Phospholipidic Colchicinoids as Promising Prodrugs Incorporated into Enzyme-Responsive Liposomes: Chemical, Biophysical, and Enzymological Aspects

Article abstract of DOI:10.1021/acs.bioconjchem.9b00051

Enzyme-responsive liposomes release their cargo in response to pathologically increased levels of enzymes at the target site. We report herein an assembly of phospholipase A2-responsive liposomes based on colchicinoid lipid prodrugs incorporated into lipid bilayer of the nanosized vesicles. The liposomes were constructed to addresses two important issues: (i) the lipid prodrugs were designed to fit the structure of the enzyme binding site; and (ii) the concept of lateral pressure profile was used to design lipid prodrugs that introduce almost no distortions into the lipid bilayer packing, thus ensuring that corresponding liposomes are stable. The colchicinoid agents exhibit antiproliferative activity in subnanomolar range of concentrations.

Full text of DOI:10.1021/acs.bioconjchem.9b00051

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Article
Phospholipidic colchicinoids as promising prodrugs incorporated into enzyme-responsive liposomes: chemical, biophysical, and enzymological aspects
Ekaterina S Shchegravina, Daria s Tretiakova, Anna S Alekseeva, Timur R. Galimzyanov, Yuri N. Utkin, Yuri A Ermakov, Elena V Svirshchevskaya, Natalia Yu Karpechenko, Valery P Chernikov, Natalia Onishchenko, Vadim V Negrebetsky, Elena L Vodovozova, Aleksey Yurievich Fedorov, and Ivan A. Boldyrev
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00051 • Publication Date (Web): 28 Feb 2019
Downloaded from http://pubs.acs.org on March 1, 2019
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Phospholipidic Colchicinoids as Promising Prodrugs Incorporated into
Enzyme-Responsive Liposomes: Chemical, Biophysical, and Enzymological
Aspects
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Ekaterina S. Shchegravina[a,b], Daria S. Tretiakova[b], Anna S. Alekseeva[b], Timur R. Galimzyanov[c,d]
Yuri N. Utkin[b], Yuri A. Ermakov[c], Elena V. Svirshchevskaya[b], Vadim V. Negrebetsky[e], Natalia Yu.
Karpechenko[f], Valery P. Chernikov[g], Natalia R. Onishchenko[b], Elena L. Vodovozova[b], Alexey Yu.
Fedorov[a], and Ivan A. Boldyrev*[b]
,
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a Lobachevsky State University of Niznhy Novgorod, 23 Gagarin Prospest, Nizhny Novgorod, 603950 Russian
Federation
b Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-
Maklaya Street, Moscow, 117997 Russian Federation, e-mail: ivan@lipids.ibch.ru
c A. N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, 31/4 Leninskii
Prospekt, Moscow, 119071 Russian Federation
d National University of Science and Technology MISiS, 4 Leninskiy Prospekt, Moscow, 119049 Russian Federation
e Pirogov Russian National Research Medical University, 1 Ostrovityanov Street, Moscow, 117997 Russian
Federation
f N.N.Blokhin National Medical Research Center of Oncology, 24 Kashirskoye Shosse, Moscow, 115478 Russian
Federation
g Scientific Research Institute of Human Morphology, 3 Tsurupa Street, Moscow, 117418 Russian Federation
Abstract: Enzyme-responsive liposomes release their cargo in response to pathologically
increased levels of enzymes at the target site. We report herein an assembly of phospholipase
A2-responsive liposomes based on colchicinoid lipid prodrugs incorporated into lipid bilayer of
the nano-sized vesicles. The liposomes were constructed to addresses two important issues: (i)
the lipid prodrugs were designed to fit the structure of the enzyme binding site; and (ii) the
concept of lateral pressure profile was used to design lipid prodrugs that introduce almost no
distortions into the lipid bilayer packing, thus ensuring that corresponding liposomes are stable.
The colchicinoid agents exhibit antiproliferative activity in sub-nanomolar range of
concentrations.
Keywords: colchicinoids • lipid prodrugs • stimuli-responsive liposomes • phospholipase A2 •
anticancer therapy
Introduction
Colchicine (1), an alkaloid isolated from Colchicum autumnale, was the first discovered
tubulin destabilizing agent. At the molecular level, it prevents self-assembly of tubulin by
distorting the interface between α/β-tubulin heterodimers1–4. Interaction between colchicine and
tubulin results in both antimitotic activity and suppression of cell motility5. In clinical practice,
colchicine has been approved for treatment of gout, familial Mediterranean fever6,7, and,
recently, for several other diseases, such as amyloidosis, progressive systemic scleroderma,
cirrhosis, and Behcet's disease8–12. The ability of colchicine to accumulate within the immune
system cells, leading to the suppression of inflammatory reactions, opened the possibility of its
application for the treatment of cardiovascular disease, such as acute pericarditis and atrial
fibrillation caused by inflammation13–16. Usage of colchicine in treatment of malignant tumors is
prevented by rather high general toxicity resulting in renal, hepatic, circulatory, and central
nervous system injury (see for example17–19).
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One way to reduce general toxicity of the drug and improve its biodistribution upon
systemic administration is to design a carrier-linked drug delivery system20–25. Liposomes have
been the first nano-sized drug delivery system to be successfully translated into clinical
practice26. To enhance the ability of a liposomal nanocarrier to target specific tissues, ligand-
targeted and stimuli-responsive liposomes have been proposed27. Ligands attached on surface of
liposomes guide them to corresponding receptor on target tissue thus enhancing liposome uptake
by the tissue, while stimuli-responsive liposomes do not release their cargo unless they are
exposed to an endogenous or exogenous trigger at the target site. Particularly, enzyme-
responsive liposomes are sensitive to pathologically increased levels of enzymes at the target
site28.
Secreted phospholipase A2 (sPLA2) level is elevated in various inflammatory diseases,
atherosclerosis, and cancers (for example, prostate, breast, and pancreatic cancers)29. This
enzyme hydrolyzes lipids specifically at the sn-2 position, which can lead to either direct release
of the active drug or degradation of liposome resulting in its unloading. Therefore, sPLA2-
responsive liposomes can be considered as promising carriers for targeted delivery of therapeutic
agents to pathological tissues (see reviews on the topic30,31). In the case of colchicinoid-type
agents, we expect that such carries should manifest anti-inflammatory, cardioprotective, and
anticancer properties at the site of inflammation or in tumor tissues.
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This manuscript addresses the design and synthesis of colchicine-based therapeutic
agents and corresponding phospholipid prodrugs, construction of enzyme-responsive therapeutic
liposomes for delivery of the colchicinoids, investigation of the drug–lipid interactions inside the
liposomal carrier, liposomes’ stability in human serum, and drug release under the effect of
PLA2.
Results and Discussion
Design of Colchicinoid-Derived Phospholipid Prodrugs
Figure 1. Structural design of a colchicinoid-containing phospholipid.
Based on the evidence of enhanced PLA2 levels at sites of inflammation and in tumors,
we present a concept of PLA2-degradable prodrug where the active compound is bound to distal
end of the fatty acid chain at sn-2 position of a phospholipid. Such a prodrug releases the active
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agent upon two cleavage events: (1) PLA2 cleaves the drug-conjugated fatty acid chain from the
phospholipid and (2) the drug is cleaved from the fatty acid chain (Figure 1).
For a drug derivative to be cleavable by PLA2, it has to fit the enzyme binding site, while
leaving the drug moiety, especially as bulky as the colchicinoid moiety, outside. Earlier the
ability of PLA2 to hydrolyze various substrates was studied32,33. In assays of hydrolysis of
phospholipids naturally occurring in humans, secretory PLA2 did not show any specificity for
individual substrate in the sn-2 position34. However, this is not true for fluorescently labeled or
other modified acyl chains35. Particularly, the active site of sPLA2 IIA has intricate shape;
therefore, a substrate should be flexible enough to adopt appropriate conformation. To reach the
same binding efficiency as natural phospholipids, a modified phospholipid should accommodate
the bulky moiety farther than 10 carbon atoms away from the beginning of the sn-2 chain. This is
supported by the data on cobra venom PLA2, which accommodates first 10 or so carbons of the
sn-2 acyl chain of phospholipids in its catalytic site, where they are exposed to a very
hydrophobic environment34. Locating of a bulky group more than 10 carbon atoms away from
the beginning of the sn-2 chain has been successfully used to improve sensitivity of PLA2
specific fluorescent probes36.
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The linker between the drug moiety and the fatty chain was chosen to promote release of
the active substance in vitro and in vivo. Particularly, ester bond is easily hydrolyzed by low-
specificity esterases found in abundance in most tissues37.
Colchicinoid Design and Synthesis of Lipid Prodrugs
The presence of an easily oxidizable 7-membered C-cycle and sensitive tropolone ether
moiety in the colchicine 1 skeleton limits the possibilities of its direct chemical modification
(Figure 2). Colchifoline 2 (Figure 2) is one of colchicine metabolites known for its strong
binding to tubulin and higher antitumor activity than that of colchicine; it possesses a
hydroxymethyl moiety in an amide side-chain suitable for easy functionalization38–40. However,
it reveals the same level of general toxicity as the parent colchicine molecule41–43. Allocolchicine
344, as well as indole- and furane-derived allocolchicine analogs45–50, have demonstrated higher
activity as tubulin polymerization and cell proliferation inhibitors compared to colchicine, while
their general cytotoxicity was relatively low. Therefore, two colchicinoids were chosen for
conjugation with a phospholipid: allocolchicine derivative bearing a hydroxymethyl group in the
amide fragment (4)40 and corresponding dihydrofurane congener of allocolchicine (5) (Figure 2).
MeO
MeO
MeO
MeO
MeO
MeO
X
X
CH2OH
O
N
N
N
H
H
H
O
O
MeO
MeO
MeO
O
CO2Me
O
OMe
Colchicine 1 (X=Me)
Colchifoline 2 (X=CH2OH)
Allocolchicine 3 (X=Me)
Allocolchifoline 4 (X=CH2OH)
5
Figure 2. Colchicine and related compounds.
Synthesis of functionalized allocolchicinoid 7 started from the conversion of commercial
colchicine 1 into deacetylallocolchicine 6 by a three-step cleavage of the acetamide group
performed in 56% yield according to a known protocol51 (Scheme 1). At the next step, the amino
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group in 6 was acylated with glycolic acid under Steglich conditions to give allocolchifoline 4 in
good yield.
