Synthesis and biological evaluation of novel mono- and bivalent ASGP-R-targeted drug-conjugates

Article abstract of DOI:10.1016/j.bmcl.2017.12.032

Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose and anticancer drug – paclitaxel (PTX). Several molecules have demonstrated high affinity towards ASGP-R and good stability under physiological conditions, significant in vitro anticancer activity comparable to PTX, as well as good internalization via ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be regarded as a promising therapeutic option against HCC.

Full text of DOI:10.1016/j.bmcl.2017.12.032

Accepted Manuscript
Synthesis and Biological Evaluation of Novel Mono- and Bivalent ASGP-R-
targeted Drug-conjugates
Rostislav A. Petrov, Svetlana Yu. Maklakova, Yan A. Ivanenkov, Stanislav A.
Petrov, Olga V. Sergeeva, Emil Yu. Yamansarov, Irina V. Saltykova, Igor I.
Kireev, Irina B. Alieva, Ekaterina V. Deyneka, Alina A. Sofronova, Anastasiia
V. Aladinskaia, Alexandre V. Trofimenko, Renat S. Yamidanov, Sergey V.
Kovalev, Victor E. Kotelianski, Timofey S. Zatsepin, Elena K. Beloglazkina,
Alexander G. Majouga
PII:
S0960-894X(17)31192-7
https://doi.org/10.1016/j.bmcl.2017.12.032
BMCL 25488
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 November 2017
12 December 2017
13 December 2017
Please cite this article as: Petrov, R.A., Maklakova, S.Y., Ivanenkov, Y.A., Petrov, S.A., Sergeeva, O.V.,
Yamansarov, E.Y., Saltykova, I.V., Kireev, I.I., Alieva, I.B., Deyneka, E.V., Sofronova, A.A., Aladinskaia, A.V.,
Trofimenko, A.V., Yamidanov, R.S., Kovalev, S.V., Kotelianski, V.E., Zatsepin, T.S., Beloglazkina, E.K., Majouga,
A.G., Synthesis and Biological Evaluation of Novel Mono- and Bivalent ASGP-R-targeted Drug-conjugates,
Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.032
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Synthesis and Biological Evaluation of Novel Mono- and Bivalent ASGP-R-targeted
Drug-conjugates
Rostislav A. Petrova, Svetlana Yu. Maklakovaa, Yan A. Ivanenkova-c,g,*, Stanislav A. Petrova, Olga V.
Sergeevaa,d, Emil Yu. Yamansarova, Irina V. Saltykovaa, Igor I. Kireevf, Irina B. Alievaf, Ekaterina V.
Deynekab, Alina A. Sofronovah, Anastasiia V. Aladinskaiab, Alexandre V. Trofimenkob, Renat S.
Yamidanovg, Sergey V. Kovaleva, Victor E. Kotelianskid, Timofey S. Zatsepina,d, Elena K. Beloglazkinaa,
Alexander G. Majougaa,c,e
.
a Lomonosov Moscow State University, Chemistry Dept, Leninskie gory, Building 1/3, GSP-1, Moscow, 119991, Russian Federation.
b Moscow Institute of Physics and Technology (State University), 9 Institutskiy lane, Dolgoprudny City, Moscow Region, 141700, Russian Federation.
c National University of Science and Technology MISiS, 9 Leninskiy pr, Moscow, 119049, Russian Federation.
d Skolkovo Institute of Science and Technology,100 Novaya st., 143025 Skolkovo, Russian Federation.
e Dmitry Mendeleev University of Chemical Technology of Russia, Miusskaya sq. 9, Moscow, 125047, Russian Federation.
f
Lomonosov Moscow State University, A.N. Belozersky Institute of Physico-Chemical Biology, Leninskye gory, house 1, building 40, Moscow, 119992, Russian
Federation
g Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences (IBG RAS), Prosp. Oktybrya 71, Ufa, Bashkortostan, 450054, Russian
Federation.
h Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
* corresponding author, e-mail: yai@chemdiv.com
ARTICLE INFO
ABSTRACT
Article history:
Received
Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into
hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of
ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular
carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic
approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-
aminogalactopyranose and anticancer drug – paclitaxel (PTX). Several molecules have
demonstrated high affinity towards ASGP-R and good stability under physiological conditions,
significant in vitro anticancer activity comparable to PTX, as well as good internalization via
ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be
regarded as a promising therapeutic option against HCC.
Received in revised form
Accepted
Available online
Keywords:
ASGP-R
targeted drug delivery
paclitaxel
2017 Elsevier Ltd. All rights reserved.
cancer
Cancer is the second leading cause of death. In 2015, it was
responsible for 8.8 million deaths worldwide. Generally, about 1
in 6 deaths is due to cancer. Hepatocellular carcinoma (HCC,
liver cancer) is the most common type of primary liver cancer
and accounts for 90% of all liver cancers (~788,000 deaths are
associated with HCC1). Many therapeutic options including
small-molecule drugs are available to date. However, they have a
number of disadvantages such as low selectivity and high toxicity
rate with a wide range of adverse side effects. Targeted drug
delivery (TDD) can be reasonably regarded as a promising route
for the modification of medicinal agents, which allows scientists
to improve the pharmacological profile of anticancer drugs,
especially highly cytotoxic molecules. Using this approach,
therapeutics can be selectively localized (or targeted) in the
desired tissue, organ or cell, to improve their therapeutic index
and efficiency by increasing its active concentration and reducing
the total amount within the organism2.
