European Journal of Medicinal Chemistry 45 (2010) 4446e4457

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Isoxazole analogs of curcuminoids with highly potent multidrug-resistant

antimycobacterial activity
Chatchawan Changtam a, Poonpilas Hongmanee b, Apichart Suksamrarn a, *
a
Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand
b
Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Curcumin (1), demethoxycurcumin (2) and bisdemethoxycurcumin (3), the curcuminoid constituents of
Received 19 May 2010 the medicinal plant Curcuma longa L., have been structurally modified to 55 analogs and anti-
Received in revised form mycobacterial activity against Mycobacterium tuberculosis has been evaluated. Among the highly active
20 June 2010
curcuminoids, the isoxazole analogs are the most active group, with mono-O-methylcurcumin isoxazole
Accepted 3 July 2010
Available online 5 August 2010
(53) being the most active compound (MIC 0.09 mg/mL). It was 1131-fold more active than curcumin (1),
the parent compound, and was approximately 18 and 2-fold more active than the standard drugs
kanamycin and isoniazid, respectively. Compound 53 also exhibited high activity against the multidrug-
Keywords:
Curcuminoids
resistant M. tuberculosis clinical isolates, with the MICs of 0.195e3.125 mg/mL. The structural require-
Chemical modification ments for a curcuminoid analog to exhibit antimycobacterial activity are the presence of an isoxazole ring
Isoxazole analogs and two unsaturated bonds on the heptyl chain. The presence of a suitable para-alkoxyl group on the
Antimycobacterial activity aromatic ring which is attached in close proximity to the nitrogen function of the isoxazole ring and
a free para-hydroxyl group on another aromatic ring enhances the biological activity.
Ó 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction major constituents of C. longa and some other Curcuma species. It

has been known for some time that curcuminoids have been used
Tuberculosis is the leading cause of mortality among all as a natural food additive. The major curcuminoid isolated from
infectious diseases worldwide and is responsible for over two C. longa is curcumin (1). The minor constituents include deme-
million deaths annually [1]. The incident of the human immuno- thoxycurcumin (2) and bisdemethoxycurcumin (3). Curcuminoids
deficiency virus (HIV) pandemic and the increased prevalence of exhibited many interesting biological activities [7], for example,
multidrug-resistant strains of Mycobacterium tuberculosis caused antioxidant activity [8,9], anti-inflammatory activity [10,11], anti-
this disease to be more complicated [2,3]. The recent increase in cancer activity [8,12,13], anti-trypanosomal activity [14] and
the number of multidrug-resistant clinical isolates of M. tubercu- anti-HIV activity [15]. In the present work, the crude curcuminoids
losis has created an urgent need for the evolution of new antitu- were separated into compounds 1, 2 and 3 and the anti-
berculosis therapeutics [4,5]. Naturally occurring compounds have mycobacterial activity of these compounds against the non-viru-
demonstrated significant activity in the in vitro assays against lent M. tuberculosis H37Ra was 100, 50 and 25 mg/mL, respectively.
M. tuberculosis [6]. The structural modification of natural products These curcuminoids displayed much lower activity than the
is one of the potential strategies for the development of new anti- standard antitubercular drugs kanamycin, which exhibited MIC of
TB drugs which are different from the drugs currently used. In the 2.5 mg/mL, and especially isoniazid and rifampin, which showed
search for compounds with anti-TB property, a number of MIC of 0.06 and 0.004 mg/mL, respectively. However, the well
medicinal plants have been investigated by our group. Among the known medicinal use of curcuminoids, the relatively less toxicity
tested extracts, the curcuminoid fraction of Curcuma longa L. was of these food ingredients and the unlimited availability of the
shown to exhibit antimycobacterial activity. Curcuminoids are the compounds from C. longa are of special interest. The unique
skeleton of curcuminoids which is different from the current
anti-TB drugs could also avoid any possible cross resistance of
* Corresponding author. Tel.: þ662 3190931; fax: þ662 3108404. the curcuminoid-derived compounds with the current anti-TB
E-mail addresses: [email protected], [email protected] (A. Suksamrarn). drugs. This work deals with the structural modification of the

0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.003
 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457 4447

curcuminoids 1e3 to analogs with exceptionally high activity to give mono-O-n-propylcurcumin (9) and di-O-n-propylcurcumin
against the multidrug-resistant (MDR) strains of M. tuberculosis. (10) in 25 and 50% yield, respectively. The spectroscopic data of 9
(see Section 5) were in agreement with the structure. The spec-
2. Chemistry troscopic data of 10 were identical to those of reported values [14].
Mono-O-(2-hydroxyethyl)curcumin (11), di-O-(2-hydroxyethyl)
2.1. Chemical modification of curcuminoids curcumin (12), mono-O-allylcurcumin (13) and di-O-allylcurcumin
(14) were prepared by appropriate alkylation of curcumin (1) using
The natural curcuminoids 1e3 obtained from C. longa were the literature procedures [14]. n-Pentylation of compound 1 was
subjected to chemical modifications for antimycobacterial achieved by reacting 1 with n-propyl iodide in acetone in the
evaluations. presence of anhydrous potassium carbonate to afford mono-O-n-
pentylcurcumin (15) and di-O-n-pentylcurcumin (16) in 34 and 40%
2.1.1. Demethylated analogs yields, respectively. The spectroscopic data of 15 and 16 (see Section
The parent curcuminoids 1 and 2 were demethylated as 5) were consistent with the structures.
described previously [14,16] to yield the corresponding demethy-
lated analogs 4, 5 and 6 (Scheme 1). 2.1.3. Acetate analogs
In order to have both the mono and diacetate derivatives for
2.1.2. Methyl ether and higher alkyl ether analogs biological evaluation, the curcuminoids 1e3 were subjected to
Alkylation of the curcuminoid 1 is outlined in Scheme 1. partial acetylation as shown in Scheme 2. Reaction of 1 with acetic
Methylation of 1 was achieved by modification of the literature anhydride in pyridineeCHCl3 afforded the monoacetate 17 and
procedure [14,17] to afford mono-O-methylcurcumin (7) and di-O- diacetate 18 in 25 and 70% yields. The spectroscopic data of 17
methylcurcumin (8) in 39 and 47%, respectively. The spectroscopic (Section 5) were in agreement with the structure, and those of 18
(IR, 1H NMR and mass spectra) data of compounds 7 and 8 were were consistent with the literature values [18]. Compounds 2 and 3
consistent with the literature values [17]. Compound 1 was sub- were similarly subjected to partial acetylation to give the corre-
jected to n-propylation by modification of the reported procedure sponding mono and diacetate analogs 19, 20, 21, 22 and 23. The
spectroscopic data of these compounds (Section 5) were consistent
with the structures and were in agreement with the reported data
[14,18,19]. The isomeric monoacetates 19 and 20 were distin-
guished by 1H NMR spectral data. Thus, the presence of acetate
group at the 40 -position was evident from the downfield shift of the
two aromatic protons, H-30 and H-50 , at d 7.11. This was different
from the 1H NMR spectrum of compound 20 that the H-30 and 50
signals appeared at d 6.81, and H-500 at d 7.02.

2.1.4. Dihydro, tetrahydro, hexahydro and octahydro analogs

Dihydrocurcumin (24) was prepared in 30% yield by zinceacetic
acid reduction of the parent curcuminoid 1. The structure of the
product 24 was confirmed by 1H NMR spectral data. The differences

Scheme 1. Demethylation and alkylation of curcuminoids 1e3. Reagents and condi-

tions: (a) BBr3, CH2Cl2, 0  C, then ambient temp.; (b) MeI, K2CO3, acetone, reflux; (c) n-
propyl iodide, K2CO3, acetone, reflux; (d) 2-bromoethanol, K2CO3, acetone, reflux; (e) Scheme 2. Acetylation of curcuminoids 1e3. Reagents and condition: (g) Ac2O, pyri-
allyl bromide, K2CO3, acetone, reflux; (f) n-pentyl iodide, K2CO3, acetone, reflux. dine-CHCl3 (1:1), ambient temp.
4448 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457

from the starting curcuminoid 1 being the absence of two olefinic 2.1.8. Isoxazole analogs of dihydro and tetrahydrocurcumins
protons of H-6 and H-7 at ca. d 6.47 and 7.57, and the presence of The isoxazole analog 40 was prepared in 63% yield by treatment
two methylene signals at d 2.64 and 2.87. Tetrahydrocurcumin (25), of dihydrocurcumin (24) with hydroxylamine hydrochloride in
hexahydrocurcumin (26) and octahydrocurcumin (27) were pyridine (Scheme 5). The structure of the product was established
prepared by catalytic hydrogenation of 1 according to the literature as 40, not the isomeric structure 41, by the spectroscopic evidence.
procedure [20,21] (Scheme 3). Thus, the 1H NMR spectrum showed a four-proton broad singlet
signal of methylene group at d 2.92 (H-100 and H-200 ), and two
2.1.5. Unsaturated and saturated mono-keto analogs olefinic protons at d 6.73 (H-10 ) and 7.19 (H-20 ). The evidence was
The enone 28, the dienones 29 and 30, the trienone 31 and the supported by the 2D NMR experiments in which the HMBC corre-
unconjugated ketone 32 were prepared by the literature methods lations between H-20 and C-3, C-2000 , C-6000 ; H-200 and C-5, C-20000 ,
[14,22,23] (Scheme 4). C-60000 were observed. The isoxazole analog 42 was similarly
prepared in 77% yield from tetrahydrocurcumin (25) (Scheme 5).
2.1.6. Pyrazole analogs Thus, treatment of 25 in pyridine with hydroxylamine hydrochlo-
The pyrazole analogues of the curcuminoids 1e3 were ride to give the intermediate monoxime, which was subsequently
prepared as outlined in Scheme 5. Using the literature procedure treated with p-toluenesulfonic acid to yield 42 in 54% overall yield
[24] with a slight modification, curcumin pyrazole (33) was from 25. The spectroscopic data of 42 were different from those of
obtained in 71% yield by treatment of 1 with hydrazine hydrate in compound 40 by the absence of olefinic signal at d 6.73 (H-10 ) and
AcOH at 50  C. Reaction of 2 and 3 with the same reagent afforded 7.19 (H-20 ) and the presence of additional two methylene signals at
the pyrazole analogs 34 and 35 in 77% and 41% yields, respectively. d 2.87e2.96 (m, 8H). The mass spectral data were also consistent
The spectroscopic data of these compounds were consistent with with its structure.
the structures and were in agreement with the reported data
[20,24]. The N-phenyl substituted pyrazole 36 was similarly 2.1.9. Alkyl ethers and methyl ethers of isoxazole analogs
prepared in 81% yield by reaction of compound 1 with phenyl- Antimycobacterial evaluation of the foregoing curcuminoid 1
hydrazine hydrate. The spectroscopic data (Section 5) were and its mono-O-n-pentyl ether analog 15 indicated that introduc-
consistent with the structure and were in agreement with the tion of an n-pentyl group resulted in 8-fold increase in activity
reported data [24]. (Table 1). In order to see whether the n-pentyl group would
enhance the activity in the case of the isoxazole analog 37, the
2.1.7. Isoxazole analogs mono-O-n-pentyl ether analog 43 and the isomeric isoxazole
The isoxazole analog 37 was prepared in 70% yield by treatment analog 44 (1:1 mixture), were prepared in 24%, together with the
of 1 with hydroxylamine hydrochloride in pyridine (Scheme 5). The di-O-n-pentyl ether analog 45 in 62% (Scheme 6). The 43/44
spectroscopic data of 37 (see Section 5) were consistent with the mixture was separated by HPLC (see detail in Section 5). The
structure and were in agreement with those reported previously structures of the isomeric pentyl ether analogs 43 and 44 were
[24]. Treatment of 2 with the same reagent afforded a 1:1 mixture distinguished by 1H and 13C NMR data (see Section 5). The present
(65%) of two isomeric isoxazoles 38a and 38b, which could not be of the pentyl group was evident from the signals of the methyl
separated by column chromatography (Scheme 5). However, group at d 0.91 (t, J ¼ 6.8 Hz, H-500000 ), two methylene groups at d 1.40
separation of the mixture was achieved by normal phase HPLC (see (m, H-300000 and H-400000 ), one methylene group at d 1.84 (m, H-200000 ),
Section 5) to give pure 38a and 38b. The structures of these iso- and another one methylene group at d 4.02 (t, J ¼ 6.4 Hz, H-100000 ).
xazoles were distinguished by 1H and 13C NMR analysis. Data The 13C NMR data of C-500000 , C-200000 , C-300000 , C-400000 and C-100000
supporting this interpretation were provided by 2D NMR experi- appeared at d 13.9, 22.4, 28.0, 28.8 and 69.0, respectively. The
ments in which the HMBC correlations between H-20 and C-3, C-2000 , evident was supported by 2D NMR experiments in which the HMBC
C-6000 ; H-200 and C-5, C-20000 , C-60000 were observed. The isoxazole correlations between H-20 and C-3, C-2000 , C-6000 ; H-200 and C-5, C-20000 ,
analog 39 was similarly prepared in 68% yield from the curcumi- C-60000 ; H-100000 and C-300000 , C-3000 were observed. Although compound
noid 3. The spectroscopic data (Section 5) were consistent with the 43 was very active, this compound encountered solubility problem
structure. during the dilution of sample solution for biological evaluation. The