Intermediate 10 necessary for the synthesis of hydrofurano-allocolchicinoid 5 is available
from colchicine 1 exploiting electrocyclic rearrangement/oxidation of colchicine into N-
acetyliodocolchinol 852. The phenolic hydroxyl underwent allylation to afford colchicinoid 9 in
63% yield. It was converted then into corresponding amine 10 (50% overall yield) via three-step
deacetylation procedure. The latter was subjected to Steglich acylation with glycolic acid
followed by catalytic intermolecular Heck reaction to afford desired hydrofurano-
allocolchicinoid 5 with exo-double bond in D-cycle in 69% yield over two steps.
One should note (see section “Сytostatic Properties”) that colchicinoid 4 shows slightly
lower level of cytotoxic activity in comparison with colchifoline 2, while colchicinoid 5
demonstrates the same range of cytotoxicity as compound 2. Both allocolchicinoids 4 and 5
inhibit proliferation of pancreatic cells at low nanomolar or even at subnanomolar
concentrations. High cytotoxic activity of mentioned colchicinoids and increased level of sPLA2
in pancreatic tumors34,53–57 are important features for creating sPLA2-responsive liposomes as a
promising tool for pancreatic cancer treatment.
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O
O
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
N
NHAc
N
e
NH2
d
a-c
O
H
H
O
HO
51%
MeO
MeO
MeO
4
MeO
O
CO2Me
CO2Me
CO2Me
1
6
7
OMe
f, g
O
O
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
NH2
NHAc
N
H
N
H
e
l,m
i-k
O
HO
O
MeO
10
49%
MeO
MeO
MeO
11
O
OR
O
O
I
I
5
8 R=H
9 R=allyl
h
Scheme 1. Synthesis of colchicinoids 7 and 11. Reaction conditions: a) Boc2O, 4-DMAP, Et3N, MeCN,
3h, 100 °C, 78%; b) MeONa, MeOH, 40 min, 40 °C, 99%; c) TFA, DCM, 1,5 h, 25 °C, 95%; d) glycolic
acid, DIC, NHS, Et3N, DCM, 20 h, 25 °C, 64%; e) 4-pentynoic acid, 2,4,6-trichlorobenzoyl chloride
(TCBC), 4-DMAP, Et3N, DCM, 24 h, 0→rt; 51% and 49% for compounds 7 and 11 respectively; f) 0,1
M HCl, AcOH, 100 °C, 3h, 98%; g) I2, KI, NaOH, H2O, 0–5 °C, 1h, 95%; h) allyl bromide, Na2CO3,
DMF, 60 °C, 24 h, 63%; i) Boc2O, 4-DMAP, Et3N, MeCN, 3h, 80 °C, than cooling to rt, 20 h, 86%; j)
MeONa, MeOH, 40 °C, 1 h, 94%; k) TFA, DCM, 30 °C, 24 h, 62%; l) glycolic acid, DIC, NHS, Et3N,
DCM, 20 h, 25 °C, 79%; m) Pd(dppf)Cl2, AcOK, DMSO, 75 °C, 20 h, 87%.
All attempts to connect colchicinoids 4 and 5 directly with lyso-PC 12 using the linkers
on the basis of dicarboxylic acids (>C12) failed. Particularly, different ways of acylchloride
formation, as well as the Steglich acylation, Yamaguchi, Mitsunobu esterification, and mixed
anhydrides, were used. It can be explained by strong coordination of carboxylic group and its
further deactivation. Therefore, allocolchicinoids 4 and 5 were functionalized by 4-pentynoic
acid applying Yamaguchi esterification giving alkyne building blocks 7 and 11 in 51 and 49%
yields, respectively (Scheme 1).
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Modified phosphatidylcholine 14 was prepared by Steglich acylation of lyso-
phosphatidylcholine (lyso-PC) 12 with 11-azidoundecanoic acid 1358 (Scheme 2) in 78% yield.
Alkynes 7 and 11 were used in the copper-mediated 1,3-dipolar cycloaddition59 with azido-
containing phosphatidylcholine building block 14 leading to target colchicinoid-containing
conjugates 15 and 16 in 56 and 62% yields, respectively. The first example of exploiting the
powerful click-conjugation tool in colchicine functionalization was performed by N. Nicolaus59.
N
10
11
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Bn
TBTA:
N
N
O
MeO
MeO
N
H
N
O
N
O
N
N
N
MeO
O
MeO
MeO
N
O
N
O
N
Bn
N
N
CO2Me
O
N
H
Bn
O
O
O
MeO
O
O
P
O
P
O
N
CO2Me
7
15
O
N
N
O
O
P
O
O
56% after isolation
O
O
DCC, DMAP
CHCl3
OH
O
O
CuSO4*5H2O,
TBTA, AscNa
DMF-H2O(1:1),
O
O
O
78%
O
O
O
HO
55 o
C
O
MeO
MeO
N
O
H
O
N
N
MeO
N
O
O
MeO
MeO
N
H
O
16
O
O
MeO
O
62% after isolation
N
N
O
O
P
O
N
O
O
N
N
N
N
11
O
O
lyso-PC 12
13
14
Scheme 2. Synthesis of colchicinoid phospholipid conjugates.
Incorporation of Colchicinoid Prodrugs into Lipid Bilayer
Inclusion of alien conjugates in the lipid bilayer may induce a disturbance of its ordered
structure, which can affect both the loading capacity of liposomal formulations and their stability
in the biological milieu. This fact, for example, has been established for taxol (another tubulin-
binding agent)60. When content of the drug in the bilayer exceeds 2.8 mol. %, taxol forms
aggregates, which results in destabilization of the lipid bilayer and precipitation of the drug .
Indeed, pharmacokinetics of the formulation in rats is consistent with losses of taxol associates
from liposomes in the circulation61.
To investigate the behavior of colchicinoid conjugates in the lipid bilayer, we focused on
the spatial and orientational distribution of colchicinoids in the lipid layer, lipid packing
differences, and prodrug-loaded liposome stability in serum.
We describe the drug moiety as an interbilayer inclusion that has shape and dimensions.
In our case it is attached to lipid host molecules and incorporated into the bilayer (Table 1). The
geometrical parameters of the interbilayer inclusions were obtained by quantum chemistry
optimization (see SI section “Quantum Chemistry”). It turned out that inclusions of both
compounds 15 and 16 had very close molecular volumes and projection areas (Table 1).
60
Table 1. Projection areas of conjugates 15 and 16
15
16
Minimal projection area, Å2
Maximal projection area, Å2
52.68
98.01
57.88
95.63
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Important characteristics of a conjugate inclusion are its spatial and orientational
distributions in the membrane leaflet (lipid monolayer). The distributions depend on the
molecular structures of the inclusion, as well as membrane properties. The lipid tail region in
monolayer is stressed, which is usually described by a so-called lateral pressure profile, i.e. the
dependence of the 2D pressure on the distance from the head-group region. The lateral pressure
inside the bilayer tends to exclude the molecule from the tightly packed areas. At the same time,
the inclusion tends to hide its hydrophobic surface from water into the hydrophobic region. The
equilibrium position is the result of the effect of these two forces.
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Lateral pressure profile can be obtained from molecular dynamics simulation (MD)62,
application of lipid probes (pyrene)63, or analytically64,65. Here, for the quantitative estimation,
lateral pressure profile produced in MD simulations66 was used. Molecular geometries obtained
by quantum chemistry optimization were used for quantitative description of the molecule shape.
The optimized molecular geometries and coordinate system location are shown in Figure 3. The
coordinate system is oriented with XY plane being parallel to the membrane plane.
Colchicinoids are connected with the lipid core through a polar linker (structures 15 and
16 on Scheme 2; Figures 1 and 3). Nitrogen and oxygen atoms of the linker are involved in
hydrogen bond formation and polar interactions with surrounding lipid head groups. Through
these interactions, the linker binds with the polar head region. From this we deduce that the
colchicinoid fragment should be integrated in the polar head region and the linker can be fixed in
the XY plain inside the polar head region. The rest of the molecule is non-polar—it should be
located inside the hydrophobic area of the membrane.
The energy of intermonolayer inclusion can be calculated as a work for expansion of the
area of lipid layer against the pressure profile
푊 = ∫VmolPdV = ∫푧2푃(푧)dz
,
(1)
푧1
where z is the coordinate along the axes parallel to average lipid tail direction, z = 0
corresponds to the lipid tail–head group connection region; P(z) is the lateral pressure profile;
integration is performed over the region occupied by the molecule in the membrane. (This
approach was recently successfully applied to the design of fluorescent lipid probes non-
disturbing lipid bilayer67.) The inclusion should rearrange into optimal layout to minimize its
energy, W. Since the area occurs in the integral as a linear term (equation 1) and the inclusion
volume is constant, the optimum position for the inclusion is to expose its largest projection area
to the minimum pressure.
Taking that colchicinoid group is bound to the polar region, energy minimization could
be achieved through rotation of the inclusion around the z-axis (angle ϕ) and XY-plane (angle ϴ).
Due to lateral homogeneity of the monolayer, rotation around the z-axis does not vary the
system’s state and energy. Therefore, we can exclude rotation around the z-axis from the analysis
and focus on the possible distributions of the colchicinoid moiety over angle ϴ (Figure 4A). The
probability distributions were calculated under the assumption that the latter is proportional to
Boltzmann factor
―푊
푒푘 푇
.
퐵
Two most preferred orientations were around ϴ = 90° and ϴ = 0° (or 180°) for both
conjugates 15 and 16. Note that the probability allocations are not totally symmetrical
(probabilities at ϴ = 0° and 180° are not equal). The latter is explained by low symmetry of
molecular structures of the inclusions. Polar groups of both conjugates (15 and 16) are arranged
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either parallel to the bilayer surface or perpendicular to it (see Figure 3B for schematic
representation). We suggest that these orientations should be in a dynamic equilibrium.
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Figure 3. A. Optimized geometry of the colchicine moiety in conjugates 15 and 16. B. Most probable
orientations of colchicine moiety in compound 16 inside the bilayer. Hydrocarbon chain structure is not
shown for clarity.
Figure 4. A. The probability to find colchicinoid moiety aligned along the tilt angle ϴ. B. Surface
pressure diagrams.
The colchicinoid moiety localizing near the lipid head groups seems unexpectable due to
presumably tight packing in that region. However, it has been shown that the bilayer surface is
not homogeneous even in single-component membranes. There are hydrophobic, hydrophilic,
and neutral areas at the bilayer surface. Hydrophobic regions are created by acyl chains of lipids
“snorkeling” near the membrane–water interface. These hydrophobic “spots” correspond to
regions between the lipid heads68 covering up to 20% of the bilayer surface according to
molecular dynamic simulations68. Thus, colchicine moieties in conjugates 15 and 16 can be
forced out towards head group region and populate these hydrophobic surface spots, which are
otherwise inhabited by hydrophobic chains.
