Indole carboxylic acid esters of melampomagnolide B are potent anticancer agents against both hematological and solid tumor cells

Article abstract of DOI:10.1016/j.ejmech.2017.05.031

A series of novel, heteroaryl carboxylic acid conjugates of the sesquiterpene melampomagnolide-B (MMB, 3) has been evaluated as antitumor agents against an NCI panel of 64 human hematopoetic and solid tumor cell lines. The indole-3-acrylic acid conjugate 7j and the indole-3-carboxylic acid conjugate 7k were found to be the most potent analogs in the series. Compounds 7j and 7k exhibited remarkable growth inhibition, with GI₅₀ values in the range 0.03–0.30?μM and 0.04–0.28?μM, respectively, against the cell lines in the leukemia sub-panel, and GI₅₀ values of 0.05–0.40?μM and 0.04–0.61?μM, respectively, against 90% of the solid tumor cell lines in the NCI panel. Compound 7a was particularly effective against the sub-panel of breast cancer cell lines with GI₅₀ values in the range <0.01–0.30?μM. Compounds 7j, 7a and its water soluble analog 7p also exhibited potent anticancer activity against rat 9L-SF gliosarcoma cells in culture. Compound 7j was the most potent compound in the series in the M9-ENL1 AML cell assay with a lethal dose concentration EC₅₀ value of 720?nM, and exhibited the greatest cytotoxicity against a collection of primary AML stem cell specimens, which included a specimen that was unresponsive to PTL, affording EC₅₀ values in the range 0.33–1.0?μM in three out of four specimens. The results from this study provide further evidence that analogs of the sesquiterpene MMB can be designed to afford molecules with significantly improved anticancer activity. Thus, both 7j and 7k are considered potential lead molecules in the search for new anticancer agents that can be used as treatments for both hematopoetic and solid tumors.

Full text of DOI:10.1016/j.ejmech.2017.05.031

Accepted Manuscript
Indole carboxylic acid esters of melampomagnolide B are potent anticancer agents
against both hematological and solid tumor cells
Shobanbabu Bommagani, Jessica Ponder, Narsimha R. Penthala, Venumadhav
Janganati, Craig T. Jordan, Michael J. Borrelli, Peter A. Crooks
PII:
S0223-5234(17)30392-6
10.1016/j.ejmech.2017.05.031
EJMECH 9461
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 January 2017
Revised Date: 8 March 2017
Accepted Date: 10 May 2017
Please cite this article as: S. Bommagani, J. Ponder, N.R. Penthala, V. Janganati, C.T. Jordan, M.J.
Borrelli, P.A. Crooks, Indole carboxylic acid esters of melampomagnolide B are potent anticancer agents
against both hematological and solid tumor cells, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.05.031.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Leave this area blank for abstract info.
Indole carboxylic acid esters of melampomagnolide B
are potent anticancer agents against both
hematological and solid tumor cells
Shobanbabu Bommagani,a Jessica Ponder,b Narsimha R. Penthala,a Venumadhav Janganati,a Craig T. Jordan,b
Michael J. Borrelli,c and Peter A. Crooksa*
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
bDivision of Hematology, University of Colorado, Aurora, CO 80045, USA
cDepartment of Radiology and Neurology University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
Original article
Indole carboxylic acid esters of melampomagnolide B are potent anticancer
agents against both hematological and solid tumor cells
Shobanbabu Bommagani,a Jessica Ponder,b Narsimha R. Penthala,a Venumadhav Janganati,a Craig T.
Jordan,b Michael J. Borrelli,c and Peter A. Crooksa*
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
b Division of Hematology, University of Colorado, Aurora, CO 80045, USA
cDepartment of Radiology and Neurology University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A
series of novel, heteroaryl carboxylic acid conjugates of the sesquiterpene
melampomagnolide-B (MMB, 3) has been evaluated as antitumor agents against an NCI panel
of 64 human hematopoetic and solid tumor cell lines. The indole-3-acrylic acid conjugate 7j and
the indole-3-carboxylic acid conjugate 7k were found to be the most potent analogs in the series.
Compounds 7j and 7k exhibited remarkable growth inhibition, GI50 values in the range 0.03-
0.30 ꢀM and 0.04-0.28 µM, respectively, against the cell lines in the leukemia sub-panel, and
exhibited GI50 values of 0.05-0.40 µM and 0.04-0.61 µM, respectively, against 90% of the solid
tumor cell lines in the NCI panel. Compound 7a was particularly effective against the sub-panel
of breast cancer cell lines with GI50 values in the range <0.01 to 0.30 µM. Compounds 7j, 7a
and its water soluble analog 7p also exhibited potent anticancer activity against rat 9L-SF
gliosarcoma cells in culture. Compound 7j was the most potent compound in the series in the
M9-ENL1 AML cell assay with an lethal dose concentration, EC50 value of 720 nM, and
exhibited the greatest cytotoxicity against a collection of primary AML stem cell specimens,
which included a specimen that was unresponsive to PTL, affording EC50 values in the range
0.33 to 1.0 µM in three out of four specimens. The results from this study provide further
evidence that analogs of the sesquiterpene MMB can be designed to afford molecules with
significantly improved anticancer activity. Thus, both 7j and 7k are considered potential lead
molecules in the search for new anticancer agents that can be used as treatments for both
hematopoetic and solid tumors.
Available online
Keywords:
Melampomagnolide B analogues
Indole carboxylic esters
Anti-cancer activity
Leukemia cell lines
9L-SF gliosarcoma cells
2009 Elsevier Ltd. All rights reserved.
1. Introduction
PTL is a well-established antileukemic agent, but has poor
drug-like properties and lacks clinical potential due to its poor
The sesquiterpene lactones are a noteworthy class of
biologically active constituents of natural products that have been
isolated from various medicinal plants. In recent years there has
been an increasing interest in these 15-carbon terpenoids and
studies on the synthesis of analogs of these natural products have
been reported and their biological activities have been
evaluated.[1-3] Parthenolide (PTL, 1, Figure 1), originally
isolated from the medicinal herb Feverfew (Tanacetum
parthenium), is one of the best-known members of the
sesquiterpene lactones, having been identified as an anticancer
agent that is effective against both hematological and solid
tumors.[4-10]
oral bioavailability. Analogs have been recently designed in
attempts to improve the drug-like properties of PTL while
retaining its antitumor properties. One such analog,
dimethlyamino parthenolide (DMAPT, 2, Figure 1), is currently
in Phase I clinical studies for evaluation as a treatment for AML
and other related leukemias.[11]
A structurally related sesquiterpene, melampomagnolide B
(MMB, 3, Figure 1), a melampolide originally isolated from
Magnolia grandiflora,[12] has similar antileukemic properties as
PTL,[13] but is somewhat less potent. Both sesquiterpenes
exhibit significantly reduced cytotoxicity against normal bone
marrow stem cells when compared to their effect on AML stem
cells.[13-15] The presence of the C-14 primary allylic hydroxyl
group in the MMB molecule (3, Figure 1) allows the medicinal
chemist the opportunity to examine the biological activity of a
wide variety of new MMB derivatives via conjugation and
___________
* Corresponding author.
E-mail address: pacrooks@uams.edu (P.A. Crooks).

Interestingly, the above pro-apoptotic actions of MMB are not
functional group transformations. Utilizing thAis CapCproEaPchT, EC-D14 MANUSCRIPT
carbamate and carbonate conjugates of MMB have recently been
shown to possess improved cytotoxicity against hematological
and solid tumor cell lines when compared to the parent
compound,[16-18] and the dimeric MMB carbamate esters (4 and
5, Figure 1) have been shown to possess promising anticancer
activities against a wide variety of both hematological and solid
human tumor cell lines.[17] The symmetrical MMB carbonate
dimer (5) also exhibits superior anticancer activity against 9LSF
gliosarcoma and M9-EML1 AML cells when compared to PTL
and DMAPT.[17]
observed in normal bone marrow stem cells.[13, 14] Thus, MMB
exhibits two selective and synergistic anticancer activities, i.e.
NF-κB inhibition and induction of oxidative stress through
interaction with the glutathione pathway.
In the current study, we have prepared and evaluated several
novel aryl carboxylic acid conjugates of MMB (7a-7o), and
compared their anticancer properties to those of PTL. The results
indicate that two indole carboxylic conjugates of MMB (7j and
7k) have been identified as strong leads in the development of
potent sesquiterpene-derived anticancer agents. These
compounds represent the most promising anticancer agents ever
reported as structural analogs of MMB and PTL.
2. Results and discussion
2.1. Rational drug design and synthesis
Based on our previous work, [16-18] a series of novel
heteroaromatic ester conjugates of MMB (7a-7o and 9) were
prepared using standard EDC coupling conditions (Steglich type
esterification) by reacting MMB (3) with an appropriate aryl
carboxylic acid (6a-6o) in the presence of EDCI and DMAP in
dichloromethane at room temperature for 8 h. The heteroaryl
carboxylic acids 6a-6o were chosen from a variety of available
indole (6a-6l), benzothiophene (6m-6n), and benzofuran
carboxylic acids, (6o) to afford the corresponding MMB
conjugates (7a-7o) in yields ranging from 54% to 74% (Scheme1
and Table 1). Indole carboxylic acids 6e and 6f were prepared
from the commercially available methyl esters of these
compounds by alkaline hydrolysis to the corresponding acids.
Conjugate 7p, a water-soluble analog of 7a, was synthesized
by initial conversion of MMB (3) to the C-13 dimethylamino
Michael adduct (8), followed by esterification of 8 with indole-2-
carboxylic acid (5a) utilizing standard EDC coupling conditions
to afford the MMB dimethylamino indole-2-carboxylate ester, 9,
in 80% yield. Compound 9 was then treated with fumaric acid to
afford compound 7p as the monofumarate salt (Scheme 2).
Fig 1. Anticancer sesquiterpene lactone PTL (1) and its
derivatives (2-5).
In mechanistic studies, we have shown that MMB inhibits the
phosphorylation of p65[13] in AML stem cells. In addition, a C-
14 biotin-conjugated derivative of MMB has been prepared and
used in elucidating the mechanism of action of MMB in AML
stem cells[13, 14, 15]. Utilizing this probe, and carrying out
subsequent streptavidine pull-down and LC/MS/MS peptide
sequencing studies, the MMB-biotin probe pulls down IKKβ in
both streptavidine/antibody and LC/MS/MS proteomic assays.
Thus, the mechanism of action of MMB involves promotion of
apoptosis by inhibition of the NFκB transcription factor complex,
thereby down-regulating anti-apoptotic genes under NFκB
control. MMB and PTL are believed to bind to both IKKβ and
RelA/p65, leading to decreased phosphorylation of IκBα and
reduced DNA binding of p65.[10, 14, 19-22]
Anticancer agents such as PTL and MMB also cause a
reduction in cellular glutathione levels, and a selective increase in
reactive oxygen species (ROS) in AML stem cells through
inhibition of key enzymes involved in glutathione biosynthesis
and catabolism.[14] In this respect, MMB-biotin pulls down
important elements of the glutathione pathway, i.e. glutathione
peroxidase (GPX1), glutathione ligase (both modulatory and
catalytic units GCLC and GCLM, respectively), and thioredoxin.
Scheme 1. Synthesis of heteroaryl carboxylic acid esters of MMB (7a-7o)
Table 1. Structures of carboxylic acid ester conjugates of melampomagnolide B (7a-7o), and product yields
S. No
Heteroaromatic acid
Product
Isolated yield (%)
1
62
6a
7a

