Discovery of potent parthenolide-based antileukemic agents enabled by late-stage P450-mediated C - H functionalization

Article abstract of DOI:10.1021/cb400626w

The sesquiterpene lactone parthenolide has recently attracted considerable attention owing to its promising antitumor properties, in particular in the context of stem-cell cancers including leukemia. Yet, the lack of viable synthetic routes for re-elaborating this complex natural product has represented a fundamental obstacle toward further optimization of its pharmacological properties. Here, we demonstrate how this challenge could be addressed via selective, late-stage sp³ C-H bond functionalization mediated by P450 catalysts with tailored site-selectivity. Taking advantage of our recently introduced tools for high-throughput P450 fingerprinting and fingerprint-driven P450 reactivity prediction, we evolved P450 variants useful for carrying out the highly regioselective hydroxylation of two aliphatic sites (C9 and C14) in parthenolide carbocyclic backbone. By chemoenzymatic synthesis, a panel of novel C9- and C14-modified parthenolide analogs were generated in order to gain initial structure-activity insights on these previously inaccessible sites of the molecule. Notably, some of these compounds were found to possess significantly improved antileukemic potency against primary acute myeloid leukemia cells, while exhibiting low toxicity against normal mature and progenitor hematopoietic cells. By identifying two 'hot spots' for improving the anticancer properties of parthenolide, this study highlights the potential of P450-mediated C-H functionalization as an enabling, new strategy for the late-stage manipulation of bioactive natural product scaffolds.

Full text of DOI:10.1021/cb400626w

Articles
pubs.acs.org/acschemicalbiology
Discovery of Potent Parthenolide-Based Antileukemic Agents
Enabled by Late-Stage P450-Mediated CH Functionalization
Joshua N. Kolev,† Kristen M. O’Dwyer,‡ Craig T. Jordan,‡,§ and Rudi Fasan*,†
†Department of Chemistry, University of Rochester, Rochester, New York 14627, United States of America
‡Department of Hematology/Oncology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14627,
United States of America
S
* Supporting Information
ABSTRACT: The sesquiterpene lactone parthenolide has recently attracted considerable attention owing to its promising
antitumor properties, in particular in the context of stem-cell cancers including leukemia. Yet, the lack of viable synthetic routes
for re-elaborating this complex natural product has represented a fundamental obstacle toward further optimization of its
pharmacological properties. Here, we demonstrate how this challenge could be addressed via selective, late-stage sp3 C−H bond
functionalization mediated by P450 catalysts with tailored site-selectivity. Taking advantage of our recently introduced tools for
high-throughput P450 fingerprinting and fingerprint-driven P450 reactivity prediction, we evolved P450 variants useful for
carrying out the highly regioselective hydroxylation of two aliphatic sites (C9 and C14) in parthenolide carbocyclic backbone. By
chemoenzymatic synthesis, a panel of novel C9- and C14-modified parthenolide analogs were generated in order to gain initial
structure−activity insights on these previously inaccessible sites of the molecule. Notably, some of these compounds were found
to possess significantly improved antileukemic potency against primary acute myeloid leukemia cells, while exhibiting low toxicity
against normal mature and progenitor hematopoietic cells. By identifying two ‘hot spots’ for improving the anticancer properties
of parthenolide, this study highlights the potential of P450-mediated C−H functionalization as an enabling, new strategy for the
late-stage manipulation of bioactive natural product scaffolds.
particularly relevant toward the development of more effective
treatments for AML and other hematologic malignancies. In
addition, PTL was found to possess notable antiproliferative
properties against many other types of human cancers,
including breast,14,15 lung,16 prostate,17 liver,18,19 brain,20
pancreas,21 and bone22 cancer.
The anticancer properties of parthenolide has been primarily
associated to its ability to inhibit the transcription factor NF-
κB,23−25 which is known to control multiple tumor-related
processes such as inflammation, proliferation, angiogenesis, and
metastasis.4 However, additional mechanisms have been
recently found to lie at the basis of the pharmacological effect
of this molecule in cancer cells, which include activation of
The past decade has witnessed an increasing interest for the
natural product class of sesquiterpene lactones, a group of
plant-derived 15-carbon terpenoids arising from the biosyn-
thetic assembly of three isoprene units and containing a lactone
ring either cis- or trans-fused to the carbocyclic skeleton.1,2
Among them, parthenolide (1, PTL, Scheme 1) has attracted
particular attention owing to its promising potential as an
anticancer agent.3,4 In recent studies, PTL was found capable of
inducing robust apoptosis in primary acute myelogenous
leukemia (AML) cells, proving to be equally effective among
all subpopulations within primary AML specimens, including
the so-called leukemia stem cells (LSCs).5−7 LSCs are believed
to play a crucial role not only in the genesis of AML8,9 but also
in the clinical relapse of AML patients following traditional
chemotherapy10 due to their reduced responsiveness to
chemotherapeutic agents that kill actively cycling cells.11−13
Thus, the LSC-targeting ability of PTL makes this molecule
Received: August 22, 2013
Accepted: October 22, 2013
© XXXX American Chemical Society
A
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Scheme 1. Chemical Structure of Parthenolide (1, PTL) and of the Oxidation Products Obtained from Reaction with P450BM3
a
Variant FL#62
a
Product distribution: 77% 2; 13% 3; 10% 4. X-ray crystal structures of 163 and 2 (this study) are shown.
p535,26 and proapoptotic Bcl-2 proteins,27 induction of
oxidative stress,18,28,29 and alteration of epigenetic mecha-
nisms.30,31
rapidly generating a panel of engineered P450 variants, derived
from the bacterial, catalytically self-sufficient CYP102A1 (B.
megaterium),50 for the highly regio- and stereoselective
oxidative activation of two sp3 C−H sites (C9 and C14) as
well as of the C1,C10 double bond in PTL carbocyclic skeleton.