Scheme 3. Reduction of curcumin (1). Reagents and condition: (h) Zinc powder, AcOH, ambient temp.; (i) H2/PdeC, EtOH.
 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457 4449

Scheme 4. Dehydration of hexahydrocurcumin (25), and dehydrogenation and catalytic hydrogenation of 28. Reagents and conditions: (j) p-TsOH, C6H6, reflux; (k) DDQ, THF; (l) H2/
PdeC, EtOH.

relatively more soluble and lower alkyl ether analogs, the mono-O- activity, we therefore synthesized the higher alkyl ether analogs of
(3,3-dimethylallyl) ether analogs 46a/46b (2:1 mixture) and the di- the parent curcuminoid 1 starting with the mono-O-n-propyl ether
O-(3,3-dimethylallyl) ether analogs 47, the mono-O-ethyl ether 9. As expected, the antimycobacterial activity of 9 was higher than
analogs 48a/48b (5:2 mixture) and the di-O-ethyl ether analogs 49, the parent compound 1; it was approximately 4-fold more active
and the acetate analogs 50, 51 and 52, were prepared (Scheme 6). than compound 1. However, the di-O-n-propyl analog 10 was very
The structures of the isomeric mono-O-(3,3-dimethylallyl) ether weakly active (MIC 200 mg/mL). The relatively nonpolar nature of
analogs 46a/46b, the mono-O-ethyl ether analogs 48a/48b, and the the alkyl group is required for high activity of the analogs. This was
monoacetates 50 and 51 were distinguished by 1H and 13C NMR evident from the relatively low activity of the mono-O-(2-hydrox-
spectral data in similar manner to those of 43 and 44. After the yethyl) analog (11) (MIC 200 mg/mL), which was more polar than
assay results were obtained, the methyl ether analogs 53, 54, 55, the n-propyl ether analog 9. The di-O-(2-hydroxyethyl) analog (12)
56a/56b (1:1 mixture) and 57 were also prepared (Scheme 6). The also showed low activity. The activity of the ether analogs was
structures of the isomeric methyl ether analogs 54 and 55, and 56a sensitive to the nature of the alkyl group, as seen from the relatively
and 56b were distinguished by 1H and 13C NMR data in similar lower activity of the mono-O-allyl ether 13 and the di-O-allyl ether
manner to those of 43 and 44 (see Section 5). The mono-deme- 14, the unsaturated ether analog of 9 and 10, than the analogs 9 and
thylated analogs 58a/58b (1:2 mixture) and the di-demethylated 10, respectively. To see whether saturated higher alkyl ether
analog 59 were also prepared (Scheme 5) and structural charac- analogs contributed to high activity of the curcuminoid, the mono
terization was achieved by similar spectroscopic spectral analysis. and di-O-n-pentyl ether analogs 15 and 16 were synthesized and
assessed for antimycobacterial activity and it was found that
3. Results and discussion compounds 15 and 16 (MIC 12.5 and 100 mg/mL) were more active
than the corresponding n-propyl ether analogs 9 and 10 (MIC 25
The antimycobacterial activity of the parent curcuminoids 1e3 and 200 mg/mL), respectively. At this point, it is concluded that in
against the non-virulent M. tuberculosis H37Ra was 100, 50 and going from the mono alkyl analogs to dialkyl analogs, a sharp
25 mg/mL, respectively (Table 1). The first type of analogs selected decrease in activity was observed.
for antimycobacterial evaluation was the demethylated analogs. We next evaluated the potency of the acetate analogs of cur-
However, the mono-O-demethylated analog 4 and the di-O- cuminoids. The results have indicated that the monoacetates 17,
demethylated analog 5 were only as active as, or even less active 19, 20 and 22 exhibited comparable activity to that of their
than, the parent compound 1 (see Table 1). The demethylated respective parent compounds 1, 2, and 3. As expected, the corre-
analog 6 was much less active than its parent compound 2. The sponding diacetate analogs 18 and 21 showed lower activity than
results have indicated that increase in polarity of the curcuminoids the monoacetate analogs 17, 19 and 20. The exception was for the
caused decrease in activity. We therefore moved to analogs which analog 23, which showed comparable activity to that of the parent
were more lipophilic than their respective parent compounds. The- compound 3.
mono-O-methyl analogs 7 was 4-fold more active than the parent The chemical modification of the curcuminoids 1e3 to the
compound 1. However, further methylation to the di-O-methyl above corresponding ether and acetate derivatives did not seem to
analog 8 resulted in decrease in activity (MIC 100 mg/mL) when give promising analogs with high antimycobacterial activity. We
compared with the mono-O-methyl analog 7 (MIC 50 mg/mL). Since therefore chose to modify the skeleton of the curcuminoids. The
it seemed that increase in lipophilicity resulted in increase in reduced analogs of curcuminoid 1, viz. 24, 25, 26 and 27, were
4450 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457

Scheme 5. Preparation of pyrazole and isoxazole analogs. Reagents and conditions: (m) NH2NH2, hydrate, AcOH; (n) phenylhydrazine hydrate, AcOH; (o) NH2OH.HCl, pyridine,
50  C; (p) (1) NH2OH.HCl, pyridine, ambient temp., (2) p-TsOH, C6H6, 60  C.

prepared and assessed for antimycobacterial activity. The assay MICs of these compounds were 200, 100, and 25 mg/mL, respec-
results indicated that, except for the tetrahydro analog 25 that was tively. The N-substituted analog 36 was also prepared, but it was
approximately 2-fold more active than the parent compound 1, only as active as compound 35.
the activity of the rest were approximately the same as (in the We then turned our attention to the five-membered cyclic NeO
case of the hexahydro analog 26) or less active (in the case of the analogs, the isoxazole analogs. Curcumin isoxazole (37) exhibited
dihydro analog 24 and the octahydro analog 27) than the parent interesting biological activities, for example, anti-inflammatory
compound 1. activity [25] and anticancer activity [26,27]. Potent anti-
We then explored the biological activity of the mono-keto mycobacterial compounds with the isoxazole functionality in the
analogs of the parent compound 1. The enone 28, the dienones 29 molecules have also been reported [28e30]. In the present work,
and 30, the trienone 31 and the unconjugated ketone 32 were the isoxazole analogs 37, 38a/38b (1:1 mixture), and 39 were
assessed for antimycobacterial activity. Except for the ketone 32 prepared from the parent compounds 1, 2, and 3, respectively, and
which exhibited comparable activity to that of the parent curcu- subjected to antimycobacterial evaluation. The results indicated
minoid 1, the rest showed 4-fold (compounds 28 and 30) and 8-fold that the isoxazole analog 37 was highly active; its MIC was 1.56 mg/
(compounds 29 and 31) more active than the parent compound 1. mL, which was about 64-fold more active than the parent curcu-
Although the above conjugated mono-keto analogs exhibited minoid 1. The isoxazole analogs 38a/38b and 39 were less active;
much improved antimycobacterial activity than that of the parent their MICs were both 12.5 mg/mL. It was worth noting that the 3,4-
compound 1, it was still less active than the standard drug kana- dioxygenated groups on both aromatic rings are required for an
mycin. We therefore changed our strategy to analogs with more isoxazole analog to exhibit high antimycobacterial activity. The
rigid heptyl chain. In order to keep the heptyl chain less flexible, isoxazole 37 was thus selected as the lead compound.
a cyclic structure was introduced into the chain. The five- In order to see the influence of unsaturation at the alkyl chain,
membered cyclic dinitrogen analog, the pyrazole analog, was first the corresponding dihydro analog 40 and the tetrahydro analog 42
chosen and the analogs 33, 34 and 35 were prepared from the were prepared and their MICs were determined. The gradual
curcuminoids 1, 2 and 3, respectively. The antimycobacterial decrease in activity in going from the fully unsaturated analog 37
activity of these cyclic analogs, however, was not promising; the (MIC 1.56 mg/mL) to the dihydro analog 40 (MIC 6.25 mg/mL) and
 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457 4451

Table 1
The in vitro activity of the curcuminoid analogs against M. tuberculosis H37Ra strain.

