Synthesis and antileukemic activities of C1-C10-modified parthenolide analogues

Article abstract of DOI:10.1016/j.bmc.2015.05.037

Parthenolide (PTL) is a sesquiterpene lactone natural product with anti-proliferative activity to cancer cells. Selective eradication of leukemic stem cells (LSCs) over healthy hematopoietic stem cells (HSCs) by PTL has been demonstrated in previous studies, which suggests PTL and related molecules may be useful for targeting LSCs. Eradication of LSCs is required for curative therapy. Chemical optimizations of PTL to improve potency and pharmacokinetic parameters have focused largely on the α-methylene-γ-butyrolactone, which is essential for activity. Conversely, we evaluated modifications to the C1-C10 olefin and benchmarked new inhibitors to PTL with respect to inhibitory potency across a panel of cancer cell lines, ability to target drug-resistant acute myeloid leukemia (AML) cells, efficacy for inhibiting clonal growth of AML cells, toxicity to healthy bone marrow cells, and efficiency for promoting intracellular reactive oxygen species (ROS) levels. Cyclopropane 4 was found to possess less toxicity to healthy bone marrow cells, enhanced potency for the induction of cellular ROS, and similar broad-spectrum anti-proliferative activity to cancer cells in comparison to PTL.

Full text of DOI:10.1016/j.bmc.2015.05.037

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antileukemic activities of C1–C10-modified
parthenolide analogues
a
a
a
a
a
Aaron M. Kempema , John C. Widen , Joseph K. Hexum , Timothy E. Andrews , Dan Wang ,
b
a
c
c
b,d
Susan K. Rathe , Frederick A. Meece , Klara E. Noble , Zohar Sachs , David A. Largaespada
,
a,b,e,
⇑
Daniel A. Harki
a
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA
b
c
d
e
Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Parthenolide (PTL) is a sesquiterpene lactone natural product with anti-proliferative activity to cancer
cells. Selective eradication of leukemic stem cells (LSCs) over healthy hematopoietic stem cells (HSCs)
by PTL has been demonstrated in previous studies, which suggests PTL and related molecules may be
useful for targeting LSCs. Eradication of LSCs is required for curative therapy. Chemical optimizations
Received 19 May 2015
Accepted 24 May 2015
Available online xxxx
of PTL to improve potency and pharmacokinetic parameters have focused largely on the
a-methylene-
Keywords:
c-butyrolactone, which is essential for activity. Conversely, we evaluated modifications to the C1–C10
Bone marrow toxicity
Cyclopropanation
Acute myeloid leukemia
Parthenolide
olefin and benchmarked new inhibitors to PTL with respect to inhibitory potency across a panel of cancer
cell lines, ability to target drug-resistant acute myeloid leukemia (AML) cells, efficacy for inhibiting clonal
growth of AML cells, toxicity to healthy bone marrow cells, and efficiency for promoting intracellular
reactive oxygen species (ROS) levels. Cyclopropane 4 was found to possess less toxicity to healthy bone
marrow cells, enhanced potency for the induction of cellular ROS, and similar broad-spectrum anti-
proliferative activity to cancer cells in comparison to PTL.
Sesquiterpene lactone
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
(AML) stem and progenitor cells without exhibiting comparable
toxicity to healthy hematopoietic stem cells (HSCs) has anointed
Sesquiterpene lactones (SL) are a diverse family of plant-
derived natural products with utilities in treating inflammatory
PTL as the prototypical member of next-generation therapies for
6
eradicating leukemic stem cells (LSCs). AML growth is hierarchical
1
,2
7,8
diseases and cancer.
Parthenolide (PTL) is a well-studied SL
and originates from LSCs. Therefore, small molecules that elimi-
3
derived from the feverfew plant Tanacetum parthenium, bearing
broad-spectrum anti-proliferative activities to a variety of cancer
types through multiple mechanisms of inhibition.4 The seminal
discovery that PTL induces apoptosis in acute myeloid leukemia
nate LSCs are expected to confer more durable and potentially
9
–13
curative therapies.
In addition to its anti-leukemic activity,
,5
PTL has been explored as a potential therapeutic for a spectrum
1
,4,14
of indications.
Chemical optimizations of PTL have been required to optimize
the natural product for in vivo applications. A Phase I dose escala-
tion trial of feverfew extract failed to achieve measurable levels of
PTL in serum and oral dosing (40 mg/kg) of PTL in mice yielded
approximately 200 nM concentrations in serum, which is not suf-
Abbreviations: PTL, parthenolide; LSC, leukemic stem cell; AML, acute myeloid
leukemia; SL, sesquiterpene lactone; HSC, hematopoietic stem cell; DMAPT,
dimethylamino-parthenolide; MelB, melampomagnolide B; ROS, reactive oxygen
2
species; PtO , platinum(IV) oxide; DME, dimethoxyethane; LogD, distribution
1
5,16
coefficient; CSC, cancer stem cell; CTL, costunolide; BM, bone marrow; DOX,
doxorubicin; AraC, cytarabine; MEM, minimum essential medium; FBS, fetal bovine
serum; DMEM, Dulbecco’s modified Eagle medium; HEPES, (4-2-hydroxyethyl)-1-
piperazineethanesulfonic acid); IMDM, Iscove’s modified Dulbecco’s medium; MTS,
ficient to confer anti-proliferative activity.
Conversion of PTL
to prodrug dimethylamino-parthenolide fumarate, DMAPT (or
LC-1), increased water solubility by ꢀ1000-fold and yielded an
analogue with substantially improved pharmacokinetic parame-
3
-(4,5-dimethylthiaol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium; SFEM, serum-free expansion medium.
ters (mice:
C
max = 25
l
M,
t
1/2 = 0.6 h; canine:
C
max = 61
l
M,
⇑
Corresponding author.
E-mail address: daharki@umn.edu (D.A. Harki).
17,18
t1/2 = 1.9 h) with oral dosing (100 mg/kg).
Hetero-Michael
http://dx.doi.org/10.1016/j.bmc.2015.05.037
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037

2
A. M. Kempema et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
addition of aliphatic amines to natural products and synthetic ana-
logues bearing -methylene- -butyrolactones has constituted a
modular strategy to enhance water solubilities through prodrug
(Fig. 1). Previous studies have found that the C1–C10 olefin of
PTL can participate in electrophilic transannular cyclizations with
a
c
the C4–C5 epoxide under Brønsted or Lewis acid conditions to
1
9–25
28,29
formation.
-methylene-
Semisynthetic modifications to PTL outside of the
-butyrolactone warhead, however, are significantly
yield guaianolide analogues.
Therefore, we synthesized
a
c
reduced analogue 3 to eliminate any potential for acid instability.
The exocyclic methylene on the -methylene- -butyrolactone of
less developed. Acid-catalyzed conversion of PTL to 5-7-5 guaiano-
a
c
lide, Micheliolide (1), has been achieved, yielding a derivative with
PTL was transiently protected by dimethylamine addition to yield
dimethylamino-PTL, which was not converted to the fumarate salt
for this application (Scheme 1). Hydrogenation of dimethylamino-
2
1,26,27
anti-proliferative activity comparable to its predecessor.
Photochemical isomerization of the C1–C10 olefin of PTL has also
been reported, yielding cis-olefin 2.2
8–31
Allylic oxidation of the
2 2
PTL free base with PtO catalyst at 50 psi H resulted in facial selec-
C1–C10 vinyl methyl group of PTL results in formation of another
natural product, Melampomagnolide B (MelB), which exhibits
comparable anti-proliferative activity as PTL, but contains an
allylic alcohol, which is useful for further transformations (e.g.,
synthesis of affinity pulldown reagents and O-functionalized ana-
tive reduction (ꢀ15:1), yielding 10R-diastereomer 3. The exocyclic
methylene was deprotected under Hofmann elimination condi-
2
0,48,49
tions (excess MeI, THF, H
2
O)
to yield 3 in 32% yield (over
three steps). The stereochemistry of the methyl group was
assigned by X-ray crystallography (SI).
3
2–36
2
logues).