Asialglycoprotein receptor (ASGP-R, C-type lectin family) is
one of the most attractive targets for the TDD of medications in
liver cells3. This is due to several key reasons: 1) an outstanding
receptor selectivity toward galactose derivatives, 2) elevated
expression level in hepatocytes and predominant localization on
the surface, 3) a relatively high exposition (over 500K receptors
per hepatocyte), and 4) receptor internalization into the cells via
clathrin-mediated endocytosis (turnover approx. 15-20 min with
or without ligands)4,5. Several comprehensive reviews describing
the architecture and functions of ASGP-R as well as ligand
properties and selectivity have recently been described3,6–9. The
receptor recognizes hydroxyl groups in 3rd and 4th positions of
galactose. The outer part of the receptor consists of three
subunits: 2×H1 (46 kDa) and H2 (50 kDa), each of them is able

to selectively bind galactose. The core structure of the subunits
includes a 40-amino-acid N-terminal cytoplasmic domain, a ∼20-
amino-acid single-pass transmembrane domain, an ∼80-amino-
acid extracellular stalk region, and an ∼140-amino-acid
functional calcium-dependent carbohydrate recognition domain
(CRD, C-type Ca2+-dependent superfamily) anchored via
intervening neck region. The monosaccharide ligands, e.g. D-
mannose, D-glucose, D-galactose and their derivatives, interact
with CRDs by direct calcium coordination. The trimeric
ensemble of the ASGP-R extracellular subunits is the most
abundant configuration observed among mammalian species and
demonstrates the highest binding affinity to endogenous ligand -
asialoorosomucoid (ASOR), whereas single CRD exhibits low
binding affinity. Therefore, it is not surprising that in contrast to
monovalent ligands, bivalent and especially trivalent analogues
show dramatically enhanced binding potency toward ASGP-R10.
However, several bivalent ligands with a comparatively high
target affinity have been reported as well11–14. The variation in the
number and structure of the galactose fragments may increase the
binding affinity and in vitro activity, which has been the subject
of many studies. However, in accordance with the results by
Prakash et al.,15 the number of galactose moieties did not have a
significant effect on activity in vivo.
As a promising galactose moiety, we have selected ligand 3
(Fig. 1), previously described by Mamidyala32, Sanhueza33 and
co-authors, that has demonstrated high binding affinity towards
the receptor. In addition, warheads 1 and 2 were proposed as
alternative cores in order to assess the effect of allyl group and
attachment point on binding.
Fig. 1. GalNAc core-heads used in this study.
Initially, the selected core-heads have been synthesized by
analogy to the approaches described previously34–36. Thus,
GalNAc building block 1, containing the azide attachment point
at C1 position, was prepared in full accordance with the synthetic
route published by Salunke and co-workers34, while sugars 2 and
3 were synthesized following a slightly modified procedure
(Scheme 1).
There are many examples of ASGP-R-targeted conjugates
(including functionalized nanoparticles) equipped by siRNA or
different small-molecule drugs targeted against several liver-
related disorders16. For example, PK2 by Pfizer was evaluated in
Phase 1/2 clinical trial in patients with primary or metastatic liver
cancer17. Doxorubicin (Dox) was attached via a lysosomally
degradable
tetrapeptide
sequence
to
N-(2-
hydroxypropyl)methacrylamide copolymers bearing the GalNAc
core-head. As a result, it was clearly demonstrated that the liver-
specific Dox delivery was effective, and a dose schedule was
recommended for Phase 2 studies with subjects suffering from
primary hepatocellular tumors. However, no additional
information has been published for this trial. Alnylam
Pharmaceuticals is the leading company within the title field18.
Several clinical trials have been proceeded, for instance against
Scheme 1. Synthesis of building blocks 2 and 3. (i) Ac2O/Py, rt, 2h; (ii)
TMSOTf/DCE, 50°C, 12h; (iii) TMSOTf, AlOH (4.5 eq.)/DCE, rt, 48h; (iv)
1M MeONa/MeOH, rt, 3h; (v) TsCl/Py, 0°C, 1h, then stirring at rt, 12h; (vi)
NaN3/DMF, 50°C, 6h; (vii) 2-azidoethanol, TMSOTf/DCE, rt, 48h; (viii) 1M
MeONa/MeOH, rt, 3h.
TTR-related
amyloidosis19–24
,
haemophilia
A/B25,26
,
Thus, the initial cyclic D-galactosamine 4 was treated with
Ac2O in pyridine to furnish fully acetylated derivative 5 in good
yield (89%). The obtained intermediate was then readily
converted into the oxazoline derivative 6 upon the treatment with
TMSOTf at 50°C (yield 92%). The subsequent ring opening in
the presence of allyl alcohol or 2-azidoethanol proceeded
smoothly and yielded compound 7 or 10, respectively. Alkaline
alkoholysis of the synthesized intermediates using MeONa
provided O-deacetylated compound 2 equipped by the extended
azide attachment point at position C1 or intermediate 8. The latter
was then easily converted into compound 9 upon the treatment
with TsCl in Py (yield 63%). The desired product 3, containing
the attachment point at position C6, was obtained from
compound 9 through the reaction with NaN3 in DMF.
