This study aimed to improve the antitumor compound furanoallocolchicinoid 2 by conjugating it with chitosan to reduce hydrophobicity and increase molecular weight. Researchers synthesized compound 4 by conjugating furanoallocolchicinoid 3 with chitosan. In vitro and in vivo testing found that compound 4 more effectively inhibited tumor cell proliferation and growth compared to compounds 2 and 3 due to its shielding from interactions with blood cells. The results demonstrate that cheap production and effectiveness of colchicine-chitosan conjugates can improve antitumor efficacy.

1. Rachel Shore
11/30/16
Journal Review for CHE350
Article
Svirshchevskaya, E. V., Gracheva, I. A., Kuznetsov, A. G., & Myrsikova, E. V. (2016).
Antitumor Activity of Furanoallocolchicinoid-Chitosan Conjugate. Medicinal Chemistry, 6(9).
doi:10.4172/2161-0444.1000401
Introduction
Colchicine is a small, non-polar molecule and inhibitor for the enzyme β-tubulin, which is
crucial for microtubule formation during mitosis [1]. The small, hydrophobic character of
colchicine allows it to penetrate cell membranes, while its β-tubulin inhibitor activity prevents
cell division, leading to the prevention of tissue growth [1]. Colchicine accumulates in tumors at
a higher rate than it accumulates in normal tissues for a few reasons. First, the high growth and
division rates of tumor cells necessitate increased blood supply, meaning that drugs in the blood
stream are taken up by tumor cells at a disproportionately high rate [2]. Second, tumor tissues
often lack the effective lymphatic drainage that normal tissues have, preventing tumor tissues
from removing drugs and other unwanted molecules [2]. Collectively, these factors and are
known as the “enhanced permeability and retention effect” and lead to a high accumulation of
colchicine in tumor cells [3].
Colchicine’s ability to permeate cell membranes, inhibit cell division, and accumulate quickly in
tumor tissues makes it a good antitumor agent [3,4]. However, colchicine has a high partition
coefficient, meaning that it partitions quickly into adjacent cell membranes and can thus bind to
various blood cells and endothelial and epithelial cells before reaching its target organ [4]. This
leads to many negative side effects because non-target tissues cannot fully undergo mitosis [4].
Chemists have found that adding hydrophilic groups to the colchicine molecule or increasing its
molecular weight can increase tissue specificity by reducing unspecific tissue partition [5]. This
prevents colchicine from affecting organs other than the target organ [5]. Since modifications of
colchicine itself have been shown to increase target specificity, drug conjugates of colchicine are
a popular area of study within antitumor drug research [5].
The Ringsdorf Model, which states that conjugating a drug to a polymer backbone and adding a
targeting moiety increase target selectivity, has been the basis for chemical modifications of
colchicine [6]. Multiple conjugates of colchicine have been synthesized, notably a compound
including polyethylene glycol (PEG), which is often used to increase polarity and molecular
weight of antitumor drugs [5]. PEG helps to prevent colchicine from interacting with plasma
albumin and decreases other unfavorable interactions, reducing overall side effects of the drug
[7]. Unfortunately, PEG molecules only have one reactive functional group that can be used for
conjugation, making it difficult to add a targeting group to the conjugate or immobilize
molecules on a polymeric backbone [8-11].

2. In terms of biocompatible polymers, chitosan is commonly used because it can be easily
manipulated to obtain a derivative of desired charge and hydrophobicity [12]. Chitosan on its
own is used to treat wounds because of its hemostatic properties [13]. Given the Ringsdorf
Model and the use of chitosan for polymers, this research on colchicine derivatives discusses the
synthesis of a series of allocolchicine analogues called furanoallocolchicinoids. The purpose of
this research was to study the in vitro and in vivo activities of a conjugation of
furanoallocolchicinoids with chitosan.
Synthesis and Testing of Furanoallocolchicinoids
In previous research,
furanoallocolchicinoid 2 was
synthesized from colchicine by
replacing a heptacyclic ring
with hexocyclic and pentacyclic
structures as shown in the
diagram below [14]. Instead of
a carbonyl group as a
substituent of the ring, an
alcohol group is used increase
target binding (fig. 1). The
activity of
furanoallocolchicinoid 2
compound was shown to be 5-10 times higher than the activity of colchicine, which had been
previously demonstrated in earlier experiments [14]. The researchers wanted to modify
furanoallocolchicinoid 2 to a new allocolchicine derivative that would show improved activity.
They synthesized the new compound, furanoallocolchicinoid 3, by dissolving compound 2 and
succinic anhydride in tetrohydrofuran, adding trimethylamine to the solution, and extracting the
resulting solution with EtOAc. The
resulting compound furanocolchicinoid 3
had a similar maximal inhibition level to
compound 2 in vitro. In addition,
compound 3 was shown to disrupt
tubulin microtubules and block mitotic
spindle formation in both 2D (cells
grown on same plane) and 3D conditions
(cells grown in 3D layers—more closely
mimicking natural tissues). In vivo, cells
from the Colo-357 cell line and W1204
cell line (originating from Wnt-1 breast
tumor), demonstrated sensitivity to
compound 3. Fig. 5 shows the effects of
Fig. 1: Furanoallocolchicinoid 2 (2) is synthesized from colchicine
(1), increasing drug activity by 5-10 times.
Fig. 2: Furanoallocolchicinoid 3 is synthesized from
furanoallocolchicinoid 2 with succinic anhydride. Compound 2
showed 66% maximal inhibition of tubulin while compound 3
showed 67% maximal inhibition of tubulin.

