Medicinal chemistry of dihydropyran-based medium ring macrolides related to aspergillides: Selective inhibition of PI3Kα

Article abstract of DOI:10.1021/jm400515c

A set of nine trans-disubstituted dihydropyran-based medium ring macrolides has been synthesized using d-glucal as chiral pool and evaluated against a panel of three human cancer cell lines and a normal cell line. The synthetic route to the targeted molecule is simple, concise, and high yielding compared to other reported methods. Bioevaluation studies have resulted in the identification of a potent cytotoxic molecule (10) exhibiting dose-dependent growth inhibition against HL-60 cell line with an IC₅₀ value of 1.10 ± 0.075 μM, which is lower than that of naturally occurring molecules of this class and of comparable activity to the synthetic drug fludarubin. Compound 10 inhibits the PI3K/AKT signaling pathway by selectively targeting the p110α subunit of PI3Kα. This leads to mitochondrial stress that causes translocation of cytochrome c from mitochondria to cytosol, which in turn activates caspase-mediated apoptotic cell death. Further in silico docking simulations of four macrolides with p110α subunits have been carried out to visualize the orientation pattern.

Full text of DOI:10.1021/jm400515c

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Article
Medicinal chemistry of dihydropyran based medium ring macrolides
related to aspergillides: selective inhibition of PI3K-alpha#
Mallikharjuna Rao Lambu, Suresh Kumar, Syed Khalid Yousuf, Deepak K
Sharma, Altaf hussain, Ajay Kumar, Fayaz Malik, and Debaraj Mukherjee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400515c • Publication Date (Web): 11 Jul 2013
Downloaded from http://pubs.acs.org on July 18, 2013
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Medicinal chemistry of dihydropyran based medium
ring macrolides related to aspergillides: selective
inhibition of PI3Kα!
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Mallikharjuna R. Lambu, 1,2 Suresh Kumar,1,2 Syed K. Yousuf,2 Deepak K. Sharma,1,2
Altaf Hussain, 1, 2 Ajay Kumar,2 Fayaz Malik,*1,2 Debaraj Mukherjee.*1,2
1Academy of Scientific and Innovative Research, 2CSIRꢀIIIM
Corresponding
authors:
Tel.:
(+) 91ꢀ011ꢀ256ꢀ9000,
Fax: (+)ꢀ91ꢀ011ꢀ256ꢀ9111.
email.dmukherjee@iiim.ac.in, fmalik@iiim.ac.in
Keywords. Macrolides, triꢀOꢀacetylꢀDꢀglucal, ring closing metathesis (RCM), apoptosis,
PI3K/AKT.
Abstract: A set of nine trans disubstituted dihydropyran based medium ring macrolides has been
synthesised using ꢀ glucal as chiral pool and evaluated against a panel of three human cancer
D
cell lines and a normal cell line. The synthetic route to the targeted molecule is simple, concise
and high yielding compare to other reported methods. Bio evaluation studies have resulted in the
identification of potent cytotoxic molecule (10) exhibiting dose dependent growth inhibition
against HLꢀ60 cell line with IC50 value of 1.10± 0.75ꢁM which is lower than the naturally
occurring molecules of this class and comparable activity to the synthetic drug fludarubin. The
compound 10 inhibits PI3K/AKT signaling pathway by selectively targeting p110α subunit of
PI3Kα. This leads to mitochondrial stress that causes translocation of cytochrome c from
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mitochondria to cytosol, which in turn activate the caspases mediated apoptotic cell death.
Further in silico docking simulations of four macrolides with p110α subunits have been carried
out to visualize the orientation pattern.
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Introduction
Bioactive natural products have had a dramatic impact on medicine and society.1 They display a
seemingly endless structural diversity but are often found only in miniscule quantities.
Inspiration from natural products has also brought new perspectives to organic synthesis and
chemical biology.2 The systematic modification of natural products through diverted total
synthesis is a powerful concept for the editing of natural products with the aim of studying
mechanistic aspects of their biological activity. This concept offers farꢀreaching opportunities for
discovery at the interface of biology and chemistry.3 Nonetheless, implementation of this type of
design require the development of new synthetic strategies in order to modify the structure at
will.4
Macrocycles represent one of the universal structural features of innumerable number of
biologically significant natural products.5 The structural motifs are highly selective and potent,
they are conformationally preorganized, enabling them to bind selectively to targets with
minimal entropic loss.6 In fact these are the smallest examples of bio molecules that exhibit
functional subunits. Aspergillides (Figure 1) comprise a novel class of medium ring (14ꢀ
membered) macrolides.7 These were isolated from a marineꢀderived fungus Aspergillus ostianus
strain 01F313 cultivated in a bromine modified medium7 and are characterised by the presence of
triꢀsubstituted tetrahydro (1 and 2 in Figure 1) and dihydrofuran (3 in Figure 1) units.