Using these catalysts, a series of novel, C9- and C14-substituted
parthenolide derivatives were made available by chemo-
enzymatic synthesis for activity evaluation in assays with
primary AML specimens. Importantly, these studies demon-
strate that both the C9 and C14 positions constitute ‘hot spots’
for improving the antileukemic potency of parthenolide, while
granting high selectivity against malignant cells over normal
mature and progenitor hematopoietic cells.
Owing to the promising anticancer properties of PTL, and in
particular to its ability to target cancer stem cells, there is
currently a high interest in re-elaborating this natural product
scaffold to obtain derivatives with enhanced potency and
improved drug-like properties. Previous efforts in this direction
have taken advantage of the reactivity of the α-methylene-γ-
lactone moiety, resulting in the preparation of various C13-
modified PTL analogs.7,32−36 However, the α-methylene-γ-
lactone moiety is also critical for mediating PTL pharmaco-
logical effects, serving as an electrophilic center in Michael-type
addition reactions with sulphydryl groups in the various cellular
components (e.g., NF-κB, IκK, glutathione) targeted by the
molecule.23,24,28,37 As a result, the tolerance of this site to
functionalization has been limited, with the corresponding C13-
substituted derivatives often exhibiting a large decrease or
complete loss of biological activity compared to PTL.7,32−36 As
an exception, a few C13-amino adducts, and in particular 11,13-
dehydro-13-dimethylamino-parthenolide (DMAPT),7,32 has
been shown to retain comparable anticancer activity to PTL,
while presenting improved oral bioavailability due to the
increased water-solubility of the amine adduct.7 Notably,
DMAPT has advanced to clinical trials for the treatment of
AML and other hematologic malignancies. Despite this
progress, improvements in PTL anticancer potency have not
been achieved to date, posing a barrier to the development of
more effective and selective parthenolide-based anticancer
agents.
RESULTS AND DISCUSSION
■
Parthenolide Oxidation via a Substrate-Promiscuous
P450BM3 Variant. In previous studies,38 we found that a
substrate-promiscuous variant of the fatty acid monooxygenase
P450BM3 (B. megaterium),50 called FL#62, is able to oxidize a
broad range of bulky compounds, including decaline-based
terpenes. Accordingly, we expected that our target compound,
parthenolide, could be equally well accepted by this P450 as a
substrate for oxidation. Gratifyingly, FL#62 was found to be
capable of efficiently oxidizing PTL, supporting more than 1000
total turnovers (TTN) and producing a mixture of three
monooxygenated products in 77:13:10 ratio as determined by
GC-MS. Structural elucidation by NMR and X-ray crystallog-
raphy revealed that the major product consisted of 1(R),10(R)-
epoxy-parthenolide (2, 77%), while the two minor products
consisted of 9(S)-hydroxy-parthenolide (3, 13%) and 14-
hydroxy-parthenolide (4, 10%), respectively (Scheme 1). The
stereochemical configuration of the newly formed oxirane ring
in 2 was determined by X-ray crystallographic analysis (Scheme
1). On the other hand, the observation of NOEs between the
9(H) and 1(H) protons allowed for the unambiguous
assignment of the S configuration to the C9 carbon in 3. In
contrast to FL#62, wild-type P450BM3 showed minimal PTL-
oxidation activity (30 TTN), producing the epoxide 2 as the
only product.
Based on our previous work,38,39 we envisioned that P450-
mediated C−H functionalization could provide a means to
expand opportunities for the functional re-elaboration of the
PTL scaffold toward this goal. Cytochrome P450 enzymes
constitute an attractive catalytic platform for the oxyfunction-
alization of unactivated C−H bonds in organic molecules,40−48
complementing and often extending beyond the scope of
chemical oxidation reagents and catalysts.49 In particular,
methodologies for high-throughput P450 active-site mapping
(“P450 fingerprinting”) and P450 reactivity prediction recently
introduced by our group have provided a way to guide and
accelerate the development of P450 catalysts with fine-tuned
regio- and stereoselectivity,38,39 a key requirement toward the
application of these biological catalysts for synthetic applica-
tions.49 Here, we demonstrate how these tools proved useful in
Compounds 2 and 3 correspond to naturally occurring
derivatives of parthenolide,51,52 whereas compound 4 has been
previously obtained as a minor product (4%) from microbial
metabolism of PTL.53 The hydroxylation products 3 and 4
were of particular interest to us, as they can provide two
valuable intermediates, not accessible via currently available
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Figure 1. (a) Crystal structure of P450BM3 heme domain in complex with N-palmitoylglycine (PDB code 1JPZ). The heme prosthetic group is
displayed in red (stick model) and the enzyme-bound substrate in green (sphere model). (b) Close-up view of the enzyme active site, in which the
amino acid residues targeted for mutagenesis in this study are highlighted.
Figure 2. Fingerprint-based prediction of parthenolide reactivity via fingerprint single component analysis (SCA). (a) Ranking of the 522 FL#62-
derived P450 variants according to their normalized activity on the decaline-based probe P4 (SI Figure S1). The 75 top-ranking (solid box) and 20
bottom-ranking (dotted box) variants are highlighted. (b) Total turnovers in PTL oxidation for the 75 top-scoring P450 variants arranged from the
least to the most active variant. TTN value for FL#62 is indicated for comparison (dotted line).
