Synthesis, cytotoxicity against human oral cancer KB cells and structure-activity relationship studies of trienone analogues of curcuminoids

Article abstract of DOI:10.1016/j.bmcl.2014.04.105

A general method for the synthesis of substituted (1E,4E,6E)-1,7- diphenylhepta-1,4,6-trien-3-ones, based on the aldol condensations of substituted 4-phenylbut-3-en-2-ones and substituted 3-phenylacrylaldehydes, was achieved. The natural trienones 4 and 5 have been synthesized by this method, together with the trienone analogues 9-20. These analogues were evaluated for their cytotoxic activity against human oral cancer KB cell line. The structure-activity relationship study has indicated that the analogues with the 1,4,6-trien-3-one function are more potent than the curcuminoid-type function. Analogues with meta-oxygen function on the aromatic rings are more potent than those in the ortho- and para-positions. Free phenolic hydroxy group is more potent than the corresponding methyl ether analogues. Among the potent trienones, compounds 11, 18 and 20 were more active than the anticancer drug ellipticine. All compounds were also evaluated against the non-cancerous Vero cells and it was found that compounds 11, 12 and 17 were much less toxic than curcumin (1); they showed high selectivity indices of 35.46, 33.46 and 31.68, respectively. These analogues are regarded as the potent trienones for anti-oral cancer study.

Full text of DOI:10.1016/j.bmcl.2014.04.105

Accepted Manuscript
Synthesis, cytotoxicity against human oral cancer KB cells and structure–ac-
tivity relationship studies of trienone analogues of curcuminoids
Thipphawan Chuprajob, Chatchawan Changtam, Ratchanaporn Chokchaisiri,
Warangkana Chunglok, Nilubon Sornkaew, Apichart Suksamrarn
PII:
S0960-894X(14)00462-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.105
BMCL 21598
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 January 2014
15 April 2014
25 April 2014
Please cite this article as: Chuprajob, T., Changtam, C., Chokchaisiri, R., Chunglok, W., Sornkaew, N., Suksamrarn,
A., Synthesis, cytotoxicity against human oral cancer KB cells and structure–activity relationship studies of trienone
analogues of curcuminoids, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/
j.bmcl.2014.04.105
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1
Synthesis, cytotoxicity against human oral cancer KB cells and structure–
activity relationship studies of trienone analogues of curcuminoids
Thipphawan Chuprajob a, Chatchawan Changtam b, Ratchanaporn Chokchaisiri c,
Warangkana Chunglok d, Nilubon Sornkaew a, Apichart Suksamrarn a,*
a Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of
Science, Ramkhamhaeng University, Bangkok 10240, Thailand
b Division of Physical Science, Faculty of Science and Technology, Huachiew
Chalermprakiet University, Samutprakarn 10540, Thailand
c Department of Chemistry, School of Science, Phayao University, Phayao 56000, Thailand
d School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si
Thammarat 80160, Thailand

* Corresponding author. Tel.: +66 2 319 0931; Fax: +66 2 319 0931.
E-mail addresses: s_apichart@ru.ac.th, asuksamrarn@yahoo.com (A. Suksamrarn).

2
ABSTRACT
A general method for the synthesis of substituted (1E,4E,6E)-1,7-diphenylhepta-1,4,6-
trien-3-ones, based on the aldol condensations of substituted 4-phenylbut-3-en-2-ones and
substituted 3-phenylacrylaldehydes, was achieved. The natural trienones 4 and 5 have been
synthesized by this method, together with the trienone analogues 9–20. These analogues were
evaluated for their cytotoxic activity against human oral cancer KB cell line. The structure–
activity relationship study has indicated that the analogues with the 1,4,6-trien-3-one function
are more potent than the curcuminoid-type function. Analogues with meta-oxygen function
on the aromatic rings are more potent than those in the ortho- and para-positions. Free
phenolic hydroxy group is more potent than the corresponding methyl ether analogues.
Among the potent trienones, compounds 11, 18 and 20 were more active than the anticancer
drug ellipticine. All compounds were also evaluated against the non-cancerous Vero cells and
it was found that compounds 11, 12 and 17 were much less toxic than curcumin (1); they
showed high selectivity indices of 35.46, 33.46 and 31.68, respectively. These analogues are
regarded as the potent trienones for anti-oral cancer study.
Keywords:
Curcuminoid; Trienone analogue; Synthesis; Cytotoxicity; KB

