Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication

Article abstract of DOI:10.1016/j.bmc.2013.12.022

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand-protein interactions and design analogues with increased affinity to Pgp Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (K_d = 4.4 μM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation

Full text of DOI:10.1016/j.bmc.2013.12.022

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of Strychnos alkaloids as MDR reversal
agents for cancer cell eradication
Surendrachary Munagala a, Gopal Sirasani a, Praveen Kokkonda a, Manali Phadke b, Natalia Krynetskaia b,
Peihua Lu c, Frances J. Sharom c, Sidhartha Chaudhury d, Mohamed Diwan M. Abdulhameed d,
Gregory Tawa d, Anders Wallqvist d, Rogelio Martinez b, Wayne Childers b, Magid Abou-Gharbia b,
Evgeny Krynetskiy b, Rodrigo B. Andrade a,
⇑
a Department of Chemistry, Temple University, Philadelphia, PA 19122, United States
b Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA 19122, United States
c Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
d Department of Defense Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, U.S. Army
Medical Research and Materiel Command, Fort Detrick, MD 21702, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that
resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed
an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular mod-
eling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand–protein
interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evalu-
ated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a
panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity.
Received 4 October 2013
Revised 30 November 2013
Accepted 8 December 2013
Available online xxxx
Keywords:
Total synthesis
Strychnos alkaloids
P-glycoprotein
ABCB1
Multidrug resistance
Resensitization
Docking
Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd = 4.4 lM), the strongest
inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines.
Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp
modulators justifies further investigation.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
were launched with optimism, as these drugs were already FDA-
approved.6 It was realized that the dosage required to inhibit Pgp
Cancer is one of the leading causes of death worldwide, with
multidrug resistance (MDR) being responsible for the failure of
chemotherapy in >90% of metastatic cancer patients.1 MDR is often
characterized by the overexpression of ATP-dependent transport-
ers, particularly P-glycoprotein (Pgp), which efflux anti-cancer
drugs (e.g., taxol, vincristine, and doxorubicin) out of cancer cells.2
In 1981, Tsuruo demonstrated that verapamil, a calcium-channel
blocker, resensitized MDR cancer cells to vincristine.3 It was later
established that verapamil inhibits Pgp activity by direct competi-
tion with Pgp substrates.4 While attempts to resensitize patient
tumors to chemotherapy via MDR reversal (i.e., Pgp inhibition)
have been largely unsuccessful to date in clinical trials, the acceler-
ated discovery of natural product Pgp inhibitors (i.e., fourth
generation) has reenergized the field.5
was toxic to the patients; however, randomized Phase III clinical
trials of cytarabine and daunorubicin in combination with cyclo-
sporine in patients with poor-risk AML was beneficial.7 In addition,
the use of quinine in combination with chemotherapy showed an
increase in complete remission rates and patient survival in Pgp-
positive MDS cases.8 The low affinity for Pgp was addressed to
some extent with second generation inhibitors (e.g., valspodar,
biricodar), but problems associated with pharmacokinetic interac-
tions (i.e., metabolism and drug clearance due to cytochrome P450
inhibition) were responsible for failure in clinical trials. The third
generation of Pgp inhibitors such as tariquidar, which were
completely synthetic in origin and obtained through methodical
combinatorial chemistry, possessed both nanomolar affinity for
Pgp and optimal pharmacokinetic parameters. To date, the limited
clinical studies of tariquidar have not conclusively shown MDR
reversal. Bates has argued that negative results from clinical trials
with Pgp inhibitors can be traced to poor clinical study design, par-
ticularly in patient selection, dosing regimen, and combinations
thereof.9 There is considerable interest in the development of
Within a decade of Tsuruo’s discovery, clinical trials with first
generation Pgp inhibitors (e.g., cyclosporine, verapamil, quinine)
⇑
Corresponding author. Tel.: +1 215 204 7155; fax: +1 215 204 9851.
E-mail address: randrade@temple.edu (R.B. Andrade).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.022
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2
S. Munagala et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
fourth generation inhibitors, which are largely natural product
based. This is not surprising considering first-generation Pgp inhib-
itors cyclosporine and quinine, which yielded successful results in
pioneering clinical trials, are, in fact, natural products.
identify and design molecules with increased affinity to Pgp within
the Strychnos alkaloid scaffold. To that end, we built a docking
model of human Pgp (Fig. 2A). We carried out a BLAST search of
the human Pgp sequence against the Protein Data Bank16 and iden-
tified the protein structure of the mouse Pgp homolog,17 with 86%
sequence similarity, as a suitable template for homology modeling.
We selected the mouse Pgp structure co-crystallized with the Pgp
inhibitor QZ59-RRR (PDB code: 3G60, Fig. 2D) as a template and
carried out homology modeling using Schrödinger’s PRIME soft-
ware,18 followed by energy minimization and refinement using
the default protocols in the software. We used Schrödinger’s Glide
software19,20 for docking grid generation and ligand docking. The
homology model was prepare for docking using the software’s Pro-
tein Preparation Wizard tool. We generated a docking grid based
on residues identified from structural and biochemical studies to
be important for Pgp substrate binding.12,21 Ligands were prepared
using the Schrodinger Glide ‘Ligprep’ tool to sample all possible
tautomers and all possible ionic states from a pH of 5.0 to 9.0.
The three highest scoring poses were stored for each ligand. Dock-
ing was carried out using Schrodinger Glide’s XS docking protocol.
