Structural modification of a specific antimicrobial lead against Helicobacter pylori discovered from traditional Chinese medicine and a structure-activity relationship study

Article abstract of DOI:10.1016/j.ejmech.2010.08.045

Psoralen (1a) was found to be a specific and potent antimicrobial lead against Helicobacter pylori (H. pylori) from a traditional Chinese medicine (TCM) in the bioassay directed isolation. A series of structurally diverse analogues of 1a were thus designed and synthesized to improve the antimicrobial potency, some of which showed more potent activities than the lead compound (1a) against H. pylori. Among them, compound 25a is 16-fold stronger (MIC = 0.39 μg/mL) than 1a (MIC = 6.25 μg/mL), and is even potent than the positive control metronidazole (MIC = 0.50 μg/mL). The in vitro antimicrobial activities against H. pylori of these structurally diverse analogues based on the scaffold of 1a have also led to an outline of structure-activity relationship.

Full text of DOI:10.1016/j.ejmech.2010.08.045

European Journal of Medicinal Chemistry 45 (2010) 5258e5264
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structural modification of a specific antimicrobial lead against Helicobacter pylori
discovered from traditional Chinese medicine and a structureeactivity
relationship study
,
,
Bang-Le Zhang a b, Cheng-Qi Fan a, Lei Dong a, Fang-Dao Wang a, Jian-Min Yue a
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park,
Shanghai 201203, PR China
b School of Pharmacy, Fourth Military Medical University, Xi’an, Shaanxi 710032, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Psoralen (1a) was found to be a specific and potent antimicrobial lead against Helicobacter pylori (H.
pylori) from a traditional Chinese medicine (TCM) in the bioassay directed isolation. A series of struc-
turally diverse analogues of 1a were thus designed and synthesized to improve the antimicrobial
potency, some of which showed more potent activities than the lead compound (1a) against H. pylori.
Received 31 May 2010
Received in revised form
2 August 2010
Accepted 22 August 2010
Available online 15 September 2010
Among them, compound 25a is 16-fold stronger (MIC ¼ 0.39
m
g/mL) than 1a (MIC ¼ 6.25
mg/mL), and is
g/mL). The in vitro antimicrobial
even potent than the positive control metronidazole (MIC ¼ 0.50
m
activities against H. pylori of these structurally diverse analogues based on the scaffold of 1a have also led
to an outline of structureeactivity relationship.
Keywords:
Anti-Helicobacter pylori lead
Structural modification
SAR study
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
infection will continue to evolve, and the discovery of alternative
agents with specific activity against H. pylori will be continuously
Helicobacter pylori (H. pylori), a ubiquitous Gram-negative spiral
bacterium, has been recognized as a vital pathogen which induces
chronic gastritis, and is associated with gastroduodenal ulcer, gastric
adenocarcinoma, and mucosa-associated lymphoid tissue (MALT)
lymphoma [1e7]. The eradication of H. pylori in the infected patients
has been demonstrated to be effective for the treatment of both
chronic active gastritis and gastroduodenal ulcer [8e10]. Treatment
regimens against H. pylori infection currently employed in the first-
line therapy are combinations of one proton pump inhibitor and/or
bismuth, and two or three antibiotics [11]. Although optimal combi-
nation regimens commonly provide high cure rates, the rising prev-
alence of drug resistance to the currently used antibiotic components
increasingly threatens to compromise the efficacy of these treatment
regimens. The high dosages of these combination therapy regimens
also disrupt the natural population of commensal microorganisms in
the gastrointestinal tract, leading to the undesired side effects
[12e14]. Therefore, therapeutic regimens directed against H. pylori
sought among synthetic compounds and natural products.
In an effort to discover antimicrobial components against H.
pylori from a traditional Chinese medicine (TCM), the ethanolic
extract of the seeds of Psoralea corylifolia, which is a well-known
TCM, namely “buguzhi”, was found to be active [15,16]. Bioassay-
directed fractionation of the ethanolic extract has led to the isolation
of a class of compounds, furocoumarins 1aec (Fig. 1) with potent
antimicrobial activities against H. pylori. Although furocoumarin
analogues 1aec are known compounds, their antimicrobial activi-
ties against H. pylori have not been reported previously. The in vitro
antimicrobial activities of these natural isolates 1aec against H.
pylori (MICs of 1aec were 6.25, 25 and 25
mg/mL, respectively)
suggested that the linear furocoumarin, psoralen (1a) as the lead
compound is more favorable than the angular furocoumarin, iso-
psoralen (1c). The antimicrobial tests of 1a against a series of
microbes including Gram-positive and Gram-negative bacteria, and
fungi indicated that it is a specific antimicrobial agent of H. pylori.
Therefore, psoralen (1a) was served as the lead structure in the
current study. Herein, we present the structural modification of
psoralen 1a, the in vitro antimicrobial evaluation of its structurally
modified analogues, and a structureeactivity relationship study.
* Corresponding author. Tel./fax: þ86 21 50806718.
E-mail address: jmyue@mail.shcnc.ac.cn (J.-M. Yue).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.045

B.-L. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 5258e5264
5259
5
4
i or ii or iii
6
7
10
9
3'
R
3
2
2'
A
B
C
O
O
O
O
O
O
O
O
O
2a-c
O
O
O
8
1a
1'
1
R
R = Br, Cl, NO2,
iv
1a: R= H,
psoralen
1c isopsoralen
1b: R= OMe, xanthotoxin
Fig. 1. The anti-H. pylori structures of furocoumarins 1aec.
v
ClH2C
RH2C
O
O
O
O
O
O
2. Results and discussion
2.1. Chemistry
3
4a-e
R = OMe, OEt, OBu-n,
OAc, NEt2
To obtain structurally diverse analogues of 1a for more potent
antimicrobial components against H. pylori, and to understand the
structureeactivity relationship of this class of compounds,
a systematic modification throughout the AeC rings of linear
furocoumarin 1a was thus conducted (Schemes 1e7), and a series
of structural analogues based on the scaffold of 1a were synthe-
sized (Tables 1e3).