To study the effect of conjugates 15 and 16 on lipid packing, we recorded surface
pressure isotherms for pure prodrugs, as well as mixed 1-palmitoyl-2-oleoylphosphatidylcholine
(POPC)–conjugate 15 and POPC–conjugate 16 monolayers (Figure 4). Both conjugates have
increased area per lipid comparing to POPC, while area per lipid of conjugate 16 is lower than
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that of conjugate 15. The higher is the content of the conjugates, the greater is the difference
between the two. However, mixtures of 10% mol. of compound 15 or 16 with POPC
demonstrate almost the same surface pressure isotherm profiles as pure POPC, which evidences
that at 10 mol. % the conjugates practically do not disturb the lipid bilayer packing.
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Calcein Release Assay
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Colchicine moieties lying on the surface of the bilayer should alter the surface properties.
Thus, lipid bilayer of liposomes can suffer from lipid–protein interactions upon contact with
human serum and protein corona formation. This in turn can limit application of liposomes as a
delivery system for drug targeting. We examined stability of liposomes with colchicine
derivatives in PBS and in human serum using calcein release assay. In the absence of any
colchicinoid derivatives, ePC liposomes demonstrated very slow release of calcein (the degree of
calcein release (CR) was approximately 3% after 24 h of incubation) (Figure 5A), which can be
explained by simple diffusion of calcein through the fluid lipid bilayer69. The single-component
liposomes incubated in serum demonstrated more intense, but still gradual calcein release, with
CR reaching 10% by the end of the incubation (Figure 5A). The result may be a consequence of
progressive degradation of the fluid lipid bilayer under the action of enzymes in serum or even
albumin adsorption on liposomes and increase of permeability of the bilayer70.
Figure 5. Leakage of calcein from liposomal formulations in 80% human serum (solid lines) or PBS
(dashed lines) at 37 °C. Plots were obtained for the following formulations: A, ePC; B, ePC–conjugate
15; C, ePC–conjugate 16. Plotted are mean ± SD values for three repetitive measurements.
Liposomes with 5% of conjugate 16 demonstrated similar CR kinetics in both PBS and
serum (Figure 5C), releasing slightly more calcein than the ePC liposomes by the end of
incubation (24 h). Addition of 5% of conjugate 15 did not change the liposome stability in PBS
(compared to ePC liposomes), but led to a dramatic change in liposome stability in serum, with
27% CR. The difference could not be explained by electrostatic forces, as ζ-potential for the
ePC–conjugate 15 and ePC–conjugate 16 formulations was almost the same (Table 2).
In order for calcein to be released from a liposome, it should pass three stages: bind the
internal bilayer surface; get across the bilayer; and desorb from the external bilayer surface.
Allocolchicinoid moieties floating on the bilayer interface change the pattern of
hydrophobic/hydrophilic area distribution on surface of liposomes. Therefore, (1) they alter the
sorption/desorption constants of calcein on the bilayer surface. It appears that conjugate 16
promotes calcein binding to the bilayer surface more readily than conjugate 15 (compare CR
plots produced in PBS; Figures 5B and 5C). (2) Allocolchicinoid moieties hinder access of
serum proteins, such as albumin, to oxygen atoms of phosphate and carbonyl groups, which are
important for initial binding of albumin to the bilayer. However, in case of the conjugate 15,
carboxymethyl group may create new possibilities for interaction thus promoting membrane
destabilization upon contact with proteins, while conjugate 16 provides no such possibility.
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Table 2. Liposome properties
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Liposomes
Size, nma
111.0 ± 0.3
111.1 ± 0.3
ζ-potential, mV
5% conj. 15–95% ePC
5% conj. 16–95% ePC
–5.4
–3.5
9
[a] See SI section on liposomes for DLS instrument reports and TEM images.
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PLA2 Responsivity
To check whether the prodrugs in liposomes are susceptible to hydrolysis with PLA2, we
subjected them to the effect of two secreted PLA2: porcine pancreas PLA2 (ppPLA2) and PLA2
from Vipera ursinii venom (vuPLA2). The former is homologous to human pancreatic PLA2 and
the latter to non-pancreatic enzyme71. As a group I PLA2, ppPLA2 has the so-called surface
loop, i.e. a helix region, which is a part of interfacial binding surface (IBS) of the enzyme (see
Figure 6). Deletion of the surface loop enhances the enzyme activity72. vuPLA2 belongs to group
II PLA2 and has no surface loop: shortened by 10 amino acids, the region has a random
structure, yet two of its amino acids are still involved in interaction with the bilayer surface.
They are Lys60 (corresponds to Tyr69 in ppPLA2) and Thr61 (Thr70).
Figure 6. Visualization of PLA2 structures. A, ppPLA2 (PDB ID 3O4M); cyan helix with indigo
selection indicate amino acid residues of interfacial binding surface and light blue selection, the active
site (His48, Tyr52, Tyr73, and Asp9973). B, vuPLA2 homolog (ammodytoxin A from Vipera ammodytes
ammodytes [https://www.uniprot.org/uniprot/P00626]; >90% homology; PDB ID 3G8G); lilac helix with
dark purple selection represents amino acid residues of IBS and magenta selection (His48, Tyr52, Tyr73,
Asp9971), the active site.
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Figure 7. Densitometry of TLC plates upon development with phosphate group reagent; areas under the
curves are proportional to relative content of POPC and the prodrugs (peak on the left) and lyso-PC (peak
on the right) upon treatment of the ePC–conjugate 15 (solid line) or ePC–conjugate 16 (dashed line)
liposomes with vuPLA2 (A, B) and ppPLA2 (C, D) for 1 (A, C) or 24 h (B, D). Figures represent relative
areas of the peaks (in %). For more information, see Supporting Information.
During the first hour of incubation of vuPLA2 with the liposomes, hydrolysis of both
ePC–conjugate 15 and ePC–conjugate 16 liposomes was well pronounced (Figure 7A). After 24
h, lyso-forms of lipids became dominating (Figure 7B, see also SI). Notably, hydrolysis of the
ePC–conjugate 16 liposomes proceeded faster than that of the ePC–conjugate 15 liposomes.
PLA2 is an interfacial enzyme that acts at the lipid–water interface. Initially PLA2 interacts with
the membrane by its interfacial binding surface (IBS) and subsequently the lipid is hydrolyzed in
the active site of the enzyme74. Thus, difference in the rate of hydrolysis between two conjugates
might reflect either differences in surface structure of the liposomes, particularly, distribution of
hydrophobicity over the surface, or hydrolysis rate of the conjugates in the active sites of the
enzymes. In other words, either the structure of conjugate 16 favors hydrolysis by PLA2 or,
when inserted in the ePC bilayer, creates an interface beneficial for PLA2 binding (compared to
conjugate 15). We assume that the two structures should be indistinguishable for PLA2, since the
colchicinoid moieties are far enough from the active site of the enzyme. This implies that
vuPLA2 binds surface of the ePC–conjugate 16 liposomes more efficiently than that of
liposomes with conjugate 15. The fact that the ePC–conjugate 15 liposomes were less stable in
serum (Figure 5) suggests that interaction with other serum proteins destabilizes liposome
membranes more actively than PLA2.
Hydrolysis of liposomes with ppPLA2 is almost negligible during the first hour (Figure
7C). Small amount of hydrolyzed lipids can be detected for the ePC–conjugate 16 sample. The
difference in hydrolysis between two samples increases after 24 h (Figure 7D). The slow rate of
hydrolysis of the prodrugs by ppPLA2, which is most likely due to the presence of the surface
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loop in the enzyme structure, suggests that the prodrug formulations will be more efficient in
treatment of conditions characterized by elevated group II and not group I (pancreatic) PLA2.
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Сytotoxic Properties of the Colchicinoids and Colchicinoid-Containing Liposomes
According to our concept, the liposomal forms of phospholipids 15 and 16 are to be
hydrolyzed selectively at the sn-2 position by PLA2. The enzyme acts at the lipid–water
interface on the surface of liposomes resulting in release of colchicine-derived fatty acids 17 and
18, respectively (see Figure 8). These compounds were synthesized for a comparative biological
assay (see details in SI). In turn, acids 17 and 18 should undergo further hydrolysis of
biodegradable ester bonds with non-specific esterases releasing intact therapeutic colchicinoids 4
and 5.
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O
O
MeO
MeO
O
hydrolysis by
esterases
NH
O
NH OH
PLA2-assisted
hydrolysis
MeO
15
MeO
OMe
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OMe
4
OH
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CO2Me
CO2Me
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PLA2-assisted
hydrolysis
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OMe
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OMe
OH
N
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N
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N
O
O
5
Figure 8. Stepwise hydrolysis of conjugates 15 and 16 by PLA2 and non-specific esterases.
Cytotoxic properties of compounds 4, 5, 15–18, and therapeutic liposomes loaded with
conjugates 15 and 16 were tested against pancreatic cell lines PANC-1, BxPC-3, Colo-357, and
HaCaT (non-transformed immortalized keratinocytes) using colchifoline 2 as a reference (see
Table 3). The choice of cell lines was due to the fact that in 90% of human pancreatic cancers
elevated PLA2 GIIA levels were detected in serum; also, 65 and 84% tissues are PLA2 GIIA
positive according to immunohistochemistry and RNA blotting of tissue extracts, respectively75.
Intact colchicinoids 4 and 5, as well as reference compound 2, demonstrated high
cytotoxic activity in nanomolar concentration range against all tested cell lines. Colchicinoid 5
appears to be the most active compound. Notably, this trend persisted for its derivatives in the
form of fatty acid 18 and the ePC–conjugate 16 liposomes, especially in the case of the PANC-1
cells (compare corresponding IC50 values with acid 17 and the ePC–conjugate 15 liposomes in
Table 3).
Lipid-conjugate colchicinoid prodrugs 15 and 16 lost 1.5–2 orders of magnitude of
activity compared to intact molecules 4 and 5, which is due to delay needed for the enzymatic
release of active agents. Fatty acids 17 and 18 exhibited intermediate or borderline cytotoxic
activity between intact therapeutic agents 4 and 5 and their lipidic conjugates 15 and 16 (Table
3). The ePC–conjugate 15 and ePC–conjugate 16 liposomes showed higher cytotoxicity in
comparison with conjugates 15 and 16 as such and even a bit higher values of activity than acids
17 and 18 after incubation with cells during 72 h (except for the BxPC-3 cells).