Compound MIC (mg/mL) Compound MIC (mg/mL)

1 100 32 100
2 50 33 200
3 25 34 100
4 200 35 25
5 100 36 25
6 200 37 1.56
7 50 38a/38b (1:1) 12.5
8 100 39 12.5
9 25 40 6.25
10 200 41 NDb
11 200 42 100
12 200 43 0.39
13 100 44 6.25
14 Inactivea 45 Inactivea
15 12.5 46a/46b (2:1) 1.56
16 100 47 NDb
17 100 48a/48b (5:2) 12.5
18 200 49 NDb
19 50 50 1.56
20 50 51 1.56
21 100 52 1.56
22 25 53 0.09
23 25 54 0.78
24 200 55 0.39
25 50 56a/56b (1:1) 100
26 100 57 Inactivea
27 Inactivea 58 6.25
28 25 59 200
29 12.5 Kanamycin 2.5
30 25 Isoniazid 0.06
31 12.5 Rifampin 0.004
a
Inactive at MIC > 200 mg/mL.
b
ND, not determined.

finally to the tetrahydro analog 42 (MIC 100 mg/mL) has led to

a conclusion that full conjugated system of the C7 chain is required
for high antimycobacterial activity of the isoxazole analogs.
Since the mono-O-n-pentyl analog 15 was 8-fold more active than
the parent compound 1, the lead compound 37 was transformed to
the corresponding mono-O-n-pentyl analogs 43 and its isomeric
analog 44, and the di-O-n-pentyl ether analog 45. It was found that
the analog 43 was highly active (MIC 0.39 mg/mL) which was about
256-fold more active than the parent compound 1. However, its
isomeric analog 4 was less active than the analog 43 (MIC of
compound 44 was 6.25 mg/mL). As expected, compound 45 was
inactive to the test. The low activity of 45 was possibly, at least partly,
due to its low solubility. Since compound 43 (and 44) has some
solubility problem upon dilution with water, which was due to the
Scheme 6. Alkylation and acetylation of isoxazole analogs 37, 38a, 38b and 39.
lipophilic nature of the n-pentyl group, we then further explored Reagents and conditions: (q) n-pentyl iodide, K2CO3, acetone, reflux; (r) 3,3-dime-
other alkyl group that exhibited comparable activity to that of thylallyl bromide, K2CO3, acetone, reflux; (s) diethyl sulfate, K2CO3, acetone, reflux; (t)
compound 43, but with higher solubility than that of compound 43. A Ac2O, pyridine; (u) MeI, K2CO3, acetone, reflux.
number of relatively less polar alkyl ether analogs, including the
mono-O-(2,2-dimethylallyl) ether analogs 46a/46b (2:1 mixture) compound 53 with that of compound 54 and the activity of
and the mono-O-ethyl ether analogs 48a/48b, and the acetate compound 43 with that of 44 have led to a conclusion that one of the
analogs 50, 51 and 52, were prepared and assessed for anti- structural requirements for a mono-O-alkylated analog of isoxazole
mycobacterial activity. However, none of them was as active as the to exhibit high antimycobacterial activity is that the alkoxyl group is
analog 43 (see Table 1). The di-O-(3,3-dimethylallyl) ether analogs 47 located at the aromatic ring which is closer to the nitrogen function
and the di-O-ethyl ether analogs 49 have not been subjected to than the oxygen function of the isoxazole ring. It should be noted
biological evaluation. We eventually discovered that the mono-O- that, despite it being a di-O-methyl ether analog which was much
methyl analog 53 exhibited the highest activity; its the MIC was less active than the mono-O-methyl ether analog, the isoxazole
0.09 mg/mL or 0.24 mM, that is 1131-fold more active than the parent analog 52 still exhibited high activity, with MIC of 0.39 mg/mL, though
curcuminoid 1 (MIC 100 mg/mL or 271.45 mM). Comparison of the MIC it was less active than its mono-O-methyl ether analog. In contrast,
of the isoxazole 53 with those of the standard drugs, it was evident the activity of 56a/56b and 57, the fully methylated analogs of the
that compound 53 was approximately 18 and 2-fold more active than parent compounds 2 and 3, was very low (see Table 1). The findings
kanamycin sulfate (MIC 2.50 mg/mL or 4.29 mM) and isoniazid (MIC have pointed out that the number of oxygenation on the aromatic
0.06 mg/mL or 0.44 mM), respectively. Its isomeric analog 54 was less rings of curcuminoids, the number and nature of the substituents on
active, the MIC of which is 0.78 mg/mL. Comparison of the activity of the oxygen function of the aromatic rings are important for high
4452 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457

Table 2 5. Experimental
The in vitro activity of the isoxazole analogs 43, 44, 53 and 54 against multidrug-
resistant M. tuberculosis compared with H37Ra strain.
5.1. General
Entry Code Isolate resistant profilea MIC (mg/mL)

43 44 53 54 Melting points were determined on an Electrothermal melting

1 H37Ra Pan sensitive 0.39 6.25 0.09 0.78 point apparatus and are uncorrected. IR spectra were recorded in
2 M3 INH, RIF, SM 0.39 1.56 0.195 0.78 KBr on a PerkineElmer FT-IR Spectrum BX spectrophotometer. 1H
3 M4 INH, RIF, EMB, SM 1.56 1.56 1.56 6.25 and 13C NMR spectra were recorded on a Bruker AVANCE 400
4 M5 INH, RIF, SM 1.56 1.56 3.125 12.5
spectrometer operating at 400 and 100 MHz, respectively. Electron
5 M6 INH, RIF 0.78 3.125 0.39 1.56
6 M8 INH, RIF, EMB, SM 3.125 6.25 3.125 12.5
impact (EI) and electrospray (ES) mass spectra were obtained using
7 M11 INH, RIF 3.125 6.25 3.125 12.5 a Finnigan Polaris Q and a Finnigan LC-Q mass spectrometer. High
8 M16 INH, RIF, EMB 1.25 1.56 1.56 12.5 resolution mass spectra were obtained using a Bruker micrOTOF
9 M21 INH, RIF, EMB, SM 3.125 6.25 3.125 12.5 mass spectrometer. Column chromatography and TLC were carried
10 M22 INH, RIF 0.78 6.25 0.39 1.56
out using Merck silica gel 60 (<0.063 mm) and precoated silica gel
11 M27 INH, RIF 0.39 1.56 0.195 0.78
12 M46 INH, RIF, EMB, SM, OFX, CIP 1.56 1.56 1.56 6.25 60 F254 plates, respectively. Spots on TLC were detected under UV
13 M48 INH, RIF, EMB, SM, OFX, CIP 3.125 3.125 1.56 12.5 light (254 nm) and by spraying with anisaldehydeeH2SO4 reagent
14 M53 INH, RIF, EMB, SM, OFX, CIP 3.125 3.125 3.125 12.5 followed by heating.
a
INH, isoniazid; RIF, rifampin; EMB, ethambutol; SM, streptomycin; OFX, oflox-
acin; CIP, ciprofloxacin. 5.2. Chemical modifications of curcuminoids

antimycobacterial activity. It should also be noted that, like the cur- The three natural curcuminoids, curcumin (1), demethox-
cuminoid analogs, demethylation of the isoxazole analogs resulted in ycurcumin (2) and bisdemethoxycurcumin (3), were obtained as
decrease in activity. This was exemplified by the low activity of the described previously [14].
isoxazoles 58a/58b and 59 (MICs 6.25 and 200 mg/mL, respectively).
The two most potent analogs, 43 and 53, together with their 5.2.1. Demethylation of compounds 1 and 2
isomers, 44 and 54, were assessed for various multidrug-resistant Compound 1 was demethylated in the same manner described
(MDR) clinical isolates M. tuberculosis (Table 2, entries 2e14). For previously [14,16] to yield mono-O-demethylcurcumin (4) (42%)
the isoniazid (INH)- and rifampin (RIF)-resistant isolates (entries 5, and di-O-demethylcurcumin (5) (33%). Compound 2 was also sub-
7, 10 and 11), the most active analog 53 were still very active (MICs jected to demethylation in similar manner to that of compound 1 to
0.195e3.125 mg/mL) whereas the analog 43 were also very active give O-demethyldemethoxycurcumin (6) (64%). The spectroscopic
(MICs 0.39e3.125 mg/mL). For the INH-, RIF-, and streptomycin (IR, 1H NMR and mass spectra) data of these analogs were consis-
(SM)-resistant isolates (entries 2 and 4), the analog 53 also tent with the reported values [14,16].
exhibited comparable activity against those of the INH- and RIF-
resistant isolates (MICs 0.195 and 3.125 mg/mL) whereas 5.2.2. Methylation of curcumin (1)
compound 43 showed similar activity (MICs 0.39 and 1.56 mg/mL). Compound 1 was subjected to methylation by modification of
For the INH-, RIF-, and ethambutol (EMB)-resistant isolates (entry the literature procedure [14,17]. Thus, compound 1 (100 mg,
8), and INH-, RIF-, EMB- and SM-resistant isolates (entries 3, 6 and 0.27 mmol) was dissolved in dry acetone (3 mL) and anhydrous
9), both the analogs 43 and 53 showed MICs in the range K2CO3 (100 mg) and MeI (0.8 mL) were added. The reaction mixture
1.56e3.125 mg/mL. More interestingly, the isolates that also resis- was refluxed for 3 h; water was added and the mixture was
tant to the second-line drugs ofloxacin (OFX) and ciprofloxacin extracted with CH2Cl2 (100 mL  2). The combined organic phase
(CIP) (entries 12e14) were also sensitive to compounds 43 and 53 was washed with H2O, dried over anhydrous Na2SO4 and the
(MICs 1.56e3.125 mg/mL). As expected, compound 44 was less solvent was removed under vacuum. The products were separated
active than its isomer 43 (MICs 1.56e6.25 mg/mL). Compound 54 by column chromatography using CH2Cl2 as eluent to yield curcu-
exhibited varying activity against different MDR isolates (MICs min mono-O-methyl ether (7) (40 mg, 39%) and curcumin di-O-
0.78e12.5 mg/mL). The isoxazole analog 53 is, therefore, the potent methyl ether (8) (50 mg, 47%). The spectroscopic (IR, 1H NMR, and
structure lead for the development of MDR antitubercular agents. mass spectra) data were consistent with the reported values [17].