3
Curcuminoids are the major constituents of turmeric (Curcuma longa L., Zingiberaceae)
and have been used for centuries as a dietary pigment, spice and traditional medicine in India
and China. The major curcuminoid isolated from this plant species is curcumin (1), with
demethoxycurcumin (2) and bisdemethoxycurcumin (3) as the minor constituents.
Curcuminoids exhibited many interesting biological activities,1 for example, antioxidant
activity,2,3 anti-inflammatory activity,4,5 anticancer activity,6,7 anti-protozoal activity8 and
anti-HIV activity.9 In clinical report, curcuminoids could be orally taken up to 12 g/day
without toxic effect in humans.10 Therefore, many researchers have used this class of
compounds to study different biological activities. Some of these studies investigated the role
of α,β-unsaturated β-diketo moiety and it was concluded that this functional group is
necessary for the biological activities.11,12 Recently, a new curcuminoid analogue, 1,7-bis(4-
hydroxyphenyl)-1,4,6-heptatrien-3-one (4) (see Fig. 1), which is structurally related to the
curcuminoid 3, has been isolated as a minor component of C. longa.13 This compound
inhibited the production of TNF-α by lipopolysaccharide-activated macrophages.14 Another
new curcuminoid analogue, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one (5),
which is structurally related to the curcuminoid 1, has later been isolated.15 It is interesting to
note that these two compounds possess a 1E,4E,6E-heptatrien-3-one functionality, which is a
rare group of naturally occurring curcuminoid analogue and has not much been studied,
especially the biological activities of this type of compounds.
Insert Figure 1 →
The structural diversity of the trienones 4 and 5 has prompted us to investigate their
biological activities. However, the scarcity of these compounds in natural sources has
prevented us from obtaining them for biological activity evaluations. In order to see whether
this group of curcuminoid analogues exhibited more interesting biological activities than
those of the curcuminoids 1–3, we decided to synthesize the natural trienones 4 and 5. Within
the heptatrien-3-one framework, the analogues containing different hydroxy and methoxy
substituents on the aromatic moieties would then be synthesized if the trienones 4 and 5
showed higher biological activity than the corresponding curcuminoids 3 and 1.
Oral cancer is a major health problem in the economically developing countries.
According to GLOBOCAN 2012, oral cancer was estimated to account for 2.7% of all
cancers.16 There were about 300,000 oral cancer cases in 2012 worldwide and about 50% of

4
patients died from this type of cancer. The five-year relative survival rate of oral cancer
patients is less than 35% in advanced stage of disease at initial diagnosis.17 Late diagnosis,
disease recurrence, metastasis and resistant to therapy may attribute to this poor survival rate.
Conventional treatments, including surgical treatment combined with radiotherapy and
chemotherapy or concomitant chemo-radiotherapy, have limited efficacy and result in
adverse systemic and cytotoxic effects on normal cells. Chemoprevention is a promising
treatment strategy for oral cancer. Appropriate chemopreventive agents should be
inexpensive, nontoxic, and target important pathways involved in the development of this
cancer. Combining chemopreventive agents and conventional therapeutic approaches may
reduce toxicities and improving treatment outcomes. The addition of cetuximab, epidermal
growth factor receptor (EGFR) targeted chemopreventive agent, to current conventional
chemotherapeutic agents (cisplatin and 5-fluorouracil) represents a standard systemic
treatment for recurrent or metastatic cancer.18,19 However, the concurrent therapy also leads
to substantial toxicities and the overall survival remains short. To date, various molecular
targeted chemopreventive agents are actively under investigation, but the only pharmacologic
strategy targeting the EGFR has been approved by regulatory agencies worldwide to treat this
oral cancer.20
Insert Table 1 →
The synthesis of the trienone 4 has been reported in seven steps in good yield.21
Compound 5 has previously been prepared by a multi-step chemical modification of
compound 1.22 However, we would like to have a general method for the synthesis of
substituted trienones, so that a wide variety of this group of compounds could be prepared in
case the trienones 4 and 5 exhibited better cytotoxic activity than the respective curcuminoids
3 and 1. The retrosynthetic analysis of (1E,4E,6E)-1,7-diarylhepta-1,4,6-trien-3-ones by cross
aldol condensation reaction has been proposed as shown in Scheme 1. Despite the fact that
this type of condensation sometimes gives low yield of products,23 it was nevertheless is a
convenient and concise procedure for the synthesis of a large number of analogues for
biological evaluation and structure–activity relationship study. Disconnection of the C4–C5
bond of the seven-carbon linker gave two key fragments, substituted 4-phenylbut-3-en-2-ones
(substituted cinnamones) and substituted 3-phenylacrylaldehydes (substituted
cinnamaldehydes). The resulting substituted 4-phenylbut-3-en-2-ones and substituted 3-