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O
O
O
O
O
O
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O
O
O
H
H
H
H
H
H
HO
HO
HO
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1
3
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Figure 1. Structures of aspergillides A (1), B (2) and C (3)
These structurally intriguing molecules exhibit cytotoxicity against mouse lymphocytic
leukemia cells (L1210) in micromolar range.8 Owing to their structural features and promising
biological properties, aspergillides have always attracted the attention of organic chemists as
targets for total synthesis.8 Although total synthesis of aspergillides A and B are well
documented along with limited SAR studies,9 there are only a few reports of total synthesis of
aspergillide C with no structural analogues known.10 In a programme directed towards the
synthesis of medium ring macrocyles,11 herein we would like to report our study towards the
synthesis of a library of medium sized macrocycles fused to a dihydropyran framework and
thereafter evaluation of most potent cytotoxic analogs against specific cancer target. Although,
the antiproliferative activity of macrolides aspergillide against different cancer cell lines has
already been reported, 9c but the molecular mechanism and the selective cancer driving targeting
pathways behind this remain unexplored. It has now become evident that several kinase
pathways including phosphatidylinositol 3ꢀkinase (PI3K) and the Raf/mitogenꢀactivated
extracellular signalꢀregulated kinase (ERK) are critical for normal human physiology, and also
commonly dysregulated in several human cancers.12 A large number of studies have shown that
the aberrant activation of PI3K/Akt is involved in many human cancers and is responsible for cell
proliferation, tumor growth and invasion of cancer cells.13 It has been suggested that the
Inhibition of the PI3K/Akt pathway through small molecules might be a promising strategy for
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cancer treatment by inhibiting tumor cell growth and proliferation.14 An effort has been made to
investigate the effect of ring size, stereo diversity and position of double bond of synthesized
macrolides on their targeting crucial tumor promoting pathways and antiproliferative activity
against different cancer cell lines and to explore the mode of action and of the most active
compound. We used in silico approach to check the binding affinities of the compounds towards
critical tumor driving p110α subunit of PI3K, which was confirmed through the wet lab
experiments with detailed mechanism of action.
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Keeping in mind the basic skeleton of aspergillide macrocycles, we proposed two types of
structural variants 4 and 5 (Figure 2).
Same stereochemistry and same group
O
O
O
H
H
RO
4
O
O
Same ring size but inverse
stereochemistry at ring junction
and shifted double bond
O
H
H
Reverse stereochemistry and different group
HO
3
R
Aspergillide C
14-membered
O
O
O
H
H
R'O
5-12
One carbon less than original,
with inverse stereochemistry at ring junction
and shifted double bond
Figure 2. Structures of proposed aspergillide based stereodivergent medium ring macrocycles
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The challenges from a synthetic point of view are construction of the bridged 2,6ꢀtransꢀ
trisubstituted pyran and cyclisation of the bridged 14ꢀmembered macrocyclic core with an olefin
at Cꢀ8/Cꢀ9, which is rare in natural product. It was observed that most reported methods centers
on synthesis of 2,6ꢀtransꢀdisubstituted pyran aldehyde/masked aldehyde derivatives (Figure 3),
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9b, 10a,10b
which can be obtained from non carbohydrate/carbohydrate precursors followed by
olefination of that aldehyde with a chiral alcohol to generate Cꢀ8/Cꢀ9 double bond and finally
macrolactonisation of that alcohol with the secoꢀacid. Although the synthesis of these class of
compound has been made, all the methods suffer from longer reaction sequence with low overall
yield. Moreover there is no scope of diversifying the macrolides as designed by us in figure 2.
.
EtO
O
CHO
OTBDS
O
CHO
O
MeOOC
ref.9b
AcO
AcO
O
OAc
OAc
O
O
MOMO
ref.10a
O
+
OAc
N
Ferrier type reaction
N
TBSO
HO
N
O
N
S
O
O
O
O2
O
+
O
ref.10b
O
O
TMSO
Oxidation
H
lactonisation
Figure 3. Prior art to the synthesis of 2,6ꢀ transꢀdisubstituted pyran aldehyde derivatives
In order to reduce the number of steps and diversify the core we have chosen ꢀglucal as chiral
D
pool for our studies considering our previous experience15 working with it. The synthetic strategy
for the medium sized ring macrolides is illustrated in scheme 1.
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Scheme 1. Proposed retrosynthetic approach for medium sized ring macrolides
5
6
7
8
9
R
1
R
R
n
n
n
O
O
3
OH
19, 27-32
O
O
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+
O
H
7
O
O
H
AcO
AcO
O
OH
H
RO
RO
O
4-10
OAc
20, 37-42
H
RO
Stereodivergent analogue
13-14-membered
Tri-O-acetyl-D-glucal (A)
18, 35-36
n = 0,1
The strategy centres on a Lewis acid mediated stereoselective Cꢀallylation of the readily
available triꢀOꢀacetylꢀ ꢀglucal (A) to fix trans stereochemistry at Cꢀ3 and C7 (oppsite
D
stereochemistry relative to parent molecule). The macrocycles could be assembled by using a
sequential EDC coupling of a alcohols of appropriate size (19, 27-32) with the corresponding
sugar derived acids (18, 35-36) and ring closing metathesis of the diꢀolefinic compounds (20, 37-
42).
Results and discussions:
Chemistry. In order to achieve trans stereochemistry at ring juncture of pyran ring we started
our investigation with αꢀCꢀallylation of triꢀOꢀacetylꢀ ꢀglucal (A). As anticipated αꢀallyl
D
glycoside (13) was obtained in good yield with high stereoselectivity (α:β, 95:5). Subsequent
regioselective protection of primary hydroxyl by tosyl group afforded 15, nucleophilic
substitution of –OTs compound with NaCN provided cyano derivative 16.
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Scheme 2. Synthesis of 14ꢀmembered macrolide 4a
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aReagents and conditions: (a) Allyl trimethylsilane (1.2 equiv .), BF3.Et2O (1.2 equiv .) ꢀ10 oC,
3h, 89%; (b) NaOMe, MeOH, rt, 0.5h, 98%; (c) Tosyl chloride (2.5 equiv .) dry pyridine, 0 oCꢀrt,
o
8h, 65%; (d) NaCN (2.0 equiv .), DMSO, 70 C, 4h, 85%; (e) Ethyl iodide (1.2 equiv .), NaH
o
(1.5 equiv.), 0 C, 2h, 90%; (f) 8N NaOH + EtOH (1:1), reflux, overnight, 75%; (g) EDC (1.2
equiv .), DMAP (0.1 equiv .), DCM, rt, 8h, 78%.