hypercholesterolemia27, HBV28, primary hyperoxaluria type I29,
etc. Recently, the company has initiated Phase 2 clinical trial
(ORION30) and Phase 1 study27 with Inclisiran (ALN-PCSSC)
against elevated low density lipoprotein cholesterol (LDL-C) and
subjects diagnosed with atherosclerotic cardiovascular disease
(ASCVD) or ASCVD-risk equivalents (e.g., diabetes and familial
hypercholesterolemia). ALN-GO1 has been entered in Phase 1/2
clinical investigation in healthy adult subjects and patients with
primary hyperoxaluria Type 1 (PH1)29. Amgen has developed
AMG-529, an ASGP-R antagonist, which is currently ongoing
Phase I clinical evaluation for the treatment of cardiovascular
diseases31. However, there are no ASGP-R-targeted drug
conjugates in clinics containing Dox or PTX attached to the
GalNAc warhead through a simple linker. This is the first report
describing a direct conjugation of PTX with the ASGP-R-specific
ligand via a relatively short and spacer of an appropriate length.
PTX can be modified at positions C2’, C7 and C1. The
functionalization of C1-OH reduces the activity, but not
significantly, while the modification of C7-OH does not
influence the binding potency. The introduction of substituents at
position C2’-OH, e.g. fluorine or alkyl, is unfavorable, while
esterification results in loss of microtubule disassembly activity
in vitro, but not cytotoxicity37. It has been reported that PTX
esters are instable under the conditions of cellular endocytosis
and susceptible to esterase-mediated hydrolysis38. Acylation of
C2’-OH proceeds readily and rapidly as compared to acylation of
C7-OH and especially C1-OH.
The [3+2] azide-alkyne cycloaddition is a convenient and
simple method for laboratory synthesis which does not affect
other functional groups of reactants. In addition, the resulting
triazole cycle can serve as a bioisosteric replacement for peptide
bond. Therefore, we introduced azido group in the galactose
moiety and alkyne fragment in PTX for their subsequent
conjugation. This arrangement was based on the fact that
galactose fragment linked via position 4 by triazole fragment has
lower affinity as compared with position 132.

The general synthetic approach to the desired TDD system is
depicted in Scheme 2. PTX was modified at the most convenient
C2’ and C7 diversity points. It should be noted that in PTX
structure the most reactive hydroxyl group is attached to C2’
atom, while –OH moiety at position С7 is much less pliable. This
allowed us to carry out the selective modification of С2’-OH
group keeping position C7 unsubstituted or obtain C2’, C7-
disubstituted analogues. We have synthesized six novel
conjugates of the molecule with selective ASGP-R ligands
described above. Thus, the initial acylation of PTX was
performed with 5-hexynoic acid in CH2Cl2 in the presence of
EDC and DMAP following the procedure described by
Pilkington-Miksa and colleague39. As a result, monovalent (C2’
modification) and bivalent (C2’ and C7 attachment) precursors
11 and 12 were obtained in good yields. Monovalent building
block was isolated using 1 eq. of the acid, while bivalent
analogue was synthesized in the presence of 2-fold excess (2 eq.)
of the reactant. Purification was performed using a routine
column chromatography in СH2Cl2:MeOH (40:1).
NMR and HRMS spectra (see SI). Thus, one (for monovalent
conjugates) or two (for bivalent ligands) characteristic singlet (or
sometimes overlapped with other signals) proton at C5 atom of
1
1,4-substituted 1,2,3-triazole ring is seen in H NMR spectra at
~7.88 ppm.
As briefly described above, trivalent ligands possess the
highest binding affinity for ASGP-R vs. mono- and, in general,
bivalent analogues. To perform
a comparative biological
evaluation, we have synthesized a conjugate of PTX and
triantennary GalNAc vector. For this purpose we equipped the
GalNAc cluster designed by Prakash and colleagues15 with azido
group following the synthetic protocol depicted in Scheme 3.
We have prepared the desired ligand using two basic units: the
GalNAc moiety 16 and the tris-tricarboxylic acid 17. The first
was
obtained
by
glycosylation
of
benzyl
(6-
hydroxyhexyl)carbamate with oxazoline 615 and subsequent
removal of the protecting group. The second was prepared
according with the protocol described previously2. The activation
of carboxylic function of the initial compound 17 was carried out
by pentafluorophenyl trifluoroacetate (Pfp-TFA). Compound 18
was obtained with good yield and high purity. The activated ester
18 (1 eq.) was treated with amine 16 (3.5 eq.) in DMF in the
presence of HOBT and DIPEA as a base. The product was
isolated with 54% yield and was subsequently subjected to
alkaline alcoholysis with MeONa/MeOH to furnish the
deacetylated product 20 (94%). The conversion was complete
and quantitative. As a result, we have obtained the ASGP-R
ligand containing azido group, which can be used for further
derivatization with small-molecule drug compounds or molecular
diagnostic tools. The final product 21 was synthesized using Cu-
catalyzed [3+2] azide-alkyne cycloaddition of intermediate 11
with azide 20 under the standard conditions (sodium ascorbate,
CuSO4, DMF/H2O). Conjugate 21 was isolated with good yield
(66%) using HPLC (H2O:MeCN) and fully characterized
(Scheme 4).