5
phenylacrylaldehydes would then be further disconnected to the substituted benzaldehydes
(Scheme 1).
Insert Scheme 1 →
The key fragment substituted 4-phenylbut-3-en-2-ones and 3-phenylacrylaldehydes used
for synthesis of the natural trienones (4, 5) and analogues (9–15 and 17–20) were prepared as
shown in Scheme 2. Condensation of the substituted benzaldehydes (6a1–d1, 6e and 6f) with
excess acetone under basic aldol condensations at room temperature gave the substituted 4-
phenylbut-3-en-2-one analogues 7a1–d1, 7e and 7f. The analogue 7c3 was obtained by
methylation of 7c1 using methyl iodide in the presence of potassium carbonate in acetone.
The synthesis of substituted 3-phenylacrylaldehydes 8a1–d1 were planned by coupling of the
substituted benzaldehydes 6a1–d1 with acetaldehyde, using aldol condensation in the same
method as that of substituted 4-phenylbut-3-en-2-one analogues. In this method, we were able
to successfully generate only the meta-hydroxy-3-phenylacrylaldehyde analogue 8c1.
However, we failed to generate the ortho- and para-3-phenylacrylaldehydes 8a1, 8b1 and
8d1. In view of these results, the ortho- and para-hydroxy groups of benzaldehydes 6a1, 6b1
and 6d1 were therefore protected as its tetrahydropyranyl (THP) ethers24 6a2, 6b2 and 6d2,
which were then condensed with acetaldehyde to yield the intermediates 8a2, 8b2 and 8d2.
Removal of the THP protecting group gave the corresponding 3-phenylacrylaldehydes 8a1,
8b1 and 8d1, respectively. Methylation of 8c1 yielded the analogue 8c3.
Insert Scheme 2 →
The trienones 4, 5 and the analogues were synthesized as shown in Scheme 3. Compound
4 was synthesized by the following two different approaches. Condensation of 7a1 with 8a1
under aldol condition gave the trienone 4 in 29% yield. Alternatively, condensation of 7a1
with the intermediate 3-phenylacrylaldehyde 8a2 gave 4a and subsequent THP deprotection
with 3M HCl yielded the trienone 4. The trienone 5 was similarly synthesized in 12% yield
from 7b1 and 8b1, or in 14% from 7b1 and 3-phenylacrylaldehyde 8b2 to yield the
intermediate 5a followed by deprotection of the THP group. All other trienone analogues, 9–
15 and 17–20, were similarly obtained from direct coupling of substituted hydroxy and/or
methoxy 4-phenylbut-3-en-2-ones (7a1–d1, 7e, 7f and 7c3) with substituted hydroxy or