After protection of free hydroxyl group as ethyl ether, compound 17 was subjected to cyanide
hydrolysis to obtain the key carboxylic acid derivative (18) required for esterification. However,
hydrolysis of 17 failed to proceed smoothly and caused degradation of starting material. Careful
optimization of hydrolysis condition (see SI table1) eventually afforded trans 2,6 disubstituted
DHP fragment 18 in satisfactory yield. EDC mediated esterification of acid 18 with optically
pure alcohol 19 generated diolefinic compound 20 ready for final macrolide synthesis. Ring
closing metathesis (RCM) of 20 in presence of 10 mol % of Grubb’s II catalyst at 40 oC failed to
yield the desired cyclisation product. Nonetheless, after rigorous optimisation of reaction
conditions, it was found that the concentration of the substrate plays a critical role in the RCM
step (Table 1). It is noteworthy that 3 mM DCM solution of 20 gave the desired RCM product 4
with 68% yield. The structure of the product 4 was determined through spectroscopic analysis.
1
The disappearance of peaks between 4.98ꢀ5.12 present in the H NMR spectrum of 20 for two
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terminal alkene –CH2’s and appearance of new peaks between 5.50ꢀ5.34 with J = 11.2 Hz (along
5
6
13
with peaks at 126.5 and 125.1 in C NMR for the newly generated alkene of 4 indicated the
7
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9
formation of macrolide 4. Small coupling constant (always <12 Hz) clearly indicated the
preponderance of Zꢀisomer. The ratio of E/Z isomers was deducible from the 1H NMR spectrum.
Table 1. Standardisation of reaction conditions for RCM reaction
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O
O
O
O
O
O
H
H
EtO
EtO
4
20
Entry Catalysta
(mol %)
Solvent
Temp.
Yield %b
(Z/E)c
(Molar conc. of (0C)
substrate)
1
10
5
1
5
5
5
5
5
5
5
5
DCM (15)
DCM (15)
DCM (15)
DCM (10)
DCM (5)
DCM (3)
DCM (3)
DCM (3)
DCM (3)
DCM (3)
Toluene (15)
40
40
40
40
40
40
30
20
0
Nil
2
Nil
3
6 (50:50)
16 (50:50)
20 (60:40)
35 (75:25)
42 (85:15)
55 (90:10)
38 (99:1)
68 (99:1)
Nil
4
5
6
7
8
9
10
11
0-20
40
aIn all cases Grubbs II generation catalyst was used. bIsolated yield after
c
column chromatography. Determined from 1H NMR .
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Encouraged by the ease in accomplishing the stereodivergent synthesis of 14ꢀmembered
macrolide 4, an aspergillide C analogue, we next directed our attention towards the synthesis of a
set of medium ring macrolides for SAR. Thus, using the same sequence of reactions but by
varying the alcohol partner, which was generated from literature procedure.16 It is noteworthy
that changing the protection from methoxy or ethoxy to PMB (35) leads to increased yield of
acid during cyanide hydrolysis. We generated the analogues of 4 with one carbon less (Figure 4).
The compound 5 shows the small coupling constant (always <12 Hz) clearly indicated the
preponderance of Zꢀisomer, the stereo chemistry at Cꢀ12 position of the macrolide 5 confirmed
by the NOSEY correlation between Hꢀ12 and Hꢀ3. This confirms proposed structures of the 13
membered macrolides.
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Compounds 11 and 12 were obtained in quantitative yield (>90) by DDQ mediated removal of
the PMBꢀprotecting group of 7 and 9 respectively
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allyl trimethylsilane
(1.1 equiv.)
OH
O
R
R
H
FeCl3 (5 mol %), DCM
1h, 90-95%
27-32
21-26
8
9
Br
Cl
F
OMe
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CF3
R
21
22
23
24
25
26
CN
COOH
O
8N NaOH:EtOH
(1:1)
O
27-32, DCM,
O
O
EDC (1.2 equiv.)
DMAP (0.1 equiv.)
rt, 8-10 h
RO
reflux, overnight
RO
O
R = OEt (17),
OMe, (33)
18, 35-36
RO
OPMB (34)
37-42
R
3 mM DCM solution of
5mol% Grubb's II
50% of starting material
consumed
O
O
O
H
H
RO
5-10
Cl
F
Br
MeO
O
O
O
O
O
O
O
O
O
H
O
O
H
O
H
H
H
H
H
H
EtO
PMBO
MeO
EtO
5
8
6
7
CF3
F
O
O
O
O
O
O
O
O
O
O
H
O
H
H
O
H
H
H
PMBO
H
H
HO
HO
MeO
12
9
11
10
Figure 4. Library of medium sized pyran based macrocycles
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Molecular modeling study:
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Docking simulations were performed to predict the binding modes of molecules with target
protein P110α subunit of PI3K. Docking studies reveals that compounds 10 and 5 having alkoxy
protection (OMe, OEt respectively) interacts with Glnꢀ859 and Tyrꢀ836 residues respectively via
hydrogen bonding at the catalytic site of the p110α (Figure 5). In addition, hydrophobic
interactions between Trpꢀ780, Ileꢀ932 and Metꢀ922 and aromatic rings of the dihydroꢀpyran
based macrocycles are driving force for P110 α binding motifs (Figure 5). However, compound
12 (having hydroxyl group at Cꢀ4) adopts different binding pattern and orients toward the Serꢀ
854 residue (Figure 5). Similarly the compound 9 which bears OꢀPMB functionality at Cꢀ4
adopts complete opposite orientation by 180o (Figure 5) thus allowing it to bind with Valꢀ850 of
hinge region instead of Glnꢀ859 or Tyrꢀ836 residues of P110α kinase domain.