4. Conclusion 5.2.3. Propylation of curcumin (1)

Compound 1 (120 mg, 0.32 mmol) was dissolved in dry acetone
Structural modifications of the natural curcuminoids, curcumin (2 mL) and anhydrous K2CO3 (40 mg) and n-propyl iodide (0.6 mL)
(1), demethoxycurcumin (2) and bisdemethoxycurcumin (3), to 55 were added. The reaction mixture was reflux for 5 h, water was
analogs have been undertaken and antimycobacterial activity added and the mixture was extracted with CH2Cl2. The combined
against M. tuberculosis has been evaluated. The isoxazole analogs organic phase was washed with H2O, dried over anhydrous Na2SO4
are the most active class of analogs, with mono-O-methylcurcumin and the solvent was removed under vacuum. The crude products
isoxazole (53) being the most active compound (MIC 0.09 mg/mL). It were isolated and purified by column chromatography using
was 1131-fold more active than the parent compound 1. Compound CH2Cl2 to yield mono-O-n-propylcurcumin (9) (34 mg, 25%) and di-
53 also exhibited high activity against the multidrug-resistant O-n-propylcurcumin (10) (72 mg, 50%). The spectroscopic data of
M. tuberculosis (MDR-TB) strains, with the MICs of 0.195e3.125 mg/ compound 10 were consistent with the reported values [14].
mL. The structural requirements for a curcuminoid analog to
exhibit antimycobacterial activity are the presence of an isoxazole 5.2.3.1. Mono-O-n-propylcurcumin (9). IR nmax: 3398, 2965, 2935,
ring and two unsaturated bonds on the heptyl chain. The presence 1618, 1563, 1508, 1258, 1133, 1030, 968, 840 cm1; 1H NMR
of a suitable para-alkoxyl group on the aromatic ring which is (400 MHz, CDCl3) d 1.03 (t, J ¼ 7.3 Hz, 3H, H-3000 ), 1.86 (sextet,
attached in close proximity to the nitrogen function of the isoxazole J ¼ 7.1 Hz, 2H, H-2000 ), 3.89 and 3.93 (each s, 2  3H, 2  OMe), 4.00
ring and a free para-hydroxyl group on another aromatic ring (t, J ¼ 6.8 Hz, 2H, H-1000 ), 5.78 (s, 1H, H-4), 5.83 (br s, 1H, OH), 6.45
enhances the biological activity. and 6.46 (each d, J ¼ 15.7 Hz, 2  1H, H-2 and H-6), 6.85 and 6.91
 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457 4453

(each d, J ¼ 8.2 Hz, 2  1H, H-50 and H-500 ), 7.03 and 7.06 (each br d, (19), mono-O00 -acetyldemethoxycurcumin (20) and di-O-acetylde-
J ¼ 1.4 Hz, 2  1H, H-20 and H-200 ), 7.15 (br d, J ¼ 8.2 Hz, 2  1H, H-60 methoxycurcumin (21) in 28%, 21% and 33% yield, respectively. The
and H-600 ), 7.57 and 7.58 (each d, J ¼ 15.7 Hz, 2  1H, H-1 and H-7); spectroscopic data of compound 21 were consistent with the
ESMS (þve): m/z (% rel. abund.) 411 [M þ H]þ (100). reported values [14].

5.2.4. 2-Hydroxyethylation and allylation of curcumin (1) 5.2.6.2. Mono-O0 -acetyldemethoxycurcumin (19). Orange amor-
Compound 1 was subjected to 2-hydroxyethylation as described phous solid; m.p. 127e129  C; IR nmax: 3757, 3758, 1762, 1750, 1718,
previously [14] to afford mono-O-(2-hydroxyethyl)curcumin (11) 1685, 1654, 1627, 1560, 1542, 1508, 1458, 1429, 1207, 1137, 968,
and di-O-(2-hydroxyethyl)curcumin (12) in 48 and 35%. Compound 1 838 cm1; 1H NMR (400 MHz, CDCl3) d 2.29 (s, 3H, OAc), 3.92 (s, 3H,
was similarly subjected to allylation [14] to afford mono-O-allylcur- OMe), 5.80 (s, 1H, H-4) and 5.85 (br s, 1H, OH), 6.47 (d, J ¼ 15.7 Hz,
cumin (13) and di-O-allylcurcumin (14) in 33 and 46%, respectively. 1H, H-6), 6.54 (d, J ¼ 15.7, 1H, H-2), 6.91 (d, J ¼ 8.2 Hz, 1H, H-500 ),
The spectroscopic data were consistent with the reported values [14]. 7.03 (br s, 1H, H-200 ), 7.11 (d, J ¼ 8.5 Hz, 2H, H-30 and H-50 ), 7.11
(obscured signal, 1H, H-600 ), 7.55 (d, J ¼ 8.5 Hz, 2H, H-20 and H-60 ),
5.2.5. Pentylation of curcumin (1) 7.59 (d, J ¼ 15.7 Hz, 1H, H-7), 7.61 (d, J ¼ 15.8 Hz, 1H, H-1); ESMS
Compound 1 was subjected to pentylation in similar manner to (þve) m/z (% rel. abund.) 403 [M þ Na]þ (29), 381 [M þ H]þ (100).
that of methylation of compound 1, but using n-pentyl iodide in
place of methyl iodide to give mono-O-n-pentylcurcumin (15) and 5.2.6.3. Mono-O00 -acetyldemethoxycurcumin (20). Yellow amor-
di-O-n-pentylcurcumin (16) in 34 and 40% yields, respectively. phous solid; m.p. 166e168  C; IR nmax: 3448, 1757, 1732, 1718, 1701,
1654, 1627, 1602, 1576, 1559, 1508, 1458, 1599, 1199, 1169, 1145,
5.2.5.1. Mono-O-n-pentylcurcumin (15). IR nmax: 3420, 2934, 2869, 1032, 961, 831 cm1; 1H NMR (400 MHz, CDCl3 þ 2 drops of CD3OD)
1625, 1582, 1511, 1465, 1424, 1262, 1136, 1032, 967, 847, 811 cm1; d 2.30 (s, 3H, OAc), 3.85 (s, 3H, OMe), 5.78 (s, 1H, H-4), 6.45 and 6.51
1
H NMR (400 MHz, CDCl3) d 0.91 (t, J ¼ 7.1 Hz, 3H, H-5000 ), 1.40 (m, (each d, J ¼ 15.8 Hz, 2H, H-2 and H-6), 6.81 (d, J ¼ 8.5 Hz, 2H, H-30
2  2H, H-3000 and H-4000 ), 1.84 (br q, 2H, H-2000 ), 3.89 and 3.93 (each s, and H-50 ), 7.02 (d, J ¼ 8.1 Hz, 1H, H-500 ), 7.09 (br s, 1H, H-200 ), 7.12 (dd,
2  3H, 30 -OMe and 300 -OMe), 4.03 (t, J ¼ 6.8 Hz, 2H, H-1000 ), 5.79 (s, J ¼ 8.1, 1.4 Hz, 1H, H-600 ), 7.42 (d, J ¼ 8.5 Hz, 2H, H-20 and H-60 ), 7.55
1H, H-4), 5.84 (br s, 1H, 400 -OH), 6.46 and 6.47 (each d, J ¼ 15.8 Hz, and 7.59 (each d, J ¼ 15.8 Hz, 2H, H-1 and H-7); ESMS (þve) m/z (%
2  1H, H-2 and H-6), 6.85 (d, J ¼ 8.2, 1H, H-50 ), 6.92 (d, J ¼ 8.2 Hz, rel. abund.), 403 [M þ Na]þ (27), 381 [M þ H]þ (100).
1H, H-500 ), 7.03 and 7.06 (each br s, 2  1H, H-20 and H-200 ), 7.10 (br d, Compound 3 was subjected to acetylation in the same manner to
J ¼ 8.2 Hz, 2  1H, H-60 and H-600 ), 7.57 and 7.58 (d, J ¼ 15.8 Hz, that of compound 1 to give mono-O-acetylbisdemethoxycurcumin
2  1H, H-1 and H-7); EIMS: m/z (% rel. abund.) 438 [M]þ (10), 420 (22) and di-O-acetylbisdemethoxycurcumin (23) in 25% and 60%
(100), 350 (29), 349 (22), 191 (36), 190 (51), 177 (38). yield, respectively. The spectroscopic (IR, H NMR and mass spectra)
data of compound 23 were consistent with the reported values [19].
5.2.5.2. Di-O-n-pentylcurcumin (16). IR nmax: 2958, 2940, 2857,
1625, 1581, 1511, 1466, 1420, 1337, 1256, 1227, 1133, 1026, 969, 845, 5.2.6.4. Mono-O-acetylbisdemethoxycurcumin (22). Yellow powder,
793 cm1; 1H NMR (400 MHz, CDCl3) d 0.91 (t, J ¼ 7.0 Hz, 2  3H, H- m.p. 178e180  C; IR nmax: 3872, 3448, 1735, 1718, 1685, 1654, 1637,
5000 and H-50000 ), 1.40 (m, 4  2H, H-3000 , H-4000 , H-30000 and H-40000 ), 1.84 1604, 1508, 1458, 1374, 1235, 1169, 972, 833 cm1; 1H NMR
(br q, 2  2H, H-2000 and H-20000 ), 3.89 (s, 2  3H, 30 -OMe and 300 - (400 MHz, CDCl3) d 2.30 (s, 3H, OAc), 5.78 (s, 1H, H-4), 6.48 and 6.54
OMe), 4.03 (t, J ¼ 6.8 Hz, 2  2H, H-1000 and H-10000 ), 5.79 (s, 1H, H-4), (each d, J ¼ 15.8 Hz, 2  1H, H-2 and H-6), 6.83 (d, J ¼ 8.3 Hz, 2H, H-
6.46 (d, J ¼ 15.8 Hz, 2  1H, H-2 and H-6), 6.85 (d, J ¼ 8.2 Hz, 2  1H, 300 and H-500 ), 7.11 (d, J ¼ 8.3 Hz, 2H, H-30 and H-50 ), 7.45 (d,
H-50 and H-500 ), 7.05 (br s, 2  1H, H-20 and H-200 ), 7.09 (br d, J ¼ 8.3 Hz, 2H, H-200 and H-600 ), 7.54 (d, J ¼ 8.3 Hz, 2H, H-20 and H-60 ),
J ¼ 8.2 Hz, 2  1H, H-60 and H-600 ), 7.58 (d, J ¼ 15.8 Hz, 2  1H, H-1 7.60 (d, J ¼ 15.8 Hz, 2H, H-1 and H-7); ESMS (þve) m/z (% rel.
and H-7); EIMS: m/z (% rel. abund.) 508 [M]þ (5), 420 (16), 344 abund.) 723 [2M þ Na]þ (83), 373 [M þ Na]þ (8), 351 [M þ H]þ (56).
(100), 234 (77), 220 (25), 177 (17).
5.2.7. Reduction of curcumin (1) with zinceacetic acid
5.2.6. Acetylation of curcuminoids 1e3 To a solution of curcumin (1) (100 mg, 0.27 mmol) in acetic acid
Ac2O (0.5 mL) was added to a solution of compound 1 (100 mg, (3 ml) was added Zn dust (20 mg) and the mixture was stirred at
0.27 mmol) in pyridineeCHCl3 (1:1, 2 ml) and the reaction mixture ambient temperature for 4 h. The reaction was worked up with H2O
was stirred at ambient temperature for 2 h. After the usual work up, and the solution was extracted with EtOAc. The organic phase was
the mixture was extracted with CH2Cl2 and the organic phase was washed with H2O, dried over anhydrous Na2SO4 and the solvent
washed with H2O, dried over anhydrous Na2SO4 and the solvent was evaporated to dryness. The crude products were purified by
was evaporated to dryness. The crude product was purified by column chromatography using n-hexaneeEtOAc (3:2) to give
column chromatography eluting with CH2Cl2 to afford mono-O- dihydrocurcumin (24) (30 mg, 30%).
acetylcurcumin (17) (28 mg, 25%) and di-O-acetylcurcumin (18)
(85 mg, 70%) as yellow amorphous solid; m.p. 163e165  C (lit. [18] 5.2.7.1. Dihydrocurcumin (24). 1H NMR (400 MHz, CDCl3 þ 2 drops
160  C). The spectroscopic (IR, 1H NMR and mass spectra) data of of CD3OD) d 2.64 (t, J ¼ 7.7 Hz, 2H, H-7), 2.87 (t, J ¼ 7.7 Hz, 2H, H-6),
compound 18 were consistent with the reported values [18]. 3.83 and 3.90 (each s, 2  3H, 2  OMe), 5.56 (s, 1H, H-4), 6.27 (d,
J ¼ 15.7 Hz, 1H, H-2), 6.66 (partially obscured signal, 1H, H-500 ), 6.68
5.2.6.1. Mono-O-acetylcurcumin (17). Orange foam: 1H NMR (s, 1H, H-200 ), 6.79 (d, J ¼ 7.8 Hz, 1H, H-600 ), 6.88 (d, J ¼ 8.2 Hz, 1H, H-
(400 MHz, CDCl3) d 2.30 (s, 3H, OAc), 3.86 and 3.93 (each s, 2  3H, 50 ), 6.98 (d, J ¼ 1.4 Hz, 1H, H-20 ), 7.04 (dd, J ¼ 8.2, 1.4 Hz, 1H, H-60 ),
2  OMe), 5.81 and 5.85 (each s, 2  1H, H-4 and OH), 6.44 (d, 7.48 (d, J ¼ 15.7 Hz, 1H, H-1); EIMS m/z (% rel. abund.): 370 [M]þ
J ¼ 15.8 Hz,1H, H-6), 6.35 (d, J ¼ 15.8 Hz,1H, H-2), 6.92 (d, J ¼ ca 8.0 Hz, (24), 352 (97), 305 (47), 177 (100), 150 (46), 137 (90), 135 (36).
H-500 ), 7.03 (br s, 2H, H-20 and H-200 ), 7.11 (br d, J ¼ ca 8.0 Hz,1H, H-50 ),
7.59 (br d, J ¼ 15.8 Hz, 2H, H-1 and H-7); EIMS m/z (% rel. abund.) 410 5.2.8. Catalytic hydrogenation of curcumin (1)
[M]þ (17), 368 (40), 350 (100), 191 (33), 190 (57), 177 (24). Tetrahydrocurcumin (25), hexahydrocurcumin (26) and octa-
Compound 2 was subjected to acetylation in the same manner hydrocurcumin (27) were prepared by catalytic hydrogenation of
to that of compound 1 to give Mono-O0 -acetyldemethoxycurcumin curcumin (1) according to the literature procedure [20,21]. Their
4454 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457