3. compound 3 on cell cycling in 2D and 3D cultures. Incubation of epithelial cells with compound
3 caused cells to accumulate in the G2/Metaphase stage of cell proliferation, indicating that
compound 3 effectively inhibited tubulin and prevented formation of a mitotic spindle. However,
the activity of the carboxylic acid group necessary for conjugation with chitosan showed an
activity comparable to the activity of colchicine (compound 1).
The final step in following the Ringsdorf Model was to add a
biocompatible polymer to reduce hydrophobicity and increase
molecular weight. As discussed earlier, chitosan (fig. 3) was the
chemical of choice for the polymer, so furanoallocolchicinoid 3 was
conjugated with a polymer of this molecule to get
furanoallocolchicinoid 4. The polymerization and conjugation
process is outlined in figure 4.
The chitosan polymer added significant molecular weight considering the individual units’
molecular weight of 40 kDa. It also reduced hydrophobicity because of the polar nature of
chitosan (due to its alcohol groups). Both epithelial cell lines (Colo-357 and W1204) were found
to have high sensitivity to the furanoallocolchicinoid-chitosan conjugate in both 2D and 3D
cultures. In vitro, compound 4 was shown to inhibit β-tubulin in three additional tumor cell lines,
and was shown to be more potent due to chitosan conjugation. Similar to compound 3,
compound 4 was also shown to induce cell accumulation in the G2/M phase of mitosis, meaning
that it successfully prevented cell proliferation (fig. 5).
Final Result
This study found the final compound, furanoallocolchinoid 4, to shield the molecule from
interactions with blood cells, thereby reducing side effects and accumulation in non-target
organs. This overall improves anti-tumor efficacy. While the in vitro results for compounds 3
and 4 are shown in figure 5, the in vivo effects of each compound on Wnt-1 breast tumor growth
in mice are shown in figure 6. The results clearly show that furanoallocolchicinoid 2 reduces
tumor growth as previously demonstrated, and that compound 4 further improves the anti-tumor
effect. While compound 2 is capable of slowing tumor growth from 4000 cubic millimeters in 40
days to about 2500 cubic millimeters in 40 days, compound 4 was able to further decelerate
Fig. 3: Chitosan to be polymerized
and used to conjugate compound 3.
Fig. 4: Polymerization and conjugation of
compound 3 with chitosan to obtain compound 4.

4. tumor growth to only 2000 millimeters in 40 days. This shows that compound 4 effectively
prevents tumor growth, and may be considered as a medication for cancer treatment.
Furthermore, this research demonstrates that cheap production and effectiveness of colchicine-
chitosan conjugates overall.
Fig. 5: Effects of compounds on cell cycles in 2D (A – D) and 3D (E – H) conditions. A and E show the cell
cycle with no colchicinoids added, and B/F, C/G, and D/H show the effects on the cell cycles caused by
compounds 1, 2, and 3 respectively. The peaks represent the cell cycling events in which cells accumulated—the
G2/M phase in this case.
Fig. 6: Effect of compounds 2 and 4 on Wnt-1 tumors in
mice. The effect of chitosan alone was also observed since
it may have other anti-tumor effects that could affect the
results.

5. Summary
The purpose of this research was to improve the compound furanoallocolchicinoid 2 synthesized
in previous antitumor research by adding a chitosan polymer to reduce hydrophobicity and
increase molecular weight in accordance with the Ringsdorf Model. It was hypothesized that the
resultant compound would better penetrate target cells and more effectively inhibit cell
proliferation, which was shown in both in vitro and in vivo studies of the compound. A complete
synthesis of the resultant compound is shown in figure 7.
Fig. 7: Summary of chemical synthesis of
furanoallocolchicinoid-chitosan conjugate

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