Two cyclic peptide inhibitors (QZ59-SSS and QZ59-RRR) that were
previously co-crystallized with mouse Pgp (Aller et al. Science
2009) were re-docked into the human Pgp model to validate that
the overall docking protocol recapitulated known ligand poses
for this receptor.
Preliminary docking results revealed that many of the top rank-
ing compounds docked to an aromatic-rich hydrophobic pocket
formed by residues F72, F728, F732, V982, and F978 (dubbed
‘site-1’ by Ding et al.21), which is shown in Figure 2. Further
analysis of the docked poses was necessary to elucidate the scaf-
fold-specificity of various binding-site regions in Pgp and identify
further analogues for synthesis possessing substituents that maxi-
mize binding affinity for a given scaffold class. Significantly, the
best docking score was obtained with the third-generation MDR
reversal agent tariquidar (score = ꢀ9.53), which is the most potent
Pgp inhibitor developed to date. The modeling also predicted that
the (R)-enantiomer of verapamil (score = ꢀ7.99) binds tighter than
the (S)-enantiomer (score = ꢀ7.16); the former was employed as a
second-generation Pgp MDR reversal agent.1 Both verapamil
(Fig. 2E) and tariquidar bound to the aromatic-rich pocket with
site-1, which was the preferred binding site for the leuconicine
base scaffold in a number of the top-ranking leuconicine A deriva-
tives. To test the validity of our Pgp homology model, we selected
the third highest docking hit 7 (score = ꢀ8.70), which is the 3,4,5-
trimethoxybenzyl analogue of leuconicine A (5), and prepared it by
synthesis (vide infra). The selected 3,4,5-trimethoxybenzyl
analogue presented an additional interaction between the elec-
tron-rich arene group and an adjacent hydrophobic pocket formed
by F343, I340, and L339 (Fig. 2).
Semi-synthetic derivatives of natural products provide a prom-
ising expansion of the oncologist’s armamentarium, with 79% of all
FDA-approved antineoplastic drugs derived from natural prod-
ucts.10 To explore the potential of natural alkaloids and their
semi-synthetic derivatives to reverse Pgp-mediated MDR, we have
synthesized and evaluated various Strychnos alkaloids and ana-
logues 1–7 for their capacity to bind Pgp and reverse MDR by (a)
measuring binding affinity to Pgp by fluorescence quenching; (b)
assessing the inhibitory effect of synthesized compounds on Pgp-
associated ATPase activity, and (c) performing cell-based assays
in two Pgp-overexpressing cell lines. Based on the structure of
the natural compound leuconicine A, we have employed molecular
modeling (i.e., docking studies) to maximize ligand-Pgp binding.
Kam and co-workers showed the MDR-inhibiting properties of
structurally novel hexacyclic Strychnos alkaloids (ꢀ)-leuconicine
A (5) and B (6), which were isolated from the Malaysian plant Leu-
conotis maingayi.11 Prior to Kam’s disclosure, we had developed a
novel bis-cyclization method for preparing the tetracyclic core of
the Strychnos alkaloids, and shortly thereafter applied this method
toward the racemic syntheses of akuammicine (1) and strych-
nine.12 Inspired by the architectural complexity of the leuconicines
coupled with their MDR-reversing properties, we initiated a syn-
thetic campaign to prepare these and related analogues to evaluate
bioactivity (Fig. 1).13
Our method allows facile, efficient access to milligram quanti-
ties of analogues of these and other Strychnos alkaloids, in addition
to the ability to test intermediates en route to the targets. We fur-
ther expanded our approach by performing the first asymmetric
syntheses of leuconicines A (5) and B (6). Herein, we describe the
application of this strategy toward the syntheses of classic
alkaloids (ꢀ)-akuammicine (1), (ꢀ)-dihydroakuammicine (2), (ꢀ)-
norfluorocurarine (3), in addition to preparing analogues (ꢀ)-dehy-
droleuconicine B (4) and (ꢀ)-3,4,5-trimethoxybenzyl leuconicine A
(7). Next, we evaluated the Pgp-binding properties of synthesized
alkaloids, along with their potential to reverse Pgp-mediated
MDR using a panel of in vitro and cell-based assays. In all evalua-
tion studies we used verapamil as our standard Pgp competitive
inhibitor.14
2. Results and discussion
2.1. Molecular modeling
A number of pharmacophores have been associated with affin-
ity for Pgp.15 We required a structural tool that would allow us to
N
N
N
H
H
H
H
Et
OMe
OMe
OMe
H
H
H
Me
Me
N
N
N
H
R
O
O
O
R
NH
O
O
1: R=OMe; (Z)-ethylidene =
(−)-akuammicine
4: R=OMe; (Z)-ethylidene =
(−)-dehydroleuconicine B
5: R=OMe; β-ethyl = (−)-leuconicine B
6: R=NH2; β-ethyl = (−)-leuconicine A
7: (−)-3,4,5-trimethoxybenzyl
2: R=OMe; β-ethyl =
(−)-dihydroakuammicine
3: R=H; (Z)-ethylidene =
(−)-norfluorocurarine
leuconicine A
Figure 1. Structures of akuammicine (1), dihydroakuammicine (2), norfluorocurarine (3), dehydroleuconicine
trimethoxybenzyl leuconicine A (7).