synthetic methods, for re-elaboration of parthenolide carbocy-
clic skeleton by chemoenzymatic means. Indeed, while chemical
oxidation of the allylic site C14 in parthenolide has been
reported (with SeO2 and t-BuOOH),54 this transformation is
accompanied by a Z→E isomerization of the 1,10-double bond,
leading to an important structural reorganization of the 10-
membered ring of the molecule.55 Interestingly, the sites
targeted by FL#62 (i.e., si face of 1,10 CC bond, pro-S
C(9)H bond, and C(14)H bond) are localized within the
same region of the molecule, as evinced from inspection of the
crystal structure of 2 (Scheme 1). While the expanded active
site of FL#62 is likely at the basis of the poor regio/
stereocontrol in the oxidation of this and other substrates,38,39
the preferential formation of 2 over 3 and 4 is likely to arise
from the higher reactivity of the electron-rich olefinic group
compared to the neighboring allylic positions, C9 and C14, to
P450-catalyzed oxidation. This electronic bias notwithstanding,
our previous success in fine-tuning the site-selectivity of
artemisinin-hydroxylating P450 variants suggested that P450
catalysts with improved site-selectivity toward each of the
positions targeted by FL#62, and in particular the two, less
activated aliphatic C−H sites, could be obtained via re-
elaboration of the enzyme active site in combination with our
recently introduced P450 fingerprint-based tools38,39 to
expedite this process.
Prediction of PTL Oxidation Reactivity via Fingerprint
Single Component Analysis (SCA). In the course of
previous work, over 500 functionally diverse P450 catalysts
derived from FL#62 were obtained via a two step process
involving (a) simultaneous site-saturation mutagenesis of
multiple ‘first-sphere’ active-site residues (i.e., 74, 78, 81, 82,
87, 181, and 184, Figure 1), followed by (b) high-throughput
mapping of the active site configuration of the resulting
engineered P450 variants by means of a panel of five
structurally diverse chromogenic probes (compounds P1−P5,
Supporting Information (SI) Figure S1). Through this process,
a collection of 522 FL#62-derived P450s featuring a unique
active site geometry and thus unique regio- and stereoselectivity
properties (as derived from the uniqueness of their fingerprint
profile)38 were thus made available for the search of more
selective PTL-oxidizing P450 catalysts in the context of this
work.
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a
Table 1. Amino Acid Mutations and Catalytic Properties of Parthenolide-Oxidizing P450 Variants
product
distribution (%)
b
amino acid substitutions
c
d
variant
78
81
82
87 180 181 184
2
3
4
TTN
product form. rate
coupling efficiency (%)
KD (μM)
FL#62
III-D4
II-C5
A
F
S
V
I
V
A
A
T
T
A
A
A
A
T
L
V
77
90
29
22
19
26
17
4
13
7
10
3
1042 98
4980 430
1370 153
1710 116
1310 71
1055 59
420 46
234 23
214 31
55.8
29.5
37.5
60.8
25.8
42.9
31.1
1.4
243
9
193 67
56 12
340 21
229 38
289 116
478 109
n.d.
T
T
T
N
N
N
68
77
80
20
2
3
59
44
27
9
2
1
XI-A11
XII-F12
II-E2
I
1
I
A
1
F
F
F
53
81
95
176 12
VII-H11
XII-D8
A
A
A
A
S
S
21
2
3
V
0
60
8
0.6
a
b
Mean values and standard deviations are calculated from triplicate experiments. Compared to P450BM3, FL#62 carries the following mutations:
c
V78A, F81S, A82 V, F87A, P142S,T175I, A180T, A184 V, A197 V, F205C, S226R, H236Q, E252G, R255S, A290 V, L353 V. Rates are measured
over initial 30 s and expressed as mole product per mole P450 per minute. Ratio between product formation rate and NADPH oxidation rate in the
d
presence of parthenolide.
We recently reported two complementary methods for
predicting P450 reactivity based on the analysis of their
fingerprints. A first one, suitable for probe-related target
substrates, utilizes the fingerprint component corresponding
to the probe most closely related, structurally, to the target
substrate as a predictor of enzyme reactivity toward this
molecule (referred to as fingerprint single component analysis,
or SCA).38 More recently, we described a more general
approach, applicable to probe-unrelated target molecules, that
relies upon the multivariate analysis of fingerprint/substrate
reactivity correlations using a randomly chosen training set of
P450 variants (fingerprint multiple component analysis, or
MCA).39 In the context of parthenolide, both approaches are
feasible, which provided us with an interesting opportunity to
compare side-by-side the performance and predictive capability
of the two methods.
largest improvement toward C9 hydroxylation (13% → 68%)
while maintaining absolute S stereoselectivity and supporting
more catalytic turnovers than FL#62 (Table 1 and SI Table
S2). On the other hand, the triple mutant II-E2 represented the
best C14-hydroxylating catalyst (10% → 53%) within this
generation of variants, supporting over 1000 TTN in
parthenolide oxidation (Table 1 and SI Table S2). Finally, a
P450 variant with improved regioselectivity and absolute
stereoselectivity for production of 1(R),10(R)-epoxy-PTL (2),
III-D4, was also found at this stage, this variant also exhibiting a
nearly 5-fold increase in catalytic activity compared to the
parent enzyme FL#62 (4980 vs 1042 TTN).
Parthenolide Reactivity Prediction via Fingerprint
Multiple Component Analysis (MCA). To compare the
relative performance of MCA vs SCA toward prediction of
parthenolide reactivity, a training set consisting of 20
parthenolide-active P450 variants, including FL#62, was first
assembled (SI Table S3). Then, a fingerprint-based predictive
model of PTL reactivity was generated by correlating the
fingerprints with the experimentally determined PTL oxidation
activities (measured in TTNs) across the training set using
multiple linear regression analysis (MLR), as described
previously (SI Figure S2a).39 Using this model, the P450
catalysts in the 522-member collection were ranked according
to their predicted PTL reactivity (SI Figure S2b). Experimental
testing of the 75 top-ranking variants revealed that 62 of them
(83%) were capable of PTL oxidation at synthetically useful
levels (TTN > 100), supporting an average of 807 TTN (SI
Figure S2c). To further probe the MCA-based predictions, the
20 bottom-ranking variants were also tested. Within this group,
only six (30%) were found to be active (avg. TTN: 163).