Such an intricate pattern of cytotoxic activity produced by various allocolchicinoid-based
preparations against different cell lines should be the result of an interplay between several
factors. (1) Interaction of the preparations with cell surface and further internalization depend
strongly on specific features of cell surface of a given cell line. (2) Also, morphologies produced
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by the allocolchicinoids (liposomes for conjugates 15 and 16 vs micelles or some other kind of
aggregates for compounds 15–18 in aqueous phase) may promote or hinder the process of
internalization. (3) Drug release inside cell is enzyme-dependent. As for PLA2, it is a surface-
dependent enzyme and liposomes present the most convenient substrate therefor, which may
ascribe for higher activity of prodrug-loaded liposomes compared to the prodrugs themselves. (4)
Decreasing the incubation time from 72 to 48 h could have limited the possibility of liposomes to
unload and decay with subsequent partial cleavage of the ester bonds in lipid conjugates 15 and
16 to release active compounds 4, 5, 17, and 18, resulting in the significant decrease in the
cytotoxicity of liposomes ePC–conjugate 15 and ePC–conjugate 16 (Table 3, results marked with
the “b” index).
Rather high cytotoxicity of conjugates 15 and 16 in liposomal form indirectly implies that
liposomes are well internalized and processed by cells with the release of the parent drugs 4 and
5, respectively76. On the other hand, considering future experiments in vivo, it is required to
augment the formulations’ performance with some stabilizing components, such as
phosphatidylinositol77,78, cholesterol79–81, PEG-derived molecules82.
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Table 3. Сytotoxicity of colchifoline 2, colchicinoids 4 and 5, conjugates 15 and 16,
corresponding fatty acids 17 and 18, and therapeutic liposomes containing phospholipidic
a
conjugates 15 and 16 (IC50 , nM)
Compound
PANC-1
6
Colo-357
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16 (16)b
3 (3)b
400 (400)b
160 (400)b
80
BxPC-3
-
HaCaT
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ePC–conj. 15, 95 : 5
liposomes
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38 (200)b
38 (200)b
38 (40)b
ePC–conj. 16, 95 : 5
liposomes
38
38 (50)b
15 (40)b
8 (40)b
ePC liposomes
Non toxic
Non toxic
Non toxic
Non toxic
[a] IC50 concentration is concentration inducing 50% inhibition of cell growth after incubation during72 h.
[b] Values in brackets are given for cell incubation during 48 h.
Conclusions
We present two novel colchicinoid-containing phospholipid prodrugs encapsulated into
phosphatidylcholine-based enzyme-responsive liposomes. The prodrugs induce minimal
distortions into lipid packing and corresponding liposomes appear to be stable in human serum.
At the same time, being exposed to elevated levels of phospholipases A2, especially in the case
of sPLA2 analog, the liposomes release colchicinoid-containing fatty acids. The latter undergo
further hydrolysis by non-specific esterases with release of the active colchicinoid species. Our
data on in vitro cytotoxicity of liposomes loaded with the new phospholipid conjugates evidence
feasibility of such a scenario. For further in vivo studies the composition of therapeutic
liposomes should be changed to improve their stability towards opsonisation and to prevent their
premature decay in biological media. In vivo investigations are to be carried out in the nearest
future.
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Experimental Section
Synthesis
General information
13
1H NMR and C NMR spectra were recorded on an Agilent DDR2 400 spectrometer at
25°C. Chemical shifts (δ) are reported in ppm for the solution of compound in MeOD, CDCl3,
and DMSO-d6 with internal reference TMS and J values in Hz. MALDI spectra were recorded
on Bruker Microflex LT spectrometer. Elemental analysis was performed using an Elementar
(Vario Micro Cube) apparatus; final compounds have a purity of >95%. Separation by column
chromatography was performed using Merck Kieselgel 60 (70–230 mesh). All reactions were
performed with commercially available reagents (Aldrich, Alfa Aesar, Acros, Serva). Solvents
were purified according to standard procedures. The petroleum spirit refers to the fraction with
distillation range 40–70 °C.
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Synthesis of intermediate compounds
(Atomic numeration is given only for NMR assignment, for details see supplementary
information.)
Synthesis of N-deacetylallocolchicine 6
Synthetic procedure was carried out according to literature protocol39.
Colchicine 1 (3.000 g, 1 eq., 7.5 mmol) was treated with 4-DMAP (1.830 g, 2 eq., 15
mmol), triethylamine (0,756 g, 1 eq., 7.48 mmol, 1.043 ml), and Boc2O (5.700 g, 3.46 eq., 26
mmol) in MeCN at 100 °C for 2.5 h; after that another portion of Boc2O (3.200 g, 1.96 eq., 14.67
mmol) was added and the solution was heated at 100°C for 1 h. When the reaction was complete,
volatiles were evaporated and the product was isolated by column chromatography with AcOEt–
acetone (4 : 1) as red-brownish foam with78% yield (2.925 g).
1H NMR (400 MHz, DMSO-d6) δ 7.27 (s, 1H, 8-H), 7.11 (d, J = 10.7 Hz, 1H, 11-H), 7.02 (d, J =
10.9 Hz, 1H, 12-H), 6.77 (s, 1H, 4-H), 4.90 (dd, J = 12.4, 6.0 Hz, 1H, 7-H), 3.87 (s, 3H, OMe),
3.83 (s, 3H, OMe), 3.77 (s, 3H, OMe), 3.54 (s, 3H, OMe), 2.69 (m, 1H, 6-H), 2.34–2.25 (m, 1H,
5-H), 2.22 (s, 3H, NAc), 1.96–1.85 (m, 2H, 5-H, 6-H), 1.49 (s, 9H, NBoc).
13C NMR (101 MHz, DMSO-d6) δ 178.31, 170.79, 163.95, 153.55, 153.43, 150.91, 148.69,
141.26, 135.20, 134.96, 134.28, 132.16, 126.03, 112.59, 108.15, 84.80, 61.27, 61.09, 60.20,
56.48, 56.27, 32.24, 29.68, 27.72.
MALDI (neg. mode): 494.4 (100%), 484.3 (52%), 474.6 (20%), 497.4 (19%).
Elemental analysis: for C27H33NO8 calcd.: C, 64.92; H, 6.66; found: C, 64.71; H, 6.82.
mp 105 °C
N-Boc-colchicine (2.000 g, 1 eq., 4 mmol) was dissolved in dry methanol, sodium
methoxide (896 mg, 4 eq., 16 mmol) was added into the solution and the mixture was stirred at
40 °C for 40 min–1 h. When the reaction was complete, saturated solution of NH4Cl (26 mL)
was added and methanol was removed under reduced pressure. The aqueous solution was
extracted with AcOEt (3 × 50 mL); combined organic layer was washed with brine and dried
over Na2SO4. Solvent was evaporated under reduced pressure and N-Boc-deacetylallocolchicine
was obtained as beige solid with 99% yield (1.813 g) and used without further purification.
1H NMR (400 MHz, DMSO-d6) δ 7.69 (d, J = 7.8 Hz, 1H, NHBoc), 7.21 (s, 1H, 8-H), 7.10 (d, J
= 10.6 Hz, 1H, 10-H), 7.02 (d, J = 10.9 Hz, 1H, 11-H), 6.76 (s, 1H, 4-H), 4.08 (dt, J = 13.7, 7.0
Hz, 1H, 7-H), 3.87 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.54 (s, 3H, OMe), 2.55
(dd, J = 13.3, 6.0 Hz, 1H, 6-H), 2.17 (td, J = 13.1, 7.0 Hz, 1H, 5-H), 2.06 – 1.93 (m, 1H, 5-H),
1.87 – 1.77 (m, 1H, 6-H), 1.32 (s, 9H, NHBoc).
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13C NMR (101 MHz, DMSO-d6) δ 178.00, 163.50, 154.50, 152.94, 151.03, 150.36, 140.69,
135.10, 134.39, 134.26, 130.51, 125.31, 112.12, 107.65, 60.83, 60.64, 56.01, 55.84, 54.90,
52.87, 40.78, 35.80, 28.14.
MALDI (pos. mode): 458.2 (M+H+, 72%), 401.2 (39%), 356.1 (11%).
Elemental analysis: for C25H31NO7 calcd.: C, 65.63; H, 6.83; found: C, 65.82; H, 6.69.
mp 151 °C.
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N-Boc-deacetylallocolchicine (1.800 g, 1 eq., 3.93 mmol) was placed into round-bottom
flask and dissolved in 30 ml DCM; 9 mL TFA were added subsequently and the solution was
stirred for 1.5 h at room temperature. The acid was neutralized by addition of NaHCO3 saturated
solution until pH became 8. The aqueous layer was extracted with DCM (3 × 70 mL), the
combined organic layer was washed with brine and dried over Na2SO4. Solvent was evaporated
under reduced pressure and N-deacetylallocolchicine 5 was obtained as pale-beige solid with
95% yield (1.407 g) and used without further purification.
1H NMR (400 MHz, DMSO-d6) δ 7.64 (s, 1H, 8-H), 7.05 (d, J = 10.6 Hz, 1H, 10-H), 6.99 (d, J =
10.7 Hz, 1H, 11-H), 6.74 (s, 1H, 4-H), 3.86 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.76 (s, 3H, OMe),
3.55 (s, 3H, OMe), 3.46 (dd, J = 10.5, 5.8 Hz, 1H, 7-H), 2.37–1.91 (m, 4H, 6-H, 5-H).
13C NMR (101 MHz, DMSO-d6) δ 178.20, 163.26, 153.82, 152.73, 150.15, 140.47, 135.68,
134.88, 133.76, 131.83, 125.40, 111.84, 107.34, 60.66, 60.53, 55.90, 55.84, 53.11, 39.64, 29.87.
MALDI (pos. mode): 357.2 (100%), 341.2 (49%), 326.1 (18%).
Elemental analysis: for C20H23NO5 calcd.: C, 67.21; H, 6.49; found: C, 67.40 2; H, 6.21.
mp 133 °C.