Recently, the Fasan laboratory has utilized P450
Cyclopropanes are unique ring systems with significant sp -
enzymes to oxygenate proximal to the C1–C10 olefin of PTL, yield-
ing alcohols at C9 and C14, which were subsequently esterified
with substituted benzoic acids to yield PTL analogues with anti-
character, thereby mimicking the electronics of double bonds.50
Such modifications can be valuable for increasing the stability of
a drug candidate. In the case of PTL, replacement of the C4–C5
epoxide with a cyclopropane significantly enhanced plasma half-
life (t1/2 = 13.9 h vs 1.6 h for PTL; testing in mouse plasma).51 To
further probe the role of the C1–C10 olefin in PTL with a struc-
turally analogous mimetic, we synthesized C1–C10-cyclo-
3
7
leukemic activities.
In this study, we examined the necessity of the PTL C1–C10 olefin
and its tolerance to structural modification with respect to sustained
anti-proliferative activities to cancer cells through the synthesis and
biochemical screening of C1–C10 modified PTL analogues. Included
among our small library of compounds are established PTL ana-
logues, such as Micheliolide (1), cis-PTL (2), and MelB, as well as
additional mechanistic probes, such as 3 (reduced C1–C10 olefin)
propanated 4. Utilizing the Furukawa modification (ZnEt
2
) to the
PTL was treated with
in a solution of DME and CH Cl , which
5
2–54
classical Simmons–Smith reaction,
pre-formed Zn(CH I)
2
2
2
2
yielded (1S,10R) 4 in 41% yield following silica gel chromatography
(Scheme 1). Interestingly, no attack to the exocyclic olefin was
observed, with the remaining mass balance consisting of mostly
unreacted PTL. The structure of 4 was assigned by X-ray crystallog-
raphy (Fig. 2 and SI). As expected, the solid-state structure of cyclo-
propane 4 was highly similar to a recently reported PTL X-ray
and
4 (cyclopropanated C1–C10 olefin). Interestingly, cyclo-
propanated analogue 4 was found to exhibit similar anti-prolifera-
tive activity to cancer cells as PTL, but conferred less toxicity to
healthy bone marrow and more potently induced cellular reactive
oxygen species (ROS), which is known to promote cell death to
AML stem cells and other cancer cells.3
8–47
51
structure with a root-mean-square deviation of 0.167 Å (Fig. 2;
alignment of structures provided in the SI). Recognizing that 4
may suffer from poor aqueous solubility akin to PTL, we synthe-
sized dimethylamine congener 5, which was converted to the
2
2
. Results and discussion
1
8
fumarate salt for consistency with DMAPT.
PTL and
.1. Design and synthesis of PTL analogues
A small library of C1–C10 olefin modified PTL analogues and
Costunolide (CTL) were purchased from commercial vendors and
the remaining analogues in our library (1, 2, MelB, and 6) were
1
8,21–26,28,29,32–35
synthesized as previously reported.
The structure
control compounds were synthesized or purchased commercially
of synthesized Micheliolide (1) was verified by X-ray crystallogra-
5
5
phy (SI) and compared to a previous report.
.2. Lipophilicity analyses
The distribution coefficients (LogD) of select C1–C10 PTL ana-
C1
C10
(
fumarate)
2
O
O
NMe2
O
O
O
O
Parthenolide (PTL)
Dimethylamino-parthenolide
(
DMAPT, LC-1)
logues were assessed through calculated and measured analyses
(Table 1). Calculated LogD values were less predictive in compar-
ison to experimentally derived measurements for PTL
OH
H
O
HO
O
O
O
O
O
O
O
Micheliolide (1)
2
Melampomagnolide B
(MelB)
H
(fumarate)
O
O
O
O
NMe2
O
4
O
5
O
O
O
3
O
O
O
O
O
6
Costunolide (CTL)
Figure 1. Structures of PTL and DMAPT, C1–C10 modified PTL analogues (1–5 and
MelB), and control compounds for biochemical assays (6 and CTL).
Scheme 1. (a) NHMe
2
, MeOH; Pt
Cl
2
O, H
2
(50 psi), EtOAc; MeI, THF, H
2
O, 45 °C, 32% (3
steps); (b) Zn(CH I) , DME, CH
2
2
2
2
, 41%; (c) NHMe
2
, MeOH; fumaric acid, 85%.
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037

A. M. Kempema et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
CCRF-CEM) and solid tumors (U-87 MG, GBM6, MCF-7, DU-145,
and NCI/ADR-RES). An established drug-resistant tumor cell line,
NCI/ADR-RES, was included in our screen to determine if PTL and
related analogues possess activity against cancer cell lines with
5
6,57
high P-glycoprotein expression.
Clinically used chemothera-
peutic drugs gemcitabine and doxorubicin are inactive (IC50
5
8
>500
l
M) against NCI/ADR-RES cells,
and cancer stem cells
(
CSCs) are known to have high expression levels of drug efflux
5
9–61
machinery.
Additionally, we included the GBM6 glioblastoma
multiforme (GBM) cell line in our primary screen because it pos-
+
62
sesses a CD133 population of cells, which is a frequently used
6
3
marker of GBM stem cells.
Screening of PTL and related analogues revealed broad-spec-
trum, low-micromolar IC50 growth inhibitory activity to all cancer
cell lines regardless of modification to the C1–C10 olefin (Table 2).
In contrast, 6, which bears a reduced exocyclic methylene on the
methylene- -butyrolactone, was found to be completely inactive
against all cell lines examined (IC50 >500 M). These data are con-
and reinforce the necessity of
-butyrolactone for anti-proliferative activity of
a-
c
l
1
8,32,64
sistent with previous reports,
the -methylene-
molecules of this class.
a
c
1
,20,23,65
CTL was found to be equipotent to
the C1–C10 modified analogues, suggesting the C4–C5 epoxide is
non-essential for activity, which is also consistent with a previous
5
1
report. All compounds except for exomethylene-reduced 6 were
active against drug-resistant NCI/ADR-RES cells (IC50 range: 9.4–
2
2.0 lM).
2
.4. Bone marrow toxicity studies
The CD34 CD38- bone marrow (BM) immunophenotype is
+
6
6,67
enriched for self-renewing stem cells.
Previous studies have
+
ꢁ
demonstrated that PTL is non-toxic to total BM and CD34 CD38
6
BM cells when dosed at 5 lM for 18 h. To assess BM toxicity of
Figure 2. X-ray crystal structures. (A) PTL18 and (B) cyclopropane 4 adopt similar
conformations in the solid-state. Root-mean-square deviation between the two
structures is 0.167 Å.
the synthesized C1–C10 PTL analogues in comparison to PTL, we
performed flow cytometry assays with human BM cells and mea-
sured cellular viability by flow cytometry using markers for apop-
tosis (Annexin V) and necrosis (7-AAD). Since PTL has been shown
Table 1
6
to elicit some overall BM toxicity at a dose of 7.5
lM for 18 h, we
Calculated LogD values (cLogD7.4
; MarvinSketch) and measured LogD values
elected to utilize a slightly higher dose to exacerbate the toxicity of
PTL so that analogues with less toxicity to BM in comparison to
PTL could be measured. Doxorubicin (DOX) was included as a pos-
itive control since it is known to elicit BM toxicity.68 The mean
overall viability of the BM specimen utilized in our study was
(
LogD7.4; Sirius Analytical, average of two measurements) at pH 7.4 for select C1–
C10 PTL analogues
Compound
cLogD7.4
LogD7.4
PTL
1
2
3
4
3.07
1.97
3.07
3.48
3.16
4.22
1.79
2.18
2.00
2.30
2.29
2.90
+
-
7
8% (Fig. 3A) and 94% for the CD34 CD38 population (Fig. 3B).
Treatment with 0.5 M DOX for 12 h resulted in a 56% reduction
in total BM viability and an 85% reduction in the primitive
l
CTL
+
-
CD34 CD38 BM population (Fig. 3). PTL treatment at 25 lM
resulted in a 48% reduction in total BM viability, whereas C1–C10
modified PTL analogues 1, MelB, and 4, as well as control analogue,
CTL, were less toxic (range: 22–26% average reduction of total BM
viable cells). PTL was found to elicit no significant toxicity to prim-
(
cLogD7.4 = 3.07 vs LogD7.4 = 1.79) and cis-PTL isomer
2
(cLogD7.4 = 3.07 vs LogD7.4 = 2.00), whereas calculated and
measured values were generally consistent for the remaining
analogues (Table 1). Both reduction and cyclopropanation of the
C1–C10 olefin increased the overall lipophilicity of the PTL
skeleton (LogD7.4 = 2.30 for 3 and LogD7.4 = 2.29 for 4). CTL, which
contains a C4–C5 olefin in place of the epoxide in PTL, was sub-
stantially more lipophilic (LogD7.4 = 2.90 for CTL vs LogD7.4 = 1.79
for PTL). The distribution coefficients of dimethylamine fumarate
salts of PTL and 4, analogues DMAPT and 5, respectively,
were not measured because their calculated LogD values
+
-
itive CD34 CD38 BM cells at 25 lM dose and the C1–C10 PTL ana-
logues were similarly non-toxic at the same concentration
(Fig. 3B). Therefore, modification to the C1–C10 olefin of PTL signif-
icantly lowers its overall toxicity to BM cells. However, the
observed toxicity of PTL to total BM would be expected to be tran-
+
-
sient since little cell death was measured in the CD34 CD38 BM
population upon PTL treatment, which is responsible for BM clonal
6
6,67
growth.