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In order to predict the most suitable analog which can be an inhibitor of P110α, we calculated the
prime ligand binding energies (ꢂG) of different synthesized analogues by end point MMGB/SA
method and compared with known PI3K inhibitors. ꢂG value of the compound 10 (ꢀ73.96
Kcal/mol) docked with P110α was found to be comparable with known PI3K inhibitor Liphagal
(ꢀ77.27 Kcal/mol). Compound 10 showed lower binding affinity towards P110β (ꢀ58.05
Kcal/mol) and P110δ (ꢀ49.78 Kcal/mol) which was further confirmed by docking simulation
study (Section D in SI).
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Compound 5
Compound 10
Compound 9
Compound 12
Figure 5. Docking simulations of the compounds 10, 5, 9, and 12 with target protein PI3Kꢀα
Biology. All the newly synthesised macrolides were assayed against various cancer cell lines
including HLꢀ60 (acute promyelocytic leukaemia), PCꢀ3 (human prostate cancer cell line), and
A375 (human amelanotic melanoma cell line) for their antiproliferative activity. It was found that
all compounds exhibited antiproliferative effects at micromolar concentrations (Table 2).
Table 2. Growth inhibitory activity of the macrocycles against various cancer cell lines
S. No. Compound IC 50 in HLꢀ60(ꢁM) IC50 in PCꢀ3(ꢁM) IC50 in A375(ꢁM) IC50 in FR2 (ꢁM)
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69.74± 1.54
78± 0.76
57± 2.67
ND
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9
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3
5.7± 0.863
10.00± 0.73
8.83± 0.87
69.9+ 0.92
10.23± 0.24
1.10± 0.75
40.23± 2.13
30.92± 0.873
81.2± 17.5
32.8± 7.6
9.16± 0.63
15.76± 1.82
2.23± 0.96
56.00+ 0.45
45.4± 1.34
7.36± 00.37
35.80± 3.23
21.5± 1.23
ND
4.9± 0.12
5.45± 2.76
4.76± 1.87
62.7± 1.12
>100
ND
ND
ND
ND
ND
≥ 100
ND
ND
ND
ND
5
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10
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4
7
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9
7
9.64± 0.35
20.76± 1.34
50.00± 0.54
ND
10
11
12
19c
29c
8
9
10
11
ND
ND
The IC50 values, defined as the drug concentration required for inhibiting cell proliferation by
50%, were calculated from the curve of concentration dependent survival percentage, itself
defined as absorbance in experimental wells compared with absorbance in control wells after
subtraction of the blank values. It was found that compound 10 is most active among others and
we concluded that HLꢀ60 cell line is more sensitive than PCꢀ3 and A375 for the compound 10.
While compound 10 shows potential cytotoxicty against cancer cell lines, we also tried to
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evaluate its selective killing by observing it’s effect on Human normal breast epithelial cell line
“FR2”. It was observed that compound 10 confer selective cytotoxicity towards cancer cells
while as its IC50 value in FR2 cells was not attained till 100 ꢁM concentration. In general,
electron withdrawing substituents in the aromatic moiety located at Cꢀ12 position lead to the
enhanced antiproliferative activity with CF3 group in compound 10 as most potent followed by
halogen substituents like F, Cl and Br in the compounds 7, 5, 6 respectively. Presence of a free
hydroxyl at Cꢀ4 decreases the activity, but smaller alkoxy substituents (Me > Et > PMB) lead to
more activity as found in 10. It is more like lipophilic driven SAR. In other word, potency may
gradually increase when the lipophilicity increases. Introduction of biphenyl group increases
lipophlicity significantly in compound 10 (clogP = 4.65) and thus improves potency. Similarly,
in compound 11, introduction of a polar OH motif with one hydrogen donor lowers lipophilicity
(clogP = 1.87) and hence potency decreases compared to compound 9 (clogP = 4.82). In order to
get some mechanistic insight of the mode of action of compound 10 in HL60 cell lines, few more
experiments such as phase contrast microscopy, Hoechst staining for nuclear morphology,
Mitochondrial membrane potential loss, Annexine V/PI staining, Cell cycle phase distribution
and western blotting analysis have been carried out.
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Phase contrast microscopy
Phase contrast microscopy was done to observe the morphological changes. Significant changes
in morphology of cell were observed. The changes increased with increasing dose of compound
10. Treatment with 5 ꢁM of compound 10 showed blebbing and shrinkage of cells (Figure 6);
the amount of apoptosis (blebbing and shrinkage) increased significantly in cells treated with 20
ꢁM of compound 10 (Figure 6).
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Figure 6. Compound 10 induced cell death in HLꢀ60. Cells were treated with indicated
concentrations of compound 10 and observed for morphological changes under microscope (1 ×
81, Olympus).
Hoechst staining for nuclear morphology
HLꢀ60 cells treated with different concentrations of compound 10 displayed pronounced
changes in nuclear morphology; the change in morphology of nucleus increased with increasing
concentrations (Figure 7).
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Figure 7. Alteration in nuclear morphology by compound 10 treatment. HLꢀ60 cells were
incubated with different concentrations of compound 10, collected at 400 g, washed once with
PBS and then stained with Hoechst 33258 for 30 min. The procedure is discussed in materials
and methods section.