Interestingly, the group of 75 top-ranking P450s as defined by
MCA largely overlapped with that based on SCA (64%) and
included the best C9- and C14-hydroxylating variants (i.e., II−
C5 and II-E2, respectively) previously identified by the latter
method. None of the 27 variants uniquely found by MCA show
further improved regioselectivity toward these positions. Based
on these results, we conclude that both methods performed
equally well in guiding the search for PTL oxidizing P450
catalysts.
The Maximum Common Substructure (MCS) algorithm56,57
is particularly well suited to assess the degree of structural
similarity between core-related molecules.58 Based on this
algorithm, the decaline-based probe P4 (Supporting Informa-
tion (SI) Figure S1) was determined to represent the best
predictor of parthenolide reactivity among the fingerprint probe
set (SMCS = 0.68 vs SMCS < 0.4 against P1, P2, P3, or P5, with
SMCS ranging from 1 (structural identity) to 0 (max. structural
dissimilarity)) via the SCA method. Accordingly, the collection
of 522 FL#62-derived P450 catalysts were ranked based on
their P4 probe activity in order to prioritize our search for PTL-
oxidizing variants with improved regioselectivity (Figure 2a).
Guided by these analyses, the 75 top-ranking P450 variants
were retrieved from the collection and tested for PTL oxidation
activity. Gratifyingly, we found that 81% of these variants (61/
75) showed PTL-oxidation activity at synthetically useful levels
(>100 TTN; average = 695 TTN, Figure 2b). In addition, a
good fraction (21%) exhibited higher PTL oxidation activity
than the parent enzyme, with four of them supporting total
turnover numbers in excess of 4000 (Figure 2b). In contrast,
90% of the predicted inactive (i.e., bottom-ranking) variants
(18/20) were found to be inactive on parthenolide, further
supporting the reliability of the SCA predictions.
The parthenolide-oxidizing P450 variants identified in this
manner exhibited in most cases diversified regioselectivity
properties as expected from their different fingerprint profiles
(SI Table S2). Most importantly, P450 catalysts with improved
regioselectivity toward each of three target oxidation sites were
found. In particular, the triple mutant II-C5 displayed the
Selective P450 Catalysts for C9 and C14 Hydrox-
ylation. To achieve further improvements in regioselectivity
for C9 hydroxylation, II-C5 was used as the parent enzyme for
the construction of a series of single mutant libraries via site-
saturation mutagenesis (NNK degenerate codon) of positions
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82, 87, 180, 181, 184, 263, and 328 (Figure 1). These positions
correspond to ‘first-sphere’ active site residues (based on the
available P450BM3 crystal structure59) that, with the exception of
residue 82, have remained unmodified as compared to FL#62.
High-throughput fingerprinting of these libraries (609 total
recombinants screened) followed by fingerprint comparative
analysis revealed the occurrence of 111 unique-fingerprint
variants. Upon screening of these variants, three P450 catalysts
with improved C9-selectivity (65−75%) were found, with the
best one, XI-A11, also exhibiting higher catalytic activity than
the parent enzyme II-C5 (1710 vs 1040 TTN, Table 1). As
expected, each of these variants carried a single active-site
mutation, namely A82T (in XI-A11), A87S, and T180A. The
latter two beneficial mutations were then introduced into XI-
A11, alone and in combination. Among the resulting variants,
XII-F12, which corresponds to XI-A11(T180A), emerged as
the best catalyst for C9-hydroxylation, being able to produce
the desired product 3 with 80% regioselectivity, absolute S-
stereoselectivity, and supporting over 1300 TTN (Table 1).
Using a similar approach, the best C14-hydroxylating variant
identified during the first step, II-E2, was selected as the starting
point toward generating P450 catalysts that could provide
selective access to the C14 aliphatic position. Conveniently, a
panel of about 50 functionally diverse, II-E2-derived active-site
variants were already available from our previous work.39 These
variants were produced by triple site-saturation mutagenesis of
active site residues 74, 181, and 184. Screening of this panel of
P450 catalysts against parthenolide led to the discovery of a
triple mutant variant, VII-H11, which showed significantly
improved regioselectivity toward C14 hydroxylation compared
to II-E2 (53% → 81%, Table 1), albeit at the expense of the
catalytic activity (420 vs 1055 TTN). In a further round of
directed evolution, additional first-sphere active residues left
untouched in VII-H11 (i.e., positions 74, 75, 87, 263, and 328)
were subjected to site-saturation mutagenesis. From these five
single mutant libraries, forty P450 variants were determined to
be catalytically active and functionally diverse based on
fingerprint analysis. Among these enzymes, XII-D8 represented
the best catalyst for C14 hydroxylation, catalyzing the
formation of 4 with excellent regioselectivity (95%, Table 1).
Interestingly, also in this case, the improvement of C14-
regioselectivity was accompanied by a reduction in the catalytic
turnover number (Table 1).
Overall, the process outlined above enabled us to achieve our
task of obtaining a panel of P450 catalysts with refined site-
selectivity for each of the sites oxidized by the initial enzyme,
FL#62, in the PTL carbocyclic scaffold (Figure 3). The fact that
this goal could be accomplished by analyzing only a minimal
fraction (1.8%) of the original engineered enzyme libraries
(>16,500 members) highlights the peculiar advantage of the
fingerprint-based analysis/prediction tools to guide this process.
Characterization of the Evolved P450 Catalysts.
