 178–179 ꢁC, ESI MS: 256.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 9.96 (s, 1H), 9.42 (s,

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J. Fu et al. / European Journal of Medicinal Chemistry 45 (2010) 2638–2643
4.1.12. (E)-N-(4-Fluorophenyl)-3-(3,4-dihydroxyphenyl)
acrylamide (10)
4.1.19. (E)-3-(3,4-Dihydroxyphenyl)-N-(3-methoxyphenyl)
acrylamidez (17)
Pale yellow powder, yield 74%, mp: 199–200 ꢁC, ESI MS: 274.1
White powder, yield 86%, mp: 199–200 ꢁC, ESI MS: 286.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d
ppm): 9.96 (s, 1H), 9.42 (s,
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.06 (s, 1H), 9.46
1H), 9.12 (s, 1H), 7.66 (d, 2H, J ¼ 8.0 Hz), 7.36 (d, 1H, J ¼ 15.6 Hz), 7.12
(d, 2H, J1 ¼ 8.0 Hz), 6.98 (d, 1H, J ¼ 2.0 Hz), 6.87 (dd, 1H, J1 ¼ 8.2 Hz,
J2 ¼ 2.0 Hz), 6.77 (d, 1H, J ¼ 8.2 Hz), 6.50 (d, 1H, J ¼ 15.6 Hz). Anal.
Calc for C15H12FNO3: C, 65.93; H, 4.43; N, 5.13%. Found: C, 65.68; H,
4.80; N, 4.97%.
(s, 1H), 9.15 (s, 1H), 7.40 (d, 1H, J ¼ 15.6 Hz), 7.30–7.35 (m, 2H), 7.20
(m, 1H), 7.12 (d, 1H, J ¼ 2.1 Hz), 6.88 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 2.1 Hz),
6.76 (d, 1H, J ¼ 8.4 Hz), 6.66 (m, 1H), 6.52 (d, 1H, J ¼ 15.6 Hz), 3.92 (s,
3H). Anal. Calc for C16H15NO4: C, 67.36; H, 5.30; N, 4.91%. Found: C,
67.02; H, 5.61; N, 5.01%.
4.1.13. (E)-N-(4-Chlorophenyl)-3-(3,4-dihydroxyphenyl)
acrylamide (11)
4.1.20. (E)-3-(3,4-Dihydroxyphenyl)-N-(4-methoxyphenyl)
acrylamide (18)
Pale yellow powder, yield 76%, mp: 195–196 ꢁC, ESI MS: 290.0
Yellow powder, yield 77%, mp: 201–202 ꢁC, ESI MS: 286.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d
ppm): 10.02 (s, 1H), 9.43
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 9.98 (s,1H), 9.39 (s,
(s, 1H), 9.14 (s, 1H), 7.60 (d, 2H, J ¼ 8.2 Hz), 7.44 (d, 2H, J1 ¼ 8.2 Hz),
7.37 (d, 1H, J ¼ 15.4 Hz), 7.00 (d, 1H, J ¼ 2.1 Hz), 6.88 (dd, 1H,
J1 ¼ 8.2 Hz, J2 ¼ 2.1 Hz), 6.78 (d, 1H, J ¼ 8.2 Hz), 6.51 (d, 1H,
J ¼ 15.4 Hz). Anal. Calc for C15H12ClNO3: C, 62.19; H, 4.17; N, 4.83%.
Found: C, 62.70; H, 4.23; N, 4.35%.
1H), 9.02 (s, 1H), 7.52 (d, 2H, J ¼ 8.2 Hz), 7.38 (d, 1H, J ¼ 15.6 Hz),
7.06 (d, 1H, J ¼ 2.1 Hz), 6.96 (d, 2H, J ¼ 8.2 Hz), 6.83 (dd, 1H,
J1 ¼ 8.5 Hz, J2 ¼ 2.3 Hz), 6.72 (d, 1H, J ¼ 8.5 Hz), 6.48 (d, 1H,
J ¼ 15.6 Hz), 3.86 (s, 3H). Anal. Calc for C16H15NO4: C, 67.36; H, 5.30;
N, 4.91%. Found: C, 67.54; H, 5.62; N, 4.72%.