Mitochondrial membrane potential loss
Mitochondrial integrity is required for cell to be functional and mitochondrial potential loss is
considered as one of the major causes of cell death. Compound 10 induced MMP loss which
increased with increasing doses. The loss was significant at 5 ꢁM treatment and further increased
to 62% when the cells were treated with 20 ꢁM of compound 10 (Figure 8A). This demonstrates
the apoptotic potential of the compound. To ascertain the role of mitochondrial potential in cell
death induced by compound 10, we checked the translocation of cytochrome c from
mitochondria to cytosol. A marked decrease in the expression of cytochrome c in mitochondrial
fraction with simultaneous increase in cytosolic fraction was observed during the period of
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treatment, showing the role of mitochondrial potential loss in apoptosis induced by compound 10
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in HLꢀ60 cells (Figure 8B).
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Figure 8. A. Compound 10 induced time dependent MMP loss in HLꢀ60 cells. HLꢀ60 cells
(.5×106) were treated for 6 h with indicated concentrations of compound 10, washed once with
PBS, and stained with RH 123. MMP was measured as discussed in materials and methods
section. B. compound 10 mediated cytochrome c release from mitochondria to cytosol. 4×106
HLꢀ60 cells were treated with indicated concentrations of compound 10 for 6 h. Cell pellet was
washed once with PBS and cytosolic and mitochondrial proteins were isolated as described in
materials and methods. Translocation of cytochrome c from mitochondria to cytosol was seen
through western blot. COX IV was used as internal control for mitochondrial fraction while β
actin was used as an internal control for cytosolic frcation.
Annexine V/PI staining
Annexin V/PI staining was performed to distinguish apoptotic and necrotic cell deaths. Cell
death induced by compound 10 was purely apoptotic as the amount of necrotic cells (PI stained)
was negligible as compared to apoptotic population (Annexin VꢀFITC stained) of cells. About
25% cells were purely apoptotic after treatment with 5 ꢁM of compound 10 (Figure 9). The
apoptosis further increased with increasing doses of compound.
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Figure 9. Cell death induced by compound 10 is apoptotic. Treated and untreated HLꢀ60 cells
were collected washed once with PBS and stained with annexine VꢀFITC/PI as explained in
materials and methods section.
Cell cycle phase distribution
HLꢀ60 cells treated with compound 10 for 6 h showed considerable increase in apoptotic
population (sub G0/G1), the apoptotic population increased from 5% in control sample to 37% in
sample treated with 20 ꢁM of compound 10 (Figure 10).
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Figure 10. Induction of G0/G1 population. HLꢀ60 cells (1×106) were seeded in 12 well plates,
treated with different concentrations of compound 10 (5 ꢁM, 10 ꢁM and 20 ꢁM) for 6 h. After
completion of time cells were collected at 400 g, washed once with PBS, and fixed in 70%
ethanol overnight. Cells were then washed once with PBS and stained with 100 ꢁg of PI for 30
min. Modfit software was used to differentiate between different phases and determine the
amount of apoptotic population.
Compound 10 induced apoptosis through the inhibition of PI3K/AKT pathway
Western blot analysis showed that compound 10 inhibited major proteins of PI3K/AKT
signaling pathway. The compound inhibited the p110α subunit of PI3K. Interestingly, P110β and
P110δ subunits of PI3K were not affected by compound 10 treatments. However, the expression
of the major downstream effectors of the pathway was diminished significantly. The effect was
more pronounced in both the phosphorylated forms of AKT (T 308) and pAKT(S 473). Also
GSK3β, substrate of AKT, was inhibited significantly, but total AKT was not affected.
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Furthermore, mTOR a downstream effectors of AKT and its substrate pP70s6k were also
inhibited in compound 10 treated cells (Figure 11A). Expression of ERK1/2 was also checked to
see the effect of the compound on this pathway. However, the compound did not affect the
expression of ERK. Inhibition of the PI3K/AKT signaling has been reported to activate caspase
320, which is considered as sign of apoptosis. Compound 10 causes caspase 3 cleavage in HLꢀ60
cells (Figure 11B), which was increased treatment dose. Compound 10 treatment was also found
cause PARPꢀ1 cleavage, which play significant role in apoptosis along with caspase 3. Hence, it
was found that compound 10 mediated inhibiton of P13K signaling pathway stimulate caspase
dependent cancer cell death.
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Figure 11. Compound 10 induces apoptosis by inhibiting PI3K/AKT pathway specifically
thorough the inhibition of p110α subunit of PI3K. A) HLꢀ60 cells (2×106) were treated with
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indicated concentrations of compound 10. Cells were lysed and western blot for different
proteins was performed as described in materials and methods. Compound 10 was found to
inhibit whole of the PI3K/AKT pathway. β actin was used as internal control. B). Compound 10
induces apoptosis. Treated and untreated cells were collected and lysed in RIPA buffer as
described earlier, western blot for PARPꢀ1 and caspase 3 was done, and significant cleavage of
PARPꢀ1 and caspase 3 was observed confirming the role of compound 10 in apoptosis. β actin
was used as internal control.
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Discussions
The present study describes the mechanism of cell death induced by compound 10 in HLꢀ60
cells. Apoptotic potential of the compound was observed through phase contrast microscopy and
Hoechst staining, which was further confirmed by annexine VꢀPI staining. Mitochondrial
membrane potential loss has been considered as a mark of cell death.17 As compound 10 induced
mitochondrial membrane potential significantly, and the treated cells were exhibiting G0/G1
arrest of cell cycle, this points to the apoptotic potential of the compound. The PI3K/AKT
18
19
pathway has been found to play a crucial role in cell proliferation,
cell survival,
and
inhibition of apoptosis.18 Compound 10 inhibited the major proteins of this pathway. AKT
pathway has been found to control the activation of caspaseꢀ3, inhibition of AKT being
associated with activation of caspaseꢀ3.20 PI3K inhibitor LY294002 has been reported to mediate
the translocation of cytochrome c from mitochondria to cytosol,21 indicating the role of PI3K
pathway in cytochrome c release and apoptosis. Similarly, it was found that compound 10 also
mediate the release of cytochrome c from mitochondria to cytosol during the cell death induced
by compound 10. Since cytochrome c play a role role in activation of caspase 3, 22 it was found
that treatment of compound 10 was able to activate the caspase3 followed by PARP1 cleavage.