Further experiments were conducted to characterize the best
P450 variants for 1,10-epoxidation (III-D4), 9(S)-hydroxylation
(XI-A11, XII-F12), and 14-hydroxylation (VII-H11, XII-D8), as
well as their evolutionary intermediates (II-C5, II-E2), with
respect to their kinetic parameters, substrate binding affinity,
and coupling efficiency. Sequencing revealed that a small set of
active site mutations (3−4), clustered within the back-end
region of the heme pocket (i.e., positions 78, 81, 82, (180);
Figure 1) were beneficial for steering the regioselectivity of the
enzyme toward hydroxylation of the C9 site or toward
epoxidation of the 1,10 double bond (Table 1). In comparison,
Figure 3. Overview of the directed evolution process leading to the
selective parthenolide-oxidizing P450 variants.
refinement of C14-regioselectivity required a larger number of
amino acid substitutions (6−7), which include modification of
the aforementioned positions as well as another cluster of
spatially close active site residues (i.e., 180, 181, 184; Figure 1).
The effect of these mutations on the substrate binding
affinity (KD) was assessed via substrate-induced heme spin shift
experiments (SI Figures S3−S4). These analyses indicated that
the parent enzyme FL#62 binds parthenolide with relatively
moderate affinity (KD = 243 μM, Table 1), although this KD
value remains within the range of those observed for wild-type
P450BM3 and some fatty acid substrates (e.g., laurate; KD = 270
μM).60 Comparable equilibrium dissociation constants (190−
290 μM) were measured for most of the improved variants,
with the exception of VII-H11, which showed a roughly 2-fold
higher KD, and XII-D8, which showed no signs of substrate-
induced heme spin shift. Thus, these data suggest that, while
being beneficial toward improving C14-selectivity, the muta-
tions in the 180/181/184 cluster somewhat weaken the enzyme
interaction with PTL.
Across the set of C9- and C14-hydroxylating variants, the
progressive increase in site-selectivity was found to be generally
accompanied by a decrease in the rate of substrate oxidation
(Table 1). Interestingly, a similar trade-off was observed in the
context of our previously reported artemisinin-hydroxylating
P450s,39 suggesting that this trend may be a common feature
within this class of enzymes. This notwithstanding, all the
improved variants, with the exception of VII-H11 and XII-D8,
were able to support higher TTN compared to the initial
enzyme FL#62, exhibiting coupling efficiencies (= ratio of
product formation rate/NADPH oxidation rate) that range
from 25% to 60% (Table 1). Interestingly, as noted above, the
improvement of site-selectivity toward C14 hydroxylation was
accompanied by a reduction in TTN, and in the case of XII-D8,
also by a pronounced reduction in coupling efficiency, likely a
result of the larger number of active site mutations accumulated
by these variants. Nevertheless, the selectivity and catalytic
efficiency of VII-H11 were well suited for synthetic purposes as
demonstrated below.
Chemoenzymatic Synthesis of PTL Derivatives. With
the strategy outlined above, three highly regio- and stereo-
selective P450 catalysts could thus be made available to explore
the importance of the 1,10-double bond for parthenolide
antileukemic activity (2) as well as provide two new points of
entry (3, 4) for further functionalization of this molecule at the
C9 and C14 sites via chemoenzymatic synthesis. To isolate
sufficient quantities of 3 and 4 for these studies, preparative-
scale reactions (100 mg PTL) were carried out using P450
E
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a
Scheme 2. Chemoenzymatic Synthesis of the C9- and C14-Substituted Parthenolide Derivatives
a
Designated as PTL-9-(#) and PTL-14-(#), respectively.
variants XII-F12 (0.26 mol %) and VII-H11 (0.32 mol %),
respectively, in the presence of a NADPH regeneration system
consisting of a thermostable phosphite dehydrogenase61 and
sodium phosphite as sacrificial reductant. VII-H11 was
preferred over the more regioselective XII-D8 because of
much higher total turnover numbers, making it a superior
catalyst for synthetic purposes. From these reactions, about 75
mg of each of the desired oxidation products could be obtained
in over 70% isolated yields.
Table 2. LD50 Values for Parthenolide (PTL) and Its
Chemoenzymatic Derivatives against the Two Primary AML
a
Specimens and Healthy Bone Marrow (BM) Cells
LD50 (μM)
LD50 (μM)
LD50 (μM)
AML123009
AML100510
bone marrow
PTL
9.7 (1)
6.1 (1)
>80
n.d.
n.d.
n.d.
>20
>50
>50
25
2
13.5 (0.7)
95 (0.1)
>100
13.9 (0.4)
17.4 (0.4)
24.2 (0.3)
6.2 (1.0)
7.2 (0.8)
3.1 (2.0)
2.7 (2.2)
4.3 (1.4)
6.3 (1.0)
3.7 (1.6)
5.1 (1.2)
>100
3
PTL-9-3
PTL-9-4
PTL-9-5
PTL-9-6
PTL-9-9
PTL-9-10
PTL-9-11
PTL-9-12
PTL-9-13
4
The isolated 9(S)-hydroxy- (3) and 14-hydroxy-PTL (4)
were then further processed to generate a panel of 9- and 14-
substituted parthenolide derivatives (Scheme 2). To this end,
direct acylation of 3 and 4 with acid chloride reagents was
chosen as a rapid means to explore the accessibility of the C9
and C14 sites to substituents of varying size (e.g., acetyl vs
benzoyl group) and to gain initial structure−activity insights on
the impact of these modifications on PTL antileukemic activity.
Based on the superior performance of the benzoylated analogs
PTL-9/14-4 (vide infra), a second set of derivatives were
prepared in which the benzoyl moiety was substituted with
polar or lipophilic groups. Finally, the activity data collected
with these compounds inspired the design and synthesis of a
third set of parthenolide analogs in which two trifluoromethyl
groups are installed at different positions of the benzoyl moiety.