6
methoxy 3-phenylacrylaldehydes (8a1, 8c1, 8d1 and 8c3) (Scheme 3). The trienone 16 was
obtained by methylation of compound 11. All synthesized analogues were purified by
crystallization or column chromatography and characterized by NMR (1H and 13C NMR, and
2D COSY, HMQC and HMBC) and mass spectroscopy (see Supplementary data).
Insert Scheme 3 →
The natural curcuminoids 1–3, the trienone analogues 4 and 5, and analogues 9–20 were
subjected to cytotoxic activity evaluation against the KB cells using resazurin microplate
assay for cancer cell growth inhibition25 and the results are presented in Table 1. Compounds
1–3 exhibited weak cytotoxicity against this cancer cell line, with the IC50 values of 21.36,
26.45 and 21.44 µM, respectively, which was much less active than ellipticine, the reference
anticancer drug, which exhibited cytotoxicity against KB cells at IC50 of 2.25 µM. The
trienone analogues 4 and 5, however, showed strong and moderate cytotoxic effects at IC50 of
5.75 and 12.23 µM, respectively. An increase in cytotoxicity of 1.7-fold from the
curcuminoid 1 to the trienone 5, and 3.7-fold from the curcuminoid 3 to the trienone 4 was
very significant; it implied that the 1,4,6-trien-3-one moiety contributed to higher cytotoxicity
against the KB cells than the 1,6-dien-3,5-dione moiety of the curcuminoids. This finding has
prompted us to further investigate cytotoxicity of other trienone analogues. The strategy of
was to modify the trienone structure by varying number and position of the oxygen functions
(hydroxyl/methoxy groups). As the trienone 4 was about 2-fold more potent than the trienone
5, it was used as the structure lead for analogues with higher cytotoxic activity. In order to
see whether substitution pattern of the oxygen function on the aromatic rings affected the
cytotoxicity of the trienone structure, compound 9, the B-ring meta-hydroxy analogue of the
trienone 4, was synthesized and it was found that this analogue was approximately 2-fold
more active than 4, with the IC50 of 2.98 µM. In order to see the effect of oxygenation
patterns on ring B compared to the para- and meta-hydroxy analogues 4 and 9, the ortho-
hydroxy analogue 10 was synthesized and it was found that its cytotoxicity (IC50 8.16 µM)
was lower than both the analogues 4 and 9. The oxygen function at the meta-position of the
B-ring is therefore essential for high cytotoxicity against KB cells. In order to see the effect
of oxygenation patterns on ring A, the high cytotoxic compound 9 was further modified by
moving the hydroxy group at the para-position on the A ring to the meta-position to give the
trienone 11 and this analogue was found to be highly toxic against the KB cells; the IC50 of

7
this compound was 1.72 µM, or 3.3-fold more active than the trienone 4. It is worth noting
that the trienone 11 was 1.3-fold more active than ellipticine. In order to see the effect of
replacement of the meta-hydroxy group on the A-ring of compound 11 by the ortho-hydroxy
group, compound 12 was synthesized and its cytotoxic activity was 1.4-fold less active than
11; its IC50 value was 2.35 µM, which was almost as active as ellipticine. As expected,
placement of the hydroxy groups at the ortho-positions on the A- and B-rings in analogue 13
resulted in decrease in activity compared with the meta- or para-dihydroxy analogue; it
exhibited moderate cytotoxicity with IC50 of 11.19 µM, that is 6.5- and 2-fold less active than
compounds 11 and 4, respectively. The results have indicated that the presence of hydroxy
group at the meta-position of the A-ring was also responsible for the increase in cytotoxicity
against KB cells. The contribution of free hydroxy group on cytotoxic activity was
demonstrated by the methyl ether 14 that showed 2.6-fold decrease in activity when
compared with compound 11. Sharp decrease in cytotoxicity was observed in the isomeric B-
ring methyl ether 15; it was 11-fold less active than compound 11. As expected, full
methylation of 11 resulted in the analogue 16 which showed weak cytotoxic activity.
In order to see the effect of an extra oxygen function on the aromatic ring, the A-ring 3-
hydroxy-4-methoxy analogue 17 was evaluated for cytotoxicity and this compound exhibited
1.7-fold less active than compound 11. The isomeric A-ring 4-hydroxy-3-methoxy analogue
18, however, exhibited unexpectedly high activity; it showed comparable cytotoxic potency
(IC50 1.70 µM) to that of compound 11. In order to compare the B-ring para-hydroxy
analogue of compound 18 and to make a complete set of the trienone analogue of
demethoxycurcumin (2), the trienone 19 was synthesized and cytotoxicity was evaluated. As
expected, it was more active (IC50 7.57 µM) than the curcuminoid 2 and was less active than
its isomeric B-ring meta-hydroxy analogue 18. Removal of the methyl group from either
compound 17 or 18 gave the corresponding trihydroxy analogue 20. Surprisingly, compound
20 exhibited the most active cytotoxicity among the trienones tested; its IC50 was 1.36 µM, or
1.7-fold more active than ellipticine.
The results have indicated that the analogues with the 1,4,6-trien-3-one function are more
potent than the curcuminoids with the 1,6-dien-3,5-dione function. Analogues with meta-
oxygen function on both aromatic rings are more potent than those in the ortho- and para-
positions. Free phenolic hydroxy group is more potent than the corresponding methyl ether
analogues.
To assess whether the cytotoxic activity described above should be attributed to general
toxicity, rather than specific cytotoxic activity of compounds to the cancer cells, these