At the final stage, we examined the cytotoxicity of the
resulting conjugates using the culture of human HCC cells
HepG2. It is well documented that, after the isolation of
hepatocytes from liver, the amount of ASGP-R on surface
decreases40. Accordingly, we have selected a stable, proliferating
HepG2 cell line containing 76K receptors per cell41. The HepG2
cell line has been considered as an appropriate system to evaluate
efficiency of TDD drug conjugates with selective ASGP-R-
Scheme 2. Synthesis of monovalent (13a-c) and bivalent (14a-c) ASGP-
R-targeted conjugates bearing PTX
During the next step, intermediates 1-3 were introduced into
the click reaction ([3+2]-azide-alkyne cycloaddition) with
building block 11 or 12 using CuI and Et3N (0.2 and 0.4 eq.,
respectively). The desired novel conjugates 13a-c and 14a-c were
obtained in moderate to good yields without any complications.
The final products were then purified using reversed-phase
HPLC (H2O/MeCN). The structures of the synthesized
compounds are in excellent agreement with the obtained 1H
Scheme 4. Synthesis of trivalent drug conjugate 21
targeted ligands42. To assess the cytotoxic effect of the
synthesized conjugates, a standard MTS test was performed. The
obtained results are summarized in Table 1. Cytotoxicity of the
unmodified PTX against HepG2 cells was reported by Luo and
Scheme 3. Synthesis of GalNAc “trident” containing azide attachment
point

co-authors43 and addressed in the current work for a comparative
assessment.
determined by Meier et al.46. Key supramolecular interactions in
QPDWGH crystal are described in 47 48
and in SI.
,
Table 1. Cytotoxicity of the obtained conjugates and PTX
against HepG2 cells
Compound CC50 (µmol) Compound CC50 (µmol)
13a
13b
13c
14a
14b
0.092 0.016
0.82 0.27
3.25 0.10
22.9 0.1
na*
14c
21
na*
0.11 0.12
0.12 0.01
0.21 0.03
PTX
PTX43
na – CC50 > 50 µmol (CC50 – cellular cytotoxicity)
The obtained conjugates were relatively stable under pH=5.0
and 7.4. HPLC-MS analysis did not reveal hydrolysis even after
24h (8 time points). The compounds were tested for binding to
ASGP-R using surface plasmon resonance (SPR), with results
reported as dissociation constants (Kd). The synthesized
compounds showed high affinity towards the receptor (Kd for the
conjugates are close to 10-9 while for the unmodified GalNAc
Kd~10-3). The main results for all the compounds evaluated
and detailed experimental protocol are presented in SI.
Fig. 3. Morphological changes observed in HepG2 cells treated
with the selected conjugates and PTX. Cells were fixed,
permeabilized, and immunostained with antibody against α-
tubulin (green); nuclei were stained with DAPI (red).
Recently, two novel X-Ray crystallographic structures of N-
[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-
dioxabicyclo[3.2.1]octan-4-yl]acetamide (I) and α-lactose in
complex with ASGP-R have been published32. We used this
crystallographic data to construct our 3D computational model of
the ASGP-R binding site. The model was developed and
internally validated in ICM-Pro Software v.3.8-5 (MolSoft49).
Then, three structures II-IV (Fig. 5) containing triazole fragment
were docked into the site to assess their binding affinity. As a
result, relatively good scorings were calculated for all the
structures docked. Structures II and III were predicted with a
similar binding affinity, in contrast to structure IV. Docking
details is presented in SI.
Nuclear and cytoskeletal morphology was analyzed by
immuno-fluorescence microscopy (see SI). The untreated cells
showed typical nuclear and cytoskeleton structures, with
formation of the normal mitotic spindles. Microtubules organized
as diffuse in cytoplasm (Fig. 2). After the model cells were
treated with PTX, characteristic morphology changes were
observed: mitoses with more than two poles; many abnormal
nuclei (micronuclei or multinuclear), altered microtubule
structure (thicker and denser microtubule bundles) (Fig. 3). The
similar cellular morphological changes were observed upon the
treatment using PTX-contained conjugates. Since esterification at
position C2’ resulted in loss of microtubule disassembly activity
in vitro, we tentatively speculate that the conjugates release PTX
presumably via esterase-mediated hydrolysis37.
HO
OH
OH
O
AcHN
O
I
H
Fig. 4. 3D in silico model of the ASGP-R Gal binding site (PDB:
5JQ1).
N
N
O
N
HO
HO
NAc
OH
II (green)
Fig. 2. The untreated HepG2 cells were fixed, permeabilized, and
immunostained with antibody against α-tubulin (Green). Nuclei
were stained with DAPI (in red).
O
O
HO
N
HO
NAc
N
N
OH
III (orange)
The active site of the CRD was firstly mutated from a Man-
binding protein (MBP) to that of ASGP-R44. The mutated protein
termed QPDWG was subsequently modified to another variant –
QPDWGH – with higher binding affinity towards GalNAc
compared to Gal. Thus, the crystal structure of QPDWGH
complexed with GalNAc [PDB: 1BCH45] has been considered as
an appropriate computational model to predict the binding to
ASGP-R. The first crystal structure of the H1 subunit was
O
O
N
N
N
HO
NAc
OH
IV (grey)
Fig. 5. The most similar to the template molecule I (yellow)
binding modes predicted for structures II-IV (superposition is
shown). The corresponding energetic score values (Kcal/mol) are:
-30, -32, and -26 for structures II-IV, respectively.