Scheme 1. Preparation of 20-substituted furocoumarin analogues from psoralen 1a.
Reagents and conditions: (i) NBS, CCl4, refluxing, 9 h; (ii) NCS, CCl4, refluxing, 9 h; (iii)
HNO3, HOAc, 0 ꢀCert; (iv) MOMCl, HOAc, rt, 36 h; (v) for 4aec: ROH (R ¼ Me, Et, n-Bu),
refluxing; for 4d: NaOAc, HOAc, Ac2O, 110 ꢀC, 3 h; for 4e: Et2NH, anhydrous K2CO3,
acetone, refluxing, 18 h.
2-hydroxysuccinic acid via Pechmann reaction, compound 18 was
produced. When reacted with 3-chloropropane-1,2-diol in the
presence of anhydrous K2CO3, 18 was converted to 19, which was
then treated with NaIO4 supported on SiO2, and the resultant was
refluxed in 1 M aqueous NaOH to yield the desired compound 20.
2.1.1. Modification of A-ring of linear furocoumarin 1a
Starting from psoralen 1a, a series of 20-substituted furo-
coumarin analogues have been prepared (Scheme 1). Psoralen 1a
was refluxed with NBS or NCS in CCl4 to yield the corresponding
halogenated derivatives 2a or 2b. Psoralen 1a was treated with
nitric acid in the presence of HOAc to produce a nitro-substituted
compound 2c. Derivative 3 was obtained by the chloromethylation
2.1.3. Modification of C-ring of linear furocoumarin 1a
A number of C-ring modified derivatives of psoralen 1a were
prepared via total synthesis (Scheme 6). Starting from 2,4-dihy-
droxybenzaldehyde, the linear furocoumarin derivatives 25aee
with different substituents at the C-4 of C-ring were prepared in
a moderate to high yields [21e23]. The preparation and the spec-
troscopic data of intermediates 21e24 were described in the liter-
ature [21].
Based on the compound 25a, C-4 substituted analogues 26e28
were further prepared (Scheme 7). Oxidation of compound 25a
with SeO2 afforded an aldehyde 26 in 69% yield. Compound 26 was
converted into compound 27a in 84% yield by treating with sodium
borohydride in MeOH at room temperature. With 27a at hand, the
linear furocoumarin analogues 27b and 28 were readily prepared.
of 1a via
a standard procedure [17] using HCHOeHCl or
MOMCleHOAc. When compound 3 was refluxed in the appropriate
alcohols, such as methanol, ethanol and n-butanol, compounds
4aec were afforded respectively. With compound 3 at hand, furo-
coumarin derivatives 4d and 4e were also prepared readily.
In addition, 30-methyl-psoralen (7) was synthesized (Scheme 2).
Starting from resorcinol and 2-hydroxysuccinic acid, 7-hydrox-
ycoumarin (5) was prepared via Pechmann reaction, which was
then converted to an
a-keto ether 6 by Williamson reaction
refluxing with bromoacetone at the presence of anhydrous K2CO3
in acetone. The desired compound 7 was finally produced by
refluxing 6 in 0.5 M aqueous NaOH for 18 h [18].
2.2. Antimicrobial evaluation
2.1.2. Modification of B-ring of linear furocoumarin 1a
Starting from xanthotoxin 1b, a series of the central aromatic B-
ring modified derivatives of linear furocoumarin 1a have been
prepared (Scheme 3). Xanthotoxin 1b was converted to 8 by using
BBr3 as the O-demethylation reagent, and then compounds 9aed
were facilely synthesized from 8. The B-ring modified analogues
11aeg were prepared from 1b according to the same conditions
used in the preparation of 4a. More additional B-ring modified
analogues 12aec, 13a and b, 14 and 15 with electron-donating or
electron-drawing groups at C-5 were also prepared (Scheme 3).
Sulfonamide or sulfonic acid ester moiety played an important
role in the design and synthesis of antibiotics. Attia and coworkers
[19,20] reported that introduction of sulfonamide or sulfonic acid
ester moieties into furocoumarins could enhance the activities
again microbes, such as Staphylococcus aureus, Escherichia coli and
Bacillus subtilis. A number of sulfonic acid esters and sulfonamides
of xanthotoxin were thus synthesized (Scheme 4). Xanthotoxin 1b
was reacted with chlorosulfonic acid in CHCl3 to yield sulfonic acid
chloride 16, which was then refluxed with the corresponding
alcohols (methanol and phenyl alcohols) or amines to give sulfonic
acid esters 17aeb or sulfonamides 17cef, respectively.
The minimum inhibitory concentrations (MICs) of lead
compounds 1aec and their synthetic analogues against H. pylori
were determined by an agar dilution method according to the
standard protocol, and commercially available antibiotic metroni-
dazole was used as the positive control. The minimum inhibitory
O
HO
i
OH
OH
HO
HO
O
O
OH
O
5
O
iii
ii
O
O
O
O
O
O
6
7
Scheme 2. Preparation of 30-methyl psoralen 7. Reagents and conditions: (i) H2SO4, rt,
30 min; 120e130 ꢀC, 2.5 h; (ii) bromoacetone, anhydrous K2CO3, acetone, refluxing,
18 h; (iii) 0.5 M aq. NaOH, refluxing, 18 h.
Moreover, 5-methyl-psoralen 20 was synthesized (Scheme 5).