Studies in our group using PTL prodrug, DMAPT, have
(
cLogD7.4 = 0.50, DMAPT; cLogD7.4 = 0.56, 5) were outside the
revealed no measurable toxicity to mice upon continuous oral dos-
ing (100 mg/kg, daily) for over four weeks.
6
4
measurable range of the assay (1–5 units).
2
.3. Cellular cytotoxicity screening
2.5. Inhibition of drug-resistant AML and toxicity to LSCs
All compounds were screened for anti-proliferative activity
against 7 cell lines representing blood lineage cancers (HL-60 and
Given the selectivity of our compounds for inhibiting growth of
blood lineage cancer cells (e.g., HL-60 and CCRF-CEM, Table 2) and
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037

4
A. M. Kempema et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
Growth inhibitory activities (IC50) of PTL, C1–C10-modified PTL analogues, and related probes to cell lines: HL-60 (acute promyelocytic leukemia), CCRF-CEM (acute
lymphoblastic leukemia), U-87 MG (glioblastoma multiforme), GBM6 (glioblastoma multiforme), MCF-7 (breast adenocarcinoma), DU-145 (prostate cancer), and NCI/ADR-RES
(ovarian cancer; adriamycin-resistant), IC50 values are mean ± SD in
l
M (n P3 analyses)
Compound
HL-60
CCRF-CEM
U-87 MG
GBM6
MCF-7
DU-145
NCI/ADR-RES
9.3 ± 3.8a
7.1 ± 0.4
9.2 ± 2.2
8.0 ± 1.1
7.5 ± 3.0
9.0 ± 2.1
4.4 ± 1.3
6.5 ± 2.7
>500
4.7 ± 1.6
1.9 ± 0.4
2.7 ± 1.1
2.2 ± 1.0
5.5 ± 1.2
2.5 ± 2.1
2.0 ± 0.6
2.9 ± 0.6
>500
8.8 ± 2.1
8.8 ± 1.9
a
3.4 ± 1.1
3.5 ± 1.1
8.5 ± 0.7
3.3 ± 0.8
4.5 ± 1.9
2.0 ± 0.3
2.3 ± 0.5
3.2 ± 0.7
>500
a
a
9.7 ± 2.8
10.4 ± 1.2
9.6 ± 1.0
7.5 ± 0.8
9.5 ± 1.9
20.1 ± 1.3
14.1 ± 1.0
16.9 ± 2.0
>500
8.9 ± 4.6a
8.4 ± 4.5
14.8 ± 4.0
6.0 ± 4.2
14.3 ± 5.9
5.5 ± 1.5
14.8 ± 6.7
13.3 ± 2.2
>500
11.4 ± 2.4b
12.3 ± 2.7
22.0 ± 5.3
9.4 ± 2.1
15.0 ± 4.9
15.6 ± 3.1
12.0 ± 2.5
11.7 ± 3.1
>500
PTL
LC-1
1
a
17.8 ± 5.0
9.1 ± 4.4
16.3 ± 6.8
7.5 ± 0.4
11.6 ± 1.4
10.5 ± 0.9
>500
2
MelB
3
4
5
6
CTL
13.0 ± 0.2
2.3 ± 0.2
9.6 ± 0.8
7.0 ± 1.8
17.5 ± 3.7
7.7 ± 2.8
17.1 ± 0.9
a
Obtained previously.58,64
Slightly lower than previously reported (57.6
b
M).58
l
B117H and B140H by loss-of-function mutations in the deoxycy-
tidine kinase gene Dck, which inhibits intracellular metabolism of
0
72
cytarabine to its 5 -monophosphate. PTL and C1–C10-modified
analogues 1, MelB, and 4, and control analogue CTL all inhibited
the growth of this panel of cell lines with low micromolar activity
(
IC50 range: 1.1–13.5 lM). No loss in potency was observed
between cytarabine-sensitive (parental) cell lines B117P and
B140P and cytarabine-resistant lines B117H and B140H for any
of the molecules tested. These data suggest that PTL analogues
have the potential to sustain anti-proliferative activities to AML
cell lines that become sensitive to cytarabine.
6
PTL is known to eradicate LSCs, and therefore, we investigated
if the C1–C10-modified PTL analogues could inhibit LSCs with sim-
ilar potency as the parent natural product. We utilized an engi-
neered leukemia cell line, TEX, for these assays. TEX cells are
ꢁ
derived from lineage depleted (Lin ) human cord blood cells trans-
duced with the fusion gene TLS-ERG. TEX cells effectively mimic
human AML by maintaining the potential for multi-lineage differ-
entiation through their heterogeneous population of cells with
hierarchical growth properties. A large population of primitive
+
CD34 cells are present in the TEX model system, which has also
been utilized in high-throughput screening for small molecule
7
3,74
inhibitors of LSCs.
MelB, 4, and CTL) revealed broad-spectrum inhibitory activity
IC50 range: 2.7–6.8 M) by metabolic viability staining following
8 h treatment (Table 4). Analysis of the LSC-enriched
Screening of PTL and related analogues (1,
(
4
l
+
-
CD34 CD38 population of TEX cells treated with 25 lM PTL, 1,
MelB, and CTL for 12 h revealed a nearly complete reduction in
cellular viability by flow cytometry analysis (cell viability range:
1
7
1
–10%, Fig. 4), with the majority of cells staining positive for
-AAD, indicating necrotic cell death. Treatment of cells with
5 lM PTL analogues yielded slightly higher amounts of viable
Figure 3. Toxicity of PTL and analogues to human bone marrow cells. BM was
dosed with DOX (0.5 M), and PTL, 1, MelB, 4, and CTL (25 M) for 12 h. Cellular
l
l
cells (cell viability range: 10–25%) with no statistical significant
differences in potencies between the analogues tested. A relatively
low dose of DOX (0.5 lM) was sufficient to reduce the viability of
CD34 CD38 TEX cells to 6%. Consequently, all of the PTL analogues
viability was then measured by flow cytometry and viable cells (%) were those not
stained by Annexin V (apoptosis) and 7-AAD (necrosis) reagents. (A) Viability of the
+
-
total bone marrow cell population and (B) viability of the CD34 CD38 population.
+
-
Data is the mean (n P3 analyses) ± SD. *p = 0.05, p 60.01, p 60.001 in compar-
**
***
ison to untreated control (U).
tested were able to induce cell death in the LSC-enriched
their lack of toxicity to healthy BM (Fig. 3), we focused subsequent
efforts on characterizing the anti-leukemic activities of our mole-
cules. Four murine AML cell lines were utilized for our initial
screen (Table 3). B117P and B140P are murine cell lines isolated
from the BXH-2 mice strain that spontaneously develops AML
due to the presence of a murine leukemia virus.69 These cells are
sensitive to cytarabine (AraC), which is used in standard-of-care
AML therapy. Continual low dosing of B117P and B140P with
cytarabine resulted in cytarabine-resistant cell lines B117H and
Table 3
Growth inhibitory activities (IC50) of PTL, C1–C10-modified PTL analogues, and
related probes to murine AML cell lines B117P, B117H, B140P, and B140H. IC50 values
are mean ± SD in
l
M (n P3 analyses)
Compound
B117P
B117H
B140P
B140H
PTL
1
MelB
4
1.1 ± 0.2
6.4 ± 1.0
2.1 ± 0.8
2.9 ± 0.6
3.5 ± 0.4
4.7 ± 1.6
8.8 ± 1.7
2.5 ± 1.0
6.4 ± 2.1
4.8 ± 0.7
5.8 ± 2.3
10.0 ± 0.4
2.9 ± 1.0
13.5 ± 2.3
3.6 ± 1.2
3.4 ± 1.1
9.3 ± 1.5
2.2 ± 0.7
5.9 ± 2.4
2.8 ± 0.7
7
0,71
CTL
B140H, respectively.