ACCEPTED MANUSCRIPT
6b
2
67
7b
6c
60
58
63
60
3
7c
4
6d
7d
5
6e
7e
6
6f
7f
7
8
65
70
6g
7g
7h
6h
9
62
6i
7i

ACCEPTED MANUSCRIPT
10
11
12
54
61
64
6j
7j
6
k
7k
6
l
7l
13
74
6m
7m
14
70
6n
7n
15
72
6o
7o
2.2. Biological Evaluation
2.2.1. In vitro growth inhibition exhibited by heteroaryl
carboxylic acid esters of MMB against a panel of human cancer
cell lines.
MMB ester conjugates 7a-7o and dimethylamine MMB ester
conjugate (7p) were submitted to the National Cancer Institute
(NCI) for anticancer screening. The compounds were first
screened at a single concentration of 10-5 M against a panel of 60
human cancer cell lines derived from nine human cancer cell
types grouped into sub-panels that contain leukemia, lung, colon,
central nervous system (CNS), melanoma, renal, ovary, breast
and prostate cancer cell lines. Compounds that showed more than
60% growth inhibition in at least eight of the 60 cell lines in the
panel were selected for a complete dose response study utilizing
the procedure described by Rubinstein et al.,[23] at five different
concentrations over the range 10−4 to 10−8 M. From the initial
Scheme 2. Synthesis of compound 7p, a water-soluble analog of the indole-2-
carboxylic acid ester of MMB (7a). Reagents: a) dimethylamine, methanol,
RT, 6 h; b) 6a, EDCI, DMAP, DCM, RT, 8 h; c) Fumaric acid, ethanol.

GI50 value of 0.38 µM against PC-3 and DU-145 cell lines in the
single dose screening data, ten analogs, 7a-7d,A7hC, 7Cj-7EmP,T7oEaDnd MANUSCRIPT
7p, were selected for five dose testing for evaluation of cancer
cell growth inhibitory properties (GI50). Dose-response curves
were generated by plotting cytotoxic effect against the log10 of
the drug concentration for each cell line. Cytotoxic effects of
each compound were determined as GI50 values, which represent
the molar drug concentration required to cause 50% growth
inhibition. The results are given in Table 2.
prostate cancer sub-panel. Of note, compounds 7j and 7k both
exhibited low nanomolar growth inhibition against CCRF-CFM
and RPMI-8226 leukemia cell lines with GI50 values of 70 nM
and 30 nM, and 50 nM and 40 nM, respectively. Compounds 7j
and 7k also potently inhibited the growth of the melanoma cell
line, LOX IMVI, with GI50 values of 50 nm and 40 nM,
respectively, and were particularly effective against the drug
All ten conjugates tested in the NCI 60 human cancer cell
screen 5-dose assays showed significantly improved anticancer
activity when compared to PTL (Table 2). Compounds 7j and 7k,
exhibited the most potent growth inhibition in the series against
both hematological and solid tumor cell lines in the human tumor
cell panel, affording GI50 values in the range 0.03-0.30 ꢀM and
0.04-0.28 µM, respectively, against the cell lines in the leukemia
sub-panel, and exhibiting GI50 values of 0.05-0.40 µM and 0.04-
0.61 µM, respectively, against 90% of the solid tumor cell lines
in the panel. Remarkable GI50 potency ranges were exhibited by
7j in the renal cancer (0.15-0.27 µM), breast cancer (0.17-0.35
µM), prostate cancer (0.32-0.39 µM), ovarian cancer (0.17-0.35
µM), melanoma (0.05-0.35 µM), and colon cancer (0.18-0.40
µM) sub-panels. Similarly, compound 7k afforded GI50 potency
ranges of 0.16-0.32 µM, 0.17-0.61 µM, 0.20-2.24 µM, 0.04-0.36
µM, and 0.19-0.47 µM in the renal cancer, breast cancer, ovarian
cancer, melanoma, and colon cancer sub-panels, and exhibited a
resistant breast cancer cell lines MDA-MB-231/ATCC (GI50
170 nM and 190 nM, respectively) and MDA-MB-468 (190 nM
and 190 nM, respectively). Although compound 7a was
=
generally less potent than either 7j or 7k in the five-dose study, it
exhibited remarkable potency against two of the cell lines in the
breast cancer cell sub-panel, affording GI50 values of <10 nM for
the BT-549 and T-47D cell lines. Compound 7a was also an
effective anticancer agent against the drug resistant breast cancer
cell line MDA-MB-468 (GI50 = 300 nM). However, the water
soluble dimethylamino analog of 7a, compound 7p, which was
evaluated as its fumarate salt, was somewhat less potent than 7a
in the human cancer cell panel assays (Table 2), and did not
exhibit the low nanomolar potency of 7a against breast cancer
cell lines BT-549, T-47D and MDA-MB-468. Nevertheless,
compound 7p did show good potency against the cell lines in the
leukemia sub-panel (EC50 range 0.24-1.59 µM) and in the colon
cancer sub-panel (EC50 range 0.29-2.24 µM).
Table 2. Growth inhibition (GI50) dataa for MMB heteroaryl ester conjugates 7a-7d, 7h, 7j-7m and 7o, and dimethylamine MMB ester conjugate 7p against a
panel of 60 human cancer cell lines
1
7a
(µM)b
7b
(µM)b
7c
(µM)b
7d
(µM)b
7h
(µM)b
7j
(µM)b
7k
(µM)b
7l
(µM)b
7m
(µM)b
7o
(µM)b
7p
(µM)b
Panel/cell line
(µM)b
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
9.16
5.71
22.6
14.3
8.20
NA
0.28
0.54
0.32
1.51
0.26
1.08
0.34
0.32
0.56
1.32
0.50
NA
0.91
2.07
2.24
2.26
2.00
2.03
0.39
0.44
1.14
0.55
0.81
0.77
0.75
1.63
2.60
1.53
2.31
1.73
0.07
0.25
0.27
0.30
0.03
0.27
0.05
0.27
0.27
0.28
0.04
0.28
0.21
1.54
0.40
0.57
0.21
0.34
0.40
1.89
0.81
1.88
0.56
0.52
0.33
1.22
1.15
1.77
0.71
1.15
0.32
0.24
0.63
1.51
0.28
1.59
Lung Cancer
A549/ATCC
EKVX
HOP-62
5.47
2.02
6.54
1.68
2.04
1.72
13.8
4.54
1.57
91.8
17.3
49.0
13.5
20.5
20.2
21.7
18.8
5.47
2.06
NA
NA
0.48
1.47
1.69
1.68
1.73
0.75
1.66
1.50
1.64
3.08
1.58
2.92
1.38
1.76
1.90
2.69
3.04
1.65
1.69
1.40
2.22
1.25
1.80
1.63
4.00
1.72
1.59
1.27
0.23
0.82
0.15
0.23
1.25
1.73
1.01
0.18
1.50
0.31
0.82
0.16
0.44
1.63
1.85
1.10
0.19
1.82
0.96
1.81
2.04
1.69
1.81
1.68
2.00
1.74
1.98
2.21
0.98
2.91
2.02
2.48
1.44
1.74
1.98
5.52
2.00
0.68
2.44
3.36
2.20
1.40
1.98
1.96
10.8
5.85
1.22
HOP-92
0.44
1.57
1.49
1.88
1.83
0.70
0.36
1.48
1.93
3.01
1.73
0.39
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
14.5
24.1
10.8
10.2
23.5
16.2
14.5
NA
2.14
0.19
0.21
0.27
2.05
0.25
1.18
1.54
0.42
0.32
0.35
1.54
0.64
1.64
2.02
1.50
1.15
1.13
1.98
1.43
1.72
1.40
0.70
1.00
1.33
2.15
1.40
1.86
1.79
0.96
1.61
1.71
1.89
1.84
0.22
0.27
0.18
0.18
0.19
0.40
0.18
0.24
0.32
0.19
0.25
0.24
0.47
0.23
1.24
1.81
1.02
0.53
0.52
1.83
0.30
1.71
1.75
0.59
0.40
0.49
1.78
0.51
1.55
2.47
0.32
1.07
0.82
2.02
0.66
1.98
1.72
0.29
0.29
0.43
2.24
1.70
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS Cancer
SF-268
25.1
28.9
18.9
70.9
48.4
41.5
1.59
1.71
1.32
1.60
1.01
1.89
1.66
2.42
1.02
1.57
NA
1.62
7.10
1.55
5.04
11.1
1.82
1.44
3.18
1.44
2.91
0.57
1.75
1.69
5.69
1.63
2.59
12.6
1.95
0.22
1.62
0.26
0.64
0.17
0.21
0.29
1.94
0.26
1.30
0.20
0.61
1.30
2.41
0.74
1.59
1.09
1.42
1.54
3.33
1.60
2.09
3.52
1.52
1.56
5.90
1.45
3.16
1.34
1.63
1.48
3.95
2.51
11.9
3.21
2.61
SF-295
SF-539
SNB-19
SNB-75
U251
1.60
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
8.24
13.8
NA
NA
16.2
0.18
1.56
0.81
0.76
1.92
0.30
1.61
1.27
0.62
1.60
1.05
1.67
1.89
1.50
1.95
0.81
1.63
1.47
1.24
1.73
1.55
1.79
1.66
1.73
1.96
0.05
0.28
0.22
0.22
0.20
0.04
0.32
0.22
0.24
0.23
0.22
1.62
1.41
1.35
1.38
0.20
1.82
1.75
1.33
1.75
1.42
1.66
1.43
1.43
1.77
0.17
2.01
1.57
1.28
2.10

SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
21.0
17.4
14.4
16.8
1.29
1.26
1.61
1.47
1.23
1.79
1.58
1.70
0.33
0.36
0.20
0.36
0.18
1.55
1.42
1.32
1.26
1.76
1.75
1.77
1.85
1.75
1.52
1.52
1.68
2.21
1.85
1.47
1.65
ACCEPTED MANUSCRIPT
1.74
1.61
1.38
1.85
2.28
1.89
1.91
1.70
1.67
1.74
2.03
1.84
0.20
0.35
0.17
Ovarian Cancer
IGROV1
21.1
17.4
21.5
34.1
17.1
15.0
54.7
NA
NA
NA
1.14
1.99
2.20
1.65
1.48
0.93
1.63
1.77
1.58
1.68
4.90
1.44
1.20
1.67
1.77
2.17
1.98
11.6
1.38
1.25
1.33
1.53
1.34
1.45
10.2
1.63
1.53
1.66
1.89
3.38
2.90
17.3
0.22
0.17
0.20
0.31
0.19
0.35
1.88
0.25
0.20
0.25
0.40
0.28
0.39
2.24
0.69
0.38
1.14
1.56
0.80
2.15
4.33
1.76
0.44
2.08
2.02
1.80
2.14
13.9
1.39
0.48
1.56
1.88
2.43
2.59
16.7
2.07
0.35
1.36
2.38
1.68
1.42
2.19
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786-0
A498
NA
7.04
NA
9.35
12.2
22.8
NA
0.37
1.74
0.34
1.08
1.07
1.74
1.39
0.88
1.27
1.63
0.81
1.71
1.14
1.77
1.28
1.12
1.78
3.35
1.46
1.20
1.47
1.85
1.83
1.20
1.51
2.65
1.35
1.19
1.38
1.70
1.75
1.21
1.60
11.9
NA
0.22
0.27
0.20
NA
0.17
0.19
0.27
0.15
0.32
0.26
0.21
NA
0.19
0.23
0.28
0.16
1.46
1.59
0.82
NA
0.58
0.71
1.10
0.77
1.52
10.1
1.50
1.46
1.23
2.06
1.55
1.40
1.81
12.9
1.44
NA
1.43
1.78
3.01
1.46
1.71
13.1
0.67
1.11
1.14
2.21
2.12
0.52
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
NA
1.46
1.90
1.64
1.41
41.9
Prostate Cancer
PC-3
DU-145
NA
NA
1.42
1.21
1.83
1.03
1.70
1.67
2.42
NA
1.92
1.67
0.32
0.39
0.38
0.38
1.47
1.01
1.83
1.05
1.76
1.18
2.37
1.49
Breast Cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
16.4
NA
NA
NA
NA
NA
0.30
1.14
NA
<0.01
<0.01
0.30
0.34
1.58
3.09
1.06
1.30
1.13
1.02
1.59
2.95
NA
1.56
1.86
0.86
1.32
2.25
1.31
1.34
1.89
1.42
0.22
0.17
0.35
0.17
0.20
0.19
0.26
0.19
0.61
0.17
0.40
0.19
0.40
1.05
2.39
0.51
1.19
0.44
0.38
1.97
2.89
NA
1.47
1.63
0.39
1.64
2.91
1.48
1.35
1.50
0.61
1.74
3.03
1.57
2.11
1.12
3.19
1.37
2.17
1.76
MDA-MB-468
NA: Not analyzed; aGI50 values <1 µM are bolded; bGI50: 50% growth inhibition defined as concentration of drug resulting in a 50% reduction in net protein
increase compared with control cells.
2.3. Antileukemic activity of heteroaryl carboxylic acid esters of
MMB (7a-7o) and dimethylamine MMB ester conjugate 7p
against M9-ENL1 AML and primary AML stem cell specimens.
To investigate further the antileukemic activity of the different
heteroaryl MMB ester conjugates (7a-7p), these compounds were
tested for antileukemic activity against M9-ENL1 acute
myelogenous leukemia cells (Table 3, Figure 2). This cell line
was obtained from transduction of human blood cells with the
leukemia-derived MLL-ENL translocation gene. Selected MMB
conjugates 7a, 7b, 7j were also screened against primary AML
stem cell specimens, which were obtained with informed consent
from human patients (Table 4; Figure 3). Dose-response curves
were generated over the concentration range 0.5-10 ꢀM to
determine the concentration resulting in 50% efficacy (EC50).
Evaluations were performed after 24 h of drug exposure using
flow cytometric analysis by labeling with 240 Annexin V and 7-
compound 7p, the water soluble analog of 7a, afforded an EC50
value of 2.8 µM against the M9-ENL1 AML cell line.
aminoactinomycin
D (7-AAD) to identify apoptotic cell
populations. Evaluations were performed using viability labeling
with trypan blue dye, as well as flow cytometric labeling with
Annexin V and propidium iodide to determine dead cell
populations. For all studies, PTL and/or MMB were included as
a reference control.
The data from the M9-ENL1 AML cell assay indicated that the
heteroaryl carboxylic acid esters of MMB (7a-7o) were 2- to 20-
fold more cytotoxic than MMB, and were generally more
cytotoxic than PTL in this assay. The most potent compound in
the series was the indole-3-acrylic acid ester conjugate of MMB,
7j (EC50 = 0.72 µM), which was approximately 9-fold more
potent compared to PTL (EC50 = 5.5µM) and greater than 20-fold
more potent when compared to the parent sesquiterpene, MMB
(EC50 = 15 µM), in this assay. Other compounds of interest were
7a, 7b, and 7k, which exhibited EC50 values of 1.1 µM, 1.8 µM
and 1.9 µM, respectively. It is noteworthy to mention that
Fig 2. EC50 (95% CI) values for PTL (1), MMB (3), and MMB heteroaryl
ester conjugates 7a-7o and dimethylamine MMB ester conjugate (7p) against
M9-ENL1 AML cells