Starting from the materials of 5-methylbenzene-1,3-diol and

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B.-L. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 5258e5264
O
i
ii
HO
O
O
O
i
O
O
O
O
OH
OH
O
OCH3
O
OH
OR
9a-d
HO
HO
O
O
OH
1b
8
O
18
R = COCH3, CH2CH3,
CH2CO2Et, CH2CO2H
OH
O
HO
CH2Cl
CH2R
ii
iii
iii
iv
O
1b
O
O
O
O
O
O
O
O
O
O
20
19
OCH3
OCH3
Scheme 5. Preparation of 5-methyl psoralen 20. Reagents and conditions: (i) H2SO4, rt,
30 min; 100 ꢀC, 1.5 h; (ii) anhydrous K2CO3, CH3CN, refluxing, 30 min; ClCH2CH(OH)
CH2OH, cat.KI, refluxing, 11 h; (iii) NaIO4/SiO2, CH2Cl2, rt; 1 M aq. NaOH, refluxing,
3.5 h.
11a-g
10
R = OMe, OEt, OPr-n, OBu , NEt2,
-n
NHPh-p-NO2, NHPh-p-OMe
R
R
The activities of C-5 and/or C-8 modified analogues of psoralen
1a against H. pylori were presented in Table 2. Most of the C-5 and/
or C-8 modified analogues of psoralen 1a exhibited decreased
activities as compared with the lead 1a. The introduction of sulfonic
acid ester or sulfonamide group did not enhance the activity, but
decreased the activity against H. pylori, which was different from
the cases of inhibition on other bacteria as reported [19,20]. Inter-
estingly, compound 12a with a nitro group at C-5 and a methoxy at
C-8 showed significantly increased activity against H. pylori
vi (R = NO2)
O
O
v
1b
O
O
O
O
OCH3
OCH3
13a R = NH2
12a-c R = NO2, Br, Cl
vii
viii (R = NO2)
13b R = -N=CH2Ph
NO2
NO2
(MIC ¼ 1.56
mg/mL, four-fold stronger than the lead 1a). While,
ix
when the nitro group at C-5 of 12a was reduced to an amine (13a),
or the methoxy at C-8 of 12a was demethylated to a hydroxy (14),
the antimicrobial activities of 13a and 14 against H. pylori were
dramatically decreased.
O
O
O
O
O
O
OAc
OH
14
15
The MICs of the C-4 modified analogues of psoralen 1a against H.
pylori were showed in Table 3. The antimicrobial activities of the
modified analogues 25a, 26, 27a, 27b and 28 against H. pylori were
potentially increased as compared with the lead 1a. Among them,
the C-4 methyl substituted compound 25a exhibited the strongest
inhibitory effect against the targeted microbe with the MIC value of
Scheme 3. Preparation of C-5/and C-8-substituted analogues of linear furocoumarin
1a. Reagents and conditions: (i) BBr3, CH2Cl2, 0 ꢀCert, 10 h; (ii) for 9a: Ac2O, DMAP, py.,
rt, 24 h; for 9b: EtI, anhydrous K2CO3, acetone, refluxing, overnight; for 9c:
BrCH2CO2Et, K2CO3, DMF, rt, 18 h; for 9d: 9c, LiOH, EtOH-H2O, 0 ꢀC; (iii) MOMCl, HOAc,
rt, 14 h; (iv) for 11aed: ROH (R ¼ Me, Et, n-Pr, n-Bu), refluxing; for 11eeg: Et2NH (p-
NO2PhNH2 or p-MeOPhNH2), K2CO3, acetone, refluxing, 3 h; (v) for 12a: HNO3, HOAc,
0
ꢀCert; for 12bec: NBS or NCS, CCl4, refluxing, 7 h; (vi) SnCl2$2H2O, refluxing, 1 h;
0.39 mg/mL, which is 16-fold potent than the lead 1a, and even
stronger than the positive control metronidazole.
(vii) PhCHO, EtOH, refluxing; (viii) BBr3, CH2Cl2, 0 ꢀCert; (ix) Ac2O, DMAP, py., rt, 24 h.
The lead 1a and its two promising anti-H. pylori derivatives 12a
and 25a were also tested against a lot of microbes, including four
strains of Gram-positive bacteria, three strains of Gram-negative
bacteria and four strains of fungi (Table 4). The antimicrobial tests
of three compounds 1a, 12a and 25a showed all inactive against the
selected eleven strains of microbes, indicating that they are potent
and specific agents against H. pylori.
concentration (MIC) was defined as the lowest concentration that
completely inhibited the visible growth of H. pylori.
The antimicrobial activities of the C-20 and C-30 modified
analogues of psoralen 1a against H. pylori were showed in Table 1.
The compounds 2a and 2b with substituents of bromine and chlo-
rine, respectively, at C-20 of psoralen 1a displayed inhibitory activity
against H. pylori at the same level as the lead compound 1a, while
compound 2c with a substituent of eNO2 at C-20 was two-fold
stronger than compound 1a. However, the other modified analogues
3 and 4aee bearing a eCH2R group at C-20 were inactive against H.
pylori. Compared with the lead 1a, the modified analogue 7 with
a methyl group at C-30 showed decreased activity against H. pylori.
The results of antimicrobial tests against H. pylori of the lead
compound and its structurally diverse derivatives (Tables 1e3)
O2N
O2N
OHC
HO
i
ii
HO
OH
OH
HO
22
OH
21
SO2R
SO2Cl
i
ii
R
O
O
O
O
O
O
OCH3
O
O
OCH3
O
O
O
iii
iv
OCH3
H
O
O
OH
OH
O
HO
17a-f
1b
16
24
23
25a: R= Me 25b: R= CF3
25c: R= Et 25d: R= n-Pr
25e: R= Ph
R = OMe, OPh, NH2, NEt2,
NHPh-p-NO2, NHPh-p-OMe
Scheme 4. Synthesis of C-5-sulphonic acid esters or sulfonamides of xanthotoxin.