Cytarabine resistance is conferred in
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037

A. M. Kempema et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 4
Growth inhibitory activities (IC50) of PTL, C1–C10-modified PTL analogues, and
Table 5
Clonal growth assay with TEX cells
related probes to TEX cells. IC50 values are mean ± SD in
l
M (n P3 analyses)
Compound
Colonies
Compound
TEX
2.5
l
M
15 lM
PTL
1
MelB
4
2.8 ± 0.2
PTL
1
MelB
4
2.6 ± 2.7
4.2 ± 2.5
5.3 ± 1.6
7.0 ± 5.1
6.9 ± 3.6
N.D.
N.D.
0.1 ± 0.3
0.6 ± 0.9
5.6 ± 4.7
2.7 ± 0.4
6.8 ± 2.3
3.8 ± 0.2
4.9 ± 0.4
CTL
CTL
0.1
lM
0.5 lM
DOX
AraC
DMSO
0.7 ± 1.3
2.6 ± 1.0
20.8 ± 9.0
N.D.
N.D.
+
ꢁ
CD34 CD38 population of TEX cells. To corroborate that the
+
-
observed inhibition of primitive CD34 CD38 TEX cells also affects
the LSC population, we performed methylcellulose clonal growth
assays of TEX cells in the presence of our compounds. As previously
mentioned, AML is characterized by hierarchical growth properties
that originate from LSCs,7 and therefore, small molecule inhibi-
tion of LSCs will prevent clonal growth of cells in methylcellulose.
Control (DMSO) treated TEX cells yield on average 20.8 clones per
assay (Table 5). Treatment with PTL or analogues 1, MelB, 4, or CTL
TEX cells were plated 6000 cells/well and dosed with DMSO (0.05%, control wells)
or compounds at the concentrations noted. Values are the mean number of
colonies ± SD (3 biological replicates) observed after 11 d growth on methylcellu-
lose. N.D., clonal growth not detected. p 60.001 in comparison to DMSO control for
,8
all samples, except 4 at 2.5 lM (p 60.01).
all potently inhibit clonal outgrowth of TEX cells at 15
with PTL and 1 yielding no measurable clones. Decreasing the
dosage to 2.5 M with the same compounds also elicits inhibition
lM dose,
l
of TEX clonal growth (range: 2.6–7.0 clone average). DOX and AraC
were both able to inhibit TEX clonal growth in our assay, with
0.5 lM treatment of both compounds completely inhibiting cell
growth. Taken together, these data demonstrate PTL and related
analogues are proficient at inhibiting LSCs, which is likely medi-
ated through their common pharmacophore, the
butyrolactone.
a-methylene-c-
2
.6. Induction of reactive oxygen species
The mechanism by which PTL eradicates cancer cell viability is
an area of substantial debate. PTL has been shown to affect a vari-
ety of cellular processes, including (among many others) inhibition
of NF-jB signaling and microtubule detyrosination, reduction in
DNA methylation, and induction of cellular ROS (reviewed in
Figure 5. Intracellular ROS induction by PTL and analogues. TEX cells were treated
with hydrogen peroxide H (100 M) and PTL, 1, MelB, 4, CTL (100, 25 M) and ROS
activity was measured by flow cytometry using CellROX Green reagent. The median
l
l
Refs. 1,2,4,5,75,76). Multiple studies have implicated ROS induc-
fluorescence intensity (MFI) of each sample was normalized to the untreated
4
0,43,46,47
*
tion as a mechanism of PTL-mediated cancer cell death.
control and averaged. Values are mean MFI ± SD (n P3 analyses). p = 0.05,
**
***
p 60.01, p 60.001 in comparison to untreated control.
Consequently, we measured changes in intracellular ROS in TEX
cells resulting from treatment with PTL, 1, MelB, 4, and CTL to rank
order the pharmacological utility of our compounds. Treatment of
TEX cells with hydrogen peroxide results in a 3.1-fold induction of
intracellular ROS after 30 min, which is consistent with a previous
study where a similar induction of ROS was measured by flow
7
7
cytometry in HL-60 cells. Dosage of TEX cells with 1 (2.9-fold),
(2.6-fold), and CTL (2.2-fold) all substantially induced ROS levels
at 100 M dose in comparison to untreated control (Fig. 5). PTL
4
l
induced ROS as well, but to a lower level (1.6-fold). No observable
induction of ROS was detected with MelB at either concentration
tested. Decreasing the concentration of compounds to 25 lM also
resulted in a significant induction of ROS levels for 1 (1.7-fold)
and 4 (1.6-fold). Consequently, these data suggest that 1 and 4
more potently induce ROS than parent natural product, PTL.
3
. Conclusions
A small library of C1–C10 PTL analogues was synthesized and
evaluated for anti-proliferative activity to cancer cells, toxicity to
healthy BM, ability to inhibit drug-resistant AML and target LSCs,
and proficiency at inducing intracellular ROS. All compounds with
the exception of 6 were capable of inducing cancer cell death with
low micromolar potency. However, Micheliolide (1) and cyclo-
propane 4 were found to inhibit the growth of drug-resistant
+
ꢁ
Figure 4. Cellular viability (%) of CD34 CD38 TEX cells treated with DOX (2.5, 0.5,
.05 M) and PTL, 1, MelB, 4, CTL (25, 15, 2.5 M). Viable cells were those not
stained by Annexin (apoptosis) and 7-AAD (necrosis) markers. Values are
0
l
l
V
*
**
***
mean ± SD (n P5 analyses). p = 0.05, p 60.01,
p 60.001 in comparison to
untreated control (U).
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037

6
A. M. Kempema et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
AML and eliminate LSCs similarly to PTL, but offer the advantages
of being less toxic to healthy BM and more potently activating ROS
in AML cells than PTL. Additionally, elaboration of 4 to its dimethy-
lamine congener 5 provided an analogous prodrug to DMAPT (LC-
(Costar 3596, Corning, Inc.). Assays were conducted in biological
triplicate and IC50 values are the mean ± SD.
4.6. Bone marrow cell culture
1
). Given the continued interest in PTL, highlighted by its first total
1
4
synthesis, and the rekindled popularity of covalent drugs in gen-
eral,
Frozen human mononuclear bone marrow cells were purchased
from AllCells (Cat. #ABM011F). These bone marrow cells were
from two donors (#5630 [Lot #BM4565] and #4887 [Lot
#BM4118]). The cells were thawed according to vendor instruc-
tions and then cultured in StemSpan SFEM (STEMCELL
Technologies, Inc.) media supplemented with StemSpan CC100
cytokine cocktail (STEMCELL Technologies, Inc.) in a humidified
7
8,79
C1–C10 modifications such as cyclopropanation may be
useful for optimizing PTL and related germacranolides for thera-
peutic applications.
4
4
. Material and methods
5
2
% CO environment at 37 °C in tissue culture flasks (Corning)
.1. LogD measurements
under normoxic conditions.
Calculated LogD values were obtained using MarvinSketch
3
4.7. Flow cytometry analysis of cytotoxicity in bone marrow and
TEX cells
(
ChemAxon). cLogD7.4 was calculated using 0.1 mol/dm elec-
-
+
+
trolyte concentrations (Cl , Na , K ) at pH 7.4. Experimental
LogD7.4 measurements were performed by Sirius Analytical. The
LogD7.4 of each sample was determined using the LDA (liquid–liq-
uid distribution chromatography) method. The data is the average
of two measurements.
Human bone marrow or TEX cells were plated in their respec-
6
tive media at 1 ꢂ 10 cells/mL (1 mL/well) in a 24-well plate for-
mat (Corning). Cells were dosed with compounds or 1%
DMSO/media and incubated for 12 h at 37 °C and 5% CO
normoxic conditions. The final DMSO concentration was 0.03%
v/v) per well. After 12 h of incubation, each sample was trans-
2
under
4
.2. Preparation of stock solutions
(
ferred into FACS tubes and centrifuged for 5 min at 800 rpm. The
supernatant was decanted and each sample was washed with cold
Compound stock solutions were prepared in DMSO (20–
1
00 mM concentrations) and stored at ꢁ20 °C when not in use.