B (4), leuconicine B (5) and leuconicine A (6) and
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S. Munagala et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
A
B
C
CH3
Se
D
E
H3C
O
O
N
CH3
CH3
CH3
N
NH
N
CN
i-Pr
HN
N
*
CH3O
CH3O
OCH3
OCH3
Se
Se
H3C
H
N
Verapamil
O
CH3
QZ59-RRR
Figure 2. (A) Homology model of Pgp-glycoprotein (green) docked with leuconicine A (6, magenta); (B) structure of 6 and close-up view of 6 docked to Pgp homology model;
(C) structure of 3,4,5-trimethoxybenzyl leuconicine A (7) and zoomed-in view of 7 docked to homology model; (D) structure of QZ59-RRR; and (E) structure of verapamil.
2.2. Chemistry
(one-pot). Alkylation with (Z)-2-iodobutenyl bromide23 and acyla-
tion with bromoacetyl chloride furnished amide 12 in 83% yield
(two steps).
Cross-metathesis of 12 and methyl acrylate employing 10 mol%
of Hoveyda–Grubbs 2nd-generation (HG-II) catalyst24 gave 13 in
80% yield. The key step was effected with AgOTf and 2,6-di-t-bu-
tyl-4-methylpyridine (DTBMP) followed by DBU in toluene
(dr = 13:1), delivering ABCE tetracycle 14 in 60% yield. Lactam
reduction was realized with thionation with Lawesson’s reagent
The syntheses of alkaloids 1ꢀ3 are outlined in Scheme 1.
Employing the asymmetric method of Yus,22 N-tosyl indole-3-car-
boxaldehyde (8) was treated with (R)-N-tert-butane-sulfinylamine
(9), Ti(OEt)4, allylbromide and In(0) to furnish sulfinamide 10 in
87% yield (dr = 10:1). Removal of the auxiliary and N-tosyl group
was accomplished by sequential treatment with 4 M HCl in diox-
ane followed by magnesium in MeOH to afford 11 in 75% yield
O
S
NH2
HN
t-Bu
CHO
b
a
N
N
H
N
Ts
Ts
10
8
11
O
X
H
Br
N
R
Me
f
N
Me
I
h
c-d
I
H
N
N
H
H
CO2Me
12: R = H
13: R = CO2Me
14: X = O
15: X = S
e
g
N
N
N
H
H
H
H
Me
j
k
I
H
Et
H
H
Me
N
N
N
H
H
H
CO2Me
O
O
R
R
(-)-dihydroakuammicine (2)
1: R=OMe=(-)-akuammicine (87%)
3: R=H =(-)-norfluorocurarine (86%)
16: R = OMe
17: R = H
i
Scheme 1. Synthesis of Strychnos alkaloids 13. Reagents and conditions: (a) (R)-N-tert-butanesulfinamide, Ti(OEt)4 then allyl bromide, In(0), THF, 87% (dr = 10:1); (b) 4 M HCl
in dioxane then Mg(0), MeOH, 75%; (c) Cs2CO3, (Z)-2-iodo-2-butenyl bromide; (d) BrAcCl, Et3N, 83% over two steps; (e) methyl acrylate, 10 mol % Hoveyda–Grubbs 2nd-
generation catalyst, CH2Cl2, 80%; (f) AgOTf, DTBMP then DBU, PhMe, 12 h, 60%; (g) Lawesson’s reagent; (h) Et3OBF4 then NaBH4 in MeOH, 80% over two steps; (i) LiNMe(OMe)
then DIBAL-H, CH2Cl2, 89% over two steps; (j) Pd(OAc)2, PPh3, Et3N; (k) PtO2, H2 (1 atm), MeOH, 85%.
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4
S. Munagala et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
to give 15 followed by Borch25 reduction to afford 16 in 80%
yield.26 Rawal’s intramolecular Heck reaction delivered
(ꢀ)-akuammicine (1) in 87% yield.27 To access (ꢀ)-norfluorocurar-
ine (3), ester 16 was converted to aldehyde 17 using Weinreb’s
method.28 Heck cyclization of 17 gave 3 in 86% yield. Finally,
(ꢀ)-dihydroakuammicine (2) was prepared by hydrogenation of 1
with Adams’s catalyst (85% yield).29
The syntheses of the leuconicines 5 and 6 are shown in
Scheme 2. The pyridone ring in 19 was prepared in 82% yield by
a novel one-pot sequential amidation-intramolecular Knovenagel
condensation via 18. Application of the Heck cyclization tactic
delivered (ꢀ)-dehydroleuconicine B (4) in 81% yield. Chemoselec-
tive reduction of the ethylidene moiety was accomplished with Ra-
Table 1
Affinity of synthetic alkaloids 1–7 and verapamil to purified Pgp, as determined by
intrinsic tryptophan fluorescence quenching
Test compound
ID
Kd
(lM)
Akauammicine
1
2
3
4
5
6
7
VER
99
92
81
16.1
13.8
14.1
4.42
2.40
Dihydroakuammicine
Norfluorocurarine
Dehydroleuconicine A
Leuconicine A
Leuconicine B
3,4,5-Trimethoxybenzyl leuconicine A
Verapamil
ney Ni in 82% yield. Weinreb aminolysis of
5
with
dimethylaluminum amide30 secured (ꢀ)-leuconicine A (5) in 91%
yield.
As previously discussed, inspection of the Pgp homology model
in Figure 2 indicated that the primary amide of leuconicine A (6)
was proximal to a hydrophobic pocket formed by residues F343,
I340, L339 and F336 (i.e., site 1). Docking studies revealed that
analogue 7 was the third highest scoring hit, and its preparation
could be realized from leuconicine A (5). To this end, we treated
5 with 3,4,5-trimethoxybenylamine and trimethylalane to afford
leuconicine A analogue 7 in 79% yield.