Table 4. EC50 (95% CI, µM) valuesa for PTL (1), and MMB heteroaryl ester
Based on the above antileukemic propertiesAofCcoCnEjugPaTtesE7Da- MANUSCRIPT
conjugates 7a, 7b, and 7j against primary AML cell specimens
7o in the M9-ENL1 AML assay, compounds 7a, 7b, and 7j were
selected for evaluation of their cytotoxicity against four primary
AML cell specimens (Figure 3). Evaluations were performed
after 24 h of drug exposure using flow cytometric analysis by
labeling with 240 Annexin V and 7-aminoactinomycin D (7-
AAD) to identify apoptotic cell populations. PTL was included as
a reference control in these assays. Dose-response curves were
generated over the concentration range 0.5-10 ꢀM to determine
the concentration resulting in 50% efficacy (EC50). In three of
these AML specimens all four MMB conjugates exhibited
improved cytotoxicity when compared to PTL. The most potent
compound in the group was 7j, which was more effective than
PTL in all four primary AML specimens, affording EC50 values
over the range 0.30-1.3 µM. Compound 7j was particularly
effective (EC50 = 1.0 µM) against primary specimen AML04,
which was obtained from an AML patient after disease relapse,
and was more resistant to the action of PTL (EC50 = 13 µM).
Com.
AML01
AML02
AML03
AML04
EC50
13 [11-15]
2.2 [2.0-2.5]
3.3 [3.0-3.8]
1.0 [0.83-1.4]
EC50
EC50
EC50
1
5.2 [4.6-6.0]
3.5 [3.1-4.0]
3.0 [2.7-3.2]
1.3 [1.2-1.5]
6.3 [5.6-7.0]
2.7 [2.6-2.8]
3.3 [3.0-3.6]
0.91 [0.82-1.1]
1.9 [1.6-2.2]
2.9 [2.1-3.8]
2.2 [1.7-3.0]
0.32 [0.24-0.53]
7a
7b
7j
aEC50 values ≤1µM are bolded.
2.4. Anticancer activity of heteroaryl carboxylate esters of MMB
(7a-7o) and dimethylamine MMB ester conjugate 7p against rat
9L-SF gliosarcoma cells
The intriguing results with the MMB conjugates 7a-7o and
dimethylamine MMB ester conjugate 7p against hematological
and solid tumor cell lines directed our attention to their potential
cytotoxicity against rat 9L-SF gliosarcoma cells in culture. For
this purpose we initially examined the activity of conjugates 7a-
7d, 7j, 7m and 7p against 9L-SF cells. Initial screening results
demonstrated that conjugates 7a, 7b, 7j and 7p were effective
cytotoxic agents against this solid tumor cell type. Prior
experience with the natural sesquiterpene PTL and its synthetic
analog DMAPT demonstrated that these compounds had potent
toxicity against cell lines derived from solid tumors when such
cells were treated at low cell densities (less than 2.7 x 104
cells/cm2)[17] but were far less toxic when these drugs were used
to treat confluent cell monolayers.[17] Consequently, the above
MMB conjugates were screened visually (microscopic
observation) for their ability to inhibit proliferation or cause cell
death in 50% confluent monolayers of rat 9L-SF gliosarcoma
cells. The MMB-Indole composite drugs that were most effective
at inducing visually detectable cell detachment and necrosis at
concentrations less than 20 µM were 7a and 7j. The MMB
conjugates 7a, 7b, and 7j were tested for their ability to render
9L-SF cells clonogenically nonviable using the colony formation
assay (Figure 4). In addition, the clonogenic toxicity of the two
components of 7a, i.e. MMB (3) and indole-2-carboxylic acid
(6a) were also tested, along with the sesquiterpene analog,
DMAPT (2); the combination of MMB plus indole-2-carboxylic
acid were tested to determine if the toxicity of 7a could be
replicated by treating the cells with MMB (3) or indole-2-
carboxylic acid (6a) alone, or in combination.
Table 3. EC50 (µM)a values for PTL (1), MMB (3), MMB heteroaryl ester
conjugates 7a-7o, and dimethylamine MMB ester conjugate 7p against M9-
ENL1 AML cells
Entry
MMB Conjugate
M9-ENL1 AML Viability
EC50 (µM)
5.5
95% Cl
[5.1-5.9]
[0.90-1.4]
[0.90-3.3]
[2.5-3.2]
[3.5-4.0]
[2.8-3.2]
[2.2-2.7]
[3.9-4.1]
[1.8-2.1]
[2.5-3.2]
[0.68-0.77]
[1.9-2.8]
[6.3-8.3]
[2.3-3.1]
[2.0-2.3]
[2.7-3.2]
[2.6-3.1]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
1.1
1.8
2.8
3.8
3.0
2.5
4.0
2.0
2.7
0.7
1.9
7.2
2.7
2.1
2.9
2.8
aEC50 values below 2µM are bolded.
Fig 3. Dose-response curves for PTL (1) and MMB heteroaryl ester
conjugates (7a, 7b and 7j) with primary AML cell specimens. MMB esters
7a, 7b, and 7j exhibited potent antileukemic activity with EC50 values in the
range 0.3-3.5 µM against four specimens of primary AML cells.
Fig 4. Dose-response Curves for Compounds 7a, 7b, 3+6a, 7p, 7j and
DMAPT against rat gliosarcoma cells (9L-SF): cell survival as a function of
drug concentration for cells treated for 48 h with the indicated drug. Each
datum point represents the mean plus standard error of the mean for three
experiments.

3.0. SAR and mechanism of action studies
EC50 value of 720 nM, and exhibited the greatest cytotoxicity
ACCEPTED MANUSCRIPT
against a small collection of primary AML stem cell specimens,
which included a specimen from a patient that was that was
unresponsive to PTL. Compounds 7j and 7a were found to be
60,000-fold and 10,000-fold, respectively, more toxic than
DMAPT (2) against rat 9L-SF gliosarcoma cells in culture, and
7a exhibited remarkable potency against breast cancer cell lines
BT-549 and T-47D with GI50 values of <10 nM for each cell line.
It is also important to note that 7p, the water-soluble analog of
7a, was shown to be equipotent with 7a in the 9L-SF colony
formation assay, indicating that a water- soluble analog of 7a can
be designed without loss of anticancer activity in this solid tumor
cell line.
The results from this study provide further evidence that
analogs of the melampolide MMB can be designed to afford
molecules with significantly improved anticancer activity. Thus,
both 7j and 7k are considered potential lead molecules in the
search for new anticancer agents that can be used as treatments
for both hematopoetic and solid tumors.
Useful data to base structure-activity information on can be
gleaned from the NCI 60 human cancer cell panel results. From
these data, it can clearly be seen that of the bicyclic aromatic
carboxylic acid conjugates of MMB (7a-7p) tested, the indole-2-
carboxylic acid (7a), indole-3-carboxylic acid (7k) and indole-3-
acrylic acid (7j) esters were potent anticancer agents, with 7j
being the most active compound in the series. Conjugation of
MMB (3) with indole-5- or indole-6-carboxylic acids (6e and 6f,
respectively) led to failure of the resulting esters 7e and 7f to
qualify for 5-dose testing. Incorporating methoxy, fluoro or
chloro substituents at the 5-position of the indole-2-carboxylate
moiety (i.e. 7b, 7c, and 7d, respectively) led to MMB conjugates
with similar properties to the unsubstituted indole-2-carboxylic
acid conjugate 7a. N-Methylation of the indole-3-carboxylic ester
of MMB afforded 7l, which had lower overall cytotoxicity than
7k against the human cancer cells in the NCI screening panel.
The MMB conjugate of indole-3-acetic acid (7g) and the
structurally related indomethacin-MMB conjugate 7i both failed
to qualify for 5-dose testing, whereas the indole-3-butyric acid
conjugate 7h was considerably less potent than 7k, indicating
that insertion of methylene moieties between the carboxylate
moiety and the indole 3-position is detrimental to anticancer
potency. The use of benzothiophene-2-carboxylic acid instead of
indole-2-carboxyic acid afforded MMB conjugate 7m, which
qualified for 5-dose testing but exhibited quite low potency
against most of the cells in the NCI panel. The related 3-
chlorobenzothiophene-2-carboxylic acid ester of MMB (7n) did
not qualify for 5-dose testing. Interestingly, the benzofuran-2-
carboxylic acid ester of MMB (7o) was only slightly less
cytotoxic than the indole-2-carboxylic acid ester 7a in the NCI
cancer screen. The most potent MMB conjugate in the series was
7j, in which the indole-3-carboxyl moiety in 7k has been
replaced with an indole-3-acrylic acid moiety. This structural
change introduces a second Michael acceptor moiety into the
structure of 7j. MMB conjugates 7j and 7k were the most active
compounds in the NCI screen. The design of the dimethylamino
Michael adduct 7p as a water-soluble analog of 7a is based on a
similar approach that was utilized previously[24] to afford
DMAPT, a water soluble analog of PTL. DMAPT is currently in
Phase 1 clinical trials for treatment of AML.[11] As is the case
with DMAPT, 7p retains the cytotoxic properties of the parent
compound 7a, but has somewhat less potency.
5. Experimental Section
5.1. Chemistry
Melting points were recorded on a Kofler hot-stage apparatus
and are uncorrected. 1H and 13C NMR spectra were recorded on a
Varian 400 MHz spectrometer equipped with
a Linux
workstation operating on vNMRj software. All the spectra were
phased, baseline was corrected where necessary, and solvent
1
signals (CDCl3) were used as reference for both H and 13C
spectra. The chemical shifts given as δ values in parts per million
(ppm) and coupling constants (J) are given in hertz (Hz). HRMS
data were obtained on an Agilent 6210 LCTOF instrument
operated in multimode. Thin-layer chromatographic (TLC)
separations were carried out on pre-coated silica gel plates (F 254
Merck). The purities of all the final compounds were determined
to be ≥95% as determined by high pressure liquid
chromatography (HPLC) on a Model 1290 Agilent HPLC-Diode
Array unit utilizing an Altima-C18 column (4.6 × 250 349mm, 5
ꢀm), a mobile phase of acetonitrile containing 30% Milli Q water
at a flow rate of 0.8 mL/min. Detection was at 210 nm. If
required, synthetic products were purified by flash column
chromatography (silica gel; methanol/dichloromethane).
5.2. Methodology for the in vitro 60 human cancer cell screen:
The methodology for the anticancer activity screening assay
was carried out at the National Cancer Institute utilizing a
reported literature procedure.[23] Briefly, RPMI 1 medium with
5% fetal bovine serum and 2 mM L-glutamine was used for
growing the NCI-60 human tumor cells. Initially, the tumor cells
were inoculated into 96-well microtiter plates in 100 ꢀL of
medium at plating densities starting from 5,000 to 40,000 cells
per well. The range of cell numbers is dependent on the doubling
time of the individual tumor cell lines. The plates were then
incubated at 37 °C for 24 h prior to the addition of the submitted
compounds. After 24 h, two microtiter plates of each tumor cell
line were fixed in situ with TCA. The optical density reading at
this point represents the cell population for each tumor cell line at
the time of compound addition (ODtzero). The MMB conjugates
were dissolved in DMSO at 400-fold concentration to the desired
final maximum test concentration and stored at −80 °C. Then, an
aliquot of the frozen concentrate was thawed and diluted to 10−4
M with medium containing 50 ꢀg/mL of gentamicin. A control
sample with just DMSO was also prepared. Aliquots of 100 ꢀL
of the different heteroaryl MMB ester conjugate dilution controls
were added to the appropriate microtiter wells containing 100 ꢀL
4. Conclusions
Evaluation of a novel series of heteroaryl esters of the
melampolide sesquiterpene MMB (7a-7p) has identified two
remarkably potent anticancer agents with activities against a wide
variety of hematological and solid human tumor cell lines. These
two compounds, the indole-3-acrylic acid conjugate 7j and the
indole-3-carboxylic acid conjugate 7k were evaluated in studies
utilizing an NCI panel of 64 human cancer cell lines, M9-ENL1
AML cells, primary AML stem cell specimens obtained with
informed consent from AML patients, and also in studies with
San Francisco variant 9L rat gliosarcoma cells (9L-SF) cells.
Compounds 7j and 7k exhibited remarkable GI50 values in the
range 0.03-0.27 ꢀM and 0.04-0.28 µM, respectively, against the
cell lines in the NCI leukemia sub-panel, and exhibited GI50
values of 0.05-0.40 µM and 0.04-0.61 µM, respectively, against
90% of the solid tumor cell lines in the NCI panel. 7j was the
most potent compound in the M9-ENL1 AML cell assay with an