identities were confirmed by 1H NMR spectroscopic data compar- flow rate: 1 mL/min, detector: 254 nm) to yield 38a (tR 13.25 min)
ison with those of the reported values [20,21]. and 38b (tR 14.39 min).

5.2.9. Synthesis of unsaturated and saturated mono-keto analogs 5.2.12.2. Demethoxycurcumin isoxazole isomer1 (38a). White solid;
The enone 28 was prepared by dehydration of hexahy- m.p. 198e200  C; IR nmax: 3406, 1645, 1606, 1590, 1513, 1433, 1277,
drocurcumin (26) by the literature method [14]. The dienones 29 and 1171, 1033, 960, 820 cm1; 1H NMR (400 MHz, CDCl3 þ 5 drops of
30, and the trienone 31 were prepared by DDQ oxidation of the enone CD3OD) d 3.91 (s, 3H, OMe), 6.38 (br s, 1H, H-4), 6.76 (d, J ¼ 16.4 Hz,
28 using the literature procedure [14]. Catalytic hydrogenation of the 1H, H-10 ), 6.81 (br d, J ¼ 8.4 Hz, 2H, H-3000 and H-5000 ), 6.87 (d,
enone 28 using palladium on charcoal as a catalyst furnished the J ¼ 8.1 Hz, 1H, H-50000 ), 6.92 (d, J ¼ 16.4 Hz, 1H, H-100 ), 6.98 (br d,
saturated ketone 32. The spectroscopic data of the synthesized J ¼ 8.1 Hz, 1H, H-60000 ), 7.05 (br s, 1H, H-20000 ), 7.07 (d, J ¼ 16.4 Hz, 1H,
compounds were consistent with the reported values [14,21]. H-200 ), 7.25 (obscured signal, 1H, H-20 ), 7.31 (br d, J ¼ 8.4 Hz, 2H, H-
2000 and H-6000 ); ESMS (ve) m/z (% rel. abund.) 334 [M  H] (100).
5.2.10. Synthesis of pyrazole analogs of the curcuminoids 1, 2 and 3
Curcumin (1) (5 g, 13.6 mmol) was dissolved in AcOH (150 ml) 5.2.12.3. Demethoxycurcumin isoxazole isomer 2 (38b). White solid;
and hydrazine hydrate (1.5 mL, 30.9 mmol) was added. The reaction m.p. 194e195  C; IR nmax: 3406, 1645, 1606, 1590, 1513, 1433, 1277,
mixture was stirred at 50  C for 24 h; water (100 mL) was then 1171, 1033, 960, 820 cm1; 1H NMR (400 MHz, CDCl3 þ 5 drops of
added and the mixture was extracted with EtOAc (150 mL  3). The CD3OD) d 3.90 (s, 3H, OMe), 6.37 (br s, 1H, H-4), 6.76 (partial
combined organic phase was washed with H2O, dried over anhy- obscured signal, 1H, H-10 ), 6.79 (br d, J ¼ 8.4 Hz, 2H, H-30000 and H-
drous Na2SO4. The solvent was evaporated and the residue was 50000 ), 6.86e6.89 (m, 2H, H-10 and H-5000 ), 6.98 (br s, 1H, H-2000 ), 7.01
purified by column chromatography using CH2Cl2eMeOH (50:2) to (br d, J ¼ 8.1 Hz, 1H, H-6000 ), 7.06 (d, J ¼ 16.4 Hz, 1H, H-200 ), 7.22
yield compound 33 (3.5 g, 71%). (obscured signal, 1H, H-20 ), 7.25 (obscured signal, 1H, H-20 ), 7.31 (br
d, J ¼ 8.4 Hz, 2H, H-2000 and H-6000 ); 13C NMR (100 MHz, CDCl3 þ 5
5.2.10.1. Curcumin pyrazole (33). Yellow crystals (from drops of CD3OD) d 55.9 (3000 -OMe), 97.5 (C-4), 108.9 (C-2000 ), 110.7 (C-
CH2Cl2eMeOH); m.p. 223e224  C (lit. [20] 215  C): 1H NMR data 10 ), 113.1 (C-100 ), 114.8 (C-5000 ), 115.7 (C-30000 , C-50000 ), 121.4 (C-6000 ),
were in agreement with those reported previously [24]; ESMS 128.6 (C-20000 , C-60000 ), 134.8 (C-20 ), 135.6 (C-200 ), 146.9 (C-3000 , C-4000 ),
(ve) m/z (% rel. abund.) 363 [M  H] (100). 157.4 (C-40000 ), 162.3 (C-5), 168.4 (C-3); ESMS (ve): m/z (% rel.
By using the same procedure for the preparation of compound abund.) 334 [M  H] (100).
33, compound 2 was converted to the corresponding pyrazole Compound 39 was prepared in 68% yield by the method
analog 34 in 77%. 1H NMR data were in agreement with those employed for compound 37.
reported previously [20].
By using the same procedure for the preparation of compound 5.2.12.4. Bisdemethoxycurcumin isoxazole (39). Needles (from
33, compound 3 was converted to the corresponding pyrazole 35 as CH2Cl2eMeOH); m.p. >250  C; IR nmax: 3341, 1637, 1604, 1514, 1450,
white solid, m.p. >250  C (lit. [20] 272e273  C) in 41%. 1H NMR data 1282, 1254, 970, 835 cm1; 1H NMR (400 MHz, CDCl3 þ 12 drops of
were in agreement with those reported previously [20]. CD3OD) d 6.35 (s, 1H, H-4), 6.72 (d, J ¼ 16.3 Hz, 1H, H-10 ), 6.77 (br d,
J ¼ 8.4 Hz, 4H, H-3000 , H-5000 , H-30000 , H-50000 ), 6.85 (d, J ¼ 16.4 Hz, 1H, H-
5.2.11. Synthesis of phenylpyrazole analog of curcumin (1) 100 ), 7.04 (d, J ¼ 16.4 Hz, 1H, H-200 ), 7.21 (d, J ¼ 16.3 Hz, 1H, H-20 ), 7.33
Compound 1 was converted to the corresponding phenyl- (d, J ¼ 8.4 Hz, 4H, H-2000 , H-6000 , H-20000 , H-60000 ); ESMS (þve): m/z (% rel.
pyrazole 36 in 81% by the same procedure for the preparation of abund.) 633 [2M þ Na]þ (100); HR-TOFMS (ESþ): m/z 306.1130
compound 33, but using phenylhydrazine in place of hydrazine [M þ H]þ; calcd for C19H15NO3 þ H, 306.1130.
hydrate. 1H NMR data were in agreement with those reported
previously [24]. 5.2.13. Synthesis of isoxazole analog of dihydrocurcumin (24) and
tetrahydrocurcumin (25)
5.2.12. Synthesis of isoxazole analog of the curcuminoids 1, 2 and 3 Starting from dihydrocurcumin (24), the isoxazole analog 40
A solution of curcumin (1) (100 mg, 0.27 mmol) in pyridine was prepared in 63% yield by the same method employed for the
(3 mL) was treated with NH2OH$HCl (100 mg) and the mixture was preparation of compound 37.
stirred at 50  C for 6 h. The reaction was worked up with H2O and
the solution was extracted with EtOAc. The organic phase was 5.2.13.1. Dihydrocurcumin isoxazole (40). White solid; m.p.
washed with H2O, dried over anhydrous Na2SO4 and the solvent 168e170  C; 1H NMR (CDCl3) d 2.92 (br s, 4H, H-100 and H-200 ), 3.84
was evaporated to dryness. The crude product was purified by and 3.92 (each s, 6H, 2  OMe), 5.94 (br s, 1H, H-4), 6.68 (br s, 1H, H-
column chromatography using CH2Cl2eMeOH (100:1.5) to give 20000 ), 6.70 (obscured signal, H-60000 ), 6.73 (d, J ¼ 16.3 Hz,1H, H-10 ), 6.84
compound 37 (70 mg, 70%). (d, J ¼ 8.5 Hz, 1H, H-50000 ), 6.89 (d, J ¼ 8.1 Hz, 1H, H-5000 ), 6.98 (br s, 1H,
H-2000 ), 7.01 (d, J ¼ 8.1 Hz, 1H, H-6000 ), 7.19 (d, J ¼ 16.3 Hz, 1H, H-20 ); 13C
5.2.12.1. Curcumin isoxazole (37). Needles (from CH2Cl2en- NMR (100 MHz, CDCl3) d 28.2 (C-6), 34.2 (C-7), 55.9 (3000 -OMe and
hexane); m.p. 177e178  C (lit. [24] m.p. 162  C); IR nmax: 3447, 1646, 30000 -OMe), 100.7 (C-4), 108.6 (C-2000 ), 110.9 (C-20000 ), 111.0 (C-2), 114.3
1606, 1575, 1513, 1433, 1369, 1277, 808, 734 cm1; 1H NMR (C-50000 ), 114.7 (C-5000 ), 121.0 (C-60000 ), 121.4 (C-6000 ), 128.2 (C-1000 ), 132.6
(400 MHz, CDCl3) d 3.93 (s, 2  3H, 2  OMe), 6.41 (s, 1H, H-4), 6.79 (C-10000 ), 134.6 (C-1), 144.0 (C-40000 ), 146.4 (C-30000 ), 146.8 (C-4000 , C-3000 ),
(d, J ¼ 16.3 Hz, 1H, H-10 ), 6.89 (br d, J ¼ 8.0 Hz, 1H, H-5000 ), 6.90 (br d, 163.6 (C-5), 168.4 (C-3); ESMS (þve): m/z (% rel. abund.) 368
J ¼ 8.0 Hz, 1H, H-50000 ), 6.94 (d, J ¼ 16.7 Hz, 1H, H-100 ), 7.01 (over- [M þ H]þ (100); HR-TOFMS (ESþ): m/z 368.1505 [M þ H]þ; calcd for
lapping signals, H-2000 , H-60000 ), 7.04 (m, 1H, H-6000 ), 7.06 (br s, 1H, H- C21H21NO5 þ H, 368.1498.
20000 ), 7.08 (d, J ¼ 16.7 Hz, 1H, H-200 ), 7.26 (d, J ¼ 16.3 Hz, 1H, H-20 ); The isoxazole analog 42 was prepared as followed. A solution of
ESMS (þve) m/z (% rel. abund.) 366 [M þ H]þ (100). 25 (116 mg, 0.31 mmol) in pyridine (3 mL) was treated with
A 1:1 mixture of 38a and 38b was prepared from 2 in 65% yield NH2OH$HCl (60 mg) and the mixture was stirred at ambient
by the method employed for compound 37. The 38a/38b mixture temperature for 1 h. The reaction was worked up with H2O and the
was further purified by normal phase HPLC (column: Luna Silica (2) solution was extracted with EtOAc. The organic phase was washed
100 A, 5 mm, 4.6  250 mm, mobile phase: CH2Cl2eMeOH (99:1), with H2O, dried over anhydrous Na2SO4 and the solvent was
 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457 4455