4.1.14. (E)-N-(4-Bromophenyl)-3-(3,4-dihydroxyphenyl)
acrylamide (12)
4.1.21. (E)-3-(3,4-Dihydroxyphenyl)-N-(2-nitrophenyl)
acrylamide (19)
Yellow powder, yield 75%, mp: 208–209 ꢁC, ESI MS: 334.0
Yellow powder, yield 74%, mp: 200–201 ꢁC, ESI MS: 301.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d
ppm): 10.11 (s, 1H), 9.45
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.16 (s, 1H), 9.54
(s, 1H), 9.16 (s, 1H), 7.56 (d, 2H, J ¼ 8.3 Hz), 7.48 (d, 2H, J ¼ 8.3 Hz),
7.38 (d, 1H, J ¼ 15.6 Hz), 7.02 (d, 1H, J ¼ 2.2 Hz), 6.70 (dd, 1H,
J1 ¼ 8.2 Hz, J2 ¼ 2.2 Hz), 6.80 (d, 1H, J ¼ 8.3 Hz), 6.52 (d, 1H,
J ¼ 15.6 Hz). Anal. Calc for C15H12BrNO3: C, 53.91; H, 3.62; N, 4.19%.
Found: C, 53.36; H, 3.21; N, 4.62%.
(s, 1H), 9.28 (s, 1H), 8.46 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 2.2 Hz), 8.18 (dd,
1H, J1 ¼ 8.3 Hz, J2 ¼ 2.1 Hz), 7.68–7.74 (m, 2H), 7.38 (d, 1H,
J ¼ 15.5 Hz), 7.05 (d, 1H, J ¼ 2.0 Hz), 6.88 (dd, 1H, J1 ¼ 8.2 Hz,
J2 ¼ 2.0 Hz), 6.78 (d, 1H, J ¼ 8.2 Hz), 6.52 (d, 1H, J ¼ 15.5 Hz). Anal.
Calc for C15H12N2O5: C, 60.00; H, 4.03; N, 9.33%. Found: C, 60.34; H,
4.38; N, 9.01%.
4.1.15. (E)-3-(3,4-Dihydroxyphenyl)-N-o-tolylacrylamide (13)
Yellow powder, yield 73%, mp: 196–197 ꢁC, ESI MS: 270.1 [M þ H]þ,
4.1.22. (E)-3-(3,4-Dihydroxyphenyl)-N-(3-nitrophenyl)
acrylamide (20)
1H NMR(300 MHz, DMSO-d6,
d ppm): 10.02(s,1H), 9.44 (s,1H), 9.18 (s,
1H), 7.42 (d,1H, J¼ 15.5Hz), 7.23–7.35(m, 3H), 7.07(m,1H), 7.00(d,1H,
J ¼ 2.0 Hz), 6.90 (dd,1H, J1 ¼8.3 Hz, J2 ¼1.9 Hz), 6.78 (d,1H, J ¼ 8.3 Hz),
6.53 (d,1H, J ¼ 15.5 Hz), 2.16 (s, 3H). Anal. Calc for C16H15NO3: C, 71.36;
H, 5.61; N, 5.20%. Found: C, 71.52; H, 5.31; N, 5.39%.
White powder, yield 72%, mp: 201–202 ꢁC, ESI MS: 301.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.12 (s, 1H), 9.51
(s, 1H), 9.14 (s, 1H), 8.66 (m, 1H), 7.88–7.90 (m, 2H), 7.58 (m, 1H),
7.39 (d, 1H, J ¼ 15.4 Hz), 7.12 (d, 1H, J ¼ 2.1 Hz), 6.85 (dd, 1H,
J1 ¼ 8.2 Hz, J2 ¼ 2.0 Hz), 6.76 (d, 1H, J ¼ 8.2 Hz), 6.49 (d, 1H,
J ¼ 15.5 Hz). Anal. Calc for C15H12N2O5: C, 60.00; H, 4.03; N, 9.33%.
Found: C, 60.25; H, 4.32; N, 9.25%.
4.1.16. (E)-3-(3,4-dihydroxyphenyl)-N-m-tolylacrylamide (14)
White powder, yield 78%, mp: 200–201 ꢁC, ESI MS: 270.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.12 (s, 1H), 9.50
(s, 1H), 9.22 (s, 1H), 7.60 (m, 1H), 7.48 (m, 1H), 7.38 (d, 1H,
J ¼ 15.6 Hz), 7.25 (m, 1H), 6.98 (d, 1H, J ¼ 2.1 Hz), 6.95 (m, 1H), 6.90
(dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 2.1 Hz), 6.79 (d, 1H, J ¼ 8.3 Hz), 6.53 (d, 1H,
J ¼ 15.6 Hz), 2.28 (s, 3H). Anal. Calc for C16H15NO3: C, 71.36; H, 5.61;
N, 5.20%. Found: C, 71.62; H, 5.52; N, 5.25%.