the cells were placed into a flask that was seeded 24 h earlier with
of medium, resulting in the required final drugAcoCncCenEtrPatTionEsDof MANUSCRIPT
10−5 M and 0 M (control). Once the MMB conjugates were
added, the microtiter plates were incubated for 48 h at 37 °C and
100% relative humidity. Cold TCA was used to terminate the
assay for adherent cells. Cells were fixed by the addition of 50 ꢀL
of cold 50% (w/v) TCA and further incubated for 1 h at 4 °C.
The supernatant was discarded, and the microtiter plates were
splashed five times with water and air-dried. Sulforhodamine B
(SRB) solution (100 ꢀL) at 0.4% (w/v) in 1% CH3COOH was
added to each well, and the plates were incubated for another 10
min at room temperature. After SRB staining, free SRB was
removed by washing five times with 1% acetic acid. Bound SRB
stain was successively dissolved with 10 mM trizma base, and
the optical density was measured at a wavelength of 515 nm. The
growth inhibitory or cytotoxicity effects of the MMB conjugates
in the above cellular assay was measured by determining the
percentage of cell growth (PG) inhibition. Optical density (OD)
measurements of SRB-derived color just before exposing the
cells to the test compound (ODtzero) and after 48 h exposure to
the test compound (ODtest) or the control vehicle (ODctrl) were
recorded.
lethally irradiated feeder cells at
a
density of 2,000
feeders/cm2.[26] No feeders were used when the number of
seeded experimental cells yielded a cell density in the flask
greater than 2,000 cells/cm2. The seeded flasks were incubated
long enough to permit formation of distinct colonies with a
longer incubation time required following drug treatments that
resulted in lower survival levels. Cell survival was normalized to
the plating efficiency (number of colonies divided by the number
of seeded cells) exhibited by control, non-treated 9L-SF cells.
5.4. Methodology for M9-ENL1 AML cells and primary AML cell
Assays with MMB conjugates 7a-7o and the fumarate salt of the
indole-2-carboxylic acid ester of dimethylamino MMB, 7p:
Compounds 7a-7p were screened for cytotoxicity against the
M9-ENL1 AML cell line, and the most active compounds then
screened against primary AML specimens as follows: M9-ENL1
AML cells were plated at a density of 106 cells/mL in alpha-
MEM culture media (Invitrogen) supplemented with 5% of
human plasma, 20% of FBS, and the cytokines SCF, IL-3, IL-7,
and FLT3 (Peprotech). For primary AML specimens, cells were
obtained from volunteer donors. Informed consent was obtained
in accordance with the Declaration of Helsinki. Cells were
isolated from the samples using Ficoll-Paque (GE Healthcare
Bio-Sciences, Pittsburgh, PA) density gradient separation. In
some cases, cells were cryopreserved in freezing medium of
Iscove modified Dulbecco medium (IMDM), 40% of fetal bovine
serum (FBS), and 10% of dimethyl sulfoxide (DMSO) or in
CryoStor CS-10 (VWR,West Chester, PA). Cells were cultured
in serum-free medium (SFM), prepared with Iscove’s MDM
supplemented with 20% of BIT 9500 serum substitute (StemCell
Technologies), LDL, and beta mercaptoethanol for 1 h before the
addition of PTL or the MMB conjugates. Drugs were diluted
from a DMSO stock into PBS such that the final concentration of
DMSO did not exceed 0.5%. Flow cytometric analysis was
performed by co-staining with Annexin V and 7-AAD, according
to manufacturer specifications, to identify the percentage of non-
apoptotic cells, which was defined as the population with
negative staining for both labels. The percentage of non-apoptotic
cells observed was normalized to that of the vehicle control, and
dose-response curves were analyzed using GraphPad Prism
software to determine EC50 values. All analyses were conducted
in triplicate.
5.3. Methodology for the colony formation assay with 9L-SF
gliosarcoma Cells:
San Francisco variant 9L, rat gliosarcoma cells (9L-SF)[25]
were cultured in DMEM/ F12 medium containing 10% iron-
supplemented calf serum and grown in a humidified 37.0 °C
incubator with atmospheric air supplemented to 5% CO2. The
9L-SF cells were seeded into 60 mm culture dishes such that they
were 50% confluent 24 h later (i.e. cell density of 6.65 × 104
cells/cm2). The medium content in each dish was 0.313 mL/cm2
of growth surface area. Compounds 7a, 7b and 7j were dissolved
in DMSO at a concentration of 12 mM and then diluted serially
into the culture medium. The maximum DMSO concentration to
which the drug- treated cells were exposed to was 0.05% (for the
10 µM concentration), and some cells were also treated with
0.05% DMSO only. The fumarate salt of DMAPT, which is a
water-soluble analog of PTL, was used in these experiments. A
2.93 mM stock solution of DMAPT was made in normal saline,
and this was diluted serially to produce the different
concentrations of DMAPT used for the clonogenic assay to test
for DMAPT toxicity. MMB (3) and indole-2-carboxylic acid (6a)
were each dissolved in DMSO to afford a concentration of 5.0
mM and serially diluted to 10 µM in cell culture medium for a
single concentration testing for clonogenic toxicity. Compound
7p, a water-soluble analog of 7a, was also included in the assay
and was utilized as the monofumarate salt. This compound was
dissolved in normal saline at a concentration of 5 mM and diluted
serially to 10 µM for comparative single concentration testing of
its clonogenic toxicity. All cultures were treated with their
respective drugs for 48 h. Cells were then trypsinized from the
dishes, diluted appropriately and seeded into 25 cm2 flasks for the
colony formation. Cell density in the assay flasks was maintained
at 2,000 cells/cm2 by adding lethally irradiated (25 Gy),
nonclonogenic 9L-SF feeder cells. All experiments were repeated
three times using three replicate flasks to determine the toxicity
of each tested drug concentration.
5.5. Comparison of NCI Screening and the Colony Formation
Assay.
It should be noted that the NCI drug screening methodology
and the colony formation assay measure very different cancer-
relevant end points of metabolic and growth inhibition and
clonogenic death, respectively. Because of this, both assays can
facilitate the selection of compounds that should be translated
forward into testing in animal models for specific cancer types,
e.g., glioblastoma, hepatocellular carcinoma, and so forth. The
NCI cancer screening program encompasses a larger number of
compounds and identifies which molecules have the greatest
toxicity for each cancer cell type. Once investigators decide
which cancer type to study in animal models, the smaller number
of compounds that are highly toxic to this cancer type, can be
tested in vitro using the colony formation assay. Arguably, the
compounds that are most effective in rendering the cells
clonogenically dead will have the greatest likelihood of treating
ectopic or orthotopic tumors successfully, and these molecules
are likely the ones that should progress to animal studies.
Following treatment, drug-containing medium was removed
from the culture dishes, and cells were rinsed three times with
fresh medium before the cells were trypsinized from the culture
dish surface, diluted appropriately, and their concentration
determined using an electronic cell counter. A previously
determined number of cells were seeded into 25 cm2 or 75 cm2
culture flasks to determine cell survival by means of colony
formation (flasks seeded in triplicate). When the seeded cell
number would result in a cell density of less than 2,000 cells/cm2,
5.6. General synthetic procedure for the synthesis of MMB ester
conjugates 7a-7o. To the stirred solution of an appropriate