8
analogues were also tested for their effect on the normal cells (Vero cells, the African green
monkey kidney cells) using green fluorescent protein (GFP) detection method26 and the
results are included in Table 1. The results have indicated that most of the trienones were
toxic against Vero cells and were more toxic than curcumin (1), which was regarded as
weakly toxic, except compounds 11, 12, 16 and 17, which were approximately 2- to 3-fold
less active than the curcuminoid 1. The selectivity indices (SIs, Table 1) of the toxic trienones
were between 0.55 and 6.17, whereas those of 1 and ellipticine were 1.64 and 2.88,
respectively. In contrast, the SIs of the non-toxic or less toxic trienones, 11, 12 and 17, were
35.46, 33.46 and 31.68, which were relatively very high. Among these three highly active
trienones with high SIs, the trienone 11 seems to be the most suitable compound for further
in-depth study.
In conclusion, we report the general method for the synthesis of the (1E,4E,6E)-1,7-
diarylhepta-1,4,6-trien-3-one based on the aldol condensations of substituted 4-phenylbut-3-
en-2-ones and substituted 3-phenylacrylaldehydes. The natural trienones 4 and 5 have been
synthesized by this method, together with the trienone analogues 9–20. These analogues were
evaluated for their cytotoxic effects against KB cell line. The results have indicated that the
analogues with the 1,4,6-trien-3-one function are more potent than the curcuminoids.
Analogues with meta-oxygen function on the aromatic rings are more potent than those in the
ortho- and para-positions. Free phenolic hydroxy group is more potent than the
corresponding methyl ether analogues. Among the potent trienones, compounds 11, 18 and
20 were more active than the anticancer drug ellipticine. All compounds were also evaluated
against the non-cancerous Vero cells and it was found that compounds 11, 12 and 17 were
much less toxic than curcumin (1); they showed high selectivity indices of 35.46, 33.46 and
31.68, respectively.
Acknowledgments
This work was supported by The Thailand Research Fund. Financial support from the
Center of Excellence for Innovation in Chemistry (PERCH-CIC), Office of the Higher
Education Commission, Ministry of Education is gratefully acknowledged.

9
Supplementary data
Supplementary data (1H, 13C NMR and DEPT spectra and mass spectroscopic data)
associated with this article, can be found, in the online version, at ….

10
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12
Captions
Figure 1. Structures of the natural curcuminoids, curcumin (1), demethoxycurcumin (2) and
bisdemethoxycurcumin (3), and the natural trienones 4 and 5.
Scheme 1. Retrosynthesis of (1E,4E,6E)-1,7-diphenylhepta-1,4,6-trien-3-one analogues.
Scheme 2. Synthesis of the substituted 4-phenylbut-3-en-2-ones 7a–f and the substituted 3-
phenylacrylaldehydes 8a–d from the aldehydes 6a–f. Reagents and conditions: (i) excess
acetone, 20% aq. NaOH, EtOH; (ii) 3,4-dihydro-2H-pyran, p-TsOH, THF; (iii) MeI, K2CO3,
acetone, rt; (iv) excess acetaldehyde, 20% aq. NaOH, EtOH, 0–10 oC; (v) p-TsOH, MeOH–
H2O.
Scheme 3. Synthesis of the trienones 4, 5, 9–15 and 17–20 from aldol condensation of the
substituted 4-phenylbut-3-en-2-ones 7a–f and the substituted 3-phenylacrylaldehydes 8a–d,
and methylation of the trienone 11 to 16. Reagents and conditions: (i) 20% aq. NaOH, EtOH,
0 oC–rt; (ii) 3M HCl; (iii) MeI, K2CO3, acetone, rt.