1
X PBS (1 mL) and centrifuged again. After centrifugation, the
Compound purities were assessed frequently by analytical
reverse-phase HPLC analysis and fresh solutions were prepared
as needed.
supernatant was decanted and the samples were stained with
Brilliant Violet 421 mouse anti-human CD34 (BD Biosciences
[
(
Cat. #562577]; 5
BD Biosciences [Cat. #555462]; 20
l
L/sample) and APC mouse anti-human CD38
L/sample) antibodies in
L total vol-
l
4
.3. Cell culture
FACS buffer (1X PBS, 2% FBS, 0.1% sodium azide; 100
l
ume/sample) for 10 minutes at 4 °C. The cells were then diluted
with FACS buffer (1 mL) and centrifuged. The supernatant was dec-
anted and stained with Annexin V-FITC (BD Biosciences [Cat.
2
All cell lines were maintained in a humidified 5% CO environ-
ment at 37 °C in tissue culture flasks (Corning) under normoxic
conditions. Adherent cells were dissociated using either Trypsin-
EDTA solution (0.25%, Gibco) or TrypLE Express solution
#
5
556420]; 5
0]; 5 L/sample) in FACS buffer (100
for 10 minutes at room temperature in the dark. The samples were
diluted with FACS buffer (300 L), and kept on ice during analysis
l
L/sample) and 7-AAD (eBioscience [Cat. #00-6993-
l
lL total volume/sample)
(
Invitrogen). HL-60, CCRF-CEM, U-87 MG, GBM6, DU-145, and
5
8,64,80
NCI/ADR-RES cells were cultured as described previously.
l
MCF-7 cells (ATCC, HTB-22) were cultured in MEM media
Cellgro) supplemented with 10% FBS (Gibco), bovine insulin
by flow cytometry using a BD Biosciences LSR II flow cytometer.
(
(
4
Greater than 5 ꢂ 10 events were measured for each sample during
0.01 mg/mL, Sigma), penicillin (100 I.U./mL, ATCC), and strepto-
analysis. All antibodies and stains were stored at 4 °C in the dark.
3,74
mycin (100
l
g/mL, ATCC). TEX cells7
were cultured in IMDM
After data collection, each sample was processed using FlowJo
containing L-glutamine (Cellgro) supplemented with 15% FBS
Gibco), Stem Cell Factor (20 ng/mL, PeproTech), Interleukin-3
2 ng/mL, PeproTech), penicillin (100 I.U./mL, ATCC), and strepto-
(
5
Tree Star; v 7.6.5). The cell viability is expressed as a mean of 3–
biological replicates ± SD. Statistical significance was determined
(
(
using unpaired t-tests (GraphPad Prism v. 5.0). An example of the
data processing is shown in Supporting information.
mycin (100
l
g/mL, ATCC).
.4. Human cancer cell line cytotoxicity assays
Alamar blue cellular cytotoxicity assays and data analyses were
4
4.8. Colony growth assay
TEX cells were added to Methocult H4230 (STEMCELL
Technologies Inc.) supplemented with penicillin (100 I.U./mL,
5
8,64,80
performed as previously described.
Suspension cell lines
3,74
(
1
HL-60, CCRF-CEM and TEX7 ) were seeded at a density of
ATCC) and streptomycin (100 lg/mL, ATCC) at a final cell density
4
0,000 cells/well in media (50
MG, GBM6, MCF-7, DU-145, and NCI/ADR-RES) were seeded at a
density of 5,000 cells/well in media (50 L) 24 h prior to treatment
lL) and adherent cell lines (U-87
of 1.2 ꢂ 10 cells/mL. Compounds were diluted in TEX cell media
and dosed to each cell suspension to obtain the respective concen-
tration. Each sample was vortexed vigorously to evenly distribute
the cells before and after compound dosing. Each sample
(1.5 mL) was plated into three wells (0.5 mL/well, three technical
replicates) of a 24-well plate (Corning) and incubated under nor-
l
with compounds in 96-well plates (Costar 3595, Corning, Inc.). IC50
values (n P3 biological replicates) are the mean ± SD.
4
.5. Murine cytotoxicity assay
moxic conditions at 37 °C, 5% CO for 11 days before scoring colo-
2
nies. The final DMSO concentration was 0.05% (v/v) per well.
Cell culture and cytotoxicity assays with murine cell lines
Colonies were counted for each well at 10ꢂ magnification with a
light microscope by two people independently and averaged for
each sample. The data is the mean number of colonies for three
biological replicate ± SD. Statistical significance was determined
using unpaired t-tests (GraphPad Prism v. 5.0).
B117P, B117H, B140P, and B140H were performed as previously
7
1,72
described.
6,000 and 44,000 cells/well for B117P, B117H, B140P, and
B140H cell lines, respectively, in media (200 L) in 96-well plates
Cells were seeded at a density of 25,000, 28,000,
3
l
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037

A. M. Kempema et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4
.9. ROS Assay
mixture was dissolved in THF (3 mL) and iodomethane was added
in excess (0.10 mL, 1.60 mmol). The reaction was allowed to stir at
RT for 2 h. The solvent and excess iodomethane were removed in
vacuo resulting in a white solid. Water (10 mL) was added and
the reaction was heated to 45 °C. Complete solvation of the yellow-
ish material resulted within minutes of heating. The reaction was
allowed to stir with heating for 3 h, and then solvent was removed
5
TEX cells were seeded in 24-well plates at 5 ꢂ 10 cells/mL
1mL per well) and incubated overnight at 37 °C and 5% CO . The
M), includ-
(
2
cells were then treated with compounds (25 and 100
l
2 2
ing H O (100 lM; positive control). The final DMSO concentration
was 0.25% (v/v) per well. Immediately after treatment with com-
pounds, CellROX Green (Invitrogen) reagent was added to the
3
in vacuo. Aqueous NaHCO (sat’d, 5 mL) was added to the reaction
appropriate samples at a final concentration of 5
were then incubated for 30 min at 37 °C and 5% CO
l
2
M. The cells
under nor-
mixture, and the product was extracted with DCM (3 ꢂ 20 mL). The
combined organic layers were washed with brine (20 mL), and
moxic conditions. Following incubation, the samples were trans-
ferred to 5 mL FACS tubes and washed twice with FACS buffer
dried with Na
raphy over SiO
3
3 as a white solid (0.014 g, 32%). H NMR (CDCl , 500 MHz): d 6.24
2
SO
4
. The reaction was purified by flash chromatog-
2
(10–50% ethyl acetate in hexanes gradient) to yield
1
(
3mL; FACS buffer: 1X PBS, 2% FBS, 0.1% sodium azide). The sam-
ples were run using a BD Biosciences LSR II flow cytometer and
(d, J = 2.8 Hz 1H), 5.53 (d, J = 2.4 Hz, 1H), 3.84 (t, J = 7.6 Hz, 1H),
3.10 (d, J = 7.6 Hz, 1H), 2.99–2.94 (m, 1H), 2.20–2.14 (m, 2H),
1.81–1.75 (m, 2H), 1.75–1.56 (m, 2H), 1.51 (s, 3H), 1.51–1.40 (m,
4
5
ꢂ 10 events were recorded for each sample. Flow cytometry
data was analyzed using FlowJo software (Tree Star; version
.6.5). Samples were run in quadruplicate with the exception of
(triplicate data). Median fluorescence intensity (MFI) values
7
2H), 1.26 (m, 2H), 1.17–1.14 (m, 2H), 0.93 (d, J = 4.8 Hz, 3H). 13
C
2
H O
2
NMR (CDCl
3
, 125 MHz): 169.7, 139.5, 119.7, 81.0, 66.4, 61.3, 43.9,
+
were obtained for each sample and were normalized to the
untreated control. Data are shown as mean MFI value ± SD.
Statistical significance was determined using unpaired t-tests
36.7, 36.1, 30.1, 27.9, 24.7, 21.3, 20.6, 19.2. HRMS (ESI ) m/z calc’d
for [C15H O +Na] 273.1461; found 273.1470. The structure of 3
22 3
+
was further confirmed by small molecule X-ray crystallography
(SI; CCDC 1033013).
(
GraphPad Prism v. 5.0).