Reagents and conditions: (i) HSO3Cl, CHCl3, 0 ꢀC; (ii) for 17a: MeOH, refluxing, 1.5 h; for
17b: PhOH, anhydrous K2CO3, CHCl3, refluxing, 4.5 h; for 17c: NH3$H2O, rt, 30 min;
60 ꢀC, 30 min; for 17def: Et2NH (p-NO2PhNH2 or p-MeOPhNH2), py., toluene, refluxing.
Scheme 6. Preparation of C-4 substituted psoralen analogues 25aee via total
synthesis. Reagents and conditions: (i) CH3NO2, HOAc-NH4OAc, 120 ꢀC, 45 min; (ii)
NaBH4, i-PrOH-THF (1:4), rt, 40 min; (iii) Nef reaction; (iv)
R ¼ Me, CF3, Et, n-Pr, and Ph), CH3SO3H.
b-ketoesters (RCOCH2CO2Et:

B.-L. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 5258e5264
5261
Table 2
CHO
Activities of C-5 and/or C-8 modified analogues of psoralen 1a.
ii
i
R1
O
O
O
O
O
R
O
O
O
O
26
25a
O
O
O
CH2Br
R2
iv
O
O
O
28
MIC (m
g/mL)a
27a: R=CH2OH
Entry
Serial number
Compounds
1
2
3
4
5
6
7
8
e
Metronidazole
R1 ¼ H, R2 ¼ H
0.50
6.25
12.5
25
>50
>50
>50
>50
>50
25
>50
>50
>50
50
>50
>50
12.5
1.56
>50
>50
25
>50
25
25
>50
>50
>50
>50
>50
>50
iii
1a
20
1b
8
9a
9b
9c
9d
R1 ¼ Me, R2 ¼ H
R1 ¼ H, R2 ¼ OMe
R1 ¼ H, R2 ¼ OH
R1 ¼ H, R2 ¼ OAc
R1 ¼ H, R2 ¼ OEt
27b: R=CH2OAc
Scheme 7. Preparation of C-4 substituted psoralen derivatives 26e28 from 25a.
Reagents and conditions: (i) SeO2, xylene, refluxing, 18 h; (ii) NaBH4, MeOH, rt, 30 min;
(iii) Ac2O, DMAP, py., rt, overnight; (iv) PBr3, PPh3, THF, rt, 2 h.
R1 ¼ H, R2 ¼ OCH2CO2Et
R1 ¼ H, R2 ¼ OCH2CO2H
R1 ¼ CH2Cl, R2 ¼ OMe
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
11a
11b
11c
11d
11e
11f
11g
12a
12b
12c
13a
13b
14
R1 ¼ CH2OMe, R2 ¼ OMe
R1 ¼ CH2OEt, R2 ¼ OMe
R1 ¼ CH2OPr-n, R2 ¼ OMe
R1 ¼ CH2OBu-n, R2 ¼ OMe
R1 ¼ CH2N(Et)2, R2 ¼ OMe
R1 ¼ CH2NHPh(p-NO2), R2 ¼ OMe
R1 ¼ CH2NHPh(p-OMe), R2 ¼ OMe
R1 ¼ NO2, R2 ¼ OMe
have offered an outline of structureeactivity relationship, which
are: (1) An electron-drawing group (eNO2) at C-20 (2c) will
enhance the activity (two-fold stronger than 1a), and an electron-
donating group (eCH2R) at C-20 will render the analogues (3 and
4aee) inactive. (2) An electron-donating methyl group at C-30 will
slightly decrease activity against H. pylori. (3) A strong electron-
drawing group (eNO2) at C-5 and an electron-donating methoxy
at C-8 (12a) will significantly increase the activity against H. pylori
(four-fold stronger than the lead 1a), on the contrary, it is detri-
mental to the antimicrobial activities (13a, 14 and other examples
in Table 2). (4) It seems that a small electron-donating group at C-
4 (25a, 26, 27a, 27b and 28) will dramatically increase the activ-
ities against H. pylori, especially the analogue 25a, which is 16-fold
R1 ¼ Br, R2 ¼ OMe
R1 ¼ Cl, R2 ¼ OMe
R1 ¼ NH2, R2 ¼ OMe
R1 ¼ -N¼CHPh, R2 ¼ OMe
R1 ¼ NO2, R2 ¼ OH
15
R1 ¼ NO2, R2 ¼ OAc
17a
17b
17c
17d
17e
17f
R1 ¼ SO3Me, R2 ¼ OMe
R1 ¼ SO3Ph, R2 ¼ OMe
R1 ¼ SO2NH2, R2 ¼ OMe
R1 ¼ SO2N(Et)2, R2 ¼ OMe
R1 ¼ SO2NHPh(p-NO2), R2 ¼ OMe
R1 ¼ SO2NHPh(p-OMe), R2 ¼ OMe
stronger (MIC ¼ 0.39
and is even slightly potent than the positive control metronida-
g/mL). Replacement of the C-4 methyl group of
m
g/mL) than the lead 1a (MIC ¼ 6.25
mg/mL),
zole (MIC ¼ 0.50
m
25a with more bulky groups (25cee) will slightly reduce the
activity against H. pylori. Furthermore, a furocoumarin derivative
25b with a trifluoromethyl group at C-4 also attenuated the
activity.
a
MIC was defined as the lowest concentration that completely inhibited the
visible growth of H. pylori.
Table 1
Table 3
Activities of C-20 or C-30 modified analogues of psoralen 1a.