Antileukemic Activity of the PTL Derivatives. To
evaluate the antileukemic activity of the novel parthenolide
derivatives, these compounds were tested against primary acute
myelogenous leukemia (AML) cells obtained with informed
consent from leukemia patients. In particular, two relapsed
refractory AML specimens were utilized, which feature both a
normal (AML100510) and a complex karyotype
(AML123009), the latter exhibiting reduced sensitivity to
PTL (LD50: 9.7 vs 6.1 μM, Table 2). Dose−response curves
were obtained by measuring the variation of cell viability at
increasing compound concentration using a previously
described assay based on cell staining with annexin-V and 7-
amino-actinomycin (7-ADD) followed by flow cytometry
analysis.62
4.1 (2.4)
6.1 (1.6)
4.8 (2.0)
6.3 (1.5)
3.5 (2.7)
4.6 (2.1)
2.3 (4.2)
2.7 (3.6)
>100
23
n.d.
44
105
n.d.
n.d.
>80
n.d.
n.d.
n.d.
37
PTL-14-3
PTL-14-4
PTL-14-5
PTL-14-6
PTL-14-9
PTL-14-10
PTL-14-11
PTL-14-12
PTL-14-13
>100
12.5 (0.5)
7.8 (0.8)
8.7 (0.7)
5.4 (1.1)
6.2 (1.0)
3.1 (2.0)
10 (0.6)
9.5 (0.6)
3.4 (1.8)
6.4 (1.5)
20.8 (0.5)
5.9 (1.7)
6.4 (1.5)
3.7 (2.6)
7.8 (1.2)
5 (1.9)
n.d.
n.d.
25
2.5 (3.9)
a
The values in parentheses indicate relative activities compared to
PTL. n.d. = not determined.
dramatic reduction in antileukemic potency (Figure 4a and SI
Figures S6 and S7). In stark contrast, the benzoylated
derivatives PTL-9-4 and PTL-14-4, were found to exhibit a
significantly improved activity (compared to PTL) against the
complex-karyotype AML cells (AML123009), as indicated by
the 2-fold lower LD50 values (Table 2). These results clearly
showed the beneficial effect of larger, aromatic substituents at
either the C9 or C14 sites toward potentiating PTL
antileukemic activity. Accordingly, a set of compounds carrying
variously substituted benzoyl groups at each of these positions
were synthesized. Notably, most of the resulting semisynthetic
derivatives were found to be 2- to 3-fold more potent than
parthenolide as illustrated by the dose−response curves in
Figure 4a and SI Figures S6 and S7, and as summarized in
Table 2. Within the C9-functionalized series, the largest
increases in potency were achieved through substitution of
the aryl moiety at the para position with fluorine (PTL-9-9), a
As illustrated in Figure 4a and SI Figure S6, 1(R),10(R)-
epoxy-PTL (2) was found to show a moderate (2-fold)
reduction in potency compared to PTL in both AML
specimens, possibly due to the slight structural change resulting
from epoxidation of the 1,10-double bond as revealed by the
crystallographic data (Scheme 1). Interestingly, a complete loss
of activity was observed with the two hydroxylated derivatives 3
and 4, clearly pointing at the deleterious effect of introducing a
polar, hydroxyl group at either the C9 or C14 site. Similarly, the
acetylated derivatives PTL-9-3 and PTL-14-3, featured a
F
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Figure 4. Biological activity for representative parthenolide (PTL) analogs. (a) Dose response curves for C9- (left panel) and C14-modified (right
panel) PTL derivatives tested against a complex-karyotype primary AML specimen (AML123009). Data for the remaining analogs with a second
primary AML specimen (AML100510) cells are provided in SI Figures S6 and S7. (b, c) Cytotoxicity of the most potent PTL analogs against (b)
total and (c) primitive (CD34+CD38−) normal bone marrow cells.
dimethylamino (PTL-9-6), or trifluoromethyl group (PTL-9-
10). A similar structure−activity trend was observed for the
C14-functionalized series of compounds, although in this case
the para-trifluoromethyl-benzoyl substituted derivative, PTL-
14-10, emerged as the most potent derivative in the context of
both AML specimens.
The beneficial effect of increasing the lipophilicity of the aryl
moiety further suggested the design of compounds PTL-9/14-
12 and PTL-9/14-13. Notably, the addition of a second
trifluoromethyl group to the benzoyl moiety brought about a
further increase in antileukemic potency for both the C9- and
C14-modified analogs and in particular against AML123009
cells. Overall, the most promising compounds within each
series, namely PTL-9-12 (LD50: 2.3 μM) and PTL-14-13
(LD50: 2.5 μM), were found to exhibit a 4.2- and 3.9-fold
enhanced cytotoxicity, respectively, against primary AML cells
compared to PTL (LD50: 9.7 μM, Table 2).
The most potent parthenolide derivatives identified in these
studies were selected for further characterization to evaluate
their selectivity against malignant over normal cells. For these
studies, normal bone marrow cells (BM cells) obtained from
healthy donors were utilized. Importantly, all these compounds,
with the exception of PTL-9-6, did not significantly impart the
viability of normal cells (Figure 4b), thus presenting the desired
high selectivity against leukemic cells. Remarkably, at a
concentration sufficient to kill 98% of primary AML cells (10
μM), compounds PTL-9-12 and PTL-9-13 were found to cause
only less than 15% reduction in the viability of normal BM cells.
For some of the most promising PTL analogs, it was also
possible to test their cytotoxic effect on the progenitor
(CD34+CD38−) cell subpopulation of the bone marrow
samples (Figure 4c). Notably, the compounds exhibited
comparably low or even lower cytoxicity than in the context
of mature BM cells.