It is not surprising that chemotherapy with anticancer drugs,
e.g. PTX, Dox or Cisplatin, is accompanied with a relatively low
specificity, non-selective biodistribution and a plethora of severe
side-effects that significantly restrict the applicability of these
drugs. One of the most promising ways to improve the efficiency
and safety of HCC chemotherapy is the TDD of drug molecules
selectively into hepatocytes through the ASGP-R-mediated
endocytosis. Thus, GalNAc-containing carriers of different types
and architectures have achieved notable successes in recent years,
especially in development of drug-ligand conjugates, ligand-
anchored nanocarriers, and nucleic acid therapeutics (gene
delivery systems). Moreover, ASGP-R-mediated targeting is
currently used for diagnostics. This includes imaging with
positron emission tomography & magnetic resonance imaging,
intracellular uptake by nuclear and magnetic resonance imaging,
identification of circulating tumor cells in HCC patients, etc. It
can also be used for assessing disorders in pre-operative50–52 and
post-operative liver functions53,54. Based on the data on the
activity of ASGP-R ligands in vivo16, we suggest more simple
and convenient route to bivalent conjugates in order to avoid
laborious synthetic pathways to trivalent ligands. We have
synthesized a small series of conjugates containing PTX as an
anticancer drug and mono- (13a-c), bi- (14a-c) and trivalent (20)
GalNAc-equipped ASGP-R ligands using click reaction. As
mentioned above, according to the available data37, the most
convenient points suitable for modification in the structure of
PTX are position C2' and position C7. There are many examples
of the conjugation of PTX through these atoms (see the
references above). Ester group has been considered as one of the
most felicitous esterase-cleavable trigger to release the drug
inside the target cells38. It should also be noted that, as a rule, an
increase in the number of anchoring moieties in the structure of
drug-conjugates corresponds to higher binding affinity33. The
performed biological evaluation with ASGP-R-expressing
HepG2 cell line has revealed monovalent conjugates 13a and 13b
as the most effective cytotoxic agents within the novel series.
Thus, compound 13a showed cytotoxic potency (CC50=0.092
µmol) comparative to a “virgin” PTX (CC50=0.12 µmol) and tri-
valent conjugate 21 (CC50=0.11 µmol), while compound 13b was
slightly less active. Computational study described above
predicted that the core-heads of compounds 13a,b and 14a,b
possessed superior binding affinity vs. the core-head of
compound 14c. However, the comparison of CC50 values,
measured in the performed cell-based assay, with the calculated
in silico scorings is valuable rather for a rough estimation. For
more exact prognosis a protein-based binding assay is urgently
needed, and we are working on it. It is well known that drug-
conjugates containing selective ligands targeted on receptors
abundantly expressed in different types of cancer cells usually
demonstrate lower systemic toxicity and side-effects in contrast
to parent drug molecule. This benefit can be achieved only in the
case of an effective endocytosis, a sufficient drug release
capacity, and an appropriate pharmacokinetic profile of such
conjugates. Anyway, the presence of GalNAc-containing
fragments in the structure of a hybrid drug molecule increases the
chances to deliver a drug specifically into the HCC nest. Indeed,
in many cases, a promising activity observed in vitro is not
displayed in vivo and vice versa. However, it should be expected
that during the subsequent in vivo trials bivalent conjugate 14a
will show lower off-target toxicity, appropriate selectivity and
high anticancer potency as compared to PTX and trivalent
analogue 20.
synthetic route including an optimized amide synthesis followed
by click reaction to obtain the desired molecules with good
yields. All the synthesized compounds were then evaluated on
their cytotoxic activity against HepG2 cells. As a result, two
monovalent conjugates 13a and 13b showed high efficiency
comparable with PTX and trivalent derivative. Moreover,
conjugates 13a-c have higher lipophilicity, lower molecular
weight and flexibility than bivalent analogues and are more
appropriate for a passive transport. Molecular docking study has
predicted the sugar moieties of the compounds (series a,b) as the
most attractive core-heads for good ASGP-R binding. During
SPR study, the conjugates demonstrated high affinity towards the
target receptor. The synthesized hybrid molecules were quite
stable and caused the similar to PTX morphological changes in
HepG2 cells. The compounds with the best anticancer activity are
now planned to advance further in in vivo trials.
Acknowledgments
The authors gratefully acknowledge Ministry of Education and
Science of the Russian Federation, government grant
20.9907.2017/VU (expert opinion, discussion, manuscript
preparation), Russian Science Foundation №17-14-01316
(synthesis and general biological evaluation, stability)
Russian Science Foundation №17-74-30012, IBG RAS Ufa (in
silico modeling), Ministry of Education and Science of the
Russian Federation in the framework of Increase
Competitiveness Program of NUST «MISiS» №K2-2016-069 (a
part of biological evaluation), and Russian fund of innovation
promotion UMNIK №8879GU/2015 (stability analysis)
References and notes
1.
2.
World Health Organization: Cancer. Fact sheet.
http://www.who.int/mediacentre/factsheets/fs297/en/ . 2017.
Maklakova SY, Kucherov FA, Petrov RA, et al. A new approach to
the synthesis of ligands of asialoglycoprotein receptor for targeted
delivery of oligonucleotides to hepatocytes. Russ Chem Bull.
2015;64(7):1655-1662. doi:10.1007/s11172-015-1056-6.
D’Souza AA, Devarajan PV. Asialoglycoprotein receptor mediated
hepatocyte targeting - strategies and applications. J Control
Release. 2015;203:126-139. doi:10.1016/j.jconrel.2015.02.022.