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As compound 10 selectively inhibited P110α subunit of the PI3K, expressions of P110β and
P110δ were not affected. So we concluded that compound 10 induce apoptosis by targeting p110
α subunit of the PI3K through mitochondrial stress.
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Conclusion
In summary, starting from triꢀOꢀacetylꢀDꢀglucal, a set of medium sized macrocycles based on
the structural framework of aspergillide has been synthesised. Short, high yielding synthetic steps
for targeted molecules with easy processing are some of the advantages of the process. Further,
in an effort to investigate the effect of ring size, stereo diversity and position of double bond of
synthesized macrolides on their antiproliferative activity against different cancer cell lines, it has
been found that one of the derivatives (compound 10) selectively inhibits tumorigenic PI3Kꢀ
α/AKT pathway and further modification in the structure of this molecules can be useful to
develop new anticancer drugs.
Materials and methods
Biology. RPMIꢀ1640, propidiumiodide (PI), 3ꢀ(4,5, ꢀdimethylthiazoleꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT), Rhodamine 123, penicillin, streptomycin, Lꢀglutamine,
Hoechstꢀ33258, pyruvic acid, camptothecin, ribonuclease A, protease inhibitor cocktail were
purchased from Sigma chemical Co. Fetal bovine serum was obtained from GIBCO Invitrogen
Corporation USA. Tween 20, AnnexinVꢀFITC apoptosis detection kit, PARPꢀ1, β actin,
caspaseꢀ3 were purchased from Santa Cruz biotechnology. AKT, pAKT (s473), pAKT , T308 ,
PII0α (s4249), P70s6k, pmTOR,pGSK3β were acquired from Cell Signaling Technology.
Cell culture, growth conditions and treatment. Human promyelocytic leukemia cells HLꢀ60,
were obtained from National Cancer Institute (NCI), Bethesda, USA. The cells were grown in
RPMIꢀ1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS),
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penicillin (100 units/ml), streptomycin (100 lg/ml), Lꢀglutamine (0.3 mg/ml), pyruvic acid (0.11
mg/ml), and 0.37% NaHCO3. Cells were grown in CO2 incubator (Thermocon Electron
Corporation, USA) at 37oC in an atmosphere of 95% air and 5% CO2 with 98% humidity.
Different derivatives of macrolides (4-12) series were dissolved in DMSO (dimethylsulfoxide)
and delivered to cell culture in complete medium.
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Cell proliferation assay. Cells were plated in 96 well plates at the density of 15000 cells per
well/200ꢁl of the medium. Culture were treated with different concentrations of different
molecules of macrolide for 48 hours, 20ꢁl of MTT (3ꢀ(4,5ꢀdimethylthiazoleꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide) (2.5mg/ml) was added to each well and incubated at 37o C for two
hours. The plates were centrifuged at 2000 rpm for 15 minutes and supernatant were discarded
and the MTT formazen crystals were dissolved in 150ꢁl of DMSO. Plates were shaken on shaker
for 3 minutes and then incubated at 37oC for five minutes. The OD measured at 570nm. OD of
control samples was equilibrated to 1 and the cell viability of control was considered to be 100%,
and accordingly, viability of other samples was calculated by using the following formula.
OD ꢊTestꢋ
% ꢀꢁꢂꢂ ꢃꢄꢅꢆꢄꢂꢄꢇꢈ ꢉ
ꢌ 100
OD ꢊControlꢋ
Hoechst 33258 staining of cells for nuclear morphology: HLꢀ60 cells were treated with
indicated concentrations of compound 10 for 6 h. Cells were centrifuged at 400 g for 5min and
washed twice with PBS. Cells were then stained with one milliliter of staining solution (Hoechst
33258, 10 ꢁg/ml of 0.01M citric acid and 0.45 M disodium phosphate containing 0.05% Tween
20) and stained for 30 min under subdued light at room temperature. After staining the cells were
resuspended in 50 ꢁl of mounting fluid (PBS:glycerol, 1/1), 10 ꢁl mounting solution, containing
cells was spread on clean glass slides and covered with the cover slips. The slides were then
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observed for any nuclear morphological alterations and apoptotic bodies under inverted
fluorescence microscope (Olympus 1X70, magnification 30X) using UV excitation.
Cell cycle analysis. Effect of compound 10 on different phases of cell cycle was assessed by
propidium iodide fluorescence. HLꢀ60 cells 1×106 per well, were incubated with different
concentrations of compound 10 (5,10,20 ꢁM) for 6 h. The cells were then washed twice with iceꢀ
cold PBS, harvested, fixed with iceꢀcold PBS in 70% ethanol and stored at ꢀ20oC overnight.
After fixation, these cells were incubated with RNAse A (0.1 mg/mL) at 37oC for 90 min, stained
with propidium iodide (100 ꢁg/mL) for 30 min on ice in dark, and then measured for DNA
content using BD FACS flow cytometer (Becton Dickinson, USA). Data were collected in list
mode and 10,000 events were analysed for FL2ꢀA vs. FL2ꢀW. Modfit software was used to
distinguish different phases of cell cycle.
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Measurment of mitochondrial membrane potential. For mitochondrial membrane potential
1×106 HLꢀ60 cells were seeded in 12 well plate and incubated with compound 10 (5 ꢁM,10 ꢁM
and 20 ꢁM) for 6 h. RH123(200 nM/ml) was added 30 minutes before termination of experiment.