evaporated to dryness. The crude product was purified by column 5.2.15. 3,3-Dimethylallylation of the isoxazole 37
chromatography using CH2Cl2eMeOH (100:1.5) to give the mon- The isoxazole 37 was subjected to 3,3-dimethylallylation in
oxime of 25 (85 mg, 70%), which was dissolved in benzene (2 mL) similar manner to that of methylation of compound 1, but using
and p-toluenesulfonic acid monohydrate (50 mg) was added. The 3,3-dimethylallyl bromide in place of methyl iodide, to afford
reaction mixture was stirred at 60  C for 1.5 h; water was added and a mixture of 46a and 46b and 47 in 25 and 73% yields, respectively.
the mixture was extracted with EtOAc. The combined organic phase
was washed with water and dried over anhydrous Na2SO4. The 5.2.15.1. Compounds 46a/46b (2:1 mixture). IR nmax: 3427, 2934,
solvent was evaporated and the residue was chromatographed 1646, 1559, 1511, 1430, 1269, 1137, 962, 810 cm1; 46a: 1H NMR
using CH2Cl2eMeOH (10:0.1) to yield 42 (62 mg, 77%). (400 MHz, CDCl3) d 1.73 and 1.76 (each s, 2  3H, 2  Me), 3.91 and
3.93 (each s, 2  3H, 2  OMe), 4.60 (br d, J ¼ 6.1 Hz, 2H, H-100000 ),
5.2.13.2. Tetrahydrocurcumin isoxazole (42). White amorphous 5.50 (m, 1H, H-200000 ), 5.74 (s, 1H, OH), 6.40 (s, 1H, H-4), 6.80 (d,
solid. 1H NMR (CDCl3, 400 MHz): d 2.87e2.91 (m, 6H), 2.96 (m, 2H) J ¼ 16.3 Hz, 1H, H-10 ), 6.86 (d, J ¼ 8.0 Hz, 1H, H-5000 ), 6.90 (d,
(H-10, H-20 , H-100, H-200 ), 3.82 and 3.83 (each s, 2  3H, 2  OMe), 5.50 J ¼ 8.0 Hz, 1H, H-50000 ), 6.95 (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.00e7.11 (m,
and 5.51 (each s, 2  1H, 2  OH), 5.67(s, 1H, H-4), 6.61e6.67 (m, 4H, 5H, H-200 , H-2000 , H-20000 , H-6000 , H-60000 ), 7.27 (d, J ¼ 16.3 Hz, 1H, H-20 );
H-2000 , H-6000 , H-20000 , H-60000 ), 6.80 and 6.81 (each d, J ¼ 7.8 Hz, H-5000 ESMS (þve): m/z (% rel. abund.) 434 [M þ H]þ (100); 46b: 1H NMR
and H-50000 ); ESMS (þve): m/z (% rel. abund.) 370 [M þ H]þ (100). (400 MHz, CDCl3) d 1.73 and 1.76 (each s, 6H, 2  Me), 3.91 and 3.93
(each s, 6H, 2  OMe), 4.60 (br d, J ¼ 6.1 Hz, 2H, H-100000 ), 5.50 (m, 1H,
5.2.14. Pentylation of the isoxazole 37 H-200000 ), 5.77 (s, 1H, OH), 6.40 (s, 1H, H-4), 6.79 (d, J ¼ 16.4 Hz, 1H, H-
The isoxazole 37 was subjected to n-pentylation in similar 10 ), 6.85 (br d, J ¼ 8.0 Hz, 1H, H-50000 ), 6.91 (br d, J ¼ 8.0 Hz, 1H, H-5000 ),
manner to that of methylation of compound 1, but using n-pentyl 6.98 (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.00e7.07 (m, 5H, H-200 , H-2000 , H-20000 ,
iodide in place of methyl iodide, to afford 43 and 44 (1:1 mixture) H-6000 , H-60000 ), 7.26 (d, J ¼ 16.3 Hz, 1H, H-20 ); 46a/46b: ESMS (þve):
and 45 in 24 and 62%, respectively. The 43/44 mixture was sepa- m/z (% rel. abund.) 434 [M þ H]þ (100).
rated by normal phase HPLC (column: Luna Silica (2) 100A, 5 mm,
4.6  250 mm, mobile phase: n-hexaneeCHCl3 (1:1), flow rate: 5.2.15.2. Compound 47. White solid; m.p. 167e186  C; IR nmax:
1.2 mL/min, detector: 254 nm) to yield 43 (tR 21.42 min) and 44 (tR 2928, 1645, 1581, 1512, 1432, 1312, 1136, 1028, 989, 818 cm1; 1H
19.02 min). NMR (400 MHz, CDCl3) d 1.73 and 1.76 (each s, 4  3H, 4  Me), 3.90
and 3.91 (each s, 2  3H, 2  OMe), 4.60 (br d, J ¼ 6.1 Hz, 4H, H-100000
5.2.14.1. Compound 43. White solid; m.p. 116e117  C; IR nmax: 3376, and H-1000000 ), 5.50 (m, 2H, H-200000 and H-2000000 ), 6.40 (s, 1H, H-4), 6.81
2937, 1644, 1597, 1509, 1467, 1428, 1279, 1225, 1163, 1140, 1032, 962, (d, J ¼ 16.3 Hz, 1H, H-10 ), 6.86 (br d, J ¼ 8.3 Hz, 2H, H-5000 and H-50000 ),
807, 734 cm1. 1H NMR (400 MHz, CDCl3) d 0.91 (t, J ¼ 6.8 Hz, 3H, H- 6.97 (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.01e7.11 (m, 5H, H-200 , H-2000 , H-20000 ,
500000 ), 1.40 (m, 4H, H-300000 and H-400000 ), 1.84 (m, 2H, H-200000 ), 3.90 and H-6000 , H-60000 ), 7.27 (d, J ¼ 16.3 Hz, 1H, H-20 ); ESMS (ve) m/z (% rel.
3.93 (each s, 2  3H, 2  OMe), 4.02 (t, J ¼ 6.4 Hz, 2H, H-100000 ), 6.40 (s, abund.) 500 [M  H] (100); HR-TOFMS (ES): m/z 500.2436
1H, H-4), 6.80 (d, J ¼ 16.3 Hz, 1H, H-10 ), 6.85 (d, J ¼ 8.0 Hz, 1H, H-5000 ), [M  H]; calcd for C31H35NO5eH, 500.2437.
6.90 (d, J ¼ 8.0 Hz, 1H, H-50000 ), 6.95 (d, J ¼ 16.5 Hz, 1H, H-100 ),
6.99e7.09 (m, 5H, H-200 , H-2000 , H-20000 , H-6000 , H-60000 ), 7.27 (d, 5.2.16. Ethylation of the isoxazole 37
J ¼ 16.3 Hz, 1H, H-20 ); 13C NMR (100 MHz, CDCl3) d 13.9 (C-500000 ), 22.4 The isoxazole 37 was subjected to ethylation in similar manner
(C-200000 ), 28.0 (C-300000 ), 28.8 (C-400000 ), 55.9 (30000 -OMe), 56.0 (3000 -OMe), to that of methylation of compound 1, but using diethyl sulfate in
69.0 (C-100000 ) , 97.6 (C-4),108.2 (C-20000 ),109.6 (C-2000 ),110.0 (C-10 ),111.2 place of methyl iodide, to afford a 5:2 mixture of 48a and 48b in 23%
(C-5000 ), 113.8 (C-100 ), 114.6 (C-50000 ), 121.0 (C-6000 ), 121.6 (C-60000 ), 128.4 and 49 in 60%.
(C-10000 ), 128.5 (C-1000 ), 134.8 (C-20 ), 135.5 (C-200 ), 146.6 (C-40000 ), 146.8
(C-30000 ), 149.6 (C-3000 , C-4000 ), 162.1 (C-5), 168.5 (C-3); ESMS (ve): m/z 5.2.16.1. Compound 48a/48b (5:2 mixture). IR nmax: 3420, 2934,
(% rel. abund.) 434 [M  H] (100); HR-TOFMS (APCIþ): m/z 436.2121 1644, 1598, 1509, 1430, 1267, 1138, 1032, 961, 808 cm1; 48a: 1H
[M þ H]þ; calcd for C26H29NO5 þ H, 436.2118. NMR (400 MHz, CDCl3) d 1.46 (t, J ¼ 6.9 Hz, 3H, H-200000 ), 3.92 and
3.93 (each s, 2  3H, 2  OMe), 4.12 (m, 2H, H-100000 ), 5.75 (s, 1H, OH),
5.2.14.2. Compound 44. White solid; m.p. 115e116  C; 1H NMR 6.40 (s, 1H, H-4), 6.80 (d, J ¼ 16.3 Hz, 1H, H-10 ), 6.86 (d, J ¼ 8.0 Hz,
(400 MHz, CDCl3) d 0.91 (t, J ¼ 6.8 Hz, 3H, H-500000 ), 1.40 (m, 4H, H- 1H, H-5000 ), 6.90 (d, J ¼ 8.0 Hz, 1H, H-50000 ), 6.95 (d, J ¼ 16.4 Hz, 1H, H-
300000 and H-400000 ), 1.84 (m, 2H, H-200000 ), 3.90 and 3.93 (each s, 2  3H, 100 ), 7.00e7.11 (m, 5H, H-200 , H-2000 , H-20000 , H-6000 , H-60000 ), 7.27 (d,
2  OMe), 4.02 (t, J ¼ 6.4 Hz, 2H, H-100000 ), 6.40 (s, 1H, H-4), 6.79 (d, J ¼ 16.3 Hz, 1H, H-20 ); 48b: 1H NMR (400 MHz, CDCl3) d 1.46 (t,
J ¼ 16.3 Hz, 1H, H-10 ), 6.85 (d, J ¼ 8.2 Hz, 1H, H-50000 ), 6.91 (d, J ¼ 6.9 Hz, 3H, H-200000 ), 3.91 and 3.94 (each s, 2  3H, 2  OMe), 4.12
J ¼ 8.0 Hz, 1H, H-5000 ), 6.97 (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.01e7.08 (m, (m, 2H, H-100000 ), 5.78 (s, 1H, OH), 6.40 (s, 1H, H-4), 6.79 (d,
5H, H-200 , H-2000 , H-20000 , H-6000 , H-60000 ), 7.26 (d, J ¼ 16.3 Hz, 1H, H-20 ); J ¼ 16.4 Hz, 1H, H-10 ), 6.85 (br d, J ¼ 8.0 Hz, 1H, H-50000 ), 6.91 (br d,
ESMS (ve): m/z (% rel. abund.) 434 [M  H] (100); HR-TOFMS J ¼ 8.0 Hz, 1H, H-5000 ), 6.98 (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.00e7.11 (m,
(APCIþ): m/z 436.2125 [M þ H]þ; calcd for C26H29NO5þH, 436.2118. 5H, H-200 , H-2000 , H-20000 , H-6000 , H-60000 ), 7.26 (d, J ¼ 16.3 Hz, 1H, H-20 );
48a/48b: ESMS (þve) m/z (% rel. abund.) 434 [M þ H]þ (100).
5.2.14.3. Compound 45. Aggregated needles (from n-hex-
aneeCH2Cl2); m.p. 130e131  C; IR nmax: 3446, 2952, 2869, 1634, 5.2.16.2. Compound 49. White plates (from CH2Cl2en-hexane);
1596, 1581, 1558, 1514, 1467, 1393, 1325, 1268, 1241, 1138, m.p. 175  C; IR nmax: 2976, 1633, 1581, 1558, 1514, 1473, 1264, 1239,
1050 cm1; 1H NMR (400 MHz, CDCl3) d 0.89 (t, J ¼ 6.9 Hz, 6H, H- 1136, 1026, 967, 800 cm1; 1H NMR (400 MHz, CDCl3) d 1.46 (t,
500000 and H-5000000 ), 1.37 (m, 8H, H-300000 , H-3000000 , H-400000 , H-4000000 ), 1.82 J ¼ 6.9 Hz, 6H, H-200000 and H-2000000 ), 3.91 and 3.92 (each s, 2  3H,
(m, 4H, H-200000 and H-2000000 ), 3.87 and 3.88 (each s, 2  3H, 2  OMe), 2  OMe), 4.12 (m, 4H, H-100000 and H-1000000 ), 6.40 (s, 1H, H-4), 6.81 (d,
4.60 (br t, J ¼ 5.4 Hz, 4H, H-100000 and H-1000000 ), 6.38 (s, 1H, H-4), 6.78 J ¼ 16.3 Hz, 1H, H-10 ), 6.85 and 6.86 (each d, J ¼ 8.0 Hz, 2H, H-5000 and
(d, J ¼ 16.4 Hz, 1H, H-10 ), 6.82 and 6.83 (each d, J ¼ 8.2 Hz, 2H, H-5000 H-50000 ), 6.97 (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.01e7.11 (m, 5H, H-200 , H-2000 ,
and H-50000 ), 6.95 (d, J ¼ 16.4 Hz, 1H, H-100 ), 6.99e7.06 (m, 5H, H-200 , H-20000 , H-6000 , H-60000 ), 7.27 (d, J ¼ 16.3 Hz, 1H, H-20 ); ESMS (þve): m/z
H-2000 , H-20000 , H-6000 , H-60000 ), 7.25 (d, J ¼ 16.3 Hz, 1H, H-20 ); ESMS (% rel. abund.) 422 [M þ H]þ (100); HR-TOFMS (ESþ): m/z 422.1974
(þve): m/z (% rel. abund.) 506 [M þ H]þ (100). [M þ H]þ; calcd for C25H27NO5 þ H, 422.1967.
4456 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457