18.1
ppm; HRMS calcd. for C24H24NO5NaCl, (M+Na)+:
heteroaryl carboxylic acid (0.15 mmol) in dry dAichCloCroEmePthTanEeD(5 MANUSCRIPT
o
mL) under an argon atmosphere at 0 C was added EDCI (0.181
mmol), and the mixture allowed to stir for 15 min. Then a
catalytic amount of DMAP (0.045 mmol) was added, followed
by addition of MMB (0.151 mmol) in dry dichloromethane (3
464.1235, found: 464.1233.
5.6.5. Indole-5-carboxylic acid ester of MMB (7e): White solid,
o
1
mp: 134-136 C; H NMR (CDCl3, 400 MHz): δ 8.54 (s, 1H),
8.38 (s,1H), 7.89 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H),
7.30 (t, J = 2.8 Hz, 1H), 6.65 (s, 1H), 6.21 (s, 1H), 5.80 (t, J = 8.8
Hz, 1H), 5.52 (s, 1H), 4.93 (d, J = 12.4 Hz, 1H), 4.75 (d, J = 12.4
Hz, 1H), 3.88 (t, J = 9.6 Hz, 1H), 3.04 (brs, 1H), 2.95 (d, J = 9.6
Hz, 1H), 2.49-2.16 (m, 6H), 1.65-1.64 (m, 1H), 1.56 (s, 3H), 1.17
(t, J = 12.8 Hz, 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ 169.6,
167.4, 138.7, 138.6, 135.4, 130.4, 127.6, 125.9, 123.9, 123.4,
121.5, 120.7, 111.0, 104.1, 81.2, 66.9, 63.4, 60.2, 42.9, 36.8,
26.0, 24.8, 24.0, 18.1 ppm; HRMS calcd. for C24H26NO5,
(M+H)+: 408.1806, found: 408.1805.
o
mL) at 0 C and the resulting mixture stirred for 8 h at room
temperature. After completion of the reaction (monitoring by
TLC), the reaction mixture was washed with water (10 ml) and
the dichloromethane layer separated, dried over anhydrous
sodium sulfate, filtered, concentrated, and purified by silica gel
column chromatography using
2
to 5% methanolic
dichloromethane as eluent to afford the corresponding heteroaryl
esters of MMB (7a-7o) as solids in 56 to 74%. Analytical data
for the above MMB esters are provided below.
5.6.1 Indole-2-carboxylic acid ester of MMB (7a): Pale yellow
1
solid, mp:141-143 °C; H NMR (CDCl3, 400 MHz): δ 8.85 (s,
5.6.6. Indole-6-carboxylic acid ester of MMB (7f): White solid,
1H), 7.70 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.34 (t, J
= 7.2 Hz, 1H), 7.22 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 6.25 (s, 1H),
5.82 (t, J = 8.8 Hz, 1H), 5.55 (s, 1H), 4.96 (d, J = 12.8 Hz, 1H),
4.75 (d, J = 12.8 Hz, 1H), 3.90 (t, J = 9.2 Hz, 1H), 2.98 (brs, 1H),
2.92 (d, J = 9.2 Hz, 1H), 2.53-2.17 (m, 6H), 1.74 (t, J = 8.0 Hz,
1H), 1.56 (s, 3H), 1.14 (t, J = 13.2 Hz, 1H) ppm; 13C NMR
(CDCl3, 100 MHz): δ 169.4, 161.7, 138.7, 137.1, 134.9, 130.9,
127.5, 126.7, 125.9, 122.8, 121.2, 120.6, 112.0, 109.2, 81.1, 67.0,
63.4, 60.1, 42.8, 36.7, 25.9, 24.5, 24.0, 18.1 ppm; HRMS m/z
calcd. for C24H26NO5, (M+H)+: 408.1806, found: 408.1809.
o
1
mp: 129-131 C; H NMR (CDCl3, 400 MHz): δ 8.62 (brs, 1H),
8.09 (s,1H), 7.92 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H),
7.39 (s, 1H), 6.19 (s, 1H), 5.82 (t, J = 8.4 Hz, 1H), 5.49 (s, 1H),
4.93 (d, J = 12.4 Hz, 1H), 4.75 (d, J = 12.4 Hz, 1H), 3.92 (t, J =
9.2 Hz, 1H), 3.14 (t, J = 8.4 Hz, 1H), 2.96 (d, J = 12.0 Hz, 1H),
2.54-2.16 (m, 6H), 1.73 (t, J = 7.2 Hz, 1H), 1.56 (s, 3H), 1.16 (t,
J = 12.0 Hz, 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ 169.8,
167.4, 138.8, 135.4, 135.2, 131.9, 130.8, 128.0, 120.9, 120.8,
120.6, 120.5, 113.6, 103.1, 81.3, 67.3, 63.4, 60.1, 43.0, 36.8,
25.9, 25.0, 24.0, 18.1 ppm; HRMS calcd. for C24H26NO5,
(M+H)+: 408.1806, found: 408.1810.
5.6.2. 5-Methoxyindole-2-carboxylic acid ester of MMB (7b):
White solid, mp:130-132 °C; 1H NMR (CDCl3, 400 MHz): δ 8.85
(s, 1H), 7.33 (d, J = 8.8 Hz, 1H), 7.13 (s, 1H), 7.06 (s, 1H), 7.00
(d, J = 8.8 Hz, 1H), 6.25 (s, 1H), 5.81 (t, J = 8.4 Hz, 1H), 5.55 (s,
1H), 4.94 (d, J = 12.4 Hz, 1H), 4.74 (d, J = 12.4 Hz, 1H), 3.90 (t,
J = 9.6 Hz, 1H), 3.85 (s, 3H), 2.99 (brs, 1H), 2.89 (d, J = 9.2 Hz,
1H), 2.52-2.15 (m, 6H), 1.73 (t, J = 8.8 Hz, 1H), 1.62 (s, 3H),
1.16 (t, J = 12.4 Hz, 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ
169.5, 161.7, 154.9, 138.7, 134.9, 132.5, 130.8, 127.8, 127.1,
120.6, 117.6, 112.9, 108.8, 102.6, 81.1, 66.9, 63.4, 60.1, 55.8,
42.8, 36.7, 25.8, 24.5, 23.9, 18.1 ppm; HRMS (ESI) m/z calcd.
for C25H28NO6, (M + H)+: 438.1911, found: 438.1920.
5.6.7. Indole-3-acetic acid ester of MMB (7g): White solid; mp:
67-69 oC; 1H NMR (CDCl3, 400 MHz): δ 8.16 (s, 1H), 7.58 (d, J
= 8.0 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H),
7.11 (d, J = 6.8 Hz, 1H), 7.07 (s, 1H), 6.13 (s, 1H), 5.61 (t, J =
7.6 Hz, 1H), 5.32 (s, 1H), 4.70 (d, J = 12.0 Hz,1H), 4.41 (d, J =
12.4 Hz,1H), 3.83 (q, J = 16.0 Hz, J = 3.6 Hz, 2H), 3.70 (d, J =
9.6 Hz,1H), 2.59-2.55 (t, J = 12.0 Hz , 1H), 2.29-2.01 (m, 7H),
1.48-1.44 (m, 4H), 0.91 (t, J = 12.4 Hz, 1H) ppm; 13C NMR
(CDCl3, 100 MHz): δ 171.8, 169.9, 139.2, 136.4, 134.9, 131.8,
127.1, 123.2, 122.6, 120.0, 119.8, 118.9, 111.8, 108.4, 81.0, 67.1,
63.0, 59.9, 42.6, 36.5, 32.0, 25.8, 25.3, 23.9, 18.1 ppm; HRMS
calcd. for C25H27NO5Na, (M+Na)+: 444.1781, found: 444.1788.
5.6.3. 5-Fluroindole-2-carboxylic acid ester of MMB (7c): White
1
solid, mp: 127-129 oC; H NMR (CDCl3, 400 MHz): δ 8.89 (brs,
1H), 7.37-7.30 (m, 2H), 7.17 (s, 1H), 7.13 (t, J = 9.6 Hz, 1H),
5.81 (s, 1H), 5.79 (t, J = 8.0 Hz, 1H), 5.55 (s, 1H), 4.95 (d, J =
12.8 Hz,1H), 4.75 (d, J = 12.4 Hz, 1H), 3.90 (t, J = 9.6 Hz,1H),
2.97 (brs, 1H), 2.91 (d, J = 9.2 Hz, 1H), 2.53-2.16 (m, 6H), 1.76
(t, J = 4.4 Hz, 1H), 1.57 (s, 3H), 1.17 (t, J = 12.8 Hz, 1H) ppm;
13C NMR (CDCl3, 100 MHz): δ 169.4, 161.4, 159.5, 157.2,
138.7, 134.7, 133.6, 131.0, 128.3, 127.7, 127.6, 120.6, 115.2,
114.9, 113.0, 112.9, 109.0, 109.0, 107.1, 106.8, 81.1, 67.1, 63.4,
60.1, 42.8, 36.7, 25.8, 24.5, 24.1, 18.1 ppm; HRMS calcd. for
C24H24NO5FNa, (M+Na)+: 448.1531, found: 448.1546.
5.6.8. Indole-3-butyric acid ester of MMB (7h): White solid, mp:
74-76 0C; 1H NMR (CDCl3, 400 MHz): δ 8.20 (s, 1H), 7.57 (d, J
= 7.6 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.17 (t, J = 6.8 Hz, 1H),
7.09 (t, J = 7.2 Hz, 1H), 6.95 (s, 1H), 6.17 (s, 1H), 5.64 (t, J = 8.4
Hz,1H), 5.44 (s, 1H), 4.61 (d, J = 12.0 Hz,1H), 4.42 (d, J = 12.4
Hz,1H), 3.83 (d, J = 9.2 Hz, 1H), 2.83-2.77 (m, 4H), 2.38-2.14
(m, 8H), 2.09 (p, J = 7.2 Hz, J = 14.4 Hz, 2H), 1.60-1.59 (m,
1H), 1.56 (s, 3H), 1.09 (t, J = 11.6 Hz, 1H) ppm; 13C NMR
(CDCl3, 200 MHz): δ 173.4, 169.6, 138.7, 136.4, 134.9, 130.8,
127.4, 121.9, 121.7, 120.4, 119.1, 118.7, 115.1, 111.3, 81.1, 66.8,
63.2, 60.1, 42.7, 36.6, 33.8, 25.7, 25.3, 24.6, 24.5, 23.8, 18.0
ppm; HRMS calcd. For C27H32NO5, (M+H)+: 450.2346, found:
450.2344.
5.6.4. 5-Chloroindole-2-carboxylic acid ester of MMB (7d):
o
1
White solid, mp: 145-147 C; H NMR (CDCl3, 400 MHz): δ
9.13 (s, 1H), 7.65 (s, 1H), 7.38 (d, J = 8.8 Hz, 1H), 7.29 (d, J =
8.8 Hz,1H), 7.14 (s, 1H), 6.25 (s, 1H), 5.78 (t, J = 8.8 Hz,1H),
5.55 (s, 1H), 4.95 (d, J = 12.8 Hz,1H), 4.76 (d, J = 12.4 Hz,1H),
3.90 (t, J = 8.8 Hz, 1H), 2.95 (brs, 1H), 2.90 (d, J = 8.8 Hz, 1H),
2.53-2.19 (m, 6H), 1.76-1.61 (m, 1H), 1.55 (s, 3H), 1.26-1.15 (m,
1H) ppm; 13C NMR (CDCl3, 100 MHz): δ 169.5, 161.5, 138.7,
135.4, 134.7, 130.9, 128.3, 128.1, 126.7, 126.3, 121.8, 120.6,
113.3, 108.4, 81.1, 67.2, 63.4, 60.1, 42.8, 36.7, 25.8, 24.4, 23.9,
5.6.9. Indomethacin ester of MMB (7i): Pale yellow solid, mp:
o
1
95-97 C; H NMR (CDCl3, 400 MHz): δ 7.67 (d, J = 8.4 Hz,
2H), 7.48 (d, J = 8.4 Hz, 2H), 6.95 (s, 1H), 6.84 (d, J = 8.8 Hz,
1H), 6.68 (d, J = 9.2 Hz,1H), 6.19 (s, 1H), 5.62 (t, J = 8.4
Hz,1H), 5.39 (s, 1H), 4.66 (d, J = 12.4 Hz,1H), 4.51 (d, J = 12.4
Hz,1H), 3.83 (s, 3H), 3.79 (t, J = 9.2 Hz, 1H), 3.69 (s, 2H), 2.73-
2.71 (m, 1H-), 2.69 (d, J = 9.2 Hz, 1H), 2.40 (s, 3H), 2.36-2.10