Cells were collected at 400g, washed once with PBS and mitochondrial membrane potential was
measured in FLꢀ1 channel of flow cytometer.
Annexin /PI staining to measure apoptosis and necrosis simultaneously. 1×106 HLꢀ60 cells
were treated with 5 ꢁM ,10 ꢁM and 20 ꢁM of compound 10 for 6 h. Cells were double stained
with annexin V/PI by using kit manufacture’s protocol (santa cruze biotechnology, sc4252). The
FACS analysis for apoptosis and necrosis was done as described earlier.23
Phase contrast microscopy. Phase contrast microscopy was done to assess the morphological
changes on cells after treatment with compound 10. Cells were incubated in six well plates and
treated with different concentration of compound 10 for 6 h, after completion of time cells were
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subjected to photography. Apoptosis was assessed by using microscope (1X70, Olympus),
photographs were taken by using DPꢀ12 camera.
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Western blot analysis. Treated and untreated HLꢀ60 cells were centrifuged at 400g at 4oC,
washed in PBS and cell pellets were lysed in RIPA buffer for preparation of whole cell lysate as
described earlier.24 Equal amount of protein (60 ꢁg) was loaded into each well for SDSꢀPAGE.
Blots were incubated with different primary antibodies, and chemiluminiscence was captured on
hyperfilm after incubating the blots in ECL plus solution.
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Preparation of mitochondrial and cytoslic lysates. 4×106 HLꢀ60 cells were seeded in 90mm
tissue culture dishes and treated with indicated concentrations of compound 10 for 6 h. cytocolic
fractions were isolated by incubating cells with lysis buffer containing digitonin for 2 mins and
centrifuging the lysates at 12000g, while mitochondrial fractions were lysed with RIPA buffer as
described early.24
Experimental section
Molecular modeling study
The docking studies of molecules were performed using the Schrodinger software suite (Maestro,
version 9.2).25 The macrocyclic compound 10 were sketched in 3D format using build panel and
were prepared for docking using LigPrep application. The Protein coordinates of PI3Kꢀalpha
(PDB ID: 2RD0) for docking study were taken from protein data bank (PDB).26 Protein was
prepared by giving preliminary treatment like adding hydrogen, adding missing residues, refining
the loop with prime and finally minimized by using OPLS2005 force field.27 Grids for molecular
docking were generated with wortmannin (from PDB ID 3HHM).28 Wortamanin coordinates
were adopted to 2RD0 and were used as the ligand to generate the grid for the docking study.
Compounds were docked using Glide module, 25 with up to three poses saved per molecule.
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Chemistry. All the reagents used were purchased from sigma Aldrich. Solvents were distilled
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before use. H and 13C NMR spectra were recorded on 200, 400 and 500 MHz spectrometers
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(Model No. D 205/ 52ꢀ2382, Avance 500) with TMS as the internal standard. Chemical shifts are
expressed in parts per million (δ ppm). Silica gel coated aluminium plates were used for TLC.
The products were purified by column chromatography on silica gel (60ꢀ120/100ꢀ200 mesh)
using petroleum ether–ethyl acetate as the eluent to obtain the pure products. Elemental analyses
were performed on Elementar. Reagents used were mostly purchased from Sigma Aldrich.
LCMS was recorded on waters (Model No. Symapt MS) and optical rotation was reported from
Perkin Elmer (Model No. 241). The molecular formulas of compounds were determined by
HRMS (agilent, Model No. 6540), and all tested compounds yielded data consistent with purity >
95% measured by by HPLC (agilent 1260 affinity)
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Synthesis of Acetic acid 2-acetoxymethyl-6-allyl-3,6-dihydro-2H-pyran-3-yl ester (13):
BF3.OEt2 (4.4 mL, 1.2 equiv. diluted with DCM until 15 mL) was injected to a solution of triꢀOꢀ
acetylatedꢀDꢀ glucal (A) (10 gm, 0.036 mmol) and allyltrimethylsilane (7.05 mL, 1.2 equiv) in
dry DCM at ꢀ10 0C. The reaction mixture was stirred for 2h till TLC monitoring indicated the
completion of the reaction. The reaction mixture was diluted with water and extracted with DCM
(3 x100 ml). The organic layers were combined and washed with aqueous sodium bicarbonate
solution and brine prior to drying over anhydrous Na2SO4. The crude oily product obtained after
filtration and concentration under reduced pressure was subjected to flash column
chromatography (1ꢀ8% EtOAc in petroleum ether) to afford 13 ( 8.31 gm, 89%). 1H NMR for αꢀ
major isomer (400 MHz, CDCl3): δ, 5.94 (ddd, J = 1.6, 2.4, 10.4 Hz, 1H), 5.85 (dd, J = 3.2, 10.0
Hz, 1H), 5.80 (dt, J =2.4, 10.4 Hz, 1H), 5.16ꢀ5.10 (m, 3H), 4.30ꢀ4.26 (m, 1H), 4.23 (dd, J =6.4,
11.6 Hz, 1H), 4.15 (dd, J =3.2, 11.6 Hz, 1H), 3.96 (ddd, J =3.2, 6.4, 12.8 Hz, 1H), 2.50ꢀ2.43 (m,
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1H), 2.36ꢀ2.29 (m, 1H), 2.09(s, 6H). C NMR (100 MHz, CDCl3): 170.7, 170.3, 133.9, 132.8,
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123.7, 117.5, 71.3, 69.7, 64.9, 62.8, 37.8, 21.0, 20.7. ESIꢀMS; 277.01 (M+Na)+ ; Anal. Cal. for,
C13H18O5: C, 61.40; H, 7.14; Found C, 61.36; H, 7.07.