Activities of C-4 modified analogues of psoralen 1a.
R2
R
R1
O
O
O
O
O
O
Entry
Serial number
Compounds
MIC (m
g/mL)a
Entry
Serial number
Compounds
MIC (m
g/mL)a
1
2
3
4
5
6
7
8
e
metronidazole
R1 ¼ R2 ¼ H
0.50
6.25
6.25
6.25
3.12
1a
2a
2b
2c
3
4a
4b
4c
4d
4e
7
1
2
3
4
5
6
7
8
e
metronidazole
R ¼ H
0.50
6.25
0.39
12.5
6.25
12.5
12.5
1.56
R1 ¼ Br, R2 ¼ H
R1 ¼ Cl, R2 ¼ H
R1 ¼ NO2, R2 ¼ H
1a
25a
25b
25c
25d
25e
26
27a
27b
28
R ¼ Me
R ¼ CF3
R1 ¼ CH2Cl, R2 ¼ H
R1 ¼ CH2OMe, R2 ¼ H
R1 ¼ CH2OEt, R2 ¼ H
R1 ¼ CH2OBu-n, R2 ¼ H
R1 ¼ CH2OAc, R2 ¼ H
R1 ¼ CH2N(Et)2, R2 ¼ H
R1 ¼ H, R2 ¼ Me
>50
R ¼ Et
50
>50
50
>50
>50
R ¼ Pr-n
R ¼ Ph
9
R ¼ CHO
R ¼ CH2OH
R ¼ CH2OAc
R ¼ CH2Br
10
11
12
9
10
11
0.78
1.56
1.56
12.5
a
a
MIC was defined as the lowest concentration that completely inhibited the
visible growth of H. pylori.
MIC was defined as the lowest concentration that completely inhibited the
visible growth of H. pylori.

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B.-L. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 5258e5264
Table 4
4.1.2. 2-Nitro-7H-furo[3,2-g]chromen-7-one (2c)
Effects of compounds 1a, 12a and 25a on other bacteria and fungi.
A mixture of furocoumarin 1a (80 mg, 0.43 mmol) and glacial
acetic acid (5 mL) was stirred strongly at 0 ꢀC, and a solution of nitric
acid (0.5 mL) in glacial acetic acid (2 mL) was then added dropwise.
The reaction mixture was stirred for 5 min, and then warmed up to
room temperature and further stirred until starting material was
fully reacted. The resulting mixture was diluted with 10 mL water,
and extracted with ethyl acetate (3 ꢂ 15 mL). The organic layer was
washed with water (3 ꢂ 10 mL) and brine (10 mL) in turn, and dried
over anhydrous Na2SO4. After removal of solvent, the residue was
purified by flash chromatography to give 2c (21 mg) in 21% yield. mp
Microbes
MIC (m
g/mL)a, b
1a
12a
25a
Staphylococcus aureus
Staphylococcus epdermidis
Micrococcus luteus
Bacillus subtilis
Escherichia coli
Shigella flexneri
Pseudomonas aeruginosa
Candida albicans
Saccaromyces sake
Trichophyton rubrum
Microsporum gypseum
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
240 ꢀC (dec.) [lit [24]. 244 ꢀC]; 1H NMR (400 MHz, DMSO-d6)
d 8.30
(d, J ¼ 0.6 Hz, 1H), 8.28 (s, 1H), 8.23 (d, J ¼ 9.6 Hz, 1H), 8.00 (d,
J ¼ 0.6 Hz, 1H), 6.55 (d, J ¼ 9.6 Hz, 1H); EI-MS m/z (%) 231 (Mþ, 100),
201 (85), 173 (55), 157 (25), 145 (30), 129 (30), 101 (25), 75 (30).
a
MIC was defined as the lowest concentration that completely inhibited the
visible growth of microbes.
b
MIC > 50 mg/mL was defined as inactive.
4.1.3. 2-(Chloromethyl)-7H-furo[3,2-g]chromen-7-one (3)
Furocoumarin 1a (300 mg, 1.61 mmol) was dissolved in 10 mL of
glacial acetic acid. And then chloromethyl ether (MOMCl, 2 mL) was
added to the mixture and stirred at room temperature for 36 h. The
reaction mixture was diluted with 20 mL water and extracted with
ethyl acetate (3 ꢂ 30 mL). The combined organic layer was washed
with water and brine, and dried over anhydrous Na2SO4. After
removal of solvent, the residue was purified by flash chromatog-
raphy over silica gel to give 221 mg of derivative 3 in 68% yield.
Compound 3: white solid, mp 164e166 ꢀC [lit [17]. 168e170 ꢀC]; 1H
3. Conclusions
In summary, a series of furocoumarin analogues based on the
specific lead compound 1a against H. pylori discovered from a TCM
(seeds of P. corylifolia) have been prepared via structural modifi-
cation or total synthesis. Compounds 2a, 2c, 12a, 25a, 26, 27a, 27b
and 28 exhibited potent activities against H. pylori. The in vitro
antimicrobial tests of these structurally diverse analogues against
H. pylori also have led to an outline of structureeactivity relation-
ship. It is noteworthy that analogue 25a is 16-fold stronger
NMR (400 MHz, CDCl3)
(s, 1H), 6.80 (s, 1H), 6.39 (d, J ¼ 10.0 Hz, 1H), 4.70 (s, 2H).
d
7.79 (d, J ¼ 10.0 Hz, 1H), 7.64 (s, 1H), 7.46
(MIC ¼ 0.39
slightly potent than the positive control metronidazole
g/mL). Compound 25a is a specific and promising
m
g/mL) than the lead 1a (MIC ¼ 6.25
mg/mL), and even
4.1.4. Analogues 4aec
(MIC ¼ 0.50
m
The chloromethyl derivative 3 (40 mg, 0.17 mmol) was dissolved
in 5 mL of appropriate alcohols (methanol, ethanol and n-butanol)
and stirred at refluxing until the starting material was consumed.