5.2.17. Acetylation of the isoxazole 37 607 cm1; 1H NMR (400 MHz, CDCl3) d 3.90 and 3.93 (each s, 2  3H
Ac2O (0.05 mL) was added to a solution of compound 37 (90 mg, and 1  3H, 3  OMe), 6.40 (s, 1H, H-4), 6.81 (d, J ¼ 16.3 Hz, 1H, H-10 ),
0.25 mmol) in pyridine (2 mL) and the reaction mixture was stirred 6.86 (br d, J ¼ 8.3 Hz, 1H, H-5000 ), 6.90 (br d, J ¼ 8.3 Hz, 1H, H-50000 ), 6.94
at ambient temperature for 1 h. The crude product which were (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.00 (br d, J ¼ 8.3 Hz, 1H, H-60000 ), 7.04e7.08
obtained by the usual work up followed by solvent extraction were (m, 4H, H-200 , H-2000 , H-20000 , H-6000 ), 7.28 (d, J ¼ 16.3 Hz, 1H, H-20 ); 13C
subjected to column chromatography eluting with CH2Cl2:MeOH NMR (100 MHz, CDCl3) d 55.9 (3000 -OMe and 30000 -OMe), 56.0 (4000 -
(100:0.8) to afford a mixture of 50 and 51 (31 mg, 30%), and 52 OMe), 97.7 (C-4),108.2 (C-20000 ),109.1 (C-2000 ),111.1 (C-10 ), 111.2 (C-5000 ),
(62 mg, 55%). The 50/51 mixture was subjected to normal phase 113.8 (C-100 ), 114.6 (C-50000 ), 121.1 (C-6000 ), 121.6 (C-60000 ), 128.5 (C-10000 ),
HPLC separation (column: Luna Silica (2) 100A, 5 mm, 4.6  250 mm, 128.6 (C-1000 ), 134.7 (C-20 ), 135.6 (C-200 ), 146.7 (C-40000 ), 146.8 (C-30000 ),
mobile phase: n-hexaneeCHCl3 (30:50), flow rate: 0.8 mL/min, 149.3 (C-3000 ), 150.2 (C-4000 ), 162.1 (C-5), 168.4 (C-3); ESMS (þve): m/z
detector: 254 nm) to yield 47 (tR 30.99 min) and 48 (tR 33.62 min). (% rel. abund.) 380 [M þ H]þ (100); HR-TOFMS (APCIþ): m/z 380.1497
[M þ H]þ; calcd for C22H21NO5 þ H, 380.1492.
5.2.17.1. Compound 50. White solid; m.p. 167e168  C; IR nmax:
3421, 1759, 1647, 1602, 1561, 1513, 1429, 1371, 1276, 1220, 1121, 1031, 5.2.18.2. Compound 54. Colorless foam; 1H NMR (400 MHz, CDCl3)
962, 828, 739 cm1; 1H NMR (400 MHz, CDCl3) d 2.30 (s, 3H, OAc), d 3.89, 3.92 and 3.94 (each s, 3  3H, 3  OMe), 6.40 (s, 1H, H-4), 6.79
3.86 and 3.94 (each s, 2  3H, 2  OMe), 6.41 (s, 1H, H-4), 6.79 (d, (d, J ¼ 16.4 Hz, 1H, H-10 ), 6.85 (br d, J ¼ 8.0 Hz, 1H, H-50000 ), 6.91 (br d,
J ¼ 16.3 Hz, 1H, H-10 ), 6.91 (d, J ¼ 8.1 Hz, 1H, H-50000 ), 7.00e7.14 (m, J ¼ 8.0 Hz, 1H, H-5000 ), 6.98 (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.04 (br s, 1H, H-
7H, H-100, H-200 , H-2000 , H-20000 , H-5000 , H-6000 , H-60000 ), 7.27 (d, J ¼ 16.3 Hz, 2000 ), 7.06e7.09 (m, 4H, H-200 , H-20000 , H-6000 , H-60000 ), 7.26 (d, J ¼ 16.3 Hz,
1H, H-20 ); 13C NMR (100 MHz, CDCl3) d 20.6 (COMe), 55.9 (3000 - 1H, H-20 ); 13C NMR (100 MHz, CDCl3) d 55.9 (3000 -OMe and 30000 -OMe),
OMe), 56.0 (30000 -OMe), 97.6 (C-4), 108.8 (C-2000 ), 110.2 (C-20000 ), 110.8 56.0 (4000 -OMe), 97.6 (C-4), 108.8 (C-2000 ), 108.9 (C-20000 ), 110.9 (C-10 ),
(C-10 ), 114.8 (C-50000 ), 116.5 (C-100 ), 120.0 (C-60000 ), 121.5 (C-6000 ), 121.6 111.2 (C-50000 ), 114.2 (C-100 ), 114.7 (C-5000 ), 120.8 (C-60000 ), 121.5 (C-6000 ),
(C-60000 ), 123.1 (C-5000 ), 128.1 (C-1000 and C-10000 ), 134.9 (C-200 ), 135.0 (C- 128.2 (C-1000 ), 129.0 (C-10000 ), 134.9 (C-20 ), 135.4 (C-200 ), 146.8 (C-4000 ),
20 ), 146.8 (C-40000 ), 147.0 (C-30000 ), 151.1 (C-3000 ), 152.2 (C-4000 ), 161.8 (C- 146.9 (C-3000 ), 149.2 (C-30000 ), 150.0 (C-40000 ), 162.1 (C-5), 168.5 (C-3);
5), 168.7 (C-3), 168.8 (COMe); ESMS (ve): m/z (% rel. abund.) 406 ESMS (þve): m/z (% rel. abund.) 380 [M þ H]þ (100); HR-TOFMS
[M  H] (100); HR-TOFMS (APCIþ): m/z 408.1449 [M þ H]þ; calcd (APCIþ): m/z 380.1501 [M þ H]þ; calcd for C22H21NO5 þ H, 380.1492.
for C23H21NO6 þ H, 408.1442.
5.2.18.3. Compound 55. White powder; m.p. 159e160  C; IR nmax:
5.2.17.2. Compound 51. White solid; m.p.159  C; 1H NMR (400 MHz, 3449, 2999, 2936, 2838, 1646, 1601, 1561, 1513, 1428, 1266, 1224,
CDCl3) d 2.30 (s, 3H, OAc), 3.87 and 3.92 (each s, 2  3H, 2  OMe), 1140,1026, 966, 807, 767, 736 cm1; 1H NMR (400 MHz, CDCl3) d 3.89
6.44 (s, 1H, H-4), 6.87 (d, J ¼ 16.3 Hz, 1H, H-10 ), 6.90 (d, J ¼ 8.1 Hz, 1H, and 3.91 (each s, 2  3H and 2  3H, 4  OMe), 6.41 (s, 1H, H-4), 6.81
H-50000 ), 6.95 (d, J ¼ 16.4 Hz, 1H, H-100 ), 6.99e7.10 (m, 6H, H-200 , H-2000 , (d, J ¼ 16.3 Hz, 1H, H-10 ), 6.84 (br d, J ¼ 8.2 Hz, 1H, H-5000 ), 6.86 (br d,
H-20000 , H-5000 , H-6000 , H-60000 ), 7.29 (d, J ¼ 16.3 Hz,1H, H-20 ); ESMS (ve): J ¼ 8.2 Hz,1H, H-50000 ), 6.98 (d, J ¼ 16.4 Hz,1H, H-100 ), 7.03e7.10 (m, 5H,
m/z (% rel. abund.) 406 [M  H] (100); HR-TOFMS (APCIþ): m/z H-200 , H-2000 , H-20000 , H-6000 , H-60000 ), 7.27 (d, J ¼ 16.3 Hz, 1H, H-20 ); 13C
408.1444 [M þ H]þ; calcd for C23H21NO6 þ H, 408.1442. NMR (100 MHz, CDCl3) d 55.8 and 55.9 (3000 -OMe, 30000 -OMe, 4000 -OMe,
40000 -OMe), 97.6 (C-4), 108.7, 109.0 (C-2000 , C-20000 ), 111.0 (C-10 ), 111.1 (C-
5.2.17.3. Compound 52. White solid; m.p. 188e189  C; IR nmax: 3015, 5000 , C-50000 ), 114.0 (C-100 ), 120.8, 121.0 (C-6000 , C-60000 ), 128.5, 128.8 (C-1000,
2924, 1753, 1638, 1601, 1566, 1514, 1470, 1396, 1207, 1225, 1203, 971, C-10000 ), 134.6 (C-20 ), 135.3 (C-200 ), 149.1, 149.8, 150.1 (C-3000 , C-30000 , C-
832, 740, 656 cm1; 1H NMR (400 MHz, CDCl3) d 2.31 (s, 2  3H, 4000 , C-40000 ), 162.0 (C-5), 168.3 (C-3); ESMS (þve): m/z (% rel. abund.)
2  OAc), 3.87 (s, 2  3H, 2  OMe), 6.47 (br s, 1H, H-4), 6.89 (d, 394 [M þ H]þ (100); HR-TOFMS (APCIþ): m/z 394.1650 [M þ H]þ;
J ¼ 16.3 Hz,1H, H-10 ), 7.02e7.15 (m, 8H, H-100, H-200 , H-2000 , H-20000 , H-5000 , calcd for C22H23NO5 þ H, 394.1649.
H-50000 , H-6000 , H-60000 ), 7.31 (d, J ¼ 16.3 Hz,1H, H-20 ); 13C NMR (100 MHz,
CDCl3) d 20.5 (COMe), 55.8 (3000 -OMe, 30000 -OMe), 98.5 (C-4),110.1,110.6 5.2.19. Methylation of a mixture of the isoxazole mixture 38a/38b
(C-2000 , C-20000 ), 119.8, 120.0 (C-6000 , C-60000 ), 123.0, 123.1 (C-5000 , C-50000 ), The 1:1 mixture of the isoxazoles 38a/38b was subjected to
134.2 (C-20 ),134.4,134.7 (C-1000, C-10000 ),135.0 (C-200 ),140.2,140.4 (C-4000 , methylation in similar manner to that of compound 37 to give a 1:1
C-40000 ), 151.3 (C-3000 , C-30000 ), 161.7 (C-5), 168.0 (C-3), 168.8 (2  COMe); mixture of 56a and 56b in 80%.
ESMS (þve): m/z (% rel. abund.) 450 [M þ H]þ (100); HR-TOFMS (ESþ):
m/z 450.1566 [M þ H]þ; calcd for C25H23NO7 þ H, 450.1553. 5.2.19.1. Compounds 56a/56b (1:1 mixture). IR nmax: 2962, 2838,
1644, 1603, 1577, 1509, 1430, 1307, 1263, 1139, 1025, 961, 821 cm1;
5.2.18. Methylation of the isoxazole 37 56a: 1H NMR (400 MHz, CDCl3) d 3.82, 3.89 and 3.92 (each s, 3  3H,
Compound 37 (1.5 g, 4.1 mmol) was dissolved in dry acetone 3  OMe), 6.41 (s, 1H, H-4), 6.83 (d, J ¼ 16.4 Hz, 1H, H-10 ), 6.86 (d,
(45 mL) and anhydrous K2CO3 (1.1 g) and MeI (4.5 mL) was added. J ¼ 8.2 Hz, 1H, H-50000 ), 6.89 (br d, J ¼ 8.4 Hz, 2H, H-3000 and H-5000 ), 6.98
The reaction mixture was reflux for 5 h. Water was added and the (d, J ¼ 16.4 Hz,1H, H-100 ), 7.03 (br s,1H, H-20000 ), 7.06e7.12 (m, 2H, H-200
mixture extracted with CH2Cl2. The combined organic phase was and H-60000 ), 7.28 (d, J ¼ 16.4 Hz, 1H, H-20 ), 7.45 (br d, J ¼ 8.4 Hz, 2H, H-
washed with H2O, dried over anhydrous Na2SO4 and the solvent was 2000 and H-6000 ); 56b: 1H NMR (400 MHz, CDCl3) d 3.82, 3.89 and 3.92
removed under vacuum. The crude products were separated and (each s, 3  3H, 3  OMe), 6.40 (s, 1H, H-4), 6.83 (d, J ¼ 16.4 Hz, 1H, H-
purified by column chromatography using CH2Cl2eMeOH (100:1) to 10 ), 6.85 (d, J ¼ 8.2 Hz, 1H, H-5000 ), 6.89 (br d, J ¼ 8.4 Hz, 2H, H-30000 and
yield a 1:1 mixture of isomeric mono-methylated analogs 53 and 54 H-50000 ), 6.97 (d, J ¼ 16.4 Hz, 1H, H-100 ), 7.03 (br s, 1H, H-2000 ), 7.06e7.12
(590 mg, 38%) and the di-methylated analog 55 (805 mg, 50%). A (m, 2H, H-200 and H-6000 ), 7.27 (d, J ¼ 16.4 Hz, 1H, H-20 ), 7.45 (br d,
mixture of 53 and 54 was separated by normal phase HPLC (column: J ¼ 8.4 Hz, 2H, H-20000 and H-60000 ); 56a/56b: ESMS (þve) m/z (% rel.
Luna Silica (2) 100A, 5 mm, 4.6  250 mm, mobile phase: n-hex- abund.) 749 [2M þ Na]þ (100).
aneeEtOAc (72:28), flow rate: 1 mL/min, detector: 254 nm) to yield
pure 53 (tR 31.38 min) and 54 (tR 32.01 min). 5.2.20. Methylation of the isoxazole 39
The isoxazole 39 was subjected to methylation in similar
5.2.18.1. Compound 53. Colorless foam; IR nmax: 3413, 2935, 2836, manner to that of compound 37 to give the corresponding dimethyl
1645, 1598, 1513, 1431, 1268, 1159, 1139, 1025, 961, 809, 736, ether analog 57 in 78%.
 C. Changtam et al. / European Journal of Medicinal Chemistry 45 (2010) 4446e4457 4457