4.1.23. (E)-3-(3,4-Dihydroxyphenyl)-N-(4-nitrophenyl)
acrylamide (21)
Colorless needle, yield 71%, mp: 199–200 ꢁC, ESI MS: 301.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.02 (s, 1H), 9.48
(s, 1H), 9.24 (s, 1H), 8.16 (d, 2H, J ¼ 8.3 Hz), 7.78 (d, 2H, J ¼ 8.3 Hz),
7.34 (d, 1H, J ¼ 15.3 Hz), 7.08 (d, 1H, J ¼ 2.1 Hz), 6.85 (dd, 1H,
J1 ¼ 8.3 Hz, J2 ¼ 2.1 Hz), 6.72 (d, 1H, J ¼ 8.3 Hz), 6.44 (d, 1H,
J ¼ 15.3 Hz). Anal. Calc for C15H12N2O5: C, 60.00; H, 4.03; N, 9.33%.
Found: C, 60.41; H, 3.93; N, 9.45%.
4.1.17. (E)-3-(3,4-Dihydroxyphenyl)-N-p-tolylacrylamide (15)
Pale yellow crystal, yield 77%, mp: 199–200 ꢁC, ESI MS: 270.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 9.96 (s, 1H), 9.42 (s,
1H), 9.16 (s, 1H), 7.57 (d, 2H, J ¼ 8.2 Hz), 7.38 (d, 1H, J ¼ 15.6 Hz), 7.12
(d, 2H, J ¼ 8.0 Hz), 7.00 (d, 1H, J ¼ 2.1 Hz), 6.90 (dd, 1H, J1 ¼ 8.2 Hz,
J2 ¼ 2.1 Hz), 6.78 (d, 1H, J ¼ 8.2 Hz), 6.53 (d, 1H, J ¼ 15.5 Hz), 2.26 (s,
3H). Anal. Calc for C16H15NO3: C, 71.36; H, 5.61; N, 5.20%. Found: C,
71.61; H, 5.41; N, 5.02%.
4.1.24. (E)-3-(3,4-Dihydroxyphenyl)-N-methyl-N-
phenylacrylamide (22)
White powder, yield 80%, mp: 190–191 ꢁC, ESI MS: 270.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 9.42 (s, 1H), 9.16 (s,
1H), 7.77 (dd, 2H, J1 ¼ 8.3 Hz, J2 ¼ 2.0 Hz), 7.50 (d, 1H, J ¼ 15.6 Hz),
7.32 (m, 2H), 7.10 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 2.0 Hz), 7.04 (d, 1H,
J ¼ 2.0 Hz), 6.80 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 1.9 Hz), 6.77 (d, 1H,
J ¼ 8.3 Hz), 6.50 (d, 1H, J ¼ 15.6 Hz), 3.42 (s, 3H). Anal. Calc for
C16H15NO3: C, 71.36; H, 5.61; N, 5.20%. Found: C, 71.50; H, 5.18; N,
5.14%.
4.1.18. (E)-3-(3,4-Dihydroxyphenyl)-N-(2-methoxyphenyl)
acrylamide (16)
Pale yellow powder, yield 75%, mp: 205–206 ꢁC, ESI MS: 286.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 10.14 (s, 1H), 9.52
(s, 1H), 9.26 (s, 1H), 7.56 (dd, 1H, J1 ¼ 8.5 Hz, J2 ¼ 2.3 Hz), 7.40 (d, 1H,
J ¼ 15.6 Hz), 7.06 (d, 1H, J ¼ 2.0 Hz), 6.99–7.05 (m, 3H), 6.90 (dd, 1H,
J1 ¼ 8.3 Hz, J2 ¼ 2.0 Hz), 6.78 (d, 1H, J ¼ 8.3 Hz), 6.54 (d, 1H,
J ¼ 15.6 Hz), 3.96 (s, 3H). Anal. Calc for C16H15NO4: C, 67.36; H, 5.30;
N, 4.91%. Found: C, 67.57; H, 5.32; N, 5.09%.