4
.10. General synthesis information
4.10.2. Cyclopropane 4
A 0.20 M solution of Zn(CH
following manner: To a stirred solution of diethyl zinc (1.0 M solu-
tion in hexanes, 4.0 mL, 4.00 mmol) in CH Cl (20 mL) and DME
(0.50 mL) at 0 °C was added diiodomethane (0.80 mL, 9.92 mmol)
under N . The mixture was stirred for 10 minutes. PTL (0.090 g,
0.36 mmol) in CH Cl (3 mL) was added dropwise over 10 min to
the Zn(CH I)
ꢃDME complex at 0 °C. The reaction was allowed to
warm to rt over 12 h. The reaction was quenched with aqueous
NH Cl (sat’d, 5 mL) and extracted with CH Cl
(3 ꢂ 20 mL). The
combined organic layers were washed with aqueous NaHCO
(sat’d, 20 mL), brine (20 mL), dried over Na SO and concentrated
2
I)
2
ꢃDME complex was made in the
Chemical reagents were typically purchased from Sigma-
Aldrich and used without additional purification unless noted.
Bulk solvents were from Fisher Scientific. PTL was purchased from
Enzo Life Sciences and CTL was purchased from Santa Cruz
2
2
2
Biotechnology. Previously reported analogues DMAPT, MelB, 1, 2
2
2
and 6 were synthesized as described.1
8,21–26,28,29,32–35
The structure
2 2
of 1 was further confirmed by small molecule X-ray crystallogra-
5
5
phy (SI; CCDC 1033012) and compared to the previous report.
Tetrahydrofuran (THF) was rendered anhydrous by passing
through the resin column of solvent purification system
MBraun). Reactions were performed under an atmosphere of dry
4
2
2
3
a
2
4
(
N
in vacuo. The crude mixture was purified using silica gel
chromatography (gradient 10–30% EtOAc in hexanes over
2
unless noted. Silica gel chromatography was performed on a
Teledyne-Isco Combiflash Rf-200 instrument utilizing Redisep R
f
15 min) to yield 4 (0.036 g, 40%) as a colorless oil and recovered
1
Gold High Performance silica gel columns (Teledyne-Isco).
Analytical HPLC analysis was performed on an Agilent 1200 series
instrument equipped with a diode array detector and a Zorbax SB-
PTL (0.037 g, 41%).
3
H NMR (400 MHz, CDCl ) d: 6.28 (d,
J = 3.7 Hz, 1H), 5.57 (d, J = 3.3 Hz, 1H), 3.96 (t, J = 9.1 Hz, 1H), 2.98
(d, J = 9.0 Hz, 1H), 2.67 (m, 1H), 2.39 (dd, J = 8.0 Hz, J = 14.7 Hz,
1H), 2.19 (dd, J = 2.3 Hz, J = 8.3 Hz, 1H), 1.95 (m, 2H), 1.70 (m,
1H), 1.40 (s, 3H), 1.28 (m, 2H), 1.09 (s, 3H), 0.85 (dd, J = 11.1 Hz,
J = 14.7, 1H), 0.64 (td, J = 6.0 Hz, J = 9.5 Hz, 1H), 0.39 (dd,
C18 column (4.6 ꢂ 150 mm, 3.5
method started with 10% CH CN (with 0.1% trifluoroacetic acid
TFA)) in H O (0.1% TFA). The 10% CH CN (with 0.1% TFA) was
increased to 85% over 22 minutes, and then increased to 95%
CH CN (with 0.1% TFA) over 2 more minutes. Nuclear magnetic res-
onance (NMR) spectroscopy was employed by using either a
lm, Agilent Technologies). The
3
(
2
3
J = 4.3 Hz, J = 9.4 Hz, 1H), -0.08 (dd, J = 4.6 Hz, J = 5.6 Hz, 1H). 13
C
3
3
NMR (100 MHz, CDCl ) d: 169.4, 139.9, 120.5, 82.7, 65.5, 60.6,
48.0, 42.3, 38.4, 25.7, 24.5, 22.3, 20.4, 18.8, 18.5, 17.1. HRMS
1
13
+
+
Bruker Avance (400 MHz for H; 100 MHz for C) or Bruker
22 3
(ESI ) m/z calc’d for [C16H O +Na] 285.1461; found 285.1470.
1
13
Ascend (500 MHz for H; 125 MHz for C) NMR operating at ambi-
ent temperature. Chemical shifts are reported in parts per million
and normalized to internal solvent peaks or tetramethylsilane.
High-resolution masses were obtained from the University of
Minnesota Department of Chemistry Mass Spectrometry lab,
employing a Bruker BioTOF II instrument.
The structure of 4 was further confirmed by small molecule
X-ray crystallography (SI; CCDC 1033014).
4.10.3. Cyclopropyl-PTL Dimethylamine Fumarate 5
To a stirred solution of 4 (0.009 g, 0.034 mmol) in MeOH (2 mL)
was added dimethylamine (2.0 M in MeOH, 1.5 mL). The reaction
was stirred for 12 h at rt. The reaction mixture was concentrated
in vacuo purified by silica gel chromatography (gradient 0–50%
EtOAc in hexanes over 10 min, then gradient 0–25% MeOH in
4
.10.1. C1–C10 Reduced 3
To a stirred solution of PTL (0.050 g, 0.201 mmol) in MeOH
(
2 mL) was added dimethylamine (2.0 M in MeOH, 1 mL). The reac-
2 2
CH Cl over 10 min) to yield the dimethylamino product as a white
tion was allowed to stir at RT overnight and then concentrated in
vacuo. The crude material was used without further purification.
solid (0.009 g). To a stirred solution of this product in THF (5 mL)
was added fumaric acid (0.0034 g, 0.029 mmol). A white precipi-
tate was observed after stirring overnight at rt. The reaction mix-
The residual material was dissolved in EtOAc (3 mL) and PtO
0.005 g, 0.022 mmol) was added. The reaction mixture was
degassed, then shaken for 8 h in a Parr shaker under an atmosphere
of H (50 psi). The mixture was then degassed, filtered through
2
(
ture was concentrated in vacuo to give the fumarate salt 5 as
1
white solid (0.0124 g, 85%). H NMR (DMSO-d
6
, 400 MHz): d 6.61
2
(s, 2H), 4.09 (t, J = 9.5 Hz, 1H), 3.04 (d, J = 9.2 Hz, 1H), 2.64 (m,
3H), 2.24 (s, 6H), 2.14 (m, 2H), 2.05 (m, 1H), 1.78 (m, 2H), 1.59
(m, 1H), 1.31 (s, 3H), 1.19 (m, 2H), 1.02 (s, 3H), 0.74 (m, 2H),
celite, and concentrated in vacuo. The crude material was taken
on to the next step without further purification. The reaction
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037

8
A. M. Kempema et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
0
.28 (dd, J = 3.8 Hz, J = 9.2 Hz, 1H), -0.18 (t, J = 4.8 Hz, 1H). 13C NMR
, 100 MHz): 176.6, 166.1, 134.1, 81.8, 64.3, 60.3, 57.2,
7.4, 45.6, 45.3, 41.7, 38.0, 24.2, 24.0, 21.6, 20.1, 18.4, 18.3, 16.7.
14. Amorim, M. H. R.; Gil da Costa, R. M.; Lopes, C.; Bastos, M. M. S. M. Crit. Rev.
Toxicol. 2013, 43, 559.
(
DMSO-d
6
15. Curry, E. A., III; Murry, D. J.; Yoder, C.; Fife, K.; Armstrong, V.; Nakshatri, H.;
4
O’Connell, M.; Sweeney, C. J. Invest. New Drugs 2004, 22, 299.
+
+
HRMS (ESI ) m/z calc’d for [C18
3
3
H30NO +H] 308.2220; found
16. Sweeney, C. J.; Mehrotra, S.; Sadaria, M. R.; Kumar, S.; Shortle, N. H.; Roman, Y.;
Sheridan, C.; Campbell, R. A.; Murry, D. J.; Badve, S.; Nakshatri, H. Mol. Cancer
Ther. 2005, 4, 1004.
08.2216.
1
7. Guzman, M. L.; Rossi, R. M.; Neelakantan, S.; Li, X.; Corbett, C. A.; Hassane, D. C.;
Becker, M. W.; Bennett, J. M.; Sullivan, E.; Lachowicz, J. L.; Vaughan, A.;
Sweeney, C. J.; Matthews, W.; Carroll, M.; Liesveld, J. L.; Crooks, P. A.; Jordan, C.