Synthesis of allocolchifoline 4
N-deacetylallocolchicine 6 (1.400 g, 1 eq., 3.91 mmol) was mixed with glycolic acid (297
mg, 1 eq., 3.91 mmol) and N-hydroxysuccinimide (341 mg, 0.76 eq., 2.97 mmol). The mixture
was dissolved in dry DCM with further addition of triethylamine (1.184 g, 3 eq., 11.73 mmol,
1.634 mL) and di-isopropylcarbodiimide (DIC) (680 mg, 1.5 eq., 5.86 mmol, 835 µL). The
mixture was stirred at room temperature for 20 h. The solvent was removed under reduced
pressure and the product was isolated using column chromatography with the mixture of DCM–
methanol (18 : 1) as eluent. Allocolchifoline 4 was obtained as pale-yellow solid with 64% yield
(1.038 g).
1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 7.7 Hz, 1H, NH), 7.17 (s, 1H, 8-H), 7.11 (d, J =
10.6 Hz, 1H, 10-H), 7.02 (d, J = 10.9 Hz, 1H, 11-H), 6.77 (s, 1H, 4-H), 5.51 (t, J = 5.8 Hz, 1H,
OH), 4.41 (dt, J = 11.6, 7.2 Hz, 1H, 7-H), 3.87 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.83–3.80 (m,
2H, 2’-H), 3.79 (s, 3H, OMe), 3.52 (s, 3H, OMe), 2.59 (dd, J = 13.2, 6.0 Hz, 1H, 6-H), 2.25–2.16
(m, 1H, 5-H), 2.14–2.03 (m, 1H, 5-H), 1.97 (dd, J = 12.7, 6.3 Hz, 1H, 6-H).
13C NMR (101 MHz, DMSO-d6) δ 177.99, 172.78, 171.39, 163.52, 152.93, 150.50, 150.45,
140.71, 135.18, 134.28, 130.76, 125.48, 112.09, 107.72, 61.39, 60.79, 60.70, 56.03, 55.86,
50.85, 29.27, 25.23.
MALDI (neg. mode): 400.2 (100%), 415.2 (88%).
Elemental analysis: for C22H25NO7 calcd.: C, 63.61; H, 6.07; found: C, 63.45 2; H, 6.24.
mp 123 °C.
Synthesis of (aR,5S)-N-(3-carboxymethyl-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]-
cycloheptene-5-yl)(pent-4-yn-1-oyl)acetamide 7
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4-Pentynoic acid (25 mg, 1.5 eq., 0.252 mmol) was treated with Yamaguchi reagent
TCBC (82 mg, 2 eq., 0.336 mmol, 53 µL) and triethylamine (85 mg, 5 eq., 0.84 mmol, 117 µL)
in dry DCM for 5 h at 0–5 °C. Than the mixture of allocolchifoline 4 (70 mg, 1 eq., 0.168 mmol)
and 4-DMAP (61 mg, 3 eq., 0.504 mmol) in DCM was added dropwise and the mixture was
stirred for 20 h at room temperature. When the reaction was complete, solvent was removed
under reduced pressure and the product was isolated using column chromatography with the
mixture of petroleum–ethyl acetate–ethanol (5 : 1 : 1) as eluent. Compound 7 was isolated as
pale-beige solid with 51% yield (42 mg).
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1H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 7.4 Hz, 1H, NH), 7.11 (d, J = 11.3 Hz, 1H, 10-H),
7.10 (s, 1H, 8-H), 7.03 (d, J = 11.1 Hz, 1H, 11-H), 6.77 (s, 1H, 4-H), 4.53 (d, J = 1.5 Hz, 2H, 2’-
H), 4.39–4.31 (m, 1H, 7-H), 3.87 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.52 (s,
3H, OMe), 2.80 (t, J = 2.6 Hz, 1H, 7’-H), 2.58 (t, J = 7.2 Hz, 3H, 4’-H, 5’-H), 2.42–2.39 (m, 1H,
5’-H), 2.27–2.17 (m, 2H, 6-H, 5-H), 2.07–1.96 (m, 1H, 5-H), 1.95–1.85 (m, 1H, 6-H).
13C NMR (101 MHz, DMSO-d6) δ 177.94, 170.87, 166.04, 163.53, 152.97, 150.42, 150.11,
140.74, 134.97, 134.45, 134.12, 130.45, 130.04, 125.32, 112.10, 82.98, 71.67, 62.79, 62.25,
60.82, 60.69, 56.06, 55.85, 51.13, 35.67, 32.41, 29.15.
MALDI (DCTB, pos. mode): 518.2 (M+Na+, 49%), 495.4 (98%), 332.2 (100%).
Elemental analysis: for C27H29NO8 calcd.: C, 65.44; H, 5.90; found: C, 65.77; H, 6.02.
mp 53 °C.
Synthesis of iodo-colchinol 8
Synthesis of iodo-colchinol 8 was carried out according to literature protocol51.
Colchicine 1 (1200 mg, 3 mmol) was dissolved in 11.5 ml of glacial acetic acid, 70 mL
0.1 N hydrochloric acid was added, and the mixture was stirred for 3 h at 100 °C. When the
reaction was over, the mixture was cooled to room temperature, neutralized with NaHCO3 to pH
6, and extracted with CHCl3 (60 mL × 3); combined organic layer was dried over Na2SO4; the
solvent was removed under reduced pressure. Colchiceine was obtained as yellowish-green foam
with 98% yield (1132 mg) and used at the next step without further purification.
1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 7.9 Hz, 1H, NHAc), 6.87 (s, 1H, 8-H), 6.54 (s, 1H, 4-
H), 5.91 (d, J = 8.1 Hz, 1H, 11-H), 5.75 (d, J = 8.1 Hz, 1H, 12-H), 4.81−4.72 (m, 1H, 7-H), 3.92
(s, 3H, OMe), 3.88 (s, 3H, OMe), 3.53 (s, 3H, OMe), 2.54−2.26 (m, 4H, 5-H, 6-H), 2.04 (s, 3H,
NHAc).
13C NMR (101 MHz, CDCl3): δ 175.59, 170.36, 153.47, 151.57, 150.69, 141.32, 136.37, 134.31,
125.71, 122.53, 119.95, 107.22, 61.27, 61.22, 55.94, 52.61, 37.26, 29.64.
mp 151 °C (lit. 150 °C).
Colchiceine (1068 mg, 2.7 mmol, 1 eq.), obtained on the previous step, was dissolved in
26 ml H2O, NaOH (1106 mg, 27 mmol, 10 eq.) was added and the mixture was cooled to 0 °C.
NaI (9.3 g, 62.1 mmol, 23 eq.) and I2 (2.057 g, 8.1 mmol, 3 eq.) were dissolved in 116 mL H2O.
This mixture was added dropwise to solution of colchiceine during 1 h with temperature control
(<5 °C). After complete addition, the mixture was stirred for 1 h at 0 °C. When the reaction was
over (TLC control), the mixture was allowed to warm to ambient temperature. After removing
the excess of iodine by addition of Na2S2O3, the mixture was acidified with HCl (conc.) to pH 2
and extracted with AcOEt (50 mL × 3); organic layers were collected and dried over Na2SO4.
The solvent was removed under reduced pressure and iodo-colchinol 8 was obtained as yellow
powder with 95% yield (1.244 g). The product was used without further purification.
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1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H, OH), 8.38 (d, J = 8.0 Hz, 1H, NHAc), 7.56 (s,
1H, 8-H), 6.86 (s, 1H, 11-H), 6.76 (s, 1H, 4-H), 4.40–4.33 (m, 1H, 7-H), 3.82 (s, 3H, OMe), 3.77
(s, 3H, OMe), 3.48 (s, 3H, OMe), 2.50–2.44 (m, 1H, 6-H), 2.16–2.07 (m, 2H, 5-H), 1.93–1.88
(m, 1H, 6-H), 1.87 (s, 3H, NHAc).
13C NMR (101 MHz, DMSO-d6) δ 168.31, 155.63, 152.22, 150.17, 142.41, 140.51, 139.18,
134.77, 126.60, 123.24, 110.05, 108.07, 81.42, 60.55, 60.53, 55.82, 48.23, 37.91, 29.98, 22.62.
mp 238 °C (lit. 238 °C)
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Synthesis of (aR,5S)-N-(3-allyloxy-4-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo-
[a,c]cycloheptene-5-yl)acetamide 9
Iodo-colchinol 8 (750 mg, 1 eq., 1.546 mmol) was treated with allyl bromide (374 mg, 2
eq., 3.09 mmol, 267 µL) and potassium carbonate (640 mg, 3 eq., 4.64 mmol) in DMF under
inert atmosphere at 60 °C. The reaction was carried out for 20 h, the solvent was removed under
reduced pressure. To the residue, 50 mL of distilled water was added and the mixture was
extracted with AcOEt (2 × 70 mL); combined organic layer was washed with brine and dried
over Na2SO4. Solvent was removed under reduced pressure and the residue was purified using
column chromatography with the mixture of petroleum–ethyl acetate–ethanol (7 : 1 : 1) as
eluent. Compound 9 was isolated as pale-beige solid with 63% yield (511 mg).
1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.5 Hz, 1H, NHAc), 7.68 (s, 1H, 8-H), 6.98 (s,
1H, 11-H), 6.78 (s, 1H, 4-H), 6.08 (ddd, J = 17.5, 10.5, 5.1 Hz, 1H, 2’-H), 5.56 (d, J = 17.2 Hz,
1H, 3’-H), 5.33 (d, J = 10.5 Hz, 1H, 3’-H), 4.66 (br.s., 2H, 1’-H), 4.49 (dt, J = 12.0, 7.9 Hz, 1H,
7-H), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.50 (s, 3H, OMe), 2.55–2.50 (m, 1H, 6-H), 2.23–
2.12 (m, 1H, 5-H), 2.12–2.00 (m, 1H, 5-H), 1.89 (s, 3H, NHAc), 1.87–1.76 (m, 1H, 6-H).
13C NMR (101 MHz, DMSO-d6) δ 168.48, 155.69, 152.46, 150.15, 142.51, 140.51, 139.43,
134.82, 133.20, 128.32, 122.85, 117.55, 108.20, 107.79, 83.24, 69.13, 60.60, 60.55, 55.82,
48.16, 38.39, 30.00, 22.64.
MALDI (DCTB, pos. mode): 523.1 (100%), 332.2 (29%), 242.3 (23%).
Elemental analysis: for C23H26INO5 calcd.: C, 52.78; H, 5.01; found: C, 52.55; H, 5.14.
mp 82 °C.