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Synthesis of Toluene-4-sulfonic acid 6-allyl-3-hydroxy-3,6-dihydro-2H-pyran-2-ylmethyl
ester (15): To a solution of 13 (8.3 gm, 0.0326 mmol) in dry methanol (80 mL) was added
NaOMe (176 mg, 0.1equiv.) and allowed to stirred for 30 minutes at room temperature.
Completion of the reaction was monitored by TLC. The reaction mixture was neutralized with
amberlyte (IR 120 H+) resin followed by filtration and concentration of the solvent under reduced
pressure. The product obtained was directly used for the next step without further purification.
The compound 14 (5.5 gm, 0.032 mmol) was dissolved in dry Pyridine (30 mL) at 0 0C, followed
by tosyl chloride (11.2gm, 1.8 equiv.) addition. The reaction mixture was stirred for 8h at rt.,
when TLC monitoring indicated the completion of the reaction. The reaction mixture was diluted
with aqueous CuSO4 solution and extracted with ethyl acetate (3 x 50 mL). The combined
organic layers were washed with brine, dried with anhydrous Na2SO4. Filtration and
concentration under reduced pressure afforded the crude product which was subjected to
purification using silica gel chromatography (5ꢀ20% EtOAc in petroleum ether) to afford 15
(6.81 gm, 65%). 1H NMR (400 MHz, CDCl3): δ 7.79ꢀ7.77 (m, 2H), 7.33ꢀ7,31 (m, 2H), 5.79ꢀ5.77
(m, 1H), 5.76 (dd, J = 0.8, 3.2 Hz, 1H), 5.74ꢀ5.71(m, 1H), 5.07(d, J =7.6 Hz, 1H), 5.04 (dd, J
=0.8, 9.2 Hz, 1H), 4.25 (ddd, J =0.8, 5.2, 11.2 Hz, 1H), 4.17 (dd, J =2.4, 11.2 Hz, 1H), 4.01ꢀ4.00
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(m, 1H), 3.66ꢀ3.65 (m, 1H), 2.42 (s, 3H), 2.35.2.33 (m, 1H), 2.24ꢀ2.15 (m, 1H). C NMR(100
MHz, CDCl3): 145.0, 134.0, 133.6, 132.7, 129.9, 129.8, 118.4, 72.0, 69.1, 62.7, 37.7, 21.6. ESIꢀ
MS; 347.382 (M+Na)+; Anal. Cal. for, C16H20O5S; C, 59.24; H, 6.21; Found C, 59.17; H, 6.15.
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Synthesis of (6-Allyl-3-hydroxy-3,6-dihydro-2H-pyran-2-yl)-acetonitrile (16). A mixture of
NaCN (1.36gm, 1.5 equiv.) and 15 (6 gm, 0.0185 mmol) in dry DMSO was refluxed at 70 0C for
4h, until complete consumption of starting material as indicated by TLC. The reaction mixture
was diluted with ferrous sulphate solution (to remove the excess of NaCN) and extracted with
diethyl ether (3 x 40 mL). The combined organic layers were washed with brine, dried with
anhydrous Na2SO4, concentrated in vacuum and purified by flash chromatography (10ꢀ25%
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EtOAc in petroleum ether) to afford 16 (2.81gm, 85%) as a colorless powdered. H NMR(500
MHz, CDCl3): δ 6.00ꢀ5.83 (m, 3H), 5.17ꢀ5.12 (m, 2H), 4.27 (d, J = 6.04 Hz, 1H), 3.97ꢀ3.93 (m,
1H), 3.75 (ddd, J = 4.19, 7.17, 14.2 Hz), 2.78 (dd, J = 4.16, 16.8 Hz), 2.66 (dd, J =7.25, 16.8 Hz),
2.46 (m, 1H), 2.32 (m, 1H).13C NMR (100 MHz, CDCl3): 133.4, 130.9, 127.8, 117.9, 72.4, 69.8,
65.8, 37.8, 21.0. ESIꢀMS; 202.206 (M+Na)+; Anal. Cal. for, C10H13NO2: C, 67.02; H, 7.31; N,
7.82; Found C, 66.97; H, 7.27; N, 7.75.
General procedure for 4-hydroxyl protection of 16 (a). A solution of 16 in dry DMF at 0 0C
was stirred for 30 minutes followed by addition of NaH (60% dispersion in mineral oil, 1.5
equiv.) and halide (RX) (RX = C2H5I, pꢀmethoxybenzyl chloride, MeI, 1.2 equiv.) and stirred for
4h at rt. The reaction was completed as indicated by TLC and was diluted with ethyl acetate (50
mL) and extracted using water. The organic portion was washed with brine, dried with anhydrous
Na2SO4, concentrated in vacuum and purified by flash chromatography.
Preparation of compound 17. Prepared by using 16 (1g, 5.58mmol), EtI (871ꢃL, 1.2equiv.)
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following general procedure a to afford 17 (1.04g, 90%). H NMR (400 MHz, CDCl3): δ, 6.00ꢀ
5.83 (m, 3H), 5.17ꢀ5.12 (m, 2H), 4.27 (d, J = 6.0 Hz, 1H), 3.97ꢀ3.93 (m, 1H), 3.75 (ddd, J = 4.1,
7.1, 14.2 Hz, 1H), 3.73ꢀ3.65 (m, 1H), 3.50ꢀ3.44 (m, 1H), 2.78 (dd, J = 4.1, 16.8 Hz, 1H), 2.66
(dd, J = 7.2, 16.8 Hz, 1H), 2.46 (m, 1H), 2.32 (m, 1H).13C NMR(100MHz, CDCl3): 133.4,
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