5.2.20.1. Compound 57. Aggregated needles (from CH2Cl2en- which prevented the color change. NIH, RIF and kanamycin (Sigma)
hexane), m.p.180e181  C; IR nmax: 2934,1646,1604,1578,1559,1510, were included as controls.
1307, 1248, 1174, 1092, 969, 819 cm1; 1H NMR (400 MHz, CDCl3)
d 3.82 (s, 2  3H, 2  OMe), 6.39 (s, 1H, H-4), 6.85 (d, J ¼ 16.4 Hz, 1H,
H-10 ), 6.90 (br d, J ¼ 8.4 Hz, 4H, H-3000 , H-5000 , H-30000 , H-50000 ), 6.97 (d, Acknowledgements
J ¼ 16.4 Hz, 1H, H-100 ), 7.10 (d, J ¼ 16.4 Hz, 1H, H-200 ), 7.28 (d,
J ¼ 16.4 Hz, 1H, H-20 ), 7.45 (d, J ¼ 8.4 Hz, 4H, H-2000 , H-6000 , H-20000 , H- This work was supported by the Research Team Strengthening
60000 ); ESMS (þve): m/z (% rel. abund.) 334 [M þ H]þ (100); HR-TOFMS Grant of the National Center for Genetic Engineering and Biotech-
(ESþ): m/z 334.1442 [M þ H]þ; calcd for C21H19NO3 þ H, 334.1443. nology, National Science and Technology Development Agency.
Support from the Center of Excellence for Innovation in Chemistry
5.2.21. Synthesis of isoxazole analogs of mono-O- (PERCH-CIC), Commission on Higher Education, Ministry of Education
demethylcurcumin (4) is gratefully acknowledged. CC acknowledges a scholarship from the
A 1:2 mixture of the isoxazole analogs 58a and 58b was Royal Golden Jubilee Ph.D. Program of The Thailand Research Fund.
prepared from 4 in 60% yield by the same method employed for the
preparation of compound 37 from compound 1.
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