After workup, the crude products were purified by flash column
chromatography over silica gel to give the desired compounds 4aec.
antimicrobial candidate against H. pylori, and a further investiga-
tion into this structure and its analogues is warranted.
4. Experimental
4.1. Chemistry
4.1.4.1. 2-(Methoxymethyl)-7H-furo[3,2-g]chromen-7-one
(4a). Yield 83%. mp 114e115 ꢀC; 1H NMR (400 MHz, CDCl3)
d 7.79 (d,
Melting points were determined on a SGW X-4 melting point
apparatus and were uncorrected. 1H NMR spectra were recorded on
a Bruker AM-400 spectrometer using TMS as internal standard. IR
spectrawere recordedona NicoletMagna750 spectrometerandonly
characteristic absorptions were reported. EI mass spectra were per-
formed on a Finnigan MAT-95 spectrometer. THF was distilled with
sodium and benzophenone prior to use. DMF was distilled with
calcium hydride prior to use. All the oxygen or moisture sensitive
reactions were carried out under nitrogen atmosphere. Flash column
chromatography was performed with silica gel (200e300 mesh).
J ¼ 9.6 Hz,1H), 7.64 (s,1H), 7.45 (s,1H), 6.74 (s,1H), 6.38 (d, J ¼ 9.6 Hz,
1H), 4.57 (s, 2H), 3.47 (s, 3H); EI-MS m/z (%) 230 (Mþ, 45), 199 (100),
171 (35), 115 (10); HREI-MS calcd for C13H10O4 230.0579, found
333.0581; Anal. Calcd for C13H10O4: C, 67.82; H, 4.38. Found: C, 67.87;
H, 4.35; IR (KBr): 3056, 2943, 1722, 1634, 1228, 1136, 756 cmꢁ1
.
4.1.4.2. 2-(Ethoxymethyl)-7H-furo[3,2-g]chromen-7-one (4b). Yield
74%. mp 104e106 ꢀC; 1H NMR (400 MHz, CDCl3)
d
7.80 (d, J ¼ 9.6 Hz,
1H), 7.61 (s,1H), 7.45 (s,1H), 6.73 (s,1H), 6.37 (d, J ¼ 9.6 Hz,1H), 4.59
(s, 2H), 3.58 (q, J ¼ 7.2 Hz, 2H), 1.23 (t, J ¼ 7.2 Hz, 3H); EI-MS m/z (%)
244 (Mþ, 30), 199 (100), 171 (30), 57 (15); HREI-MS calcd for
C14H12O4 244.0736, found 244.0741; IR (KBr): 3122, 2976, 1716,
4.1.1. 2-Bromo-7H-furo[3,2-g]chromen-7-one (2a) and 2-chloro-
7H-furo[3,2-g]chromen-7-one (2b)
1630, 1582, 1132, 1076, 758 cmꢁ1
.
A mixture of furocoumarin 1a (22 mg, 0.12 mmol), NBS or NCS
(1.1 eq, 0.13 mmol) and anhydrous CCl4 (5 mL) was stirred in refluxing
under nitrogen atmosphere for 9 h. After workup, the crude products
were purified by flash chromatography to give the desired compound
2a or 2b. 2a: yield 32%. mp 99e101 ꢀC; 1H NMR (400 MHz, CD3OD)
4.1.4.3. 2-(Butoxymethyl)-7H-furo[3,2-g]chromen-7-one (4c). Yield
80%. mp 85e86 ꢀC; 1H NMR (400 MHz, CDCl3)
d
7.79 (d, J ¼ 9.6 Hz,
1H), 7.62 (s, 1H), 7.45 (s, 1H), 6.72 (s, 1H), 6.37(d, J ¼ 9.6 Hz,1H), 4.60
(s, 2H), 3.57 (t, J ¼ 6.6 Hz, 2H), 1.43e1.37 (m, 4H), 0.92 (t, J ¼ 7.4 Hz,
3H); EI-MS m/z (%) 272 (Mþ, 30), 199 (95), 171 (35), 149 (30), 57
(35); HREI-MS calcd for C16H16O4 272.1049, found 272.1046; IR
d
7.94 (d, J ¼ 9.8 Hz, 1H), 7.27 (s, 1H), 7.10 (s, 1H), 6.39 (s, 1H), 6.37 (d,
J ¼ 9.8 Hz, 1H); EI-MS m/z (%) 266 (10), 264 (Mþ, 10), 236 (10), 186
(100), 158 (60), 130 (15), 102 (15); HREI-MS calcd for C11H5BrO3
263.9422, found 263.9418; IR (KBr): 3047,1740,1633,1390,1101,1020,
959, 825 cmꢁ1 2b: yield 25%. mp 132e134 ꢀC; 1H NMR (400 MHz,
(KBr): 3012, 1708, 1630, 1576, 1161, 1110 cmꢁ1
.
4.1.5. (7-Oxo-7H-furo[3,2-g]chromen-2-yl)methyl acetate (4d)
A mixture of chloromethyl derivative 3 (50 mg, 0.21 mmol),
sodium acetate (25 mg), glacial acetic acid (2 mL) and acetic
anhydride (2 mL) was heated to 110 ꢀC and stirred for 3 h. After
cooling down to room temperature, the reaction mixture was
CD3OD)
d
7.93 (d, J ¼ 9.6 Hz, 1H), 7.28 (s, 1H), 7.12(s, 1H), 6.40 (s, 1H),
6.34 (d, J ¼ 9.6 Hz,1H); EI-MS m/z (%) 220 (Mþ,15), 203 (90),190 (100),
175 (35), 161 (45), 147 (25); HREI-MS calcd for C11H5ClO3 219.9927,
found 219.9931; IR (KBr): 2924,1709,1632,1564,1122, 829, 732 cmꢁ1
.