Taken together, these results showed that functionalizations
at the C9/C14 sites are not only beneficial in enhancing PTL
cytotoxicity but also that such effect is exerted with high
selectivity in the context of leukemic cells. As a result, the
LD50(BM)/LD50(AML) ratio of the original compound could
be improved by several folds by means of these chemo-
enzymatic manipulations (e.g., 19 (PTL-9-12) and 39 (PTL-9-
13) as compared to 8 for PTL with BM and AML123009 cells,
Table 1). Given the relatively broad spectrum of cellular
proteins/processes affected by PTL in cancer cells,5 it is
possible that the functional modification at the C9/C14 sites
may cause a shift in the protein-targeting profile of the
molecule, in a way that affect preferentially the proliferation of
malignant cells.
Conclusion. In conclusion, we have developed efficient,
P450-based chemoenzymatic routes to access novel derivatives
of the sesquiterpene lactone parthenolide with potent activity
and high selectivity against AML cells. From a methodological
point of view, this study demonstrates the efficiency of our
P450 fingerprint-based tools as a way to accelerate the
development of P450 oxidation catalysts with refined regio-
and stereoselectivity, a current bottleneck toward the
exploitation of these enzymes for synthetic applications. Since
the aliphatic positions targeted by the engineered P450s
developed here have so far remained inaccessible to chemical
manipulation, this work also highlights the potential of P450-
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PTDH (2 μM), NADP+ (150 μM), and sodium phosphite (50 mM).
The reaction mixture was stirred for 12 h at 4 °C. The crude product
was extracted with dichloromethane (3 × 80 mL). The collected
organic layers were dried with sodium sulfate, concentrated under
vacuum, and purified by flash chromatography (hexanes/ethyl acetate:
1/2) to afford 4 (77 mg, 72% (90% of theoretical maximum)) and 2
(17 mg, 16% (79% of theoretical maximum)). 14-hydroxy-
parthenolide (4): 1H NMR (500 MHz, CDCl3): δ 1.31 (s, 3 H),
1.32−1.38 (m, 1 H), 1.82−1.1.92 (m, 1 H), 2.09-2.16 (m, 1 H), 2.20−
2.32 (m, 3 H), 2.50 (dq, 1 H, J = 5.0, 13.4 Hz), 2.82−2.90 (m, 3 H),
3.92 (t, 1 H, J = 8.7 Hz), 4.16 (d, 1 H, J = 11.3 Hz), 4.46 (d, 1 H, J =
11.8 Hz), 5.43 (dd, 1 H, J = 4.1, 12.4 Hz), 5.68 (d, 1 H, J = 3.2 Hz),
6.39 (d, 1 H, J = 3.8 Hz). 13C NMR (125 MHz, CDCl3): δ =16.9, 23.7,
31.4, 36.3, 47.3, 59.8, 61.1, 66.2, 82.4, 121.4, 129.0, 137.8, 139.2, 169.3.
HRMS (ESI) calcd for C15H20O4 [M+H]+ m/z: 265.1440; found:
265.1440; [α]D23 = −61.7° (c: 0.13 g 100 mL−1, CH2Cl2).
General Procedure for Synthesis of Parthenolide Deriva-
tives. To a solution of compound 3 (or 4) in 3 mL of anhydrous
dichloromethane under argon atmosphere was added 4-dimethylami-
nopyridine (1 equiv.), triethylamine (5 equiv.), and the corresponding
acid chloride (5 equiv.). Reaction was stirred at RT until complete
disappearance of the starting material (ca. 2 h). At this point, the
reaction mixture was added with saturated sodium bicarbonate
solution (5 mL) and extracted with dichloromethane (3 × 5 mL).
The combined organic layers were dried over sodium sulfate,
concentrated under reduced pressure, and the ester product was
isolated by silica gel flash chromatography (5 to 40% ethyl acetate in
hexanes). NMR and MS characterization data for these compounds are
provided as Supporting Information.
mediated C−H functionalization as a new, enabling strategy for
the late-stage diversification and optimization of complex,
bioactive molecules. A particularly relevant result from the
present studies is the discovery that the C9 and C14 sites
represent two ‘hot spots’ for potentiating the antileukemic
activity of parthenolide. Notably, the improvements in
anticancer activity against AML cells could be achieved without
increasing their cytotoxicity against normal hematopoietic cells,
thereby effectively enhancing the therapeutic index of the
molecule. These findings have important implications. On one
hand, two parallel routes (i.e., via C9 or C14 functionalization)
have now become available toward further optimization of
parthenolide as a therapeutic agent. In this regard, the tolerance
of each of these sites to variously substituted aryl substituents is
very promising, as it suggests that a variety of functional groups
may be explored at these positions toward this goal.
Furthermore, our results raise intriguing questions regarding
the biochemical mechanisms at the basis of the improved
cytoxicity of the compounds in the context of malignant cells.
Since parthenolide is known to target a variety of cellular
components, it is plausible that the newly introduced
functionalizations at the C9/C14 sites may shift the protein-
targeting profile of the molecule in a way that affects
preferentially the proliferation of malignant cells. We envision
that future investigation of these aspects could provide valuable
insights toward the development of effective pharmacological
strategies for selective targeting of leukemia stem cells.
Cell Isolation and Culture. Primary human AML and normal
bone marrow (BM) cells were obtained from volunteer donors.
Informed consent was obtained in accordance with the Declaration of
Helsinki. All manipulation and analysis of human specimens was
approved by the University of Rochester Institutional Review Board.
In some cases, cells were cryopreserved in freezing medium of Iscove
modified Dulbecco medium (IMDM), 40% fetal bovine serum (FBS),
and 10% dimethylsulfoxide (DMSO) or in CryoStor CS-10 (VWR,
West Chester, PA). Cells were cultured in serum-free medium
(SFM)19 for 1 h before the addition of parthenolide or its derivatives.