Synthesis of (aR,5S)-N-(3-Allyloxy-4-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo-
[a,c]cycloheptene-5-yl)amine 10
Into Schlenk flask #1 compound 9 (496 mg, 1 eq., 0.95 mmol) and 4-DMAP (115 mg, 1 eq., 0.95
mmol) were put, the flask was filled with argon and acetonitrile was added under inert
atmosphere. Into Schlenk flask #2 Boc2O (826 mg, 4.6 eq., 3.79 mmol) was placed and
acetonitrile was added under inert atmosphere. A half of Schlenk #2 contents was added into the
first mixture followed by triethylamine (191 mg, 2 eq., 1.89 mmol, 264 µL) pouring. The
mixture was stirred for 3 h at 75 °C, then the second part of Schlenk #2 contents was added, the
solution was cooled to ambient temperature and stirred overnight. When the reaction was
complete, solvent was evaporated and the residue was purified using column chromatography
with the mixture of petroleum–ethyl acetate–ethanol (12 : 1 : 1) as eluent. (aR,5S)-N-(3-
Allyloxy-4-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-5-yl)(tert-
butoxycarbamoyl)acetamide was obtained as reddish foam with 86% yield (509 mg).
1H NMR (400 MHz, DMSO-d6) δ 7.71 (s, 1H, 8-H), 6.93 (s, 1H, 11-H), 6.79 (s, 1H, 4-H), 6.10–
6.02 (m, 1H, 2’-H), 5.49 (d, J = 17.2 Hz, 1H, 3’-H), 5.31 (d, J = 10.6 Hz, 1H, 3’-H), 5.13 (dd, J
= 9.1, 2.5 Hz, 1H, 7-H), 4.62 (dd, J = 34.5, 10.7 Hz, 2H, 1’-H), 3.83 (s, 3H, OMe), 3.78 (s, 3H,
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OMe), 3.44 (s, 3H, OMe), 2.68–2.57 (m, 2H, 6-H, 5-H), 2.28 (s, 3H, NAc), 2.18–2.04 (m, 2H, 5-
H, 6-H), 1.50 (s, 9H, NBoc).
13C NMR (101 MHz, DMSO-d6) δ 155.54, 153.30, 152.58, 150.36, 140.66, 140.19, 139.47,
134.67, 133.22, 128.54, 122.84, 117.37, 109.17, 108.18, 83.87, 83.73, 69.14, 69.11, 63.36,
60.61, 60.60, 55.80, 36.22, 34.64, 30.10, 27.46.
MALDI (DCTB, pos. mode): 623.2 (100%), 332.3 (27%), 486.6 (19%).
Elemental analysis: for C25H34INO7 calcd.: C, 53.94; H, 5.50; found: C, 53.81; H, 5.62.
mp 69 °C.
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(aR,5S)-N-(3-Allyloxy-4-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]-
cycloheptene-5-yl)(tert-butoxycarbamoyl)acetamide (485 mg, 1 eq., 0.78 mmol) was treated
with MeONa (13 mg, 0.3 eq., 0.23 mmol) in methanol at 40 °C for 1 h. When the reaction was
complete, the solvent was evaporated and the product was isolated using short-column silica-gel
chromatography (petroleum–ethyl acetate–ethanol, 15 : 1 : 1 as eluent) with 94% yield (425 mg)
as a beige oil.
1H NMR (400 MHz, DMSO-d6) δ 7.66 (s, 1H, 8-H), 7.52 (d, J = 8.5 Hz, 1H, NHBoc), 6.99 (s,
1H, 11-H), 6.77 (s, 1H, 4-H), 6.10 (ddd, J = 15.5, 10.2, 4.9 Hz, 1H, 2’-H), 5.55 (d, J = 15.1 Hz,
1H, 3’-H), 5.32 (d, J = 10.5 Hz, 1H, 3’-H), 4.64 (tt, J = 13.3, 6.5 Hz, 2H, 1’-H), 4.17 (dt, J =
15.8, 8.0 Hz, 1H, 7-H), 3.83 (s, 3H, OMe), 3.77 (s, 3H, OMe), 3.54 (s, 3H, OMe), 2.46 (d, J =
5.7 Hz, 1H, 6-H), 2.16 (dd, J = 12.3, 6.3 Hz, 1H, 5-H), 2.00 (dt, J = 19.8, 9.8 Hz, 1H, 5-H),
1.92–1.82 (m, 1H, 6-H), 1.35 (s, 9H, NHBoc).
13C NMR (101 MHz, DMSO-d6) δ 155.66, 154.74, 152.49, 150.08, 142.85, 140.49, 139.44,
134.84, 133.05, 128.25, 122.75, 117.60, 108.12, 107.77, 83.21, 77.91, 69.11, 60.66, 60.50,
55.80, 50.13, 38.31, 29.99, 28.19.
Elemental analysis: for C26H32INO6 calcd.: C, 53.71; H, 5.55; found: C, 53.66; H, 5.70.
MALDI (DCTB, pos. mode): 581.4 (100%), 242.3 (58%), 332.2 (29%), 396.2 (17%).
(aR,5S)-N-(3-Allyloxy-4-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]-
cycloheptene-5-yl)tert-butoxycarbamate (425 mg, 1 eq., 0.73 mmol), obtained at the previous
step, was dissolved in a mixture of DCM (30 mL) and concentrated hydrochloric acid (8 mL)
was added. The solution was stirred overnight at 30 °C. When the reaction was complete, the
mixture was neutralized with NaHCO3 sat. solution and extracted with DCM (3 × 50 mL),
combined organic layer was washed with brine and dried over Na2SO4. Solvent was removed
under reduced pressure and the residue was purified using column chromatography with the
mixture of petroleum–ethyl acetate–ethanol (6 : 1 : 1) as eluent. Compound 10 was isolated as
beige oil with 62% yield (218 mg).
1H NMR (400 MHz, DMSO-d6) δ 7.70 (s, 1H, 8-H), 7.32 (s, 1H, 11-H), 6.80 (s, 1H, 4-H), 6.13
(ddd, J = 15.5, 10.0, 4.7 Hz, 1H, 2’-H), 5.58 (d, J = 15.1 Hz, 1H, 3’-H), 5.32 (d, J = 10.6 Hz, 1H,
3’-H), 4.70 (s, 2H, 1’-H), 3.83 (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.69 (dd, J = 11.2, 6.7 Hz, 1H,
7-H), 3.54 (s, 3H, OMe), 2.56–2.50 (m, 1H, 6-H), 2.45–2.33 (m, 1H, 5-H), 2.10–1.99 (m, Hz,
1H, 5-H), 1.90–1.82 (m, 1H, 6-H).
13C NMR (101 MHz, DMSO-d6) δ 155.66, 152.57, 150.11, 140.85, 140.52, 139.58, 134.73,
133.10, 128.24, 122.48, 117.52, 108.41, 108.17, 83.75, 69.31, 60.66, 60.51, 55.86, 50.47, 29.84,
28.21.
Elemental analysis: for C21H24INO4 calcd.: C, 52.40; H, 5.03; found: C, 52.23; H, 5.17.
MALDI (DCTB, pos. mode): 481.0 (100%), 332.2 (45%), 465.0 (36%).
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Synthesis of (aR,5S)-N-(9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo-[a,c]cycloheptene-5-yl-
[2,3-f]-3’-methyleno-2’-hydrofuranyl)2-hydroxyacetamide 5
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Substrate 10 (200 mg, 1 eq., 0,41 mmol), glycolic acid (32 mg, 1 eq., 0,41 mmol), and N-
hydroxysuccinimide (36 mg, 0.76 eq., 0.31 mmol) were dissolved in dry DCM under inert
atmosphere. Et3N (124 mg, 3 eq., 1.23 mmol, 171 µL) and DIC (58 mg, 1.5 eq., 0.62 mmol, 99
µL) were subsequently added and the mixture was stirred at room temperature for 20 h. After the
completion of the reaction, the solvent was removed under reduced pressure and the residue was
purified using column chromatography with the mixture of petroleum–ethyl acetate–ethanol (8 :
1 : 1) as eluent. Acylated product was isolated as beige oil with 79% yield (175 mg).
1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 8.8 Hz, 1H, NH), 7.67 (s, 1H, 8-H), 6.98 (s, 1H,
11-H), 6.79 (s, 1H, 4-H), 6.08 (ddd, J = 15.6, 10.4, 5.1 Hz, 1H, 2’-H), 5.56 (dd, J = 17.3, 1.4 Hz,
1H, 3’-H), 5.52 (s, 1H, OH), 5.32 (d, J = 10.6 Hz, 1H, 3’-H), 4.67 (d, J = 11.7 Hz, 2H, 13-H),
4.60–4.53 (m, 1H, 7-H), 3.83 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.50 (s, 3H, OMe), 2.55–2.51
(m, 1H, 6-H), 2.14–2.02 (m, 3H, 6-H, 5-H).
13C NMR (101 MHz, DMSO-d6) δ 171.32, 155.61, 152.48, 150.18, 142.28, 140.52, 139.32,
134.89, 133.17, 128.31, 122.86, 117.67, 108.23, 108.16, 83.28, 69.12, 61.56, 60.60, 60.58,
55.83, 47.77, 37.97, 30.03.
MALDI (DCTB, pos. mode): 412.1 (100%), 562.0 (M+Na+, 77%), 539.0 (69%), 465.0 (38%).
Elemental analysis: for C23H26INO6 calcd.: C, 51.22; H, 4.86; found: C, 51.33; H, 4.99.
mp 124 °C
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Substrate, obtained at the previous step (170 mg, 1 eq., 0.32 mmol) was treated with
Pd(dppf)Cl2 (11 mg, 0.05 eq., 0.016 mmol) and AcOK (93 mg, 3 eq., 0,945 mmol) in DMSO.
The mixture was stirred for 20 h at 75 °C. When the reaction was over, distilled water (50 mL)
was added to the mixture and the resulting solution was extracted with AcOEt (3 × 70 mL).
Combined organic layer was washed with brine and dried over Na2SO4. Solvent was removed
under reduced pressure and the residue was purified using column chromatography with the
mixture of petroleum–ethyl acetate–ethanol (9 : 1 : 1) as eluent. Compound 5 was isolated as
beige oil with 87% yield (114 mg).
1H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 8.8 Hz, 1H, NH), 7.41 (s, 1H, 8-H), 6.88 (s, 1H,
11-H), 6.78 (s, 1H, 4-H), 5.49 (d, J = 6.0 Hz, 1H, 10-H), 5.46 (t, J = 3.1 Hz, 1H, OH), 5.14 (d, J
= 2.7 Hz, 2H, 9-H), 5.02 (d, J = 2.5 Hz, 1H, 10-H), 4.61–4.52 (m, 1H, 7-H), 3.84 (d, J = 1.8 Hz,
2H, 13-H), 3.83 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.48 (s, 3H, OMe), 2.19–1.99 (m, 4H, 5-H, 6-
H).