B.-L. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 5258e5264
5263
diluted with 10 mL water and extracted with ethyl acetate
(3 ꢂ 20 mL) to obtain the crude product, which was purified by
flash column chromatography over silica gel to give 4d (42 mg, in
76% yield): white crystal, mp 156e158 ꢀC; 1H NMR (400 MHz,
4.1.10. (7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl acetate (27b)
A mixture of 27a (19 mg, 0.09 mmol), DMAP (1 mg) and one
drop of acetic anhydride in 2 mL of anhydrous pyridine was stirred
at room temperature overnight under nitrogen atmosphere, and
5 mL of icy water was then added. The reaction mixture was
extracted with ethyl acetate (3 ꢂ 10 mL), and the organic layer was
washed with brine and dried over anhydrous Na2SO4. After removal
of solvent, the crude product was purified by flash column chro-
matography over silica gel eluted with petroleum ether and
acetone (4:1) to give 27b (18 mg, in 79% yield): mp 199e201 ꢀC; 1H
CDCl3)
d
7.80 (d, J ¼ 9.6 Hz, 1H), 7.66 (s, 1H), 7.47 (s, 1H), 6.83 (s, 1H),
6.39 (d, J ¼ 9.6 Hz, 1H), 5.22 (s, 2H), 2.15 (s, 3H); EI-MS m/z (%) 258
(Mþ, 12), 216 (20), 199 (35), 149 (100); HREI-MS calcd for C14H10O5
258.0528, found 258.0515; IR (KBr): 3082, 1722, 1630, 1273, 1161,
889, 763 cmꢁ1
.
4.1.6. 2-(Diethylamino)methyl-7H-furo[3,2-g]chromen-7-one (4e)
A mixture of chloromethyl derivative 3 (25 mg, 0.11 mmol),
anhydrous potassium carbonate (20 mg, 0.14 mmol), diethylamine
NMR (400 MHz, CDCl3)
d
7.73 (s, 1H), 7.71 (d, J ¼ 2.4 Hz, 1H), 7.52 (s,
1H), 6.85 (d, J ¼ 2.4 Hz,1H), 6.49 (s, 1H), 5.37 (s, 2H), 2.23 (s, 3H); EI-
MS m/z (%) 258 (Mþ, 80), 216 (100), 188 (40), 171 (60), 89 (30);
HREI-MS calcd for C14H10O5 258.0528, found 258.0527; IR (KBr):
(8.3 mg,12 mL) and anhydrous acetone (5 mL) was stirredat refluxing
overnight under nitrogen atmosphere. After workup, the crude
products were purified by flash column chromatography over silica
gel to afford 15 mg 4e in 52% yield. mp 82e83 ꢀC; 1H NMR (400 MHz,
3084, 1720, 1632, 1277, 1161, 1078, 899, 766, 552 cmꢁ1
.
4.1.11. 5-(Bromomethyl)-7H-furo[3,2-g]chromen-7-one (28)
CDCl3)
d
7.78 (d, J ¼ 9.4 Hz, 1H), 7.58 (s, 1H), 7.44 (s, 1H), 6.62 (s, 1H),
A mixture of 27a (36 mg, 0.17 mmol) and PPh3 (52 mg,
0.20 mmol) in anhydrous THF (3 mL) was stirred under 0 ꢀC, and
54 mg of PBr3 (0.20 mmol) was added dropwise. The reaction
mixture was warmed up to room temperature and further stirred for
2 h, and then 5 mL of water was added. The reaction mixture was
extracted with ethyl acetate (3 ꢂ 15 mL), and the organic layer was
washed with brine and dried over anhydrous Na2SO4. After removal
of solvent, the residuewas purified byflash column chromatography
over silica gel to give 28 (34 mg, in 73% yield): mp 217e219 ꢀC; 1H
6.36 (d, J ¼ 9.4 Hz, 1H), 3.80(s, 2H), 2.62 (q, J ¼ 7.2 Hz, 4H), 1.11 (t,
J ¼ 7.2 Hz, 6H); EI-MS m/z (%) 271 (Mþ, 15), 256 (10), 199 (100), 171
(10); HREI-MS calcd for C16H17NO3 271.1208, found 271.1202; IR
(KBr): 2970, 1724, 1709, 1630, 1128, 930 cmꢁ1
.
4.1.7. 3-Methyl-7H-furo[3,2-g]chromen-7-one (7)
According to the method reported in the literature [18], the
desired furocoumarin derivative 7 was prepared in 44% yield.
Compound 7: mp 186e187 ꢀC [lit [18]. 188 ꢀC]; 1H NMR (300 MHz,
NMR (400 MHz, DMSO-d6)
d
8.20 (s, 1H), 8.13 (d, J ¼ 2.1 Hz,1H), 7.78
CDCl3)
d
7.81 (d, J ¼ 9.6 Hz, 1H), 7.56 (s, 1H), 7.47 (q, J ¼ 1.2 Hz, 1H),
(s,1H), 7.14 (dd, J ¼ 2.1 Hz, J ¼ 1.3 Hz,1H), 6.71 (s,1H), 4.95 (s, 2H); EI-
MS m/z (%) 280 (80), 278 (80),199 (75),171 (100),115 (20); HREI-MS
calcd for C12H7BrO3 277.9579, found 277.9582; IR (KBr): 3083, 2922,
7.41 (d, J ¼ 0.4 Hz, 1H), 6.37 (d, J ¼ 9.6 Hz, 1H), 2.27 (dd, J ¼ 1.2 Hz,
J ¼ 0.4 Hz, 3H).