T. Blood 2007, 110, 4427.
4
.11. Analysis of X-ray structures
The X-ray structures of 4 and PTL51 were analyzed for root-
mean-square deviation (RMSD) and graphics were rendered using
1
8. Neelakantan, S.; Nasim, S.; Guzman, M. L.; Jordan, C. T.; Crooks, P. A. Bioorg.
Med. Chem. Lett. 2009, 19, 4346.
8
1,82
19. Hejchman, E.; Haugwitz, R. D.; Cushman, M. J. Med. Chem. 1995, 38, 3407.
UCSF Chimera.
Thermal ellipsoids were drawn at the 50% prob-
20. Woods, J. R.; Mo, H.; Bieberich, A. A.; Alavanja, T.; Colby, D. A. MedChemComm
013, 4, 27.
ability level. RMSD was calculated for all non-hydrogen atoms.
2
2
1. Zhang, Q.; Lu, Y.; Ding, Y.; Zhai, J.; Ji, Q.; Ma, W.; Yang, M.; Fan, H.; Long, J.;
Tong, Z.; Shi, Y.; Jia, Y.; Han, B.; Zhang, W.; Qiu, C.; Ma, X.; Li, Q.; Shi, Q.; Zhang,
H.; Li, D.; Zhang, J.; Lin, J.; Li, L.-Y.; Gao, Y.; Chen, Y. J. Med. Chem. 2012, 55, 8757.
2. Nasim, S.; Crooks, P. A. Bioorg. Med. Chem. Lett. 2008, 18, 3870.
Acknowledgments
2
We thank Ezra Menon and Margaret Olson (University of
Minnesota, UMN) for assistance with anti-proliferative activity
experiments and Victor G. Young, Jr. of the Department of
Chemistry, X-ray Crystallographic Laboratory (UMN) for solving
the X-ray structures of 1, 3, and 4. We thank Professor Michael
Verneris (UMN) for assistance with flow cytometry analysis and
NIH P30 CA77598, which supports the Flow Cytometry shared
resource of the Masonic Cancer Center (UMN). Professor John
Dick (University of Toronto) is gratefully acknowledged for the
gift of the TEX cell line. GBM6 cells were a gift from the late
Professor John Ohlfest (UMN). This research was supported by
grants from the UMN, Academic Health Center (Seed Grant
23. Hwang, D.-R.; Wu, Y.-S.; Chang, C.-W.; Lien, T.-W.; Chen, W.-C.; Tan, U.-K.; Hsu,
J. T. A.; Hsieh, H.-P. Bioorg. Med. Chem. 2006, 14, 83.
2
2
4. Matsuda, H.; Toguchida, I.; Ninomiya, K.; Kageura, T.; Morikawa, T.; Yoshikawa,
M. Bioorg. Med. Chem. 2003, 11, 709.
5. Srivastava, S. K.; Abraham, A.; Bhat, B.; Jaggi, M.; Singh, A. T.; Sanna, V. K.;
Singh, G.; Agarwal, S. K.; Mukherjee, R.; Burman, A. C. Bioorg. Med. Chem. Lett.
2006, 16, 4195.
2
2
6. Castañeda-Acosta, J.; Fischer, N. H.; Vergas, D. J. Nat. Prod. 1993, 56, 90.
7. An, Y.; Guo, W.; Li, L.; Xu, C.; Yang, D.; Wang, S.; Lu, Y.; Zhang, Q.; Zhai, J.; Fan,
H.; Qiu, C.; Qi, J.; Chen, Y.; Yuan, S. PLoS One 2015, 10, e0116202.
8. Neukirch, H.; Guerriero, A.; D’Ambrosio, M. Eur. J. Org. Chem. 2003, 3969.
9. Neukirch, H.; Kaneider, N. C.; Wiedermann, C. J.; Guerriero, A.; D’Ambrosio, M.
Bioorg. Med. Chem. 2003, 11, 1503.
2
2
30. Long, J.; Zhang, S.-F.; Wang, P.-P.; Zhang, X.-M.; Yang, Z.-J.; Zhang, Q.; Chen, Y. J.
Med. Chem. 2014, 57, 7098.
3
1. Dell’Agli, M.; Galli, G. V.; Bosisio, E.; D’Ambrosio, M. Bioorg. Med. Chem. Lett.
009, 19, 1858.
32. Kwok, B. H. B.; Koh, B.; Ndubuisi, M. I.; Elofsson, M.; Crews, C. M. Chem. Biol.
001, 8, 759.
2
010.01); UMN, Leukemia Research Fund—Danny Thompson
2
Memorial Golf Tournament; Children’s Cancer Research Fund,
Minneapolis, MN; American Cancer Society (IRG-58-001-52-
IRG68); Hyundai Hope on Wheels, Hope Grant Award; The V
Foundation for Cancer Research, V Scholar Award to D.A.H.; and
startup funds to D.A.H. from the UMN. J.C.W. thanks the UMN,
College of Pharmacy for a Bighley Graduate Fellowship. D.W.
acknowledges the American Heart Association for a predoctoral
fellowship (11PRE7240035).
2
33. Macías, F. A.; Velasco, R. F.; Álvarez, J. A.; Castellano, D.; Galindo, J. C. G.
Tetrahedron 2004, 60, 8477.
34. Nasim, S.; Pei, S.; Hagen, F. K.; Jordan, C. T.; Crooks, P. A. Bioorg. Med. Chem.
2011, 19, 1515.
3
3
5. El-Feraly, F. S. Phytochemistry 1984, 23, 2372.
6. Janganati, V.; Penthala, N. R.; Madadi, N. R.; Chen, Z.; Crooks, P. A. Bioorg. Med.
Chem. Lett. 2014, 24, 3499.
37. Kolev, J. N.; O’Dwyer, K. M.; Jordan, C. T.; Fasan, R. ACS Chem. Biol. 2014, 9, 164.
3
8. Raj, L.; Ide, T.; Gurkar, A. U.; Foley, M.; Schenone, M.; Li, X.; Tolliday, N. J.;
Golub, T. R.; Carr, S. A.; Shamji, A. F.; Stern, A. M.; Mandinova, A.; Schreiber, S.
L.; Lee, S. W. Nature 2011, 475, 231.
Supplementary data
3
4
9. Schumacker, P. T. Cancer Cell 2006, 10, 175.
0. Skalska, J.; Brookes, P. S.; Nadtochiy, S. M.; Hilchey, S. P.; Jordan, C. T.; Guzman,
M. L.; Maggirwar, S. B.; Briehl, M. M.; Bernstein, S. H. PLoS One 2009, 4, e8115.
CCDC (1033012–1033014) contains the supplementary crystal-
lographic data for compounds 1, 3, and 4 in this paper. These data
can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
Supplementary data (NMR spectra, HPLC characterization data,
and additional details of biochemical assays and X-ray crystallo-
graphic structures) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.05.037.
41. Lagadinou, E. D.; Sach, A.; Callahan, K.; Rossi, R. M.; Neering, S. J.; Minhajuddin,
M.; Ashton, J. M.; Pei, S.; Grose, V.; O’Dwyer, K. M.; Liesveld, J. L.; Brookes, P. S.;
Becker, M. W.; Jordan, C. T. Cell Stem Cell 2013, 12, 329.
4
2. Diehn, M.; Cho, R. W.; Lobo, N. A.; Kalisky, T.; Dorie, M. J.; Kulp, A. N.; Qian, D.;
Lam, J. S.; Ailles, L. E.; Wong, M.; Joshua, B.; Kaplan, M. J.; Wapnir, I.; Dirbas, F.
M.; Somlo, G.; Garberoglio, C.; Paz, B.; Shen, J.; Lau, S. K.; Quake, S. R.; Brown, J.
M.; Weissman, I. L.; Clarke, M. F. Nature 2009, 458, 780.
43. Shanmugam, R.; Kusumanchi, P.; Cheng, L.; Crooks, P. A.; Neelakantan, S.;
Matthews, W.; Nakshatri, H.; Sweeney, C. J. Prostate 2010, 70, 1074.
44. Trachootham, D.; Alexandre, J.; Huang, P. Nat. Rev. Drug Discov. 2009, 8, 579.
45. Adams, D. J.; Boskovic, Z. V.; Theriault, J. R.; Wang, A. J.; Stern, A. M.; Wagner, B.
K.; Shamji, A. F.; Schreiber, S. L. ACS Chem. Biol. 2013, 8, 923.