13C NMR (101 MHz, DMSO-d6) δ 171.23, 162.48, 152.21, 150.35, 143.65, 143.27, 140.57,
134.81, 126.56, 124.31, 123.62, 122.00, 108.05, 105.61, 99.64, 74.92, 61.52, 60.59, 60.56,
55.83, 48.12, 37.75, 29.98.
MALDI (pos. mode): 411.0 (100%), 379.0 (11%), 353.0 (7%).
Elemental analysis: for C23H25NO6 calcd.: C, 67.14; H, 6.12; found: C, 67.27; H, 6.02.
mp 157 °C.
Synthesis of (aR,5S)-N-(9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo-[a,c]cycloheptene-5-yl-
[2,3-f]-3’-methyleno-2’-hydrofuranyl)(2-pent-4-oyl)acetamide 11
4-Pentynoic acid (36 mg, 1.5 eq., 0.364 mmol) was treated with Yamaguchi reagent
TCBC (118 mg, 2 eq., 0.486 mmol, 76 µL) and triethylamine (123 mg, 5 eq., 1.215 mmol, 169
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µL) in dry DCM for 5 h at 0–5 °C. Then the mixture of compound 5 (100 mg, 1 eq., 0.243
mmol) and 4-DMAP (89 mg, 3 eq., 0.729 mmol) in DCM was added dropwise and the mixture
was stirred for 20 h at room temperature. After completion of the reaction, solvent was removed
under reduced pressure and the product was isolated using column chromatography with the
mixture of petroleum–ethyl acetate–ethanol (10 : 1 : 1) as eluent. Compound 11 was isolated as
pale-beige solid with 49% yield (58 mg).
1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 8.2 Hz, 1H, NH), 7.42 (s, 1H, 8-H), 6.85 (s, 1H,
11-H), 6.78 (s, 1H, 4-H), 5.47 (br.s, 1H, 10-H), 5.15 (s, 2H, 9-H), 5.03 (br.s, 1H, 10-H), 4.56 (d,
J = 3.2 Hz, 2H, 2’-H), 4.52–4.46 (m, 1H, 7-H), 3.83 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.48 (s,
3H, OMe), 2.81 (s, 1H, 7’-H), 2.60 (t, J = 7.2 Hz, 2H, 4’-H), 2.42–2.39 (m, 2H, 5’-H), 2.22–1.97
(m, 3H, 6-H, 5-H), 1.97–1.88 (m, 1H, 6-H).
13C NMR (101 MHz, DMSO-d6) δ 172.88, 170.96, 165.93, 162.53, 152.24, 150.34, 143.24,
143.22, 140.60, 134.65, 126.51, 124.21, 123.75, 122.12, 108.06, 105.36, 99.74, 83.53, 82.99,
74.92, 71.67, 71.31, 60.58, 55.82, 48.58, 37.92, 33.03, 32.47.
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MALDI (DCTB, pos. mode): 491.1 (100%), 394.2 (37%), 354.2 (26%)
Elemental analysis: for C28H29NO7 calcd.: C, 68.42; H, 5.95; found: C, 68.23; H, 6.07.
mp 67 °C.
1.3 Synthesis of target conjugates 15 and 16
Synthesis of 1-palmitoyl-2-(11-azidoundecanoyl)-sn-glycero-3-phosphocholine 14
11-Azidoundecanoic acid (125 mg, 6 eq., 0.546 mmol), 1-palmitoyl-2-hydroxy-sn-
glycero-3-phosphocholine (Avanti Polar Lipids, Inc.) (45 mg, 1 eq., 0.091 mmol), and 4-DMAP
(67 mg, 6 eq., 0.546 mmol) were dissolved in dry CHCl3. DIC (92 mg, 8 eq., 0.728 mmol, 113
µL) was added subsequently and the mixture was stirred for 36 h at room temperature. Then
solvent was removed and the product was isolated using column chromatography with the
mixture of CHCl3–EtOH (6 : 4) → CHCl3–EtOH–H2O (10 : 10 : 1) as a colorless oil with 78%
yield (50 mg).
1H NMR (400 MHz, CD3OD) δ 5.20 (dd, J = 6.2, 2.9 Hz, 1H, 2-H), 4.39 (dd, J = 12.0, 3.1 Hz,
1H, 4-H), 4.23 (br.s, 2H, 5-H), 4.13 (dd, J = 12.0, 6.9 Hz, 1H, 4-H), 3.98 (t, J = 6.2 Hz, 2H, 1-
H), 3.61 – 3.56 (m, 2H, 3-H), 3.24 (t, J = 6.9 Hz, 2H, 2’-H), 3.19 (s, 9H, NMe3), 2.29 (q, J = 7.7
Hz, 4H, 2’’-H, 11’’-H), 1.61 – 1.53 (m, 6H, 3’-H, 3’’-H, 10’’-H), 1.32 – 1.20 (m, 36H, alkyl
chains), 0.85 (t, J = 6.7 Hz, 3H, 16’-H).
13C NMR (101 MHz, CD3OD) δ 174.46, 174.05, 70.89, 70.81, 66.88, 66.82, 64.23, 64.18, 63.08,
59.65, 59.60, 54.47, 51.90, 34.64, 34.52, 32.37, 30.13, 30.11, 30.09, 29.95, 29.90, 29.82, 29.80,
29.75, 29.72, 29.58, 29.52, 29.27, 27.16, 25.34, 25.33, 23.10, 14.29.
31P NMR (162 MHz, CD3OD) δ –0.80.
General procedure for conjugation
Alkyne-source (1.2 eq.) and azido-containing phosphatydylcholine 14 (1 eq.) were
dissolved in DMF under inert atmosphere. To this mixture, solution of CuSO4 · 5H2O (0.2 eq.),
sodium ascorbate (0.2 eq.), and tris(benzyltriazolylmethyl)amine (TBTA) (0.4 eq.) in water was
added dropwise. The resulting solution was stirred for 2 h at 55 °C. After completion of the
reaction, solvents were removed under reduced pressure and the target conjugate was isolated
using column chromatography with the mixture of CHCl3–EtOH (6 : 4) → CHCl3–EtOH–H2O
(10 : 10 : 1) as eluent.
Compound 15
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Isolated as white solid with 56% yield.
1H NMR (400 MHz, CD3OD) δ 7.58 (s, 1H, 18-H), 7.54 (d, J = 1.4 Hz, 1H, NH), 7.37 (d, J =
10.8 Hz, 1H, 10-H), 7.32 (s, 1H, 8-H), 7.01 (d, J = 10.9 Hz, 1H, 11-H), 6.61 (s, 1H, 4-H), 5.20
(dd, J = 6.5, 2.9 Hz, 1H, 2’-H), 4.63 (d, J = 6.6 Hz, 1H, 4’-H), 4.41 – 4.37 (m, 1H, 4’-H), 4.28
(d, J = 7.1 Hz, 2H, 5’-H), 4.23 (s, 2H, 13-H), 4.13 (dd, J = 12.0, 6.8 Hz, 1H, 7-H), 3.98 (s, 3H,
OMe), 3.95 (d, J = 9.4 Hz, 2H, 1’-H), 3.90 (s, 3H, OMe), 3.89 (s, 3H, OMe), 3.59 (s, 3H, OMe),
3.58 (d, J = 3.2 Hz, 2H, 3’-H), 3.20 (s, 9H, NMe3), 3.04 (d, J = 4.6 Hz, 2H, 15-H), 2.81 (t, J =
6.9 Hz, 2H, 16-H), 2.59 (dd, J = 13.7, 6.3 Hz, 1H, 11b-H), 2.39 (dd, J = 13.2, 6.5 Hz, 1H, 11b-
H), 2.29 (dt, J = 13.8, 7.3 Hz, 6H, 3a-H, 3b-H, 10b-H), 2.21 (dd, J = 12.5, 6.3 Hz, 1H, 2a-H),
2.13–2.06 (m, 1H, 2a-H), 1.87–1.82 (m, 2H, 2b-H), 1.59–1.55 (m, 4H, 5-H, 6-H), 1.27–1.23 (m,
36H, alkyl chains), 0.85 (br.s, 3H, 16a-H).
13C NMR (101 MHz, CD3OD) δ 174.51, 174.07, 172.65, 168.48, 165.67, 154.28, 153.07, 151.53,
141.95, 137.86, 136.87, 135.16, 133.12, 126.01, 117.97, 117.92, 114.28, 108.12, 78.32, 72.39,
70.88, 70.79, 66.88, 66.78, 64.55, 63.12, 63.03, 61.75, 61.68, 59.97, 59.90, 56.77, 56.44, 54.48,
52.86, 51.07, 49.50, 49.29, 49.07, 36.52, 34.66, 34.55, 33.64, 32.45, 30.65, 30.31, 30.20, 30.16,
30.01, 29.87, 29.82, 29.75, 29.64, 29.56, 29.46, 26.92, 25.39, 23.17, 20.93, 14.31.
31P NMR (162 MHz, CD3OD) δ –0.64.
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HRMS: found: 1200.6859
mp 47 °C.
Compound 16
Isolated as white solid with 62% yield.
1H NMR (400 MHz, CD3OD) δ 7.57 (d, J = 5.5 Hz, 1H, NH), 7.55 (s, 1H, 18-H), 7.46 (s, 1H, 8-
H), 6.73 (s, 1H, 11-H), 6.62 (s, 1H, 4-H), 5.37 (br.s, 1H, 10-H), 5.22–5.19 (m, 1H, 2’-H), 5.09 (s,
2H, 9-H), 4.97 (s, 1H, 10-H), 4.76–4.71 (m, 1H, 4’-H), 4.55 (dd, J = 4.5, 1.4 Hz, 1H, 4’-H), 4.39
(t, J = 9.1 Hz, 1H, 7-H), 4.23 (t, J = 6.9 Hz, 4H, 15-H, 16-H), 4.14 (dd, J = 11.9, 6.8 Hz, 2H, 1’-
H), 3.97 (s, 2H, 13-H), 3.90 (s, 3H, OMe), 3.88 (s, 3H, OMe), 3.58 (br.s, 2H, 3’-H), 3.52 (s, 3H,
OMe), 3.19 (s, 9H, NMe3), 3.09–3.05 (m, 2H, 5’-H), 2.85–2.81 (m, 2H, 11b-H), 2.54–2.42 (m,
2H, 2a-H), 2.31–2.27 (m, 6H, 2b-H, 3b-H, 10b-H), 1.84–1.79 (m, 2H, 3a-H), 1.59–1.55 (m, 4H,
5-H, 6-H), 1.32–1.16 (m, 36H, alkyl chains), 0.85 (t, J = 4.6 Hz, 3H, 16a-H).