2.3. In vitro binding assay
As MDR reversal agents act by binding and inhibiting Pgp, we
quantified the affinity of alkaloids 1ꢀ7 for binding to purified
Pgp. Specifically, we employed an established intrinsic tryptophan
fluorescence quenching method to obtain Kd values.31 The results
Figure 3. The effect of compounds 1–7 (1 mM) on Pgp ATPase activity was assessed
in the presence of recombinant human Pgp using Pgp-Glo assay system, as
described in the experimental section. The results were normalized to basal activity
in the absence of a test compound. Compound 7 had the strongest inhibitory effect
on ATPase activity.
are shown in Table 1. Kd values ranged from ꢁ100
low affinity compounds akauammicine (1) and its dihydro
analogue 2 to 4.4 M for 3,4,5-trimethoxybenzyl leuconcicine A
(7), which had an affinity comparable to that of verapamil. The
alkaloids fell into three groups: low affinity (80–100 M for
compounds 1–3), medium affinity (14–16 M for compounds
lM for the
l
(7) interact with adjacent hydrophobic pockets in site-1 that likely
contribute to their increased potency.
l
l
2.4. In vitro Pgp-associated ATPase assay
4–6) and higher affinity for compound 7 and verapamil. Binding
affinity correlated well with aromatic ring content and molecular
weight (i.e., lipophilicity).1
Overall, the predicted binding poses of the tested compounds
were highly similar, primarily interacting with the aforementioned
aromatic-rich region of site-1 in Pgp. However unlike alkaloids
1–6, both verapamil and 3,4,5-trimethoxybenzyl leuconicine A
In parallel with binding experiments, we tested the effect of
synthesized compounds 1–7 on Pgp ATPase activity associated
with the transport function of Pgp using the Pgp-Glo assay. In this
analysis, ATP is consumed by Pgp ATPase activity to promote trans-
membrane transport of the test compound. The remaining ATP is
N
N
N
H
H
H
Me
Me
Me
I
I
I
H
H
H
b
a
N
H
N
N
CHO
CHO
17
O
O
CO2Me
CO2Me
18
19
N
N
N
H
H
H
H
H
Et
Et
H
OMe
OMe
OMe
c
e
H
H
Me
N
N
N
O
O
NH
O
R
CO2Me
O
O
4
5: R = OMe = (
6: R = NH2 = (
−
)-leuconicine B
)-leuconicine A
d
−
(−
)-3,4,5-trimethoxybenzyl
leuconicine B (7)
Scheme 2. Syntheses of leuconicines A (6), B (5) and 3,4,5-trimethoxybenzyl leuconicine A (7). Reagents and conditions: (a) methyl malonyl chloride, Et3N, CH2Cl2, 82%; (b)
Pd(OAc)2, PPh3, Et3N, 81%; (c) Raney Ni, 82%; (d) NH3, AlMe3, 91%; (e) 3,4,5-trimethoxybenzylamine, AlMe3, 79%.
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S. Munagala et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Figure 4. KB-V20C (Panel A) and KB-MDR (Panel B) cell growth in the presence of compounds 1–7, or verapamil (Ver). Panel
C
shows cytotoxic effect of
3,4,5-trimethoxybenzyl Leuconicine (7) in KB-V20C (D) and KB-MDR (N) cell lines.
then detected by luciferase-generated luminescence signal. The
assay provides a rapid method to detect inhibitory or stimulatory
effects of test compounds at high concentrations (1 mM). Figure 3
summarizes the changes in Pgp ATPase activity in the presence of
verapamil and test compounds 1–7 normalized to basal activity. As
shown in Figure 3, test compounds manifest weak (1, 3, 4, 5) to
moderate (6) stimulatory effects similar to that of verapamil, while
compounds 2 and 7 had an inhibitory effect on ATPase activity. The
Pgp-Glo in vitro assay helped to perform the fast preliminary
screen of synthesized compounds and correctly identified com-
pound 7 as a promising strong inhibitor of Pgp-associated ATPase
activity.
establish the pharmacological characteristics of test compounds,
their cytostatic/cytotoxic effects were first evaluated by the
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay in two cancer cell lines overexpressing Pgp.32 Incubation of
KB-MDR or KB-V20C cells with test compounds 1–7 resulted in a
concentration-dependent decrease of viable cells (Fig. 4A and B).
Importantly, KB-MDR was more resistant to all tested compounds
than KB-V20C. Compound 7 had the highest cytotoxic effect on
KB-V20C cells (IC50 = 0.38 0.032 lM, Fig. 4C). Therefore, in all
subsequent MDR reversal experiments we used compounds 1–7
at concentration that resulted in no more than a 50% decrease of
viable cells after a four day incubation: 1–3
1–6, and 0.07–1.0 M for compound 7.
lM for test compounds
l
2.5. MDR reversal activity in drug-resistant tumor cell lines
To estimate the MDR reversal effect of alkaloids 1–7 on the
sensitivity of Pgp-overexpressing cells KB-V20C or KB-MDR to vin-
cristine and doxorubicin, we treated cells with alkaloids 1–7 at fi-
Finally, we estimated cytotoxic/cytostatic effects, and the
reversal effect of test compounds on the sensitivity of MDR cell
lines to the anticancer drugs vincristine and doxorubicin. To
nal concentrations 0.07–3.0
lM for 1 h. Treatment with 1 lM
verapamil was used as a positive control, and the negative control
Figure 5. Reversal of sensitivity to vincristine in the presence of compound 7. Verapamil was used as positive control. Panel A: KB-V20C, 1
compound 7. Panel B: KB-MDR, 10 M verapamil, and 1 M compound 7.