4.1.25. (E)-3-(3,4-Dihydroxyphenyl)-N,N-diphenylacrylamide (23)
White powder, yield 83%, mp: 191–192 ꢁC, ESI MS: 332.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 9.48 (s, 1H), 9.18 (s,

J. Fu et al. / European Journal of Medicinal Chemistry 45 (2010) 2638–2643
2643
1H), 7.52–7.58 (m, 8H), 7.46 (d, 1H, J ¼ 15.6 Hz), 7.18 (dd, 2H,
J1 ¼ 8.3 Hz, J2 ¼ 2.2 Hz), 7.02 (d, 1H, J ¼ 2.2 Hz), 6.80 (dd, 1H,
J1 ¼ 8.3 Hz, J2 ¼ 2.2 Hz), 6.76 (d, 1H, J ¼ 8.3 Hz), 6.52 (d, 1H,
J ¼ 15.6 Hz). Anal. Calc for C21H17NO3: C, 76.12; H, 5.17; N, 4.23%.
Found: C, 76.06; H, 5.24; N, 4.61%.
compounds were incorporated in specified quantity of sterilized
liquid medium (MH medium for antibacterial activity and RPMI-
1640 medium for antifungal activity). A specified quantity of the
medium containing the test compound was poured into micro-
titration plates. Suspension of the microorganism was prepared to
contain approximately 105 cfu/mL and applied to micro-
timicrotitration plates with serially diluted compounds in DMSO to
be tested and incubated at 37 ꢁC for 24 h and 48 h for bacteria and
fungi, respectively. After the MICs were visually determined on
4.1.26. (E)-N-Ethyl-3-(4-hydroxyphenyl)acrylamide (24)
White powder, yield 75%, mp: 139–140 ꢁC, ESI MS: 192.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 9.32 (s, 1H), 8.91 (s,
1H), 7.55 (d, 2H, J ¼ 8.2 Hz), 7.47 (d, 1H, J ¼ 15.8 Hz), 6.67 (d, 1H,
J ¼ 8.2 Hz), 6.36 (d, 1H, J ¼ 15.9 Hz), 4.02 (q, 2H, J ¼ 6.8 Hz), 1.18 (t,
3H, J ¼ 7.0 Hz). Anal. Calc for C11H13NO2: C, 65.09; H, 6.85; N, 7.32%.
Found: C, 65.42; H, 8.55; N, 7.42%.
each of the microtitration plates, 50 mL of PBS containing 2 mg of
MTT/mL was added to each well. Incubation was continued at room
temperature for 4–5 h. The content of each well was removed, and
100
mL of isopropanol containing 5% 1 mol/L HCl was added to
extract the dye. After 12 h of incubation at room temperature, the
optical density (OD) was measured with a microplate reader at
550 nm.
4.1.27. (E)-3-(4-Hydroxyphenyl)-N-phenylacrylamide (25)
Pale yellow crystal, yield 75%, mp: 197–198 ꢁC, ESI MS: 240.1
[M þ H]þ, 1H NMR (300 MHz, DMSO-d6,
d ppm): 9.44 (s, 1H), 9.24 (s,
1H), 7.61 (d, 2H, J1 ¼ 8.2 Hz, J2 ¼ 2.0 Hz), 7.55 (d, J ¼ 8.2 Hz), 7.48 (d,
1H, J ¼ 15.6 Hz), 7.32 (m, 2H), 7.19 (dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 2.0 Hz),
6.68 (d, 1H, J ¼ 8.3 Hz), 6.54 (d, 1H, J ¼ 15.6 Hz). Anal. Calc for
C15H13NO2: C, 75.30; H, 5.48; N, 5.85%. Found: C, 75.65; H, 5.62; N,
5.64%.
Acknowledgements
The authors are very grateful to the editor and anonymous
reviewers for their invaluable comments and suggestions. This
work was supported by the Jiangsu National Science Foundation
(No. BK2009239 ) and Anhui National Science Foundation (No.
070416274X).
4.2. Crystallographic studies
X-ray single-crystal diffraction data for compound 15 were
collected on a Bruker SMART APEX CCD diffractometer at 293 (2) K
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The antibacterial activities of the synthesized compounds were
tested against B. subtilis ATCC 6633, E. coli ATCC 35218, P. fluorescens
ATCC 13525 and S. aureus ATCC 6538 using Mueller–Hinton (MH)
medium. The antifungal activities of the compounds were tested
against A. niger ATCC 16404, C. albicans ATCC 10231, and T. rubrum
ATCC 16404, using RPMI-1640 medium. The MICs of the test
compounds were determined by a colorimetric method using the
dye MTT [14]. A stock solution of the synthesized compounds
(50 mg/mL) in DMSO was prepared and graded quantities of the test