Spiess M. The asialoglycoprotein receptor: a model for endocytic
transport receptors. Biochemistry. 1990;29(43):10009-10018.
doi:10.1021/bi00495a001.
3.
4.
5.
6.
Stockert J. The asialoglycoprotein receptorꢀ: relationships between
structure, function, and expression. Physiol Rev. 1995;75(3):591-
609.
Huang X, Leroux JC, Castagner B. Well-defined multivalent
ligands for hepatocytes targeting via asialoglycoprotein receptor.
Bioconjug Chem. 2017;28(2):283-295.
doi:10.1021/acs.bioconjchem.6b00651.
7.
Hu J, Liu J, Yang D, Lu M, Yin J. Physiological roles of
asialoglycoprotein receptors (ASGPRs) variants and recent
advances in hepatic-targeted delivery of therapeutic molecules via
ASGPRs. Protein Pept Lett. 2014;21(10):1025-1030.
Wang Y, Du H, Zhai G. Recent advances in active hepatic targeting
drug delivery system. Curr Drug Targets. 2014;15(6):573-599.
Roggenbuck D, Mytilinaiou MG, Lapin S V., Reinhold D, Conrad
K. Asialoglycoprotein receptor (ASGPR): a peculiar target of liver-
specific autoimmunity. Autoimmun Highlights. 2012;3(3):119-125.
doi:10.1007/s13317-012-0041-4.
8.
9.
10.
Biessen EA, Beuting DM, van Berkel TJC, Roelen HCPF, van de
Marel GA, van Boom JH. Synthesis of cluster galactosides with
high affinity for the hepatic asialoglycoprotein receptor. J Med
Chem. 1995;38(9):1538-1546. doi:10.1021/jm00009a014.
Lee K, Rafi M, Wang X, et al. In vivo delivery of transcription
factors with multifunctional oligonucleotides. Nat Mater.
2015;14(7):701-706. doi:10.1038/nmat4269.
11.
12.
Summarizing, we have synthesized a series of novel mono-
and bivalent small-molecule conjugates of the selective ASGP-R
ligands with PTX. We have validated a convenient and versatile
Chang WY, Kao HW, Wang HE, et al. Synthesis and biological
evaluation of technetium-99m labeled galactose derivatives as
potential asialoglycoprotein receptor probes in a hepatic fibrosis

mouse model. Bioorganic Med Chem Lett. 2013;23(23):6486-6491.
doi:10.1016/j.bmcl.2013.09.012.
Effect of ALN-PCSSC Treatment on Low Density Lipoprotein
Cholesterol (LDL-C) (ORION). First received: November 3, 2015.
Last updated: May 5, 2017. Current status: This study is ongoing,
but not recruiting participants.
13.
14.
Valentijn ARPM, Van Der Marel GA, Sliedregt LAJM, Van Berkel
TJC, Biessen EA, Van Boom JH. Solid-phase synthesis of lysine-
based cluster galactosides with high affinity for the
31.
32.
33.
ClinicalTrials.gov identifier: NCT03170193. AMG 529 20160338
First in Human Study. First received: May 25, 2017. Last updated:
NA. Current status: this study is currently recruiting participants.
Mamidyala SK, Dutta S, Chrunyk BA, et al. Glycomimetic ligands
for the human asialoglycoprotein receptor. J Am Chem Soc.
2012;134(4):1978-1981. doi:10.1021/ja2104679.
asialoglycoprotein receptor. Tetrahedron. 1997;53(2):759-770.
doi:10.1016/S0040-4020(96)01018-6.
Biessen EA, Valentijn AR, De Vrueh RL, et al. Novel
hepatotrophic prodrugs of the antiviral nucleoside 9-(2-
phosphonylmethoxyethyl)adenine with improved pharmacokinetics
and antiviral activity. Faseb J. 2000;14(12):1784-1792.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=
PubMed&dopt=Citation&list_uids=10973928.
Prakash TP, Yu J, Migawa MT, et al. Comprehensive structure-
activity relationship of triantennary N-acetylgalactosamine
conjugated antisense oligonucleotides for targeted delivery to
hepatocytes. J Med Chem. 2016;59(6):2718-2733.
Sanhueza CA, Baksh MM, Thuma B, et al. Efficient liver targeting
by polyvalent display of a compact ligand for the
asialoglycoprotein receptor. J Am Chem Soc. 2017;139(9):3528-
3536. doi:10.1021/jacs.6b12964.
15.
34.
35.
Salunke SB, Babu NS, Chen C-T. Iron(III) chloride as an efficient
catalyst for stereoselective synthesis of glycosyl azides and a
cocatalyst with Cu(0) for the subsequent click chemistry. Chem
Commun. 2011;47(37):10440. doi:10.1039/c1cc13370e.
Wang Q, Ekanayaka SA, Wu J, Zhang J, Guo Z. Synthetic and
immunological studies of 5’-N-phenylacetyl sTN to develop
carbohydrate-based cancer vaccines and to explore the impacts of
linkage between carbohydrate antigens and carrier proteins.
Bioconjug Chem. 2008;19(10):2060-2067. doi:10.1021/bc800243f.
Wong CH, Hendrix M, Manning DD, Rosenbohm C, Greenberg
WA. A library approach to the discovery of small molecules that
recognize RNA: use of a 1,3-hydroxyamine motif as core. J Am
Chem Soc. 1998;120(33):8319-8327. doi:10.1021/ja980826p.