lM verapamil, and 70 nM
l
l
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S. Munagala et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 2
Cytotoxicity (IC50) of vincristine in KB-V20C and KB-MDR cell lines* over-expressing Pgp in the presence of compounds 1–7
Test compound
ID
Test compound, (
lM)
Vincristine, IC50, (
lM)
IC50, Fold change, VER/test compound
KB-V20C
KB-MDR
KB-V20C
KB-MDR
No inhibitor
Verapamil
Akuammicine
0
1
1
3
1
3
1
3
1
3
1
3
1
3
0.07
0.14
0.28
1
0.05 0.021
0.003 0.0014
0.02 0.005
0.007 0.0012
0.04 0.011
0.017 0.005
0.02 0.008
0.01 0.005
0.02 0.002
0.01 0.004
0.02 0.002
0.01 0.001
0.012 0.004
0.005 0.001
0.005 0.001
0.003 0.001
0.002 0.001
Toxic
1
0.41
VER
1
1.5 0.63
0.6 0.03
0.4 0.03
0.6 0.03
0.4 0.05
0.7 0.11
0.7 0.06
0.7 0.03
0.4 0.05
0.4 0.05
0.22 0.01
0.4 0.03
0.3 0.01
0.7 0.16
0.6 0.07
0.4 0.05
0.016 0.005
1.0
0.2
0.4
0.1
0.2
0.2
0.3
0.2
0.3
0.2
0.3
0.3
0.6
0.6
1.0
1.5
n/a
1.0
2.5
3.8
2.5
3.8
2.1
2.1
2.1
3.8
3.8
6.8
3.8
5.0
2.1
2.5
3.8
93.8
Dihydroakuammicine
Norfluorocurarine
2
3
4
5
6
19,20-Dehydro-
Leuconicine B
Leuconicine B
Leuconicine A
3,4,5-
Trimethoxybenzyl
Leuconicine A
7
*
Parental cell line KB is highly sensitive to vincristine: IC50 = 0.003 lM.
contained no inhibitors. Next, 3-fold serial dilutions of doxorubi-
cin, or vincristine were added to the cells. After a four-day incuba-
tion, cell viability was determined by MTT assay. The IC50 values
for vincristine and doxorubicin were calculated by fitting the
sigmoid Emax model to a plot of cell viability versus drug concentra-
tion (Fig. 5).
used as a lead to further optimize the Pgp inhibitory activity/cyto-
toxicity of the Strychnos scaffold.
4. Experimental section
4.1. Chemistry
Table 2 summarizes the results of MDR reversal experiments in
two cell lines incubated with serial dilutions of vincristine in the
absence of Pgp inhibitor, in the presence of verapamil (positive
control), or different concentrations of compounds 1–7.
Importantly, compound 7 at a concentration of 70 nM increased
the sensitivity of KB-V20C cells to vincristine by an order of mag-
4.1.1. General
All reactions containing water or air sensitive reagents were
performed in oven-dried glassware under nitrogen or argon.
Tetrahydrofuran and dichloromethane were passed through two
columns of neutral alumina. Toluene was passed through one
column of neutral alumina and one column of Q5 reactant. Trieth-
ylamine was distilled from CaH2 prior to use, and 4 Å molecular
sieves were activated by flame-drying under vacuum. AgOTf was
azeotroped with dry toluene prior to use. Compounds 4–6 and
10–17 were prepared according to the procedures of Andrade.9
(Z)-2-Iodobutenyl bromide was prepared according to the proce-
dure of Rawal.19 All other reagents were purchased from commer-
cial sources and used without further purification. All solvents for
work-up procedures were used as received. Flash column chroma-
tography was performed with ICN Silitech 32–63 D 60 Å silica gel
with the indicated solvents. Thin layer chromatography was
performed on Analtech 60F254 silica gel plates. Detection was per-
formed using UV light, KMnO4 stain, PMA stain and subsequent
heating. 1H and 13C NMR spectra were recorded at the indicated
field strength in CDCl3 at rt. The purity of each tested compounds
(>95%) was determined on an Agilent 1200 LC/MS instrument
using a Kinetex 2.6u C18 column (30 ꢂ 2.1 mm, with a flow rate
of 1 mL/min and detection at 254 nm) employing a 5–100% aceto-
nitrile/water/0.1% formic acid gradient.
nitude, and at 1
lM concentration, it increased the sensitivity of
KB-MDR cells to vincristine about 60-fold. At 1
l
M concentration,
compound 7 was approximately 90 times more potent as a MDR
reversal agent by ‘sensitivity index’ (ratio of IC50 for verapamil to
that of compound 7, as shown in the Table 2, right column).
Similarly, in the presence of compound 7 at 1
of KB-MDR cells to doxorubicin increased about 20-fold (IC50
5.7 2.35 M vs 0.3 0.13 M). Interestingly, verapamil and com-
pound 7 demonstrated similar Pgp affinity (Kd of 2.4 and 4.4 M,
lM, the sensitivity
=
l
l
l
respectively, Table 1). The distinct effects of verapamil and com-
pound 7 on Pgp-mediated MDR suggest that their Pgp binding
could invoke different molecular events. We speculate that while
verapamil is a competitive inhibitor of Pgp, compound 7 is possibly
an allosteric regulator of Pgp transport activity. This hypothesis
needs further experimental investigation.