13
Table 1
The structure and cytotoxicity of trienones against KB and Vero cells and selectivity index
Compound/Structure
SIc
Cytotoxicity (IC50, µM)
KBa
Verob
35.05
O
O
O
O
O
O
O
O
O
O
O
MeO
HO
OMe
OH
21.36
26.45
21.44
5.75
1.64
1
2
3
4
5
9
MeO
HO
CDd
CDd
9.65
6.78
10.81
-
OH
OH
OH
-
HO
HO
1.68
0.55
3.63
MeO
HO
OMe
OH
12.23
2.98
OH
HO
OH
8.16
1.72
12.18
60.99
78.64
19.60
8.00
1.49
35.46
33.46
1.75
10
11
12
13
14
15
16
17
HO
HO
OH
OH
OH
OH
O
2.35
O
OH
11.19
4.47
O
MeO
HO
OH
1.79
O
O
O
OMe
OMe
OH
19.06
20.10
2.90
5.68
0.30
MeO
54.37
91.86
2.70
HO
31.68
MeO
O
O
O
MeO
HO
OH
1.70
7.57
10.49
14.36
6.17
1.90
18
19
MeO
HO
OH
OH
HO
HO
1.36
2.25
6.97
6.48
5.13
2.88
20
Ellipticine
a Cancer of the oral cavity.
b Green African monkey kidney cells.
c Selectivity index = IC50 for normal cell line/IC50 for cancerous cell line.
d Could not be determined due to fluorescence behavior of the compound.

14
O
O
O
1
1
7
7
5
R1
R2
R1
R2
3'
3"
4"
3
3
5
HO
OH
HO
OH
4'
4: R1 = R2 = H
1: R1 = R2 = OMe
5: R1 = R2 = OMe
2: R1 = OMe, R2 = H
3: R1 = R2 = H
Figure 1. Structures of the natural curcuminoids, curcumin (1), demethoxycurcumin (2) and
bisdemethoxycurcumin (3), and the natural trienones 4 and 5.

15
O
O
O
R
R'
R
R'
5
Aldol
H
B
A
4
A
B
condensation
Substituted
4-phenylbut-3-en-2-one
(Substituted cinnamone)
Substituted
3-phenylacrylaldehyde
(Substituted cinnamaldehyde)
Aldol
condensation
Aldol
condensation
O
R
O
R'
H
H
A
B
Substituted
benzaldehyde
Substituted
benzaldehyde
Scheme 1. Retrosynthesis of (1E,4E,6E)-1,7-diphenylhepta-1,4,6-trien-3-one analogues.

16
R1
O
7a1, 78%
7b1, 88%
7c1, 67%
7d1, 91%
7e, 98%
7f, 47%
R2
R3
(i)
(iii)
7c3: R1 = R3 = H, R2 = OMe, 85%
R1
R1
R2
R3
CHO
R2
R3
CHO
(v)
8a1, 63%
8b1, 62%
8d1, 51%
(iv)
6a1: R1 = R2 = H, R3 = OH
6b1: R1 = H, R2 = OMe, R3 = OH
6c1: R1 = R3 = H, R2 = OH
6d1: R1 = OH, R2 = R3 = H
6e: R1 = H, R2 = OH, R3 = OMe
6f: R1 = H, R2 = R3 = OH
R1
R2
R3
R1
CHO
8c1, 67%
(iii)
8c3: R1 = R3 = H, R2 = OMe, 97%
(ii)
R1
R2
R3
CHO
R2
R3
CHO
(iv)
8a2, 67%
8b2, 18%
8d2, 10%
6a2: R1 = R2 = H, R3 = OTHP, 68%
6b2: R1 = H, R2 = OMe, R3 = OTHP, 59%
6d2: R1 = OTHP, R2 = R3 = H, 37%
Scheme 2. Synthesis of the substituted 4-phenylbut-3-en-2-ones 7a–f and the substituted 3-
phenylacrylaldehydes 8a–d from the aldehydes 6a–f. Reagents and conditions: (i) excess
acetone, 20% aq. NaOH, EtOH; (ii) 3,4-dihydro-2H-pyran, p-TsOH, THF; (iii) MeI, K2CO3,
acetone, rt; (iv) excess acetaldehyde, 20% aq. NaOH, EtOH, 0–10 oC; (v) p-TsOH, MeOH–
H2O.