Fu Y, Li S, Zu Y, et al. Medicinal chemistry of paclitaxel and its
analogues. Curr Med Chem. 2009;16(30):3966-3985.
doi:10.2174/092986709789352277.
doi:10.1021/acs.jmedchem.5b01948.
16.
17.
Ivanenkov YA, Maklakova SY, Beloglazkina EK, et al.
Development of liver cell-targeted drug delivery systems:
experimental approaches. Russ Chem Rev. 2017;86(8):750-776.
doi:10.1070/RCR4707.
Seymour BLW, Ferry DR, Anderson D, et al. Hepatic drug
targeting: Phase I evaluation of polymer-bound doxorubicin. J Clin
Oncol. 2009;20(6):1668-1676. doi:10.1200/jco.20.6.1668.
Alnylam Pharmaceuticals. http://www.alnylam.com/.
ClinicalTrials.gov identifier: NCT02595983. The study of an
investigational drug, revusiran (ALN-TTRSC), for the treatment of
transthyretin (TTR)-mediated amyloidosis in patients whose disease
has continued to worsen following liver transplant. First received:
October 30, 2015. Last updated: July 27, 2016. Current status:
ongoing, but not recruiting participants.
36.
18.
19.
37.
38.
39.
Böhme D, Beck-Sickinger AG. Controlling toxicity of peptide-drug
conjugates by different chemical linker structures. ChemMedChem.
2015;10(5):804-814. doi:10.1002/cmdc.201402514.
20.
ClinicalTrials.gov identifier: NCT02292186. A extension study to
evaluate revusiran (ALN-TTRSC) in patients with transthyretin
(TTR) cardiac amyloidosis. First received: November 10, 2014.
Last updated: July 18, 2016. Current status: ongoing, but not
recruiting participants.
Pilkington-Miksa M, Arosio D, Battistini L, et al. Design,
synthesis, and biological evaluation of novel cRGD–paclitaxel
conjugates for integrin-assisted drug delivery. Bioconjug Chem.
2012;23(8):1610-1622. doi:10.1021/bc300164t.
21.
22.
ClinicalTrials.gov identifier: NCT01981837. Phase 2 study to
evaluate ALN-TTRSC (revusiran) in patients with transthyretin
(TTR) cardiac amyloidosis. First received: November 6, 2013. Last
updated: February 4, 2016. Current status: has been completed.
ClinicalTrials.gov identifier: NCT01814839. A Phase 1, single- and
multi-dose, dose escalation study to evaluate the safety, tolerability,
pharmacokinetics, and pharmacodynamics of subcutaneously
administered ALN-TTRSC (Revusiran) in healthy volunteers. First
received: March 18, 2013. Last updated: February 4, 2016. Current
status: has been completed.
40.
41.
Schwartz AL, Ashwell G. The hepatic asialoglycoprotein receptor.
Crit Rev Biochem. 1984;16(3):207-233.
doi:10.3109/10409238409108716.
Li Y, Huang G, Diakur J, Wiebe L. Targeted delivery of
macromolecular drugs: asialoglycoprotein receptor (ASGPR)
expression by selected hepatoma cell lines used in antiviral drug
development. Curr Drug Deliv. 2008;5(4):299-302.
doi:10.2174/156720108785915069.
42.
Zhang X, Ng HLH, Lu A, et al. Drug delivery system targeting
advanced hepatocellular carcinoma: current and future.
Nanomedicine Nanotechnology, Biol Med. 2016;12(4):853-869.
doi:10.1016/j.nano.2015.12.381.
Luo D, Cheng SC-S, Xie H, Xie Y. Effects of Bcl-2 and Bcl-X L
protein levels on chemoresistance of hepatoblastoma HepG2 cell
line. Biochem Cell Biol. 2000;78(2):119-126. doi:10.1139/o00-008.
Kolatkar AR, Weis WI. Structural basis of galactose recognition by
C-type animal lectins. J Biol Chem. 1996;271(12):6679-6685.
doi:10.1074/jbc.271.12.6679.
23.
ClinicalTrials.gov identifier: NCT02319005. ENDEAVOUR: Phase
3 multicenter study of revusiran (ALN-TTRSC) in patients with
transthyretin (TTR) mediated familial amyloidotic cardiomyopathy
(FAC). First received: December 12, 2014. Last updated: August
12, 2016. Current status: ongoing, but not recruiting participants.
ClinicalTrials.gov identifier: NCT02797847. A safety and
tolerability study of an investigational drug, ALN-TTRSC02, in
healthy subjects. First received: May 18, 2016. Last updated: June
8, 2016. Current status: recruiting participants.
ClinicalTrials.gov identifier: NCT02554773. An open-label
extension study of an investigational drug, ALN-AT3SC, in
patients with moderate or severe hemophilia A or B. First received:
September 15, 2015. Last updated: July 19, 2016. Current status:
recruiting participants.
43.
44.
45.
24.
25.
Kolatkar AR, Leung AK, Isecke R, Brossmer R, Drickamer K,
Weis WI. Mechanism of N -acetylgalactosamine binding to a C-
type animal lectin carbohydrate-recognition domain. J Biol Chem.
1998;273(31):19502-19508. doi:10.1074/jbc.273.31.19502.
Meier M, Bider MD, Malashkevich VN, Spiess M, Burkhard P.
Crystal structure of the carbohydrate recognition domain of the H1
subunit of the asialoglycoprotein receptor. J Mol Biol.