3. Conclusion
We have employed a highly efficient and effective synthetic
route to prepare Strychnos alkaloids and their derivatives thereof
and employed docking studies to design an analogue with en-
hanced potency compared to prototype molecules. Results of
in vitro and cell-based assays identified 3,4,5-trimethoxybenzyl
leuconicine B (7) as a potent Pgp inhibitor and MDR reversal agent.
At a concentration of 70 nM, this compound increased sensitivity
of Pgp-expressing KB-V20C cells to vincristine around 10-fold,
4.1.2. (ꢀ)-Akuammicine (1)
Palladium(II) acetate (7.0 mg, 0.0313 mmol) and PPh3 (16.4 mg,
0.0626 mmol) were added to
a
solution of 16 (47 mg,
0.1044 mmol) in Et3N (5 mL). The reaction mixture was purged
with Argon for 10 min, heated to 90 °C (oil bath) and stirred for
3.5 h. After cooling to rt, the mixture was diluted with CH2Cl2
(25 mL), washed with brine (10 mL), dried (Na2SO4) and filtered.
The solvent was concentrated under reduced pressure, and the res-
idue was purified by flash column chromatography eluting with
MeOH/CH2Cl2 (0.4:9.6 ? 1:9). The material was washed with a
solution of 25% aq NaOH (10 mL), which afforded 24 mg (71%) of
and at 1 lM it increased sensitivity of Pgp-expressing KB-MDR
cells to doxorubicin about 90-fold. Our data on MDR reversal in
the human cancer cells overexpressing Pgp suggest that the poten-
tial of compound 7 as a strong inhibitor of vincristine efflux from
tumor cells needs to be further investigated. Compound 7 will be
Please cite this article in press as: Munagala, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.022

S. Munagala et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
1 as white solid whose NMR spectra (1H and 13C) were identical
4.3. General procedure for in vitro Pgp-associated ATPase assay
with reported literature values.33 The optical rotation for synthetic
material was: ½a D20
ꢃ
ꢀ769 (c 0.4, CHCl3) whereas the literature value
The inhibitory or stimulatory effect of a test compound on ATP-
ase activity of human recombinant Pgp was assessed in vitro using
the Pgp-Glo Assay System (Promega, WI). Briefly, recombinant hu-
man Pgp membranes were incubated with ATP in the presence of
0.3 mM sodium vanadate (an ATPase inhibitor), 0.5 mM verapamil
(and ATPase stimulator), or 0.3–1 mM of the test compounds. The
reaction was initiated by adding ATP to a final concentration
5 mM. After 40 min incubation at 37 °C, the reaction was stopped
by addition of ATP detection reagent, and the amount of residual
ATP was estimated by measuring luciferase-generated biolumines-
cence. All reactions were replicated three times, in two indepen-
dent experiments. The difference in sample luminescence was
used to calculate the effect of a test compound on Pgp ATPase
activity.
was: ½a 2D0
ꢃ
ꢀ737 (c 0.5, CHCl3).34
4.1.3. (ꢀ)-Norfluorocurarine (2)
Palladium(II) acetate (24.1 mg, 0.107 mmol) and PPh3 (56.2 mg,
0.214 mmol) were added to a solution of 17 (150 mg, 0.357 mmol)
in Et3N (18 mL). The reaction mixture was purged with Argon for
20 min, heated to 90 °C (oil bath) and stirred for 3.0 h. After cooling
to rt, the mixture was diluted with CH2Cl2 (25 mL), washed with
brine (10 mL), dried (Na2SO4) and filtered. The solvent was concen-
trated under reduced pressure, and the residue was purified by
flash column chromatography eluting with MeOH/CH2Cl2
(0.4:9.6 ? 1:9). The material was washed with a solution of 25%
aq NaOH (10 mL), which afforded 90 mg (86%) of 2 as yellow liquid
whose NMR spectra (1H and 13C) were identical with reported lit-
erature values.35 The optical rotation for synthetic material was:
4.4. General procedure for MDR reversal assay
½
a 2D0
ꢃ
ꢀ1184 (c 0.5, CHCl3) whereas the literature value was: ½a D20
ꢃ
ꢀ1230 (c 0.5, CHCl3).31
Growth inhibition assays were performed in three cell lines, the
parental human oral epidemoid carcinoma KB cells, and two lines
(KB-MDR and KB-V20C) overexpressing Pgp. KB-MDR was gener-
ated by infection of KB with a retroviral vector carrying the human
mdr-1 gene, and was grown in the presence of 37 nM doxorubicin.