Cell Viability Assays. Apoptosis assays were performed as
described.62 Briefly, after 24 h of treatment, normal BM cells and
AML specimens were stained for the surface antibodies CD38-
allophycocyanin (APC), CD34-PECy7, and CD123-phycoerythin
(Becton Dickinson, San Jose, CA) for 15 min. Cells were washed in
cold PBS and resuspended in 200 μL of annexin-V buffer (0.01 M
HEPES/NaOH, 0.14 M NaCl, and 2.5 mM CaCl2). Annexin-V−
fluorescein isothiocyanate (FITC) and 7-aminoactinomycin (7-AAD;
Molecular Probes, Eugene, OR) were added, and the tubes were
incubated at RT for 15 min then analyzed on a BD LSRII flow
cytometer (BD Biosciences, San Jose, CA). Analyses for phenotypi-
cally described stem cell subpopulations were performed by gating
CD34+/CD38− populations. Viable cells were scored as Annexin-V
negative/7-AAD negative. The percent viability data provided are
normalized to untreated control specimens.
METHODS
■
For complete description of the materials and methods including
reagents, cloning procedures, protein expression and purification,
fingerprint-based analyses, and enzyme characterization, see Support-
ing Information.
Preparative-Scale Synthesis of 9(S)-, and 14-Hydroxy-
partenolide. To prepare 3, purified P450 variant XII-F12 (final
concn.: 2.5 μM; 0.26 mol %) was dissolved in 400 mL 50 mM
phosphate buffer (pH 8.0) in the presence of parthenolide (100 mg,
final concn.: 0.95 mM), PTDH (2 μM), NADP+ (150 μM), and
sodium phosphite (50 mM). The reaction mixture was stirred for 12 h
at 4 °C. The crude product was extracted with dichloromethane (3 ×
80 mL). The collected organic layers were dried with sodium sulfate,
concentrated under vacuum, and purified by flash chromatography
(hexanes/ethyl acetate: 1/2) to afford 3 (75 mg, 70% (88% of
theoretical maximum)) and 2 (16 mg, 15% (75% of theoretical
maximum)). 9(S)-hydroxy-parthenolide (3): 1H NMR (500 MHz,
CDCl3): δ 1.34 (s, 3 H), 1.76 (s, 3 H), 1.97−2.06 (m, 1 H), 2.15−2.27
(m, 4 H), 2.50 (dq, 1 H, J = 5.2, 12.4 Hz), 2.70 (d, 1 H, J = 8.7 Hz),
2.83−2.90 (m, 1 H), 3.86 (t, 1 H, J = 8.5 Hz), 4.27 (dd, 1 H, J = 2.2,
10.5 Hz), 5.42 (d, 1 H, J = 11.3 Hz), 5.69 (d, 1 H, J = 3.2 Hz), 6.36 (d,
1 H, J = 3.6 Hz); 13C NMR (125 MHz, CDCl3): δ 10.9, 17.4, 23.9,
36.1, 38.0, 44.5, 61.4, 66.2, 79.7, 81.5, 121.6, 126.5, 136.6, 138.3, 168.8;
HRMS (ESI) calcd for C15H20O4 [M+H]+ m/z: 265.1440; found:
265.1433; [α]2D3 = −83.9° (c: 0.43 g 100 mL−1, CH2Cl2). The 9(S)
configuration of 3 was assigned based on the observed strong NOE
signal (2.5%) between the 9(H) proton and 1(H) proton. 1(R),10(R)-
epoxy-parthenolide (2): 1H NMR (500 MHz, CDCl3): δ 1.34 (s, 3 H),
1.36−1.42 (m, 4 H), 1.45−1.56 (m, 1 H), 1.56−1.65 (m, 1 H), 2.01−
2.29 (m, 4 H), 2.47 (dd, 1 H, J = 8.1, 14.0 Hz), 2.70−2.76 (m, 1 H),
2.85 (d, 1 H, J = 12.2 Hz), 2.90 (d, 1 H, J = 8.9 Hz), 3.93 (t, 1 H, J =
8.9 Hz), 5.62 (d, 1 H, J = 3.0 Hz), 6.33 (d, 1 H, J = 3.0 Hz); 13C NMR
(125 MHz, CDCl3): δ 17.0, 17.5, 24.0, 26.0, 35.1, 40.1, 47.6, 60.7,
63.7, 64.6, 81.8, 101.2, 121.4, 138.8, 168.9. HRMS (ESI) calcd for
C15H20O4 [M+H]+ m/z: 265.1440; found: 265.1441; [α]2D3 = −71.0°
(c: 0.24 g 100 mL−1, CH2Cl2).
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemical structures of probes P1−P5, oligonucleotide
sequences, additional data corresponding to the fingerprint-
based predictions and characterization of P450 variants, results
from spin-shift experiments, characterization data for parthe-
nolide derivatives. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
To prepare 4, purified P450 variant VII-H11 (final concn.: 3 μM;
0.32 mol %) was dissolved in 400 mL 50 mM phosphate buffer (pH
8.0) in the presence of parthenolide (100 mg, final concn.: 0.95 mM),
Corresponding Author
*E-mail: fasan@chem.rochester.edu.
H
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Present Address
§Division of Hematology, University of Colorado, Aurora,
Colorado 80045, United States of America
Author Contributions
R.F. and C.T.J. conceived the project. J.N.K. conducted all the
experiments concerning the engineering of the P450 catalysts
and synthesis of the parthenolide derivatives. K.M.O.
performed all the cellular and biochemical experiments
concerning the biological activity of the compounds. R.F.
wrote the manuscript with input from all the other coauthors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institute of Health
R01 grant GM098628 awarded to R.F. C.T.J. was supported by
a grant from the Leukemia and Lymphoma Society (TRP 6230-
11). MS instrumentation was supported by the National
Science Foundation grants CHE-0840410 and CHE-0946653.
The authors are grateful to Bill Brennessel for assistance with
the X-ray crystallographic analyses.
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