The compounds 25aee were synthesized according to the
method reported by our group [21] or the literature procedures
[22,23].
1726, 1632, 1389, 1157, 883, 577 cmꢁ1
.
4.2. In vitro antimicrobial evaluation
4.1.8. 7-Oxo-7H-furo[3,2-g]chromene-5-carbaldehyde (26)
4.2.1. In vitro antimicrobial assay against H. pylori
A mixture of furocoumarin analogue 25a (150 mg, 0.75 mmol),
SeO2 (291 mg, 2.62 mmol, 3.5 eq) and anhydrous xylene (5 mL) was
stirred at refluxing under nitrogen atmosphere for 18 h. After
cooling down to room temperature, the reaction mixture was fil-
trated and wished with EtOAc. After removal of solvents, the
residue was purified by flash column chromatography over silica
gel to afford 110 mg of compound 26 (in 69% yield): 1H NMR
The antibacterial activities against H. pylori SS1 (Sydney Strain 1)
were tested according to the protocols described in the literature
[25]. The minimum inhibitory concentrations (MICs) of compounds
against H. pylori were determined by agar dilution method using
Columbia Blood Agar Base (Oxoid) containing 7% of defibrinated
sheep blood. In brief, all the compounds were dissolved in a limited
amount of dimethylsulphoxide (DMSO) and various concentrations
of two-fold diluted compounds were prepared, and were dispersed
into the culture medium. To the well-prepared agar plates, H. pylori
cells suspended in saline at the density of 108 CFU/mL were inocu-
lated and incubated at 37 ꢀC for 96 h under an atmosphere of 5% O2,
10% CO2 and 85% N2. The blank control (H. pylori culture in the agar
plates with a limited amount of DMSO) and the positive control (H.
pylori culture in the agar plates with various concentrationsof double
diluted metronidazole) were incubated under the same condition.
The amount of DMSO was kept at a level that did not inhibit bacterial
growth under the same condition. The minimum inhibitory
concentration (MIC) was defined as the lowest concentration that
completely inhibited the visible growth of H. pylori. All measure-
ments were repeated three times under the same condition.
(400 MHz, DMSO-d6)
d
10.16 (s, 1H), 8.79 (s, 1H), 8.11 (d, J ¼ 2.0 Hz,
1H), 7.80 (s, 1H), 7.15 (d, J ¼ 2.0 Hz, 1H), 7.13 (s, 1H); EI-MS m/z (%)
214 (Mþ, 100), 185 (30), 158 (40), 129 (10), 102 (10); HREI-MS calcd
for C12H6O4 214.0266, found 214.0269; IR (KBr): 3115, 2975, 1724,
1696, 1636, 1108, 857 cmꢁ1
.
4.1.9. 5-(Hydroxymethyl)-7H-furo[3,2-g]chromen-7-one (27a)
Aldehyde 26 (90 mg, 0.42 mmol) in 5 mL of anhydrous MeOH
was stirred, and 32 mg of NaBH4 (0.84 mmol) was added in batches
over 10 min. The reaction solution was kept stirring at room
temperature for an additional 30 min, and 5 mL of 0.5 M HCl was
added dropwise at ꢀC. The resultant mixture was extracted with
ethyl acetate (3 ꢂ 20 mL), and the organic layer was washed with
brine (3 ꢂ 10 mL) and dried over anhydrous Na2SO4. After removal
of the solvent under reduced pressure, the residue was purified by
flash column chromatography over silica gel to afford 27a (76 mg,
4.2.2. In vitro antimicrobial assay against Gram-positive and Gram-
negative bacteria
84% yield): mp 206e208 ꢀC; 1H NMR (400 MHz, CD3OD)
d
7.95 (s,
The in vitro antibacterial activities against Gram-positive
S. aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228,
Micrococcus luteus ATCC 9341 and B. subtilis CMCC 63501, and
Gram-negative E. coli ATCC 25922, Shigella flexneri F2a and Pseu-
domonas aeruginosa ATCC 14502 were conducted by following the
manipulations described in the literature [26]. The microbial cells
were suspended in Mueller Hinton broth to form a final density of
1H), 7.86 (d, J ¼ 2.2 Hz, 1H), 7.55 (s, 1H), 6.96 (d, J ¼ 2.2 Hz, 1H), 6.55
(s, 1H), 4.94 (d, J ¼ 1.2 Hz, 2H); EI-MS m/z (%) 216 (Mþ, 100), 188
(60), 171 (30), 159 (35), 131 (45); HREI-MS calcd for C12H8O4
216.0423, found 216.0422; IR (KBr): 3442, 2928, 1705, 1631, 1161,
1084, 1036, 864, 773 cmꢁ1; Anal. Calcd for C12H8O4: C, 66.67; H,
3.73. Found: C, 66.71; H, 3.72.

5264
B.-L. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 5258e5264
5 ꢂ 10ꢁ5e10ꢁ6 CFU/mL and incubated at 37 ꢀC for 18 h under
aerobic conditions with the respective compounds and positive
control, which have been dissolved in DMSO. The blank controls of
microbial culture were incubated with limited DMSO under the
same condition. DMSO was determined not to be toxic at a limited
amount under the experimental conditions.
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Financial support of National Natural Science Foundation (Grant
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Experimental procedures, physical and spectral data of
compounds 8e20, and the elemental analyses of the potent antimi-
crobialcompoundsagainstH. pyloriassociatedwiththisarticlecanbe
found in the online version at doi:10.1016/j.ejmech.2010.08.045.