17
R1
O
R4
1
7
5
R2
R3
R5
R6
(i)
+
7a-f
8a-d
3
7a1 + 8a1 → 4: R1 = R2 = R4 = R5 = H, R3 = R6 = OH, 29%, 32% from 8a2
7a1 + 8a2 → 4a: R1 = R2 = R4 = R5 = H, R3 = OH, R6 = OTHP
(ii)
7b1 + 8b1 → 5: R1 = R4 = H, R2 = R5 = OMe, R3 = R6 = OH, 12%, 14% from 8b2
7b1 + 8b2 → 5a: R1 = R4 = H, R2 = R5 = OMe, R3 = OH, R6 = OTHP
7a1 + 8c1 → 9: R1 = R2 = R4 = R6 = H, R3 = R5 = OH, 33%
(ii)
7a1 + 8d2 → 10a: R1 = R2 = R5 = R6 = H, R3 = OH, R4 = OTHP
(ii)
10: R1 = R2 = R5 = R6 = H, R3 = R4 = OH, 14% from 8d2
7c1 + 8c1 → 11: R1 = R3 = R4 = R6 = H, R2 = R5 = OH, 58%
7d1 + 8c1 → 12: R1 = R5 = OH, R2 = R3 = R4 = R6 = H, 57%
7d1 + 8d1 → 13: R1 = R4 = OH, R2 = R3 = R5 = R6 = H, 85%
(iii)
7c3 + 8c1 → 14: R1 = R3 = R4 = R6 = H, R2 = OMe, R5 = OH, 14%
7c1 + 8c3 → 15: R1 = R3 = R4 = R6 = H, R2 = OH, R5 = OMe, 61%
16: R1 = R3 = R4 = R6 = H, R2 = R5 = OMe, 95%
7e + 8c1 → 17: R1 = R4 = R6 = H, R2 = R5 = OH, R3 = OMe, 33%
7b1 + 8c1 → 18: R1 = R4 = R6 = H, R2 = OMe, R3 = R5 = OH, 45%
7b1 + 8a1 → 19: R1 = R4 = R5 = H, R2 = OMe, R3 = R6 = OH, 20%
7f + 8c1 → 20: R1 = R4 = R6 = H, R2 = R3 = R5 = OH, 10%
Scheme 3. Synthesis of the trienones 4, 5, 9–15 and 17–20 from aldol condensation of the
substituted 4-phenylbut-3-en-2-ones 7a–f and the substituted 3-phenylacrylaldehydes 8a–d,
and methylation of the trienone 11 to 16. Reagents and conditions: (i) 20% aq. NaOH, EtOH,
0 oC–rt; (ii) 3M HCl; (iii) MeI, K2CO3, acetone, rt.

18
Graphical_abstract_REVISED
Synthesis, cytotoxicity against human oral cancer KB cells and
structure–activity relationship studies of trienone analogues of curcuminoids
Thipphawan Chuprajob, Chatchawan Changtam, Ratchanaporn Chokchaisiri,
Warangkana Chunglok, Nilubon Sornkaew, Apichart Suksamrarn*
R4
O
O
R5
R6
HO
OH
R1
11
R2
R3
CHO
7
R7
IC50 1.72 µM (1.3-fold more
active than ellipticine).
Selectivity index is 35.46.
R8
R9
CHO
6
8