2H), 6.23 (d, J = 3.6 Hz, 1H), 5.86 (t, J = 8.4 Hz, 1H), 5.55 (d, J
(m, 6H), 1.54-1.53 (m, 1H), 1.50 (s, 3H), 1.30-A1.2C8 C(mE, 1PHT) EppDm; MANUSCRIPT
13C NMR (CDCl3, 100 MHz): δ 170.6, 169.4, 168.4, 156.2,
139.5, 138.7, 136.2, 134.7, 133.8, 131.4, 130.9, 130.7, 130.6,
129.4, 120.4, 115.3, 112.4, 111.4, 101.9, 81.0, 67.1, 63.3, 60.0,
55.9, 53.6, 42.7, 36.7, 30.7, 25.7, 24.6, 23.9, 18.2 ppm; HRMS
calcd. for C34H34NO7NaCl, (M+H)+: 626.1916, found: 626.1908.
= 2.8 Hz, 1H), 4.91 (dd, J = 12.4 Hz, J = 27.2 Hz, 2H), 3.90 (d, J
= 9.6 Hz, 2H), 2.99 (t, J = 3.2 Hz, 1H), 2.92 (d, J = 9.2 Hz,1H),
2.53-2.17 (m, 6H), 1.76 (m, 1H), 1.57 (s, 3H), 1.18 (t, J = 12.8
Hz, 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ 169.5, 161.2,
138.8, 138.8, 137.2, 134.6, 131.7, 128.7, 128.0, 125.9, 125.5,
124.2, 123.0, 120.7, 81.2, 67.9, 63.5, 60.2, 43.0, 36.8, 25.9, 24.7,
24.1, 18.2 ppm; HRMS calcd. for C24H24O5SCl, (M+H)+:
459.1028, found: 459.1035.
5.6.10. Indole-3-acrylicacid ester of MMB (7j): White solid, mp:
o
1
137-139 C; H NMR (CDCl3, 400 MHz): δ 8.61 (brs, 1H), 7.93
(d, J = 15.6 Hz, 1H), 7.88 (brs, 1H),7.50 (d, J = 2.8 Hz, 1H), 7.42
(brs, 1H), 7.29-7.27 (m, 2H), 6.45 (d, J = 16.0 Hz,1H), 6.26 (d, J
= 3.6 Hz, 1H), 5.79 (t, J = 8.4 Hz,1H), 5.58 (d, J = 3.2 Hz,1H),
4.81 (d, J = 12.4 Hz,1H), 4.62 (d, J = 12.8 Hz,1H), 3.88 (t, J =
6.04 Hz,1H), 3.09-3.07 (m, 1H), 2.94 (d, J = 9.2 Hz, 1H), 2.48-
2.20 (m, 6H), 1.72-1.69 (m, 1H), 1.58 (s, 3H), 1.42-1.33 (m, 1H)
ppm; 13C NMR (CDCl3, 100 MHz): δ 169.8, 168.1, 139.5, 138.7,
137.3, 135.4, 130.5, 129.9, 125.2, 123.4, 121.5, 120.7, 120.4,
113.3, 112.2, 112.1, 81.4, 66.8, 63.4, 60.3, 42.8, 36.7, 26.0, 24.8,
23.9, 18.1 ppm; HRMS calcd. for C26H27NO5Na, (M+Na)+:
456.1781, found: 456.1790.
5.6.15. Benzofuran-2-carboxylic acid ester of MMB (7o): White
o
1
solid, mp: 59-61 C; H NMR (CDCl3, 400 MHz): δ 7.69 (d, J =
8.0 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.55 (s, 1H), 7.49 (t, J =
6.8 Hz, 1H), 7.33 (t, J = 7.6 Hz,1H), 6.22 (d, J = 3.6 Hz,1H),
5.85 (t, J = 8.4 Hz, 1H), 5.54 (d, J = 3.2 Hz,1H), 5.0 (d, J = 12.4
Hz, 1H), 4.75 (d, J = 12.0 Hz, 1H), 3.91 (t, J = 9.2 Hz, 1H), 3.09
(brs, 1H), 2.95 (d, J = 6.8 Hz, 1H), 2.54-2.25 (m, 6H), 1.75 (t, J =
9.6 Hz, 1H), 1.57 (s, 3H), 1.18 (t, J = 12.4 Hz, 1H) ppm; 13C
NMR (CDCl3, 100 MHz): δ 169.6, 159.5, 156.0, 145.2, 138.9,
134.7, 132.2, 128.2, 127.0, 124.2, 123.1, 120.5, 114.7, 112.6,
81.2, 68.0, 63.5, 60.2, 43.0, 36.8, 26.1, 25.0, 24.1, 18.2 ppm;
HRMS calcd. for C24H25O6, (M+H)+: 409.1646, found: 409.1648.
5.6.11. Indole-3-carboxylic acid ester of MMB (7k): White solid,
1
mp:140-142 °C; H NMR (CDCl3, 400 MHz): δ 8.82 (s, 1H)
8.16-8.14 (m, 1H), 7.91 (s,1H), 7.45-7.43 (m, 1H), 7.29-7.27 (m,
2H), 6.18 (s, 1H), 5.80 (t, J = 8.4 Hz, 1H), 5.49 (s, 1H), 4.92 (d, J
= 12.4 Hz, 1H), 4.80 (d, J = 12.8 Hz, 1H), 3.89 (t, J = 9.2 Hz,
1H), 3.03 (t, J = 9.2 Hz, 1H), 2.95 (d, J = 9.2 Hz, 1H), 2.51-2.14
(m, 6H), 1.70-1.65 (m, 1H), 1.56 (s, 3H), 1.14 (t, J = 13.2 Hz,
1H) ppm; 13C NMR (CDCl3, 100 MHz): δ 169.5, 164.6, 138.8,
137.3, 135.7, 135.4, 130.1, 126.6, 123.1, 122.2, 121.5, 120.4,
110.1, 106.5, 81.2, 65.8, 63.4, 60.1, 42.8, 36.8, 25.9, 24.8, 23.9,
18.1 ppm; HRMS (ESI) m/z calcd. for C24H25NO5Na (M + Na)+:
430.1625, found: 430.1620.
5.7. Synthesis of dimethylaminomelampomagnolide B (8):
Melampomagnolide B (1 mmol), was dissolved in methanol (5
ml) at room temperature. Added dimethylamine solution in
methanol (0.18 ml, 2.5 M in methanol) and stirred the reaction
mass at room temperature for 6 hrs, until the completion of
reaction by TLC (TLC mobile phase, dichloromethane:
methanol= 98:2). Reaction mass was concentrated under reduced
pressure to remove methanol. Obtained crude product was
purified by silica column chromatography using 2% methanolic
1
dichloromethane. Gummy mass; H NMR (CDCl3, 400 MHz): δ
5.60 (t, J = 7.6 Hz, 1H), 4.16 (dd, J = 12.8 Hz, J = 32.0 Hz, 2H),
3.88 (t, J = 9.2 Hz, 1H), 2.82 (d, J = 9.2 Hz, 1H), 2.76-2.70 (m,
2H), 2.51-2.42 (m, 6H), 2.30-2.15 (m, 7H), 2.15-2.08 (m, 2H),
1.64-1.61 (m, 1H), 1.52 (s, 3H), 1.11(t, J = 11.2 Hz, 1H) ppm;
13C NMR (CDCl3, 100 MHz): δ 176.7, 140.9, 127.4, 81.6, 66.2,
63.9, 59.9, 57.3, 45.5, 44.1, 42.1, 36.9, 27.5, 25.8, 23.7, 17.9
ppm. HRMS calcd. for C17H28NO4, (M+H)+: 310.2047, found:
310.2037.
5.6.12. N-Methylindole-3-carboxylic acid ester of MMB (7l):
White solid, mp: 109-111 °C; H NMR (CDCl3, 400 MHz): δ
1
8.14 (d, J = 7.2 Hz, 1H), 7.75 (s, 1H), 7.37-7.30 (m, 3H), 6.18 (s,
1H), 5.80 (t, J = 8.4 Hz, 1H), 5.50 (s, 1H), 4.92 (d, J = 12.4 Hz,
1H), 4.75 (d, J = 12.4 Hz, 1H), 3.88 (m, 4H), 3.05 (t, J = 9.2 Hz,
1H), 2.92 (d, J = 9.2 Hz, 1H), 2.51-2.15 (m, 6H), 1.70-1.68 (m,
1H), 1.55 (s, 3H), 1.15 (t, J = 13.2 Hz, 1H) ppm; 13C NMR
(CDCl3, 100 MHz): δ 169.5, 164.6, 138.8, 137.3, 135.6, 135.4,
130.0, 126.6, 123.1, 122.2, 121.5, 120.4, 110.0, 106.5, 81.2, 65.8,
63.4, 60.1, 42.8, 36.8, 33.6, 25.9, 24.8, 23.9, 18.1 ppm; HRMS
(ESI) m/z calcd. for C25H27NO5Na, (M + Na)+: 444.1781, found:
444.1777.
5.8. Synthesis of the indole-2-carboxylic acid ester of
dimethylamino MMB (9): To a stirred solution of indole-2-
carboxylic acid (6a, 0.15 mmol) in dry dichloromethane (5 mL)
o
under an argon atmosphere at 0 C was added EDCI (0.181
mmol), and the mixture allowed to stir for 15 min. Then a
catalytic amount of DMAP (0.045 mmol) was added, followed
by addition of dimethylaminomelampomagnolide B (8, 0.15
5.6.13. Benzothiophene-2-carboxylic acid ester of MMB (7m):
o
1
White solid, mp: 71-73 C; H NMR (CDCl3, 400 MHz): δ 8.06
(s, 1H), 7.89-7.85 (m, 2H), 7.49-7.40 (m, 2H), 6.24 (d, J = 2.8
Hz, 1H), 5.83 (t, J = 8.0 Hz,1H), 5.55 (d, J = 2.0 Hz, 1H), 4.93
(d, J = 12.0 Hz,1H), 4.77 (d, J = 12.8 Hz,1H), 3.90 (t, J = 9.6
Hz,1H), 2.99 (brs, 1H), 2.92 (d, J = 9.2 Hz, 1H), 2.53-2.17 (m,
6H), 1.73-1.69 (m, 1H), 1.57 (s, 3H), 1.17 (t, J = 13.2 Hz, 1H)
ppm; 13C NMR (CDCl3, 100 MHz): δ 169.53, 162.7, 142.4,
138.8, 138.8, 134.8, 131.4, 131.1, 127.4, 125.8, 125.3, 122.9,
120.7, 81.2, 67.7, 63.5, 60.2, 43.0, 36.8, 25.9, 24.77, 24.10, 18.2
ppm; HRMS calcd. for C24H25O5S, (M+H)+: 403.0909, found:
403.0917.
o
mmol) in dry dichloromethane (3 mL) at 0 C and the resulting
mixture stirred for 8 h at room temperature. After completion of
reaction (monitoring by TLC), the reaction mixture was washed
with water (10 ml) and the dichloromethane layer separated,
dried over anhydrous sodium sulfate, filtered, concentrated, and
purified by silica gel column chromatography using 2 to 5%
methanolic dichloromethane as eluent to afford the corresponding
indole-2-carboxylic acid ester of dimethylamino MMB (9) as
white solid, mp: 114-116 oC; 1H NMR (CDCl3, 400 MHz): δ 8.92
(s, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.35 (t,
J = 7.2 Hz, 1H), 7.23 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 5.72 (t, J =
8.8 Hz, 1H), 5.03 (d, J = 12.8 Hz, 1H), 4.85 (d, J = 12.8 Hz, 1H),
3.90 (t, J = 9.6 Hz, 1H), 2.83 (d, J = 9.2 Hz,1H), 2.79 (dd, J = 4.8
Hz, J = 12.8 Hz, 1H), 2.69 (dd, J = 5.2 Hz, J = 12.8 Hz, 1H),
5.6.14. 3-Chlorobenzothiophene-2-carboxylic acid ester of MMB
o
1
(7n): White solid, mp: 69-71 C; H NMR (CDCl3, 400 MHz): δ
7.99 (d, J = 6.8 Hz, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.54-7.50 (m,