References and notes
4
6. Pei, S.; Minhajuddin, M.; Callahan, K. P.; Balys, M.; Ashton, J. M.; Neering, S. J.;
Lagadinou, E. D.; Corbett, C.; Ye, H.; Liesveld, J. L.; O’Dwyer, K. M.; Li, Z.; Shi, L.;
Greninger, P.; Settleman, J.; Benes, C.; Hagen, F. K.; Munger, J.; Crooks, P. A.;
Becker, M. W.; Jordan, C. T. J. Biol. Chem. 2013, 288, 33542.
1
2
.
.
Merfort, I. Curr. Drug Targets 2011, 12, 1560.
Ghantous, A.; Gali-Muhtasiv, H.; Vuorela, H.; Saliba, N. A.; Darwiche, N. Drug
Discov. Today 2010, 15, 668.
47. Wen, J.; You, K.-R.; Lee, S.-Y.; Song, C.-H.; Kim, D.-G. J. Biol. Chem. 2002, 277,
38954.
3
4
.
.
Knight, D. W. Nat. Prod. Rep. 1995, 12, 271.
Ghantous, A.; Sinjab, A.; Herceg, Z.; Darwiche, N. Drug Discov. Today 2013, 18,
48. Jain, T. C.; Banks, C. M.; McCloskey, J. E. Tetrahedron 1976, 32, 765.
49. Lee, K.-H.; Furukawa, H.; Huang, E.-S. J. Med. Chem. 1972, 15, 609.
50. de Meijere, A. Angew. Chem., Int. Ed. Engl. 1979, 18, 809.
51. Long, J.; Ding, Y.-H.; Wang, P.-P.; Zhang, Q.; Chen, Y. J. Org. Chem. 2013, 78,
10512.
52. Furukawa, J.; Kawabata, N.; Nishimura, J. Tetrahedron Lett. 1968, 31, 3495.
53. Simmons, H. E.; Smith, R. D. J. Am. Chem. Soc. 1958, 80, 5323.
54. Simmons, H. E.; Smith, R. D. J. Am. Chem. Soc. 1959, 81, 4256.
55. Acosta, J. C.; Fronczek, F. R.; Fischer, N. H. Acta Crystallogr., Sect. C 1991, 47,
2702.
8
94.
5
6
7
.
.
.
Kreuger, M. R.; Grootjans, S.; Biavatti, M. W.; Vandenabeele, P.; D’Herde, K.
Anti-Cancer Drugs 2012, 23, 883.
Guzman, M. L.; Rossi, R. M.; Karnischky, L.; Li, X.; Peterson, D. R.; Howard, D. S.;
Jordan, C. T. Blood 2005, 105, 4163.
Lapidot, T.; Sirard, C.; Vormoor, J.; Murdoch, B.; Hoang, T.; Caceres-Cortes, J.;
Minden, M.; Paterson, B.; Caligiuri, M. A.; Dick, J. E. Nature 1994, 367, 645.
Hope, K. J.; Jin, L.; Dick, J. E. Nat. Immunol. 2004, 5, 738.
Crews, L. A.; Jamieson, C. H. M. Cancer Lett. 2013, 338, 15.
8
9
.
.
1
1
1
1
0. Jordan, C. T. Sci. Transl. Med. 2010, 2, 31ps21.
1. Dick, J. E. Blood 2008, 112, 4793.
2. Horton, S. J.; Huntly, B. J. P. Haematologica 2012, 97, 966.
3. Guzman, M. L.; Allan, J. N. Stem Cells 2014, 32, 844.
56. Batist, G.; Tulpule, A.; Sinha, B. K.; Katki, A. G.; Myers, C. E.; Cowan, K. H. J. Biol.
Chem. 1986, 261, 15544.
57. Ke, W.; Yu, P.; Wang, J.; Wang, R.; Guo, C.; Zhou, L.; Li, C.; Li, K. Med. Oncol.
2011, 28, S135.
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037

A. M. Kempema et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
5
5
6
6
6
8. Wen, B.; Hexum, J. K.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Org. Lett.
013, 15, 2644.
9. Hirschmann-Jax, C.; Foster, A. E.; Wulf, G. G.; Nuchtern, J. G.; Jax, T. W.; Gobel,
U.; Goodell, M. A.; Brenner, M. K. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14228.
0. Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Nat. Rev.
Cancer 2013, 13, 714.
1. Sotiropoulou, P. A.; Christodoulou, M. S.; Silvani, A.; Herold-Mende, C.;
Passarella, D. Drug Discov. Today 2014, 19, 1547.
2. Swaminathan, S. K.; Olin, M. R.; Forster, C. L.; Cruz, K. S.; Panyam, J.; Ohlfest, J.
R. J. Immunol. Methods 2010, 361, 110.
71. Yin, B.; Kogan, S. C.; Dickins, R. A.; Lowe, S. W.; Largaespada, D. A. Exp. Hematol.
2006, 34, 631.
72. Rathe, S. K.; Moriarity, B. S.; Stoltenberg, C. B.; Kurata, M.; Aumann, N. K.;
Rahrmann, E. P.; Bailey, N. J.; Melrose, E. G.; Beckmann, D. A.; Liska, C. R.;
Largaespada, D. A. Sci. Rep. 2014, 4, 6048.
73. McDermott, S. P.; Eppert, K.; Notta, F.; Isaac, M.; Datti, A.; Al-awar, R.; Wrana,
J.; Minden, M. D.; Dick, J. E. Blood 2012, 119, 1200.
74. Warner, J. K.; Wang, J. C. Y.; Takenaka, K.; Doulatov, S.; McKenzie, J. L.;
Harrington, L.; Dick, J. E. Leukemia 2005, 19, 1794.
75. Mathema, V. B.; Koh, Y.-S.; Thakuri, B. C.; Sillanpää, M. Inflammation 2012, 35,
560.
76. Paj a˛ k, B.; Orzechowski, A.; Gajkowska, B. Folia Histochem. Cytobiol. 2008, 46,
129.
2
6
6
3. Andrews, T. E.; Wang, D.; Harki, D. A. Drug. Deliv. Transl. Res. 2013, 3, 121.
4. Hexum, J. K.; Becker, C. M.; Kempema, A. M.; Ohlfest, J. R.; Largaespada, D. A.;
Harki, D. A. Bioorg. Med. Chem. Lett. 2015, 25, 2493.
6
6
5. Kitson, R. R. A.; Millemaggi, A.; Taylor, J. K. Angew. Chem., Int. Ed. 2009, 48, 9426.
6. Bhatia, M.; Wang, J. C. Y.; Kapp, U.; Bonnet, D.; Dick, J. E. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 5320.
7. Larochelle, A.; Vormoor, J.; Hanenberg, H.; Wang, J. C. Y.; Bhatia, M.; Lapidot, T.;
Moritz, T.; Murdoch, B.; Xiao, X. L.; Kato, I.; Williams, D. A.; Dick, J. E. Nat. Med.
77. Tartier, L.; McCarey, Y. L.; Biaglow, J. E.; Kochevar, I. E.; Held, K. D. Cell Death
Differ. 2000, 7, 1002.
78. Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. Nat. Rev. Drug Disc. 2011, 10, 307.
79. Potashman, M. H.; Duggan, M. E. J. Med. Chem. 2009, 52, 1231.
80. Hexum, J. K.; Tello-Aburto, R.; Struntz, N. B.; Harned, A. M.; Harki, D. A. ACS
Med. Chem. Lett. 2012, 3, 459.
6
6
1
996, 2, 1329.
8. Sundman-Engberg, B.; Tidefelt, U.; Paul, C. Cancer Chemother. Pharmacol. 1998,
2, 17.
81. Meng, E. C.; Pettersen, E. F.; Couch, G. S.; Huang, C. C.; Ferrin, T. E. BMC
Bioinform. 2006, 7, 339.
4
6
7
9. Bedigian, H. G.; Taylor, B. A.; Meier, H. J. Virol. 1981, 39, 632.
0. Largaespada, D. A.; Shaughnessy, J. D., Jr.; Jenkins, N. A.; Copeland, N. G. J. Virol.
82. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.;
Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605.
1
995, 69, 5095.
Please cite this article in press as: Kempema, A. M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.037