KB-V20C cell line was developed from the parental KB cells by
stepwise selection for resistance with increasing concentrations
of vincristine (VCR), and was grown in the presence of 20 nM
VCR. All three cell lines were a generous gift from Dr. Yung-Chi
Cheng (Yale University School of Medicine) and were maintained
in RPMI1640 medium supplemented with 10% FBS, antibiotic/anti-
mycotic, and cytotoxic drug (DOX or VCR) where indicated. Cell
growth and viability were measured by flow cytometry with Guava
Personal Cell Analyzer (PCA, Guava Technologies, Hayward, CA)
using ViaCount reagent (Millipore, Hayward, CA), as described ear-
lier.37 Cells were seeded in 96-well plate at 1000 cells (KB-MDR) or
2000 cells (KB-V20C) per well and treated with test compounds at
4.1.4. (ꢀ)-19,20-Dihydroakuammicine (3)
Excess PtO2 (10 mg) was added to a solution of (ꢀ)-akuammi-
cine (1) (11 mg, 0.0341 mmol) in CH3OH (4 mL). The reaction
mixture was stirred for 12 h under a H2 atmosphere, filtered
through a short bed of Celite (pre-wet with CH3OH) and the solvent
was evaporated under reduced pressure. The residue was purified
by flash column chromatography eluting with MeOH/CH2Cl2
(0.5:9.5), to give 10.5 mg (95%) of 3 as light yellowish oil whose
NMR spectra
(1H and 13C) were identical with reported literature values.36
The optical rotation for synthetic material was: ½a D20
ꢃ
ꢀ508 (c
0.55, CH3OH) whereas the literature value was: ½a D20
ꢃ
ꢀ568 (c
0.175, CH3OH).32
4.1.5. (ꢀ)-3,4,5-Trimethoxybenzyl leuconicine A (7)
To a stirred solution of Me3Al (0.31 mL, 2 M in toluene,
concentrations 1, 3, 10 lM for four days. For the MTT (3-(4,5-
0.63 mmol) in CH2Cl2 (3 mL) at ꢀ15 °C was added 3,4,5-trimeth-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay (CellTiter
96 cell proliferation kit, Promega, WI), cells (1000–2000 cells per
well) were plated into 96-well plates, and cultured for 4 days in
varying concentrations of the test compounds. After incubation,
MTT reagent was added to each well, and endpoint data collected
by a SpectraMax M2 microplate spectrophotometer (Molecular De-
vice, CA) according to the manufacturer’s instructions. The IC50 val-
ues were calculated using GraphPad Prism (GraphPad Software
Inc., CA) by fitting a sigmoid Emax model to the cell viability versus
drug concentration data, as determined in duplicate from three
independent experiments.
oxybenzyl amine (93 lL 0.55 mmol). The reaction mixture was
stirred for 20 min then warmed to rt. Stirring was continued for
an additional 1 h. (ꢀ)-Leuconicine B (5) (40 mg, 0.11 mmol) dis-
solved in CH2Cl2 (2 mL) was added, and the reaction mixture was
refluxed for overnight. After cooling to rt, the reaction was
quenched with aq 1 N HCl (2 mL), stirred for 30 min, and extracted
with CHCl3 (3 ꢂ 10 mL). The combined organic layers were washed
with brine (1 ꢂ 10 mL), and dried over Na2SO4. The solvent was
concentrated under reduced pressure, and the residue was purified
by flash column chromatography eluting with MeOH/CH2Cl2
(1.0:9.0) to give 42 mg (79%) of 7. ½a D25
ꢃ
ꢀ424 (c 0.9, CHCl3); IR
(neat) 3760, 3010, 2980, 1760, 1691, 1100, 730 cmꢀ1
;
1H NMR
5. Disclaimer
(400 MHz) d 10.07 (t, J = 5.6 Hz, 1H), 8.41 (dd, J = 8, 0.8 Hz, 1H),
8.25 (s, 1H), 7.39–7.33 (m, 2H), 7.29 (dd, J = 7.6, 1.2 Hz, 1H), 6.61
(s, 2H), 4.60 (t, J = 6.6Hz, 2H), 4.02 (t, J = 2.4 Hz, 1H), 3.80 (s, 6H),
3.75 (s, 3H), 3.11–3.08 (m, 1H), 2.95 (dd, J = 6.2, 3.2 Hz, 1H),
2.86–2.79 (m, 3H), 2.13 (dt, J = 12.4, 2.8 Hz, 1H), 1.94–1.80 (m,
3H), 1.44–1.40 (m, 1H), 1.30 (dt, J = 13.6, 3.2 Hz, 1H), 1.25–1.18
(m, 1H), 0.99 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz) d 164.3,
162.0, 160.7, 153.6, 145.2, 140.9, 140.3, 134.8, 128.4, 127.3,
120.6, 120.5, 117.7, 116.2, 105.0, 77.9, 62.5, 61.0, 56.3, 55.8, 54.7,
51.8, 45.2, 44.0, 39.0, 36.5, 31.6, 29.9, 26.7, 11.7; HRMS(FAB) calc’d
for C32H35N3O5+H = 542.2655, found 542.0376.
The opinions, findings, conclusions, or other recommendations
expressed herein are the private views of the authors and do not
necessarily reflect the views of the U.S. Department of Defense or
the U.S. Defense Threat Reduction Agency. This paper has been
approved for public release with unlimited distribution.
Acknowledgments
We are grateful to Dr. Yung-Chi Cheng (Yale University School
of Medicine) for the generous gift of KB, KB-V20C, and KB-MDR cell
lines. We thank Dr. Richard Pederson (Materia, Inc.) for catalyst
support. Finally, this research was supported by the National Sci-
ence Foundation (CHE-1111558) and the Drug Discovery Initiative
(DDI) Grant from the Moulder Center for Drug Discovery, Temple
University (RA, recipient). Additional funding was provided by
4.2. General procedure for in vitro binding assay
To obtain Kd values, an established intrinsic tryptophan fluores-
cence quenching method was used as described elsewhere.27
Please cite this article in press as: Munagala, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.022

8
S. Munagala et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
15. Pearce, H. L.; Safa, A. R.; Bach, N. J.; Winter, M. A.; Cirtain, M. C.; Beck, W. T.
Proc. Natl. Acad. Sci. 1989, 86, 5128.
the Defense Threat Reduction Agency through projects TMTI0
004.09.BH.T (AW, recipient).
16. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
17. Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.; Harrell, P. M.;
Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G. Science 2009, 323, 1718.
18. Jacobson, M. P.; Pincus, D. L.; Rapp, C. S.; Day, T. J.; Honig, B.; Shaw, D. E.;
Friesner, R. A. Proteins 2004, 55, 351.
19. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
Supplementary data
Supplementary data (1H and 13C NMR spectra of 1, 2, 3, and 7)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2013.12.022.
20. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W.
T.; Banks, J. L. J. Med. Chem. 2004, 47, 1750.
21. Ding, P. R.; Tiwari, A. K.; Ohnuma, S.; Lee, J. W.; An, X.; Dai, C. L.; Lu, Q. S.; Singh,
S.; Yang, D. H.; Talele, T. T.; Ambudkar, S. V.; Chen, Z. S. PLoS ONE 2011, 6,
e19329.
22. Gonzalez-Gomez, J. C.; Medjahdi, M.; Foubelo, F.; Yus, M. J. Org. Chem. 2010, 75,
6308.
23. Rawal, V. H.; Michoud, C. J. Org. Chem. 1993, 58, 5583.
24. Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H. J. Am. Chem. Soc. 2000,
122, 8168.
References and notes
1. Eckford, P. D. W.; Sharom, F. J. Chem. Rev. 2009, 109, 2989.
2. Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer 2002, 2, 48.
3. Tsuruo, T.; Iida, H.; Tsukagoshi, S.; Sakurai, Y. Cancer Res. 1981, 41, 1967.
4. Futscher, B. W.; Foley, N. E.; GleasonGuzman, M. C.; Meltzer, P. S.; Sullivan, D.
M.; Dalton, D. S. Int. J. Cancer 1996, 66, 520.
5. Palmeira, A.; Sousa, E.; Vasconcelos, M. H.; Pinto, M. M. Curr. Med. Chem. 2012,
19, 1946.
6. Szakács, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M.
Nat. Rev. Drug Disc. 2006, 5, 219.
7. List, A. F.; Kopecky, K. J.; Head, D. R.; Persons, D. L.; Slovak, M. L.; Dorr, R.;
Karanes, C.; Hynes, H. E.; Doroshow, J. H.; Shurafa, M.; Appelbaum, F. R. Blood
2001, 98, 3212.
8. Wattel, E.; Solary, E.; Hecquet, B.; Caillot, D.; Ifrah, N.; Brion, A.; Milpied, N.;
Janvier, M.; Guerci, A.; Rochant, H.; Cordonnier, C.; Dreyfus, F.; Veil, A.; Hoang-
Ngoc, L.; Stoppa, A. M.; Gratecos, N.; Sadoun, A.; Tilly, H.; Brice, P.; Lioure, B.;
Desablens, B.; Pignon, B.; Abgrall, J. P.; Leporrier, M.; Dupriez, B.; Guyotat, D.;
Lepelley, P.; Fenaux, P. Adv. Exp. Med. Biol. 1999, 457, 35.
9. Tamaki, A.; Ierano, C.; Szakács, G.; Robey, R. W.; Bates, S. E. Essays Biochem.
2011, 50, 209.
10. Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem. Rev. 2009, 109, 3012.
11. Gan, C.-Y.; Low, Y.-Y.; Etoh, T.; Hayashi, M.; Komiyama, K.; Kam, T.-S. J. Nat.
Prod. 2009, 72, 2098.
12. Sirasani, G.; Andrade, R. B. Org. Lett. 2009, 11, 2085.
13. Sirasani, G.; Andrade, R. B. Org. Lett. 2011, 13, 4736.
14. Woodland, C.; Koren, G.; Wainer, I. W.; Batist, G.; Ito, S. Can. J. Physiol.
Pharmacol. 2003, 81, 800.
25. Borch, R. F. Tetrahedron Lett. 1968, 9, 61.
26. Raucher, S.; Klein, P. Tetrahedron Lett. 1984, 21, 4061.
27. Rawal, V. H.; Michoud, C. Tetrahedron Lett. 1991, 32, 1695.
28. Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.
29. Bonjoch, J.; Solé, D.; García-Rubio, S.; Bosch, J. J. Am. Chem. Soc. 1997, 119, 7230.
30. Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977, 18, 4171.
31. Liu, R.; Siemiarczuk, A.; Sharom, F. J. Biochemistry 2000, 9, 14927.
32. Burton, J. A. Methods in Molecular Medicine Vol. 110: Chemosensitivity: Vol. 1.
In In Vitro Assays; Blumenthal, R. D., Ed.; Humana Press: Totowa NJ, 2005; pp
69–78.
33. Jones, S. B.; Simmons, B.; Mastracchio, A.; MacMillan, D. W. C. Nature 2011, 475,
183.
34. Henry, A. T.; Sharp, M. T. J. Chem. Soc. 1927, 1950.
35. Stauffacher, D. Helv. Chim. Acta 1961, 44, 2006.
36. Amat, M.; Coll, M. D.; Bosch, J.; Espinosa, E.; Molins, E. Tetrahedron: Asymmetry
1997, 8, 935.
37. Phadke, M.; Krynetskaia, N.; Krynetskiy, E. Anticancer Drugs 2013, 24, 366–374.
Please cite this article in press as: Munagala, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.022