13C NMR (101 MHz, CD3OD) δ 174.46, 174.00, 172.77, 168.31, 155.43, 153.06, 141.97, 141.57,
136.90, 135.85, 129.18, 121.39, 115.92, 108.28, 106.30, 94.51, 84.37, 78.27, 70.70, 70.63,
66.69, 64.81, 63.36, 62.85, 61.62, 61.32, 60.20, 56.40, 56.25, 54.44, 51.16, 50.21, 50.16, 49.71,
49.50, 49.29, 49.07, 48.86, 38.78, 34.62, 34.52, 33.50, 32.42, 31.08, 30.42, 30.17, 30.13, 29.98,
29.84, 29.79, 29.68, 29.61, 29.50, 29.34, 26.79, 25.36, 25.32, 25.01, 23.14, 20.85, 14.30.
31P NMR (162 MHz, CD3OD) δ –4.57.
HRMS: found: 1196.6882
mp 41 °C.
Synthesis and characterization of carboxyl-containing compounds for comparative analysis are
given in supplementary information
Quantum Chemistry
Geometries of colchicinoids were optimized by the Hartree–Fock method with the def2-
SVP basis set. Calculations were carried out using the ORCA software83. Calculations were
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performed for compounds 4 and 5 on the assumption that the resulting colchicinoid structures
will be equivalent to those in 15 and 16.
Van der Waals distances listed in84 were used to calculate the volumes, dimensions, and
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projections for the rigid part of the colchicinoid molecule (without the HO–CH2–CO– fragment).
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Monolayer Surface Area Experiments
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In the monolayer experiments, we used 0.5 mg/mL POPC solution in chloroform–
methanol (5 : 1, v/v) (Merck KGaA and Macron Fine Chemicals, correspondingly) or the POPC
solution mixed with 0.3 mg/mL colchicinoid solution in chloroform. To create lipid film, we
used a Hamilton syringe to place the lipid solution on the surface of 10 mM KCl (Sigma-
Aldrich) prepared in triple distilled water. To let the solvent evaporate, the measurements were
performed in about 20 min (room temperature of about 20 °C) after applying the lipid film.
Experimental pressure–area diagrams were obtained simultaneously with the Volta potential
measurements. The experiments were performed using a Microtrough XS equipment operating
under FilmWareX-4.0 software (both Kibron Inc. Finalnd) with a PTFE trough and two POM
barriers. Before the measurements, a cleaning procedure was performed, and the system was
tested for the stability of pressure (within 0.1 mN/m) and potential (within 10 mV) over the
entire area of the trough. We used the compression speed of 10 mm2/min.
Liposome Preparation and Characteristics
Liposome Preparation
Liposomes (large unilamellar vesicles) were prepared as described earlier85. Briefly, lipid
films were obtained by co-evaporation of aliquots of stock solutions in chloroform–methanol (2 :
1) in round-bottom flask on a rotary evaporator, with subsequent drying for 45 min at 5 Pa. The
resulting compositions were ePC–conjugate 15, 95 : 5; ePC–conjugate 16, 95 : 5; ePC, 100 %
(by mol). Then lipid films were hydrated in phosphate buffered saline (PBS, pH 7.4) for MTT
experiments or TrisHCl/NaCl buffer for TLC (pH = 8.2, with addition 5 mM Ca2+), subjected to
seven cycles of freezing/thawing (liquid nitrogen/+40 °C), and extruded 20 times through
polycarbonate membrane filters (Nucleopore, USA) with a pore size of 100 nm on a Mini-
extruder by Avanti. Concentrations of 15 and 16 were controlled by UV spectrophotometry after
liposome disruption with ethanol (λ15 max = 350 nm, ε ~ 10200 M –1 cm –1, λ16 max = 250 nm, ε
~ 16500 M –1 cm –1).
To prepare liposomes with self-quenched calcein, lipid films were hydrated in PBS with
80 mM calcein and processed as described above. After extrusion, non-encapsulated calcein was
removed by size exclusion chromatography on a Sephadex G-50 column (1.3 × 18 cm)
equilibrated in PBS: 200-μL aliquots of calcein-containing liposome dispersions were applied
onto the column, the void volume (4.5 mL) was discarded, and then 150–200 μL fractions were
collected. Three or four peak liposomal fractions were combined. 15, 16, and calcein
concentrations were evaluated by measuring UV spectra of decomposed liposomal dispersions in
−1
−1
ethanol (λ max calcein 504 nm, ε ~74,000 M cm ). Liposome dispersions were stored at
+4°C and used for experiments within 5 days.
Zeta Potential
For reliable measurements, liposome samples were prepared as described earlier85.
Briefly, lipid films were hydrated in 10 mM KCl, 1 mM KH2PO4, 1mM K2HPO4 solution (pH
7.35), and after freezing–thawing procedure they were extruded 20 times through 200 nm
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polycarbonate membrane filters to gain liposome diameter of 200 nm. Zeta potential values were
obtained using ZetaPALS analyzer (Brookhaven Instruments Corp., Holtsville, NY; provided by
the CoreFacility of the Institute of Gene Biology, Russian Academy of Sciences). Samples of
liposomes (1.5 mL, 1 mg/mL total lipids) were equilibrated for 1 min in cuvettes before 10 runs
of 25 cycles per sample were performed at 25 °C. Zeta potential values were calculated using
Smoluchowski approximation.
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Dynamic Light Scattering and TEM
Liposome size upon preparation was controlled in diluted suspensions (1 mg total
lipids/mL PBS) by dynamic light scattering using a ZetaPALS analyzer (Brookhaven
Instruments Corp., Holtsville, NY; provided by the CoreFacility of the Institute of Gene Biology,
Russian Academy of Sciences) equipment in at least ten runs per sample.
The liposome suspensions were studied under a Jeol JEM-100-CX-II transmission electron
microscope. To prepare a sample, a droplet of dispersion was placed on a copper carbon-coated
grid and left for 2 min. The excess of liquid was then carefully removed with an edge of a filter
paper and the grid was stained with a droplet of 2% uranyl acetate (aqueous solution) for 2 min.
After the removal of the stain droplet, the grid was air-dried. Transmission electron microscopy
was performed using an acceleration voltage of 80 kV.
PLA2 Hydrolysis
General Information
Liposomes (ePC; ePC–conjugate 15; ePC–conjugate 16) with total lipid concentration 5
mM were treated with PLA2s from Viperae ursinii and porcine pancreas. Enzyme concentration
in every sample was 1 μM. Liposome suspension was incubated with PLA2 for 15, 30, 60 min, or
24 h. Then PLA2 activity was inhibited by 8-fold excess of EDTA, compared to Ca2+
concentration. The samples were diluted with ethanol, evaporated on a rotary evaporator, and
vacuum dried for 4 h at 3 Pa. Lipid components were extracted with the mixture of CHCl3 and
CH3OH, 1 : 1, using ultrasound treatment.
Insoluble material was pelleted and discarded (800 g, 10 min). Supernatant was analyzed by
TLC on aluminium sheets precoated with silica gel (Kieselgel 60, Merck, Darmstadt, Germany).
Eluent: CHCl3–CH3OH–NH4OH 28%, 65 : 25 : 4. Detection: molybdenum blue reagent by
Vaskovsky86.
Cytotoxicity Determination
Cell Cultures
Pancreatic cell lines BxPC-3, PANC-1, COLO-357, and HaCaT were grown in DMEM
medium supplemented with 10% fetal calf serum (FCS), pen-strep-glut (all from PanEco,
Moscow, Russian Federation). All cell lines used were routinely tested for mycoplasma.
Adherent cells were detached using 0.05% trypsin–EDTA (PanEco, Moscow), counted, and sub-
cultured. Twenty four hours before assays, cells were seeded in appropriate plates (96- or 24-
well plates), adjusted to 3 × 105 cells/mL, and incubated overnight to achieve standardized
growth conditions.
MTT Assay
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Cytotoxic effect of the allocolchicinoids was estimated by a standard 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) test as was described earlier87.
The compounds were dissolved in dimethylsulfoxide to 20 mM concentration and stored at –20
°C until the assay. Different dilutions of the compounds in the DMEM medium from 20 M to
0.1 nM were prepared separately and transferred in 100 µL to the plates with the cells. Liposome
dispersions containing equivalent amounts of conjugates 15 and 16 were prepared similarly.
Non-treated cells served as controls. Plates were incubated for 72 h. For the last 6 h, 5 mg/ml of
MTT was added in the amount of 10 µL to each well. After the incubation, culture medium was
removed and 100 L dimethylsulfoxide was added to each well. Plates were incubated at
shaking for 15 min to dissolve the formed formazan product. Optical density was read on
spectrophotometer Titertek (UK) at 540 nm. Results were analyzed in Excel package
(Microsoft). Cytotoxic concentration giving 50% of the maximal toxic effect (IC50) was
calculated from the titration curves.
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Acknowledgements
Synthesis, PLA2 responsivity studies, liposome stability investigations, and cytotoxicity
studies were supported by the Russian Foundation of Basic Research (project no. 17-33-50121).
Biophysical investigations (conjugate 15 and 16 geometry calculations, lateral stress profile
calculations, and monolayer experiments) were supported by the Russian Science Foundation
(project no. 17-79-20440). The Russian Foundation of Basic Research grant (19-04-00242)
used for monolayer equipment maintenance is also acknowledged.
Author Contributions
I.B. designed the study. I.B., Ek.S., A.F., and E.V. designed conjugates 15and 16. Ek.S.
synthesized both 15 and 16 molecules. Ek.S., D.T., and A.A. performed PLA2 responsivity
studies. Y.U. isolated enzymes. I.B. performed quantum chemistry calculations T.G. calculated
the lateral stress profile. Y.E. performed monolayer experiments. N.K., El.S. performed MTT
tests. A.A. and V.C. performed TEM analysis. All authors contributed to data analysis and
manuscript writing.
Supporting Information
Complete characteristics of synthesized compounds; parameters of quantum chemistry
calculations; liposome characterization; PLA2 hydrolysis raw data. The Supporting Information
is available free of charge on the ACS Publications website at DOI:
xxx.
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