2000;300(4):857-865. doi:10.1006/jmbi.2000.3853.
46.
26.
27.
ClinicalTrials.gov identifier: NCT02035605. A Phase 1 study of an
investigational drug, ALN-AT3SC, in healthy volunteers and
hemophilia A or B patients. First received: January 13, 2014. Last
updated: February 12, 2016. Current status: recruiting participants.
ClinicalTrials.gov identifier: NCT02314442. A Phase 1 study of an
investigational drug, ALN-PCSSC, in subjects with elevated low
density lipoprotein cholesterol (LDL-C). First received: December
5, 2014. Last updated: December 16, 2015. Current status: has been
completed.
47.
48.
Wu J. Targeting hepatocytes for drug and gene delivery emerging
novel approaches and applications. Front Biosci. 2002;7(4):A806.
doi:10.2741/A806.
D’Souza AA, Jain P, Galdhar CN, Samad A, Degani MS,
Devarajan P V. Comparative in silico–in vivo evaluation of ASGP-
R ligands for hepatic targeting of curcumin gantrez nanoparticles.
AAPS J. 2013;15(3):696-706. doi:10.1208/s12248-013-9474-6.
Molsoft. www.molsoft.com. 2017.
28.
29.
30.
ClinicalTrials.gov identifier: NCT02826018. A study of ALN-HBV
in healthy adult volunteers and non-cirrhotic patients with chronic
hepatitis B virus (HBV) infection. First received: July 5, 2016. Last
updated: July 6, 2016. Current status: recruiting participants.
ClinicalTrials.gov identifier: NCT02706886. Study of ALN-GO1 in
healthy adult subjects and patients with primary hyperoxaluria Type
1. First received: March 3, 2016. Last updated: March 8, 2016.
Current status: recruiting participants.
49.
50.
Hwang EH, Taki J, Shuke N, et al. Preoperative assessment of
residual hepatic functional reserve using 99mTc-DTPA-galactosyl-
human serum albumin dynamic SPECT. J Nucl Med.
1999;40(10):1644-1651.
http://www.ncbi.nlm.nih.gov/pubmed/10520704.
51.
Iimuro Y, Kashiwagi T, Yamanaka J, et al. Preoperative estimation
of asialoglycoprotein receptor expression in the remnant liver from
CT/ 99m Tc-GSA SPECT fusion images correlates well with
ClinicalTrials.gov identifier: NCT02597127. Trial to Evaluate the

postoperative liver function parameters. J Hepatobiliary Pancreat
Sci. 2010;17(5):673-681. doi:10.1007/s00534-010-0264-6.
Yumoto Y, Yagi T, Sato S, et al. Preoperative estimation of
remnant hepatic function using fusion images obtained by 99mTc-
labelled galactosyl-human serum albumin liver scintigraphy and
computed tomography. Br J Surg. 2010;97(6):934-944.
doi:10.1002/bjs.7025.
52.
53.
54.
Yoshida M, Shiraishi S, Sakaguchi F, et al. Fused 99m-Tc-GSA
SPECT/CT imaging for the preoperative evaluation of
postoperative liver function: can the liver uptake index predict
postoperative hepatic functional reserve? Jpn J Radiol.
2012;30(3):255-262. doi:10.1007/s11604-011-0041-8.
Hashida M, Hirabayashi H, Nishikawa M, Takakura Y. Targeted
delivery of drugs and proteins to the liver via receptor-mediated
endocytosis. J Control Release. 1997;46(1-2):129-137.
doi:10.1016/S0168-3659(96)01577-5.

Graphical Abstract
Synthesis and Biological Evaluation of Novel Mono- and
Leave this area blank for abstract info.
Bivalent ASGP-R-targeted Drug-conjugates
Rostislav A. Petrova, Svetlana Yu. Maklakovaa, Yan A. Ivanenkova-c,g*,
Stanislav A. Petrova, Olga V. Sergeevaa,d, Emil Yu. Yamansarova, Irina V.
Saltykovaa, Igor I. Kireevf, Irina B. Alievaf, Ekaterina V. Deynekab, Alina A.
Sofronovah, Anastasiia V. Aladinskaiab, Alexandre V. Trofimenkob, Renat S.
Yamidanovg, Sergey V. Kovaleva, Victor E. Kotelianskid, Timofey S.
Zatsepina,d, Elena K. Beloglazkinaa, Alexander G. Majougaa,c,e
.
a Lomonosov Moscow State University, Chemistry Dept, Leninskie gory, Building 1/3,
GSP-1, Moscow, 119991, Russian Federation.
b Moscow Institute of Physics and Technology (State University), 9 Institutskiy lane,
Dolgoprudny City, Moscow Region, 141700, Russian Federation.
c National University of Science and Technology MISiS, 9 Leninskiy pr, Moscow,
119049, Russian Federation.
d Skolkovo Institute of Science and Technology,100 Novaya st., 143025 Skolkovo,
Russian Federation.
e Dmitry Mendeleev University of Chemical Technology of Russia, Miusskaya sq. 9,
Moscow, 125047, Russian Federation.
f Lomonosov Moscow State University, A.N. Belozersky Institute of Physico-Chemical
Biology, Leninskye gory, house 1, building 40, Moscow, 119992, Russian Federation
g Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of
Sciences (IBG RAS), Prosp. Oktybrya 71, Ufa, Bashkortostan, 450054, Russian
Federation.
h Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics,
Moscow, Russia.