[3] W.W. Ma, Q.Q. Shi, Y.H. Ding, J. Long, Q. Zhang, Y. Chen,
2.54-2.44 (m, 4H), 2.38-2.28 (m, 2H), 2.24 (s, A6HC) C2.2E3-P2.T13E(Dm, MANUSCRIPT
Synthesis of micheliolide derivatives and their activities against
AML progenitor cells, Molecules 18 (2013) 5980-5992.
[4] D.W. Knight, Feverfew: chemistry and biological activity, Nat.
Prod. Rep. 12 (1995) 271-276.
2H), 1.65-1.61(m, 1H), 1.56 (s, 3H), 1.13 (t, J = 12.4 Hz, 1H)
ppm; 13C NMR (CDCl3, 100 MHz): δ 177.0, 161.6, 137.0, 135.7,
128.8, 127.5, 127.1, 125.7, 122.7, 121.1, 112.0, 109.0, 81.3, 66.8,
64.0, 60.0, 58.3, 45.8, 44.5, 43.0, 37.0, 31.7, 27.0, 24.7, 23.9,
18.1 ppm; HRMS calcd. for C26H33N2O5, (M+H)+: 453.2384,
found: 453.2336.
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Bernstein, Modulation of cell surface protein free thiols: a potential
novel mechanism of action of the sesquiterpene lactone parthenolide,
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5.9. The fumarate salt of the indole-2-carboxylic acid ester of
dimethylamino MMB (7p): Compound 9 (50 mg, 0.11 mmol) was
dissolved in ethanol under an argon atmosphere, and fumaric acid
(0.11 mmol in 0.5 ml ethanol) in ethanol solution was added
drop- wise while stirring the reaction mixture for 30 minutes at
room temperature. The resulting white precipitate was filtered
and dried under vacuum to afford 7p (37 mg) in 60% yield.
o
1
White solid, mp: 184-186 C; H NMR (CD3OD, 400 MHz): δ
7.65 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 7.2
Hz, 1H), 7.20 (s, 1H), 7.10 (t, J = 7.6 Hz, 1H), 6.70 (s, 2H), 5.79
(t, J = 8.0 Hz, 1H), 5.02 (d, J = 12.4 Hz, 1H), 4.76 (d, J = 12.8
Hz, 1H), 4.18 (t, J = 9.2 Hz, 1H), 3.30-3.23 (m, 2H), 3.01-3.00
(m, 1H), 2.91 (d, J = 9.2 Hz, 1H), 2.78 (s, 6H), 2.64-2.39 (m,
4H), 2.25-2.14 (m, 3H), 1.80-1.75 (m, 1H) 1.58 (s, 3H), 1.10 (t, J
= 13.2 Hz, 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ 178.0,
170.4, 163.3, 139.2, 136.6, 135.9, 131.0, 128.6, 128.3, 126.2,
123.2,, 121.5, 113.3, 109.7, 83.4, 67.8, 64.5, 61.5, 57.4, 44.6,
44.5, 44.0, 43.1, 37.8, 26.8, 25.3, 24.5, 17.9 ppm; CHN analysis
calcd. for C30H38N2O10.H2O; C, 61.42%, H, 6.53%, N, 4.78%, O,
27.27%; Found: C, 61.25%, H, 6.56%, N, 4.43%, O, 27.27%.
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Acknowledgments
We are grateful to the NIH/National Cancer Institute (grant #
R01 CA158275), the UAMS Translational Research Institute
(TRI), grant UL1TR000039 through the NIH National Center for
Research Resources, the National Center for Advancing
Translational Sciences, the UAMS Department of Radiology for
partial support of this research, to the Arkansas Research
Alliance for an Arkansas Scholar award, and to the NCI drug-
screening program. The authors also thank Laura J. Bernock,
M.S. for her technical assistance.
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Conflict of Interest Statement
The authors declare the following competing financial
interest(s): The University of Arkansas for Medical Sciences
(UAMS) holds patents on the molecules described in this study.
A potential royalty stream to SB, JP, NRP, VJ, CTJ, and PAC
may occur consistent with UAMS policy.
Appendix A. Supplementary data
1H-NMR, 13C NMR, and HRMS spectra for synthetic products
are provided in the Supplementary Information. Supplementary
data related to this article can be found at http://....
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ACCEPTED MANUSCRIPT
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Design and synthesis of novel MMB-indole carboxylic acid ester analogs
Screening of analogs for anticancer activity against NCI-60 cancer cell panel
Potent growth inhibitory effects of analogs on human cancer cell lines
Potent anti-leukemic activity of analogs against cell specimens from AML patients
Potent antiproliferative effects against rat 9L-SF gliosarcoma cells